Evaluation of cytochrome P450 probe substrates commonly used by the pharmaceutical industry to study in vitro drug interactions

Abstract

Pharmaceutical industry investigators routinely evaluate the potential for a new drug to modify cytochrome p450 (p450) activities by determining the effect of the drug on in vitro probe reactions that represent activity of specific p450 enzymes. The in vitro findings obtained with one probe substrate are usually extrapolated to the compound's potential to affect all substrates of the same enzyme. Due to this practice, it is important to use the right probe substrate and to conduct the experiment under optimal conditions. Surveys conducted by reviewers in CDER indicated that the most common in vitro probe reactions used by industry investigators include the following: phenacetin O-deethylation for CYP1A2, coumarin 7-hydroxylation for CYP2A6, 7-ethoxy-4-trifluoromethyl coumarin O-dealkylation for CYP2B6, tolbutamide 4'-hydroxylation for CYP2C9, S-mephenytoin 4-hydroxylation for CYP2C19, bufuralol 1'-hydroxylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, and testosterone 6 beta-hydroxylation for CYP3A4. We reviewed the validation information in the literature on these reactions and other frequently used reactions, including caffeine N3-demethylation for CYP1A2, S-mephenytoin N-demethylation for CYP2B6, S-warfarin 7'-hydroxylation for CYP2C9, dextromethorphan O-demethylation for CYP2D6, and midazolam 1'-hydroxylation for CYP3A4. The available information indicates that we need to continue the search for better probe substrates for some enzymes. For CYP3A4-based drug interactions it may be necessary to evaluate two or more probe substrates. In many cases, the probe reaction represents a particular enzyme activity only under specific experimental conditions. Investigators must consider appropriateness of probe substrates and experimental conditions when conducting in vitro drug interaction studies and when extrapolating the results to in vivo situations.