In vitro and in vivo antileishmanial activity of ß-acetyl-digitoxin, a cardenolide of Digitalis lanata potentially useful to treat visceral leishmaniasis.

Current treatments of visceral leishmaniasis face limitations due to drug side effects and/or high cost, along with the emergence of parasite resistance. Novel and low-cost antileishmanial agents are therefore required. We report herein the antileishmanial activity of ß-acetyl-digitoxin (b-AD), a cardenolide isolated from Digitalis lanata leaves, assayed in vitro and in vivo against Leishmania infantum. Results showed direct action of b-AD against parasites, as well as efficacy for the treatment of Leishmania-infected macrophages. In vivo experiments using b-AD-containing Pluronic® F127 polymeric micelles (b-AD/Mic) to treat L. infantum-infected mice showed that this composition reduced the parasite load in distinct organs in more significant levels. It also induced the development of anti-parasite Th1-type immunity, attested by high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and specific IgG2a antibodies, in addition to low IL-4 and IL-10 contents, along with higher IFN-γ-producing CD4+ and CD8+ T-cell frequency. Furthermore, low toxicity was found in the organs of the treated animals. Comparing the therapeutic effect between the treatments, b-AD/Mic was the most effective in protecting animals against infection, when compared to the other groups including miltefosine used as a drug control. Data found 15 days after treatment were similar to those obtained one day post-therapy. In conclusion, the results obtained suggest that b-AD/Mic is a promising antileishmanial agent and deserves further studies to investigate its potential to treat visceral leishmaniasis.

TITLE: Activité antileishmaniale in vitro et in vivo de la ß-acétyl-digitoxine, un cardénolide de Digitalis lanata potentiellement utile pour traiter la leishmaniose viscérale. ABSTRACT: Les traitements actuels de la leishmaniose viscérale font face à des limitations dues aux effets secondaires des médicaments et/ou à leur coût élevé, ainsi qu'à l'émergence d'une résistance parasitaire. Des agents antileishmaniaux nouveaux et peu coûteux sont donc nécessaires. Nous rapportons ici l'activité antileishmaniale de la ß-acétyl-digitoxine (b-AD), un cardénolide isolé à partir de feuilles de Digitalis lanata, mesurée in vitro et in vivo contre Leishmania infantum. Les résultats ont montré une action directe de la b-AD contre les parasites, ainsi qu'une efficacité pour le traitement des macrophages infectés par Leishmania. Des expériences in vivo utilisant des micelles polymériques Pluronic® F127 contenant de la b-AD (b-AD/Mic) pour traiter des souris infectées par L. infantum ont montré que cette composition réduisait à des niveaux plus significatifs la charge parasitaire dans différents organes, ainsi que le développement d'une immunité antiparasitaire de type Th1, attestée par les taux élevés d'IFN-γ, d'IL-12, de TNF-α, de GM-CSF, de nitrite et d'anticorps IgG2a spécifiques, en plus des faibles taux d'IL-4 et IL-10, ainsi qu'une fréquence plus élevée des cellules T CD4+ and CD8+ productrices d' IFN-γ. De plus, une faible toxicité a été trouvée dans les organes des animaux traités. En comparant l'effet thérapeutique des traitements, b-AD/Mic était le plus efficace pour protéger les animaux contre l'infection, par rapport aux autres groupes comprenant la miltefosine utilisée comme contrôle médicamenteux. Les données trouvées 15 jours après le traitement étaient similaires à celles obtenues un jour après le traitement. En conclusion, les résultats obtenus suggèrent que b-AD/Mic est un agent antileishmanial prometteur et mérite des études supplémentaires pour étudier son potentiel à traiter la leishmaniose viscérale.

Acarbose presents in vitro and in vivo antileishmanial activity against Leishmania infantum and is a promising therapeutic candidate against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) is mainly hampered by drug toxicity, long treatment regimens and/or high costs. Thus, the identification of novel and low-cost antileishmanial agents is urgent. Acarbose (ACA) is a specific inhibitor of glucosidase-like proteins, which has been used for treating diabetes. In the present study, we show that this molecule also presents in vitro and in vivo specific antileishmanial activity against Leishmania infantum. Results showed an in vitro direct action against L. infantum promastigotes and amastigotes, and low toxicity to mammalian cells. In addition, in vivo experiments performed using free ACA or incorporated in a Pluronic® F127-based polymeric micelle system called ACA/Mic proved effective for the treatment of L. infantum-infected BALB/c mice. Treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes when compared to the controls, as well as the development of antileishmanial Th1-type humoral and cellular responses based on high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibodies. In addition, ACA or ACA-treated animals suffered from low organ toxicity. Treatment with ACA/Mic outperformed treatments using either Miltefosine or free ACA based on parasitological and immunological evaluations performed one and 15 days post-therapy. In conclusion, data suggest that the ACA/Mic is a potential therapeutic agent against L. infantum and merits further consideration for VL treatment.

