Progress and challenges in the discovery of macrofilaricidal drugs

Transcription

1 Review For reprint orders, please contact Progress and challenges in the discovery of macrofilaricidal drugs Expert Rev. Anti Infect. Ther. 9(8), (2011) Timothy G Geary 1 and Charles D Mackenzie 2 1 Institute of Parasitology, McGill University, Lakeshore Road, te-anne-de-bellevue QC, 9X 3V9, Canada 2 Department of Pathobiology and Diagnostic Investigation, Michigan tate University, East Lansing, MI 48824, UA Author for correspondence: Tel.: Fax: timothy.g.geary@mcgill.ca Control of human filarial infections currently depends on chemotherapeutic strategies predominantly directed at microfilariae. Doxycycline therapy in an extended daily dose regimen sterilizes and kills adult stages, but the utility of this drug for routine field use remains an issue of concern. o macrofilaricidal drugs with efficacy after one or two doses are available for use, delaying the achievement of the elimination or eradication of onchocerciasis and lymphatic filariasis. Moxidectin, a macrocyclic lactone, is currently in clinical trials for onchocerciasis. A few other drugs that have already been approved for use in veterinary practice or in human medicine for other indications are available for investigation. Early drug discovery pipelines are poorly populated and the process of macrofilaricide discovery and development remains highly challenging. In particular, the lack of convenient, validated animal models in an antifilarial drug discovery pathway is an unresolved issue. Keywords: anthelmintic filarial flubendazole lymphatic filariasis macrocyclic lactone nematode onchocerciasis Filarial infections are widespread in humans and animals throughout much of the world. These infections are caused by a phylogenetically related group of parasitic nematodes, which can be distinguished by two features of the life cycle: they include a vector-mediated trans mission step (involving an arthropod intermediate host), and the adult stages reside in host internal tissues rather than in the GI tract. This life cycle imposes chemotherapeutic and control challenges distinct from those that characterize the more widespread and numerous species of nematodes that parasitize the GI tract in humans and animals, and filariases have proven in some respects to be more difficult to control with chemotherapy. Most prominent among the human infections are those caused by nchocerca volvulus, the agent of river blindness or onchocerciasis, and a group of infections termed the lymphatic filariases (LF), or elephantiasis; the most prevalent parasite in this group is Wuchereria bancrofti, with infections caused by species in the genus Brugia (Brugia malayi and Brugia timori) contributing approximately 10% of cases. Although very large drug-based elimination campaigns have been underway for many years for both kinds of infections and have been remarkably successful at reducing both pathology and transmission [1 3], the number of humans infected remains well over 100,000,000. The vectors for onchocerciasis are black flies in the genus imulium, which breed in river water in tropical areas. LF is transmitted by mosquitoes in several genera, including Anopheles and Culex. In veterinary medicine, the overwhelming majority of attention has been paid to infections of dogs (and to a lesser extent cats) with the heartworm, Dirofilaria immitis. This nematode is also transmitted by mosquitoes (in many genera) and occurs throughout the temperate and tropical regions of the globe [4,5]. Chemoprophylaxis of heartworm infections with macrocyclic lactones (MLs) is a mainstay of veterinary practice in the UA and parts of Europe, and is a prominent contributor to income in the animal health pharmaceutical industry. For this indication, a single monthly dose of one of several MLs is sufficient to kill early larval stages of D. immitis, preventing the development of the adult stages, which are the pathogenic forms [5]. In humans, the prototype ML ivermectin (IVM) is the mainstay of current onchocerciasis control programs and is an essential contributor to LF elimination programmes in onchocerciasis areas when given /ERI Expert Reviews Ltd I

2 Review Geary & Mackenzie once or twice yearly at a dose of 150 µg/kg. In areas in which LF, but not onchocerciasis, is found, diethylcarbamazine (DEC) is used as the primary filaricide at a dose of 6 mg/kg as the citrate salt, again once yearly. Because of the profound adverse events seen when DEC is used in onchocerciasis patients, the drug is contraindicated for this use [2]. Albendazole (standard dose of 400 mg/person) is coadministered with these two agents in the LF elimination programs. Macrocyclic lactones are phenomenally effective and safe drugs for this indication [6]. Their activity is characterized by two remarkable actions: these drugs remove microfilariae from the circulation or skin for an extended period of time (6 9 months in human filariases), and sterilize adult worms for equally long periods after a single dose. As the half-life of IVM in humans and animals is less than 48 h, the basis for this unusually prolonged action is independent of the continued presence of the drug in host fluids. ne possibility is that the drug binds with extremely high affinity to its receptor (effectively irreversibly); if receptor turn-over is very low in adult parasites, recovery from the drug may require a prolonged period [7 9]. A significant deficiency of IVM is the lack of, or only very slowly apparent, lethal activity against adult stages of filarial nematodes (macrofilariae) in vivo [1,2]. Although DEC can be adulticidal in LF, the adverse consequences of a macrofilaricidal regimen preclude this as a routine strategy (see below). As adult filariae can live in the host for at least 10 years (the limit of the lifespan is unknown), the lack of a reliable and safe macrofilaricide means that control programs must be maintained for decades, even in the presence of reduced transmission. Despite the impressive gains made in easing the incidence and pathology of filariases in humans and companion animals, it is unlikely that eradication, or even stable elimination, can be achieved globally without a safe and effective macrofilaricide [10 12]. The need for such a drug is the main driving force for discovery programs in this area. It must be realized that the requirements for a macrofilaricide vary among the filariases; in the case of canine heartworm disease, a nonarsenical compound with a simpler regimen and less toxicity is needed in cases of failed or neglected prophylaxis. For LF, as in heartworm, the adult stage is the cause of pathology, and elimination of these stages is an essential aspect of adequate treatment. In onchocerciasis, the pathogenic stage is the microfilariae (mf), and agents that kill or permanently sterilize the adult stages would act primarily to supplement ongoing control efforts by reducing the duration