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The filarial nematode Onchocerca volvulus causes onchocerciasis (river blindness), a neglected tropical disease affecting 21 million people, mostly in Sub-Saharan Africa. Targeting the endosymbiont Wolbachia with antibiotics leads to permanent sterilization and killing of adult worms. The gold standard to assess Wolbachia depletion is the histological examination of adult worms in nodules beginning at 6 months post-treatment. However, nodules can only be used once, limiting the time points to monitor Wolbachia depletion. A diagnostic to longitudinally monitor Wolbachia depletion from microfilariae (MF) at more frequent intervals wOvftsZ to quantify Wolbachia from as few as one MF that had migrated from skin biopsies and compared quantification using circular and linearized plasmids or synthetic dsDNA (gBlock®). qPCR for MF from the rodent nematode Litomosoides sigmodontis was used to support the reproducibility and validate the principle. The qPCR using as few as 2 MF from O. volvulus and L. sigmodontis reproducibly quantified Wolbachia. Use of a linearized plasmid standard or synthesized dsDNA resulted in numbers of Wolbachia/MF congruent with biologically plausible estimates in O. volvulus and L. sigmodontis MF. The qPCR assay yielded a median of 48.8 (range 1.5–280.5) Wolbachia/O. volvulus MF. The qPCR is a sensitive tool for quantifying Wolbachia in a few MF from skin biopsies and allows for establishing the qPCR as a surrogate parameter for monitoring Wolbachia depletion in adult worms of new antiwolbachial candidates.

Oxfendazole is one of the lead macrofilaricidal candidates for the treatment of onchocerciasis and lymphatic filariasis. Originally, oxfendazole was developed for the veterinary market, where it is mainly used to treat intestinal helminth infections. In humans, oxfendazole was proven to be safe in multiple ascending dose studies. Furthermore, previous experimental studies demonstrated that the benzimidazoles class is active in animals and humans against filarial nematodes. In the present study, we have compared the efficacy of oxfendazole isomers with the commercially available racemic mixture Dolthene against the rodent filaria Litomosoides sigmodontis in female BALB/c mice. Treatment with either the isomers or Dolthene led to a reduction of the adult worm burden by 94-98% following the ten-day treatment and by 72% (oxfendazole (-)), 85% (oxfendazole (+)) and 91% (Dolthene) following the five-day treatment. No statistically significant differences in the macrofilaricidal efficacy against L. sigmodontis were observed for both isomers and Dolthene. Metabolites of oxfendazole are fenbendazole and fenbendazole sulfone. Two hours after treatment with Dolthene and both oxfendazole isomers, fenbendazole sulfone, but rarely fenbendazole, was detected. The oxfendazole (-) isomer was metabolised at the highest rate to fenbendazole sulfone. Furthermore, oxfendazole isomers have a comparable pharmacokinetic profile in dogs. In conclusion, our data does not point at the development of a single isomer for future use in humans.

Infections with helminths affect more than one billion people worldwide. Despite an urgent need there is no vaccine available that would confer long lasting protection against helminth infections. Previous studies indicated that a vaccination with irradiated infective L3 reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as adjuvant improves the efficacy of a vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in increased neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-β RNA levels at the site of injection. To investigate the in vivo impact on parasite clearance, BALB/c mice received 3 subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Serum analysis before the challenge infection confirmed the induction of L. sigmodontis-specific antibodies in response to the immunization and serum from immunized mice significantly reduced larval motility in vitro with naïve cells. 63 days after the challenge infection, vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a significantly greater reduction in adult worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). Further, the treatment of L. sigmodontis infection with 3pRNA alone, but not poly(I:C), resulted in a reduced worm burden, supporting the therapeutic potential for the activation of RIG-I with 3pRNA. In conclusion, our data demonstrate that the additional activation of nucleic acid sensing immune receptors boosts the immune response and provides better protection against L. sigmodontis. Thus, the use of nucleic acid receptor agonists as vaccine adjuvants represents a promising novel strategy to improve the efficacy of vaccines against filariae and potentially of other helminths.Author SummaryFilarial nematodes can cause debilitating diseases such as onchocerciasis and lymphatic filariasis that present a major public health burden in the tropics and subtropics, putting more than a billion people at risk of infection. Filarial diseases are transmitted to humans by insect vectors as they take a blood meal. The WHO classifies both filarial infections as neglected tropical diseases and aims to eliminate the transmission of onchocerciasis and eliminate lymphatic filariasis as public health problem by 2030. However, up to date there is no vaccination available that could support the efforts to eliminate filarial diseases and potentially helminth infections in general. Here, we used the well-established murine model for filarial infection, Litomosoides sigmodontis, to test the use of nucleic acid receptor agonists as vaccine adjuvants to enhance local immune responses. We found that infection with L. sigmodontis induces type I IFN and our vaccine strategy enhances the production of type I IFN resulting in increased parasite-specific immune responses and enhanced worm clearance. In summary, our study provides a promising novel approach for a vaccination strategy using cytosolic RNA receptor agonists.

