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Antileishmanial activity of ezetimibe: inhibition of sterol biosynthesis, in vitro synergy with azoles 1 and efficacious in experimental cutaneous leishmaniasis 2 3 Running title: Antileishmanial activity of ezetimibe 4 5 Valter Viana Andrade-Netoa, Edézio Ferreira Cunha-Júniora, Marilene Marcuzzo do Canto-6 Cavalheiroa, Geórgia Correa Atellab, Talita de Almeida Fernandesc, Paulo Roberto Ribeiro Costac, 7 Eduardo Caio Torres-Santosa* 8 9 aLaboratório de Bioquímica de Tripanosomatídeos, Instituto Oswaldo Cruz, FIOCRUZ, Rio de 10 Janeiro, Brazil. 11 bInstituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil. 12 cLaboratório de Química Bioorgânica, Instituto de Pesquisas de Produtos Naturais, Universidade 13 Federal do Rio de Janeiro, Brazil. 14 15 * Corresponding author. Tel: +55-21-3865-8247; E-mail: ects@ioc.fiocruz.br 16 17 18 19 20

AAC Accepted Manuscript Posted Online 6 September 2016Antimicrob. Agents Chemother. doi:10.1128/AAC.01545-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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ABSTRACT 21 Leishmaniasis affects mainly low-income populations in tropical regions. Radical innovation in 22 drug discovery is time- and money-consuming, causing severe restrictions to launch new chemical 23 entities to treat neglected diseases. Drug repositioning is an attractive strategy to attend a specific 24 demand more easily. In this project, we have evaluated the antileishmanial activity of 30 drugs 25 currently in clinical usage for different morbidities. Ezetimibe, clinically used to reduce intestinal 26 cholesterol absorption in dyslipidemic patients, killed promastigotes of L. amazonensis, with an 27 IC50 of 30 μM. Morphological analysis revealed that ezetimibe caused the parasites to become 28 rounded with multiple nuclei and flagellae. Analysis by GC/MS showed that promastigotes treated 29 with ezetimibe had a lower amount of C14-demethylated sterols and accumulated cholesterol and 30 lanosterol. Then, we evaluated the combination of ezetimibe with well-known antileishmanial 31 azoles. The Fractional Inhibitory Concentration Index (FICI) indicated synergy when ezetimibe is 32 associated with ketoconazole or miconazole. Ezetimibe activity was confirmed against intracellular 33 amastigotes, with an IC50 of 20 μM, and reduced the IC90 of ketoconazole and miconazole from 34 11.3 and 11.5 μM to 4.14 and 8.25 μM, respectively. Following, ezetimibe confirmed its activity in 35 vivo, decreasing the lesion development and parasite load in murine cutaneous leishmaniasis. We 36 concluded that ezetimibe has promising antileishmanial activity and should be considered in 37 association with azoles in further preclinical and clinical studies. 38 39 Keywords: Sterol biosynthesis, Leishmania amazonensis, azoles, ezetimibe, drug repositioning. 40 41

