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OATP1B1, OATP1B3, AND MRP2 ARE INVOLVED IN HEPATOBILIARY TRANSPORT OF OLMESARTAN, A NOVEL ANGIOTENSIN II BLOCKER

Drug Metabolism and Pharmacokinetics Research Laboratories, Sankyo Co., Ltd., Tokyo, Japan (R.N.-H., D.N., K.K., Y.Y., T.T., T.I.); and Division of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan (T.A.)

(Received December 12, 2005; accepted February 17, 2006)

	Abstract

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Hepatic uptake and biliary excretion of olmesartan, a new angiotensin II blocker, were investigated in vitro using human hepatocytes, cells expressing uptake transporters and canalicular membrane vesicles, and in vivo using Eisai hyperbilirubinemic rats (EHBR), inherited multidrug resistance-associated protein (mrp2)-deficient rats. The uptake by human hepatocytes reached saturation with a Michaelis constant (K_m) of 29.3 ± 9.9 µM. Both Na⁺-dependent and Na⁺-independent uptake of olmesartan by human hepatocytes were observed. The uptake by Na⁺-independent human liver-specific organic anion transporters OATP1B1 and OATP1B3 expressed in Xenopus laevis oocytes was also saturable, with K_m values of 42.6 ± 28.6 and 71.8 ± 21.6 µM, respectively. The Na⁺-dependent taurocholate-cotransporting polypeptide expressed in HEK 293 cells did not transport olmesartan. The cumulative biliary excretion in EHBR was one-sixth compared with that in Sprague-Dawley rats. ATP-dependent uptake of olmesartan was observed in both human canalicular membrane vesicles (hCMVs) and MRP2-expressing vesicles. An MRP inhibitor, MK-571 ([[[3-[2-(7-chloro-2-quinolinyl)ethenyl]phenyl][3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid) completely inhibited the uptake of olmesartan by hCMVs. In conclusion, the hepatic uptake and biliary excretion of olmesartan are mediated by transporters in humans. OATP1B1 and OATP1B3 are involved in hepatic uptake, at least in part, and MRP2 plays a dominant role in the biliary excretion.

The hydrophilic nature of olmesartan as indicated by log D at pH 7.0 (–1.2; in-house data) and a high ratio of the ionized form at pH 7.4 as indicated by the pKa value (4.3; in-house data) were considered to limit greatly permeation of olmesartan through the cell membrane by passive diffusion. Therefore, the high fecal excretion of olmesartan via biliary excretion observed in vivo in humans was thought to be a consequence of the basolateral uptake and biliary excretion of olmesartan mediated by transporters in a concerted manner. In the case of rats, a previous study reported that the biliary excretion of olmesartan is mediated by multidrug resistance-associated protein 2 (mrp2) based on low biliary excretion in Eisai hyperbilirubinemic rats (EHBR), which are inherited mrp2-deficient rats, compared with Sprague-Dawley rats (Takayanagi et al., 2005). However, the transporters involved in the hepatobiliary transport of olmesartan in humans have not been fully investigated yet.

Na⁺-dependent and Na⁺-independent hepatic basolateral transport systems are known to be responsible for the clearance of various endogenous and exogenous substances from the systemic circulation (Meier, 1988; Tiribelli et al., 1990). As Na⁺-independent transporters, OATP1B1 (also known as OATP-2, OATP-C, LST-1, and SLC21A6) (Abe et al., 1999) and OATP1B3 (also known as OATP-8, LST-2, and SLC21A8) (Abe et al., 2001) are well known transporters that are specifically expressed on the basolateral membrane of human hepatocytes (Abe et al., 2001) and are involved in the hepatic uptake of various of endogenous and xenobiotic substances. On the other hand, Na⁺-dependent taurocholate-cotransporting polypeptide (NTCP) is the most relevant Na⁺-dependent transporter identified as a hepatic uptake transporter so far (Trauner and Boyer, 2003). With regard to the transporters involved in biliary excretion, it is known that P-glycoprotein (ABCB1), MRP2 (ABCC2), the bile salt export protein (ABCB11), and the breast cancer resistance protein (ABCG2) are predominantly expressed on canalicular membrane (Chan et al., 2004).

