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Xenobiotic biotransformation is an important modulator of anthelmintic drug potency and a potential mechanism of anthelmintic resistance. Both the free-living nematode Caenorhabditis elegans and the ruminant parasite Haemonchus contortus biotransform benzimidazole drugs by glucose conjugation, likely catalysed by UDP-glycosyltransferase (UGT) enzymes. To identify C. elegans genes involved in benzimidazole drug detoxification, we first used a comparative phylogenetic analysis of UGTs from humans, C. elegans and H. contortus, combined with available RNAseq datasets to identify which of the 63 C. elegans ugt genes are most likely to be involved in benzimidazole drug biotransformation. RNA interference knockdown of 15 prioritized C. elegans genes identified those that sensitized animals to the benzimidazole derivative albendazole (ABZ). Genetic mutations subsequently revealed that loss of ugt-9 and ugt-11 had the strongest effects. The "ugt-9 cluster" includes these genes, together with six other closely related ugts. A CRISPR-Cas-9 deletion that removed seven of the eight ugt-9 cluster genes had greater ABZ sensitivity than the single largest-effect mutation. Furthermore, a double mutant of ugt-22 (which is not a member of the ugt-9 cluster) with the ugt-9 cluster deletion further increased ABZ sensitivity. This additivity of mutant phenotypes suggest that ugt genes act in parallel, which could have several, not mutually exclusive, explanations. ugt mutations have different effects with different benzimidazole derivatives, suggesting that enzymes with different specificities could together more efficiently detoxify drugs. Expression patterns of ugt-9, ugt-11 and ugt-22 gfp reporters differ and so likely act in different tissues which may, at least in part, explain their additive effects on drug potency. Overexpression of ugt-9 alone was sufficient to confer partial ABZ resistance, indicating increasing total UGT activity protects animals. In summary, our results suggest that the multiple UGT enzymes have overlapping but not completely redundant functions in benzimidazole drug detoxification and may represent "druggable" targets to improve benzimidazole drug potency., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Anthelmintics are drugs used for controlling pathogenic helminths in animals and plants. The natural compound betaine and the recently developed synthetic compound monepantel are both anthelmintics that target the acetylcholine receptor ACR-23 and its homologs in nematodes. Here, we present cryo-electron microscopy structures of ACR-23 in apo, betaine-bound, and betaine- and monepantel-bound states. We show that ACR-23 forms a homo-pentameric channel, similar to some other pentameric ligand-gated ion channels (pLGICs). While betaine molecules are bound to the classical neurotransmitter sites in the inter-subunit interfaces in the extracellular domain, monepantel molecules are bound to allosteric sites formed in the inter-subunit interfaces in the transmembrane domain of the receptor. Although the pore remains closed in betaine-bound state, monepantel binding results in an open channel by wedging into the cleft between the transmembrane domains of two neighboring subunits, which causes dilation of the ion conduction pore. By combining structural analyses with site-directed mutagenesis, electrophysiology and in vivo locomotion assays, we provide insights into the mechanism of action of the anthelmintics monepantel and betaine., (© 2024. The Author(s).)

Breast cancer resistance protein/ATP-binding cassette subfamily G2 (BCRP/ABCG2) is an ATP-binding cassette efflux (ABC) transporter expressed in the apical membrane of cells in tissues, such as the liver, intestine, kidney, testis, brain, and mammary gland. It is involved in xenobiotic pharmacokinetics, potentially affecting the efficacy and toxicity of many drugs. In this study, the role of ABCG2 in parasiticide monepantel (MNP) and its primary metabolite, monepantel sulfone (MNPSO 2 )'s systemic distribution and excretion in milk, was tested using female and male wild-type and Abcg2 -/- mice. Liquid chromatography coupled with a tandem mass spectrometer (LC-MS/MS) was used for the analysis in a 10-min run time using positive-mode atmospheric pressure electrospray ionization (ESI+) and multiple reaction monitoring (MRM) scanning. For the primary metabolite tested, milk concentrations were 1.8-fold higher in wild-type mice than Abcg2 -/- female lactating mice (P = 0.042) after intravenous administration of MNP. Finally, despite the lack of a difference between groups, we investigated potential differences in MNP and MNPSO 2 's plasma and tissue accumulation levels between wild-type and Abcg2 -/- male mice. In this study, we demonstrated that MNPSO 2 milk levels were affected by Abcg2, with potential pharmacological and toxicological consequences, contributing to the undesirable xenobiotic residues in milk., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Doramectin, an essential animal anthelmintic, is synthesized through the fermentation process of Streptomyces avermitilis. This study delves into the transcriptomic profiles of two strains, namely the doramectin-producing wild-type S. avermitilis N72 and its highly doramectin-producing mutant counterpart, S. avermitilis XY-62. Comparative analysis revealed 860 up-regulated genes and 762 down-regulated genes in the mutant strain, notably impacting the expression of key genes pivotal in doramectin biosynthesis, including aveA1, aveA2, aveA3, aveA4, aveE, and aveBI. These findings shed light on the molecular mechanisms underpinning the heightened doramectin production in S. avermitilis XY-62, presenting promising avenues for optimizing doramectin production processes., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Recently, a S168T variant in the acetylcholine receptor subunit ACR-8 was associated with levamisole resistance in the parasitic helminth Haemonchus contortus. Here, we used the Xenopus laevis oocyte expression system and two-electrode voltage-clamp electrophysiology to measure the functional impact of this S168T variant on the H. contortus levamisole-sensitive acetylcholine receptor, L-AChR-1.1. Expression of the ACR-8 S168T variant significantly reduced the current amplitude elicited by levamisole compared to acetylcholine, with levamisole changing from a full to partial agonist on the recombinant L-AChR. Functional validation of the S168T mutation on modulating levamisole activity at the receptor level highlights its critical importance as both a mechanism and a marker of levamisole resistance., Competing Interests: Declaration of competing interest The authors report no conflict of interest for this work., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Parasitic nematodes are globally important and place a heavy disease burden on infected humans, crops, and livestock, while commonly administered anthelmintics used for treatment are being rendered ineffective by increasing levels of resistance. It has recently been shown in the model nematode Caenorhabditis elegans that the sensory cilia of the amphid neurons play an important role in resistance toward macrocyclic lactones such as ivermectin (an avermectin) and moxidectin (a milbemycin) either through reduced uptake or intertissue signaling pathways. This study interrogated the extent to which ciliary defects relate to macrocyclic lactone resistance and dye-filling defects using a combination of forward genetics and targeted resistance screening approaches and confirmed the importance of intraflagellar transport in this process. This approach also identified the protein trafficking pathways used by the downstream effectors and the components of the ciliary basal body that are required for effector entry into these nonmotile structures. In total, 24 novel C. elegans anthelmintic survival-associated genes were identified in this study. When combined with previously known resistance genes, there are now 46 resistance-associated genes that are directly involved in amphid, cilia, and intraflagellar transport function., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Background: Haemonchus contortus (H. contortus) is the most common parasitic nematode in ruminants and is prevalent worldwide. H. contortus resistance to albendazole (ABZ) hinders the efficacy of anthelmintic drugs, but little is known about the molecular mechanisms that regulate this of drug resistance. Recent research has demonstrated that long noncoding RNAs (lncRNAs) can exert significant influence as pivotal regulators of the emergence of drug resistance., Results: In this study, transcriptome sequencing was conducted on both albendazole-sensitive (ABZ-sensitive) and albendazole-resistant (ABZ-resistant) H. contortus strains, with three biological replicates for each group. The analysis of lncRNA in the transcriptomic data revealed that there were 276 differentially expressed lncRNA (DElncRNA) between strains with ABZ-sensitive and ABZ-resistant according to the criteria of |log2Foldchange|≥ 1 and FDR < 0.05. Notably, MSTRG.12969.2 and MSTRG.9827.1 exhibited the most significant upregulation and downregulation, respectively, in the resistant strains. The potential roles of the DElncRNAs included catalytic activity, stimulus response, regulation of drug metabolism, and modulation of the immune response. Moreover, we investigated the interactions between DElncRNAs and other RNAs, specifically MSTRG.12741.1, MSTRG.11848.1, MSTRG.5895.1, and MSTRG.14070.1, involved in regulating drug stimulation through cis/trans/antisense/lncRNA‒miRNA-mRNA interaction networks. This regulation leads to a decrease (or increase) in the expression of relevant genes, consequently enhancing the resistance of H. contortus to albendazole. Furthermore, through comprehensive analysis of competitive endogenous RNAs (ceRNAs) involved in drug resistance-related pathways, such as the mTOR signalling pathway and ABC transporter signalling pathway, the relevance of the MSTRG.2499.1-novel-m0062-3p-HCON&#95;00099610 interaction was identified to mainly involve the regulation of catalytic activity, metabolism, ubiquitination and transcriptional regulation of gene promoters. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) validation indicated that the transcription profiles of six DElncRNAs and six DEmRNAs were consistent with those obtained by RNA-seq., Conclusions: The results of the present study allowed us to better understand the changes in the lncRNA expression profile of ABZ-resistant H. contortus. In total, these results suggest that the lncRNAs MSTRG.963.1, MSTRG.12741.1, MSTRG.11848.1 and MSTRG.2499.1 play important roles in the development of ABZ resistance and can serve as promising biomarkers for further study., (© 2024. The Author(s).)

