title: Determination of human hepatic cytochrome P4501A2 activity in vitro use of tacrine as an isoenzyme-specific probe.
abstract: Oxidative metabolism of the cognition activator tacrine (1,2,3,4-tetrahydro-9-aminoacridine) is thought to be catalyzed by cytochrome P4501A2 (CYP1A2). In this study, the use of tacrine as a specific substrate to measure CYP1A2 activity in vitro was investigated. Tacrine metabolism was assessed in 16 human liver microsomal samples. Initially, the percentage conversion of tacrine to stable metabolites (i.e. 1-, 2-, 4-, and 7-hydroxytacrine) at a single time point was correlated with levels of CYP1A2 apoprotein. Apoprotein was detected by immunoquantification using a monospecific CYP1A2 antipeptide antibody. Significant correlations were seen between CYP1A2 content and the degree of 1-hydroxylation (r = 0.81, p < 0.001), 7-hydroxylation (r = 0.70, p < 0.001), and metabolism to all stable products (r = 0.82, p < 0.001). The major metabolite formed in all livers was 1-hydroxytacrine. The conversion of tacrine to this metabolite was examined in more detail. The rate of formation varied from 19.2 pmol min-1 mg-1 to 101.0 pmol min-1 mg-1. There was a significant correlation (r = 0.84, p < 0.001) between the rate of formation and CYP1A2 levels. Tacrine metabolism was also compared with the rate of formation of 3-methylxanthine, from theophylline, a reaction previously shown to be catalyzed by CYP1A2. Significant correlations were found between 3-methylxanthine formation and all quantified tacrine metabolites. The rate of 3-methylxanthine generation also correlated with CYP1A2 apoprotein levels. It is concluded, therefore, that tacrine is a valuable probe for the determination of human hepatic CYP1A2 activity in vitro.
authors.affiliation: Department of Pharmacology and Therapeutics, University of Liverpool, UK.
authors.forename: V
authors.initials: V
authors.lastname: Spaldin
authors.forename: S
authors.initials: S
authors.lastname: Madden
authors.forename: D A
authors.initials: DA
authors.lastname: Adams
authors.forename: R J
authors.initials: RJ
authors.lastname: Edwards
authors.forename: D S
authors.initials: DS
authors.lastname: Davies
authors.forename: B K
authors.initials: BK
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D001711:Biotransformation; D019388:Cytochrome P-450 CYP1A2; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis*; D006801:Humans; D015151:Immunoblotting; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D018697:Nootropic Agents / Q000493:pharmacokinetics; D010088:Oxidoreductases / Q000032:analysis*; D010726:Phosphodiesterase Inhibitors / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D013619:Tacrine / Q000493:pharmacokinetics; D013806:Theophylline / Q000378:metabolism
chemical_list: D007527:Isoenzymes; D018697:Nootropic Agents; D010726:Phosphodiesterase Inhibitors; D013619:Tacrine; D003577:Cytochrome P-450 Enzyme System; D013806:Theophylline; D010088:Oxidoreductases; D019388:Cytochrome P-450 CYP1A2