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Organic Anion Transporter 2–Mediated Hepatic Uptake

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Organic Anion Transporter 2–Mediated Hepatic Uptake Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2018/08/22/jpet.118.252049.DC1 1521-0103/367/2/322–334$35.00 https://doi.org/10.1124/jpet.118.252049 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 367:322–334, November 2018 Copyright ª 2018 by The American Society for Pharmacology and Experimental Therapeutics Organic Anion Transporter 2–Mediated Hepatic Uptake Contributes to the Clearance of High-Permeability–Low- Molecular-Weight Acid and Zwitterion Drugs: Evaluation Using 25 Drugs s Emi Kimoto, Sumathy Mathialagan, Laurie Tylaska, Mark Niosi, Jian Lin, Anthony A. Carlo, David A. Tess, and Manthena V. S. Varma Medicine Design, Worldwide Research and Development, Pfizer Inc., Groton, Connecticut Downloaded from Received July 12, 2018; accepted August 15, 2018 ABSTRACT High-permeability–low-molecular-weight acids/zwitterions [i.e., drugs (21 of 25) showed active uptake by plated human hepa- extended clearance classification system class 1A (ECCS 1A) tocytes, with rifamycin SV (pan-transporter inhibitor) reducing drugs] are considered to be cleared by metabolism with a the uptake by about 25%–95%. Metabolic turnover was esti- jpet.aspetjournals.org minimal role of membrane transporters in their hepatic clearance. mated for 19 drugs after a few showed no measurable substrate However, a marked disconnect in the in vitro-in vivo (IVIV) depletion in liver microsomal incubations. IVIV extrapolation translation of hepatic clearance is often noted for these drugs. using in vitro data was evaluated to project human hepatic Metabolic rates measured using human liver microsomes and clearance of OAT2-alone substrates considering 1) uptake trans- primary hepatocytes tend to underpredict. Here, we evaluated port only, 2) metabolism only, and 3) transporter-enzyme interplay the role of organic anion transporter 2 (OAT2)–mediated hepatic (extended clearance model). The transporter-enzyme interplay uptake in the clearance of ECCS 1A drugs. For a set of 25 ECCS approach achieved improved prediction accuracy (average fold 1A drugs, in vitro transport activity was assessed using error 5 1.9 and bias 5 0.93) compared with the other two at ASPET Journals on September 29, 2021 transporter-transfected cells and primary human hepatocytes. approaches. In conclusion, this study provides functional All but two drugs showed substrate affinity to OAT2, whereas evidence for the role of OAT2-mediated hepatic uptake in four (bromfenac, entacapone, fluorescein, and nateglinide) also determining the pharmacokinetics of several clinically important showed OATP1B1 activity in transfected cells. Most of these ECCS 1A drugs. Introduction this class of drugs. That is, metabolic rates measured by substrate depletion and/or metabolite formation in the incuba- We recently proposed a framework called extended clearance tions of human liver microsomes and human hepatocytes classification system (ECCS) to predict the rate-determining considerably underpredict human hepatic clearance of ECCS 1A clearance mechanism of drugs or new molecular entities using drugs (Obach, 1999; Brown et al., 2007; Bowman and Benet, simple molecular properties, including ionization state and 2016; Wood et al., 2017). Several previous studies discussed molecular weight and in vitro membrane permeability (Varma plausible causes for IVIV disconnect in hepatic clearance as et al., 2015; El-Kattan and Varma, 2018). According to this measured using hepatocytes and liver microsomes, which include validated system, ECCS class 1A drugs (i.e., high-permeabili- permeation rate limitation, diffusion through unstirred water – , ty low-mol. wt. 400-Da acids/zwitterions) are thought to be layer, suboptimal substrate concentration, reagent handling, and cleared primarily by metabolism as the rate-determining step. assay methods (Hallifax and Houston, 2012; Poulin et al., 2012; However, despite the predominance of cytochrome 450 (P450)- Bowman and Benet, 2016; Wood et al., 2017). Although the 9 and/or uridine 5 -diphospho-glucuronosyltransferase (UGT)- causes are thought to be multifactorial, empirical correction of mediated metabolic pathways, many studies have indicated a prediction bias has been suggested as a pragmatic approach for – marked disconnect in the in vitro in vivo (IVIV) translation for pharmacokinetic predictions in drug discovery (Hallifax and Houston, 2012; Poulin et al., 2012; Wood et al., 2017). https://doi.org/10.1124/jpet.118.252049. Membrane transporters play a key role in the absorption, s This article has supplemental material available at jpet.aspetjournals.org. distribution, clearance, and elimination of drugs (Shitara ABBREVIATIONS: AFE, average fold error; CLint, intrinsic clearance; CLint, h, intrinsic hepatic clearance; CLint, met, intrinsic metabolic clearance; CLplasma, h, plasma hepatic clearance; ECCS, extended clearance classification system; HBSS, Hanks’ balanced salt solution; HLM, human liver microsome; IVIV, in vitro-in vivo; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MDCK, Madin-Darby canine kidney (cells); OAT, organic anion transporter; OATP, organic anion-transporting polypeptide; P450, cytochrome P450; PBPK, physiologically based pharmacokinetic; PSactive, active uptake clearance; PSpassive, passive clearance; REF, relative expression factor; SLC, solute carrier; UGT, uridine 59-diphospho- glucuronosyltransferase. 