Vol. 29, Issue 4, 460-465, April 2001

Flunitrazepam Metabolism by Cytochrome P450s 2C19 and 3A4

Tansel Kilicarslan, Robert L. Haining, Allan E. Rettie, Usanda Busto, Rachel F. Tyndale, and Edward M. Sellers

Departments of Pharmacology (T.K., U.B., R.F.T., E.M.S.), Psychiatry (E.M.S.), and Medicine (E.M.S.), Sunnybrook and Women's College Health Sciences Center (E.M.S.), the Center for Addiction and Mental Health (T.K., U.B., R.F.T., E.M.S.) and Faculty of Pharmacy (U.B.), University of Toronto, Toronto, Canada; and Department of Medicinal Chemistry (R.L.H., A.E.R.), University of Washington, Seattle, Washington

We have identified CYP2C19 and CYP3A4 as the principal cytochrome P450s involved in the metabolism of flunitrazepam to its major metabolites desmethylflunitrazepam and 3-hydroxyflunitrazepam. Human CYP2C19 and CYP3A4 mediated the formation of desmethylflunitrazepam with K_m values of 11.1 and 108 µM, respectively, and 3-hydroxyflunitrazepam with K_m values of 642 and 34.0 µM, respectively. In human liver microsomes (n = 4) formation of both metabolites followed biphasic kinetics. Desmethylflunitrazepam formation was inhibited 31% by S-mephenytoin and 78% by ketoconazole, suggesting involvement 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 CYP2C19 and CYP3A4 selectively inhibited formation of desmethylflunitrazepam by 26 and 45%, respectively, while anti-CYP3A4 antibodies reduced 3-hydroxyflunitrazepam formation by 80%. Our data also suggest that CYP1A2, -2B6, -2C8, -2C9, -2D6, and -2E1 are not involved in either of these metabolic pathways. We estimate that the relative contributions of CYP2C19 and CYP3A4 to the formation of desmethylflunitrazepam in vivo are 63 and 37%, respectively, at therapeutic flunitrazepam concentrations (0.03 µM). We conclude that the polymorphic enzyme CYP2C19 importantly mediates flunitrazepam demethylation, which may alter the efficacy and safety of the drug, while CYP3A4 catalyzes the formation of 3-hydroxyflunitrazepam.