Abstract

The use of in vitro data for quantitative predictions of transporter-mediated elimination in vivo requires an accurate estimation of the transporter Michaelis-Menten parameters, V_max and K_m, as a first step. Therefore, the experimental conditions of in vitro studies used to assess hepatic uptake transport were optimized regarding active transport processes, nonspecific binding, and passive diffusion (P_dif). A mechanistic model was developed to analyze and accurately describe these active and passive processes. This two-compartmental model was parameterized to account for nonspecific binding, bidirectional passive diffusion, and active uptake processes based on the physiology of the cells. The model was used to estimate kinetic parameters of in vitro transport data from organic anion-transporting peptide model substrates (e.g., cholecystokinin octapeptide deltorphin II, fexofenadine, and pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters [mean coefficient of variations (CVs) for V_max and K_m were 19 and 23%, respectively] compared with the conventional kinetic method of transport data analysis (mean CVs were 58 and 115%, respectively, using this method). Furthermore, permeability was found to be highly temperature-dependent in Chinese hamster ovary (CHO) control cells and artificial membranes (parallel artificial membrane permeability assay). Whereas for some compounds (taurocholate, estrone-3-sulfate, and propranolol) the effect was moderate (1.5–6-fold higher permeability at 37°C compared with that at 4°C), for fexofenadine a 16-fold higher passive permeability was seen at 37°C. Therefore, P_dif was better predicted if it was evaluated under the same experimental conditions as V_max and K_m, i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37°C, instead of a parallel control evaluation at 4°C.