Terapias alternativas e combinadas para o tratamento da Leishmaniose Cutânea Americana: uma abordagem experimental in vitro e in vivo

A leishmaniose é uma doença tropical causada por protozoários do gêneroLeishmania que afeta 12 milhões de pessoas em 98 países. Seu tratamentoconta com um restrito arsenal terapêutico e exige a administração defármacos tóxicos por longos períodos. Na busca por novas terapias, oreposicionamento de fármacos e a associação terapêutica têm sidoaplicados com sucesso para doenças negligenciadas. O presente estudoteve como objetivo a avaliação in vitro, ex vivo e in vivo do potencial antiLeishmania(Leishmania) amazonensis dos fármacos amitriptilina, econazol,sertralina e triclosan, bem como o estudo de associações terapêuticas invitro e/ou ex vivo e mecanismo de ação in vitro dos fármacos amitriptilina etriclosan. Os resultados demonstraram que todos os fármacos estudados apresentaram atividade contra formas promastigotas e amastigotasintracelulares de L. (L.) amazonensis, com valores de Concentração Efetiva50% que variam de 1,50 a 51,48 µM. Os resultados obtidos a partir das associações entre os fármacos estudados e fármacos padrões foram classificados como aditivos ou indiferentes. Por meio da investigação domecanismo de ação leishmanicida, foi possível concluir que a mitocôndria é uma organela alvo do fármaco amitriptilina, enquanto que o fármaco triclosaninduz danos à membrana plasmática parasitária. Quando tratados comeconazol por via oral (10 mg/kg/dia por 28 dias consecutivos) ou triclosanpor via tópica (creme 1% por 14 dias consecutivos), houve uma redução de75 a 89% da carga parasitária dos camundongos infectados com L. (L.) amazonensis. Os resultados obtidos contribuem para a investigação de novas alternativas para o tratamento da leishmaniose cutânea e sugerem que novos estudos utilizando associação ou coadministração dessesfármacos com fármacos padrões podem ser promissores em modelosanimais.

Leishmaniasis is a tropical disease caused by protozoa of the genusLeishmania that affects 12 million people in 98 countries. There is a limitedtherapeutic arsenal and the treatment requires the administration of toxicdrugs for long periods. In the search for new therapies, the drug repositioningand therapeutic association have been successfully applied to neglecteddiseases. The aim of the present study was to evaluate in vitro, ex vivo andin vivo anti-Leishmania (Leishmania) amazonensis potential of the drugsamitriptyline, econazole, sertraline and triclosan, as well as the study of invitro and / or ex vivo therapeutic associations and mechanism of action of thedrugs amitriptyline and triclosan. The results showed that all studied drugshave activity against L. (L.) amazonensis promastigotes and intracellularamastigotes, with 50% Effective Concentration values ranging from 1.50 to51.48 μM. The results obtained from the combination between the studieddrugs and standard drugs were classified as additives or indifferent. Throughthe investigation of the leishmanicial mechanism of action, it was possible toconclude that the mitochondria is a target organelle of the drug amitriptyline,whereas the drug triclosan induces damage to the parasitic plasmamembrane. When treated with oral econazole (10 mg/kg/day for 28consecutive days) or triclosan topically (1% cream for 14 consecutive days),there was a 75 - 89% reduction in the parasite load of the mice infected withL. (L.) amazonensis. The results obtained contribute to the investigation ofnew alternatives for the treatment of cutaneous leishmaniasis and suggestthat new studies using association or coadministration of these drugs withstandard drugs may be promising in animal models.

Efficacy of sertraline against Trypanosoma cruzi: an in vitro and in silico study

Drug repurposing has been an interesting and cost-effective approach, especially for neglected diseases, such as Chagas disease. Methods: In this work, we studied the activity of the antidepressant drug sertraline against Trypanosoma cruzi trypomastigotes and intracellular amastigotes of the Y and Tulahuen strains, and investigated its action mode using cell biology and in silico approaches. Results: Sertraline demonstrated in vitro efficacy against intracellular amastigotes of both T. cruzi strains inside different host cells, including cardiomyocytes, with IC50 values between 1 to 10 µM, and activity against bloodstream trypomastigotes, with IC50 of 14 µM. Considering the mammalian cytotoxicity, the drug resulted in a selectivity index of 17.8. Sertraline induced a change in the mitochondrial integrity of T. cruzi, resulting in a decrease in ATP levels, but not affecting reactive oxygen levels or plasma membrane permeability. In silico approaches using chemogenomic target fishing, homology modeling and molecular docking suggested the enzyme isocitrate dehydrogenase 2 of T. cruzi (TcIDH2) as a potential target for sertraline. Conclusions: The present study demonstrated that sertraline had a lethal effect on different forms and strains of T. cruzi, by affecting the bioenergetic metabolism of the parasite. These findings provide a starting point for future experimental assays and may contribute to the development of new compounds.(AU)

