title: CYP3A4 is the major CYP isoform mediating the in vitro hydroxylation and demethylation of flunitrazepam.
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 microM), CYP2B6 (K(m) = 101 microM), CYP2C9 (K(m) = 50 microM), CYP2C19 (K(m) = 60 microM), and CYP3A4 (K(m) = 155 microM), and 3-hydroxylated by cDNA-expressed CYP2A6 (K(m) = 298 microM) and CYP3A4 (K(m) = 286 microM). The 3-hydroxylation pathway was predominant in liver microsomes, accounting for more than 80% of intrinsic clearance compared with the N-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 (IC(50) = 0.11 microM) and ritonavir (IC(50) = 0.041 microM). Formation of DM FNTZ was inhibited to 40% of control velocity by 2.5 microM ketoconazole and to 30% of control by 2.5 microM ritonavir. Neither 3-OH FNTZ nor DM FNTZ formation was inhibited to less than 85% of control activity by alpha-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.
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Ave., Boston, Massachusetts, USA.
authors.forename: L M
authors.initials: LM
authors.lastname: Hesse
authors.forename: K
authors.initials: K
authors.lastname: Venkatakrishnan
authors.forename: L L
authors.initials: LL
authors.lastname: von Moltke
authors.forename: R I
authors.initials: RI
authors.lastname: Shader
authors.forename: D J
authors.initials: DJ
authors.lastname: Greenblatt
mesh_terms: D014151:Anti-Anxiety Agents / Q000378:metabolism*; D001571:Benzoflavones / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005445:Flunitrazepam / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007654:Ketoconazole / Q000494:pharmacology; D007700:Kinetics; D008745:Methylation; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009853:Omeprazole / Q000494:pharmacology; D011802:Quinidine / Q000494:pharmacology; D019438:Ritonavir / Q000494:pharmacology; D013426:Sulfaphenazole / Q000494:pharmacology; D014162:Transfection
chemical_list: D014151:Anti-Anxiety Agents; D001571:Benzoflavones; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; C069164:N-desmethylflunitrazepam; D013426:Sulfaphenazole; C011512:alpha-naphthoflavone; D005445:Flunitrazepam; C117725:3-hydroxyflunitrazepam; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; D011802:Quinidine; D009853:Omeprazole; D019438:Ritonavir; D007654:Ketoconazole