The repositioned drugs disulfiram/diethyldithiocarbamate combined to benznidazole: Searching for Chagas disease selective therapy, preventing toxicity and drug resistance

Abstract

Chagas disease (CD) affects at least 6 million people in 21 South American countries besides several thousand in other nations all over the world. It is estimated that at least 14,000 people die every year of CD. Since vaccines are not available, chemotherapy remains of pivotal relevance. About 30% of the treated patients cannot complete the therapy because of severe adverse reactions. Thus, the search for novel drugs is required. Here we tested the benznidazole (BZ) combination with the repositioned drug disulfiram (DSF) and its derivative diethyldithiocarbamate (DETC) upon Trypanosoma cruzi in vitro and in vivo. DETC-BZ combination was synergistic diminishing epimastigote proliferation and enhancing selective indexes up to over 10-fold. DETC was effective upon amastigotes of the BZ- partially resistant Y and the BZ-resistant Colombiana strains. The combination reduced proliferation even using low concentrations (e.g., 2.5 µM). Scanning electron microscopy revealed membrane discontinuities and cell body volume reduction. Transmission electron microscopy revealed remarkable enlargement of endoplasmic reticulum cisternae besides, dilated mitochondria with decreased electron density and disorganized kinetoplast DNA. At advanced stages, the cytoplasm vacuolation apparently impaired compartmentation. The fluorescent probe H₂-DCFDA indicates the increased production of reactive oxygen species associated with enhanced lipid peroxidation in parasites incubated with DETC. The biochemical measurement indicates the downmodulation of thiol expression. DETC inhibited superoxide dismutase activity on parasites was more pronounced than in infected mice. In order to approach the DETC effects on intracellular infection, peritoneal macrophages were infected with Colombiana trypomastigotes. DETC addition diminished parasite numbers and the DETC-BZ combination was effective, despite the low concentrations used. In the murine infection, the combination significantly enhanced animal survival, decreasing parasitemia over BZ. Histopathology revealed that low doses of BZ-treated animals presented myocardial amastigote, not observed in combination-treated animals. The picrosirius collagen staining showed reduced myocardial fibrosis. Aminotransferase de aspartate, Aminotransferase de alanine, Creatine kinase, and urea plasma levels demonstrated that the combination was non-toxic. As DSF and DETC can reduce the toxicity of other drugs and resistance phenotypes, such a combination may be safe and effective.

Keywords: Chagas disease; Diethyldithiocarbamate; Trypanosoma cruzi; chemotherapy; disulfiram; drug combination; repositioning.

Figure 1

Evaluation of the activity of sodium diethyldithiocarbamate (DETC) and benznidazole (BZ) in the in vitro proliferation of Trypanosoma cruzi epimastigote forms (10⁷ parasites/mL, Y strain). The proportions of the combinations were: 5 x IC₅₀ DETC (A); 5 x IC₅₀ BZ (B), 4 x IC₅₀ DETC + IC₅₀ BZ (C), 3 x IC₅₀ DETC + 2 x IC₅₀ BZ (D), 2 x IC₅₀ DETC + 3 x IC₅₀ BZ (E) and IC₅₀ DETC + 4 x IC₅₀ BZ (F). After 24h incubation, the inhibitory effects were determined using Alamar Blue.

Figure 2

Representative isobologram demonstrating the synergistic interaction between DETC and BZ upon T. cruzi (Y strain) epimastigote forms, cultured for 24 h, based on IC₅₀ values.

Figure 3

Evaluation of the in vitro proliferation of T. cruzi epimastigote forms (Y strain), challenged with DETC and BZ combined or alone. 10⁵ parasites were incubated for 5 days with 5 µM DETC, 5 µM BZ and the combination of 2.5 µM each compound. The effects of the compounds were evaluated by daily quantitation of parasites by light microscopy. Inset displays 72-120 h data in a different scale. The combined drugs effectivity was significant, (*p < 0.05; **p < 0.01), despite the reduced concentrations, as compared to the parasites treated with BZ by the 2-way ANOVA. Data represent the mean ± SD (n = 3).

Figure 4

Scanning electron microscopy of T. cruzi epimastigote forms (Y strain). (A) Untreated control, showing normal parasite morphology. B–F) Parasites treated with 200 µM DETC for 24 h, showing cellular disorganization (B, C) and plasma membrane discontinuities (D–F, arrowheads), associated with cell body rounding and volume reduction. Bars correspond to 1 µm.

Figure 5

Transmission electron microscopy of T. cruzi epimastigotes (Y strain) incubated with 200 µM DETC for 24 h. Contrary to control cells (A, arrows – endoplasmic reticulum cisternae; m- mitochondria; N- nuclei), DETC treated (B–G) displayed remarkably enlarged endoplasmic reticulum cisternae (B–E*), often replacing an even distorting nuclei (B, C). Some parasites displayed electroluscent nuclear matrix with aggregated peripheral dense chromatin (C, arrow). Mitochondria were enlarged and generally presented reduced electrondensity (D–F) and eventually presenting kinetoplast (k) DNA (kDNA) disorganized fibers (F- arrows). Some cells displaying condensed mitochondria presenting swollen cristae (C, G-arrows) were also observed. In the final stage of treatment, parasites showed loss of normal compartmentation (G).