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INTRODUCTION 42 Leishmaniasis is a non-contagious infectious disease caused by parasites of the genus Leishmania 43 and is usually transmitted by sandflies belonging to the genera Phlebotomus and Lutzomyia. 44 According to the species of Leishmania involved, the infection can affect the skin and mucous 45 membranes (cutaneous leishmaniasis - CL) or the internal organs (visceral leishmaniasis - VL) (1, 46 2). Official estimates point an annual incidence of approximately 300.000 VL cases and 1.0 million 47 CL cases worldwide (3). The basis of chemotherapy of leishmaniasis has its origin in the work of 48 Vianna (1912), whom successfully treated a patient with emetic tartar (trivalent antimonial), a 49 formulation broadly used at that time to treat infectious diseases (4). From this empirical approach, 50 pentavalent antimonials were developed in the 1940´s and have saved thousands of lives for several 51 decades. Other drugs have been repositioned to treat leishmaniasis, such as amphotericin B, 52 pentamidine and more recently, miltefosine. Unfortunately, the therapeutic arsenal for leishmaniasis 53 has become quite limited and outdated. Antimonials have many toxic effects including cardiac, 54 hepatic, pancreatic and renal toxicity and should be used with caution and with clinical and 55 laboratory monitoring in patients with heart disease and liver disease (5). Miltefosine, the only oral 56 drug licensed, was the most important advance for the treatment of leishmaniasis in recent years. 57 Currently, it is the treatment of choice of program elimination of visceral leishmaniasis in India and 58 recently was approved by FDA for all forms of this disease, but cases of resistance have been 59 reported with increased failure of treatment (6-9). This scenario is a reflex of the difficulty of 60 launching new chemical entities as drugs for this disease. Radical innovation in drug discovery is a 61 time- and money-consuming process. Drug repositioning is an approach very used in the past and 62 that can be useful nowadays to address specific demands in areas of public health, such as neglected 63 and orphan diseases (10-12). This strategy offers the advantage of working with compounds that 64 have already been proved to be safe and to have a favorable pharmacokinetic profile in humans. 65 Thereby, we selected a small set of drugs currently in clinical usage for several morbidities for 66 testing against Leishmania amazonensis, the causal agent of cutaneous diffuse leishmaniasis. 67

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Ezetimibe, the most active in the initial screening, was then evaluated about its mode of action and 68 its effectiveness in association with well-known leishmanicidal azoles in vitro and in vivo. 69 MATERIALS AND METHODS 70 Drugs. Ezetimibe was extracted from sixty Ezetrol® 10 mg tablets (Merck Sharp & Dohme Corp., 71 Kenilworth, NJ, USA), using dichloromethane in the proportion of 1:0.3 (wt/v). The mixture was 72 incubated for 30 min under agitation and filtrated afterwards. The solvent was evaporated, yielding 73 200 mg. The extracted product was dissolved in deuterated chloroform and analyzed in a 400 MHz 74 1H-NMR (Fig. S1). All other drugs were provided by the Sigma–Aldrich Corp. (St Louis, MO, 75 USA). The drugs were dissolved in a 10 mM stock of dimethylsulfoxide (DMSO) or PBS and 76 stored at -20 C. 77 Maintenance and cultivation of parasites. Leishmania amazonensis promastigotes (strain 78 MHOM/BR/77/LTB 0016) were maintained at 26°C in RPMI medium (Sigma- Aldrich Corp., St. 79 Louis, MO, US) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, 100 80 U/ml penicillin and 5 mg/ml hemine. Subcultures were performed twice a week until the tenth 81 passage. Afterwards, old cultures were discarded and fresh parasites were obtained from infected 82 BALB/c mice. 83 Promastigote morphology. L. amazonensis promastigotes (1.0 x 106/mL) were grown at 10, 20 84 and 40 μM ezetimibe. After 72 hours, the parasites were washed twice with PBS, fixed, and stained 85 using the Instant Prov hematological dye system (Newprov, Curitiba, Brazil). 86 Antipromastigote activity. Assays were performed with promastigotes of L. amazonensis at 26°C 87 in RPMI medium without phenol red (Sigma- Aldrich Corp., St. Louis, MO, USA) supplemented as 88 described above. The tests were performed in 96-well plates, with an initial inoculum of 1.0 x 106 89 cells/ml and compound concentrations ranging to 24 μM for ketoconazole and miconazole, and to 90 100 μM for all other drugs. In association experiments, ezetimibe concentrations were 2.5, 5 and 10 91 μM. Plates were incubated at 26°C for 72 hours. After this period, parasite growth was evaluated by 92 adding 10% tetrazolium salt (MTT) (5 mg/ml) per well. The plates were incubated at 26°C for a 93

