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IN VITRO AND IN VIVO CORRELATION OF THE INHIBITORY EFFECT OF CYCLOSPORIN A ON THE TRANSPORTER-MEDIATED HEPATIC UPTAKE OF CERIVASTATIN IN RATS

School of Pharmaceutical Sciences, Showa University, Tokyo, Japan (Y.Sh., H.S.), Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan (M.H., Y.Su.); ADME/TOX Institute, Daiichi Pure Chemical, Inc., (Y.A.); and School of Pharmaceutical Sciences, Kitasato University, Tokyo, Japan (T.I.)

(Received March 18, 2004; accepted September 14, 2004)

	Abstract

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Previously, we have shown that the inhibition of the transporter-mediated hepatic uptake of cerivastatin (CER) by cyclosporin A (CsA) could, at least partly, explain a pharmacokinetic interaction between these drugs in humans. In the present study, we have examined the effect of CsA on the in vivo disposition of CER in rats and the in vitro uptake of [¹⁴C]CER in isolated rat hepatocytes in an attempt to evaluate the effect of inhibition of transporter-mediated hepatic uptake on the in vivo CER disposition. The steady-state plasma concentration of CER increased 1.4-fold when coadministered with CsA up to a steady-state blood concentration of 4 µM. Studies of [¹⁴C]CER uptake into isolated rat hepatocytes showed saturable transport, with the saturable portion accounting for more than 80% of the total uptake. CsA competitively inhibited the uptake of [¹⁴C]CER with a K_i of 0.3 µM. The IC₅₀ for the uptake of [¹⁴C]CER in the absence and presence of rat plasma was 0.2 and 2.3 µM, respectively. The in vivo hepatic uptake of [¹⁴C]CER evaluated by the liver uptake index method was also inhibited by CsA in a dose-dependent manner. On the other hand, CsA did not inhibit the metabolism of [¹⁴C]CER in rat microsomes. The in vitro and in vivo correlation analysis revealed that this pharmacokinetic interaction between these drugs in rats could be quantitatively explained by the inhibition of transporter-mediated hepatic uptake. Thus, this drug-drug interaction in rats is predominantly caused by the transporter-mediated uptake process.

The AUC of CER was also increased when coadministered with cyclosporin A (CsA) (Mück et al., 1999). We have previously shown that this clinically relevant DDI is, at least partly, caused by the inhibition by CsA of the transporter-mediated hepatic uptake but not its metabolism (Shitara et al., 2003). CsA inhibited the transporter-mediated uptake of CER in cryopreserved human hepatocytes and in organic anion-transporting polypeptide 1B1 (OATP1B1, formerly referred to as OATP2/OATP-C)-expressing cells (Shitara et al., 2003) with the inhibition constant (K_i) of 0.2 to 0.7 µM. Although this K_i value was higher than the unbound concentration of CsA in the circulating blood in clinical situations (at most 0.1 µM), it was lower than, or similar to, the estimated maximum unbound concentration of CsA at the inlet to the liver (0.66 µM) (Ito et al., 1998a,b; Kanamitsu et al., 2000; Hirota et al., 2001). Based on this analysis, we believe that inhibition of the transporter-mediated uptake also takes place in clinical situations and causes a DDI. However, it is impossible to measure the unbound concentration of CsA at the inlet to the liver and extrapolate from an in vitro inhibition study to the situation of in vivo DDI.

In the present study, to extrapolate the inhibition by CsA of the transporter-mediated uptake of CER from the in vitro to the in vivo situation, we analyzed the effect of CsA on the in vitro uptake of CER and in vivo disposition of CER in rats. Hirayama et al. (2000) examined the saturable uptake of CER in a primary culture of rat hepatocytes, and it was also taken up into human hepatocytes in a saturable manner (Shitara et al., 2003). In addition, the Oatp family transporters are conserved in rats, whereas OATP1B1 is, at least partly, responsible for the hepatic uptake of CER in humans (Shitara et al., 2003). Among the Oatps, Oatp1a1 (Oatp1), 1a4 (Oatp2), and 1b2 (Oatp4) are localized in the liver (Jacquemin et al., 1994; Noé et al., 1997; Cattori et al., 2000), although it is unknown which of them is the counterpart of OATP1B1. OATP/Oatp family transporters have similar substrate and inhibitor specificities, with some exceptions (Meier et al., 1997; Hagenbuch and Meier, 2003). In fact, CsA, an inhibitor of human OATP1B1, also inhibits rat Oatp1a1 and 1a4, although its effect on Oatp1b2 is unknown (Shitara et al., 2002). These facts suggest that the rat appears to be a good animal model for measuring the hepatic uptake of CER, although the molecular mechanisms governing the hepatic uptake in rats and humans are different. However, the metabolic profiles of CER in humans and rats are different. Both in rats and in humans, CER is mainly excreted in the form of metabolites. In humans, CER is mainly metabolized to M1 and M23, with a lesser amount of M24 by CYP2C8 and 3A4, whereas it is metabolized into many different products in rats (Mück et al., 1999; Mück, 2000; Boberg et al., 1998). Also, in rats, the isoform of cytochrome P450 responsible for its metabolism is unknown. Therefore, there is an interspecies difference between rats and humans in the molecular mechanism of the disposition and elimination of CER. However, as far as the transporter-mediated hepatic uptake is concerened, rats can be used as test models for extrapolating from in vitro to in vivo situations.

