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Twenty diseases are recognized as neglected tropical diseases (NTDs) by World Health Assembly resolutions, including human filarial diseases. The end of NTDs is embedded within the Sustainable Development Goals for 2030, under target 3.3. Onchocerciasis afflicts approximately 20.9 million people worldwide with > 90% of those infected residing in Africa. Control programs have made tremendous efforts in the management of onchocerciasis by mass drug administration and aerial larviciding; however, disease elimination is not yet achieved. In the new WHO roadmap, it is recognized that new drugs or drug regimens that kill or permanently sterilize adult filarial worms would significantly improve elimination timelines and accelerate the achievement of the program goal of disease elimination. Drug development is, however, handicapped by high attrition rates, and many promising molecules fail in preclinical development or in subsequent toxicological, safety and efficacy testing; thus, research and development (R&D) costs are, in aggregate, very high. Drug discovery and development for NTDs is largely driven by unmet medical needs put forward by the global health community; the area is underfunded and since no high return on investment is possible, there is no dedicated drug development pipeline for human filariasis. Repurposing existing drugs is one approach to filling the drug development pipeline for human filariasis. The high cost and slow pace of discovery and development of new drugs has led to the repurposing of “old” drugs, as this is more cost-effective and allows development timelines to be shortened. However, even if a drug is marketed for a human or veterinary indication, the safety margin and dosing regimen will need to be re-evaluated to determine the risk in humans. Drug repurposing is a promising approach to enlarging the pool of active molecules in the drug development pipeline. Another consideration when providing new treatment options is the use of combinations, which is not addressed in this review. We here summarize recent advances in the late preclinical or early clinical stage in the search for a potent macrofilaricide, including drugs against the nematode and against its endosymbiont, Wolbachia pipientis.

The neglected tropical disease onchocerciasis, or river blindness, is caused by infection with the filarial nematode Onchocerca volvulus. Current estimates indicate that 17 million people are infected worldwide, the majority of them living in Africa. Today there are no non-invasive tests available that can detect ongoing infection, and that can be used for effective monitoring of elimination programs. In addition, to enable pharmacodynamic studies with novel macrofilaricide drug candidates, surrogate endpoints and efficacy biomarkers are needed but are non-existent. We describe the use of a multimodal untargeted mass spectrometry-based approach (metabolomics and lipidomics) to identify onchocerciasis-associated metabolites in urine and plasma, and of specific lipid features in plasma of infected individuals (O. volvulus infected cases: 68 individuals with palpable nodules; lymphatic filariasis cases: 8 individuals; non-endemic controls: 20 individuals). This work resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine (CCG) as biomarker for O. volvulus. During the targeted validation study, metabolite-specific cutoffs were determined (inosine: 34.2 ng/ml; hypoxanthine: 1380 ng/ml; CCG: 29.7 ng/ml) and sensitivity and specificity profiles were established. Subsequent evaluation of these biomarkers in a non-endemic population from a different geographical region invalidated the urine metabolite CCG as biomarker for O. volvulus. The plasma metabolites inosine and hypoxanthine were confirmed as biomarkers for filarial infection. With the availability of targeted LC-MS procedures, the full potential of these 2 biomarkers in macrofilaricide clinical trials, MDA efficacy surveys, and epidemiological transmission studies can be investigated., Author summary Today’s diagnosis of infection with the filarial parasite Onchocerca volvulus mainly depends on the microscopic analysis of skin biopsies and serological testing. The work presented here describes the use of multiple mass spectrometry-based screening methods (metabolomics and lipidomics) to search for biomarkers indicative of infection with Onchocerca volvulus. This resulted in the identification of elevated concentrations of the plasma metabolites inosine and hypoxanthine as biomarkers for filarial infection, and of the urine metabolite cis-cinnamoylglycine as biomarker for O. volvulus. Further evaluation of these biomarkers in a geographically distinct non-endemic population however invalidated the use of urine cis-cinnamoylglycine. These findings are of utmost importance as it not only opens new avenues in the development of non-invasive diagnostic tools for filarial infections, but also emphasizes the need for evaluation and validation of newly discovered biomarkers in different populations from different geographies.