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further 1 hour, and formazan crystals were dissolved by adding 80 μl DMSO each well. The 94 reaction was analyzed in a spectrophotometer at a 570 nm wavelength. IC50 values were obtained by 95 non-linear regression using Graphpad Prism 6 software (GraphPad Software, Inc., La Jolla, USA). 96 The fractional inhibitory concentration (FICI) for the analysis of synergy was calculated as follows: 97 FICI = IC50 of drug A in combination/IC50 of drug A alone + IC50 of drug B in combination/IC50 of 98 drug B alone. The interpretation of FICI, according to published guidelines are: synergy (FICI ≤ 99 0.5), antagonism (FICI > 4.0) and ‘no interaction’ (FICI > 0.5–4.0) (13). 100 Toxicity to macrophages. 101 Mouse peritoneal macrophages in 96-well plates were treated with ezetimibe, ketoconazole and 102 miconazole alone or in combination for 72 hours at 37°C. After this period, the cell viability was 103 evaluated by adding 10% tetrazolium salt (MTT) (5 mg/ml) to each well. The plates were incubated 104 at 37°C for another four hours, and the resulting formazan crystals were dissolved by adding 80 μl 105 DMSO to each well. The plates were then read at 570 nm. The results were expressed as the 106 percentage of viable cells compared to the untreated control. 107 Antiamastigote activity. Peritoneal macrophages from BALB/c mice were infected with 108 promastigotes of L. amazonensis (stationary growth phase) at a 3:1 parasite-to-macrophage ratio 109 and incubated for 4 h in Lab-Tek chambers (Nunc, Roskilde, Denmark) and kept at 37°C. After 4 110 hours, the chambers were washed, and the cultures were treated with the azole derivatives alone or 111 combined with ezetimibe in supplemented RPMI medium for 72 hours. For initial screening, the 112 concentration of drugs was fixed in 25 μM and a threshold of at least 50% of inhibition was 113 stablished. A drug-activity curve was done with ezetimibe, the only drug that reached the threshold, 114 with concentration ranging to 40 μM. In the association assays, the concentration of ezetimibe was 115 fixed at 20 μM, while the concentration of ketoconazole and miconazole ranged to 16 and 32 μM, 116 respectively. After incubation, the slides were stained, and the infection rate was determined by 117 counting under a light microscope. The infection rate was calculated using the formula: % infected 118 macrophages x number of amastigotes / number of total macrophages. 119

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Extraction of lipids. Lipids from promastigotes of L. amazonensis were extracted using the method 120 of Bligh and Dyer (1959) (14). Briefly, samples were pelleted, and a solution of methanol, 121 chloroform and water (2:1:0.5 v/v) was added. After stirring the mixture for 1 hour, the samples 122 were centrifuged for 20 min at 3,000 rpm and the supernatant, containing the lipids was separated 123 from the precipitate. The precipitate was subjected to a second extraction under the same 124 conditions. The supernatants were combined, and chloroform/water (1:1 v/v) was added. After 40 125 seconds of stirring, the material was centrifuged (3.000 rpm/30 min) again. The lower layer 126 (organic) containing the lipids was then separated with the aid of a glass syringe and transferred to a 127 1.5 ml tube resistant to organic solvents (Axygen Scientific, Inc., Union City, CA, USA). The 128 solvent was evaporated under N2 flux, and the lipids were analyzed by gas chromatography- mass 129 spectrometry (GC/MS), as described below. 130 Analysis of the sterol profile by gas chromatography coupled with mass spectrometry 131 (GC/MS). Promastigotes of L. amazonensis were cultured with 10, 20 or 40 μM ezetimibe or in 132 culture medium alone. After 72 hours, 1x108 parasites of each culture were washed 3 times in cold 133 PBS (pH 7.5), and the sterols were extracted as described above. The samples were injected into a 134 GCMS - QP2010 Ultra Machine (Shimadzu Scientific Instruments, Tokyo, Japan). After injection, 135 the column temperature was maintained at 50°C for 1 minute and then increased to 270°C at a rate 136 of 10°C/min and finally to 300°C at a rate of 1°C/min. The flow of the carrier gas (He) was kept 137 constant at 1.1 ml/min. The temperatures of the injector and detector were 250°C and 280°C, 138 respectively (15). 139 Ethics Statement 140 Studies in L. amazonensis-infected BALB/c mice were performed in accordance with protocols 141 approved by the Ethics Committee for Animal Use of the Instituto Oswaldo Cruz (L026/2015). 142 In vivo assay. To assess the in vivo activity of the combination of ezetimibe and ketoconazole, 143 BALB/c mice (9 animals per group) were infected on the right ear with 2x106 promastigotes of L. 144 amazonensis in stationary phase. The treatment started ten days after infection. The animals were 145

