Pitavastatin is a highly potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis (Aoki et al., 1997; Kajinami et al., 2003). Previously, Kimata et al. (1998) have demonstrated that [¹⁴C]pitavastatin is selectively distributed to the liver in rats with the liver/plasma concentration ratio of more than 53. Shimada et al. (2003) have recently reported that the uptake of pitavastatin by rat hepatocytes is saturable and temperature dependent, suggesting that pitavastatin might be transported by carrier-mediated systems. Pitavastatin is scarcely metabolized in human liver microsomes (Fujino et al., 2003) and is excreted into the bile in unchanged form (Kojima et al., 2001). In humans, the fraction excreted in urine was less than 2% (Kajinami et al., 2003). Therefore, when considering the pharmacokinetics of pitavastatin, we should focus on the hepatic clearance of unchanged pitavastatin.

Statins reduce the plasma level of low-density lipoprotein cholesterol and triglycerides in a dose-dependent manner, whereas one of the severe adverse effects, rhabdomyolysis, also appears to be dose-dependent (Davidson et al., 1997). Since liver is a major clearance organ as well as a pharmacological target organ of pitavastatin, it is essential to clarify the uptake mechanism of pitavastatin by hepatocytes to predict the pharmacological and toxicological effects.

At the present time, several transporters are thought to be involved in the hepatic uptake of a variety of drugs in human liver. Na⁺-taurocholate cotransporting polypeptide, organic anion transporting polypeptide (OATP) 2 (OATP1B1, OATP-C/LST-1), OATP8 (OATP1B3, LST-2), OATP-B (OATP2B1), organic anion transporter (OAT) 2, and organic cation transporter 1 are localized on the basolateral membrane of human hepatocytes (Hagenbuch and Meier, 1996, 2003; Muller and Jansen, 1997). In particular, OATP2 and OATP8 are selectively expressed in the human liver and exhibit broad substrate specificities, which suggest that they play an important role in the hepatic uptake of several anionic endogenous compounds and drugs (Hagenbuch and Meier, 2003). OATP2 accepts statins including pravastatin, cerivastatin, and rosuvastatin as substrates (Hsiang et al., 1999; Brown et al., 2001; Nakai et al., 2001; Sasaki et al., 2002; Shitara et al., 2003a). The substrate specificity of OATP2 commonly overlaps that of OATP8, and several compounds can be bisubstrates of both OATP2 and OATP8. However, there are some differences as far as substrate recognition and transcriptional regulation are concerned (Hagenbuch and Meier, 2003; Kullak-Ublick et al., 2004). Therefore, it is essential to evaluate their quantitative contribution to the total hepatic uptake to estimate the overall hepatic clearance for individuals when there are changes in expression level or function caused by pathological conditions, single-nucleotide polymorphisms, and transporter-mediated drug-drug interactions. This kind of information will help us predict the distribution of pitavastatin in the target organ, as well as the plasma concentrations, subsequent pharmacological effects, and adverse events under these conditions. However, so far, no studies have been published describing how to estimate the contribution of each uptake transporter quantitatively in human liver.

In the present study, we analyzed the involvement of the transporters, OATP2 and OATP8, in the hepatic uptake of pitavastatin along with estradiol 17β-d-glucuronide (E₂17βG), a typical substrate of both OATP2 and OATP8 (Cui et al., 2001; Ismair et al., 2001). In addition, we developed a methodology for determining the contribution of each transporter to the hepatic uptake of test compounds by comparing their uptake clearance with that of reference compounds (a selective substrate for OATP2 (estrone-3-sulfate) and OATP8 (cholecystokinin octapeptide) in expression systems and human hepatocytes. We also used another approach by directly comparing expression levels of OATP2 and OATP8 in expression systems and hepatocytes using Western blot analysis and compared the results obtained by these two methods.

