Veterinary Parasitology 84 (1999) 275–295

Frontiers in anthelmintic pharmacology

Timothy G. Gearya,∗, Nicholas C. Sangsterb, David P. Thompsonaa Animal Health Discovery Research, Pharmacia& Upjohn, 301 Henrietta Street, Kalamazoo,

MI 49007-4940, USAb Department of Veterinary Anatomy and Pathology, School of Veterinary Medicine, University of Sydney,

Sydney, NSW 2006, Australia

Abstract

Research in anthelmintic pharmacology faces a grim future. The parent field of veterinary par-asitology has seemingly been devalued by governments, universities and the animal industry ingeneral. Primarily due to the success of the macrocyclic lactone anthelmintics in cattle, problemscaused by helminth infections are widely perceived to be unimportant. The market for anthelminticsin other host species that are plagued by resistance, such as sheep and horses, is thought to be toosmall to sustain a discovery program in the animal heallth pharmaceutical industry. These atti-tudes are both alarming and foolish. The recent history of resistance to antibiotics provides morethan adequate warning that complacency about the continued efficacy of any class of drugs for thechemotherapy of an infectious disease is folly. Parasitology remains a dominant feature of veteri-nary medicine and of the animal health industry. Investment into research on the basic and clinicalpharmacology of anthelmintics is essential to ensure chemotherapeutic control of these organismsinto the 21st century. In this article, we propose a set of questions that should receive priorityfor research funding in order to bring this field into the modern era. While the specific questionsare open for revision, we believe that organized support of a prioritized list of research objectivescould stimulate a renaissance in research in veterinary helminthology. To accept the status quo isto surrender. ©1999 Elsevier Science B.V. All rights reserved.

Keywords:Anthelmintic; Resistance; Nematode;C. elegans

1. Introduction

Despite the annual use of millions of doses of anthelmintics on production and companionanimals, we remain without answers to many important questions about the pharmacologyof these drugs. These questions persist on many levels, from the most basic aspects of

∗ Corresponding author. Tel.: +1-616-833-0916; fax: +1-616-833-1149; e-mail: timothy.g.geary@am.pnu.com

0304-4017/99/$ – see front matter ©1999 Elsevier Science B.V. All rights reserved.PII: S0304-4017(99)00042-4

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molecular pharmacology, to fundamental aspects of drug discovery strategies, to clinicalpharmacology. It is sobering to recognize that our understanding of how anthelminticswork is still incomplete, that the molecular bases for anthelmintic resistance remain largelyobscure, that we have only a tenuous grasp of the pharmacodynamic principles that underlietheir efficacy, and that our ability to discover novel anthelmintics is waning.

The end of the 20th century, in which modern chemotherapy was born, is an opportune,if trite, time to attempt to identify areas of critical importance for research on anthelminticpharmacology. By advancing a strategic research agenda designed to improve the discov-ery and use of anthelmintics, greater interest and financial support may be generated forthis field. The message must be broadcasted that billions of humans suffer from and lacktreatment for helminthiases, while (ironically) treatment and prevention of these infectionsconstitutes a major component of veterinary practice. The contemporary and unwarrantedcomplacency that is diminishing support for veterinary parasitology from many govern-ments and universities rests on the current efficacy of a single class of anthelmintic drugs,the macrocyclic lactone anthelmintics (avermectins and milbemycins, AM). Parasitic wormshave not disappeared; should resistance to these compounds spread (as may be inevitable)in cattle and companion animals, we would be once more at the mercy of the worms. Itis folly to trust in the hope that AM resistance in cattle will not worsen; this lesson hasbeen learned the hard way for infections like tuberculosis, malaria and HIV. We need toaggressively advance the case that parasitology is a key component of academic teachingand research and that the introduction of novel anthelmintics is a priority for the animalhealth industry.

What follows is the product of the authors’ opinions; others would almost certainlypropose different key areas for research. Nonetheless, one way to advance the case foranthelmintic research is to establish a platform from which reasoned discussion can belaunched. Perhaps financial and institutional support for studies on anthelmintic pharma-cology in the context of veterinary parasitology will be enhanced by broad adoption of aprioritized set of experimental goals. Our intent is to propose a series of urgent questions thatrequire answers before chemotherapeutic control of veterinary helminths can be secured.

2. Pharmacodynamics

Pharmacodynamics may be thought of as the nexus of efficacy and pharmacokinetics.Attaining a therapeutic effect requires that a drug be in the right place for enough timeand in sufficient concentration to exert the desired response. For most classes of drugs,this ‘sufficient concentration’ can be determined by measuring drug potency in in vitromodel systems. Anthelmintic discovery and development, and our ability to improve throughrational means the use of currently available drugs, suffer from our immature understandingof each of the components of pharmacodynamics. We lack an accepted model system thatcan be used to accurately identify drug concentrations required to deny parasite survival invivo and cannot confidently predict the host compartment(s) in which this concentrationmust be reached to ensure efficacy. Although much research has been done in this area (e.g.,Lanusse and Prichard, 1993; Baggot and McKellar, 1994), more is needed.

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2.1. How do we determine the ‘sufficient concentration’ of an anthelminti?

Most fields of chemotherapy enjoy in vitro test systems that can be used to accuratelypredict concentrations of drugs required for efficacy in vivo. For instance, the survival ofbacteria exposed to drugs in culture can be measured to predict the concentration needed intissues or blood for therapeutic success. Antibiotic-induced inhibition of bacterial survivaland/or reproduction is easy to measure and generally translates directly into drug effectsthat can be expected in vivo. Time-dependence can also be factored into the system, so thata more realistic simulation of the in vivo situation can be generated.

Unfortunately, this essential component of pharmacodynamics cannot easily be addressedfor helminths. While several options are available for measuring anthelmintic potency invitro, and each can illuminate important aspects of anthelmintic action, all have significantdisadvantages for predicting drug concentrations required for in vivo efficacy.Since nema-todes are the primary targets for anthelmintic chemotherapy, most effort has been focusedon them. The primary systems developed for this purpose include cultures of the free-livingnematodeCaenorhabditis elegansand of the adult and larval stages of parasitic nematodes(particularly ruminant trichostrongyloids).

