ACCELERATED COMMUNICATION Targeted Disruption of Murine Organic Anion-Transporting Polypeptide 1b2 (oatp1b2/Slco1b2) Significantly Alters Disposition of Prototypical Drug Substrates Pravastatin and Rifampin

Organic anion-transporting polypeptides (OATP) 1B1 and 1B3 are widely acknowledged as important and rate-limiting to the hepatic uptake of many drugs in clinical use. Accordingly, to better understand the in vivo relevance of OATP1B transporters, targeted disruption of murine Slco1b2 gene was carried out. It is noteworthy that Slco1b2( / ) mice were fertile, developed normally, and exhibited no overt phenotypic abnormalities. We confirmed the loss of Oatp1b2 expression in liver using real-time polymerase chain reaction, Western Blot analysis, and immunohistochemistry. Expression of Oatp1a4 and Oatp2b1 but not Oatp1a1 was greater in female Slco1b2( / ) mice, but expression of other non-OATP transporters did not significantly differ between wild-type and Slco1b2( / ) male mice. Total bilirubin level was elevated by 2-fold in the Slco1b2( / ) mice despite the fact that liver enzymes ALT and AST were normal. Pharmacological characterization was carried out using two prototypical substrates of human OATP1B1 and -1B3, rifampin and pravastatin. After a single intravenous dose of rifampin (1 mg/kg), a 1.7-fold increase in plasma area under the concentration-time curve (AUC) was observed, whereas the liver-to-plasma ratio was reduced by 5-fold, and nearly 8-fold when assessed at steady-state conditions after 24 h of continuous subcutaneous infusion in Slco1b2( / ) mice. Likewise, continuous subcutaneous infusion at low (8 g/h) or high (32 g/h) dose rates of pravastatin resulted in a 4-fold lower liver-plasma ratio in the in Slco1b2( / ) mice. This is the first report of altered drug disposition profile in the Slco1b2 knockout mice and suggests the utility of this model for understanding the in vivo role of hepatic OATP transporters in drug disposition. Carrier-mediated membrane transport is now widely appreciated as a critical determinant of drug uptake, distribution, and elimination (Ho and Kim, 2005). The organic aniontransporting polypeptide (OATP) transporters belong to a large family of membrane proteins that mediate the sodiumindependent cellular uptake of a variety of amphipathic compounds, including hormones, bile acids, eicosanoids, environmental toxins, and many drugs in clinical use today (Tirona and Kim, 2007). Members of this family are expressed in a variety of organs of relevance to drug disposition or response, such as liver, kidney, brain, and intestine (Marzolini et al., 2004). One subset of the OATP transporters that has received This work was supported in part by United States Public Health Service grant GM54724 (R.B.K.) and the Deutsche Forschungsgemeinschaft grant ME 3090/1-1 (H.E.M.z.S). H.Z. and H.E.M.z.S. contributed equally to this work. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.108.046458. ABBREVIATIONS: OATP/Oatp, organic anion-transporting polypeptide; ES, embryonic stem; bp, base pair(s); PCR, polymerase chain reaction; kb, kilobase pair(s); F, forward; R, reverse; FBS, fetal bovine serum; PBS, phosphate-buffered saline; TBS-T, Tris-buffered saline/Tween 20; LC-MS/MS liquid chromatography/tandem mass spectrometry; AUC, area under the concentration-time curve; SLC, solute carrier; VSS, volume of distribution at steady state. 0026-895X/08/7402-320–329$20.00 MOLECULAR PHARMACOLOGY Vol. 74, No. 2 Copyright © 2008 The American Society for Pharmacology and Experimental Therapeutics 46458/3353442 Mol Pharmacol 74:320–329, 2008 Printed in U.S.A. 320 at A PE T Jornals on Jne 1, 2017 m oharm .aspeurnals.org D ow nladed from significant attention in relation to hepatic drug uptake is the human OATP1B subfamily. There are two members, OATP1B1 (SLC21A6, OATP-C, OATP2, LST-1) and OATP1B3 (SLC21A8, OATP-8, LST-2) in humans, which share 80% sequence homology (Hagenbuch and Meier, 2004). Both transporters have been described to be highly expressed in human liver (Abe et al., 1999, 2001; König et al., 2000; Ho et al., 2006) and are localized to the basolateral membrane, facilitating the hepatocellular uptake of endoand xenobiotic substrates before their metabolism and efflux from the liver (Shimizu et al., 2005). In rodents, there is only one member of the Oatp1b subfamily. This