title: In vitro evaluation of the effects of anti-fungals, benzodiazepines and non-steroidal anti-inflammatory drugs on the glucuronidation of 19-norandrosterone: implications on doping control analysis.
abstract: We have studied whether the phase II metabolism of 19-norandrosterone, the most representative metabolite of 19-nortestosterone (nandrolone), can be altered in the presence of other drugs that are not presently included on the Prohibited List of the World Anti-Doping Agency. In detail, we have evaluated the effect of non-prohibited drugs belonging to the classes of anti-fungals, benzodiazepines, and non-steroidal anti-inflammatory drugs on the glucuronidation of 19-norandrosterone. In vitro assays based on the use of either pooled human liver microsomes or specific recombinant isoforms of uridine diphosphoglucuronosyl-transferase were designed and performed to monitor the formation of 19-norandrosterone glucuronide from 19-norandrosterone. Determination of 19-norandrosterone (free and conjugated fraction) was performed by gas chromatography - mass spectrometry after sample pretreatment consisting of an enzymatic hydrolysis (performed only for the conjugated fraction), liquid/liquid extraction with tert-butylmethyl ether, and derivatization to form the trimethylsilyl derivative. In parallel, a method based on reversed-phase liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization with acquisition in selected reaction monitoring mode was also developed to identify the non-prohibited drugs considered in this study. Incubation experiments have preliminarily shown that the glucuronidation of 19-norandrosterone is principally carried out by UGT2B7 (39%) and UGT2B17 (31%). Inhibition studies have shown that the yield of the glucuronidation reaction is reduced in the presence of the anti-fungals itraconazole, ketoconazole, and miconazole, of the benzodiazepine triazolam and of the non-steroidal anti-inflammatory drugs diclofenac and ibuprofen, while no alteration was recorded in the presence of all other compounds considered in this study. Copyright © 2015 John Wiley & Sons, Ltd.
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197, Rome, Italy.|Dipartimento di Chimica e Tecnologia del Farmaco, 'Sapienza' Università di Roma, Piazzale Aldo Moro 5, 00185, Rome, Italy.
authors.forename: Amelia
authors.initials: A
authors.lastname: Palermo
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197, Rome, Italy.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Alessi
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197, Rome, Italy.|Dipartimento di Medicina Sperimentale, 'Sapienza' Università di Roma, Viale Regina Elena 324, 00161, Rome, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Botrè
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197, Rome, Italy.
authors.forename: Xavier
authors.initials: X
authors.lastname: de la Torre
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197, Rome, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Fiacco
authors.affiliation: Laboratorio Antidoping, Federazione Medico Sportiva Italiana, Largo Giulio Onesti, 1, 00197, Rome, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Mazzarino
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000378:metabolism*; D000935:Antifungal Agents / Q000378:metabolism*; D001569:Benzodiazepines / Q000378:metabolism*; D002853:Chromatography, Liquid; D004300:Doping in Sports; D004347:Drug Interactions; D004962:Estranes / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D020719:Glucuronides / Q000378:metabolism*; D014453:Glucuronosyltransferase / Q000378:metabolism; D006801:Humans; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism; D015778:Minor Histocompatibility Antigens / Q000378:metabolism; D020033:Protein Isoforms / Q000378:metabolism; D015813:Substance Abuse Detection; D053719:Tandem Mass Spectrometry
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D000935:Antifungal Agents; D004962:Estranes; D020719:Glucuronides; D015778:Minor Histocompatibility Antigens; D020033:Protein Isoforms; D001569:Benzodiazepines; C036015:19-norandrosterone; C491474:UGT2B7 protein, human; D014453:Glucuronosyltransferase; C500513:UGT2B17 protein, human
keywords: 19-norandrosterone; anti-doping analysis; anti-fungals; benzodiazepines; drug-drug interactions; masking strategy; non-steroidal anti-inflammatory drugs