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Standardization, Screening and Clinical Standardization, Screening and Clinical Evaluation of Estrogenic Evaluation of Estrogenic IsoflavonesIsoflavones in Red in Red
Clover (Clover (TrifoliumTrifolium pratensepratense) for Women) for Women’’s Healths Health
Richard B. van BreemenRichard B. van BreemenUIC/NIH Center for Botanical Dietary Supplements ResearchUIC/NIH Center for Botanical Dietary Supplements Research
Department of Medicinal Chemistry and Department of Medicinal Chemistry and PharmacognosyPharmacognosyUniversity of Illinois College of Pharmacy, Chicago, ILUniversity of Illinois College of Pharmacy, Chicago, IL
Core BAnalytical Core
Richard van BreemenDejan Nikolic
Project 1Botanical Standardization
Norman FarnsworthGuido Pauli
Project 2Bioassay Development
Judy Bolton
Project 3Metabolism & Bioavailability
Richard van Breemen
Clinical Evaluation TeamS. Geller, S. Banuvar
L. Schulman
Core AAdministration
N. Farnsworth, E. KrauseR. van Breemen, S. Hedayat
AdvisoryCommittee
UIC/NIH Center for Botanical Dietary
Supplements Research
Director, Norman R. FarnsworthCo-Director, Richard B. van Breemen
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Steps Required Prior to Clinical Assessment of Botanical Dietary Supplements
1. Acquire plant material• Verify identity; taxonomic/microscopic/PCR• Record geographical origin of field specimens or point of
origin of cultivated material• Check for pesticides; herbicides; heavy metals• Check microbial content
2. Establish/select appropriate bioassays3. Bioassay several types of extracts in vitro and in
vivo.4. Identify active constituents
• Bioassay-guided isolation or ultrafiltration LC-MS• Chemical characterization (MS, NMR, etc.)
Steps Required Prior to Clinical Studies 5. Use appropriate analytical method(s) such as
TLC, HPLC, GC, GC-MS, LC-MS, etc., to standardize the botanical product.
6. Carry out biological standardization.7. Stability studies are required for the
standardized product8. Pharmacologic studies are required for the
standardized product• Metabolism (including interactions with cytochrome P450
enzymes)• Pharmacokinetics• Toxicity• Mechanism of Action
3
Risks of Estrogen Replacement Therapy Using Equine Estrogens
Hormone replacement therapy (HRT) in menopausal women is associated with increased risks of certain cancers, stroke and dementia.*The leading HRT products Premarin™ and Prempro™ contain equine estrogens such as equilenin.Equilenin is metabolized to the toxic metabolite 4-hydroxyequilenin that is oxidized to quinoids that alkylate biopolymers and promote oxidative stress through redox cycling.
* Shumaker et al. 2003 JAMA, 2651-2662; Wassertheil-Smoller et al. 2003 JAMA 2673-2684
Alternatives to HRT: Screening Botanicals for Estrogens
Botanical specimens were obtained by field collection, cultivation or from suppliers.Organic and aqueous extracts of each plant were prepared.Extracts were screened using ultrafiltration LC-MS for ligands to estrogen receptor (ER)-α and ER-β.Ligands to ER-α and ER-β were identified in Trifolium pratense L. (red clover) and Humuluslupulus (hops) but not in black cohosh (Cimicifugaracemosa).Black cohosh was determined to have serotoninergic activity.
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• Red clover was grown at the University of Illinois Pharmacognosy Field Station (Downers Grove, IL) and identified by a taxonomist.
• Voucher specimens were prepared.• Chemical standardization was carried out using
HPLC-UV and LC-MS.• PCR analysis was carried out using Trifolium
pratense and related species.• Extraction and hydrolysis of red clover for the
clinical trial were carried out at PureWorldBotanicals (S. Hackensack, NJ) under GMP.
