title: Biotransformation of amitriptyline by Cunninghamella elegans.
abstract: A fungal biotransformation system as an in vitro model for mammalian drug metabolism was investigated. Amitriptyline, a widely used antidepressant, was effectively biotransformed within 72 hr by the filamentous fungus, Cunninghamella elegans. Eight major metabolites in HPLC elution order (11-hydroxyamitriptyline N-oxide, 11-hydroxynortriptyline, 11-hydroxyamitriptyline, 10-hydroxyamitriptyline, 3-hydroxyamitriptyline, 2-hydroxyamitriptyline, nortriptyline, and amitriptyline N-oxide) were produced at estimated molar ratios of 2:1:10:0.6:0.1:1.2.5:0.5, respectively. These metabolites were isolated by HPLC and identified by UV/MS analyses, as well as NMR spectroscopic analysis for most of these metabolites. In some cases, they were also compared with authentic standards. Glucose, culture age, and substrate concentration significantly affected the extent of amitriptyline metabolism. Kinetic studies indicated that nortriptyline and 11-hydroxyamitriptyline were produced as initial major metabolites. The hydroxylated metabolite was excreted from mycelia, but amitriptyline and its N-demethylated metabolite, nortriptyline, were not. An 18O2 labeling experiment showed that the oxygen atoms in 11-hydroxyamitriptyline and 2-hydroxyamitriptyline were derived from molecular oxygen. The cytochrome P450 inhibitors SKF 525-A (1.5 mM), metyrapone (2.0 mM), and 1-aminobenzotriazole (1.0 mM) inhibited the biotransformations of amitriptyline by 50, 75, and 95%, respectively. A microsomal preparation was shown to catalyze the 11-hydroxylation of amitriptyline, which was inhibited by SKF 525-A and carbon monoxide. The similarities of amitriptyline metabolism in C. elegans and in humans and rats are discussed.
authors.affiliation: National Center for Toxicological Research, U.S. Food and Drug Administration.
authors.forename: D
authors.initials: D
authors.lastname: Zhang
authors.forename: F E
authors.initials: FE
authors.lastname: Evans
authors.forename: J P
authors.initials: JP
authors.lastname: Freeman
authors.forename: B
authors.initials: B
authors.lastname: Duhart
authors.forename: C E
authors.initials: CE
authors.lastname: Cerniglia
mesh_terms: D000639:Amitriptyline / Q000493:pharmacokinetics*; D000929:Antidepressive Agents, Tricyclic / Q000493:pharmacokinetics*; D001711:Biotransformation; D002474:Cell-Free System; D002851:Chromatography, High Pressure Liquid; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009090:Mucorales / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet
chemical_list: D000929:Antidepressive Agents, Tricyclic; D065607:Cytochrome P-450 Enzyme Inhibitors; D000639:Amitriptyline; D003577:Cytochrome P-450 Enzyme System; D005947:Glucose