Albendazole (ABZ), a benzimidazole-based anthelmintic, is widely used to treat helminth infections. The extensive and improper use of ABZ may cause drug residues in animal-origin food and anthelmintics resistance, which potentially threaten human health. Meanwhile, albendazole sulfoxide (ABZSO), a metabolite of ABZ, also exhibits toxic effects. Therefore, the detection of ABZ and ABZSO in animal-derived food is significantly necessary. Herein, a dual-emission europium fluorescent sensor (EuUHC-30) was rationally designed and constructed. EuUHC-30 exhibits high selectivity and sensitivity towards ABZ and ABZSO with a detection limit of 0.10 and 0.13 μM, respectively. Furthermore, EuUHC-30 was successfully applied for quantification of ABZ and ABZSO in milk and pig kidney, which were verified by HPLC analysis. Moreover, a smartphone-assisted EuUHC-30 fluorescent paper sensor was fabricated for the practical determination of ABZ and ABZSO in real food. Overall, this work provides a visual, rapid, and intelligent method for the detection of ABZ and ABZSO in animal-origin food., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Patients with end‑stage metastatic disease have limited treatment options and those diagnosed with triple negative breast cancer (Her2, Estrogen receptor, Progesterone receptor) have a poor prognosis. Using a triple negative mammary tumor model selected for brain metastasis (4T1Br4) in the mouse, treatment options that may increase survival when therapeutics are applied at post‑metastasis were assessed. Anti‑parasitic benzimidazoles (BZs) destabilize microtubules, inhibit metabolic pathways, reduce cell proliferation, and induce apoptosis in tumor cells. Co‑administration of two BZs was selected, oxfendazole (OFZ) and parbendazole (PBZ), shown to overcome resistance development in anthelmintic effects by imposing metabolic delay to assess if multiple BZ approach is also suitable to enhance anticancer effects. It has been previously reported that treatment of mammary tumor‑bearing mice at an early stage with chitin microparticles (CMPs) decreased tumor growth and metastases by enhancing both innate M1 macrophage and TH1 adaptive immune response. Oral administration of CMPs was previously revealed to affect the gut in intestinal inflammation. A combination BZ (OFZ/PBZ) and CMP treatment was tested to target tumor development and metastasis and effects were compared in response to monotherapies of the same compounds or to untreated mice. The results demonstrated increased survival, decreased tumor cell proliferation, decreased metastasis in lungs and brain, increased levels of fecal SCFAs butyric, acetic, propionic and valeric acids with increased butyric and propionic acid levels in brain biopsies in combination treated compared with untreated mice. At the primary tumor, SCFA receptor FFAR2 expression was increased in combination treatment compared with untreated mice, suggestive of a non‑invasive cancer phenotype. The superior cytotoxic effects of OFZ/PBZ were confirmed as opposed to single treatment with OFZ or PBZ using 3D spheroids generated from a human breast cancer cell line, MDA‑MB‑468. These data are compelling for treatment option possibility even at late stages of metastasized breast cancer.

Anthelmintic (AH) compounds are used to control gastrointestinal nematodes (GINs) in livestock production. They are only partially metabolized in animals ending in animal excreta whose use as manures leads to AH dispersal in agricultural soils. Once in soil, AHs interact with soil microorganisms, with the outcome being either detrimental, or beneficial. We aimed to disentangle the mechanisms of these complex interactions. Two soils previously identified as « fast » or « slow», regarding the degradation of albendazole (ABZ), ivermectin (IVM), and eprinomectin (EPM), were subjected to repeated applications at two dose rates (1, 2 mg kg -1 and 10, 20 mg kg -1 ). We hypothesized that this application scheme will lead to enhanced biodegradation in «fast » soils and accumulation and toxicity in «slow » soils. Repeated application of ABZ resulted in different transformation pathways in the two soils and a clear acceleration of its degradation in the «fast » soil only. In contrast residues of IVM and EPM accumulated in both soils. ABZ was the sole AH that induced a consistent reduction in the abundance of total fungi and crenarchaea. In addition, inhibition of nitrification and reduction in the abundance of ammonia-oxidizing bacteria (AOB) and archaea (AOA) by all AHs was observed, while commamox bacteria were less responsive. Amplicon sequencing analysis showed dose-depended shifts in the diversity of bacteria, fungi, and protists in response to AHs application. ABZ presented the most consistent effect on the abundance and diversity of most microbial groups. Our findings provide first evidence for the unexpected toxicity of AHs on key soil microbial groups that might have to be considered in a regulatory context., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Haemonchus contortus (H. contortus) has developed resistance to nearly all available anthelmintic medications. Hence, alternative strategies are required to counter anthelmintic resistance. The present study investigated the anthelmintic potential of Bacillus thuringiensis (B. thuringiensis) against H. contortus. Bacterial spp were identified by conventional methods and confirmed by PCR; In addition, PCR amplification of the bacterial 16S rRNA gene detected B. thuringiensis at 750 base pairs (bps). The amplified products were sequenced, and the sequence data were confirmed using the Basic Local Alignment Tool (BLAST), which showed a significant alignment (97.98%) with B. thuringiensis and B. cereus. B. thuringiensis were selected to isolate purified crystal proteins (toxins), The protein profile confirmed by SDS-PAGE showed three prominent bands at 70, 36, and 15 kDa. In addition, the larval development of H. contortus was examined in vitro using two different treatments. Purified crystal protein diluted in 10 mM NaCl at a concentration of 2 mg/ml significantly reduced (P < 0.001) larval development by 75.10% compared to 1 × 10 8  CFU/ml spore-crystal suspension reduced (43.97%). The findings of in vitro experiments indicated that purified crystal protein was more toxic to the H. contortus larva than the spore-crystal suspension and control group. Moreover, To test the antinematodal effects of B. thuringiensis toxins in vivo, we chose 12 male goats (6 months old) and reared these animals in parasite-free conditions. We performed Fecal egg count reduction tests (FECRT) on samples collected before and after treatment at various times denotes 48 h post-treatment with Purified crystal proteins was significantly decreased (842 ± 19.07) EPG compared to 24 (2560 ± 233.66) and 12 h (4020 ± 165.22). Similarly, after 48 h of treatment, the FECRT of the Spores-crystal mix was reduced (2920 ± 177.20) EPG followed by 24- and 12-h denotes (4500 ± 137.84) and (4760 ± 112.24), respectively. Results of the above experiment suggested that purified crystal proteins have more anthelmintic potential in vivo. Current findings determine that B. thuringiensis toxin against H. contortus could be used in small ruminants to counter anthelmintic resistance. This study also suggested that future research structured on these proteins' pharmacokinetics and mode of action., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Fenbendazole (FBZ), a benzymidazole (BZD) anthelmintic drug, is used for in-feed medication in pigs. BZD-containing drugs may induce cytochrome P450 isozymes (CYPs), particularly those members of the CYP1A subfamily. The current research evaluated the plasma and liver availability and metabolism of FBZ and its metabolites, oxfendazole (OFZ) and fenbendazole sulphone (FBZSO 2 ), after the administration of the parent drug in feed, and characterized the effect of the sustained administration of the anthelmintic on the catalytic activities of xenobiotic metabolizing enzymes in pig liver. Five female Landrace piglets remained untreated (controls), and other six were treated with a pre-mix of FBZ, combined with feed, for 9 consecutive days as usually is recommended. Blood samples were collected from each treated animal up to day 9 and analyzed by HPLC; all animals were slaughtered for preparation of liver microsomes. Plasma concentration ratios OFZ/FBZ and FBZSO 2 /OFZ increased significantly (p < 0.05) from the beginning to the end of drug exposure, which may indicate an enhanced conversion of FBZ into its metabolites. FBZ represented 45.8 ± 3.4% of the total anthelmintic molecules in liver tissue. Increased CYP1A-dependent 7-ethoxy (24.5-fold, p = 0.0032) and 7-methoxyresorufin (17.2-fold, p = 0.0006) O-dealkylase activities was observed in liver microsomes from FBZ-treated animals. In addition, a 64% increase (p = 0.042) in the rate of FBZ S-oxidation was observed in pigs treated with the anthelmintic drug compared to that measured in untreated animals. Thus, the continuous FBZ administration may accelerate its own in vivo hepatic metabolism through the CYP1A pathway., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The anthelmintic paraherquamide A acts selectively on the nematode L-type nicotinic acetylcholine receptors (nAChRs), but the mechanism of its selectivity is unknown. This study targeted the basis of paraherquamide A selectivity by determining an X-ray crystal structure of the acetylcholine binding protein (AChBP), a surrogate nAChR ligand-binding domain, complexed with the compound and by measuring its actions on wild-type and mutant Caenorhabditis elegans nematodes and functionally expressed C. elegans nAChRs. Paraherquamide A showed a higher efficacy for the levamisole-sensitive [L-type (UNC-38/UNC-29/UNC-63/LEV-1/LEV-8)] nAChR than the nicotine-sensitive [N-type (ACR-16)] nAChR, a result consistent with in vivo studies on wild-type worms and worms with mutations in subunits of these two classes of receptors. The X-ray crystal structure of the Ls -AChBP-paraherquamide A complex and site-directed amino acid mutation studies showed for the first time that loop C, loop E, and loop F of the orthosteric receptor binding site play critical roles in the observed L-type nAChR selective actions of paraherquamide A. SIGNIFICANCE STATEMENT: Paraherquamide A, an oxindole alkaloid, has been shown to act selectively on the L-type over N-type nAChRs in nematodes, but the mechanism of selectivity is unknown. We have co-crystallized paraherquamide A with the acetylcholine binding protein, a surrogate of nAChRs, and found that structural features of loop C, loop E, and loop F contribute to the L-type nAChR selectivity of the alkaloid. The results create a new platform for the design of anthelmintic drugs targeting cholinergic neurotransmission in parasitic nematodes., Competing Interests: No author has an actual or perceived conflict of interest with the contents of this article., (Copyright © 2023 by The Author(s).)

