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Research ArticleMetabolism, Transport, and Pharmacogenomics

In Vitro and In Vivo P-Glycoprotein Transport Characteristics of Rivaroxaban

Mark Jean Gnoth, Ulf Buetehorn, Uwe Muenster, Thomas Schwarz and Steffen Sandmann
Journal of Pharmacology and Experimental Therapeutics July 2011, 338 (1) 372-380; DOI: https://doi.org/10.1124/jpet.111.180240

Abstract

Rivaroxaban, an oral, direct factor Xa inhibitor, has a dual mode of elimination in humans, with two-thirds metabolized by the liver and one-third renally excreted unchanged. P-glycoprotein (P-gp) is known to be involved in the absorption, distribution, and excretion of drugs. To investigate whether rivaroxaban is a substrate of P-gp, the bidirectional flux of rivaroxaban across Caco-2, wild-type, and P-gp-overexpressing LLC-PK1 cells was investigated. Furthermore, the inhibitory effect of rivaroxaban toward P-gp was determined. Rivaroxaban exhibited high permeability and polarized transport across Caco-2 cells. Rivaroxaban was shown to be a substrate for, but not an inhibitor of, P-gp. Of a set of potential P-gp inhibitors, ketoconazole and ritonavir, but not clarithromycin or erythromycin, inhibited P-gp-mediated transport of rivaroxaban, with half-maximal inhibitory concentration values in the range of therapeutic plasma concentrations. These findings are in line with observed area under the plasma concentration-time curve increases in clinical drug-drug interaction studies indicating a possible involvement of P-gp in the distribution and excretion of rivaroxaban. In vivo studies in wild-type and P-gp double-knockout mice demonstrated that the impact of P-gp alone on the pharmacokinetics of rivaroxaban is minor. However, in P-gp double-knockout mice, a slight increase in brain concentrations and decreased excretion into the gastrointestinal tract were observed compared with wild-type mice. These studies also demonstrated that brain penetration of rivaroxaban is fairly low. In addition to P-gp, a further transport protein might be involved in the secretion of rivaroxaban.

Introduction

Rivaroxaban is a novel, oral, direct Factor Xa inhibitor (Roehrig et al., 2005) that has been approved in the European Union and many other countries for the prevention of venous thromboembolism (VTE) in adult patients undergoing elective total hip or knee replacement surgery, and it is in advanced clinical development for the prevention and treatment of a variety of other thromboembolic disorders. Rivaroxaban is a highly selective Factor Xa inhibitor, which inhibits human free Factor Xa [enzyme inhibition constant (Ki) 0.4 nM], as well as prothrombinase-bound Factor Xa, independently of antithrombin (Perzborn et al., 2005).

In humans, rivaroxaban is almost completely absorbed and has a dual mode of elimination: two-thirds of the drug are metabolized in the liver, with half then being eliminated renally and the other half eliminated by the hepatobiliary route (Weinz et al., 2009). The final one-third undergoes direct renal excretion as unchanged active substance in the urine, mainly via active renal secretion (Kubitza et al., 2010). Coadministration of rivaroxaban with the cytochrome P450 3A4 (CYP3A4)/P-glycoprotein (P-gp)/breast cancer resistance protein (BCRP) inhibitors ketoconazole (Schwab et al., 2003; Gupta et al., 2004) and ritonavir (Kumar et al., 2003; Schwab et al., 2003; Gupta et al., 2004) led to a 2.6- and 2.5-fold increase, respectively, in mean rivaroxaban area under the plasma concentration-time curve (AUC) and decreased renal excretion (http://www.xarelto.com/html/downloads/Xarelto_Summary_of_Product_Characteristics_May2009.pdf) (W. Mueck, manuscript in preparation). In contrast to the two drugs cited above, coadministration of clarithromycin and erythromycin led to a less pronounced increase in the AUC and maximum plasma concentration (Cmax) in humans (http://www.xarelto.com/html/downloads/Xarelto_Summary_of_Product_Characteristics_May2009.pdf) (W. Mueck, manuscript in preparation).

P-gp (MDR1, ABCB1) is a multidrug resistance protein that is expressed in many organs and tissues, especially in the main excretory organs in humans (van de Vrie et al., 1998). Thus, P-gp could be involved in the excretion of rivaroxaban.

The current study was designed to determine the permeability of rivaroxaban across relevant physiological cell layers and assess the contribution of active transport processes mediated by P-gp to the pharmacokinetics, especially the excretion, of rivaroxaban in more detail. Caco-2 cells are a widely used model for studying permeability and transport characteristics of drugs in vitro (Hidalgo et al., 1989; Grès et al., 1998). To investigate whether P-gp is involved in the active transport of rivaroxaban, LLC-PK1 cells overexpressing human P-gp (L-MDR1 cells) were used (Schinkel et al., 1995).

In addition, animal studies in vivo in male wild-type mice [mdr1a/1b(+/+,+/+)] and P-gp double-knockout mice [mdr1a/1b(−/−,−/−)] were performed to evaluate the impact of P-gp on the pharmacokinetics of rivaroxaban, as described previously (Schinkel et al., 1994, 1995, 1997).

The International Transporter Consortium and the United States Food and Drug Administration (FDA) recommend that the inhibitory potential and substrate characteristics of new drug candidates toward P-gp should be determined (Zhang et al., 2006). Therefore, the inhibitory potential of rivaroxaban on the P-gp-mediated drug transport of probe substrates was determined in vitro. Furthermore, the inhibitory potential of various comedications on the P-gp-mediated transport of rivaroxaban was determined to investigate the observed drug-drug interactions in more detail.