2.1.1. C. elegansThat cultures of this free-living nematode have utility in anthelmintic screening was orig-

inally proposed almost 20 years ago (Simpkin and Coles, 1981). While discovery of newanthelmintic templates using a primaryC. elegansscreen has not been notably successful(see Geary et al., 1999), this organism has provided an exceptionally valuable model forresearch on the basic pharmacology of anthelmintic drugs (Rand and Johnson, 1995; Brglinet al., 1998). It is easy to detect drug effects in cultures of this nematode by monitoringbehavior, survival and/or reproduction. Drugs that reduce motility or survival, such as lev-amisole and AM class, can be detected in these cultures at low concentrations (Simpkinand Coles, 1981; our unpublished data), and their potency againstC. elegansis a reason-able predictor of potency against parasitic nematodes in culture. However, the correlation isnot universal. Morantel and pyrantel, which act like levamisole at nematode acetylcholinereceptors (Martin, 1997), are 50- and 100-fold less potent than levamisole, respectively,againstC. elegans(Simpkin and Coles, 1981). Differences in in vivo potency between thetetrahydropyrimidines and levamisole are much less marked. In addition, benzimidazolestypically show low potency and delayed onset of activity againstC. elegans(Simpkin andColes, 1981; our unpublished data). Finally, closantel, a salicylanilide with excellent activ-ity againstH. contortusin vivo, is only very weakly active againstC. elegans(E.M. Thomasand D.P. Thompson, unpublished observations).

Perhaps the most serious disadvantage of the use ofC. elegansas a model to estimateintrinsic potency of anthelmintics against parasitic nematodes is the profound difference inlife styles that can be tolerated by a free-living nematode in culture compared to a parasitetrying to survive in a hostile host environment. Subtle drug-induced alterations in behaviormay result in expulsion of worms from a host, but may be difficult to detect inC. elegans.Therefore, while the system can usually (but not always) be manipulated to detect knownanthelmintics, it is poorly suited to characterize the intrinsic potency of new compoundswith unknown mechanisms of action.

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2.1.2. Adult parasites in cultureParasitic nematodes cannot yet be raised in continuous culture, though maturation of

larvae to egg-laying adults has been attained (e.g., Stringfellow, 1986). The ability to raiseparasites in the laboratory outside of a host would be of enormous benefit to studies on thebasic biology of these organisms and the effects of drugs on them; the absence of a suitableculture system is a major impediment to such research. Further investment in this area isneeded to produce a breakthrough. Since the adult stage is a primary target for chemotherapy,it would be most desirable to be able to determine the intrinsic potency of anthelminticsagainst them. Available systems for maintaining adult stages in culture, following isolationfrom the host, seem to be inevitably plagued by a continuous drop in viability, complicatingthe interpretation of drug toxicity tests. Some species, such asHaemonchus contortus, areless robust in culture (Geary et al., 1993) than others, such asTrichostrongylus colubriformis(Rapson et al., 1985; Jenkins et al., 1986) andNippostrongylus brasiliensis(Rapson et al.,1987).

As for C. elegans, it is easy to identify drugs that quickly paralyze adult parasites; eithervisual observation or an automated ‘motility meter’ (Bennett and Pax, 1987) can be used.However, not all anthelmintics (e.g., benzimidazoles) affect nematode motility in the time-frame in which drug testing occurs. Alternative endpoints, such as pharyngeal functionand ATP levels (Geary et al., 1993), may be useful in this regard, but the manipulationsare laborious. Assays that detect anthelmintic activity of the benzimidazoles have beendeveloped, including measurement of the secretion of acetylcholinesterase (e.g., Rapsonet al., 1987), which also detects other anthelmintics, and migration (Petersen et al., 1997).

An assay measuring aggregation ofT. colubriformisis sensitive to very low concentrationsof the broad-spectrum anthelmintics (Jenkins et al., 1986). Due to the difficulty of obtaininglarge numbers of these parasites and the long duration of the test, such an assay is not suitablefor random screening. However, it is simple to perform and may offer a workable option fora quasi-universal endpoint for determination of anthelmintic potency. Whether the test canbe modified to provide steadily decreasing drug concentrations over time (thus mimickingthe in vivo situation) remains to be seen. This type of modification could permit correlationof mortality (efficacy) with peak concentration and time of exposure, data that are useful ininterpreting antibacterial pharmacodynamics.

2.1.3. Larval stages of parasitesAlthough several endpoints have been developed for anthelmintic assays in larvae of

parasitic nematodes, the most broadly useful appears to be one of the numerous variationsof a larval development assay (LDA). It has long been recognized that low concentrationsof broad-spectrum anthelmintics affect hatching, development or motility of nematodes.Using this approach, the rank order of potency of a series of anthelmintic AM analogswas correlated with in vivo efficacy of the same compounds against adult parasites of thesame species (Gill and Lacey, 1998). Furthermore, comparisons between drug-resistant and-susceptible isolates are transferable between the in vitro larval development and efficacyagainst adults of the same isolates in vivo. Unfortunately, rank order of potency amongunrelated anthelmintic classes in LDA tests is not well-correlated with in vivo potency (seeIbarra and Jenkins, 1988). As developing larvae are not the traditional targets of anthelmintic

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chemotherapy, and stage-dependent differences in target expression are possible, no variantof the LDA seems robust enough to use as a general assay for measuring intrinsic activityof experimental anthelmintics against adults.

2.2. What is the right place?

Most chemotherapy is targeted at organisms (or tumors) that exist in the blood or invarious tissues (or both). Measurement of drug levels in these compartments allows one tocompare pharmaceutically achievable concentrations with those required to kill or disablethe target in culture. Our understanding of the processes that control tissue distributionof drugs is maturing to the point that delivery strategies can be designed to enhance drugconcentrations in specific compartments, if necessary. It is usually possible to design drugassays that can be used to decide if these strategies are effective. Drug is usually acquiredby the target organism via some form of diffusion across the cell surface; no matter how thedrug is administered (orally, parenterally or topically), the compartment in which it mustappear is the relevant site in which to measure concentration. For orally administered drugs,the concept of bioavailability is straightforward: drug which is not absorbed from the GItract following dosing is lost to the host. Only drug that is absorbed can have a therapeuticeffect (outside of the GI tract itself, which is rarely targeted).

