Miltefosine (hexadecylphosphocholine [HePC]) is the first orally active drug approved for the treatment of visceral leishmaniasis. In order to investigate the biochemical modifications occurring in HePC-resistant (HePC-R) Leishmania donovani promastigotes, taking into account the lipid nature of HePC, we investigated their fatty acid and sterol metabolisms. We found that the content of unsaturated phospholipid alkyl chains was lower in HePC-R parasite plasma membranes than in those of the wild type, suggesting a lower fluidity of HePC-R parasite membranes. We also demonstrated that HePC insertion within an external monolayer was more difficult when the proportion of unsaturated phospholipids decreased, rendering the HePC interaction with the external monolayer of HePC-R parasites more difficult. Furthermore, HePC-R parasite membranes displayed a higher content of short alkyl chain fatty acids, suggesting a partial inactivation of the fatty acid elongation enzyme system in HePC-R parasites. Sterol biosynthesis was found to be modified in HePC-R parasites, since the 24-alkylated sterol content was halved in HePC-R parasites; however, this modification was not related to HePC sensitivity. In conclusion, HePC resistance affects three lipid biochemical pathways: fatty acid elongation, the desaturase system responsible for fatty acid alkyl chain unsaturation, and the C-24-alkylation of sterols.Leishmania spp. are the protozoan parasites responsible for leishmaniases, a family of diseases including a variety of clinical manifestations classically labeled as visceral, cutaneous, and mucocutaneous leishmaniases (9). Chemotherapy is the main tool for the control of leishmaniasis; however, the standard treatments, including pentavalent antimonials, amphotericin B (AmB), and paromomycin, are toxic and expensive.

The phospholipid analogue miltefosine or hexadecylphosphocholine (HePC) is a drug of high interest in the treatment for fatal visceral leishmaniasis (VL) due to Leishmania donovani particularly because of its activity by oral route. In this study, the interaction of HePC with a monolayer of beta-palmitoyl-gamma-oleyl-phosphatidylcholine (POPC) as membrane model or sterol (ergosterol or cholesterol) was investigated. At a constant pressure of 25 mN/m, the adsorption kinetics of HePC into the monolayers showed that HePC molecules are inserted into the monolayer of lipids as monomers until the critical micellar concentration (CMC). At HePC concentrations superior to the CMC, the micelles of HePC are deployed at the interface as groups of monomers into the POPC or sterol monolayer. The study of mixture of HePC/(POPC or sterol), spread at the air-water interface, shows that a simple miscibility between HePC and POPC is observed, whereas a high condensation appears between HePC and sterols showing a high affinity between HePC and sterols. In addition, HePC does not act as detergent disturbing membrane integrity.