Background Current treatment options for onchocerciasis are sub-optimal, prompting research and development of a safe cure (macrofilaricide). Onchocerca ochengi, a parasite of cattle, is used as a close surrogate for the human parasite O. volvulus in a murine model for pre-clinical screening of macrofilaricides. Skin from naturally infected cattle have been used in previous studies as a reliable source of parasite material. However, there is limited knowledge on how source-related factors such as the microfilaridermia status of the cattle, the nodule load and nodular worm viability may affect survival of male O. ochengi worms implanted in the rodent hosts. Such relationships were investigated in this study. Methods Dermal tissue and nodules were obtained from Gudali cattle, dissected and cultured to obtain migrating microfilariae (mf) and male worms. Emerged male worms were implanted into SCID mice and Gerbils (Meriones unguiculatus) and recovery rates were determined upon 42 days post implantation. Finally, nodules were processed for histology and embryogram analyses to assess the nodular worm viability and fertility, respectively. Results Of the 69 cattle sampled, 24 (34.8%) were mf+ and 45 (65.2%) were mf–. The mean nodule loads were 180.5 ± 117.7 (mf+) and 110.6 ± 102.7 (mf-) (p = 0.0186). The mean male worm harvest from nodules were 76.8 ± 120.3 and 47.2 ± 33.4 (p = 0.2488) for mf+ and mf– cattle, respectively. The number of male worms per 100 nodules were 57/100 and 46/100 nodules for mf+ and mf– cows, respectively. Female worms from nodules of mf– cows had higher counts of both normal and abnormal embryos with higher proportions of dead nodular worms evinced by histology compared to those from mf+ cows. A total of 651 worms were implanted into mice and gerbils, out of which 129 (19.81%) were recovered. Logistic regression analysis indicated that the microfilaridermia status of the cattle (presence of mf) (OR = 4.3319; P = 0.001) is the single most important predictor of the success of male worm recovery after implantation into rodents. Conclusion Microfilaridermic cattle provide a promising source of adult O. ochengi. Male worms from this group of cattle have a better success rate of survival in a murine implant model. Nevertheless, in the programmatic point of view, amicrofilaridermic Gudali cattle would still constitute an important source of O. ochengi male worms with relatively good viability after implantation into rodents., Graphical abstract Image 1, Highlights • There is a need of developing safe macrofilaricide for human onchocerciasis using cattle Onchocerca ochengi. • Skin from naturally infected cattle have been used as a reliable parasite source for development of murine pre-clinical screening models. • The role of source-related factors on O. ochengi production and the success of pre-clinical screening models is assessed. • Worms from positive microfilaridermic cattle are successfully recovered after their implantation into rodents.

While flubendazole has been used as a macrofilaricide in humans and animals for some 40 years, work in vitro and in preclinical models over the last decade has suggested its potential use as an anticancer agent. This article reviews recent studies in a range of tumor types indicating novel functions for flubendazole in its control of processes associated with tumor growth, spread and renewal including ferroptosis, autophagy, cancer stem-like cell killing and suppression of intratumoral myeloid-derived suppressor cell accumulation and programmed cell death protein 1. Flubendazole’s potential use in clinical oncology will require further understanding of its mechanistic roles, range of inhibition of cancer types, capacity for adjunctive therapy and possible reformulation for enhanced solubility, bioavailability and potency.

Onchocerciasis (river blindness), caused by the filarial worm Onchocerca volvulus, is a neglected tropical disease mostly affecting sub-Saharan Africa and is responsible for >1.3 million years lived with disability. Current control relies almost entirely on ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. Here, we evaluated emodepside, a semi-synthetic cyclooctadepsipeptide registered for deworming applications in companion animals, for activity against adult filariae (i.e., as a macrofilaricide). We demonstrate the equivalence of emodepside activity on SLO-1 potassium channels in Onchocerca volvulus and Onchocerca ochengi, its sister species from cattle. Evaluation of emodepside in cattle as single or 7-day treatments at two doses (0.15 and 0.75 mg/kg) revealed rapid activity against microfilariae, prolonged suppression of female worm fecundity, and macrofilaricidal effects by 18 months post treatment. The drug was well tolerated, causing only transiently increased blood glucose. Female adult worms were mostly paralyzed; however, some retained metabolic activity even in the multiple high-dose group. These data support ongoing clinical development of emodepside to treat river blindness., Author summary Onchocerciasis (river blindness), caused by the parasitic worm Onchocerca volvulus, is a devastating neglected tropical disease affecting sub-Saharan Africa with an overall impact of >1.3 million years lived with disability. Current control relies mainly on a single drug, ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. The identification of a drug that can safely eliminate adult worms (i.e., a macrofilaricide) is a major research objective for onchocerciasis. We evaluated the anthelminthic activity of emodepside, a veterinary wormer, in cattle infected with a close relative of O. volvulus (Onchocerca ochengi) before conducting pharmacokinetic modelling to estimate drug distribution in humans. Emodepside as single or 7-day treatments at two doses produced rapid activity against O. ochengi microfilariae, prolonged suppression of female worm fecundity, and paralysed most female worms by 18 months, although some remained metabolically active even in the multiple high-dose treatment group. The drug was well tolerated, causing only transiently increased blood glucose. Thus, emodepside shows slow but significant efficacy against adult O. ochengi in naturally infected cattle, meeting the criteria for a safe macrofilaricidal drug. Our data support the ongoing clinical development of emodepside for the treatment of human onchocerciasis.

