New insights for an old drug: difluoromethylornithine boosts the trypanocidal action of benznidazole on Trypanosoma cruzi in vitro and in vivo

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi , is currently a global health problem. Its multiple forms of transmission, which favor the spread of the infection, and the limited treatment options—still restricted to only two drugs—highlight the urgent need for research and development in the field. Despite certain disadvantages, the potent trypanocidal activity of benznidazole (BNZ) and nifurtimox explains their continued use notwithstanding their high toxicity. Reducing BNZ doses through combination with another antiparasitic drug is an attractive strategy to improve tolerability while maintaining efficacy. In this work, we explored the anti- T. cruzi effect of BNZ in combination with difluoromethylornithine (DFMO), a repurposed drug currently used for the treatment of certain cancers and other pathologies. Our data showed that DFMO increases parasite susceptibility to BNZ, reducing the IC50 by half compared to BNZ monotherapy in vitro across strains from three different T. cruzi genetic lineages. Subsequently, we examined the effect of the combination in vivo . We found that mice treated with a 10-fold lower dose of BNZ combined with DFMO displayed a lower parasitemia peak and reduced tissue parasitosis during the acute stage, as well as less parasite reactivation in organs after immunosuppression in the chronic stage, compared to BNZ monotherapy. Overall, these findings support the BNZ/DFMO combination as a proof of concept for Chagas disease therapy, offering a viable strategy to mitigate BNZ-related toxicity through dose reduction without compromising therapeutic efficacy.