Production and Standardization
Production and Standardization
The extract intended for clinical use was analyzed for heavy metals, herbicides and pesticidesMicrobial content was tested to be within acceptable limitsThe clinical extract was standardized biologically by Project 2.Chemical standardization was carried out and was set to a total of 15% isoflavones(by weight).
5
Ultrafiltration LC-MS Screening of Trifolium pratense L. (Red clover) for Estrogens
Extract Bioassay Active Extract
HPLC Fractionation
Bioassays
......Characterization of Active Compounds
PUF-LC-MS
Natural Product Drug Discovery PUF-LC-MS vs Bioassay Guided Fractionation
6
Pulsed Ultrafiltration-Mass Spectrometric Screening for Ligands of ER-α and ER-β
2. UltrafiltrationSeparation
2a. Wash Unbound to Waste
3. LC-MS Identification
m/z
1. Binding
2b. Elute Bound Ligands into Trapping Column
++
++
++
+
+
+ +++
+
++
+
Preincubate Extract and Estrogen Receptor
Ligand-ER Complexes
Unbound Compounds
+
+++
+
HPLC Pump
Injector
Elute desaltedligands into MS
Sun et al. 2005 J. Am. Soc. Mass Spectrom. 16:272-279.
Ultrafiltration LC-MS Screening of Trifolium pratense L. (Red Clover) Extract (10 mg/L)
5.0 10.0 15.0 20.0Retention time (min)
0
100
50
LC-M
S re
spon
se
0
100
50 m/z 253
m/z 269m/z 267
m/z 283
Int. Std.Daidzein
Genistein Formononetin
Biochanin A
ER-β (0.667 μM)ER-α (0.667 μM)Control
Computer Reconstructed Mass
Chromatograms
TIC
Liu, et al. 2001 J. Agric. Food Chem. 49:2472.
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Estrogenic Isoflavones in Red Clover Used for Chemical Standardization of the Dietary
Supplement for the Clinical Trials
O
O
OH
OH
O
O
OH
OCH3
O
O
OH
OCH3
OH
O
O
OH
OHOH
Daidzein Formononetin
Genistein Biochanin A
Biological Standardization and Mechanism of Action
ER-α and ER-βCompetitive binding assays using [3H]-estrodiol
and recombinant estrogen receptorsInduction of alkaline phosphatase activity and up-regulation of progesterone receptor mRNA in Ishikawa (endometrial) cellsAssays of estrogenicity and anti-estrogenicity
Estrogenic properties in ovariectomized ratsUterotrophic effects were confirmed in vivo.
Extracts were assayed for estrogenicity
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Estrogenic Activities of Red Clover Standardized Extract and Pure Isoflavones
ER-α IC50μg/mL or μM
ER-β IC50μg/mL or μM
AP Induction EC50 μg/mL or μM
PR mRNA Fold Induction
E2 0.021 0.015 0.00014 47 DMSO N/A N/A N/A 1.0
Red Clover 18 2.0 1.9 30Daidzein 17 1.2 0.53 2.0
Formononetin 104 60 N/A 1.9
Biochanin A 35 4.1 4.6 1.4
Genistein 0.3 0.02 0.33 3.0
Metabolism, Bioavailability and Toxicity Screening
• No toxic compounds or electrophilic metabolites were found in vitro or in vivo for the standardized red clover extract.
• In comparison, electrophilic and potentially toxic metabolites of equine estrogens contained in Prempro have been reported such as 4-hydroxy-equilenin.
• No heavy metal or pesticide contaminants were detected in the standardized botanical extracts.
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Preclinical Studies of Metabolism, Toxicity and Intestinal Absorption
• Ultrafiltration tandem mass spectrometry and human liver microsomes were used to screen the red clover extract for reactive metabolites. None were detected.
• Human hepatocytes and liver microsomes were used to generate isoflavone phase I and II metabolites which were identified using LC-MS-MS.
• The cytochrome P450 ezymes responsible for phase I O-demethylation were identified.
