1

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

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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).

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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

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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

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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

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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

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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

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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

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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