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Artemisia BioMedical , Inc The Discovery of Artemisinin. Better by Natural Design ™. March 2014 PNWBIO, Seattle. (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. - PowerPoint PPT Presentation
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Artemisia BioMedical, IncArtemisia BioMedical, IncThe Discovery of ArtemisininThe Discovery of Artemisinin
March 2014PNWBIO, Seattle
Better by Natural Design™
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(Re-)Discovery of Artemisinin(Re-)Discovery of Artemisinin
Being considered for the Nobel Prize in MedicineBeing considered for the Nobel Prize in Medicine The Nobel rules specify no more than three winners The Nobel rules specify no more than three winners
and no posthumous prizes, either.and no posthumous prizes, either. Tu Youyou awarded 2011 Lasker~DeBakey Clinical Tu Youyou awarded 2011 Lasker~DeBakey Clinical
Medical Research Award for discovering artemisinin Medical Research Award for discovering artemisinin and its use in treating malariaand its use in treating malaria
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Science in China 1950-1960Science in China 1950-1960
Stopped interacting with the WestStopped interacting with the West Younger scientists did not have access training Younger scientists did not have access training
comparable to that found in Europe and the UScomparable to that found in Europe and the US Major project was the development of atomic and Major project was the development of atomic and
hydrogen weapons and later artificial satelliteshydrogen weapons and later artificial satellites Medical research projects were fewMedical research projects were few Facilities were crude when compared to Western labsFacilities were crude when compared to Western labs
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Cultural RevolutionCultural Revolution
The Great Proletarian Cultural Revolution started in The Great Proletarian Cultural Revolution started in 19661966
Intellectuals, including scientists, were publicly Intellectuals, including scientists, were publicly humiliated and forced to labor on collective farms.humiliated and forced to labor on collective farms.
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Cultural RevolutionCultural Revolution
Scientific publications within China were stoppedScientific publications within China were stopped Books and journals were recycled for their paperBooks and journals were recycled for their paper Science was no longer taught at middle schoolsScience was no longer taught at middle schools Western training was cause for imprisonment or Western training was cause for imprisonment or
banishment to the countrysidebanishment to the countryside However, the military protected scientists working on However, the military protected scientists working on
projects of national importanceprojects of national importance
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1960’s Vietnam1960’s Vietnam
Late 1950’s program by WHO to eradicate malaria in Late 1950’s program by WHO to eradicate malaria in Southeast Asia had failedSoutheast Asia had failed
There was Plasmodium falciparum resistance to all There was Plasmodium falciparum resistance to all known anti-malarials including chloroquineknown anti-malarials including chloroquine
Soldiers on both sides were infected with malaria Soldiers on both sides were infected with malaria (roughly ½ at any given time)(roughly ½ at any given time)
In 1966, experts were sent by the USA and China to In 1966, experts were sent by the USA and China to study the problemstudy the problem
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Malaria Transmission CycleMalaria Transmission Cycle
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Vietnam Was an Ideal Malaria Vietnam Was an Ideal Malaria Breeding GroundBreeding Ground
Vast tunnel network containing ants, poisonous Vast tunnel network containing ants, poisonous centipedes, snakes, and mosquitoscentipedes, snakes, and mosquitos
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China Offers to Help AllyChina Offers to Help Ally
Ho Chi Min asked Chairman Mao Zedong for help.Ho Chi Min asked Chairman Mao Zedong for help. Malaria was also a problem in southern ChinaMalaria was also a problem in southern China Hundreds of thousands of Chinese troops were in Hundreds of thousands of Chinese troops were in
VietnamVietnam
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Prophylaxis FirstProphylaxis First
In 1966, scientists for the Chinese military began In 1966, scientists for the Chinese military began reviewing existing antimalarial drugs for the reviewing existing antimalarial drugs for the prevention of malaria.prevention of malaria.
Three were selected and pairs were used in Three were selected and pairs were used in combination tablets to use prophylactically against combination tablets to use prophylactically against malaria.malaria.
A new cure was still needed.A new cure was still needed.