View larger version (14K): [in this window] [in a new window]   FIG. 1. Structure of olmesartan medoxomil (A), [3H]olmesartan (B), and [14C]olmesartan (C). T and asterisk denote the positions of the tritium and carbon labels, respectively.

In this study, to assess the involvement of transporters in the hepatobiliary transport of olmesartan, we carried out experiments in vitro using human hepatocytes, various transporter expression systems, and human canalicular membrane vesicles (hCMVs), and in vivo using an EHBR animal model.

	Materials and Methods

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Materials. [¹⁴C]Olmesartan (specific activity, 1.3 MBq/mg) and [³H]olmesartan (specific activity, 79 Ci/mmol) were synthesized at Amersham Pharmacia Biotech Ltd. (Tokyo, Japan) (Fig. 1). Olmesartan was synthesized at Sankyo Organic Chemicals Co., Ltd. (Tokyo, Japan). Cryopreserved human hepatocytes were purchased from In Vitro Technologies Inc. (Baltimore, MD). [³H]Estradiol 17ß-D-glucuronide (E2-17G) was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). hCMVs were purchased from Tissue Transformation Technologies Inc. (Edison, NJ). Human MRP2-expressing vesicles were purchased from SOLVO Technologies Inc. (Budapest, Hungary). Percoll was purchased from Amersham Pharmacia Biotech (Uppsala, Sweden). Collagenase A was purchased from Roche Diagnostics GmbH (Mannheim, Germany). Human liver polyadenylated RNA was purchased from BD Biosciences (Palo Alto, CA). Pentobarbital (Nembutal) was purchased from Dainippon Pharmaceutical Co., Ltd. (Osaka, Japan). MK-571 was purchased from Cayman Chemical Company (Ann Arbor, MI). All other chemicals used were of reagent grade.

Animals. Mature Xenopus laevis females were purchased from Hamamatsu Kyozai Co., Ltd. (Shizuoka, Japan) and maintained in a controlled environment (Goldin, 1992). Male Sprague-Dawley rats and EHBR (7 weeks old) were purchased from SLC Co., Ltd. (Shizuoka, Japan). Animal experiments were carried out according to the guidelines provided by the Institutional Animal Care and Use Committee of Sankyo Co., Ltd.

Uptake by Human Hepatocytes. Cryopreserved human hepatocytes (In Vitro Technologies Inc., Baltimore, MD) were thawed and added into an L-15 medium. After centrifugation (50g, 3 min), nonviable cells were removed by Percoll density centrifugation (100g, 10 min) (Groothuis et al., 1995). The viable cells were suspended in Krebs-Henseleit buffer (118 mM NaCl, 5 mM KCl, 1.1 mM MgSO₄, 2.5 mM CaCl₂, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 10 mM glucose, and 10 mM HEPES, pH 7.4), saturated with O₂/CO₂ (95:5). Viability of the cells was verified by a trypan blue exclusion test, and the cells with viability of not less than 85% were used. The cell suspension was preincubated at 37°C for 5 min. Then, radiolabeled compounds were added to start the uptake experiments. At designated times, the incubation mixture was collected and transferred to a centrifuge tube containing an alkaline solution layer with a silicone oil layer overlaid on top of it. Then, the tube was centrifuged to precipitate the cells into the alkaline layer through the silicone oil layer to terminate the uptake reaction (Yamazaki et al., 1993). After the cells were solubilized in the alkaline layer, the bottom of the tube containing the solubilized cell layer was sliced off with a razor blade, and the contents were transferred into a vial for liquid scintillation counting. After the addition of 10 ml of a liquid scintillation fluid (Hionic-Fluor; Packard Bioscience, Groningen, The Netherlands) to the vial, the radioactivity was determined using a Packard TriCarb 2200 CA liquid scintillation analyzer (Packard Instrument Company, Meriden, CT). To examine whether or not the olmesartan uptake by the human hepatocytes is sodium-dependent, the cells were incubated with a choline buffer. The composition of the choline buffer was the same as that of the Krebs-Henseleit buffer, except that the sodium chloride and sodium bicarbonate were replaced with choline chloride and choline bicarbonate, respectively.

