REGIOSELECTIVE BIOTRANSFORMATION OF MIDAZOLAM BY MEMBERS OF THE HUMANCYTOCHROME-P450-3A (CYP3A) SUBFAMILY

The capabilities of cytochrome P4503A4 (CYP3A4), CYP3A5, and fetal hep atic microsomes containing CYP3A7 to metabolize midazolam were investi gated using human hepatic microsomes and purified CYP3A4 and CYP3A5. U nder initial rate conditions and high substrate concentration (400 mu M midazolam), variability among eighteen human liver microsomal sample s was 30- and 16- fold far 1'- and 4-hydroxylation of midazolam, respe ctively. Exclusion of two samples isolated from patients previously ad ministered barbiturates reduced the inter-individual variability to 10 .5- and 6.0-fold for 1'- and 4-hydroxylation, respectively. Six fetal hepatic microsomal samples showed 10-fold variation in both 1'-hydroxy midazolam and 4-hydroxymidazolam formation rates. The rates of formati on of 4-hydroxymidazolam and 1'-hydroxymidazolam from midazolam by adu lt samples containing only CYP3A4 and by fetal liver samples were high ly correlated (r(2) = 0.99 and 0.97, P < 0.01, respectively). The rate s of formation of 1'-hydroxymidazolam and 4-hydroxymidazolam from mida zolam (400 mu M) by adult samples that contained only CYP3A4 were corr elated significantly (P < 0.01) with the ability of the samples to N-d emethylate erythromycin (r(2) = 0.95 and 0.92, respectively), 6 beta-h ydroxylate testosterone (r(2) = 0.96 and 0.96, respectively), and the CYP3A4 content of the samples (r(2) = 0.89 and 0.86, respectively). Mi crosomal samples containing CYP3A5 in addition to CYP3A4 exhibited a s ignificantly greater ratio of 1'-hydroxymidazolam to 4-hydroxymidazola m compared with samples containing only CYP3A4 or CYP3A7 (P < 0.001). Purified CYP3A5 in a reconstituted system, consisting of dilauroylphos phatidylcholine, cytochrome b(5), and NADPH-cytochrome P450 reductase, and an NADPH-regenerating system displayed a 2-fold greater rate of 1 '-hydroxymidazolam formation and a similar rate of 4-hydroxymidazolam formation compared with a reconstituted system with CYP3A4. In conclus ion, CYP3A4, CYP3A5, and fetal microsomes containing CYP3A7 catalyze 1 '- and 4-hydroxylation of midazolam with the ratio of these metabolite s indicative of the CYP3A form.