Naturally azole-resistant Leishmania braziliensis promastigotes are rendered susceptible in the presence of terbinafine: comparative study with azole-susceptible Leishmania mexicana promastigotes

Abstract

Leishmania braziliensis (isolate 2903) was naturally resistant to ketoconazole or the bis-triazole D0870, inhibitors of sterol C-14 demethylase, which produced only moderate effects on the proliferation of promastigotes at 10 microM. In contrast, Leishmania mexicana (isolate NR) was extremely susceptible to the azoles, as complete growth arrest and cell lysis were induced by incubation of the parasites with 0.05 microM concentrations of the drugs for 72 h. The opposite response was observed with terbinafine, an inhibitor of squalene epoxidase: L. braziliensis 2903 was three times more susceptible to the drug than L. mexicana NR (MICs of 5 and 15 microM, respectively). However, when the L. braziliensis stock was grown in the presence of 1 microM terbinafine, which by itself produced only marginal (< 10%) effects on growth, it became highly susceptible to the azoles, with an MIC of 0.03 microM. Analysis of cellular free sterols by high-resolution capillary gas chromatography coupled to mass spectrometry showed that 14-methyl sterols can support normal growth of L. braziliensis 2903 but not of L. mexicana NR. On the other hand, the higher susceptibility of the L. braziliensis isolate to terbinafine was correlated with a massive accumulation of squalene in the presence of the allylamine while no significant effects on L. mexicana sterol composition were observed at drug concentrations up to 1 microM. Thus, the > 300-fold increase in the susceptibility of L. braziliensis promastigotes to azoles in the presence of terbinafine was attributed to the combined effect of squalene and the methylated sterol precursors on the physical properties of the cell's membranes, leading to the loss of cell viability. Combination therapy with azoles and terbinafine in the treatment of human L. braziliensis infections deserves further study.

Targeting Ergosterol Biosynthesis in Leishmania donovani: Essentiality of Sterol 14alpha-demethylase

“…donovani infantum [48], to our knowledge there has been no clinical trial of azoles for visceral leishmaniasis. Persistent Leishmania growth in the presence of azoles has been tied to tolerance to 14-methyl sterol accumulation in parasite membranes [14,18,49] as well as increased exogenous cholesterol incorporation [18]. …”

Antileishmanial Phenylpropanoids from the Leaves ofHyptis pectinata(L.) Poit

“…Cells were also cultured in a medium without compounds and vehicle (basal growth control) or with DMSO 0.1% (vehicle control). After 48 h, the extracellular load of L. braziliensis promastigotes was estimated by counting the promastigotes in Schneider's medium with a CELM automatic cell counter (model CC530) [21]. …”

Design, synthesis and in vitro trypanocidal and leishmanicidal activities of novel semicarbazone derivatives

“…Cells were also cultured in a medium free of compounds or vehicle (basal growth control) or with DMSO 0.1% (vehicle control). After 48 h, extracellular load of L. major promastigotes was estimated by counting the parasites in Schneider's medium in a CELM automatic cell counter (model CC530) [41]. Data obtained from experiments were expressed as the mean ± S.E.M.…”

“…The cytotoxicity of novel semicarbazone derivatives and pentamidine against promastigotes was determined [29,40,41]. Stationary phase L. major promastigotes were plated in 96-well vessels (Nunc) at 10 5 cells per well, in Schneider's medium, supplemented with 10% FBS and 2% human urine.…”