• The intestinal permeability of the red clover isoflavones was investigated using human intestinal epithelial Caco-2 cells
Mas
s sp
ectro
met
er re
lativ
e re
spon
se
Retention time (min)4.0 8.0 12.0 16.0 20.0 24.0 28.0 32.0 36.0 40.0
0
100
%
Gen
iste
in Biochanin A
0
%
Prunetin
Gen
iste
in
Oxi
datio
n pr
oduc
t0
100
%
Oxi
datio
n pr
oduc
t
Oxi
datio
n pr
oduc
t
Dad
izei
n
Formononetin
LC-MS Detection of Metabolites of Formononetin, Prunetin and Biochanin A
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Phase I Metabolism of Biochanin A, Prunetinand Formonetin to Genistein and Daidzein
Biochanin A5,7-Dihydroxy-4’-methoxyisoflavone
6
7
8 12
3
453’
4’
5’6’
2’A
B
O
OOCH3
OH
HO
Prunetin5,4’-Dihydroxy-7-methoxyisoflavone
5
12
7
8
3
46
2’
3’
4’
5’6’
A
B
O
OOH
OH
H3CO
Genistein5,7,4’-Trihydroxyisoflavone
O
OOH
OH
HO
Daidzein7,4’-Dihydroxyisoflavone
O
OOH
HO
Formononetin7-Hydroxy-4’-methoxyisoflavone
8 1
37
65 4
2’
3’
4’
5’
6’
A
B
2O
OOCH3
HO
CYP3A4, CYP2A6
CYP3A4
CYP2A6
CYP3A4, CYP2A6
0
20
40
60
80
100
120
140
160
Inhibitors of cytochrome P450 enzymes
Per
cent
of a
ctiv
ity
Con
trol
Ket
ocon
azol
e (3
A4)
Fura
fylli
ne(1
A2)
Qui
nidi
ne (2
D6)
TCP
(2A
6,2C
19,2
B6)
Om
epra
zole
(2C
19)
DD
C (2
E1)
Ore
phen
adin
e(2
B6)
Sul
phen
azol
e(2
C9)
Que
certi
n(2
C8)
Inhibition of O-Demethylation of Formononetin by Selective Inhibitors of Cytochrome P450
11
HBSS + Compound
HBSS
Apical to Basolateral
Caco-2 Cell Monolayer System to Study Absorption Across Human Intestinal Mucosa
HBSS + Compound
HBSS
Basolateral to Apical
Uptake of compounds across intestinal mucosa is determined by a combination of processes including Paracellular diffusion, Transcellular diffusion, Facilitated transport, and Metabolism.Facilitated transport (i.e.,P-glycoprotein) can be probed using specific inhibitors and measuring transport rates in opposite directionsUse of mass spectrometry enhances the amount of information available concerning substrate metabolism and allows multiple compounds to be studied simultaneously.
LC-MS Analysis of Red Clover Isoflavones in the Caco-2 Culture Medium
Reversed Phase HPLC and Negative Ion APCI
0
20000 m/z 253
0
20000
010000
3 4 10 110
10000
5 6 7 8 9
Daidzein
Formononetin
Genistein
Biochanin A
Internal Std
m/z 267
m/z 269
m/z 283
Retention time (min)
Mas
s sp
ectro
met
er S
IM re
spon
se
12
Apparent Permeability Coefficients of Red Clover Isoflavones through the Caco-2 Monolayer
1 Papp = Apparent Permeability Coefficients, cm/sec (×10-5)2 AP→BL = Apical to Basolateral transport3 BL →AP = Basolateral to Apical transportData are expressed as mean ± SD, n = 3
Papp1
cm/sec (×10-5)Daidzein50 μM
Genistein50 μM
Formononetin50 μM
Biochanin A 50 μM
AP to BL2 3.10 ± 0.48 4.06 ± 0.65 4.62 ± 0.23 5.47 ± 0.23
BL to AP3 2.76 ± 0.19 4.56 ± 0.12 4.29 ± 0.13 5.11 ± 0.25
Caco-2 Monolayer Papp Coefficients of Red Clover Isoflavones from a Capsule Extract
1 Papp = Apparent Permeability Coefficients, cm/sec (×10-5)2 AP→BL = Apical to Basolateral transport3 BL →AP = Basolateral to Apical transportData are expressed as mean ± SD, n = 3
Papp1
cm/sec (×10-5)Daidzein Genistein Formononetin Biochanin A
AP to BL2 4.36 ± 0.07 4.36 ± 0.23 5.78 ± 0.23 4.95 ± 0.24
BL to AP3 4.22 ± 0.36 5.05 ± 0.33 5.50 ± 0.24 5.15 ± 0.25
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Caco-2 Cell Monolayer System to Study Metabolism of Red Clover Isoflavones
• The metabolism of genistein, daidzein, biochanin A, and formononetin by the intestinal mucosa was investigated by incubating each isoflavone at 50 μM with a Caco-2 cell monolayer for 4 hr at 37 ºC.
