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Research ArticleArticle

Effect of Coadministration of Single and Multiple Doses of Rifampicin on the Pharmacokinetics of Fexofenadine Enantiomers in Healthy Subjects

Hiroyuki Kusuhara, Masatomo Miura, Norio Yasui-Furukori, Kenta Yoshida, Yumiko Akamine, Miyu Yokochi, Shinya Fukizawa, Kazuaki Ikejiri, Kayoko Kanamitsu, Tsukasa Uno and Yuichi Sugiyama
Drug Metabolism and Disposition January 2013, 41 (1) 206-213; DOI: https://doi.org/10.1124/dmd.112.048330

Abstract

The effect of rifampicin on the pharmacokinetics of fexofenadine enantiomers was examined in healthy subjects who received fexofenadine alone or with single or multiple doses of rifampicin (600 mg). A single coadministered dose of rifampicin significantly decreased the oral clearance (CLtot/F) and renal clearance (CLr) of S- and R-fexofenadine by 76 and 62%, and 73 and 62%, respectively. Even after multiple doses, rifampicin significantly decreased these parameters, although the effect on the CLtot/F was slightly blunted. Multiple doses of rifampicin abolished the difference in the CLtot/F of fexofenadine enantiomers, whereas the stereoselectivity in the CLr persisted. Rifampicin inhibited the uptake of fexofenadine enantiomers by human hepatocytes via organic anion transporter (OAT) OATP1B3 and its basal-to-apical transport in Caco-2 cells, but not OAT3-mediated or multidrug and toxic compound extrusion 1 (MATE1)–mediated transport. The plasma-unbound fraction of S-fexofenadine was 1.8 times higher than that of R-fexofenadine. The rifampicin-sensitive uptake by hepatocytes was 1.6 times higher for R-fexofenadine, whereas the transport activities by OATP1B3, OAT3, MATE1, or P-glycoprotein were identical for both enantiomers. S-fexofenadine is a more potent human histamine H1 receptor antagonist than R-fexofenadine. In conclusion, rifampicin has multiple interaction sites with fexofenadine, all of which contribute to increasing the area under the curve of fexofenadine when they are given simultaneously, to surpass the effect of the induction of P-glycoprotein elicited by multiple doses.

Introduction

Drug–drug interactions (DDIs) involving metabolism and/or excretion processes alter the pharmacokinetics of victim drugs, and consequently their pharmacological/adverse effects. They are one of the leading causes of withdrawal of new drugs from the market. Recent progress in the study of membrane transport has expanded our understanding of the mechanisms underlying pharmacokinetic DDIs involving transporters (Giacomini et al., 2010; Yoshida et al., 2012).

Fexofenadine, a selective and nonsedative histamine H1 receptor antagonist, is one of the most frequently used treatments for seasonal allergic rhinitis and chronic urticaria. Because fexofenadine is hardly metabolized by cytochrome P450, transporters play an indispensable role in fexofenadine pharmacokinetics. Actually, it is a substrate of OATP1A2, OATP1B3, OATP2B1, OAT3, MATE1, BSEP, MRP2, MRP3, and P-glycoprotein (P-gp) (Cvetkovic et al., 1999; Nozawa et al., 2004; Shimizu et al., 2005; Tahara et al., 2005, 2006; Matsushima et al., 2008a, 2009; Tian et al., 2008). A number of drug interaction studies have been conducted in healthy subjects where fexofenadine was used as victim drug. These studies show the impact of inhibition of P-gp-mediated efflux in the small intestine by erythromycin, itraconazole, ketoconazole, ritonavir and verapamil on the systemic exposure of fexofenadine (Dresser et al., 2002; Tachibana et al., 2009) and the impact of inhibition of OAT3 and MATE1 by probenecid and cimetidine, respectively, on the urinary excretion and systemic exposure of fexofenadine (Yasui-Furukori et al., 2005; Tahara et al., 2006; Matsushima et al., 2009). However, the impact of inhibition of hepatic elimination on fexofenadine pharmacokinetics remains to be evaluated although hepatic elimination is the major elimination pathway of fexofenadine from the systemic circulation (Lappin et al., 2010).

We suggested that OATP1B3, a multispecific organic anion transporter (OAT) predominantly expressed in the liver, mediates the hepatic uptake of fexofenadine based on in vitro experiments (Shimizu et al., 2005). In addition, a pharmacogenomic study focused on the genotypes of OATP1B1, another hepatic uptake transporter, suggests that OATP1B1 also mediate the hepatic uptake of fexofenadine (Niemi et al., 2005). Rifampicin, which is used to treat tuberculous meningitis, is well known to induce drug-metabolizing enzymes and transporters through the activation of the nuclear receptor pregnane X receptor, and consequently to lower the systemic exposure of victim drugs. Indeed, Hamman et al., (2001) reported that multiple doses of rifampicin markedly reduced the area under the curve (AUC) of fexofenadine in healthy subjects. On the other hand, simultaneous administration of rifampicin increases the systemic exposure of organic anion transporting polypeptide (OATP) substrates, such as atorvastatin, glibenclamide, and pravastatin, by inhibiting their hepatic uptake by OATP1B1 (Lau et al., 2007; Zheng et al., 2009; Maeda et al., 2011). Because the Ki value of rifampicin for OATP1B3 is similar to that for OATP1B1 (Matsushima et al., 2008b), rifampicin will inhibit OATP1B3 and consequently increase the AUC of its substrate drugs when both drugs are given simultaneously. In the previous study conducted by Hamman et al., because the final dose of rifampicin was given at bedtime, inhibition of OATPs by rifampicin became negligible when fexofenadine was given to the subjects. Furthermore, fexofenadine is administered therapeutically as a racemic mixture of S- and R-enantiomers. The plasma concentration of S-fexofenadine in humans is about 1.5 times lower than that of the corresponding R-fexofenadine, although the underlying mechanism of this difference remains unknown (Robbins et al., 1998; Miura et al., 2007). Notably, the impact of DDI on the CLtot/F of fexofenadine is stereoselective: a single dose of itraconazole or multiple doses of carbamazepine blunted the stereoselective pharmacokinetics of fexofenadine enantiomers (Tateishi et al., 2008; Sakugawa et al., 2009; Akamine et al., 2012).