In contrast to viruses, bacteria, fungi and tumors, target helminths parasitize (often si-multaneously) virtually all host compartments, including blood (Dirofilaria immitis, Schis-tosoma spp.), tissues (Dictyocaulus viviparus, Fasciola hepatica) and the gastrointestinaltract from stomach to anus. Broad-spectrum anthelmintics must reach sufficient concen-tration to exert an effect in each of these compartments, preferably using a single deliverystrategy.

2.2.1. What does bioavailability mean for anthelmintics?The standard concepts of bioavailability clearly apply to parasites that infect blood or

tissues other than the GI tract. For intestinal inhabitants, the situation is more complex. Itis instructive to compare the spectrum of two anthelmintics with distinct and limited tis-sue distribution patterns. Closantel, a salicylanilide, is essentially restricted in distributionto blood; it is highly bound to serum proteins (albumin) and has a very long eliminationhalf-life. Among the GI parasites, only the hematophagous species, especiallyH. contortus,are susceptible to the drug. The closely related but non-hematophagous species,T. colubri-formis, is not affected by closantel in vivo. In culture, closantel is active against adult stagesof both species at similar concentrations (Bacon et al., 1998; E.M. Thomas and D.P. Thomp-son, unpublished observations). Why closantel is ineffective in vivo against hookworms,which are voracious blood feeders, is unclear.

At the opposite end of the spectrum, the pamoate salt of the tetrahydripyrimidine pyrantelis so insoluble as to be essentially restricted in distribution to the gut; very little is absorbedinto the bloodstream (e.g., Bjorn et al., 1996). Although only used in non-ruminants, pyrantelpamoate exhibits a respectable spectrum of action against GI nematodes. As expected, thepamoate salt is inactive against tissue-dwelling parasites.

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Taken together, these observations suggest that anthelmintic concentration in the plasmacompartment is at best an indirect measurment of bioavailability vis a vis GI worms. Ob-viously, when an anthelmintic is administered topically or parenterally, transfer to the cir-culatory compartment is an essential first step in attaining bioavailabilty for GI parasites.However, transfer of the drug from plasma to the GI compartment is required for broad spec-trum activity. Routes by which parenterally-administered anthelmintics reach the gut arenot well understood. Biliary secretion, diffusion from tissue fluids to gut fluids and direct,transporter-mediated excretion all probably contribute to some extent to in situ bioavail-ability of anthelmintics. These same processes effectively recycle drug absorbed from thegut after oral dosing, returning the anthelmintic to the site of action. It must be rememberedthat target parasites inhabit the gut from stomach to rectum. Does drug delivery from theplasma to the gut occur along its length, or are large intestinal worms only exposed to drugintroduced farther upstream? Within the GI tract, we cannot yet define the importance ofGI subcompartments, such as the tissue itself, secretions (e.g., mucus), and lumen fluids,as repositories for drug acquisition by parasites. How patterns of anthelmintic distributionin the GI tract influence spectrum of action remains to be determined.

It should be noted that considerable progress is being made in unraveling the pharmacoki-netic behavior of anthelmintics in GI compartments (e.g., Lanusse et al., 1998). However,this clearly remains a fertile area for more research. Of particular interest are experimentsrecently reported by Lanusse’s group in Argentina, in which the distrubution of albendazolewas simultaneously measured in host and parasite (Alvarez et al., 1998) and which provideda basis for understanding differences among AM drugs by measuring concentrations in var-ious tissue compartments (Lifschitz et al., 1998). Once a culture system can be developedto provide an accurate measure of intrinsic potency, progress in understanding anthelminticpharmacodynamics should be extremely rapid.

2.2.2. How do worms acquire drug in situ?Tissue dwelling parasites follow the paradigm developed for most applications of chemother-

apy: the targets are exposed to drug in the tissue of residence. The extent of anthelminticexposure is determined by diffusion from the plasma compartment, or, in the case of blood-dwelling parasites, directly by plasma levels.

How drugs enter worms parasitizing the gut is more difficult to conceptualize. Inhabitantsof the gut exhibit a wide range of feeding behaviors, from nearly exclusive hematophagy, totissue grazing, to consumption of material from the lumen, to an intracellular niche (adultTrichinella spiralis). The main route of acquisition of broad-spectrum anthelmintics bynematodes appears to be via trans-cuticular diffusion as opposed to oral ingestion (Ho etal., 1994; Sims et al., 1996). This concept is consistent with the hypothesis that anthelminticsmust be in the compartment of residence in order to exert broad spectrum activity. However,the trans-cuticular route has not been rigorously proven. If it is true, then measurementsof the physicochemical properties of an anthelmintic, coupled with an undertanding of itspharmacokinetic behavior in the gut, would enable one to predict the concentration versustime profile attained within the parasites in any given section of the tract (see Ho et al., 1994).More experiments that measure drug uptake by worms in situ (Rothwell and Sangster, 1997;Alvarez et al., 1998) could provide a powerful test of the validity of this approach.

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3. Resistance

Anthelmintic resistance is an undeniably catastrophic problem in some segments of theanimal health market, especially in small ruminants (Conder and Campbell, 1995; Waller,1997). Resistance arises in nematode populations (and other targets of chemotherapy) as aresult of selection with anthelmintics. Survivors of treatment reproduce and pass on allelesthat confer resistance to their offspring. The important consequence of resistance is that itcan lead to treatment failure and persistence of the detrimental effects of parasitism. Theessential question to be answered is whether this problem will eventually overwhelm other,currently less affected segments of that market.

