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 The US FDA draft guidance has recently recommended to use the lower IC₅₀ values after inhibitor-preincubation to assess OATP1B1 and OATP1B3-mediated DDIs.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.

Figure 1.

Effects of pretreatment with EVR on substrate transport in human SCH. Model-estimated fold change and associated SE of accumulation of (A) [³H]CCK-8 (1 μM, 1.5 min), (C) [3H]rosuvastatin (0.5 μM, 2 min), and (E) [³H]Pitavastatin (1 μM, 0.5 min) vs. CTL in human SCH in the absence of EVR after pretreatment with EVR at the indicated concentrations for 1 h followed by washing. *p<0.05 vs. CTL by mixed-effect model (n=3 hepatocytes donors at each concentration in triplicate). B, D, and F. The IC₅₀ values were determined by fitting dose-response curves to the data by nonlinear regression analysis in co-incubation and pre+co-incubation as determined in the Materials and Methods. Solid (co-incubation) and dashed lines (pre+co-incubation) represent the fitted lines. Data are expressed as percentage of vehicle control in co-incubation (closed circles) and pre+co-incubation (open circles) scenarios. Data represent mean ± SEM (n=3 hepatocytes donors in triplicate).

Figure 2.. Effects of pretreatment with SIR on substrate transport in human SCH.

Model-estimated fold change and associated SE of accumulation of (A) [³H]CCK-8 (1 μM, 1.5 min), (C) [³H]rosuvastatin (0.5 μM, 2 min), and (E) [³H]Pitavastatin (1 μM, 0.5 min) vs. CTL in human SCH in the absence of SIR after pretreatment with SIR at indicated concentrations for 1 h, followed by washing. *p<0.05 vs. CTL by mixed-effect model (n=3 hepatocytes donors at each concentration in triplicate). B, D, and F. The IC₅₀ values were determined by fitting dose-response curves to the data by nonlinear regression analysis in co-incubation and pre+co-incubation as determined in the Materials and Methods. Solid (co-incubation) and dashed lines (pre+co-incubation) represent the fitted lines. Data are expressed as percentage of vehicle control in co-incubation (closed circles) and pre+co-incubation (open circles) scenarios. Data represent mean ± SEM (n=3 hepatocytes donors in triplicate for B and D), or mean ± range (n=2 hepatocytes donors in triplicate for F).

Figure 3.

Inhibitory effects of dasatinib on [³H]rosuvastatin transport under co-incubation and pre+co-incubation in human SCH. [³H]rosuvastatin accumulation (0.5 μM, 2 min) was determined in the presence of dasatinib at the indicated concentration without (co) (A) or with (pre+co) a 1-h pre-incubation with dasatinib. IC₅₀ values were determined by fitting dose-response curves to the data by nonlinear regression analysis as described in the Materials and Methods. Solid (co-incubation) and dashed lines (pre+co-incubation) represent the fitted lines. Data are expressed as percentage of vehicle control in co-incubation (closed circles) and pre+co-incubation (open circles) scenarios. Data are percent of control expressed as mean ± SEM (n=3 hepatocytes donors in triplicate for pre+co-incubation) and mean ± range (n=2 hepatocytes donors in triplicate for co-incubation). A and B were plotted on different scales to indicate that different hepatocytes donors were used in co- and pre+co-incubation (Table S3).

Figure 4.. Effects of BSP on substrates transport in human SCH.

Model-estimated fold change and associated SE of accumulation of (A) [³H]CCK-8 (1 μM, 1.5 min), (B) [³H]rosuvastatin (0.5 μM, 2 min), and (C) [³H]pitavastatin (1 μM, 0.5 min) vs. CTL in human SCH. Co-incubation, the indicated substrate was co-incubated with vehicle control or BSP (100 μM). Pre-incubation, human SCH was preincubated with BSP (100 μM) or vehicle control for 1 h; after washing, substrate accumulation was determined in the absence of inhibitor BSP. All experiments were conducted in triplicate (n=2 and 3 hepatocytes donors in A and C, respectively; in B, n=2 and 3 hepatocytes donors for co-incubation and pre-incubation, respectively). *p<0.05 vs. CTL by mixed-effect model.

Figure 5.. Effects of rifampicin on substrates transport in human SCH.

Model-estimated fold change and associated SE of accumulation of (A) [³H]CCK-8 (1 μM, 1.5 min) and (B) [³H]rosuvastatin (0.5 μM, 2 min) vs. CTL in human SCH. Cells were preincubated with 50 μM rifampicin for 1 h, then co-incubated with 50 μM rifampicin and substrate [³H]CCK-8 (1 μM, 1.5 min) (A) or [3H]rosuvastatin (0.5 μM, 2 min) (B). Experiments were conducted in triplicate. N=2 for CCK-8, N=3 for rosuvastatin. *p<0.05 vs. CTL by mixed-effect model.