We designed this clinical study to investigate the effect of coadministered rifampicin on the pharmacokinetics of fexofenadine enantiomers in healthy subjects who receive single and multiple doses of rifampicin, and we conducted in vitro studies to support the clinical data.

Materials and Methods

[3H]Estrone-3-sulfate (54.3 Ci/mmol) and [pyridinyl-5-3H]-pyrilamine ([3H]-mepyramine, 20 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA), and [3H]CCK-8 (97.5 Ci/mmol) from Amersham Biosciences UK Ltd. (Little Chalfont, Buckinghamshire, UK). R- and S-fexofenadine were purchased from Toronto Research Chemicals Inc. (ON, Canada), and cetirizine was purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals used were commercially available and of analytical grade. Cryopreserved human hepatocytes (TDH and 03-013) were purchased from In Vitro Technologies Inc. (Baltimore, MD) and Life Technologies (Durham, NC), respectively, and kept in liquid nitrogen until use.

Clinical Study in Healthy Subjects.

Clinical study was conducted at Hirosaki University. Twelve healthy Japanese volunteers (eight men and four women) were enrolled in this study after giving informed written consent. Each subject was physically normal according to clinical examination and routine laboratory testing and had no history of significant medical illness or hypersensitivity to any drugs. Their mean values for age and body weight were 23.8 ± 5.6 years (range, 20–41 years) and 55.7 ± 7.8 kg (range, 42–70 kg), respectively. The Ethics Committee of Hirosaki University School of Medicine approved this study.

This randomized open-label study consisted of two (control and 6-day treatment) phases and four study days. In the control phase, the volunteers received 60 mg of fexofenadine hydrochloride (Allegra; Aventis Pharma Ltd, Tokyo, Japan) at 8:00 AM after an overnight fast. In the treatment phase, the volunteers received 600 mg rifampicin (Rifadin; Daiichi-Sankyo Pharmaceutical, Tokyo, Japan) at 8:00 AM for 6 days. On days 1, 3, and 6, each subject was simultaneously administered 60 mg of fexofenadine hydrochloride with rifampicin after an overnight fast. The order of the two phases was randomly assigned for each volunteer. Six volunteers started the control phase first, and their treatment phase was started more than 24 hours after the last blood sampling of the control phase. The remaining subjects started the treatment phase first, and their control phase was started more than 2 weeks after the last sampling. The volunteers did not take any other medication or consume fruit juice for at least 7 days before each phase, and no food or beverages were allowed until 3 hours after fexofenadine administration.

Blood samples (10 ml each) were drawn before and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, and 24 hours after fexofenadine administration. Time points for blood sampling follow those previously reported (Yasui-Furukori et al., 2005). A spot of urine was collected as a blank sample before fexofenadine administration; thereafter, the urine was collected until 24 hours after administration. Plasma and urine concentrations of fexofenadine were determined by the high-pressure liquid chromatography method developed in our laboratory (Miura et al., 2007). Plasma and urine samples from the treatment phase did not have any interfering peak for the assay, and the plasma and urine samples before fexofenadine administration had no detectable fexofenadine peak.

The maximum plasma concentration (Cmax) and the time to reach Cmax (tmax) were determined directly from the observed data. The elimination rate constant (λz) was obtained by linear regression analysis by use of at least three sampling points of the terminal log-linear declining phase to the last measurable concentration. The elimination half-life (t1/2) was calculated as 0.693 divided by λz. The area under the plasma concentration–time curve from time zero to infinity (AUC0–∞) was calculated using AUC (0–12) + Clast/λz, where Clast was the plasma drug concentration at 12 hours. The oral clearance (CLtot/F), apparent volume of distribution (Vd/F), and renal clearance (CLr) were obtained using the following equations:Embedded ImageandEmbedded Image

Determination of the Uptake of Fexofenadine by cDNA-Transfected Cells.

OATP1B1-, OATP1B3-, OAT3-, MATE1-, and MATE2-K-expressing HEK293 cell lines were constructed previously (Deguchi et al., 2004; Hirano et al., 2004; Matsushima et al., 2009). The transport study was performed as described previously (Hirano et al., 2004). Briefly, the cells were incubated with Krebs-Henseleit buffer (118 mM NaCl, 23.8 mM NaHCO3, 4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, 12.5 mM HEPES, 5.0 mM glucose, and 1.53 mM CaCl2 adjusted to pH 7.4) containing radiolabeled or nonradiolabeled compounds at 37°C in the presence or absence of inhibitors. Uptake was terminated by the addition of ice-cold buffer after removal of the incubation buffer. The fexofenadine concentration associated with the cell specimens was determined by liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis.

Determination of the Uptake of Fexofenadine by Cryopreserved Human Hepatocytes.

The uptake of fexofenadine was determined in cryopreserved human hepatocytes as reported previously, with minor modifications (Shitara et al., 2003). Briefly, hepatocytes were suspended in Krebs-Henseleit buffer to give a cell density of 1.0 × 106 viable cells/ml for the uptake study. After preincubation at 37°C for 3 minutes, uptake was initiated by adding labeled and unlabeled substrates to the cell suspension, and was terminated at the designated time by separating the cells from the substrate solution using a layer of oil mixture (density 1.015, a mixture of silicone oil and mineral oil; Sigma-Aldrich) and centrifugation for 10 s in a tabletop centrifuge (10,000g; Beckman Microfuge E; Beckman Coulter, Inc., Fullerton, CA). During this process, the hepatocytes passed through the oil layer into the lower layer of 2 N NaOH for estrone-3-sulfate and CCK-8 or 5 M ammonium acetate for fexofenadine. The amount of substrate in each compartment was measured with a liquid scintillation counter or LC–MS/MS.

