Expression and Transport Function of Drug Uptake Transporters in

Aug 16, 2012 - ... Hideki Aizaki , Maiko Okada , Masaya Sugiyama , Soichi Kojima , Yasuhito Tanaka ... Kenta Yoshida , Kazuya Maeda , Yuichi Sugiyama...
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Expression and Transport Function of Drug Uptake Transporters in Differentiated HepaRG Cells Naoki Kotani,† Kazuya Maeda,† Yasuyuki Debori,† Sandrine Camus,‡ Ruoya Li,‡ Christophe Chesne,‡ and Yuichi Sugiyama*,§ †

Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, the University of Tokyo, 3-1, 7-Chome Hongo, Bunkyo-ku, Tokyo 113-0033, Japan ‡ Biopredic International, 14 rue Jean Pecker, 35000 Rennes, France § Sugiyama Laboratory, RIKEN Innovation Center, RIKEN Research Cluster for Innovation, 1-6 Suehiro-Cho, Tsurumi-ku, Yokohama 230-0045, Japan ABSTRACT: HepaRG cells have the ability to differentiate into hepatocyte-like cells. Many papers have shown that these hepatocyte-like cells share several functional properties with intact human hepatocytes. However, although previous studies have indicated the partial maintenance of mRNA expression of drug transporters, their expression and function have not been quantitatively characterized. In the present study, the mRNA and protein expression levels and transport activities of hepatic uptake transporters, organic anion transporting polypeptides (OATPs) and Na+-taurocholate cotransporting polypeptide (NTCP) in HepaRG cells were compared with those in cryopreserved human hepatocytes. The mRNA expression levels of OATP1B1, OATP1B3, OATP2B1, and NTCP in HepaRG cells were 22−38%, 2−15%, 82−113%, and 191−247% of those in human hepatocytes, respectively. The relative protein expression of these transporters was comparable with their mRNA expression. We observed saturable uptake of typical substrates of NTCP and OATPs except for cholecystokinin octapeptide (OATP1B3-selective substrate), and Na+-dependent uptake of taurocholate was confirmed. Their relative uptake clearances were well explained by their mRNA and protein expression levels. Additionally, inhibition potencies of 12 OATP1B1 inhibitors were investigated both in HepaRG cells and in OATP1B1expressing HEK293 cells to demonstrate the usefulness of HepaRG cells for the characterization of OATP1B1-mediated drug− drug interactions. The Ki values in both cell lines were comparable and showed significant correlation. These results suggest that the hepatic uptake transport function of OATP and NTCP transporters was relatively well maintained in HepaRG, although OATP1B3 function was too low to be detected. KEYWORDS: HepaRG cells, organic anion transporting polypeptide (OATP), Na+-taurocholate cotransporting polypeptide (NTCP), hepatic uptake, cryopreserved human hepatocytes, drug−drug interaction

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INTRODUCTION

show large interbatch variability in their metabolic and transport activities.7,8 Thus, it is generally recommended to screen the transport activity of typical substrates in each batch of human hepatocytes and to perform screening in advance of routine assays. Moreover, even if ideal batches of hepatocytes can be determined, the number of hepatocytes obtained from one donor is limited. Under such situations, it is difficult to get reproducible results with different batches of human hepatocytes and they remain too expensive to be used for large-scale screening at the early stage of drug development. Therefore, there is a great need for alternative in vitro models of human hepatocytes that could be applied to the prediction of hepatic clearance in humans.

The liver is one of the most important organs for eliminating drugs from circulating blood. Many endogenous and exogenous compounds undergo hepatic uptake, metabolism, and biliary excretion, and thus the hepatic clearance and subsequent pharmacokinetics of drugs are sometimes affected by the alteration of the functions of hepatic uptake transporters, cytochrome P450s (CYPs), and efflux transporters.1−3 Therefore, the quantitative prediction of hepatic clearance of drugs in humans by in vitro experiments is clearly of great importance. In recent decades, numerous studies have suggested that drug transporters play an important role in clinical pharmacokinetics, and the importance of the accurate prediction of in vivo drug disposition from in vitro methods has been rapidly recognized, especially in the early stage of drug development.4 Currently, cryopreserved human hepatocytes can be obtained from several commercial sources and they are good in vitro tools for predicting the hepatic transport properties of drugs in humans.5,6 However, human hepatocytes © 2012 American Chemical Society

Received: Revised: Accepted: Published: 3434

April 2, 2012 July 26, 2012 August 16, 2012 August 16, 2012 dx.doi.org/10.1021/mp300171p | Mol. Pharmaceutics 2012, 9, 3434−3441