In the present study, we examined the inhibitory effect of CsA on the in vivo plasma CER concentration at steady state after intravenous administration, as well as the hepatic uptake of CER and the in vitro uptake of CER in isolated rat hepatocytes and rat Oatp1a1-expressing cells. The data obtained in the in vivo study were compared with those obtained in the in vitro study.

	Materials and Methods

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Reagents and Animals. [¹⁴C]CER (2.03 GBq/mmol) and unlabeled CER were kindly provided by Bayer Healthcare AG (Wuppertal, Germany). CsA was purchased from Sigma-Aldrich (St. Louis, MO), and all other reagents were of analytical grade. Male Sprague-Dawley (SD) rats were purchased from Nihon SLC (Hamamatsu, Japan).

Determination of CER Plasma Concentrations in Rats. The studies were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. CER (1.32 and 4.95 µM for administration by bolus and infusion, respectively) was dissolved in saline. CsA was dissolved in a mixture of Cremophor EL (Sigma-Aldrich) and 94% ethanol (Wako, Osaka, Japan) (containing 0.65 g of Cremophor EL/ml), and subsequently diluted with 10 volumes of saline. Under light ether anesthesia, the right and left femoral veins of male SD rats weighing from 220 to 260 g were cannulated with polyethylene tubing (PE-50; BD Biosciences, Franklin Lakes, NJ). To avoid enterohepatic recirculation, which increases the inhibitor concentration in the portal vein and at the inlet to the liver, the bile duct was also cannulated with polyethylene tubing (PE-10: BD Biosciences). CER (1.32 nmol/kg) and CsA (0, 1.16, and 3.99 µmol/kg) were administered intravenously as a bolus via the right and left femoral veins, respectively, followed by infusion of CER (4.95 nmol/h/kg) and CsA (0, 0.175, and 0.599 µmol/h/kg). At 5 h after infusion, 400 µl of blood was collected from the tail vein, and EDTA (1 mg/ml) was added. At this time point, the plasma and blood concentrations of CER and CsA had both reached steady state (data not shown). The samples were stored at -20°C until analysis. Plasma concentrations of CER were determined by a validated method using atmospheric pressure ionization liquid chromatography-tandem mass spectrometry. To a 50-µl sample, 500 µl of 1 M phosphate buffer (pH 5.5) and 5 ml of diethyl ether/dichloromethane (2:1) were added; the sample was shaken for 10 min and centrifuged. Then, the organic layer was evaporated to dryness under N₂ gas at 40°C and the residue was dissolved in a 200-µl mobile phase. A 20-µl portion of each sample was then subjected to atmospheric pressure ionization liquid chromatography-tandem mass spectrometry. A Shimadzu 10A high performance liquid chromatography (HPLC) system (Shimadzu, Kyoto, Japan) combined with a model API 365 MS/MS (Applied Biosystems/MDS Sciex, Foster City, CA) equipped with a Turbo IonSpray probe was used. The analytes were separated on an Inertsil ODS-3 column (5 µm, 2.1 mm i.d.x150 mm; GL Sciences Inc., Tokyo, Japan) using a mobile phase (acetonitrile/0.2% formic acid, 60:40, v/v) at a flow rate of 0.2 ml/min. Selected reaction monitoring was used to detect the analytes and internal standard (positive mode, m/z 460.6/356.1). The quality control sample showed an analytical variance of less than 8.8%. Blood concentrations of CsA were determined by radioimmunoassay using CYCLO-Trac (DiaSorin, Stillwater, MN), with quality control samples that were included in the kit and confirmed the precision of assay.

Uptake of [¹⁴C]CER into hepatocytes. Isolated rat hepatocytes were prepared from SD rats weighing from 220 to 250 g by the collagenase perfusion method described previously (Yamazaki et al., 1993). Isolated hepatocytes (viability >90%) were suspended in Krebs-Henseleit buffer (KHB), adjusted to 4.0 x 10⁶ viable cells/ml, and stored on ice. Before the uptake study, hepatocytes were incubated at 37°C for 3 min and the uptake reaction was started by adding an equal volume of KHB prewarmed at 37°C containing 0.6 µM [¹⁴C]CER (final concentration, 0.3 µM) with unlabeled CER or CsA. At 0.5 and 2 min, the reaction was terminated by separating the cells from the substrate solution. For this purpose, an aliquot of 100 µl of incubation mixture was collected and placed in a centrifuge tube (250 µl) containing 50 µl of 2 N NaOH under a layer of 100 µl of oil (density, 1.015; a mixture of silicone oil and mineral oil, Sigma-Aldrich), and, subsequently, the sample tube was centrifuged for 10 s using a tabletop centrifuge (10,000g; Beckman Microfuge E; Beckman Coulter, Fullerton, CA). During this process, the hepatocytes pass through the oil layer into the alkaline solution. After an overnight incubation at room temperature to dissolve the cells in alkali, the centrifuge tube was cut and each compartment was transferred to a scintillation vial. The compartment containing dissolved cells was neutralized with 50 µl of 2 N HCl, mixed with scintillation cocktail (Clearsol II; Nakalai Tesque, Kyoto, Japan), and the radioactivity was determined in a liquid scintillation counter (LS6000SE; Beckman Coulter). For the estimation of the inhibitory effects of CsA, it was added at the same time as [¹⁴C]CER. In the present investigation, the uptake study was also conducted in incubation buffer containing 90% rat plasma to evaluate the effect of plasma protein. For this purpose, isolated hepatocytes were suspended in KHB, adjusted to 2.0 x 10⁷ cells/ml, and prewarmed at 37°C before the uptake study. The reaction was started by addition of 9 volumes of rat plasma containing 0.25 µM [¹⁴C]CER.

