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

Abstract

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 (IC 50 and CC 50 , 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.

Parasitological and immunological evaluation of a quinoline derivative salt incorporated into a polymeric micelle formulation against Leishmania infantum infection

“…Treatments were performed by subcutaneous route, while miltefosine was administered by oral route. The concentration of products used for the treatments was based on previous studies, where quinoline derivatives were also evaluated as antileishmanial agents (Tavares et al 2020 ; Freitas et al 2021 ; Reis et al 2021 ; Mendonça et al 2022 ). In addition, the parasitological and immunological assays were performed 1 and 15 days after treatment.…”

“…5% CO 2 ) and, subsequently, levels of IFN-γ, GM-CSF, IL-12p70, IL-4 and IL-10 were measured in culture supernatant through a capture ELISA using commercial kits (BD Pharmingen, San Diego, CA, USA). Nitrite levels were also determined in the same supernatant by Griess reaction (Sigma-Aldrich, USA) (Freitas et al 2021 ).…”

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

“…After washing twice with PBS, cells were added to a black 96-well plate and fluorescence intensity was measured in a fluorometer (FL × 800, BioTek Instruments, Inc., Winooski, VT, USA), with excitation and emission wavelengths of 528 nm and 600 nm, respectively. Parasites treated with carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP; 5.0 μM) for 10 min were used as a positive control, while untreated parasites were used as a negative control [ 25 ].…”

“…Fluorescence intensity was measured in a spectrofluorometer (Varioskan ® Flash, Thermo Scientific, USA), with excitation and emission wavelengths of 485 and 528 nm, respectively. H 2 O 2 -treated parasites (4000 μM; Sigma-Aldrich, USA) were used as a positive control, while untreated parasites were used as a negative control [ 25 ].…”

“…Levels of anti- Leishmania IgG1 and IgG2a isotype antibodies were measured in serum samples of the treated animals, which were obtained one and 15 days after treatment, as described in [ 25 ]. Briefly, SLA was added as an antigen to the plates, at a concentration of 1.0 μg per well, for 16 h at 4 °C.…”