Artemisinin- an innovative cornerstone for anti- malaria therapy 성균관대학교 약학대학 천연물약품화학연구실 석박통합 7 기 김충섭

About malaria Market development through supply chain enlargement of A. annua Ⅰ III Contents From Artemisia annua to artemisinin and Coartem ® Ⅱ Market development through supply chain enlargement of artemisinin Ⅳ

About malaria Ⅰ 1.Symptoms & Infections 2.Epidemiology & Economic effects 3.Research 4.Prevention 5.Treatment

센터의 필요성 Ⅰ. About malaria About malaria Malaria Symptoms - Flu-like symptoms - High-fever, intermittent fever - Coma, death Infections - Blood parasite Plasmodium - Anopheles mosquitoes Epidemiology - Often in Africa million infection/year - 1 children/30 sec dies Economic effects - USD 12 billion/year - No economic development - No tourism Research - About infection cycle and vector control - Four Nobel prizes (1902, 04, 27, 48) Prevention - Insecticide-treated bed nets - Indoor Residual Spraying - Treat mosquitoes’ breeding areas with insecticides Treatment - Artemisinin-based Combination Therapies (ACT) - ‘Roll Back Malaria’ (RBM) by WHO, IRBD, UNDP, and UNICEF - Global Fund to Fight AIDS, Tuberculosis and Malaria (GFATM)

From Artemisia annua to artemisinin and Coartem ® 1.Antimalarial activity of Qinghao (A. annua L.) 2.Artemisinin from A. annua L. 3.Congeners of artemisinin 4.Development of ACT – Coartem ® Ⅱ

Ⅱ. From Artemisia annua to artemisinin and Coartem 1. Antimalarial activity of Qinghao (A. annua L.) (1950s) Drug resistance to chloroquine (1960s) Malaria out of control (Vietnam War) Soldiers infected (1970s) 40,000 compounds and extracts screened in China Drug resistance Screening Qinghao (Artemisia annua L., 개똥쑥 ) Hemorrhoids (168 BC) Hemorrhoids (168 BC) Intermittent fever (1596 AD) Intermittent fever (1596 AD) Fever (300s AD) Fever (300s AD) Remedies for Qinghao (Yin) + Internal heat (Yang) Qinghao (Yin) + Internal heat (Yang) Sulphadoxine- pyrimethamine, Mefloquine developed

Artemisinin Arteannuin Qinghaosu Artemisinin Arteannuin Qinghaosu Ⅱ. From Artemisia annua to artemisinin and Coartem 2. Artemisinin from A. annua L. Amounts - ↑Leaves, small green stems, buds, flowers and seeds - ↓Stems, roots Amounts - ↑Leaves, small green stems, buds, flowers and seeds - ↓Stems, roots Sesquiterpene s Artemisia annua L. Structure elucidation - NMR, MS and IR - Sesquiterpene lactone - C 15 H 22 O 5 - X-ray crystallography - 1,2,4-trioxane ring (activity) Structure elucidation - NMR, MS and IR - Sesquiterpene lactone - C 15 H 22 O 5 - X-ray crystallography - 1,2,4-trioxane ring (activity) Essential oils Clinical trials - Parasite decreased 95% in 16h (chloroquine: 40h) - Effective in resistant spp. and cerebral form Clinical trials - Parasite decreased 95% in 16h (chloroquine: 40h) - Effective in resistant spp. and cerebral form

Ⅱ. From Artemisia annua to artemisinin and Coartem 2. Artemisinin from A. annua L. SERCA: sarco-endoplasmic reticulum Ca 2+ -ATPas e Nat. Chem. Biol. 3, (2007) (from hemoglobin)

3. Congeners of artemisinin Total syntheses - First reported in Not commercially viable Characteristics - Thermally stable (~150 ℃ ) - Poorly soluble in water and oil - Peroxide: essential for activity Dihydroartemisinin - More potent - Solubility in water ↓ - Stability ↓ Artemether - Soluble in oil - Available by I.M. - Short t 1/2 Arteether - Soluble in oil - Available by I.M. Sodium artesunate - Soluble in water - Available by I.V. - Short t 1/2 OZ277 (RBx11160) - More potent - Long lasting - Structurally simple - Scale-up - Reach market in 2009 (with piperaquine) Ⅱ. From Artemisia annua to artemisinin and Coartem ®

3. Congeners of artemisinin Total syntheses - First reported in Not commercially viable Characteristics - Thermally stable (~150 ℃ ) - Poorly soluble in water and oil - Peroxide: essential for activity OZ277 (RBx11160) - More potent - Long lasting - Structurally simple - Scale-up - Reach market in 2009 (with piperaquine) Artemether - Soluble in oil - Available by I.M. - Short t 1/2 Sodium artesunate - Soluble in water - Available by I.V. - Short t 1/2 - Approved as new malarial drugs in China ( ) - Added to Essential Medicines List (1997, 2000) - Approved as new malarial drugs in China ( ) - Added to Essential Medicines List (1997, 2000) Ⅱ. From Artemisia annua to artemisinin and Coartem ®

