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Third World Conference on

Physico-Chemical Methods in

Drug Discovery

and Development

Programme

&

Book of Abstracts

Dubrovnik, Croatia, September 22-26, 2013

Third World Conference on Physico-Chemical Methods in Drug Discovery and Development Dubrovnik, Croatia, September 22-26, 2013

Programme & Book of Abstracts Published by

International Association of Physical Chemists E-mail: [email protected], URL: http://www.iapchem.org For Publisher Zoran Mandić Editor Zoran Mandić Design, Page Making and Computer Layout Aleksandar Dekanski Circulation 150 Copies

mailto:[email protected]

http://www.iapchem.org/

The Organizing Committee:

Alex Avdeef, In-ADME Research, US

Biserka Cetina-Čižmek, PLIVA, Croatia

Vesna Gabelica Marković, Galapagos, Croatia

Rolf Hilfiker, Solvias, Switzerland

Zoran Mandić, University of Zagreb, Croatia

Krisztina Takács-Novák, Semmelweis University, Hungary

Kin Tam, University of Macau, China

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CONTENTS

CONFERENCE PROGRAMME _______________________________________________ I

ORAL PRESENTATIONS

Physico-chemical profiling for Successful Lead Optimisation: Minimising the Risk of Attrition

Alan P. Hill _____________________________________________________________ 3

The role of physicochemical properties in pathways based toxicology risk assessment Paul J. Russell __________________________________________________________ 4

Integrated physico chemical methods for effective drug candidate selection Vesna Gabelica Marković _________________________________________________ 5

Uses of physicochemical properties to estimate the pharmacokinetics and pharmacodynamics of antibacterial fluoroquinolones

Kin Tam, Gergely Völgyi, Gábor Vizserálek, Krisztina Takács-Novák, Alex Avdeef ____ 6

High throughput determination log Po/w/pKa/log Do/w of drugs by combination of UHPLC and CE methods

Martí Rosés, Joan Marc Cabot, Elisabet Fuguet, Xavier Subirats __________________ 7

Applications of in situ Concentration Monitoring: Release from Nanoparticles, Supersaturation, Dual Component Separation and more …

Konstantin Tsinman, Oksana Tsinman, Alex Avdeef ____________________________ 8

The importance of pKa values in pharmaceutical science John Comer ____________________________________________________________ 9

The absolute importance of applicability domain in QSAR: A new in silico multiprotic pKa prediction tool with significantly improved prediction accuracy and new functi-onality for PhysChem, MedChem, CompChem, and Cheminformatics applications

Robert Fraczkiewicz, Robert D. Clark, Alexander Hillisch, Andreas H. Göller, Ursula Krenz, Rolf Schoenneis, Mario Lobell _________________________________ 10

On-line Chemical Modelling Environment – database and models for physico-chemical properties

Igor V. Tetko, Sergey Novotarskyi, Iurii Sushko, Robert Körner, Ahmed Abdelaziz ___ 11

Multivariate analysis of hydrophobic parameters Stefan Dove ___________________________________________________________ 12

Biorelevant in vitro performance testing of orally administered drug products: What and Why

Christos Reppas ________________________________________________________ 13

Usage of Caco-2 Permeability Studies for Biowaiving and Bioequivalence Applications of Immediate Released Generic Drug Formulations

Seval Korkmaz _________________________________________________________ 14


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Aceclofenac microspheres: Optimization of formulation using IV-Optimal statistical Design

Rameshwar K. Deshmukh, Jitendra B. Naik __________________________________ 15

Salt Solubility Determination, Complicated by the Presence of Drug Aggregates, Drug-Buffer Precipitates, and Co-precipitated Solids

Alex Avdeef ___________________________________________________________ 16

Diagnosing solubility limitations – the example of hydrate formation Jörg Berghausen _______________________________________________________ 17

Molecular Dynamics, Physical Stability and Solubility Advantage Z. Wojnarowska, K. Grzybowska, L. Hawelek, M. Dulski, M. Paluch, K. J. Paluch, L. Tajber, W. Sawicki ____________________________________________________ 18

High-throughput gravimetric moisture sorption in pharmaceutical preformulation U. J. Griesser, D. E. Braun ________________________________________________ 19

DVS: Application within a generic pharmaceutical company Jan Smith _____________________________________________________________ 20

Advanced Raman techniques in new drugs research and development Simona Cîntă Pînzaru, C. A. Dehelean, A. Fălămas, M. Steiner ___________________ 21

Co-Crystals: A tool to fine-tune solid-state properties Rolf Hilfiker ___________________________________________________________ 22

Determination of nanocrystals size distribution in polymer-drug systems M. Grassi, A Piccolo, M. Abrami, S. Fiorentino, R. Farra, G. Grassi, D. Hasa, D. Voinovich, B. Perissutti, I. Colombo ______________________________________ 23

Mechanochemistry of drugs E. V. Boldyreva ________________________________________________________ 24

X-ray diffraction methods in drug design Jindřich Hašek _________________________________________________________ 25

Relaxation dynamics and crystallization study of sildenafil in the liquid and glassy states

M. Paluch, K. Grzybowska, K. Kołodziejczyk, A. Grzybowski, Z. Wojnarowska, L. Hawelek, J. D. Zioło ___________________________________________________ 26

In vitro - in vivo extrapolation of transporters S. Neuhoff ____________________________________________________________ 27

Affinity- Mass Spectrometry and Ion Mobility- Mass Spectrometry: New Tools for Elucidating Structures, Reaction Pathways and Interactions of “Misfolding” - Aggregating Proteins

Michael Przybylski _____________________________________________________ 28

Application of PAT methods in the development and quality control of Orally Disintegrating Tablets

Tamás Sovány, Ildikó Oláh, Péter Kása jr., Klára Pintye-Hódi ____________________ 29

Fragment-Based Lead Generation in Drug Discovery Geoff Holdgate ________________________________________________________ 30

3rd World Conference on Physico-Chemical Methods in Drug Discovery and Development

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Forensic Intelligence for Medicine Anti-Counterfeiting Dégardin Klara, Roggo Yves, Margot Pierre _________________________________ 31

AFM Nanoimaging and nanomechanics for nanomedicine Ratnesh Lal, Srinivasan Ramachandran, Fernando Teran Arce, Preston Landon _____ 32

G-quadruplex DNA and DNA origami: Insights from atomic force microscopy Robert Henderson ______________________________________________________ 33

Structural dynamics of single molecules using Atomic Force Microscopy Jean-Luc Pellequer,

Shu-wen W. Chen,

Michael Odorico, Jean-Marie Teulon,

Pierre Parot, Rui C. Chaves _______________________________________________ 34

Online Bioaffinity-Mass Spectrometry: New Tool for Simultaneous Detection, Structure Determination and Affinity Quantification of Protein-Ligand Interactions from Biological Material

Michael Przybylski, Stefan Slamnoiu, Mihaela Stumbaum, Camelia Vlad, Kathrin Lindner, Christiaan Karreman, Marcel Leist, Bastian Hengerer ____________ 35

POSTER PRESENTATIONS

A new in silico multiprotic pKa prediction tool with significantly improved prediction accuracy and new functionality for PhysChem, MedChem, CompChem, and Cheminformatics applications

Robert Fraczkiewicz, Robert D. Clark, Alexander Hillisch, Andreas H. Göller, Ursula Krenz, Rolf Schoenneis, Mario Lobell _________________________________ 39

Challenges with development of a melting point model using public data: not all errors are the same!

Igor V. Tetko, Luc Patiny, Larisa Charochkina, Iurii Sushko, Ahmed Abdelaziz, Abdullah M. Asiri ______________________________________________________ 40

Lipophilicity assessment by Microemulsion Electrokinetic Chromatography (MEEKC) Xavier Subirats, Hui-Ping Yuan, Martí Rosés _________________________________ 41

Evaluation of log Po/w values of drugs from chromatographic measurements and from molecular structure calculations

Juan M. Pallicer, Martí Rosés, Clara Ràfols, Elisabeth Bosch, Rosalia Pascual, Adriana Port __________________________________________________________ 42

Prediction of human skin permeation of neutral compounds through chromatographic measurements

Martí Rosés, Elisabet Fuguet, Sara Soriano-Meseguer, Marta Hidalgo-Rodríguez ___ 43

PAMPA experimental measurements and QSAR model for membrane permeability of full pH range in human intestinal

Mare Oja, Uko Maran ___________________________________________________ 44

Development, characterization and permeability study on a model PAMPA membrane with polymer supported lipid bilayer

Gábor Vizserálek, Bálint Sinkó, Tamás Bozó, Krisztina Takács-Novák _____________ 45


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Buccal-PAMPA model: monosolvent or tissue specific lipid membrane systems? Preliminary study

Judit Müller, György T. Balogh ____________________________________________ 46

An investigation on Caco-2 Permeability Studies of Metformin or Gliclazide Alone and Gliclazide Plus Metformin

Seval Korkmaz, Banu Erdem ______________________________________________ 47

In vitro evaluation of penetration of nanonized alaptide from semi-solid formulations Josef Jampilek, Radka Opatrilova, Aneta Cernikova, Lenka Coufalova, Jiri Dohnal ___ 48

Biorelevant solubility study of poorly soluble neutral and ionizable drugs Gergely Völgyi, Vera Szőke, Péter Horváth, Krisztina Takács-Novák ______________ 49

Effect of plasdone polymers on the solubility enhancement of some acidic and basic drugs

Elisenda Fornells, Meritxell Mañé, Elisabet Fuguet, Clara Ràfols, Elisabeth Bosch, Rebeca Ruiz, Karl J. Box, John Comer _________________________ 50

Dynamic dissolution method provides rank-order prediction of in vivo performance for immediate release formulations of a BCS class II drug

Martin Čulen, Jiří Dohnal ________________________________________________ 51

Solubility and thermodynamic properties of methylxanthines Aneta. Pobudkowska, Urszula Domańska, Justyna. A. Kryska ___________________ 52

Evaluation of drug polymorphic difference on dissolution behavior Nataša Mrvičin, Vlatka Džale, Ivana Sorić, Ana Martinčić, Biserka Cetina-Čižmek ___ 53

Investigation of Xylazine hydrochloride phase transition from polymorphs Z and M to polymorph A

Kristīne Krūkle-Bērziņa, Kirils Oļehnovičs, Andris Actiņš ________________________ 54

Experimental and computational study of tegafur polymorphs and organic solvents interaction

Raitis Bobrovs, Andris Actiņš _____________________________________________ 55

The preparation of various forms of furosemide and analysis of their structures A. A. Beloborodova, V. S. Minkov, E. V. Boldyreva ____________________________ 56

Study and formulation of promazine hydrochloride–loaded nanoparticles Halayqa Mohammed, Urszula Domańska ___________________________________ 57

Domoic acid biotoxin from seawater environment: Raman and SERS characterization and detection

Csilla Müller, Karina Weber, Dana Cialla, Jürgen Popp, Iva Pozniak, Branko Glamuzina, Simona Cîntă Pînzaru ___________________________________ 58

Characterization of Antazoline Oxalate and Antazoline Maleate Agnese Dravniece, Kristīne Krūkle-Bērziņa, Andris Actiņš _______________________ 59

Characterization of Antazoline Base Kārlis Bērziņš, Ilze Grante, Andris Actiņš ____________________________________ 60


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Thermal study of antipsychotic active substance aripiprazole and its mixtures with different excipients

Adriana Fulias, Florin Soponar, Mihaela Maria Şandru, Balazs Szaniszlo, Niculina Bădicu, Ionut Ledeti _____________________________________________ 61

Synthesis, thermoanalytical behavior and EGA study of a highly specific anti-prostate cancer compound - betulonic acid

Adriana Fulias, Gabriela Vlase, Titus Vlase, Vasile Bercean, Ionut Ledeti __________ 62

Thermal behavior of antiemetic and gastroprokinetic agent metoclopramide hydrochloride and compatibility study with pharmaceutical excipients

Adriana Fulias, Gabriela Vlase, Titus Vlase, Ionut Ledeti _______________________ 63

Comparative thermal behavior of synthetic thyroid hormone L-thyroxine and L-thyroxine sodium salt hydrate vs. pharmaceutical formulations

Ionut Ledeti, Gabriela Vlase, Titus Vlase, Adriana Fulias _______________________ 64

Thermoanalytical study on methotrexate – active substance and tablets Ionut Ledeti, Gabriela Vlase, Titus Vlase, Georgeta Simu, Paul Albu, Adriana Fulias _ 65

Compatibility study of immunosuppressive agent methotrexate with some pharmaceutical excipients

Ionut Ledeti, Gabriela Vlase, Titus Vlase, Alina Heghes, Laura Sbârcea, Adriana Fulias _________________________________________________________ 66

Solid-state characterization of antiarrhythmic agent amiodarone hydrochloride under non-isothermal conditions. Compatibility study with some pharmaceutical excipients

Ionut Ledeti, Gabriela Vlase, Titus Vlase, Lucretia Udrescu, Adriana Fulias _________ 67

Inclusion complexes between pentacyclic triterpenes and amphiphilic cyclodextrins: synthesis and physico-chemical and pharmacological analysis

Codruta Soica, Cristina Trandafirescu, Florin Borcan, Corina Danciu, Stefana Avram, Rita Ambrus, Istvan Zupko, Ionut Ledeti, Adriana Fulias, Cristina Dehelean ________ 68

Liposomes for the drug delivery of Carboranes Temidayo Olusanya ____________________________________________________ 69

Drug release from redox sensitive poly(aspartic acid) based hydrogels Dávid Juriga, Rita A. Bauer, Miklós Zrínyi ___________________________________ 70

Hydroxyapatite scaffolds use in sustained release of ibuprofen – synthesis and characterization

Joanna Kołodziejek, Magdalena Szubert, Adam Voelkel ________________________ 71

Influence of HME downstream process options on in vitro release of low soluble drug substance in one formulation

Dunja Gamilec _________________________________________________________ 72

Evaluation of the performance and PK of a DPI formulation for in-vivo testing Valentina Bagnacani, Alessandro Fioni, Franco Bassani, Milco Lipreri, Paola Caruso, Marta Bellini, Francesco Amadei, Giandomenico Brogin, Silvia Catinella __________ 73


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Effect of ovocystatin polymerization on inhibitory activity towards legumain Krzysztof Gołąb, Katarzyna Juszczyńska, Bogusława Konopska, Maria Warwas, Jakub Gburek _________________________________________________________ 74

Study on micellization of sodium 3-dehydro-cholate by calorimetry and NMR Bojana Vasiljević, Etelka Tombácz, Dušan Škorić, László A. Király, Janoš Čanadi ____ 75

Pyrene Fluorescence Probe Method for Determination of Micelles Applying Low Energetic IR-Radiation

V. V. Komarov,, A. M. Popova, L. Schmidt, H. Jungclas _________________________ 76

Application of experimental design for the development of polymeric micro-nanoparticles

Rameshwar K. Deshmukh, Jitendra B. Naik __________________________________ 77

FTIR spectroscopic investigation of specific and non-specific interactions on oxo bile acid methyl esters

Janoš Čanadi, Branislav Jović, Branko Kordić, Dušan Škorić _____________________ 78

Aggregation behaviour of oxo bile acids – Tween 40 binary systems Dejan Ćirin, Mihalj Poša, Ana Sebenji ______________________________________ 79

Physico-chemical properties of poloxamer P 237 – Tween 80 or sodium dodecyl sulfate binary systems

Dejan Ćirin, Veljko Krstonošić, Mihalj Poša __________________________________ 80

Thermodynamics of the interactions between both bacterial and human ribosomal RNA with some antibiotic aminoglycosides

Javier Alguacil, Jordi Robles, Clara Ràfols, Elisabeth Bosch ______________________ 81

Liver membrane proteins sequestered on hemoglobin affinity matrix Jakub Gburek, Krzysztof Gołąb, Katarzyna Juszczyńska, Bogusława Konopska ______ 82

Estimation of the possible interactions between antioxidant drugs and membranes Fátima Paiva-Martins, Carina Silva, Marlene Costa, Paula Gameiro, Sonia Losada-Barreiro, Carlos Bravo-Díaz, Laurence S. Romsted _________________ 83

Human serum albumin binding of 2-[(carboxymethyl)sulfanyl]-4-oxo-4-arylbutanoic acids

Ilija N. Cvijetić, Dušan D. Petrović, Tatjana Ž. Verbić, Ivan O. Juranić, Branko J. Drakulić ______________________________________________________________ 84

Synthetic Cannabinoids as an Alternative to Cannabis: GC-MS, LC-MS/MS and LC-HRMS analyses

Zbynek Oktabec, Vera Maresova, Marie Balikova, Monika Zidkova ______________ 85

Organic synthesis of the pharmaceuticals with crystallization and determining of the morphological characteristics of fexofenadine-hydrochloride

Božo Banjanin, Rade Biočanin, Sonja Stefanov _______________________________ 86

AUTHOR INDEX ___________________________________________________ 87

Conference Programme


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Sunday, September 22nd - morning 09:00 – 10:00 Participant registration, Conference Hall, Hotel Princess Royal

10:00 – 12:00 & 16:00 – 18:00

Course 1, Lecture Room “Neptun”, Hotel Neptun QSAR analysis - principles, methods and applications Stefan Dove, University of Regensburg, Germany

10:00 – 12:00 & 16:00 – 18:00

Course 3, Lecture Room “Lokrum”, Hotel Neptun ABC of Modelling Transporter Data - Principles and Practical Know How Sibylle Neuhoff, Simcyp Ltd, UK

15:00 – 19.30 Participant registration, Conference Hall, Hotel Princess Royal

20:00 - Welcome party, Taraca Lounge bar

Monday, September 23rd (Royal Banquet Hall, Hotel Princess Royal) 08:00 Poster mounting, Princess Room 1, In front of the Royal Banquet Hall

09:00 Opening Ceremony

Chairs: Biserka Cetina Čižmek and John Comer

09:15 OP 01

Alan Hill, GSK, UK Physico-chemical profiling for Successful Lead Optimisation: Minimising the Risk of Attrition

10:55 OP 02

Paul J. Russell, Unilever, UK The role of physicochemical properties in pathways based toxicology risk assessment

10:30 OP 03

Vesna Gabelica Marković, Fidelta Ltd, Croatia Integrated physico chemical methods for effective drug candidate selection

10:45 Coffee break

11:00 OP 04

Kin Tam, Gergely Völgyi, Gábor Vizserálek, Krisztina Takács-Novák, Alex Avdeef, University of Macau, China Uses of physicochemical properties to estimate the pharmacokinetics and pharmacodynamics of antibacterial fluoroquinolones

11:40 OP 05

Martí Rosés, Joan Marc Cabot, Elisabet Fuguet, Xavier Subirats, Universitat de Barcelona, Spain High throughput determination log Po/w/pKa/log Do/w of drugs by combination of UHPLC and CE methods

12:20 OP 06

Konstantin Tsinman, Oksana Tsinman, Alex Avdeef, in-ADME Research, USA Applications of in situ Concentration Monitoring: Release from Nanoparticles, Supersaturation, Dual Component Separation and more …

13:00 Lunch


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Sunday, September 22nd - afternoon Chairs: Alex Avdeef and Kin Tam

15:00 OP 07

John Comer, Sirius Analytical Ltd., UK The importance of pKa values in pharmaceutical science

15:40 OP 08

Robert Fraczkiewicz, Robert D. Clark, Alexander Hillisch, Andreas H. Göller, Ursula Krenz, Rolf Schoenneis, Mario Lobell, Simulations Plus, USA & Bayer, Germany The absolute importance of applicability domain in QSAR: A new in silico multiprotic pKa prediction tool with significantly improved prediction accuracy and new functionality for PhysChem, MedChem, CompChem, and Cheminformatics applications

16:20 OP 09

Igor V. Tetko,Sergey Novotarskyi, Iurii Sushko, Robert Körner, Ahmed Abdelaziz, Helmholtz Zentrum München - German Research Center for Environmental Health, Germany On-line Chemical Modelling Environment – database and models for physico-chemical properties

17:00 Coffee break

17:20 OP 10

Stefan Dove, Universtiy of Regensburg, Germany Multivariate analysis of hydrophobic parameters

17:50 OP 11

Christos Reppas, National and Kapodistrian Collaborators of Athens, Greece Biorelevant in vitro performance testing of orally administered drug products: What and Why

18:20 OP 12

Seval Korkmaz, Abdi Ibrahim, Turkey Usage of Caco-2 Permeability Studies for Biowaiving and Bioequivalence Applications of Immediate Released Generic Drug Formulations

18:50 OP 13

Rameshwar K. Deshmukh, Jitendra B. Naik, University Institute of Chemical Technolgy, India Aceclofenac microspheres: Optimization of formulation using IV-Optimal statistical Design

19:20 End of Monday's session


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Tuesday, September 24th (Royal Banquet Hall, Hotel Princess Royal) Chairs: Rolf Hilfiker and Mario Grassi

09:00 OP 14

Alex Avdeef, in-ADME Research, USA Salt Solubility Determination, Complicated by the Presence of Drug Aggregates, Drug-Buffer Precipitates, and Co-precipitated Solids

09:40 OP 15

Jörg Berghausen, Novartis Institutes for Biomedical Research, Switzerland Diagnosing solubility limitations – the example of hydrate formation

10:15 OP 16

Z. Wojnarowska, K. Grzybowska, L. Hawelek, M. Dulski, M. Paluch, K.J. Paluch, L. Tajber, W. Sawicki, University of Silesia, Poland Molecular Dynamics, Physical Stability and Solubility Advantage from Amorphous Indapamide Drug

10:45 Coffee break

11:05 OP 17

Ulrich Griesser, Doris E. Braun, University of Innsbruck, Austria High-throughput gravimetric moisture sorption in pharmaceutical preformulation

11:45 OP 18

Jan Smith, Synthon BV, The Netherlands DVS: Application within a generic pharmaceutical company

12:20 OP 19

Simona Cîntă Pînzaru, C. A. Dehelean, A. Fălămas, M. Steiner, Babes-Bolyai University, Romania Advanced Raman techniques in new drugs research and development

13:00 Lunch

Chairs: Ulrich Griesser

15:00 OP 20

Rolf Hilfiker, Solvias AG, Switzerland Co-Crystals: A tool to fine-tune solid-state properties

15:40 OP 21

M. Grassi, A Piccolo, M. Abrami, S. Fiorentino, R. Farra, G. Grassi, D. Hasa, D. Voinovich, B. Perissutti, I. Colombo, Trieste University, Italy Determination of nanocrystals size distribution in polymer-drug systems

16:20 Coffee break

16:40 OP 22

Elena Boldyreva, Novosibirsk State University, Russia Mechanochemistry of drugs

17:20 OP 32

Jindřich Hašek, Institute of Biotechnology, Czech Republic X-ray diffraction methods in drug design

17:55 OP 24

M. Paluch, K. Grzybowska, K. Kołodziejczyk, A. Grzybowski, Z. Wojnarowska, L. Hawelek, J. D. Zioło, University of Silesia, Poland Relaxation dynamics and crystallization study of sildenafil in the liquid and glassy states

18.30 Poster session (until 23:00)


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Wednesday, September 25th (Royal Banquet Hall, Hotel Princess Royal) 08:00 Poster dismounting

Chairs: Vesna Gabelica Marković and Predrag Novak

09:00 OP 25

Sibylle Neuhoff, Simcyp (a Certara company), UK In vitro - in vivo extrapolation of transporters

09:40 OP 26

Michael Przybylski, University of Konstanz, Germany Affinity- Mass Spectrometry and Ion Mobility- Mass Spectrometry: New Tools for elucidating structures, reaction pathways and interactions of “misfolding” - aggregating proteins

10:20 OP 27

Tamás Sovány, Ildikó Oláh, Péter Kása jr., Klára Pintye-Hódi, University of Szeged, Hungary Application of PAT methods in the development and quality control of Orally Disintegrating Tablets

10:50 Coffee break

11:10 OP 28

Geoff Holdgte, AstraZeneca, UK Fragment-Based Lead Generation in Drug Discovery

11:50 OP 29

Dégardin Klara, Roggo Yves, Margot Pierre, F. Hoffmann-La Roche, Switzerland Forensic Intelligence for Medicine Anti-Counterfeiting

12:30 Lunch

14:00 Excursion + Social Dinner


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Thursday, September 26th

(Lecture room Coral, Hotel Neptun) Joint PCMDDD-3 & COST Action TD 1002 session

Chairs: Vesna Svetličić and Pierre Parot

09:00 OP 30

Ratnesh Lal, Srinivasan Ramachandran, Fernando Teran Arce, Preston Landon, University of California San Diego, USA AFM Nanoimaging and nanomechanics for nanomedicine

09:40 OP 31

Robert Henderson, University of Cambridge, UK G-quadruplex DNA and DNA origami: Insights from atomic force microscopy

10:20 Coffee break

10:35 OP 32

Jean-Luc Pellequer, Shu-wen W. Chen, Michael Odorico, Jean-Marie Teulon, Pierre Parot, Rui C. Chaves, CEA, France Structural dynamics of single molecules using Atomic Force Microscopy

11:15 OP 33

Seminar/Workshop Michael Przybylski, Stefan Slamnoiu, Mihaela Stumbaum, Camelia Vlad, Kathrin Lindner, Christiaan Karreman, Marcel Leist, and Bastian Hengerer, University of Konstanz, Germany Online Bioaffinity-Mass Spectrometry: New Tool for Simultaneous Detection, Structure Determination and Affinity Quantification of Protein-Ligand Interactions from Biological Material

12:00 Conference closing

Oral Presentations

3nd World Conference on Physico-Chemical Methods in Drug Discovery and Development

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Physico-chemical profiling for Successful Lead Optimisation: Minimising the Risk of Attrition

Alan P. Hill

GlaxoSmithKline, Stevenage UK Physicochemical measurements are key enablers for successful medicinal chemistry. The availability of physicochemical data early in the drug discovery process can highlight potential issues, which if unresolved could result in the progression of candidate drug molecules with sub optimal properties resulting in high late stage attrition. The physchem teams at GSK have developed assays and models to support our medicinal chemistry efforts. Providing the appropriate physicochemical data, at the right stage of the lead optimisation process, allows chemistry teams to readily incorporate molecular modifications to address potential issues. Examples will be given, demonstrating where in-house properties (both calculated and measured) have been used to guide chemistry projects away from potentially detrimental physicochemical and biological characteristics, ensuring that candidate drug molecules possess more desirable attributes and consequently minimising the likelihood of late stage attrition.

