Search

Glutamate-gated chloride channels (GluCls) are unique to invertebrates and are targeted by macrocyclic lactones. In this study, we cloned an AVR-14B GluCl subunit from adult Brugia malayi , a causative agent of lymphatic filariasis in humans. To elucidate this channel’s pharmacological properties, we used Xenopus laevis oocytes for expression and performed two-electrode voltage-clamp electrophysiology. The receptor was gated by the natural ligand L-glutamate (effective concentration, 50% [ EC 50 ] = 0.4 mM) and ivermectin (IVM; EC 50 = 1.8 nM). We also characterized the effects of nodulisporic acid (NA) on Bma -AVR-14B and NA-produced dual effects on the receptor as an agonist and a type II positive allosteric modulator. Here we report characterization of the complex activity of NA on a nematode GluCl. Bma -AVR-14B demonstrated some unique pharmacological characteristics. IVM did not produce potentiation of L-glutamate–mediated responses but instead, reduced the channel’s sensitivity for the ligand. Further electrophysiological exploration showed that IVM (at a moderate concentration of 0.1 nM) functioned as an inhibitor of both agonist and positive allosteric modulatory effects of NA. This suggests that IVM and NA share a complex interaction. The pharmacological properties of Bma -AVR-14B indicate that the channel is an important target of IVM and NA. In addition, the unique electrophysiological characteristics of Bma -AVR-14B could explain the observed variation in drug sensitivities of various nematode parasites. We have also shown the inhibitory effects of IVM and NA on adult worm motility using Worminator. RNA interference (RNAi) knockdown suggests that AVR-14 plays a role in influencing locomotion in B. malayi .

Filariae are vector-borne nematodes responsible for an enormous burden of disease. Human lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori, and onchocerciasis (caused by Onchocerca volvulus) are neglected parasitic diseases of major public health significance in tropical regions. To date, therapeutic efforts to eliminate human filariasis have been hampered by the lack of a drug with sufficient macrofilaricidal and/or long-term sterilizing effects that is suitable for use in mass drug administration (MDA) programs, particularly in areas co-endemic with Loa loa, the causative agent of loiasis. Emodepside, a semi-synthetic cyclooctadepsipeptide, has been shown to have broad-spectrum efficacy against gastrointestinal nematodes in a variety of mammalian hosts, and has been approved as an active ingredient in dewormers for cats and dogs. This paper evaluates, compares (where appropriate) and summarizes the in vitro effects of emodepside against a range of filarial nematodes at various developmental stages. Emodepside inhibited the motility of all tested stages of filariae frequently used as surrogate species for preclinical investigations (Acanthocheilonema viteae, Brugia pahangi, Litomosoides sigmodontis, Onchocerca gutturosa, and Onchocerca lienalis), human-pathogenic filariae (B. malayi) and filariae of veterinary importance (Dirofilaria immitis) in a concentration-dependent manner. While motility of all filariae was inhibited, both stage- and species-specific differences were observed. However, whether these differences were detected because of stage- and/or species-specific factors or as a consequence of variations in protocol parameters among the participating laboratories (such as purification of the parasites, read-out units, composition of media, incubation conditions, duration of incubation etc.) remains unclear. This study, however, clearly shows that emodepside demonstrates broad-spectrum in vitro activity against filarial nematode species across different genera and can therefore be validated as a promising candidate for the treatment of human filariases, including onchocerciasis and lymphatic filariasis., Graphical abstract Image 1, Highlights • Emodepside causes concentration dependent paralysis on filariae. • Findings confirm the broad-spectrum nematicidal profile of emodepside. • Data reveal emodepside as a promising drug candidate for human filariasis.