Expression of OATP1B1 and OATP1B3 in X. laevis Oocytes and Uptake by Oocytes. In vitro synthesis of OATP1B1 and OATP1B3 cRNA was performed using cloned cDNAs of OATP1B1 and OATP1B3, as described previously (Abe et al., 1999, 2001). X. laevis oocytes were prepared according to a conventional procedure described previously (Tokui et al., 1999). Briefly, defolliculated oocytes were microinjected with 50 ng of transcribed cRNA or human liver polyadenylated RNA and were cultured for 3 days at 18°C, with daily replacements of modified Barth's medium [88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 0.3 mM Ca(NO₃)₂, 0.41 mM CaCl₂, 0.82 mM MgSO₄, and 15 mM HEPES, pH 7.6]. Uptake of radiolabeled substrate was examined in a medium containing 100 mM choline chloride, 2 mM KCl, 1 mM CaCl₂, 1 mM MgCl₂, and 10 mM HEPES, pH 7.5. Oocytes were incubated in 100 µl of the same medium containing a radiolabeled substrate at room temperature. Uptake was terminated by addition of 3 ml of ice-cold uptake buffer, and the oocytes were washed five times. The water-injected oocytes were used as control. Each single oocyte was solubilized in 0.5 ml of 10% (w/w) sodium dodecyl sulfate, and 4 ml of scintillation fluid (Pico-Fluor; Packard Bioscience) was added. The radioactivity was determined using the Packard TriCarb 2200 CA liquid scintillation analyzer.

Uptake Study Using Human NTCP-Transfected HEK-293 Cells. We isolated a human NTCP (hNTCP) clone and introduced the vector including hNTCP into HEK-293 cells. Transfected HEK-293 cells were seeded at 1.0 x 10⁵ cells/ml/well on collagen I-coated dishes (BD Biosciences) and cultured for 3 days at 37°C in humidified O₂/CO₂ (95:5). The cells were first washed with DMEM containing 0.2% bovine serum albumin and then incubated with 0.8 ml of DMEM containing 0.2% bovine serum albumin containing 7.5 nM [³H]olmesartan or 75 nM [³H]taurocholate at 37°C. At 5, 15, and 30 min after incubation, the uptake was stopped by three washes with ice-cold phosphate-buffered saline. The cells were then solubilized with 0.5% NaOH, and the radioactivity taken up by the cells was measured by the Packard TriCarb 2200 CA liquid scintillation analyzer.

In Vivo Rat Study. Male Sprague-Dawley rats and EHBR were anesthetized by intraperitoneal administration of pentobarbital (Nembutal; 50 mg/kg), and the common bile duct was cannulated with polyethylene tubing (PE-10) to collect bile samples. [¹⁴C]Olmesartan (1 mg/kg) was injected intravenously via the caudal vein. At designated times, blood samples were collected from the jugular vein, and the plasma was immediately separated by centrifugation. The bile samples were collected at 0 to 0.25, 0.25 to 0.5, 0.5 to 1.0, 1.0 to 1.5, and 1.5 to 2.0 h after administration. Urine samples were collected from the urinary bladder, which was excised at 2 h after administration. The total radioactivity values in the plasma, urine, and bile samples were measured by the Packard TriCarb 2200 CA liquid scintillation analyzer. The radioactivity of [¹⁴C]olmesartan, extracted from the plasma, urine, and bile samples with ethanol, was analyzed by silica gel thin-layer chromatography in combination with a BioImage Analyzer (BAS-2000; Fuji Photo Film Co., Ltd., Tokyo, Japan).

