Benznidazole

Benznidazole (Sigma, St Louis, MO) and Posaconazole (purified from oral suspension Noxafil) were solubilized in 10% solutol (Kolliphor, Sigma, St Louis, MO), and administered once a day orally, at 100 mg/kg for Benznidazole, and 20 mg/kg for Posaconazole. A solution of 10% solutol (just the vehicle) was administered to control, T. cruzi- infected groups. The treatment of mice acutely infected with T. cruzi Sylvio X10/7 strain started 6 days post-infection (dpi). For analysis at the chronic stage of infection, mice infected with T. cruzi Brazil strain were treated starting at 60 days post infection. Mice were treated for 21 days. In both approaches, survival, general health, and the weight of mice was monitored for one year after the end of treatment.

Benznidazole (Sigma-Aldrich Co. LLC, USA) and nifurtimox (Sigma-Aldrich) were dissolved in DMSO and dispensed into 96 well microplate, and T. cruzi axenic amastigotes in LIT medium (1 × 10⁶ cells in 100 μL) was added to each well. The final concentration of DMSO was 0.5%. After 48 h of incubation at 37°C under 5% CO₂ in a humidified incubator, 10 μL of resazurin solution (Sigma-Aldrich) was added at a final concentration of 3 mM[24 (link)]. The plates were incubated for additional 5 h, and the reaction was stopped by addition of 50 μL 3% SDS. Amount of resorufin was quantitated by scanning the microplate by SpectraMax Gemini fluorescent plate reader (Molecular Devices, LLC., USA) at ex. 560 nm/em. 590 nm. EC50 was calculated by fitting the dose response curves with non-linear regression analysis, using "(inhibitor) vs. normalized response" model of GraphPad Prism7 software (GraphPad Software Inc., USA).

A selectivity index (SI) was calculated and is defined as the RAW IC₅₀ value divided by the T. cruzi IC₅₀ value (IS = CC₅₀/IC₅₀), which expresses the safety index of the tested substance³⁷ (link). Benznidazole (Sigma-Aldrich, Milan, Italy) was kept as a positive control drug for the cytotoxicity assay on RAW 264.7.