Albendazole (ABZ), a broad-spectrum anthelmintic drug frequently used in livestock against parasitic worms (helminths), enters the environment mainly via faeces of treated animals left in the pastures or used as dung for field fertilization. To obtain information about the subsequent fate of ABZ, the distribution of ABZ and its metabolites in the soil around faeces along with uptake and effects in plants were monitored under real agricultural conditions. Sheep were treated with a recommended dose of ABZ; faeces were collected and used to fertilize fields with fodder plants. Soil samples (in two depths) and samples of two plants, clover (Trifolium pratense) and alfalfa (Medicago sativa), were collected at distances 0-75 cm from the faeces for 3 months after fertilization. The environmental samples were extracted using QuEChERS and LLE sample preparation procedures. The targeted analysis of ABZ and its metabolites was conducted by using the validated UHPLC-MS method. Two main ABZ metabolites, ABZ-sulfoxide (anthelmintically active) and ABZ-sulfone (inactive), persisted in soil (up to 25 cm from faeces) and in plants for three months when the experiment ended. In plants, ABZ metabolites were detected even 60 cm from the faeces and abiotic stress was observed in the central plants. The considerable distribution and persistence of ABZ metabolites in soil and plants amplify the negative environmental impact of ABZ documented in other studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Praziquantel (PZQ) is the drug of choice for the treatment of all forms of schistosomiasis, although its mechanisms of action are not completely understood. PZQ acts largely on adult worms. This narrative literature review describes what is known about the mechanisms of action of PZQ against schistosomes from in vitro and in vivo studies and highlights the molecular targets in parasites and immune responses induced in definitive hosts by this drug. Moreover, new therapeutic uses of PZQ are discussed. Studies have demonstrated that in addition to impacting voltage-operated Ca2 + channels, PZQ may interact with other schistosome molecules, such as myosin regulatory light chain, glutathione S-transferase, and transient receptor potential channels. Following PZQ administration, increased T regulatory type 1 (Tr1) cell differentiation and decreased inflammation were observed, indicating that PZQ promotes immunoregulatory pathways. Although PZQ is widely used in mass drug administration schemes, the existence of resistant parasites has not been proven; however, it is a concern that should be constantly investigated in human populations. In addition, we discuss studies that evaluate health applications of PZQ (other than helminth infection), such as its effect in cancer therapy and its adjuvant action in vaccines against viruses., Competing Interests: Conflicts of interest The authors declare there are no conflicts of interest., (Copyright © 2022. Published by Elsevier B.V.)

Fenbendazole (FBZ), a benzimidazole carbamate anthelmintic, has attracted attention for its antitumor activity. This study examined the metabolic characteristics of FBZ in humans compared with those in dogs. The phase I metabolites were identified in liver microsomal incubates using liquid chromatography-mass spectrometry (MS)-based untargeted metabolomics approaches. Seven metabolites of FBZ were identified by principal component analysis and orthogonal partial least square-discriminant analysis based on the global ion variables of the FBZ incubation groups. The chemical structure of the FBZ metabolites was suggested by examining the MS/MS spectrum and isotope distribution pattern. Cytochrome P450 (CYP) 1A1, CYP2D6, and CYP2J2 were the major isozymes responsible for the FBZ metabolism. No differences in the types of metabolites produced by the two species were noted. Multivariate analysis of human and dog incubation groups showed that five metabolites were relatively abundant in humans and the other two were not. In summary, the phase I metabolic profile of FBZ and the comparative metabolism between humans and dogs were examined using an untargeted metabolomics approach. This study suggests a successful investigation of FBZ metabolism in humans for conducting safety assessments regarding drug repositioning.

The ubiquitin-mediated pathway has been comprehensively explored in the free-living nematode Caenorhabditis elegans, but very little is known about this pathway in parasitic nematodes. Here, we inferred the ubiquitination pathway for an economically significant and pathogenic nematode - Haemonchus contortus - using abundant resources available for C. elegans. We identified 215 genes encoding ubiquitin (Ub; n = 3 genes), ubiquitin-activating enzyme (E1; one), -conjugating enzymes (E2s; 21), ligases (E3s; 157) and deubiquitinating enzymes (DUBs; 33). With reference to C. elegans, Ub, E1 and E2 were relatively conserved in sequence and structure, and E3s and DUBs were divergent, likely reflecting functional and biological uniqueness in H. contortus. Most genes encoding ubiquitination pathway components exhibit high transcription in the egg compared with other stages, indicating marked protein homeostasis in this early developmental stage. The ubiquitination pathway model constructed for H. contortus provides a foundation to explore the ubiquitin-proteasome system, crosstalk between autophagy and the proteasome system, and the parasite-host interactions. Selected E3 and DUB proteins which are very divergent in sequence and structure from host homologues or entirely unique to H. contortus and related parasitic nematodes may represent possible anthelmintic targets., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Gaining insight into molecular signalling pathways of socioeconomically important parasitic nematodes has implications for understanding their molecular biology and for developing novel anthelmintic interventions., Methods: Here, we evaluated the use of a human antibody-based microarray to explore conserved elements of the signalome in the barber's pole worm Haemonchus contortus. To do this, we prepared extracts from mixed-sex (female and male) adult worms and third-stage larvae (L3s), incubated these extracts on the antibody microarray and then measured the amounts of antibody-bound proteins ('signal intensity')., Results: In total, 878 signals were classified into two distinct categories: signals that were higher for adults than for larvae of H. contortus (n = 376), and signals that were higher for larvae than for adults of this species (n = 502). Following a data-filtering step, high confidence ('specific') signals were obtained for subsequent analyses. In total, 39 pan-specific signals (linked to antibodies that recognise target proteins irrespective of their phosphorylation status) and 65 phosphorylation-specific signals were higher in the adult stage, and 82 pan-specific signals and 183 phosphorylation-specific signals were higher in L3s. Thus, notably more signals were higher in L3s than in the adult worms. Using publicly available information, we then inferred H. contortus proteins that were detected (with high confidence) by specific antibodies directed against human homologues, and revealed relatively high structural conservation between the two species, with some variability for select proteins. We also in silico-matched 763 compound structures (listed in the DrugBank and Kinase SARfari public databases) to four H. contortus proteins (designated HCON&#95;00005760, HCON&#95;00079680, HCON&#95;00013590 and HCON&#95;00105100)., Conclusions: We conclude that the present antibody-based microarray provides a useful tool for comparative analyses of signalling pathways between/among developmental stages and/or species, as well as opportunities to explore nematocidal target candidates in H. contortus and related parasites., (© 2022. The Author(s).)

