Avaliação de técnicas miniaturizadas de preparação de amostras em estudos estereosseletivos de biotransformação e metabolismo in vitro
AGRADECIMENTOS Agradeço a Deus e mais uma vez a meus pais; Ao professor Anderson por me aceitar mais uma vez em seu laboratório e por toda a orientação; Agradeço à minha amiga Fernanda de Lima Moreira por todos os ensinamentos e ajuda durante o desenvolvimento do meu projeto; Á Marcela Armelim Bortoleto pela paciência, amizade e auxílio; Meu agradecimento, em especial, à professora Cristiane Mazetto de Gaitani por ceder espaço em seu laboratório e tornar possível a realização de etapas deste
... jeto; Aos professores: Mônica T. Pupo e Niegi A.J.C. Furtado; Ao técnico do LABMETS: Thiago Cavassani por todo seu auxílio; A Faculdade de Ciências Farmacêuticas pelos auxílios na pesquisa e oportunidade de realização deste projeto; Ao Departamento de Química da Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto; As agências de fomento: CAPES pela bolsa de estudos e FAPESP pelo auxílio financeiro; Aos demais colegas de laboratório pelo auxílio e companheirismo. Palavras-chave: Técnicas de microextração; Estudos enantiosseletivos de metabolismo in vitro iii ABSTRACT BOCATO, M. Z. Evaluation of miniaturized sample preparation techniques in enantioselective biotransformation and in vitro metabolism studies. 2016. 174 pages. PhD Thesis. Faculty of Pharmaceutical Sciences of Ribeirao Preto -University of Sao Paulo, Ribeirao Preto, 2016. Nowadays miniaturized extraction techniques are widely used in many sectors of analytical chemistry because they present several advantages such as: the ability to extract analytes in levels of trace employing minimal or none amounts of organic solvents; facility of automation and speed in the extraction procedure. New methodologies with the aim of producing pure enantiomers of drugs marketed as racemates are also very promising. In this context, this study aimed to evaluate the miniaturized sample preparation techniques, Solid Phase Microextraction (SPME), Hollow Fiber Liquid Phase Microextraction (HF-LPME) and Dispersive Liquid-Liquid Microextraction (DLLME) in extraction of drugs and their metabolites: oxcarbazepine (OXC) and tetrabenazine (TBZ) from complex matrices such as microsomal medium and liquid culture medium for subsequent application in stereoselective in vitro metabolism using human liver microsomes (HLMs) and in biotransformation studies employing fungi as catalytic agent. Prior to the biotransformation and the in vitro metabolism studies, all the proposed methods were validated and the results were in agreement with the official guidelines. Initially, an enantioselective capillary electrophoresis method was developed for the simultaneous determination of OXC and its metabolites in liquid culture medium. The chiral separation was carried out using phosphated β-cyclodextrin (P-β-CD) 1% (w/v) as the chiral selector in tris-phosphate 10 mmol L -1 pH 2.5 buffer solution. The effective length of the capillary was 20 cm, the applied voltage was 20 kV and the temperature of analysis was 15 °C. For this method, no miniaturized sample preparation technique was effective in extracting these analytes from the culture medium. Therefore, liquid-liquid extraction using methyl tert-butyl ether as solvent extractor as employed. The biotransformation studies showed enantioselectivity in the formation of licarbazepine (LIC) by two fungus species. The specie Glomerella cingulata (VA1) biotransformed OXC with 100% of enantiomeric fraction (EF) for the (S)-(+)-LIC enantiomer while the fungus Beuveria bassiana (ATCC 7159) metabolized with EF of 79% for the (S)-(+)-LIC enantiomer. Next, another method by capillary electrophoresis was also developed in this work. This new method was employed for the enantioselective analysis of diHTBZ metabolites after in vitro microsomal metabolism of the drug TBZ. Additionally, this method was used to analyze the diHTBZ metabolites after TBZ biotransformation by fungi. The chiral separation of diHTBZ metabolites was performed by using carboxymethyl-β-cyclodextrin (CM-β-CD) 1% (w/v) as the chiral selector added to trisphosphate buffer solution 80 mmol L -1 pH 2.5. The effective length of the capillary was 20 cm and the applied voltage was +15 kV. The analysis temperature was 15 °C. Among the miniaturized sample preparation techniques evaluated for the extraction of diHTBZ metabolites from both matrices, human liver microsomal and in liquid culture iv medium, DLLME showed to be the most adequate. Therefore, using microsomal medium as matrix 75 µL dichloromethane as solvent extractor and 150 µL acetone as disperser solvent was used. The in vitro metabolism of TBZ showed a kinetic profile of inhibition by substrate and demonstrated a diastereo-and enantioselective metabolism. These studies showed also that the enantiomers of the diastereomers of the diHTBZ were catalyzed mainly by CYP2C19 and the predicted clearance suggests that the metabolism by the liver is the major pathway for the elimination of TBZ. For the last, for fungal biotransformation studies with TBZ, 75 µL was used as extracting solvent of dichloromethane and 150 µL acetone as solvent disperser for the DDLME procedure. Preliminary biotransformation studies TBZ demonstrated a diastereoisomerism for all evaluated fungi, and additionally for some species of fungi, showed enantioselectivity in the formation of isomers. The fungus Chaetomiun globusum (VR10) metabolized both isomers of diHTBZ, and the production of metabolites was diastereoselective with majority formation of the trans-stereoisomer diHTBZ and enantioselectivity only in the production of cis-stereoisomer diHTBZ. The species Glomerella cingulata (VA1), Mucor rouxii, and Beuveria bassiana (ATCC 7159), metabolized diastereomerically and also enantiosselectivelly both metabolites of diHTBZ. The fungus Mucor rouxii showed an interesting biotransformation profile, with the majority training enantiomers (E1) of cis-and trans-diastereoisomers and majority formation of trans-diHTBZ metabolite. The results presented in this study showed that only DLLME was effective in extracting the TBZ from microsomal and liquid culture medium. For analytes with very basic features such as the OXC, the other evaluated microextraction techniques were not effective under the conditions employed in this study due mainly to the difficulty of keeping these analytes in the molecular form.