Materials and Methods

Materials. Pitavastatin (monocalcium bis[(3R,5S,6E)-7-[2-cyclopropyl-4-(4-fluorophenyl)-3-quinolyl]3,5-dihydroxy-6-hepteonate]) was synthesized by Nissan Chemical Industries (Chiba, Japan). [Fluorobenzene-U-¹⁴C]pitavastatin (11.7 mCi/mmol) was synthesized by Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). E₂17βG and estrone-3-sulfate (E-sul) (45 and 46 Ci/mmol, respectively) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA), whereas cholecystokinin octapeptide (CCK-8) (77 Ci/mmol) was purchased from Amersham Biosciences UK, Ltd.. Unlabeled E₂17βG, E-sul, and CCK-8 were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were of analytical grade and commercially available.

Construction of Stably Transfected HEK293 Cells Expressing Human OATP2 and OATP8. The human OATP2 gene was isolated by PCR using the human liver cDNA purchased from Takara Bio Inc. (Shiga, Japan). The first half (1–1125) of the gene was amplified using a forward primer containing a KpnI site (5′-GGGGTACCATGGACCAAAATCAACATTTGAAT-3′) and a reverse primer (5′-GTTAGCCTTAGATGAAGGCTGACC-3′). The second half (1041–2076) of the gene was amplified using a forward primer (5′-ACAAGTAAGCAGCTATATTGGTGC-3′) and a reverse primer containing a NotI site (5′-GGGCGGCCGCTTAACAATGTGTTTCACTATCTG-3′). Each PCR product was TA cloned into the pGEM vector (Promega, Madison, WI), respectively. Then, the pGEM vector containing the first half was digested with KpnI and Eco105 I, and the pGEM vector with the second half was digested with Eco105 I and NotI. Subsequently, the first and second halves of the OATP2 cDNA were ligated into KpnI and NotI sites of the pcDNA3.1 (+) (Invitrogen, Carlsbad, CA) to obtain the full-length human OATP2 gene.

The human OATP8 gene was isolated by PCR using human liver cDNA purchased from BD Biosciences Clontech (Palo Alto, CA) as a template. The C-terminal fragment of the OATP8 gene was amplified using a forward primer (5′-AGAGTCAGCATCTTCAGAG-3′) and a reverse primer (5′-TTTTCCTTTTGCGGCCGCATTGTCAGTGAAAGACCAGG-3′) and TA cloned into pGEM vector. The N-terminal fragment was amplified using a forward primer containing a HindIII site and Kozak sequence just before the start codon (5′-CCCAAGCTTCCGCCATGGACCAACATCAAC-3′) and a reverse primer (5′-CTTCGGTCTGTGTAGTTTAG-3′). To subclone the full length of the OATP8 cDNA, fragment 1 was prepared by digesting the pGEM vector containing the C-terminal fragment with HindIII and NotI, and fragment 2 was created by digesting the pGEM vector containing the N-terminal fragment with HindIII. Then, fragments 1 and 2 were ligated into linearized pcDNA3.1 (+) digested with HindIII and NotI.

OATP2- and OATP8-expressing HEK293 cells and control cells were constructed by the transfection of expression vector and control pcDNA3.1 vector, respectively, into cells using FuGENE6 (Roche Diagnostics, Indianapolis, IN), according to the manufacturer's instruction and the selection by 800 μg/ml Antibiotic G418 sulfate (Promega, Madison, WI) for 3 weeks.

Cell Culture. Transporter-expressing or vector-transfected HEK293 cells were grown in Dulbecco's modified Eagle's medium low glucose (Invitrogen) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B at 37°C with 5% CO₂ and 95% humidity. Cells were then seeded in 12-well plates at a density of 1.5 × 10⁵ cells/well. For the transport study, the cell culture medium was replaced with culture medium supplemented with 5 mM sodiumbutyrate for 24 h before transport assay to induce the expression of OATP2 and OATP8.