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Background Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 μg/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 μg/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.(AU)

Ergosterol isolated from the basidiomycete Pleurotus salmoneostramineus affects Trypanosoma cruzi plasma membrane and mitochondria

Abstract Background Major drawbacks of the available treatment against Chagas disease (American trypanosomiasis) include its toxicity and therapeutic inefficiency in the chronic phase of the infection, which makes it a concern among neglected diseases. Therefore, the discovery of alternative drugs for treating chronic Chagas disease requires immediate action. In this work, we evaluated the mushroom Pleurotus salmoneostramineus in the search for potential antiparasitic compounds. Methods Fruit bodies of the basidiomycete Pleurotus salmoneostramineus were triturated and submitted to organic solvent extraction. After liquid-liquid partition of the crude extract, three fractions were obtained and the bioguided fractionation study was conducted to isolate the active metabolites. The elucidation of the chemical structure was performed using GC-MS and NMR techniques. The biological assays for antiparasitic activity were carried out using trypomastigotes of Trypanosoma cruzi and murine macrophages for mammalian cytotoxicity. The mechanism of action of the isolated compound used different fluorescent probes to evaluate the plasma membrane permeability, the potential of the mitochondrial membrane and the intracellular levels of reactive oxygen species (ROS). Results The most abundant fraction showing the antiparasitic activity was isolated and chemically elucidated, confirming the presence of ergosterol. It showed anti-Trypanosoma cruzi activity against trypomastigotes, with an IC50 value of 51.3 g/mL. The compound demonstrated no cytotoxicity against mammalian cells to the maximal tested concentration of 200 g/mL. The mechanism of action of ergosterol in Trypanosoma cruzi trypomastigotes resulted in permeabilization of the plasma membrane, as well as depolarization of mitochondrial membrane potential, leading to parasite death. Nevertheless, no increase in ROS levels could be observed, suggesting damages to plasma membrane rather than an induction of oxidative stress in the parasite. Conclusions The selection of naturally antiparasitic secondary metabolites in basidiomycetes, such as ergosterol, may provide potential scaffolds for drug design studies against neglected diseases.

Mecanismo de ação de fármacos sintéticos e associações terapêuticas em Leishmania (L) infantum / Mechanism of action of synthetic drugs and therapeutic associations in Leishmania (L) infantum

A leishmaniose visceral (LV) é uma doença negligenciada endêmica em 68 países e que se tornou um grande problema na área da saúde pública. Essa doença afeta principalmente populações menos favorecidas, onde o acesso, os cuidados médicos e a terapia medicamentosa são limitados. Visando buscar novas alternativas para o tratamento da LV, o reposicionamento de fármacos oferece uma grande oportunidade para introdução de novas terapias. Por meio desta abordagem, foi realizada em Leishmania (Leishmania) infantum: i) a triagem de oito fármacos nitrogenados, sendo eles: bifonazol, clotrimazol, cloxazolam, econazol, hidroxizina, loratadina, nitazoxanida e quetiapina; ii) o estudo da ação letal dos fármacos loratadina e nitazoxanida, e iii) o estudo in vitro da associação do fármaco nitazoxanida com fármacos padrões. Para isto, os possíveis mecanismos de ação foram estudados por meio de ensaios fluorimétricos, visando avaliar alteração de permeabilidade de membrana plasmática, alteração do potencial de membrana mitocondrial, produção de espécies reativas de oxigênio, exposição de fosfatidilserina, assim como o estudo das alterações ultraestruturais. Os resultados mostram que todos os fármacos apresentaram atividade contra formas promastigotas, com concentração efetiva 50% (CE50) entre 2 e 167 μM; apenas o econazol e a nitazoxanida eliminaram os amastigotas intracelulares, com CE50 de 11 a 22 μM, respectivamente. O fármaco loratadina alterou a permeabilidade da membrana plasmática do parasita, causando poros e levando o parasita a morte. O estudo da ação letal da nitazoxanida em Leishmania sugere que o fármaco tenha como alvo a mitocôndria, considerando a despolarização do potencial de membrana mitocondrial e aumento da produção de espécies reativas de oxigênio, resultando em estresse oxidativo e morte celular. Além disso, a presença de marcadores específicos como a exposição de fosfatidilserina e alterações...