This is a lecture by Dr. Jerry McLaughlin about his research into extracts of pawpaw plants, annonaceous acetogenins, in vitro, in vivo, mechanism of action, and toxicity in mice.

1. Paw Paw and Cancer: From Discovery to Clinical Trials Jerry L. McLaughlin, B.S.(Pharm.), M.S., Ph.D, R.Ph. Emeritus Professor of Pharmacognosy Purdue University West Lafayette, IN

2. Disclaimer The following information is for educational purposes only and is not intended for the mitigation, cure, or treatment of medical conditions.

10. Bullatacin (from Annona bullata ) (Hiu et al., Journal of Natural Products , 52 , 463-477, 1989) Major Functional Groups of the Annonaceous Acetogenins

11. Compounds A B C D E Trilobacin (1) threo trans erythro cis threo Asimicin (2) threo trans threo trans threo Bullatacin (3) threo trans threo trans erythro Bullatacinone (4) threo trans threo trans erythro Stereochemical Aspects of the Major Paw Paw Acetogenins Rieser et al., Journal of the American Chemical Society 114 , 10203-10213, 1992 Gu et al., Journal of Organic Chemistry , 59 , 5162-5172, 1994

14. Plant Part Extracted LC 50 in ppm % yield Twigs 0.042 (0.20-0.09) 1.78 Unripe fruit 0.060 (0.03-0.08) 5.11 Root wood 0.060 (0.03-0.08) 1.27 Seed 0.065 (0.03-0.10) 4.03 Stem bark (7-88 ) 0.077 (0.04-0.12) 2.71 Stem bark (10-90) 0.102 (0.06-0.15) 1.96 Stem bark (10-90) 0.104 (0.03-0.20) 2.52 Root bark 0.135 (0.09-0.21) 2.72 Stem, wood, & leaves 0.202 (0.14-0.31) 1.6 Stem wood 4.86 (0.37-11.93) 0.9 Leaves 53.6 (33-82) 4.36 Brine Shrimp Lethality and % Yields from plant parts of Asimina triloba Conclusion: twigs are a renewable source of biomass

15. Bioactivity of Asimina triloba Fractions against Pests Fraction 1000 100 10 1 1 1 F017 100 90 0 0 0 100 F018 50 0 0 0 0 0 F019 100 100 70 0 100 100 F020 100 100 100 50 100 100 Blowfly larvae (%) C. elegans (%) Mosquito larvae (ppm) *The results are corrected for control deaths by Abbott’s formula Testing conducted at Eli Lilly and Co. Greenfield, IN (Alhofahi et al., Insecticides of Plant Origin , Amer. Chem. Soc., Washington, D.C., 1989, pp.(25-43) % Mortality*

19. Biological Activities of Maryland (WMREC) Paw Paw Plantation 135 individual Asimina triloba twig samples collected 26 Sept. 1995 Grand BST Mean= 1.2ppm SE=0.30ppm High Producers Low Producers Tree Sample BST-LC 50 (ppm) Tree Sample BST-LC 50 (ppm) 4-11 0.033+/-0.0010 2-32 32 +/- 12 5-82 0.053+/-0.015 1-19 12+/- 4.7 4-70 0.056+/-0.0354 4-46 10 +/- 0.55 2-88 0.063+/-0.020 5-14 8.4 +/- 2.5 Individual paw paw trees can vary 1000x in twig potency; thus, bioassay of materials is essential.

20. Two sites of cellular action: (depletes ATP) Lewis et al., Pesticide Biochemistry and Physiology , 45 , 12-23, 1993. Ahammadsahib et al., Life Sciences , 53 , 1113-1120, 1993. Hollingworth et al., Biochemical Society Transactions , 22 , 230-233, 1994. Schuler et al., Proceedings of the National Academy of Sciences USA , 96, 4149-4155, 1999. Morre et al., Life Sciences , 56 , 343-348, 1995.