3.1. Is the selection and spread of anthelmintic resistant nematodes inevitable?

3.1.1. Host factorsWhile devastating anthelmintic resistance is wide-spread among nematodes that para-

sitize sheep, resistance in cattle nematodes has been less frequently observed. However,resistance has appeared to all broad-spectrum anthelmintics in at least some species of cat-tle parasites (Conder and Campbell, 1995). Recently, ivermectin-resistantCooperiaspp.have been identified in New Zealand (Vermunt et al., 1995, 1996) and UK (Coles et al.,1998). Although plausible hypotheses have been advanced to explain the slower develop-ment of resistance in cattle parasites compared to parasites in sheep and goats, none has yetbeen proven. The important question is whether (or perhaps when) anthelmintic resistancein cattle will develop to the extent observed in small ruminants. We have insufficient datato answer this question, and a high priority for research should be experiments designed toprovide those data.

Treatment frequency may be the most important variable in this regard; it is unlikelythat physiological differences between small and large ruminants or their parasites underliethe phenomenona. However, there are no data of which we are aware to show that treatingcattle more frequently results in more rapid development of anthelmintic resistance. Onlywhen we can track the frequency of resistance alleles in worm populations will we be ableto show how treatment schedules result in changes in allele distribution. As described inSection 3.2, probes to permit such experiments are largely lacking. It is foolish to assumethat cattle parasites are somehow intrinsically unable to develop anthelmintic resistance; itis imperative that the matter be experimentally addressed as soon as possible.

3.1.2. Parasite factorsAre all nematode populations equally susceptible to the development of resistance? Many

interconnected factors influence the probability that a population of resistant organisms willbe selected by drug pressure. Such factors include the relative distribution of the populationin refugia (on pasture) versus in host during treatment; the absolute number of treatedreproductive individuals and the stage of treated individuals; the reproductive potential ofthe treated population (No. of eggs/female); and the inherent genetic diversity of the treatedpopulation. Perhaps the most basic factor is the genetic diversity of the pressured population;drug exposure does not create resistant organisms, but instead selects for the survival of

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individuals carrying an allele of a gene (or genes) that reduces susceptibility to the drug.Drug resistance can arise only if the resistant allele is present in the treated population. Themore genetically diverse a species, the more likely a resistance allele will be present.

In this regard, it is significant to note that genetic variation within populations of tri-chostrongyloid nematodes (from which most reports of anthelmintic resistance derive) isunexpectedly large (Grant, 1994). Studies of mitochondrial DNA sequences suggest largevariation within and between hosts (Blouin et al., 1992, 1995), which indicates that theeffective population size of these parasites is enormous. This condition is ideal for thespread of anthelmintic resistance alleles. Confirmation of these findings is evident in thevery large number of alleles found in a gene encodingb-tubulin in H. contortus(Beechet al., 1994) and in other trichostrongyloids (C.A. Winterrowd and T.G. Geary, unpublisheddata). Importantly, this pronounced genetic heterogeneity is found in species that parasitizecattle as well as sheep.

Examples of resistance are known for other helminths, includingFasciola, cyathostomes,ascarids, hookworms and strongyles (Conder and Campbell, 1995). In contrast, there areno documented examples of anthelmintic resistance in filariae. Little is known about thegenetic heterogeneity of non-trichostrongyloid nematode populations, although some datasuggest that heterogeneity is of the same magnitude as in free-living invertebrates (Nadler,1990). Estimating the genetic diversity of more species of parasitic nematodes, using toolsdeveloped for the trichostrongyloids, should be a high priority for research. Combined witha consideration of population exposure to treatment, diversity data should help us to predictthe risk of anthelmintic resistance for other species.

3.2. Can we detect and monitor resistance before it becomes clinically aparent?

Anthelmintic resistance is currently tracked with bioassays that measure the susceptibilityof larval stages to drugs or reductions in fecal egg count after treatment (see Sangster,1996). Neither method can detect resistant organisms until they have reached reasonableabundance, at which time a switch to an alternative treatment may be unable to preserve theutility of the original compound. Resistance might be managed better, and would certainlybe better understood in an epidemiological sense, if we had molecular probes sensitiveenough to detect low incidence of resistance alleles in populations. Such probes must bederived from a thorough understanding of the molecular pharmacology of anthelminticresistance. Unfortunately, this area of science is not yet well developed. We urgently needto fund research into the molecular mechanisms of resistance.

Such research should begin from a consideration of the question, “How many geneticloci underlie resistance to the major classes of anthelmintics?” Genetic mechanisms cov-ering the range from recessive to dominant, sex-linked to autosomal, and from single tomultigenic have been described (see Sangster, 1996), which may vary for a given drug indifferent species (Sangster et al., 1998a). The occurrence history, mathematical modelingand intuition all indicate that dominant inheritance will accelerate the selection and spreadof resistance alleles. Obviously, for any new drug the genetic mechanism of resistance isimpossible to predict, although independent occurrences of resistance in a species and fora drug appear to be consistent.

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There is still debate over how many genes (and which ones) are involved in the commonresistances. Part of the problem lies in the experimental limitations of genetic experimentsemploying mass matings of diverse populations of parasitic nematodes. We usually lackinbred populations of worms and the experiments are technically tedious and expensive.Consequently, few such studies have been done. For benzimidazole resistance inH. contor-tus, classical genetics suggest that multiple genes are responsible. However, it is possiblethat a single gene is correlated with resistance and that genetic variability between wormsgives the appearance that several genes are involved. On the other hand, patterns of inher-itance which suggest that single genes are responsible for resistance may actually be dueto a cluster of genes or a reflection of selection from a genetically restricted population inwhich the full range of potential resistance genes is not present. Nevertheless, a priority forresearch should be to identify probes to detect single, predominant resistant alleles. Probesof this sort are likely to be the most useful for studying the molecular epidemiology ofresistance. They can only be derived when we understand resistance mechanisms at themolecular level.

