Utilization of CYP3A‐Specific Inhibitors to Study Cytochrome P450 CYP3A7 Activity in Neonatal Human Liver Microsomes

Abstract

CYP3A7 is a member of the cytochrome P450 (CYP) 3A enzyme sub‐family that accounts for ~50% of the total CYP content in neonate and fetal livers. The amount of other CYP3A enzymes, CYP3A4 and CYP3A5, expressed in neonates is low but highly variable, and therefore, the activity of individual enzyme isoforms is difficult to differentiate. Recent studies report that CYP3cide, clobetasol propionate, and azamulin are specific inhibitors for CYP3A4, CYP3A5, and CYP3A4/5, respectively. However, the inhibitory effects towards CYP3A7 were not investigated for the latter two compounds, and the use of these compounds to study CYP3A7 activity has not been reported/investigated. Thus, we sought to characterize these compounds further and utilize them to isolate and study the activity of CYP3A7 in neonatal human liver microsomes. We have examined three substrate probes—dibenzylfluorescein, luciferin‐PPXE, and midazolam—to determine the IC50 values and mechanism‐based inactivation parameters (Ki, Kinact) of the purported isoform specific inhibitors. We discovered that azamulin was unable to selectively inhibit any of the CYP3As, but CYP3cide and clobetasol propionate exhibited IC50values for CYP3A4 and CYP3A5 two to three orders of magnitude lower than CYP3A7. CYP3cide and clobetasol propionate were further tested in mixtures of CYP3A4, CYP3A5, and CYP3A7 to mimic the neonatal human liver microsome environment and ascertain their effectiveness in isolating CYP3A7 activity. We were able to inhibit CYP3A4 without disturbing CYP3A7 activity using CYP3cide at concentrations below 5 μM. The majority of CYP3A5 activity was inhibited using low clobetasol propionate concentrations, but using higher concentrations also inhibited CYP3A7 activity due to its lower selectivity. These findings provide valuable insight regarding testing conditions to investigate the metabolism of new drug candidates and help determine drug safety in neonates.