The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16−18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10–14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days., Graphical abstract Image 1, Highlights • CBR417 and CBR490 provide long-term effects in 2 chronic filaria jird models. • CBR417 and CBR490 deplete >99% Wolbachia in B. pahangi and L. sigmodontis filariae. • CBR417 and CBR490 clear L. sigmodontis microfilariae after 10–14 weeks. • CBR417 and CBR490 inhibit filarial embryogenesis in both models. • Suboptimal doses do not maintain reduction of microfilariae and Wolbachia.

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., Author summary Onchocerciasis and lymphatic filariasis represent two debilitating filarial diseases that belong to the neglected tropical diseases. The current efforts to eliminate those diseases is hampered by the lack of short-course macrofilaricidal drugs, i.e. drugs that kill the adult worms, or regimens that are proven to be safe for both diseases. In the present study we demonstrate that the anthelmintic drug oxfendazole, currently used in veterinary medicine against intestinal helminths, has excellent efficacy in the Litomosoides sigmodontis rodent model of filariasis. Oxfendazole caused complete clearance of adult filariae after a short oral regimen in vivo. Oxfendazole was not directly active against the circulating filarial progeny, the microfilariae, suggesting that drug-induced serious adverse events due to the clearance of microfilariae are unlikely. Human dose was predicted based on the efficacy in the rodent model, the calculation estimated a low efficacious dose, which has already been shown to be safe in phase 1 clinical trials. Thus, oxfendazole represents a promising drug candidate for the treatment of human filarial diseases such as onchocerciasis and lymphatic filariasis.

Current efforts to eliminate the neglected tropical diseases onchocerciasis and lymphatic filariasis, caused by the filarial nematodes Onchocerca volvulus and Wuchereria bancrofti or Brugia spp., respectively, are hampered by lack of a short-course macrofilaricidal–adult-worm killing–treatment. Anti-wolbachial antibiotics, e.g. doxycycline, target the essential Wolbachia endosymbionts of filariae and are a safe prototype adult-worm-sterilizing and macrofilaricidal regimen, in contrast to standard treatments with ivermectin or diethylcarbamazine, which mainly target the microfilariae. However, treatment regimens of 4–5 weeks necessary for doxycycline and contraindications limit its use. Therefore, we tested the preclinical anti-Wolbachia drug candidate Corallopyronin A (CorA) for in vivo efficacy during initial and chronic filarial infections in the Litomosoides sigmodontis rodent model. CorA treatment for 14 days beginning immediately after infection cleared >90% of Wolbachia endosymbionts from filariae and prevented development into adult worms. CorA treatment of patently infected microfilaremic gerbils for 14 days with 30 mg/kg twice a day (BID) achieved a sustained reduction of >99% of Wolbachia endosymbionts from adult filariae and microfilariae, followed by complete inhibition of filarial embryogenesis resulting in clearance of microfilariae. Combined treatment of CorA and albendazole, a drug currently co-administered during mass drug administrations and previously shown to enhance efficacy of anti-Wolbachia drugs, achieved microfilarial clearance after 7 days of treatment at a lower BID dose of 10 mg/kg CorA, a Human Equivalent Dose of 1.4 mg/kg. Importantly, this combination led to a significant reduction in the adult worm burden, which has not yet been published with other anti-Wolbachia candidates tested in this model. In summary, CorA is a preclinical candidate for filariasis, which significantly reduces treatment times required to achieve sustained Wolbachia depletion, clearance of microfilariae, and inhibition of embryogenesis. In combination with albendazole, CorA is robustly macrofilaricidal after 7 days of treatment and fulfills the Target Product Profile for a macrofilaricidal drug., Author summary Infections with filarial roundworms can cause the disfiguring human neglected tropical diseases onchocerciasis and lymphatic filariasis. Treatment of these diseases is limited, as there is no well-tolerated treatment available that kills the adult worms after a short-term regimen. Thus, mass drug administrations (MDA) are performed with drugs that temporarily clear the microfilariae, the filarial offspring, to inhibit the transmission of the disease. As these MDA treatments have to be given 1–2 times per year for many years, the goal to eliminate onchocerciasis and lymphatic filariasis is hampered. In the present study we investigated a novel preclinical candidate for the treatment of filariasis. Corallopyronin A (CorA) is a natural compound that clears the essential Wolbachia endobacteria of filariae. Using the Litomosoides sigmodontis rodent model of filariasis we demonstrated that 2 weeks of CorA treatment clears Wolbachia endosymbionts in vivo, leading to a maintained clearance of microfilariae by inhibition of filarial embryogenesis. Combination therapy of CorA with the MDA drug albendazole allowed lower CorA doses and shortened treatment to 7 days. More importantly, it also led to the death of the adult filariae. Portfolios (Target Product Profiles) of new drugs against filariae should show adult killing efficacy like CorA.