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The role of physicochemical properties in pathways based toxicology risk assessment

Paul J. Russell

Safety and Environmental Assurance Centre, Unilever, Colworth Science Park, Shanbrook, Bedfordshire, UK, MK44 1LQ

Consumer and environmental safety decisions are based on exposure and hazard data interpreted using risk assessment approaches. The Adverse Outcome Pathway (AOP) conceptual framework [1] has been presented as a logical sequence of events or processes within biological systems which can be used to understand adverse effects and refine the current risk assessment practice. Although originally described as an approach to ecotoxicology risk assessment, this is now being extended into the human safety area. The AOP shifts the risk assessment focus from traditional toxicological apical endpoints to the development of a mechanistic understanding of a chemical’s effect at a molecular and cellular level. In order to obtain this level of mechanistic detail, chemistry in all its disciplines has a key role to play [2]. Physico-chemical properties used in measurement and effective predictive techniques will be important in understanding chemical characterisation, free concentration and exposure at the site of interest. This is particularly relevant at the first interaction between a chemical and a biological system (or the molecular initiating event, MIE) which can initiate a series of subsequent biochemical events. Physico-chemical inputs will be vital in developing structure-based toxicological alerts and informing predictive models for pathways based risk assessments. The challenge is identifying which parameters are key for a successful model and determining the most appropriate ways to measure or predict them through an integrated wet/dry approach. www.tt21c.org [1] G.T. Ankley et al, Adverse outcome pathways: a conceptual framework to support ecotoxicology

research and risk assessment, Environmental Toxicology and Chemistry. 29 (2010) 730–741. [2] S. Gutsell and P.J. Russell, The role of chemistry in developing understanding of adverse

outcome pathways and their application in risk assessment, Toxicology Research. (2013) DOI: 10.1039/C3TX50024A.

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Integrated physico chemical methods for effective drug candidate selection

Vesna Gabelica Marković

Fidelta Ltd, Zagreb, Croatia Drug discovery and development is under pressure to meet the demands for high quality compounds. In the early phase of discovery it is well known that identity and physicochemical properties of potent molecules is unavoidable. In the late stage of discovery and development additional properties such as stability, impurity profile and solid form are required for candidate selection. There is a list of different methods for measurement of properties however Fidelta’s approach is to use the maximum of different techniques by transferring methods and integrating the obtained data. In this presentation a few examples of using physicochemical methods, such as stability of compounds, identification and isolation of degradation products and measurement of physicochemical properties will be described.

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Uses of physicochemical properties to estimate the pharmacokinetics and pharmacodynamics of antibacterial fluoroquinolones

Kin Tam, Gergely Völgyi*, Gábor Vizserálek*, Krisztina Takács-Novák*, Alex Avdeef**

Faculty of Health Science, University of Macau, Macau, China *Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary

**in-ADME Research, New York, USA Fluoroquinolones (FQs) are important antimicrobial agents, with a broad anti-infectious spectrum against a number of both Gram-positive and Gram-negative bacteria. Norfloxacin is the first FQ that was approved in 1986. Since then, newer generations of quinolone antibacterial agents have been developed. There is a continuing need for new antimicrobial agents to deal with emerging bacterial infections. While developing novel bacterial targets may be a little more challenging and time consuming, one alternative approach could be to continue modifying existing classes of successful antibiotics, such as FQs, to enhance their clinical efficacy [1]. In the development of new FQs, two important considerations would be useful to assess the effectiveness of the compounds. These are: (1) the microbiological potency, which is described by the minimum inhibitory concentration (MIC), and (2) the pharmacokinetics of the selected compound, which is described by the area under the concentration–time curve (AUC). A number of FQs spanning from second to fourth generations of quinolone have been selected in the present study. The physiochemical properties of these FQs, namely parallel artificial membrane permeability and partition coefficient, were determined in our laboratories [2]. It has been demonstrated that permeability and partition coefficient are useful descriptors for predicting AUC and MIC data of the selected antibacterial FQs. We have shown that the AUC/MIC ratios predicted from our models are in good agreement with the literature values. With the aid of these models, the AUC/MIC prediction could be carried out well ahead of any pharmacokinetics/pharmacodynamics studies, and microbiological evaluations, which would help the development of new efficacious FQs. [1] Bush, K., Pucci, M.J., 2011. New antimicrobial agents on the horizon. Biochemical Pharmacology

82, 1528–1539. [2] Völgyi, G., Vizserálek, G., Takács-Novák, K., Avdeef, A., Tam, K.Y., 2012. Predicting the exposure

and antibacterial activity of fluoroquinolones based on physicochemical properties. European Journal of Pharmaceutical Sciences 47, 21-27.

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High throughput determination log Po/w/pKa/log Do/w of drugs by combination of UHPLC and CE methods

Martí Rosés, Joan Marc Cabot, Elisabet Fuguet, Xavier Subirats

Departament de Química Analítica - Institut de Biomedicina, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona

The drug discovery and development process requires the high-throughput determination of physicochemical parameters of drugs and drug candidates. Parameters measuring lipophilicity, acidity, solubility or protein interaction must be determined in a fast way for a high number of compounds of potential interest to select the most appropriate for further development. The most important physical property affecting biological activity of substances is their lipophilicity expressed as the logarithm of the octanol-water partition ratio (log Po/w). The knowledge of the log D (the effective log P at a particular pH) is crucial in drug discovery. Years ago, Valkó et al. developed a generic fast gradient HPLC method, based on the calculation of the chromatographic hydrophobicity index (CHI) from the gradient retention times, in order to measure lipophilicity. We have enhanced the correlations between CHI and log Po/w and adapted the rapid gradient HPLC method to UHPLC obtaining excellent resolution and repeatability in a short analysis time (<4min). The log Po/w can be easily obtained from these CHI measurements. Unfortunately, these correlations are only valid for non-ionized compounds. Consequently, we have combined the UHPLC-CHI/lop Po/w method with the fast high-throughput method for pKa determination. The IS-CE method is based in the use of internal standard (IS) of pKa similar to the test compound to determine the pKa of the test compound by capillary electrophoresis (CE). Mobilities of standards and test are measured in two pH buffers, where they are partially or totally ionized, respectively. The difference between the pKas of test and standard is easily calculated from the ratios of the mobilities differences between test and standard. Two CE runs are enough for each pKa. Both methods have been combined in order to describe, as a high throughput alternative, the log D/pH profile sample compound. This communication describes the routine imple-mentation of the method in physicochemical measurement laboratories in order to get the lipophilicity profiles of bioactive compounds and compares and contrasts the log P, pKa and log D profiles obtained with the ACD/Labs predictions and with the log P and log D7.4 (log D at physiological pH) experimental values determined by the usual shake-flask method.

[1] K. Valkó, C.M. Du, C.D. Bevan, D.P. Reynols, M.H. Abraham, Rapid-Gradient HPLC for Measuring Drug Interections with Immobilitzed Artificial Membrane: Comparison with Other Lipophility Measures, J. Pharm. Sci. 89 (2000) 1085-1096.

[2] J.M. Cabot, E. Fuguet, C. Ràfols, M. Rosés, Determination of acidity constants by the capillary electrophoresis internal standard method. IV. Polyprotic compounds, J. Chromatrogr. A 1279 (2013) 108-116.

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Applications of in situ Concentration Monitoring: Release from Nanoparticles, Supersaturation, Dual Component Separation and more …

Konstantin Tsinman, Oksana Tsinman, Alex Avdeef*

Pion Inc., USA; *in-ADME Research, USA

PURPOSE: Presented studies demonstrate benefits of in situ concentration monitoring for multiple applications essential during the drug development phase. Among others, the case studies include the investigation of supersaturation behavior for amorphous formulations in bio-relevant media, a method for in situ measurements of free API being released from nanoparticles, estimation of effective particle sizes from powder dissolution profiles and a combined dissolution-permeability assay with dynamic concentration assessment. METHODS: The drug solution concentration versus time was evaluated in 1-20 mL of media using µDISS Profiler™ (Pion Inc.). An eight channel dispensing module add-on can be programmed to change the media during the run, e.g., by delivering specially formulated concentrate transforming SGF into FaSSIF. Concentration of API in the presence of nanoparticles was measured using zero intercept method (ZIM). In ZIM, zero-crossing points of second derivative spectrum are used to separate the contributions of free API and nanoparticles in the overall signal. The Dissolution-Permeability (DP) apparatus is an add-on option to µDISS Profiler consisting of three pairs of temperature controlled side-by-side permeability chambers mounted on the stirring platform. Each pair is separated by user-specified membrane, e.g., filter supported GIT-optimized artificial membrane. RESULTS: Solubility of amorphous Piroxicam in pH 5.0 buffer and in FeSSIF was up to 10 times higher than in case of crystalline material (~ 20 µg/mL). Its re-precipitation kinetics in buffer was dose dependent with fully dissolved quasi-stable state (> 24 h) for 70 µg/mL solution and quick re-precipitation for 200 µg/mL loads. Release of naproxen from nanosuspension was qualitatively different from other forms while nano-sized naproxen solubility was similar to its micro-sized forms (18.6±0.7 µg/mL) also confirmed by the dialysis experiment. Estimated from the dissolution curves particle sizes agreed well with independent particle size measurements. The flux of danazol permeating from pH 5.0 buffer into blood-simulating environment was determined to be 103*10

-6 µg*cm

-2*s

-1. Although solubility of

danazol in FeSSIF was ~ 40 times higher, its flux increased only ~4.5 times. CONCLUSIONS: The real time concentration monitoring approach helps with fundamental understanding of processes that may be relevant in vivo providing rational basis to formulation design and development. Novel ZIM method overcomes challenges associated with solid separation of nanoparticles from solution allowing in situ measurement of free API concentration in the presence of nanoparticles. DP apparatus extends capability of µDISS Profiler allowing investigation of a peculiar interplay between solubility and permeability in the complex media.

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The importance of pKa values in pharmaceutical science

John Comer

CSO, Sirius Analytical Ltd. Pharmaceutical scientists already understand that pKa is an important property that influences lipophilicity, solubility and dissolution, as well as other ADMET properties. Although these effects are well documented, there has been continued interest in this field, with a steady stream of new publications featuring pKa in medicinal chemistry having been published since the first IAPC meeting in 2009. This presentation will review some of these publications and discuss their conclusions in the light of what we know is possible from an experimental point of view.

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The absolute importance of applicability domain in QSAR: A new in silico multiprotic pKa prediction tool with significantly improved

prediction accuracy and new functionality for PhysChem, MedChem, CompChem, and Cheminformatics applications

Robert Fraczkiewicz, Robert D. Clark, Alexander Hillisch*, Andreas H. Göller*, Ursula Krenz*, Rolf Schoenneis*, Mario Lobell*

Simulations Plus, Lancaster, CA 93534, USA * Bayer Pharma AG, Global Drug Discovery, Wuppertal, Germany

Ultimately, QSAR models are useful if they help push forward the boundary of scientific research. One of the crucial utilitarian aspects, the applicability domain (AD) of a QSAR model (understood in terms of chemical space coverage), does not receive as much atten-tion as specific modeling methodologies or descriptor sets. In this work we show that at least in terms of pharmaceutical applications, the AD should have high priority. In an un-paralleled joint collaboration, Simulations Plus and the Bayer Pharma AG have developed a new in silico pKa prediction tool which is currently in use at Bayer and will be a part of the upcoming release 7.0 of the Simulations Plus ADMET Predictor software. The new model has been trained with curated sets of ~14000 literature pKa values (~11000 compo-unds, representing literature chemical space) and ~19500 pKa values experimenttally determined at Bayer Pharma (~16000 compounds, representing industry chemical space). We compare and quantify differences between the two domains. The new model is also compared to one developed exclusively within the literature AD (“old model”, version 6.1 of ADMET Predictor). Model validation was performed with ~31000 new pKa values measured at Bayer. On the largest and most difficult test subset with >16000 pKa values (all from measurements on new compounds after training with the first Bayer training set) the old model achieved a mean absolute error (MAE) of 0.72, a root mean square error (RMSE) of 0.94, and a squared correlation coefficient (R

2) of 0.87. The new model achieved

significantly improved prediction statistics, with MAE=0.50, RMSE=0.67 and R2=0.93.

At Bayer, the new pKa prediction model has been integrated into Pipeline Pilot and the PharmacophorInformatics platform PIx used by scientists at Bayer Pharma. Different output formats have been defined which allow customized use by Medicinal Chemists, Physical Chemists, and Computational Chemists. The fast calculation speed (>100,000 compounds/h [CPU: Intel Xeon L5420, 2.5 GHz]) allows Computational Chemists to automatically modify the protonation state of structures to reflect their dominant charge state(s) at a given pH (default 7.4) in order to prepare large libraries for high-throughput docking applications (e.g. virtual screening). The mathematical modeling methodology has been published elsewhere [1]. All the models mentioned above are resolved with respect to all possible ionization microequili-bria. In this work, we also introduce novel concepts of Averaged Site Protonation and Sin-gle Proton Midpoint (pK50) as measures of individual ionizable group acidities in multi-protic molecules.

[1] R. Fraczkiewicz, In Silico Prediction of Ionization, in: B. Testa, H. van de Waterbeemd (Eds.), Comprehensive Medicinal Chemistry II, Elsevier, Oxford, UK, 2006, Vol. 5, pp. 603-626.

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On-line Chemical Modelling Environment – database and models for physico-chemical properties

Igor V. Tetko*,**, Sergey Novotarskyi**, Iurii Sushko**, Robert Körner**,

Ahmed Abdelaziz**

*Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH), Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany

**Chemistry Department, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah 21589, Saudi Arabia and eADMET GmbH, Lichtenbergstraße 8,

D-85748 Garching, Germany

The Online Chemical Modeling Environment1 is a web-based platform (http://www.och-

em.eu) that aims to automate and simplify the typical steps required for QSAR modeling. It is a logical extension of Virtual Computational Chemistry Laboratory (VCCLAB) http://www.vcclab.org project,

2 which has been dramatically extended with new ideas

and functionality. The platform consists of two major subsystems: the database of experimental measurements and the modeling framework. As compared to other similar systems, OCHEM is not intended to re-implement the existing tools or models but rather to invite the original authors to contribute their results, make them publicly available, and share them with other users and to become members of the growing research community. Our intention is to make OCHEM a widely used platform to perform the QSPR/QSAR studies online and share it with other users on the Web. In this poster we will exemplify data and models, which are available on the OCHEM for physico-chemical properties, i.e. melting point, logP, solubility in water and in DMSO, boiling point, vapour pressure, etc. The importance of the accuracy of predictions will be demonntsrated for model to predict solubility in DMSO.

3 The identification of

overrepresented structural fragments, atutomatic outlier detection, modeling of chemical mixtures feature nets, will be described and demonstrated using the on-line tools. This study was partly supported by the European Union Marie Curie Initial Training Network project “Environmental Chemoinformatics” (ECO), grant agreement number 238701, and the GO-Bio 1B BMBF project iPRIOR, grant agreement number 315647. 1) Sushko I, et al Online chemical modeling environment (OCHEM): web platform for data storage,

model development and publishing of chemical information. J Comput Aided Mol Des. 2011 Jun;25(6):533-54.

2) Tetko IV, et al Virtual computational chemistry laboratory--design and description. J Comput Aided Mol Des. 2005 Jun;19(6):453-63.

3) Tetko I.V, et al Development of Dimethyl Sulfoxide Solubility Models Using 163 000 Molecules: Using a Domain Applicability Metric to Select More Reliable Predictions. J Chem Inf Model. 2013, DOI: 10.1021/ci400213d

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http://www.ochem.eu/

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Multivariate analysis of hydrophobic parameters

Stefan Dove

Univ. Regensburg, Inst. Pharmacy, 93040 Regensburg, Germany

Following the IUPAC definition, hydrophobicity is the association of non-polar groups or molecules in an aqueous environment which arises from the tendency of water to exclude non-polar molecules. The pioneering work of Corwin Hansch and Albert Leo provided the basis for the consideration of hydrophobic effects in QSAR analysis and, by this, to quantitatively characterize the transport of bioactive molecules in biosystems and "hydrophobic binding" to targets. More than 20,000 log P values of solutes in different solvent/water partition systems were compiled till 1995, and hydrophobic substituent

constants were derived from log P (n-octanol/water) for various aromatic series [1, 2]. Multivariate approaches like principal component or factor analysis may help to explain these data and to discriminate between intrinsic hydrophobicity and polar contributions like hydrogen bonds, inductive and mesomeric effects. Multivariate analyses of log P measured for 18 solutes in six solvent/water systems and of

-values for 14 meta- and para-substituents, respectively, on eight aromatic scaffolds were performed (re-analysis of previous work [3, 4]). The data are reproduced within experimental errors by two principal components (three in the case of the para-substituents). Additionally to an intrinsic or average hydrophobic component, log P values depend on the water solubility of the solvent and hydrogen bonding capabilities of both

the solute and the solvent. Analysis of -values also results in a component quantifying

intrinsic hydrophobicity. The -scale of the commonly used benzene series is compressed by electron-attracting functional groups (in, e.g., nitrobenzenes) and spread by electron releasing groups (in, e.g., anilines). A second component is related to inductive and mesomeric effects and correlates with electronic substituent constants. The data indicate

that electron attracting functional groups increase the -values of electron releasing substituents and vice versa. In conclusion, intramolecular interactions with functional groups affect the hydrophobic "constants" of substituents, so that additivity rules underlying the computation of log P are violated. [1] C. Hansch, A. Leo, Substituent constants for correlation analysis in chemistry and biology, Wiley

& Sons, New York, 1979. [2] C. Hansch, A. Leo, D. Hoekman, Exploring QSAR; Vol. 2: Hydrophobic, electronic and steric

constants, ACS, 1995. [3] R. Franke, S. Dove, R. Kühne, Hydrophobicity and hydrophobic interactions I. On the physical

nature of aromatic hydrophobic substituent constants, Eur. J. Med. Chem. 14 (1979), 363-372. [4] S. Dove, R. Franke, R. Kühne, Principal component analysis of partition coefficients in different

solvent systems, in: J. Knoll, F. Darvas (Eds.), Chemical Structure - Biological Actvity Relationships: Quantitative Approaches, Akademiai Kiado, Budapest, 1980, pp. 247-255.

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Biorelevant in vitro performance testing of orally administered drug products: What and Why

Christos Reppas

Department of Pharmaceutical Technology, Faculty of Pharmacy, National and Kapodistrian Collaborators of Athens, Greece

In the development of new Active Pharmaceutical Ingredients (APIs) as well as new formulations of existing products (including generic products) it is of great interest to be able to predict to what extent the API can be absorbed from the gastrointestinal (GI) tract and how formulation may affect the absorption profile. Typical aspects of GI physiology which can influence drug absorption are the composition of the GI fluids (which affects various processes including release from the dosage form and stability of the API), GI motility and hydrodynamics (transit characteristics of the dosage form, release from the dosage form etc), permeability of the GI mucosa to the API as a function of location in the GI tract, and gut wall metabolism. Of these, formulations effects are typically targeted at optimizing the site and pattern of drug release, so the focus of biorelevant testing in this case is the composition and hydrodynamics of the GI fluids at intended site(s) of release – so-called biorelevant dissolution testing. The baseline performance of the API in terms of its solubility and dissolution characteristics under the intended conditions of release, should also be established to serve as a reference against which formulation efforts can be evaluated for their ability to optimize performance. The degree of simulation of lumenal composition and hydrodynamics for in vitro data to be physiologically relevant (or, biorelevant) varies with the dosage form, the API, and the specific estimation to be made or the specific process to be simulated. In this presentation, the physiological conditions in the GI tract relevant to API and formulation performance will be highlighted and description of in vitro test conditions which can be used to simulate these will be made. In addition, the usefulness of biorelevant media in measuring lumenal solubility at various locations of the gastrointestinal lumen and in evaluating the impact of formulation and of food on the absorption of lipophilic APIs will be presented. The potential importance of medium composition on the apparent intestinal permeability coefficient of lipophilic APIs that are administered as lipid dosage forms will be addressed. The usefulness of biorelevant media in the evaluation of lumenal processes other than dissolution will finally be discussed, i.e. the evaluation of lumenal disintegration of tablets and capsules, the evaluation of binding of the API onto lumenal materials, and the evaluation of the stability of the API at various locations in the gut.

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Usage of Caco-2 Permeability Studies for Biowaiving and Bioequivalence Applications of Immediate Released Generic Drug Formulations

Seval Korkmaz

1 Abdi İbrahim İlaç A.Ş. R&D Center, 34555 Esenyurt- İstanbul, TURKEY

[email protected]

BCS (Biopharmaceutics Classification System) is a system to classify drugs to the four different classes on the basis of their solubility and permeability characteristics. It is possible to apply to biowaiving by demonstrating its permeability and solubility profiles of either API (active pharmaceutical ingradient) or formulations. BCS-based biowaivers are applicable for immediate-release solid oral dosage formulations containing one or more of the API(s) mentioned above if the required data ensure the similarity of the submitted pharmaceutical product and the appropriate pharmaceutically equivalent comparator product. Caco-2 permeability method is one of the common in vitro model to test intestinal permeability charecteristics of drugs. While they are used especially to identification of API’s permeability profile, at the otherhand it is possible to verify similarity of generic drug formulation to the original or reference drug. We aimed to explain and show data on usage of BCS data for generic drug applications. It is possible to apply for biowaiving not only for class I and/or class III drugs but also extentions make possible to apply for biowaiving of certain BCS class II drugs such as weak asids or bases. Biowaiving rules, importance of polymorphism and usage of Caco-2 studies for preparation of biowaiver files and estimation of bioequivalence test results will be explain during the current presentation.

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Aceclofenac microspheres: Optimization of formulation using IV-Optimal statistical Design

Rameshwar K. Deshmukh, Jitendra B. Naik*

University Institute of Chemical Technolgy, North Maharashtra University, Jalgaon, Maharashtra, India-425 001

In the present study, polymeric microspheres containing aceclofenac was formulated by single emulsion [oil-in-water (o/w)] solvent evaporation method using response surface methodology by employing a IV-optimal design using the Design-Expert® software (Version-8.0.7.1). Two biocompatible polymers, ethylcellulose, and Eudragit® RS100 were used in combination. Two formulation variables and one process variables were investigated by statistical experimental design in order to enhance the encapsulation efficiency of the microspheres. Amount of PVA (surfactant) and amount of polymers in combination were selected as formulation variables and speed of the stirrer as process variable. The resultant microspheres were characterized for their size, morphology, encapsulation efficiency, and in vitro drug release. Imaging of particles was performed by field emission scanning electron microscopy. The drug polymer interactions were investigated by Fourier Transform Infrared spectroscopy (FTIR), and X-ray powder diffractometry (XRPD). Graphical and mathematical analysis of the design showed the polyvinyl alcohol (Surfactant) and speed of the stirrer were a significant effect and identified as the significant factors determining the encapsulation efficiency of the microspheres. A quadratic mathematical model equation fitted to the data was used to predict the encapsulation efficiency in the optimal region. Optimized formulation of microspheres was prepared using optimal process variables setting in order to evaluate the optimization capability of the models generated according to IV-optimal design. Low magnitude of error and significant value of R

2 proves the high prognostic ability of the

design. The encapsulation efficiency of the microspheres was found to be 61.86% to 84.07%. The absence of drug polymer interactions was confirmed by FTIR spectroscopy. XRPD revealed the molecular dispersion of drug within microspheres formulation. The microspheres were found to be discrete, spherical with smooth surface. Perfect prolonged drug release profile (18.55% - 62.79%) in 12 hours was achieved by a combination of polymers. In conclusion, polymeric microspheres containing aceclofenac can be successfully prepared using the technique of IV-optimal experimental design, and these results helped in finding the optimum formulation variables for enhancement of encapsulation efficiency of microspheres.