Uptake of CER into Rat Oatp1a1-Expressing Cells. The rat Oatp1a1-expression vector was constructed as described previously (Kouzuki et al., 1999). Rat Oatp1a1-expressing HEK293 cells and control cells were constructed by the transfection of expression vector and control pcXN2 vector, respectively, into cells using FuGENE6 (Roche Diagnostics, Indianapolis, IN), according to the manufacturer's instruction, and selection by 800 µg/ml antibiotic G418 sulfate (Promega, Madison, WI) for 3 weeks. Oatp1a1-expressing cells and control cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, 1000 U/ml penicillin G sodium, 1 mg/ml streptomycin sulfate, 2.5 µg/ml amphotericin B and 200 mg/liter G418 sulfate. For the uptake study, cells were seeded on 12-well plates coated with poly-L-lysine/poly-L-ornithine at 1.2 x 10⁵ cells/well and cultured. After 2 days, culture medium was replaced with the same culture medium containing 10 mM sodium butyrate (Wako) for the induction of Oatp1a1 expression (Schroeder et al., 1998), followed by culturing for one more day. Before the uptake study, cells were washed twice with ice-cold KHB. Then the ice-cold KHB was replaced with KHB at 37°C followed by prewarming at 37°C for 10 min. The uptake reaction was started by the replacement of KHB with a solution containing 0.3 µM [¹⁴C]CER, with unlabeled CER or CsA. The reaction was terminated by removing the substrate solution by suction and, subsequently, cells were washed twice with ice-cold KHB after 5 min, since we confirmed the linearity of the uptake for at least 5 min. Cells were dissolved in 500 µl of 0.1 N NaOH overnight, followed by neutralization with 500 µl of 0.1 N HCl. Then, 800-µl aliquots were transferred to scintillation vials, and the radioactivity associated with cells and the medium was determined (LS6000SE). In addition, 50 µl of cell lysate was used for the protein assay by the Lowry method with bovine serum albumin as a standard (Lowry et al., 1951).

Protein Binding of CER in Rat Plasma. To estimate the fraction not bound to plasma protein, 3 µM [¹⁴C]CER was added to rat plasma supplemented with 10% KHB and incubated for 30 min at 37°C. After that, the sample underwent ultrafiltration (Amicon Centrifree; Millipore Corporation, Bedford, MA). The radioactivity in the 90% plasma and filtrate was determined (LS6000SE) and the plasma protein unbound fraction was calculated. In a pilot study, no significant difference of protein binding was confirmed up to its concentration of 1000 µM.

Liver Uptake Index (LUI) Method. Under light ether anesthesia, the femoral vein of male SD rats (weighing from 220 to 280 g) was cannulated with polyethylene tubing (PE-50). Before the LUI study, a 2 ml/kg bolus of CsA (0, 2.4, 4.8, and 9.6 mg/kg) was administered via the femoral vein. At 5 min after intravenous administration of CsA, approximately 100 µl of blood was collected from the jugular vein for determination of the concentration of CsA. [¹⁴C]CER and [³H]inulin dissolved in rat plasma (approximately 18.5 and 100 kBq/ml/kg for [¹⁴C]CER and [³H]inulin, respectively) containing a 1:1500 dilution of CsA solution, which was used for the bolus intravenous administration, was rapidly injected into the portal vein immediately after ligation of the hepatic artery. After 18 s of bolus administration of radiolabeled compounds, which is long enough for the bolus to pass completely through the liver but short enough to prevent recirculation of the isotope (Partridge et al., 1985), the portal vein was cut and the liver was excised. The excised liver was minced, and approximately 100 mg of sample was transferred to a scintillation vial and dissolved in a solubilizing agent (Soluene-350; PerkinElmer Life and Analytical Sciences, Boston, MA) at 55°C, followed by the addition of liquid scintillation cocktail (Hionic-Fluor; PerkinElmer Life and Analytical Sciences). Also, 100 µl of injectate was transferred to a scintillation vial and liquid scintillation cocktail (Hionic-Fluor) was added. After that, the radioactivity taken up by the liver and in the injectate was determined in a liquid scintillation counter (LS6000SE).