of treatment needed to achieve elimination. Therapeutic challenges Are macrofilaricidal effects inevitably associated with host pathology? As noted, prospects for successful conclusion of filariasis control and elimination programs in the near- to mid-term would be markedly enhanced by the availability of a safe and effective macrofilaricide. The two most significant drugs available for filarial control both seem to act through immune-mediated mechanisms [13 16], which may contribute to the pathological consequences observed following killing of adult and larval stages. There has historically been concern about the necessity of slowly killing adult stages to avoid these complications, but it has not yet been possible to conclusively determine if these sequelae are due to parasite death per se or to the involvement of host immune responses in the killing; the situation may also differ among the various filariases. DEC has detectable but incomplete macrofilaricidal effects in bancroftian LF, which are accompanied by the development of inflammatory lesions and nodules at the site of adult worm death [17]; this effect is not even observed with intensive treatment with IVM [18]. Whether it reflects a direct or indirect (immunemediated) mechanism is uncertain. uramin, a macrofilaricide no longer in use owing to serious drug toxicity, killed adult worms slowly and did not appear to cause serious adverse effects (AEs) based on worm death [19]. Arguments that killing adult worms en masse would necessarily generate immune-related toxicity (as a sort of anaphylaxis) are inconsistent with the fact that there is continual turn-over (death) of mf and that exposures to even low antigen concentrations, as would be found from dying mf or from slowly dying adult worms, should be enough to trigger significant immune responses. ince a safe, effective macrofilaricide with acute activity in humans is not available, it is not possible on the basis of data to set a bar for rapidity of killing as part of a Target Product Profile; the consequences will have to be determined empirically. onetheless, new drugs that kill adult filariae independently of host immune responses might have added value in this context, even if the immune consequences associated with the presence of dead worms cannot be entirely avoided. In this context, it should be noted that the introduction of large amounts of protein released from the bacterial Wolbachia symbiont following acute adult death could contribute to the adverse events attributed to chemotherapy [20]. owever, the killing of massive numbers of mf within a few days after IVM treatment should also dump large amounts of Wolbachia antigen into the circulation, but this regimen is not generally associated with adverse events. This area warrants additional research in animal models. Can control of. volvulus parasites be safely accomplished in loaisis areas? A major impediment to the eradication of onchocerciasis is the occurrence of AEs, including fatalities, after the administration of IVM to patients coinfected with the filarial parasite Loa loa, particularly those who harbor high loads of L. loa mf [14,21,22]. While L. loa is not considered to be seriously pathogenic and is not currently a target of control programs, it coexists with. volvulus in significant parts of Africa. IVM is microfilaricidal against L. loa and it is thought that this action is responsible for the AEs, although the pathogenic mechanism remains unclear [23]. imple quantitative tests for L. loa are not available, and as a consequence, IVM must be used carefully in loaisis areas. A new agent that can be safely deployed in such areas is urgently needed. Is resistance to MLs a credible threat to control? The final therapeutic challenge in this area is the possible emergence of resistance to the microfilaricidal effects of IVM (and related MLs in heartworm infections). A phenotype of early reappearance of. volvulus mf in the skin of IVM-treated Ghanaian patients has been reported [24,25], and genetic changes in. volvulus populations exposed to IVM pressure are suggestive of drug selection [26]. Proof that the genotypic and phenotypic changes are linked 682 Expert Rev. Anti Infect. Ther. 9(8), (2011)

3 Progress & challenges in the discovery of macrofilaricidal drugs Review is difficult to obtain in light of the minimal response to IVM evident in microfilariae in culture and the lack of an animal model in which putatively resistant parasites can be experimentally characterized (see below). If a resistance genotype is present and leads to abrogation of IVM-induced repression of transmission and/or pathology, an alternative to the current yearly dosing paradigm needs to quickly be identified. The availability of a convenient macrofilaricide, not affected by mechanisms that confer resistance to MLs, would ameliorate the consequences of IVM-resistant parasite populations. Concern has arisen over cases of apparent loss-of-efficacy of ML heartworm preventatives [27]. Genetic and phenotypic investigations of parasites obtained from such cases support the possibility that populations of D. immitis resistant to these drugs have been selected in the UA [28]. Although these observations require confirmation and experimental validation, they suggest that alternatives to standard ML prophylaxis therapy for heartworm prevention may be needed quickly, along with better strategies for curing infected companion animals. Can drugs be developed for case management as well as for mass drug administration (MDA) deployment, or must a new macrofilaricide meet the requirements for both indications? Therapy of extant filariases in developed regions (for instance, in returning tourists, diplomats, soldiers and so on) can be managed with doxycycline therapy. In disease-endemic countries, especially in areas to which it is difficult to deliver medical care, more acute regimens will probably be needed. afety considerations for a drug only used in proven infections, administered under medical supervision, may be less stringent than for a drug meant to be administered to populations without definitive diagnosis. Would a drug unsuited for MDA be of sufficient value to warrant development? Can a macrofilaricide with some toxicity concerns be incorporated into current control strategies? Existing macrofilaricidal drugs uramin (Figure 1), the original macrofilaricide, was proven to be too toxic for use and is no longer ethical or appropriate for this indication. The severe toxicity seems to have been owing to the nonspecific nature of these compounds and not to the killing of filariae per se, although some adverse effects attributable to the killing of filariae were noted [29,30]. The only safe and highly effective macrofilaricidal strategy currently available for humans is the use of antibiotics. Antibiotics that eliminate the Wolbachia bacterial symbiont from adult filariae, especially doxycycline (Figure 2), have useful