Onchocerciasis Drug Discovery: In Vitro Evaluation of FDA-Approved Drugs against Onchocerca gutturosa in Gambia.

Onchocerciasis treatment and control relies mainly on the use of ivermectin which has high activity against the microfilarial stage of Onchocerca volvulus but limited activity against the long-lived, tissue dwelling adult nematodes. As this neglected tropical disease has now been targeted for elimination, there is an urgent need for new drugs to combat these parasites, ideally with macrofilaricidal activity. In this study, we have examined the anti-Onchocerca activity of a range of existing FDA-approved drugs with a view to repurposing, which can lead to rapid and relatively inexpensive development. From the Pharmakon-1600 library, 106 drugs were selected and tested against O. gutturosa adult male parasites using a concentration of 1.25 × 10-5 M in an in vitro 5-day standard assay to assess motility and viability (using MTT/formazan colorimetry). The findings revealed that 44 drugs produced marginal/moderate activity (50-99% motility and/or MTT reductions) including cefuroxime sodium, methenamine, primaquine phosphate and rivastigmine tartrate, while 23 drugs produced good activity (100% motility reductions and significant MTT reductions), including atovaquone, isradipine, losartan, rifaximin, cefaclor and pyrantel pamoate. Although this study represents only a first step, some of the identified hits indicate there are potential anti-Onchocerca drug candidates worthy of further investigation.

Discovery of Substituted Di(pyridin-2-yl)-1,2,4-thiadiazol-5-amines as Novel Macrofilaricidal Compounds for the Treatment of Human Filarial Infections.

Filarial diseases, including lymphatic filariasis and onchocerciasis, are considered among the most devastating of all tropical diseases, affecting about 145 million people worldwide. Efforts to control and eliminate onchocerciasis are impeded by a lack of effective treatments that target the adult filarial stage. Herein, we describe the discovery of a series of substituted di(pyridin-2-yl)-1,2,4-thiadiazol-5-amines as novel macrofilaricides for the treatment of human filarial infections.

Filarial nematode phenotypic screening cascade to identify compounds with anti-parasitic activity for drug discovery optimization.

Filarial diseases, including lymphatic filariasis and onchocerciasis, are considered among the most devastating of all tropical diseases, affecting over 86 million people worldwide. To control and more rapidly eliminate onchocerciasis requires treatments that target the adult stage of the parasite. Drug discovery efforts are challenged by the lack of preclinical animal models using the human-pathogenic filariae, requiring the use of surrogate parasites for Onchocerca volvulus for both ex vivo and in vivo evaluation. Herein, we describe a platform utilizing phenotypic ex vivo assays consisting of the free-living nematode Caenorhabditis elegans, microfilariae and adult filariae of the bovine filariae Onchocerca lienalis and Onchocerca gutturosa, respectively, as well as microfilariae and adult filariae of the feline filariae Brugia pahangi, the rodent filariae Litomosoides sigmodontis and the human-pathogenic filariae Brugia malayi to assess activity across various surrogate parasites. Utilization of those surrogate nematodes for phenotypic ex vivo assays in order to assess activity across various parasites led to the successful establishment of a screening cascade and identification of multiple compounds with potential macrofilaricidal activity and desirable physicochemical, MW = 200-400 and low lipophilicity, logP <4, and pharmacokinetic properties, rat and human liver S9 stability of ≥70% remaining at 60 min, and AUC exposures above 3 µM h. This platform demonstrated the successful establishment of a screening cascade which resulted in the discovery of potential novel macrofilaricidal compounds for futher drug discovery lead optimization efforts. This screening cascade identified two distinct chemical series wherein one compound produced a significant 68% reduction of adult Litomosoides sigmodontis in the mouse model. Successful demonstration of efficacy prompted lead optimization medicinal chemistry efforts for this novel series.

Evaluation of the in vitro susceptibility of various filarial nematodes to emodepside.

