ABSTRACT
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% [EC50] = 0.4 mM) and ivermectin (IVM; EC50 = 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.
Subject(s)
Brugia malayi , Chloride Channels , Indoles , Animals , Brugia malayi/drug effects , Brugia malayi/genetics , Brugia malayi/metabolism , Chloride Channels/drug effects , Chloride Channels/genetics , Chloride Channels/metabolism , Glutamic Acid/metabolism , Indoles/pharmacology , Ivermectin/pharmacology , LigandsABSTRACT
Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important "classical" anthelmintics like levamisole and pyrantel, as well as "novel" anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.
Subject(s)
Anthelmintics/pharmacology , Helminth Proteins/metabolism , Indoles/pharmacology , Nematoda/metabolism , Oxepins/pharmacology , Phenylenediamines/pharmacology , Receptors, Nicotinic/metabolism , Animals , Helminth Proteins/genetics , Nematoda/genetics , Receptors, Nicotinic/genetics , Xenopus laevisABSTRACT
BACKGROUND: Drugs currently used for controlling onchocerciasis and lymphatic filariasis (LF) are mainly microfilaricidal, with minimal or no effect on the adult worms. For efficient management of these diseases, it is necessary to search for new drugs with macrofilaricidal activities that can be used singly or in combination with existing ones. Daniellia oliveri and Psorospermum febrifugum are two plants commonly used in the local management of these infections in Bambui, a township in the North West Region of Cameroon, but there is currently no documented scientific evidence to support their claimed anthelmintic efficacy and safety. The aim of this study was to provide evidence in support of the search for means to eliminate these diseases by screening extracts and chromatographic fractions isolated from these plants for efficacy against the parasitic roundworms Onchocerca ochengi and Brugia pahangi. METHODS: The viability of O. ochengi adult worms was assessed using the MTT/formazan assay. Fully confluent monkey kidney epithelial cells (LLC-MK2) served as the feeder layer for the O. ochengi microfilariae (mfs) assays. Viability of the mfs was assessed by microscopic examination for mean motility scoring (relative to the negative control) every 24 h post addition of an extract. The Worminator system was used to test the effects of the extracts on adult B. pahangi motility, and mean motility units were determined for each worm. Cytotoxicity of the active extracts on N27 cells was assessed using the MTS assay. RESULTS: Extracts from D. oliveri and P. febrifugum were effective against the adult roundworms O. ochengi and B. pahangi. Interestingly, extracts showing macrofilaricidal activities against O. ochengi also showed activity against O. ochengi mfs. The hexane stem bark extract of D. oliveri (DOBHEX) was more selective for adult O. ochengi than for mfs, with a half maximal and 100% inhibitory concentration (IC50 and IC100, respectively) against adult O. ochengi of 13.9 and 31.3 µg/ml, respectively. The in vitro cytotoxicity of all active extracts on N27 cells showed selective toxicity for parasites (selectivity index > 1). Bioassay-guided fractionation of the extracts yielded fractions with activity against adult B. pahangi, thus confirming the presence of bioactive principles in the plant extracts. CONCLUSIONS: Our study supports the use of D. oliveri and P. febrifugum in the traditional treatment of onchocerciasis and LF. The further purification of active extracts from these plants could yield lead compounds for filarial drug discovery and development.
Subject(s)
Clusiaceae/chemistry , Fabaceae/chemistry , Filaricides/pharmacology , Onchocerca/drug effects , Plant Extracts/pharmacology , Animals , Cameroon , Cell Line , Haplorhini , Humans , Onchocerca/growth & development , Onchocerciasis/drug therapy , Onchocerciasis/parasitology , Plant Bark/chemistryABSTRACT
BACKGROUND: The current treatment of onchocerciasis relies on the use of ivermectin which is only microfilaricidal and for which resistant parasite strains of veterinary importance are increasingly being detected. In the search for novel filaricides and alternative medicines, we investigated the selective activity of crude extracts of Margaritaria discoidea and Homalium africanum on Onchocerca ochengi, a model parasite for O. volvulus. These plants are used to treat the disease in North West Cameroon. METHODS: Sixteen crude extracts were prepared from various parts of M. discoidea and H. africanum using different organic solvents. The filaricidal activities were determined in vitro. Cytotoxicity of the active extracts was assessed on monkey kidney epithelial cells in vitro and the selectivity indices (SI) of the extracts determined. Acute toxicity of the promising extracts was investigated in mice. RESULTS: Four out of the 16 extracts showed microfilaricidal activity based on motility reduction, whereas, none showed macrofilaricidal activity based on the MTT/formazan assay. The methylene chloride extract of H. africanum leaves (HLC) recorded the lowest IC50 of 31.25 µg/mL and an IC100 of 62.5 µg/mL. The SI for the active extracts ranged from 0.5 - 2.63. No form of acute toxicity was observed in mice. Phytochemical analysis revealed the presence of anthraquinones, sterols and terpenoids in the promising extracts. CONCLUSIONS: The non-polar extracts of M. discoidea and H. africanum are potential sources of new microfilaricidal lead compounds, and the results support their use in traditional medicine.