activity against the major pathogenic species of humans and companion animals. The contribution of the bacterial symbiont to parasite physiology has not been entirely unraveled, but it is clear that removal of these prokaryotic organisms impairs both the reproduction and viability of adult filariae. The magnitude of these effects is dependent on the regimen, especially the duration of treatment, and the species of parasite [31]. Daily doses of doxycycline ( mg) for 4 6 weeks are generally sufficient to permanently sterilize female. volvulus, with significant but not necessarily complete killing of adult worms [32]. As the pathology of onchocerciasis is due to the microfilariae, permanent sterilization is equivalent to removal of the adults. imilar regimens are effective in LF [31], although lethal effects in adults are important in these infections, as the adult stage is the cause of pathogenesis. The same is true for canine heartworm infections, in which prolonged regimens of doxycycline have been shown to sterilize adult worms, but are not associated with full lethality [4]. The addition of IVM to doxycycline is reported to increase adulticidal efficacy in this parasite [33,34]. Importantly, L. loa parasites do not harbor Wolbachia symbionts and are not susceptible to doxycycline; the drug can be used safely in loaisis areas to treat onchocerciasis [32,35]. The challenge is to reduce the duration of therapy needed for efficacy; it does not appear that other available antibiotics provide an advantage in this regard over doxycycline, or that higher doses lead to faster therapeutic resolution. The problem may lie in the apparently sluggish lifestyle of these symbionts; as is seen in TB and leprosy chemotherapy, prolonged dosing may be needed to clear slowly dividing Wolbachia. Doxycycline therapy can certainly be useful in some settings, such as areas of suspected resistance to IVM, but it is not clear how long a regimen can be tolerated in the context of disease elimination programmes. A targeted discovery program focused on evaluating methods to minimize the duration of therapy required, and to test alternative antibiotics and antibiotic strategies to identify more quickly acting agents, is highly worthwhile in this context [101]. Ivermectin (Figure 3) has been shown to kill young D. immitis adults and even established parasites if administered at monthly intervals for prolonged periods in a so-called slow-kill paradigm [36]. owever, even these regimens are incompletely efficacious. There is little evidence for clinically useful acute macrofilaricidal efficacy of IVM in either onchocerciasis [2] or LF [11,12,18,37,38], although it is possible that chronic treatment may shorten the lifespan of adult. volvulus. Diethylcarbamazine (Figure 4) has definite, but incomplete, macrofilaricidal effects in LF [11,17,37], but these are not increased by therapy more intense in dose or duration than the current once-yearly regimen [37]. The killing of adult worms in situ is associated with inflammation and pain in the surrounding tissues, which can be significant. While the addition of a 6-week course of doxycycline to a single dose of DEC enhances the apparent microfilaricidal efficacy and reduces the incidence of adverse events associated with microfilarial killing [20,39,40], no evidence has been presented that this regimen is macrofilaricidal, and it is unlikely that a 6-week course of the antibiotic will be widely adopted for control programs. Albendazole (Figure 5) is combined with DEC or IVM for LF control. There is evidence for some enhanced microfilaricidal effects with the combinations, but the doses of albendazole used in MDA programs do not have clinically significant macrofilaricidal effects [16,37,38], nor do the combinations. Experimental use in much more intensive regimens was reported to have some macrofilaricidal effects in LF, but was associated with adverse inflammatory-related reactions attributed to the killing of adult worms [37]. There is no evidence for clinically useful macrofilaricidal efficacy of albendazole in onchocerciasis [38]

4 Review Geary & Mackenzie - 3 C C C 3 C C C 6a + C Figure 1. uramin. The standard treatment for adult heartworm infections remains the supported use of the arsenical melarsomine (Figure 6) [4,5]. Treatment is generally given in divided doses, sometimes with a period of rest between consecutive courses. upportive care is needed and the treatment is difficult to tolerate in heavily infected or symptomatic animals. ome evidence suggests that an initial clearance of Wolbachia from D. immitis via a course of doxycycline can reduce the pathology associated with worm killing by melarsomine [40]. There is no evidence from human studies that other anthelmintic drugs currently used in human or veterinary medicine have as much therapeutic potential at tolerated doses and regimens as macrofilaricides. Drugs currently in development Although prolonged exposure to MLs is reported to cause a gradual decline in the viability of D. immitis adults [36], conclusive evidence for high adulticidal efficacy of IVM in human filariases has not been reported. IVM is typically not adulticidal in the animal models in which it has been tested. owever, interest has been focused on moxidectin (MX; milbemycin) (Figure 3) as a filaricide. Figure 2. Doxycycline. C 3 C 3 C 3 2 Cl ½ 2 ½C 3 C 2 This ML lacks the oleandrose substituent of the avermectins, but has actions that are generally similar to other drugs in this class. MX has a considerably longer half-life than IVM in mammals [41] and is not as good a substrate for mammalian P-glycoprotein drug pumps as IVM is [42]. In part because of this pharmacological profile, MX has been advanced as a possible macrofilaricide for onchocerciasis [102]. Advancement of the molecule into development was also apparently predicated on unpublished data that are reputed to show macrofilaricidal activity in an animal model, but it is not possible to analyze the potency, efficacy and antifilarial spectrum of action of a drug without access to these data. It should be noted that doses of IVM administered at bi-weekly intervals, a regimen that would be expected to at least partially mimic the more prolonged exposure attainable with MX, failed to show macrofilaricidal activity in humans [19], and repeated high doses of IVM sterilize, but do not kill, adult nchocerca ochengi in cattle [43]. In any case, Phase II trials of MX in onchocerciasis patients are underway, with results of nodule examinations as an index of parasite killing expected in Resistance may also be a driver. As discussed above, recent data show a phenotype in Ghana that can be interpreted as early repopulation of skin by mf. Typically, the reappearance of mf in skin following a single dose of IVM is not evident until approximately 6 months [24], whereas some individuals in a local area in Ghana exhibited mf rebound as early as 3 months post-treatment [24]. Evidence has also recently been generated that suggests the appearance of ML-resistant populations of D. immitis in the Mississippi delta region of the UA [28]. In veterinary applications, resistance to one ML is typically associated with resistance to other members of the class, as measured by shifts to the right in dose- or concentration-response curves [44]. owever, depending on the mechanism of resistance in filariae to MLs, which remains to be rigorously proven, it is possible that therapeutic doses of MX may retain activity against IVM-resistant populations. 684 Expert Rev. Anti Infect. Ther. 9(8), (2011)

