Abstract Background The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro. Methodology/Principal Findings We found that ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance. Conclusions/Significance Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation.

Author Summary Leishmaniasis represents a major international health problem, has a high morbidity and mortality rate, and is classified as an emerging and uncontrolled disease by the World Health Organization. The migration of population from endemic to nonendemic areas, and tourist activities in endemic regions are spreading the disease to new areas. Unfortunately, treatment of leishmaniasis is far from satisfactory, with only a few drugs available that show significant side-effects. Here, we show in vitro and in vivo evidence for the antileishmanial activity of the ether phospholipid edelfosine, being effective against a wide number of Leishmania spp. causing cutaneous, mucocutaneous and visceral leishmaniasis. Our experimental mouse and hamster models demonstrated not only a significant antileishmanial activity of edelfosine oral administration against different wild-type Leishmania spp., but also against parasites resistant to pentavalent antimonials, which constitute the first line of treatment worldwide. In addition, edelfosine exerted a higher antileishmanial activity and a lower proneness to generate drug resistance than miltefosine, the first drug against leishmaniasis that can be administered orally. These data, together with our previous findings, showing an anti-inflammatory action and a very low toxicity profile, suggest that edelfosine is a promising orally administered drug for leishmaniasis, thus warranting clinical evaluation.

Citation: Varela-M RE, Villa-Pulgarin JA, Yepes E, Müller I, Modolell M, Muñoz DL, et al. (2012) In Vitro and In Vivo Efficacy of Ether Lipid Edelfosine against Leishmania spp. and SbV-Resistant Parasites. PLoS Negl Trop Dis 6(4): e1612. https://doi.org/10.1371/journal.pntd.0001612 Editor: Jayne Raper, New York University School of Medicine, United States of America Received: November 7, 2011; Accepted: February 27, 2012; Published: April 10, 2012 Copyright: © 2012 Varela-M et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Spanish Ministerio de Ciencia e Innovación (SAF2008-02251; SAF2011-30518; RD06/0020/1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union; and TRA2009-0275), European Community's Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986), Junta de Castilla y León (CSI052A11-2; GR15-Experimental Therapeutics and Translational Oncology Program) and Spain-UK International Joint Project grant from The Royal Society-CSIC (2004GB0032). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: FM is co-founder of Apointech and a member of its scientific advisory board. REV, JAVP and EY are employees of Apointech. The other authors disclose no potential conflicts of interest.

Introduction The impact of the leishmaniases on human health has been grossly underestimated for many years, and this complex of diseases has been classified by the World Health Organization (WHO) as one of the most neglected tropical diseases [1]. During the last decade, endemic areas have been spreading and a sharp increase in the number of leishmaniasis cases has been recorded. The WHO classifies leishmaniasis as a category 1 disease (“emerging and uncontrolled”), and there is an urgent need to develop new therapeutic drugs and approaches. Currently, about 350 million people in 98 countries around the world are at risk, and an estimated 12 million people are infected [1]. Despite progress in the diagnosis and treatment, leishmaniasis remains a major public health problem, particularly in tropical and sub-tropical developing countries. Published figures indicate an estimated incidence of two million new cases per year, with 1.5 million cases of self-healing, but disfiguring, cutaneous leishmaniasis, and 500,000 cases of life-threatening visceral leishmaniasis [1], [2]. Approximately 60,000 people die from visceral leishmaniasis each year, a rate surpassed among parasitic diseases only by malaria; and a loss of about 2.4 million disability-adjusted life years (DALYs) throughout the world has been calculated as the total disease burden of leishmaniasis [1]–[3]. Furthermore, a number of reports have emphasized the increasing importance of visceral leishmaniasis as an opportunistic infection among HIV-positive patients in areas where both infections are endemic [4]. The chemotherapy currently available for the leishmaniases is far from satisfactory and presents several problems, including toxicity, many adverse side-effects, high costs and development of drug resistance [2], [5]. Two pentavalent antimonial (SbV) compounds, sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime), were first introduced in the 1940's and have