Subject(s)
Filaricides/pharmacology , Magnoliaceae/chemistry , Onchocerca/drug effects , Onchocerciasis/drug therapy , Plant Extracts/pharmacology , Animals , Anthraquinones/analysis , Cattle , Cell Line , Female , Filaricides/therapeutic use , Haplorhini , Kidney/cytology , Male , Mice , Mice, Inbred BALB C , Onchocerciasis/parasitology , Onchocerciasis/veterinary , Phytosterols/analysis , Plant Extracts/therapeutic use , Terpenes/analysisABSTRACT
The ongoing and widespread emergence of resistance to the existing anti-nematodal pharmacopeia has made it imperative to develop new anthelminthic agents. Historically, plants have been important sources of therapeutic compounds and offer an alternative to synthetic drugs. Monoterpenoids are phytochemicals that have been shown to produce acute toxic effects in insects and nematodes. Previous studies have shown nicotinic acetylcholine receptors (nAChRs) to be possible targets for naturally occurring plant metabolites such as carvacrol and carveol. In this study we examined the effects of monoterpenoid compounds on a levamisole sensitive nAChR from Oesophagostomum dentatum and a nicotine sensitive nAChR from Ascaris suum. We expressed the receptors in Xenopus laevis oocytes and used two-electrode voltage-clamp to characterize the effect of various compounds on these cys-loop receptors. At 100⯵M the majority of these compounds acted as antagonists. Interestingly, further experiments revealed that both 0.1⯵M and 10⯵M menthol potentiated acetylcholine and levamisole responses in the levamisole sensitive receptor but not the nicotine sensitive receptor. We also investigated the effects of 0.1⯵M menthol on the contractility of A. suum somatic muscle strips. Menthol produced significant potentiation of peak contractions at each concentration of acetylcholine. The positive allosteric modulatory effects of menthol in both in vivo and in vitro experiments suggests menthol as a promising candidate for combination therapy with cholinergic anthelmintics.
Subject(s)
Anthelmintics/pharmacology , Levamisole/pharmacology , Menthol/pharmacology , Receptors, Nicotinic/drug effects , Allosteric Regulation , Animals , Ascaris suum/drug effects , Ascaris suum/genetics , Cholinergic Agents/pharmacology , Female , Monoterpenes/pharmacology , Oesophagostomum/drug effects , Oesophagostomum/genetics , Receptors, Nicotinic/genetics , Xenopus laevisABSTRACT
Parasitic nematode infections are treated using anthelmintic drugs, some of which target nicotinic acetylcholine receptors (nAChRs) located in different parasite tissues. The limited arsenal of anthelmintic agents and the prevalence of drug resistance imply that future defense against parasitic infections will depend on the discovery of novel targets and therapeutics. Previous studies have suggested that Ascaris suum ACR-16 nAChRs are a suitable target for the development of antinematodal drugs. In this study, we characterized the pharmacology of the Ancylostoma caninum ACR-16 receptor using two-electrode voltage-clamp electrophysiology. This technique allowed us to study the effects of cholinergic agonists and antagonists on the nematode nAChRs expressed in Xenopus laevis oocytes. Aca-ACR-16 was not sensitive to many of the existing cholinomimetic anthelmintics (levamisole, oxantel, pyrantel, and tribendimidine). 3-Bromocytisine was the most potent agonist (> 130% of the control acetylcholine current) on the Aca-ACR-16 nAChR but, unlike Asu-ACR-16, oxantel did not activate the receptor. The mean time constants of desensitization for agonists on Aca-ACR-16 were longer than the rates observed in Asu-ACR-16. In contrast to Asu-ACR-16, the A. caninum receptor was completely inhibited by DHßE and moderately inhibited by α-BTX. In conclusion, we have successfully reconstituted a fully functional homomeric nAChR, ACR-16, from A. caninum, a model for human hookworm infections. The pharmacology of the receptor is distinct from levamisole-sensitive nematode receptors. The ACR-16 homologue also displayed some pharmacological differences from Asu-ACR-16. Hence, A. caninum ACR-16 may be a valid target site for the development of anthelmintics against hookworm infections.
