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Annual Meeting of the German Pharmaceutical Society – DPhG

Pharmaceutical Sciences We live interdisciplinarity

Munich, GermanyOctober 4 – 7, 2016at Ludwig-Maximilians-University


Munich, Germany October 4 – 7, 2016 at Ludwig-Maximilians-University

www.2016.dphg.de

ISBN 978-3-9816225-3-9

DPhG

Annual Meeting 2016 – Conference Book

Conference BookPharmaceutical SciencesWe live interdisciplinarity

Annual Meeting of the GermanPharmaceutical Society 2016 – DPhG

Printed by:

Munich, Germany, Oktober 2016

ISBN 978-3-9816225-3-9

REPRODUKT digital GmbHStahlgruberring 2081829 München

Annual Meeting of the German

Pharmaceutical Society – DPhG

Conference Book


Munich, Germany

October 05 – 07, 2016

at Ludwig-Maximilians-University

www.2016.dphg.de

Institutional Sponsors

Förderer der DPhG-Jahrestagung 2016

DPhG Annual Meeting 2016 Conference Book • 1

TABLE OF CONTENTS

CONFERENCE COMMITTEES .................................................................................................................................. 2

WELCOME ADDRESS ................................................................................................................................................ 3

GENERAL INFORMATION ........................................................................................................................................ 4

LOCATIONS ................................................................................................................................................................. 6

CONFERENCE PROGRAM OVERVIEW .................................................................................................................. 9

1 PLENARY LECTURES ............................................................................................................................................ 17

2 SCIENTIFIC LECTURES ....................................................................................................................................... 25

2.1 Materials for Drug Delivery .................................................................................................................. 26 2.2 Ion Channels in Health and Disease .................................................................................................... 30 2.3 Progress in Drug Synthesis ................................................................................................................... 34 2.4 New Research, New Researchers I ...................................................................................................... 38 2.5 New Approaches in Gene and Stem Cell Therapy ........................................................................... 44 2.6 MS Based Drug Screening ..................................................................................................................... 48 2.7 The Diversity in Pharmaceutical Biology ........................................................................................... 51 2.8 The Quality of Therapeutic Proteins .................................................................................................. 55 2.9 Anti-Infectives .......................................................................................................................................... 59 2.10 New Research, New Researchers II ..................................................................................................... 62 2.11 Clinical Pharmacy .................................................................................................................................... 68 2.12 Advances in Drug Formulation and Biopharmaceutics ................................................................... 72 2.13 Fighting Depression ................................................................................................................................ 76 2.14 Interface Tumor/Inflammation ............................................................................................................ 79 2.15 Industrial Pharmacy ................................................................................................................................ 83

3 POSTERS ................................................................................................................................................................. 87

3.1 Analytics .................................................................................................................................................... 88 3.2 Inflammation ............................................................................................................................................ 97 3.3 Cancer/Inflammation .......................................................................................................................... 105 3.4 Biotechnology/Protein Drugs ............................................................................................................. 115 3.5 Clinical Pharmacy ................................................................................................................................. 118 3.6 Drug Design/Medicinal Chemistry ................................................................................................... 123 3.7 GPCR/Ion Channels .............................................................................................................................. 142 3.8 Natural Compounds/Chemical Biology ........................................................................................... 145 3.9 Biopharmaceutics ................................................................................................................................. 147 3.10 Pharmaceutical Technology and Biomaterials ............................................................................... 152 3.11 Pharmacology ....................................................................................................................................... 165 3.12 Regulatory Sciences/Industrial Drug Development ...................................................................... 169 3.13 Antiinfectives ........................................................................................................................................ 171 3.14 Neurological Disorders ....................................................................................................................... 174 3.15 Drug Screening ..................................................................................................................................... 175 3.16 Other Topics .......................................................................................................................................... 177

AUTHOR INDEX ..................................................................................................................................................... 182

2 • DPhG Annual Meeting 2016 Conference Book

CONFERENCE COMMITTEES

Scientific committee: Prof. Dr. Stefan Laufer Prof. Dr. Andreas Link Dr. Olaf Queckenberg Prof. Dr. Christoph Friedrich Prof. Dr. Peter Gmeiner Prof. Dr. Jochen Klein Prof. Dr. Peter Langguth Prof. Dr. Kristina Friedland Prof. Dr. Angelika Vollmar Prof. Dr. Hermann Wätzig Prof. Dr. Thomas Efferth Prof. Dr. Ulrike Holzgrabe

Prof. Dr. Ulrich Jaehde Prof. Dr. Heyo Kroemer Prof. Dr. Irmgard Merfort Prof. Dr. Klaus Mohr Prof. Dr. Peter Ruth Prof. Dr. Manfred Schubert-Zsilavecz Prof. Dr. Andrea Sinz Prof. Dr. Holger Stark Prof. Dr. Dieter Steinhilber Prof. Dr. Werner Weitschies Prof. Dr. Gerhard Winter

Organization committee: Dr. Lars Allmendinger Prof. Dr. Martin Biel Prof. Dr. Franz Bracher Dr. Simone Braig Laura Engelke Prof. Dr. Wolfgang Frieß PD Dr. Dr. Christian Grimm Dr. Verena Hammelmann Katharina Heimberger Dr. Sandra Hemmers Dr. Marco Keller

Dr. Ulrich Lächelt PD Dr. Johanna Liebl Dr. Jörg Pabel Prof. Dr. Franz Paintner Dr. Selma Speith-Kölbl Prof. Dr. Angelika Vollmar Prof. Dr. Ernst Wagner Prof. Dr. Christian Wahl-Schott Prof. Dr. Klaus Wanner Prof. Dr. Gerhard Winter


WELCOME ADDRESS

As President of the Deutsche Pharmazeutische Gesellschaft (DPhG) and as congress Chairman of this meeting it is our pleasure to welcome you in Munich as attendees of our 2016 Annual Meeting.

Although our conference should be seen as a continuation of many successful annual meetings held by our society over the last years, we want to invite you to jointly move one step further with respect to emphasizing the interdisciplinarity of our profession and our pharmaceutical sciences. We have put that vision not only into our conference title but also let it come to life within our sessions and plenary lectures. You will notice that the themes touched in the 6 plenary lectures are all extended in one session linked to the subject, indicating that the structure of the meeting is not arbitrarily set but clearly laid out to certain focus topics. The poster session with its truly colorful variety of topics from all disciplines has long been a signature part of the meeting and we have added more time to allow all of us to see the posters without being distracted by too many parallel events. The number of posters we received fills the capacity of the available premises at the Chemistry and Pharmacy Campus in Großhadern with three sessions.

Another focus we, the President and the Chairman, share is the strong appreciation of the work of our young academic researchers. In particular with our German academic systems in mind, we know that the decision for an academic career is risky and seems often less rewarding in the early years compared to other careers. It is therefore of utmost importance to support those who pursue such an academic path. They carry the future of the pharmaceutical sciences on their shoulders. Consequently we have assigned two full prime time sessions, chaired by the President and the Vice-President, to young researchers and their work.

Many thanks go to the local organizers, the scientific chairs of the sessions, to the DPhG sections and scientific advisory board who all have worked together almost seamlessly to set the stage for this event. Besides the science and networking during the conference, we invite you to spend some time before or after in Munich and around in beautiful Upper Bavaria.

Our social event in the world famous Augustinerkeller with its typical Bavarian style may allow us all to relax with old and also new-found friends. At that point we want to express our gratitude to our General secretary Dr. Stein, who worked hard behind the scenes to ensure that the logistics of such a conference finally work well.

The conference book you hold in your hand carries all the necessary information on schedules, abstracts, poster-dates etc. and may also serve as a reference, later when you are back home.

We hope we have a fruitful meeting and a good time together

Stefan Laufer, President Gerhard Winter, Chairman


GENERAL INFORMATION

The Annual DPhG Meeting 2016 takes place at the faculty of chemistry and pharmacy (Butenandtstraße 5-13) in building F of the Ludwig-Maximilians-University Munich. LANGUAGE

The Conference language is English, no simultaneous translation will be provided. INSTRUCTIONS FOR USING CONFERENCE WLAN

If your institution is member of the “eduroam” community, you can use the wireless network “eduroam”. The configuration of your device should be the same as instructed by your home institution. Please use your account and the domain of your home institution. If your institution is not member of the “eduroam” community, you can obtain a guest account and a password at the Conference Office. WLAN: mwn-events USER-NAME: DPhGLMU2016 CONFERENCE OFFICE

The Conference office is located at the Leipelt-Foyer in the Conference building F. Opening hours: Wednesday, October 5th, 2016: 9:30 – 19:00 Thursday, October 6th, 2016: 8:00 – 19:00 Friday, October 7th, 2016: 8:00 – 14:00 LIABILITY

The organizers of the conference cannot be held responsible for any loss, theft, damage or injury to any person or property during the Conference, whatever the cause may be. The liability of persons and enterprises providing means of transportations or other services remains unaffected. Each congress participant and accompanying person takes part in all tours at his/her own risk.


ABSTRACT AND POSTER NUMBERS

Each abstract has a unique identifier, a letter-number combination. Letters refer to the conference topic, a contribution was assigned to (plenary lectures are identified by the letter “P”, scientific lectures by the letters “SL”, and poster presentations by the letters “POS”). Please note that in case of poster presentations the abstract number is identical with the poster number. Please refer to the author’s index on page 182 for direct access to specific abstracts. For all speakers: please hand over your presentation as a Power Point file at the conference desk until 8.30 am (for morning sessions) or 12.30 pm (for afternoon sessions). POSTER SESSIONS

Topics: Analytics, Inflammation, Cancer/Inflammation, Biotechnology/Protein Drugs, Clinical Pharmacy, Drug Design/Medicinal Chemistry, GPCR/Ion Channels, Natural Compounds/Chemical Biology, Biopharmaceutics, Pharmaceutical Technology and Biomaterials, Pharmacology, Regulatory Sciences/Industrial Drug Development, Antiinfectives, Neurological Disorders, Drug Screening, Other Topics. The posters were assigned to three poster sessions. Check the list below in which session you are presenting your poster. Presenting authors are requested to be present at their poster during the poster session.

Poster Session I: POS.47 – POS.70, POS.92 - POS.118, POS.158 – POS.170 and POS.223 – POS.237

Poster Session II: POS.26 – POS.45, POS.119 – POS.145, POS.171 – POS.199 and POS.217 – POS.219

Poster Session III: POS.1 – POS.23, POS.71 – POS.91, POS.146 – POS.157, POS.200 – POS.216 and POS.220 – POS.222

CONFERENCE DINNER

Separate registration necessary (special fee). Please refer to the Conference Office for registration and details. The Conference dinner will take place at “Augustinerkeller”, Arnulfstr. 52, 80335 Munich. BADGES:

Badges will be issued to all registered participants and enable access to all scientific sessions.

Poster session I Poster session II Poster session III

Session

Wednesday, October 5th, 2016, 15:00 – 15:30 and

18:00 – 21:00

Thursday, October 6th, 2016, 11:30 – 13:00 and

15:30 – 16:00

Friday, October 7th, 2016,

11:30 – 13:00

Set-up Wednesday,

October 5th, 2016, before 13:00

Thursday, October 6th, 2016,

before 10:00


before 10:00

Dismantling Wednesday,

October 5th, 2016, after 21:00

Thursday, October 6th, 2016,

after 18:30


after 15:00


Waldhüterstraße Großhadern

Pas ing

Hörsaalbere ich

LOCATIONS

Ludwig-Maximilians-University, campus Großhadern: The Congress will take place at Ludwig-Maximilians-University Munich (Campus Großhadern), faculty of chemistry and pharmacy, Butenandtstr. 5-13, building F (ground floor and basement), 81377 Munich. Arriving by plane and public transport: From the Munich airport take the suburban train S8 or S1 to Marienplatz. Change to the subway U6 in the direction of Klinikum Großhadern and get off at Großhadern. Take the rear extit and there take the left stairway. You are now on Würmtalstraße. Now you can walk along Würmtalstraße until the campus comes into view (large modern buildings) on the left (about 10 to 15 minutes walk). To arrive by bus from the subway station Großhadern, follow the sign bus 160 in the direction of Pasing or bus 268 in the direction of Gräfelfing. Exit the bus at Waldhüterstraße. Arriving by train and public transport: Munich Central station (München Hauptbahnhof) is connected to all international long-distance routes. From there, take the subway U1 or U2 to Sendlinger Tor. Change to the subway U6 in the direction of Klinikum Großhadern and get off at Großhadern. Take the rear extit and there take the left stairway. You are now on Würmtalstraße. Now you can walk along Würmtalstraße until the campus comes into view (large modern buildings) on the left (about 10 to 15 minutes walk). To arrive by bus from the subway station Großhadern, follow the sign bus 160 in the direction of Pasing or bus 268 in the direction of Gräfelfing. Exit the bus at Waldhüterstraße.

Figure 1: How to arrive to the Annual Meeting of the DPhG from the subway station Großhadern: Dashed line: arriving by foot / Continuous line: arriving by bus (picture from MVV, with kind permission)


Arriving by car: From the Nürnberg highway (A9): get onto the Mittlerer Ring B2R (direction Autobahn Lindau, A96), then onto the Lindau highway to the exit Blumenau, keep left onto Waldwiesenstraße until you reach the subway station Großhadern. From there, you will find several parking areas as described below.

From the Stuttgart highway (A8): from the end of the highway in Obermenzing turn off to Pasing, from Pasing drive in the direction of Gräfelfing, then turn left to Großhadern. From there, you will find several parking areas as described below.

From the Salzburg (A8) or Garmisch (A95) highways: drive onto the Mittlerer Ring B2R in the direction of Großhadern and Stuttgart. At Luise-Kiesselbach-Platz turn left towards Großhadern. Follow Waldfriedhofstraße, then Würmtalstraße until the subway station Großhadern. From there, you will find several parking areas as described below.

Parking areas: The only parking possibilities close to the conference building are located at Klinikum Großhadern. The daily parking fee is € 7,-. At the subway station Großhadern enter Sauerbruchstraße and follow the signs towards Klinikum Großhadern. Take the second street to your right hand side entering Marchioninistraße. Here you will find several parking areas.

If you are using the navigation system of your mobile phone, scan the following QR-codes. They will lead you to google maps, where the address of the parking areas and the congress building is already inserted.

Parking possibilities

(how to get to the

parking areas)

Building F, Butenandtstraße 11

(how to get to the

campus Großhadern)

Figure 2: Front view of the congress building F

Figure 3: Plan of the congress building F with its lecture halls


Augustiner-Keller (Conference Dinner, Thursday, 6th October, 19.30): Arnulfstraße 52, 80335 München Arriving from Central Station: The subways U1, U2, U4 and U5 and suburban trains S1 – S8 are leading to Central Station. Take the northern exit towards Arnulfstraße and keep left. After 650 m you will find Augustiner-Keller on your right hand side. Arriving from Hackerbrücke: The suburban trains S1 – S8 are leading to Hackerbrücke. Exit Hackerbrücke, enter Grassnerstraße and keep right. After 300 m you will find Augustiner-Keller on your left hand side. If you are using the navigation system of your mobile phone, scan the following QR-code. It will lead you to google maps, where the address of Augustiner-Keller is already inserted.

Augustiner-Keller

(how to get to the Augustiner-Keller)

Vorsymposium der Fachgruppe Geschichte der Pharmazie (Ehrensaal des Deutschen Museums):

Arriving from Isartor:

All suburban trains in the direction of Ostbahnhof are leading to Isartor. There, follow the signs “Deutsches Museum” and exit the station onto Zweibrückenstraße. Follow Zweibrückenstraße until you reach to the river Isar. There turn right and follow Erhardtstraße. Take the first bridge to your left (Boschbrücke) and enter the main entrance of the Deutsches Museum. There will be a Meeting Point where a representative of “Fachgruppe der Geschichte der Pharmazie” will guide you to the lecture hall (Ehrensaal des Deutschen Museums).

CONFERENCE PROGRAM OVERVIEW



Pre-Meeting Program

Tuesday, October 4th

Vorsymposium der Fachgruppe „Geschichte der Pharmazie“: Pharmazie in München Ort: Ehrensaal des Deutschen Museums, München

14:00 – 14:15

Begrüßung durch den Vorsitzenden der FG Geschichte der Pharmazie Prof. Dr. Christoph Friedrich, Marburg

und des Generaldirektors des Deutschen Museums Prof. Dr. Wolfgang M. Heckl

14:15 – 15:00

Zur Entwicklung des Hochschulfaches Pharmazie an der Universität München Dr. Doris Winter, München

15:00 – 15:45

Zur Entwicklung des Apothekenwesens in München Dr. Gerhard Gensthaler, München

15:45 – 16:15

Kaffeepause

16:15 – 17:00

Zur Geschichte der pharmazeutischen Industrie in München: Pharmazeutische Familienunternehmen – Know How, Flexibilität und eigene Marken

Dr. Ursula Lang, Seefeld

17:00 – 17:45

Die pharmaziehistorische Sammlung des Deutschen Museums München Dr. Florian Breitsameter, München

19:00 – 21:30

Treffen Arbeitsgemeinschaft Katastrophenpharmazie Ort: Campus Großhadern, Haus F, Butenandt-HS

20:00 – 21:30

Notfall- und Katastrophenpharmazie - Die Arbeit des Apothekers in der internationalen Katastrophenhilfe

Dr. Carina Vetye-Maler, Buenos Aires



Wednesday, October 5th Main Symposium (Congress language English)

10:30 – 12:00

Sitzung Verband der Professoren an Pharmazeutischen Hochschulinstituten (Konferenz der Fachbereiche Pharmazie), Leitung: Prof. Dr. Bernd Clement (Buchner-HS)

13:00 – 13:30

Opening of the Annual DPhG Meeting 2016 (Liebig-HS) Pharmaceutical Sciences, We live interdisciplinarity

13:30 – 14:15

Plenary lecture 1, Achim Göpferich, The challenge of delivering biologics (Liebig-HS) P.1

14:15 – 15:00

Plenary lecture 2, Jörg Striessnig, L-Type Ca2+ channels in brain disorders? New targets for old drugs? (Liebig-HS) P.2

15:00 – 15:30

Poster viewing (Cancer/Inflammation, Drug Design/Medicinal Chemistry I, Biopharmaceutics, Other Topics) and coffee break

SHORT TALKS (parallel sessions I)

15:30 – 17:00

SL1 (Buchner-HS) SL2 (Butenandt-HS) SL3 (Willstätter-HS)

Materials for Drug Delivery Chairs: W. Frieß, W. Weitschies

Ion Channels in Health and Disease Chairs: R. Lukowski, P. Ruth

Progress in Drug Synthesis Chairs: P. Gmeiner, M. Heinrich

15:30 SL.01 Ferdinand Brandl: Hydrogels for controlled release of protein therapeutics

15:30 SL.05 Thomas Wieland: Phosphorylation of histidine residues by nucleoside diphosphate kinase B: A novel mechanism to regulate ion channel activity in disease

15:30 SL.09 Burghard König: Making and breaking of chemical bonds with light - Visible light photocatalysis and photochromic molecular switches

15:55 SL.02 Anne Seidlitz: Evaluation of different polymers for implants produced via fused deposition modeling

15:55 SL.06 Alexander Dietrich: TRPC channels in lung function and disease

15:55 SL.10 Michael Müller: Diversity-oriented synthesis of pharmaceuticals

16:20 SL.03 Peter Wich: Biopolymers as multifunctional materials for nanoparticulate drug delivery

16:20 SL.07 Achim Schmidtko: Pain control by calcium-activated potassium channels

16:20 SL.11 Nicolas Blanchard: Chemical synthesis of mycolactone analogs - insights into human mycobacterium ulcerans infection

16:40 SL.04 Gregor Fuhrmann: Extracellular vesicles as smart carriers for small molecule drugs

16:45 SL.08 Christian Schmidt: Noradrenergic and serotonergic compounds to target narcoleptic episodes in a mouse model

16:45 SL.12 Christian Ducho: Inhibitors of the bacterial translocase MraY as potential novel antibiotics

17:00 – 18:00

Meetings der DPhG-Fachgruppen (in den Hörsälen)

Fachgruppe Pharm./Med. Chemie P. Gmeiner (Liebig)

Fachgruppe Pharm. Biologie A. Vollmar (Buchner)

Fachgruppe Pharma-kologie J. Klein (Butenandt)

Fachgruppe Pharm. Technologie P. Langguth (Wieland)

Fachgruppe Klinische Pharmazie K. Friedland (Willstätter)

Fachgruppe Industrie-pharmazie C. Küster (Baeyer)

18:00 – 21:00

Poster viewing (Cancer/Inflammation, Drug Design/Medicinal Chemistry I, Biopharmaceutics, Other Topics) and welcome reception



Thursday, October 6th

9:00 – 9:45

Plenary lecture 3, Eckhard Wolf, Genetically tailored pigs as organ donors and models for medical research (Liebig-HS) P.3

SHORT TALKS (parallel sessions II)

10:00 – 11:30


New Research, New Researchers I Chairs: S. Laufer, A. Link

New Approaches in Gene and Stem Cell Therapy Chairs: M. Biel, E. Wagner

MS based Drug Screening Chairs: M. Lämmerhofer, K. Wanner

10:00 SL.13 Julia Engert (Pharmaceutical Technology): Application of a dried H1N1 vaccine by epidermal powder immunization in piglets using a novel pyrotechnically driven applicator elicits antigen-specific antibodies

10:00 SL.19 Volker Busskamp: Evaluating microRNA-based therapies in stem cell-derived retinal organoids

10:00 SL.23 Gabriella Massolini: Frontal affinity chromatography and mass spectrometry: a perfect fit in drug discovery

10:15 SL.14 Alexander Titz (Med. Chem.): The virulence factor LecB varies in clinical isolates: consequences for ligand binding and drug discovery

10:25 SL.20 Caroline Le Guiner: Localized vs systemic gene therapy using rAAV vectors: achievements and remaining challenges; the example of Duchenne Muscular Dystrophy gene therapy

10:30 SL.15 Christian Grimm (Pharmacology): From mucolipidosis type IV to ebola: Insights into function and pharmacology of endolysosomal TRP channels

10:30 SL.24 Johannes Ottl: MS in pharmaceutical drug discovery

10:45 SL.16 Astrid Kahnt (Biochemistry and Pharmacology): Molecular mechanisms of lipoxin and resolvin biosynthesis

10:50 SL.21 Hildegard Büning: From viruses to designer nanoparticles – tailoring adeno-associated viruses for gene therapy 11:00 SL.17

Andreas Koeberle (Biochemistry): Insights from functional lipidomics into the long-term regulation of kinases by vitamin A

11:00 SL.25 Kai Scheffler: High resolution mass spectrometry of antibody drug conjugates using the orbitrap mass analyzer 11:10 SL.22

Stylianos Michalakis: Gene therapy for human achromatopsia

11:15 SL.18 Finn Hansen (Med. Chem): α-Aminoxy peptides: from membranolytic anticancer foldamers to the first in class peptidomimetic Hsp90 C-terminal domain dimerization inhibitors



11:30 – 13:00

Poster viewing (Inflammation, Drug Design/Medicinal Chemistry II, Pharmaceutical Technology and Biomaterials, Neurological Disorders) and lunch break

13:00 – 13:45

Plenary lecture 4, Hans-Georg Rammensee, Impact of the immunogenic landscape of cancers on immunotherapy (Liebig-HS) P.4

SHORT TALKS (parallel sessions III)

14:00 – 15:30


The Diversity in Pharmaceutical Biology Chairs: I. Merfort, T. Efferth

The Quality of Therapeutic Proteins Chair: H. Wätzig

Anti-Infectives Chair: R. Hartmann

14:00 SL.26 Thomas Efferth: Beyond malaria: The clinical anticancer activity of artesunate

14:00 SL.30 Martin Schiestl: Analytical tools in the biosimilar development

14:00 SL.34 Rolf Müller: Innovative antibiotics from microorganisms: Some case studies

14:30 SL.27 Rudolf Bauer: Application of plant metabolomics in herbal drug research

14:30 SL.31 Christoph Scherübl: Technical challenges for development and manufacturing of biopharmaceuticals

14:35 SL.35 Shahriar Mobashery: New antibiotics for the post-antibiotic era 15:00 SL.28

Jennifer Herrmann: Cystobactamids: Novel gyrase inhibitors from myxobacteria that inhibit multi-resistant pathogens

15:00 SL.32 Ellen Köpf: How does pH Affect interfacial antibody behaviour and aggregation upon shaking?

15:10 SL.36 Ralph Holl: LpxC inhibitors – a novel class of antibiotics

15:15 SL.29 Leonard Kaysser: Genome mining-guided drug discovery: from proteasome to protease inhibitors

15:15 SL.33 Stefan Krimmer: Rational design of thermodynamic and kinetic binding profiles by optimizing surface water networks coating protein bound ligands

15:30 – 16:00

Poster viewing (Inflammation, Drug Design/Medicinal Chemistry II, Pharmaceutical Technology and Biomaterials, Neurological Disorders) and coffee break



SHORT TALKS (parallel sessions IV)

16:00 – 17:30


New Research, New Researchers II Chairs: A. Link, S. Laufer

Clinical Pharmacy Chair: K. Friedland, C. Wahl-Schott

Advances in Drug Formulation and Biopharmaceutics Chairs: P. Langguth, H. Rein

16:00 SL.37 Steffen Lüdeke (Pharmaceutical Analytics): How polysaccharide superstructure impacts hydrogel properties: A Raman optical activity study

16:00 SL.43 Carsten Culmsee: Evidence-based evaluation system for OTC drugs

16:00 SL.47 Peter Kleinebudde: Thoughts on a manufacturing classification system

16:15 SL.38 Dominique Lunter (Pharmaceutical Technology): Skin penetration analysis by confocal Raman microspectros-copy – potentials and pitfalls

16:25 SL.44 Dorothea Strobach: Influence of over-the-counter drugs and prescription-only-medication on male fertility

16:25 SL.48 Christel Müller-Goymann: Formulation development for topical treatment of tinea pedis and onychomycosis

16:30 SL.39 Sarah Hedtrich (Pharmacology): Influence of Th2 cytokines on the cornified envelope, tight junction proteins and ß-defensins in filaggrin-deficient skin equivalents

16:50 SL.45 Hartmut Derendorf: Evidence-based dose finding using modeling and simulation on earth and in space

16:50 SL.49 Richard Hirsch: Improving the quality of split tablets

16:45 SL.40 Karin von Schwarzenberg (Pharmaceutical Biology): Tumor selectivity of V-ATPase inhibition is based on differential regulation of AMPK

17:15 SL.46 Sebastian Wicha: The value of pharmacometrics in development, optimisation and clinical use of anti-infective therapies

17:10 SL.50 Jozef Al-Gousous: Toward biopredictive dissolution for enteric coated dosage forms

17:00 SL.41 Oliver Koch (Med. Chem.): Analysing the framework of protein ligand interactions: Ligand-sensing cores and privileged scaffolds

17:15 SL.42 Pierre Koch (Med. Chem.): A fluorescence polarization-based competition binding assay for detecting compounds interacting with inactive mitogen-activated protein kinases…

17:45 – 18:30

Anke Krüger, Stefan Wulle: PubPharm - der Fachinformationsdienst Pharmazie: Präsentation der neuen Rechercheplattform für die Pharmazie (POS.231) (Buchner-HS)

19:30 Conference dinner (Augustiner-Keller, Arnulfstr. 52, 80335 München)



Friday, October 7th

9:00 – 9:45

Plenary lecture 5, Florian Holsboer, Personalized therapy of depression – the future has begun (Liebig-HS) P.5

SHORT TALKS (parallel sessions V)

10:00 – 11:30


Fighting Depression Chairs: F. Paintner, K. Wanner

Interface Tumor/ Inflammation Chairs: R. Fürst, O. Werz

Industrial Pharmacy Chairs: O. Queckenberg, C. Olbrich

10:00 SL.51 Felix Hausch: FKBP51 inhibitiors - a pharmacological concept to enhance stress resilience

10:00 SL.54 Alexandra Kiemer: The mRNA binding protein p62/IGF2BP2 as a promoter of metaflammation and hepatocellular carcinoma

10:00 SL.58 Hubertus Rehbaum: Introduction of continuous manufacturing from an engineering perspective

10:30 SL.52 Theo Rein: The stress protein FKBP51 shapes antidepressant pharmacology

10:30 SL.55 Oliver Werz: Targeting monocytes and macrophages for intervention with inflammation-related cancer

10:20 SL.59 Thomas De Beer: Model based PAT implementation in pharmaceutical manufacturing processes

11:00 SL.53 Thomas Kirmeier: Reengineering in the field of Psychopharmacology: Learning from successful models

11:00 SL.56 Tessa Lühmann: Clickable IL-4 cytokines to induce M2 macrophage polarization

10:45 SL.60 Armin Schweiger: Modular, continuous API production units by INVITE

11:15 SL.57 Dian-Jang Lee: Oligoaminoamide-based siRNA carriers for in vivo tumor targeting and gene silencing

11:10 SL.61 Axel Zeitler: Advancing process understanding in film coating by in-line terahertz pulsed imaging, optical coherence tomography and discrete element modelling

11:30 – 13:00

Poster viewing (Analytics, Biotechnology/Protein Drugs, Clinical Pharmacy, GPCR/Ion Channels, Natural Compounds/Chemical Biology, Pharmacology, Regulatory Sciences/Industrial Development, Antiinfectives, Drug Screening) and lunch break

13:00 – 13:45

Plenary lecture 6, Dario Neri, Antibody-cytokine fusion proteins for the treatment of cancer and of chronic inflammation: from the bench to Phase III clinical trials (Liebig-HS) P.6

14:00 – 15:00

Award Session and Closing (Liebig-HS)

15:00 – 18:00

Gemeinsame Sitzung von Repräsentanten der Deutschen Pharmazeutischen Gesellschaft und des Verbandes der Professoren an Pharmazeutischen Hochschulinstituten (Konferenz der Fachbereiche Pharmazie), separate Einladung (Butenandt-HS)


Post-Meeting Program

Saturday, October 8th

15:00 – 18:30

Tag der Offizinpharmazie: FG Allgemeinpharmazie der DPhG in Kooperation mit der Deutschen Gesellschaft für Schmerzmedizin (DGS) und der Bayerischen Landesapothekerkammer (BLAK) Ort: Ludwig-Maximilians-Universität München, Fakultät für Chemie und Pharmazie, Butenandtstraße 5-13, Haus F (Buchner-Hörsaal)

15:00 Prof. Dr. Stefan Laufer, Präsident der DPhG Prof. Dr. Gerhard Winter, Congress Chairman Begrüßung und Einführung

15:15 Dr. med. Johannes Horlemann, Vizepräsident der Deutschen Gesellschaft für Schmerzmedizin, DGS Differentialtherapie und Beratung zu Opiaten

16:15 Prof. Dr. Theo Dingermann, Institut für Pharmazeutische Biologie, Goethe-Universität in Frankfurt am Main Cannabis – Die öffentliche Apotheke wird sich kümmern müssen

17:00 Pause mit Kaffee und Imbiss

17:30 Prof. Dr. Dieter Steinhilber, Institut für Pharmazeutische Chemie, Goethe-Universität Frankfurt am Main Prof. Dr. Theo Dingermann, Institut für Pharmazeutische Biologie, Goethe-Universität Frankfurt am Main Keith Richards und Osteoarthrose – wenn die Gelenke schmerzen

ANMELDUNG Eine Anmeldung zum Tag der Offizinpharmazie in Kooperation mit der Deutschen Gesellschaft für Schmerzmedizin ist nicht erforderlich. Die Veranstaltung ist kostenfrei. www.dphg.de/apo16


1 PLENARY LECTURES

PLENARY LECTURES


P.1 The Challenge of Delivering Biologics Göpferich, A. Pharmaceutical Technology, Department of Chemistry and Pharmaceutical Sciences, University of Regensburg, Universitätsstrasse 31, 93040 Regensburg, Germany

Over recent years drug products of biological origin gained continuously of importance since they improved numerous therapies from cancer to ocular diseases such as age related macular degeneration. With respect to pharmaceutical applications proteins and nucleic acids are the most prominent examples for such drugs that became known as biologics. On the one hand they have a high therapeutic potential but on the other hand they also come with a number of caveats with respect to formulating them to medicinal products that stem from their structure and their physicochemical properties [1]: some of the molecules are of a size that does not allow for an unhindered distribution in the organism [2], they carry a multitude of electrical charges [3] which is a handicap for crossing biological barriers and they often tend to be of limited chemical and physical stability to mention just a few. To make them available for therapy, biologics have frequently been formulated as aqueous solutions for parenteral injection since it is in many cases the safest and simplest way of application. But even then a number of problems have to be solved. Aqueous solutions of antibodies for example often suffer from high viscosities due to their macromolecular character [4] and are frequently subject to aggregate formation [5] that have been reported to be immunogenic in some cases [6]. However, even though aqueous solutions are the most straightforward way to make biologics available for therapy, there are a number of scenarios in which the controlled release oft such compounds over extended periods of time could be highly advantageous. In such cases, the materials need to be processed to delivery systems, which is quite challenging. For proteins there are two general options that we can choose from. Biologics such as proteins can simply be embedded into matrix materials of hydrophobic character from which they can be released by degradation and erosion of the material in combination with diffusion. While this approach has definitely its merits, the interaction of proteins with the matrix material is frequently causing problems due to the presence of phase boundaries, degradation products as well as the changing physicochemical environment. Hydrogels are a promising alternative as carrier materials. Their advantage is a high water content that does not compromise protein stability as much as hydrophobic materials. In some cases, the interaction oft a polymer backbone with a biologic can even become part of the hydrogel structure such as in the case of antibodies that interact via electrostatic interactions with polymer chains [3]. However, also hydrogels have drawbacks. Loading proteins into their three-dimensional network can be difficult and is often solved by simply soaking hydrogel matrices in concentrated protein solutions. Also controlling the release of proteins can be sometimes challenging with respect to extensive premature release, the duration of release that is often too short and the release kinetics that can frequently not be tailored to the intended application. Nucleic acids finally ‘suffer’ from their highly negative charge density. We usually intend to overcome this drawback by formulating them with oppositely charged cationic materials to nanostructures that allow for cellular uptake via endocytotic processes. Nanoparticles offer a number of advantages for the delivery of biologics. However, they suffer from similar problems as the macromolecules they intend to deliver. Size and charge make it difficult to bring them to a target site in sufficient quantities. In many cases a poor target structure affinity and low target cell specificity seem to be problems that deserve more attention [7]. The talk will elucidate the need for delivering biologics beyond the injection of aqueous solutions. Concomitantly it will it will expand on some of the problems that have to be dealt with when formulating delivery systems for biologics. A special focus will, thereby, be on materials for protein delivery and on nanoparticles as well as hydrogels as carrier systems. References: 1. Mitragotri, S., Burke, P. A. & Langer, R. Nat. Rev. Drug. Discov. 2014, 13: 655–672. 2. Schweizer, D., Serno, T. & Goepferich, A. Europ. J. Pharm. and Biopharm. 2014, 88: 291–309. 3. Schweizer, D. et al. Biomacromolecules 2013, 14: 75–83. 4. Yadav, S., et al. J. Pharm. Sci. 2010, 99: 1152–1168. 5. Mahler, H.-C., et al. J. Pharm. Sci. 2009, 98: 2909–2934. 6. Moussa, E. M. et al. J. Pharm. Sci. 2016, 105: 417–430. 7. Wilhelm, S. et al. Nat, Rev. Mat. 2016, 1: 1–12.

PLENARY LECTURES


P.2 L-Type Ca2+ Channels in Brain Disorders? New Targets for Old Drugs? Striessnig, J.1,2 ; Ortner, N. J.1,2 ; Pinggera, A.1,2 ; Negro, G.1,2 ; Mackenroth, L.3 ; Hoferm, N.1,2 ; Tuluc, P.1,2 ; Dougalis, A.4 ; Duda, J.4 ; Ciossek, T.5 ; Draheim, H. J.5 ; Liss, B.4 1 Pharmacology and Toxicology, Institute of Pharmacy and 2 Center for Molecular Biosciences, Univ. of Innsbruck, Innsbruck, Austria; 3 Institute for Clinical Genetics, Technical University, Dresden; 4 Institute of Applied Physiology, University of Ulm, Ulm, Germany; 5 Boehringer Ingelheim Pharma GmbH & Co KG, CNS Research, Biberach an der Riß, Germany.

Organic Ca2+-channel blockers ("Ca2+ antagonists", e.g. the dihydropyridines amlodipine, nimodipine, isradipine) are clinically used for first-line treatment of hypertension. They inhibit ion permeation through voltage-gated L-type Ca2+-channels (LTCCs, Cav1) in arterial smooth muscle and the heart. However, LTCCs are not only expressed in muscle cells of the cardiovascular system but are also key regulators of Ca2+-dependent signaling processes in many other electrically excitable cells, including neurons and (neuro-) endocrine cells (1-3 for review). Cav1.2 and Cav1.3 LTCC isoforms are expressed in the brain, both located postsynaptically at dendrites and the cell soma (1-3). They control neuronal excitability, synaptic morphology and couple synaptic activity to gene transcription. Through their distinct biophysical properties Cav1.2 and Cav1.3 contribute in different ways to various forms of learning, memory, emotional and drug-taking behaviours (1,3). No CNS side effects have been reported during antihypertensive therapy with brain-permeable LTCC blockers. However, recent evidence from human genetics strongly suggests an important role of enhanced LTCC function for neuropsychiatric disease risk (1). Timothy syndrome is a rare disease in which the mutation-induced increase in Cav1.2 channel activity (CACNA1C gene) causes not only long QT syndrome and other organ abnormalities but also autism (1). Genome-wide association studies revealed a strong association between intronic SNPs in CACNA1C and susceptibility for various psychiatric disorders, including bipolar disorder, schizophrenia and major depression. One of these SNPs (rs1006737) leads to increased Cav1.2 activity in fibroblast-derived induced neurons (3). We (4) and others (5) have recently discovered somatic mutations in the α1-subunit of Cav1.3 (CACNA1D) LTCCs which cause increased Cav1.3 activity and excess aldosterone production in aldosterone producing adenomas. When present germline, two of these mutations cause a severe congenital syndrome with primary aldosteronism, seizures and neurodevelopmental deficits at early age (PASNA, 5). Moreover, we identified very similar de novo missense mutations in the pore-forming 1-subunit of Cav1.3 channels in three unrelated patients with sporadic autism and intellectual disability (6). These mutations are all located in the channel's activation gate and cause pronounced gating changes leading to a strong gain of channel function. Together these data suggest that increased Cav1.2 and Cav1.3 channel activity can substantially contribute to neuropsychiatric disease risk in humans. This makes already existing brain-permeable LTCC inhibitors, such as isradipine or nimodipine, promising therapeutics in individuals carrying LTCC risk genes. We therefore investigated the sensitivity of Cav1.2 and Cav1.3 channels to the dihydropyridine LTCC blocker isradipine in HEK293 cells stably expressing these channels. IC50 values were obtained during activity patterns simulating either neuronal firing patterns or arterial smooth muscle - like depolarizations. We found that during neuronal-like activity isradipine is a significantly weaker inhibitor of Cav1.3 than of Cav1.2 and that inhibition of Cav1.3 is splice-variant dependent. Isradipine inhibited Cav1.2 channels during arterial smooth muscle – like depolarizations with an IC50 of 1.5 nM, which is 2-fold lower than for neuronal Cav1.2 and 4.5 - 11-fold lower than for neuronal Cav1.3 splice variants. Our data indicate that inhibition of neuronal Ca2+ channel with existing LTCC blockers could be a novel option for treatment of neuropsychiatric disease. However, chronic treatment may require high maintenance doses likely to cause hypotension. Acknowledgments: Support: Austrian Science Fund (FWF) F44020; P27809; W1101-B12; DFG LI 1754/1

References: 1. Striessnig, J. et al., Wiley Interdiscip. Rev. Membr. Transp. Signal. 2014, 3 (2): 15–38. 2. Ortner, N.J. and Striessnig, J. Channels (Austin) 2016, 10 (1): 7-13. 3. Striessnig, J. et al. Curr. Mol. Pharmacol. 2015, 8 (2): 110-122. 4. Azizan et al. Nat. Genet. 2013, 45 (9): 1055-1060. 5. Scholl et al. Nat. Genet. 2013, 45 (9): 1050-1054. 6. Pinggera, A. et al. Biol. Psychiatry 2015, 77 (9): 816-822.

PLENARY LECTURES


P.3 Genetically tailored pigs as organ donors and models for medical research Wolf, E.1,2,3; Klymiuk, N.2; Renner, S.2,3; Reichart, B.4 1 Gene Center, LMU Munich, Feodor-Lynen-Str. 25, D-81377 Munich, Germany 2 Center for Innovative Medical Models (CiMM), LMU Munich, D-85764 Oberschleißheim, Germany 3 German Center for Diabetes Research (DZD), D-85764 Oberschleißheim, Germany 4 Walter-Brendel-Center for Experimental Medicine, LMU Munich, D-81377 Munich, Germany

The number of donated human organs and tissues for patients with terminal organ failure falls far short of the need, a situation that threatens the life of many potential recipients, especially in Germany. Alternative techniques such as xenotransplantation (and stem cell therapies) are therefore urgently needed. The DFG-funded Transregional Collaborative Research Center 127 “Biology of Xenogeneic Cell, Tissue and Organ Transplantation – from Bench to Bedside” is a unique Consortium encompassing basic research on xenogeneic immune mechanisms, generation and evaluation of novel genetically (multi-)modified donor pigs, and preclinical porcine islet, heart valve, and heart transplantation studies. Hearts of triple-modified donor pigs lacking the major xenoantigen Gal-alpha1,3-Gal and expressing the human complement regulator hCD46 as well as human thrombomodulin [1] survived after heterotopic abdominal transplantation into baboons for up to 945 days [2], demonstrating that cardiac xenotransplantation may become a realistic option. Since T-cell mediated rejection is considered as the major barrier for long-term survival of pancreatic islet xenografts, we generated transgenic pigs expressing the T-cell co-stimulation blocking molecule LEA29Y under the control of the porcine INS promoter. After transplantation under the kidney capsule of diabetic NOD-SCID Il2rg-/- (NSG) mice, LEA29Y expressing porcine neonatal islet cell clusters (NICCs) restored glucose control and were – in contrast to wild-type NICCs – not rejected by transplanted human blood mononuclear cells. Only very low levels of LEA29Y were detectable in the circulation of mice grafted with transgenic islets, supporting the concept of local immune modulation by LEA29Y [3]. The LEA29 transgene was backcrossed to an Gal-alpha1,3-Gal deficient, hCD46 transgenic background to facilitate efficacy studies of multi-modified NICCs in diabetic nonhuman primates. Genetically modified pigs cannot only serve as tissue and organ donors, but also as tailored models of human diseases. For instance, genetically engineered pig models may help to bridge the gap between basic research and clinical studies in (pre)diabetic patients. Type 2 diabetic patients exhibit a reduced insulinotropic action of the incretin hormone glucose-dependent insulinotropic polypeptide (GIP). To mimic this disturbance in a large animal model, we generated transgenic pigs expressing a dominant-negative GIP receptor (GIPRdn) in the pancreatic islets. GIPRdn transgenic pigs exhibit a blunted insulinotropic action of GIP, a progressive deterioration of glucose control due to delayed and – at later stages – quantitatively reduced insulin secretion, and an impairment of physiological age-related expansion of beta-cell volume [4]. GIPRdn transgenic pigs thus provide a unique opportunity to screen for biomarker candidates during the pre-diabetic period [5] and to test therapeutic strategies targeting the glucagon-like peptide 1 (GLP1) receptor [6]. Missense mutations in the INS gene have been identified as common cause of permanent neonatal diabetes mellitus, also referred to as mutant INS gene-induced diabetes of youth (MIDY). We produced a transgenic MIDY pig line expressing INSC94Y. MIDY pigs show early-onset clinical diabetes mellitus, reduced body weight gain and beta-cell volume associated with a marked reduction of insulin secretory granules and severe dilation of the endoplasmic reticulum in the beta-cells [7]. MIDY pigs can be used for insulin treatment studies or for testing the efficacy of gene or cell therapies as well as islet transplantation. Secondary lesions of diabetes mellitus are another interesting area of research. We thus established the Munich MIDY pig biobank as a unique resource for studying systemic consequences of chronic hyperglycemia. Acknowledgments: Funded by the Deutsche Forschungsgemeinschaft (TRR127: Biology of xenogeneic cell, tissue and organ transplantation – from bench to bedside) and by the German Center for Diabetes Research (DZD).

References: 1. Wuensch, A. et al.: Transplantation 2014, 97(2): 138-47. 2. Mohiuddin, M.M. et al.: Nat Commun 2016, 7: 11138. 3. Klymiuk, N., Wolf-van Bürck, L. et al.: Diabetes 2012, 61(6): 1527-32. 4. Renner, S. et al.: Diabetes 2010, 59(5): 1228-38. 5. Renner, S. et al.: Diabetes 2012, 61(8): 2166-75. 6. Streckel, E. et al.: J Transl Med 2015, 13: 73.

PLENARY LECTURES


P.4 Impact of the immunogenic landscape of cancers on immunotherapy Rammensee, H.-G. Department of Immunology, University of Tuebingen, Auf der Morgenstelle 15, D-72076 Tuebingen, Germany

We have analyzed tumor samples of intermediate mutation load, in particular hepatocellular carcinomas, renal cell carcinomas, ovarian carcinomas and several leukemia types, by exome sequencing for somatic mutations and by mass spectrometry analysis for the identification of HLA ligands. Careful attention was given to HLA ligands possibly containing mutations; sequences containing nonsynonymous mutations on DNA level were subjected to prediction of peptides fitting to the relevant HLA class I molecules. High score predicted peptides were synthesized and used as references to identify the expected tumor peptide by mass spectrometry. In no instance we were able to find mutated peptides presented by tumor HLA molecules, although we had shown that the approach works with tumor cell lines. These findings are in accordance with the assumption that many human tumors expressing an intermediate or low number of somatic mutations do not express immunogenic mutated antigens visible for T cells. In contrast, in analysing the entire detectable landscape of HLA ligands on these tumor samples, consisting of 1000 through 5000 peptides per sample, we do find dozens to hundreds of peptides in germline sequence with apparently tumor specific expression, based on the absence of these peptides on adjacent autologous benign tissue and absence on a large number of normal tissue samples from all organs and tissue types available for analysis, all, of course, within the sensitivity limits of our technology. A fraction of these peptides is derived from gene products with tumor specific expression (like cancer testis antigens), another fraction, however, is derived from ubiquitously expressed proteins, pointing to tumor specific altered RNA or protein processing. The population of these tumor peptides is highly different between patients. Many of these tumor specific peptides are immunogenic, as tested by in vitro priming experiments with human T cells from healthy donors. We analyzed patients for the presence of antigen experienced T cells specific for these tumor peptides and not only found that such T cells exist in a fraction of patients but also that this fraction of patients shows a better overall survival. We conclude that 1.) Germline sequence HLA ligands with tumor specific expression should be efficient as targets for personalized antigen specific immunotherapy and 2.) Although the generally accepted view at present is that checkpoint inhibition only works via mutation specific T cells, we would not be surprised if in addition T cells against immunogenic germline sequence HLA ligands with individualized tumor specific expression will also be found if one looks for these.

PLENARY LECTURES


P.5 Personalized therapy of depression – the future has begun Holsboer, F. HMNC Brain Health, Maximilianstraße 34, 80539 Munich, Germany

Biomedical progress has shown that diagnostic entities are not necessarily reflecting uniform disease mechanisms. Oncology heralded the birth of personalized medicine on a large scale demonstrating that specific target engagement is superior to a “one-size-fits” approach. Personalized treatment of depression is obviously more complex than in oncology as our knowledge how the disease evolves and how antidepressants act is limited. Studies of causes underlying depression focused on genetic risk, e.g. polymorphisms and environmental factors, e.g. stressors. The complexity is reflected by the absence of gene loci that confer vulnerability and the fact that stress is an unspecific risk factor for all psychiatric disorders. With that in mind, a gene test and biomarker-guided, personalized medicine was deemed as futile undertaking. Two examples will show that this perception is premature, if not wrong. The central master hormone, coordinating the whole range of adaptations needed to cope with stress is corticotropin-releasing hormone, CRH. If elevated over a longer period depression might precipitate, which resulted in pharmaceutical efforts to block the CRH/CRHR1 signaling pathway. CRHR1 antagonist failed to be superior to placebo in large clinical trials, which led industry to terminate CRHR1 programs. The negative results where unsurprising as only a fraction of patients has a CRH-based pathology. Research into identification, who might respond, has led to a gene test and to a sleep-EEG-based biomarker that predict CRHR1 response. While translation of these findings into clinical application will take time, another gene test demonstrates that personalized treatment of depression is already available: Antidepressants need to enter the brain but their passage is controlled by a transporter molecule P-glycoprotein (Pgp) that is encoded by the ABCB1-gene. ABCB1-gene variants determine if a given antidepressant that is a Pgp-substrate will be effective, or whether a higher dose is needed, or whether switching to an antidepressant that is not a Pgp-substrate, is appropriate. These examples demonstrate that personalized approaches in depression therapy are no longer utopian.

PLENARY LECTURES


P.6 Antibody-cytokine fusion proteins for the treatment of cancer and of chronic inflammation: from the bench to Phase III clinical trials Neri, D.1

1 Institute of Pharmaceutical Sciences, ETH Zürich, Vladimir-Prelog-Weg-4, CH-8093 Zürich (Switzerland)

Antibodies can be used to deliver bioactive molecules (drugs, cytokines, photosensitizers, radionuclides, etc.) to the tumor environment, thus sparing normal tissues. The antibody-based targeting of certain modified extracellular matrix (e.g. splice isoforms of fibronectin and of tenascin-C) is particularly attractive, as these antigens represent accessible, abundant and selective tumor-associated antigens [1-6]. In this lecture, I will present an overview of preclinical and clinical product development activities, performed by my group in collaboration with Philogen (www.philogen.com), for the development of antibody-cytokine fusion proteins (also termed “immunocytokines). A number of products, featuring IL2 or TNF as pro-inflammatory payloads, have moved to advanced clinical trials in oncology, while other immunocytokines (based on anti-inflammatory payloads) are currently being considered for the treatment of rheumatoid arthritis, inflammatory bowel disorders and other conditions. Acknowledgment: The research activity of the group Neri is financed by the ETH Zürich, the Swiss National Science Foundation, the European Research Council (ERC Grant “Zauberkugel”) and the Swiss Federal Commission for Technology and Innovation (KTI).

References: 1. Neri, D. & Bicknell, R.. Nature Rev. Cancer. 2005, 5, 436-446 2. Neri, D. & Supuran, C. Nature Rev. Drug Discov., 2011, 10, 767-777 3. Neri, D. & Pasche, N. Drug Discov. Today, 2012, 17, 583-590 4. Gutbrodt, K. et al. Science Transl. Med., 2013, 5, 201ra118 5. Hemmerle T. et al. Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 12008-12012 6. Bootz, F. & Neri, D. Drug Discov. Today, 2016, 21, 180-189


2 SCIENTIFIC LECTURES

SCIENTIFIC LECTURES


2.1 Materials for Drug Delivery Chairs: W. Frieß, W. Weitschies

SL.01

Hydrogels for controlled release of protein therapeutics Brandl, F. P. 1,2; Gregoritza, M.1 1 University of Regensburg, Department of Pharmaceutical Technology, 93040 Regensburg, Germany 2 Present address: BASF SE, 67056 Ludwigshafen, Germany

Hydrogels have many favorable properties that make them attractive for applications in controlled protein release [1,2]. For example, the preparation procedure is beneficial for preserving protein stability, since protein denaturating conditions (i.e., use of organic solvents, extreme temperature and/or pH values, high shear forces etc.) can be avoided. The mobility of incorporated proteins can be restricted by the polymer network; the hydrogel then forms a reservoir from which the protein is slowly released. Depending on the size of the protein and the average network mesh size, the release can be controlled by diffusion, swelling, degradation, or a combination of these mechanisms. And finally, in situ forming hydrogels can be administered by minimally invasive methods; these are injected in liquid form and turn into a viscoelastic drug depot at the site of application. However, despite the favorable properties of hydrogels, it is often challenging to guarantee the stability and availability of encapsulated proteins [1,2]. The stability of proteins depends on the method of drug loading, the polymer concentration, the type of the cross-linking reaction, the initiator concentration, and the use of stabilizing agents (e.g., radical scavengers). The availability of proteins is influenced by the method of drug loading, the type and molecular mass of the polymer, the polymer concentration, the cross-linking density, the hydrogel degradation rate, and the affinity of the payload for the carrier. While many cross-linking reactions can be used for the preparation of hydrogels (e.g., radical polymerization, Michael-type addition, “click” reactions etc.), only few reactions do not compromise the stability and availability of incorporated proteins. For example, the Diels–Alder reaction has been used for the preparation of hydrogels [3,4,5]. The reaction readily proceeds in water without catalyst or initiator; moreover, the cross-linking process is reversible which allows the preparation of degradable materials. However, side reactions with nucleophilic groups of proteins (e.g., cysteine or lysine residues) can affect their stability and availability [6]. In this talk, possible methods of protein loading and different principles of protein release will be discussed. Several cross-linking methods will be introduced that can be used for applications in controlled protein release. And last but not least, strategies will be presented to protect proteins during cross-linking. References: 1. Lin, C.-C.; Anseth, K.S.: Pharm. Res. 2009, 26(3): 631–643. 2. Vermonden, T.; Censi, R.; Hennink, W.E.: Chem. Rev. 2012, 112(5): 2853–2888. 3. Kirchhof, S. et al.: J. Mater. Chem. B 2013, 1(37): 4855–4864. 4. Kirchhof, S. et al.: J. Mater. Chem. B 2015, 3(3): 449–457. 5. Kirchhof, S. et al.: Eur. J. Pharm. Biopharm. 2015, 96: 217–225. 6. Hammer, N. et al.: Macromol. Biosci. 2015, 15(3): 405–413.

MATERIALS FOR DRUG DELIVERY


SL.02

Evaluation of different polymers for implants produced via fused deposition modeling Kempin, W.; Bogdahn, M.; Weitschies, W.; Seidlitz, A. Biopharmaceutics and Pharmaceutical Technology, Institute of Pharmacy, EMA University of Greifswald, Felix-Hausdorff-Str. 3, 17487 Greifswald, Germany

3D printing of medicinal products and medical devices is a promising alternative to conventional dosage form manufacturing [1], especially when the therapeutic outcome can be improved by individually adapting the shape of the dosage form to the patients’ needs. This would be especially benefical when the placement of an implant in a natural passage or conduit is intended. Fused depositon modeling (FDM) is a 3D printing technique in which a polymer filament is introduced into the printhead, heated, extruded through the nozzle and deposited on the coordinates defined in the machine instructions (G-code). In order to print drug-loaded implants using this technique with commonly designed machines it is mandatory to produce filaments containing the drug and the polymer and if necessary further excipients. In this work we evaluated the formation of filaments with different polymers and the model drug quinine. The tested polymers included poly-L-lactide (PLLA), polycaprolactone (PCL), polydioxanone (PDO), polyhydroxybutyrate (PHB), poly(ethylene-vinyl acetate) (PEVA), ethyl acrylate methyl methacrylate copolymer (Eudragit® RS), and ethyl cellulose (EC under addition of triacetin). The polymers and quinine were mixed using a solvent casting technique at a quinine content of 5 % of total solids. The obtained films were formed into filaments using hot-melt extrusion using a self-constructed extruder (nozzle diameter 3 mm). Filaments that possessed the properties required for printing, e.g. homogeneity and acceptable consistency of diameter, were further tested regarding their printability using a Multirap M420 printer (nozzle diameter 0.5 or 0.35 mm). Model implants were printed as hollow cylinders with a designated height of 3 mm and an outer diameter of 4 or 5 mm. Model drug release of the successfully printed implants was assessed in an incubation setup with phosphate buffered saline solution (PBS) pH 7.4 over several weeks. Quinine content of the samples obtained was determined via fluorescence spectrometry. Filaments and model implants were successfully produced using PLLA, PCL, Eudragit® RS and EC. Implants based on PEVA were printed but showed visible shape variations and thus drug release was not investigated. The other polymers could not be formed into filaments either because no homogenous mixtures of the polymer and the model substance could be produced or since no suitable temperature was identified at which the blends were extrudable but not too liquid to be adequately deposited. Exemplaric images of a filament and model implant composed of PCL and quinine are given in figure 1. The optimum temperature at the noozle was very different for the different polymers ranging from 53 °C (PCL) to 164 °C (PLLA). Under addition of quinine to Eudragit® RS the process temperature for FDM was reduced compared to the pure polymer indicating a plasticizing effect of the model drug. Quinine release from the implants also showed marked differences depending on the type of polymer used. After 8 weeks, releases ranged from 3 % (Eudragit® RS) to 76 % (PCL) of total drug load. The addition of 10 % polyethyleneglycol as a pore forming agent to the model implant composed of PCL and quinine was able to moderately accelerate the release behaviour. FDM is a very promising technique for the preparation of drug-loaded implants adapted to patient anatomy. In this pilot study several polymers were identified that could be used to manufacture model implants containing the model drug quinine and yielded a wide array of release behaviours. More research is needed to identify suitable compositions and processing parameters for implant applications. These parameters are expected to change with every change of the compositon, e.g. changes in processing temperature caused by concentration-dependent plasicizing effects of the active pharmaceutical ingredients as well as excipients. Also it must be kept in mind that drug as well as polymer might (partially) degrade due to the high temperatures used for processing.

Acknowledgments: Financial support of the conducted research by the German federal ministry of education and research within “RESPONSE” is gratefully acknowledged. Furthermore, the authors thank L.-C. Koster and C. Franz for their assistance.

References: 1. Lee Ventola, C.: P T. 2014, 39(10): 704–711.

SCIENTIFIC LECTURES


SL.03

Biopolymers as Multifunctional Materials for Nanoparticulate Drug Delivery Wich, P. R. Johannes Gutenberg Universität-Mainz, Institut für Pharmazie und Biochemie, Staudingerweg 5, 55128 Mainz, Germany

Biopolymers, such as polysaccharides and proteins show a remarkable versatility as multifunctional materials for therapeutic applications. They can be easily modified with the toolkit of bioorganic chemistry and are particularly attractive because of their degradability and biocompatibility [1]. We present a chemical modified polysaccharide (acetal-modified dextran) that can be formulated into nano- und microparticles using a variety of common emulsion-based techniques (A). It is possible to transport proteins, DNA and RNA, as well as small hydrophobic drugs. Ac-DEX particles can release their encapsulated payload under mild acidic conditions including those found in sites of inflammation, tumor tissue, or endocytic vesicles. In addition, the degradation rate can be easily tuned within the time scale of relevant cellular processes. The low-toxicity and payload versatility makes Ac-DEX particles an ideal platform for a wide range of biotherapeutic delivery applications including immunotherapy[2] and gene delivery [3,4]. We also present a new universal approach for the preparation of a new class of protein-based nanoparticles for the delivery of therapeutic payloads (B). The lipophilic surface modification of proteins allows the use of solvent evaporation techniques for the formation of nanoparticles. Unlike previous approaches, we preserve the native structure of the proteins and our particles are stable without denaturation or crosslinking of the biopolymers. The material shows low toxicity at high concentrations and successfully delivers drugs, for example chemo-therapeutics to cancer cells [5,6].

References: 1. Wich, P. R.: Nachrichten aus der Chemie 2015, 63 (2): 128–138. 2. Broaders, K. E. et al.: Proc. Natl. Acad. Sci. USA, 2009, 106: 5497–5502. 3. Cohen, J. L. et al.: Bioconj. Chem. 2011, 13: 1902–1905. 4. Wich, P. R. et al.: Aust. J. Chem. 2012, 1: 15–19. 5. Radi, L. et al.: Med. Chem. Commun. 2016, Advance Article, DOI: 10.1039/c5md00475f. 6. Fach, M; Radi, L; Wich, P. R.: J. Am. Chem. Soc. 2016, Article ASAP, DOI: 10.1021/jacs.6b06243.

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SL.04

Extracellular vesicles as smart carriers for small molecule drugs Fuhrmann, G.1,2; Serio, A.2; Stevens, M. M.2 1 Helmholtz Centre for Infection Research, Helmholtz Institute for Pharmaceutical Sciences Saarland, Saarland University, Campus Building E8.1, 66123 Saarbrücken, Germany

2 Imperial College London, Department of Materials, Department of Bioengineering, Prince Consort Road, SW7 2AZ London, United Kingdom

Extracellular vesicles (EVs) are cell-derived lipid membrane particles decorated with surface and membrane proteins [1]. EVs are nature’s way to deliver information as they transfer protein and nucleic acid based cargoes selectively to their target cell. Moreover, EVs feature a naturally derived composition; can potentially bypass complement activation and coagulation factors leading to reduced immunogenicity and increased stability in biological fluids. In addition, they often transit to their specific target cell rendering them promising candidates for drug delivery applications. In order to harness these properties, new ways of encapsulation of drugs into EVs need to be developed. Although EVs have been investigated to deliver RNA-based therapeutics [2], their use as carriers for small molecule drugs has not been studied in detail.

Figure 1. (a) Chemical structure of hydrophobic porphyrin. (b) Size and morphology of EVs analysed by nanoparticle tracking analysis and electron microscopy. (c) Cell uptake of EV and liposome loaded porphyrin, or free drug (1 μM) in MDA or HUVEC cells (*p<0.05, **p<0.01 vs. free drug, ANOVA, Tukey post-hoc test). In this work, we discuss the potential of EVs as smart drug delivery systems [3]. EVs from various cell types (endothelial HUVEC, stem MSC and cancer MDA cells) were isolated and compared regarding size distribution and morphology. Subsequently, they were loaded with model compounds (porphyrins) of different hydrophobicities (Fig. 1a). Porphyrins are currently investigated as potent drugs for photodynamic therapy (i.e., light activatable cytotoxic drug) but their cellular uptake under physiological conditions is often poor. Here, we show successful loading of porphyrins into EVs using various active and passive encapsulation techniques and assessed their therapeutic efficiency. First, EVs from various cell types showed an average diameter of 171-197 nm (single population) as confirmed by TEM (Fig. 1b). Subsequently, EVs were loaded with hydrophobic porphyrin at high ratios and more efficiently than into standard liposomes. EV-mediated delivery of encapsulated porphyrin significantly increased its cellular uptake in cancer and endothelial cells. EVs from MDA and HUVEC cells were more efficient to bring the hydrophobic porphyrin into the cells compared to MSC EVs but all EVs induced a significantly better drug uptake in cancer cells compared with free or liposome encapsulated porphyrin which indicated their potential for drug delivery applications (Fig. 1c). Upon light illumination, cells that had previously taken up EV-encapsulated porphyrin had a significantly increased cumulative risk to undergo cell death compared to free drug and control cells. Our results indicate that EVs are promising carriers that are able to overcome physiological barriers and deliver drugs with high efficiency and at the cellular level. Acknowledgments: This work was supported by the European Union (Marie Curie Intra-European Fellowship for GF) and the German Academic Exchange Service (Postdoctoral Fellowship for GF).

References: 1. Fuhrmann, G.; Herrmann, I.K.; Stevens M.M.: Nano Today. 2015, 10(3): 397-409. 2. El Andaloussi, S. et al.: Nat. Rev. Drug Discov. 2013, 5: 347-357. 3. Fuhrmann, G. et al.: J. Control Release. 2015, 205: 35-44.

a b c

SCIENTIFIC LECTURES


2.2 Ion Channels in Health and Disease Chairs: R. Lukowski, P. Ruth

SL.05

Phosphorylation of Histidine Residues by Nucleoside Diphosphate Kinase B: A Novel Mechanism to Regulate Ion Channel Activity in Disease Wieland, T.1; Zhou, X. B.2; Feng, Y. X.1; Ruth, P.3 1 Institute of Experimental and Clinical Pharmacology and Toxicology and 2 1st Medical Clinic, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 3 Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, Tübingen, Germany

Reversible phosphorylation of amino acid side chains in proteins is a frequently used mechanism in cellular signal transduction and alterations of such phosphorylation patterns are very common in cardiovascular diseases. They reflect changes in the activities of the protein kinases and phosphatases involving signaling pathways. Reversible histidine phosphorylation, a well-known regulatory signal in lower organisms, has been largely neglected as it has been generally assumed that histidine phosphorylation is of minor importance in vertebrates. During the last decade, it has become evident that the nucleoside diphosphate kinase isoform B (NDPK B), an ubiquitously expressed enzyme involved in nucleotide metabolism, and a highly specific phosphohistidine phosphatase (PHPT-1) form a regulatory histidine protein kinase /phosphatase system in mammals. At least two ion channels substrates of NDPK B are known, the intermediate conductance potassium channel SK4 and the Ca2+ conducting TRP channel family member, TRPV5. In both proteins the phosphorylation of a specific histidine residue regulates channel activity. As SK4 channel is involved in pathological VSMC proliferation, we assessed function and expression of SK4 channels in VSMC from injured mouse carotid arteries by patch-clamping and real-time PCR. ISK4 was detectable in VSMC from injured but not from uninjured arteries correlating with the occurrence of the proliferative phenotype. Direct application of NDPK-B to the membrane of inside-out patches increased ISK4 whereas NDPKB did not alter currents in VSMC obtained from injured vessels of SK4-deficient mice. The NDPK-B-induced increase in ISK4 was prevented by PHPT-1 indicating that ISK4 is regulated via histidine phosphorylation in proliferating VSMC: Moreover genetic NDPK-B ablation reduced ISK4 by 50% suggesting a constitutive activation of ISK4 in proliferating VSMC. In line, neointima formation after wire-injury of the carotid artery was substantially reduced in mice deficient in SK4 channels or NDPK-B. Our data therefore show that NDPK-B to SK4 signaling is required for neointima formation. Targeting this interaction via e.g. activation of PHPT-1 may thus provide clinically meaningful effects in vasculoproliferative diseases such as atherosclerosis and post angioplasty restenosis.

ION CHANNELS IN HEALTH AND DISEASE


SL.06

TRPC channels in lung function and disease Dietrich, A.1; Hofmann, K.1; Fiedler, S.1; Weber, J.1; Gudermann, T.1; Königshoff, M.2

1 Walther-Straub-Institute for Pharmacology and Toxicology, Member of the German Center for Lung Research (DZL), LM-University of Munich, Munich, Germany 2 Comprehensive Pneumology Center, Member of the German Center for Lung Research (DZL), Munich, Germany

Classical transient receptor potential (TRPC) channels are highly expressed in different lung tissues, but their physiological function was still elusive. By construction of TRPC-deficient mouse models and analysis of lung function in comparison to wild-type animals, essential roles of these channels were identified. Moreover, signal transduction cascades for TRPC activation and pathways induced by TRPC dependent cation influx, which are important for smooth muscle cell proliferation1 and contraction2 as well as endothelial barrier function3 were dissected by the same approach. Recently, we focused on another pathophysiological role in the lung. Pulmonary fibrosis (PF) is a progressive lung disease ultimately leading to death with no effective therapeutic options.4 Next to other unknown factors PF may be induced by drugs like bleomycin. One of the pathological features of PF is the formation of myofibroblast foci and the deposition of extracellular matrix (ECM). Many different cell types are suggested to transdifferentiate into these activated myofibroblasts e.g. alveolar epithelial cells type I and II, resident and peripheral fibroblasts. Although the mechanism of PF is not fully understood yet, the profibrotic transforming growth factor β (TGF-β) is considered to play a crucial role. TRPC6 is an unselective cation channel which might contribute to PF since it is known to play an important role in myofibroblast transdifferentiation and wound healing in cardiac and dermal fibroblasts.5 To study a potential role of TRPC6 in the development of PF we analyzed lung function, gene and protein expression in wild-type (WT) and TRPC6-deficient (Trpc6-/-) lungs utilizing a bleomycin-induced PF-model. WT mice died more frequently than TRPC6-deficient mice during the fibrotic phase. Moreover, collagen accumulation in bleomycin-treated Trpc6-/--lungs was indistinguishable to PBS-treated control animals, while treated WT mice showed increased collagen levels. To analyze TRPC6 function on a cellular level, we isolated primary murine lung fibroblasts (PMLFs) from mice of both genotypes and incubated them with TGF-β. Most interestingly, barrier function and contraction of a collagen matrix was significantly reduced in Trpc6-/--PMLFs in clear contrast to WT cells, which also showed increased TRPC6-expression after exposure to TGF-β. Therefore, defining TRPC6 function might help to identify pharmacological targets for new therapeutic options in PF. References: 1. Malczyk, M. et al., American journal of respiratory and critical care medicine 188, 1451-1459, doi:10.1164/rccm.201307-1252OC (2013). 2. Weissmann, N. et al., Proc Natl Acad Sci U S A 103, 19093-19098, doi:0606728103 [pii]10.1073/pnas.0606728103 (2006). 3. Weissmann, N. et al., Nature communications 3, 649, doi:10.1038/ncomms1660 (2012). 4.King, T. E., Jr., Pardo, A. & Selman, M., Lancet 378, 1949-1961, doi:10.1016/S0140-6736(11)60052-4 (2011). 5. Davis, J., Burr, A. R., Davis, G. F., Birnbaumer, L. & Molkentin, J. D., Developmental cell 23, 705-715, doi:10.1016/j.devcel.2012.08.017 (2012).

SCIENTIFIC LECTURES


SL.07

Pain control by calcium-activated potassium channels Schmidtko, A.1 1 Pharmakologisches Institut für Naturwissenschaftler, Goethe-Universität, Fachbereich Biochemie, Chemie und Pharmazie, 60438 Frankfurt am Main, Germany

Potassium (K+) channels are the most populous and widely distributed class of ion channels, governed by at least 77 genes in humans. Accumulating research has highlighted a prominent involvement of different K+ channel subunits in pain processing, and they are increasingly recognized as potential targets for treatment of pain. K+ channels are organized into voltage-gated, two-pore, inwardly rectifying, and Ca2+-activated (KCa) channels. Based on their single-channel conductance and sequence homology of transmembrane cores, KCa channels are further divided into KCa1.1, KCa2.1-3, KCa3.1, KCa4.1-2, and KCa5.1 subunits. Recent studies demonstrated a distinct expression pattern of KCa subunits in various cell populations of the nociceptive system. Moreover, behavioral studies in knockout mice and pharmacological modulation of KCa channels revealed that certain KCa subunits are directly gated to pain-relevant stimuli, whereas others are specifically activated during the processing of chronic inflammatory or neuropathic pain. Here I will present some of the pain-relevant functions of KCa channels with focus on KCa1.1, KCa3.1 and KCa4.1. These data suggest that targeting of KCa subunits with dominant roles in pain processing could yield novel analgesic treatments.

ION CHANNELS IN HEALTH AND DISEASE


SL.08

Noradrenergic and serotonergic compounds to target narcoleptic episodes in a mouse model Schmidt, C.; Leibiger, J.; Fendt, M. Symptoms of narcolepsy are excessive daytime sleepiness, REM sleep disturbance, nighttime wakefulness and cataplexy. The latter is characterized by a sudden loss of muscle tone triggered by high emotions and is often accompanied with falls. In human, a deficiency of orexin/hypocretin neurons are the underlying basis of these symptoms. Beyond other drugs noradrenergic as well as serotonergic antidepressants are used to treat the cataplectic attacks. Using a mouse model of narcolepsy (orexin-deficient mice), we here compared the effects of the selective norepinephrine reuptake inhibitor reboxetine (doses 0.05 – 5.0 mg/kg) and the selective serotonin reuptake inhibitor escitalopram (doses 1.0 – 9.0 mg/kg) on narcoleptic episodes. Furthermore, the effects of noradrenergic ɑ1-receptor stimulation agonism and antagonism were evaluated. After treatment with reboxetine, escitalopram, prazosin or cirazoline the number and duration of narcoleptic episodes were measured and analyzed. Additionally, the effects of the compounds on locomotor activity were evaluated in an open field test. Our data demonstrated that reboxetine reduces narcoleptic episodes in orexin-deficient mice more effective than escitalopram. Prazosin and cirazoline had no significant effects on narcoleptic episodes. Treatment with cirazoline but no other drug reduced significantly the locomotor activity in orexin-deficient mice. The results of our study support the idea that selective noradrenergic reuptake inhibitors should be used for the treatment of narcolepsy.

SCIENTIFIC LECTURES


2.3 Progress in Drug Synthesis Chairs: P. Gmeiner, M. Heinrich

SL.09

Making and breaking of chemical bonds with light - Visible light photocatalysis and photochromic molecular switches König, B. Fakultät für Chemie und Pharmazie, Universität Regensburg, D-93040 Regensburg, Germany

Light is a fascinating reagent for chemistry as it provides energy to drive reactions, but leaves no trace. In visible light photoredox catalysis [1] coloured redox active dyes, such as ruthenium(trisbipyridine) [2], eosin Y [3] or rhodamine 6G [4], convert the absorbed light into redox energy allowing photoinduced electron transfer reactions. We discuss recent examples using the method for metal free cross-coupling reactions [5] in organic synthesis and the late stage functionalization of complex molecules [6].

Light can also be used to control the conformation of bioactive molecules. Photo-switchable enzyme inhibitors for sirtuins illustrate this application [7].

Acknowledgement: We thank the Deutsche Forschungsgemeinschaft (GRK 1626 and GRK 1910) for financial support.

References: 1. König, B. (Ed.) Chemical Photocatalysis (De Gruyter) 2013. 2. Ravelli, D.; Protti S.; Fagnoni, M. Chem. Rev. 2016, DOI: 10.1021/acs.chemrev.5b00662 3. Hari, D. P.; König, B. Chem. Commun. 2014, 50, 6688. 4. Ghosh, I.; König, B. Angew. Chem. Int. Ed. 2016, 55, 7676. 5. Ghosh, I.; Ghosh, T.; Bardagi, J. I.; König, B. Science 2014, 346, 725. 6. Brachet, E.; Ghosh, T.; Ghosh, I.; König, B. Chem. Sci. 2015, 6, 987 7. Falenczyk, C.; Schiedel, M.; Karaman, B.; Rumpf, T.; Kuzmanovic, N.; Grøtli, M.; Sippl, W.; Jung, M.; König, B. Chem. Sci. 2014, 5, 4794.

PROGRESS IN DRUG SYNTHESIS


SL.10

Diversity-oriented synthesis of pharmaceuticals Müller, M. Institute of Pharmaceutical Sciences, Albertstr. 25, University of Freiburg, 79104 Freiburg

Diversity-oriented strategies are widespread in biosynthesis, or it may be more appropriate to say that biosynthesis is the archetype of diversity-oriented synthesis. We are working towards the elucidation and application of diversity-oriented aspects of biosynthesis and biocatalysis, like stereoselective phenolic coupling, chorismate-derived microbial products [1] or the use of multi-purpose biocatalysts for C–C bond formation [2].

O

OH

CO2H

CO2H

Chorismat

O

CO2H

CO2H

Isochorismat

OH

OH

CO2H

ShikimatBiosynthese

O

NH2

CO2H

CO2H

ADC

O

CO2H

CO2H

ADIC

NH2OH

OH

CO2H

2,3-trans-CHD

3,4-trans-CHDOH

OH

CO2H

NH2

OH

CO2H

2,3-t rans-CHA

3,4-trans-CHA

NH2

CO2oder

GlucoseO

HO2COH

CO2H

SHCHC

By using biosynthetic strategies we introduced novel biocatalytic and biomimetic syntheses of small bioactive molecules like HMG-CoA reductase inhibitors, antiviral compounds and antibiotics[3]. The long-term goal of this project is the identification of advantageous trajectories for the synthesis of putative new bioactives without the need for the synthesis of huge compound libraries. References: 1. J. Bongaerts, S. Esser, V. Lorbach, et al., Angew. Chem. 2011, 123, 7927–7932. 2. M. Müller, Adv. Synth. Catal. 2012, 354, 3161–3174. 3. M. Müller, Current Opin. Biotechnol. 2004, 15, 591–598.

SCIENTIFIC LECTURES


SL.11

Chemical synthesis of mycolactone analogs - insights into human Mycobacterium ulcerans infection Blanchard, N. Université de Strasbourg, CNRS, Laboratoire de Chimie Moléculaire, 25 rue Becquerel 67087 Strasbourg, France, E-Mail: [email protected]

Buruli ulcer is a necrotizing skin disease present in more than thirty countries in the world, located mainly in Western and Central Africa but also in Australia and now Japan [1]. To date no specific treatment of Buruli ulcer has been developed which correlates with the dramatic lack of understanding of the associated chemical and biological mechanisms. This infection is caused by Mycobacterium ulcerans that secretes a toxin called mycolactone. This macrolide is the first polyketide isolated from a human pathogen. For the past years, we have been involved in a research program at the frontier of chemical synthesis and immunology (in close collaboration with the Institut Pasteur of Paris for the latter part), aiming at better understanding the mode of action of these human toxins [2,3]. We have devised a modular synthetic approach of mycolactone-derived probes that contributed to the discovery of the first structure-activity relationship of these exotoxins. Based on this knowledge, structurally related fluorescent hybrids were prepared and used in confocal microscopy. Overall, these informations were crucial to the discovery and confirmation of the first proteic target of mycolatones, a long-standing goal in this research area [4].

References: 1. Review: N. Blanchard et al., Nat. Prod. Rep. 2013, 30, 1527-1567. 2. (a) N. Blanchard et al., Chem. Eur. J. 2011, 17, 14413-14419; (b) N. Blanchard et al., J. Med. Chem. 2014, 57 7382–7395; (c) N. Blanchard et al., In Strategies and Tactics in Organic Synthesis, Harmata, M., Ed. Academic Press 2015; 11, pp 85-117. 3. For selected contributions from other groups, see: (a) C. A. Brown, V. K. Aggarwal, Chem. Eur. J. 2015, 21, 13900-13903; (b) K.-H. Altmann et al., Chem. Eur. J. 2011, 17, 13017-13031; (c) Y. Kishi, Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 6703-6708; (d) E.-i Negishi et al., Chem. Eur. J. 2011, 17, 4118-4130. 4. Demangel, C. et al., Science - Trans. Med. 2015, 7, 289ra285.

Small building blocks

(3 or 4 carbon atoms)

Diverted Total

Synthesis

Mycolactones analogs(with areas of structural altera ons)

O

Me

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Me Me

OH OH

O

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The first Structure‐Ac vity Rela onships Finding the cri cal mo fs for func onal interac ons is decisive for the development of a therapeu c solu on

The development of fluorescent hybrids for cellular localiza onA modular synthe c strategy based on click chemistry has been successful in this regard

Iden fica on of a proteic target of mycolactoneThe use of simplified deriva ves and fluorescent probes allowed the iden fica on of a proteic target of mycolactone for the first me

Key Biological Ques ons Answered by our work

Me

Me

OHHO

HO

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PROGRESS IN DRUG SYNTHESIS


SL.12

Inhibitors of the bacterial translocase MraY as potential novel antibiotics Ducho, C.1

1 Saarland University, Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Campus C2 3, 66123 Saarbrücken, Germany

Emerging resistances of bacterial strains, e.g. methicillin-resistant Staphylococcus aureus (MRSA), towards established antibiotics cause an urgent need for the development of novel antibacterial agents. One approach to achieve this goal is the systematic investigation of naturally occurring antibiotics with new or previously unexploited modes of action. Muraymycins (e.g. 1-4, see Figure) belong to the class of nucleoside-peptide antibiotics and were isolated from a Streptomyces sp. as a collection of 19 compounds [1]. They inhibit the bacterial membrane protein translocase I (MraY), a key enzyme in the intracellular part of peptidoglycan biosynthesis and therefore an attractive target for new antibacterial drugs [2,3].

We have developed a modular tripartite synthetic approach for the preparation of muraymycins and their analogues, e.g. 5'-deoxy muraymycin C4 5 (see Figure) [4-8]. This tripartite route and also an alternative dipartite route were employed for the synthesis of a series of muraymycin analogues, including 5'- and 6'-epimers [9]. Using a fluorescence-based in vitro assay [10], the inhibitory activity against MraY was determined, with IC50 values of several analogues being in the nM to low M range. These results as well as further studies on the properties of synthetic muraymycin analogues [11,12] will be presented. Acknowledgements: Deutsche Forschungsgemeinschaft (DFG, SFB 803 "Functionality controlled by organization in and between membranes"), Fonds der Chemischen Industrie (FCI, Sachkostenzuschuss) for funding.

References: 1. McDonald, L. A. et al.: J. Am. Chem. Soc. 2002, 124(35): 10260-10261. 2. Winn, M. et al.: Nat. Prod. Rep. 2010, 27(2): 279-304. 3. Wiegmann, D. et al.: Beilstein J. Org. Chem. 2016, 12: 769-795. 4. Spork, A. P. et al.: Tetrahedron: Asymmetry 2010, 21(7): 763-766. 5. Spork, A. P. et al.: J. Org. Chem. 2011, 76(24): 10083-10098. 6. Büschleb, M. et al.: Amino Acids 2012, 43(6): 2313-2328. 7. Ries, O. et al.: Beilstein J. Org. Chem. 2014, 10, 1135-1142. 8. Spork, A. P. et al.: Chem. Eur. J. 2014, 20(47): 15292-15297. 9. Spork, A. P.; Ducho C.: Synlett 2013, 24(3): 343-346. 10. Brandish, P. E. et al.: J. Biol. Chem. 1996, 271(13): 7609-7614. 11. Rodolisa, M. T. et al.: Chem. Commun. 2014, 50(86): 13023-13025. 12. Ries, O. et al.: Med. Chem. Commun. 2015, 6(5): 879-886.

SCIENTIFIC LECTURES


2.4 New Research, New Researchers I Chairs: S. Laufer, A. Link

SL.13

Application of a dried H1N1 vaccine by epidermal powder immunization in piglets using a novel pyrotechnically driven applicator elicits antigen-specific antibodies Engert, J.1; Anamur, C.1; Engelke, L.1; Fellner, C.2; Lell, P.2; Henke, S.3; Stadler, J. 4; Zöls, S.4; Ritzmann, M.4; Winter, G.1 1 Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilians-University Munich, Butenandtstr. 5, D-81377 Munich, Germany 2 PyroGlobe GmbH, Hauptstr. 15, D-85276 Hettenshausen, Germany 3 IIS Innovative Injektionssysteme GmbH, Lohmannstr. 2, D-56626 Andernach, Germany 4 Klinik für Schweine, Ludwig-Maximilians-University Munich, Sonnenstr. 16, D-85764 Oberschleißheim, Germany

Epidermal powder immunization (EPI) has become a promising emerging approach in the search for new, needle-free vaccination strategies [1]. The principle of EPI relies on the acceleration of vaccine particles to high speed which are administered into the skin, thereby avoiding the use of needles and syringes [2-4]. Ideally, vaccine particles are deposited in the epidermis which lacks blood vessels and nerve endings, thereby making the administration pain free and in consequence may improve patient compliance. In this work, we present the results of an in vivo study in piglets using a dried influenza model vaccine which was applied by EPI using a novel pyrotechnically driven applicator. A liquid influenza vaccine (Pandemrix®) was first concentrated by tangential flow filtration, then transformed into a dry powder by collapse freeze-drying and subsequently cryo-milled to obtain antigen-loaded sugar particles in the desired size range of 20 – 80 µm. The vaccine powder was attached to a membrane of a novel pyrotechnical applicator using oily components with or without adjuvants. Upon actuation of the applicator, particles were accelerated to high speed as determined by a high speed camera setup. Piglets were immunized twice using either the novel pyrotechnical applicator or classical intramuscular injection with the commercial product. Blood samples of the animals were collected directly before vaccine administration and at various time points during the study and analysed for antigen-specific H1N1 IgG antibodies by an enzyme-linked immunosorbent assay. Our study shows that the administration of a dry, cryo-milled powder vaccine into piglets using a novel pyrotechnically driven applicator is possible. The speed and impact of the particles is sufficient to breach the stratum corneum of piglet skin. Importantly, the administration of the vaccine powder with or without adjuvants leads to an increase in the H1N1-specific IgG antibody titre in vivo compared to a negative control containing no antigen. The study provides evidence that it is possible to combine the advantages of a storage stable dry vaccine with a pain free injection technique. Acknowledgements: The authors would like to thank PD Dr. Wolfgang Rebel (WOREB-TOX Consulting, Neustadt, Germany) and Dr. Monir Majzoub-Altweck (LMU Munich, Germany) for assistance with sample analysis. This work was supported by a grant from the Federal Ministry for Education and Research Germany (BMBF), grant number FKZ 13N11315 – 13N11319.

References: 1. Mitragotri S.: Nat. Rev. Immunol. 2005. 5(12): 905-916. 2. Chen D et al.: Expert. Rev. Vaccines 2002. 1(3): 265-276. 3. Chen D et al.: Cell Res. 2002. 12(2): 97-104. 4. Dean HJ et al.: Vaccine 2004. 23(5): 681-686.

NEW RESEARCH, NEW RESEARCHERS I


SL.14

The virulence factor LecB varies in clinical isolates: consequences for ligand binding and drug discovery Titz, A.1 1 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus E8.1, 66123 Saarbrücken, Germany

P. aeruginosa causes a substantial number of nosocomial infections and is the leading cause of death of cystic fibrosis patients. This Gram-negative bacterium is highly resistant against antibiotics and further protects itself by forming a biofilm. Moreover, a high genomic variability among clinical isolates complicates therapy. Its lectin LecB is a virulence factor and necessary for adhesion and biofilm formation. We analyzed the sequence of LecB variants in a library of clinical isolates and demonstrate that it can serve as a marker for strain family classification. LecB from the highly virulent model strain PA14 presents 13% sequence divergence with LecB from the well characterized PAO1 strain. These differences might result in differing ligand binding specificities and ultimately in reduced efficacy of drugs directed towards LecB. Despite several amino acid variations at the carbohydrate binding site, glycan array analysis showed a comparable binding pattern for both variants. A common high affinity ligand could be identified and after its chemoenzymatic synthesis verified in a competitive binding assay: an N-glycan presenting two blood group O epitopes (H-type 2 antigen). Molecular modeling of the complex suggests a bivalent interaction of the ligand with the LecB tetramer by bridging two separate binding sites. This binding rationalizes the strong avidity (35 nM) of LecBPA14 to this human fucosylated N-glycan. Biochemical evaluation of a panel of glycan ligands revealed that LecBPA14 demonstrated higher glycan affinity compared to LecBPAO1 including the extraordinarily potent affinity of 70 nM towards the monovalent human antigen Lewisa. The structural basis of this unusual high affinity ligand binding for lectins was rationalized by solving the protein crystal structures of LecBPA14 with several glycans.

References: 1. R. Sommer, S. Wagner, A. Varrot, C. M. Nycholat, A. Khaledi, S. Haussler, J. C. Paulson, A. Imberty, A. Titz, Chem. Sci., 2016, doi:10.1039/C6SC00696E

SCIENTIFIC LECTURES


SL.15

From mucolipidosis type IV to Ebola: Insights into function and pharmacology of endolysosomal TRP channels Grimm, C.1; Chen, C. C.1; Chao, Y. K.1; Plesch, E.2; Keller, M.2; Bracher, F.2; Wahl-Schott, C.1, Biel, M.1 1 Department of Pharmacy - Center for Drug Research and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität, Butenandtstraße 5-13, 81377 München, Germany

2 Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität, Butenandtstraße 5-13, 81377 München, Germany

Endosomes and lysosomes are cell organelles involved in transport, breakdown and secretion of proteins, lipids, and other macromolecules. Dysfunction of the endolysosomal system can cause storage disorders such as mucolipidoses, sphingolipidoses, or neuronal ceroid lipofuscinoses, but is also implicated in the development of metabolic diseases, neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, retinal and pigmentation disorders, infectious diseases and cancer. Endolysosomal cation channels are highly critical for the complex regulation of the multiple endolysosomal trafficking and transport processes including endo- and exocytotic events as well as the regulation of calcium, sodium, proton and other ionic concentrations within the endolysosomal vesicle lumen. With a combined approach comprising endolysosomal patch-clamp electrophysiology, imaging techniques, molecular biology techniques, and transgenic mouse models, we have recently contributed to the elucidation of the biophysical properties and physiological roles of some of the ion channels found in endosomes and lysosomes such as TRPML channels (also called mucolipins) and Two-pore channels (TPCs). Thus, we have generated (TRPML3, TPC2) or made use of (TRPML1, TPC1) knockout mouse models to study the pathophysiological consequences of the loss of these cation channels for the endolysosomal system. We found that loss of TPC2 makes mice more susceptible for the development of hypercholesterinemia and fatty liver hepatitis, we have explored the role of TPCs in viral infections such as Ebola virus infection and we have studied the role of TPCs for the migration of cancer cells and the formation of metastases. We have further developed small molecule tools to biophysically characterize channel properties and to explore options for a small molecule treatment of human patients carrying mutations in TRPML1 which cause the lysosomal storage disease mucolipidosis type IV. In summary, we provide novel insights into the function and pharmacology of different endolysosomal cation channels and we present novel technical approaches to characterize these proteins functionally such as selective early endosomal versus late endosomal/ lysosomal patch clamp techniques. References: 1. Grimm, C. et al, Chemistry and Biology, 2010, 17(2): 135-148. 2. Cang, C. et al, Cell, 2013, 152: 778-790. 3. Grimm, C. et al, Nature Communications, 2014, 5: 4699. 4. Chen, C.-C. et al, Nature Communications, 2014, 5: 4681. 5. Sakurai, Y. et al, Science, 2015, 347: 995-998.



SL.16

Molecular mechanisms of lipoxin and resolvin biosynthesis Lehmann, C.1; Cumbana, R.2; Ebert, R.2; Toewe, A.3; Angioni, C.3; Ferreirós, N.3; Geisslinger, G.1,3; Parnham, M. J.1; Steinhilber, D.2; Kahnt, A. S.2 1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology, Theodor Stern Kai 7, 60596 Frankfurt/Main, Germany 2 Goethe-University, Institute of Pharmaceutical Chemistry, ZAFES, Max-von-Laue-Str. 9, D-60438 Frankfurt/Main, Germany 3 Goethe University, Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, ZAFES, Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germany

The resolution of inflammation is an active process controlled by endogenous specialized pro-resolving lipid mediators (SPM) which are formed by various immune cells from polyunsaturated fatty acids during the course of an acute inflammatory response by the concerted action of different lipoxygenases, cyclooxygenase-2 and cytochrome P450 enzymes. Lipoxins and resolvins represent two members of this still growing family of SPM. However, the molecular mechanisms underlying cellular lipoxin and resolvin biosynthesis in humans are not completely understood. But this knowledge is of great importance for the development of pro-resolutionary pharmacotherapies as well as a better comprehension of potential resolution-toxic effects induced by already existing anti-inflammatory therapies. Therefore, we aimed to investigate lipoxin and D-type resolvin formation in primary immune cells such as neutrophils, macrophages, platelets and monocytic cell lines in detail. We found that various enzymes known to be involved in the biosynthesis of pro-inflammatory leukotrienes such as FLAP, cPLA2α, LTA4 hydrolase and LTC4 synthase also influence transcellular lipoxin and resolvin formation [1]. In addition, we studied differently polarized and stimulated macrophages as single cell model for lipoxin and resolvin biosynthesis. Here, polarisation and activation considerably rearranged the enzymatic layout of the cells leading to substantial changes in lipid mediator patterns. References: 1. Lehmann, C. et al., FASEB J. 2015 (12):5029-43.

SCIENTIFIC LECTURES


SL.17

Insights from functional lipidomics into the long-term regulation of kinases by vitamin A Pein, H.1; Voelkel, M.1; Schneider, F.1; Rossi, A.2; Koeberle, S. C.3; Loeser, K.1; Morrison, H.3; Sautebin, L.2; Werz, O.1; Koeberle, A.1 1 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, 07743 Jena, Germany 2 Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy 3 Leibniz Institute of Age Research - Fritz-Lipmann-Institute, 07745 Jena, Germany

Functional lipidomics combines comprehensive lipid profiling with target identification and lead discovery to reveal novel strategies for pharmacological intervention. Using this approach, we found that phosphatidylcholines with polyunsaturated fatty acids (PUFA-PC) oscillate during the cell cycle. PUFA-PC suppresses cell proliferation by inhibiting membrane binding and thus activation of the survival kinase Akt [1] and is apparently part of a feed-forward mechanism of apoptosis. By screening a library of nutrients, natural products and drugs for selective effects on the phospholipidome and lipid mediator production, we identified vitamin A as long-term regulator of Akt through modulation of PUFA-PC. The pleiotropic effects of retinol (vitamin A) on adult physiology and embryonic development are mediated through the active metabolite all-trans retinoic acid (RA). Bound to retinoic acid receptors (RARs), RA controls transcription but also fine-tunes the expression of RA target genes by activating kinases such as Akt. The mechanisms for long-term regulation of Akt by vitamin A are incompletely understood. We have shown by lipidomic profiling that retinol and RA deplete NIH-3T3 fibroblasts from phosphatidylcholines (PC) with polyunsaturated fatty acids, in particular linoleic acid (18:2), and concomitantly induce long-term Akt activation. Moreover, we found that the cellular ratio of 18:2-PC determines the activation state of Akt, and ascribed the effects of vitamin A on lipid composition and Akt signaling to retinoic acid X receptor (RXR) activation by using selective agonists. Administration of vitamin A to mice decreased 18:2-PC levels in brain (but not in other tissues) and in parallel enhanced basal Akt activation, which was attributed to astrocytes rather than to neurons in dissociated cortical cultures. Our study reveals how vitamin A is likely to regulate long-term Akt signaling: binding to nuclear receptors, modulating the membrane lipid composition and subsequently enhancing Akt membrane translocation and thus activation. We anticipate this cascade to be key for brain homeostasis in light of the well-established roles of vitamin A, polyunsaturated fatty acids as well as Akt for brain physiology. References: 1. Koeberle, A. et al.: Proc. Natl. Acad. Sci. U. S. A. 2013, 110(7): 2546-2551.



SL.18

α-Aminoxy peptides: from membranolytic anticancer foldamers to the first in class peptidomimetic Hsp90 C-terminal domain dimerization inhibitors Diedrich, D.1; Bhatia, S.2; Frieg, B.1; Stein, S.3; Bopp, B.4; Lang, F.2; Ernst, T.5; Rodrigues Moita, A. J.1; Rüther, A.6; Lüdeke, S.6; Kassack, M. U.1; Hochhaus, A.5; Borkhardt, A.2; Jose, J.4; Kurz, T.1; Gohlke, H.1; Hauer, J.2; Hansen, F. K.1

1 Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany. 2 Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich Heine University Düsseldorf, Medical Faculty, Düsseldorf, Germany. 3 Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Germany. 4 Institute for Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Münster, Germany. 5 Klinik für Innere Medizin II, Universitätsklinikum Jena, Erlanger Alle 101, 07747 Jena. 6 Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, Freiburg, Germany.

α-Aminoxy peptides are peptidomimetic foldamers with high proteolytic and conformational stability. We have recently developed an improved synthetic route to α-aminoxy peptides by the straightforward use of a combination of solution-phase and solid-phase supported methods.[1] In particular long-chained oligomers showed a remarkable anticancer activity against a panel of cancer cell lines.[1] Preliminary mode of action studies revealed that the anticancer activity of long-chained derivatives is at least in parts related to membranolysis whereas α-aminoxy hexapeptides did not possess membranolytic properties.[1] By X-ray crystallography, 2D NMR experiments, MD simulations, and CD spectroscopy we identified a 28-helical conformation as the preferred secondary structure of α-aminoxy peptides.[1] Molecular modeling studies revealed that this 28-helix can mimic the spatial arrangement of peptide side chains in α-helices.[1] Based on these results we hypothesized that α-aminoxy peptides can serve as α-helix mimetics and thus could be a suitable scaffold to inhibit protein-protein interactions (PPIs). To validate this hypothesis, we selected the C-terminal dimerization of the heat shock protein 90 (Hsp90) as a proof of concept PPI target. Hsp90 has been discovered as a promising target to combat cancer and several inhibitors targeting the ATP binding pocket in the N-terminal domain (NTD) of Hsp90 are currently under clinical investigation.[2] Unfortunately, all N-terminal Hsp90 inhibitors trigger a survival mechanism in cancer cells referred to as the heat shock response (HSR), which represents a major efficacy problem in clinical use. In contrast to Hsp90 NTD inhibitors, compounds that act at the C-terminal domain (CTD) of Hsp90 do not initiate the unfavorable HSR.[2] One novel approach to inhibit the CTD of Hsp90 is to target the Hsp90 CTD dimerization interface.[3,4] However, only a few peptidic CTD dimerization inhibitors have been described so far.[4] We now utilized the knowledge about the folding propensity of α-aminoxy peptides[1] and recently discovered hot spots at the Hsp90 CTD dimerization interface[3] to design and synthesize the first peptidomimetic Hsp90 CTD dimerization inhibitors. The hit compound 1 (Cbz-NOPhe-NOPhe-NOIle-NOIle-NOLeu-NOLeu-NH2), an α-aminoxy hexapeptide, exhibited promising anti-proliferative and cytotoxic activity in several human myeloid leukemic cell line models including imatinib resistant cell lines. The specificity of 1 to Hsp90 was determined by a Hsp90-dependent luciferase refolding assay and further confirmed by analysis of the downstream signalling. Furthermore, binding of 1 to the CTD of Hsp90 was demonstrated by MST measurements with the purified recombinant CTD of Hsp90. Most notably, in vivo proof of concept studies demonstrated the efficacy of 1 at 0.5 mg/kg in a K-562-Luciferase Xenograft tumor model. Compound 1 reduced the tumor burden significantly with respect to tumor weight (1: 0.2 ± 0.01g vs vehicle: 1.26 ± 0.44g (p = 0.04)). Immunoblot analysis of tumor samples derived from mice treated with 1 revealed the absence of HSR. Taken together, we have developed the first in class peptidomimetic Hsp90 CTD dimerization inhibitors. References: 1. Diedrich, D. et al.: Chem. Eur. J. 2016: in press. 2. Wang, Y.; McAlpine, S. R.: Future Med. Chem. 2015, 7(2): 87–90. 3. Ciglia, E. et al.: PLOS ONE 2014, 9(4): e96031. 4. Bopp, B. et al.: BBA – Gen. Subjects 2016, 1860(6): 1043–1055.

SCIENTIFIC LECTURES


2.5 New Approaches in Gene and Stem Cell Therapy Chairs: M. Biel, E. Wagner

SL.19

Evaluating microRNA-based therapies in stem cell-derived retinal organoids Busskamp, V. Center for Regenerative Therapies Dresden (CRTD), Dresden, Germany

The outer segments of cones serve as light detectors for daylight color vision, and their dysfunction leads to several human blindness conditions. Here we show that the cone-specific disruption of the DGCR8 locus in adult mice led to the loss of miRNAs and the loss of outer segments, resulting in photoreceptors with significantly reduced light responses. However, the number of cones remained unchanged. The loss of the outer segments occurred gradually over one month, and during this time the genetic signature of cones decreased. Re-expression of the sensory-cell-specific miR-182 and miR-183 prevented the loss of outer segments. These miRNAs were also necessary and sufficient for the formation of inner segments, connecting cilia and short outer segments, as well as light responses in stem-cell-derived retinal cultures. Our results show that miR-182- and miR-183-regulated pathways are necessary for cone outer segment maintenance in vivo and functional outer segment formation in vitro.

NEW APPROACHES IN GENE AND STEM CELL THERAPY


SL.20

Localized vs systemic gene therapy using rAAV vectors: achievements and remaining challenges; the example of Duchenne Muscular Dystrophy gene therapy Le Guiner, C. Atlantic Gene Therapies, INSERM UMR 1089, Nantes, France

Among vector systems that allow efficient in vivo gene transfer, recombinant Adeno Associated Virus vectors (rAAV) hold great promise and are currently evaluated in multiple clinical trials for the treatment of inherited diseases. In particular, gene-therapy of muscle diseases rapidly gained attention because delivery of rAAV vectors of several serotypes results in very efficient transduction of skeletal muscles. Duchenne Muscular Dystrophy (DMD) is an example of a devastating muscle disorder without strongly effective treatment, which could benefit from the reconstitution of a deficient protein after rAAV-mediated gene transfer. DMD is a X-linked inherited muscle-wasting disease primarily affecting young boys with a prevalence of 1:5,000. The disease is caused by loss-of-function mutations in the gene encoding for the Dystrophin protein and is characterized by systemic, progressive, irreversible and severe loss of muscle function. Using a large network of laboratories with complementary skills, we demonstrated the efficacy of two different rAAV-based gene therapy strategies for DMD, in which the vector was injected in the GRMD (Golden Retriever Muscular Dystrophy) dog model, after one single locoregional intravenous injection in a forelimb. In GRMD dogs, such intravenous perfusion, in which the limb is transiently isolated from the general circulation by an atraumatic tourniquet, allowed diffuse transduction of the skeletal muscles of the limb, with significant normalisation of histological, NMR imaging and spectroscopy parameters and muscle strength, without deleterious immune responses. These results paved the way for a human trial of the upper-limb of non-ambulatory DMD patients. This locoregional approach was essential at a time when no one had been able to show therapeutic efficacy of rAAV vectors for muscular diseases beyond one isolated muscle. This proof of efficacy in a whole forelimb, as well as the significant increase in production capacity of rAAV vectors, recently opened the way to an injection in the whole body. The goal is to reach the entire muscles of the body including the heart and the diaphragm, and thus hope for a real treatment of systemic muscle diseases, like DMD. We then systemically administered one of our therapeutic rAAV vectors by a single intravascular injection in several GRMD dogs. Such injection led to long-term transduction of distant muscle groups and extended lifespan (up to 2 years) of the treated GRMD dogs. When using high doses of the therapeutic rAAV vector profound improvement of multiple clinical features was observed and no toxicity or deleterious immune consequences were observed. The specific development and the recent results of these translational projects will be presented, with a particular focus of the remaining challenges regarding rAAV vector delivery for muscle diseases.

SCIENTIFIC LECTURES


SL.21

From viruses to designer nanoparticles - tailoring adeno-associated viruses for gene therapy Büning, H.1,2,3 1 Center for Molecular Medicine Cologne (CMMC), University of Cologne, Robert-Koch-Str. 21, 50931 Cologne, Germany 2 German Center for Infection Research (DZIF), partner sites Bonn-Cologne and Hannover-Braunschweig-Berlin 3 Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany

Vectors based on the replication-defective parvovirus adeno-associated virus (AAV) have emerged as one of the leading gene transfer systems for in vivo gene therapy. As of yet, AAV vectors demonstrated an excellent safety profile and clinical success has been reported for a variety of disorders. The main focus of AAV’s clinical application is long-term gene transfer into post-mitotic tissues such as liver, muscle, brain or the eye. Furthermore, AAV vectors do show promise as tools for transient modification of proliferating cells and as templates in gene correction approaches, and have recently entered the area of vaccine development. However, since AAV vectors use common receptors for cell binding and cellular uptake, target as well as off-target cells are transduced when AAV vectors are applied in vivo. Besides increasing the vector dose needed to achieve therapeutic efficacy, transgene expression in off-target cells may result in undesired adverse events. Furthermore, locally as well as systemically applied AAV particles are transported via the blood to the liver, where they accumulate. Since the native tropism is mainly defined by the viral capsid, capsid engineering is exploited to overcome these drawbacks and optimize the AAV vector system for clinical use. Using AAV serotype 2 (AAV-2), the so far best characterized serotype, as basis, we have developed strategies to expand AAV's tropism by overcoming pre- and/or post entry barriers enabling thereby transduction of cell types refractory to transduction with natural serotypes. The same strategies are explored to re-directed AAV vectors towards a receptor of choice tackling the challenge of off-target transduction. As the capsid also represents the main target of the host immune system, we exploited capsid engineering for developing AAV vector variants that transduce their target cells despite the presence of neutralizing antibodies. Furthermore, we could demonstrate the potency of capsid engineering in augmenting immunogenicity of AAV vector-based vaccines.

NEW APPROACHES IN GENE AND STEM CELL THERAPY


SL.22

Gene therapy for human achromatopsia Michalakis, S. Department Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München

The past decade has seen major developments in the field of ocular gene therapy. The use of recombinant adeno-associated virus (rAAV) vectors has “jump started” the field and led to the development of very promising gene therapies for the treatment of previously untreatable genetic diseases of the eye like achromatopsia (ACHM). ACHM is an autosomal recessive vision disorder, which occurs approximately in one out of every 30,000 births. Patients with ACHM suffer from severely impaired daylight vision, characterized by poor visual acuity, photophobia, nystagmus (involuntary rapid eye movements), and lack of the ability to discriminate colors. Currently, six disease causing genes have been identified including the two genes encoding the cone-specific cyclic nucleotide gated channel subunits CNGA3 and CNGB3. Up to 80% of ACHM patients carry mutations in either CNGA3 or CNGB3. CNGB3 mutations are more prevalent in Europe and United States whereas CNGA3 is the most frequent disease gene in the Middle East and China. Within the collaborative research consrotium RD-CURE (http://www.rd-cure.de), which was established in 2012 between scientists and clinicians from the LMU Munich and the University Eye Hospital Tübingen we developed and evaluated a therapeutic strategy for ACHM based on rAAV vector-mediated CNGA3 gene supplementation. These activities led to the first human gene therapy trial for achromatopsia (https://clinicaltrials.gov; NCT02610582). In this presentation I will provide an overview on the preclinical development, toxicology and biodistribution studies and on the ongoing phase I/II clinical trial. Acknowledgement: This work was supported by the Tistou and Charlotte Kerstan Foundation.

SCIENTIFIC LECTURES


2.6 MS Based Drug Screening Chairs: M. Lämmerhofer, K. Wanner

SL.23

Frontal affinity chromatography and mass spectrometry: a perfect fit in drug discovery Massolini, G.; Calleri, E.; Temporini, C.; Brusotti, G. Dipartimento di Scienze del Farmaco, Viale Taramelli 12 Università degli Studi di Pavia, 27100 Pavia, Italy

Molecular interactions are the engine of biological processes, they determine the efficacy of drugs and therapeutics. Thus, considerable efforts have been devoted to manage the screening of a large number of compounds for ligands of biologically relevant targets in order to accelerated drug discovery processes. Frontal affinity chromatography (FAC) using immobilized receptor-based stationary phases represents a novel screening method among the affinity-based screening strategies to quickly prioritize compound hits for further biological testing and holds particular appeal due to its simplicity, wealth of approaches for coupling biomolecules, and modularity that supports the application of a range of detectors. In particular, FAC coupled to mass spectrometry (FAC-MS) has been shown to be a viable screening tool that has been y applied to a wide range of biological targets. Aside from the utility of the frontal analysis method to provide the opportunity to rank-order binding strengths in a single experiment as each compound has a unique m/z value, the technique provides precise and accurate Kd measurements on single ligands. In the presentation some examples of the power of FAC-MS approach [1-5], developed in our laboratory, using membrane and cytosolic/Nuclear receptors will be discussed together with the key parameters to be considered for the development of a reliable bioaffinity tool. References: 1. Calleri E. et al : J. Med. Chem. 2010, 53 : 3489–3501 2. Calleri E et al. :Journal of Chrom. A, 2012, 1232 : 84– 92 3. Temporini C. et al. : Anal Bioanal Chem, 2013 405: 837–845 4. Temporini C. et al. Journal of Chrom. A, 2013, 1284 : 36– 43 5. de Moraes M.C. et al J Chrom A 1338 (2014)

MS BASED DRUG SCREENING


SL.24

MS in pharmaceutical drug discovery Ottl, J. Novartis Pharma NIBR, Basel, Switzerland

Application of biophysics/label-free detection technologies in drug discovery has gained more and more interest in the past years. This lecture will shed some light on the current state of the art use of biophysics suitable for higher throughput industrial lead/drug discovery applications. Key focus of the presentation is the utilization of mass spectrometry (MS) as biophysical method at early stages of the drug discovery process in miniaturized high throughput formats. In our group we are successfully applying MS-based screening to detect and qualify the binding of low molecular weight (LMW) lead candidates to their respective target proteins. In projects we mostly follow one of two typical application routes: the detection of the LMW cpd after being presented to the target protein or the detection of the protein/compound complex formed when reactive LMW cpds are exposed to target proteins. Case studies show how our application is impacting the early stages of drug discovery and how they are augmenting other technologies and approaches.