Filariae are vector-borne nematodes responsible for an enormous burden of disease. Human lymphatic filariasis, caused by Wuchereria bancrofti, Brugia malayi, and Brugia timori, and onchocerciasis (caused by Onchocerca volvulus) are neglected parasitic diseases of major public health significance in tropical regions. To date, therapeutic efforts to eliminate human filariasis have been hampered by the lack of a drug with sufficient macrofilaricidal and/or long-term sterilizing effects that is suitable for use in mass drug administration (MDA) programs, particularly in areas co-endemic with Loa loa, the causative agent of loiasis. Emodepside, a semi-synthetic cyclooctadepsipeptide, has been shown to have broad-spectrum efficacy against gastrointestinal nematodes in a variety of mammalian hosts, and has been approved as an active ingredient in dewormers for cats and dogs. This paper evaluates, compares (where appropriate) and summarizes the in vitro effects of emodepside against a range of filarial nematodes at various developmental stages. Emodepside inhibited the motility of all tested stages of filariae frequently used as surrogate species for preclinical investigations (Acanthocheilonema viteae, Brugia pahangi, Litomosoides sigmodontis, Onchocerca gutturosa, and Onchocerca lienalis), human-pathogenic filariae (B. malayi) and filariae of veterinary importance (Dirofilaria immitis) in a concentration-dependent manner. While motility of all filariae was inhibited, both stage- and species-specific differences were observed. However, whether these differences were detected because of stage- and/or species-specific factors or as a consequence of variations in protocol parameters among the participating laboratories (such as purification of the parasites, read-out units, composition of media, incubation conditions, duration of incubation etc.) remains unclear. This study, however, clearly shows that emodepside demonstrates broad-spectrum in vitro activity against filarial nematode species across different genera and can therefore be validated as a promising candidate for the treatment of human filariases, including onchocerciasis and lymphatic filariasis.

Development of emodepside as a possible adulticidal treatment for human onchocerciasis-The fruit of a successful industrial-academic collaboration.

Current mass drug administration (MDA) programs for the treatment of human river blindness (onchocerciasis) caused by the filarial worm Onchocerca volvulus rely on ivermectin, an anthelmintic originally developed for animal health. These treatments are primarily directed against migrating microfilariae and also suppress fecundity for several months, but fail to eliminate adult O. volvulus. Therefore, elimination programs need time frames of decades, well exceeding the life span of adult worms. The situation is worsened by decreased ivermectin efficacy after long-term therapy. To improve treatment options against onchocerciasis, a drug development candidate should ideally kill or irreversibly sterilize adult worms. Emodepside is a broad-spectrum anthelmintic used for the treatment of parasitic nematodes in cats and dogs (Profender and Procox). Our current knowledge of the pharmacology of emodepside is the result of more than 2 decades of intensive collaborative research between academia and the pharmaceutical industry. Emodepside has a novel mode of action with a broad spectrum of activity, including against extraintestinal nematode stages such as migrating larvae or macrofilariae. Therefore, emodepside is considered to be among the most promising candidates for evaluation as an adulticide treatment against onchocerciasis. Consequently, in 2014, Bayer and the Drugs for Neglected Diseases initiative (DNDi) started a collaboration to develop emodepside for the treatment of patients suffering from the disease. Macrofilaricidal activity has been demonstrated in various models, including Onchocerca ochengi in cattle, the parasite most closely related to O. volvulus. Emodepside has now successfully passed Phase I clinical trials, and a Phase II study is planned. This Bayer-DNDi partnership is an outstanding example of "One World Health," in which experience gained in veterinary science and drug development is translated to human health and leads to improved tools to combat neglected tropical diseases (NTDs) and shorten development pathways and timelines in an otherwise neglected area.

Oxfendazole mediates macrofilaricidal efficacy against the filarial nematode Litomosoides sigmodontis in vivo and inhibits Onchocerca spec. motility in vitro.