Subject(s)
Ancylostoma/metabolism , Anthelmintics/pharmacology , Helminth Proteins/drug effects , Receptors, Nicotinic/drug effects , Ancylostomiasis , Animals , Cholinergic Agents/pharmacology , Helminth Proteins/analysis , Helminth Proteins/metabolism , Receptors, Nicotinic/analysis , Receptors, Nicotinic/metabolismABSTRACT
Zolvix® is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel is a positive allosteric modulator of DEG-3/DES-2 type nicotinic acetylcholine receptors (nAChRs) in several nematode species. The drug has been reported to produce hypercontraction of Caenorhabditis elegans and Haemonchus contortus somatic muscle. We investigated the effects of monepantel on nAChRs from Ascaris suum and Oesophagostomum dentatum heterologously expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp electrophysiology, we studied the effects of monepantel on a nicotine preferring homomeric nAChR subtype from A. suum comprising of ACR-16; a pyrantel/tribendimidine preferring heteromeric subtype from O. dentatum comprising UNC-29, UNC-38 and UNC-63 subunits; and a levamisole preferring subtype (O. dentatum) comprising UNC-29, UNC-38, UNC-63 and ACR-8 subunits. For each subtype tested, monepantel applied in isolation produced no measurable currents thereby ruling out an agonist action. When monepantel was continuously applied, it reduced the amplitude of acetylcholine induced currents in a concentration-dependent manner. In all three subtypes, monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 from A. suum was particularly sensitive to monepantel inhibition (IC50 values: 1.6⯱â¯3.1â¯nM and 0.2⯱â¯2.3⯵M). We also investigated the effects of monepantel on muscle flaps isolated from adult A. suum. The drug did not significantly increase baseline tension when applied on its own. As with acetylcholine induced currents in the heterologously expressed receptors, contractions induced by acetylcholine were antagonized by monepantel. Further investigation revealed that the inhibition was a mixture of competitive and non-competitive antagonism. Our findings suggest that monepantel is active on multiple nAChR subtypes.
Subject(s)
Aminoacetonitrile/analogs & derivatives , Ascaris suum/drug effects , Nicotinic Antagonists/pharmacology , Oesophagostomum/drug effects , Receptors, Nicotinic/drug effects , Acetylcholine/pharmacology , Aminoacetonitrile/pharmacology , Animals , Anthelmintics/pharmacology , Ascaris suum/cytology , Electrophysiology/methods , Muscles/drug effects , Muscles/physiology , Oesophagostomum/cytology , Oocytes , Receptors, Nicotinic/genetics , Xenopus laevis/geneticsABSTRACT
Anthelmintics are some of the most widely used drugs in veterinary medicine. Here we review the mechanism of action of these compounds on nematode parasites. Included are the older classes of compounds; the benzimidazoles, cholinergic agonists and macrocyclic lactones. We also consider newer anthelmintics, including emodepside, derquantel and tribendimidine. In the absence of vaccines for most parasite species, control of nematode parasites will continue to rely on anthelmintic drugs. As a consequence, vigilance in detecting drug resistance in parasite populations is required. Since resistance development appears almost inevitable, there is a continued and pressing need to fully understand the mode of action of these compounds. It is also necessary to identify new drug targets and drugs for the continued effective control of nematode parasites.