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Salt Solubility Determination, Complicated by the Presence of Drug Aggregates, Drug-Buffer Precipitates, and Co-precipitated Solids

Alex Avdeef

in-ADME Research, USA

PURPOSE: To consider three challenging topics in the determination of salt solubility: (a) in many cases (esp. for hydrophobic molecules), the log S vs. pH curve cannot be described by the Henderson-Hasselbalch equation, (b) it is often necessary to add excessive amount of drug in order to form a saturated salt solution, so ionic strength may be as high as 5 M and the activities of ions cannot be predicted with the Debye-Hückel equation, and (c) under gastric pH conditions, (pH ~ 1), accurate measurement of pH is difficult. METHODS: A novel solubility-pH analysis computer program, pDISOL-X

TM, has been developed, which

can determine ionization constants (pKa), intrinsic solubility (S0), salt solubility products (Ksp), and aggregation/complexation constants (Kn). The unique algorithm does not require any explicitly derived solubility equations based on extensions of the Henderson-Hasselbalch equation. Rather, the program internally derives implicit equations, given a set of equilibria and constants (which are then iteratively refined by weighted nonlinear regression). E.g., the effects of drug-salt precipitates, -aggregates, -complexes, -bile salts, can be tested. Buffer details are part of the calculation. The ionic strength and buffer capacity are rigorously calculated (using the Stokes and Robinson 1948 hydration theory, slightly modified by Bockris and Reddy in 1973), at each step, and equilibrium constants are accordingly adjusted for ionic strength variation. The Avdeef-Bucher (1978) four-parameter calibration equation is automatically compensated for very low pH solutions. RESULTS: The 25 basic drugs whose log S-pH profiles (in 0.15 M phosphate solutions) reported by Bergström et al. (2004) were re-analyzed. It was possible to rationalize quantitatively the solubility-pH profiles of all 25 drugs, in terms of phosphate or chloride precipitates in acidic solutions and the free base precipitates in alkaline solutions, often in the presence of an aggregated drug species. Considerably different intrinsic solubility constants were found, compared to those originally reported, for the drugs celiprolol, desipramine, fendiline, haloperidol, procyclidine, terazosin, and thioridazine. The least soluble molecule was amiodarone, which analyzed to have an intrinsic solubility of about 2 pg/mL, a salt solubility of 0.82 mg/mL at the Gibbs-pKa 5.43, corresponding to the species BH.H2PO4(s), and a strong presence of the cationic aggregate, (BH)5. CONCLUSIONS: With the aid of the new data analysis algorithm, it was evident that phosphate buffers can dramatically influence the solubility profiles of sparingly-soluble drugs, and clearly, the Bergström et al. data could not be explained by the simple Henderson-Hasselbalch equation. With sparingly-soluble drugs, such complexities may be common, but are not easy to analyze. pDISOL-X could be a helpful new tool in such instances.

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Diagnosing solubility limitations – the example of hydrate formation

Jörg Berghausen

Novartis Institutes for Biomedical Research, Novartis Campus, CH-4002 Basel

Solubility remains one of the key challenges in many drug discovery projects. Thus, it’s essential to support the lead finding and optimization efforts by appropriate solubility data. In this contribution, current ideas for solubility characterization supporting drug discovery efforts at Novartis are outlined. In silico solubility prediction remains challenging and therefore a screening assay is used as a first filter, followed by selected follow-up assays to reveal what causes the low solubility of a specific compound or chemotype. Results from diagnosing the underlying reason for solubility limitation are discussed. As lipophilicity and crystal lattice forces are regarded as main contributors to limiting solubility, changes in solid state are important to be recognized. Solubility limitation by various factors will be presented and the impact of the solid-state is exemplified by compounds that are able to form hydrates.

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Molecular Dynamics, Physical Stability and Solubility Advantage from Amorphous Indapamide Drug

Z. Wojnarowska, K. Grzybowska, L. Hawelek, M. Dulski, M. Paluch, K. J. Paluch*, L. Tajber*, W. Sawicki**

Institute of Physics, University of Silesia, ul. Uniwersytecka 4, 40-007 Katowice, Poland *School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland

**Department of Physical Chemistry, Medical University of Gdansk, Hallera 107, 80-416, Gdansk, Poland

Indapamide (IND) is a sulphonamide diuretic drug used in the treatment of hypertension. Despite having a slightly different chemical structure than thiazides (e.g. hydrochloro-thiazide), its mechanism of action remains similar. It increases the urine volume by increasing the renal excretion of sodium, chlorine, potassium and magnesium ions. However, the commercial form of IND available on the market works very weakly. The most probable reason is its low solubility (75 mg/l) and consequently poor bioavailability. Thus, the question arise how to improve the solubility of indapamide? This study for the first time investigates physicochemical properties of amorphous indapamid drug (IND). The solid-state properties of the vitrified, cryo- and ball-milled IND samples were analyzed using X-ray powder diffraction (XRD), mass spectrometry, infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and broadband dielectric spectroscopy (BDS). These analytical techniques enabled us (i) to confirm the purity of obtained amorphous samples, (ii) to describe the molecular mobility of IND in the liquid and glassy state, (iii) to determine the parameters describing the liquid-glass transition i.e. Tg and dynamic fragility, (iv) to test the chemical stability of amorphous IND in various temperature conditions and finally (v) to check the long-term physical stability of the amorphous samples. These studies were supplemented by density functional theory (DFT) calculations and solubility studies of the amorphous IND in 0.1 M HCl, phosphate buffer (pH = 6.8), and water (25 and 37 °C). Acknowledgement: The authors Z.W., K.G., M.P. and W.S. are deeply grateful for the financial support by the National Science Centre within the framework of the Opus3 project (Grant No. DEC-2012/05/B/NZ3/03233).

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High-throughput gravimetric moisture sorption in pharmaceutical preformulation

U. J. Griesser, D. E. Braun

Institute of Pharmacy/Pharmaceutical Technology, University of Innsbruck, Innrain 52c, A-6020 Innsbruck, Austria

Moisture is one of the key factors in decisions related to processing, packaging, storage and shelf-life of pharmaceutical products since many active pharmaceutical ingredients and most excipients tend to absorb moisture or undergo moisture induced changes, resulting in products with altered chemical and physical properties. Therefore gravimetric moisture sorption analysis is an integral part of the instrumentation in a modern preformulation lab. The major problem connected with the investigation of moisture dependent changes is the often slow kinetics of the reactions which is particularly true for hydrateon/dehydra-tion processes that involve significant structural changes. Such slow processes are particu-larly observed in stoichiometric hydrates or biopolymers and it is not unusual that the re-cording of a complete equilibrium moisture sorption isotherm requires many weeks. In or-der to utilize the analytical potential of moisture sorption studies a series of commercially available single sample moisture balances or a multisample instrument is highly useful. The aim of this presentation is to overview important applications of high-throughput moisture sorption and address the potential, strategies and limits of this method. Applications include the detection of amorphous content or residual solvent. Multisample instruments, where all samples are exposed to the same atmospheric conditions, are particularly suited to discover batch-to-batch variations in drug compounds, excipients or formulations. The high affinity of water molecules to amorphous regions, highly reactive sites or polar impurities may allow a quick detection of subtle differences between batches that may be hardly detectable with other analytical methods. However, the investigation of the stability and phase transition behaviour of hydrates

1 is one of the

most ingriguing and challenging applications of gravimetric moisture sorption analysis and will be illustrated in more detail using selected examples. Understanding the underlying mechanisms of such reactions requires also the use of a series of complementary analytical techniques, which ideally allow measurements under controlled temperature and water activity/water vapour pressure conditions. [1] U.J. Griesser, Importance of Solvates, in: R. Hilfiker (Ed.), Polymorphism in fine chemical and

Pharmaceutical Industry, Wiley, New York, 2006, pp. 211 – 234.

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DVS: Application within a generic pharmaceutical company

Jan Smith

Synthon BV, Physchem Characterisation Platform, Analytical Research Group (ARG) Nijmegen The Netherlands,

In pharmaceutical industry DVS is considered a helpful and versatile tool to determine interaction with moisture. Synthon is especially interested in early detection of moisture related chemical or physical changes (i.e. hydrate formation

1, salt disproportionation

3,

deliquescence1, phase change

2,4) to determine storage and handling conditions of both

active pharmaceutical ingredients and pharmaceutical dosage forms under development. In this lecture a few examples of the daily use of the the Pro Umid SPSx-1µ DVS within Synthon are presented.

[1] Airaksinen S. Role of Excipients in Moisture Sorption and Physical Stability of Solid

Pharmaceutical Formulations. Academic Dissertation.5 A.D.; [2] Atassi F, Almaya A, and Aburub A. Effect of Storage Conditions on Compaction Behavior of Two

Grades of Spray-Dried Lactose. Pharmaceutical Development and Technology 2008; 13: 277-282 [3] Guerrieri P and Taylor L. Role of Salt and Excipient Properties on Disproportionation in the Solid-

State. Pharmaceutical Research 2009; 26: 2015-2026 [4] Vollenbroek J, Hebbink GA, Ziffels S, and Steckel H. Determination of low levels of amorphous

content in inhalation grade lactose by moisture sorption isotherms. International Journal of Pharmaceutics 2010; 395: 62-70

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Advanced Raman techniques in new drugs research and development

Simona Cîntă Pînzaru, C. A. Dehelean*, A. Fălămas, M. Steiner

Babes-Bolyai University, Bopmedical Physics, Theoretical and Molecular Spectroscopy Dept., Kogalniceanu 1, RO 400084 Cluj-Napoca, Romania;

e-mail: [email protected] *Victor Bbaes-University of Medicine and Pharmacy Faculty of Pharmacy,

Eftimie Murgu Square no.2, 300041, Timisoara, Romania

We present here a complete Raman spectroscopy-based approach in pentacyclic triterpe anticancer drugs chain implementation, from natural resources identification to final formulations, characterization, evaluation and finally testing on animal models (Fig. 1). In the first step, we demonstrate how Raman spectroscopy could directly evaluate the triterpene content in birk bark natural resources collected from Romanian flora. Secondly, we employed Raman spectroscopy to characterise and differentiate the extraction products from natural birck bark upon optimised extraction protocols. Thirdly, we describe three pharmaceutical formulations based on pentacyclic triterpenes from natural extracts using Raman techniques. Because of the pentacyclic triterpenes low solubility we employed: i) encapsulation in various cyclodextrins; ii) nanoemulsion formulation and iii) semisolid ointments. Their Raman characterization allowed to draw conclusion on the structure-function relationship. Finally, we applied the extraction products as well as the triterpene based pharmaceutical formulations on mice models with melanoma induced in three ways, by UV exposure, through chemical promoters (TPA-DMBA) or through B16 melanoma cell injection, respectively, to evaluate their anticancer activity. Both in vivo and ex-vivo confocal Raman, as well as surface enhanced Raman scattering (SERS) techniques have been applied to investigate the molecular changes along the malignancy and treatment in animal skin [1]. SERS signal from tissue allowed achieving very good reproducibility and revealed clear differences associated with pharmaceutical treatment efficiency.

Fig. 1. Graphycal sketch of new drug identification, extraction evaluation, formulation and testing on animal models using Raman spectroscopy techniques.

[1]. S. Cinta Pinzaru, A. Falamas, and C. A. Dehelean, (2013), Molecular conformation changes along

the malignancy revealed by optical nanosensors. J. Cell Mol Med, 17: 277–286. doi: 10.1111/jcmm.12006.

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Co-Crystals: A tool to fine-tune solid-state properties

Rolf Hilfiker

Solvias AG, Department Solid-State Development, Römerpark 2, CH-4303 Kaiseraugst, Switzerland; [email protected]

Solid-state properties play a big role in active substances all the way from research to manufacture of the final product. In this presentation we will focus on two selected aspects of the solid-state development process. In an early phase, when the optimal solid form of an API has to be chosen, co-crystals may present an attractive alternative to the parent molecule or salts. We will discuss what the thermodynamic requirements for co-crystal formation are and what strategies are promising to identify co-crystals experimentally. In particular, the importance of phase diagrams for the design of successful co-crystal screens will be highlighted. A case study will show how physical properties can be improved by choosing a suitable co-crystal. In a later phase, the chosen form (polymorph, hydrate, co-crystal or salt) has to be produced on a large scale. This requires the development of a crystallization process, where a sizeable number of parameters, such as yield, speed, purity and particle size have to be optimized while ensuring that the correct form is obtained consistently. An example of a substance which can exist in two polymorphs with very similar free energy will be presented. We will show how online tools such as a Lasentec probe can be a very useful aid to achieve the desired aim.

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Determination of nanocrystals size distribution in polymer-drug systems

M. Grassi, A Piccolo, M. Abrami, S. Fiorentino, R. Farra, G. Grassi*, D. Hasa**, D. Voinovich**, B. Perissutti**, I. Colombo

Trieste University, Dept of Eng and Arch, Via A. Valerio 6/A, I-34127, Trieste, Italy *Trieste University, Dept of Life Sci, Cattinara Hospital, P.le Europa 1, I-34127, Trieste, Italy

**Trieste University, Dept of Chem and Pharm Sci, P.le Europa 1, I-34127 Trieste, Italy

Drug bioavailability improvement still remains one of the most important challenges in the pharmaceutical field as, at present, about 40% of the drugs being in the development pipelines are poorly soluble, up to 60% of compounds coming directly from synthesis are poorly soluble and 70% of the potential drug candidates were discarded due to low bioavailability related with poor solubility in water [1,2]. Accordingly, different approaches have been developed to increase drug solubility and one of the most promising implies the reduction of crystals size in the nano-range. Indeed, it is well known that while melting temperature and enthalpy decrease with crystal size, solubility increases when crystals size is reduced [3]. As nanocrystals are basically unstable (at least in the lifetime requested to a pharmaceutical formulation), the use of a stabilizer (usually a polymer) is needed. Thus, the final formulation is essentially composed by polymeric particles embedding drug nano-crystals. As the in vitro and in vivo behaviour of the drug-polymer system strongly depends on nanocrystals dimensions, the determination of their size distribution is of paramount importance. Aim of this work is to develop a mathematical model able to determine drug nanocrystals size distribution resorting to differential scanning calorimeter data referring to the drug-polymer system. This thermodynamic model generalizes the Brun approach (working for an isolated crystal) [4] as it takes into account that nano-crystals melting can occur in the presence of an amorphous drug phase, whose abundance depends on the process (for example, solvent swelling, mechanochemical activation, supercritical fluids) conditions adopted for the formation of the polymer-drug composite. Finally, the results descending from this approach are compared with those deriving from another well-known approach (X Rays Powder Diffraction) in the case of two poorly water soluble drugs (nifedipine and griseofulvine) co-ground with polyvinylpirrolidone in a 1/5 w/w ratio. This comparison underlines the reliability of the proposed thermodynamic approach. [1] C. Lipinski, Poor aqueous solubility: an industry wide problem in drug discovery. Am. Pharm. Rev.

5(2002) 82-85. [2] E. R. Cooper, Nanoparticles: a personal experience for formulating poorly water soluble drugs. J.

Control. Release 141(2010) 300–302. [3] N. Coceani, L. Magarotto, D. Ceschia, I. Colombo, M. Grassi, Theoretical and experimental

analysis of drug release from an ensemble of polymeric particles containing amorphous and nano-crystalline drug. Chem. Eng. Sci. 71(2012) 345-355.

[4] M. Brun, A. Lallemand, J. Quinson, C. Eyraud, Changent d’etat liquid – solide dans les milieu poreux, J. De Chim. Phys. 70(1973), 979-989.

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Mechanochemistry of drugs

E. V. Boldyreva

Dept. Solid State Chemistry, Novosibirsk State University and Institute of Solid State Chemistry and Mechanochemistry SB RAS

Mechanical treatment is widely used in processing pharmaceuticals, grinding and tabletting serving as examples of typical procedures used in most technologies. These procedures can provoque various changes not only in the size of particles, but also in the crystal structure, the type and concentration of defects present. As a result, physical and chemical properties can change, which are relevant for manufacturing, storage, therapeutic use and marketing of the drug. It is important to understand and to control these processes, to avoid unexpected and not-controllable transformations. Mechanical treatment can be also applied on purpose, to improve the characteristics of a drug, for example, to increase its dissolution rate, or to produce a new compound, a new composite, polymorph, or an amorphous sample. In this case the types of mechanical treatment can be very different, ranging from continuous loading to very short-pulse treatment. In particular, one can vary the ratio of pressure vs. shear, change the pulse frequency giving the system different time for relaxation, use liquid or solid additives, change the temperature of treatment, etc. As a result, the outcome of the process can be quite different. In this contribution I am giving a comprehensive introduction in mechanochemistry and illustrate the main concepts of this emerging field of science and technology using examples relevant for pharmaceuticals. The study was supported by a grant from the Russian Ministry of Education and Science No. 14.B37.21.1093 and an Integration Project of the SB RAS No 108.

[1] E.V. Boldyreva, Chem. Soc. Rev., 2013, DOI: 10.1039/C3CS60052A

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X-ray diffraction methods in drug design

Jindřich Hašek

Institute of Biotechnology, Academy of Sciences, Vídeňská 1083, Praha 4, Czech Republic Protein crystallography [PX] (i.e. direct imaging of complex macromolecular systems in sub-atomic resolution) is traditionally used for determination of enzyme-inhibitor, and receptor-ligand interactions. PX facilitates the rational design of more efficient drugs. However, the use of the PX molecular snapshots is not straightforward. Correct prediction of thermodynamic functions (e.g. inhibition constants Ki) includes relatively easy calcula-tion of enthalpy terms (interaction energies), but also the ”entropy related terms” de-pending on the whole molecular system dynamics. It can be estimated in PX by the multi-solution refinement of protein structure - the water perturbed refinement in our design. The unconstrained molecular dynamics simulations [1] are often problematic because the enzymatic processes (1) are slow [ms], (2) can proceed via intermediate states or (3) by alternative paths. Simplified simulations (e.g. forced withdrawal of inhibitor) or simplified protein dynamics can provide better insight on the system behavior. However, this approach requires apriory estimate of the reaction path and of the system behavior during reaction, as:

molecular model of the reaction including transport of water molecules on interfaces

recognition of possible multi-step mechanism of reaction increasing stability

recognition of the shock energy absorbing elements increasing stability of the system (e.g. conformational flexibility and mobility of bulky side chains)

correct recognition of paths for energy dissipation from protein to solution

recognition of the shock energy dumping elements in the protein skeleton

recognition of changes in low frequency vibration modes induced by protein mutations (e.g. stiffness of structure elements induced by far mutations).

The X-rays analysis is used also (1) in rational drug delivery to its target using MAb-like targeting, (2) for analysis of molecular coatings of drug-complexes against degradation [2], (3) for targeted delivery of low molecular drugs bound to special polymers, (4) for checking the results of chemical reactions in preparation of macromolecular drugs (e.g. substituent positions on the protein surface). Another use of X-rays is in preparation of solid formulations composed from mixtures of more components. Diffraction methods can resolve whether the active substance mixed into the polymer matrix is completely dissolved, or forms colloid particles, nanocrystals, or microcrystals. It allows fine tuning of formulations for controlled delivery and activation of drugs (enzymatically, by the change of pH, by heat, by external magnetic field, etc.). Commonly used practice is a reliable determination of chirarity and absolute configuration of drug molecules, and the identification of different crystalline forms of drugs by X-rays. The study is supported by the project BIOCEV CZ.1.05/1.1.00/02.0109 from the ERDF. [1] Spiwok V. et al. (2005) J. Comp.Aided.Mol.Design 19, 887-901. [2] Hašek J. (2006) Z. Crystallogr. 23, 613-618.

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Relaxation dynamics and crystallization study of sildenafil in the liquid and glassy states

M. Paluch*, K. Grzybowska*, K. Kołodziejczyk*, A. Grzybowski*, Z. Wojnarowska*, L. Hawelek*

,**, J. D. Zioło***

*Institute of Physics, University of Silesia, ul. Uniwersytecka 4, 40-007 Katowice, Poland **Institute of Non Ferrous Metals, ul. Sowinskiego 5, 44-100 Gliwice, Poland ***Poznan University of Medical Sciences, Faculty of Medicine I, Fredry 10,

61-701 Poznan, Poland

In this presentation, the results from the investigation on the physical stability and molecular dynamics of amorphous sildenafil in both the liquid and glassy states are reported. We have established that the amorphous sildenafil is resistant to recrystal-lization at temperatures below the glass transition temperature Tg during the experimental period of its storage (i.e., above 70 days), however, it easily undergoes cold crystallization at T>Tg. To determine the crystallization mechanism, the isothermal and non-isothermal studies of the cold crystallization kinetics of the drug are performed by using the broadband dielectric spectroscopy (BDS) and the differential scanning calorimetry (DSC), respectively. The cold crystallization mechanism has been found to be similar in both the isothermal and non-isothermal cases. This mechanism has been analyzed from the point of view of the molecular mobility of sildenafil investigated in the supercooled liquid and glassy states by using the BDS measurements in the wide temperature range. This analysis has been enriched with a new approach based on a recently reported measure of dynamic heterogeneity given by a four-point dynamic susceptibility function. No tendency to recrystallization of glassy sildenafil at T<Tg is also discussed in relation to molecular dynamics of sildenafil in the glassy state. The relatively small molecular mobility reflected in one secondary relaxation as well as the predicted large time scale of structural relaxation of glassy sildenafil suggest that amorphous sildenafil should not recrystallize during its long-term storage at room temperatures. .

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In vitro - in vivo extrapolation of transporters

S. Neuhoff

Simcyp (a Certara company)

A general overview on how to scale intestinal, hepatic and/or renal transporters to in vivo PK, what we need, what we have and what we can and cannot do! For key drug transporters the availability of high quality in vitro data and suitable in vitro-in vivo scaling approaches allows the use of robust physiologically-based pharmacokinetic models to predict the transporter mediated drug-drug interaction (DDI) potential of new drug candidates. To model a transporter-based interaction, the function and location of the transporter(s) (apical/basolateral) as well as their direction of transport (uptake/efflux) and the relevant concentrations of substrate and inhibitor need to be accounted for. In addition to being saturated and/or inhibited, transporters may also be induced or suppressed due to disease or co-medications. This presentation will provide an overview of the experimental methodology used and its pitfalls in the process of in vitro-in vivo extrapolation (IVIVE) of transporter data with respect to DDIs. In addition it will be demonstrated how these approaches have been used to accurately predict the extent of transporter-mediated DDIs from in vitro data for some specific, clinically relevant cases.

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Affinity- Mass Spectrometry and Ion Mobility- Mass Spectrometry: New Tools for elucidating structures, reaction pathways and

interactions of “misfolding” - aggregating proteins

Michael Przybylski

Departments of Chemistry and Biology, and Steinbeis Research Center for Biopolymer Chemistry & Biomolecular Mass Spectrometry, University of Konstanz,

78457 Konstanz, Germany A large variety of cellular processes are based on the formation and dynamics of multi- and supramolecular protein assemblies, and several diseases, previously thought to be unrelated, such as cancer and neurodegenerative diseases, are characterised by “misfolded” protein aggregates. Chemical structures and reaction pathways of pathophysiological aggregates are only poorly characterised at present. “Soft-ionisation” mass spectrometry (MS), such as HPLC-electrospray-MS, is often unsuitable to direct analysis of reaction pathways and intermediates in aggregation. Recently, ion mobility- MS (IM-MS) has been emerging as a new tool for analysis of protein aggregation due to its concentration- independent gas phase separation capability. First applications of IM-MS to the in vitro oligomerization of α-synuclein (αSyn) and ß-amyloid (Aß), key proteins for Parkinson’s disease and Alzheimer’s disease, enabled the identification of hitherto unknown degradation and aggregation products. Time- dependent studies of the in vitro oligomerization- aggregation of αSyn provided the first identification of a specific autoproteolytic fragmentation, previously observed by gel electrophoresis but not identified, particularly a highly aggregation-prone fragment by cleavage at Val71/Thr72 in the ß-breaking triplett Val-Val-Thr in the central aggregation domain [1]. The corresponding recombinant αSyn(72-140) fragment showed substantially faster aggregation and high neurotoxicity compared to the intact protein. Recently, the development of combined (online) affinity- MS methods [2] enabled first direct (“top-down”) structural studies in vivo, such as from brain homogenate. Applications of affinity-MS will be discussed using epitope-specific αSyn- and ß-amyloid (Aß)- antibodies [3] for the characterization of oligomers and interactions of Aß, αSyn and ß-glucocerebrosidase, the target enzyme for Gaucher’s Disease, a neurological lysosomal storage disorder [4]. These results indicate ion mobility- MS and affinity- MS as powerful tools for the molecular elucidation of structures and intermediates of polypeptide aggregation. Corresponding structures thus obtained provide a basis for (i), the detailed study of oligomerization- aggregation pathways; (ii), the design of peptides capable of inhibiting or modifying aggregation; and (iii), the development of specific methods for quantitative protein determinations in biological fluids. [1] Vlad, C. et al., (2011) ChemBiochem. 12, 2740-2744. [2] Dragusanu, M., et al. (2010) J. Am. Soc. Mass Spectrom. 21, 1643-1648. [3] McLaurin, J., et al. (2002) Nature Med. 8: 1263-1269 [4] Przybylski, M. et al./Univ. Konstanz & Centogene (2012) Eur. & US Patent Applications.

