Altered Oral Bioavailability and Pharmacokinetics of P-Glycoprotein Substrates by Coadministration of Biochanin A

Altered Oral Bioavailability and Pharmacokinetics of P-Glycoprotein Substrates by Coadministration of Biochanin A SEAN X. PENG, DAVID M. RITCHIE, MARTIN COUSINEAU, EARL DANSER, ROBERT DEWIRE, JANE FLODEN Johnson & Johnson Pharmaceutical Research & Development, 1000 Route 202, Raritan, New Jersey 08869

Received 25 October 2005; revised 16 April 2006; accepted 17 April 2006 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20664

ABSTRACT: Effects of coadministration of dietary supplement biochanin A (BA) on the pharmacokinetics of three P-glycoprotein substrates, paclitaxel, digoxin, and fexofenadine, were investigated in rats. With BA coadministration, the oral bioavailability and peak plasma concentration were markedly increased by 3.77- and 2.04-fold for paclitaxel, 1.75- and 1.71-fold for digoxin, but were reduced by 0.694- and 0.429-fold for fexofenadine, respectively. Paclitaxel is a Pgp and CYP3A substrate, the drastic increase in systemic exposure may be attributed to the synergistic inhibition of Pgp and CYP3A by BA in the intestine. Digoxin is a substrate for Pgp, CYP3A, and Oatp2. BA may suboptimally inhibit Pgp and CYP3A, resulting in a moderate increase in oral bioavailability of digoxin. Fexofenadine is a substrate for Pgp, Oatp1, Oatp2, and Oatp3. BA appears to preferentially inhibit Oatp3 over Pgp in the intestine, leading to the decreased oral absorption of fexofenadine. No significant changes in mean residence time and terminal half-life were observed for all drugs, suggesting a negligible effect of BA on their hepatic/ renal elimination. These findings demonstrate the importance of interplay among uptake/efflux transporters and metabolizing enzymes. The enhanced oral absorption by BA coadministration may be exploited to improve oral bioavailability of Pgp and CYP3A substrate compounds. ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:1984–1993, 2006

Keywords: bioavailability; oral absorption; P-glycoprotein; organic anion-transporting polypeptide; CYP enzymes; biochanin A; paclitaxel; digoxin; fexofenadine; coadministration

INTRODUCTION A significant number of people regularly consume dietary supplements, hoping to improve their health and prevent or fight certain diseases. However, many ingredients in the dietary supplements are not thoroughly studied and may cause severe interactions with certain drugs. The concomitant administration of food and drug may significantly alter the oral bioavailability of the drug, causing either harmful or beneficial effects. These food–drug interactions can be damaging to Correspondence to: Sean X. Peng (Telephone: 215-6287070; Fax: 215-540-4878; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 95, 1984–1993 (2006) ß 2006 Wiley-Liss, Inc. and the American Pharmacists Association

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the patients taking interacting medications, leading to adverse side effects or even life-threatening conditions due to increased drug levels above the toxicity threshold or decreased drug levels below the effective concentration. On the other hand, the interaction of dietary supplements with drug may be exploited as a way to improve pharmacokinetic properties of the coadministered drug. In drug discovery and development, many orally administered drug candidates are potent, safe, selective, and affordable, but they do not exhibit desired pharmacokinetic profiles. When all other attempts such as modifying dosage formulations, changing particle morphology, and making prodrugs, have failed to improve the drug’s pharmacokinetic properties, coadministration of another drug or molecule to affect the pharmacokinetics of