3.2.1. Molecular biology of anthelmintic resistanceAmong broad-spectrum anthelmintics, we know most about benzimidazole resistance

at a molecular level. At least two isotypes ofb-tubulin are found in trichostrongyloidnematodes andC. elegans(Savage and Chalfie, 1991; Geary et al., 1992). InC. elegans,benzimidazole resistance is asociated with selection of animals that do not express a specificb-tubulin isotype, termedben-1(Driscoll et al., 1989). The function of the ‘missing’ isotypeis presumably taken over by a benzimidazole-insensitive isotype, termedtub-1(Savage andChalfie, 1991). Of interest is thatben-1differs fromtub-1in having phe at amino acid residue200 instead of tyr, a change associated with benzimidazole resistance in other organisms.

Studies in trichostrongyloid parasites reveal similar allele selection. Strong allelic se-lection (including selection of null mutants) has been observed at one of theb-tubulinisotypes inH. contortus(see Roos et al., 1993) andT. colubriformis(Grant and Mascord,1996). However, selection of a distinctb-tubulin allele with a phe-to-tyr mutation at po-sition 200 has been associated with benzimidazole resistance inH. contortus(Kwa et al.,1994),T. colubriformis(Kwa et al., 1994),Teladorsagia circumcincta(Elard et al., 1996)andTrichostrongylus axei(C.A. Winterrowd and T.G. Geary, unpublished observations).Importantly, expression of the mutantH. contortusallele inC. elegansconfers benzimida-zole resistance on the transgenic animals, proving the connection between genotype andphenotype (Kwa et al., 1995).

As the genetic data suggest that benzimidazole resistance is a multigenic trait in tri-chostrongyloids, it appears that the selection of a F200Y mutant is only partly responsiblefor the complete phenotype; further selection at bothb-tubulin loci also occurs. The ques-tion to be answered is whether the F200Y mutant is necessary for the resistant phenotype;does its abundance in a nematode population indicate the propensity for benzimidazoleresistance to develop?

Recently, a PCR assay that can detect the presence of the benzimidazole-resistant and-sensitive alleles of thisb-tubulin gene in single specimens ofT. circumcinctawas described(Elard et al., 1999). This assay has tremendous potential utility for monitoring resistance

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allele in wild-type populations of this parasite in order to determine how fequency changeswith drug exposure. If a similar assay can be developed for a cattle parasite, controlledanthelmintic exposures could reveal the variables which differentiate the rate of resistancedevelopment in sheep compared to cattle. Such experiments would permit analysis of therisk of anthelmintic resistance in cattle from a data-based approach.

Less is known about the molecular pharmacology of resistance to other broad-spectrumanthelmintics. Resistance to levamisole, pyrantel and morantel inC. elegansis primarilyassociated with mutations in genes that encode subunits of the nicotinic acetylcholine recep-tor (Fleming et al., 1996, 1997). Resistant mutants show phenotypes ranging from highlyuncoordinated to essentially wild-type (Lewis et al., 1980a, b). Binding of [H3]-meta-aminolevamisole (MAL) to these mutants varied compared to wild-type animals, from dra-matic reduction to essentially unchanged (Lewis et al., 1987). While highly uncoordinatedphenotypes are incompatible with survival in parasitic nematodes, the existence of pseudo-wild-type levamisole-resistant mutants ofC. eleganssuggests that clues to this genotype inparasites may be found in studies of the free-living worm. Studies on levamisole-resistantH. contortusshow that [H3]MAL binding is subtly altered compared to wild-type animals(Sangster et al., 1998b), which is not inconsistent with results from some of theC. elegansmutants.

Sequence analysis of theC. elegansgenome has revealed the presence of about 40 pre-dicted acetylcholine receptor subunits (Bargmann, 1998). Electrophysiological evidence inOesophagostomum dentatumsuggests that these subunits associate to form channels withdistinct characteristics (Martin et al., 1997). Changes in subunit composition could resultin retention of function, but loss of levamisole sensitivity. These findings must be extendedto other nematodes, and their significance in resistance reconciled with the retention ofhigh-affinity levamisole binding sites inH. contortus(Sangster et al., 1998b).

Much work has been done on AM resistance inC. elegans, though little has been pub-lished; a useful summary is available (Blaxter and Bird, 1997). Low-level resistance isassociated with over 20 genetic loci in this animal. Examples include two genes encodingproteins involved in electrical coupling of excitable cells,unc-7andunc-9(Boswell et al.,1990; Starich et al., 1993; Barnes and Hekimi, 1997). These mutants are probably not rele-vant for parasites. Phenotypes ofunc-7andunc-9worms include movement disorders andegg-laying deficiencies; both would work against selection of parasites after AM exposure.Many of the AM resistance genes inC. eleganshave phenotypes that include amphid dys-function (Blaxter and Bird, 1997). As pharyngeal function is the most sensitive target forAM in nematodes (Avery and Horvitz, 1990; Geary et al., 1993) and amphidial neuronsinfluence pharyngeal function, this connection may be functional. However, it is not clearthat amphidial dysfunction is compatible with survival in a parasite’s niche. Complete pub-lication of the many mutations found to underlie AM resistance inC. eleganswould greatlyfacilitate studies to determine which, if any, are relevant for parasites.

AM anthelmintics act by opening glutamate-gated chloride channels in nematodes (Cullyet al., 1996). InC. elegans, loss-of-function mutations in one of these avermectin receptorgenes (avr-15) cause AM resistance (Dent et al., 1997), but this is not true for others(Vassilatis et al., 1997). Evidence for an association between alleles of a gene encodinga glutamate-gated chloride channel and AM resistance inH. contortus(Blackhall et al.,1998a) is consistent with a mechanism of resistance due to changes in the drug target.

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However, most attention has recently been focused on the possible role of drug exportpumps in AM resistance (see next section).

3.2.2. What is the role of nematode p-glycoproteins?Members of a large family of proteins that can transport compounds from inside to the

outside of cells are thought to play a role in resistance to various kinds of chemotherapeuticagents. These proteins, referred to as P-glycoproteins or multiple drug resistant (mdr) pro-teins, are often mutated or overexpressed in resistant cells or organisms. Initially discoveredin tumor cells, and then in a number of parasitic protozoa, the precise role of P-glycoproteinsin mediating drug resistance in clinical settings is largely unknown. Strategies designed toinhibit the function of P-glycoproteins by co-administration of drugs that block the mdrpumps (such as verapamil) have not proven widely effective in the clinic. Original accep-tance of a role for P-glycoproteins in malaria, based on verapamil-dependent reversal ofchloroquine resistance in vitro, seems now to be a much more complicated situation thanoriginally conceived (Chow and Volkman, 1998).