Background: Lymphatic filariasis (LF) is a parasitic disease that causes permanent disability (elephantiasis). Currently used antifilarial drugs are failing to control LF and there is resurgence in some areas. Looking for new antifilarial leads, we found that Calotropis procera plant parts have been used in traditional medicine for alleviating elephantiasis but the antifilarial activity is not known. Objective: In the present study, the antifilarial activity of ethanolic extract (A001) and its hexane fraction (F001) of C. procera flowers was investigated using the human filarial parasite Brugia malayi. Method: A001 and F001 were tested for antifilarial activity using motility and 3-(4,5-dimethylthiazol-2- yl)-2,5 diphenyltetrazolium bromide (MTT) assays (in vitro) and in the rodent models B. malayi- Meriones unguiculatus and B. malayi-Mastomys coucha. In the rodent models, A001 and F001 were administered orally for 5 consecutive days, and the adult worm burden and course of microfilaraemia were determined. Results: Both A001 and F001 showed microfilaricidal and macrofilaricidal activity in vitro. In animal models, A001 killed ~49-54% adult worms. In M. coucha model, F001 killed 12-60% adult worms in a dose (125-500 mg/kg) dependent manner; A001 and F001 suppressed microfilaraemia till days 91 and 35 post initiation of treatment, respectively. HPTLC revealed 0.61% lupeol, 0.50% β-sitosterol and 1.50% triacontanol in F001. Conclusion: Flowers of C. procera have definite microfilaricidal and macrofilaricidal activities. Whether this activity is due to lupeol, β-sitosterol and triacontanol found in the hexane fraction remains to be investigated. This is the first report on the antifilarial efficacy of flowers of the plant C. procera.

Flubendazole (FLBZ) is a potent and efficacious macrofilaricide after parenteral administration. Studies in animal models and one trial in patients infected with Onchocerca volvulus revealed that FLBZ elicits minimal effects on microfilariae (mf). Severe complications after ivermectin (IVM) treatment of patients with high Loa loa microfilaraemia are of great concern. We examined the potential of FLBZ to rapidly kill L. loa mf, the phenomenon proposed to underlie the complications. Mf of L. loa were exposed to FLBZ, its reduced metabolite, albendazole, or IVM in vitro. Viability of L. loa mf was unaffected by FLBZ (10 μM, 72 hours); similar results were obtained with mf of Brugia malayi. We also measured the effects of FLBZ on transmission of mf. Aedes aegypti were fed FLBZ-exposed B. malayi mf and dissected 24 hours or 14 days postfeeding to count mf that crossed the midgut and developed to infective L3. FLBZ impaired the ability of mf to cross the midgut, regardless of duration of exposure (≥ 2 hours). FLBZ also prevented the development of mf to L3s, irrespective of duration of exposure or concentration. FLBZ is not microfilaricidal under these conditions; however, it blocks transmission. These results support the possibility that FLBZ may be a useful macrofilaricide in loiasis regions and may limit transmission from treated individuals.

Background Onchocerciasis currently afflicts an estimated 15 million people and is the second leading infectious cause of blindness world-wide. The development of a macrofilaricide to cure the disease has been hindered by the lack of appropriate small laboratory animal models. This study therefore, was aimed at developing and validating the Mongolian gerbil, as an Onchocerca ochengi (the closest in phylogeny to O. volvulus) adult male worm model. Methodology/Principal findings Mongolian gerbils (Meriones unguiculatus) were each implanted with 20 O. ochengi male worms (collected from infected cattle), in the peritoneum. Following drug or placebo treatments, the implanted worms were recovered from the animals and analyzed for burden, motility and viability. Worm recovery in control gerbils was on average 35%, with 89% of the worms being 100% motile. Treatment of the gerbils implanted with male worms with flubendazole (FBZ) resulted in a significant reduction (p = 0.0021) in worm burden (6.0% versus 27.8% in the control animals); all recovered worms from the treated group had 0% worm motility versus 91.1% motility in control animals. FBZ treatment had similar results even after four different experiments. Using this model, we tested a related drug, oxfendazole (OFZ), and found it to also significantly (p = 0.0097) affect worm motility (22.7% versus 95.0% in the control group). Conclusions/Significance We have developed and validated a novel gerbil O. ochengi adult male worm model for testing new macrofilaricidal drugs in vivo. It was also used to determine the efficacy of oxfendazole in vivo., Author summary Onchocerciasis is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus. The only drug currently used for the treatment of this disease and reduction of transmission is ivermectin. However, this drug kills only the microfilarial stage of the parasite, and not the adult worm, and cases of parasite resistance to the drug have been recorded. Therefore, there is an urgent need to find an efficacious macrofilaricidal drug that targets the adult worms. To identify an effective macrofilaricide, suitable small laboratory animal models are needed for routine drug screening and/or validation in vivo. To develop such a model, we implanted Mongolian gerbils with adult O. ochengi male worms; O. ochengi is the closest filarial relative of O. volvulus. Gerbils appeared to be permissive to O. ochengi male worms and when treated with flubendazole, a known macrofilaricidal drug, the drug significantly decreased worm burden, motility and viability when compared to vehicle-treated animals. This model was also used to test the efficacy of oxfendazole, a drug belonging to same class as flubendazole, and it also showed potent activity on O. ochengi male worms. The gerbil O. ochengi male worm model is now available to screen new candidate macrofilaricides.