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the drug candidate is an attractive alternative. In this respect, the coadministration of a safe dietary supplement that is an inhibitor of known drug transporters or metabolizing enzymes interacting with the drug candidate can be a promising approach. Biochanin A (Fig. 1) is a principal flavonoid in red clover extracts that are marketed and sold as dietary supplements. It reportedly possesses many desirable pharmacological properties such as anticancer, anti-inflammation, and antioxidation, which is beneficial to human health.1 As a primary member of flavonoids that are rich in human diet and are naturally occurring in fruits, vegetables, nuts, tea, and red wine, biochanin A has been found to exhibit inhibitory activities against Pglycoprotein (Pgp),2,3 a major efflux transporter protein responsible for the poor absorption of many drugs. It is well known that flavonoids intake can significantly influence the pharmacokinetics of many orally and intravenously administered drugs.4,5 One of the well-documented food–drug interactions is the altered oral bioavailability of many marketed drugs from coadministered grapefruit juice.5 It has been shown that flavonoid-rich grapefruit juice inhibits not only drug efflux transporters such as Pgp and drug metabolizing enzymes such as cytochrome P450s (CYPs), but also drug uptake transporters such as organic anion transporting polypeptides (OATPs).5 However, it is still not clear which components in grapefruit juice are responsible for the observed food–drug interactions. The interplay among influx/efflux transporters, such as OATPs and Pgp, and cytochrome P450 isozymes, such as CYP3A4, is well recognized to play a significant role in limiting oral absorption and therefore bioavailability of their substrate drugs.6 Since biochanin A is a Pgp inhibitor and may potentially inhibit other drug transporters and metabolizing enzymes, the effect of coadministration of biochanin A on the oral bioavailability and pharmacokinetics of known Pgp substrates is the subject of current investigation. This potential food–drug interaction is especially important for people who are regularly taking flavonoid-rich dietary supplements or for people who are asked to take medications with a regular meal at the same time to improve compliance to the treatment regimen. Pgp is a member of the adenosine triphosphate (ATP)-binding cassette family and is a 170-kDa plasma membrane-associated protein, present in small intestine, liver, kidney, and blood-brain DOI 10.1002/jps

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barrier in both rodents and humans.7,8 The presence of Pgp in animal and human organs and tissues alters the pharmacokinetic properties of many Pgp substrate drugs. Paclitaxel, digoxin, and fexofenadine (see Fig. 1) were chosen for this study as they are the three widely used and accepted Pgp probe substrates for in vitro and in vivo studies to evaluate the potential Pgp inhibitory activities of other drug candidates.9 These substrate drugs were used here to investigate the effects of coadministered biochanin A on their oral bioavailability and pharmacokinetics. Paclitaxel is a natural occurring anticancer agent for the treatment of ovarian, breast, and nonsmall cell lung cancers. Digoxin is a cardiac glycoside and is commonly used to treat arrhythmia and congestive heart failure. Fexofenadine is a nonsedating antihistamine drug widely used for the treatment of seasonal allergy. Both digoxin and fexofenadine have minimal systemic metabolism, which is a desired property as a Pgp probe substrate. All three probe drugs are neither Pgp inhibitors nor inducers, another important property to simplify our data interpretation.10 However, paclitaxel and digoxin are good substrates for rat CYP3A1/2. In addition, fexofenadine is a substrate for human OATP-A and OATP-B, as well as rat Oatp1, Oatp2, and Oatp3, while digoxin is a substrate for human OATP-8 and rat Oatp2.11 Since Pgp, CYP3A, and Oatp3 are found to be highly expressed in the small intestine,11 the effects of coadministration of biochanin A on the interplay among these transporters and metabolizing enzymes can significantly alter the oral absorption and pharmacokinetic properties of these Pgp probe substrates. The main objective of this study was to characterize the effects of biochanin A on the oral absorption and pharmacokinetics of coadministered Pgp probe substrates.

MATERIALS AND METHODS Materials Paclitaxel, digoxin, fexofenadine, biochanin A, and ammonium acetate were purchased from Sigma (St. Louis, MO). Polyethylene glycol 400 and solutol HS15 were obtained from BASF (Mount Oliver, NJ) and acetonitrile from EMD Chemicals (Gibbstown, NJ). Ethanol and D5W (5% dextrose in water) were supplied from VWR (Bridgeport, NJ). All other chemicals used were of reagent grade or better. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

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Figure 1. Chemical structures of biochanin A and three Pgp substrates: paclitaxel, digoxin, and fexofenadine.

Animals

In Vivo Studies

Male Sprague–Dawley rats (240–280 g) from Ace Animals (Boyertown, PA) were used in all in vivo studies with four rats in each dosing group. The animals were fed a standard laboratory rodent diet (Purina Mills, St. Louis, MO) and housed in individual cages on a 12-h light and 12-h dark cycle with room temperature maintained at 22
38C and relative humidity at 50
20%. Rats were fasted overnight before dosing with food returned after 6 h blood samples were obtained. Water was provided ad libitum throughout the study.

