In vitro study of amphotericin-B and clomipramine combination upon Leishmania braziliensis.

STRAUSS MARIANA; LOZANO ESTEBAN; AGUILAR JAVIER; KONIGHEIM BRENDA; AMELIA ENZO; LO PRESTI SILVINA; BAZÁN CAROLINA; BÁEZ ALEJANDRA; VELÁZQUEZ DANIELA; PAGLINI PATRICIA; RIVAROLA WALTER

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

Current drugs for the treatment of Leishmaniasis show disadvantages due to toxicity and high cost. Among these, Amphotericin-B (AMPHO), which causes the death of the parasites due to alterations in their cell membranes, also has adverse effects upon the host. Consequently, the search for new drugs, focused on molecular targets that are present in the parasite but absent in the host, continues. Clomipramine (CLO) is a potent inhibitor of trypanothione reductase, an enzyme exclusive to the Kinetoplastida order. The aim of the present work was to analyze the in vitro combined effect of AMPHO and CLO upon L. braziliensis promastigotes. We performed a cytotoxicity assay on Vero cells by incubating 2.5x105 cells/mL with AMPHO = 3.25?50 μg/mL and CLO = 7.8? 250 μg/mL for 4 days, and a combined effect assay in which 3x106 promastigotes/mL were incubated with AMPHO = 0.002?0.032 μg/mL and CLO = 0.3?2 μg/mL. We determined the IC50 values, calculated the Combination Index (CI: CI>1 antagonist, CI=1 additive, CI<1 synergist) and isobolograms were graphed. The effect of the drugs on the ultrastructure of L. braziliensis was evaluated by transmission electron microscopy. IC50 values were 0.018 μg/mL for AMPHO and 1.098 μg/mL for CLO upon promastigotes, which were 1033.3 and 104.8 times lower than the cytotoxic concentration, respectively. IC50 values upon Vero cells were 18.6 μg/mL for AMPHO and 126.34 μg/mL for CLO. CI upon Vero cells was 1.04 indicating additive effect and upon L. braziliensis was 0.18 indicating a markedly synergistic effect. IC50 values of the combinations upon L. braziliensis were located below the additivity line in the isobolograms, indicating a synergistic effect between AMPHO and CLO. The ultrastructural analysis of treated parasites revealed signs of autophagy, which was evidenced by increased autophagosomes. The association of AMPHO and CLO can therefore be considered as a more effective and safer potential strategy for Leishmaniasis pharmacotherapy.