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Application of PAT methods in the development and quality control of Orally Disintegrating Tablets

Tamás Sovány, Ildikó Oláh, Péter Kása jr., Klára Pintye-Hódi

University of Szeged, Department of Pharmaceutical Technology, H-6720, Eötvös u. 6., Szeged, Hungary

The application of the Process Analytical Technology (PAT) methods in the development of various dosage forms has increasing importance in the pharmaceutical industry. The continous quality control of products with noninvasive analitycal techniques improves safety and efficacy of the production. The aim of the present study was to investigate the capability of at-line NIR measurements to identify the differtences in the compostion and to predict the mechanical properties of various Orally Disintegrating Tablets (ODTs). Calcium gluconate/cholecalciferol ODTs were compressed according to a 2

2 full factorial design with 4 different compositions, using

composite excipients and physical powder mixtures. Compression pressure and compression speed were used as input factors, and breaking hardness and the disintegration time of the products were the optimization parameters. The product properties were checked with conventional and alternative methods and with NIR measurements. The identification of minor differences in the composition was based on peak shifts and differences in the shape or in the relative intensities of the identical peaks. The results suggest that qualitative differences could be identified in the composition, but the determination of the quantitative differences in the composition requires further experiments. Despite of the above mentioned difficulties the expectable mechanical properties of the tablets could be quantified and predicted from the results of NIR measurements. A linear relationship exists between the breaking hardness of the tablets and the peak intensities. The prediction of the disintegration time based on NIR data is also possible, since this property could be derived from the breaking hardness using a composition independent exponential function. As conclusion the combination of the different investigation methods helps to understand the differences between the behaviour of the different compostions and enhances the selection of the most suitable composition in the early stage of the product development. Acknowledgement: The work was supported by the European Union and co-funded by the European Social Fund. Project title: “Broadening the knowledge base and supporting the long term professional sustainability of the Research University Centre of Excellence at the University of Szeged by ensuring the rising generation of excellent scientists.” Project number: TÁMOP-4.2.2/B-10/1-2010-0012

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Fragment-Based Lead Generation in Drug Discovery

Geoff Holdgate

Structure & Biophysics, Discovery Sciences, AstraZeneca UK Fragment-Based Lead Generation (FBLG) is an attractive approach to drug discovery, where screening of a small library of low molecular weight (<250 Da) compounds represents better sampling of chemical space than traditional HTS. Fragment hits typically have low affinity and require biophysical methods for detection, but when binding data is combined with structural information these initial hits can often be rapidly optimised. In large Pharma, where multiple hit identification processes may be undertaken, fragment information can be combined with hits identified from other screening approaches to improve potency, reduce chemical liabilities or explore new binding modes. Fragment screening has historically been undertaken using NMR, but more recently SPR has emerged as a viable approach to fragment screening due to advances in sensitivity and throughput. These improvements, combined with low protein consumption mean that a fragment library can be screened in just a few days with limited resource. At AstraZeneca, a toolbox of methods is employed, coupled with dedicated fragment libraries for fragment efficient screening. Examples will be presented of how this has enabled us to identify novel chemical equity for drug-hunting projects.

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Forensic Intelligence for Medicine Anti-Counterfeiting

Dégardin Klara, Roggo Yves*, Margot Pierre

Institut de Police Scientifique, Université de Lausanne, Switzerland *F. Hoffmann-La Roche, Basel, Switzerland,

Medicine counterfeiting is a crime that has increased in recent years and now involves the whole world. Health and economic repercussions have led pharmaceutical industries and agencies to develop many measures to protect genuine medicines and differentiate them from counterfeits. Detecting counterfeit is chemically relatively easy, but much more information can be gained f rom the analyses in a forensic intelligence perspective. Analytical data can feed criminal investigation and law enforcement by detecting and understanding the criminal phenomenon. Profiling seizures using chemical and packaging data constitutes a strong way to detect organised production and industrialised form of criminality. Research is on going and constitutes the focus of this paper. Thirty-three seizures of a commonly counterfeited type of capsule have been studied. The results of the packaging and chemical analyses were gathered within an organised database. Strong linkage was found between the seizures at the different production steps, indicating the presence of a main counterfeit network dominating the market. The interpretation of the links with circumstantial data provided information about the production and the distribution of counterfeits coming from this network. This forensic intelligence perspective has been generalized to other types of products. This may be the only reliable approach to help the understanding of the organised crime phenomenon behind counterfeiting and to enable efficient strategic and operational decision making in an attempt to dismantle counterfeit networks.

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AFM Nanoimaging and nanomechanics for nanomedicine

Ratnesh Lal, Srinivasan Ramachandran, Fernando Teran Arce, Preston Landon

Bionegineering Department, PFBH 219, University of California San Diego (UCSD), 9500 Gilman Drive, La Jolla, CA 92093, USA; [email protected]

Bio-nanotechnology is directed toward measuring, monitoring and manipulating fundamental biological processes at nano-to-micro spatial and temporal scales. Their relevance spans from single molecule to system level understanding of the normal physiology and pathological human diseases. Atomic force microscopy (AFM) and AFM-based nano-tools allow visualization and manipulation of multiscale living biological systems with the potential to unravel structures and functions currently beyond the scope of existing tools. For example, AFM has provided 3D molecular scale images of polymorphic structures adopted by amyloid peptides as the ion channels that is believed to be at the core of Alzheimer’s disease (AD) and other neurodegenerative and protein-misfolding diseases. This information enables us to screen library of small molecules in drug discovery process. The versatility of AFM application lies in its ability to be integrated with an array of complementary tools and techniques, including nanopore devices and electrical recording and imaging tools to provide combined functional and structural information about ion channels, receptors, and synaptic connections that govern almost all human activity. Development of AFM-mechanics based nanosensors to monitor cell and tissue functions can be used to diagnose the onset of disease as well as for monitoring therapeutic efficacy: functionalized micro-cantilever is an excellent sensor for detecting inter-molecular interactions at single molecular level. Using arrays of these nanosensors, one can design high-throughput assay for identifying multiple biomarkers as well as for detecting molecular events and constituents of any system level understanding. My talk will discuss potentials for AFM and AFM-mechanics-based nanomedicine discoveries.

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G-quadruplex DNA and DNA origami: Insights from atomic force microscopy

Robert Henderson

Department of Pharmacology, University of Cambridge, UK DNA is normally considered only in its classical double-helical form, but the chemical nature of the bases means that DNA can, in nature and in artificial structures adopt other forms. In this presentation I will talk about work we have conducted using atomic force microscopy (AFM) imaging, of two ‘alternative’ forms of DNA, namely the G-quadruplex structure and scaffolds made from DNA origami. G-quadruplex DNA possesses relatively long guanine repeats, which are abundant in the genome. Under appropriate conditions these connect in groups of 4 - the ‘G-quadruplex’, having the potential of producing interruptions in the normal helical structure of DNA. G-quadruplexes occurring in nature are associated with a number of pathological conditions and are particularly prominent in telomeres. This is important because in telomeres, quadruplexes tend to inhibit telomerase - the enzyme that is involved in maintaining the length of the telomere, and hyperactivity of telomerase is commonly implicated in cancer. This means that there is therapeutic potential for agents that might stabilise quadruplexes. AFM provides an ideal tool to study directly, at the molecular level the effects of agents that influence G-quadruplex stability. DNA origami was developed in the latter part of the last decade as a potential tool to build biological nanostructures. Because of the structural predictability afforded by A-T and C-G binding, it is possible to design and make 2- and 3-dimensional structures from plasmid DNA by introducing short oligonucleotides that pair with specific base sequences in the plasmid, so, folding the DNA into predetermined shapes. The origami structures can bear chemically modified oligonucleotides, allowing the attachment at specific positions on the origami of other macromolecules. We are using DNA origami structures, imaged by AFM, as scaffolds to investigate the cAMP-protein kinase A-phosphodiesterase signalling system. By altering the positions of the molecules relative to each other we hope to throw light on the importance of the spatial distribution of the proteins involved in this pathway.

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Structural dynamics of single molecules using Atomic Force Microscopy

Jean-Luc Pellequer*, Shu-wen W. Chen*

,**,

Michael Odorico*, Jean-Marie Teulon*,

Pierre Parot*, Rui C. Chaves*

*CEA, iBEB, Department of Biochemistry and Nuclear Toxicology, F-30207 Bagnols sur Cèze, France; [email protected]

**13 Avenue de la Mayre, 30200 Bagnols sur Cèze, France

Molecular recognition between a receptor and a ligand requires a certain level of flexibility in macromolecules. However, the current techniques in structural biology are hampered by global molecular flexibility, which is often characterized by domain-to-domain move-ments and intrinsic protein disordering. New approaches need to be developed for study-ing dramatic changes in protein conformations. We have been building a protocol for re-constructing large protein 3D structures using Atomic Force Microscopy topographic ima-ges

1,2. The protocol named AFM-Assembly performs a docking of individual macromole-

cular constituent units beneath AFM topographic surfaces of single molecules. Then, dock-ed molecular constituting units are assembled using an exhaustive combinatorial appro-ach. A ranking score is developed to compare the topography of final assembled structu-res to that of AFM image. This protocol has been applied on antibodies (IgGs) and human coagulation factor Va (FVa). In both cases, high-resolution AFM images of single molecules were obtained and full structure reconstructions were performed. Regarding antibodies, when compared with three X-ray structures of antibodies present in the PDB database, results indicate that several arrangements of reconstructed subunits are comparable with those of known structures. Nevertheless, no reconstructed structure superimposes adequately to any particular X-ray structure, a direct consequence of the antibody flexibility. Regarding human FVa, results suggest that there is a large structural flexibility in the orientation of the C1 and C2 domains relative to the pseudo-trimeric A domains. In conclusion, multi-scale protein reconstruction as obtained with the above protocol op-ens new possibilities for AFM in structural biology. This approach opens new road for clas-sically difficult proteins that are large in size and particularly flexible (or partly disordered).

Overview of the four different steps of the AFM-Assembly protocol that aims at reconstructing large macromolecules using structure constituents (subunits) and AFM topographic images

1 Trinh M-H, Odorico M, Pique ME, Teulon J-M, Roberts VA, Ten Eyck LF, Getzoff ED, Parot P, Chen S-wW and Pellequer J-L, Structure 20 (2012)113-120.

2 Chaves RC, Teulon J-M, Odorico M, Parot P, Chen S-wW and Pellequer J-L () J. Mol. Recognit, 2013, Submitted

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Online Bioaffinity-Mass Spectrometry: New Tool for Simultaneous Detection, Structure Determination and Affinity Quantification of Protein-Ligand

Interactions from Biological Material

Michael Przybylski*, Stefan Slamnoiu*, Mihaela Stumbaum*,**, Camelia Vlad*

,**,

Kathrin Lindner*, Christiaan Karreman*, Marcel Leist*, Bastian Hengerer***

*Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Departments of Chemistry and Biology, University of Konstanz, Germany,

[email protected]

**SAW Instruments GmbH, Bonn, Germany ***Boehringer Ingelheim Pharma GmbH & Co. KG,

ZNS Research Dept., Biberach, Germany

Bioaffinity analysis using biosensors such as surface plasmon resonance has become an established technique for the detection and quantification of biomolecular interactions. However, a principal limitation of biosensors is their lack of providing structure analysis of affinity-bound ligands. We have developed an online combination of a surface acoustic wave (SAW) biosensor with electrospray ionization mass spectrometry (SAW-ESI-MS) that enables the simultaneous structure determination and affinity quantification of biopolymer ligands, dissociated from the biopolymer- ligand complex on a gold chip. Key tool of the SAW-MS combination is an interface that provides sample concentration and in-situ desalting for the MS analysis of the ligand eluate. Recent applications of the online bioaffinity- MS show broad bioanalytical potential for direct interaction studies from biological material, as diverse as antigen-antibody and lectin- carbohydrate complexes; affinity binding constants (KD) are determined from milli- to nanomolar ranges [1,2]. We report here first applications of the online- SAW- MS to the direct top-down structural characterization of proteolytic intermediates and oligomers in the aggregation of Parkinson’s Disease key protein, alpha-synuclein (αSyn) [3] from brain homogenate. [1] Petre, A, et al. (2012) J. Am. Soc. Mass Spectrom., 23, 1831-11840. [2] Moise, A., et al., (2011) J. Am. Chem. Soc. 133, 14844-14847. [3] Vlad, C. et al., (2011) ChemBiochem. 12, 2740-2744.

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The absolute importance of applicability domain in QSAR: A new in silico multiprotic pKa prediction tool with significantly improved

prediction accuracy and new functionality for PhysChem, MedChem, CompChem, and Cheminformatics applications

Robert Fraczkiewicz, Robert D. Clark, Alexander Hillisch*, Andreas H. Göller*, Ursula Krenz*, Rolf Schoenneis*, Mario Lobell*

Simulations Plus, Lancaster, CA 93534, USA, * Bayer Pharma AG, Global Drug Discovery, Wuppertal, Germany

Ultimately, QSAR models are useful if they help push forward the boundary of scientific research. One of the crucial utilitarian aspects, the applicability domain (AD) of a QSAR model (understood in terms of chemical space coverage), does not receive as much attention as specific modeling methodologies or descriptor sets. In this work we show that at least in terms of pharmaceutical applications, the AD should have high priority. In an unparalleled joint collaboration, Simulations Plus and the Bayer Pharma AG have developed a new in silico pKa prediction tool which is currently in use at Bayer and will be a part of the upcoming release 7.0 of the Simulations Plus ADMET Predictor software. The new model has been trained with curated sets of ~14000 literature pKa values (~11000 compounds, representing literature chemical space) and ~19500 pKa values experiment-tally determined at Bayer Pharma (~16000 compounds, representing industry chemical space). We compare and quantify differences between the two domains. The new model is also compared to one developed exclusively within the literature AD (“old model”, versi-on 6.1 of ADMET Predictor). Model validation was performed with ~31000 new pKa values measured at Bayer. On the largest and most difficult test subset with >16000 pKa values (all from measurements on new compounds after training with the first Bayer training set) the old model achieved a mean absolute error (MAE) of 0.72, a root mean square error (RMSE) of 0.94, and a squared correlation coefficient (R

2) of 0.87. The new model achieved

significantly improved prediction statistics, with MAE=0.50, RMSE=0.67 and R2=0.93.

At Bayer, the new pKa prediction model has been integrated into Pipeline Pilot and the PharmacophorInformatics platform PIx used by scientists at Bayer Pharma. Different output formats have been defined which allow customized use by Medicinal Chemists, Physical Chemists, and Computational Chemists. The fast calculation speed (>100,000 compounds/h [CPU: Intel Xeon L5420, 2.5 GHz]) allows Computational Chemists to automatically modify the protonation state of structures to reflect their dominant charge state(s) at a given pH (default 7.4) in order to prepare large libraries for high-throughput docking applications (e.g. virtual screening). The mathematical modeling methodology has been published elsewhere [1]. All the models mentioned above are resolved with respect to all possible ionization microequilibria. In this work, we also introduce novel concepts of Averaged Site Protonation and Single Proton Midpoint (pK50) as measures of individual ionizable group acidities in multiprotic molecules.

[1] R. Fraczkiewicz, In Silico Prediction of Ionization, in: B. Testa, H. van de Waterbeemd (Eds.), Comprehensive Medicinal Chemistry II, Elsevier, Oxford, UK, 2006, Vol. 5, pp. 603-626.

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Challenges with development of a melting point model using public data: not all errors are the same!

Igor V. Tetko*,***, Luc Patiny****, Larisa Charochkina*****, Iurii Sushko***,

Ahmed Abdelaziz***, Abdullah M. Asiri**

*Helmholtz Zentrum München - German Research Center for Environmental Health (GmbH), Ingolstädter Landstraße 1, D-85764 Neuherberg, Germany;

**Chemistry Department, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah 21589, Saudi Arabia

***eADMET GmbH, Lichtenbergstraße 8, D-85748 Garching, Germany ****Chemical Information, Ecole Polytechnique Fédérale de Lausanne, BCH.5121,

CH-1015 Lausanne, Switzerland *****Institute of Bioorganic and Petrochemistry, Murmanksya 1, Kyiv, Ukraine

The challenges with development of models using datasets collected from public literature will be exemplified using melting point model. Such data frequently have mistakes due to errors in chemical structures, incorrectly reported values (i.e., Celsius instead of Kelvin, incorrect sign, reporting boiling or flash point instead of melting point, values for salt forms instead of those for pure compounds, predicted values instead of experimental ones, etc.) All these mistakes can dramatically influence performance of developed models. When using a proper validation protocol, these mistakes can frequently pop-up as predictions with large errors. However, large errors can be also observed for chemicals, which are structurally different to those used in the training set. These latter compounds represent valuable information for models and should be kept in the training set. Thus, there is a need to differentiate both types of compounds. I will show how the use of consensus modeling combined with statistical criteria allows automatic identification and exclusion of problematic compounds. This approach can be recommended for an automatized model development for large and noisy datasets (e.g., tens thousands of compounds) in particular if manual curation of such data is too expensive or is unfeasible one. This study was partly supported by the European Union Marie Curie Initial Training Network project “Environmental Chemoinformatics” (ECO), grant agreement number 238701, and the GO-Bio 1B BMBF project iPRIOR, grant agreement number 315647.

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Lipophilicity assessment by Microemulsion Electrokinetic Chromatography (MEEKC)

Xavier Subirats, Hui-Ping Yuan, Martí Rosés

Departament de Química Analítica – Institut de Biomedicina, Universitat de Barcelona Martí i Franquès 1-11, 08028 Barcelona

Since the capacity of a specific compound to penetrate a lipid membrane and reach a proposed target is a key factor of a potential drug candidate, lipophilicity is a fundamental physicochemical property in the drug discovery process. The most widely used lipophilicity index is the partition ratio between the immiscible phases 1-octanol and water, traditionally determined by shake-flask procedures. Unfortunately these reference methods are excessively time consuming for screening purposes, require a high purity, and a relatively high amount of sample. In order to overcome these limitations several new techniques have been developed [1], being the automated chromatographic methods the ones providing a highest throughput. Especially remarkable is the Microemulsion Electrokinetic Chromatography (MEEKC) due to its versatility, resolution power, wide range of applicability [2], and relatively low running costs. Lipophilicity assessment by MEEKC is based in the partition of the compounds between the aqueous phase of the microemulsion and its oil droplets, and the great potential of this technique relies on its tuneability. The buffer and the wide pH range of the background electrolyte, the addition of organic solvents to the microemulsion or the selection of an appropiate surfactant for the stabilization of the oil droplets, among others, can be appropriately tunned in order to obtain a successful analysis. In the present work, the optimization of MEEKC for the determination of lipophilicity of neutral compounds with a log Po/w comprised between -0.8 and 5.1 has been achieved. Up to 10% of acetonitrile was added to the microemulsion with the aim of improving the separation resolution and the measurable log Po/w range. The studied pH values were the physiological 7.4, 10 and 12 in all cases less than 20 min per analysis were needed. It is noteworthy that this investigation proves the feasibility of MEEKC for the lipophilicity assessment of basic compounds at extreme pH environments, which are out of reach of chromatographic methods due to the rapid degradation of the stationary phase. [1] B. Sethi, M. Soni, S. Kumar, G.D. Gupta, S. Mishra, R. Singh, Lipophilicity Measurement Through

Newer Techniques, J. Pharm. Res. 3 (2010) 345-351. [2] W.W. Buchberger, Microemulsion Electrokinetic Chromatography, in P. Schmitt-Kopplin (Ed.),

Methods in Molecular Biology, vol. 384, Humana Press Inc, Totowa, NJ, 2008, p. 717-733.

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Evaluation of log Po/w values of drugs from chromatographic measurements and from molecular structure calculations

Juan M. Pallicer, Martí Rosés, Clara Ràfols, Elisabeth Bosch, Rosalia Pascual*, Adriana Port*

Departament de Química Analítica and Institut de Biomedicina (IBUB), Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona

*ESTEVE, Baldiri i Reixac, 4-8, 08028 Barcelona;

Optimal lipophilicity in compounds should be targeted in the early phases of drug research since it contributes on individual ADMET (absorption, distribution, metabolism, elimination and toxicology)

1,2, including blood-brain barrier penetration and clearance

3.

In previous studies a simple chromatographic method combined with several molecular descriptors (two and thre-dimensional models) derived from the structure of the drug to determine the 1-octanol/water partition coefficient (logPo/w) was reported

4,5. Predictive

software to estimate lipophilicity has also evolved as a master piece in the structure design for new chemical entities. There are classical and well accepted programs to predict log P values such as the BioByte-ClogP or more recently developed ones as the GALAS algorithm

6 offered by ACD. In this work, a set of 103 drugs with different structures

and functionalities have been selected in order to evaluate the accuracy of the log Po/w values obtained through computational programs and the ones derived from chromatographic measurements. [1] Henchoz Y, Bard B, Guillarme D, Carrupt PA, Veuthey JL, Martel S 2009. Analytical tools for the

physicochemical profiling of drug candidates to predict absorption/distribution. Anal. Bioanal Chem. 394: 707-729

[2] Waring MJ, 2010. Lipophilicity in drug discovery. Expert Opin. Drug Discov. 5: 235-248 [3] Kerns EH, Di L, 2004. Physicochemical profiling: overview of the screens. Drug Discov. Today

Technol. 1: 343-348 [4] Pallicer JM, Calvet C, Port A, Rosés M, Ràfols C, Bosch E, 2012. Critical evaluation of a new

chromatographic method to assess the lipophilicity of acidic, basic and amphoteric drugs. J. Chromatogr. A 1240: 113-122

[5] Pallicer JM, Pascual R, Port A, Rosés M, Ràfols C, Bosch E, 2013. The contribution of the hydrogen bond acidity on the lipophilicity of drugs estimated from chromatographic measurements. Eur. J. Pharm. Sci. 48: 484-493

[6] Mannhold R, Poda GI, Ostermann C, Tetko IV, 2009. Calculation of molecular lipophilicity: state-of-the-art and comparison of logP methods on more than 96000 compounds. J. Pharm. Sci. 98: 861-893

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Prediction of human skin permeation of neutral compounds through chromatographic measurements

Martí Rosés, Elisabet Fuguet, Sara Soriano-Meseguer, Marta Hidalgo-Rodríguez

Departament de Química Analítica - Institut de Biomedicina, Universitat de Barcelona, C/ Martí i Franquès 1-11, 08028, Barcelona, Catalonia, Spain, [email protected]

Dermal absorption from exposure to chemicals in aqueous solution is a key process in drug delivery studies of pharmaceutical and cosmetic interest, as well as in dermal toxicology. It is typically described by skin-water permeability coefficients (Kp). However, direct measurements of Kp are usually difficult, expensive, time-consuming and ethically questionable. Hence, there is a need for methods capable of predicting Kp values in a simple, economic, fast and ethical way. In this sense, chromatographic systems can provide practical and sustainable alternatives based on the estimation of Kp data from chromatographic retention measurements (k). In the present work, several chromatographic systems of micellar electrokinetic chromatography and high-performance liquid chromatography, besides the reference octanol-water partition system, have been evaluated by a systematic procedure [1] to know their suitability to emulate human skin permeation of neutral solutes. It mainly consists of predicting the overall precision achieved in the correlations of Kp and k data, which is contribution of three factors: the precision of Kp values, the precision of k values, and the dissimilarity between the two correlated systems. Both predictions and experimental tests have shown that only good performances are obtained if the volume of the solute (V) is included as additional variable in the correlations, i.e. Kp against k and V. This fact agrees with other author’s studies and illustrates the main roles of both lipophilicity and size of the solute to penetrate through the skin. With volume correction, the HPLC systems based on C18 columns are the most suitable to provide precise estimations of Kp values. Therefore, a methodology has been established to predict Kp data through HPLC measurements in a C18 column. It has been validated by training and test sets and successfully applied to the prediction of Kp values of several hormones and pesticides. The precision achieved by this methodology is even better than that obtained by other approaches (e.g. calculation from structure) [2]. [1] M. Hidalgo-Rodríguez, E. Fuguet, C. Ràfols, M. Rosés, Estimation of biological properties by

means of chromatographic systems: evaluation of the factors that contribute to the variance of biological-chromatographic correlations, Anal. Chem. 82 (2010) 10236-10245.