Metabolism of CER and Testosterone in Rat Microsomes. Before the metabolism study, rat liver microsomes (final concentration 0.5 mg of protein/ml; BD Gentest, Woborn, MA) were incubated at 37°C for 10 min in 100 mM potassium phosphate buffer (pH 7.4) containing 3.3 mM MgCl₂, 3.3 mM glucose 6-phosphate, 0.4 U/ml glucose-6-phosphate deydrogenase, 1.3 mM NADPH, and 0.8 mM NADH. A 500-µl volume of incubation mixture was transferred to a polethyelene tube, and [¹⁴C]CER (final 0.25 µM) or testosterone (final concentration 30 µM; Wako) was added to initiate the reaction with or without inhibitors. After incubation at 37°C for a designated time, the reaction was terminated by the addition of 500 µl of ice-cold acetonitrile and 20 µl of ice-cold methanol for the metabolism of [¹⁴C]CER and testosterone, respectively, followed by centrifugation. To measure the metabolic rate of [¹⁴C]CER, the supernatant was collected and concentrated to approximately 20 µl in a centrifugal concentrator, followed by thin-layer chromatography. The separation was carried out on a silica gel 60F₂₅₄ plate (Merck, Darmstadt, Germany) using a suitable mobile phase (toluene/acetone/acetic acid, 70:30:5, v/v/v). The intensity of the bands for intact [¹⁴C]CER separated by TLC was determined using the BAS 2000 system (Fuji Film, Tokyo, Japan). To measure the metabolic rate of testosterone, 6ß- and 16-hydroxytestosterone in the incubation mixture were determined by an HPLC-UV method. To a 100-µl volume of supernatant, 100 µl of internal standard (10 µg/ml phenacetin) was added followed by HPLC (VP-5 system; Shimadzu). The analyte was separated on a C18 column (Cosmosil 5C18-AR, 5 mm, 4.6 mm i.d. x 250 mm; Nakalai Tesque) at 45°C. The mobile phase consisted of a mixture of solvent A (20% tetrahydrofuran, 80% water) and solvent B (methanol). A 20-min linear gradient from 20% B to 30% B was applied at a flow rate of 1.0 ml/min. The products were detected by their absorbance at 254 nm and quantified by comparison with the absorbance of a standard curve for 6ß- and 16-hydroxytestosterone.

Data Analysis. The time courses of the uptake of [¹⁴C]CER into hepatocytes were expressed as the uptake volume [µl/10⁶ viable cells] for the radioactivity taken up into cells [dpm/10⁶ cells] divided by the concentration of radioactivity in the incubation buffer [dpm/µl]. The initial uptake velocity of [¹⁴C]CER was calculated from a slope of the uptake volume versus time plot obtained at 0.5 and 2 min and expressed as the uptake clearance (CL_uptake; µl/min/10⁶ cells). The time courses of the uptake of [¹⁴C]CER into rat Oatp1-expressing cells and vector-transfected cells were also expressed as the uptake volume (µl/mg protein) for the radioactivity in the cell lysate (dpm/mg protein) divided by the concentration of radioactivity in the incubation buffer (dpm/µl). Rat Oatp1-mediated uptake was calculated by using the uptake volume at 5 min in rat Oatp1-expressing cells and vector-transfected cells and expressed as the uptake clearance (CL_uptake; µl/min/mg protein), i.e., the CL_uptake in rat Oatp1-expressing cells minus that in vector-transfected cells.

The kinetic parameters for the uptake of [¹⁴C]CER were calculated using the following equation:

(1)

where v₀ is the initial uptake rate (pmol/min/mg protein), S is the substrate concentration (µM), K_m is the Michaelis constant (µM), V_max is the maximum uptake rate (pmol/min/mg protein), and P_dif is the nonsaturable uptake clearance (µl/min/mg protein).

The uptake clearance in the isolated hepatocytes obtained in the presence of CsA was fitted to the following equation to calculate the inhibitor concentration to produce a 50% reduction (IC₅₀) in the uptake of [¹⁴C]CER.

(2)

where CL_uptake(+CsA) is the CL_uptake estimated in the presence of CsA, CL_uptake(control) is the CL_uptake estimated in the absence of CsA, CL_uptake(resistant) is the CL_uptake which is not affected by CsA, and I is the CsA concentration. In the presence of 90% rat plasma, these parameters were calculated based on the total concentration, not the estimated free concentration, of substrate (CER) and inhibitor (CsA). Parameters calculated based on the total concentrations of CER or CsA are referred to as CL_uptake,app, K_m,app, P_dif,app and IC_50,app.

To determine the inhibition constant (K_i), the initial uptake rate of [¹⁴C]CER into rat hepatocytes determined in the presence and absence of CsA was fitted to the following equations in which competitive inhibition was assumed:

(3)

These equations were fitted to the data obtained in the present study using a computerized version of the nonlinear least-squares method, WinNonlin (Pharsight, Mountain View, CA), to obtain the kinetic parameters or inhibition constant with a computer-calculated standard error of each estimate (computer-calculated S.E.), which means the precision of the estimated parameter, but not an estimate of inter-rat variability. The input data were weighted as the reciprocal of the observed values, and the Gauss Newton (Levenberg and Hartley) method was used as the fitting algorithm.