There is an urgent global need for a safe macrofilaricide drug to accelerate elimination of the neglected tropical diseases onchocerciasis and lymphatic filariasis. From an anti-infective compound library, the macrolide veterinary antibiotic, tylosin A, was identified as a hit against Wolbachia. This bacterial endosymbiont is required for filarial worm viability and fertility and is a validated target for macrofilaricidal drugs. Medicinal chemistry was undertaken to develop tylosin A analogs with improved oral bioavailability. Two analogs, A-1535469 and A-1574083, were selected. Their efficacy was tested against the gold-standard second-generation tetracycline antibiotics, doxycycline and minocycline, in mouse and gerbil infection models of lymphatic filariasis (Brugia malayi and Litomosoides sigmodontis) and onchocerciasis (Onchocerca ochengi). A 1- or 2-week course of oral A-1535469 or A-1574083 provided >90% Wolbachia depletion from nematodes in infected animals, resulting in a block in embryogenesis and depletion of microfilarial worm loads. The two analogs delivered comparative or superior efficacy compared to a 3- to 4-week course of doxycycline or minocycline. A-1574083 (now called ABBV-4083) was selected for further preclinical testing. Cardiovascular studies in dogs and toxicology studies in rats and dogs revealed no adverse effects at doses (50 mg/kg) that achieved plasma concentrations >10-fold above the efficacious concentration. A-1574083 (ABBV-4083) shows potential as an anti-Wolbachia macrolide with an efficacy, pharmacology, and safety profile that is compatible with a short-term oral drug course for treating lymphatic filariasis and onchocerciasis.

Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

Benzimidazole anthelmintics have long been employed for the control of soil-transmitted helminth infections. Flubendazole (FBZ) was approved in 1980 for the treatment of gastrointestinal nematode infections in both veterinary and human medicine. It has also long been known that parenteral administration of FBZ can lead to high macrofilaricidal efficacy in a variety of preclinical models and in humans. As part of an effort to stimulate the discovery and development of new macrofilaricides, particularly for onchocerciasis, research has recently been devoted to the development of new formulations that would afford high oral bioavailability of FBZ, paving the way for potential clinical development of this repurposed drug for the treatment of human filariases. This review summarizes the background information that led to this program and summarizes some of the lessons learned from it.

Significance Onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) are neglected tropical diseases that cause severe disability and affect more than 157 million people globally. Current control efforts are hindered by the lack of a safe macrofilaricidal drug that can eliminate the parasitic adult nematodes safely. A clinically validated approach for delivering macrofilaricidal activity is to target the Wolbachia bacterial endosymbiont of the causative nematodes. This first-in-class and highly potent and specific anti-Wolbachia preclinical candidate molecule, AWZ1066S, has the potential to significantly impact current global onchocerciasis and lymphatic filariasis elimination programs and reduce elimination time frames from decades to years., Onchocerciasis and lymphatic filariasis are two neglected tropical diseases that together affect ∼157 million people and inflict severe disability. Both diseases are caused by parasitic filarial nematodes with elimination efforts constrained by the lack of a safe drug that can kill the adult filaria (macrofilaricide). Previous proof-of-concept human trials have demonstrated that depleting >90% of the essential nematode endosymbiont bacterium, Wolbachia, using antibiotics, can lead to permanent sterilization of adult female parasites and a safe macrofilaricidal outcome. AWZ1066S is a highly specific anti-Wolbachia candidate selected through a lead optimization program focused on balancing efficacy, safety and drug metabolism/pharmacokinetic (DMPK) features of a thienopyrimidine/quinazoline scaffold derived from phenotypic screening. AWZ1066S shows superior efficacy to existing anti-Wolbachia therapies in validated preclinical models of infection and has DMPK characteristics that are compatible with a short therapeutic regimen of 7 days or less. This candidate molecule is well-positioned for onward development and has the potential to make a significant impact on communities affected by filariasis.

