Article pubs.acs.org/molecularpharmaceutics
Characterization of Organic Anion Transporting Polypeptide (OATP) Expression and Its Functional Contribution to the Uptake of Substrates in Human Hepatocytes Emi Kimoto,† Kenta Yoshida,‡ Larissa M. Balogh,† Yi-an Bi,† Kazuya Maeda,‡ Ayman El-Kattan,† Yuichi Sugiyama,§ and Yurong Lai*,† †
Pfizer Global Research and Development, Department of Pharmacokinetics, Dynamics, and Metabolism, Groton, Connecticut 06340, United States ‡ Department of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan § Sugiyama Laboratory, RIKEN Innovation Center, RIKEN Research Cluster for Innovation, Yokohama, Japan ABSTRACT: Since the substrate specificities of OATP1B1, 1B3, and 2B1 are broad and overlapping, the contribution of each isoform to the overall hepatic uptake is of concern when assessing transporter-mediated drug−drug interactions (DDIs) or genetic polymorphism impact in the clinic. Herein, we quantitatively measured OATP proteins in cryopreserved hepatocytes, sandwich-cultured human hepatocytes (SCHH), and the liver, and examined the relationship with functional uptake of OATP substrates in an effort to identify the OATP isoform(s) contributing to the hepatic uptake of pitavastatin. The modulation of OATP expression in SCHH was found to be lot-dependent. However, OATP protein measurements averaged from 5 lots of SCHH were comparable to that of suspended hepatocytes. All three OATP transporters in suspended hepatocytes and SCHH were significantly lower than those in the liver. In SCHH, the uptake of CCK-8 and pravastatin was found to be associated with the expression of OATP1B3 and OATP1B1, respectively. In suspended hepatocytes, OATP1B1 appeared to show a positive trend with respect to the uptake of pitavastatin, which suggests a selective contribution of OATP1B1 to pitavastatin transport and thus an OATP quantitative protein expression−activity relationship. While the passive diffusion of rosuvastatin in SCHH was consistent across hepatocyte lots, the passive diffusion of pitavastatin varied over a broad range (>4fold) in suspended hepatocytes and was inversely correlated with transporter-mediated uptake, presumably due to cell membrane alterations imparted by cryopreservation. Collectively, SCHH maintains OATP protein expression and membrane integrity and, if feasible when considering research goals, would be considered a superior tool for the characterization of in vitro transport parameters without the complication of membrane leakage. KEYWORDS: organic anion transporting polypeptide (OATP), human hepatocytes, LC−MS/MS, quantitative protein expression−activity relationship (QPEAR)
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(CYP) enzymes, CYP2C8 and 3A4.3 While itraconazole, a potent CYP inhibitor, caused a 1.15- to 1.27-fold increase in the area under the plasma concentration−time curve (AUC) for cerivastatin in healthy subjects,4,5 cotreatment with cyclosporine A, a potent OATP inhibitor, resulted in a 3.8-fold increase in the AUC for cerivastatin in kidney transplant recipients.6 The difference in PK modulation caused by OATP or CYP inhibitors indicates that hepatic uptake mediated by OATP proteins accounts for the drug−drug interactions (DDIs).7 As a result, it should be emphasized that net inhibition of hepatic uptake can result in DDIs and unexpected toxicity.8,9
INTRODUCTION The liver is the primary organ for xenobiotic elimination wherein the hepatic sinusoidal drug transport process includes passive diffusion and/or uptake mediated by hepatic uptake transporters. Liver transporters residing on the sinusoidal membrane of human hepatocytes include organic anion transporting polypeptides (OATP1B1, 1B3, and 2B1), Na+taurocholate cotransporting polypeptide (NTCP), organic anion transporter 2 (OAT2), and organic cation transporter 1 (OCT1).1 Based on the concept of extended clearance, drug uptake is the rate limiting step if passive diffusion into the liver is significantly lower than intrinsic clearance mediated by metabolism and/or biliary elimination, and therefore affects drug pharmacokinetics (PK) and tissue disposition.2 This principle is valid even when compounds are extensively metabolized. For example, cerivastatin is an OAPT1B1 substrate that is also metabolized by two cytochrome P450 © 2012 American Chemical Society
Received: Revised: Accepted: Published: 3535
July 12, 2012 September 25, 2012 October 19, 2012 October 19, 2012 dx.doi.org/10.1021/mp300379q | Mol. Pharmaceutics 2012, 9, 3535−3542
Molecular Pharmaceutics
Article