The data obtained in the LUI study were expressed as %LUI [%], which represents the ratio of the hepatic extraction of [¹⁴C]CER to that of [³H]inulin. The %LUI was obtained by the following equation:

(4)

where, [¹⁴C]CER and [³H]inulin taken up by the liver was measured based on the radioactivity associated with the liver [dpm/mg tissue] and [¹⁴C]CER and [³H]inulin in the injectate were measured based on their concentrations [dpm/µl].

Statistical comparisons among multiple groups were carried out using Dunnett's test.

	Results

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In Vivo Study. The steady-state plasma concentrations of CER and the blood concentrations of CsA in rats 5 h after their intravenous infusion are shown in Table 1. The plasma concentration of CER significantly increased as the blood concentration of CsA increased (1.40- and 1.44-fold for 1.2 and 3.0 µM CsA; Table 1). The total body clearance (CL_tot) of CER was significantly reduced from 1.51 to 1.09 and 1.05 [l/h/kg] at steady-state blood concentrations of 1.2 and 3.0 µM CsA, respectively.

Uptake into Isolated Rat Hepatocytes. The uptake of CER into isolated rat hepatocytes incubated with KHB is shown in Fig. 1a. Kinetic analyses revealed that the K_m, V_max, and P_dif for the uptake of CER into isolated rat hepatocytes were 9.20 ± 2.24 µM, 1510 ± 330 pmol/min/10⁶ viable cells, 35.2 ± 4.2 µl/min/10⁶ viable cells (mean ± computer-calculated S.E.), respectively. The saturable component of the hepatic uptake, estimated by V_max/K_m, accounted for 82.4%. Uptake studies were also conducted in the presence of 90% rat plasma to investigate the effect of plasma protein binding. The apparent CL_uptake of CER was reduced in the presence of plasma (Fig. 1b). The kinetic parameters were 16.1 ± 2.4 µM, 481 ± 53 pmol/min/10⁶ viable cells, and 2.37 ± 0.22 µl/min/10⁶ viable cells (mean ± computer-calculated S.E.) for K_m,app, V_max,app, and P_dif,app, respectively (Fig. 1b). The saturable component accounted for 92.6% (Fig. 1b). Correcting for the CER-free fraction (4.32 ± 0.24%) gave unbound K_m and P_dif values of 0.696 µM and 0.102 µl/min/10⁶ viable cells, respectively.

View larger version (18K): [in this window] [in a new window]   FIG. 1. The uptake of CER in isolated rat hepatocytes in the absence or presence of 90% rat plasma. Uptake of CER in isolated rat hepatocytes was examined in KHB (a) or 90% rat plasma containing KHB (b). The uptake rate versus CER concentration plot is shown. In the presence of 90% rat plasma, the data are shown with () and without () correction for CER plasma protein binding. Each point represents the mean ± S.E. (n = 3, three cell preparations). Solid lines, dotted lines, and dashed lines represent the fitted lines for total, nonsaturable, and saturable transport of CER. Eadie-Hofstee plot for the uptake of CER is also shown.

Inhibition of the Uptake of [¹⁴C]CER into Isolated Rat Hepatocytes by CsA. The uptake of [¹⁴C]CER was examined in the presence of CsA (Fig. 2). CsA inhibited the uptake of CER into isolated rat hepatocytes in a concentration-dependent manner (Fig. 2a). The IC₅₀ value was 0.198 ± 0.028 µM (mean ± computer-calculated S.E.). In the presence of 90% rat plasma, CsA inhibited the uptake of CER; however, the apparent IC₅₀ values based on the total, but not free, concentration of CsA (IC_50,app) was increased. The IC_50,app estimated in the presence of 90% rat plasma was 2.32 ± 0.33 µM (mean ± computer-calculated S.E.) (Fig. 2b). To determine the K_i value, kinetic analysis was performed in the presence of 0.1 and 0.3 µM CsA (Fig. 3). As shown in Fig. 3a, CsA affected the K_m value of CER rather than the V_max value, suggesting that CsA competitively inhibits the uptake of CER. This suggestion was also supported by a Lineweaver-Burk plot (Fig. 2b). Based on the competitive inhibition model (eq. 3), the K_i value of CsA was estimated to be 0.180 ± 0.023 µM (mean ± computer-calculated S.E.).

View larger version (12K): [in this window] [in a new window]   FIG. 2. Inhibitory effect of CsA on the uptake of [14C]CER in isolated rat hepatocytes. The inhibitory effect of CsA on the uptake of CER (0.3 µM) in isolated rat hepatocytes was examined in the absence (a) or presence (b) of 90% rat plasma. Each point represents the mean ± S.E. (n = 3, three cell preparations). Solid lines represent the fitted lines. The asterisk represents a statistically significant difference from control shown by Dunnett's test (*, p < 0.05; **, p < 0.01).