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Project 523Project 523
Leaders from the Chinese Military, the Ministry of Leaders from the Chinese Military, the Ministry of Health and the National Commission on Science and Health and the National Commission on Science and Technology met on May 23, 1967Technology met on May 23, 1967
Coordinating office located in the Military Academy Coordinating office located in the Military Academy of Medical Sciencesof Medical Sciences
Participating institutions were spread across China, Participating institutions were spread across China, involving hundreds of individuals, from Beijing, involving hundreds of individuals, from Beijing, Shanghai, Yunnan, Shangdong to Guangdong and Shanghai, Yunnan, Shangdong to Guangdong and GuangxiGuangxi
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Project 523Project 523
Composed of:Composed of:The General Logistics Department of the Chinese People’s The General Logistics Department of the Chinese People’s Liberation Army Liberation Army The State Science and Technology Commission of ChinaThe State Science and Technology Commission of ChinaMinistry of Public Health of PRC Ministry of Public Health of PRC Ministry of Chemical Industry of PRCMinistry of Chemical Industry of PRCCommission of Science and Technology for National DefenseCommission of Science and Technology for National DefenseChinese Academy of Sciences andChinese Academy of Sciences andThe General Pharmaceutical Industry Corporation The General Pharmaceutical Industry Corporation ~Five hundred scientific workers from sixty+ institutions~Five hundred scientific workers from sixty+ institutions
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Project 523 AimsProject 523 Aims
New drugs for drug-resistant falciparum malariaNew drugs for drug-resistant falciparum malaria Combine traditional Chinese and modern Western Combine traditional Chinese and modern Western
medicinemedicine Emphasized discovery of new drugs from traditional Emphasized discovery of new drugs from traditional
Chinese medicineChinese medicine Drugs should be safe with few adverse effectsDrugs should be safe with few adverse effects Drugs should be highly effective with rapid onset of Drugs should be highly effective with rapid onset of
actionaction Administered infrequently for preventionAdministered infrequently for prevention
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Two Approaches UsedTwo Approaches Used
Copy Western drugs and synthesize new derivativesCopy 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 Investigate anti-malarial drugs frequently used in traditional Chinese medicine and folklore medicinetraditional 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
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Synthesis of New DrugsSynthesis of New Drugs
Take existing drugs and make new family membersTake existing drugs and make new family members
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ArtemisiaArtemisia annua Lannua L
Qinghao grows in south and north ChinaQinghao grows in south and north China 168 BC - "Fifty-two Prescriptions” 168 BC - "Fifty-two Prescriptions”
from Han Dynasty Tombsfrom Han Dynasty Tombs 340 AD - antimalarial application was 340 AD - antimalarial application was
first described in first described in Zhou Hou Be Ji Fang Zhou Hou Be Ji Fang ("Handy Therapies for Emergencies")("Handy Therapies for Emergencies")
Crushed plants and crude plant extracts Crushed plants and crude plant extracts were used by Chinese farmers in the were used by Chinese farmers in the 1950s 1950s and 1960s to treat malariaand 1960s to treat malaria
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Beijing Institute of Materia MedicaBeijing Institute of Materia Medica
1970 – scientists reviewed ancient and current 1970 – scientists reviewed ancient and current records and identified 808 anti-malaria candidatesrecords and identified 808 anti-malaria candidates
~100 candidates tested at the Military Academy of ~100 candidates tested at the Military Academy of Sciences in a mouse model of malariaSciences in a mouse model of malaria
Mouse model transferred to BeijingMouse model transferred to Beijing Soak Qinghao in water, compress the plant and drink Soak Qinghao in water, compress the plant and drink
the juice to treat malariathe juice to treat malaria Replaced ethanol extraction with ether extraction Replaced ethanol extraction with ether extraction
(lower boiling point)(lower boiling point)
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Cooler ExtractionCooler Extraction
Success rate with ether extraction material was 100% Success rate with ether extraction material was 100% cure rate in mice with P bergheicure rate in mice with P berghei
The neutral portion of the extract was identified as The neutral portion of the extract was identified as containing the active elementcontaining the active element
Tested in animals and a few healthy volunteers and Tested in animals and a few healthy volunteers and deemed safedeemed safe
1972 – tested in 9 P vivax patients and 11 P 1972 – tested in 9 P vivax patients and 11 P falciparium patients, failed in 2 P falciparium patientsfalciparium patients, failed in 2 P falciparium patients
Project head office requested purified active elementProject head office requested purified active element
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Clinical FailureClinical Failure
Purified extract from Beijing Institute showed cardiac Purified extract from Beijing Institute showed cardiac toxicity in animalstoxicity in animals
Tested on 3 researchers and did not show toxicityTested on 3 researchers and did not show toxicity Field tested in 8 patients, 6 had normalized Field tested in 8 patients, 6 had normalized
temperature, but 2 patients had cardiac toxicity, and 2 temperature, but 2 patients had cardiac toxicity, and 2 showed no signs of cureshowed no signs of cure
