Background:The treatment of leishmaniasis relies mostly on parenteral drugs with potentially serious adverse effects. Additionally, parasite resistance in the treatment of leishmaniasis has been demonstrated for the majority of drugs available, making the search for more effective and less toxic drugs and treatment regimens a priority for the control of leishmaniasis. The aims of this study were to evaluate the antileishmanial activity of raloxifene in vitro and in vivo and to investigate its mechanism of action against Leishmania amazonensis.Methodology/Principal Findings:Raloxifene was shown to possess antileishmanial activity in vitro against several species with EC50 values ranging from 30.2 to 38.0 μM against promastigotes and from 8.8 to 16.2 μM against intracellular amastigotes. Raloxifene's mechanism of action was investigated through transmission electron microscopy and labeling with propidium iodide, DiSBAC2(3), rhodamine 123 and monodansylcadaverine. Microscopic examinations showed that raloxifene treated parasites displayed autophagosomes and mitochondrial damage while the plasma membrane remained continuous. Nonetheless, plasma membrane potential was rapidly altered upon raloxifene treatment with initial hyperpolarization followed by depolarization. Loss of mitochondrial membrane potential was also verified. Treatment of L. amazonensis - infected BALB/c mice with raloxifene led to significant decrease in lesion size and parasite burden.Conclusions/Significance:The results of this work extend the investigation of selective estrogen receptor modulators as potential candidates for leishmaniasis treatment. The antileishmanial activity of raloxifene was demonstrated in vitro and in vivo. Raloxifene produces functional disorder on the plasma membrane of L. amazonensis promastigotes and leads to functional and morphological disruption of mitochondria, which culminate in cell death. © 2014 Reimão et al.85 Alvar, J., Velez, I.D., Bern, C., Herrero, M., Desjeux, P., Leishmaniasis Worldwide and Global Estimates of Its Incidence (2012) PLoS One, 7, pp. e35671Croft, S.L., Olliaro, P., Leishmaniasis chemotherapy-challenges and opportunities (2011) Clin Microbiol Infect, 17, pp. 1478-1483Dorlo, T.P., Balasegaram, M., Beijnen, J.H., de Vries, P.J., Miltefosine: a review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis (2012) J Antimicrob Chemother, 67, pp. 2576-2597Croft, S.L., Sundar, S., Fairlamb, A.H., Drug resistance in leishmaniasis (2006) Clin Microbiol Rev, 19, pp. 111-126(2010) Control of the leishmaniasis: Report of a meeting of the World Health Organization Expert Committee on the Control of Leishmaniases, , WHO. Geneva: WHOCroft, S.L., Seifert, K., Yardley, V., Current scenario of drug development for leishmaniasis (2006) Indian J Med Res, 123, pp. 399-410Ekins, S., Williams, A.J., Krasowski, M.D., Freundlich, J.S., In silico repositioning of approved drugs for rare and neglected diseases (2011) Drug Discov Today, 16, pp. 298-310Osborne, C.K., Zhao, H., Fuqua, S.A., Selective estrogen receptor modulators: structure, function, and clinical use (2000) J Clin Oncol, 18, pp. 3172-3186Cohen, I., Endometrial pathologies associated with postmenopausal tamoxifen treatment (2004) Gynecol Oncol, 94, pp. 256-266Heringa, M., Review on raloxifene: profile of a selective estrogen receptor modulator (2003) Int J Clin Pharmacol Ther, 41, pp. 331-345Ko, S.S., Jordan, V.C., Treatment of osteoporosis and reduction in risk of invasive breast cancer in postmenopausal women with raloxifene (2011) Expert Opin Pharmacother, 12, pp. 657-674Miguel, D.C., Yokoyama-Yasunaka, J.K., Andreoli, W.K., Mortara, R.A., Uliana, S.R., Tamoxifen is effective against Leishmania and induces a rapid alkalinization of parasitophorous vacuoles harbouring Leishmania (Leishmania) amazonensis amastigotes (2007) J Antimicrob Chemother, 60, pp. 526-534Miguel, D.C., Zauli-Nascimento, R.C., Yokoyama-Yasunaka, J.K., Katz, S., Barbieri, C.L., Tamoxifen as a potential antileishmanial agent: efficacy in the treatment of Leishmania braziliensis and Leishmania chagasi infections (2009) J Antimicrob Chemother, 63, pp. 365-368Eissa, M.M., Amer, E.I., El Sawy, S.M., Leishmania major: activity of tamoxifen against experimental cutaneous leishmaniasis (2011) Exp Parasitol, 128, pp. 382-390Miguel, D.C., Yokoyama-Yasunaka, J.K., Uliana, S.R., Tamoxifen is effective in the treatment of Leishmania amazonensis infections in mice (2008) PLoS Negl Trop Dis, 2, pp. e249Lainson, R., Shaw, J.J., New World Leishmaniasis - The Neotropical Leishmania species (1998) Topley & Wilson's Microbiology and Microbial Infections, pp. 241-266. , In: Kreier JP WD, editor. London: F.E.G. CoxReimao, J.Q., Trinconi, C.T., Yokoyama-Yasunaka, J.K., Miguel, D.C., Kalil, S.P., Parasite burden in Leishmania (Leishmania) amazonensis-infected mice: Validation of luciferase as a quantitative tool (2013) J Microbiol Methods, 93, pp. 