5 Progress & challenges in the discovery of macrofilaricidal drugs Review Ivermectin-related severe adverse events in high-microfilaremia loaisis patients seriously complicate elimination strategies (see earlier), and the possibility that adverse events would be less of a concern with MX could also be a factor in its adoption for clinical use. Unfortunately, we lack experimental models capable of accurately predicting the risk of such events or biomarkers that could be used in humans as surrogate indicators of the propensity of MX (or any other microfilaricide) to exhibit severe toxicity in patients bearing high L. loa loads. It should be stressed that activity against filariae was not a prime driver in the selection of ML derivatives for development. Many thousands of MLs were prepared during the expansion of this series following the commercial success of IVM in veterinary medicine, but very few have been subjected to testing in animal models of filariasis. In this regard, it is worth noting that selamectin, for example, was chosen for development at Pfizer on the basis of superior activity against arthropod ectoparasites [45]. It is certainly possible that a ML derivative with superior macrofilaricidal activity could be found in a focused screen if the pharmaceutical companies holding these collections were invited to contribute material for testing. Low hanging fruit The quickest transition to human use of a macrofilaricide can be accomplished with compounds that are already approved for use in other human diseases or in veterinary medicine. Compounds in this category will already have a considerable package of toxicology data, as well as information and expertise in formulation, manufacturing and pharmacology (both basic and clinical), which facilitates a relatively rapid path to clinical trials in human filariasis patients. A B C 3 3 C Figure 3. Macrocyclic lactones. (A) Ivermectin. (B) Moxidectin. C 3 for cats and an oral dosage form for dogs. These products suggest that there is a basic level of mammalian safety and that systemic delivery is possible. To date, registration for use in ruminants has 3 C C 3 C 3 C 3 C 3 Emodepside The prototype cyclic depsipeptide anthelmintic PF1022A (Figure 7) exhibited potent activity against gastrointestinal (GI) nematodes in various animal hosts, but was considerably less potent in ruminants than in monogastric species [46,47]. Efforts to enhance potency of the class for use in livestock (cattle and sheep) led to the development of a (bis)-morpholino derivative, emodepside (Figure 7). Emodepside was introduced into the veterinary market for the treatment and prevention of companion animal GI nematode infections; it is administered as a topical preparation C 3 3 C C 3 Figure 4. Diethylcarbamazine

6 Review Geary & Mackenzie C 3 C 2 C 2 C C 3 C 3 F C 3 Figure 5. Benzimidazoles. (A) Albendazole. (B) Thiabendazole. (C) Mebendazole. (D) Flubendazole. (E) xfendazole. not occurred. In cattle, emodepside is highly efficacious against GI nematodes and the lungworm Dictyocaulus viviparus at single doses of <1 mg/kg [48]. everal pathways for total synthesis of PF1022A have been reported (for a review, see [46]), but it is not apparent that this route can be developed for the production of sufficient amounts of the drug at an acceptable cost-of-goods. owever, much work on the generation of semi-synthetically modified derivatives of the prototype, which is isolated from fermentation of fungus in the genus Rosellinia, has been reported [46,49]. Initial work on the antifilarial activity of PF1022A and emodepside [50] showed that topical and oral dosing in rodent models of filariases had potent and rapid microfilaricidal effects. Macrofilaricidal effects were obtained against some species (Litomosoides sigmodontis) with high doses of emodepside (100 mg/ kg per day 5 days), but the drug was ineffective against B. malayi adults. Even so, single doses of mg/kg produced severe defects in intrauterine microfilariae, which may contribute to the long-lasting suppression of microfilarial counts observed. More recent data document exceptionally potent effects of emodepside against adult nchocerca gutterosa adults in culture (sub-nm 2 2 Figure 6. Melarsomine. As 2 2 concentrations) [51], while it was orders of magnitude less potent against adult B. pahangi in similar experiments. It was also highly potent in a mouse model of nchocerca lienalis mf. These data demonstrate that anthelmintics can distinguish among even closely related filarial species, for unknown reasons. More importantly, they argue that emodepside should be evaluated in animal models of human onchocerciasis, such as the cattle-. ochengi model, to determine whether it should be evaluated in human onchocerciasis. Whether the microfilaricidal activity of this class would preclude the use of emodepside in loaisis areas must be experimentally determined. The potential availability of a toxicology data package submitted for approval for use in companion animals, as well as the associated expertise in formulation, manufacturing and pharmacology, puts emodepside in the top ranks of the low hanging fruit category for further development for use against onchocerciasis. Concerns include the possible microfilaricidal activity of the drug, with potential implications for use in loaisis regions. owever, as we do not know the mechanistic basis for the IVM-related AE, we cannot judge a priori whether any agent that kills mf is equally likely to cause these reactions in patients bearing coinfections. Mechanism of action studies revealed that the most likely target for this drug in nematodes is a member of the Ca 2+ - and voltage-gated K + channel family. In the free-living nematode Caenorhabditis elegans, loss-of-function mutations in the gene encoding the L-1 K + channel generate high-level resistance to the drug [52]. Evidence that emodepside directly gates this channel has not been reported, but the available data strongly support the hypothesis that the anthelmintic action of this class is primarily mediated by effects on L-1. An important consideration for the class of cyclodepsipeptide anthelmintics is that the selection of emodepside for development was not based on activity against adult filariae. Therefore, it would be worthwhile to characterize additional 686 Expert Rev. Anti Infect. Ther. 9(8), (2011)

