Artemisia BioMedical , Inc The Discovery of Artemisinin

1. Artemisia BioMedical, IncThe Discovery of Artemisinin Better by Natural Design™ March 2014 PNWBIO, Seattle

2. (Re-)Discovery of Artemisinin • Being considered for the Nobel Prize in Medicine • The Nobel rules specify no more than three winners and no posthumous prizes, either. • TuYouyou awarded 2011 Lasker~DeBakey Clinical Medical Research Award for discovering artemisinin and its use in treating malaria

3. Science in China 1950-1960 • Stopped interacting with the West • Younger scientists did not have access training comparable to that found in Europe and the US • Major project was the development of atomic and hydrogen weapons and later artificial satellites • Medical research projects were few • Facilities were crude when compared to Western labs

4. Cultural Revolution • The Great Proletarian Cultural Revolution started in 1966 • Intellectuals, including scientists, were publicly humiliated and forced to labor on collective farms.

5. Cultural Revolution • Scientific publications within China were stopped • Books and journals were recycled for their paper • Science was no longer taught at middle schools • Western training was cause for imprisonment or banishment to the countryside • However, the military protected scientists working on projects of national importance

6. 1960’s Vietnam • Late 1950’s program by WHO to eradicate malaria in Southeast Asia had failed • There was Plasmodium falciparum resistance to all known anti-malarials including chloroquine • Soldiers on both sides were infected with malaria (roughly ½ at any given time) • In 1966, experts were sent by the USA and China to study the problem

7. Malaria Transmission Cycle

8. Vietnam Was an Ideal Malaria Breeding Ground • Vast tunnel network containing ants, poisonous centipedes, snakes, and mosquitos

9. China Offers to Help Ally • Ho Chi Min asked Chairman Mao Zedong for help. • Malaria was also a problem in southern China • Hundreds of thousands of Chinese troops were in Vietnam

10. Prophylaxis First • In 1966, scientists for the Chinese military began reviewing existing antimalarial drugs for the prevention of malaria. • Three were selected and pairs were used in combination tablets to use prophylactically against malaria. • A new cure was still needed.

11. Project 523 • Leaders from the Chinese Military, the Ministry of Health and the National Commission on Science and Technology met on May 23, 1967 • Coordinating office located in the Military Academy of Medical Sciences • Participating institutions were spread across China, involving hundreds of individuals, from Beijing, Shanghai, Yunnan, Shangdong to Guangdong and Guangxi

12. Project 523 Composed of: • The General Logistics Department of the Chinese People’s Liberation Army • The State Science and Technology Commission of China • Ministry of Public Health of PRC • Ministry of Chemical Industry of PRC • Commission of Science and Technology for National Defense • Chinese Academy of Sciences and • The General Pharmaceutical Industry Corporation • ~Five hundred scientific workers from sixty+ institutions

13. Project 523 Aims • New drugs for drug-resistant falciparum malaria • Combine traditional Chinese and modern Western medicine • Emphasized discovery of new drugs from traditional Chinese medicine • Drugs should be safe with few adverse effects • Drugs should be highly effective with rapid onset of action • Administered infrequently for prevention

14. Two Approaches Used • Copy Western drugs and synthesize new derivatives • P. falciparum was resistant to existing drugs • Modifying drugs would make them effective • Combination therapy with existing and new drugs • Investigate anti-malarial drugs frequently used in traditional Chinese medicine and folklore medicine • Malaria had been endemic to China for 3,000 years • Different regions may have unique treatments • Back to the future approach

15. Synthesis of New Drugs • Take existing drugs and make new family members

16. Artemisiaannua L • Qinghao grows in south and north China • 168 BC - "Fifty-two Prescriptions” from Han Dynasty Tombs • 340 AD - antimalarial application was first described in Zhou Hou Be JiFang ("Handy Therapies for Emergencies") • Crushed plants and crude plant extracts were used by Chinese farmers in the 1950s and 1960s to treat malaria

17. Beijing Institute of MateriaMedica • 1970 – scientists reviewed ancient and current records and identified 808 anti-malaria candidates • ~100 candidates tested at the Military Academy of Sciences in a mouse model of malaria • Mouse model transferred to Beijing • Soak Qinghao in water, compress the plant and drink the juice to treat malaria • Replaced ethanol extraction with ether extraction (lower boiling point)

18. Cooler Extraction • Success rate with ether extraction material was 100% cure rate in mice with P berghei • The neutral portion of the extract was identified as containing the active element • Tested in animals and a few healthy volunteers and deemed safe • 1972 – tested in 9 P vivax patients and 11 P falciparium patients, failed in 2 P falciparium patients • Project head office requested purified active element

19. Clinical Failure • Purified extract from Beijing Institute showed cardiac toxicity in animals • Tested on 3 researchers and did not show toxicity • Field tested in 8 patients, 6 had normalized temperature, but 2 patients had cardiac toxicity, and 2 showed no signs of cure • Results not shared with project head office

