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. 2021:28:38.
doi: 10.1051/parasite/2021036. Epub 2021 Apr 14.

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

Camila S Freitas  1 , Daniela P Lage  1 , João A Oliveira-da-Silva  1 , Rafaella R Costa  1 , Débora V C Mendonça  1 , Vívian T Martins  1 , Thiago A R Reis  1 , Luciana M R Antinarelli  2 , Amanda S Machado  1 , Grasiele S V Tavares  1 , Fernanda F Ramos  1 , Rory C F Brito  3 , Fernanda Ludolf  1 , Miguel A Chávez-Fumagalli  4 , Bruno M Roatt  3 , Gabriela S Ramos  5 , Jennifer Munkert  6 , Flaviano M Ottoni  5 , Priscilla R V Campana  5 , Mariana C Duarte  7 , Denise U Gonçalves  1 , Elaine S Coimbra  2 , Fernão C Braga  5 , Rodrigo M Pádua  5 , Eduardo A F Coelho  7
Affiliations

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

Camila S Freitas et al. Parasite. 2021.

Abstract
in English, French

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 β-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 β-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 β-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.

Keywords: Drug repositioning; Miltefosine; Toxicity; Treatment; Visceral leishmaniasis; β-acetyl-digitoxin.

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Figures

Figure 1
Figure 1
Chemical structure of β-acetyl-digitoxin.
Figure 2
Figure 2
Evaluation of mitochondrial membrane potential. Leishmania infantum (107 cells) were incubated alone (control) or in the presence of b-AD (41.93 μM, corresponding to two times the IC50 value) for 24 h at 25 °C. Parasites were then incubated with MitoTracker Red CM-H2XROS for 30 min and in the dark. Cells were washed twice with PBS and transferred to a black 96-well plate, when the fluorescence intensity was evaluated using a fluorometer. Promastigotes treated with carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 5.0 μM) were used as a positive control, while those untreated were used as a negative control (control). Bars represent the mean plus standard deviation of the groups. (**) indicates a statistically significant difference as compared to the control (p < 0.0001).
Figure 3
Figure 3
Production of reactive oxygen species. Leishmania infantum (107 cells) were incubated alone (control) or in the presence of b-AD (41.93 μM) for 24 h at 25 °C. Parasites were then washed twice in PBS and incubated with 20 μM cell-permeant 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) for 30 min and in the dark. Fluorescence was measured in a fluorometer, with excitation and emission wavelengths of 485 and 528 nm, respectively. H2O2 (4000 μM; Sigma-Aldrich, USA)-treated parasites were used as a positive control, while untreated parasites were used as a negative control (control). Bars represent the mean plus standard deviation of the groups. (***) indicates a statistically significant difference as compared to the control (p < 0.0001).
Figure 4
Figure 4
Organ parasitism evaluated in the treated animals by a limiting dilution technique. Mice (n = 12 per group) were infected with L. infantum and, 1 and 15 days after the distinct treatment schedules, they (n = 6 per group in each time) were euthanized and their livers, spleens, bone marrow (BM) and draining lymph nodes (dLNs) were collected to evaluate the parasite load, by a limiting dilution technique. Bars represent the mean ± standard deviation of the groups. (*) indicates a significant difference as compared to the saline and B/Mic groups (p < 0.05). (**) indicates a statistically significant difference as compared to the miltefosine group (p < 0.05). (***) indicates a statistically significant difference as compared to the b-AD group (p < 0.05).
Figure 5
Figure 5
Splenic parasitism estimated by quantitative PCR. Leishmania infantum-infected mice (n = 6 per group) were submitted to the distinct treatment regimens and, 15 days post-therapy, they were euthanized and their spleens were collected to evaluate parasitism by qPCR. Results were expressed as the number of parasites per total DNA, and bars represent the mean ± standard deviation of the groups. (*) indicates a significant difference as compared to the saline and B/Mic groups (p < 0.05). (**) indicates a statistically significant difference as compared to the miltefosine group (p < 0.05). (***) indicates a statistically significant difference as compared to the b-AD group (p < 0.05).
Figure 6
Figure 6
