Portal de Periódicos da CAPES

Ivermectin: An Anthelmintic, an Insecticide, and Much More

2020; Elsevier BV; Volume: 37; Issue: 1 Linguagem: Inglês

10.1016/j.pt.2020.10.005

1471-5007

Richard J. Martin, Alan P. Robertson, Shivani Choudhary,

Parasite Biology and Host Interactions

Ivermectin and analogs are remarkable broad-spectrum anthelmintics and insecticides, but resistance is now a real concern. Resistance mechanisms have been proposed but they do not appear to account for the resistance seen in parasitic nematodes.Ivermectin is very effective for controlling microfilaria at low doses but it has limited effects on adult filaria for unclear reasons.Ivermectin is a positive allosteric modulator of glutamate-gated chloride channels found in both nematodes and insects, and it binds to the channels in their lipid phase.Ivermectin and analogs also modulate other ion channels and have effects on the mammalian host brain when the blood–brain barrier is impaired.Preliminary repositioning studies of ivermectin show antiviral, antimalarial, antimetabolic, and anticancer effects at concentrations higher than anthelmintic concentrations in tissue culture. Here we tell the story of ivermectin, describing its anthelmintic and insecticidal actions and recent studies that have sought to reposition ivermectin for the treatment of other diseases that are not caused by helminth and insect parasites. The standard theory of its anthelmintic and insecticidal mode of action is that it is a selective positive allosteric modulator of glutamate-gated chloride channels found in nematodes and insects. At higher concentrations, ivermectin also acts as an allosteric modulator of ion channels found in host central nervous systems. In addition, in tissue culture, at concentrations higher than anthelmintic concentrations, ivermectin shows antiviral, antimalarial, antimetabolic, and anticancer effects. Caution is required before extrapolating from these preliminary repositioning experiments to clinical use, particularly for Covid-19 treatment, because of the high concentrations of ivermectin used in tissue-culture experiments. Here we tell the story of ivermectin, describing its anthelmintic and insecticidal actions and recent studies that have sought to reposition ivermectin for the treatment of other diseases that are not caused by helminth and insect parasites. The standard theory of its anthelmintic and insecticidal mode of action is that it is a selective positive allosteric modulator of glutamate-gated chloride channels found in nematodes and insects. At higher concentrations, ivermectin also acts as an allosteric modulator of ion channels found in host central nervous systems. In addition, in tissue culture, at concentrations higher than anthelmintic concentrations, ivermectin shows antiviral, antimalarial, antimetabolic, and anticancer effects. Caution is required before extrapolating from these preliminary repositioning experiments to clinical use, particularly for Covid-19 treatment, because of the high concentrations of ivermectin used in tissue-culture experiments. Ivermectin is a mixture of more than 80% 22,23-dihydroavermectin B1a and B1b (Figure 1). It is a remarkably potent anthelmintic and insecticide when given orally at therapeutic doses of 150 or 200 μg/kg to ruminants, pigs, horses, or humans where it yields Cmax plasma concentrations of 11–54 ng/ml or 13–63 nM [1.Canga A.G. et al.The pharmacokinetics and interactions of ivermectin in humans – A mini-review.AAPS J. 2008; 10: 42-46Crossref PubMed Scopus (148) Google Scholar,2.González Canga A. et al.The pharmacokinetics and metabolism of ivermectin in domestic animal species.Vet. J. 2009; 179: 25-37Crossref PubMed Scopus (142) Google Scholar]. It is generally safe, with acute LD50 (see Glossary) toxicities seen at 24 000 μg/kg in monkeys [3.Merck Stomectol safety data sheet.2019: 7Google Scholar] and 80 000 μg/kg in beagles [4.Bauck S. Ivermectin toxicity in small animals.Can. Vet. J. 1987; 28: 563-564PubMed Google Scholar]; it has a wide spectrum of action against gastrointestinal (GI) parasitic nematodes, lungworms, lice, and mange, but it is not effective against cestodes or trematodes. It is, however, very effective at low plasma concentrations against microfilaria, and it impairs adult filaria fertility for long periods without killing most adult filaria. Here we review ivermectin’s mechanism of action as an anthelmintic, as an insecticide, and recent studies that have sought to reposition ivermectin as an antiviral, antimalarial, and antidiabetic drug (Table 1).Table 1Experimental Settings for Studying the Effects of Ivermectin on Ion Channels, FXR, NHR-8, Viruses, Malaria, Cancer, and AsthmaTargetExperimental preparationConcentration: ng/ml (nM)Refs and commentAVR-15b Haemonchus contortus channel openingHEK293 expressedEC50 19 (22 nM)[28.Atif M. et al.Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus.PLoS Pathog. 2017; 13e1006663Crossref PubMed Scopus (13) Google Scholar]Abamectin on GluCls of Ascaris suum pharynx inhibitionDissected whole worm pharynxEC50 350 (400 nM)[37.Puttachary S. et al.Derquantel and abamectin: effects and interactions on isolated tissues of Ascaris suum.Mol. Biochem. Parasitol. 2013; 188: 79-86Crossref PubMed Scopus (20) Google Scholar]Brugia malayi mf protein release, ES inhibition by Bma-AVR-14A GluClWhole Brugia and microfilaria<88 (<100 nM)[31.Moreno Y. et al.Ivermectin disrupts the function of the excretory–secretory apparatus in microfilariae of Brugia malayi.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 20120-20125Crossref PubMed Scopus (92) Google Scholar]Downregulation of B. malayi meiosis genesWhole Brugia<88 (<100 nM)[8.Ballesteros C. et al.The effects of ivermectin on Brugia malayi females in vitro: a transcriptomic approach.PLoS Negl. Trop. Dis. 2016; 10e0004929PubMed Google Scholar]A. suum GABA ion-channel inhibitionDissected preparation<175 (<200 nM)[27.Martin R.J. Pennington A.J. A patch-clamp study of effects of dihydroavermectin on Ascaris muscle.Br. J. Pharmacol. 1989; 98: 747-756Crossref PubMed Scopus (62) Google Scholar]Histamine-gated Cl channel activationExpressed channel from Drosophila melanogaster875 (1000 nM) used[33.Zheng Y. et al.Identification of two novel Drosophila melanogaster histamine-gated chloride channel subunits expressed in the eye.J. Biol. Chem. 2002; 277: 2000-2005Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar]pH-gated Cl channel activationSarcoptes scabiei oocyte expressed receptorSingle conc 10 000(11 429 nM)[34.Mounsey K.E. et al.Molecular characterisation of a pH-gated chloride channel from Sarcoptes scabiei.Invertebr. Neurosci. 2007; 7: 149-156Crossref PubMed Scopus (37) Google Scholar]Nematode nAChRsExpressed channels and A. suum preparationEC50 368 (420 nM)[35.Abongwa M. et al.Curiouser and curiouser: the macrocyclic lactone, abamectin, is also a potent inhibitor of pyrantel/tribendimidine nicotinic acetylcholine receptors of gastro-intestinal worms.PLoS One. 2016; 11e0146854Crossref PubMed Scopus (11) Google Scholar,37.Puttachary S. et al.Derquantel and abamectin: effects and interactions on isolated tissues of Ascaris suum.Mol. Biochem. Parasitol. 2013; 188: 79-86Crossref PubMed Scopus (20) Google Scholar]FXR activation and transcriptionFXR and ivermectin Crystal structure and mice, serum glucose and cholesterol decrease1300 μg/kg per day for 14 days[69.Jin L. et al.The antiparasitic drug ivermectin is a novel FXR ligand that regulates metabolism.Nat. Commun. 2013; 4: 1937Crossref PubMed Scopus (81) Google Scholar, 70.Jin L. et al.Selective targeting of nuclear receptor FXR by avermectin analogues with therapeutic effects on nonalcoholic fatty liver disease.Sci. Rep. 2015; 517288Crossref PubMed Scopus (24) Google Scholar, 71.Wang Y.D. et al.Farnesoid X receptor antagonizes nuclear factor kappaB in hepatic inflammatory response.Hepatology. 2008; 48: 1632-1643Crossref PubMed Scopus (360) Google Scholar, 72.Wang Y.D. et al.FXR: a metabolic regulator and cell protector.Cell Res. 2008; 18: 1087-1095Crossref PubMed Scopus (220) Google Scholar]Repurpose or find more potent analogNHR-8 transcription factorNHR-8 of Caenorhabditis elegans and H. contortusKnockout in C. elegans reduced ~2✕ the EC50 for the larval development assay (from 1.63 to 0.96 nM)[104.Ménez C. et al.The transcription factor NHR-8: A new target to increase ivermectin efficacy in nematodes.PLoS Pathog. 