Comparison of Cytochrome P450 3A4 and 3A7 with Azole Inhibitors

Cytochrome P450 (CYP) 3A enzymes metabolize up to 50% of human drugs. While CYP3A4 is the major adult enzyme, CYP3A7 is the major form through 6–12 months of development. Though 87% identical and frequently assumed to be functionally very similar, it has recently become clear that there are some significant differences between CYP3A4 and CYP3A7 that may affect fetal and infant drug metabolism. This knowledge gap motivates a direct study of CYP3A7 metabolism and inhibition to better inform drug usage in very young patients. Azoles can be effective for common fungal infections in infants, but can also be rather nonselective CYP inhibitors. In addition to the clinical relevance of these compounds, azoles constitute a useful series of CYP3A active site probes. This study systematically evaluated the interactions of a series of azoles with purified, recombinant human CYP3A4 and CYP3A7. Binding studies revealed that each of the following azoles demonstrated spectral features typical for direct coordination to the heme iron (type II binding) with parallel rank order affinities for the two enzymes: from subnanomolar K d values for clotrimazole, to moderate affinity for liarozole and ketoconazole, and low affinity for imidazole. In contrast, fluconazole and fosfluconazole demonstrated more unusual binding characteristics that differed between the two CYP3A enzymes. Fluconazole binding to CYP3A7 resulted in only increasing absorbance, migrating at higher concentrations to wavelengths associated with heme coordination. In contrast, fluconazole binding to CYP3A4 was characteristic of water displacement from the heme (type I) at low concentrations, but converted to heme coordination at high concentrations, suggesting that CYP3A4 may bind fluconazole in different orientations, perhaps depending on whether one fluconazole molecule or two are bound in the active site simultaneously. This would be consistent with the established capacity of CYP3A4 to bind more than one azole molecule simultaneously. Fosfluconazole, a more water‐soluble phosphorylated version of fluconazole, demonstrated a red‐shifted type I spectrum with CYP3A4, but with CYP3A7 yielded only increasing absorbance consistent with water displacement from the iron. Overall, these observations establish differences between CYP3A7 and CYP3A4 interactions with fluconazole and fosfluconazole, prompting further evaluation by comparative inhibition studies and structural studies. Such differences may underlie differential metabolism of common drugs at different life stages and inform dosing in infants vs. adults. Support or Funding Information National Institutes of Health R37GM076343 (EES)