Liquid chromatography/mass spectrometric identification of benzimidazole anthelminthics metabolites formed ex vivo by Dicrocoelium dendriticum

Abstract

With further use of chemical agents in the control of parasitic infections, an increased number of drug resistance occurrences to antiparasitic drugs has been reported. Induction of enzymes responsible for detoxification of given drugs can contribute to drug resistance development in a parasitic organism. The identification of formed metabolites allows the characterization of the enzymes participating in biotransformation and possibly in drug resistance development. The objective of our work was to find and identify phase I and phase II metabolites of the anthelminthic drugs albendazole, flubendazole and mebendazole formed in ex vivo incubations by the parasitic helminth Dicrocoelium dendriticum, a parasite of ruminants and other grazing animals, using liquid chromatography/mass spectrometric (LC/MS) techniques. In the ex vivo study, approximately 50 living D. dendriticum adults were incubated in 5 mL RPMI-1640 medium in the presence of 10.0 micromol L(-1) benzimidazole drug (5% CO(2), 38 degrees C) for 24 h. The bodies of the parasite were then removed from the medium. After homogenization of parasites, both parasite homogenates and medium from the incubation were separately extracted using solid-phase extraction. The extracts were analyzed using LC/MS with electrospray ionization. The results showed that D. dendriticum enzymatic systems are capable of phase I oxidation and reduction as well as phase II conjugation reactions. Detected phase I metabolites comprised albendazole sulfoxide, reduced flubendazole and reduced mebendazole. As for phase II metabolites, methyl derivatives of both reduced flubendazole and reduced mebendazole were observed.

Metabolism of albendazole, ricobendazole and flubendazole in Haemonchus contortus adults: Sex differences, resistance-related differences and the identification of new metabolites

“…Several new worm-specific metabolites, which were not found in hosts, were described. Moreover, significant differences in anthelmintics metabolism between nematodes and flukes were observed ( Cvilink et al, 2008 , Cvilink et al, 2009 ).…”

“…Most of these new metabolites were surprising, as these metabolic reactions of anthelmintics have not as of yet been described in nematodes. The carbamate hydrolysis of ABZ and FLU has been described in pigs ( Bartikova et al, 2010 ), while the methylation of anthelmintics has been reported only in flukes ( Cvilink et al, 2009 ). The formation of acetylglucosides of the anthelmintics has been identified only in plants ( Podlipna et al, 2013 , Stuchlikova et al, 2016 ).…”

The activity of drug-metabolizing enzymes and the biotransformation of selected anthelmintics in the model tapeworm Hymenolepis diminuta

“…The measurements in positive and negative ion mode were carried out according to the protocol of Cvilink et al . (2009 c ) with minor changes of mode values. The following optimized ion source parameters were set for BZD drug analysis during sample data acquisition in positive ion mode (negative ion mode values in parentheses): spray voltage, 5·3 kV (−3·6 kV); capillary voltage, 3·0 V (−7·0 V); heated capillary temperature, 200°C; tube lens offset voltage, 40·0 V (−15·0 V); sheath gas flow rate, 20·0 arbitrary units; auxiliary gas flow rate, 10·0 arbitrary units.…”

“…The screening for metabolites was carried out according to the protocol of Cvilink et al . (2009 c ). The structures of metabolites were tentatively assessed by the combination of their corresponding full-scan spectra, MS n spectra and chromatographic behaviour.…”

Similarities and differences in the biotransformation and transcriptomic responses of Caenorhabditis elegans and Haemonchus contortus to five different benzimidazole drugs

“…In an in vitro assay that examined oxidation of albendazole, the resistant strains of F. hepatica have been shown to produce more of the oxidized product than sensitive strains (Solana et al, 2001). The liver fluke D. dendriticum was found to biotransform albendazole to albendazole sulphoxide, and mebendazole and flubendazole were either reduced, or reduced and methylated (Cvilink et al, 2009a, 2009b). F. hepatica metabolizes triclabendazole (TCBZ) into sulfoxide (TCBZ-SO) and sulphone (TCBZ-SO2), these metabolized products were found at higher concentrations in resistance strains (Solana et al, 2001; Mottier et al, 2004; Robinson et al, 2004).…”