interactions between the flavonoid biochanin a and p-glycoprotein substrates in rats: in vitro and...

Interactions Between the Flavonoid Biochanin A andP-Glycoprotein Substrates in Rats: In Vitro and In Vivo

SHUZHONG ZHANG, KAZUKO SAGAWA, ROBERT D. ARNOLD, ELAINE TSENG,XIAODONG WANG, MARILYN E. MORRIS

Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences,University at Buffalo, State University of New York, Amherst, New York 14260

Received 15 January 2009; revised 25 April 2009; accepted 26 April 2009

Published online 4 June 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21827

Shuzhong ZFriars Rd, San

Kazuko SagaPoint Rd, Groto

Robert D. Armaceutical andAthens, GA 306

Elaine TsengRd, MS 8220-43

Xiaodong WaMail Stop HWPennington, NJ

Corresponde2842 ext. 230; FE-mail: memorr

Journal of Pharm

� 2009 Wiley-Liss

430 JOURNA

ABSTRACT: The purpose of this study was to investigate the in vitro and in vivointeractions between flavonoids and P-glycoprotein (P-gp) substrates. The inhibitoryeffects of flavonoids on P-gp were determined by accumulation studies in P-gp-over-expressing MCF-7/ADR cells using daunomycin (DNM) as a model substrate. Morin,phloretin, biochanin A, chalcone, and silymarin significantly increased DNM accumula-tion by greater than 2.5-fold, suggesting they are P-gp inhibitors. To explore potentialin vivo interactions of flavonoids with P-gp, the effect of biochanin A on the pharma-cokinetics of the P-gp substrates doxorubicin, cyclosporine A, and paclitaxel wasinvestigated. In contrast to the in vitro results, intraperitoneal or oral administrationof biochanin A did not significantly change the pharmacokinetics of doxorubicin andcyclosporine A. Moderate interaction was observed between biochanin A and paclitaxel,resulting in lower AUC values after both i.v. and oral administration of paclitaxel.The disconnect between the in vitro and in vivo data suggests that P-gp interactionsmediated by biochanin A may be limited due to its poor bioavailability and rapidclearance. It is also possible that other transporters or metabolizing enzymesare more important in the in vivo disposition of doxorubicin, cyclosporine A,and paclitaxel than P-gp. � 2009 Wiley-Liss, Inc. and the American Pharmacists Association

J Pharm Sci 99:430–441, 2010

Keywords: ABC transporters; pharam
cokinetics; P-glycoprotein; drug transport;oral absorption
hang’s present address is Pfizer, Inc., 5920Diego, CA 92108.wa’s present address is Pfizer, Inc., Eastern

n, CT 06340.nold’s present address is Department of Phar-Biomedical Sciences, University of Georgia,

02-2342.’s present address is Pfizer, Inc., Eastern Point34, Groton, CT 06340.ng’s present address is Bristol Myers Squibb,8A 1.18, 311 Pennington Rocky-Hill Rd,08534.

nce to: Marilyn E. Morris (Telephone: 716-645-ax: 716-645-3693;[email protected])

aceutical Sciences, Vol. 99, 430–441 (2010)

, Inc. and the American Pharmacists Association

L OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUA

INTRODUCTION

Flavonoids have attracted considerable scientificand public interest in recent years due to theirhealth-promoting and beneficial pharmacologicalactivities including antioxidant, antiviral, anti-carcinogenic, and anti-inflammatory properties.1,2

Biochanin A (Fig. 1) is a naturally occurringflavonoid and a major component of the widelyconsumed herbal product red clover extracts.3

Biochanin A has been shown to inhibit carcinogen-induced mammary and lung tumor growth4,5 andreduce the incidence of prostate tumors inxenograft mice.6 Red clover extracts are com-monly used to prevent or relieve post-menopausal

RY 2010

Figure 1. Effect of flavonoids on [3H]DNM accumulation in MCF-7/ADR cells. Theaccumulation of [3H]daunomycin (0.05mM) in MCF-7/ADR cells was determined at theend of a 2-h incubation with flavonoids (100mM). Verapamil is a P-gp inhibitor and wasincluded as a positive control. The results were normalized by protein concentration andare presented as mean�SD (n� 6). �p< 0.05, ��p< 0.01, compared with the accumula-tion in the control group.

FLAVONOIDS AND P-GLYCOPROTEIN 431

symptoms such as hot flashes and bone loss, and tomaintain prostate health.7–9 Red clover extractssuch as Promensil (Novogen, Inc., Samford, CT)are now commercially available as dietary supple-ments, which contain approximately 26 mg ofbiochanin A per tablet.10

Flavonoid–drug interactions have been increas-ingly reported due to the popular use of flavonoid-containing dietary supplements. The mechanismsunderlying the majority of these interactionsare attributed to the modulation of cytochromeP450 (CYP) enzymes and phase II (conjugation)enzymes responsible for the detoxification ofcarcinogens.11 Recently, P-glycoprotein (P-gp)has also been identified as an important playerresponsible for the interactions between flavo-noids and clinically important P-gp substrates.For example, oral coadministration of theflavonoids quercetin and flavone significantlyincreased the bioavailability of paclitaxel in ratsin a dose-dependent manner.12,13 In some cases,these flavonoid–drug interactions can be seriousor even life threatening. This is especially truewhen flavonoids or dietary supplements are usedconcomitantly with narrow therapeutic indexdrugs, which was exemplified by the interaction

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between quercetin and digoxin in pigs.14 In arecent clinical study, oral administration ofquercetin (5 mg/kg, 30 min or 3 days pretreat-ment) significantly increased the area underthe plasma concentration–time curve (AUC) ofcyclosporine by 36% and 47%, respectively.15

Interestingly, in a preclinical study, the oraladministration of quercetin (50 mg/kg) to pigs andrats resulted in significant decreases in the AUCof cyclosporine by 56% and 43%, respectively.16

All these studies indicated that P-gp-mediatedflavonoid–drug interactions could occur in vivo.However, the reasons for the observed contra-dictory results due to different dosing regimensand different species remain largely unknown.Since cyclosporine A is also a drug with anarrow therapeutic index,17 it is important tocharacterize and understand its interaction withdifferent flavonoid inhibitors abundant in dietarysupplements.

