Clomipramine and Benznidazole Act Synergistically and Ameliorate the Outcome of Experimental Chagas Disease

Abstract

Chagas disease is an important public health problem in Latin America, and its treatment by chemotherapy with benznidazole (BZ) or nifurtimox remains unsatisfactory. In order to design new alternative strategies to improve the current etiological treatments, in the present work, we comprehensively evaluated the in vitro and in vivo anti-Trypanosoma cruzi effects of clomipramine (CMP) (a parasite-trypanothione reductase-specific inhibitor) combined with BZ. In vitro studies, carried out using a checkerboard technique on trypomastigotes (T. cruzi strain Tulahuen), revealed a combination index (CI) of 0.375, indicative of a synergistic effect of the drug combination. This result was correlated with the data obtained in infected BALB/c mice. We observed that during the acute phase (15 days postinfection [dpi]), BZ at 25 mg/kg of body weight/day alone decreased the levels of parasitemia compared with those of the control group, but when BZ was administered with CMP, the drug combination completely suppressed the parasitemia due to the observed synergistic effect. Furthermore, in the chronic phase (90 dpi), mice treated with both drugs showed less heart damage as assessed by the histopathological analysis, index of myocardial inflammation, and levels of heart injury biochemical markers than mice treated with BZ alone at the reference dose (100 mg/kg/day). Collectively, these data support the notion that CMP combined with low doses of BZ diminishes cardiac damage and inflammation during the chronic phase of cardiomyopathy. The synergistic activity of BZ-CMP clearly suggests a potential drug combination for Chagas disease treatment, which would allow a reduction of the effective dose of BZ and an increase in therapeutic safety.

FIG 1

(A) Scheme of the checkerboard test employed. White wells show the combinations of drug concentrations that suppress parasitemia. Light gray wells show the combinations of drugs that reduced parasitemia levels, and dark gray wells show the minimal concentration of each drug that suppresses parasitemia by itself. (B) Isobologram of the combinations of CMP and BZ. The points below the dotted line indicate a synergistic effect. The calculated CI is also displayed. (C) Representative immunofluorescence images of infected BMDM treated with BZ at 8 µg/ml (BZ 8), BZ at 2 µg/ml (BZ 2), or BZ and CMP at 2 µg/ml each (BZ 2 + CMP 2). (D) Quantitative analysis of antiamastigote activity. The error bars show the standard error of the mean. ***, P < 0.001.

FIG 2

Effects of BZ alone and in combination with CMP, at a fixed dose, on parasitemia in infected mice. The values were obtained at 15 dpi. Asterisks indicate a significant difference between the indicated groups (***, P < 0.001). ND, parasites not detected.

FIG 3

Effects of BZ alone and in combination with CMP at a fixed dose in infected mice at 15 dpi. (A) Relative spleen weight. ***, P < 0.001; **, P < 0.01; ns, no significant difference. (B) Relative percentage of CK-MB enzyme with respect to total CK in plasma. The differences between groups were not statistically significant. (C) De Ritis index. The differences between groups were not statistically significant. The error bars show the standard error of the mean.

FIG 4

Effects of BZ at a fixed dose in combination with CMP at different doses in infected mice at 15 dpi. (A) Parasitemia levels. Asterisks indicate a significant difference between the indicated group and the control (***, P < 0.001). ND, parasites not detected. (B) Relative spleen weight. Asterisks indicate the significant differences between the analyzed group and the control of infected/nontreated animals (INT) (***, P < 0.001). (C) Relative percentage of CK-MB enzyme in plasma. The asterisk indicates the significant differences between the tested group and INT (*, P < 0.05). (D) De Ritis index. The differences between groups were not statistically significant. The error bars show the standard error of the mean.

FIG 5

Effects of BZ alone or in combination with CMP in the experimental model of chronic Chagas disease (90 dpi). (A) Relative heart weight. (B) Relative percentage of CK-MB enzyme in plasma. (C) Representative histological sections of noninfected and infected heart tissue stained with hematoxylin and eosin (H&E) from mice treated with BZ alone or BZ-CMP (400× magnification). (D) Inflammatory index of myocardial tissue. The inflammatory index was calculated according to the details outlined in Materials and Methods. (E) Parasite DNA detected by real-time PCR in cardiac tissues. Asterisks indicate the significant differences between the indicated groups. ND, not detected. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant difference.

FIG 6

Effects of BZ alone and in combination with CMP in noninfected mice. (A) Relative liver weight. (B) Levels of GPT activity in plasma. *, P < 0.05; **, P < 0.01.