SCIENTIFIC LECTURES


SL.25

High Resolution Mass Spectrometry of Antibody Drug Conjugates Using the Orbitrap Mass Analyzer Scheffler, K. Thermo Fisher Scientific, Dreieich, Germany

The complexity of modern therapeutic proteins presents a great analytical challenge. Most often a whole set of analytical techniques is used to characterize these proteins. With mass spectrometry alone several complementary methods are required to analyze protein drugs on the intact protein and on the peptide levels. Native mass spectrometry on the intact protein level allows also for the analysis of molecules which rely on non-covalent interactions to preserve critical structural features, such as antibody-drug conjugates (ADC). In addition the use of 100% aqueous buffers in native MS analysis produces lower charge states detected at higher m/z values compared to analysis under denaturing condition and thus improves mass separation of heterogeneous mixtures. Recent technical advancement on the benchtop Orbitrap mass spectrometry platform offers now complete characterization of the complex conjugates composed of small molecule drugs attached to antibodies on a single instrument. In this presentation data obtained from two different types of ADCs are presented and relevant analysis workflows as well as challenges are discussed.

THE DIVERSITY IN PHARMACEUTICAL BIOLOGY


2.7 The Diversity in Pharmaceutical Biology Chairs: I. Merfort, T. Efferth

SL.26

Beyond malaria: The clinical anticancer activity of artesunate Efferth, T. 1 Department of Pharmaceutical biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz, Germany. E-mail: [email protected]

More than a decade ago, we initiated a research program on the molecular pharmacology of phytochemicals derived from Chinese medicinal herbs. Bioactive plant extracts have been fractionated by chromatographic techniques. We isolated bioactive compounds and elucidated their chemical structures by nuclear magnetic resonance and mass spectrometry. A promising compounds was artemisinin from Artemisia annua L. and its semisynthetic compound artesunate. Artemisinin and artesunate are anti-malarial drugs. Our data indicated profound activity against cancer cells, but also against various viruses, Schistosoma, Trypanosoma, and even plant crown gall tumors. To elucidate the molecular mode of actions against cancer, we applied molecular biological and pharmacogenomic approaches in vitro and in vivo. Different signaling pathways were identified not only in cancer cells but also in cells infected with viruses, e.g. HCMV, HSV1 and others. To translate the experimental results in cell lines and animals to the bedside, we report on the compassionate use of artesunate in single cancer patients as well as on our efforts to initiate several clinical phase I/II trials in veterinary tumors as well as in human cervix or colorectal carcinoma. These pilot studies indeed indicate that artesunate is not only useful as antimalarial drug, but also exerts activity against cancer and viral diseases. Clinical results will also be presented that not only artesunate as semisynthetic chemical derivative of artemisinin, but also herbal extracts from Artemisia annua are active in veterinary and human tumor patients. Artesunate represents an illustrative example for the therapeutic potential of medicinal herbs and drugs derived from traditional Chinese medicine. References: 1. Efferth et al.: Journal of Molecular Medicine 2002;80:233-42. 2. Efferth: Drug Resistance Updates 2005;8:85-97. 3. Efferth et al.: Molecular Cancer Therapeutics 2008;7:152-61. 4. Li et al.: Cancer Research 2008;68:4347-51. 5. Shapira et al.: Clinical Infectious Diseases 2008;46:1455-7. 6. Efferth et al.: Clinical Infectious Diseases 2008;47:804-11. 7. Krishna et al.: Ebiomedicine 2014;2:82-90. 8. Saeed et al.: Pharmacological Research 2016;110:216-226.

SCIENTIFIC LECTURES


SL.27

Application of plant metabolomics in herbal drug research Bauer, R.1 1 Institute of Pharmaceutical Sciences, University of Graz, Universitaetsplatz 4, 8010 Graz, Austria

In recent years, plant metabolomics has increasingly been used for the identification of active constituents in medicinal plants. LC-HRMS based metabolomics and multivariate data analyses allow correlation of the chemical profiles of herbal extracts and their pharmacological activities, and the prediction of compounds which contribute to the activity of the extract. Within the FWF funded national research network (NFN) “Drugs from nature targeting inflammation”, the metabolic profile of ethanolic extracts from 36 Lonicera samples has been analyzed by UHPLC-ESI-HRMS using a Q ExactiveTM Hybrid Quadrupole Orbitrap-MS (Thermo Fisher). In parallel the extracts have been tested for inhibitory effects on NO production in RAW 264.7 macrophages, IL-8 expression in HUVECs, and NF-κB activation in HEK 293 cells, as well as their effect on the activation of PPAR-β/δ in HEK 293 cells. The LC-MS data were processed in an untargeted approach by MZmine 2 [1]. Peaks were identified and quantified by Lipid Data Analyzer [2]. The abundance of the peaks was linked to the pharmacological activity of the extracts using SIMCA 13 [3]. Correlations were modelled using orthogonal partial least squares-discriminant analysis (OPLS-DA) and the results were visualized by means of the S-plot. This led to the identification of compounds which were predicted to be most relevant for the in vitro anti-inflammatory activity in the particular bioassays [4]. The metabolic potential of the human microbiome also needs to be considered for the explanation of the activity of herbal extracts. Intestinal microbiota may metabolize herbal constituents and form active compounds, and herbal medicine may help to balance the gut ecosystem [5].Metabolomics can be used to compare herbal extracts before and after incubation and to identify new metabolites [6]. Quality of control of herbal medicines is currently mainly based on specifications of pharmacopoeias including assays on single marker compounds. More holistic methods, like metabolic profiling with multivariate data analysis may be applied in the future. Acknowledgments: We gratefully acknowledge the funding provided by the Austrian Science Fund (FWF) for projects S107- B13 (NFN: Drugs from Nature Targeting Inflammation) and P26148 “High-Throughput Identification of Lipid Molecular Species in LC-MS/MS Data”, and the support by Dr. Kenneth Bendix Jensen, NAWI Graz Central Lab "Environmental, Plant & Microbial Metabolomics".

References: 1. Pluskal, T. et al.: BMC Bioinformatics 2010, 11: 395. 2. Hartler, J. et al.: Bioinformatics 2011, 27: 572-577. 3. Whelehan, O.P. et al. : Chemometr. Intell. Lab. 2006, 84: 82–87. 4. Waltenberger, B. et al.: Monatshefte für Chemie. 2016,147: 479-491. 5. David, L.A. et al.: Nature 2014, 505: 559-563. 6. Selma, M.V. et al.: Food Funct. 2014, 5: 1779-1784.

THE DIVERSITY IN PHARMACEUTICAL BIOLOGY


SL.28

Cystobactamids: Novel gyrase inhibitors from myxobacteria that inhibit multi-resistant pathogens Herrmann, J.1,2; Müller, R.1,2 1 Helmholtz Centre for Infection Research (HZI), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Saarland University, Campus E8 1, 66123, Saarbrücken, Germany 2 German Centre for Infection Research (DZIF), partner site Hannover-Braunschweig

Infectious diseases caused by bacterial pathogens remain a major health issue, not only restricted to developing countries. Especially antibiotics with activity against hard-to-treat and multidrug-resistant pathogens of the so-called ESKAPE panel (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) are urgently needed. Cystobactamids are a group of newly discovered myxobacterial natural products isolated from Cystobacter spp. that show strong antibacterial activity against many Gram-negative and Gram-positive pathogens in the submicromolar range. Based on analysis of the biosynthesis gene cluster and self-resistance determinants in the natural producer organism, the molecular target of this compound class was identified. Cystobactamids target bacterial type IIa topoisomerases and their mechanism of inhibition resembles that of fluoroquinolone (FQ) antibiotics such as ciprofloxacin. However, only limited FQ cross-resistance was observed and we conclude that the cystobactamid binding site on gyrase might be overlapping but is not identical to that of ciprofloxacin. The basic structure of the cystobactamids, that consist of a central amino acid linker region flanked by substituted p-amino benzoic acid units, provides a new scaffold for the generation of innovative antibiotic drugs to combat infections caused by Gram-negative and Gram-positive pathogens. Intriguingly, in our search for alternative myxobacterial producer strains we discovered some new natural analogs with even improved antibacterial spectrum. This enabled an initial structure-activity-relationship (SAR) study that now aids to the rational design of second generation cystobactamids by means of chemical synthesis. Preclinical studies are currently being pursued with two frontrunner molecules with the overall aim to provide a first proof-of-concept for the in vivo efficacy of the cystobactamids.

References: 1. Baumann, S. et al.: Angew. Chem. Int. Ed. Engl. 2014, 53(52): 14605-14609.

SCIENTIFIC LECTURES


SL.29

Genome mining-guided drug discovery: from proteasome to protease inhibitors Kaysser, L.1,2; Zettler, J. 1,2; Zubeil, F.3; Leipoldt, F. 1,2; Wolf, F.1,2: Schorn, M.4; Bauer, J.1,2; Bendel, T.1,2; Kulik, A.5; Dorrestein, P. C.6; Moore, B. S.4,6; Gross, H.1,2; Grond, S.3

1 Dept. Pharmaceutical Biology, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany 2 German Centre for Infection Research (DZIF), partner site Tübingen 3 Inst.. Organic Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany 4 Scripps Institution of Oceanography, UC San Diego, 9500 Gilman Drive, La Jolla CA, 92093-0204, USA 5 Interfaculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany 6 Skaggs School of Pharmacy, UC San Diego, 9500 Gilman Drive, La Jolla CA, 92093-0751, USA

Proteases and protease-like enzymes are ubiquitous in all aspects of life and have thus been established as therapeutic targets for various diseases such as cancer, hypertension, thrombosis, diabetes as well as viral and bacterial infections. Notably, a substantial number of pharmaceutical relevant protease inhibitors have been developed or at least been inspired by natural products. We recently identified the biosynthetic gene clusters of two bacterial epoxyketone proteasome inhibitors, eponemycin and epoxomicin.[1] The latter served as a lead compound for the development of carfilzomib (Kyprolis®) which has been approved by the European Commission for the treatment of multiple myeloma in 2015. Bioinformatic analysis and genetic engineering of the gene clusters as well as precursor feeding studies showed that a hybrid non-ribosomal peptide synthetase/polyketide synthase (NRPS/PKS) machinery and a conserved acyl-CoA dehydrogenase are responsible for the building of the characteristic epoxyketone warhead in these compounds.[2] Genome mining revealed a number of homologous gene clusters in various bacteria. Interestingly, one of these gene clusters encoded for the production of the hydroxamate metalloproteinase inhibitor matlystatin. Biosynthetic investigations of this molecule are ongoing and current results will be presented. Acknowledgments: This work was funded by a Feodor Lynen Research Fellowship from the Aelxander von Humboldt-Foundation to L.K., a DFG Research Grant KA 3071/4-1 to L.K. and a PhD Stipend from the Ministry of Science Baden-Wuerttemberg to F.W.

References: 1. Schorn, M. et al.: ACS Chem. Biol. 2014, 9(1): 301-309. 2. Zettler, J. et al.: Chembiochem 2016, 17(9): 792-798.

THE QUALITY OF THERAPEUTIC PROTEINS


2.8 The Quality of Therapeutic Proteins Chair: H. Wätzig

SL.30

Analytical tools in the biosimilar development Schiestl, M. 1 Sandoz GmbH, Biochemiestrasse 10, A-6250 Kundl

The evolvement in analytical tools over the last decades provided ever increasing insights in the nature of biopharmaceuticals. From an industry perspective, this evolvement facilitated the development of manufacturing processes. It allowed us to move from the former way of development where a ‘process defined the product’ to a nowadays more systematic, i.e. Quality by Design approach which gains better process and product understanding. This knowledge lead to better control strategies with an increased process robustness. It also facilitated the ability to change processes without altering the product quality. The central role of analytics becomes especially striking when we look at biosimilars, which are products containing essentially the same active pharmaceutical ingredients as their reference products [1]. The focus in biosimilar development is the demonstration of similarity, which can be done best and in the most sensitive manner by analytical tools. Already the early manufacturing process development requires a wide panel of protein characterization methods for making a biosimilar candidate. Otherwise it is unlikely to achieve the normally very narrow development target which is defined by the measured variability of the reference product. The presentation will show practical examples highlighting the role of analytics in biosimilar development. References: 1. European Commission Consensus Information Document 2013 “What you need to know about Biosimilar Medicinal Products“ http://ec.europa.eu/DocsRoom/documents/8242/attachments/1/translations/en/renditions/native

SCIENTIFIC LECTURES


SL.31

Technical challenges for development and manufacturing of biopharmaceuticals Scherübl, C.1 1 Boehringer Ingelheim Pharma GmbH & Co. KG, Biopharma Fill & Finish Germany, Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany

The characteristics of biopharmaceuticals pose several challenges for drug product development and manufacturing. The presentation summarizes the main challenges (e.g. aseptic filling, aggregation, light sensitivity and interaction with packaging material) and shows how they are addressed within routine drug product manufacturing.

THE QUALITY OF THERAPEUTIC PROTEINS


SL.32

How does pH Affect Interfacial Antibody Behaviour and Aggregation upon Shaking? Köpf, E.1; Schroeder, R.2; Brezesinski, G.3; Friess, W.1 1 Ludwig-Maximilians-Universitaet Muenchen, Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, 81377 Munich, Germany 2 AbbVie Deutschland GmbH & Co. KG, 67061, Ludwigshafen am Rhein, Germany 3 Max-Planck Institute of Colloids and Interfaces, Department of Interfaces, 14476 Potsdam, Germany

In the development of protein pharmaceuticals the high tendency to form aggregates requires special consideration. Specifically, the liquid-air interface is known to negatively impact protein stability and to trigger aggregate and particle formation. Although considerable effort has been put into the understanding of the mechanisms behind the link between the interfacial behaviour and the formation of particles is still poorly understood. In this study three antibodies (mAB1, mAB2, human IgG) were investigated for the impact of pH on their liquid-air interfacial protein film characteristics, as well as on protein-protein interactions and therefore protein stability in terms of aggregation. The formation of particles strongly depends not only on the protein itself, but also on pH [1]. Surface pressure measurements indicate a time- and concentration dependent adsorption [e.g. Πmax = 18.5 mN/m for IgG after 4h]. Equilibrium surface pressure as well as interfacial film compressibility investigated in a Langmuir trough is not considerably influenced by pH except for pH values of ≤ 2. This can be explained by pH-induced changes in secondary structure (Fig. 1). Moreover, with decreasing pH the numbers of particles formed upon shaking become less (Fig. 2).

Infrared Reflectance Absorbance (IRRA) spectra substantiate the presence of the IgG molecules at the interface by their amide bands. Both, adsorption and compression do not induce any conformational changes. Brewster Angle Microscopy (BAM) shows a continuous but heterogeneous protein distribution over the interface (Fig. 3) [2].

Submerse AFM of the interfacial film reveals inhomogeneities, similar to BAM results but at different scale, with areas of telescoped material which appear upon compression. Determination of B22 via DLS [3] points out that protein-protein interactions depend on the IgG itself and that B22 decreases with increasing pH values. Therefore, attractive forces between IgG molecules increase with increasing pH thereby causing increased numbers of particles after 48h shaking. Hence, the combinatorial use of the different methods described provides comprehensive insight into interfacial protein behaviour on the one hand, and on interaction parameters in solution on the other. Protein pharmaceuticals are exposed to liquid-air interfaces at many points during development, production and storage. Choosing an appropriate formulation pH is of crucial importance to improve protein stability against interface related stress. References: 1. Jayaraman M., et al., Eur. J. Pharm. Biopharm. 2014, 87: 299–309 2. Rodrigueznino M., et al., Food Hydrocoll. 2005, 19 (3): 417–428 3. Menzen T. and Friess W., J. Pharm. Sci. 2014, 103 (2): 445–455

SCIENTIFIC LECTURES


SL.33

Rational Design of Thermodynamic and Kinetic Binding Profiles by Optimizing Surface Water Networks Coating Protein Bound Ligands Krimmer, S. G.1; Cramer, J.1 ; Betz, M.1 ; Fridh, V.2 ; Karlsson, R.2 ; Heine, A.1 ; Klebe, G.1 1 Institute of Pharmaceutical Chemistry, University of Marburg, Marbacher Weg 6, 35032 Marburg, Germany 2 GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden

In any biological system, the binding reaction between an inhibitor and its target protein takes place in water. Therefore, water molecules have to be considered as additional binding partners besides protein and inhibitor. Nevertheless, the influence of water molecules on protein–ligand binding is still poorly understood. With the aim to study the thermodynamic influence of the rearrangement of water molecules around a newly formed protein-ligand complex, we analysed a series of eight congeneric thermolysin inhibitors exhibiting side-chains of increasing size and hydrophobicity (from methyl to a phenylethyl) by high-resolution crystallography and isothermal titration calorimetry [1]. Across the eight complexes, the crystal structures revealed water networks of different degrees of completeness adjacent to the protein-bound ligands. The observed structural differences correlated remarkably well with the differences observed between the thermodynamic signatures of complex formation. The establishment of a well-ordered, pronounced water network resulted in an increase in ∆H°, whereas the disruption of a water network resulted in an increase in –T∆S°. The inhibitor with the highest affinity exhibited a medium-sized hydrophobic side-chain stabilizing a pronounced water network, resulting in an highly favourable ∆H° overcompensating losses in –T∆S°. Based on these observations, we designed new inhibitors with the aim to further improved the water network stabilization and thereby boost affinity [2]. Prior to ligand synthesis, we validated the newly designed ligands by predicting the putative water networks by MD simulations. Afterwards, the new ligands were synthesized, and structurally and thermodynamically characterized. Furthermore, we also determined their binding kinetics by surface plasmon resonance. As a result, one of the new ligands showed the most pronounced water network, and, consequently, the highest affinity with an overall improvement of 1.5 orders of magnitude. Moreover, due to the pronounced water network caging the protein-bound ligand, a decreased dissociation rate constant was determined for this ligand. References: 1. Krimmer, S.G. et al.: Chem. Med. Chem. 2014, 9, 833-846 2. Krimmer, S.G. et al.: submitted

ANTI-INFECTIVES


2.9 Anti-Infectives Chair: R. Hartmann

SL.34

Innovative antibiotics from microorganisms: Some case studies Müller, R.1 1 Helmholtz-Institutut für Pharmazeutische Forschung, Saarland

Natural products continue to be a major source for novel antibiotic lead structures often exhibiting new mode of action(s). In this presentation I will describe our efforts to identify innovative sources of antibiotics and the path from microbial extracts to promising lead structures for pharmaceutical development. As a first example for entirely new structures I will describe the recently identified cystobactamides as a new class of natural products with broad activity against gram-negative ESKAPE pathogens. The ESKAPE panel of bacteria represents the currently most difficult to treat causative and often multiresistant agents of nosocomial infections. The current status of our efforts to further develop and characterize the cystobactamid gyrase inhibitors in preclinical studies will be presented. As second example an update on our efforts to identify novel tuberculosis agents will be given. Despite modern antibiotics and the development of a curative regimen for this devastasting disease, tuberculosis remains a worldwide problem and the emergence of drug-resistant Mycobacterium tuberculosis has prioritised the need for new drugs. We show that new and optimised derivatives from Streptomyces-derived griselimycin are highly active against M. tuberculosis, both in vitro and in vivo. After identification of the griselimycin biosynthetic gene cluster we were able to clarify the biosynthesis of the biosynthetic precursor methyl-proline which is incorporated into the natural product at the site of metabolic lability. This finding opened up oportunities to improve the ratio of metabolically stable versus unstable griselimycin derivatives. Based on self-resistance studies in Streptomyces and genomic analyses of resistant mycobacteria, we found that griselimycins inhibit the DNA polymerase sliding clamp DnaN; these interactions were characterised by surface plasmon resonance and crystal structure analysis. Furthermore, we discovered that infrequent resistance to griselimycin is associated with highly unusual target amplification in mycobacteria. Our results demonstrate that griselimycin and its derivatives have high translational potential for tuberculosis, validate DnaN as an antimicrobial target and capture the process of antibiotic pressure-induced target amplification.

SCIENTIFIC LECTURES


SL.35

New Antibiotics for the Post-Antibiotic Era Mobashery, S. Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 USA

-Lactam antibiotics are the most important antibiotics targetting the bacterial cell wall. However, their utility has been compormised due to broad resistance by bacteria to these antibiotics. Yet, their targets, penicillin-binding proteins (PBPs) still remain important targets for antibiotics. The importance of PBPs, especially the high-molcular-mass variants, is due to their critical functions in biosynhesis of bacterial cell wall. But equally important, these proteins decorate the surface of the plasma membrane, hence access by antibiotics is often less of a problem than is for the cytoplasmic targets. I will describe an in silico search for novel classes of antibiotics carried out with the X-ray structure of the PBP2a from methicillin-resistant Staphylococcus aureus (MRSA). MRSA is a global scourge, infections by which afflicate 100,000 individuals annually in the USA alone. A significant proportion of these cases leads to mortality. Resistance to -lactam antibiotics in these organisms is overencompassing, including penicillins, cephalosporins, carbapenems, among others. I will describe discovery of the oxadiazole and quinazolinone classes of antibacterials, which target PBPs in MRSA. The lead compounds were elaborated synthetically into a library of several hundred members, which were screened for antibacterial activity. Both classes of antibiotics target bacterial cell-wall biosynthesis, they exhibit favorable pharmacokinetic properties, they are efficacious in a rodent model of MRSA infection and they are orally bioavailable. Both classes of compounds hold great promise in addressing clinical needs in treating infections by MRSA.

ANTI-INFECTIVES


SL.36

LpxC inhibitors – a novel class of antibiotics Holl, R.1,2

1 Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, 48149 Münster, Germany 2 Institut für Organische Chemie, Universität Hamburg, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany

Due to the constantly increasing number of multidrug resistant bacteria, the successful treatment of bacterial infections with the currently available antibiotics is becoming more and more difficult. Therefore, novel antibacterial compounds addressing so far unexploited bacterial targets, thereby circumventing established mechanisms of resistance, are urgently required [1,2]. A promising strategy to combat infections caused by multidrug resistant Gram-negative bacteria is the inhibition of LpxC, a Zn2+-dependent deacetylase, which was validated as an antibacterial drug target. LpxC catalyzes the first irreversible step of lipid A biosynthesis in Gram-negative bacteria, the deacetylation of UDP-3-O-[(R)-3-hydroxymyristoyl]-N-acetylglucosamine. Lipid A, the hydrophobic membrane anchor of lipopolysaccharides in the outer membrane of Gram-negative bacteria, is essential for growth and viability of the majority of Gram-negative bacteria. As the inhibition of lipid A biosynthesis is lethal to these bacteria, LpxC inhibitors represent a promising new class of antibiotics [3]. The potent LpxC inhibitor CHIR-090, containing a hydroxamate moiety to chelate the catalytic Zn2+-ion and a hydrophobic region to mimic the fatty acyl chain of the enzyme’s natural substrate, was chosen as lead compound for the development of C-furanosidic and proline-derived LpxC inhibitors as well as of the respective open-chain derivatives [4,5]. To access the envisaged compounds, chiral-pool syntheses were elaborated. Disc diffusion assays against various clinically important Gram-negative bacteria were performed to reveal the antibacterial properties of the synthesized compounds. Additionally, their inhibitory activity of was determined in an LpxC enzyme assay. References: 1. Projan, S., J. et al.: Curr. Opin. Microbiol. 2002, 5, 463-465. 2. Cooper, M. A. et al.: Nature 2011, 472, 32. 3. Kalinin, D. V. et al.: Curr. Top. Med. Chem. 2016, 16, 2379-2430. 4. Tangherlini, G. et al.: Bioorg. Med. Chem. 2016, 24, 1032-1044. 5. Müller, H. et al.: Eur. J. Med. Chem. 2016, 110, 340-375.

SCIENTIFIC LECTURES


2.10 New Research, New Researchers II Chairs: A. Link, S. Laufer

SL.37

How polysaccharide superstructure impacts hydrogel properties: A Raman optical activity study Lüdeke, S.1 ; Rüther, A.;1 ; Forget, A.2; Roy, A.3; Carballo, C.3; Dukor, R. K.3; Nafie, L. A.3; Johannessen, C.4; Shastri, V. P.5 1 Inst. of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25,79104 Freiburg, Germany 2 Future Industries Insitute, University of South Australia, MM Building, 5095 Mawson Lakes, Australia 3 BioTools, Inc., 17546 Beeline Hwy, Jupiter, FL, USA 4 Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium 5 Institute for Macromolecular Chemistry, University of Freiburg, Stefan-Meier-Str. 31, 79104 Freiburg, Germany

Hydrogels are cross-linked, water swollen polymers with numerous applications, particularly for biomedical and pharmaceutical purposes [1]. The choice of a hydrogel for a specific application is determined by its mechanical properties. Polysaccharide hydrogels are particularly advantageous, because they are non-toxic and biocompatible but achieving defined stochastic roughness and stiffness is often not trivial. We have shown that polysaccharide backbone carboxylation alters the chain organization of agarose-derived hydrogels, thereby leading to much softer hydrogels, which can be used to mimic the biomechanical properties of the extracellular matrix [2]. We applied Raman optical activity (ROA), a spectroscopic technique used previously to study polysaccharides [3], on gel samples of agarose and agarose with different degrees of carboxylation (28, 60, and 93%). The carboxylation-dependent spectra showed features that could be clearly attributed to higher order structure. By employing matrix least squares global fitting [4] we were able to identify two spectral species that interconvert into each other as a function of chemical modification. Comparison to ROA spectra from quantum chemical calculations allowed to assign them to double helical structure in agarose and β-strand-like conformation in the carboxylated derivatives. Our results suggest that 40% of the polysaccharide are already single stranded in native agarose and that carboxylation fully inhibits chain organization as double strands. This suggests that the rigidity and stiffness of agarose hydrogels depends on the presence of double helix structure.

References: 1. Peppas, N. A., et al.: Eur. J. Pharm. Biopharm 2000, 50(1): 27–46. 2. a) Forget, A., et al.: Proc. Natl. Acad. Sci. U. S. A. 2013, 110(32): 12887–12892. b) Forget, A., et al.: Macromol. Rapid Commun. 2015, 36(2): 196–203. 3. Bell, A. F., et al.: J. Raman Spectrosc. 1995, 26(12): 1071–1074. 4. Rüther, A., et al.: J. Phys. Chem. B 2014, 118(14): 3941–3949.

NEW RESEARCH/NEW RESEARCHERS II


SL.38

Skin penetration analysis by confocal Raman microspectroscopy – potentials and pitfalls Lunter, D.1 1 Department of Pharmaceutical Technology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany

In the course of the development of new dermal dosage forms, one major aspect that needs to be evaluated is the dermal absorption of the active from the dosage form. The use of confocal Raman microspectroscopy (CRM) to investigate this aspect has gained increasing attention over the past years. Among the potentials of CRM are the ability to perform penetration analysis in vivo as well as ex vivo with no or only little sample preparation. High spatial resolution and high chemical sensitivity enable label free detection. Two methods for CRM skin penetration analysis are currently used: depth profiling and 2D imaging. The former is the acquisition of spectra along a line perpendicular to the skin surface and subsequent calculation of the relative amount of the active across the depth of the skin that results in a penetration-depth-profile. The latter is the acquisition of spectra across a 2D image plane and subsequent calculation of the relative concentration of the active in each image point. It can be used to generate a color coded image of the distribution of the active within the scanned area. I investigated the feasibility of depth profiling and imaging to investigate the effect of penetration enhancers on the dermal penetration of a model active (procaine HCl) from a model formulation (hydrophilic gel) [1]. Both methods give similar results on the extent of penetration enhancement while the imaging approach is able to give more detailed information on the localization of the active with respect to the skin constituents. As CRM has not yet become a standard technique in dermo-pharmaceutical research consensus on optimal parameters for the conduct of skin penetration studies remains yet to be established. Factors like objective numerical aperture, pinhole size, laser wavelength and removal of the formulation from the skin surface strongly impact the results. Consequently are among the pitfalls: the use of inadequate microscope configuration (objectives and pinholes) and the insufficient removal of the formulation from the skin surface. I thus investigated the effect of the use different objectives and pinholes on the outcome of CRM depth profiling experiments. Regarding objective configuration, I found that simply by varying the objective and pinhole penetration depths-values varied between 10 and > 50 µm for the same sample. The effect of sample preparation was found to be even more pronounced. If the formulation was left on the skin or simply wiped off, the amounts of active which were erroneously detected in the skin were an order of magnitude higher than the actual penetrated amount [2]. To exclude such bias careful evaluation of the methods variables needs to be performed prior to any penetration study.

Acknowledgements: PD Dr. Martin Schenk is acknowledged for the donation of pig ears. This project was supported by the European Social Fund and by the Ministry of Science, Research and the Arts Baden-Wuerttemberg

References: 1. Lunter D, Daniels R, J. Biomed. Opt., 2014, 19(12): 126015-126019. 2. Lunter D, Skin Pharma. Physiol., 2016, 29: 92–101.

SCIENTIFIC LECTURES


SL.39

Influence of Th2 cytokines on the cornified envelope, tight junction proteins and ß-defensins in filaggrin-deficient skin equivalents Hönzke, S.1; Schäfer-Korting, M.1 ; Hedtrich, S.1 1 Institute of Pharmacy, Pharmacology & Toxicology, Königin-Luise-Straße 2+4, 14195 Berlin, Germany

Atopic dermatitis (AD) is a chronic inflammatory skin disease which is characterized by an impaired skin barrier function. In 2006, mutations in the filaggrin gene (FLG) were identified as a major predisposing factor for the manifestation of AD [1]. Aside from barrier deficiencies, AD is characterized by over-shooting Th2-mediated inflammatory processes and impaired innate immunity such as reduced expression of antimicrobial peptides (AMP) [2]. The Th2 cytokines IL-4 and IL-13 significantly contribute to the pathogenesis of AD, but their effects on the skin barrier and particularly the interdependencies with FLG deficiency are not yet fully understood. In this study, the influence of FLG knockdown on the expression of the AMP´s human β-defensin 1-3 and skin barrier proteins under normal and inflammatory conditions was evaluated. Histological examination revealed a thickening of the viable epidermis in the skin models following IL-4 and IL-13 treatment; FLG knockdown amplified this effect (FLG+ 89.5 ± 9.9 µm vs. FLG+/IL-4/13 128.8 ± 22.1 µm and FLG- 109.8 ± 10.2 µm vs. FLG-/IL-4/13 160.7 ± 30.8 µm). Additionally, supplementation of FLG- models with IL-4/IL-13 resulted in a major shift towards higher pH values (pH 6.37 ± 0.03) compared the normal skin models (pH 5.45 ± 0.05). Furthermore, we observed a compensatory 3-fold upregulation of involucrin and occludin in FLG- models, which was considerably disturbed by IL-4/13 exposure. Concordantly, these cytokines significantly reduced the expression of the skin barrier proteins FLG and involucrin in normal skin models. Most interestingly, for the first time we detected significantly (~ 5-fold) higher expression of HbD2 in FLG- models. This was particularly noteworthy because HbD2 is known to be upregulated through bacteria or inflammation but not by a genetic defect [2]. Interestingly, this up-regulation was markedly reduced under inflammatory conditions. In conclusion, these results indicate that defects in the epidermal barrier and cutaneous innate immune response are not primarily linked to filaggrin deficiency but are rather secondarily induced by Th2 inflammation [3]. Acknowledgments: Financial support by the Collaborative Research Center 1112 for the projects C02 is gratefully acknowledged.

References: 1. Palmer, C.N., et al. Nat Genet, 2006. 38(4) 2. Kopfnagel, V., J. Harder, and T. Werfel. Curr Opin Allergy Clin Immunol, 2013. 13(5) 3. Hönzke, S., et al. J Invest Dermat, 2016. 136 (3)



SL.40

Tumor selectivity of V-ATPase inhibition is based on differential regulation of AMPK von Schwarzenberg, K.1; Menche, D.2; Müller, R.3; Vollmar, A. M.1 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich 2 Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Gerhard - Domagk-Str.1, 53121 Bonn, Germany 3 Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO 151150, Universitätscampus E8 1, 66123 Saarbrücken, Germany

Altered tumor metabolism is a hallmark of cancer directly linked to tumor progression. A key player involved in metabolic adaption is the AMP activated protein kinase-1 (AMPK) which is often deregulated in tumors but its role for tumor cell survival is controversial discussed. The recent finding that the vacuolar H+-ATPase (V-ATPase) is involved in AMPK activation during glucose deprivation promoted a role of the V-ATPase in controlling cellular metabolism and represents an interesting new option for targeting cancer. Here we show that AMPK is differentially regulated in tumor and non-tumor cells upon V-ATPase inhibition. The V-ATPase inhibitor archazolid lead to an increased phosphorylation as well as lysosomal localization of AMPK in non-tumor cells – in contrast to tumor cells where no regulation by archazolid was observed. Induction of AMPK by archazolid was shown to have a pro-survival role as silencing of AMPK in non-malignant cells decreased growth rate of the cells whereas induction of AMPK in tumor cells protected them from archazolid induced cytotoxicity. These effects where accompanied by a distinct metabolic regulation concerning ATP level, glucose uptake, ROS production and NADPH level in tumor and non-tumor cells. The pro-survival effects could be attributed to AMPK ability to maintain redox homeostasis by inhibiting ROS production and maintaining NADPH level. Therefore this work identifies a novel role for V-ATPase in metabolic regulation and presents the AMPK as a key protein in tumor-specific cytotoxicity of V-ATPase inhibition.

SCIENTIFIC LECTURES


SL.41

Analysing the framework of protein ligand interactions: Ligand-sensing cores and privileged scaffolds Koch, O. TU Dortmund University, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Straße 6, 44227 Dortmund, Germany

Binding site comparison is a widely established approach for the identification of similar ligand binding, which is typically based on the comparison of potential protein-ligand interaction patterns [1]. However, it was shown that the possible binding pocket space is limited [2] and that similar spatial arrangement of secondary structure elements around the ligand binding site (‘ligand-sensing cores’) can recognize similar scaffolds independent of the overall fold (see Figure 1) [3]. We are focusing on the development of different tools, to get a better insight into the underlying assumption and to analyse how to use this information for computational molecular design.

Fig. 1: Glycogen synthase kinase 3 and trypanothione synthetase show a similar ligand-sensing core and known inhibitors that share a similar paullone scaffold A python based workflow was developed that utilise Scaffold Hunter [4] to analyse bioactivity databases for privileged scaffolds that belong to ligands binding to completely different targets. Using this tool, a scaffold binding to different targets was identified and used to find new inhibitors for one of the proteins (BRD4) with IC50s in the low micromolar range. Biochemical validation using two orthogonal assays and crystallisation studies proved the BRD4 binding of ligands similar to known ligands of the other protein target. So, a new relationship between BRD4 and another protein target was discovered. Overall, this example underlines the basic concept of privileged scaffolds and how to use this information in drug discovery workflows. An automated method to determine ligand-sensing cores of otherwise unrelated proteins was also developed. The current implementation allows the calculation of an all-against-all comparison of predicted binding site within all known protein structures (>100,000) on a current workstation within a reasonable time. In addition, for the identification of similar ligand-sensing cores of one selected binding site whole proteins can be used, neglecting the drawbacks of automated binding site identification methods. The final aim is a database with all known ligand-sensing cores that will hopefully be available soon for general use. The underlying approach and newly developed computational tools will be discussed in detail and promising results will be presented to demonstrate the usefulness. References: 1. Ehrt C, Brinkjost T, Koch O., J. Med. Chem.. 2016, 59: 4121-4151. 2. Skolnick, J., et al., Bioorg. Med. Chem. Lett. 2015, 25:1163-1170. 3. Koch, O., Future Med. Chem. 2011, 3: 699-708 4. Klein, K., Koch, O., Kriege, N., Mutzel, P., Schäfer, T., Mol .Inf. 2013, 32: 964–975.



SL.42

A fluorescence polarization-based competition binding assay for detecting compounds interacting with inactive mitogen-activated protein kinases and development of covalent inhibitors of c-Jun N-terminal kinase 3 Koch, P.1 1 Eberhard Karls Universität Tübingen, Institute of Pharmaceutical Sciences, Department of Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tübingen, Germany.

The two mitogen-activated protein kinases (MAPK) c-Jun N-terminal kinase 3 (JNK3) and p38α MAPK have emerged as attractive drug targets due to their implication in several pathologic conditions such as neurodegenerative diseases and inflammation.[1-3] Biological evaluation of inhibitors for these two kinases is generally carried out through activity assays, although these methods are often expensive and time consuming. Fluorescence polarization (FP)-based competition binding assays were developed for both enzymes using probe 2 (JNK3: Kd = 3.0 nM; p38α MAPK: Kd = 5.7 nM) obtained by labelling of our potent dual JNK3/p38α MAPK inhibitor 1 (JNK3: IC50 = 24 nM; p38α: IC50 = 17 nM).[4] The assays were validated with known inhibitors of the two enzymes and results showed good correlation with data obtained from activity assays. The developed JNK3-FP assay represents the first example of an FP-based binding assay for this protein kinase and was recently employed to study the targeting of the gatekeeper of JNK3 with halogen bonds.[5] These features, together with the viability of both FP binding assays for the high throughput screening format, makes the assays suitable as fast and inexpensive pre-screening protocols for JNK3 and p38α MAPK inhibitors. In continuing efforts to enhance both, JNK3 selectivity and activity of our pyridinylimidazole-based kinase inhibitors, we successfully applied the approach of covalent targeting of JNK3 described by Zhang et al.[6] to our dual JNK3/p38α inhibitor 1 (Figure 1). It is crucial for the linker between the hinge-binding motif and the electrophilic warhead to comprise the optimal length and angle in order to orient the warhead ideally for the nucleophilic attack of the thiol to occur and simultaneously retain the original binding mode of the scaffold. Therefore, we synthesized a broad variety of linkers, altering the meta- and para-substitution pattern on both phenyl rings. Covalent JNK3 inhibitor 3 was finally identified as a potent JNK3 inhibitor showing an IC50-value in the low nanomolar range and displaying 735-fold selectivity against p38α MAPK (JNK3: IC50 = 2 nM; p38α: IC50 = 1,543 nM). Compound 3 is metabolic stable when exposed to human liver microsomes and displays a good selectivity profile in a screening against 410 kinases.

Fig.1: Pyridinylimidazole-based dual JNK3/p38α MAPK inhibitor 1 as a lead compound for development of FP-probe 2 and covalent JNK3 inhibitor 3. References: 1. Manning, A. M.: Nat. Rev. Drug Discov., 2003, 2, 554-565. 2. Koch, P. et al.: J. Med. Chem. 2015, 58, 72-95 3. Cuenda, A.: Biochim. Biophys. Acta, 2007, 1773, 1358-1375 4. Ansideri, F. et al.: Anal. Biochem, 2016, 503, 28-40. 5. Lange, A. et al.: J. Am. Chem. Soc. 2015, 137, 14640-14652. 6. Zhang, T et al.: Chem. Biol. 2012, 19, 140-154.

SCIENTIFIC LECTURES


2.11 Clinical Pharmacy Chair: K. Friedland; C. Wahl-Schott

SL.43

Evidence-based evaluation system for OTC drugs Achenbach, J.1; Culmsee, C.1 1 Institut für Pharmakologie und Klinische Pharmazie, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032 Marburg

Background and Objectives: Methods of evidence-based medicine and pharmacy are becoming more and more important. Especially the field of self-medication has been neglected in terms of evidence-based principles so far. In community pharmacies there is often not enough time to perform literature searches on over-the-counter (OTC) drugs during daily practice. So far, a system that provides transparent, fast and evidence-based evaluations of OTC drugs to support counselling and scientifically-based information on self-medication is not available. The aim of this project is to develop an evaluation and information system for OTC drugs by using the indication migraine. This system has to provide a clear structure and easy orientation. Moreover, it has to present detailed data that are necessary to practice evidence-based and individual counselling. Methods and Results: In order to structure the evaluation system aspects of the “Analytic Hierarchy Process” (AHP) have been applied [1]. This method developed by Thomas L. Saaty is used to support decision problems by defining the problem or goal (e.g. an effective and safe migraine therapy) and by structuring the problem in a hierarchy (e.g. the single efficacy and safety criteria for different drugs used in this indication). Furthermore, the AHP provides the possibility to include individual patient preferences. A systematic procedure including systematic literature searches has been used in order to evaluate efficacy and safety criteria. Moreover, parts of the “Grading of Recommendation, Assessment, Development and Evaluation (GRADE)-system” have been integrated into the evaluation system. Thus, it is possible to incorporate quality ratings of the included studies and to provide a transparent and reproducible presentation of the data. Results of the evaluations for the different drugs are shown in structured and easily accessible “Summary-of-Findings-(SoF)-tables”. Conclusion: The system provides evidence-based data as well as transparent and reproducible results of the efficacy and safety evaluations of different drugs used in the self-medication of acute migraine headaches. This way it can support pharmacists to provide evidence-based counselling on OTC drugs. References: 1. Saaty, T.L.: EJOR 1990, 48: 9–26.

CLINICAL PHARMACY


SL.44

Influence of over-the-counter drugs and prescription-only-medication on male fertility Strobach, D. University Hospital Munich, LMU

The number of couples seeking consultation for infertility problems has steadily increased over the past decades. It is assumed that male infertility concerns at least 50% of all affected couples. Male infertility is a multifactorial state. Among other risk factors, drugs can adversely affect male fertility and sexual function. But, epidemiological data on potential adverse drug reactions (ADRs) on male fertility and reproduction are sparse. In addition, information on these effects for a specific drug is often not easy accessible. Overall, the awareness of potential ADRs on male fertility and reproduction is often lacking. Drugs may impair male fertility by direct gonadotoxic effects, alteration of the hypothalamus-pituitary-gonadal (HPG) axis, impairment of ejaculation and erectile function, and libido. Both, over-the-counter drugs and prescription-only-medications can cause relevant effects. The presentation will include epidemiological data on drug use in men seeking fertility evaluation recently analysed by our group. Exemplarily, cases of an ongoing prospective study will be presented. Commonly used drug classes with potential negative impact on male fertility and sexual function will be discussed. In addition, the problem of information gathering on potential ADRs on male fertility and sexual function will be addressed.

SCIENTIFIC LECTURES


SL.45

Evidence-based Dose Finding Using Modeling and Simulation on Earth and in Space Derendorf, H. University of Florida, Gainesville, United States

The cost of drug development has exploded in recent years and risen to a level that soon will no longer be affordable to society. The public expectation of drug safety and guaranteed therapeutic success has become unrealistic. As a result, the number of new drug approvals can be expected to go down in the near future, a trend that is already noticeable in drug classes with low market potential due to short term therapeutic use, e.g. antibiotics. One reason for the high cost of drug development are many unnecessary studies where the results could have been predicted with reasonable certainty. PK/PD modeling is a tool that can be used to collect and integrate all the available information about a drug candidate and its class in order to make rational decisions on studies that will decrease the uncertainty of the compound. It is based on quantitative data on drug exposure and response and particularly well suited to address the question of dose finding and optimization. In the drug development process, it bridges the complete cycle from discovery to clinical use. The advantage of this approach is to define objective go/no-go decision criteria for the development process rather than relying on subjective empirical decisions. There is no way that today all developing questions can be answered by experimental evidence, and modeling and simulation is a powerful alternative approach. The presentation will feature a number of recent examples from our group where we contributed to dose optimization and development of better medicines. The examples will include some recent work in collaboration with NASA to explore if doses of sleeping medications and antibiotics will need to be modified on the planned mars missions.

CLINICAL PHARMACY


SL.46

The value of pharmacometrics in development, optimisation and clinical use of anti-infective therapies. Wicha, S. G.1 1 Department of Pharmaceutical Biosciences, Uppsala Universitet, Husargatan 3, 75124 Uppsala, Sweden

Resistant infective organisms are on the rise and render our anti-infective armamentarium progressively ineffective. WHO has recently been alarmed by this development and prognoses worse-clinical outcomes world-wide due to a potentially upcoming back-transition into the pre-antibiotic era, and calls for leadership to stimulate the development of new anti-infective agents and foster the rationale use of existing drug entities [1]. The science of pharmacometrics represents an intersection between mathematics and statistics on the one hand, and pharmacy, medicine and physiology on the other hand aiming at quantitative characterisation of biological systems, such as bacteria or drug behaviour in humans by mathematical models. The presentation will outline three examples how pharmacometrics can help addressing the abovementioned WHO calls. The first example [2] will focus on a novel translational prediction approach in tuberculosis research, which can be utilised to streamline the development of new anti-tuberculosis drugs. In this approach, in vitro experiments can be utilised by means of mathematical modelling and simulation to predict animal dose fractionation studies as well as the result of phase IIb clinical trials and hence can support informed decision making for first-in-man dose selection and study design. The second example [3,4] outlines how combination regimens can be comprehensively evaluated by pharmacometric techniques to optimise for synergistic drug effects and avoid unfavourable combinatory effects such as antagonism or evolvement of bacterial resistance. Also, a comparison to conventional interaction assessment is made that can generate misleading conclusions. The third example [5] illustrates how the value of pharmacometric models and pharmacokinetic-pharmacodynamic relationships can be translated to the bedside to personalise anti-infective therapy by using a newly developed, open-access web-application (TDMx software, www.TDMx.eu, developed by the author). In summary, the presentation will display application fields of pharmacometrics from drug development to clinical treatment with anti-infective drugs, illustrating the diversity of clinical pharmacy research within the pharmaceutical sciences. References: 1. World Health Organisation: Antimicrobial Resistance Global Report on Surveillance 2014. 2. Wicha, S.G. et al.: 25th European Congress on Clinical Microbiology and Infectious Diseases, Amsterdam, The Netherlands 2016. 3. Wicha, S.G. et al.: Pharm. Res 2015, 32(7): 2410-2418. 4. Wicha, S.G. et al.: 25th Population Approach Group Europe Meeting, Lisbon, Portugal 2016. 5. Wicha, S.G. et al.: Int. J. Antimicrob. Agents.2015, 45(4): 442-444.

SCIENTIFIC LECTURES


2.12 Advances in Drug Formulation and Biopharmaceutics Chairs: P. Langguth, H. Rein

SL.47

Thoughts on a Manufacturing Classification System Kleinebudde, P. 1 Heinrich-Heine-University, Institute of Pharmaceutics and Biopharmaceutics, Universitätsstr. 1, 40225 Düsseldorf, Germany

The Biopharmaceutical Classification System (BCS) became popular during the last 20 years. It is based on the solubility and permeability of APIs. The BCS was modified several times and can be used for many purposes including regulatory issues. Taking the BCS characteristics into account, it is of interest to select an appropriate manufacturing technology for a certain API. For a new API it is of interest to select a manufacturing technology as early as possible. The question is, if there are any API properties, which can guide to choose an appropriate technology. For oral solid dosage forms an approach was made to compile first ideas for a Manufacturing Classificaton System (MCS) [1]. There is a clear link between the MCS and BCS as the reproducible production of a dosage form that does not impede dissolution and subsequent absorption is critical. Having a tablet product in mind four major routes for manufacturing were identified: (a) direct compression, (b) dry granulation, (c) wet granulation, (d) other technologies like spray drying or melt extrusion before tableting. The costs of production, the number of process steps and the stresses (shear, moisture, heating) applied to the API during production increase from (a) to (d). Some thoughts are provided for the construction of a MCS. The drug load in the dosage form is of major importance. At high drug load the behaviour during production is dominated by the API. It is more difficult to compensate for impaired properties like poor flowability or tabletability. The concept of percolation theory may be helpful to classify the drug loading based on percolation thresholds. The developability of an API is determined by several different properties like the dose, particle size, morphology, surface area, shape or density. Other parameters like flowability, segregation tendency, mechanical behaviour or surface adhesion are complicating the description. The first attempts to select a production route include a number of properties of the API. A major goal is to ideally select few properties based on first principles to construct a simple but useful MCS. The discussion is going on in the MCS group. Formulations on the market are analysed with respect to APi properties and production routes. Based on this, a simplified MCS based on two factors is proposed as an umbrella: Dose of the API in the proposed formulation and BCS class of that API i.e. BCS Class 1/3 vs BCS Class 2/4. Acknowledgments: Michael Leane, Kendall Pitt, Gavin Reynolds and the MCS discussion group for lively and constructive discussions on the topic.

References: 1. Leane, M. et al.: Pharm. Dev. Technol. 2015 20: 12-21.

ADVANCES IN DRUG FORMULATION AND BIOPHARMACEUTICS


SL.48

Formulation development for topical treatment of tinea pedis and onychomycosis Müller-Goymann, C.; Täuber, A. Institut Pharmazeutische Technologie TU Braunschweig, Mendelssohnstr. 1, 38106 Braunschweig, Germany

Superficial fungal skin infections (e.g., tinea pedis) belong to the most common infections worldwide. The most prevalent trigger is the dermatophyte fungus Trichophyton rubrum. Due to the fungus’ ability to feed on keratin, it is mostly located in the human nail plate and the horny layer of the skin, i.e., the stratum corneum (SC). The treatment is usually done with topical formulations containing active antifungal ingredients. Since the fungi may enter the nail plate via fissures and gaps, tinea pedis often goes along with fungal nail infections (onychomycosis) being considerably more difficult to treat. A convenient approach supporting the patient’s compliance would be a single formulation treating both diseases simultaneously. Hitherto, no such formulation is marketed due to the distinct barrier properties of nail and skin. The nail is considered as a hydrophilic gel membrane with an additional lipophilic pathway, whereas the skin represents a lipophilic partition membrane.

We developed such a simultaneous formulation from hydrophilic and lipophilic compounds and incorporated an antifungal active ingredient (API). Variations in composition led to different consistencies (liquid to semi-solid). The antifungal efficacy of the formulations was tested in a novel in vitro model, in which human SC was infected with T. rubrum. After 6 days of incubation, a variety of liquid formulations indicated complete fungal growth inhibition, whereas a marketed antifungal cream for the treatment of tinea pedis as a reference did not inhibit fungal growth completely. Moreover, a loosening of the tight microstructure of the SC was proven by DSC measurements aiding drug penetration and resulting in a better fungal growth inhibition. One-year stability studies at 30 °C proved API contents > 95 % (with one exception) and unchanged macroscopic appearance during storage. The semi-solid formulations did not show any phase separation phenomena, while some of the liquid formulations indicated reversible creaming. Combining these data with previously published results (1-6), in vitro antifungal efficacy against T. rubrum on infected SC as well as on artificial nail models was proven. In vitro permeation studies across SC and nail models indicated promising permeation behaviour for a variety of formulations. Therefore, we suggest a simultaneous antifungal therapy as a future therapy approach. References: 1. Täuber A, Müller-Goymann CC: Mol. Pharm. 2014, 11(7):1991-1996. 2. Täuber A, Müller-Goymann CC: Int. J. Pharm. 2015, 489(1-2):73-82. 3. Täuber A, Müller-Goymann CC: Int. J. Pharm. 2015, 494(1):304-311. 4. Täuber A, Müller-Goymann CC: Akt. Dermatologie 2015, 41:1-7. 5. Täuber A, Müller-Goymann CC: Int. J. Pharm. 2016, 505(1-2):20-23. 6. Täuber A, Müller-Goymann CC: 2015 http://atlasofscience.org/one-for-two-one-medicine-against-two-diseases/

Infected stratum corneum model Successfully treated stratum corneum model

Treatment

Formula( on*

PET.foil*

Polyamide*ring*

Stratum*corneum*with*filter*as*backing*

T. rubrum

SCIENTIFIC LECTURES


SL.49

Improving the quality of split tablets Hirsch, R.1 1 TH Köln – University of Applied Sciences, Claudiusstr. 1, 50678 Cologne, Germany

Tablets are split prior to ingestion for many reasons, varying from therapeutical need to purely economical considerations. The pharmaceutical quality of the split tablet – notabliy uniformity of content – will, however, decrease, even if stability and bioavailability (with their impact on efficacy and safety) are not affected. In order to minimise the mass variance of split tablets, Wenz Blister-Verpackungstechnik in cooperation with TH Köln is developing a device for the exact cutting of tablets, which will eventually enable pharmacies or facilities of medicinal or geriatric care to provide optimally split tablets to their customers. The development of the splitting device is accompanied by a finite element modeling of the breaking process in scored and unscored tablets. Critical parameters on breakability that should be considered during formulation development are crushing strength, porosity, and height of the tablets. Their impact on the variance introduced by the splitting process is investigated as is the influence of the plasticity and elasticity of the excipients. The concept of an effective particle number is introduced in order to characterise the homogeneity of the tableting mass. It can be used to predict the uniformity of content of the fragments from their mass variance. With this information the probability of acceptance of the relevant pharmacopoeial tests can be calculated for any formulation candidate. Acknowledgements: The author wishes to thank the AiF Projekt GmbH (grant no. KF3275402US4) for financial support.

ADVANCES IN DRUG FORMULATION AND BIOPHARMACEUTICS


SL.50

Toward biopredictive dissolution for enteric coated dosage forms Al-Gousous, J.1.; Amidon, G. L.2; Langguth, P.1 1 Insititute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudinger Weg 5, 55099 Mainz, Germany. 2 Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan 48109, USA.

The currently established methodologies for in vitro release testing of enteric-coated dosage forms suffer from poor biopredictivity because of the too high buffer capacity of the employed buffer systems, which makes successful development of such products challenging [1]. Therefore, in this work, we undertook the development of a new dissolution testing method with improved biopredictivity for this type of dosage forms in the fasted state. Two commercially available enteric-coated aspirin products were used as model formulations: Aspirin Protect 300 mg (Bayer AG, Germany) , and Walgreens Aspirin 325 mg (LNK International, USA). The new method was developed by selecting the phosphate buffer resulting in a tablet disintegration performance that most closely matches that in a physiological bicarbonate buffer. The fact that the pH and the buffer molarity along the small intestine are far from being constant was also accounted for. This was done by introducing an algorithm where the pH and the buffer molarity of the medium were increased by adding a calculated amount of a concentrated Na2HPO4 solution to the dissolution vessel for products that release less than 75% of the drug within one hour of starting the test in buffer. Dissolution performance using the accordingly developed method was compared to that observed when using two well-established dissolution methods: the United States Pharmacopoeia (USP) method and blank Fasted State Simulated Intestinal Fluid (FaSSIF). The resulting dissolution profiles were convoluted using GastroPlus software to obtain predicted pharmacokinetic profiles. A pharmacokinetic study on 12 healthy human volunteers, in the fasted state, was performed to evaluate the predictions made by the different dissolution setups for the two aforementioned model formulations. The novel method provided the best prediction, by a relatively wide margin, for the difference between the lag times of the two tested formulations indicating its being able to predict the post-gastric emptying onset of drug release with reasonable accuracy. The post-lag time absorption rate predictions were evaluated using the Cmax/AUC0-24 ratio as an absorption rate metric. Regarding this metric’s predictions, the new method predicted the two products' performances relative to each other very accurately (only 1.05% prediction error in this regard). This prediction was superior to those made by the currently established methods (6.18% and 18.64% prediction errors for the USP and blank FaSSIF methods respectively). The reason behind the method’s success in this regard was found not to rest solely on its lower buffer capacity but also on its accounting for the changing pH and buffer molarity values faced by the dosage form as it traverses the small bowel. As for the new method’s predictions for the individual products’ values in absolute terms, they were borderline (22.58% maximum prediction error) but superior to those made by the currently established methods (maximum prediction errors of 41.67% for the USP method and 33.97% for the blank FaSSIF method). Therefore, the new method has been shown to be of improved biopredictivity compared to so far established methods, and so it could make successful development of enteric-coated products easier. Acknowledgments: The German Academic Exchange Service (DAAD) is acknowledged for supporting Jozef Al-Gousous with a scholarship and the PHATI foundation for support. We also thank Dr. Brian Krieg Jung and Mr. Uwe for their help in setting up the bicarbonate system. We thank ACDIMA Biocenter (Amman, Jordan) for conducting the pharmacokinetic study Additional thanks goes to Dr. Michael Bolger for helpful discussion on GastroPlus. This work has been contributed to Innovative Medicines Joint Undertaking (www.imi.europa.eu) as a sideground.

References: 1. Al-Gousous J, Langguth P. Dissolut. Tech. 2015, 22(3): 6-8.

SCIENTIFIC LECTURES


2.13 Fighting Depression Chairs: F. Paintner, K. Wanner

SL.51

FKBP51 Inhibitiors - a Pharmacological Concept to Enhance Stress Resilience Hausch, F.1 1 Technical University Darmstadt, Alarich-Weiss-Str. 4, 64287 Darmstadt, Germany

FKBP51, encoded by the FKBP5 gene, has attracted considerable attention due to its robust genetic and epigenetic association with numerous psychiatric disorders as well as stress-related endophenotypes. Several animal models confirmed FKBP51 as a key regulator of stress endocrinology. In addition, we and others could recently show that FKBP51 also contributes to chronic inflammatory pain and to diet-induced obesity. Drug discovery for FKBP51 has been hampered by the inability to pharmacologically differentiate against the highly homologous functional counter-player FKBP52 and all known FKBP ligands are unselective.[1] Here, we present the discovery of the first potent and highly selective inhibitors of FKBP51, SAFit1 and SAFit2.[2] This novel class of ligands achieves selectivity for FKBP51 by an induced-fit mechanism that is much less favorable for FKBP52. By using these ligands we demonstrate that selective inhibition of FKBP51 enhances neurite outgrowth in vitro and reduces glucocorticoid secretion, improves stress-coping, alleviates pain pathology and protectes from weight gain in vivo.[3] We thus propose FKBP51 inhibitors as a novel pharmacological concept to enhance resilience against overshooting stress that could be useful for the treatment of depression, obesity of chronic inflammatory pain.

Acknowlegements: This work was supported by the m4 Award 2011 from the Bayerische Staatsministerium für Wirtschaft, Infrastruktur, Verkehr und Technologie

References: 1. Pomplun et al., Angew. Chem. Int. Ed., 2015, 54, 345-8. 2. Gaali et al., Nat. Chem. Biol., 2015, 11, 33-37. 3. Maiaru et al., Sci. Transl. Med. 2016, 8, 325ra19

FIGHTING DEPRESSION


SL.52

The stress protein FKBP51 shapes antidepressant pharmacology Gassen, N. C.1; Stepan, J.2; Balsevich,G.2; Hartmann, J.2; Genewsky, A.2; Hafner, K.1; Schmidt, M. V.2; Eder, M.2; Rein, T.1 1 Max Planck Institute of Psychiatry, Department of Translational Research in Psychiatry, Kraepelinstr. 10, Munich 80804, Germany; 2 Max Planck Institute of Psychiatry, Department of stress neurobiology and neurogenetics, Kraepelinstr. 10, Munich 80804, Germany

FK506 binding protein (FKBP) 51 has been implicated in antidepressant response in several genetic studies. Initially, we had characterized FKBP51 as potent regulator of the glucocorticoid receptor and thereby also of the stress hormone axis [1]. Our recent research deciphered several novel FKBP51-directed molecular pathways that might explain the observed FKBP51 dependency of antidepressant action. These pathways include GSK3beta signalling, DNA methyltransferase 1 dependent epigenetic processes and autophagy [2-5]. We here present novel mechanistic actions of FKBP51 that characterize this remarkably multifunctional protein as scaffolder organizing a protein complex that regulates the stability of the autophagy driver Beclin1. This complex can be targeted by small molecules. The novel pharmacological treatment induces autophagy as indicated by several markers. In addition, it produces antidepressant-like behavioural effects in mice as well as antidepressant-like effects in synaptic function in slices and in vivo. Further experimental evidence suggests that the underlying mechanism involves autophagy-like molecular machineries for membrane fusion at synapses. Together, these results provide a novel route to autophagy, reveal a particular form of neuronal autophagy, and identify novel compounds for autophagic therapy in depression and several other diseases. References: 1. Rein, T.: Bioessays 2016, in press [doi: 10.1002/bies.201600050]. 2. Gassen, N.C. et al.: PLoS Medicine 2014, 11(11):e1001755. doi: 10.1371. 3. Gassen, N.C. et al.: Autophagy 2015, 11(3): 578-580. 4. Gassen, N.C. et al.: Sci. Signal. 2015, 8(404):ra119. doi: 10.1126/scisignal.aac7695 5. Gassen, N.C. et al.: Mol. Psychiatry 2016, 21(2):277-89.

SCIENTIFIC LECTURES


SL.53

Reengineering in the field of Psychopharmacology: Learning from successful models Kirmeier, T. Max Planck Institute of Psychiatry; HMNC Brain Health

Antidepressants are commonly used to treat depressive symptoms not only within major depressive disease (MDD) but also within many other psychiatric disorders. The remission rate of depressive symptoms within MDD upon antidepressant treatment has been reported to lie between 58 % and 67 %. Genetic variability is suspected to account significantly for individual response rates on antidepressants’ treatment. It is widely accepted that not a single genetic marker but the combination of various genetic markers account for the clinical effects upon antidepressant treatment. Despite numerous pharmacogenetic and pharmacogenomic attempts determining genetic markers for antidepressant response no breakthrough has been achieved. More recently, awareness emerged that in addition to genetic variation also the identity of direct drug target structures may be necessary to unravel pharmacogenetic effects upon drug treatment. The aim of this research program was to develop a chemical tool allowing for the identification of direct interaction partner of antidepressant. We were able to develop such a chemical tool (azidobupramine) characterized by two additional chemical groups, one for photoaffinity labelling and the other one for click chemistry. We could show that azidobupramine is characterized by equal affinities to the monoamine transporters as those found for clinically effective substances. Furthermore, it was possible to demonstrate that the intracellular distribution pattern of azidobupramine corresponds to that one of clinically active substances. Finally, we were able to select risk genes for pharmacogenomic analyses according to their probability to directly interact with antidepressants. According to our results, most promising risk genes for antidepressant response rates belong to protein families involved in provision of energy. With this study, we are the first using direct interaction partner of antidepressant as candidate genes for pharmacogenetic analysis. We think that our results may not only contribute to the discovery of yet unidentified mechanisms of MDD but also help to identify novel therapeutic options in the treatment of MDD.

INTERFACE TUMOR/INFLAMMATION


2.14 Interface Tumor/Inflammation Chairs: R. Fürst, O. Werz

SL.54

The mRNA binding protein p62/IGF2BP2 as a promoter of metaflammation and hepatocellular carcinoma Kiemer, A. K. 1 Saarland University, Department of Pharmacy, Pharmaceutical Biology, Campus C2 3, 66123 Saarbrücken

Hepatocellular carcinoma (HCC) represents the third-leading cause of cancer-related death worldwide. Hepatitis B and C infections and alcohol-related liver disease frequently lead to fibrosis and cirrhosis representing risk factors for the development of HCC. A substantial increase in non-alcoholic fatty liver disease (NAFLD) linked to the metabolic sydrome significantly contributes to the rising incidence of HCC (1). NAFLD represents an epidemic, which affects an estimated 10 to 20 million people in Germany. When metabolic disturbances are accompanied by inflammatory processes, i.e. when metaflammation occurs, the condition is classified as non-alcoholic steatohepatitis (NASH). NASH patients have an estimated annual risk to develop HCC of 2.6% (1). The IGF2 mRNA binding protein p62/IMP2-2 was identified in 1999 as an autoantigen in an HCC patient and was observed to exhibit an oncofetal expression pattern, i.e. the healthy adult liver expresses no p62. In order to investigate the role of elevated p62 we generated transgenic mice expressing p62 only in the liver. These animals developed steatosis and showed a highly elevated expression of the metabolic growth factor Igf2 (2). Igf2 proved to be causative for lipid deposition in p62 transgenic animals (3-4). The hepatic lipid composition in transgenic animals was characterized by a distinct increase of elongated (i.e. C18) fatty acids and of free cholesterol, both representing hallmarks of hepatic metaflammation (3;5-6). Interestingly, fatty acid elongation seems to be a characteristic feature for NASH and human NASH-associated HCC, while playing no role in virus-induced human HCC (7-8). In a NASH feeding model p62 amplified hepatic lipid accumulation and accelerated inflammation and fibrosis development (9). The actions of p62 as an inducer and promoter of metaflammation are directly linked to carcinogenic and tumor-promoting actions of p62: p62 induces the generation of reactive oxygen species and genomic instability (10). In a murine HCC model, p62 accelerates hepatocarcinogenesis; in human HCC, p62/IMP2 expression strongly correlates with markers of an aggressive tumor type. What is more, p62 facilitates chemoresistance in hepatoma cells by activating ERK as survival pathway (11). Taken together, p62 represents a pivotal inducer and promoter of hepatocarcinogenesis by amplifying metaflammation and therapy resistance. Antagonizing p62 actions might therefore represent an interesting therapeutic target for the prevention and treatment of NASH-associated HCC. Acknowledgments: The project was funded, in part, by the Deutsche Krebshilfe (107751), the Else Kröner-Fresenius-Stiftung (2012_A250), and the BMBF (01KU1216F, Deutsches EPigenom Programm DEEP). Dr. Sonja M. Kessler was supported by an EASL Dame Sheila Sherlock Fellowship and a Bank Austria Visiting Scientists Programme Fellowship; Dr. Stephan Laggai obtained a post-graduate fellowship from Saarland University. Dr. Yvette Simon was awarded the 2014 Apotheker Jacob Award.

References: 1. Malek, N.P. et al.: Dtsch Arztebl Int 2014; 111: 101-106 2. Tybl, E. et al.: J Hepatol 2011 54: 994-1001 3. Laggai, S. et al.: J Lipid Res 2014 55: 1087-1097 4. Kessler, S.M. et al.: Front Physiol 2016 7: 147 5. Laggai, S. et al.: World J Hepatol 2013 10: 558-567 6. Simon, Y. et al.: World J Gastroenterol 2014 20: 17839-17850 7. Kessler, S.M. et al.: Cancer Res 2014 74: 2903-2904 8. Kessler, S.M. et al.: Int J Mol Sci 2014 15: 5762-5773 9. Simon, Y. et al.: Gut 2014 63: 861-863 10. Kessler, S.M. et al.: Cell Death Dis 2015 6: e1894 11. Kessler, S.M. et al.: Am J Physiol - Gastroint Liver Physiol 2013 304: G328-G336

SCIENTIFIC LECTURES


SL.55

Targeting monocytes and macrophages for intervention with inflammation-related cancer Werz, O. Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, 07743 Jena, Germany

Monocytes are peripheral blood leukocytes that can differentiate into various subsets of macrophages and dendritic cells. These cells play essential roles in innate immunity and protect the host against pathogenic microorganisms, but are also important for cancer immunosurveillance. Aberrant activation of monocytes or macropahges, however, may lead to numerous events (e.g. secretion of cytokines, growth factors, lipid mediators, and reactive oxygen species) that can promote inflammation-related disorders such as atherosclerosis or cancer. For example, when monocytes become activated they may infiltrate tumors and differentiate towards tumor-associated macrophages thereby promoting the persistence of an inflammatory milieu and thus, differentially contribute to various phases of the cancer process. In fact, pharmacological suppression of monocyte infiltration and differentiation into macrophages by trabectedin inhibited the production of interleukin-6 within the tumor microenvironment and is considered as clinically relevant approach for ovarian cancer therapy [1]. In this presentation, novel pharmacological concepts using natural products will be presented that target monocytes and different macrophage subsets (M1 and M2). Focus is placed on two types of natural compounds that had been described as anticancer agents with therapeutic potential: (i) the actin-targeting agent chondramide and (ii) the vacuolar-type ATPase inhibitor archazolid. Chondramide induces G-actin nucleation in macrophages and thereby predominantly depletes pro-tumoural M2 while promoting the tumour-suppressive M1 phenotype, associated with a greater likelihood of a protective anti-tumour immune response. Archazolid suppresses the secretion of pro-inflammatory cytokines and the formation of lipid mediators in primary monocytes related to cancer [2], and selectively increases tumor necrosis factor-α release from M1 but not from M2. Our results propose induction of G-actin nucleation and interference with v-ATPase as novel and promising pharmacological approaches for simultaneously targeting macrophage subtypes, in addition to cancer cells. References: 1. Tang, X. et al., Immunology, 2015, 138:93-104 2. Scherer, O. et al., Biochem. Pharmacol., 2014, 91:490-500

INTERFACE TUMOR/INFLAMMATION


SL.56

Clickable IL-4 cytokines to induce M2 macrophage polarization Lühmann, T.1; Spieler, V.1; Werner, V.1; Fiebig, J.2; Müller, T. D.2; Meinel, L.1

1 Institute of Pharmacy and Food Chemistry, University of Würzburg, 97074, Germany 2 Lehrstuhl für Botanik I, University of Würzburg, 97070, Germany

Introduction: Regulation of macrophage (Mφ) polarization and phenotypic plasticity opens exciting new treatment strategies against inflammatory diseases, including impaired wound healing, rheumatoid arthritis or arteriosclerosis [1,2]. Although interleukin-4 (IL-4) effectively triggers M2-Mφ polarization thereby providing benefit for tissue repair and regeneration processes, its systemic use is constrained by dose-limiting toxicity. We addressed this demand by deploying genetic code expansion, thereby integrating unnatural amino acids (uAA) with either azide or alkyne functionalities into the IL-4 backbone at position 42 during protein synthesis in Escherichia coli (E. coli). These modifications enable controlled and site-specifc immobilisation of IL-4, aiming at sustained and localized activity. Methods: Human IL-4 and IL-4 muteins were expressed in E.coli BL21 (DE3) and were purified using cationic exchange chromatography Characterisation was performed by MALDI-MS, ESI-LC-MS/MS, SPR and RP-HPLC. Bioactivity of IL-4 and analogues was determined by proliferation of TF-1 cells and by using a secreted alkaline phosphatase (SEAP) STAT-6 reporter gene assay. NHS modified agarose particles were decorated with azide or dibenzooctyl functionalities for immobilisation of IL-4 either via copper catalyzed or copper free click chemistry [3,4]. Monocytes were isolated from human peripheral blood mononuclear cells obtained from blood buffy coats. Monocytes were differentiated into Mφ with M-CSF-1 (unpolarized, M0) and further treated with a combination of M-CSF-1 and IL-4 muteins or wild-type IL-4 for M2 polarization or with a cocktail of LPS and IFNγ for M1 polarization, respectively. Degree of Mφ polarization was assessed by RT-PCR for clicked agarose surfaces decorated with IL-4 and for respective controls (physio-adsorbed IL-4), respectively. Results and Discussion: Alkyne and azide functionalized IL-4 muteins were successfully expressed in the presence of 3 mM uAA and were purified by cationic exchange chromatography in an analogue manner to wild-type IL-4. The correct incorporation of the uAA at position 42 was confirmed by MALDI-MS and ESI-MS analysis and peptide mapping after trypsin digest. The soluble IL-4 muteins were as active as the wild-type protein in respect to TF-1 cell proliferation, SEAP stimulation and high affinity (IL-4Rα receptor) as well as low affinity receptor (IL-13Rα1 / common gamma chain) interaction. M2 polarization of Mφ as analysed by M2 marker gene upregulation was similarly in the presence of soluble IL-4 muteins compared to the wild type IL-4. Copper catalyzed (CuAAC) and copper free strain promoted (SPAAC) 1,3-dipolar azide alkyne cycloadditions were used to site-selectively anchor IL-4 to agarose surfaces. These surfaces had sustained IL-4 activity as demonstrated by TF-1 cell proliferation and M2 but not M1 polarization of M-CSF generated human Mφ. Site-directed anchoring of ‘clickable IL-4 muteins’ on surfaces by defined bioorthogonal chemistry can provide sustained immune modulating stimuli. The presented approach herein provides a blueprint for the engineering of cytokine-activated surfaces profiled for sustained and spatially controlled activity. Acknowledgments: Support by DFG (grant ME 3920/3-1 ‘Macrophage plasticity deployed for efficient bone (re-) generation’) is gratefully acknowledged.

References: 1. Mosser, D.M. and J.P. Edwards: Nat. Rev. Immunol. 2008, 8(12):958–69. 2. Chazaud, B: Immunobiology. 2014, 219:172–178. 3. Lühmann, T. et al: ACS Biomater. Sci. Eng. 2015, 1(9):740–746. 4. Gutmann, M. et al: ChemBioChem. 2016, 17: 866–875.

SCIENTIFIC LECTURES


SL.57

Oligoaminoamide-based siRNA carriers for in vivo tumor targeting and gene silencing Lee, D. J.1,2; Kessel, E.1,2; He, D.1,2; Klein, P. M.1; Lächelt, U.1,2; Wagner, E.1,2 1 Department of Pharmacy & Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 Munich, Germany 2 Nanosystems Initiative Munich, Schellingstraße 4, 80799 Munich, Germany

Successful applications of RNAi-based cancer therapy depend upon efficient intracellular delivery of siRNA and effective knockdown of targeted transcripts. To achieve this, oligoaminoamide-based polycationic oligomers, which can form polyplexes with anionic siRNA by electrostatic interaction, have shown great potential as siRNA carrier [1]. However, delivery of siRNA with specificity to the tumor site remains a major limitation [2]. In two different approaches, we have synthesized a series of sequence-defined oligomers which include a cationic oligoaminoamide core, cysteines (as bioreversible disulfide-forming units), and polyethylene glycol chain (for shielding surface charges) coupled to a terminal ligand to target folate receptor (FR)-overexpressing tumors [1-3]. First, to enhance the targeted antitumor effect, the antifolate drug methotrexate (MTX) was employed as both targeting ligand for FR-mediated uptake and as an anticancer agent as it is toxic to the target cells by blocking de novo thymidylate and purine synthesis [4]. MTX-conjugated polyplexes are spherical nanoparticles with a hydrodynamic diameter of 6.5 nm (Fig. A). Treatments with these polyplexes containing EG5 siRNA in FR-expressing tumor cells triggered knockdown of EG5 gene and caused augmented cytotoxicity. Intratumoral administration of the MTX-based polyplexes showed significantly enhanced tumoral retention (168 h) compared to the non-targeted polyplexes (48 h), and mediated the longer survival time (>70 days) than untreated controls (24 days) in tumor-bearing mice (Fig. B) [5]. Second, to increase the polyplex stability for systemic delivery, we optimized the physicochemical properties of polyplexes by combinatorial optimization of PEGylated folate-conjugated oligomer (for FR targeting and shielding of surface charges) and 3-arm thiol-oligomer (for size modification and particle stability) (Fig. C). For uni-directional fast coupling between the two groups of oligomers, we activated the cysteine thiol groups of one of the oligomers with 5,5’-dithio-bis(2-nitrobenzoic acid) to achieve a fast chemical linkage through disulfide formation with the free thiol groups of the other oligomer. These targeted combinatorial polyplexes (TCPs) are homogeneous spherical particles with favorable size and strong siRNA binding activity. TCPs were internalized into cells by FR-mediated endocytosis (Fig. D & E; arrowheads: FR), triggered significant eGFP-luciferase marker gene silencing, and transfection with antitumoral EG5 siRNA suppressed cell proliferation in FR-expressing tumor cells. Moreover, the most promising formulation TCP1 after i.v. administration in tumor-bearing mice exhibited siRNA delivery into the tumor, reducing the EG5 gene expression by 46% at mRNA level (Fig. F) [6]. Therefore, we developed highly functionalized and defined siRNA carrier systems, which could be a potential strategy for RNAi-based cancer therapeutics.

Acknowledgments: The study was supported by DFG Excellence Cluster Nanosystems Initiative Munich (NIM).

References: 1. Lächelt, U., Wagner, E.: Chem. Rev. 2015, 115 (19), 11043-78. 2. Lee, D.J. et al.: Methods Mol. Biol. 2015, 1206:15-27. 3. Dohmen, C. et al.: ACS Nano. 2012, 6(6): 5198-208. 4. Lächelt, U. et al.: Mol. Pharm. 2014, 11(8): 2631-9. 5. Lee, D.J. et al.: Biomaterials. 2016, 77, 98-110. 6. Lee, D.J. et al.: J. Control. Release. 2016, DOI: 10.1016/j.jconrel.2016.06.011.

INDUSTRIAL PHARMACY


2.15 Industrial Pharmacy Chairs: Q. Queckenberg, F. Kramer

SL.58

Introduction of Continuous Manufacturing from an Engineering Perspective Rehbaum, H.1

1 Dr. Rehbaum Consulting, Berlin 10117, Germany

Driven by initiatives of national regulatory authorities, pharmaceutical companies and international societies, continuous manufacturing for secondary manufacturing of solid dosage forms has gained increasing interest in the pharmaceutical industry. Early adopters were among both pharmaceutical manufacturers and equipment suppliers, resulting in different approaches as either single unit operations or turnkey solutions for continuous manufacturing. However, continuous manufacturing implies a change of the mindset for all participating parties. This leads to different expectations and understandings, especially when pharmaceutical companies are approaching equipment suppliers to jointly evaluate technologies and continuous processes. In his presentation, Dr. Rehbaum will report about hands-on experience and examples from his work as technology consultant for the pharmaceutical industry, targeting especially pharmaceutical companies currently in preparation to engage into continuous manufacturing.

SCIENTIFIC LECTURES


SL.59

Model based PAT implementation in pharmaceutical manufacturing processes De Beer, T.1 1 Ghent University, Department of Pharmaceutical Analysis, Laboratory of Pharmaceutical Process Analytics & Technology (LPPAT), Ottergemsesteenweg 460, B-9000 Ghent, Belgium

Process Analytical Technology (PAT) refers to a toolbox used to ensure that quality is built into products herewith improving process understanding, increasing efficiency, and decreasing costs. PAT is getting more and more adopted by the pharmaceutical industry, as stimulated by the regulatory authorities. The PAT toolbox contains process analyzers, multivariate analysis tools, modelling and simulation tools, process control tools, and continuous improvement/knowledge management/information technology systems. The integration and implementation of these tools is complex, and has resulted in uncertainty with respect to both regulation and validation. The paucity of staff knowledgeable in this area may complicate adoption. This presentation will especially focus – by means of cases studies - on the challenges related to the correct implementation of process sensors (e.g., interfacing of measurement probes) in pharmaceutical process environments allowing the obtain representative and meaningful real-time measurement. Case studies will demonstrate how model based analysis (e.g., based on computational fluid dynamics) may contribute to optimal process sensor implementation.

INDUSTRIAL PHARMACY


SL.60

Modular, Continuous API Production Units by INVITE Schweiger, A. INVITE GmbH, CHEMPARK Bldg. W32, D-51368 Leverkusen, Germany

Modular continuous production plants are a promising approach to face the challenges of volatile markets, shorter product lifecycles and diversification of the product range. Recent public funded projects successfully demonstrated the technical and economic potential of small scale modularly built production plants [1]. Small scale continuous production addresses various business sectors with different boundary conditions like specialties, fine chemicals and pharmaceuticals manufacturing. Through modularization, a flexible infrastructure is provided in order to achieve versatile production plants. This is required to be competitive with currently applied batch technology. The flexibility within the modular concept is achieved by modularization on different technical and information layers. Furthermore methods such as standardization, process intensification and scalability reinforce the continuous modular plant concept. The improved efficiency by transformable multi-purpose plants is strength of the modular concept, as well as the ability of capacity expansion by numbering-up the modular equipment. Additionally, the modularization allows accelerating the engineering by reusing know-how which leads to faster development times [2]. To enable the reuse of engineering information a systematic modularization approach is pursued at INVITE. This approach divides a continuous process into Process Equipment Design modules (PED). A PED represents at least one unit operation including the peripheral components. The PEDs ensure the documentation during engineering and plant lifecycle and contain for example typical engineering documents such as P&ID as well as safety and reliability assessments. Stored in a data base, the created PEDs can be reused over several projects. The physical module is then built following technical and geometrical guidelines to provide the functional modularization. This allows the reuse, replace and combination of single modules. Each module is designed as an autonomous module, decoupled from the overall system, which allows independent replacement, cleaning or maintenance. If a standalone or decentralized production is desirable, the modules can be integrated into a Process Equipment Container (PEC). These containers provide a fully integrated infrastructure to build up a mobile production environment. During the F³ Factory project a modular and flexible continuous production of an active pharmaceutical ingredient was demonstrated at the INVITE research centre. A multi-step synthetic batch process was partly transferred to the modular continuous infrastructure. This led to significant reduction in processing steps, reaction time and solvents used. Additionally, a reduction in design and installation costs as well as apparatus costs could be achieved [1]. The subsequent research project CONSENS (Integrated Control and Sensing) is now focussing to advance the continuous production of high-value products by introducing novel online sensing equipment and closed loop control of the key product parameters. For example, an online concentration measurement by Medium Resolution Nuclear Magnetic Resonance Spectroscopy (MR-NMR) will be integrated into the modular plant concept. Especially with regard to the pharmaceutical manufacturing, continuous modular plants can enable an efficient manufacturing with high quality. In spite of some regulatory uncertainties, the FDA even encourages the development towards continuous manufacturing [3], so that small scale modular plants can be seen as a suitable strategy of the future pharma production. References: 1. F³ Factory: Flexible, Fast and Future Production Processe - Final Report. http://www.f3factory.com/scripts/pages/en/newsevents/F3_Factory_final_report_to_EC.pdf (accessed July 20, 2016) 2. Bramsiepe, C.; Schembecker, G.: CIT 2012, 84 (5), 581-587 3. Chatterjee, S.: FDA Perspective on Continuous Manufacturing, IFPAC Annual Meeting, January 2012, 4. Kessler, S.M. et al.: Front Physiol 2016 7: 147 5. http://www.fda.gov/downloads/AboutFDA/CentersOffices/OfficeofMedicalProductsandTobacco/CDER/UCM341197.pdf (accessed July 20, 2016)

SCIENTIFIC LECTURES


SL.61

Advancing process understanding in film coating by in-line terahertz pulsed imaging, optical coherence tomography and discrete element modelling Lin, H.1; Dong, Y.2; Markl, D.3; Pei, C.4; Williams, B. M.5; Zheng, Y.5; Shen, Y. C. 2; Elliott, J.A.5; Zeitler, J. A.3

1 Department of Engineering, Lancaster University, Lancaster LA1 4YW, UK 2 Department of Electrical Engineering and Electronics, University of Liverpool, Liverpool L69 3GJ, UK 3 Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge CB2 3RA, UK 4 Department of Materials Science and Metallurgy, University of Cambridge, Cambridge CB3 0FS, UK 5 Department of Eye and Vision Science, University of Liverpool, Liverpool LT7 8TX, UK

We have recently developed the measurement technologies to acquire coating thickness measurements simultaneously in-line using two independent sensing modalities: terahertz pulsed imaging (TPI) and optical coherence tomography (OCT). Both techniques are sufficiently fast to resolve the coating thickness of individual pharmaceutical tablets in situ during the film coating operation and both techniques are direct structural imaging techniques that do not require multivariate calibration. The TPI sensor is suitable to measure films of > 50 μm and can penetrate thick layers even in the presence of pigments over a wide range of excipients. Due to the long wavelength of terahertz radiation it is not affected by scattering due to dust within the coater. In contrast, OCT can resolve coating layers as thin as 10 μm and is capable of measuring the intra-tablet coating uniformity as well as the inter-tablet coating thickness distribution within the coating pan. However, the OCT technique is less robust when it comes to the compatibility with excipients, dust and potentially the maximum coating thickness that can be resolved. Using a custom built laboratory scale perforated pan coating unit the coating thickness measurements were acquired independently by the TPI and OCT sensors throughout a film coating run. Results of the in-line TPI and OCT measurements were compared against one another and validated with off-line TPI and weight gain measurements. Compared to other process analytical technology sensors the TPI/OCT sensors can resolve the inter-tablet thickness distribution based on sampling a significant fraction of the populations of tablets in the process. By combining two complementary sensing modalities it was possible to seamlessly monitor the coating process over the range of film thickness of 20 to > 250 μm. To complement the in-line measurements and to further challenge the validity of the measurement data we have developed discrete element models (DEM) of the film coating operation at exactly the same process scale as the experimental work. Using the DEM simulations we were able to numerically evaluate the mixing of tablets in the coater and the mass transfer of the coating onto the tablet cores. The DEM results allowed us to check whether the chosen sensor locations and measurement geometry are suitable to measure a representative sample of the total tablet population in the coater and what process parameters have the most pronounced effect on the coating quality. Acknowlegements: We would like to acknowledge financial support from the U.K. Engineering and Physical Sciences Research Council (EP/L019787/1 and EP/L019922/1). The authors acknowledge BASF for providing the materials used in this study, Colorcon Ltd. (Dartford, UK) for coating process recommendations, Huettlin GmbH (Bosch Packaging Technology, Schopfheim, Germany) for advice on the coating unit design and the staff of the electronics and mechanical workshops at the Department of Chemical Engineering and Biotechnology, University of Cambridge for building the lab scale coater.


3 POSTERS

POSTERS


3.1 Analytics

POS.1

Investigating the Interaction of an adhesion protein P-selectin with heparinoids using affinity capillary electrophoretic and computational methods Mozafari, M.1; Balasupraminiam, S.1; El Deeb, S.1; Wätzig, H.1 1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstrasse 55, 38106 Braunschweig, Germany

P-selectin is a transmembrane protein located in the granules of platelets and the Weibel-Palade bodies of endothelial cells. P-selectin imparts rolling of leukocytes on activated endothelial cells as well as interaction of platelets with leukocytes after binding to its nature ligand called PSGL-1 [1,2]. This binding is dependent on the presence of calcium ions [3,4]. Studies have indicated that plasma P-selectin levels are higher in disorders associated with arterial thrombosis [5,6]. Hence it is important to investigate the binding affinities of this protein to potential inhibitors. Therefore, heparin and pentosan polysulfate sodium (PPS) were investigated for their binding affinity to P-selectin. PPS is a highly sulfated semi synthetic polysaccharide which has shown to exhibit numerous pharmacological activities [7,8]. For the purpose of binding studies, a fast and precise affinity capillary electrophoresis method has been developed and applied for the investigation of the interactions between P-selectin and heparinoids in the presence and absence of calcium ions. The normalized mobility ratios (∆R/Rf) were used to evaluate the binding affinities [9,10]. It was found that P-selectin more strongly interacts with heparinoids in the presence of calcium ions. The half-maximal concentration of heparinoids to affect P-selectin mobility was estimated to be 3 mg/L. Acknowledgments: We gratefully acknowledge bene pharmaChem for providing the PPS substances and the financial support, Furthermore we thank PolymicroTM Technologies for providing the capillaries used in this study.

References: 1. Danese, S. et al.: Digest. Liver Dis. 2005, 37 (11): 811–818. 2. Lorant, D. E. et al.: J. Clin. Invest. 1993, 92 (2): 559–570. 3. Barondes, S. H. et al.: Trends Biochem. Sci. 1988, 13 (12): 480–482. 4. Ernst, B. et al.: Nat. Rev. Drug Discov. 2009, 8 (8): 661–677. 5. Merten, M. et al.: Z. Kardiol. 2004, 93 (11): 855–863. 6. Zhu, H. et al.: Med. Chem. Commun. 2013, 4 (7): 1066–1072. 7. Abdel-Haq, H., Bossu, E.: J. Chromatogr. A. 2012, 1257: 125–130. 8. Dürüst, N., Meyerhoff, M. E.: Anal. Chim. Acta. 2001, 432 (2): 253–260. 9. Redweik, S. et al.: Electrophoresis. 2013, 34 (12): 1812–1819. 10. Alhazmi, H. A. et al.: J. Pharm. Biomed. Anal. 2015, 107: 311–317.

POS.2 Using affinity capillary electrophoresis to investigate the concentration-dependant binding of heparinoids to albumins Mozafari, M.1; El Deeb, S.1; Wätzig, H.1 1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstrasse 55, 38106 Brunswick, Germany.

Serum albumins are the most studied proteins over the years. They are predominant protein component in blood plasma. Albumins are important for transport of a variety of endogenous and exogenous substances in blood. They have many physiological functions; for instance, they bind a diversity of hydrophobic ligands such as fatty acids and steroids; but also small molecules and some metal ions bind to albumins [1-3]. Binding the drugs to serum albumins is one of the most important pharmacokinetic determinants. This might have an impact on the lifetime of a drug in plasma [1-3]. Hence, it is important to find out how strong the drugs interact with albumins. For this reason, an affinity capillary electrophoretic method has been applied due to its numerous benefits such as short analysis duration and usage of small amounts of samples [4-6]. The evaluation of binding affinities was carried out using normalized mobility ratios (∆R/Rf) [4-6].

The Interactions of various heparinoids (unfractionated and a low molecular weight heparin), as well as pentosan polysulfate sodium (PPS), with human and bovine albumins (BSA and HSA) have been studied. The experiments were performed at 23°C and 37°C. Both BSA and HSA interact more strongly with PPS than with unfractionated and low molecular weight heparins. For PPS, interactions can already be observed at low mg/L concentrations (3mg/L), and saturation is already obtained at approximately 20 mg/L. Unfractionated heparin showed almost no interactions with BSA at 23°C, but weak interactions at 37°C at higher heparin concentrations. The peak shapes also changed at higher concentrations at 37°. This was the most substantial difference due to the temperature. In most cases the binding data were similar at both temperatures. However, temperature-dependant changes in the peak shapes have been observed for heparin and PPS. Furthermore, HSA showed a characteristic splitting in two peaks especially after interacting with PPS, which is probably attributable to the formation of two species or conformational change of HSA after interacting with PPS. The successive experiments and the presentation of the electropherograms, ordered by increasing heparinoid concentrations, seems to be very powerful to understand peak splitting phenomena and differences between heparinoids and their albumin interactions. Marked differences have been found. Acknowledgments: We gratefully acknowledge bene pharmaChem for providing the PPS substances and the financial support, Furthermore we thank PolymicroTM Technologies for providing the capillaries used in this study.

References: 1. Rezaei-Tavirani, M. et al.: J. Biochem. Molec. Biol. 2006, 39 (5): 530-536. 2. Fanali, G. et al.: Molec. Aspects of Med. 2012, 33: 209–290. 3. Gelamo, E.L. et al.: Biochim. Biophys. Acta. 2002, 1594: 84-99. 4. Redweik, S. et al.: Electrophoresis. 2013, 34 (12): 1812–1819. 5. Alhazmi, H. A. et al.: J. Pharm. Biomed.l Anal. 2015, 107:311–317. 6. Mozafari, M. et al.: Electrophoresis. 2015, 36: 2665–2669.

POS.3 Determination of the Enantiomeric Purity of Nipecotic Acid as 1-(7-Nitrobenzo[c][1,2,5]oxadiazol-4-yl) Derivatives Schmidt, S. K.1; Höfner, G.1; Wanner, K. T.1 1 Department für Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377 München, Deutschland

Nipecotic acid is an important precursor for the synthesis of novel enantiopure inhibitors of γ-aminobutyric acid transporters (GATs) [1,2], the inhibitory activity of which is frequently found to differ quite substantially. For example, the (S)-enantiomer DDPM-1457 is known to be more potent at the GABA transporter subtype mGAT4 than its (R)-enantiomer [(S): pIC50 = 5.87 ± 0.08; (R): pIC50 = 4.33 ± 0.05] [3,4] (Figure 1). With the data of pharmacological testing being dependent on the enantiopurity of the studied samples, the knowledge of the precise enantiopurity of nipecotic acid serving as starting material for the synthesis of new GAT inhibitors is of fundamental importance. Therefore, an HPLC method for the determination of the enantiopurity of nipecotic acid (piperidine-3-carboxylic acid) was established. Derivatization of nipecotic acid prior to chromatography appeared promising to guarantee high wavelengths for UV-Vis detection ensuring a selective way of determination of the individual enantiomers of the analyte and as a consequence thereof the determination of high ee values. With 4-fluoro-7-nitrobenzo[c][1,2,5]oxadiazole, a derivatization reagent was found fulfilling this requirement. It enabled efficient separation of both enantiomers of nipecotic acid as 1-(7- [c][1,2,5]oxadiazol-4-yl) derivatives on a ChiralPak ID-3 stationary phase (Daicel, Illkirch, France) and reliable UV-Vis quantification at 490 nm (Figure 1). The established HPLC based method has been validated regarding specificity, linearity, quantification limit (QL), accuracy, and precision. In addition, by spiking of highly enantiopure samples of nipecotic acid derivatives with small amounts of racemic mixture, the newly developed method has been shown to even enable the detection of slight changes of ee values and to be well suited for the accurate determination of very high enantiomeric excesses.

ANALYTICS


N

OH

O

NO

N

NO2

N

OH

O

MeO

MeO

OMe

1-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)nipecotic acid

DDPM-1457

NO

N

NO2

F

4-fluoro-7-nitrobenzo[c][1,2,5]oxadiazole

Fig. 1: mGAT4 inhibitor DDPM-1457, derivatization reagent 4-fluoro-7-nitrobenzo[c]-[1,2,5]oxadiazole and 1-(7-nitrobenzo[c][1,2,5]oxadiazo-4-yl)nipecotic acid.

References: 1. Herein, the nomenclature for the murine GABA transporters (mGAT1-mGAT4) is used. 2. Krogsgaard-Larsen, P. et al.: Epilepsy Research 1987, 1(2): 77–93. 3. Kragler A., Höfner G., Wanner K. T.: Eur. J. Med. Chem. 2008, 43: 2404–2411. 4. Pabel, J. et al.: Chem. Med. Chem. 2012, 7(7): 1245–1255.

POS.4 Determination of pesticide exposure of Podarcis muralis by a micro-QuEChERS approach and GC-MS/MS Stöckelhuber, M.1; Müller, C.1; Mingo, V.2; Lötters, S.2; Wagner, N.2, Bracher, F.1 1 Department für Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 München, Germany 2 Department für Biogeographie, Universität Trier, Universitätsring 15, 54296 Trier, Germany

Pesticides are widely applied in a variety of different ways during the production of foods to control the growth of weeds and fungi or to prevent crop damage by insects, mites, rodents and other pests [1]. Although many species within the European Union are strictly protected in all member states due to the Habitats Directive, detrimental effects of pesticide use are possible within [2] and especially outside protected areas [3]. This is in particular remarkable as negative effects, which may lead to a regional diversity loss, have already been identified in laboratory and mesocosm studies. In reptiles, as one of the various threatened vertebrate groups, ongoing worldwide population declines are recognized. The causes for these declines are highly assorted, and it is believed that among all factors, habitat loss and degradation is the major factor in industrialized countries, followed by use of agrochemicals in their habitats. Although effects of pesticides on reptiles have been reviewed to some degree, and different studies have shown evidence of potential strong effects on reptile wildlife, there is still a great lack of data. Especially, toxicity data concerning squamates is scarce, and data on effects of pesticides in species' natural habitats even more so [3]. To evaluate the exposure risk of the lizard Podarcis muralis the residue unit dose of its prey animals at different times after exposition has to be determined. In the last few years a new multiresidue analytical method for the determination of pesticides, the QuEChERS concept (Quick, Easy, Effective, Cheap, Rugged and Save) has become very popular in the analysis of pesticides in fruits and vegetables [4,5]. QuEChERS methodology (used for extraction and clean-up steps) has been applied with success on diverse food matrices [6]. However, the prey animals of Podarcis muralis (mostly insects and spiders) possess another tissue composition than plant material with the result that the method had to be adapted to the current matrix. Therefore different cleaning procedures and extraction steps have been tested to obtain satisfying recoveries and precision. Solving the problem with the minimal available tissue material (100 – 1000 mg), a so called micro-QuEChERS approach was developed and combined with the powerful gas chromatography triple quadrupole system (GC-MS/MS). This combination allows the simultaneous analysis of pesticides in animal tissue with minute amounts of sample. The method was fully validated according to SANCO/12571/2013 [7]. Finally, the samples of the prey animals have been prepared with the new micro-QuEChERS approach and analyzed with GC-MS/MS to assess the oral exposure risk of Podarcis muralis. References: 1. Boes, E. et al.: Procedia Chemistry 2015, 16:229 – 236. 2. Wagner, N. et al. Biol. Conserv. 2015, 191: 667-673. 3. Mingo V., Lötters S., Wagner N.: Environ. Pollut. 2016, 215:164-169. 4. Müller, C.; Bracher, F.; Plößl, F.: Chromatographia 2011, 73:807-811. 5. Anastassiades, M. et al.: J. AOAC Int. 2003, 86 (2):412-431. 6. Castillo, M.; González, C.; Miralles, A.: Anal. Bioanal. Chem. 2011, 400 (5):1315–1328.

7. SANCO Guideline SANCO/12571/2013

POS.5 Determination of plasma protein binding for ephedrine and stereoisomers Volpp, M.1; Holzgrabe, U.1 1 University of Würzburg, Institute for Pharmacy and Food Chemistry, 97074 Würzburg, Germany

(-)-Ephedrine and its diastereomer (+)-pseudoephedrine, both natural occurring alkaloids from Ephedra species, have been used in therapy for centuries [1] and are regaining popularity in the last few years with new over-the-counter combination drugs containing pseudoephedrine. None the less there is scarcely any information on their plasma protein binding in literature. The substances’ plasma protein binding is an important parameter from a pharmacokinetic and pharmacodynamic point of view and can play a crucial role in therapy. A few publications determined a binding constant for either ephedrine or pseudoephedrine to bovine or human serum albumin (BSA or HSA). A drawback of all publications was the application of different methods, like capillary electrophoresis [2], microdialysis [3] or ultracentrifugation [4], for their experiments, resulting in a wide range of the extend of albumin binding. All of these studies weren’t conducted under physiological conditions, impeding any kind of direct comparison. These circumstances lead us to conduct different experiments, determining the plasma protein binding for (-)-ephedrine, (+)-pseudoephedrine and their diastereomers. Since it has been shown that the affinity to albumin, the main transport protein in plasma, can vary for the drug’s different stereoisomers, e.g. propranolol [5], we included all stereoisomers of those two alkaloids in order to find out whether their binding to albumin is enantioselective. The methods used to determine protein binding were continuous and discontinuous ultrafiltration in order to have orthogonal methods to verify our findings. The experiments were conducted in phosphate buffered saline, pH 7.4 with human serum, bovine or human serum albumin, and ultrafiltration filters (MWCO 10kDa) made of regenerated cellulose. To separate and quantify the enantiomers simultaneously, the samples obtained by discontinuous ultrafiltration were analysed using capillary electrophoresis and a chiral selector. Results of different methods will be discussed. References: 1. Chen, K.K.; Kao, C.H.: J. Am. Pharm. Assoc. 1926, 15(8):625-639. 2. Ye, N. et al.: J. Chromatogr. Sci. 2007, 45(5):246-250. 3. Yang, R. et al.: Luminescence 2011, 26(5):374-379. 4. Till, A.E.;Benet, L.Z.: J. Pharmacol. Exp. Ther. 1979, 211(3):555-560. 5. Albani, F. et al.: Br. J. Clin. Pharmacol. 1984, 18(2):244-246.

POS.6

Chiral separation of phenethylamines by capillary electrophoresis using tetrabutylammonium chloride as background electrolyte additive Wahl, J.1; Holzgrabe, U.1 1 University of Würzburg, Institute of Pharmacy and Food Chemistry, Am Hubland, 97074 Würzburg, Germany

The enantiomers of a chiral drug can have different pharmacodynamic, pharmacokinetic and toxicological properties [1-3]. Thus, especially new drugs are launched as pure enantiomers. However, the isomeric purity has to be proven. Capillary electrophoresis (CE) is often used for the chiral separation of drugs and pharmaceutical products. Phenethylamines, like ephedrine, pseudoephedrine, methylephedrine and norephedrine are active components extracted from ephedrae herba [4]. They are used as ingredients of cold medicines and abused as starting material for the synthesis of methamphetamine [5]. In the past few years a great interest was drawn towards ionic liquids (ILs) in analytical separation techniques. Ionic liquids are defined as (semi-)organic salts with a melting point below 100 °C. One special advantage of ILs is that they can be designed as requested. It is possible to modify the melting point, the viscosity, the water-solubility/-

POSTERS


miscibility and the electrochemical behavior by altering the combination of cations and anions. Ionic and/or proton donor-acceptor interactions between analyte and IL are possible interactions facilitating new kinds of separation mechanisms. In capillary electrophoresis ILs can be used as main electrolyte, as electrolyte additive and for dynamic coating of the capillary surface. ILs possess many properties making them excellent additives in CE background electrolytes (BGE). The most important property is the charge of the dissolved ions in BGE enabling the cations to interact with deprotonated silanol groups on the capillary surface and thereby modifying the electroosmotic flow. The enantioseparation of four phenethylamines (ephedrine, pseudoephedrine, methylephedrine, norephedrine) was investigated by adding different concentrations of tetrabutylammonium chloride (TBAC) to a phosphate buffer containing β-cyclodextrin as chiral selector [6]. By increasing the concentration of TBAC an enhancement of the chiral resolution was found. Due to an adsorption of the IL cations to the capillary surface, an increase of migration times was observed by increasing the concentration of TBAC in the BGE. To ensure reproducible migration times different rinsing procedures (acidic, basic, organic) were examined. The best results have been observed for a basic rinsing step using electric voltage instead of pressure. To further optimize the chiral separation method, phosphate buffers in a range from 50 to 100 mM and from pH 2.0 to pH 3.0 have been tested. Furthermore the concentration of TBAC was varied from 0 - 200 mol/L and different electrical voltages and capillary temperatures have been investigated. For three compounds (ephedrine, pseudoephedrine, methylephedrine) a baseline resolution was achieved. The best enantioseparation for all analytes was found using a 75 mM phosphate buffer pH 2.5 containing 0.125 mol/L TBAC. References: 1. Bialer, M.: Adv. Drug Deliver. Rev. 2012, 64: 887-895. 2. Duggan, K. C. et al.: Nat. Chem. Biol. 2011, 7: 803-809. 3. Blaschke, G. et al.: Arznei.-Forsch. 1979, 29: 1640-1642. 4. Liu, Y. M.; Sheu, S. J.: J. Chromatogr. 1992, 600: 370-372. 5. Mikuma, T. et al.: Forensic Sci. Int. 2015, 249C: 59-65. 6. Holzgrabe, U.; Wahl, J.: Capillary Electrophoresis - Methods and Protocols 2016, article in press.

POS.7

Development of quinine-derived chiral stationary phases for hyphenated enantioselective liquid chromatography-mass spectrometry Woiwode, U.1; Zimmermann, A.1; Sievers-Engler, A.1; Lämmerhofer, M.1 1 Institute of Pharmaceutical Sciences, Pharmaceutical (Bio)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany

State-of-the-art analytical HPLC and UHPLC for research and quality control is more and more combined with mass spectrometry (MS) to provide comprehensive analytical results within a minimum of time. Despite the vast amount of data obtainable by MS, MS/MS and MSn, respectively, there is virtually no possibility to discriminate enantiomers by this way of detection alone. Accordingly, separation on the LC-stage is required to access chiral compounds analytically, especially those of pharmaceutical relevance. In this attempt the cinchona alkaloid quinine was used as a backbone to produce chiral selector molecules by formation of C-9-carbamates with aliphatic or aromatic residues and immobilized to silica supports. However, selector bleeding from the column has a pronounced influence on ionization efficiency in positive MS mode when comparing, for example, to alkyl silanols cleaved from RP-phases. To obtain highly stable chiral stationary phases, a new immobilization strategy using a siloxane polymer as carrier of several selector molecules was applied. In contrast to commonly used direct immobilization of the selector via a single thioether bridge there is a higher stability of the polymer-type phase due to redundancy of linkages between the polymer and the actual surface of the support [1]. The chiral stationary phases were further modified by a strongly acidic endcapping. Free thiol-groups on the polymeric film were oxidized to form sulfonic acid residues. This caused secondary repulsive interactions for acidic compounds and rendered milder elution conditions in polar organic elution mode, while maintaining separation performance. LC-MS compatible conditions including reduced mobile phase ion strength and lower flow rate were successfully implemented and gave consistent chiral separation results for sub-nanogram sample amounts within short cycle times. The new

stationary phases also showed individual characteristics under achiral RP- and HILIC-test conditions due to the influence of secondary interactions emerging from silica surface, polymeric film and endcapping. Its multi-purpose usability and its compatibility with modern mass spectrometers such as QTOF and Ion Trap instruments make these new chiral stationary phases a promising tool for various applications, especially in pharmaceutical analytics. References: 1. Zimmermann, A. et al.: J. Chromatogr. A 2016, 1436: 73-83.

POS.8

Characterization and Application of Stable-bond Reversed-phase/Weak Anion-exchange Mixed-mode Silica in Pharmaceutical Research Bäurer, S.1; Zimmermann, A.1; Horak, J.1; Lämmerhofer, M.1 1 Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany

Nowadays, the use of High Performance Liquid Chromatography (HPLC) coupled with several methods of detection is the state of the art in instrumental analysis for quality control of pharmaceutical drugs as well as in research of biomolecules, drug development and synthesis control. For chemically differing analyte mixtures, as well as for mixtures of very similar polar and ionic compounds, complementary methods to the well known and often used reversed phase (RP) chromatography become more and more necessary. Mixed Mode (MM) chromatography combines multiple complementary interaction principles due to the multiple interaction sites of the immobilized selectors. Reversed phase/weak anion exchangers (RP/WAX) stationary phases have already demonstrated to be nicely suitable for the analysis of drugs, metabolites and peptides [1, 2]. The stability of the covalently bonded selectors often turns out to be problematic due to their hydrolysis and detachment from the silica surface, also known as column bleeding. On the one hand, in the case of coupling with very sensitive detection methods, the ageing process of the modified silica is detected and will limit the use of the separation material in combination with this detection mode. On the other hand, the decreasing selector surface coverage causes an alteration of retention times and can affect adversely the separation. Hence, a more stable immobilization strategy has been developed which uses a multiple crosslinked poly(3-mercaptopropyl)methylsiloxane layer on the silica surface for immobilization of the selector via thiol-ene click chemistry. Recently, it has been proven to be greatly MS compatible [3]. The RP/WAX stationary phases obtained via polysiloxane layer coating have been characterized according to their MM character and anion exchange capacity for evaluation of the influence of this stabilization strategy. Further investigations utilizing this promising stabilized RP/WAX silica were done to overcome challenging separation problems, which were previously successfully solved by comparable brush-type RP/WAX stationary phases. References: 1. Hinterwirth, H. et al.: J. Sep. Sci. 2010, 33(21): 3273-82. 2. Zimmermann, A. et al.: J. Chromatogr. A. 2014, 1354: 43-55. 3. Zimmermann, A. et al.: J. Chromatogr. A. 2016, 1436: 73-83.

