Phase 1 Clinical Trial:

1. World Congress of Chinese Medicine 2006 Shangri-La, Hong Kong 25 November 2006 Phase 1 Clinical Trial: Pharmacokinetics/Pharmacodynamics of estrogenic compounds from Epimedium(淫羊藿) Professor Yong Eu Leong Obstetrics & Gynecology National University of Singapore

2. Estrogens are necessary for normal female sexual health Estrogens 14 28 1 Day of menses

3. Large clinical study From US NIH (Women Health Initiative) recommends NO role for long term hormone replacement therapy in menopausal women • Associated with increased hazard ratios (Rousouw, 2002) for: • Coronary heart disease (29% increase), • Stroke (20% increase) • Breast cancer (26% increase), • Pulmonary embolism (13% increase )

4. hPR hAR 甾体激素受体家族 TAD DBD LBD hER a hER b hGR hMR

5. Differential recruitment of co-activators and co-repressors to the transcription complex is the basis of Selective Hormone Receptor Modulator activity Steroid-responsive cell nucleus Gene transcription & protein production HRE Steroid-responsive gene

6. Sensitive and specific bioassays based on permanently transfected human cell lines measure summated activity of Steroid receptors nucleus Gene transcription & luciferase production HRE Steroid-responsive gene

7. Herbal extract Estrogenic activity (fold increase over vehicle) Activity compared to estradiol (1nM) (%) Vehicle (EtOH) 1.0 - Estradiol 1nM 25.0 100 A. sinensis (Dang gui)当归 0.7 2.6 P. kingianum (Huang jing)黄精 0.8 2.7 C. orchioides (Xian mao)仙茅 0.9 2.9 M. officinalis (Ba ji tian)巴戟天 1.0 3.9 R. astragali (Tong ji li)潼蒺藜 1.1 4.2 L. chinense (Gou qi zi)枸杞子 1.3 5 C. chinensis (Tu si zi)菟丝子 1.6 7.6 D. opposita (Shan yao)山药 2.8 8 A. asphodeloides (Zhi mu)知母 4.2 18 P. corylifoloa (Bu gu zi)补骨子 5.9 23.1 Epimedium （Yin Yang Huo)淫羊藿 13.7 54.7 Screening of herbs for estrogenic activity using ERa and ERE-LUC reporter gene in Hela cells

8. Epimedium agonistic activity specific on Estrogen Receptor

9. Epimedium species （淫羊藿） • Yang tonic Shen Nong Ben Cao Jing (ca 200BC) • “ to reinforce the kidney yang, strengthens bones and tendons..” • Dried aerial parts are boiled with water or steeped in alcohol • Used as part of complicated formulations with other herbs • In-vitro studies indicate pharmacological effects on immune, CVS, CNS, respiratory, skeletal systems. E. grandiflorum

10. Isolation and elucidation of bioactive compounds using bioassay-guided fractionation ES crude extract Polyamide MeOH HexaneDCM EtOAc MeOH H20 MeOH Diol C18 Bioassay HPLC HPLC MS / NMR

11. Genistein (Isoflavone) C26 H29 N O Tamoxifen Why do we need another phyto-estrogen?Novel prenyl moiety with interesting SERM propertiesProprietary bioactive extractbased on content ofnovel Breviflavone B Breviflavone B (flavone) Yong et al, USP No: 2003/0170292 A1

12. Epimedium extract stops growth of breast cancer cells by down-regulating estrogen receptor protein Tamoxifen Vehicle Epimedium & Tamoxifen Epimedium1mg/ml Epimedium10mg/ml Epimedium100mg/ml

13. Taxonomic authentication • Traditional herbalists do not differentiate among Epimedium species, but rather mix species together as Herba Epimedii • Variable number of species: 20-50 • Not cultivated, but harvested in the wild • Materials on sale not taxonomically defined but are mixtures of many species • Taxonomically identified Epimedium species from different locations in China by Dr Guo Bao-Lin, Institute for Medicinal Plant Development (IMPLAD), Chinese Academy of Sciences, Beijing

14. Fluorescent Amplified Fragment Length Polymorphisms (AFLP) • Based on DNA extracted from authenticated samples • 16 selective primer combinations • AFLP fingerprints were visualized on a polyacrylamide gel using the ABI PRISM 377 DNA sequencer and the data processed using Genotype software. • Data from 194 AFLP bands converted into a phylogenetic tree

