Dados do Trabalho

Título

Shedding light on cutaneous leishmaniasis treatment: in vitro profiling of repositioned and novel compounds targeting amastigotes ex vivo and various mammalian cell types

Introdução

<p>Chemotherapy for cutaneous leishmaniasis is hindered by high costs and adverse effects, compromising the quality of life for approximately 1 million people worldwide. Developing new safe and selective leishmanicidal agents is crucial and can be effectively guided by <em>in vitro</em> assays targeting clinically relevant amastigotes and various cell types.</p>

Objetivo (s)

<p>Thus, we evaluated the activity of POD-1, methylene blue (MB), new methylene blue (NMB) and its complexes (NMB-B and NMB-P) on <em>ex vivo</em> amastigotes of <em>Leishmania amazonensis </em>LTB0016 (106 parasites/mL) and mammalian cells (L929, HEpG2, VERO, J774.G8 and MØ - 105 cells/mL).</p>

Material e Métodos

<p>Parasites and cells were treated or untreated (negative control - C-) with the compounds under serially diluted concentrations in 96-well plates. Cell viability after 24 hours was assessed using the Alamar Blue assay to determine IC50, CC50 and selectivity index (SI). Miltefosine (MT) was used as a reference compound. Drug-treated parasites (with IC50) were further analyzed by flow cytometry using 7-AAD, TMRE and H2DCFDA markers.</p>

Resultados e Conclusão

<p>The compounds significantly inhibited parasite proliferation in a dose-dependent manner compared to the C- (p&lt;0.0001). The IC50 values for POD-1, MB, NMB, NMB-B, NMB-P and MT were 0.45, 15.65, 2.66, 3.16, 0.46 and 0.97 μM, respectively. &nbsp;The ranging of CC50 (μM) / SI values of the compounds for each cell type were: L929 (from 112.71/116.19 to &gt;1.000/2,222.00), HEpG2 (from 34.24/10.83 to &gt;1.000/2,222.00), VERO (from 44.73/16.81 to &gt;1,000/2,222.00), J774.G8 (from 68.50/25.75 to &gt;1,000/2,222.00) and MØ (from 31.27/11.75 to 102.93/106.11). Flow cytometry revealed 25.33% to 61.05% 7AAD+ events with statistical significance for all compounds (p&lt;0.05). Compared to C-, POD-1 caused the greatest reduction in ΔΨm (74%) and NAM the least (43%). For ROS production, increases ranged from 0.79-fold with NAM to 1.24-fold with POD-1. The data indicate a hit-to-lead profile for the tested compounds with IC50&lt;4 μM (except for MB) and SI&gt;10. Notably, POD-1 and NAM-P exhibiting submicromolar inhibitory concentrations, even lower than MT. The observed leishmanicidal effect may be attributed to ROS-induced mitochondrial damage. Low to moderate toxicity in mammalian cells highlights the importance of <em>in vitro</em> assays across diverse cell types for optimal selection before progressing to <em>in vivo</em> and clinical studies.</p>

Palavras Chave

Leishmaniasis; Chemotherapy; Repositioned drugs; Methylene blue