All studies were reviewed and approved by the Johnson & Johnson Institutional Animal Care and Use Committee. Paclitaxel, digoxin, and fexofenadine were prepared in 30% PEG400:20% solutol:50% water for oral dosing via gavage needle at 20 mg/kg and in 10% ethanol:10% solutol:80% D5W for intravenous bolus injection via tail vein at 2 mg/kg. Biochanin A was also prepared in 30% PEG400:20% solutol:50% water for oral coadministration (15 min prior to drug dosing) at 100 mg/kg with paclitaxel, digoxin, and fexofenadine, respectively.

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All blood samples were taken via the saphenous vein at 0 (predose), 0.5, 1, 2, 4, 6, 8, and 24 h after oral dosing, and at 0 (predose), 15, and 30 min and 1, 2, 4, 6, 8, and 24 h following intravenous administration. Blood samples were collected in Becton Dickinson (Franklin Lakes, NJ) Microtainer tubes coated with EDTA as the anticoagulant and were centrifuged at 15000g for 3 min to obtain plasma samples. All plasma samples were stored at 708C until analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS).

Analytical Assay Procedures The plasma samples were treated with two volumes of acetonitrile containing an internal standard, vortexed and centrifuged; the resulting supernatants were analyzed by LC-MS/MS. LCMS/MS analysis of samples was performed using two Shimadzu LC-10AD VP pumps (Columbia, MD), a Leap HTS PAL autosampler (Carrboro, NC), and an AB/MDS Sciex API 4000 triple quadrupole mass spectrometer (Concord, Ontario, Canada). Analytes were separated on a Waters (Milford, MA) Atlantis dC18 column (4.6  50 mm, 5 mm), using a 1.5-min fast gradient elution from 5%B to 100%B (A: 10 mM ammonium acetate; B: acetonitrile) at a flow rate of 1.5 mL/min. All analytes were ionized in the TurboIonSpray interface in either the positive or negative ion mode and detected using selected reaction monitoring. All ion source and tandem MS instrument parameters for the analytes were optimized for high sensitivity with optimal transitions from m/z 854 to 286 for paclitaxel, m/z 779 to 85 for digoxin, m/z 502 to 484 for fexofenadine, and m/z 285 to 152 for biochanin A. The calibration standards ranged from 0.5 ng/mL to 10 mg/mL. The precision and accuracy of the quality control samples at three concentration levels (low, middle, and high) were within 15%RSD and 15%RE, respectively.

Pharmacokinetic Data Analysis Pharmacokinetic parameters were determined based on a noncompartmental approach using WinNonlin Professional version 4.0 (Pharsight Corp., Mountain view, CA). The terminal elimination half-life (t1/2) was calculated as ln2/lz using the slope (lz) from linear regression analysis of the terminal phase of the plasma concentration-time curve on a semilog scale. The DOI 10.1002/jps

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area under the plasma concentration-time curve (AUC0–1) was determined by noncompartmental analysis using the linear trapezoidal rule and extrapolated to infinity as Clast/lz using the last measurable concentration (Clast) and terminal slope (lz). The plasma concentration at time zero (C0) following intravenous administration was estimated by linear extrapolation from the first two time points after dosing. The mean residence time (MRT) was obtained by dividing the area under the first moment curve (AUMC0–1) by AUC0–1, while the mean absorption time (MAT) was the difference in MRT between oral and intravenous administrations. The systemic plasma clearance (CLp) was calculated as intravenous dose divided by AUC0–1, and the volume of distribution at steady state (Vss) was determined as the product of CLp and MRT. The absolute oral bioavailability (F) was calculated as the percentage ratio of mean dose-normalized oral AUC0–1 to dose-normalized intravenous AUC0–1. A twocompartment model with first order absorption and elimination from the central compartment was also used for paclitaxel data to obtain absorption rate constant (ka) along with other parameters such as the elimination rate constant (ke), the distribution and elimination half-lives (t1/2a and t1/2b), and the intercompartmental transfer rate constants (k12 and k21). Statistical analysis was performed using a two-tailed unpaired t-test. A p-value of less than 0.05 was considered statistically significant.

