RESUMO
Zebrafish (Danio rerio) water tank (ZWT) approach was investigated as an alternative model for metabolism studies based on six different experiments with four model compounds. Sibutramine was applied for the multivariate optimization of ZWT conditions, also for the comparison of the metabolism among ZWT, humans and mice, beyond for the role of CYP2B6 in ZWT. After the optimization, 18 fish and 168 hours of experiments is the minimum requirement for a relevant panel of biotransformation products. A comparison among the species resulted in the observation of the same hydroxylated metabolites, with differences in metabolites concentration ratio. However, the ZWT allowed tuning of the conditions to obtain a specific metabolic profile, depending on the need. In addition, by utilizing CYP2B6 inhibition, a relevant ZWT pathway for the demethylation of drugs was determined. The stereospecificity of the ZWT metabolism was investigated using selegiline and no racemization or inversion transformations were observed. Moreover, the investigation of metabolism of cannabimimetics was performed using JWH-073 and the metabolites observed are the same described for humans, except for the hydroxylation at the indol group, which was explained by the absence of CYP2C9 orthologs in zebrafish. Finally, hexarelin was used as a model to evaluate studies by ZWT for drugs with low stability. As a result, hexarelin displays a very fast metabolization in ZWT conditions and all the metabolites described for human were observed in ZWT. Therefore, the appropriate conditions, merits, and relevant limitations to conduct ZWT experiments for the investigation of drug metabolism are described.
Assuntos
Preparações Farmacêuticas/metabolismo , Peixe-Zebra/metabolismo , Adulto , Animais , Antidepressivos/metabolismo , Antidepressivos/urina , Biotransformação , Ciclobutanos/metabolismo , Ciclobutanos/urina , Citocromo P-450 CYP2B6/metabolismo , Inibidores do Citocromo P-450 CYP2B6/farmacologia , Feminino , Humanos , Hidroxilação , Indóis/metabolismo , Indóis/urina , Masculino , Camundongos , Modelos Animais , Naftalenos/metabolismo , Naftalenos/urina , Oligopeptídeos/metabolismo , Oligopeptídeos/urina , Preparações Farmacêuticas/urina , Selegilina/metabolismo , Selegilina/urina , Peixe-Zebra/urina , Proteínas de Peixe-Zebra/metabolismoRESUMO
A method for identifying the metabolites of sibutramine 1-(4(chlorophenyl)-N,N-dimethyl-alpha-(2-methylpropyl))cyclobutanemethanamine) in urine, utilizing a double derivatization strategy, with N-methyl-N-(trimethylsilyl)-trifluoroacetamide and N-methyl-bis-(trifluoroacetamide), in gas chromatography/mass spectrometry is proposed. This methodology results in mass spectra with at least three fragments in abundance superior to 20%, attending the World Anti-Doping Agency identification criteria for qualitative assays. The characterization of the derivatives was obtained through two ionization modes: Chemical Ionization and Electron Impact ionization, both in full scan mode. Sibutramine was administered to 5 (five) volunteers and the excretion profile followed for 92h. Routine analytical, hydroxy-cyclobutane-bis-nor-sibutramine which becomes the more abundant metabolite in the first 10h and hydroxy-isopropyl-bis-nor-sibutramine which becomes the most abundant after 40h, were proposed for doping monitoring.