Determination of Transcellular Transport of Fexofenadine across Caco-2 Cells.

Briefly, Caco-2 cells were cultured on Transwell membrane inserts (6.5 mm diameter, 0.4-μm pore size; Corning Life Sciences, Lowell, MA) for 25–30 days. Cells were first washed with Krebs-Henseleit buffer at 37°C. Subsequently, fexofenadine and inhibitors (1 ml) were added to the basolateral compartment to start the experiments. Simultaneously, inhibitors were added to the apical compartment (250 μl). After 90 minutes of incubation, 100 μl of the buffer in the apical compartment was collected.

Quantification of Fexofenadine and Celiprolol by LC–MS/MS.

Concentrations of fexofenadine and celiprolol were measured on an AB SCIEX QTRAP 5500 mass spectrometer (Applied Biosystems, Foster City, CA) equipped with a Prominence high-pressure liquid chromatography system (Shimadzu Corporaton, Kyoto, Japan) operated in the positive electrospray ionization mode. Chromatographic separation was achieved on an Inertsil ODS-4 column (30 mm × 2.1 mm internal diameter, 3 μm; GL Science, Tokyo, Japan) in binary gradient mode at a flow rate of 0.4 ml/min. The mobile phase comprised 0.1% formic acid and acetonitrile. The acetonitrile concentration was initially 5 or 20%; it was then linearly increased to 80% or 90% over 1 or 2 minutes, and maintained for a further 0.5 or 1.2 minutes. Finally, the column was re-equilibrated with acetonitrile at a concentration of 5 or 20% for 1 minute. Mass-to-charge transitions were 502.4–466.0 for fexofenadine, and 380.1–251.2 for celiprolol, respectively.

Membrane Preparation and Binding Assay.

Human histamine H1 receptor (D28481.1) was cloned from a human cerebral cortex cDNA library. Transient transfection of HEK293 cells was performed using pcDNA3.1(+) (Life Technologies Corporation, Carlsbad, CA) and FuGENE HD (Roche, Roche Applied Science, Penzberg Upper Barvaria, Germany) according to the manufacturers’ protocols. The [3H]-pyrilamine binding assay was performed as described previously (Ohuchi et al., 1998; Gillard and Chatelain, 2006). Briefly, membranes prepared from the cDNA transfectants (25 μg of protein) were incubated for 60 minutes at 37°C in 0.5 ml of 50 mM Na2/K phosphate buffer (pH 7.4) containing [3H]-mepyramine (3 nM) and competing drugs. Samples were rapidly filtered by vacuum through 0.1% polyethylenimine-pretreated 24-mm glass fiber filters (Whatman GF/C; Whatman, Clifton, NJ), and the filters were washed 4 times with 2 ml of ice-cold Na2/K phosphate buffer. Nonspecific binding was defined as the amount of [3H]-pyrilamine bound in the presence of 10 μM cetirizine. Radioactivity retained on the filter was measured in a liquid scintillation counter (LS 6000SE; Beckman Instruments, Fullerton, CA). Data were analyzed according to the model proposed by Motulsky and Mahan (1984).

Statistical Analysis.

The data are presented as means ± standard error (S.E.). The Student’s two-tailed nonpaired t test and a one-way analysis of variance followed by Dunnett’s post hoc test were used to identify significant differences between groups where appropriate. P < 0.05 was considered significant.

Results

Effect of Single and Multiple Doses of Rifampin on the Pharmacokinetics of Fexofenadine.

No clinically undesirable signs or symptoms attributable to the administration of fexofenadine and rifampicin were recognized during the study. All subjects completed the study successfully according to the protocol.

Plasma concentrations of fexofenadine enantiomers in healthy subjects who received fexofenadine alone or with single or multiple doses of rifampicin are shown in Fig. 1. The plasma concentration of fexofenadine enantiomers at last sampling point (24 after the administration) was below the lower limit of quantification. The pharmacokinetic parameters of fexofenadine enantiomers are summarized in Table 1. There was no sex difference in the pharmacokinetic parameters of fexofenadine enantiomers (data not shown). A single dose of rifampicin significantly decreased the CLtot/F and CLr by 77 and 62%, respectively, for S-fexofenadine, and by 73 and 61%, respectively, for R-fexofenadine. Rifampicin also significantly decreased the CLtot/F and CLr for S-fexofenadine by 66 and 54%, respectively and for R-fexofenadine by 57 and 54%, respectively, after multiple doses. Individual data are shown in Fig. 2. The CLtot/F and CLr of S-fexofenadine were 1.5 and 1.7 times larger than those of R-fexofenadine (Table 1). Multiple doses of rifampicin abolished the difference in the CLtot/F of fexofenadine enantiomers, whereas the stereoselectivity in the CLr persisted both after single and multiple doses. The unbound fraction of fexofenadine in human plasma, measured using equilibrium dialysis, was stereoselective: 1.8 times higher for S-fexofenadine than for R-fexofenadine (0.235 ± 0.019 versus 0.134 ± 0.005 [mean ± S.E.]).

Fig. 1.
Fig. 1.

Effect of single and multiple doses of rifampicin on the plasma concentrations of fexofenadine enantiomers in healthy subjects. Healthy volunteers received fexofenadine alone (circles) or with rifampicin as a single dose (squares) or multiple doses (triangles). Plasma concentrations of R- and S-fexofenadine (closed and open symbols, respectively) were determined by high-performance liquid chromatography. Each point represents the mean ± S.E. (n = 12). R-FEX, R-fexofenadine; RIF, rifampicin; S-FEX, S-fexofenadine.

View this table:
TABLE 1

Pharmacokinetic parameters of the pharmacokinetics of fexofenadine enantiomers in control and rifampicin-treated subjects

Data are shown as mean (95% confidence interval); tmax data are shown as median (range).

Fig. 2.
Fig. 2.