POS.9

Development of a bioreactor based on papain immobilized on gold nanoparticles for efficient protein digestion Liu, S.1; Höldrich, M.1; Lämmerhofer, M.1 1 University of Tübingen, Institute of Pharmaceutical Sciences, Auf der Morgenstelle 8, 72076 Tübingen, Germany

Immobilized enzyme on nanostructure materials has gathered increasing attention in recent years. Compared with free enzyme in solution, immobilized enzyme has many outstanding qualities such as high catalytic efficiency, flexible control of reaction, easy removal after reaction, no contamination for product and repetitive usage. Due to the significant physical properties of the gold nanoparticle (GNP), it

ANALYTICS


becomes a popular substrate which can provide a large surface-to-volume ratio for the immobilization of enzyme. In our study, surface modified GNPs were achieved via layer-by-layer (LBL) process with alternative cationic polyallylamine and anionic poly(acrylic acid). Afterwards, papain was immobilized on the GNPs via the covalent amide coupling between the amino groups on papain and the terminal carboxylic groups of the GNPs by using EDC and sulfo-NHS. The resultant papain-functionalized gold nanoparticles were characterized by surface plasmon resonance (SPR) band, dynamic light scattering (DLS) and zeta potential.[1] A new technology Resonant Mass Measurement (RMM) was applied for determining the average number of papain immobilized on per GNP by precise measurement of the single nanoparticle mass in the range of femtogram to attogram.[2] The activity of the immobilized enzyme was estimated by determination of the Michaelis-Menten parameter (Km, Vmax and Kcat) with the standard chromogenic substrate Nα-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BApNA).[3] Further, the standard protein solution was digested by the gold nanobiocatalyst to the peptides, and further analysed by using UHPLC-ESI-QTof-MS/MS, which demonstrated the applicability of the bioreactor based on papain functionalized GNPs. The immobilized papain not only has higher activity recovery and better stability, but also can be easily isolated from the reaction medium by easy centrifugation steps for reusage. Acknowledgments: We acknowledge Dr. Markus Epe, Malvern GmbH (Herrenberg, Germany).

References: 1. Hinterwirth, H. et al.: Anal. Chem. 2013, 85: 8376-8384. 2. Nejadnik, M. R.; Jiskoot, W.: J. Pharm. Sci. 2015, 104: 698-704. 3. Hinterwirth, H.; Linder,W.; Lämmerhofer, M.: Anal. Chim. Acta. 2012, 733: 90-97.

POS.10

Application of near infrared spectroscopy for content determination in semi-solid pharmaceutical formulations Schlegel, L.1; Schubert-Zsilavecz, M.1,2; Abdel-Tawab, M.1

1 Zentrallaboratorium Deutscher Apotheker (Central laboratory of German pharmacists), Carl-Mannich-Str. 20, 65760 Eschborn, Germany 2 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany

Over the last decades near infrared spectroscopy (NIRS) has become a valuable tool in quality control and is widely used in the pharmaceutical industry for identification and qualification purposes of active pharmaceutical ingredients (APIs) and excipients. It has also gained in importance in the course of the FDA PAT initiative for determination of critical process parameters such as moisture content and blend uniformity [1-3]. Use of NIRS for API quantification has been described for solid formulations including tablets, capsules or powder mixtures [4] and also for fluids [4-6], while little data for the application to semi-solid formulations [4] is available. Since affordable NIR spectrometer with limited wavelength range are increasingly used in pharmacies and, furthermore, there is a rising need for economic and practical methods in quality control of extemporaneous mixtures the question arises whether those NIR spectrometer are suitable for API quantification in creams and ointments. Therefore, we have evaluated the applicability of NIRS on preparations with different frequently used APIs in hydrophilic and lipophilic creams as well as in water-free lipid systems. Due to the fact that NIRS is not suitable for analysis of very small drug amounts, APIs were chosen that are used in dermatologically effective concentrations above 1 % such as salicylic acid, urea, erythromycin and metronidazole. We studied six different formulations each containing one of the aforementioned drugs using NIR transflectance measurements and developed quantitative models for content determination of common concentrations. To generate appropriate fitting methods with low prediction errors concerning RMSEP and SEP as well as with acceptable coefficients of determination we used partial least squares regression (PLS) and multiple spectral pretreatments. Finally, we successfully validated one method based on the criteria of the EMA Guideline [7]. The results show that the application of NIRS for API quantification in semi-solid preparations is possible in general, but accuracy and variance depend on the active ingredient and its concentration range.

Acknowledgements: We thank HiperScan GmbH, Dresden, Germany for providing the NIR spectrometer and calibration software.

References: 1. Blanco, M. et al.: Analyst 1998, 123(8): 135R–150R. 2. Reich, G.: Adv. Drug Deliv. Rev. 2005, 57(8): 1109–43 3. Jamrógiewicz, M.: JPBA 2012, 66: 1–10. 4. Roggo, Y. et al.: JPBA 2007, 44(3): 683–700. 5. Silva, M. et al.: Talanta 2012, 89: 342–51. 6. Lê, L.M. et al.: Talanta 2014, 119(36): 361–6. 7. European Medicines Agency: Guideline on the use of near infrared spectroscopy by the pharmaceutical industry and the data requirements for new submissions and variations 2014 (URL:http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2014/06/WC500167967.pdf)

POS.11

2D protein analysis – a part of the SynFoBiA project at the Center of Pharmaceutical Engineering Maul, K. J.1,2; Hahne, T.1; Wätzig, H.1,2 1 TU Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany 2 PVZ – Center of Pharmaceutical Engineering, Franz Liszt Straße 35a, 38106 Braunschweig, Germany

Biopharmaceuticals become more and more important in the therapy of different illnesses. Thus it is important to develop new and cheap ways to produce them. Like all active pharmaceutical ingredients the quality has to be very high. Proteins are, in contrast to most small molecules, not that stable and may also form aggregates. To ensure the required quality a precise and fast analysis is mandatory, especially to detected unwanted impurities. For this purpose a 2D separation with a combination of LC and CE was developed. In the first dimension a strong anion exchanger (SAX) column was used. Fractions were collected each 80 sec. These were then analyzed with capillary gel electrophoresis (CGE). To determine the suitability of this method standard protein samples were prepared. They contain Myoglobin, β-Lactoglobulin, Ovalbumin, BSA and a monoclonal Antibody. Six samples were prepared. Each was then analyzed with SAX five times and 23 fractions were collected each time. Afterwards each collection was analyzed with CGE. The 2D-separation technique was applied to a real sample. This sample was produced within the Center of Pharmaceutical Engineering (PVZ) project “New synthesis and formulation of poorly soluble API’s and sensible biopharmaceuticals” (SynFoBiA: Neuartige Synthese- und Formulierungsverfahren für schwerlösliche Arzneistoffe und empfindliche Biopharmazeutika). The PVZ combines pharmaceutics, chemical engineering, bioengineering and micro engineering, which is a unique German collaboration placed at the TU Braunschweig. Institutes from the TU Clausthal and the Leibniz Universität Hanover also participate. Acknowledgements: We thank Polymicro for providing the capillaries used in the CGE and our cooperation partners which provided the real samples.

POS.12

Characterization of Adsorption Phenomena of Antibody Conjugates Duerr, C.1; Seifert, I.1; Friess, W.1 1 Ludwig-Maximilians-Universität München, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Butenandtstr. 5, 81377 Munich, Germany

Introduction: Antibody-drug conjugates are becoming a vital tool in effective cancer treatment. Due to the added hydrophobic payload, antibody-drug conjugates may show different characteristics than the naïve mAb [1]. Since the amount of protein lost or unfolded due to adsorption to container, processing material or the air-liquid interface is a critical factor [2], the adsorption to glass and the air-liquid interface was analyzed for a model antibody conjugate and the naïve mAb. Moreover, the mechanical stress stability of the samples was investigated in this study. Materials and Methods: A therapeutic mAb (2 mg/mL) in 10 mM phosphate, 145 mM NaCl PBS at pH 7.2, was mixed with (NHS)-Fluorescein in DMF. The same therapeutic mAb in 100 mM NaHCO3 buffer at pH 9.0 was mixed with Eosin-ITC in DMSO [3]. The

POSTERS


products were purified and buffer exchanged to 10 mM phosphate, 145 mM NaCl PBS pH 7.2 or 6.5. 0.5 mg/mL samples were analyzed on ServalytTM PrecotesTM gels pH 6-9 and stained with Serva Blue W to determine the isoelectric point. The adsorption behavior of 2 mg/mL samples was analyzed on a SiO2 surfaced quartz crystal chip using a QCM apparatus qCell T, resembling a glass surface. Colloidal stability was measured using a Zetasizer APS at protein concentrations between 0.2 and 10 mg/mL and calculated according to [4].To determine the surface pressure at the air-liquid interface, the samples were analyzed using a Teflon multiwellplate on a MicroTroughX. A mechanical stress study was performed to analyze the continuous formation of a new air-liquid interface by shaking 0.2 mg/mL samples at 500 rpm for 24 hours. The samples were analyzed for visual appearance, turbidity at 350 nm (Nanodrop 2000) and subvisible particles (PAMAS SVSS-25). Results and Discussion: The degree of labelling was 6 for both model mAb conjugates. The calculated hydrophobicity is 2.55 (NHS-Fluorescein) and 4.52 (Eosin-ITC) resp., indicating that hydrophobic molecules were attached to the mAb and that the Eosin-conjugates are more hydrophobic than the Fluorescein-conjugates. The isoelectric points were decreased for the conjugates (5.3-7.4) compared to the naïve mAb (7.6-8.0). Consequently, at the pH values tested, the mAb is slightly net positively charged, whereas the mAb conjugates are negatively charged or neutral. In QCM measurements, the mAb conjugates showed a smaller frequency shift and decreased total adsorbed and irreversible adsorbed masses at pH 7.2, as the charge interaction between the net neutrally charged mAb conjugates and the negatively charged chip surface is decreased compared to the slightly positively charged naïve mAb. The surface pressure was not increased for the mAb conjugates compared to the naïve mAb, showing that the adsorption to the air-liquid interface is not changed with conjugation. Colloidal stability displayed net attraction for the mAb conjugates and net repulsion for the mAb, indicating that the mAb conjugates are more prone to aggregation. After the mechanical stress, the visual appearance of all samples remained unchanged. The turbidity showed a more pronounced increase for the conjugates. Moreover, the number of subvisible particles was significantly increased for the conjugates compared to the naïve mAb, illustrating that aggregates have formed. Differences between the two mAb conjugates or an effect of the pH value could not be identified. Thus, the antibody conjugates are less resistant to mechanical stress. The formation of particles is not due to an increased adsorption to the air-liquid interface or to the glass surface, but because of net attractive interactions of the mAb conjugate molecules. Conclusion: The conjugation of negatively charged hydrophobic molecules to the mAb has slightly decreased protein adsorption to glass imitating SiO2 chips. Adsorption to the air-liquid interface was not increased for the conjugates compared to the naïve mAb. However, the mAb conjugates were more sensitive to shaking stress and showed net attractive interaction. Acknowledgments: Coriolis Pharma, Aftahy, K., Quraeschi, M.

References: 1. Wakankar, A.A. et al., Bioconj Chem. 2010, 21(9): 1588-1595. 2. Dixit, N., Maloney, K.M., Kalonia, D.S., Int. J. Pharm. 2011, 412: 201-27. 3. Molecular Probes by Life Technologies, Amine-Reactive Probes, MAN001774, 2013, Revision 2.0. 4. Menzen, T.; Friess, W.; J. Pharm. Sci. 2014.103(2): 445-455.

POS.13

Investigation of the Possible Anticancer Properties of the Rhodium(I) N-Heterocyclic Carbene Complexes Wölker, J.1,2; Ott, I.1,2 1 Institute of Medicinal and Pharmaceutical Chemistry, Technische Universität Braunschweig, Beethovenstrasse 55, D-38106 Braunschweig, Germany 2 Center of Pharmaceutical Engineering (PVZ), Technische Universität Braunschweig, Franz-Liszt Strasse 35a, D-38106 Braunschweig, Germany

N-heterocyclic Carbene moieties, especially the “Arduengo-type” metal complexes, have recently started to play an important role as new potential anticancer agents. Although the most extensively investigated derivatives of this class of compounds contain gold(I) and silver(I) metal ions, series of rhodium(I) N-heterocyclic carbene (NHC) complexes with 1,5-cyclooctadiene (COD) and a halido ligand (X, Fig. 1), have also shown potential as antitumoral agents.[1,2] The complexes of the type

Rh(I)(NHC)(COD)Cl triggered antiproliferative effects against MCF-7 (human breast adenocarcinoma) and HT-29 (colon carcinoma) cells and inhibited the enzyme thioredoxin reductase (TrxR),[1,2] which is overexpressed in tumor cells. It was confirmed, that the complexes with iodide (X = I) exhibited higher biological activity as their chloro-substituted (X = Cl) equivalents.[2] Rhodium-complexes were also proven to interact with the DNA.[3,4] Scheme 1 shows the newly synthesized rhodium-based complexes, which were characterized by 1H-NMR, 13C-NMR, elemental analysis and mass spectrometry. In our ongoing studies we evaluate the chemical and biological properties of the new complexes with a focus on variations in the X ligand. The current results will be presented on the poster.

Fig. 1: Rhodium(I)-NHC-complexes

Acknowledgments: The financial support of the Center of Pharmaceutical Engineering (PVZ) is gratefully acknowledged.

References: 1. L. Oehninger, L.N. Küster, C. Schmidt et al.: Chem. Eur. J., 2013, 19: 17871 – 17880. 2. L. Oehninger, S. Spreckelmeyer, P. Holenya et al.: J. Med. Chem., 2015, 58: 9591−9600. 3. J. R. McConnell, D. P. Rananaware, D. M. Ramsey et al.: Bioorg. Med. Chem. Lett., 2013, 23: 2527–2531. 4. C. L. Kielkopf, K. E. Erkkila, B. P. Hudson et al.: Nature Struct. Biol., 2000, 7: 117-121.

POS.14/SL.17 Insights from functional lipidomics into the long-term regulation of kinases by vitamin A Pein, H.1; Voelkel, M.1; Schneider, F.1; Rossi, A.2; Koeberle, S. C.3; Loeser, K.1; Morrison, H.3; Sautebin, L.2; Werz, O.1; Koeberle, A. 1 1 Department of Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, 07743 Jena, Germany 2 Department of Pharmacy, University of Naples Federico II, 80131 Naples, Italy 3 Leibniz Institute of Age Research - Fritz-Lipmann-Institute, 07745 Jena, Germany

For Abstract see Short Lectures SL.17

POS.15 Gradual depolymerization of fucoidan from Fucus vesiculosus L. and its effect on the pharmacological profile Lahrsen, E.1; Schoenfeld, A.1; Alban, S.1 1 Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstrasse 76, 24118 Kiel, Germany

The fucose-containing sulfated polysaccharides (syn. “fucoidans”) from brown algae exhibit a wide range of bioactivities and are therefore considered promising candidates for health-supporting and medicinal applications [1]. The past three decades, research on isolation, molecular characterization, and screening of in vitro and in vivo pharmacological activities has significantly increased. Regarding in vivo application, the usually high-molecular mass of native fucoidans may, however, be associated with unfavourable biopharmaceutical properties and possibly undesired effects so that it seems reasonable to develop fucoidan derivatives with reduced size. The aim of the presented study was to establish a suitable depolymerization method for fucoidans without concomitant desulfation and to examine the activity profile of the resulting depolymerized fractions. For this, fucoidan extracted from Fucus vesiculosus L. with an weight-averaged molecular weight (Mw) of 38.2 kDa, a degree of sulfation (DS) of 0.63 and a fucose content of 83.1 % (mol/mol), was used [2]. The two most suitable methods turned out to be hydrothermal depolymerisation at 120 °C and degradation with hydrogen peroxide for different durations. In this way, 19 fractions with Mw down to 10.3 and

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4.9 kDa, respectively, were obtained. Chemical characterization revealed no changes of their chemical composition and no elimination of sulfate groups. The impact of degradation on the pharmacological profile was examined by the following in vitro activities: (1) elastase activity inhibition, (2) inhibition of the classical pathway-activated haemolytic complement activity, (3) inhibition of the classical pathway-activated C5a generation, (4) potentiation of the complement factor C1s inhibition by the serine protease inhibitor C1 inhibitor (C1inh) as well as the undesired effects (5) activation of Factor XII, and (6) anticoagulant activity in the activated partial thromboplastin time (APTT) assay. The activities of the native fucoidan and its fractions were compared either by calculating the IC50, e.g. elastase inhibition assay, or by other characteristic values, i.e. concentration for doubling coagulation time (DC). Table 1 presents the activity loss of four exemplary fractions compared to native fucoidan.

Fuc Mw 38.2 Fuc Mw 34.4 Fuc Mw 20.6 Fuc Mw 15.9 Fuc Mw 10.3

Elastase inhibition

IC50

(µg/mL) 0.48 0.53 0.61 1.18 4.27 Activity loss1

0.0 % 8.5 % 20.0 % 59.1 % 88.7 %

Complement inhibition (haemolysis)

IC50


0.0 % 4.9 % 48.1 % 77.8 % 94.5 %

Complement inhibition (C5a generation)

IC50


0.0 % 14.5 % 23.8 % 45.7 % 70.8 %

C1inh potentiation %2 24.84 25.75 26.28 20.75 9.87 Activity loss1

0.0 % 0.0 % 0.0 % 16.5 % 60.3 %

Factor XII activation %3 25.46 22.38 12.28 13.24 9.32 Activity loss1

0.0 % 12.1 % 51.8 % 48.0 % 63.4 %

Anticoagulant activity DC (µg/mL) 16.70 19.47 18.63 32.14 76.00 Activity loss1

0.0 % 14.2 % 10.4 % 48.0 % 78.0 %

Table 1: Mw dependent activities of native fucoidan and degraded fucoidan fractions 1 calculated in relation to the native fucoidan, 2 sample conc. 6.25 µg/mL 3 in relation to Pathromtin SL® at sample conc. 25.0 µg/mL

All the activities decreased with decreasing Mw, but both the extent and the molecular weight range of activity loss and thus the overall shape of the molecular weight dependent activity loss was different for any individual activity. For example, whereas the inhibitory effect on the haemolytic complement activity was lost at Mw of 10.3 kDa, the C1inh potentiation was only reduced at Mw < 20 kDa and at most by 60 %. In conclusion, the activities of the fucoidan from Fucus vesiculosus l. proved to be dependent on the Mw. However, the impact of the Mw on the various activities turned out to differ resulting in modified pharmacological profiles of the fucoidan fractions. References: 1. Ruocco, N. et al.: Molecules 2016, 21(5): pii: E551. 2. Schneider, T. et al.: Glycobiology 2015, 25(8): 812–824.

POS.16 Development and validation of enzyme-linked immunosorbent assays for the determination of aldosterone and renin concentrations in small sample volumes - a paediatric-tailored approach Schaefer, J.1; Burckhardt, B. B.1; Tins, J.1; Bartel, A.; Läer, S.1 1 Institute of Clinical Pharmacy and Pharmacotherapy, Heinrich-Heine-University Duesseldorf, Universitaetsstr. 1, 40225 Duesseldorf, Germany

Background: The LENA (Labeling of Enalapril from Neonates up to Adolescents) project investigates a novel age-appropriate enalapril formulation to improve the pharmacotherapy of heart failure in young children. To gain a better understanding of the underlying disease and to evaluate the impact of the angiotensin-converting enzyme inhibitor enalapril in the diseased population, pharmacokinetic and humoral parameters require systematic investigation. As blood volume in children is limited, validated bioanalytical assays for small sample volumes are required. Objective: Development and validation according to current international EMA (European Medicines Agency) and FDA (Food and Drug Administration) guidelines of paediatric-tailored bioanalytical

assays for the determination of aldosterone and renin concentrations in small sample volumes of serum and plasma [1,2]. Methods: Based on commercial available enzyme-linked immunosorbent assay (ELISA) kits for the determination of aldosterone or renin concentrations, assays for the determination in small sample volumes were improved. For aldosterone, a competitive assay was used, while the renin assay used was based on a sandwich immunoassay [3,4]. The validation plan covers the following parameters: calibration curve, accuracy, precision, analytical run procedures, stability, matrix effect, dilutional linearity, parallelism, reproducibility and incurred sample reanalysis, processed sample stability, determination of hemolyzed blood samples. Results: Two enzyme-linked immunosorbent assays for the determination of aldosterone or renin concentrations in small sample volumes (40 µl) were developed. Regarding both assays, the parameters calibration curve, accuracy, precision, analytical run procedures, reproducibility and incurred sample reanalysis, processed sample stability and determination of hemolyzed blood samples were successfully validated. All validation runs comply with current bioanalytical guidelines of EMA and FDA [1,2]. For aldosterone, the assay ranges from 31.3 pg/ml to 1000.0°pg/ml, while the renin assay ranges from 4.0 pg/ml to 128.0 pg/ml. On the basis of six independent assay runs, within-run as well as between-run accuracy and precision were demonstrated. For both assays, five concentration levels were used for the evaluation of between-run accuracy and precision. Regarding accuracy, the relative error of the different concentration levels ranged from -3.8 % to -0.8 % (aldosterone) and from -3.1 % to +3.0°% (renin). Precision, reported as coefficient of variation, ranged from 4.6 % to 8.5 % (aldosterone) and from 4.7°% to 10.7 % (renin). Regarding both assays, hemolyzed samples showed no effect on accurate sample determination within the calibration range of the assays. Conclusion: The developed enzyme-linked immunosorbent assays allow a precise and accurate determination according to current bioanalytical guidelines of EMA and FDA. This reliable determination in very small sample volumes provides the basis for sophisticated investigations in children. Acknowledgements: The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n°602295 (LENA).

References: 1. EMA. Guideline on Bioanalytical Method Validation. EMA, Committee for Medicinal Products for Human Use, London, UK 2011. 2. US FDA. Guidance for Industry: Bioanalytical Method Validation. (Draft Guidance) US Department of Health and Human Services, US FDA, Center for Drug Evaluation and Research, Rockville, MD, USA 2013. 3. Aldosterone ELISA Kit (Ref. EIA-5298), DRG Instruments GmbH, Germany 4. Renin ELISA Kit (Ref. EIA-5125), DRG Instruments GmbH, Germany

POS.17

Serum and cerebrospinal fluid concentrations of vancomycin in neurosurgical critically ill patients with central nervous system infections Blassmann, U.1; Roehr, A. C.2; Frey, O. R.2; Vetter-Kerkhoff, C.1; Thon, N.3; Briegel, J.4; Huge, V.4

1 Pharmacy, University Hospital of Munich, Marchioninistr. 15, 81377 Munich, Germany 2 Pharmacy, Hospital of Heidenheim, Schlosshaustr. 100, 89522 Heidenheim, Germany 3 Neurosurgery, University Hospital of Munich, Marchioninistr. 15, 81377 Munich, Germany 4 Anesthesiology, University Hospital of Munich, Marchioninistr. 15, 81377 Munich, Germany

Background: Combination therapy with meropenem and vancomycin is recommended for hospital-acquired central nervous system (CNS) infections [1]. The limited penetration of vancomycin into the cerebrospinal fluid (CSF) is well known. However, only limited data exist on the disposition of vancomycin in critically ill patients with CNS infections and non-inflamed meninges [2]. The aim of this study was to describe concentrations of vancomycin in serum and CSF in critically ill patients with external CSF drainage and proven or suspected CNS infections. Material/methods: This was an observational pharmacokinetic (PK) study in neurosurgical critically ill patients with proven or suspected CNS infection receiving vancomycin. Serial blood and CSF samples are taken and analysed by using an in vitro chemiluminescent micro particle immunoassay (ARCHITECT iVancomycin assay, Abbott; measuring range: 0.24 mg/l – 100.00 mg/l). Pharmacokinetic parameters are

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analysed by a one compartment model. The primary pharmacokinetic/pharmacodynamic targets are the area under the concentration curve (AUC) divided by the minimum inhibitory concentration (MIC) value of 400 in serum and concentrations above the MIC of suspected pathogens throughout the entire dosing interval in CSF (100 % T>MIC). According to EUCAST 67 % of staphylococci display an MIC of 1 mg/l, 13 % display an MIC of ≤ 0.5 mg/l. Variables are described with median values [interquartile range]. Results: Ten patients (mean age 54, mean weight 73) were enrolled. A total of 110 serum samples and 106 CSF samples were analysed. The median of peak and trough concentration in serum was 24.97 [19.96–29.86] mg/l and 8.66 [6.60-10.99] mg/l, respectively. The median AUC24 in serum was 394.77 [337.93 – 450.89] mg/l. The median of corresponding peak and trough concentration in CSF was 1.60 [0.24-2.11] mg/l and 1.12 [0.24-2.50] mg/l, respectively. In CSF, 31 % of the samples remained below the detection limit. Assuming an MIC of 0.5 mg/l all patients achieved an AUC/MIC value >400. Assuming an MIC of 1 mg/l 50 % of all sampling days achieved AUC/MIC > 400. In CSF, 56 % of all concentrations reached 1 mg/l and 32 % of all concentrations reached 2 mg/l. Conclusions: Vancomycin demonstrated adequate CSF concentrations for high susceptible staphylococci/methicillinresistant staphylococcus aureus. With the high inter-individual PK variability observed, therapeutic drug monitoring in CSF might be an option to optimize vancomycin dosing in critically ill patients with CNS infections. Higher serum level targets or switching to alternative antibiotics should be considered, if CSF concentrations are lacking. Acknowledgements: Stiftung Patient und Klinische Pharmazie, Lesmüllerstiftung

References: 1. Tunkel, A.R. et al.: Clin. Infect. dis. 2004; 39(9): 1267-1284. 2. Nau, R. et al.: Clin. Microb. Rev. 2010; 23(4): 858-83.

POS.18

Development of Novel Inhibitors Targeting Elastase (LasB) from Pseudomonas aeruginosa Kany, A. M.1; Sikandar, A.2; De Mello Martins, A. G. G.1; Eberhard, J.1; Hamed, M.1; Haupenthal, J.1; Köhnke, J.2; Hartmann, R. W. 1,3 1 Helmholtz Institute for Pharmaceutical Research Saarland, Department of Drug Design and Optimization, Campus E8.1, 66123 Saarbrücken, Germany 2 Helmholtz Institute for Pharmaceutical Research Saarland, Department of Structural Biology of Biosynthetic Enzymes, Campus E8.1, 66123 Saarbrücken, Germany 3 Saarland University, Pharmaceutical and Medicinal Chemistry, Campus E8.1, 66123 Saarbrücken, Germany

The increasing appearance of bacteria that are resistant to commonly used antibiotics poses a threat to public health and makes the development of novel antibiotics an urgent necessity [1]. Targeting bacterial virulence factors is a new approach in the field of antibacterial drug development. Virulence factors are known to play a pivotal role during the infection process of pathogenic bacteria [2]. The harmful zinc metalloprotease elastase (LasB) is such a virulence factor, secreted by Pseudomonas aeruginosa. This pathogen is responsible for severe lung infections, especially in immunocompromised and cystic fibrosis patients[3]. LasB enables the bacteria to colonize a niche in the host, to evade the host immune response and to obtain nutrition from the infected cells [4]. Furthermore, it is involved in the formation of P. aeruginosa biofilm by proteolytic activation of nucleoside diphosphate kinase (NDK) [5]. Consequently, this protease represents a prime target for novel inhibitors which attenuate the aforementioned virulence mechanisms. In order to identify novel active and selective LasB inhibitors, we performed a fluorescence-based functional screening of a focused protease inhibitor library as well as of a number of fragments. Inhibition was crosschecked by additional LC-MS-based analysis of the fluorescence assay to detect compounds whose apparent inhibition was only due to quenching of fluorescence. We discovered a sulfonic acid derivative and a series of mercaptoacetamides as inhibitors of LasB. Treatment of PA14 wild type cultures with the sulfonic acid led to a reduction of biofilm volume. Using cell based and in vivo assays, the biological effects of LasB inhibition by our hits will be further investigated. Cocrystallization of these compounds with LasB is currently ongoing and will serve as a basis for chemical optimization regarding potency and selectivity against human matrix-metalloproteases (MMPs).

References: 1. World Health Organization, Antimicrobial Resistance: Global Report on Surveillance, 2014, http://www.who.int/drugresistance/documents/surveillancereport/en/ 2. Rasko, D.A.; Sperandio, V.: Nat. Rev. Drug Discovery. 2010, 9: 117-128. 3. Aloush, V. et al.: Antimicrob. Agents Chemother. 2006, 50: 43-38. 4. Wretlind, B.; Pavlovskis, O.R.: Rev. Infect. Dis. 1983, 5(5): 998-1004. 5. Kamath, S. et al.: Mol. Microbiol. 1998, 30(5): 933-941.

POS.19

MicroScale thermophoretic investigation of deferiprone interaction with selected biometals Asmari, M.1; Kleusch, C.2; Michalcova, L.; El Deeb, S.1

1 Institute of Medicinal and Pharmaceutical Chemistry, TU Braunschweig, Beethovenstraße 55, 38106 Braunschweig, Germany 2 NanoTemper Technologies GmbH, Flössergasse 4, 81369 Munich, Germany 3 Department of Biochemistry, Faculty of Science, Masaryk University, Kamenice 5, berno 62500, Czech Republic

MicroScale Thermophoresis (MST) method has been developed and applied for investigating the interaction of deferiprone (iron-chelator) with Cu2+, Zn2+, Ni2+, Mn2+, Co2+, Mg2+, Ca2+ and Fe3+.The experiments were performed on Monolith NT. LabelFree MicroScale Thermophoresis system. Pretest scanning indicated good fluorescence intensity of deferiprone proper for label free experiments. Different concentrations of the intended metals in the range of 0.048 - 500.0 µM were titrated against 100 µM fixed concentration of deferiprone dissolved in 0.1 M Tris buffer at pH 7.4. MST measurements were performed in standard capillaries at 50% Excitation-power and 20% MST-power. The results indicated significant interactions of deferiprone with Cu2+, Zn2+, Ni2+, Co2+ and Fe3+. The data fitted to Hill equation curves with Hill coefficients of 1.5, 3.2, 1.5, 1.6, and 1.8 for Cu2+, Zn2+, Ni2+, Co2+ and Fe3+, respectively, thus indicating more than 1:1 stoichiometry. EC50 values for the binding of Cu2+, Zn2+, Ni2+, Co2+ and Fe3+ to deferiprone where calculated to be 38.1, 39.5, 101.1, 51.1 and 20.6 µM, respectively. No binding of deferiprone with Mg2+, Ca2+ and Mn2+ were observed. The technique shows a fast and simple approach to study the binding of deferiprone to different biometals.

POS.20 MS Uptake Assays for the Serotonin Transporter (rSERT) Währa, M.1; Ackermann, T.1; Höfner, G.1; Wanner, K. T.1

1 Department Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, D-81377 Munich, Germany

Uptake experiments are the fundamental assay type for characterization of substrate transport and evaluation of inhibitor profiles at transport proteins. Most of the assays commonly used for this purpose are based on substrates labeled by radioisotopes or fluorophores. The last years showed a steady development of uptake assays based on unlabeled substrates quantified by LC-MS/MS. This concept e.g. named MS Uptake Assays has been applied to different transporters like the GABA transporters (hGAT1, hGAT2, hGAT3 and BGT1) [1] as well as the multidrug and toxin extrusion transporters 1 and 2 (MATE 1 and MATE2) and the organic cation transporter 2 (OCT2) [2]. In the present study, this strategy should be used to set up MS based transport assays for the Serotonin Transporter (SERT) employing 1-methyl-4-phenylpyridinium (MPP+) as substrate. A sensitive LC-ESI-MS/MS quantification method for MPP+ (m/z 170 → 128) using five times deuterated MPP+ (d5-MPP+, m/z 175 → 133) as internal standard was developed and validated according to the FDA guideline for bioanalytical methods. Applying this quantification method, MS Uptake Assays for rSERT (stably expressed in HEK293 TREX cells) were established following the procedure shown in the scheme below.

ANALYTICS


Using this procedure, the KM-value of MPP+ for rSERT could be determined in saturation experiments. The obtained result is in good accordance with those of radio uptake assays. Finally, inhibitory potencies for a series of reference compounds were evaluated with the established MS Uptake Assays. The results derived therefrom correlate well with the affinities found in binding assays for hSERT. [3] References: 1. Schmitt, S., Höfner, G., Wanner K.T.: Anal. Chem. 2014, 86(15): 7575-7583. 2. Vath M et al.: J. Chromatogr. B. 2014, 967: 211-218. 3. Grimm, S.H., Höfner, G., Wanner, K.T.: Chem. Med. Chem. 2015, 10(6):1027-1039.

POS.21

Multimeric phosphonic acids as anchor molecules to generate stable antifouling surfaces on metals Klitsche, F.1; Maison, W.1 1 University of Hamburg, Institute of Pharmacy, Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, 20146 Hamburg, Germany

The stable modification of surfaces becomes increasingly important especially in the fields of clinical hygiene and implant medicine [1, 2]. Bacterial infections during hospitalization and implantation cause high costs for the health care systems and have often serious consequences for the patients. In this context, strategies to prevent bacterial attachment to surfaces the so-called antifouling are attractive to decrease microbial loading. There are two main approaches used to generate antifouling surfaces: on the one hand an active approach employing surface-bound antibiotics, cations or proteases and on the other hand a repelling strategy with biopassive coatings based on polyethylene glycol (PEG), polyglycerols or zwitterions which inhibit protein adsorption to the surface [3-7]. These effector moieties need to be strongly bound to the surface via appropriate anchor molecules [8]. Herein we report the binding properties of different adamantane-based phosphonic acids. These anchors can be immobilized on clinically relevant metal surfaces such as titanium and zirconium. We report a proof-of-concept study on different metal nanoparticles as model systems for larger metal surfaces. The obtained coated nanoparticles are characterized via different analytical methods (e.g. solid-state 31P MAS-NMR, EDX, XRD, TGA and FT-IR-spectroscopy). Moreover the stability of the coated particles towards different pH-values and time periods is examined.

Acknowledgements: We kindly like to thank A. Hensel from the Institute of Physical Chemistry at the University of Hamburg for the analysis of the samples via TEM, EDX and XRD, Dr. Young Joo Lee from the Institute of Inorganic Chemistry at the University of Hamburg for conducting the solid-state NMR investigations and Dr. Silvia Gross from the Dipartimento di Scienze Chimiche at the Università degli Studi di Padova, Italy for the synthesis of the ZnS and ZnO nanoparticles.

References: 1. Darouiche R. O., N.: Engl. J. Med. 2004, 350(14): 1422-1429. 2. Costerton J. W., Stewart P. S., Greenberg E. P.: Science 1999, 284(5418): 1318-1322. 3. Banerjee I., Pangule R. C., Kane R. S.: Adv. Mater. 2011, 23(6): 690-718. 4. Lejars M., Margaillan A., Bressy C.: Chem. Rev. 2012, 112(8): 4347-4390. 5. Sisson A. L., Haag R.: Soft Matter 2010, 6(20): 4968-4975. 6. Khalil, F. et al.: Colloids Surf. B 2014, 117(2014): 185-192. 7. Shao Q.; Jiang S.: Adv. Mater. 2015, 27(1): 15-26. 8. Pujari S. P. et al.: Angew. Chem. 2014, 126(25): 6438-6474.

POS.22

Total phenolic and tannins determination: A modification of Ph. Eur. 2.8.14 suitable for high-throughput screenings Wiesneth, S.1; Heilmann, J.1; Jürgenliemk, G.1 1 Institute of Pharmaceutical Biology, University of Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany

To get an overview of the natural compounds’ composition of an herbal drug or extract thereof, very often convention methods are used to estimate the concentration of major groups of secondary plant ingredients. A principle mostly being used for phenolic compounds is the Folin-Ciocalteu’s reagent method described in the European Pharmacopeia method 2.8.14 (Ph. Eur.) [1] as not only the total phenolic content can be determined by this method, but also the tannin concentration, irrespective of the type of tannins (hydrolyzable or condensed tannins) [1]. Unfortunately, it is hardly possible to handle a large number of samples in an appropriate time using this complex method. To work more efficiently, the aim of the present study was to simplify this procedure. By changing the wavelength [2], the amounts of reagents [3], minituarizing the setting to microtiter scale and changing the time interval in between pipetting the assay and the measurement, the Ph. Eur. method was optimized for a larger number of experiments. Calibration curves and time kinetics with different phenolic compounds (catechol, gallic acid, (+)-catechin, tannic acid, salicylic acid, ferulic acid) were determined in addition to the molar extinction coefficient by extrapolation of each calibration curve and thus, to examine possible correlations between the reaction’s stoichiometry and the absorption. Also a complete data set of ascorbic acid, a non phenolic compound, was generated in order to check its response to this assay. The method was validated concerning its repeatability, robustness, linearity and reproducibility. Using this method, at least 120 samples can be handled per day by one person for the estimation of the total phenolic as well as tannin content. Acknowledgments: Many thanks are due to M. Jünger for performing the experiments concerning this project (Institute of Pharmaceutical Biology, University of Regensburg).

References: 1. 2.8.14: Gerbstoffe in pflanzlichen Drogen. Europäisches Arzneibuch 8. Ausgabe (Deutscher Apotheker Verlag) 2015; 383. 2. Glasl, H.: DAZ 1983, 123, 1979–1987. 3. Li, H. et al.: Food Chemistry 2007, 102, 771–776.

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POSTERS


POS.23

The molybdoenzyme mARC detoxifies trimethylamine N-oxide, a risk factor for cardiovascular disease Schneider, J.1; Girreser, U.1; Havemeyer, A.1; Tyl-Bielicka, A.2; Pysniak, K.2; Ramotowska, E.2; Mikula, M.2; Clement, B.1

1 Christian-Albrechts-University Kiel, Department of Pharmaceutical and Medicinal Chemistry, Gutenbergstraße 76, 24118 Kiel, Germany 2 Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Department of Genetics, Wilhelma Konrada Roentgena 5, 02-781 Warsaw, Poland

Cardiovascular disease (CVD) is the leading cause of morbidity worldwide. Therefore, it is of the utmost importance to learn more about its genesis. Within this context, trimethylamine N-oxide (TMAO), the physiological metabolite of dietary phosphatidylcholine, is in the center of interest. As the metabolic profile of TMAO in plasma correlates with the risk for CVD due to a pro-atherosclerotic mechanism [1], any process that leads to decreased TMAO plasma levels could possibly reduce the risk for CVD. TMAO is an oxidation product of hepatic flavin monooxygenase (FMO), thus its reduction to the precursor trimethylamine (TMA) is an obvious option to diminish its plasma concentration. So far, no enzyme catalyzing this reaction was identified. The recently in our lab discovered mitochondrial amidoxime reducing component mARC, the fourth molybdenum containing enzyme in mammals [2], could potentially catalyze this reduction. Along with the heme-containing cytochrome b5 (CYB5) and its flavin-containing cytochrome b5 reductase (CYB5R), mARC forms an N-reductive enzyme system which is able to reduce a variety of N-hydroxylated compounds. The human genome encodes for two mARC proteins, hmARC1 and hmARC2. So far, the physiological role of mARC remains mainly unknown [2-3]. Biotransformation assays including the reconstituted recombinant N-reductive enzyme system and TMAO as substrate can easily reveal if a reduction to the metabolite TMA is of significance. The volatile character as well as the lack of a chromophore of TMA poses particular challenges for this task. Hence, we developed an LC/MS/MS-method involving a dedicated and reliable sample preparation comprising the derivatization of TMA to a non-volatile compound by quaternization of the amine. With this newly developed analytical tool we investigated the in vitro formation of TMA through the reconstituted N-reductive enzyme system. We found that hmARC1 but not hmARC2 reduces TMAO. Moreover, we show that murine liver homogenates of wild type mice and mARC2(-/-) knock-out mice reduce TMAO without significant difference in specific activity. These data suggest that only one mARC isoform participates in the detoxification of TMAO. Our results prove that mARC reduces TMAO, thus represents the counterpart to FMO and plays a role in the prevention of CVD. Furthermore, these findings also propose a physiological function of the molybdoenzyme mARC. Acknowledgements: We thank A. Meier1 for preparing the murine liver homogenates.

References: 1. Wang Z. et al., Nature 2011, 472(7341): 57-63. 2. Havemeyer A. et al., J. Biol. Chem. 2006, 281(46): 34796-34802. 3. Gruenewald S. et al., J. Med. Chem. 2008, 51(24): 8173-8177.

POS.24/SL.37 How polysaccharide superstructure impacts hydrogel properties: A Raman optical activity study Lüdeke, S.1; Rüther, A.1; Forget, A.2; Roy, A.3; Carballo, C.3; Dukor, R. K.3; Nafie, L. A.3; Johannessen, C.4; Shastri, V. P.5 1 Inst. of Pharmaceutical Sciences, University of Freiburg, Albertstr. 25,79104 Freiburg, Germany 2 Future Industries Insitute, University of South Australia, MM Building, 5095 Mawson Lakes, Australia 3 BioTools, Inc., 17546 Beeline Hwy, Jupiter, FL, USA 4 Department of Chemistry, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium 5 Institute for Macromolecular Chemistry, University of Freiburg, Stefan-Meier-Str. 31, 79104 Freiburg, Germany


INFLAMMATION


3.2 Inflammation

POS.25/SL.16 Molecular mechanisms of lipoxin and resolvin biosynthesis Lehmann, C.1; Cumbana, R.2; Ebert, R.2; Toewe, A.3; Angioni, C.3; Ferreirós, N.3; Geisslinger, G.1,3; Parnham, M. J.1; Steinhilber, D.2; Kahnt, A. S.2 1 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and Pharmacology, Theodor Stern Kai 7, 60596 Frankfurt/Main, Germany 2 Goethe-University, Institute of Pharmaceutical Chemistry, ZAFES, Max-von-Laue-Str. 9, D-60438 Frankfurt/Main, Germany 3 Goethe University, Institute of Clinical Pharmacology, Pharmazentrum Frankfurt, ZAFES, Theodor Stern Kai 7, D-60590 Frankfurt/Main, Germany


POS.26

First insights into the action of the carbazole derivative C81 and the role of its primary target BMP2K in the activated vascular endothelium Bischoff, I.1; Dai, B.2; Strödke, B.3; Knapp, S.4,5; Bracher, F.3; Fürst, R.1 1 Institute of Pharmaceutical Biology, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany 2 Pharmaceutical Biology, Center for Drug Research, University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany 3 Department of Pharmacy - Center for Drug Research, University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany 4 Institute for Pharmaceutical Chemistry, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany 5 Nuffield Department of Clinical Medicine, University of Oxford, Old Road Campus, Headington, Oxford OX3 7BN, United Kingdom

Chronic inflammatory diseases, such as psoriasis or rheumatoid arthritis, are characterized by constant leukocyte infiltration and ongoing angiogenesis in the inflamed tissue. As current anti-inflammatory pharmacotherapy is not always satisfying, there is a great need for the discovery of new drug leads and targets. The synthetic carbazole derivative C81 acts as a kinase inhibitor. Results of a thermal shift assay revealed that C81 shows by far the highest binding affinity to the BMP-2-inducible kinase (BMP2K/BIKE) and the adaptor-associated kinase 1 (AAK1). Both kinases belong to the Numb-associated kinase (NAK) family, which has been linked to various biological functions, such as osteoblast differentiation or receptor-mediated endocytosis. Since the vascular endothelium crucially regulates inflammatory processes, we hypothesized that these kinases might play a pathophysiological role in the inflammation-activated endothelium. The functional role of both kinases has not been characterized in the endothelium so far. Therefore, we aimed to analyze the pharmacological potential of C81 and, as a starting point, to investigate the role of BMP2K in angiogenic and inflammatory processes in the vascular endothelium. Initial experiments show that only high concentrations of C81 affected the viability of human umbilical vein endothelial cells (HUVECs) after 24 hours of treatment (IC50: 171 μM). Longer incubation periods (72 h) reduced the proliferation of a human microvascular endothelial cell line (HMEC-1) with an IC50 of 7 μM. C81 treatment (10 μM) resulted in a reduced migratory capacity of HMEC-1. A tube formation assay on Matrigel® demonstrated that C81 significantly impaired the formation of capillary-like structures in a concentration-dependent manner. In addition, C81 (3 μM) reduced the formation of VEGF-induced sprouts in HUVEC spheroids. Interestingly, the analysis (Western blot) of signaling molecules in HUVECs that play a crucial role in cell proliferation (e.g. ERK, Akt) revealed that these pathways are not influenced, neither by C81 treatment nor by knock-down of BMP2K (RNAi). In regard to inflammatory processes, C81 treatment and BMP2K silencing of HUVECs decreased the adhesion of THP-1 cells, a monocytic cell line, onto the activated endothelial cells. As the interaction of leukocytes is mainly mediated by cell adhesion molecules (CAMs), the effect of C81 or BMP2K silencing on their expression was analyzed (flow cytometry, qPCR). While the expression of CAMs was strongly decreased after C81 treatment, the knock-down of BMP2K did not markedly affect their expression. Physiologically, leukocytes are recruited to the site of inflammation by chemotactic cues. The expression of CX3CL1, a

chemoattractant for leukocytes produced by endothelial cells, was concentration-dependently down-regulated by C81 (qPCR). Furthermore, neither C81 treatment nor BMP2K silencing led to the reduction of TNFα-induced IκBα degradation (Western blot) or p65 translocation into the nucleus (microscopy). Interestingly, silencing of BMP2K resulted in a marked decrease of TNFα-induced COX-2 expression (Western blot). Our study provides first insights into the anti-inflammatory and anti-angiogenic potential of the carbazole derivative C81 in vitro. Since the inhibition of BMP2K seems to be responsible only for some actions of C81, we will investigate the role of AAK1 in these processes. The precise role of BMP2K/AAK1 in angiogenic and inflammatory endothelial processes as well as the involved pathways during BMP2K/AAK1 silencing and C81 treatment will be further elucidated.

POS.27

Vioprolide A interferes with pro-inflammatory processes in human endothelial cells Luong, B.1; Bischoff, I.1; Müller, R.2; Fürst, R.1

1 Institute of Pharmaceutical Biology, Biocenter, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany 2 Helmholtz Institute for Pharmaceutical Research Saarland, Department of Microbial Natural Products and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany

Myxobacteria have been shown to be a rich source of potentially useful, bioactive secondary metabolites [1,2]. A myxobacterial library of 259 purified compounds was screened for their effects on the TNFα-induced ICAM-1 expression and on the serum-induced migration and proliferation. One of the compounds identified was vioprolide A, a cyclic peptide obtained from the myxobacterium Cystobacter violaceus [3]. Aim of this study was to characterize the effects of vioprolide A on the tumor necrosis factor α (TNFα)-activated endothelium and to gain first insights into the underlying mode of action. First, we investigated the impact of vioprolide A on the viability of primary human umbilical vein endothelial cells (HUVECs). Concentrations up to 10 nM did not significantly influence the metabolic activity (CTB assay), the apoptosis rate (sub-diploid DNA content) or the LDH release. The combined treatment with 10 nM Vio A and 10 ng/ml TNFα slightly decreased the metabolic activity by about 11 % without influencing the apoptosis rate. Proinflammatory cytokines such as TNFα trigger the recruitment of leukocytes by an increased surface expression of leukocyte and endothelial cell adhesion molecules [4]. Vioprolide A (10 nM) was able to strongly reduce the TNFα-induced leukocyte adhesion (THP-1, Jurkat, PBMC). Flow cytometry analysis revealed that 30 minutes preincubation with 10 nM vioprolide A diminished the TNFα-induced surface expression of the endothelial cell adhesion molecules ICAM-1, VCAM-1 and E-selectin by more than 50 %. Quantitative real-time PCR showed that vioprolide A also strongly downregulated the TNFα-induced mRNA levels of all three adhesion molecules by about 80-90 %. The TNFα-induced expression of these cell adhesion molecules is mainly regulated by the transcription factor NFκB [4]. Therefore, we analysed the NFκB promotor activity using a dual luciferase reporter gene assay. TNFα stimulated the NFκB promotor activity and pretreatment with 10 nM vioprolide A led to a fulminant decrease of this induction by about 90 %. To get a deeper insight into the TNFα-related pathways that are influenced by vioprolide A, we performed a transcriptome analysis using the MACE (massive analysis of cDNA ends) and RNA-sequencing technique. We found 395 and 557 genes that were upregulated by TNFα in HUVECs after 6 and 16 hours, respectively. From these genes, 133 (6 hours) and 239 genes (16 hours) were downregulated by preincubation with vioprolide A for 30 minutes. Within these genes, we identified further NFκB targets such as the chemokines CX3CL1 and monocyte chemoattractant protein 1 (MCP-1) or genes related to gene expression such as CBP/p300 activating peptide and interferon regulatory factor 1 (IRF1). Taken together, vioprolide A inhibits the TNFα-induced leukocyte adhesion and surface expression of ICAM-1, VCAM-1 and E-selectin probably via deactivation of the NFκB signalling pathway. Vioprolide A may interfere with NFκB-regulating molecules, such as CBP/p300 or IRF-1, to exert these effects. The in vivo relevance of these findings as well as the underlying mechanisms are currently studied.

POSTERS


Acknowledgments: This work was supported by the German Research Foundation (DFG, FOR 1406, FU 691/9-2).

References: 1. Reichenbach H.: J. Ind. Microbiol. Biotechnol. 2001, 27(3): 149-156. 2. Weissmann K.J., Müller R.: Nat. Prod. Rep. 2010, 27(9): 1276-1295. 3. Schummer D. et al.: Liebigs Ann./Recl.1996, 6:971-978. 4. Newton K., Dixit V.M.: Cold Spring Harb. Perspect. Biol. 2012, 4(3).

POS.28

Anti-inflammatory and cytoprotective effects of Hypericum extract STW 3-VI Schwendler, A.1; Abdel-Aziz, H.2; Kelber, O.3; Hüther, J.1; Bonaterra, G. A.1; Cordes, A.1; Kinscherf, R.1 1 Dept. of Medical Cell Biology, Philipps-University Marburg, Marburg, Robert-Koch-Str. 8, 35032 Marburg, Germany 2 Medicial Affairs, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany 3 Innovation & Development, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany

Glutamate toxicity and inflammation play an integral role in a variety of disorders as depression. Additionally, innumerable evidence indicates that adaptation to chronic stress involves response from both, the neuroendocrine and immune systems. Depression has also been linked to an inflammatory response since proinflammatory cytokines like interleukin 6 (IL-6) and tumor necrosis factor (TNF) are increased in depressed patients. In this context, the combined antioxidant and anti-inflammatory properties [1] of St. John's wort (Hypericum perforatum L.) extract are suggested to contribute to the antidepressant effects [2] by normalization of an overactive hypothalamic-pituitary-adrenal axis [3]. Thus, the aim of our investigations was to determine the effects of STW 3-VI on protection of differentiated mouse hippocampal HT22 cells from the cytotoxic effects of glutamate or NMDA and the possible anti-inflammatory properties on LPS-activated macrophages (MΦ). Differentiated HT22 cells were pre-treated with STW 3-VI to investigate the protective effects against glutamate or NMDA cytotoxicity. The anti-inflammatory properties of STW 3-VI were evaluated by quantification of the TNF release on LPS activated PMA-differentiated THP 1 MΦ using ELISA assay and the mRNA expression of TNF and IL-6 by qRT-PCR. Glutamate or NMDA (0.1mM) induced 30% cytotoxicity in HT22 cells. Pre-incubation (24h) with 10μg/ml of STW 3-VI improved the viability by 30% compared to the control. Pre-treatment (48h) of LPS activated MΦ with STW 3-VI (60, 80 or 100 μg/ml) induced a significant lowering (54%, 64% and 53%) of TNF release. QRT-PCR revealed that 48 h pre-treatment with 60 or 80 μg/ml STW 3-VI inhibited the mRNA expression of IL-6 and TNF respectively by LPS-activated MΦ. In conclusion, STW 3-VI protects hippocampal cells from glutamate or NMDA induced cytotoxicity and activates the anti-inflammatory defense by inhibition of the cytokine production by MΦ. These effects are in accordance with the therapeutic use of STW3-VI in depression. Acknowledgements: The study received financial support from Steigerwald Arzneimittelwerk GmbH

References: 1. Breyer, A et al.: Phytomedicine 2007, 14: 250-255 2. Denke, A et al.: Drug Res 2000, 50 (5): 415-419 3. Gastpar, M; Singer, A; Zeller, K.: Pharmacopsychiatry 2006, 39: 66-75 4. Grundmann, O; Kelber, O; Butterweck, V.: Planta Medica 2006, 72: 1366-1371

POS.29

Mode of action of a phytomedicine, STW 5, in an experimental model of Crohn´s disease Khayyal, M. T.1; Wadie, W.1; Abdallah, D.1; Schneider, M.2; Efferth, T.2; Kelber, O.3; Abdel-Aziz, H.4 1 Department of Pharmacology, Faculty of Pharmacy, Cairo University, Kasr-El-Aini Street, 11562 Cairo, Egypt 2 Department of Pharmaceutical Biology, Institute of Pharmacy and Biochemistry, Johannes Gutenberg University, Staudinger-Weg 5, 55128 Mainz, Germany 3 Medicial Affairs, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany 4 Innovation & Development, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany

STW 5 is a standardized multi-component herbal preparation consisting of hydro-alcoholic extracts of bitter candytuft, lemon balm, chamomile, caraway, peppermint, angelica, milk thistle, celandine, and licorice. It has been used effectively in functional dyspepsia [1] and irritable bowel syndrome [2] and showed efficacy in experimental dextran sodium sulfate induced colitis as a model for ulcerative colitis [3]. The present study was conducted to investigate its potential usefulness in 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) induced colitis as a model of Crohn’s disease. Colitis was induced by instilling TNBS in the colon of male Wistar rats under light ether anesthesia. In a prophylactic setting, STW 5 was given orally one week before induction of colitis and continued for three days onwards. After 24 h rats were sacrificed. In the curative setting, STW 5 was given orally 48 h after colitis induction daily for one week and the rats sacrificed 24 h later. Mucosal colonic damage was assessed macroscopically. Sulfasalazine was used as a reference drug. Colon homogenates and serum samples were used to assess levels of inflammatory and oxidative stress parameters. Immuno-histochemical staining of colon sections was used to assess calprotectin and IL-17A levels, reported to be implicated indicators for IBD in man. TNBS colitis led to severe ulcerative damage, inhibition of reduced glutathione and a rise in myeloperoxidase in colon homogenates. Relevant cytokines TNFα, IL-1β, ICAM-1 were elevated as well as LT-B4 and PGE2. Immuno-histochemical examination showed a rise in calprotectin and IL-17A. Pre-treatment with STW 5 prevented such effects in a similar manner to sulfasalazine. In the curative setting, STW 5 tended to normalize changes in reduced glutathione and myeloperoxidase and in ulcerative indices induced by TNBS. Present findings provide good supportive evidence for the potential usefulness of STW 5 in Crohn’s disease. References: 1. Schmulson, M. J.: Nat Clin Pract Gastroenterol Hepatol 2008; 5: 136-137 2. Madisch, A. et al.: Aliment Pharmacol Ther 2004; 19: 271‐279 3. Wadie, W. et al.: Int J Colorectal Dis 2012; 27: 1445‐1453

POS.30

Development of smart cell-free and cell-based assay systems for investigation of leukotriene C4 synthase (LTC4S) activity and evaluation of inhibitors Liening, S.1; Scriba, G. K.1; Rummler, S.2; Weinigel, C.2; Kleinschmidt, T. K.3; Haeggström, J. Z.3; Werz, O.1; Garscha, U.1 1 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Germany, 2 Institute of Transfusion Medicine, Jena University Hospital, Jena, Germany, 3 Division of Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Cysteinyl-leukotrienes (cys-LT) are powerful pro-inflammatory mediators that cause bronchoconstriction in anaphylaxis and asthma. They are formed by 5-lipoxygenase (5-LOX) from arachidonic acid (AA) yielding the unstable leukotriene A4 (LTA4) that is subsequently conjugated with glutathione (GSH) by LTC4 synthase (LTC4S), an integral membrane protein that belongs to the superfamily of membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG), to form LTC4. The tripeptide side chain of LTC4 is then cleaved in two successive steps to form LTD4 and LTE4. These cys-LTs are recognized by GPCRs. Cys-LT receptor antagonists as well as LTC4S inhibitors have been developed, but only the former have reached the market. One reason might be the high structural homology of LTC4S to related enzymes of the MAPEG family. One the other hand a convenient test systems to identify potential LTC4S inhibitors was missing due to instability of exogenously added LTA4 as substrate. The main purpose of our work was to establish cell-free and cell-based assay systems based on in situ-generated LTA4 that allow studying LTC4S activity and investigating LTC4S inhibitors. Co-incubations of microsomes from HEK293 cells stably expressing LTC4S together with isolated 5-LOX efficiently converted exogenous AA to LTC4 (~1.3 µg/200 µg protein). Stimulation of HEK293 cells co-expressing 5-LOX and LTC4S with Ca-ionophore A23187 and 20 µM AA leads to a strong LTC4 formation (~ 250 ng/106 cells). MK-886 consistently inhibited LTC4 formation in the assay types (IC50 = 3.2 and 3.1 µM, respectively) and we successfully confirmed TK04a as potent LTC4S inhibitor in these assay systems (IC50 = 17 and 300 nM, respectively). Further, we

INFLAMMATION


demonstrated transcellular LTC4 biosynthesis between human neutrophils or 5-LOX-expressing HEK293 cells that produce LTA4 from AA and HEK293 cells expressing LTC4S that transform LTA4 to LTC4. Summarizing, we established cell-free and cell-based HEK 293 systems for evaluating LTC4S inhibitors. Our assay approaches are advantageous as the substrate LTA4 is generated in situ and are suitable for studying enzymatic functionality of LTC4S including site-directed mutations.

POS.31

Thermodynamic properties of leukotriene A4 hydrolase inhibitors Wittmann, S.1; Kalinowsky, L.1; Kramer, J. S.1; Blöcher, R.1; Knapp, S.1; Steinhilber, D.1; Pogoryelov, D.2; Proschak, E.1; Heering, J.3,* 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Street 9, 60438 Frankfurt, Germany 2 Institute of Biochemistry, Goethe University Frankfurt, Max-von-Laue Street 9, 60438 Frankfurt, Germany 3 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Med-icine and Pharmacology TMP, Theodor-Stern-Kai 7, D-60596 Frankfurt am Main, Germany * Author to whom all correspondence should be addressed. E-mail: [email protected]

The leukotriene A4 hydrolase (LTA4H) is a bifunctional enzyme, containing a peptidase and a hydrolase activity [1]. The hydrolase activity is responsible for the conversion of leukotriene A4 to pro-inflammatory leukotriene B4 Both activities having opposing functions regulating inflammatory processes [2,3]. The hydrolase activity is responsible for the conversion of leukotriene A4 to pro-inflammatory leukotriene B4, and hence, selective inhibitors of the hydrolase activity are of high pharmacological interest [4]. The size of the binding pocket complicates the development of potent inhibitors. We therefore worked out the thermodynamic properties of five known LTA4H inhibitors. The apo structure was solved by X-ray crystallography and utilizing an in silico method we determined the position of stabilized water molecules. From the occupancy of different regions within the binding pocket we predicted an individual profile for the selected inhibitors. A variety of biochemical and biophysical methods was then applied to evaluate the derived mapping of the binding pocket, which in the future could facilitate inhibitor development. References: 1. Haeggström, J. Z. et al, (1990) Leukotriene A4 hydrolase: an epoxide hydrolase with peptidase activity. Biochem. Biophys. Res. Commun. 173, 431–7. 2. Rao, N. L. et al, (2007) Anti-inflammatory activity of a potent, selective leukotriene A4 hydrolase inhibitor in comparison with the 5-lipoxygenase inhibitor zileuton. J. Pharmacol. Exp. Ther. 321, 1154–60. 3. Wells, J. M. et al, (2014) An aberrant leukotriene A4 hydrolase-proline-glycine-proline pathway in the pathogenesis of chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 190, 51–61. 4. Stsiapanava, A. et al, (2014) Binding of Pro-Gly-Pro at the active site of leukotriene A4 hydrolase/aminopeptidase and development of an epoxide hydrolase selective inhibitor. Proc. Natl. Acad. Sci. U. S. A. 111, 4227–32.

POS.32

5-Lipoxygenase-activating protein (FLAP) rescues activity of 5-lipoxygenase mutations that delay nuclear membrane association and disrupt product formation Garscha, U.1; Gerstmeier, J.1; Newcomer, M. E.2; Romp, E.1; Werz, O.1

1 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University, 07743, Jena, Germany 2 Department of Biological Sciences, Louisiana State University, Baton Rouge, Louisiana

Leukotrienes (LT) are pro-inflammatory lipid mediators that are formed from arachidonic acid (AA) via the 5-lipoxygenase (5-LOX) pathway and are pronounced in asthma, allergy and cardiovascular diseases. AA is converted by 5-LOX first to 5(S)-hydroperoxyeicosatetraenoic acid (5-HPETE) and subsequently to LTA4. In cellulo, 5-LOX receives its substrate from the membrane-embedded 5-LOX-activating protein (FLAP) for product formation, and inhibition of FLAP or genetic knock-down blocks LT formation. Beside the absolute necessity of FLAP for 5-LOX activity in the cell, the crystal structure of 5-LOX revealed an active site that is concealed by two residues, F177 and Y181 referred to as

“FY-cork” [1]. We examined the influence of these residues on 5-LOX activity, substrate access, membrane binding, and interaction with FLAP in intact HEK293 cells expressing 5-LOX in the absence and presence of FLAP [2]. Uncapping the 5-LOX active site by mutation of F177 and/or Y181 to less bulkier alanine (5-LOX-F177A, 5-LOX-Y181A, 5-LOX-F177/Y181A) resulted in delayed and diminished 5-LOX membrane association in A23187-stimulated cells. Additionally, for 5-LOX-F177A and 5-LOX-F177/Y181A, the formation of 5-LOX products was dramatically reduced relative to 5-LOX-wild-type (wt). Strikingly, co-expression of FLAP in A23187-activated HEK293 cells effectively restored formation of 5-H(p)ETE by these same 5-LOX mutants (≈ 60-70% 5-LOX-wt levels) but not of LTA4 hydrolysis products. Substitution of Y181 by less bulkier residues as phenylalanine or alanine, allows dioxygenation at carbon 5 and generated 5-H(p)ETE at levels comparable to 5-LOX-wt but mainly prevented LT formation. Again, co-expression of FLAP partially restored LTA4 hydrolysis product formation by 5-LOX-Y181A. Together, the data suggest that amino acids (F177 and Y181) obstructing access to the active site are essential for membrane association. Additionally, protein-lipid (5-LOX/membrane) as well as protein-protein (5-LOX/FLAP) interaction promote LT formation in the cell. Acknowledgments: Financial support was provided by Deutsche Forschungsgemeinschaft (DFG) within the SFB1127: Chemical Mediators in complex Biosystems.

References: 1. Gilbert, N. et al. Science 2011, 331: 217-219 2. Gerstmeier, J.; Garscha, U. et al. FASEB J. 2016, 30(5):1892-900

POS.33

Particulate matter (PM2.5) from biomass combustion alters the methylation profile of genes related to cancer Heßelbach, K.1; Kim, G.-J.2; Flemming, S.2; Dornhof, R.1; Häupl, T.3; Günther, S.2; Merfort, I.1; Humar, M.1 1 Department of Pharmaceutical Biology and Biotechnology, Albert-Ludwigs University of Freiburg, 79104 Freiburg, Germany 2 Pharmaceutical Bioinformatics, Albert-Ludwigs University of Freiburg, 79104 Freiburg, Germany 3 Department of Rheumatology and Clinical Immunology, Charité University Hospital Berlin, 10117 Berlin, Germany

Biomass combustion is increasingly used as a renewable, CO2 neutral alternative energy source. However, biomass combustion significantly contributes to indoor air pollution and emissions by biomass generated PM might be regionally comparable or even higher than traffic related emissions. Nowadays, it is generally known that anthropogenic PM in ambient air is a major health hazard [1] resulting in a variety of diseases, including chronic obstructive pulmonary disease (COPD), acute respiratory infections, fibrosis, and lung cancer [2, 3]. Here, particles less than 2.5 μm in diameter (PM2.5) are considered to be most harmful, as they penetrate deeply into the lungs and adversely affect bronchioles or alveoli. However, it is still not completely understood how particles emitted during biomass combustion affect human health. Recent reports indicate that the occurrence of these diseases is closely connected to epigenetic aberrations, such as alterations in CpG residue methylation, histone modifications and changes in micro(mi)RNA expression [4]. Here, we analyzed the effect of PM2.5 using human epithelial bronchial alveolar cells (BEAS-2B) as a model on the genome-wide methylation of CpG nucleotides by an Illumina Methylation450K BeadChip array and linked the results with the impact on the transcriptome by an Affymetrix Human Genome U133 Plus 2.0 Array. We filtered 155 genes which were either hyper- or hypomethylated and simultaneously transcriptionally differently regulated. From these genes, 66 were related to lung diseases, especially to lung cancer. Our results give first insights that epigenetic aberrations induced by chronic exposure to PM may be involved in the development of various lung diseases. Acknowledgments: The presented work is part of an interdisciplinary EU-funded research project (see http://www.biocombust.eu), supported in part through the Interreg IV Program “Oberrhein” (project C35 BIOCOMBUST).

References: 1. Lelieveld J. et al.: Nature. 2015, 525(7569): 367-71. 2. Anderson JO., Thundiyil JG., Stolbach A.: J Med Toxicol. 2012, 8(2): 166-75. 3. Raaschou-Nielsen O. et al.: Lancet Oncol. 2013, 14(9): 813-22. 4. Heerboth S. et al.: Genet Epigenet. 2014, 6: 9-19.

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POS.34

Eculizumab, the only complement inhibitor in the clinic is not always efficient: mechanistic evidence for incomplete inhibition under eculizumab and how the problem can be fixed Harder, M. J.1; Simmet, T.1; Schmidt, C. Q.1; 1 Ulm University, Institute of Pharmacology of Natural Products & Clinical Pharmacology, Ulm, Germany

Eculizumab inhibits the terminal and lytic pathway of complement by blocking the activation of the complement protein C5. The terminal pathway (TP) of the complement cascade generates the anaphylatoxin C5a and the plasma membrane penetrating membrane attack complex and, thus, holds the most inflammatory potential of the whole complement cascade. Treatment with eculizumab shows remarkable clinical benefits in the complement mediated diseases atypical haemolytic uraemic syndrome (aHUS) and paroxysmal nocturnal haemoglobinuria (PNH). However, several reports demonstrate that activation of C5 is not always completely suppressed in patients even in presence of excess amounts of eculizumab. This suggests that residual C5 activity may limit the drug’s therapeutic benefit under certain conditions. Therefore, we challenge the prevailing dogma that C5 inhibitory agents, like the therapeutic antibody eculizumab, completely inhibit the TP and set out to elucidate the underlying molecular mechanism responsible for the observed residual TP activity in presence of C5 inhibitors. We show in several experiments ex vivo that C5 inhibition by eculizumab and other C5 inhibitory agents is generally susceptible to incomplete suppression of the TP. By using biophysical and several cell based studies we demonstrate that it is the surface density of the deposited complement opsonin C3b that directs the level of this residual lytic complement activity. For example, we show in a clinical relevant model of autoimmune mediated haemolysis that residual lytic activity under C5 inhibition occurs on human erythrocytes after forceful complement activation via alloantibodies directed against blood group antigens. The level of such residual C5 activity directly correlated with the strength/titer of the complement activating alloantibodies. This indicates that the therapeutic benefit of eculizumab is especially impaired in clinical situations that are characterised by a particular forceful activation of complement which actually would require a particular efficient suppression of the complement system. We found two potential solutions how this clinical problem of residual C5 activity, e.g. after ischemia reperfusion injury or during antibody mediated transplant rejection, may be alleviated in the future. In ex vivo studies on patient material we demonstrate that inhibition of the TP by simultaneous employment of two different C5 inhibitors can completely block the lytic complement activity even after very forceful complement activation. Alternatively, a biotherapeutic fusion molecule derived from novel protein-engineering approaches of several natural complement inhibitors also proved to be extremely potent in suppressing inflammatory TP activities after powerful initiation of the complement cascade. In conclusion, with our study we advance the mechanistic understanding of complement C5 activation by demonstrating that high cell surface densities of C3b opsonins can override the blocking effects of different C5 inhibitors. We demonstrate that such inflammatory, residual TP activity under C5 inhibition can be circumvented by simultaneously employing two orthogonal C5 inhibitors or by using a novel, engineered fusion protein that efficiently blocks all three complement activation pathways. Thus, our study not only provides the rational explanation for the clinically observed phenomenon of residual TP activity under eculizumab therapy, which has significant implications for anti-C5 therapy in general, but also indicates potential future therapeutic avenues that allow efficient blockage of complement even after very forceful activation of this innate immune cascade.

POS.35

6‐Arylamino‐3,4‐dihydroisoquinolin‐1(2H)‐ones as new pharmacophores for linear hinge binders inducing the glycine-flip Praefke, B. A.1; Laufer, S. A.1 1 University of Tuebingen, Faculty of Science, Pharmaceutical and Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tuebingen, Germany

Various diseases can be linked to dysregulated protein kinase activity [1]. Achieving selectivity with ATP-competitive kinase inhibitors is difficult, due to highly conserved ATP-binding sites. We previously developed highly potent and selective carbonyl based linear hingebinding p38α MAPK inhibitors inducing a so called “glycine-flip”, a rotation of Gly110 in the hinge region, resulting in the formation of two hydrogen bonds from the carbonyl oxygen to the amide-NH of Gly110 and Met109 [2,3]. As only 46 of the known 518 protein kinases bear a glycine in the equivalent position, targeting the glycine-flip represents a way to increase kinome selectivity [4].

Derived from our dibenzosuberone, dibenzoxepinone and benzosuberone scaffolds we developed a new hinge binding motif by replacing the ketone by an amide group. We expect an increase in electron density of the amide oxygen to strengthen the bidentate hydrogen bonds to the flipped glycine and the adjacent amino acid. The arylamino moiety was decorated with various substituents in order to cover a broad range of electronic and lipophilic properties to explore the hydrophobic region I of kinases possessing a glycine in the hinge region. References: 1. G. Manning et al.: Science. 2002, 298(5600), 1912-1934. 2. S. C. Koeberle et al.: J. Med. Chem. 2012, 55(12), 5868-5877. 3. B. Baur et al., J. Med. Chem. 2013, 56(21), 8561-8578. 4. K. E. Martz et al., J. Med. Chem. 2012, 55(17), 7862-7874.

POS.36

Chemical Tuning of Anthranilamides Towards Selective PPARδ Agonism Heitel, P.1; Proschak, E.1; Schubert-Zsilavecz, M.1; Merk, D.1 1 Goethe University of Frankfurt, Max-von-Laue-Straße 9, 60438 Frankfurt, Germany

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor family that function as ligand-activated transcription factors [1]. PPAR activation by endogenous ligands - fatty acids and eicosanoids - leads to the expression of various genes involved in proliferation of liver peroxisomes, metabolic regulation of lipid and glucose homeostasis, as well as inflammation [2-5]. In mammals, three subtypes have been identified which differ in expression and physiological function. Whereas PPARα and PPARγ agonists have been extensively studied because of hypolipidemic and antidiabetic properties, the physiological role of PPARδ (also referred to as PPARβ) remained unknown for a long time. By now, it has been figured out that PPARδ is ubiquitously expressed and plays a pivotal role in fatty acid oxidation in key metabolic tissues such as skeletal

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muscle [6]. Besides, PPARδ activation exhibits anti-inflammatory effects and hence gained interest as therapeutic target. However, in contrast to PPARα and PPARγ, no PPARδ ligand has been approved as drug so far. Although first clinical trials with PPARδ agonist GW501516 demonstrated promising results such as decreased plasma triglyceride levels, elevated HDL levels and enhanced insulin sensitivity in obese patients [7-8], GW501516 promoted the growth of intestinal adenomas [9]. In initial studies, we have already shown that anthranilamides are promising candidates to overcome the need for selective PPARδ agonists [10]. In the process, compound 1 proved to be selective over PPARα and PPARγ, showing a low micromolar EC50 value on PPARδ. Starting from computational docking of 1 into the PPARδ ligand binding domain (LBD), we investigated the acidic head group, substitution of the aromatic moieties, as well as introduction of heteroaromatic systems to exploit the interaction between the ligand and the binding pocket. In this structure-activity relationship (SAR) study, we chemically optimize the potency of anthranilamide 1 in several cycles to come up with a selective, nanomolar PPARδ agonist, which is tested in a PPAR-Gal4 transactivation assay for each subtype. Further research including in vivo investigations will reveal whether this compound class is suited as novel strategy for treatment of metabolic syndrome. Acknowledgements: Financial support by the Else Kröner-Fresenius-Stiftung, Translational Research Innovation - Pharma (TRIP) is gratefully acknowledged.

References: 1. Gronemeyer, H.; Gustafsson, J.-Å.; Laudet, V.: Nat. Rev. Drug Discov. 2004, 3(11): 950–964. 2. Forman, B. M.; Chen, J.; Evans, R. M.: Proc. Natl. Acad. Sci. 1997, 94(9): 4312–4317. 3. Xu, H. E. et al.: Mol. Cell 1999, 3(3): 397–403. 4. Keller, H. et al.: Proc. Natl. Acad. Sci. U.S.A. 1993, 90(6): 2160–2164. 5. Kliewer, S. A. et al.: Proc. Natl. Acad. Sci. 1997, 94(9): 4318–4323. 6. Neels, J. G.; Grimaldi, P. A.: Physiol. Rev. 2014, 94(3): 795–858. 7. Sprecher, D. L. et al.: Arterioscler. Thromb. Vasc. Biol. 2007, 27(2): 359–365. 8. Risérus, U. et al.: Diabetes 2008, 57(2): 332–339. 9. Gupta, R. A. et al.: Nat. Med. 2004, 10(3): 245–247. 10. Merk, D. et al.: Bioorg. Med. Chem. 2015, 23(3): 499–514.

POS.37

Facing non-alcoholic steatohepatitis with multi-target agents Schmidt, J.1; Rotter, M.1; Weiser, T.1; Wittmann, S.1; Kaiser, A.1; Weizel, L.1; Proschak, E.1; Merk, D.1 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt; [email protected]

Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) arising from western diet and lifestyle evolve as serious health burden with alarming incidence.[1] NAFLD and NASH are characterized by accumulation of fat in liver subsequently causing inflammation and fibrosis and are strongly associated with the metabolic syndrome.[1] Although the high prevalence of NAFLD and NASH elicited intensive research for novel treatment options there is still no satisfying pharmacological therapy.[2] Several molecular targets have been identified as potentially suitable for NAFLD/NASH treatment. Promising clinical data has been reported for elafibranor[3], an agonist of the peroxisome proliferator-activated receptors (PPAR) α and δ as well as for obeticholic acid[4] which activates the farnesoid X receptor (FXR). Additionally, the inhibition of a number of enzymes including stearyl-CoA desaturase 1 (SCD1)[5] and soluble epoxide hydrolase (sEH)[6,7] proved effective in treating NASH in vivo. In light of the multifactorial nature of NASH, modulation of more than one target might provide a superior therapeutic effect. Especially, combination of FXR activation that revealed anti-steatotic and anti-fibrotic effects in clinical trials with inhibition of sEH generating anti-inflammatory effects promises synergistic activity. The nuclear receptor FXR acts as intracellular bile acid sensor and liver protector. Its activation has various beneficial metabolic effects and via induction of small heterodimer partner (SHP) as well as sterol regulatory element binding protein 1c (SREBP1c) reduces liver fat content.[8] sEH is an enzyme of the arachidonic acid cascade located in the CYP pathway and catalyzes the degradation of anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). Therefore, sEH inhibition hinders EET degradation and has anti-inflammatory properties.[7]

To exploit the concept of dual FXR/sEH modulation for NASH treatment we developed dual agents with partial FXR agonistic and sEH inhibitory potency. Initially, we merged known pharmacophores[9,10] for both targets to generate our lead compound that exhibited moderate FXR activation and sEH inhibition at 50 µM in vitro. By systematic exploration of the structure-activity relationship (SAR) of the compound class on both targets, we optimized the potency for partial FXR activation and sEH inhibition to low nanomolar values and finally used this knowledge to generate compounds with the desired dual activity and well-balanced nanomolar potency. The most promising compounds were intensively trialed in vitro for FXR target gene induction, selectivity, metabolic stability and toxicity. The compounds revealed a favorable profile and pilot in vivo data is encouraging. In summary, we report the first class of dual FXR agonists/sEH inhibitors and based on favorable in vitro and in vivo properties, further exploration of the concept and the compound class is warranted. References: 1. Rinella, M.: JAMA 2015, 313(22): 2263–73. 2. Gawrieh, S.; Chalasani, N.: Semin. Liver Dis. 2015, 35(3): 338–48. 3. Ratziu, V. et al.: Gastroenterology 2016, 150(5): 1147–1159. 4. Neuschwander-Tetri, B. et al.: Lancet 2014, 385(9972): 956–65. 5. Zhang, Z; Dales, N.; Winther, M.: J. Med. Chem. 2014, 57(12): 5039–56. 6. Liu, Y. et al.: PLoS One 2012, 7(6): e39165. 7. He, J. et al.: J. Diabetes 2016, 8(3): 305–13. 8. Arab, J. et al.: Hepatology 2016, doi:10.1002/hep.28709. 9. Merk, D. et al.: J. Med. Chem. 2014, 57(19): 8035–55. 10. Blöcher, R. et al.: J. Med. Chem. 2016, 59(1): 61–81.

POS.38

1-HeteroaryIpropan-2-one inhibitors of cytosolic phospholipase A2with improved metabolic stability Althaus, J.; Hake, T.; Subeska, A.; Hanekamp, W.; Fabian, J.; Lehr, M. Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstr. 48, 48149 Münster, Germany

Cytosolic phospholipase A2 (cPLA2) catalyzes the first step in the biosynthesis of pro-inflammatory lipid mediators such as prostaglandins, leukotrienes, and platelet activating factor (PAF) by cleaving membrane phospholipids in arachidonic acid and lyso-phospholipids. Therefore, inhibition of cPLA2 is considered to be an attractive target for the design of new anti-inflammatory drugs. Recently, we have found that the indole-5-carboxylic acid derivative 1 is a potent inhibitor of cPLA2 in cell free systems as well as in intact cells.1 However, the compound only showed a low bioavailability in mice after peroral administration. One reason for this behaviour appears to be its excessive in vivo metabolism. Primarily, the reactive ketone group of the propan-2-one scaffold is reduced to an alcohol and the carboxylic acid moiety of the indole ring system is converted into an ester glucuronide. In the present study we tried to increase the metabolic stability of this kind of compounds, e.g. by introduction of methyl substituents into the α-position of the reactive ketone functionality and by transformation of the aromatic carboxylic acid group to an aliphatic one, respectively.2 The effects of these structural variations on cPLA2α inhibitory potency as well as on phase I and phase II metabolic stability in rat liver homogenate are described.

References: 1. Ludwig, J. et al.: J. Med. Chem. 2006, 49: 2611-2620. 2. Schwarzkopf, J. et al.: Med. Chem. Res. 2014, 23: 5250-5262.

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POS.39

Tuning Selectivity: Novel Covalent-Reversible JAK3-Inhibitors with High Isoform Specifity Targeting a Nitrile Induced Arginine Pocket Forster, M.1; Chaikuad, A.2,4; Bauer, S. M.1; Holstein, J.3; Gehringer, M.1,5; Pfaffenrot, E.1; Ghoreschi, K.3; Knapp, S.2,4; Laufer, S. A.1 1 Department of Medicinal Chemistry, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany 2 Nuffield Department of Clinical Medicine, Structural Genomics Consortium and Target Discovery Institute, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford, OX3 7DQ, United Kingdom 3 Department of Dermatology, University Medical Center, Eberhard-Karls-University Tuebingen, Liebermeisterstr. 25, 72076 Tuebingen, Germany 4 Present address: Institute for Pharmaceutical Chemistry, Johann Wolfgang Goethe-University and Buchmann Institute for Molecular Life Sciences, Max-von-Laue-Strasse 9, D-60438 Frankfurt am Main, Germany 5 Present address: Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology (ETH) Zurich, Vladimir-Prelog-Weg 1-5/10, 8093 Zurich, Switzerland

In the last decade, Janus kinases (JAKs) have evolved to targets of high interest in the development of anti-inflammatory and oncologic agents. This family of cytosolic tyrosine kinases, consisting of four isoforms (JAK1, JAK2, JAK3 and TYK2), has a variety of crucial functions in many important signal pathways. While the other isoforms are ubiquitously expressed in different tissues, JAK3 is solely expressed in cells of the lymphoid lineage. Therefore, it plays a major role in the development of immunocompetent cells like T-cell or natural killer cells. Because of this isolated role, selective inhibition of JAK3 is supposed to be a promising strategy to achieve immunosuppression with less adverse effects [1]. However, the sufficiency of specific JAK3 inhibition is heavily debated, since it always is co-localized with JAK1 at the transmembranic γc-cytokine receptors [2,3]. To finally resolve this enigma, highly JAK3-selective chemical probes are required.

The current gold standard to investigate JAK dependent signalling is Tofacitinib. Unless it has an excellent kinome wide selectivity, it suffers from a poor selectivity within the JAK-family, being a potent inhibitor of all four isoforms [4]. Starting from a tricyclic analogue of Tofacitinib with a simplified cyclohexyl side chain, we were able to develop a new class of highly potent and selective JAK3 inhibitors. By targeting a JAK3 specific cysteine residue near the ATP binding pocket following a covalent-reversible approach, our inhibitors demonstrate both, an outstanding isoform specifity (400-, 2700- and 3600-fold JAK3 over JAK1, JAK2 and TYK2, respectively) as well as a high kinome wide selectivity. We were able to prove the covalent-reversible binding mode of our compounds with protein Xray crystallography and simultaneously discovered a novel binding pocket. This cavity is unique to our structures and is induced by interactions of the nitrile substituent of the inhibitor with arginine residues of the enzyme. The high potency and selectivity of this inhibitor class are also successfully carried over to cellular models resulting in a selective inhibition of JAK3 dependent signalling in functional T-cells. Therefore, these compounds are suitable to serve as molecular probes to elucidate the role of selective JAK3 inhibition. References: 1. Ghoreschi, K., Laurence, A., O’Shea, J.J.: Nat. Immunol. 2009, 4(10): 356-360 2. Haan, C. et al.: Chem. Biol. 2011, 3(18): 314-323 3. Thorarensen, A. et al.: ACS Chem. Biol. 2014, 9(7): 1552–1558 4. Thoma, G., Drückes, P., Zerwes, H.G.: Bioorg. Med. Chem. Lett. 2014, 19(24): 4617-4621

POS.40

Enhancement of binding interactions between hydrophobic region I and deep pocket-p38 MAP Kinase inhibitors with outstanding potencies Wentsch, H. K.1; Walter, N. M.1; Mayer-Wrangowski, S. C.2; Rauh, D.2; Laufer, S. A.1 1 Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard Karls Universitaet Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany

2 Faculty of Chemistry-Chemical and Biology, Technische Universitaet Dortmund, Otto-Hahn-Strasse 6, 44227 Dortmund, Germany

The fundamental role of p38 mitogen-activated protein kinases (MAP kinases) in the biosynthesis of proinflammatory cytokines like IL-1β and TNFα underlines their importance as therapeutic targets for the treatment of (auto)inflammatory diseases [1], cancer [2] and neurodegenerative diseases [3]. Although, a plethora of p38α MAP kinase inhibitors arising from manifold structural classes have been developed over the last decades, no inhibitor has launched to the market yet. Thus, there is still an urgent need for promising clinical candidates with improved in vivo efficacies and reduced side effects [4]. For p38 MAP kinase inhibitors the major challenges like outstanding potency, due to low ATP competitiveness, combined with excellent selectivity have already been solved [5, 6].

Fig. 1: Binding mode of compound 1 in p38α MAP kinase (PDB code: 3UVQ)

Our lead compound 1 (figure 1) showed an excellent IC50 value (IC50 = 1 nM) with respect to p38 MAP kinase, but unfortunately only a moderate inhibitory activity in a human whole blood TNFα release assay (IC50 = 280 nM). One reason might be a short target residence time due to the high intracellular ATP concentration and a competitive binding mechanism. Based on the X-ray structure (PDB code: 3UVQ) [6] we synthesized compounds which form the same interactions to the enzyme and can moreover enhance the interactions between hydrophobic region I and the deep pocket of the enzyme. Both are valid strategies to overcome the shortcomings mentioned above. Furthermore, we pursued the strategy of parallel synthesis of a dibenzooxepinone and a dibenzosuberone scaffold to compare their affinity to the enzyme and their activity in whole blood tests as well as to avoid potential metabolic disadvantages of the dibenzosuberone scaffold. Finally, we synthesized a variety of dibenzepinones following the intentional binding mode and were able to improve the inhibitory potency on the isolated enzyme down to the picomolar range and achieved IC50 values in whole blood system in the low double-digit nanomolar range. References: 1. Player, M. R. Curr Top Med Chem, 2009, 9, 598. 2. Tsai, C. J.; Nussinov, R. Semin Cancer Biol, 2013, 23, 235-42. 3. Anton, R. et al.: PLoS One, 2014, 9, e95641.54. 4. Zhang, J. et al.: T. in Pharm.Science 2007, 28, 286-295. 5. Baur, B. et al.: J Med Chem, 2013, 56, 8561-8578. 6. Fischer, S. et al.: J Med Chem, 2013, 56, 241-53.

INFLAMMATION


POS.41

In-situ forming gel devices as local depot therapeutic for rheumatoid arthritis Mohammadi, M.1; Li, Y.2; Abebe, D.3; Xie, Y.; Kandil, R.; Kraus, T.; Gomez-Lopez, N.; Fujiwara, T.; Merkel, O. M. 1 Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilian University of Munich, Butenandtstr. 5-13, 81377 Munich, Germany 2 Department of Pharmaceutical Sciences, Wayne State University, 259 Mack Ave, Detroit, MI 48201, USA 3 Department of Chemistry, The University of Memphis, Memphis, TN 38152, USA 4 Department of Obstetrics and Gynecology & Immunology and Microbiology, School of Medicine, Wayne State University, CS Mott Center for Human Growth and Development, 275 E. Hancock, Detroit, Michigan 48201 5 Department of Oncology, Molecular Therapeutics Program, Wayne State University, 4100 John R St, Detroit, MI 48201, USA

For new treatment options in rheumatoid arthritis (RA) folic acid (FA) coupled three layered micelles (3LM) were developed to encapsulate nucleic acids. Their application as locally implantable, targeted, macrophage-specific RNA interference (RNAi)-based delivery system could therefore revolutionize RA therapy [1, 2]. 3LM were formed from triblock copolymers of PLLA-PEI-PLLA and PLLA-PEG-PLLA in a three-step procedure [3]. Their structure and DNA entrapment in the core were determined by staining DNA with silver nitrate and TEM detection [4]. Hydrodynamic diameters and zeta potentials were measured by dynamic light scattering and laser Doppler anemometry. DNA release in neutral and acidic pH was detected by modified SYBR Gold assays [3]. Fluorescein labeled folic acid was used for flow cytometric detection of the expression of functional folic receptor β in LPS-activated and resting macrophages. For targeting of activated macrophages, folic acid (FA) was attached to the PEG-chain of a PLLA-PEG diblock affording PLLA-PEG-FA. 3LM were formed with 75%PLLA-PEG-FA and 25%FA-PEG-PLLA in the outer polymer shell for optimal targeting conditions. After RAW264.7 and primary macrophages were activated with LPS [5] or left resting they were treated with targeted and non-targeted 3LM loaded with fluorescently labeled DNA to optimize the uptake rate or GFP-Plasmid for investigating transgene expression, quantified by flow cytometry. Thermoresponsive and injectable hydrogels as depot formulation were formed by stereocomplexing 3LM which contain PLLA-PEG-PLLA in the outer core with PDLA-PEG-PDLA [6]. To examine the stability and DNA release of the hydrogels under physiologic and inflamed conditions, hydrogels were exposed to different conditions. The core-corona structure and efficient DNA entrapment in the core were confirmed by TEM. The sizes were found to be less than 200 nm, and the encapsulation efficiency of DNA was optimized based on the ratio of the PEI block in PLLA-PEI-PLLA per DNA [3]. 3LM were stable at neutral pH but released DNA in an acidic environment [3]. FR-overexpressing activated cells, as successfully identified by internalization of FA-fluorescein, showed significantly higher uptake of targeted 3LM and GFP expression in vitro and ex vivo than resting macrophages. Stereocomplexes of 3LM form hydrogels above their phase transition temperature. In the physiologic environment almost no DNA was released and in an acidic environment most DNA was encapsulated in 3LM while the mass of the gel degraded. Our findings confirm that FA-3LM are taken up by activated macrophages via folate receptor mediated endocytosis and thus could become a promising delivery system for receptor-mediated drug or gene delivery and novel therapy of rheumatoid arthritis in an in situ forming gel formulation. References: 1. WHO, Chronic diseases and health promotion in: Chronic rheumatic conditions, 2015 , Geneva. 2. Gordon S.,Taylor P.R: Nat Rev Immunol, 2005 5: 953-964. 3. Abebe D.G. et al.: Macromolecular Bioscience, 2015 1828 - 1836. 4. Zheng M. et al.: ACS Nano, 2012 6: 9447-9454. 5. Funk J.L. et al.: Atherosclerosis, 1993 98: 67-82. 6. Abebe D.G., Fujiwara T.: Biomacromolecules, 2012 13: 1828-1836.

POS.42

Transferrin-Polyethylenimine Nanoparticles for T Cell targeted siRNA Delivery as Novel Anti-inflammatory Asthma Therapy Kandil, R.1; Xie, Y.2; Kim, N. H.2; Nadithe, V.2; Thakur, A.2,3; Lum, L. G.2,3; Bassett, D. J. P.2; Merkel, O. M.1,2,3 1 Department of Pharmacy, Pharmaceutical Technology and Biopharmacy, Ludwig-Maximilians-Universität München, 81377 München 2 Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201 3 Karmanos Cancer Institute, Detroit, MI 48201

Asthma is a major public health problem as the disease affects 235 million people worldwide, and in a considerable portion of patients it is still not sufficiently controlled. Therefore, novel efficient anti-inflammatory therapies with minimum side effects are urgently needed. The disorder is characterized by infiltration of immune cells, including T helper 2 cells (Th2), a type of activated T cells (ATC), in the lung, causing chronic inflammation of the airways. Various underlying cascades are orchestrated via the secretion of Th2 cytokines, such as IL-4, IL-5, and IL-13. [1] All the more, than downregulation of these single cytokines, the therapeutic interference with decisive transcription factors involved in this disease process, such as GATA-3, is a promising approach to early-on undermine pathologic pathways in asthma. [2] Since RNA interference was shown to induce transient and reversible knockdown [3], it offers an auspicious therapeutic base for the silencing of clinically relevant genes. However, the lack of efficient biocompatible siRNA carrier systems to overcome extra- and intracellular barriers still impedes the clinical translation. To achieve the particularly challenging transfection of T lymphocytes, we designed a novel nano-sized ATC targeted delivery system composed of Transferrin-N-Succinimidyl-3-(2-Pyridyldithio)-Propionate-Polyethylenimine (Tf-SPDP-PEI). By utilizing the physiological iron transport molecule Transferrin as a ligand, we both achieve cell internalization and active targeting, as its endocytosis-mediating receptor is overexpressed by activated but not by naïve T cells. The incorporated disulfide bond is stable extracellularly, but can easily be cleaved in the endosome, releasing the particles inside of the cells. Following the successful synthesis of the Tf-SPDP-PEI conjugate, size and zeta potential of conjugate-siRNA polyplexes were measured by dynamic light scattering (DLS) and condensation efficiency was determined by SYBR Gold assay. Particle uptake and siRNA delivery were examined using flow cytometry and knockdown of GAPDH, a universal housekeeping gene, was quantified by real-time PCR. Prepared siRNA polyplexes featured favorable sizes of less than 200 nm, slightly negative zeta potentials and comparable siRNA condensation rates with particles composed of non-modified PEI. The designed conjugate was proven to successfully deliver siRNA to both human primary ATCs and murine T cells in a murine asthma model without causing considerable adverse effects in the latter. Furthermore, significant reduction of GAPDH expression was demonstrated in vitro after delivery of respective siRNA in conjugate nanoparticles to human ATCs compared with PEI alone. In conclusion, our biocompatible targeted delivery system holds great promise to be an innovative therapeutic option to improve asthma control in the future. Acknowledgements: ERC Starting Grant (StG-2014-LS7-637830), NIH Boost Grant and Wayne State Start-Up Grant to Olivia Merkel

References: 1. Ray, A. and L. Cohn: J. Clin. Inves. 1999, 104(8): 985-93. 2. Sel, S. et al.: J. Allergy. Clin. Immunol. 2008, 121(4): 910-916 e5. 3. Kole, R. et al.: Nat. Rev. Drug. Discov. 2012, 11(2): 125-40.

POS.43/SL.39 Influence of Th2 Cytokines on the Cornified Envelope, Tight Junction Proteins and ß-Defensins in Filaggrin-Deficient Skin Equivalents Hönzke, S.1; Schäfer-Korting, M.1; Hedtrich, S.1 1 Institute of Pharmacy, Pharmacology & Toxicology, Königin-Luise-Straße 2+4, 14195 Berlin, Germany

For abstract see Short Lecture SL.39

POSTERS


POS.44

A new class of selective and potent inhibitors of human delta 24-dehydrocholesterol reductase Müller, C.1; Hemmers, S.1; Schreiber, F.1; Körner, A.2; Mirakaj, V.2; Giera, M.3; Bracher, F.1 1 Department für Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 München, Germany 2 Universitätsklinikum Tübingen, Eberhards Karls Universität Tübingen, Waldhörnlestraße 22, 72072 Tübingen, Germany 3 Leiden University Medical Center, Center for Proteomics and Metabolomics, Albinusdreef 2, 2300 RC Leiden, The Netherlands

In the 1960’s the pharma company William S. Merrell Co marketed triparanol (Mer-29) as a hypolipidemic drug inhibiting the enzyme Δ24-dehydrocholesterol reductase (24-DHCR). Inhibition of this enzyme leads to reduction of cholesterol levels and accumulation of desmosterol, the substrate of 24-DHCR ultimately leading to cholesterol. However, harmful side effects led to a withdrawal of the drug authorisation for triparanol [1]. Besides this early setback in the use of 24-DHCR inhibitors, several promising applications for such enzyme inhibitors have recently been claimed [2,3]. Spann et al. [2] observed in a murine atherosclerosis model that foam cells, evolving from transforming macrophages, surprisingly presented an intrinsically anti- instead of pro-inflammatory phenotype. The phenotype was attributed to the intra-cellular accumulation of desmosterol. Other studies showed that 24-DHCR inhibitors could play a crucial role in HCV infection [3]. These findings led us to focus our research on the development of potent and selective 24-DHCR inhibitors [4,5]. We used a whole cell assay in combination with targeted GC-MS based sterol pattern analysis for the characterization of target enzyme and selectivity system for the analysis of the obtained sterol pattern under inhibitor treatment. Furthermore, IC50 values reflecting inhibition of total cholesterol biosynthesis were established. Here we present lathosterol side chain esters as novel non-toxic, selective and potent 24-DHCR inhibitors with IC50 values in the sub-nanomolar range. Furthermore, we demonstrate that the well-known and established 24-DHCR inhibitors are not selective, and significantly less potent. The newly developed inhibitors are highly useful tools for further studies in the field of 24-DHCR inhibition.

References: 1. Rozman, D., Monstory, K.: Pharmacol. Ther. 2010, 127(1):19-40. 2. Spann, N.J. et al.: Cell 2012, 151(1):138-152. 3. Takano, T. et al.: J. Hepatol. 2011, 55(3):512-521. 4. Giera, M., Plössl, F., Bracher, F.: Steroids 2007, 73(8):633-642. 5. Müller, C. et al.: Steroids 2013, 78(5):483-493.

CANCER/INFLAMMATION


3.3 Cancer/Inflammation

POS.46/SL.40 Tumor selectivity of V-ATPase inhibition is based on differential regulation of AMPK von Schwarzenberg, K.1; Menche, D.2; Müller, R.3; Vollmar, A. M.1 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich 2 Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn, Gerhard - Domagk-Str.1, 53121 Bonn, Germany 3 Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, PO 151150, Universitätscampus E8 1, 66123 Saarbrücken, Germany


POS.47

New Inhibitors of Golgi alpha-mannosidase II Irsheid, L.1; Borek, C.1; Guilherme dos Santos, M.1; Weickert, A.2; Seibel, J.2; Engels, B.2; Stauber, R.3; Schirmeister, T.1 1 Germany Institute of Pharmacy and Biochemistry, University of Mainz, Germany 2 Institute of Physical and Theoretical Chemistry, University of Würzburg, Germany 3 Molecular and Cellular Oncology, University Medicine Center Mainz, Germany

Golgi α- mannosidase II (GMII) plays a crucial role in the N-glycosylation pathway. In various tumor cell lines, the distribution of the N-linked sugars on the cell surface is modified and correlates with the progression of tumor metastasis(1). GMII therefore is a molecular target for anticancer agents and its inhibition has shown to induce tumor repression(2). GMII, a member of the family 38 glycoside hydrolases, cleaves two mannose units (α-(1,3) and α-(1,6)) of the intermediate GlcNAcMan5(GlcNAc)2.The active site of the enzyme consists of two aspartate residues and a zinc cation. GMII acts as a retaining glycosidase and cleaves the sugars in a two-step-SN2-mechanism resulting in a covalent glycosyl-enzyme complex. The mechanism preserves the configuration of the anomeric C-atom(3,4). Several natural product-based or synthetic inhibitors have been investigated. However, the clinical use of the known potent inhibitor swainsonine is restricted due to the side effects resulting from inhibition of the closely related lysosomal α-mannosidases(5). We performed two virtual screenings with a library of about five million purchasable compounds to identify new chemotypes inhibiting the GMII. The docking studies were carried out on the known active site and on a potential allosteric binding site identified by molecular modeling. The most promising candidates were purchased and subjected to enzyme inhibition assays yielding several weakly active compounds. The aim of our work is to derivatize the most active compounds in order to increase the inhibitory effect and elucidate the structure-activity relationships. Furthermore we synthesize derivatives based on a central D-mannose core as promising lead structures for a selective, covalent GMII-inhibition. References: 1. Fujita, T. et al.: Org. Lett. 2004, 6 (5): 827-830. 2. Van den Elsen, Y. M. H.; Kuntz, D. A.; Rose,D. R.: The EMBO Journal 2001, 20 (12): 3008-3017. 3. Petersen, L. et al.: J. Am. Chem.Soc. 2010, 132: 8291-8300. 4. Zhong, W. et al.: J. Am. Chem.Soc. 2008, 130: 8975-8983. 5. Cheng, T.-J. R. et al: Chem. Asian J. 2013, 8: 2600-2604.

POS.48

Latent Heparanase – Impact on angiogenic mediators within the metastatic niche Hoß, S. G.1; Grundmann, M.2; Ilan, N.3; Vlodavsky, I.3; Bendas, G.1 1 University of Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry II, An der Immenburg 4, 53121 Bonn, Germany 2 University of Bonn, Institute for Pharmaceutical Biology, Section for Molecular-, Cellular- and Pharmacobiology, Nussallee 6, 53115 Bonn, Germany 3 Technion-Israel Institute of Technology, Vascular and Cancer Biology Research Center Rappaport Faculty of Medicine and Research Institute, P.O. Box 9649, 31096 Haifa, Israel

The endo-ß-D-glucuronidase heparanase cleaves heparan sulfate (HS) chains which are part of so called heparan sulfate proteoglycans (HSPG). HSPGs consist of a protein core with attached linear HS-chains and comprise versatile functions regarding the integration of the individual cell in and communication with its environment. HSPGs are able to bind macromolecules within the extracellular matrix (ECM), e.g. collagen and fibronectin and have adhesive functions during migration. In addition, HS-residues form a scaffold for diverse active moieties and growth factors, such as vascular endothelial growth factor (VEGF). [1] HS cleavage by heparanase enzymatic activity releases these factors and brings them to action. Therefore, tumor cells make use of heparanase activity during metastatic progression inducing an ECM-remodelling for transmigration purpose. Consequently, heparanase expression and activity is considered as a bad prognostic factor in cancer. [2] In addition to the active heparanase, the enzymatically inactive precursor form called “latent heparanase” displays activities independent of HS-cleavage, but obviously related to HS binding. These activities include the induction of multiple signalling events, fostering integrin binding [2] or an increased secretion of VEGF by tumor cells [3], which plays a key role in establishing the early metastatic niche. Aims of the study: The molecular mechanism by which VEGF-induction is realized by latent heparanase is actually unknown and forms the pivotal question of our current studies. VEGF release in tumors has been related to thrombin activity and thrombin receptor overexpression [4], however, this pathway has not been associated with heparanase, and a “heparanase receptor” is unknown. Results: Western blot analysis revealed that there is a substantial expression of thrombin receptors in our MV3 human melanoma cell model system. Therefore we followed the effects of latent heparanase and the stimulation of thrombin receptors by TRAP-6 (thrombin receptor activating peptide 6) on VEGF-expression by analyzing the culture supernatants after defined incubation periods via VEGF-ELISA. Both interventions led to a significant increase in the VEGF level and hence join latent HPSE and thrombin receptor activation in giving an angiogenic stimulus. Besides this view on functional consequences of latent heparanase, an innovative biosensor technique allowed us to get insight into the intracellular signalling, applied for the first time in the heparanase research. The sensor device measures the dynamic mass redistribution in the cytoplasm resulting from a cell (receptor) activation. Thus, a ligation of receptors in the cell membrane and a triggered rearrangement of mass as a downstream effect can be recorded in a real time sensorgram. We applied TRAP-6 in different concentrations and obtained a typical, in part complex concentration dependent DMR-response curve. The application of TRAP-6 together with a thrombin receptor antagonist led to a decrease in DMR-response and verified the obtained signals to be specifically evoked by thrombin receptor activation. Interestingly latent heparanase also generated a concentration dependent DMR-curve and therefore latent heparanase downstream effects can be monitored and analysed using this method. Co-incubation with thrombin receptor antagonist did not influence the DMR-signal in a consistent manner, and therefore a direct impact of latent heparanase on thrombin receptors seems up to now not to be obvious. The elucidation of a potential heparanase / receptor link leading to a VEGF release remains the matter of further investigations. In light of ongoing clinical trials using heparanase inhibitors in cancer and considering the established VEGF-blocking strategies in clinics, an interrelation between both appears promising regarding the elucidation of involved mechanistic patterns and might reveal new targets for anti-angiogenic therapeutic options. References: 1. Vlodavsky, I. et al.: Nat. Med. 1999, 5(7): 793–802. 2. Gerber U. et al.: Semin. Thromb. Hemost. 2015, 41(2): 244-254. 3. Zetser, A. et al.: Cancer Res. 2006, 66(3): 1455–1463. 4. Dorsam R. T., Gutkind J. S.: Nat. Rev. Cancer 2007, 7(2): 79-94.

POSTERS


POS.49

Fucoidans inhibit inflammation and tumor metastasis supporting processes mediated by IL-8 or C5a Liewert, I.1; Ehrig, K.1; Alban, S.1 1 Pharmaceutical Institute, Christian-Albrechts-University of Kiel, Gutenbergstraße 76, 24118 Kiel, Germany

Heparins are known to exhibit anti-inflammatory and antimetastatic activities, however their application in inflammatory diseases and cancer is limited due their immanent bleeding risk. Fucose-containing sulfated polysaccharides (syn. “fucoidans”) isolated from brown algae, which are currently considered promising candidates for health-supporting and medicinal applications [1], showed anti-inflammatory and antimetastatic activity in vivo as well, but considerably reduced anticoagulant effects [2,3]. However, there is only limited knowledge about their mode of actions and structure-dependent effects. Attractive targets for both anti-inflammatory and antitumor agents are the anaphylatoxin C5a and interleukin 8 (IL-8) which are both closely linked to inflammatory processes and tumor progression [4,5,6,7,8]. The aim of the current study was to investigate the affinity of two structurally distinct fucoidans from Saccharina latissima (S.l.-SP) and Fucus vesiculosus (F.v.-SP) [9,10], as well as heparin (UFH) as reference, to IL-8 and C5a and their effects on downstream processes initiated in polymorphonuclear neutrophils (PMN) as cell type critically involved in many inflammatory diseases. In a competitive SPC-ELISA [11], both fucoidans, but not UFH, displayed high affinity to IL-8 as well as C5a and S.l.-SP bound about 4 times stronger than F.v.-SP to both stimuli. The impact of this binding on the PMN activation was examined by means of two intracellular signalling processes, i.e. phosphorylation of the protein kinases Erk1 and Erk2 and intracellular calcium release. According to Western Blot analyses, both fucoidans inhibited the Erk phosphorylation, whereby the inhibition was more pronounced in IL-8- than in C5a-stimulated PMN. Further, both fucoidans concentration-dependently reduced the strong intracellular calcium release induced by IL-8 as well as C5a, whereas UFH showed only moderate effects. Contrary to the binding assay, F.v.-SP was significantly more active than S.l.-SP. As typical reaction of activated PMN, the IL-8- and C5a-induced PMN chemotaxis was assessed using modified Boyden chambers. Similar to the Erk phosphorylation, chemotaxis to IL-8 was effectively inhibited by both fucoidans and up to 30 % by UFH, whereas migration towards C5a was only reduced to about 50 % by both fucoidans and not at all by heparin. Strikingly, S.l.-SP and F.v.-SP displayed distinct shapes of the concentration-dependent curves for inhibition of chemotaxis towards IL-8 with more pronounced inhibition by S.l.-SP at <10 µg/ml and a stronger effect of F.v.-SP at higher concentrations. In conclusion, both examined fucoidans showed to interfere with IL-8- and C5a-induced stimulation of PMN by binding to these activators. They proved to be considerably more active than heparin, however, their activity profiles differed. This suggests that further mechanisms may be involved in their inhibitory effects on PMN. Acknowledgements: We thank Cornelia Rodde for her excellent technical assistance.

References: 1. Ruocco, N. et al.: Molecules 2016, 21(5): 2. Fitton, J., Stringer, D., Karpiniec, S.: Mar Drugs 2015, 13(9): 5920–5946. 3. Kwak, J.-Y.: Mar Drugs 2014, 12(2): 851–870. 4. Manthey, H. et al.: The International Journal of Biochemistry & Cell Biology 2009, 41(11): 2114–2117. 5. Guo, R.-F., Ward, P.: Annu Rev Immunol 2005, 23: 821–852. 6. Darling, V. et al.: Expert Rev Clin Immunol 2015, 11(2): 255–263. 7. Harada, A. et al.: J Leukoc Biol 1994, 56(5): 559–564. 8. Campbell, L., Maxwell, P., Waugh, D.: Pharmaceuticals (Basel) 2013, 6(8): 929–959. 9. Ehrig, K., Alban, S.: Mar Drugs 2014, 13(1): 76–101. 10. Schneider, T. et al.: Glycobiology 2015, 25(8): 812–824. 11. Alban, S., Gastpar, R.: Journal of Biomolecular Screening 2001, 6(6): 393–400.

