CYP3A4 is the major CYP isoform mediating the in vitro hydroxylation and demethylation of flunitrazepam

The kinetics of flunitrazepam (FNTZ) N-demethylation to desmethylflunitraze pam (DM FNTZ), and 3-hydroxylation to 3-hydroxyflunitrazepam (3-OH FNTZ), w ere studied in human liver microsomes and in microsomes containing heterolo gously expressed individual human CYPs. FNTZ was N-demethylated by cDNA-exp ressed CYP2A6 (K-m = 1921 muM), CYP2B6 (K-m = 101 muM), CYP2C9 (K-m = 50 mu M), CYP2C19 (K-m = 60 muM), and CYP3A4 (K-m = 155 muM), and 3-hydroxylated by cDNA-expressed CYP2A6 (K-m = 298 muM) and CYP3A4 (K-m = 286 muM). The 3- hydroxylation pathway was predominant in liver microsomes, accounting for m ore than 80% of intrinsic clearance compared with the N-demethylation pathw ay. After adjusting for estimated relative abundance, CYP3A accounted for t he majority of intrinsic clearance via both pathways. This finding was supp orted by chemical inhibition studies in human liver microsomes. Formation o f 3-OH FNTZ was reduced to 10% or less of control values by ketoconazole (I C50 = 0.11 muM) and ritonavir (IC50 = 0.041 muM). Formation of DM FNTZ was inhibited to 40% of control velocity by 2.5 muM ketoconazole and to 30% of control by 2.5 mM ritonavir. Neither 3-OH FNTZ nor DM FNTZ formation was in hibited to less than 85% of control activity by alpha -naphthoflavone (CYP1 A2), sulfaphenazole (CYP2C9), omeprazole (CYP2C19), or quinidine (CYP2D6). Thus, CYP-dependent FNTZ biotransformation, like that of many benzodiazepin e derivatives, is mediated mainly by CYP3A. Clinical interactions of FNTZ w ith CYP3A inhibitors can be anticipated.