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treated with ezetimibe (10 mg/kg/day), ketoconazole (100 mg/kg/day), miltefosine (20 mg/kg/day) 146 and the combination of ezetimibe + ketoconazole (10 mg/kg/day + 100 mg/kg/day) by oral route. 147 The animals were treated 5 days per week in a total of 20 doses. Negative controls were also 148 similarly treated with PBS. The ear measurement was recorded once a week. After the treatment 149 period, the animals were euthanized for determination of parasite load and toxicological analysis. 150 Serum levels of urea (URE), albumin (ALB), alanine aminotransferase (ALT), aspartate 151 aminotransferase (AST), creatinine (CREA), total bilirubin (BIL), creatine kinase (CK) and 152 cholesterol (CHO) were measured in a Vitros 250 equipment (Ortho Clinical - Johnson & Johnson) 153 using dry chemistry methodology. 154 155 RESULTS 156 Antileishmanial screening. The complete list of drugs and the IC50 for promastigotes and 157 intracellular amastigotes of L. amazonensis can be found in the Table 1. Among all tested 158 compounds, only glimepiride, domperidone and ezetimibe showed activity against promastigotes, 159 with IC50 of 54, 51 and 30 μM, respectively. For intracellular amastigotes, the clinically relevant 160 stage of the parasite, only ezetimibe showed IC50 lower than the arbitrated threshold of 25 μM, 161 reaching to 20 μM. Ezetimibe also drew attention by provoking striking morphological changes on 162 the promastigotes of L. amazonensis. We observed that, in a concentration dependent manner, the 163 parasites became rounded and displayed multiple nuclei and flagellae, mainly at 40 μM (Fig. 1). 164 Ezetimibe interferes with sterol biosynthesis in L. amazonensis. The morphological alterations 165 seen in ezetimibe-treated promastigotes induced us to investigate whether this drug interferes with 166 sterol biosynthesis of the parasite. Table 2 shows an analysis of the relative amount of each sterol 167 indicated by GC/MS. Promastigotes treated with miconazole, an inhibitor of C14-demethylase, 168 showed a decrease in the ergostane-derived sterols ergosta-5,7,24-trien-3β-ol (dehydroepisterol) (3) 169 and ergosta-7,24-dien-3β-ol (episterol) (4). Miconazole also induced the accumulation of 170 exogenous cholesterol collected from culture medium and an increase in the methylated sterols 171

