CAFFEINE AS A PROBE FOR HUMAN CYTOCHROMES-P450 - VALIDATION USING CDNA-EXPRESSION, IMMUNOINHIBITION AND MICROSOMAL KINETIC AND INHIBITOR TECHNIQUES

被引：109

作者：

TASSANEEYAKUL, W ^{[1
]}

MOHAMED, Z ^{[1
]}

BIRKETT, DJ ^{[1
]}

MCMANUS, ME ^{[1
]}

VERONESE, ME ^{[1
]}

TUKEY, RH ^{[1
]}

QUATTROCHI, LC ^{[1
]}

GONZALEZ, FJ ^{[1
]}

MINERS, JO ^{[1
]}

机构：

[1] FLINDERS UNIV S AUSTRALIA,MED CTR,DEPT CLIN PHARMACOL,BEDFORD PK,SA 5042,AUSTRALIA

来源：

PHARMACOGENETICS | 1992年 / 2卷 / 04期

关键词：

D O I：

10.1097/00008571-199208000-00004

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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 K(m) values for the high- and low-affinity components of the demethylations ranged from 0.13-0.31 mM and 19.2-30.0 mM, respectively. cDNA-expressed CYP1A2 catalysed all three CA demethylations, and the apparent K(m) for CA 3-demethylation (the major metabolic pathway in humans) by the expressed enzyme was similar to the K(m) 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 0-deethylation, with the apparent K(i) (0.080 mM) closely matching the apparent K(m) (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.

引用

页码：173 / 183

页数：11

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