Synergistic effect of clomipramine and amphotericin on Leishmania amazonensis promastigotes

Abstract

The search for new therapies involves the use of drug combinations (F) effective for conditions with similar pathogen-host biology and with molecular targets present in the parasite, but absent in the host, such as trypanothione reductase in parasites of the genus Leishmania. An F inhibitor of this enzyme is clomipramine (Clo), used for its antidepressant action. The commercial formulations of the F are associated with excipients, which can be eliminated by simple methods and thus achieve their purification. The objective of this work was focused on analyzing the "in vitro" combined therapy of Amphotericin-B (Anf) and purified Clo on L. amazonensis promastigotes.

Purification of Clo was carried out taking into account its high solubility in dichloromethane. 3x10⁶ promastigotes/mL were incubated with concentrations of F alone (n=3) and combined (n=3) (Anf=0.02-0.62 μg/mL and Clo=0.48-15.6 μg/mL ). After 72 h, the parasite count was performed and the 50% inhibitory concentration (IC50) and the Combination Index (CI: antagonistic IC>1; IC=1 additive; IC<1 synergistic) were determined. Likewise, the isobologram corresponding to the IC50 values ​​of each of the F and their combinations was performed. The comparison of the treatments was made with an ANOVA and the Bonferroni post hoc test.

The IC50 values ​​of each F were: Anf=0.28 μg/ml and Chlo=1.27 μg/ml, being significantly lower than the cytotoxic concentration in Vero Cells. Of the concentrations studied, the combination of Chlo with 0.02 (IC50=1.11 μg/ml); 0.04 (IC50=0.80 μg/ml) and 0.08 (IC50=0.78 μg/ml) of Anf were below the additivity line, evidencing a synergistic effect. These data coincided with the IC values ​​obtained for each concentration respectively: 0.95; 0.78 and 0.89.

The use of purified F improved the antiparasitic effect allowing the reduction of Chlo concentration. The synergistic effect observed is consistent with the results found in previous studies on Trypanosoma cruzi and L. braziliensis.