title: Flunitrazepam oxidative metabolism in human liver microsomes: involvement of CYP2C19 and CYP3A4.
abstract: The aims were to examine the kinetics of the oxidative metabolism of flunitrazepam in vitro when flunitrazepam was dissolved in dimethylformamide and acetonitrile, and to determine which cytochrome P450 isoform(s) are involved. The kinetics of the formations of 3'-hydroxyflunitrazepam and desmethyl-flunitrazepam were non-linear and best estimated using the Hill equation. Inhibition of their formation was studied using specific chemical inhibitors, expressed enzyme systems and specific antibodies. Ks, Vmax, Clmax and n (slope factor) for the formation of 3'-hydroxyflunitrazepam and desmethylflunitrazepam had ranges of 165-338 and 179 391 microM, 22-81 and 3-10 nmol x mg protein(-1) x h(-1), 6-17 and 0.9-1.9 microl x mg protein(-1) x h(-1), and 2.3-3.6 and 1.6-2.6 respectively when dimethylformamide was the organic solvent. When acetonitrile was the solvent, Ks, Vmax, Clmax and n (slope factor) for the formation of 3'-hydroxyflunitrazepam and desmethylflunitrazepam had ranges of 173-231 and 74-597 microM, 35-198 and 2.7-48 nmol.mg protein(-1) x h(-1), 1347 and 0.7-6.3 microl.mg protein(-1) x h(-1), and 1.5-3.6 and 1.1-2.7 respectively. CYP2C19, CYP3A4 and CYP1A2 mediated the formation of both 3'-hydroxyflunitrazepam and desmethylflunitrazepam. Investigators need carefully to consider the choice of organic solvent to avoid false CYP identification.
authors.affiliation: Department of Clinical and Experimental Pharmacology, University of Adelaide, Australia. jcoller@medicine.adelaide.edu.au
authors.forename: J K
authors.initials: JK
authors.lastname: Coller
authors.forename: A A
authors.initials: AA
authors.lastname: Somogyi
authors.forename: F
authors.initials: F
authors.lastname: Bochner
mesh_terms: D000097:Acetonitriles; D000328:Adult; D000368:Aged; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001189:Aryl Hydrocarbon Hydroxylases; D001571:Benzoflavones / Q000494:pharmacology; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004126:Dimethylformamide; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005445:Flunitrazepam / Q000378:metabolism*; D005838:Genotype; D006801:Humans; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008875:Middle Aged; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D012997:Solvents
chemical_list: D000097:Acetonitriles; D000911:Antibodies, Monoclonal; D001571:Benzoflavones; D012997:Solvents; C011512:alpha-naphthoflavone; D005445:Flunitrazepam; D004126:Dimethylformamide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C045793:CYP2C19 protein, human; C104464:CYP3A protein, human; D065731:Cytochrome P-450 CYP2C19; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C032159:acetonitrile