Background Loiasis, an often-neglected tropical disease, is a threat to the success of lymphatic filariasis and onchocerciasis elimination programmes in rainforest areas of the central and western Africa. Its control and even its elimination might be possible through the use of a safe macrofilaricide, a prophylactic drug, or perhaps a vaccine. This present study evaluated the effect of flubendazole (FLBZ) on the development of Loa loa L3 in vitro and in vivo. Methods Infective stages of L. loa were isolated and co-cultured in Dulbecco’s Modified Eagle’s Medium in the presence of monkey kidney epithelial cells (LLC-MK2) feeder cells. FLBZ and its principal metabolites, reduced flubendazole (RFLBZ) and hydrolyzed flubendazole (HFLBZ), were screened in vitro at concentrations 0.05, 0.1, 0.5, 1 and 10 μg/ml. The viability of the parasites was assessed microscopically daily for 15 days. For in vivo study, a total of 48 CcR3 KO mice were infected subcutaneously with 200 L. loa L3 and treated with 10 mg/kg FLBZ once daily for 5 consecutive days. Twenty-four animals were used as control and received L3 and vehicle. They were dissected at 5, 10, 15 and 20 days post-treatment for worm recovery. Results The motility of L3 larvae in vitro was reduced from the second day of incubation with drugs at in vivo plasma concentration levels, with a strong correlation found between reduced motility and increased drug concentration (Spearman’s rho = -0.9, P < 0.0001). Except for HFLBZ (0.05 μg/ml and 0.01 μg/ml), all concentrations of FLBZ, HFLBZ and RFLBZ interrupted the moulting of L. loa infective larvae to L4. In vivo, regardless of the experimental group, there was a decrease in parasite recovery with time. However, at each time point this reduction was more pronounced in the group of animals treated with FLBZ compared to equivalent control. Parasites were recovered from the flubendazole-treated groups only on day 5 post-inoculation at an average rate of 2.1%, a value significantly lower (Mann-Whitney U-test, U = 28, P = 0.0156) than the average of 31.1% recovered from the control group. Conclusions This study reveals the ability of flubendazole to inhibit the development of L. loa L3 both in vitro and in vivo, and in addition validates the importance of in vitro and animal models of L. loa as tools for the development of drugs against loiasis. Electronic supplementary material The online version of this article (10.1186/s13071-018-3282-x) contains supplementary material, which is available to authorized users.

River blindness and lymphatic filariasis are two filarial diseases that globally affect millions of people mostly in impoverished countries. Current mass drug administration programs rely on drugs that primarily target the microfilariae, which are released from adult female worms. The female worms can live for several years, releasing millions of microfilariae throughout the course of infection. Thus, to stop transmission of infection and shorten the time to elimination of these diseases, a safe and effective drug that kills the adult stage is needed. The benzimidazole anthelmintic flubendazole (FBZ) is 100% efficacious as a macrofilaricide in experimental filarial rodent models but it must be administered subcutaneously (SC) due to its low oral bioavailability. Studies were undertaken to assess the efficacy of a new oral amorphous solid dispersion (ASD) formulation of FBZ on Brugia pahangi infected jirds (Meriones unguiculatus) and compare it to a single or multiple doses of FBZ given subcutaneously. Results showed that worm burden was not significantly decreased in animals given oral doses of ASD FBZ (0.2–15 mg/kg). Regardless, doses as low as 1.5 mg/kg caused extensive ultrastructural damage to developing embryos and microfilariae (mf). SC injections of FBZ in suspension (10 mg/kg) given for 5 days however, eliminated all worms in all animals, and a single SC injection reduced worm burden by 63% compared to the control group. In summary, oral doses of ASD formulated FBZ did not significantly reduce total worm burden but longer treatments, extended takedown times or a second dosing regimen, may decrease female fecundity and the number of mf shed by female worms., Author summary Safe and effective macrofilaricidal drugs are critically needed to treat onchocerciasis and lymphatic filariasis, which affect over 54 million people worldwide. Flubendazole (FBZ) in its current commercial formulations is an effective anthelminthic for intestinal soil transmitted helminth (STH) infections but not for filarial infections due to its low bioavailability. The purpose of this study was to assess the efficacy of a new amorphous solid dispersion (ASD) formulation of FBZ given orally to jirds (Meriones unguiculatus) infected with the filarial nematode Brugia pahangi and compare it to FBZ (in suspension) given subcutaneously as a single or multiple dose. Our results indicated that treatment with ASD FBZ did not significantly reduce the total number of worms. However, doses as low as 1.5 mg/kg caused ultrastructural damage to the early stages of developing embryos. No worms were recovered from jirds given 10 mg/kg SC injections of FBZ (suspension) for 5 days when necropsied 68 days after the first-dose, and the number of mf at necropsy was significantly decreased compared to the control group. Animals given only a single 10 mg/kg SC injection had a 63% decrease in the number of adult worms. These animals also had fewer female worms and mf compared to the control group suggesting that even a single SC injection of FBZ had an effect on female survival and fecundity. TEM of worms recovered from jirds given low doses of the ASD formulated FBZ (1.5 mg/kg) suggested that female fecundity and mf production were reduced, but longer treatments, longer takedown times or a second dosing regimen of ASD FBZ may be needed to significantly decrease the total worm burden.