since been used as first-line chemotherapeutic agents against all forms of leishmaniasis through parenteral administration. Although SbV, administered by intramuscular or intravenous route, remains the first-line drug for the treatment of leishmaniasis worldwide, its efficacy is becoming increasingly lower [6], and highly depends on Leishmania species and distinct endemic regional variations, even within the same country. Resistance is now common in India, and rates of resistance have been shown to be higher than 60% in parts of the state of Bihar, in north-east India [7], [8]. In addition, the incidence of adverse effects, including myalgia, arthralgias, pancreatitis, nephrotoxicity, hepatotoxicity, and cardiotoxicity [1], [2], [9], makes the search for new alternative medicines to SbV an urgent issue, and a number of drugs are now in clinical trials [10]. Intravenous infusion of liposomal amphotericin B (AmBisome) is at present the most effective anti-Leishmania drug [2], [11], but its relatively high cost makes it unaffordable in several poor areas of the world where the disease is more prevalent [2]. In addition, the requirement for long periods of parenteral administration, frequently requiring hospitalization, has also limited the clinical use of amphotericin B. Miltefosine (Impavido) is a new oral agent that has shown high cure rates in visceral leishmaniasis in India (L. donovani; 94% cure) [12], and in cutaneous leishmaniasis in Colombia (L. panamensis; >90% cure) [13]. However, a recent therapeutical trial has revealed a limited potential of miltefosine for the treatment of American cutaneous leishmaniasis, with an unsatisfactory cure rate of 69.8% in Colombia [14]. Furthermore, this percentage fell to 49% when miltefosine was administered to patients with lesions caused by L. braziliensis, which comprise more than 60% of cutaneous leishmaniasis in Colombia [14]. Additional recent clinical trials in Brazil showed a cure rate of miltefosine for the treatment of cutaneous leishmaniasis caused by L. braziliensis of 75% [15], and for the treatment of cutaneous leishmaniasis caused by L. guyanensis of 71% [16]. Miltefosine treatment also led to approximately 70% cure rate for mucosal leishmaniasis due to L. braziliensis in Bolivia [17], [18]. Moreover, the miltefosine cure rate was approximately 53% for cutaneous leishmaniasis (33% for L. braziliensis infection, and 60% for L. mexicana infection) in Guatemala [13], [19], [20], and a cure rate of 63% was reported for L. tropica in Afghanistan [20]. These figures contrast with cure rates of more than 82% in the treatment of visceral leishmaniasis (kala-azar) in India [21], [22] and Bangladesh [23]. These data point out the great variability in the outcome depending on the geographical area for reasons that are not well understood. In addition, miltefosine commonly induces gastrointestinal side-effects, such as anorexia, nausea, vomiting and diarrhea, that sometimes lead to drop out from treatment [1], [2], [22]. Miltefosine is potentially teratogenic and should not be administered to pregnant women [1], [2], for whom adequate contraception should be guaranteed during treatment and for up to 3 months afterwards [1], given the teratogenic potential of miltefosine in animal models [24]. An additional concern is the rapid in vitro generation of resistance to miltefosine [25]–[27] that could limit its clinical use. Thus, these studies reinforce the need to search for new therapeutic alternatives in the treatment of leishmaniasis. Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine, ET-18-OCH 3 ) is a promising antitumor ether lipid drug [28]–[30], which is not mutagenic and acts by activating apoptosis through its interaction with cell membranes [31]–[34]. In addition to its antitumor activity, edelfosine has been shown to exert in vitro antiparasitic activity against different species of Leishmania parasites [35]–[37]. Edelfosine has been considered the prototype molecule of a rather heterogeneous family of synthetic compounds collectively known as alkyl-lysophospholipid analogs (ALPs), that comprise the above clinically relevant miltefosine as well as perifosine, which also shows anti-Leishmania activity [38], [39]. Although the mechanism of action of miltefosine against Leishmania parasites remains to be fully elucidated, there are some reports showing that the ability of this compound to promote an apoptosis-like cell death is critical for its leishmanicidal activity [40], [41]. Because edelfosine has been shown to have a higher proapototic activity than both miltefosine and perifosine in human cancer cells [29], [30], [33], we have carried out here a comprehensive in vitro and in vivo study, investigating the putative anti-Leishmania traits of edelfosine, as compared to other ALPs, using different Leishmania species as well as mouse and hamster experimental models.