Nematode parasites of humans and livestock pose a significant burden to human health, economic development, and food security. Anthelmintic drug resistance is widespread among parasites of livestock and many nematode parasites of humans lack effective treatments. Here, we present a nitrophenyl-piperazine scaffold that induces motor defects rapidly in the model nematode Caenorhabditis elegans. We call this scaffold Nemacol and show that it inhibits the vesicular acetylcholine transporter (VAChT), a target recognized by commercial animal and crop health groups as a viable anthelmintic target. We demonstrate that it is possible to create Nemacol analogs that maintain potent in vivo activity whilst lowering their affinity to the mammalian VAChT 10-fold. We also show that Nemacol synergizes with the anthelmintic ivermectin to kill C. elegans. Hence, Nemacol represents a promising new anthelmintic scaffold that acts through an identified viable anthelmintic target.One sentence summaryA small molecule screen identifies a vesicular acetylcholine transporter inhibitor scaffold that incapacitates parasitic nematodes

Despite being considered one of the most pathogenic helminth infections of companion animals, members of macrocyclic lactone class are the only drugs available for the prevention of heartworm disease caused by Dirofilaria immitis. Alarmingly, heartworm prevention is at risk; several studies confirm the existence of macrocyclic lactone resistance in D. immitis populations across the United States. To safeguard the long term prevention and control of this disease, the identification and development of novel anthelmintics is urgently needed. To identify novel, resistance-breaking drugs, it is highly desirable to: Unfortunately, none of the three above statements can be answered sufficiently for D. immitis and most of our hypotheses derive from surrogate species and/or in vitro studies. Therefore, the present study aims to improve our fundamental understanding of the neuromuscular system of the canine heartworm by establishing new methods allowing the investigation of body wall and pharyngeal muscle responses and their modulation by anthelmintics. We found that the pharynx of adult D. immitis responds to both ivermectin and moxidectin with EC50s in the low micromolar range. We also demonstrate that the somatic muscle cells have robust responses to 30 μM acetylcholine, levamisole, pyrantel and nicotine. This is important preliminary data, demonstrating the feasibility of electrophysiological studies in this important parasite., Graphical abstract Image 1, Highlights • We have developed new preparations to record drug responses from adult heartworm. • These preparations allow investigation of ion-channel responses in adult parasites. • Somatic muscle cell patch-clamp recordings show responses to levamisole & pyrantel. • Pharyngeal current-clamp recordings show responses to ivermectin & moxidectin.

Nematode parasites of humans, livestock and crops pose a significant burden on human health and welfare. Alarmingly, parasitic nematodes of animals have rapidly evolved resistance to anthelmintic drugs, and traditional nematicides that protect crops are facing increasing restrictions because of poor phylogenetic selectivity. Here, we present a pipeline that exploits multiple motor outputs of the model nematode C. elegans for nematicide discovery. This pipeline yielded multiple compounds that selectively kill and/or immobilize diverse nematode parasites. We focus on one compound that induces violent convulsions and paralysis that we call Nementin. We find that Nementin agonizes neuronal dense core vesicle release, which in turn agonizes cholinergic signaling. Consequently, Nementin synergistically enhances the potency of widely-used non-selective acetylcholinesterase inhibitors (AChEIs), but in a nematode-selective manner. Nementin therefore has the potential to reduce the environmental impact of toxic AChEI pesticides used to control nematode infections and infestations.Significance StatementParasitic nematodes pose a considerable burden to human health and food security. Small molecules that have traditionally been used to control these parasites have either been banned because of toxicity concerns or are being rendered ineffective because of the evolution of resistance. Significant gaps in our nematicidal toolkit are therefore becoming an alarming problem. Here, we describe our discovery of Nementin, a small molecule that disrupts the nematode nervous system but is ineffective against non-targeted organisms. We find that Nementin also enhances the activity of non-selective pesticides but does so in a nematode-selective manner. Hence, Nementin is an innovative solution to combat parasitic nematodes in a safe and phylum-selective manner.One-Sentence SummaryA C. elegans-based screening pipeline identifies a selective nematicide that also potentiates acetylcholinesterase inhibitors.