Results not shared with project head officeResults not shared with project head office
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Relay RaceRelay Race
1972 – Work at Beijing Institute duplicated in 1972 – Work at Beijing Institute duplicated in Shandong and Yunnan provinces using their local Shandong and Yunnan provinces using their local artemisia plantsartemisia plants
Both groups were able to make purified artemisininBoth groups were able to make purified artemisinin Monomer isolated at Shandong Institute of Chinese Monomer isolated at Shandong Institute of Chinese
Materia Medica by Wei Zhenxing did not show Materia Medica by Wei Zhenxing did not show cardiac toxicitycardiac toxicity
Luo Zeyuan from the Yunnan Institute of Materia Luo Zeyuan from the Yunnan Institute of Materia Medica got active isomers from ether extractMedica got active isomers from ether extract
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Progress in YunnanProgress in Yunnan
Yunnan group found their extract cleared P berghei Yunnan group found their extract cleared P berghei rapidly from mice and did not effect heart, liver, or rapidly from mice and did not effect heart, liver, or kidneyskidneys
They identified a variant of Artemisia annua that had They identified a variant of Artemisia annua that had the highest amounts of artemisinin from local plantsthe highest amounts of artemisinin from local plants
They identified the Youyang area in the Sichuan They identified the Youyang area in the Sichuan Province as having plants with 10-15 the Province as having plants with 10-15 the concentration of artemisininconcentration of artemisinin
Material was provided to other research institutionsMaterial was provided to other research institutions
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1974 – Clinical Success1974 – Clinical Success
Group at Beijing could not produce purified materialGroup at Beijing could not produce purified material Shandong group compared crude with purified Shandong group compared crude with purified
artemisinin extracts in 26 patients with P vivaxartemisinin extracts in 26 patients with P vivax All patients were cleared of parasites and no evidence All patients were cleared of parasites and no evidence
of cardiac toxicity was seenof cardiac toxicity was seen Most patients had recurrence of parasites within 28 Most patients had recurrence of parasites within 28
daysdays Tried combining artemisinin with prophylactic tablet Tried combining artemisinin with prophylactic tablet
to prevent recurrenceto prevent recurrence
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1974 – Clinical Success Pt 21974 – Clinical Success Pt 2
Material from the Yunnan group was used to treat P Material from the Yunnan group was used to treat P falciparumfalciparum
The first 3 patients responded very quickly to The first 3 patients responded very quickly to artemisinin; faster than existing therapiesartemisinin; faster than existing therapies
14 P falciparum patients including 3 with malignant 14 P falciparum patients including 3 with malignant malaria were treated and 12 showed clearance of the malaria were treated and 12 showed clearance of the parasites by 20 hoursparasites by 20 hours
Parasite development was arrested at the tiny-ring Parasite development was arrested at the tiny-ring form stageform stage
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Findings Made PublicFindings Made Public
1972 Artemisia annua (sweet wormwood) extract 1972 Artemisia annua (sweet wormwood) extract shown to cure malariashown to cure malaria
Several groups of scientists at different institutions Several groups of scientists at different institutions needed to determine structure of active ingredientneeded to determine structure of active ingredient
1977 first Chinese paper describing Qinghaosu but 1977 first Chinese paper describing Qinghaosu but omitting antimalarial actionomitting antimalarial action
1982 first paper in English and available outside of 1982 first paper in English and available outside of ChinaChina
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Artemisinin and DerivativesArtemisinin and Derivatives
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Results of Project 523Results of Project 523
Chemical compounds were developed for the acute Chemical compounds were developed for the acute and emergency treatment of malaria, prevention of and emergency treatment of malaria, prevention of malaria, and eradication of malariamalaria, and eradication of malaria
More than 10,000 chemicals synthesized, 40,000 More than 10,000 chemicals synthesized, 40,000 chemical samples were screened, 1,000 possible chemical samples were screened, 1,000 possible active substances identified, 38 underwent preclinical active substances identified, 38 underwent preclinical screening, 29 approved for clinical trialsscreening, 29 approved for clinical trials
14 drugs passed all testing and were used14 drugs passed all testing and were used
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1979 Discovery of Qinghaosu Award1979 Discovery of Qinghaosu Award
Ministry of Health nominated 6 institutionsMinistry of Health nominated 6 institutions Academy of Chinese Traditional Medicine; Ministry of HealthAcademy of Chinese Traditional Medicine; Ministry of Health Shandong Institute of Chinese Traditional MedicineShandong Institute of Chinese Traditional Medicine Yunnan Institute of Materia MedicaYunnan Institute of Materia Medica Institute of Biophysics of the Chinese Academy of SciencesInstitute of Biophysics of the Chinese Academy of Sciences Shanghai Institute of Organic Chemistry of the Chinese Shanghai Institute of Organic Chemistry of the Chinese
Academy of SciencesAcademy of Sciences Guangzhou College of Traditional Chinese MedicineGuangzhou College of Traditional Chinese Medicine
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Were the Chinese First?Were the Chinese First?