Effect of single and multiple doses of rifampicin on the CLtot/F and CLr of fexofenadine enantiomers in healthy subjects. CLtot/F and CLr of fexofenadine enantiomers were calculated as described in Materials and Methods. The plots represent the CLtot/F and CLr of S- and R-fexofenadine for individual subjects; the points representing the same individual are connected to one another by dotted lines. The horizontal line in each column indicates the mean values. *P < 0.05, **P < 0.01, and ***P < 0.001 between control phase and rifampicin phase.

Effect of Rifampicin on the Uptake of R- and S-Fexofenadine by OATP1B3.

The uptake of fexofenadine enantiomers was determined in two batches of cryopreserved human hepatocytes (lot TDH and lot 03-013), which were characterized by the difference in the uptake of an OATP1B3 reference substrate, CCK-8 (Fig. 3A). The uptake of both R- and S-fexofenadine was higher in lot TDH than in lot 03-013. Rifampicin significantly inhibited the uptake of fexofenadine only in lot TDH. The level of uptake of fexofenadine that remained in the presence of rifampicin was similar in the two lots. The rifampicin-sensitive uptake of R-fexofenadine was 1.6 times higher than that of S-fexofenadine.

Fig. 3.
Fig. 3.

Effect of rifampicin on the uptake of R- and S-fexofenadine by cryopreserved hepatocytes (A) and by OATP1B3-HEK (B). (A) cryopreserved human hepatocytes (lot TDH and lot 03-013) were incubated at 37°C for 2 minutes (estrone-3-sulfate 18 nM and CCK-8 10 nM) or 5 minutes (fexofenadine, 5 μM) in the presence and absence of rifampicin (RIF) at designated concentrations. (B) mock vector-transfected and OATP1B3-overexpressing HEK293 cells were incubated at 37°C for 10 minutes with fexofenadine enantiomers (5 μM) in the presence and absence of rifampicin at designated concentrations. *Significantly different (P < 0.05) from the uptake in control groups. Each bar represents the mean ± S.E. (n = 3 or 4). Where bars are not shown, the S.E. is contained within the limits of the symbol.

The effect of rifampicin on the uptake of fexofenadine enantiomers was determined in HEK293 cells expressing OATP1B3 (OATP1B3-HEK) (Fig. 3B). The uptake of fexofenadine enantiomers was significantly greater in OATP1B3-HEK than in control mock vector-transfected cells. Rifampicin inhibits OATP1B3-mediated uptake of fexofenadine enantiomers, and the specific uptake by OATP1B3 was abolished at 2 µM. There was no detectable stereoselectivity in the transport activities by OATP1B3 and the inhibition potency of rifampicin. The uptake of fexofenadine enantiomers (microliters per milligram of protein per 5 minutes) was similar in mock vector-transfected and OATP1B1-HEK cells, at 1.92 ± 0.19 and 2.17 ± 0.15 for R-fexofenadine, and 1.78 ± 0.11 and 2.00 ± 0.18 for S-fexofenadine, respectively (mean ± S.E., n = 3–6).

Effect of Rifampicin on the Uptake of R- and S-Fexofenadine by OAT3 and MATE1.

The uptake of fexofenadine enantiomers was also determined in OAT3- and MATE1-HEK in the presence and absence of rifampicin or the representative inhibitors (benzylpenicillin for OAT3 and tetraethylammonium for MATE1 and MATE2-K) (Fig. 4). Compared with mock vector-transfected cells, OAT3- and MATE1-HEK cells showed significantly greater uptake of fexofenadine enantiomers, which was markedly inhibited by the representative inhibitors, but not by rifampicin at 80 µM.

Fig. 4.
Fig. 4.

Effect of rifampicin on the uptake of R- and S-fexofenadine by OAT3- and MATE1- and MATE2-K HEK. Mock vector-transfected and OAT3-, MATE1-, and MATE2-K-overexpressing HEK293 cells were incubated at 37°C for 5 minutes with fexofenadine enantiomers (5 μM) in the presence or absence of rifampicin or representative inhibitors (benzylpenicillin 10 mM for OAT3, tetraethylammonium 10 mM for MATE1 and MATE2-K) at designated concentrations. *P < 0.05 and **P < 0.01 versus R- or S-fexofenadine uptake by mock vector-transfected cells. †P < 0.01 versus R- or S-fexofenadine uptake by cells expressing the corresponding transporter (OAT3 or MATE1) without inhibitors. Each bar represents the mean ± S.E. (n = 3 or 4).

Effect of Rifampicin on the Basal-to-Apical Transport of R- and S-Fexofenadine in Caco-2 Cells.

The basal-to-apical transport of fexofenadine across a monolayer of Caco-2 cells was 7.1-fold higher than the apical-to-basal transport (Papp 4.97 ± 0.16 × 106 versus 0.70 ± 0.01 × 106 cm/s). Verapamil significantly inhibited this basal-to-apical transport by 61%, but did not affect the apical-to-basal transport (data not shown). Celiprolol was used as the reference P-gp substrate (Fig. 5A). Rifampicin significantly inhibited the transport of R- and S-fexofenadine with similar potency (Fig. 5B). It also inhibits the basal-to-apical transport of celiprolol, but the inhibition potency for celiprolol was lower than that for fexofenadine enantiomers (Fig. 5). There was no detectable stereoselectivity in the basal-to-apical transport of R- and S-fexofenadine (Fig. 5).

Fig. 5.
Fig. 5.

Effect of rifampicin on the basal-to-apical transport of celiprolol (A) and R- and S-fexofenadine (B) across a monolayer of Caco-2 cells. Basal-to-apical transport of (A) celiprolol (2 μM) and (B) fexofenadine enantiomers (4.5 μM) for 90 minutes across a monolayer of Caco-2 cells was determined at 37°C in the presence or absence of rifampicin at designated concentrations. The cyclosporine A (Cys A) concentration in (A) was 20 μM. *Significantly different (P < 0.05) from the transport in control groups. Each bar represents the mean ± S.E. (n = 3).

Stereoselectivity of R- and S-Fexofenadine Binding to Human H1 Receptor.