Figure 6

Detection of cellular reactive oxygen species (ROS) in T. cruzi epimastigote forms (Y strain) using the H₂DCFDA probe accessed by fluorescence microscopy (B, D). (A, B) - Control and (C, D)- 10µM DETC-treated. (A, C) - phase contrast images. Note intense and compartmented staining in DETC-treated parasites. Magnification - 400X.

Figure 7

Measurement of lipid peroxidation in T. cruzi epimastigote forms (Y strain) by determination of thiobarbituric acid reactive substances (TBARS). Parasite cells treated with 10 µM DETC and 10 µM BZ combination for 24 h presented a significantly (*p < 0.05, ANOVA and Dunn’s post-test) increased lipoperoxidation. Bars represent mean ± SD (n = 4).

Figure 8

Effect of DETC and BZ on the concentration of low mol. wt. thiols of T. cruzi epimastigote forms (Y strain), determined colorimetrically by the Ellman reaction. DETC reduced thiol levels dose-dependently (A). The DETC + BZ combination significantly potentiated thiol depletion (B). Data represent mean ± SD. *p < 0.05 and ***p < 0.001 (ANOVA and Dunn’s post-test; n = 5).

Figure 9

Inhibitory effect of DETC on the proliferation of T. cruzi amastigote forms in Y (A) and Colombiana (B) strains, after incubations of 120 h and 24 h, respectively. * p < 0.05, **p < 0.01 and *** indicates p < 0.001 ANOVA and Dunn’s post-test. Bars represent the mean ± SD (n=3).

Figure 10

Effect of DETC alone or combined with BZ on the T. cruzi-macrophage interaction. BALB/c mice peritoneal macrophages were infected with Colombiana strain bloodstream trypomastigote forms 1:10 phagocyte/parasite ratio. Control cultures (A) showing several parasitized macrophages as well as larger number of intracellular forms per phagocyte (arrowheads) than cells treated with the combination 5 µM DETC + 5 µM BZ (B). Magnification 200X. The association index in cultures treated with 10 µM BZ or the 5 µM DETC + 5 µM BZ combination after 24 hours of incubation (C) was significantly (***p < 0.001, ANOVA and Dunn’s post-test) reduced. A slightly greater inhibition was observed in cultures incubated with the combination, despite the reduced concentrations. Bars represent mean ± SD.

Figure 11

Parasitemia of Swiss Webster mice infected with T. cruzi blood trypomastigotes, Y strain (A, B) and Colombiana (C). The animals were infected with 10⁴ blood trypomastigotes/mL, strain Y, treated for 60 days with vehicle; low doses of BZ (20 mg/kg); DETC (20 mg/kg) or a combination of BZ + DETC (20:20 mg/kg), orally. *p < 0.05 and **p < 0.01 ANOVA test and Dunn’s post-test (A). Parasites treated with vehicle; BZ (50 mg/kg); DSF (50 mg/kg); combination of BZ + DSF (50:50 mg/kg); and BZ + DSF (50:100 mg/kg), administered for 10 consecutive days (B, C, p > 0.05). Insert shows data in a different scale. The parasites were counted daily (A) or weekly (B, C) on Neubauer chambers and the graphs were plotted in GraphPad Prism, 7.

Figure 12

Monitoring the cumulative survival of mice infected intraperitoneally with 10⁴ T. cruzi blood trypomastigote/mL (Y strain) of and treated with 10 mg/kg/day of DETC and BZ alone or in combination, p.o. daily for 60 consecutive days. Note that animals treated with the combination had a survival rate of 60%, whereas the group treated with BZ was 10% and the control group died on the 14^th day. ***p < 0.001 ANOVA and Tukey post-test.

Figure 13

Histopathological analysis (H&E) of murine myocardium T. cruzi (Y strain)-infected mice, treated for 60 consecutive days, p.o. with vehicle (A); 10 mg/kg/d BZ-treated and (B) treated with the of 10 mg/kg/d DETC combined to 10 mg/kg/d BZ (C). Amastigote nests were observed in A and B (arrowheads). Skeletal muscle of mice infected with Colombiana strain, for 100 d and treated for 30 consecutive days orally with 20 mg/kg/d BZ (D), 20 mg/kg/d DSF (E) or combination of 10 mg/kg/d each (F). Picro Sirius red staining was employed to demonstrate fibrosis. The combination using lowered concentrations was associated with reduced fibrosis (F). Magnification: 200X.

Figure 14

Systemic toxicity of mice treated with DSF and BZ, alone and in combination, by determining blood levels of creatine kinase (A), urea (B), alanine aminotransferase (C) and aspartate aminotransferase (D); after 20 mg/kg BZ or DSF and the 10 mg/kg/d BZ + 10 mg/kg/d DSF. Contrary to isolated drugs, the combination, employing lowered dosages, did not increase plasma levels.