POS.50

Integrin activation leads to increased chemoresistance against cisplatin, doxorubicin and mitoxantrone in breast cancer cell lines MCF-7 and MDA-MB-231 Baltes, F.; Piva, M. B. R.; Schlesinger, M.; Bendas, G. University of Bonn, Pharmaceutical Institute, An der Immenburg 4, 53121 Bonn, Germany

Background: The development of resistance against chemotherapeutics is the major obstacle in the clinical treatment of tumor diseases. The molecular mechanisms of chemoresistance are versatile and in many cases of multifactorial origin. The contact formation of tumor cells with extracellular matrices has been shown to contribute to a lower sensitivity for chemotherapy. Thus, this interaction between cells and their microenvironment has been indicated as “environment mediated drug resistance” (EM-DR) that can further be divided into “cell adhesion mediated drug resistance” (CAM-DR) and “soluble factor mediated drug resistance” (SFM-DR) depending on the molecules involved [1]. Integrins are heterodimeric transmembrane receptors that take an important role in communication between cells and their microenvironment. Consequently, their contribution to CAM-DR is highly probable but has not explicitly been shown for breast cancer cells. Aim/objectives: The aim of this study was to investigate and quantify the effect of EM-DR in breast cancer cell lines MCF-7 and MDA-MB-231 depending on different integrin activation levels. Methods: Sensitivity against cytostatic drug was measured by MTT assay. Cells were seeded in triplicates, inoculated with either cisplatin, doxorubicin or mitoxantrone and incubated for 72 h. The resulting dose-effect curves were used to get EC50 as a parameter for cell sensitivity. The assay was performed at least threefold for each treatment. By treating the cells additionally with integrin influencing agents, the receptors’ effect was measured. Integrins were activated by cell ligation on extracellular matrices such as collagen and fibronectin. Furthermore, manganese(II)-cations were added to allosterically activate integrins by binding to their MIDAS structure [2]. In order to inhibit integrins we used low molecular weight heparins (e.g. tinzaparin [3]) or a monoclonal integrin antibody (natalizumab). To interfere with the intracellular integrin signaling pathway, FAK inhibitor 1,2,4,5-benzenetetramine tetrahydrochloride or PI3K/mTOR inhibitor BEZ235 was applied. Changes in the expression of extracellular and intracellular molecules were investigated by Western Blot and FACS. Results: The integrin activation by ECM ligation declines the sensitivity against the cytostatic drugs cisplatin, doxorubicin or mitoxantrone in both MCF-7 and MDA-MB-231 cells. Apart from binding to extracellular matrices, the allosteric activation by manganese(II)-cations displayed the highest resistance increasing effect. Since both, blockade of integrin binding and interference with integrin downstream signaling enhance sensitivity, a clear functional involvement of cellular integrins in resistance formation can be suggested. However, differences in the cellular response to the different cytostatics and their relation to integrins became evident suggesting more complex mechanisms than a simple integrin-resistance way. Conclusions: The data suggest that there is a molecular resistance mechanism based on integrin activation in breast cancer cell lines MCF-7 and MDA-MB-231 that exceeds simple physical adhesion processes. This sheds a new light on integrins as potential targets to overcome tumor cells’ resistance to cytostatic treatments. References: 1. Meads, M. B.; Gatenby, R. A.; Dalton, W. S.: Nat Rev Cancer. 2009, 9(9): 665–74. 2. Chen, J.; Salas, A.; Springer, T.A.: Nat Struct Biol. 2003, 10(12): 995–1001. 3. Schlesinger, M. et al.: Thromb Haemost. 2009, 102(5): 816–22.

CANCER/INFLAMMATION


POS.51

The anti-metastatic properties of the tubulin-binding agent pretubulysin could be based on the trapping of tumor cells to the endothelium Schwenk, R.1; Stehning, T.1; Bischoff, I.1; Ullrich, A.2; Kazmaier, U.2; Fürst, R.1 1 Institute of Pharmaceutical Biology, Biocenter, Goethe-University, 60438 Frankfurt, Germany 2 Institute of Organic Chemistry, Saarland University, 66123 Saarbrücken, Germany

Tubulin-binding agents are the most widely used anti-cancer drugs. Due to the side effects and the development of resistances, the discovery of new agents is still of importance [1]. Recently, pretubulysin (PT), a naturally occurring precursor of the myxobacterial compound tubulysin, was identified as a novel tubulin-binding compound [2]. Within the DFG research group FOR 1406, PT was characterized as an anti-tumoral, anti-angiogenic and vascular-disrupting compound [3,4,5]. Moreover, PT was also found to inhibit the formation of metastases in vivo [6]. Aim of the present study was to gain insights into the mechanisms underlying this anti-metastatic effect by investigating the influence of PT on the interaction of endothelial and tumor cells in vitro. Treatment of primary human endothelial cells (HUVECs) with PT strongly increased the adhesion of breast cancer cells (MDA-MB-231) onto HUVECs, but limited their transmigration through the endothelium (Transwell assay). Based on this data, the gene expression of presumably involved adhesion molecules was determined by qRT-PCR: ICAM-1, VCAM-1, E-selectin, N-cadherin, and galectin-3. Moreover, the chemokine system CXCL-12/CXCR4 was analyzed. We found that the mRNA level of endothelial N-cadherin was upregulated by PT. While the total protein expression of N-cadherin was enhanced in PT-treated HUVECs, its surface expression was only marginally influenced by PT (Western blot, flow cytometry). In line with this, blocking endothelial N-cadherin by a neutralizing antibody revealed that this protein is not involved in PT-evoked tumor cell adhesion. Interestingly, PT strongly augmented the mRNA expression of CXCL12 in HUVECs (qRT-PCR), whereas its protein expression and endothelial secretion was only slightly enhanced by PT (Western blot, ELISA). However, the secretion of CXCL12 (and cytokine secretion in general) is of no importance for the PT-evoked tumor cell adhesion, since there was no difference in tumor cell adhesion when culture medium was changed or not before tumor cells were added. Moreover, an autocrine action of CXCL12 could be excluded, since inhibition of the CXCL12 receptor CXCR4 on endothelial cells with plerixafor did not influence cancer cell adhesion. By microscopic analyses, we found that PT treatment causes transient gaps in the HUVEC monolayer, where tumor cells prefer to adhere. Since β1-integrins on the tumor cells could mediate interactions between cancer cells and extracellular matrix proteins in the gaps (e.g. collagen), their influence in cell adhesion and transmigration assays was examined. Both the PT-evoked increase in cell adhesion and the decrease in transmigration was completely abolished when β1-integrins were blocked on MDAs by a neutralizing antibody. These results indicate that the anti-metastatic action of pretubulysin might be based on the trapping of tumor cells on the endothelium. Whether this effect is also relevant in vivo, will be analyzed in future studies using intravital microscopy. Acknowledgments: This work was supported by the German Research Foundation (DFG, FOR 1406, FU 691/9-2).

References: 1. Dumontet, C.; Jordan, M. A.: Nat. Rev. Drug Disc., 2010, 9(10): 790-803 2. Ullrich, A. et al.: Angew. Chem. Int. Ed. Engl., 2009, 48(24): 4422-4425 3. Herrmann, J. et al.: PLoS ONE, 2012, 7(5): e37416 4. Rath, S. et al.: Br. J. Pharmacol., 2012, 167(5): 1048-1061 5. Kretzschmann, V. K. et al.: Arterioscler. Thromb. Vasc. Biol., 2014, 34(2): 294-303 6. Braig, S. et al.: Cell Death Dis., 2014, 5: e1001

POS.52

A novel screening method to identify inhibitors of PPIs based on the Autodisplay technology Bopp, B.1; Ciglia, E.2; Hansen, F. K.2; Neundorf, I.3; Hochscherf, J.3; Kurz, T.2; Niefind, K.3; Gohlke, H.2; Jose, J.1

1 Westfälische Wilhelms-Universität Münster, PharmaCampus, Institut für Pharmazeutische und Medizinische Chemie, Corrensstraße 48, D-48149 Münster

2 Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf 3 Department für Chemie, Institut für Biochemie, Universität zu Köln, Zülpicher Straße 47, D-50674 Köln

The inhibition of critical protein-protein interactions (PPIs) has become increasingly important in drug discovery. Although, targeting PPIs is considered as difficult, more and more success stories demonstrate the feasibility of this approach. The lack of assays to investigate the inhibition of PPIs leads to the necessity of new methods to overcome this hurdle. A novel method based on the Autodisplay technology facilitates to screen for small molecules that inhibit PPIs in vitro. As PPIs can be mainly divided into two classes, heteromeric and homomeric interaction, two different targets were chosen to develop a flow cytometric screening assay based on the Autodisplay technology: the heterotetrameric kinase CK2 [1] and the homodimeric chaperone Hsp90 [2]. As proof of principle, Autodisplay and subsequent flow cytometric analysis were used to demonstrate the inhibition of CK2/CK2 interaction. In this case, purified CK2 was labelled with a fluorophore and binding to CK2 displayed on the surface of E. coli cells was examined via flow cytometry. High cellular fluorescence intensity indicated binding of CK2 to CK2. Addition of a cyclic peptide derived from the C-terminal CK2 segment, known to inhibit this interaction, led to lowered fluorescence intensity, indicating inhibition of the CK2CK2 interaction [3]. For inhibition of a homomeric interaction, the homodimer Hsp90 was displayed on the surface of E. coli. As only dimerized Hsp90 is able to bind client proteins such as p53, the next step was to proof dimerization of Hsp90 on the surface of E. coli. For this purpose, purified and fluorophore labelled p53 was added to cells displaying Hsp90 followed by flow cytometric analysis. The addition of labelled p53 to these cells led to an increased cellular fluorescence intensity, indicating binding of labelled p53 to surface displayed and dimerized Hsp90. In consequence inhibition of dimerization should lead to reduced cellular fluorescence intensity. Addition of peptides derived from the C-terminal dimerization interface of Hsp90 was used to inhibit the homomeric interaction. Flow cytometric analysis revealed blocking of the dimerization of Hsp90 with these peptides with IC50 values in the low micromolar range [4]. With this new method we were able to screen and identify a new class of peptidomimetic compounds that inhibit dimerization of Hsp90 with IC50 values in the low µM range. The novel screening method based on the Autodisplay technology facilitates screening of inhibitors for both, heteromeric and homomeric interactions, as shown for CK2 and Hsp90. It is simple to adapt for the screening of PPI inhibitors for other targets, making this technique a valuable addition to the still limited arsenal of ex vivo assays to investigate the inhibition of PPIs. References: 1. Niefind, K. et al.: EMBO J. 2001 , 20, 5320-31. 2. Wegele, H. et al.: Rev. Phyiol. Biochem. Pharmacol. 2004, 151, 1-44. 3. Raaf, J. et al.: (2013) ACS Chem. Biol. 2013, 8, 901-907. 4. Bopp, B.; Ciglia, E. et al.: BBA – Gen. Subjects 2016, 1860(6): 1043–1055.

POS.53

Identification of clinically relevant patient factors on the pharmacokinetics of tamoxifen based on study data of two clinical trials: Exploratory data analysis Richter, T.1; Klopp-Schulze, L.1; Lintermans, A.2; Van Asten, K.2; Jongen, L.2; Blomme, C.3; Poppe, A.3; Neven, P.2,3; Joerger, M.4; Kloft, C.1

1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universität Berlin, Germany, 2 KU Leuven - University of Leuven, University Hospitals Leuven, Department of Oncology, Leuven, Belgium 3 KU Leuven - University of Leuven, University Hospitals Leuven, Department of Gynecology and obstetrics, Leuven, Belgium 4 Dept. of Medical Oncology & Hematology, Cantonal Hospital St. Gallen, Switzerland

Background and objective: Breast cancer is one of the 10 most frequent causes of death in Germany with 22 deaths per 100 000 inhabitants (2.1% of all inhabitants) in 2014. Tamoxifen (TAM), a selective estrogen-receptor modulator (SERM), is used for adjuvant, neoadjuvant and metastatic setting with a dose of 20 mg daily. A high

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inter-individual variability in the pharmacokinetics (PK) of TAM and its metabolites has been observed1. Potent metabolites of TAM with a high receptor affinity are (Z)-endoxifen (ENDX) and (Z)-4-hydroxytamoxifen (100-fold higher than TAM), predominantly metabolised by CYP2D6.2 The current investigation aims to identify influential patient factors which could explain the high inter-individual variability found in the PK of TAM and its metabolites and guide towards TAM dose individualisation. Material and Methods: The two multi-centre studies included 391 adult, female patients, treated with 20 mg once daily. In both studies only patients with postmenopausal status and estrogen receptor-positive (ER+) cancer were eligible. The primary objective of CYPTAM 2 (NCT00965939, npatients = 247, treatment: neoadjuvant or metastatic) was to assess the relation between ENDX plasma concentrations and radiological treatment response to tamoxifen based on the RECIST criteria 1.0 3, whereas the primary objective of CYPTAM 3 (NCT00966043, npatients = 144, treatment: adjuvant) was to assess a change in endometrial thickness. In both studies, SNP genotyping of various cytochrome P450 enzymes involved in TAM metabolism, such as CYP2D6, was performed. Besides that, they differed in the number of blood samples taken for PK analysis (129 samples in CYPTAM 3 and 441 samples in CYPTAM 2). An exploratory statistical and graphical data analysis was conducted in R (Version 3.2.4): To assess similarities and differences in the two studies, continuous and categorical covariates, such as age, BMI and genotype, were statistically compared. For this purpose, study populations were stratified based on categorical covariates (e.g. treatment setting) into subgroups to identify potential patterns. Results: The patient characteristics between the subgroups of the studies, defined as “setting: adjuvant”, “setting: neoadjuvant” and “setting: metastasised”, differed considerably. Approximately 75% of the patients in CYPTAM 2 suffered from a metastasised adenocarcinoma with a median age of 68 years (range: 50 – 88 years) and a median BMI of 26.8 kg/m2 (range:16.6 – 50.7 kg/m2); the other 25% of the patients of CYPTAM 2 study were treated in a neoadjuvant setting with a median age of 82 years (range: 49 – 96 years) and median BMI of 26.3 kg/m2 (range: 18.4 – 42.4 kg/m2). Patients of the CYPTAM 3 study, with a median age of 60 years (range: 41 – 85 years) and median BMI of 26.1 kg/m2 (range: 18.4 – 39.2 kg/m2) were treated in an adjuvant setting. High inter-patient variability was observed for ENDX concentrations: In the CYPTAM 3 study the median concentration was 11.8 ng/mL (range: 0.77 - 31.8 ng/mL) after 3 - 6 months of treatment; in the CYPTAM 2 study the median concentrations were similar with 12.4 ng/mL (range: 0.70 – 35.1 ng/mL) and 11.5 ng/mL (range: 2.20 – 30.5 ng/mL) in the metastasised and neoadjuvant setting after 2 months of treatment, respectively. Conclusions: Overall, a high inter-individual variability in TAM ( 16-fold) and ENDX concentrations ( 50-fold) was observed across the two studies. Interestingly, ENDX concentrations showed similar distributions (median, range) in the different treatment settings. The results of this exploratory data analysis will be used to assist a subsequent covariate analysis utilising a population modelling approach. The PK model considering the clinically relevant covariates shall support individual treatment decisions. References: 1. Schroth, W. et al.: J. Clin. Oncol., 2007, 25(33): 5187-5193. 2. Mürdter, T. E. et al.: Clin. Pharm. Ther., 2011, 89(5): 708-717. 3. Eisenhauer, E. A. et al.: Eur. J. Cancer., 2009, 45(2): 228-247.

POS.54

Melatonin-Tamoxifen Hybrid Ligand as Potent Agent against Breast Cancer Zlotos, D. P.1; Marzouk, M. A.1; Hasan, M.2; Darveau, T.2; Guarinini, L.2; Browne, E.2; Witt-Enderby, P. A.2 1 The German University in Cairo, Dept. of Pharmaceutical Chemistry, New Cairo City, 11835 Cairo, Egypt 3 Duquesne University, Division of Pharmaceutical Sciences, School of Pharmacy, 421 Mellon Hall, Pittsburgh, PA, 15282, USA

Melatonin-tamoxifen hybrid ligands 1a-e have been recently reported to be promising new agents in the prevention and treatment of breast cancer.1 In particular, in vitro studies using MCF-7 (ER+/PR+), MDA-MB-231 (triple negative) and MMCs (HER2+/ER-/PR-) cancer cells the

hybrid ligand 1c decreased proliferation and migration. In mice, the hybrid ligand 1d was uterine protective whereas tamoxifen alone or melatonin and tamoxifen (unlinked) stimulated uterine tissue similar to the effects of 17-estradiol alone. Moreover, 1d was shown to bind with equal affinity to estrogen receptors and melatonin MT1 receptors similar to tamoxifen or melatonin alone; however, at concentrations greater than 1M, 1d increased melatonin receptor and estrogen receptor binding sites suggesting a dual but unique mechanism of action. Here, we describe a new synthetic approach toward compounds 1a-e and in vitro anti-cancer action of the hybrid ligand 1c with respect to cancer cell proliferation, viability, and invasion. The findings provide a new promising perspective for the treatment of breast cancer.

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Acknowledgments: Würzburg University, Prof. Dr. Ulrike Holzgrabe

References: 1. Witt-Enderby, P.A. et al. US Patent 8785201. 2014

POS.55

Photoactivation of Pt(IV)-anticancer complexes coupled to upconverting nanoparticles Perfahl, S.1; Natile, M.2; Natile, G.3; Bednarski, P. J.1

1 Institute of Pharmacy, University of Greifswald, 17487 Greifswald, Germany 2 CNR- IENI, Department of Chemical Sciences, University of Padua, 35131 Padua, Italy 3 Department of Chemistry, University of Bari, 70125 Bari, Italy

Photoactivated chemotherapy (PACH) has been attracting attention as a potential novel therapy of cancer. In particular, Pt(IV) complexes have been utilized in this approach.[1,2] However, the need for relatively short wavelength light for activation of transition metal complexes to cytotoxic species limits wider applications because only superficial tumors can be treated due to the short penetration distances of light into tissues. By converting two or more longer wavelength photons into one photon of shorter wavelength, upconverting nanoparticles (UCNP) promise to help overcome this problem. Thus, light of longer wavelength, such as near infrared (NIR) radiation, could penetrate deeper into tumor tissues, become upconverted by nanoparticles to shorter wavelength, and selectively activate a nearby metal complex to a reactive anticancer drug. In this poster, we present two strategies to develop photoactivatable diiodido-Pt(IV) diamines coupled in various ways to UCNPs (Figure). Diiodido-Pt(IV) diamines were the first Pt complexes described that can be photoactivated to cytotoxic species[3], thus making them interesting candidates for UCNP assisted PACH. Our results show that NIR is able to facilitate the release of reactive Pt species from both forms of the modified UCNPs, which platinate calf thymus DNA. Moreover, NIR potentiates the cytotoxic activity of UCNPs in cell culture.[4]

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Acknowledgements:This work was supported by European Research Council (BioIncmed 247450) and the Italian Ministero dell’Università (PON01_01078 and FIRB RINAME RBAP114AMK).

References: 1. Bednarski, P. J.; Mackay, F. S.; Sadler, P. J.: Anti-Cancer Agents Med.Chem. 2007, 7: 75-93. 2. Farrer, N. J.; Salassa, L.; Sadler P. J.: Dalton Trans 2009: 10690-10701. 3. Kratochwil, N. A. et al.:J. Med. Chem. 1996, 39: 2499-2507. 4. Perfahl, S. et al.: Mol. Pharmaceut. (in press).

POS.56

Lung Cancer: EGFR Inhibitors with Low Nanomolar Activity against a Therapy-Resistant L858R/T790M/C797S Mutant Günther, M.1; Juchum, M.1; Kelter, G.2; Fiebig, H.2; Laufer, S. A.1 1 University of Tuebingen, Faculty of Science, Pharmaceutical and Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tuebingen, Germany 2 Cell Biology & Compound Screening Oncotest GmbH, Am Flughafen 12 – 14, 79108 Freiburg, Germany

The treatment of non-small-cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) inhibitors is made challenging by acquired resistance caused by somatic mutations [1]. Third generation EGFR inhibitors (WZ4002, Osimertinib) have been designed to overcome resistance, mediated by the T790M mutation, through covalent binding to the Cys797 residue of the enzyme. These inhibitors are effective against most clinically relevant EGFR mutations, however their high dependence on this particular interaction means that additional mutation of Cys797 results in poor inhibitory activity, which leads to tumor relapse in initially responding patients [2,3].

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Based on a selectivity screening of a highly potent reversible p38 inhibitor [4], we identified EGFR inhibition as an off-target effect of this compound. High potency, as well as moderate physicochemical properties and cellular activity against p38, led us to pick this compound as a lead structure for further improvements in terms of mutant EGFR inhibition. With this concept, we have successfully developed highly potent reversible and irreversible T790M EGFR inhibitors that showed picomolar IC50-values in an enzyme assay and down to 18 nM GI50 in a double mutant (L858R/T790M) cellular assay. In contrast to classic third generation EGFR inhibitors, some of these compound showed high inhibitory activity in the low nanomolar range against the therapy-resistant L858R/T790M/C797S EGFR triple mutant [5]. References: 1. Juchum, M. et al.: Drug Resist. Updat. 2015, 20: 12-18. 2. Piotrowska, Z. et al.: Cancer Discov. 2015, 5: 713-722. 3. Thress, K.S. et al.: Nat. Med. 2015, 21: 560-562. 4. Selig, R. et al.: J. Med. Chem. 2012, 55: 8429-8439. 5. Günther, M. et al.: Angew. Chem. Int. Ed., 2016, in press, doi:10.1002/anie.201603736

POS.57

Alkoxyurea-based histone deacetylase inhibitors increase cisplatin chemosensitivity Stenzel, K.1; Hamacher, A.1; Hansen, F. K.1; Gertzen, C. G. W.1; Leven, M.1; Marek, L.1; Senger, J.3; Marek, M.4; Romier, C.4; Jung, M.3; Gohlke, H.1; Kassack, M. U.1; Kurz, T.1

1 Institut für Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universität, Universitätsstr. 1, 40225 Düsseldorf, Germany. 2 Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104 Freiburg, Germany. 3 IGBMC, Universite de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch Cedex, France.

Histone deacetylase inhibitors (HDACis) belong to an emerging class of anticancer compounds which cause growth arrest and apoptosis of several tumor cells.[1] It is increasingly recognized that the combination of HDACis with established anticancer drugs (e.g. cisplatin) provides synergistic effects in the treatment of hematological and solid tumors, probably generated through HDACi-mediated increased accessibility of DNA.[2,3] Starting from LMK235, a HDACi with HDAC4 and 5 preference[4], we reasoned that the enlargement of the cap group and the connecting unit should lead to a novel type of HDACi with HDAC6 preference (Figure 1). The lead structure LMK214 (N-Hydroxy-6-((3-(quinolin-3-yl)ureido)oxy)hexanamide) of the novel compound library showed potent inhibition of HDAC6 and no inhibition of HDAC4 and 8 up to a concentration of 10 µM. Based on these preliminary results we performed a docking study in order to understand the selectivity profile of compound LMK214 and to design improved analogues. Here we describe the synthesis of new potent hydroxamate-based HDAC6 preferential inhibitors with a novel alkoxyurea connecting unit linker region. A microwave-assisted protocol allowed the systematic variation of the cap moiety. The biological evaluation of the target compounds included cellular HDAC and MTT assays on Cal27 (human tongue squamous carcinoma cell line) and A2780 (human ovarian cancer cell line) as well as their cisplatin resistant sublines. Some compounds showed improved effects on inhibition of cellular HDACs in a whole-cell HDAC assay compared to vorinostat. Based on their antiproliferative effects and HDAC inhibition, the three most potent compounds were selected for isoform profiling against human HDAC1, HDAC6 and HDAC8. The most promising compound demonstrated excellent selectivity over HDAC8 (SI: 550), moderate selectivity over HDAC1 (SI: 15) and highly improved HDAC6 inhibition of 2.8 nM. Furthermore, drug combination studies with cisplatin revealed for all three selected compounds a markedly enhancement of cisplatin-induced cytotoxicity and a synergistic antitumor effect with CI < 0.9 in the cell lines Cal27 and Cal27 CisR. These effects were more pronounced for the cisplatin resistant subline Cal27 CisR.

Fig. 1.: Strategy and target compounds

Acknowledgments: J. Senger and M. Jung thank the Deutsche Forschungsgemeinschaft (DFG) for funding. (Ju295/9-2 within SPP1463, Ju295/10-2 within CRU201; Ju295/13-1).

References: 1. Witt, O. et al.: Cancer Lett. 2009, 277(1): 8–21. 2. Ong, P.-S. et al.: Int. J. Oncol. 2012, 40(5): 1705–1713. 3. Stiborova, M. et al.: Curr Med Chem. 2012, 19(25): 4218–38. 4. Marek, L. et al.: J. Med. Chem. 2013, 56(2): 427–436.

POS.58/SL.18 α-Aminoxy peptides: from membranolytic anticancer foldamers to the first in class peptidomimetic Hsp90 C-terminal domain dimerization inhibitors Diedrich, D.1; Bhatia, S.2; Frieg, B.1; Stein, S.3; Bopp, B.4; Lang, F.2; Ernst, T.5; Rodrigues Moita, A. J.1; Rüther, A.6; Lüdeke, S.6; Kassack, M. U.1; Hochhaus, A.5; Borkhardt, A.2; Jose, J.4; Kurz, T.1; Gohlke, H.1; Hauer, J.2; Hansen, F. K.1

1 Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Düsseldorf, Germany. 2 Department of Pediatric Oncology, Hematology and Clinical Immunology, Heinrich Heine University Düsseldorf, Medical Faculty, Düsseldorf, Germany. 3 Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Germany. 4 Institute for Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westphalian Wilhelms-University, Münster, Germany. 5 Klinik für Innere Medizin II, Universitätsklinikum Jena, Erlanger Alle 101, 07747 Jena. 6 Institute of Pharmaceutical Sciences, University of Freiburg, Albertstraße 25, Freiburg, Germany.


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POS.59

A fluorescent oxaliplatin analogue sheds light on the mechanism of action of the drug and the mechanisms of resistance Kalayda, G. V.1; Bosman, I.1; Gollos, S.2; Kullmann, M.1; Hamacher, A.3; Sarin, N.1; Galanski, M.4; Kassack, M. U.3; Müller, C. E.2 1 Department of Clinical Pharmacy, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 2 Department of Pharmaceutical Chemistry I, Institute of Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 3 Institute of Pharmaceutical and Medicinal Chemistry, University of Düsseldorf, Universitätsstr. 1, 40225 Düsseldorf, Germany 4 Institute of Inorganic Chemistry, University of Vienna, Währinger Strasse 42, 1090 Vienna, Austria

Oxaliplatin is a third-generation platinum-based anticancer drug distinguished by its activity in colorectal cancer, which is intrinsically resistant to the other platinum chemotherapeutics cisplatin and carboplatin. The development of acquired chemoresistance in the course of treatment with oxaliplatin is, however, common. The mechanism of action of cisplatin and the mechanisms of cancer cell resistance to this drug have been extensively studied. In contrast, only little is known about oxaliplatin. This study focused on the investigation of the cellular uptake and intracellular transport of oxaliplatin using an analogue featuring a fluorescent tag. We have successfully synthesized and fully characterized an oxaliplatin derivative CFDA-oxPt bearing a carboxyfluorescein-diacetate in the position 4 of the cyclohexane ring. In the sensitive human ileocecal colorectal adenocarcinoma cell line HCT-8 and in oxaliplatin-resistant variant HCT-8ox, the cytotoxicity of CFDA-oxPt was lower compared to the parent drug as revealed using an MTT-based assay (oxPt, HCT-8: EC50 = 4.87 µM; oxPt, HCT-8ox: EC50 = 92.4 µM; CFDA-oxPt, HCT-8: EC50 = 26.5 µM; CFDA-oxPt, HCT-8ox: EC50 = 865 µM). Nevertheless, oxaliplatin-resistant HCT-8ox cells were found cross-resistant to CFDA-oxPt. The platinum-free label CFDA was not toxic up to 1 mM in both cell lines. These results indicate that CFDA-oxPt represents a suitable model to investigate the mechanism of action of oxaliplatin and mechanisms of resistance. The study of the cellular processing of CFDA-oxPt using confocal laser scanning microscopy revealed extensive accumulation of the drug in the plasma membrane of the sensitive cells without any nuclear accumulation visible. In oxaliplatin-resistant cells, CFDA-oxPt localized to the plasma membrane and vesicular structures. In order to further investigate the differences between the sensitive and oxaliplatin-resistant cells, cellular accumulation of oxaliplatin was studied in more detail. Passive diffusion was found an important component of oxaliplatin uptake in both cell lines. Inhibition of copper transporter 1 (CTR1) by CuSO4 led to a significant reduction of oxaliplatin accumulation in HCT-8 and HCT-8ox cells as measured by atomic absorption spectrometry. Atropine, a potent inhibitor of organic cation transporter 1 (OCT1) significantly decreased oxaliplatin accumulation but only in the sensitive cell line. Inhibition of organic cation transporter 2 (OCT2) with cimetidine significantly reduced oxaliplatin accumulation in sensitive and resistant cells. Decynium-22, an inhibitor of organic cation transporter 3 (OCT3) had no effect on oxaliplatin accumulation in both cell lines. Plasma membrane accumulation of CFDA-Pt is being further investigated using co-localization experiments between the oxaliplatin analogue and the transport proteins mentioned above. CTR1 is abundantly present in both cell lines but its expression is not significantly different between sensitive and resistant cells as revealed by qRT-PCR and Western Blot. Also the expression of organic cation transporters is similar in both cell lines. OCT2 is expressed to a marginal extent in HCT-8 and HCT-8ox cells. Thus, the oxaliplatin derivative with a fluorescent tag (CFDA-oxPt) has revealed plasma membrane accumulation in both cell lines and additional vesicular accumulation in oxaliplatin-resistant cells. The latter may indicate the mechanism of oxaliplatin resistance. CTR1 and OCT1 are likely involved in oxaliplatin accumulation in the sensitive cells but only CTR1 appears to mediate active uptake in resistant cells. The contribution of OCT2 appears to be marginal. Acknowledgements: The authors acknowledge the financial support by the Deutsche Forschungsgemeinschaft.

POS.60

Targeting cell membrane characteristics by soraphen A: A novel therapeutic option to fight cancer Stoiber, K.1,4; Winzi, M.2; Pernpeintner, C.3,4; Koeberle, A. 5; Ulrich, M.1; Werz, O.5; Müller, R.6; Zahler, S.1; Lohmüller, T.3,4; Guck, J.2; Feldmann, J.3,4; Vollmar, A. M.1,4; Braig, S.1 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich, Munich, Germany. 2 Biotechnology Center, Technische Universität Dresden, Tatzberg 47/49, 01307, Dresden, Germany 3 Photonics and Optoelectronics Group, Department of Physics and Center for Nanoscience, Ludwig-Maximilians-University of Munich, Amalienstr. 54, 80799, Munich, Germany. 4 Nanosystems Initiative Munich (NIM), Schellingstraße 4, 80799, Munich, Germany. 5 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, Philosophenweg 14, 07743 Jena, Germany. 6 Department of Microbial Natural Products, Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany.

Albeit cellular membranes exert important functions in signaling, transport and recycling processes, targeting membrane characteristics by using pharmacologically attractive compounds in anti-cancer therapy has not been addressed so far. Here we employed the acetyl-CoA carboxylase inhibitor soraphen A as a tool to study the effects of a disturbed phospholipid homeostasis on membrane characteristics and its functional consequences on cancer progression. By using state-of-the-art biophysical methodologies such as real-time deformability cytometry, optical tweezers measurements of giant plasma membrane vesicles and fluorescence recovery after photobleaching analysis we could demonstrate that the membrane deformability and fluidity of cancer cells are disturbed after soraphen A treatment. Sophisticated imaging techniques such as proximity ligation assays revealed that soraphen A abrogates recycling processes and modulates the dimerization of growth factor receptors located in the membrane. Consequently, HER2, Src and EGFR receptor signaling cascades are inhibited leading to diminished breast tumor growth in vitro and in vivo. Thus, soraphen A might represent a promising lead substance for anti-cancer agents interfering with the so far undescribed field of membrane properties.

POS.61

Targeting cholesterol metabolism in hepatocellular carcinoma – V-ATPase inhibition as a novel therapeutic option Bartel, K.1; Winzi, M.2; Ulrich, M.1; Koeberle, A.3; Menche, D.4; Werz, O.3; Müller, R.5; Guck, J.2; Vollmar, A. M.1; von Schwarzenberg, K.1 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich 2 Biotechnology Center, Technische Universität Dresden 3 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich Schiller University Jena 4 Kekulé Institute of Organic Chemistry and Biochemistry, University of Bonn 5 Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University

Hepatocellular carcinoma (HCC) is still a major cause of cancer-related death worldwide, however therapy options are limited, leaving a pressing need for new therapies. Regarding HCC, cellular lipid and cholesterol metabolism is gaining interest, as high cholesterol levels are beneficial for proliferation tumor progression [1,2]. Cholesterol is essential for membrane-related mitogenic signaling and increased cell softness of cancer cells, which increases malignancy and invasive potential. Despite intensive research , targeting cholesterol metabolism effectively still remains challenging. We provide evidence, that the vacuolar ATPase (V-ATPase) inhibitor archazolid (arch), which was recently shown to interfere with cholesterol metabolism, has a major impact on the mechanical phenotype of HCC. Archazolids restrictes cellular access to free chol caused by lysosomal trapping through V-ATPase inhibition. Thereby it increases cell stiffness and membrane polarity selectively in HCC, while leaving hepatocytes unaffected and as a consequence inhibits membrane-related Ras signaling leading to impaired proliferation in vitro and in vivo. Hence, V-ATPase inhibition represents a novel, central link between cell biophysical properties and proliferative signaling in malignant HCC cells, providing the basis for an attractive and innovative strategy against HCC.

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Fig.1: Proposed mechanism of action of archazolid in HCC. (A) Under physiological conditions low-density lipoprotein (LDL) binds to its receptor (LDL-R) and is internalized. In the acidic environment of the lysosome, provided by the V-ATPase LDL dissociates from the receptor and is cleaved. Free cholesterol is released and integrated into membranes. Ras is a membrane-bound small GTPase mainly activated in cholesterol-enriched membrane microdomains. Ras in turn promotes proliferation and survival. (B) Archazolid inhibits the V-ATPase and thereby acidification of the endo-lysosome leading to cholesterol accumulation. Cholesterol is depleted from the membrane and cholesterol-enriched microdomains are disrupted, changing membrane properties. As a counsequence Ras activation and downstream signalling is disrupted, leading to reduced proliferation.

Acknowledgements:This work was supported by the DFG research funds 1406 SCHW 1781/1-1 and AV 376/18-1 and by HTCR, a non-profit foundation under German civil law, which facilitates research with human tissue by providing an ethical and legal framework for prospective sample collection and the Alexander von Humboldt foundation (Alexander von Humboldt Professorship to JG).

References: 1. Gorin, A.; Gabitova, L; Astsaturov, I.: Current opinion in pharmacology 2012, 12: 710-716, doi:10.1016/j.coph.2012.06.011. 2. Fages, A. et al.: BMC medicine 2015, 13: 242, doi:10.1186/s12916-015-0462-9.

POS.62

Migration Inhibition by Actin Binding Compounds Beyond Simple Actin Polymerization Moser, C.1; Kazmaier, U.2; Zahler, S.1; Vollmar, A. M.1 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilian University, Butenandtstraße 5-13, 81377 Munich, Germany 2 Institute for Organic Chemistry, Saarland University, 66041 Saarbrücken, Germany

An astonishing number of natural compounds interfere with the cytoskeleton of mammalian cells. Compounds targeting the microtubules like vinca-alkaloids or taxanes, are extensively studied and used for cancer therapy. In contrast, knowledge about pharmacological properties of actin binding drugs is poor and drugs interfering with actin are far from clinical use. On the other hand, the role of actin cytoskeleton in cancer related processes such as migration and invasion as well as apoptosis and proliferation of tumor cells is well accepted. Unfavourable cytotoxicity or other limitations such as the supply of respective natural compounds might be a reason, which, however, could be overcome by synthetically accessible natural compounds. One promising option along this line might be the use of Miuraenamide (Miu), a cyclic depsipeptide isolated from a halophilic myxobacterium, previously synthesized by the Kazmaier group [1] and shown to induce actin hyperpolymerization and disruption of the cytoskeleton (CSK). Our working hypothesis was that actin binding agents are able to evoke distinct pharmacological effects beyond an obvious interruption of the actin CSK, when administered in subtoxic concentrations over time. We proposed that a subtoxic concentration of Miu leads to inhibition of migration of invasive tumor cells, such as ovarian cancer cell, driven by changes in transcriptional activity. In fact, first experiments showed an inhibition of cell migration after 72h Miu treatment, at a subtoxic concentration, without obvious disruption of the actin CSK. Interestingly, this inhibition was independent of cancer cell adhesion, spreading and motility. Of note, migration was only inhibited in the setting of a chemotactic Boyden chamber assay, where the cells have to squeeze through narrow pores, but not in mechanical deformation independent 2D scratch assay. Moreover the examination of the transcription level of Miu treated ovarian cancer cells indicated significantly regulated genes, which are supposed to be important in migration, e.g., structural proteins and integrins. In sum we have evidence that actin binding agents such as Miu are able to induce distinct changes in transcriptional activity, when employed in concentrations which do not evoke an instant and pronounced effect on actin polymerisation. Whether these changes in

gene expression are responsible for a specific effect on migration of tumor cells, will be subject of future investigations. Acknowledgments: This work was supported by the Deutsche Forschungsgemeinschaft (FOR 1406)

References: 1. Karmann, L. et al.: Angew. Chem. Int. Ed. Engl. 2015, 54(15): 4502-7.

POS.63

Effects of the actin binding compound Miuraenamide A on mechanosensitive gene expression in endothelial cells Gegenfurtner, F. A.1; Müller, R.2; Kazmaier, U.3; Vollmar, A. M.1; Zahler, S.1 1 Ludwig-Maximilians-University Munich, Department of Pharmacy, Institute of Pharmaceutical Biology, Germany, Email: [email protected] 2 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) and Institute of Pharmaceutical Biotechnology, Saarland University Campus, 66123 Saarbrücken, Germany 3 Institute for Organic Chemistry, Saarland University, 66041 Saarbrücken, Germany

Apart from its classic structural and transport-related tasks in the cell, the actin cytoskeleton recently emerges as an important regulatory element of gene expression in a variety of mammalian cells. For example, it has been reported that nuclear actin participates in chromatin remodeling and directly binds to RNA Polymerases I, II and III. Furthermore, actin is involved in the regulation of mechanosensitive transcription factors such as MRTF-A and the Hippo-YAP axis[1]. Regarding the formation of new blood vessels, this is particularly important since the actin cytoskeleton undergoes constant structural remodeling in response to changing parameters of the cellular microenvironment. Consequently, both MRTF and YAP are being discussed as potential key players in angiogenesis[2,3]. By using the myxobacterial actin-binding compound Miuraenamide A as a chemical tool, we examine the functional role of nuclear actin and the consequences on gene expression in endothelial cells with a focus on the mechanosensitive transcription factors MRTF and YAP. We show that Miuraenamide A is a potent inducer of MRTF-A nuclear translocation and subsequent SRF target gene expression in HUVEC. In contrast, both, the Hippo-YAP axis, which shares a variety of target genes with the SRF-MRTF pathway, and basal transcriptional activity remain largely unaffected by our compound. On a structural level, we demonstrate that stimulation with Miuraenamide A causes pronounced nuclear deformation accompanied by the formation of nucleolar actin aggregates and an increase in heterochromatic regions. In summary, we characterize Miuraenamide A as an activator of mechanosensitive signaling pathways with a marked preference for the MRTF-SRF axis in endothelial cells. We furthermore find that Miuraenamide A provokes the remodeling of chromatin and nuclear actin structures, suggesting a regulatory influence of actin on gene expression beyond the modulation of MRTF or YAP, which will be the subject of further investigations. Acknowledgments:This work was funded by the DFG

References: 1. Hendzel; M. J.: Curr Opin Cell Biol 2014, 28: 84-9. 2. Franco, C. A. et al.: Development 2013. 3. Choi, H. J. et al.: Nat. Commun. 2015.

POS.64/SL.04 Extracellular vesicles as smart carriers for small molecule drugs Fuhrmann, G.1,2; Serio, A.2; Stevens, M. M.2 1 Helmholtz Centre for Infection Research, Helmholtz Institute for Pharmaceutical Sciences Saarland, Saarland University, Campus Building E8.1, 66123 Saarbrücken, Germany

2 Imperial College London, Department of Materials, Department of Bioengineering, Prince Consort Road, SW7 2AZ London, United Kingdom


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POS.65

Protein-based nanoparticles for drug delivery applications Fach, M.1; Radi, L.1; Wich P. R.1 1 Johannes Gutenberg Universität-Mainz, Institut für Pharmazie und Biochemie, Staudingerweg 5, 55128 Mainz, Germany

Modern drug delivery systems for therapeutic applications are very similar in size and shape to biological nanostructures with comparable functions, like exosomes or viruses. Polymer-based particles are in particular interesting as drug, vaccine and gene delivery systems because they can be easily chemically modified [1] but they often lack biocompatibility and degradability. Alternatives are nature’s biopolymers that are readily accessible, can be obtained in high quantity, are structurally well defined and trigger in most cases only a low reaction of the immune system. [2] Especially proteins have interesting properties as structurally well-defined biopolymers for the formation of nanoparticles. They have several advantages over synthetic polymers, including biodegradability, non-antigenicity, stability, binding capacity and toxicity. Because of these unique properties, protein-based nanocarriers are promising candidates for the delivery of drugs and genetic material. [3] Here, we present a new approach for the preparation of protein-based nanoparticles for the delivery of hydrophobic therapeutics. We are creating a protein-polymer conjugate that can be used in solvent evaporation methods for the formation of stable nanoparticles. We obtain empty and drug-loaded nanoparticles with a size of around 100 nm that are stable in different physiological environments. The produced nanoparticles are non-toxic but do not effect of the activity of the encapsulated drug. Additionally, we transferred the method of NP preparation to various proteins in different sizes showing that this method can be universally applied to any protein of choice. [4,5] This novel method for nanoparticle preparation extends the range of biocompatible materials beyond current known synthetic polymers and polysaccharides and potentially opens up new strategies for therapeutic applications.

References: 1. Sapsford, K. E. et al.: Chem. Rev. 2013 113(3), 1904-2074. 2. Zolnik, B. S. et al.: Endocrinology 2010 151(2), 458-465. 3. Elzoghby, A. O. et al.: J. Controlled Release 2012 157(1), 38-49. 4. Radi, L. et al.: Med. Chem. Commun. 2016, Advance Article, DOI: 10.1039/c5md00475f. 5. Fach, M; Radi, L; Wich, P. R.: J. Am. Chem. Soc. 2016, Article ASAP, DOI: 10.1021/jacs.6b06243.

POS.66

L-tyrosine-modification of low molecular weight PEIs increases siRNA knockdown efficacies in vitro and in vivo Ewe, A.1; Przybylski, S.2; Burkhardt, J.2; Aigner, A.1

1 Rudolf-Boehm-Institute for Pharmacology and Toxicology, Clinical Pharmacology; Leipzig University, Härtelstrasse 16-18, D-04107 Leipzig, Germany 2 Fraunhofer Institute for Cell Therapy and Immunology (IZI), Perlickstrasse 1, D-04103 Leipzig, Germany

The non-viral delivery of small RNA molecules like siRNAs for specific gene silencing still poses a major hurdle for their use in vivo. Their successful therapeutic application is thus dependent on the development of save and efficient delivery strategies. Polyethylenimines (PEIs) are cationic polymers that are well established for the delivery of nucleic acids, but major concerns have been raised with regard to their limited efficacy and inherent toxicity. On the other hand, the presence of reactive amines in PEI molecules allows for chemical modifications, and thus offers new opportunities for more efficient and safer delivery vectors. One promising strategy is based on the introduction of amino acids. Recently, amino acid modifications of the more toxic 25 kDa branched PEI have shown improvement for nucleic acid delivery.

However, PEIs of lower molecular weight and hence higher biocompatibility have not been explored so far. We synthesized a series of three novel tyrosine-modified 2 kDa, 5 kDa and 10 kDa branched PEIs (termed “PxY” with x = molecular weight of parent PEI), and for comparison the 25 kDa branched tyrosine-modified PEI, via an amide linkage. All tyrosine-modified PEIs were initially characterized for optimal siRNA complexation conditions, complex size and zeta potential. Biological activities were first determined in reporter cell lines stably expressing luciferase or eGFP. All PEIs benefitted from the tyrosine modification, with knockdown efficacies of ~ 80 % for the P5Y, P10Y, P25Y and 50 % even for P2Y. Importantly, the tyrosine modification strongly enhanced the knockdown efficacies and required lower amounts of the polymer as compared to the parent PEIs. Another critical aspect regarding PEI/siRNA complexes is their colloidal instability in buffers and the presence of serum. Notably, incubation of the tyrosine-PEI/siRNA complexes in the presence of different serum concentrations revealed the absence of particle aggregation. In addition, we also identified suitable conditions, e.g. freezing and lyophilisation, for long-term storage without loss of biological activity. Beyond reporter cell lines, the use of our tyrosine-modified PEI derivatives was extended towards the therapeutically more relevant siRNA-mediated targeting of the endogenous anti-apoptotic oncogene survivin in different carcinoma cell lines. Transfection experiments led to a marked >60% reduction of cell proliferation over negative control transfected cells upon siSurvivin delivery mediated by the 5 kDa to 25 kDa modified PEIs. This was based on a >70% survivin knockdown, as determined by RT-qPCR. The profound survivin knockdown thus also demonstrated the biological efficacy of tyrosine-PEI/siRNA complexes in targeting endogenous, (patho-) physiologically overexpressed target genes. The therapeutic application of non-viral RNAi in vivo requires the delivery of intact siRNAs into the target tissue. Initially, the P10Y was selected and comprehensively characterized in a pre-clinical in vivo study in tumour xenograft-bearing mice. The intact delivery of P10Y/siRNA complexes into various tissues and the xenograft tumours upon systemic (intraperitoneal or intravenous) injection was demonstrated in a radioactive biodistribution assay. Toxicity studies demonstrated that the repetitive administration of P10Y/siRNA caused no adverse effects such as hepatotoxicity, immunostimulation, alterations in the immunophenotype or weight loss. Finally, the therapeutic potential of the P10Y-mediated delivery of siRNAs was analysed in a melanoma xenograft tumour mouse model. The systemic application of P10Y/siRNA complexes targeting survivin demonstrated profound inhibition of tumour growth and down-regulation of the target oncogene. Taken together, we demonstrate that the simple modification of PEI with the aromatic amino acid L-tyrosine strongly enhances the biological activity of low molecular weight PEIs in vitro compared to their parent PEIs. The modification prevents serum-induced particle aggregation as one major critical factor for in vivo applications. The first in vivo study with the 10 kDa derivative (P10Y) demonstrates the high biocompatibility in mice, the efficient delivery of intact siRNA and the applicability of P10Y/siRNA complexes as RNAi therapeutic. Acknowledgements: This work was in part supported by grants from the Saxonian Ministry of Science and Art (SMWK) and the Deutsche Krebshilfe (grant number 11616) to A.A., and by the German Ministry of Education and Research (BMBF, grant # 1315883).

References: 1. Creusat, G.; Zuber, G.: Chembiochem : a European journal of chemical biology 2008, 9: 2787-2789. 2. Ewe, A. et al.: J Control Release 2016, 230:13-25. 3. Ewe, A. et al.: Drug Deliv Transl Res 2016 [Epub ahead of print]

POS.67 Inhibition of Cdk5 – a novel strategy to address cancer stem cells Mandl, M. M.1; Zhang, S.1; Ulrich, M.1; Schmoeckel, E.2; Mayr, D.2; Vollmar, A. M.1; Liebl, J.1 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-University of Munich (LMU), Munich, Germany 2 Institute of Pathology, Ludwig-Maximilians-University of Munich (LMU), Munich, Germany

Cancer stem cells (CSCs) represent a major problem in tumor therapy as they account for chemoresistance, tumor recurrence and metastasis. However, strategies to address CSCs are limited. In this context, we

CANCER/INFLAMMATION


hypothesize the serine/threonine kinase cyclin dependent kinase 5 (Cdk5) as a particular interesting and promising player to explore. Cdk5 exerts pivotal functions in the neuronal system and is associated with neurodegenerative diseases. During the last years, important non-neuronal functions of Cdk5 have been elucidated and recent studies indicate Cdk5 as a promising target for anti-cancer therapy. Although Cdk5 has recently been associated with epithelial-mesenchymal transition (EMT), a role of Cdk5 in CSCs has not been described yet. In fact, here we suggest a function of Cdk5 in CSCs by showing that knockdown and pharmacological inhibition of Cdk5 impaired tumorsphere formation and reduced tumor establishment in vivo. Conversely, Cdk5 overexpression promoted tumorsphere formation which was in line with increased expression of Cdk5 in human breast cancer tissues as shown by staining of a human TMA. In order to understand how Cdk5 inhibition affects tumorsphere formation, we identify a role of Cdk5 in detachment induced cell death: Cdk5 inhibition induced apoptosis in tumorspheres by stabilizing the transcription factor Foxo1 which results in increased levels of the pro-apoptotic protein Bim. In summary, our study elucidates a Cdk5-Foxo1-Bim pathway in cell death in tumorspheres and suggests Cdk5 as a potential target to address CSCs.

POS.68

Disturbing mitochondrial proteostasis by inhibition of Clp complex as an anti-cancer strategy Mandl, M. M.1; Gronauer, T.2; Fetzer, C.2; Stahl, M.2; Vollmar, A. M.1; Sieber, S. A.2; Liebl, J.1 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians-Universität München (LMU), Butenandtstraße 5-13, Munich, D-81377, Germany 2 Center for Integrated Protein Science at the Department of Chemistry, Technische Universität München, Lichtenbergstraße 4, Garching, D-85747, Germany

The mitochondrial matrix protease Clp is implicated in ageing and disease. By regulating the mitochondrial proteostasis and the mitochondrial unfolded protein response (UPRmt) Clp is a key player for the maintenance of mitochondrial function. Although it has been found lately that Clp subunit P (ClpP) is upregulated in a majority of Acute Myeloid Leukemia cells and its activity has been linked to cisplatin resistance, our knowledge of the function of Clp complex in cancer is still limited. Neither general mechanisms of action of its inhibition, nor the signaling from mitochondria to nucleus and substrates in mammalian cells, especially in cancer, have been investigated yet. According, the aim of the present study was to characterize the function of Clp as a potential new target for anti-cancer therapy. First, we found ClpP and ClpX subunits to be expressed in various cancer cell lines including myeloid and lymphatic leukemia, breast and liver derived cancer cell lines. To evaluate its therapeutic relevance, we used novel Clp complex inhibitors, performed profound pharmacological testing in several functional cell-based assays and found that Clp inhibition affected cell viability of leukemia, breast and liver cancer cells. Additionally, as an early event, ATP production was decreased pointing to a direct effect on mitochondria. Furthermore, Clp inhibition led to an induction of apoptosis and exerted chemosensitizing effects in leukemia cells towards chemotherapeutics including Imatinib and Etoposid. In order to validate Clp as a therapy target and to characterize the mechanism of action of the inhibitors, we observed the induction of mitochondrial ROS production and common hallmarks of the intrinsic apoptosis mechanism, like the loss of mitochondrial membrane potential, PARP cleavage or Caspase activation. Clp inhibition was also found to be effective in patient-derived leukemia cells. Currently, we are focusing on the general mode of action of Clp inhibition, such as, for instance, the impact on the mitochondrial proteome using stable isotope labeling with amino acids in cell culture (SILAC). In addition, identification of peptides released from mitochondria under stress conditions and Clp inhibition using a mass spectrometry-based approach should expand knowledge about UPRmt signaling and specific Clp substrates. Moreover, the establishment of an in vivo model should further promote Clp inhibition as a promising strategy in cancer therapy and may even improve treatment of resistant cancer cells.

POS.69

Inhibition of Cdk5 – a novel strategy to improve Sorafenib response in HCC therapy Ardelt, M. A.1; Fröhlich, T.2; Lehr, T.3; Wojtyniak, J.-G.3; Arnold, G. J.2; Vollmar, A. M.1; Liebl, J.1 1 Department of Pharmacy, Pharmaceutical Biology, Ludwig-Maximilians University, 81377 Munich, Germany 2 Laboratory for Functional Genome Analysis, LAFUGA, Gene Centre, LMU, 81377 Munich, Germany 3 Clinical Pharmacy, Saarland University, 66123 Saarbrücken, Germany

Introduction: For the treatment of advanced hepatocellular carcinoma (HCC) Sorafenib is the only approved systemic therapy. Unfortunately patients only gain a survival benefit of about 3 months and therefore there is a great need for novel systemic treatment option or combinational treatments.[1] Recently we identified cyclin dependent kinase 5 (Cdk5), an atypical kinase, as a novel target in HCC therapy: We found that Cdk5 was frequently overexpressed in HCC and rendered HCC cells sensitive for the treatment with DNA damaging agents by regulating DNA damage response.[2] The aim of this study was to investigate whether Cdk5 inhibition could also sensitize HCC cells for Sorafenib treatment and thereby enhance the tumour growth inhibition and to elucidate the corresponding mechanism. Methods: To evaluate the impact of Cdk5 inhibition on Sorafenib treatment we used the pharmacological Cdk5 inhibitors roscovitine and dinaciclib as well as genetic downregulation of Cdk5 via shRNA in different cell based assays and mouse xenograft models. For the investigation of Cdk5 function and signalling in the context of Sorafenib treatment we used a proteomics screen of whole cell lysates of Sorafenib treated HCC cells with or without Cdk5 knockdown. Results: In fact, the combination of Sorafenib with a functional ablation of Cdk5 synergistically inhibited the proliferation and clonogenic survival of HCC cells in vitro. This synergism could also be confirmed in a xenograft mouse model, where a combinational therapy resulted in a significantly reduced tumour growth rate. Moreover, we could show that Cdk5 inhibition not only reduced overall migration but also prevented the induced cancer cell migration caused by sub therapeutic concentrations of Sorafenib. The proteomics screen revealed on the one hand, that Cdk5 knockdown cells seem to have deregulated metabolic activity and on the other hand that the epidermal growth factor receptor (EGFR) is connected to Cdk5. Our further investigations could show that Cdk5 is involved in EGFR signalling and trafficking and prevents the activation of the PI3K/Akt pathway, which was shown to be caused by compensatory activation of EGFR.[3] Conclusion: The pharmacological inhibition of Cdk5 with drugs, which are already clinically used in the treatment of other diseases, offers a new concomitant therapy option for patients treated with Sorafenib. The combination has the benefit of not only inhibiting tumour cell growth to a greater extent but also impeding tumour cell motility compared to Sorafenib single treatment. To conclude, our work provides evidence for Cdk5 inhibition as a new promising strategy to improve the therapeutic effect of Sorafenib in HCC. Acknowledgements: Financial support by DFG (VO376/17-1) is gratefully acknowledged.

References: 1. Llovet, J. M. et al.: N Engl J Med 2008, 358: 378-390. 2. Ehrlich, S. M. et al.: J Hepatol 2015, 63: 102-113. 3. Blivet-Van Eggelpoel, M. J. et al.: J Hepatol 2012, 57: 108-115.

POS.70

Characterization of Sorafenib Resistance and Rebound Growth in Hepatocellular Carcinoma Cells Meßner, M.1; Ardelt, M. A.1; Meyer, K.2; Fröhlich, T.2; Vollmar, A. M.1; Liebl, J.1

1 Department of Pharmacy, Pharmaceutical Biology, LMU, Butenandtstr. 5-13, 81377 Munich, Germany 2 Laboratory for Functional Genome Analysis, LAFUGA, Gene Centre, LMU, Feodor-Lynen-Straße 25, 81377 Munich, Germany

Introduction: Sorafenib represents the current standard of care for patients with advanced stage hepatocellular carcinoma (HCC) based on two recent phase III trials that reveal an overall survival benefit of three months compared to placebo [1]. Nevertheless, its use is hampered by the occurrence of drug resistance. Further, up to 80% of patients

POSTERS


treated with sorafenib suffer from side effects necessitating dose reduction, short-term “drug holidays” or permanent treatment termination [2]. In order to improve sorafenib therapy, the aim of the study was to elucidate the molecular basis for this drug resistance in general. In addition we studied the effect of sorafenib withdrawal as rebound phenomena have been reported in the context of anti-angiogenic therapy abrogation. Methods: To investigate the molecular mechanisms involved in sorafenib resistance we developed a sorafenib resistant human liver cell line in which we studied morphology, protein expression as well as proliferative and migratory potential under sorafenib exposure and withdrawal conditions. The effects of sustained sorafenib exposure were additionally investigated on protein level by means of a mass spectrometry-based proteomics analysis. Results: The sorafenib resistant human liver cells changed their morphology to elongated spindle-shaped cells, lost E-Cadherin and showed high expression of N-Cadherin and Vimentin, indicating epithelial-to-mesenchymal transition but yet the migratory potential was decreased. Further they showed increased apoptosis, a G1-phase cell cycle arrest and evasive PI3K (phosphatidylinositol 3-kinase)/Akt pathway activation. Proteomics screen revealed on the one hand a strong induction of the translational machinery and on the other hand reduced fatty acid metabolic processes. Remarkably, following withdrawal of sorafenib, the resistant cells showed rebound growth, a phenomenon also found in patients. As broad cross-resistance is presumed to occure, this cell model was used to investigate sensitivity towards a variety of chemotherapeutics and pathway inhibitors in order to elucidate an alternative treatment or combination therapy. Conclusion: Our work demonstrated that several mechanisms are involved in the acquired resistance to sorafenib, such as crosstalks involving the PI3K/Akt pathway and epithelial-to-mesenchymal transition. However, the exact mechanisms accounting for sorafenib resistance remains unclear and further investigations are needed to improve our understanding and help to find effective strategies for overcoming sorafenib resistance in HCC. Importantly, this study revealed that halting antiangiogenic therapy with sorafenib could lead to rapid tumor growth rebounds, influencing clinical practice with regard to dosing schedules and presurgical intervention strategies. References: 1. Llovet, J. M. et al.: N Engl J Med. 2008, 359: 378-390. 2. Abou-Alfa, G. K. et al.: J Clin Onkol. 2006, 24: 4293-4300.

BIOTECHNOLOGY/PROTEIN DRUGS


3.4 Biotechnology/Protein Drugs

POS.71

NADPH Cofactor regeneration on the cell surface: an important step to pharmaceutical applications of surface displayed phase I P450 enzymes Schüürmann, J.; Lindhorst, F.; Jose, J. Institute of Pharmaceutical and Medicinal Chemistry, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstraße 48, D 48149 Münster, Germany

The use of purified enzymes for preparative applications is still held back by costly production and purification processes, stability problems and reusability issues. In this regard, whole cell biocatalysts offer significant advantages. Theses catalysts however are restricted to cell permeable substrates and products. One solution to circumvent mass transfer problems and possible cross reactions with intracellular enzymes is the presentation of enzymes on the cell surface. Our group was recently able to display human cytochrome P450 phase I enzymes CYP1A2 and CYP3A4 in an active form on the cell surface of E. coli using the Autodisplay technique [1,2]. Therefore the passenger domain of a native autotransporter was replaced by the peptide or protein of choice, which was subsequently presented at the cell surface [3]. A current project focusses on the display of CYP102A1 from Bacillus megaterium for pharmaceutical applications. Surface exposure of the enzyme was confirmed by protease accessibility tests, flow-cytometry analysis and activity measurements. CYP102A1, also known as BM3 is one of the best studied cytochrome P450s to date and can be engineered to accept many pharmaceutical relevant substances [4]. In a proof of principle study we analyzed a small library of substances with the known R47L F87V L188Q triple mutant of CYP102A1 displayed at the cell surface of E. coli. However, the project also elucidated a major challenge for the use of the technology in pharmaceutical applications. Bringing an enzyme to the cell surface blocks the access to intracellular cofactors, which are too expensive to be added stoichiometrically. In an approach to conserve the advantages of a surface displayed catalyst, we used Autodisplay as well to present various dehydrogenases on the surface of E.coli cells for the regeneration of the essential cofactor NADPH. References: 1. Quehl, P et al. Microb Cell Fact 2016, 15:1-15 2. Schumacher S and Jose J J Biotechnol 2012, 161(2):113-120 3. Schüürmann, J et al. Appl Microbiol Biotechnol 2014, 98:8031-8046 4. Whitehouse C, Bell S and Wong L. Chem Soc Rev 2012, 41:1218-1260

POS.72

Long-term release of Ranibizumab from lipid based implants for intravitreal treatment of age-related macular degeneration (AMD) Engert, J.1; Vollrath, M.; Winter, G.1 1 Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University, Butenandtstrasse 5-13, 81377 Munich, Germany

Introduction: Age-related macular degeneration (AMD) is an eye disease characterized by a loss of the central vision causing visual impairment and blindness [1]. The main characteristic of the more severe neovascular form of AMD (wet AMD) is an abnormal blood vessel formation leading to the presence of blood in the back of the eye region [2]. According to the limitations of the repeated intravitreal injections of anti-VEGFs, the development of insertable devices providing long-term release of anti-VEGF drugs is in the focus of current research [3]. In our study, solid lipid implants (SLIs) were investigated for the sustained release of the recombinant monoclonal antibody fragment Ranibizumab (Lucentis®) for intraocular delivery. It has already been demonstrated that SLIs are a suitable platform for long-term protein release since a sustained release of an IgG1 over 190 days was described [4]. Additionally, protein stability of the released fractions was

analysed showing good physical and conformational stability [5]. Since lipids are biodegradable and biocompatible [6] they represent a promising delivery platform for AMD treatment. Material and Methods: Triglycerides Dynasan® D118 and H12 were kindly donated by Cremer Oleo (Hamburg, Germany). Hydroxy-β-cyclodextrine (HP-β-CD) was a gift of Wacker Chemie AG (Burghausen, Germany). Ranibizumab (Lucentis®) is a fab-fragment with a molecular weight of about 49 kDa. Ranibizumab was formulated with HP-β-CD at a 1:1 ratio [m/m] before lyophilization. SLIs were prepared by twin-screw extrusion as described before [6] using a ZE-5 extruder from Three-Tec (Seon, Switzerland). A mixture of H12, D118 and 10% protein lyophilizate was fed to the extruder and extrusion was performed at 35°C. Implants had a semicircle shaped form which is suitable for intravitreal use and contained 1.65mg protein per implant. For in-vitro release studies, implants were placed into 2.0 ml centrifugation tubes and incubated at 37°C in a horizontal shaker (40rpm) in PBS pH 7.4. The release medium was completely exchanged at predetermined time points and protein concentration was analyzed UV-metrically at 280 nm. Monomer content was determined via SE-HPLC using a Waters 2695 Separations Module with a Waters 2487 Dual λ Absorbance Detector (Waters, Milford, USA). The flow rate was adjusted to 0.5ml/min and 50µl were injected onto a TSKgel G3000SWXL size exclusion column (300mm x 7.8mm; Tosoh Bioscience, Tokyo, Japan). The running buffer consisted of 50mM sodium phosphate with 300mM NaCl and 0.05% NaN3 and was adjusted to pH 7.0. Results: A sustained release of Ranibizumab was achieved over approximately 110 days. The release profile is characterized a triphasic release behaviour without an initial burst. An initial phase for the first two weeks where 40% of the incorporated protein was released followed by a linear phase ranging from week 2 to week 14 releasing 50% of encapsulated protein. During the last release phase, lasting from week 14 to 18, only small amounts of protein were released (approx. 3%). In total, nearly 90% of incorporated protein was released. The monomer content of the released protein fractions was measured over 18 weeks showing no loss of monomer compared to Ranibizumab bulk material showing that the fab-fragment is physically stable within the lipidic matrix over time. Conclusion: We successfully demonstrated that twin-screw extruded SLIs are a suitable technology platform for the long-term release of a therapeutically relevant protein providing a sustained release of 14 weeks in linear manner. Good physical stability of the fab-fragment indicates sufficient protein stability within the lipidic matrix. Therefore, this approach is a promising platform for intravitreal delivery of anti-VEGF drugs for AMD treatment. References: 1. Wong, W.L. et al.: Lancet Glob. Health 2014, 2: e106-e116 2. Lim, L.S. et al.:Lancet 2012, 379: 1728 – 1738 3. Lovett, M.L. et al.: Eur. J. Pharm. Biopharm.2015, 95: 271-278 4. Vollrath, M. et al.: Poster 10th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology, Glasgow, Scotland, UK, 2016 5. Vollrath, M. et al.: Poster AAPS Annual Meeting and Exposition, Orlando, FL, USA, 2015 6. Sax, G. et al.: J. Control. Release, 2012, 2: 195–202

POS.73

Protein powder suspensions based on perfluorodecalin Marschall, C.1; Yasin, A.2; Witt, M.2; Friess, W.1 1 LMU Munich, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Butenandtstr.5, 81377, Munich, Germany 2 Novaliq GmbH, Im Neuenheimer Feld 515, 69120 Heidelberg,Germany

Introduction: Currently there is a growing demand for highly concentrated protein formulations for subcutaneous injection. However, conventional aqueous formulations have different challenges such as increased viscosity and potentially decreased protein stability. Protein powder suspensions in non-aqueous vehicles represent a promising alternative. Herein the protein is present in a solid state offering the chance of higher protein stability compared to an aqueous solution. Furthermore the suspensions may provide lower viscosity compared to the respective aqueous solution depending on the protein-protein-interaction in water [1]. The choice of potential suspension vehicles is limited as traditional non-aqueous liquids such oils show an inherent high viscosity [2]. A potential alternative is perfluorodecalin. Previous

POSTERS


investigations by Knepp et al. showed high protein stability of suspended lyophilizates in this vehicle over one year at elevated temperatures comparable to the same lyophilizate stored at -80°C [3]. Materials and methods: A lab scale spray-drier was used to produce lysozyme powder, formulated with trehalose as a stabilizer, at a high protein to stabilizer ratio. Process parameters were chosen according to Schuele et al. [4]. Feed solutions for spray-drying contained different total solid contents. The obtained powders were analysed by differential scanning calorimetry, laser diffraction and scanning electron microscopy. Suspensions were prepared in perfluorodecalin at different powder contents up to 500 mg/ml using a dispersing device. The rheological properties of the suspensions were investigated using a cone-plate rheometer. Results: Spray-drying showed good powder yields (>80%). Increasing the total solid content in the feed solution led to larger particle diameters up to 30 µm. The powder particles showed a typical spherical morphology. The prepared suspensions showed a shear-thinning behaviour and the viscosities were remarkably low even at high powder concentrations. The true density of the prepared powder was lower than the perfluorodecalin density leading to flotation of the powder. Suspensions showed good redispersion behaviour as well as good suspension stability during short term storage at room temperature and elevated temperature. Conclusion: Overall the suspensions benefit from perfluorodecalin’s exceptional physical properties such as its low viscosity resulting in low suspension viscosity and good redispersibility even at higher powder concentrations. Further work will show long term suspension stability of the prepared protein powder suspensions in perfluorodecalin. Also other, more relevant proteins with lower protein stability, such as monoclonal antibodies, will be formulated as suspensions in order to confirm the advantages of protein powder suspensions and to investigate long-term protein stability. References: 1. M. a Miller, J. D. Engstrom, B. S. Ludher, and K. P. Johnston, “NIH Public Access,” vol. 26, no. 2, pp. 1067–1074, 2011. 2. Y.-F. M. Mayumi Bowen, Nick Armstrong, “Investigating High-Concentration Monoclonal Antibody Powder Suspension in Nonaqueous Suspension Vehicles for Subcutaneous Injection,” J. Pharm. Sci., vol. 101, no. 12, pp. 4433 – 4443, 2012. 3. A. M. et al. V. M. Knepp, “Stability of nonaqueous suspension formulations of plasma derived factor IX and recombinant human alpha interferon at elevated temperatures,” Pharm. Res., vol. 15, no. 7, pp. 1090–1095, 1998. 4. S. Schüle, T. Schulz-Fademrecht, P. Garidel, K. Bechtold-Peters, and W. Frieß, “Stabilization of IgG1 in spray-dried powders for inhalation,” Eur. J. Pharm. Biopharm., vol. 69, no. 3, pp. 793–807, 2008.

POS.74/SL.32

How does pH Affect Interfacial Antibody Behaviour and Aggregation upon Shaking? Koepf, E.1; Schroeder, R.2; Brezesinski, G.3; Frieß, W.1 1 Ludwig-Maximilians-Universitaet, Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, 81377 Munich, Germany 2 AbbVie Deutschland GmbH & Co. KG, 67061 Ludwigshafen am Rhein, Germany 3 Max-Planck Institute of Colloids and Interfaces, Department of Interfaces, 14476 Potsdam, Germany


POS.75

Suitability testing of novel polymer syringes for long term storage of biopharmaceuticals Werner, B. P.1; Winter, G.1 1 Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University, Butenandtstr. 5, 81377 Munich, Germany, [email protected], phone: +49 89 2180 77035

Prefilled syringes receive more and more interest for the administration of biopharmaceuticals, because of an easy and quick drug administration in combination with an increased patient compliance [1,

2, 4, 5]. So far, the material of choice for prefilled syringe barrels has been glass [2, 4]. These glass syringes need silicone oil as a lubricant for a smoot injection. However, silicone oil can contribute to the particle count of the solution. Either it sheds into the solution or can foster protein aggregation. Particles, especially silicone oil droplets with protein attached, in general compromise the safety and efficacy of the drug [1, 4, 5]. Therefore, silicon oil free, alternative materials for prefilled syringes are necessary. A suitable material can be cyclic olefin polymer (COP). This material in combination with special coated stoppers works well without silicone oil for lubrication [5]. COP syringes possess a major drawback, namely providing a lower gas barrier than glass [4]. This might limit their usage for the storage of biopharmaceuticals, because many of those are sensitive to oxidation [3]. However, oxygen barrier properties can be modified. One option is the usage of multilayer syringes with a layer incorporated displaying high oxygen barrier properties. Yet, these systems are expensive and not yet available in high numbers. A cheap solution would be a tight aluminum bag as secondary packaging which would also protect from light. Another cost-effective option would be an oxygen impermeable label, as a label is necessary anyway. Even without additional measures stability could be achieved, if the effect of oxygen permeation on the particular protein is negligible. The suitability of such novel COP based syringes as a long term packaging material for biopharmaceuticals is evaluated systematically in this study. The study focuses especially on the issue of oxygen permeation and its consequences. To this end, two model proteins of high therapeutic relevance were investigated. Further, the study comprises the low cost options of an aluminum bag which is filled with nitrogen and a tight label. Finally we benchmarked the COP syringes against commonly available glass syringes. The data of the ongoing study suggest a superiority of the COP syringes regarding particle count. MicroFlow Imaging reveals clearly the impact of silicone oil from glass syringes on the particle burden of the solution. Depending on the syringe setup, differences regarding chemical stability of the proteins are visible. Ion exchange and reversed phase chromatography show the highest content of oxidized protein for COP syringes without any alterations. However, the storage of COP syringes in nitrogen filled aluminum bags or with tight labels leads to lower amounts of oxidized species than in glass syringes. The impact of a higher oxygen permeability of the COP barrel in comparison to glass syringes can easily be overcome by low cost modifications. Acknowledgments: The authors thank West Pharmaceutical Services Deutschland GmbH & Co KG, Eschweiler, Germany and Gerresheimer Bünde GmbH, Bünde, Germany for the supply of syringe material.

References: 1. Depaz, R.A., et al.: J Pharm Sci 2014, 103: 1384-1393. 2. Krayukhina, E., et al.: J Pharm Sci 2015, 104: 527-535. 3. Manning, M.C., et al.: Pharm Res 27, 2010, 544-575. 4. Nakamura, K., et al.: PDA J Pharm Sci Technol 2015: 69, 88-95. 5. Yoshino, K., et al.: J Pharm Sci 2014, 103: 1520-1528.

POS.76

Is bedside filtration an effective tool to diminish risks associated with protein aggregates? Werner, B. P.1; Winter, G.1 1 Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University, Butenandtstr. 5, 81377 Munich, Germany, [email protected], phone: +49 89 2180 77035

In the last decade much progress has been made in formulating stable and easy to handle protein drug products despite the complex structure of proteins and their chemical and physical instability [2, 3]. Nevertheless, the chance remains that protein aggregates are formed during transportation, storage or handling. Generally, these protein particles could endanger the safety and efficacy of the protein drug [2]. Hence, these aggregates may present a hazard especially for immunosuppressed and intensive care patients [1]. The potential of protein aggregates to induce immune responses is known since the 1950s and is nowadays broadly accepted [4, 7]. For several proteins, like adalimumab, insulin or human growth hormone the formation of antidrug antibodies is observed [8]. The consequences of this antidrug antibody formation can be negligible and minor but also very severe, like the neutralization of an endogenous protein [5]. Today, the FDA and other regulatory bodies have increased the pressure on drug

BIOTECHNOLOGY/PROTEIN DRUGS


manufacturers which now have to supply comprehensive documentation regarding particle burden of protein solutions. An easy and practicable approach to protect patients from any risks associated with protein aggregates could be bedside filtration. In our unique and comprehensive survey, we identified more than 50 products of more than 300 marketed products which are already filtered during preparation and administration (Figure 1) [8]. Considering these findings and the positive benefits associated with bedside filtration, like reduced occurrence of thrombi [6] or sepsis, we like to suggest contemplating a broader application of final bedside filtration.

Fig. 1: Already 15.9% of all protein drug products listed in the Rote Liste® are filtered during preparation or administration of the drug. Adapted from Werner, B.P. and Winter, G. [8].

We critically assessed several practical aspects arising from a broader application of final bedside filtration. First, ejection of a high concentrated protein solution is still possible after filter attachment, whereas the filter has only a minor impact on the ejection force. Second, the so far investigated filters do not contribute to the particle burden of the solution, but are highly capable in reducing the amount of protein particles. Further, protein adsorption or structural changes of the protein caused by filtration are not observed. Overall, our data shows that bedside filtration during protein drug preparation and administration can be a powerful and effective tool to guard patients from potentially negative effects associated with protein aggregates. References: 1. Bethune, K., et al.: Nutrition 2001, 17: 403-408. 2. Kessler, M., et al.: Dial Transplant 2006, 21: v9-v12. 3. Liu, L., Randolph, T.W., Carpenter, J.F.: J Pharm Sci. 2012, 101(8): 2952-9. 4. Mahler, H.C., et al.: J Pharm Sci 2009, 98 (9): 2909-2934. 5. Rosenberg, A. AAPS J. 2006, 8 (3): E501-E507. 6. Schellekens, H. Best Pract Res Clin Haematol 2005, 18: 473-480. 7. van Lingen, R.A., et al.: Acta Paediatr 2004, 93 (5): 658-662. 8. Wang, W., et al.: Int J Pharm 2012, 431: 1-11. 9. Werner, B.P., Winter, G.: Int J Pharm 2015, 496: 250-267.

POS.77

Kunitz-type inhibitors targeting matriptase-2: Promising therapeutics for iron overload diseases Beckmann, A.-M.1; Maurer, E.1; Mangold, M.1; Furtmann, N.1, 2; Bajorath, J.2; Walter, J.3; Becker-Pauly, C.4; Gütschow, M.1; Stirnberg, M.1 1 Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 2 Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and Medicinal Chemistry, University of Bonn, Dahlmannstr. 2, 53113 Bonn, Germany 3 Department of Neurology, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany 4 Institute of Biochemistry, Unit for Degradomics of the Protease Web, Christian Albrechts University, Rudolf-Höber-Str. 1, 24118 Kiel, Germany

The type II transmembrane serine protease (TTSP) matriptase-2 (gene name: TMPRSS6) is a critical stimulator of iron absorption by negatively regulating hepcidin, the key hormone of iron homeostasis [1,2]. By cleaving the BMP co-receptor hemojuvelin matriptase-2 suppresses BMP/SMAD signalling thereby downregulating the expression of hepcidin [3]. Consequently, this protease attracted much attention as a target to treat primary and secondary iron overload diseases associated with deficiency in hepcidin. Kunitz-type serine protease inhibitors are natural inhibitors containing at least one Kunitz-domain binding with high affinity to TTSPs. As stand-alone domains with intrinsic stability, Kunitz-domains can be used as scaffolds for engineering protease inhibitors to be employed as biopharmaceuticals. In this study, a strategy based on the Kunitz-type inhibitors hepatocyte growth factor activator inhibitor (HAI)-1, HAI-2 and the amyloid

precursor protein (APP) was implemented. The kinetics of matriptase-2 inhibition by Kunitz-type inhibitors were analyzed and the intracellular downstream effects of matriptase-2 inhibition were investigated by applying a set of different assays using human liver cell systems. Co-immunoprecipitation revealed the formation of matriptase-2/inhibitor complexes. Western blotting and reporter gene assays were employed to analyze the impact on matriptase-2-mediated hemojuvelin cleavage and hepcidin expression. Binding modes of the matriptase-2/Kunitz-type inhibitor complexes were suggested by molecular modeling. In conclusion, we have characterized Kunitz-type inhibitors as powerful, slow-binding inhibitors of matriptase-2 with HAI-2 being the most effective inhibitor. Thus, HAI-2 provides an excellent framework for the development of a drug inhibiting matriptase-2 as a therapeutic agent in iron overload diseases. References: 1. Finberg, K. E. et al.: Nat. Genet. 2008, 40(5): 569-571. 2. Du, X. et al.: Science 2008, 320(5879): 1088-1092. 3. Silvestri, L. et al.: Cell. Metab. 2008, 8(6): 502-511.

POS.78/SL.56 Clickable IL-4 cytokines to induce M2 macrophage polarization Lühmann, T.1; Spieler, V.1; Werner, V.1; Fiebig, J.2; Mueller, T. D.2; Meinel, L.1

1 Institute of Pharmacy and Food Chemistry, University of Würzburg, 97074, Germany 2 Lehrstuhl für Botanik I, University of Würzburg, 97070, Germany


POS.79/SL.03 Biopolymers as Multifunctional Materials for Nanoparticulate Drug Delivery Wich, P. R. Johannes Gutenberg Universität-Mainz, Institut für Pharmazie und Biochemie, Staudingerweg 5, 55128 Mainz, Germany


POSTERS


3.5 Clinical Pharmacy

POS.80

Implementing preferred generic prescribing – community pharmacies’ view Breiholz, S.1; Eickhoff, C.1; Felberg, M.1; Klintworth, D.1; Müller, U.1; Strunz, A. K.1; Schulz, M.1 1 Institute: Department of Medicine, ABDA – Federal Union of German Associations of Pharmacists, 10117 Berlin, Germany

In 2011, the ABDA and the Federal Association of Statutory Health Insurance Physicians (KBV) developed a concept that aims to improve outcomes of drug therapy in patients with polymedication [1]. Against this background, a project named ARMIN (Arzneimittelinitiative Sachsen-Thüringen, www.arzneimittelinitiative.de) was developed and contracted for at first five years in two states, Saxony and Thuringia. ARMIN consists of three components: (1) prescription of active ingredients (INN) instead of brand products, (2) a medication catalogue to select appropriate drugs based on guidelines, and (3) medication management by pharmacists and physicians utilizing an electronic medication plan. The preferred generic prescribing by physicians within this project was implemented in July 2014. It aims to increase transparency for the patient and to reduce medication errors, considering the multitude of rebate contracts for generics between statutory health insurances and pharmaceutical companies. Firstly, we developed a dataset including appr. 190 suitable active ingredients, defined in terms of active substances and formulations. Secondly, we specified how the description has to be filled in by the physician including a new six-figured code named WG14. This code leads to a list of all fitting brand products when entered into the local software of any German pharmacy. In February 2016, 1,552 community pharmacies were asked anonymously to evaluate the acceptance of this concept (Survey Monkey®). Pharmacies were asked to assess the concept of generic prescribing in general. Among others, participants were asked to indicate advantages and disadvantages of the concept for both pharmacies and patients. By the end of March 2016, a total of 628 pharmacies participated in the survey (response rate 40.5%). 69 % (n=433) assessed generic prescribing as useful or very useful. On average, the general concept of preferred generic prescribing achieved a score of 2.4 on a scale of 1–6 (1=very good to 6=very bad). Due to pharmacies major advantages for patients are an enhancement of medication adherence and patient safety by avoiding unnecessary switching of the generic product dispensed. Prescribing an active ingredient instead of a brand name was highly acknowledged by German pharmacies. Routine use of this concept in combination with an electronic medication plan has the potential to enhance medication adherence and patient safety. References: 1 ABDA-KBV-Modell/ARMIN: http://www.abda.de/themen/positionen-und-initiativen/armin/

POS.81 Pharmaceutical care provided by community pharmacists for adolescents with type 1 diabetes mellitus – follow up of DIADEMA Deters, M. A.1; Obarcanin, E.1; Läer, S.1

1 Institue of Clinical Pharmacy and Pharmacotherapy Heinrich-Heine-University Duesseldorf

Background: Diabetes mellitus is one of the most common diseases in childhood and the incidence increased over the past 20 years about 3-4% [1]. Micro- and macrovascular complications, due to poor metabolic control, can lead to long-term complications such as high blood pressure [2]. Especially in adolescence lower medication adherence is a huge risk [2]. The DIADEMA study, a randomized controlled trial, has shown that community pharmacists can have a positive impact on therapy adjustment and glycemic control of adolescent diabetes patients [3].

Method: A quantitative statistical analysis of the pharmaceutical care documented in the case-report-forms from 39 DIDEMA patients over the study period of 6 months was conducted. The case report forms were analysed according to the following parameters: Self-monitoring of blood glucose (SMBG), daily insulin injections, average fasting blood glucose levels, insulin- therapy-adherence, daily insulin dose and number of patients, which are following their nutrition plan or doing exercise or having hypoglycemic episodes. Inconsistent and imprecise data were excluded from the statistical analysis. A Wilcoxon-signed-rank test or fisher-extact test with a significance level of alpha=0.05 were used to evaluate the differences between the study visits from every month compared to the pre-study parameter characteristics. Results: The statistical analysis revealed that pharmaceutical care provided by community pharmacists result in more frequent SMBG, a greater amount of patients complying to their individual nutrition plan and injecting the correct insulin dose. Also, the insulin-therapy adherence increased and fasting blood glucose levels were lowered. However, all these positive changes did not lead to an increase in hypoglycemic episodes. Conclusion: The results show that pharmaceutical care can positively alter disease specific parameters and improve patient behaviour to cope with the daily burden of type I diabetes mellitus in adolescents. With this, the pharmacist as a health-communicator can substantially support these individual adolescents by translating current treatment guideline into daily practice, giving answers to nutrition questions, reviewing the individual nutrition plan and thus set realistic goals for the next visit. Consequently, better glycemic control should minimize the risk of short- and long-term diabetes related complications such as retinopathy or blindness for these young individuals [2]. References: 1. Tamayo T, Rosenbauer J, Wild SH et al. Diabetes Res. Clin. Pract. 2014; 103:206-217. 2. ISPAD Clinical Consensus Guidelines 2014; Pediatric Diabetes 2014:15(Suppl. 20):1-290. 3. Obarcanin E, Krüger M, Müller P, Nemitz V et al. Int. J. Clin. Pharm. 2015: 37(5):790-8.

POS.82 The impact of the implementation of an electronic prescribing software on the drug therapy safety in clinical practice Ebbing, L.1; Kunze, T.1; Eisend, S.2

1 Christian-Albrechts-University Kiel, Department of Clinical Pharmacy, Gutenbergstraße 76, 24118 Kiel, Germany 2 University Medical Centre Schleswig-Holstein, Department Pharmacy, Arnold-Heller-Str. 3, 24105 Kiel, Germany

A complex medication process can increase the amount of medication errors, which might lead to a higher risk of adverse drug reactions. Computerized physician order entry systems (CPOE-systems) may reduce medication errors - particularly prescribing errors - but may also increase them and thereof carry the risk of leading to patients harm [1]. The scientific observation of the implementation of electronic prescribing software is highly recommended [2]. In April 2014 the pharmaceutical employees of the University medical centre Schleswig-Holstein started the rollout of the prescribing software MEONA with integrated clinical decision support system (CDSS). The objective of this study is to determine whether the implementation of MEONA has any impact upon the quantity and severity of medication errors. Through visitation of various wards the operating cycles of the medical and nursing personnel inside the medication process were documented and critical processes were identified. A medication error registration and classification system for prescription, transcription and dispensing process was established. On four chosen wards over a period of each four weeks total with handwritten prescriptions and four weeks after the implementation of the CPOE the occurrence of medication errors was recorded. Drug-drug interactions were evaluated as one category of prescription errors by using pharmazie.com which is based on the ABDA-database. First results didn’t show any significant reduction - or increase - in amount or severity of interactions, although the CDSS contains an interaction-warning system. The evaluation of further medication error categories is still in progress.

CLINICAL PHARMACY


References: 1. Bundesministerium für Gesundheit. Aktionsplan 2013/2015 zur Verbesserung der Arzneimitteltherapiesicherheit in Deutschland. Bonn, 2013. 2. AOK Bundesverband. HTA-Gutachten zur Beurteilung der Wirksamkeit von CPOE-Verfahren zur Erhöhung der Arzneimitteltherapiesicherheit. Bremen, 2009.

POS.83 Semi-mechanistic PK/PD modelling to characterise long-term deterioration of neutropenia in cancer patients Henrich, A.1,2; Joerger, M.3; Kraff, S.4; Jaehde, U.4; Huisinga, W.5; Parra-Guillen, Z. P.1; Kloft, C.1

1 Dept. Clinical Pharmacy & Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr. 31, 12169 Berlin, Germany 2 and Graduate Research Training program PharMetrX 3 Dept. of Oncology & Haematology, Cantonal Hospital St. Gallen, Rorschacher Strasse 95, 9007 St. Gallen, Switzerland 4 Dept. Clinical Pharmacy, Institute of Pharmacy, Universitaet Bonn, An der Immenburg 4, 53121 Bonn, Germany 5 Institute of Mathematics, Universitaet Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam Golm, Germany

Background: Paclitaxel (PTX) exhibits complex pharmacokinetics (PK), including non-linearity and high inter-individual variability. In addition, severe toxicity, especially neutropenia (pharmacodynamics (PD)) is observed, resulting in a narrow therapeutic window. The combination of those two factors increase the need for dose-individualisation and therapeutic drug monitoring. For the sparse PTX and neutrophil concentrations of the clinical trial “CEPAC-TDM”, a previously published PK/PD model [1] was evaluated to assess whether it was able to describe the new data: Regarding PK, a slight underprediction of the PTX plasma concentrations was observed [2]; regarding PD, worsening neutropenia over repeated chemotherapy cycles was not captured by the PK/PD model. This behaviour was hypothesised to be due to bone marrow exhaustion (BME) [2]. Aim/Objectives: Based on the results of the external model evaluation, the first aim was to refine the PK model for the population in the CEPAC-TDM trial. In a second step the PK/PD model was extended to be able to describe long-term neutropenia by incorporating the hypothesised BME effect. Methods: In the CEPAC-TDM trial, non-small cell lung cancer patients (n=183) were treated with PTX (doses according to a published algorithm [1]) in combination with a carbo- or cisplatin every 3 weeks for up to 6 cycles. PTX plasma concentrations (PK) were measured ~ 24 h after drug administration, while neutrophil concentrations (PD) were quantified on day 1 and 15±2 of each treatment cycle. For model optimisation a stepwise analysis was performed: First, parameter estimates from the published PK model were used as prior information utilised as prior information for re-estimation of the PK parameters with the new data assuming the same model structure. Second, the hypothesised BME was implemented in a semi-mechanistic way, by extending the original Friberg et al. model [3]. For this purpose, one compartment accounting for slowly proliferating stem cells was added as a first step in the maturation chain of neutropoiesis. For all modelling activities NONMEM 7.3 in combination with PsN 4.4 was used. Results: An improved prediction of PTX concentrations was achieved for the CEPAC-TDM study population. Further, the optimised PK/PD model, including the BME effect, was able to describe the observed long-term worsening of neutropenia over the treatment cycles. For this PK/PD model a high PD parameter precision (RSE < 10%) was achieved. The proliferation rate constant of the progenitor cells was 3.7-fold higher than the one of the stem cells. Conclusions: An adequate description of PTX PK was achieved by combining previous knowledge from the former rich data with the sparse CEPAC-TDM data. To account for long-term neutropenia, a mechanistically plausible PK/PD model was developed that adequately described the hypothesised BME. Ultimately, the model shall be used to further improve individualised dosing of PTX to reduce toxicity. Further, due to the mechanistic approach, the structure of the PK/PD model can also be beneficial for optimisation of long-term therapy of cancer patients with cytotoxic drugs exhibiting neutropenia as dose-limiting toxicity. References: 1. Joerger, M. et al.: Clin. Pharmacokinet. 2012, 51(9): 607–17. 2. Henrich, A. et al.: PAGE 24 2015, Abstr. 3460. 3. Friberg, L. E. et al.: J. Clin. Oncol. 2002, 20(24): 4713–21

POS.84 Fostering interprofessional teamwork among pharmacy and medical students: A new patient-centred clinical approach Hopf, Y. M.1,2; Andraschko, M.1; Wahl-Schott, C.3; Fischer, M. R 2

1

University Hospital Munich, Department of Pharmacy, Marchioninistr. 15, 81377 Munich, Germany 2 University Hospital Munich, Institut für Didaktik und Ausbildungsforschung in der Medizin, Ziemssenstr. 1, 80336 Munich, Germany 3 Ludwig-Maximilians-University, Department of Pharmacy, Pharmacology of Natural Sciences, Butenandtstr. 5-13, 80336 Munich, Germany

The WHO considers interprofessional education (IPE) as a prerequisite for optimal patient care [1]. Studies have shown that IPE improves teamwork and encourages/promotes/facilitates understanding and regard between different professions during working life [2-4]. The standards published by the German Pharmaceutical Society (DPhG) regarding education in the field of clinical pharmacy specifically request IPE sessions for pharmacy and medical students [5]. The Schools of Pharmacy and Medicine at the Ludwig-Maximilians-University (LMU) in Munich partnered to foster a new IPE programme by joined and simultaneous bedside teaching in clinical pharmacy. The goals of this new IPE programme is to stimulate and encourage interprofessional teamwork amongst undergraduates, to create an early opportunity to foster communication for medical and pharmacy students, and to increase clinical experience for pharmacy students. Preregistration medical students (post exam) were teamed up with pharmacy students in their final year. The pharmacy students shadowed the medical students in their respective clinical settings. Students’ teams were based in all clinical settings, medical as well as surgical. Evaluation was done by pre/post testing of students’ perception of IPE utilising SPICE 2D. Qualitative methods were used to evaluate additionally views and opinions regarding IPE after attending the course. Ethical approval was obtained from the LMU ethics committee. The pilot with 15 participants took place during summer term 2016, since then further 110 students took part in the programme. SPICE 2D evaluations in all groups showed an increase in appreciation of interprofessional teamwork. Further statements from students after the IPE programme showed that participants in general enjoyed the interprofessional experience. Pharmacy students valued the “real life” experience with discussion based upon real patients rather than case studies. Medical students enjoyed in particular the exchange with a different profession as well as the interest in patient care as displayed by the pharmacy students. One particular point of criticism was that pharmacy students were not able to apply their theoretical/university knowledge within a new clinical context. Medical students were disappointed as they hoped for more input from the pharmacy students for example in terms of interactions, pharmacy students were often frustrated by seemingly too complex patient cases. Both student groups though wish for a continuation of the programme. The IPE programme is set to be anchored in both curricula, pharmacy and medicine. Extended evaluation will take place another term in order to implement and trial recommendations from previous term evaluations. In addition to questionnaire evaluations two focus groups will be conducted with the participants of summer term 2016. Acknowledgements: We would like to thank Dr Patricia Raes, Dr Yvonne Kosanke and Professor Angstwurm for their help in recruiting medical students.

References: 1. World Health Organization. Learning together to work together for health-Report of a WHO Study Group on Multiprofessional Education of Health Personnel: the Team Approach. World Health Organization Technical Report Series 769, 1988. Geneva: WHO. 2. Olson R, Bialocerkowski A. Med. Educ. 2014;48:236-246. 3. Stößel U KK, Kaba-Schönstein L. GMS Z. Med. Ausbild. 2006;23:Doc34. 4. Zwarenstein M, Goldman J, Reeves S. The Cochrane database Syst. Rev. 2009;3:CD000072. 5. Jaehde U (für die Fachgruppe Klinische Pharmazie der Deutschen Pharmazeutischen Gesellschaft, DPhG). DAZ 2004, 144;15: 1743-1746.

POSTERS


POS.85/SL.43 Evidence-based evaluation system for OTC drugs Achenbach, J.1; Culmsee, C.1 1 Institut für Pharmakologie und Klinische Pharmazie, Philipps-Universität Marburg, Karl-von-Frisch-Straße 1, 35032 Marburg


POS.86 STW 5 in functional gastrointestinal diseases: Metaanalysis of clinical trials Müller, J.1; Rabini, S.1; Abdel-Aziz, H.1; Kelber, O.2; Storr, M.3; Kraft, K. 4 1 Medical Affairs, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany 2 Innovation & Development, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany 3 Zentrum für Endoskopie Starnberg, Oßwaldstraße 1, 82319 Starnberg, Germany, und Medizinische Klinik und Poliklinik II, Klinikum der Universität München, Campus Großhadern, Marchioninistraße 15, 81377 München, Germany 4 Zentrum für Innere Medizin der Universitätsmedizin Rostock, Lehrstuhl für Naturheilkunde, Ernst-Heydemann-Straße 8, 18057 Rostock, Germany

Introduction: The gastro-prokinetics, such as metoclopramide, domperidone and cisapride, had been introduced to the therapy of functional gastrointestinal diseases in the 70s and 80s, and for a long time they seemed to be standard therapeutics in this indication. Since they are no longer available for the use in this indication due to restrictions of their marketing authorizations based on rare but severe side effects, other well proven therapeutic options gain increased attention. One of these is STW 5 (Iberogast), for which more than 5 decades of therapeutic experience in more than 60 Mio patients are available. Aims & Methods: Whether also the available clinical data comply to modern standards for a proof of efficacy was now tested by a meta-analysis of the randomized placebo-controlled double blind trials available in the therapeutic indication functional dyspepsia. The individual datasets from all trials were entered and demografic data and primary endpoints were evaluated (ANCOVA). Results: The primary outcome variable, the validated gastrointestinal symptom score (GIS) [1], as well as the therapeutic dose (3 x 20 drops/day) were identical in all trials, so allowing a uniform evaluation. The full analysis set (FAS) included 557 patients (272 resp. 285 for placebo resp. verum). The mean age (48 resp. 49 years), the mean body size (in both groups 168.7 cm), the mean body weight (72.0 resp. 72.2 kg), the BMI (25.35 resp. 25.54), the gender disitribution (67.3 resp. 69.5 % females), the duration of the disease at the time of inclusion and the baseline of the GIS (11.6 resp. 11.5 points) were very well comparable between both groups. For the primary variable GIS the difference between placebo and verum after 28 days of treatment showed a highly significant (p< 0.0001) difference between placebo and verum (6.7 resp. 4.7 points). Conclusions: This meta analysis therefore clearly shows the efficacy of STW 5 (Iberogast) according to present standards of evidence-based medicine (EBM). In addition it emphasizes the high quality of the trials, which is indicated e.g. by the very good balance between both patient groups. Additional insights can be expected from further analyses, as e.g. the evaluation of different subgroups with specific predominant symptoms or demographic properties. References: 1. Adam et al. Aliment Pharmacol Ther 2005; 22: 357-363

POS.87 Phytomedicines as standard therapy - wishful thinking or reality? Kelber, O.1; Kraft, K.2 1 Innovation & Development, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany 2 Zentrum für Innere Medizin der Universitätsmedizin Rostock, Lehrstuhl für Naturheilkunde, Rostock, Germany

Phytomedicines as standard therapeutics – this has been the case long ago, once upon a time, when chemically defined medicines had not yet been discovered. So is the common view. But it is worthwhile to ask, whether this is also true today. Obviously, today, certain circumstances are needed to make this true. A recent example is the therapy of functional gastrointestinal diseases. Prokinetics had become standard therapeutics in the 1980ies and 1990ies, i.e. substances like cisapride, metoclopramide and domperidon, which act via serotonin receptors. Since 2000 cisapride, since 2014 metoclopramide and domperidon are no longer available in this indication due to rare but severe side effects. The phytomedicine Iberogast, which had been shown to be equivalent to metoclpramide already in 1984, and to cisapride in 2002, and which had been included to the medical therapy guidelines in Germany already in 1999 resp. 2001, has now in many cases taken the place of the former standard therapeutics. Another example is St. John´s wort, which was included to the German national medical therapy guideline on unipolar depression 2009 after equivalence to chemical standards had been shown in clinical trials repeatedly. Also e.g. ginkgo has good reputation in its indication, the therapy of dementia. But as more than 80 % of all phytomedicines are used within the frame of self-medication, also the recommendation by the pharmacist has to be taken into account when looking for standard therapeutics. Here e.g cough and cold phytomedicines like primrose root/thyme combinations or marshmallow root extract are used widely. But here the way to become standard therapeutics might not yet have been finalized. Taken together, the uptake of phytomedicines to medicinal guidelines and their acceptance as standard therapeutics in daily practice rep. in the recommendations by pharmacists has in some cases already taken place in the last years. But there is still much more to be done in this field within the next few years.

POS.88 Does pre-coating of probes enable in vitro and in vivo microdialysis investigation of anidulafungin? Weiser, C.1; Zeitlinger, M.2; Kloft, C.1 1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstraße 31, 12169 Berlin, Germany 2 Dept. of Clinical Pharmacology, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria

Objectives: Microdialysis (µD) is a minimally invasive technique, which can serve in clinical settings to determine the drug concentrations directly at the target-site e.g. in the interstitial fluid of various organs and tissue. This technique enables the subsequent collection of unbound drug molecules, defined as the pharmacologically active fraction of the drug, due to the diffusion through a semipermeable membrane. Anidulafungin (AFG) is a lipophilic echinocandin antifungal drug and a treatment option for invasive candidiasis in non-neutropenic adult patients and shows high adsorption on probe material as membrane and tubing [1]. An in vitro µD investigation was performed to investigate the influence of a (pre-)coating approach of µD probes to block the potential binding sites prior and during the actual in vitro µD investigation with AFG. Hence, the influence of (pre-)coating the probe with AFG containing perfusate was investigated with regard to the relative recovery (RR) of AFG in the µDialysate. Methods: Two 8 h in vitro µD-investigations were performed consecutively with the same CMA71 probes (100 kDa cut-off) and as perfusate and probe-surrounding medium a mixture of Ringer`s solution (RS) and human serum albumin solution (HSA) (0.5%, v/v). The AFG concentration in the probe-surrounding medium was 1 µg/mL for (i) uncoated probes and (ii) AFG coated probes. In-between (i) and (ii), probes were pre-coated with the AFG containing perfusate (1 µg/mL) in AFG-free probe-surrounding medium for 16 h. Coating was continued during the actual recovery investigation. Microdialysate (µDialysate) was collected (nprobes=2) in 40 min intervals over each 8 h investigation as well as 3 40 min samples at the end of the in-between pre-coating period (flow rate of 1 µL/min). Quantification of AFG was performed with a developed and validated HPLC assay (XBridge BEH C18 column (50 x 3.0 mm, 2.5 µm), isocratic method with mobile phase: methanol and ammonium dihydrogen phosphate 80:20 (v/v)). The concentration of AFG in the µDialysate samples and the RR were

CLINICAL PHARMACY


calculated and then results from (i) and (ii) were compared with each other. Results: The mean (n=7 each) AFG concentration of the probe-surrounding medium and of the perfusate of probe 1 was 1.139 µg/mL (CV 3.96%) and 1.149 µg/mL (CV 8.80%) and of probe 2 was 1.103 µg/mL (CV 4.17%) and 1.181 µg/mL (CV, %: 2.51), respectively. AFG was not detectable in the first µDialysate (0-40 min collection interval) sample of both uncoated probes for (i). RR of AFG in µDialysate during the 8 h of investigation for (i) increased over time from 1.57% to 32.5% (probe 1) and from 0.6% to 34.6% (probe 2) (n=11 µDialysate samples per probe). Compared to that, the AFG RR in µDialysate samples of (ii) also increased over time and ranged from 62.2% to 107% (probe 1) and from 49.6% to 112% (probe 2) (n=12 µDialysate samples per probe). After subtracting the mean AFG concentration from the µDialysate in the 3 40 min in-between pre-coating period of 0.552 µg/mL (probe 1) and 0.511 µg/mL (probe 2) from the AFG concentration from (ii), the RR ranged from 13.7% to 58.9% (probe 1) and from 7.72% to 69.9% (probe 2), respectively. Conclusions: The comparison of RR in the two investigations showed higher RR values in the pre-coated than in the uncoated probes. A plausible reason is that due to pre-coating, AFG molecules from perfusate (partly) block the potential binding sites of diffused AFG molecules from the probe-surrounding medium. Still, RR of AFG from pre-coated probes increased during the 8 h investigated. With these findings we conclude that pre-coating of probes with AFG in perfusate helps blocking potential binding sites of AFG and increase the RR values but a constant RR value is not reached earlier than for uncoated probes, hence limiting its use in clinical µD studies in humans. References: 1. Weiser et al.: 26th ECCMID, Amsterdam, Netherlands. 2016: EV0652

POS.89 Treatment of catheterised ICU patients with levofloxacin- How in silico models help to streamline in vitro investigations of treatment efficacy at the target site. Goebgen, E. B.; Hartung, N.; Seeger, J.; Schaeftlein, A.; Kloft, C. Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr. 31, 12169 Berlin, Germany

Objectives: Intensive care unit (ICU) patients are at risk of bacterial infections due to their impaired immune system. Catheterisation increases this risk with decreased susceptibility to antibiotics, as catheterisation is highly linked to biofilm formation. Patients suffering from ventilator-associated pneumonia are often catheterised and are treated with antibiotics, e.g. the fluoroquinolone levofloxacin (LEV), with high renal elimination active against most probable pathogens. Currently two dosing regimens of LEV are given in the ICU setting: 500 mg bid i.v. and 750 mg qd i.v.. To investigate the pharmacodynamic (PD) efficacy in urine of the different dosing regimens in vitro, the concentration-time profile of LEV in urine is needed. However, data in literature is only considering cumulated antibiotic amount from collection intervals between 1.5 to 4 hours. Hence, this study aimed at using the concentration of the collection intervals to adapt an existing population pharmacokinetic (populationPK) model to simulate the urinary concentration-time profile of LEV in silico in order to streamline in vitro investigations. Methods: LEV is known to rapidly penetrate into tissue and to be linearly eliminated via the kidneys. These properties are accounted for in the 2 compartmental populationPK model [1] with a fast distribution rate constant (k12=2.78 h-1) from the central (V1=21.7 L) to the peripheral (V2=64.4 L) compartment and a back-distribution rate constant k21 (0.936 h-1). From V1, LEV is eliminated linearly (k13=0.512 h-1). The mean fraction of LEV excreted unchanged in urine was fixed to 0.761 [2]. We extended the populationPK model by a urinary tract compartment with the remaining urine volume in the bladder (V3=0.15 L) and the LEV excretion rate constant (k34) to the catheter bag as further parameters. The collected volume of urine (Vu) in 24 hours was fixed to 1.71 (±0.64) L or 2.35 L, respectively, representing the mean (standard deviation) or the maximum value [2]. Based on that, the rate constant k34 was derived from Vu eliminated per hour divided by V3. Simulations using these PK parameters were performed and compared with the data by Pea et al.. Finally the parameters were used to simulate the concentration-time profile of LEV

in urine of both dosing regimens for the first 24 hours after start of the treatment. Results: The results from the simulations performed with the adapted populationPK model showed a similar shape in the cumulated urine amounts published by Pea et al. (329.1, 388.6, 266.0 and 168 mg/L) for the four different collection intervals: 0-2, 2-4, 4-8 and 8-12 hours [2]. For the mean Vu fixed to 1.71 L the concentrations for the four different intervals were 460, 590, 480 and 320 mg/L. The maximum Vu of 2.35 L resulted in smaller concentrations of 360, 450, 345 and 220 mg/L for the different collection intervals and described the observed data more adequately. For the simulations of the urine concentration-time profiles of LEV, we fixed the Vu to 2.35 L. The regimen 500 mg LEV bid resulted in a maximum LEV urine concentration around 425 mg/L after 14 hours and a minimal concentration of 125 mg/L after 12 hours before the next LEV dose. The other regimen 750 mg qd showed a maximum LEV concentration of 480 mg/L after 2 hours and a minimum concentration of 50 mg/L after 24 hours before the next dose. Conclusions: The urine concentrations from the simulations performed with the adapted populationPK model were in agreement with the urine concentrations by Pea et al.. Since we are interested in the patient population at risk for therapy failure, we performed simulations with the higher Vu. The simulated concentration-time profiles of LEV were overall plausible with regard to their maximal and minimal concentrations at the respective time-points. The current investigation showed how in silico models streamline in vitro experiments exploring published clinical data to investigate treatment efficacy at the target site, i.e. the antibiotic effect on bacteria in catheterised ICU patients. References: 1. Schaeftlein, A. et al.: Abstract 2359, PAGE 21, Venice, Italy 2012. 2. Pea, F. et al.: Journal of Chemotherapy 2003, 15(6): 563-567.

POS.90 Identification of phase-II metabolites from serum samples of a human pharmacokinetic study with willow bark (Salicis cortex) Untergehrer, M.1; Knuth, S.1; Jürgenliemk, G.1; Heilmann, J.1 1 Faculty for Chemistry and Pharmacy; Pharmaceutical Biology; University of Regensburg; Universitätsstr. 31; D-93053 Regensburg; Germany

Willow bark (Salicis cortex, Salix ssp., Salicaceae) is used against low back pain and mild osteoarthritic and rheumatic complains as well as against minor articular pain, fever associated with common cold and headache. Whereas the efficacy of willow bark was shown in several clinical studies, the principle of action is not fully understood. Although the anti-inflammatory active compound salicylic acid is known as a metabolite of salicylic alcohol derivatives, its plasma-concentration is much too low to explain the efficacy of the whole willow bark extract [1]. To broaden the knowledge about the metabolisation of absorbed compounds from a willow bark extract, serum samples of a human pharmacokinetic study (10 volunteers, 12 h fasting time, controlled diet poor in phenolics, 13 blood withdrawals over a period of 24 h) [2] were analysed by LC-ESI-MS. A library of 116 possible phase-II metabolites concerning salicylic alcohol derivatives, flavonoids, and proanthocyanidins was used to identify possible metabolisation products. In the serum samples, phase-II metabolites of naringenin (2x glucuronide, 2x sulfate, 2x glucuronide-sulfate), eriodictyol (3x glucuronide, 1x sulfate), taxifolin (1x sulfate), catechin (1x sulfate, 1x glucuronide-sulfate), ferulic acid (1x sulfate), hydroxyphenyl-propionic acid (1x sulfate), saligenin (1x glucuronide, 1x sulfate), salicylic acid (1x sulfate, 1x free, 1x salicyluric acid), and catechol (1x glucuronide, 1x sulfate) could be identified. As taxifolin, ferulic acid, and hydroxyphenyl-propionic acid were not present in the extract, they could be metabolisation products of eriodictyol or naringenin and coumaric acid or C-ring cleaved flavonoids, respectively, which could be found in the willow bark preparation. No phase-II metabolites of procyanidins and no genuine flavonoid glycosides could be detected in all serum samples. These results confirm the demand to use not only isolated compounds from plant extracts for in vitro tests but also to consider their metabolisation products to increase the relevance of obtained pharmacological data [3].

