Pre-incubation with OATP1B1 and OATP1B3 inhibitors potentiates inhibitory effects in physiologically relevant sandwich-cultured primary human hepatocytes

Abstract

Organic anion transporting polypeptides (OATP)1B1 and OATP1B3 are liver-specific transport proteins that express on the basolateral membrane of human hepatocytes and mediate hepatic uptake of many drugs, including statins. They are important determinants of transporter-mediated drug-drug interactions (DDIs). It has been reported that pre-incubation with some OATP1B1 and OATP1B3 inhibitors potentiates the inhibitory effects, yielding reduced IC₅₀ values. The US FDA draft guidance has recently recommended to use the lower IC₅₀ values after inhibitor-preincubation to assess OATP1B1 and OATP1B3-mediated DDIs. However, it remains unknown whether the potentiation effects of inhibitor-preincubation on IC₅₀ values occur in a physiologically relevant cell model. The current study was designed to determine the IC₅₀ values of OATP1B1 and OATP1B3 inhibitors everolimus (EVR), sirolimus (SIR), and dasatinib against OATP1B substrates in physiologically relevant primary human hepatocytes with or without inhibitor-preincubation and to compare the OATP-mediated DDI prediction using data from primary human hepatocytes and that reported previously in transporter-expressing cell lines. Primary human hepatocytes were cultured in a sandwich configuration. Accumulation of [³H]-CCK-8 (1 µM, 1.5 min), [³H]-rosuvastatin (0.5 µM, 2 min) and [³H]-pitavastatin (1 µM, 0.5 min) was determined in human sandwich-cultured hepatocytes (SCH) in the presence of vehicle control or an inhibitor with or without inhibitor-preincubation at designated concentrations, and was utilized to determine the IC₅₀ values for these inhibitors. R-value models were used to predict OATP-mediated DDIs. Pre-incubation with EVR at a clinically relevant concentration of 0.2 µM significantly reduced accumulation of [³H]-CCK-8 and [³H]-rosuvastatin even after washing. Reduced IC₅₀ values following inhibitor pre-incubation were observed for all three inhibitors using [³H]-CCK-8 and [³H]-rosuvastatin as substrates in human SCH. The IC₅₀ values after inhibitor-preincubation were lower or comparable in transporter-expressing cell lines compared with that in human SCH. For dasatinib, R-values from both cell lines and human SCH were greater than the US FDA cut-off value of 1.1. For EVR, R values from cell lines were 1.23 and were lowered to near 1.1 (1.08-1.09) in human SCH. For SIR, R values from either cell type were less than the cut-off values of 1.1. In conclusion, the current study is the first to report that pre-incubation with OATP1B inhibitors potentiates inhibitory effects in physiologically relevant primary human hepatocytes, supporting the rationale of the current US FDA draft guidance of including an inhibitor-preincubation step when assessing OATP-mediated DDIs in vitro. IC₅₀ values after inhibitor-preincubation in transporter-expressing cell lines may be used for DDI prediction for the purpose of mitigating false-negative OATP-mediated DDI prediction.

Keywords: Drug interactions; Drug transport; Hepatic transport; Hepatocytes; Organic anion-transporting polypeptide; Pharmacokinetics; Physiologically based pharmacokinetic modeling; Transporters.