Received for publication December 1, 2005.
Revised January 27, 2006.
Accepted for publication January 31, 2006.

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

Abstract

As modulation of P-glycoprotein (Pgp) through inhibition or induction can lead to drug-drug interactions by altering intestinal, CNS, 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 interactions studies, the utility of five probe substrates (calcein-AM, colchicine, digoxin, prazosin and vinblastine) was evaluated by inhibiting their Pgp-mediated transport across MDR1-MDCKII 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 IC₅₀ values typically within 3- to 5-fold of each other. However, several notable differences in the IC₅₀ values were observed. Digoxin and prazosin were the most sensitive probes (e.g., lowest IC₅₀ 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. Further, this study demonstrates that it may not be necessary to generate IC₅₀ 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.

Key words: ABC transporters, cytochrome P450, drug efflux, drug interactions, drug transport, hepatic transport, in vivo probes, membrane transport, p-glycoprotein, transporters

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