2019; 15e1007598Crossref PubMed Scopus (15) Google Scholar]Low level of resistanceDengue; West Nile virus; Venezuelan equine encephalitis virus inhibitionExperiments used tissue culture and showed virus inhibition was associated with the inhibition of transport by nuclear transporter IMPα/β1IC50s 875–3500(1000–4000 nM)[55.Wagstaff K.M. et al.An AlphaScreen®-based assay for high-throughput screening for specific inhibitors of nuclear import.J. Biomol. Screen. 2011; 16: 192-200Crossref PubMed Scopus (88) Google Scholar, 56.Caly L. et al.Nuclear trafficking of proteins from RNA viruses: potential target for antivirals?.Antivir. Res. 2012; 95: 202-206Crossref PubMed Scopus (52) Google Scholar, 57.Wagstaff K.M. et al.Ivermectin is a specific inhibitor of importin α/β-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus.Biochem. J. 2012; 443: 851-856Crossref PubMed Scopus (313) Google Scholar, 58.Lundberg L. et al.Nuclear import and export inhibitors alter capsid protein distribution in mammalian cells and reduce Venezuelan equine encephalitis virus replication.Antivir. Res. 2013; 100: 662-672Crossref PubMed Scopus (81) Google Scholar, 59.Jans D.A. et al.Inhibitors of nuclear transport.Curr. Opin. Cell Biol. 2019; 58: 50-60Crossref PubMed Scopus (51) Google Scholar, 60.Lv C. et al.Ivermectin inhibits DNA polymerase UL42 of pseudorabies virus entrance into the nucleus and proliferation of the virus in vitro and in vivo.Antivir. Res. 2018; 159: 55-62Crossref PubMed Scopus (38) Google Scholar, 61.Ketkar H. et al.Lack of efficacy of ivermectin for prevention of a lethal Zika virus infection in a murine system.Diagn. Microbiol. Infect. Dis. 2019; 95: 38-40Crossref PubMed Scopus (21) Google Scholar]More potent analog required to be effectiveSARS-Covid-2 inhibitionTissue culture4375 (5000 nM),~ 100×[107.Caly L. et al.The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro.Antivir. 2020; 178Google Scholar]Plasmodium inhibitionCulture: inhibition of Plasmodium in liver inhibits nuclear import of signal recognition particles of P. falciparumIC50 ~438 (~500 nM)[65.Panchal M. et al.Plasmodium falciparum signal recognition particle components and anti-parasitic effect of ivermectin in blocking nucleo-cytoplasmic shuttling of SRP.Cell Death Dis. 2014; 5: e994Crossref PubMed Scopus (32) Google Scholar, 66.Mendes A.M. et al.Inhibition of Plasmodium liver infection by ivermectin.Antimicrob. Agents Chemother. 2017; 61 (e02005-16): 1-7Crossref Scopus (27) Google Scholar, 67.Singh L. et al.Molecular design and synthesis of ivermectin hybrids targeting hepatic and erythrocytic stages of Plasmodium parasites.J. Med. Chem. 2020; 63: 1750-1762Crossref PubMed Scopus (4) Google Scholar]More potent analog required to be effective.Ivermectin has effects on mosquitoesCancerTissue culture and mice experiments by different mechanisms<8000 nM[76.Antoszczak M. et al.Old wine in new bottles: Drug repurposing in oncology.Eur. J. Pharmacol. 2020; 866172784Crossref PubMed Scopus (21) Google Scholar,77.Zhang X. et al.Inhibition of TMEM16A Ca.Pharmacol. Res. 2020; 156104763Crossref PubMed Scopus (8) Google Scholar,79.Melotti A. et al.The river blindness drug Ivermectin and related macrocyclic lactones inhibit WNT-TCF pathway responses in human cancer.EMBO Mol. Med. 2014; 6: 1263-1278Crossref PubMed Scopus (66) Google Scholar, 80.Draganov D. et al.Modulation of P2X4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death.Sci. Rep. 2015; 516222Crossref PubMed Scopus (76) Google Scholar, 81.Chen L. et al.Ivermectin suppresses tumour growth and metastasis through degradation of PAK1 in oesophageal squamous cell carcinoma.J. Cell. Mol. Med. 2020; 24: 5387-5401Crossref PubMed Scopus (3) Google Scholar, 82.Ashraf S. Prichard R. Ivermectin exhibits potent anti-mitotic activity.Vet. Parasitol. 2016; 226: 1-4Crossref PubMed Scopus (10) Google Scholar, 83.Dominguez-Gomez G. et al.Ivermectin as an inhibitor of cancer stem-like cells.Mol. Med. Rep. 2018; 17: 3397-3403PubMed Google Scholar,105.Nappi L. et al.Ivermectin inhibits HSP27 and potentiates efficacy of oncogene targeting in tumor models.J. Clin. Invest. 