Depletion of Wolbachia endosymbionts of human pathogenic filariae using 4–6 weeks of doxycycline treatment can lead to permanent sterilization and adult filarial death. We investigated the anti-Wolbachia drug candidate ABBV-4083 in the Litomosoides sigmodontis rodent model to determine Wolbachia depletion kinetics with different regimens. Wolbachia reduction occurred in mice as early as 3 days after the initiation of ABBV-4083 treatment and continued throughout a 10-day treatment period. Importantly, Wolbachia levels continued to decline after a 5-day-treatment from 91.5% to 99.9% during a 3-week washout period. In jirds, two weeks of ABBV-4083 treatment (100mg/kg once-per-day) caused a >99.9% Wolbachia depletion in female adult worms, and the kinetics of Wolbachia depletion were recapitulated in peripheral blood microfilariae. Similar to Wolbachia depletion, inhibition of embryogenesis was time-dependent in ABBV-4083-treated jirds, leading to a complete lack of late embryonic stages (stretched microfilariae) and lack of peripheral microfilariae in 5/6 ABBV-4083-treated jirds by 14 weeks after treatment. Twice daily treatment in comparison to once daily treatment with ABBV-4083 did not significantly improve Wolbachia depletion. Moreover, up to 4 nonconsecutive daily treatments within a 14-dose regimen did not significantly erode Wolbachia depletion. Within the limitations of an animal model that does not fully recapitulate human filarial disease, our studies suggest that Wolbachia depletion should be assessed clinically no earlier than 3–4 weeks after the end of treatment, and that Wolbachia depletion in microfilariae may be a viable surrogate marker for the depletion within adult worms. Furthermore, strict daily adherence to the dosing regimen with anti-Wolbachia candidates may not be required, provided that the full regimen is subsequently completed., Author summary Onchocerciasis and lymphatic filariasis represent debilitating neglected tropical diseases that are caused by parasitic filarial nematodes. Current efforts to eliminate onchocerciasis are hampered by the lack of drugs that lead to permanent sterilization of the adult worms or provide a macrofilaricidal effect, i.e. kill the adult worms. In the past, doxycycline has been shown to deplete Wolbachia endosymbionts of filarial nematodes, leading to permanent sterilization and macrofilaricidal efficacy in filariae causing both onchocerciasis and lymphatic filariasis. However, contraindications and a requirement for at least 4 weeks of doxycycline treatment impair its broader use, creating a need for drugs with a shorter treatment regimen and potentially fewer contraindications. ABBV-4083 is such an anti-Wolbachia candidate that was efficacious in several filarial animal models and has recently been tested in a phase 1 clinical trial. The present studies addressed several points that are important for subsequent phase 2 clinical trials, namely the comparison of once vs. twice-per-day treatments, the impact of missed treatments, and a comparison of the kinetics of Wolbachia depletion in adult worms and microfilariae, the latter of which has the potential to be a surrogate indicator to avoid the necessity of surgically removing nodules with adult worms at repeated time points.

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.