P-glycoproteins are also present in nematodes; the first experiments identified fourpgpgenes inC. elegans(Lincke et al., 1992). It is now apparent that there are 14pgp genesin this genome, though two may be pseudogenes (Broeks, 1997). The P-glycoproteins arefound primarily in the apical membrane of cells in the digestive and excretory systems(Lincke et al., 1993; Broeks et al., 1995), suggesting that they may export compoundsfrom the inside of worms to the external milieu. Indeed, deletion of two of these genesleads to susceptibility to toxins such as colchicine and chloroquine (pgp-1; Broeks et al.,1995) and heavy metals (pgp-1andpgp-3, as well as a different multidrug resistance gene,mrp-1; Broeks et al., 1996). However, to our knowledge, none of theC. elegansavermectinresistance genes maps to any of thepgploci, nor do any of the loci associated with levamisoleor benzimidazole resistance. Deletion of these twopgpgenes has no apparent phenotypein unchallenged animals, supporting the hypothesis that their role is to protect the wormsfrom environmental poisons (Broeks et al., 1995). This role leads to the suspicion thatparasitic nematode P-glycoproteins may mediate anthelmintic resistance, even though nodata have been published demonstrating that theC. elegans pgpmutants are more sensitiveto anthelmintics than wild-type worms.

The parasiteH. contortuscontains at least fourpgpgenes (Sangster, 1994; Xu et al., 1998;Kwa et al., 1998; Sangster et al., 1999). Data supporting the association ofH. contortuspgpgenes with avermectin resistance are available (Xu et al., 1998; Blackhall et al., 1998b;Sangster et al., 1999), but this finding is not universal (Kwa et al., 1998). Negative findingsin these experiments are not conclusive, however. All employed partial cDNA clones tocharacterize a genomic locus; detection of a polymorphism requires use of a restriction en-zyme that detects a variable site which falls within the region covered by the partial cDNA.Alternatively, and as is the case inC. elegans, only a subset of these genes may be involvedin resistance to a particular anthelmintic. Finally, it must be considered whether the associa-tion of pgppolymorphism with AM resistance is an artefact of selection pressure. Parasiticnematodes show an enormous amount of genetic diversity (Section 3.1). Drug exposureprotocols that permit survival of a very limited number of organisms, which then repro-duce, will automatically result in a skewed distribution of alleles compared to unselected

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populations, due to the drug-induced bottleneck. Whether or not this phenomenon explainsthe apparent polymorphisms inpgpgenes associated with AM resistance inH. contortusis unknown. A conclusive demonstration that parasite P-glycoproteins transport AM re-mains to be provided, as does evidence that inhibition of P-glycoprotein function in vitrocan reverse the phenotype of AM resistance, which is clearly evident in culture. Prelimi-nary data indicating that verapamil can partially reverse AM resistance inH. contortus(Xuet al., 1998) require confirmation. However, the possibility that alterations inmdr functionunderlie at least some aspects of AM resistance is intriguing, and it appears that ivermectinis a substrate for at least some mammalian P-glycoproteins (Schinkel et al., 1994; Pouliotet al., 1997). On a cautionary note, it is also clear that parasitic nematodes exhibit morethan one type of AM resistance (Gill and Lacey, 1998; Gill et al., 1998).

Whetherpgp gene polymorphisms are involved in resistance to other anthelmintics isunknown. A closantel-resistant strain ofH. contortusaccumulated less drug than wild-typestrains (Rothwell and Sangster, 1997), but it has not been proven that this phenomenonis mediated by a P-glycoprotein mutation. No polymorphism in severalpgp genes fromH. contortuswas associated with closantel resistance (Kwa et al., 1998; Sangster et al.,1999), though, in light of the presence of multiplepgpgenes in this parasite and the use ofpartial cDNA clones as probes, these were not definitive studies. A report that benzimidazoleresistance in larval stages ofH. contortuswas partially reversed by verapamil, a knowninhibitor of P-glycoprotein function (Beugnet et al., 1997) is dubious. In this paper, thecalculation of resistance factors was not corrected for the verapamil control. Althoughbenzimidazoles are substrates for P-glycoproteins in other systems (Nare et al., 1994), whichsuggests that an association between nematodepgpalleles and benzimidazole resistance isnot out of the question, the conclusions of Beugnet et al. (1997) require confirmation beforean alternative to the knownb-tubulin allelic variation/isotype deletion model (Roos et al.,1993; Kwa et al., 1994) can be accepted.

4. Drug discovery

Only three classes of broad-spectrum anthelmintics are available; we define class as agroup of compounds that are related on the basis of common structure, common mechanismof action or cross-resistance. These include the benzimidazoles, macrocyclic lactones andthe acetylcholine receptor agonists levamisole, pyrantel and morantel. In contrast, at least 10distinct classes of antibacterials with more or less broad spectrum activity are available. Theabundance of antibacterial classes available for veterinary medicine (relative to anthelminticclasses) is undeniably due to the relative importance of these kinds of drugs in humanmedicine. This discrepancy has many roots, not the least of which is the fact that drugconsumers in the developed world suffer very commonly from bacterial infections, whichare undeniably serious if untreated. The developed world suffers rarely from helminthinfections (which are, with some notable exceptions, not usually life-threatening). Instead,helminthiases in humans are found mostly in impoverished regions. Whether the endeavorsof the pharmaceutical industry should be directed by charitable attention to diseases ofpatients who cannot pay for treatment is not an appropriate topic for this venue. It is worthnoting that western governments spend little for research on helminths, reflecting the low

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threat to their citizenry and the low priority of veterinary diseases. Therefore, few academiclaboratories work toward identifying new targets for anthelmintic discovery.

