**5. Miltefosine effects on lipid metabolism**

APLs, including miltefosine, have been found to interact with membrane lipids and affect lipid metabolism [75-77], these actions having been suggested being involved in their mechanism of action. In lipid monolayer studies, miltefosine molecules were inserted into the monolayer of lipids as monomers until the critical micellar concentration, and a high condensation was found between miltefosine and sterols showing a high affinity between miltefosine and sterols [78]. However, miltefosine did not act as detergent disturbing membrane integrity [78]. *Leishmania* parasites have high levels of ether-phospholipids [79-83], and these are mainly found in the glycosylphosphatidylinositol-anchored glycolipids and glycoproteins present on the surface of the parasites [84-86]. Because edelfosine and other APLs are ether lipids, it could be suggested that the biosynthesis of ether lipids occurring in the glycosomes of *Leishmania* might be affected. Miltefosine and edelfosine did not affect enzymes involved in early steps in ether lipid biosynthesis in *L. mexicana* promastigotes, including dihydroxyacetonephos‐ phate acyltransferase, *sn*-l-acyl-2-*lyso*-glycero-3-phosphocholine and *sn*-l-alkyl-2-*lyso*-glyc‐ ero-3-phosphocholine acyltransferases activities [87]. However, both miltefosine and edelfosine affected the later metabolism of alkyl-phosphatidylcholine intermediates by inhibiting the glycosomal located alkyl-specific-acyl-CoA acyltransferase in a dose-dependent manner with an inhibitory concentration of 50 µM, thus suggesting these drugs can perturb ether-lipid remodelling [87]. However, the fact that inhibition of alkyl-specific-acyl-CoA acyltransferase required drug concentrations higher than those showing cytotoxicity to *L. mexicana* (IC50, 14 µM and 18 µM for miltefosine and edelfosine, respectively) [87] challenges the putative involvement of this pathway as the primary target of these drugs. In addition, the role of glycosylphosphatidylinositols and ether phospholipids in the survival of *Leishmania* amastigotes is questioned by the viability of *L. major* null mutants for alkyldihydroxyaceto‐ nephosphate synthase (ADS), the first committed step of ether lipid synthesis. These mutants lacked all ether phospholipids, including plasmalogens, lipophosphoglycan (LPG), and smaller glycosylphosphatidylinositols (GIPLs) [88].

Treatment of *L. donovani* promastigotes with 10 µM miltefosine significantly reduced the phosphatidylcholine content and enhanced the phosphatidylethanolamine content in parasite membranes, suggesting a partial inactivation of phosphatidylethanolamine-N-methyltrans‐ ferase [89]. Phospholipase D activity was not affected by miltefosine, whereas the enhancement of the lysophosphatidylcholine content could be ascribed to phospholipase A2 activation. No effect was observed in the fatty acid alkyl chain length or the fatty acid unsaturation rate upon miltefosine treatment, whereas a two-fold increase was detected in the amount of cholesterol within the membranes [89]. Because cholesterol is not biosynthesized by the *Leishmania* parasite, but is taken from the external medium, it might be envisaged that miltefosine promotes cholesterol uptake in promastigotes perhaps by the condensation effect between miltefosine and cholesterol [78]. In contrast, a strong reduction of about two times in the C24 alkylated sterol content was detected in miltefosine-treated membranes, even though the level of the final C24 sterol alkylating product, ergosterol, the predominant plasma membrane sterol in fungi and *Leishmania*, was not changed [89].

Interestingly, the content of unsaturated phospholipid alkyl chains was lower in miltefosineresistant parasite plasma membranes than in those of the wild type, suggesting a lower fluidity of miltefosine-resistant parasite membranes, and rendering the miltefosine interaction with the external monolayer of miltefosine-resistant parasites more difficult. Miltefosine-resistant parasite membranes displayed a higher content of short alkyl chain fatty acids, suggesting a partial inactivation of the fatty acid elongation enzyme system in miltefosine-resistant parasites, and the C24-alkylated sterol content was halved in miltefosine-resistant parasites, but this modification was not related to miltefosine sensitivity [90]. Thus, miltefosine resistance affects three lipid biochemical pathways: fatty acid elongation, the desaturase system respon‐ sible for fatty acid alkyl chain unsaturation, and the C-24-alkylation of sterols. [90]. Because of the differences detected in the lipid composition of miltefosine-treated *Leishmania* and miltefosine-resistant parasites, it could be hypothesized that continuous *in vitro* drug pressure affects the regulation of *Leishmania* lipid metabolism [89], but the real implication of these actions on parasite killing remains a topic of much debate.