22. Oberlies et al., Cancer Letters , 115 , 173-179, 1997 Oberlies et al., Journal of Medicinal Chemistry , 40 , 2102-2106, 1997 Johnson et al., in Biologically Active Natural Products , CRC Press, Boca Raton, 1999, pp.173-183 Diagram of the P-170 glycoprotein multidrug resistant pump

23. J. McAlpine, Abbott Laboratories (unpublished data)

24. Oberlies et al., Cancer Letters , 96 ,55-62, 1995

25. In vivo anticancer activities of two paw paw acetogenins against mice bearing L1210 murine leukemia Product Survival 1st run (% T/C) Weight change (g/mouse) Survival 2nd run (% T/C) Bullatacin 50.0 138 138 -0.4 Bullatacinone 400 144 1.1 Taxol 15000 139 -2.5 Dose* (  g/kg) Conclusion: 300 times as potent as taxol in vivo Acetogenins cause much less weight loss than taxol Studies performed at the Upjohn Co. Ahammadsahib et al., Life Sciences , 53 , 1113-1120, 1993

26. Anti-tumor efficacy of Paw Paw compounds against A2780 human ovarian carcinoma implanted in athymic mice Product Dose (mg/kg) TGI(%) Wt. Change g/mouse Bullatacin 0.10 68 -1.5 0.05 67 -0.3 0.025 35 0.1 Bullatacinone 0.50 33 -0.7 0.25 24 0.1 0.125 52 -0.1 Bullatalicin 2.00 63 -0.9 1.00 75 -0.1 0.50 42 -0.2 Cisplatin 5.00 78 -1.1 V.Control -0.3 TGI%= tumor groth inhibition %; animals were tested (ip) daily for 10 days starting day one after tumor implantation. Studies performed by the Upjohn Company. Ahammadsahib et al., Life Sciences , 53 , 1113-1120, 1993 Conclusion: Acetogenins approach the effectiveness of cisplatin with less weight loss

27. DNA laddering proves apoptosis Geahlen, Purdue University(unpublished data)

28. Over 100 Scientific papers on the chemistry and biology of the paw paw compounds This work was honored on the cover of J. Nat. Prod. For the first 6 months of 1999. The zebra swallowtail butterfly concentrates the acetogenins as a protection from predation by birds Martin et al., Journal of Natural Products , 62 , 2-4, 1999 Alali et al., Journal of Natural Products , 62 , 504-540, 1999 (our 5 th comprehensive review)

29. 4 Mechanisms by Which Paw Paw Regulates Cells 1. Slows or stops ATP (adenosine triphosphate) production, thus energy is depleted. Apoptosis results in programmed cell death a. At NADH: ubiquinone oxidoreductase (mitochondrial) b. At NADH: oxidase (tumor cell membranes) 2. ATP is a direct and indirect precursor of DNA and RNA, thus cell reproduction is slowed or stopped; this effect blocks viral replication (cold sores, shingles, etc.) 3. Angiogenesis in endothelial cells requires ATP, thus , blood vessel formation is slowed or stopped 4. The multiple-drug resistance (MDR) pump requires ATP, thus drug resistant cells are slowed or stopped . In doing so it helps restore the function of refractive chemotherapy.

31. Clinical Results: Breast Cancer Patient #3 - Tumor size reduced and was not detected with MRI. Patient #8 – 7 months Chemo + paw paw: Tumor almost completely disappeared. Biopsy after radical mastectomy showed no tumors in 14 lymph nodes—patient is cancer free Patient #12 Stage IV 6 weeks of paw paw: 50% reduction in tumor markers (160  80), size of tumors reduced significantly

32. Clinical Results: Prostate Cancer Patient #48 – Over 6 months PSA went from 36  33  30 Patient #53 – reduced pressure on prostate, reduced frequency of urination Patient #56- Stage IV: distinct reduction in tumor masses (25% reduction verified by CT scan) after only 6 weeks Patient #75 PSA of 4.5  1.7 after 6-8 weeks.

35. Clinical Results: Stage IV Melanoma (lung metastases) Patient #52 In just 4 days on paw paw patient could breathe without “burning” sensation in his lungs Strength returned and now can ride a bike, walk Two lipomas on arm have decreased in size Toe-nail fungus (had for 10 years) is going away