• Metabolites were identified using LC-MS and LC-MS-MS.
• Unlike hepatic metabolism involving cytochrome P450 enzymes, no O-demethylation or other Phase I metabolites were detected.
• Phase II glucuronides and sulfates were observed.
10.0 20.0 30.0 40.0 50.0 60.0 70.00
1000
1000
100m/z 429
m/z 333
Daidzein
Daidzein sulfates
Daidzein glucuronide
Retention time (min)
Total ion chromatogram
Neg
ativ
e io
n el
ectro
spra
y re
lativ
e re
spon
se
LC-MS Analysis of Daidzein Metabolites after Incubation with Caco-2 Cells
14
Retention time (min)
Neg
ativ
e io
n el
ectro
spra
y M
S re
lativ
e re
spon
se
LC-MS Analysis of Formononetin Metabolites after Incubation with Caco-2 Cells
10.0 20.0 30.0 40.0 50.0 60.0 70.00
1000
100m/z 443
Formononetin glucuronide
FormononetinTotal ion chromatogram
15 women were recruited and placed on dietary restrictions for 3 weeks.Subjects were randomized into 3 groups and administered a single oral dose of red clover capsules containing 40 mg, 80 mg or 120 mg isoflavones.Women were monitored hourly in the GCRC for the first 24 h. Side effects were monitored for 1 week.Blood samples were drawn at baseline, hourly for the first 12 h, then at 24, 36, 48, 72, 96, 120, and 144 h.Urine was collected for the first 24 h.Estrogenic isoflavones in serum and urine were measured using LC-MS
Clinical Assessment of Red CloverPhase 1 Clinical Design
15
Phase 1 Toxicity Results for the Red Clover Extract
No acute indications of toxicity were detected
• No nausea or emesis• No acute discomfort• No changes in blood pressure or heart rate• No clinical chemistry abnormalities
LC-MS Time-concentration Curves of Isoflavones in Serum of Women Receiving 40 mg Red Clover Extract
Daidzein
0
20
40
60
80
100
120
0 20 40 60 80 100 120 140 160
Time (hour)
Con
cent
ratio
n (n
g/m
l) RC1RC2RC3RC4RC5
Genistein
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120 140 160Time (hour)
Con
cent
ratio
n (n
g/m
l)
RC1RC2RC3RC4RC5
Biochanin A
0
5
10
15
20
25
0 20 40 60 80 100 120 140 160
Time (hour)
Con
cent
ratio
n (n
g/m
l)
RC1RC2RC3RC4RC5
Formononetin
0
5
10
15
20
25
30
0 20 40 60 80 100 120 140 160
Time (hour)
Con
cent
ratio
n (n
g/m
l)
RC1RC2RC3RC5
16
Linear Regression Analysis of AUC and Cmax vs. Dose For Daidzein, Genistein, Biochanin A and Formononetin
Key: Daidzein Genistein Biochanin A Formononetin
AUC vs DoseA
UC
(hr x
ng/
ml)
Cmax vs Dose
Dose (mg)
Cm
ax (n
g/m
l)
0
1000
2000
3000
4000
0 50 100 1500
40
80
120
160
200
20 60 80 100 120 14040
R2 = 0.9950 Daidzein, 0.9737 Genistein0.9557 Biochanin A, 0.8096 Formononetin
R2 = 0.8907 Daidzein, 0.9325 Genistein0.8012 Biochanin A, 0.8660 Formononetin
Dose (mg)
0
2
4
6
8
10
12
20 70 120
Dose of red clover isoflavones (mg)
Qua
ntity
in u
rine
(mg)
Daidzein
Genistein
Biochanin A
Formononetin
24-Hour Urinary Excretion of Daidzein, Genistein, Biochanin A and Formononetin
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Clinical Assessment
88 peri-menopausal women4 arms (22 women per arm)
Black cohoshRed cloverPremproPlacebo