Transport Study Using Transporter Expression Systems. The transport study was carried out as described previously (Sugiyama et al., 2001). Uptake was initiated by adding Krebs-Henseleit buffer containing radiolabeled and unlabeled substrates after cells had been washed twice and preincubated with Krebs-Henseleit buffer at 37°C for 15 min. The Krebs-Henseleit buffer consisted of 118 mM NaCl, 23.8 mM NaHCO₃, 4.8 mM KCl, 1.0 mM KH₂PO₄, 1.2 mM MgSO₄, 12.5 mM HEPES, 5.0 mM glucose, and 1.5 mM CaCl₂ adjusted to pH 7.4. The uptake was terminated at a designated time by adding ice-cold Krebs-Henseleit buffer after removal of the incubation buffer. Then, cells were washed twice with 1 ml of ice-cold Krebs-Henseleit buffer, solubilized in 500 μl of 0.2 N NaOH, and kept overnight at 4°C. Aliquots (500 μl) were transferred to scintillation vials after adding 250 μl of 0.4 N HCl. The radioactivity associated with the cells and incubation buffer was measured in a liquid scintillation counter (LS6000SE; Beckman Coulter, Fullerton, CA) after adding 2 ml of scintillation fluid (Clear-sol I; Nacalai Tesque, Kyoto, Japan) to the scintillation vials. The remaining 50 μl of cell lysate was used to determine the protein concentration by the method of Lowry with bovine serum albumin as a standard.

Transport Study Using Human Cryopreserved Hepatocytes. This experiment was performed as described previously (Shitara et al., 2003a). Cryopreserved human hepatocytes were purchased from In Vitro Technologies (Baltimore, MD). In this experiment, we selected three batches of human hepatocytes (Lot. OCF, 094, and ETR) that show relatively high uptake amount of E₂17βG and E-sul among eight independent batches of hepatocytes. Immediately before the study, the hepatocytes (1-ml suspension) were thawed at 37°C, then quickly suspended in 10 ml of ice-cold Krebs-Henseleit buffer and centrifuged (50g) for 2 min at 4°C, followed by removal of the supernatant. This procedure was repeated once more to remove cryopreservation buffer, and then the cells were resuspended in the same buffer to give a cell density of 1.0 × 10⁶ viable cells/ml for the uptake study. The number of viable cells was determined by trypan blue staining. To measure the uptake in the absence of Na⁺, sodium chloride and sodium bicarbonate in Krebs-Henseleit buffer were replaced with choline chloride and choline bicarbonate. Prior to the uptake studies, the cell suspensions were prewarmed in an incubator at 37°C for 3 min. The uptake studies were initiated by adding an equal volume of buffer containing labeled and unlabeled substrates to the cell suspension. After incubation at 37°C for 0.5, 2, or 5 min, the reaction was terminated by separating the cells from the substrate solution. For this purpose, an aliquot of 80 μl of incubation mixture was collected and placed in a centrifuge tube (450 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). During this process, hepatocytes passed through the oil layer into the alkaline solution. After an overnight incubation in alkali to dissolve the hepatocytes, the centrifuge tube was cut and each compartment was transferred to a scintillation vial. The compartment containing the dissolved cells was neutralized with 50 μlof 2 N HCl, mixed with scintillation cocktail, and the radioactivity was measured in a liquid scintillation counter.

Antiserum and Western Blot Analysis. As shown in previous reports, anti-OATP2 and anti-OATP8 sera were raised in rabbits against a synthetic peptide consisting of the 21 and 15 carboxylterminal amino acids, respectively, of OATP2 and OATP8 coupled to keyhole limpet hemocyanine at its N terminus via an additional cysteine (Konig et al., 2000a,b). Crude membrane fractions were prepared from human hepatocytes and transporter-expressing HEK293 cells as described previously (Sasaki et al., 2002). The crude membrane fractions were diluted with 3× Red loading buffer (Bio-Labs, Hertfordshire, UK) and loaded onto a 7% SDS-polyacrylamide gel with a 4.4% stacking gel. Proteins were electroblotted onto a polyvinylidene diflouride membrane (Pall, East Hills, NY) using a blotter (Trans-blot; Bio-Rad, Hercules, CA) at 15 V for 1 h. The membrane was blocked with Tris-buffered saline containing 0.05% Tween 20 (TBS-T) and 5% skimmed milk for 1 h at room temperature. After washing with TBS-T, the membrane was incubated with anti-OATP2 serum (dilution 1:500) or anti-OATP8 serum (dilution 1:1000). The membrane was incubated with a horseradish peroxidase-labeled anti-rabbit IgG antibody (Amersham Biosciences UK, Ltd.) diluted 1:2000 in TBS-T for 1 h at room temperature followed by washing with TBS-T. The band was detected, and its intensity was quantified using an image analyzer (LAS-1000 plus; Fuji Film, Tokyo, Japan).

