Assessment of the main metabolism pathways for the flukicidal compound triclabendazole in sheep.

Triclabendazole (TCBZ) is an halogenated benzimidazole (BZD) compound worldwide used to control immature and adult stages of the liver fluke Fasciola hepatica. The purpose of this investigation was to characterize in vitro the patterns of hepatic and ruminal biotransformation of TCBZ and its metabolites in sheep. TCBZ parent drug was metabolized into its sulphoxide (TCBZSO), sulphone (TCBZSO2) and hydroxy derivatives by sheep liver microsomes. The same microsomal fraction was also able to oxidize TCBZSO into TCBZSO2 and hydroxy-TCBZSO (HO-TCBZSO). TCBZ sulphoxidation was significantly (P < 0.001) inhibited after inactivation of the flavin-monooxygenase (FMO) system (77% inhibition) as well as in the presence of the FMO substrate methimazole (MTZ) (71% inhibition). TCBZ sulphoxidative metabolism was also reduced (24% inhibition, P < 0.05) by the cytochrome P450 inhibitor piperonyl butoxide (PB). The rate of TCBZSO conversion into TCBZSO2 was also significantly inhibited by PB (55% inhibition), MTZ (52% inhibition) and also following FMO inactivation (58% inhibition). The data reported here indicate that the FMO is the main enzymatic pathway involved in TCBZ sulphoxidation (ratio FMO/P450 = 3.83 +/- 1.63), although both enzymatic systems participate in a similar proportion in the sulphonation of TCBZSO to form the sulphone metabolite (ratio FMO/P450 = 1.31 +/- 0.23). Additionally, ketoconazole (KTZ) did not affect TCBZ sulphoxidation but decreased (66% inhibition, P < 0.05) the formation of TCBZSO2. Similarly, inhibition of TCBZSO2 production was observed after incubation of TCBZSO in the presence of KTZ and erythromycin (ETM). Conversely, thiabendazole (TBZ) and fenbendazole (FBZ) did not affect the oxidative metabolism of both incubated substrates. The sheep ruminal microflora was able to reduce the sulphoxide (TCBZSO) into the parent thioether (TCBZ). The ruminal sulphoreduction of the HO-TCBZSO derivative into HO-TCBZ was also demonstrated. The rate of sulphoreduction of HO-TCBZSO was significantly (P < 0.05) higher than that observed for TCBZSO. The metabolic approach tested here contributes to the identification of the different pathways involved in drug biotransformation in ruminant species. These findings on the pattern of hepatic and ruminal biotransformation of TCBZ and its main metabolites are a further contribution to the understanding of the pharmacological properties of widely used anthelmintics in ruminants. Comprehension of TCBZ metabolism is critical to optimize its flukicidal activity.

Enantiomeric determination of pantoprazole in human plasma by multidimensional high-performance liquid chromatography.

Multidimensional HPLC is a powerful tool for the analysis of samples of a high degree of complexity. This work reports the use of multidimensional HPLC by coupling a RAM column with a chiral polysaccharide column to the analysis of Pantoprazole in human plasma by direct injection. The enantiomers from the plasma samples were separated with high resolution on a tris(3,5-dimethoxyphenylcarbamate) of amylose phase after clean-up by a RAM BSA octyl column. Water was used as solvent for the first 5 min in a flow-rate of 1.0 ml/min for the elution of the plasmatic proteins and then acetonitrile-water (35:65 v/v) for the transfer and analysis of pantoprazole enantiomers, which were detected by UV at 285 nm. Analysis time was 28 min with no time spent on sample preparation. A good linear relationship was obtained in the concentration range of 0.20 to 1.5 microg/ml for each enantiomer. Inter and intra-day precision and accuracy were determined by one low (0.24 microg/ml), one medium (0.70 microg/ml) and one high (1.3 microg/ml) plasma concentration and gave a C.V. varying from 1.80 to 8.43% and accuracy from 86 to 92%. Recoveries of pantoprazole enantiomers were in the range of 93.7-101.2%. The validated method was applied to the analysis of the plasma samples obtained from ten Brazilian volunteers who received an 80 mg oral dose of racemic pantoprazole and was able to quantify the enantiomers of pantoprazole in all clinical samples analyzed.