International Journal for Parasitology 40 (2010) 1–13

Contents lists available at ScienceDirect

International Journal for Parasitology

journal homepage: www.elsevier .com/locate / i jpara

Invited Review

Unresolved issues in anthelmintic pharmacology for helminthiases of humans

Timothy G. Geary a,*, Katherine Woo b, James S. McCarthy c, Charles D. Mackenzie d, John Horton e,Roger K. Prichard a, Nilanthi R. de Silva f, Piero L. Olliaro g, Janis K. Lazdins-Helds g,1,Dirk A. Engels h, Donald A. Bundy i

a Institute of Parasitology, McGill University, Montreal, QC, Canadab Institute for One World Health, Sand Francisco, CA, USAc Queensland Institute of Medical Research, University of Queensland, Qld, Australiad Department of Pathology, Michigan State University, East Lansing, MI, USAe Tropical Projects, 24 The Paddock, Hitchin SG4 9EF, UKf Department of Parasitology, Faculty of Medicine, University of Kelaniya, Sri Lankag UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), World Health Organization, Geneva, Switzerlandh Department of Control of Neglected Diseases, World Health Organization, Geneva, Switzerlandi Human Development Network, The World Bank, Washington, DC, USA

a r t i c l e i n f o

Article history:Received 28 August 2009Received in revised form 31 October 2009Accepted 2 November 2009

Keywords:AnthelminticsSchistosomiasisSoil-transmitted helminthsOnchocerciasisLymphatic filariasisMass drug administrationPharmacology

0020-7519/$36.00 � 2009 Australian Society for Paradoi:10.1016/j.ijpara.2009.11.001

* Corresponding author. Address: Institute of Par21111 Lakeshore Road, Ste-Anne-de-Bellevue, Que., C398 7612; fax: +1 514 398 7857.

E-mail address: timothy.g.geary@mcgill.ca (T.G. Ge1 Present address: Novartis Vaccine Academy, Siena,

a b s t r a c t

Helminth infections are an important constraint on the health and development of poor children andadults. Anthelmintic treatment programmes provide a safe and effective response, and increasingnumbers of people are benefitting from these public health initiatives. Despite decades of clinical expe-rience with anthelmintics for the treatment of human infections, relatively little is known about theirclinical pharmacology. All of the drugs were developed initially in response to the considerable marketfor veterinary anthelmintics in high- and middle-income countries. In contrast, the greatest burdencaused by these infections in humans is in resource-poor settings and as a result there has been insuffi-cient commercial incentive to support studies on how these drugs work in humans, and how they shouldbest be used in control programmes. The advent of mass drug administration programmes for the controlof schistosomiasis, lymphatic filariasis, onchocerciasis and soil-transmitted helminthiases in humansincreases the urgency to better understand and better monitor drug resistance, and to broaden thecurrently very narrow range of available anthelmintics. This provides fresh impetus for developing acomprehensive research platform designed to improve our understanding of these important drugs, inorder to bring the scientific knowledge base supporting their use to a standard equivalent to that of drugscommonly used in developed countries. Furthermore, a better understanding of their clinicalpharmacology will enable improved therapy and could contribute to the discovery of new products.

� 2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction

Helminth parasitism remains an underappreciated scourge ofhumans in most of the developing world. As many as two billionindividuals harbour these parasites, with millions typically simulta-neously infected with filariae, hookworms, whipworm, large round-worms and/or schistosomes (Brooker et al., 2006; Hotez et al., 2008),all of which often result in chronic, debilitating morbidity. Duringthe past few years, the renewed acknowledgement of the burden im-posed by these infections has led to mass drug administration (MDA)

sitology Inc. Published by Elsevier

asitology, McGill University,anada H9X 3V9. Tel.: +1 514

ary).Italy.

programmes for the control and possible elimination of the majorhuman helminths, which are underway or under consideration inmost economically poor regions of the world (Molyneux et al.,2005). The drugs (anthelmintics) involved are in many cases donatedby pharmaceutical companies or are available as relatively cheapgeneric preparations. Billions of doses have been taken by humans,but aside from the longstanding Mectizan Donation Program foronchocerciasis and the Global Program for the Elimination of Lym-phatic Filariasis (GPELF), this has rarely been done in a systematicfashion. In general, these drugs are safe and at least moderatelyeffective. However, compared with the knowledge base that sup-ports drugs used in wealthier countries and despite notable effortsin this area (e.g., de Silva et al., 1997; Albonico et al., 1999, 2004; Hor-ton, 2000; Dayan, 2003; Utzinger and Keiser, 2004; Danso-Appiahet al., 2009), many gaps remain in our understanding of the pharma-cology of drugs used or advocated for MDA programmes for hel-

Ltd. All rights reserved.

2 T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13

minth parasites. These gaps represent a challenge to the continuedsuccessful use of these medicines, as they leave us ill-equipped tounderstand or monitor the emergence of drug resistance and con-strain the search for new treatment options. Furthermore, they pres-ent an ethical dilemma: why are we satisfied with a lower volume ofresearch-based knowledge about drugs used for poor people than forthose used in richer countries, and apparently content with a verylimited number of drugs which do not meet all of our needs in termsof efficacy?

Several factors suggest an urgent need to identify and closethese gaps. First, the available pharmacopeia for human helminthinfection is exceptionally restricted. For some of the most commoninfections, only one drug is available for human use and the rangefor even the best served infections is undeniably sub-optimal;understanding more about how current drugs work may illumi-nate ways to improve this situation. Second, there is no guaranteethat the current state of affairs, especially with regard to the so-farlimited development and spread of anthelmintic resistance in hu-mans, will continue. We understand relatively little about the vari-ables that govern how resistance to anthelmintic drugs may beselected and spread in humans. More research in this area maypay dividends as MDA programmes are enlarged. Finally, whilethere is substantial experience in and understanding of the epide-miology of these infections, best-practices for MDA programmesalso need to be based on sound scientific understanding of the ba-sic and clinical pharmacology of the drugs used.

In an attempt to highlight how much and/or how little weknow, we here indicate some gaps in knowledge about the phar-macology of anthelmintic drugs commonly used in humans andidentify important areas for research to address these issues.

2. Antifilarials

2.1. Macrocyclic lactones (MLs)

This class, of which ivermectin is the only example currentlyapproved for use in humans, has revolutionized the treatment ofnematode infections in livestock, companion animals and, in hu-mans, filarial infections. Ivermectin is an exceptionally potentand usually very safe drug used for the control of onchocerciasisand lymphatic filariasis in MDA campaigns. It has been donatedby Merck & Co. for these indications and has changed the prospectsfor management of these diseases (Greene et al., 1989; Brown,2002; Geary, 2005). It is generally given once or twice yearly in asingle dose to clear microfilariae from the periphery (skin or blood)and suppress their reappearance. MLs, including ivermectin, arevery potent and highly efficacious against adult and larval gastro-intestinal nematodes and lungworms in animals, but are much lesseffective against adult stages of filarial nematodes, which greatlyprolongs the time required for MDA programmes to progress toeradication.

2.1.1. Mechanism of action: microfilariaeThe paradox is that ivermectin and other MLs lack evident

activity against microfilariae in culture at pharmacologically rele-vant concentrations; how do these drugs cause a rapid removalof this stage from skin or blood of patients? Ivermectin is an ago-nist of ligand-gated Cl� channels, with particular activity againstglutamate-gated Cl� channels (GluCls) in invertebrates, a mecha-nism first discovered by studies in the free-living nematode Caeno-rhabditis elegans (for a review, see Martin et al., 1997). It is possiblethat the drug also acts on gamma-aminobutyric acid (GABA)-gatedCl� channels in parasitic nematodes, although this remains to beexperimentally confirmed as pharmacologically relevant. The con-sequences of this action on parasitic nematodes resident in the

gastrointestinal tract and lungs of mammals include inhibition ofmotion and feeding, which lead to their death and removal fromthe host.

In contrast, microfilariae of most species pathogenic for humansare apparently unaffected by ivermectin in culture until the con-centration reached is far higher than found in treated patients(Bennett et al., 1993). The rapid disappearance of microfilariaefrom skin or blood under the influence of ivermectin is thus notcaused by the mechanisms operating in other nematodes (paraly-sis or starvation). It is reasonable to assume that the drug also tar-gets GluCls in this stage of the life-cycle. However, we do not knowhow many GluCl subunit genes are expressed in microfilariae, inwhat tissue(s) they are expressed or what the functional conse-quences of such activation are. Ivermectin may interfere with thephysiological (perhaps secretory) processes that microfilariae em-ploy for protection against host immune responses; the possibilityhas been raised that the human immune response is a variablecontrolling the parasitological response to the drug in onchocerci-asis patients (Ali et al., 2002), but supporting evidence is lacking.Understanding how ivermectin (and other MLs) cause the elimina-tion of microfilariae is an important goal. One potential phenotypeof ivermectin resistance in filariae is insensitivity of microfilariaeto the drug; a genotype that endows this effect would confer anenormous advantage in transmission. Knowledge of the mecha-nism(s) of action of ivermectin in microfilariae would focus atten-tion for resistance monitoring on likely genes. As repeated skinsnips for resistance monitoring (the standard method used to eval-uate drug responses) is difficult to deploy on a large scale, molec-ular or other tools will be needed for this purpose.

2.1.2. Mechanism of action: adult femalesIvermectin causes a prolonged suppression of microfilarial pro-

duction and release from adult females in infected humans, an ef-fect that persists for months longer than drug levels aremeasurable in blood or tissues. It is puzzling, then, that ivermectinhas little or no effect at pharmacologically relevant concentrationson microfilarial release from adult females of several filarial spe-cies in culture, even during prolonged exposure (Bennett et al.,1993). Gastrointestinal and lung nematodes may also show drugeffects on reproductive function but these must be acute in nature,as adult worms are rapidly removed from the host by the drug.Caenorhabditis elegans unc-9 mutants share phenotypes of ivermec-tin resistance and an egg-laying defect (Barnes and Hekimi, 1997).

Since inhibition of feeding in this species causes an egg-laying(egl) defect, it is difficult to detect additional effects, if any, of iver-mectin on egg-laying. GluCls are not known to be expressed inreproductive tissues of this species or in filariae (indeed, nothingis known of their tissue-level expression in filariae). It is the inabil-ity of ivermectin to cause mortality in adult filariae that allows thisunusual and poorly understood effect to become evident.

It is apparent that selection of females in which the period ofdrug-induced reproductive quiescence is reduced is a potentialmechanism for ivermectin resistance (Osei-Atweneboana et al.,2007). Again, it is essential to understand the biology underlyingthis effect in order to focus a molecular investigation that couldilluminate the set of alleles that could be monitored as an indexof drug resistance. As the current programme for control of filaria-ses in humans is based primarily on this action of ivermectin(which permits once yearly dosing), it is arguably scandalous thatwe have developed no scientific comprehension of the pharmaco-logical basis for its activity. It is also important to note that thelong-term sterilization observed with the related drug milbemycinoxime in the canine heartworm Dirofilaria immitis is apparentlyprimarily due to effects on the reproductive competence of maleworms (Lok et al., 1995). Possible species differences in ML phar-macology among filariae would only complicate the picture.

T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13 3

2.1.3. Lack of macrofilaricidal activityIvermectin has limited effects on the survival of adult filariae in

humans or other animals, at least acutely, despite the fact that thisdrug is extremely potent and highly efficacious against almost allspecies of non-filarial adult nematodes except human hookworms.The lack of robust, well-documented macrofilaricidal activity in thecurrent regimen is a significant lacuna in the nematocidal spec-trum of the drug. As with microfilariae, ivermectin has no apparenteffects on the motility of most adult filariae in culture at pharma-cologically relevant concentrations (Pax et al., 1988; Satti et al.,1988). The availability of fully efficacious macrofilaricides wouldsimplify control and perhaps enable eradication of human filaria-ses, and thus discovery of such drugs remains a continued researchfocus, despite the lack of success over many years. The characteris-tics of neuromuscular physiology that distinguish filariae fromother nematodes remain to be identified. Do fundamental differ-ences in the distribution or physiological function of ligand-gatedCl� channels in these parasites preclude the possibility that drugsacting at these sites can exert prominent macrofilaricidal effects?Are GluCls in somatic muscle of adult filariae simply less intrinsi-cally sensitive to MLs, or less important in neuromuscular coordi-nation in filarial nematodes? Identifying the basis for differentialdrug sensitivity in adult filariae compared with other nematodespecies may provide a lead for the development of other MLs moreuseful than ivermectin itself. It should also be noted that availabledata are insufficient to conclude that paralysis of somatic bodywall muscle per se can cause the death of adult filariae in situ.