The objective of this study was to investigate theinteractions between P-gp substrates and theflavonoid biochanin A. Three clinically importantP-gp substrates (Fig. 1), namely doxorubicin, ananthracycline antibiotic with broad-spectrumantineoplastic activity,18 cyclosporine, a widely

NAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010

432 ZHANG ET AL.

used immunosuppressant drug,17 and paclitaxel,a naturally occurring anticancer agent,19 wereused in this study.

MATERIALS AND METHODS

Chemicals and Reagents

Biochanin A, cyclosporine A, paclitaxel, creme-phor, and hydroxypropyl-b-cyclodextrin (HPbCD)were purchased from Sigma–Aldrich (St. Louis,MO). Doxorubicin was a generous gift fromSicor (Rome, Italy). [3H]daunomycin (DNM,16 Ci/mmol) was purchased from Perkin-ElmerLife Sciences (Waltham, MA). Saline was pur-chased from Henry-Schein (Melville, NY). RPMI1640, fetal bovine serum, L-glutamine, penicillin,and streptomycin were purchased from Invitrogen(Carlsbad, CA). All other reagents or solventsused were either analytical or high-performanceliquid chromatography (HPLC) grade and werepurchased from Fisher Scientific (Springfield,NJ).

Cell Culture and Accumulation Studies

P-gp-overexpressing MCF-7/ADR cells20 withpassage number between 16 and 24 were grownin RPMI 1640 supplemented with 10% fetalbovine serum, 2 mM L-glutamine, penicillin(100 U/mL), and streptomycin (100mg/mL). Cellswere cultured in 75-cm2 flasks at 378C in ahumidified 5% CO2/95% air atmosphere. At the90% confluence, cells were trypsinized and seededinto 35 mm2 Petri dishes for accumulation studies.Experiments were performed 2–3 days afterseeding.

For the accumulation studies, cell culturemedium was removed from the Petri dishes andcells were washed twice with uptake buffer(137 mM NaCl, 5.4 mM KCl, 2.8 mM CaCl2,1.2 mM MgCl2, 10 mM HEPES, pH 7.4). Onemicroliter of uptake buffer containing 0.05mM of[3H]DNM and 100mM of flavonoid was added tothe dish and incubated for 2 h. Verapamil, a P-gpinhibitor, was used as a positive control inthe studies, at a concentration of 100mM. Theuptake of [3H]DNM was stopped by aspirating theincubation buffer and washing the cells threetimes with ice-cold stopping solution (137 mMNaCl, 14 mM Tris–base, pH 7.4). The cells werethen solubilized using 1 mL of 0.3 N sodiumhydroxide solution containing 1% sodium dodecyl

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010

sulfate. The radioactivity in 150mL aliquots of celllysates was determined by liquid scintillationcounting (1900 CA, Tri-Carb liquid scintillationanalyzer; Perkin-Elmer Life Sciences). The accu-mulation in MCF7/ADR cells was normalizedby the protein concentration determined bybicinchoninic acid protein assay (Pierce Chemical,Rockford, IL).

Animals and Surgery

Male Sprague–Dawley (SD) rats (bodyweight �250 g) were purchased from Harlan(Indianapolis, IN) and Charles River Laboratories(Wilmington, MA). Animals were kept in atemperature and humidity-controlled environ-ment with a 12-h light–dark cycle and receiveda standard diet with water ad libitum. Animalswere acclimatized to this environment for at least1 week before experiments. All animal procedureswere performed in accordance with InstitutionalAnimal Care and Use Committee guidelinesand followed approved protocols. Two or 3 daysprior to the study day, rats were anesthetized(ketamine 90 mg/kg and xylazine 10 mg/kg, i.m.injection) and implanted with a cannula (Micro-Renathane1, type MRE-040, 0.040 OD� 0.025ID, Braintree Scientific, Inc., Braintree, MA) inthe right jugular vein. The cannula was flushedwith saline containing 50 IU/mL heparin dailyuntil the study day. The rats were fasted over-night for the oral pharmacokinetic studies.

Pharmacokinetic Interaction Studies

Biochanin A and Doxorubicin Interaction Study

Doxorubicin was dissolved in saline at a concen-tration of 5 mg/mL. Biochanin A was prepared in75% DMSO at a concentration of 20 mg/mL. In thecontrol group, rats were pretreated with 75%DMSO (Biochanin A vehicle, 5 mL/kg, i.p.) 5 minprior to the intravenous administration ofdoxorubicin (7.5 mg/kg). In the treatment group,Biochanin A in 75% DMSO was given intra-peritoneally at 5 mL/kg (i.e., 100 mg/kg) 5 minbefore the intravenous administration of doxo-rubicin (7.5 mg/kg). Blood samples (150mL) weretaken at 5, 15, 30 min and 1, 2, 4, 8, 12, 24, 48, 72,and 96 h. The plasma was separated from thewhole blood by centrifugation at 2000g for 5 min at48C. All plasma samples were stored at �808C

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until analysis by LC-MS/MS. Six or seven ratswere used for each group.

Biochanin A and Cyclosporine A Interaction Study

The i.v. formulation of Cyclosporine A was pre-pared in ethanol/cremephor (1/1), then dilutedsixfold with saline to a final concentration of 3 mg/mL. The oral formulation was made using olive oilat a concentration of 5 mg/mL. The oral formula-tion of biochanin A was made in 25% HPbCD at aconcentration of 125 mg/mL.

For the intravenous interaction study, controlrats were pretreated with 25% HPbCD (BiochaninA vehicle, 2 mL/kg, p.o.) 1 h prior to the intrave-nous administration of cyclosporine A (3 mg/kg).In the treatment group, biochanin A in HPbCDwas given orally at 2 mL/kg (i.e., 250 mg/kg) 1 hbefore the intravenous administration of cyclos-porine A (3 mg/kg). For the oral interactionstudy, control rats were pretreated with 25%HPbCD (biochanin A vehicle, 2 mL/kg, p.o.)10 min prior to the oral administration of cyclos-porine A (10 mg/kg). In the treatment group,biochanin A in HPbCD (2 mL/kg or 250 mg/kg)was given orally 10 min before the oral adminis-tration of cyclosporine A (10 mg/kg). Rats werefasted overnight and food returned after the 4 hblood sample. Rats had free access to waterthroughout the study. The blood samples weretaken at 5, 10, 15, and 30 min and 1, 2, 4, 6, 8, 12,24, 36, and 48 h for the i.v. study, and at 15 and30 min and 1, 2, 4, 6, 8, 12, 24, 36, and 48 h for theoral study. Three rats were used for each group.