Uptake by hCMVs and MRP2-Expressing Membrane Vesicles. hCMVs were purchased from Tissue Transformation Technologies Inc. Human MRP2-expressing vesicles were purchased from SOLVO Technologies Inc. A transport experiment was performed using a rapid filtration technique described in a previous report (Ishikawa et al., 1990). Transport medium (10 mM Tris, 250 mM sucrose, and 10 mM MgCl₂, pH 7.4) containing a test compound was preincubated at 37°C for 3 min and was added to a suspension of the membrane vesicles (ca. 10–50 µg of protein) in the presence or absence of 5 mM ATP and an ATP-regenerating system (10 mM creatine phosphate and 100 µg/ml creatine phosphokinase). The mixture was incubated at 37°C for a designated time, and the transport reaction was stopped by the addition of 1 ml of ice-cold stop buffer (250 mM sucrose, 0.1 M NaCl, and 10 mM Tris-HCl, pH 7.4). The reaction mixture was immediately filtered through a 0.45-µm GVWP filter (Millipore Corp., Bedford, MA) and washed twice with 5 ml of the stop buffer. Each filter was placed in a vial with 10 ml of scintillation fluid (Pico-Fluor), and the radioactivity was determined using the Packard TriCarb 2200 CA liquid scintillation analyzer.

Determination of Kinetic Parameters. The kinetic parameter for olmesartan uptake by human hepatocytes was calculated according to the following equation:

(1)

where V₀ is the initial uptake rate (pmol/min/10⁶ cells), V_max is the maximum uptake rate (pmol/min/10⁶ cells), K_m is the Michaelis constant (micromolar), P_dif is the nonspecific uptake clearance (ml/min/10⁶ cells), and S is the radiolabeled olmesartan concentration in the medium (micromolar). The K_m value and P_dif value were calculated by an iterative nonlinear least-squares method using WinNonlin (version 4.01; Pharsight Corporation, Mountain View, CA).

The OATP1B1- and OATP1B3-mediated uptake values of olmesartan were calculated by subtracting the uptake in water-injected oocytes from those in OATP1B1 and OATP1B3 cRNA-injected oocytes, respectively. The kinetic parameter was calculated according to the following equation:

(2)

where V₀ is the initial uptake rate (pmol/min/oocyte), V_max is the maximum uptake rate (pmol/min/oocyte), K_m is the Michaelis constant (micromolar), and S is the radiolabeled olmesartan concentration in the medium (micromolar). The uptake data were fitted to eq. 2 by an iterative nonlinear least-squares method using WinNonlin. All kinetic parameters obtained are shown as mean ± S.D.

View larger version (21K): [in this window] [in a new window]   FIG. 2. Concentration dependence of initial uptake of [3H]olmesartan by human hepatocytes. Olmesartan was incubated in a concentration range from 3.13 to 200 µM at 37°C up to 2 min. Experiments were conducted using hepatocytes from three different donors. Data show one representative result. The solid line represents the least-squares fit of the data to eq. 1.

(3)

where A_bile(0–2h) is the amount of olmesartan excreted into the bile by 2 h, and AUC_(0–2h) is the area under the plasma concentration time curve from time 0 to 2 h calculated by the trapezoidal rule using WinNonlin.

Estimation of in Vivo Hepatic Clearance from in Vitro Data. To estimate the in vivo hepatic clearance based on the in vitro hepatocyte uptake data, the permeability-surface area product for influx (PS_influx) and hepatic intrinsic clearance (CL_H,u,int) were calculated according to the following equation under linear conditions:

(4)

where Cn is the number of hepatocyte cells (1.0 x 10⁸ cells/g liver) (Olinga et al., 1993), and W_L is human liver weight (1800 g/body) (Davies and Morris, 1993). Hepatic intrinsic clearance was estimated by the following equation:

(5)

where CL_bile is intrinsic biliary clearance, and PS_efflux is the permeability-surface area product for efflux. In eq. 5, CL_H,u,int was considered to be equal to PS_influx, assuming that PS_efflux is much lower than CL_bile. Finally, the hepatic clearance was estimated according to the equation below, taking to consideration that olmesartan does not enter red blood cells (Yoshida and Kohzuki, 2004).

