title: In vitro enantioselective human liver microsomal metabolism and prediction of in vivo pharmacokinetic parameters of tetrabenazine by DLLME-CE.
abstract: A new capillary electrophoresis method for the enantioselective analysis of cis- and trans- dihydrotetrabenazine (diHTBZ) after in vitro metabolism by human liver microsomes (HLMs) was developed. The chiral electrophoretic separations were performed by using tris-phosphate buffer (pH 2.5) containing 1% (w/v) carboxymethyl-β-CD as background electrolyte with an applied voltage of +15kV and capillary temperature kept at 15°C. Dispersive liquid-liquid microextraction was employed to extract the analytes from HLMs. Dichloromethane was used as extraction solvent (75μL) and acetone as disperser solvent (150μL). The method was validated according to official guidelines and showed to be linear over the concentration range of 0.29-19.57μmolL(-1) (r=0.9955) for each metabolite enantiomer. Within- and between-day precision and accuracy evaluated by relative standard deviation and relative error were lower than 15% for all enantiomers. The stability assay showed that the analytes kept stable under handling, storage and in metabolism conditions. After method validation, an enantioselective in vitro metabolism and in vivo pharmacokinetic prediction was carried out. This study showed a stereoselective metabolism and the observed kinetic profile indicated a substrate inhibition behavior. DiHTBZ enantiomers were catalyzed mainly by CYP2C19 and the predicted clearance suggests that liver metabolism is the main route for TBZ elimination which supports the literature data.
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903, Ribeirão Preto, SP, Brazil.
authors.forename: Mariana Zuccherato
authors.initials: MZ
authors.lastname: Bocato
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903, Ribeirão Preto, SP, Brazil.
authors.forename: Fernanda
authors.initials: F
authors.lastname: de Lima Moreira
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brazil.
authors.forename: Nayara Cristina Perez
authors.initials: NCP
authors.lastname: de Albuquerque
authors.affiliation: Departamento de Ciências Farmacêuticas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, 14040-903, Ribeirão Preto, SP, Brazil.
authors.forename: Cristiane Masetto
authors.initials: CM
authors.lastname: de Gaitani
authors.affiliation: Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, Ribeirão Preto, SP, Brazil. Electronic address: deoliveira@usp.br.
authors.forename: Anderson Rodrigo Moraes
authors.initials: ARM
authors.lastname: de Oliveira
mesh_terms: D065731:Cytochrome P-450 CYP2C19 / Q000378:metabolism; D004355:Drug Stability; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D059627:Liquid Phase Microextraction / Q000379:methods*; D008862:Microsomes, Liver / Q000378:metabolism*; D015203:Reproducibility of Results; D013237:Stereoisomerism; D013747:Tetrabenazine / Q000031:analogs & derivatives / Q000378:metabolism / Q000493:pharmacokinetics*
chemical_list: C032811:dihydrotetrabenazine; C045793:CYP2C19 protein, human; D065731:Cytochrome P-450 CYP2C19; D013747:Tetrabenazine
keywords: Capillary electrophoresis; Chiral analysis; Dispersive liquid-liquid microextraction; Enantioselective in vitro metabolism; Tetrabenazine