Abstract: Benzimidazoles are frequently and widely used veterinary anthelmintics. Unfortunately, an administration of these anthelmintics does not always result in the expected therapeutic success. Many host-related factors modify pharmacokinetic behavior and efficacy of a chosen anthelmintic. Pharmacokinetics of anthelmintics varies among animals of different species, sex and age. Also diseases, medication, feed and environmental conditions can significantly affect behavior of anthelmintics and resultant drug efficacy in animals. The presented review gathers information, gained in last 20years, on factors which bring about the variability in performance of benzimidazole anthelmintics in food-producing animals. It is focused particularly on differences in absorption and metabolism of these anthelmintics as these stages of the pharmacokinetic process seem to be the most important for the overall anthelmintic efficacy. The consequences of abnormalities and alterations in pharmacokinetics of benzimidazole anthelmintics are summarized and discussed. [Copyright &y& Elsevier]

Veterinary anthelmintics excreted from treated animals pass to soil, subsequently to plants and then to their consumers. This circulation might have various consequences, including drug-resistance promotion in helminths. The present study was designed to follow the effect of the environmental circulation of the common anthelmintic drug albendazole (ABZ) in real farm conditions on the parasitic nematode Haemonchus contortus in vivo. Two fields with fodder plants (clover and alfalfa) were fertilized, the first with dung from ABZ-treated sheep (at the recommended dosage), the second with dung from non-treated sheep (controls). After a 10-week growth period, the fresh fodder from both fields was used to feed two groups of sheep, which were infected with H. contortus. Eggs and adult nematodes from the animals of both groups were isolated, and various parameters were compared. No significant changes in the eggs' sensitivity to ABZ and thiabendazole were observed. However, significantly increased expression of several cytochromes P450 and UDP-glycosyl transferases as well as increased oxidation and glycosylation of ABZ and ABZ-sulfoxide (ABZ-SO) was found in the exposed nematodes. These results show that ABZ environmental circulation improves the ability of the helminths to deactivate ABZ., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Ion channels are specialized multimeric proteins that underlie cell excitability. These channels integrate with a variety of neuromuscular and biological functions. In nematodes, the physiological behaviors including locomotion, navigation, feeding and reproduction, are regulated by these protein entities. Majority of the antinematodal chemotherapeutics target the ion channels to disrupt essential biological functions. Here, we have summarized current advances in our understanding of nematode ion channel pharmacology. We review cys-loop ligand gated ion channels (LGICs), including nicotinic acetylcholine receptors (nAChRs), acetylcholine-chloride gated ion channels (ACCs), glutamate-gated chloride channels (GluCls), and GABA (γ-aminobutyric acid) receptors, and other ionotropic receptors (transient receptor potential (TRP) channels and potassium ion channels). We have provided an update on the pharmacological properties of these channels from various nematodes. This article catalogs the differences in ion channel composition and resulting pharmacology in the phylum Nematoda. This diversity in ion channel subunit repertoire and pharmacology emphasizes the importance of pursuing species-specific drug target research. In this review, we have provided an overview of recent advances in techniques and functional assays available for screening ion channel properties and their application., (Copyright © 2021. Published by Elsevier Ltd.)

Praziquantel (PZQ) is a pyrazino-isoquinoline compound with broad spectrum of activity against parasitic trematodes and cestodes, and a key veterinary drug in the parasitic disease control field. However, PZQ residues caused by non-conforming or excessive use in food-producing animals may pose a serious threat to human health. Herein, a simple, sensitive and reproducible LC-MS/MS method was developed for the simultaneous determination of praziquantel and trans - and cis -4-hydroxypraziquantel in black goat tissues to guide the reasonable use of PZQ. The mean recoveries for three target analytes were 71.2 ∼ 117.6%, and the limits of quantification were 1.0 μg/kg. Twenty-five healthy black goats were administered a single dose of praziquantel tablets at a dose of 35 mg/kg of body weight for residue elimination study, The results revealed that praziquantel and 4-hydroxypraziquantel were rapidly depleted in goat tissues and the elimination half-lives did not exceed 1 day in all tissues except for muscle and lung. It provides guidance for the establishment of maximum residue limit of praziquantel in goat.

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma. Mono-therapeutic treatment of this disease with the drug praziquantel, presents challenges such as inactivity against immature worms and inability to prevent reinfection. Importantly, ion channels are important targets for many current anthelmintics. Transient receptor potential (TRP) channels are important mediators of sensory signals with marked effects on cellular functions and signaling pathways. TRPML channels are a class of Ca 2+ -permeable TRP channels expressed on endolysosomal membranes. They regulate lysosomal function and trafficking, among other functions. Schistosoma mansoni is predicted to have a single TRPML gene (SmTRPML) with two splice variants differing by 12 amino acids. This study focuses on exploring the physiological properties of SmTRPML channels to better understand their role in schistosomes. In mammalian cells expressing SmTRPML, TRPML activators elicit a rise in intracellular Ca 2+ . In these cells, SmTRPML localizes both to lysosomes and the plasma membrane. These same TRPML activators elicit an increase in adult worm motility that is dependent on SmTRPML expression, indicating a role for these channels in parasite neuromuscular activity. Suppression of SmTRPML in adult worms, or exposure of adult worms to TRPML inhibitors, results in tegumental vacuolations, balloon-like surface exudates, and membrane blebbing, similar to that found following TRPML loss in other organisms. Together, these findings indicate that SmTRPML may regulate the function of the schistosome endolysosomal system. Further, the role of SmTRPML in neuromuscular activity and in parasite tegumental integrity establishes this channel as a candidate anti-schistosome drug target., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2022 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Entomopathogenic Photorhabdus bacteria (Enterobacteriaceae: Gamma-proteobacteria), the natural symbionts of Heterorhabditis nematodes, are a rich source for the discovery of biologically active secondary metabolites (SMs). This study describes the isolation of three nematicidal SMs from in vitro culture supernatants of the Arizona-native Photorhabdus luminescens sonorensis strain Caborca by bioactivity-guided fractionation. Nuclear magnetic resonance spectroscopy and comparison to authentic synthetic standards identified these bioactive metabolites as trans -cinnamic acid ( t -CA), (4 E )-5-phenylpent-4-enoic acid (PPA), and indole. PPA and t- CA displayed potent, concentration-dependent nematicidal activities against the root-knot nematode (Meloidogyne incognita) and the citrus nematode ( Tylenchulus semipenetrans ), two economically and globally important plant parasitic nematodes (PPNs) that are ubiquitous in the United States. Southwest. Indole showed potent, concentration-dependent nematistatic activity by inducing the temporary rigid paralysis of the same targeted nematodes. While paralysis was persistent in the presence of indole, the nematodes recovered upon removal of the compound. All three SMs were found to be selective against the tested PPNs, exerting little effects on non-target species such as the bacteria-feeding nematode Caenorhabditis elegans or the entomopathogenic nematodes Steinernema carpocapsae, Heterorhabditis bacteriophora, and Hymenocallis sonorensis . Moreover, none of these SMs showed cytotoxicity against normal or neoplastic human cells. The combination of t- CA + PPA + indole had a synergistic nematicidal effect on both targeted PPNs. Two-component mixtures prepared from these SMs revealed complex, compound-, and nematode species-dependent interactions. These results justify further investigations into the chemical ecology of Photorhabdus SMs, and recommend t- CA, PPA and indole, alone or in combinations, as lead compounds for the development of selective and environmentally benign nematicides against the tested PPNs. IMPORTANCE Two phenylpropanoid and one alkaloid secondary metabolites were isolated and identified from culture filtrates of Photorhabdus l. sonorensis strain Caborca. The three identified metabolites showed selective nematicidal and/or nematistatic activities against two important plant parasitic nematodes, the root-knot nematode (Meloidogyne incognita) and the citrus nematode ( Tylenchulus semipenetrans ). The mixture of all three metabolites had a synergistic nematicidal effect on both targeted nematodes, while other combinations showed compound- and nematode-dependent interactions.