Significance Filarial nematode infections, caused by Wuchereria bancrofti , Brugia malayi (elephantiasis), and Onchocerca volvulus (river blindness) infect 150 million of the world’s poorest populations and cause profound disability. Standard treatments require repetitive, long-term, mass drug administrations and have failed to interrupted transmission in certain sub-Saharan African regions. A drug cure using doxycycline, which targets the essential filarial endosymbiont Wolbachia , is clinically effective but programmatically challenging to implement due to long treatment durations and contraindications. Here we provide proof-of-concept of a radical improvement of targeting Wolbachia via identification of drug synergy between the anthelmintic albendazole and antibiotics. This synergy enables the shortening of treatment duration of macrofilaricidal anti- Wolbachia based treatments from 4 wk to 7 d with registered drugs ready for clinical testing.

Despite the well established ivermectin activity against microfilaria, the success of human filariasis control programmes requires the use of a macrofilaricide compound. Different in vivo trials suggest that flubendazole (FLBZ), an anthelmintic benzimidazole compound, is a highly efficacious and potent macrofilaricide. However, since serious injection site reactions were reported in humans after the subcutaneous FLBZ administration, the search for alternative pharmaceutical strategies to improve the systemic availability of FLBZ has acquired special relevance both in human and veterinary medicine. The goal of the current experimental work was to compare the pharmacokinetic plasma behavior of FLBZ, and its metabolites, formulated as either an aqueous hydroxypropyl- β -cyclodextrin-solution (HPBCD), an aqueous carboxymethyl cellulose-suspension (CMC) or a Tween 80-based formulation, in pigs. Animals were allocated into three groups and treated (2 mg/kg) with FLBZ formulated as either a HPBCD-solution (oral), CMC-suspension (oral) or Tween 80-based formulation (subcutaneous). Only trace amounts of FLBZ parent drug and its reduced metabolite were measured after administration of the different FLBZ formulations in pigs. The hydrolyzed FLBZ (H-FLBZ) metabolite was the main analyte recovered in the bloodstream in pigs treated with the three experimental FLBZ formulations. The oral administration of the HPBCD-solution accounted for significantly higher (P, Graphical abstract, Highlights • The pharmacokinetics of three different FLBZ formulations was assessed in pigs. • Hydrolyzed-FLBZ was the main metabolite detected in pigs given the formulations. • Traces of FLBZ and reduced-FLBZ were measured after administration of FLBZ. • Oral administration of FLBZ-HPBCD solution resulted in a high systemic H-FLBZ exposure. • Similar FLBZ plasma exposure was observed after parenteral FLBZ-Tween 80 and HPBCD administration.