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14α-methylergosta-8,24(28)-dien-3β-ol (2) and 4α-14α-dimethylergosta-8,24(28)-dien-3β-ol 172 (obtusifoliol) (5). Treatment with ezetimibe also decreased dehydroepisterol and episterol (C14-173 demethylated sterols) and promoted the accumulation of cholesterol (1) and lanosterol (C14-174 methylated sterol) (7). We also observed the accumulation of an unknown sterol (6). 175 Ezetimibe potentiates the effect of azoles. Considering the significative alteration in sterol pattern 176 of the parasites after ezetimibe treatment, the antileishmanial activity of this drug was also 177 evaluated in combination with azoles (ketoconazole and miconazole) in promastigotes and 178 amastigotes of L. amazonensis. To graphically evaluate the interaction of ezetimibe with azoles in 179 promastigotes, the IC50 of the drugs alone and in combination was plotted as isobolograms, and to 180 estimate the type of interaction (synergy, antagonism or neutral), the fractional inhibitory 181 concentration index (FICI) was calculated. As expected, miconazole and ketoconazole were active 182 against the promastigotes, with IC50 of 2.5±0.1 and 2.7±0.1 μM, respectively. The IC50 of the azoles 183 decreased when in combination with ezetimibe, generating fully concave isobolograms (Fig. 2). The 184 calculation of FICI resulted in values of 0.4 for both combinations, confirming the synergism. Due 185 to the experimental difficulty of performing a “checkerboard” approach to draw isobolograms for 186 antiamastigote activity, we fixed the concentration of ezetimibe at the IC50 (20 µM) (Fig. 3A) and 187 varied the concentration of the azoles to calculate the IC90 (concentration that inhibits 90% of the 188 parasites) of the combination. When associated with ezetimibe, the IC90 of ketoconazole and 189 miconazole were reduced from 11.3±0.2 μM and 11.5±0.1 μM to 4.14±0.3 μM and 8.25±0.2 μM, 190 respectively (Figs. 3B and C). Furthermore, ezetimibe, miconazole, ketoconazole alone or 191 combined showed no toxic effects to macrophages (Fig. 4A-E). Figure 5 shows representative 192 micrographs of infected macrophages treated with ezetimibe alone and combined with ketoconazole 193 and miconazole that have normal morphology and a reduced parasite load (Fig. 5). 194

For in vivo assays, BALB/c mice were infected with L. amazonensis and treated with 195 ezetimibe (10 mg/kg/day) and ketoconazole (100 mg/kg/day) alone or in combination by oral route. 196 As can be seen in Figure 6A, ketoconazole and ezetimibe were able to individually control the 197

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lesion development. When both drugs were combined, the lesion growth was more effectively 198 controlled. All treatments reduced the parasite load significantly (Fig. 6A – insert). Nonetheless, no 199 significant differences in serum levels of urea, albumin, ALT, AST, creatinine, bilirubin and 200 creatine kinase were observed in treated and untreated animals, demonstrating that the treatment 201 was not toxic to the mammalian host. Furthermore, no significant change in cholesterol level in 202 serum was observed (Figs. 6B-I). 203 DISCUSSION 204

Thirty drugs in clinical usage were included in this study. Instead of focusing in 205 antimicrobial agents, we selected compounds belonging to a wide spectrum of pharmacological 206 classes (Table 1). There is no previous data in literature about antileishmanial activity of 207 glimepiride and ezetimibe but, interestingly, domperidone has been used in clinical trials in 208 naturally infected dogs, aiming veterinary use (16, 17). However, in those studies, no intrinsic 209 antileishmanial activity was assigned to domperidone. The therapeutic effect of this drug in dogs 210 has been attributed to its immunomodulatory activity (17). 211 Among the active drugs, ezetimibe drew attention due to its property of causing dramatic 212 morphological alterations in the promastigotes (Fig. 1). These morphological changes are consistent 213 with those previously described as consequence of drug interference in sterol metabolism of 214 trypanosomatids (18-20). In fact, we observed that ezetimibe causes alterations in the sterol 215 composition of the parasites compatible with inhibition of C14-demethylase (21-23). Curiously, 216 ezetimibe is a potent inhibitor of the intestinal cholesterol absorption mediated by the Niemann–217 Pick C1-like 1 protein (NPC1L1) and it is clinically used to reduce cholesterolemia (24). 218 Nevertheless, no inhibition in the human sterol biosynthesis pathway has been described for 219 ezetimibe. However, the sterol pathway of the trypanosomatids has diverged sufficiently to allow 220 selective pharmacological inhibition. Thereby, besides its effect on transport of cholesterol in 221 humans, it is possible that ezetimibe also could interfere with sterol biosynthesis in Leishmania. 222

