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Comparison of the metabolism of mephedrone in cultured and in freshly isolated primary rat hepatocytes

Alanazi, Ibrahim and Grant, M. Helen and Watson, David and Henderson, Catherine (2016) Comparison of the metabolism of mephedrone in cultured and in freshly isolated primary rat hepatocytes. Applied in Vitro Toxicology, 2 (4). p. 241. ISSN 2332-1539

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Mephedrone (4-MMC) is a drug of abuse, which was controlled in 2010, and has been associated with several fatalities among users. In this study, the in vitro metabolism of 4-MMC in cultured Sprague-Dawley rat hepatocytes was characterised and Phase I and II metabolites quantified after specific periods of time in culture - 6, 24, and 48 hours. The cells were extracted and metabolites analysed using zwitterionic hydrophilic interaction (ZIC(r)-pHILIC) column and an exactive high resolution mass spectrometer instrument. Metabolism of 4-MMC yielded in 14 metabolites. These metabolites were structurally characterised on the basis of accurate mass analyses having been previously characterized by LC-MSn. The major identified metabolic routes for 4-MMC were found to be (i) 4’-methyl group oxidation and (ii) b-keto group reduction. The metabolism was compared to the published data of the mephedrone metabolism in freshly isolated rat hepatocytes. Three metabolites were not identified in culture compared to the previously published metabolism data in freshly isolated cells. Metabolite 4-carboxy-mephedrone was found to be the most prominent metabolite after 6 and 24 hours incubation, whereas nor-mephedrone was the predominant metabolite after 48 hours incubation. These data suggest that primary cultures of rat hepatocytes can be used to screen for metabolites of drugs like mephedrone to find out if the toxicity observed in users is related to formation of reactive metabolites and that the hepatocytes maintain their metabolic competency after 48 hours in culture.