Combined "in vitro" treatment of Amphotericin-B and Clomipramine on promastigotes of Leishmania braziliensis

Abstract

Leishmaniasis, an infectious disease caused by protozoa of the genus Leishmania, is a public health problem around the world.  Amphotericin-B, a drug used for its treatment, is often toxic to patients. The search for new drugs should identify molecular targets in the parasite and absent in the host. Trypanotione reductase is an exclusive enzyme of the order Kinetoplastide, which makes it useful as a molecular target. Tricyclic antidepressants, such as clomipramine (CLO) are good inhibitors of this enzyme. The objective of this work was to analyze the combined “in vitro” treatment of Amphotericin-B (ANF) and CLO on promastigotes of L. braziliensis.

We performed a cytotoxicity (MTT) test on Vero cells, incubating 2.5x105 cells/ml with ANF = 25-400µg/ml and CLO = 25-1000 µg/ml and a combined effect test (checkerboard) in which 3x106promastigotes/ml were incubated with ANF = 0.002-0.032µg/ml and CLO = 0.3-2µg/ml.  We determined the values ​​of the 50% inhibitory concentration (IC50) of both drugs, evaluated the effect of the combination calculating the Combination Index (CI: CI> 1 antagonistic; CI = 1 additive; CI <1 synergic) and plottedisobolograms from the IC50 values ​​of each of the drugs and the combinations. The ultrastructural alterations in the leishmanias were evaluated with electron microscopy.

IC50 values upon promastigotes were 0.018μg/ml for ANF and 1.098μg/ml for CLO, being 25 and 108 times lower than the cytotoxic concentration, respectively. IC50 values ​​upon Vero cells were 0.44μg/ml for ANF and 119μg/ml for CLO. The CI value of 0.18 indicates a synergistic effect. The IC50 of the combinations were located below the line of additivity in the isobolograms, which indicates a synergistic effect between ANF and CLO, coinciding with the result obtained with the CI. These results coincide with those observed in the microphotographs, since the greatest alterations corresponded to the leishmanias treated with the combination.

The observed synergistic effect may be the result of the complementary mechanisms of action of both drugs, which would potentiate the lethal effect, causing the death of the parasite at lower concentrations than the monotherapy and without obvious toxic effects to the host.

  Leishmaniasis; Amphotericin-B; clomipramine; trypanothione reductase