Design, data analysis, and simulation of in vitro drug transport kinetic experiments using a mechanistic in vitro model.
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 degrees C compared with that at 4 degrees C), for fexofenadine a 16-fold higher passive permeability was seen at 37 degrees 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 degrees C, instead of a parallel control evaluation at 4 degrees C.
Poirier
Agnès
A
F. Hoffmann-La Roche Ltd., Non-Clinical Development, Drug Safety, Basel, Switzerland.
Lavé
Thierry
T
Portmann
Renée
R
Brun
Marie-Elise
ME
Senner
Frank
F
Kansy
Manfred
M
Grimm
Hans-Peter
HP
Funk
Christoph
C
D000465:Algorithms; D000818:Animals; D001693:Biological Transport, Active; D016466:CHO Cells; D003198:Computer Simulation; D006224:Cricetinae; D003412:Cricetulus; D004058:Diffusion; D004970:Estrone / Q000031:analogs & derivatives / Q000378:metabolism; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism; D000077340:Fluvastatin; D022781:Hepatocytes / Q000378:metabolism*; D007211:Indoles / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008567:Membranes, Artificial; D008954:Models, Biological; D009281:Naphthalenes / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D027361:Organic Anion Transporters / Q000235:genetics; D010539:Permeability; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D010599:Pharmacokinetics; D010880:Piperidines / Q000378:metabolism; D011804:Quinolines / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012844:Sincalide / Q000378:metabolism; D013696:Temperature; D016593:Terfenadine / Q000031:analogs & derivatives / Q000378:metabolism
D005229:Fatty Acids, Monounsaturated; D007211:Indoles; D008567:Membranes, Artificial; D009281:Naphthalenes; D009842:Oligopeptides; D027361:Organic Anion Transporters; D004364:Pharmaceutical Preparations; D010880:Piperidines; D011804:Quinolines; C060209:deltorphin II, Ala(2)-; D004970:Estrone; D000077340:Fluvastatin; D016593:Terfenadine; C093230:fexofenadine; D012844:Sincalide; C108475:pitavastatin; C017296:estrone sulfate; C087198:napsagatran