Protein Modelling and Molecular Docking Analysis of Fasciola hepatica ß-Tubulin's Interaction Sites, with Triclabendazole, Triclabendazole Sulphoxide and Triclabendazole Sulphone.

PURPOSE: Fasciola hepatica is a globally distributed trematode that causes significant economic losses. Triclabendazole is the primary pharmacological treatment for this parasite. However, the increasing resistance to triclabendazole limits its efficacy. Previous pharmacodynamics studies suggested that triclabendazole acts by interacting mainly with the ß monomer of tubulin. METHODS: We used a high-quality method to model the six isotypes of F. hepatica ß-tubulin in the absence of three-dimensional structures. Molecular dockings were conducted to evaluate the destabilization regions in the molecule against the ligands triclabendazole, triclabendazole sulphoxide and triclabendazole sulphone. RESULTS: The nucleotide binding site demonstrates higher affinity than the binding sites of colchicine, albendazole, the T7 loop and pßVII (p < 0.05). We suggest that the binding of the ligands to the polymerization site of ß-tubulin can lead a microtubule disruption. Furthermore, we found that triclabendazole sulphone exhibited significantly higher binding affinity than other ligands (p < 0.05) across all isotypes of ß-tubulin. CONCLUSIONS: Our investigation has yielded new insight on the mechanism of action of triclabendazole and its sulphometabolites on F. hepatica ß-tubulin through computational tools. These findings have significant implications for ongoing scientific research ongoing towards the discovery of novel therapeutics to treat F. hepatica infections.

A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance.

Fasciola hepatica infection is responsible for substantial economic losses in livestock worldwide and poses a threat to human health in endemic areas. The mainstay of control in livestock and the only drug licenced for use in humans is triclabendazole (TCBZ). TCBZ resistance has been reported on every continent and threatens effective control of fasciolosis in many parts of the world. To date, understanding the genetic mechanisms underlying TCBZ resistance has been limited to studies of candidate genes, based on assumptions of their role in drug action. Taking an alternative approach, we combined a genetic cross with whole-genome sequencing to localise a ~3.2Mbp locus within the 1.2Gbp F. hepatica genome that confers TCBZ resistance. We validated this locus independently using bulk segregant analysis of F. hepatica populations and showed that it is the target of drug selection in the field. We genotyped individual parasites and tracked segregation and reassortment of SNPs to show that TCBZ resistance exhibits Mendelian inheritance and is conferred by a dominant allele. We defined gene content within this locus to pinpoint genes involved in membrane transport, (e.g. ATP-binding cassette family B, ABCB1), transmembrane signalling and signal transduction (e.g. GTP-Ras-adenylyl cyclase and EGF-like protein), DNA/RNA binding and transcriptional regulation (e.g. SANT/Myb-like DNA-binding domain protein) and drug storage and sequestration (e.g. fatty acid binding protein, FABP) as prime candidates for conferring TCBZ resistance. This study constitutes the first experimental cross and genome-wide approach for any heritable trait in F. hepatica and is key to understanding the evolution of drug resistance in Fasciola spp. to inform deployment of efficacious anthelmintic treatments in the field.

Evidence of sequestration of triclabendazole and associated metabolites by extracellular vesicles of Fasciola hepatica.

Fascioliasis is a neglected zoonotic disease that infects humans and ruminant species worldwide. In the absence of vaccines, control of fascioliasis is primarily via anthelminthic treatment with triclabendazole (TCBZ). Parasitic flatworms, including Fasciola hepatica, are active secretors of extracellular vesicles (EVs), but research has not been undertaken investigating EV anthelmintic sequestration. Adult F. hepatica were cultured in lethal and sub-lethal doses of TCBZ and its active metabolites, in order to collect EVs and evaluate their morphological characteristics, production and anthelmintic metabolite content. Transmission electron microscopy demonstrated that F. hepatica exposed to TCBZ and its metabolites produced EVs of similar morphology, compared to non-TCBZ exposed controls, even though TCBZ dose and/or TCBZ metabolite led to measurable structural changes in the treated F. hepatica tegument. qNano particle analysis revealed that F. hepatica exposed to TCBZ and its metabolites produced at least five times greater EV concentrations than non-TCBZ controls. A combined mass spectrometry and qNano particle analysis confirmed the presence of TCBZ and the TCBZ-sulphoxide metabolite in anthelmintic exposed EVs, but limited TCBZ sulphone was detectable. This data suggests that EVs released from adult F. hepatica have a biological role in the sequestration of TCBZ and additional toxic xenobiotic metabolites.

Pharmacologic interaction between oxfendazole and triclabendazole: In vitro biotransformation and systemic exposure in sheep.

