An In Vitro/In Vivo Model to Analyze the Effects of Flubendazole Exposure on Adult Female Brugia malayi

Abstract

Current control strategies for onchocerciasis and lymphatic filariasis (LF) rely on prolonged yearly or twice-yearly mass administration of microfilaricidal drugs. Prospects for near-term elimination or eradication of these diseases would be improved by availability of a macrofilaricide that is highly effective in a short regimen. Flubendazole (FLBZ), a benzimidazole anthelmintic registered for control of human gastrointestinal nematode infections, is a potential candidate for this role. FLBZ has profound and potent macrofilaricidal effects in many experimental animal models of filariases and in one human trial for onchocerciasis after parental administration. Unfortunately, the marketed formulation of FLBZ provides very limited oral bioavailability and parenteral administration is required for macrofilaricidal efficacy. A new formulation that provided sufficient oral bioavailability could advance FLBZ as an effective treatment for onchocerciasis and LF. Short-term in vitro culture experiments in adult filariae have shown that FLBZ damages tissues required for reproduction and survival at pharmacologically relevant concentrations. The current study characterized the long-term effects of FLBZ on adult Brugia malayi by maintaining parasites in jirds for up to eight weeks following brief drug exposure (6-24 hr) to pharmacologically relevant concentrations (100 nM-10 μM) in culture. Morphological damage following exposure to FLBZ was observed prominently in developing embryos and was accompanied by a decrease in microfilarial output at 4 weeks post-exposure. Although FLBZ exposure clearly damaged the parasites, exposed worms recovered and were viable 8 weeks after treatment.

Fig 1. Effect of FLBZ on recovery of exposed and transplanted adult B. malayi male and female worms from the peritoneal cavity of naïve jirds.

A. Five day, four week, or eight week maintenance in jirds after transplantation following 24 hr in culture. Data is a combination of two experiments. In experiment one 20 worms (10 male and ten female) were transplanted into each of three jirds for all treatment groups and maintained for 5 days or 4 weeks. Thirty worms (15 male and 15 female) were transplanted into each of three jirds per treatment group in experiment two and maintained for 4 weeks or 8 weeks. The 4 week data from each experiment did not differ significantly, therefore, they were combined for presentation purposes. B. Worm recovery eight weeks after transplantation following 6, 12, or 24 hr exposure to FLBZ in vitro. Thirty worms (15 male and 15 female) were transplanted into each of three jirds per treatment group, following the appropriate duration of in vitro exposure to FLBZ. Bars indicate the mean recovery of worms, both male and female, from each jird in a treatment group. Lines represent the standard deviation.

Fig 2. Effects of FLBZ on recovery of microfilariae from the peritoneal cavity of jirds.

A. Five day, four week, or eight week maintenance after transplantation into naïve jirds following 24 hr culture. Data is a combination of two experiments. Microfilariae were enumerated from the peritoneal wash of each of three jirds which had 10 female worm transplanted in experiment one (5 day and 4 week maintenance) and 15 females in experiment two (4 and 8 week maintenance). Data were corrected for number of females transplanted. The 4 week data from each experiment did not differ significantly, therefore, they were combined for presentation purposes. B. Recovery eight weeks post-transplantation following 6, 12, or 24 hr exposure to FLBZ in vitro. Microfilariae were enumerated from peritoneal washes of each of three jirds which had 15 females worms transplanted, following the appropriate duration of in vitro exposure to FLBZ. Bars indicate the mean mf counts from each jird in a treatment group. Lines represent the standard deviation.

Fig 3. Effects of FLBZ on B. malayi embryogenesis.

A. Embryograms for untreated and FLBZ-treated (24 hr exposure) females recovered four weeks post-transplantation. Two to four worms per treatment group were used for embryogram analysis. Data shown are % of oocytes, early and late morulae, pretzels and stretched mf in worm homogenates. Significantly more early developing embryos were observed in treated groups (P < 0.001). B. Proportion of degenerating intrauterine stages.

Fig 4. Long-term effects of FLBZ on developing embryos.

Exposure period was 24 hr in culture prior to re-implantation. Time to recovery after transplant was 4 weeks. A. Oocytes, DMSO control; B. Morulae, DMSO control; C. Oocytes, 100 nM; D. Early Morulae, 1 μM; E. Late Morulae, 100 nM; F. Microfilariae, 10 μM.