View larger version (15K): [in this window] [in a new window]   FIG. 3. The concentration-dependent uptake of CER in the presence or absence of CsA. The uptake of CER in isolated rat hepatocytes was examined in the presence of 0 (), 0.1 (), and 0.3 () µM CsA. The uptake rate versus the CER concentration plot (a) and a Lineweaver-Burk plot (b) are shown. Each symbol represents mean ± S.E. of three independent experiments. In a, thick solid, dotted, and dashed lines represent fitted curves for the total transport of CER in the presence of 0, 0.1, and 0.3 µM CsA, respectively. Thin solid, dotted, and dashed lines represent fitted curves for the saturable transport of CER in the presence of 0, 0.1, and 0.3 µM CsA, respectively. A chain double-dashed line represents the fitted curve for the nonsaturable transport of CER. In b, solid, dotted, and dashed lines represent fitted lines in the presence of 0, 0.1, and 0.3 µM CsA, respectively.

Uptake of [¹⁴C]CER into Rat Oatp1a1-Expressing cells. The rat Oatp1a1-mediated uptake of [¹⁴C]CER, estimated by its uptake in Oatp1a1-expressing cells minus that in control cells, is shown in Fig. 4. The K_m and V_max for the rat Oatp1-mediated uptake of CER were 6.42 ± 1.16 µM and 33.4 ± 4.2 pmol/min/mg protein (mean ± computer-calculated S.E.), respectively. Rat Oatp1a1-mediated uptake was also inhibited by CsA (Table 2).

View larger version (20K): [in this window] [in a new window]   FIG. 4. The rat Oatp1a1-mediated uptake of CER. The rat Oatp1a1-mediated uptake rate of CER, which represented the uptake in Oatp1-expressing cells minus that in control cells, was plotted versus the CER concentration. Each symbol represents mean ± S.E. of three independent experiments. Solid line represents the fitted curve. The Eadie-Hofstee plot for the Oatp1a1-mediated uptake is also shown.

LUI Study. The hepatic uptake of CER in vivo was also examined by an LUI study in rats (Fig. 5). The %LUI (eq. 4) was reduced following coadministration of CsA in a concentration-dependent manner up to 4 µM (Fig. 5). The reduction in %LUI was 66.9, 54.0, and 49.7% of the control for CsA blood concentrations of 2.0, 2.7, and 4.0 µM, respectively (Fig. 5).

View larger version (11K): [in this window] [in a new window]   FIG. 5. Inhibitory effect of CsA on the hepatic uptake of [14C]CER in rats in vivo. Inhibitory effect of CsA on the hepatic extraction of [14C]CER normalized by that of [3H]inulin (%LUI) during a single pass after intraportal bolus injection in rats was examined. %LUI was calculated from eq. 4. Concentration of CsA was measured just before the LUI study. Each point represents the mean ± S.E. (n = 4 and 8, with and without coadministration of CsA, respectively). The asterisk represents a statistically significant difference from control shown by Dunnett's test (*, p < 0.05).

Metabolism of [¹⁴C]CER. The metabolism of [¹⁴C]CER in rat microsomes was examined. After a 2 h-incubation, approximately 74% of [¹⁴C]CER remained unchanged in the absence of inhibitors. The metabolism of [¹⁴C]CER was not inhibited by CsA up to a concentration of 30 µM, whereas it was significantly inhibited by 0.2 µM ketoconazole (to 24.8% of the control) (Fig. 6a). The metabolism of testosterone in rat liver microsomes was also examined, and the formation rates of 6ß- and 16-hydroxylation were, respectively, 996 ± 22 and 1520 ± 10 pmol/min/mg protein (mean ± S.E.) in the absence of inhibitors (Fig. 6b). Both 6ß- and 16-hydroxylations were significantly inhibited by CsA (Fig. 6b). Ketoconazole (0.2 µM) significantly inhibited 6ß-hydroxylation of testosterone, whereas it did not inhibit 16-hydroxylation (Fig. 6b).

View larger version (12K): [in this window] [in a new window]   FIG. 6. Metabolic stability of CER and testosterone 6ß- and 16-hydroxylation in rat liver microsomes in the presence or absence of CsA or ketoconazole. In a, the effects of CsA and ketoconazole on the metabolism of CER in rat liver microsomes were examined. In b, those for testosterone 6ß- () and 16-hydroxylation () were examined. The metabolic rate in the presence or absence of inhibitors is shown in each figure. Each bar represents the mean ± S.E. (n = 3). The asterisk represents a statistically significant difference from control shown by Dunnett's test (*, p < 0.05; **, p < 0.01).