In this context, it may also be worth considering the possibilitythat alternative regimens of ivermectin or the use of moxidectin,an ML with different pharmacokinetic properties, might lead to en-hanced macrofilaricidal effects. Repeated monthly doses of iver-mectin are the basis for a ‘soft kill’ regimen for the treatment ofadult D. immitis infections in dogs (McCall, 2005). Could a dailyor alternate day regimen of ivermectin for several weeks or monthsexert such an effect? The bovine-Onchocerca ochengi model couldbe an appropriate system in which to test the macrofilaricidal po-tential of ML exposure when continuously maintained for variousperiods of time, although six monthly doses of 500 lg/kg ivermec-tin or doramectin were not macrofilaricidal in this model (de CBronsvoort et al., 2005). In addition, six doses of ivermectin at 2-week intervals were ineffective against adult Onchocerca volvulus(Duke et al., 1991). Moxidectin has a considerably longer plasmahalf-life than ivermectin (Cotreau et al., 2003) and it is possiblethat somewhat different results may be obtained with it, althoughdata in support of this hypothesis are absent from the peer-re-viewed literature.

2.1.4. Ivermectin-associated encephalopathyRelatively rare but extremely serious and sometimes fatal ad-

verse events have appeared in patients co-infected with O. volvulusand Loa loa when treated with the standard dose of ivermectin. Thebasis for this toxicity in humans has yet to be resolved. The currentlack of a rational therapeutic alternative constitutes a seriousimpediment to the possibility of eliminating onchocerciasis andlymphatic filariasis in Central Africa, as there is extensive overlapof regions endemic for loaisis with those for onchocerciasis andlymphatic filariasis. The adverse events observed in a small num-ber of patients preclude the routine use of ivermectin for onchocer-ciasis control in loaisis co-endemic regions. There is a clearcorrelation between the density of L. loa microfilariae and the riskof experiencing a severe adverse event following the administra-tion of ivermectin (Gardon et al., 1997; Scientific Working Group,2003; Twum-Danso and Meredith, 2003). However, while the toxicsyndrome is clearly drug-related, its pathogenesis remains obscure(Mackenzie et al., 2003, 2007). Additional research is urgentlyneeded to identify the pharmacological basis for ivermectin-asso-

ciated severe adverse events. The research-based ability to identifypatients at risk with a simple procedure could permit resumptionof uncomplicated ivermectin distribution in areas where loaisis isco-endemic. Failure to control the infection in these regions wouldprovide a ready source of parasites for recolonization of previouslycleared areas.

2.1.5. MLs as anthelminticsIt is evident that we are far from understanding how ivermectin

works as an antifilarial agent. While the drug is highly efficaciousat very low doses against almost all non-filarial nematodes in ani-mals, it is ineffective when used as a single agent against hook-worms in humans and little better against Trichuris spp. in mosthosts (Brown, 2002; Wen et al., 2008). In contrast, it is quite a gooddrug for human ascariasis (Brown, 2002; Reddy et al., 2007; Keiserand Utzinger, 2008). Its relative inefficacy against hookworms andTrichuris trichiura is a major weakness in the human anthelminticpharmacopoeia; developing a better understanding of the basisfor the sub-optimal efficacy of this drug against these parasitescould lead to new tactics to improve clinical results.

2.2. Optimal dose of ivermectin for control of lymphatic filariasis

The efficacy of ivermectin (with or without the addition ofalbendazole) for suppression of microfilaremia in lymphatic filari-asis at currently used doses is significantly lower than found withdiethylcarbamazine (DEC) plus albendazole (Plaisier et al., 2000;Tisch et al., 2005). The efficacy of single-dose ivermectin in lym-phatic filariasis improves as the dose is increased from 150 to400 lg/kg (Plaisier et al., 2000). This is not the case for ivermectinin onchocerciasis, in which the standard 150 lg/kg regimen ap-pears to be as effective as higher doses (Greene et al., 1989; Gardonet al., 2002). While it is not clear that 400 lg/kg ivermectin wouldsignificantly enhance the success of lymphatic filariasis MDA pro-grammes based on ivermectin plus albendazole (Plaisier et al.,2000; Brown et al., 2000, although see also Brown, 2002), it is rea-sonable to ask whether the choice of a less efficacious dose hasimplications for resistance. The decision to recommend the ‘onchodose’ of ivermectin for use in lymphatic filariasis control pro-grammes was presumably made on the basis of a range of factors,of which absolute efficacy was not given primacy. While it is clearthat the current programme is successful, it is nonetheless worthconsidering whether the choice of a sub-optimal dose may favourthe development of resistance to the drug and thus jeopardise thelong-term effectiveness of the control programmes. Sub-optimaldosing (under-dosing) is believed to play a significant role in thedevelopment and spread of anthelmintic resistance in veterinarysettings (Conder and Campbell, 1995) and may become a factorin human parasite populations as broad-coverage control pro-grammes in humans accelerate (Geerts and Gryseels, 2001). At aminimum, modelling exercises appear to be warranted (Smithet al., 1999).

Another potential concern is recent evidence that ivermectinmay select for genetic changes in the nematode b-tubulin gene thatare responsible for resistance to benzimidazole anthelmintics inveterinary nematodes (Eng et al., 2006; Mottier and Prichard,2008). MDA for lymphatic filariasis, which is dependent on re-peated co-administration of ivermectin and albendazole, maytherefore enhance selection pressure for benzimidazole resistancenot only in lymphatic filariae, but also in soil-transmitted hel-minths which are collaterally affected by this co-administrationregimen. Some initial modelling of this possibility has been under-taken (Schwab et al., 2007). While there is evidence that ivermec-tin exposure exerts selective pressure on b-tubulin alleles in O.volvulus (Eng et al., 2006), it is not known whether it selects on thisgene in soil-transmitted nematodes of humans.

4 T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13

2.3. Albendazole in combination chemotherapy for lymphatic filariasiscontrol

A 400 mg dose of albendazole is routinely included with annualtreatments of DEC or ivermectin in lymphatic filariasis control pro-grammes. The activity of the benzimidazole component in this reg-imen is uncertain, and whether combination therapy confersbenefits over DEC or ivermectin alone remains controversial (Ksh-irsagar et al., 2004; Critchley et al., 2005; Tisch et al., 2005; Olsen,2007). Meta-analyses fail to show a prolonged benefit in microfil-aricidal or macrofilaricidal effects of the albendazole-containingcombinations compared with DEC or ivermectin alone, althoughapparent enhancement of activity in regimens including albenda-zole has been reported in individual studies (Gyapong et al.,2005). It is possible that the activity of albendazole against nema-tode parasites of the gastrointestinal tract (see below) enhancescommunity acceptance of the treatment regimen, offering health(and visually tangible) benefits by reducing these infections, thusmotivating participation (CD Mackenzie, unpublished observa-tions). However, treatment for soil-transmitted nematodes is a dis-tinct therapeutic goal in the context of filariasis controlprogrammes. Although albendazole in this regimen has an excel-lent safety record, the question remains whether it should be rou-tinely included in lymphatic filariasis control programmes becauseof its possible effect on filariae.

2.4. Unresolved issues with DEC

The same basic concerns exist for DEC, the mainstay for thetreatment of lymphatic filariasis in most parts of the world (otherthan areas co-endemic for onchocerciasis, where it is contraindi-cated). It is almost certain that ivermectin and DEC do not sharea common mechanism of action at the molecular level. However,it is striking that, 60 years after its introduction, we have no provenunderstanding of the molecular mechanism of action of DEC, thesingle most important drug for treatment of lymphatic filariasis,with action against all life-cycle stages present in humans (L3, L4,adults and microfilariae). In contrast to ivermectin, we do not haveeven a candidate receptor for DEC that is supported by experimen-tal data. DEC rapidly clears microfilariae from the skin or circula-tion of patients and causes approximately 40% mortality of adultparasites at clinical doses (Mackenzie and Kron, 1985; Noroeset al., 1997). It is not even clear whether repeated doses can killall adult worms. As for ivermectin, pharmacologically relevant con-centrations of DEC have no apparent effect on many species ofmicrofilariae in culture. The drug may instead act by altering thehost-parasite interface at the level of the immune response, en-abling the host to ‘see’ the parasite and, by attracting effector cellssuch as eosinophils, leads to the demise of the parasite (Greene,1986). This action may involve parasite products that interact withhost prostanoid systems (Maizels and Denham, 1992). Immune-dependent effects of DEC may account for the differential activityof the drug against different species of filariae in different hosts,and against adult stages of some but not all species. Recent insightinto DEC effects on Brugia malayi microfilariae was gained in mice;pharmacological and genetic evidence implicated host arachido-nate- and nitric oxide-dependent pathways in mediating the antif-ilarial effects of the drug (McGarry et al., 2005). Although how thedrug interacts with these pathways at the receptor level remainsunknown, and whether the results are extendable to chronic filar-ial infections in humans must still be established, these resultsidentify a promising system for further experiments and definemechanism-related hypotheses that can be tested in humans.

Nonetheless, DEC action against nematodes involves direct ef-fects on both the worm and the host. The latter, in many cases, isimportant in the primary activity against the parasite (e.g., induc-

ing host mechanisms that lead to the destruction and degenerationof the parasite), and in the secondary phase in which the host re-sponds to the initial burst of parasite killing by prolonging the ef-fect of the treatment, often by a suppressive effect on parasitemultiplication. Research is needed to better understand the mech-anisms involved in these two (or more) phases. The biochemicaland cellular changes occurring in worms under drug pressure arepoorly understood. Although there has been renewed interest insome areas of parasite biology (e.g., Wolbachia in filarial parasites),much more can be done, especially with the much improved tech-niques now available for morphological and biochemical analyses.

Outside of areas endemic for onchocerciasis, DEC remains thedrug of choice for the control (and perhaps elimination) of lympha-tic filariasis. Like ivermectin, it is given annually to tens of millionsof people. Unfortunately, little recent work has been done on thebasic pharmacology of this compound (though see McGarry et al.,2005; Junnila et al., 2007). Although it is well-tolerated andcontinues to be effective, it is scientifically necessary to investigatefurther how it works against filariae to provide a better under-standing of its activity in people. Understanding how this drugalerts the immune system to kill the parasite may also identifypathways that account for host-parasite specificity and may ratio-nally identify parasite protein targets for vaccine development.

On the clinical side, it is worth considering whether there areadvantages to the longer-course therapy previously recommendedfor DEC treatment of lymphatic filariases (12–14 days) comparedwith the current 1-day regimen, particularly with regard toaddressing the symptoms associated with acute attacks.

The ban on DEC in onchocerciasis regions is appropriate giventhe disastrous effects it has on the eye in infected individuals (Birdet al., 1980); in fact, much of the onchocercal blindness in Africaand Latin America seen before the era of ivermectin may have beendue to, or at least greatly exacerbated by, the use of DEC, for morethan 30 years the standard treatment for this infection. However,studies of low dose regimens of DEC (reviewed in Mackenzie andKron, 1985) show that these adverse effects may be lessened orcontrolled by altering the dose and duration of DEC treatment.The efficacy of DEC-salt in lymphatic filariasis with an apparentlymore benign adverse effect profile may support the feasibility ofthis approach. The use of less noxious regimens of DEC in oncho-cerciasis endemic areas should be carefully re-evaluated as a pos-sible alternative treatment in selected areas in case ivermectinloses its efficacy due to the development of resistant parasite pop-ulations. At the very least, much more research into the mecha-nisms of action of DEC, and particularly its effects on the host, isrequired.