2020; 130: 699-714Crossref PubMed Scopus (13) Google Scholar]High concentrations required for effectsAsthmaMice: reduction of cellular and humoral responses to antigen in asthma model2000 μg/kg[89.Yan S. et al.Anti-inflammatory effects of ivermectin in mouse model of allergic asthma.Inflamm. Res. 2011; 60: 589-596Crossref PubMed Scopus (51) Google Scholar]High μg/kg dose Open table in a new tab Ivermectin and its analogs are positive allosteric modulators (PAMs) that selectively open inhibitory glutamate-gated chloride ion channels in the membranes of pharyngeal muscles, motor nerves, female reproductive tracts, and the excretory/secretory (ES) pores of nematodes and of muscle and nerves of insects and crustaceans [5.Campbell W.C. Ivermectin and Abamectin. Springer-Verlag, 1989Crossref Google Scholar,6.Vercruysse J. Macrocyclic Lactontes in Antiparasitic Therapy. CABI, 2002Crossref Google Scholar]. The effect is: (i) inhibition of pharyngeal pumping (Figure 2) when the pharyngeal muscle is the target; (ii) inhibition of motility when motor nerves are the main target; (iii) inhibition of egg or microfilaria release when the female reproductive tract is the target site; and (iv) loss of host immunosuppression when the ES pore cannot open to release host immunosuppressants. Figure 2A–E shows a two-micropipette recording of the effects of glutamate on membrane potential and the conductance of pharyngeal muscle in Ascaris suum. When glutamate is added to the preparation, glutamate-gated chloride (GluCl) ion channels open, the resistance of the pharyngeal muscle decreases, and the membrane potential hyperpolarizes as Cl– enters the pharynx (Figure 2D,E). This is reversible on washing. Ivermectin behaves similarly, but is slower in onset, and because it is lipophilic (sticky) it does not wash off from the preparation after application (Figure 2F). When ivermectin is applied to active pumping pharyngeal muscle cells of Trichostrongylus colubriformis, the electropharyngeogram shows the reduction in electrical activity (Figure 2G). Although equivalent recordings from the nerves, reproductive tract, and the ES pore of parasitic nematodes have not been made it is expected that similar observations would be made from all their tissues that have GluCl channels. The different tissues in the different parasitic nematodes have molecularly diverse GluCl channels and therefore different sensitivities to ivermectin. The most ivermectin-sensitive tissues, and the phenotypic effects of ivermectin, vary with the parasite species. Li et al. [7.Li B.W. et al.High level expression of a glutamate-gated chloride channel gene in reproductive tissues of Brugia malayi may explain the sterilizing effect of ivermectin on filarial worms.Int. J. Parasitol. Drugs Drug Resist. 2014; 4: 71-76Crossref PubMed Scopus (20) Google Scholar] used in situ hybridization to localize tissue expression of the Brugia malayi GluCl channel gene avr-14 (Bma-AVR-14) in adult filarial worms. Splice variant subunits of Bma-AVR-14 were expressed in female worms in the ovary, in developing embryos, in the lateral hypodermal chords, and in the uterus wall adjacent to stretched microfilariae. The genes were also expressed in adult male worms in the spermatogonia, in the vas deferens, and in the somatic muscles adjacent to the vas deferens. The tissues of the ovary and other reproductive cells are nonexcitable, so the GluCl channels may serve a paracrine function, inhibiting the growth and development of these tissues like other ligand-gated ion channels in the nonexcitable tissues of vertebrates. The activation of GluCl channels in the reproductive tracts of nematodes, by a paracrine function, could change gene expression producing the longer-term inhibitory effects of ivermectin on the developing embryos and reproductive tissues of filaria and suppression of microfilaria production by female worms. The significance of the effect of ivermectin on the reproductive tract was also shown by RNAseq analysis that revealed downregulation of meiosis genes in female B. malayi by low (100 nM–1 μM) concentrations of ivermectin [8.Ballesteros C. et al.The effects of ivermectin on Brugia malayi females in vitro: a transcriptomic approach.PLoS Negl. Trop. Dis. 2016; 10e0004929PubMed Google Scholar]. The evidence of GluCl channel expression in the nematode reproductive tract of filaria may explain the effects of ivermectin reducing their fertility but not why this effect is so slow in onset. It is pointed out that this effect is prolonged and persists long after the ivermectin has been cleared from the host and is no longer detectable. GluCl ion channels consist of a ring of five protein subunits that may arrange in homogeneous or heterogeneous combinations (Figure 3C ). In the free-living nematode Caenorhabditis elegans, there are six GluCl genes encoding channel subunits – avr-14, avr-15, glc-1, glc-2, glc-3, and glc-4 – that contribute to the ivermectin sensitivity in C. elegans [9.Cully D.F. et al.Cloning of an avermectin-sensitive glutamate-gated chloride channel from Caenorhabditis elegans.Nature. 