A major impediment to eliminate lymphatic filariasis and onchocerciasis is the lack of effective short-course macrofilaricidal drugs or regimens that are proven to be safe for both infections. In this study we tested oxfendazole, an anthelmintic shown to be well tolerated in phase 1 clinical trials. In vitro, oxfendazole exhibited modest to marginal motility inhibition of adult worms of Onchocerca gutturosa, pre-adult worms of Onchocerca volvulus and Onchocerca lienalis microfilariae. In vivo, five days of oral treatments provided sterile cure with up to 100% macrofilaricidal efficacy in the murine Litomosoides sigmodontis model of filariasis. In addition, 10 days of oral treatments with oxfendazole inhibited filarial embryogenesis in patent L. sigmodontis-infected jirds and subsequently led to a protracted but complete clearance of microfilaremia. The macrofilaricidal effect observed in vivo was selective, as treatment with oxfendazole of microfilariae-injected naïve mice was ineffective. Based on pharmacokinetic analysis, the driver of efficacy is the maintenance of a minimal efficacious concentration of approximately 100 ng/ml (based on subcutaneous treatment at 25 mg/kg in mice). From animal models, the human efficacious dose is predicted to range from 1.5 to 4.1 mg/kg. Such a dose has already been proven to be safe in phase 1 clinical trials. Oxfendazole therefore has potential to be efficacious for treatment of human filariasis without causing adverse reactions due to drug-induced microfilariae killing.

Whence river blindness? The domestication of mammals and host-parasite co-evolution in the nematode genus Onchocerca.

The genus Onchocerca includes 34 described species and represents one of the largest genera of the filarial nematodes within the family Onchocercidae. Representative members of this genus are mainly parasites of ungulates, with some exceptions such as Onchocerca lupi and Onchocerca volvulus, infecting carnivores and/or humans. For a long time, the evolutionary relationships amongst onchocercids remained poorly studied, as the systematics of this genus was impaired by the high morphological variability of species included in the taxon. Although some molecular phylogenies were developed, these studies were mainly focused on bovine Onchocerca spp. and O. volvulus, including assessments of Wolbachia endosymbionts. In the present study, we analysed 13 Onchocerca spp. from a larger host spectrum using a panel of seven different genes. Analysis of the coxI marker supports its usefulness for the identification of species within the genus. The evolutionary history of the genus has been herein revised by multi-gene phylogenies, presenting three strongly supported clades of Onchocerca spp. Analyses of co-evolutionary scenarios between Onchocerca and their vertebrate hosts underline the effect of domestication on Onchocerca speciation. Our study indicates that a host switch event occurred between Bovidae, Canidae and humans. Cophylogenetic analyses between Onchocerca and the endosymbiotic bacterium Wolbachia indicate the strongest co-evolutionary pattern ever registered within the filarial nematodes. Finally, this dataset indicates that the clade composed by O. lupi, Onchocerca gutturosa, Onchocerca lienalis, Onchocerca ochengi and O. volvulus derived from recent speciation.

Repurposing of approved drugs from the human pharmacopoeia to target Wolbachia endosymbionts of onchocerciasis and lymphatic filariasis.

Lymphatic filariasis and onchocerciasis are debilitating diseases caused by parasitic filarial nematodes infecting around 150 million people throughout the tropics with more than 1.5 billion at risk. As with other neglected tropical diseases, classical drug-discovery and development is lacking and a 50 year programme of macrofilaricidal discovery failed to deliver a drug which can be used as a public health tool. Recently, antibiotic targeting of filarial Wolbachia, an essential bacterial symbiont, has provided a novel drug treatment for filariasis with macrofilaricidal activity, although the current gold-standard, doxycycline, is unsuitable for use in mass drug administration (MDA). The anti-Wolbachia (A·WOL) Consortium aims to identify novel anti-Wolbachia drugs, compounds or combinations that are suitable for use in MDA. Development of a Wolbachia cell-based assay has enabled the screening of the approved human drug-pharmacopoeia (∼2600 drugs) for a potential repurposing. This screening strategy has revealed that approved drugs from various classes show significant bacterial load reduction equal to or superior to the gold-standard doxycycline, with 69 orally available hits from different drug categories being identified. Based on our defined hit criteria, 15 compounds were then selectively screened in a Litomosoides sigmodontis mouse model, 4 of which were active. These came from the tetracycline, fluoroquinolone and rifamycin classes. This strategy of repurposing approved drugs is a promising development in the goal of finding a novel treatment against filariasis and could also be a strategy applicable for other neglected tropical diseases.

Characterization of the Ca2+-gated and voltage-dependent K+-channel Slo-1 of nematodes and its interaction with emodepside.