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(77 Ci/mmol) was purchased from GE Healthcare BioSciences (Waukesha, WI). [3H]-Olmesartan (79 Ci/mmol) and unlabeled olmesartan were kindly donated by Daiichi Sankyo Co., Ltd. (Tokyo, Japan). [3H]-Pitavastatin (16.0 Ci/mmol) and unlabeled pitavastatin were kindly donated by Kowa Co., Ltd. (Tokyo, Japan). Unlabeled E217βG, E1S, and CCK-8 were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals were of analytical grade and commercially available. Cell Culture. Plated differentiated HepaRG cells from Biopredic International (Rennes, France) were provided in a 24-well plate format (HPR203-MW24) and were cultured according to the manufacturer’s normal protocol. The passage numbers of HepaRG cells we used were between 13 and 20. The cells were cultured at the density of 0.27−0.46 × 106 cells/ well in 24-well plate. OATP1B1-expressing and vector-transfected control HEK293 cells were constructed previously.19 They were grown in Dulbecco’s modified Eagle’s medium (low glucose) (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen) and 1% antibiotic−antimycotic solution (Invitrogen) at 37 °C under 5% CO2 and 95% humidity. Determination of mRNA Expression Levels of Hepatic Uptake Transporters. The mRNA levels of OATP1B1, OATP1B3, OATP2B1, NTCP, and human glyceraldehyde 3phosphate dehydrogenase (hGAPDH) were quantified using a quantitative real-time polymerase chain reaction (qPCR) method. Total RNA was isolated from cultured HepaRG cells or cryopreserved human hepatocytes using Isogen (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer’s instructions. Reverse transcription was performed after DNase I (Takara Bio, Shiga, Japan) treatment at 37 °C for 30 min qPCR was performed using SYBR Premix Ex Taq (Takara Bio) and a LightCycler system (Roche Diagnostics, Mannheim, Germany). For qPCR, the following primers were used: OATP1B1, forward 5′-ACT TGG AGG CAC CTC ACA TG-3′ and reverse 5′-CCT ACA TGA CCC ACG TGT GC-3′; OATP1B3, forward 5′-GGG TGA ATG CCC AAG AGA TA-3′ and reverse 5′-ATT GAC TGG AAA CCC ATT GC-3′; OATP2B1, forward 5′-CTT CAT CTC GGA GCC ATA CC-3′ and reverse 5′-GCT TGA GCA GTT GCC ATT G-3′; NTCP, forward 5′-ACT GGT CCT GGT TCT CAT TCC-3′ and reverse 5′-GTG GCA ATC AAG AGT GGT GTC-3′; and GAPDH, forward 5′-AAT GAC CCC TTC ATT GAC-3′ and reverse 5′-TCC ACG ACG TAC TCA GCG C-3′. For the mRNA quantification, an external standard curve for each target gene was generated by using serially diluted solutions of its conventional PCR product as a template for qPCR. This conventional PCR product was purified by agarose electrophoresis, and its DNA content was measured using PicoGreen dsDNA Quantification Reagent (Invitrogen). Using Light Cycler software, mRNA expression levels of OATP1B1, OATP1B3, OATP2B1, and NTCP were determined and normalized to that of hGAPDH. Western Blot Analysis. Crude membrane fraction was prepared from cultured HepaRG cells or cryopreserved human hepatocytes as reported previously.20 After the crude membrane fraction was suspended in phosphate buffered saline (PBS), it was immediately frozen in liquid N2 and stored at −80 °C until used. The protein concentration of the crude membrane fractions prepared from HepaRG cells or human hepatocytes was determined by the method of Lowry (1951) with bovine serum albumin as a standard.21 The membrane fraction was dissolved in 3×SDS sample buffer (New England

Recently, a new human hepatocarcinoma cell line, HepaRG cells, which can differentiate into hepatocyte-like cells, has become available.9,10 In recent reports, several liver-specific properties of differentiated HepaRG cells were investigated. These differentiated hepatocyte-like cells express various metabolic enzymes including CYPs, uridine 5′-diphosphoglucuronosyltransferases and glutathione S-transferases as abundantly as do human hepatocytes, in contrast to other human hepatoma-derived cell lines such as HepG2 cells. Moreover, long-term stable expression and function of CYP3A4 in the HepaRG cell line has been confirmed.11 A good relationship between the CYP3A4 induction potency of drugs obtained from in vitro assays using HepaRG cells and that from an in vivo study was also observed.12 McGill et al. reproduced several phenomena in acetaminophen-induced liver injury by using an in vitro experimental system with HepaRG cells such as a reduction of intracellular reduced glutathione and an increase in the covalent binding of active metabolites of acetaminophen to intracellular protein, which demonstrated the usefulness of HepaRG cells for toxicity screening.13 Several studies demonstrated that HepaRG cells are susceptible to hepatitis virus infection,10,14 and demonstrated the utility of the cells as a surrogate infection system. These studies suggested that differentiated HepaRG cells share several functional properties with intact human hepatocytes. The mRNA expression of hepatic uptake transporters, such as Na+-taurocholate cotransporting polypeptide (NTCP) and organic anion transporting polypeptide (OATP), was detected in HepaRG cells to a similar extent to that in human hepatocytes described in previous reports.15,16 Conversely, in many other human hepatocarcinoma cell lines such as HepG2 and Fa2N-4, the expression of these uptake transporters was very low or undetectable compared with in human hepatocytes.17,18 This evidence suggests that HepaRG cells possess unique characteristics in transporter expression, and it is expected that HepaRG cells can be used as an alternative in vitro system for the screening of transporter substrates and inhibitors to an assay with human hepatocytes. However, there is very little information regarding the expression and the function of hepatic uptake transporters in the hepatocyte-like cells differentiated from HepaRG cells so far, and their utility in the evaluation of transporter-mediated transport remains unknown. In the present study, we investigated the mRNA and protein expression levels of the hepatic uptake transporters, NTCP and OATPs, in HepaRG cells and compared them with those in cryopreserved human hepatocytes. The uptake activities of these transporters were assessed using an uptake assay with a typical NTCP substrate, taurocholate, and several OATP substrates, estrone-3-sulfate, estradiol-17β-D-glucuronide, cholecystokinin octapeptide, taurocholate, pitavastatin, and olmesartan. An inhibition study using 12 OATP1B1 inhibitors in both HepaRG cells and OATP1B1-expressing HEK293 cells was also performed to determine whether this cell line can be applied to the evaluation of OATP1B1-mediated drug−drug interactions.