Transient transfection of human H1 receptor cDNA induces specific binding of [3H]-mepyramine with a Kd and Bmax of 3.7 ± 0.5 nM and 3.8 ± 0.1 pmol/mg protein, respectively. The competition of fexofenadine with [3H]-mepyramine binding was examined, through which the Ki values of R- and S-fexofenadine were determined to be 30 ± 3 and 13 ± 3 nM, respectively (Fig. 6A). Kinetic analyses of the inhibition [3H]-mepyramine binding by fexofenadine enantiomers were also performed (Fig. 6B). Based on the dissociation of [3H]-mepyramine (koff = 1.43 min–1), the association constant (kon) and dissociation constant (koff) of R- and S-fexofenadine were calculated to be 0.86 ± 0.13 and 0.92 ± 0.02 ml/nmol per minutes, and 0.032 ± 0.008 and 0.017 ± 0.002 min–1, respectively.

Fig. 6.
Fig. 6.

Evaluation of R- and S-fexofenadine binding to human H1 receptor. (A) competition of R- and S-fexofenadine against [3H]- mepyramine binding to membranes expressing human H1 receptor was determined. (B) [3H]-mepyramine association kinetics were determined in the presence or absence of R- and S-fexofenadine (200 and 100 nM, respectively). Solid lines represent the fitted line. Each symbol represents the mean ± S.E. (n = 3).

Discussion

This study examined the effect of single and multiple doses of rifampicin on the pharmacokinetics of fexofenadine enantiomers in healthy subjects to demonstrate the impact of OATP1B3 inhibition.

A single dose of rifampicin significantly decreased the CLtot/F and CLr of fexofenadine enantiomers. Based on the reported contribution of urinary excretion to systemic elimination [38% (Lappin et al., 2010)], the magnitude of the interaction of rifampicin with the CLtot/F of fexofenadine cannot be explained only by the reduction in CLr. Coadministration of rifampicin also inhibits the hepatic elimination of fexofenadine and/or increases its bioavailability. Because of a negligible hepatic first-pass effect, the bioavailability of fexofenadine, which was reported to be 0.30 at its therapeutic dose (Lappin et al., 2010), is attributable to its absorption and solubility in the intestine. Given that rifampicin increases the bioavailability of fexofenadine, the inhibition of intestinal efflux can be the underlying mechanism.

To evaluate the relevance of the inhibition of hepatic elimination by rifampicin, in vitro inhibition studies were conducted in cryopreserved human hepatocytes and cDNA-transfected cells. Two batches of cryopreserved hepatocytes were used in this study. The two batches of hepatocytes showed very similar transport activity of the OATP1B1 reference substrate, but hepatocytes from batch number 03-013 showed 5-fold lower transport activity of the OATP1B3 reference substrate than batch TDH (Fig. 3A). The uptake of fexofenadine by TDH hepatocytes, but not by 03-013 hepatocytes, was inhibited by rifampicin (Fig. 3A), suggesting that rifampicin-sensitive uptake of fexofenadine in the hepatocytes could be related to their OATP1B3 activity. We speculate that OATP1B3 makes a greater contribution than OATP1B1 to rifampicin-sensitive uptake in hepatocytes. Although the specific uptake of fexofenadine was not observed in OATP1B1- and OATP2B1-expressing HEK cells, this does not exclude their involvement in the hepatic uptake of fexofenadine enantiomers. In fact, a transcellular transport study using OATP1B1 and MRP2 double transfectants clearly showed that transfection of OATP1B1 is required to mediate directional transport of fexofenadine in MDCK cells (Matsushima et al., 2008a). Specific uptake of fexofenadine by OATP2B1 was reported previously in OATP2B1-expressing Xenopus laevis oocytes, and HEK293 cells (Nozawa et al., 2004; Imanaga et al., 2011). Because rifampicin was given orally, the in vivo relevance of the interactions in hepatic elimination should be evaluated using the unbound rifampicin concentration at the inlet to the liver, which was estimated to be a maximum of 4.6 μM at the dose of 600 mg (Maeda et al., 2011). Given the in vitro inhibition potency of rifampicin (Fig. 3B), this concentration is sufficient to cause a marked inhibition of OATP1B3 in vivo. Assuming a negligible effect of rifampicin on intestinal absorption, the hepatic clearance of fexofenadine enantiomers is decreased to 4% of the control values by coadministration of rifampicin.

Although the CLr of fexofenadine was significantly decreased by rifampicin (Table 1), this effect is not explained by an inhibition of the candidate transporters OAT3 and MATE1 (Tahara et al., 2006; Matsushima et al., 2009), because rifampicin did not show any inhibitory effect on these transporters (Fig. 4). The absence of an effect of rifampicin on OAT3 is consistent with clinical data showing that a simultaneous dose of rifampicin did not affect the renal clearance of another OAT3 substrate, pravastatin (Maeda et al., 2011). Tubular secretion of fexofenadine may involve other unknown transporters that are sensitive to rifampicin. Based on the observation that probenecid, a potent inhibitor of OAT3, inhibited the tubular secretion of fexofenadine almost completely (Yasui-Furukori et al., 2005; Tahara et al., 2006), the site of interaction with rifampicin may be the luminal efflux process. Further studies are necessary to elucidate the mechanism of this DDI.

In addition to the hepatic and renal elimination, the effect of rifampicin on the CLtot/F of fexofenadine may involve inhibition of P-gp in the small intestine. We found that rifampicin inhibited the basal-to-apical transport of fexofenadine enantiomers in Caco-2 cells (Fig. 5). Tachibana et al., (2009) proposed an apparent volume of the small intestine lumen based on published DDI data to associate the DDI risk for P-gp and CYP3A4 (11–28 liters and 2.8–9.4 liters, respectively) with the doses of inhibitors. When a volume of 11–28 liters is used, the estimated concentration of rifampicin in the lumen is 26–66 μM, which is high enough to cause a moderate inhibition of P-gp–mediated efflux of fexofenadine in the small intestine. Notably, the effect of rifampicin on P-gp will be substrate dependent, as the same concentration did not affect the basal-to-apical transport of celiprolol (Fig. 5).