Human onchocerciasis (river blindness), caused by the filarial nematode Onchocerca volvulus, has been successfully controlled by a single drug, ivermectin, for over 25 years. Ivermectin prevents the disease symptoms of severe itching and visual impairment by killing the microfilarial stage, but does not eliminate the adult parasites, necessitating repeated annual treatments. Mass drug administration with ivermectin does not always break transmission in forest zones and is contraindicated in individuals heavily co-infected with Loa loa, while reports of reduced drug efficacy in Ghana and Cameroon may signal the development of resistance. An alternative treatment for onchocerciasis involves targeting the essential Wolbachia symbiont with tetracycline or its derivatives, which are adulticidal. However, implementation of antibiotic therapy has not occurred on a wide scale due to the prolonged treatment regimen required (several weeks). In the bovine Onchocerca ochengi system, it has been shown previously that prolonged oxytetracycline therapy increases eosinophil counts in intradermal nodules, which kill the adult worms by degranulating on their surface. Here, in an "immunochemotherapeutic" approach, we sought to enhance the efficacy of a short, sub-lethal antibiotic regimen against O. ochengi by prior immunotherapy targeting onchocystatin, an immunomodulatory protein located in the adult female worm cuticle. A key asparagine residue in onchocystatin was mutated to ablate immunomodulatory activity, which has been demonstrated previously to markedly improve the protective efficacy of this vaccine candidate when used as an immunoprophylactic. The immunochemotherapeutic regimen was compared with sub-lethal oxytetracycline therapy alone; onchocystatin immunotherapy alone; a gold-standard prolonged, intermittent oxytetracycline regimen; and no treatment (negative control) in naturally infected Cameroonian cattle. Readouts were collected over one year and comprised adult worm viability, dermal microfilarial density, anti-onchocystatin IgG in sera, and eosinophil counts in nodules. Only the gold-standard antibiotic regimen achieved significant killing of adult worms, a profound reduction in microfilarial load, and a sustained increase in local tissue eosinophilia. A small but statistically significant elevation in anti-onchocystatin IgG was observed for several weeks after immunisation in the immunotherapy-only group, but the antibody response in the immunochemotherapy group was more variable. At 12 weeks post-treatment, only a transient and non-significant increase in eosinophil counts was apparent in the immunochemotherapy group. We conclude that the addition of onchocystatin immunotherapy to a sub-lethal antibiotic regimen is insufficient to induce adulticidal activity, although with booster immunisations or the targeting of additional filarial immunomodulatory proteins, the efficacy of this strategy could be strengthened.

The use of a microfilaricidal drug for the control of onchocerciasis and lymphatic filariasis necessitates prolonged yearly dosing. Prospects for elimination or eradication of these diseases would be enhanced by availability of a macrofilaricidal drug. Flubendazole (FLBZ), a benzimidazole anthelmintic, is an appealing candidate macrofilaricide. FLBZ has demonstrated profound and potent macrofilaricidal effects in a number of experimental filarial rodent models and one human trial. Unfortunately, FLBZ was deemed unsatisfactory for use in mass drug administration (MDA) campaigns due to its markedly limited oral bioavailability. However, a new formulation that provided sufficient bioavailability following oral administration could render FLBZ an effective treatment for onchocerciasis and LF. This study characterized the effects of FLBZ and its reduced metabolite (FLBZ-R) on filarial nematodes in vitro to determine the exposure profile which results in demonstrable damage. Adult female Brugia malayi were exposed to varying concentrations of FLBZ or FLBZ-R (100 nM–10 μM) for up to five days, after which worms were fixed for histology. Morphological damage following exposure to FLBZ was observed prominently in the hypodermis and developing embryos at concentrations as low as 100 nM following 24 h exposure. The results indicate that damage to tissues required for reproduction and survival can be achieved at pharmacologically relevant concentrations., Graphical abstract, Highlights • Detrimental effects observed in tissues required for development and survival. • Damage occurs at concentrations ≥100 nM. • Hypodermal and embryonic tissues exhibited the most severe damage. • Oocytes and early morula are the most affected embryonic tissues.

Background Mass drug administration (MDA) with ivermectin is the cornerstone of efforts to eliminate human onchocerciasis by 2020/2025. The feasibility of elimination crucially depends on the effects of multiple ivermectin doses on Onchocerca volvulus. A single ivermectin (standard) dose clears the skin-dwelling microfilarial progeny of adult worms (macrofilariae) and temporarily impedes the release of such progeny by female macrofilariae, but a macrofilaricidal effect has been deemed minimal. Multiple doses of ivermectin may cumulatively and permanently reduce the fertility and shorten the lifespan of adult females, but rigorous quantification of these effects necessitates interrogating longitudinal data on macrofilariae with suitably powerful analytical techniques. Methods Using a novel mathematical modelling approach, we analysed―at an individual participant level―longitudinal data on viability and fertility of female worms from the single most comprehensive multiple-dose clinical trial of ivermectin, comparing three-monthly with annual treatments administered for three years, in Cameroon. Results Multiple doses of ivermectin have a partial macrofilaricidal and a modest permanent sterilising effect after 4 or more consecutive treatments, even at routine MDA doses (150 g/kg) and (annual) frequencies. The life expectancy of adult O. volvulus is reduced by approximately 50% and 70% after three years of annual or three-monthly (quarterly) exposures to ivermectin. Conclusions Our quantification of macrofilaricidal and sterilising effects of ivermectin should be incorporated into transmission models informing onchocerciasis elimination efforts in Africa and residual foci in Latin America. It also provides a framework to assess macrofilaricidal candidate drugs currently under development.