The elimination of filarial diseases such as onchocerciasis and lymphatic filariasis is hampered by the lack of a macrofilaricidal–adult worm killing–drug. In the present study, we tested the in vivo efficacy of AN11251, a boron-pleuromutilin that targets endosymbiotic Wolbachia bacteria from filarial nematodes and compared its efficacy to doxycycline and rifampicin. Doxycycline and rifampicin were previously shown to deplete Wolbachia endosymbionts leading to a permanent sterilization of the female adult filariae and adult worm death in human clinical studies. Twice-daily oral treatment of Litomosoides sigmodontis-infected mice with 200 mg/kg AN11251 for 10 days achieved a Wolbachia depletion > 99.9% in the adult worms, exceeding the Wolbachia reduction by 10-day treatments with bioequivalent human doses of doxycycline and a similar reduction as high-dose rifampicin (35 mg/kg). Wolbachia reductions of > 99% were also accomplished by 14 days of oral AN11251 at a lower twice-daily dose (50 mg/kg) or once-per-day 200 mg/kg AN11251 treatments. The combinations tested of AN11251 with doxycycline had no clear beneficial impact on Wolbachia depletion, achieving a > 97% Wolbachia reduction with 7 days of treatment. These results indicate that AN11251 is superior to doxycycline and comparable to high-dose rifampicin in the L. sigmodontis mouse model, allowing treatment regimens as short as 10–14 days. Therefore, AN11251 represents a promising pre-clinical candidate that was identified in the L. sigmodontis model, and could be further evaluated and developed as potential clinical candidate for human lymphatic filariasis and onchocerciasis., Author summary Onchocerciasis and lymphatic filariasis are human filarial tropical diseases, which can cause blindness and severe dermatitis (onchocerciasis) or lymphedema and hydrocele (lymphatic filariasis). Current strategies to eliminate these diseases include the mass drug administration (MDA) of drugs that target the progeny of the filariae, the microfilariae, and temporarily inhibit filarial embryogenesis and, therefore, the transmission of the disease. However, MDA has several limitations that delay the goal of elimination including the lack of a drug with a short term regimen and a potent macrofilaricidal effect. As an alternative approach, the antibiotic doxycycline has been proven to be effective in depleting Wolbachia endosymbionts from adult filariae, which then leads to permanent sterilization and death of the adult worms. Due to contraindications for doxycycline and prolonged treatment regimen of at least 4 weeks, there is an urgent need for new anti-filarial drugs with an improved safety profile and shorter regimens. The current study demonstrates that the boron-pleuromutilin derivative AN11251 provides an excellent in vivo anti-Wolbachia depletion in the Litomosoides sigmodontis filarial mouse model that is superior to doxycycline and comparable to rifampicin, allowing for regimens as short as 10–14 days. Combination with doxycycline for 7 days had no significant beneficial effect on efficacy, achieving Wolbachia reductions of more than 97%. Therefore, AN11251 shows potent anti-Wolbachia activity in the L. sigmodontis mouse model and may also present an alternative pre-clinical candidate for filariasis treatment.

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

Filarial parasites can be targeted by antibiotic treatment due to their unique endosymbiotic relationship with Wolbachia bacteria. This finding has led to successful treatment strategies in both, human onchocerciasis and lymphatic filariasis. A 4–6 week treatment course using doxycycline results in long-term sterility and safe macrofilaricidal activity in humans. However, current treatment times and doxycycline contraindications in children and pregnant women preclude widespread administration of doxycycline in public health control programs; therefore, the search for shorter anti-wolbachial regimens is a focus of ongoing research. We have established an in vivo model for compound screening, using mice infected with Litomosoides sigmodontis. We could show that gold standard doxycycline treatment did not only deplete Wolbachia, it also resulted in a larval arrest. In this model, combinations of registered antibiotics were tested for their anti-wolbachial activity. Administration of rifamycins in combination with doxycycline for 7 days successfully depleted Wolbachia by > 2 log (>99% reduction) and thus resulted in a significant reduction of the treatment duration. Using a triple combination of a tetracycline (doxycycline or minocycline), a rifamycin and a fluoroquinolone (moxifloxacin) led to an even greater shortening of the treatment time. Testing all double combinations that could be derived from the triple combinations revealed that the combination of rifapentine (15mg/kg) and moxifloxacin (2 x 200mg/kg) showed the strongest reduction of treatment time in intraperitoneal and also oral administration routes. The rifapentine plus moxifloxacin combination was equivalent to the triple combination with additional doxycycline (>99% Wolbachia reduction). These investigations suggest that it is possible to shorten anti-wolbachial treatment times with combination treatments in order to achieve the target product profile (TPP) requirements for macrofilaricidal drugs of no more than 7–10 days of treatment., Author summary Over the past years, more attention has been brought to neglected tropical diseases including lymphatic filariasis and onchocerciasis. The latter are caused by helminthic parasites and lead to chronic and debilitating symptoms and present a major health burden that also affects the economy of endemic countries. It has been suggested that disease elimination may be possible but an accelerated implementation of proven and cost-effective interventions are needed if the targets for elimination are to be achieved. Recently, an indirect mode of action has been identified, targeting bacterial Wolbachia endosymbionts within the filariae, which also kills the adult parasites, an advantage over the drug currently used for mass drug administration, i.e. ivermectin. Doxycycline has been successfully used in clinical trials, however due to its long regimen as well as restrictions of use in children and pregnant women new drugs or drug combinations are required that overcome these obstacles. Here, we present the filarial parasite Litomosoides sigmodontis as suitable model for the preclinical testing of anti-wolbachial drugs against filariae and show that combinations of already registered drugs with anti-wolbachial efficacy are able to reduce the treatment time dramatically.

