Combinations of the azaquinazoline anti-Wolbachia agent, AWZ1066S, with benzimidazole anthelmintics synergise to mediate sub-seven-day sterilising and curative efficacies in experimental models of filariasis.

Lymphatic filariasis and onchocerciasis are two major neglected tropical diseases that are responsible for causing severe disability in 50 million people worldwide, whilst veterinary filariasis (heartworm) is a potentially lethal parasitic infection of companion animals. There is an urgent need for safe, short-course curative (macrofilaricidal) drugs to eliminate these debilitating parasite infections. We investigated combination treatments of the novel anti-Wolbachia azaquinazoline small molecule, AWZ1066S, with benzimidazole drugs (albendazole or oxfendazole) in up to four different rodent filariasis infection models: Brugia malayi-CB.17 SCID mice, B. malayi-Mongolian gerbils, B. pahangi-Mongolian gerbils, and Litomosoides sigmodontis-Mongolian gerbils. Combination treatments synergised to elicit threshold (>90%) Wolbachia depletion from female worms in 5 days of treatment, using 2-fold lower dose-exposures of AWZ1066S than monotherapy. Short-course lowered dose AWZ1066S-albendazole combination treatments also delivered partial adulticidal activities and/or long-lasting inhibition of embryogenesis, resulting in complete transmission blockade in B. pahangi and L. sigmodontis gerbil models. We determined that short-course AWZ1066S-albendazole co-treatment significantly augmented the depletion of Wolbachia populations within both germline and hypodermal tissues of B. malayi female worms and in hypodermal tissues in male worms, indicating that anti-Wolbachia synergy is not limited to targeting female embryonic tissues. Our data provides pre-clinical proof-of-concept that sub-seven-day combinations of rapid-acting novel anti-Wolbachia agents with benzimidazole anthelmintics are a promising curative and transmission-blocking drug treatment strategy for filarial diseases of medical and veterinary importance.

Eosinophils Are an Endogenous Source of Interleukin-4 during Filarial Infections and Contribute to the Development of an Optimal T Helper 2 Response.

INTRODUCTION: Interleukin-4 (IL-4) is a central regulator of type 2 immunity, crucial for the defense against multicellular parasites like helminths. This study focuses on its roles and cellular sources during Litomosoides sigmodontis infection, a model for human filarial infections. METHODS: Utilizing an IL-4 secretion assay, investigation into the sources of IL-4 during the progression of L. sigmodontis infection was conducted. The impact of eosinophils on the Th2 response was investigated through experiments involving dblGATA mice, which lack eosinophils and, consequently, eosinophil-derived IL-4. RESULTS: The absence of eosinophils notably influenced Th2 polarization, leading to impaired production of type 2 cytokines. Interestingly, despite this eosinophil deficiency, macrophage polarization, proliferation, and antibody production remained unaffected. CONCLUSION: Our research uncovers eosinophils as a major source of IL-4, especially during the early phase of filarial infection. Consequently, these findings shed new light on IL-4 dynamics and eosinophil effector functions in filarial infections.

The efficacy of the benzimidazoles oxfendazole and flubendazole against Litomosoides sigmodontis is dependent on the adaptive and innate immune system.

Filarial nematodes can cause debilitating diseases such as lymphatic filariasis and onchocerciasis. Oxfendazole (OXF) is one promising macrofilaricidal candidate with improved oral availability compared to flubendazole (FBZ), and OXF is currently under preparation for phase 2 clinical trials in filariasis patients. This study aimed to investigate the immune system's role during treatment with OXF and FBZ and explore the potential to boost the treatment efficacy via stimulation of the immune system. Wild type (WT) BALB/c, eosinophil-deficient ΔdblGata1, IL-4r/IL-5-/-, antibody-deficient µMT and B-, T-, NK-cell and ILC-deficient Rag2/IL-2rγ-/- mice were infected with the rodent filaria Litomosoides sigmodontis and treated with an optimal and suboptimal regimen of OXF and FBZ for up to 5 days. In the second part, WT mice were treated for 2-3 days with a combination of OXF and IL-4, IL-5, or IL-33. Treatment of WT mice reduced the adult worm burden by up to 94% (OXF) and 100% (FBZ) compared to vehicle controls. In contrast, treatment efficacy was lower in all immunodeficient strains with a reduction of up to 90% (OXF) and 75% (FBZ) for ΔdblGata1, 50 and 92% for IL-4r/IL-5-/-, 64 and 78% for µMT or 0% for Rag2/IL-2rγ-/- mice. The effect of OXF on microfilariae and embryogenesis displayed a similar pattern, while FBZ's ability to prevent microfilaremia was independent of the host's immune status. Furthermore, flow cytometric analysis revealed strain-and treatment-specific immunological changes. The efficacy of a shortened 3-day treatment of OXF (-33% adult worms vs. vehicle) could be boosted to a 91% worm burden reduction via combination with IL-5, but not IL-4 or IL-33. Our results suggest that various components of the immune system support the filaricidal effect of benzimidazoles in vivo and present an opportunity to boost treatment efficacy.

Potential of Nucleic Acid Receptor Ligands to Improve Vaccination Efficacy against the Filarial Nematode Litomosoides sigmodontis.

