Broadening the spectrum of ivermectin: Its effect on Trypanosoma cruzi and related trypanosomatids

Abstract

Chagas disease is an endemic American parasitosis, caused by Trypanosoma cruzi. The current therapies, benznidazole (BZN) and nifurtimox (NFX), show limited efficacy and multiple side effects. Thus, there is a need to develop new trypanocidal strategies. Ivermectin (IVM) is a broad-spectrum antiparasitic drug with low human and veterinary toxicity with effects against T. brucei and Leishmania spp. Considering this and its relatively low cost, we evaluate IVM as a potential repurposed trypanocidal drug on T. cruzi and other trypanosomatids. We found that IVM affected, in a dose-dependent manner, the proliferation of T. cruzi epimastigotes as well as the amastigotes and trypomastigotes survival. The Selectivity Index for the amastigote stage with respect to Vero cells was 12. The IVM effect was also observed in Phytomonas jma 066 and Leishmania mexicana proliferation but not in Crithidia fasciculata. On the epimastigote stage, the IVM effect was trypanostatic at 50 μM but trypanocidal at 100 μM. The assays of the drug combinations of IVM with BNZ or NFX showed mainly additive effects among combinations. In silico studies showed that classical structures belonging to glutamate-gated Cl channels, the most common IVM target, are absent in kinetoplastids. However, we found in the studied trypanosomatid genomes one copy for putative IMPα and IMPβ, potential targets for IVM. The putative IMPα genes (with 76% similarity) showed conserved Armadillo domains but lacked the canonical IMPβ binding sequence. These results allowed us to propose a novel molecular target in T. cruzi and suggest IVM as a good candidate for drug repurposing in the Chagas disease context.

Keywords: Chagas disease; Trypanosoma cruzi; drug combination; drug repurposing; ivermectin; trypanocidal drug.

Figure 1

Cell proliferation recovery assay to test the effects of ivermectin (IVM) when exposed for short periods of time. T. cruzi epimastigotes were preincubated with (A) IVM 50 μM (4xEC50_72h) or (B) IVM 100 μM (8xEC50_72h) for 30 min, 1 or 3 h and then transferred to a drug-free medium for 8 days of culture. Untreated control: 0 μM IVM (0.5% dimethyl sulfoxide); positive control: 50 or 100 μM IVM during the 8 days of culture. Results are expressed as the number of epimastigotes (10⁶)/ml, counted in a hemocytometer chamber. ***p<0.001, *p<0.05, ANOVA, Dunnett test.

Figure 2

Comparison between the in vitro and predicted effects of drug combinations on T. cruzi epimastigote proliferation. A combination matrix was designed with IVM (0–50 µM) and benznidazole (BZN; 0–100 µM) or nifurtimox (NFX; 0–5 µM), and the drug combination effect was evaluated on the culture proliferation of epimastigotes at day 4. The results obtained in the in vitro culture assay were compared against the synergy scores predicted by applying the Bliss independence model for: (A) IVM-NFX and (B) IVM-BZN combined simultaneously; (C) IVM-NFX and (D) IVM-BZN with the preincubation of IVM for 1 h; (E) NFX-IVM and (F) BZN-IVM with preincubation of the trypanocidal drug for 1 h; and (G) by applying Loewe additivity model for the combination of NFX-BZN simultaneously. The scale of numbers and colors represents the type of effect obtained according to the comparison between in vitro and model-predicted results; the scale spans from statistically significant synergistic interaction (positive values—blue color, p<0.01) to statistically significant antagonism (negative values—red color, p<0.01).