Combined therapy “in vitro" of Amphotericin-B and Clomipramine on promastigotes of Leishmania amazonensis

Abstract

Abstract: 

Leishmaniasis is a disease caused by parasites of the Leishmania genus. Amphotericin-B (Anf) is one of the main drugs used for its treatment, however cases of toxicity and resistance have been reported. The search for new therapies involves the use of drug combinations, which have been shown to be effective for conditions of similar host-pathogen biology and the identification of molecular targets present in the parasite, but absent in the host, such as trypanethione reductase in Leishmania. An inhibitor drug of this enzyme is clomipramine (Clo), traditionally used for its antidepressant action. The objective of this work was to analyze the combined therapy "in vitro" of Anf and Clo on promastigotes of  L. amazonensis.

Were incubated 5x106 promastigotes / mL with concentrations of the drugs alone and in combination (Anf = 0.02-0.62 μg / mL and Clo = 0.48-7.8 μg / mL). These concentrations were previously shown to be non-cytotoxic in Vero (CV) cells. After 72 h, the parasite count was performed and the 50% inhibitory concentration (IC50) and the Combination Index (IC: IC> 1 antagonistic; IC = 1 additive; IC <1 synergistic) were determined. Finally, the isobologram corresponding to the IC50 values ​​of each of the drugs and their combinations was performed.

The IC50 values ​​of each drug were: Anf = 0.18 µg / ml and Clo = 6 µg / ml, being 103 and 21 times lower than the cytotoxic concentration in CV. Of the concentrations studied, the combination of Clo (0.48-7.8 μg / mL) with 0.02; 0.04 and 0.08 of Anf were located below the additivity line, which indicates a synergistic effect, coinciding with the value obtained from CI = 0.61.

These tests show an increase in the antiparasitic effect at low concentrations when using combined therapies, allowing to reduce the toxic effects on the host. The synergistic effect observed is consistent with the results found in previous studies in Trypanosoma cruzi and L. braziliensis.