The cyclooctadepsipeptide emodepside and its parent compound PF1022A are broad-spectrum nematicidal drugs which are able to eliminate nematodes resistant to other anthelmintics. The mode of action of cyclooctadepsipeptides is only partially understood, but involves the latrophilin Lat-1 receptor and the voltage- and calcium-activated potassium channel Slo-1. Genetic evidence suggests that emodepside exerts its anthelmintic activity predominantly through Slo-1. Indeed, slo-1 deficient Caenorhabditis elegans strains are completely emodepside resistant. However, direct effects of emodepside on Slo-1 have not been reported and these channels have only been characterized for C. elegans and related Strongylida. Molecular and bioinformatic analyses identified full-length Slo-1 cDNAs of Ascaris suum, Parascaris equorum, Toxocara canis, Dirofilaria immitis, Brugia malayi, Onchocerca gutturosa and Strongyloides ratti. Two paralogs were identified in the trichocephalids Trichuris muris, Trichuris suis and Trichinella spiralis. Several splice variants encoding truncated channels were identified in Trichuris spp. Slo-1 channels of trichocephalids form a monophyletic group, showing that duplication occurred after the divergence of Enoplea and Chromadorea. To explore the function of a representative protein, C. elegans Slo-1a was expressed in Xenopus laevis oocytes and studied in electrophysiological (voltage-clamp) experiments. Incubation of oocytes with 1-10 µM emodepside caused significantly increased currents over a wide range of step potentials in the absence of experimentally increased intracellular Ca2+, suggesting that emodepside directly opens C. elegans Slo-1a. Emodepside wash-out did not reverse the effect and the Slo-1 inhibitor verruculogen was only effective when applied before, but not after, emodepside. The identification of several splice variants and paralogs in some parasitic nematodes suggests that there are substantial differences in channel properties among species. Most importantly, this study showed for the first time that emodepside directly opens a Slo-1 channel, significantly improving the understanding of the mode of action of this drug class.

Anti-Wolbachia drug discovery and development: safe macrofilaricides for onchocerciasis and lymphatic filariasis.

Anti-Wolbachia therapy delivers safe macrofilaricidal activity with superior therapeutic outcomes compared to all standard anti-filarial treatments, with the added benefit of substantial improvements in clinical pathology. These outcomes can be achieved, in principle, with existing registered drugs, e.g. doxycycline, that are affordable, available to endemic communities and have well known, albeit population-limiting, safety profiles. The key barriers to using doxycycline as an mass drug administration (MDA) strategy for widespread community-based control are the logistics of a relatively lengthy course of treatment (4-6 weeks) and contraindications in children under eight years and pregnancy. Therefore, the primary goal of the anti-Wolbachia (A·WOL) consortium is to find drugs and regimens that reduce the period of treatment from weeks to days (7 days or less), and to find drugs which would be safe in excluded target populations (pregnancy and children). A secondary goal is to refine regimens of existing antibiotics suitable for a more restricted use, prior to the availability of a regimen that is compatible with MDA usage. For example, for use in the event of the emergence of drug-resistance, in individuals with high loiasis co-infection and at risk of severe adverse events (SAE) to ivermectin, or in post-MDA 'endgame scenarios', where test and treat strategies become more cost effective and deliverable.

Integrated dataset of screening hits against multiple neglected disease pathogens.

New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach.

Challenges in drug discovery for novel antifilarials.

Control programmes are at present focused on the elimination of onchocerciasis and lymphatic filariasis as public health problems in countries where they are endemic. The availability of effective drugs used in combination (diethylcarbamazine, albendazole and ivermectin) has paved the way for the implementation of Mass Drug Administration (MDA) campaigns. Considerable progress in the implementation of MDA programmes had led to significant reductions in transmission and morbidity. However, new drugs are needed to overcome the threat of resistance to existing microfilaricides as well as to identify new macrofilaricides. This paper discusses the existing screening tools available for antifilarial drug discovery and efforts towards optimising their use through the Helminth Drug Initiative.

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro.

BACKGROUND: The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay. METHODS: Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared. RESULTS: Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 x 10(-5) M) was the most effective anti-mycobacterial agent; clofazimine (1.25 x 10(-5) M and 3.13 x 10(-6) M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 x 10(-5) M) was filaricidal, but minocycline was more effective and at a lower concentration (5 x 10(-5) M and 1.25 x 10(-5) M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline. CONCLUSION: The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode.