Standardization, Screening and Clinical Evaluation of Estrogenic Isoflavones in Red Clover (Trifolium pratense) for Women’s Health

Richard B. van Breemen UIC/NIH Center for Botanical Dietary Supplements Research Department of Medicinal Chemistry and Pharmacognosy University of Illinois College of Pharmacy, Chicago, IL

Core A Administration Advisory N. Farnsworth, E. Krause Committee R. van Breemen, S. Hedayat

Core B Analytical Core Richard van Breemen UIC/NIH Center for Dejan Nikolic Project 1 Botanical Dietary Botanical Standardization Norman Farnsworth Supplements Guido Pauli Project 2 Research Bioassay Development Judy Bolton

Project 3 Director, Norman R. Farnsworth Metabolism & Bioavailability Co-Director, Richard B. van Breemen Richard van Breemen

Clinical Evaluation Team S. Geller, S. Banuvar L. Schulman

1 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 bioassays 3. 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 product 8. Pharmacologic studies are required for the standardized product • Metabolism (including interactions with cytochrome P450 enzymes) • Pharmacokinetics • Toxicity • Mechanism of Action

2 Risks of Estrogen Replacement Therapy Using Equine Estrogens

ƒ Hormone replacement therapy (HRT) in menopausal women is associated with increased risks of certain cancers, stroke and dementia.* ƒ The leading HRT products Premarin™ and Prempro™ contain equine estrogens such as equilenin. ƒ 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

ƒ Botanical specimens were obtained by field collection, cultivation or from suppliers. ƒ Organic and aqueous extracts of each plant were prepared. ƒ Extracts were screened using ultrafiltration LC-MS for ligands to estrogen receptor (ER)-α and ER-β. ƒ Ligands to ER-α and ER-β were identified in Trifolium pratense L. (red clover) and Humulus lupulus (hops) but not in black cohosh (Cimicifuga racemosa). ƒ Black cohosh was determined to have serotoninergic activity.

3 Production and Standardization

• 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 PureWorld Botanicals (S. Hackensack, NJ) under GMP.

Production and Standardization

ƒ The extract intended for clinical use was analyzed for heavy metals, herbicides and pesticides ƒ Microbial content was tested to be within acceptable limits ƒ The clinical extract was standardized biologically by Project 2. ƒ Chemical standardization was carried out and was set to a total of 15% isoflavones (by weight).

4 Ultrafiltration LC-MS Screening of Trifolium pratense L. (Red clover) for Estrogens

Natural Product Drug Discovery PUF-LC-MS vs Bioassay Guided Fractionation

Extract Bioassay Active Extract

HPLC Fractionation PUF-LC-MS Bioassays

Characterization of ... Active Compounds ...

5 Pulsed Ultrafiltration-Mass Spectrometric Screening for Ligands of ER-α and ER-β

Unbound HPLC Compounds Pump 2a. Wash Unbound to Waste + + + + + + + + + + + + + + + + Injector + + + 2b. Elute Bound Ligands + + into Trapping Column Preincubate Extract and Ligand-ER Complexes Estrogen Receptor Elute desalted ligands into MS 2. Ultrafiltration 1. Binding Separation

3. LC-MS Identification Sun et al. 2005 J. Am. Soc. Mass Spectrom. 16:272-279. m/z

Ultrafiltration LC-MS Screening of Trifolium pratense L. (Red Clover) Extract (10 mg/L)

100 ER-β (0.667 μM) m/z 269 m/z 283 ER-α (0.667 μM) m/z 267 TIC Control 50 m/z 253

0

100 Biochanin A Computer Genistein Formononetin

LC-MS response Reconstructed Mass Chromatograms 50 Int. Std. Daidzein

0 5.0 10.0 15.0 20.0 Retention time (min) Liu, et al. 2001 J. Agric. Food Chem. 49:2472.

6 Estrogenic Isoflavones in Red Clover Used for Chemical Standardization of the Dietary Supplement for the Clinical Trials

HO O HO O

O O OH OCH3 Daidzein Formononetin

HO O HO O

OH O OH OH O OCH3 Genistein Biochanin A

Biological Standardization and Mechanism of Action Extracts were assayed for estrogenicity

ƒ ER-α and ER-β Competitive binding assays using [3H]-estrodiol and recombinant estrogen receptors ƒ Induction of alkaline phosphatase activity and up-regulation of progesterone receptor mRNA in Ishikawa (endometrial) cells Assays of estrogenicity and anti-estrogenicity ƒ Estrogenic properties in ovariectomized rats Uterotrophic effects were confirmed in vivo.

7 Estrogenic Activities of Red Clover Standardized Extract and Pure Isoflavones

ER-α IC50 ER-β IC50 AP Induction PR mRNA μg/mL or μM μg/mL or μM EC50 μg/mL or μM 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 30 Daidzein 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.

8 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

LC-MS Detection of Metabolites of Formononetin, Prunetin and Biochanin A

100 Formononetin

% Oxidation product product Oxidation Dadizein Oxidation product product Oxidation 0

Prunetin

% Genistein Oxidation product Oxidation

0 100 Biochanin A

% Genistein Mass spectrometer relative response

0 4.0 8.0 12.0 16.0 20.0 24.0 28.0 32.0 36.0 40.0 Retention time (min)

9 Phase I Metabolism of Biochanin A, Prunetin and Formonetin to Genistein and Daidzein

8 1 HO O 8 1 2 H CO 3 O 2 7 A 2’ 7 3 A 2’ 6 3 3’ 6 5 4 3’ B 5 4 4’ B OH O 4’ 6’ OH O 6’ 5’ OCH3 OH Biochanin A Prunetin 5’ 5,7-Dihydroxy-4’-methoxyisoflavone 5,4’-Dihydroxy-7-methoxyisoflavone

HO O CYP3A4, CYP2A6 CYP3A4, CYP2A6

OH O Genistein OH 5,7,4’-Trihydroxyisoflavone 81 HO O 2 HO O 7 A 2’ 3 6 3’ CYP3A4 54 B 4’ O 6’ CYP2A6 OCH3 O 5’ OH Formononetin Daidzein 7-Hydroxy-4’-methoxyisoflavone 7,4’-Dihydroxyisoflavone

Inhibition of O-Demethylation of Formononetin by Selective Inhibitors of Cytochrome P450

160

140

120 )

100 3A4

80 Quinidine (2D6) Quinidine Furafylline (1A2) Control 60 DDC (2E1) ,2C19,2B6) Ketoconazole ( Ketoconazole Percent of activity

40 2A6 Quecertin (2C8) Sulphenazole (2C9) Omeprazole (2C19) Orephenadine (2B6) 20 TCP (

0 Inhibitors of cytochrome P450 enzymes

10 Caco-2 Cell Monolayer System to Study Absorption Across Human Intestinal Mucosa

ƒ Uptake of compounds across intestinal mucosa is determined by a combination of processes including Paracellular diffusion, Transcellular diffusion, Facilitated transport, and Metabolism. ƒ Facilitated transport (i.e.,P-glycoprotein) can be probed using specific inhibitors and measuring transport rates in opposite directions ƒ Use of mass spectrometry enhances the amount of information available concerning substrate metabolism and allows multiple compounds to be studied simultaneously.

HBSS + Compound HBSS

HBSS HBSS + Compound

Apical to Basolateral Basolateral to Apical

LC-MS Analysis of Red Clover Isoflavones in the Caco-2 Culture Medium Reversed Phase HPLC and Negative Ion APCI

20000 m/z 253 Daidzein Internal Std 0

20000 m/z 267 Formononetin 0

m/z 269 Genistein 10000 0

m/z 283 Biochanin A 10000 Mass spectrometer SIM response 0 3 4 5678910 11 Retention time (min)

11 Apparent Permeability Coefficients of Red Clover Isoflavones through the Caco-2 Monolayer

Papp1 Daidzein Genistein Formononetin Biochanin A cm/sec (×10-5) 50 μM 50 μM 50 μM 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

1 -5 Papp = Apparent Permeability Coefficients, cm/sec (×10 ) 2 AP→BL = Apical to Basolateral transport 3 BL →AP = Basolateral to Apical transport Data are expressed as mean ± SD, n = 3

Caco-2 Monolayer Papp Coefficients of Red Clover Isoflavones from a Capsule Extract

Papp1 Daidzein Genistein Formononetin Biochanin A cm/sec (×10-5)

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

1 -5 Papp = Apparent Permeability Coefficients, cm/sec (×10 ) 2 AP→BL = Apical to Basolateral transport 3 BL →AP = Basolateral to Apical transport Data are expressed as mean ± SD, n = 3

12 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.