Biochanin A and Paclitaxel Interaction Study

The i.v. formulation of paclitaxel was prepared inethanol/cremephor (1/1), then diluted sixfold withsaline to a final concentration of 1 mg/mL. Theoral formulation was made using olive oil at aconcentration of 2 mg/mL. The oral formulation ofbiochanin A was made in 25% HPbCD at aconcentration of 125 mg/mL.

For the intravenous interaction study, controlrats were pretreated with 25% HPbCD (BiochaninA vehicle, 2 mL/kg, p.o.) 1 h prior to the intra-venous administration of paclitaxel (1 mg/kg). Inthe treatment group, biochanin A in HPbCD wasgiven orally at 2 mL/kg (i.e., 250 mg/kg) 1 h beforethe intravenous administration of paclitaxel(1 mg/kg). For the oral interaction study, controlrats were pretreated with 25% HPbCD (biochaninA vehicle, 2 mL/kg, p.o.) 10 min prior to the oraladministration of paclitaxel (4 mg/kg). In the


treatment group, biochanin A in HPbCD (2 mL/kg or 250 mg/kg) was given orally 10 min beforethe oral administration of paclitaxel (4 mg/kg).Rats were fasted overnight and food returnedafter the 4 h blood sample. Rats had free access towater throughout the study. The blood sampleswere taken at 5, 10, 15, and 30 min and 1, 2, 4, 6, 8,12, 24, 36, and 48 h for the i.v. study, and at 15 and30 min and 1, 2, 4, 6, 8, 12, 24, 36, and 48 h for theoral study. Three rats were used for each group.

Liquid Chromatography-Tandem MassSpectrometry Analysis

Doxorubicin Analysis

Total doxorubicin was extracted from plasma andquantified by liquid chromatography-tandemmass spectroscopy (LC-MS/MS), as describedpreviously.21 In brief, plasma samples werediluted fivefold in extraction solvent (60% acet-onitrile and 40% 5 mM ammonium acetate pH3.5). Plasma samples were cooled in an ice waterbath for 10 min, mixed intermittently, and cen-trifuged for 10 min at 15,000g at 48C. Thedeproteinized supernatant was recovered andanalyzed immediately by LC-MS/MS. The mobilephase consisted of 40% acetonitrile and 60% 5 mMammonium acetate, pH 3.5. An electrosprayionization source was used on an ABI/SciexAPI3000 triple quadruple mass spectrometer(ABI, Inc., Foster City, CA) following chromato-graphic separation using an Agilent HPLC system(Model 1100; Palo Alto, CA). The concentration ofdoxorubicin in plasma samples was calculatedfrom a standard curve prepared using standardand quality control samples prepared in blankplasma and extracted as described previously.21

The ions measured for doxorubicin were 544/361.The assay was linear over the concentrationrange of 0.125–10,000 nM and is selective fordoxorubicin.

Cyclosporine A and Paclitaxel Analysis

The plasma cyclosporine A and paclitaxel con-centrations were determined following the addi-tion of 200mL of 50/50 methanol/acetonitrile to50mL samples. Twenty microliters of paclitaxel(250 ng/mL) or cyclosporine A (500 ng/mL) wasalso added as the internal standard to cyclo-sporine A and paclitaxel plasma samples, respec-tively. The mixture was centrifuged, and 150mL ofthe supernatant was removed. After a second


434 ZHANG ET AL.

centrifugation, 20mL of supernatant was injectedonto the column. The analysis was carried out on aPE Sciex API3000 triple quadruple mass spectro-meter with a turbo ion spray source linked to aShimadzu LC10 liquid chromatography systemequipped with a Supelcosil LC-1column (C18,4.6 mm� 50 mm i.d., 5mm). The source tem-perature was set at 5008C. The ionization wasset at positive mode. The mobile phase consistedof (A) methanol, and (B) 10 mM ammoniumacetateþ 1% 2-propanol. The sample was elutedusing a gradient from 0% to 95% A at 0.75 mL/min.The ions measured for cyclosporine A andpaclitaxel were 1202/100 and 854/569, respec-tively. The retention time for cyclosporine A andpaclitaxel was 2.5 and 2.3 min, respectively.The assay was linear over the concentrationrange of 1–5000 ng/mL for both cyclosporineA and paclitaxel.

Biochanin A Analysis

The plasma biochanin A concentrations weredetermined following the addition of 200mL of50/50 methanol/acetonitrile to 25mL samples.The mixture was centrifuged, and 150mL ofthe supernatant was removed. After a secondcentrifugation, 20mL of supernatant was injectedonto the column. The analysis was carried out on aPE Sciex API3000 triple quadruple mass spectro-meter with a turbo ion spray source linked to aShimadzu LC10 liquid chromatography systemequipped with a Supelcosil LC-1column (C18,4.6 mm� 50 mm i.d., 5mm). The source tem-perature was set at 5008C. The ionization wasset at negative mode. The mobile phase consistedof (A) methanol, and (B) 10 mM ammoniumacetateþ 1% 2-propanol. The sample was elutedusing a gradient from 0% to 95% A at 0.75 mL/min.The ions measured for biochanin A were 283/239.The assay was linear over the concentration rangeof 1–5000 ng/mL.

Pharmacokinetic Parameters

The plasma concentration data were analyzed bynoncompartmental analysis using WinNonlinversion 2.1 (Pharsight, Mountain View, CA).The area under the plasma concentration–timecurve (AUC) was calculated using the log-lineartrapezoidal rule. The elimination half-life (t1/2)was calculated by the equation: t1/2¼ 0.693/l,where l was estimated from the terminal slope ofthe plasma concentration versus time curve. The


clearance (CL) was determined from Dose/AUC.The volume of distribution at steady state (Vss)was determined by Vss¼CL� (AUMC/AUC),where AUMC is the area under the first momentcurve (Concentration� time vs. time curve). Oralbioavailability ( F) was calculated by the ratioof dose-normalized AUCs following oral andintravenous administration.

Statistical Analysis

Statistical analysis was conducted using a Stu-dent’s t-test or one-way ANOVA (Prism 3.0 soft-ware, GraphPad, San Diego, CA) followed by aDunnett’s post hoc test. p values <0.05 wereconsidered statistically significant.