Materials and Methods

Reference Compounds and Chemicals

Rivaroxaban (Roehrig et al., 2005) and the internal standard 5-chloro-N-([(5S)-2-oxo-3-[4-(3-oxomorpholin-4-yl)phenyl]-1,3-oxazolidin-5-yl]methyl)thiophene-2-carboxamide were synthesized at Bayer Schering Pharma AG (Wuppertal, Germany). Analytical-grade reagents and solvents were obtained from commercial sources. Digoxin was purchased from Digoxin Alfa Aesar GmbH Co KG (Karlsruhe, Germany). Atenolol, antipyrine, cimetidine, dipyridamole, ketoconazole, ivermectin, sulfasalazine, and vinblastine were purchased from Sigma-Aldrich (Deisenhofen, Germany). Fluvastatin was purchased from Calbiochem (San Diego, CA) and ritonavir was from United States Pharmacopeia (Rockville, MD). Cell culture media, fetal bovine serum, and antibiotics were purchased from Gibco-Invitrogen (Karlsruhe, Germany).

Caco-2 Cell Culture

Caco-2 cells ACC 169 were purchased from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (Braunschweig, Germany). Cells were plated at a density of 0.8 × 106 cells per flask (150 cm2) and grown for 7 days in Dulbecco's modified Eagle's medium supplemented with 5 ml/500 ml of nonessential amino acids, 1 mM sodium pyruvate, 10% fetal bovine serum, 50 ml/500 ml of streptomycin, and 50,000 IU/500 ml of penicillin in 8% CO2 (Caco-2 growth medium).

The transport buffer consisted of 500 ml of Hanks' balanced salt solution (HBSS) without phenol red, supplemented with 3.9 ml of glucose solution (2.27 M; final concentration in the assay 17.6 mM) and 5 ml of HEPES (1 M; final concentration in the assay 9.8 mM).

Cells were seeded at a density of 4 × 104 cells per well in 24-well microporous polycarbonate insert filter plates (0.4-μm pore size) (Corning Costar plates; Corning Life Sciences, Lowell, MA) and grown for 15 days in Caco-2 growth medium. The medium was replaced every 3 to 4 days. Before running the assay, the culture medium was replaced by HBSS buffer supplemented with 10 mM HEPES and 20 mM glucose. The different compounds tested were dissolved in dimethyl sulfoxide (DMSO) and diluted with transport buffer to respective final concentrations (final DMSO concentration was always 1%). These solutions were added to either the apical or the basolateral compartment. After 2 h of incubation at 37°C, samples were taken from both compartments and, after the addition of ammonium acetate buffer and acetonitrile, analyzed by liquid chromatography (LC)-tandem mass spectrometry (MS/MS) (API 4000; Applied Biosystems, Darmstadt, Germany). To confirm the confluence of the cell monolayer in each well, transepithelial electrical resistance (TEER) was measured for each individual well before (0 h) and after (2 h) the permeability studies using an STX 100 TEER electrode (World Precision Instruments, Berlin, Germany). Monolayers with TEER values greater than 700 Ω/cm2 were used.

L-MDR1 Cell Culture

LLC-PK1 wild-type and MDR1-overexpressing L-MDR1 cells (Schinkel et al., 1995) were purchased from the Dutch Cancer Institute (Amsterdam, The Netherlands).

LLC-PK1 and L-MDR1 cells were seeded at a density of 12 × 106 cells per 150-cm2 flask and grown for 7 days in medium 199 supplemented with 2 mM glutamine, 10% fetal bovine serum, 10 mg/500 ml of streptomycin, and 10,000 IU/500 ml of penicillin. To ensure a constant expression level of transport protein, L-MDR1 cells were grown in the presence of 640 nM vincristine.

LLC-PK1 and L-MDR1 cells were seeded at a density of 2 × 105 cells per well in 96-well culture plates with microporous polycarbonate inserts (0.4-μm pore size) (Corning Costar plates; Corning Life Sciences) and grown for 4 days in the same medium as used for cell cultures but without vincristine. The medium was replaced every 2 days. Before running the assay, the culture medium was replaced by HBSS buffer supplemented with 10 mM HEPES. The different compounds tested were dissolved in DMSO and diluted with transport buffer to the respective final test concentrations (final DMSO concentration was always 1%). For inhibitor studies, the inhibitor was added at the appropriate concentration. For IC50 determination the inhibitor was tested at seven concentrations plus a control (without inhibitor). These solutions were added to either the apical or basolateral compartment of the insert filter plates. After 2-h incubation at 37°C, samples were taken from both compartments and, after the addition of ammonium acetate buffer and acetonitrile, were analyzed by LC-MS/MS. To confirm the confluence of the cell monolayer in each well, TEER was measured at 0 and 2 h using an STX 100 TEER electrode (World Precision Instruments). Monolayers with TEER values greater than 200 Ω/cm2 were used. All values were determined using three wells.

Data Evaluation

Calculation of the Apparent Permeability Coefficient Values for Rivaroxaban.

The apparent permeability coefficient (Papp) was calculated using eq. 1: Embedded Image where Papp is the apparent permeability, Vr is the volume of medium in the receiver chamber, C0 is the concentration of the test drug in the donor chamber at t = 0 h, S is the surface area of the monolayer, C2 is the concentration of the test drug in the acceptor chamber after 2 h of incubation, and t is the incubation time. For reference compounds, peak heights were used instead of concentrations.

The efflux ratio was defined by eq. 2: Embedded Image where Papp(B–A) and Papp(A–B) represent the apparent permeability of the test compound from the basolateral to apical and apical to basolateral sides of the cellular monolayer, respectively.

The S.D. for the efflux ratio was calculated using eq. 3: Embedded Image where SD Papp(B–A) and SD Papp(A–B) represent the standard deviations of the apparent permeability of test compound from the basolateral to apical and apical to basolateral side of the cellular monolayer, respectively.

Calculation of Half-Maximal Inhibitory Concentration Values.

To calculate IC50 values, curves were fitted to data by nonlinear regression. Calculation was performed by a sigmoid Emax model using eq. 4: Embedded Image

In Vivo Animal Studies with Wild-Type and P-gp Double-Knockout Mice

Animal studies were conducted in accordance with the German Animal Protection Act (Deutsches Tierschutzgesetz). Male wild-type mice [FVB mdr1a/1b(+/+,+/+)] and P-gp double-knockout mice [FVB mdr1a/1b(−/−,−/−)] were purchased from Taconic Farms (Germantown, NY). Animals were fed and given water ad libitum before and during the experiments.

Tritiated digoxin, a well characterized substrate of P-gp (Mayer et al., 1996; Kawahara et al., 1999; Schwab et al., 2003), was prepared in accordance with the formulation (0.5 mg/kg, 5 ml/kg) published by Leusch et al. (2002) and administered intravenously to male wild-type and P-gp double-knockout mice. Tritiated digoxin was used as a P-gp probe substrate to demonstrate the suitability of the transgenic animal model (Schinkel et al., 1997; Fromm et al., 1999). Plasma concentrations and extent of blood-brain barrier penetration of total radioactivity were investigated at 5, 15, 30, 60, 120, 240, and 420 min after dosing in both types of mice. Total radioactivity is a good predictor for unchanged digoxin concentrations because digoxin is only marginally metabolized in mice. Furthermore, the stability of the [3H]-radiolabel (formation of tritiated water) was investigated by determination of total radioactivity concentrations in wet-weighed and freeze-dried samples of brain tissue. Volatile radioactivity was expected to evaporate as tritiated water during the freeze-drying process.

Rivaroxaban was administered intravenously (1 mg/kg, 5 ml/kg, plasma formulation) to male wild-type and P-gp double-knockout mice as a single bolus dose by direct injection into the lateral tail vein or orally (3 mg/kg, 5 ml/kg, 60% polyethylene glycol/40% demineralized water, v/v) by gavage using a calibrated glass syringe. In both experimental sets, the plasma concentrations and the extent of blood-brain barrier penetration of unchanged compound were investigated in wild-type and P-gp double-knockout mice at 15, 30, and 60 min after dosing. In addition, the excretion of rivaroxaban into the gastrointestinal tract (GIT) of wild-type and P-gp double-knockout mice was investigated after intravenous administration of rivaroxaban.

In all sets of experiments, blood samples were collected into heparinized syringes after exsanguination by a cut through the carotid artery under deep isoflurane [Isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-ethane); Delta Select, Dreieich, Germany] anesthesia (approximately 2.5% v/v). Blood cells were separated from plasma by centrifugation, and the plasma was stored for further analyses. After exsanguination, brain and GIT with contents were removed, homogenized, and used for further analysis.

Analytical Methods

Radioactivity concentrations in the biological samples were determined by liquid scintillation counting (LSC). Plasma samples were pipetted into Ultima Gold (PerkinElmer Life and Analytical Sciences, Waltham, MA) scintillation cocktail and measured by LSC. Aliquots of the solid samples (brain tissue and GIT) were combusted using a PerkinElmer Life and Analytical Sciences oxidizer. The tritiated water formed by this process was trapped in Carbo-Sorb (PerkinElmer Life and Analytical Sciences) and topped up with scintillation cocktail (Permafluor E+; PerkinElmer Life and Analytical Sciences) before LSC analysis.

Rivaroxaban concentrations were determined in plasma, brain, and GIT samples by a validated LC-MS/MS method. A stable, isotope-labeled internal standard was used (2H5, 15N). After precipitation of plasma or tissue homogenate proteins with acetonitrile containing the internal standard, followed by centrifugation, the supernatant was injected directly onto the LC-MS/MS system. A Hypersil 120 ODS, 5-μm particle size, 20 × 4.6-mm internal diameter column (Thermo Fisher Scientific, Waltham, MA) was used for separation. The columns were operated at ambient temperature. The mobile phase consisted of 0.01 M ammonium acetate solution, pH 6.8/acetonitrile (90:10 v/v) and acetonitrile, using a gradient elution. The flow rate was set at 0.5 ml/min. The TurboIonspray device of the AB Sciex API 4000 mass spectrometer (Applied Biosystems) was maintained at 450°C with an ionization voltage of 4 kV and an ion spray gas flow of 8 l/min. The nebulizing gas (N2) and curtain gas (N2) flows were set at 40 units, and the declustering potential was at 71 V for rivaroxaban and 81 V for the internal standard.

The dwell time was 200 ms, and mass analyzers Q1 and Q3 were operated at unit (approximately 0.8 atomic mass units) and low (approximately 2 atomic mass units) mass resolution. The mass spectrometer was programmed to admit the protonated parent ion masses [M+H]+ at m/z 435.9 for rivaroxaban and m/z 442.2 for the internal standard via the first quadrupole filter (Q1). Collision-induced fragmentation at Q2 (collision energy 41 eV) yielded the product ions at Q3 of m/z 144.9 for rivaroxaban and the internal standard. Peak height ratios of rivaroxaban and the internal standard obtained from selective reaction monitoring of the analytes (m/z 435.9 → 144.9)/(m/z 442.2 → 144.9) were used for the construction of calibration lines using log/log linear least-squares regression of the plasma concentrations and measured peak height ratios.

Rivaroxaban concentrations were determined in plasma, brain, and GIT samples by a validated LC-MS/MS assay using an AB Sciex API 4000 mass spectrometer equipped with a TurboIonspray Interface (Applied Biosystems), a CTC-PAL autosampler (CTC Analytics, Zwingen, Switzerland), and an Agilent 1100 system (Aglient Technologies, Waldbronn, Germany).

Method validation and analysis of the study samples were performed in compliance with the FDA guidelines on Bioanalytical Method Validation (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070107.pdf). Samples were analyzed after the addition of acetonitrile containing an internal standard. The lower limit of quantitation was determined to be 0.5 μg/l, and the analytical range was between 0.2 μg/l and 0.5 mg/l using a sample volume of 200 μl.

Concentrations of reference compounds were determined by LC-MS/MS. Samples were subjected to high-performance LC performed on an Agilent 1200 LC system (Agilent Technologies). The mobile phase consisted of 10 mM ammonium acetate (pH 3.0 or 6.8) and acetonitril. A linear gradient from 20 to 90% acetonitril (v/v) within 2 min was applied. Tandem mass spectrometry was performed on an API 3000 or 4000 triple-quadrupole mass spectrometer (Applied Biosystems) connected to the high-performance LC system through a TurboIonspray interface.

Student's t test was used for the analysis of the statistical significance.

Results

Cell Permeability of Rivaroxaban across Caco-2 Cell Monolayers.

The in vitro permeability of rivaroxaban for the apical (A) to basolateral (B) direction (A-B) and for the counter direction (B-A) across Caco-2 cells was investigated. The highest concentration tested (92 μM; 10 mg dissolved in 250 ml of water) was calculated based on the highest dose used in the VTE prevention studies (Mueck et al., 2008). In addition, the permeability at 10- and 100-fold lower concentrations was investigated. Before rivaroxaban testing, the assay used for the studies was validated in accordance with the FDA guidelines for the Biopharmaceutics Classification System (http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm070246.pdf), by determining Papp values for 22 reference compounds (data not shown; concentrations were determined by LC-MS/MS). Then, with the same batch of cells used to determine the permeability for rivaroxaban, the Papp values for 10 reference compounds across Caco-2 cells were determined again (Table 1).

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TABLE 1

Papp values for the bidirectional transport of 10 reference compounds and rivaroxaban across Caco-2 cell monolayers after 2-h incubation at 37°C (n = 3)

The Papp A-B values were 8.9 ± 0.1, 8.0 ± 0.6, and 7.5 ± 0.5 × 10–6 cm/s at rivaroxaban concentrations of 0.92, 9.2, and 92 μM, respectively. At the same concentrations, the Papp B-A values were significantly higher, leading to efflux ratios of 5.5 to 6.8 (Table 1). This efflux was not saturable within the concentration range tested. However, despite the observed efflux, the permeability of rivaroxaban was in the range of that of quinidine, a highly permeable compound (Table 1). The addition of ivermectin (5 μM), a strong P-gp, and weak Bcrp inhibitor (Schwab et al., 2003; Muenster et al., 2008) completely blocked the efflux of rivaroxaban at a concentration of 9.2 μM (Table 1).

Efflux ratios for strong substrates of efflux pumps, such as vinblastine (efflux ratio 110) and sulfasalazine (efflux ratio 160), were considerably higher than the ratios calculated for rivaroxaban (Table 1). Total recovery of rivaroxaban was >80% for both directions, showing that rivaroxaban was not trapped inside the cells.

Permeability of Rivaroxaban across L-MDR1 Cells.

The substrate properties of rivaroxaban for P-gp and the effect of several drugs on rivaroxaban transport were studied in L-MDR1 cells, which overexpress human P-gp. Efflux ratios of rivaroxaban across L-MDR1 cells at concentrations of 0.5, 1, 10, and 100 μM decreased from 15.9 to 9.78 (Table 2). This slight decrease in the efflux ratio at higher concentrations of rivaroxaban suggested concentration dependence. Efflux of rivaroxaban (2 μM) across L-MDR1 cells was almost completely blocked in the presence of the P-gp/Bcrp inhibitor ivermectin (Choo et al., 2000; Muenster et al., 2008) (Table 2).

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TABLE 2

Papp values of rivaroxaban (0.5–100 μM) across P-gp overexpressing L-MDR1 cells in the absence or presence of an inhibitor and across wild-type LLC-PK1 cells after 2-h incubation at 37°C (n = 3)

In LLC-PK1 cells (wild type), an efflux ratio of 1.5 ± 0.27 was observed at a rivaroxaban concentration of 0.5 μM (Table 2). In the presence of 5 μM ivermectin, the efflux ratio of rivaroxaban across LLC-PK1 cells was reduced slightly to 0.61 ± 0.16, indicating that LLC-PK1 cells might express endogenous P-gp at a low level. In summary, the results indicated that rivaroxaban is a moderate P-gp substrate.

