Abstract
A refined cytochrome P450 (P450) enzyme IC50 shift assay for more accurately screening CYP3A time-dependent inhibitors (TDIs) is presented. In contrast to the regular IC50 shift assay, in which only one pair of P450 inhibition curves is generated, this modified method generates two pairs of inhibition curves; one pair of curves is created from human liver microsomal incubations with the test article in the presence or absence of NADPH (curves 1 and 2) (same as the traditional assay), and the other pair is created from new microsomal incubations with extract (compound/metabolites) of previous incubations (curves 3 and 4). To assess the true CYP3A time-dependent inhibition, we propose a new parameter, the vertical IC50 curve shift (VICS), represented by vertical shift difference between the two sets of curves divided by inhibitor concentration at which maximal vertical shift of curves 1 and 2 is observed. A shift in the curves 1 and 2 could mean a time-dependent inhibition or formation of a more active inhibitory metabolite(s). The new method provides more reliable characterization of the shift as a result of a true TDI- or metabolite-mediated reversible inhibition. Nine known TDI drugs were evaluated using this refined shift assay. The derived VICS values correlated well with the reported kinact/KI values derived via the conventional dilution assay method. Thus, the refined assay can be used to identify a true TDI and quantitatively assess the inactivation potential of TDIs in a high-throughput fashion. This assay can be invaluable to screen for true P450 TDIs in the early drug discovery.
Footnotes
↵1 Drug Metabolism and Pharmacokinetics, Preclinical and Clinical Development Sciences, Biogen Idec, Inc., 14 Cambridge Street, Cambridge, MA 02142.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.111.038208.
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ABBREVIATIONS:
- DDI
- drug-drug interaction
- TDI
- time-dependent inhibitor
- MBI
- mechanism-based inhibitor
- VICS
- vertical IC50 curve shift
- CImax
- inhibitor concentration that produced maximal vertical shift in the IC50 curves
- P450
- cytochrome P450.
- Received January 14, 2011.
- Accepted March 10, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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