In Vitro P-Glycoprotein Inhibition Assays for Assessment of Clinical Drug Interaction Potential of New Drug Candidates: A Recommendation for Probe Substrates

Abstract

ABSTRACT:Because modulation of P-glycoprotein (Pgp) through inhibition or induction can lead to drug-drug interactions by altering intestinal, central nervous system, renal, or biliary efflux, it is anticipated that information regarding the potential interaction of drug candidates with Pgp will be a future regulatory expectation. Therefore, to be able to utilize in vitro Pgp inhibition findings to guide clinical drug interaction studies, the utility of five probe substrates (calcein-AM, colchicine, digoxin, prazosin, and vinblastine) was evaluated by inhibiting their Pgp-mediated transport across multidrug resistance-1-transfected Madin-Darby canine kidney cell type II monolayers with 20 diverse drugs having various degrees of Pgp interaction (e.g., efflux ratio, ATPase, and calcein-AM inhibition). Overall, the rank order of inhibition was generally similar with IC 50 values typically within 3-to 5-fold of each other. However, several notable differences in the IC 50 values were observed. Digoxin and prazosin were the most sensitive probes (e.g., lowest IC 50 values), followed by colchicine, vinblastine, and calcein-AM. Inclusion of other considerations such as a large dynamic range, commercially available radiolabel, and a clinically meaningful probe makes digoxin an attractive probe substrate. Therefore, it is recommended that digoxin be considered as the standard in vitro probe to investigate the inhibition profiles of new drug candidates. Furthermore, this study shows that it may not be necessary to generate IC 50 values with multiple probe substrates for Pgp as is currently done for cytochrome P450 3A4. Finally, a strategy integrating results from in vitro assays (efflux, inhibition, and ATPase) is provided to further guide clinical interaction studies.P-glycoprotein (Pgp) is a member of the ATP-binding cassette superfamily of transport proteins and is expressed in numerous tissues such as the luminal membrane of the small intestine and blood-brain barrier, and the apical membranes of excretory organs such as liver and kidney (Ayrton and Morgan, 2001). Pgp has broad substrate recognition, which can affect the pharmacokinetics, efficacy, safety, and target organ specificity of drugs. As a result, drug-drug interactions resulting from inhibition or induction of Pgp are a recognized clinical concern (Englund et al., 2004;Balayssac et al., 2005) recently highlighted in the Food and Drug Administration (FDA) concept paper "Drug Interaction Studies-Study Design, Data Analysis, and Implications for Dosing and Labeling" (FDA, 2004).Despite many years of investigation, considerable uncertainty remains about the number of drug binding sites within Pgp and their mutual relationships. It is postulated that the transmembrane regions of Pgp form a large binding pocket (Sharom et al., 1998;Lugo and Sharom, 2005) composed of amino acid residues from multiple transmembrane segments Clarke, 2001, 2002). Recent experiments investigating drug binding (Martin et al., 2000), fluorescent dye uptake (Shapiro and Ling, 1997;Lugo ...

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