Background: Helminth infections in animals to be consumed by humans are an important medical and public health problem. Pharmaceutical research has focused on developing new anthelmintic drugs for parasite control in these animals. However, the incorrect use of anthelmintics can leave residues in animal products intended for human consumption. Their determination is therefore crucial in terms of food safety., Results: In this work, a simple and sensitive method has been developed for the analysis of anthelmintic drugs in milk. The method involves extraction of the analytes using a QuEChERS (quick, easy, cheap, effective, rugged, and safe) method, and separation and determination by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The use of a core-shell column significantly reduced the analysis time compared with conventional columns. The method was validated and applied to the analysis of different commercial milk samples: whole, semi-skimmed and skimmed cows' milk, and goats' milk. None of the benzimidazoles studied was found in the samples analyzed, so these were spiked with the analytes at three concentration levels (10, 50, and 100 μg kg -1 )., Conclusions: The proposed method provided high sensitivity compared with other methods for the determination of anthelmintics in milk samples, at concentration levels well below the established maximum residue limit (MRLs) values. The proposed method is simple, easy, precise, accurate, and leads to good recovery levels. It can be used successfully for the routine analysis. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Background: Alveolar echinococcosis (AE) is a chronic zoonosis caused by the larval form of Echinococcus multilocularis (E. multilocularis). Current chemotherapy against AE has relied on albendazole and mebendazole, which only exhibit parasitostatic and not parasiticidal efficacy. Therefore, novel compounds for the treatment of this disease are needed., Methods: Phosphoglucose isomerase (PGI) assays were used for compound screening of seven neonicotinoids. The anti-parasitic effects of thiacloprid were then evaluated on E. multilocularis metacestode vesicles, germinal cells and protoscoleces in vitro. Human foreskin fibroblasts (HFF) and Reuber rat hepatoma (RH) cells were used to assess cytotoxicity. Glucose consumption in E. multilocularis protoscoleces and germinal cells was assessed by measuring uptake of 2-deoxyglucose (2-DG). Molecular docking was used to evaluate the potential binding sites of thiacloprid to acetylcholine receptors. In vivo efficacy of thiacloprid was evaluated in mice by secondary infection with E. multilocularis. In addition, ELISA and flow cytometry were used to evaluate the effects of cytokines and T lymphocyte subsets after thiacloprid treatment. Furthermore, collagen deposition and degradation in the host lesion microenvironment were evaluated., Results: We found that thiacloprid is the most promising compound, with an IC 50 of 4.54 ± 1.10 μM and 2.89 ± 0.34 μM, respectively, against in vitro-cultured E. multilocularis metacestodes and germinal cells. Thiacloprid was less toxic for HFF and RH mammalian cell lines than for metacestodes. In addition, thiacloprid inhibited the acetylcholinesterase activity in protoscoleces, metacestodes and germinal cells. Thiacloprid inhibited glucose consumption by protoscoleces and germinal cells. Subsequently, transmission electron microscopy revealed that treatment with thiacloprid damaged the germinal layer. In vivo, metacestode weight was significantly reduced following oral administration of thiacloprid at 15 and 30 mg/kg. The level of CD4 + T lymphocytes in metacestodes and spleen increased after thiacloprid treatment. Anti-echinococcosis-related cytokines (IL-2, IL-4, IL-10) were significantly increased. Furthermore, thiacloprid inhibited the expression of matrix metalloproteinases (MMPs 1, 3, 9, 13) and promoted collagen deposition in the host lesion microenvironment., Conclusions: The results demonstrated that thiacloprid had parasiticidal activity against E. multilocularis in vitro and in vivo, and could be used as a novel lead compound for the treatment of AE., (© 2021. The Author(s).)

In the present study, fifteen benzimidazolyl-2-hydrazones 7a-7o of fluoro-, hydroxy- and methoxy-substituted benzaldehydes and 1,3-benzodioxole-5-carbaldehyde were synthesized and their structure was identified by IR, NMR, and elemental analysis. The compounds 7j 2-(3-hydroxybenzylidene)-1-(5(6)-methyl-1H-benzimidazol-2-yl)hydrazone and 7i 2-(3-hydroxybenzylidene)-1-(1H-benzimidazol-2-yl)hydrazone have exerted the strongest anthelmintic activity (100% after 24 h incubation period at 37 °C) against isolated muscle larvae of Trichinella spiralis in an in vitro experiment. The in vitro cytotoxicity assay towards MCF-7 breast cancer cells and mouse embryo fibroblasts 3T3 showed that the studied benzimidazolyl-2-hydrazones exhibit low to moderate cytotoxic effects. The ability of the studied benzimidazolyl-2-hydrazones to modulate microtubule polymerization was confirmed and suggested that their anthelmintic action is mediated through inhibition of the tubulin polymerization likewise the other known benzimidazole anthelmitics. It was also shown that the four most promising benzimidazolyl-2-hydrazones do not affect significantly the AChE activity even at high tested concentration, thus indicating that they do not have the potential for neurotoxic effects. The binding mode of compounds 7j and 7n in the colchicine-binding site of tubulin were clarified by molecular docking simulations. Taken together, these results demonstrate that for the synthesized benzimidazole derivatives the anthelmintic activity against T. spiralis and the inhibition of tubulin polymerization are closely related., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The high-affinity choline transporter CHT1 mediates choline uptake, the rate-limiting and regulatory step in acetylcholine synthesis at cholinergic presynaptic terminals. CHT1-medated choline uptake is specifically inhibited by hemicholinium-3, which is a type of choline analog that acts as a competitive inhibitor. Although the substrate choline and the inhibitor hemicholinium-3 are well-established ligands of CHT1, few potent ligands other than choline analogs have been reported. Here we show that tetrahydropyrimidine anthelmintics, known as nicotinic acetylcholine receptor agonists, act as competitive inhibitors of CHT1. A ligand-dependent trafficking assay in cell lines expressing human CHT1 was designed to search for CHT1 ligands from a collection of biologically active compounds. We found that morantel as well as other tetrahydropyrimidines, pyrantel and oxantel, potently inhibits the high-affinity choline uptake activity of CHT1 in a competitive manner similar to the inhibitor hemicholinium-3. They also inhibit the high-affinity choline transporter from the nematode Caenorhabditis elegans. Finally, tetrahydropyrimidines potently inhibit the high-affinity choline uptake in rat brain synaptosomes at a low micromolar level, resulting in the inhibition of acetylcholine synthesis. The rank order of potency in synaptosomes is as follows: morantel > pyarantel > oxantel (K i  = 1.3, 5.7, and 8.3 μM, respectively). Our results reveal that tetrahydropyrimidine anthelmintics are novel CHT1 ligands that inhibit the high-affinity choline uptake for acetylcholine synthesis in cholinergic neurons., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Parascaris univalens is a pathogenic parasite of foals and yearlings worldwide. In recent years, Parascaris spp. worms have developed resistance to several of the commonly used anthelmintics, though currently the mechanisms behind this development are unknown. The aim of this study was to investigate the transcriptional responses in adult P. univalens worms after in vitro exposure to different concentrations of three anthelmintic drugs, focusing on drug targets and drug metabolising pathways., Methods: Adult worms were collected from the intestines of two foals at slaughter. The foals were naturally infected and had never been treated with anthelmintics. Worms were incubated in cell culture media containing different concentrations of either ivermectin (10 -9  M, 10 -11  M, 10 -13  M), pyrantel citrate (10 -6  M, 10 -8  M, 10 -10  M), thiabendazole (10 -5  M, 10 -7  M, 10 -9  M) or without anthelmintics (control) at 37 °C for 24 h. After incubation, the viability of the worms was assessed and RNA extracted from the anterior region of 36 worms and sequenced on an Illumina NovaSeq 6000 system., Results: All worms were alive at the end of the incubation but showed varying degrees of viability depending on the drug and concentration used. Differential expression (Padj < 0.05 and log2 fold change ≥ 1 or ≤ - 1) analysis showed similarities and differences in the transcriptional response after exposure to the different drug classes. Candidate genes upregulated or downregulated in drug exposed worms include members of the phase I metabolic pathway short-chain dehydrogenase/reductase superfamily (SDR), flavin containing monooxygenase superfamily (FMO) and cytochrome P450-family (CYP), as well as members of the membrane transporters major facilitator superfamily (MFS) and solute carrier superfamily (SLC). Generally, different targets of the anthelmintics used were found to be upregulated and downregulated in an unspecific pattern after drug exposure, apart from the GABA receptor subunit lgc-37, which was upregulated only in worms exposed to 10 -9  M of ivermectin., Conclusions: To our knowledge, this is the first time the expression of lgc-37 and members of the FMO, SDR, MFS and SLC superfamilies have been described in P. univalens and future work should be focused on characterising these candidate genes to further explore their potential involvement in drug metabolism and anthelmintic resistance.