Ivermectin presents effective and selective antileishmanial activity in vitro and in vivo against Leishmania infantum and is therapeutic against visceral leishmaniasis.

Treatment for visceral leishmaniasis (VL) is hindered mainly by the toxicity and/or high cost of therapeutic drugs. In addition, parasite resistance has been registered. Thus, there is an urgent need for the identification of novel, effective and low-cost antileishmanial agents. Since drug discovery is a long and expensive process, drug repositioning for treatment of leishmaniasis should be considered. In the present study, Ivermectin (IVE), a broad-spectrum drug used for treatment of parasitic diseases, was evaluated in vitro and in vivo against Leishmania infantum species. Results in vitro showed that IVE presented 50% Leishmania and macrophage inhibitory concentrations (IC50 and CC50, respectively) of 3.64 ± 0.48 µM and 427.50 ± 17.60 µM, respectively, with a selectivity index (SI) of 117.45; whereas Amphotericin B (AmpB), which was used as control, showed IC50 and CC50 values of 0.12 ± 0.05 µM and 1.06 ± 0.23 µM, respectively, with a corresponding SI of 8.90. Treatment with IVE effectively reduced the infection percentage and parasite burden in infected and treated macrophages and displayed a prophylactic activity by inhibiting macrophage infection with pre-treated parasites. Furthermore, preliminary studies suggested that IVE targets the parasite's mitochondria. Activity of IVE in its free format or incorporated into Pluronic® F127-based polymeric micelles (IVE/Mic) was also evaluated in vivo as a treating drug for L. infantum-infected BALB/c mice. Miltefosine was used as a control. Results showed that Miltefosine, IVE and IVE/Mic-treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as well as development of an antileishmanial Th1-type immune response one and 15 days after treatment. Notably, IVE/Mic showed a better parasitological and immunological response in comparison to other alternative treatments. In conclusion, results suggest that IVE/Mic could be considered in future studies as a therapeutic alternative to treat VL.

Digitoxigenin presents an effective and selective antileishmanial action against Leishmania infantum and is a potential therapeutic agent for visceral leishmaniasis.

Treatment for visceral leishmaniasis (VL) is hampered mainly by drug toxicity, their high cost, and parasite resistance. Drug development is a long and pricey process, and therefore, drug repositioning may be an alternative worth pursuing. Cardenolides are used to treat cardiac diseases, especially those obtained from Digitalis species. In the present study, cardenolide digitoxigenin (DIGI) obtained from a methanolic extract of Digitalis lanata leaves was tested for its antileishmanial activity against Leishmania infantum species. Results showed that 50% Leishmania and murine macrophage inhibitory concentrations (IC50 and CC50, respectively) were of 6.9 ± 1.5 and 295.3 ± 14.5 µg/mL, respectively. With amphotericin B (AmpB) deoxycholate, used as a control drug, values of 0.13 ± 0.02 and 0.79 ± 0.12 µg/mL, respectively, were observed. Selectivity index (SI) values were of 42.8 and 6.1 for DIGI and AmpB, respectively. Preliminary studies suggested that the mechanism of action for DIGI is to cause alterations in the mitochondrial membrane potential, to increase the levels of reactive oxygen species and induce accumulation of lipid bodies in the parasites. DIGI was incorporated into Pluronic® F127-based polymeric micelles, and the formula (DIGI/Mic) was used to treat L. infantum-infected mice. Miltefosine was used as a control drug. Results showed that animals treated with either miltefosine, DIGI, or DIGI/Mic presented significant reductions in the parasite load in their spleens, livers, bone marrows, and draining lymph nodes, as well as the development of a specific Th1-type response, when compared with the controls. Results obtained 1 day after treatment were corroborated with data corresponding to 15 days after therapy. Importantly, treatment with DIGI/Mic induced better parasitological and immunological responses when compared with miltefosine- and DIGI-treated mice. In conclusion, DIGI/Mic has the potential to be used as a therapeutic agent to protect against L. infantum infection, and it is therefore worth of consideration in future studies addressing VL treatment.