Bioactivation of Diclofenac in Vitro and in Vivo: Correlation to Electrochemical Studies
| Autor: | Tommy N. Johansen, Claus Cornett, Steen Honoré Hansen, Christian Skonberg, Ulrik Jurva, Kim G. Madsen, Jørgen Olsen |
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| Rok vydání: | 2008 |
| Předmět: |
Diclofenac
Magnetic Resonance Spectroscopy Stereochemistry Metabolite Oxidative phosphorylation Toxicology Hydroxylation chemistry.chemical_compound Tandem Mass Spectrometry In vivo Electrochemistry Animals Humans Cells Cultured Chromatography Molecular Structure Chemistry General Medicine Glutathione Metabolism Rats stomatognathic diseases Microsome Oxidation-Reduction Drug metabolism |
| Zdroj: | Chemical Research in Toxicology. 21:1107-1119 |
| ISSN: | 1520-5010 0893-228X |
| DOI: | 10.1021/tx700419d |
| Popis: | Diclofenac is widely used in the treatment of, for example, arthritis and muscle pain. The use of diclofenac has been associated with hepatotoxicity, which has been linked to the formation of reactive metabolites. Diclofenac can be metabolized to 4'-OH- and 5-OH-diclofenac, both of which are able to form quinone imines capable of reacting with, for example, GSH and nucleophilic groups in proteins. Electrochemistry has been shown to be a suitable tool for mimicking some types of oxidative drug metabolism and for studying the formation of reactive metabolites. In these studies, the electrochemical oxidation of diclofenac to a +16 Da metabolite was shown to be identical to a synthetic standard of 5-OH-diclofenac. Furthermore, two different experimental designs were investigated with respect to the electrochemical oxidation of 4'-OH- and 5-OH-diclofenac. In the first approach, the oxidized sample was collected in an aqueous solution of GSH, whereas in the other approach, GSH was added to the sample before the oxidation was performed. From these electrochemical oxidations, a range of GSH conjugates of 4'-OH- and 5-OH-diclofenac were observed and characterized by MS/MS. This allowed the development of sensitive LC-MS methods in order to detect the GSH conjugates from in vivo (rat bile) and in vitro (human liver microsomes (HLM), rat liver microsomes (RLM), and rat hepatocytes) samples. A wide range of mono-, di-, and triglutathionyl conjugates were detected in the in vitro and in vivo samples. It was also observed that 5-OH-diclofenac formed GSH conjugates with RLM and HLM without addition of NADPH, whereas GSH conjugate formation of 4'-OH-diclofenac was NADPH-dependent. This indicated that 5-OH-diclofenac was prone to auto-oxidation. The oxidation potentials of the two hydroxy metabolites were determined by cyclic voltammetry. A difference of 69 mV was observed between the two oxidation potentials, which in part may explain the extent of auto-oxidation for 5-OH-diclofenac. In conclusion, it was shown that electrochemical oxidation was capable of mimicking the metabolic hydroxylation of diclofenac to 5-OH-diclofenac. Furthermore, electrochemical oxidation was used to generate a range of GSH conjugates of 4'-OH- and 5-OH-diclofenac and a number of these conjugates were also detected in metabolism studies with microsomes (HLM/RLM) and freshly isolated rat hepatocytes, and in vivo in rat bile. |
| Databáze: | OpenAIRE |
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