The aim of the current work was to evaluate a potential pharmacokinetic interaction between the flukicide triclabendazole (TCBZ) and the broad-spectrum benzimidazole (BZD) anthelmintic oxfendazole (OFZ) in sheep. To this end, both an in vitro assay in microsomal fractions and an in vivo trial in lambs parasitized with Haemonchus contortus resistant to OFZ and its reduced derivative fenbendazole (FBZ) were carried out. Sheep microsomal fractions were incubated together with OFZ, FBZ, TCBZ, or a combination of either FBZ and TCBZ or OFZ and TCBZ. OFZ production was significantly diminished upon coincubation of FBZ and TCBZ, whereas neither FBZ nor OFZ affected the S-oxidation of TCBZ towards its sulfoxide and sulfone metabolites. For the in vivo trial, lambs were treated with OFZ (Vermox® oral drench at a single dose of 5 mg/kg PO), TCBZ (Fasinex® oral drench at a single dose of 12 mg/kg PO) or both compounds at a single dose of 5 (Vermox®) and 12 mg/kg (Fasinex®) PO. Blood samples were taken to quantify drug and metabolite concentrations, and pharmacokinetic parameters were calculated by means of non-compartmental analysis. Results showed that the pharmacokinetic parameters of active molecules and metabolites were not significantly altered upon coadministration. The sole exception was the increase in the mean residence time (MRT) of OFZ and FBZ sulfone upon coadministration, with no significant changes in the remaining pharmacokinetic parameters. This research is a further contribution to the study of metabolic drug-drug interactions that may affect anthelmintic efficacies in ruminants.

Total determination of triclabendazole and its metabolites in bovine tissues using liquid chromatography-tandem mass spectrometry.

A reliable LC-MS/MS analytical method for the determination of residual triclabendazole and its principal metabolites (triclabendazole sulfoxide, triclabendazole sulfone and keto-triclabendazole) in bovine tissues was developed, in which triclabendazole and its metabolites are oxidized to keto-triclabendazole as a marker residue. The method involves sample digestion with hot sodium hydroxide, thus releasing the bound residues of various triclabendazole metabolites in bovine tissues. The target compounds are extracted from the digest mixture with ethyl acetate, defatted by liquid-liquid partitioning using n-hexane and acetonitrile, then oxidized with hydrogen peroxide in a mixture of ethanol and acetic acid. The reaction mixture is cleaned up using a strong cation exchange cartridge (Oasis MCX) and the analytes are quantified using LC-MS/MS. The optimal conditions for the complete oxidation of triclabendazole and its metabolites to keto-triclabendazole are an incubation time of 16 h and a temperature of 90 °C. The developed method was evaluated using three bovine samples: muscle, fat, and liver. Samples were spiked with triclabendazole and its principal metabolites at 0.01 mg/kg and at the Japanese Maximum Residue Limits (MRLs) established for each sample. The validation results show excellent recoveries (81-102%) and precision (<10%) for all target compounds. The limit of quantification (S/N ≥ 10) of the developed method is 0.01 mg/kg. These results suggest the developed method is applicable to quantifying residual triclabendazole in bovine tissues in compliance with the MRLs established by the Codex Alimentarius and EU and Japanese regulations, and thus the proposed method will be a useful tool for the regulatory monitoring of residual triclabendazole and its metabolites.

In Vitro Drug-Drug Interaction Potential of Sulfoxide and/or Sulfone Metabolites of Albendazole, Triclabendazole , Aldicarb, Methiocarb, Montelukast and Ziprasidone.

BACKGROUND: The use of polypharmacy in the present day clinical therapy has made the identification of clinical drug-drug interaction risk an important aspect of drug development process. Although many drugs can be metabolized to sulfoxide and/or sulfone metabolites, seldom is known on the CYP inhibition potential and/or the metabolic fate for such metabolites. OBJECTIVE: The key objectives were: a) to evaluate the in vitro CYP inhibition potential of selected parent drugs with sulfoxide/sulfone metabolites; b) to assess the in vitro metabolic fate of the same panel of parent drugs and metabolites. METHODS: In vitro drug-drug interaction potential of test compounds was investigated in two stages; 1) assessment of CYP450 inhibition potential of test compounds using human liver microsomes (HLM); and 2) assessment of test compounds as substrate of Phase I enzymes; including CYP450, FMO, AO and MAO using HLM, recombinant human CYP enzymes (rhCYP), Human Liver Cytosol (HLC) and Human Liver Mitochondrial (HLMit). All samples were analysed by LC-MS-MS method. RESULTS: CYP1A2 was inhibited by methiocarb, triclabendazole, triclabendazole sulfoxide, and ziprasidone sulfone with IC50 of 0.71 µM, 1.07 µM, 4.19 µM, and 17.14 µM, respectively. CYP2C8 was inhibited by montelukast, montelukast sulfoxide, montelukast sulfone, tribendazole, triclabendazole sulfoxide, and triclabendazole sulfone with IC50 of 0.08 µM, 0.05 µM, 0.02 µM, 3.31 µM, 8.95 µM, and 1.05 µM, respectively. CYP2C9 was inhibited by triclabendazole, triclabendazole sulfoxide, triclabendazole sulfone, montelukast, montelukast sulfoxide and montelukast sulfone with IC50 of 1.17 µM, 1.95 µM, 0.69 µM, 1.34 µM, 3.61 µM and 2.15 µM, respectively. CYP2C19 was inhibited by triclabendazole and triclabendazole sulfoxide with IC50 of 0.25 and 0.22, respectively. CYP3A4 was inhibited by montelukast sulfoxide and triclabendazole with IC50 of 9.33 and 15.11, respectively. Amongst the studied sulfoxide/sulfone substrates, the propensity of involvement of CY2C9 and CYP3A4 enzyme was high (approximately 56% of total) in the metabolic fate experiments. CONCLUSION: Based on the findings, a proper risk assessment strategy needs to be factored (i.e., perpetrator and/or victim drug) to overcome any imminent risk of potential clinical drug-drug interaction when sulfoxide/sulfone metabolite(s) generating drugs are coadministered in therapy.