Onchocerciasis and loiasis are caused by infection with filarial worms. Severe skin and eye lesions, and blindness are found in onchocerciasis, and the disease is an important public health problem in endemic areas. Loiasis causes Calabar swelling and ‘eye worm,’ whose public health significance is yet to be clarified. Vector control and ivermectin treatment have been used to successfully control or eliminate onchocerciasis, but the risk of severe adverse reactions impedes ivermectin distribution in areas with loiasis. Research has played a key role in optimizing onchocerciasis control. Onchocerciasis is being progressively eliminated and with a macrofilaricide its eradication could be a reality.

Lymphatic filariasis is a disfiguring disease caused by parasitic worms that destroy the human lymphatic system leading to substantial morbidity. The current drug of choice for the treatment of filariasis is diethylcarbamazine and ivermectin with albendazole which are only effective against the microfilaria, leaving the adult worm unaffected, requiring the development of "adulticidal drugs." Thirty amino substituted 2-hydroxy/5-hydroxy/2-methyl-1,4-naphthoquinones were synthesized via the reac- tion of 2-hydroxy/5-hydroxy/2-methyl-1,4-naphthoquinones with different primary and secondary amines. Compounds 1-30 were evaluated for in vitro antifilarial activity against the adult bovine filarial worm Setaria digitata as assessed by worm motility and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) reduction assays. The mutagenecity, tumerogenecity, irritantancy, repro- ductive toxicity, drug score, druglike, and cLogP properties were calculated using OSIRIS property predictor. Ten compounds showed macrofilaricidal activity with ED50 values ranging between 0.086 and 7.6 mM. Taking into account the biological effects and the promising drug-like profiles of these com- pounds, these represent valid leads for the development of antifilarial agents against adult filarial worm. Drug Dev Res 74 : 216-226, 2013. © 2013 Wiley Periodicals, Inc.

In a placebo controlled field trial, the effects of doxycycline (200mg/day) for 23 days followed by doxycycline (200mg/day) in combination with albendazole (ABZ) (400mg/day) for 7 days on depletion of Wolbachia endobacteria from Wuchereria bancrofti and microfilaricidal activity were studied in 68 patients (34 males and 34 females) from West Bengal, India. The drugs in combination (i.e., doxycycline+ABZ) provided the best efficacy by totally eliminating the circulating microfilaria (mf) (in 42% cases) on day 365 with (99.8%, P

Although of limited veterinary significance, Onchocerca ochengi has become famous as a natural model or 'analogue' of human onchocerciasis (river blindness), which is caused by Onchocerca volvulus. On the basis of both morphological and molecular criteria, O. ochengi is the closest extant relative of O. volvulus and shares several key natural history traits with the human pathogen. These include exploitation of the same group of insect vectors (blackflies of the Simulium damnosum complex) and formation of collagenous nodules with a similar histological structure to human nodules. Here, we review the contribution of this natural system to drug and vaccine discovery efforts, as well as to our basic biological understanding of Onchocerca spp., over the past quarter-century.

The use of microfilaricidal drugs for the control of onchocerciasis and lymphatic filariasis (LF) necessitates prolonged yearly dosing. Prospects for elimination or eradication of these diseases would be enhanced by the availability of a macrofilaricidal drug. Flubendazole (FLBZ), a benzimidazole anthelmintic, is an appealing candidate. FLBZ has demonstrated potent macrofilaricidal effects in a number of experimental rodent models and in one human trial. Unfortunately, FLBZ was deemed unsatisfactory for use in mass drug administration campaigns due to its limited oral bioavailability. A new formulation that enables sufficient bioavailability following oral administration could render FLBZ an effective treatment for onchocerciasis and LF. Identification of drug-derived effects is important in ascertaining a dosage regimen which is predicted to be lethal to the parasite in situ. In previous histological studies, exposure to FLBZ induced damage to tissues required for reproduction and survival at pharmacologically relevant concentrations. However, more precise and quantitative indices of drug effects are needed. This study assessed drug effects using a transcriptomic approach to confirm effects observed histologically and to identify genes which were differentially expressed in treated adult female Brugia malayi. Comparative analysis across different concentrations (1 μM and 5 μM) and durations (48 and 120 h) provided an overview of the processes which are affected by FLBZ exposure. Genes with dysregulated expression were consistent with the reproductive effects observed via histology in our previous studies. This study revealed transcriptional changes in genes involved in embryo development. Additionally, significant downregulation was observed in genes encoding cuticle components, which may reflect changes in developing embryos, the adult worm cuticle or both. These data support the hypothesis that FLBZ acts predominantly on rapidly dividing cells, and provides a basis for selecting molecular markers of drug-induced damage which may be of use in predicting efficacious FLBZ regimens., Highlights • Genes involved in development and cuticle synthesis were differentially expressed. • Downregulation of adult cuticle genes suggests an alternate mechanism of action. • One gene was identified as a possible FLBZ-specific marker of damage.