7 Progress & challenges in the discovery of macrofilaricidal drugs Review analogs in vitro and in animal models to determine whether any others may be a better candidate than emodepside. Flubendazole Benzimidazoles (BZs) have been important components of chemotherapeutic strategies for the control of parasitic nematodes in livestock, companion animals and humans since the introduction of thiabendazole (Figure 5) almost 50 years ago. Currently, albendazole (Figure 5) and mebendazole (Figure 5) are used for GI nematodes in humans; albendazole is also used as an adjunct anthelmintic in LF control programs (see previously), though the evidence that it exerts significant antifilarial effects at the doses used is equivocal [16]. Albendazole is also used for the treatment of cestode infections in humans, including tissue stages, such as neurocysticercosis. For these indications, albendazole is administered at high doses in prolonged courses [53]; it also has some activity against liver flukes (Fasciola hepatica) in ruminants at lower doses. owever, it has not demonstrated significant macrofilaricidal activity in humans at standard therapeutic doses (see above). Whether higher doses, such as those used for systemic helminthiases, could provide useful macrofilaricidal effects in onchocerciasis is unknown. As noted, high doses of albendazole do have activity against adult worms in LF, but these effects are associated with sequelae that will probably preclude the adoption of these regimens for this indication. Flubendazole (Figure 5) is an intriguing candidate macrofilaricide. It is marketed for the treatment of parasitic nematodes in the GI tract of humans, in a formulation that provides very low bioavailability. owever, parenterally administered flubendazole provides very high efficacy against adult filariae of multiple species in several hosts [54], including against. volvulus in humans [55]. Efficacy in this trial was accompanied by serious injection site reactions, precluding the further development of that formulation. In animals, parenteral administration apparently provides a drug depot that generates a prolonged, low concentration exposure to the parasites. Whether equal efficacy can be obtained with oral administration of the drug in a vehicle that provides appreciable bioavailability is unknown, but is of obvious relevance. It has recently been shown that flubendazole prepared in a hydroxypropyl-b-cyclodextrin vehicle was active against a tissue cestode, Echinococcus granulosus, in mice [56]. This provides a starting point for the evaluation of flubendazole in animal filariasis models in an oral dosing regimen that provides much better bioavailability than currently possible. Although the drug is approved for human use, increased systemic exposure associated with a macrofilaricide Figure 7. Cyclic depsipeptides. (A) PF1022. (B) Emodepside. R 2 indication would require a new registration, with the toxicological and potentially manufacturing studies associated with that process. Whether a parenteral dosage form would be tolerated in the field depends on the ability of the drug to kill macrofilariae in a limited dosing regimen; it could be a useful adjunct to end-stage elimination situations and in loaisis areas even if not deliverable in an oral preparation, provided that it is safe. Concerns about toxicity center on the effects of this class of drugs (and all agents that target tubulin as a mechanism of action) as embryotoxins; by interfering with cell division, the anthelmintic benzimidazoles typically cause aneuploidy in cells in culture and fetotoxicity in animal studies. Flubendazole is no different in this regard [57 59], but albendazole, which has similar effects, is used to treat systemic helminthiases in humans, suggesting that this toxicity may not be evident at therapeutic doses or may be manageable by restricting its use in women of child-bearing age. Concerns have also been raised about the possible neurotoxicity of flubendazole. In part, this concern stems from studies carried out on a prodrug of flubendazole, UMF- 078, which was synthesized as part of a campaign to develop flubendazole derivatives that could be delivered orally. This drug was efficacious against adult stages of the bovine parasite. ochengi, phylogenetically related to. volvulus and also residing in nodules, at single intramuscular doses of 150 mg/kg [60]. R

8 Review Geary & Mackenzie Approximately 10% of UMF-078 is converted to flubendazole. eurotoxicity was seen in treated cows and in dogs after multiple treatments with UMF-078, but it is not clear whether this is due to the prodrug or to flubendazole. Parenteral administration of flubendazole in animal models of filariasis has not been associated with neurotoxicity and a toxicity study reported no such effects in mice given 200 mg/kg flubendazole by intraperitoneal injection daily for 14 days [59]. In addition, the fact that flubendazole has been reported to have activity against human neurocysticercosis (40 mg/kg per day for 10 days in two divided doses) without evidence of such neurotoxicity [61] suggests that systemic exposure to this drug per se is not the cause of effects seen with UMF-078. owever, it should also be noted that the LD 50 of flubendazole in mice has been reported to be mg/kg, and some neurotoxicity was observed [103]. Thus, the development of flubendazole will require thorough evaluation of the margin of safety in both areas, although no data that would preclude development have been published. Because of the therapeutic and commercial success of BZs as veterinary anthelmintics, many analogs were prepared by animal health companies; the BZ structural template has also been exploited for other indications and mechanisms of action. owever, no data are available to suggest that any would present therapeutic or pharmaceutical advantages over flubendazole as a macrofilaricide. As noted previously, high-dose, prolonged therapy with albendazole [53], and also flubendazole [61] and oxfendazole (Figure 5) [62], can be used to control at least some tissue helminths; whether either is macrofilaricidal in similar regimens has not been fully evaluated. In this regard, it should be emphasized that the MDA programs associated with LF elimination strategies could generate relevant data on macrofilaricidal effects of albendazole in combination with DEC or IVM if field results were analyzed appropriately. This type of post hoc data should be gathered in an effort to, at the very least, guide end-game protocols. ew anthelmintics itazoxanide itazoxanide (Figure 8) was patented in 1975 as a drug with activity against tapeworms. It has an exceptionally broad spectrum of action, with at least some efficacy against GI protozoa and nematodes, some flatworms and several bacteria and viruses [63]. Its mechanism of action is not known precisely, but it may act as a metabolic poison by collapsing transmembrane proton