Kinetic Analyses. Ligand uptake was expressed as the uptake volume (microliters per milligram of protein), given as the amount of radioactivity associated with the cells (disintegrations per minute per milligram of protein) divided by its concentration in the incubation medium (disintegrations per minute per microliter). Specific uptake was obtained by subtracting the uptake into vector-transfected cells from the uptake into cDNA-transfected cells. Kinetic parameters were obtained using the following equation: where v is the uptake velocity of the substrate (picomoles per minute per milligram of protein), S is the substrate concentration in the medium (micromolar), K_m is the Michaelis constant (micromolar), V_max is the maximum uptake rate (picomoles per minute per milligram of protein), and P_dif is the nonsaturable uptake clearance (microliters per minute per milligram of protein). Fitting was performed by the nonlinear least-squares method using a MULTI program (Yamaoka et al., 1981), and the Damping Gauss Newton Method algorithm was used for fitting. The input data were weighted as the reciprocal of the observed values.

To determine saturable hepatic uptake clearance in human hepatocytes, we first determined the hepatic uptake clearance [CL_{(2min–0.5min)}] (microliters per minute per 10⁶ cells) by calculating the slope of the uptake volume (V_d) (microliters per 10⁶ cells) between 0.5 and 2 min (eq. 2). The saturable component of the hepatic uptake clearance (CL_hep) was determined by subtracting CL_{(2 min–0.5 min)} in the presence of 100 μM substrate (excess) from that in the presence of 1 μM substrate (tracer) (eq. 3). where CL_{(2min–0.5min),tracer} and CL_{(2min–0.5 min),excess} represent CL_{(2min–0.5min)} estimated in the presence of 1 and 100 μM substrate, respectively.

Estimation of Uptake Clearance in Human Hepatocytes from cDNA Transfectants. The use of this method for estimating the contribution of each molecule to the overall reaction [relative activity factor (RAF) method] has been described for cytochrome P450 (Crespi and Penman, 1997), and it has also been applied to renal uptake transporters (Hasegawa et al., 2003). Based on this strategy, we estimated the contribution of OATP2 and OATP8 to the overall uptake by human hepatocytes. Because E-sul and CCK-8 could be selective substrates for OATP2 and OATP8, respectively, they were used as reference compounds for OATP2- and OATP8-mediated uptake (Cui et al., 2001; Ismair et al., 2001). The ratio of the uptake clearance of reference compounds in human hepatocytes to that in the expression system was calculated and defined as R_{act, OATP2} and R_{act, OATP8}. The uptake clearance by OATP2 and OATP8 was separately calculated by multiplying the uptake clearance of the test compounds (pitavastatin and E₂17βG) in transporter-expressing cells (CL_OATP2,test and CL_OATP8,test) by R_{act, OATP2} and R_{act, OATP8}, respectively, as described in the following equations:

In addition, the ratio of the expression levels of OATP2 and OATP8 in human hepatocytes (per 10⁶ cells) to transporter-expressing cells (per milligram of protein) (R_{exp, OATP2} and R_{exp, OATP8}) could be calculated from the intensity of specific bands in Western blot analysis and the amount of crude membrane prepared from each cell type as follows. The relative expression level per 10⁶ hepatocytes or milligram of protein in HEK293 whole cells was given by multiplying the band density per unit protein amount in crude membrane of each batch of hepatocytes or transporter-expressing HEK293 cells by protein amount in crude membrane obtained by 10⁶ hepatocytes or 1 mg of whole-cell protein in HEK293 transfectants. The R_exp value was calculated as the relative expression level per 10⁶ hepatocytes divided by that per milligram of protein in HEK293 cells. The OATP2- and OATP8-mediated hepatic uptake of test compounds (pitavastatin and E₂17βG) was calculated from the following equation:

