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Validation of cell-based OATP1B1 assays to assess drug transport and the potential for drug–drug interaction to support regulatory submissions

Transporters are carrier proteins that may influence pharmacokinetic, pharmacodynamic, and toxicological characteristics of drugs. The development of validated in vitro transporter models is imperative to support regulatory submissions of drug candidates. This study is focused on utilizing human embryonic kidney (HEK) 293 cell cultures genetically transfected with the human organic anion transporting polypeptides (OATP) 1B1 transporter to identify substrates and inhibitors in drug development. The kinetics of OATP1B1-mediated uptake of [3H]-oestradiol 17β-glucuronide and inhibition of uptake by rifamycin SV were used to determine Km, Vmax, and IC50 values over a range of passage numbers to investigate accuracy and precision. The mean Km and Vmax values were found to be 6.3 ± 1.2 μM and 460 ± 96 pmol min−1 mg−1, respectively. The mean IC50 value for rifamycin SV was 0.23 ± 0.07 μM on uptake of 1 μM [3H]-oestradiol 17β-glucuronide. These data were similar to previously reported values (accuracy greater than 82%), reproducible (precision less than 29%) and exhibited low standard deviations (SDs) obviating the need to study test compounds on more than one occasion. [3H]-oestrone 3-sulfate and [3H]-pravastatin exhibited concentration-dependent OATP1B1 uptake, and statistically significant differences were observed at each concentration between uptake rates of HEK293-OATP1B1 and HEK293-MOCK cells (uptake ratios greater than or equal to 3). Propranolol showed no positive uptake ratio. Bezafibrate and gemfibrozil exhibited concentration-dependent inhibition of OATP1B1-mediated uptake of [3H]-oestradiol 17β-glucuronide with mean IC50 values of 16 and 27 μM, respectively. Based on the validation results, acceptance criteria to identify a test compound as a substrate and/or inhibitor using these specific cell lines were determined. These validated OATP1B1 assays were robust, reproducible, and suitable for routine in vitro evaluation of candidate drugs.