TY - JOUR T1 - Diazepam Metabolism by <span class="sc">c</span>DNA-Expressed Human 2C P450<span class="sc">s</span> JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 133 LP - 139 VL - 25 IS - 2 AU - Frank Jung AU - Toby H. Richardson AU - Judy L. Raucy AU - Eric F. Johnson Y1 - 1997/02/01 UR - http://dmd.aspetjournals.org/content/25/2/133.abstract N2 - The present study provides a detailed kinetic analysis of diazepam metabolism by all four known members of the human P4502C subfamily expressed from their cDNAs in Escherichia coli. Both P4502C18 and P4502C19 were found to be low KM diazepam N-demethylases with apparentKM values of 24 ± 4 μM and 21 ± 3 μM, respectively. These values closely resemble the lowKM component of diazepamN-demethylase activity exhibited by human liver microsomes. In addition, P4502C19 also catalyzed diazepam 3-hydroxylation with aKM value of 21 ± 9 μM. Although P4502C8 was essentially inactive in catalyzing diazepam metabolism, P4502C9 catalyzed the N-demethylation with a relatively highKM of 80 ± 15 μM and an overall 3- to 6-fold lower catalytic efficiency, compared with P4502C18 and P4502C19, respectively. At a substrate concentration of 10 μM, diazepamN-demethylation in a panel of human liver microsomes was inhibited 42 ± 12% (mean ± SD, N = 6) by a polyclonal anti-CYP2C antibody. In the same experiment, 3-hydroxylation remained unaffected (&lt;10% inhibition). 1 μM of the CYP3A inhibitor ketoconazole inhibited 37 ± 19% of theN-demethylation and 86 ± 5% of 3-hydroxylation. Estimates of relative contributions to diazepamN-demethylation of P4502C9 (8 ± 4%), P4502C18 (&lt;2%), and P4502C19 (33 ± 14%) and to diazepam 3-hydroxylation of P4502C19 (9 ± 3%) based on the kinetic parameters of the recombinant enzymes and on specific contents of the individual 2C P450s determined in immunoblots are consistent with the inhibition data. In conclusion, these data confirm that both P4502C19 and P4503A are major contributors to human liver microsomal diazepamN-demethylation at low substrate concentrations, whereas P4503A is the major enzyme responsible for 3-hydroxylation. The American Society for Pharmacology and Experimental Therapeutics ER -