Materials and Methods Ethics statement Animal procedures in this study complied with the Spanish (Real Decreto RD1201/05) and the European Union (European Directive 2010/63/EU) guidelines on animal experimentation for the protection and humane use of laboratory animals, and were conducted at the accredited Animal Experimentation Facility (Servicio de Experimentación Animal) of the University of Salamanca (Register number: PAE/SA/001). Procedures were approved by the Ethics Committee of the University of Salamanca (protocol approval number 48531). Drugs Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) was from INKEYSA (Barcelona, Spain) and Apointech (Salamanca, Spain). Miltefosine (hexadecylphosphocholine) was from Calbiochem (Cambridge, MA). Perifosine (octadecyl-(1,1-dimethyl-piperidinio-4-yl)-phosphate) and erucylphosphocholine ((13Z)-docos-13-en-1-yl 2-(trimethylammonio)ethyl phosphate) were from Zentaris (Frankfurt, Germany). Stock sterile solutions of the distinct ALPs (2 mM) were prepared in RPMI-1640 culture medium (Invitrogen, Carlsbad, CA), supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin (GIBCO-BRL, Gaithersburg, MD) as previously described [28]. Leishmania cells and culture conditions The following Leishmania strains were used in this study: L. amazonensis (MHOM/Br/73/LV78), L. braziliensis (MHOM/CO/88/UA301), L. donovani (MHOM/IN/80/DD8), L. infantum (MCAN/ES/96/BCN150), L. major LV39 (MRHO/SU/59/P), L. mexicana (MHOM/MX/95/NAN1), and L. panamensis (MHOM/CO/87/UA140). Leishmania promastigotes were grown in RPMI-1640 culture medium, supplemented with 10% FBS, 2 mM glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at 26°C. Promastigotes were treated with the indicated compounds during their logarithmic growth phase (1.5×106 parasites/ml) at 26°C. Late stationary promastigotes were obtained after incubation of the parasites for 5–6 days with starting inocula of 1×106 parasites/ml. Leishmania axenic amastigotes were obtained at pH 5.0 in Schneider's culture medium following a stepped temperature increase to 30, 31 and 32°C, except for L. infantum amastigotes, which were exposed to 34, 36 and 37°C, as previously described [42]. Growth inhibition assay The antileishmanial activity in promastigotes and axenic amastigotes was determined by using the XTT (sodium 3,3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) cell proliferation kit (Roche Molecular Biochemicals, Mannheim, Germany) as previously described [42], [43]. Cells were resuspended in FBS-containing RPMI-1640 culture medium (1.5×106 cells/ml for promastigotes, and 2×106 cells/ml for axenic amastigotes), and plated (100 µl/well) in 96-well flat-bottomed microtiter plates at 26°C, in the absence and in the presence of different concentrations of the indicated ALPs. After 72-h incubation at 26°C, IC 50 (half-maximal inhibitory concentration) values, defined as the drug concentration causing 50% inhibition in cell proliferation with respect to untreated controls, were determined for each compound. Measurements were done in triplicate, and each experiment was repeated four times. Analysis of apoptosis-like cell death by flow cytometry One and a half million Leishmania spp. promastigotes or axenic amastigotes were treated in the absence and in the presence of the indicated concentrations of ALPs for different incubation times. Then, parasites were pelleted by centrifugation (1000× g) for 5 min, and analyzed for apoptosis-like DNA breakdown by flow cytometry following a protocol previously described [44]. Quantitation of apoptotic-like cells was monitored as the percentage of cells in the sub-G 0 /G 1 region (hypodiploidy) in cell cycle analysis [44], [45], using a fluorescence-activated cell sorting (FACS) Calibur flow cytometer (Becton Dickinson, San Jose, CA) equipped with a 488 nm argon laser. WinMDI 2.8 software was used for data analysis. Intracellular distribution of fluorescent edelfosine analog in L. panamensis–infected J774 macrophages The mouse macrophage-like cell line J774, grown in RPMI-1640 culture medium, supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/ml streptomycin, at 37°C in humidified 95% air and 5% CO 2 , was infected overnight at the exponential growth phase (3×105 cells/ml) with stationary-phase L. panamensis promastigotes, at a macropage/promastigote ratio of 1/10 in complete RPMI-1640 culture medium. Non-internalized promastigotes were removed by 2–3 successive washes with PBS. Then, uninfected and L. panamensis-infected J774 macrophages were incubated for 1 h with 10 µM of the fluorescent edelfosine analog all-(E)-1-O-(15′-phenylpentadeca-8′,10′,12′,14′-tetraenyl)-2-O-methyl-rac-glycero-3-phosphocholine (PTE-ET) [34], [46], [47] (kindly provided by F. Amat-Guerri and A.U. Acuña, Consejo Superior de Investigaciones Científicas, Madrid, Spain) in complete RPMI-1640 culture medium. In addition, J774 cells were also incubated first with 10 µM PTE-ET for 1 h, then washed with PBS and infected with L. panamensis in the darkness for 6 h. Samples were fixed with 1% formaldehyde, and analyzed with a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss GmbH, Oberkochen, Germany) (40× magnification). Assessment of intracellular parasitic load in macrophage-like cells J774 cells were infected with L. panamensis promastigotes as above. The number of intracellular viable parasites was assessed by incubating infected cells with RPMI-1640 medium containing 0.008% SDS to gently disrupt macrophage plasma membrane, followed by addition of RPMI-1640 culture medium containing 20% FBS to stop further lysis. Samples were then sequentially diluted in 96-well plates containing biphasic Novy-MacNeal-Nicolle (NNN) medium. Plates were incubated at 26°C for 20 days, and examined weekly under an inverted Nikon TS-100 microscope (Nikon, Kanagawa, Japan) to evaluate the presence of viable motile promastigotes. The reciprocal of the highest dilution found positive for parasite growth was considered to be the concentration of parasites. Determination of nitric oxide (NO) by the nitrite assay Macrophage-like J774 cells were plated in complete RPMI-1640 culture medium at a concentration of 1×106 cells/well in 24-well culture plates (Costar, Cambridge, MA), and let them adhere for 2 h at 37°C in 5% CO 2 . Non-adhering cells were removed by gentle washing with complete RPMI-1640 culture medium. Adherent J774 cells were incubated in the absence (negative control), or in the presence of 10 µg/ml lipopolysaccharide (Sigma, St. Louis, MO) (LPS; positive control) or of different concentrations of edelfosine. After 18-h incubation at 37°C in 5% CO 2 , supernatants were collected, centrifuged at 500× g for 10 min, and stored at −80°C until analysis. NO release was indirectly measured using a colorimetric assay based on the Griess reaction. Triplicate 100-µl aliquots of cell culture supernatants were incubated with 50 µl of freshly prepared Griess reagent (1% sulfanilamide, 0.1% naphthylethylene diamide dihydrochloride, and 2.5% orthophosphoric acid) for 15 min at room temperature, and then absorbance of the azo-chromophore was measured at 550 nm. Nitrite concentration was determined by using sodium nitrite as a standard. All samples were assayed against a blank comprising complete RPMI-1640 culture medium incubated for 18 h on the same plates as the samples, but in the absence of cells. All reagents were purchased from Sigma. Results were expressed in nanomoles of nitrite per 106 macrophages. Evaluation of antileishmanial activity in mouse and hamster models Six-week-old female BALB/c mice (18–20 g) and four-week-old male Syrian golden hamsters (Mesocricetus auratus) (about 120 g) (Charles River Laboratories, Lyon, France), kept in a pathogen-free facility and handled according to institutional guidelines, complying with the Spanish legislation under a 12/12-h light/dark cycle at a temperature of 22°C, received a standard diet and water ad libitum. Mice were inoculated s.c. into their left hind footpad (in a total volume of 50 µl PBS) with 2×106 infective stationary-phase promastigotes, whereas hamsters, previously anesthetized with inhaled Forane, were inoculated intradermally in the nose with 1×106 stationary-phase promastigotes in a volume of 50 µl PBS. When inflamation was evident (about 1 week in mice, and 6 weeks in hamsters, after inoculation), animals were randomly assigned into cohorts of 7 animals each, receiving a daily oral administration (through a feeding needle) of edelfosine (15 mg/kg for mice, and 26 mg/kg for hamsters, in water), or an equal volume of vehicle (water). In mice, the footpad thickness was measured with calipers every week, and compared with the uninfected right hind footpad to obtain the net increase in footpad swelling. In hamsters, nose swelling was measured with calipers every week, and compared