2.5. Role of anti-Wolbachia chemotherapy in filariasis control

Prolonged treatment (daily dosing for 4–6 weeks) with antibiot-ics such as tetracyclines or rifampicin kills or permanently steril-izes adult filariae, including those affecting humans (Taylor et al.,2005; Hoerauf, 2008). It may be possible to shorten the durationof treatment by combining antibiotics or by finding faster-actingagents through screening programmes already underway (e.g.,http://www.a-wol.net/). However, it is unclear whether this situa-tion differs conceptually from that encountered with tuberculosisor leprosy, in which slowly dividing bacteria require prolongedtherapy for elimination. Despite significant research efforts, short-er regimens (e.g., 1 or 2 weeks) have not been successful in clinicaltrials for these infections. It is also worthwhile in this context toreach a consensus about the role(s) of anti-Wolbachia therapy infilariasis control programmes (see http://www.a-wol.com). Theymay be very useful in local elimination campaigns in which annualsuppressive therapy programmes are not likely to be sustainable orwhere resistance to available agents is a threat (Osei-Atweneboana

T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13 5

et al., 2007; Taylor et al., 2009). An anti-Wolbachia regimen may beof great importance in loaisis co-endemic regions, as L. loa do notharbour the symbionts and thus would be unaffected by drugswith this mechanism, potentially avoiding the severe adverseevents (SAE) that now complicate onchocerciasis control in theseareas. In these contexts, we need to define the duration of dailydosing that is minimally acceptable for antibiotic-based therapy.We must also decide how to protect vulnerable populations inwhich currently available tetracyclines are contraindicated forsafety reasons (children and potentially pregnant women). Anti-Wolbachia therapy may be more suitable for individual or smallgroup treatments such as those needed during the final stages ofelimination.

While efforts to refine and optimise existing regimens arehighly deserving of funding, it is also worthwhile to approach thesituation from a research base. For example, developing a morethorough understanding of the physiological role of the symbiontin filarial worms may reveal novel targets for chemotherapeuticintervention. In pursuit of this objective, we need to better com-prehend why killing the symbiont affects worm viability. It wouldbe interesting, for example, to profile these effects at both theultrastructural and metabolomic levels.

2.6. Defining a discovery path for macrofilaricides

Interest in discovery of a macrofilaricide has waxed and wanedover the past several decades (Ginger, 1986; Gutteridge et al.,1990; Mak et al., 1991; Sharma, 1993; Townson et al., 2007). Be-cause of advances in the control of onchocerciasis and lymphaticfilariasis achieved with currently available drugs, the prospectsfor an accelerated path to elimination would be much enhancedif a safe and effective macrofilaricide was available. This hasspurred new funding, in both public and private sectors, in supportof macrofilaricide discovery (Nwaka and Hudson, 2006). However,the path from the identification of ‘hit’ compounds through testingin animal models prior to evaluation in humans remains ill-de-fined. Current paradigms test compounds in rodent models, usingeither non-native or native species of filarial worms. There aredog and primate models of lymphatic filariasis (Mak et al., 1991),and a cattle model for nodular species using O. ochengi (Treeset al., 2000) could be used as a pre-clinical filter between rodentsand humans. It is important to recognise that testing in rodentstypically entails evaluation of compounds in models that may bepoorly predictive of activity in more physiologically relevanthost-parasite systems. Furthermore, the lack of known, useful mac-rofilaricides means that these models cannot be fully validated.Whether additional models should be included as filters prior tothe evaluation of compounds in humans is a question that shouldbe carefully considered by all stakeholders involved in macrofilari-cide discovery.

It is instructive to consider emodepside as a model. This semi-synthetic cyclic depsipeptide is a novel anthelmintic that worksprimarily by opening a Ca2+-activated K+ channel in nematodes(Guest et al., 2007). It is licensed for use in cats and dogs for thetreatment of gastrointestinal nematodiases. Recent studies haveshown that it is exceptionally potent against Onchocerca spp. inmice and in culture, although it is not very potent against Brugiapahangi (Townson et al., 2005). How should this compound be ad-vanced? It perhaps represents the optimal early candidate macro-filaricide as it has a known mechanism of action, availableinventory and at least some accumulated safety data. These attri-butes make it far more advanced that most compounds obtainedthrough screening programmes. It is logical that it be advancedinto human evaluation as quickly as possible, but the path to thatgoal, and perhaps regulatory approval, remains to be defined.While the commercial interests of the manufacturer of the drug

(Bayer) must be addressed, significant and important progress indeveloping models for interactions between animal health compa-nies (and other pharmaceutical interests) and programmes for dis-covery of antiparasitic drugs for human indications has alreadybeen made (Nwaka and Hudson, 2006).

Another compound of interest as a macrofilaricide is flubendaz-ole. It was shown to be effective in humans in an injectable formu-lation for killing adult O. volvulus (Dominguez-Vazquez et al., 1983;Mackenzie, unpublished observations). Flubendazole has a markedlethal effect on many stages of Brugia in jirds and cats (Denhamet al., 1979; Surin and Denham, 1990). In the cat, a single100 mg/kg s.c. dose was macrofilaricidal, and a single dose of25 mg/kg was similarly effective in Brugia-infected jirds. Fluben-dazole appears to be the most macrofilaricidal of the benzimidaz-oles in animal models (Zahner and Schares, 1993), for unknownreasons. Injection of the drug in the vehicle used in humans causedsevere pain and injection site reactions, leading to termination ofthe development effort for flubendazole as a macrofilaricide. Likeother benzimidazoles, flubendazole has very poor bioavailabilityfollowing oral administration. Whether research could identify im-proved formulations that generate efficacious plasma levels formacrofilaricidal activity, or vehicles that permit safe parenteraldosing, are objectives worth pursuing.

2.7. Next steps to close knowledge gaps pertaining to antifilarial drugs

(i) Funding should be targeted to research aimed at discoveringthe mechanisms of antifilarial action (including adults andmicrofilariae) of ivermectin and DEC. Based on these data,the actions of other MLs (e.g., moxidectin) may be betterpredicted prior to or during development. Data obtained inthese studies may also inform research on possible mecha-nisms of resistance, which could in turn facilitate monitoringof parasite populations for the appearance and spread ofthese traits.

(ii) Dose optimisation studies for ivermectin in lymphatic filari-asis should be extended and confirmed to demonstratewhether the current regimen provides maximal health andparasite control benefits, or to support changes in the exist-ing programme if warranted.

(iii) Biomarkers predictive of worm burden need to be identifiedto simplify epidemiological studies and clinical trials for newmacrofilaricide development. Current methods used to esti-mate worm burden and drug response in lymphatic filariasisand onchocerciasis are inadequate.

(iv) The pathology of ivermectin-associated encephalopathy inconcurrent onchocerciasis and loaisis needs to be resolvedat a mechanistic level. Can a simple diagnostic procedurebe developed to prevent the administration of ivermectinto humans at risk for the development of this severe adverseevent?

(v) The lack of a macrofilaricidal drug complicates attempts toeliminate filariases of humans. The path for discovery anddevelopment of macrofilaricides requires standardizationand dedicated funding, despite the inability to expect com-mercial rewards on the normal pharmaceutical scale. Carefulattention needs to be paid in particular to the animal modelsneeded for testing prior to clinical trials in humans. Progresstowards ‘go/no go’ decisions for development of advancedcandidates (moxidectin, emodepside, flubendazole) shouldbe as rapid as possible.

(vi) It should be kept in mind that control efforts must be sup-ported by a platform of basic research; this is the philosoph-ical basis underpinning the funding of most Westerngovernment-funded research programmes (such as the

Table 1Efficacy of anthelmintics against soil-transmitted helminths in humans (adapted fromKeiser and Utzinger, 2008).

Drug Dose Species Cure rate: mean(95% CIa)

Albendazole 400 mg Ascaris lumbricoides 88 (79–93)Trichuris trichiura 28 (13–39)Hookworms 72 (59–81)

Mebendazole 500 mg A. lumbricoides 95 (91–97)T. trichiura 36 (16–51)Hookworms 15 (1–27)

Pyrantel pamoate 10 mg/kg A. lumbricoides 88 (79–93)Hookworms 31 (19–42)

a CI, confidence interval.

6 T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13

National Institutes of Health in the USA or the MedicalResearch Council in the UK). As noted above, this shouldinclude basic research in pharmacology (and the role of hostdefense systems in drug actions). However, it is prudent toassume that the best laid plans will often lead to failure,and much more research is needed to bolster new effortsfor the development of tools for control by illuminatingthe basic biology and physiology of the parasites and theircrucial interface with their human hosts. This kind ofresearch will open unanticipated avenues for the develop-ment of diagnostics and therapies, including vaccines anddrugs. The lack of basic, fundamental research is a major rea-son we find ourselves still wrestling to gain or consolidatecontrol of these widespread infections.

3. Anthelmintics for soil-transmitted helminths (STH)

3.1. Optimal dosing regimens for benzimidazoles for treatment ofhuman STH infections

Benzimidazoles such as albendazole and mebendazole are themainstay of therapy to control human infections with Ascaris lumb-ricoides, T. trichiura and hookworms. A great deal of clinical experi-ence suggests that a single-dose regimen with either albendazole(400 mg) or mebendazole (500 mg) provides good to excellent effi-cacy against A. lumbricoides, but single doses are less efficaciousagainst hookworms (although Ancylostoma duodenale appears tobe generally more sensitive to benzimidazoles than is Necator amer-icanus) and T. trichiura (Bennett and Guyatt, 2000; Olsen, 2007;Reddy et al., 2007; Keiser and Utzinger, 2008). Pyrantel pamoate, anon-benzimidazole cholinergic agonist, has been less studied butappears to have similar efficacy. Efficacy is determined by measuringthe change in nematode faecal egg output in patients before andafter treatment. Data are usually expressed as cure rate (treatmentleads to a complete absence of eggs in faeces at a specified time afterdosing) or as egg reduction rate (overall percentage reduction ineggs/gram of faeces before and after treatment). A complicating fac-tor is that methods for detecting eggs and analysing data are not fullyharmonised (see www.who.int/neglected_diseases/preventive_chemotherapy/anthelminthic_drug_efficacy/en/index.html). Curerates are a reasonable substitute for direct measurement of adultworm killing (assessed at necropsy in animals), but egg counts arean inadequate measure of adult worm abundance in veterinaryand probably in human parasitic infections (Conder and Campbell,1995; Kotze and Kopp, 2008). Egg output from hookworms is densitydependent; there is an inverse correlation between adult wormnumber and egg production per female (Anderson and Schad,1985). It is thus problematic to define nematocidal efficacy based so-lely on egg counts. The difficulty in precisely determining efficacy ofanthelmintics in humans confounds the detection of resistance andcomplicates attempts to define the optimal ways in which to deploythem for MDA. It must be noted in this context that measuring eggcounts in stool samples is essential in the current situation and re-mains the only available method for describing the epidemiologyof STH infections in humans. Research into improvements intomethods to measure egg counts is promising (Bergquist et al.,2009), but epidemiological monitoring requires different precisionthan required to estimate worm burden in trials of new compoundsor improved regimens for existing drugs. Biomarker-based methods,preferably relying on proteins released from adult worms, may pro-vide a better opportunity for such tests.

In any case, it appears that efficacy in humans is sub-optimal, atleast in comparison with what is expected in veterinary applica-tions. A recent meta-analysis (Keiser and Utzinger, 2008) reportedcure rates in humans for single-dose regimens of albendazole,mebendazole and pyrantel pamoate (Table 1; and see Reddy

et al., 2007). Levamisole has been studied much less intensivelyin humans but the available data suggest that it is not markedlybetter than the others (Keiser and Utzinger, 2008), although the re-sults of meta-analyses based on a small number of studies must bejudged cautiously. Nonetheless, based on these considerations,only single-dose mebendazole against A. lumbricoides would gainregulatory approval based on efficacy standards employed for vet-erinary registration.