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EXPERIMENTAL SECTION Chemicals and Reagents. [3H]-Taurocholate (4.6 Ci/ mmol), [3H]-estrone-3-sulfate (E1S) (46 Ci/mmol), and [3H]estradiol-17β-D-glucuronide (E217βG) (45 Ci/mmol) were purchased from PerkinElmer Life and Analytical Sciences (Boston, MA), and [3H]-cholecystokinin octapeptide (CCK-8) 3435

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three batches of human hepatocytes (lots HH206, purchased from KAC Co. Ltd. (Kyoto, Japan); 609, purchased from XenoTech (Lenexa, KS); and TDH, purchased from In Vitro Technologies. Inc. (Baltimore, MD)). Immediately before the study, the hepatocytes (1 mL suspension) were thawed at 37 °C, 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 density of 1.0 × 106 viable cells/mL for the uptake study. The number of viable cells was determined by trypan blue staining. Only those hepatocytes with greater than 85% viability were used for further experiments. Before 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 radiolabeled and unlabeled taurocholate or E217βG to the cell suspension. In the uptake study, 1 μM taurocholate and 0.1 μM E217βG were used as tracer concentrations, and 1 mM taurocholate and 100 μM E217βG were used as excess concentrations. After incubation at 37 °C for 0.5 and 2 min, the reactions were terminated by separating the cells from the buffer. 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 (10000g; Beckman Microfuge E; Beckman Coulter, Fullerton, CA). During this process, hepatocytes passed through the oil layer into the alkaline solution. After 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 μL of 2 N HCl, mixed with scintillation cocktail, and the radioactivity was measured in a liquid scintillation counter. Determination of the Uptake Clearance of the Compounds. Uptake clearance (CLuptake) was determined by calculating the slope of the distribution volume (Vd) (μL/mg protein), given as the amount of radioactivity associated with the cells (dpm/mg protein) divided by its concentration in the incubation medium (dpm/μL), between 0.5 and 2 min (eq 1). In this time range, the time-dependent linear uptake was confirmed for each compound:

Biolabs, Ipswich, MA) and loaded onto a 7.5% SDS− polyacrylamide electrophoresis gel with a 3.75% stacking gel. Molecular weights were determined using prestained protein markers (New England Biolabs). Proteins were transferred electrophoretically to a polyvinylidene difluoride membrane (Pall Corporation, East Hills, NY) using a blotter (Transblot; Bio-Rad, Hercules, CA) at 15 V for 90 min. The membrane was blocked with Tris-buffered saline containing 0.05% Tween 20 (TTBS) and 5% skimmed milk for 1 h at room temperature. After washing with TTBS, the membrane was incubated overnight at 4 °C in TTBS with 500-fold diluted antiOATP1B1, anti-OATP1B3, anti-NTCP antiserums, and 10000-fold diluted anti-β-actin antiserum (C4 antibody, Millipore Biomedicals, Solon, OH). Anti-OATP1B1 and antiOATP1B3 rabbit antiserums were produced previously by ourselves.19 As described in the previous reports, anti-NTCP antiserum was raised in rabbits against a synthetic peptide consisting of the 12 carboxyterminal amino acids of NTCP coupled to keyhole limpet hemocyanin at its N terminus via an additional cysteine.22,23 For the detection of each transporter, the membrane was incubated with 5000-fold diluted donkey anti-rabbit IgG conjugated with horseradish peroxidase (GE Healthcare, Uppsala, Sweden), and for the detection of β-actin, the membrane was incubated with 5000-fold diluted goat antimouse IgG conjugated with horseradish peroxidase (GE Healthcare) for 1 h in TTBS. The immunoreactive band was detected using an ECL Advance Western Blotting Detection Kit (Amersham Biosciences, Piscataway, NJ). Uptake Study Using Plated Differentiated HepaRG Cells. The uptake study was conducted as described previously.19 Uptake was initiated by the addition of 0.5 mL of Krebs−Henseleit buffer containing radiolabeled substrates with unlabeled substrates, after the cells had been washed twice and preincubated with Krebs−Henseleit buffer at 37 °C for 10 min. This 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 and was adjusted to pH 7.4. Uptake was terminated at the designated times by the addition of 1 mL of ice-cold Krebs−Henseleit buffer after the removal of the incubation buffer. The cells were then rinsed twice with 1 mL of the ice-cold buffer, and solubilized with 0.5 mL of 0.2 N NaOH overnight at room temperature. Aliquots (0.5 mL) were transferred to scintillation vials after adding 0.25 mL of 0.4 N HCl. The radioactivity associated with the cells and incubation buffer was measured in a liquid scintillation counter (Tri-Carb 3100TR; PerkinElmer Life and Analytical Sciences) after adding 3 mL of scintillation fluid (Clear-sol I; Nacalai Tesque, Kyoto, Japan) to the scintillation vials. In the uptake study, 1 μM taurocholate, 0.1 μM E1S, 0.1 μM E217βG, 0.1 μM CCK-8, 0.1 μM olmesartan, and 0.1 μM pitavastatin were used as tracer concentrations, and 1 mM taurocholate, 100 μM E1S, 100 μM E217βG, 100 μM CCK-8, 100 μM olmesartan, and 100 μM pitavastatin were used as excess concentrations. The protein concentration was determined using the Lowry method with bovine serum albumin as a standard as described previously.21 To measure the uptake of the compounds in the absence of sodium ions, the sodium chloride and sodium bicarbonate in Krebs− Henseleit buffer were replaced with choline chloride and choline bicarbonate, respectively. Uptake Study Using Cryopreserved Human Hepatocytes. An uptake study using human cryopreserved hepatocytes was performed as described previously.19 We selected

CLuptake =

Vd,2min − Vd,0.5min 2−0.5

(1)

Saturable uptake clearance was estimated as the difference between the in vitro uptake clearance at a tracer concentration (CLuptake,tracer: μL/min/mg protein) and an excess concentration (CLuptake,excess): saturable uptake clearance = CL uptake,tracer − CL uptake,excess (2)

