Investigation in vitro of effect of protein restrict in the metabolism of albendazole in rats and growth inhibition studies in human tumor cells by enantiomers of albendazole sulphoxide

This work presents an in vitro investigation of protein restriction in the metabolism of albendazole (ABZ). The study was conducted with microsomal fractions obtained from Wistar rats using a High Performance Multidimensional Liquid Chromatography method which was developed and fully validated for the determination of the ABZ metabolites albendazole sulphoxide (ABZ-SO), albendazole sulphone (ABZ-SO2) and albendazole 2-aminesulphone (ABZ-SO2-NH2). The compounds were extracted from the biological matrix using a C8-RAM-BSA column (5.0 x 0.46 cm i.d.) and analyzed on a chromatographic chiral column containing amylose tris(3,5- dimethylphenylcarbamate) (15.0 x 0.46 cm i.d.) with a runtime of 35 min. The results of the biotransformation experiments showed that the protein restriction influenced the oxidative metabolism of ABZ. The production of (+) and (-)-ABZ-SO was higher in the control animals. Aditionally, the production of ABZ-SO enantiomers was enantioselective, where the (-)-ABZ-SO was formed in greater amounts than the (+)- ABZ-SO in control animals. However, this enantioselectivity was not observed when the ABZ biotransformation was conducted with microsomal fractions obtained from protein restriction animals. Four chiral polysaccharide phases were also evaluated for the enantioseparation of ABZ-SO in normal and polar organic elution modes. Among the tested conditions, the tris(3,5-dimethylphenylcarbamate) of amylose and MeOH 100% as mobile phase was selected to perform a semipreparative HPLC separation of ()-ABZ-SO. The pure enantiomers obtained were used in in vitro assays to evaluate the growth inhibitory effects on human tumor cells. Inhibition of cell growth was observed to be more pronounced with the (+)-ABZ-SO compared to (-)-ABZ-SO, particularly in MCF-7 cells where the inhibition was about four times more efficient.Universidade Federal de Minas GeraisEste trabalho apresenta estudos sobre influência da restrição proteica no metabolismo in vitro do albendazol (ABZ). O estudo foi realizado com emprego de frações microssomais de fígados de ratos Wistar, através de um método por Cromatografia Líquida Multidimensional de Alta Eficiência, desenvolvido e validado para a determinação dos metabólitos do ABZ: o albendazol-sulfóxido (ABZ-SO), albendazolsulfona (ABZ-SO2) e o albendazol-2-amino-sulfona (ABZ-NH2-SO2). Os compostos foram extraídos da matriz biológica através do emprego de uma coluna C8-RAM-BSA (5,0 x 0,46 cm d.i.) e analisados em uma coluna cromatográfica quiral tris(3,5- dimetilfenilcarbamato) de amilose (15,0 x 0,46 cm d.i.), em um tempo total de 35 min. Os dados de biotransformação demonstraram que a restrição proteica influenciou o metabolismo oxidativo do ABZ, onde a produção do (+) e (-)-ABZ-SO foi maior nos animais controles. Adicionalmente, a obtenção dos enantiômeros do ABZ-SO foi enantiosseletiva, onde o (-)-ABZ-SO foi produzido em maior quantidade do que o (+)- ABZ-SO em animais controle, porém, essa enantiosseletividade não foi observada quando da análise do metabolismo do ABZ em frações microssomais obtidas de fígados de ratos submetidos a restrição proteica. Ainda neste trabalho, foi realizada a avaliação de quatro fases quirais de polissacarídeos, no modo normal e polar orgânico, para a separação dos enantiômeros do ABZ-SO. Dentre as condiçõess avaliadas, a fase quiral tris(3,5-dimetilfenilcarbamato) de amilose e fase móvel composta por MeOH 100% foram empregadas para realizar a separação em escala semipreparativa do ()- ABZ-SO. Os enantiômeros puros obtidos foram utilizados em ensaios in vitro de inibição do crescimento de células tumorais humanas. Os resultados destes testes mostraram que a inibição do crescimento celular foi aproximadamente 4 vezes maior com o (+)-ABZ-SO do que com o (-)-ABZ-SO, principalmente nas células MCF-7

Quantification of albendazole metabolites in bovine plasma by high performance liquid chromatography, using direct injection of samples