ABSTRACT
Nematode parasite infections pose a significant threat in human and veterinary medicine. At least a third of the world's population is at risk from nematode parasite infections. These infections not only cause health problems, but also cause loss of livestock production and hence, economic losses. Anthelmintic drugs are the mainstay by which control of nematode parasite infections is achieved. Many of the currently available anthelmintics act on nicotinic acetylcholine receptors (nAChRs). However, the detailed mode of action (MOA) of these anthelmintics is not clearly understood. Elucidation of the MOA of anthelmintics is highly desirable; an in-depth knowledge of the MOA will better inform on mechanisms of resistance development and on ways to slow down or overcome resistance. The cholinomimetic anthelmintic, morantel, has a complex MOA involving the activation and block of levamisole-sensitive single nAChR channels (L-type nAChR or L-nAChR). More recently, morantel has been demonstrated to activate Haemonchus contortus and Parascaris equorum ACR-26/ACR-27 nAChRs expressed in Xenopus laevis oocytes. Previous studies in our laboratory, however, have shown morantel does not activate the nicotine-sensitive nAChR (N-type nAChR or N-nAChR), Ascaris suum ACR-16 (Asu-ACR-16). In this study, we used two-electrode voltage-clamp (TEVC) electrophysiology to investigate the inhibitory effects of morantel, on expressed Asu-ACR-16 nAChRs in X. laevis oocytes. Our results show that morantel acts as a non-competitive antagonist on Asu-ACR-16. This non-competitive antagonism by morantel was further demonstrated to be voltage-sensitive. We conclude based on our findings that morantel is a non-competitive voltage-sensitive open channel blocker of Asu-ACR-16.
Subject(s)
Anthelmintics/pharmacology , Ascaris suum , Morantel/pharmacology , Nicotinic Antagonists/pharmacology , Receptors, Nicotinic/drug effects , AnimalsABSTRACT
Nematode parasites may be controlled with drugs, but their regular application has given rise to concerns about the development of resistance. Drug combinations may be more effective than single drugs and delay the onset of resistance. A combination of the nicotinic antagonist, derquantel, and the macrocyclic lactone, abamectin, has been found to have synergistic anthelmintic effects against gastro-intestinal nematode parasites. We have observed in previous contraction and electrophysiological experiments that derquantel is a potent selective antagonist of nematode parasite muscle nicotinic receptors; and that abamectin is an inhibitor of the same nicotinic receptors. To explore these inhibitory effects further, we expressed muscle nicotinic receptors of the nodular worm, Oesophagostomum dentatum (Ode-UNC-29:Ode-UNC-63:Ode-UNC-38), in Xenopus oocytes under voltage-clamp and tested effects of abamectin on pyrantel and acetylcholine responses. The receptors were antagonized by 0.03 µM abamectin in a non-competitive manner (reduced Rmax, no change in EC50). This antagonism increased when abamectin was increased to 0.1 µM. However, when we increased the concentration of abamectin further to 0.3 µM, 1 µM or 10 µM, we found that the antagonism decreased and was less than with 0.1 µM abamectin. The bi-phasic effects of abamectin suggest that abamectin acts at two allosteric sites: one high affinity negative allosteric (NAM) site causing antagonism, and another lower affinity positive allosteric (PAM) site causing a reduction in antagonism. We also tested the effects of 0.1 µM derquantel alone and in combination with 0.3 µM abamectin. We found that derquantel on these receptors, like abamectin, acted as a non-competitive antagonist, and that the combination of derquantel and abamectin produced greater inhibition. These observations confirm the antagonistic effects of abamectin on nematode nicotinic receptors in addition to GluCl effects, and illustrate more complex effects of macrocyclic lactones that may be exploited in combinations with other anthelmintics.