Jackson (Muskegon, MI) and Mallinckrodt Baker (Phillipsburg, NJ), respectively. Pitavastatin, pravastatin, and rosuvastatin were purchased from Sequoia Research Products (Pangbourne, U.K.). Formic acid, ammonium bicarbonate (ABC), dithiothreitol (DTT), iodoacetamide (IAA), deoxycholate (DOC), Dulbecco’s phosphate-buffer saline, Krebs−Henseleit buffer, and all other chemicals were purchased from Sigma (St. Louis, MO). The Protein Lobind tubes and plates were purchased from Eppendorf North America (Hauppauge, NY), and the siliconized pipet tips were obtained from VWR International (Bridgeport, NJ). Cryopreserved human hepatocyte lot 109 was purchased from BD Biosciences (Woburn, MA), and Hu4163, Hu4165 (Hu4163 and Hu4165 are prepared from same donor), and Hu4241 were purchased from CellzDirect (Pittsboro, NC). RCP, BOB, and all other lots were purchased from Celsis IVT (Baltimore, MD). Freshly frozen human liver tissues were obtained from the Pfizer tissue bank (Groton, CT). Sandwich-Cultured Human Hepatocytes (SCHH). Plateable cryopreserved hepatocytes (109, Hu4165, Hu4241, RCP, and BOB) were thawed and plated as described previously.14 Briefly, hepatocytes were thawed in a water bath at 37 °C and placed on ice. The cells were then poured into In VitroGro-HT medium at 37 °C at a ratio of one vial/50 mL in a conical tube. The cells were centrifuged at 50g for 3 min and resuspended at 0.7−0.85 × 106 cells/mL in In VitroGro-CP medium. Cell viability was determined by trypan blue exclusion. On day 1, hepatocyte suspensions were plated in collagencoated 24-well plates at a density of 0.35−0.425 × 106 cells/ well in a volume of 0.5 mL/well. After 18 to 24 h of incubation at 37 °C, cells were overlaid with ice-cold 0.25 mg/mL Matrigel in In VitroGro-HI medium at 0.5 mL/well. Cultures were maintained in In VitroGro-HI medium that was refreshed every 24 h. Determination of Uptake Clearance in SCHH. The determination of uptake clearance (CLuptake) in SCHH was conducted as described previously.19 On day 5 of SCHH, the hepatocytes were first rinsed twice with Ca2+ HBSS buffer and then preincubated for 10 min with Ca2+ HBSS buffer in the absence or presence of 100 μM rifamycin SV. After aspiration of the preincubation buffer, 0.5 mL of incubation buffer containing substrate was added in the absence or presence of rifamycin SV. The uptake was terminated at a designated time by adding 0.5 mL of ice cold Ca2+ HBSS buffer after removal of the incubation buffer. Cells were then washed three times with 0.5 mL of ice cold Ca2+ HBSS buffer. The hepatocytes were lysed with methanol containing the internal standard for LC− MS/MS quantification. CLuptake and passive diffusion (CLpassive) were obtained from an initial rate analysis with a linear regression fit up to 1.5 min. The experiments were optimized to determine initial uptake rates in the hepatocyte models.19 Determination of Uptake Clearance in Suspended Hepatocytes. CL uptake in suspended hepatocytes was determined using cryopreserved human hepatocytes by a fast filtration approach using either a cell harvester (Micro96 Harvester, Molecular Devices, Sunnyvale, CA) or oil-spin separation as previously described.20,21 For the cell harvester approach, cryopreserved hepatocytes were thawed in a water bath at 37 °C and then placed on ice, after which the hepatocytes were resuspended in Krebs−Henseleit buffer. Cell viability was determined by trypan blue exclusion, and the suspension was diluted to 2 × 106 cells/mL. 50 μL aliquots of cells were placed in test plates and prewarmed in the 37 °C
OATP1B1, 1B3, and 2B1 are coexpressed on the sinusoidal membrane of hepatocytes and play an important role in regulating the hepatic intracellular concentrations of endogenous compounds and xenobiotics. Because the substrate specificity of OATP transporters is broad and overlapping, it is uncertain as to whether the inhibition of one OATP isoform would actually cause significant PK changes for substrate drugs when a compensatory function from other OATP isoforms is possible. However, determining the relative contribution of OATP isoforms to the overall hepatic uptake of a given compound is a challenging task that has yet to be adequately addressed. For example, Niemi et al. has reviewed that OATP1B1 expression is associated with PK for a number of statins.10,11 These statins are transported by OATP1B1, however, the clinical effect of solute carrier organic anion transporter family member 1B1 (SLCO1B1) polymorphisms on PK and adverse events for the individual statins suggests that the contribution of OATP1B1 to hepatic uptake varies.12 Given that several statins are also substrates of OATP1B3 and 2B1,13 it is important to assess the OATP isoform contribution to uptake for substrates when evaluating transporter DDIs or genetic