Ancylostoma caninum is the most prevalent intestinal nematode of dogs, and has a zoonotic potential. Multiple-drug resistance (MDR) has been confirmed in a number of A. caninum isolates, including isolate Worthy 4.1F3P, against all anthelmintic drug classes approved for hookworm treatment in dogs in the United States (US). The cyclooctadepsipeptide emodepside is not registered to use in dogs in the US, but in a number of other countries/regions. The objective of this study was to evaluate the efficacy of emodepside + praziquantel, as well as three commercial products that are commonly used in the US for treatment of hookworms, against a suspected (subsequently confirmed) MDR A. caninum isolate Worthy 4.1F3P. 40 dogs infected on study day (SD) 0 with 300 third-stage larvae, were randomly allocated to one of five treatment groups with eight dogs each: pyrantel pamoate (Nemex®-2), fenbendazole (Panacur® C), milbemycin oxime (Interceptor®), emodepside + praziquantel tablets and non-treated control. Fecal egg counts (FEC) were performed on SDs 19, 20, 22, 27, 31 and 34. All treatments were administered as per label requirements on SD 24 to dogs in Groups 1 through 4. Two additional treatments were administered on SDs 25 and 26 to dogs in Group 2 as per label requirements. Dogs were necropsied on SD 34 and the digestive tract was removed/processed for worm recovery and enumeration. The geometric mean (GM) worm counts for the control group was 97.4, and for the pyrantel pamoate, fenbendazole, milbemycin oxime, and emodepside + praziquantel groups were 74.8, 72.0, 88.9, and 0.4, respectively. These yielded efficacies of 23.2%, 26.1%, and 8.8%, and 99.6%, respectively. These data support previous findings of the MDR status of Worthy 4.1F3P as treatments with pyrantel pamoate, fenbendazole and milbemycin oxime lacked efficacy. In sharp contrast, Worthy 4.1F3P was highly susceptible to treatment with emodepside + praziquantel., Graphical abstract Image 1

Onchocerciasis (river blindness), caused by the filarial worm Onchocerca volvulus, is a neglected tropical disease mostly affecting sub-Saharan Africa and is responsible for >1.3 million years lived with disability. Current control relies almost entirely on ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. Here, we evaluated emodepside, a semi-synthetic cyclooctadepsipeptide registered for deworming applications in companion animals, for activity against adult filariae (i.e., as a macrofilaricide). We demonstrate the equivalence of emodepside activity on SLO-1 potassium channels in Onchocerca volvulus and Onchocerca ochengi, its sister species from cattle. Evaluation of emodepside in cattle as single or 7-day treatments at two doses (0.15 and 0.75 mg/kg) revealed rapid activity against microfilariae, prolonged suppression of female worm fecundity, and macrofilaricidal effects by 18 months post treatment. The drug was well tolerated, causing only transiently increased blood glucose. Female adult worms were mostly paralyzed; however, some retained metabolic activity even in the multiple high-dose group. These data support ongoing clinical development of emodepside to treat river blindness., Author summary Onchocerciasis (river blindness), caused by the parasitic worm Onchocerca volvulus, is a devastating neglected tropical disease affecting sub-Saharan Africa with an overall impact of >1.3 million years lived with disability. Current control relies mainly on a single drug, ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. The identification of a drug that can safely eliminate adult worms (i.e., a macrofilaricide) is a major research objective for onchocerciasis. We evaluated the anthelminthic activity of emodepside, a veterinary wormer, in cattle infected with a close relative of O. volvulus (Onchocerca ochengi) before conducting pharmacokinetic modelling to estimate drug distribution in humans. Emodepside as single or 7-day treatments at two doses produced rapid activity against O. ochengi microfilariae, prolonged suppression of female worm fecundity, and paralysed most female worms by 18 months, although some remained metabolically active even in the multiple high-dose treatment group. The drug was well tolerated, causing only transiently increased blood glucose. Thus, emodepside shows slow but significant efficacy against adult O. ochengi in naturally infected cattle, meeting the criteria for a safe macrofilaricidal drug. Our data support the ongoing clinical development of emodepside for the treatment of human onchocerciasis.

