Desenvolvimento de uma estratégia de avaliação in vitro aplicável à verificação do potencial anti- Trypanosoma cruzi de candidatos a fármacos

Abstract

The failure observed in clinical studies with the most promising trypanosomal drug candidates to date has shown the need for greater understanding of the factors that interfere in the selection and analysis of new drug candidates for Chagas disease. However, the translational value of screening strategies in the field of in vitro experimentation has been little explored. In this sense, the objectives of the present study are to evaluate the influence of the host cell type on the anti-T. cruzi activity of drugs and to support the standardization of an in vitro strategy of high translational value for identifying the therapeutic potential of new molecules. Two cell lines were used, Vero and H9c2, infected by the Y strain of T. cruzi, incubated or not with drugs of recognized trypanosomicidal action in vitro and in vivo, benznidazole, reference drug, and those selected in clinical studies, fex -sulfone, ravuconazole and posaconazole. This model was used mainly for the determination of the EC-50 and EC-90 values of the drugs comparatively in the two types of cell phones in 48-hour trials using 24-well plates. Subsequently, a plate test was developed to verify the suppression and recrudescence of parasitism after prolonged in vitro treatment. To standardize this strategy, the same cell models and drugs were used, however, as cells were kept in 90mm plates and treated for 7 days, when they were subjected to washout. The plates were evaluated daily by optical microscopy up to 12 days after treatment for detection of parasitism reactivation. After that period, cells were collected to determine the parasitic load by qPCR. The results obtained suggest that the activity of the drugs is influenced by the cell type, with the EC-50 values at least twice lower for the Vero strain. This profile was confirmed in the plaque assay, in which it was possible to detect suppression of parasitism during treatment with all drugs and reactivation after treatment with nitroimidazoles in both cell types. However, reactivation in H9c2 occurred previously, and the parasitic load detected by post- incubation qPCR with nitroimidazoles and ravuconazole was significantly lower in Vero

cells. The results obtained suggest that the host cell type influences the response of the parasite to the drugs and allowed the identification of an in vitro strategy applicable to the selection of candidates for the treatment of Chagas disease before progressing to preclinical studies in vivo.