gradients. This drug, similar to the structurally related closantel and the b-ketoamide class [64], may require a mobile proton for activity, accounting for its extremely broad spectrum of activity. itazoxanide has not been proven to exhibit useful antifilarial activity in vivo [65], although concentrations of approximately 10 ug/ml were active against adult B. malayi in culture; this is in the range observed in humans following oral dosing. The basis for the lack of in vivo activity is unknown, but again may reflect the lack of validated models for macrofilaricide discovery and the possible loss of normal viability when worms are removed from a host for long-term culture (see later). Monepantel Monepantel (Figure 9) is the marketed representative of a new class of anthelmintics termed the aminoacetonitrile derivatives (AADs) [66]. These drugs have potent actions on a class of nicotinic acetylcholine receptors (nachrs) not affected by the classic cholinergic anthelmintics and are very safe and highly efficacious against a broad range of GI and lung nematodes in ruminants [67]. Their activity against filariae has not yet been reported, but preliminary searches of the B. malayi genome suggest that the receptor type that seems to be the primary target of this class may not be present [68]. Derquantel Derquantel (Figure 10), 2-desoxoparaherquamide, is the first marketed member of the paraherquamide/marcfortine class of anthelmintics [69]. Unique among anthelmintics, it is a receptorlevel antagonist at nematode (and mammalian) nachrs. imilar to the AADs, this class exhibits potent and broad-spectrum activity against trichostrongylid nematodes of clinical importance in ruminants [70]. Its potential as a macrofilaricide has not been reported. Tribendimidine Tribendimidine (Figure 11) is under development as an anthelmintic for use against GI nematodes and other helminths of humans [71]. The parent drug is a nachr agonist in nematodes [72] and is metabolized to deacylated amidantel, which itself has potent nematocidal activity [73]. Amidantel and related nicotinic agonist compounds have not been found to exhibit robust macrofilaricidal activity, and the potential of tribendimidine for this indication remains to be demonstrated. Early leads in drug discovery creens of compounds for macrofilaricidal activity, although generally carried out on a small scale and in whole-organism viability platforms, have identified some actives. The field has recently been reviewed [74,75]. The primary problem in this area is the difficulty in translating in vitro actives to in vivo actives, as discussed later. This is particularly the case for hits derived from whole-organism screens of random compound collections; it is quite easy to identify compounds that kill nematodes in culture at a concentration of 10 µm (a standard screening point), but progressing such hits to true leads (activity at nontoxic doses against parasites in an animal) has proven to be remarkably challenging. This has been the experience even in the animal health industry, which has screened millions of compounds and extracts against nematodes, especially C. elegans, in culture, with very few new products to show for the effort [16,76]. It must be acknowledged that at least the prototype of every commercially available anthelmintic, including the newer classes, was discovered in animals infected with parasites. In the macrofilaricide arena, no compound or series of compounds derived from random screening against worms in culture has displayed the combination of potency, efficacy and selectivity in animal models of filariasis to warrant further investment in development. Thus, in the 688 Expert Rev. Anti Infect. Ther. 9(8), (2011)

9 Progress & challenges in the discovery of macrofilaricidal drugs Review judgment of the authors, it is not useful to re-review them here. Indeed, many series first identified in the 1990s remain described as potential leads, but have not fostered additional work [75]. Current discovery paths A strategy for antifilarial drug discovery in the context of the Tropical Diseases Research etwork of the W has been described [77]. Efforts to screen chemical libraries at even a reasonably high throughput are focused on microfilarial stages of. lienalis collected from local cattle skins; compounds active against these organisms in culture are then screened in mice that have been implanted with mf (either. lienalis or. gutterosa), and in jirds infected with Brugia pahangi. Compounds active in the nchocerca microfilariae in vivo test are then assayed against adult stages of. gutterosa (adult males) in culture (and possibly against other adult nchocerca spp. in culture also). Following testing to optimize pharmacological properties and toxicological evaluation, compounds would be suitable for testing in humans. Compounds active in the jird/b. pahangi model would be tested in dogs infected with D. immitis; positives would be processed for pharmacology and toxicology profiling before being advanced to human trials. ther groups have chosen different screening streams and different animal models [54]. The utility of any of these screens has not been rigorously established in comparative studies; again, the lack of a safe and effective acutely dosed macrofilaricide for human use prevents typical validation studies. As a result, there is no standard path for macrofilaricide discovery and development. The situation is complicated by the fact that convenient laboratory animal models do an uncertain (at best) job of replicating the tissue situations of human diseases; meaning that the pathology associated with the different parasites in their particular niches varies greatly across the filariae, and this variation may effect a drug s action on that worm at that site. It would obviously be best if we could study drug effects on adult worms in lymphatic vessels or in nodules, situations not readily attained in most rodent models. igh-throughput screens against adult filariid species in culture, particularly target species, are precluded by the lack of routine availability of species that only grow in humans and the high labor costs associated with recovery of model adult parasites from infected hosts. Alternatives include, as noted, screens against mf, which presume that different developmental stages share the functional biology of essential drug targets. Given the inability to sustain a screen with adult stages, this is the only alternative available for whole-organism based antifilarial screening. In the veterinary arena, C. elegans was used for many years as a surrogate organism for parasites in wholeorganism screens [78,79]. These efforts were largely fruitless; no new anthelmintic class was brought to market based on activity against this organism [80]. Recent data show that the accumulation of xenobiotics in this worm is much more limited than expected based on physical chemical characteristics [81], suggesting that it