with the nose size before inoculation and treatment. Evolution index of the lesion was calculated as size of the lesion during treatment (mm)/size of the lesion before treatment. Animal body weight and any sign of morbidity were monitored. Drug treatment lasted for 28 days, and animals were killed following institutional guidelines, 24 h after the last drug administration. After the killing of the animals, the parasite burden in the infected tissues was determined by limiting dilution assays as previously described [48]. Biopsies were washed 3 times with PBS supplemented with 100 units/ml penicillin and 100 µg/ml streptomycin (GIBCO-BRL), and then incubated overnight (12 h) at 4°C with PBS containing 100 units/ml of penicillin and 100 µg/ml streptomycin. Following overnight incubation, biopsies were washed 2–3 times with PBS supplemented with the above antibiotics, and then a weighed piece of the infected area was homogenized in 1 ml PBS containing antibiotics using a sterile glass Potter-Elvejhem type tissue grinder. Homogenate was diluted at a final concentration of 0.1 mg/ml in Schneider's culture medium, containing 100 units/ml penicillin and 100 µg/ml streptomycin; and then serial dilutions were made in triplicate in 96-well plates containing biphasic Novy-MacNeal-Nicolle (NNN) medium. Plates were incubated at 26°C for 20 days, and examined weekly under an inverted Nikon TS-100 microscope to evaluate the presence of viable promastigotes. The reciprocal of the highest dilution found positive for parasite growth was considered to be the concentration of parasites per mg of tissue. Total parasite load was calculated using the total weight of the respective infected organ. Induction of in vitro resistance to Glucantime in L. panamensis promastigotes Parasites cultured in Schneider's culture medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 µg/ml streptomycin at 26°C for 5 days, were washed twice with PBS, and centrifuged at 1000× g for 10 min at room temperature. Parasites were then resuspended at 2×106 promastigotes/ml in Schneider's culture medium, and incubated at 26°C for 5 days with 4 mg/ml Glucantime (Aventis Pharma, Sao Paulo, Brazil), which corresponded to its IC 50 value, previously assessed by the XTT technique. Drug-containing culture medium was changed every 4–6 days, depending on parasite growth, and parasites were washed with PBS, analyzed by XTT assay, and resuspended again at 2×106 parasites/ml. This procedure was repeated until parasite viability in the presence of the drug was over 80%. Then, after achieving this viability rate, this process was repeated three times, with increasing concentrations of SbV, up to reaching a final concentration of 37 mg/ml. The volume of drug solution used in each passage was controlled not to exceed 10% of the total volume of culture medium. Assessment of L. panamensis resistance to SbV in the hamster animal model The level of SbV resistance was further assessed by infection of golden hamsters with the above in vitro-generated SbV-resistant (SbV-R) L. panamensis parasites, growing in the presence of 37 mg/ml SbV, as well as with wild-type susceptible L. panamensis, followed by treatment with Glucantime. Hamsters were divided into two groups, eight animals infected with the resistant strain and eight animals infected with the susceptible strain. Each group was inoculated intradermally on the nose with 1×106 stationary-phase promastigotes in a volume of 50 µl PBS. These animals were previously anesthetized with ketamine (50 mg/ml) and xylazine (5 mg/kg) intraperitoneally. About six weeks after infection, lesions were evident in both animal groups, and animals were treated daily with 40 mg/kg Glucantime, intramuscularly using a 27-gauge needle, for ten days. Evolution of the lesions and drug efficacy were monitored as above. Induction of in vitro resistance to ALPs in different Leishmania species ALP-resistant Leishmania strains were generated as indicated above for SbV-resistant parasites. Drugs were initially incubated at their corresponding IC 50 values, and then drug concentration was gradually increased. Parasites were considered resistant when they could grow at a drug concentration of 30 µM. Statistical analysis Data are shown as mean ± SD. Between-group statistical differences were assessed using the Mann-Whitney or the Student's t test. A P-value of <0.05 was considered statistically significant.