RESULTS Effects of Biochanin A on Pharmacokinetics of Paclitaxel The pharmacokinetic parameters of paclitaxel are listed in Table 1 and its plasma concentration-time curves are shown in Figure 2. The total plasma clearance of paclitaxel in the rat was moderate at 24 mL/min/kg while its volume of distribution at steady state was quite high at 11 L/kg. As indicated in Table 1, the coadministration of biochanin A (BA) at 100 mg/kg significantly increased the AUC0–1 of paclitaxel from 583 to 2204 ng  h/mL at an oral dose of 20 mg/kg, representing a 3.77-fold change (about 277% gain). The Cmax was also markedly changed from 89 to 182 ng/mL, a 2.04-fold increase. In addition, the time to reach maximum plasma concentration (tmax) was prolonged from 1 to 6 h while the JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

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Table 1. Pharmacokinetic Parameters of Paclitaxel after Intravenous and Oral Administrations in Rats (Mean
SD)

Dose (mg/kg) AUC0–1 (ng  h/mL) C0 (ng/mL) Cmax (ng/mL) tmax (h) ka (h1) MRT (h) t1/2 (h) CLp (mL/min/kg) Vss (L/kg) F (%)

IV

PO (without BA)

PO (with BA)

Fold Change

2.0 1447
292 4295
831

20 583
93

20 2204
928a

þ3.77

89.3
15.2 1.01
0 1.83
0.52 6.89
1.90 4.35
0.94

182
47a 6.03
0a 0.158
0.066a 8.01
0.81 4.06
0.93

þ2.04

4.03
1.1

15.2
7.0a

þ3.77

5.41
2.05 11.7
2.9 23.6
3.8 11.2
3.7

a Statistically significant difference between oral treatments with and without biochanin A, p < 0.05.

absorption rate constant (ka) was reduced from 1.83 to 0.158/h based on a two-compartment model with first order absorption and elimination from the central compartment. The concomitant

administration of biochanin A did not significantly affect the MRT and terminal half-life (t1/2). The oral bioavailability of paclitaxel was significantly enhanced from 4 to 15% in the rat.

Figure 2. Plasma concentration-time curves of three Pgp substrates administered intravenously (*) and orally in the presence (&) and absence (~) of biochanin A: paclitaxel (a), digoxin (b), and fexofenadine (c). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

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Effects of Biochanin A on Pharmacokinetics of Digoxin The total plasma clearance of digoxin in the rat was moderate at 25 mL/min/kg, as determined from the plasma concentration-time profile of intravenously administered digoxin (shown in Tab. 2 and Fig. 2). The steady-state volume of distribution was large at 6 L/kg. As shown in Table 2, the coadministration of biochanin A at 100 mg/kg significantly altered AUC0–1 of digoxin from 3894 to 6823 ng  h/mL at an oral dose of 20 mg/kg, a 1.75-fold change (a 75% increase). The Cmax was also markedly elevated from 313 to 536 ng/mL, up 1.71-fold. However, the concomitant administration of biochanin A did not significantly affect the time to reach maximum plasma concentration (tmax), MRT, and terminal half-life (t1/2). The oral bioavailability of digoxin was significantly raised from 28 to 50% in the rat.

Effects of Biochanin A on Pharmacokinetics of Fexofenadine As shown in Table 3, the total plasma clearance and the volume of distribution at steady state of fexofenadine were moderate at 21 mL/min/kg and 1.4 L/kg, respectively. The coadministration of biochanin A changed the AUC0–1 of fexofenadine from 411 to 285 ng  h/mL, a 0.693-fold decrease (about 31% loss). The Cmax was also significantly lowered from 90 to 39 ng/mL, representing a 0.429-fold reduction (a 57% decrease). The coadministration of biochanin A did not show any significant effect on tmax, MRT, and t1/2. Consis-

1989

tent with a decrease in AUC0–1, the oral bioavailability of fexofenadine was reduced from 2.6 to 1.8%.