The inhibitory potential of various drugs that could be coadministered in the clinical setting on the P-gp-mediated transport of rivaroxaban was investigated (Fig. 1). The influence of drugs on the active transport of rivaroxaban was investigated at a rivaroxaban concentration of 1 μM (Cmax = 286 nM after oral administration of rivaroxaban 10 mg once daily) (Mueck et al., 2008).

Fig. 1.
Fig. 1.

Inhibition of the P-glycoprotein-mediated efflux of rivaroxaban at a concentration of 1 μM in L-MDR1 cells by different protease inhibitors, azoles, erythromycin, clarithromycin, atorvastatin, and amiodarone (10 μM, except saquinavir 20 μM) after 2-h incubation at 37°C. All values were normalized to the control. *, p < 0.05; **, p < 0.01.

The addition of HIV protease inhibitors, such as ritonavir, atazanavir, indinavir, and saquinavir, all of which are known to be P-gp inhibitors, at 10 μM (a concentration in the range or above clinically relevant plasma concentrations) (Hamzeh et al., 2003; Cook et al., 2004; McCance-Katz et al., 2007), did not significantly (p > 0.05) reduce the efflux. The strongest inhibition within this class of compounds was observed for ritonavir. In the presence of ritonavir, the efflux was reduced to 76 ± 13.5% of the control value (Fig. 1). Therefore, an IC50 value was determined for ritonavir toward the P-gp-mediated efflux of rivaroxaban. The calculated IC50 value was 27.9 ± 10.3 μM (Table 3).

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TABLE 3

IC50 values of several drugs on the P-gp-mediated efflux of rivaroxaban (1 μM) across L-MDR1 cells after 2-h incubation at 37°C (IC50 values are based on seven concentrations + control) (n = 3)

The addition of 10 μM antifungal azoles, such as ketoconazole, itraconazole, miconazole, and clotrimazole, all of which are known P-gp inhibitors, significantly reduced (p < 0.05) the efflux ratio of rivaroxaban across L-MDR1 cells compared with the control (Fig. 1). Therefore, the inhibitory effect was determined at additional concentrations. All of the azoles tested showed a concentration-dependent inhibition of the efflux ratio of rivaroxaban. Itraconazole showed the lowest IC50 of 0.16 ± 0.082 μM, followed by ketoconazole, clotrimazole, and miconazole, with IC50 values above 10 μM for the latter two (Table 3).

In contrast to protease inhibitors and azoles, clarithromycin, erythromycin, and atorvastatin did not inhibit the efflux of rivaroxaban at a concentration of 10 μM (Fig. 1). For erythromycin and atorvastatin, a statistically significant increase in the efflux ratio was observed.

The IC50 values for the P-gp inhibitors ivermectin, quinidine, verapamil, and cyclosporin A were 0.25 ± 0.12, 4.3 ± 1.7, 4.3 ± 1.9, and 2.34 ± 0.83 μM, respectively (Table 3).

Amiodarone showed a concentration-dependent inhibition of the efflux of rivaroxaban (Fig. 1). However, based on our data, the IC50 value was estimated to be in the range of 10 to 15 μM.

Inhibitory Potential of Rivaroxaban on P-gp-Mediated Drug Transport.

Because rivaroxaban is a substrate of P-gp, it might also be an inhibitor of P-gp. Therefore, the inhibitory effect of rivaroxaban on the P-gp-mediated efflux of the probe substrates dipyridamole and digoxin (Schwab et al., 2003) was determined in L-MDR1 cells (Tables 4 and 5).

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TABLE 4

Inhibitory effect of rivaroxaban and ritonavir on the efflux ratio of 1 μM dipyridamole in L-MDR1 cells after 2-h incubation at 37°C (n = 3)

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TABLE 5

Inhibitory effect of rivaroxaban on the efflux ratio of 25 μM digoxin in L-MDR1 cells after 2-h incubation at 37°C (n = 3)

At a concentration of 1 μM dipyridamole showed an efflux ratio of 37.6 ± 3.2. This is in accordance with previously published data (Polli et al., 2001). The addition of rivaroxaban up to a concentration of 100 μM did not affect the efflux of dipyridamole (Table 4). In contrast to rivaroxaban, ritonavir, a moderate P-gp inhibitor (Choo et al., 2000; Kumar et al., 2003), showed a concentration-dependent inhibition of the efflux ratio of dipyridamole in L-MDR1 cells (Table 4) with an IC50 of 11.1 μM.

Digoxin at a concentration of 25 μM showed an efflux ratio of 12.1 ± 4.6 in L-MDR1 cells, which is in agreement with values published previously (Schwab et al., 2003). Similar to dipyridamole, the addition of rivaroxaban up to a concentration of 100 μM had no effect on the efflux ratio of digoxin (Table 5). Thus in vitro rivaroxaban exhibited no inhibitory effect on P-gp-mediated drug transport up to concentrations of 100 μM.

In Vivo Animal Studies with Digoxin in Wild-Type and P-gp Double-Knockout Mice.