Inhibition Study Using Plated Differentiated HepaRG Cells and OATP1B1-Expressing HEK293 Cells. OATP1B1expressing or vector-transfected HEK293 cells were seeded 72 h before the transport study in 12-well plates coated with poly5 L-lysine and poly-L-ornithine at a density of 1.5 × 10 cells per well. For the transport study, the cell culture medium was replaced with culture medium supplemented with 5 mM sodium butyrate 24 h before the transport assay to induce the 3436

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OATP1B1 in HepaRG was partially maintained, and that of OATP1B3 was significantly lower than that in human hepatocytes, whereas those of OATP2B1 and NTCP were comparable or even higher. The relative expression levels of OATP1B1, 1B3, 2B1, and NTCP in differentiated HepaRG cells were 22.2−38.3%, 2.01−15.4%, 81.7−113%, and 191− 247% of those in human hepatocytes, respectively. Protein Expression Levels of Hepatic Uptake Transporters in Differentiated HepaRG Cells. The protein expression levels of OATP1B1, 1B3, and NTCP in differentiated HepaRG cells and cryopreserved human hepatocytes were evaluated by Western blot analyses (Figure 2). The

expression of exogenous transporters in HEK293 cells. The uptake study was conducted as described above. For the evaluation of the effect of OATP1B1 inhibitors on the cellular uptake of E217βG, OATP1B1 inhibitors were also added to the incubation buffer containing labeled and unlabeled E217βG (0.1 μM) at the beginning of the uptake assay. Determination of Inhibition Constants. The data obtained from the inhibition studies can be fitted to the following equation to calculate the inhibition constants (Ki): CLuptake(+inhibitor) =

CLuptake(control) 1 + I /K i

+ Pdif

(3)

where CLuptake(+inhibitor) and CLuptake(control) represent the uptake clearance determined in the presence or absence of the inhibitor, Pdif represents the nonsaturable uptake clearance, and I represents the initial inhibitor concentration in the media. Fitting was performed using the nonlinear least-squares method with the Damping Gauss−Newton algorithm in the MULTI program.24

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RESULTS Comparison of mRNA Expression Levels of Hepatic Uptake Transporters between Differentiated HepaRG Cells and Cryopreserved Human Hepatocytes. To confirm the mRNA expression of hepatic uptake transporters in hepatocyte-like cells differentiated from HepaRG cells, mRNA levels of OATP1B1, 1B3, 2B1, and NTCP were quantified using a qPCR method. The mRNA expression level of each transporter was normalized to that of hGAPDH and compared with that in three different batches of cryopreserved human hepatocytes (Figure 1). The relative expression of

Figure 2. Protein expression levels of hepatic uptake transporters in differentiated HepaRG and cryopreserved human hepatocytes. Protein expression levels of OATP1B1, OATP1B3, NTCP, and β-actin (internal control) were evaluated using the crude membrane fraction of differentiated HepaRG cells and cryopreserved human hepatocytes by Western blot analyses.

expression of β-actin was also detected as an internal control. Expression of OATP1B1 and NTCP protein could also be observed in hepatocyte-like cells differentiated from HepaRG cells, and the protein expression level of OATP1B1 in HepaRG was lower than that in human hepatocytes, and that of NTCP was higher. On the other hand, OATP1B3 protein expression could not be detected in differentiated HepaRG cells. Uptake of Transporter Ligands in Differentiated HepaRG Cells. To investigate the functions of hepatic uptake transporters in differentiated HepaRG cells, an uptake study was performed using taurocholate (typical ligand of NTCP), and E1S, E217βG, CCK-8, pitavastatin, and olmesartan (typical ligands of OATPs) (Figure 3). Saturable uptake of taurocholate, E1S, E217βG, pitavastatin, and olmesartan could be observed in HepaRG cells. Taurocholate uptake could not be detected in the absence of Na+ in HepaRG cells, confirming its Na+-dependent uptake. However, we did not observe any significant uptake of CCK-8 into HepaRG cells. Subsequently, uptake clearances (CLuptake) of these compounds were calculated from their initial uptake and compared with those in human hepatocytes (Table 1). CLuptake of taurocholate in HepaRG cells was 1.98-fold higher than the average of the CLuptake values from three different batches of cryopreserved human hepatocytes, and those of OATP substrates, E1S, E217βG, pitavastatin, and olmesartan, in HepaRG cells were 0.198−0.602-fold of those in cryopreserved human hepatocytes. Inhibitory Effects of OATP1B1 Inhibitors on E217βG Uptake in Differentiated HepaRG Cells and OATP1B1Expressing HEK293 Cells. Inhibitory effects of 12 OATP1B1

Figure 1. mRNA expression levels of hepatic uptake transporters in differentiated HepaRG and cryopreserved human hepatocytes. Relative mRNA expression levels of OATP1B1 (A), OATP1B3 (B), OATP2B1 (C), and NTCP (D) in differentiated HepaRG cells and three different batches (HH206, 609, TDH) of cryopreserved human hepatocytes, normalized to the expression level of hGAPDH are shown (open bar, HepaRG cells; solid bar, human hepatocytes). Data represent mean ± SE (n = 3). **: p < 0.01 (Student’s t-test). 3437

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Figure 3. Uptake activities of typical transporter substrates and their Na+-dependency in differentiated HepaRG cells. The time-dependent uptake of [3H]-taurocholate (A), [3H]-E1S (B), [3H]-E217βG (C), [3H]-CCK-8 (D), [3H]-pitavastatin (E), and [3H]-olmesartan (F) were observed in differentiated HepaRG cells in the presence or absence of Na+ in the incubation buffer at a tracer or excess concentration (solid square, Na+(+)tracer concentration; open square, Na+(−)-tracer concentration; solid circle, Na+(+)-excess concentration). In the case of taurocholate, 1 μM and 1 mM were used, while for other compounds, 0.1 μM and 100 μM were used as tracer concentration and excess concentration, respectively. Data represent mean ± SE (n = 3).

