ASPECTS OF THE DISCOVERY AND DEVELOPMENT OF PLANT-

DERIVED DRUGS

A. Douglas Kinghorn, Ph.D., D.Sc. Professor and Jack L. Beal Chair

College of Pharmacy The Ohio State University

Second FDA/PQRI Conference on Advancing

Product Quality, North Bethesda, MD (October 5-7, 2015)

OUTLINE OF PRESENTATION Rationale for the Search for New

Drugs from Higher Plants. General Approaches to Drug

Discovery from Tropical Plants in an Academic Environment.

Development of Silvestrol from Aglaia foveolata (a Potential Anticancer Agent) and Pentalinonsterol from Pentalinon andrieuxii (a Potential Antileishmanial Agent).

Summary and Conclusions.

KEY QUESTIONS TO BE ANSWERED

In natural product drug discovery screening programs in an academic environment, how, why, and when may highly promising compounds be identified?

What types of collaborative investigations can then be pursued in order to develop these lead compounds further?

RATIONALE FOR THE SEARCH

FOR NEW DRUGS FROM HIGHER

PLANTS

SMALL-MOLECULE NATURAL PRODUCT BASED DRUGS (JANUARY 1, 1981 − SEPTEMBER 9, 2012)

Total number of small molecule approved drugs world-wide was 1115 “N_all” refers to sum of N, NB and ND codes “S*_all” refers to sum of S* and S*/NM codes

(Newman and Cragg 2012)

NATURAL PRODUCT-DERIVED DRUGS INTRODUCED 2000-2008

[Chin et al., AAPS J., 8 (2), E239 (Article 28), 2006; http://www.aapsj.org; Butler, in Natural Product Chemistry for Drug Discovery, eds. A.D. Buss and M.S. Butler, RSC: Cambridge, U.K., 2010; p. 321]

Source Organism Type Number Terrestrial Plants (apomorphine HCl, arteether, dronabinol/cannabidiol (mixture), galanthamine HBr, lisdexamfetamine, methylnatrexone Br, nitisinone, tiotropium Br)

8

Terrestrial Microorganisms (amrubicin HCl, anidulafungin, biapenem, caspofungin acetate, cefditoren pivoxil, ceftobiprole medocaril, daptomycin, doripenem, ertapenem, everolimus, fumagillin, gentumazumab ozogamicin, ixabepilone, micafungin Na, miglustat, mycophenolate Na, pimecrolimus, pitavastatin, retapamulin, rosuvastatin Ca, telithromycin, temsirolimus, tigecycline, zotarolimus)

24

Marine Organism (trabectidin, ziconotide)

2

Terrestrial Animals (bivalirudin; exenatide; synthetic versions of natural forms)

2

Year Generic Name Natural Lead Compound Trade Name Indication

2012 Ingenol mebutate Ingenol-3-angelate Picato® Actinic keratosis

2012 Lorcaserin hydrochloride Ephedrine Belviq® Obesity

2012 Omacetaxine mepesuccinate

Homoharring-tonine Synribo® Chronic myeloid

leukemia

2012 Crofelemer Croton lechleri oligomeric pro-anthocyanidins

Fulyzaq®

HIV/AIDS anti-retroviral-associated diarrhea

2013 ado-Trastuzumab emtansine (protein-bound)

Maytansinea Kadcyla® Breast cancer

2013 Ospemifene Phytoestrogens Osphena® Menopause-associated dyspareunia

PLANT NATURAL PRODUCTS AND DERIVATIVES APPROVED BY THE U.S. FDA (2012 TO MID-2013)

aAlthough first isolated from a plant, maytansine is now regarded as a microbial product.

GENERAL APPROACHES TO

COLLABORATIVE DRUG DISCOVERY

FROM TROPICAL PLANTS IN AN

ACADEMIC ENVIRONMENT

Lead Identification

Lead Optimization

Lead Development

Drug Candidates

Natural Products Discovery Medicinal Chemistry Molecular Modeling

Target-based Bioassays Cell-based Bioassays

In Vivo Bioassays

Medicinal Chemistry Combinatorial Chemistry

Pharmacology, Toxicology Pharmacokinetics, ADME

Drug Delivery

PLANT NATURAL PRODUCT DRUG DISCOVERY AND DEVELOPMENT

Clinical Trials

Organism collection (after development of intellectual property agreements). Preparation of extracts (using standardized extraction scheme). Initial bioassays (cell-based and target-based). Biostatistics; data management; dereplication of leads; lead prioritization). Bioactivity-directed fractionation (= isolation of active compounds from biomass using a decision tree based solely on bioactivity). Structure elucidation of bioactive compounds. Scale up and analogue development of lead compounds. Advanced bioassays; data management, biostatistics). Lead optimization; pharmaceutical development.