Animal studies in vivo in male wild-type mice [mdr1a/1b(+/+,+/+)] and P-gp double-knockout mice [mdr1a/1b(−/−,−/−)] were performed to evaluate the impact of P-gp on the pharmacokinetics of rivaroxaban. Tritiated digoxin was used as the reference substrate for P-gp (Schinkel et al., 1997; Fromm et al., 1999). Total radioactivity is a good predictor for unchanged digoxin concentrations, because digoxin is only marginally metabolized in mice. Furthermore, the stability of the [3H]-radiolabel of digoxin was demonstrated with brain samples during freeze-drying (data not shown), in which no loss of volatile radioactivity could be identified.

The plasma concentrations of total radioactivity after intravenous administration of [3H]digoxin (0.5 mg/kg) were higher in P-gp double-knockout mice compared with wild-type mice at all investigated time points (Fig. 2), resulting in plasma concentration ratios of 1.2 at 5 min to 7.7 at 420 min after administration (Table 6). This finding was even more pronounced for brain concentrations of digoxin, resulting in brain concentration ratios of 4.3 at 5 min to 65.1 at 420 min after administration (Table 6). In contrast to digoxin plasma concentrations, brain concentrations in P-gp double-knockout mice but not in wild-type mice increased until the end of the observation period (Fig. 2).

Fig. 2.
Fig. 2.

Equivalent concentrations of [3H]digoxin in plasma and brain after intravenous administration (0.5 mg/kg) to male wild-type and mdr1a/1b(−/−,−/−) mice.

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TABLE 6

Plasma, brain, and brain-to-plasma concentration ratios of [3H]digoxin radioactivity in wild-type and P-gp double-knockout mice after intravenous administration of 0.5 mg/kg [3H]digoxin (arithmetic means) (n = 3 animals per time point)

Because the plasma concentrations were elevated in P-gp double-knockout mice, it was more appropriate to compare the brain-to-plasma concentration ratios rather than the absolute brain concentrations in P-gp double-knockout and wild-type mice. The brain-to-plasma concentration ratios increased from 0.03 at 5 min to 0.50 at 420 min in wild-type mice and from 0.096 at 5 min to 4.15 at 420 min in P-gp double-knockout mice (Table 6). To adequately describe the impact of P-gp on drug distribution, the quotient of the brain-to-plasma ratios in P-gp double-knockout mice versus wild-type mice was calculated. The resulting quotient of the brain-to-plasma concentration ratios ranged from 3.6 to 13.5 (Table 6).

In Vivo Studies with Rivaroxaban in Wild-Type and P-gp Double-Knockout Mice.

Plasma and brain concentrations of unchanged rivaroxaban were slightly higher in P-gp double-knockout mice than in wild-type mice after intravenous and oral administration of the test compound (Fig. 3).

Fig. 3.
Fig. 3.

Concentration of rivaroxaban in plasma and brain after intravenous administration (1 mg/kg) (A) and oral administration (3 mg/kg) (B) of rivaroxaban to male wild-type and P-gp double-knockout mice.

Brain concentrations of rivaroxaban were very low compared with the plasma concentrations, indicating a low blood-brain barrier penetration of rivaroxaban in both types of mice. After intravenous administration of rivaroxaban (1 mg/kg), the brain-to-plasma concentration ratio in wild-type mice was approximately 0.027 (15 and 30 min after dosing) and in P-gp double-knockout mice it was between 0.053 and 0.068 (15–60 min after dosing) (Table 7).

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TABLE 7

Brain-to-plasma concentration ratios of rivaroxaban in wild-type and P-gp double-knockout mice after intravenous administration of 1 mg/kg rivaroxaban (arithmetic means) and oral administration of 3 mg/kg rivaroxaban (arithmetic means) (n = 3 animals per time point)

The brain-to-plasma concentration ratios of rivaroxaban in P-gp double-knockout mice were 1.9- to 2.3-fold higher compared with wild-type mice in the time interval up to 30 min after administration (Table 7). In the same observation period, the P-gp double-knockout mice excreted less rivaroxaban into the GIT than the wild-type mice (Table 8).

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TABLE 8

Concentrations (μg/l) of rivaroxaban in the gastrointestinal tract including contents of wild-type and P-gp double-knockout mice after intravenous administration of 1 mg/kg rivaroxaban (arithmetic means and S.D.) (n = 3 animals per time point)

Likewise, after oral administration of rivaroxaban (3 mg/kg), the brain-to-plasma concentration ratio in wild-type mice was approximately 0.015 (15–60 min after dosing) and between 0.025 and 0.047 in P-gp double-knockout mice (15–60 min after dosing). Thus, the brain-to-plasma concentration ratios in P-gp double-knockout mice were slightly higher, 1.6-fold at 15 min to 3.2-fold at 60 min, compared with wild-type mice (Table 7), confirming the observation made after intravenous administration.

The quotients of the brain-to-plasma concentration ratios determined in P-gp double-knockout versus wild-type mice of approximately 2 to 3 were considerably lower than the respective ratios between 3.6 and 13.5, as determined for the strong P-gp substrate digoxin. This indicated that rivaroxaban is a rather weak substrate for P-gp.

Discussion

In humans, rivaroxaban exhibits a dual mode of elimination, with two-thirds being metabolized in the liver through oxidative biotransformation and hydrolysis at the amide bonds (Lang et al., 2009) and one-third being excreted unchanged by the kidneys through active renal secretion and glomerular filtration (Kubitza et al., 2010). These studies were performed to investigate the role of P-gp on active secretion of rivaroxaban in more detail, with special emphasis on studies and the role of P-gp.

