Flunitrazepam metabolism by cytochrome P450S2C19 and 3A4

We have identified CYP2C19 and CYP3A4 as the principal cytochrome P450s inv olved in the metabolism of flunitrazepam to its major metabolites desmethyl flunitrazepam and 3-hydroxyflunitrazepam. Human CYP2C19 and CYP3A4 mediated the formation of desmethylflunitrazepam with K-m values of 11.1 and 108 mu M, respectively, and 3-hydroxyflunitrazepam with K-m values of 642 and 34.0 muM, respectively. In human liver microsomes (n = 4) formation of both met abolites followed biphasic kinetics. Desmethylflunitrazepam formation was i nhibited 31% by S-mephenytoin and 78% by ketoconazole, suggesting involveme nt of both CYP2C19 and CYP3A4. Formation of 3-hydroxyflunitrazepam was also significantly inhibited by ketoconazole (94%) and S-mephenytoin (18%). In support of these chemical inhibition data, antibodies directed against CYP2 C19 and CYP3A4 selectively inhibited formation of desmethylflunitrazepam by 26 and 45%, respectively, while anti-CYP3A4 antibodies reduced 3-hydroxyfl unitrazepam formation by 80%. Our data also suggest that CYP1A2, -2B6, -2C8 , -2C9, -2D6, and -2E1 are not involved in either of these metabolic pathwa ys. We estimate that the relative contributions of CYP2C19 and CYP3A4 to th e formation of desmethylflunitrazepam in vivo are 63 and 37%, respectively, at therapeutic flunitrazepam concentrations (0.03 muM). We conclude that t he polymorphic enzyme CYP2C19 importantly mediates flunitrazepam demethylat ion, which may alter the efficacy and safety of the drug, while CYP3A4 cata lyzes the formation of 3-hydroxyflunitrazepam.