Regioselective biotransformation of midazolam by members of the human cytochrome P450 3A (CYP3A) subfamily.
The capabilities of cytochrome P4503A4 (CYP3A4), CYP3A5, and fetal hepatic microsomes containing CYP3A7 to metabolize midazolam were investigated using human hepatic microsomes and purified CYP3A4 and CYP3A5. Under initial rate conditions and high substrate concentration (400 microM midazolam), variability among eighteen human liver microsomal samples was 30- and 16- fold for 1'- and 4-hydroxylation of midazolam, respectively. Exclusion of two samples isolated from patients previously administered 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'-hydroxymidazolam and 4-hydroxymidazolam formation rates. The rates of formation of 4-hydroxymidazolam and 1'-hydroxymidazolam from midazolam by adult samples containing only CYP3A4 and by fetal liver samples were highly correlated (r2 = 0.99 and 0.97, P < 0.01, respectively). The rates of formation of 1'-hydroxymidazolam and 4-hydroxymidazolam from midazolam (400 microM) by adult samples that contained only CYP3A4 were correlated significantly (P < 0.01) with the ability of the samples to N-demethylate erythromycin (r2 = 0.95 and 0.92, respectively). 6 beta-hydroxylate testosterone (r2 = 0.96 and 0.96, respectively), and the CYP3A4 content of the samples (r2 = 0.89 and 0.86, respectively). Microsomal samples containing CYP3A5 in addition to CYP3A4 exhibited a significantly greater ratio of 1'-hydroxymidazolam to 4-hydroxymidazolam compared with samples containing only CYP3A4 or CYP3A7 (P < 0.001). Purified CYP3A5 in a reconstituted system, consisting of dilauroylphosphatidylcholine, cytochrome b5, 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 conclusion, CYP3A4, CYP3A5, and fetal microsomes containing CYP3A7 catalyze 1'- and 4-hydroxylation of midazolam with the ratio of these metabolites indicative of the CYP3A form.
Gorski
J C
JC
Department of Pharmacy Practice, School of Pharmacy, Purdue University, West Lafayette, IN 47907.
Hall
S D
SD
Jones
D R
DR
VandenBranden
M
M
Wrighton
S A
SA
D001711:Biotransformation; D019392:Cytochrome P-450 CYP2E1; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D005333:Fetus / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology*; D008874:Midazolam / Q000031:analogs & derivatives / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology
D007527:Isoenzymes; C042940:4-hydroxymidazolam; D003577:Cytochrome P-450 Enzyme System; C040081:1-hydroxymethylmidazolam; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; D008874:Midazolam