Discussion Our results show the in vitro and in vivo antileishmanial activity of edelfosine against different Leishmania species. The ability of edelfosine to kill distinct Leishmania spp. promastigotes and amastigotes is in general higher than other ALPs, and the antileishmanial activity of ALPs ranked edelfosine>perifosine>miltefosine>erucylphosphocholine. Edelfosine also shows a higher capacity to induce an apoptosis-like cell death in Leishmania than miltefosine (Impavido), which has been approved as the first oral drug active against visceral leishmaniasis [2]. However, recent studies have challenged the efficacy of miltefosine against some cutaneous leishmaniasis [13]–[15], [17]–[20], and relapse cases of miltefosine-treated parasites have been reported in visceral and diffuse cutaneous leishmaniasis [82]–[84] as well as in HIV-positive patients [85], [86]. Here, we have found that edelfosine shows an outstanding activity against a wide number of Leishmania spp. causing cutaneous, mucocutaneous and visceral leishmaniasis. Edelfosine was able to kill parasites in both promastigote and amastigote forms through an apoptosis-like process that involved DNA degradation, as assessed by an increase in the percentage of cells with a hypodiploid DNA content. Leishmania parasites infect macrophages wherein they reside and replicate in a fusion competent vacuole (parasitophorous vacuole). Interestingly, edelfosine efficiently killed intracellular parasite amastigotes inside macrophages, without affecting the host cells. This killing activity on intracellular parasites seems to be mainly due to a direct action of the drug on the parasite, as edelfosine was unable to induce NO generation in macrophages, while a fluorescent edelfosine analog accumulated in the intracellular parasites within macrophages. Our data also reveal a remarkable antileishmanial activity of edelfosine in several in vivo assays using mouse and hamster animal models infected with L. major, L. panamensis or L. braziliensis. To our knowledge this is the first study using hamsters as animal models for the in vivo evaluation of ALPs against cutaneous leishmaniasis. In addition, both in vitro and in vivo evidence showed that edelfosine was very effective against SbV-resistant Leishmania parasites. This is of importance as pentavalent antimonials Glucantime and Pentostam are being used in the treatment of leishmaniasis for over more than six decades, and still they are the first line drugs of choice and the traditional treatment worldwide. However, resistance to pentavalent antimonials is emerging as a result of their widespread use. A stark example of SbV resistance is well documented in Bihar (India), which houses approximately 90% of Indias's cases of visceral leishmaniasis, representing about 50% of the world's cases, and where resistance ended the usefulness of SbV more than a decade ago [2]. A major potential drawback in the use of miltefosine could be the relatively rapid generation of drug resistance in vitro. We have found here that generation of drug resistance required longer incubation times of Leishmania spp. with edelfosine than with miltefosine. Furthermore, whereas miltefosine generated drug resistance in L. donovani following a 40-day treatment, no resistance to edelfosine was detected after 100-day incubation. It is worthwhile to note that miltefosine treatment has been reported to be unsatisfactory against infections caused by L. braziliensis [13]–[15], [17]–[20], whereas here we have found a remarkable antiparasitic activity of edelfosine in L. braziliensis-infected hamsters. In addition, edelfosine offers a number of additional advantages as compared to miltefosine, such as the fact that edelfosine shows a potent anti-inflammatory action [87], and no apparent toxicity [87]. Leishmania parasites enter first neutrophils through the regulation of granule fusion processes that prevents any deleterious action on the parasite [88]. Leishmania parasites use polymorphonuclear neutrophils as intermediate hosts before their ultimate delivery to macrophages, following engulfment of parasite-infected neutrophils, and in this way Leishmania can escape the host immune system [89]. A significant part of the destruction caused by cutaneous leishmaniasis is due to severe inflammation at the site of infection in the skin, leading to ulceration [90]. Neutrophils are recruited into the site of infection during cutaneous leishmaniasis [91], [92], and accumulation of neutrophils have been linked to tissue damage [93]. Edelfosine induces L-selectin (CD62L) shedding, and thus prevents neutrophil extravasation to the inflammation or infection site [87]. On these grounds, leishmaniasis could be ameliorated by oral treatment of edelfosine, which could reduce the parasite burden, by direct parasite killing, as well as the ulcerative process and subsequent scar formation, by a reduction in the recruitment of neutrophils into the site of infection. A serious drawback of miltefosine is its teratogenic effects [24], however no studies have been conducted so far for a putative teratogenic action of edelfosine. The studies reported here provide compelling evidence for the potent antileishmanial activity of edelfosine, which together with the low toxicity profile displayed by this ether lipid and its anti-inflammatory activity, warrants further clinical evaluation as a possible alternative treatment against leishmaniasis.

Acknowledgments We are indebted to P. Kropf and B. G. Sierra for excellent and skillful assistance in the initial stages of this study.

Author Contributions Conceived and designed the experiments: FM REV JAV-P. Performed the experiments: REV JAV-P EY IM DLM JL-A. Analyzed the data: REV JAV-P IM MM SMR CEM JL-A AM IDV FM. Contributed reagents/materials/analysis tools: IM IDV FM. Wrote the paper: FM REV JAV-P.