POSTERS


References: 1. Nahrstedt, A. et al.; Wien Med. Wochenschr. 2007, 157: 348-351. 2. Knuth, S. et al.: Planta Med. 2013, 79: 1489-1494. 3. Kroon, P. A. et al.: Am. J. Clin. Nutr. 2004, 80: 15-21.

POS.91 Evaluation of kinetic and dynamic parameters of oral L-homoarginine supplementation in young volunteers Cordts, K.1,2; Schönhoff, M.1; Hoppe, J.3; Ortland, I.4; Hummel, F. C.3; Gerloff, C.3; Jaehde, U.4; Jagodzinsk, A. 2,5; Böger, R. H. 1,2; Atzler, D.1,2,6; Choe, C.2,3; Schwedhelm, E.1,2 1 Institute of Clinical Pharmacology and Toxicology, UKE, Martinistr. 52, 20246 Hamburg, Germany 2 DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany 3 Department of Neurology, UKE, Martinistr. 52, 20246 Hamburg, Germany 4 Institute of Pharmacy, Department of Clinical Pharmacy, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 5 Department of General and Interventional Cardiology, University Heart Center Hamburg-Eppendorf, Martinistr. 52, 20246 Hamburg, Germany 6 Vascular Biology, Institute for Stroke and Dementia Research, Klinikum der Universität München, Ludwig Maximilians-University of Munich

Background: L-Homoarginine (L-hArg) is a naturally occurring amino acid that differs from L-arginine by an additional methylene group. Formation of L-hArg from L-lysine and L-arginine is catalyzed by the L-arginine:glycine amidinotransferase (reviewed in Atzler et al. 2015). Median (25th; 75th percentile) plasma L-hArg concentration in healthy humans is 1.88 (1.47; 2.41) µM, and L-hArg is a natural component of pulses, e.g. Lathyrus species. L-hArg competes with L-arginine as a weak substrate for endothelial nitric oxide synthase and weakly inhibits arginase. Low L-hArg plasma concentrations have been associated with a higher risk for cardiovascular, cerebrovascular, and kidney diseases as well as for cardiovascular and all-cause mortality. The aim of this study was to investigate kinetic and dynamic properties of single and multiple oral doses of 125 mg L-hArg in twenty young healthy subjects. Subjects: Twenty apparently healthy Asian-Caucasian volunteers (11 female, 9 male) completed this study. The median (25th; 75th percentile) age was 28.5 (24.3; 48.8) years and the body mass index was 24.1 (22.9; 25.7) kg/cm2. Median hArg plasma concentration at baseline examination was 2.98 (2.32; 3.62) µmol/L. Study design: Participants received either 125 mg L-hArg (Wellnest International Ltd., West Sussex, UK) or placebo once daily in the morning for four weeks each in a randomized, double-blind, placebo-controlled cross-over design, interrupted by wash-out phases of four weeks. Primary endpoint was the evaluation of kinetic parameters, i.e. maximal concentration (Cmax), time-to-peak (Tmax), and area under the plasma concentration-time curve (AUC0-24hrs) after single and multiple doses of L-hArg. As secondary endpoints routine laboratory parameters, plasma L-arginine and asymmetric dimethylarginine (ADMA) concentrations, pulse wave velocity (PWV), augmentation index (AIx), flow-mediated vasodilatation (FMD), and transcranial magnetic stimulation (TMS) were evaluated at baseline and four weeks after each supplementation period. Results: Maximal L-hArg plasma concentrations were reached one hour after ingestion (Tmax). Oral single and multiple doses of 125 mg L-hArg increased baseline L-hArg plasma concentration by 8.74�4.46 [95% confidence intervals 6.65; 10.9] and 17.3 �4.97 [14.9; 19.6] µmol/L (Cmax), respectively. AUC0-24hrs were 63.5 �28.8 [50.0; 76.9] and 225 �78.5 [188; 2624] µmol/L*h for single and multiple doses, respectively. Blood glucose was increased after L-hArg supplementation (p<0.05). Alkaline phosphatase activity was increased after placebo treatment and at four weeks of follow-up (p<0.05 for both). Other laboratory parameters, L-arginine, ADMA, PWV, AIx, FMD, TMS did not change significantly after four weeks of L-hArg ingestion. Adverse events were equally distributed in both study arms. Summary and Conclusion: Oral supplementation with 125 mg L-hArg once daily for four weeks is safe and suitable to elevate L-hArg plasma concentrations in humans. With this study we paved the way for larger, prospective clinical studies to investigate the benefit of L-hArg supplementation in patients with cardiovascular or metabolic disease.

Acknowledgments: We gratefully acknowledge the contributions to sample and data collection made by volunteers and appreciate the excellent medical and technical assistance of A. Dehn, S. Griesbach, M. Kastner, J. Lockowandt, A. Steenpass, and J. Wiener. Dr Atzler acknowledges the support of the European Community under a Marie Curie Intra-European Fellowship for Career Development and Dr Choe was funded by an Else Kröner Memorial Stipendium from the Else Kröner-Fresenius-Stiftung. This work was funded by LMU Munich‘s Institutional Strategy LMUexcellent within the framework of the German Excellence Initiative (DA).

References: Atzler D et al.: Curr. Opin. Clin. Nutr. Metab. Care. 2015, 18(1):83-8.

DRUG DESIGN/MEDICINAL CHEMISTRY


3.6 Drug Design/Medicinal Chemistry

POS.92

Discovery of orally available dual sEH/PPARγ modulators for simultaneous treatment of hyperglycemia and hypertension in metabolic syndrome Blöcher, R.1; Lamers, C.1; Wittmann, S. K.1; Merk, D.1; Hartmann, M.1; Weizel, L.1; Diehl, O.1; Brüggerhoff, A.1; Boß, M.2; Kaiser, A.1; Schader, T.1; Göbel, T.1; Grundmann, M.3; Angioni, C.4; Heering, J.5; Abdul, H. K.6; Geisslinger, G.4; Wurglics, M.1; Kostenis, E.3; Brüne, B.2; Steinhilber, D.1; Imig, J. D.6; Schubert-Zsilavecz, M.1; Kahnt, A. S.1; Proschak, E.1 1 Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Strasse 9, D-60438 Frankfurt a. M., Germany. 2 Institute of Biochemistry I, Goethe-University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt a. M., Germany. 3 Institute of Pharmaceutical Biology, Rheinische Friedrich-Wilhelms-Universität Bonn, Nussallee 6, D-53115 Bonn, Germany. 4 Institute of Clinical Pharmacology, Goethe-University Frankfurt, Theodor-Stern-Kai 7, D-60590 Frankfurt a. M., Germany. 5 Project Group Translational Medicine and Pharmacology TMP, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Theodor-Stern-Kai 7, D-60590 Frankfurt a. M., Germany. 6 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA

The cardiometabolic syndrome (MetS) is a multifactorial disease cluster consisting of dyslipidemia, cardiovascular disease, type 2 diabetes mellitus and obesity. Pharmacological intervention in the MetS is dependent on numerous drugs, thus polypharmacy is an obvious problem in the treatment of MetS patients. This study focuses on the dual target approach to accomplish a more efficient therapy for MetS. The two targets addressed by dual ligand design are the soluble epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). In vivo studies could demonstrate that even though an inhibitor of sEH or PPARγ agonist have benefits when used individually, the combination is more beneficial for the multidisease features in cardiometabolic syndrome [1]. Using a split-and-combine strategy we designed a library of dual sEH/PPARγ modulators and proved that both targets can be simultaneously addressed by a merged pharmacophore [2,3]. In a follow-up study, we designed lead-like merged N-benzyl benzamides which were able to modulate sEH and PPARγ. Structure activity relationship studies on both targets were performed resulting in an equipotent submicromolar (IC50 (sEH) = 0.3 µM/ EC50 (PPARγ) = 0.3 µM) propionic acid N-benzyl benzamide derivative. Evaluation in vitro and in vivo displayed good ADME properties qualifying the novel dual modulator as pharmacological tool compound for long term animal models of MetS [4]. 8-week evaluation in spontaneously hypertensive obese rats (SHROB), a rat model of MetS, demonstrated excellent efficacy including simultaneous reduction of blood pressure, improvement of glucose tolerance, and organ protection. These results could be confirmed in an 8-week curative study in ZSF1 rat model of MetS.

Acknowledgments: This work was supported by the Else-Kröner-Fresenius Foundation and Deutsche Forschungsgemeinschaft (DFG; Sachbeihilfe PR1405/1-2; SFB 1039 Teilprojekt A07). R.B., O.D. and M.B thanks to the graduate college Translational Research Inovation Pharma (TRIP) for the PhD fellowship. The abstract figure is Courtesy of University of California Television.

References:

1. Imig J.D. Exp Biol Med 2012, 237, 1402. 2. Blöcher R. et al. J. Med. Chem. 2012, 55, 10771-10775 3. Blöcher R, et al. Med. Chem. Comm. 2016, 7, 1209-1216 4. Blöcher R. et al. J. Med. Chem. 2016, 59, 61-81

POS.93

First 17β-Hydroxysteroid Dehydrogenase Type 14 Inhibitors and their 3D-Structures in Complex with the Enzyme: Important Tools for the Characterization of a new Protein Marchais-Oberwinkler, S.1; Braun, F.1; Bertoletti, N.1; Möller, G.2; Adamski, J.2,3,4; Salah, M.5; Van Koppen, C. J. 5; Heine, A.1; Klebe, G.1 1 Philipps University Marburg, Institute for Pharmaceutical Chemistry, Marbacher Weg 6, 35037 Marburg, Germany 2 Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Experimental Genetics, Genome Analysis Center, Ingolstaedter Landstraße 1, 85764 Neuherberg, Germany 3 Lehrstuhl für Experimentelle Genetik, Technische Universität München, 85350 Freising-Weihenstephan, Germany 4 German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany 5 ElexoPharm GmbH, Campus A12, 66123 Saarbrücken, Germany

17β-HSD141 belongs to the SDR family and oxidizes the position 17 of estradiol and 5-androstenediol using NAD+ as cofactor. As inhibitors are useful tools to characterize enzymes, the goal of this study was to identify the first inhibitors of 17β-HSD14. In a preliminary study a library of 17β-HSD1 and 17β-HSD2 inhibitors, selected with respect to scaffold diversity, was tested for 17β-HSD14 inhibition. The most interesting hit was taken as starting point for chemical modification of the initial lead applying a ligand-based approach. The designed compounds were synthesized and tested for 17β-HSD14 inhibitory activity with a fluorescence-based assay using the recombinant purified protein, estradiol as substrate and NAD+ as cofactor. The best inhibitors identified will be presented as well as their binding mode in the enzyme’s active site, which was elucidated after analysis of the crystallographic structures2. The selectivity of the best compounds towards 17β-HSD1 and 17β-HSD2 will also be addressed. References: 1. Lukacik, P.; Keller, B.; Bunkoczi, G.; et al. Biochem. J. 2007, 402(3): 419–427 2. Bertoletti, N.; Braun, F.; Lepage, M.; et al. J. Med. Chem. 2016, DOI: 10.1021/acs.jmedchem.6b00293.

POS.94

Design and synthesis of a second-generation clickable, photoreactive cholesterol analogue Bartl, N.1; Bracher, F.1 1 Department of Pharmacy – Center for Drug Research, Ludwigs-Maximilians-University, Butenandtstr. 5-13, D-81377 Munich, Germany

Cholesterol is the most abundant sterol lipid in mammalian cells. It and its metabolites play a major role in regulating diverse biological processes. Alterations in these processes are presumed to be associated with various diseases like arteriosclerosis or Alzheimer’s disease. Located in membranes, cholesterol is an essential structural component, but also acts by direct interaction with membrane proteins [1]. Very limited knowledge exists about this regulating interaction, mainly because it is not based on a classical ligand-receptor interaction [1,2]. In order to identify and examine more closely these sterol-binding proteins, we designed and synthesized a novel cholesterol-based molecule as a chemoproteomic tool. The synthesized molecule bears a photoreactive diazirine group for photoaffinity labeling (PAL) as well as a terminal alkyne moiety for the copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) to conjugate any reporter azide (containing e.g. biotin or fluorescent dyes) via “click-chemistry”. To ensure that the sterol core is fully recognized by the target membrane proteins, we left the tetracyclic sterol structure untouched. Both the diazirine and the alkyne moieties were incorporated into the side chain, avoiding hydrolysis-labile functional groups and exceeding the size of the original cholesterol side chain too much. These two advantages, a native steroid core and hydrolytic stability, make this new molecule superior to earlier described clickable, photoreactive sterol probes [3] and suitable for bioorthogonal in vivo studies in any compartment of cells. References: 1. Song, Y. et al.: Protein Sci. 2014, 23(1): 1-22 2. Peng, T. et al.: Curr. Opin. Chem. Biol. 2014, 21: 144-153 3. Hulce, J. et al.: Nature Methods 2013, 10(3): 259-264

POSTERS


POS.95

New approaches to β-carbolines via indole-2-Weinreb amides Kamlah, A.; Lirk, F.; Bracher, F. Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians-University, Butenandtstr. 5-13, 81377 Munich, Germany

β-Carbolines are a large and structurally diverse class of alkaloids from plants, microorganisms, and marine organisms, showing a broad range of biological activities. Numerous approaches to 1-substituted β-carbolines have been worked out over the decades, and most of these approaches start from tryptophan or tryptamine derivatives. Unfortunately, tryptamines bearing additional substituents on the benzene ring are poorly available, and consequently, 1-substituted β-carbolines with additional residues on ring C are accessible only via multistep procedures. In recent projects, we identified highly substituted β-carbolines as lead structures for the inhibition of the protein kinase DYRK1A [1] and as promising new antivirals based on the inhibition of the kinase CLK1 [2]. For being able to perform comprehensive analysis of structure-activity relationships in these chemotypes, we worked out new approaches to polysubstituted β-carbolines. As starting materials we selected indole-2-carboxylates, which are, with a broad variety of substituents on the benzene ring, readily available via established methods. Conversion of these compounds into Weinreb amides opened the opportunity to introduce variable residues R’ utilizing respective organometallic compounds, and completion of ring A (pyridine moiety) of the target compounds was accomplished with different building blocks.

References: 1. Rüben K. et al.: PLOS ONE 2015, 10(7):e0132453. 2. Karlas A. et al.: Nature Communications 2016, 7:11320.

POS.96

Synthetic approach to novel 4-substituted 3-arylideneindolin-2-ones as NAD+ dependent histone deacetylase (sirtuin) inhibitors Ong, N.1; Swyter, S.2; Jung, M.2; Bracher, F.1

1 Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-University, Butenandtstr. 5-13, 81337 Munich, Germany 2 Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104 Freiburg i. Br., Germany

Previous work on 3-arylidene-indolin-2-ones in our group yielded two compounds showing promising biological activities on Sirt2 [1].

Docking studies indicated that Sirt2 contains additional space in binding pocket that could be covered by an additional substituent at C-4 of the indoline-2-one moiety. This prompted us to introduce diverse substituents at said position for SAR studies. As the established synthetic pathway was not suitable for synthetic modifications at the said position we strived to develop a new synthetic approach allowing the introduction of diverse substituents.

All synthesized molecules were submitted to a homogenous fluorescence based assay for Sirt2 inhibition. Acknowledgments: This project was funded by DFG-Förderung GRK1976.

References: 1. K. Huber et al., J. Med. Chem. 2009, 53: 1383–1386.

POS.97

Development of benzothiazoles as dual 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 inhibitors Cheung, S.-Y.1; Werner, M. 2; Werz, O.2; Schubert-Zsilavecz, M.1, Hanke, T.1 1 Goethe University of Frankfurt am Main, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany 2 Friedrich-Schiller University of Jena, Philosophenweg 14, 07743 Jena, Germany

Prostaglandins (PGs) and leukotrienes (LTs) are powerful bioactive lipid mediators that have a large number of biological actions in the human body [1], [2]. The common precursor of PGs and LTs is arachidonic acid (AA). The 5-lipoxygenase (5-LO) and the microsomal prostaglandin E2 synthase-1 (mPGES-1) are both enzymes within the arachidonic acid cascade. 5-LO is the initial enzyme which catalyzes the conversion of AA to the corresponding LTs; whereas the mPGES-1 is responsible for the transformation of PGH2 into PGE2 which is one of the most prominent mediators of inflammation, pain and fever. A valuable pharmacological approach for anti-inflammatory therapy is the dual inhibition of 5-LO and mPGES-1. In contrast to the traditional NSAIDs the dual inhibition of PGs and LTs might be superior over single interference with PGs in terms of anti-inflammatory effectiveness as well as regarding reduced side effects [3]. In the post area of selective COX-2 inhibitors different approaches for dual inhibition of PGs and LTs have been pursued, like dual COX/LO, dual COX-2/LTA4-Hydrolase or dual 5-LO/mPGES-1 inhibitors [4, 5, 6, 7]. Within the dual 5-LO/mPGES-1 inhibitors the pirinixic acid derivatives are the most advanced one. However pirinixic acid derivatives are well known compounds with many various biological activities especially PPARα and PPARγ activation [8]. Therefore, in this series we replaced the central scaffold of the pirinixic acid, the chlorinated pyrimidine core, by a benzothiazole, which was identified by a virtual screening approach [9]. Here we present the synthesis and in vitro pharmacological characterization of the benzothiazole derivatives and we were able to identify compounds, which are about equally potent to the most potent pirinixic acid derivatives. References: 1. Funk, C. D.: Science 2001, 294(5548): 1871−1875. 2. Samuelsson, B., Morgenstern, R., Jakobsson, P. J.: Pharmacol Rev 2007, 59(3): 207–224. 3. Koeberle, A., Werz, O.: Curr. Med. Chem. 2009, 16(32): 4274–4296. 4. Celotti, F., Laufer, S.: Pharmacol Res. 2001, 43(5): 429-436. 5. Chen, Z. et al.: J Med Chem 2011, 54(10): 3650-3660. 6. Koeberle, A. et al.: J Med Chem. 2008, 51(24): 8068-8076. 7. Hanke, T. et al.: J Med Chem. 2013, 56(22): 9031-9044. 8. Merk, D. et al.: Future Med Chem. 2015, 7(12): 1597-1616. 9. Waltenberger, B. et al.: J. Med. Chem. 2011, 54(9): 3163–3174.

POS.98

Novel antifungal 2-arylindoles Luber, M.1; Stadler, M.1; Bracher, F.1 1 Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians-University, Butenandtstr. 5-13, 81377 Munich, Germany

In the course of a project aimed at the optimization of indole-derived inhibitors of the protein kinase CLK1, a series of 2-arylindoles was



prepared. Routine testing of all intermediates in our standard agar diffusion assay for antifungal and antibacterial activities gave surprising results for some of these indoles, as well as their synthetic precursors (hydrazones). High in vitro activity against some fungi (Yarrowia lipolytica, Candida glabrata, Hyphopichia burtonii) was observed, but the compounds did not show any antibacterial effects against gram-negative and gram-positive bacteria. The hydrazones were excluded from further investigations due to their chemical instability and proposed toxicity. A systematic investigation of structure-activity relationships was performed with the 2-arylindole chemotype. A systematic variation of the residues on the benzene moiety of the indole as well as the (hetero)aryl substituent at C-2 of the indole was performed, and selected chlorine-substituted compounds were shown to exhibit antifungal activity comparable to or better than the reference drug clotrimazole. In addition to our experimental work we found out that the antifungal activity appears to be in relationship with theoretically calculated Mulliken charges. The optimized structure parameters and the Mulliken charges of each 2-(hetero)arylindole have been calculated at the HF/STO-3G, B3LYP/STO-3G, B3LYP/6-31G(d), B3LYP/6-31G(d,p), B3LYP/6-311G(d) and B3LYP/6-311G(d,p) levels of theory, using the Gaussian03 software package [1,2].

References: 1. Chana, A. et al.: Chem. Res. Toxicol. 2002, 15(2): 1514-1526. 2. Buyukuslu, H. et al.: Spectrochimica Acta Part A 2010, 75(4): 1362-1369.

POS.99 A novel approach to 2-substituted quinolin-4(1H)-ones via nickel boride-mediated reductive ring transformation of (2-nitrophenyl)isoxazoles Lohrer, B.1; Bracher, F.1 1 Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-University, Butenandtstr. 5-13, 81377 Munich, Germany

2-Substituted quinolin-4(1H)-ones represent a promising class of antimicrobial compounds. Their N-alkylated analogs for example show antimycobacterial activity [1,2]. Another interesting biological property is their essential function in an intercellular communication system of Pseudomonas aeruginosa called quorum sensing, e.g. by 2-heptylquinolin-4(1H)-one (HHQ) [3].

NH

O

2-Heptylquinolin-4(1H)-one (HHQ)

Here we report a novel method for the synthesis of 2-substituted quinolin-4(1H)-ones starting from 5-(2-nitrophenyl)isoxazoles, which are readily available from 2-nitrophenylacetylenes via 1,3-dipolar cycloaddition. Key step is the simultaneous reduction of the nitro group and reductive ring cleavage of the isoxazole, followed by spontaneous cyclization to give the quinolin-4(1H)-one. Nickel boride was identified as the most suitable reductant for this transformation.

In contrast to standard methods (Conrad-Limpach and related syntheses), which build up the quinolin-4(1H)-one ring system via intramolecular ring acylation, this protocol avoids the formation of mixtures of isomers if additional substituents are present on the benzene ring. References: 1. Wube, A. A. et al.: Eur. J. Med. Chem. 2011, 46(6): 2091-2101. 2. Wube, A. et al.: Molecules 2012, 17(7): 8217-8240. 3. Jerry Reen, F. et al.: Org. Biomol. Chem. 2012, 10(44): 8903-8910.

POS.100/SL.33 Rational design of thermodynamic and kinetic binding profiles by optimizing surface water networks coating protein bound ligands Krimmer, S. G.1; Cramer, J.1; Betz, M.1; Fridh, V.2; Karlsson, R.2; Heine, A.1; Klebe, G.1 1 Institute of Pharmaceutical Chemistry, University of Marburg, Marbacher Weg 6, 35032 Marburg, Germany 2 GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden


POS.101 Structure and Fragment Based Lead Discovery for New Potential h17β-HSD14 Inhibitors. Bertoletti, N.1; Zara, L.1, Metz, A.1; Heine, A.1; Klebe, G.1, Marchais-Oberwinkler, S.1 1 Philipps University Marburg, Institute for Pharmaceutical Chemistry, Marbacher Weg 6, 35032 Marburg, Germany

Human 17β-hydroxysteroid dehydrogenase type 14 (h17β-HSD14) is the latest identified 17β-HSD member of the Short-chain Dehydrogenase-Reductase super family (SDR) [1,2]. In vitro, h17β-HSD14 catalyses the NAD+ dependent oxidation at position 17 of estradiol (E2) and 5-androstene-3β,17β-diol (5-diol) to estrone and dehydroepiandrosterone, respectively. Northern blot experiments revealed that h17β-HSD14 is predominantly expressed in placenta, liver and brain [1]. As it is expressed in the brain, it may become a potential target for the treatment of neuronal diseases, which are estradiol level dependent. Two variants of h17β-HSD14 are known. The first one was isolated from the retina and contains a serine at position 205 (S205). An allelic variant differs only by a threonine at this position (T205), and was identified from melanotic melanoma cells. The in vitro turnover of both variants of h17β-HSD14 for E2 and 5-diol is equal. Recently, we reported and characterized the apo (S205), holo (S205 and T205) and ternary complex crystal structures of h17β-HSD14 with estrone as well as with a nonsteroidal inhibitor (T205) [3]. In addition, we initiated a fragment-based lead discovery (FBLD) campaign by screening a 96 fragment library containing successful detected crystallographic screening hits and assembled considering the “Rule of 3” as a guideline. FBLD can explore the chemical space with respect to scaffold diversity capable of binding to a protein binding pocket. The small size of the fragments makes them suitable for optimizing activity and selectivity for a target. We believe that crystallographic fragment screening is a promising approach which may lead to the identification of more hits than other biophysical screening methods, especially for those that have weak binding affinity [4], while providing essential structural information about binding modes. We will present the binding mode of the crystallographic hits from our fragment screen against h17β-HSD14 and will we discussed how this information can be used for the improvement of our inhibitors.

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References: 1. Lukacik, P. et al.: Biochem. J. 2007, 402(3): 419–427. 2. Marchais-Oberwinkler, S. et al.: J. Steroid Biochem. Mol. Biol. 2011, 125(1-2): 66–82. 3. Bertoletti et al.: J. Med. Chem. 2016, Article ASAP; doi: 10.1021/acs.jmedchem.6b00293 4. Schiebel et al.: ACS Chem Biol. 2016, 11(6):1693–1701.

POS.102 Pyridoisothiazolones as covalent modifiers of Lysine Acetyltransferase Activity Simon, R. P.1; Furdas, S.1; Gajer, J.1; Sippl, W.2; Jung, M.1 1 Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, 79104 Freiburg, Germany 2 Institute of Pharmacy, Martin-Luther-Universität Halle-Wittenberg, 06120 Halle, Germany

Lysine acetyltransferases (KATs) are epigenetic modifiers that catalyze the transfer of acetyl groups from their cofactor acetyl-CoA to the ε-amino group of lysines in histones and other protein substrates. Aberrant activity of these enzymes has been correlated to the manifestation of several diseases, including cancer, inflammation, and neurodegenerative disorders.1, 2 Therefore, the development of small molecule modulators of KAT activity may provide useful tools for probing the precise implication of these enzymes in pathogenesis and assess their potential as possible drug targets.3, 4 Our group has developed N-substituted pyridoisothiazolones as covalent inhibitors of lysine acetyltransferases by combining virtual screening and molecular docking studies using the structural information of the hPCAF (KAT2B) catalytic domain. Initial structure-activity-relationship studies focused on the substitution pattern of the isothiazolone nitrogen atom. We were able to show that N-phenyl derivatives inhibit KAT activity unselectively in low micromolar concentrations, whereas N-benzyl analogs were selective for hCBP (KAT3A).5 The inhibitory potency of these modulators was confirmed in cellular assays as well as in a neuroblastoma xenograft mouse model.6 To further improve the initial pyridoisothiazolone lead scaffold, a functionalized derivative was developed that allows derivatization of the pyridine moiety. In this way, a small library of pyridine-substituted compounds was synthesized and tested for inhibition of recombinant hPCAF using a heterogeneous antibody-based assay with time-resolved fluorescence readout in order to deduce initial structure-activity-relationships. Further optimization of this promising class of compounds will possibly yield modulators with improved selectivity and physicochemical properties to serve as epigenetic tools. References: 1. Portela, A.; Esteller, M. Epigenetic modifications and human disease. Nat. Biotechnol. 2010, 28, 1057-1068. 2. Dekker, F. J.; van den Bosch, T.; Martin, N. I. Small molecule inhibitors of histone acetyltransferases and deacetylases are potential drugs for inflammatory diseases. Drug Discovery Today 2014, 19, 654-660. 3. Simon, R. P.; Robaa, D.; Alhalabi, Z.; Sippl, W.; Jung, M. KATching-Up on Small Molecule Modulators of Lysine Acetyltransferases. J. Med. Chem. 2016, 59, 1249-70. 4. Arrowsmith, C. H.; Bountra, C.; Fish, P. V.; Lee, K.; Schapira, M. Epigenetic protein families: a new frontier for drug discovery. Nat. Rev. Drug Discovery 2012, 11, 384-400. 5. Furdas, S. D.; Hoffmann, I.; Robaa, D.; Herquel, B.; Malinka, W.; Świątek, P.; Akhtar, A.; Sippl, W.; Jung, M. Pyrido- and benzisothiazolones as inhibitors of histone acetyltransferases (HATs). MedChemComm 2014, 5, 1856-1862. 6. Gajer, J. M.; Furdas, S. D.; Grunder, A.; Gothwal, M.; Heinicke, U.; Keller, K.; Colland, F.; Fulda, S.; Pahl, H. L.; Fichtner, I.; Sippl, W.; Jung, M. Histone acetyltransferase inhibitors block neuroblastoma cell growth in vivo. Oncogenesis 2015, 4, e137.

POS.103 Synthesis and biological evaluation of novel benzylamine-type antifungals Krauss, J.; Stadler, M.; Bracher, F. 1 Department of Pharmacy LMU Munich

A series of benzylamine derived antifungals with isooctyl side chain and alkyl or aryl ethers was synthesized and evaluated for antifungal activity as novel ergosterol biosynthesis inhibitors (SBI). An isooctyl side chain was introduced for imitating the alkyl side chain of ergosterol and its precursors, alkyl or aryl ethers should imitate the rings A and B of ergosterol.

The target compounds were prepared from appropriately substituted benzaldehydes and isooctylamines by reductive amination and subsequent precipitation with hydrogen chloride. The antifungal activity was evaluated in an agar diffusion assay and a microdilution assay against Candida glabrata, Yarrowia lipolytica, and Aspergillus niger compared to clotrimazole and terbinafine.

References: 1. Nussbaumer, P. et. al.: J. Med. Chem. 1993, 36: 2115-2120. 2. Krauss, J. et. al. Arch. Pharm. Chem. Life Sci. 2014, 347: 1-8.

POS.104 The Involvement of the Mitochondrial Amidoxime Reducing Component (mARC) in the Reductive Metabolism of Hydroxamic Acids Ginsel, C.1; Plitzko, B.1; Jakobs, H. H.1; Froriep, D.1; Rothert, M.1; Stolfa, D. A.3; Jung, M.3; Mendel, R. R.2; Bittner, F.2; Havemeyer, A.1; Clement, B.1 1 Christian-Albrechts-University Kiel, Department of Pharmaceutical Chemistry, Gutenbergstraße 76, 24118 Kiel, Germany 2 Department of Plant Biology, Braunschweig University of Technology, 38023 Braunschweig, Germany 3 Albert-Ludwigs-Universität Freiburg, Institut für Pharmazeutische Wissenschaften, Albertstr. 25, D-79104 Freiburg, Germany

The mitochondrial amidoxime reducing component mARC is a recently in our lab discovered molybdenum enzyme in mammals which in concert with the electron transport proteins cytochrome b5 and NADH cytochrome b5 reductase catalyzes the reduction of N-oxygenated structures [1]. This three component enzyme system plays a major role in N-reductive drug metabolism and is thus the counterpart of cytochrome P450s and flavin-containing monooxygenases (FMO) catalysed N-oxygenations [2]. Belonging to the group of N-hydroxylated structures hydroxamic acids are also potential substrates of the mARC-system (see figure 1). Hydroxamic acids show a variety of pharmacological activities and are therefore often found in drug candidates [3,4]. On the other hand they can also exhibit toxic properties as it is the case for many aryl hydroxamic acids formed during the metabolism of arylamides[5,6]. Thus, the metabolic stability of new hydroxamic acid containing drug candidates as well as the detoxification of toxic hydroxamic acid metabolites are of significant interest. Biotransformation assays with recombinant human proteins, subcellular porcine tissue fractions as well as in HEK293 cells were performed. The mARC-dependent reduction of the model compound benzhydroxamic acid could be demonstrated. The N-reductive conversion of the approved histone deacetylase inhibitor Vorinostat (SAHA) however, was demonstrated to be rather low, thereby reflecting the relatively high metabolic stability and oral bioavailability of this compound. The toxic N-hydroxylated metabolite of the analgesic phenacetin, NOH-phenacetin[5], is not reduced by the mARC-system under the chosen conditions. This confirms the high toxicity of this component, as it needs to be detoxified by other pathways. As a consequence, for the evaluation of the metabolic stability of new hydroxamic acid containing drug candidates or for the evaluation of the potential risc for toxic metabolites it should be obligatory to monitor the N-reductive metabolism by the mARC-system. This study underlines the hypothesis that mainly those N-oxygenated compounds exhibit toxicity which are not easily reduced by the mARC enzyme system. So far our lab could demonstrate the reduction of several N-oxygenated structures (N-hydroxyamidines, N-hydroxyguanidines, oximes, N-oxides, hydroxylamines, sulfhydroxamic acids). These functional groups are reduced significantly irrespective of the complete structure of the molecule. In contrast to this for hydroxamic acids the reduction rate might be low or undetectable depending on the structure. Further



structure-activity studies will be necessary for the investigation of these influences. This might be helpful for the design of drug candidates avoiding deactivation by a mARC catalysed reduction.

Acknowledgments: We thank Melissa Zietz and Sven Wichmann (Christian-Albrechts-University Kiel, Germany) for technical assistance.

References: 1. Havemeyer, A. et al.: J Biol Chem 2006, 281(46), 34796-802. 2. Gruenewald, S. et al.: J Med Chem 2008, 51(24), 8173-77. 3. Ratner, M.: Nat Biotechnol. 2014, 32(9), 853-4 4. Barb, A., Zhou P.: Curr Pharm Biotechnol. 2008, 9(1), 9-15 5. Vaught, J. el al.: Cancer Res. 1981, 41(9), 3424-9 6. Hinson, J., Mitchell J.: Drug Metab Dispos. 1976, 4(5), 430-5

POS.105 Improvement of anthranilic acid derivates as a dual agonist of FXR and PPARα Gellrich, L. M.1; Schubert-Zsilavecz, M.1; Merk, D.1 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt a. M., Germany; [email protected]

The prevalence of non-alcoholic fatty liver disease (NAFLD), one of the most common liver disease, is rising constantly and currently amounts to 20-30% in the general population of Western countries [1,2]. The disease is particularly associated with obesity and insulin resistance and constitutes the hepatic manifestation of the metabolic syndrome [2]. 5-20% of NAFLD patients develop a non-alcoholic steatohepatitis (NASH) which may lead to fibrosis and later on to liver cirrhosis and hepatocellular carcinoma [1]. There are no approved drugs for the treatment of NASH. The current therapy consists of Vitamin E and thiazolidinediones. Regarding the drug pipeline Obeticholic acid, a farnesoid X receptor (FXR) agonist, is the leading clinical candidate in phase III [3,4]. Additionally, GFT505, a dual peroxisome proliferator-activated receptor (PPAR) α/δ agonist, successfully completed a phase IIb study [2,4]. Therefore, the nuclear receptors FXR and PPARα are interesting targets for new active ingredients to treat NASH. FXR is a bile acid receptor, whose activation decreases hepatic gluconeogenesis and reduces circulating triglycerides [3]. The main function of FXR is to control bile acid and cholesterol regulation. It was observed that the hepatic expression of the receptor in NAFLD patients is decreased [2]. PPARα upregulates ß-oxidation and induces enzyme systems, that protect cells against oxidative stress und suppress inflammation [5]. The receptor is activated by fatty acids and enhances their clearance in the liver. PPARα knockout mice develop increased hepatic steatosis and PPARα expression is low in patients with NAFLD. This is among other things a consequence of a low FXR level, because FXR is linked to the PPARα expression [2,7]. During the investigation of anthranilic acid derivates it was revealed that some agents of this substance group show a dual agonism of FXR and PPARα [6]. Since the bioavailability of these agents is not very drug-like, we substituted functional groups that are unimportant for the receptor binding with more hydrophilic groups. The tert-butyl group of the lead compound was replaced with some aliphatic amine components, e.g. diethylamine or cyclic amine components, e.g. azetidine. Furthermore, we aimed an approximation of the EC50 of FXR and PPARα, thus the carboxylic acid and the methyl group at the head group of the anthranilic acid were modified. Changes at the methyl group at the head group assimilate the activation of FXR and PPARα and improve the maximum activation at FXR and PPARα in general. Elongation of the carboxylic acid at the head group and implementation of hetero atoms is another opportunity to improve the receptor activation.

References: 1. Bellentani, S. et al.: Dig Dis. 2010, 28: 155-161. 2. Cave, M. C. et al.: Biochim. Biophys. Acta 2016, 1874: 30041-30044 3. Neuschwander-Tetri, B. A. et al.: Lancet. 2015, 385: 956-965. 4. Jahn, D. et al.: Dig Dis. 2016, 34(4): 356-363. 5. Tanaka, N. et al.: Biochim. Biophys. Acta 2015, 1852: 1242-1252. 6. Merk, D.& Lamers C. et al.: Bioorganic & Medicinal Chemistry 2015, 23: 499-514. 7. Lalloyer, F. et al.: Arteriosclerosis thrombosis and vascular biology. 2011, 31: 1573-1579.

POS.106 Pan retinoid X receptor agonist causes side-effects of lipid-lowering agent pirinixic acid Pollinger, J. C.1; Heitel, P.1; Kalinowsky, L.1; Proschak, E.1; Schubert-Zsilavecz, M.1; Merk, D.1 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt a. M., Germany; [email protected]

Retinoid X receptors (RXR) are part of the superfamily of nuclear receptors and many of these nuclear receptors, including e.g. the farnesoid X receptor (FXR) and the peroxisome proliferator-activated receptors (PPAR), form heterodimers with RXR as counterpart. These heterodimers can be further divided into two categories: permissive and non-permissive heterodimers. The permissive heterodimers can be activated by agonists of either partner and also the simultaneous presence of both RXR and partner receptor agonists resulting in a synergistic activating effect [1]. Due to this, RXR plays a key role in the modulation of numerous physiological functions mediated by nuclear receptors [2]. Although this seems to be a chance of influencing many diseases, a pan-agonistic effect of RXR has only reached clinical approval in the treatment of a few diseases such as cutaneous kaposi’s sarcoma or chronic hand eczema with alitretinoin as active ingredient [3]. A selective agonist for one of the three subtypes has not been discovered so far but holds therapeutic potential for multiple indications. Pirinixic acid is a dual PPAR α and γ agonist and has shown robust cholesterol lowering effects in rodent models [4]. But although its activity is similar to that of fibrates, pirinixic acid was never applied to humans, which might be explained by pleiotropic side effects [5]. We have discovered that pirinixic acid is a partial agonist of all three subtypes of retinoid X receptors and report a preliminary structure-activity relationship (SAR) for the agonistic interaction of pirinixic acid and RXR. We have characterized our compound library of pirinixic acid derivatives in reporter gene assays for all RXR subtypes and observed a significantly different SAR for RXR than for PPAR activation. Especially the 2,3-xylidino and the 4-chlorophenyl residues caused activity on RXR. These findings might explain the side effects of pirinixic acid observed in preclinical development. Furthermore, our data indicates pirinixic acid as valuable lead compound to develop novel RXR modulators. References: 1. Evans, R.M., Mangelsdorf, D.J.:Cell 2014, 157(1): 255-266 2. Pérez, E. et al.: Biochimica et Biophysica Acta 2012, 1821(1): 57-69 3. Bubna, A.: Indian J Dermatol 2015, 60(5): 520 4. Santilli, A., Scotese, A., Tomarelli, R.: Experientia 1974, 30(10): 1110-1111 5. Merk, D. et al.: Future Med. Chem. 2015, 7 (12): 1597-1616

POS.107 Synthesis of folic acid derivatives for prostate cancer imaging Peric, N.1; Maison, W.1

1 Pharmaceutical and Medicinal Chemistry, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany

Targeting tumor specific cell surface epitopes, so called tumor markers, with small molecules can lead to improved tools for cancer diagnosis and therapy. Elevated levels of prostate specific membrane antigen (PSMA) are used as a tumor marker for prostate cancer [1]. PSMA is a glycosylated type-II membrane protein that is present in high density on the surface of malignant prostate cancer cells. Its expression increases with clinical stage, thus making it an extremely useful tumor marker [2]. Both, small molecules and antibodies have been shown to be useful for PSMA targeting and have been used successfully for tumor imaging and immune therapy. Phosphinic acids like GPI, for example, can be

POSTERS


used as modular ligands for the targeting of prostate cancer [3]. GPI binds with nanomolar affinity to PSMA and permits conjugation of effector molecules like dyes without altering the binding properties [4]. However, GPI has suboptimal binding properties in vivo and needs to be improved for imaging applications in animals or humans. GPI has been developed as a transition state analogue of the native PSMA substrate N-acetylaspartylglutamate (NAAG). In addition, PSMA has been found to act as a folate hydrolase and does thus recognize folylpolyglutamates in the same binding pocket as NAAG [5]. The poster highlights our efforts to design and synthesize improved modular PSMA ligands. We have combined properties of the known ligand GPI with structural elements of folate and a conjugation site for effector molecules according to docking studies. The synthesis involves the arylation of benzylamines, which are notoriously difficult, yet highly important, starting materials for Pd-catalyzed arylations of the Buchwald Hartwig type.

Acknowlegments: We kindly want to thank to Dr. Thomas Lemcke from Institute for Pharmaceutical and Medicinal Chemistry, University of Hamburg, for performing molecular modeling studies.

References: 1. Hilgenfeld R. et al.: EMBO J. 2006, 25(6), 1375-1384. 2. Heston W. D. et al.: Urology 2001, 57(6), 1179-1113. 3. Coward J.K. et al.: J. Org. Chem. 2005, 70(17), 6757-6774. 4. Maison W. et al.: J. Med. Chem. 2009, 52(2), 544-550. 5. Carter, R. E., Feldman, A. R., Coyle, J. T.: PNAS 1996, 93(2), 749-753.

POS.108 Medicinal Inorganic and Boron-Organic Chemistry: From Boronic Acids to Boron Clusters Scholz, M. S. University of Bonn, Pharmaceutical Institute, An der Immenburg 4, 53121 Bonn, Germany

Pharmaceuticals based on inorganic elements are rare. However, these inorganic elements offer intriguing properties, which can be of high value for the application in drug molecules. Besides lithium and platinum complexes, boron compounds are becoming more and more popular. This is clearly underlined by the recent acquisition of Anacor Pharmaceuticals by Pfizer for 5.2 billion dollars. Anacor Pharmaceuticals is specialized on boron compounds and is developing boronic acid drugs, which already reached the market or will be approved soon. The biological activity of boronic acids is attributed to the electron-deficient nature of the element boron. This feature also gives rise to versatile cluster compounds. These boron clusters begin to enter the field of drug development.[1] In addition to boronic acids, our research addresses the chemistry and biological activity of 12-vertex dicarba-closo-dodecaborane (in short carborane) clusters (Fig. 1).

Fig. 1: Carborane Isomers.

Earlier studies integrated carboranes in known bioactive lead structures.[2-6] Now, we create unique carborane-based compound libraries and screen for new applications. We develop chemical procedures to modify the cluster compounds at specific vertex positions, both at the cluster carbon and at the cluster boron atoms. We

focus on those functional groups that give access to carborane building blocks. Reacting these building blocks with organic compounds allows us to create diverse organic-inorganic hybrid-libraries. The resulting hybrid compounds are promising tools to study the applicability of carboranes as pharmacophores and identify unprecedented biological activities. References: 1. Scholz, M. et al.: Chem. Rev. 2011, 111: 7035-7062. 2. Scholz, M. et al.: ChemMedChem 2009, 4: 746-748. 3. Scholz, M. et al.: ChemMedChem 2011, 6: 89-93. 4. Scholz, M. et al.: Eur. J. Med. Chem. 2011, 46: 1131-1139. 5. Scholz, M. et al.: Bioorg. Med. Chem. 2012, 20: 4830-4837. 6. Neumann, W. et al.: ChemMedChem 2016, 11: 175-178.

POS.109 Investigation of structure-affinity relationships for novel GluN2A selective ligands based on the lead compound TCN-201 Müller, S. L.1; Schreiber, J.1; Wünsch, B.1; Seebohm, G.2; 1 PharmaCampus, Department of Pharmaceutical and Medicinal Chemistry, Corrensstraße 48, 48149 Münster, Germany 2 Institute for Genetics of Heart Diseases, Domagkstraße 3, 48149 Münster, Germany

NMDA receptors, ligand gated ion-channels, are expressed in the central nervous system as well as in the periphery. Due to the involvement of GluN2A subunit containing NMDA receptors in diseases like anxiety, depression, sinus arrhythmia and reduction of heart contractility, the development of selective GluN2A antagonists is envisaged. [1,2] The sulfonamide 1 (TCN-201) is the lead compound for the synthesis of GluN2A selective NMDA ligands. It shows a moderate GluN2A affinity (IC50 = 158 nM) and additionally a high selectivity towards GluN2B containing receptors (GluN2A : GluN2B > 300). [3] To increase the activity and to investigate structure-affinity relationships for this compound class, 1 was modified in a systematic way. The biological activity of the novel derivatives was investigated electrophysiologically, i.e. the inhibition of the ion flux through GluN2A containing receptors was recorded.

References: 1. Boyce-Rustay, J. M., Holmes, A.: Neuropsychopharmacology 2006, 31: 2405-2414. 2. Bogdanova, A., Institute of Veterinary Physiology, University Zurich. 3. Bettini, E. et al.: J. Pharmacol. Exp. Ther. 2010, 335: 636–644.

POS.110 Templated assembly of protein-binding fragments as a method to reveal inhibitors of enteroviral 3C proteases Tauber, C.1; Becker, D.1; Rademann, J.1 1 Institute of Pharmacy, Medicinal Chemistry, Freie Universität Berlin, Königin-Luise-Straße 2+4, 14195 Berlin, Germany

Using small-molecule fragments as starting points for the development of drugs is a tempting method that is able to provide ligands with improved potency, ligand efficiency, and selectivity. Unfortunately, it is usually restricted by the detection of these low affinity inhibitors. Our group was able to circumvent this by establishing dynamic ligation screening (DLS) [1]. Here a chemical reaction between fragments occurs whereas the equilibrium is shifted by addition of the protein as a template. Furthermore, DLS uses classical bioassays for the detection of favourable fragment combinations. The approach works for various nucleophiles such as amines, thiols and alcohols reacting with electrophilic fragments on a protein surface [2]. Now, we extended the concept for the assembly of irreversible inhibitors of Coxsackie virus B3 3C protease [3]. A protein-binding nucleophile 1



reacts reversibly with a bis-electrophilic warhead 2, thereby positioning the second electrophile in close proximity to the active site. The attack of the nucleophilic thiolate of the viral cysteine protease (blue) results in the covalent, irreversible deactivation of the enzyme. The assessment of the fragment combinations was based on measuring the enzyme inactivation rate via FRET-assay and detection of covalent protein modification in mass spectrometry (Figure 1).

Derivatives of the covalently bound fragments were designed, synthesized and tested for their inhibitory potential. Additionally, a crystal structure gave insight in the binding mode and proved assumptions of former docking studies. The protein-reactive electrophile was modified to various Michael acceptors and optimization yielded a compound with submicromolar half-maximal inhibition as a potent broad-spectrum inhibitor against enteroviral proteases. Further endeavours will concentrate on investigating the compatibility of diverse biselectrophilic fragments with this assay with regard to their reactivity, chain length, and reversibility. References: 1. Schmidt,M.F. et al.: Angew. Chem. Int. Ed. 2008, 47: 3275 –3278. 2. Burda, E.; Rademann, J.: Nat. Commun. 2014, 5:5170. 3. Becker,D. et al.: Nat. Commun. 2016, in revision.

POS.111 Development of a new synthetic route for 3-hydroxy-N’-arylidenepropanehydrazonamides as potential antiplasmodial compounds Knaab, T. C.1; Held, J.2; Mordmüller, B.2; Kurz, T.1 1 Institute for Pharmaceutical and Medicinal Chemistry, Heinrich Heine University, Universitätsstraße 1, 40225 Düsseldorf, Germany 2 Institute for Tropical Medicine, Eberhard Karls University, Wilhelmstraße 27, 72074 Tübingen, Germany

Malaria is still one of the most frequent causes of death in low income tropical countries. Globally, there were approximately 214 million malaria cases and 438 000 deaths in 2015 caused by the infections with protozoan parasites of the genus Plasmodium.[1] The limited number of antimalarial drugs and increasing parasite resistance leads to an urgent need for new antimalarial drugs. The arylamino alcohol quinine was the first antimalarial drug in clinical use and today its analogue lumefantrine is part of the first-line antimalarial therapy (artemisinine-based combination therapy (ACT): lumefantrine & artemether).[2] Recently, we developed a new chemical class of antiplasmodial compounds which mimics the general structure of arylamino alcohols. The lead structure, a 3-hydroxy-N’-arylidenpropanehydrazonamide derivative (see figure 1) demonstrated in vitro activity in single digit nanomolar range against P. falciparum strain 3D7 along with moderate in vivo activity in the P. berghei mouse model.[3] Initially, a classical Pinner reaction was used as a key step to provide imidate hydrochlorides as precursor for subsequent amidrazone synthesis.[4] Here we present an optimized synthetic protocol that enables a higher degree of chemical diversity for subsequent structure-activity relationship (SAR) studies. Notably, trimethylaluminum [5] promoted amidation of β-hydroxynitriles provided β-hydroxamidines, while amidrazones where accessible using hydrazine hydrate instead of an amine.

Fig. 1: Intended structure modification of the lead structure

References: 1. WHO, World Health Organization: World Malaria Report, 2015. 2. WHO, World Health Organization: Guideline, 2015. 3. Leven, M. et al.: J. Med. Chem., 2014, 57(19): 7971-7976. 4. Khankischpur, M., Kurz, T.: Synthesis, 2009, (23): 4068-4074. 5. Korbad, B. L.; Lee, S.: Bull. Korean Chem. Soc., 2013, 34(4): 1266-1268.

POS.112 Development of novel non-steroidal Farnesoid X Receptor (FXR) Antagonists Schmidt, J.1; Schubert-Zsilavecz, M.1; Merk, D.1 1 Institute of Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt a. M., Germany; [email protected]

The nuclear farnesoid X receptor (FXR) is a ligand-activated transcription factor, which acts as cellular sensor for bile acids and is primarily expressed in liver, kidney and intestine [1-4]. It takes part in the self-regulation of bile acids with the result that bile acid synthesis is blocked and their metabolism is enhanced when high levels of toxic bile acids occur. Furthermore, FXR is involved in many other metabolic systems such as glucose and lipid homeostasis and seems to have anti-inflammatory effects as well [4]. Since both, FXR activation with obeticholic acid (OCA) and whole-body FXR knockout mouse models showed a metabolic improvement in obese mice, FXR holds promise in the treatment of obesity and metabolic syndrome. However, the site of FXR activation appears to be essential for these effects [5]. Due to recent studies, inhibition of intestinal FXR activity through glycine-β-muricholic acid (Gly-β-MCA) improved obesity, non-alcoholic fatty liver disease (NAFLD) and insulin resistance [5]. In obese mouse models, oral treatment with Gly-β-MCA prevented weight gain and also reduced absolute fat mass. In addition, blood glucose levels were reduced and insulin sensitivity improved. To prove the site of action, intestine specific FXR-knockout mice were equally treated and did not benefit from Gly-β-MCA [5-8]. Altogether, the study suggests significant therapeutic value of intestine specific FXR antagonism. However, the intestinal stability and selectivity of Gly-β-MCA are questionable and potent non-steroidal and selective FXR antagonists are required to prove beneficial effects of FXR antagonism. In an in-house library screening, we discovered a benzamidophenylacetic acid derivative as lead compound for the development of non-steroidal FXR antagonists. It already offers respectable FXR antagonistic potency with a submicromolar IC50 value. Its structure can be divided into two building blocks, which allows a systematic, antagonistic structure-activity relationship (SAR) compilation for each block. Our first investigations focused on the position and size of the acidic group while the benzamido moiety was unaltered. By variation of the substitution pattern, we could improve potency to a two-digit nanomolar IC50 value. Since spatial extension is promising to enhance antagonistic potency, we systematically methylated all free positions of the scaffold to discover additional space for structural expansion. With this systematic strategy, we draw a broad SAR of benzamidophenylacetic acids as novel class of highly potent FXR antagonists. Further structural optimization will not only focus on antagonistic potency but also consider solubility, metabolic stability and toxicity to develop a novel FXR antagonistic tool compound for a more specific evaluation of FXR antagonism as therapeutic concept. References: 1. Seol, W., Choi, H. S., Moore, D. D.: Mol. Endocrinol. 1995, 9(1): 72–85. 2. Forman, B. M. et al.: Cell 1995, 81(5): 687–693. 3. Parks, D. J. et al.: Science 1999, 284(5418): 1365–1368. 4. Luciano Adorini, L., Pruzanski, M., Shapiro, D.: Drug Discov. Tod. 2012, 17(17): 988-997. 5. Jiang, C., et al.: Nat. Commun. 2015, 6: 1–18. 6. Lamers, C., Schubert-Zsilavecz, M., Merk, D.: Curr. Top. Med. Chem. 2014, 14(19): 2188–2205. 7. Merk, D., Steinhilber, D., Schubert-Zsilavecz, M.: Future Med. Chem. 2012, 4(8): 1015–1036. 8. Huang, H., et al.: ChemMedChem. 2015, 10: 1184–1199.

POSTERS


POS.113 Development of new versatile building blocks for the pharmacokinetic optimization of PET-tracers Theiler, S.1; Wünsch, B. 1; Faust, A.2; Höltke, C.3

1 Institut für pharmazeutische und medizinische Chemie, Corrensstraße 48, 48149 Münster 2 European Institute for Molecular Imaging – EIMI, Waldeyerstraße 15, 48149 Münster 3 Institut für Klinische Radiologie, Universitätsklinikum Münster Albert-Schweitzer-Campus 1 / A16, 48149 Münster

A fluorescent probe1 and a number of PET-tracers2,3 have been developed to image the endothelin A (ET-A) receptor which is a potential target for medical imaging as it is overexpressed in connection with many kinds of diseases, like cancer, inflammation and (cardio)vascular disorders. In comparison to the fluorescent probe, the PET-tracers exhibit rather poor in vivo performance e.g. low accumulation in target tissue. This discrepancy is believed to result from the molecular structure of the fluorophore. Small-molecular PET-tracers usually consist of rather lipophilic structures whereas fluorescent probes consist of large heteroaromatic systems being e.g. negatively charged due to hydrophilic sulfonic acid groups. The aim of this project is to synthesize versatile building blocks mimicking the molecular structure of fluorescent dyes in order to enhance the hydrophilicity and hence the in vivo performance of the PET-tracers. First constructs were designed to contain an aromatic system with one or more sulfonic acid groups (fig.1). This building block can then be bound to a variety of ligands, including the developed endothelin receptor antagonist. It contains a carboxylic acid group (blue, fig.1) where a ligand can be bound via an amide bond and an alkyne group (red, fig.1) where 18F as the PET radionuclide can be introduced via a cycloaddition reaction with [18F]fluoroethyl azide.

Fig. 1: Naphthalene-based building block for PET-tracers, n=1-2.

First radiosyntheses were performed successfully. Future studies will examine the influence of different charges on the pharmacokinetic behavior in vivo. Therefore, building blocks with positive charges (quarternary ammonium) or polar neutral compounds (polyalcohols) will be developed. References: 1. Höltke, C. et al. Bioconjug. Chem. 2007, 18 (3): 685–94. 2. Höltke, C. et al. Bioorg. Med. Chem. 2009, 17 (20): 7197–208. 3. Michel, K. et al. J. Med. Chem. 2011, 54 (4): 939–48.

POS.114/SL.42 A fluorescence polarization-based competition binding assay for detecting compounds interacting with inactive mitogen-activated protein kinases and development of covalent inhibitors of c-Jun N-terminal kinase 3 Koch, P.1 1 Eberhard Karls Universität Tübingen, Institute of Pharmaceutical Sciences, Department of Medicinal Chemistry, Auf der Morgenstelle 8, 72076 Tübingen, Germany.


POS.115/SL.41 Analysing the framework of protein ligand interactions: Ligand-sensing cores and privileged scaffolds Koch, O. TU Dortmund University, Faculty of Chemistry and Chemical Biology, Otto-Hahn-Straße 6, 44227 Dortmund, Germany


POS.116 Synthesis of 1- and 8-pyridinyl-substituted imidazo[1,5-a]quinoxalines as potential PDE10A-ligands for positron emission tomography (PET) Franz, L.1; Scheunemann, M.2; Wagner, S.2; Lang, M.1; Brust, P.2; Briel, D.1 1 University of Leipzig, Institute of Pharmacy, Bruederstraße 34, 04103 Leipzig, Germany 2 Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiopharmaceutical Cancer Research, Permoserstraße 15, 04318 Leipzig, Germany

Phosphodiesterases (PDE´s) are second messenger hydrolysing enzymes and important regulators of signal transduction mediated by these molecules. PDE10A, a cAMP and cGMP sensitive hydrolase, is primarily expressed in the striatum and was identified as drug target for the therapy of diverse disorders in the central nervous system (CNS) [1] like schizophrenia or chorea huntington [2]. Recently, 1-arylimidazo[1,5-a]quinoxalines have been reported to be potent and selective inhibitors of PDE10A [3]. In terms of a potential use as 18F-labelled PET imaging agent new fluorine-containing substituted derivatives were synthesized. It has been shown that the methoxy substituted inhibitors are prone to metabolic oxidation, which leads to a loss of inhibitory potency or ability to cross the blood brain barrier [3]. To improve the metabolic stability of inhibitors the methoxy function in position 6 was exchanged by chlorine in the first synthesis step by electrophilic substitution. An electron deficient system was generated in step 2 by oxidation of the amine to a nitro function to allow the nucleophilic aromatic substitution of fluorine by 4-methylimidazole in step 3. Afterwards, the amine was recovered by acidic reduction with elementary iron in step 4 and acetylated in step 5. The Cyclisation in step 6 to create the quinoxaline was realized by a Bischler-Napieralski-reaction, the bromination of position 1 by electrophilic substitution in step 7.

NH2

F

X

NH2

F

X

ClNO2

F

X

ClNO2

N

X

Cl NNH2

N

X

Cl N

NHAcN

X

Cl N

step 2 step 3 step 4

NCS NaBO3 · 4 H2O HN N Fe

step 1

step 5

NN

NCl

X

18

6

POCl3

step 6

Ac2OX = -CF3

-BrN

NN

Cl

X

18

6

NBS

step 7Br

The derivatization of the quinoxalines was focused on position 1 and 8. Finally, fluor-containing pyridinyl-groups were introduced by Suzuki-Coupling with the corresponding boronic acid at the brominated positions to afford the monosubstituted (1, 2) and disubstituted (3) pyridinyl-derivatives. All compounds were characterized by high performance liquid chromatography, nuclear magnetic resonance spectroscopy and mass spectrometry. It is expected that the new chlorinated derivatives have the same pharmaceutical effects as their methoxy analogues [4].

N

NN

Cl

CF3

18

6

N

F

N

NN

Cl

Br

18

6

N

F

N

NN

Cl1

86

N

FN

F

(1) (2) (3)

Acknowledgments: Thanks are to J. Ortwein for HPLC analysis, Dr. L. Hennig for recording and analysis of NMR data and Dr. J. Preidl for LC-MS analysis.



References: 1. Liras, S.; Bell, A.S. Phosphodiesterases and Their Inhibitors (Wiley-VCH) 2014. 2. Schmidt, C.J. et al.: J. Pharm. Exp. Ther. 325 (2008) 681–690. 3. Malamas, M. et al.: J. Med. Chem. 54 (2011) 7621–7638. 4. Wagner, S. Eur. J. Med. Chem. 107 (2016) 97–108.

POS.117 Triazole-type ABCG2 modulators: approaches to improve drug-like properties Antoni, F.1#; Stark, S.2#; Scholler, M.1; Bernhardt, G.1; König, B.2; Buschauer, A.1 1 Institute of Pharmacy, 2 Institute of Organic Chemistry, University of Regensburg, D-93040 Regensburg; #Authors contributed equally

The efflux transporter breast cancer resistance protein (BCRP, ABCG2) is associated with the chemoresistance of malignant tumors. In addition, ABCG2 is highly expressed at the blood-brain barrier preventing the entry of numerous xenobiotics including drugs into the central nervous system. Previously, UR-MB108 [1, 2] was synthesized and characterized as a highly potent, selective and chemically stable ABCG2 inhibitor.

However, this compound shows high lipophilicity and low water solubility. Aiming at improving drug-like properties, the tetrahydroisoquinoline moiety was replaced by a series of different saturated heterocycles. Furthermore, the quinoline moiety was replaced by smaller aromatic heterocycles. This approach resulted in modulators of lower molecular weights with activities in the three-digit nM range. References: 1. Bauer, S. Quinoline carboxamides as modulators of breast cancer resistance protein (ABCG2): Investigations on potency, selectivity, mechanism of action, cytotoxicity, stability and drug-like properties. Ph.D. thesis, University of Regensburg, 2014; http://epub.uni-regensburg.de/29589/. 2. Bause, M. Synthesis of melanoma inhibitory activity protein inhibitors, ABCG2 transporter modulators, and neurotensin mimetics. Ph.D. thesis, University of Regensburg, 2015.

POS.118 A fragment-based drug design approach for the optimization of LpxC inhibitors Agoglitta, O.1,2; Martin, B.1; Kalinin, D.1,3; Köhler, J.1; Holl, R.1,3

1 Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Corrensstraße 48, 48149 Münster, Germany 2 NRW Graduate School of Chemistry, University of Münster, Wilhelm-Klemm-Str. 10, 48149 Münster, Germany 3 Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), University of Münster, Germany

Multidrug resistant Gram-negative bacteria are becoming a global threat. E.g., the treatment of healthcare-associated infections is steadily getting more and more challenging, due to the increasing number of multidrug resistant bacteria. Additionally, the number of available and effective antibiotics is limited. Novel antimicrobial agents addressing so far unexploited bacterial targets are therefore urgently needed [1]. Being the main and fundamental component of the outer monolayer of the outer membrane of Gram-negative bacteria, lipopolysaccharides (LPS) ensure an extremely effective permeability barrier towards chemical attacks. The inhibition of the enzymes being involved in the

biosynthesis of lipid A, the lipophilic membrane anchor of LPS, represents therefore a promising strategy to combat Gram-negative bacteria. The enzyme LpxC, catalyzing the first committed step of lipid A biosynthesis, has been explored as an optimal target for novel antibiotics, due to its fundamental role for growth and viability of Gram-negative bacteria and its lack of homology towards mammalian enzymes [2]. A fragment-based drug design approach was used to develop novel LpxC inhibitors. Using NMR spectroscopy, a library of fragments was screened in the presence of a known inhibitor and the LpxC enzyme. In ligand-observed NMR experiments interligand NOEs were detected [3]. The hits found were used to optimize the inhibitory activity of a known LpxC inhibitor. References: 1. Theuretzbacher, U.: Int. J. Antimicrob. Agents. 2012, 39: 295-299 2. Barb, A. W. et al.: PNAS. 2007, 104(47): 18433-18438 3. Harner, M. J. et al.: J. Biomol. NMR. 2013, 56(2): 65-75

POS.119

Synthetic phthalimide conjugation strategies for PROTAC development Steinebach, C.1; Gütschow, M.1 1 University of Bonn, Pharmaceutical Institute, An der Immenburg 4, D-53121 Bonn, Germany

Proteolysis targeting chimeras (PROTACs) are used to link an E3 ubiquitin ligase to a specific target protein. These molecules have two recognition motifs separated by a linker: one terminal moiety recognizes specific target proteins and the other one binds to an E3 ubiquitin ligase. Ubiquitination of these proteins ultimately leads to their elimination via the ubiquitin/proteasome system (UPS). PROTAC technology in combination with an immunomodulatory drug (IMiD) as a low-molecular weight CRL4CRBN recruiter was first reported in 2015 for BET protein degradation in vitro and in vivo.1 A further heterodimer for the degradation of oncogenic BCR-ABL via the E3 ubiquitin ligase complex CRL4CRBN was described in 2016.2 Recently, PROTAC technology was proven to inhibit the growth of a solid tumor in vivo.3 From that perspective, it was considered to have the potential for a platform technology in drug research.4

Herein we describe the development and synthesis of linker strategies for phthalimide conjugation in view of PROTAC technology, which has most recently received much attention in medicinal chemistry. We evaluated literature known and new synthetic entries towards bifunctional probes and examined protein data bank files with respect to phthalimide-cereblon (CRBN) complexes. To prepare various thalidomide derivatives containing functional groups e.g. nitro, amino, hydroxy and carboxy, one-pot syntheses were applied starting from commercially available phthalic anhydrides. These derivatives were investigated for the attachment of linkers by using different coupling techniques. Furthermore, our study includes new protecting techniques for the glutarimide moiety, which were needed in case of electrophilic alkylation reactions at the non-glutarimide part of the molecule. Our rational design of phthalimide-conjugated linker building blocks includes different synthetic entries towards heterodimeric compounds. The synthetic implementation is presented herein. Our work is aimed at generating further examples which could hijack E3 ligases for the degradation of target proteins as a new therapeutic strategy. Acknowledgments: University of Bonn, Pharmaceutical Institute, Gilberg, E.

POSTERS


References: 1. Winter, G. E. et al.: Science 2015, 348(6241): 1376-81. 2. Lai, A. C. et al.: Angew. Chem. Int. Ed. Engl. 2016, 55(2):807-10. 3. Raina, K. et al.: Proc. Natl. Acad. Sci. USA. 2016, 113(26):7124-9. 4. Deshaies, R. J.: Nat. Chem. Biol. 2015, 11(9): 634-5.

POS.120

Synthesis and biological evaluation of neuroprotective 7-O-esters of the flavonolignan silibinin Schramm, S.1; Huang, G.1; Kling, B.2; Heilmann, J.2; Decker, M.1 1 Pharmazeutische und Medizinische Chemie, Institut für Pharmazie und Lebensmittelchemie, Julius-Maximilians-Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany 2 Lehrstuhl für Pharmazeutische Biologie, Institut für Pharmazie, Universität Regensburg, Universitätsstraße 31, D-93053 Regensburg, Germany

It is well established that oxidative stress, the formation of reactive oxygen species (ROS), and subsequent neurotoxicity are key processes in the pathophysiology of Alzheimer’s disease (AD). [1] There are reports that standardized extracts of Silybum marianum, containing the silymarin complex and its main component silibinin (diastereomeric mixture of Silybin A 1a and Silybin B 1b, Scheme 1) and chemical derivatives of silibinin might exhibit neuroprotective properties. [2,3] Ferulic acid represents a naturally occurring phenolic acid, that is a potent radical scavenger, measured in the physicochemical oxygen-radical absorbance capacity (ORAC) assay, [4] but it has been shown to lack neuroprotective activity in vitro after impairment of HT-22 cells with tert-butyl hydroperoxide. [5] To combine the potent antioxidant properties of silibinin 1 with ferulic acid, and structurally closely related compounds, and to produce putatively neuroprotective silibinin esters with antioxidant activity, 7-O-esters of silibinin 1 (2-5, Scheme 1) were synthesized. In order to establish SARs for the esters synthesized, their radical scavenging properties as well as their neuroprotective effects have been determined.

O

R2R1

O

R2R1

O

R2R1 2 a-c) R1 = R2 = R3 = H

3 a-c) R1 = OMe, R2 = OH, R3 = H4 a-c) R1 = OMe, R2 = OH, R3 = OMe5 a-c) R1 = R2 = O2CH(CH3)2 , R3 = H

R3 R3 R3

OHO

OH OOH

O

OOH

OMe

OH

OO

OH OOH

O

OOH

OMe

OH

R

R =

11a: silybin A (2R, 3R, 10R, 11R)1b: silybin B (2R, 3R, 10S, 11S)

**

**

a b c

2-5

Reagents and conditions: (I) acid, oxalyl chloride, CH2Cl2, rt; (II) silibinin 1, NEt3, THF, rt Scheme 1: Reaction conditions and structures of synthesized 7-O-esters (2-5)

For the synthesis of the 7-O-esters several esterfication methods were investigated to get the desired regioselectivity without the use of a time consuming protecting group strategy. One of the first methods applied previously was Mitsunobu esterification [3], which in fact yielded 7-O-feruloylsilibinin (3a, Scheme 1) rather than the described 23-O-ester. 7-Esterification can also be achieved using acyl chlorides in basic conditions. [6] As this alternative method does not show as many unwanted side products [7] and is devoid of tedious chromatographic purification that are necessary for Mitsunobu reactions, we decided to use acyl chlorides for the syntheses of the 7-O-esters. Also compounds 3 and 4 could be obtained using the respective acid chlorides, which were prepared in situ without protection of the hydroxyl groups (Scheme 1). This procedure gave the 7-O-esters 2-5 consistently. To investigate how stable the esters are under assay conditions, stability of 3a was determined via LCMS. To determine the radical scavenging properties of esters 2-5, the ORAC assay, which assesses their antioxidant physicochemical properties, was employed. Neuroprotective effects of the compounds were investigated towards glutamate-induced oxidative stress in HT-22 hippocampal neurons. Additionally, putative self-cytotoxic effects of the compounds were studied. References: 1. Palmer, A.M.: Trends Pharmacol. Sci. 2011, 32: 141–147. 2. Yang, L.X. et al.: J. Med. Chem. 2009, 52: 7732–7752. 3. Wang, F. et al.: Bioorg. Med. Chem. 2009, 17: 6380–6389. 4. Fang, L. et al.: Bioorg. Med. Chem. Lett. 2008, 18: 2905–2909. 5. Kling, B. et al.: J. Nat. Prod. 2014, 77: 446–454. 6. Gažák, R. et al.: J. Med. Chem. 2011, 54, 7397–7407. 7. Huang, G. et al.: Beilstein J. Org. Chem. 2016, 12: 662–669.

POS.121

New oxetane derivatives designed as p38 MAP kinase inhibitors Rodrigues de Sá Alves, F.; Laufer, S. A. Institute of Pharmacy, Department of Pharmaceutical and Medicinal Chemistry, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076, Tübingen

Aberrant kinase activity is implicated in a variety of human diseases, principally those involving inflammatory responses, such as rheumatoid arthritis. They could be treated through modulation of cytokines related to the protein kinase pathway such as tumor necrosis factor-a (TNF-α) and interleukin-1β (IL-1β), whose biosynthesis and release are regulated by the activation of the p38α MAPK [1-2]. Laufer and co-workers have been reporting the design and pharmacological evaluation of potent p38α MAPK inhibitors bearing the dibenzepinone, dibenzoxepine, dibenzosuberone and benzosuberone scaffolds [3-4]. In order to discover new analogs of benzosuberones, we proposed the isosteric replacement of the carbonyl group by an oxetane moiety to fulfill the two selectivity requirements, the ‘linear binding’ mode and the glycine–flip at Gly110. Moreover, an oxetane group can elicit deep changes in aqueous solubility, lipophilicity, metabolic stability, and conformational preference when replacing commonly employed functionalities like gem-dimethyl and carbonyl groups [5-6].

Acknowledgments: University of Tübingen, DAAD-CNPq

References: 1. Melnikova, I.; Golden, J:. Nat. Rev. Drug. Discov. 2004, 3(10): 993-994. 2. Laufer, S. A. et al.: J. Med. Chem. 2003, 46(15): 3230–3244. 3. Laufer, S. A. et al.: J. Med. Chem. 2006, 49(26): 7912–7915. 4. Karcher, S. C.; Laufer, S. A.: J. Med. Chem. 2009, 52(6): 1778–1782. 5. Koeberle, S. C. et al.: Nat. Chem. Biol. 2012, 8(2): 141–143. 6. Wuitschik, G. et al.: J. Med. Chem. 2010, 53(8): 3227-3246.

POS.122

Characterization of diazenylaryl sulfonic acids as neuraminidase inhibitors Hoffmann, A.1; Richter, M.1; von Grafenstein, S.2; Walther, E.1; Xu, Z.1; Schumann, L.1; Grienke, U.3; Mair, C. E.3; Kramer, C.2; Rollinger, J. M.3; Liedl, K. R.2; Schmidtke, M.1; Kirchmair, J.2,4

1 Jena University Hospital, Department of Virology and Antiviral Therapy, Hans-Knoell-Straße 2, 07745 Jena, Germany

2 University of Innsbruck, Centre for Chemistry and Biomedicine (CCB), Institute of General, Inorganic and Theoretical Chemistry, Innrain 82, 6020 Innsbruck, Austria

3 University of Vienna, Department of Pharmacognosy, Althanstraße 14, 1090 Vienna, Austria

4 University of Hamburg, Center for Bioinformatics, Bundesstraße 43, 20146 Hamburg, Germany

Neuraminidases (NAs) are valid targets to combat not only influenza but also secondary Streptococcus pneumoniae infections causing pneumonia [1-3]. This study reports on the identification of diazenylaryl sulfonic acids as inhibitors of influenza A NAs and S. pneumoniae NA (NanA) by virtual screening. Several reports link diazenylaryl sulfonic acids to antiviral activity and the inhibition of influenza NA (e.g. [4]) but their function and potency have never been thoroughly characterized. We applied different biochemical and cell-based assays that confirmed the inhibitory activity of several of the 17 tested diazenylaryl sulfonic acids against influenza A virus NA and, in addition, revealed their inhibitory activity against NanA. For the most active compound, NSC65847 from the National Cancer Institute’s (Bethesda, MD) compound library, the measured Ki values were <1 µM for both viral and pneumococcal NAs. The compound also inhibited N1 virus variants containing NAI resistance-conferring substitutions. Via enzyme kinetics and nonlinear regression modeling, NSC65847 was suggested to impair the viral NA as well as NanA with a mixed-type inhibition mode. Diazenylaryl sulfonic acids appear to be binding to NAs in various different orientations. Given its antiviral and antipneumococcal activity,



NSC65847 was identified as a novel starting point for the development of dual-acting NA inhibitors.

Acknowledgments: We thank the National Cancer Institute of the National Institute of Health (NIH) for the provision of free samples of compounds for testing and the Cambridge Crystallographic Datacenter (CCDC) for providing us with a complimentary license for GOLD for the purpose of this study. This work was funded by research grants P23051 and P24587 of the Austrian Science Fund (FWF), the European Social Fund together with Thuringian Ministry of Economy, Labour and Technology (2011FGR0137).

References: 1. Grienke, U. et al.: Sci. Rep. 2016, 27156. 2. Walther, E. et al.: Int. J. Med. Microbiol. 2015, 305(3): 289-97. 3. Walther, E. et al.: Front. Microbiol. 2016, 7: 357. 4. Cheng, L. S. et al.: J. Med. Chem. 2008, 51(13):3878-3894.

POS.123

Identifying triazole-type quinoline carboxamide derived modulators of ABCG2 as inhibitors using an ATPase assay Scholler, M.1; Stark, S.2; Bause, M.2; Bernhardt, G.1; König, B.2; Buschauer, A.1 1 Institute of Pharmacy, University of Regensburg, 93040 Regensburg, Germany 2 Institute of Organic Chemistry, University of Regensburg, 93040 Regensburg, Germany

The breast cancer resistance protein (BCRP, ABCG2) is an efflux transporter highly expressed at the blood-brain barrier, preventing xenobiotics including cytostatic drugs to enter the central nervous system (CNS). In addition, ABCG2 overexpression by malignant cells is associated with poor survival of cancer patients. Co-adminstration of ABCG2 inhibitors with chemotherapeutics, which are substrates of this transporter, harbours the potential to overcome multidrug-resistance and to reach effective concentration of cytostatics in the CNS. Previously, our group reported on selective carboxamide-type ABCG2 modulators with activities in the two-digit nanomolar range [1, 2]. Cellular uptake assays such the Hoechst 33342 assay do not discriminate between ABCG2 substrates and inhibitors. To investigate whether modulators of general structure 1 are substrates or act as ABCG2 inhibitors, an ATPase assay was established. The human ABCG2 was expressed in Sf9 cells by baculoviral infection [3] and membranes were prepared. ABCG2 expression was confirmed by Western blot, and transporter activity was proven in a vesicular transport assay, using the fluorescent ABCG2 substrate lucifer yellow. To quantify the inhibition of the efflux pump, the liberation of inorganic phosphate upon transporter dependent ATP hydrolysis was measured photometrically [4]. When exploring different substrates, sulfasalazine gave the highest signal-to-noise ratio.

Transport driven ATP hydrolysis decreased in the presence of the investigated carboxamide-type modulators in a concentration dependent manner, demonstrating that the investigated quinolone carboxamides are inhibitors, not substrates. The same holds for the reference compound fumitremorgin C (2). Compared to the latter (IC50 1.8 µM), the quinoline derivatives were considerably more potent as ABCG2 inhibitors (IC50 values < 100 nM) in the ATPase assay. The

data were in good agreement with those obtained from the Hoechst 33342 assay.

References: 1. Kühnle, M. et al.: J. Med. Chem. 2009, 52, 1190-1197. 2. Bauer, S. et al.: ChemMedChem 2013, 8, 1773-1778. 3. Glavinas, H. et al.: Drug. Metab. Dispos. 2007, 35(9), 1533-1542. 4. Sakardi, B.; et al.: J. Biol. Chem. 1992, 267(7), 4854-4858.

POS.124

Applying machine-learning algorithms and high-throughput screening (HTS) on twelve targets for identifying potentially active compounds and reducing animal toxicity testing Sayed, A.1,2; Spahn-Langguth, H.3; Schramm, K. W. 1,4 1Center for Life Sciences, Technical University Munich, Emil-Erlenmeyer-Forum 2, 85354 Freising, Germany 2Rosettastein Consulting, Goethestrasse 2, 85354 Freising, Germany 3Department of Pharmaceutical Sciences/Pharmaceutical Chemistry, Karl-Franzens-University, Universitätsplatz 1/I, 8010 Graz, Austria 4Molecular EXposomics, German Research Center for Environmental Health, Helmholtz Zentrum München, Neuherberg, Germany

The regulatory necessity to fill information gaps about chemicals’ toxicity is increasing worldwide. The need for filling such gaps while reducing toxicity testing in animals is also becoming more predominant in risk assessment. Programs run by the multiple European and US agencies are encouraging the use of alternative approaches for toxicity estimation. Recent legislations are accepting in silico approaches for predicting toxicological outcomes. As more data is being generated through High throughput screening (HTS) technologies, statistical in silico approaches are getting more popular as an alternative method for predictive toxicology. We present an automated workflow to construct and analyze Quantitative Structure Activity Relationship (QSAR) for predicting the outcome of in vitro profiling of chemicals. We describe the results of QSAR modeling efforts within Tox21 Data Challenge, which calculated the best balanced accuracy across all molecular pathway endpoints as well as the highest scores for ATAD5 and mitochondrial membrane potential disruption. Twelve molecular pathway endpoints were investigated, which were selected on the basis of toxicological relevance. The targets were experimentally screened as part of the Tox21 program and the resulting data library made accessible for competitors by the Tox21 Data Challenge organizers. Tox21 represents a multi- agency effort that uses HTS assays for toxicity modeling and prediction in the US. The US Environmental Protection Agency (EPA), The National Institutes of Health (NIH), The National Center for Advancing Translational Sciences (NCATS), The National Institutes of Environmental Health Sciences/National Toxicology Program (NIEHS/NTP) and the Food and Drug Administration (FDA) cooperate in screening chemical substances for some selected potential toxic effects. The data may then be used, with the assistance of in silico techniques, for providing an alternative for expensive, time- consuming, and ethically-questioned animal testing. In this study we constructed models for 12 targets using a consensus of selected associative neural networks models from 10 descriptor packages. Automated QSPR workflow systems, OCHEM, the analytics platform, KNIME and the statistics software, CRAN R, were used to conduct the analysis and develop consensus models. All models were constructed using bootstrap aggregation. We also investigated multiple approaches for constructing consensus models and set criteria for including a model into consensus voting. The resulting consensus models yielded a balanced accuracy as high as 88.1% ± 0.6 for mitochondrial membrane disruptors. Such high balanced accuracy, in combination with applicability domain estimation, encourages the use of in silico modeling to guide future selection of chemical libraries for screening. The comprehensive statistics of all models are publicly available online at https://github.com/amaziz/Tox21-Challenge-Publication while the developed consensus models can be accessed at http://ochem.eu/article/98009.

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POS.125

3D-QSAR analysis of Dual Leucine Zipper Kinase Inhibitors Brinker, C.1; Lemcke, T.1 1 Institute of Pharmacy, University of Hamburg, Bundesstr. 45, 20146 Hamburg, Germany

The dual leucine zipper kinase (DLK) is a MAP3-kinase which plays a crucial role in different MAPK signaling pathways, activating downstream kinases like p38 and JNK. DLK is especially expressed in the peripheral and central nervous system being required for neurodegeneration and axon regeneration, and in the insulin-producing beta cells, inhibiting the transcription factors CREB and MafA, important for insulin gene transcription and insulin secretion. Therefore, an overexpression of DLK contributes to a loss of beta-cell function and inferentially to diabetes mellitus. A well-established way to intervene in kinase signal transmission is the inhibition by ATP competitive small molecule inhibitors. A major drawback of known inhibitors is the lack of DLK selectivity and suitable ADME properties. [1] In connection with a project to identify new selective inhibitors of DLK by structure-based virtual screening, 3D-QSAR models were generated by a set of structurally various inhibitors known from literature. Docking of these structures to the ATP-binding site of different DLK X-ray structures[2] led to an alignment of likely biological active binding conformations. This alignment was used to generate CoMFA and CoMSIA models with different field combinations. Thorough model evaluation by crossvalidation, test set prediction and statistical analysis (R², R²cv, Q²,…) provided valid 3D-QSAR models which will be presented on this poster. [3] These models will be used in the postprocessing and compound selection steps of the above mentioned virtual screening workflow.

Training set compound GNE-3511 in the ATP-binding site of DLK (5ceo)

References: 1. E. Oetjen, T. Lemcke, Expert Opinion on Therapeutic Patents 2016, 26, 607–616. 2. S. Patel et al., J. Med. Chem. 2015, 58, 8182–8199. 3. a) R. D. Cramer, D. E. Patterson, J. D. Bunce, J. Am. Chem. Soc. 1988, 110, 5959–5967; b) G. Klebe, U. Abraham, T. Mietzner, J. Med. Chem. 1994, 37, 4130–4146.

POS.126

Characterization of 3-amido-benzhydroxamates as modulators of epigenetic targets for the treatment of schistosomiasis Heimburg, T.1; Chakrabarti, A.2; Melesina, J.1; Robaa, D.1; Hauser, A. T.2; Schmidtkunz, K.2; Marek, M.3; Romier, C.3; Pierce, R.4; Erdmann, F.1; Schmidt, M.1; Jung, M.2; Sippl, W.1

1 Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck –Straße 4, 06120 Halle (Saale), Germany 2 Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104 Freiburg, Germany 3 IGBMC, Universite de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch Cedex, France 4 Center for Infection and Immunity of Lille (CIIL), Université Lille Nord de France, Institut Pasteur de Lille, 1 rue Professeur Calmette, 59019 Lille Cedex, France

Schistosomiasis is one of the major human neglected parasitic diseases[1]. At the moment, there is no licensed vaccine available against Schistosomiasis and for Praziquantel, the one drug which is effective against all schistosome species, reduced efficiency and drug resistance are reported[2]. Currently inhibitors of human epigenetic enzymes are investigated as novel anti-cancer drugs and have the

potential to be used as new anti-parasitic agents[3]. Here, we report that Schistosoma mansoni histone deacetylase 8 (SmHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. SmHDAC8 is a zinc depending enzyme. Linkerless aromatic hydroxamates were identified by screening the ZINC-Database for small molecules containing zinc-chelating groups. Further investigation of these compounds revealed the induction of apoptosis and mortality in schistosomes. Crystal structures of smHDAC8 with several linkerless hydroxamates could be resolved[4,5]. Based on structural data and modeling studies structure activity relationship analysis was established and used to guide the further synthesis[6]. This approach resulted in a series of novel 3-amido-benzhydroxamates as nanomolar inhibitors of SmHDAC8 with good selectivity regarding the major human HDACs (HDAC1 and HDAC6) also inhibitory effects in phenotypic screening and promising pharmacokinetic properties.

References: 1. Hotez PJ, Pecoul B: PLoS Negl Trop. 2010, 4: e718 2. Doenhoff MJ, Cioli D, Utzinger J: Curr Opin Infect 2008, 21: 659-667 3. Andrews KT, Haque A, Jones MK: Immunol Cell Biol 2012, 90: 66-77 4. Marek, M. et al.: PLOS Pathogens. 2013, 9: e1003645 5. S. Kannan, J. Melesina, A. Hauser et al.: J. Chem. Inf. and Mod., 2014, 54: 3005-3019 6. T. Heimburg, A. Chakrabarti et al.: J. Med. Chem., 2016, 59: 2423-2435

POS.127

Synthesis and in vitro characterization of hydroxamic acids as small molecule inhibitors for epigenetic parasitic targets Bayer, T.1; Melesina, J.1; Chakrabarti, A. 2; Marek, M.3; Romier, C.3; Erdmann, F.1; Schmidt, M.1; Jung, M.2; Sippl, W.1 1 Institut für Pharmazie, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeck –Straße 4, 06120 Halle (Saale), Germany 2 Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104 Freiburg, Germany 3 IGBMC, Université de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch Cedex, France

Schistosomiasis, also known as bilharzia is caused by a blood-dwelling fluke of the genus Schistosoma. It uses a fresh water snail as an intermediate host and is transmitted through contaminated water [1]. Taking into account that an estimate of 258 million people are infected worldwide among who 280 000 die annually schistosomiasis is one of the most important parasitic diseases after malaria [2, 3]. Praziquantel is an anthelminthic which is effective against all human forms of schistosomiasis. Without any effective vaccine available and the excessive use of praziquantel for the treatment of infected individuals as well as for preventive treatment the problem of resistant schistosome strains is arising [1]. Histone deacetylases (HDACs) take an important part in epigenetics since the state of acetylation of the histones correlates with transcriptional control [4]. The schistosome histone deacetylase 8 (smHDAC8) was recently identified as a potential target for antiparasitic therapy and a large scale screening identified several hydroxamic acids as active compounds on HDACs [5, 6]. Based on a screening hit J1075 (3 - Chloro benzothiophene - 2 -hydroxamic acid) a series of hydroxamic acids with different scaffolds and different substitution patterns were synthesized and characterized. Here we present the synthesis of these compounds and their phenotypical and in vitro effects on schistomomes.



Acknowledgements: SEtTReND, A-ParaDDisE

References: 1. Deribew, K. et al.: Int. J. Med. Med. Sci., 2013, 5 (3), 131-139 2. Steinmann, P. et al.: LancetInfectDis, 2006, 6 (7): 411-425 3. WHO Fact sheet Schistosomiasis, updated February 2016 4. KrennHrubec K. et al.: Bioorg Med Chem Lett. 2007, 17 (10): 2874 - 2878 5. Kannan, S. et al.: J. Chem. Inf. Model., 2014, 54 (10), 3005–3019 6. Marek, M. et al.: PLoS path. 2013, 9 (9), e1003645

POS.128

Selective histone deacetylase 6 (HDAC6) inhibitors using the phenothiazine system as cap group framework Vögerl, K.1; Senger, J.2; Jung, M.2; Bracher, F.1 1 Department of Pharmacy, LMU Munich, 81377 Munich, Germany, 2 Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, 79104 Freiburg, Germany

Histone deacetylases catalyze the removal of acetyl groups from lysine residues in histone and additionally in several non-histone proteins [1]. Tubastatin A has previously been described as potent and highly selective inhibitor of histone deacetylase 6 (HDAC6), a HDAC isoenzyme mainly involved in the acetylation status of non-histone proteins. Structure-based drug design combined with homology modeling techniques were used to develop this selective HDAC6 inhibitor consisting of a tetrahydro-γ-carboline as a surface recognition cap group, a p-tolyl group linker, forcing the cap to lie against the catalytic channel rim and bearing the zinc-binding group (ZBG) [2]. Inspired by this work we investigated various alternative polycyclic cap group frameworks. Based on the phenothiazine system in conjunction with an aromatic hydroxamic acid we developed a new chemotype of selective HDAC6 inhibitors with enhanced potency.

References: 1. Dallavalle, S. et al.: Biochemical Pharmacology 2012, 84, 756-765 2. Butler, K. V. et al.: Journal of the American Chemical Society 2010, 132, 10842-10846

POS.129

Investigation about the Schistosoma mansoni sirtuin 2 (smSirt2) deacylase activity and its inhibitors Monaldi, D.1; Schiedel, M.1; Marek, M.2; Romier, C.2; Jung, M.1

1 Institute of Pharmaceutical Sciences, Albert-Ludwigs-Universität Freiburg, Albertstr. 25, 79104, Freiburg, Germany. 2 Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, Illkirch, France.

Schistosoma mansoni sirtuin 2 (smSirt2) is a NAD+-dependent lysine deacetylase which has been recently identified as therapeutic target for the treatment of schistosomiasis, a parasitic disease caused by the flatworm Schistosoma mansoni.[1] Phylogenetic analysis have defined smSirt2 as orthologous of the human Sirtuin 2 (hSirt2)[1] and even if a crystal structure of SmSirt2 has been not solved yet, homology models have reported high similarity with the human isoform.[2]

Several in vivo and in vitro studies have recently showed the ability of hSirt2 to catalyze long chain deacylation of lysine residues from histone and non histone proteins.[3,4] As consequence of the similarity with smSirt2 the investigation regarding the ability of the parasitic enzyme to catalyze deacylation of lysine residues and its results are reported. Furthermore since the treatment of schistosomiasis relies on the only available drug Praziquantel,[5] a screening of diverse compound libraries has been used as strategy for finding novel smSirt2 inhibitors and developing new drugs against schistosomiasis. Acknowledgements: We thank our collaborators from the CO-factors RTG1976 (Deutsche Forschungsgemeinschaft) and A-ParaDDisE (EU FP-7 Health no. 602080) consortium for funding and support

References: 1. Lancelot, J. et al. Schistosoma mansoni Sirtuins: characterization and potential as chemotherapeutic targets. PLoS Negl. Trop. Dis. 2013, 9: e2428. 2. Schiedel, M. et al. Fluorescence-based screening assays for the NAD+-dependent histone deacetylase smSirt2 from Schistosoma mansoni. J. of Biomol. Screen. 2015, 20: 112-121. 3. Feldman, J.L. et al. Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins. J.Bio.Chem. 2013, 288: 31350-31356. 4. Teng, Y. et al. BNature/ Sci. Rep. 2015, 5: 8529. 5. Wilson, M.S. et al. Immunophatology of schistosomiasis. Immunol. Cell. Biol. 2007, 85: 148-154.

POS.130

Structure-guided development of D2R/NTS1R heterodimer-selective ligands Möller, D. Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich-Alexander Universität Erlangen-Nürnberg, Schuhstraße 19, 91052 Erlangen, Germany

Dopamine D2 receptors (D2Rs), which belong to family A of G protein-coupled receptors (GPCRs), regulate numerous physiological functions and are involved in the pathophysiology of severe neuropsychiatric disorders including schizophrenia and Parkinson’s disease. Along with an increasing number of other GPCRs, D2Rs have been proven to not only exist as isolated entities but to form both homo- and heterodimers within the plasma membrane. Among receptors interacting with D2Rs in the central nervous system (CNS), the neurotensin receptor subtype 1 (NTS1R) together with its endogenous ligand neurotensin has gained substantial interest over the past decades. Both GPCRs are known to be closely associated and highly co-localized in vivo. The specific targeting of heterodimers with bivalent ligands represents a powerful approach for the development of novel tool compounds and drugs for GPCRs. Driven by the binding energy of two recognition elements, a carefully designed bivalent ligand bridging the orthosteric binding sites of two adjacent protomers should exhibit extremely high binding affinity, and therefore high tissue selectivity for heterodimer-expressing cells over areas that express only one individual receptor. The growing number of recent high-resolution GPCR x-ray crystal structures and the increase in computational power available for homology modeling and molecular dynamics enabled us to design a collection of bi-orthosteric ligands addressing D2R/NTS1R heterodimers. By means of solid state-supported synthesis NT(8-13), the active fragment of neurotensin, was linked to three different D2R-specific pharmacophores employing -amino acid-functionalized polyethylene glycol (PEG) spacers (Fig. 1). Within this series of D2R/NTS1R-recognizing heterobivalent ligands, only compounds with a sufficient spacer size exhibited affinities in the low picomolar range and up to 10,000-fold selectivity for D2R/NTS1R-coexpressing cells compared to cells expressing D2R only. The preferential recognition of the D2R/NTS1R heterodimer over monomeric binding modes was confirmed in binding studies with membranes from striatal tissue, underpinning the biological relevance of the approach.

POSTERS


Fig. 1: Overview of the D2R/NTS1R heterodimer geometry modelled based on recent x-ray crystal structures of the closely related D3R, the NT(8-13)-bound NTS1R and the synthesized series of heterobivalent ligands targeting D2R/NTS1R.

Using bivalent ligands containing a D2R-agonist substructure, we could demonstrate that Gi/Go-promoted D2R signaling was attenuated in the D2R/NTS1R-coexpressing cells, while the compounds behaved as full D2R agonists in absence of NTS1R. In contrast, our bivalent ligands showed atypical dose-response curves in a -arrestin recruitment assay and were able to significantly enhance -arrestin recruitment to the D2R/NTS1R heterodimer. In summary our results suggest a highly selective binding profile and distinct signalling behavior for our bi-orthosteric heterobivalent ligands compared to monovalent congeners addressing D2R or NTS1R, thereby opening a promising perspective towards novel tools for GPCRs. References: 1. Hübner, H., Schellhorn, T., Gienger, M., Schaab, C., Kaindl, J., Leeb, L., Clark, T., Möller, 2. D.*, Gmeiner, P.* Nat. Commun., 2016, doi: 10.1038/ncomms12298, in press.

POS.131

Small molecule activators of TRPML channels Plesch, E.1; Keller, M.1; Chao, Y. K.2; Chen, C. C.2; Grimm, C.2#; Bracher, F.1# 1 Department of Pharmacy - Center for Drug Research, Ludwig-Maximilians-Universität, Butenandtstraße 5-13, 81377 München, Germany 2 Department of Pharmacy - Center for Drug Research and Center for Integrated Protein Science Munich (CIPSM), Ludwig-Maximilians-Universität, Butenandtstraße 5-13, 81377 München, Germany # shared last

TRPML channels, also called mucolipins, are endolysosmal cation channels. Mutations of TRPML1 in humans can cause the neurodegenerative lysosomal storage disorder mucolipidosis type IV while TRPML3, when mutated, causes deafness and pigmentation defects. The discovery of small molecule activators in a recent high-throughput screen [1] has significantly fostered and facilitated TRPML channel and thus endolysosomal ion channel research. Currently available TRPML channel agonists are non-selective and activate all three TRPML channel isoforms [1]. Through systematic chemical modifications of a selected lead structure, we have now designed potent and selective TRPML channel agonists. Our lead structure SN2 [1] potently activates human and mouse TRPML3 but lacks selectivity for mouse TRPML1 and human TRPML1 and TRPML2 (Figure 1). With the aim to further improve the potency and selectivity profile of SN2-type compounds, we set out to synthesize and test >50 chemically modified versions of SN2.

These modifications comprise variations of the substitution pattern of the aryl-ring, variations of the aliphatic norbornane ring system, aromatisation of isoxazoline to an isoxazole fragment, introduction of polar substituents as well as replacement of the isoxazol(in)e ring by other heterocycles. Following synthesis, we tested the compounds in overexpressing HEK293 cells using calcium imaging. We thus identified novel compounds with an increased efficacy for both mouse and human

TRPML3 and increased selectivity either for human or for mouse TRPML3. References: 1. Grimm, C. et al, Chemistry and Biology, 2010, 17(2): 135-148.

POS.132

Development of a G-protein biased mu opioid agonist with reduced side effects Dengler, D.1; Manglik, A.2; Lin, H.3; Aryal, D. K.4; McCorvy, J.4; Corder, G.5; Levit, A.3; Kling, R. C.1; Bernat, V.1; Hübner, H.1; Huang, X. P.4; Sassano, M. F. 4; Giguere, P. M.4; Löber, S.1; Duan, D.2; Scherrer, G.5; Kobilka, B. K.2; Roth, B. L.4; Shoichet, B. K.3; Gmeiner, P.1 1 Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander University, Schuhstraße 19, 91052 Erlangen, Germany 2 Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA 94305, USA 3 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, USA 4 Department of Pharmacology, UNC Chapel Hill Medical School, Chapel Hill, NC 27514, USA 5 Department of Anesthesiology, Stanford University School of Medicine, Stanford, CA 94305 USA.

Morphine and related opioids are well-known traditional analgesics and play until today an essential role in pain therapy. Their powerful analgesic effect is mainly referred to their ability to activate the μ opioid receptor (μOR). While the wide range of μOR agonists are alleviating pain extremely efficacious, they all suffer from similar adverse effects like respiratory suppression, constipation, sedation, physical dependence and tolerance. The side effects often limit the dose for opioid analgesics resulting in an inadequate pain management. Recent studies with morphine in β-arrestin-2 knock out mice suggest that the analgesic effect results from stimulation of the G-protein pathway, while many side effects may be an outcome of activated β-arrestins. Thus, functionally selective μOR agonists with bias towards G-protein promoted signaling are required. [1] The determination of the μOR crystal structure [2] provides an opportunity to seek μOR ligands with new chemotypes via structure-based approaches. In silico screening of over three million commercially available compounds against the orthosteric binding pocket and further structure-based optimization of initial docking hits led us to the identification of a novel μOR agonist scaffold. Starting from the originally determined hit as an isomeric mixture, we were able to develop an optically pure, subtype specific and G-protein biased μOR agonist (PZM21) by an appropriate synthetic strategy. Based on the crystal structure of the μOR in the active state [3], the binding mode of PZM21 could be established using SAR and molecular dynamics studies involving diffusible and covalently binding analogs. PZM21 shows an unique efficacy profile in mice models. The compound generates substantial analgesia with only little suppression of respiration compared to morphine. Results of mouse open field locomotion and conditioned place preference experiments indicate that PZM21 may have less reinforcing activity than classic opioids. Uniquely, PZM21 seems also to be able to differentiate between the affective component of pain and reflexive behaviors. Hence, this novel scaffold may serve both as a probe to investigate μOR signaling and as a therapeutic lead for a new class of opioid analgesics devoid of many of the dose-limiting side effects. [4] References: 1. L.M. Bohn et al., Science 1999, 286: 2495-2498 2. A. Manglik et al., Nature 2012, 485: 321-326 3. W. Huang et al., Nature 2015, 524: 315-321 4. A. Manglik et al., Nature 2016, accepted for publication



POS.133

Identification and in vitro characterization of GPCR ligands by a covalent docking approach Stößel, A.1; Fish, I.2; Hübner, H.1; Shoichet, B. K.2; Gmeiner, P.1 1 Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander University, Schuhstraße 19, 91052 Erlangen, Germany 2 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158, USA

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and remain a highly relevant drug target. Covalent molecular probes have proven to be useful tools to gain further insights in structural and functional properties of GPCRs. They have shown enhanced potency or selectivity towards the targeted protein and have demonstrated the ability to stabilize ligand-receptor complexes for crystallization. [1] Recently, a new approach (DOCKovalent) for the screening of large virtual libraries of electrophilic small molecules was developed and applied to discover covalent ligands for different enzymatic systems. [2] We successfully adopted this structure-based virtual screening method to identify covalent molecular probes for three different class A GPCRs. Therefore, a library of approximately 800,000 commercially available or synthetically accessible small molecules bearing a reactive group (Michael system or alkyl halide) were virtually screened against the µ-opioid receptor, the ß2-adrenergic receptor (H2.64C mutant) and the 5HT2A receptor (S2.61G, T2.64C double mutant). The high-ranking candidates were selected for experimental validation in a radioligand depletion assay indicating an irreversible blocking of the orthosteric binding site. The identified hits were further characterized by kinetic and functional studies, and used as templates for an additional screening round of close analogs. Taking advantage of this concept, we have identified specific covalent hits indicating that the covalent docking approach can be applied prospectively on several targets and may have broad utility for the discovery of covalent ligands or fragments for GPCRs.

References: 1. Weichert, D.; Gmeiner, P.: Covalent Molecular Probes for Class A G Protein-Coupled Receptors: Advances and Applications. ACS Chem Biol 2015, 10(6), 1376-1386. 2. London, N. et al.: Covalent docking of large libraries for the discovery of chemical probes. Nat Chem Biol 2014, 10(12), 1066-72.

POS.134

Pharmacological tools for the immune-stimulatory orphan G protein-coupled receptor 84 Köse, M.1; Schiedel, A. C.1; Brandt, S.1; Hoffmann, K.2; von Kügelgen, I.2; Müller, C. E.1 1 PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 2 PharmaCenter Bonn, Department of Pharmacology, University of Bonn, Sigmund-Freud-Straße 25, 53127 Bonn, Germany

The G protein-coupled receptor (GPCR) 84 (GPR84) is a Gi-coupled class A GPCR which is highly expressed in the bone marrow and in microglial cells; and upregulated under inflammatory conditions. GPR84 can be activated by medium-chain fatty acids (MCFAs, 9-14 carbon chain length) leading to an enhanced cytokine (IL-12 p40, IL-8 and TNF-α) production in myeloid cells.1-3 Recent in vivo experiments have indicated that GPR84 may play an important role in myeloid leukemia4, neuropathic pain5 and Alzheimer´s disease.6 Thus, GPR84 might be a promising drug target, e.g., for the treatment of inflammatory diseases. Besides MCFAs, which activate GPR84 in the micromolar concentration range, only very few ligands are available. However, selective pharmacological tools are required for further evaluation of GPR84 as a potential new drug target.

In an attempt to address this need, we performed an antagonist screening campaign investigating compounds from our in-house compound library in both cAMP accumulation and β-arrestin recruitment assays at the human GPR84 recombinantly expressed in Chinese hamster ovary (CHO) cells. As a result, we identified several hit compounds which blocked the receptor, the most potent compound showing an IC50 value of 1.40 µM in cAMP assays (versus 20 µM decanoic acid corresponding to its EC80). The new antagonists represent useful pharmacological tools to further investigate the (patho)physiological roles of GPR84. Furthermore, these structures will serve as starting points for optimization and development of more potent GPR84 antagonists. Moreover, in order to gain a better understanding of the signalling pathways involved in the immune response triggered by MCFAs, we evaluated a series of MCFAs as well as hydroxylated MCFAs in cAMP and β-arrestin assays. The results revealed that the structure-activity relationships of the MCFAs obtained in cAMP assays differ from those observed in β-arrestin assays suggesting biased signalling of the natural agonists. In contrast to the non-hydroxylated MCFAs, the hydroxylated fatty acids showed higher activity in cAMP assays than in -arrestin assays, leading to the assumption that the hydroxylated MCFAs are Gi-biased. This may have implications with regard to the modulation of inflammatory responses by MCFAs. References: 1. Wang, J et al.: J. Biol. Chem. 2006, 281: 34457-34464. 2. Bouchard, C et al. Glia 2007 55: 790-800. 3. Suzuki, M et al.: J. Biol. Chem. 2013, 288: 10684-10691. 4. Dietrich, P.A. et al.: Blood 2014, 124(22): 3284-3294. 5. Nicol, L.S.C. et al.: J. Neurosci. 2015, 35(23): 8959-8969. 6. Audoy-Rémus, J. et al.: Brain Behav. Immun. 2015, 46: 112-120.

POS.135

Identification of sphingosine-1-phosphate receptor ligands using a combined approach of molecular dynamics and pharmacophore-based virtual screening Bermudez, M.1; Leutz, S.1; Nguyen, T. N.1; Wolber, G.1 1 Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2, 14195 Berlin, Germany

Belonging to the class of G protein-coupled receptors (GPCRs)1, sphingosine-1-phosphate receptors (S1PRs) represent promising thus challenging targets for the treatment of various inflammatory diseases and autoimmune disorders2. However, only few orally bioavailable ligands are known to date and only fingolimod is therapeutically used for the treatment of multiple sclerosis. The aim of this study is to discover novel S1PR ligands by computer-aided drug design as both pharmacological tools and potential drug candidates. To date only one out of 5 S1PR subtypes has been crystallized3. Starting from the crystal structure of the S1PR 1 subtype we developed a structure-based virtual screening workflow that combines chemical feature-based 3D-pharmacophores and conformational sampling by molecular dynamics (MD) simulations4,5. Due to the availability of a crystal structure we initially focused on the S1PR 1 subtype and identified 10 potential ligands with high chemical diversity. In the second step we investigate structural differences between the 5 subtypes by means of homology modeling, docking and MD simulations. This information will be essential for the future development of ligands with subtype specific effects. Taken together we present and discuss the virtual screening work-flow, model validation, experimental challenges and the MD-based enhancement of the static pharmacophore models that were used to study S1PRs. References: 1. M. Bermudez, G.Wolber, Bioorg. Med. Chem., 2015, 14, 3907-3912. 2. R. Proia and T. Hla, J. Clin. Invest., 2015, 125, 1379-1387 3. M.Hanson et al., Science, 2012, 335, 851-855 4. A. Bock and M.Bermudez et al.: J. Biol. Chem., 2016, in press, doi: 10.1074/jbc.M116.735431 5. M. Bermudez et al.: Drug Discov. Today, 2016, in press, doi: 10.1016/j.drudis.2016.07.001

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POS.136

Design and synthesis of OH-substituted benzo[7]annulen-7-amines as GluN2B selective NMDA receptor ligands Temme, L.1; Wünsch, B.1 1 PharmaCampus, Department of Pharmaceutical and Medicinal Chemistry, WWU Münster, Corrensstr. 48, 48149 Münster, Germany

The function of the ionotropic NMDA receptor is important for synaptic plasticity. Whilst its hyperactivity leads to diseases like Alzheimer´s and Parkinson´s disease, its hypoactivity is for example involved in the development of schizophrenia. Therefore it is necessary to reconstitute the function of the NMDA receptor. Several strategies have been employed to reduce the hyperactivity and therefore prevent the resulting damaging of neurons (excitotoxicity). Whereas open channel blockers lead to a total block of the NMDA receptor and therefore cause unwanted side effects like hallucinations, GluN2B selective NMDA receptor antagonists reduce the excitotoxicity by stabilizing the agonist-bound receptor in a desensitized state and preserving the physiological function. [1]

Based on the lead structure ifenprodil a conformational restriction approach was persued with the aim to overcome its low selectivity. [2] Encouraged by the promising results this approach was applied on a distinct and but also highly potent GluN2B antagonist, Ro 25-6981. The aminoalcohol 1 shows high GluN2B affinity (Ki=16 nM), inhibitory activity (IC50=12 nM) and selectivity towards σ1 and σ2 receptors. The phenol 2 is even 10 times more affine (Ki=1.6 nM). [3, 4] Additionally, molecular docking studies showed promising interactions between the OH moieties of 1 and 2 and crucial amino acids in the binding pocket of the receptor. [5] Therefore further compounds with structure 3 were designed to combine the promising properties of 1 and 2 with the objective to increase affinity, selectivity and inhibitory activity. The poster will show the synthesis of ligands 3, their GluN2B affinity and further relationships between the structure and the GluN2B affinity.

Acknowledgements: Cells-in-Motion Cluster of Excellence in Münster, Prof. Wolfgang Sippl and his working group in Halle

References: 1. Zhu, A. et al.: Cell 2016, 165: 704–714. 2. Fischer, J. et al.: Pharmacol. Exp. Ther. 1997, 283: 1285–1292. 3. Benner, A. et al.: ChemMedChem 2014, 9, 741–751. 4. Gawaskar, S. et al.: Bioorg. Med. Chem 2014, 22, 6638–6646. 5. The docking studies were performed in cooperation with Prof. Dr. Wolfgang Sippl, University of Halle.