Table 1. Comparison of Hepatic Uptake of Taurocholate, E217βG, E1S, Pitavastatin, and Olmesartan in Differentiated HepaRG Cells and Cryopreserved Human Hepatocytesa

correlation coefficient value was r = 0.9021, and it was statistically significant (p < 0.05).

inhibitors on E217βG uptake in differentiated HepaRG cells and OATP1B1-expressing HEK293 cells were evaluated to confirm whether OATP1B1-mediated drug−drug interactions could be determined in this manner. The inhibition constants (K i values) of cyclosporin A, rifampicin, atorvastatin, pravastatin, olmesartan, valsartan, ritonavir, saquinavir, clarithromycin, erythromycin, ketoconazole, and gemfibrozil were obtained from an inhibition study using both HepaRG cells and OATP1B1-expressing HEK293 cells (Figure 4). The Ki values obtained from both cell types were significantly correlated, and Ki values of eight out of 12 inhibitors for OATP1B1 could be accurately evaluated from the inhibition assay using HepaRG cells within a 3-fold difference (Table 2). Spearman’s

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DISCUSSION

The new human hepatocarcinoma cell line, HepaRG, has been developed as an alternative in vitro model of human hepatocytes.9,10 Although the expression and activity of metabolic enzymes in differentiated HepaRG cells have already been characterized, there is little information about those of hepatic uptake transporters.16,25 Thus, in this report, the expression and transport activity of hepatic uptake transporters in HepaRG cells were quantitatively evaluated and compared 3438

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We also endeavored to check the protein expression of these uptake transporters in HepaRG by Western blot analysis. Although the expression of OATP1B3 was undetectable, the level of protein expression of OATP1B1 and NTCP in differentiated HepaRG cells could be confirmed (Figure 2). The relative expression level of OATP1B1 was less than half of that in human hepatocytes, while NTCP expression was more than 2-fold higher than that in human hepatocytes, in HepaRG cells. This tendency can be explained by the mRNA expression of these transporters, suggesting that the difference in the relative expression of uptake transporter proteins is mainly determined at the transcriptional level. As for NTCP, protein expression of NTCP apparently varied so much among different batches of human hepatocytes, while its mRNA expression was similar. One of the possible reasons for such a discrepancy is that the band density of NTCP by Western blot with the use of our antiserum was not strong enough for us to accurately quantify the protein expression of NTCP from our results of Western blot. It was reported that the differentiated HepaRG cells include two different types of cell populations, hepatocyte-like cells and biliary epithelial-like cells.26 Thus, if only hepatocyte-like cells were purified from the differentiated HepaRG cells, OATP1B1 expression per unit cellular protein amount could well be maintained at the same level as it is in normal human hepatocytes. We also tried to estimate the protein expression level of OATP2B1 by Western blot analysis, but unfortunately we could not clearly detect the OATP2B1 protein since our anti-OATP2B1 rabbit antiserum made by ourselves did not produce a clear band both in HepaRG cells and in human hepatocytes at the moment. Next, we attempted to investigate the transport activities of hepatic uptake transporters in differentiated HepaRG cells by using several transporter substrates. Saturable uptake of taurocholate, E1S, E217βG, pitavastatin, and olmesartan was observed in HepaRG cells, whereas we could not observe a significant uptake of CCK-8 (Figure 3). Although taurocholate is a substrate of both OATPs and NTCP, it has been reported that taurocholate is mainly taken up into human hepatocytes in a Na+-independent manner, suggesting that NTCP plays a major role in the hepatic uptake of taurocholate.27 We found that taurocholate uptake was almost nonexistent in the absence of Na+ in the buffer (Figure 3D), which indicates that NTCP also mainly functions as an uptake transporter in hepatocytelike cells differentiated from HepaRG cells. CCK-8 is wellknown as an OATP1B3-selective substrate,28 so the loss of CCK-8 uptake activity in HepaRG cells was considered to be reasonable because of the quite low expression of OATP1B3 at both the mRNA and protein levels (Figures 2, 3). Previous reports indicated that both OATP1B1 and OATP1B3 can accept E217βG, pitavastatin, and olmesartan as substrates.19,29 In HepaRG cells, because of the lack of OATP1B3 expression, uptake of these compounds should be attributed to OATP1B1 function. As shown in Table 1, CLuptake of taurocholate in HepaRG cells was 1.98-fold higher, while that of OATP transporter substrates (E1S, E217βG, pitavastatin, and olmesartan) was lower compared with that in cryopreserved human hepatocytes. The intrabatch difference in the uptake of taurocholate in human hepatocytes was not consistent with that in the protein expression level by Western blot. On the other hand, both the mRNA expression of NTCP and uptake activity of taurocholate were not so much varied among different batches of hepatocytes. So we think that we may not accurately evaluate the protein expression of NTCP from our

Figure 4. Correlation of Ki values of 12 OATP1B1 inhibitors obtained from inhibition studies using differentiated HepaRG cells and OATP1B1-expressing HEK293 cells. Ki values of (1) cyclosporin A, (2) rifampicin, (3) atorvastatin, (4) pravastatin, (5) olmesartan, (6) valsartan, (7) ritonavir, (8) saquinavir, (9) clarithromycin, (10) erythromycin, (11) ketoconazole, and (12) gemfibrozil for E217βG uptake estimated by using OATP1B1-expressing HEK293 cells are shown on the X-axis, and those obtained by using differentiated HepaRG cells are shown on the Y-axis. Data represent mean ± computer-calculated SD. Solid and dotted lines in this graph represent 1:1 correlation and 1:3 and 3:1 correlations.