Results

Uptake of E₂17βG, E-sul, CCK-8, and Pitavastatin by Transporter-Expressing Cells. The saturation kinetics of E₂17βG, E-sul, CCK-8, and pitavastatin by OATP2- and OATP8-expressing cells and vector-transfected HEK293 are shown as Eadie-Hofstee plots in Fig. 1. Pitavastatin as well as E₂17βG were significantly taken up into both OATP2- and OATP8-expressing HEK293 cells compared with vector-transfected cells (Fig. 1, A and D). The transfection of OATP2 resulted in an increase in the uptake of E-sul but did not affect the uptake of CCK-8, whereas transfection of OATP8 resulted in an increase in the uptake of CCK-8 but not E-sul (Fig. 1, B and C). The concentration dependence of the uptake of pitavastatin, E₂17βG, and E-sul could be explained by a one-saturable component (Fig. 1, A, B, and D). However, a saturable and a nonsaturable component could be accounted for even in the specific uptake of CCK-8 determined by subtraction of the uptake by vector-transfected cells from that by OATP8-expressing cells (Fig. 1C). Their kinetic parameters are summarized in Table 1. The rank order in the uptake clearance by OATP2 was E-sul > pitavastatin > E₂17βG, whereas that by OATP8 was pitavastatin > CCK-8 > E₂17βG.

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Fig. 1.

Eadie-Hofstee plots of the uptake of [³H]E₂17βG, [³H]E-sul, [³H]CCK-8, and [¹⁴C]pitavastatin by OATP2- and OATP8-expressed HEK293 cells. Squares and circles represent the uptake in OATP2- and OATP8-expressed cells, respectively. The concentration dependence of OATP2- and OATP8-mediated uptake of [³H]E₂17βG (A), [³H]E-sul (B), [³H]CCK-8 (C), and [¹⁴C]pitavastatin (D) is shown as Eadie-Hofstee plots. The uptake of [³H]E₂17βG, [³H]E-sul, [³H]CCK-8, and [¹⁴C]pitavastatin for 5, 0.5, 2, and 2 min, respectively, was determined at various concentrations (0.1–30 μM). The OATP2- and OATP8-mediated transport was obtained by subtracting the uptake in vector-transfected cells from that in OATP2- or OATP8-expressing cells. Lines represent the fitted curves obtained by nonlinear regression analysis. Each point represents the mean ± S.E. (n = 3).

Uptake of E₂17βG, E-sul, CCK-8, and Pitavastatin by Human Cryopreserved Hepatocytes. Typical time profiles of the uptake of E₂17βG, E-sul, CCK-8, and pitavastatin in one batch of human hepatocytes (Lot. OCF) are shown in Fig. 2. Time-dependent uptake of all ligands was observed at 1 μM, and it was decreased in the presence of 100 μM unlabeled ligands in all batches of hepatocytes examined in the present study (data not shown). The uptake clearance of these substrates in each donor is listed in Table 2. The uptake clearance by human hepatocytes was in the order E-sul > pitavastatin > E₂17βG > CCK-8.

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Fig. 2.

Time profiles of the uptake of [³H]E₂17βG, [³H]E-sul, [³H]CCK-8, and [¹⁴C]pitavastatin by human hepatocytes (Lot. ETR). The uptake of these substrates for 0.5, 2, and 5 min was determined at two concentrations (closed circle, 1 μM; open circle, 100 μM) at 37°C. Each point represents the mean ± S.E. (n = 3).

The Effect of Sodium Ion on the Uptake of Pitavastatin by Human Cryopreserved Hepatocytes. To determine whether the uptake of pitavastatin into hepatocytes is Na⁺ dependent or not, we investigated the pitavastatin uptake in the presence and absence of Na⁺ in human hepatocytes. As shown in Fig. 3, replacement of Na⁺ with choline in the transport buffer had no effect on the uptake of pitavastatin in human hepatocytes.

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Fig. 3.

Time profiles of the uptake of [¹⁴C]pitavastatin by human hepatocytes (Lot. OCF) in the presence and absence of Na⁺. The uptake of pitavastatin for 0.5, 2, and 5 min was determined at two concentrations (closed symbol, 1 μM; open symbol, 100 μM) at 37°C. Circles and triangles indicate the pitavastatin uptake in the presence and absence of Na⁺, respectively. Each point represents the mean ± S.E. (n = 3).

