Short-course, oral flubendazole does not mediate significant efficacy against Onchocerca adult male worms or Brugia microfilariae in murine infection models

Abstract

The Onchocerca ochengi adult implant and Brugia malayi microfilariemic Severe-Combined Immunodeficient (SCID) mouse models are validated screens to measure macrofilaricidal and microfilaricidal activities of candidate onchocerciasis drugs. The purpose of this study was to assess whether 5 daily sub-cutaneous (s.c.) injections of standard flubendazole (FBZ) suspension (10mg/kg), a single s.c. injection (10mg/kg) or 5 daily repeated oral doses of FBZ amorphous solid dispersion (ASD) formulation (0.2, 1.5 or 15mg/kg) mediated macrofilaricidal efficacy against O. ochengi male worms implanted into SCID mice. The direct microfilaricidal activity against circulating B. malayi microfilariae of single dose FBZ ASD formulation (2 or 40 mg/kg) was also evaluated and compared against the standard microfilaricide, ivermectin (IVM). Systemic exposures of FBZ/FBZ metabolites achieved following dosing were measured by pharmacokinetic (PK) bioanalysis. At necropsy, five weeks following start of FBZ SC injections, there were significant reductions in burdens of motile O. ochengi worms following multiple injections (93%) or single injection (82%). Further, significant proportions of mice dosed following multiple injections (5/6; 83%) or single injection (6/10; 60%) were infection negative (drug-cured). In comparison, no significant reduction in recovery of motile adult O. ochengi adult worms was obtained in any multiple-oral dosage group. Single oral-dosed FBZ did not mediate any significant microfilaricidal activity against circulating B. malayi mf at 2 or 7 days compared with >80% efficacy of single dose IVM. In conclusion, multiple oral FBZ formulation doses, whilst achieving substantial bioavailability, do not emulate the efficacy delivered by the parenteral route in vivo against adult O. ochengi. PK analysis determined FBZ efficacy was related to sustained systemic drug levels rather than achievable Cmax. PK modelling predicted that oral FBZ would have to be given at low dose for up to 5 weeks in the mouse model to achieve a matching efficacious exposure profile.

Fig 1

O. ochengi male worm burdens 5 weeks following indicated treatments for experiments (A) and (B). Bars are median recovered numbers per group. Treatment with s.c. injected FBZ 10mg/kg qd 5x mediated a significant reduction in the retrieval of viable O. ochengi worms (Kruskal-Wallis ANOVA = 15.45, Dunn’s tests untreated vs FBZ 10mg/kg qd 5x P<0.05*).

Fig 2

Average O. ochengi metabolic activity per mouse where motile worms were present, five weeks post-dosing with indicated treatments for experiment A (A) or B (B). Data points are mean formazan optical densities @490nm from worms recovered from each animal. Bars are mean +/-SEM per treatment group. Variation between untreated and treatment groups in experiment A was not significantly different (1way ANOVA). Metabolic activity was significantly reduced following FBZ oral 15mg/kg qd x 5 days (Unpaired T-test, *P<0.05) in experiment B.

Fig 3. Average B. malayi microfilariaemias in cardiac blood seven days post-dosing with indicated treatments.

Data points are microfilariaemias per mouse. Bars are mean +/-SEM per treatment group. Variation between untreated and treatment groups was significantly different (1way ANOVA F = 4.6, P = 0.019). Significant differences compared with vehicle controls, evaluated by Dunnett’s tests, are indicated * (P<0.05).

Fig 4. Comparison between oral PK profile (dashed red lines) and s.c. injection PK profile (solid black line) in CB.

17 SCID mice as assessed from PK modelling of exposure data. (Top left) FBZ PK after 10mg/kg qd 5x s.c. FBZ or 15mg/kg qd 5x oral FBZ. (Top right) H-FBZ PK after 10mg/kg qd 5x s.c. FBZ or 15mg/kg qd 5x oral FBZ. (bottom left) R-FBZ PK after 10mg/kg qd 5x s.c. or 15mg/kg qd 5x oral FBZ. (bottom right) Simulated FBZ PK profile after 10mg/kg qd 5x s.c. or 5 week, 4 times daily (qds) regimen of 0.5mg/kg oral FBZ.