Towards improved antileishmanial therapiesdelivery issues and application to berberine

Resumo

This thesis has two independent parts. The first part is devoted to “basic research” and was addressed to evaluate the effect of VL infection in the BD of hard (polystyrene, PS) and soft-matter (PLGA) nanocarriers. The rationale is that VL produces serious morphological alterations in the liver, spleen and bone marrow (BM), wherein infected macrophages and other cells of the mononuclear phagocyte system (MPS) reside, being at both organ and cellular level the natural destination of most types of NP. We optimized fluorescently labelled NP to this purpose. Our in vivo imaging, immunofluorescence and flow cytometry results revealed that VL caused several organ and cellular alterations that tend to modify NP organ sequestration, their cellular uptake, and produce changes in the pattern of NP cellular distribution. These results extended what was previously described for AmBisome® to other types of NP. Moreover, we observed that NP cellular distribution was highly specific for each type of NP. We ignore whether the observed lower NP accumulation in the infected organs is significant enough to have an effect in their efficacy. However, these quantitative changes occur at the same time that NP redistributed among different phagocytic cells. As the efficacy of antileishmanial drugs depends on the drug accumulation in the infected host cells, NP cellular distribution, that is a “hallmark” of each NP and that can be profoundly affected by VL infection, should be correlated with their efficacy. As we proceed with this “basic research” and with the need for better treatments for Leishmaniasis, the second part of this thesis was focused on the quaternary isoquinoline alkaloid Berberine (BER) and how pharmaceutical technology can be used to fully exploit its antileishmanial potential. We chose this compound attracted by its low IC50 (around 1 µM, for L. infantum and L. major amastigotes) and its high selectivity index (SI of 115 and 89 µM, for L. infantum and L. major, respectively). However, BER rapid metabolism and excretion and its inadequate tissue distribution to target Leishmania spp. parasites, hider its application against VL. Thus, BER was encapsulated in liposomes to overcome these limitations, and their potential as antileishmanial treatment was evaluated in a murine model of VL. In vitro, the loading of BER in liposomes enhanced more than 7-fold its SI by decreasing its cytotoxicity. In vivo, in L. infantum infected BALB/c mice, BER-LP reduced the parasitaemia in liver and spleen by 99.2 and 93.5%, respectively. This improvement was explained by the higher accumulation of the drug in these organs. Unfortunately, their i.p. administration significantly reduced the plasma triglycerides levels. Finally, we evaluated the efficacy of BER in the cutaneous form of the disease, namely in CL caused by L. major. As drug delivery systems (DDS) have shown limited advantages after topical application, the drug was formulated in a conventional cream. The formulation combined the incorporation of a permeation enhancer, and the ion-pairing complexation of BER with a compound containing phosphate groups, in order to promote arrival of enough drug to dermal macrophages. After 35 days of treatment, lesions were significantly lower (8.5 times) than control lesions in infected non-treated mice and the parasite burden decreased 99.9%, in agreement with BER dermal accumulation at a higher concentration than the IC50 for enough time to kill the parasites.