322 OAT2-Mediated Hepatic Uptake Clearance 323 et al., 2006; Giacomini et al., 2010; El-Kattan and Varma, In Vitro Transport Studies Using Transporter-Transfected 2018). Emphasis has been given to hepatic uptake clearance Cells. HEK293 cells singly transfected with OATP1B1 or OAT2 and 5 mediated by organic anion transporting polypetides (OATPs), wild-type cells were seeded at a density of 0.5–1.2 Â 10 cells/well on – where many high-molecular-weight acids/zwitterions (ECCS BioCoat 48- or 96-well poly-D-lysine coated plates (Corning Inc., class 1B/3B) are shown to be substrates (Giacomini et al., Corning, NY), grown in DMEM containing 10% fetal bovine serum, 1% sodium pyruvate, 1% nonessential aminoacids, and 1% GlutaMAX 2010; Shitara et al., 2013; Varma et al., 2015, 2017b). OAT2, a for 48 hours at 37°C, 90% relative humidity, and 5% CO2. In addition, member of solute carrier 22A (SLC22A7), is expressed in the OAT2-HEK cells were supplemented with 1% gentamycin and liver and kidney and is known to transport several xenobiotics hygromycin B (50 mg/ml) and OATP1B1-HEK cells were supple- and endogenous compounds (e.g., creatinine and cGMP) mented with 1% HEPES. (Lepist et al., 2014; Shen et al., 2017). Although protein Methods adopted for uptake studies are similar to those previously abundance of OAT2 in the human liver is relatively similar reported by our group (Bi et al., 2017, 2018a; Mathialagan et al., 2017). to that of other major uptake transporters, such as OATP1B, Stock solutions of all compounds were made in DMSO. Cells were Na1-taurocholate cotransporting polypeptide, and organic rinsed three times with warm uptake buffer (Hanks’ balanced salt cation transporter (OCT)1 (Nakamura et al., 2016; Vildhede solution (HBSS) with 20 mM HEPES, pH 7.4), followed by incubating m m et al., 2018), little is known about its role in the clinical test compounds at 1 M (or 10 M in some cases with analytical sensitivity issues) with a final concentration of 1% DMSO. At the end pharmacokinetics of drugs (Shen et al., 2017). We recently of incubation (2 minutes), the cellular uptake was terminated by identified the role of OAT2-mediated hepatic uptake in the washing the cells four times with ice-cold transport buffer, and then Downloaded from clearance of tolbutamide and R- and S-warfarin, which were the cells were lysed with 0.2 ml of 1% NP-40 in water (radiolabeled previously assumed to be cleared by metabolism involving compounds) or methanol containing internal standard (nonlabeled P450 2C9/19 (Bi et al., 2018a,b). Mechanistic in vitro studies compounds). Transporter functionality was validated using in vitro and physiologically based pharmacokinetic (PBPK) modeling probe substrates: 0.5 mM[3H]-cGMP (OAT2) or 0.5 mM rosuvastatin and simulations demonstrated the significance of OAT2- (OATP1B1) as described previously (Bi et al., 2017; Mathialagan et al., CYP2C interplay in their clinical pharmacokinetics (Bi et al., 2018). Intracellular accumulation was determined either by mixing 2018,b). Given that these drugs are high-permeability–low- the cell lysate with scintillation fluid followed by liquid scintillation jpet.aspetjournals.org molecular-weight acids, we hypothesized that OAT2-mediated analysis (PerkinElmer Life Sciences) for radiolabeled compounds or by liquid chromatography-tandem mass spectrometry (LC-MS/MS) hepatic uptake contributes to the clearance of ECCS class 1A analysis for nonlabeled compounds. The total cellular protein content drugs. was determined using a Pierce BCA Protein Assay kit according to the The main objective of this investigation was to evaluate the manufacturer’s specifications. The uptake ratio was calculated as a role of transporter-mediated hepatic uptake in the pharma- ratio of accumulation in the transfected cells to the accumulation in cokinetics of high-permeability–low-molecular- weight acids wild-type cells. and zwitterions. To this end, for a set of about 25 ECCS class Determination of Uptake Clearance Using Short-Term at ASPET Journals on September 29, 2021 1A drugs (Varma et al., 2015), in vitro transport activity was
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