may not be a faithful mimic of drug permeation into and accumulation in adult nematode parasites. Figure 8. itazoxanide. This finding also suggests that use of C. elegans as a primary anthelmintic screen may have missed important actives [82]; there is every reason to devise new and improved screening methods for this purpose. The adoption of a mechanism-based high-throughput screening strategy for a macrofilaricide remains a viable option, especially in consideration with the limits imposed by organism availability on the ability to screen high numbers of compounds in wholeorganism screens and the potential limits of C. elegans as a model organism for this purpose. Although target-based screens have not delivered candidate antiparasitic drugs in advanced development for any indication, there are valid reasons to believe that such a strategy can be useful in this situation [78,79,83,84]. In the context of adult filariids, it must be noted that very few attempts at targetbased discovery have been reported. The narrower phylogenetic distance embodied in these parasites compared with the spectrum required for veterinary nematocides is another factor that favors increased investment in this area. Finally, the ability to invest chemical and biological resources on compounds with a known mechanism of action as the leads progress through animal models of uncertain predictive value is a strong advantage; one can correlate pharmacokinetic exposure with antiparasitic consequence to decide on the value of continuing or dropping a particular series and target. A significant hurdle to overcome is the selection and validation of macrofilaricide targets. We know little about the biology and physiology of parasitic nematodes compared with vertebrates or prokaryotes [79]. Bioinformatics-based approaches to identifying targets [85 88] can identify proteins that could serve F F CF 3 Figure 9. Monepantel. * + CF

10 Review Geary & Mackenzie in mechanism-based screens, but techniques to validate those targets by RA interference [89] suffer from the lack of general efficacy of this strategy in parasitic species [90] and the fact that this approach only identifies those targets for which loss-of-function (inhibition) impairs worm viability. It cannot mimic the effects of gain-of-function agents (agonists), even though these comprise the majority of available anthelmintics. It cannot reveal agents that disrupt the host parasite interface to favor the host, an action that may underlie the efficacy of IVM against mf [9]. We clearly need additional research on target identification and prioritization in adult filariids to advance the case for high-throughput screening, and community resources may be of great assistance in this effort [86]. Discovery & development challenges As noted, the discovery path to new macrofilaricides is plagued by significant impediments. Prominent among them are: the lack of an existing safe and effective macrofilaricide to use for validating models; the lack of a readily available adult filariid to use in moderately high-throughput whole-organism assays; the inability to assess in vivo activity against the two most important parasites,. volvulus and W. bancrofti in convenient animal models until human trials are completed; the lack of a validated animal model in which harmful consequences of drug action against L. loa can be detected; and a requirement for prolonged post-treatment periods prior to the evaluation of macrofilaricidal efficacy in humans. Lack of convenient animal models for human parasites remains a serious obstacle to antifilarial discovery. either. volvulus nor W. bancrofti can be maintained in animals that Figure desoxoparaherquamide. Figure 11. Tribendimidine. can be used in laboratories for basic drug discovery studies. While primate models for these parasites have been reported for both onchocerciasis and LF [91,92], none is amenable for routine use in early-stage drug discovery. Without safe, selective and effective macrofilaricides (in simple regimens), it is not possible to predict with confidence whether compounds active against adult filariae in culture or even in accessible animal models will be effective against the target species in humans. trategies to address this issue can help to ameliorate concerns (see below), but cannot eliminate the need for postdevelopment validation in human clinical trials. The lack of proven macrofilaricides (preferably several, from distinct classes) limits our ability to validate animal models and a screening path. In this regard, the potential utility of the cattle-. ochengi model should be emphasized. Although this model is not useful for routine screening of actives from preliminary screens, it has been used to characterize the antifilarial actions of many drugs, including antibiotics [93]. A case can be made that this model should be a required gate to advance novel drugs to human testing; a drug that failed to act as a macrofilaricide in this model (assuming adequate drug exposure) perhaps should not be tested in humans. This opinion is based on the extraordinarily long duration of human macrofilaricide trials in onchocerciasis, which precludes facile dose optimization, and the desire to limit the risk of testing new drugs in humans to only those with strong evidence to support an expectation of efficacy. Loaisis is not easily modeled outside of a baboon; this parasite has no convenient laboratory hosts. A major impediment to continental elimination in Africa is the large area in which coinfections with L. loa and. volvulus occur; AEs after IVM administration in some individuals who harbored high levels of L. loa mf have made ML-based MDAs inadvisable in these areas. As we remain ignorant of the pathological basis for these AEs, it is not possible to predict the likelihood of their occurrence with a new drug. It would be highly desirable to have an animal model in which the propensity of a new macrofilaricide to cause Loa-related pathology could be evaluated. In its absence, the safest way forward is to develop a macrofilaricide that is devoid of microfilaricidal activity. Determining whether a new compound has efficacy against adult filariae in naturally infected human populations is not a simple task. The assessment of activity against adult. volvulus is currently determined by nodulectomy and histological evaluation of worm sections up to 18 months post-treatment (although timepoints at 3 and 6 months may be included, with efficacy being detected sooner). This makes optimization of dose and treatment schedules a time-consuming process. This strategy was validated to some extent through the use of suramin, an old macrofilaricide no longer used owing to unacceptable toxicity. Efficacy against adult LF parasites is even harder to detect, as surgical removal of adults from lymph vessels for direct evaluation of viability is not a routine procedure. The current standard is to image the motility of adult parasites in lymph vessel nests by ultrasound (the worm dance sign). owever, in the absence of a proven macrofilaricide, this measurement has not been rigorously 690 Expert Rev. Anti Infect. Ther. 9(8), (2011)