More than two-hundred-million people are infected with filariae worldwide. However, there is no vaccine available that confers long-lasting protection against filarial infections. Previous studies indicated that vaccination with irradiated infective L3 larvae reduces the worm load. This present study investigated whether the additional activation of cytosolic nucleic acid receptors as an adjuvant improves the efficacy of vaccination with irradiated L3 larvae of the rodent filaria Litomosoides sigmodontis with the aim of identifying novel vaccination strategies for filarial infections. Subcutaneous injection of irradiated L3 larvae in combination with poly(I:C) or 3pRNA resulted in neutrophil recruitment to the skin, accompanied by higher IP-10/CXCL10 and IFN-ß RNA levels. To investigate the impact on parasite clearance, BALB/c mice received three subcutaneous injections in 2-week intervals with irradiated L3 larvae in combination with poly(I:C) or 3pRNA prior to the challenge infection. Vaccination with irradiated L3 larvae in combination with poly(I:C) or 3pRNA led to a markedly greater reduction in adult-worm counts by 73% and 57%, respectively, compared to the immunization with irradiated L3 larvae alone (45%). In conclusion, activation of nucleic acid-sensing immune receptors boosts the protective immune response against L. sigmodontis and nucleic acid-receptor agonists as vaccine adjuvants represent a promising novel strategy to improve the efficacy of vaccines against filariae and potentially other helminths.

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.

A qPCR to quantify Wolbachia from few Onchocerca volvulus microfilariae as a surrogate for adult worm histology in clinical trials of antiwolbachial drugs.

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 < 6 months post-treatment would accelerate clinical trials of antiwolbachials. We developed a TaqMan qPCR amplifying the single-copy gene 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.

Corallopyronin A for short-course anti-wolbachial, macrofilaricidal treatment of filarial infections.

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.

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.

Macrophages Mediate Increased CD8 T Cell Inflammation During Weight Loss in Formerly Obese Mice.

Even after successful weight reduction, obese adults tend to quickly regain the lost weight. This raises the question of whether weight loss improves the underlying chronic adipose tissue inflammation characteristic of obesity. In order to improve our understanding of the mechanisms that reshape metabolic organs during weight loss, we investigated the macrophage and T cell function of the liver and adipose tissue on reversing high fat diet (HFD) mice to normal control diet (NCD). Obese mice that were switched to NCD showed an improvement in their metabolic profile that included enhanced glucose and insulin tolerance, decreased cholesterol, triglyceride, serum glutamic-oxaloacetic transaminase (SGOT), and serum glutamic pyruvic transaminase (SGPT) levels that were comparable to NCD controls. However, despite weight loss, increased frequencies, but not total numbers, of IL-17+ and IL-22+ CD4+ T cells, IFN-γ+ and TNF+ CD8+ T cells and IL-17+ and IL-22+ CD8+ T cells were observed in the adipose tissue of mice switched from HFD to NCD compared to NCD and even HFD fed mice. Further, in the liver, IFN-γ+ and TNF+ CD8+ T cell, IL-17+ and IL-22+ CD8+ T cell, macrophage frequencies and their expression of antigen presenting molecules were increased. To determine if macrophages are the major determinants of the sustained inflammation observed during weight loss, we depleted macrophages, which significantly reduced IFN-γ+, TNF+, IL-17+, and IL-22+ CD8+ T cell frequencies in the liver and the adipose tissue. In conclusion, we show that although weight loss improves the metabolic profile, there is an active and ongoing CD8+ T cell inflammation in liver and adipose tissue mediated by macrophages.

In vivo efficacy of the boron-pleuromutilin AN11251 against Wolbachia of the rodent filarial nematode Litomosoides sigmodontis.

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.

Short-course quinazoline drug treatments are effective in the Litomosoides sigmodontis and Brugia pahangi jird models.

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.

In vivo kinetics of Wolbachia depletion by ABBV-4083 in L. sigmodontis adult worms and microfilariae.

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.

Preclinical development of an oral anti-Wolbachia macrolide drug for the treatment of lymphatic filariasis and onchocerciasis.

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.

Discovery of ABBV-4083, a novel analog of Tylosin A that has potent anti-Wolbachia and anti-filarial activity.

There is a significant need for improved treatments for onchocerciasis and lymphatic filariasis, diseases caused by filarial worm infection. In particular, an agent able to selectively kill adult worms (macrofilaricide) would be expected to substantially augment the benefits of mass drug administration (MDA) with current microfilaricides, and to provide a solution to treatment of onchocerciasis / loiasis co-infection, where MDA is restricted. We have identified a novel macrofilaricidal agent, Tylosin A (TylA), which acts by targeting the worm-symbiont Wolbachia bacterium. Chemical modification of TylA leads to improvements in anti-Wolbachia activity and oral pharmacokinetic properties; an optimized analog (ABBV-4083) has been selected for clinical evaluation.

Boron-Pleuromutilins as Anti- Wolbachia Agents with Potential for Treatment of Onchocerciasis and Lymphatic Filariasis.

A series of pleuromutilins modified by introduction of a boron-containing heterocycle on C(14) of the polycyclic core are described. These analogs were found to be potent anti- Wolbachia antibiotics and, as such, may be useful in the treatment of filarial infections caused by Onchocerca volvulus, resulting in Onchocerciasis or river blindness, or Wuchereria bancrofti and Brugia malayi and related parasitic nematodes resulting in lymphatic filariasis. These two important neglected tropical diseases disproportionately impact patients in the developing world. The lead preclinical candidate compound containing 7-fluoro-6-oxybenzoxaborole (15, AN11251) was shown to have good in vitro anti- Wolbachia activity and physicochemical and pharmacokinetic properties providing high exposure in plasma. The lead was effective in reducing the Wolbachia load in filarial worms following oral administration to mice.

Macrofilaricidal efficacy of single and repeated oral and subcutaneous doses of flubendazole in Litomosoides sigmodontis infected jirds.

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.

Combinations of registered drugs reduce treatment times required to deplete Wolbachia in the Litomosoides sigmodontis mouse model.

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.