	Discussion

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The present study shows that CsA increases the plasma concentration of CER in rats in a dose-dependent manner (Table 1). It also shows that CsA inhibits the uptake of CER in rat hepatocytes, whereas it does not affect the in vitro metabolism of CER in microsomes (Figs. 2 and 6). Also in humans, CsA increases the plasma concentration of CER (Mück et al., 1999) and it potently inhibits the uptake of CER in hepatocytes with a minimal effect on the microsomal metabolism (Shitara et al., 2003). Therefore, the effect of CsA in rats appears to be similar to that in humans, although the molecular mechanisms of drug elimination in rats and humans are basically different. CsA inhibits the uptake of CER in rat hepatocytes with an unbound IC₅₀ value similar to that in human hepatocytes (0.2-0.7 µM: Shitara et al., 2003). However, the in vivo effect of CsA on the disposition of CER was different between rats and humans, and the increase in the steady-state plasma concentration of CER in rats was minimal compared with that in humans. In fact, 3 µM blood CsA increased the steady-state plasma concentration of CER in rats only 1.4-fold (Table 1), whereas, in humans, CsA increased the AUC and the maximum plasma concentration of CER 3.8- and 5.0-fold, respectively, when the maximum blood concentration of CsA was approximately 1 µM (Mück et al., 1999). This difference in the severity of the interaction by these drugs between rats and humans may be explained by the different dosage regimen and experimental system. In rats, CsA, the inhibitor, was given intravenously and the biliary-excreted CsA was eliminated via cannulation without returning to the body via enterohepatic circulation, whereas, in humans, it was given orally and biliary excreted, with the CsA being transported to the portal vein and the inlet to the liver via enterohepatic circulation. In this case, the concentration of CsA at the inlet to the liver in humans may be higher than that in the circulating blood (Ito et al., 1998a,b). Therefore, in humans, a higher concentration of CsA at the inlet to the liver compared with that in the circulating blood may potently inhibit the hepatic uptake of CER, although its concentration in the circulating blood is lower in humans than in rats. However, there may be other mechanisms governing the DDI, and/or renal failure or kidney transplantation in patients may also have affected the plasma concentration of CER in the clinical case reported by Mück et al. (1999).

In vitro uptake studies in isolated hepatocytes revealed saturable transport of CER in rat hepatocytes both in the absence and presence of rat plasma (Fig. 1). Saturable transport of CER in primary cultured rat hepatocytes has already been reported by Hirayama et al. (2000), although their CL_uptake (44.4 µl/min/mg protein calculated by V_max/K_m) was lower than that in the present study (165 µl/min/10⁶ viable cells calculated by V_max/K_m), assuming that 10⁶ cells from our studies correspond to 1 mg of protein. This may be due to differences in the experimental system (i.e., isolated and primary cultured hepatocytes), since the transporter function can be affected by the primary culture (Ishigami et al., 1995). In the present study, we have found that the transporter-mediated uptake accounted for more than 80% and 90% of the total hepatic uptake in the absence and presence of plasma, respectively, at concentrations of CER lower than the K_m (Fig. 1). The large saturable portion of the uptake of [¹⁴C]CER in hepatocytes was similar to that in humans (70-80%; Shitara et al., 2003), suggesting that transporters play an important role in the hepatic uptake and disposition of CER both in rats and humans. Kinetic parameters, i.e., K_m, V_max, and P_dif, based on the free, or estimated free, concentrations of CER were different in the presence and absence of rat plasma. This difference was due to the different range of CER free concentrations under these two sets of experimental conditions (Fig. 1).

In the present study, the Oatp1a1-mediated uptake of [¹⁴C]CER was examined (Fig. 4). In our pilot studies, it was shown that other Oatp family transporters in rats, i.e., Oatp1a4 and 1b2, also accepted CER as a substrate (data not shown). However, we performed the kinetic analysis and inhibition study using CsA only in Oatp1a1-expressing cells because the highest saturable transport was observed in these cells among all the Oatp family transporter-expressing cells we possess. Kinetic analyses revealed that CER was taken up into rat Oatp1a1-expressing cells with a K_m value (6.4 µM; Fig. 3) similar to that in isolated rat hepatocytes (9.2 µM; Fig. 1). Thus, Oatp family transporter(s) appears to be responsible for the hepatic uptake of CER in rats, whereas OATP1B1, at least partly, mediates its hepatic uptake in humans (Shitara et al., 2003). The inhibition by CsA of the Oatp1a1-mediated uptake of CER was also examined (Table 2). It was found that the Oatp1a1-mediated uptake of CER was inhibited by CsA in a concentration-dependent manner (Table 2), also supporting the involvement of Oatp transporter(s) in the hepatic uptake of CER.

We investigated the inhibitory effect of CsA on the uptake of CER in rat hepatocytes in the presence of 90% rat plasma, which was similar to in vivo conditions. In the presence of 90% rat plasma, the IC_50,app value was approximately 12 times higher than the IC₅₀ value in its absence (Fig. 2). Lemaire and Tillement (1982) reported that approximately 90% of CsA is bound to plasma proteins, mainly lipoprotein in rats. Taking this into consideration, the IC₅₀ value, based on the estimated free concentration of CsA in the presence of 90% rat plasma, was calculated to be 0.232 µM, which is close to the IC₅₀ value obtained in the study without rat plasma. The LUI study confirmed that the hepatic CER uptake measured in vivo was also affected by CsA (Fig. 5). When the blood concentration of CsA was 4 µM, the hepatic uptake of CER in the LUI study was reduced to 50% of the control value (Fig. 5), suggesting that the IC₅₀ value for the in vivo hepatic uptake of CER was approximately 4 µM, which was similar to the IC_50,app value estimated in the presence of plasma (Fig. 2b).