Moxidectin (MOX) is a widely used anthelmintic drug for the treatment of internal and external parasites in food-producing and companion animals. Transformation products (TPs) of MOX, formed through metabolic degradation or acid hydrolysis, may pose a potential environmental risk, but only few were identified so far. In this study, we therefore systematically characterized electro- and photochemically generated MOX TPs using high-resolution mass spectrometry (HRMS). Oxidative electrochemical (EC) TPs were generated in an electrochemical reactor and photochemical (PC) TPs by irradiation with UV-C light. Subsequent HRMS measurements were performed to identify accurate masses and deduce occurring modification reactions of derived TPs in a suspected target analysis. In total, 26 EC TPs and 59 PC TPs were found. The main modification reactions were hydroxylation, (de-)hydration, and derivative formation with methanol for EC experiments and isomeric changes, (de-)hydration, and changes at the methoxime moiety for PC experiments. In addition, several combinations of different modification reactions were identified. For 17 TPs, we could predict chemical structures through interpretation of acquired MS/MS data. Most modifications could be linked to two specific regions of MOX. Some previously described metabolic reactions like hydroxylation or O-demethylation were confirmed in our EC and PC experiments as reaction type, but the corresponding TPs were not identical to known metabolites or degradation products. The obtained knowledge regarding novel TPs and reactions will aid to elucidate the degradation pathway of MOX which is currently unknown. Graphical abstract.

Combinations of bioactive phytochemicals with synthetic compounds have been suggested as promissory tools for the improvement of nematode control in livestock. Bioactive phytochemicals may interfere with the activity of drug-metabolizing enzymes and delay the metabolic conversion of anthelmintics into less potent metabolites.This research assessed the effect of the monoterpene thymol (TML) on the in vitro hepatic metabolism of the anthelmintic albendazole (ABZ) in sheep.Liver microsomes from four (4) Texel lambs were incubated with ABZ (50 µM) in absence or in presence of TML (0.05-10 mM).The concentration of TML producing a 50% decrease in ABZ S-oxygenation (IC50) was 13.5 mM. The FMO-dependent S-oxygenation of ABZ was markedly inhibited by the monoterpene (54.1 ± 11.6%, p  < .01). In agreement with this observation, TML produced a marked inhibition of benzydamine (BZ) N-oxidase, a specific FMO activity.The CYP-dependent production of the sulfoxide metabolite (ABZSO) was less affected, being 25.3 ± 17.5 lower ( p  < .05) in presence of TML. Additionally, TML completely abolished the specific CYP1A1-dependent enzyme activity 7-ethoxyresorufin O-deethylase.Overall, the results presented here show that, in addition to its own anthelmintic affect, TML may potentiate ABZ anthelmintic activity by preventing its metabolic conversion into a less active metabolite.

Veterinary drugs enter the environment in many ways and may affect non-target organisms, including plants. The present project was focused on the biotransformation of ivermectin (IVM), one of the mostly used anthelmintics, in the model plant Arabidopsis thaliana. Our results certified the ability of plants to uptake IVM by roots and translocate it to the aboveground parts. Using UHPLC-MS/MS, six metabolites in roots and only the parent drug in rosettes were found after 24- and 72-h incubation of A. thaliana with IVM. The metabolites were formed only via hydroxylation and demethylation, with no IVM conjugates detected. Although IVM did not induce changes in the activity of antioxidant enzymes in A. thaliana rosettes, the expression of genes was significantly affected. Surprisingly, a higher number of transcripts, 300 and 438, respectively, was dysregulated in the rosettes than in roots. The significantly affected genes play role in response to salt, osmotic and water deprivation stress, in response to pathogens and in ion homeostasis. We hypothesize that the above described changes in gene transcription in A. thaliana resulted from disrupted ionic homeostasis caused by certain ionophore properties of IVM. Our results underlined the negative impact of IVM presence in the environment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Aim: The study sought to determine the effect of ketoconazole (KTZ) on the pharmacokinetics of praziquantel (PZQ) and on the formation of its major hydroxylated metabolites, cis- and trans-4-OH-PZQ, and X-OH-PZQ in healthy subjects., Methods: Two treatments were evaluated by single-dose PK studies; the reference treatment was a 20 mg/kg dose of praziquantel given alone. The test treatment was a 20 mg/kg dose of praziquantel given in combination with 200 mg of ketoconazole. The study had a balanced and randomised cross-over design. Serial blood samples were collected between 0 and 12 h after each drug administration. PZQ, and cis- and trans-4-OH-PZQ and X-OH-PZQ concentrations in plasma were determined by LC-MS. A non-compartmental approach was used for pharmacokinetic analysis. Data were analysed using ANOVA and assessment of the 90% confidence interval of the geometric means of the log-transformed PK parameters obtained for each treatment., Results: The pharmacokinetics of PZQ following the two treatments, PZQ alone and PZQ + KTZ, were not equivalent based on the assessment of the 90% CI of the geometric mean ratios of the AUC and C max (α = 0.05). The geometric mean ratios of the AUC and C max were found to be 176.8% and 227% respectively. The 90% CI of the AUC and C max were found to be 129.8%-239.8% and 151.4%-341.4% respectively. The AUC of PZQ was increased by 75% with KTZ co-administration (3516 vs 6172 ng h/ml) (p < 0.01). Meanwhile, the mean AUC of trans-4-OH-PZQ increased by 67% (61,749 ng h/ml vs 103,105 ng h/ml) (p < 0.01). X-OH-PZQ levels were reduced by about 57% (semi-quantified as 7311 ng h/ml vs 3109 ng h/ml by using trans-4-OH as standards) (p < 0.01) with KTZ co-administration., Conclusions: The relative bioavailability of praziquantel was increased by concomitant KTZ administration. KTZ preferentially inhibited the formation of X-OH-PZQ rather than 4-OH-PZQ, confirming in vitro data which implicates CYP3A4 in the formation of X-OH-PZQ rather than 4-OH-PZQ. The 4-hydroxylation of PZQ was shown to be the major metabolic pathway of PZQ, as evidenced by larger quantities of 4-OH-PZQ produced, thus explaining the modest albeit significant effect of ketoconazole on PZQ pharmacokinetics.

Praziquantel (PZQ) is one of the most widespread anthelmintic drugs. However, the frequent insufficient application of PZQ after oral administration is associated with its low solubility, penetration rate, and bioavailability. In the present study, the permeation of PZQ through a 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC) membrane was investigated to probe glycyrrhizin-assisted transport. Glycyrrhizin (or glycyrrhizic acid, GA), a natural saponin, shows the ability to enhance the therapeutic activity of various drugs when it is used as a drug delivery system. However, the molecular mechanism of this effect is still under debate. In the present study, the transport rate was measured experimentally by a parallel artificial membrane permeation assay (PAMPA) and molecular dynamics (MD) simulation with DOPC lipid bilayers. The formation of the noncovalent supramolecular complex of PZQ with disodium salt of GA (Na2GA) in an aqueous solution was proved by the NMR relaxation technique. PAMPA experiments show a strong increase in the amount of the penetrating praziquantel molecules in comparison with a saturated aqueous solution of pure drug used as a control. MD simulation of PZQ penetration through the bilayer demonstrates an increase in permeability into the membrane in the presence of a glycyrrhizin molecule. A decrease in the free energy barrier in the middle of the lipid bilayer was obtained, associated with the hydrogen bond between PZQ and GA. Also, GA reduces the local bilayer surface resistance to penetration of PZQ by rearranging the surface lipid headgroups. This study clarifies the mechanism of increasing the drug's bioavailability in the presence of glycyrrhizin.

Targeting chokepoint enzymes in metabolic pathways has led to new drugs for cancers, autoimmune disorders and infectious diseases. This is also a cornerstone approach for discovery and development of anthelmintics against nematode and flatworm parasites. Here, we performed omics-driven knowledge-based identification of chokepoint enzymes as anthelmintic targets. We prioritized 10 of 186 phylogenetically conserved chokepoint enzymes and undertook a target class repurposing approach to test and identify new small molecules with broad spectrum anthelmintic activity. First, we identified and tested 94 commercially available compounds using an in vitro phenotypic assay, and discovered 11 hits that inhibited nematode motility. Based on these findings, we performed chemogenomic screening and tested 32 additional compounds, identifying 6 more active hits. Overall, 6 intestinal (single-species), 5 potential pan-intestinal (whipworm and hookworm) and 6 pan-Phylum Nematoda (intestinal and filarial species) small molecule inhibitors were identified, including multiple azoles, Tadalafil and Torin-1. The active hit compounds targeted three different target classes in humans, which are involved in various pathways, including carbohydrate, amino acid and nucleotide metabolism. Last, using representative inhibitors from each target class, we demonstrated in vivo efficacy characterized by negative effects on parasite fecundity in hamsters infected with hookworms.