MAJOR STAGES IN NATURAL PRODUCT DRUG DISCOVERY

(Clark, In Foye’s Principles of Medicinal Chemisry, 5th Edn., Williams, D.A.; Lemke, T.L., Eds., Lippincott Williams & Wilkins: Baltimore, 2002, p. 24)

CONVENTION ON BIOLOGICAL DIVERSITY (CBD)

A treaty with 42 Articles dictating codes of behavior in the study and sustainable use of biological diversity (http://www.biodiv.org/).

This was signed by >150 countries during the 1992 Earth Summit in Rio de Janeiro (“Rio Convention”).

The U.S.A. signed in 1993, but never ratified this treaty. In practice, this means the government must follow the treaty, but there is no binding effect on private citizens.

Source countries have sovereign right over their genetic resources (e.g., mutually agreed terms, prior informed consent, equitable sharing of benefits).

(Cordell, in Natural Product Chemistry for Drug Discovery, eds. A.D. Buss and M.S. Butler, RSC: Cambridge, U.K., 2010; p. 81)

NUMBERS OF ORGANISMS FOR DRUG DISCOVERY

Eubacteria (bacteria), cyanobacteria (blue-green algae) 4,000a

Archaea (halobacteria, cyanogens) Protoctista (e.g., protozoa, diatoms, “algae”, including “red algae” and “green algae”)

80,000

Plantae (mosses and liverworts, ferns, seed plants)b 270,000 Fungi (e.g., molds, lichens, yeasts, mushrooms)c 72,000

Animalia (e.g., mesozoa, sponges, jellyfish, corals, flatworms, roundworms, sea urchins, mollusks, segmented worms, arthropods, insects, fish, amphibians, birds, mammals)d-f

1,320,000

a Figures are species described taxonomically to date in each group. b Plants are the second largest group of classified organisms, representing 15% of the known biodiversity. c Only a relatively small proportion (5%) of the estimated 1.5 m fungi have been classified taxonomically to date. d The largest numbers of organisms are the arthropods, inclusive of insects (ca. 950,000 species). e Of the 28 major animal phyla, 26 are found in a marine environment. f Over 200,000 species of invertebrate animals and algal species occur in the sea.

(Tan et al., Curr. Drug Targets 7, 265, 2006)

SECONDARY METABOLITES FROM VASCULAR PLANTS (TRACHEOPHYTES; HIGHER PLANTS)

Altogether there are an estimated 200,000 secondary metabolites (“natural products”) (Dixon and Strack, Phytochemistry 62, 815, 2003).

Of these, the major groups are estimated as isoprenoids (ca. 80,000 compounds), phenolics (ca. 40,000 compounds), and alkaloids (ca. 30,000 compounds).

More secondary metabolites have been isolated from plants than other types of organisms.

An average leaf may contain >30,000 phytochemicals (Turi et al., J. Nat. Prod. 78, 953, 2015).

Specialized defensive secondary metabolites are associated with higher plants, such as gallotannins, proanthocyanidins, and resveratrol oligomers.

NUMBER OF NEW NATURAL PRODUCT COMPOUNDS PUBLISHED IN THE

JOURNAL OF NATURAL PRODUCTS OVER THE DECADE 2005-2014a,b

Terrestrial Microbes/

Fungi

Terrestrial Plantsc

Terrestrial Animals

Marine / Aquaticd

Otherse

1,869 (14.5%)

7,760 (60.0%)

117 (0.9%)

3,138 (24.3%)

33 (0.3%)

a From a total of 12,917 new small-molecule compounds. Percentages of the total are shown in parentheses. b Annual totals of new compounds reported were: 2005, 1,200; 2006, 1,276; 2007, 1,269; 2008, 1,388; 2009, 1,505; 2010, 1,369; 2011, 1,303; 2012, 1,049; 2013, 1,156; 2014, 1,402. c Including higher and lower plants (vascular and non-vascular). d Including animals, microorganisms, and plants. e For example, propolis of different geographical origins.