Western Blot Analysis. The relative expression levels of OATP2 and OATP8 in crude membrane from transfected cells and hepatocytes were investigated by Western blot analyses. An antiserum against OATP2 recognized approximately 80-kilodalton proteins in the crude membrane fractions prepared from human hepatocytes and OATP2-expressing cells, respectively (Fig. 4A). Anti-OATP8 antiserum could detect the specific band of approximately 120-kilodalton proteins in the crude membrane fractions prepared from human hepatocytes and OATP8-expressing cells, respectively (Fig. 4B). The molecular weights of OATP2 and OATP8 in the human hepatocytes were almost the same as those in OATP2- and OATP8-expressing cells, respectively. No expression of OATP2 or OATP8 was observed in vector-transfected HEK293 cells (Fig. 4, A and B). Figure 4, C and D showed the linear relationship between the applied amount of crude membrane obtained from transporter-expressing cells and human hepatocytes and the intensity of the specific band measured by digital densitometer. The slope of the regression line in Fig. 4, C and D reflected the relative expression level of each transporter in the transfectants and hepatocytes.

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Fig. 4.

Western blot analysis of OATP2 and OATP8. Crude membrane fractions (5, 10, and 20 μg) prepared from OATP2- and OATP8-expressed HEK293 cells and human hepatocytes (Lot. 094) were loaded and separated by SDS-PAGE (7% separating gel). The sample indicated “Human liver” means the crude membrane vesicles prepared from a human frozen liver block as a positive control. OATP2 and OATP8 were detected by preimmune antisera raised against the carboxyl terminus of human OATP2 (A) and OATP8 (B), respectively. Comparison of the relative expression levels of OATP2 (C) and OATP8 (D) between transfectants and hepatocytes is shown. The x-axis and y-axis represent the amount of crude membrane obtained from transfectants and human hepatocytes and the intensity of the specific band in Western blot analysis, respectively. Closed circles and open circles indicate the band density of human hepatocytes (Lot. 094) and OATP2- (C) or OATP8- (D) expressing HEK293 cells, respectively.

Estimation of Uptake Clearance and Contribution of OATP2 and OATP8 in Human Hepatocytes. We calculated the estimated uptake clearance of OATP2 and OATP8 in human hepatocytes by two approaches (Tables 3 and 4). In the first approach, by comparing the uptake clearance of reference compounds (E-sul for OATP2 and CCK-8 for OATP8) in transfectants and hepatocytes, we were able to calculate the ratio of the clearance (R_act) and estimated clearance of pitavastatin and E₂17βG mediated by OATP2 and OATP8 based on the RAF method (Table 3). In the second approach, we compared the intensity of specific bands of transfectants and hepatocytes in Western blot analysis. We could obtain 62.1 and 96.7 μg of protein in crude membrane from 1 mg of whole cell protein in OATP2- and OATP8-expressing HEK293 cells, respectively, whereas 178, 89, and 82 μg of protein in crude membrane were obtained from 10⁶ hepatocytes of Lot. OCF, 094, and ETR, respectively. When the band density per unit protein amount in crude membrane of OATP2- or OATP8-expressing HEK293 cells was defined as 1, the relative expression level of OATP2 or OATP8 per unit protein amount in crude membrane of hepatocytes of Lot. OCF, 094, and ETR was 1.01, 1.10, and 0.673 (per microgram) for OATP2 and 0.659, 1.01, and 0.872 (per microgram) for OATP8, respectively. Using these values, the relative ratio of the expression level (R_exp) and estimated clearance could be calculated (Table 4). The estimation by the two approaches suggested that both pitavastatin and E₂17βG are taken up into human hepatocytes predominantly by OATP2 (Tables 3 and 4).

Discussion

In the present study, we have clarified that pitavastatin is taken up into human hepatocytes via OATP2 and OATP8, and we also investigated the transport properties of pitavastatin using transporter-expressing HEK293 cells and human cryopreserved hepatocytes to estimate the contribution of these transporters to the total uptake in human hepatocytes.