[2] M. Hidalgo-Rodríguez, S. Soriano-Meseguer, E. Fuguet, C. Ràfols, M. Rosés, Evaluation of the suitability of chromatographic systems to predict human skin permeation of neutral compounds, Eur. J. Pharm. Sci. (2013) http://dx.doi.org/10.1016/j.ejps.2013.04.005

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PAMPA experimental measurements and QSAR model for membrane permeability of full pH range in human intestinal

Mare Oja, Uko Maran

Institute of chemistry, University of Tartu, Ravila 14A, Tartu 50411, Estonia

Intestinal absorption largely determines transport of oral administred drugs. Transport through intestinal epithelium is either active or passive and it is hypothesized that approximately 90% of drugs are transported passively. Absorption is commonly described using two in vitro experimental membrane permeability assays: parallel artificial membrane permeability assay (PAMPA) and cell-based assay. Current work focuses on PAMPA method, which is able to describe correctly passive transport for majority of drugs and drug-like compounds. Experimental values for PAMPA have been previously used to develop in silico descriptive and predictive models for membrane permeability. To the best of our knowledge up to now in silico models for the permeability have been developed for single pH, while in human intestinal pH varies in the range ~3 to ~8. This finding serves ground for present study. The aim is to developing QSAR model that accounts of membrane permeability of human intestinal in wide range of pH. As literature lacks of systematic PAMPA experimental values for different pH-s the respective measurements were carried out. Membrane permeability values were measured for 70 chemical compounds using PAMPA method, where artificial membrane was formed using solution of 1% lecithin in dodecane. The time-dependence over 48 hours and four pH-s (3, 5, 7.4 and 9) were measured for all compounds. Membrane permeability values were calculated using iso- and gradient effective membrane permeability equations. Measured compounds were structurally diverse and belonged to different classes of chemicals. The resulting database contained 280 datapoints, which were further used for in silico modelling. For modelling the highest membrane permeability values for each compound from the range of pH-s were selected. QSAR model was developed using step-wise forward selection of best molecular descriptors into the multi-linear regression model. Descriptors and octanol-water partition coefficient were calculated using CODESSA-Pro program and XlogP3 methods, respectively. Model diagnostics and applicability domain analysis was performed. As result QSAR model using membane permeability values of different pH was developed, it was statistically significant and able to predict membrane permeability values on wide pH range, 3 to 9. Descriptors included in the model were logarithmic octanol-water partition coefficient and hydrogen bond surface area, which both are mechanistically related to the membrane permeability. Analysis of applicability domain revealed some statistical outliers and structurally deviating points. Most likely the deviation may result from limited solubility in DMSO/water solution or accuracy of calculated octanol-water partition coefficient. Acknowledgement: This research was supported by European Social Fund’s Doctoral Studies and Internationalisation Programme DoRa, which is carried out by Foundation Archimedes.

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Development, characterization and permeability study on a model PAMPA membrane with polymer supported lipid bilayer

Gábor Vizserálek*, Bálint Sinkó*,**, Tamás Bozó***, Krisztina Takács-Novák*

*Department of Pharmaceutical Chemistry, Semmelweis University, Budapest **SinkoLAB Scientific, Budapest

***Department of Biophysics and Radiation Biology, Semmelweis University, Budapest

Measuring the exact permeability value of the potentially active compounds in early phase of drug development is essential. For this purpose, parallel artificial membrane permeability assay (PAMPA) was introduced. PAMPA (a 96-well microtiter plate based technology) was aimed to serve as a rapid, high throughput in vitro method for the evaluation of passive transcellular permeability. Models for gastrointestinal, blood-brain barrier and skin permeation study were developed previously. The artificial membrane in these methods - except the Skin PAMPATM - contains phospholipid(s) dissolved in organic solvents, generally in n-dodecane, therefore multilamellar bilayers are formed by the membrane components inside the filter channels. The direct contact between the membrane and the filter may cause the decrease of mobility of the lipids and fluidity of the membrane, which may results in deviations from the permeation through biological membranes. The aim of our work was to develop a new PAMPA model with single lipid bilayer system, which is suitable stable and non-fragile for usage in HT approach. For this reason, a polymer supported membrane was prepared in every single well, where the lipid bilayer was supported by a hydrated polymeric cushion. Polydopamine film, which is known as multifunctional and versatile polymer coating, was served as supporting layer. Two hydrophil filter plates with pore size 0.45 and 0.22 μm were used for this study. The polydopamine film and the deposited lipid bilayer were characterized by atomic force microscopy (AFM). Permeability measurement was performed using a large set of drugs with diverse acid-base properties and structures. Correlation analysis was carried out between permeability data obtained by other non-bilayer PAMPA systems and the permeability values measured in this model. Our results are shown and discussed in this poster.

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Buccal-PAMPA model: monosolvent or tissue specific lipid membrane systems? Preliminary study

Judit Müller*,**, György T. Balogh**

*Compound Profiling Laboratory, Gedeon Richter Plc., Gyömrői u. 19-21.,

H-1475 Budapest, Hungary **Department of Organic Chemistry and Technology, Budapest University of Technology

and Economics, Budafoki u. 8., H-1111 Budapest, Hungary

The peroral and parenteral routes are both used for administration of drugs. The buccal route is important since it is non-invasive and drug delivery across oral tissue offers several advantages. In drug discovery there has been an increasing interest in in silico and high-throughput in vitro methods for predicting both in vivo pharmacokinetic (PK) and pharmacodynamic properties. Permeability is one of the most important physico-chemical properties to predict in vivo PK parameters. Parallel artificial membrane permeability assay (PAMPA) is a widely used tool to mimic absorption. Using different lipid compositions and wide range of pH, PAMPA can be able to estimate blood-brain barrier penetration [1], gastrointestinal absorption and skin permeation [2]. The aim of the present study is to develop an in vitro non-cell based buccal permeability assay. The correlation was investigated between ex vivo measurements and PAMPA effective permeability (Pe) using different membrane systems. [1] L. Di, E. H. Kerns, K. Fan, O. J. McConnell, G. T. Carter, High throughput artificial membrane

permeability assay for blood-brain barrier, Eur. J. Med. Chem. 38 (2003) 223-232. [2] B. Sinkó, T. M. Garrigues, G. T. Balogh, Z. K. Nagy, O. Tsinman, A. Avdeef, K. Takács-Novák, Skin-

PAMPA: A new method for fast prediction of skin penetration, Eur. J. Pharm. Sci. 45 (2012)698-707.

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An investigation on Caco-2 Permeability Studies of Metformin or Gliclazide Alone and Gliclazide Plus Metformin

Seval Korkmaz, Banu Erdem

Abdi İbrahim İlaç A.Ş. R&D Center, 34555 Esenyurt- İstanbul, Turkey [email protected]

Diabetes mellitus type II is one of the common metabolic disorders is characterized by high blood glucose level in the context of insulin resistence and relative insulin deficiency. The most frequently used initial pharmacological agent for the treatment diabetes; metformin is an oral antidiabetic drug in the biguanidine class. Sulphonylurea agents, such as gliclazide, are the most commonly used add-on treatment in patients not adequately controlled with metformin monotherapy. Current study was proposed to identfy permeability profile of metformin and gliclazide alone and gliclazide plus metformin. Caco-2 studies were performed for intestinal permeability characheristics of these two drugs and samples were evaluated by using LC/MSMS method and BCS classification of them identified. At the end of current study, permeability profile and permeability constants of metformin and gliclazide and gliclazide plus metformin evaluated. New LC/MSMS method was developed and presented.

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In vitro evaluation of penetration of nanonized alaptide from semi-solid formulations

Josef Jampilek, Radka Opatrilova, Aneta Cernikova, Lenka Coufalova, Jiri Dohnal

Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 61242 Brno, Czech Republic,

[email protected]

Alaptide, (S)-8-methyl-6,9-diazaspiro[4.5]decane-7,10-dione, was discovered in the 1980s at Prague by Kasafirek et al. and was characterized as a melanocyte-stimulating hormone inhibiting factor (MIF) [1], nevertheless alaptide also showed a significant curative effect in different therapeutic areas on experimental animal models [2]. An influence of alaptide on epidermal regeneration was investigated in a number of tests. In vivo experiments were performed using domestic pigs, to which alaptide was applied on experimental injury, and faster skin regeneration was observed after alaptide application. Similarly, alaptide accelerated curing of experimental skin injuries in rats. Alaptide also demonstrated very low acute toxicity [3].

NH

H3C

OHN

O

(S)-alaptide: (S)-8-methyl-6,9-diazaspiro[4.5]decan-7,10-dione

*

Unfavourable property of the compound is its poor water-solubility and consequent

insufficient membrane permeation. As preparation of nanoparticles is an effective tool for

increasing solubility, nanonized alaptide was prepared from micronized alaptide using a

nanomill. Various semi-solid formulations were prepared from this modified alaptide and

the penetration of nanonized alaptide through full thickness dorsal skin from porcine ear

was evaluated [4].

This study was supported by the Czech Science Foundation – GACR P304/11/2246. [1] E. Kasafirek, J. Vanzura, I. Krejci, J. Krepelka, A. Dlabac, M. Valchar (SPOFA & VUFB). 2,5-

Piperazinedione derivs. Belgian patent Belg. 897843, 1984 & Czechoslovakian patent CS 231227, 1986.

[2] S. Radl, E. Kasafirek, I. Krejci. Alaptide. Drugs Fut. 15(1990) 445-447. [3] E. Kasafirek, L. Korbova, J. Kohout, M. Jiraskova, I. Krejci, A. Galatik (SPOFA & VUFB). Preparation

for local therapy of cutaneous and mucosal lesions. Czechoslovakian patent CS 276270, 1992. [4] J. Jampilek, R. Opatrilova, A. Rezacova, Z. Oktabec, J. Dohnal (FaF VFU Brno). Alaptide: Methods

of effecting its solubility, membrane penetration and pharmaceutical compositions for human and veterinary applications. PTC/CZ2012/000074, 2012.

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Biorelevant solubility study of poorly soluble neutral and ionizable drugs

Gergely Völgyi, Vera Szőke, Péter Horváth, Krisztina Takács-Novák

Department of Pharmaceutical Chemistry, Semmelweis University, Budapest, Hungary

Knowledge of the physico-chemical parameters (ionization, solubility, lipophilicity and permeability) of drug molecules is essential in the estimation of their pharmacokinetic properties. Among these parameters, solubility is used to predict the absorption of orally administered drugs from the gastrointestinal tract. However, solubility is influenced by many factors, such as pH, temperature and the composition of the gastrointestinal tract, and therefore the importance of solubility measurement in biorelevant media (BRM) has increased. In this poster the biorelevant solubility of four drugs (rivaroxaban, furosemide, papaverine and niflumic acid) is presented. The equilibrium solubility of these molecules was determined in simulated gastric fluid (SGF pH 1.2), in simulated intestinal fluid fasted state (FaSSIF pH 6.5) and fed state (FeSSIF pH 5.0) and their corresponding blank buffers at a temperature of 37°C using saturation shake-flask method. The concentration was measured by optimized HPLC analysis. The solubilizing effect of bile acid/lipid micelles as additive components of BRM was expressed with the solubility ratio (SR: SBRM/Sblank buffer) and the food effect was estimated from SFeSSIF/SFaSSIF coefficient. It was concluded that ionization plays primarily role in solubility of compounds which undergo ionization in BRM. The solubilizing effect in FaSSIF was marginal for the neutral compound (rivaroxaban) and for molecules are anionic at pH 6.5 (furosemide and niflumic acid). The higher concentration of solubilizing agents in FeSSIF improved the solubility of papaverine carrying positive charge and niflumic acid being partially zwitterionic at pH 5.0 [1]. [1 K. Takács-Novák, V. Szőke, G. Völgyi, P. Horváth, R. Ambrus, P. Szabó-Révész, Biorelevant

solubility of poorly soluble drugs: Rivaroxaban, furosemide, papaverine and niflumic acid, J. Pharm. Biomed. Anal. 83 (2013) 279-285.

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Effect of plasdone polymers on the solubility enhancement of some acidic and basic drugs

Elisenda Fornells*, Meritxell Mañé*, Elisabet Fuguet*,**, Clara Ràfols*

,**,

Elisabeth Bosch*,**, Rebeca Ruiz***, Karl J. Box***, John Comer***

*Departament de Química Analítica, Universitat de Barcelona, Barcelona (Spain) **Institut de Biomedicina de la Universitat de Barcelona, (IBUB)

***Sirius Analytical Ltd., Forest Row, East Sussex, (UK)

Medicinal chemists devote much effort to the enhancement of drug solubility in gastric tract conditions since it is a major factor in drug bioavailability. One promising way to improve drug solubility is the addition of polymers able to stabilize the supersaturated drug solution. In this work, intrinsic solubility and supersaturation profiles of two diprotic acidic compounds (benzthiazide and bendroflumethiazide) and several basic molecules (bupivacaine, ciproheptadine, haloperidol, propanolol, maprotiline and papaverine) have been determined in the presence and absence of polyvinylpyrrolidone polymers, generically known as plasdones. When different plasdones (K-12, K-17, K-25, K-29/32, K-90, and S-630) are added to benzthiazide, supersaturated solutions are created. The plasdone with lower polymerization degree (K-12) provides the higher degree of supersaturation. When plasdones are added to bendroflumethiazide a different behaviour is observed. Thus, solubility increases almost fifteen-fold but supersaturated solutions are not formed. Finally, six basic pharmaceutical drugs have been tested too. However, none of them changes its behaviour significantly when plasdones are added to the drug solution, so only bupivacaine has been selected for further studies. To investigate the nature of the drug-plasdone interaction itself, three different studies (solubility, X-ray and HPLC) have been done. They support the hypothesis that plasdones interact with drugs through different mechanisms. In case of bendroflumethiazide some kind of complex or aggregate between the drug and plasdones is formed. In case of benzthiazide, supersaturated solutions are formed when plasdones are added, but after a certain time, the solution returns to its equilibrium state. Results obtained for the selected bases show a slight interaction of bupivacaine with the tested plasdones whereas the other basic compounds do not interact with them.

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Dynamic dissolution method provides rank-order prediction of in vivo performance for immediate release formulations of a BCS class II drug

Martin Čulen, Jiří Dohnal

Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1-3, 612 42 Brno, Czech Republic

The current research and development (R&D) field could benefit from dissolution methods that could easily provide accurate predictions of in vivo performance of drug products. In the presented study, a physiologically relevant dissolution model, simulating fasted gastrointestinal tract (GIT), based on a four-compartmental instrument, was tested. The model mimicked the GIT in terms of transit times, peristaltic mixing, dynamic change of the medium, and a biorelevant volume and composition of the medium, including enzymes and bile salts. Three developed generic immediate release formulations of a weak acid BCS class II drug were used for the experiment. These were previously tested in separate bioequivalence studies along with a reference product. The dissolution method provided correct rank-order prediction for the three generic products; one bioequivalent, one having lower and one higher bioavailability. The method furthermore revealed important difference in the dissolution behavior of the reference and the tested products, in stomach compartment, resulting from the use of a buffering agent in the reference tablet. The buffering agent raised the pH in stomach compartment to values around 7 and significantly improved dissolution of the active substance contained. A classical USP 2 dissolution experiment, using 900 ml of medium, failed to detect this effect and resulted in incorrect rank-order prediction. The presented dynamic dissolution model offers many advantages over the contemporary instruments, mainly in means of biorelevancy, and could be a valuable new tool in research and development.

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Solubility and thermodynamic properties of methylxanthines

Aneta. Pobudkowska, Urszula Domańska, Justyna. A. Kryska

Department of Physical Chemistry, Faculty of Chemistry, Warszaw University of Technology, Poland

Xanthine derivates belong to alkaloids which are described as natural bases having nitrogen atoms in molecular structure. Like other molecules of this major group Xanthine derivatives property have strong physiological effect on human and animal organism. The most famous substance of this group is Caffeine - the tea and coffee culture has a several centuries long age. Two other derivatives of xanthine – Theophylline and Theobromine have a long history of use in the treatment of asthma [1]. Use of natural Xanthine derivates in medicine is complicated with their physical properties. The aim of this study was to evaluate the physio-chemical properties of the drugs. The study involved three drugs: 7-(β-Hydroxyethyl) theophylline, Theobromine and Theophylline in binary systems with solvents. The solvents used were: water, ethanol, and 1 - octanol. Score of the solubility of these substances is being important for their dissolution effect inside the cell, the transportation by bod’s fluids and the penetration possibility of lipid membranes. Pharmaceuticals were presented to the group of methylxanthines, which in their structure contain purine. Solubility of 7 (β-Hydroxyethyl) theophylline and Theophylline were tested using synthetic method [2]. In case of Theobromine, which solubility is very low in mentioned solvents, the spectrophotometric method has been used to measure its solubility [3]. After designating phase diagrams of each of the solute in the bipolar system, experimental points have been correlated with the equations: Wilson, NRTL, UNIQUAC. Results show that Theophylline and its derivatives have the best solubility from all pharmaceutics. Another method also used during this study was differential scanning calorimetry (DSC), which allowed to designate the thermal properties of pharmaceuticals. These are parameters such as the melting temperature, enthalpy of melting, glass transition temperature. It was found that it is not possible to determine the melting temperature and enthalpy of Theobromine. Last property tested was constant acidity, to this end, the spectrophotometric method of Bates – Schwarzenbach [4]. Both awareness and knowledge of values of the drug pKa solubility is important in its production. This allows the selection of a suitable solvent and allows estimation of the correct dose and its capacity to absorb in human body. [1] J.J. Johnston, Evaluation of cocoa- and coffee-derived methylxanthines as toxicants for the

control of pest coyotes, J. Agric Food Chem. 53 (2005) 4069-4075. [2] U. Domańska, A. Pobudkowska, A. Pelczarska, Ł. Żukowski, Modelling, solubility and pKa of five

sparingly soluble drugs, Int. J. Pharm., 403 (2011) 115-122. [3] U. Domańska, A. Pobudkowska, A. Pelczarska, M. Winiarska-Tusznio, Solubility and pKa of selekt

pharmaceutical in water, etanol, and 1-octanol, J. Chem. Therm., 42 (2010) 1465-1472. [4] U. Domańska, A. Pobudkowska, A. Pelczarska, Solubility of sparingly soluble drug derivatives of

anthranilic acid, J. Phys. Chem. B, 115 (2011) 2547-2554.

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Evaluation of drug polymorphic difference on dissolution behavior

Nataša Mrvičin, Vlatka Džale, Ivana Sorić, Ana Martinčić, Biserka Cetina-Čižmek

PLIVA Croatia Ltd., Research and Development, Prilaz baruna Filipovića 29, Zagreb, Croatia

The purpose of our work is to develop drug product using API in anhydrous polymorphic form which needs to match in vivo behavior of reference drug (RLD) in monohydrate form. Initial solubility results indicate the significant difference in solubility between these two polymorphic forms. That could have impact on in vivo drug performance. Therefore, two different formulations (formulation I and II) have been developed, one of them with functional excipients that modify drug dissolution. pH solubility profile was determined for both polymorphic forms (anhydrous and monohydrate form) by solvent saturated method in physiological pH range (aqueous buffers at pH 1.2 – 6.8). Results show higher solubility of anhydrous form than monohydrate form especially at pH 4.0. Therefore, dissolution testing was performed in pH 4.0 dissolution media using paddle apparatus with the rotational speed at 50 rpm, volume 900 ml. Dissolution results of two different formulations compared with referent product show that formulation II has similar dissolution profile with referent product although it contains anhydrous form of drug substance. Formulation I which is similar in composition to the referent product, with exception of drug substance in anhydrous form, shows higher dissolution profile in the most discriminatory dissolution medium pH 4.0. Both developed formulations will be evaluated on in vivo performance.

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Investigation of Xylazine hydrochloride phase transition from polymorphs Z and M to polymorph A

Kristīne Krūkle-Bērziņa, Kirils Oļehnovičs, Andris Actiņš

University of Latvia, Faculty of Chemistry, LV-1045, Kr. Valdemāra iela 48, Riga, Latvia Xylazine hydrochloride (2-(2,6-xylidino)-5,6-dihydro-4H-1,3-thiazine hydrochloride) is an adrenergic α-agonist used as a sedative, analgesic, and muscle relaxant in veterinary medicine [1]. Xylazine hydrochloride crystallizes in four polymorphs (A, M, Z and X) and several pseudopolymorphs. The form A is the most stable of the polymorphs at temperatures above 50

oC. The transition of polymorphs X, M and Z to polymorh A is

observed at temperature above 50 oC [2, 3]. From all of the possible Xylazine

hydrochloride polymorph phase transitions the kinetic of the solid-state phase transition from X to A is the most studied one. The kinetic of the polymorph transitions can be affected by many factors, such as temperature, humidity, mechanical stress, compression [4], crystalline defects and particle size [5]. In this study the phase transition from Xylazine hydrochloride polymorphs Z and M to A form was investigated by powder x-ray diffractometery. The effect of the temperature, relative humidity, amount of the polymorph A in the initial sample and other parameters on the phase transition rate was investigated. Both transitions were also investigated by powder x-ray diffractometer in in situ mode. The kinetical model and rate constant for all experimental phase transition data sets were determined. The activation energy of phase transition was calculated using two methods – modelistic method and isoconversional methods. [1] European pharmacopeia, 4th edition. Council of Europe, Strasbourg, (2001) 2152-2153. [2] Bērziņš, A., Actiņš, A., Veldre, K. Lat. J. Chem. 3 (2008) 263-269. In Latvian. [3] Bērziņš A., Krūkle K., Actiņš A, Kreišmanis J. P. Pharm. Dev. Technol. 15 (2010) 217-222. [4] Kishi Y., Matsuoka M. Crystal Growth & Design. 10 (2010) 2916-2920. [5] Terblanche R., Liebenberg W., De Villiers M., Lötter A. Drug. Dev. Ind. Pharm. 5 (2000) 531-537.

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Experimental and computational study of tegafur polymorphs and organic solvents interaction

Raitis Bobrovs, Andris Actiņš

University of Latvia, Department of Chemistry, Kr. Valdemāra iela 48, LV-1013, Rīga, Latvija

Pharmaceutical compounds are mostly produced in defined crystalline forms that are commonly crystallized from solutions. Polymorphs usually have different, precisely known dissolution rates and bioavailability, but crystal size and shape can affect these properties. Therefore, the crystal shape is monitored during drug manufacturing process and a lot of work in the area of crystal engineering has been devoted to controlling the crystal habit and determination of crystal properties [1,2]. The solvent used for the crystallization of an organic compound can have a large effect on the resulting crystal shape and morphology [3-5]; therefore solvent effects on crystal surface have been comprehensively studied. Most of the research regarding solvent effects on surfaces has been performed with solutions, but an identical situation should occur in solvent vapor atmosphere as well. Desorption behavior of organic solvents from α and β tegafur (5-fluoro-1-(tetrahydro-2-furyl)uracil), as well as molecular mechanics calculation for these systems has been studied in this work. Solvent desorption from samples stored at 95 % relative solvent vapor pressure was studied in isothermal conditions at 30 °C. The results of this study demonstrated that solvent desorption did not differ significantly for both phases, and solvent desorption in all cases occurred faster from samples with the largest particle size, and solvent desorption in most cases occurred in two steps. Theoretical calculations were performed to predict α and β tegafur crystal morphology and to calculate the surface and solvent interaction energies. Solvent desorption data matched the calculated interaction energies – desorption was faster for those solvents with low surface-solvent interaction energy. Molecular mechanics calculations indicated that interaction energy increases upon increasing the solvent molecule volume, and solvent adsorption was most energetically favourable on the (011) and (10-1) crystal faces of β tegafur, as well as the (010) face of α tegafur. The solvent and surface interaction effect on actual crystal shape has been discussed. Scanning electron micrographs showed that small tegafur particles recrystallized to larger particles during sample exposure to solvent vapor atmosphere. [1] H.G. Brittain, Polymorphism in Pharmaceutical Solids, second ed., Informa Healthcare, 2009. [2] R. Hilfiker, Polymorphism in the pharmaceutical industry, Wiley-VCH, Weinhei, 2006. [3] M. Lahav, L. Leiserowitz, The effect of solvent on crystal growth and morphology, Chem. Eng. Sci.

56 (2001) 2245–2253. [4] C. Stoica, P. Verwer, H. Meekes, P. Van Hoof, F.M. Kaspersen, E. Vlieg, Understanding the effect

of a solvent on the crystal habit, Cryst. Growth Des. 4 (2004) 765–768. [5] J. Schöll, D. Bonalumi, L. Vicum, M. Mazzotti, M. Müller, In situ monitoring and modeling of the

solvent-mediated polymorphic transformation of L-glutamic acid, Cryst. Growth Des. 6 (2006) 881–891.