11 Progress & challenges in the discovery of macrofilaricidal drugs Review validated as a surrogate for direct and timely detection of dead worms. An area of considerable need for both indications is thus a parasite molecule that could serve as a biomarker to track the presence and abundance of viable adults; while antigen tests for these infections are available, they have not been developed for this application. In this regard, proteins shown to be secreted by adult B. malayi in culture, but not by mf, might be of interest [94 96]. This kind of surrogate marker would greatly facilitate the development of new macrofilaricides and clinical comparison of drugs and regimens to optimize therapeutic use. Ways forward Primary screening of chemical libraries remains an area of concern. The relatively low throughput tolerated in whole nematode screens that employ parasites means that compound collections should be selected prior to assay; selection can be based on activity against related organisms (other nematodes, helminths or invertebrates) or on the basis of mechanism of action (e.g., collections of kinase inhibitors or G protein-coupled receptor ligands) [76,84,97 99]. This strategy enables follow-up testing in labor-intensive secondary screens because of the intrinsic chemical-pharmacological merit embodied in selected libraries compared with truly random libraries. Target-based approaches, though unable to bear fruit for some time owing to the need for intensive investment in development, should be accelerated. A consensus path for development in animal models is also needed. Given the dearth of effective macrofilaricidal molecules, a candidate should be evaluated in as many animal models as possible. Information on exposure (pharmacokinetics) and efficacy should be obtained to allow correlation of these parameters. Coupled with in vitro experiments to determine intrinsic potency, these data can be used to characterize cross-species variation in sensitivity and may direct development for one or all types of filariases. We are still in the learning phase with regard to macrofilaricide discovery and development, and need as much data as can be generated to guide future advances. Expert commentary & five-year view Mass drug administration programs are well underway for onchocerciasis and lymphatic filariasis and will continue to provide marked reductions in transmission and pathology of these infections in many areas. Goals for incidence reduction are unlikely to be globally reached within 5 years to a level that will allow cessation of MDA campaigns, especially in regions with concurrent infections with. volvulus and L. loa. In addition, the prevalence of these infections, especially onchocerciasis, in conflict regions and among migrating populations complicates efforts to attain global control. Although the benefits of IVM and DEC in controlling microfilarial levels in onchocerciasis and LF are of enormous value, the antifilarial armamentarium has undeniable gaps. Discovery of new macrofilaricidal drugs has been hampered by the lower priority assigned to this indication among the many rampant neglected tropical diseases and by the lack of a major market for such a drug in the animal health industry. The development path is also a challenge, as human clinical trials have end points that typically require month monitoring in what can be challenging circumstances. The lack of safe, acutely effective macrofilaricides has made validation of in vitro and in vivo screening systems difficult, and there is no universally accepted pathway for discovery and development of these compounds. Furthermore, it is not evident that the anatomical distribution and basic biology of the pathogens underlying onchocerciasis and LF are sufficiently conserved to permit a simple screening approach for drugs that would be useful for both indications. As current global efforts to control LF transmission may allow the eradication or elimination of this disease without introduction of a new macrofilaricide, most of the emphasis is now placed on onchocerciasis, which is unfortunately more difficult to accurately model in laboratory animals. The proven value of relatively prolonged courses of antibiotics, such as doxycycline, in sterilizing and killing adult filariids that harbor Wolbachia symbionts is a notable step forward. Antibiotic regimens can assist in the treatment of individual patients, or for control of locally important infections (potential IVM resistance or re-emergence in a previously cleared zone), but it is not clear that extended daily dosing is compatible with routine and broad field use in control operations. The historical path for the introduction of a new anthelmintic for humans is via transfer of approved veterinary drugs. uch drugs bring to the human development process a dossier that includes some relevant toxicology, formulation and manufacturing data. Three drugs from this source are of interest in this regard. The macrofilaricidal potential of moxidectin in onchocerciasis will soon be reported and a decision will have to be made whether to incorporate it into control programs based on this activity or its efficacy as a prolonged-duration microfilaricide. It is unlikely that another new drug will have progressed into registration as a macrofilaricide within the next 5 years, given that none is currently in active development. It is possible that flubendazole, a benzimidazole currently approved for the treatment of GI nematode infections in humans and some animal species, could be developed as a macrofilaricide. This drug has shown efficacy as a macrofilaricide in onchocerciasis in a single human trial, although it was administered via a parenteral route in that case. The approved formulation for human use provides almost no systemic bioavailability, and development work will require careful pharmacodynamic and toxicodynamic analyses. It is also possible that the semi-synthetic cyclic depsipeptide emodepside could be in advanced trials for a macrofilaricide indication within the next 5 years. This drug is approved for oral administration to dogs for the control of GI nematodes and has been shown to be extremely potent against adult nchocerca spp. in culture. Whether it will be sufficiently safe and affordable for use in onchocerciasis patients remains to be seen, but it represents an attractive opportunity for introduction into human practice that is not matched at this point by any other veterinary anthelmintic. Beyond these agents, a small number of earlier-stage candidates warrant additional investigation, and new efforts are needed to enable whole-organism and mechanism-based screening approaches to the discovery of new macrofilaricides. ow these efforts will 691