Hamman et al., (2001) reported that multiple doses of rifampicin significantly increased the CLtot/F of fexofenadine. It is well known that multiple doses of rifampicin induce expression of P-gp in the small intestine (Shimizu et al., 2006) and consequently lower bioavailability of P-gp substrates including fexofenadine (Hamman et al., 2001; Akamine et al., 2012). Rifampicin also induces OATP1B1 expression in primary cultured human hepatocytes (Sahi et al., 2006), and this may contribute to reducing the AUC of fexofenadine. In this study, even after multiple doses, when the two drugs were given simultaneously, rifampicin significantly decreased the CLtot/F of fexofenadine although the magnitude of the interaction on the CLtot/F was slightly blunted compared with that after a single dose (Fig. 2). Thus, the impact on inhibition of drug transporters by rifampicin surpasses its impact on induction of P-gp and OATP1B1. Multiple doses of rifampicin decrease its AUC; however, the Cmax of rifampicin in subjects given multiple doses over 6 days was decreased by at most one-third of the corresponding value on day 1 (Acocella, 1978) and remained high enough to cause a significant inhibition of the hepatic uptake transporter. Thus, during rifampicin therapy, attention must be paid to the intervals of administration of rifampicin and victim drugs, particularly OATP substrates, because these determine the direction of the DDI (i.e., an increase versus a decrease in the systemic exposure of victim drugs).

Consistent with previous reports (Miura et al., 2007; Tateishi et al., 2008; Sakugawa et al., 2009; Akamine et al., 2012), the pharmacokinetics of fexofenadine showed stereoselectivity (Table 1). Notably, the stereoselectivity in CLtot/F persisted after a single dose of rifampicin, but diminished after multiple doses, whereas the stereoselectivity in CLr persisted after both single and multiple doses (Table 1). We found stereoselectivity for the unbound fractions of fexofenadine enantiomers in the plasma. The fact that the magnitude of the difference in the unbound fraction of fexofenadine enantiomers (1.8) is similar to the difference in CLr (1.7) excludes stereoselectivity in the intrinsic tubular secretion clearance. This is in good agreement with in vitro data (Fig. 3). The stereoselectivity of the CLtot and F remains debatable as the CLtot and F could not be separately evaluated in this study. Stereoselectivity was not observed in the uptake of fexofenadine enantiomers by OATP1B3 (Fig. 3). Thus, the in vivo hepatic uptake clearance, which is the product of the unbound fraction in the plasma and intrinsic influx clearance, will show stereoselectivity. Explanation of the stereoselective effect on the CLtot/F of multiple doses of rifampicin requires the involvement of an additional stereoselective factor that is induced under treatment with multiple doses of rifampicin. In Caco-2 cells, the basal-to-apical transport of fexofenadine enantiomers was identical, indicating similar activities of P-gp for fexofenadine enantiomers (Fig. 5). OATP2B1 is considered to mediate the uptake of fexofenadine from the intestinal lumen to epithelial cells (Imanaga et al., 2011). However, since specific uptake of fexofenadine enantiomers by OATP2B1 could not be detected in our OATP2B1-expressing cells (data not shown), the stereoselectivity of the transport activity by OATP2B1 remains unknown. Separate evaluation of CLtot and F is necessary to elucidate the mechanism underlying the stereoselectivity of fexofenadine pharmacokinetics.

The impact of the stereoselectivity on the pharmacological effect of fexofenadine is debated. Taking the unbound fraction into consideration, the AUC of the unbound plasma fexofenadine enantiomers is similar. Although Robbins et al., (1998) reported that the two enantiomers are pharmacologically identical, in vitro binding assays demonstrated that the inhibition constant of S-fexofenadine against mepyramine binding to H1 receptor is 2-fold lower than that of R-fexofenadine (Fig. 6). Therefore, S-fexofenadine shows larger receptor occupancy, and consequently makes a greater contribution to the pharmacological response than R-fexofenadine. Multiple doses of rifampicin diminished the difference in the AUC of fexofenadine enantiomers, thereby increasing the contribution of S-fexofenadine to the pharmacological effect.

In conclusion, rifampicin interacts with fexofenadine at multiple sites in the body, with a simultaneous dose inhibiting the hepatic and renal transporters (OATP1B3 and an unknown transporter, respectively), and presumably the intestine efflux transporter P-gp, all of which contribute to increasing the AUC of fexofenadine to surpass the effect of the induction of P-gp elicited by multiple doses.

Authorship Contributions

Participated in research design: Kusuhara, Yasui-Furukori, Akamine, Uno, Sugiyama.

Conducted experiments: Miura, Yasui-Furukori, Yoshida, Yokochi, Fukizawa, Ikejiri, Kanamitsu.

Performed data analysis: Kusuhara, Miura, Yasui-Furukori, Akamine, Uno, Sugiyama.

Wrote or contributed to the writing of the manuscript: Kusuhara, Uno, Sugiyama.

Footnotes

  • This study was supported partly by a Grant-in-Aid for Scientific Research (S) [Grant 24229002] and a Grant-in-Aid for Scientific Research (B) [Grant 23390034] from the Japan Society for the Promotion of Science, Japan, and partly by a Grant-in-Aid for Scientific Research on Innovative Areas HD-Physiology [Grant 23136101] from the Ministry of Education, Science, and Culture of Japan.

  • dx.doi.org/10.1124/dmd.112.048330.

Abbreviations

AUC
area under the curve
CCK-8
cholecystokinin octapeptide
CLr
renal clearance
CLtot
total oral clearance
DDI
drug-drug interaction
LC–MS/MS
liquid chromatography–tandem mass spectrometry
MATE
multidrug and toxic compound extrusion
OAT
organic anion transporter
OATP
organic anion transporting polypeptide
  • Received August 1, 2012.
  • Accepted October 26, 2012.