Milutin StefanovicMilutin Stefanovic
1972, first publication1972, first publication
describing artemisinindescribing artemisinin
No mention of malariaNo mention of malaria
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Structure and MechanismStructure and Mechanism
1993 - malaria parasite survives in its host by 1993 - malaria parasite survives in its host by consuming approximately 25% of the hemoglobin in consuming approximately 25% of the hemoglobin in the host's red blood cells and stores ironthe host's red blood cells and stores iron
Artemisinin comes in contact with the iron converting Artemisinin comes in contact with the iron converting it into a toxic chemical, releasing a free radical that it into a toxic chemical, releasing a free radical that destroys the parasitedestroys the parasite
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Artemisinin ActionsArtemisinin Actions
Kills malaria parasitesKills malaria parasites- Heme-derived iron reacts with artemisinin in food vacuole
to produce lethal amounts of ROS
Kills cancer cellsKills cancer cells- Free iron reacts with artemisinin in lysosomes to produce
ROS
- Death by activated programmed cell death pathways (PCD)
Inhibits angiogenesisInhibits angiogenesis
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Cancer Cells and IronCancer Cells and Iron
All cells need iron for growthAll cells need iron for growth Normal cells control iron uptake by neg feedbackNormal cells control iron uptake by neg feedback Cancer cells accumulate iron beyond needsCancer cells accumulate iron beyond needs Iron becomes a target for anticancer therapiesIron 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
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Iron and CancerIron and Cancer
Cancer cell lysosomes contain higher levels of redox Cancer cell lysosomes contain higher levels of redox active ironactive iron- Nat Rev Cancer. 2005 Nov;5(11):886-97
Breast cancer cells have higher transferrin receptor Breast cancer cells have higher transferrin receptor expression levels and reduced ferroportin levelsexpression levels and reduced ferroportin levels- Sci Transl Med. 2010 Aug 4;2(43):43ra56. doi:
10.1126/scisignal.3001127
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LysosomesLysosomes
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Free Radical ReactionsFree Radical Reactions
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Creation of ROS is Iron DependentCreation of ROS is Iron Dependent
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+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
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Programmed Cell Death PathwaysProgrammed Cell Death Pathways
Lysosomes accumulate free iron and artemisininLysosomes accumulate free iron and artemisinin ROS from artemisinin causes membrane ROS from artemisinin causes membrane
permeabilizationpermeabilization Hydrolytic enzymes are released from the lysosome Hydrolytic enzymes are released from the lysosome
lumen into the cytosollumen into the cytosol Cathepsins activate pro-apoptotic proteinsCathepsins activate pro-apoptotic proteins BH3 interacting-domain death agonist and caspase 8 BH3 interacting-domain death agonist and caspase 8
activate mitochondrial outer membrane activate mitochondrial outer membrane permeabilization and apoptosispermeabilization and apoptosis
J Biol Chem. 2011 Feb 25;286(8):6587-601J Biol Chem. 2011 Feb 25;286(8):6587-601
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Artemisinin Multiple Anticancer Effects
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Firestone GL, Sundar SN. Anticancer activities of artemisinin and its bioactive derivatives. Expert Rev Mol Med. 2009 Oct 30;11:e32.
Multi-targeted, Selectively Promiscuous™
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NCI In Vitro Cancer Screening
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Trioxane Dimers exhibit cancer cell cytotoxicity superior to gold standard chemotherapy in NCI screening panel
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Effect of Art Derivative Effect of Art Derivative in vivoin vivo
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Questions?Questions?
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