Design, Data Analysis, and Simulation of in Vitro Drug Transport Kinetic Experiments Using a Mechanistic in Vitro Model

Abstract

ABSTRACT:The use of in vitro data for quantitative predictions of transportermediated 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 twocompartmental 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.

Kinetic Characterization of Rat Hepatic Uptake of 16 Actively Transported Drugs

“…However, in reality, the intracellular free concentration may differ markedly as a result of transporter activity and, hence, affect the assessment of clearance and drug-drug interactions Webborn et al, 2007;Brown et al, 2010;Watanabe et al, 2010). Intracellular binding and enzyme transporter interplay are further sources of complexity in elucidating in vitro-in vivo relationships (Lam et al, 2006;Poirier et al, 2008).…”

“…Despite the increasing number of in vitro transporter studies, a lack of consistency in rat and human in vitro uptake methodology and data is apparent, particularly in the assessment and modeling of passive permeation, drug efflux, and nonspecific binding (Kitamura et al, 2008;Paine et al, 2008;Poirier et al, 2008;Watanabe et al, 2009a). In many cases, uptake is limited to use of a single low-and highsubstrate concentration to estimate saturable and nonsaturable uptake, respectively.…”

“…There are a number of approaches to determining hepatic uptake in vitro; however, the common principle involves separation of cells and media and the monitoring of drug concentrations in either matrix or both matrices (Petzinger and Fuckel, 1992;Hallifax and Houston, 2006;Poirier et al, 2008). In the present study, drug associated with the hepatocytes was measured after separation from free drug by layering the suspension of drug and hepatocytes over silicone/mineral oil followed by rapid centrifugation Ϫ the oil-spin method (Petzinger and Fuckel, 1992), a method used by several groups (Ishigami et al, 1995;Nakai et al, 2001;Nezasa et al, 2003;Shimada et al, 2003;Hallifax and Houston, 2006) to characterize hepatic uptake by a clearance term for active transport, CL active , together with K m and V max and a passive permeability parameter, P diff .…”

Comparative Use of Isolated Hepatocytes and Hepatic Microsomes for Cytochrome P450 Inhibition Studies: Transporter-Enzyme Interplay

“…Because accumulation of drugs in hepatocytes may occur via active uptake processes and/or intracellular binding, a concentration difference may exist between hepatocyte and hepatic microsomal incubations. Whether the drug is a bound or free entity within the cell is of importance; intracellular binding to sites not involved in the metabolic process may be of little consequence, as the free concentration within the cell will be in equilibrium with the external incubation media concentration (Grime and Riley, 2006;Poirier et al, 2008). However, in the case of uptake transporters, raising the cellular concentration in excess of the incubation medium concentration will result in more drug available to the enzyme.…”

Prediction of Hepatic Clearance in Human From In Vitro Data for Successful Drug Development

“…13) and unbound drug concentrations in the hepatocytes. Compartment-model-based analysis has been further extended to the mechanistic assessment of Michelis-Menten parameters (K m and V max ) responsible for the transporter-mediated hepatic uptake, bidirectional passive diffusion, and nonspecific binding which directly reflect those obtained from in vitro experiments (140). These mechanistic approaches can provide an integrated tool not only for the accurate predictions of pharmacokinetics but also for the predictions of potential drug-drug interactions by transporter(s) and/or CYP(s) and of efficacy by liver-targeted drug candidates.…”