Benzimidazoles and pyrantel pamoate were originally regis-tered for use in veterinary medicine and are still used to treatascarids, hookworms and whipworms in livestock, horses and pets.Pertinent to their use in humans, it is important to note that thebenzimidazoles, when used alone, are administered as two or threeconsecutive daily doses to monogastric animals, a regimen thatreliably provides >95% efficacy against most of these parasites. Incontrast, horses (which are also monogastric) are treated with sin-gle doses of benzimidazoles, which achieve good efficacy againstascarids and the other parasitic species (small and large strongyles,and pinworms, but not hookworms) commonly found in equids.Based on available data (see below), it appears that humans mayresemble dogs more than horses in this regard: two or threesequential daily doses may be needed to achieve full efficacyagainst the prevalent worms. Pyrantel pamoate at a single doseof 5 mg/kg (dogs) or 20 mg/kg (cats) is highly efficacious againstascarids and hookworms but lacks appreciable activity againstwhipworms; the basis for the discrepancy in activity of singledoses against hookworms in pets compared with humans remainsunexplained. Whether two or three sequential doses of pyrantelpamoate in humans would result in higher efficacy against hook-worms and whipworms is unclear.

As noted, neither drug class is reliably effective against whip-worms in monogastric animals in single doses, although repeateddosing of benzimidazoles does achieve full efficacy in dogs. Thismay be due in part to the predilection of these species to live inthe lower colon (which, as a compartment, is difficult to accessfor most drugs). However, it has recently been shown that thepolymorphism in b-tubulin (Y200F) that underlies benzimidazoleresistance in nematode parasites of animals (Kwa et al., 1994) isprevalent in at least some populations of T. trichiura (Diawaraet al., 2009). This may also be an important factor in the poorand variable efficacy of albendazole and mebendazole against thisspecies in humans. It should also be noted that a rigorous evalua-tion of the geographic variation in efficacy of these drugs againstgastrointestinal nematodes is lacking; differences in allele fre-quency in populations across the globe could contribute to differ-ences in efficacy.

The single-dose regimen of albendazole is more efficaciousagainst hookworms (particularly N. americanus) in humans thansingle-dose mebendazole. However, 100 mg mebendazole dosedon two consecutive days appears to be as effective as single-dose

Fig. 1. Relative efficacy of albendazole in humans for the treatment of nematodi-ases. Y axis: % efficacy. X axis: dose in mg/kg, with number of repeated doses (daily).Summary from Horton (2000).

T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13 7

albendazole for hookworm infection in humans (although species-specific differences in efficacy in this regard are not often analysed)and is better than single doses of either drug against whipworms(Bennett and Guyatt, 2000; Horton, 2000). Repeated doses ofalbendazole, but not higher single doses, achieve much better effi-cacy against hookworms than the single 400 mg dose, whereasboth increased single doses and repeated doses improve efficacyagainst whipworms (Fig. 1; Horton, 2000). Additional studies com-paring different doses and regimens are still needed to identifyoptimal treatment strategies for MDA applications.

Differences in chemosensitivity between N. americanus andAncylostoma ceylonicum (which may be similar in this regard toA. duodenale) have been noted for both ivermectin and benzimi-dazoles (Behnke et al., 1993; Richards et al., 1995; Horton, 2000)and warrant further investigation. In both cases, N. americanus ismore refractory. Failure to identify the hookworms present to thespecies level may thus confound analysis of efficacy in clinicalstudies. The basis for the difference in sensitivity of these two spe-cies to ivermectin is profound and has been experimentally inves-tigated; it appears to be intrinsic (Richards et al., 1995), but a fullexplanation for that observation is not available. The basis forthe difference in sensitivity to benzimidazoles has not been simi-larly investigated.

3.2. Challenges in dose selection in humans

These data pose important questions for the expansion of sin-gle-dose MDA programmes to control gastrointestinal nematodesin humans. First, it is clear that minimally acceptable efficacy goalshave neither been established nor achieved. The relationship be-tween worm burden and health consequences in humans has beeninadequately documented, so it is unclear whether the target forefficacy should be 70%, 90% or 95% reduction in mean worm bur-dens in order to achieve optimal gains in health. This is compli-cated further by the demonstrable species-specific differences inefficacy that characterise currently used drug regimens. Does theability to control A. lumbricoides with very high efficacy outweighthe considerably poorer results typically obtained for hookwormsand T. trichiura? Veterinary medical regulations require efficacyof 95% against a particular species for label inclusion. A corre-sponding absence of efficacy standards for drugs for inclusion inan MDA programme in humans is unsettling. Mediocre efficacyof anthelmintics against some of the human intestinal helminthsmight be justified on the basis that even modest reductions inworm burdens may alleviate some of the debilitating effects of par-asitism (although, as noted, this possibility remains to be rigor-ously quantified). On the other hand, mediocre efficacy could

challenge the long term sustainability of control programmes ifconcrete reductions in parasitism (at least of hookworms andwhipworms) cannot be reliably demonstrated.

Research in this arena suggests that gains in livestock produc-tivity are apparent if even relatively low worm burdens (non-symptomatic or sub-clinical parasitism) are removed by chemo-therapy (Gross et al., 1999; Vercruysse and Claerebout, 2001; Ball-weber, 2006). Do humans with even rather low levels of intestinalnematodes experience lower health status compared with unin-fected people? There is some evidence that this may be the case,particularly in people infected with multiple parasites (Forresteret al., 1998; Stephenson et al., 2000a,b; Drake and Bundy, 2001;Ezeamama et al., 2005; Engels and Savioli, 2006; Pullan and Brook-er, 2008), but more work in this area is clearly needed. AdoptingMDA strategies that reduce but do not eliminate intestinal nema-todes is a viable option if the intervention has clear health benefits.One must ask whether the benefits would be greater if largerreductions in worm burden were achieved. Finally, repeated treat-ments with low efficacy regimens can be expected to have rela-tively modest effects on environmental contamination andlikelihood of reinfection. Would long-term control objectives bebetter served if more efficacious treatments were used?

Implementing clinical trials to identify optimal dosing strate-gies is a challenging task. Routinely used assay methods for adultworm numbers are insufficiently sensitive and reliable to accu-rately measure drug efficacy. Better clinical indices of the health ef-fects of different levels of worm burden are needed, combined withbetter indicators of worm numbers (species-specific) that can beused to measure drug effects in trials or MDA programmes. Inthe absence of reliable and convenient assays for anthelmintic effi-cacy in humans, trials will be much more complicated and conten-tious than they should be. A priority for a research platform built tosupport the optimisation of MDA programs would include devel-opment of biomarker-based quantitative, species-specific assaysfor adult worm burdens as a key component.

Furthermore, the use of insufficiently efficacious dosage regi-mens is the functional equivalent of under-dosing, a clear risk fac-tor for rapid emergence and spread of drug resistance (Conder andCampbell, 1995; Smith et al., 1999; Geerts and Gryseels, 2001). Inaddition to efficacy of the drug used, a key variable in the selectionand spread of drug-resistant populations of parasites includes thefrequency of treatment (continuity of pressure) and the proportionof the population exposed to selective pressure. Parasites not ex-posed to the drug are said to be ‘in refugia’ (van Wyk, 2001) andrepresent a source of wild-type alleles that dilute resistance allelesin interbreeding populations in situations in which drug (selective)pressure is incomplete or intermittent. Recent use of benzimidaz-oles in regions with high intestinal nematode burdens has beenat best irregular, with pressure applied once or twice per year,and to a limited segment of the infected population (typicallyschool-aged children). As MDA programmes relying on albenda-zole and mebendazole become more widespread and intense, thecombination of under-dosing, continuous pressure and inadequaterefugia could lead to more rapid development of benzimidazoleresistance in whipworms and hookworms. There is already someevidence that populations of Necator spp. have been selected for al-leles of b-tubulin associated with benzimidazole resistance in vet-erinary species (Diawara, A. Development of DNA assays for thedetection of single nucleotide polymorphisms associated withbenzimidazole resistance, in human soil-transmitted helminths.McGill University MSc Thesis, 2008). Resistance to benzimidazolesgenerally extends to all members of this drug class (Conder andCampbell, 1995). Selection of mebendazole-resistant intestinalnematodes, for example, would be expected to limit the utility ofalbendazole in the same areas. Given that possibility, a re-evalua-tion of the regimen used for these drugs in human MDA pro-

8 T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13

grammes is warranted. Concerns relating to this general dilemmahave been put forward and should serve as a basis for discussionson monitoring in humans for possible anthelmintic resistance inthe field (Albonico et al., 2004).

Based on data in companion animals, several research avenuescould be considered. An initial step should be the rigorous evalua-tion of pharmacokinetic parameters vis-à-vis efficacy, especiallysince co-administration of benzimidazoles with food in humansand animals is known to have profound effects on drug absorptionfollowing oral dosing (Dayan, 2003). In addition, a two-dose regi-men of mebendazole rather than a single dose could be consideredfor these programmes. A related question is whether a two-doseregimen of albendazole or a single, higher dose would have suffi-ciently greater efficacy against hookworms than the single-doseregimen currently employed in humans to warrant a change inprotocol (including appropriate analyses of safety of higher dosesor drug combinations). It is obviously necessary to develop a strat-egy that maximises community compliance and penetration. Atthe same time, expanded MDA programmes will be sustainableonly to the extent that resistance does not compromise outcomes;treatment strategies that promote the development of resistanceare self-defeating in that regard.

Choosing optimal dosage regimens is further complicated bypossible influences of nutrition and population genetics, whichcan have large impacts on pharmacokinetic parameters. For mosttherapeutic indications, it is possible to correlate pharmacokineticparameters (half-life, mean compartment level, maximum concen-tration) with efficacy, but these analyses are very difficult for manyanthelmintics used against gastrointestinal nematodes, as it is notentirely clear which compartment should be measured (blood ver-sus intestinal lumen) and this may vary among different classes ofanthelmintics. For example, some parasites may acquire drug byfeeding on blood (hookworms) while others do not (ascarids, whip-worms), and it is not known whether drug in solution in the lumenor dissolved in the mucous layer is the major route of delivery fortranscuticular absorption. Benzimidazoles present particular chal-lenges, as they achieve very low blood levels, are metabolized toderivatives with variable bioactivity and may undergo enterohe-patic recirculation (Dayan, 2003). It has not been possible to iden-tify a pharmacokinetic parameter for any of these drugs that can beused to reliably predict efficacy against STH.

3.3. Drug combinations for STH

A strategy commonly employed in chemotherapy (e.g., HIV/AIDS, cancer, malaria and tuberculosis) is the use of combinationsof drugs, which enhance efficacy and/or retard the emergence ofresistance. Similarly, combination chemotherapy is commonlyused for parasite control in pets to maximise owner convenienceand to enhance efficacy. Many combination anthelmintic productshave been developed for the small ruminant market in Australiaand New Zealand in an effort to circumvent widespread drug resis-tance. While this strategy has intuitive appeal for the treatment ofgastrointestinal nematodes (and indeed benzimidazoles are usedin combination for filariasis control), it has yet to be establishedin MDA programmes. Challenges include the fact that there is apaucity of potential partner drugs with acceptably compatiblepharmacokinetic properties (although this is less of a concern ifhigh efficacy is achieved with single dose administrations). Moreimportantly, few studies document additive or synergistic effectsof anthelmintic combinations, indicating that considerable invest-ment in formulation research and manufacture may be requiredprior to clinical trials for registration if a single pill/tablet dosageform is desired.

The limited data available suggest that a combination of levam-isole and mebendazole at standard doses is much more efficacious

than either drug alone against hookworms (Albonico et al., 2003);that a combination of ivermectin plus albendazole is highly effica-cious against whipworms (Ismail and Jayakody, 1999; Belizarioet al., 2003; Olsen, 2007), that a combination of mebendazole pluslevamisole (and oxantel plus pyrantel pamoate) was more effica-cious against whipworms than either drug alone (Zu et al., 1992)and that a combination of pyrantel pamoate plus albendazolewas more active against hookworms than either drug alone (Zhanget al., 1998); the latter two studies used dosage forms that com-bined the active ingredients, an important step for demonstratingmanufacturability. This is crucial for control programmes, whereco-packaged individual products have inherent disadvantages. Sin-gle-product combination preparations can only be used whereweight-based dosing is not a problem, or where weight-to-dose ra-tio of the components is very similar. In this context, it must alsobe stressed that manufacturing and registration requirements forgeneric products used as anthelmintics must be stringently ap-plied, otherwise gains in efficacy possible with combinations maynot be realised.