DEVELOPMENT OF SILVESTROL

(A POTENTIAL ANTICANCER AGENT)

AND PENTALINONSTEROL

(A POTENTIAL ANTILEISHMANIAL

AGENT)

(Photograph by Jimmy W. Crawford, RTI)

DRS. M.E. WALL (LATE) AND M.C. WANI: CO-DISCOVERERS OF TAXOL AND

CAMPOTHECIN

Current Collaboration is between: The Ohio State University (OSU), Columbus, OH;

University of Illinois at Chicago (UIC), Chicago, IL; University of North Carolina at Greensboro (UNC-G), NC;

Mycosynthetix Inc., NC; Eisai Inc., Andover, MA

Both grants funded by the United States National Cancer

Institute, NIH a,bPrincipal Investigators: G.A.Cordell (UIC; 1990-1992);

A.D. Kinghorn (UIC/OSU; 1992-present)

National Cooperative Drug Discovery Group (NCDDG) Granta (U19 CA52956) (1990-1995; 1995-2000; 2000-2006)

Program Project Grantb (P01 CA125066) (2007-2013; 2014-2019)

[Most recent review: Kinghorn et al., Pure Appl. Chem. 81, 1051, 2009]

COLLABORATIVE PROJECTS ON THE DISCOVERY OF NATURAL PRODUCT ANTICANCER AGENTS

Anticancer Plant Collections 1990-2011 Total plant accessions obtained 6,599 Representing: 2,609 species 1,466 genera 222 families Over 200 recollections

Some tropical forests support more tree species in 0.5 km2 than in all of North America or Europe (Burslem, 2001).

About 120,000 endemic species in tropical areas regarded as threatened due to massive habitat loss (Pitman & Jørgensen, 2002).

Source countries have sovereign right over their genetic resources (e.g., mutually agreed terms, prior informed consent, equitable sharing of benefits).

PLANT COLLECTIONS AT UIC (DRS. NORMAN FARNSWORTH AND DOEL SOEJARTO)

FORMER RAID PROGRAM OF THE UNITED STATES NATIONAL CANCER INSTITUTE

Rapid Access to Intervention Development (http://dtp.nci.nih.gov/docs/raid/raid_index.html).

A program designed to facilitate translation to the clinic of novel, scientifically meritorious therapeutic interventions originating in the academic community.

Contracts leading to preclinical development leading to filing of an IND.

Required submission of a formal proposal and peer review.

HO H

H

H COOH

H

BETULINIC ACID: A NCDDG COMPOUND THAT ENTERED CLINICAL TRIALS AT THE

UNIVERSITY OF ILLINOIS AT CHICAGO

• Betulinic acid was isolated from Ziziphus mauritania as a selective, non-toxic antimelanoma agent (Pisha et al., Nature Med. 1, 1046, 1995).

• Preclinical development of betulinic acid was supported through the RAID program of NCI (cycles III and VI, T.K. Das Gupta, Principal Investigator).

• Has entered Phase I/II clinical trials as a 20% ointment for topical treatment of dysplastic nevus syndrome with the potential to transform to melanoma (http://clinicaltrials.gov/; NCT00346502).

PROJECT 1 – The Ohio State University (OSU)

Tropical Plants, Biological Testing A.D. Kinghorn, PL

E.J. Carcache de Blanco D.M. Lucas

CORE A - UIC Biological Testing J.E. Burdette, CD*

S.M. Swanson (University of Wisconsin)

SCHEME OF ORGANIZATION FOR PROGRAM PROJECT 2P01 CA125066-07

EXTERNAL ADVISORY COMMITTEE William H. Gerwick (Scripps Institute of Oceanography, San Diego)

Susan B. Horwitz (Yeshiva University) George R. Pettit (Arizona State University)

William C. Rose (formerly Bristol-Myers Squibb, Princeton, N.J.)

PROJECT 2 – University of Illinois at Chicago (UIC) Aquatic Cyanobacteria

Plant Collections J. Orjala, PL D.D. Soejarto

PROJECT 3 – University of North Carolina – Greensboro (UNC-G)

Filamentous Fungi, Biological Testing N.H. Oberlies, PL

C. J. Pearce (Mycosynthetix Inc.) B.R. Stockwell (Columbia University)

M.C. Wani (Consultant) (RTI)

CORE B – OSU Medicinal Chemistry;

Pharmacokinetics J.R. Fuchs, CD

M.A. Phelps

CORE C - OSU Administration/Biostatistics

A.D. Kinghorn, CD L.-H. Xu, X. Zhang Y. Shen (Eisai Inc.)