The macrocyclic lactone anthelmintics are the only class of drug currently used to prevent heartworm disease. Their extremely high potency in vivo is not mirrored by their activity against Dirofilaria immitis larvae in vitro, leading to suggestions that they may require host immune functions to kill the parasites. We have previously shown that ivermectin stimulates the binding of canine peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) to D. immitis microfilariae (Mf). We have now extended these studies to moxidectin and examined the ability of both drugs to stimulate canine PBMC and PMN attachment to Mf from multiple strains of D. immitis, including two that are proven to be resistant to ivermectin in vivo. Both ivermectin and moxidectin significantly increased the percentage of drug-susceptible parasites with cells attached at very low concentrations (100 nM) were required to increase the percentage of the two resistant strains, Yazoo-2013 and Metairie-2014, with cells attached. Moxidectin increased the percentage of the two resistant strains with cells attached at lower concentrations (, Graphical abstract Image 1, Highlights • Ivermectin promotes attachment of PMN and PBMC to D. immitis microfilariae in vitro. • Moxidectin has a similar effect. • Higher ivermectin concentrations are needed if Mf of ML-resistant strains are used. • Moxidectin is more effective at promoting cell attachment to resistant Mf. • Neither PMN nor PBMC attachment does not result in parasite death in vitro.

Summary Eosinophils mediate protection against filarial nematodes. Our results demonstrate that eosinophil extracellular traps (EETosis) are induced by microfilariae and infective L3 larvae of Litomosoides sigmodontis. These extracellular DNA traps inhibit microfilariae motility in a DNA- and contact-dependent manner in vitro. Accordingly, microfilariae-injection triggers DNA release in an eosinophil-dependent manner in vivo and microfilariae covered with DNA traps are cleared more rapidly. Using dectin-1, we identify the required receptor for the microfilariae-induced EETosis, whereas signaling via other C-type lectin receptors, prior priming of eosinophils, and presence of antibodies are not required. The DNA released upon microfilariae-induced EETosis is mainly of mitochondrial origin, but acetylated and citrullinated histones are found within the traps. We further demonstrate that the presented DNA-dependent inhibition of microfilariae motility by eosinophils represents a conserved mechanism, as microfilariae from L. sigmodontis and the canine heartworm Dirofilaria immitis induce ETosis in murine and human eosinophils.

Current mass drug administration (MDA) programs for the treatment of human river blindness (onchocerciasis) caused by the filarial worm Onchocerca volvulus rely on ivermectin, an anthelmintic originally developed for animal health. These treatments are primarily directed against migrating microfilariae and also suppress fecundity for several months, but fail to eliminate adult O. volvulus. Therefore, elimination programs need time frames of decades, well exceeding the life span of adult worms. The situation is worsened by decreased ivermectin efficacy after long-term therapy. To improve treatment options against onchocerciasis, a drug development candidate should ideally kill or irreversibly sterilize adult worms. Emodepside is a broad-spectrum anthelmintic used for the treatment of parasitic nematodes in cats and dogs (Profender and Procox). Our current knowledge of the pharmacology of emodepside is the result of more than 2 decades of intensive collaborative research between academia and the pharmaceutical industry. Emodepside has a novel mode of action with a broad spectrum of activity, including against extraintestinal nematode stages such as migrating larvae or macrofilariae. Therefore, emodepside is considered to be among the most promising candidates for evaluation as an adulticide treatment against onchocerciasis. Consequently, in 2014, Bayer and the Drugs for Neglected Diseases initiative (DNDi) started a collaboration to develop emodepside for the treatment of patients suffering from the disease. Macrofilaricidal activity has been demonstrated in various models, including Onchocerca ochengi in cattle, the parasite most closely related to O. volvulus. Emodepside has now successfully passed Phase I clinical trials, and a Phase II study is planned. This Bayer–DNDi partnership is an outstanding example of “One World Health,” in which experience gained in veterinary science and drug development is translated to human health and leads to improved tools to combat neglected tropical diseases (NTDs) and shorten development pathways and timelines in an otherwise neglected area., Author summary Onchocerca volvulus is the causative agent of human river blindness, and current elimination programs rely on the use of ivermectin to kill microfilariae. Since no adulticidal drug is available and adult worms have a life span of up to 15 years, elimination programs need to be sustained over several decades. Emodepside is an anthelmintic that is licensed as a dewormer for cats and dogs. Due to its ability to eliminate nematodes located in various extraintestinal host tissues, including migrating larvae and adult filarial worms, it is considered to be an excellent candidate for the treatment of onchocerciasis. Intense collaboration between academia and the pharmaceutical industry has led to a deep understanding of the novel mode of action of the drug and of its parasite target spectrum. Phase I clinical trials with emodepside have demonstrated its safety and adulticide activity against the closely related cattle parasite Onchocerca ochengi. Currently, Phase II clinical trials are planned to confirm that emodepside, developed initially to improve animal health, has also the potential to improve human health by tackling a very important neglected tropical disease (NTD).