Significant uptake of pitavastatin via OATP2 and OATP8 was observed compared with control cells, and this became saturated on increasing its concentration in the medium (Fig. 1). It has been shown that, in a series of statins, pravastatin, cerivastatin, and rosuvastatin are substrates of OATP2 (Brown et al., 2001; Nakai et al., 2001; Sasaki et al., 2002; Shitara et al., 2003a). The K_m value of pitavastatin for OATP2 is comparable with that of cerivastatin (K_m = 4.3 μM) (in-house data) and rosuvastatin (K_m = 7.3 μM) (Brown et al., 2001) and smaller than that of pravastatin (K_m = 24.3 μM) (Sasaki et al., 2002).

We demonstrated here for the first time that, as far as statins are concerned, pitavastatin is a substrate for OATP8 as well as OATP2 (Fig. 1D). K_m values of pitavastatin for OATP2 and 8 were comparable (Fig. 1; Table 1); therefore, we were not able to determine which transporter is mainly responsible for its uptake in human hepatocytes only by saturation kinetics in human hepatocytes.

It has been reported that E-sul and CCK-8 are selective substrates of OATP2 and OATP8, respectively (Cui et al., 2001; Ismair et al., 2001). We confirmed these findings in our experimental systems (Fig. 1). We also confirmed that E₂17βG was a good substrate for both OATP2 and OATP8 by using transporter-expressing cells (Fig. 1) (König et al., 2000a, b). As shown in Table 1, the K_m values of E₂17βG, E-sul, and CCK-8 were comparable with the reported values (Ismair et al., 2001; Tamai et al., 2001).

Next, we performed the uptake study in human cryopreserved hepatocytes. Shitara et al. (2003b) reported that large interbatch differences in uptake activity were observed in human cryopreserved hepatocytes probably due to differences in the conditions of isolation of the hepatocytes and cryopreservation as well as the interindividual variability in the expression and function of transporters. So, we carried out a study using three batches of hepatocytes prepared from three independent donors. In all three batches examined in the present study, pitavastatin, E₂17βG, E-sul, and CCK-8 were taken up in a time-dependent and saturable manner (Fig. 2).

We examined the uptake of pitavastatin in the presence and absence of Na⁺ to determine whether Na⁺-dependent uptake of pitavastatin was observed in human hepatocytes. In this experiment, we confirmed that the batch (Lot. OCF) used in the present study exhibited Na⁺-dependent uptake of taurocholate (data not shown) to the same extent as shown in the previous report (Shitara et al., 2003b). However, the uptake of pitavastatin by hepatocytes did not change regardless of the presence of Na⁺, suggesting that pitavastatin is mainly taken up in a Na⁺-independent way, suggesting that Na⁺-taurocholate-cotransporting polypeptide is not mainly involved in hepatic uptake of pitavastatin. Previous reports demonstrated that both OATP2 and OATP8 can transport substrates in an Na⁺-independent manner (König et al., 2000a,b), and these Na⁺-independent transporters are involved in hepatic uptake of pitavastatin.

To estimate the contribution of OATP2 and OATP8 to the hepatic uptake of pitavastatin, we developed a method using reference compounds that were selective substrates of OATP2 or OATP8. Crespi and Penman (1997) proposed the RAF method using the ratio of the metabolic activity in human liver microsomes divided by the activity in each isoform-specific cytochrome P450 reaction. Applying the RAF concept to transporter research, Hasegawa et al. (2003) calculated the contribution of Oat1 and Oat3 to the renal uptake in rats by comparing the uptake clearance of reference compounds (p-aminohippurate for Oat1 and benzylpenicillin for Oat3) in kidney slices and Oat1- and Oat3-transfected cells. According to this method, assuming that both for OATP2 and OATP8, the intrinsic clearance per unit molecule in human hepatocytes is the same as that in the expression system, the ratio of the transport activity of reference compounds in human hepatocytes to that in transporter-expressing cells (R_act) should correspond to the relative expression level of each transporter. Multiplying R_act by the uptake clearance of pitavastatin and E₂17βG in each transfectant, we can estimate the OATP2- and OATP8-mediated portion of the uptake of pitavastatin and E₂17βG in hepatocytes. From our estimation described above, the sum of the calculated OATP2- and OATP8-mediated uptake was almost identical to the experimentally observed uptake clearances in the cases of both pitavastatin and E₂17βG (Tables 2 and 3). Therefore, the uptake of pitavastatin and E₂17βG by human hepatocytes could be fully explained by OATP2 and OATP8, and more than 87% of their total hepatic uptake could be accounted for by OATP2 in all three independent batches of human cryopreserved hepatocytes (Tables 2 and 3).

