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 Source Organism Type Number Terrestrial Plants 8 (apomorphine HCl, arteether, dronabinol/cannabidiol (mixture), galanthamine HBr, lisdexamfetamine, methylnatrexone Br, nitisinone, tiotropium Br) Terrestrial Microorganisms 24 (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) Marine Organism 2 (trabectidin, ziconotide) Terrestrial Animals 2 (bivalirudin; exenatide; synthetic versions of natural forms) [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] PLANT NATURAL PRODUCTS AND DERIVATIVES APPROVED BY THE U.S. FDA (2012 TO MID-2013)
Natural Lead Year Generic Name Trade Name Indication Compound Ingenol-3- 2012 Ingenol mebutate Picato® Actinic keratosis angelate Lorcaserin 2012 Ephedrine Belviq® Obesity hydrochloride Omacetaxine Homoharring- Chronic myeloid 2012 Synribo® mepesuccinate tonine leukemia HIV/AIDS anti- Croton lechleri retroviral- 2012 Crofelemer oligomeric pro- Fulyzaq® associated anthocyanidins diarrhea ado-Trastuzumab 2013 emtansine (protein- Maytansinea Kadcyla® Breast cancer bound) Menopause- 2013 Ospemifene Phytoestrogens Osphena® associated dyspareunia 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
PLANT NATURAL PRODUCT DRUG DISCOVERY AND DEVELOPMENT
Natural Products Discovery Medicinal Chemistry Molecular Modeling
Target-based Bioassays Cell-based Bioassays Lead Identification In Vivo Bioassays
Medicinal Chemistry Lead Optimization Combinatorial Chemistry
Pharmacology, Toxicology Pharmacokinetics, ADME Lead Development Drug Delivery
Drug Candidates Clinical Trials MAJOR STAGES IN NATURAL PRODUCT DRUG DISCOVERY
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.
(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) 80,000 Protoctista (e.g., protozoa, diatoms, “algae”, including “red algae” and “green algae”) 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, 1,320,000 roundworms, sea urchins, mollusks, segmented worms, arthropods, insects, fish, amphibians, birds, mammals)d-f 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 Terrestrial Terrestrial Marine / Otherse Microbes/ Plantsc Animals Aquaticd Fungi 1,869 7,760 117 3,138 33 (14.5%) (60.0%) (0.9%) (24.3%) (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) DRS. M.E. WALL (LATE) AND M.C. WANI: CO-DISCOVERERS OF TAXOL AND CAMPOTHECIN
(Photograph by Jimmy W. Crawford, RTI) COLLABORATIVE PROJECTS ON THE DISCOVERY OF NATURAL PRODUCT ANTICANCER AGENTS
National Cooperative Drug Discovery Group (NCDDG) Granta (U19 CA52956) (1990-1995; 1995-2000; 2000-2006) Program Project Grantb (P01 CA125066) (2007-2013; 2014-2019)
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) [Most recent review: Kinghorn et al. , Pure Appl. Chem. 81, 1051, 2009] PLANT COLLECTIONS AT UIC (DRS. NORMAN FARNSWORTH AND DOEL SOEJARTO)
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). Anticancer Plant Collections 1990-2011 Total plant accessions obtained 6,599 Representing: 2,609 species 1,466 genera 222 families Over 200 recollections FORMER RAID PROGRAM OF THE UNITED STATES NATIONAL CANCER INSTITUTE
Rapid Access to Intervention Development (http://dtp.nci.nih.gov/docs/raid/raid_index.ht ml). 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. BETULINIC ACID: A NCDDG COMPOUND THAT ENTERED CLINICAL TRIALS AT THE UNIVERSITY OF ILLINOIS AT CHICAGO
H
H COOH
H HO H
• 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). 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 1 – The Ohio PROJECT 2 – University of State University (OSU) Illinois at Chicago (UIC) PROJECT 3 – University of North Tropical Plants, Aquatic Cyanobacteria Carolina – Greensboro (UNC-G) Biological Testing Plant Collections Filamentous Fungi, Biological Testing A.D. Kinghorn, PL J. Orjala, PL N.H. Oberlies, PL E.J. Carcache de Blanco D.D. Soejarto C. J. Pearce (Mycosynthetix Inc.) D.M. Lucas B.R. Stockwell (Columbia University) M.C. Wani (Consultant) (RTI)
CORE A - UIC CORE B – OSU CORE C - OSU Biological Testing Medicinal Chemistry; Administration/Biostatistics J.E. Burdette, CD* Pharmacokinetics A.D. Kinghorn, CD S.M. Swanson J.R. Fuchs, CD L.-H. Xu, X. Zhang (University of Wisconsin) M.A. Phelps 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
O OCH3 OCH3 OH OH HO O H O OH COOCH3 O Silvestrol
OCH3
X-ray Structure by Drs. Bernard Santarsiero and Andrew Mesecar (UIC)
Drs. Bang Yeon Hwang and Baoning Su (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
Cell linea Compound Lu1 LNCaP MCF-7 HUVEC Silvestrol 1.2 1.5 1.5 4.6 Methyl 163 325 Not 203 rocaglateb determined 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 Hollow fiber: Murine P-388 leukemia: 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%.