RESULTS

Effect of Flavonoids on DNM Accumulationin MCF-7/ADR Cells

Western analysis of MDR/ADR and MCF-7sensitive cells has previously been reported fromour laboratory. MCF-7/ADR cells express P-gp,but not the efflux proteins multidrug resistance-associated protein 1 (MRP1, ABCC1) or breastcancer resistance protein (BCRP, ABCG2).22,23

MCF-7 sensitive cells do not express P-gp, MRP1,or BCRP.23 MCF-7/ADR cells were simulta-neously incubated with various flavonoids(100mM) or the flavonoid vehicle and [3H]DNMfor 2 h (Fig. 1). In the control group, theuptake of [3H]DNM was determined as 0.52�0.23 pmol/mg protein. Verapamil, a P-gp inhibitorused as the positive control, significantlyincreased [3H]DNM accumulation to 1.30�0.39 pmol/mg protein (p< 0.01). The flavonoidsmorin, phloretin, biochanin A, chalcone, andsilymarin all significantly increased [3H]DNMaccumulation by twofold or higher. Biochanin Awas the most effective inhibitor, with a fourfoldincrease of [3H]DNM (2.07� 0.76 pmol/mg pro-tein, p< 0.01). Concentration-dependent studiesdemonstrated P-gp inhibition at concentrationsof 10–30mM for biochanin A and silymarin.24

All other flavonoids such as hesperetin, myricetin,naringenin, galangin, kaempferol, genistein,chrysin, naringin, and luteolin had no significanteffects on the [3H]DNM accumulation in MCF-7/ADR cells. We confirmed the flavonoid effects onP-gp-mediated transport using a second P-gpsubstrate [3H]vinblastine (data not shown). We

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also previously reported that biochanin A cansignificantly increase DNM accumulation inMDA435/LCC6MDR1 cells with high expressionof P-gp, and that biochanin A has no effect onDNM accumulation in MCF-7 sensitive cells thatlack P-gp expression.22 All together, these resultssuggest that biochanin A increases the accumula-tion of DNM through inhibition of P-gp.

Effects of Biochanin A on the Pharmacokineticsof Doxorubicin

To explore the potential in vivo interactions offlavonoid inhibitors with P-gp, biochanin A wasselected as a model flavonoid inhibitor due toits potent in vitro P-gp inhibitory activity andextensive herbal product use. The pharmacoki-netics of the P-gp substrate doxorubicin wascharacterized with and without the coadministra-tion of biochanin A. Doxorubicin pharmacoki-netics, after intravenous administration, could bedescribed by a biexponential profile, exhibiting arapid decrease at the early time points followed bya slower decrease beyond 8 h. In the control rats,the AUC and CL were determined as 196�82.7mg mL�1 min and 43.9� 16.3 mL min�1 kg�1,respectively. In addition, doxorubicin demon-strated a long terminal half-life t1/2 (49.7�15.6 h) and a large steady-state volume ofdistribution (Vss) (158� 95.3 L/kg). The coadmi-nistration of a 100 mg/kg dose of biochanin A byintraperitoneal injection had no significant effectson the pharmacokinetics of doxorubicin. The AUC,t1/2, CL, and Vss were 224� 100mg mL�1 min,45.6� 15.4 h, 40.3� 18.5 mL min�1 kg�1, and118� 86.2 L/kg, respectively (Tab. 1).

Table 1. Summary of the PharmacokineticParameters of Doxorubicin

Parameters

Doxorubicin (7.5 mg/kg, i.v.)

Control(n¼ 7)

Biochanin A,100 mg/kg, i.p.

(n¼ 6)

AUC (mg mL�1 min) 196� 82.7 224� 100t1/2 (h) 49.7� 15.6 45.6� 15.4CL (mL min�1 kg�1) 43.9� 16.3 40.3� 18.5Vss (L/kg) 158� 95.3 118� 86.2

The pharmacokinetic parameters were obtained by non-compartmental analysis using WinNonlin. Data are presentedas mean�SD. The number of animals for each group is spe-cified in parentheses after the corresponding group names.


Effects of Biochanin A on the Pharmacokinetics ofCyclosporine A

The interaction of biochanin A with P-gp wasalso explored by using cyclosporine A as a P-gpsubstrate. As shown in Table 2, after intravenousadministration, the AUC and clearance of cyclos-porine A in control rats were 4960� 983 ng mL�1 hand 619� 113 mL h�1 kg�1, respectively. Thesteady-state volume of distribution Vss was large(5.66� 1.90 L/kg) and the terminal half-life t1/2

was determined as 6.92� 0.56 h. The oraladministration of biochanin A at a 250 mg/kgdose had no significant effects on the pharmaco-kinetics of cyclosporine A. The AUC, t1/2, CL, andVss were 7050� 1080 ng mL�1 h, 9.68� 1.69 h,432� 66.3 mL h�1 kg�1, and 4.91� 1.02 L/kg,respectively (Tab. 2).

For the oral interaction study, the Cmax and tmax

of cyclosporine A after oral administration incontrol rats were 214� 116 ng/mL and 4.33�0.58 h, respectively. The AUC and apparent oralclearance (CL/F) of cyclosporine A were 2280�801 ng mL�1 h and 4780� 1740 mL h�1 kg�1, res-pectively. The terminal half-life t1/2 was similar tothat determined after intravenous administration(10.5� 4.84 h). The oral coadministration of bio-chanin A at 250 mg/kg had no significant effectson the pharmacokinetics of cyclosporine A. TheCmax and tmax of cyclosporine A in biochaninA-treated rats were 251� 112 ng/mL and 4.00�2.00 h, respectively. The AUC, t1/2, and CL/F were2290� 375 ng mL�1 h, 6.29� 1.01 h, and 4440�683 mL h�1 kg�1, respectively (Tab. 2). There wasno significant difference in cyclosporine A bio-availability between the control and biochanin Acoadministered rats (13.8� 4.84% and 9.74�1.60%, respectively).

Effects of Biochanin A on the Pharmacokineticsof Paclitaxel

The interaction of biochanin A with P-gp wasalso explored by using paclitaxel as a P-gpsubstrate. As shown in Figure 2 and Table 3,after intravenous administration, the AUC andclearance of paclitaxel in control rats were275� 15.5 ng mL�1 h and 3.65� 0.21 L h�1 kg�1,respectively. The steady-state volume of distri-bution Vss was 13.7� 1.17 L/kg, which was con-sistent with previously reported values.25 Theterminal half-life t1/2 was determined as3.97� 0.98 h. Compared to the control group,the oral administration of biochanin A at a


Table 2. Summary of the Pharmacokinetic Parameters of Cyclosporine A

Parameters

Cyclosporine A (3 mg/kg, i.v.) Cyclosporine A (10 mg/kg, p.o.)