It is therefore an important research question whether combi-nations of levamisole or oxantel, or pyrantel pamoate with alben-dazole or mebendazole for MDA programmes targeted at intestinalnematodes, would enhance efficacy and minimise the risk of selec-tion of resistant populations. It is appropriate and ethical in thecontext of global health-related MDA programmes to advocatecombination drug regimens for gastrointestinal nematodes, inkeeping with the situation for artemisinin-based therapy for ma-laria, to attain these therapeutic aims. Choosing a combination todevelop should be based on clinical trials and considerations ofthe challenges faced in manufacture and toxicity (clinical trialsfor registration typically require using the drug in its final form,so manufacturing issues would have to be addressed first). Alterna-tively, the periodic use of pyrantel pamoate or levamisole insteadof albendazole or mebendazole may reduce the reproductiveadvantage of any nematodes that do become resistant to the benz-imidazoles, slow selection for benzimidazole resistance, and main-tain the life of the current front line anthelmintics for STH.However, this strategy would not address the sub-optimal efficacyof benzimidazoles against hookworms and whipworms.

3.4. Anthelmintics on the near horizon

Because anthelmintic resistance plagues livestock production,particularly for small ruminants but also for cattle and monogastricanimals (Conder and Campbell, 1995; Kaplan, 2004), efforts havebeen devoted to the discovery of new anthelmintics for veterinarymarkets. These drugs may find application in the treatment of hu-man gastrointestinal nematodiases in the future. Nitazoxanide issuch a compound; originally patented as a cestocide, it has beenreported to have some activity in a very wide variety of humaninfections including viral, bacterial, protozoal, nematode, trema-tode and cestode pathogens (Fox and Saravolatz, 2005). It is ap-proved in the USA for treatment of Giardia and Cryptosporidiuminfections in humans. It has not proven sufficiently superior in cost,efficacy or safety to replace benzimidazoles as first-line agents forMDA programmes. Whether nitazoxanide would be adopted forthis use if benzimidazole resistance appears, or as an alternativeto benzimidazoles in any case, is unknown.

Emodepside, which, as noted above, has intriguing activityagainst some filariae in the laboratory, is a more recent introduc-tion. This drug is marketed in combination with praziquantel asa broad spectrum anthelmintic for companion animals; althoughthere are no publicised plans to register the drug for human use,it perhaps could, if necessary, play a role in the control of STH ifdevelopment to registration is feasible.

T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13 9

Tribendimidine has been developed in China specifically for useas a human anthelmintic (Xiao et al., 2005) with an unusuallybroad spectrum of action, including nematodes and cestodes (inthis regard being somewhat similar to nitazoxanide, although itis structurally unrelated). This drug reportedly has a good safetyprofile but it is unclear whether it has advantages over albendazoleor pyrantel pamoate for the treatment of common gastrointestinalnematodes (Steinmann et al., 2008). Like nitazoxanide, anddepending upon availability and registration in the target coun-tries, tribendimidine could be of use as a secondary drug for thisindication with potential as a benzimidazole replacement if resis-tance emerges. Like the benzimidazoles, however, much remainsto be learned about how to use it optimally for nematode infec-tions. It has recently been learned that the drug shares a mecha-nism of action with levamisole and pyrantel as a nicotiniccholinergic agonist in nematodes (Hu et al., 2009); whether it willbe superior to those drugs for human use will thus depend on fac-tors of clinical pharmacology.

Finally, monepantel has been introduced as a new anthelminticfor sheep, arising from a series of novel cholinergic agonists thattarget receptors distinct from those affected by levamisole orpyrantel pamoate (Kaminsky et al., 2008, 2009). Because it retainsactivity against strains of parasites resistant to all other anthelmin-tics, it could be an appealing addition to the pharmacopeia for STHinfections in humans. However, its activity against these specieshas not been reported, nor is it clear that development for humanuse is under consideration at this time.

3.5. Towards optimal drug regimens for MDA programmes: next stepsand research priorities

We have sought to identify the broad range of pharmacologicalissues that should be better understood if MDA programmes are tobe designed, implemented and monitored according to appropriatepractice for human public health programs. Here, we attempt toprioritize this list. Given our currently inadequate understandingof the pharmacology of anthelmintics in humans, all of these issuesdeserve urgent research attention.

(i) Establish minimally acceptable clinical endpoints as efficacygoals to guide the choice of a drug and dosing regimen for imple-mentation of intensive MDA programmes in endemic areas. Tosupport this goal, develop more accurate estimates of adult wormburdens in humans using biomarkers such as parasite coproanti-gens. Investigate alternative measures to quantitate the health im-pacts of intestinal nematode infections in humans that can be usedto correlate worm burdens with clinical sequelae and the beneficialconsequences of efficacious chemotherapy (what percentage ofworm clearance is needed to maximise acute health benefits in apopulation?). Both adults and children should be included in theanalysis.

(ii) Establish 400 mg albendazole and 500 mg mebendazole sin-gle dose base-line efficacy in relevant populations and use thesestudies to develop standardised protocols for future comparativedrug efficacy studies. Modify dose level and frequency of treatmentto determine whether these drugs can meet the standards for rel-evant parasite species established in (i).

(iii) A logistical goal for global MDA programmes may be a sin-gle, fixed dose combination of anthelmintics with broad spectrumactivity against the common helminth species. In this regard, theveterinary field offers an informative example, with the combina-tion of pyrantel pamoate + febantel + praziquantel commerciallyavailable for use in dogs in the USA and other countries. The com-bination of two distinct nematocidal compounds offers an attrac-tive option for MDA programmes that would likely bothmarkedly enhance efficacy against hookworms and whipwormsand retard the development of resistance in intestinal nematode

populations in a single pill format. Manufacturing in the veterinarysector provides single tablet doses for dogs up to �25 kg. It is pos-sible that tablet size could be increased for use in adult humans iffebantel was approved. Alternatively, investigation of a mebenda-zole or albendazole combination with pyrantel pamoate could cir-cumvent the need to register febantel for human use (althoughapproval of the combination of already registered compoundswould still be needed). Whether manufacture of solid formulationsof such combinations is feasible remains to be demonstrated.

Combination anthelmintic products registered for use in smallruminants are not formulated in preparations suitable for humansbut instead are available as liquids for oral drenching or topicaladministration. Some aspects of the manufacturing process forthese products may be transferable to the solid dosage forms morecommonly used in human MDA programs, but this also remains tobe demonstrated.

(iv) Develop a better understanding of the relationship betweenpharmacokinetic parameters and efficacy for anthelmintics used tocontrol gastrointestinal and tissue nematodes. Can we find a phar-macokinetic marker such as area under the curve (AUC) or maxi-mal serum concentration (Cmax) that can be used to guide thechoice of dose or treatment frequency to maximise efficacy in dif-ferent populations? Is there such a marker that correlates withtoxicity?

(v) Develop reliable culture systems that can be used to esti-mate the intrinsic potency of anthelmintics against adult stagesof gastrointestinal and tissue nematodes. Such assays will be re-quired to monitor, however crudely, the possible appearance ofdrug-resistant parasite populations until and unless molecular-le-vel tests are made available. Egg hatch assays adapted from veter-inary applications for use in monitoring anthelmintic resistance inhumans (Albonico et al., 2005; Kotze et al., 2005, 2009) may beuseful in this regard, although they target different stages of thelife-cycle and lack precision.

(vi) Evaluate in relevant clinical settings whether either nita-zoxanide or tribendimidine can substitute for available drugs inMDA campaigns if needed.

4. Chemotherapy of schistosomiasis

Although ‘anthelmintics’ is a term used to describe drugs thatare effective against nematodes, trematodes or cestodes, theseorganisms are separated by an enormous evolutionary and phylo-genetic gulf. Several species of schistosomes, together with othertrematode species, continue to present large and serious globalhealth problems; schistosome infections outnumber those fromother trematode species by a considerable margin and are the fo-cus of this review. No single drug available today has utility forthe treatment or prevention of nematode and trematode infectionsin humans. Although albendazole has some activity against the li-ver fluke Fasciola hepatica in ruminants, it lacks useful activityagainst schistosomes in humans or in animal models. Thus, theconcerns raised in this section are focused on a completely differ-ent set of drugs.

4.1. Pharmacological gaps: praziquantel

Control of all forms of schistosomiasis is currently dependentupon a single drug, praziquantel. Praziquantel was developed inthe early 1970s for veterinary practice, as is true of all availableanthelmintics for humans. The drug is now produced by variousmanufacturers and is used both in human and animal health ona large scale.

Much remains to be learned about drug disposition for prazi-quantel; gaps in this area mean that decisions about dosing must

10 T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13

be based upon efficacy (which is poorly estimated by egg counts inthe absence of a biomarker for adult parasites) instead of a phar-macokinetic marker. Since estimates of efficacy are based onreductions in egg shedding rather than on precise measures ofadult worm kill, optimisation of dosing schedules and regimens re-mains problematic. Most available data on drug disposition comefrom studies in adult healthy volunteers and are not recent; basicpharmacokinetic information is lacking in the target population.No correlation between a pharmacokinetic parameter (e.g., AUCor Cmax) and efficacy in humans is available to guide improved dos-ing options.

Praziquantel is rapidly and extensively absorbed after oraladministration (for review see Dayan, 2003) and is rapidly elimi-nated. There is great variability in pharmacokinetics among indi-viduals, which remains partly unexplained (see below). Exposureof the parasite in the blood stream increases exponentially withdose, although again, variability is high (Leopold et al., 1978).Importantly, no published studies have directly compared pharma-cokinetic parameters of the two most commonly used doses (40versus 60 mg/kg) in patients (see http://apps.who.int/tdr/svc/news-events/news/praziquantel-dosing).

Bioavailability is increased by administration of the drug withfood, in particular low fat (Mandour et al., 1990) and carbohydrates(Castro et al., 2000) in healthy adults, but the extent of exposure inthese two experiments using 40 mg/kg was very different and nocorrelation between increased absorption and efficacy (or the inci-dence of adverse effects) has been formally drawn. There is noinformation on parasite exposure achieved in target populations(primarily school-aged children and adolescents) when the drugis given in routine conditions in different settings. More researchon the influence of food on the pharmacokinetics of the drug andcommunity acceptance/compliance is also warranted.

Praziquantel has a high hepatic extraction rate with first-passeffects and is metabolized by cytochrome P450 (CYP 1A2, 2C19and 3A4; Li et al., 2003). Metabolites, the most abundant of whichis the 4-hydroxycyclohexyl-carbonyl analogue, are inactive. Theextent to which pharmacogenetic variation influences the pharma-cokinetics and consequent efficacy of the drug remains uncertain.

Commercially available praziquantel is a racemic mixture oftwo stereoisomers. One, the L-isomer, is responsible for the schist-osomicidal activity, while the D-isomer is inactive but as toxic asthe L-isomer (Cioli et al., 1995). It is important to confirm and ex-tend these findings, as a stereospecific dosage form could enhancecommunity enthusiasm and compliance with current MDA pro-grammes. Reduction of tablet size could allow co-formulation ofthe active isomer with other drugs for broadened MDA purposes.However, issues around compatibility of chemistry, manufacturingprocesses, cost-of-goods, registration and commercial viability ofcombination products, as well as the active isomer by itself, remainto be resolved. Better knowledge of how the isomers interact withputative drug targets is also needed. Praziquantel affects primarilyadult worms and not juvenile forms (Utzinger and Keiser, 2004).This stage specificity may in part explain underperformance incontrol programmes if infection has occurred less than 4–8 weeksprior to treatment, but the significance of this stage specificity forstudies on the mechanism of action of the drug should not beunderestimated (Valle et al., 2003).

Praziquantel has a broad spectrum of activity encompassingseveral other helminths and is used in other indications in humanand veterinary medicine. We do not know whether extensive useof the drug, particularly in domestic animals, may eventually erodeparasite sensitivity and select for resistant worms that can betransmitted to humans. This breadth of spectrum may pose a prob-lem in regions with a significant incidence of neurocysticercosis; itis possible that single doses of the drug, in the absence of concur-rent steroid therapy, may stimulate or exacerbate parasite-related

seizures, although epidemiological evidence to support this possi-bility is lacking. Nonetheless, the worrisome incidence of humaninfection with Taenia solium in parts of the world already endemicfor schistosomiasis (Garcia et al., 2007) suggests that the concernshould be addressed with alacrity.

