CYP 3 A 4 Substrate Selection and Substitution in the Prediction of Potential Drug-Drug Interactions

The complexity of in vitro kinetic phenomena observed for CYP3A4 substrates (homoor heterotropic cooperativity) confounds the prediction of drug-drug interactions, and an evaluation of alternative and/or pragmatic approaches and substrates is needed. The current study focused on the utility of the three most commonly used CYP3A4 in vitro probes for the prediction of 26 reported in vivo interactions with azole inhibitors (increase in area under the curve ranged from 1.2 to 24, 50% in the range of potent inhibition). In addition to midazolam, testosterone, and nifedipine, quinidine was explored as a more “pragmatic” substrate due to its kinetic properties and specificity toward CYP3A4 in comparison with CYP3A5. Ki estimates obtained in human liver microsomes under standardized in vitro conditions for each of the four probes were used to determine the validity of substrate substitution in CYP3A4 drug-drug interaction prediction. Detailed inhibitor-related (microsomal binding, depletion over incubation time) and substrate-related factors (cooperativity, contribution of other metabolic pathways, or renal excretion) were incorporated in the assessment of the interaction potential. All four CYP3A4 probes predicted 69 to 81% of the interactions with azoles within 2-fold of the mean in vivo value. Comparison of simple and multisite mechanistic models and interaction prediction accuracy for each of the in vitro probes indicated that midazolam and quinidine in vitro data provided the best assessment of a potential interaction, with the lowest bias and the highest precision of the prediction. Further investigations with a wider range of inhibitors are required to substantiate these findings. CYP3A4 is the most abundant human P450 enzyme, metabolizing a wide range of structurally diverse therapeutic agents; hence, it is the target for many drug-drug interactions (DDI). A recent Food and Drug Administration report (Yuan et al., 2002) has shown testosterone to be the most commonly used in vitro CYP3A4 probe (50% of reported studies) in contrast to midazolam (15–20% of in vitro estimates of CYP3A4 activity) whereas nifedipine, felodipine, and erythromycin were used in less than 10% of studies. The existence of different substrate subgroups for CYP3A4 was based on correlation and cluster analysis of CYP3A4 inhibition data for a range of modifiers (Kenworthy et al., 1999). This was substantiated by others (Stresser et al., 2000) and further investigated in a number of detailed mechanistic kinetic studies (Houston et al., 2003) that indicated the existence of distinct and preferential binding domains for each substrate subgroup, namely, midazolam, testosterone, and nifedipine. Due to the substrate-differential response observed for CYP3A4, the recommended approach to CYP3A4 DDI analysis is the use of multiprobes (Tucker et al., 2001; Bjornsson et al., 2003) where the lowest inhibition constant (Ki) obtained indicates the “worst case scenario” for a potential interaction. However, an inappropriate selection of a probe substrate may lead to false positive or negative prediction of