Role of Oatp2b1 in Drug Absorption and Drug-Drug Interactions

Abstract

The organic anion transporting polypeptide (OATP)2B1 is localized on the basolateral membrane of hepatocytes and is expressed in enterocytes. Based on its distribution pattern and functional similarity to OATP1B-type transporters, OATP2B1 might have a role in the absorption and disposition of a range of xenobiotics. Although several prescription drugs, including hydroxymethylglutaryl-coenzyme A-CoA reductase inhibitors (statins) such as fluvastatin, are OATP2B1 substrates in vitro, evidence supporting the in vivo relevance of this transporter remains limited, and most has relied on substrate-inhibitor interactions resulting in altered pharmacokinetic properties of the victim drugs. To address this knowledge deficit, we developed and characterized an Oatp2b1-deficient mouse model and evaluated the impact of this transporter on the absorption and disposition of fluvastatin. Consistent with the intestinal localization of Oatp2b1, we found that the genetic deletion or pharmacological inhibition of Oatp2b1 was associated with decreased absorption of fluvastatin by 2- to 3-fold. The availability of a viable Oatp2b1-deficient mouse model provides an opportunity to unequivocally determine the contribution of this transporter to the absorption and drug-drug interaction potential of drugs. SIGNIFICANCE STATEMENT: The current investigation suggests that mice deficient in Oatp2b1 provide a valuable tool to study the in vivo importance of this transporter. In addition, our studies have identified novel potent inhibitors of OATP2B1 among the class of tyrosine kinase inhibitors, a rapidly expanding class of drugs used in various therapeutic areas that may cause drug-drug interactions with OATP2B1 substrates.

Fig. 1.

Development of OATP2B1-deficient (OATP2B1^−/−) mouse model. (A) Conformation of transgenic allele in Slco2b1^{tm1a(KOMP)Wtsi}. FRT, flippase recombinase target; En2 SA, splice acceptor of mouse Engrailed2 Exon2; IRES, internal ribosome entry site; LacZ, gene encoding β-galactosidase; pA, polyadenylation signal; loxP, sequence targeted by Cre recombinase; hBactP, human β-actin gene promotor; neo, neomycin resistnace. (B) Illustration of breeding strategy. Flp, mice expressing flippase; Cre, mice expressing Cre recombinase. (C) For genotyping of the rederived mice, PCRs detect a fragment of loxP (283 bp) that is downstream to the targeted region, indicating the success of embryo rederivation (1, 3, 4, and 6). ddH₂O, double-distilled H₂O. (D) For genotyping of resulting mice from F₂, PCRs detect a fragment of Oatp2b1 exon 4 (wild-type primers, wild-type: 463 bp) or the entire exon 4 region (knockout primers, wild-type: 1318 bp, knockout: 616 bp). (E) mRNA expression was assessed by real-time qPCR, and levels were undetectable in major organs of Oatp2b1-deficient mice. LI, large intestine; SI, small intestine.

Fig. 2.

Pharmacokinetic profiles of fluvastatin in mice. (A) Plasma concentration-time profiles of fluvastatin in male wild-type mice and male Oatp2b1-knockout (Oatp2b1^−/−) mice after oral fluvastatin administration through gavage (n = 5). (B) Plasma concentration-time profiles of fluvastatin in female wild-type mice and female Oatp2b1^−/− mice after oral fluvastatin administration (n = 5). (C) Fluvastatin in liver 4 hours after oral administration (n = 5). (D) Plasma concentration-time profile of fluvastatin in male wild-type mice and male Oatp2b1^−/− mice after tail vein injection of fluvastatin. Slopes of best-fit lines were 0.36 ml/h vs. 0.42 ml/h (n = 5, P > 0.05). (E) Plasma concentration-time profile of fluvastatin in female wild-type mice and female Oatp2b1^−/− mice after tail vein injection of fluvastatin. Slopes of best-fit lines were 0.36 ml/h vs. 0.32 ml/h (n = 5, P > 0.05).

Fig. 3.

Expression of transporters and enzymes of putative relevance to fluvastatin pharmacokinetics as determined by real-time qPCR. Relative mRNA expression of the Oatp1b2, Abcc2 (Mrp2), Cyp2c65, and Cyp2c66 genes was determined in livers (A and C) and small intestine (B and D) collected from male (A and B) and female (C and D) wild-type mice or male and female Oatp2b1-knockout (Oatp2b1^−/−) mice. Data are presented as means (bars) and S.E.M. (error bars) of four observations per genotype and sex. Expression values were normalized to the housekeeping gene, glyceraldehye-3-phosphate dehydrogenase. *P < 0.05 vs. wild-type; ****P < 0.0001 vs. wild-type.

Fig. 4.

Oatp2b1-mediated fluvastatin-erlotinib interaction. (A) Influence of TKIs on E3S uptake in HEK293 cells overexpressing human OATP2B1. Both HEK293-OATP2B1 and HEK293-control vector cells were coincubated with designated amount of inhibitors and [³H]-E3S (2.5 µM) for 5 minutes. Uptake values were normalized to that observed for the DMSO group. Data are presented as means (bars) and S.E.M. (error bars) of at least two experiments performed in triplicate (n = 6–12 for TKIs; n = 69 for DMSO). (B) Comparison between uptake of fluvastatin (0.2 μM) in the presence or absence of erlotinib (10 μM) in cells overexpressing OATP2B1 or Oatp2b1. (C and D) Plasma concentration-time profile of fluvastatin in mice pretreated with vehicle or erlotinib (100 mg/kg) in female wild-type mice (C) and female Oatp2b1-knockout (Oatp2b1^−/−) mice (D) (n = 4). (E) Fluvastatin concentration in liver 4 hours after oral administration (n = 4).