A final cause of the dearth of available anthelmintic classes is the strong economicpressure under which livestock producers work. The AM anthelmintics combine excellentspectrum with safety, duration and potency. The demands of the marketplace generallyrequire competitor drugs of novel classes to be equally good, which in this case is a veryhigh bar. This demand arises, at least in part, from the intimidating costs of drug development(see Hennessy, 1997). The consequence of this pressure is that we are now often forced torely on a single anthelmintic class to control helminthiases. If AM resistance spreads, as hasresistance to other broad-spectrum anthelmintics, novel drugs with fewer clinical attributeswill become commercially viable. Unfortunately, such pressures may keep novel productsfrom the market too long to forestall or limit AM resistance by inclusion in rotational useschemes.

As industrial drug discovery moves beyond the era of whole-organism screening, weface a real problem in keeping anthelmintic discovery in pace with paradigms developedfor other areas of drug discovery (Geary et al., 1999), at least in part because we lackwell-characterized, validated targets for mechanism-based screening. It is essential thatpriority be placed on basic research devoted to the physiology and biochemistry of parasitichelminths. Further erosion of the pool of researchers able to conduct such studies couldeventually eliminate the ability to discover anthelmintics in the pharmaceutical industry.

4.1. What does the pipeline look like?

What new anthelmintics are on the horizon? We are aware of only three new classesthat hold promise for veterinary applications, the diketopiperazines (paraherquamides andmarcfortines), cyclic depsipeptides (PF1022A) and nitazoxanide. Unfortunately, we haveno information that a compound from any of these classes is in clinical development. Itappears that at least 5 years will pass before a new anthelmintic class is available on themarket.

How many companies still have an anthelmintic discovery program? The chances fordiscovery of a novel anthelmintic are related to the number of groups looking for one. Thefactors described above, in addition to the recent merger mania in animal health companies,have reduced the number with healthy anthelmintic discovery programs to no more than10 (based on our informal survey). Fewer have developed a mechanism-based approachto anthelmintic discovery. As we discuss elsewhere (Geary et al., 1999), whole-organismscreens, the traditional mainstay of antiparasitic drug discovery programs, are increasinglydifficult to accomodate under the modern pharmaceutical discovery paradigm. This is be-cause mechanism-based screens need knowledge of biology, and the translation from targetdescription to the development of screens is necessarily slow and expensive. We stronglyadvocate increased support for basic research programs focused on nematode biology. Thereis a real danger that more anthelmintic discovery programs will be discarded. Identificationof new molecular targets for anthelmintics in non-industrial laboratories will help keep suchprograms functioning.

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4.1.1. DiketopiperazinesAnthelmintic activity associated with paraherquamide was first reported in 1990, nearly

a decade ago (Ostlind et al., 1990; Shoop et al., 1990). The compound has excellent activityagainst almost all of the major nematode parasites of sheep (Shoop et al., 1990) and cattle(Shoop et al., 1992a) and appears to be safe in these species. However, dogs exhibit toxicityeven with very low doses of paraherquamide (Shoop et al., 1991). Marked species-dependenttoxicity makes development of the molecule problematic (Shoop et al., 1992b), since itwould be difficult to estimate human risk associated with residues or with inadvertentexposure (i.e., during manufacture or dosing). Nonetheless, the impressive intrinsic potencyof paraherquamide against trichostrongyles in vitro (Gill and Lacey, 1993), coupled with theexcellent potency against these organisms in target animals (Shoop et al., 1990, 1991) makethe paraherquamide template a valuable starting point for discovery. That the compoundapparently lacks activity against arthropods and flatworms would make it difficult to competeagainst the AM class in terms of spectrum. However, reports that paraherquamide is morepotent against larval stages of ivermectin-resistant trichostrongyles (Gill and Lacey, 1993,1998) suggest that a compound which shares the paraherquamide mechanism of actionmay have particular value as part of a rotation to limit the development and spread of AMresistance in these important target parasites. It must be noted that it remains to be seen if thispattern extends to adult stages of parasites in vitro or in vivo (see Thompson et al., 1996).

A key development to support continued investigation of this class is the identification ofthe molecular target for paraherquamide toxicity in nematodes. Despite notable advancesin this regard (see Thompson et al., 1996, for review), we have little indication of what theparaherquamide receptor is. While this should be a priority area for basic research, it mustbe stressed that development of a novel compound discovered in a mechanism-based screenwill take at least 5 years, and a screen for paraherquamide-like compounds has yet to bedeveloped, as far as we know.

4.1.2. Cyclic depsipeptidesAnthelmintic activity of the novel natural product PF1022A has recently been reported

(Sasaki et al., 1992). This molecule demonstrates exceptional potency against a varietyof nematodes in vitro and in certain animals (see Conder et al., 1995; Thompson et al.,1996). Activity does not extend to flatworms or arthropods. The mechanism of action ofPF1022A has not been proven; although radiolabelled PF1022A binds to GABAergic sitesin nematodes (Chen et al., 1996), the potency of this binding is somewhat lower than thepotency of the compound against target parasites in culture (e.g., Conder et al., 1995).Electrophysiological and biochemical data also suggest that the site of action of PF1022Aremains to be elucidated (Geyner et al., 1996; Martin et al., 1996).

Although PF1022A is efficacious when given orally to non-ruminant animals (Sasakiet al., 1992; Conder et al., 1995), parenteral delivery is problematic (Conder et al., 1995).These problems are more serious in ruminant animals, in which both parenteral and oraldelivery are ineffective for broad-spectrum activity (unpublished observations). The basisfor this problem is not understood, although it appears that part of the explanation may liein the complicated pharmaceutical chemistry of the molecule (Kachi et al., 1998). Untilthese difficulties are resolved, one should not expect PF1022A to appear on the market.