Genistein is a type of isoflavonoid found predominantly in leguminous plants. Genistein has diverse biological activities, such as anthelmintic and antioxidant effects, as well as inhibitory effects on the growth of several cancers. In addition, genistein is well known as a phytoestrogen. In this study, we attempted to biologically synthesize genistein from either p -coumaric acid or naringenin using Escherichia coli as a biotransformation host. Four genes, Os4CL , PeCHS , RcIFS , and OsCPR , were used for genistein production. To functionally express RcIFS and OsCPR, two members of the cytochrome P450 family, in E. coli , the membrane-binding anchor domain of each gene was removed, and RcIFS and OsCPR were translationally fused to generate an RcIFS-OsCPR hybrid. Os4CL and PeCHS, or the RcIFS-OsCPR hybrid, were then transformed into E. coli BL21(DE3). Using these strains, we optimized our culture system at a laboratory scale in terms of the cell density, concentrations of substrate and isopropyl-β-D-thiogalactoside, temperature, and culture medium. Under the optimized culture conditions, genistein was produced at up to 35 mg/l and 18.6 mg/l using naringenin and p -coumaric acid, respectively.

Fenbendazole, a broad spectrum anthelmintic used especially in veterinary medicine, may impact non-target organisms in the environment. Nevertheless, information about the effects of fenbendazole in plants is limited. We investigated the biotransformation of fenbendazole and the effect of fenbendazole and its metabolites on gene expression in the model plant Arabidopsis thaliana. High-sensitive UHPLC coupled with tandem mass spectrometry, RNA-microarray analysis together with qPCR verification and nanoLC-MS proteome analysis were used in this study. Twelve fenbendazole metabolites were identified in the roots and leaves of A. thaliana plants. Hydroxylation, S-oxidation and glycosylation represent the main fenbendazole biotransformation pathways. Exposure of A. thaliana plants to 5 μM fenbendazole for 24 and 72 h significantly affected gene and protein expression. The changes in transcriptome were more pronounced in the leaves than in roots, protein expression was more greatly affected in the roots at a shorter period of exposure (24 h) and in leaf rosettes over a longer period (72 h). Up-regulated (>2-fold change, p < 0.1) proteins are involved in various biological processes (electron transport, energy generating pathways, signal transduction, transport), and in response to stresses (e.g. catalase, superoxide dismutase, cytochromes P450, UDP-glycosyltransferases). Some of the proteins which were up-regulated after fenbendazole-exposure probably participate in fenbendazole biotransformation (e.g. cytochromes P450, UDP-glucosyltransferases). Finally, fenbendazole in plants significantly affects many physiological and metabolic processes and thus the contamination of ecosystems by manure containing this anthelmintic should be restricted., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Recently, we have discovered that the registered pesticide, tolfenpyrad, unexpectedly and potently inhibits the development of the L4 larval stage of the parasitic nematode Haemonchus contortus with an IC 50 value of 0.03 μM while displaying good selectivity, with an IC 50 of 37.9 μM for cytotoxicity. As a promising molecular template for medicinal chemistry optimization, we undertook anthelmintic structure-activity relationships for this chemical. Modifications of the left-hand side (LHS), right-hand side (RHS), and middle section of the scaffold were explored to produce a set of 57 analogues. Analogues 25, 29, and 33 were shown to be the most potent compounds of the series, with IC 50 values at a subnanomolar level of potency against the chemotherapeutically relevant fourth larval (L4) stage of H. contortus. Selected compounds from the series also showed promising activity against a panel of other different parasitic nematodes, such as hookworms and whipworms.

Clopidogrel bisulphate (CB) is a first line antiplatelet drug for treatment of myocardial infarction and stroke. Yet, its efficacy is limited by its poor solubility in intestinal pH, its main site of absorption. The main aim of this study is to enhance the intestinal release of CB by loading in cubosome nanoparticles. Glyceryl monooleate (GMO) based CB loaded cubosomes were prepared using a 3 3 factorial design to study the effect of polyvinyl alcohol (PVA), poloxamer 407 (PL407) concentrations and ratio of CB to the disperse phase on the average particle size, entrapment efficiency (%EE), in vitro release at 15 min (%Q 15 ), and their morphology using transmission electron microscopy (TEM). The release of the optimized formula was compared in buffer transition media (pH 1.2 for 2 h then pH 6.8 for 6 h) to free drug to study the effect of the changing pH in the gastrointestinal tract (GIT) on CB release. The antihaemostatic properties of the optimized formula were compared to the commercial product Plavix ® using bleeding time (BT) model in rabbits. The prepared cubosomes were in the nano range (115±6.47 to 248±4.63 nm) with high %EE (91.22±4.09% to 98.98±3.21%). The optimized formula showed significantly higher (p<0.05) CB release in intestinal pH and preserved the high% released (95.66±1.87%) in buffer transition release study compared to free drug (66.82±4.12%) as well as significantly (p<0.05) higher antihaemostatic properties with longer BT (628.47±6.12 s) compared to Plavix ® (412.43±7.97 s). Thus, cubosomes proved to be a successful platform to enhance the intestinal release of CB and improve its absorption.

The cys-loop superfamily of ligand-gated ion channels are well recognized as important drug targets for many invertebrate specific compounds. With the rise in resistance seen worldwide to existing anthelmintics, novel drug targets must be identified so new treatments can be developed. The acetylcholine-gated chloride channel (ACC) family is a unique family of cholinergic receptors that have been shown, using Caenorhabditis elegans as a model, to have potential as anti-parasitic drug targets. However, there is little known about the function of these receptors in parasitic nematodes. Here, we have identified an acc gene (hco-acc-1) from the sheep parasitic nematode Haemonchus contortus. While similar in sequence to the previously characterized C. elegans ACC-1 receptor, Hco-ACC-1 does not form a functional homomeric channel in Xenopus oocytes. Instead, co-expression of Hco-ACC-1 with a previously characterized subunit Hco-ACC-2 produced a functional heteromeric channel which was 3x more sensitive to acetylcholine compared to the Hco-ACC-2 homomeric channel. We have also found that Hco-ACC-1 can be functionally expressed in C. elegans. Overexpression of both cel-acc-1 and hco-acc-1 in both C. elegans N2 and acc-1 null mutants decreased the time for worms to initiate reversal avoidance to octanol. Moreover, antibodies were generated against the Hco-ACC-1 protein for use in immunolocalization studies. Hco-ACC-1 consistently localized to the anterior half of the pharynx, specifically in pharyngeal muscle tissue in H. contortus. On the other hand, expression of Hco-ACC-1 in C. elegans was restricted to neuronal tissue. Overall, this research has provided new insight into the potential role of ACC receptors in parasitic nematodes., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Nematodes exhibit a vast array of cys-loop ligand-gated ion channels with unique pharmacologic characteristics. However, many of the structural components that govern the binding of various ligands are unknown. The nematode cys-loop GABA receptor uncoordinated 49 (UNC-49) is an important receptor found at neuromuscular junctions that plays an important role in the sinusoidal movement of worms. The unique pharmacologic features of this receptor suggest that there are structural differences in the agonist binding site when compared with mammalian receptors. In this study, we examined each amino acid in one of the main agonist binding loops (loop E) via the substituted cysteine accessibility method (SCAM) and analyzed the interaction of various residues by molecular dynamic simulations. We found that of the 18 loop E mutants analyzed, H142C, R147C, and S157C had significant changes in GABA EC 50 and were accessible to modification by a methanethiosulfonate reagent (MTSET) resulting in a change in I GABA In addition, the residue H142, which is unique to nematode UNC-49 GABA receptors, appears to play a negative role in GABA sensitivity as its mutation to cysteine increased sensitivity to GABA and caused the UNC-49 receptor partial agonist 5-aminovaleric acid (DAVA) to behave as a full agonist. Overall, this study has revealed potential differences in the agonist binding pocket between nematode UNC-49 and mammalian GABA receptors that could be exploited in the design of novel anthelmintics., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Avermectins, are pesticides of fermentation products with high efficacy and low toxicity and play important roles in food and agricultural product safety, animal and human health. Ivermectins, the derivatives of avermectins, are used to treat Onchocerciasis, also known as River Blindness. Thanks to these discoveries, more than 200 million Africans are lucky to be exempted from blindness. Great efforts have been made on better understanding of the microbial production system of avermectins through genetic engineering in China. Starting from scratch, Chinese avermectin industry has experienced a comprehensive technological innovation with significantly improved yield, titer and productivity of avermectins, and became the exclusive supplier for the global market. The success of avermectin industry innovation in China not only shed lights on the intelligent yield improvement of other microbial natural product drugs, but also gained wide international recognition. We provide here an overview of the discovery and development of basic and applied research of avermectins, especially historical evolution of avermectins made in China, which will benefit industrial development of microbial drugs in China.