Cellular response generated in the treated animals. BALB/c mice (n = 12 per group) were infected with L. infantum promastigotes and submitted to distinct treatment schedules. 1 and 15 days post-therapy, they were euthanized and their spleens were collected and splenocytes were unstimulated (medium) or stimulated with L. infantum SLA (50.0 μg/mL), for 48 h at 37 °C in 5% CO2. Levels of IFN-γ, IL-4, IL-10, IL-12 and GM-CSF were quantified in the cell supernatant, one (A) and 15 (B) days post-treatment, by capture ELISA. Bars represent the mean ± standard deviation of the groups. (*) indicates a significant difference as compared to the saline and B/Mic groups (p < 0.05). (**) indicates a statistically significant difference as compared to the miltefosine group (p < 0.05). (***) indicates a statistically significant difference as compared to the b-AD group (p < 0.05). (#) indicates a statistically significant difference as compared to the miltefosine, b-AD and b-AD/Mic groups (p < 0.05).
Figure 7
Figure 7
T-cell subtypes involved in the IFN-γ production. Splenocytes of mice treated with miltefosine, b-AD or b-AD/Mic (n = 6 per group) were cultured in DMEM (medium) or stimulated with SLA (50.0 μg/mL) in the presence of anti-CD4 or anti-CD8 antibody, for 48 h at 37 °C in 5% CO2. IFN-γ levels were quantified in the cell supernatant by a capture ELISA. Bars represent the mean ± standard deviation of the groups. (*) indicates a statistically significant difference as compared to the use of anti-CD4 or anti-CD8 antibody (p < 0.05). (**) indicates a statistically significant difference as compared to the use of anti-CD8 antibody (p < 0.05).
Figure 8
Figure 8
Investigation of intracytoplasmic cytokine-producing T cell frequency. Cytometry flow was performed to evaluate the IFN-γ, TNF-α and IL-10-producing T CD4+ and CD8+ cell frequency in splenocytes of mice treated, 15 days post-therapy. Results were calculated by the ratio between SLA-stimulated versus unstimulated cultures (SLA/CC ratio), and reported as cytokine indexes for CD4+ and CD8+ T cells. Bars represent the mean plus standard deviation of the groups. (*) indicates a significant difference as compared to the saline and B/Mic groups (p < 0.05). (**) indicates a statistically significant difference as compared to the miltefosine group (p < 0.05). (***) indicates a statistically significant difference as compared to the b-AD group (p < 0.05).
Figure 9
Figure 9
Nitrite production evaluated by Griess reaction. The cellular supernatants used to quantify cytokines in both periods of time after treatments were also used to evaluate SLA-specific nitrite production. Bars represent the mean ± standard deviation of the groups. (*) indicates a significant difference as compared to the saline and B/Mic groups (p < 0.05). (**) indicates a statistically significant difference as compared to the miltefosine group (p < 0.05). (***) indicates a statistically significant difference as compared to the b-AD group (p < 0.05). (#) indicates a statistically significant difference as compared to the miltefosine, b-AD and b-AD/Mic groups (p < 0.05).
Figure 10
Figure 10
Antibody response produced in the treated animals. Serum samples were collected of mice infected and later treated, 1 and 15 days post-therapy, and levels of anti-parasite IgG1 and IgG2a isotypes were investigated by ELISA. Bars represent the mean ± standard deviation of the groups. (*) indicates a significant difference as compared to the saline and B/Mic groups (p < 0.05). (**) indicates a statistically significant difference as compared to the miltefosine group (p < 0.05). (***) indicates a statistically significant difference as compared to the b-AD group (p < 0.05). (#) indicates a statistically significant difference as compared to the miltefosine, b-AD and b-AD/Mic groups (p < 0.05).
Figure 11
Figure 11
In vivo cytotoxicity. To investigate the in vivo toxicity of the therapeutics, levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatine kinase muscle brain fraction (CK-MB) enzymes were quantified in serum samples of mice infected and treated, 1 and 15 days post-therapy. Samples of naive (non-infected and non-treated) mice were used as control. Bars represent the mean ± standard deviation of the groups. (*) indicates a significant difference as compared to the saline and B/Mic groups (p < 0.05). (**) indicates a statistically significant difference as compared to the miltefosine group (p < 0.05). (***) indicates a statistically significant difference as compared to the b-AD group (p < 0.05). (#) indicates a statistically significant difference as compared to the miltefosine, b-AD and b-AD/Mic groups (p < 0.05).
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