Elise Saunier Bionovo, Inc. NAMS 2009 Identification of Tissue-Selective ER  agonists from Plants

Endocrine changes during the menopausal transition, rather than the aging process, are related to changes in body weight and fat distribution. In age-matched women: higher percentage of fat tissue in peri- and post- menopausal women (p<0.001) compare to premenopausal and postmenopausal + HRT women Perimenopausal and postmenopausal women show a shift to a central, android fat distribution. Regional distribution shows higher fat distribution in the trunk (p<0.001) and lower fat in the legs (p<0.05) for the postmenopausal women Overweight and obesity are associated with increased morbidity and mortality, with central distribution of body fat recognized as an independent predictor of cardiovascular disease in women Can be counteracted by HRT Weight Gain during Menopause 2

Fat redistribution during menopause *p<0.05 (vs. premenopause value); **p<0.01 (vs premenopause value) 3 Genazzani & Gambacciani studied the modifications of body weight and fat distribution from 2529 women

Mammary Gland Uterus Breast Cancer Endometrial Cancer Adipose Tissue Decreased Obesity Decreased Metabolic Syndrome ER  Estrogen in HT Estrogen in HT ER  Estrogen in HT Estrogens in HT are not Tissue Selective 4

Adipose Tissue Decreased Obesity Decreased Metabolic Syndrome ER  Tissue Selective ER α -estrogens 5 Tissue Selective ER α Agonist Tissue Selective ER α Agonist Mammary Gland Uterus Breast Cancer Endometrial Cancer ER  Tissue Selective ER α Agonist SAFERSAFER

To identify tissue selective ER  agonists in plants  Methods 1. Screened over 50 plant extracts for activation of an ERE reporter gene 2. Selected two plant extracts for testing in mice by gavage: #3 from Radix Glycyrrhiza #39 from Radix Pueraria 3.Ovariectomized mice were pre-fed for 50 days with HFD before started treatment with E2, #3 and #39 for 42 days 4.Measured weights of body, abdominal fat, MG and uterus 5.Determine gene expression profiles in abdominal fat, MG and uterus Goal 6

7 Plant extract #3 and #39 induce ERE-Luciferase reporter expression in vitro Plant Extracts have ERα Activity *p<0.05 vs. control ERE-ER α tk-Luc EC50 = 16 μg/ml EC50 = 5.5 μg/ml Binding assay

8 Consumption of a high fat diet increases body weight in mice E2 administration reverses the weight gain Plant extract #3 and #39 also reverse weight gain Decrease in Body Weight by the Plant Extracts *p<0.05 vs. control

9 #3 and #39 mimic estradiol’s effect on the abdominal fat Plant Estrogens Decrease Abdominal Fat Abdominal Fat *p<0.05 vs. control

10 Estradiol treatment induces a massive increase in uterus weight #3 and #39 do not mimic estradiol’s effect on the uterus Uterus Weight is not Increased by Plant Extracts UterusUterus *p<0.05 vs. control

11 Estradiol reduces the mammary gland weight but induces development of ducts (elongation, branching) Mammary gland is not affected by the Plant Extracts Mammary Gland control39 3 E2 While the weight of the mammary gland is diminished by the plant extracts, the ducts do not develop. *p<0.05 vs. control

12 #3 and #39 mimic estradiol’s effect on the abdominal fat #3 and #39 DO NOT mimic estradiol’s effect on the uterus Plant Extracts have Tissue Specific Estrogenic Effects Abdominal Fat UterusUterus Mammary Gland To understand molecular basis for tissue specific effects gene expression profile by microarray

13 Compared to estradiol, #3 and #39 regulate a comparable number of genes in the abdominal fat Gene regulation by the Plant Extracts is Tissue Specific Mammary Gland UterusUterus Abdominal Fat However, little or no genes are induced or repressed in the Uterus and MG, when compare to E2 treatment.

E2 # # Mammary Gland E2 #39 #3 Tissue Specific Activity of Plant Estrogens 1980 # E2#39 UterusUterus Abdominal Fat E2, #3 and 39 regulate genes in the fat E2 is the major gene regulator in the uterus and the mammary gland 158 Inflammatory genes repressed by E2, #3 and #39

Mammary Gland Uterus Breast Cancer Endometrial Cancer Adipose Tissue Decreased Obesity Decreased Metabolic Syndrome ER  Estrogen in HT Estrogen in HT ER  Estrogen in HT Estrogens in HT are not Tissue Selective 15 E2 669 Fat MG Uterus modifies gene expression in all 3 tissues

Adipose Tissue Decreased Obesity Decreased Metabolic Syndrome ER  Tissue Selective ER α -estrogens 16 Tissue Selective ER α Agonist Tissue Selective ER α Agonist #3 MG Fat Uterus # MG Fat Uterus Plant Estrogens #3 and #39 have one major target abdominal FAT = GOOD NEWS Much less or no effect on MG and Uterus

Plant extracts mimic the beneficial effect of estradiol on body weight and repression of inflammatory genesPlant extracts mimic the beneficial effect of estradiol on body weight and repression of inflammatory genes Plant extracts have little or no effect on the mammary gland and uterusPlant extracts have little or no effect on the mammary gland and uterus Animal data support the goal to use these plants in clinical trials to safely treat obesity in postmenopausal womenAnimal data support the goal to use these plants in clinical trials to safely treat obesity in postmenopausal women These studies demonstrate that it is possible to discover tissue selective ER  agonists. Might have a more favorable clinical profile than estrogens used currently in classic HT for menopausal symptomsThese studies demonstrate that it is possible to discover tissue selective ER  agonists. Might have a more favorable clinical profile than estrogens used currently in classic HT for menopausal symptoms Summary