DISCUSSION Biochanin A has been shown to inhibit Pgpmediated efflux in Caco-2 cells.2,3 The aim of the present study is to investigate the effects of biochanin A on the oral bioavailability and pharmacokinetics of three model Pgp substrates: paclitaxel, digoxin, and fexofenadine. Paclitaxel is a substrate for Pgp efflux transporter and CYP3A metabolizing enzymes.12,13 Biochanin A inhibits Pgp, leading to enhanced oral absorption of paclitaxel through rat small intestine. It has been reported that genistein, a metabolite of biochanin A, has shown inhibitory activity against rat CYP3A.14 Biochanin A can be converted to genistein in the small intestine by the P450 isozymes and therefore indirectly inhibits rat CYP3A. As many other flavonoids can inhibit various P450 isozymes,4 biochanin A may also directly inhibit CYP3A in the rat intestine, leading to the increased peak plasma concentration and oral bioavailability of paclitaxel. Therefore, the significantly enhanced bioavailability of paclitaxel could be due to the synergistic inhibition of both CYP3A and Pgp in the rat intestine. When the plasma concentration-time curves of paclitaxel with and without coadministration of biochanin A are compared in Figure 2, the level of paclitaxel in the presence of biochanin A is low initially and then slowly increases to reach a maximum at a

Table 2. Pharmacokinetic Parameters of Digoxin after Intravenous and Oral Administrations in Rats (Mean
SD)

Dose (mg/kg) AUC0–1 (ng  h/mL) C0 (ng/mL) Cmax (ng/mL) tmax (h) MRT (h) t1/2 (h) CLp (mL/min/kg) Vss (L/kg) F (%)

IV

PO (without BA)

PO (with BA)

Fold Change

2.0 1368
248 1833
189

20 3894
518

20 6823
730a

þ1.75

313
61 5.33
1.15 10.8
2.6 7.26
1.85

536
95a 5.01
1.41 10.7
1.0 7.04
1.03

þ1.71

28.5
6.2

49.9
10.1a

þ1.75

4.31
2.27 10.1
3.3 25.0
4.8 6.08
2.25

a Statistically significant difference between oral treatments with and without biochanin A, p < 0.05.

DOI 10.1002/jps

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Table 3. Pharmacokinetic Parameters of Fexofenadine after Intravenous and Oral Administrations in Rats (Mean
SD)

Dose (mg/kg) AUC0–1 (ng  h/mL) C0 (ng/mL) Cmax (ng/mL) tmax (h) MRT (h) t1/2 (h) CLp (mL/min/kg) Vss (L/kg) F (%)

IV

PO (without BA)

PO (with BA)

Fold Change

2.0 1611
256 3669
234

20 411
31

20 285
43a

0.693

90.2
20.5 1.33
0.58 4.27
0.67 4.96
0.50

38.7
3.0a 3.00
1.73 5.63
0.70 4.13
0.29

0.429

2.55
0.5

1.77
0.4a

0.694

1.14
0.05 5.23
0.70 21.0
3.1 1.43
0.16

a

Statistically significant difference between oral treatments with and without biochanin A, p < 0.05.

later time point. The increase in the time to reach maximum plasma concentration, tmax, from 1 to 6 h, may indicate significant effects of biochanin A on the interplay between the interactions of paclitaxel with Pgp and CYP3A along the rat intestinal tract, where CYP3A is more concentrated in the duodenum while Pgp expresses more in the ileum.15 The lower concentrations of paclitaxel at the beginning and a smaller absorption rate constant observed for paclitaxel with biochanin A treatment could be attributed to the reduced solubility of paclitaxel in the presence of biochanin A in the GI tract as both compounds are quite hydrophobic. The plasma concentrationtime profile of biochanin A revealed a secondary peak (ca 50 ng/mL) at about 6 h postdose due to enterohepatic recirculation (data not shown), which is consistent with the tmax observed for paclitaxel with coadministration of biochanin A. Therefore, the increased levels of biochanin A lead to increased inhibition of CYP3A and Pgp, which in turn resulted in higher concentrations of paclitaxel after 3 h and a delayed tmax. Because the plasma levels of paclitaxel with coadministration of biochanin A are much greater after about 3 h, the AUC and therefore oral bioavailability are significantly increased. It appears that the concomitant and synergistic inhibition of both Pgp and CYP3A in the rat intestine is a plausible explanation for the large increase in oral bioavailability of paclitaxel. It is likely that the inhibitory selectivity and potency (e.g., Ki or IC50) of biochanin A for Pgp and CYP3A may overlap with the substrate binding selectivity and affinity (e.g., Km or kcat/Km) of paclitaxel for Pgp and CYP3A, leading to this marked increase in oral absorption. A lack of significant changes in MRT and terminal JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

