Metabolic Activation of Diclofenac by Human Cytochrome P450 3A4: Role of 5-Hydroxydiclofenac
| Autor: | Margaret R. Davis, Michael R. Marchick, George A. Doss, Lance R. Pohl, Sijiu Shen |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Male
Diclofenac Stereochemistry Toxicology Sulfaphenazole Gas Chromatography-Mass Spectrometry Mixed Function Oxygenases Rats Sprague-Dawley chemistry.chemical_compound Cytochrome P-450 Enzyme System Cytochrome P-450 CYP3A medicine Animals Humans Troleandomycin chemistry.chemical_classification biology Anti-Inflammatory Agents Non-Steroidal Cytochrome P450 General Medicine Metabolism Glutathione Rats stomatognathic diseases Enzyme Steroid 16-alpha-Hydroxylase chemistry Biochemistry Steroid Hydroxylases Microsomes Liver Microsome biology.protein Aryl Hydrocarbon Hydroxylases medicine.drug |
| Zdroj: | Chemical Research in Toxicology. 12:214-222 |
| ISSN: | 1520-5010 0893-228X |
| Popis: | Cytochrome P450 2C11 in rats was recently found to metabolize diclofenac into a highly reactive product that covalently bound to this enzyme before it could diffuse away and react with other proteins. To determine whether cytochromes P450 in human liver could catalyze a similar reaction, we have studied the covalent binding of diclofenac in vitro to liver microsomes of 16 individuals. Only three of 16 samples were found by immunoblot analysis to activate diclofenac appreciably to form protein adducts in a NADPH-dependent pathway. Cytochrome P450 2C9, which catalyzes the major route of oxidative metabolism of diclofenac to produce 4'-hydroxydiclofenac, did not appear to be responsible for the formation of the protein adducts, because sulfaphenazole, an inhibitor of this enzyme, did not affect protein adduct formation. In contrast, troleandomycin, an inhibitor of P450 3A4, inhibited both protein adduct formation and 5-hydroxylation of diclofenac. These findings were confirmed with the use of baculovirus-expressed human P450 2C9 and P450 3A4. One possible reactive intermediate that would be expected to bind covalently to liver proteins was the p-benzoquinone imine derivative of 5-hydroxydiclofenac. This product was formed by an apparent metal-catalyzed oxidation of 5-hydroxydiclofenac that was inhibited by EDTA, glutathione, and NADPH. The p-benzoquinone imine decomposition product bound covalently to human liver microsomes in vitro in a reaction that was inhibited by GSH. In contrast, GSH did not prevent the covalent binding of diclofenac to human liver microsomes. These results suggest that for appreciable P450-mediated bioactivation of diclofenac to occur in vivo, an individual may have to have both high activities of P450 3A4 and perhaps low activities of other enzymes that catalyze competing pathways of metabolism of diclofenac. Moreover, the p-benzoquinone imine derivative of 5-hydroxydiclofenac probably has a role in covalent binding in the liver only under the conditions where levels of NADPH, GSH, and other reducing agents would be expected to be low. |
| Databáze: | OpenAIRE |
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