Table 2. Ki Values of 12 OATP1B1 Inhibitors Obtained from an Inhibition Study Using Differentiated HepaRG Cells and OATP1B1-Expressing HEK293 Cellsa Ki valueb (μM) inhibitor

HepaRG

HEK-OATP1B1

cyclosporin A rifampicin atorvastatin pravastatin olmesartan valsartan ritonavir saquinavir clarithromycin erythromycin ketoconazole gemfibrozil

0.319 ± 0.167 0.391 ± 0.099 0.0384 ± 0.0114 20.7 ± 14.9 11.2 ± 5.3 5.47 ± 3.74 0.518 ± 0.277 4.54 ± 2.79 16.9 ± 5.4 23.0 ± 7.5 69.6 ± 37.2 89.5 ± 18.6

0.194 ± 0.056 0.278 ± 0.018 0.320 ± 0.087 31.9 ± 3.6 9.76 ± 1.89 4.19 ± 0.40 0.724 ± 0.191 0.907 ± 0.181 3.44 ± 0.57 5.39 ± 0.66 35.3 ± 9.5 31.5 ± 3.8

Data represent mean ± computer-calculated SD. bSubstrate: E217βG (0.1 μM). a

with those in cryopreserved human hepatocytes. Moreover, the utility of this cell line in the prediction of transporter-mediated drug−drug interactions was also investigated. In most of the previously described human hepatocarcinoma cell lines, such as HepG2 or Fa2N-4 cells, the expression of hepatic uptake transporters is almost completely lost.17,18 Thus, we attempted first to check mRNA expression levels of uptake transporters in differentiated HepaRG cells. From the results of qPCR, mRNA expression levels of OATP1B1, OATP2B1, and NTCP in HepaRG cells were seen to be comparable to those in human cryopreserved hepatocytes, but that of OATP1B3 was significantly lower (Figure 1). Although we used completely different batches of cryopreserved human hepatocytes, the relative expression levels of OATP1B1, 1B3, and 2B1 between HepaRG cells and human hepatocytes were consistent with those in a previous report.15 3439

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Article

OATP1B3 expression and function were severely depleted for unknown reasons. Regarding the evaluation of OATP1B1mediated drug−drug interactions, inhibition potencies of compounds could be estimated from an inhibition assay with differentiated HepaRG cells. Our results indicate that HepaRG cells partially express hepatic uptake transporters and that, unlike other hepatocarcinoma cells, they have the ability to actively take up transporter substrates.

Western blot because of the low band density as indicated above. The rank order of the CLuptake of OATP substrates was almost consistent with that in normal human hepatocytes (Table 1). As we mentioned above, because HepaRG cells contain two different kinds of cell populations, it is possible that we also underestimate the uptake activity per cellular protein amount as well as the expression level of each uptake transporter in hepatocyte-like cells differentiated from HepaRG cells. Moreover, it was reported that HepaRG cells possess the SLCO1B1*5 (Val174Ala) SNP (cf. HepaRG data sheet), which is known to decrease the uptake function of OATP1B1.1 Considering this information, it is reasonable to assume that the uptake activity of OATP1B1 in differentiated HepaRG cells should be lower than that in the intact human hepatocytes, though we could not exclude the possibility that the intrinsic transcription efficiency of OATP1B1 mRNA was decreased in HepaRG cells with unknown mechanisms. Regarding the uptake activity mediated by OATP2B1, since OATP2B1specific substrates or inhibitors have not been reported so far, we could not have appropriate methods to specifically measure the OATP2B1 function in HepaRG cells and contribution of OATP2B1 to the overall uptake of compounds in HepaRG cells remains to be clarified. To further confirm the function of OATP1B1 in HepaRG cells and also to evaluate the utility of this cell line for the prediction of transporter-mediated drug−drug interactions, we compared the Ki values of 12 OATP1B1 inhibitors obtained from both HepaRG cells and OATP1B1-expressing HEK293 cells. We chose E217βG as an OATP1B1-selective substrate in this study. E217βG is a substrate of both OATP1B1 and OATP1B3,19 but as mentioned above, it would be an OATP1B1-specific substrate in HepaRG cells because of the loss of OATP1B3 expression and function. Although the expression of OATP2B1 was also detected in HepaRG cells (Figure 1), we confirmed that significant uptake of E217βG in OATP2B1-expressing HEK293 cells was not observed (data not shown). The Ki values of these 12 OATP1B1 inhibitors obtained from OATP1B1-expressing HEK293 cells were consistent with the reported K i or K m values for OATP1B1.29−32 Our results indicate that Ki values obtained from OATP1B1-expressing cells are comparable to the Ki values obtained by using HepaRG cells (Figure 4), and the Ki values of eight out of 12 inhibitors for OATP1B1 could be accurately estimated within a 3-fold difference. These results suggest that inhibition potencies of compounds for OATP1B1mediated uptake can be estimated by inhibition studies using differentiated HepaRG cells without OATP1B1 expression systems. From our results, the calculated Ki values obtained from HepaRG cells have larger SD values compared with those obtained from OATP1B1-expressing HEK293 cells. One of the possible reasons for this is the lower saturable uptake of E217βG in HepaRG cells compared with normal human hepatocytes because of the lower expression and SNP of OATP1B1. If an OATP1B1-selective substrate that shows higher CLuptake could be used for an inhibition assay, the accuracy of Ki value prediction for compounds could improve. In conclusion, we have investigated the expression and function of the hepatic uptake transporters, NTCP and OATPs, which have important roles in the disposition of bile acids and drugs in human liver, in a novel human hepatocarcinoma cell line, HepaRG. In HepaRG cells, mRNA and protein expression of NTCP and OATPs was confirmed, and their function was almost proportional to their protein expression levels. However,