For more than four decades, the free-living nematode Caenorhabditis elegans has been extensively used in anthelmintic research. Classic genetic screens and heterologous expression in the C. elegans model enormously contributed to the identification and characterization of molecular targets of all major anthelmintic drug classes. Although these findings provided substantial insights into common anthelmintic mechanisms, a breakthrough in the treatment and control of parasitic nematodes is still not in sight. Instead, we are facing increasing evidence that the enormous diversity within the phylum Nematoda cannot be recapitulated by any single free-living or parasitic species and the development of novel broad-spectrum anthelmintics is not be a simple goal. In the present review, we summarize certain milestones and challenges of the C. elegans model with focus on drug target identification, anthelmintic drug discovery and identification of resistance mechanisms. Furthermore, we present new perspectives and strategies on how current progress in C. elegans research will support future anthelmintic research., Graphical abstract Image 1

Filariae are parasitic nematodes that are transmitted to their definitive host as third-stage larvae by arthropod vectors like mosquitoes. Filariae cause diseases including: lymphatic filariasis with distressing and disturbing symptoms like elephantiasis; and river blindness. Filarial diseases affect millions of people in 73 countries throughout the topics and sub-tropics. The drugs available for mass drug administration, (ivermectin, albendazole and diethylcarbamazine), are ineffective against adult filariae (macrofilariae) at the registered dosing regimen; this generates a real and urgent need to identify effective macrofilaricides. Emodepside, a veterinary anthelmintic registered for treatment of nematode infections in cats and dogs, is reported to have macrofilaricidal effects. Here, we explore the mode of action of emodepside using adult Brugia malayi, one of the species that causes lymphatic filariasis. Whole-parasite motility measurement with Worminator and patch-clamp of single muscle cells show that emodepside potently inhibits motility by activating voltage-gated potassium channels and that the male is more sensitive than the female. RNAi knock down suggests that emodepside targets SLO-1 K channels. We expressed slo-1 isoforms, with alternatively spliced exons at the RCK1 (Regulator of Conductance of Potassium) domain, heterologously in Xenopus laevis oocytes. We discovered that the slo-1f isoform, found in muscles of males, is more sensitive to emodepside than the slo-1a isoform found in muscles of females; and selective RNAi of the slo-1a isoform in female worms increased emodepside potency. In Onchocerca volvulus, that causes river blindness, we found two isoforms in adult females with homology to Bma-SLO-1A and Bma-SLO-1F at the RCK1 domain. In silico modeling identified an emodepside binding pocket in the same RCK1 region of different species of filaria that is affected by these splice variations. Our observations show that emodepside has potent macrofilaricidal effects and alternative splicing in the RCK1 binding pocket affects potency. Therefore, the evaluation of potential sex-dependent effects of an anthelmintic compound is of importance to prevent any under-dosing of one or the other gender of nematodes once given to patients., Author summary Emodepside is an anthelmintic drug that is used for treatment of nematode infections in cats and dogs. A potassium channel, SLO-1, has been implicated as a target for emodepside in the nematode model Caenorhabditis elegans. The anthelmintic effects of emodepside has been demonstrated against filariae, but its molecular mode of action requires further elucidation. Here, we show that emodepside activates SLO-1 channels in the human filaria, Brugia malayi, one of the causative agents of lymphatic filariasis. Emodepside is more potent on male than on the female B. malayi. Worms where slo-1 was knocked down were less susceptible to emodepside. Alternative splicing of the slo-1 gene in the region encoding the RCK1 domain, results in the expression of SLO-1F and SLO-1A in female worms and SLO-1F alone in male worms. SLO-1F expressed in Xenopus laevis oocytes was more sensitive to emodepside than SLO-1A or a combination of SLO-1A and SLO-1F. Selective knockdown of slo-1a in female worms increased their emodepside sensitivity, so their sensitivity resembled male worms. Emodepside binds to the RCK1 region of SLO-1 in in-silico protein interaction studies of the SLO-1 sequences of different filariod species. This is the first report demonstrating a gender-specific effect of emodepside correlating directly to the expression of alternatively spliced isoforms of SLO-1. This study emphasizes that the gender-dependent effects should always be considered when evaluating the efficacy profile of an anthelmintic drug candidate.

