Mode of action of a formulation containing hydrazones and saponins against leishmania spp. Role in mitochondria, proteases and reinfection process.

Toxicity and poor adherence to treatment that favors the generation of resistance in the Leishmania parasites highlight the need to develop better alternatives. Here, we evaluated the in vitro effectiveness of hydrazone derived from chromanes 2-(2,3-dihydro-4H-1-benzothiopyran-4-ylidene) hydrazide (TC1) and 2-(2,3-dihydro-4H-1-benzopyran-4-ylidene) hydrazide (TC2) and the mixture of triterpene saponin hederagenin-3-O-(3,4-O-diacetyl-ß-D-xylopyranosyl-(1à3)-a-L- rhamnopyranosyl-(1à2)-a-L-arabinofuranoside, hederagenin-3-O-(3,4-O-diacetyl-a-L- arabinopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside and, hederagenin-3-O-(4-O-acetyl-ß-D-xylopyranosyl-(1à3)-a-L-rhamnopyranosyl-(1à2)-a-L-arabinofuranoside from Sapindus saponaria (SS) on L. braziliensis and L. pifanoi. Mixtures of TC1 or TC2 with saponin were formulated for topical application and the therapeutic effectiveness was evaluated in the model for cutaneous leishmaniasis (CL) in golden hamster. The mode of action of these compounds was tested on various parasite processes and ultrastructural parasite modifications. TC1, TC2 and SS showed moderate cytotoxicity when tested independently but toxicity was improved when tested in combination. The compounds were more active against intracellular Leishmania amastigotes. In vivo studies showed that combinations of TC1 or TC2 with SS in 1:1 ratio (w/w) cured 100% of hamsters with no signs associated with toxicity. The compounds did cause changes in the mitochondrial activity of the parasite with a decrease in ATP levels and depolarization of membrane potential and overproduction of reactive oxygen species; nevertheless, these effects were not related to alterations in membrane permeability. The phagolysosome ultrastructure was also affected impacting the survival of Leishmania but the function of the lysosome nor the pH inside the phagolysosome did not change. Lastly, there was a protease inhibition which was directly related to the decrease in the ability of Leishmania to infect and multiply inside the macrophage. The results suggest that the combination of TC1 and TC2 with SS in a 1:1 ratio is capable of curing CL in hamsters. This effect may be due to the ability of these compounds to affect parasite survival and the ability to infect new cells.

Antileishmanial Activity of a Calophyllum brasiliense Leaf Extract.

Calophyllum brasiliense (Clusiaceae) is a tree that grows mainly in the Atlantic Forest in Brazil. Its leaves possess antibacterial activity, cytotoxic activity against certain tumor cell lines, and antimicrobial activity in BALB/c mice infected with Leishmania (Leishmania) amazonensis.Aiming to identify ultrastructural changes and DNA fragmentation in Leishmania (Viannia) braziliensis, promastigotes were treated with a concentration of the dichloromethane extract and coumarin (-) mammea A/BB from C. brasiliense leaves that inhibited 50 % of the parasites (IC50), and were evaluated by transmission and scanning electron microscopy. Ultrastructural changes showed different levels of mitochondrial alterations, including mitochondrial swelling and a reduction in the density of the mitochondrial matrix. Induced DNA fragmentation, as observed by TUNEL, suggested that the extract and coumarin (-) mammea A/BB induced apoptosis-like cell death. These results suggest that the combination of C. brasiliense extract and coumarin (-) mammea A/BB can be considered a promising candidate for the development of new antiprotozoal agents, because of its significant leishmanicidal activity.

Leishmanicidal Activity of (+)-Phyllanthidine and the Phytochemical Profile of Margaritaria nobilis (Phyllanthaceae).

The effects of the Securinega alkaloid (+)-phyllanthidine on Leishmania (L.) amazonensis and the first chemical investigation of Margaritaria nobilis L.f. (Phyllanthaceae) are described. Treating the parasites with this alkaloid caused a dose-dependent reduction in promastigote growth of 67.68% (IC50 82.37 µg/mL or 353 µM) and in amastigote growth of 83.96% (IC50 49.11 µg/mL or 210 µM), together with ultrastructural alterations in the promastigotes. No cytotoxic effect was detected in mammalian cells (CC50 1727.48 µg/mL or CC50 5268 µM). Classical chromatographic techniques and spectral methods led to the isolation and identification of betulinic acid, kaempferol, corilagin, gallic acid and its methyl ester, besides (+)-phyllanthidine from M. nobilis leaves and stems. Margaritaria nobilis is another source of the small group of Securinega alkaloids, together with other Phyllanthaceae (Euphorbiaceae s.l.) species. The low toxicity to macrophages and the effects against promastigotes and amastigotes are suggestive that (+)-phyllanthidine could be a promising antileishmanial agent for treating cutaneous leishmaniasis.

In vitro leishmanicidal activity of 1,3-disubstituted 5-nitroindazoles.

The antiprotozoal activity of some indazole-derived amines (2, 3, 5-8) as well as that of some simple structurally related 3-alkoxy-1-alkyl-5-nitroindazoles (1, 4) against promastigote and amastigote forms of Leishmania infantum and Leishmania braziliensis is reported. In some cases, these compounds showed in vitro activities against the different morphological forms of Leishmania similar to or higher than those of the reference drug glucantime; this fact, along with low unspecific cytotoxicities against macrophages shown by some of them, led to good selectivity indexes (SI). The high efficiency of some 5-nitroindazoles against the mentioned protozoa was confirmed by further in vitro studies on infection rates. Complementary analyses by (1)H NMR of the changes on the metabolites excreted by parasites after treatment with the more active indazole derivatives in many cases showed the decreased excretion of succinate and increased levels of acetate, lactate and alanine, as well as, in some cases, the appearance of glycine and pyruvate as new metabolites. Damage caused by indazoles at the glycosomal or mitochondrial level are consistent with these metabolic changes as well as with the huge ultrastructural alterations observed by transmission electron microscopy (TEM), especially affecting the mitochondria and other cytoplasmic organelles.

[Effect of intralesional treatment with emetine hydrochloride on Leishmania (Viannia) braziliensis in hamsters]. / Efecto del tratamiento intralesional con clorhidrato de emetina sobre Leishmania (Viannia) braziliensis en hámsteres.

The therapeutic effect of the emetine hydrochloride alkaloid administered intralesionally was compared with that of standard parenteral treatment with Glucantime in outbred male hamsters experimentally infected with 4 x 10(3) amastigotes of Leishmania (Viannia) braziliensis. Both chemotherapeutic agents reduced significantly (P < 0.01) the average lesion sizes in experimental animals in comparison with those untreated. The alkaloid infiltration was found to be as effective as the antimonial injection for clinical resolution. The ultrastructural effects on the Leishmania parasites exposed to emetine were observed mainly in the inner cytoplasm, which appeared disorganized, pycnotic and with loss of morphological definition; however, any known emetine hydrochloride action mechanism factor could not be directly related with ultrastructure effects detected on leishmanial parasites. Smears, conventional histopathology, culture in NNN medium and indirect immunoperoxidase method showed viable amastigotes in nodules and/or scars of all the evaluated hamsters 75 to 230 days after the end of treatment. These findings suggest that measurement of the size of cutaneous leishmania lesions does not appear to be a valid criterion for evaluating the efficiency of chemotherapy in experimental LT. Detection of leishmania parasites in the lesion scars, supports the hypothesis that man could be considered as an domestic reservoir.