(6)

where Q_H and fp represent the hepatic plasma flow rate (0.75 l/min) (Sandker et al., 1994) and free fraction of olmesartan in plasma, respectively (fp, 0.01, protein binding of olmesartan over 99%) (Yoshida and Kohzuki, 2004).

Statistical Analysis. Data were processed using Microsoft Excel (Microsoft, Redwood, WA) to calculate means and standard deviations. Student's t test with 95% confidence (p < 0.05) was performed on the data from the two treatment groups to determine any significant differences between the means.

	Results

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Uptake by Human Hepatocytes. Uptake of [³H]olmesartan by human hepatocytes increased linearly against time up to 2 min (data not shown). Thus, the initial uptake velocity was calculated by linear regression using the data points at 0.5 and 2 min. Substitution of Na⁺ with choline⁺ in the medium decreased the uptake of olmesartan as shown in Table 1. We observed a significant difference in the uptake between Na⁺-dependent and Na⁺-independent conditions using the donor 2 hepatocytes. However, in the donor 1, we did not observe a significant difference because of interexperiment variation, although a tendency of Na⁺ dependence was observed (P = 0.058). Taken together, we concluded that there was Na⁺-dependent hepatic uptake of olmesartan. The uptake became saturated with an increasing concentration of [³H]olmesartan in the medium (Fig. 2). Kinetic parameters for uptake of olmesartan in the presence of Na⁺ were a K_m value of 29.3 ± 9.9 µM, V_max of 72.9 ± 65.8 pmol/min/10⁶ cells, and P_dif, the nonspecific uptake clearance, of 0.6 ± 0.4 µl/min/10⁶ cells, respectively. The permeability-surface area product PS_influx was estimated to be approximately 15 (l/h/liver) according to eq. 4 under linear conditions. The hepatic clearance CL_{H,in vitro} estimated by eq. 6 was approximately 0.36 l/h.

View larger version (18K): [in this window] [in a new window]   FIG. 3. Effect of Na+ on uptake of [3H]olmesartan (2 µM) (A), [3H]estradiol-17ß-D-glucuronide (14.6 µM) (B), and [3H]taurocholate (320 µM) (C) in oocytes injected with human liver polyadenylated RNA. Uptake was measured in medium containing choline chloride [Na+(–)] or sodium chloride [Na+(+)] (see Materials and Methods). Human liver polyadenylated RNA was preincubated for 15 min at 42°C before the injection. Uptake was measured after 2-h incubation. Data are expressed as the mean ± S.E. of five to six uptake measurements. poly A RNA, human liver polyadenylated RNA.

The uptake of olmesartan by OATP1B1- and OATP1B3-expressing oocytes at 10 µM were 2.9 ± 1.0 and 15.6 ± 10.0 times higher than that by the water-injected oocytes, respectively. The uptake by OATP1B1- and OATP1B3-injected oocytes was linear against time up to 30 and 60 min, respectively (data not shown). The OATP1B1- and OATP1B3-mediated uptake of olmesartan was concentration-dependent and reached saturation with K_m values of 42.6 ± 28.6 and 71.8 ± 21.6 µM, respectively (Fig. 4).

View larger version (19K): [in this window] [in a new window]   FIG. 4. Concentration-dependent uptake of [3H]olmesartan by OATP1B1 (A) and OATP1B3 (B) cRNA-injected oocytes. Olmesartan was incubated in a concentration range from 1 to 300 µM. , uptake in OATP1B1 or OATP1B3 cRNA-injected oocytes. , uptake in water-injected oocytes. , OATP1B1- or OATP1B3-mediated uptake. Uptake was measured in the medium without sodium chloride. Uptake was measured after 30-min and 1-h incubation for OATP1B1- and OATP1B3-injected oocytes, respectively. Data are expressed as the mean ± S.E. of five to six uptake measurements in the one representative preparation of the three separate oocyte preparations.