16. Taxonomic classification of Epimedium Species (Boot-strap analysis of Phylogenetic tree constructed from AFLP genetic polymorphisms )

17. Estrogenic activity of main Epimedium species

18. Concentrations of minor flavonoid aglycones correlate with estrogenic activity

19. Preclinical Pharmacology of complex mixtures • Additive, synergistic and/or antagonistic effects • Platform technology required that can measure summated pharmacokinetic/pharmacodynamic parameters • PK/PD data essential for dosing and formulation

20. Pre-clinical Pharmacokinetics/Pharmacodynamics using estrogen-responsive bioassays • Administration of Control (sesame oil), estradiol (10mg/ml), Epimedium extract Test for estrogenic activity with the bioassay Obtain serum and precipitated proteins Collection of blood at specific time points.

21. Pharmacokinetics/Pharmacodynamics of Epimedium in Sprague–Dawley rats using ERa reporter gene assays

22. Pharmacokinetic/Pharmacodynamic modeling of Estrogen activity of Epimedium in Sprague-Dawley rats *p < 0.05, **p < 0.01,***p < 0.001; Student’s t test vs placebo

23. HUMAN CLINICAL TRIAL Estrogen Receptor Activity in Serum after Oral Ingestion of Estradiol Valerate and a Decoction of Epimedium pubescens

24. Washout period Baseline Epimedium E2V Randomized Washout period Baseline E2V Epimedium Day 1 2 3 4 9 10 11 CLINICAL TRIAL Pharmacokinetic/Pharmacodynamic study 1) validate bioassays against gold standard LC-MS/MS2) Evaluate estrogenic activity of Epimedium decoction • Outcome parameters: • Concentration of estrone (E1) in serum • Concentration of estradiol (E2) in serum • Estrogenic bioactivity in serum as measured by ERa and ERb reporter gene bioassays and MCF-7 cell proliferation

25. Standard Pharmacokinetics:Measurement of estrogens (E2 and E1) by LC-MS/MS after administration of pro-drug Estradiol Valerate (E2V)

26. ER-alpha Driven Cell-based Bioassay Objectives • 96 well plate, 10,000 cells/well. • 20% stripped pooled human serum containg 8 pM of E2 determined by LC-MS. • 6 unknown E2 standard samples for quality control and calculating intra- and inter CVs.

27. Novel Combined Pharmacokinetics /Pharmacokinetics:Measurement of estrogenic effects by ERa-driven bioassays after administration of pro-drug Estradiol Valerate (E2V)

28. Validation of ERa bioassay: Good correlations with “gold standard”LC-MS-MS measurement of estradiol (E2)

29. Novel method for measuring combined Pharmacokinetics /Pharmacokinetics :Two peaks of Epimedium action

30. Icarin (Inactive) Icaritin (active) Hydrolysis in GIT sugar + Epimedium exert delayed estrogenic action through metabolism of a pro-drug. Early action (4 hours) Intestinal metabolism of major flavonoid sugars Results in bioactive new flavonoid aglycones late action (24 hours) Action of late appearing novel metabolites ?

31. Novel Combined Pharmacokinetics /Pharmacokinetics:Measurement of estrogenic effects by ERa-driven bioassays after administration of pro-drug Estradiol Valerate and Epimedium decoction

32. CONCLUSION: Randomized control PK/PD study of a taxonomically, genetically, chemically and biologically standardized Epimedium decoction donePharmacokinetics of Estrone and Estradiol in serum of Singapore males after E2V follow that reported for post-menopausal womenHigh correlation between estrogen receptor-alpha and MCF-7 bioactivity and estradiol concentrations and the assay can measure summated E1/E2 activityEstrogen receptor activity can be detected in Serum after Oral Ingestion of a Decoction of Epimedium pubescens and Epimedium may have value for menopausal hormone replacement therapy Mechanisms underlying novel PK/PD patterns detected by ERa bioassay are under investigation.

