Dados do Trabalho

Título

Combination makes strength: an in vitro study of innovative associations of compounds with synergistic efficacy for cutaneous leishmaniasis treatment

Introdução

Combined therapy holds promise for treating leishmaniases due to its potential to reduce toxicity, adverse effects, treatment duration and resistance, while additionally increasing selectivity and cure rates.

Objetivo (s)

In this study, we evaluated the in vitro combination activity of IC50 values (μM) for POD-1 (317.06) with Methylene Blue - MB (61.44), New Methylene Blue - NMB (5.42) and their derived complexes, NMB-B (5.48) and NMB-P (2.84) on promastigote forms of Leishmania amazonensis PH8 (106 parasites/mL).

Material e Métodos

We employed the fixed proportions method (1:4, 2:3, 3:2, 4:1), serial dilution (1:2) in 96-well microplates and the Alamar Blue viability assay after 24 hours to determine the IC50 of the compound combinations. Subsequently, the FIC25, 50 and 75, as well as x̅ΣFICs50, were calculated to classify the combinations (≤ 0.5 = synergistic; > 0.5 or ≤ 4.0 = additive; > 4.0 = antagonistic). Parasites, both treated and untreated (negative control), were analyzed using flow cytometry with Annexin V-FITC/PI, TMRE and DHE markers.

Resultados e Conclusão

All 48 combinations inhibited parasite proliferation in a concentration-dependent manner, with IC50 values up to 39 times lower than those of the individual compounds. In association, the lowest inhibitory concentrations for POD-1, MB, NMB, NMB-B and NMB-P were 8.23, 3.13, 0.87, 1.07 and 0.15 μM, respectively. Based on FICs, none of the combinations were antagonistic, three were additive and the remaining were synergistic (n=45). Notable synergy (≤ 0.1) was observed in 4 combinations of POD-1: MB and in 12 of POD-1: NMB-P. Analysis by x̅ΣFICs50 demonstrated that the association of POD-1 with MB and NMB-P was synergistic, while with NMB and NMB-B it was additive, with no antagonism observed. Flow cytometry analysis revealed that 68.34% to 94.15% of parasites treated with the combinations were dead. Approximately 7% to 50% of events were Annexin+ PI- and around 20% to 87% were PI+, suggestive of late apoptosis or necrosis. No reduction in ΔΨm or increase in ROS production was observed compared to the negative control, presumably due to the high death rate. Our results corroborate the potential of combined therapy for the treatment of cutaneous leishmaniasis by reducing compound doses and subsequent cellular toxicity, with a greater effect on the target organism. This encourages the development of further studies and clinical applications.

Palavras Chave

Chemotherapy; Drug combination; Leishmaniasis therapy; Repositioned drugs