Biliary Excretion in Rats. [¹⁴C]Olmesartan was administered intravenously to bile-fistula Sprague-Dawley rats and EHBR at a dose of 1 mg/kg. EHBR showed a higher plasma level of olmesartan than Sprague-Dawley rats (Fig. 5A), and the AUC_(0–2h) in EHBR was approximately 2 times higher than that in Sprague-Dawley rats (Table 2). The cumulative biliary excretion of radioactivity in Sprague-Dawley rats was 68.12 ± 11.81% of the dose by 2 h, being significantly higher than the biliary excretion in EHBR (11.45 ± 3.73%). The CL_bile value in EHBR was approximately one-tenth of that in Sprague-Dawley rats (Table 2). Only olmesartan (more than 94% of total radioactivity) was observed as a metabolite in the bile by thinlayer chromatography (data not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 5. Plasma concentrations of Sprague-Dawley rats () and EHBR () (A) and cumulative biliary and urinary excretion (B) of total radioactivity after intravenous administration of [14C]olmesartan to Sprague-Dawley rats (, bile; , urine) and EHBR (, bile; , urine) at a dose of 1 mg Eq of [14C]olmesartan/kg. Each point represents the mean ± S.D. (n = 4).

Uptake by hCMVs and Human MRP2-Expressing Vesicles. The uptake of [³H]olmesartan (0.021 µM) and [³H]E2-17G (0.25 µM) by hCMVs in the presence of ATP was significantly higher than that in the absence of ATP. ATP-dependent uptake of both compounds was almost linear against time up to 30 min (Fig. 6). ATP-dependent uptake of [³H]olmesartan (0.021 µM) and [³H]E2-17G (0.25 µM) was also observed in human MRP2-expressing vesicles (Fig. 7). MK-571 (50 µM), an MRP inhibitor, completely inhibited the uptake of [³H]olmesartan (0.021 µM) and [³H]E2-17G (0.25 µM) by hCMVs (Fig. 8).

View larger version (12K): [in this window] [in a new window]   FIG. 6. Time courses of uptake of [14C]olmesartan (A) and [3H]estradiol-17ß-D-glucuronide (B) by human canalicular membrane vesicles. Membrane vesicles (50 µg of protein for olmesartan and 10 µg for E2-17G) were incubated at 37°C for 30 min in 200 µl of transport buffer containing 50 µM [14C]olmesartan or 0.25 µM [3H]E2-17G with () or without () 5 mM ATP and an ATP-generating system. The data represent the mean of duplicate uptake measurements using vesicles of a single donor.

View larger version (13K): [in this window] [in a new window]   FIG. 7. Uptake of [3H]olmesartan and [3H]estradiol-17ß-D-glucuronide by human MRP2-expressing vesicles. MRP2-expressing vesicles were incubated at 37°C for 30 min in 200 µl of transport buffer containing [3H]olmesartan (0.021 µM) and [3H]E2-17G (0.25 µM) with [ATP(+)] or without [ATP(–)] 5 mM ATP and an ATP-generating system. The data represent the mean of duplicate uptake measurements.

View larger version (14K): [in this window] [in a new window]   FIG. 8. Effect of MK-571 on uptake of [3H]olmesartan (A) and [3H]estradiol-17ß-D-glucuronide (B) by human canalicular membrane vesicles. Human canalicular membrane vesicles were incubated at 37°C for 30 min in 200 µl of transport buffer containing [3H]olmesartan (0.021 µM) and [3H]estradiol-17ß-glucuronide (0.25 µM) with [ATP(+)] or without [ATP(–)] 5 mM ATP and an ATP-generating system. MK-571(+) and MK-571(–) indicate the presence or absence of 50 µM MK-571 in the transport buffer, respectively. The data represent the mean of duplicate uptake measurements using vesicles of a single donor.