95-101Arruda, D.C., D'Alexandri, F.L., Katzin, A.M., Uliana, S.R., Leishmania amazonensis: biosynthesis of polyprenols of 9 isoprene units by amastigotes (2008) Exp Parasitol, 118, pp. 624-628Zamboni, D.S., Rabinovitch, M., Nitric oxide partially controls Coxiella burnetii phase II infection in mouse primary macrophages (2003) Infect Immun, 71, pp. 1225-1233Zauli-Nascimento, R.C., Miguel, D.C., Yokoyama-Yasunaka, J.K., Pereira, L.I., Pelli de Oliveira, M.A., In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B (2010) Trop Med Int Health, 15, pp. 68-76Munafo, D.B., Colombo, M.I., A novel assay to study autophagy: Regulation of autophagosome vacuole size by amino acid deprivation (2001) J Cell Sci, 114, pp. 3619-3629Fernandes, M.C., Da Silva, E.N., Pinto, A.V., de Castro, S.L., Menna-Barreto, R.F., A novel triazolic naphthofuranquinone induces autophagy in reservosomes and impairment of mitosis in Trypanosoma cruzi (2012) Parasitology, 139, pp. 26-36Pinheiro, R.O., Nunes, M.P., Pinheiro, C.S., D'Avila, H., Bozza, P.T., Induction of autophagy correlates with increased parasite load of Leishmania amazonensis in BALB/c but not C57BL/6 macrophages (2009) Microbes Infect, 11, pp. 181-190Luque-Ortega, J.R., Rivas, L., Characterization of the leishmanicidal activity of antimicrobial peptides (2010) Methods Mol Biol, 618, pp. 393-420de Morais-Teixeira, E., Damasceno, Q.S., Galuppo, M.K., Romanha, A.J., Rabello, A., The in vitro leishmanicidal activity of hexadecylphosphocholine (miltefosine) against four medically relevant Leishmania species of Brazil (2011) Mem Inst Oswaldo Cruz, 106, pp. 475-478Biederbick, A., Kern, H.F., Elsasser, H.P., Monodansylcadaverine (MDC) is a specific in vivo marker for autophagic vacuoles (1995) Eur J Cell Biol, 66, pp. 3-14Besteiro, S., Williams, R.A., Morrison, L.S., Coombs, G.H., Mottram, J.C., Endosome sorting and autophagy are essential for differentiation and virulence of Leishmania major (2006) J Biol Chem, 281, pp. 11384-11396Custodio, J.B., Moreno, A.J., Wallace, K.B., Tamoxifen inhibits induction of the mitochondrial permeability transition by Ca2+ and inorganic phosphate (1998) Toxicol Appl Pharmacol, 152, pp. 10-17Taurin, S., Allen, K.M., Scandlyn, M.J., Rosengren, R.J., Raloxifene reduces triple-negative breast cancer tumor growth and decreases EGFR expression (2013) Int J Oncol, 43, pp. 785-792Wang, Q., Lu, L., Gao, X., Wang, C., Wang, J., Effects of raloxifene on voltage-dependent T-type Ca2+ channels in mouse spermatogenic cells (2011) Pharmacology, 87, pp. 70-80Benaim, G., Garcia-Marchan, Y., Reyes, C., Uzcanga, G., Figarella, K., Identification of a sphingosine-sensitive Ca2+ channel in the plasma membrane of Leishmania mexicana (2013) Biochem Biophys Res Commun, 430, pp. 1091-1096Palit, P., Ali, N., Oral Therapy with Amlodipine and Lacidipine, 1,4-Dihydropyridine Derivatives Showing Activity against Experimental Visceral Leishmaniasis (2008) Antimicrob Agents Chemother, 52, pp. 374-377Tempone, A.G., Taniwaki, N.N., Reimao, J.Q., Antileishmanial activity and ultrastructural alterations of Leishmania (L.) chagasi treated with the calcium channel blocker nimodipine (2009) Parasitol Res, 105, pp. 499-505Reimao, J.Q., Scotti, M.T., Tempone, A.G., Anti-leishmanial and anti-trypanosomal activities of 1,4-dihydropyridines: In vitro evaluation and structure-activity relationship study Bioorg Med Chem, 18, pp. 8044-8053Morishima, S., Shibata, M.A., Ohmichi, M., Otsuki, Y., Raloxifene, a selective estrogen receptor modulator, induces mitochondria-mediated apoptosis in human endometrial carcinoma cells (2008) Med Mol Morphol, 41, pp. 132-138Bursch, W., Ellinger, A., Kienzl, H., Torok, L., Pandey, S., Active cell death induced by the anti-estrogens tamoxifen and ICI 164 384 in human mammary carcinoma cells (MCF-7) in culture: the role of autophagy (1996) Carcinogenesis, 17, pp. 1595-1607de Medina, P., Silvente-Poirot, S., Poirot, M., Tamoxifen and AEBS ligands induced apoptosis and autophagy in breast cancer cells through the stimulation of sterol accumulation (2009) Autophagy, 5, pp. 1066-1067de Macedo-Silva, S.T., de Oliveira Silva, T.L., Urbina, J.A., de Souza, W., Rodrigues, J.C., Antiproliferative, Ultrastructural, and Physiological Effects of Amiodarone on Promastigote and Amastigote Forms of Leishmania amazonensis (2011) Mol Biol Int, 2011, p. 876021Bera, A., Singh, S., Nagaraj, R., Vaidya, T., Induction of autophagic cell death in Leishmania donovani by antimicrobial peptides (2003) Mol Biochem Parasitol, 127, pp. 23-35Lilly, E., Product monograph: Raloxifene hydrochloride (2008) TorontoReagan-Shaw, S., Nihal, M., Ahmad, N., Dose translation from animal to human studies revisited (2008) FASEB J, 22, pp. 659-661Bonano, V.I., Yokoyama-Yasunaka, J.K.U., Miguel, D.C., Jones, S.A., Dodge, J.A., Discovery of Synthetic Leishmania Inhibitors by Screening of a 2-Arylbenzothiophene Library (2014) Chem Biol Drug Des, 83, pp. 289-296