This work presents the development and validation of a sensitive and enantioselective multidimensional high performance liquid chromatography (HPLC) method for determination of albendazole metabolites: albendazole sulphoxide, chiral compound, albendazole sulphone and albendazole 2-aminesulphone in bovine plasma. The compounds, (±)- albendazole sulphoxide, albendazole sulphone and 2-aminesulphone were extracted from bovine plasma using a octyl restricted access media bovine serum albumin column (C8-RAM-BSA) (5.0 x 0.46 cm i.d.) and analyzed on a chiral column containing amylose tris(3,5-dimethylphenylcarbamate) coated onto APS-Nucleosil support, as stationary phase (15.0 x 0.46 cm i.d.). The chromatographic separations of all target compounds were achieved using a mobile phase composed of phosphate buffer (0.01 M; pH 7.5):acetonitrile (60:40 v/v), flow rate of 0.5 mL/min and fluorescence detection at 290 nm and 320 nm, excitation and emission, respectively. The calibration curves were linear in the concentration range of 40.00-1280 ng/ml for each albendazole sulphoxide enantiomer, 10.0-320 ng/mL for albendazole sulphone and 10.0-320 ng/mL for albendazole 2-aminesulphone. The validated method showed linearity, precision, accuracy and adequate sensitivity, allowing it to be appropriate for pharmacokinetics studies after administration of albendazole by different routes. Moreover, two metabolites of albendazole, the albendazole sulphoxide and albendazole sulphone, were synthesized by oxidation reaction using the aquo(N-oxo of piridine) molybdenum (VI) oxo diperoxo complexe.Universidade Federal de Minas GeraisEste trabalho apresenta o desenvolvimento e validação de um método, sensível e enantiosseletivo, por Cromatografia Líquida Multidimensional de Alta Eficiência, para a determinação dos metabólitos do albendazol: o albendazol-sulfóxido, metabólito quiral, albendazol-sulfona e albendazol-2-aminosulfona, em plasma bovino. Os compostos (±)-albendazol-sulfóxido, albendazolsulfona e 2-amino-sulfona foram extraídos do plasma bovino através do emprego de uma coluna extratora da fase hidrofóbica C8, do tipo fase de acesso restrito de albumina sérica bovina (C8-RAM-BSA) (5,0 x 0,46 cm d.i.) e analisados em uma coluna quiral tris(3,5-dimetilfenilcarbamato) de amilose em sílica APS-Nucleosil (15,0 x 0,46 cm d.i.). A separação cromatográfica de todos os compostos de interesse foi obtida através do uso de uma fase móvel composta por tampão fosfato (0,01 M; pH 7,5):acetonitrila (60:40 v/v), com vazão de 0,5 mL/min e detecção por fluorescência, λexc e λem de 290 e 320 nm, respectivamente. As curvas de calibração foram lineares nas faixas de 40,00-1280 ng/mL para cada enantiômero do albendazol-sulfóxido, de 10,00-320,0 ng/mL para o albendazolsulfona e de 20,00-320,0 ng/mL para o albendazol-2-amino-sulfona. O método validado mostrou linearidade, precisão, exatidão e sensibilidade adequada para ser utilizado em estudos farmacocinéticos após administração do albendazol por diferentes vias. Ainda neste trabalho, foi realizada a síntese de dois dos metabólitos do albendazol, o albendazol-sulfóxido e albendazol-sulfona, através da oxidação do sulfeto com emprego do complexo aquo(N-óxido de piridina) oxo diperoxo molibdênio (VI)

Multimilligram enantioresolution of sulfoxide proton pump inhibitors by liquid chromatography on polysaccharide-based chiral stationary phase

The enantiomers of sulfoxide proton pump inhibitors - omeprazole, lansoprazole, rabeprazole and Ro 18-5364 - were enantiomerically separated by liquid chromatography at multimilligram scale on a poly saccharide-based chiral stationary phase using normal and polar organic conditions as mobile phase. The values of the recovery and production rate were significant for each enantiomer; better results were achieved using a solid-phase injection system. However, this system was applied just for the enantionteric separation of omeprazole to demonstrate the applicability of this injection mode at milligram scale. The chiroptical characterization of the compounds was performed using a polarimeter and a circular dichroism detector. The higher enantiomeric purity obtained for the isolated enantiomers suggests that the methods here described should be considered as a simple and rapid way to obtain enantiomeric pure standards for analytical purpose. (C) 2007 Elsevier B.V. All rights reserved

Identification of corynebacterium bovis by MALDI-mass spectrometry

Corynebacterium bovis is a mastitis-causing microorganism responsible for economic losses related to decrease in milk production. The aim of the study was identify Corynebacterium spp. strains recovered from milk samples of subclinical mastitis by using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Samples were collected during a 10-mo mastitis-monitoring program in a high-production dairy farm. In this study, 80 strains were analyzed; from these 54 (67.5%) were identified at species level as Corynebacterium bovis, 24 (31.2%) isolates were identified at the genus level as Corynebacterium spp., and only 1 (1.35%) isolated had unreliable identification. Results demonstrated that MALDI-MS could be an important technique for the identification of Corynebacterium spp. in milk. © 2017 American Dairy Science Association.Corynebacterium bovis is a mastitis-causing microorganism responsible for economic losses related to decrease in milk production. The aim of the study was identify Corynebacterium spp. strains recovered from milk samples of subclinical mastitis by using m100642874289FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2012/24213-

Modulation of long-chain Acyl-CoA synthetase on the development, lipid deposit and cryosurvival of in vitro produced bovine embryos.

In this study, we evaluated the modulation effect of long-chain Acyl-CoA synthetase during early embryo development. Bovine embryos were cultured in four groups: positive modulation (ACS+) with GW3965 hydrochloride, negative modulation (ACS-) with Triacsin C, association of both modulators (ACS±), and control. Embryo development rates were not altered (P>0.05) by treatments. Embryonic cytoplasmic lipid content increased in ACS+ but reduced in ACS- compared to the control (P 0.05) between groups; however, an increased apoptotic cells percentage was found in ACS- compared to control. Twenty-four hours after warming, ACS+ and control grade I embryos presented the best hatching rates, whereas the ACS+ group equaled the hatching rates between their embryos of grades I, II and III 48 hours after warming. The relative abundance of transcripts for genes associated with lipid metabolism (ACSL3, ACSL6, ACAT1, SCD, and AUH), heatshock (HSP90AA1 and HSF1), oxidative stress (GPX4), and angiogenesis (VEGF), among other important genes for embryo development were affected by at least one of the treatments. The treatments were effective in modulating the level of transcripts for ACSL3 and the cytoplasmic lipid content. The ACS- was not effective in increasing embryonic cryosurvival, whereas ACS+ restored survival rates after vitrification of embryos with low quality, making them equivalent to embryos of excellent quality