Category: Preclinical Development
Purpose: The human organic anion-transporting polypeptide 2B1 (OATP2B1; SLC21A9) is localized to the basolateral membrane of hepatocytes and the brush-border membrane of small-intestinal enterocytes. Based on its distribution pattern and functional similarity to OATP1B-type transporters, it has been hypothesized that OATP2B1 might influence the absorption and disposition properties of a wide range of xenobiotic agents. Although several prescription drugs, including statins such as fluvastatin, have been identified as OATP2B1 substrates in vitro, the lack of a useful in vivo model system has limited our understanding of the pharmacological role of this transporter. Here we described the generation of transgenic OATP2B1-deficient mouse model and its application in some pilot PK studies.
Methods: OATP2B1-overexpressing cells were generated by transfecting HEK293 cells with an empty vector (VC) or a construct containing human OATP2B1 or mouse OATP2B1 cDNA. Transporter function was evaluated in vitro by measuring the comparative cellular uptake of [3H]estrone-3-sulfate (ES) in VC cells and OATP2B1-overexpressing cells. Candidate substrates and inhibitors were identified based on the difference in intracellular accumulation of radioactivity. OATP2B1-knockout (KO) mice were obtained from the Slco2b1tm1a(KOMP)Wtsi clone, and derived chimera and subsequent progeny were mated to C57BL/6N for germline production and colony expansion. Mouse genotypes were confirmed by PCR, and OATP2B1 gene deletion was verified by qPCR on major organs. The pharmacokinetic profiles of fluvastatin, a known OATP2B1 substrate, and sorafenib, a known OATP2B1 inhibitor, were determined through serial blood collection and subsequent LC-MS/MS analysis. Noncompartmental pharmacokinetic parameters were calculated using WinNolin 6.2 software (Pharsight).
Results: An approximately four-fold increase in intracellular accumulation of radiolabeled ES (2.5 µM) was observed in the OATP2B1 overexpressing cells compared to VC cells (34.8 ± 2.45 pmol/mg vs 8.38 ± 0.341 pmol/mg, respectively). We then identified several drugs as candidate OATP2B1 substrates, including multiple tyrosine kinase inhibitors (TKIs) Knockout of OATP2B1 in organs from KO mice was confirmed at the gene level (P< 0.01), and consistent with the intestinal localization of OATP2B1, we found that the absorption rate of fluvastatin was decreased compared with that in WT mice (Tmax: 0.5 h vs 1 h; Cmax: 1.05 µg/mL vs 2.19 µg/mL). No gender differences in mRNA expression levels were detected. Among 24 different agents tested, sorafenib was identified as one of the most potent OATP2B1 inhibitors in the class of TKIs. However, the pharmacokinetic properties of sorafenib and its main metabolites were not significantly affected by OATP2B1 deficiency.
Conclusion: Our studies have identified novel potent inhibitors of OATP2B1 among TKIs, a rapidly expanding class of drugs used in various therapeutic areas. The availability of a viable, well-characterized OATP2B1-deficient mouse model provides a previously unavailable opportunity to unequivocally determine the contribution of this transporter to the absorption and drug-drug interaction potential of drugs.