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AUTHOR INFORMATION

Corresponding Author

*Sugiyama Laboratory, RIKEN Innovation Center, RIKEN Research Cluster for Innovation, Yokohama Bio Industry Center 2F, 1-6 Suehiro-Cho, Tsurumi-ku, Yokohama 230-0045, Japan. Phone: +81-45-506-1814. Fax: +81-45-506-1800. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We sincerely thank Kowa Co. Ltd. and Daiichi Sankyo Co., Ltd., for kindly providing us with radiolabeled and unlabeled pitavastatin and olmesartan, respectively. This study was partly supported by Grants-in-Aid for Research on Publicly Essential Drugs and Medical Devices from the Ministry of Health, Labor and Welfare of Japan and a Grant-in-Aid for Scientific Research (A) [20249008] from the Japan Society for the Promotion of Science (JSPS), and Research Grants by the Mandom International Research Grants on Alternative to Animal Experiments.

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ABBREVIATIONS USED Ki, inhibition constant; OATP, organic anion transporting polypeptide; NTCP, Na+-taurocholate cotransporting polypeptide; E1S, estrone-3-sulfate; E217βG, estradiol-17β-D-glucuronide; CCK-8, cholecystokinin octapeptide; SNP, single nucleotide polymorphism; hGAPDH, human glyceraldehyde 3-phosphate dehydrogenase; qPCR, quantitative real-time polymerase chain reaction

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REFERENCES

(1) Ieiri, I.; Higuchi, S.; Sugiyama, Y. Genetic polymorphisms of uptake (OATP1B1, 1B3) and efflux (MRP2, BCRP) transporters: implications for inter-individual differences in the pharmacokinetics and pharmacodynamics of statins and other clinically relevant drugs. Expert Opin. Drug Metab. Toxicol. 2009, 5, 703−29. (2) Maeda, K.; Sugiyama, Y. Impact of genetic polymorphisms of transporters on the pharmacokinetic, pharmacodynamic and toxicological properties of anionic drugs. Drug Metab. Pharmacokinet. 2008, 23, 223−35. (3) Shou, M.; Hayashi, M.; Pan, Y.; Xu, Y.; Morrissey, K.; Xu, L.; Skiles, G. L. Modeling, prediction, and in vitro in vivo correlation of CYP3A4 induction. Drug Metab. Dispos. 2008, 36, 2355−70. (4) Giacomini, K. M.; Huang, S. M.; Tweedie, D. J.; Benet, L. Z.; Brouwer, K. L.; Chu, X.; Dahlin, A.; Evers, R.; Fischer, V.; Hillgren, K. M.; Hoffmaster, K. A.; Ishikawa, T.; Keppler, D.; Kim, R. B.; Lee, C. A.; Niemi, M.; Polli, J. W.; Sugiyama, Y.; Swaan, P. W.; Ware, J. A.; Wright, S. H.; Yee, S. W.; Zamek-Gliszczynski, M. J.; Zhang, L. Membrane transporters in drug development. Nat Rev. Drug Discovery 2010, 9, 215−36. (5) Abe, K.; Bridges, A. S.; Brouwer, K. L. Use of sandwich-cultured human hepatocytes to predict biliary clearance of angiotensin II receptor blockers and HMG-CoA reductase inhibitors. Drug Metab. Dispos. 2009, 37, 447−52.

