Abstract

The kinetics of flunitrazepam (FNTZ) N-demethylation to desmethylflunitrazepam (DM FNTZ), and 3-hydroxylation to 3-hydroxyflunitrazepam (3-OH FNTZ), were studied in human liver microsomes and in microsomes containing heterologously expressed individual human CYPs. FNTZ was N-demethylated by cDNA-expressed CYP2A6 (K_m = 1921 μM), CYP2B6 (K_m = 101 μM), CYP2C9 (K_m = 50 μM), CYP2C19 (K_m = 60 μM), and CYP3A4 (K_m = 155 μM), and 3-hydroxylated by cDNA-expressed CYP2A6 (K_m = 298 μM) and CYP3A4 (K_m = 286 μM). The 3-hydroxylation pathway was predominant in liver microsomes, accounting for more than 80% of intrinsic clearance compared with theN-demethylation pathway. After adjusting for estimated relative abundance, CYP3A accounted for the majority of intrinsic clearance via both pathways. This finding was supported by chemical inhibition studies in human liver microsomes. Formation of 3-OH FNTZ was reduced to 10% or less of control values by ketoconazole (IC₅₀ = 0.11 μM) and ritonavir (IC₅₀ = 0.041 μM). Formation of DM FNTZ was inhibited to 40% of control velocity by 2.5 μM ketoconazole and to 30% of control by 2.5 μM ritonavir. Neither 3-OH FNTZ nor DM FNTZ formation was inhibited to less than 85% of control activity by α-naphthoflavone (CYP1A2), sulfaphenazole (CYP2C9), omeprazole (CYP2C19), or quinidine (CYP2D6). Thus, CYP-dependent FNTZ biotransformation, like that of many benzodiazepine derivatives, is mediated mainly by CYP3A. Clinical interactions of FNTZ with CYP3A inhibitors can be anticipated.