We have also examined the effect of CsA on the metabolism of CER in rat liver microsomes (Fig. 5). Only 26% of [¹⁴C]CER was metabolized following a 2-h incubation in rat liver microsomes, whereas more than 50% was metabolized in human liver microsomes within 45 min (Shitara et al., 2003), suggesting slower metabolism in rat liver microsomes compared with their human counterparts. Since this metabolism of [¹⁴C]CER was not significantly inhibited by CsA up to its concentration of 30 µM (Fig. 5), microsomal metabolism was not the mechanism for the pharmacokinetic interaction between CER and CsA in rats examined in the present study.

The results obtained in the in vitro studies should be quantitatively discussed in relation to those in vivo. Without administration of CsA, the CL_tot was estimated to be 1.51 l/h/kg (Table 1). In the case of CER, the urinary excretion is negligible (Boberg et al., 1998) and, therefore, the CL_tot is close to the hepatic clearance (CL_H). Assuming a well stirred model, the CL_H can be described by the following equation (Miyauchi et al., 1993; Yamazaki et al., 1996):

(5)

where Q_H is the hepatic blood flow and f_b is the blood unbound fraction. The CL_int,all represents the overall intrinsic hepatic clearance, which includes membrane permeability, metabolism, and biliary excretion, as described by the following equation:

(6)

where PS_u,influx and PS_u,efflux represent the membrane permeability clearance of the unbound drug for the influx and efflux process from outside and inside the cells, respectively, and CL_int represents the "exact" intrinsic clearance which includes metabolism and/or biliary excretion of the unbound drug. When the CL_tot is 1.51 l/h/kg, the f_b · CL_int,all is calculated to be 3.04 l/h/kg from eq. 6, assuming the hepatic blood flow rate is 3 l/h/kg. As shown in eq. 6, the CL_int,all will be reduced in proportion to the decrease in PS_u,influx. In the presence of 1.2 and 3.0 µM CsA, the PS_u,influx of CER fell to 66 and 44% of the control, respectively, when the hepatocytes were incubated in the presence of rat plasma (Fig. 2b), and, therefore, the in vivo f_b · CL_int,all is reduced to 2.01 and 1.34 l/h/kg (i.e., 66 and 44% of the control), which gives a predicted CL_H value of 1.20 and 0.93 l/h/kg, respectively, from eq. 5. This predicted CL_H is comparable with the CL_H observed in the present in vivo study (1.09 and 1.05 l/h/kg, respectively) (Table 1), suggesting that the pharmacokinetic interaction between CER and CsA in rats can be quantitatively explained by inhibition of the transporter-mediated uptake of CER.

In conclusion, the increased plasma concentration of CER in rats when coadministered with CsA can be quantitatively explained by inhibition of transporter-mediated uptake. The present study strongly suggests that the inhibition of the transporter-mediated uptake in the liver affects the drug disposition. Also, in humans, it is possible that inhibition of the transporter-mediated uptake of drugs may lead to a clinically relevant DDI.

	Acknowledgments

 
We are grateful to Bayer AG and Bayer Yakuhin for kindly providing us with radiolabeled and unlabeled CER. We are grateful to Kazuya Maeda and Wakaba Yamashiro at the University of Tokyo for kindly providing us rat Oatp1a1-expressing HEK293 cells and for technical advice. We also appreciate Pharsight Corporation for providing us a license for WinNonlin as part of the Pharsight Academic License (PAL) program.

	Footnotes

 
This study was, in part, supported by the Grant-in-Aid for Young Scientists (B) provided by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Y.Sh.), Grant-in-Aid for the Advanced and Innovational Research program in Life Sciences (Y.Su.) and for the 21st Century Center of Excellence program provided by the Ministry of Education, Culture, Sports, Science and Technology, Japan (Y.Su.).

ABBREVIATIONS: CER, cerivastatin; AUC, area under the plasma concentration-time curve; CL_H, hepatic clearance; CL_int, intrinsic hepatic clearance; CL_int,all, overall intrinsic hepatic clearance; CL_tot, total body clearance; CL_uptake, uptake clearance; CsA, cyclosporin A; CYP2C8, cytochrome P450 2C8; DDI, drug-drug interaction; f_b, blood unbound fraction; HEK, human embryonic kidney; HPLC, high performance liquid chromatography; IC₅₀, inhibitor concentration to produce a 50% reduction in the transport; KHB, Krebs-Henseleit buffer; K_i, inhibition constant; K_m, Michaelis constant; LUI, liver uptake index; OATP/Oatp, organic anion transporting polypeptide; P_dif, nonsaturable uptake clearance; PS_u,efflux, membrane permeability clearance of the unbound drug for the efflux process; PS_u,influx, membrane permeability clearance of the unbound drug for the influx process; SD rat, Sprague-Dawley rat; V_max, maximum uptake rate.

Address correspondence to: Dr. Yuichi Sugiyama, Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: sugiyama{at}mol.f.u-tokyo.ac.jp

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