Received for publication February 4, 2008.
Revised July 17, 2008.
Accepted for publication September 19, 2008.

Design, data analysis and simulation of in vitro drug transport kinetic experiments using a mechanistic in vitro model

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, non-specific 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 non-specific 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 (OATP) model substrates (e.g. CCK8, deltorphin II, fexofenadine, pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters (mean coefficient of variation for V_max and K_m were respectively 19% and 23%) as compared to the conventional kinetic method of transport data analysis (mean CV% were respectively 58% and 115% using this method). Furthermore, permeability was found to be highly temperature-dependent in CHO control cells and artificial membranes (PAMPA). While for some compounds (taurocholate, estrone-3-sulfate, propranolol) the effect was moderate (1.5 to 6-fold higher permeability at 37°C compared to 4°C), for fexofenadine a 16 fold higher passive permeability was seen at 37°C. Therefore, P_dif was better predicted if evaluated under the same experimental conditions as V_max and K_m, i.e. in a single incubation of CHO over-expressed cells or rat hepatocytes at 37°C, instead of a parallel control evaluation at 4°C.

Key words: ABC transporters, active transport, cellular transport, hepatocytes, liver physiology/models, mathematical modeling, membrane transport, organic anion transport