Abstract
Mechanism-based cytochrome P450 inactivation is defined as a time- and NADPH-dependent inactivation that is not reversible upon extensive dialysis. Current methodologies use dilution approaches to estimate the rate of inactivation and offer limited mechanistic insight and are significantly influenced by experimental conditions. We investigated the potential of progress curve analysis because this experimental design allows investigation of both the reversible (Kiapp) and irreversible (Ki, Kinact) components of the reaction mechanism. The human liver microsomal CYP1A2 inactivation kinetics of resveratrol, oltipraz, furafylline, and dihydralazine (Fig. 2) were evaluated. The inactivation results for furafylline (Ki, 0.8 μM; Kinact, 0.16 min–1) are within 2-fold to pub-lished data (Ki, 1.6 μM; Kinact, 0.19 min–1). Resveratrol and dihydralazine results are within a 4.3-fold range of published data, which compares well with ranges of estimates of these parameters across publications (e.g., furafylline has estimates ranging of Ki from 1.6 to 22.3 μM and Kinact from 0.19 to 0.87 min–1). This range of estimates highlights the potential caveats surrounding the existing methodologies that have been previously discussed in depth. In addition to these inactivation parameters, we have been able to demonstrate a variation in balance of reversible versus irreversible inhibition within these inactivators. Oltipraz and resveratrol have Kiapp values similar to their Ki, indicating a significant early onset reversible inhibition, whereas furafylline and dihydralazine are dominated by irreversible inactivation. This approach allows a more mechanistic investigation of an inactivator and in the future may improve the prediction of clinical drug-drug interactions.
Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.107.017236.
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ABBREVIATIONS: DDI, drug-drug interaction; P450, cytochrome P450; MBI, mechanism-based inactivation.
- Received June 13, 2007.
- Accepted September 5, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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