Haemonchus contortus is one of the most economically important parasites infecting small ruminants worldwide. This nematode has shown a great ability to develop resistance to anthelmintic drugs, calling for the development of alternative control approaches. Because lectins recognize and bind to specific carbohydrates and glycan structures present in parasites, they can be considered as an alternative to develop new antiparasitic drugs. Accordingly, this work aimed to investigate the anthelmintic effect of Canavalia brasiliensis (ConBr) lectin against H. contortus and to evaluate a possible interaction of ConBr with glycans of this parasite by molecular docking. ConBr showed significant inhibition of H. contortus larval development with an IC 50 of 0.26 mg mL -1 . Molecular docking assays revealed that glycans containing the core trimannoside [Man(α1-3)Man(α1-6)Man] of H. contortus interact in the carbohydrate recognition domain of ConBr with an interaction value of MDS = -248.77. Our findings suggest that the inhibition of H. contortus larval development is directly related to the recognition of the core trimannoside present in the glycans of these parasites. This work is the first to report on the structure-function relationships of the anthelmintic activity of plant lectins., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Praziquantel (PZQ) is an inexpensive, low toxicity BCS II class anthelmintic drug used for the treatment of neglected tropical diseases. In earlier papers a mechanochemical activation has been used to induce physical transformations on the drug which would ameliorate its solubility and hence its bioavailability and a systematic study of the effects of varying temperature, frequency and time of milling on drug melting enthalpy and drug recovery was given. In this communication, the focus is on the degradation products that are formed during this mechanical treatment of Praziquantel. In the cogrinding process with povidone and crospovidone several degradation products are formed. Different degradation products are formed, which depend on the type of polymer rather than the process conditions. Two of the most prominent degradation products were identified and their structure proposed on the basis of information obtained from GC-MS, UPLC-MS and 1 H NMR techniques., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Parasitic nematodes infect over 1/4 th of the human population and are a major burden on livestock and crop production. Benzimidazole class anthelmintics are widely used to treat infections, but resistance is a widespread problem. Mutation of genes encoding the benzimidazole target β-tubulin is a well-established mechanism of resistance, but recent evidence suggests that metabolism of the drugs may also occur. Our objective was to investigate contributions of the detoxification-response transcription factor SKN-1 to anthelmintic drug resistance using C. elegans. We find that skn-1 mutations alter EC 50 of the common benzimidazole albendazole in motility assays by 1.5-1.7 fold. We also identify ugt-22 as a detoxification gene associated with SKN-1 that influences albendazole efficacy. Mutation and overexpression of ugt-22 alter albendazole EC 50 by 2.3-2.5-fold. The influence of a nematode UGT on albendazole efficacy is consistent with recent studies demonstrating glucose conjugation of benzimidazoles., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Triclabendazole is the first-line drug of choice to treat and control fasciolasis, a neglected parasitic human disease. It is a class II/IV compound according to the Biopharmaceutics Classification System. Thus, the aim of this study was to improve aqueous solubility and dissolution rate of triclabendazole complexed with 2-hydroxylpropyl-β-cyclodextrin (HP-β-CD) and methyl-β-cyclodextrin (Me-β-CD) at 1:1 and 1:2 M ratio. The impact of storage on the solubility, dissolution profile, and solid-state properties of such complexes was also investigated. Drug-carrier interactions were characterized by infrared spectroscopy, differential scanning calorimetry, X-ray diffractometry, and scanning electron microscopy. The solubility of triclabendazole improved up to 256- and 341-fold using HP-β-CD and Me-β-CD, respectively. In particular, the drug complexed with Me-β-CD showed a positive deviation from linearity, suggesting that its solubility increases with an increasing concentration of Me-β-CD concentration in a nonlinear manner. The drug dissolution was found to be improved through complex formation with HP-β-CD and Me-β-CD. In particular, the 1:2 M ratio complexes exhibited higher dissolution than the corresponding 1:1 M ratio complexes. The physicochemical characterization of the systems showed strong evidence of amorphous phases and/or of the formation of an inclusion complex. Stored at 25 °C, 60% RH for 24 months, drug complexed with β-cyclodextrins (CDs) at 1:2 M ratio remained amorphous. Based on these findings, it is postulated that the formation of triclabendazole-CD inclusion complexes produced significant enhancement in both the dissolution and solid-state properties of the drug, which may lead to the development of triclabendazole novel formulations with improved biopharmaceutical characteristics.

In the course of screening for microbes with nematicidal activity, we found that Enterobacter asburiae HK169 displayed promising nematicidal activity against the root-knot nematode Meloidogyne incognita , along with plant growth-promoting properties. Soil drenching of a culture of HK169 reduced gall formation by 66% while also increasing root and shoot weights by 251% and 160%, respectively, compared with an untreated control. The cell-free culture filtrate of the HK169 culture killed all juveniles of M. incognita within 48 h. In addition, the nematicidal activity of the culture filtrate was dramatically reduced by a protease inhibitor, suggesting that proteolytic enzymes contribute to the nematicidal activity of HK169. In order to obtain genomic information about the HK169 isolate related to its nematicidal and plant growth-promoting activities, we sequenced and analyzed the whole genome of the HK169 isolate, and the resulting information provided evidence that the HK169 isolate has nematicidal and plant growth-promoting activities. Taken together, these observations enable the future application of E. asburiae HK169 as a biocontrol agent for nematode control and promote our understanding of the beneficial interactions between E. asburiae HK169 and plants.

Mebendazole (MBZ) was developed as a broad-spectrum anthelmintic but has recently shown efficacy as an anticancer agent. The use of MBZ for cancer, however, is challenging due to its poor solubility leading to poor bioavailability. Herein, we developed a prodrug approach with various N-linked promoieties including acyloxymethyl, aminoacyloxymethyl, and substituted phosphonooxymethyl in attempt to improve these characteristics. Compound 12, containing an (((((isopropoxycarbonyl)oxy)methoxy)phosphoryl)oxy)methyl promoiety, showed a >10 000-fold improvement in aqueous solubility. When evaluated in mice, 12 displayed a 2.2-fold higher plasma AUC 0- t and a 1.7-fold improvement in brain AUC 0- t with a calculated oral bioavailability of 52%, as compared to 24% for MBZ-polymorph C (MBZ-C), the most bioavailable polymorph. In dogs, 12 showed a 3.8-fold higher plasma AUC 0- t with oral bioavailability of 41% compared to 11% for MBZ-C. In summary, we have identified a prodrug of MBZ with better physicochemical properties and enhanced bioavailability in both mice and dog.

Schistosoma mansoni, like other trematodes, expresses a number of unusual calcium binding proteins which consist of an EF-hand domain joined to a dynein light chain-like (DLC-like) domain by a flexible linker. These proteins have been implicated in host immune responses and drug binding. Three members of this protein family from S. mansoni (SmTAL1, SmTAL2 and SmTAL3) have been well characterised biochemically. Here we characterise the remaining family members from this species (SmTAL4-13). All of these proteins form homodimers and all except SmTAL5 bind to calcium and manganese ions. SmTAL9, 10 and 11 also bind to magnesium ions. The antischistosomal drug, praziquantel interacts with SmTAL4, 5 and 8. Some family members also bind to calmodulin antagonists such as chlorpromazine and trifluoperazine. Molecular modelling suggests that all ten proteins adopt similar overall folds with the EF-hand and DLC-like domains folding discretely. Bioinformatics analyses suggest that the proteins may fall into two main categories: (i) those which bind calcium ions reversibly at the second EF-hand and may play a role in signalling (SmTAL1, 2, 8 and 12) and (ii) those which bind calcium ions at the first EF-hand and may play either signalling or structural roles (SmTAL7, 9, 10 and 13). The remaining proteins include those which do not bind calcium ions (SmTAL3 and 5) and three other proteins (SmTAL4, 6 and 11). The roles of these proteins are less clear, but they may also have structural roles., (Copyright © 2018 Elsevier B.V. All rights reserved.)