Non-ribosomal peptide synthetases are complex multimodular biosynthetic machines that assemble various important and medically relevant peptide antibiotics. An interesting subgroup comprises the cyclodepsipeptide synthetases from fungi synthesizing cyclohexa- and cyclo-octadepsipeptides with antibacterial, anthelmintic, insecticidal, and anticancer properties; some are marketed drugs. We exploit the modularity of these highly homologous synthetases by fusing the hydroxy-acid-activating module of PF1022 synthetase with the amino-acid-activating modules of enniatin and beauvericin synthetase, thus yielding novel hybrid synthetases. The artificial synthetases expressed in Escherichia coli and the fungus Aspergillus niger yielded new cyclodepsipeptides, thus paving the way for the exploration of these derivatives for their bioactivity.

Heartworm disease is a zoonotic vector-borne disease caused by Dirofilaria immitis mainly affecting canids. Infectious third-stage larvae (L3) are transmitted to the definitive hosts via culicid mosquitoes; adult nematodes reside in the pulmonary arteries and in the right heart releasing unsheathed first-stage larvae (microfilariae) into the bloodstream leading to chronic and sometimes fatal disease. So far, early innate immune reactions triggered by these different D. immitis stages in the canine host have scarcely been investigated. Therefore, D. immitis microfilariae and L3 were analyzed for their capacity to induce neutrophil extracellular traps (NETs) in canine polymorphonuclear neutrophils (PMN). Overall, scanning electron microscopy analysis revealed both larval stages as strong inducers of canine NETosis. Co-localization of PMN-derived extracellular DNA with granulocytic histones, neutrophil elastase, or myeloperoxidase in parasite-entrapping structures confirmed the classical characteristics of NETosis. Quantitative analyses showed that both larval stages triggered canine NETs in a time-dependent but dose-independent manner. Moreover, parasite-induced NET formation was not influenced by the parasites viability since heat-inactivated microfilariae and L3 also induced NETs. In addition, parasite/PMN confrontation promoted significant entrapment but not killing of microfilariae and L3. Both, NETosis and larval entrapment was significantly reversed via DNase I treatments while treatments with the NADPH oxidase inhibitor diphenyleneiodonium failed to significantly influence these reactions. Interestingly, different types of NETs were induced by microfilariae and L3 since microfilarial stages merely induced spread and diffuse NETs while the larger L3 additionally triggered aggregated NET formation.