References

  1. ↵
    1. Acocella G
    (1978) Clinical pharmacokinetics of rifampicin. Clin Pharmacokinet 3:108–127.
    OpenUrlPubMed
  2. ↵
    1. Akamine Y,
    2. Miura M,
    3. Yasui-Furukori N,
    4. Kojima M,
    5. Uno T
    (2012) Carbamazepine differentially affects the pharmacokinetics of fexofenadine enantiomers. Br J Clin Pharmacol 73:478–481.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Cvetkovic M,
    2. Leake B,
    3. Fromm MF,
    4. Wilkinson GR,
    5. Kim RB
    (1999) OATP and P-glycoprotein transporters mediate the cellular uptake and excretion of fexofenadine. Drug Metab Dispos 27:866–871.
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Deguchi T,
    2. Kusuhara H,
    3. Takadate A,
    4. Endou H,
    5. Otagiri M,
    6. Sugiyama Y
    (2004) Characterization of uremic toxin transport by organic anion transporters in the kidney. Kidney Int 65:162–174.
    OpenUrlCrossRefPubMed
  5. ↵
    1. Dresser GK,
    2. Bailey DG,
    3. Leake BF,
    4. Schwarz UI,
    5. Dawson PA,
    6. Freeman DJ,
    7. Kim RB
    (2002) Fruit juices inhibit organic anion transporting polypeptide-mediated drug uptake to decrease the oral availability of fexofenadine. Clin Pharmacol Ther 71:11–20.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Giacomini KM,
    2. Huang SM,
    3. Tweedie DJ,
    4. Benet LZ,
    5. Brouwer KL,
    6. Chu X,
    7. Dahlin A,
    8. Evers R,
    9. Fischer V,
    10. Hillgren KM,
    11. et al.,
    12. International Transporter Consortium
    (2010) Membrane transporters in drug development. Nat Rev Drug Discov 9:215–236.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Gillard M,
    2. Chatelain P
    (2006) Changes in pH differently affect the binding properties of histamine H1 receptor antagonists. Eur J Pharmacol 530:205–214.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Hamman MA,
    2. Bruce MA,
    3. Haehner-Daniels BD,
    4. Hall SD
    (2001) The effect of rifampin administration on the disposition of fexofenadine. Clin Pharmacol Ther 69:114–121.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Hirano M,
    2. Maeda K,
    3. Shitara Y,
    4. Sugiyama Y
    (2004) Contribution of OATP2 (OATP1B1) and OATP8 (OATP1B3) to the hepatic uptake of pitavastatin in humans. J Pharmacol Exp Ther 311:139–146.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Imanaga J,
    2. Kotegawa T,
    3. Imai H,
    4. Tsutsumi K,
    5. Yoshizato T,
    6. Ohyama T,
    7. Shirasaka Y,
    8. Tamai I,
    9. Tateishi T,
    10. Ohashi K
    (2011) The effects of the SLCO2B1 c.1457C &gt; T polymorphism and apple juice on the pharmacokinetics of fexofenadine and midazolam in humans. Pharmacogenet Genomics 21:84–93.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Lappin G,
    2. Shishikura Y,
    3. Jochemsen R,
    4. Weaver RJ,
    5. Gesson C,
    6. Houston B,
    7. Oosterhuis B,
    8. Bjerrum OJ,
    9. Rowland M,
    10. Garner C
    (2010) Pharmacokinetics of fexofenadine: evaluation of a microdose and assessment of absolute oral bioavailability. Eur J Pharm Sci 40:125–131.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Lau YY,
    2. Huang Y,
    3. Frassetto L,
    4. Benet LZ
    (2007) effect of OATP1B transporter inhibition on the pharmacokinetics of atorvastatin in healthy volunteers. Clin Pharmacol Ther 81:194–204.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Maeda K,
    2. Ikeda Y,
    3. Fujita T,
    4. Yoshida K,
    5. Azuma Y,
    6. Haruyama Y,
    7. Yamane N,
    8. Kumagai Y,
    9. Sugiyama Y
    (2011) Identification of the rate-determining process in the hepatic clearance of atorvastatin in a clinical cassette microdosing study. Clin Pharmacol Ther 90:575–581.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Matsushima S,
    2. Maeda K,
    3. Hayashi H,
    4. Debori Y,
    5. Schinkel AH,
    6. Schuetz JD,
    7. Kusuhara H,
    8. Sugiyama Y
    (2008a) Involvement of multiple efflux transporters in hepatic disposition of fexofenadine. Mol Pharmacol 73:1474–1483.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Matsushima S,
    2. Maeda K,
    3. Ishiguro N,
    4. Igarashi T,
    5. Sugiyama Y
    (2008b) Investigation of the inhibitory effects of various drugs on the hepatic uptake of fexofenadine in humans. Drug Metab Dispos 36:663–669.
    OpenUrlAbstract/FREE Full Text
  16. ↵
    1. Matsushima S,
    2. Maeda K,
    3. Inoue K,
    4. Ohta KY,
    5. Yuasa H,
    6. Kondo T,
    7. Nakayama H,
    8. Horita S,
    9. Kusuhara H,
    10. Sugiyama Y
    (2009) The inhibition of human multidrug and toxin extrusion 1 is involved in the drug-drug interaction caused by cimetidine. Drug Metab Dispos 37:555–559.
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Miura M,
    2. Uno T,
    3. Tateishi T,
    4. Suzuki T
    (2007) Pharmacokinetics of fexofenadine enantiomers in healthy subjects. Chirality 19:223–227.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Motulsky HJ,
    2. Mahan LC
    (1984) The kinetics of competitive radioligand binding predicted by the law of mass action. Mol Pharmacol 25:1–9.
    OpenUrlAbstract
  19. ↵
    1. Niemi M,
    2. Kivistö KT,
    3. Hofmann U,
    4. Schwab M,
    5. Eichelbaum M,
    6. Fromm MF
    (2005) Fexofenadine pharmacokinetics are associated with a polymorphism of the SLCO1B1 gene (encoding OATP1B1). Br J Clin Pharmacol 59:602–604.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Nozawa T,
