New therapeutic approches for human African trypanosomiasis

Abstract

African trypanosomiasis is a parasitic disease with a devastating socio-economic impact in Sub-Saharan Africa through the direct infection of humans and livestock. Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. The disease is fatal if left untreated. Current chemotherapy relies only on five drugs that have many limitations, ranging from problems with oral absorption, acute toxicities and the emergence of trypanosomal resistance, which is a major concern owing to the absence of a vaccine or therapeutic alternatives. All known mechanisms of drug resistance are associated with functional loss of surface transporters except nifurtimox. Here we report the trypanocidal activity of nicotinamide, a soluble compound of vitamin B3. Nicotinamide treatment results in T. brucei cell growth inhibition and dymorphism of the flagellar pocket, the site for both endocytosis and exocitosis and the subjacent endocytic compartment. Protease activity assays show that nicotinamide inhibits directly the lysosomal protease cathepsin B and subsequently blocks endocytosis causing programmed cell death. Finally, an in vivo study shows that nicotinamide has an alleviating effect on T. brucei-infected mice. The results presented here support the possible use of nicotinamide in HAT therapy. New approaches in currents chemotherapies are focused on the use of drug delivery systems or immune tools. Nanobodies (Nbs) are small antibody fragments, with unique antigen recognition properties, derived from heavy chain camelid antibodies through recombinant gene technology. These can be used to target biologically active components. In this context, we have prepared pentamidine-loaded nanoparticles of chitosan polymer coated by a specific nanobody that target the surface of T. brucei. Nanobody-coated pentamidine-loaded chitosan nanoparticles bind to the trypanosome surface and the drug is taken up by endocytosis instead of its classical membrane transporters. The new formulation was significantly more efficient than free pentamidine in killing trypanosomes. In vitro studies revealed that the half-maximal inhibitory concentration (IC50) of pentamidine-loaded in nanobody-coated PEGlycated chitosan nanoparticles was 14 fold lower than free pentamidine. An in vivo experiment in murine model of the acute phase of African trypanosomiasis determined that the curative dose of pentamidine-loaded in nanobody-chitosan nanoparticles was 100 fold lower than pentamidine alone. Moreover, a pentamidine resistant cell line was obtained by growing the parasites in increasing concentration of pentamidine. Genetic and functional assays revealed that the resistance mechanism to pentamidine in this cell line was due to mutation in aquaglyceroporin 2, a cell surface transporter. An in vitro study showed that this cell line was not resistant to pentamidine-loaded nanobody-coated chitosan nanoparticles. The development of trypanocidal drugs loaded in chitosan nanoparticles coated by specific nanobodies against trypanosome invariant surface antigens may reduce the minimal curative dose of all these drugs, minimize drug toxicity and circumvent drug resistance. Furthermore, the possibilities offered by this nanobody-based system are enormous and could be adapted to load any compound with a reported trypanocidal action, for instance nicotinamide. Finally, the promising results obtained with this new formulation open a range of new potential therapies with application to other diseases.