The mechanism(s) of action of praziquantel is incompletelyunderstood. In vitro, the drug elicits a rapid contraction of schisto-somes, associated with Ca2+ influx and tegumental damage (Dayet al., 1992). The most popular hypothesis is that the drug opensa particular type of voltage-gated Ca2+ channel (Cambvar; Jeziorskiand Greenberg, 2006), leading to a massively increasing Ca2+ influx,muscle contraction and tegument disruption; this latter effect ex-poses antigens to immune attack and may account for the observa-tion that the in vivo efficacy of the drug depends to some extent onimmune system functions (Doenhoff et al., 1991).

Although the proposed target is supported by experimental evi-dence, other mechanisms may also be involved. For instance, thechannel appears to be expressed in immature stages of the parasitebut the clinical activity is largely restricted to adult worms (Valleet al., 2003). Importantly, exposure to cytochalasin D protectsschistosomes from praziquantel-induced lethality but does notabrogate the drug-induced Ca2+ influx, suggesting that these ef-fects are functionally decoupled (Doenhoff et al., 2008).

Whether praziquantel resistance exists is being debated (seeBotros and Bennett, 2007). It is clear that patients in certain areas(Northern Senegal, Egypt) did not respond to standard, repeated orincreased doses of praziquantel, although subsequent testing insome of the same villages many years later found no resistant iso-lates despite continued drug pressure. It is thought that praziquan-tel-resistant alleles incur a fitness deficit that militates againststable invasion into the population. Apparent resistance can be in-duced experimentally by repeated exposure to sub-optimal dosesof praziquantel in mice infected with Schistosoma mansoni (Fallonand Doenhoff, 1994), although the extent of resistance is not ashigh as observed with other antiparasitic drugs and the phenotypecan revert during passage in the absence of drugs. These character-istics make it difficult to design a genomics- or proteomics-basedapproach to monitor the evolution and spread of stable praziquan-tel resistance in human-derived parasite populations. Nonetheless,the potential threat to public health from such strains, should theyeventually evolve and spread, is enormous, which justifies contin-ued research in the area.

4.2. Other drugs

Praziquantel replaced older drugs primarily due to its increasedefficacy in simpler regimens against the three major Schistosomaspp. that infect humans. However, the drug is not ‘perfect’: it is inef-fective against immature stages and presents some problems incompliance. In addition, reliance upon a single agent could poten-tially lead to the development of resistance. If resistance becomesa problem, re-introduction of older drugs may be worthwhile.Two in particular, oxamniquine and/or metrifonate, may still havea role (Danso-Appiah et al., 2009), although they are species-spe-cific (S. mansoni and Schistosoma haematobium, respectively) andtheir commercial production is not guaranteed. More research isneeded to assess their suitability in the current context, possiblyin combination with praziquantel. They could, at least as temporarymeasures, retard the spread of praziquantel-resistant strains andprovide therapeutic relief (for a review, see Cioli et al., 1995).

Other compounds have been advanced or advocated for schisto-somiasis (Ribeiro-dos-Santos et al., 2006), but more work is neededto determine if any holds real promise for further development.New drug candidates are clearly needed, but screening protocolswhich rely on testing of compounds in cultures of the schistosom-ula stage (Ramirez et al., 2007) are inadequate for truly high-

T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13 11

throughput screening and a model trematode equivalent to C. ele-gans for this purpose has not been developed.

In the interim, several promising leads have been identified,including the antimalarial artemisinin analogues and their synthetictrioxane and trioxolane derivatives (Caffrey, 2007; Keiser and Utzin-ger, 2007). Interestingly, the antimalarial drug mefloquine and itsanalogues have also been shown to have schistosomicidal activity(Keiser et al., 2009). Any shared mechanism of action that underliestheir effects on schistosomes and malaria parasites are still unclear.Whether the adoption of antimalarial medications for schistosomi-asis is feasible in light of concerns about drug resistance inPlasmodium falciparum remains to be seen; perhaps selectivelyschistosomicidal analogues can be developed. It should also berecognised that the use of artemisinin analogues, and perhaps mef-loquine, for the treatment of malaria may have additional benefits inareas co-endemic for schistosomiasis (Keiser and Utzinger, 2007).

It is possible that drug combinations could prove useful, inwhich shortcomings of individual agents (such as stage- or spe-cies-dependence) could be offset by a partner drug which fillsthe missing parts of the spectrum and possibly provides mutualprotection against the development of resistance. The adoption ofa combination strategy for antimalarial chemotherapy has beenmotivated by the need to delay the development and spread ofresistant strains. Despite this precedent, the development chal-lenges should not be underestimated. In addition, if an antimalarialdrug is combined with praziquantel for schistosomiasis control,concerns about the consequences of additional, perhaps non-cura-tive, exposure of P. falciparum to essential antimalarial drugs (a riskfactor for the development of resistance) may be have to be dis-proven or minimised.

4.3. Priorities for research to close the schistosomiasis gaps

(i) Develop a better understanding of the relationship betweenpraziquantel pharmacokinetics and efficacy against schisto-somes to guide optimisation of dosing strategies.

(ii) Develop biomarkers that correlate with adult worm burdento facilitate studies designed to optimise praziquantel treat-ment strategies and the development of new schistosomi-cidal drugs. Current methods, which rely on egg counts,are essential for epidemiological monitoring and give a gen-eral indication of drug efficacy, but are insufficiently corre-lated with adult worm numbers to offer the precisionneeded for rigorous clinical trials.

(iii) Develop and validate models for the evaluation of candidateschistosomicides and combinations thereof for potentialtesting in humans.

(iv) Develop a better understanding of the mechanism(s) ofaction of praziquantel and better tools to assess geneticchanges in exposed parasite populations that may foretellresistance. Can significant operational advantages be rea-lised if the L-isomer as a pure compound is introduced intocontrol programs?

(v) Standardise trial design to allow cross-trial comparisons andmeta-analysis of clinical studies.

(vi) Systematically evaluate the effect of antimalarial drugs cur-rently used within the framework of artemisinin-based com-bination therapy against schistosomiasis, first in vivo andthen, if warranted, in clinical trials.

5. Conclusion

Major gaps remain in our understanding of the pharmacology ofanthelmintics in humans. While experience gives us some confi-dence about the safety and efficacy of drugs in current use, this re-view shows that there are urgent requirements – on ethical andscientific grounds – to develop a fuller understanding of the phar-

macology of these drugs if they are to continue to be given to tensof millions of people every year. The current and planned MDAprogrammes should be supported by a research platform that in-forms and optimises the field use of available drugs and providesa sound basis in biology for longer term enhancements of control.Knowledge generated by such a platform is also an essential contri-bution to research designed to improve the efficacy of currentanthelmintics and to discover and develop new anthelmintics.Knowledge is also required to illuminate mechanisms of resistanceto these drugs and thus contribute to monitoring resistance andperhaps slowing its emergence. There is no doubt that large-scaleanthelmintic treatment programmes are required to help childrenand adults lead fuller lives and achieve their full capacity, but it isequally clear that pharmacological research is urgently required toensure that these programs are effective and safe today and intothe future.

Acknowledgements

This paper was produced as a result of a Joint World Bank/World Health Organization Meeting on ‘‘Monitoring of DrugEfficacy in Large Scale Treatment Programmes for Human Helmin-thiases”, held in Washington, DC, USA at the World Bank, 31 Octo-ber–2 November 2007. This paper reflects the personal views ofthe authors and should not be interpreted to represent official pol-icies or positions of their respective institutions.

References

Albonico, M., Crompton, D.W., Savioli, L., 1999. Control strategies for humanintestinal nematode infections. Adv. Parasitol. 42, 277–341.

Albonico, M., Bickle, Q., Ramsan, M., Montresor, A., Savioli, L., Taylor, M., 2003.Efficacy of mebendazole and levamisole alone or in combination againstintestinal nematode infections after repeated targeted mebendazole treatmentin Zanzibar. Bull. WHO 81, 343–352.

Albonico, M., Engels, D., Savioli, L., 2004. Monitoring drug efficacy and earlydetection of drug resistance in human soil-transmitted nematodes: a pressingpublic health agenda for helminth control. Int. J. Parasitol. 34, 1205–1210.

Albonico, M., Wright, V., Ramsan, M., Haji, H.J., Taylor, M., Savioli, L., Bickle, Q., 2005.Development of the egg hatch assay for detection of anthelminthic resistance inhuman hookworms. Int. J. Parasitol. 35, 803–811.

Ali, M.M.M., Mukhtar, M.M., Baraka, O.Z., Homeida, M.M.A., Kheir, M.M., Mackenzie,C.D., 2002. Immunocompetence may be important in the effectiveness ofMectizan (ivermectin) in the treatment of human onchocerciasis. Acta Trop. 84,49–53.

Anderson, R.M., Schad, G.A., 1985. Hookworm infections and faecal egg counts: ananalysis of the biological basis of variation. Trans. R. Soc. Trop. Med. Hyg. 79,812–825.

Ballweber, L.R., 2006. Endoparasite control. Vet. Clin. North Am. Food Anim. Pract.22, 451–461.

Barnes, T.M., Hekimi, S., 1997. The Caenorhabditis elegans avermectin resistance andanesthetic response gene unc-9 encodes a member of a protein familyimplicated in electrical coupling of excitable cells. J. Neurochem. 69, 2251–2260.

Behnke, J.M., Rose, R., Garside, P., 1993. Sensitivity to ivermectin and pyrantel ofAncylostoma ceylonicum and Necator americanus. Int. J. Parasitol. 23, 945–952.

Belizario, V.Y., Amarillo, M.E., de Leon, W.U., de los Reyes, A.E., Bugayong, M.G.,Macatangay, B.J.C., 2003. A comparison of the efficacy of single doses ofalbendazole, ivermectin and diethylcarbamazine alone or in combinationsagainst Ascaris and Trichuris spp.. Bull. WHO 81, 35–42.

Bennett, A., Guyatt, H., 2000. Reducing intestinal nematode infection: efficacy ofalbendazole and mebendazole. Parasitol. Today 16, 71–74.

Bennett, J.L., Williams, J.F., Dave, V., 1993. Pharmacology of ivermectin. Parasitol.Today 4, 226–228.

Bergquist, R., Johansen, M.V., Utzinger, J., 2009. Diagnostic dilemmas inhelminthology: what tools to use and when? Trends Parasitol. 25, 151–156.

Bird, A.C., El Sheikh, H., Anderson, J., Fuglsang, H., 1980. Changes in visual functionand in the posterior segment of the eye during the treatment of onchocerciasiswith diethylcarbamazine citrate. Br. J. Ophthalmol. 64, 191–200.

Botros, S.S., Bennett, J.L., 2007. Praziquantel resistance. Curr. Opin. Drug Disc. 2(Suppl. 1), S35–S40.

Brooker, S., Clements, A.C.A., Bundy, D.A.P., 2006. Global epidemiology, ecologyand control of soil-transmitted helminth infections. Adv. Parasitol. 62,221–261.

Brown, K.R., 2002. The use of macrocyclic lactones to control parasites of humans.In: Vercruysse, J., Rew, R.S. (Eds.), Macrocyclic Lactones in AntiparasiticTherapy. CABI Publishing, NY, pp. 405–412.

12 T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13

Brown, K.R., Ricci, F.M., Ottesen, E.A., 2000. Ivermectin: effectiveness in lymphaticfilariasis. Parasitology 121 (Suppl.), S133–S146.

Caffrey, C.R., 2007. Chemotherapy of schistosomiasis: present and future. Curr.Opin. Chem. Biol. 11, 433–439.

Castro, N., Medina, R., Sotelo, J., Jung, H., 2000. Bioavailability of praziquantelincreases with concomitant administration of food. Antimicrob. AgentsChemother. 44, 2903–2904.

Cioli, D., Pica-Mattoccia, L., Archer, S., 1995. Antischistosomal drugs: past,present. . .and future? Pharmacol. Ther. 68, 35–85.

Conder, G.A., Campbell, W.C., 1995. Chemotherapy of nematode infections ofveterinary importance, with special reference to drug resistance. Adv. Parasitol.35, 1–84.

