title: CYP2C9-mediated metabolic activation of losartan detected by a highly sensitive cell-based screening assay.
abstract: Drug-induced hepatotoxicity is a major problem in drug development, and reactive metabolites generated by cytochrome P450s are suggested to be one of the causes. CYP2C9 is one of the major enzymes in hepatic drug metabolism. In the present study, we developed a highly sensitive cell-based screening system for CYP2C9-mediated metabolic activation using an adenovirus vector expressing CYP2C9 (AdCYP2C9). Human hepatocarcinoma HepG2 cells infected with our constructed AdCYP2C9 for 2 days at multiplicity of infection 10 showed significantly higher diclofenac 4'-hydroxylase activity than human hepatocytes. AdCYP2C9-infected cells were treated with several hepatotoxic drugs, resulting in a significant increase in cytotoxicity by treatment with losartan, benzbromarone, and tienilic acid. Metabolic activation of losartan by CYP2C9 has never been reported, although the metabolic activations of benzbromarone and tienilic acid have been reported. To identify the reactive metabolites of losartan, the semicarbazide adducts of losartan were investigated by liquid chromatography-tandem mass spectrometry. Two CYP2C9-specific semicarbazide adducts of losartan (S1 and S2) were detected. S2 adduct formation suggested that a reactive metabolite was produced from the aldehyde metabolite E3179, but a possible metabolite from S1 adduct formation was not produced via E3179. In conclusion, a highly sensitive cell-based assay to evaluate CYP2C9-mediated metabolic activation was established, and we found for the first time that CYP2C9 is involved in the metabolic activation of losartan. This cell-based assay system would be useful for evaluating drug-induced cytotoxicity caused by human CYP2C9.
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Iwamura
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Fukami
authors.forename: Hiroko
authors.initials: H
authors.lastname: Hosomi
authors.forename: Miki
authors.initials: M
authors.lastname: Nakajima
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yokoi
mesh_terms: D000959:Antihypertensive Agents / Q000378:metabolism* / Q000494:pharmacology / Q000633:toxicity; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001553:Benzbromarone / Q000378:metabolism; D001711:Biotransformation; D016861:Cyclooxygenase Inhibitors / Q000378:metabolism; D065729:Cytochrome P-450 CYP2C9; D004008:Diclofenac / Q000378:metabolism; D057809:HEK293 Cells; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D019808:Losartan / Q000378:metabolism* / Q000494:pharmacology / Q000633:toxicity; D051267:NF-E2-Related Factor 2 / Q000378:metabolism; D012663:Semicarbazides / Q000378:metabolism; D012680:Sensitivity and Specificity; D013997:Time Factors; D014528:Uricosuric Agents / Q000378:metabolism
chemical_list: D000959:Antihypertensive Agents; D016861:Cyclooxygenase Inhibitors; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D012663:Semicarbazides; D014528:Uricosuric Agents; D004008:Diclofenac; D001553:Benzbromarone; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; D019808:Losartan