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METABOLISM, TRANSPORT, AND PHARMACOGENOMICS

Impact of Impurity on Kinetic Estimates from Transport and Inhibition Studies

Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, Maryland (P.G., J.E.P.); and Departamento de Farmacia, Facultad de Química, Pontificia Universidad Católica de Chile, Santiago, Chile (P.G.)

Although in vitro transport/inhibition studies are commonly performed on impure drug candidates to screen for pharmacokinetic properties in early development, quantitative guidelines concerning acceptable impurity levels are lacking. The broad goal was to derive models for the effect of impurity on transport and inhibition studies and identify the maximum allowable impurity level that does not bias assay results. Models were derived, and simulations were performed to assess the impact of impurity on substrate properties K_t and J_max and inhibition K_i. Simulation results were experimentally challenged with a known amount of impurity, using the intestinal bile acid transporter as a model system. For substrate uptake studies, glycocholate served as substrate and was contaminated with either a very strong, strong, or moderate impurity (i.e., taurolithocholate, chenodeoxycholate, or ursodeoxycholate, respectively). For inhibition studies, taurocholate and glycocholate together was the substrate/inhibitor pair, where glycocholate was contaminated with taurolithocholate. There was high agreement between simulation results and experimental observations. It is not surprising that, in the inhibition assay, potent impurity caused test compound to appear more potent than the true potency of the test compound (i.e., reduced inhibitory K_i). However, results in the transport scenario surprisingly indicated that potent impurity did not diminish test compound potency but, rather, increased substrate potency (i.e., reduced Michaelis-Menten substrate K_t). In general, less than 2.5% impurity is a reasonable target, provided the impurity is less than 10-fold more potent than test compound. Study results indicate that careful consideration of possible impurity effect is needed when quantitative structure-activity relationship analysis cannot explain high compound potency from transport or inhibition studies.