POS.137

Novel Hybrid Inhibitors of Cholinesterases as Potential Anti-Alzheimer Agents Messerer, R.1; Dallanoce, C.2; Matera, C.2; Wehle, S.1; Flammini, L.3; Barocelli, E.3; Decker, M.1; Sotriffer, C.1; De Amici, M.2; Holzgrabe, U.1 1 Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany 2 Dipartimento di Scienze Farmaceutiche, Sezione di Chimica Farmaceutica “Pietro Pratesi”, Università degli Studi di Milano, Via Mangiagalli 25, 20133 Milano, Italy 3 Dipartimento di Farmacia, Università degli Studi di Parma, Parco Area delle Scienze, 27/A, 43124 Parma, Italy

Alzheimer`s disease (AD) is the most prominent form of dementia affecting worldwide about 9% of the population aged over 65 and the number of patients will rise to estimated 81.1 million by 2040 [1]. AD is a multifactorial disease and several theories about its pathogenesis are discussed, mostly including the ß-amyloid cascade [2], the -protein hyperphosphorylation hypothesis [3], oxidative stress, free radical formation and neuroinflammation [4]. Today’s medication is based on the cholinergic hypothesis, asserting that a decline of acetylcholine (ACh) in the brain leads to cognitive and memory deficits. The level of

ACh is regulated by cholinesterases (ChEs), namely acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). A therapeutic approach is to inhibit these two hydrolytic enzymes. Well known AChE inhibitors are tacrine, donepezil, galantamine and rivastigmine, with tacrine being the most potent derivative in the series [5]. Unfortunately, the use of tacrine is currently limited by its serious hepatotoxicity [6] and the clinical effectiveness of AChEIs is still under debate [7]. It is therefore desirable to develop new and highly effective drugs for AD. Promising candidates are bipharmacophoric hybrid compounds in which the inhibitory potency of tacrine can be combined with other pharmacological benefits, such as reduced hepatotoxicity [8]. In this context, compound 1, consisting of tacrine and the muscarinic superagonist iperoxo [9] (imitating the endogenous ligand ACh) linked by a C10 polymethylene spacer, was found to have excellent anticholinesterase activities for both AChE (IC50 = 0.155 nM) from electric eel and isolated rat brain as well as for BChE (IC50 = 1.797 nM) from equine serum, respectively. Additionally, this hybrid shows less cytotoxicity than the corresponding tacrine-related dimers. Docking experiments provide a structural model to rationalize the inhibitory power towards AChE. Compound 1 is a promising candidate for further drug development investigations.

Acknowledgments: Thanks are due to the Institute for Molecular Infection Biology of the University of Würzburg (Germany) for evaluation of the cytotoxicity of the tacrine-related compounds.

References: 1. Deutsche Alzheimer Gesellschaft, Factsheet 2014. 2. Hardy, J.: J. Neurochem. 2009, 110(4): 1129-1134. 3. Bulic, B. et al.: Angew. Chem. Int. Ed. Engl. 2009, 48(10): 1740-1752. 4. Gella A., Durany, N.: Cell Adh. Migr. 2009, 3(1): 88-93. 5. Grimmer T., Kurz A.: Drugs Aging 2006, 23(12): 957-967. 6. Watkins, P.B. et al.: JAMA: J. Am. Med. Assoc. 1994, 271(13): 992-998. 7. Munoz-Torrero D.: Curr. Med. Chem. 2008, 15(24), 2433-2455. 8. Nepovimova E. et al.: J. Med. Chem. 2015, 58(22): 8985-9003. 9. Schrage R. et al.: Br. J. Pharmacol. 2013, 169(2): 357-370.

POS.138

Synthesis, receptor-subtype-selectivity and species-selectivity of dimeric hetarylpropylguanidine-type-histamine H2 receptor agonists (hH1,2,3,4R, gpH1,2R, rH2R) Pockes, S.1; Buschauer, A.1; Elz, S.1 1 Institute of Pharmacy, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany

Since the 1970s 3-(1H-imidazol-4-yl)propylguanidine (SK&F-91,486 (1)[1]) is known as the prototypic pharmacophore of highly potent histamine H2-receptor (H2R) agonists of the guanidine class of compounds including, e.g., impromidine and arpromidine.[2] In order to understand more of the structure-activity relationships of alkylated analogues of SK&F-91,486, we characterized 78 newly synthesized derivatives including several bivalent compounds (e.g., 2-5) by using different pharmacological in-vitro methods.[3] The already mentioned dimers were equipped with different spacer lenghts (C3-C12) and various heteroaromatic 5-ring systems. We replaced the initial imidazol-4-yl group of SK&F-91,486 (1) by the bioisosteric 2-amino-4-methylthiazol-5-yl and 2-aminothiazol-5-yl moieties, respectively, to study their influence on histamine receptor subtype selectivity. The potential H2R agonists were subjected to a broad screening procedure utilizing radioligand binding assays with membranes of Sf9 cells[4] (hH1,2,3,4R). Compounds were also functionally characterized in the [35S]GTPS assay (hH2R, Sf9 cell membranes).[5] Receptor subtype selectivity vs. hH1,3,4R was determined for selected derivatives also using this technique. The most promising dimers (2-5) were also tested with gpH2R- and rH2R-Sf9 cell membranes in the [35S]GTPS assay to characterize the species profile of the ligands.



Organ bath studies (gpH1R (ileum), gpH2R (right atrium)) yielded functional data in a more physiological environment. The major part of the new SK&F-91,486 analogues displayed partial or full agonism via hH2R, rH2R, and gpH2R, respectively. The most potent analogue, bivalent thiazole-type bisguanidine 5, was a partial agonist (Emax = 88%) and 250-times (pEC50 = 8,56) as potent as histamine vis-à-vis the gpH2R. Attempts to antagonize the positive chronotropic effect of (partial) agonists by preincubation with cimetidine, or by adding a cimetidine bolus at the end of the concentration-response curve, respectively, were successful and furnished pA2 values for the antagonist (5.87 – 6.38) which are in accordance with literature data.

References: 1. Parsons, M.E. et al.; Agents Actions 1975, 5, 464. 2. Buschauer, A.; J. Med. Chem. 1989, 32, 1963-1970. 3. Pockes, S.; Dissertation, Univ. Regensburg 2015. 4. Seifert, R. ; J. Pharmacol. Exp. Ther. 2003, 305, 1104-1115. 5. Schneider, E.H.; Seifert, R.; Pharmacol. Ther. 2010, 128, 387-418.

POS.139

Computational simulations guided by experiments - a strong alliance for GPCR research Bermudez, M.1; Wolber, G.1 1 Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Straße 2, 14195 Berlin, Germany

G protein coupled receptors (GPCRs) are integrative and highly dynamic signaling machines, transferring information across membranes via multiple signaling pathways [1]. Driven by the hypothesis form follows function the complexity of GPCR signaling roles requires a large conformational ensemble of distinct receptor states. Over the last decade, specific receptor-ligand complexes were determined by crystallography [2] providing an indispensable structural view on this protein class. However, these crystal structures represent single static conformations of highly flexible proteins. For a better understanding of receptor functionality we need functional models that consider GPCRs as dynamic entities. By means of computational simulations and 3D-pharmacophore analysis we show a dynamic view on GPCR signaling [3,4]. Taking muscarinic acetylcholine receptors as model systems we link conformational characteristics to distinct receptor functions [5]. Guided by pharmacological experiments, we developed mechanistic models to show that partial agonists for the muscarinic M2 receptor stabilize distinct fractions of inactive agonist-bound receptors [6]. Using bitopic ligands as pharmacological tools, we clearly identified a dualsteric and a purely allosteric binding mode stabilizing active and inactive receptor states, respectively. Modulation of the resulting ligand binding ensembles by various means reveals that agonist’s preference for inactive receptor complexes decreases its overall efficacy. Our findings suggest a more general role of ligand binding ensembles in determining agonist efficacy and may pave the way towards the rational design of partial agonists. Independently from this effect, we demonstrate on a molecular level how specific signaling pathways can be addressed in a ligand-dependent and predictable manner. Our data indicate a restriction of the conformational flexibility based on the chemical structure of a ligand resulting in a shift of the physiologically imprinted signaling preference. This chemically encoded conformational selection potentially represents a rational explanation for biased signaling, not only for different G proteins, but also for other signaling pathways including β-arrestins or GPCR-interacting proteins. References: 1. A. Bock et al.: Trends Pharmacol. Sci., 2014, 35, p630–638. 2. C. Piscitelli et al.: Mol. Pharmacol., 2015, 88, 536-551. 3. M. Bermudez, G.Wolber, Bioorg. Med. Chem., 2015, 14, 3907-3912. 4. M. Bermudez et al.: Drug Discov. Today, 2016, in press, doi: 10.1016/j.drudis.2016.07.001 5. M. Bermudez et al.: Mol. Inform., 2015, 8, 526-530. 6. A. Bock and M.Bermudez et al.: J. Biol. Chem., 2016, in press, doi: 10.1074/jbc.M116.735431

POS.140

A divergent synthesis of oxoaporphine and oxoisoaporphine alkaloids via direct metalation of alkoxyisoquinolines Melzer, B.1; Bracher, F.1 1 Department of Pharmacy – Center for Drug Research, Ludwig-Maximilians University, Butenandtstr. 5-13, D-81377 Munich, Germany

The tetracyclic oxoaporphine and oxoisoaporphine alkaloids are a small class in the wide field of benzylisoquinoline alkaloids. Due to their antibacterial, antifungal, anticancer, antiangiogenic and other biological activities they are of high pharmaceutical relevance [1,2]. Herein we describe a novel and divergent synthetic approach to oxoaporphine and oxoisoaporphine alkaloids starting from alkoxyisoquinolines. Direct metalation as key-step using the Knochel-Hauser base (TMPMgCl·LiCl) led to 1-magnesiated isoquinolines [3,4], which in turn afforded 1-iodoisoquinolines by quenching with iodine or aryl(isoquinolin-1-yl)carbinols upon trapping with aromatic aldehydes [4], respectively. Photochemical cyclization of ortho-bromo aryl(isoquinolin-1-yl)carbinols under reductive conditions gave the oxoaporphine alkaloids lysicamine and oxoglaucine. For the synthesis of oxoisoaporphine alkaloids, the required intermediate 1-arylisoquinolines were obtained by palladium-catalyzed Suzuki cross-coupling of the 1-iodoisoquinolines with the appropriate arylboronic acid pinacol ester building blocks. Subsequent hydrolysis of the ester to a carboxylic acid, directly followed by Friedel-Crafts type cyclization with Eaton’s reagent afforded the 6-O-demethylated oxoisoaporphines 6-O-demethylmenisporphine and dauriporphinoline. Re-methylation of these alkaloids using Kunitomo’s protocol [5] with methyl iodide and silver oxide gave the alkaloids menisporphine and dauriporphine.

References: 1. Stévigny, C., Bailly, C., Quetin-Leclercq, J.: Curr. Med. Chem.: Anti-Cancer Agents 2005, 5(2): 173-182. 2. Liu, Y. et al.: Curr. Top. Med. Chem. 2013, 13(17), 2116-2126. 3. Metzger, A., Schade, M. A., Knochel, P.: Org. Lett. 2008, 10(6), 1107-1110. 4. Melzer, B., Bracher, F.: Org. Biomol. Chem. 2015, 13, 7664-7672. 5. Kunitomo, J., Satoh, M.: Tetrahedron 1983, 39(20), 3261-3265.

POS.141

Total synthesis of Rubrolide R and S Schacht, M.; Boehlich, G. J.; De Vries, J.; Schützenmeister, N. Universität Hamburg, Pharmaceutical and Medicinal Chemistry, Bundesstraße 45, D-20146 Hamburg

The increasing emerge and distribution of resistant pathogens primary demands the discovery and development of novel antimicrobial agents and therapeutic approaches [1]. A promising source in this subject are represented by rubrolides, which are biological active metabolite from marine derived organisms [2]. In 2014 rubrolide R 1 and S 2 have been isolated from the marine-derived fungus Aspergillus terreus (OUCMDZ-

NH

N

HN

NH2

NH

(1) (2-5)

NH

NH

NH

NH

n

R1

NH

NH

R2

N

HN

S

N CH3

H2NS

NH2N = C= A = B

2

3

4

5

A

A

B

C

A

A

B

B

Nr. R2R1

8

12

8

8

n

POSTERS


1925).[3] Both rubrolide structures reveal biological activity against influenza A and rubrolide S 2 has also shown activity against the tobacco mosaic virus.[4] The total synthesis of these two novel natural products is essential for further biological investigations, especially in the field of antiviral as well as antibiotic testings. The rubrolide scaffold can be dissected into three building blocks. A short synthesis avoiding toxic tin compounds and unnecessary protecting groups has been established. The key steps of the synthesis are a Suzuki-Miyaura cross coupling [5] and a vinylogous Knoevenagel condensation [6].

References: 1. European Center for Disease Prevention and Control Antimicrobial surveillance in Europe 2013, Annual Reprort oft he European Resistance Surveillance Network (EARS-Net), 2014. 2. S. Miao et al.: J. Org Chem. 1991, 56, 6275-6260. 3. W. Zhu et al.: J. Antibiot. 2014, 67, 315-318. 4. Q.-F. Hu et al.:, Heterocycles 2014, 89, 2177-2183. 5. J. Boukouvalas, L.C. McCann, Tetrahedron Lett 2010, 51, 4636-4639. 6. J. Boukouvalas, F. Maltais, N. Lachance, Tetrahedron Lett. 1994, 43, 7897-7900.

POS.142

GluN2B selective antagonists: Enantiomerically pure tetrahydro-3-benzazepines derived from (S)-DOPA Rath, S.1; Wünsch, B.1 1 PharmaCampus, Institut für Pharmazeutische und Medizinische Chemie, Westfälische Wilhelms-Univ. Münster, D 48149 Münster, Germany

Due to overaging global population neurodegenerative diseases like Alzheimer’s disease and Parkinson’s disease have attracted major attention. Thus the development of selective therapeutic drugs for treatment is of high interest. A promising approach is to address the GluN2B subunit of the NMDA receptor. Overactivation of the NMDA receptor leads to excitotoxicity which is associated with neurodegenerative diseases. Ifenprodil served as promising lead compound for the design and development of novel GluN2B antagonists. Although ifenprodil shows high affinity (Ki = 10 nM, IC50 = 13.3 nM) its selectivity is rather low.[1,2,3,4] Based on the ifenprodil structure our group developed antagonists 1 with a 3-benzazepine scaffold. Several compounds of type 1 possess high GluN2B affinity and antagonistic activity. The phenyl butyl derivative 1a (R1 = CH3, R2 = H, R3 = (CH2)4Ph) with a Ki value of 5.4 nM and an IC50 value of 360 nM belongs to the most active and selective GluN2B antagonists.[5] Based on these encouraging results, a chiral pool synthesis of diastereo- and enantiomerically pure 3-benzazepines 2 starting from (S)-DOPA was envisaged. The additional substituents in position 1 and 4 should allow the fine tuning of the GluN2B affinity. Moreover, the introduction of [18F] to generate a tracer for positron emission tomography (PET) is possible. Evaluation of binding affinity is currently in progress.

References: 1. Kemp, J. A., McKernan, R. M.; Nat. Neurosci. 2002, 5, 1039–1042. 2. Stark, H., Graßmann, S., Reichert, U., Pharm. UZ 2000, 29, 159–166. 3. Schepmann, D. et al., J. Pharm. Biomed. Anal. 2010, 53, 603–608. 4. Borza, I., Domány, G., Curr. Top. Med. Chem. 2006, 6, 687–695. 5. Tewes, B. et al., ChemMedChem 2010, 5, 687–695.

POS.143

Investigation of the hemagglutinin cleaving type-II transmembrane proteases TMPRSS2 and matriptase as potential targets for influenza treatment Keils, A.1; Böttcher-Friebertshäuser, E.2; Magdolen, V.3; Steinmetzer, T.1 1 Institute of Pharmaceutical Chemistry, Philipps University Marburg, Marbacher Weg 6, 35037 Marburg, Germany 2 Institute of Virology, Philipps University Marburg, Hans-Meerwein-Str. 3, 35043 Marburg, Germany 3 Department of Gynecology, Technical University of Munich, Ismaninger Str.22, 81675 München, Germany

The activation of the surface glycoprotein hemagglutinin (HA) by host proteases is indispensable for influenza virus infection. HA is synthesized as a precursor protein (HA0) and cleaved into the subunits HA1 and HA2 either in the trans-Golgi network (TGN) or on the cell surface. Whereas the HA of highly pathogenic avian influenza viruses (HPAIV) is cleaved by furin-like proprotein convertases, the HA of the seasonal and pandemic human viruses as well as low pathogenic avian influenza viruses (LPAIV) is activated by trypsin-like serine proteases, such as TMPRSS2 and matriptase. Therefore, these proteases emerged as potential targets for influenza treatment [1]. TMPRSS2 and matriptase belong to the type II transmembrane serine proteases (TTSPs), which contain an N-terminal cytosolic segment, followed by a transmembrane domain, a variable “stem region” and a C-terminal extracellular protease domain of the chymotrypsin S1-fold. The TTSPs are synthesized as single chain zymogens and are activated by autocatalytic cleavage after an arginine or lysine residue, with the P4/P’4 cleavage site RQAR↓VVGG for matriptase and RQSR↓IVGG in case of TMPRSS2. Through a conserved disulfide bond, linking the protease domain with the “stem region”, these TTSPs are likely to remain membrane-bound following activation. Active matriptase can be detected on the cell surface and after shedding also in the extracellular space. In contrast, TMPRSS2 activity is principally limited to the inside of the cell where it accumulates in the TGN. Using an expression plasmid encoding the protease domain with an N-terminally located histidine-tag, followed by an enterokinase cleavage site, matriptase was conveniently expressed in form of inclusion bodies in E.coli. After refolding, purification, and activation by enterokinase sufficient material for kinetic measurements and structure determinations could be obtained. In contrast, the preparation of active TMPRSS2 using E. coli expression systems is problematic, detailed information about TMPRSS2 remains rare and is mainly based on subcellular full-length expression in mammalian cell systems. Therefore, we are working on an appropriate mammalian expression system for TMPRSS2 using different constructs to obtain active protease for further biochemical characterization and the development of new inhibitors. References: 1. Garten W. et al.: EurJ Cell Biol. 2015 94(7-9): 375-83

POS.144

CRISPR/Cas9 Bid knockout reveals a key role for Bid-mediated mitochondrial damage in ferroptosis Jelinek, A.1; Neitemeier, S.1; Hoffmann, L.1; Ganjam, G. K.1; Culmsee, C.1 1 Institute of Pharmacology and Clinical Pharmacy, Biochemical-Pharmacological Center, Philipps-University Marburg, Karl-von-Frisch-Straße 1, 35043 Marburg, Germany

Targeted genome engineering by CRISPR/Cas9 is an evolving tool for generating specific knockout cell lines by DNA cleavage and introduction of insertion or deletion mutations. In this study, we exploited this tool to generate a Bid (BH3-interacting domain death agonist) knockout cell line in neuronal HT-22 cells. Bid determines regulated cell death in paradigms of oxidative stress by glutamate toxicity (oxytosis) in neurons, where its activation and mitochondrial translocation mediates mitochondrial damage, subsequent release of Apoptosis inducing factor (AIF) and cell death. In the present study we generated a Bid CRISPR/Cas9-knockout cell line to elucidate the potential role of BID in a model of ferroptosis in HT-22 cells. Ferroptosis has recently been characterized as an iron-dependent form of oxidative



stress induced cell death [1] and involves cystine/glutamate antiporter (Xc-) inhibition, impairment of GpX4 (glutathione peroxidase 4) activity and subsequently increased lipid peroxidation. So far, however, the key mechanisms mediating cell death as a consequence of ROS accumulation and potential involvement of mitochondrial death pathways in paradigms of ferroptosis have not been reported. In order to investigate mechanisms of ferroptosis in neuronal HT-22 cells, we applied erastin as a pharmacological inhibitor of Xc- and analyzed glutathione depletion (GSH assay), lipid peroxidation (BODIPY - FACS analysis), Bid translocation to the mitochondria (confocal fluorescent microscopy), mitochondrial ROS formation (MitoSOX - FACS analysis), mitochondrial morphology (fluorescence microscopy), and cell death (Annexin V/PI - FACS analysis). In this paradigm of ferroptosis in neuronal cells we detected time-dependent GSH depletion, followed by increases in lipid peroxidation and mitochondrial impairments which all preceded cell death. Using the CRISPR/Cas9-Bid-knockout HT-22 cell line we found that Bid knockout prevented erastin-induced cell death as well as upstream events, e.g. lipid peroxidation, loss of mitochondrial membrane potential (TMRE - FACS analysis), mitochondrial ROS production as well as mitochondrial fission. In contrast, erastin-mediated early glutathione depletion was not affected by Bid knockout. In addition, we analyzed the effects of the established pharmacological inhibitors ferrostatin-1, liproxstatin-1 and the BID inhibitor BI-6c9 on paradigms of mitochondrial parameters in wild-type cells compared to the effects in Bid-knockout cells after induction of ferroptosis with erastin. BI-6c9 inhibited erastin-induced morphological changes and also prevented cell death. Similar protective effects were achieved in a concentration-dependent manner with ferrostatin-1 and liproxstatin-1. FACS analysis demonstrated that the applied inhibitors abolished lipid peroxide formation and reduced mitochondrial ROS production in both conditions of oxidative cell death, i.e. erastin-induced ferroptosis and glutamate-induced oxytosis. In conclusion, the present study exposes the mitochondrial transactivation of BID as a key molecular link between oxidative stress and mitochondrial pathology in the model of ferroptosis considerably resembling established characteristics of cell death in paradigms of oxytosis in neuronal cells. References: 1. Dixon, S. J. et al; Cell 2012, 149: 1060–1072.

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3.7 GPCR/Ion Channels

POS.146 Dualsteric compounds to induce signaling pathway selectivity in CHO-M1 cells Bödefeld, T.1; Matera, C.2; Dallanoce, C.2; Messerer, R.3; Holzgrabe, U.3; De Amici, M.2; Mohr, K.1; Schrage, R.1 1 Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, Gerhard-Domagk- Straße 3, 53121 Bonn, Germany; 2 Department of Pharmaceutical Sciences, University of Milan, Via Mangiagalli 25, 20133 Milan, Italy; 3 Institute of Pharmaceutical and Medicinal Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

Muscarinic acetylcholine receptors (mAChRs) belong to the large superfamily of G protein-coupled receptors (GPCRs). Stimulation of GPCRs leads to a conformational change in the receptor protein allowing the receptor to transduce extracellular stimuli onto intracellular adaptor proteins which then further propagate the signal inside the cell [1]. GPCR-induced signaling can be the result of the interplay between rather complex molecular events as several GPCRs can activate multiple different adaptor proteins, for instance different classes of heterotrimeric G proteins. Additionally, GPCRs can be targeted by compounds not only via the binding site for the endogenous messenger (orthosteric binding site) but also via distinct druggable “allosteric” sites [2]. In the present work, we investigated the influence of spatial restriction of the allosteric vestibule located in the extracellular loops of the receptor protein on muscarinic M1 acetylcholine receptor (M1 mAChR) -mediated signaling pathways. To this end, we employed several dualsteric compounds which are able to simultaneously occupy the orthosteric and an allosteric binding site of mAChRs, thereby constraining flexibility of the extracellular vestibule which constitutes the common allosteric binding site [3]. At the muscarinic M2 receptor, such spatial restriction of the allosteric area of the receptor protein has been demonstrated to allow selective activation of particular signaling pathways [2]. Here, we want to transfer this principle to the muscarinic M1 subtype, which preferentially signals into Gq/11-mediated pathways, but can also promiscuously stimulate Gs and Gi proteins [4]. We investigated M1 receptor-mediated signaling induced by dualsteric compounds consisting of highly affinitive and efficacious agonist iperoxo [5] as an orthosteric building block linked to an allosteric phthalimide (phth) moiety or a bulkier naphthalimide (naph) residue through alkyl chains of different lengths (6, 7, 8 and 10 methylene units). Gq/11- and Gs-dependent signaling pathways were analyzed in HTRF-based IP1 and cAMP accumulation assays, respectively. We found that, in general, Gs protein activation in CHO-M1 cells was highly sensitive to the restriction of spatial flexibility of the extracellular receptor area, because application of the bulky allosteric naph residue compromised M1 receptor-mediated cAMP production to a greater extent than IP1 accumulation. Moreover, compounds with a rather short linker length (i.e. C6) displayed weaker potency and efficacy for both Gq/11- and Gs-mediated signaling than dualsteric ligands with elongated linker chains (i.e. C8). In particular, iper-6-phth was a partial agonist for the Gq/11 pathway, but totally lost affinity for Gs-mediated signaling, whereas iper-7-phth was a good partial agonist for both pathways under investigation. For compounds carrying the rather voluminous naph moiety as allosteric residue an even stronger impact of a short linker length was observed. Indeed, iper-6-naph acted as a neutral antagonist on both pathways investigated up to a concentration of 10 µM, although it was able to bind to the M1 mAChRs with an affinity in the low micromolar range. Interestingly, previous studies demonstrated that this ligand is a partial agonist at M2 and M3 subtypes of mAChRs [2 and unpublished data]. Dualsteric compounds with 7, 8 and 10 polymethylene spacer chains were all good partial agonists for Gq and moderate partial agonists for Gs protein activation. We conclude that a C6 linker connecting the allosteric and the orthosteric moiety compromises receptor flexibility to the greatest extent and thus has the strongest impact on M1-mediated signaling. Taken together, we present iper-6-naph as the first dualsteric compound with functional subtype selectivity at mAChRs, as it is a rather good partial agonist at M2 [2] and M3 receptors, but clearly acts as a neutral antagonist at the M1 subtype. Furthermore, our findings show that it is possible to control M1-receptor mediated signaling by restriction of the conformational flexibility in the allosteric area of the receptor protein.

References: 1. Magalhaes, A., Dunn, H., Ferguson, S.: Br. J. Pharmacol. 2011, 165(6): 1717-36. 2. Bock, A. et al.: Nat. Commun. 2012, 3:1044 doi: 10.1038/ncomms2028. 3. Antony, J. et al.: FASEB J. 2009, 23:442-450. 4. Thomas, R. et al.: J. Pharmacol. Exp. Ther. 2008, 327(2):365-74. 5. Schrage, R. et al.: Br. J. Pharmacol. 2013, 169(2):357-70.

POS.147 Differential modulation by allosteric receptor epitopes of dualsteric ligand binding and signaling at human muscarinic M1 acetylcholine receptors Holze, J.1; Klöckner, J.2; Holzgrabe, U.2; Decker, M.2; Mohr, K.1; Tränkle, C.1 1 Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, Gerhard-Domagk-Str. 3, 53121 Bonn, Germany 2 Pharmaceutical and Medicinal Chemistry, Institute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, 97074 Würzburg, Germany

The five muscarinic receptor subtypes are metabotropic GPCRs, with the M1 being most abundantly expressed in the central nervous system [1]. Recently, we investigated the bitopic/dualsteric hybrid ligands JK 550 and JK 537 (cf. Table 1) containing the orthosteric binding block iperoxo and BQCA-derivated compounds as allosteric moieties [2]. It is known that dualsteric ligands are able to bind in two different orientations to the receptor which are characterizable by different binding constants: the dualsteric binding pose which stabilizes the active state with the affinity measure Kactive and the allosteric binding pose preferring the inactive state with Kinactive [3]. Based on the operational model of agonism for dynamic ligands [4] we have shown earlier that increasing the spacer length (n = 3 to n = 5, cf. Table 1) between the orthosteric and the allosteric moiety favored the active dualsteric orthosteric/allosteric receptor binding pose at the human hM1 receptor [2]. To elucidate these findings we studied the binding and signaling properties of JK 550 and JK 537 in intact live Flp-InTM-Chinese hamster ovary cells (Flp-InTM-CHO) stably expressing either hM1 wild-type or hM1 receptors single point mutated at two allosteric epitopes. In detail, receptor binding experiments were carried out applying [3H]N-methylscopolamine ([3H]NMS, 0.2 nM) and, functionally, receptor-mediated signaling was quantified as compound induced IP1-accumulation via FRET measurement in wild-type hM1, hM1-Y179A and hM1 E401A cells. Data analysis based on the operational model of agonism for dynamic ligands (e.g. Kactive, Kinactive) [4] or a four parameter logistic equation (Emax).

For both hybrids the binding experiments revealed that in the allosteric M1-E401A mutation, Kinactive was significantly increased compared to the M1-wt resulting in a significant increase in the orientation ratio Rpose,binding (= -log(Kactive/Kinactive) (cf. Table). Vice versa, for JK 550 but not JK 537 in the allosteric M1-Y179A mutation, Kinactive was significantly decreased compared to the M1-wt resulting in a significant decrease of the orientation ratio Rpose,binding. When the global regression data analysis was extended to also include the functional signaling data, in no case the Rpose,function values were different from the corresponding Rpose,binding

values. The lower value of Kinactive in the M1-Y179A mutation for JK 550 resulted in a shortfall of its maximal receptor occupancy (max. occ.) in the dualsteric pose to 45% compared to 93% in M1-wt. Functionally, this translated into a significant decrease of the maximum effect Emax from 53% in M1-wt to 16% in M1-Y179A, and it strongly reduced max*, i.e. the efficacy of JK 550 at 100 % receptor occupancy in the active pose. Interestingly, the longer hybrid JK 537 did not show decreased Rpose values in the M1-Y179A mutant. However, the - compared to the M1-wt - identical maximum receptor occupancy of the dualsteric pose of JK 537, i.e. 97 vs. 94%, did not translate into any IP1 signaling (cf. Table). In conclusion, our findings reveal that Y179 in the extracellular loop 2 (ECL2) of the hM1 receptor plays a key role for the binding orientation and the extent of signaling induced by a given dualsteric ligand.

GPCR/ION CHANNELS


References: 1. Bymaster, F.P. et al.: Neurochem. Res. 2003, 28(3-4), 437-442. 2. Chen, X. et al.: J. Med. Chem. 2015, 58(2), 560-76. 3. Bock, A. and Mohr, K.: Drug Discov. Today Technol. 2013, 10(2), 245-252. 4. Bock, A. et al.: Nat. Chem. Biol. 2014, 10(1), 18-20.

POS.148 Knockdown of the psychiatric susceptibility gene Cacna1c mediates protection of mitochondria against glutamate-induced stress in neuronal cells Michels, S.1; Ganjam, G. K.1; Martins, H.2; Braun, M. D.3; Kisko, T. M.3; Schwarting, R. K. W.3; Wöhr, M.3; Schratt, G.2; Culmsee, C.1 1 Institute for Pharmacology and Clinical Pharmacy, Philipps-University, Karl-von-Frisch-Str. 1, 35043 Marburg, Germany 2 Institute of Physiological Chemistry, Philipps-University, Karl-von-Frisch-Str. 1, 35043 Marburg, Germany 3 Experimental and Physiological Psychology, Philipps-University, Gutenbergstr. 18, 35037 Marburg, Germany

Several genome wide association studies have identified CACNA1C as one of the strongest genetic risk factors for affective disorders [1,2]. It has recently been shown that the main SNP rs1006737 is associated with increased mRNA expression of CACNA1C [3]. However, its role in disease pathogenesis is still largely unknown [4]. CACNA1C codes for the α1C subunit of CaV1.2, which is the major L-type voltage-gated calcium channel in the brain, and underlies key neuronal functions such as dendritic development, cell survival, and synaptic plasticity [5]. Furthermore, mitochondrial dysfunction is also linked to psychiatric disorders and associated with the deregulation of intracellular calcium levels [6]. The aim of the present study is to investigate the effects of siRNA-mediated knockdown of Cacna1c gene expression on mitochondrial function combined with glutamate-induced oxidative stress in immortalized mouse hippocampal HT-22 cells. We analyzed cell viability and mitochondrial parameters using real-time impedance measurements, colorimetric and luminescence-based assays, and flow cytometry with different fluorescent dyes. We found that the downregulation of Cacna1c mRNA levels significantly protected the neuronal HT-22 cells from glutamate-induced cell death. In detail, glutamate challenged HT-22 cells transfected with Cacna1c siRNA showed reduced mitochondrial ROS formation and diminished rise in mitochondrial Ca2+ levels compared to control. Moreover, loss of mitochondrial membrane potential upon glutamate treatment was attenuated in Cacna1c siRNA transfected cells. Confirming these results, inhibition of L-type calcium channels with isradipine also prevented glutamate-mediated excitotoxicity in primary rat cortical neurons. Accordingly, several current publications suggest L-type calcium channel antagonists as an approach to innovative pharmacotherapy of mood disorders [7,8]. The molecular mechanisms underlying the effects of Cacna1c on mitochondrial performance remain to be elucidated. Acknowledgments: Supported by DFG FOR2107

References: 1. Green, E. et al.: Mol. Psychiatry. 2010, 15(10): 1016-22. 2. Ferreira, M. et al.: Nat. Genet. 2008, 40(9): 1056-8. 3. Yoshimizu, T. et al.: Mol. Psychiatry. 2015, 20(2): 162-9. 4. Harrison, P.: Curr. Opin. Neurobiol. 2016, 36: 1-6. 5. Bhat, S. et al.: Prog. Neurobiol. 2012, 99(1): 1-14. 6. Manji, H. et al.: Nat. Rev. Neurosci. 2012, 13(5): 293-307. 7. Cipriani, A. et al.: Mol. Psychiatry advance online publication. 2016 8. Zamponi, G.: Nat. Rev. Drug. Discov. 2016, 15(1): 19-34.

POS.149 Autistic and anxiety-related behaviors of Slack and Fmr1 knockout mice Bausch, A.1; Zerfass, P.1; Nann, Y.1; Dieter, R.1; Ruth, P.1; Lukowski, R.1 1 Pharmakologie, Toxikologie & Klinische Pharmazie, Institut für Pharmazie, Universität Tübingen

Background: Sodium-activated Slack channels are highly expressed throughout the brain and modulate firing patterns and general

excitability of many types of neurons. Recent findings have shown that the Fragile X mental retardation protein (FMRP) directly activates Slack channels. Deletion or mutation of FRMP results in Fragile X syndrome (FXS), the most common form of inherited intellectual disability and autism in humans. A mouse model of FXS, the Fmr1 knockout mouse, displays some phenotypical features of the human FXS. Interestingly, Na+-activated K+ currents are decreased in Fmr1-deficient mice suggesting that Slack may contribute to the neuronal abnormalities in FXS. Experimental Procedures: To identify phenotypes of the Fmr1 knockout mouse that may rely on decreased Slack activity, we compared autistic and anxiety-related phenotypes of Slack- and Fmr1-deficient mice, using well-established behavioral experimental tests (e.g. elevated-plus maze test, open field test, nest building test and social interaction tests). Results: We did not find any altered anxiety levels in Fmr1-deficient mice, but our findings reveal a trend to decreased anxiety-related behaviors of Slack-deficient mice in all anxiety tests. Fmr1 knockouts build nests of poor quality and show lack of preference for social novelty in a three-chamber social interaction test while performance of Slack knockouts is normal in these experimental setups. Conversely, Slack-deficient mice show social dominance in a tube test version whereas there is no difference between WT and Fmr1 knockout mice. Conclusion: We could not detect any similarities between autism- and anxiety-related phenotypes of Slack- and Fmr1-deficient mice. Our findings do not support the hypothesis of a contribution of Slack to these behavioral deficits seen in FXS.

POS.150 Approach to Map the P2X7 Receptor Interactome by Protein Cross-Linking Mass Spectrometry Stocklauser, R.1; Schmidt, A.2; Zhang, J.1; Imhof, A.2; Nicke, A.1

1 Walter Straub Institute of Pharmacology and Toxicology, LMU Munich, Germany 2 Munich Center of Integrated Protein Science, Biomedical Center, LMU Munich, Germany

The P2X7 receptor (P2X7Rs) is a trimeric ATP-gated cation channel. It is involved in inflammatory processes by triggering the release of pro-inflammatory cytokines and has been shown to contribute to a variety of pathologies, such as neurodegenerative processes and inflammatory bowel disease. Therefore, it represents a promising target for the development of novel drugs. In contrast to all other P2XR family members, it has a low affinity for extracellular ATP and a long cytoplasmic C-terminal domain, which is supposed to be crucial for targeting of the receptor to the plasma membrane, interactions with other proteins as part of a larger signalling complex and its ability to induce effects such as the formation of large pores and membrane blebbing. Despite its importance as a drug target, the proteins involved in its localization, P2X7R signalling and functional modulation under physiological conditions are still unclear. To address this issue we generated a P2X7R BAC transgenic mouse model, in which the receptor is fused via a streptag-heptahistidyl-linker to an EGFP reporter protein. This BAC transgenic approach permits P2X7R-EGFP expression at near physiological or moderate over-expression levels under its endogenous promotor. Biochemical and immunohistochemical experiments show that expression patterns and levels of P2X7R-EGFP correlate with endogenous P2X7Rs in different tissues. The complex glycosylation of the protein as well as specific membrane localization indicate its correct folding and integration into the plasma membrane. Using the P2X7R-EGFP fusion protein as a bait, endogenous P2X7R subunits are efficiently and specifically co-purified by immunoprecipitation against EGFP, confirming their coassembly. Although immunoprecipitation-coupled mass spectrometry (IP-MS) is nowadays the standard for the identification of protein complexes, protein-protein interactions with high dissociation rates are typically lost due to extensive washing. Especially for membrane proteins, detergents are essential to efficiently extract the protein complex from the plasma membrane and therefore impairing its native environment. Chemical crosslinking of proteins prior to the actual purification process reduces the loss of transient interactions. To further analyse the P2X7R interactome, we adapted this approach to our P2X7R BAC transgenic mouse model. Initial biochemical experiments show that the P2X7R can be crosslinked using the membrane permeable homo-bi-functional NHS-ester disuccinimidyl suberate (DSS), leading to the formation of stable di- and trimers of endogenous P2X7R and the P2X7R-EGFP

POSTERS


fusion protein. The optimization of crosslinking conditions is ongoing and will be presented. Data obtained from protein crosslinking coupled with mass spectrometry (XL-MS) experiments will not only be used to identify novel interaction partners, but also to define intra- and intermolecular interaction domains and will therefore provide a vital insight in the organization and function of the P2X7R signalling complex.

NATURAL COMPOUNDS/CHEMICAL BIOLOGY


3.8 Natural Compounds/ Chemical Biology

POS.152 The acetyl-CoA carboxylase (ACC) inhibitor soraphen A blocks the migration of primary endothelial cells by shifting the cell membranes’ composition of fatty acids Glatzel, D.1; Koeberle, A.2; Werz, O.2; Ashtikar, M.3, Müller, R.4; Fürst, R.1 1 Institute of Pharmaceutical Biology, Biocenter, Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany 2 Chair of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, University Jena, Philosophenweg 14, 07743 Jena, Germany 3 Fraunhofer Institute for Molecular Biology and Applied Ecology (IME), Project Group Translational Medicine and Pharmacology (TMP), Goethe-University, Max-von-Laue-Str. 9, 60438 Frankfurt am Main, Germany 4 Helmholtz Institute for Pharmaceutical Research Saarland, Department of Microbial Natural Products and Department of Pharmaceutical Biotechnology, Saarland University, Campus E8.1, 66123 Saarbrücken, Germany

Acetyl-CoA carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacterial and eukaryotic cells, i.e. the conversion (carboxylation) of acetyl-CoA into malonyl-CoA. ACC-generated malonyl-CoA functions as a substrate for de novo lipogenesis and acts as an inhibitor of mitochondrial β-oxidation of fatty acids. Because of its role in lipid metabolism this enzyme has become an interesting target in drug discovery in the field of metabolic diseases and cancer. Despite this interest in ACC, no attention has as yet been given to the role of ACC in endothelial cells. We aimed to investigate the role of ACC in endothelial cell migration, a functional key aspect of angiogenesis. We used the ACC inhibitor soraphen A, a polyketidic natural compound isolated from the myxobacterium Sorangium cellulosum, as well as an RNAi-based approach to inhibit the function of ACC. Primary human umbilical vein endothelial cells (HUVECs) were used as in vitro model. First, we analyzed the action of soraphen A on cell viability. The compound did neither lower the metabolic activity of HUVECs up to a concentration of 100 μM after 24 and 48 h (CTB assay) nor increase in the apoptosis rate after 24, 48, or 72 h up to 100 μM. Measuring adenosine triphosphate (ATP) levels revealed that 30 μM soraphen A does not alter the ATP levels in HUVECs after 24 h treatment. In contrast, a 48 h treatment significantly lowered the ATP levels by 12 %. Also gene silencing of ACC1 in HUVECs attenuated the ATP levels by 11 %. Mitochondrial membrane potential (MMP) assays showed decreased MMP levels (10 %) in soraphen A-treated cells after 24 h. Interestingly, the compound inhibited the migration of endothelial cells in a wound healing/scratch assay by 65 %. Gene silencing of ACC1 in HUVECs strongly decreased endothelial migration, whereas a knockdown of ACC2 had no influence. Furthermore, Boyden chamber assays revealed that soraphen A can also lower chemotactic migration by 34 %. Since actin rearrangement is necessary for migratory processes, we analyzed the F-actin cytoskeleton (microscopy) and found that soraphen A decreases the number of filopodia by 60 %, but did not influence stress fiber formation. Soraphen A inhibited the activation of the migration-regulating kinase Erk (Western blot analysis), whereas the activation of Akt was not influenced. Liquid chromatography-mass spectrometry (LC-MS) showed that soraphen A leads to a shift of the membrane lipid composition of HUVECs. Most notably, the levels of phosphatidylglycerol (PG) were lowered by 54 %. To establish a causal link between the reduction of PG levels and the observed migration-inhibiting action of soraphen A, a rescue experiments was performed: Substitution of dioleoylphosphatidylglycerol (10 μM and 100 μM, multilamellar vesicle) completely reversed the action of soraphen A. Moreover, soraphen A-treated cells also showed a shift in the degree of membrane lipid desaturation. Among the saturated fatty acids, the levels of the species phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) were decreased by 10 %, 6.5 %, and 15 %, respectively. In contrast, soraphen A increased PI levels within the class of monounsaturated fatty acids (MUFA) by 37 % as well as PC and PE within the group of polyunsaturated fatty acids (PUFA) by 55 % and 21 %. Interestingly, the treatment of HUVECs with the PUFA linolenic acid (100 μM) mimicked the effect of soraphen A and lowered

the endothelial migration by 60 %. Oleic acid as a representative of MUFAs had no influence. In summary, we could show for the first time that the enzyme ACC plays a crucial role in the migration of endothelial cells. Inhibition or knockdown of ACC1 strongly inhibited the migratory process. This inhibition is clearly based on the shift of the fatty acid composition of the endothelial cell membrane. Acknowledgement: This work was supported by the German Research Foundation (DFG, FOR 1406, FU 691/9-2).

POS.153 Crystallization and structure elucidation of plant type III polyketide synthase enzymes Khalil, E.1; Lukat, P. 2; Liu, B.1; Beerhues, L.1

1 Institut für Pharmazeutische Biologie, Technische Universität Braunschweig, Mendelssohnstrasse 1, 38106 Braunschweig, Germany 2 Struktur und Funktion der Proteine, Helmholtz-Zentrum für Infektionsforschung, Inhoffenstrasse 7, 38124 Braunschweig, Germany

Among the huge number of natural products distributed all over the world, i.e. in bacteria, fungi and plants, polyketides provide one of the most important families. They include many powerful drugs such as lovastatin, tetracycline and daunorubicin and their broad biological activities make them good candidates for new drug discovery. They are biosynthesized through a serious of decarboxylative condensation reactions by a group of enzymes called polyketide synthase (PKS) enzymes, which are further divided into three main groups: type I, type II and type III PKSs [1].Type III PKSs have, compared to the other types, drawn much attention, as they are smaller and simpler but still maintain the catalytic function of polyketide chain elongation and cyclization. In 1999, chalcone synthase (CHS) from Medicago sativa (alfalfa) was the first enzyme of this class that was crystallized and still is the most well studied [2]. Recently, many CHS-like enzymes have been crystallized, which revealed the structural basis for the functional diversity of this enzyme family. In this study, we have successfully solved the structures of two important enzymes of the same family, benzophenone synthase (BPS) and biphenyl synthase (BIS). BPS and BIS were cloned and expressed in our workgroup from cell cultures of Hypericum androsaemum and Malus domestica, respectively: Both enzymes utilize benzoyl-CoA as starter substrate, which is a rare starter unit for type III PKSs. After reaction with three molecules of malonyl CoA, BPS forms 2,4,6-trihydroxybenzophenone via a Claisen condensation mechanism, but BIS forms 3,5-dihydroxybiphenyl using an aldol condensation mechanism. Biphenyls are compounds with antimicrobial activity and represent the phytoalexins of the Rosaceae plant family, which includes important fruit trees like apple and pear. The production of these compounds helps the plants to resist microbial diseases. Furthermore, benzophenones and polyprenylated benzophenones are important biological compounds, exemplified by guttiferone F which exhibits antimicrobial and anti-HIV effects [3, 4]. In this project, we were also able to solve the structure of a BPS single site mutant which converted the wild type enzyme into a new type III PKS, called phenylpyrone synthase (PPS), which produces 6-phenyl-4-hydroxy-2-pyrone as a major product after only two condensation reactions with malonyl-CoA [5]. Our work will help understand the variations in the sequential condensation and cyclization mechanisms of type III PKSs, leading to product variability although using the same starter substrates.

Acknowledgments: This work was supported by a scholarship from the Libyan government to Ebtisam Khalil and a grant from the Deutsche Forschungsgemeinschaft (DFG).

References: 1. Shen B: J. Curr Opin Chem Biol. 2003, 7: 285-295. 2. Ferrer, JL. et al.: J. Nat Struct Biol. 1999, 6: 775-784 3. Chizzali, C. et al.: J. Plant physiol. 2012, 158: 864-875 4. Liu, B. et al.: J. Plant J. 2003, 34: 847-855 5. Klundt, T. et al.: J. Biol. Chem. 2009, 284 (45): 30957-30964

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POS.154 Permeation characteristics of hypericin is influenced by Hypericum perforatum extract Butterweck, V.1; Verjee, S.1; Kelber, O.3; Abdel-Aziz, H.3 1 Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences Northwestern Switzerland, Gründenstrasse 40, 4132 Muttenz, Swizzerland 2 Innovation & Development, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany 3 Medical Affairs, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany

Hypericin is a polycyclic quinone found in Hypericum perforatum L. Although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound [1], presumably, because hypericin is a highly lipophilic compound which is poorly soluble in aqueous solutions. Recently we have shown [2] that quercitrin and isoquercitrin, but not hyperoside, quercetin or rutin increased the uptake of hypericin in Caco-2 cells. The major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the Caco-2 cell system when combined with extract matrix. The permeation characteristics of hypericin (5 μM) in absence or presence of H. perforatum extract STW 3-VI (7.5, 29 and 58 μg/ml) were studied in the absorptive direction. Following application of hypericin to the apical side of the monolayer only negligible amounts of the compound were found in the basolateral compartment. The amount of hypericin in the basolateral compartment increased concentration-dependent in the presence of extract matrix (from 0 to 7.5 %). The majority of hypericin was found after cell extraction (44% in absence and 76% in presence of the extract). The precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. The experiments with the extract STW 3-VI as a matrix showed that besides flavonoids there seem to be further compounds in the extract (e.g. phenolic acids or proanthocyanidins) which substantially improve the permeation characteristics of hypericin. References: 1. Butterweck V et al., Planta Med 2003 69 (3):189-192 2. Verjee S et al. Planta Med 2015; 81: 1111-1120

POS.155/SL.29 Genome mining-guided drug discovery: from proteasome to protease inhibitors Kaysser, L.1,2; Zettler, J. 1,2; Zubeil, F.3; Leipoldt, F. 1,2; Wolf, F.1,2; Schorn, M.4; Bauer, J.1,2; Bendel, T.1,2; Kulik, A.5; Dorrestein, P. C.6; Moore, B. S.4,6; Gross, H.1,2; Grond, S.3

1 Dept. Pharmaceutical Biology, University of Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany 2 German Centre for Infection Research (DZIF), partner site Tübingen 3 Inst.. Organic Chemistry, University of Tübingen, Auf der Morgenstelle 18, 72076 Tübingen, Germany 4 Scripps Institution of Oceanography, UC San Diego, 9500 Gilman Drive, La Jolla CA, 92093-0204, USA 5 Interfaculty Institute of Microbiology and Infection Medicine Tübingen (IMIT), University of Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany 6 Skaggs School of Pharmacy, UC San Diego, 9500 Gilman Drive, La Jolla CA, 92093-0751, USA


POS.156/SL.14 The virulence factor LecB varies in clinical isolates: consequences for ligand binding and drug discovery Titz, A.1 1 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Campus E8.1, 66123 Saarbrücken, Germany


POS.157 The heritage of 19th and early 20th century explorers: historical diaries, letters and manuscripts as tools to identify pharmaceutically active natural compounds Helmstädter, A. Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt am Main, Germany

Diaries, letters and publications of pioneering explorers sent form Europe throughout the world in the 19th and early 20th century, are containing information about the medicinal uses of plants now and then. This has been already shown for the botanist Berthold Seemann (1825-1871) [1], the anthropologist Mellville William Hilton-Simpson (1881-1938) [2], the physician Arthur William Francis Kerr (1877-1942), and the reverend Joseph John Freeman (1794-1851). It is well known that historical sources may help to unveil traditional uses of medicinal plants which may then serve as promising guidelines for phytopharmaceutical screening and the search for new lead structures [3]. These sources include historical herbals [4], the heritage of Christian missionaries [5] and many others. It has been shown that the heritage of pioneering explorers is serving that purpose as well. Reports have been screened and the information given has been compared with state-of-the-art ethnopharmacological, phytochemical, and pharmaceutical knowledge in order to (i) estimate the degree of reliability of the historical records and (ii) to identify medicinal plants traditionally used but scarcely investigated with modern methods. Obviously, the sources investigated exert a significant degree of reliability, as many uses and indications could be scientifically confirmed. On the other hand, many plants described as medicinally useful have never been investigated so far. Thus it seems reasonable to prefer these plants in phytochemical and pharmacological screening programs as investigations should lead to useful results with higher probability than screening at random. Several suggestions can already be made. Acknowledgments: This study was partially funded by the Wellcome Trust (Grant No. WT106016AIA). Studies were done at the Archives of Kew Gardens,The Royal Anthropological Institute of Great Britain, London, and the Pitt Rivers Museum, Oxford.

References: 1. Helmstädter, A.: Ethnopharmacological information from the botanical correspondence of Berthold Seemann (1825-1871). Pharmazie 2015, 70(9): 616-626. 2. Helmstädter, A.: Ethnopharmacology in the work of Melville William Hilton-Simpson (1881-1938) – historical analysis and current research opportunities. Pharmazie 2016, 71(6): 352-360. 3. Buenz, E.J.; Schnepple, D.J.; Bauer, B.A. et al.: Techniques: Bioprospecting historical texts by hunting for new leads in old tomes. Trends Pharmacol. Sci. 2004, 25(9): 494-498. 4. Adams, M.; Berset, C.; Kessler, M.; Hamburger, M.: Medicinal herbs for the treatment of rheumatic disorders – a survey of European herbals form the 16th and 17th century. J. Ethnopharmacol. 2009, 121(3): 343-359. 5. Anagnostou, S.: Forming, transfer and globalization of medical-pharmaceutical knowledge in south east Asian mission (17th to 18th c.) – historical dimensions and modern perspectives. J. Ethnopharmacol. 2015, 167: 78-85.

BIOPHARMACEUTICS


3.9 Biopharmaceutics

POS.158

Physicochemical performance and skin penetration kinetics of different acyclovir formulations analysed by confocal Raman microscopy Jung, N.1; Namjoshi, S. N.2; Mohammed, Y. H.2; Grice, J. E.1; Raney, S. G.3; Roberts, M. S.2,4; Windbergs, M.1 1 Helmholtz-Institute for Pharmaceutical Research Saarland and Saarland University, Campus E 8.1, 66123 Saarbrücken, Germany 2 Therapeutics Research Centre, University of Queensland, BrisbaneQld 4072, Australia 3 U.S. Food and Drug Administration, 10903 New Hampshire Avenue, Silver Spring, Maryland 20993, USA 4 University of South Australia, 101 Curie St, Adelaide SA 5001, Australia

For development and quality control of topically administered formulations, in vitro analysis of penetration of drugs and excipients into and through the skin is indispensable. Generally, this involves in vitro permeation testing (IVPT) using Franz diffusion cells with subsequent drug quantification by HPLC. As this procedure is destructive, drug quantification is limited to the endpoint of the study or requires high numbers of individual Franz cells which are dismantled at predefined time points. Further, such tests do not allow to investigate interactions of the formulation with the skin sample. Therefore, novel non-destructive analytical approaches are highly valuable alternatives. In this study, confocal Raman microscopy was used as a chemically selective and non-destructive technology to assess the penetration of six different commercially available acyclovir creams into excised human skin. The creams were rubbed into full thickness skin samples with a circular motion simulating the clinical “in use” situation. After predefined time intervals, the cream was gently removed from the skin surface and confocal Raman microscopy was used to track the compounds of the formulations within the skin tissue. The depth of penetration of the cream components was determined by acquiring virtual cross sections in xz-direction throughout the skin sample, as well as line scans in the z-direction downward. While no significant difference in penetration depth among the creams was found after 4 hours, the reference products showed a relatively higher accumulation in the stratum corneum after 24 hours, In addition, deeper penetration into the upper epidermis could be detected for the reference products as compared to the test drug products. The data acquired with confocal Raman microscopy correlated well with findings from IVPT studies performed with the same formulations in static Franz diffusion cells using heat separated human epidermis samples. As physicochemical characteristics of the formulations are prone to influence the skin penetration kinetics, the different formulations were investigated by confocal Raman microscopy with respect to their compound distribution and phase behavior. Interestingly, the dispensing process was found to influence the phase behaviour and consequently the skin penetration kinetics. Based on the data, confocal Raman microscopy shows a high potential to serve as an innovative approach for label-free and non-destructive analysis of physicochemical formulation characteristics as well as the skin penetration behaviour of drugs as well as of excipients. Acknowledgments: Funding for this project was made possible, in part, by the Food and Drug Administration through grant U01FD005226-01. The views expressed in this abstract do not reflect the official policies of the U.S. Food and Drug Administration or the U.S. Department of Health and Human Services; nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government.

POS.159

Baclofen carbamate prodrugs: Physicochemical properties and membrane permeabilities in cell monolayer-based studies on intestinal and CNS transport Pendl, E.1,2; Hänggi, S.2; Mosad, S.3; Niess, R.3; Imanidis, G.2; Abdelaziz, A.4; Spahn-Langguth, H.1,2,3 1 Department of Pharmaceutical Sciences/Pharmaceutical Chemistry, Karl-Franzens-University, Universitätsplatz 1/I, 8010 Graz, Austria 2 Institute of Pharma Technology, University of Applied Life Sciences, Gründenstrasse 40, 4132 Muttenz/Basel, Switzerland 3 Faculty of Pharmacy & Biotechnology, German University in Cairo - GUC, Main Entrance El-Tagamoa El-Khames, New Cairo City, Egypt 4 Life Sciences Center Weihenstephan, Technical University Munich, Emil-Erlenmeyer-Forum 2, 85354 Freising, Germany

Baclofen (BAC), a specific GABAB-receptor agonist, has successfully been used for decades for the treatment of spastic disorders. However, the fraction of an oral or i.v. dose that reaches the CNS is negligible, since BAC does not sufficiently permeate through the blood-brain barrier (BBB). As a consequence intrathecal administration is needed to obtain appropriate biophase levels. More lipophilic analogues (e.g. ester derivatives) usually exhibit considerably higher membrane permeability than parent BAC. Hence, as potential CNS-targeting prodrugs, BAC methyl- and propyl-ester carbamates were studied in vivo in intravenously dosed rats, where - as opposed to results with the respective esters - a significant amount of BAC was detected in brain tissues following prodrug dosage, particularly in the case of the methyl carbamate. The purpose of the current studies, mainly comparing the methyl- vs. the propyl carbamate, was the characterization of physicochemical properties in-silico and in-vitro as well as membrane permeabilities in cell monolayer-based models for intestinal and CNS transport. Test systems used were Caco-2 cell monolayers for studies on intestinal permeabilities as well as hCMEC/3D cell monolayers as model for the BBB. Solubility studies yielded a significant solubility decrease with increasing lipophilicities (BAC, 25 mg/ml, BAC methyl carbamate, 0.41 mg/ml, 2-propyl carbamate, 0.09 mg/ml) with the butyl carbamate being very poorly soluble, experimental logP values ranged from -0.56 for BAC, 1.5 for BAC methyl carbamate to 3.4 for BAC 2-propyl carbamate. Membrane permeability studies confirmed that BAC and BAC carbamates have no relevant affinity to P-glycoprotein, while BAC, yet not the ester carbamates, appears to be a substrate for the taurine transporter. Apparent apical-to-basolateral permeabilities for BAC methyl carbamate in Caco-2 cell monolayers were appr. 20-fold higher than for BAC, apparent permeabilities in the BBB membrane model for the methyl carbamate were double as high as for parent BAC with a tendency to increase with higher lipophilicities. In summary, for BAC in Caco-2 cells the impact of the taurine transporter was lower than expected and that of the P-glycoprotein transporter basically nonexistent. Moreover, no relevant impact of transport systems was detected so far in the permeability studies with ester carbamates up to logP = 3.4. Hence, neither lipophilicities nor apparent permeabilities or transporter affinities would oppose against CNS targeting, yet support the concept that ester carbamates may represent suitable BAC prodrugs.

POS.160

A novel disintegration testing setup for solid oral dosage forms including adjustable hydrodynamics and pressure forces Rach, R.1; Nawroth, T.1; Langguth, P.1

1 Institute of Pharmacy and Biochemistry, Department of Pharmaceutical Technology and Biopharmacy, Johannes Gutenberg-University, 55128 Mainz, Germany

Disintegration testing is a widely used technique to characterize and analyze solid dosage forms, not only during formulation development but also as a tool for quality control. The disintegration apparatus described in PhEur and USP is commonly utilized. However, the device was introduced 50 years ago and it is not surprisingly, that the knowledge about the human gastrointestinal tract conditions, e.g., gastric motility and media composition, has vastly increased over the last decades and the significant impact of those parameters on the disintegration of orally administered dosage forms and the dissolution of drugs was acknowledged [1]. It is hence obvious that the compendial device cannot reflect the current status of biopharmaceutical science, as there were no significant changes implemented since. Therefore, it is highly necessary to develop a biopredictive disintegration tester that accounts for hydrodynamics, movement pattern and pressure forces. As our group presented previously, hydrodynamics and forces in the compendial disintegration tester are highly variable and non-controllable [2]. The need for disintegration testing with modifiable movement patterns and pressure forces that accounts for hydrodynamics and mechanical forces under controllable conditions motivated the development and introduction of an in house build device with a modified probe chamber and programmable moving unit. This work shows the results of disintegration studies in the modified device with various immediate release (IR) solid dosage forms. The

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influence of pressure forces on disintegration time was investigated in combination with different hydrodynamics. Distinct IR formulations were used with diverse disintegration mechanisms, in particular mechanical force dependent, as well as independent formulations. The aim was to demonstrate the effect of applying pressure on the dosage form during the disintegration process. The studies were performed and analyzed with the help of a DoE software (MODDE). These studies are part of a project that aims to develop orally administered formulations with maximum bioavailabilities with the help of a biopredictive in vitro disintegration tool. However, to validate the modified disintegration setup and define biopredictive operating conditions an increased knowledge about in vivo disintegration times is needed. References: 1. Koziolek, M. et al.: Mol. Pharmaceutics 2013, 10(5): 1610–1622. 2. Kindgen, S. et al.: Journal of Pharmaceutical Sciences 2015, 104(9): 2956–2968.

POS.161

Nature’s red remedy towards microorganisms Griffin, S.1,2,3; Nasim, M. J.1; Estevam, E. C.1; Denezhkin, P.1; Lilischkis, R.4; Keck, C. M.2; Schäfer, K. H.3; Jacob, C.1

1 Division of Bioorganic Chemistry, School of Pharmacy, Saarland University, D-66123 Saarbuecken, Germany 2 Institute of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg, Marburg 35032, Germany 3 Department of Biotechnology, University of Applied Sciences, Kaiserslautern, 66482 Zweibruecken, Germany 4 Department of Information Technology and Microsystem Technology, University of Applied Sciences, Kaiserslautern, 66482 Zweibruecken, Germany

Introduction: Staphylococcus carnosus has the potential of producing elemental chalcocogen nanoparticles [1-3]. S. carnosus excels its trade, in particular, producing elemental selenium nanoparticles. These deposits of selenium are, however, toxic for the host microorganisms and ultimately result in cell mortality. Besides the unwanted toxicity against the host organisms, these nanoparticles might be beneficial for the treatment of other diseases caused by microorganisms [4-6]. Aim: Therefore, the aim of this study was to investigate if bacterial based selenium nanoparticles possess anti-microbial activity against various different microorganisms and to investigate if mechanically generated selenium particles could show a similar efficacy. Methods: Bacterially cultivated nanoparticles were obtained by incubation of S. carnosus in media containing various concentrations of Na2SeO3. Series of centrifugation and sonication steps were performed for the isolation of elemental selenium. Mechanically generated particles were obtained by bead milling. The size and morphology was analysed using dynamic light scattering, laser diffraction and light microscopy. The anti-microbial activity was performed against S. carnosus, Escherichia coli, Saccharomyces cerevisiae and Steinernema feltiae. Results: In the recent studies, different strains of S. carnosus have shown the capability of reducing inorganic selenites into elemental selenium particles [7-8]. Keeping this in view bacterial selenium nanoparticles were produced and cultivated with considerable efficacy. The particles possessed a size of about 440 nm. The results of the antimicrobial experiments showed that bacterial particles were more effective against the gram positive host. The bacterial and yeast assays showed considerable activity while the least activity was seen with the nematodes. The mechanically modified particles were, in comparison, less effective over all, which may be related to the quality (i.e. larger size) of these particles. Conclusions: In short, the study shows effective production and cultivation of selenium nanoparticles from S. carnosus. Apart from this, the toxicities against the host as well as other common microorganisms have demonstrated a possible application in the field of medicine and agriculture [9]. The natural particles were shown to be more toxic against the test microorganism, while the mechanically manufactured particles were less efficient, which might be due to the larger size. Therefore, future studies will aim at producing smaller sized particles to improve the efficacy. Ultimately, the bacterially cultivated selenium nanoparticles are an effective natural source in the fight against diseases caused by microorganisms. Acknowledgments: The authors acknowledge the financial support from the University of Saarland and the CAPES Foundation, Ministry of Education of Brazil.

References: 1. Debieux. C. M. et al.: Proceedings of the National Academy of Sciences of the United States of America, 2011, 108: 13480-13485 2. Baesman. S.M. et al.: Extremophiles, 2009, 13: 695-705. 3. Borghese. R. et al.: J Hazard Mater, 2014, 269: 24-30. 4. Aruguete. D.M. et al.: Environ Sci-Proc Imp, 2013, 15: 93-102. 5. Shamim. A.N. et al.: Abstr Pap Am Chem S, 2009, 237: 66-66. 6. Estevam. E.C. et al.: Molecules, 2015, 20: 13894-13912. 7. Biswas. K.C. et al.: J Microbiol Meth, 2011, 86: 140-144. 8. Hunter. W.J. et al.: Curr Microbiol, 2008, 57: 83-88. 9. Ramya. S. et al.: J Trace Elem Med Biol, 2015, 32: 30-39.

POS.162

Friction-related protein particle formation in compounding and filling steps Brückl, L.1,2; Hahn, R.2; Schröder, T.1; Sergi, M.1; Sonderegger, C.1; Scheler, S.1 1 Department of Pharmaceutical Development, Sandoz GmbH, 6336 Langkampfen, Austria 2 Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, 1190 Vienna, Austria

Protein aggregation and particle formation by physical degradation is a common concern during the compounding and filling steps of biopharmaceuticals. Many factors are currently under discussion to affect the physical stability of proteins during compounding and filling, however, the contribution of each factor and their interactions are still unclear. A highly debated topic is protein denaturation and subsequent aggregation or particle formation by the effect of shear, which is an omnipresent factor as it occurs in form of velocity gradients in moving liquids. However, recent studies using in situ and real-time circular dichroism spectroscopy could show that the structural conformation of representative proteins in solution is unaltered by shear rates up to at least 104 s-1 [1]. Thus, it becomes clear that the stability of proteins in free solution is most likely unaffected during processing and that investigations should focus on interfaces, which impart enough energy to unfold proteins upon adsorption. As the mechanism of protein aggregation at air/liquid interfaces has already been characterized, we are focusing here on friction-related protein degradation at solid surfaces. Friction at solid surfaces occurs at various spots in the manufacturing of biopharmaceuticals, e.g. inside sliding bearings of bottom mounted magnetic type stirrers which are nowadays widely used for compounding. For the first time custom designed small scale bearings, made of different materials, in combination with an IgG1 antibody as model protein were used as a realistic model for lab scale investigation of the degradation mechanism inside the bearing. In the course of various experiments we could exclude protein denaturation caused by frictional heat generation, by the possible presence of extraordinary high shear rates in free solution as well as heterogeneous nucleation of IgG1 particles on SiC micro/nano particles shed from the bearing. Abrasion of adsorbed proteins [2] by contact sliding was identified as prevailing protein degradation mechanism and was quantified by an increase in turbidity and by monomer loss. The time course of protein degradation in the bearing was found to be saturable and to follow Michaelis-Menten kinetics. Interestingly, the time a molecule requires for conformational change after surface adsorption in relation to the touching frequency of the bearing was found to be most probably the crucial parameter determining the dependence of protein degradation from the stirring rate. Furthermore, protein degradation was highly dependent on combinations of the material of the bearing and excipients. Thus, a test system was introduced which allowed to systematically study these effects with a wider variety of materials and to investigate the effectiveness of stabilizers for different surface materials. The test system consists of spheres of a certain size and material, which are placed inside a sample-containing, rotating container under the exclusion of air. The rotational movement of the container causes friction between the spheres, which is finally leading to protein degradation and particle formation. Results from the test system using IgG1 and recombinant human growth hormone confirmed a protective effect of polysorbate 80 against friction-mediated particle formation. Polysorbate 80 most likely acts by a reduction of protein adsorption, showing higher protection in combination with a highly hydrophobic sliding material (PTFE) in contrast to silicon carbide and glass. Interestingly, monomer loss in presence of Fe3+-ions, which were introduced upon contact sliding of stainless steel, was even facilitated in

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the presence of polysorbate 80, cancelling out its protective effect against abrasion. Finally, a comparison of degradation products from various stresses by infrared spectroscopy (ATR-FTIR) revealed a high similarity between friction-related degradation products in general. Therefore, abrasion of adsorbed proteins is very likely the prevailing physical degradation mechanism in processing steps where contact sliding occurs. Summarizing, our findings can directly be applied in the optimization of production steps, the future design of compounding vessels and the test system can be used as tool for formulation development Acknowledgments: Biophysical Characterization group in Oberhaching (Sandoz)

References: 1. Brückl, L. et al.: Journal of Pharmaceutical Sciences 2016, 105 (6): 1810-1818. 2. Sediq, A., Duijvenvoorde, R., Jiskoot, W. : Journal of Pharmaceutical Sciences 2016, 105 (2) : 519-529

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Engineering of Bioinspired Nanoparticles for Drug Delivery Haryadi, B. M.1; Winter, G.1; Engert, J.1 1 Pharmazeutische Technologie und Biopharmazie, Ludwig-Maximilians-Universität (LMU) München, Butenandtstraße 5, 81377 München Germany

Nanoparticles have gained a lot of interest in drug delivery. Besides size and charge, nowadays other parameters, such as geometry, surface properties, deformability, and degradability, are considered for nanoparticle advancement [1]. Recent reports in the literature show that ellipsoidal, cell membrane-attired, and soft nanoparticles were least phagocytized by macrophages and last longer in the circulation system [2-4]. By implementing all parameters in one system, we developed bioinspired nanoparticles. A mechanical stretching technique for fabricating elongated polylactic acid (PLA) or poly(lactic-co-glycolic acid) (PLGA) nanoparticles with diameters and lengths around 200 and 700 nm, respectively, was applied. For functionalization of nanoparticles, a top-down biomimetic method was applied by smothering biodegradable polymeric nanoparticles with natural erythrocyte membranes, inclusive of membrane lipids and associated membrane proteins. The structure, size and surface zeta potential and protein analysis of the particles were examined by transmission electron microscopy, dynamic light scattering, as well as gel electrophoresis, consecutively. The bioinspired nanoparticles may offer a distinctive and elegant way in drug delivery.

Fig. 1: Transmission electron micrograph of bare ellipsoidal (above) and erythrocyte membrane-coated (below) elongated nanoparticles.

Acknowledgements: DAAD (Deutscher Akademischer Austauschdienst) is highly thanked for B.M.H.’s doctoral scholarship. The authors are grateful to Barbara Kneidl and PD Dr. med. Lars Lindner (Arbeitsgruppe Liposomen, Medizinische Klinik und Poliklinik III, Klinikum der LMU München) for use of Liposofast-Basic. Dr. Wei Zhang (Department of Pharmaceutical Biotechnology, LMU München) is kindly acknowledged for providing negative staining material for TEM analysis. The authors would like to thank Dr. Markus Döblinger (Department of Chemistry and Center for NanoScience (CeNS), LMU München) for assistance with TEM investigations.

References: 1. Blanco, E. et al.: Nature Biotechnol. 2015, 33 (9), 941-951. 2. Mathaes, R. et al.: Int. J. Pharm. 2014, 465, 159-164. 3. Hu, C. et al.: Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 10980-10985. 4. Anselmo, A. et al.: ACS Nano. 2015, 9, 3169-3177.

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Is the unintended targeting of polymeric and lipid Nano-DDS to ovarian and adrenal tissues an artefact or a common phenomenon? Mäder, K.1; Lucas, H.1; Weiss, V.1; Chytil, P.2; Etrych, T.2; Kressler, J.3, Müller, T.4 1 Institute of Pharmacy, Martin Luther University Halle-Wittenberg, W. Langenbeckstr. 4, 06120 Halle (Saale), Germany 2 Institute of Macromolecular Chemistry AS CR, v.v.i., Heyrovský Sq. 2, 162 06 Prague 6, Czech Republic, 3 Institute of Chemistry, Martin Luther University Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany 4 Dept. of Internal Medicine IV, Martin Luther University Halle-Wittenberg, Ernst Grube Str. 40, 06120 Halle (Saale), Germany

Nanoscaled drug delivery systems (Nano-DDS) are made from different materials (e.g. lipids and polymers). They offer unique opportunities to optimize the efficiency of drug delivery. One of the main goals includes the accumulation of the drug loaded carrier in the target tissue (e.g. in tumor tissue or in inflamed or infected tissue). It is well known, that many Nano-DDS show unintended accumulation in the liver, spleen and lung. Unfortunately, small organs are often neglected because of the efforts to separate them and the problem of poor drug content due to the small size. However, despite their small size these organs possess important functions. Using the dye DIR and optical imaging, we described previously, that several Nano-DDS might accumulate in the ovaries [1]. Our findings were confirmed with other Nano-DDS (lipid nanodispersions) by the group of Benoit, who used two different probes and independent detection techniques (NIR and γ-counting) [2]. Furthermore, Merian et al. showed that triple labeled (3H, 14C and fluorescence) lipid nanodispersions accumulate on the adrenals and ovaries [3]. In order to understand this surprising accumulation better and to increase the evidence of our findings on polymeric Nano-DDS, we extended our efforts to other polymeric nanoparticles and also to polymer micelles. We designed also double labelled nanoparticles, where one dye was covalently linked and one highly lipophilic dye was non covalently dissolved in the polymer matrix (Fig. 1 left). Ex vivo optical images show that both dyes accumulate in the adrenals and ovaries (Fig. 1 right).

Fig.1: Left: Principle structure of double labelled nano-particles. DY-676 is covalently bound to the amphiphilic HPMA derivative. The highly lipophilic dye DiR is dissolved in the FA-PGA core of the nanoparticles. Right: Ex- vivo images of ovaries with fallopian tubes (top, a-c) and kidneys with adrenals (bottom, d-f). From left to right are shown respectively: (a,d) Intensity weighted single spectrum images of DiR. (b,e) Intensity weighted single spectrum images of DY-676. (c,f) Unmixed composite images with overlaid signals (red color: DY-676; green color: DIR).

Our on different nanoscaled drug carriers indicate that polymeric micelles, polymer nanoparticles, nanocapsules and even nanoemulsions might accumulate in the ovaries and the adrenals. This observation has been made on different mice strains and on rats. Other groups made similar observations with lipid nano-carriers [2, 3]. Our current results indicate no clear impact of the particle size and the zeta potential. Further studies are necessary to evaluate the key parameters for this accumulation in order to design Nano-DDS which either avoid this uptake or to design specific carriers for ovarian or adrenal drug delivery. Acknowledgements: The support from the German Research Foundation (DFG grants MA 1648/7 and MA1648/8) and the Grant Agency of the Czech Republic (grant No. P207/12/J030) is gratefully acknowledged.

References: 1. Schädlich, A. et al. J. Contr. Rel. 2012, 160, 105–112. 2. Hirsjärvi, S. et al., Int. J. Pharm. 2013, 453, 594–600. 3. Mérian, J.; et al. J. Nucl. Med.2013, 54,1996-2003.

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POS.165

Layer-by-Layer Stabilization of Cylindrical Microparticles Using DNA Möhwald, M.; Pourasghar, M.; Schneider, M. Department of Pharmacy, Biopharmaceutics and Pharmaceutical Technology, Saarland University, Campus A4 1, 66123 Saarbrücken, Germany

In recent years many novel shapes have been introduced for particulate drug delivery systems. Changing the geometry of particles allows the creation of new drug release profiles, improved targeting and altered cell interactions [1 – 3]. Therefore innovative engineering techniques have to be established, allowing full control of shape and size as well as the incorporation of active compounds. We previously introduced a bottom-up, template-assisted engineering technique for the production of cylindrical, nano-composed microparticles [4, 5]. Within a shape-defining template, nanoparticles of different materials can be interconnected by alternating polymer deposition (Layer-by-Layer), leading to cylindrical micron-sized particulates. For the attachment of the polymers, electrostatic interactions can be the driving forces. In this recent study, we applied amorphous silica nanoparticles (SNPs) providing negative surface charges. For the polymer coating we chose chitosan (CHT) as a biocompatible and biodegradable polyelectrolyte providing positive charges due to tertiary amines. As the counterpart we used a herring testes DNA (HT-DNA) with negative charges due to the phosphate-sugar backbone. The resulting polymer coating after the deposition of six double-layers (CHT/HT-DNA counted as one double-layer) can be observed in Figure 1.

Fig. 1: Electron microscopy (SEM) visualization of nano-composed microparticles stabilized by chitosan and HT-DNA building up six double-layers; A] After the mechanical manipulation of the particles, the polymer matrix interconnecting the nanoparticles can be observed; B] A close-up of the particles shows polymer deposition in fine layers on the nanomaterial

The coating approach including HT-DNA lead to stable, cylindrical microparticles representing the geometry of the applied template. The electron microscopy visualization clearly shows the polymers building up a matrix immobilizing the SNPs to a corn-like architecture. Due to the biodegradability of chitosan, a release of the DNA can be achieved potentially qualifying the system as a novel delivery platform for nucleic-acid based drugs. References: 1. Sarah L. Clark et al.: Ionic Effects of Sodium Chloride on the Templated Deposition of Polyelectrolytes Using Layer-by-Layer Ionic Assembly, Macromolecules 1997, 30: 7237–7244 2. S. S. Shiratori, M. F. Rubner, pH-Dependent Thickness Behavior of Sequentially Adsorbed Layers of Weak Polyelectrolytes, Macromolecules 2000, vol. 33: 4213–4219 3. S. E.A. Gratton et al.: Nanofabricated particles for engineered drug therapies: A preliminary biodistribution study of PRINT™ nanoparticles, J. Control. Release 2007, vol. 121: 10–18 4. C. Tscheka et al.: Macrophage uptake of cylindrical microparticles investigated with correlative microscopy, Eur. J. Pharm. Biopharm. 2015, vol. 95: 151-155 5. D. Kohler et al.: Template-assisted polyelectrolyte encapsulation of nanoparticles into dispersible, hierarchically nanostructured microfibers, Adv. Mater. 2011; vol. 23: 1376–1379

POS.166/SL.57 Oligoaminoamide-based siRNA carriers for in vivo tumor targeting and gene silencing Lee, D. J.1,2; Kessel, E.1,2; He, D.1,2; Klein, P. M.1; Lächelt, U.1,2; Wagner, E.1,2 1 Department of Pharmacy & Center for NanoScience, Ludwig-Maximilians-Universität München, Butenandtstraße 5-13, 81377 Munich, Germany 2 Nanosystems Initiative Munich, Schellingstraße 4, 80799 Munich, Germany


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Stabilization of Oligonucleotide-loaded Gelatin Nanoparticles by Lyophilization Geh, K. J.1; Hubert, M.2; Winter, G.1

1 Ludwig-Maximilians-Universität München, Munich, 81377, Germany 2 Umeå University, Umeå, 901 87, Sweden

Introduction: In several studies, gelatin nanoparticles (GNPs) loaded with immunomodulatory cytosine-phosphate-guanosine oligonucleo-tides (CpG-ODNs) showed excellent clinical effects in the treatment of recurrent airway obstruction in horses1-3. We could establish a large scale production of GNPs4 in order to supply extensive clinical studies in equine patients. A topical application is ensured by the administration of CpG-GNPs via inhalation after nebulization. However, one main disadvantage is the maximal colloidal stability of 48 h of CpG-loaded GNPs in dispersion. Therefore, the purpose of this study was to develop of a freeze-dried CpG-GNP formulation in order to allow long-term storage. Methods: GNPs were prepared according to one-step desolvation4 as a modification of the common two-step desolvation method. After cationization, GNPs were loaded with CpG-ODNs. Sucrose and trehalose were used as drying adjuvants and lyophilisation of the formulations was performed according to Zillies et al.5. Samples were stored at room temperature as well as at 2-8°C. Formulations were characterized in terms of particle size and size distribution (PDI) using dynamic light scattering, loading efficiency (UV spectroscopy) and CpG-ODN stability using matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). Results: GNPs showed particle sizes between 225 nm and 245 nm and PDI values below 0.37 after loading with CpG-ODNs. Interestingly, after reconstitution of the lyophilized samples, particle sizes (208 – 236 nm) and PDI values (< 0.28) decreased (Figure 1). This phenomenon may be caused by the formation of a porous structure in the lyophilisates and subsequent shrinking of the particles, which is not completely reversible following rehydration. Particle sizes and PDI values remained stable upon storage for 6 months at room temperature or at 2-8°C. No difference between the drying agents sucrose and trehalose could be seen. MALDI MS data revealed that the integrity CpG-ODNs was not affected by the freeze drying process no the storage conditions (Figure 2). Furthermore, loading efficiencies of the samples were not impaired by lyophilization or storage and remained above 90%.

Conclusions: Lyophilization has been demonstrated as a suitable method to stabilize CpG-ODN-loaded GNPs. The drying procedure had no negative impact on particle characteristics or CpG integrity. MALDI MS could successfully be used to investigate the stability of CpG-ODNs bound to the particle surface. Furthermore, stability of the lyophilized CpG-GNPs at different storage conditions (room temperature and 2-8°C) could be shown for 6 months. Bioactivity of freeze-dried formulations is currently being assessed in an in vitro assay using equine cells from bronchoalveolar lavage. References: 1. Klier, J., et al., Journal of Veterinary Internal Medicine, 2015. 29(1): p. 286-293. 2. Klier, J., et al., Pharm Res, 2012. 29(6): p. 1650-1657. 3. Klier, J., et al., Equine Veterinary Journal, 2015. 47(S48): p. 26-26. 4. Geh, K.J. in 10th World Meeting on Pharmaceutics, Biopharmaceutics and Pharmaceutical Technology. 2016. Glasgow, GB. 5. Zillies, J.C., et al., European Journal of Pharmaceutics and Biopharmaceutics, 2008. 70(2).

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POS.168/SL.50 Toward Biopredictive Dissolution for Enteric Coated Dosage Forms Al-Gousous, J.1; Amidon, G. L.2; Langguth, P.1 1 Insititute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, Staudinger Weg 5, 55099 Mainz, Germany. 2 Department of Pharmaceutical Sciences, University of Michigan, Ann Arbor, Michigan 48109, USA.


POS.169

Thermoresponsive nanogel mediated protein replacement therapy restores skin-barrier function to full-thickness skin models derived from Transglutaminase 1 deficient patients Yealland, G.1; Plank, R.2,3; Obst, K.1; Miceli, M.4; Molina, M.4; Eckl, K.5,2,3; Calderón, M.4; Hennies, H. C.2,5; Hedtrich, S.1

1 Institute of Pharmaceutical Sciences Freie Universität Berlin, Germany; 2 Dept. of Biological Sciences University of Huddersfield, UK; 3 Medical University of Innsbruck, Austria; 4 Institute of Chemistry & Biochemistry Freie Universität Berlin, Germany; 5 Dept of Biology, Edge Hill University, UK; 6 Cologne Centre for Genomics, University of Cologne, Germany

Mutations in the Transglutaminase 1 (TGase1) gene, TGM1, cause autosomal recessive congenital ichthyosis (ARCI), a rare skin cornification disorder characterised by severe epidermal scaling and significant impairment to skin-barrier function. Current therapies offer only symptomatic relief. Delivery of functional TGase1 to the viable epidermis might rectify those dysfunctions caused by TGM1 mutations. Whilst topical application would be the most direct route of administration, proteins are unable to penetrate into the skin due to their physicochemical properties, calling for the use of novel delivery strategies. Full-thickness skin models were generated from fibroblasts and keratinocytes derived from ARCI patients possessing TGM1 mutations [1]. Relative to healthy controls these demonstrated increased permeability to testosterone and clear histopathological aberrations analogous to those seen in ARCI. As an experimental protein replacement therapy, thermoresponsive-hyperbranched polyglycerol-poly(N-isopropylacrylamide) nanogels (NG) were loaded with TGase1 and applied topically to skin models [2]. These were subsequently exposed to a temperature gradient emulating that found across the epidermal layers of human skin. Following four applications over nine days (1, 5 or 10µg/cm2 TGase1 applied in 500µg/cm2 NG), skin-barrier function was improved in a manner proportional to TGase1 concentration (Fig 1). Improvements were markedly greater than those seen following application of TGase1 in PBS alone (Fig 2), in keeping with previous work demonstrating enhanced protein delivery to the viable epidermis by NG [2]. Application of unloaded NG saw no effect to skin-barrier function. An immunohistochemical activity assay confirmed the delivery of functional TGase1 to the SC and outer viable epidermis in NG, but not PBS treated skin models. Assessment of NG interactions with cell monolayers demonstrated uptake of unloaded NG in healthy keratinocytes in a concentration dependant manner, which could be attenuated by endocytic inhibition. No apparent cytotoxic induction was seen in healthy keratinocytes or fibroblasts incubated with TGase1 loaded NG, as assessed by MTT, LDH release and BrDU incorporation assays. Mutant TGM1 skin models demonstrate deficient skin-barrier properties that can be restored by topical application of functional TGase1 encapsulated within thermo-responsive NG. Given their apparent biocompatibility, the use of thermo-responsive NG for topical protein replacement therapies presents a promising avenue in the treatment of severe congenital skin disorders such as ARCI.

Fig. 1 and 2: Skin model permeability to Testosterone following treatment with (1) TGase1 dissolved in PBS or (2) TGase1 loaded in thermo-responsive NG

Acknowledgments: Financial support by the German Research Foundation (HE 7440/2-2 and HE 3119/9-1), Bundesministerium für Bildung und Forschung (NanoMatFutur 13N12561) and the Austrian Science Fund (2259-B26) is gratefully acknowledged.

References: 1. Eckl, K.M.: et al.: J. Invest. Dermatol., 2011, 131(9): 1938–1942. 2. Witting, M.: et al.: Nanomed. Nanotech., Biol. Med., 2015, 11(5): 1179–1187

POS.170

alpha-Synuclein at the Blood-Brain Barrier in Parkinson`s Disease Mahringer, A.; Fricker, G. Department of Pharmaceutical Technology and Biopharmaceutics, Ruprecht-Karls University of Heidelberg, Heidelberg, Germany

Localised at the brain microvessel endothelium, the blood-brain barrier (BBB) provides a precise homeostatic neuronal environment and means a key determinant in drug transport to the brain. Solute carriers, efflux transporters (P-glycoprotein (P-gp), Breast Cancer Resistance Protein (Bcrp), Multidrug Resistance Protein 4 (Mrp4)), tight junctions (TJs, Occludin, Claudin-5, Zonula occludens (ZO-1)) as well as endocytotic processes selectively deliver essential nutrients to the brain or remove neurotoxic agents. These BBB elements respond to a variety of regulatory signals making them susceptible to profound changes that occur during CNS diseases or pharmacotherapy [1]. In Parkinson`s Disease, alpha-Synuclein (αS) is a 14.4 kDa neuropeptide that forms neurotoxic deposits in dopaminergic neurons and is prone to aggregation into fibrils or Lewy bodies [2]. Particularly, the A53T mutant conformation leads to an early-onset of motor dysfunction and cognitive impairment symptoms. It has been shown recently that αS is present in cerebral blood vessels of cerebral amyloid angiopathy patients as well as in the cerebrospinal fluid (CSF) and blood plasma [3,4]. The following project provides insights into changes of BBB elements evoked by αS in Parkinson`s disease and allows conclusions on the involvement of pathophysiologically altered BBB clearance mechanisms in αS brain accumulation. Their restoration to healthy control levels could imply new targets in the therapy of Parkinson`s disease and extend established neurologic treatments to a vascular approach. A biphasic effect of both human native and A53T mutant αS monomers on the expression and function of the efflux transporters P-gp, Bcrp and Mrp4 as well as of TJs in an in vitro and ex vivo model of porcine brain capillary endothelial cells, isolated rat brain capillaries as well as in A53T αS transfected rats (SD-Tg(SNCA*A53T)268Cjli) was determined in the present study: Both native and mutant αS isoforms significantly increased P-gp, Bcrp and Mrp4 RNA, protein-expression and -function at lower concentrations after short-term incubation which turned into a decline at higher concentrations and after exposure for 48 to 72h (1-1000ng/ml, 1-72h); this process was accompanied by an initial tightening of the BBB at low αS concentrations but followed by a gradual opening at higher concentrations after long-term incubation (Occludin). In parallel to the up-regulation of the efflux transporters an increased RAGE (receptor of advanced glycation end-products) expression was observed, which emerged together with the secretion of inflammatory TNFα and the induction of NFκB. Additionally, low αS concentrations caused a transient reduction (1-24h) of LRP1 (low density lipoprotein receptor-related protein 1) expression in capillary endothelial cells. Transport experiments across the BBB in vitro indicated an increased efflux rate and extent of both native and mutant αS monomers from the brain to the blood compartment relative to their uptake. This was also confirmed by the calculation of Kp,uu in vitro. Inhibitors of clathrin-mediated endocytosis decreased the passage from brain to blood and vice versa implicating a receptor-mediated mechanism (e.g. RAGE, LRP1). Last, αS uptake was higher in Parkinson rats after tail vein injection which can be linked to the pathological modifications at the BBB. References: 1. Danemann R., Prat A.: The Blood-Brain Barrier (Cold Spring Harb. Perspect. Biol.) 2015. 2. Peelaerts W. et al.: Nature 2015, 522(7556): 340-344. 3. Tamo W. et al.: Neurosci. Lett. 2005, 326: 5-8. 4. Simonsen A.H. et al.: Biomark. Med. 2016, 10(1): 19-34.

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3.10 Pharmaceutical Technology and Biomaterials

POS.171

Molecular interactions in pharmaceutical compounds Edkins, K.1 1 School of Medicine, Pharmacy and Health, Durham University, University Boulevard, Stockton-on-Tees, TS17 6BH, United Kingdom

Small organic molecules, especially in the pharmaceutical sciences, tend to crystallise in a plethora of different crystal forms, either as pure compounds or with the inclusion of solvent molecules. Due to their changed physico-chemical characteristics, such as melting point, compressibility, solubility and thus bioavailability, and physical and chemical stability, different crystal forms can pose a problem to the manufacture of medicines.[1] It is thus crucial to understand the crystallisation behaviour and manufacturability of these compounds in order to avoid problems in the life-time of the medicine and costly recalls comparable to ritonavir[2] or rotigotine.[3] Bioactive molecules and pharmaceuticals typically have multiple functional groups, enabling them to interact with receptors and thus show pharmacological action. In the solid-state, the interactions through these functional groups are the driving forces of molecular recognition. By applying X-ray and neutron diffraction methods as well as thermoanalysis, vapour sorption and spectroscopic analysis in combination with computational techniques, we are probing the strong and weak interactions within the crystal forms and during the crystallisation in order to understand and predict their characteristics. Using the pharmaceutical compounds Piroxicam, Theophylline and Diatrizoic acid, the influence of the molecular interactions within the crystal structure of their respective hydrated crystal forms on the observed macroscopic characteristics will be discussed. A series of tetrahydrocarbazolone derivatives are used as model compounds to investigate the destabilising effect of steric bulk on the predominant hydrogen bonding interaction in the solid-state and solution. Recent interest in medicinal chemistry in using bio-isosteric replacement as tool for lead structure optimisation has led to the replacement of functional groups with more or less related substituents without change of overall pharmacological effect. However, the implications that a changed substituent have on the solid-state characteristics of the new compound are rarely investigated. We are systematically elucidating the substituent influence on the crystal form landscape in order to predict changes in processability and manufacturability of novel drug compounds. A series of triphenylimidazole derivatives are used as model compounds to reveal the influence of halogen substitution on solvent inclusion as well as the relationship of the respective desolvated crystal forms. References: 1. Hilfiker R, Polymorphism: In the Pharmaceutical Industry (Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany), 2006. 2. Chemburkar SR et al., Org. Proc. R. & D. 2000, 4, 413-417. 3. Rietveld IB and Ceolin R, J. Pharm. Sci. 2015, 104, 4117-4122.

POS.172

Monoterpenyl and related glucosides – surfactants with added value in pharma and cosmetics? Mohr, C1; Dauer, K2.; Schwab, W.3; Huang, F.-C.3; Hoffmann, T.3; Fischer, T.3; Keck, C. M.1

1 Institute of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg, 35032 Marburg, Germany 2 Applied Pharmacy, University of Applied Sciences Kaiserslautern-Campus Pirmasens, 66953 Pirmasens, Germany 3 Biotechnology of Natural Products, Technische Universität München, 85354 Freising, Germany

Introduction: Terpenes and especially monoterpenes as well as aryl alcohols are secondary plant metabolites with manifold applications in the fields of food, healthcare and cosmetics [1]. Monoterpenes and

phenols are major components in volatile oils which are used as fragrances, insect repellents, pheromones or aroma in many different products [2]. The pharmacological action of monoterpenes is very broad, including antimicrobial [3], anti-inflammatory and anticancer activity [4]. In the late 1960s glucoconjugated forms of monoterpenes were firstly detected in rose petals. These conjugates represent nonvolatile precursors of the monoterpenes and open the possibility to retard the release of the actives in all fields of their application [5]. Despite this, the use of these compounds has been hindered until know, because only recently an innovative method to produce these compounds by biotechnological means was developed [5]. Potential applications of these compounds were recently reviewed by Schwab et al., also suggesting a potential use as surfactant, due to its related chemical structure to decyl glucoside, a surfactant frequently used in cosmetic industry [5]. Aim: Therefore, the aim of this study was to investigate whether monoterpenyl glucosides can be used as surfactants, which would make these compounds to actives with added value in pharma, food and cosmetics. Materials and methods: Two monoterpenyl glucosides, geraniol glucoside and thymol glucoside, as well as 2-phenylethanol glucoside were investigated. The possibility to form an emulsion was investigated by producing emulsions consisting of 10% (w/w) oil (Miglyol 812) and water to up to 100% (w/w). The amount of surfactant was varied from 1% to 5%, respectively. The emulsions obtained were analysed regarding droplet size and physical stability by using light microscopy and macroscopic observations. The emulsion quality was compared to emulsions produced with Tween 80 as stabilizer. The CMC was investigated by determining the surface tension at different concentrations using a tensiometer equipped with a wilhelmy plate. As references the stabilizers Sulfopon 1216g (MW 330) and Tween 80 (MW 1310) were used. Results: The emulsions produced with Tween 80 resulted in droplet sizes of about 10 µm, independent on the concentration of stabilizer used. The emulsions showed a slight physical instability, i.e. a slight creaming was observed after one day of storage. In addition a strong foam formation during production of the emulsions was observed. The emulsions produced with geraniol glucoside as stabilizer showed similar results, indicating a good emulsification efficacy. Emulsions stabilized with 2-phenylethanol glucoside resulted in emulsions with similar sizes to the control, however, a phase separation shortly after production was observed. Emulsification with thymol glucoside stabilizer did not lead to homogeneously sized emulsions. Interestingly, the amount of foam created during production was directly correlated to the ability to form emulsions, i.e. most of the foam was created by Tween 80 and geraniol glucoside, some foam was created by 2-phenylethanol glucoside and no foam was created with thymol glucoside. Measurements of the CMC values for Sulfopon and Tween 80 confirmed the results obtained from the literature, with values of about 50µM/l and 1mM/l (i.e. 0.03%), respectively. The CMC of geraniol glucoside was found to be about 10mM/l (i.e. 0.3%). The CMC of 2-phenylethanol glucoside is even higher and no CMC was detected for thymol glucoside, because the solubility of thymol glucoside was < 1%. However, for all compounds a strong reduction in the surface tension, even at low concentrations, was observed. The highest reduction in surface tension was obtained for Sulfopon and geraniol glucoside (about 30mN/m), followed by thymol glucoside, Tween and 2-phenylethanol glucoside, respectively. Conclusion: Results confirm that monoterpene glucosides possess amphiphilic properties. Depending on their chemical structure they might be used as emulsifiers, stabilizers or wetting agents. Therefore, this class of excipients might be used in the future for the production of innovative healthcare products, combining a medical or cosmeceutical and processing action in only one compound. In addition with other actives they might lead to synergistic effects, i.e. an improved medical or cosmeceutical activity and/or less required excipients. The possibility of tailor-made release profiles from these compounds further increases the scientific interest in this kind of compounds, suggesting more research in this field. References: 1. Raskin, I. et al.: Trends Biotechnol. 2002, 20(12): 522–531. 2. Isman, M.: Crop Prot. 2000, 19(8-10): 603-608. 3. Park, S. et al.: Anaerobe 2012, 18(3): 369–372. 4. Salminen, A. et al.: Cell. Mol. Life Sci. 2008, 65(19): 2979–2999. 5. Schwab, W. et al.: Appl. Microbiol. Biotechnol. 2015, 99:165–174.

PHARMACEUTICAL TECHNOLOGY AND BIOMATERIALS


POS.173

Is a particle counter an alternative to laser diffractometry for size analysis? Stumpf, F.1; Ibraimi, M.1,2; Paulke, B.3; Keck, C. M.1 1 Institute of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg, 35032 Marburg, Germany 2 Institute of Pharmaceutical Technology, Anadolu University, 26470 Eskisehir, Turkey 3 Fraunhofer-Institute for Applied Polymer Research, 14476 Potsdam-Golm, Germany

Introduction: Nanosized drug carriers, i.e. liposomes, nanoemulsions, lipid nanoparticles, nanocrystals or polymeric nanoparticles, possess special properties when compared to larger particles. Besides the development, formulation and application of these carriers, size characterization is one of the major fields of interest. Size characterization aims not only on the analysis of the main size, but also on the size distribution and on the detection of possible larger particles. Especially the detection of possible larger particles is highly important, because larger particles might influence the physical stability of the nanosized formulation (e.g. Ostwald-ripening). Therefore, methods which detect larger particles highly reliable are highly important. In most cases microscopic techniques and laser diffraction are used for such purposes. The advantages and disadvantages of these techniques were investigated in previous studies [1-3]. The aim of this study was to investigate if a particle counter, normally used for counting particles in infusion systems, can also be used for this purpose. Materials and methods: Latex samples with different sizes (2-3µm and 3-6µm) were analysed separately and as mixture (1:1) using different sizing technologies. Laser diffraction measurements were performed using a Mastersizer X and a Mastersizer 3000 (Malvern Instruments, UK). Particle counting was performed using a PAMAS SVSS (Pamas Partikelmess- und Analysesysteme, Germany). Particles were further characterized using light microscopy (Zeiss, Germany) and raster electron microscopy (Hitachi High-Technologies Europe GmbH, Germany). The results obtained were compared to each other. Results: Microscopic analysis confirmed the size of the samples (Fig. 1). In addition, a few agglomerates of the particles in the range from about 10 µm – 100 µm were detected. Results obtained from the Mastersizer 3000 revealed monodisperse distributions when analysed with the universal mode using Mie theory and correct optical properties for latex particles. Modification of the results, e.g. analysis in the special “spherical particles” mode and/or without the additional backscattering

Fig. 1: REM images of mixed latex sample, containing particles from 2µm – 6µm.

technique (blue light) led to more accurate results, which were in agreement with the results obtained from microscopic observations. However, the larger agglomerates were not detected by this technique. The reason for this might be the stirrer of the instrument which might have provided enough forces to destroy the agglomerates during the measurement. Results obtained from the Mastersizer X were similar to the results from the Mastersizer 3000 with optimized size analysis, i.e. the results showed bi- or trimodal distributions, which corresponded to the results obtained from microscopy. In contrast to the results obtained from the Mastersizer 3000, results obtained from the mastersizer X revealed a small fraction of larger sized particles, indicating that the Mastersizer X was more appropriate to detect a few larger particles within a small sized main population. The reason might the smaller sample cell of the Mastersizer X which uses only a small magnetic stirrer. Thus, the agglomerates were not destroyed during the measurement. The results obtained from the Pamas instrument were highly depended on the concentration used for the measurement. Most appropriate dilutions were found to be in the range from 1:10,000 to 1:20,000. By using these dilutions, similar results to the Mastersizer X,

i.e. bi- oder trimodal distribution and larger agglomerates, were obtained, indicating that the particle counter is able to provide useful information of the size and size distribution of microsized particles. Conclusion: Results confirm that the Mastersizer 3000 is a highly accurate instrument for size analysis. However, as shown in previous studies, results proved again that the size results can be highly misleading if parameters for analysis are incorrect. Another hazard of this method is the possibility that larger particles might not be detected by this method. The Mastersizer X and the particle counter showed accurate results and led to reliable information about the presence of larger particles. Therefore, especially if the presence of larger particles should be investigated, these techniques are recommended. Advantages of the particle counter are significantly lower costs of the equipment, a simple setup of the instrument and fast measurement procedures. Acknowledgments: Mirsad Ibraimi thanks ERASMUS for providing the scholarship. We thank Matthias Wojcik for the REM images.

References: 1. Keck, C. M., Müller, R. H. Int. J. Pharm. 2008, 355 (1-2): 150-63. 2. Keck, C. M. Int. J. Pharm. 2010, 390 (1):3-12. 3. Acar Kübart, S., Keck, C. M., J Pharm Tech Drug Res 2013, 2(17) dx.doi.org/10.7243/2050.

POS.174

Sortase-A mediates VHH – conjugation to differently PEGylated liposomes Wöll, S.1,2; Schiller, S.1; Scherließ, R.2

1Merck KGaA, Frankfurter Straße 250, 64293 Darmstadt, Germany 2Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany

Introduction: Liposomes are widely used as drug delivery system (DDS) in research for cancer therapy. Enclosing cytotoxic compounds into physiologically indifferent carriers releasing their cargo at the site of action increases safety and efficacy of anti-cancer drugs [1]. Equipping the DDS with ligands binding to specific proteins on tumour cells enables a targeted delivery. Common chemical coupling approaches make use of any of the reactive side chains of amino-acids (e.g. maleimide or EDC-NHS chemistry), leading to an undefined binding and decreased activity of the targeting moiety [2]. A sterically defined conjugation in non-paratop regions of an antibody would ensure preservation of activity as well as steric accessibility of the antigen binding regions. Therefore, we make use of a Sortase-A mediated directional transpeptidation of a LPETG (leucine-proline-glutamate-threonine-glycine) modified anti-eGFP single-domain antibody (VHH) to pentaglycine-modified liposomes, where the antibody is conjugated to the outer leaflet with high steric selectivity [3]. Four liposome concepts are tested for enzyme reaction with regard to develop a DDS platform with various in-vivo behaviour controlled by PEGylation status and surface charge (Fig 1). Conjugation and activity of the antibody is confirmed by an eGFP-binding assay.

Fig. 2: Liposome formulation concepts

Methods: Liposomes were prepared by a continuous solvent injection technique. Formulations consisted of DPPC, Cholesterol, DSPEmpeg-2000 or DPPG and DMA-G5 or DMA-PEG-G5 in a molar ratio of 59.4 : 34.65 : 4.95 : 1. Physical properties were controlled by dynamic light scattering and Laser Doppler electrophoresis, and pentaglycine content was determined using RP-HPLC ELSD. After incubation with Sortase-A and anti-eGFP VHH, liposomes were purified using dialysis. Reaction success was confirmed by mass spectroscopy. Binding ability was shown using HP-SEC by determination of unbound eGFP in a liposome – target protein mixture. Results and Conclusion: Liposomes were prepared in a size range suitable for in-vivo administration (<200 nm, PDI < 0.25) and functionalized with the targeting ligand while retaining physicochemical properties (Fig. 2). Mass spectroscopy confirmed conjugations of anti-eGFP VHH with a plane and a PEG-spaced pentaglycine modified lipid (Table 1). HP-SEC showed activity of the targeting ligand on the surface of the liposomes and revealed superior binding of eGFP to

POSTERS


Fig. 3: Physicochemical properties of VHH modified liposomes

Table 1: Mass spectroscopy of targeting ligands

targeted formulations compared to unspecific incubated DDS. The present work therefore shows a possible liposomal platform for the investigation of therapeutic VHHs with respect to the different in-vivo properties (circulation time, cellular uptake) of the drug delivery system due to the adaptable surface properties.

Conjugation Product

Expected Mw Determined Mw

DMA-VHH 14179 14179

DMA-PEG-VHH 15836 15842

Acknowledgements: The authors thank Lee Kim Swee, Christopher Bachran, Matthias Schröder and Lena Conrad of BioMedX for providing proteins and helpful discussion about the experiments.

References: 1. Barenholz, Y.: J. Control. Release, 2012, 160 (2), 117-134. 2. Saha, B., T.H. Evers; M.W. Prins: Analytical chemistry 2014, 86 (16), 8158-8166. 3. Ta, H.T. et al: Circ. Res. 2011, 109 (4), 365-373.

POS.175

Development of a New Depot Formulation for Peptide Delivery Yordanova, Y.1; Kauffold, J.2; Zaremba, W.3; Frieß, W.1 1 LMU Munich, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Butenandtstr.5-13, 81377 Munich, Germany, 2 University of Leipzig, Faculty of Veterinary Medicine, An den Tierkliniken 29, 04103 Leipzig, Germany 3 Veyx Pharma GmbH, Söhreweg 6, 34639 Schwarzenborn, Germany

Introduction: A new oil based parenteral sustained release formulation of Gonadorelin [6-D-Phe] in a multi-dose container for the manipulation of the reproduction cycle in the swine was developed [1]. In a previous study the correlation between viscosity and release rate from pure oil mixtures composed of castor oil and medium chain triglycerides (MCT) was evaluated. In vitro and in vivo data showed that a sustained release of the peptide can be achieved. However, a relatively high initial burst release of the peptide from the pure oil mixtures was observed. Furthermore, the rather high viscosity at 25 °C and Newtonian flow behavior posed difficulties upon multiple dose application. Therefore, a second generation of oil suspensions was composed of aluminium distearate (AlSt) gelled MCT with the addition of wetting agents phospholipon® 90H (PL 90H), Tween® 80 and Span® 80 as well as the resuspendibility enhancer Kolliphor® ELP (KP ELP). These components improved considerably the applicability, resulting from the shear-thinning behavior of the formulation compared to pure oil mixtures.The current study aims to evaluate the use of suspended particles made of polymer and peptide incorporated in the developed vehicle containing MCT, 3% (m/m) AlSt, 1% (m/m) PL 90H, 5% (m/m) KP ELP in order to obtain a self-emulsifying sustained/controlled release formulation. Materials and methods: Tested polymers: Hydroxy-propylmethylcellulose (HPMC) K4M, sodium carboxymethylcellulose (CMC) 7LP EP, sodium carboxymethylcellulose (CMC) 7M8SF, hydroxypropylcellulose (HPC), 2-hydroxypropyl-β-cyclodextrin HP7 (Ashland, D-Düsseldorf) and hydroxyethyl starch (HES) 200/0.5 for parenteral use (Fresenius Kabi, A-Linz). Solutions of 0.2% peptide and 0.2% or 2% polymer mixtures were freeze dried using a Christ Epsilon 2-6 D freeze-dryer (Martin Christ Gefriertrocknungsanlagen GmbH, D-Osterode) (100 mTorr; primary drying: 60 h, -25°C; secondary drying: 12 h, 35°C). The lyophilisates were cryo ground with a Retsch Cryo Mill (Retsch Technology, D-Haan) and suspended in the oil vehicle using an Ultra-Turrax T-10 basic (IKA-Labortechnik, D-Staufen) for 5 min at 2000 rpm. The in vitro release experiments were performed in an incubated

shaker 3031 (GFL, D- Burgwedel) at 39 °C and 60 rpm, using a visking dialysis tubing MWCO 12-14 kD in PBS pH=7.4. Samples were analyzed by RP-HPLC with UV detection at 220 nm. Results and Conclusion: The peptide release requires the penetration of aqueous body fluids and dissolution at the muscle tissue/oil vehicle interface. The incorporation of HPMC, CMC and HPC at the higher concentration counteracts the encapsulation caused by the AlSt gelled MCT, thus increasing the diffusion of water into the oil depot. In addition to this, the HPC: peptide complex (10:1) showed a nearly complete and sustained release of the peptide, reaching approximately 70% after 14 days. In contrast, the reference formulation without polymer showed an incomplete release of the peptide which levelled off at 45% after 14 days. Upon contact with water nanostructures are formed. HPC is able to adsorb at the oil/water interface to the area of the nanostructures that are not fully covered by PL90H, thus affecting the release [2]. This creates a diffuse layer around the structures promoting a steric repulsive effect between the formed nanodroplets and therefore contributes to their stability and the complete release of the peptide from the oil depot [3]. Furthermore, the oil vehicle containing the 2% to 0.2% HPC to peptide particles exhibited a zero-shear viscosity of 443 mPas at 39°C after a linear ramp shearing, 1 to 500 s -1 for 3 min, which ultimately reduces the spreading of the oil depot after application. Further in vitro studies are required in order to elucidate whether the observed effect on the release can be correlated to the polymer chain length or to a specific polymer class. References: 1. Beckjunker, J. and Kauffold, J.: Anim Reprod Sci 2007, 97:84-93. 2. Mezdour, S.:Colloids Surfaces A Physicochem Eng Asp 2008, 331:76–83. 3. Karlberg, M., Thuresson, K., Lindman, B.: Colloids Surfaces A Physicochem Eng Asp 2005, 262:158–167.