NCI PROJECT OFFICER Yali Fu

NExT PROGRAM OF THE UNITED STATES NATIONAL CANCER INSTITUTE (NCI)

The NCI Experimental Therapeutics (NExT) Program “is designed to enable and foster the clinical translation and successful commercialization of novel therapeutic interventions, either synthetic, natural product, or biologic, arising from academic, private or governmental entities” (http://next.cancer/gov).

Proposals for drug discovery and development resources and expertise are examined by an extramural panel.

The panel reviews the applications for scientific merit and feasibility, novelty, and clinical need.

Proposals may be selected for entry into various points in the NeXT pipeline (e.g., lead development, preclinical toxicology, and phase 0/I clinical trials).

STRUCTURAL CHARACTERIZATION OF SILVESTROL FROM AGLAIA FOVEOLATA

Drs. Bang Yeon Hwang and Baoning Su

O

OH OHO

OCH3

COOCH3

OCH3O

OHO

OCH3

HOH

Silvestrol

X-ray Structure by Drs. Bernard Santarsiero and Andrew Mesecar (UIC)

(Hwang et al., J. Org. Chem. 69, 3350, 2004; ibid., 69, 6156)

AGLAIA FOVEOLATA (MELIACEAE) COLLECTED IN INDONESIA

Aglaia foveolata Pannell (Meliaceae), is a tree growing in lowland forests in Borneo (Brunei, Indonesia, Malaysia), with edible fruits.

This plant (fruits and twigs) was collected initially in Kalimantan, Indonesia in 2000, but misidentified as Aglaia silvestris (M. Rohmer) Merrill.

The voucher specimen for the original collection was re-identified by Dr. Christine Pannell, Daubeny Herbarium, University of Oxford as Aglaia foveolata Pannell (Hwang et al., J. Org. Chem. 69, 6956, 2004).

IN VITRO CYTOTOXICITY OF SILVESTROL

Compound Cell linea

Lu1 LNCaP MCF-7 HUVEC Silvestrol 1.2 1.5 1.5 4.6 Methyl rocaglateb

163 325 Not determined

203

Paclitaxelc 2.3 4.7 0.7 105.5 Camptothecinc 28.7 28.7 28.7 258.6 a ED50 values (nM) Lu1 = human lung cancer; LNCaP =

hormone-dependent human prostate cancer; MCF-7 = human breast cancer; HUVEC = human umbilical vein endothelial cells.

b Isolated in previous work from Aglaia rubiginosa (Rivero-Cruz et al., J. Nat. Prod., 67, 343, 2004).

c Used as positive control. (Hwang et al., J. Org. Chem. 69, 3350, 2004)

EFFECT OF SILVESTROL IN THE IN VIVO HOLLOW FIBER TEST AND IN THE MURINE P-388 LEUKEMIA

MODEL

Active at maximum tolerated dose of 2.5 mg/kg/inj, given by intraperitoneal injection daily for five consecutive days (qd×5) in ip P388 model.

Achieved maximum lifespan increase corresponding to T/C of 150%.

Hollow fiber: Murine P-388 leukemia:

Inactive (T/C = 100%) in iv P388 leukemia model when administered by either the iv or ip route using a daily times five schedule (qd×5).

Active (T/C = 129%) in same tumor model when injected iv on a twice-daily schedule (2qd×5) using the same cumulative dose (2 mg/kg/day).

(Hwang et al., J. Org. Chem. 69, 3350, 2004)

ISOLATION OF SILVESTROL FROM A RECOLLECTION OF AGLAIA FOVEOLATA FROM INDONESIA

MeOH Extract

Residue Hexanes Extract Add H2O, partition with CHCl3

CHCl3 Extract

Partially Detannified CHCl3 Extract

Aqueous Extract

Dissolve in mixture of MeOH-H2O (9:1) Defat with hexanes

Wash with 1% NaCl

Extract with MeOH

Plant Material

An aliquot (500 g, 5% w/w of the total dried methanol extract) was used for work-up

A 40-45 kg recollection of Aglaia foveolata stem bark was collected in the Fall of 2007, in Kalimantan

This extract was active against the HT-29 cell line (IC50 = 0.4 µg/ml)