	Discussion

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We believe that cryopreserved hepatocytes are useful for extrapolation of in vivo hepatic clearance based on the in vitro data as described below. In vivo CL_H was calculated to be 0.86 l/h based on the total clearance and the renal clearance after intravenous administration of olmesartan to healthy male subjects (Laeis et al., 2001). The CL_H value estimated from the in vitro uptake data collected using cryopreserved hepatocytes in the present study (CL_{H,in vitro}) was 0.36 l/h, assuming that the back flux of olmesartan from the liver to plasma is negligible compared with the biliary excretion of olmesartan and was approximately one-third of that of the in vivo CL_H calculated using clinical data. The discrepancy between in vivo CL_H and CL_{H, in vitro} would be generated by a decrease in the transport activity as a result of freezing and/or thawing of the cryopreserved human hepatocytes. Shitara et al. (2003) also demonstrated a decrease in the transport activity of hepatocytes during cell isolation and/or cryopreservation. However, a 3-fold range in predicting or extrapolating the pharmacokinetic parameters in vivo based on in vitro data is considered as an acceptable allowance.

Uptake of olmesartan by human hepatocytes had both Na⁺-dependent and Na⁺-independent fractions as indicated by the facts that the substitution of sodium ion with choline in the uptake medium led to a decrease in the uptake of olmesartan (Table 1), and the uptake under Na⁺-free conditions was higher than the uptake via passive diffusion. Supporting this view, the oocytes injected with human liver polyadenylated RNA also transported olmesartan in both Na⁺-dependent and Na⁺-independent manners (Fig. 3).

As for the Na⁺-independent uptake of olmesartan, both OATP1B1- and OATP1B3-expressed oocytes transported olmesartan (Fig. 4). OATP1B1- and OATP1B3-mediated uptake of olmesartan reached saturation with K_m values of 42.5 and 70.1 µM, respectively (Fig. 4). These values were comparable with the K_m values obtained from human hepatocytes, indicating that OATP1B1 and OATP1B3, at least in part, contribute to the uptake of olmesartan into human liver.

As for the Na⁺-dependent uptake of olmesartan, NTCP-transfected HEK-293 cells did not transport olmesartan. Only NTCP has been identified as an Na⁺-dependent basolateral hepatic transporter (Lecureur et al., 2000), although it has not been reported to transport xenobiotic substrates so far. Thus, there is a possibility that olmesartan is taken up by unknown Na⁺-dependent hepatic basolateral transporter(s) other than NTCP. Akhteruzzaman et al. (1999) have also reported that Na⁺-dependent fraction is involved in hepatic uptake of an anionic cyclopentapeptide, BQ-123, but NTCP did not transport it.

According to the results obtained in the present study, MRP2 quite likely plays a dominant role in the biliary excretion of olmesartan as indicated by the fact that the biliary excretion of olmesartan was low in the experiment using EHBR, which have inherited mrp2 deficiency, compared with Sprague-Dawley rats (Fig. 5). The AUC_(0–2h) in EHBR was ca. 2 times higher than that in Sprague-Dawley rats, although the CL_bile value in EHBR was ca. one-tenth of that in Sprague-Dawley rats (Table 2). It has not been clarified why the AUC_(0–2h) in EHBR was not elevated very much when the CL_bile decreased by almost one-tenth of control without any changes in the cumulative accumulation in urine, but we speculate that accumulation of olmesartan in the liver after uptake could be responsible for the relatively small increase in the plasma exposure. In humans, olmesartan was transported by human MRP2-expressing vesicles (Fig. 7), and an MRP inhibitor, MK-571, completely inhibited the uptake of olmesartan by hCMVs (Fig. 8). Although MK-571 is not a specific inhibitor for MRP2, MK-571 is known to inhibit MRP1, MRP2, and MRP4 (Letschert et al., 2005; Wu et al., 2005), whereas only MRP2 is reported to be expressed on the canalicular membrane (Chan et al., 2004). On the other hand, Takayanagi et al. (2005) reported that olmesartan might be a substrate for P-glycoprotein in rats. However, the results obtained in the present study indicate that P-glycoprotein is not a dominant transporter involved in the biliary excretion of olmesartan.