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The preparation of various forms of furosemide and analysis of their structures

A. A. Beloborodova*, V. S. Minkov*,**, E. V. Boldyreva*

,**

*Dept. Solid State Chemistry, Novosibirsk State University, Russia **Institute of Solid State Chemistry and Mechanochemistry SB RAS, Russia

The studies of polymorphism of molecular crystals of biologically active substances and drugs are important for understanding the relationship between the preparative methods and the resulting structure and properties. They find applications in optimizing the preparation of pharmaceuticals, in design of novel products and in improving their efficacy and reactivity. Production of any drug involves not only the invention and synthesis of a new drug substance (based on the knowledge of the targeted biochemical mechanism and modern synthetic and computational technologies), but also the development and manufacturing of dosage forms, the properties of which can depend on the preparative method. Development of new drug forms is also of great importance for patent protection agencies, in particular, for production of generics. The development of new formulations is not always related to significant changes in the chemical composition of a drug substance itself, but often involves only variations in crystal structure, shape and size of particles, crystallinity (often aiming at an amorphous state), and may also include adding the excipients. The cost of developing new forms of known drugs is significantly lower than that of introducing a new substance on the market. In the present work Furosemide, an anthranilic acid derivative, was studied. Given the importance of this compound as an active pharmaceutical ingredient in many medications (as well as being used as a diuretic), it was interesting to attempt to understand the effects of preparative methods on the crystallisation of this compound. Various polymorphs and solvates of Furosemide were obtained by different crystallisation techniques. Their structures and properties were characterised by single-crystal and powder X-ray diffraction. The study was supported by a grant from the Russian Ministry of Education and Science No. 14.B37.21.1093 and an Integration Project of the SB RAS No 108.

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Study and formulation of promazine hydrochloride–loaded nanoparticles

Halayqa Mohammed, Urszula Domańska

Department of Physical Chemistry, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland

In recent years, significant effort has been devoted to develop nanotechnology for drug delivery. Nanotechnology focuses on formulating therapeutic agents in biocompatible nanocomposites such as nanoparticles, nanocapsules or micellar systems [1,2]. In this work the formulation of a polymeric nanoparticls (NPs) as the drug delivery system has been studied. Promazine hydrochloride (PMZ), hydrophobic molecule, was intrapped into biodegradable poly(DL-lactide-co-glycolide) (PLGA) by using emulsion-solvent evaporation method. PMZ was capsulated into nanoparticles with theoretical drug loading (DLth) varying from 10 to 30% (w/w). The effects of sonication time, PLGA concentration in organic phase and the pH of aqueous phase were studied. After lyophilization of promazine hydrochloride-loaded nanoparticles, the average size, charge, and polydispersity index were 365 ± 21 nm, −22.4 ± 5.07 mV, and 0.194 ± 0.2, respectively. The maximum drug encapsulation efficiency and loading capacity were 61.4 ± 4.1% (w/w) and 16.3 ± 1.6 mg/100 mg nanopowder, respectively. Scanning electron microscopy studies showed spherical and smoth shape of drug-loaded nanoparticles. Acknowledgement: Funding for this research was provided by the Warsaw University of Technology. [1] J. Panyama, V. Labhasetwara, Adv. Drug Deliv. Rev. 55 (2003) 329–347. [2] C. Astete, C. M. Sabliov, J. Biomater. Sci. Polymer Edn, 17 (2006) 247–289.

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Domoic acid biotoxin from seawater environment: Raman and SERS characterization and detection

Csilla Müller, Karina Weber*, Dana Cialla*, Jürgen Popp*, Iva Pozniak**, Branko Glamuzina**, Simona Cîntă Pînzaru

Babeş-Bolyai University, Biomedical Physics, Theoretical and Molecular Spectroscopy Department, 400084 Cluj Napoca, Romania

*Friedrich Schiller University, Institute of Physical Chemistry and Abbe Center of Phothonics, 07743 Jena, Helmholtzweg 4, Germany and Institute for Photonic Technology,

Albert-Einstein-Strasse 9, 07745 Jena, Germany **University of Dubrovnik, Department for Aquaculture, Ćira Carića 4,

20000 Dubrovnik, Croatia

Domoic acid (DA), C15H21NO6, is a marine biotoxin released during the blooming period of phytoplankton algae (Nitzschia navis-varingica) that can accumulate in shellfish, such as mussels, oysters, limpets, clams and scallops, during filter feeding [1,2,3]. When accumu-lated in high concentration by shellfish, domoic acid can be passed on to humans via con-sumption of the contaminated seafood, producing severe intoxication known as amnesic shellfish poisoning (ASP). In more severe cases, neurological symptoms develop within 48 hours from investion disorientation and loss of short memory. People poisoned with very high doses of the toxin can die. DA intoxication has also resulted in the mortality of hundreds of marine birds, mammals and fish in many different parts of the world [1]. The European Union has established a permitted level of 20 mg DA/kg in shellfish [2]. Traditional methods (HPLC) to detect DA in algae and in food could provide DA concentration levels below those of concern in food, being expensive, time consuming and unavailable for in situ and real time detection.[3] Since surface enhanced Raman spectroscopy (SERS) combines a fingerprint specificity with an increased sensitivity by several orders of magnitude, this technique is a powerful candidate for the detection of DA with less techniqual effort than traditional methods [4]. In this paper we present the characterization and detection of DA, in a large pH and concentration range using Raman and SERS spectroscopy assisted by theoretical DFT calculations. Complete vibrational characterization is provided in order to correctly assign the SERS signal in various conditions regarding the pH or concentration range. We can detect the DA up to 0.33 ppm (3.3×10

-7 mol l

-1) using simple chemically reduced silver

nanoparticles. Additionally, an in situ detection scheme of DA was proposed in seawater environment, taking into account the specific seawater SERS signal and its partial interference with those of DA.

[1] Q. Wu, W.H. Nelson, J.M. Treubing Jr., P.R. Brown, P. Hargraves, M. Kirs, M. Feld, R. Desari, R. Manoharan, E.B. Hanlon, Anal. Chem. 72 (2000) 1666-1671.

[2] Y. Djaoued, S. Balaji, S. Priya, Spectrochim. Acta, Part A. 67 (2007) 1362-1369. [3] Y. Yao, W.H. Nelson, P. Hargraves, J. Zhang, Appl. Spectrosc. 51 (1997) 785-791. [4] Dana Cialla, Anne März, René Böhme, Frank Theil, Karina Weber, Michael Schmitt, Jürgen Popp,

Anal. Bioanal. Chem. 403 (2012) 27–54.

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Characterization of Antazoline Oxalate and Antazoline Maleate

Agnese Dravniece, Kristīne Krūkle-Bērziņa, Andris Actiņš

University of Latvia, Faculty of Chemistry, LV-1045, Kr. Valdemāra iela 48, Riga, Latvia Antazoline base (C17H19N3, N(4,5-dihydro-1H-imidazol-2-ylmethyl)-N-(phenylmethyl)-aniline), an imidazoline derivative, is an antihistamine used in treatment of nasal congestion and in eye drops because of its anticholinergic properties [1]. Introducing and researching new salts is an opportunity to improve drug substance properties [2, 3]. Therefore several antazoline salts have been obtained in order to perform polymorph screening [4], however, only antazoline oxalate (AO) and antazoline maleate (AM) are considered for additional characterization. New polymorphs of antazoline salts have been found and characterized by powder X-ray diffractometry (PXRD), single crystal X-ray diffractometry (SCXRD), differential scanning calorimetry (DSC), differential thermal analysis (DTA) and thermogravimetry (TG) as well as infrared spectrometry (IS) and Karl Fischer titration. Antazoline salts have been crystallized in ten different solvents as well as obtained by co-grinding salt components in a ball mill with or without a solvent. Anhydrous form and solvate of AO and two polymorphs of AM have been found. The stability of obtained crystalline forms as well as the solid state phase transition kinetics of AM polymorphs have been studied. Solubility of AM polymorphs in different temperatures has been determined. The crystal structures of thermodynamically stable forms of antazoline oxalate (AO-α) and antazoline maleate (AM-β) have been determined and analysed. AM crystallization in various solvents is found to be the optimal method for AM-α formation whereas co-grinding AM salt components has been defined as optimal method for AM-β preparation.

[1] Figus M. et al. Treatment of allergic conjunctivitis: results of a 1-month, single-masked

randomized study. Eur J Ophthalmol., 20, (2011) 811. [2] Giron D. Characterisation of salts of drug substances, J. Therm. Anal. Cal. Vol. 73. Kluwer

Academic Publishers, Dordrecht (2003) 441. [3] Stahl P.H., Wermuth C.G. (Eds.) Handbook of Pharmaceutical Salts. Wiley-VCH, Zurich (2002)

374. [4] Dravniece A., Actins A., Krukle-Berzina K. New crystalline forms of antazoline. 12th International

Conference on Pharmacy and Applied Physical Chemistry, Austria, 06.05.2012-09.05.2012.

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Characterization of Antazoline Base

Kārlis Bērziņš, Ilze Grante, Andris Actiņš

University of Latvia, Faculty of Chemistry, LV-1045, Kr. Valdemāra iela 48, Riga, Latvia Antazoline base (C17H19N3, N(4,5-dihydro-1H-imidazol-2-ylmethyl)-N-(phenylmethyl)-aniline), an imidazoline derivative, is an antihistamine drug with anticholinergic properties used in treatment of nasal congestion and allergic conjunctivitis [1]. The study of the carbon dioxide effect on drug solutions in ambient conditions is essential in pharmaceutical industry. This kind of research greatly contributes to phisico-chemical profiling of drug itself, as well as benefits manufacturing, obtaining new salts and/or polymorphs of the substance [2]. Protonation constants of antazoline base were determined in inert and oxygen atmosphere by potentiometric titrations in semi-aqueuos solutions. Amorphous phase of antazoline base was obtained as by-product during titration and significant shift of stoichiometric point was observed. Correlation between content of amorphous phase in dry matter of titrand and stirring time before the titration has been described and characteriezed by powder X-ray diffractometry (PXRD), differential thermal analysis (DTA) and thermogravimetry (TG) as well as infrared spectrometry (IS) and nuclear magnetic resonance (NMR). Effect of protonation on UV/VIS spectra of antazoline was investigated. Finally, a new mathematical model describing titration curves and stoichiometric point dependence on stirring time before titration in ambient conditions has been developed. [1] Figus M. et al. Treatment of allergic conjunctivitis: results of a 1-month, single-masked

randomized study. Eur J Ophthalmol., 20, (2011) 811. [2] Richard A., Ymėn S., Ymėn I. (Eds.) Solid State Characterization of Pharmaceuticals, First Edition.

Blackwell Publishing Ltd., (2011) 506

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http://en.wikipedia.org/wiki/Allergy

http://en.wikipedia.org/wiki/Conjunctivitis


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Thermal study of antipsychotic active substance aripiprazole and its mixtures with different excipients

Adriana Fulias, Florin Soponar*, Mihaela Maria Şandru**, Balazs Szaniszlo**, Niculina Bădicu**, Ionut Ledeti

University of Medicine and Pharmacy “Victor Babeş”, Faculty of Pharmacy, Eftimie Murgu Square 2, Timişoara, RO-300041, Romania

*University of Bucharest, Faculty of Chemistry, Department of Analytical Chemistry, 90 Panduri Avenue, Bucharest 050663, Romania

**Research and Development Center, S.C. Polipharma Industries S.R.L., 156 Alba Iulia Street, Sibiu 550052, Romania

Aripiprazole (ARP) is a partial dopamine agonist of the second generation class of atypical antipsychotics with additional antidepressant properties which has been used in the treatment of schizophrenia, bipolar disorder and major depressive disorder. In the present study, the physico-chemical properties of ARP were fully characterized using five analytical techniques such as Thermogravimetric analysis, DSC, FT-IR, Powder X-ray diffraction, and SEM. The DSC curve shows a sharp endothermic event at 141.2 °C which corresponds to the melting (an endothermic effect with no mass loss ΔH=-77.28 J/g), and the TG/DTG curve presents one decomposition event between 210 °C and 490 °C with a mass loss of 90% and DTGpeak= 302 °C.

Fig. 1. The thermoanalytical curves DSC/TG/DTG obtained in N2 at β=10 °C·min

-1 for ARP

A compatibility study involving ARP and 18 pharmaceutical excipients generally used in pharmaceutical formulations was performed. Binary physical mixtures of ARP with each excipient were prepared in a 1:1 (w/w) ratio. After preparation, the samples were analyzed immediately and the results reveal solid-state interaction with Tablettose 80, Mg and Ca stearate, Rylo MD50, Kollidon 90 and 30 and stearic acid. Such interactions between the drugs and excipients, observed in the DSC curves, should result in the partial or complete disappearance of the reactant phases and appearance of new phases. These changes can be inferred from X-ray diffraction patterns and from FT-IR spectroscopy [1].

Acknowledgements: This work was supported by a grant from the University of Medicine and Pharmacy “Victor Babeş” Timişoara (Grant 15250/19.12.2012 to Adriana Fuliaş).

[1] A. Fulias, I. Ledeţi, G. Vlase, T. Vlase, Physico-chemical solid-state characterization of pharmaceutical pyrazolones, J. Pharm. Biomed. Anal. 81–82 (2013) 44–49.

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Synthesis, thermoanalytical behavior and EGA study of a highly specific anti-prostate cancer compound - betulonic acid

Adriana Fulias, Gabriela Vlase*, Titus Vlase*, Vasile Bercean**, Ionut Ledeti


*West University of Timisoara, Research Centre for Thermal Analysis in Environmental Problems, Pestalozzi Street 16, Timişoara, RO-300115, Romania

**“Politehnica" University of Timişoara, Faculty of Industrial Chemistry and Environmental Engineering, Carol Telbisz 6, Timişoara, RO-300001, Romania

Betulonic acid [lup-20(29)-en-3-oxo-28-oic] is a pentacyclic triterpenoid derived from betulinol, that possess valuable biological properties such as antiviral, antitumor, anti-inflammatory, antimicrobial, hepatoprotective, as well as immunostimulant activities [1]. Even if betulonic acid (BetoA) was not studied as much as betulinic acid, studies have shown that it has a highly specific anti-prostate cancer activity in in vitro cell cultures [2]. In this study, we present the synthesis of betulonic acid following a modified Jones’ oxidation reaction using as starting material betulinol. Physico-chemical properties of BetoA are in good agreement with the one mentioned in literature. Following the synthesis of high-purity BetoA, it’s thermal behaviour and FT-IR study of gaseous mixtures evolved during the decomposition which occurs under heating in oxidative and inert atmospheres were studied [3].

0 100 200 300 400 500

0

20

40

60

80

100

en

do

Mass lo

ss / %

Temperature/ oC

TG

-0.14

-0.12

-0.10

-0.08

-0.06

-0.04

-0.02

0.00

0.02

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-5

0

5

10

15

20

25

30

35

HF

DT

G/ m

g.m

in-1

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/ m

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Fig. 1. Structure of BetoA and thermoanalytical curves obtained in air at β=10°C·min

-1


[1] N. Melnikova, I. Burlova et al., A Practical Synthesis of Betulonic Acid Using Selective Oxidation of Betulin on Aluminium Solid Support, Molecules, 17 (2012) 11849-11863.

[2] B.B. Saxena, L. Zhu et al., Boc-lysinated-betulonic acid: a potent, anti-prostate cancer agent, Bioorg. Med. Chem. 14(18) (2006) 6349-6358.

[3] A. Fulias, I. Ledeţi, G. Vlase, T. Vlase, Physico-chemical solid-state characterization of pharmaceutical pyrazolones, J. Pharm. Biomed. Anal. 81–82 (2013) 44–49.

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Thermal behavior of antiemetic and gastroprokinetic agent metoclopramide hydrochloride and compatibility study with pharmaceutical excipients

Adriana Fulias, Gabriela Vlase*, Titus Vlase*, Ionut Ledeti


West University of Timisoara, Research Centre for Thermal Analysis in Environmental Problems, Pestalozzi Street 16, Timişoara, RO-300115, Romania

Metoclopramide (MET) is an antiemetic and gastrokinetic agent, currently used in several pharmaceutical formulations. It is used to treat vomiting and nausea, it facilitates the gastric motility in people with delayed gastric emptying, and as a treatment for the retention of fluid in the stomach. In this work, the thermal behaviour of MET was studied by drawing up the TG/DTG/HF curves in air atmosphere at β= 10 °C·min

-1. The data on a

possible drug-excipient interaction were obtained from the thermoanalytical study of mixtures of this active compound with talc, magnesium stearate (MS), SiO2, manitol, microcrystalline cellulose (MC), sorbitol and polyvinylpyrrolidone. The information on the thermal-induced events were corroborated with the FT-IR spectra of the solid samples (before and after heating), respectively with the ones obtained by evolved gases analysis (EGA). The processes between 60 and 112 °C correspond to the decomposition of the metoclopramide hydrocloride (HCl loss), which are followed by melting (THF melting=183 °C) and by decomposition at higher temperatures (200-450 °C).

Fig. 1. Structure and thermoanalytical curves obtained in air at β=10°C·min-1

for MET

According to the thermal curves, especially HF curves, one can say that all tested excipients, less MC, SiO2 and MS, present compatibility with MET. This fact is supported by the differences between the values of THF melting and of the enthalpies of melting (ΔH), which were confirmed also from X-ray diffraction patterns and from FT-IR spectroscopy.


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Comparative thermal behavior of synthetic thyroid hormone L-thyroxine and L-thyroxine sodium salt hydrate vs. pharmaceutical formulations

Ionut Ledeti, Gabriela Vlase*, Titus Vlase*, Adriana Fulias



Levothyroxine (L-thyroxine or T4), is a synthetic form of the thyroid hormone thyroxine which is currently used to treat thyroid hormone deficiency, and as a preventer for the recurrence of thyroid cancer. Today, most patients are treated with levothyroxine, or L-thyroxine sodium salt hydrate, which are currently formulated as numerous brand names, as well as generic versions [1]. Our study deals with the physico-chemical characterization of synthetic thyroid hormone L-thyroxine and L-thyroxine sodium salt hydrate vs. common pharmaceutical formulations (Merck Euthyrox 100® and Merck Euthyrox 200®), which are usually commercialised on the Romanian pharmaceutical market. Samples were fully characterized using five analytical techniques such as Heat Flow (HF), Thermogravimetric analysis (TG/DTG) and FT-IR spectroscopy. Comparative with L-thyroxine where mass loss begins at 203 °C (DTGmax = 237.6 °C), the sodium salt presents a more complex way of decomposition. By the analysis of TG curve corresponding to hydrated sodium thyroxine, one can notice a multistadial degradation process which begins at considerable lower temperatures (37 °C). A similar thermal behaviour can be observed in the case of pharmaceutical formulations, namely Euthyrox 100® and Euthyrox 200® tablets.

Fig. 1. The thermoanalytical curves TG/DTG obtained in air at β=10°C·min

-1

Acknowledgements: This work was supported by a grant from the University of Medicine and Pharmacy “Victor Babes” Timisoara (Grant 15250/19.12.2012 to Adriana Fulias).

[1] . Nakajima, M. Yamada et al. Resistance to thyroid hormone due to a novel thyroid hormone receptor mutant in a patient with hypothyroidism secondary to lingual thyroid and functional characterization of the mutant receptor, Thyroid. 20(8) (2010) 917-926.

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Thermoanalytical study on methotrexate – active substance and tablets

Ionut Ledeti, Gabriela Vlase*, Titus Vlase*, Georgeta Simu, Paul Albu**, Adriana Fulias



**„Vasile Goldis” Western University of Arad, Faculty of Medicine, Pharmacy and Dental Medicine, Liviu Rebreanu Street 86 Arad, RO-310414, Romania

Methotrexate (MTX) is an antineoplastic agent used in the therapy of numerous types of cancers, as well in the treatment of some autoimmune disorders, especially rheumatoid arthritis. It is an inhibitor of tetrahydrofolate dehydrogenase and prevents the formation of tetrahydrofolate, necessary for synthesis of thymidylate, an essential component of DNA [1]. TG/DTG and Heat Flow (HF) data were used to determine the thermal parameters of methotrexate active substance and tablets. A tablet with 2.5 mg active substance was analysed. The TG curves of MTX active substance and tablet displayed three and five thermal decomposition processes, respectively. Analysis of the HF data showed some chemical interactions between methotrexate active substance and the excipients of tablets, suggested by alterations in the melting point of methotrexate and the modification of enthalpies of melting value (ΔH).

0 100 200 300 400 500

40

50

60

70

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do

Mass lo

ss / %

Temperature/ oC

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-0.4

-0.3

-0.2

-0.1

0.0

0.1

DTG

-5

-4

-3

-2

-1

0

1

2

3

4

HF DT

G/ m

g.m

in-1

HF

/ m

W

Fig. 1. Structure and thermoanalytical curves obtained in air at β=10 °C·min-1

for MTX

Acknowledgements: This work was supported by a grant from the University of Medicine and Pharmacy “Victor Babeş” Timişoara (Grant 15250/19.12.2012 to Adriana Fuliaş). [1] S.L. Hider, I.N. Bruce, W. Thomson, The pharmacogenetics of methotrexate, Rheumatology

(Oxford). 46 (2007) 1520-1524.

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Compatibility study of immunosuppressive agent methotrexate with some pharmaceutical excipients

Ionut Ledeti, Gabriela Vlase*, Titus Vlase*, Alina Heghes, Laura Sbârcea, Adriana Fulias



Even if methotrexate (MTX) was primary developed as a chemotherapeutic agent for the treatment of several types of neoplasms, recent studies has shown that can be successfully used in autoimmune disorders, especially rheumatoid arthritis. As an antirheumatic agent, methotrexate is administered weekly in doses up to 10

3 lower than

those required for the treatment of malignancy (5–25 mg/week versus 5000 mg/week). Studies had revealed that an early administration in the course of the disease, proved for MTX a positive result, comparable to the one of the biologic agents recently introduced for the treatment of rheumatoid arthritis [1]. The aim of this study was to characterize and evaluate the thermal behavior of MTX and its binary mixtures with magnesium stearate (MgS), microcrystalline cellulose (MC), starch (St), talc and PVP by TG/ DTG/HF, FT-IR and XRD. The HF curve for MTX showed an endothermic event from 50 to 110 °C, probably due to the formation of a cyclic anhydride by water elimination from the two carboxyl groups followed by the melting endothermic event from 170 to 196 °C (HFmax= 185 °C) and the decomposition peaks between 230-300 °C.

Fig. 1. HF curves obtained in air at β=10 °C·min-1

and FT-IR spectra for MTX and binary mixtures

Acknowledgements: This work was supported by a grant from the University of Medicine and Pharmacy “Victor Babeş” Timişoara (Grant 15250/19.12.2012 to Adriana Fuliaş). [1] B.N. Cronstein, Low-Dose Methotrexate: A Mainstay in the Treatment of Rheumatoid Arthritis".

Pharmacol. Rev. 57(2) (2005) 163–172.

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Solid-state characterization of antiarrhythmic agent amiodarone hydrochloride under non-isothermal conditions. Compatibility study with some

pharmaceutical excipients

Ionut Ledeti, Gabriela Vlase*, Titus Vlase*, Lucretia Udrescu, Adriana Fulias University of Medicine and Pharmacy “Victor Babeş”, Faculty of Pharmacy,

Eftimie Murgu Square 2, Timişoara, RO-300041, Romania *West University of Timisoara, Research Centre for Thermal Analysis in Environmental

Problems, Pestalozzi Street 16, Timişoara, RO-300115, Romania Amiodarone (AM) is used in the treatment of a wide range of cardiac tachyarrhythmias, including both ventricular and supraventricular (atrial) arrhythmias [1]. Even if AM shows several side-effects, including abnormalities in thyroid function, it is used in arrhythmias that are otherwise difficult to treat with other medication [2]. This study aimed to evaluate drug-excipient compatibility of binary mixtures (1:1, w/w), initially by thermoanalytical techniques (TG/DTG/HF), and subsequently, if there were any interaction evidence, by complementary techniques (FT-IR and XRD). For the analyses of the binary mixtures by HF, it was selected polyvinylpyrrolidone (PVP), microcrystalline cellulose (MC), starch (St), talc and anhydrous lactose (LA). The compatibility study is based on changes in the thermal profile of AM, especially its fusion parameters (Tonset and ΔH). A shifting of melting event was observed in the case of the mixture with LA (Tonset = 154 °C), but the additional analyses by FT-IR and XRD showed no interaction evidence. Whatever, the LA should be avoided in the formulation process of AM hydrochloride.

Fig. 1. Structure and thermoanalytical curves obtained in air at β=10 °C·min-1

for AM

Acknowledgements: This work was supported by a grant from the University of Medicine and Pharmacy “Victor Babeş” Timişoara (Grant 15250/19.12.2012 to Adriana Fuliaş). [1] P.J. Kudenchuk, L.A. Cobb et al. Amiodarone for resuscitation after out-of-hospital cardiac arrest

due to ventricular fibrillation. N. Engl. J. Med. 341(12) (1999) 871–878. [2] E.L. Batcher, X.C. Tang et al. Thyroid function abnormalities during amiodarone therapy for

persistent atrial fibrillation Am. J. Med. 120(10) (2007) 880–885.

