Efic?cia da combina??o de ravuconazol ou seu pr?-f?rmaco E1224 com o metab?lito sulfona do fexinidazol no tratamento da infec??o experimental por Trypanosoma cruzi.

Programa de P?s-Gradua??o em Ci?ncias Biol?gicas. N?cleo de Pesquisas em Ci?ncias Biol?gicas, Pr?-Reitoria de Pesquisa de P?s Gradua??o, Universidade Federal de Ouro Preto.O objetivo do presente estudo foi avaliar o efeito do tratamento com o derivado sulfona do fexinidazol (SfN) em combina??o com o E1224 (pr?-f?rmaco do ravuconazol- Rv) na infec??o experimental por T. cruzi. Ap?s a determina??o dos valores de IC-50 de SfN e Rv sobre a infec??o de c?lulas H9c2 pela cepa Y, foi determinada a natureza da intera??o entre esses f?rmacos in vitro. As c?lulas infectadas foram incubadas com os diferentes f?rmacos isoladamente ou combinados em propor??es fixas. SfN e Rv n?o apresentaram intera??o in vitro na elimina??o de formas amastigotas, resultando em efeito aditivo (m?dia de ?FIC= 1,08?0,7). Adicionalmente, as combina??es de f?rmacos n?o induziram citotoxicidade em c?lulas H9c2 na aus?ncia de infec??o. A partir desses dados foi investigado o efeito in vivo das combina??es usando modelo murino de infec??o aguda. Nessa etapa camundongos Swiss f?meas foram inoculados com formas tripomastigotas sangu?neas da cepa Y de T. cruzi. Ap?s a detec??o da parasitemia os animais foram tratados com doses sub?timas de E1224 (2,5 e 5 mg por Kg de peso corporal-mg/Kg) e fex-sulfona (25 e 50mg/Kg), isoladamente ou em combina??o por 20 dias. Foi tamb?m avaliado o efeito do tratamento na resposta imune humoral, por meio da detec??o de anticorpos IgG anti-T-cruzi aos 180 dias ap?s o tratamento. Os resultados mostraram que todos os tratamentos foram bem tolerados e eficientes em proteger os animais da mortalidade que acometeu 100% dos animais infectados e n?o tratados. A subdoses de SfN e E1224 quando combinadas foram mais eficientes em induzir cura do que quando usadas em monoterapia. Nos grupos tratados com E1224 nas doses de 2,5 e 5 mg/Kg, foi observada cura em 2/7 (28,5%) e 3/7 (42,8%) dos animais, respectivamente. No caso dos tratamentos com SfN, foi observada reativa??o natural da parasitemia em todos os animais. De forma interessante, nos grupos de animais tratados com ambas as combina??es, 85,7% de cura foi observada, percentual pr?ximo do obtido com a utiliza??o de doses ?timas de cada f?rmaco. Esses resultados demonstraram o benef?cio resultante da administra??o combinada de SfN e E1224 in vivo e indicaram que combina??es aditivas in vitro devem ser submetidas a avalia??es in vivo.The drugs used to treat Chagas? disease, benznidazole and nifurtimox, have limitations related to variable efficacy, long treatment courses and toxicity. In the last years, a number of compounds have been evaluated in preclinical studies, however, few of them were able to induce parasitological cure. New safe and effective treatments are urgently needed. Nowadays, combination therapy is the most promising strategy to treat Chagas disease. In this way, the aim of this study was to evaluate the effect of drug combinations using fexinidazole-sulfone and ravuconazole (or E1224, the ravuconazole prodrug) upon T. cruzi infection in vitro and in vivo. First, the nature of Rv and SfN interaction was assessed in a standard 72hr assay. H9c2 cells infect with Y T. cruzi strain were incubed with the drugs isolated and in combination using a modified fixed ratio. To classify the interactions, the fractional inhibitory concentration (FIC) and sum FICs ?FIC were calculated to each combination. The drug combinations were considered synergistic for ?FIC4. The SfN/Rv interaction in vitro can be classified as an additive effect,(mean ?FIC= 1,08?0,7). Considering theses results the in vivo effect of the same combinations using a murine model of acute T. cruzi infection was verified. Female Swiss mice were infected with blood trypomastigotes of Y strain. Oral treatment of infected animals was administered at the detection of parasitemia, 4thday post-inoculation. Suboptimal doses of SfN (25 and 50 mg per kilogram of bodyweight-mg/Kg) and E1224 (5 and 2.5mg/Kg) were administered in monotherapy or combinations for 20 days. The results demonstrated that all treatments were well tolerated and protected the mice of mortality. The combined treatments were more efficient in induce parasitological cure than the same doses in monotherapy. The treatments with sulfone at different doses were unable to induce parasitological cure, while in mice treated with E1224 at 2.5 and 5.0 mg/Kg a cure rate of 28,5% and 42,8% was obtained, respectively. On the other hand, the combined treatments using different doses induced 85.7% of cure in T. cruzi infected mice. Interesting, the combined therapy using suboptimal doses of each compound induce cure indexes near to detected among animals that received each drug alone, but in the optimal doses. These results showed the beneficial effects of combination therapy using SfN and E1224 and indicated that in vitro effect can be predictive of the in vivo interaction between different compound

Antitrypanosomal activity of fexinidazole metabolites, potential new drug candidates for Chagas disease.

This study was designed to verify the in vivo efficacy of sulfoxide and sulfone fexinidazole metabolites following oral administration in a murine model of Chagas disease. Female Swiss mice infected with the Y strain of Trypanosoma cruzi were treated orally once per day with each metabolite at doses of 10 to 100 mg/kg of body weight for a period of 20 days. Parasitemia was monitored throughout, and cures were detected by parasitological and PCR assays. The results were compared with those achieved with benznidazole treatment at the same doses. Fexinidazole metabolites were effective in reducing the numbers of circulating parasites and protecting mice against death, compared with untreated mice, but without providing cures at daily doses of 10 and 25 mg/kg. Both metabolites were effective in curing mice at 50 mg/kg/day (30% to 40%) and 100 mg/kg/day (100%). In the benznidazole- treated group, parasitological cure was detected only in animals treated with the higher dose of 100 mg/kg/day (80%). Single-dose pharmacokinetic parameters for each metabolite were obtained from a parallel group of uninfected mice and were used to estimate the profiles following repeated doses. Pharmacokinetic data suggested that biological efficacy most likely resides with the sulfone metabolite (or subsequent reactive metabolites formed following reduction of the nitro group) following administration of either the sulfoxide or the sulfone and that prolonged plasma exposure over the 24-h dosing window is required to achieve high cure rates. Fexinidazole metabolites were effective in treating T. cruzi in a mouse model of acute infection, with cure rates superior to those achieved with either fexinidazole itself or benznidazole