Avaliação da citotoxicidade do nifur0mox, fexinidazol-sulfona, amiodarona e alopurinol frente à infecção por Trypanosoma cruzi

Abstract

Research on anti-Trypanosoma cruzi chemotherapy has advanced significantly in recent years, culminating in the evaluation of drug candidates in phase II clinical trials for the treatment of chronic Chagas disease. However, due to limitations related to the efficacy or tolerability of candidates, there is still no satisfactory treatment for the chronic phase of the infection. The results obtained with fexinidazole, one of the most promising candidates, are particularly intriguing; it is a nitroimidazole that showed high trypanocidal efficacy and tolerability in preclinical and phase I clinical studies, but when administered to Chagas disease patients, it induced relevant neutropenia and hepatotoxicity. Although the factors involved in the observed therapeutic failures are widely discussed, studies and information available in the literature on the toxic effects of trypanocidal drugs, even those in clinical use, are scarce. Therefore, the aim of this study was to investigate the cytotoxicity profile of drugs with trypanocidal activity using different cell types and incubation times, in the absence or presence of T. cruzi infection. For this purpose, candidates already evaluated in non-clinical and clinical studies were chosen – fexinidazole-sulfone (the active metabolite of fexinidazole), amiodarone, and allopurinol, in addition to nifurtimox, which is one of the reference drugs in the treatment of Chagas disease. H9c2 and HepG2 cells were incubated with the drugs for two different periods, 72 and 120 hours, in the absence and presence of T. cruzi Y strain infection. The viability of host cells was assessed by resazurin reduction compared to untreated and uninfected cells. Additionally, viability assays with Sulforhodamine B and proliferation evaluation under prolonged treatment (192 hours) were performed for fexinidazole-sulfone by determining the CPD (cumulative population doubling). The results of the resazurin assays showed that the toxicity profile varied according to cell type and drug, suggesting a relationship with the mechanisms of action and physiology of the cells. Fexinidazole-sulfone showed low interference with the viability of both cell types, with significant reduction in cell viability from 1mM onwards. Amiodarone exhibited concentration and time-dependent toxic effects, with CC-50 values ranging from 15µM to 41µM. Nifurtimox induced greater toxicity in H9c2 cells, while allopurinol was significantly more toxic for HepG2 cells. Parasite infection did not exert a significant influence on the viability profile of treated cells, likely due to the anti-T. cruzi activity of the drugs at the evaluated concentrations. The determination of HepG2 cell viability using Sulforhodamine B showed a time and concentration-dependent cytotoxic profile for fexinidazole-sulfone, especially at concentrations from 1mM onwards. CPD determination for the same cell type revealed a significant reduction in cell proliferation from 96 hours of treatment with fexinidazolesulfone, particularly at concentrations of 1.0 and 2.0 mM. These results demonstrate, in the context of anti-T. cruzi pharmacological development, the importance of using prolonged treatment time to evaluate drug cytotoxicity and may assist in designing experiments that contribute to increasing the translational value of preclinical studies of new drug candidates.