Control(n¼ 3)

Biochanin A250 mg/kg, p.o.

(n¼ 3)Control(n¼ 3)


(n¼ 3)

AUC (ng mL�1 h) 4960� 983 7050� 1080 2280� 801 2290� 375t1/2 (h) 6.92� 0.56 9.68� 1.69 10.5� 4.84 6.29� 1.01CL or CL/F (mL h�1 kg�1) 619� 113 432� 66.3 4780� 1740 4440� 683Vss (L/kg) 5.66� 1.90 4.91� 1.02 — —Cmax (ng/mL) — — 214� 116 251� 112tmax (h) — — 4.33� 0.58 4.00� 2.00F (%) — — 13.8� 4.84 9.74� 1.60

The pharmacokinetic parameters were obtained by noncompartmental analysis using WinNonlin. Data are presented asmean�SD. The number of animals for each group is specified in parentheses after the corresponding group names.

Figure 2. (A) Plasma concentration versus time pro-files of paclitaxel after an intravenous bolus (1 mg/kg),1 h following the oral administration of control vehicle(*) or biochanin A (250 mg/kg, *). (B) Plasma concen-tration versus time profiles of paclitaxel after oralcoadministration of paclitaxel (4 mg/kg) with controlvehicle (*) or biochanin A (250 mg/kg, *). Plasmalevels of paclitaxel were determined by LC-MS/MS.The data are presented as mean�SD, n¼ 3 for eachgroup.


436 ZHANG ET AL.

250 mg/kg dose significantly decreased the AUC(177� 19.4 ng mL�1 h vs. 275� 15.5 ng mL�1 h incontrol, p< 0.01) and increased clearance(5.69� 0.66 L h�1 kg�1 vs. 3.65� 0.21 L h�1 kg�1

in control, p< 0.01). However, no significantchanges were observed for t1/2 (2.54� 0.29 h)and Vss (15.2� 0.32 L/kg) (Tab. 3).

For the oral interaction study, the Cmax and tmax

of paclitaxel after oral administration incontrol rats were 22.7� 14.2 ng/mL and 1.50�0.87 h, respectively. The AUC and apparentoral clearance (CL/F) of paclitaxel were 79.8�57.1 ng mL�1 h and 68.3� 40.2 L h�1 kg�1, respec-tively. The oral coadministration of biochanin Aat 250 mg/kg had no significant effects on thepharmacokinetics of paclitaxel. The Cmax and tmax

of paclitaxel in biochanin A-treated rats were10.0� 6.93 ng/mL and 1.33� 0.58 h, respectively.The AUC and CL/F were 33.5� 16.6 ng mL�1 hand 149� 93.4 L h�1 kg�1, respectively (Tab. 3).There was no significant difference in paclitaxelbioavailability between the control and biochaninA coadministered rats (7.27� 5.20% and 4.74�2.34%, respectively).

The plasma concentrations of biochanin Afollowing oral administration were also deter-mined. As shown in Figure 3, the Cmax and tmax ofbiochanin A were 54.0� 23.6 ng/mL and6.50� 1.26 h, respectively. The plasma concentra-tions of biochanin A throughout the study were inthe range of 10–100 ng/mL (Fig. 3).

DISCUSSION

In recent years, with the increased public interestin alternative medicines, the use of herbal pre-

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Table 3. Summary of the Pharmacokinetic Parameters of Paclitaxel

Parameters

Paclitaxel (1 mg/kg, i.v.) Paclitaxel (4 mg/kg, p.o.)

Control(n¼ 3)


(n¼ 3)Control(n¼ 3)


(n¼ 3)

AUC (ng mL�1 h) 275� 15.5 177� 19.4� 79.8� 57.1 33.5� 16.6t1/2 (h) 3.97� 0.98 2.54� 0.29 1.47� 0.26 1.84� 1.70CL or CL/F (L h�1 kg�1) 3.65� 0.21 5.69� 0.66� 68.3� 40.2 149� 93.4Vss (L/kg) 13.7� 1.17 15.2� 0.32 — —Cmax (ng/mL) — — 22.7� 14.2 10.0� 6.93tmax (h) — — 1.50� 0.87 1.33� 0.58F (%) — — 7.27� 5.20 4.74� 2.34

The pharmacokinetic parameters were obtained by noncompartmental analysis using WinNonlin. Data are presented asmean�SD. The number of animals for each group is specified in parentheses after the corresponding group names.

�p< 0.05, compared to the corresponding control group.


parations and dietary supplements containinghigh doses of flavonoids for health maintenancehas become very popular,26 raising the potentialfor interactions with conventional drug therapies.However, to date, very limited information isavailable on the pharmacokinetic interactionsbetween flavonoids and clinically importantdrugs. Therefore, additional studies are neededto better understand the flavonoids–drug inter-actions and to utilize these interactions for atherapeutic benefit or to avoid potential adversereactions. In the present study, the effects ofbiochanin A, a naturally occurring flavonoid, onthe pharmacokinetics of doxorubicin, cyclosporineA, and paclitaxel were investigated.

To determine the P-gp inhibitory activityof flavonoids, the effects of 14 flavonoids on

Figure 3. Plasma concentration versus time profileof biochanin A following the oral administration ofa dose of 250 mg/kg. The data are presented asmean�SD, n¼ 4.


[3H]DNM accumulation were investigated inP-gp-overexpressing MCF-7/ADR cells. The fla-vonoids morin, phloretin, biochanin A, chalcone,and silymarin all significantly increased[3H]DNM accumulation by twofold or higher.Among these flavonoids, biochanin A was the mostpotent inhibitor, with a fourfold increase in[3H]DNM accumulation. We confirmed theseresults in the same cell line with a second P-gpsubstrate, 3H-vinblastine (results not shown).These findings were consistent with our previousreport that biochanin A inhibited P-gp-mediatedefflux of digoxin and vinblastine in Caco-2 cells24

and can significantly decrease the cytotoxicity ofdoxorubicin in MDA435/LCC6 cells.