The anthelmintic cyclooctadepsipeptide emodepside is effective against nematodes showing resistance against established drug classes. Emodepside exerts its nematicidal effects mainly through its validated target, the tetrameric voltage- and calcium-activated potassium channel SLO-1. Two slo-1 genes were described in Trichuris muris. Alternative splicing is known to alter SLO-1 properties. Here, 16 T. muris splice variants for slo-1.1 and three variants for slo-1.2 were identified in addition to previously described variants. Splice variants caused by intron retentions and/or exon exclusions encode varyingly truncated subunits. Depending on the subunit composition, channels might have altered physiological and pharmacological properties including different modulation by calcium and/or voltage or reduced emodepside susceptibility which might lead to emodepside resistance as observed in Caenorhabditis elegans expressing only similarly truncated Slo-1. The comprehensive characterisation of splice variants is a prerequisite for functional analysis and confirmed conservation of remarkable differences found between both slo-1 paralogs in Trichuris suis.

The anthelmintic cyclooctadepsipeptide emodepside is effective against nematodes showing resistance against established drug classes. Emodepside exerts its nematicidal effects mainly through its validated target, the tetrameric voltage- and calcium-activated potassium channel SLO-1. Two slo-1 genes were described in Trichuris muris. Alternative splicing is known to alter SLO-1 properties. Here, 16 T. muris splice variants for slo-1.1 and three variants for slo-1.2 were identified in addition to previously described variants. Splice variants caused by intron retentions and/or exon exclusions encode varyingly truncated subunits. Depending on the subunit composition, channels might have altered physiological and pharmacological properties including different modulation by calcium and/or voltage or reduced emodepside susceptibility which might lead to emodepside resistance as observed in Caenorhabditis elegans expressing only similarly truncated Slo-1. The comprehensive characterisation of splice variants is a prerequisite for functional analysis and confirmed conservation of remarkable differences found between both slo-1 paralogs in Trichuris suis.

The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among species. Most importantly, this study showed for the first time that emodepside directly opens a Slo-1 channel, significantly improving the understanding of the mode of action of this drug class., Author Summary Emodepside is an anthelmintic introduced into the market as an ingredient of different dewormers for cats and dogs, and is the only member of the cyclooctadepsipeptide class which has been commercialized. The voltage-gated and calcium-activated potassium channel Slo-1 has been implicated in the mode of action of emodepside, but evidence for direct emodepside-induced changes on the properties of Slo-1 channels has not been reported. Emodepside is active against a broad spectrum of parasitic nematodes, but the putative target Slo-1 has only been described for the model nematode Caenorhabditis elegans, and for relatively closely related strongylids. Here, Slo-1 channels of ascarids, filariae and trichocephalids are described. Unexpectedly, in the latter group two distinct Slo-1 channels are encoded in their genomes. The C. elegans Slo-1a channel was expressed in Xenopus laevis oocytes. After membrane depolarization without experimentally increasing intracellular Ca2+, no Slo-1 specific currents were observed. In contrast, very large Slo-1-specific currents were observed after preincubation of oocytes with emodepside, suggesting that the drug opens the C. elegans Slo-1a channel irreversibly. This is the first report demonstrating direct interaction of a cyclooctadepsipeptide with a Slo-1 channel, which substantially enhances our understanding of the mode of action of this drug class.

SUMMARYThe cyclooctadepsipeptide PF1022A and the aminophenylamidines amidantel, deacylated amidantel (dAMD) and tribendimidine were tested as examples for drug classes potentially interesting for development as anthelmintics against human helminthiases. These compounds and levamisole were tested alone and in combination to determine their efficacy against the rat hookworm Nippostrongylus brasiliensis. After three oral treatments, intestinal worms were counted. Drug effects on parasite morphology were studied using scanning electron microscopy (SEM). Plasma pharmacokinetics were determined for tribendimidine and dAMD. All drugs reduced worm burden in a dose-dependent manner, however amidantel was significantly less active than the other aminophenylamidines. Combinations of tribendimidine and dAMD with levamisole or PF1022A at suboptimal doses revealed additive effects. While PF1022A caused virtually no changes in morphology, levamisole, dAMD and tribendimidine caused severe contraction, particularly in the hind body region. Worms exposed to combinations of PF1022A and aminophenylamidines were indistinguishable from worms exposed only to aminophenylamidines. After oral treatment with tribendimidine, only the active metabolite dAMD was detectable in plasma and concentrations were not significantly different for oral treatment with dAMD. The results support further evaluation of cyclooctadepsipeptides alone and in combination with cholinergic drugs to improve efficacy. Combining these with registered drugs may help to prevent development of resistance.