To validate our estimation, we directly compared the expression level of OATP2 and OATP8 between transfectants and hepatocytes by Western blot analysis and calculated the ratio of the expression level in these cells (R_exp) as described above. As a result of our estimation using R_exp values, the sum of the predicted OATP2- and OATP8-mediated uptake of pitavastatin and E₂17βG was 5 to 10 times higher than their observed clearance in hepatocytes (Tables 2 and 4), a finding that is not consistent with our earlier estimation based on the relative uptake clearance of the reference compounds. There are two possibilities to account for this discrepancy. Firstly, the recovery of each transporter protein in samples for Western blot analysis in transfectants and hepatocytes may be different. Secondly, the total amount of protein in the whole-cell crude membrane, which we measured in the present study, may not indicate the expression level of a functional transporter on the cell surface. On the other hand, our results indicated that the ratio of the R_exp values for OATP2 and OATP8 was comparable with that of the R_act values and therefore, the estimated contribution of each transporter was almost the same when using the two calculation approaches. The contribution of OATP2 to the hepatic uptake of pitavastatin and E₂17βG estimated from the R_exp value also indicated more than 75 and 85% of the overall uptake in all three batches of hepatocytes, respectively, suggesting that the results obtained by the two approaches are consistent, and OATP2 appears to be mainly responsible for the hepatic uptake of pitavastatin and E₂17βG although both compounds are partly taken up by OATP8.

Alcorn et al. (2002) have reported that the mRNA level of OATP-B is almost the same as that of OATP2 in human liver. Strictly speaking, E-sul is not a selective substrate of OATP2 because some reports have indicated that it can be taken up by OATP-B and OATP8 (Kullak-Ublick et al., 2001; Kobayashi et al., 2003; Ismair et al., 2003), although we could not observe significant uptake of E-sul via OATP8 (Fig. 1), and Cui et al. (2001) reported that OATP8-expressing HEK293 cells did not significantly take up E-sul. However, we believe that E-sul can be used as a specific substrate of OATP2 in hepatocytes for the following reason. Previous reports have demonstrated that CCK-8 can be transported by OATP8, but not OATP-B and OATP2 (Ismair et al., 2001), and that E₂17βG can be a substrate of OATP2 and OATP8, but not OATP-B (Kullak-Ublick et al., 2001; Tamai et al., 2001). Taking these facts into consideration, we believe that the hepatic uptake of E₂17βG can be almost completely accounted for by OATP2 because the contribution of OATP8 to the overall hepatic uptake of E₂17βG was negligible in our estimation based on the uptake clearance of CCK-8 (Table 3). On the other hand, the uptake clearance of E₂17βG in human hepatocytes was almost comparable with the OATP2-mediated uptake clearance of E₂17βG estimated from the uptake clearance of E-sul in human hepatocytes and the OATP2 expression system. Therefore, we believe that E-sul is taken up into human hepatocytes mainly by OATP2.

In clinical situations, Shitara et al. (2003a) suggested that the drug-drug interaction between cerivastatin and cyclosporin A is mediated by OATP2. Nishizato et al. (2003) reported that OATP-C*15 polymorphism affects plasma concentration of pravastatin. Therefore, OATP2 may play a major role in the hepatic uptake of statins, and we should pay attention to functional change of OATP2 in pharmacokinetics of statins.

In conclusion, we have evaluated the contribution of OATP2 and OATP8 to the hepatic uptake of pitavastatin and E₂17βG by transporter-expressing HEK293 cells and human hepatocytes. When we estimated their contribution by two approaches using the uptake of transporter-selective substrates and the relative expression level measured by Western blot analysis, most of the total hepatic clearance of pitavastatin and E₂17βG could be accounted for by OATP2. These methods will be useful for rapidly identifying the separate contributions of OATP2 and OATP8 to the hepatic uptake.