We therefore chose to investigate potentialinteractions that might occur due to inhibitionof Pgp by biochanin A. Biochanin A is the majorisoflavone in red clover (Trifolium pratense), butnot present in soy foods. Red clover isoflavoneextracts, such as Promensil (Novogen, Inc.), arecommercially available as dietary supplements forrelieving post-menopausal symptoms such as hotflashes, bone loss and for maintaining men’sprostate health, and as such are widely used.Concurrent studies in the laboratory investigatedthe pharmacokinetics and bioavailability of bio-chanin A. These studies demonstrated very lowbioavailability, suggesting that significant inter-actions, if they occur, might involve intestinalPgp.27 However, biochanin A has been reported toinhibit chemical-induced tumor carcinogenesisand prevent tumor growth after implantation inanimal xenograft models,4,28 suggesting in vivoefficacy in these animal models even after oraladministration.


438 ZHANG ET AL.

To further explore the potential in vivo inter-actions between biochanin A and P-gp substrates,the pharmacokinetics of doxorubicin, cyclosporineA, and paclitaxel was characterized with andwithout the administration of biochanin A.29,30

The pharmacokinetics of doxorubicin was chara-cterized by a rapid decrease in plasma concentra-tions, followed by a slow elimination phase.Consistent with the literature,31–34 doxorubicinexhibited a long terminal half-life t1/2 (49.7�15.6 h) and a large steady-state volume ofdistribution Vss (158� 95.3 L/kg), indicating asignificant tissue distribution of doxorubicin inrats.35,36 The coadministration of biochanin A(100 mg/kg, i.p.) had no significant effects on thepharmacokinetics of doxorubicin. One explana-tion for this lack of interaction in vivo might bedue to the rapid and extensive metabolism ofbiochanin A into genistein and glucuronide/sulfate conjugates of biochanin A and genisteinin the liver.27,37 The bioavailability of biochanin Aafter i.p. administration has been demonstrated tobe 23% for a 50 mg/kg dose (in 75% DMSO/water)in rats.27

The potential in vivo interaction of biochaninA with cyclosporine A or paclitaxel was alsoinvestigated in this study. In these studies,biochanin A was administered as high doses byoral administration, in order to mimic the normalroute of administration of biochanin A and toexamine possible effects on drug absorption andbioavailability, mediated by P-gp or by othermechanisms. Cyclosporine A had a long terminalhalf-life t1/2 (6.92� 0.56 h) and a large steady-state volume of distribution Vss (5.66� 1.90 L/kg),indicating a significant tissue distribution ofcyclosporine A in rats. This is consistent withthe highly lipophilic nature of cyclosporine A(log P¼ 4.3).38 The oral administration of bio-chanin A (250 mg/kg) increased the AUC of i.v.cyclosporine A (3 mg/kg, i.v.) by 1.42-fold, onaverage, but this was not statistically significant.Oral administration of BCA was used in order toinvestigate the possibility of intestinal interac-tions. All other pharmacokinetic parameters weresimilar to those in the control group. The lack ofinteraction following administration of biochaninA (p.o.) and cyclosporine A (i.v.) is likely due to thelow bioavailability of biochanin A. In a previousstudy, the bioavailability of biochanin A wasshown to be poor (1.2% at a 50 mg/kg dose).27

Consistent with this, the plasma concentrations ofbiochanin A following a 250 mg/kg oral dose in thisstudy were in the 10–100 ng/mL range (equivalent


to 0.035–0.35mM). Given that the in vitro inhibi-tion potency of biochanin A is in the micromolarrange (Ki� 30mM in inhibiting P-gp-mediatedefflux of digoxin in Caco-2 cells),22,24 and the EC50

value for increasing the accumulation of DNM inMCF-7/ADR cells is 10.2� 1.44mM [Nguyen T.D.and Morris M.E., unpublished data], it is notsurprising that oral administration of biochanin Ahad minimal effects on P-gp-mediated systemicelimination of intravenously administered cyclos-porine A. Surprisingly, coadministration of oralbiochanin A had no significant effects on thepharmacokinetics of orally administered cyclos-porine A (10 mg/kg, p.o.). Following the oraladministration of biochanin A at a 250 mg/kgdose, high intestinal concentrations of biochaninA, in the mM range, would be expected; theseconcentrations are likely sufficient to inhibitintestinal P-gp activity. Although the in vivointeraction between biochanin A and cyclosporinehas not been previously investigated, to ourknowledge, the oral administration of quercetin(5 mg/kg as capsules), in a clinical study, signifi-cantly increased the AUC of cyclosporine (300 mgper subject) by 36%.15 In contrast, the oral admi-nistration of quercetin (50 mg/kg in glycofurol) topigs and rats resulted in significant decreases inthe AUC of cyclosporine (10 mg/kg) by 56% and43%, respectively.16 The flavonoid baicalein(112 mg/kg) has also been reported to increasethe Cmax and AUC of cyclosporine in rats.39

In this investigation, the oral administration ofbiochanin A (250 mg/kg) significantly decreasedthe AUC and increased the clearance of intra-venously administered paclitaxel (1 mg/kg, i.v.)(Tab. 3). The exact mechanisms underlying theeffects of biochanin A on the clearance ofpaclitaxel are unknown. An increase in clearancewould only occur due to P-gp, if P-gp activity wasstimulated, increasing the biliary or urinaryelimination of paclitaxel. Although coadmi-nistration of oral biochanin A had no significanteffects on the pharmacokinetics of orally adminis-tered cyclosporine A, the average plasma concen-trations and AUC of paclitaxel after a 4 mg/kgoral dose were decreased, similar to that seenafter i.v. administration. In a recent study, theoral administration of biochanin A at a dose of100 mg/kg significantly increased the bioavail-ability of P-gp substrates paclitaxel and digoxin inrats.25 The flavonoids genistein, a metabolite ofbiochanin A, and morin have also been reported toincrease the AUC of paclitaxel in rats.40,41 Theexact reasons for these differences are not clear,

DOI 10.1002/jps


but it might be due to the strain/speciesdifferences, the dosing vehicle, the methods ofadministration (concomitant or separate admin-istration), and the doses used. An alternativeexplanation for this might be due to the involve-ment of other drug transporters or metabolizingenzymes in the disposition of paclitaxel. Manyflavonoids have been shown to modulate thecytochrome P450 (CYP) system,11 and bothcyclosporine A and paclitaxel are known to beCYP3A substrates.42,43 Biochanin A is an effectiveinhibitor of DMBA-induced DNA damage inMCF-7 cells by inhibiting CYP1A1 and CYP1B144

and inhibits tamoxifen a-hydroxylation in femalerat liver microsomes in vitro via inhibition ofCYP1A2.45 Inhibition of CYP3A4 has not beenreported for biochanin A, although the demethy-lated metabolite of biochanin A, genistein, hasbeen reported to decrease CYP3A4 activityin vitro.46 Inhibition of other uptake or effluxtransporters may also be important: biochanin Acan inhibit rat Oatp3 and human OATP1B1,25,47

as well as BCRP (ABCG2);23 additionally,the glucuronide and sulfate conjugates of bio-chanin A may also contribute to the inhibition ofBCRP and MRP2. However, a role of thesetransporters in the transport of paclitaxel, aswell as cyclosporine, has not been reported.Further studies are needed to understand thedifferences reported for in vivo flavonoid–druginteractions for P-gp substrates.

