Abstract 4928: Role of hepatic organic anion-transporting polypeptides in methotrexate uptake and clearance

Abstract

Abstract Methotrexate (MTX) is a folate antimetabolite drug extensively used in patients with malignant and autoimmune diseases. Organic anion-transporting polypeptides 1A/1B (OATP1A/1B) are drug uptake transporters that are primarily expressed in pharmacokinetically relevant tissues and are known for broad substrate specificity, including many drugs in clinical use. While MTX is a known substrate for the efflux transporter MRP2, significantly less is known regarding its interactions with drug transporters that could mediate its cellular uptake and clearance. Previous studies have suggested MTX to be a substrate for OATP transporters. We sought to undertake a comprehensive evaluation of OATP transporters to the in vitro and in vivo disposition of MTX. In vitro, using a recombinant vaccinia-based method, we screened an array of OATPs for MTX transport in HeLa cells, indicating that human OATP1A2, OATP1B1, OATP1B3 and their rat orthologs, Oatp1a4, Oatp1a5 and Oatp1b2, were capable of significant MTX uptake (p < 0.01). In addition, an assessment of a panel of single nucleotide polymorphisms in SLCO1A/1B revealed that two nonsynonymous polymorphisms (NSPs; 516A>C and 404A>T) in SLCO1A2, seven NSPs including 521T>C and 388A>G+521T>C in SLCO1B1, and six NSPs in SLCO1B3 were associated with significantly impaired MTX transport. Transcellular vectorial transport (basal→apical) studies of MTX conducted in MDCKII cells stably expressing hepatic OATP1B transporters revealed both OATP1B1 and OATP1B3 transport MTX. There was significantly increased MTX in the apical compartments of MDCKII-OATP1B/MRP2 cells compared to control cells (p < 0.05) at all time points. Furthermore, MTX translocated into the apical compartments was significantly higher in MDCKII-OATP1B/MRP2 cells than MDCKII-OATP1B cells (p < 0.05) at late time points, reflecting active efflux mediated by MRP2. Compared to control cells, intracellular accumulation of MTX was significantly higher in both MDCKII-OATP1B and MDCKII-OATP1B/MRP2 cells up to 2h post incubation (p < 0.05), reflecting active uptake by OATP1B at early time points. Subsequently, in vivo MTX transport studies showed that plasma MTX AUC was 2.8-fold higher and clearance was reduced by 64% in Slco1a/1b-/- versus wild-type (WT) mice (p < 0.01). The liver-to-plasma ratio of MTX was 9-fold higher in WT mice compared to Slco1a/1b-/- mice (p < 0.01) but no difference between WT and humanized TG mice, collectively suggesting hepatic OATP1B transporters are important for hepatic clearance of MTX. In conclusion, we demonstrate significant roles for OATP1B in transporter-mediated uptake and disposition of MTX. Moreover, significantly impaired MTX transport by OATP1A/1B variants may have important toxicological and therapeutic ramifications. Accordingly, these findings reveal important new insights into the relevance of the hepatic OATP1B transporters to the clinical pharmacology of MTX. Citation Format: Hannah H. Lee, Brenda F. Leake, Richard H. Ho. Role of hepatic organic anion-transporting polypeptides in methotrexate uptake and clearance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4928.