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Inclusion complexes between pentacyclic triterpenes and amphiphilic cyclodextrins: synthesis and physico-chemical and pharmacological analysis

Codruta Soica, Cristina Trandafirescu, Florin Borcan, Corina Danciu, Stefana Avram, Rita Ambrus*, Istvan Zupko**, Ionut Ledeti, Adriana Fulias, Cristina Dehelean

Faculty of Pharmacy, University of Medicine and Pharmacy V. Babes, Timisoara, Romania *Department of Pharmaceutical Technology, University of Szeged, Szeged, Hungary

**Department of Pharmacodynamics and Biopharmacy, University of Szeged, Szeged, Hungary

Ursolic and oleanolic acids, two isomers with pentacyclic triterpene structure, have been proven as therapeutically active compounds, with antineoplazic and anti-inflammatory activity [1]. Given their low water solubility which is supposed to be the cause of their inadequate bioavailability [2], attempts have been made in order to improve hydrosolubility, under the form of cyclodextrin complexes [3]. The present study focuses on inclusion complexation with amphiphilic cyclodextrins, prepared according to the literature [4] and capable of forming nanostructures with high water solubility and loading capacity of the active compounds. Inclusion complexes were analyzed using thermal analysis, X-ray diffraction and scanning electron microscopy (SEM, fig. 1); nanostructures were in vitro tested using MTT test on several tumour cell lines, showing an improved antineoplazic activity in comparison with the active compounds alone.

a

b

Figure 1. SEM pictures of: a) ursolic acid and b) complexed ursolic acid

In conclusion, cyclodextrin complexation using amphiphilic derivatives may be a powerful tool for future formulations in anticancer therapy.

Acknowledgements: This work was supported by UEFISCDI, PN II – Capacităţi, CT-163/22.04.2013, Module III - bilateral projects Romania-Hungary; contract 674/22.04.2013

[1] M.N. Laszczyk. Pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy, Planta Med. 75 (2009) 1549-1560

[2] D. Gao, S. Tang, Q. Tong, Oleanolic acid liposomes with polyethylene glycol modification: promising antitumor drug delivery, Int J Nanomedicine. 7 (2012) 3517-26.

[3] O. Cerga, F. Borcan, R. Ambrus, I. Popovici, Syntheses of new cyclodextrin complexes with oleanolic and ursolic acids, Journal of Agroalimentary Processes and Technologies. 17 (2011) 405-409

[4] E. Bilensoy, A.A. Hincal. Recent advances and future directions in amphiphilic cyclodextrin nanoparticles, Expert Opin Drug Deliv. 6 (2009) 1161-73

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http://www.ncbi.nlm.nih.gov/pubmed?term=Gao%20D%5BAuthor%5D&cauthor=true&cauthor_uid=22848175

http://www.ncbi.nlm.nih.gov/pubmed?term=Tang%20S%5BAuthor%5D&cauthor=true&cauthor_uid=22848175

http://www.ncbi.nlm.nih.gov/pubmed?term=Tong%20Q%5BAuthor%5D&cauthor=true&cauthor_uid=22848175

http://www.ncbi.nlm.nih.gov/pubmed/22848175

http://www.ncbi.nlm.nih.gov/pubmed?term=Bilensoy%20E%5BAuthor%5D&cauthor=true&cauthor_uid=19705965

http://www.ncbi.nlm.nih.gov/pubmed?term=Hincal%20AA%5BAuthor%5D&cauthor=true&cauthor_uid=19705965

http://www.ncbi.nlm.nih.gov/pubmed/19705965


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Liposomes for the drug delivery of Carboranes

Temidayo Olusanya

University of Portsmouth,St.Micheal’s Building, School of Pharmacy and Biomedical Sciences, White Swan Road, Portsmouth, Hampshire, United Kingdom, PO1 2DT.

Aimed towards the development and effective delivery of antitumour agents, this programme of research targets the evaluation of new boronated lipophilic cations for use in the neutron capture therapy of cancer through the development of delivery vehicles that will facilitate the transport of these agents to their site of action. The work will involve the formulation of a series of carborane-modified molecular structures that possess subcellular (mitochondrial and/or lysosomal) targeting properties and high tumour/normal cell selectivity. Integral to this aspect of the work is the study of the transport properties of boronated agents through hydrophobic barriers, such as cell membranes and membranes of subcellular organelles. The second facet of the project will involve the preparation and evaluation of new carriers that can overcome reticulo-endothelial phagocytosis to facilitate drug delivery through the blood-brain barrier via liposome-mediated transport; this study will be centred on biodegradable materials that possess moderately hydrophilic surfaces, and which can be used for the transport of boronated chemotherapeutic agents across the blood-brain barrier. Successful completion of the programme of work will yield a range of well-characterised formulations, the performance and toxicity of which will have been evaluated in vitro. The datasets generated by this study will be of benefit to those working towards the development of effective drug targeting technologies that are not impeded by the blood-brain barrier. The implications in the treatment of brain cancer are significant: agents will be developed that show high tumour selectivity and increased ease of administration, at a low toxicity cost. In the long term, the concept could be extended to several cancer therapy strategies involving the selective destruction of other subcellular organelles (endoplasmic reticulum, Golgi apparatus).

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Drug release from redox sensitive poly(aspartic acid) based hydrogels

Dávid Juriga, Rita A. Bauer, Miklós Zrínyi

Semmelweis University, Department of Biophysics and Radiation Biology, Laboratory of Nanochemistry, [email protected]

The investigation of polymer gels has become a major field in pharmaceutics and medical-biology in the past decades. Our particular interest is to develop biocompatible and biodegradable smart poly(aspartic acid) (PASP) hydrogels that can be used as promising new candidates for controlled drug delivery. These soft materials are proved to respond various environmental parameters, such as pH, temperature or redox potential [1]. We have developed a novel preparation method for synthesis of chemically cross-linked and functionalized polysuccinimide (PSI) and poly(aspartic acid) (PASP) gels. This synthesis route broadly extends the chemical variability of the PASP based polymers and hydrogels. Dual cross-linked poly(aspartic acid) hydrogels were prepared by using two different cross-linking agents simultaneously, one of them was the diaminobutane (DAB) and the other was cystamine (CYS). DAB provides stable cross-links, whereas CYS cross-linker contains disulfide bonds, which can be cleaved by reduction using dithiotreitol (DTT) [2]. The cleavage of disulfide cross-links results in abrupt swelling as well as rapid release of loaded drug molecules. The drug releasing profile was investigated from gel beads using Metoprolol as model drug. These gels would be promising new candidates for controlled drug delivery. [1] T. Gyenes, V. Torma, B. Gyarmati, M. Zrínyi: Synthesis and swelling properties of novel pH-

sensitive poly(aspartic acid) gels, Acta Biomaterialia, 4. (2008) 733-744.

[2] M. Zrínyi, T. Gyenes, D. Juriga, K. Ji-Heung: Volume change of double cross-linked poly(aspartic acid) hydrogels induced by cleavage of one of the crosslinks, Acta

Biomaterialia 9. (2013) 5122-5131.

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Hydroxyapatite scaffolds use in sustained release of ibuprofen – synthesis and characterization

Joanna Kołodziejek, Magdalena Szubert, Adam Voelkel

Poznan University of Technology, Institute of Chemical Technology and Engineering, Pl. M. Skłodowskiej-Curie 2, 60-965 Poznań, e-mail: [email protected]

Hydroxyapatite (HAp) Ca10(PO4)6(OH)2 is one of the most stable forms of the calcium phosphates and the major inorganic component of bone. In form of scaffolds design should stimulate new bone growth resulting at the end state, in native bone tissue with no trace of the scaffold. Hydroxyapatite is commercially available but its limitation is associated with low mechanical strength, fracture toughness and low thermal stability. Hydroxyapatite implants can also function as drug carriers which avoids the post-operative complications, reducing the formation of inflammation. These problems and issues are now widely developed but has not yet elaborated a universal drug carrier. Hydroxyapatite drug carriers due to their mineralogical and chemical similarity to natural bone after the release of the drug does not have to removed from the body, so there is no need for revision surgery [1,2]. The aim of the study was obtaining scaffolds based on hydroxyapatite ceramics, both unmodified and modified polymer materials for use as active substance carrier. The aim of the project was also assessement of active substance release from the scaffolds. Inverse gas chromatography (IGC) is one of the methods used to study the physicochemical properties of materials. In this method the examined material is placed in the chromatographic column and its properties are concluded basing on retention behavior of carefully selected test compounds [3]. From the drug carrier interaction point of view, this method allows to determine different parameters: the dispersive component of the surface free energy exhibiting the activity of the tested material, KA and KD parameters expressing the acidity and basicity of the surface and Flory-Huggins interaction parameter expressing the magnitude of interactions between the components of the carrier and an active agent. It is important to assess the impact of the estimated parameters on the release of a model active pharmaceutical ingredient (API) from scaffolds, and to find the optimal ranges of parameter values to achieve the expected period of time and/or rate of release of the studied API. These studies was financed by DS-MK 32-404/2013. [1] X. Zhang, W. Zhang, Z. Yang, Z. Zhang, Nanostructured hollow spheres of hydroxyapatite:

preparation and potential application in drug delivery, Front. Chem. Sci. Eng. 6 (2012) 246–252. [2] J. Andersson, E. Johannessen, S. Areva, N. Baccile, T. Azaıs, M. Linden, Physical properties and in

vitro bioactivity of hierarchical porous silica–HAP composites, J. Mater. Chem. 17 (2007) 463–468.

[3] I.M. Grimsey, J.C. Feeley, P. York, Analysis of the surface energy of pharmaceutical powders by inverse gas chromatography, J. Pharm. Sci. 91 (2012) 571-583.

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Influence of HME downstream process options on in vitro release of low soluble drug substance in one formulation

Dunja Gamilec

Formulation Scientist, GR&D Formulation, Pliva Croatia Ltd.

Hot-melt extrusion (HME) is new and emerging technology in pharmaceutical dosage form production. The process consists of feeding materials in powdered form which melts under elevated temperature and pumping the melt with rotating screw through a die to form a product of uniform shape. The product is cooled upon exit. Although it has various applications, mainly it serves as tool for preparing solid dispersions for solubility enhancement. The drug substance used for preparing solid dispersions is cathegorized as BCS class IV, having low solubility as well as low permeability. Formulation also comprised pharmaceutical grade polymer and solubilizer. Solid dispersion was prepared using HME and alternate downstream processing option: chill rolls and pelletizer. Results showed that using chill roll increased in vitro dissolution profiles of tested formulation. This can be explained by higher dispersion level when melt is rapidly cooled upon exit ('quenched') by using chill rolls. Slow cooling when using pelletizer can cause phase separation and larger API paricles/domains which can lead to smaller dissolution extent.

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Evaluation of the performance and PK of a DPI formulation for in-vivo testing

Valentina Bagnacani, Alessandro Fioni, Franco Bassani, Milco Lipreri, Paola Caruso, Marta Bellini, Francesco Amadei, Giandomenico Brogin, Silvia Catinella

Chiesi Farmaceutici S.p.A., Parma, Italy [email protected]

Inhalation therapy is increasingly being pursued to deliver locally [1] or systemically [2] drugs for different purposes. The inhalation systems can be classified in three main categories: dry powder inhalers (DPI), metered dose inhalers (MDI) and nebulizers. DPI are free from anti-environmental propellants, easy to use, highly stable, use passive breath activation and have a high dose delivery [3]. For the PK and PD studies conducted during the pre-clinical phases of development of an inhaled drug, the dry powder can be administered via endotracheal delivery to rodents. This methodology enables controlled administration directly into the respiratory tract, with a high predictability in terms of delivered dose. The aim of this study was to create a workflow to characterize the in-vitro performance and evaluate the PK of a DPI formulation for animal administration, by using a small number of animals. In a first phase, three DPI formulations of a test compound at different doses were prepared with appropriate excipients and their physico-chemical properties were characterized. The performance of the administration was also investigated studying the in vitro delivered doses by the Penn Century DP-4 insufflator. In a second phase, the formulations were administered by the device to rats (n= 4 for each formulation) and the compound deposition into trachea and lungs was assessed 15 min after administration; moreover, a PK experiment was performed using catheterized rats (n= 4) in order to determine compound plasma levels and systemic exposure after endotracheal administration. [1] R.W. Chapman, A. House, H. Jones, J. Richard, C. Celly, D. Prelusky, P. Ting, J.C. Hunter, J. Lamca,

J.E. Phillips, Effect of inhaled roflumilast on the prevention and resolution of allergen-induced late phase airflow obstruction in Brown Norway rats, Eur. J. Pharmacol. 571 (2007) 215-221.

[2] M. Morello, C.L. Krone, S. Dickerson, E. Howerth, W.A. Germishuizen, Y.L. Wong, D. Edwards, B.R. Bloom, M.K. Hondalus, Dry-powder pulmonary insufflation in the mouse for application to vaccine or drug studies, Tuberculosis (Edinb.). 89 (2009) 371-377.

[3] N.R. Labiris and M.B. Dolovich, Pulmonary drug delivery. Part II: The role of inhalant delivery devices and drug formulations in therapeutic effectiveness of aerosolized medications, British Journal of Clinical Pharmacology. 56 (2003) 600-612.

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Effect of ovocystatin polymerization on inhibitory activity towards legumain

Krzysztof Gołąb, Katarzyna Juszczyńska, Bogusława Konopska, Maria Warwas, Jakub Gburek

Department of Pharmaceutical Biochemistry, Wroclaw Medical University, Wroclaw Poland

Legumain, also known as asparaginyl endopeptidase (AEP) is a lysosomal cysteine protease. It contributes to turnover of proteins in lysosomes together with cathepsins. Otherwise it plays important role in exogenous antigen processing and presentation by MHCII complex. The enzyme activates TLR-9 and thus contributes to the innate immune response mediated by dendritic cells. Additionally legumain participates in the activation of cathepsins B, H and L and progelatinase A. Overexpression of the legumain is associated with enhanced tissue invasion and metastasis in many tumors like ovarian, breast, lung, prostate and colorectal cancer. Therefore legumain and cathepsins are potentially target for anticancer therapy. `The inhibitory of the protein center for legumain differs from that interacting with papain-like proteases. The antipapain activity of ovocystatin is reduced by aggregation. During this process the 3D domain swapping occurs. It is interesting if the polymerization of ovocystatin also influence on antilegumain activity. Ovocystatin was aggregated by heating at 90°C and the aggregates were separated by size exclusion chromatography. The separated polymers were studied. Inhibitor activity towards papain and legumain was determined by colorimetric and fluorymetric assay, respectively. In all polymeric forms i.e. dimer, tetramer and octamer the antipapain activity was lost. In contrast, the antilegumain activity was preserved in dimeric form. Its seems that 3D domain swapping occurring during dimerization affects the inhibitory center for papain but not for legumain. Further polymerization also destroys the second inhibitory center due to induced conformational changes.

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Study on micellization of sodium 3-dehydro-cholate by calorimetry and NMR

Bojana Vasiljević, Etelka Tombácz*, Dušan Škorić, László A. Király*, Janoš Čanadi

Department of Chemistry, Biochemistry and Environmental Protection, University of Novi Sad Faculty of Sciences, 21000 Novi Sad, R. Serbia

*Department of Physical Chemistry and Materials Sciences, University of Szeged Faculty of Science and Informatics, Szeged, Hungary

One of the main characteristics of oxo bile acids is micelle formation, which occurs in aqueous solutions of sodium salts of these compounds. Their ability to form micelles is characterized by critical micelle formation concentration (CMC). Since oxo bile acids possess very interesting physico-chemical properties for pharmacological application, investigation of these properties, especially micellization, became very important [1]. Isotherm titration microcalorimetry (ITC) is mostly used to investigate the thermodynamics of micellization, which is mainly an entropy driven process due to hydrophobic interactions. Calorimetric measurements of micellization equilibria allow us to calculate the parameters of micelle formation, i.e., cmc values and thermodynamic

data: enthalpy change (micH), change of Gibbs free energy (micG) and entropy term

(TmicS). Technique that is also convenient for micellization studies is NMR measurement of T1 relaxation time (spin-lattice relaxation). T1 relaxation is in connection with molecular motion and with the environment of measured nucleus, so the changes caused by micellization are easily monitored by T1 measurement. Micellization of oxo bile acid sodium salts is already investigated by NMR spectroscopy [2]. In this work the heat of dilution for micellar solution of sodium 3-dehydro-cholate was measured at different temperatures. The cmc values and some thermodynamic parameters of micellization were calculated. Using standard inversion recovery experiment, proton T1 relaxation is measured for different concentrations of oxo bile acid sodium salt solutions in D2O. Relaxation time dependence from concentration of oxo bile acid salt indicates the critical micellar concentration (CMC) of sodium 3-dehydro-cholate. Financial support of AP of Vojvodina Republic of Serbia No: 114-451-3690/2011-01 for IPA-CBC HUSRB/1002/214/193 is gratefully acknowledged. [1] M. Poša, QSPR study of the effect of steroidal hydroxyl and oxo substituents on the critical

micellar concentration of bile acids, Steroids, 76 (2011) 85-93. [2] M. Poša, V. Guzsvany, J. Csanadi, Determination of critical micellar concentrations of two

monoketo derivatives of cholic acid, Colloids and surfaces B: Biointerfaces, 74 (2009) 84-90. [3] A. B. Pahi, D. Varga, Z. Kiraly, A. Mastalir, Thermodynamics of micelle formation of the

ephedrine-based chiral cationic surfactant DMEB in water, and the intercalation of DMEB in montmorillonite, Colloids and Surfaces A: Physicochem. Eng. Aspects, 319(2008) 77–83.

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Pyrene Fluorescence Probe Method for Determination of Micelles Applying Low Energetic IR-Radiation

V. V. Komarov*,**, A. M. Popova*

,**, L. Schmidt**, H. Jungclas**

*Lomonosov Moscow State University, Skobeltzin Institute of Nuclear Physics, 119992, Moscow, Russia

**Chemistry Department Philipps-University Marburg D-06421, Marburg, Germany. [email protected]

Fluorescence of a donor-acceptor nanoaggregate by microwave IR-radiation is considered. We assume, that this nanoaggregate consists of two dipole-dipole interacting parts: an aromatic molecule as acceptor and a hydrophobic sector of an amphyphilic polymer as a donor. It was shown, that the fluorescence of this nanoaggregate occurs by the condition, that the donor contains a substructure of the type СnH2n, which serves as antenna for IR-radiation. This antenna can accumulate vibrational energy as a sum of collective vibrational excitations (excimols). The aromatic acceptor molecule has no permanent dipole momentum and can not be excited by microwave IR radiation. In the field of the donor IR antenna this molecule can obtain the induced dipole momentum. Due to dipole-dipole interaction between the donor and the acceptor in the considered nanoaggregate, a part of the energy accumulated in the IR antenna is transmitted to the acceptor molecule. Since this energy is equal to the energy of the electronic excited state in the visible part of aromatic molecule absorption spectrum, electronic excitation and the following fluorescence is possible. As an example we consider the fluorescence of the nanoaggregate consisting of the donor molecule with СnH2n substructures and the acceptor i.e. the aromatic pyrene molecule. The external IR radiation has a frequency of 1.1· 10

14 s

-1, which is equal to the excimol frequency in the donor IR antenna. Photo

physics of these nanoaggregates, where pyrene is an acceptor, can be used in different applications, e.g. in the fluorescence probe methods analysing the micellization of the block copolymer in different solutions.

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Application of experimental design for the development of polymeric micro-nanoparticles

Rameshwar K. Deshmukh, Jitendra B. Naik

University Institute of Chemical Technology, North Maharashtra University, Jalgaon, Maharashtra, India-425 001

In this study, diclofenac sodium loaded micro-nanoparticles were prepared by single emulsion [oil-in-water (o/w)] solvent evaporation method. Three formulation independent variables were investigated by 2

3 experimental designs by using the Design-Expert

®

software (Version-8.0.7.1) and evaluated for their particle size, morphology, encapsulation efficiency, drug polymer interactions and in vitro drug release. The physicochemical characteristics of the particles were assessed using Fourier transform infrared spectroscopy (FTIR), X-ray powder diffractometry (XRPD), and field emission scanning electron microscopy (FESEM). Graphical and mathematical analysis of the design showed that all the three independent variables were a significant effect on the encapsulation efficiency of micro-nanoparticles. A polynomial equation fitted to the data was used to predict the encapsulation efficiency in the optimal region. Low magnitude of error, significant values of R

2, and validation of the optimization study proves the high

prognostic ability of the design. The particles were found to be discrete, spherical with smooth surface. The absence of drug polymer interactions was confirmed by FTIR spectroscopy. XRPD revealed the molecular dispersion of the drug within the micro-nanoparticles formulation. Perfect prolonged drug release profile was achieved by using ethylcellulose polymer. Enhancement of encapsulation efficiency of polymeric micro-nanoparticles containing diclofenac sodium can be successfully obtained with an application of experimental design technique.

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FTIR spectroscopic investigation of specific and non-specific interactions on oxo bile acid methyl esters

Janoš Čanadi, Branislav Jović, Branko Kordić, Dušan Škorić

Department of Chemistry, Biochemistry and Environmental Protection, University of Novi Sad Faculty of Sciences, 21000 Novi Sad, R. Serbia

Investigation of hydrogen bond interactions on oxo bile acid molecules is very important due to the fact that these bile acid derivatives possess promotional action on the transport of drugs trough cell membrane. Some studies of oxo bile acid hydrogen bonded complexes are already known[1]. IR measurements can be used for determining equilibrium constants for 1 : 1 hydrogen bonded complexes formed between bile acid derivative and an appropriate proton acceptor [2,3]. In our study the proton donor ability of hydroxyl groups, as well as the proton acceptor properties of the carbonyl groups, of some selected bile acid derivative was investigated. Specific and non-specific interactions were investigated on methyl-3-oxo-12α-hydroxy-5β-cholanoat and methyl-3-oxo-7α-hydroxy-5β-cholanoat and obtained results for reactivity of hydroxyl groups in positions 7 and 12 were compared. Results for correlation with Gutmann and Kamlet-Taft solvatochromic equations were obtained for the following organic solvents: acetonytrile, tetrahydrofuran, diethyl ether, benzene, toluene, 1,4-dioxane, chloroform, dichloromethane, t-butanol, carbontetrachloride and 1,2-dichloroethane. Investigation on formation constant of hydrogen bond complex built on hydroxyl group was carried out with Becker’s methode for 1,4-dioxane, tetrahydrofuran and acetonytrile. Financial support by the IPA-CBC HUSRB/1002/214/193, AP of Vojvodina Republic of Serbia 114-451-3690/2011-01 for conducting the research is gratefully acknowledged. [1] M. Poša, V. Guzsvany, J. Csanadi, S. Keverešan, K. Kuhajda, Formation of hidrogen-bonded

complexes between bile acids and lidocaine in the lidocaine transfer from an aqueous phase to chloroform, Eur. J. Pharm. Sci., 34 (2008) 281-292.

[2] A. Nikolić, B. Jović, S. Petrović, FTIR spectroscopic study of hydrogen bonding and solvent induced frequency shifts of N-tert-butylacetamide, J. Mol. Struct., 1044 (2013) 140-143.

[3] B. Jović, A. Nikolić, B. Holló, Intermolecular hydrogen bonding between N-substituted caproamides and tetrahydrofuran, J. Struct. Chem., 54 (2) (2013) 431-436.

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Aggregation behaviour of oxo bile acids – Tween 40 binary systems

Dejan Ćirin, Mihalj Poša, Ana Sebenji

Department of Pharmacy, Faculty of Medicine, Hajduk Veljkova 3, Novi Sad, Serbia Oxo derivatives of bile salts are potentially beneficial for the development of new pharmaceutical formulations due to their ability to promote penetration of certain drugs across biological barriers. They are also less toxic than α axial hydroxy derivatives of bile acid anions. However, these bile salts have low solubilisation power since they are very hydrophilic. In this study, three oxo derivatives of bile salts (Na salt of 7-oxodeoxycholic acid, 12-

oxolithocholic acid and 3, 12-dioxo-5-cholanoic acid) were mixed with Tween 40, commonly used solubiliser in pharmaceutical formulations, in order to improve properties of oxo derivatives of bile salts. The aggregation behaviour of three different bile salt−Tween 40 binary mixtures has been characterized using tensiometry and analysed in the viewpoint of the regular solution theory. The critical micelle concentration values of investigated mixtures were found to be significantly lower than of the individual oxo bile salts. According to the calculated interaction parameter β, strong synergism was noticed in oxo bile salts – Tween 40 binary mixtures. The strongest synergism was detected in sodium

salt of 3,12-dioxo-5-cholanoic acid and Tween 40 mixtures. This synergistic behaviour is most probably consequence of the formation of hydrogen bonds between polyoxyethylene groups of the polar head of Tween 40 and equatorial oxygen atoms of bile acid anions. Namely, oxo and OH equatorial groups of bile salts are in more favorable position for hydrogen bonding than axial groups if bile salt molecules are tangentially positioned on the surface of mixed micelles with hydrophobic side turned toward the hydrophobic domain of the aggregates. Therefore, the synergism is more pronounced if the steroid skeleton contains a larger number of equatorialy positioned oxygen atoms. Acknowledgment: This study was financially supported by the Provincial Secretariat for Science and Technological Development, AP Vojvodina, Republic of Serbia, Grant No. 114-451-2113/2011-02.