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Molecular Pharmaceutics

Article

(6) Watanabe, T.; Kusuhara, H.; Maeda, K.; Kanamaru, H.; Saito, Y.; Hu, Z.; Sugiyama, Y. Investigation of the rate-determining process in the hepatic elimination of HMG-CoA reductase inhibitors in rats and humans. Drug Metab. Dispos. 2010, 38, 215−22. (7) Garcia, M.; Rager, J.; Wang, Q.; Strab, R.; Hidalgo, I. J.; Owen, A.; Li, J. Cryopreserved human hepatocytes as alternative in vitro model for cytochrome p450 induction studies. In Vitro Cell. Dev. Biol.: Anim. 2003, 39, 283−7. (8) Shitara, Y.; Li, A. P.; Kato, Y.; Lu, C.; Ito, K.; Itoh, T.; Sugiyama, Y. Function of uptake transporters for taurocholate and estradiol 17beta-D-glucuronide in cryopreserved human hepatocytes. Drug Metab. Pharmacokinet. 2003, 18, 33−41. (9) Antherieu, S.; Chesne, C.; Li, R.; Camus, S.; Lahoz, A.; Picazo, L.; Turpeinen, M.; Tolonen, A.; Uusitalo, J.; Guguen-Guillouzo, C.; Guillouzo, A. Stable expression, activity, and inducibility of cytochromes P450 in differentiated HepaRG cells. Drug Metab. Dispos. 2010, 38, 516−25. (10) Gripon, P.; Rumin, S.; Urban, S.; Le Seyec, J.; Glaise, D.; Cannie, I.; Guyomard, C.; Lucas, J.; Trepo, C.; Guguen-Guillouzo, C. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15655−60. (11) Aninat, C.; Piton, A.; Glaise, D.; Le Charpentier, T.; Langouet, S.; Morel, F.; Guguen-Guillouzo, C.; Guillouzo, A. Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells. Drug Metab. Dispos. 2006, 34, 75−83. (12) Kaneko, A.; Kato, M.; Sekiguchi, N.; Mitsui, T.; Takeda, K.; Aso, Y. In vitro model for the prediction of clinical CYP3A4 induction using HepaRG cells. Xenobiotica 2009, 39, 803−10. (13) McGill, M. R.; Yan, H. M.; Ramachandran, A.; Murray, G. J.; Rollins, D. E.; Jaeschke, H. HepaRG cells: a human model to study mechanisms of acetaminophen hepatotoxicity. Hepatology 2011, 53, 974−82. (14) Ndongo-Thiam, N.; Berthillon, P.; Errazuriz, E.; Bordes, I.; De Sequeira, S.; Trepo, C.; Petit, M. A. Long-term propagation of serum hepatitis C virus (HCV) with production of enveloped HCV particles in human HepaRG hepatocytes. Hepatology 2011, 54, 406−17. (15) Kanebratt, K. P.; Andersson, T. B. Evaluation of HepaRG cells as an in vitro model for human drug metabolism studies. Drug Metab. Dispos. 2008, 36, 1444−52. (16) Le Vee, M.; Jigorel, E.; Glaise, D.; Gripon, P.; GuguenGuillouzo, C.; Fardel, O. Functional expression of sinusoidal and canalicular hepatic drug transporters in the differentiated human hepatoma HepaRG cell line. Eur. J. Pharm. Sci. 2006, 28, 109−17. (17) Cui, Y.; Konig, J.; Nies, A. T.; Pfannschmidt, M.; Hergt, M.; Franke, W. W.; Alt, W.; Moll, R.; Keppler, D. Detection of the human organic anion transporters SLC21A6 (OATP2) and SLC21A8 (OATP8) in liver and hepatocellular carcinoma. Lab. Invest. 2003, 83, 527−38. (18) Hariparsad, N.; Carr, B. A.; Evers, R.; Chu, X. Comparison of immortalized Fa2N-4 cells and human hepatocytes as in vitro models for cytochrome P450 induction. Drug Metab. Dispos. 2008, 36, 1046− 55. (19) Hirano, M.; Maeda, K.; Shitara, Y.; Sugiyama, Y. Contribution of OATP2 (OATP1B1) and OATP8 (OATP1B3) to the hepatic uptake of pitavastatin in humans. J. Pharmacol. Exp. Ther. 2004, 311, 139−46. (20) Sasaki, M.; Suzuki, H.; Ito, K.; Abe, T.; Sugiyama, Y. Transcellular transport of organic anions across a double-transfected Madin-Darby canine kidney II cell monolayer expressing both human organic anion-transporting polypeptide (OATP2/SLC21A6) and Multidrug resistance-associated protein 2 (MRP2/ABCC2). J. Biol. Chem. 2002, 277, 6497−503. (21) Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951, 193, 265−75. (22) Konig, J.; Cui, Y.; Nies, A. T.; Keppler, D. Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. J. Biol. Chem. 2000, 275, 23161−8.

(23) Konig, J.; Cui, Y.; Nies, A. T.; Keppler, D. A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. Am. J. Physiol. 2000, 278, G156−64. (24) Yamaoka, K.; Tanigawara, Y.; Nakagawa, T.; Uno, T. A pharmacokinetic analysis program (multi) for microcomputer. J. Pharmacobio-Dyn. 1981, 4, 879−85. (25) Le Vee, M.; Gripon, P.; Stieger, B.; Fardel, O. Down-regulation of organic anion transporter expression in human hepatocytes exposed to the proinflammatory cytokine interleukin 1beta. Drug Metab. Dispos. 2008, 36, 217−22. (26) Parent, R.; Marion, M. J.; Furio, L.; Trepo, C.; Petit, M. A. Origin and characterization of a human bipotent liver progenitor cell line. Gastroenterology 2004, 126, 1147−56. (27) Hagenbuch, B.; Dawson, P. The sodium bile salt cotransport family SLC10. Pfluegers Arch. 2004, 447, 566−70. (28) Ismair, M. G.; Stieger, B.; Cattori, V.; Hagenbuch, B.; Fried, M.; Meier, P. J.; Kullak-Ublick, G. A. Hepatic uptake of cholecystokinin octapeptide by organic anion-transporting polypeptides OATP4 and OATP8 of rat and human liver. Gastroenterology 2001, 121, 1185−90. (29) Yamada, A.; Maeda, K.; Kamiyama, E.; Sugiyama, D.; Kondo, T.; Shiroyanagi, Y.; Nakazawa, H.; Okano, T.; Adachi, M.; Schuetz, J. D.; Adachi, Y.; Hu, Z.; Kusuhara, H.; Sugiyama, Y. Multiple human isoforms of drug transporters contribute to the hepatic and renal transport of olmesartan, a selective antagonist of the angiotensin II AT1-receptor. Drug Metab. Dispos. 2007, 35, 2166−76. (30) Chen, C.; Mireles, R. J.; Campbell, S. D.; Lin, J.; Mills, J. B.; Xu, J. J.; Smolarek, T. A. Differential interaction of 3-hydroxy-3methylglutaryl-coa reductase inhibitors with ABCB1, ABCC2, and OATP1B1. Drug Metab. Dispos. 2005, 33, 537−46. (31) Gui, C.; Wahlgren, B.; Lushington, G. H.; Hagenbuch, B. Identification, Ki determination and CoMFA analysis of nuclear receptor ligands as competitive inhibitors of OATP1B1-mediated estradiol-17beta-glucuronide transport. Pharmacol. Res. 2009, 60, 50− 6. (32) Hirano, M.; Maeda, K.; Shitara, Y.; Sugiyama, Y. Drug-drug interaction between pitavastatin and various drugs via OATP1B1. Drug Metab. Dispos. 2006, 34, 1229−36. (33) Camenisch, G.; Umehara, K. Predicting human hepatic clearance from in vitro drug metabolism and transport data: a scientific and pharmaceutical perspective for assessing drug-drug interactions. Biopharm. Drug Dispos. 2012, 33, 179−94.

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