Rivaroxaban demonstrated high permeability in the Caco-2 cell model, and it is unlikely that overall absorption will be affected by the weak efflux observed in vitro. Nevertheless, at a solubility of 8 mg/l (Roehrig et al., 2005) rivaroxaban shows high oral bioavailability (Kubitza et al., 2005). Similar findings have been reported for several other highly permeable substrates for efflux pumps (Toyobuku et al., 2003).

Rivaroxaban showed directed efflux in P-gp-overexpressing LLC-PK1 (L-MDR1) cell monolayers. The observed efflux ratio in L-MDR1 cells was significantly higher than in Caco-2 cells, which is in accordance with the higher expression levels of P-gp in L-MDR1 cells (Troutman and Thakker, 2003). No significant directed efflux of rivaroxaban was observed in wild-type LLC-PK1 cells, which means that the efflux of rivaroxaban observed in L-MDR1 cells is not influenced by an endogenously expressed transport protein. Furthermore, in the presence of 5 μM ivermectin, a known P-gp inhibitor (Muenster et al., 2008), the efflux of rivaroxaban across L-MDR1 cells was reduced to the same efflux ratio observed in LLC-PK1 cells. Based on these data, rivaroxaban can be classified as a moderate P-gp substrate (Polli et al., 2001).

With regard to drug-drug interactions as a result of the inhibition of transport proteins, the International Transporter Consortium recommends the investigation of the effect of known inhibitors and potentially coadministrated drugs on the relevant excretion pathways of new chemical entities (International Transporter Consortium, et al., 2010). Zhang et al. (2006) suggested that for an assessment of the drug-drug interaction potential not only the systemic maximal drug concentration (I1) but also the theoretical intestinal drug concentration (I2) has to be taken into account. Because there were no significant differences of the efflux ratios in L-MDR1 cells in the presence of ivermectin compared with LLC-PK1 cells, the studies to evaluate inhibitory effect of P-gp inhibitors used only L-MDR1 cells.

Of the drugs investigated, ketoconazole and ritonavir, two potent inhibitors of P-gp/BCRP and CYP3A4 (Kumar et al., 2003; Schwab et al., 2003; Gupta et al., 2004), showed significant inhibitory effects on the efflux ratio of rivaroxaban across L-MDR1 cells at therapeutic concentrations (IC50 9.0 ± 3.4 and 27.9 ± 10.3 μM, respectively). Based on these data, a clinical effect on the AUC of rivaroxaban is predicted because of inhibition of the excretion of rivaroxaban when these drugs are coadministrated. This is in line with the 2.6- to 2.5-fold AUC increase observed clinically after coadministration of rivaroxaban with ketoconazole or ritonavir, combined with a decreased renal excretion (http://www.xarelto.com/html/downloads/Xarelto_Summary_of_Product_Characteristics_May2009.pdf) (W. Mueck, manuscript in preparation). However, it has to be taken into account that this effect might not solely be a result of the inhibition of P-gp but also from an inhibition of other clearance rivaroxaban pathways, such as CYP3A4- and CYP2J2-mediated metabolism (Lang et al., 2009) or the involvement of other transport proteins. The directed efflux of rivaroxaban across P-gp-overexpressing L-MDR1 cells was not inhibited by the addition of the P-gp substrates clarithromycin, atorvastatin, or erythromycin but was in fact increased by the latter two. This increase could be the result of a trans-stimulation of P-gp or an uptake transport protein, which has been reported for the transport of vinblastine (Nakayama et al., 2000).

Clinically, the AUC increase of rivaroxaban after coadministration of clarithromycin and erythromycin was less pronounced, which was predicted by the in vitro findings that P-gp is not inhibited, and thus this excretion pathway still functions.

The inhibitory effect of itraconazole was observed at much lower concentrations, with an IC50 value of 0.160 ± 0.082 μM. However, with itraconazole, the active transport of rivaroxaban was not completely blocked, even at the highest concentration of 50 μM. Therefore, a less pronounced inhibitory potential is assumed for itraconazole. For all other azoles and protease inhibitors, IC50 values were significantly higher and above therapeutic Cmax concentrations. Therefore, the drug-drug interaction potential of these drugs was classified as low, especially compared with ritonavir and ketoconazole. In addition, significant inhibition of the directed efflux of rivaroxaban was achieved by addition of the P-gp substrate/inhibitor amiodarone only at supratherapeutic concentrations.

Other known moderate to strong P-gp inhibitors, such as verapamil, cyclosporin A, and ivermectin (Rautio et al., 2006), showed concentration-dependent inhibitory effects on the efflux of rivaroxaban. However, under clinical conditions for these drugs, maximal plasma concentrations were below the observed IC50 values.

With regard to theoretical gastrointestinal concentrations (I2) of these P-gp inhibitors, only a low drug-drug interaction risk resulting from the inhibition of P-gp is assumed because the higher concentrations compared with the maximal plasma concentrations are present only in the intestine and portal vein. Because of the high bioavailability of rivaroxaban and the minor influence of the liver and intestine on the excretion of rivaroxaban, only 7% of the dose is excreted unchanged by the hepatobiliary route (Weinz et al., 2009), and the relevance of P-gp within these organs is less pronounced. Thus, compared with ketoconazole and ritonavir, these drugs should have a significantly lower potential for drug-drug interactions with rivaroxaban.