In summary, our results demonstrated that theflavonoid biochanin A significantly increased[3H]DNM accumulation in MCF-7/ADR cells,suggesting it is a P-gp inhibitor, consistent withthe results of other studies in other cell linesand with other substrates.22,24 Different from thein vitro results, biochanin A at doses of 100 mg/kg(i.p.) or 250 mg/kg (p.o.) did not significantly alterthe pharmacokinetics of the P-gp substratesdoxorubicin and cyclosporine A. Moderate inter-action was observed between biochanin A andpaclitaxel. The disconnect between the in vitroand in vivo data suggests that P-gp interactionsmediated by biochanin A may be limited due to itspoor bioavailability and rapid clearance, due tothe high permeability properties of doxorubicin,cyclosporine A, and paclitaxel, or the involvementof other transporters or metabolizing enzymes inthe disposition of these P-gp substrates. It is likelythat biochanin A may exert multiple effects on thedisposition of these Pgp substrates, includinginhibition of metabolism, as well as effects onmultiple transporters.


ACKNOWLEDGMENTS

Supported in part by Pfizer, Inc. and by the SusanG. Komen Foundation BCTR0601385.

REFERENCES

1. Havsteen BH. 2002. The biochemistry and medicalsignificance of the flavonoids. Pharmacol Ther 96:67–202.

2. Middleton E, Jr., Kandaswami C, Theoharides TC.2000. The effects of plant flavonoids on mammaliancells: Implications for inflammation, heart disease,and cancer. Pharmacol Rev 52:673–751.

3. Delmonte P, Rader JI. 2006. Analysis of isoflavonesin foods and dietary supplements. J AOAC Int 89:1138–1146.

4. Gotoh T, Yamada K, Yin H, Ito A, Kataoka T,Dohi K. 1998. Chemoprevention of N-nitroso-N-methylurea-induced rat mammary carcinogenesisby soy foods or biochanin A. Jpn J Cancer Res 89:137–142.

5. Lee YS, Seo JS, Chung HT, Jang JJ. 1991. Inhibi-tory effects of biochanin A on mouse lung tumorinduced by benzo(a)pyrene. J Korean Med Sci 6:325–328.

6. Rice L, Samedi VG, Medrano TA, Sweeney CA,Baker HV, Stenstrom A, Furman J, ShiverickKT. 2002. Mechanisms of the growth inhibitoryeffects of the isoflavonoid biochanin A on LNCaPcells and xenografts. Prostate 52:201–212.

7. Atkinson C, Compston JE, Day NE, Dowsett M,Bingham SA. 2004. The effects of phytoestrogenisoflavones on bone density in women: A double-blind, randomized, placebo-controlled trial.Am J Clin Nutr 79:326–333.

8. Booth NL, Piersen CE, Banuvar S, Geller SE, Shul-man LP, Farnsworth NR. 2006. Clinical studies ofred clover (Trifolium pratense) dietary supplementsin menopause: A literature review. Menopause 13:251–264.

9. Risbridger GP, Wang H, Frydenberg M, Husband A.2001. The in vivo effect of red clover diet on ventralprostate growth in adult male mice. Reprod FertilDev 13:325–329.

10. Howes JB, Sullivan D, Lai N, Nestel P, Pomeroy S,West L, Eden JA, Howes LG. 2000. The effects ofdietary supplementation with isoflavones from redclover on the lipoprotein profiles of post menopausalwomen with mild to moderate hypercholeste-rolaemia. Atherosclerosis 152:143–147.

11. Moon YJ, Wang X, Morris ME. 2006. Dietary fla-vonoids: Effects on xenobiotic and carcinogen meta-bolism. Toxicol In Vitro 20:187–210.

12. Choi JS, Jo BW, Kim YC. 2004. Enhanced paclit-axel bioavailability after oral administration of


440 ZHANG ET AL.

paclitaxel or prodrug to rats pretreated withquercetin. Eur J Pharm Biopharm 57:313–318.

13. Choi JS, Choi HK, Shin SC. 2004. Enhanced bioa-vailability of paclitaxel after oral coadministrationwith flavone in rats. Int J Pharm 275:165–170.

14. Wang YH, Chao PD, Hsiu SL, Wen KC, Hou YC.2004. Lethal quercetin-digoxin interaction in pigs.Life Sci 74:1191–1197.

15. Choi JS, Choi BC, Choi KE. 2004. Effect ofquercetin on the pharmacokinetics of oral cyclos-porine. Am J Health Syst Pharm 61:2406–2409.

16. Hsiu SL, Hou YC, Wang YH, Tsao CW, Su SF, ChaoPD. 2002. Quercetin significantly decreased cyclos-porin oral bioavailability in pigs and rats. Life Sci72:227–235.

17. Beauchesne PR, Chung NS, Wasan KM. 2007.Cyclosporine A: A review of current oral and intra-venous delivery systems. Drug Dev Ind Pharm 33:211–220.

18. Minotti G, Menna P, Salvatorelli E, Cairo G, GianniL. 2004. Anthracyclines: Molecular advances andpharmacologic developments in antitumor activityand cardiotoxicity. Pharmacol Rev 56:185–229.

19. Altmann KH, Gertsch J. 2007. Anticancer drugsfrom nature—Natural products as a unique sourceof new microtubule-stabilizing agents. Nat ProdRep 24:327–357.

20. Tseng E, Kamath A, Morris ME. 2002. Effect oforganic isothiocyanates on the P-glycoprotein- andMRP1-mediated transport of daunomycin andvinblastine. Pharm Res 19:1509–1515.