1994; 371: 707-711Crossref PubMed Scopus (542) Google Scholar, 10.Cully D.F. et al.Molecular biology and electrophysiology of glutamate-gated chloride channels of invertebrates.Parasitology. 1996; 113: S191-S200Crossref PubMed Scopus (73) Google Scholar, 11.Vassilatis D.K. et al.Genetic and biochemical evidence for a novel avermectin-sensitive chloride channel in Caenorhabditis elegans. Isolation and characterization.J. Biol. Chem. 1997; 272: 33167-33174Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 12.Dent J.A. et al.avr-15 encodes a chloride channel subunit that mediates inhibitory glutamatergic neurotransmission and ivermectin sensitivity in Caenorhabditis elegans.EMBO J. 1997; 16: 5867-5879Crossref PubMed Scopus (255) Google Scholar, 13.Dent J.A. et al.The genetics of ivermectin resistance in Caenorhabditis elegans.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2674-2679Crossref PubMed Scopus (285) Google Scholar, 14.Horoszok L. et al.GLC-3: a novel fipronil and BIDN-sensitive, but picrotoxinin-insensitive, L-glutamate-gated chloride channel subunit from Caenorhabditis elegans.Br. J. Pharmacol. 2001; 132: 1247-1254Crossref PubMed Scopus (78) Google Scholar]. The GluCl channel subunit gene family is divergent in parasitic nematodes and varies between the species. In the intestinal parasite of sheep, Haemonchus contortus, and in the human hookworms Ancylostoma ceylanicum and Necator americanus, there are no glc-1 nor avr-15 orthologsi. glc-1 is not present and is also missing in the filarial parasites B. malayi and Onchocerca volvulus. In H. contortus the subunit genes known to be present are: avr-14, glc-2, glc-3, glc-4, glc-5, and glc-6 [15.Glendinning S.K. et al.Glutamate-gated chloride channels of Haemonchus contortus restore drug sensitivity to ivermectin resistant Caenorhabditis elegans.PLoS One. 2011; 6: e22390Crossref PubMed Scopus (49) Google Scholar]. Thus, different nematode species have different GluCl channel gene homologs. The sensitivity to ivermectin varies between the different homomeric and heteromeric GluCl channels. A few amino acid changes in the sequence of any of the subunits may alter the ivermectin sensitivity of the channel significantly. It is not surprising that there are striking differences, even between closely related species: A. ceylanicum is much more sensitive to ivermectin than the new world hookworm, N. americanus, both in vitro and in vivo [16.Richards J.C. et al.In vitro studies on the relative sensitivity to ivermectin of Necator americanus and Ancylostoma ceylanicum.Int. J. Parasitol. 1995; 25: 1185-1191Crossref PubMed Scopus (44) Google Scholar,17.Behnke J.M. et al.Sensitivity to ivermectin and pyrantel of Ancylostoma ceylanicum and Necator americanus.Int. J. Parasitol. 1993; 23: 945-952Crossref PubMed Scopus (26) Google Scholar]. Other explanations for the difference in sensitivity are also possible. In many insects, a single GluCl channel gene, GluClα, is present but its transcripts are modified by mRNA splicing and editing [18.Semenov E.P. Pak W.L. Diversification of Drosophila chloride channel gene by multiple posttranscriptional mRNA modifications.J. Neurochem. 1999; 72: 66-72Crossref PubMed Scopus (65) Google Scholar, 19.Jones A.K. et al.The cys-loop ligand-gated ion channel gene superfamily of the parasitoid wasp, Nasonia vitripennis.Heredity (Edinb). 2010; 104: 247-259Crossref PubMed Scopus (51) Google Scholar, 20.Jones A.K. Sattelle D.B. The cys-loop ligand-gated ion channel gene superfamily of the red flour beetle, Tribolium castaneum.BMC Genom. 