title: Caffeine as a probe for human cytochromes P450: validation using cDNA-expression, immunoinhibition and microsomal kinetic and inhibitor techniques.
abstract: The molecular basis for the use of caffeine (CA; 1,3,7-trimethylxanthine) as a probe for specific human cytochromes P450 has been investigated. The CA 1-, 3- and 7-demethylations (to form theobromine, paraxanthine and theophylline, respectively) all followed biphasic kinetics in human liver microsomes. Mean apparent Km values for the high- and low-affinity components of the demethylations ranged from 0.13-0.31 nM and 19.2-30.0 mM, respectively. cDNA-expressed CYP1A2 catalysed all three CA demethylations, and the apparent Km for CA 3-demethylation (the major metabolic pathway in humans) by the expressed enzyme was similar to the Km for the high-affinity liver microsomal CA 3-demethylase. IC50 values for inhibition of the CA demethylations by alpha-naphthoflavone were similar for both expressed CYP1A2 and the high-affinity microsomal demethylases. Moreover, CA was a competitive inhibitor of expressed CYP1A2 catalysed phenacetin O-deethylation, with the apparent Ki (0.080 mM) closely matching the apparent Km (0.082 mM) for CA 3-demethylation by the expressed enzyme. Expressed CYP1A1 was additionally shown to catalyse the 3-demethylation of CA, although activity was lower than that observed for CYP1A2. While these data indicate that CYP1A2 is responsible for the high-affinity component of human liver CA 3-demethylation, two limitations associated with the use of CA as an in vitro probe for CYP1A2 activity have been identified: (i) CA 3-demethylation reflects hepatic CYP1A2 activity only at appropriately low substrate concentrations; and (ii) CA is a non-specific CYP1A substrate and CYP1A1 may therefore contribute to CA 3-demethylase activity in tissues in which it is expressed. An anti-CYP3A antibody essentially abolished the 8-hydroxylation of CA to form trimethyluric acid, suggesting formation of this metabolite may potentially serve as a marker of CYP3A isozyme(s) activity.
authors.affiliation: Department of Clinical Pharmacology, Flinders Medical Centre, Bedford Park, Adelaide, Australia.
authors.forename: W
authors.initials: W
authors.lastname: Tassaneeyakul
authors.forename: Z
authors.initials: Z
authors.lastname: Mohamed
authors.forename: D J
authors.initials: DJ
authors.lastname: Birkett
authors.forename: M E
authors.initials: ME
authors.lastname: McManus
authors.forename: M E
authors.initials: ME
authors.lastname: Veronese
authors.forename: R H
authors.initials: RH
authors.lastname: Tukey
authors.forename: L C
authors.initials: LC
authors.lastname: Quattrochi
authors.forename: F J
authors.initials: FJ
authors.lastname: Gonzalez
authors.forename: J O
authors.initials: JO
authors.lastname: Miners
mesh_terms: D001571:Benzoflavones / Q000494:pharmacology; D015415:Biomarkers; D002110:Caffeine / Q000378:metabolism*; D019388:Cytochrome P-450 CYP1A2; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008862:Microsomes, Liver / Q000201:enzymology; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D001571:Benzoflavones; D015415:Biomarkers; D065607:Cytochrome P-450 Enzyme Inhibitors; D002110:Caffeine; C011512:alpha-naphthoflavone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D019388:Cytochrome P-450 CYP1A2; D010089:Oxidoreductases, N-Demethylating