Subjected to separation over a Si gel column (CH2Cl2−acetone, 20:1 to 100% acetone)

Subfractions F01-F08 Chromatography of active fraction F07 (7 g; IC50 = <0.016 µg/ml) led to the purification of silvestrol (100 mg)

Large-scale column chromatography of a recollection of Aglaia foveolata, from Indonesia, leading to purified silvestrol (conducted for the United States National Cancer Institute)

Dr. Thomas McCloud, SAIC-Frederick, MD

Incubation Time (hours)

B Cells

0

20

40

60

80

100 120

0 24 48 72 Incubation Time (hours)

T Cells

0

20

40

60

80

100

120

0 24 48 72

EFFECTS OF SILVESTROL ON B AND T CELLS IN WHOLE BLOOD FROM CLL

PATIENTS

80 nM Silvestrol 1 µM 2-F-ara A

% L

ive

Cel

ls R

elat

ive

to U

ntre

ated

(Lucas et al., Blood 113, 4656, 2009)

The remaining three mice appeared normal 12+ weeks post-engraftment, 6 weeks after the last treatment.

SILVESTROL SIGNIFICANTLY IMPROVED SURVIVAL IN AN ACUTE LYMPHOBLASTIC LEUKEMIA XENOGRAFT MOUSE MODEL

0

20

40

60

80

100

0 5 10 15 20 25 30 35 40 45

Days Post Engraftment

% S

urvi

val

Control (N=13)

Silvestrol (N=14)

1.5 mg/kg i.p. M, W, F; started 1 wk post-engraftment Treatment stopped

85

Median survival difference P = 0.002

(Lucas et al., Blood 113, 4656, 2009)

Pelletier and co-workers have shown that silvestrol is a translation

inhibitor by targeting eukaryotic initiation factor (eIF) 4A (Cencic et al., PLoS ONE 4(4), e5223, 2009).

In recent work, using biotinylated 5′′′-epi-silvestrol, a specific interaction was shown with eIF4AI and eIF4AII (Chambers et al., Org. Lett. 15, 1406, 2013).

SILVESTROL: ACTIVITY AS A TRANSLATION INHIBITOR

(Lucas et al., Curr. Drug Targets 11, 811, 2010)

O

OH

COOMe

OMe

O

OMeO

OHOOH

OH

OMe

SILVESTROL: A POTENTIAL NEW THERAPEUTIC AGENT FOR B-CELL MALIGNANCIES

At The Ohio State University, silvestrol has shown promising in vivo activity in models of acute lymphoblastic leukemia, acute myeloid leukemia, EBV-driven lymphoma, and mantle cell lymphoma as well as interesting immunomodulatory activity (Lucas et al., Blood 113, 4656, 2009; Alinari et al., Clin. Cancer Res. 18, 4600, 2012; Alahkar et al., J. Hematol. Oncol. 6, 21, 2013; Patton et al., Oncotarget 6, 2693, 2015).

The mechanism of antileukemic action was further investigated through a NCI/NIH SPORE (P50) award (Dr. J. C. Byrd, PI) (work by Drs. M.R. Grever and D.M. Lucas; 2009-2015).

Silvestrol has been undergoing preclinical toxicology development through the NCI NExT pipeline (P.I., M.R. Grever).

AGREEMENT FOR DEVELOPMENT OF SILVESTROL FOR POTENTIAL TREATMENT OF B-CELL MALIGNANCIES

In June 2012, an agreement was signed to jointly develop silvestrol between The Ohio State University and the Sarawak Biodiversity Center, with the immediate goal of conducting preclinical toxicology.

This slide shows the two other faculty participants in this work at Ohio State, Drs. Michael Grever (top left) and David Lucas (top right), and Dr. Rita Manurung, former Chief Operating Officer, Sarawak Biodiversity Center, Kuching, Sarawak, Malaysia (bottom).

Silvestrol may be sourced from Aglaia stellatopilosa Pannell obtained from Sarawak.

ATTRIBUTES OF SILVESTROL AS A CANDIDATE ANTICANCER AND IMMUNOMODULATORY COMPOUND The compound is a new composition of matter

representing a relatively unstudied structural type. Silvestrol has shown efficacy at non-toxic doses in a

range of murine in vivo models germane to human cancer, including some rare diseases that are difficult to treat. It acts differentially on different immune subsets.