Cotreau, M.M., Warren, S., Ryan, J.L., Fleckenstein, L., Vanapali, S.R., Brown, K.R.,Rock, D., Chen, C.Y., Schwertschlag, U.S., 2003. The antiparasitic moxidectin:safety, tolerability, and pharmacokinetics in humans. J. Clin. Pharmacol. 43,1108–1115.

Critchley, J., Addis, D., Ejera, H., Gamble, C., Garner, P., Gelband, H., 2005.Albendazole for the control and elimination of lymphatic filariasis: systematicreview. Trop. Med. Int. Health 10, 818–825.

Danso-Appiah, A., Garner, P., Olliaro, P., Utzinger, J., 2009. Treatment of urinaryschistosomiasis: methodological issues and research needs identified through aCochrane systematic review. Parasitology 136, 1–13.

Day, T.A., Bennett, J.L., Pax, R.A., 1992. Praziquantel: the enigmatic anthelmintic.Parasitol. Today 8, 342–344.

Dayan, A.D., 2003. Albendazole, mebendazole and praziquantel. Review of non-clinical toxicity and pharmacokinetics. Acta Trop. 86, 141–159.

de C Bronsvoort, B.M., Renz, A., Tchakouté, V., Tanya, V.N., Ekale, D., Trees, A.J., 2005.Repeated high doses of avermectins cause prolonged sterilization, but do notkill, Onchocerca ochengi adult worms in African cattle. Filaria J. 4, 4–8.

de Silva, N., Guyatt, H., Bundy, D., 1997. Anthelmintics: a comparative review oftheir clinical pharmacology. Drugs 53, 769–788.

Denham, D.A., Samad, R., Cho, S.Y., Suswillo, R.R., Skippins, S.C., 1979. Theanthelmintic of flubendazole on Brugia pahangi. Trans. R. Soc. Trop. Med. Hyg.73, 673–676.

Diawara, A., Drake, L.J., Suswillo, R.R., Kihara, J., Bundy, D.A.P., Scott, M.E., Halpenny,C., Stothard, J.R., Prichard, R.K., 2009. Assays to detect b-tubulin codon 200polymorphism in Trichuris trichiura and Ascaris lumbricoides. PLoS Negl. Trop.Dis. 3, e397.

Doenhoff, M.J., Modha, J., Lambertucci, J.R., McLaren, D.J., 1991. The immunedependence of chemotherapy. Parasitol. Today 7, 16–18.

Doenhoff, M.J., Cioli, D., Utzinger, J., 2008. Praziquantel: mechanism of action,resistance and new derivatives for schistosomiasis. Curr. Opin. Inf. Dis. 21, 659–667.

Dominguez-Vazquez, A., Taylor, H.R., Greene, B.M., Ruvalcaba-Macias, A.M., Rivas-Alcala, A.R., Murphy, R.P., Beltran-Hernandez, F., 1983. Comparison offlubendazole and diethylcarbamazine in treatment of onchocerciasis. Lancet 1(8317), 139–143.

Drake, L.J., Bundy, D.A., 2001. Multiple helminth infections in children: impact andcontrol. Parasitology 122 (Suppl.), S73–S81.

Duke, B.O.L., Pacqué, M.C., Muñoz, B., Greene, B.M., Taylor, H.R., 1991. Viability ofadult Onchocerca volvulus after six 2-weekly doses of ivermectin. Bull. WHO 69,163–168.

Eng, J.K.L., Blackhall, W.J., Osei-Atweneboana, M.Y., Bourguinat, C., Galazzo, D.,Beech, R.N., Unnasch, T.R., Awadzi, K., Lubega, G.W., Prichard, R.K., 2006.Ivermectin selection on b-tubulin: evidence in Onchocerca volvulus andHaemonchus contortus. Mol. Biochem. Parasitol. 150, 229–235.

Engels, D., Savioli, L., 2006. Reconsidering the underestimated burden caused byneglected tropical diseases. Trends Parasitol. 22, 363–366.

Ezeamama, A.E., Friedman, J.F., Olveda, R.M., Acosta, L.P., Kurtis, J.D., Mor, V.,McGarvey, S.T., 2005. Functional significance of low-intensity polyparasitehelminth infections in anemia. J. Infect. Dis. 192, 2160–2170.

Fallon, P.G., Doenhoff, M.J., 1994. Drug-resistant schistosomiasis: resistance topraziquantel and oxamniquine induced in Schistosoma mansoni in mice is drugspecific. Am. J. Trop. Med. Hyg. 51, 83–88.

Forrester, J.E., Bailar III, J.C., Esrey, S.A., Jose, M.V., Castillejos, B.T., Ocampo, G., 1998.Randomised trial of albendazole and pyrantel in symptomless trichuriasis inchildren. Lancet 352, 1103–1108.

Fox, L.M., Saravolatz, L.D., 2005. Nitazoxanide: a new thiazolide antiparasitic agent.Clin. Inf. Dis. 40, 1173–1180.

Garcia, H.H., Moro, P.L., Schantz, P.M., 2007. Zoonotic helminth infections ofhumans: echinococcosis, cysticercosis and fascioliasis. Curr. Opin. Inf. Dis. 20,489–494.

Gardon, J., Gardon-Wendel, N., Demanga-Ngangue, Kamgno, J., Chippaux, J.P.,Boussinesq, M., 1997. Serious reactions after mass treatment of onchocerciasiswith ivermectin in an area endemic for Loa loa infection. Lancet 350, 18–22.

Gardon, J., Boussinesq, M., Kamgno, J., Gardon-Wendel, N., Demanga-Nganque,Duke, B.O., 2002. Effects of standard and high doses of ivermectin on adultworms of Onchocerca volvulus: a randomized controlled trial. Lancet 360, 203–210.

Geary, T.G., 2005. Ivermectin 20 years on: maturation of a wonder drug. TrendsParasitol. 21, 530–532.

Geerts, S., Gryseels, B., 2001. Anthelmintic resistance in human helminths: a review.Trop. Med. Int. Health 6, 915–921.

Ginger, C.D., 1986. Advances in filarial chemotherapy and screening. Parasitol.Today 2, 38–40.

Greene, B.M., 1986. Diethylcarbamazine. Antimicrob. Agents Ann. 1, 258–264.

Greene, B.M., Brown, K.R., Taylor, H.R., 1989. Use of ivermectin in humans. In:Campbell, W.C. (Ed.), Ivermectin and Avermectins. Springer-Verlag, NY, pp.311–323.

Gross, S.J., Ryan, W.G., Ploeger, H.W., 1999. Anthelmintic treatment of dairy cowsand its effect on milk production. Vet. Rec. 144, 581–587.

Guest, M., Bull, K., Walker, R.J., Amliwala, K., O’Connor, V., Harder, A., Holden-Dye, L.,Hopper, N.A., 2007. The calcium-activated potassium channel, SLO-1, isrequired for the action of the novel cyclo-octadepsipeptide anthelmintic,emodepside, in Caenorhabditis elegans. Int. J. Parasitol. 37, 1577–1588.

Gutteridge, W.E., Hudson, A.T., Jenkins, D.C., 1990. The Wellcome Foundation/WorldHealth Organisation Onchocerciasis Chemotherapy Project Joint Programme todiscover and develop a macrofilaricide for onchocerciasis. Acta Leidensia 59,343–360.

Gyapong, J.O., Kumaraswami, V., Biswas, G., Ottesen, E.A., 2005. Treatmentstrategies underpinning the global programme to eliminate lymphaticfilariasis. Exp. Opin. Pharmacother. 6, 179–200.

Hoerauf, A., 2008. Filariasis: new drugs and new opportunities for lymphaticfilariasis and onchocerciasis. Curr. Opin. Inf. Dis. 21, 673–681.

Horton, J., 2000. Albendazole: a review of anthelmintic efficacy and safety inhumans. Parasitology 121 (Suppl.), S113–S132.

Hotez, P.J., Brindley, P.J., Bethony, J.M., King, C.H., Pearce, E.J., Jacobson, J., 2008.Helminth infections: the great neglected tropical diseases. J. Clin. Invest. 118,1311–1321.

Hu, Y., Xiao, S.-H., Aroian, R.V., 2009. The new anthelmintic tribendimidine is an L-type (levamisole and pyrantel) nicotinic acetylcholine receptor agonist. PLoSNegl. Trop. Dis. 3, e499.

Ismail, M.M., Jayakody, R.L., 1999. Efficacy of albendazole and its combination withivermectin or diethylcarbamazine (DEC) in the treatment of Trichuris trichiurainfections in Sri Lanka. Ann. Trop. Med. Parasitol. 93, 501–504.

Jeziorski, M.C., Greenberg, R.M., 2006. Voltage-gated calcium channel subunits fromplatyhelminths: potential role in praziquantel action. Int. J. Parasitol. 36, 625–632.

Junnila, A., Bohle, S., Prichard, R., Perepichka, I., Spina, C., 2007. Fluorescentdiethylcarbamazine analogs: sites of accumulation in Brugia malayi. Bioconjug.Chem. 18, 1818–1823.

Kaminsky, R., Ducray, P., Jung, M., Clover, R., Rufener, L., Bouvier, J., Weber, S.S.,Wenger, A., Wieland-Berghausen, S., Goebel, T., Gauvry, N., Pautrat, F., Skripsky,T., Froelich, O., Komoin-Oka, C., Westlund, B., Sluder, A., Mäser, P., 2008. A newclass of anthelmintics effective against drug-resistant nematodes. Nature 452,176–180.

Kaminsky, R., Mosimann, D., Sager, H., Stein, P., Hosking, B., 2009. Determination ofthe effective dose rate for monepantel (AAD 1566) against adult gastro-intestinal nematodes in sheep. Int. J. Parasitol. 39, 443–446.

Kaplan, R.M., 2004. Drug resistance in nematodes of veterinary importance: a statusreport. Trends Parasitol. 20, 477–481.

Keiser, J., Utzinger, J., 2007. Artemisinins and synthetic trioxolanes in the treatmentof helminth infections. Curr. Opin. Inf. Dis. 20, 605–612.

Keiser, J., Utzinger, J., 2008. Efficacy of current drugs against soil-transmittedhelminth infections. Systematic review and meta-analysis. J. Am. Med. Assoc.299, 1932–1948.

Keiser, J., Chollet, J., Xiao, S.H., Mei, J.Y., Jiao, P.Y., Utzinger, J., Tanner, M., 2009.Mefloquine – an aminoalcohol with promising antischistosomal properties inmice. PLoS Negl. Trop. Dis. 3, e350.

Kotze, A.C., Coleman, G.T., Mai, A., McCarthy, J.S., 2005. Field evaluation ofanthelmintic drug sensitivity using in vitro egg hatch and larval motilityassays with Necator americanus recovered from human clinical isolates. Int. J.Parasitol. 35, 445–453.

Kotze, A.C., Kopp, S.R., 2008. The potential impact of density dependent fecundity onthe use of the faecal egg count reduction test for detecting drug resistance inhuman hookworms. PLoS Negl. Trop. Dis. 2, e297.

Kotze, A.C., Lowe, A., O’Grady, J., Kopp, S.R., Behnke, J.M., 2009. Dose–response assaytemplates for in vitro assessment of resistance to benzimidazole and nicotinicacetylcholine receptor agonist drugs in human hookworms. Am. J. Trop. Med.Hyg. 81, 163–170.

Kshirsagar, N.A., Gogtay, N.J., Garg, B.S., Deshmukh, P.R., Rajgor, D.D., Kadam, V.S.,Kirodian, B.G., Ingole, N.S., Mehendale, A.M., Fleckenstein, L., Karbwang, J.,Lazdins-Held, J.K., 2004. Safety, tolerability, efficacy and plasma concentrationsof diethylcarbamazine and albendazole co-administration in a field study in anarea endemic for lymphatic filariasis in India. Trans. R. Soc. Trop. Med. Hyg. 98,205–217.

Kwa, M.S., Veenstra, J.G., Roos, M.H., 1994. Benzimidazole resistance in Haemonchuscontortus is correlated with a conserved mutation at amino acid 200 in b-tubulin isotype 1. Mol. Biochem. Parasitol. 63, 299–303.

Leopold, G., Ungethüm, W., Groll, E., Diekmann, H.W., Nowak, H., Wegner, D.H.G.,1978. Clinical pharmacology in normal volunteers of praziquantel, a new drugagainst schistosomes and cestodes. Eur. J. Clin. Pharmacol. 14, 281–291.