The in vivo studies with digoxin in wild-type [mdr1a/1b (+/+,+/+)] and P-gp double-knockout [mdr1a/1b(−/−,−/−)] mice demonstrated that the model is in good accordance with earlier reports (Mayer et al., 1996). The observed increase in brain digoxin concentrations in the observation period in P-gp double-knockout mice might be the result of digoxin also being a substrate of mouse oatp2, which mediates uptake into the brain (van Montfoort et al., 2002). In the presence of P-gp, this uptake is masked by P-gp-mediated efflux.

In vivo studies in wild-type and P-gp double-knockout mice confirmed the in vitro findings that rivaroxaban is a weak substrate for P-gp. Thus, the impact of P-gp alone on the pharmacokinetics of rivaroxaban should be regarded as low, and inhibition of P-gp will only have a marginal effect on the pharmacokinetics of rivaroxaban. The quotients of the brain-to-plasma ratios of rivaroxaban in P-gp double-knockout versus wild-type mice were between 1.6 and 3.2. The corresponding ratios for the known P-gp substrate digoxin were between 3.6 and 13.5, which is considerably higher. Furthermore, the excretion of rivaroxaban into the GIT was only slightly lower in P-gp double-knockout mice compared with wild-type mice. It has been reported that this model is not suitable to determine the impact of P-gp on renal excretion (Mayer et al., 1996), especially because it is known that renal excretion of rivaroxaban in rodents is less pronounced compared with humans (Weinz et al., 2009).

The weak impact of P-gp on rivaroxaban pharmacokinetics might be the result of rivaroxaban being a substrate for not only P-gp but also for other transport proteins. It is known that P-gp and BCRP show overlapping substrate specificity, and both transport proteins are expressed in human liver and kidneys (van de Vrie et al., 1998; Huls et al., 2008), thus both transport proteins could play an important role in the active (transport protein-mediated) renal elimination of rivaroxaban. Thus, the knockout of one transport protein might be substituted for by another one. Therefore, a decrease in the renal clearance of rivaroxaban might only occur when strong inhibitors of P-gp, such as ketoconazole and ritonavir, are given at doses resulting in high plasma concentrations (≥10 μM).

To study the in vitro inhibitory potential of rivaroxaban on P-gp-mediated drug transport, the influence of the drug on the efflux ratio of two known P-gp substrates, digoxin and dipyridamole, was investigated. In studies with a set of reference inhibitors it could be shown that the higher concentration of digoxin compared with dipyridamole had no influence on the IC50 values (data not shown).

Rivaroxaban (1–100 μM) did not have any significant effect on the efflux ratios of digoxin or dipyridamole up to concentrations of 10 μM, whereas the addition of known P-gp inhibitors almost completely blocked the active transport of these drugs. The maximal plasma concentrations of rivaroxaban in VTE prevention studies reached 0.3 μM (Mueck et al., 2006); therefore, no clinically relevant drug-drug interactions are expected for rivaroxaban resulting from the inhibition of P-gp.

In conclusion, rivaroxaban shows a high permeability in Caco-2 cells and is a P-gp substrate. P-gp could be involved in the pharmacokinetics of rivaroxaban in humans. Only strong P-gp inhibitors given at high doses might result in drug-drug interactions caused by inhibition of the transport protein-mediated elimination of rivaroxaban. Rivaroxaban does not inhibit P-gp and, therefore, will not cause drug-drug interactions by inhibiting this transport protein.

Authorship Contributions

Participated in research design: Gnoth, Buetehorn, Muenster, and Sandmann.

Conducted experiments: Gnoth and Muenster.

Performed data analysis: Gnoth, Buetehorn, Muenster, Schwarz, and Sandmann.

Wrote or contributed to the writing of the manuscript: Gnoth, Buetehorn, Schwarz, and Sandmann.

Acknowledgments

We thank Chris Thomas, who provided editorial support with funding from Bayer Schering Pharma AG and Johnson & Johnson Pharmaceutical Research and Development, L.L.C., and Birgit Grieshop, Mark Twele, and Karsten Ickenroth for technical support.

Footnotes

  • This work was supported by Bayer Schering Pharma AG and Johnson & Johnson Pharmaceutical Research and Development, L.L.C.

  • Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

    doi:10.1124/jpet.111.180240.

  • ABBREVIATIONS:

    VTE
    venous thromboembolism
    AUC
    area under the plasma concentration-time curve
    Cmax
    maximum plasma concentration
    DMSO
    dimethyl sulfoxide
    FDA
    United States Food and Drug Administration
    Ki
    enzyme inhibition constant
    CYP3A4
    cytochrome P450 3A4
    GIT
    gastrointestinal tract
    HBSS
    Hanks' balanced salt solution
    LC
    liquid chromatography
    IC50
    half-maximal inhibitory concentration
    MS/MS
    tandem mass spectrometry
    LSC
    liquid scintillation counting
    Papp
    apparent permeability coefficient
    P-gp
    P-glycoprotein
    TEER
    transepithelial electrical resistance
    BCRP
    breast cancer resistance protein
    A
    apical
    B
    basolateral.

  • Received February 1, 2011.
  • Accepted April 21, 2011.

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Research ArticleMetabolism, Transport, and Pharmacogenomics

In Vitro and In Vivo P-Glycoprotein Transport Characteristics of Rivaroxaban

Mark Jean Gnoth, Ulf Buetehorn, Uwe Muenster, Thomas Schwarz and Steffen Sandmann
Journal of Pharmacology and Experimental Therapeutics July 1, 2011, 338 (1) 372-380; DOI: https://doi.org/10.1124/jpet.111.180240
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