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Identification of Tissue-Selective ERa
agonists from Plants
Elise Saunier
Bionovo, Inc.
NAMS 2009
Weight Gain during Menopause
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 postmenopausal 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
2
Fat redistribution during menopause
Genazzani & Gambacciani studied the modifications of body weight and fat
distribution from 2529 women
*p<0.05 (vs. premenopause value); **p<0.01 (vs premenopause value)
3
Estrogens in HT are not Tissue Selective
Estrogen
in HT
Estrogen
in HT
Estrogen
in HT
ERa
ERa
Adipose Tissue
Mammary Gland
Uterus
Decreased Obesity
Decreased Metabolic Syndrome
Breast Cancer
Endometrial Cancer
4
Tissue Selective ERα-estrogens
Tissue Selective
ERα Agonist
Tissue Selective
ERα Agonist
Tissue Selective
ERα Agonist
ERa
ERa
Adipose Tissue
Mammary Gland
Uterus
Decreased Obesity
Decreased Metabolic Syndrome
Breast Cancer
Endometrial Cancer
SAFER
5
Goal
To identify tissue selective ERa 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
6
Plant Extracts have ERα Activity
Plant extract #3 and #39 induce ERE-Luciferase reporter expression in vitro
4
ERE-ERα tk-Luc
RLU
3
*
*
2
* *
*
*
* *
* *
* *
Control
E2
#3
#39
1
0
10-8 0.2 0.5
1
2.5
10
20 100 0.2 0.5
EC50 = 16 μg/ml
Binding assay
2.5
10
20 100
EC50 = 5.5 μg/ml
5.8
5.8
5.6
5.6
5.4
5.4
5.2
5.2
5.0
5.0
4.8
4.8
0.1
1
10
100
concentration (ug/ml)
*p<0.05 vs. control
1
7
1000
0.1
1
10
100
concentration (ug/ml)
1000
Decrease in Body Weight by the Plant Extracts
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
% weight change
10
0
vehicle
-10
-20
*
E2
*
*
*
*
*
3
**
*
*
*
-30
7
14
21
28
days in HFD
*p<0.05 vs. control
8
**
*
35
*
**
42
39
Plant Estrogens Decrease Abdominal Fat
#3 and #39 mimic estradiol’s effect on the abdominal fat
Abdominal
AbdominalFat
fat
weight (g)
2.5
2
* *
1.5
39
*
0
*p<0.05 vs. control
E2
3
1
0.5
vehicle
9
Uterus Weight is not Increased by Plant Extracts
Estradiol treatment induces a massive increase in uterus weight
#3 and #39 do not mimic estradiol’s effect on the uterus
Uterus
Uterus
weight (mg)
300
*
vehicle
200
E2
3
100
39
0
*p<0.05 vs. control
10
Mammary gland is not affected by the Plant
Extracts
Estradiol reduces the mammary gland weight but induces
development of ducts (elongation, branching)
While the weight of the mammary gland is diminished by the plant
extracts, the ducts do not develop.
MammaryGland
gland
Mammary
control
E2
3
39
weight (g)
2
1.5
*
1
0.5
*
*
vehicle
E2
3
39
0
*p<0.05 vs. control
11
Plant Extracts have Tissue Specific
Estrogenic Effects
#3 and #39 mimic estradiol’s effect on the abdominal
Abdominal fat
Fat
#3 and #39 DO NOT mimic estradiol’s effect on the uterus
Uterus
Mammary Gland
To understand molecular basis for tissue specific effects
gene expression profile by microarray
12
Gene regulation by the Plant Extracts is Tissue
Specific
Compared to estradiol, #3 and #39 regulate a comparable number of genes in
the abdominal fat
800
400
Abdominal Fat
0
-400
-800
E2
#3
#39
However, little or no genes are induced or repressed in the Uterus and MG,
when compare to E2 treatment.
Uterus
Mammary Gland
1500
1000
500
0
-500
-1000
-1500
400
200
0
-200
-400
E2
#3
E2
#39
13
#3
#39
Tissue Specific Activity of Plant Estrogens
Abdominal Fat
E2, #3 and 39 regulate
genes in the fat
#39
E2
349
45
158 Inflammatory
genes repressed by
E2, #3 and #39
847
239
413
48
338
Uterus
#3
Mammary Gland
E2
#39
E2
167
1980
304
81 112
11
25
E2 is the major gene
regulator in the uterus
and the mammary gland
#3
14
#39
469
1012
0
#3
Estrogens in HT are not Tissue Selective
Estrogen
in HT
Estrogen
in HT
Estrogen
in HT
ERa
E2
ERa
Uterus
Fat
669
338
99
Adipose Tissue
1749
73
79
228
Mammary Gland
Uterus
MG
modifies gene expression in all
3 tissues
Decreased Obesity
Decreased Metabolic Syndrome
Breast Cancer
Endometrial Cancer
15
Tissue Selective ERα-estrogens
Tissue Selective
ERα Agonist
Tissue Selective
ERα Agonist
Fat
Uterus
848
Plant
Estrogens
39
190
#3
ERa
0
MG
#3 and #39 have one major target
abdominal FAT = GOOD NEWS
Adipose Tissue
Much less or no effect on MG and
Uterus
Uterus
Fat
734
Decreased Obesity
Decreased Metabolic Syndrome
#39
16
306
358
6
16
MG
Summary
•Plant 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 uterus
•Animal 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 ERa agonists. Might have a
more favorable clinical profile than estrogens used
currently in classic HT for menopausal symptoms