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4.1.3. NitazoxanideWhile nitazoxanide has shown activity against protozoa and helminths in humans, includ-

ing tapeworms and nematodes (e.g., Cabello et al., 1997; Doumbo et al., 1997; Abaza et al.,1998), its potency and efficacy against important veterinary nematodes are too low to makeit an attractive veterinary anthelmintic (Euzeby et al., 1980). Indeed, the compound is ofinterest primarily for its reputed activity against the apicomplexan parasiteCryptosporid-ium parvum, which is highly pathogenic in immunosuppressed humans and is currentlyuntreatable. The apparent range of spectrum of nitazoxanide is unprecedented and intrigu-ing. How the compound acts against both worms and protozoa has not been deduced; if weknew the mechanism, a tremendous opportunity to discover analogs with improved potencyand efficacy could be at hand. However, such a discovery program would be unlikely toproduce a new entry in the marketplace for at least 5 years. It is sobering to note that it hasbeen almost two decades since anthelmintic activity of nitazoxanide was first reported; it isdifficult to call this ‘a new class’.

4.2. How will new anthelmintics be found?

The era of whole organism screening as a tool for drug discovery is essentially over (Gearyet al., 1999). There has been a massive switch to mechanism-based, ultra high-throughputscreening (UHTS) as the strategy of current choice in the pharmaceutical industry. Thisswitch has been accompanied by a huge investment in the infrastructure (primarily robotics,miniaturized read-out tools and sample handling equiment) necessary to centralize screeningoperations. Therapeutic areas that are unable to benefit from the investment in UHTS may notbe included in the screening operation of the parent company.The key component of UTHSprograms is the drug target, preferably available as a cloned gene. Our current understandingof the biochemistry of parasitic helminths and of the mechanisms of action of knownanthelmintics identifies several targets that could be formatted for UTHS (Thompson et al.,1996; Geary et al., 1999). However, considering that all available anthelmintics have beendiscovered through empirical whole-organism screens, it should be clear that an unknown(and probably very large) number of good anthelmintic targets remains to be discovered.Knowledge of the fundamental biology of helminths is woefully lacking; we comprehendvery little of how these animals work as organisms. Investment in basic research in parasitebiology will have an enormous impact on the development of new control strategies; indeed,without this investment, new anthelmintics may never be discovered.

An illuminating comparison can be made with ectoparasiticide discovery. In contrast tothe situation with anthelmintics, several new classes of ectoparasiticides have recently beenintroduced. In part, this has been driven by the enormous market for companion animalflea control, revealed by the first of the new products, lufenuron. The field also benefitedfrom the availability of systems for maintaining the complete life cycle of fleas in thelaboratory in the absence of a host; it is relatively simple to test compounds for effectson adult behavior and viability in assays that provide accurate estimates of the potencyof compounds in clinical settings. In addition, compounds that inhibit development canreadily be identified in host-free settings. New drugs that block development have beenshown to have important therapeutic benefits, a strategy that was not fully appreciated until

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the compounds became available. Could the search for novel anthelmintics be fulfilled in asimilar fashion? Although a chemotherapeutic strategy based on larval control has not beenchampioned, data from experiments with nematophagous fungi directly demonstrate thatreduction of pasture contamination can have therapeutic benefits (e.g., Larsen et al., 1997).Research that finally provides a host-free life cycle of a parasitic nematode may similarlystimulate unanticipated advances in anthelmintic discovery.

5. What does theC. elegans genome mean for parasites?

Definition of the entire nucleotide sequence of theC. elegansgenome has recently beenannounced (C. elegans Sequencing Consortium, 1998). The nearly 100 million base pairsthat comprise the information resource of this organism include over 19 000 genes. Althoughabout half the genes encode proteins of known function, much remains to be learned aboutthe biology of this intensely studied worm. Of particular benefit is the availability of aphysical map of the genome in concert with the nucleotide sequence, and mutational strate-gies that can identify essential genes and define the function of novel genes (see Janke etal., 1997). Although its relationship to parasites was not an important factor in selectingC. elegansas a model for developmental biology, the fact that the first metazoan creatureto have a defined genome is a nematode has enormous consequences (even if unintended)for studies of its pathogenic relatives (Blaxter, 1998). The value of this resource for an-thelmintic discovery cannot be overestimated (Geary et al., 1999). However, the genomeproject is only the first step. The complexity of studyingC. elegansgenes in the context ofa parasitic lifestyle is problematic. On more pragmatic levels, the utility of the genome isreadily apparent. The ability to express parasite genes inC. elegansprovides a powerful testof resistance mechanisms (e.g., Kwa et al., 1995). Work inC. eleganshas already identifiedcandidates for resistance alleles in parasites (e.g.,avr-15, tub-1, lev-1), with more to beuncovered. It is now possible to create transgenicC. elegansthat express mutations foundin parasites to see if they behave as predicted. Advanced strategies for knocking out ortemporarily disabling genes inC. elegansallow one to validate them as drug targets priorto screening (Geary et al., 1999). TheC. elegansgenome sequence is a magnificent giftthat is highly relevant for studies of parasitic nematodes. It is imperative that the veterinaryhelminthology community take advantage of it.

6. The future

Based on our consideration of the state of anthelmintic chemotherapy, we propose thefollowing goals for concerted effort and investment. The order in which they appear followsthe sequence of this article, but they should be considered equally important.1. Develop a culture system for nematodes that permits the accurate in vitro determination

of the intrinsic potency of diverse kinds of known and experimental anthelmintics.2. Deepen the understanding of anthelmintic pharmacodynamics so that valid correlations

can be drawn between compartment pharmacokinetics and intrinsic potency. We need

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to be able to predict the benefits of modifications in formulation or delivery based onthese factors.

3. Identify the molecular mechanisms of anthelmintic resistance, particularly for the AMclass. Implement a molecular epidemiology program based on detection of resistancealleles in single worms to monitor the development and spread of resistance.

4. Identify the mechanisms of anthelmintic action of the paraherquamides, cyclic dep-sipeptides and nitazoxanide. The targets of these compounds can be used in mechanism-based screens for novel (and more attractive) compounds that act at validated sites.

5. Integrate theC. elegansgenome with studies of parasite biology. The enormous value ofthis resource demands our most serious attention. In part, this program should includegenomics studies of nematodes significant to veterinary medicine.

6. Figure out how worms work as organisms. Our ignorance of helminth biology is vast(e.g., Geary et al., 1995). The future of parasite control depends on developing a betterunderstanding of the foe.

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