21. Arnold RD, Slack JE, Straubinger RM. 2004. Quan-tification of doxorubicin and metabolites in ratplasma and small volume tissue samples by liquidchromatography/electrospray tandem mass spec-troscopy. J Chromatogr B Analyt Technol BiomedLife Sci 808:141–152.

22. Zhang S, Morris ME. 2003. Effects of the flavonoidsbiochanin A, morin, phloretin, and silymarin onP-glycoprotein-mediated transport. J PharmacolExp Ther 304:1258–1267.

23. Zhang S, Yang X, Morris ME. 2004. Flavonoidsare inhibitors of breast cancer resistance protein(ABCG2)-mediated transport. Mol Pharmacol 65:1208–1216.

24. Zhang S, Morris ME. 2003. Effect of the flavonoidsbiochanin A and silymarin on the P-glycoprotein-mediated transport of digoxin and vinblastine inhuman intestinal Caco-2 cells. Pharm Res 20:1184–1191.

25. Peng SX, Ritchie DM, Cousineau M, Danser E,Dewire R, Floden J. 2006. Altered oral bioavaila-bility and pharmacokinetics of P-glycoproteinsubstrates by coadministration of biochanin A.J Pharm Sci 95:1984–1993.

26. Sparreboom A, Cox MC, Acharya MR, Figg WD.2004. Herbal remedies in the United States:


Potential adverse interactions with anticanceragents. J Clin Oncol 22:2489–2503.

27. Moon YJ, Sagawa K, Frederick K, Zhang S, MorrisME. 2006. Pharmacokinetics and bioavailability ofthe isoflavone biochanin A in rats. AAPS J 8:E433–E442.

28. Moon YJ, Shin BS, An G, Morris ME. 2008. Biocha-nin A inhibits breast cancer tumor growth in amurine xenograft model. Pharm Res 25:2158–2163.

29. Endres CJ, Hsiao P, Chung FS, Unadkat JD. 2006.The role of transporters in drug interactions. EurJ Pharm Sci 27:501–517.

30. Tsuji A, Tamai I, Sakata A, Tenda Y, Terasaki T.1993. Restricted transport of cyclosporin A acrossthe blood-brain barrier by a multidrug transporter,P-glycoprotein. Biochem Pharmacol 46:1096–1099.

31. Robert J, Gianni L. 1993. Pharmacokinetics andmetabolism of anthracyclines. Cancer Surv 17:219–252.

32. Gabizon A, Shmeeda H, Barenholz Y. 2003. Phar-macokinetics of pegylated liposomal doxorubicin:Review of animal and human studies. Clin Phar-macokinet 42:419–436.

33. Camaggi CM, Comparsi R, Strocchi E, Testoni F,Angelelli B, Pannuti F. 1988. Epirubicin and dox-orubicin comparative metabolism and pharmacoki-netics. A cross-over study. Cancer ChemotherPharmacol 21:221–228.

34. Robert J. 1988. Pharmacokinetics of new anthra-cyclines. Bull Cancer 75:167–174.

35. Wang JC, Liu XY, Lu WL, Chang A, Zhang Q, GohBC, Lee HS. 2006. Pharmacokinetics of intrave-nously administered stealth liposomal doxorubicinmodulated with verapamil in rats. Eur J PharmBiopharm 62:44–51.

36. Jacquet P, Averbach A, Stuart OA, Chang D, Sugar-baker PH. 1998. Hyperthermic intraperitonealdoxorubicin: Pharmacokinetics, metabolism, andtissue distribution in a rat model. Cancer Che-mother Pharmacol 41:147–154.

37. Peterson TG, Ji GP, Kirk M, Coward L, Falany CN,Barnes S. 1998. Metabolism of the isoflavones gen-istein and biochanin A in human breast cancer celllines. Am J Clin Nutr 68:1505S–1511S.

38. Cheng WP, Gray AI, Tetley L, Hang Tle B, Schat-zlein AG, Uchegbu IF. 2006. Polyelectrolyte nano-particles with high drug loading enhance the oraluptake of hydrophobic compounds. Biomacromole-cules 7:1509–1520.

39. Lai MY, Hsiu SL, Hou YC, Tsai SY, Chao PD.2004. Significant decrease of cyclosporine bioavail-ability in rats caused by a decoction of the rootsof Scutellaria baicalensis. Planta Med 70:132–137.

40. Li X, Choi JS. 2007. Effect of genistein on thepharmacokinetics of paclitaxel administered orallyor intravenously in rats. Int J Pharm 337:188–193.

DOI 10.1002/jps


41. Choi BC, Choi JS, Han HK. 2006. Altered pharma-cokinetics of paclitaxel by the concomitant use ofmorin in rats. Int J Pharm 323:81–85.

42. van Herwaarden AE, Smit JW, SparidansRW, Wagenaar E, van der Kruijssen CM,Schellens JH, Beijnen JH, Schinkel AH. 2005.Midazolam and cyclosporin a metabolism intransgenic mice with liver-specific expression ofhuman CYP3A4. Drug Metab Dispos 33:892–895.

43. Gustafson DL, Long ME, Bradshaw EL, Merz AL,Kerzic PJ. 2005. P450 induction alters paclitaxelpharmacokinetics and tissue distribution withmultiple dosing. Cancer Chemother Pharmacol56:248–254.

44. Chan HY, Wang H, Leung LK. 2003. The red clover(Trifolium pratense) isoflavone biochanin A modu-


lates the biotransformation pathways of 7,12-dimethylbenz[a]anthracene. Br J Nutr 90:87–92.

45. Chen J, Halls SC, Alfaro JF, Zhou Z, Hu M. 2004.Potential beneficial metabolic interactions betweentamoxifen and isoflavones via cytochrome P450-mediated pathways in female rat liver microsomes.Pharm Res 21:2095–2104.

46. Foster BC, Vandenhoek S, Hana J, Krantis A,Akhtar MH, Bryan M, Budzinski JW, RamputhA, Arnason JT. 2003. In vitro inhibition of humancytochrome P450-mediated metabolism of markersubstrates by natural products. Phytomedicine 10:334–342.

47. Wang X, Wolkoff AW, Morris ME. 2005. Flavonoidsas a novel class of human organic anion-transporting polypeptide OATP1B1 (OATP-C)modulators. Drug Metab Dispos 33:1666–1672.


interactions between the flavonoid biochanin a and p-glycoprotein substrates in rats: in vitro and...

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