POS.176

SmartLipids® - 3rd generation lipid nanoparticles with high loading capacity for lipophilic actives Bacher, L.1; Arntjen, A.1; Hespeler, D.2; Nguyen, T. M. H.1,2, Keck, C. M.3

1 Applied Pharmacy, Hochschule Kaiserslautern-Campus Pirmasens, 66953 Pirmasens, Germany 2 Institute of Pharmaceutics, Biopharmaceutics and NutriCosmetics, Freie Universität Berlin, 12169 Berlin, Germany 3 Institute of Pharmaceutics and Biopharmaceutics, Philipps-Universität Marburg, 35032 Marburg, Germany

Introduction: Geranylgeranyl-2-propanol (GGP) is a terpenic alcohol and acts as a metabolism facilitator by regulating the activity of various cellular proteins and their location in different cellular compartments [1]. The mechanism of action is not fully understood. However, present data prove the high potency of this compound to reduce oxidative stress at the cellular level [1]. Oxidative stress is a main cause of aging and known to promote many diseases. Therefore, GGP might be a potent active compound to prevent and treat various oxidative stress related diseases in the future. The compound is lipophilic and possesses poor aqueous solubility, leading to poor bioavailability. Thus, the aim of this study was to formulate GGP as smartLipids to improve its bioactivity. Materials and Methods: SmartLipids are referred to be the third generation of lipid nanoparticles. Similar to the first and second generation of the lipid nanoparticles, they improve the bioavailability of poorly soluble compounds. However, due to their chaotic smartLipid matrix they enable a higher loading capacity of the active compound [2,3]. Until now only limited data are available for such smartLipid matrices, which should be composed of many different solid and liquid lipids. In this study eight solid waxes with different chemical natures (microcrystalline waxes, paraffin waxes, plant waxes) were blended and used as a solid lipid matrix. Different amounts of various semisolid and liquid waxes were systematically added to the solid matrix and the mixtures were evaluated in regard to homogeneity, hardness and appearance. Suitable mixtures were transferred into smartLipids by hot high pressure homogenization using Plantacare 2000 as stabilizer. The most suitable formulation, leading to small sized particles with a narrow size distribution and a sufficient physical stability, was used for the development of the GGP-loaded smartLipids. Results: The mix of eight different solid waxes led to very hard and bridle mixtures. Addition of semisolid and/or liquid waxes up to 50% (w/w) improved the processability of the blends, decreased the



hardness and increased the stickiness. Most appropriate blends consisted of 50% solid lipid phase and 50% semisolid and/or liquid waxes. For some mixtures blends consisting of even 70% liquid and semisolid wax were found to be appropriate for further investigations. Homogenisation of these blends revealed small sized smartLipids with sizes below 300 nm. Matrices containing semisolid paraffin waxes formed agglomerates, indicating that these types of waxes might not be suitable to be used in smartLipid matrices. The best formulations were obtained from a blend containing equal parts of eight solid waxes (3.75% each) and a blend of lanolin alcohol, mineral oil, diisopropyl dilinoleate and isopropyl lanolate, contributing to 70% (w/w) of the smartLipid matrix. The loading capacity of GGP in the matrix was determined to be 28% (w/w). The production of smartLipids with 28% GGP loaded into the matrix resulted in particles with a size of about 160 nm and a polydispersity index < 0.2. However, the zeta potential was slightly different, when compared to the non-loaded formulation, indicating an overloading of the matrix, i.e. excess GGP might be located at the outside of the matrix. These particles remained stable for only one week, further indicating the overload of the matrix with GGP. In contrast, formulations containing 25% GGP possessed a similar small size, possessed a zeta potential similar to the non-loaded particles and remained physically stable for at least 6 months. Conclusion: GGP-loaded smartLipids were successfully developed in this study and are now available for further in vitro and in vivo studies. The GGP-smartLipid matrix contains twelve different lipids and enables a loading capacity of 28% for GGP. This data supports the theory of the smartLipid concept, i.e. a chaotic lipid matrix increases the loading capacity of lipophilic actives. Results also showed that smartLipids should be produced with a drug load that is slightly below the maximum loading capacity of the lipid matrix. Therefore a complete encapsulation of the active compound into the matrix can be improved, which decreases expulsion of the active compound during storage and increases the physical stability of the formulation. Acknowledgments: The project was partly funded by ZIM KF2161909SK4.

References: 1. Fournial A et al., EP2555743 A2, 2010. 2. Ruick R, PhD-Thesis, Freie Universität Berlin, 2015. 3. Junmahasathien, T. PhD-Thesis, Freie Universität Berlin, 2015.

POS.177/SL.38 Skin penetration analysis by confocal Raman microspectroscopy – potentials and pitfalls Lunter, D.1 1 Department of Pharmaceutical Technology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany


POS.178

Film-Forming Formulations with Sustained Skin Penetration of an Antipruritic Drug Heck, R.1; Lunter, D.1; Daniels, R.1 1 Department of Pharmaceutical Technology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany

Chronic pruritus is a common symptom accompanying various chronic skin diseases. Conventionally, it is treated with antihistamines and local anesthetics. However, these drugs often cannot provide sufficient relief. As an alternative, capsaicinoids can be used. Their long-lasting antipruritic effect is caused by continuous stimulation of transient receptor potential vanilloid 1 (TRPV1) at the epidermal pain conducting fibers. To achieve this, currently available formulations need to be applied 4-6 times a day. This is inconvenient and results in poor patient compliance. The aim of our study was thus to develop a film-forming formulation with sustained penetration for dermal use making it easy to treat large areas of affected skin over a long period. Film-forming formulations were prepared by incorporating an oily drug solution into mesoporous silica and subsequently incorporating these carriers into a plasticized film-forming polymer dispersion. To

investigate skin penetration from these formulations, ex vivo penetration studies were carried out using postauricular porcine skin. The drug penetration was compared to that from a conventional semisolid formulation. After incubation, the stratum corneum was removed by skin surface biopsy. Deeper skin layers were segmented via cryosectioning. The drug in the different skin layers was extracted and quantified by HPLC-UV/VIS. The penetration experiments showed that film-forming formulations are able to ensure a continuous penetration of the drug into the skin while reaching therapeutically suitable drug levels in the viable epidermis. This implies that film-forming formulations may allow more convenient dosage regimens and hence may improve the patients’ compliance. Acknowledgements: PD Dr. Martin Schenk is acknowledged for the donation of pig ears. This project was supported by the European Social Fund and by the Ministry of Science, Research and the Arts Baden-Wuerttemberg

POS.179

Advanced ex vivo test models elucidate the impact of lack of substantivity on cutaneous absorption of drugs Hermann, S.1; Schmidberger, M.1; Daniels, R.1; Lunter, D.1 1 Department of Pharmaceutical Technology, University of Tuebingen, Auf der Morgenstelle 8, 72076 Tuebingen, Germany

Skin diseases are usually treated using topical formulations that contain one or more active substances. Frequently, multiple applications per day are necessary, as up to 90 % of the formulation (and thus of the active) are withdrawn from the skin by contact with the environment. They are then no longer available for penetration into the skin and the effectiveness of the treatment may thereby be impaired. It would thus be advantageous to develop formulations with enhanced substantivity. Such formulations would stay on the treated site for a longer period of time and would thus reduce the application frequency and enhance patient compliance. However, methods that effectively characterize the substantivity are still lacking. We thus developed ex vivo methods that probe the substantivity of topical formulations by simulating skin-to-skin or clothing-to-skin contact and enable the determination of the amount of formulation that is withdrawn from the skin due to the contact. Furthermore, we simulated the impact of skin-to-clothing contact on cutaneous absorption ex vivo. Three different types of formulations were used to validate the experimental setups: A conventional semisolid cream [1] and two sustained release formulations. The first sustained release formulation was an oil-in-silicone oil-emulsion [2] and the second one a film forming formulation [3]. Nonivamide was used as a model drug. It was shown that the methods are capable of detecting differences in substantivity between the three model formulations. Furthermore, the lack of substantivity was found to lead to reduced permeation of the drug through the skin. Additionally, the extent to which the formulation was removed from the skin due to lack of substantivity was found to correlate very well with the reduced amount that permeated the skin [4]. Acknowledgements: PD Dr. Martin Schenk is acknowledged for the donation of pig ears. This project was supported by the European Social Fund and by the Ministry of Science, Research and the Arts Baden-Wuerttemberg

References: 1. Bundesvereinigung Deutscher Apotheker, Hydrophile Capsaicinoid Creme 0,025 % / 0,05 % / 0,1 % (NRF 11.125), Neues Rezeptur Formularium, D-Eschborn, 2010. 2. Rottke M, Lunter D, Daniels R, Eur J Pharm Biopharm, 2014 86: 260-266. 3. Heck R, D Lunter and R Daniels, Skin Forum 14th annual meeting, 2014. 4. Hermann S, Daniels R, Lunter D, Pharm Dev Technol 2016, DOI: 10.3109/10837450.2015.1135346.

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Premix membrane emulsification for the preparation of colloidal lipid emulsions as drug delivery systems Gehrmann, S.; Bunjes, H. Technische Universität Braunschweig, Institut für Pharmazeutische Technologie & Zentrum für Pharmaverfahrenstechnik, Mendelssohnstraße 1, 38106 Braunschweig, Germany

An increasing number of new drug substances are poorly water soluble and thus difficult to effectively administer to patients. A promising option

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for the administration of such substances is loading them into colloidal lipid emulsions. Premix membrane emulsification is a possibility to prepare these colloidal carriers with the required size and a narrow particle size distribution. In this process, a coarse pre-emulsion is extruded through the pores of a membrane, yielding smaller emulsion droplets [1]. In order to gain deeper understanding of the parameters influencing the routine applicability of premix membrane emulsification the effect of different emulsifiers and membrane filter materials on the quality of the resulting emulsions was studied. An instrumented small scale membrane extruder [2] was used in the experimental setup which allowed precise process control. The emulsions consisted of 20% Miglyol 812 (MCT) (or peanut oil in some cases) stabilized with 5% surfactant in double distilled water containing 0.01% thiomersal as preservative. Sodium dodecyl sulfate (SDS), poloxamer 188, Tween 80, tyloxapol, and sucrose laurate were used as surfactants. After dispersion with an Ultra-Turrax the pre-emulsions were processed 21 times through different disposable 200 nm membrane filters (polycarbonate (PC), polyester (PE), polyethersulfone (PES), polysulfone (PS), polyvinylidene fluoride (PVDF), nylon and cellulose acetate (CA)) at a flow rate of 0.25 ml/s. For some additional investigations a 200 nm alumina membrane was employed. The process led to different particle sizes and size distributions indicating that the polymeric membrane materials could be classified into groups. The first group led to nanoparticles with all emulsifiers under investigation (nylon, CA). The second group (PC, PS and PVDF) yielded submicron particles only with SDS. Membranes out of PES and PE were between the two groups, whereby the PES membrane tended to be part of the second group and PE of the first group. As confirmed by scanning electron microscopy the track-etched PC and PE membranes have a uniform pore size of 200 nm, whereas the other membranes have a more sponge-like membrane structure containing pores of 1-2 µm diameter in some cases. The differences in emulsion quality can thus not solely be explained by the membrane structure but the effect may include an interaction between emulsifier and membrane surface. To identify potential correlations, contact angles between emulsifier solution and membrane material were measured. The combinations of emulsifier solution and membrane material with a contact angle less than 49° led to MCT emulsions with a median particle size below 500 nm in all cases, whereas the particle size was always above 1 µm for contact angles higher than 55°. Basically analogous results were obtained with peanut oil emulsions albeit with a slightly shifted transition area between successful and unsuccessful formulation/ membrane combinations. The wetting of the membrane with the continuous phase of the emulsion seems, therefore, to be of special importance. Additional experiments with an alumina membrane confirmed this assumption as this setup did not only lead to colloidal emulsions in all cases but also to the best results with all emulsifiers except for poloxamer. Taking the correlation between wetting properties of the formulation with the membrane into consideration colloidal lipid emulsions could thus be obtained with all emulsifiers under investigation. Membranes from CA, nylon and, in particular, alumina, appear particularly promising for routine use as they can be successfully be applied with many different formulations. Acknowledgments: This study was performed in the context of the DFG research group 856 mikroPART. We thank Simone Schulze (TU Braunschweig, Institute for Chemical and Thermal Process Engineering) for taking the SEM images.

References: 1. Joseph, S., Bunjes, H.: J. Pharm. Sci., 2012, 101(7): 2479-2489. 2. Gehrmann, S., Bunjes, H.: Chem. Eng. J., 2016, 284: 716-723.

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Evaluation of solvent-shift generated surfactant-free and lecithin-stabilized triolein nanoparticles Glud, K.1; Kuntsche, J.1 1 University of Southern Denmark, Campusvej 55, Odense M 5230, Denmark

Solvent shifting is a method to precipitate compounds by introducing a solution containing the compound into a non-solvent. Under conditions resulting in high Reynolds numbers (Re), the solvent shifting method usually yields nanoparticles (NPs) in the lower nanometer size range. Such conditions can be obtained both by rapid mixing using hand-

operated impinging jet mixers [1], multi-inlet vortex devices [2] or even a simple Y-junction mixing setup [3] and by using microfluidic devices [4]. The aim of this study was to evaluate the solvent shifting method for the preparation of triolein (TO) nanoemulsions without and with lecithin (E80S) as stabilizer by using a simple Y-junction mixing setup. 1 ml of ethanol precursors containing 1-20 mg/ml TO without and with lecithin (0-10 mg/ml Lipoid E80S at fixed TO concentration of 5 mg/ml) were rapidly mixed with 19 ml water preserved with 0.02% w/v sodium azide via a Y-junction (r = 0.25 mm). Under these conditions (mixing time was about 8 s), the Re number was about 5000. All samples were prepared in triplicate, stored at 4-8 °C and analyzed by dynamic light scattering (DLS, average size and zeta potential), asymmetrical flow field-flow fractionation coupled to multi-angle laser light scattering (AF4/MALLS, size distribution), optical microscopy and single particle optical sizing (detection and quantification of µm-sized particles). The size of surfactant-free TO NPs increased with increasing TO concentration in the ethanol precursor with diameters (DLS) between 40 and 300 nm. Polydispersity indices (PDI) were below or close to 0.1 indicating rather narrow size distributions, which was confirmed by AF4/MALLS. Moreover, no particles in the µm-size range were detected by optical microscopy. The nanoemulsions were stable for up to 30 days. The stability of such surfactant-free oil droplets has been suggested to be by adsorption of OH- ions onto the droplet surface [5]. Addition of lecithin resulted in smaller NPs with decreasing size with increasing lecithin concentration (Figure 1). Interestingly, addition of lecithin in low concentration caused a destabilization of the emulsion droplets (Figure 1). The most stable formulation was obtained at an E80S/TO mass ratio of 0.20 (droplet size about 60 nm, PDI around 0.18). Calculations predict that a TO droplet with a diameter of about 60 nm would be covered completely with a lecithin monolayer at a mass ratio of 0.24, assuming a spherical particle shape, a lecithin monolayer thickness of 2 nm and a TO density of 0.91 mg/ml. Taken into account that lecithin is not a pure substance, the calculated ratio is overall in good agreement with the experimental results. A sudden increase of the PDI was observed at E80S/TO mass ratios ≥ 1 where the formation of mixed structures or vesicles may be expected. In conclusion, TO nanoemulsions with droplet sizes in the lower nm-size range and rather narrow size distributions were obtained by a simple, low-energy preparation process. However, as the solvent shifting method results in highly diluted dispersions, on-going work focusses on the evaluation of up-concentration of the nanoemulsions in addition to investigations on the NP morphology and drug incorporation.

Acknowledgements: The authors thank the Danish National Research Foundation, Denmark, for financial support of the project.

References: 1. Han, J. et al.: J Pharm Sci-Us. 2012, 101(10):4018-23. 2. Liu, Y. et al.: Chem Eng Sci. 2008, 63(11):2829-42. 3. Gillian, J.M. et al: Chem Eng Commun. 2008, 195(12):1553-74. 4. Zhigaltsev I.V. et al.: Langmuir. 2012, 28(7):3633-40. 5. Marinova, K.G. et al.: Langmuir. 1996, 12(8): 2045-51.

Fig. 1: Z-average diameters and PDI of the nanoemulsions of independently prepared samples (n = 3). The TO concentration in the ethanol precursor was 5 mg/ml.



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Delivery system for the targeting of ocular neovascularizations Haunberger, A.1; Göpferich, A.1 1 Institute of Pharmacy, Department of Pharmaceutical Technology, University of Regensburg, Universitätsstr. 31, 93053 Regensburg, Germany

Background: The proliferative forms of age-related macular degeneration (AMD) and diabetic retinopathy (DR) are both among the leading causes of blindness in the industrialized world. The newly formed vessels, which are sprouting from the choroid in AMD and from the retinal capillaries in DR respectively, are often leaky, which causes edema and haemorrhage. Also tensile stress and the breakdown of the blood-retina barrier affect the surrounding tissue and can lead to fast visual impairment. Today´s therapies like laser coagulation or anti-VEGF-therapeutics often suffer from unsatisfying outcomes and are not free from side effects [1, 2]. In our approach, we focus on a combination of Cyclosporin A (CsA) and Itraconazole (Itra), as these active substances selectively and synergistically inhibit endothelial cell proliferation and different steps of angiogenesis [3]. The two therapeutics were incorporated in lipid nanocapsules (LNC); to enable an accumulation of these nanoparticles in proliferating vessels, the surface of the nanocapsules was decorated with the cyclic peptide c(RGDfC) (cyclo(Arg-Gly-Asp-D-Phe-Cys)), which is a ligand of the αvβ3-integrin. This receptor is up-regulated in proliferating endothelial cells [4] and the presentation of c(RGDfC) on the surface of nanoparticles even leads to their accumulation in normal retinal and choroidal capillaries [5]. Methods: CsA and Itra were encapsulated into LNC via two different methods during the nanoparticle preparation in a phase-inversion process [6]. The particle size was determined by dynamic light scattering (DLS). For the quantification of the encapsulation efficiencies for CsA and Itra, respectively, the LNC were purified via ultrafiltration and size exclusion chromatography to remove non-encapsulated drug. The CsA and Itra content of purified and non-purified LNC was determined via HPLC. For the c(RGDfC)-decoration of nanocapsules, the peptide was coupled to a PEGylated phospholipid which was then incorporated into the LNC shell via post insertion [7]. To confirm the enhanced binding to endothelial cells, human dermal microvascular endothelial cells were incubated with c(RGDfC)-carrying and blank fluorescently labeled LNC and the binding was quantified via flow cytometry. Results: We were able to incorporate CsA and Itra in lipid nanocapsules with high encapsulation efficiencies (about 70 % for CsA and 100 % for Itra, respectively). The size of the prepared LNC was about 50 nm and did not change due to drug encapsulation. The c(RGDfC)-modified nanocapsules showed 5-6 times increased binding to human dermal microvascular endothelial cells. Acknowledgments: This work was supported by the German Research Foundation (DFG; GO 565/18-1).

References: 1. Hagedorn, C.; Adelmann, R.: Age-Relate Macular Degeneration in Diabetic Retinopathy in Tombrain-Tink,, Barnstable (Ed.): Ocular Angiogenesis (Humana Press) 2006. 2. Ma, J.; Zhang, S.: Endogenous Angiogenic Inhibitors in Diabetic Retinopathy in Tombrain-Tink,, Barnstable (Ed.): Ocular Angiogenesis (Humana Press) 2006. 3. Nacev, B.; Liu, J.: PLoSONE 2011, 6(9): e24793. 4. Chavakis, E. et al.: Diabetologia 2002, 45(2):262-267. 5. Pollinger, K. et al.: pnas 2013, 110(15):6115-6120. 6. Heurtault, B. et al.: Pharm. Res. 2002, 19(6):875-880. 7. Hirsjärvi, S. et al.: Eur. J. Pharm. Biopharm. 2014, 87(1):152-9.

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Complex formation between short peptide sequences and DNA for nucleic acid delivery Haas, V.1; Goepferich, A.1

1 Department of Pharmaceutical Technology University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany

Introduction: The development of nonviral vectors for the application of gene and RNAi therapies is of great interest. Lipidic or polymeric carriers are widely used for such drug delivery systems and led to significant research for improvements [1,2]. In this study small monocationic peptides were used for the complex formation with nucleic

acids as further nonviral vectors. This concept is based on the hypothesis that a minimal positive charge of the carrier is associated with less toxic effects on cells. The formation of the complex with monocationic peptides is envisioned to be induced via electrostatic interactions between the negatively charged phosphate groups of nucleic acid and the positive charge at the side chain of the peptide. The complex should be further stabilized via hydrophobic interactions between the aromatic side chains of neighbouring peptide molecules. Material and Methods: DNA/peptide complex formation was induced by mixing DNA (eurofins) and peptides (synPeptide, Peptide 1: Ac-Lys-Gly-Phe-Leu-Trp-Leu-Phe-Ser, Peptide 2: Ac-Lys-Gly-Phe-Leu-Trp-Leu-Phe-Cys) in different NP ratios (NP ratio is the number of negatively charged phosphate groups of DNA to the number of positively charged amino groups of the peptide) and incubation at room temperature. The complex formation was determined via gel retardation assay with a 1% agarose gel stained with ethidium bromide. For the visualization of the complex transmission electron microscopy was used (Libra 120, ZEISS). Results and Discussion: Two peptides consisting of eight amino acids which only differ in the carboxy terminus were used for the complex formation with a short double stranded DNA sequence (22bp). The peptides were used in different concentrations yielding different NP ratios. In a gel retardation assay complex formation was detected at every NP ratio, but only at higher NP ratios (10, 20, 30) the whole DNA was bound in a complex (data not shown). Also the measurements with the transmission electron microscope showed a difference between a pure peptide (rodlike filaments) solution and a DNA-peptide-mixture (helical strands), which is a proof for complex formation (Figure 1).

Fig. 1: Images of transmission electron microscopy with negative staining. A: complex between DNA and peptide 2 (scale bar 100 nm); B: pure peptide 2 solution (scale bar 100 nm).

Conclusion: Both peptides are suitable for the formation of a complex with DNA. Therefore they are potentially convenient for the use as a new drug delivery system for nucleic acids. Acknowledgments: Margit Schimmel

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Influence of surfactants and formulations on stratum corneum lipids Zhang, Z.1; Lunter, D.1 1 Department of Pharmaceutical Technology, Auf der Morgenstelle 8, 72076 Tübingen, Germany

Stratum corneum (SC), which consists of corneocytes and intercellular lipids, plays an essential role as a barrier to the external environment. However, treatment with surfactants on SC can induce lipid extraction and other property changes, leading to barrier defect. To investigate these influences, basic cream DAC was chosen as model formulation. It contains two kinds of non-ionic surfactants: glycerol mono stearate 60 (GMS) and macrogol-20-glycerol mono stearate (PEG20GMS). Water and sodium lauryl sulfate (SLS) were used as negative and positive control. In this study, GMS, PEG20GMS, GMS and PEG20GMS mixture, SLS solution and basic cream DAC as well as its oil phase (OP) and aqueous phase (AP) were applied onto excised porcine skin during incubation in Franz diffusion cells. Subsequently, SC was isolated according to ref [1]. Dehydrated SC sheets were subjected to confocal Raman microscopy (CRM) to detect SC lipid content and differential scanning calorimetry (DSC) measurements to evaluate SC lipid ordering. A pyranine fluorescence staining technique [2] was also conducted to visualize the barrier impairment.

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Various kinds of surfactants show different capabilities of barrier damage. SLS exhibits the strongest ability among all the studied surfactants. It withdraws 38% (w/w) lipids from SC and reduces the second lipid thermal transition temperature by 13 °C (average mean value). Fluorescence staining experiment reveals the brightest image for SLS, also indicating the least SC barrier integrity after treatment. GMS and PEG20GMS mixture exhibits stronger ability compared to either component by extracting 35% (w/w) lipids and shifting thermal transition to lower temperature by 7 °C. Interestingly, OP shows stronger barrier destroying capability than AP from both CRM and DSC results, although this difference is not obvious in fluorescence staining. However, basic cream DAC does not show significant difference in contrast with water treated samples. This might imply that, barrier impairing ability of surfactants are weakened while they interact with other formulation components, e.g. due to emulsification. Acknowledgments: PD Dr. Martin Schenk is acknowledged for the donation of pig ears. This project was supported by the European Social Fund and by the Ministry of Science, Research and the Arts Baden-Wuerttemberg and the China Scholarship Council.

References: 1. Kligrnan, A. M. and Christophers, E.: Arch. Dermatol. 1963. 88(6): 702-705. 2. Pagnoni, A., Kligman, A., Stoudemayer, T.: J. Cosmet. Sci. 1998. 49: 33-38.

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Numerical analysis of powder flow in a lab-scale tableting machine Hildebrandt, C.1; Gopireddy, S. R.2; Scherließ, R.1; Urbanetz, N. A.2 1 Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany 2 Daiichi-Sankyo Europe GmbH, Pharmaceutical Development, Luitpoldstrasse 1, 85276 Pfaffenhofen, Germany

Related to the fact that tablets constitute the top sold drug delivery system, the demand of high production rates while guaranteeing quality is increasing over the last decades. One major challenge in tablet production and from a patient safety point of view is the accurate content and content uniformity of the active pharmaceutical ingredient (API) in the final dosage form. Out of the different stages in the tableting unit operation, namely die filling, compaction and ejection, die filling represents the most crucial step since it is the last stage to control and specify the mean and range of tablet mass and API content. Numerical simulations offer an alternative solution to process analytical tools in pharmaceutical formulation development to investigate critical quality attributes (CQA) in the tableting unit operation. In this study, the die filling process from an open feeder of a lab-scale rotary tablet press was investigated using the Discrete Element Method (DEM) to elucidate the CQA with respect to process parameters as well as material properties by which the process understanding is being enhanced. The DEM considers particles as individual elements, thus collisional forces between the particles as well as with the geometry are computed to find all the particles trajectories. The particle motion is captured by the DEM as particles travel from the feeder, represented by an inclined chute connected to a rectangular box, to the dies of the turret.

Fig. 1: Visualization of the particle discharge from the feeding box in the die (view from right to left). The die table moves at a constant rotational speed of 20 rpm in the anti-clockwise direction (indicated by the arrow). Particles are coloured according to their velocity (red: high, blue: low). In Fig. 1 the process of die filling is visualized by colouring the particles according to their velocities. As soon as the die enters the bottom of the feeder (Fig. 1, t = 0.00 s), the particles slowly start to fall into the die mainly due to gravity. During the filling process, the top right corner of the feeder (portion of the box in which particles move with highest velocity, Fig. 1, t = 0.05 s) is getting emptied, which implies that the powder filled into the die is mostly composed of particles originating from this region. This difference in particle motion causes a confinement of particles on one side of the feeder and empty space on the other side hence may bear the risk of segregation.

The process conditions such as die table speed, die diameter as well as material characteristics e.g. particle size and size distribution, cohesion, friction and restitution coefficients were systematically changed to evaluate their influence on the die filling process. The results give both qualitative (powder flow pattern from the feeder to the dies) and quantitative (tablet mass and mass variation, mass flow rate etc.) insights into the die filling process. The numerical results indicate that faster die table speeds and smaller die diameters give rise to lower tablet mass with significantly increased mass variation, which could eventually increase the challenges in meeting the specifications. On the other hand, cohesion reduces the tablet mass and increases its variation. The friction between the particles and geometry influences the particle rearrangement in the feeder thus the dead zone is less prevalent. In summary, this study gives references to the CQA of die filling from an open feeder of a rotary tablet press and emphasizes the importance of blend characterization in early stage of formulation development to ensure a high tableting process performance. In addition, such numerical studies will support product quality control concurrently with established means of pharmaceutical development.

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Investigation of powder flowability in a model die filling process Fritsch, A. K.1,2; Hildebrandt, C.3; Gopireddy, S.R.1; Rackl, M.2; Urbanetz, N. A.1 1 Pharmaceutical Development, Daiichi-Sankyo Europe GmbH, Luitpoldstr. 1, 85276 Pfaffenhofen a. d. Ilm, Germany 2 Department of Materials Handling, Material Flow and Logistics, Technical University of Munich, Boltzmannstr. 15, 85748 Garching, Germany 3 Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany

Tablets constitute the most common pharmaceutical dosage form inter alia because of high patient compliance and most commonly produced using a rotary tablet press, which consists of three distinct stages. One of them is the die filling in which the powder blend is deposited into the die or cavity of the turret formed by lowering the lower punch. The amount of powder collected in the die depends on the material properties and process conditions, and it is the main factor which controls the tablet production rate. Due to its relevance, interest in understanding the die filling process has drawn more and more attention over the last years. The current study investigates the die filling process experimentally using a model die filling system. In this system, the die translates

Fig. A. The die filling ratio over the die speed. The dashed lines indicate where the fitted curve starts to descend, and thus mark the critical die speed. Fig. B. Mean particle diameter (x50) plotted over vc. The equation shows the linear relationship between vc and x50, and dashed lines indicate the 95% confidence interval band.

underneath the fixed shoe at a constant speed which can be varied from 50 to 500 mm/s simulating a simplified tablet press. Several commonly used excipients in direct compression formulations were investigated, comprising fillers/binders of different grades of lactose, mannitol and microcrystalline cellulose. The powder mass which was deposited at increasing die speeds was weighed, normalized to the maximum possible filling level, determined by the bulk density, and plotted against the die speed, and it is shown in Fig. A. A general trend of exponentially decreasing die filling at increasing die speeds was observed. The highest die speed, at which the filling ratio first starts to decrease, is called the critical die speed (vc). The vc value for a given material can be extracted from Fig. A as indicated by the dashed lines. For all tested materials, the vc value was determined and plotted against the mean particle diameter (x50), see Fig. B. The results illustrate that there is an almost perfect linear correlation between the



mean particle diameter and the critical die speed. Furthermore, the results reveal that a hundred percent increase in particle size would facilitate at least double the critical die speed. In this given setup, a minimum particle size threshold (34.2 µm) exists at which no complete die filling will be possible. Such a simple model die filling system helps in characterizing the powder flowability specific to the tableting process. Hence it supports the product and process development by e.g. formulation optimization through providing an estimation about a particle size distribution that yields a higher production rate.

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The hen’s egg test as a model for optical detection of particle behavior under dynamic flow conditions Warncke, P.1; Müller, R.2; Stranik, O.2; Schlenk, F.1; Werner, S.1; Malsch, D.2; Bergemann, C.3; Fritzsche, W.2; Fischer, D.1 1 Department of Pharmaceutical Technology, Institute of Pharmacy, Friedrich-Schiller-University Jena, Otto-Schott-Str. 41, 07745 Jena, Germany 2 Leibniz Institute of Photonic Technology, Department of Nanobiophotonics, Albert-Einstein-Str. 9, 07745 Jena, Germany 3 Chemicell GmbH, Eresburgstraße 22-23, 12103 Berlin, Germany

Iron oxide particles are attracting high interest for applications such as magnetic drug targeting, hyperthermia or MRI imaging. Differences between in vitro tests under static conditions and the dynamic conditions in the blood stream were suggested to be responsible for a limited transfer of 2D-cell culture data to the in vivo situation. Blood flow may compromise the interaction between nanoparticles and target cells, especially for particles with low binding affinities. Furthermore, cell culture systems do not sufficiently represent the complexity of interactions between nanomaterials and the different blood components relevant for systemic administration. To mimic the behaviour of particles with and without the presence of a magnetic field in vivo, the chick embryo was used as an easy accessible, vascular network system using optical microscopy. Different fluorescent iron oxide particles and magnetic silica beads (both chemicell GmbH, Berlin) with varying surface characteristics were investigated regarding their flow characteristics and optical properties. Particle stability in blood, the tendency to agglomerate and hemocompatibility were tested in red blood cell assays. The in vivo situation was investigated with the shell less HET-CAV (hen’s egg test-chick area vasulosa) model. After systemic injection of particles, the blood vessel systems were screened under an optical microscope (AxioImager Z1.m, Carl Zeiss, Jena) operated in the dark field or the fluorescence mode. A PCO-Sensicam connected to the microscope was used to record particle behavior in blood vessels. A magnetic field was generated by a cylindrical NdFeB magnet next to the objective. Data analysis was realized with TrackMate, a versatile SPT plugin for the image processing package Fiji. The HET-CAV model with its planar area vasculosa allowed the optical inspection of the whole blood vessel system. Particles with emission wavelengths of the fluorescent dye >500 nm were optimal for optical microscopy due to the background fluorescence of the embryonic tissue. Moreover, particle sizes of 1 µm in combination with a 10x objective were optimal for single particle tracking. The hemocompatibility testing of the particles indicated their suitability for administration directly into the systemic circulation. None of the particles did show any detectable disturbance of the erythrocyte membranes nor erythrocyte aggregation up to 10-fold higher concentration than in the ex ovo model. Tracking analysis allowed the detection of particle flow profiles depending on the particle coating, agglomeration behavior, and the dimensions and type of the blood vessels. In the presence of a magnetic field a hydrodynamic resistance was caused that led to a decreasing velocity of the particles near the vascular wall and strong agglomeration caused by magnetic forces. After removal of the field the agglomerates started to move again with a partial disintegration of the agglomerates. As in vitro in microfluidic channels these effects were not visible, they were suggested as surface interactions of particles with the complex blood environment depending on the coating of the particles. In conclusion, the shell-less HET-CAV model is a suitable dynamic model by offering a planar surface making the entire vascular network accessible. Transport of magnetic particles under the influence of a magnetic field can be observed with fluorescence microscopy.

Acknowledgments: The work was supported by the Federal Ministry of Education and Research (BMBF 03X0104D and E - NanoMed and BMBF 03XP0003 - NanoBEL).

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Suitability of Microemulsions as Vehicle System for Dermal Protein Application

Albold, D. 1; Scherließ, R.1 1 Department of Pharmaceutics and Biopharmaceutics, Kiel University, Grasweg 9a, 24118 Kiel, Germany

Introduction: Formulating a protein for dermal application (e.g. vaccination or intra-dermal effects) is challenging due to the protein`s molecular weight, hydrophilicity and sensitivity to external conditions, which mostly results in a short product shelf-life [1]. Here pre-microemulsion concentrates are promising topical formulations. Due to their spontaneous formation upon addition of water they facilitate a reduced contact time between the API and aqueous phase. Lipoid® S LPC 80 was chosen as surfactant with high self-emulsifying power, besides its enhanced skin penetration effects and good tolerability [2, 3]. Successful penetration of the protein-loaded microemulsion (ME) will be supported by a microneedle system, stimulating a local irritation. Therefore, an ideal formulation should balance between good skin tolerability and high drug stability.

Aim of the Study: The present study focused on stability of a model protein in a ME-system analysing changes in structure and activity. Based on tolerability studies with 3D-human skin models the “natural”-based phospholipid-ME (PL) will then be compared with a formulation including well-known PEGylated surfactants (PEG).

Methods: The ME were prepared in a fixed ratio (surfactants:lipophilic phase:aqueous phase) containing catalase as model protein. After preparing the final formulations, changes in protein structure were analysed at predefined time points via nanoDSF (NanoTemper technologies). This technique is an advanced differential scanning fluorimetry technology which offers the opportunity to detect smallest conformation changes in biomolecules, measured as shifts in thermal unfolding transition temperature (Tm). Under same conditions the catalase activity was examined, utilising the ability of active enzyme to degrade hydrogen peroxide (assay modified after Beers and Sizer [4]).

Results & Conclusion: Fig. 1 demonstrates that both formulations have an obvious influence on protein structure and their activity. Changes of catalase conformation were detectable as shift in the Tm compared to Tm of a stable solution (Fig. 1 B, C). Directly after preparation and 24 hr later the protein showed a reduced Tm and loss of activity in both systems (Fig. 1 A). At 3 hr later the Tm of PL-ME turned back close to the active maximum, corresponding to an increasing activity (Fig. 1 B, blue curve). In comparison, the PEG formulation revealed higher structural changes, resulting in lower melting curves (Fig.1 C) but in a similar activity profile. According to first findings the observed rise in activity within 3 hrs could be explained with an equilibration time of protein in its new environment. Further studies with other proteins will examine this phenomenon and characterise in more detail the time dependency of protein activity in a ME system. NanoDSF technology offers quick and reproducible measurements in presence of lipophilic ME ingredients. Does a chromatographic method, like size exclusion chromatography, produce additional and comparable results? This and more questions will be discussed in the present study with a final focus on assessment of ME as vehicle system for proteins along with tolerability studies on 3D-human skin models.

Fig.1: Comparison of catalase activity (A) and nanoDSF of PL (B) and PEG (C). All measurements were performed in comparison to 100 % catalase activity (red lines). B and C show on y-axis the first derivative of F350/F330 data, next to temperature changes from 20 °C up to 95 °C on x-axis.

Acknowledgements: The authors would like to thank NanoTemper Technologies GmbH, Lipoid GmbH and Gattefossé for their support. This project is financed by the Phospholipid Research Center.

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References: 1. Frokjaer, S.: Nature Reviews Drug Discovery 2005, 4(4): 298-306. 2. Kogan, A., Garti, N.: Adv. Coll. Interface Sci. 2006, 123: 369-385. 3. Paolino, D. et al.: Int. J. Pharm. 2002, 244(1): 21-31. 4. Beers, R.F.Jr., Sizer, I.W.: J. Biol. Chem. 1952, 195: 133-140.

POS.189/SL.13 Application of a dried H1N1 vaccine by epidermal powder immunization in piglets using a novel pyrotechnically driven applicator elicits antigen-specific antibodies Engert, J.1; Anamur, C.1; Engelke, L1; Fellner, C.2; Lell, P.2; Henke, S.3; Stadler, J.4; Zöls, S.4; Ritzmann, M.4; Winter, G.1

1 Department of Pharmacy, Pharmaceutical Technology & Biopharmaceutics, Ludwig-Maximilians-University Munich, Butenandtstr. 5, D-81377 Munich, Germany 2 PyroGlobe GmbH, Hauptstr. 15, D-85276 Hettenshausen, Germany 3 IIS Innovative Injektionssysteme GmbH, Lohmannstr. 2, D-56626 Andernach, Germany 4 Klinik für Schweine, Ludwig-Maximilians-University Munich, Sonnenstr. 16, D-85764 Oberschleißheim, Germany


POS.190

DIPOs, DIOGs and ISFOGs as novel direct injectable and biodegradable drug delivery systems for parenteral controlled release Mäder, K.1; Windorf, M.1; Kutza, J.1, Weiss, V.1; Rodrigues, A. G.1; Wersig, T.1; Kressler, J.2 1 Institute of Pharmacy, Martin Luther University Halle-Wittenberg, W. Langenbeckstr. 4, 06120 Halle (Saale), Germany 2 Institute of Chemistry, Martin Luther University Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120 Halle (Saale), Germany

The demand for optimized parenteral controlled release is increasing both for local and systemic acting drugs. Currently, the selection of the material is very limited. Polyesters made from lactic and glycolic acid are mainly used (PLA and PLGA). However, due to the high acidity of the monomers, autocatalytic degradation processes occur which often leads to complex release kinetics and acidic microenvironments (e.g. pH values 2). The acidic microenvironment might lead to the drug degradation and the formation of covalent linkages between drugs and polymer degradation products. To decrease production complexity and costs, it would be desirable to inject a drug loaded solution which forms a depot after injection. Clinically used in situ implants involve the use of organic solvents (e.g. NMP) which is undesirable. Therefore, the development of new biodegradable and direct injectable drug carriers with reduced monomeric microacidity and the avoidance or decrease of organic solvents is highly desirable. We developed direct injectable polymers (DIPOs), direct injectable oleogels (DIOGs) and in situ forming oleogels (DIOGs) as alternative systems. Our DIPOs systems are based on linear biodegradable polyester backbones with free OH-groups, which are obtained via enzymatically synthesis by the reaction of polyols with dicarboxylic acids. Furthermore, the modification of the free OH-groups with fatty acids provides a versatile platform for biodegradable drug carriers with tuneable properties, including the lipophilicity and the physical state [1]. In addition to their use as nano- and microparticles, we synthesized hydrophobic liquid polymers, which can be directly injected [2]. The polymers were well tolerated and provided in preclinical studies a controlled release over several weeks (Fig. 1).

Fig.1: Multispectral optical in vivo Images of the DIR loaded liquid DIPO formulations after s.c. injection in mice. The lipophilic fluorescent dye DIR was released over several weeks from fatty acid modified Poly-(glycerol-adipate). The modification degree of the polymeric backbone with the fatty acid was different between (a) and (b). Therefore, the release kinetics can be controlled by the DIPO composition.

As alternatives to polymeric systems, we also developed lipid based oleogels as alternative systems [3]. The main component of the oleogel is a pharma-grade oil (e.g. sesame or peanut oil, MCT). The oil is solidified by an oleogelator (e.g. hydroxy-stearic acid). Direct injectable oleogels (DIOGs) contain no organic solvents. It is possible to incorporate proteins via an emulsification process and to obtain a prolonged release in vivo (Fig. 2), [3].

Fig.2: Mutispectral Optical Imaging of in vivo albumin release (ALEXA Fluor R 680-BSA) from DIOGs. The DIOGs contained no organic solvent and were s.c. injected into mice. The release rate is determined by the selected oil and the oleogelator concentration.

In situ forming Oleogels (ISFOGs) contain a lower percentage of a biocompatible organic solvent (e.g. NMP). The oleogel is formed in situ by diffusion of the NMP into the outer environment which causes a precipitation of the oleogelator.Compared to DIOGs, ISFOGs permit higher concentrations of the oleogelator and therefore, stronger gels. The results of our preclinical in vivo studies show, that ISFOGs biodegrade with high reproducibility over several weeks. In conclusion, DIPOs, DIOGs and ISFOGs are promising carriers for parenteral controlled drug release. References: 1. Weiss, V.M. et al.; J.Contr.Rel. 2012, 158, 156–164 2. Mäder, K. et al., DE102014005782 (A1) 3. Mäder, K.; Kutza,, J.; Windorf, M; DE102013018193 (A1)

POS.191

Needle-free injection of vesicular phospholipid gels Breitsamer, M.1; Winter, G.1

1 Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig-Maximilians-University, Butenandtstr. 5, 81377 Munich, Germany

Vesicular phospholipid gels (VPGs) are highly concentrated semisolid aqueous dispersion of phospholipids in buffer [1]. They have been used in the past as depot formulations for the sustained release of proteins and peptides [2-4]. Their composition and manufacture is extremely elegant and cheap and with release rates over several weeks they represent a most desirable functionality. The only drawback of such formulations is the fact that VPGs have a high viscosity, which is influenced by the amount of phospholipids incorporated into the formulation and further by the encapsulated drug [5]. The viscosity influences the release behaviour of drugs from the VPG and compromises the easy administration of the formulations through a thin s.c. needle. A practical and elegant solution to overcome administration problems for such highly viscous drug formulations is the use of needle-free injection devices, which, like the Biojector 2000, have been approved by the FDA and other regulatory agencies. In the present study several VPG formulations composed of egg lecithin and PBS buffer pH 7.4 with a total phospholipid amount between 25% and 55% were prepared with a Dual Asymmetric Centrifuge (DAC). Methylene blue (MB) and Sulforhodamine B (SRB) were used as dyes. Viscosity of the formulations was measured with a rotational rheometer and injectability with a conventional needle and syringe into pig skin was measured with a Texture Analyzer. A Biojector 2000 with disposable needle-free syringes was used for the administration of VPGs into different in vitro models. Gelatin blocks were used as models with high transparency for first investigations of the injectability of the formulations with the needle free-injector. Further, the skin of a piglet (post mortem injections) was used for injections of VPGs. The injection depth was measured directly after administration of the VPGs with the Biojector 2000 by cutting through the injection site and measurement with a scale. Injection forces for the injection of VPGs into pig skin showed, expectedly, increasing forces with increasing phospholipid concentration, reaching maximum forces of about 50N for the formulation with 50% phospholipids. The formulation with 55%



phospholipids was not injectable. MB-VPGs with a phospholipid content of 25% to 55% were successfully injected into gelatin blocks with the Biojector 2000. We observed decreasing injection depth with increasing amount of phospholipids. Further, we were able to inject SRB-VPGs with phospholipid amounts of 25% to 50% into pig skin with the needle-free injector. The VPGs with 25% to 45% phospholipids penetrated into the subcutaneous and muscle tissue and formed a longish shaped depot, while injections of formulations with 50% phospholipids were intradermal and partially subcutaneous and formed a spherical shaped depot. Again we could observe that injection depth decreased with increasing phospholipid amount. Needle-free injection significantly simplifies the injection of highly viscous VPG formulations or makes it even possible. With that, previous restrictions in the use of VPGs can be overcome and the full potential of this controlled release concept can be used in the future. Acknowledgments: Lipoid GmbH, Frigenstraße 4, 67065 Ludwigshafen, Germany; Veterinary Clinic for Swine, Ludwig-Maximilians-University Munich, Sonnenstr. 16, 85764 Oberschleißheim, Germany.

References: 1. Brandl, M. et al: Chemistry and Physics of Lipids, 1997, 87(1): 65-72. 2. Tian, W. et al: Journal of Controlled Release, 2010, 142(3): 319-325. 3. Buchmann, S. et al: BMC Musculoskeletal Disordes, 2015, 16: 82. 4. Zhang,Y. et al: Drug Development and Industrial Pharmacy, 2015, 42(7): 1042-1049. 5. Neuhofer, C., Dissertation, LMU München, 2015.

POS.192

Simulation of the biodegradation of iron oxide and iron oxide-silica nanoparticles in artificial body fluids Rabel, M.1; Cialla-May, D.2; Müller, R.2; Kurland, H.-D.3; Thamm, J.1; Bergemann, C.4; Grüttner, C.5; Müller, F. A.3; Fischer, D.1 1 Friedrich Schiller Universtity, Department of Pharmaceutical Technology, Otto-Schott-Strasse 41, 07745 Jena, Germany 2 Leibniz Institute of Photonic Technology, Albert-Einstein-Strasse 9, 07745 Jena, Germany 3 Friedrich Schiller University, Otto Schott Institute of Materials Research, Löbdergraben 32, 07743 Jena, Germany 4 chemicell GmbH, Eresburgstrasse 22-23. 12103 Berlin, Germany 5 micromod Partikeltechnologie GmbH, Friedrich-Barnewitz-Strasse 4, 18119 Rostock, Germany

Iron oxide based magnetic nanoparticles have attracted interest in many medical and biological applications such as contrast agents for MRI, stem-cell tracking or magnetic drug targeting. Since the biodegradation of nanoparticles is a critical parameter that effects potential risks and limitations for biological purposes it is necessary to develop models to simulate the degradation behaviour of magnetic nanoparticles in biological systems. In this study, nanoparticles with varying core materials (iron oxide or iron oxide in a closed silica matrix), biodegradable and biopersistent coatings (starch, polyethylene glycol, glucuronic acid, polyethylene imine, dextran, silica) as well as different surface charges were used. These nanoparticles were extensively characterized physicochemically regarding their hydrodynamic size, surface charge, morphology and magnetic properties using photon correlation spectroscopy, laser Doppler anemometry, vibrating sample magnetometry and electron microscopy. The nanoparticles were aged and degraded in different simulation fluids mimicking the lysosomal compartment at different stages (artificial lysosomal fluid, ALF, pH = 4.5 and 5.5) and the plasma compartment (simulated body fluid, SBF, pH = 7.4) [1] over a period of up to 28 days. Degradation and changes of the core and the shell were evaluated by iron quantification, electron microscopy, Raman and infrared spectroscopy as well as magnetic measurements. All tested nanoparticles showed comparable hydrodynamic diameters in aqueous dispersion with different neutral, cationic and anionic surface charges depending on the coating material, and with controlled stability in artificial fluids. The degradation of the core-shell nanoparticles was found to be dependent on the surface type, core materials and the pH and composition of the degradation medium. At pH 7.4 in SBF all nanoparticles remained unchanged over 28 days regarding aggregation, zeta potential, magnetism and core-shell structure. In lysosomal fluids at pH 4.5 and 5.5 the degradation was found to increase with decreasing pH. The core degradation in acidic media seemed to be dependent on the biodegradability, water permeability and hydration, as well as the acid/base character of the shell. Furthermore, the degradation of the core correlated with a decrease of the magnetization. Infrared spectroscopy was utilized to evaluate the

depletion of surface material assuming that degradation is linked to a loss or degradation of the surface coating. In addition, the fate of the nanoparticles was followed by Raman spectroscopy. In conclusion, the combination of the different techniques constitute an in vitro prediction model that simulates the biodegradation of iron oxide based magnetic nanoparticles. Acknowledgements: The authors would like to thank A. Mohn and J. Grabow for their excellent technical assistance. We acknowledge the Federal Ministry of Education and Research (project 03XP0003) for financial support.

References: 1. Marques, M. R. C. et al.: Dissolution Technol. 2011, 18(3): 15-28.

POS.193

smartPearls® for dermal delivery – control of loading procedure with amorphous actives Pyo, S. M.1; Keck, C. M.2; Müller, R. H.1 1 Freie Universität Berlin, Institut für Pharmazie, Kelchstr. 31, 12169 Berlin, Germany 2 PharmaSol GmbH, Stubenrauchstr. 66, 12161 Berlin, Germany

Loading of mesoporous materials (silica, e.g. Neusilin) has been used to keep poorly soluble drugs in the amorphous state, to increase their oral bioavailability (e.g. CapsMorph® technology [1]). The amorphous state proved to be physically stable for more than 7 years of storage. Re-crystallization is hindered by the space restriction within the pores (2-50 nm). Bioavailability is increased due to the increased saturation solubility Cs, and fast dissolution velocity dc/dt of the amorphous actives (no dissolution limitation). Recently this approach has been transferred to dermal delivery, the smartPearls® [2]. Surprisingly the amorphous state remained after incorporating into dermal formulations, despite the presence of the solute water (low solubility solute, but still potentially sufficient to generate crystallization). The penetration into human skin was similar or even superior to nanocrystals, e.g. cyclosporin A. The technology can be applied to cosmetics, consumer care and pharma products. There are several procedures for loading the actives into the pores, e.g. co-grinding, solvent methods (immersion, wetness impregnation) or loading with supercritical carbon dioxide. The wetness impregnation method is a simple process; drug solution is added to the porous material, blended and then subsequently evaporated (typically in multiple steps). This leads to precipitation of the active inside the pores, and partially on the surface. Preferred is the localization in the pores to avoid potential re-crystallization on the surface in case too much active is surface located. The pore volumes are relatively high, e.g. up to about 1.7ml per g carrier, being equivalent to a maximum theoretical loading of about 60-70% w/w by an assumed drug density of 1.5g/cm3. However, before obtaining this theoretical maximum, crystal formation occurs in practice above a “critical threshold”. Thus the loading process needs to be controlled to obtain a pure amorphous product, verifiable by x-ray diffraction and differential scanning calorimetry (DSC). Parameters of the loading procedure itself can also affect the critical threshold, e.g. amount of solvent added per step, active concentration in the solvent. Industrially friendly is loading with high solvent amounts and high active concentrations, however bearing the increased risk of surface localization of active during evaporation and resulting in crystal formation. In this study Syloid® SP53D 12008 was loaded with coenzyme Q10 (Q10) as model active, using the impregnation method. Ethyl acetate containing 15% Q10 was used for loading, the amount of solvent used in each step was equal to 100%, and alternatively only to 50% theoretical pore filling. The 50% filling should lead to faster uptake by the excess pore volume and less surface localization. However, industrially it requires the double number of addition steps and thus is more costly. With Q10 in ethyl acetate it was found that both loading methods lead to almost similar loading of about 40% in the amorphous state, as shown by x-ray and DSC. In a second study, to assess potential localization of active on the carrier surface, light microscopy was applied. As model active curcumin was used, because of its strong yellowish color easy to detect. The microscopic appearance after each solvent addition and evaporation step was investigated. First, minor surface localization was detected, and the microscopic pictures can be finally assessed by comparing with x-ray and DSC data. In conclusion, a control of the loading procedure is essential not only to ensure loading in the amorphous state, but also to identify loading parameters which are most economical. In this case loading with

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maximum solvent amount still leads to an amorphous product. Combination of x-ray, DSC and light microscopy proved sensible, because first detectable surface localization does not lead already to crystallization (i.e. too thin surface film of active on carrier). Using the 3 methods, the surface coverage can be identified still not leading to detectable crystallization. References: 1. Wei, Q., Keck, C. M., Müller, R. H.: Int. J. Pharm. 2015, 482(1-2): 11–20. 2. Monsuur, F., Höfer, H. H., Keck, C. M.: US provisional patent application no. 62050587, 2014.

POS.194

smartPearls® for dermal delivery of amorphous actives – effect of loading concentration on stability & release Jin, N.1; Pyo, S. M.1; Keck, C. M.2; Müller, R. H.1 1 Freie Universität Berlin, Institut für Pharmazie, Kelchstr. 31, 12169 Berlin, Germany 2 PharmaSol GmbH, Stubenrauchstr. 66, 12161 Berlin, Germany

Amorphous drugs have well known advantages for drug delivery (bioavailability increase etc.), but often face the problem of physical stability during shelf life of the formulations. The amorphous state could be nicely stabilized by encapsulating the drugs in mesoporous materials (silica, e.g. Neusilin [1]), or macropores of small size, due to the space restriction in the pores. Physical stabilities of up to 7 years are reported. Materials used are e.g. various silicas (e.g. Syloid, company Grace), having the advantage of regulatory acceptance. The activities since 2008 focused solely on the oral administration route, to increase bioavailability (e.g. CapsMorph technology [2]); in 2014 the technology was transferred to dermal delivery [3]. An identical or superior dermal penetration was shown compared to both nanocrystals and even amorphous nanoparticles, e.g. cyclosporine A [4]. The more intensive use of the porous particle technology for drug delivery is just emerging since a few years, thus there is still a lack of basic knowledge how properties of the materials (e.g. pore size, functionalization of pore surface, etc.) or parameters of the loading procedure affect important pharmaceutical properties (e.g. drug loading, physical stability, solubility properties, release, etc.). In this study, the wetness impregnation method was used to load azithromycin (AZ) as model active onto Syloid SP53D 11920 particles (Grace, US). The active was dissolved in ethanol 96%, using two concentrations of AZ for the loading (50% saturated solution Cs (Cs50%), and 25% saturated solution (Cs25%)). The AZ solutions were admixed in multiple steps, followed by subsequent drying in an oven. The formulations were stored at room temperature, and the physical stability of the amorphous state monitored also by x-ray. The achieved drug loading was determined by HPLC. The saturation solubilities of the formulations prepared with Cs50% and Cs25% solutions were determined in a shaking test and the dissolution velocities in a release test. With both drug concentrations Cs50% and Cs25% in the loading solution, a similar drug loading of about 30% was achieved, determined by HPLC. Also the saturation solubility Cs was identical for both formulations (1310 µg/ml and 1302 µg/ml, resp.). Based on this, one could decide to use the higher concentrated loading solution, because this reduces the number of loading cycles (3 vs. 7 loading steps). However, the properties such as stability and release were clearly different. Re-crystallization was observed already after 1 week with the Cs50% formulation, whereas the Cs25% formulation remained stable for more than 1 year stored at room temperature. Also the release velocities were different, being faster for the Cs50% formulation, especially in the first 40 minutes. Based on this, the use of lower concentrated loading solutions seems to be sensible for the production of formulations with higher physical long-term stability. This might be explainable by preferential localization of the active in the pores, whereas in higher concentrated solutions the beginning evaporation after solvent addition to the silica leads to more surface localization of the drug. Surface-localized drug is more prone to form crystals, compared to the one in the space-restricted pores. This higher surface localization can also explain the faster release observed for the Cs50% formulation. These results provide a direction for optimal loading of porous materials. They show also the opportunity to modulate release by different loading parameters, and highlight the need for more intensive systematic investigations for better understanding of basic mechanisms of this novel delivery system.

References: 1. Wei, Q., Keck, C. M., Müller, R. H.: Int. J. Pharm. 2015, 482(1-2): 11–20. 2. Nolte, M. et al.: PCT/EP2009/057688, 2009. 3. Monsuur, F., Höfer, H. H., Keck, C. M.: US provisional patent application no. 62050587, 2014. 4. Staufenbiel, S. et al.: 1st European Conference on Pharmaceutics 2015, #42: 26.

POS.195

Overcoming the mucus barrier – Antibiotic nanocapsules as drug delivery system for the treatment of pulmonary infections in Cystic Fibrosis Torge, A.1,2; Wagner, S.3; Chaves, P. S.2; Oliveira, E. G.2; Titz, A.3; Beck, R. C. R.2; Schneider, M.1 1 Department of Pharmacy, Biopharmaceutics and Pharmaceutical Technology, Saarland University, Campus A4 1, 66123 Saarbrücken, Germany 2 Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), Av. Ipiranga, 2752, 90610-000 Porto Alegre, RS, Brazil 3 Helmholtz Institute for Pharmaceutical Research Saarland (HIPS), Chemical Biology of Carbohydrates, Campus E8 1, 66123 Saarbrücken, Germany

The treatment of pulmonary infections is an essential part of Cystic Fibrosis therapy, as recurrent lung infections caused by biofilm forming bacteria are associated with a decreased lung function and lower survival rates [1]. However, effectiveness of inhaled antibiotics is limited due to low diffusion rates and a possible deactivation of the drug in the highly viscous mucus [2, 3]. To overcome this biological barrier we loaded ciprofloxacin into lipid-core nanocapsules with a core consisting of oleic acid and sorbitan monostearate and a poly-ε-caprolactone shell stabilized by polysorbate. Ciprofloxacin-loaded nanocapsules were prepared by interfacial deposition of the polymer, yielding 180 nm particles with a drug encapsulation efficiency of 86.9%. Permeability was found to be increased inside respiratory horse mucus compared to a suspension of the drug. The nanocapsules showed a sustained drug release. The minimum inhibitory concentrations against Pseudomonas aeruginosa and Staphylococcus aureus were similar for the drug-loaded nanocapsules and the free ciprofloxacin. Thus, antibacterial activity is not affected by encapsulation. Interestingly, formation of biofilm-like aggregates was observed upon treatment of S. aureus with the free drug, while aggregate formation was avoided when bacteria were incubated with ciprofloxacin-loaded nanocapsules. In comparison to the free ciprofloxacin, our drug-loaded nanocapsules show several advantages: (1) higher permeability through respiratory mucus, (2) sustained drug release and (3) a biofilm-preventing effect. The combination of these benefits might enable a highly effective antibiotic therapy at a reduced dosing frequency. Acknowledgments: The DAAD and BMBF (FiDel project, FKZ 13N12530) are thanked for financial support.

References: 1. Ahlgren H.G. et al.: Bmc. Pulm. Med., 2015, 15:67, 1-6. 2. Levy J.: J. Pediatr., 1986, 108(5), 841-846. 3. Bhat P.G., Flanagan D.R., Donovan M.D.: J. Pharm. Sci.,1996, 85(6), 624-630.

POS.196

Microfluidic analysis for the testing of penetration and efficacy of antibiotic loaded PLGA micro- and nanoparticles in biofilms Ernst, J.1; Klinger-Strobel, M.2; Arnold, K.1, Markarewicz, O.2; Pletz, M. W.2; Fischer, D.1 1 Department of Pharmaceutical Technology, Friedrich-Schiller-University Jena, Otto-Schott Str. 41, 07745 Jena, Germany 2 Center for Infectious Diseases and Infection Control, Jena University Hospital, Erlanger Allee 101, 07747 Jena, Germany

Biofilm embedded bacteria benefit from the protective environment and thus reveal in strongly reduced susceptibility to antibiotics. Herein, biodegradable micro- and nanoparticular carriers loaded with antimicrobials are a promising approach to overcome the biofilm barrier [1]. For analyzing antibiotic activity, static biofilm models are widely used but suffer from some disadvantages e.g. no continuous nutrients supply or missing physiological shear flow. This can be avoided by the use of dynamic models like microfluidic flow-chamber systems



simulating in vivo conditions more closely. This study was aiming to develop poly(lactic-co glycolic acid) (PLGA) - PEG-PLGA nanoparticles (NP) and microparticles (MP) for the hydrophilic drug tobramycin (Tb) to test penetration and antimicrobial activity in a novel microfluidic technique for biofilms of Burkholderia cepacia. Particles loaded with Tb were prepared by a double-emulsion evaporation method using the Ultra-Turrax T25 (IKA-Werke, Staufen, Germany) for homogenization [2]. Additionally, drug-free particles were prepared as controls. Obtained MP and NP were characterized by scanning electron microscopy, dynamic light scattering and zetapotential measurement (Zetasizer Nano ZS, Malvern Instruments, Herrenberg, Germany). The drug content was analyzed by HPLC. For biofilm experiments a microfluidic system (BioFlux 48-well microfluidic plates, Fluxion Biosciences, South San Francisco, USA) was used to evaluate penetration and efficiency of antibiotic loaded particle suspensions in B. cepacia biofilms in a non-static environment. Moreover, 24-Well Tissue Culture Chambers (Sarstedt AG, Nümbrecht, Germany) were used as static model for comparison with the microfluidic system. To track the particles PLGA was covalently labelled with 7-amino-4-methyl-3-coumarinylacetic acid (AMCA) [3]. These blue fluorescent particles could be clearly distinguished from LIVE/DEAD (green/red) stained bacteria by confocal laser scanning microscope (cLSM; LSM510, Carl Zeiss Jena GmbH, Jena, Germany). In vitro cell viability was analyzed by CellTiterGlo® assay with the human lung endothelial cell line A-549 (ACC107, DSMZ). The particles exhibited mean hydrodynamic diameters between 700 nm and 1000 nm for MP resp. ca. 250 nm for NP with narrow size distributions. Tb incorporation increased the zetapotentials to about -10 mV compared to drug-free particles of about -30 mV. The cLSM experiments showed that MP and NP were distributed in deep layers of the biofilms indicating their penetration ability. In contrast to static chamber slide experiments, the microfluidic system contained an array of microfluidic flow channels arranged on a well plate format with each flow channel connected to an input well and an output well on the plate. To begin an experiment, bacterial cells were loaded into the input wells. The BioFlux Pressure Interface coupled to the top of the well plate applied a controlled pneumatic pressure which drives the fluid through the channels. The flow profile in BioFlux plates was found to be uniform and laminar. Surprisingly, despite a fast drug release Tb loaded NP and MP showed superior penetration and efficacy in biofilm-embedded microbes compared to the free Tb or a blend of Tb and particles. The blends had no antimicrobial activity on B. cepacia indicating that only Tb loaded particles can reach the biofilm embedded bacteria. No cytotoxicity of NP or MP in A-549 cells could be observed up to a concentration of 1 mg/mL and 24 h incubation time. In conclusion, the microfluidic system BioFlux could be successfully established for biofilm experiments utilizing LIVE/DEAD staining of the bacteria and blue fluorescent labelling of the polymeric carrier under more physiological conditions compared to static models. For both models, non-static and static, penetration in biofilm embedded bacteria and antimicrobial activity could be effectively tested. Moreover, it could be shown that biodegradable polymers such as PLGA and PEG-PLGA exhibit a great potential as micro- and nano-carrier systems for the encapsulation of antibiotics to improve their deposition and bacterial killing in deeper biofilm layers to provide therapeutic benefit in biofilm-associated chronic pulmonary B. cepacia infections, like cystic fibrosis. Acknowledgments: This work was supported by the Deutsche Forschungsgemeinschaft (DFG), grants PL 320/3-1 and FI 899/4-1.

References: 1. Klinger-Strobel, M. et al., Expert Opin. Drug Deliv. 2015, 12:1351-74. 2. Ungaro, F. et al., J. Control. Release 2012, 157:149-59. 3. Klinger-Strobel, M.; Int. J. Nanomed. 2016, 11:575-83.

POS.197

Bacterial nanocellulose as gene activated matrix Pötzinger, Y.1; Rahnfeld, L.1; Kralisch, D.1; Fischer, D.1 1 Friedrich-Schiller-University, Department of Pharmaceutical Technology, Otto-Schott-Straße 41, 07745 Jena, Germany

The biomedical application of bacterial nanocellulose (BNC) is currently under intensive investigation due to the high purity, biocompatibility and special mechanical properties of this material. Its applicability as tissue or cartilage replacement, as implant or wound dressing is particularly studied [1]. By loading different active ingredients, e.g. antiseptics, the

potential of BNC as drug delivery system for such applications could be demonstrated [2]. For the same reason, flat BNC fleeces should be loaded with nucleic acids and the applicability as GAM (“gene activated matrix”) should be examined. The main focus was the evaluation of the DNA loading efficiency and the release behaviour. Additionally, the ability of the BNC fleeces to protect DNA against nucleases as well as the biological compatibility and activity should be examined. BNC fleeces were biosynthesized by static cultivation of Komagataeibacter xylinus in 24-well plates [3]. After alkaline purification and washing until pH neutrality, BNC fleeces were loaded with plasmid DNA (pDNA, pGL3 or pSV-β-Gal) using an injection or reswelling method. In some cases, the pDNA was complexed with linear poly(ethylene imine) (2.5 kDa) to polyplexes in a N/P ratio of 20 [4]. BNC fleeces were characterized regarding morphology and physicochemical properties using scanning electron microscopy or by measuring the compression stability. Release behaviour was investigated under agitated conditions in PBS pH 7.4 at 70 rpm and room temperature. Protection against nucleases was tested by incubation of loaded BNC samples in a DNase I solution (1.5 U/µg DNA) for different time periods, inactivating DNase I by heating this mixture to 70 °C and extraction of the pDNA. Integrity of pDNA was analysed afterwards using agarose gel electrophoresis. Additionally the biocompatibility was investigated by local application of loaded BNC fleeces onto the chick area vasculosa (CAV) of fertilized hens eggs ex ovo [5]. The transfection efficiency was evaluated in vitro in dependence of the incubation time using CHO-K1 cells. By optimizing the applied loading methods, different parameters could be identified to control the efficiency and speed of loading. Thus, the BNC fleeces could be loaded with up to 50 µg pDNA using injection or reswelling techniques. Neither the three-dimensional network structure nor the mechanical properties of BNC were affected. The release behaviour could be controlled in dependence of the loading strategy. Either a fast release with an initial burst within the first 24 h or a long-term release up to 336 h with an almost linear release profile could be obtained. Loaded BNC samples were able to protect pDNA against nucleases effectively over 8 h (injection loaded BNC) or 72 h (reswelling loaded BNC), respectively. After local application of loaded BNC on the CAV of shell-less hen’s eggs no harmful influences could be observed. In transfection experiments with CHO-K1 cells, different transfection efficiencies were obtained, in accordance to the release behaviours. By means of this study it could be shown for the first time that BNC is applicable as GAM for local gene delivery purposes. By loading BNC with pDNA or polyplexes using different loading techniques, the release kinetics could be specifically modified and the pDNA could be protected against degradation by nucleases. The excellent biocompatibility of BNC was not affected by loading with pDNA or polyplexes and the transfection efficiency could be demonstrated in vitro as a first proof-of-concept. Acknowledgments: We would like to thank JeNaCell GmbH for providing the K. xylinus culture.

References: 1. Jorfi, M., Foster, E.J.: J. Appl. Polym. Sci. 2015, 132(14). 2. Moritz, S., et al.: Int. J. Pharm. 2014, 471(1–2): 45-55. 3. Kralisch, D., et al.: Biotechnol. Bioeng. 2010, 105(4): 740-7. 4. Fischer, D., et al.: Pharm. Res. 1999, 16(8): 1273-1279. 5. Müller, R., et al.: J. Magn. Magn. Mater. 2015, 380: 61-65.

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Spray drying microencapsulation of polyethylenimine (PEI)-based nanoparticles into a poly(vinyl alcohol) matrix Schulze, J.1; Aigner, A.1 1 Rudolf Boehm Institute for Pharmacology and Toxicology, Clinical Pharmacology, Leipzig University, Härtelstrasse 16-18, D-04107 Leipzig, Germany

The nucleic acid-based treatment of diseases offers great opportunities, even in the case of targets considered as otherwise undruggable. Due to its potential applications, the interest in gene therapy, and consequently in gene delivery, has grown continuously over the past years. Among non-viral delivery systems, polyethylenimine (PEI) is a promising candidate due to its high biological activity. The combination of this cationic polymer with a lipid delivery system further improves the properties of the formed nanoparticles (NPs), by combining the beneficial features of a lipid system and the advantages of PEI.

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An important problem related to polyplexes and lipopolyplexes is their tendency to aggregate, resulting in a significant loss of biological activity. As a consequence of their short shelf life, nanoparticles must be prepared freshly prior use and administered within a short time frame. Addressing this issue, we developed a spray drying method to embed PEI-based polyplexes or lipopolyplexes in a microparticulate poly(vinyl alcohol) formulation. With spray drying, we use an efficient, well-established and facile tool to produce large amounts of our NiMDS (Nanoparticles-in-Microparticle Delivery System). In the present study, we show the feasibility of a spray drying process for the generation of PVA microparticles containing PEI-based nanoparticles, and analyze their physicochemical properties and biological efficacies. A crucial point of embedding NPs in a polymeric matrix was to investigate whether this is accompanied by changes in nanoparticle size or surface charge upon their release from the polymeric matrix. The characterization of the size of released nanoparticles shows no effect of this manufacturing procedure on lipopolyplexes (LPP). In contrast, the size of encapsulated polyplexes (PP) slightly increases, indicating a corona effect of the PVA during the spray drying process. For both, LPP and PP, a decrease of the zeta potential was observed. Regarding the biological activity, we demonstrate that the encapsulated nanoparticles are not only just as efficient as their unformulated counterparts, but show even better transfection results. Another desirable feature achieved with this easy-to-handle nanoparticle-in-microparticle delivery system (NiMDS) is its long-term storability. Even upon storage of the dry powder up to three months at room temperature, the biological activity remains high. Additionally, the cytotoxicity of PEI nanoparticles was profoundly reduced after their embedding into the PVA matrix and subsequent release. The microparticles, forming the PVA matrix, show a mean diameter of 3 – 5 µm, a preferable particle size for pulmonary administration. Notably, we successfully performed an in vivo study in which BALB/c ByJ mice were exposed to the microparticulate powder in a self-constructed inhalation chamber, showing promising transfection results. Taken together, spray drying proves to be a suitable method for producing high amounts of this easy-to-handle formulation and also turns out to improve the potency and biocompatibility of PEI-based DNA nanoparticles. We further demonstrate that these NiMDS allow long-term storability of the embedded polyplexes / lipopolyplexes.

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A modular ratiometric fluorescent probe for detection of extracellular reactive oxygen species Andina, D.1; Brambilla, D.1; Leroux, J. C. 1; Luciani, P.1,2 1 Institute of Pharmaceutical Sciences, ETH Zurich, Vladimir-Prelog-Weg 1-5/10, 8093, Zurich, Switzerland 2 Institute of Pharmacy, Friedrich Schiller University Jena, Lessingstraße 8, 07743, Jena, Germany

A well-established first line defense mechanism during acute and chronic inflammation is the oxidative burst, during which reactive oxygen species (ROS) are released. The specific detection of the extracellular radicals has, however, remained a challenge. A diagnostic tool to monitor ROS-rich areas quantitatively and selectively might help to understand the progress of inflammation and evaluate the efficacy of antioxidant therapies. Thus, a new modular ratiometric fluorescent probe to detect extracellular ROS was designed. The contrast agent consists of a ROS-sensitive dye connected to a negatively charged PEG-polyamino acid-peptide nucleic acid (PNA) and a ROS-insensitive dye coupled to the complementary PNA strand (Figure 1). Hydrocyanines were shown to selectively detect superoxide and hydroxyl radicals [1] and were, therefore, used in this project as ROS-sensitive probe. The ROS-insensitive dye (Chromis dye series, Cyanagen) allows to calibrate the system and to trace the probe even when in a ROS-free environment. Hybridization of the two labelled complementary strand yielded a ratiometric fluorescent probe responsive to ROS in cell-free assays as well as in a stimulated colon carcinoma cell line.

Fig. 1: A hydrocyanine-spacer-PNA is hybridized with a ROS-insensitive dye connected to the complementary PNA strand.

References: 1. Kundu K, et al. Angew. Chem.-Int. Edit. 2009, 48: 299-303.

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3.11 Pharmacology

POS.200 Cardiomyocyte cGMP and mitochondrial BK channels exert cardioprotection against ischemia/reperfusion injury Frankenreiter, S.1, Mohr, E.1; Kniess, A.1; Ruth, P.1; Krieg, T.2; Friebe, A.3; Lukowski, R.1 1 Department of Pharmacology, Toxicology and Clinical Pharmacy, Institute of Pharmacy, University of Tuebingen, Tuebingen, Germany 2 Department of Medicine, University of Cambridge, Cambridge, United Kingdom 3 Department of Physiology, Julius Maximilian University, Wuerzburg, Germany

Background: The Ca2+- and voltage-activated potassium channel of big-conductance (BK), encoded by the KCNMA1 gene, is usually present at the plasma membrane of cells. However, in cardiomyocytes (CMs) BK channels are localized exclusively at the inner mitochondrial membrane (mitoBK) [1]. By studying hearts obtained from BK-null mice (BK-KO) in an ex vivo Langendorff perfusion setup we and others previously found evidence for mitoBKs as infarct-limiting factors [1;2]. It is well established that canonical BK channels are directly stimulated by the cyclic guanosine-3',5'-monophosphate (cGMP)/cGMP-dependent protein kinase type I pathway; however, it is unclear whether cardioprotection afforded by cardiomyocyte cGMP in vivo requires mitoBK. Methods: Using the Cre/loxP recombination system we generated animals with a CM-restricted deletion of BK channels (CMBK-KO) [3] and of the nitric oxide (NO)-sensitive guanylyl cyclase (CMsGC-KO) [4]. The susceptibility of the conditional BK and sGC mutants to ischemia/reperfusion (I/R) injury was compared to age- and litter-matched controls (CMBK-CTR and CMsGC-CTR) as well as to global BK-KO and BK wild-type (BK-WT) mice. An open chest in situ model of myocardial infarction was applied to determine differences in infarct size at baseline and upon ischemic pre-/postconditioning (iPre/iPost) or pharmacological interventions using the BK blocker paxilline and the BK opener NS11021 as well as cGMP modulating drugs like the PDE5-inhibitor sildenafil or the sGC activator cinaciguat. As additional cardiovascular parameters we assessed blood pressure and heart function of the conditional mutants. Results: After 30 min of ischemia followed by 120 min of reperfusion the infarct size between CMsGC-KO and their control hearts did not differ. I/R, however, provoked significantly more cardiac damage in global BK-KO and CMBK-KO mice than in age- and litter-matched BK-WTs and CMBK-CTRs, respectively. This could also be shown by an increase in apoptosis in the CMBK-KO hearts. The BK blocker paxilline increased cardiac damage, whereas the NS compound provoked a reduction in infarct size in CMBK-CTR but not in CMBK-KO hearts. Short repetitive episodes of ischemia applied directly after infarction (iPost) significantly reduced the myocardial damage in WT and all CTR mice, whereas the protective effect of this intervention was much less pronounced in hearts that lacked functional BK channels in CMs and was completely absent in mice lacking CM NO-sGC or global BK channel expression. Interestingly, cardioprotection elicited either by sildenafil or by cinaciguat seem to require mitoBK as well as NO-sGC in CM. Finally, CMBK-KO mice presented with a reduced blood pressure and a minor impairment of the left ventricular contractility seen under physiological conditions. Conclusion: The presented findings suggest lack of BK channels in CMs as a cause for mild cardiac dysfunctions. Additionally, absence of CM mitoBK renders the heart more susceptible to I/R injury. Cardioprotection elicited by the BK opener NS11021 suggests that BK channels may be promising drug targets that interfere with the causes and/or consequences of myocardial ischemia. Interestingly, both CM NO-sGC and BK are important to allow the protective signaling events triggered either by short, repetitive episodes of ischemia or by drugs modulating the cGMP signaling pathway. Further studies are needed to elucidate whether cardioprotection via NO-GC and BK are linked by a common cGMP pathway in the CM itself. References: 1. Singh, H. et al.: Proc Natl Acad Sci USA 2013, 110(26): 10836-10841. 2. Soltysinska, E.: PLoS One 2014, 9(7). 3. Agah, R.: J Clin Invest, 1997, 100(1): 169-179. 4. Takefuji, M. et al.: Circulation 2012, 126(16), 1972-1982.

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Crtc1-deficient mice show reduced cardiac function which is ameliorated by isoprenaline treatment Morhenn, K.1,2; Geertz, B.3; Eschenhagen, T.2,3; Oetjen, E.1,2,4 1 Department of Clinical Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany 2 DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck 3 Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany 4 Institute of Pharmacy, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany

Cardiac hypertrophy leads to heart failure, one of the common causes for hospitalization in the western world. Chronic -adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, as evidenced by the therapeutic success of β-adrenoceptor antagonists. The cAMP Regulated Transcriptional Coactivator 1 (CRTC1) is regulated by increases in cAMP and calcium/calcineurin, as elicited by -adrenergic signaling, both known to participate in the development of cardiac hypertrophy [1]. Our previous data show an increase in CRTC1 protein content in hearts of mice and humans under conditions of maladaptive hypertrophy. Mice globally deficient in Crtc1 show signs of hypertrophy indicated by a higher ratio of heart weight to tibia length and increased myocyte size while they show no signs of fibrosis. In the present study, cardiac morphology and function of Crtc1-deficient mice were echocardiographically assessed. Our findings show a decreased cardiac function in Crtc1-deficient mice compared to their wild type littermates measured by a decrease in ejection fraction, fractional shortening and cardiac output by 38±9%, 40±9% and 36±10%, respectively. Left ventricular volume and left ventricular inner diameter were increased in Crtc1-deficient mice during systole and diastole. These parameters indicate a systolic dysfunction in Crtc1-deficient mice. Wall thickness increase between diastole and systole was reduced in Crtc1-deficient mice indicating a reduced contractile function. As a model for β-adrenergic induced cardiac hypertrophy, isoprenaline was administered to Crtc1-deficient mice and their wild type littermates for seven days (30 g/day/g bodyweight); control mice received NaCl. Long term isoprenaline treatment ameliorated the decreased cardiac function in Crtc1-deficient mice: In Crtc1-deficient mice, the increase in ejection fraction, fractional shortening and cardiac output between NaCl or isoprenaline treated mice was 1.28 fold, 1.25 fold and 2 fold higher, respectively, compared to wild type littermates. Meanwhile, mRNA expression of Crtc1 showed a tendency to increase in wild type mice after isoprenaline treatment, suggesting a further increase after longer isoprenaline treatment. mRNA expression of collagen 11, collagen 31 or the profibrotic Ctgf was not increased after isoprenaline treatment in wild type nor in Crtc1-deficient mice but showed a tendency to an increase as well. In conclusion, our data indicate a systolic dysfunction with a reduced contractile function in Crtc1-deficient mice which is ameliorated by long term isoprenaline treatment. This might suggest, that the loss of CRTC1 contributes to cardiac dysfunction. Considering that propranolol inhibits CRTC1 activation [own unpublished data], reduced cardiac function after treatment with -adrenoceptor antagonists might in part be due to impaired CRTC1 signaling. Acknowledgments: The Crtc1-deficient mice were a kind gift by Jean-René Cardinaux, Center for Psychiatric Neuroscience, Prilly-Lausanne, Switzerland.

References: 1. Screaton, RA. et al.: Cell 2004, 119(1): 61-74

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Distinct cardiac metabolic shifts in two mouse models of cardiac hypertrophy Gundler, A. L.1,2; Morhenn, K.1,2; Oetjen, E.1,2,3

1 Department of Clinical Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany

2 DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck 3 Institute of Pharmacy, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany

Heart failure is one of the major reasons for hospitalization, and maladaptive hypertrophy plays a pivotal role in the pathogenesis of heart failure. The condition of a higher energy demand in cardiac

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hypertrophy provokes changes in the metabolism of cardiomyocytes. The heart returns to a fetal like pattern of using energy supplies. Oxidation of fatty acids decreases while the usage of glucose to provide ATP increases. This metabolic shift provides enough energy in the short term, however, in the long term it seems to aggravate the development of heart failure [1,2]. Our previous data show that mice deficient in CRTC1 (cAMP Regulated Transcriptional Coactivator 1) develop cardiac hypertrophy and dysfunction. Moreover, humans and mice under conditions of maladaptive hypertrophy exhibit elevated CRTC1 protein content in the heart, e. g. the Mybpc3-targeted knock-in (KI) mice, a common model for HCM (hypertrophic cardiomyopathy). To investigate the role of CRTC1 in cardiac metabolism, Crtc1-deficient (KO) mice were studied and compared to their wild-type (WT) littermates as well as to the KI mice. To investigate the state of cardiac metabolism, mRNA levels of transcription factors and enzymes for different metabolic pathways were analyzed via reverse transcription-qPCR. The mRNA content of two transcription factors contributing to the expression of enzymes for the β-oxidation, PGC-1α (peroxisome proliferator activated receptor γ coactivator 1-α) and PPARα (peroxisome proliferator activated receptor α), was reduced by 57.3±5.7% and 48.7±7.3%, respectively, in KI mice, but remained unchanged in KO mice compared to wild-type mice. There was no change in the mRNA levels of GLUT1 (glucose transporter 1) and CPT1B (carnitine palmitoyltransferase 1B). Both proteins are involved in the uptake of either glucose or fatty acids. However, the insulin dependent glucose transporter GLUT4 (glucose transporter 4) was reduced in KI mice by 43.8±3.8%. As a general marker for energy demand, the mRNA content of AMPK (AMP-activated protein kinase) was examined. The mRNA expression of the γ2-subunit of AMPK was increased by 31.9±7.1% in KO mice compared to WT mice, but did not differ in the KI mice. These findings suggest a shift away from β-oxidation in the KI mice. However, the mRNA expression of GLUT4 was reduced as well. It might be conceivable that the higher requirement for energy, complied with higher use of glucose in the KI mice over a long time period could have led to alterations in glucose metabolism. Although the KO mice seem to have an augmented need for energy as well, changes in the mRNA levels of the tested markers were not observed. Further investigations might show how CRTC1 regulates cardiac metabolism. Acknowledgments: We thank Jean René Cardinaux (Center for Psychiatric Neuroscience, Site Cery, 1008 Prilly-Lausanne, Switzerland) for the kind gift of the KO mice and Lucie Carrier (Department of Experimental Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Martinistraße 52, 20246 Hamburg, Germany) for the kind gift of the KI mice.

References: 1. Neubauer, S.: N Engl J Med. 2007, 356(11): 1140-51. 2. Taegtmeyer, H.: Ann N Y Acad Sci. 2010, 1188:191-8.

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Disrupting the interaction between calcineurin and the dual leucine zipper kinase (DLK) increases kinase activity Duque Escobar, J.1,3; Lemcke, T.2; Oetjen, E.1,2,3 1 Department of Clinical Pharmacology and Toxicology, Cardiovascular Research Center, University Medical Center Hamburg Eppendorf, Martinistr. 52, 20246 Hamburg, Germany 2 Institute of Pharmacy, University of Hamburg, Bundesstraße 45, 20146 Hamburg, Germany 3 DZHK (German Center for Cardiovascular Research), Partner Site Hamburg/Kiel/Lübeck

Diabetes mellitus type 2 incidence and prevalence are still increasing worldwide. Decrease in -cell mass and function are the most important factors for the clinical manifestation of type 2 diabetes. The structurally distinct immunosuppressive drugs tacrolimus and cyclosporin A inhibit the calcium-calmodulin dependent phosphatase calcineurin thereby decreasing insulin gene transcription and inducing -cell apoptosis [1, 2]. In addition, both drugs enhance the activity of the mitogen-activated protein kinase kinase kinase 12 (DLK, dual leucine zipper kinase) [2, 3]. Our Previous data show that mutation of V364 to A within the putative calcineurin interaction domain 362-LPVP-365 (LxVP) resulted in an increase of DLK kinase activity. In the present study, the direct regulation of DLK by calcineurin was investigated. To study the effect of DLK V364A DLK mutant on -cell apoptosis, expression vectors for flag epitope tagged DLK wild-type and DLK V364A mutant were transiently transfected into the -cell line HIT. Double immunocytochemistry was performed to detect DLK transfected

cells and cleaved caspase-3 positive cells as a marker for initiated apoptosis. DLK V364A mutant and wild-type transfected HIT cells were treated with the calcineurin inhibitors cyclosporin A, tacrolimus and hydrogen peroxide as a reactive oxygen species. Treatment of DLK wild-type with the proinflammatory cytokine TNF increases -cell apoptosis and nuclear translocation [5] and was used as a positive control. In cells transfected with the DLK V364A mutant an increase in -cell apoptosis and nuclear translocation compared to cells transfected with DLK wild-type was observed. TNF and the calcineurin inhibitors increased apoptosis and nuclear translocation in DLK wild-type transfected cells, but they were not able to increase apoptosis and nuclear translocation in DLK V364A mutant transfected cells. To investigate whether DLK and calcineurin interact, a protein protein interaction assay was performed. Recombinant produced MBP (maltose binding protein) fused DLK proteins and 6x-His tagged calcineurin protein were purified by affinity chromatography. In the protein protein interaction assay purified MBP fused DLK wild-type protein interacted with 6x-His tagged calcineurin protein compared to MBP (negative control), whereas the DLK V364A mutant did not. These findings show that DLK interacts with the phosphatase calcineurin via the V364 within the LxVP-type calcineurin interaction domain. This is the same region which calcineurin interacts with cyclosporin A and tacrolimus leading to inhibition of phosphatase activity [4]. These results show that the mutation of V364 to A within DLK interferes with calcineurin-immunosuppressant binding on calcineurin. Disruption of the DLK-calcineurin interaction increased DLK kinase activity, and induced its nuclear translocation and -cell apoptosis. Taken together with our previous data, these results suggest that DLK dephosphorylation by calcineurin contributes to the phosphatase’s -cell protective effect. In addition, a novel mechanism for regulating DLK activity was detected. References: 1. Oetjen, E. et al.: Mol. Pha. 2003, 63(6): 1289-95 2. Plaumann, S. et al.: Mol. Pha. 2008, 73(3): 652-9 3. Oetjen, E. et al.: Diabetologia 2006, 49(2): 227-236 4. Rodríguez, A. et al.: Mol. Cell. 2009, 33(5): 616-26 5. Walbach, M. et al.: Cell. Signal. 2016, 28(4): 272-83

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High throughput screening (HTS) of “Nature Bank” fractions at ecto-nucleotide pyrophosphatase1 and 3 (NPP1 and 3) to identify novel drug-like inhibitors Hinz, S.1; Lee, S.1; Serive, B.2; Mak, T.2; Vial, M.2; Böttcher, S.2; Di Capua, A.2; Scott, S.2; Pham, N. B.2; Quinn, R.J.2; Müller, C. E.1 1 PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, 53121 Bonn, Germany 2 Eskitis Institute for Drug Discovery, Griffith University, 46 Don Young Rd, Nathan QLD 4111, Australia

The ecto-nucleotide pyrophosphatase 1 and 3 (NPP1 and NPP3) are able to modulate purinergic signaling through the hydrolysis of nucleotides yielding AMP, which is further degraded to adenosine by ecto-5’-nucleotidase (CD73).[1] NPP1 is expressed in osteoclasts, chondrocytes, and adipose tissue, and has been reported to be upregulated in human gliomas. NPP1 inhibitors have therefore been proposed as new therapeutic targets for the treatment of cancer. Most of the known NPP1 inhibitors are nucleotide derivatives and analogs which are difficult to develop as orally bioavailable drugs. Others, e.g., oxadiazole or biscoumarine derivatives, display low affinity and lack selectivity.[2] NPP3 is abundantly expressed in basophils and mast cells,[3] and has been reported to be overexpressed in bile duct and colorectal cancers.[4,5] Therefore, NPP3 inhibitors have been aimed for the treatment of allergy and cancer. For NPP3 no potent and selective inhibitor has been published so far. Our goal has been to identify and develop small-molecule heterocyclic compounds with high potency and selectivity for either NPP1 or NPP3.[2,6] At the Eskitis Institute for Drug Discovery in Brisbane over 200,000 lead-like enhanced fractions obtained from extracts of endemic plants and marine organisms with drug-like properties potentially suitable for peroral application are available. Each fraction contains only 4-5 individual compounds, which display the desired lipophilicity and solubility profile. These fractions are part of a library, the so-called Nature Bank, which can be utilized for high throughput screening at pharmacological targets.[7,8] Because natural products have remained

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to constitute an important source of drugs, especially in the development of anticancer and antimicrobial agents, we set up a joint project between University of Bonn and Griffith University (Brisbane, Australia) to discover NPP1 and NPP3 inhibitors. To this end, a spectrophotometric HTS assay was developed, established and validated, by which the “Nature Bank” fractions were screened for NPP-1 and -3 inhibition. The developed HTS assay led to the identification of several fractions with high inhibitory potency at NPP1 or NPP3. The next step will be to isolate single active compounds, elucidate their structures, synthesize and optimize them. Acknowledgments: We thank the Heinrich-Hertz foundation for support

References: 1: Zimmermann, H., Zebisch, M., Sträter, M.: Purinergic Signal. 2012, 8(3): 437-502. 2: Chang, L. et al.: J. Med. Chem. 2014, 57(23): 10080-10100. 3: Stefan, C., Jansen, S., Bollen, M.: Trends Biochem. Sci. 2005, 30(10): 542-550. 4: Yano, Y. et al.: Int. J. Mol. Med. 2003, 3(5): 763-767. 5: Yano, Y. et al.: Cancer Lett. 2004, 207(2): 139-147. 6: Lee, S. et al.: Bioorg. Med. Chem. 2016, 24(14): 3157-3165. 7: Camp, D. et al.: Comb. Chem. High Throughput Screen. 2014, 17(3): 201-209. 8: Harvey, L.A., Edrada-Ebel, R., Quinn, R. J.: Nat. Rev. Drug Discov. 2015, 14(2): 111-129.

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Cysteine-rich LIM-only protein 4 (CRP4), a novel cardiac cGMP/cGKI effector protein in Angiotensin II induced myocardial remodeling Straubinger, J.1; Deng, L.1; Boldt, K.2; Krattenmacher, D.3; Ruth, P.1; Just, S.3; Lukowski, R.1 1 Institute of Pharmacy, Department of Pharmacology, Toxicology and Clinical Pharmacy, University of Tübingen, Germany 2 Institute for Ophthalmic Research, Molecular Biology of Retinal Degenerations, and Medical Proteome Center, University of Tuebingen, Germany 3 Molecular Cardiology, University of Ulm, Germany

Background: Cardiac hypertrophy is an adaptive response of the heart to many cardiovascular disorders including hypertension, myocardial infarction, defects of the valves and mutations in proteins of the sarcomere. Elevated levels of cardiac cyclic guanosine-3',5'-monophosphate (cGMP) activate cGMP-dependent protein kinase I (cGKI), which reportedly opposes detrimental cardiac remodeling induced by growth-promoting neurohormonal signals and stresses like Angiotensin II (Ang II) via Gαq-coupled receptors [1,2]. However, the molecular details underlying beneficial effects of cGMP/cGKI remain largely elusive. Hence we investigated the function of cardiac cysteine-rich LIM-only protein 4 (CRP4), a known phosphorylation target of cGKI in smooth muscle cells [3,4]. Additionally, CPR4 is a highly related homologue of the muscle LIM protein CRP3/MLP, which has been linked to dilated (DCM) and hypertrophic (HCM) cardiomyopathies in mice and humans [5]. Methods: We investigated the role of CRP4 in neurohormonal induced heart hypertrophy upon Ang II infusions (2 mg/kg/d) using CRP4 knockout (KO), wild type (WT) and heterozygous (HET) littermates. The extent of cardiac remodeling processes was defined by examining changes in the heart weight to the body weight (HW/BW), as well as the growth response of the myocytes and the determination of myocardial fibrosation. Alterations in cardiovascular performance were assessed by echocardiography and telemetric blood pressure measurements. Hypertrophic marker genes, putative effects of Ang II on components of the cGMP/cGKI pathway and the expression pattern of other members of highly related LIM proteins were analyzed in total mRNA and protein preparations isolated from healthy and hypertrophic ventricles. Since LIM domains are generally considered as protein binding interfaces, we used mass spectrometry supported Co-Immunoprecipitation (Co-IP) studies to identify interaction partners of CRP4 at baseline and upon hypertrophic Ang II-stimulation. Moreover, the importance of CRP4 in cardiac development was determined by morpholino-induced knockdown experiments in zebrafish larvae as an appropriate model for studying inherited cardiovascular defects. Results & Conclusion: CRP4 is abundantly expressed in the cardiac muscle of WT mice, whereas mRNA and protein levels were significantly reduced in HET hearts and absent from KO muscles. HW/BW ratios of all three genotypes did not differ at baseline, however, cardiomyocyte size and heart ratios were elevated in CRP4 HET and KO animals in response to the Ang II infusions. Irrespective of the blood pressure, CRP4-deficient mice developed a HCM-like phenotype with a

left ventricular outflow tract obstruction. Moreover, interstitial fibrosis was significantly stimulated by Ang II in CRP4-KO and HET hearts, whereas the production of anti-fibrotic factors such as BNP was diminished. Besides a direct interaction of CRP4 and cGKI, Co-IPs identified CRP4 to interact with numerous sarcomeric and cytoskeletal proteins such as cardiac myosin-binding protein C3 (Mybpc3) troponin I and troponin T, α-actinin 2, myosin 7 and vinculin, which are well known genetic hot spots to cause HCM and/or DCM in humans as well. Together, the increased susceptibility of CRP4-deficient hearts to chronic Ang II exposure identifies CRP4 as a novel anti-hypertrophic/-fibrotic factor regulated by cGMP/cGKI. The loss of CRP4 might be at least partly compensated by a cardiac-restricted amplification of the highly related LIM-protein CRIP1 (cysteine-rich intestinal protein 1). CRIP1 upregulation seems to protect CRP4-deficient hearts from an even more severe outcome, since the knockdown of CRIP1 (and CRP4, respectively) in zebrafish larvae results in massive cardiac abnormalities and premature death. So far, our studies discovered two novel LIM proteins acting as modifiers of the phenotypic characteristics of cardiac remodeling. References: 1. Lukowski, R. et al.: Proc. Natl. Acad. Sci. USA 2010, 107 (12): 5646-5651. 2. Patrucco, E. et al.: Proc. Natl. Acad. Sci. USA 2014, 111(35): 12925-12929. 3. Huber, A. et al.: J. Biol. Chem. 2000, 275 (8):5504-5511. 4. Zhang, T. et al.: J. Biol. Chem. 2007 282 (46): 33367-33380. 5. Geier, C. et al.: Circ. Res. 2003 107 (10):1390-1395.

POS.206

Acid sensing Ion channels as targets in gastroesophageal reflux disease Ulrich-Merzenich, G.1; Sherbakova, A.2; Kelber, O.3; Abdel-Aziz, H.4 1 Medizinische Klinik III, UKB, Friedrich Wilhelms-Universität Bonn, Germany 2 Department of Forestry, Volga State Technical University, Yoshkar-Ola, Russia 3 Innovation & Development, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany 4 Medical Affairs, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstr. 5, 64295 Darmstadt, Germany

Cytokines are relevantly involved in the signal transmission of the intestinal immune system. In earlier studies in a rat model it was shown that modulators of cytokines, as especially IL-8, play a decisive role in the pathogenesis of reflux esophagitis. IL-8 is a small heparine binding protein, which triggers activation and migration of neutrophiles fom the blood to the tissue, so having a pro-inflammatory effect. By measuring the release of IL-8 from HET-1A cells the anti-inflammatory action of the herbal combination medicine STW 5 was compared to its components. STW 5 is used in functional dyspepsia and irritable bowel syndrome and is a combination of extracts from Iberis amara (L.), Mentha piperita (L.), Matricaria camomilla (L.), Glycyrrhiza glabra (L.), Angelica archangelica (L.), Carum carvi (L.), Silybum marianum (L.) Gaertn., Melissa officinalis (L.) und Chelidonium majus (L.). Synchronized HET1A cells (20.000) were incubated with 4 concentrations each of STW 5 or the component extracts in equivalent concentrations for 18 h, alone or concomitantly with the pro inflammatory stimulator capsaicin (80µM). The IL-8 release was enhanced by the incubation with capsaicin from 46.3±5 to 85.8 ±14pg/ml (p<0.005). Treatment of the cells with the component extracts led to an IL-8 release between 20.4 ± 8 and 441 ± 177 pg/ml. The viability of the cells was not influenced by the experimental conditions. The highest IL-8 release was observed for the Iberis amara extract. When incubated together with capsaicin it caused an IL-8 relese of up to 625±121 pg/ml. In contrast, the combination STW 5 (59µg/ml), which contains Iberis amara in an equivalent concentration led to an IL-8 release of 55.3±2 pg/ml. STW 5 accordingly suspended the release of Il-8 induced by capsaicin as well as by specific single extracts. The mechanisms leading to a reduction of IL-8 release, which is an undesired pharmacological effect in this model, by STW 5, but not by some of its component extracts, is reduced, and is intended to be reduced further on. Acknowledgments: A. Shcherbakova was supported by DAAD. The study was supported by a research grant of Steigerwald Arzneimittelwerk GmbH, a part of Bayer group.

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References: 1. Abdel-Aziz H, Schneider M, Neuhuber W, Kassem AM, Khailah S, Müller J, Gamaleldeen H, Khairy A, Khayyal MT, Efferth T, Ulrich-Merzenich G. Mol Med. 2015. doi:10.2119/molmed.2015.00098

POS.207

Plasma Trough Concentrations of Azole Antimycotics in Lung Transplant Recipients Stelzer, D.1,2; Weber, A.2; Ihle, F.1; Ceelen, F.1; Zimmermann, G.1; Kneidinger, N.1; Schramm, R.3; Winter, H.4; Zoller, M.5; Vogeser, M.6; Andraschko, M.2, Behr, J.1, Neurohr, C.1

1 Department of Internal Medicine V, LMU-Munich, Marchioninistrasse 15, 81377 Munich, 2

Germany, Comprehensive Pneumology Center, Member of the German Center for Lung Research DZL 3 Hospital Pharmacy, LMU-Munich, Marchioninistrasse 15, 81377 Munich, Germany 4 Department of Cardiac Surgery, LMU-Munich, Marchioninistrasse 15, 81377 Munich, Germany 5 Department of Thoracic Surgery, LMU-Munich, Marchioninistrasse 15, 81377 Munich, Germany 6 Department of Anesthesiology, LMU-Munich, Marchioninistrasse 15, 81377 Munich, Germany Institute of Laboratory Medicine, LMU-Munich, Marchioninistrasse 15, 81377 Munich, Germany

Background: The purpose of this retrospective study was to analyze azole plasma trough levels (APL) in patients following lung transplantation. Methods: From July 2012 to July 2015 all measured APL of itraconazole, voriconazole, posaconazole liquid and posaconazole tablets in lung transplant recipients of the Munich Lung Transplant Program were evaluated. Target APL were defined as follows: itraconazole: ≥700ng/ml, voriconazole: 1000-5500ng/ml and posaconazole: ≥700ng/ml (prophylaxis) and ≥1000ng/ml (therapy). Results: 806 APL of 173 lung transplant recipients were evaluated (95 (55%) male, 119 (69%) double-LTx, age 51.4±13.4 years, underlying disease: lung fibrosis (n=70; 40%), chronic obstructive pulmonary disease (n=47; 27%), cystic fibrosis (n=31; 18%), pulmonary hypertension (n=6; 3%) and other (n=19; 11%)). Median APL for itraconazole, voriconazole, posaconazole liquid and posaconazole tablets are shown in Table 1. The highest median APL were achieved with posaconazole tablets and voriconazole. The lowest median APL were observed for posaconazole liquid, which were below the target APL for therapy and prophylaxis. Voriconazole and posaconazole tablets showed the greatest percentage of APL above the target threshold, whereas the lowest number of APL above the target APL was noted for posaconazole liquid. Table 1: Mean and median APL

indication azole daily dose (mg)

n (pat)

mean (±SD) (ng/ml)

median (ng/ml)

min (ng/ml)

max (ng/ml)

APL > TPL

prophylaxis ITR 400 79 1155 (±852) 1055 20 4203 62%

VOR 400 4 1826 (±846) 2107 600 2800 85%

POS-Liq

600 13 808 (±596) 592 50 1933 49%

POS-Tab 300 7 2709 (±2906) 2123 50 8698 76%

therapy ITR 400 10 779 (±506) 801 30 1669 50%

VOR 400 60 2173 (±2061) 1628 20 11878 70%

POS-Liq

800 31 930 (±682) 765 30 2424 38%

POS-Tab

300 25 2509 (±1495) 2107 405 4843 82%

ITR=itraconazole; VOR=voriconazole; POS-Liq=posaconazole liquid; POS-Tab=posaconazole tablets; n=number; pat=patient; SD=standard deviation; APL=azole plasma trough levels; min=minimum APL; max=maximum APL; TPL=target plasma trough level

Conclusions: Our study showed that achieved APL and APL above the target level in lung transplant recipients vary considerably between the different azoles analyzed. Even if most patients treated with voriconazole or posaconazole tablets reached APL above the target threshold, up to 30% of these APL were still below the required target levels for therapy and prophylaxis. Therefore, therapeutic drug monitoring should be integrated in the post lung transplantation management, to identify patients at risk for low APL.

REGULATORY SCIENCES/INDUSTRIAL DRUG DEVELOPMENT


3.12 Regulatory Sciences/Industrial Drug Development

POS.208 Commissioned testings of ACE inhibitors in the former GDR during the 1980s Kreiss, C. According to present guidelines, ACE Inhibitors (Ramipril, Enalapril etc.) still play an important role for the therapy of essential hypertension and congestive / chronic heart failure up to this day. That fact is reflected by the impressive number of more than 5 billion DDD (defined daily doses) alone in Germany. The first federal registration of Captopril in 1980 (Europe), respectively in 1981 (USA), by the US based company Squibb was soon followed by further developments of various international pharmaceutical companies. Many of these substances were tested in numerous clinical studies that were conducted worldwide with a large number of patients, regarding the safety, efficacy and the therapeutic value of this medication in comparison to placebo and various established standard treatments. Some of these clinical studies (mostly phase III) with ACE inhibitors were conducted in various medical facilities / hospitals of the GDR during the 1980s. These commissioned testings ("Auftragsuntersuchungen") were mirrored by massive media coverage during the last years, especially in Germany. The following exemplary analysis of these clinical studies was conducted at the Institute for History of Pharmacy (Marburg) and tries to investigate the question if the selection of the involved patients, the applied inclusion / exclusion criteria, the form of 'informed consent', the individual study design and practical aspects met the German / international standards of the 1980s. Another focus lies on the federal regulations of the GDR, concerning medicinal products and the approval of clinical studies on human subjects by the governmental agency ZGA ("Zentraler Gutachterausschus für Arzneimittelverkehr"). The source materials that were used for the analysis of these commissioned testings of ACE inhibitors consist mainly of documents originating from the federal archive of Germany (Bundesarchiv) and represent files of the ministry of health of the former GDR as well as of exemplary CRF (case report forms) that were provided through the archive of the former pharmaceutical company Hoechst for a research project that was conducted at the institute of history of medicine at the Charité in Berlin.

POS.209 Ethanol exposure in children: Food as by far more relevant source than phytomedicines Kelber, O.1; Hittinger, M.2; Gorgus, E.2; Schrenk, D.2 1 Innovation & Development, Phytomedicines Supply and Development Center, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany 2 Food Chemistry and Toxicology, University of Kaiserslautern, Erwin-Schrödinger-Straße 52, 67663 Kaiserslautern, Germany

Introduction: Liquid dosage forms of medicinal products are well suitable for children, as they allow adapting the dose to the age group. But in many cases, especially also in herbal products, they contain ethanol (Fig. 1), which has repeatedly been triggering critical questions.

Fig. 1: Liquid forms of phytomedicines may contain ethanol, which is, due to its solubility in water and its advantages as solvent of for the extraction of herbal drugs and as excipient, recommended by all leading pharmacopoeias.

Aims & Methods: The aim was therefore to assess to which extent phytomedicines contribute to the ethanol exposition in this age group, in comparison to the normal uptake with usual food items. By evaluation of data from the use of phytomedicines in liquid form and by generation of a scenario for the exposure by food items based on new analytical data, exposition values for a 6 years old child were estimated. Results: When using phytomedicines, a 6 years old child is exposed to amounts of ethanol between 70 and 180 mg with a single dose. Whith 3 times daily dosing this is 210 - 540 mg. Related to a body weight [b.w.] of 20 kg, this is 10.1 – 27.0 mg/kg b.w. [1]. An evaluation of side effects of these medicinal products, collected in non-interventional studies in more than 50.000 children, and of the spontaneous reports from their use in about 3 Mio. children, dit not reveal ethanol related side effects. For evaluation of the uptake of ethanol with food items commonly used in children, the ethanol content of these items was determined by gas chromatography. E.g. in fruit juices, up to 770 mg/L were found, in bakery products up to 1200 mg/100 g [3]. Based on these data a scenario for the mean ethanol exposure was developed, using data on nutritional habits from USA and Germany. The resulting mean ethanol exposure was 10.3 mg/kg b.w.. Assuming an exposure in the upper range of this scenario, it was 12.5 – 23.3 mg ethanol per kg b.w.. Conclusion: According to these data, the ethanol uptake with phytomedicines in children is in the same order of magnitude like everydays exposure with usual food items. From this point of view, it is conclusive that ethanol related side effects, which are not observed by the average daily ethanol intake via food, can likewise not be expected by the use of phytomedicines authorized for the use in children. The exposure to ethanol resulting from the use of these products therefore is no cause for toxicological concerns. Acknowledgments: This contribution is dedicated to Prof. Dr. Dr. h. c. mult. Heinz Schilcher, Immenstadt/Bayern, Germany, who to our great regret deceased on 17 June 2015. His textbook Leitfaden Phytotherapie [4] gives a useful summary of the subject of ethanol in herbal medicinal products for children.