Daily dosing for 1 yearPrimary endpoint: reduction of hot flashes
Phase 2 Clinical Trial of Safety and EfficacyRandomized, Double-blind, Placebo-controlled
Phase 2 Clinical Trial – to date
• > 500 women have been screened• 60/88 women currently enrolled• Only 1 withdrawal (subject moved out of
area)• No serious side effects reported• All biochemical parameters, e.g., liver
enzymes, have been normal
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Conclusions
Metabolism of Red Clover Isoflavones
The abundant isoflavone formononetin is O-demethylatedby CYP3A4 and CYP2A6 to form the more estrogenic daidzein.The abundant red clover isoflavone biochanin A and less abundant prunetin are O-demethylated by CYP3A4 and CYP2A6 to form the more estrogenic genistein.Red clover isoflavones can be conjugated in the intestine and in the liver to form monoglucuronides or monosulfateswhich are excreted in the urine and possibly in the bile.
ConclusionsPharmacokinetics of Red Clover Isoflavones
• AUC and Cmax increased linearly with dose• T1/2 of genistein and daidzein >12 h and much longer
than formononetin and biochanin A• Serum concentrations of genistein and daidzein >>
biochanin A and formononetin due to metabolic conversion instead of lower intestinal absorption
• Urinary recovery of genistein and daidzein >100% due to metabolic conversion of formononetin and biochanin A to genistein and daidzein
Phase II study of red clover and black cohosh vs. Prempro and placebo is in progress.
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ConclusionsStandardization of Botanical Dietary Supplements
Material should be botanically authenticatedActive constituents should be identifiedComposition should be chemically standardizedActivity should be biologically standardizedShould be tested for contamination by pesticides, herbicides, microbes, and heavy metalsShould investigate PK and metabolism of active constituentsShould test for botanical-drug interactionsClinical trials of safety and efficacy needed
Acknowledgements
NIH grant P50 AT00155 from the Office of Dietary Supplements, the National Institute of General Medical Sciences, the Office for Research on Women’s Health, and the National Center for Complementary and Alternative Medicine
ODS and NCCAM for supplemental funding for the phase 2 clinical trial
• Pharmavite and PureWorld for extract preparation and dosage form development
• Thermo Electron for LC-MSn instrumentation
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Acknowledgements
UIC/NIH Botanical Center Faculty
• Norman Farnsworth• Guido Pauli• Sam Hedayat• Richard Derman• Stacie Geller• Lee Shulman• Suzanne Banuvar• Doel Sojarto• Judy Bolton• Dejan Nikolic
Postdoctoral Fellows• Chuck Gu• Luke Chadwick
Graduate Students• Yongkai Sun• Yongmei Li• Wenzhong Liang• Ben Johnson• Nancy Booth• Cassia Overk• Joanna Burdette
UIC/NIH Center Faculty, Fellows, Students and Staff, Summer 2005
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