20. Relay Race • 1972 – Work at Beijing Institute duplicated in Shandong and Yunnan provinces using their local artemisia plants • Both groups were able to make purified artemisinin • Monomer isolated at Shandong Institute of Chinese MateriaMedica by Wei Zhenxing did not show cardiac toxicity • Luo Zeyuan from the Yunnan Institute of MateriaMedica got active isomers from ether extract

21. Progress in Yunnan • Yunnan group found their extract cleared P berghei rapidly from mice and did not effect heart, liver, or kidneys • They identified a variant of Artemisia annua that had the highest amounts of artemisinin from local plants • They identified the Youyang area in the Sichuan Province as having plants with 10-15 the concentration of artemisinin • Material was provided to other research institutions

22. 1974 – Clinical Success • Group at Beijing could not produce purified material • Shandong group compared crude with purified artemisinin extracts in 26 patients with P vivax • All patients were cleared of parasites and no evidence of cardiac toxicity was seen • Most patients had recurrence of parasites within 28 days • Tried combining artemisinin with prophylactic tablet to prevent recurrence

23. 1974 – Clinical Success Pt 2 • Material from the Yunnan group was used to treat P falciparum • The first 3 patients responded very quickly to artemisinin; faster than existing therapies • 14 P falciparum patients including 3 with malignant malaria were treated and 12 showed clearance of the parasites by 20 hours • Parasite development was arrested at the tiny-ring form stage

24. Findings Made Public • 1972 Artemisia annua(sweet wormwood) extract shown to cure malaria • Several groups of scientists at different institutions needed to determine structure of active ingredient • 1977 first Chinese paper describing Qinghaosu but omitting antimalarial action • 1982 first paper in English and available outside of China

25. Artemisinin and Derivatives

26. Results of Project 523 • Chemical compounds were developed for the acute and emergency treatment of malaria, prevention of malaria, and eradication of malaria • More than 10,000 chemicals synthesized, 40,000 chemical samples were screened, 1,000 possible active substances identified, 38 underwent preclinical screening, 29 approved for clinical trials • 14 drugs passed all testing and were used

27. 1979 Discovery of Qinghaosu Award • Ministry of Health nominated 6 institutions • Academy of Chinese Traditional Medicine; Ministry of Health • Shandong Institute of Chinese Traditional Medicine • Yunnan Institute of MateriaMedica • Institute of Biophysics of the Chinese Academy of Sciences • Shanghai Institute of Organic Chemistry of the Chinese Academy of Sciences • Guangzhou College of Traditional Chinese Medicine

28. Were the Chinese First? MilutinStefanovic 1972, first publication describing artemisinin No mention of malaria

29. Structure and Mechanism • 1993 - malaria parasite survives in its host by consuming approximately 25% of the hemoglobin in the host's red blood cells and stores iron • Artemisinin comes in contact with the iron converting it into a toxic chemical, releasing a free radical that destroys the parasite

30. Artemisinin Actions • Kills malaria parasites • Heme-derived iron reacts with artemisinin in food vacuole to produce lethal amounts of ROS • Kills cancer cells • Free iron reacts with artemisinin in lysosomes to produce ROS • Death by activated programmed cell death pathways (PCD) • Inhibits angiogenesis

31. Cancer Cells and Iron • All cells need iron for growth • Normal cells control iron uptake by neg feedback • Cancer cells accumulate iron beyond needs • Iron becomes a target for anticancer therapies • Block iron uptake • Scavenge iron from blood • Use iron to trigger free radical reactions • Cancer cells deficient in free radical scavengers

32. Iron and Cancer • Cancer cell lysosomes contain higher levels of redox active iron • Nat Rev Cancer. 2005 Nov;5(11):886-97 • Breast cancer cells have higher transferrin receptor expression levels and reduced ferroportin levels • SciTransl Med. 2010 Aug 4;2(43):43ra56. doi: 10.1126/scisignal.3001127

33. Lysosomes

34. Free Radical Reactions

35. Creation of ROS is Iron Dependent +iron -iron Cells treated with 0 (purple), 10 (green), 25 (red), 50 (blue), and 100 mM (orange) DHA. Cells on right treated with iron chelator desferroxamine. H Kenji, http://hdl.handle.net/2115/53216

36. Programmed Cell Death Pathways • Lysosomes accumulate free iron and artemisinin • ROS from artemisinin causes membrane permeabilization • Hydrolytic enzymes are released from the lysosome lumen into the cytosol • Cathepsins activate pro-apoptotic proteins • BH3 interacting-domain death agonist and caspase 8 activate mitochondrial outer membrane permeabilizationand apoptosis • J Biol Chem. 2011 Feb 25;286(8):6587-601

37. Artemisinin Multiple Anticancer Effects Multi-targeted, Selectively Promiscuous™ Firestone GL, Sundar SN. Anticancer activities of artemisinin and its bioactive derivatives. Expert Rev Mol Med. 2009 Oct 30;11:e32.

38. NCI In Vitro Cancer Screening • Trioxane Dimers exhibit cancer cell cytotoxicity superior to gold standard chemotherapy in NCI screening panel

39. Effect of Art Derivative in vivo

40. Questions?