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
Plant Material A 40-45 kg recollection of Aglaia foveolata stem bark Extract with MeOH was collected in the Fall of An aliquot (500 g, 5% w/w of 2007, in Kalimantan the total dried methanol MeOH Extract extract) was used for work-up Dissolve in mixture of MeOH-H2O (9:1) Defat with hexanes
Hexanes Extract Residue
Add H2O, partition with CHCl3
This extract was active against the CHCl3 Extract Aqueous Extract HT-29 cell line Wash with 1% NaCl (IC50 = 0.4 µg/ml) Partially Detannified CHCl3 Extract 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 EFFECTS OF SILVESTROL ON B AND T CELLS IN WHOLE BLOOD FROM CLL PATIENTS
B Cells T Cells 120 120 100 100 80 80 60 60 40 40 20 20 0 0 0 24 48 72 0 24 48 72
% Live % Live Cells Relative to Untreated Incubation Time (hours) Incubation Time (hours)
80 nM Silvestrol 1 µM 2-F-ara A (Lucas et al., Blood 113, 4656, 2009) SILVESTROL SIGNIFICANTLY IMPROVED SURVIVAL IN AN ACUTE LYMPHOBLASTIC LEUKEMIA XENOGRAFT MOUSE MODEL
100 Median survival difference P = 0.002 80 Control (N=13) 60 Silvestrol (N=14)
40 % Survival % 20 1.5 mg/kg i.p. M, W, F; started 1 wk post-engraftment Treatment stopped 0 0 5 10 15 20 25 30 35 40 45 85 Days Post Engraftment
The remaining three mice appeared normal 12+ weeks post- engraftment, 6 weeks after the last treatment. (Lucas et al., Blood 113, 4656, 2009) SILVESTROL: ACTIVITY AS A TRANSLATION INHIBITOR 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).
(Lucas et al., Curr. Drug Targets 11, 811, 2010)
SILVESTROL: A POTENTIAL NEW THERAPEUTIC AGENT FOR B-CELL MALIGNANCIES O OMe OMe OH OH HO O O OH COOMe O
OMe 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).
ACKNOWLEDGMENTS (STUDIES ON PLANT ANTICANCER AGENTS) Chemistry Biological Testing Prof. Geoffrey A. Cordell (University of Dr. Hee-byung Chai (UIC; OSU) Illinois at Chicago, UIC) Dr. Robert A. Baiocchi (OSU) Prof. James R. Fuchs (The Ohio State Dr. Stuart Emanuel [Bristol-Myers University, OSU) Squibb (B-MS)] Prof. Andrew D. Mesecar (UIC) Dr. Craig R. Fairchild [Bristol- Prof. Bernard D. Santarsiero (UIC) Myers Squibb (B-MS)] Dr. David J. Newman (NCI-Frederick) Prof. Michael R. Grever (OSU) Dr. Thomas G. McCloud (SAIC-Frederick) Dr. David M. Lucas (OSU) Dr. Bang Yang Hwang (UIC) Prof. John M. Pezzuto (UIC) Dr. Li Pan (OSU) Dr. William C. Rose (B-MS) Dr. Angela A. Salim [(OSU)] Prof. Steven M. Swanson (UIC) Dr. Baoning Su (UIC; OSU) Dr. Robert Wild (B-MS)
Plant Collection/Taxonomy Prof. Norman R. Farnsworth (UIC) Funding obtained from NIH/NCI Prof. Djaja D. Soejarto (UIC) CA52956 and P01 CA125066 (to A.D. Dr. Caroline M. Pannell (University of Kinghorn) is gratefully acknowledged Oxford) Dr. Leonardus B.S. Kardono (Indonesia) Dr. Soedarsono Riswan (Indonesia) BACKGROUND TO LEISHMANIASIS 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.
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. United States
Mexico
Riparian forest near Hopelchen, Campeche http://www.ecoyuc.com/ (Mexico) C. M. Lezama-Dávila and A. P. Isaac-Márquez 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 Air-dried Roots of P. andrieuxii (by Y. Deng) 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).
(Lezama-Davilla et al., Fitoterapia 78, 255, Aerial Parts of P. andrieuxii 2007) www.nybg.org/.../Pentalinon_andrieuxii.html 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
R R R
17 H H H O O O H H H H H H O HO HO O 17 R = 13 R 1 R = 7 R =
O O O
14 R = = = = 3 R 8 R 18 R OCH3 19 R = H
O OH 4 R = 9 R = H COOH
H H H 5 R = 10 R = O AcO 20 15 H
H 20 = = OH O 6 R 11 R 21 O H O H O H OH H Active compound 12 R = H H O O New compound O H3CO 2 16 (Li et al., Phytochemistry 82, 128, 2012) SYNTHESIS OF PENTALINONSTEROL (1) FROM PREGNENOLONE (2) 1. TBSCl, O imidazole, O DMF R 95% 2. LDA, HMPA, THF, -78°C; Br HO 2 TBSO 4 3 78% 1. Tebbe reagent toluene, THF 50% 2. TBAF, THF 94% 21 22 20 R 17 , Al(OiPr)3 N-Me piperidone 3 89% O 1 HO 5 = pentalinonsterol R
Drs. James R. Fuchs and Dalia Abdelhamid (The Ohio State University) (Gupta et al., ACS Inf. Dis., 2015, under revision) 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)
INVESTIGATORS INVOLVED IN THE ANTILEISMANIAL PROJECT ON PENTALINON ANDREUXII FROM MEXICO AND FUNDING SOURCES Phytochemical Studies – The Biological Testing and Ohio State University Mechanistic Studies – The Prof. A. Douglas Kinghorn Ohio State University Dr. Li Pan Prof. Abhay R. Satoskar Dr. Ben Naman Prof. K.M. Ainslie Synthetic Studies – The Ohio Prof. N.L. “Pari” Parinandi State University Dr. Claudio Lezama-Dávila Prof. James R. Fuchs Dr. Gaurav Gupta Dr. Dalia Abdelhamid Collaborator (Taxonomy and Endophytic Fungal Cultivation Supply of Plant Material – Studies – Mycosynthetix, Inc., Universidad Autónoma de Hillsborough, NC Campeche, Mexico Dr. Cedric J. Pearce Dr. Angélica P. Isaac-Márquez
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) 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