half-life (t1/2), may suggest that the coadministration of biochanin A has a negligible effect on the systemic (hepatic/renal) elimination of paclitaxel. This can be rationalized based on the opposing effects of inhibiting Pgp and CYP3A in the liver and/or kidney, where inhibition of metabolizing enzymes increases systemic AUC while inhibition of apical efflux transporters reduces drug levels owing to the increased exposure to the enzymes. The systemic plasma clearance of paclitaxel is moderate (24 mL/min/kg) relative to hepatic blood flow but the steady-state volume of distribution is high (11 L/kg), indicating an extensive tissue distribution of the drug. In humans, digoxin has been found to be a good substrate for OATP-8 and Pgp, but not for CYP3A4.11,16 In rats, however, digoxin is a good substrate for Oatp2, Pgp, and CYP3A.16,17 Since Oatp2 is not expressed in the small intestine,18 biochanin A can only inhibit Pgp and CYP3A in the rat intestine, leading to an additive effect on the enhancement of oral absorption of digoxin. The magnitude of increase in AUC or Cmax by coadministration of biochanin A is smaller with digoxin than with paclitaxel, which may be related to the difference in how oral absorption is limited between these two drugs—Pgp efflux versus CYP3A metabolism, although diffusional clearance may also play a role. It has been suggested that the increase in AUC of digoxin in the rat by oral coadministration of a dual Pgp and CYP3A inhibitor, ketoconazole, is due to the preferential inhibition of Pgp over CYP3A in the small intestine.19 It is likely in this study that the oral absorption of digoxin in the rat is differentially limited by CYP3A and Pgp with CYP3A being a predominant factor, and biochanin A may exhibit DOI 10.1002/jps

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preferential inhibition of Pgp over CYP3A, leading to a smaller magnitude of increase in AUC of digoxin than that of paclitaxel. It appears that the binding selectivity and affinity of biochanin A as an inhibitor may not coincide with those of digoxin as a substrate for Pgp and CYP3A4, resulting in a less than optimal increase in oral absorption. The lack of effects of coadministration of biochanin A on the time to reach maximum concentration (tmax), terminal half-life (t1/2), MRT, and thus MAT, may indicate a minimal effect of biochanin A on the systemic elimination of digoxin. It has been well-documented that fruit juices including grapefruit juice inhibit OATPs in addition to their inhibitory activities against Pgp and CYP3A4 in the intestine.5 It has also been demonstrated recently that biochanin A and its metabolite genistein can inhibit OATP-C uptake transporter.20 Therefore, it is possible that biochanin A may also have inhibitory activities against other human OATPs and rat Oatps such as Oatp2. Because Oatp2, Pgp, and CYP3A are all highly expressed in the liver, the inhibition of Oatp2 and CYP3A in the liver would increase digoxin levels while the inhibition of Pgp in the liver would instead decrease plasma digoxin levels. The absence of significant changes in tmax, MRT, and t1/2 may represent a balanced interplay of interactions of biochanin A with the uptake transporter (Oatp2), efflux pump (Pgp), and metabolizing enzymes (CYP3A) in the liver. It is also possible that biochanin A levels in the systemic circulation were not high enough to affect the interactions of digoxin with those influx/efflux transporters and CYP3A isozymes. The moderate systemic clearance of digoxin (25 mL/min/kg) is comparable with the literature values,19,21 whereas the large value of volume of distribution at steady state (6 L/kg) may indicate a significant tissue distribution of digoxin in the rat. Fexofenadine is a substrate for Pgp, Oatp1, Oatp2, and Oatp3 in rats and a substrate for OATP-A and -B in humans.11 Although Oatp1, Oatp2, and Oatp3 are all expressed in rat liver, kidney, and brain, only Oatp3 is highly expressed in the rat small intestine.18 Ample evidences have suggested that many inhibitors of Pgp are also inhibitors of OATPs and CYP3A4.22 As discussed previously, biochanin A may inhibit many rat Oatp uptake transporters, as do other fruit juices such as grapefruit juice,23,24 orange juice and apple juice.25 Therefore, it is reasonable to postulate that biochanin A may inhibit rat Oatp1, Oatp2 and Oatp3. In this case, biochanin A may preferenDOI 10.1002/jps

