Arylimidamide DB766, a potential chemotherapeutic candidate for Chagas' disease treatment

Abstract

Chagas' disease, a neglected tropical illness for which current therapy is unsatisfactory, is caused by the intracellular parasite Trypanosoma cruzi. The goal of this work is to investigate the in vitro and in vivo effects of the arylimidamide (AIA) DB766 against T. cruzi. This arylimidamide exhibits strong trypanocidal activity and excellent selectivity for bloodstream trypomastigotes and intracellular amastigotes (Y strain), giving IC(50)s (drug concentrations that reduce 50% of the number of the treated parasites) of 60 and 25 nM, respectively. DB766 also exerts striking effects upon different parasite stocks, including those naturally resistant to benznidazole, and displays higher activity in vitro than the reference drugs. By fluorescent and transmission electron microscopy analyses, we found that this AIA localizes in DNA-enriched compartments and induces considerable damage to the mitochondria. DB766 effectively reduces the parasite load in the blood and cardiac tissue and presents efficacy similar to that of benznidazole in mouse models of T. cruzi infection employing the Y and Colombian strains, using oral and intraperitoneal doses of up to 100 mg/kg/day that were given after the establishment of parasite infection. This AIA ameliorates electrocardiographic alterations, reduces hepatic and heart lesions induced by the infection, and provides 90 to 100% protection against mortality, which is similar to that provided by benznidazole. Our data clearly show the trypanocidal efficacy of DB766, suggesting that this AIA may represent a new lead compound candidate to Chagas' disease treatment.

FIG. 1.

Chemical structure of DB766.

FIG. 2.

Activity of DB766 upon bloodstream and intracellular forms of Trypanosoma cruzi (Y strain) in vitro. (A, B) Activity of DB766 upon bloodstream trypomastigotes during the treatment at 4°C with the drug diluted with 96% of mouse blood (A) and during the treatment at 37°C with the drug diluted in the culture medium (B). The percentages of dead parasites were measured after 2 h and 24 h of treatment. (C to F) Activity of DB766 on T. cruzi-infected cardiomyocytes (C), with illustrations showing untreated cultures (D) and cultures treated for 48 h with 0.04 μM (E) and 10.6 μM (F) doses of DB766. Arrows indicate intracellular forms. Bar = 1 μM.

FIG. 3.

Fluorescent and transmission electron microscopy analyses of the effect of DB766 on trypomastigotes of T. cruzi. Differential interference contrast (A) and fluorescent (B) analyses showing intracellular localization within bloodstream trypomastigotes of T. cruzi after treatment for 1 h at 37°C with 10 μg/ml DB766. Note the localization of this arylimidamide within the kDNA (arrow) and nuclei (asterisk). (C to F) Transmission electron microscopy showing mitochondrial alteration, with swollen cristae (arrows) in bloodstream-treated parasites (E, F), while untreated parasites display characteristic morphology (C, D). N, nuclei; K, kDNA. Bar = 0.5 μM.

FIG. 4.

Effect of DB766 in T. cruzi infection (Y strain) in vivo. The activity of 25 and 50 mg/kg/day DB766 (i.p.) and 100 mg/kg/day benznidazole (by p.o. gavage) was evaluated upon T. cruzi infection in male Swiss mice inoculated with 10⁴ bloodstream trypomastigotes, using therapy regimens of 2 doses (5 and 8 dpi) (A, B) and 10 daily doses (starting at 5 dpi) (C, D). Parasitemia curves (A to C) and mortality rates (B to D) are presented. par, parasites.

FIG. 5.

Effect of DB766 on T. cruzi infection (Colombian strain) in vivo. Activity of 25 and 50 mg/kg/day DB766 and 100 mg/kg/day benznidazole upon parasitemia curves of T. cruzi infection in female C3H mice inoculated with 10² (A) and 10³ (B, C) bloodstream trypomastigotes using a regimen of 10 daily doses (starting at parasitemia onset). DB766 was administrated by i.p. (A, B) and p.o. (C) routes, while Bz (A to C) was given only by gavage.

FIG. 6.

Effect of daily administration of DB766 on T. cruzi infection in vivo. Activity of 100 mg/kg/day DB766 (i.p. and p.o.) and benznidazole (p.o.) upon T. cruzi infection in male C3H mice inoculated with 5 × 10³ bloodstream trypomastigotes (Colombian strain) using 10 daily doses, starting at the parasitemia onset. (A) Parasitemia curve; (B) mortality rates; (C) cardiac parasitism; (D) cardiac inflammation; (E) plasma CK levels; (F) cardiac frequency; (G) plasma GPT levels; (H) ponderal curve. U/l, units/liter.

FIG. 7.

Effect of e.o.d. treatment of DB766 on T. cruzi infection in vivo. Activity of 100 mg/kg/day DB766 (i.p.) and benznidazole (p.o.) upon T. cruzi infection in male Swiss mice inoculated with 10⁴ bloodstream trypomastigotes (Y strain) and treated e.o.d., starting at the parasitemia onset. (A) Parasitemia curve; (B) mortality rates; (C) cardiac parasitism; (D) cardiac inflammation; (E) plasma CK levels; (F) plasma GPT levels; (G) ponderal curve.