UTILITY OF RECOMBINANT ENZYME KINETICS IN PREDICTION OF HUMAN CLEARANCE : IMPACT OF VARIABILITY , CYP 3 A 5 , AND CYP 2 C 19 ON CYP 3 A 4 PROBE SUBSTRATES

A systematic kinetic analysis of the metabolism of five benzodiazepines (low to high clearance compounds) was performed in CYP3A4, CYP3A5, and CYP2C19 baculovirus-expressed recombinant systems. The data obtained in the expression systems were scaled and compared with human liver microsomal predicted clearance and observed in vivo values, using either cytochrome P450 relative activity factors (RAFs) or the relative abundance approach. Interindividual variability, both in content (CYP3A4, CYP3A5) and activity (CYP3A4, CYP3A5, and CYP2C19), were incorporated in the clearance prediction by bootstrap analysis. These resampling Monte Carlo-based simulations were performed to justify any distribution assumptions in the generated range of the predicted clearance due to a limited sample size. This approach allowed extrapolation of the recombinant clearance data to specific population groups and investigation of the role of “minor” forms like CYP3A5 and CYP2C19 in comparison to the most prolific CYP3A4. The use of quinidine 3-hydroxylation and alprazolam 1 hydroxylation as RAF markers for CYP3A4 and CYP3A5 activity, respectively, and the incorporation of variability improved the clearance prediction of the selected benzodiazepines (apart from flunitrazepam) to within 2-fold of the in vivo value. Clearance estimates from the immunoquantified protein levels were approximately 8-fold lower in comparison to the RAF approach. The differences observed in the benzodiazepine metabolite pathway ratios between CYP3A4 and CYP3A5, particularly for 1 to 4-hydroxymidazolam and alprazolam, provided a useful measure of interindividual differences within the CYP3A family. Recombinant cytochrome P450 enzymes (rP450s) are a convenient and successful in vitro tool for the identification of the metabolic pathway(s) and the quantification of the individual P450 enzyme(s) contributing to the overall metabolism of a drug (Crespi, 1995; Ito et al., 1998, Nakajima et al., 2002). In a large number of cases, CYP3A enzymes are found to be the enzyme of paramount importance. However, recent studies have placed emphasis on other CYP3A family members, notably CYP3A5, which demonstrate either similar or reduced metabolic activity in comparison to CYP3A4 (Williams et al., 2002; Patki et al., 2003), and the clinical significance of hepatic and intestinal CYP3A5 is currently under debate (Paine et al., 1997; Lin et al., 2002; Thummel, 2003; Westlind-Johnsson et al., 2003; Wong et al., 2004; Xie et al., 2004). Recombinant enzymes also represent an alternative in vitro metabolic system to hepatic microsomes or hepatocytes to predict the in vivo clearance of drugs (Ito et al., 1998; Hirota et al., 2001). This approach offers an advantage in allowing incorporation and customization of interindividual variation of P450 expression, frequently a confounding element in human prediction from hepatic microsomes, particularly for CYP3A substrates. In many microsomal prediction studies, interindividual variability issues are ignored because predictions are based on kinetic data generated in a limited number of livers or using a “representative” liver pool rather than a characterized liver bank. Approaches to introduce population variability in P450 expression into in vivo predictions from hepatic tissues have yet to be