DIAZEPAM METABOLISM BY HUMAN LIVER-MICROSOMES IS MEDIATED BY BOTH S-MEPHENYTOIN HYDROXYLASE AND CYP3A ISOFORMS

1 The primary metabolism of diazepam was studied in human liver micros omes in order to investigate the kinetics and to identify the cytochro me P450 (CYP) isoforms responsible for the formation of the main diaze pam metabolites, temazepam and N-desmethyldiazepam. 2 The formation ki netics of both metabolites were atypical and consistent with the occur rence of substrate activation. A sigmoid V-max model equivalent to the Hill equation was used to fit the data. The degree of sigmoidicity wa s greater for temazepam formation than for N-desmethyldiazepam formati on, so that the ratio of desmethyldiazepam:temazepam formation increas ed as the substrate (diazepam) concentration decreased. 3 alpha-Naphth oflavone activated both reactions but with a greater effect on temazep am formation than on N-desmethyldiazepam formation. In the presence of 25 mu M alpha-naphthoflavone the kinetics for both pathways were appr oximated by Michaelis-Menten kinetics. 4 Studies with a series of CYP isoform selective inhibitors and with an inhibitory anti-CYP2C antibod y indicated that temazepam formation was carried out mainly by CYP3A i soforms, whereas the formation of N-desmethyldiazepam was mediated by both CYP3A isoforms and S-mephenytoin hydroxylase.