The presently known mechanism of action of the compound is unusual (an inhibitor of protein translation, acting specifically at eukaryotic initiation factor eIF4AI/II).

Sensitizes tumors to other agents, so its use in combination therapy may be feasible.

Silvestrol has overall favorable pharmacokinetics when injected ip or iv in mice, and may be formulated in hydroxypropyl-β-cyclodextrin.

Intellectual property (IP) protection has been established. A reliable supply of the compound is available (obtained

from Aglaia stellatopilosa grown in Sarawak, Malaysia).

Funding obtained from NIH/NCI CA52956 and P01 CA125066 (to A.D. Kinghorn) is gratefully acknowledged

Chemistry Prof. Geoffrey A. Cordell (University of Illinois at Chicago, UIC) Prof. James R. Fuchs (The Ohio State University, OSU) Prof. Andrew D. Mesecar (UIC) Prof. Bernard D. Santarsiero (UIC) Dr. David J. Newman (NCI-Frederick) Dr. Thomas G. McCloud (SAIC-Frederick) Dr. Bang Yang Hwang (UIC) Dr. Li Pan (OSU) Dr. Angela A. Salim [(OSU)] Dr. Baoning Su (UIC; OSU)

Biological Testing Dr. Hee-byung Chai (UIC; OSU) Dr. Robert A. Baiocchi (OSU) Dr. Stuart Emanuel [Bristol-Myers Squibb (B-MS)] Dr. Craig R. Fairchild [Bristol-Myers Squibb (B-MS)] Prof. Michael R. Grever (OSU) Dr. David M. Lucas (OSU) Prof. John M. Pezzuto (UIC) Dr. William C. Rose (B-MS) Prof. Steven M. Swanson (UIC) Dr. Robert Wild (B-MS)

Plant Collection/Taxonomy Prof. Norman R. Farnsworth (UIC) Prof. Djaja D. Soejarto (UIC) Dr. Caroline M. Pannell (University of Oxford) Dr. Leonardus B.S. Kardono (Indonesia) Dr. Soedarsono Riswan (Indonesia)

ACKNOWLEDGMENTS (STUDIES ON PLANT ANTICANCER AGENTS)

This is a protozoan vector borne parasitic disease (transmitted by a sand fly).

About 12 million people infected worldwide, with two million new cases annually, and 350 million people at risk.

Severe public health problem in parts of East Africa and the Middle East, the Indian subcontinent, and Central and South America.

Existing drug therapy has several drawbacks including the development of resistance, toxicity, and poor compliance.

BACKGROUND TO LEISHMANIASIS

http://www.who.int/leishmaniasis/disease_ epidemiology/en/index.html

APPROACH TO NOVEL DRUG DISCOVERY FOR LEISHMANIASIS Screening of plant materials from Mexico. Isolation and characterization of bioactive constituents. Development of efficient synthetic methods to prepare the active

compounds and derivatives for MOA, SAR, and optimization studies.

http://www.ecoyuc.com/ Riparian forest near

Hopelchen, Campeche (Mexico)

C. M. Lezama-Dávila and A. P. Isaac-Márquez

Mexico

United States

SCREENING OF PENTALINON ANDRIEUXII ROOTS FOR ANTILEISHMANIAL ACTIVITY Traditionally in Campeche, Mexico,

skin lesions from cutaneous leishmaniasis are treated topically by Mayan healers with an infusion of the roots of Pentalinon andrieuxii B.F. Hansen & Wunderlin (Apocynaceae). The inner part of the roots of is then tightly fixed to the skin lesions. This procedure is repeated daily until the wound heals.

In a preliminary laboratory study, in vitro inhibitory activity against Leishmania mexicana amastigotes was found for the root hexane extract of P. andreuxii (PARE).

Air-dried Roots of P. andrieuxii (by Y. Deng)

Aerial Parts of P. andrieuxii www.nybg.org/.../Pentalinon_andrieuxii.html

(Lezama-Davilla et al., Fitoterapia 78, 255, 2007)

EFFECT OF PENTALINON ANDRIEUXII ROOT HEXANE EXTRACT (PARE) ON THE

LEISHMANIA MEXICANA PARASITE IN VIVO A preliminary in vivo experiment with the root extract was performed using 10 week old male C57BL/6 mice, which were infected with L. mexicana promastigotes in the ear dermis.

Mice were treated with 10 µg of PARE dissolved in 50 µl of DMSO/PBS. The extract was applied on the infected ear daily for 21 days [note the lymphocytes and macrophages (white cells)].

