Miltefosine and Benznidazole Combination Improve Anti- Trypanosoma cruzi In Vitro and In Vivo Efficacy

Abstract

Drug repurposing and combination therapy have been proposed as cost-effective strategies to improve Chagas disease treatment. Miltefosine (MLT), a synthetic alkylphospholipid initially developed for breast cancer and repositioned for leishmaniasis, is a promising candidate against Trypanosoma cruzi infection. This study evaluates the efficacy of MLT as a monodrug and combined with benznidazole (BZ) in both in vitro and in vivo models of infection with T. cruzi (VD strain, DTU TcVI). MLT exhibited in vitro activity on amastigotes and trypomastigotes with values of IC_{50 =} 0.51 µM (0.48 µM; 0,55 µM) and LC_{50 =} 31.17 µM (29.56 µM; 32.87 µM), respectively. Drug interaction was studied with the fixed-ration method. The sum of the fractional inhibitory concentrations (ΣFICs) resulted in ∑FIC= 0.45 for trypomastigotes and ∑FIC= 0.71 for amastigotes, suggesting in vitro synergistic and additive effects, respectively. No cytotoxic effects on host cells were observed. MLT efficacy was also evaluated in a murine model of acute infection alone or combined with BZ. Treatment was well tolerated with few adverse effects, and all treated animals displayed significantly lower mean peak parasitemia and mortality than infected non-treated controls (p<0.05). The in vivo studies showed that MLT led to a dose-dependent parasitostatic effect as monotherapy which could be improved by combining with BZ, preventing parasitemia rebound after a stringent immunosuppression protocol. These results support MLT activity in clinically relevant stages from T. cruzi, and it is the first report of positive interaction with BZ, providing further support for evaluating combined schemes using MLT and exploring synthetic alkylphospholipids as drug candidates.

Keywords: Chagas disease; Trypanosoma cruzi; benznidazole; drug combination chemotherapy; drug repositioning; miltefosine.

Figure 1

In vitro activity of miltefosine (MLT) and benznidazole (BZ) combination on (A-B) trypomastigotes and intracellular amastigote development (C-D) of Trypanosoma cruzi (VD strain). (A) LC₅₀ value from BZ combined with variable concentrations of MLT.(B) LC₅₀ value from MLT combined with variable concentrations of BZ. (C) IC₅₀ value from BZ combined with variable concentrations of MLT. (D) IC₅₀ value from MLT combined with variable concentrations of BZ. Values are expressed in µM and reported as the mean concentration and the 95% confidence interval (IC95%).

Figure 2

Effect of miltefosine, benznidazole, or nifurtimox on parasitemia course in BALB/cJ mice infected with Trypanosoma cruzi (VD strain). Values are expressed as mean trypomastigotes/mL (± SD) in peripheral blood from experimental groups according to the days after infection (dpi). Mice were inoculated with 500 trypomastigotes of the VD strain of Trypanosoma cruzi, and treatment started at parasitemia onset (8th dpi). During treatment and up to 10 days post-treatment, parasitemia was evaluated by fresh blood examination (FBE) to determine parasitemia rebound. Animals with negative parasitemia were submitted to immunosuppression consisting of four doses of cyclophosphamide (CYP; 200 mg/kg; ip route), separated by one week. Parasitemia was evaluated during the CYP cycle and up to 7 days after the last dose. The asterisks indicate significant differences compared to NT and MLT 25 mg/kg/day groups (Kruskal-Wallis, p < 0.05). Grey shading indicates the treatment period. Arrows indicate CYP administration cycle. NT, infected non-treated. MLT, miltefosine. BZ, benznidazole. NFX, nifurtimox.

Figure 3

Effect of miltefosine (alone or in combined regimes), benznidazole, or nifurtimox on amastigote nest burden in BALB/cJ mice infected Trypanosoma cruzi, (VD strain). Mice with the presence or absence of amastigote nests in skeletal muscle (A) and heart samples (B). NT, infected non-treated. MLT, miltefosine. BZ, benznidazole. NFX, nifurtimox.