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Physico-chemical properties of poloxamer P 237 – Tween 80 or sodium dodecyl sulfate binary systems

Dejan Ćirin, Veljko Krstonošić, Mihalj Poša

Department of Pharmacy, Faculty of Medicine, Hajduk Veljkova 3, Novi Sad, Serbia It was shown in many studies that mixing of structurally diverse amphiphiles can produce blends having improved properties than their individual constituents. Hydrophilic poloxamers are widely investigated excipients for the development of novel pharmaceutical formulations of hydrophobic drugs. However, hydrophilic poloxamers have higher critical micelle concentration (cmc) values than hydrophobic triblock copolymers, indicating lower solubilisation power. Therefore, in this study, poloxamer P 237, as a model of hydrophilic poloxamer, was mixed with two commonly used surfactants in the pharmaceutical industry, Tween 80 and sodium dodecyl sulfate (SDS) to develop novel mixtures having potentially improved properties. The cmc values were obtained for poloxamer P 237 – Tween 80 and poloxamer P 237 – SDS binary systems by tensiometric measurements, using a du Nouy ring method. In this study, the mole fraction of poloxamer in investigated mixtures varied from 0.1 to 0.9. The experimentally obtained cmc values of all surfactant mixtures were significantly lower than the cmc value of poloxamer. The results were also assessed in the viewpoint of the regular solution theory. According to the high negative values of the calculated interaction parameter β, strong synergism was noticed in poloxamer P237- SDS mixtures. This is most probably due to flexible, saturated, alkyl tails of SDS which are easily incorporated in the hydrophobic core of mixed micelles together with polyoxypropylene hydrophobic part of the triblock copolymer. However, calculated values of the interaction parameter β for poloxamer P237- Tween 80 mixtures were positive, indicating that these mixed micelles are thermodynamically less stable than their individual i.e., pure micelles. This behaviour is most probably due to sterically rigid cis configuration of oleic tail of Tween 80 which doesn’t fit easily in the hydrophobic interior together with polyoxypropylene part of poloxamer P 237.

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Thermodynamics of the interactions between both bacterial and human ribosomal RNA with some antibiotic aminoglycosides

Javier Alguacil*, Jordi Robles*,**, Clara Ràfols**

,***, Elisabeth Bosch**

,***

*Departament de Química Orgànica, Universitat de Barcelona, **Institut de Biomedicina de la Universitat de Barcelona (IBUB)

***Departament de Química Analítica, Universitat de Barcelona The energetic quantities closely related to the drug-target interactions (variations of the enthalpy, ΔH, Gibbs free binding energy, ΔG, and entropy, ΔS) are of growing interest in drug discovery studies. This is because they provide a comprehensive view of the different types of molecular forces that drive the binding processes. Most medicinal chemistry strategies gravitate towards optimization of ΔG and, therefore, Kb which stands for the interaction binding constant. The relationship between these quantities is expressed by the following well known equations: ΔG=ΔH-TΔS and Kb=1/Kd, where T is the temperature and Kd the dissociation constant related to ΔG by means of Kd=e

- ΔG/RT

. In this work, interaction thermodynamic parameters of two aminoglycoside antibiotics, neomycin and paromomycin, as well as some neomycin derivatives with both bacterial and human ribosomal RNAs have been carefully measured by isothermal titration calorimetry (ITC). This technique is able to provide accurate values of all mentioned thermodynamic quantities as well as the interaction stoichiometry from only one well planed titration. Obtained results show: a) when using bacterial RNA, the first interaction involves a 1:1 stoichiometry for all essayed compounds and Kb values lay in the range 10

6-10

8. Neomycin shows the higher

value and its dinucleotide conjugates (Neo-AA and Neo-TT) the lowest ones. A second interaction is clearly observed in all instances with a 2.2-3.8 stoichiometry ratio and binding constant values about 10

5. For all aminoglycosides the main interaction seems to

be driven by the entropic term. b) when using human RNA, two interactions are evident too and, in some cases, higher order interactions are suggested by the titration curve shape. Dinucleotide-neomycin conjugates were excluded due to their low affinity for the target. The first interaction occurs at 1-1.5 molar ratio and Neomycin shows the higher Kb whereas Paromomycin and diPNA conjugates (Neo-aa and Neo-tt) show slightly lower values. The second interaction involves a 2-3 molar ratio being Kb about 10

5 except the Neo-aa conjugate which is about

106. It should be noticed that the main interaction is driven by the entropic term for

neomycin and paromomycin but by the enthalpic one for the two diPNA conjugates included in this study.

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Liver membrane proteins sequestered on hemoglobin affinity matrix

Jakub Gburek, Krzysztof Gołąb, Katarzyna Juszczyńska, Bogusława Konopska

Department of Pharmaceutical Biochemistry, Wroclaw Medical University, Wroclaw, Poland

Intravascular hemolysis of red blood cells is a physiological phenomenon, as well as complication of many pathological states, including infectious, autoimmune, or hereditary disorders. Hemoglobin (Hb) released from the cells is partially bound by plasma haptoglobin (Hp). Efficient removal of free hemoglobin from the circulation and its complex with haptoglobin (Hp-Hb) is essential for the preservation of homeostasis of the organism because of toxicity and the oxidizing properties of the protein and its degradation products. In recent years, the hemoglobin receptor was identified in Kupffer cells (CD163/HbSR). Expression of the protein is limited to the line of mononuclear phagocytic cells, and is not displayed on the hepatocytes. Thus, its role in gross liver clearance of Hb / Hp-Hb is rather small.The aim of our study was isolation and characterization of the hepatocyte surface receptor responsible for the main hemoglobin uptake by the liver. The membrane protein fraction of hepatocytes were isolated from living hepatocytes or frozen liver tissue with an specific extraction method without the use of detergent by means of Native Membrane Protein Isolation Kit (Millipore, UK). Hemoglobin binding proteins were isolated by affinity chromatography on a hemoglobin matrix. Analysis of the obtained eluates by SDS-PAGE and CBB staining showed the presence of three major protein bands corresponding to a molecular mass of about 40, 50 and 160 kDa. These proteins were identified by mass spectrometry (LC-MS/MS) and Mascot analysis. The main components of the eluate were carbamoyl-phosphate synthase, ATP synthase subunit α, ATP synthase subunit β and paraoxonase. Further study in vitro are needed in order to clarify the role of these proteins in the binding of hemoglobin under physiological conditions.

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Estimation of the possible interactions between antioxidant drugs and membranes

Fátima Paiva-Martins, Carina Silva, Marlene Costa, Paula Gameiro, Sonia Losada-Barreiro*, Carlos Bravo-Díaz*, Laurence S. Romsted**

Universidade de Porto, Fac. de Ciências, Dpto de Química e Bioquímica, Portugal *Universidad de Vigo, Fac. Química, Dpto Química Física, Vigo, Spain

**Dep of Chemistry and Chemical Biology, the State University of New Jersey, NJ, USA

Covalent modification of antioxidants through lipophilization is an important field of research aiming at developing antioxidants with improved efficacy. However, due to insufficient knowledge on how hydrophobicity affects antioxidant activity, lipophilization strategies have been largely based on empirism. The body of knowledge regarding hydrophobicity has been dramatically redefined as unexpected results were recently published. Using a broad range of lipophilized antioxidants assessed in dispersed lipids models and cultured cells, it has been demonstrated that the antioxidant activity increases progressively with increasing chain length up to a critical point, beyond which the activity of the compounds dramatically decreases [1]. Taking into account this nonlinear phenomenon, also known as cut-off effect, antioxidant drug designers now have to seek the critical chain length to synthesize the optimal drug in a rational manner. In compartmentalized systems such as emulsions and biomembranes, surface-activity (hence hydrophobicity) is generally regarded as advantageous. Surface-activity allows the antioxidant to be fusogenic and to have a good affinity with lipid water interfaces. Accordingly, lipophilisation appears as a crucial step in the design of new antioxidant additives and drugs. In this work, a large series of caffeates of C1-C16 fatty acids with increasing log P values (0.3 - 2.5) were synthetized. A recent kinetic method grounded in the pseudophase model for thermodynamically stable microemulsions was employed to estimate the partition constants of compounds between an oil phase, an interfacial phase of Tween 20 and a water phase [2]. We also determined the influence of fatty acid length on the compound location in liposomes. Interestingly, very similar results were obtained in both systems. With the increase of log P, more caffeate was found at the emulsion system interphase and more interaction with the membrane bilayer in the liposome system was observed but only for caffeates with an alkyl chain up to C8. Apparently, the determination of the caffeate concentration at the interphase of an O/Tween 20/buffer system gives a better estimation than log P of the possible partition of antioxidants in biomembranes. [1] C. Bayrasy, B. Chabi, M. Laguerre, J. Lecomte, E. Jublanc, P. Villeneuve, C. Wrutniak-Cabello, G.

Cabello, Boosting antioxidants by lipophilization: A strategy to increase cell uptake and target mitochondria, Pharma Res, (2013), DOI: 10.1007/s11095-013-1041-4.

[2] S. L. Barreiro, C. Bravo-Díaz, F. Paiva-Martins, L.S. Romsted, Maxima in antioxidant distributions and efficiencies with increasing hydrophobicity of gallic acid and its alkyl esters. The pseudophase model interpretation of the “Cutoff Effect” J. Agric. Food Chem. (2013), doi.org/10.1021/jf400981x.

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Human serum albumin binding of 2-[(carboxymethyl)sulfanyl]-4-oxo-4-arylbutanoic acids

Ilija N. Cvijetić, Dušan D. Petrović,* Tatjana Ž. Verbić,* Ivan O. Juranić,** Branko J. Drakulić**

Innovation Center, Faculty of Chemistry, University of Belgrade, Studentski Trg 16 *Faculty of Chemistry, University of Belgrade, Studentski Trg 16

**Department of Chemistry-IChTM, University of Belgrade, Njegoševa 12, 11000 Belgrade, Serbia

Title compounds (Fig. 1) exert antiproliferative activity toward human tumor cells and significant selectivity (tumor vs. healthy cells) in vitro [1]. Within the frame of physico-chemical profiling, human serum albumin (HSA) binding [2, 3] of some congeners is reported in this communication. As revealed from fluorescent spectroscopy with warfarin and ibuprofen as the binding site probes, the preferable binding site of compound 1 (Fig. 1, R = 4-tert-Bu), taken as an example, is Sudlow site I [4]. Static quenching mechanism was assumed. Binding constants (Kb) and the number of binding sites (n) are calculated according to Stern-Volmer’s equation (Eq. 1, Fig. 2) and shown in Table 1.

0log log log

b

F FK n Q

F Eq. 1

Förster’s energy resonance transfer (FRET) experiments were used to estimate the distance between Trp214 and compound 1. Obtained results were further evaluated by molecular docking and molecular dynamics simulations.

Acknowledgement: Ministry of Education, Science, and Technological Development of Serbia (Grant 172035), the European Commission (FP7 project HP-SEE (No. 261499)), and FP7 RegPot project FCUB ERA GA (No. 256716) supported this work.

[1] B.J. Drakulić, Z.D. Juranić, T.P. Stanojković, I.O. Juranić, 2-[(Carboxymethyl) sulfanyl]-4-oxo-4-arylbutanoic acids selectively suppressed proliferation of neoplastic human HeLa cells. A SAR/QSAR study, J. Med. Chem. 48 (2005) 5600-5603.

[2] K. Yamasaki, V.T.G. Chuang, T. Maruyama, M. Otagiri, Albumin–drug interaction and its clinical implication, Biochim. Biophys. Acta - Gen. Subjects (2013) in press.

[3] R. Punith, A.H. Hegde, S. Jaldappagari, Binding of an Anti-inflammatory Drug Lornoxicam with Blood Proteins: Insights from Spectroscopic Investigations, J. Fluoresc. 21 (2011) 487–495.

[4] G. Sudlow, D.J. Birkett, D.N. Wade, Further characterization of specific drug binding sites on human serum albumin, Mol. Pharmacol. 12 (1976) 1052–1061.

Figure 1. General structure of

examined compounds

-6.2 -6.0 -5.8 -5.6 -5.4 -5.2 -5.0 -4.8 -4.6-1.6

-1.4

-1.2

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

log[

(Fo-F

)/F]

HSA-warfarin + 1

HSA-ibuprofen + 1

HSA + 1

log[Q] Figure 2. Stern-Volmer plots

Table 1 logKb (M) Kb (M) n

HSA+1 4.455 2.85·104 0.943 HSA-ibupr+1 4.178 1.51·104 0.902 HSA-warf+1 2.194 1.56·102 0.623

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Synthetic Cannabinoids as an Alternative to Cannabis: GC-MS, LC-MS/MS and LC-HRMS analyses

Zbynek Oktabec*,**, Vera Maresova**, Marie Balikova**, Monika Zidkova**

*Department of Toxicology, Institute of Forensic Medicine and Toxicology, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague,

Ke Karlovu 2, 128 08 Prague 2, Czech Republic **Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences Brno, Palackého 1-3, 612 42 Brno, Czech Republic

Nearly half (45%) of the students (age 16) had used cannabis in lifetime, which is more than twice the ESPAD average (19%). The primary psychoactive compound of Cannabis sativa is Δ

9-tetrahydrocannabinol (THC) which exhibits a partial agonistic activity at CB1

receptors (found mainly in the brain, in basal ganglia and in the limbic system, including hippocampus, in the cerebellum and in both male and female reproductive systems,…) and CB2 receptors (found predominantly in the immune system with the greatest density in the spleen, in the peripheral nervous system). After identification of these receptors and identification of endocannabinoid system and endocannabinoid molecules at the end of the 20

th century, John W. Huffman and his team synthetized first generation

of synthetic cannabinoids (JWH-018, JWH-073,JWH-210,…) as a radiolabeled ligands for cannabinoid receptors binding studies and endocannabinoid system research purposes. These synthetic cannabinoids are often full agonists and are selective for one subtype of endocannabinoid system and thus possessed different and more potent properties. Nowadays, there are tens of different synthetic cannabinoids with varied chemical structure and some of these substances are the drugs of abuse worldwide; also they are recognized by some authorities as illicit drugs. They are marketed as “legal alternative to cannabis”, “legal highs”, and/or “herbal incense”, mostly by internet stores. As of April 2011 the most widely abused synthetic cannabinoids were scheduled by the Czech Department of Justice on list of controlled substances, thus the identification and quantification of these illicit substances in blood and urine samples and in other materials are the important aims for law enforcement authorities and clinical laboratories. The study is supported by grant MV VG 20122015075 and MV VG 20122015080.

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Organic synthesis of the pharmaceuticals with crystallization and determining of the morphological characteristics of fexofenadine-hydrochloride

Božo Banjanin, Rade Biočanin*, Sonja Stefanov*

Faculty of Science, University of Tuzla, Bosnia and Herzegovina *Centre for the Strategic Examination of National Security, Belgrade, Serbia

[email protected] Piperidine belong in group sixmembered heterocyclic amines and its often used in organic sinthesys including sinthesys of pharmaceutical active compounds. Fexofenadine contains several functional groups with hydrogen-bonding capacity and normally exists as a zwitte-rion. Fexofenadine forms sizeable single crystals more readily in a solvated rather then a pure form, as co-crystallisation with protic solvents allows for better optimization of the hydrogen-bonding potential. Fexofenadine Hydrochloride is piperidine derivatives and pharmaceutic active compound. Fexofenadine is antihistamine and it works by preventing the activation of H1-receptor-containing cells by histamine. Systematic name is 4-[1-hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]butyl]-2,2-imethylbenzeneacetic acid x HCl.

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Author Index

A Abdelaziz, A .......................................... 11, 40 Abrami, M ................................................... 23 Actiņš, A .................................... 54, 55, 59, 60 Albu, P......................................................... 65 Alguacil, J .................................................... 81 Amadei, F .................................................... 73 Ambrus, R ................................................... 68 Asiri, AA ...................................................... 40 Avdeef, A ............................................ 6, 8, 16 Avram, S ...................................................... 68

B Bădicu, N ..................................................... 61 Bagnacani, V ............................................... 73 Balikova, M ................................................. 85 Balogh, GT................................................... 46 Banjanin, B .................................................. 86 Bassani, F .................................................... 73 Bauer, RA .................................................... 70 Bellini, M ..................................................... 73 Beloborodova, AA ....................................... 56 Bercean, V ................................................... 62 Berghausen, J .............................................. 17 Bērziņš, K .................................................... 60 Biočanin, B .................................................. 86 Bobrovs, R ................................................... 55 Boldyreva, EV ........................................ 24, 56 Borcan, F ..................................................... 68 Bosch, E .......................................... 42, 50, 81 Box, KJ ......................................................... 50 Bozó, T ........................................................ 45 Braun, DE .................................................... 19 Bravo-Díaz, C............................................... 83 Brogin, G ..................................................... 73

C Cabot, JM ...................................................... 7 Čanadi, J ................................................ 75, 78 Catinella, S .................................................. 73 Cernikova, A ................................................ 48 Cetina-Čižmek, B ......................................... 53 Charochkina, L ............................................. 40 Chaves, RC ................................................... 34 Chen S-wW .................................................. 34 Cialla, D ....................................................... 58 Cîntă Pînzaru, S ........................................... 58 Ćirin, D .................................................. 79, 80 Clark, RD ................................................ 10, 39 Colombo, I ................................................... 23 Comer, J .................................................. 9, 50 Costa, M ...................................................... 83 Coufalova, L................................................. 48 Čulen, M ...................................................... 51 Cvijetić, IN ................................................... 84

D Danciu, C ..................................................... 68 Dehelean, CA ......................................... 21, 68 Deshmukh, RK ....................................... 15, 77 Dohnal, J................................................ 48, 51 Domańska, U ......................................... 52, 57 Dove, S ........................................................ 12 Drakulić, BJ .................................................. 84 Dravniece, A ................................................ 59 Dulski, M ..................................................... 18 Džale, V ....................................................... 53

E Erdem, B ...................................................... 47


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F Fălămas, A................................................... 21 Farra, R ....................................................... 23 Fioni, A ........................................................ 73 Fiorentino, S ............................................... 23 Fornells, E ................................................... 50 Fraczkiewicz, R ...................................... 10, 39 Fuguet, E ........................................... 7, 43, 50 Fulias, A ................... 61, 62, 63, 64, 65, 67, 68 Fulias, F ....................................................... 66

G Gabelica Marković, V .................................... 5 Gameiro, P .................................................. 83 Gamilec, D................................................... 72 Gburek, J ............................................... 74, 82 Glamuzina, B ............................................... 58 Gołąb, K ................................................ 74, 82 Göller, AH ............................................. 10, 39 Grante, I ...................................................... 60 Grassi, G ...................................................... 23 Grassi, M ..................................................... 23 Griesser, UJ ................................................. 19 Grzybowska, K ...................................... 18, 26 Grzybowski, A ............................................. 26

H Hasa, D ........................................................ 23 Hašek, J ....................................................... 25 Hawelek, L ............................................ 18, 26 Heghes, A .................................................... 66 Henderson, R .............................................. 33 Hengerer, B ................................................. 35 Hidalgo-Rodríguez, M ................................. 43 Hilfiker, R .................................................... 22 Hill, AP .......................................................... 3 Hillisch, A .............................................. 10, 39 Holdgate, G ................................................. 30 Horváth, P ................................................... 49

J Jampilek, J ................................................... 48 Jović, B ........................................................ 78 Jungclas, H .................................................. 76 Juranić, IO ................................................... 84 Juriga, D ...................................................... 70 Juszczyńska, K ....................................... 74, 82

K Karreman, C ................................................ 35 Kása, P jr. ..................................................... 29 Király, LA ..................................................... 75 Klara, D ........................................................ 31 Kołodziejczyk, K ........................................... 26 Kołodziejek, J ............................................... 71 Komarov, VV ............................................... 76 Konopska, B .......................................... 74, 82 Kordić, B ...................................................... 78 Korkmaz, S............................................. 14, 47 Körner, R ..................................................... 11 Krenz, U ................................................. 10, 39 Krstonošić, V ............................................... 80 Krūkle-Bērziņa, K ................................... 54, 59 Kryska, JA .................................................... 52

L Lal, R ............................................................ 32 Landon, P .................................................... 32 Ledeti, I ............. 61, 62, 63, 64, 65, 66, 67, 68 Leist, M ....................................................... 35 Lindner, K .................................................... 35 Lobell, M ............................................... 10, 39 Losada-Barreiro, S ....................................... 83

M Mañé, M...................................................... 50 Maran, U ..................................................... 44 Maresova, V ................................................ 85 Martinčić, A ................................................. 53 Minkov, VS .................................................. 56 Mohammed, H ............................................ 57 Mrvičin, N .................................................... 53 Müller, C...................................................... 58 Müller, J ...................................................... 46

N Naik, JB .................................................. 15, 77 Neuhoff, S ................................................... 27 Novotarskyi, S ............................................. 11

O Odorico, M .................................................. 34 Oja, M ......................................................... 44 Oktabec, Z ................................................... 85


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Oláh, I ......................................................... 29 Oļehnovičs, K .............................................. 54 Olusanya, T ................................................. 69 Opatrilova, R ............................................... 48

P Paiva-Martins, F .......................................... 83 Pallicer, JM .................................................. 42 Paluch, KJ .................................................... 18 Paluch, M .............................................. 18, 26 Parot, P ....................................................... 34 Pascual, R .................................................... 42 Patiny, L ...................................................... 40 Pellequer, J-L............................................... 34 Perissutti, B ................................................. 23 Petrović, DD ................................................ 84 Piccolo, A .................................................... 23 Pierre, M ..................................................... 31 Pintye-Hódi, K ............................................. 29 Pînzaru, SC .................................................. 21 Pobudkowska, A ......................................... 52 Popova, AM ................................................ 76 Popp, J ........................................................ 58 Port, A ......................................................... 42 Poša, M ................................................. 79, 80 Pozniak, I ..................................................... 58 Przybylski, M ......................................... 28, 35

R Ràfols, C .......................................... 42, 50, 81 Ramachandran, S ........................................ 32 Reppas, C .................................................... 13 Robles, J ...................................................... 81 Romsted, LS ................................................ 83 Rosés, M ..................................... 7, 41, 42, 43 Ruiz, R ......................................................... 50 Russell, PJ...................................................... 4

S Şandru, MM ................................................ 61 Sawicki, W ................................................... 18 Sbârcea, L .................................................... 66 Schmidt, L ................................................... 76 Schoenneis, R ........................................ 10, 39 Sebenji, A .................................................... 79 Silva, C......................................................... 83 Simu, G ....................................................... 65 Sinkó, B ....................................................... 45

Škorić, D ................................................ 75, 78 Slamnoiu, S.................................................. 35 Smith, J ........................................................ 20 Soica, C ........................................................ 68 Soponar, F ................................................... 61 Soriano-Meseguer, S ................................... 43 Sorić, I ......................................................... 53 Sovány, T ..................................................... 29 Stefanov, S .................................................. 86 Steiner, M ................................................... 21 Stumbaum, M ............................................. 35 Subirats, X ............................................... 7, 41 Sushko, I ................................................ 11, 40 Szaniszlo, B .................................................. 61 Szőke, V ....................................................... 49 Szubert, M ................................................... 71

T Tajber, L ...................................................... 18 Takács-Novák, K ................................ 6, 45, 49 Tam, K ........................................................... 6 Teran Arce, F ............................................... 32 Tetko, IV ................................................ 11, 40 Teulon, J-M ................................................. 34 Tombácz, E .................................................. 75 Trandafirescu, C .......................................... 68 Tsinman, K ..................................................... 8 Tsinman, O .................................................... 8

U Udrescu, L ................................................... 67

V Vasiljević, B ................................................. 75 Verbić, TŽ .................................................... 84 Vizserálek, G ............................................ 6, 45 Vlad, C ......................................................... 35 Vlase, G ......................... 62, 63, 64, 65, 66, 67 Vlase, T .......................... 62, 63, 64, 65, 66, 67 Voelkel, A .................................................... 71 Voinovich, D ................................................ 23 Völgyi, G .................................................. 6, 49

W Warwas, M .................................................. 74 Weber, K ..................................................... 58 Wojnarowska, Z .................................... 18, 26


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Y Yuan, H-P .................................................... 41 Yves, R ......................................................... 31

Z Zidkova, M .................................................. 85 Zioło, JD ....................................................... 26 Zrínyi, M ...................................................... 70 Zupko, I ....................................................... 68


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