2007; 8: 327Crossref PubMed Scopus (107) Google Scholar]. The presence of gene splicing adds an additional layer of diversity to the GluCl channel and its sensitivity to ivermectin. An example of the effects of ivermectin in insects is seen with Drosophila melanogaster, where GluClα mediates sensitivity to ivermectin. The presence of GluCl channel targets in insects and arthropod parasites allows ivermectin to be used for the treatment of scabies and lice in humans [21.Wendel K. Rompalo A. Scabies and pediculosis pubis: an update of treatment regimens and general review.Clin. Infect. Dis. 2002; 35: S146-S151Crossref PubMed Scopus (69) Google Scholar]. Interestingly, ivermectin also has inhibitory effects on mosquitoes, and its use has been suggested to reduce the mosquito vectors in malaria [22.Kobylinski K.C. et al.The effect of oral anthelmintics on the survivorship and re-feeding frequency of anthropophilic mosquito disease vectors.Acta Trop. 2010; 116: 119-126Crossref PubMed Scopus (69) Google Scholar]. In humans, it has been shown to be effective for treatment of head lice, (Pediculus humanus capitis), scabies (Sarcoptes scabiei) prevention of myiasis, and biting by mosquitoes [23.Ashour D.S. Ivermectin: From theory to clinical application.Int. J. Antimicrob. Agents. 2019; 54: 134-142Crossref PubMed Scopus (16) Google Scholar]. In animals, a range of preparations of ivermectin are used and are effective against lice, mites, screw worms, bot flies, and warbles. Each subunit of the pentameric GluCl ion channel is about 500 amino acids long with an N terminal and C terminal on the extracellular surface (Figure 3A). Each subunit has four transmembrane α-helices: M1, M2, M3, and M4. The five subunits of the GluCl channel may be identical (homogeneous) or nonidentical (heterogeneous). Glutamate binds to the orthosteric agonist sites (Figure 4C ) which are located at the interface between two subunits [24.Degani-Katzav N. et al.Subunit stoichiometry and arrangement in a heteromeric glutamate-gated chloride channel.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E644-E653Crossref PubMed Scopus (10) Google Scholar]. There are two to five orthosteric agonist sites on each channel, depending on the subunit composition of the GluCl channel. Figures 3C and 4C show the orthosteric binding sites where glutamate binds to the channel. When glutamate binds, the channel opens, and each channel opening can be seen under single-channel patch-clamp recording of the A. suum pharynx (Figure 3D, top trace) with current pulses of about 2 pA (2 × 10–12 A) as the channel changes from the closed state to the open state. The site of action of ivermectin is an allosteric site (Figures 3B,C and 4C), a location that is different to the physiologic glutamate ligand-binding site. Ivermectin binds to the GluCl channel within the transmembrane region of the ion channel (Figure 3B). FRAP (fluorescence recovery after photobleaching) experiments – using fluorescent bodipy (boron-dipyrromethene) ivermectin [25.Martin R.J. Kusel J.R. On the distribution of a fluorescent ivermectin probe (4'' 5,7-dimethyl-bodipy proprionylivermectin) in Ascaris membranes.Parasitology. 1992; 104: 549-555Crossref PubMed Scopus (17) Google Scholar] quenched with trypan blue – show that ivermectin locates in the outer membrane of the phospholipid bilayer to exert its effects on the chloride channels (Figure 3B). The activation of the GluCl channels is progressive and slow, increasing over minutes following application of ivermectin to the preparation (Figure 2F), indicating diffusion into the lipid membrane and diffusion to the ion channel binding site. Crystal structures of homopentameric GluCl channels [26.Hibbs R.E. Gouaux E. Principles of activation and permeation in an anion-selective Cys-loop receptor.Nature. 2011; 474: 54-60Crossref PubMed Scopus (736) Google Scholar] provide further details of the molecular binding sites of glutamate and ivermectin to the receptor within the outer phospholipid layer of the membrane. The pentameric GluCl channels form barrel-like staves around their central anion-selective pore. Recall that each of the pentameric subunits has four α-helix transmembrane regions, M1, M2, M3, and M4 (Figure 3A). Ivermectin can insert deeply into the subunits to make contact with the M2 pore-lining α-helix and the M2 and M3 loops because it is lipophilic (Figure 3B,C). This stabilizes the open-pore conformation of the ion channel, thereby increasing the time the ion channel stays open (Figure 3B,D). Single-channel experiments in A. suum [27.Martin R.J. Pennington A.J. A patch-clamp study of effects of dihydroavermectin on Ascaris muscle.Br. J. Pharmacol. 