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tially inhibit Oatp3 over Pgp in the rat small intestine, leading to the reduced oral absorption and therefore decreased oral bioavailability. Recent studies on the preferential inhibition of human OATPs and rat Oatps over Pgp efflux transporter by fruit juices during oral absorption of fexofenadine support this hypothesis.25,26 Here, the inhibition of Pgp in the small intestine enhances oral absorption, while the inhibition of Oatp3 decreases the uptake of fexofenadine. The decreased AUC and Cmax of fexofenadine with coadministration of biochanin A suggest that fexofenadine is preferentially absorbed by Oatpmediated uptake with biochanin A being a more potent inhibitor of Oatps than Pgp. These observations are consistent with the reported effects of several fruit juices on the oral bioavailability of fexofenadine in rats and humans.25,26 The lack of significant changes in t1/2 and MRT may indicate that biochanin A has negligible effects on hepatic elimination or systemic clearance of fexofenadine. This could result from the opposing effects of inhibition of Pgp and Oatps by biochanin A in the liver and/or kidney, where inhibition of Pgp decreases fexofenadine levels whereas inhibition of Oatps increases them due to the reduced exposure of fexofenadine to the metabolizing enzymes. Fexofenadine showed a moderate systemic plasma clearance (21 mL/min/kg) with a medium steady-state volume of distribution (1.4 L/ kg), indicating a similar drug binding between plasma and tissues and thus a uniform distribution of the drug throughout the body. In all cases, biochanin A has no statistically significant effects on the MRTs and terminal halflives of the three substrates, suggesting that biochanin A has negligible effects on hepatic elimination or systemic clearance of the substrates. It is apparent that the interplay among uptake transporters, efflux pumps, and metabolizing enzymes modulates oral absorption and systemic elimination of their substrate drugs. Therefore, the concomitant administration of transporter/ enzyme inhibitors such as biochanin A may significantly alter the oral bioavailability and pharmacokinetic properties of their substrate drugs. This interplay can be either harmful or beneficial. As can be postulated from this investigation, biochanin A can be used as an excipient in the oral formation to increase the oral absorption and bioavailability of the drugs that are the substrates for Pgp and CYP3A such as paclitaxel and digoxin. The main advantage of using biochanin A instead of other Pgp and P450 inhibitor JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

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drugs is that biochanin A not only is very safe but also has other therapeutic properties such as anticancer and anti-inflammation properties.

CONCLUSIONS The concomitant administration of naturally occurring dietary supplement biochanin A significantly modulates the oral bioavailability and pharmacokinetics of three Pgp substrate drugs, paclitaxel, digoxin, and fexofenadine. Oral absorption and bioavailability of paclitaxel and digoxin are markedly increased (3.77- and 1.75fold increase in bioavailability for paclitaxel and digoxin, respectively) while those of fexofenadine are significantly reduced (a 0.694-fold decrease in bioavailability). The observed effect of increased oral absorption by coadministration of biochanin A may be beneficial in that biochanin A can be formulated in the dosage forms to improve the oral bioavailability of the drugs that are the substrates for Pgp and CYP3A. Compared with coadministration of other marketed inhibitor drugs, biochanin A not only is a much safer additive but also possesses beneficial properties for human health such as anticancer and antiinflammation properties. However, if biochanin A is digested in a human diet or as a human dietary supplement, the higher levels of paclitaxel and digoxin could lead to adverse side effects as a result of this food–drug interaction. The same applies to fexofenadine where the oral bioavailability was decreased by coadministration of biochanin A, which could lead to ineffectiveness of the drug. The effects of coadministration of biochanin A on hepatic elimination or systemic clearance appear to be negligible as no statistically significant changes in MRT and terminal elimination half-life were observed for all three Pgp substrate drugs. The findings of this study have demonstrated the importance of the interplay among different uptake/efflux transporters and metabolizing enzymes. Therefore, precaution should be exercised when taking biochanin A-containing food or dietary supplements along with the drugs that are the substrates for Pgp/ OATP transporters and the CYP3A4 isozyme.

ACKNOWLEDGMENTS We thank Leslie Chalecki and Casey Cattell for their assistance in animal studies. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006

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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 9, SEPTEMBER 2006