33. Acknowledgements Yong Lab: Shen Ping, Gong YH, Yap Sook Peng, Wong Shih Peng, Liu MeiHui, Li Jun, Guo Bao Lin, IMPLAD, Chinese Academy of Medical Sciences, Beijing Hong Yan, Temasek Laboratories, Singapore Stephen Wise, Lilly NUS-Center for Clinical Pharmacology, Singapore Lawrence Lee, Johns Hopkins University, USA FUNDING NUS academic research fund NHG NMRC NIH NCCAM, USA

34. Species from this genetic branch had highest estrogenic activity

35. Taxonomic classification of Epimedium Species (Boot-strap analysis of Phylogenetic tree constructed from AFLP genetic polymorphisms )

36. Concentrations of minor flavonoid aglycones correlate with estrogenic activity

37. A1 A1 A2 A2 SL SL FG FG I I FH FH ZH ZH L1 L1 L2 L2 B1 B1 B3 B3 B2 B2 B4 B4 II P8 P8 P3 P3 P2 P2 P4 P4 P5 P5 P6 P6 P7 P7 S3 S3 LT LT ME1 ME1 ME2 ME2 PA PA M1 M1 M2 M2 WU WU P1 P1 variable trunk ZB ZB DA DA DO DO S2 S2 E koreanum trunk S1 S1 K4 K4 K2 K2 K1 K1 K3 K3 0.70 0.70 0.75 0.75 0.80 0.80 0.85 0.85 0.90 0.90 Similarity Index Similarity Index Correlations between genetic relatedness, minor flavonoid aglycones and estrogenic activity

38. Standardization of botanical raw materials Bioresponse characterization important in addition to taxonomic, genetic and chemical profiling

39. Preclinical Pharmacology of complex mixtures • Additive, synergistic and/or antagonistic effects • Platform technology required that can measure summated pharmacokinetic/pharmacodynamic parameters • PK/PD data essential for dosing and formulation

40. Pre-clinical Pharmacokinetics/Pharmacodynamics using estrogen-responsive bioassays • Administration of Control (sesame oil), estradiol (10mg/ml), Epimedium extract Test for estrogenic activity with the bioassay Obtain serum and precipitated proteins Collection of blood at specific time points.

41. Pharmacokinetics/Pharmacodynamics of Epimedium in Sprague–Dawley rats ERa reporter gene bioassay Breast cancer cells growth

42. Pharmacokinetic/Pharmacodynamic modeling of Estrogen activity of Epimedium in animal models: Area under the Curve (AUC) studies indicate differences between oral and injected samples *p < 0.05, **p < 0.01,***p < 0.001; Student’s t test vs placebo

43. HUMAN CLINICAL TRIAL Selective Estrogen Receptor Modulator (SERM) Activity in Serum after Oral Ingestion of Estradiol Valerate and a Decoction of Epimedium pubescens

44. Objectives Objectives • Validation of estrogen sensitive cell-based bioassays after oral ingestion of estradiol valerate. • To assess in an exploratory manner the estrogenic activity in serum after oral ingestion of a decoction of Epimedium pubescens.

45. Washout period Baseline Epimedium E2V Randomized Washout period Baseline E2V Epimedium Day 1 2 3 4 9 10 11 CLINICAL TRIAL Pharmacokinetic/Pharmacodynamic study design • Outcome parameters: • Concentration of estrone (E1) in serum • Concentration of estradiol (E2) in serum • Estrogenic bioactivity in serum as measured by ERa and ERb reporter gene bioassays and MCF-7 cell proliferation

46. E2 and E1 PK summary

47. Summated E2/E1 activity • ER-alpha correlated well with E2 and E1, adding E1 into ER-alpha / E2 regression model improved R to 0.83, LR test p<0.0001, E1/E2 coefficient ratio 0.13 • ER-beta correlated better with E1, adding E2 to the model did not improve R significantly, E1/E2 coefficient ratio 2.3 • MCF-7 correlated with E2, adding E1 into the model did not improve model at all • Saturation seen with ER-beta and MCF-7

48. ER-beta correlations

49. MCF-7 correlations

50. Individualized activity • Stratifying by subject ID improved correlations significantly for ER-alpha, ER-beta and MCF-7 • Further stratifying by treatment improved correlations significantly for ER-alpha • Suggests assay picks up intra-individual differences in pharmacodynamic responses to E2 and E1 concentrations