Li, X.Q., Björkman, A., Andersson, T.B., Gustafsson, L.L., Masimirembwa, C.M., 2003.Identification of human cytochrome P(450)s that metabolise anti-parasiticdrugs and predictions of in vivo drug hepatic clearance from in vitro data. Eur. J.Clin. Pharmacol. 59, 429–442.

Lok, J.B., Knight, D.H., Selavka, C.M., Eynard, J., Zhang, Y., Bergman, R.N., 1995.Studies of reproductive competence in male Dirofilaria immitis treated withmilbemycin oxime. Trop. Med. Parasitol. 46, 235–240.

Mackenzie, C.D., Kron, M.A., 1985. Diethylcarbamazine: a review of its action inonchocerciasis, lymphatic filariasis and inflammation. Trop. Dis. Bull. 82, R1–R37.

T.G. Geary et al. / International Journal for Parasitology 40 (2010) 1–13 13

Mackenzie, C.D., Geary, T.G., Gerlach, J.A., 2003. Possible pathogenic pathways in theadverse clinical events seen following ivermectin administration toonchocerciasis patients. Filaria J. 2 (Suppl. 1), S5.

Mackenzie, C., Geary, T., Prichard, R., Boussinesq, M., 2007. Where next with Loa loaencephalopathy? Data are badly needed. Trends Parasitol. 23, 237–238.

Maizels, R.M., Denham, D.A., 1992. Diethylcarbamazine (DEC):immunopharmacological interactions of an anti-filarial drug. Parasitology 105(Suppl.), S49–S60.

Mak, J.W., Navaratnam, V., Ramachandran, C.P., 1991. Experimental chemotherapyof lymphatic filariasis. A review. Ann. Trop. Med. Parasitol. 85, 131–137.

Mandour, M.E., el Turabi, H., Homeida, M.M., el Sadig, T., Ali, H.M., Bennett, J.L.,Leahey, W.J., Harron, D.W., 1990. Pharmacokinetics of praziquantel in healthyvolunteers and patients with schistosomiasis. Trans. R. Soc. Trop. Med. Hyg. 84,389–393.

Martin, R.J., Robertson, A.P., Bjorn, H., 1997. Target sites of anthelmintics.Parasitology 114 (Suppl.), S111–S124.

McCall, J.W., 2005. The safety-net story about macrocyclic lactone heartwormpreventatives: a review, an update and recommendations. Vet. Parasitol. 133,197–206.

McGarry, H.F., Plant, L.D., Taylor, M.J., 2005. Diethylcarbamazine activity againstBrugia malayi microfilariae is dependent on inducible nitric-oxide synthase andthe cyclooxygenase pathway. Filaria J. 4, 4.

Molyneux, D.H., Hotez, P.J., Fenwick, A., 2005. ‘‘Rapid-impact interventions”: how apolicy of integrated control for Africa’s neglected tropical diseases could benefitthe poor. PLoS Med. 2, e336.

Mottier, M.L., Prichard, R.K., 2008. Genetic analysis of a relationship betweenmacrocyclic lactone and benzimidazole anthelmintic selection on Haemonchuscontortus. Pharmacogenet. Genomics 18, 129–140.

Noroes, J., Dreyer, G., Santos, A., Mendes, V.G., Medeiros, Z., Addiss, D., 1997.Assessment of the effect of diethylcarbamazine on adult Wuchereria bancroftiin vivo. Trans. R. Soc. Trop. Med. Hyg. 91, 78–81.

Nwaka, S., Hudson, A., 2006. Innovative lead discovery strategies for tropicaldiseases. Nat. Rev. Drug Disc. 5, 941–955.

Olsen, A., 2007. Efficacy and safety of drug combinations in the treatment ofschistosomiasis, soil-transmitted helminthiasis, lymphatic filariasis andonchocerciasis. Trans. R. Soc. Trop. Med. Hyg. 101, 747–758.

Osei-Atweneboana, M.Y., Eng, J.K., Boakye, D.A., Gyapong, J.O., Prichard, R.K., 2007.Prevalence and intensity of Onchocerca volvulus infection and efficacy ofivermectin in endemic communities in Ghana: a two-phase epidemiologicalstudy. Lancet 369, 2021–2029.

Pax, R.A., Williams, J.F., Guderian, R.H., 1988. In vitro motility of isolated adults andsegments of Onchocerca volvulus, Brugia pahangi and Acanthocheilonema viteae.Trop. Med. Parasitol. 39 (Suppl.), 450–455.

Plaisier, A.P., Stolk, W.A., van Oortmarssen, G.J., Habbema, J.D.F., 2000. Effectivenessof annual ivermectin treatment for Wuchereria bancrofti infection. Parasitol.Today 16, 298–302.

Pullan, R., Brooker, S., 2008. The health impact of polyparasitism in humans: arewe under-estimating the burden of parasitic diseases? Parasitology 135, 783–

794.Ramirez, B., Bickle, Q., Yousif, Y., Fakorede, F., Mouries, M.-A., Nwaka, S., 2007.

Schistosomes: challenges in compound screening. Exp. Opin. Drug Disc. 2(Suppl. 1), S53–S61.

Reddy, M., Gill, S.S., Kalkar, S.R., Wu, W., Anderson, P.J., Rochon, P.A., 2007. Oral drugtherapy for multiple neglected tropical diseases. A systematic review. J. Am.Med. Assoc. 298, 1911–1924.

Ribeiro-dos-Santos, G., Verjovski-Almeida, S., Leite, L.C.C., 2006. Schistosomiasis – acentury searching for chemotherapeutic drugs. Parasitol. Res. 99, 505–521.

Richards, J.C., Behnke, J.M., Duce, I.R., 1995. In vitro studies on the relative sensitivityto ivermectin of Necator americanus and Ancylostoma ceylonicum. Int. J. Parasitol.25, 1185–1191.

Satti, M.Z., VandeWaa, E.A., Bennett, J.L., Williams, J.F., Conder, G.A., McCall, J.W.,1988. Comparative effects of anthelmintics on motility in vitro of Onchocercagutterosa, Brugia pahangi and Acanthocheilonema viteae. Trop. Med. Parasitol. 39(Suppl.), 480–483.

Schwab, A.E., Churcher, T.S., Schwab, A.J., Basáñez, M.-G., Prichard, R.K., 2007. Ananalysis of the population genetics of potential multi-drug resistance inlymphatic filariasis due to combination chemotherapy. Parasitology 134,1025–1040.

Scientific Working Group on Serious Adverse Events in Loa loa Endemic Areas, 2003.Report of a scientific working group on serious adverse events followingMectizan treatment of onchocerciasis in Loa loa endemic areas. Filaria J. 2(Suppl. 1), S2.

Sharma, S., 1993. Drugs for filariasis: four decades of research. Adv. Drug Res. 24,199–260.

Smith, G., Grenfell, B.T., Isham, V., Cornell, S., 1999. Anthelmintic resistancerevisited: under-dosing, chemoprophylactic strategies, and matingprobabilities. Int. J. Parasitol. 29, 77–91.

Steinmann, P., Zhou, X.N., Du, Z.W., Jiang, J.Y., Xiao, S.H., Wu, Z.X., Zhou, H., Utzinger,J., 2008. Tribendimidine and albendazole for treating soil-transmittedhelminths. Strongyloides stercoralis and Taenia spp.: open-label randomizedtrial. PLoS Negl. Trop. Dis. 2, e322.

Stephenson, L.S., Latham, M.C., Ottesen, E.A., 2000a. Malnutrition and parasitichelminth infections. Parasitology 121 (Suppl.), S23–S38.

Stephenson, L.S., Holland, C.V., Cooper, E.S., 2000b. The public health significance ofTrichuris trichiura. Parasitology 121 (Suppl.), S73–S95.

Surin, J., Denham, D.A., 1990. Comparative susceptibility to anthelmintics of Brugiapahangi in jirds infected by different methods. J. Helminthol. 64, 232–238.

Taylor, M.J., Bandi, C., Hoerauf, A., 2005. Wolbachia bacterial endosymbionts offilarial nematodes. Adv. Parasitol. 60, 247–286.

Taylor, M.J., Awadzi, K., Basáñez, M.-G., Biritwum, N., Boakye, D., Boatin, B., Bockarie,M., Churcher, T.S., Debrah, A., Edwards, G., Hoerauf, A., Mand, S., Matthews, G.,Osei-Atweneboana, M., Prichard, R.K., Wanji, S., Adjei, O., 2009. Onchocerciasiscontrol: vision for the future from a Ghanaian perspective. Parasit. Vectors 2, 7.

Tisch, D.J., Michael, E., Kazura, J.W., 2005. Mass chemotherapy options to controllymphatic filariasis: a systematic review. Lancet Infect. Dis. 5, 514–523.

Townson, S., Freeman, A., Harris, A., Harder, A., 2005. Activity of thecyclooctadepsipeptide emodepside against Onchocerca gutterosa, Onchocercalienalis and Brugia pahangi. Am. J. Trop. Med. Hyg. 73 (Suppl.), 93.

Townson, S., Ramirez, B., Fakorede, F., Mouries, M.-A., Nwaka, S., 2007. Challenges indrug discovery for novel antifilarials. Exp. Opin. Drug Disc. 2 (Suppl. 1), S63–S73.

Trees, A.J., Graham, S.P., Renz, A., Bianco, A.E., Tanya, V., 2000. Onchocerca ochengiinfections in cattle as a model for human onchocerciasis: recent developments.Parasitology 120 (Suppl.), S133–S142.

Twum-Danso, M.A.Y., Meredith, S.E.O., 2003. Variation in incidence of seriousadverse events after onchocerciasis treatment with ivermectin in areas ofCameroon co-endemic for loaisis. Trop. Med. Int. Health 8, 820–831.

Utzinger, J., Keiser, J., 2004. Schistosomiasis and soil-transmitted helminthiasis:common drugs for treatment and control. Exp. Opin. Pharmacother. 5, 263–285.

Valle, C., Troiani, A.R., Festucci, A., Pica-Mattoccia, L., Liberti, P., Wolstenholme, A.,Francklow, K., Doenhoff, M.J., Cioli, D., 2003. Sequence and level of endogenousexpression of calcium channel b subunits in Schistosoma mansoni displayingdifferent susceptibilities to praziquantel. Mol. Biochem. Parasitol. 130, 111–115.

van Wyk, J.A., 2001. Refugia – overlooked as perhaps the most potent factorconcerning the development of anthelmintic resistance. Onderstepoort J. Vet.Res. 68, 55–67.

Vercruysse, J., Claerebout, E., 2001. Treatment vs. non-treatment of helminthinfections in cattle: defining the threshold. Vet. Parasitol. 98, 195–214.

Wen, L.Y., Yan, X.L., Sun, F.H., Fang, Y.Y., Yang, M.J., Lou, L.J., 2008. A randomized,double-blind, multicenter clinical trial on the efficacy of ivermectin againstintestinal nematode infections in China. Acta Trop. 106, 190–194.

Xiao, S.H., Hui-Ming, W., Tanner, M., Utzinger, J., Chong, W., 2005. Tribendimidine: apromising, safe and broad-spectrum anthelmintic agent from China. Acta Trop.94, 1–14.

Zahner, H., Schares, G., 1993. Experimental chemotherapy of filariasis: comparativeevaluation of the efficacy of filaricidal compounds in Mastomys coucha infectedwith Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi and B.Pahangi. Acta Trop. 52, 221–266.

Zhang, D., Zhang, X., Tang, Z., Chen, C., Chen, Y., Yang, W., Jin, M., Huang, C., Yang, G.,Long, Y., 1998. Field trials on the efficacy of albendazole composite againstintestinal nematodiasis. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong BingZa Zhi 16, 1–5.

Zu, L.Q., Jiang, Z.X., Yu, S.H., Ding, X.M., Bin, X.H., Yang, H.F., Zhu, H.Q., Kuang, C.S.,Chen, Q.W., Zhou, C.H., 1992. Treatment of soil-transmitted helminth infectionsby anthelmintics in current use. Zhongguo Ji Sheng Chong Xue Yu Ji ShengChong Bing Za Zhi 10, 95–99.