title: Imipramine alters the sterol profile in Leishmania amazonensis and increases its sensitivity to miconazole.
abstract: BACKGROUND
  Imipramine, a tricyclic antidepressant widely used clinically, has other pharmacological effects, such as antileishmanial activity. Tricyclic antidepressants interact with lipid bilayers, and some studies have shown that imipramine inhibits methyltransferases. Leishmania spp. produces compounds with an ergostane skeleton instead of a cholesterol skeleton, and the inhibition of enzymes of the sterol biosynthesis pathway is an interesting therapeutic target. Among these enzymes, C-24 methyltransferase has been suggested to play an essential role, as its inhibition kills the parasites. In this context, we investigated whether imipramine alters the biosynthesis of sterols in L. amazonensis and evaluated the efficacy of imipramine alone and in combination with miconazole, a classical inhibitor of another step in this pathway.
  
  
  METHODS
  To analyze the interference of imipramine with sterol metabolism, promastigotes of L. amazonensis were cultured with medium alone, 15 or 30 μM imipramine or 4 μM miconazole, and their lipids were extracted with methanol/chloroform/water (1:0.5:0.4 v/v) and analyzed by GC/MS. To assess the antileishmanial activity of the treatments, promastigotes of L. amazonensis were incubated with various concentrations of imipramine up to 100 μM and up to 24 μM miconazole. Promastigotes were also treated with the combination of imipramine and miconazole at concentrations up to 12.5 μM of imipramine and 24 μM of miconazole. Parasite growth was evaluated by the MTT assay. The fractional inhibitory concentration index (FICI) was calculated to determine whether there were synergistic effects. Peritoneal macrophages with and without L. amazonensis infection were treated with miconazole (0 - 16 μM) or imipramine (0 to 50 μM) for 72 hours. For assays of the combined treatment in amastigotes, the concentration of imipramine was fixed at 12.5 μM and various concentrations of miconazole were used up to 16 μM. The infection rate was determined by counting the infected macrophages under a light microscope.
  
  
  RESULTS
  Promastigotes treated with imipramine accumulated cholesta-5,7,22-trien-3β-ol and cholesta-7-24-dien- 3β-ol, sterols that normally increase after treatment with classical inhibitors of C-24 methyltransferase. The IC50 of miconazole in promastigotes decreased when it was used in combination with imipramine, resulting in an additive effect, with a FICI value of 0.83. Imipramine also showed activity against intracellular amastigotes and enhanced the activity of miconazole, without apparent toxicity to the host cells.
  
  
  CONCLUSIONS
  Imipramine was confirmed to have antileishmanial activity in both forms of the parasite, affecting the sterol biosynthesis of the organisms. Using imipramine in combination with azoles may be advantageous for the treatment of leishmaniasis.
authors.affiliation: Laboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Avenida Brasil 4365, Manguinhos, Rio de Janeiro, Brazil.
authors.forename: Valter Viana
authors.initials: VV
authors.lastname: Andrade-Neto
authors.affiliation: Laboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Avenida Brasil 4365, Manguinhos, Rio de Janeiro, Brazil.
authors.forename: Thaís Martins
authors.initials: TM
authors.lastname: Pereira
authors.affiliation: Laboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Avenida Brasil 4365, Manguinhos, Rio de Janeiro, Brazil.
authors.forename: Marilene do
authors.initials: Md
authors.lastname: Canto-Cavalheiro
authors.affiliation: Laboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Avenida Brasil 4365, Manguinhos, Rio de Janeiro, Brazil. ects@ioc.fiocruz.br.
authors.forename: Eduardo Caio
authors.initials: EC
authors.lastname: Torres-Santos
mesh_terms: D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D004357:Drug Synergism; D005562:Formazans / Q000032:analysis; D008401:Gas Chromatography-Mass Spectrometry; D007099:Imipramine / Q000494:pharmacology*; D007894:Leishmania mexicana / Q000187:drug effects* / Q000254:growth & development / Q000378:metabolism*; D017737:Macrophages, Peritoneal / Q000469:parasitology; D051379:Mice; D008825:Miconazole / Q000494:pharmacology*; D021261:Parasitic Sensitivity Tests; D013261:Sterols / Q000032:analysis*; D013778:Tetrazolium Salts / Q000032:analysis / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism
chemical_list: D000981:Antiprotozoal Agents; D005562:Formazans; D013261:Sterols; D013778:Tetrazolium Salts; D013844:Thiazoles; C079782:MTT formazan; D008825:Miconazole; C022616:thiazolyl blue; D007099:Imipramine
keywords: Imipramine; Leishmania; Sterols