The present study investigates the in vitro efficacy of derivatives of the cyclooctadepsipeptides and the aminophenylamidines, which are promising candidates for the evaluation of the treatment of human soil-transmitted helminthiases. The effects of emodepside and PF1022A as well as of amidantel, deacylated amidantel and tribendimidine were evaluated in a concentration range between 0.01 and 100 μg/ml against third-stage larvae (L3) and adult worms of Nippostrongylus brasiliensis and first-stage larvae (L1) of Trichinella spiralis. Furthermore, drug combinations of PF1022A plus deacylated amidantel or tribendimidine and of tribendimidine plus levamisole were tested for any potential additive or even synergistic interactions. Emodepside had a significantly lower EC(50) value than PF1022A in the T. spiralis (0.02788 vs. 0.05862 μg/ml) and the N. brasiliensis (0.06188 vs. 0.1485 μg/ml) motility assays but not in the acetylcholine esterase secretion assay with adult N. brasiliensis (0.05650 vs. 0.06886 μg/ml). While amidantel showed only minimal or at best partial inhibition of nematode motility and acetylcholine esterase secretion, tribendimidine was nearly as potent as deacylated amidantel. Whereas deacylated amidantel had a significantly lower EC(50) than tribendimidine in the N. brasiliensis L3 motility assay (0.05492 vs. 0.2080 μg/ml), differences were not significant in the T. spiralis L1 motility assay (0.7766 vs. 1.145 μg/ml). Surprisingly, none of the combinations showed improved efficacy when compared to the individual drugs including levamisole/tribendimidine, which have previously been reported to act synergistically against Ancylostoma ceylanicum.

Tribendimidine has been registered for the treatment of human soil transmitted helminthiases in China. In the model nematode Caenorhabditis elegans it is an agonist of L-subtype nicotinic acetylcholine receptors and therefore shares its mode of action with levamisole and pyrantel. Besides its broad spectrum of nematicidal efficacy, tribendimidine is efficacious against several trematodes and has been attributed to have anti-cestodal effects. However, there are few published data available for the latter. The efficacy of tribendimidine and its nematicidal metabolite deacylated amidantel against Hymenolepis microstoma were examined for their anti-cestodal potential. Doses of 50 and 100mg/kg body weight deacylated amidantel and 10, 25, 50, and 100mg/kg tribendimidine were administered orally on three consecutive days to mice experimentally infected with eight cysticercoids. Necropsy was performed and the worm burdens were determined one day after the last treatment. Furthermore, levamisole was used in combination with tribendimidine (100mg/kg levamisole plus 10 and 25mg/kg tribendimidine, respectively) and alone (50 and 100mg/kg) to investigate any possible interactions of the partner compounds against cestodes. Tribendimidine showed a very high efficacy at dosages of 50mg/kg or higher. Surprisingly, deacylated amidantel led to no reduction of the worm burden in any of the treatments. Combinations of levamisole with tribendimidine did not augment the effects of tribendimidine alone and as expected levamisole alone also showed no anti-cestodal activity. To our knowledge, this study shows for the first time activity of tribendimidine against a cestode in a controlled laboratory study. Due to the excellent cure rates observed here, multiple tribendimidine treatments might be considered as useful scheme for treatments of cestode, nematode and trematode infections although this would significantly increase both costs and management efforts. Moreover, the differences between tribendimidine and deacylated amidantel indicate at least a strong difference in sensitivity of H. microstoma or a strong difference in drug availability.