    2. Imai K,
    3. Nezu J,
    4. Tsuji A,
    5. Tamai I
    (2004) Functional characterization of pH-sensitive organic anion transporting polypeptide OATP-B in human. J Pharmacol Exp Ther 308:438–445.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Ohuchi Y,
    2. Yanai K,
    3. Sakurai E,
    4. Fukui H,
    5. Yanagisawa T,
    6. Watanabe T
    (1998) Histamine-induced calcium mobilization in single cultured cells expressing histamine H1 receptors: a relationship between its sensitivity and the density of H1 receptors. Int J Mol Med 1:355–360.
    OpenUrlPubMed
  22. ↵
    1. Robbins DK,
    2. Castles MA,
    3. Pack DJ,
    4. Bhargava VO,
    5. Weir SJ
    (1998) Dose proportionality and comparison of single and multiple dose pharmacokinetics of fexofenadine (MDL 16455) and its enantiomers in healthy male volunteers. Biopharm Drug Dispos 19:455–463.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Sahi J,
    2. Sinz MW,
    3. Campbell S,
    4. Mireles R,
    5. Zheng X,
    6. Rose KA,
    7. Raeissi S,
    8. Hashim MF,
    9. Ye Y,
    10. de Morais SM,
    11. et al.
    (2006) Metabolism and transporter-mediated drug-drug interactions of the endothelin-A receptor antagonist CI-1034. Chem Biol Interact 159:156–168.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Sakugawa T,
    2. Miura M,
    3. Hokama N,
    4. Suzuki T,
    5. Tateishi T,
    6. Uno T
    (2009) Enantioselective disposition of fexofenadine with the P-glycoprotein inhibitor verapamil. Br J Clin Pharmacol 67:535–540.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Shimizu M,
    2. Fuse K,
    3. Okudaira K,
    4. Nishigaki R,
    5. Maeda K,
    6. Kusuhara H,
    7. Sugiyama Y
    (2005) Contribution of OATP (organic anion-transporting polypeptide) family transporters to the hepatic uptake of fexofenadine in humans. Drug Metab Dispos 33:1477–1481.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Shimizu M,
    2. Uno T,
    3. Sugawara K,
    4. Tateishi T
    (2006) Effects of single and multiple doses of itraconazole on the pharmacokinetics of fexofenadine, a substrate of P-glycoprotein. Br J Clin Pharmacol 62:372–376.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Shitara Y,
    2. Itoh T,
    3. Sato H,
    4. Li AP,
    5. Sugiyama Y
    (2003) Inhibition of transporter-mediated hepatic uptake as a mechanism for drug-drug interaction between cerivastatin and cyclosporin A. J Pharmacol Exp Ther 304:610–616.
    OpenUrlAbstract/FREE Full Text
  28. ↵
    1. Tachibana T,
    2. Kato M,
    3. Watanabe T,
    4. Mitsui T,
    5. Sugiyama Y
    (2009) Method for predicting the risk of drug-drug interactions involving inhibition of intestinal CYP3A4 and P-glycoprotein. Xenobiotica 39:430–443.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Tahara H,
    2. Kusuhara H,
    3. Fuse E,
    4. Sugiyama Y
    (2005) P-glycoprotein plays a major role in the efflux of fexofenadine in the small intestine and blood-brain barrier, but only a limited role in its biliary excretion. Drug Metab Dispos 33:963–968.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Tahara H,
    2. Kusuhara H,
    3. Maeda K,
    4. Koepsell H,
    5. Fuse E,
    6. Sugiyama Y
    (2006) Inhibition of oat3-mediated renal uptake as a mechanism for drug-drug interaction between fexofenadine and probenecid. Drug Metab Dispos 34:743–747.
    OpenUrlAbstract/FREE Full Text
  31. ↵
    1. Tateishi T,
    2. Miura M,
    3. Suzuki T,
    4. Uno T
    (2008) The different effects of itraconazole on the pharmacokinetics of fexofenadine enantiomers. Br J Clin Pharmacol 65:693–700.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Tian X,
    2. Zamek-Gliszczynski MJ,
    3. Li J,
    4. Bridges AS,
    5. Nezasa K,
    6. Patel NJ,
    7. Raub TJ,
    8. Brouwer KL
    (2008) Multidrug resistance-associated protein 2 is primarily responsible for the biliary excretion of fexofenadine in mice. Drug Metab Dispos 36:61–64.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Yasui-Furukori N,
    2. Uno T,
    3. Sugawara K,
    4. Tateishi T
    (2005) Different effects of three transporting inhibitors, verapamil, cimetidine, and probenecid, on fexofenadine pharmacokinetics. Clin Pharmacol Ther 77:17–23.
    OpenUrlCrossRefPubMed
  34. ↵
    1. Yoshida K,
    2. Maeda K,
    3. Sugiyama Y
    (2012) Transporter-mediated drug—drug interactions involving OATP substrates: predictions based on in vitro inhibition studies. Clin Pharmacol Ther 91:1053–1064.
    OpenUrlCrossRefPubMed
  35. ↵
    1. Zheng HX,
    2. Huang Y,
    3. Frassetto LA,
    4. Benet LZ
    (2009) Elucidating rifampin’s inducing and inhibiting effects on glyburide pharmacokinetics and blood glucose in healthy volunteers: unmasking the differential effects of enzyme induction and transporter inhibition for a drug and its primary metabolite. Clin Pharmacol Ther 85:78–85.
    OpenUrlCrossRefPubMed
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Effect of Rifampicin on Fexofenadine Pharmacokinetics

Hiroyuki Kusuhara, Masatomo Miura, Norio Yasui-Furukori, Kenta Yoshida, Yumiko Akamine, Miyu Yokochi, Shinya Fukizawa, Kazuaki Ikejiri, Kayoko Kanamitsu, Tsukasa Uno and Yuichi Sugiyama
Drug Metabolism and Disposition January 1, 2013, 41 (1) 206-213; DOI: https://doi.org/10.1124/dmd.112.048330
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