4. Development of ACT – Coartem ® Drug resistance to available agents Combination therapy - Successful cure rates in other cases Combination therapy - Successful cure rates in other cases Combination in antimalaria therapy - Chlorproguanil-dapsone (LapDap) - Dihydroartemisinin/piperaquine (DHA/PPQ) - Artesunate-amodiaquine (AS-AQ) - Artesunate-mefloquine (AS-S/P) - Artesunate/ayronaridine (AS/PRN) Combination in antimalaria therapy - Chlorproguanil-dapsone (LapDap) - Dihydroartemisinin/piperaquine (DHA/PPQ) - Artesunate-amodiaquine (AS-AQ) - Artesunate-mefloquine (AS-S/P) - Artesunate/ayronaridine (AS/PRN) Coartem ® (Artemether/lumefantrine) Artemether Lumefantrine - Only pre-qualified & fixed dose ACT - Cure rates 95 % - Effective on multidrug resistance - Registered in 81 countries million treatments

Market development through supply chain enlargement of A. annua 1.Major challenge in establishing a supply chain 2. Market situation for A. Annua 3.Plantation efforts to meet market demands 4.Geographical split of the supply market 5.Efforts to improve A. annua biomass Ⅲ

Ⅲ. Market development through supply chain enlargement of A. annua 1. Major challenge in establishing a supply chain Market development Time needed for season - Planting - Cultivating - Harvesting Time needed for season - Planting - Cultivating - Harvesting Major challenge Weather conditions - Meteorological impacts Damage by insects Supply and demand - Oversupply → price ↓ → business less attractive → production reduced

Ⅲ. Market development through supply chain enlargement of A. annua 2. Market situation for A. annua In 2004 Unexpected demand for artemisinin In 2004 Unexpected demand for artemisinin In 2001 Coartem ® launched In 2001 Coartem ® launched Until 2003 Sufficient amounts of A. annua Until 2003 Sufficient amounts of A. annua Whole supply chain had to built up Market complexity↑ - Harvest earlier → artemisinin content↓ - Unsuitable storage → loss of biomass - Extraction, purification x → poor quality Market complexity↑ - Harvest earlier → artemisinin content↓ - Unsuitable storage → loss of biomass - Extraction, purification x → poor quality African countries needed ACTs Exceed supply capacity

Ⅲ. Market development through supply chain enlargement of A. annua 3. Plantation efforts to meet market demands YearCoartem Artemisinin required [kg] Artemisinin planned for [kg] Amount of biomass [kg] Required area [hectares] Remark s ,00053 oversupply , , oversupply ,000 3,382,0001,230 Max. market could supply ,000124,00025,000,0009, ,000150,00030,100,00011, ,000150,00030,100,00011,000 Table 1. Demand planning for A. annua and artemisinin in 2004 Local governments Local governments Communities Mutual agreement Farmers Planning – hardly possible - How many kg of biomass may be harvested per hectare? - What would be the artemisinin content achieved with high yield seeds on larger plantations? - No experience of how the huge volumes of biomass may be dried and processed Planning – hardly possible - How many kg of biomass may be harvested per hectare? - What would be the artemisinin content achieved with high yield seeds on larger plantations? - No experience of how the huge volumes of biomass may be dried and processed

Ⅲ. Market development through supply chain enlargement of A. annua 3. Plantation efforts to meet market demands Farmers Novartis Extraction company Communities Contract Timely delivery Seeds Seminars Economic effects Biomass managers Meet market demands

Ⅲ. Market development through supply chain enlargement of A. annua 4. Geographical split of the supply market Novartis 중국 베트남 - Tanzania, Kenya, Uganda - Long experience with cultivation - Larger areas up to 200 hectares were planted - Irrigation on a large scale 동아프리카 Reduced agricultural risks

Ⅲ. Market development through supply chain enlargement of A. annua 5. Efforts to improve A. annua biomass High yield seeds (being investigated) Artemisinin content ↑ Plantation area ↓ Drying efforts ↓ Transport- ation Storage Reduced time & cost

Market development through supply chain enlargement of artemisinin 1.Capacity for extraction and purification: in Major side products (Waxes) 3.Other side products 4.Extraction and purification 2004 onwards Ⅳ

1. Capacity for extraction and purification: in 2003 Ⅳ. Market development through supply chain enlargement of artemisinin Dried leaves Hexane or Petroleum ether Reflux several times Lipophilic substances (waxes) EtOAc Charcoal & recrystallization Charcoal & recrystallization Artemisinin Solvent – recovered Silica gel – partially regenerate Without automation

2. Major side products (Waxes) Ⅳ. Market development through supply chain enlargement of artemisinin Waxes - Mixture of long- chain apolar lipids - Form a protective coating on plant leaves and fruits - In animals, algae, fungi and bacteria - Not chemically homogeneous group - Water-resistant - In crude artemisinin: aliphatic alcohols and hydrocarbons Identification - Microscope: difference between the samples with or without waxes can be observed - IR spectroscopy: quantification by calibration - GC-FID: quatification

3. Other side products Ⅳ. Market development through supply chain enlargement of artemisinin More than 150 natural products A. annua HPLC-UV-MSHPLC-UV -Extinction coefficients ( 흡광계수 )

4. Extraction and purification 2004 onwards Ⅳ. Market development through supply chain enlargement of artemisinin Analytical method (TLC-FID) - Accurate - Quickly - Results with and automated evaluation Purification method - Chromatography → crystallization. Waxes co-precipitating. Plugging during filtrations. With major related substance (dehydro-artemisinin) Synthetic production - Semi-synthesis (artemisinic acid → artemisinnin) - Cost non-competitive