(Lezama-Davilla et al., Phytother. Res. 20, 909, 2014)

ANTILEISHMANIAL STEROLS ISOLATED FROM PENTALINON ANDRIEUXII ROOTS

Active compound

New compound

1 R

=

3 R

=

4 R

=

5 R

=

6 R

=

R

HO

7 R

=

8 R

=

9 R

=

10 R

=

11 R

=

12 R

=

R

13 R

=

14 R

=

R

O

OH O

OH O

H

H

H

H

H

H

15

16

H

H

OH3CO

OHO

OO

20

H

H

2

OO

OO

R

17

18 R

= OCH319

R =

H

21

17

H

H

COOH

20

H

H

H H

HHH H HO HO

O

OO

AcO

O

O

(Li et al., Phytochemistry 82, 128, 2012)

SYNTHESIS OF PENTALINONSTEROL (1) FROM PREGNENOLONE (2)

Drs. James R. Fuchs and Dalia Abdelhamid (The Ohio State University)

(Gupta et al., ACS Inf. Dis., 2015, under revision)

HO TBSO

O OR

HO

R

O

1. TBSCl,imidazole,

DMF

2. LDA, HMPA,THF, -78°C;

1. Tebbe reagent

2. TBAF, THF

Al(OiPr)3,

toluene, THF

Br23

1

95%

78%

50%

94%

N-Me piperidone89%

2021

17

22

3

R =

4

5

pentalinonsterol

ADDITIONAL WORK PERFORMED ON PENTALINONSTEROL

Pentalinonsterol (>30 mg) was synthesized from pregnenolone in five synthetic steps.

In an in vivo BALB/c mouse model of visceral leishmaniasis induced by Leishmania donovani, i.v. treatment with liposomal encapsulated pentalinonsterol (2.5 mg/kg) led to a significant reduction in parasite burden in the in the liver and spleen.

Infected mice showed a strong host-protective TH1 immune response with IFN-γ production and the formation of matured hepatic granulomas, when treated with the liposomal encapsulated pentalinonsterol.

This test compound caused dose-dependent changes in the content of fatty acid lipids in L. donovani promastigotes.

Pentalinonsterol shows potential for development as a therapy for the treatment of visceral leishmaniasis.

Intellectual property protection through The Ohio State University has been obtained.

(Gupta et al., ACS Inf. Dis., 2015, under revision)

Funding by grant RC-4 AI092624 was obtained from NIAID, NIH (A.R. Satoskar, PI, A.D. Kinghorn, Co-PI; 2010-2013). Earlier support was obtained

from NIH grants R21 AI076309, R21 AT04160, and R56 AI090803 (A.R. Satoskar, PI). Current funding is from the U.S. Army Medical Research

(W81XWH-14-2-0168; A.R. Satoskar, P.I.; 2014-2017)

Phytochemical Studies – The Ohio State University Prof. A. Douglas Kinghorn Dr. Li Pan Dr. Ben Naman

Biological Testing and Mechanistic Studies – The Ohio State University Prof. Abhay R. Satoskar Prof. K.M. Ainslie Prof. N.L. “Pari” Parinandi Dr. Claudio Lezama-Dávila Dr. Gaurav Gupta Collaborator (Taxonomy and Supply of Plant Material – Universidad Autónoma de Campeche, Mexico Dr. Angélica P. Isaac-Márquez

Endophytic Fungal Cultivation Studies – Mycosynthetix, Inc., Hillsborough, NC Dr. Cedric J. Pearce

Synthetic Studies – The Ohio State University Prof. James R. Fuchs Dr. Dalia Abdelhamid

INVESTIGATORS INVOLVED IN THE ANTILEISMANIAL PROJECT ON PENTALINON ANDREUXII

FROM MEXICO AND FUNDING SOURCES

SUMMARY AND CONCLUSIONS

Tropical plants are more biodiverse than temperate plants, and thus hold the potential of offering greater chemical diversity for natural products drug discovery.

Plant collections to access plant genetic material for drug discovery must cover the source country in terms of benefit-sharing agreements.

Efforts to harness plant compounds as potential cancer chemotherapeutic and antileishmanial agents from an academic perspective require a collaborative multidisciplinary approach with open and frequent communications.

COLLEGE OF PHARMACY AND OSU COMPREHENSIVE CANCER

CENTER