Flubendazole (FBZ) is highly efficacious against filarial nematodes after parenteral administration and presents a promising macrofilaricidal drug candidate for the elimination of onchocerciasis and other filariae. In the present study the efficacy of a newly developed bioavailable amorphous solid dispersion (ASD) oral formulation of FBZ was investigated in the Litomosoides sigmodontis jird model. FBZ was administered to chronically infected, microfilariae-positive jirds by single (40mg/kg), repeated (2, 6 or 15mg/kg for 5 or 10 days) oral (OR) doses or single subcutaneous (SC) injections (2 or 10mg/kg). Jirds treated with 5 SC injections at 10mg/kg served as positive controls, with untreated animals used as negative controls. After OR doses, FBZ is rapidly absorbed and cleared and the exposures increased dose proportionally. SC administered FBZ was slowly released from the injection site and plasma levels remained constant up to necropsy eight weeks after treatment end. Increasing single SC doses caused less than dose-proportional exposures. At necropsy, all animals receiving 1x or 5x 10mg/kg SC FBZ had cleared all adult worms and the 1x 2mg/kg SC treatment had reduced the adult worm burden by 98%. 10x 15mg/kg OR FBZ reduced the adult worm burden by 95%, whereas 1x 40mg/kg and 5x 15mg/kg OR reduced the worm burden by 85 and 84%, respectively. Microfilaremia was completely cleared at necropsy in all animals of the SC treatment regimens, while all oral FBZ treatment regimens reduced the microfilaremia by >90% in a dose and duration dependent manner. In accordance, embryograms from female worms revealed a FBZ dose and duration dependent inhibition of embryogenesis. Histological analysis of the remaining female adult worms showed that FBZ had damaged the body wall, intestine and most prominently the uterus and uterine content. Results of this study demonstrate that single and repeated SC injections and repeated oral administrations of FBZ have an excellent macrofilaricidal effect., Author summary Onchocerciasis and lymphatic filariasis are debilitating human diseases that are caused by filarial nematodes leading to blindness and severe dermatitis (onchocerciasis) or lymphedema (elephantiasis) and hydroceles. Current mass drug administration (MDA) programs are restricted to the use of drugs that target the filarial offspring, the microfilariae, and lead to a temporary sterilization of the female adult worms that requires annual to bi-annual MDA for the life span of the fertile adult worms. With lower endemicity, the cost-effectiveness of community-directed MDA is reduced, requiring alternative treatment strategies to ultimately eliminate these filarial infections. Therefore, new drugs targeting the adult worms are required for achieving elimination of those filarial diseases, to provide options in areas of drug resistance, and as an approach in areas of Loa loa co-endemicity, where life-threatening severe adverse events may occur through the use of microfilaricidal drugs. Such new drugs should have a macrofilaricidal effect, i.e. kill the adult worms or lead to a permanent sterilization. Furthermore, these drugs should be preferably administered as an oral formulation or by a single parenteral administration. In the current study we tested a new oral formulation of flubendazole using the rodent filarial nematode Litomosoides sigmodontis, and have demonstrated excellent macrofilaricidal efficacy.