1989; 98: 747-756Crossref PubMed Scopus (62) Google Scholar] show that low concentrations of dihydroavermectin produce progressive opening of channels, giving rise to a ‘staircase’ effect. The conductance of these channels was 9–15 pS. Similar conductances were observed with expressed GluCl channels from H. contortus [28.Atif M. et al.Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus.PLoS Pathog. 2017; 13e1006663Crossref PubMed Scopus (13) Google Scholar]. Nematode parasites can modulate the immune response of their host by releasing a complex mixture of immune modulatory compounds [29.King I.L. Li Y. Host–parasite interactions promote disease tolerance to intestinal helminth infection.Front. Immunol. 2018; 9: 2128Crossref PubMed Scopus (14) Google Scholar,30.Maizels R.M. Toxocara canis: molecular basis of immune recognition and evasion.Vet. Parasitol. 2013; 193: 365-374Crossref PubMed Scopus (78) Google Scholar] and thereby survive within the host. Loss of the ability to release the immune modulators would contribute to the elimination of the parasite. Moreno et al. [31.Moreno Y. et al.Ivermectin disrupts the function of the excretory–secretory apparatus in microfilariae of Brugia malayi.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 20120-20125Crossref PubMed Scopus (92) Google Scholar] have pointed out that ivermectin, at submicromolar concentrations, produces a rapid loss of microfilaria in host blood but fails to affect their motility in culture. An explanation for this contradictory observation is that the ivermectin-sensitive GluCl channels of microfilaria are localized on a muscle that surrounds the microfilarial ES vesicle; release of immune-modulator proteins from the ES vesicle is therefore under the control of GluCl channels. Moreno et al. [31.Moreno Y. et al.Ivermectin disrupts the function of the excretory–secretory apparatus in microfilariae of Brugia malayi.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 20120-20125Crossref PubMed Scopus (92) Google Scholar] showed that ivermectin reduced protein release from the ES apparatus and that the loss of the ES substances could reduce the ability of the parasite to secrete proteins that allow evasion of the host immune system. Observations [32.Vatta A.F. et al.Ivermectin-dependent attachment of neutrophils and peripheral blood mononuclear cells to Dirofilaria immitis microfilariae in vitro.Vet. Parasitol. 2014; 206: 38-42Crossref PubMed Scopus (26) Google Scholar] with Dirofilaria. immitis microfilariae support this loss of immune modulation by the parasite and show that ivermectin produces attachment of mononuclear cells and neutrophils. These observations imply a direct effect, that is, that ivermectin prevents the parasites’ ability to release substances that inhibit the host’s immune response. The effect of ivermectin on ES substances in nonfilarial nematodes remains to be explored. Although it is often assumed that ivermectin only acts therapeutically as a PAM on different GluCl channels of nematodes, insects, and arthropods, it also has PAM and inhibitory negative allosteric (NAM) effects on other ion channels. These actions may support the actions of ivermectin on GluCl channels. It has inhibitory effects on γ-aminobutyric acid (GABA) channel conductances at low concentrations of <0.2 μM in A. suum [27.Martin R.J. Pennington A.J. A patch-clamp study of effects of dihydroavermectin on Ascaris muscle.Br. J. Pharmacol. 1989; 98: 747-756Crossref PubMed Scopus (62) Google Scholar], but at higher (10 μM) concentrations it has potentiating effects. Ivermectin also acts as a PAM on the histamine-gated Cl– channels in D. melanogaster [33.Zheng Y. et al.Identification of two novel Drosophila melanogaster histamine-gated chloride channel subunits expressed in the eye.J. Biol. Chem. 2002; 277: 2000-2005Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar], the pH-gated Cl– channel in Sarcoptes scabiei [34.Mounsey K