References: 1. Kelber O et al. PharmInd. 2008, 70: 1124-7 2. Kelber O et al. Wien Med Wochenschr 2016, DOI: 10.1007/s10354-016-0474-x 3. Gorgus E et al. J Analyt Toxicol. 2016, DOI: 10.1093/jat/bkw046 4. Schilcher H et al. Leitfaden Phytotherapie (Elsevier Urban & Fischer) 2016

POS.210 Similarity question of nanomedicines: Physicochemical comparison of parenteral iron generic products with their innovators Schnorr, J.1; Fütterer, S.1; Langguth, P.1 1 Pharmaceutical Technology and Biopharmaceutics, J. Gutenberg-University Mainz

Parenteral iron complexes have a pivotal role in the treatment of anaemia in patients with chronical kidney diseases [1]. Although all marketed parenteral iron complexes are based on a nano-sized ferric iron oxide core surrounded by a stabilising, varying carbohydrate as gluconate, dextran or saccharate, they show clear differences in clinical [2, 3] and non-clinical studies [4-6]. Even for generic iron products with identical carbohydrate fractions, clinical differences have been reported [7]. The interchangeability of iron products is therefore questionable.The purpose of this investigation is to compare generic parenteral iron

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products with their originators. Ferrlecit® (HFeD) was tested against Sodium Ferric Gluconate Complex in Sucrose Injection (HFeD_G), Dexferrum® (FeG) against Ironate® (FeG_G). Products were compared with respect to particle size (DLS, TEM), particle and core morphology (XRPD, Mößbauer Spectroscopy,TEM), charge (ζ –potential) and in vitro liberation of labile iron in plasma (Ferrozine assay). For HFeD and HFeD differences were obtained in size (DLS), charge, particle morphology (TEM) and in vitro liberation of iron in plasma. In case of ferrozine assay HFeD_G liberated 41,8% more labile iron in the simulated 500mg dose than HFeD. FeG and FeG_G differentiate in size (DLS), particle shape (TEM) and labile iron content. Taken together, these results suggest that there may be clinical relevant differences between the tested generics and originators. References: 1. Macdougall, I. C.: J. Ren. Care 2009, 35, Suppl. 2: 8–13. 2. Hayat A.: Clin. Med. Res. 2008, 6 (3-4), p. 93–102. 3. Okam, M. M. et al.: Am. J. Hematol. 2012, 87 (11), p. 123-124. 4. Fütterer, S. et al.: J. Pharm. Biomed. Anal. 2013, 86, p. 151–160. 5. Neiser, S. et al. : Biometals 2015, 28(4), p. 615‐635 6. Spicher, K. et al.: Regul. Toxicol. Pharmacol. 2015,3 (1), p. 65–72. 7. Stein, J.; Dignass, A.; Chow, K. U.: Curr. Med. Res. Opin. 2012, 28 (2), p. 241–243. .

ANTIINFECTIVES


3.13 Antiinfectives

POS.211

Preventing new resistance development in antibiotic therapy: Which concentration of levofloxacin should Escherichia coli be exposed to? Goebgen, E. B.1; Kloft, C.1

1 Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr. 31, 12169 Berlin, Germany

Objectives: Increasing resistance to antibiotics is one of the challenges we are currently facing. In 2013, 10% to <25% of the tested isolates of Escherichia coli (E. coli), one of the pathogens known for increasing resistances, expressed resistance to fluorquinolones in Germany [1]. Thus, there is a clear need of dosing strategies which are (i) capable of eradicating the bacteria and furthermore (ii) prevent new resistance development. As predictors, determining the pharmacodynamic (PD) potency of antibiotics, critical thresholds are used. The two major thresholds are: the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), also known as 4x MIC [2]. Therefore, this in vitro study aimed for testing different thresholds for the fluorquinolone levofloxacin (LEV) for their ability to ensure eradication of the bacterial load without any regrowth. Methods: As preliminary investigation, the MIC of the clinical E. coli isolate used was determined using the standard CLSI microdilution method [3]. The main investigations were performed in an in vitro infection model (IVIM) with absence (growth control (GC)) or presence (time-kill curve (TKC)) of constant antibiotic concentrations of 1, 2, 2.5, 3, 3.5 and 4x the MIC of the clinical E. coli isolate used. The static IVIM consisted of a cell-culture flask filled with cation-adjusted Mueller-Hinton Broth (CA-MHB). At the start of the experiment, 100 µL of bacteria suspended in 0.9% NaCl were added to 8.9 mL CA-MHB and pre-incubated at 37 °C for 2 hours to yield an inoculum of 106 colony forming units (CFU)/mL at time point zero. Subsequently, 1 mL antibiotic solution or 1 mL CA-MHB was added to TKC or GC, respectively, which was followed by incubation for 24 hours. Samples (n≥6) were taken according to a predefined sampling schedule. Bacterial concentrations were determined using the droplet plate counting method, explained in [4]. The LEV concentrations were measured using a fluorescence assay at the start and at the end of the experiments [1]. All experiments were performed in triplicate and graphically evaluated with bacterial or LEV concentration plotted versus time in RStudioTM. Results: Preliminary investigations found a LEV MIC for the tested isolate of 4 µg/mL which translated into antibiotic concentrations of 4, 8, 10, 12, 14 and 16 µg/mL in the TKC. All performed GCs showed a maximum bacterial load around 1010 CFU/mL at the end of the experiments. LEV concentrations remained stable over the whole experiment, without relevant degradation. Initially, all TKC resulted in an approximately 3 log10-fold reduction of bacterial load after 2 hours. For TKC with up to 2x MIC, the initial bacterial killing was followed by regrowth which resulted in a final bacterial load of approximately 109 CFU/mL after 24 hours. After an initial reduction, the bacterial concentration of the intermediate antibiotic concentration of 2.5x MIC persisted at a concentration of 10³ CFU/mL. Antibiotic concentrations higher than 2.5x MIC were capable of fully eradicating the bacteria with bacterial concentrations of ≤10² CFU/mL after 24 hours. Conclusions: Overall the GCs were in agreement with previous results and confirmed that optimal growth conditions for the bacteria were chosen. The regrowth present in TKC with 1 and 2x MIC seemed to indicate a development of secondary resistance, which was prevented in the TKC with higher concentrations (2.5-4x MIC). Since the intermediate LEV concentration (2.5x MIC) did not fully eradicate the bacteria, a concentration of at least 3x MIC should be used to ensure both eradication and prevent further resistance development. These investigations showed that the threshold MBC is capable of preventing an increase in resistance and eradicate the bacterial load in 24 hours in an in vitro setting. Further investigations are needed to confirm this finding for the in vivo setting.

References: 1. Bartels, K. Diploma thesis (FU Berlin) 2015. 2. Abbanat, D. et al.: Antimicrobial Pharmacodynamics in Theory and Clinical Practice, 2nd ed. (Informa Healthcare USA, Inc.) 2007. 3. CLSI: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that grow aerobically (ISBN 1-56238-784-7 [Electronic]). 4. Schwalbe, R.; Steele-Moore, L.; Goodwin, A. C.: Antimicrobial susceptibility testing protocols (CRC Press) 2007.

POS.212

Influence of the peptide chain release factor methyltransferase PrmC on virulence associated metabolic pathways of Pseudomonas aeruginosa PA14 Depke, T.1; Häußler, S.2,3; Brönstrup, M.1 1 Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany 2 Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany 3 Institute of Molecular Bacteriology, Twincore, Centre for Clinical and Experimental Infection Research, Hannover, Germany

Metabolomics is an interdisciplinary field of study that applies analytical chemistry and multivariate data analysis to examine the entirety, certain subsets or characteristic profiles of small molecules (<1500Da) in biological systems under specific conditions [1]. These metabolite profiles represent a “snapshot” of cellular biochemistry that is very useful for molecular phenotyping and understanding of biochemical reactions to various stimuli. We apply a metabolomics approach using gas chromatography and ultra-high performance liquid chromatography coupled to (tandem) mass spectrometry to examine virulence and quorum sensing related metabolic processes in the opportunistic Gram-negative pathogen Pseudomonas aeruginosa. We aim to achieve a broad coverage of the P. aeruginosa metabolome that enables us to comprehensively characterise changes in metabolite levels upon knock-out of virulence associated genes such as pqsE and prmC which are potential targets for anti-virulence drugs. The peptide chain release factor methyltransferase PrmC is essential for P. aeruginosa virulence without being directly involved in virulence factor or quorum sensing signal biosynthesis. Deficiency in PrmC leads to increased stop codon readthrough and a phenotype with strongly reduced pathogenicity. It has been reported that prmC knock-out strains produce significantly lower amounts of the redox-active small-molecule virulence factors from the phenazine class while maintaining an intact quorum sensing system [2]. Our studies reveal that PrmC deficiency leads to decreased levels of alkylquinolone quorum sensing signal molecules in the cell but not in the growth medium suggesting that PrmC affects non-quorum sensing functions of alkylquinolones making it an even more interesting protein to study in the context of virulence regulation and anti-virulence strategies. Besides these new insights into the role of PrmC in P. aeruginosa virulence, we discovered several previously undescribed members of the alkylquinolone family of quorum sensing signal molecules and developed an untargeted LC-MS/MS method that allows for the simultaneous analytical characterisation of various small molecule virulence factors and quorum sensing signal molecules. Acknowledgments: This work is supported by a scholarship of the Studienstiftung des deutschen Volkes (German National Academic Foundation) providing financial and non-material support to T.D.

References: 1. Patti, G.J., O. Yanes, and G. Siuzdak: Nat. Rev. Mol. Cell Biol. 2012, 13(4): 263-269 2. Pustelny, C., et al: Environ. Microbiol. 2013, 15(2): 597-609.

POS.213/SL.46 The value of pharmacometrics in development, optimisation and clinical use of anti-infective therapies. Wicha, S. G.1 1 Dept. of Pharmaceutical Biosciences, Uppsala Universitet, Husargatan 3, 75124 Uppsala, Sweden


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POS.214

The effect of levofloxacin 500 mg bid and 750 mg qd against resistant Escherichia coli in the in vitro dynamic infection model Seeger, J.1; Goebgen, E. B.1; Kloft, C.1

Dept. of Clinical Pharmacy and Biochemistry, Institute of Pharmacy, Freie Universitaet Berlin, Kelchstr. 31, 12169 Berlin, Germany

Background and objectives: Optimising the dosing regimens of anti-infectives is one strategy to face the challenge of increasing antimicrobial resistances. Escherichia coli (E. coli) is one of the most prevalent causative pathogens for nosocomial infections of the urinary tract and the emergence of fluoroquinolone resistant strains is a widespread problem all over Europe. Still, levofloxacin (LEV) is a commonly used agent for the treatment of complicated urinary tract infections (UTIs). For the treatment of severe UTIs, known to be caused by less susceptible strains, a 500 mg twice a day (bid) dosing regimen is recommended [1]. However, a dosing regimen of 750 mg once a day (qd) is used in the US [2]. This in vitro investigation aimed to explore whether a 750 mg qd LEV regimen is superior to a 500 mg bid LEV regimen in eradicating resistant E. coli and preventing the emergence of new resistances. For this, the in vitro dynamic infection model (IVIM) was used to imitate human pharmacokinetic (PK) profiles and to observe their pharmacodynamic (PD) effects, such as time-kill behaviour and emergence of resistances. Material and methods: The used IVIM was developed and validated for LEV against E. coli at our department [3, 4]. To mimic the concentration-time (C-t) profiles of LEV in urine resulting from a 500 mg bid and a 750 mg qd regimen, an adapted previously published LEV PK-model was used [5]. The experimental settings, such us pump rates, have been calculated in RStudio™. After 2 hours of pre-incubation, E. coli was exposed to the two different LEV C-t profiles for 24 hours. Samples were taken at predefined time points in order to determine LEV concentrations by a fluorescence assay [4]. For simultaneous bacteria quantification, an automatic plate counting method using the schuett colonyQuant® was developed and validated according to the EMA guideline on bioanalytical method validation [6]. In preliminary investigations and after each experimental run, the minimal inhibitory concentration (MIC) of the isolate or the surviving bacteria, respectively, was assessed using the microdilution method. Results: The automatic counting method was successfully validated in agreement with the EMA guideline for up to 100 colony forming units of E. coli, but failed for higher colony counts. Therefore manual counting was used for bacteria quantification. The initial MIC of the investigated E. coli strain was 8 mg/L, while the MIC increased for bacteria which survived the exposition to the 500 mg bid regimen. The used experimental settings were able to mimic the C-t profiles of the two dosing regimens. In both cases, C was achieved after 2 – 4 hours and LEV was able to initially reduce the bacteria load about ≥ 3log10-fold. However, in the 500 mg bid regimen, bacterial eradication was followed by regrowth. On the contrary, the C-t profile resulting from the 750 mg qd regimen inhibited the bacterial growth for the entire 24 hours. Conclusion: Discrimination between the efficacy of the investigated C-t profiles in eradicating resistant bacteria and preventing the development of secondary resistance was possible by mimicking them in the IVIM. A 500 mg bid LEV regimen was not fully capable of eradicating a resistant E. coli. The observed regrowth and the increase in MIC value after the performed experiment indicates the emergence of secondary resistance mechanisms, such as the expression of efflux pumps. The investigation points towards a superiority of the 750 mg qd LEV regimen in preventing the development of secondary resistances. Therefore, the once a day dosing of the high LEV concentration seems to be a promising dosing regimen for further investigations in the in vivo setting. References: 1. Pea, F. et al.: Journal of Chemotherapy 2003, 15 (1120-009X): 563-567. 2. Anderson, V. R., Perry, C. M.: Drugs 2008, 68 (4): 535-565. Gloede-Michael, J.: Pharmacodynamic in vitro studies contributing to the rational use of linezolid in infections by vancomycin resistant Enterococcus faecium. Diss. 2011. 3. Bartels, K.: Validierung des dynamischen In-vitro -Infektionsmodells zur Bestimmung des pharmakodynamischen Effekts von Levofloxacin auf Escherichia coli. Diss. 2015. 4. Schaeftlein, André: Neue Wege in der Modellierung von Mikrodialysatdaten im Menschen : Charakterisierung der klinischen ADMER-Prozesse von Moxifloxacin , Levofloxacin und Linezolid in Gesunden und Hochrisikopopulationen. Diss. 2013.

5. EMEA, Committee for Medicinal Products for Human Use: Guideline on bioanalytical method validation. 2012, 44 (July 2011).

POS.215/SL.12 Inhibitors of the bacterial translocase MraY as potential novel antibiotics Ducho, C.1

1 Saarland University, Department of Pharmacy, Pharmaceutical and Medicinal Chemistry, Campus C2 3, 66123 Saarbrücken, Germany


POS.216

Antimicrobial β2,2-amino acid derivatives eliminate Staphylococcus aureus biofilms by membrane disruption and biomass removal Ausbacher, D.1; Lorenz, L.2; Goeres, D. M.2; Stewart, P. S.2; Strøm, M. B.1; Vuorela, P. M.3; Fallarero, A.3 1 Natural Products and Medicinal Chemistry Research Group, Department of Pharmacy, UiT - The Arcitc University of Norway, Tromsø, 9037, Norway 2 Center for Biofilm Engineering, Montana State University, Bozeman, Mt, 59717, USA 3 Pharmaceutical Design and Discovery Group, Division of Pharmaceutical Biosciences, University of Helsinki, Helsinki, 00014, Finland

Bacterial biofilms account for up to 80% of all human microbial infections (1). In patients, medical devices like catheters, artificial heart valves or prosthetic joints are prone to bacterial attachment and the development of biofilms. Antibiotic treatment of biofilm infections is challenging and high intrinsic tolerance to antibiotics leads to insufficient reduction of the biofilm, recurrence of infection and increased risk for the development of resistance (2). Promising compounds for fighting biofilm bacteria are antimicrobial peptides due to their rapid and membrane disruptive mode of action. However, antimicrobial peptides have the disadvantage of being easily degraded by proteolytic enzymes of microbes or the human body. Based on the pharmacophore of short antimicrobial peptides, we have synthesized small amphipathic β2,2-amino acid derivatives (3). These derivatives have a molecular weight below 500 Da, are proteolytically stable and potent against gram-positive bacteria including antibiotic resistant strains like MRSA and MRSE. The activity spectrum also comprises Staphylococcus aureus (S. aureus) biofilms and both in vitro and microscopy studies showed that derivatives A1 - A3 are promising lead derivatives (4).

In the present study, we investigated how our small, antimicrobial peptide mimicking derivatives act on S. aureus biofilms. We used 96-well plate based assays and fluorescence microscopy to investigate kill and reduction of biofilm bacteria treated with A1 - A3. Furthermore, we applied a CDC biofilm reactor system to prepare biofilms of a green fluorescent protein (GFP) expressing S. aureus strain on glass coupons for flow cell studies. We constructed a dye leakage indicator system by utilizing expression of the 27 kDa GFP and the small dye filmtracerTM calcein-red (Mw 790 Da). Real-time confocal laser scanning microscopy was used to observe the impact of different treatments including our lead derivative A3. Our results indicated that the β2,2-amino acid derivatives both kill and remove S. aureus biofilms. Observations of membrane disintegration in biofilm bacteria and the dye leakage experiments suggested that treatment results in membrane collapse without preceding formation of pores. The high potency based on kill and removal of biofilm bacteria make β2,2-amino acid promising lead molecules to fight recalcitrant biofilm infections.

ANTIINFECTIVES


Acknowledgements: The authors thank Betsey Pitts for excellent technical assistance. The project was supported by the Research Council of Norway “fellesløftet” grant 214493/F20 and the personal overseas research grant for D.A.

References: 1. Römling, U., Balsalobre, C., J. Intern. Med. 2012, 272(6): 541-561. 2. Stewart, P.S. Pharmaceuticals, 2015, 8(3): 504-511. 3. Hansen, T., et al. J. Med. Chem., 2011, 54(3): 858-868. 4. Ausbacher, D. et al. Biofouling, 2014, 30(1)

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3.14 Neurological Disorders

POS.217

Knockdown of glucose-regulated protein 75 (Grp75) protects against glutamate toxicity in neuronal HT22 cells

Honrath, B.1; Culmsee, C.1; Dolga, A.2 1 Institute of Pharmacology and Clinical Pharmacy, University of Marburg, Karl-von-Frisch Strasse 1, 35043 Marburg, Germany 2 Department of Molecular Pharmacology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands

Glucose-regulated protein 75 (Grp75) is a protein with multiple cellular functions that has been linked to neurodegenerative disorders such as Parkinsons’ disease [1], and in tumor pathology as a prognostic marker for the differentiation of neuroblastoma [2]. Grp75 is ubiquitously expressed and is localized in the cytosol, at the endoplasmic reticulum and in the mitochondria [3]. It has been associated with the mitochondrial protein import machinery and mitochondrial integrity, cell death induction, activation of MAPK signaling pathways, and the formation of ER-mitochondrial contact sites. In tumor and neuronal cell lines, Grp75 depletion was shown to induce oxidative stress and mitochondrial fragmentation, and to increase the sensitivity to various cell death inducers [1, 4, 5, 6]. On the contrary, knockdown of Grp75 was also shown to be protective against α-synuclein toxicity in neuronal cells [7]. Thus, the exact function of this protein in different types of cells remains elusive. In this study, we investigated effects of genetic Grp75 ablation in neuronal HT22 cells challenged with toxic extracellular glutamate concentrations. Following glutamate exposure, we analyzed cell viability by real-time cellular impedance measurements, evaluated mitochondrial morphology, and investigated mitochondrial parameters such as mitochondrial ROS formation or mitochondrial calcium overload using specific fluorescent dyes for flow cytometric analysis. We show that siRNA-mediated knockdown fully preserved cell viability and blocked major hallmarks of glutamate-induced mitochondrial damage such as mitochondrial fragmentation, the formation of mitochondrial ROS and mitochondrial calcium overload. Our results indicate a distinct role for Grp75 in the response of HT22 cells to glutamate-induced oxidative stress and highlight its neuroprotective potential. References: 1. Burbulla, L. et al.: J. Hum. Mol. Gen., 2010, 19(22): 4437-4452. 2. Hsu, W. et al.: J. Clinical Cancer Res., 2008, 14(19): 6237-6245. 3. Ran, Q. et al.: Biochem. Biophys. Res. Comm., 2000, 1: 174-179. 4. Burbulla, L. et al.: Cell death & disease, 2014, 5:e1180. 5. Jin, J. et al.: Mol Cell Proteom, 2006, 5(7): 1193-1204 6. Kawai, A. et al.: J Cell Sc, 2001, 114: 3656-3574. 7. Liu, F.-T. et al.: Brain Res, 2015, 1604: 52-61.

POS.218/SL.08 Noradrenergic and serotonergic compounds to target narcoleptic episodes in a mouse model Schmidt, C.; Leibiger, J.; Fendt, M. For abstract see Short Lecture SL.08

POS.219

Oxidative stress activates mechanisms of regulated necrosis via CYLD

Eisenbach, I.1; Hoffmann, L.1; Ganjam, G. K.1; Culmsee, C.1 1 Institute for Pharmacology and Clinical Pharmacy, Biochemical-Pharmacological Center Marburg, University of Marburg, Karl-von-Frisch Straße 1, 35032 Marburg, Germany

Oxidative stress is regarded as a major trigger for neuronal dysfunction and death in the ageing brain and in multiple neurodegenerative disorders. How oxidative stress mediates neuronal death and whether the associated mechanisms are accessible for therapeutic intervention strategies is not clarified. Increasing evidence suggests, however, that

oxidative stress triggers molecular mechanisms of regulated necrosis that involve the activation of receptor interacting protein 1 (RIP1) independently of death receptor activation. Here, we show in neuronal HT-22 cells that erastin-mediated formation of reactive oxygen species (ROS) [1] triggers mechanisms of regulated necrosis independent of TNF-signaling. In this model system of ferroptosis, erastin promotes glutathione depletion and lipid peroxidation followed by activation of RIP1 and subsequent RIP1-RIP3 necrosome formation which is regarded as a hallmark of regulated necrosis [2]. Silencing of RIP1 by siRNA or by the RIP1 inhibitor necrostatin-1 prevented erastin-induced cell death. In contrast, the ferroptosis inhibitor ferrostatin-1 failed to protect cells against TNF-induced classical necroptosis, a form of programmed cell death that is mediated RIP-kinases downstream of death receptor activation (e.g. tumor necrosis factor receptor TNFR) [2, 3]. Recently, a genome-wide siRNA screen linked cylindromatosis (CYLD) to RIP1/RIP3-dependent necroptosis [4]. In the present paradigm of ferroptosis, CYLD depletion promoted neuronal survival and decreased RIP1-RIP3 complex formation. Furthermore, increasing evidence suggests a role of mitochondria in erastin-induced cell death pathways. Here, we revealed the involvement of mitochondrial fission regulating dynamin-related protein 1 (DRP-1) since genetic depletion or pharmacological inhibition of DRP-1 protected HT-22 cells against erastin toxicity. Additionally, knockout of CYLD via the CRISPR/Cas9 system prevented erastin-induced DRP-1 translocation to mitochondria, suggesting that CYLD and DRP-1 can link mechanisms of regulated necrosis to mitochondrial pathways of cell death in paradigms of oxidative stress. References: 1. Dixon SJ et al., Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012, 149(5): 1060-72 2. Vandenabeele P et al., Molecular mechanisms of necroptosis: an ordered cellular explosion. Nature Rev. Mol. Cell Biol. 2010, 11: 700-714 3. Vanden Berghe T et al., Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat Rev Mol Cell Biol. 2014, 15 (2): 135-147 4. Hitomi J et al., Identification of a molecular signaling network that regulates a cellular necrotic cell death pathway. Cell. 2008, 135: 1311-1323

DRUG SCREENING


3.15 Drug Screening

POS.220

TiDEC: Enabling Chemistry for DNA-encoded Small Molecule Screening Libraries Brunschweiger, A. Department for Chemistry and Chemical Biology, TU Dortmund, Otto-Hahn-Str. 6, 44227 Dortmund, Germany

DNA-encoded small molecule screening libraries (DELs) have found widespread use in drug research.1 DELs consist of DNA-encoded molecules. These are chimeric structures composed of a drug-like compound connected to a PCR-amplifiable DNA strand that identifies the small molecule by its sequence. Encoding with DNA allows for pooling of large numbers of molecules and to screen them efficiently for bioactive compounds by selection. DELs are synthesized through alternated organic preparative chemistry and encoding steps performed in combinatorial manner. Chemical methodology applied to the synthesis of DELs must be strictly DNA-compatible, and is currently restricted to mostly carbonyl reactions such as amide bond formation, Pd-catalyzed C-C cross-coupling reactions, and “click” reactions.1,2 This restriction defines a challenge in the field: The development of synthesis methodology for DELs is desperately needed to expand chemical space covered by these libraries.1 Rich sources of drug-like heterocyclic structures are Brønsted acid-, and transition metal ion-catalyzed reactions. However, strong acids cleave the glycosidic bond of purine nucleosides, and also many transition metal ion catalysts cause depurination through a tautomery-based mechanism. Thus, we developed a DEL synthesis strategy based on a hexathymidine sequence, called “hexT”, serving as an adapter oligonucleotide: TiDEC, oligothymidine-initiated DNA-Encoded Chemistry.3 This adapter tolerated a surprisingly broad spectrum of catalytic systems, and allowed for subsequent ligation of coding DNA sequences. Among the catalysts that are now available for DEL synthesis are acidic organocatalysts, and transition metal ions such as Au(I), Ag(I), and Cu(I). Our strategy opened access to substituted and functionalized heterocyclic scaffold structures as DNA-conjugates from simple, readily available starting materials. For example, application of transition metal catalysts furnished DNA-heterocycle conjugates through [3+2] cycloaddition reactions. Some of the newly synthesized DNA-conjugated heterocycles display structural motifs found in natural products, while others represent core structures of clinical candidates or approved drugs. All heterocyclic scaffolds were designed to enable subsequent DNA-encoded combinatorial library synthesis by well-described, robust reactions.

The TiDEC approach to DNA-encoded libraries: a) conjugation of starting materials to the hexT adapter by amide bond formation; b) heterocycle formation by acid or coinage metal catalysis; c) cleavage, purification, and characterization of the products; d) encoding by T4 DNA ligation. References: 1. Salamon, H. et al.: ACS Chem. Biol. 2016, 11(2):296-307. 2. Klika-Skopic, M. et al.: MedChemComm, 2016, DOI: 10.1039/C6MD00243A. 3. Brunschweiger, A. et al.: Eur. Pat. Appl. 15202448.5.

POS.221

Simultaneous MS Binding Assays for Monoamine Transporters Neiens, P.1; De Simone, A.2; Höfner, G.1; Wanner, K. T.1 1 Department Pharmazie – Zentrum für Pharmaforschung, Ludwig-Maximilians-Universität München, Butenandtstr. 5 - 13, 81377 Munich, Germany 2 Department for Life Quality Studies, Alma Mater Studiorum – University of Bologna, Corso D’Augusto 237, 47921 Rimini, Italy

Binding assays are an important tool in the early drug development process to characterize the affinity of a compound towards a biological target. In classic binding assays, radioligands that can be quantified by means of scintillation counting are used as reporter ligands. This procedure offers a high sensitivity, but no selectivity as only the radioisotope is detected. Recently, MS Binding Assays, a powerful alternative to radioligand binding assays employing unlabelled reporter ligands, which are quantified by LC-MS/MS, have been established. The strategy of MS Binding Assays has already been applied successfully for a variety of targets, such as GPCRs and neurotransmitter transporters [1, 2, 3]. Besides its high sensitivity, the use of mass spectrometry for the detection of reporter ligands also provides an unrivalled selectivity that can be used to quantify multiple reporter ligands simultaneously. In this study, we want to present an MS Binding Assay for the simultaneous characterization of binding affinities at the three monoamine transporters hDAT, hNET, and hSERT, which are important targets for drugs addressing affective disorders. An important requirement for the simultaneous MS Binding Assays is the use of reporter ligands which label the target selectively even in the presence of related targets. Therefore, 4-(2-benzhydryloxyethyl)-1-(4-fluorobenzyl)piperidin-3-ol (D-84) [4] was chosen as a reporter ligand for hDAT, (+)-(S,S)-Reboxetine for hNET [5], and (S)-Citalopram for hSERT [6], respectively. For these three markers an LC-ESI-MS/MS quantification method was developed and validated according to the FDA guideline for bioanalytical method validation. In the binding assay, membrane fractions made from HEK cells stably expressing the targets were incubated simultaneously with the three markers and competitors if needed. After the incubation, the target-marker complexes were separated from the unbound markers by a simple filtration step. The bound markers were then liberated from the target and quantified with the established LC-MS/MS method.

With this approach, we were able to carry out saturation experiments to characterize the binding affinities of D-84 towards hDAT, of (+)-(S,S)-Reboxetine towards hNET and of (S)-Citalopram towards hSERT simultaneously in presence of all three targets. Analogously, it was possible to determine the binding affinities of a competitor towards each one of the three monoamine transporters together in a single experiment. The binding affinities of the three markers as well as the ones of test compounds employed as competitors determined in this way, were in good agreement with literature data. These results prove that MS Binding Assays enable the characterization of binding affinities at multiple targets simultaneously. References: 1. Zepperitz, C.; Höfner, G.; Wanner, K.T.: ChemMedChem 2006, 1(2): 208 – 217. 2. Grimm, S.H.; Höfner, G.: Wanner, K.T.: ChemMedChem 2015, 10(6): 1027 – 1039. 3. Massink, A. et al.: Purinergic Signal. 2015, 11(4): 581 – 594. 4. Ghorai, S.K. et al.: J. Med. Chem. 2003, 46(7): 1220 – 1228. 5. Hajós, M. et al.: CNS Drug Rev. 2004, 10(1): 23 – 44. 6. Hyttel, J. et al.: J. Neural. Trans. 1992, 88(2): 157 – 160.

POS.222

Compound screening and assay development for sirtuins Swyter, S.1; Rumpf T1; Enzensperger, C.2; Einsle, O.3; Jung, M.1

1 Institut für Pharmazeutische Wissenschaften, Albert-Ludwigs-Universität, Albertstraße 25, 79104 Freiburg, Germany 2 Institut für Organische Chemie und Makromolekulare Chemie, Friedrich-Schiller-Universität Humboldtstr. 10, 07743 Jena, Germany

3 Institut für Biochemie, Albert-Ludwigs-Universität, Albertstraße 25, 79104 Freiburg, Germany

Sirtuins are an evolutionary conserved family of NAD+-dependent Lysine deacylases (KDAC) [1]. There are seven human isotypes which differ in their subcellular localization, their enzymatic activities as well as in their deacylation substrates. The main enzymatic activity of Sirt1-3 is the deacetylation, but also the deacylation of longer fatty acids has been observed. They deacylate a wide range of protein substrates such as p53, α-Tubulin or Acetyl-CoA-Synthetase. A dysregulation of the

POSTERS


cellular acetylation level has been associated with human diseases e.g. cancer, neurodegerenative and metabolic diseases which makes a modulation of sirtuin activity a promising strategy for pharmaceutical intervention [2, 3], For the screening of potential modulators of sirtuins, a well functional assay system feasible for high throuput-screening (HTS) is indispensable. So far, there are some well-established assay systems for measuring the binding or activity potential of sirtuins in complex with ligands. However, all these protocols deal with labeled substrate or are challenging in the performance for HTS [4, 5]. Here we present the development of a homogeneous, label free assay bases on a peptide substrate for Sirt2 & 3. It is performable for the measurement of the deacylation activity of acetylated and myristoylated peptides. To prove the comparability of this assay we screened indole derivatives bases on EX-527 [6] in comparison to a well-known assay and show insights into the binding of a discovered analogue of EX-527. Acknowledgments: We thank the DFG for funding (RTG 1976, http://www.cofactor-diversity.uni-freiburg.de/RTG)

References: 1. Trapp J, Jung M, Curr. Drug Targets, 2006, 7(11): 1553-60 2. Hoffmann G et al, J. Biol. Chem., 2014, 289(8): 5208-16 3. Feldman JL, Baeza J, Denu JM, .J Biol. Chem., 2013, 288(43): 31350-6 4. Heltweg B, Trapp J, Jung M, Methods, 2005, 36(4): 332-7 5. Schiedel M, et al, J. Biomol. Screen., 2015, 20(1): 112-21 6. Napper A D, et al, J. Med. Chem., 2005, 48(25): 8045-54

OTHER TOPICS


3.16 Other Topics

POS.223

Doctoral theses of pharmacists in the 19. century using the example of the Ludoviciana in Gießen Sakkas, A.-S.1; Friedrich, C. 1 Institute for the History of Pharmacy, Philipps-Universität Marburg, Roter Graben 10, 35032 Marburg, Germany

To obtain a doctoral degree was an ambitious goal for the pharmacists of their time, not only in order to leave their craft for science but also as strive for a fulfilled life. Not to stay away too long from their normal business, pharmacists might as well be able to achieve the desired degree by providing a handwritten or already printed treatise in absentia. For more than 80 years (1781–1860) the University of Gießen was exactly the centre for this kind of doctoral degrees. This poster explains in short the promotion practise at the time of Justus Liebig (1803–1873), who was ordinary in chemistry from 1825 to 1852 in Gießen. For scholars of Liebig, it was absolutely sufficient to provide an essay printed in a scientific journal yet. For example Wilhelm Mettenheimer (1802-1864) who had been a participant in the pharmaceutical laboratory of Liebig, obtained his doctoral degree with the already published report ‘Chemische Untersuchung der Soole zu Theodorshalle bei Kreutznach‘. Justus Liebig himself confirmed the relevance of the results, because the investigation was executed under his scientific surveillance. Mettenheimer however, as free citizen of Frankfurt (Main), didn’t have to dispute, similar to Carl Leverkus (1804-1889). Karl Friedrich Oppermann (1805-1872) and Friedrich Kodweiß (1803-1866), as well as Mettenheimer, wanted to be examined in chemistry and natural sciences. While three years ago, the question was discussed, whether it would be possible to obtain the philosophical doctoral degree with only examination subjects in the field of natural sciences, it was no longer a big problem with Oppermann and Kodweiß. The candidates in question however, caused a stir with their wish for a mutual examination. Synchronized examinations were approved of all professors, as neither legal custom, nor relevant rules stood against them. The early practise to doctorate on selected universities like Gießen, was a good opportunity for pharmacists to get a PhD without high school graduation and partially in absentia. References: 1. Universitätsarchiv Gießen, Phil O 18, 1827. Die Prüfung und Doctorpromotion des Pharmaceuten J. Fr. Wilhelm Mettenheimer aus Frankfurt a. M. betr[effend]. 2. Universitätsarchiv Gießen, Phil O 18, 1830. Betr[ifft] Promotionen 1830. 3. Mettenheimer, Friedrich von: Die Geschichte der Familie Mettenheimer. Frankfurt am Main 1897. 4. Friedrich, Christoph: Apotheker Carl Leverkus. In: Pharmazeutische Zeitung 149 (2004), S. 3864–3866. 5. Schwinges, Rainer Christoph (Hrsg.): Examen, Titel, Promotionen. Akademisches und staatliches Qualifikationswesen vom 13. bis zum 21. Jahrhundert. Basel 2007 (Veröffentlichungen der Gesellschaft für Universitäts- und Wissenschaftsgeschichte; 7).

POS.224

History of German 'Pharmacy Visitation' (Apothekenvisitation) from the Mediterranean beginnings in the 13th century to the end of Second World War Horstmann, R. D.1; Friedrich, C.1 1 Institute for the History of Pharmacy, Philipps-Universität Marburg, Roter Graben 10, 35032 Marburg, Germany

Until nowadays, pharmacy supervisions in the form of (mostly) unexpected ‘Visitations’ are still part of German pharmacies workaday life. This special kind of control is one of the oldest sections of European government administrations and accompanied pharmacists all along ever since the ‘Constitutions of Melfi’, decreed between 1231 and 1241 by Friedrich II (1194–1250), sovereign of the Kingdom of the Two Sicilies, who initialised the legitimate separation of physicians and pharmacists. Originally intended to control their implementation, these regulatory decrees became the basis for an official supervision of

pharmacies, which was first managed by special magistrates, subsequently by physicians and finally by pharmacists. The aim of this dissertation was to investigate, compare and analyse the historical development of ‘Visitationes’, which spread in the form of regularities for pharmacists and monitoring their compliance over Mediterranean metropolises like Venice, Marseille or Avignon to German speaking territories, where mainly free imperial cities like Nuremberg, Munich or Cologne enacted comparable instructions for pharmacists. The spreading of this supervision, its configuration and organisation was analysed in more than 50 geographical sites, covering the time between the 14th and 18th century, separated in intervals of a hundred years each. In this way a differentiated, tesselated picture of pharmacists and 'Visitation' legislation arose, outlining not only the structure of authorities, frequency and organisation of revisions, but also practical arrangement and assignment of tasks meaning controlling of licence, certificate of proficiency, appropriateness of staff, trainees level of knowledge just as local orderliness by total inspection of property, check-up of instruments, dispensatories and taxes and valid printed regulations for pharmacists. Above all of prime importance was the control of quality and identity of repository and the correct filling in and configuration of the visitation record. The analysis of the supervision situation of pharmacies in many different German towns and regions until the end of the 18th century was central point of the project initially, whereas with the beginning of the 19th century the Prussian conditions of visitation became particularly relevant. Based on archive materials of ‘Rheinprovinz’, the new Prussian province after 1815, intensive research work was conducted concerning the organisation and execution of pharmacy 'Visitation' in Prussia until the end of World War Two, always applying the earlier notified system of analyzing bit by bit the historical facts of this topic, which is characterised by a large range of details.

POS.225

Rudolf Schmitz - From being a student of pharmacy to having become the first full professor of the history of pharmacy Löhnert, A.1; Friedrich, C.1 1 Institute for the History of Pharmacy, Philipps-Universität Marburg, Roter Graben 10, 35032 Marburg, Germany

The poster describes the scientific career of the life of the pharmacist-historian Rudolf Schmitz (1918–1992). During the research titled “Rudolf Schmitz (1918–1992) and his scientific school in Marburg” it has to be made clear inhowfar Rudolf Schmitz has succeeded in founding a scientific school of the history of pharmacy in Marburg. Five aspects according to the methodic analysis of schools of pharmacy by Christoph Friedrich are to be examined. These are the headmaster, the scientific research programme, the pupils, the style of work as well as the scientific and social appreciation. Among these the headmaster and his scientific research programme are the most important points of this examination. Biographical data as well as the scientific progress of Rudolf Schmitz at the Philipps-University Marburg are presented here. A description of his childhood and his time at different schools in North Rhine-Westphalia during the 1920s and 1930s, of his studies and graduation in the 1940s and 1950s up to his postdoctoral lecture qualification in the year 1957 and his nomination to the first full professor for the history of pharmacy in 1967 should give a deeper insight. So this poster tries to give a summary of the way that Rudolf Schmitz had taken during his lifetime period being a student of pharmacy up to becoming the first full professor of the history of pharmacy and today being one of the most important and acknowledged pharmacist-historians of the 20th century.

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POS.226

Patient experience with a Althaea officinalis L. root extract in dry cough – results from a pharmacy survey during cough season 2015/2016 Fink, C.1; Elsässer, P.1; Rabini, S.1; Abdel-Aziz, H.1 1 Pytomedicines Supply and Development Center, Innovation & Development, Bayer Consumer Health, Steigerwald Arzneimittelwerk GmbH, Havelstraße 5, 64295 Darmstadt, Germany

Measuring the customer experience and satisfaction is the key factor to customer knowledge and understanding. Especially in the area of non-prescribed over-the-counter (OTC) medication, the experiences and needs of customers are the key resource for information. A cough syrup with aqueous extracts of Althaea officinalis (STW42) is available on the German market for more than 15 years. After a successful survey on consumer experience with a solid dosage form (lozenges) containing Althea officinalis extract [1], the aim of this project is to collect data on the liquid syrup. Cough patients were recruited directly at the point of sales. If patients were willing to participate in the survey, a questionnaire for the acquisition of data was handed over by the pharmacist. The data collected were demographic data, usage patterns, customer recommendation and satisfaction, general assessment. Additionally, effectiveness in treating cough and cough related symptoms as well as tolerability were evaluated. Acceptance of the survey by the pharmacists was good: Patients were recruited in 152 German pharmacies during the cough and cold season 2015/2016. 561 patients returned the questionnaire. More than 90% of the patients complained about cough during daily life and work. Most subjects started the therapy with STW42 after duration of cough of 3-6 days (45.8%). Nearly 44% of the patients were repurchasers and 68% used the Althaea officinalis extract as only medication to treat the cough. Most subjects (78.5 %, n = 405) got knowledge of STW42 from their pharmacist. 17.8 % (n= 92) of the subjects used STW42 syrup due to previous good experience. 67.3 % (n = 346) of the subjects were female. The average age was 40.7 ± 16.3 years. The duration of treatment was recorded for up to 7 days according to the questionnaire. The highest proportion of subjects (34%, n = 165) took the syrup for a period of 7 days. 24.3% (n= 118) took the syrup for 4 days. The average dose per day was 31.9 mL. 70.3% of the subjects used the Althaea officinalis extract as only medication to treat their dry cough. 91.7% of the subjects discontinued the therapy with STW42 because they were free of symptoms. 81.4% (n= 394) of the subjects reported an improvement of the symptom "irritative cough“ until the end of their therapy with STW42. The symptoms "scratching in the throat" and "pain in the throat" improved by 74.4% and 68.3%, respectively. 305 (72.4%) subjects reported that the feeling of dryness in the throat improved, too. 58.6 % of subjects perceived an alleviation of their symptoms within 10 minutes after taking STW42. Most subjects (42%) reported duration of action between 2-4 hours. Most subjects assessed the global effectiveness of the syrup as either “very good” (29.1 %, n = 150) or “good” (54.6 %, n = 281). The majority of subjects assessed the tolerability of STW42 as either “very good” (56,9 %, n = 285) or “good” (41,9 %, n = 210), and only 2 subject assessed the tolerability as “bad”, no subject as “very bad”. According to these results more than 84.9% of the subjects assessed their global satisfaction with STW42 as either "very pleased" or “pleased". Before treatment with STW42 the condition of the subjects due to the disease was assessed as "good" by 8.4% and after the therapy by 64.2%. 87.1% (n= 446) would buy the syrup again. Collecting consumer data via pharmacy surveys occurs as a suitable tool for receiving important insights in customer experience with OTC products. The results reveal a very good acceptance and evaluation of STW42: All cough and cough related symptoms improved well over the treatment period of 7 days. The outcome of this study well reflects application of STW42 and indicates its high value in the treatment of dry cough. References: 1. Rabini, S. et al.: Planta med. 2015, 81: 1424.

POS.227

The pharmacy sector in Baden-Württemberg from 1945 to 1961 Denninger, I.1; Friedrich, C.1 1 Institute for the History of Pharmacy, Philipps-Universität Marburg, Roter Graben 10, 35032 Marburg, Germany, [email protected]

The end of World War II and the division of Germany into different occupation zones meant serious consequences for the German pharmacy sector. The legislation for the pharmacy sector passed on to the federal states which were initially dependent in their legislative measures on the respective occupying powers. The introduction of unlimited freedom of establishment in the American occupation zone spread great unrest amongst the German pharmacists. This issue remained at the center of intensive discussions for many years. The reaction of the French military government on the decree of the freedom of establishment in the American zone was intently observed by the pharmacists in Baden. The problems of pharmacies in postwar Germany were very different ones, depending on the location. In the cities they suffered heavily from the war-related destruction, while in rural areas the supplies of finished medical products and raw materials for the recipes were difficult to maintain. The biggest challenge for the owner of the International Pharmacy in Karlsruhe was the reconstruction of completely destroyed pharmacy. the operation initially continued in a neighboring building as a emergency pharmacy. The required medicines, serums, vaccines, baby food, sugar, oil and alcohol could immediately be procured from the close manufacturers and wholesalers. The Rats-Apotheke in Waldshut, located far off any pharmaceutical wholesaler had always been producing a major part of their medicines. For this purpose, the pharmacy owned a large stock of raw materials owned and operated its own medicinal plant garden. References: Literature is available from the author on request.

POS.228

Continuous exposure to fine particulate matter (PM2.5) from biomass combustion induces stress response, autophagy and cell death in human bronchial epithelial BEAS-2B cells Dornhof, R.1; Maschowski, C.2; Osipova, A.1; Gieré, R.3; Merfort, I.1; Humar, M. 1 1 Institute of Pharmaceutical Sciences, Pharmaceutical Biology and Biotechnology, Albert-Ludwigs University Freiburg, 79104 Freiburg, Germany 2 Institute of Earth and Environmental Sciences, Albert-Ludwigs University Freiburg, 79104 Freiburg, Germany 3 Department of Earth and Environmental Science, University of Pennsylvania, 19104-6316 Philadelphia, USA

Fine particulate matter (PM2.5) from biomass combustion can adversely affect human health [1,2]. However, cellular effects have remained elusive. In the present study potential molecular mechanisms were investigated after intermediate- and long-term exposure of human bronchial epithelial BEAS-2B cells to PM2.5. We observed a three-phase response: in the initial phase PM2.5 did not affect cellular survival or proliferation. However, it triggered an activation of the stress response p38 MAPK which, along with RhoA GTPase and HSP27, mediated morphological changes in epithelial cell monolayers, including actin cytoskeletal rearrangements and paracellular gap formation. The p38 inhibitor SB203580 prevented phosphorylation of HSP27 and ameliorated morphological changes. During an intermediate phase, PM2.5 triggered proliferative regression and activation of an adaptive stress response necessary to maintain energy homeostasis, including AMPK, repression of translational elongation, and autophagy. In the final phase of exposure, accumulation of intracellular PM2.5 promoted lysosomal destabilisation and cell death, which was dependent on lysosomal hydrolases, p38 MAPK and included caspase-3 activation, but not the inflammasome and pyroptosis. All these events may impair epithelial barriers and contribute to PM-associated lung and systemic diseases.

OTHER TOPICS


Acknowledgments: The presented work is part of an interdisciplinary EU-funded research project (see http://www.biocombust.eu), supported in part through the Interreg IV Program “Oberrhein” (project C35 BIOCOMBUST).

References: 1. Lelieveld, J. et al.: Nature 2015, 525(7569): 367-371. 2. Smith, K.R. et al.: Annu Rev Public Health 2014, 35: 185-206.

POS.229

Oxycodon – a hundred year’s history Unger, M.; Helmstädter, A. Institute of Pharmaceutical Chemistry, Goethe University, Frankfurt, Germany

Exactly a hundred years ago, in 1916, the opioid derivative oxycodon was synthesized by the Jewish chemists Martin Freund (1863-1920) and Edmund Jakob Speyer (1878-1942) at the University of Frankfurt. Freund, head of the chemical laboratory of the “Physikalischer Verein” in Frankfurt since 1895, became professor of chemistry at the newly founded Goethe University in 1914. Speyer who did his PhD in 1901 at the University of Heidelberg under the supervision of Emil Knoevenagel, qualified as ‘Privatdozent’ in Frankfurt (1915, habilitation thesis Beiträge zur Kenntnis des Thebains und seiner Derivate) and worked together with Freund very closely. However, he became Professor not before 1932. In 1933 already, he lost this position due to his Jewish faith and eventually he died in the Lodz Ghetto where he had been deported to, in 1942. From April 1917 onwards, oxycodon was marketed as a substitute for morphine under the trade name Eukodal by the company E. Merck, Darmstadt. The product became immediately successful despite its great potential to induce addiction. In was marketed until 1990. Interestingly, in recent years the substance was re-introduced into the German marked as an analgesic, also in combination with naloxone for the prevention of opioid-induced obstipation. After 100 years, the substance is still a corner-stone of pain treatment as being listed in the WHO treatment guidelines. Acknowlegements: We sincerely thank the Division Corporate History, E. Merck, Darmstadt (Head: Dr. Sabine Bernschneider-Reif) for supporting this study.

POS.230

The Institute of experimental therapy The first research department of the Marburgian Behringwerk The construction work on Emil von Behring´s (1854–1917) private laboratory began in 1896 on the Schlossberg of Marburg. Besides the academic works, Behring dedicated his laboratory to his private research in particular in the field of tuberculosis. A significant part of the institute´s financing was drawn from a contract on Behring´s innovation development Diphtherieheilserum (diphtheria serum) with the “Farbwerke Hoechst vorm. Meister Lucius & Brüning“. In 1903 Behring entered into contract with the Marburg-based company Dr. Siebert & Dr. Ziegenbein on production and distribution of tetanus serum and vaccine against bovine tuberculosis. A year later he and the two mentioned pharmacists, Carl Siebert (1863–1931) and Hans Ziegenbein (1867–1920), founded the Behringwerk Marburg/Lahn. Within all these contracts the Schlossberg laboratory played a key role on the progress and research of the institute and later on the newly established company. From 1904 to 1927 it served as research department of the Behringwerk (from 1914 Behringwerke). The institute´s scientists were an interdisciplinary team and had background in human and veterinary medicine, chemistry and pharmacy. The studies by Behring and his staff have been published in Beiträge zur experimentellen Therapie.

POS.231

New tools for pharmacy-specific literature search developed by PubPharm - the Specialised Information Service Pharmacy Krüger, A. T.1; Keßler, K.1; Ghammad, Y.2; Wulle, S.1; Balke, W.-T.2; Stump, K.1 1 Universitätsbibliothek, Technische Universität Braunschweig, Pockelsstraße 13, 38106 Braunschweig, Germany 2 Institut für Informationssysteme, Technische Universität Braunschweig, Mühlenpfordtstraße 23, 38106 Braunschweig, Germany

The Specialised Information Service (SIS) Pharmacy (Fachinformationsdienst Pharmazie) aims at sustainably improving the supply of literature for academic pharmaceutical research in Germany. Therefore new search tools are being developed which are providing full text access to pharmaceutical journals free of charge. The project is being funded by the German Research Foundation (Deutsche Forschungsgemeinschaft) since January 1st 2015. A special feature of the project is the collaboration between Braunschweig University Library and the Institute for Information Systems at Braunschweig University, through which cutting edge informatics research is being incorporated into search tools. A core aspect is the user-centric implementation of an extensible and customizable research platform. The PubPharm discovery system was developed by the SIS Pharmacy. It is a search engine that allows pharmacy-specific literature search. The underlying database index goes well beyond Medline (PubMed) data to provide a comprehensive set of publications for pharmaceutical research. Hence, using one single query, many different literature resources can be found, for example, interesting Medline articles, articles in chemical journals (that are not contained in Medline), pharmaceutical e-books and PhD theses. The PubPharm discovery system contains an availability check which, for users from universities in Germany, is personalized based on location. Through this, the full text of many publications can be directly accessed. The database also contains authority data, which allows query expansion and thereby retrieval of additionally relevant documents. For example, if the search query is the brand name of a pharmaceutical drug, also publications containing only the INN or IUPAC name will be retrieved. The PubPharm discovery system can be found here: www.pubpharm.de In addition to the personalized availability check, SIS Pharmacy offers full text access to pharmaceutical journals that are not usually provided by many university libraries. These more than 50 journals include, for example, “Expert Opinion on Drug Delivery” and “Current Medicinal Chemistry”. Researchers from pharmaceutical institutes at universities in Germany can register for gaining electronic full text access free of charge. These journals provided via SIS-licences are also included in the PubPharm search system. The PubPharm search tools undergo a continuous development process. One additional application is structure search. It will be possible to search for a compound not only by its (textual) name but also by its molecular structure using a formula editor. The structure search function will be implemented within the next year. Research at the Institute for Information Systems aims at developing innovative search tools. If they proof to be beneficial, they will be incorporated into the retrieval mechanisms of the PubPharm system. One innovative application is a search function based on Natural Language Processing. It might provide a new way to find similar documents or compounds with similar effects. Functions of other search tools are for example based on similar key words, citations or molecular structures. The proposed innovative search function takes the whole context of a document into account and can find similar documents which do not contain the same or similar key words, compounds or molecular structures. More information about SIS Pharmacy and the PubPharm system can be found in the PubPharm blog: https://blogs.tu-braunschweig.de/pubpharm/

POSTERS


POS.233

The PILOT-Study - part of the LENA project A dry-run as training-concept for paediatric clinical studies with complex sampling requirements Ciplea, A. M.1; Burckhardt, B. B.1; Ablonczy, L.2; Breur, J. M. P. J.3; Burch, M.4; Dalinghaus, M.5; De Wildt, S. N.5; Kleine, K.6; Klingmann, I.7; Swoboda, V.8; Szatmári, A.2; Läer, S.1 1 Department of Clinical Pharmacy and Pharmacotherapy, Heinrich- Heine-University, Universitätsstr.1 40225 Düsseldorf, Germany 2 Gottsegen György Hungarian Institute of Cardiology, Haller utca 29 1095 Budapest, Hungary 3 Wilhelmina Children's Hospital/University Medical Center Utrecht, Pediatric Heart Center, Lundlaan 6 3584 EA Utrecht, The Netherlands 4 Great Ormond Street Hospital for Children NHS Trust, Great Ormond Street WC1N 3JH London, United Kingdom 5 Erasmus Universitair Medisch Centrum Rotterdam,‘s, Gravendijkwal 230 3015CE Rotterdam and Radboud University, Comeniuslaan 4, 6525 HP Nijmegen, The Netherlands 6 Simply Quality – Dr. Karl Kleine, Johannes-Damrich-Str.4, 82362 Weilheim i. OB, Germany 7 Pharmaplex bvba, Avenue Saint-Hubert 51, 1970 Wezembeek-Oppem, Belgium 8 Medical University of Vienna, Spitalgasse 23 1090 Vienna, Austria

Objectives: The LENA project (Labeling of Enalapril from Neonates up to Adolescents) aims to improve the healthcare of children with heart failure by an enalapril orodispersible mini-tablet. In LENA, the Renin-Angiotensin-Aldosterone-System (RAAS) in children will be investigated to improve the understanding of RAAS maturation and ACE-inhibitor effects in children. The drug, its metabolite and the humoral parameters investigated in serum and plasma face stability problems and thus require an immediate sample processing and a strict adherence to a rigorous sampling protocol. Aim: To perform training and an evaluation (PILOT Study) to demonstrate successful preparation and performance of sampling as required in the LENA paediatric trials for the medical staff. Methods: The medical staff involved in the trials was trained in a tutorial and hands-on workshop concerning small-volume blood sampling of drug concentration and time-critical, sensitive humoral parameters in paediatric patients under optimal conditions. One to four months after passing this training, the involved 5 clinical centres were evaluated by performing a PILOT Study as an on-site study in their familiar environment. The participating centres conducted a procedure resembling the LENA trials sampling routine for regular study-visits on three adult volunteers, covering the sampling procedure, sample preparation, storage and shipment by utilizing equipment and documentation matching the paediatric trials. The medical staff draw blood samples from three healthy volunteers to determine the drug concentration and the humoral parameters aldosterone, renin, plasma renin activity and angiotensin I. To mimic the drug concentrations without administering the drug itself, the blood collection tubes were pre-spiked with two different mixtures of enalapril and its metabolite enalaprilat, creating predefined concentrations when filled with whole blood. The obtained drug concentrations were compared to reference values, obtained under optimal conditions at the bioanalytical laboratory. Results: In the initial performance of the PILOT-Study the centres obtained 94% of the scheduled samples with sufficient volume for analysis. The humoral parameters, being especially prone to degradation where detectable in all samples, with values beyond the lower reference level of physiological concentrations. The acceptable values of concentrations for enalapril/enalaprilat where initially missed by one centre, resulting in a re-run which was then successful. Conclusions: As the accuracy of pre-analytical sample preparation is crucial to the projects objectives and a key element for quality and reliability of gathered data, an upfront check of the team member’s ability to obtain these high-quality samples, is necessary. The concept of a training and evaluation (PILOT-Study) has shown to be a suitable approach for detecting improper sample handling. Acknowledgements:The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement n°602295 (LENA)

POS.234

Conjugation and labeling of bioactive peptides via Nω-carbamoylated arginines Keller, M.1; Kuhn, K. K.1; Einsiedel, J.2; Hübner, H.2; Bernhardt, G.1; Cabrele, C.3; Vanderheyden, P. M. L.4; Gmeiner, P.2; Buschauer, A.1

1 Institute of Pharmacy, University of Regensburg Universitätsstr. 31, D-93053 Regensburg, Germany 2 Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Schuhstrasse 19, D-91052 Erlangen, Germany 3 Department of Molecular Biology, University of Salzburg, Billrothstrasse 11, A-5020 Salzburg, Austria 4 Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium

Conjugation of fluorophores or radionuclide-bearing moieties to biologically active peptides is widely applied to prepare pharmacological tools as well as cancer diagnostics and therapeutics. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for versatile peptide conjugation, arginine, bearing a strongly basic guanidine group, has been neglected. We introduced a new, widely applicable strategy to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety.[1] Appropriately protected Nω-carbamoylated L-arginine derivatives (Fmoc-Arg(Boc)-OH, cf. Figure) can be incorporated into peptides in the position of interest according to Fmoc solid phase peptide synthesis. This was demonstrated, e.g. for peptide ligands of angiotensin II (AngII), neurotensin (NT) and neuropeptide Y (NPY) receptors. Synthesized precursor peptides, bearing a primary amino group at the modified arginine side chain, were conjugated and labeled in a versatile manner by analogy with procedures established for lysine residues. The modified peptides proved to be chemically stable (buffer, pH 7.4). Tritiated AT1, NTS1 and NPY Y4 receptor ligands (Kd values < 2 nM) were economically accessible by acylation of the precursor peptides with commercially available succinimidyl [3H]propanoate. Retained high receptor affinities of fluorescently labeled AngII and NT(8−13) analogues revealed that conjugation to space-filling moieties such as fluorophores via Nω-carbamoylated arginines was tolerated as well. This work demonstrates that the arginine position in biologically active peptides can be used to prepare radio- and fluorescence labeled molecular and pharmacological tools.

Acknowledgments: This work was funded by the Deutsche Forschungsgemeinschaft (Sachbeihilfe KE 1857 / 1-1, Graduiertenkolleg 1910)

References: 1. Keller, M. et al.: J. Med. Chem. 2016, 59: 1925-1945.

POS.235

Is cyanide a significant metabolite of Tofacitinib citrate? Geese, H.1; Pfaffenrot, E.2; Laufer, S. A.2; Clement, B.1 1 Department of Pharmaceutical and Medicinal Chemistry, Christian-Albrechts-Universität, Gutenbergstraße 76, 24118 Kiel, Germany 2 Department of Pharmaceutical and Medicinal Chemistry, Eberhard-Karls-University Tuebingen, Auf der Morgenstelle 8, 72076 , Tuebingen, Germany

Tofacitinib (CP-690,550) was one of the first orally available Janus kinase (JAK) inhibitors approved by the US Food and Drug Administration (FDA) for the treatment of moderate to severe rheumatoid arthritis [1]. Metabolism studies of Tofacitinib were conducted in human subjects. It was previously shown that Tofacitinib is a P450 substrate and mainly metabolized by CYP3A4/3A5 and CYP2C19 [2]. The identified metabolites included a carboxylic acid (4) metabolite of Tofacitinib, but the mechanism of the formation of this

OTHER TOPICS


metabolite was not investigated [2,3]. With respect to the structure of tofacitinib containing a cyano group with α–hydrogen atoms, a formation of cyanide by these enzymes is possible as outlined in figure 1. The goal of this study was to investigate a potential formation of cyanide by an initial attack of P450 enzymes (figure1). To determine cyanide a modified pyridine-barbituric acid method was used [4]. Cyanide formation was induced by incubating Tofacitinib with a mixture of porcine or human liver microsomes and NADPH as a cofactor of CYP450. As a result potentially formed cyanide reacts with cholarmine-T. After adding the pyridine-barbituric acid reagent a violet product is formed, which could be detected by spectrophotometry. By these incubations the formation of cyanide could be demonstrated. Cyanide formation is time-dependent, after 15 min the amount of cyanide decreased. So the highest concentration after incubation with porcine liver microsomes was 40 ± 5,3 pmol CN /pmol CYP and with human liver microsomes 23 ± 2,4 pmol CN /pmol CYP. Studies performed with recombinant isoenzymes identified CYP3A4 and CYP2D6 as the main catalysts for cyanide formation. Ketoconazole as an unspecific inhibitor of P450 enzymes was added to the incubation mixture in different concentrations. A decrease of the cyanide formation was detected at a concentration of 50 µM ketoconazol. By blocking an α–hydrogen atom in a Tofacitinib derivative (replacement of an α–hydrogen atom with isopropyl) no cyanide was detected. In conclusion the amount of cyanide is formed by metabolic transformation of Tofacitinib is low and might be not of toxicological relevance. Additionally, thiocyanate synthetase converts up to 0, 5 mg/kg body weight per hour cyanide into thiocyanate and the lethal dosis of cyanide in the whole blood is 2, 97 mg/l [5,6]. Nevertheless, El Ghawabi and his group examined the chronic poisoning after a longterm cyanide exposure to industrial smoke [7]. The most frequent symptoms (78-81%) were headache, weakness and changings in taste and smell. Additionally, chronic cyanide toxicitiy by oral intake leads unequivocally to goitre, tropical ataxic neuropathy and spastic paresis [8].

Fig. 1: Potential formation of cyanide by CYP450

References: 1. West, K.: Curr Opin Investig Drugs 2009, 10(5):491-504. 2. FDA: Pharmacology Review 2011, Application Number 203214Orig1s000, 64-94. 3. Dowty, M. E. et al.: Drug Metabol and Dispos 2014, 42: 759-773. 4. Asmus, E. et al.: Fresenius´Z. Anal. Chem. 1953, 138: 414-422. 5. Reichl, F.X.: Taschenatlas der Toxikologie 2002, 2: 134. 6. Hall, A.H. et al.: Annals of emerg. Medic. 1986, 1096: 115- 122. 7. El Ghawabi, S. H. et al: British Journ of Industrial Med. 1975, 32: 215-219. 8. Okolie, N. P. et al: Food and Chemical Toxicol. 1999, 37(7): 745-750.

POS.236

Oral Turinabol. Die Entstehungsgeschichte eines Anabolikums Möckel, A. Institut für Geschichte der Pharmazie Marburg

POS.237

Exam behavior of newly registered candidates in the 4 partial exams of the 1st phase of the pharmaceutical state exam 2006-2016: Pulling through, nonattendance, approved sick note Spahn-Langguth, H.1; Winter, N.1; Shahla, H.1 1 Institut für medizinische und pharmazeutische Prüfungsfragen, Große Langgasse 8, 55116 Mainz, Germany

Within the pharmaceutical state exam the 1st state exam -usually- represents a series of 4 subsequent written (MCQ) exams. The nationwide written state exams are taking place semiannually and simultaneously. Due to the broadness and depth of the respective 4 subject groups, candidates need to show continuous and persistent performance on four subsequent days. Data available in the period since 2006, i.e., after finalization of the 2-year-transition to the currently valid exam specifications of the German AAppO, were the basis for the retrospective analyses. Out of the group of a total of approximately 19 K newly registered candidates, an average of 0.29 % did not attend any of the 4 individual exams, while 90.11 % attended all 4 exams. And out of this subgroup, 64.4 % were able to pass all 4 exams at once. When candidates were not able to take part on one day or skipped one day (average total absence in at least 1/4 and up to 4/4 exams, 9.89 %), then out of this latter subgroup 14 % (1003/7168) were approved withdrawals, 86 % (6165/7168) were non-approved = failed. The following tendencies (preferences) were detected for absence in only 1 out of 4 subject groups (total 4.95 %, i.e., appr. 1/2 out of the above given 9.89 %): Day 1 = "Chemistry", 0.67 %, Day 2 = "Pharmaceutical and human biology", 1.91 %; Day 3 = "Physics, physical chemistry, drug formulation", 1.13 %; Day 4 = "Drug analysis", 1.24 %. When candidates attended one day only and were absent in 3/4 exams (1.01 out of the 9.89 %), the total of 1.01 subdivides onto the 4 exams as follows: Day 1 - 0.69 % > Day 4 - 0.16 % > Days 2 & 3 - 0.08 % each. The average overall grade achieved by the candidates who passed all 4 exams in phase 1 within their 1st trial was 3.13 (max grade, 1.0; min grade, 4.0) with no significant positive or negative trend over the included 10-years period.

POSTERS


AUTHOR INDEX

A

Abdallah, D. ................................................................... 98

Abdelaziz, A. ................................................................ 147

Abdel‐Aziz, H. ................................ 98, 120, 146, 167, 178

Abdel‐Tawab, M. ........................................................... 91

Abdul, H. K. .................................................................. 123

Abebe, D. ..................................................................... 103

Ablonczy, L. ................................................................. 180

Achenbach, J. ......................................................... 68, 120

Ackermann, T. ............................................................... 94

Adamski, J. ................................................................... 123

Agoglitta, O.................................................................. 131

Aigner, A. ............................................................. 112, 163

Alban, S.................................................................. 92, 106

Albold, D. ..................................................................... 159

Al‐Gousous, J. ........................................................ 75, 151

Althaus, J. .................................................................... 101

Amidon, G. L. ......................................................... 75, 151

Anamur, C. ............................................................. 38, 160

Andina, D. .................................................................... 164

Andraschko, M. ................................................... 119, 168

Angioni, C. ....................................................... 41, 97, 123

Antoni, F. ..................................................................... 131

Ardelt, M. A. ................................................................ 113

Arnold, G. J. ................................................................. 113

Arnold, K. ..................................................................... 162

Arntjen, A. ................................................................... 154

Aryal, D. K. ................................................................... 136

Ashtikar, M. ................................................................. 145

Asmari, M. ..................................................................... 94

Atzler, D. ...................................................................... 122

Ausbacher, D. .............................................................. 172

B

Bacher, L. ..................................................................... 154

Bajorath, J. ................................................................... 117

Balasupraminiam, S. ...................................................... 88

Balke, W.‐T. ................................................................. 179

Balsevich,G. ................................................................... 77

Baltes, F. ...................................................................... 106

Barocelli, E. .................................................................. 138

Bartel, A. ........................................................................ 93

Bartel, K. ...................................................................... 110

Bartl, N. ....................................................................... 123

Bassett, D. J. P. ............................................................ 103

Bauer, J. ................................................................. 54, 146

Bauer, R. ........................................................................ 52

Bauer, S. M. ................................................................. 102

Bäurer, S. ....................................................................... 90

Bausch, A. .................................................................... 143

Bause, M. ..................................................................... 133

Bayer, T. ...................................................................... 134

Beck, R. C. R. ................................................................ 162

Becker, D. .................................................................... 128

Becker‐Pauly, C. .......................................................... 117

Beckmann, A.‐M. ......................................................... 117

Bednarski, P. J. ............................................................ 108

Beerhues, L. ................................................................ 145

Behr, J. ........................................................................ 168

Bendas, G. ........................................................... 105, 106

Bendel, T. .............................................................. 54, 146

Bergemann, C. ..................................................... 159, 161

Bermudez, M....................................................... 137, 139

Bernat, V. .................................................................... 136

Bernhardt, G. ............................................... 131, 133, 180

Bertoletti, N. ....................................................... 123, 125

Betz, M. ................................................................. 58, 125

Bhatia, S. ............................................................... 43, 109

Biel, M. .......................................................................... 40

Bischoff, I. ............................................................. 97, 107

Bittner, F. .................................................................... 126

Blanchard, N. ................................................................. 36

Blassmann, U. ............................................................... 93

Blöcher, R. ............................................................. 99, 123

Blomme, C. .................................................................. 107

Bödefeld, T. ................................................................. 142

Boehlich, G. J. .............................................................. 139

Bogdahn, M. .................................................................. 27

Böger, R. H. ................................................................. 122

Boldt, K. ....................................................................... 167

Bonaterra, G. A. ............................................................ 98

Bopp, B. .......................................................... 43, 107, 109

Borek, C. ...................................................................... 105

Borkhardt, A. ......................................................... 43, 109

Bosman, I. ................................................................... 110

Boß, M......................................................................... 123

Böttcher, S. ................................................................. 166

Böttcher‐Friebertshäuser, E. ....................................... 140

Bracher, F. . 40, 89, 97, 104, 123, 124, 125, 126, 135, 136,

139

Braig, S. ....................................................................... 110

Brambilla, D................................................................. 164

Brandl, F. P. ................................................................... 26

Brandt, S. .................................................................... 137

Braun, F. ...................................................................... 123

Braun, M. D. ................................................................ 143

Breiholz, S. .................................................................. 118

Breitsamer, M. ............................................................ 160

Breur, J. M. P. J. ........................................................... 180

Brezesinski, G. ....................................................... 57, 116

Briegel, J. ....................................................................... 93

Briel, D. ....................................................................... 130

Brinker, C. ................................................................... 134

Brönstrup, M. .............................................................. 171

Browne, E. ................................................................... 108

Brückl, L. ...................................................................... 148

Brüggerhoff, A. ............................................................ 123

Brüne, B. ..................................................................... 123

Brunschweiger, A. ....................................................... 175

Brusotti, G. .................................................................... 48

Brust, P. ....................................................................... 130

AUTHOR INDEX


Büning, H. ...................................................................... 46

Bunjes, H. .................................................................... 155

Burch, M. ..................................................................... 180

Burckhardt, B. B. .................................................... 93, 180

Burkhardt, J. ................................................................ 112

Buschauer, A. ...................................... 131, 133, 138, 180

Busskamp, V. ................................................................. 44

Butterweck, V. ............................................................. 146

C

Cabrele, C. ................................................................... 180

Calderón, M. ................................................................ 151

Calleri, E. ........................................................................ 48

Carballo, C ............................................................... 62, 96

Ceelen, F. ..................................................................... 168

Chaikuad, A. ................................................................ 102

Chakrabarti, A. ............................................................. 134

Chao, Y. K. .............................................................. 40, 136

Chaves, P. S. ................................................................ 162

Chen, C. C. ............................................................. 40, 136

Cheung, S.‐Y. ............................................................... 124

Choe, C. ....................................................................... 122

Chytil, P. ...................................................................... 149

Cialla‐May, D. .............................................................. 161

Ciglia, E. ....................................................................... 107

Ciossek, T. ...................................................................... 19

Ciplea, A. M. ................................................................ 180

Clement, B. .................................................... 96, 126, 180

Corder, G. .................................................................... 136

Cordes, A. ...................................................................... 98

Cordts, K. ..................................................................... 122

Cramer, J. .............................................................. 58, 125

Culmsee, C. .................................... 68, 120, 140, 143, 174

Cumbana, R. ............................................................ 41, 97

D

Dai, B. ............................................................................ 97

Dalinghaus, M. ............................................................. 180

Dallanoce, C. ........................................................ 138, 142

Daniels, R. .................................................................... 155

Darveau, T. .................................................................. 108

Dauer, K. ...................................................................... 152

De Amici, M. ........................................................ 138, 142

De Beer, T. ..................................................................... 84

De Mello Martins, A. G. G. ............................................. 94

De Simone, A. .............................................................. 175

De Vries, J. ................................................................... 139

De Wildt, S. N. ............................................................. 180

Decker, M. ................................................... 132, 138, 142

Denezhkin, P. ............................................................... 148

Deng, L. ........................................................................ 167

Dengler, D. ................................................................... 136

Denninger, I. ................................................................ 178

Depke, T. ..................................................................... 171

Derendorf, H. ................................................................. 70

Deters, M. A. ............................................................... 118

Di Capua, A. ................................................................. 166

Diedrich, D. ........................................................... 43, 109

Diehl, O. ...................................................................... 123

Dieter, R. ..................................................................... 143

Dietrich, A. .................................................................... 31

Dolga, A. ...................................................................... 174

Dong, Y. ......................................................................... 86

Dornhof, R. ............................................................ 99, 178

Dorrestein, P. C. .................................................... 54, 146

Dougalis, A. ................................................................... 19

Draheim, H. J. ................................................................ 19

Duan, D. ...................................................................... 136

Ducho, C. ............................................................... 37, 172

Duda, J. ......................................................................... 19

Duerr, C. ........................................................................ 91

Dukor, R. K. ............................................................. 62, 96

Duque Escobar, J. ........................................................ 166

E

Ebbing, L. ..................................................................... 118

Eberhard, J. ................................................................... 94

Ebert, R. .................................................................. 41, 97

Eckl, K. ......................................................................... 151

Eder, M. ........................................................................ 77

Edkins, K. ..................................................................... 152

Efferth, T. ................................................................ 51, 98

Ehrig, K. ....................................................................... 106

Eickhoff, C. .................................................................. 118

Einsiedel, J. .................................................................. 180

Einsle, O. ..................................................................... 175

Eisenbach, I. ................................................................ 174

Eisend, S. ..................................................................... 118

El Deeb, S. ............................................................... 88, 94

Elliott, J.A. ..................................................................... 86

Elsässer, P. .................................................................. 178

Elz, S. ........................................................................... 138

Engelke, L .................................................................... 160

Engelke, L. ..................................................................... 38

Engels, B. ..................................................................... 105

Engert, J. ................................................ 38, 115, 149, 160

Enzensperger, C. ......................................................... 175

Erdmann, F. ................................................................. 134

Ernst, J. ........................................................................ 162

Ernst, T. ................................................................. 43, 109

Eschenhagen, T. .......................................................... 165

Estevam, E. C. .............................................................. 148

Etrych, T. ..................................................................... 149

Ewe, A. ........................................................................ 112

F

Fabian, J. ..................................................................... 101

Fach, M. ...................................................................... 112

Fallarero, A. ................................................................. 172

Faust, A. ...................................................................... 130

Felberg, M. .................................................................. 118

Feldmann, J. ................................................................ 110

POSTERS


Fellner, C. .............................................................. 38, 160

Fendt, M. ............................................................... 33, 174

Feng, Y. X. ...................................................................... 30

Ferreirós, N. ............................................................. 41, 97

Fetzer, C. ..................................................................... 113

Fiebig, H. ...................................................................... 109

Fiebig, J. ................................................................. 81, 117

Fiedler, S. ....................................................................... 31

Fink, C. ......................................................................... 178

Fischer, D. ............................................ 159, 161, 162, 163

Fischer, M. R. ............................................................... 119

Fischer, T. .................................................................... 152

Fish, I. .......................................................................... 137

Flammini, L. ................................................................. 138

Flemming, S. .................................................................. 99

Forget, A. ................................................................. 62, 96

Forster, M. ................................................................... 102

Frankenreiter, S. .......................................................... 165

Franz, L. ....................................................................... 130

Frey, O. R. ...................................................................... 93

Fricker, G. .................................................................... 151

Fridh, V. ................................................................. 58, 125

Friebe, A. ..................................................................... 165

Friedrich, C. ......................................................... 177, 178

Frieg, B. ................................................................. 43, 109

Frieß, W. .............................................................. 116, 154

Friess, W. ......................................................... 57, 91, 115

Fritsch, A. K. ................................................................. 158

Fritzsche, W. ................................................................ 159

Fröhlich, T. ................................................................... 113

Froriep, D. .................................................................... 126

Fuhrmann, G. ......................................................... 29, 111

Fujiwara, T. .................................................................. 103

Furdas, S. ..................................................................... 126

Fürst, R. ......................................................... 97, 107, 145

Furtmann, N. ............................................................... 117

Fütterer, S. ................................................................... 169

G

Gajer, J. ........................................................................ 126

Galanski, M. ................................................................. 110

Ganjam, G. K. ............................................... 140, 143, 174

Garscha, U. .............................................................. 98, 99

Gassen, N. C................................................................... 77

Geertz, B. ..................................................................... 165

Geese, H. ..................................................................... 180

Gegenfurtner, F. A. ...................................................... 111

Geh, K. J. ...................................................................... 150

Gehringer, M. .............................................................. 102

Gehrmann, S. ............................................................... 155

Geisslinger, G. .................................................. 41, 97, 123

Gellrich, L. M. .............................................................. 127

Genewsky, A. ................................................................. 77

Gerloff, C. .................................................................... 122

Gerstmeier, J. ................................................................ 99

Ghammad, Y. ............................................................... 179

Ghoreschi, K. ............................................................... 102

Giera, M. ...................................................................... 104

Gieré, R. ...................................................................... 178

Giguere, P. M. ............................................................. 136

Ginsel, C. ..................................................................... 126

Girreser, U. .................................................................... 96

Glatzel, D. .................................................................... 145

Glud, K. ........................................................................ 156

Gmeiner, P. .................................................. 136, 137, 180

Göbel, T. ...................................................................... 123

Goebgen, E. B. .............................................. 121, 171, 172

Goepferich, A. ............................................................. 157

Goeres, D. M. .............................................................. 172

Gohlke, H. ...................................................... 43, 107, 109

Gollos, S. ..................................................................... 110

Gomez‐Lopez, N. ......................................................... 103

Göpferich, A. ......................................................... 18, 157

Gopireddy, S. R............................................................ 158

Gorgus, E. .................................................................... 169

Gregoritza, M. ............................................................... 26

Grice, J. E. .................................................................... 147

Grienke, U. .................................................................. 132

Griffin, S. ..................................................................... 148

Grimm, C. .............................................................. 40, 136

Gronauer, T. ................................................................ 113

Grond, S. ............................................................... 54, 146

Gross, H. ................................................................ 54, 146

Grundmann, M. ................................................... 105, 123

Grüttner, C. ................................................................. 161

Guarinini, L. ................................................................. 108

Guck, J. ........................................................................ 110

Gudermann, T. .............................................................. 31

Guilherme dos Santos, M. ........................................... 105

Gundler, A. L. .............................................................. 165

Günther, M. ................................................................ 109

Günther, S. .................................................................... 99

Gütschow, M. ...................................................... 117, 131

H

Haas, V. ....................................................................... 157

Haeggström, J. Z. ........................................................... 98

Hafner, K. ...................................................................... 77

Hahn, R. ....................................................................... 148

Hahne, T. ....................................................................... 91

Hake, T. ....................................................................... 101

Hamacher, A. .............................................................. 110

Hamed, M. .................................................................... 94

Hanekamp, W. ............................................................ 101

Hänggi, S. .................................................................... 147

Hanke, T. ..................................................................... 124

Hansen, F. K. .................................................. 43, 107, 109

Harder, M. J. ................................................................ 100

Hartmann, J. .................................................................. 77

Hartmann, M. .............................................................. 123

Hartmann, R. W. ........................................................... 94

Hartung, N. .................................................................. 121

Haryadi, B. M. ............................................................. 149

Hasan, M. .................................................................... 108

Hauer, J. ................................................................ 43, 109

Haunberger, A. ............................................................ 157

AUTHOR INDEX


Haupenthal, J. ................................................................ 94

Häupl, T. ........................................................................ 99

Hausch, F. ...................................................................... 76

Hauser, A. T. ................................................................ 134

Häußler, S. ................................................................... 171

Havemeyer, A. ....................................................... 96, 126

He, D. ..................................................................... 82, 150

Heck, R. ........................................................................ 155

Hedtrich, S. .................................................... 64, 103, 151

Heering, J. .............................................................. 99, 123

Heilmann, J. ........................................................... 95, 121

Heimburg, T. ................................................................ 134

Heine, A. ........................................................ 58, 123, 125

Heitel, P. .............................................................. 100, 127

Held, J. ......................................................................... 129

Helmstädter, A. ................................................... 146, 179

Hemmers, S. ................................................................ 104

Henke, S. ............................................................... 38, 160

Hennies, H. C. .............................................................. 151

Henrich, A. ................................................................... 119

Hermann, S. ................................................................. 155

Herrmann, J. .................................................................. 53

Hespeler, D. ................................................................. 154

Heßelbach, K. ................................................................ 99

Hildebrandt, C. ............................................................ 158

Hinz, S. ......................................................................... 166 Hirsch, R. ....................................................................... 74

Hittinger, M. ................................................................ 169

Hochhaus, A. ......................................................... 43, 109

Hochscherf, J. .............................................................. 107

Hoferm, N. ..................................................................... 19

Hoffmann, A. ............................................................... 132

Hoffmann, K. ............................................................... 137

Hoffmann, L. ........................................................ 140, 174

Hoffmann, T. ............................................................... 152

Hofmann, K. ................................................................... 31

Höfner, G. ........................................................ 88, 94, 175

Höldrich, M. ................................................................... 90

Holl, R. ................................................................... 61, 131

Holsboer, F. ................................................................... 22

Holstein, J. ................................................................... 102

Höltke, C. ..................................................................... 130

Holze, J. ....................................................................... 142

Holzgrabe, U. ................................................. 89, 138, 142

Honrath, B. .................................................................. 174

Hönzke, S. .............................................................. 64, 103

Hopf, Y. M. ................................................................... 119

Hoppe, J. ...................................................................... 122

Horak, J. ......................................................................... 90

Horstmann, R. D. ......................................................... 177

Hoß, S. G. ..................................................................... 105

Huang, F.‐C. ................................................................. 152

Huang, G. ..................................................................... 132

Huang, X. P. ................................................................. 136

Hubert, M. ................................................................... 150

Hübner, H. ................................................... 136, 137, 180

Huge, V. ......................................................................... 93

Huisinga, W. ................................................................ 119

Humar, M. ............................................................. 99, 178

Hummel, F. C. .............................................................. 122

Hüther, J. ....................................................................... 98

I

Ibraimi, M.................................................................... 153

Ihle, F. ......................................................................... 168

Ilan, N. ......................................................................... 105

Imanidis, G. ................................................................. 147

Imhof, A. ..................................................................... 143

Imig, J. D. ..................................................................... 123

Irsheid, L. ..................................................................... 105

J

Jacob, C. ...................................................................... 148

Jaehde, U. ........................................................... 119, 122

Jagodzinsk, A. I. ........................................................... 122

Jakobs, H. H. ................................................................ 126

Jelinek, A. .................................................................... 140

Jin, N. .......................................................................... 162

Joerger, M. .......................................................... 107, 119

Johannessen, C. ....................................................... 62, 96

Jongen, L. .................................................................... 107

Jose, J. .................................................... 43, 107, 109, 115

Juchum, M................................................................... 109

Jung, M. ........................................ 124, 126, 134, 135, 175

Jung, N. ....................................................................... 147

Jürgenliemk, G. ..................................................... 95, 121

Just, S. ......................................................................... 167

K

Kahnt, A. S. ....................................................... 41, 97, 123

Kaiser, A. ............................................................. 101, 123

Kalayda, G. V. .............................................................. 110

Kalinin, D. .................................................................... 131

Kalinowsky, L. ........................................................ 99, 127

Kamlah, A. ................................................................... 124

Kandil, R. ..................................................................... 103

Kany, A. M. .................................................................... 94

Karlsson, R. ............................................................ 58, 125

Kassack, M. U. ................................................ 43, 109, 110

Kauffold, J. .................................................................. 154

Kaysser, L. ............................................................. 54, 146

Kazmaier, U. ........................................................ 107, 111

Keck, C. M. ........................... 148, 152, 153, 154, 161, 162

Keils, A. ........................................................................ 140

Kelber, O. ....................................... 98, 120, 146, 167, 169

Keller, M. ........................................................ 40, 136, 180

Kelter, G. ..................................................................... 109

Kempin, W. ................................................................... 27

Kessel, E. ............................................................... 82, 150

Keßler, K. ..................................................................... 179

Khalil, E. ....................................................................... 145

Khayyal, M. T. ................................................................ 98

Kiemer, A. K. .................................................................. 79

Kim, G.‐J. ....................................................................... 99

POSTERS


Kim, N. H. ..................................................................... 103

Kinscherf, R. ................................................................... 98

Kirchmair, J. ................................................................. 132

Kirmeier, T. .................................................................... 78

Kisko, T. M. .................................................................. 143

Klebe, G. ........................................................ 58, 123, 125

Klein, P. M. ............................................................ 82, 150

Kleine, K. ...................................................................... 180

Kleinebudde, P. ............................................................. 72

Kleinschmidt, T. K. ......................................................... 98

Kleusch, C. ..................................................................... 94

Kling, R. C. .................................................................... 136

Klinger‐Strobel, M. ...................................................... 162

Klingmann, I. ................................................................ 180

Klintworth, D. .............................................................. 118

Klitsche, F. ..................................................................... 95

Klöckner, J. .................................................................. 142

Kloft, C. ................................. 107, 119, 120, 121, 171, 172

Klopp‐Schulze, L. ......................................................... 107

Klymiuk, N. .................................................................... 20

Knaab, T. C. .................................................................. 129

Knapp, S. .......................................................... 97, 99, 102

Kneidinger, N. .............................................................. 168

Kniess, A. ..................................................................... 165

Knuth, S. ...................................................................... 121

Kobilka, B. K. ................................................................ 136

Koch, O. ................................................................. 66, 130

Koch, P. .................................................................. 67, 130

Koeberle, A. ............................................. 42, 92, 110, 145

Koeberle, S. C. ......................................................... 42, 92

Koepf, E. ...................................................................... 116

Köhler, J. ...................................................................... 131

Köhnke, J. ...................................................................... 94

König, B. ........................................................ 34, 131, 133

Königshoff, M. ............................................................... 31

Köpf, E. .......................................................................... 57

Körner, A. .................................................................... 104

Köse, M. ....................................................................... 137

Kostenis, E. .................................................................. 123

Kraff, S. ........................................................................ 119

Kraft, K. ........................................................................ 120

Kralisch, D. ................................................................... 163

Kramer, C. .................................................................... 132

Kramer, J. S. ................................................................... 99

Krattenmacher, D. ....................................................... 167

Kraus, T. ....................................................................... 103

Krauss, J. ...................................................................... 126

Kreiss, C. ...................................................................... 169

Kressler, J. ............................................................ 149, 160

Krieg, T. ....................................................................... 165

Krimmer, S. G. ....................................................... 58, 125

Krüger, A. T. ................................................................. 179

Kuhn, K. K. ................................................................... 180

Kulik, A. .................................................................. 54, 146

Kullmann, M. ............................................................... 110

Kuntsche, J. .................................................................. 156

Kunze, T. ...................................................................... 118

Kurland, H.‐D. .............................................................. 161

Kurz, T. ................................................... 43, 107, 109, 129

Kutza, J. ....................................................................... 160

L

Lächelt, U. ............................................................. 82, 150

Läer, S. ........................................................... 93, 118, 180

Lahrsen, E. ..................................................................... 92

Lamers, C..................................................................... 123

Lämmerhofer, M. .......................................................... 90

Lang, F. .................................................................. 43, 109

Lang, M. ...................................................................... 130

Langguth, P. ........................................... 75, 147, 151, 169

Laufer, S. A. .................................. 100, 102, 109, 132, 180

Le Guiner, C. .................................................................. 45

Lee, D. J. ................................................................ 82, 150

Lee, S. .......................................................................... 166 Lehmann, C. ............................................................ 41, 97

Lehr, M. ....................................................................... 101

Lehr, T. ........................................................................ 113

Leibiger, J. ............................................................. 33, 174

Leipoldt, F. ............................................................ 54, 146

Lell, P. .................................................................... 38, 160

Lemcke, T. ........................................................... 134, 166

Leroux, J. C. ................................................................. 164

Leutz, S. ....................................................................... 137

Levit, A. ....................................................................... 136

Li, Y. ............................................................................. 103

Liebl, J. ................................................................ 112, 113

Liedl, K. R. .................................................................... 132

Liening, S. ...................................................................... 98

Liewert, I. .................................................................... 106

Lilischkis, R. ................................................................. 148

Lin, H. .................................................................... 86, 136

Lindhorst, F. ................................................................ 115

Lintermans, A. ............................................................. 107

Lirk, F. .......................................................................... 124

Liss, B. ........................................................................... 19

Liu, B. .......................................................................... 145

Liu, S. ............................................................................. 90

Löber, S. ...................................................................... 136

Loeser, K .................................................................. 42, 92

Lohmüller, T. ............................................................... 110

Löhnert, A. .................................................................. 177

Lohrer, B...................................................................... 125

Lorenz, L. ..................................................................... 172

Lötters, S. ...................................................................... 89

Luber, M. ..................................................................... 124

Lucas, H. ...................................................................... 149

Luciani, P. .................................................................... 164

Lüdeke, S. ................................................... 43, 62, 96, 109

Lühmann, T. .......................................................... 81, 117

Lukat, P. ...................................................................... 145

Lukowski, R. ................................................. 143, 165, 167

Lum, L. G. .................................................................... 103

Lunter, D. ....................................................... 63, 155, 157

Luong, B. ....................................................................... 97

M

Mackenroth, L. .............................................................. 19

AUTHOR INDEX


Mäder, K. ............................................................. 149, 160

Magdolen, V. ............................................................... 140

Mahringer, A. .............................................................. 151

Mair, C. E. .................................................................... 132

Maison, W. ............................................................ 95, 127

Mak, T. ......................................................................... 166

Malsch, D. .................................................................... 159

Mandl, M. M. ....................................................... 112, 113

Manglik, A. .................................................................. 136

Mangold, M. ................................................................ 117

Marchais‐Oberwinkler, S. .................................... 123, 125

Marek, M. ............................................................ 134, 135

Markarewicz, O. .......................................................... 162

Markl, D. ........................................................................ 86

Marschall, C. ................................................................ 115

Martin, B. .................................................................... 131

Martins, H. ................................................................... 143

Marzouk, M. A. ............................................................ 108

Maschowski, C. ............................................................ 178

Massolini, G. .................................................................. 48

Matera, C. ............................................................ 138, 142

Maul, K. J. ...................................................................... 91

Maurer, E. .................................................................... 117

Mayer‐Wrangowski, S. C. ............................................ 102

Mayr, D. ....................................................................... 112

McCorvy, J. .................................................................. 136

Meinel, L. ............................................................... 81, 117

Melesina, J. .................................................................. 134

Melzer, B. .................................................................... 139

Menche, D. .................................................... 65, 105, 110

Mendel, R. R. ............................................................... 126

Merfort, I. .............................................................. 99, 178

Merk, D. ....................................... 100, 101, 123, 127, 129

Merkel, O. M. .............................................................. 103

Messerer, R. ........................................................ 138, 142

Meßner, M. ................................................................. 113

Metz, A. ....................................................................... 125

Meyer, K. ..................................................................... 113

Miceli, M. .................................................................... 151

Michalakis, S. ................................................................. 47

Michalcova, L. ................................................................ 94

Michels, S. ................................................................... 143

Mikula, M. ..................................................................... 96

Mingo, V. ....................................................................... 89

Mirakaj, V. ................................................................... 104

Mobashery, S. ................................................................ 60

Mohammadi, M. .......................................................... 103

Mohammed, Y. H. ........................................................ 147

Mohr, C. ....................................................................... 152

Mohr, E. ....................................................................... 165

Mohr, K. ....................................................................... 142

Möhwald, M. ............................................................... 150

Molina, M. ................................................................... 151

Möller, D...................................................................... 135

Möller, G. .................................................................... 123

Monaldi, D. .................................................................. 135

Moore, B. S. ........................................................... 54, 146

Mordmüller, B. ............................................................ 129

Morhenn, K. ................................................................ 165

Morrison, H. ............................................................ 42, 92

Mosad, S. .................................................................... 147

Moser, C. ..................................................................... 111

Mozafari, M. .................................................................. 88

Mueller, T. D. .............................................................. 117

Müller, C. .............................................................. 89, 104

Müller, C. E. .................................................. 110, 137, 166

Müller, F. A. ................................................................. 161

Müller, J. ..................................................................... 120

Müller, M. ..................................................................... 35

Müller, R. ..... 53, 59, 65, 97, 105, 110, 111, 145, 159, 161

Müller, R. H. ........................................................ 161, 162

Müller, S. L. ................................................................. 128

Müller, T. ..................................................................... 149

Müller, T. D. .................................................................. 81

Müller, U. .................................................................... 118

Müller‐Goymann, C. ...................................................... 73

N

Nadithe, V. .................................................................. 103

Nafie, L. A. ............................................................... 62, 96

Namjoshi, S. N. ............................................................ 147

Nann, Y. ....................................................................... 143

Nasim, M. J. ................................................................. 148

Natile, G. ..................................................................... 108

Natile, M. .................................................................... 108

Nawroth, T. ................................................................. 147

Negro, G. ....................................................................... 19

Neiens, P. .................................................................... 175

Neitemeier, S. ............................................................. 140

Neri, D. .......................................................................... 23

Neundorf, I. ................................................................. 107

Neurohr, C. .................................................................. 168

Neven, P. ..................................................................... 107

Newcomer, M. E. .......................................................... 99

Nguyen, T. M. H. ......................................................... 154

Nguyen, T. N. ............................................................... 137

Nicke, A. ...................................................................... 143

Niefind, K. ................................................................... 107

Niess, R. ....................................................................... 147

O

Obarcanin, E. ............................................................... 118

Obst, K. ........................................................................ 151

Oetjen, E. ............................................................ 165, 166

Oliveira, E. G. ............................................................... 162

Ong, N. ........................................................................ 124

Ortland, I. .................................................................... 122

Ortner, N. J. ................................................................... 19

Osipova, A. .................................................................. 178

Ott, I. ............................................................................. 92

Ottl, J. ............................................................................ 49

P

Parnham, M. J. ........................................................ 41, 97

POSTERS


Parra‐Guillen, Z. P. ....................................................... 119

Paulke, B. ..................................................................... 153

Pei, C. ............................................................................. 86

Pein, H. .................................................................... 42, 92

Pendl, E. ....................................................................... 147

Perfahl, S. .................................................................... 108

Peric, N. ....................................................................... 127

Pernpeintner, C. .......................................................... 110

Pfaffenrot, E. ....................................................... 102, 180

Pham, N. B. ................................................................... 166 Pierce, R. ..................................................................... 134

Pinggera, A. ................................................................... 19

Piva, M. B. R. ............................................................... 106

Plank, R. ....................................................................... 151

Plesch, E. ............................................................... 40, 136

Pletz, M. W. ................................................................. 162

Plitzko, B. ..................................................................... 126

Pockes, S. ..................................................................... 138

Pogoryelov, D. ............................................................... 99

Pollinger, J. C. .............................................................. 127

Poppe, A. ..................................................................... 107

Pötzinger, Y. ................................................................ 163

Pourasghar, M. ............................................................ 150

Praefke, B. A. ............................................................... 100

Proschak, E. ................................... 99, 100, 101, 123, 127

Przybylski, S. ................................................................ 112

Pyo, S. M. ............................................................. 161, 162

Pysniak, K. ..................................................................... 96

Q

Quinn, R. J. .................................................................. 166

R

Rabel, M. ..................................................................... 161

Rabini, S. .............................................................. 120, 178

Rach, R. ........................................................................ 147

Rackl, M. ...................................................................... 158

Rademann, J. ............................................................... 128

Radi, L. ......................................................................... 112

Rahnfeld, L. .................................................................. 163

Rammensee, H.‐G. ......................................................... 21

Ramotowska, E. ............................................................. 96

Raney, S. G................................................................... 147

Rath, S. ........................................................................ 140

Rauh, D. ....................................................................... 102

Rehbaum, H. .................................................................. 83

Reichart, B. .................................................................... 20

Rein, T. ........................................................................... 77

Renner, S. ...................................................................... 20

Richter, M. ................................................................... 132

Richter, T. .................................................................... 107

Ritzmann, M. ......................................................... 38, 160

Robaa, D. ..................................................................... 134

Roberts, M. S. .............................................................. 147

Rodrigues de Sá Alves, F. ............................................. 132

Rodrigues Moita, A. J. ............................................ 43, 109

Rodrigues, A. G............................................................ 160

Roehr, A. C. ................................................................... 93

Rollinger, J. M. ............................................................ 132

Romier, C. ........................................................... 134, 135

Romp, E. ........................................................................ 99

Rossi, A. ................................................................... 42, 92

Roth, B. L. .................................................................... 136

Rothert, M. .................................................................. 126

Rotter, M. .................................................................... 101

Roy, A. ..................................................................... 62, 96

Rummler, S. ................................................................... 98

Rumpf, T. ..................................................................... 175

Ruth, P. ................................................... 30, 143, 165, 167

Rüther, A. ................................................... 43, 62, 96, 109

S

Sakkas, A.‐S. ................................................................ 177

Salah, M. ..................................................................... 123

Sarin, N. ....................................................................... 110

Sassano, M. F. ............................................................. 136

Sautebin, L. ............................................................. 42, 92

Sayed, A. ..................................................................... 133

Schacht, M. ................................................................. 139

Schader, T. .................................................................. 123

Schaefer, J. .................................................................... 93

Schaeftlein, A. ............................................................. 121

Schäfer, K. H. ............................................................... 148

Schäfer‐Korting, M. ............................................... 64, 103

Scheffler, K. ................................................................... 50

Scheler, S. .................................................................... 148

Scherließ, R. ................................................. 153, 158, 159

Scherrer, G. ................................................................. 136

Scherübl, C. ................................................................... 56

Scheunemann, M. ....................................................... 130

Schiedel, A. C. .............................................................. 137

Schiedel, M. ................................................................ 135

Schiestl, M. .................................................................... 55

Schiller, S. .................................................................... 153

Schirmeister, T. ........................................................... 105

Schlegel, L. .................................................................... 91

Schlenk, F. ................................................................... 159

Schlesinger, M. ............................................................ 106

Schmidberger, M......................................................... 155

Schmidt, A. .................................................................. 143

Schmidt, C. ............................................................ 33, 174

Schmidt, C. Q. .............................................................. 100

Schmidt, J. ........................................................... 101, 129

Schmidt, M. ................................................................. 134

Schmidt, M. V. ............................................................... 77

Schmidt, S. K ................................................................. 88

Schmidtke, M. ............................................................. 132

Schmidtko, A. ................................................................ 32

Schmidtkunz, K. ........................................................... 134

Schmoeckel, E. ............................................................ 112

Schneider, F. ........................................................... 42, 92

Schneider, J. .................................................................. 96

Schneider, M. ................................................. 98, 150, 162

Schnorr, J. ................................................................... 169

AUTHOR INDEX


Schoenfeld, A. ............................................................... 92

Scholler, M. ......................................................... 131, 133

Scholz, M. S. ................................................................ 128

Schönhoff, M. .............................................................. 122

Schorn, M. ............................................................. 54, 146

Schrage, R. ................................................................... 142

Schramm, K. W. ........................................................... 133

Schramm, R. ................................................................ 168

Schramm, S. ................................................................. 132

Schratt, G. .................................................................... 143

Schreiber, F. ................................................................. 104

Schreiber, J. ................................................................. 128

Schrenk, D. .................................................................. 169

Schröder, T. ................................................................. 148

Schroeder, R. ......................................................... 57, 116

Schubert‐Zsilavecz, M. ............ 91, 100, 123, 124, 127, 129

Schulz, M. .................................................................... 118

Schulze, J. .................................................................... 163

Schumann, L. ............................................................... 132

Schützenmeister, N. .................................................... 139

Schüürmann, J. ............................................................ 115

Schwab, W. .................................................................. 152

Schwarting, R. K. W. .................................................... 143

Schwedhelm, E. ........................................................... 122

Schweiger, A. ................................................................. 85

Schwendler, A. ............................................................... 98

Schwenk, R. ................................................................. 107

Scott, S. ........................................................................ 166 Scriba, G. K. ................................................................... 98

Seebohm, G. ................................................................ 128

Seeger, J. ............................................................. 121, 172

Seibel, J. ....................................................................... 105

Seidlitz, A. ...................................................................... 27

Seifert, I. ........................................................................ 91

Senger, J. ..................................................................... 135

Sergi, M. ...................................................................... 148

Serio, A. ................................................................. 29, 111

Serive, B. ...................................................................... 166

Shahla, H ..................................................................... 181

Shastri, V. P. ............................................................ 62, 96

Shen, Y. C. ...................................................................... 86

Sherbakova, A. ............................................................. 167

Shoichet, B. K. ...................................................... 136, 137

Sieber, S. A. ................................................................. 113

Sievers‐Engler, A. ........................................................... 90

Sikandar, A. ................................................................... 94

Simmet, T. ................................................................... 100

Simon, R. P. ................................................................. 126

Sippl, W. .............................................................. 126, 134

Sonderegger, C. ........................................................... 148

Sotriffer, C. .................................................................. 138

Spahn‐Langguth, H. ..................................... 133, 147, 181

Spieler, V. .............................................................. 81, 117

Stadler, J. ............................................................... 38, 160

Stadler, M. ........................................................... 124, 126

Stahl, M. ...................................................................... 113

Stark, S. ................................................................ 131, 133

Stauber, R. ................................................................... 105

Stehning, T. .................................................................. 107

Stein, S. .................................................................. 43, 109

Steinebach, C. ............................................................. 131

Steinhilber, D. ............................................ 41, 97, 99, 123

Steinmetzer, T. ............................................................ 140

Stelzer, D. .................................................................... 168

Stepan, J. ....................................................................... 77

Stevens, M. M. ...................................................... 29, 111

Stewart, P. S. ............................................................... 172

Stirnberg, M. ............................................................... 117

Stöckelhuber, M. ........................................................... 89

Stocklauser, R. ............................................................. 143

Stoiber, K. .................................................................... 110

Stolfa, D. A. ................................................................. 126

Storr, M. ...................................................................... 120

Stößel, A. ..................................................................... 137

Stranik, O. ................................................................... 159

Straubinger, J. ............................................................. 167

Striessnig, J. ................................................................... 19

Strobach, D. .................................................................. 69

Strödke, B. ..................................................................... 97

Strøm, M. B. ................................................................ 172

Strunz, A. K. ................................................................. 118

Stump, K. ..................................................................... 179

Stumpf, F. .................................................................... 153

Subeska, A. .................................................................. 101

Swoboda, V. ................................................................ 180

Swyter, S. ............................................................ 124, 175

Szatmári, A. ................................................................. 180

T

Täuber, A. ...................................................................... 73

Tauber, C. .................................................................... 128

Temme, L. ................................................................... 138

Temporini, C. ................................................................. 48

Thakur, A. .................................................................... 103

Thamm, J. .................................................................... 161

Theiler, S. .................................................................... 130

Thon, N. ........................................................................ 93

Tins, J. ........................................................................... 93

Titz, A. ............................................................ 39, 146, 162

Toewe, A. ................................................................ 41, 97

Torge, A. ...................................................................... 162

Tränkle, C. ................................................................... 142

Tuluc, P.......................................................................... 19

Tyl‐Bielicka, A. ............................................................... 96

U

Ullrich, A. ..................................................................... 107

Ulrich, M. ............................................................ 110, 112

Ulrich‐Merzenich, G. ................................................... 167

Unger, M. .................................................................... 179

Untergehrer, M. .......................................................... 121

Urbanetz, N. A. ............................................................ 158

POSTERS


V

Van Asten, K. ............................................................... 107

Van Koppen, C. J. ......................................................... 123

Vanderheyden, P. M. L. ............................................... 180

Verjee, S. ..................................................................... 146

Vetter‐Kerkhoff, C. ........................................................ 93

Vial, M. .......................................................................... 166 Vlodavsky, I. ................................................................ 105

Voelkel, M. .............................................................. 42, 92

Vögerl, K. ..................................................................... 135

Vogeser, M. ................................................................. 168

Vollmar, A. M. ........................ 65, 105, 110, 111, 112, 113

Vollrath, M. ................................................................. 115

Volpp, M. ....................................................................... 89

von Grafenstein, S. ...................................................... 132

von Kügelgen, I. ........................................................... 137

von Schwarzenberg, K. .................................. 65, 105, 110

Vuorela, P. M. .............................................................. 172

W

Wadie, W. ...................................................................... 98

Wagner, E. ............................................................. 82, 150

Wagner, N. .................................................................... 89

Wagner, S. ........................................................... 130, 162

Wahl, J. .......................................................................... 89

Wahl‐Schott, C. ...................................................... 40, 119

Währa, M. ..................................................................... 94

Walter, J. ..................................................................... 117

Walter, N. M. ............................................................... 102

Walther, E. ................................................................... 132

Wanner, K. T. ................................................... 88, 94, 175

Warncke, P. ................................................................. 159

Wätzig, H. ................................................................ 88, 91

Weber, A. .................................................................... 168

Weber, J. ....................................................................... 31

Wehle, S. ..................................................................... 138

Weickert, A. ................................................................. 105

Weinigel, C. ................................................................... 98

Weiser, C. .................................................................... 120

Weiser, T. .................................................................... 101

Weiss, V. .............................................................. 149, 160

Weitschies, W. ............................................................... 27

Weizel, L. ............................................................. 101, 123

Wentsch, H. K. ............................................................. 102

Werner, B. P. ............................................................... 116

Werner, M. .................................................................. 124

Werner, S. ................................................................... 159

Werner, V. ............................................................. 81, 117

Wersig, T. .................................................................... 160

Werz, O. .......................... 42, 80, 92, 98, 99, 110, 124, 145

Wich P. R. .................................................................... 112

Wich, P. R. ............................................................. 28, 117

Wicha, S. G. ........................................................... 71, 171

Wieland, T. .................................................................... 30

Wiesneth, S. .................................................................. 95

Williams, B. M. .............................................................. 86

Windbergs, M. ............................................................ 147

Windorf, M. ................................................................. 160

Winter, G. ............................... 38, 115, 116, 149, 150, 160

Winter, H. .................................................................... 168

Winter, N. ................................................................... 181

Winzi, M. ..................................................................... 110

Witt, M. ....................................................................... 115

Witt‐Enderby, P. A. ..................................................... 108

Wittmann, S. ......................................................... 99, 101

Wittmann, S. K. ........................................................... 123

Wöhr, M. ..................................................................... 143

Woiwode, U. ................................................................. 90

Wojtyniak, J.‐G. ........................................................... 113

Wolber, G. ........................................................... 137, 139

Wolf, E. .......................................................................... 20

Wolf, F. .................................................................. 54, 146

Wölker, J. ...................................................................... 92

Wöll, S. ........................................................................ 153

Wulle, S. ...................................................................... 179

Wünsch, B. ........................................... 128, 130, 138, 140

Wurglics, M. ................................................................ 123

X

Xie, Y. .......................................................................... 103

Xu, Z. ........................................................................... 132

Y

Yasin, A. ....................................................................... 115

Yealland, G. ................................................................. 151

Yordanova, Y. .............................................................. 154

Z

Zahler, S. ............................................................. 110, 111

Zara, L. ......................................................................... 125

Zaremba, W. ............................................................... 154

Zeitler, J. A. ................................................................... 86

Zeitlinger, M. ............................................................... 120

Zerfass, P. .................................................................... 143

Zettler, J. ............................................................... 54, 146

Zhang, J. ...................................................................... 143

Zhang, S. ...................................................................... 112

Zhang, Z. ...................................................................... 157

Zheng, Y. ....................................................................... 86

Zhou, X. B. ..................................................................... 30

Zimmermann, A. ........................................................... 90

Zimmermann, G. ......................................................... 168

Zlotos, D. P. ................................................................. 108

Zoller, M. ..................................................................... 168

Zöls, S. ................................................................... 38, 160

Zubeil, F. ................................................................ 54, 146


Geschäftsführer und Leiter der Geschäftsstelle Apotheker Dr. Michael Stein DPhG Geschäftsstelle Varrentrappstraße 40-42 60486 Frankfurt Tel.: +49-(0)69-71915960 Fax: +49-(0)69-719159629 Email: [email protected] http://www.dphg.de Univ.-Prof. Dr. Gerhard Winter Ludwig-Maximilians-Universität München Institut für Pharmazeutische Technologie und Biopharmazie Butenandtstraße 5-13 81377 München, GERMANY Phone: +49-(0)89–218077022 Fax: +49-(0)89–218077020 Email: [email protected] 15.09.2016


NOTES

The compact mass spectrometer expression CMS

enables chemists to monitor reactions and identify

compounds directly on their own bench.

• ASAP® (Atmospheric Solids Analysis Probe) allows

analysis without sample preparation, takes only

seconds and is mostly favoured over the

traditional flow injection.

• Plate Express™ provides an easy way of obtaining

mass spectra directly from TLC plates. The

extraction and analysis of separated compounds

takes less than a minute and is entirely

automated.

• expression CMS is also ready to connect to any

HPLC/UPLC-, Prep/Flash/SFC-system of any

vendor and provides full mass directed

purification and fractionation capability.

Chemists appreciate the independency in their MS-

analysis and the non-restricted flexibility of the system

as it saves them valuable time and improves the

laboratory efficiency.

Please contact us with your questions or requests for

further information: [email protected]

mailto:[email protected]

ww

w.2016.dphg.de



Munich, GermanyOctober 4 – 7, 2016at Ludwig-Maximilians-University


Munich, Germany October 4 – 7, 2016 at Ludwig-Maximilians-University

www.2016.dphg.de

ISBN 978-3-9816225-3-9

DPhG

Annual Meeting 2016 – Conference Book

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