polymorphism effects in the clinic. Sandwich-cultured human hepatocytes (SCHH), which repolarize to develop intact bile pockets and provide a threedimensional orientation and proper localization of transporters, represent an alternative in vitro model to assess hepatic disposition.14,15 Previously, we reported the ability to use sandwich-cultured rat hepatocytes to quantitatively predict biliary elimination and proposed the use of a scaling factor obtained from the quantitative expression profiles of hepatobiliary transporters for the prediction of biliary clearance.16,17 In order to extrapolate in vivo from in vitro parameters, it is necessary to elucidate the difference in protein expression between in vitro systems and those in the liver, and further to establish quantitative protein expression−activity relationships (QPEAR) for OATP transport. In the present study, the aims are (i) to determine the protein expression of OATP1B1, 1B3, and 2B1 in in vitro hepatocyte models and human liver using LC−MS/MS-based protein quantification, and (ii) to study the correlation between quantitative protein expression and functional activity in order to identify OATP isoforms that contribute to hepatic uptake. The results are fundamental to obtain empirical scaling factors for incorporation into physiologically based pharmacokinetic (PBPK) models that are applied in the prediction of human PK and DDIs.18
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MATERIALS AND METHODS Materials and Reagents. In VitroGro-HT (thawing), In VitroGro-CP (plating), and In VitroGro-HI (incubation) hepatocyte media were purchased from Celsis In Vitro Technologies Inc. (IVT) (Baltimore, MD). Hanks balanced salt solution (HBSS) and Williams’ medium E were purchased from Invitrogen (Carlsbad, CA). BioCoat 24-well plates and Matrigel were purchased from BD Biosciences (Bedford, MA). The ProteoExtract Native Membrane Protein Extraction Kit (M-PEK) was purchased from Calbiochem (San Diego, CA). The BCA Protein Assay Kit was purchased from Pierce Biotechnology (Rockford, IL). All research grade peptide standards were synthesized and purified by New England Peptide (Gardner, MA). Sequencing grade modified trypsin was purchased from Promega (Madison, WI). HPLC grade acetonitrile (ACN) and water were obtained from Burdick & 3536
dx.doi.org/10.1021/mp300379q | Mol. Pharmaceutics 2012, 9, 3535−3542
Molecular Pharmaceutics
Article
added to the samples at a 20:1 protein:trypsin ratio and digested at 37 °C overnight. The DOC concentration was reduced to 1% w/v during the digestion. The digestion was terminated by acidification with an equivalent volume of 0.2% formic acid containing a cocktail of external stable isotopelabeled peptide internal standards, or 0.2% formic acid containing a cocktail of unlabeled peptide standards. Samples were centrifuged at 14000g for 5 min to effectively pellet the acid-precipitated DOC and then concentrated in a SpeedVac (Thermo Fisher Scientific, Waltham, MA) prior to LC−MS/ MS analysis. LC−MS/MS Analysis of Membrane Protein. Analyses were conducted as described previously.22 Briefly, a 10 μL sample was injected onto a Kinetex C18 column (2.6 μm, 100 Å, 100 × 3.0 mm, Phenomenex, Torrance, CA) and eluted by a mobile phase with initial conditions of 5% solvent B for 5 min, followed by a linear gradient of 5% solvent B to 30% solvent B over 20 min (solvent A, 100% H2O with 0.1% formic acid; solvent B, 100% ACN with 0.1% formic acid) at a flow rate of 500 μL/min. The multiple reaction monitoring (MRM) acquisition methods were constructed with three tuned transitions and the optimal declustering potentials, collision energies, and collision cell exit potentials determined for each peptide with a 4.5 kV spray voltage, 10 eV entrance potential, and 450 °C source temperature. The analytes and isotopelabeled internal standards were quantified using Analyst 1.4.2 (MDS SCIEX, Ontario, Canada). The minimum signal-to-noise ratio considered for quantification was 10:1. Final quantifications are representative of the mean of three transitions measured for each peptide. When more than one of the three MRM channels could not be quantified, the mean was not determined. Statistical Analysis. A one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison was conducted to compare OATP proteins in the liver and hepatocytes. The correlation between uptake and OATP protein level was determined by Spearman’s correlation test. A paired t test was conducted to compare OATP protein in plateable hepatocytes, pre- and post-sandwich culture. The data was analyzed using Graphpad Prism version 5.01 for windows (GraphPad Software, San Diego, CA). A p-value