Because transporters were found to be involved in the hepatobiliary transport of olmesartan, we need to take into consideration the effects of SNPs of certain transporters, such as MRP2, OATP1B1, and OATP1B2, on the pharmacokinetics of olmesartan in humans. Recently, many genetic variants of OATP1B1 (Tirona et al., 2001; Nozawa et al., 2002; Nishizato et al., 2003; Niemi et al., 2004) and MRP2 (Ito et al., 2001; Itoda et al., 2002; Suzuki and Sugiyama, 2002; Hirouchi et al., 2004) have been reported. The SNPs on these transporters have been demonstrated to alter the transport activity in in vitro studies using transfected cells (Hirouchi et al., 2004; Iwai et al., 2004). Several mutations on the coding sequence of MRP2 have been reported in patients with Dubin-Johnson syndrome and some of which led to abolishing the function of MRP2 (Keitel et al., 2003; Ito et al., 2005). Recently, a reduction in the methotrexate elimination half-life was observed in patients with Dubin-Johnson syndrome (Hulot et al., 2005). Thus, there is a possibility that a defect of MRP2 gene may affect the pharmacokinetics of olmesartan. Monitoring of such MRP2-deficient patients may be necessary in clinical situations.

As for OATP1B1 SNPs, Niemi et al. (2004, 2005) reported that SNPs on OATP1B1 affect the pharmacokinetics of pravastatin and repaglinide. In the case of pravastatin, the mean maximum concentration of pravastatin in heterozygous carriers of OATP1B1*15B (containing the 388A>G and 521T>C variants) was approximately 2 times higher than that in noncarriers of OATP1B1*15B (Niemi et al., 2004).

Although there is a possibility of OATP1B1 SNPs somewhat affecting the pharmacokinetics of olmesartan, we believe that OATP1B1 SNPs will not have a great impact on olmesartan pharmacokinetics for the following reason. Olmesartan, as pravastatin, has dual clearance pathways, that is, hepatobiliary and urinary excretion pathways. Regarding the hepatic uptake of pravastatin, this compound is taken up by the liver only in an Na⁺-independent manner. In contrast to pravastatin, however, olmesartan is taken up in both Na⁺-dependent and Na⁺-independent manners. Therefore, even if one of the clearance pathways is impaired, the other could compensate for the impaired pathway. Thus, the extent of the increase in bodily exposure of olmesartan caused by OATP1B1 SNPs is considered to be small.

In addition to the hepatobiliary excretion, there may be a transporter-mediated urinary excretion system for olmesartan. Urinary excretion of olmesartan was approximately 40% of the dose in humans after administration of radiolabeled olmesartan (Laeis et al., 2001). In vivo renal clearance (0.53 l/h) (Davies and Morris, 1993) was higher than the glomerular filtration rate corrected by plasma-free fraction, suggesting the involvement of secretion even though no experimental data are currently available. Further investigation is necessary to clarify renal handling of olmesartan.

In conclusion, OATP1B1 and OATP1B3 contribute to the hepatic uptake of olmesartan, at least in part. MRP2 plays a dominant role in the biliary excretion of olmesartan.

	Footnotes

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

doi:10.1124/dmd.105.008888.

ABBREVIATIONS: AUC, area under the plasma concentration time curve; mrp/MRP, multidrug resistance-associated protein; EHBR, Eisai hyperbilirubinemic rat(s); OATP, organic anion-transporting polypeptide; NTCP, Na⁺-dependent taurocholate-cotransporting peptide; SNP, single nucleotide polymorphism; h, human; CMV, canalicular membrane vesicle; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's medium; E2-17G, estradiol-17ß-D-glucuronide; MK-571, [[[3-[2-(7-chloro-quinolinyl)ethenyl]phenyl][3-(dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid; CL, clearance; PS, permeability-surface area product; BQ-123, cyclo(D-Trp-D-Asp-L-Pro-D-Val)-L-Leu.

Address correspondence to: Dr. Taro Tokui, Drug Metabolism and Pharmacokinetics Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-Ku, Tokyo, 140-8710, Japan. E-mail: tokuit{at}sankyo.co.jp

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