Subject(s)
Indoles/administration & dosage , Ivermectin/analogs & derivatives , Nematoda/drug effects , Oxepins/administration & dosage , Phenylenediamines/antagonists & inhibitors , Pyrantel/antagonists & inhibitors , Receptors, Nicotinic/drug effects , Acetylcholine/chemistry , Allosteric Site , Animals , Anthelmintics/administration & dosage , Cloning, Molecular , Dose-Response Relationship, Drug , Gastrointestinal Tract/parasitology , Gene Expression Regulation , Haemonchus/metabolism , Helminthiasis/drug therapy , Intestinal Diseases, Parasitic/drug therapy , Ivermectin/administration & dosage , Nicotinic Antagonists/administration & dosage , Oocytes/cytology , Oocytes/parasitology , Patch-Clamp Techniques , Xenopus laevisABSTRACT
Soil-transmitted helminth infections in humans and livestock cause significant debility, reduced productivity and economic losses globally. There are a limited number of effective anthelmintic drugs available for treating helminths infections, and their frequent use has led to the development of resistance in many parasite species. There is an urgent need for novel therapeutic drugs for treating these parasites. We have chosen the ACR-16 nicotinic acetylcholine receptor of Ascaris suum (Asu-ACR-16), as a drug target and have developed three-dimensional models of this transmembrane protein receptor to facilitate the search for new bioactive compounds. Using the human α7 nAChR chimeras and Torpedo marmorata nAChR for homology modeling, we defined orthosteric and allosteric binding sites on the Asu-ACR-16 receptor for virtual screening. We identified four ligands that bind to sites on Asu-ACR-16 and tested their activity using electrophysiological recording from Asu-ACR-16 receptors expressed in Xenopus oocytes. The four ligands were acetylcholine inhibitors (SB-277011-A, IC50, 3.12 ± 1.29 µM; (+)-butaclamol Cl, IC50, 9.85 ± 2.37 µM; fmoc-1, IC50, 10.00 ± 1.38 µM; fmoc-2, IC50, 16.67 ± 1.95 µM) that behaved like negative allosteric modulators. Our work illustrates a structure-based in silico screening method for seeking anthelmintic hits, which can then be tested electrophysiologically for further characterization.
Subject(s)
Ascaris suum/anatomy & histology , Ascaris suum/drug effects , Ascaris suum/metabolism , Drug Discovery/methods , Nicotinic Agonists/metabolism , Nicotinic Agonists/pharmacology , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/metabolism , Allosteric Regulation , Allosteric Site/genetics , Animals , Ascaris suum/genetics , Binding Sites/genetics , Butaclamol/pharmacology , Computer Simulation , Drug Delivery Systems , Fluorenes/metabolism , Fluorenes/pharmacology , Humans , Inhibitory Concentration 50 , Isonipecotic Acids/metabolism , Isonipecotic Acids/pharmacology , Ligands , Models, Molecular , Nicotinic Agonists/chemistry , Nitriles/pharmacology , Oocytes , Patch-Clamp Techniques , Tetrahydroisoquinolines/pharmacology , Torpedo/genetics , Torpedo/physiology , Xenopus/geneticsABSTRACT
BACKGROUND AND PURPOSE: Control of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR-16, from a nematode parasite. EXPERIMENTAL APPROACH: Using RT-PCR, molecular cloning and two-electrode voltage clamp electrophysiology, we localized acr-16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr-16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested. KEY RESULTS: Amino acid sequence comparison with vertebrate nAChR subunits revealed ACR-16 to be most closely related to α7 receptors, but with some striking distinctions. acr-16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, bephenium, tribendimidine and pyrantel) did not activate Asu-ACR-16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3-bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu-ACR-16 was insensitive to α-bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu-ACR-16 had lower calcium permeability than α7 receptors. CONCLUSIONS AND IMPLICATIONS: We suggest that ACR-16 has diverse tissue-dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.
Subject(s)
Ascaris suum/metabolism , Nicotinic Antagonists/pharmacology , Receptors, Nicotinic/metabolism , Amino Acid Sequence , Animals , Ascaris suum/drug effects , Ascaris suum/genetics , Female , Nicotinic Antagonists/chemistry , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/geneticsABSTRACT
'The best way to predict the future is to create it.' When we look at drugs that are used to control parasites, we see that new knowledge has been created (discovered) about their modes of action. This knowledge will allow us to predict combinations of drugs which can be used together rationally to increase the spectrum of action and to slow the development of anthelmintic resistance. In this paper we comment on some recent observations of ours on the modes of action of emodepside, diethylcarbamazine and tribendimidine. Emodepside increases the activation of a SLO-1 K(+) current inhibiting movement, and diethylcarbamazine has a synergistic effect on the effect of emodepside on the SLO-1 K(+) current, increasing the size of the response. The combination may be considered for further testing for therapeutic use. Tribendimidine is a selective cholinergic nematode B-subtype nAChR agonist, producing muscle depolarization and contraction. It has different subtype selectivity to levamisole and may be effective in the presence of some types of levamisole resistance. The new information about the modes of action may aid the design of rational drug combinations designed to slow the development of resistance or increase the spectrum of action.