transporter, termed oatp1b2 (Slco1b2, Lst-1, oatp4), has been identified in rat and mouse and is thought to be the closest ortholog of both human OATP1B1 and -1B3 (Cattori et al., 2000; Choudhuri et al., 2000; Ogura et al., 2000). Although a number of in vitro and cell-based model systems for the study of OATP transporters exit, a murine Slco knockout mouse model has not yet been reported. The lack of such a model has prevented the delineation of the overall contribution of a given Oatp transporter to the organ-specific elimination of drugs shown to be substrates of multiple Oatp transporters in vitro. Because the mouse genome contains only one Slco1b subfamily member, we hypothesized that the targeted disruption of this transporter should allow for a better delineation and extrapolation of the in vivo relevance of human OATP1B1 and OATP1B3 to the hepatic uptake of substrate drugs. Accordingly, we now report on the generation of a Slco1b2 knockout mouse model. In addition to assessing the relative impact of this transporter to alterations of liver function or phenotype, we wanted to evaluate the utility of this model in terms of clarifying the relevance of Oatp1b2 in the hepatic elimination of prototypical substrates of human OATP1B1 and OATP1B3, such as rifampin and pravastatin. Indeed, studies from a number of laboratories, including ours, suggest that OATP1B1 is a major transporter for rifampin (Tirona et al., 2003). Likewise, HMG-CoA) reductase inhibitors (statins) have also been shown to be highly dependent on OATP1B1 and OATP1B3 to exert their effects on intracellular hepatic HMG-CoA reductase and thereby reduce cardiovascular events associated with hypercholesterolemia and atherosclerosis (Schachter, 2005). Pravastatin has been widely studied as a substrate for OATP transporters including OATP1B1 and recent studies have linked single nucleotide polymorphisms (SNPs) in SLCO1B1 as a major predictor of pravastatin pharmacokinetics (Nakai et al., 2001; Ho et al., 2007). We now provide data that suggest loss of Oatp1b2 (Slco1b2) does not result in a major phenotype in terms of viability or liver function, but the ability to eliminate drugs such as pravastatin and rifampin is significantly compromised. Taken together, our findings suggest that the Slco1b2( / ) mouse model may prove to be a useful in vivo model for assessing the contribution of OATP1B transporters to the hepatic uptake of drugs. Materials and Methods Materials. Rifampin, diclofenac, dimethyl sulfoxide, and pravastatin were obtained from Sigma (St. Louis, MO). Atorvastatin was obtained from Pfizer Global Research and Development (Ann Arbor, MI). Ammonium formate and all other chemical reagents were obtained from Sigma. Isoflurane (IsoFlo) was obtained from Abbott Laboratories (Abbott Park, IL). All other compounds used were reagents grade. Targeting of Slco1b2 in Mouse ES Cells. The targeting vector was designed using the Slco1b2 genomic locus form the Celera mouse genomic sequence database and the mRNA sequence (NM_178235). As shown in Fig. 1, homologous recombination resulted in the deletion of a 6772-bp fragment containing exon 10 to 12 of the mouse Slco1b2 genomic sequence and insertion of a neomycin resistance cassette. The 5 and the 3 regions of homology were amplified from DBA1/lacJ mouse genomic DNA using the Expand High Fidelity PCR system (Roche, Laval, QC, Canada). The primer pairs 5 -OAC-6141F, 5 -ggatccgagttgtcttttctgagtgttagg-3 ; 5 -OAC9873R, 5 -ggatccatgacagttgtctgctcattgc-3 ; and 3 -OAC-17027F, 5 ctcgagatgtttggctgagagcatctgc-3 ; and 3 -OAC-21988R, 5 -gcggccgctcatcttcatagcaaccctttcc-3 (F, forward; R, reverse) were used to amplify 4139and 4962-bp fragments of the genomic DNA, respectively. The resulting PCR fragments were cloned into the pCR 2.1TOPO vector (Invitrogen, Carlsbad, CA). After sequence verification, the homologous fragments were cloned into the backbone of the targeting vector plasmid that contained both a neomycin (neo) and a thymidine kinase expression cassette (see Fig. 1). Subsequently, the linearized Slco1b2 targeting vector was electroporated into DBA1/ lacJ ES cells. Those cells were cultured in the presence of G418 (positive selection attributable to neomycin cassette) and ganciclovir (negative selection attributable to thymidine kinase cassette) for selecting the correctly targeted ES cells. tk 5’Homology 3’Homology