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

Abstract

Haemonchus contortus (family Trichostrongylidae, Nematoda), a hematophagous gastrointestinal parasite found in small ruminants, has a great ability to develop resistance to anthelmintic drugs. We studied the biotransformation of the three benzimidazole anthelmintics: albendazole (ABZ), ricobendazole (albendazole S-oxide; RCB) and flubendazole (FLU) in females and males of H. contortus in both a susceptible ISE strain and resistant IRE strain. The ex vivo cultivation of living nematodes in culture medium with or without the anthelmintics was used. Ultrasensitive UHPLC/MS/MS analysis revealed 9, 7 and 12 metabolites of ABZ, RCB and FLU, respectively, with most of these metabolites now described in the present study for the first time in H. contortus. The structure of certain metabolites shows the presence of biotransformation reactions not previously reported in nematodes. There were significant qualitative and semi-quantitative differences in the metabolites formed by male and female worms. In most cases, females metabolized drugs more extensively than males. Adults of the IRE strain were able to form many more metabolites of all the drugs than adults of the ISE strain. Some metabolites were even found only in adults of the IRE strain. These findings suggest that increased drug metabolism may play a role in resistance to benzimidazole drugs in H. contortus.

UDP-glycosyltransferase family in Haemonchus contortus: Phylogenetic analysis, constitutive expression, sex-differences and resistance-related differences

“…6 ). This finding is in agreement with previously mentioned studies of anthelmintic metabolism and of UGT activity toward the model substrate p -nitrophenol, when the amount of glucosides formed ex vivo and in vitro was higher in H. contortus resistant strains than in the sensitive strain ( Vokral et al, 2012 ), ( Stuchlíková et al, 2018 ). An important aspect is that the ISE and IRE isolates included in our study have a similar genetic background as described above ( Kwa et al, 1993 ) ( Roos et al, 2004 ).…”

“…In the free living nematode C. elegans , conjugates of the anthelmintic drug albendazole (ABZ) with glucose were identified ( Laing et al, 2010 ). Similarly, in H. contortus all glycosylated metabolites of anthelmintics that have been identified are conjugates of glucose or another hexose ( Stuchlíková et al, 2018 ). Therefore, we believe that Hc _UGTs use UDP-glucose (hexose) as the major sugar donor rather than UDP-glucuronic acid, hence the binding topology could be slightly different.…”

“…This discrepancy can be attributed on one hand to the lack of replicates in the preliminary RNA-seq and big variation in biological replicates in several qPCR on the other hand. In our recent metabolism experiments, females and males were tested separately and showed major sex-differences in the metabolism of all anthelmintics tested ( Stuchlíková et al, 2018 ). Mostly, a higher amount of metabolites were found in females than in males and three detected metabolites of flubendazole ( N -glycosides) were female-specific.…”

“…In helminths, detoxification of benzimidazole anthelmintics via glycosylation has been observed ( Laing et al, 2010 ; Vokral et al, 2012 , 2013 ). In addition, anthelmintic glycosidation was more potent in resistant H. contortus strains than in a susceptible one, suggesting involvement of UGTs in resistance to benzimidazole anthelmintics in this nematode ( Stuchlíková et al, 2018 ; Vokral et al, 2012 , 2013 ). Recently, involvement of C. elegans ugt-22 (UGT16C1) in albendazole efficacy was reported ( Fontaine and Choe, 2018 ).…”

Sub-lethal doses of albendazole induce drug metabolizing enzymes and increase albendazole deactivation in Haemonchus contortus adults

“…The H. contortus adults were pre-incubated in medium without ABZ (control group) and with ABZ at 0.01, 0.1 and 1.0 µM concentrations (affected groups) for 18 h. The H. contortus adults were then placed in fresh medium without ABZ (blanks) or with 10 µM ABZ and incubated for 12 h. Qualitative and semi-quantitative analyses of ABZ metabolites in homogenate from the nematodes and in medium was performed using UHPLC/MS. The metabolites were identified based on their fragmentation ions as described previously [10]. The designation and description of the metabolites is presented in Table 2.…”