title: Human arylacetamide deacetylase hydrolyzes ketoconazole to trigger hepatocellular toxicity.
abstract: Ketoconazole (KC), an antifungal agent, rarely causes severe liver injury when orally administered. It has been reported that KC is mainly hydrolyzed to N-deacetyl ketoconazole (DAK), followed by the N-hydroxylation of DAK by flavin-containing monooxygenase (FMO). Although the metabolism of KC has been considered to be associated with hepatotoxicity, the responsible enzyme(s) remain unknown. The purpose of this study was to identify the responsible enzyme(s) for KC hydrolysis in humans and to clarify their relevance to KC-induced toxicity. Kinetic analysis and inhibition studies using human liver microsomes (HLM) and recombinant enzymes revealed that human arylacetamide deacetylase (AADAC) is responsible for KC hydrolysis to form DAK, and confirmed that FMO3 is the enzyme responsible for DAK N-hydroxylation. In HLM, the clearance of KC hydrolysis occurred to the same extent as DAK N-hydroxylation, which indicates that both processes are not rate-limiting pathways. Cytotoxicity of KC and DAK was evaluated using HepaRG cells and human primary hepatocytes. Treatment of HepaRG cells with DAK for 24h showed cytotoxicity in a dose-dependent manner, whereas treatment with KC did not show due to the low expression of AADAC. Overexpression of AADAC in HepaRG cells with an adenovirus expression system elicited the cytotoxicity of KC. Cytotoxicity of KC in human primary hepatocytes was attenuated by diisopropylfluorophosphate, an AADAC inhibitor. In conclusion, the present study demonstrated that human AADAC hydrolyzes KC to trigger hepatocellular toxicity.
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan. Electronic address: tatsuki@p.kanazawa-u.ac.jp.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Fukami
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan.
authors.forename: Azumi
authors.initials: A
authors.lastname: Iida
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan.
authors.forename: Keigo
authors.initials: K
authors.lastname: Konishi
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Nakajima
mesh_terms: D065767:Activation, Metabolic / Q000187:drug effects; D000935:Antifungal Agents / Q000009:adverse effects / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D056486:Chemical and Drug Induced Liver Injury / Q000201:enzymology* / Q000378:metabolism / Q000473:pathology; D065692:Cytochrome P-450 CYP3A Inhibitors / Q000009:adverse effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D006900:Hydroxylation / Q000187:drug effects; D007531:Isoflurophate / Q000494:pharmacology; D007654:Ketoconazole / Q000009:adverse effects / Q000031:analogs & derivatives / Q000378:metabolism* / Q000633:toxicity; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008875:Middle Aged; D010105:Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D000935:Antifungal Agents; D065692:Cytochrome P-450 CYP3A Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D007531:Isoflurophate; C035996:deacylketoconazole; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); C569302:AADAC protein, human; D002265:Carboxylic Ester Hydrolases; D007654:Ketoconazole
keywords: AADAC; Drug hydrolysis; FMO3; Hepatocellular toxicity; Ketoconazole