LC-MS Analysis of Daidzein Metabolites after Incubation with Caco-2 Cells

100 m/z 429 Daidzein glucuronide

0 100 m/z 333 Daidzein sulfates

0 100 Total ion chromatogram Daidzein

0 Negative ion electrospray relative response 10.0 20.0 30.0 40.0 50.0 60.0 70.0 Retention time (min)

13 LC-MS Analysis of Formononetin Metabolites after Incubation with Caco-2 Cells 100 m/z 443 Formononetin glucuronide

0 100 Total ion chromatogram Formononetin

Negative ion electrospray MS relative response 0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 Retention time (min)

Clinical Assessment of Red Clover Phase 1 Clinical Design

ƒ 15 women were recruited and placed on dietary restrictions for 3 weeks. ƒ 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. ƒ Women were monitored hourly in the GCRC for the first 24 h. Side effects were monitored for 1 week. ƒ Blood samples were drawn at baseline, hourly for the first 12 h, then at 24, 36, 48, 72, 96, 120, and 144 h. ƒ Urine was collected for the first 24 h. ƒ Estrogenic isoflavones in serum and urine were measured using LC-MS

14 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

140 120 RC1 Daidzein 120 Genistein 100 RC1 RC2 RC2 100 RC3 80 RC3 80 RC4 RC4 RC5 60 RC5 60 40 40 20

20 (ng/ml) Concentration Concentration (ng/ml) Concentration 0 0 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 Time (hour) Time (hour)

25 30

20 Biochanin A 25 Formononetin RC1 RC1 15 RC2 20 RC2 RC3 15 10 RC4 RC3 RC5 10 RC5 5 Concentration (ng/ml) Concentration 5 Concentration (ng/ml) Concentration 0 0 0 20 40 60 80 100 120 140 160 0 20 40 60 80 100 120 140 160 Time (hour) Time (hour)

15 Linear Regression Analysis of AUC and Cmax vs. Dose For Daidzein, Genistein, Biochanin A and Formononetin

AUC vs Dose Cmax vs Dose 200 4000 160 3000 120 2000 80

1000 40 Cmax (ng/ml) AUC (hr x ng/ml) x (hr AUC 0 0 050100 150 20 40 60 80 100 120 140 Dose (mg) Dose (mg)

R2 = 0.9950 Daidzein, 0.9737 Genistein R2 = 0.8907 Daidzein, 0.9325 Genistein 0.9557 Biochanin A, 0.8096 Formononetin 0.8012 Biochanin A, 0.8660 Formononetin

Key: Daidzein Genistein Biochanin A Formononetin

24-Hour Urinary Excretion of Daidzein, Genistein, Biochanin A and Formononetin

12 Daidzein

10 Genistein Biochanin A 8 Formononetin

6

4

Quantity in Quantity urine (mg) 2

0 20 70 120 Dose of red clover isoflavones (mg)

16 Clinical Assessment Phase 2 Clinical Trial of Safety and Efficacy Randomized, Double-blind, Placebo-controlled ƒ 88 peri-menopausal women ƒ 4 arms (22 women per arm) ƒ Black cohosh ƒ Red clover ƒ Prempro ƒ Placebo ƒ Daily dosing for 1 year ƒ Primary endpoint: reduction of hot flashes

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

17 Conclusions

Metabolism of Red Clover Isoflavones

ƒ The abundant isoflavone formononetin is O-demethylated by CYP3A4 and CYP2A6 to form the more estrogenic daidzein. ƒ The abundant red clover isoflavone biochanin A and less abundant prunetin are O-demethylated by CYP3A4 and CYP2A6 to form the more estrogenic genistein. ƒ Red clover isoflavones can be conjugated in the intestine and in the liver to form monoglucuronides or monosulfates which are excreted in the urine and possibly in the bile.

Conclusions

Pharmacokinetics 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.

18 Conclusions Standardization of Botanical Dietary Supplements ƒ Material should be botanically authenticated ƒ Active constituents should be identified ƒ Composition should be chemically standardized ƒ Activity should be biologically standardized ƒ Should be tested for contamination by pesticides, herbicides, microbes, and heavy metals ƒ Should investigate PK and metabolism of active constituents ƒ Should test for botanical-drug interactions ƒ Clinical trials of safety and efficacy needed

Acknowledgements

ƒ 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 ƒ 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

19 Acknowledgements

UIC/NIH Botanical Postdoctoral Fellows Center Faculty • Chuck Gu • Norman Farnsworth • Luke Chadwick • Guido Pauli • Sam Hedayat Graduate Students • Richard Derman • Yongkai Sun • Stacie Geller • Yongmei Li • Lee Shulman • Wenzhong Liang • Suzanne Banuvar • Ben Johnson • Doel Sojarto • Nancy Booth • Judy Bolton • Cassia Overk • Dejan Nikolic • Joanna Burdette

UIC/NIH Center Faculty, Fellows, Students and Staff, Summer 2005

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