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It is assumed that the sterol biosynthetic pathway is essential for Leishmania, because 223 inhibition of diverse enzymes of this pathway such as, HMG-CoA reductase, squalene epoxidase, 224 C-14 demethylase and C-24 methyltransferase, results in parasite death (25-30). Drug combination 225 in the treatment of infectious diseases is an interesting approach. Drugs acting in the same pathway 226 can be associated to improve their activity (31). Thus, we evaluated the activity of ezetimibe in 227 combination with ketoconazole or miconazole. This association showed synergistic effect on 228 promastigotes (Fig. 2) and increased the activity of these azoles on intracellular amastigotes of L. 229 amazonensis (Figs. 3B and C). 230

The next step was to evaluate the leishmanicidal activity of ezetimibe alone or combined 231 with ketoconazole in vivo (Figs. 6A and B). Ezetimibe and ketoconazole alone reduced the lesion 232 growth and parasite load equally in murine model of cutaneous leishmaniasis. The combination was 233 more efficacious in controlling the lesion development, but no difference was observed in parasite 234 burden. Ketoconazole was chosen to be evaluated in vivo because it showed better result on 235 intracellular amastigotes, when in combination with ezetimibe (Fig. 3C). Moreover, clinical trials 236 have demonstrated that ketoconazole could be advantageous for patients with New World species of 237 Leishmania (32). Oral ketoconazole combined with intra-lesional injections of meglumine 238 antimoniate (Glucantime) has been shown to be more effective than Glucantime alone in the 239 treatment of localized cutaneous leishmaniasis (33). Furthermore, ketoconazole (100 mg/kg/day) 240 reduced splenic parasite load in murine model of visceral leishmaniasis caused by L. infantum, and 241 in the lower dose of 50 mg/kg/day, potentiated the effect of meglumine antimoniate (34). 242 In summary, our screening of FDA approved drugs pointed out ezetimibe as a promising 243 antileishmanial agent. It has antileishmanial activity in vitro and in vivo, supposedly by its effect on 244 parasite sterol biosynthesis, and acts synergistically with azoles. These data suggest that ezetimibe 245 could be useful, in association or not with azoles, in further treatments for leishmaniasis. 246 247

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ACKNOWLEDGMENTS The authors thank the Program for Technological Development in 248 Tools for Health-PDTIS, FIOCRUZ for evaluating the serum toxicological markers. 249 250 FOOTNOTE PAGE 251 FUNDING: This work was supported by the Conselho Nacional de Desenvolvimento Científico e 252 Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) 253 and the Programa Estratégico de Apoio à Pesquisa em Saúde (PAPES/FIOCRUZ; grant 254 407680/2012-8 to E. C. T. S.). 255 256 CONFLICT OF INTEREST 257 All authors: No reported conflict of interest. 258 259 REFERENCES 260 1. Handler MZ, Patel PA, Kapila R, Al-Qubati Y, Schwartz RA. 2015. Cutaneous and 261 mucocutaneous leishmaniasis: Differential diagnosis, diagnosis, histopathology, and management. J 262 Am Acad Dermatol. 73(6):911-26. http://doi:10.1016/j.jaad.2014.09.014. 263 2. Savoia D. 2015. Recent updates and perspectives on leishmaniasis. J Infect Dev Ctries. 9(6):588-264 96. http://dx.doi:10.3855/jidc.6833. 265 3. Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer, M. 2012. 266 Leishmaniasis worldwide and global estimates of its incidence. Plos One 7:e3567. 267 http://dx.doi:10.1371/journal.pone.0035671. 268 4. Vianna G. 1912. Comunicação à Sessão de 24 de abril de 1912 da Sociedade Brasileira de 269 Dermatologia. Arch Bras Med. 1:36–38. 270 5. Oliveira LF, Schubach AO, Martins MM, Passos SL, Oliveira RV, Marzochi MC, Andrade 271 CA. 2011. Systematic review of the adverse effects of cutaneous leishmaniasis treatment in the 272 New World. Acta Trop.118:87-96. http://dx.doi:10.1016/j.actatropica.2011.02.007. 273

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22. Berman JD, Holz GG, Beach DH. 1984. Effects of ketoconazole on growth and sterol 324 biosynthesis of Leishmania-mexicana promastigotes in culture. Mol Biochem Parasitol. 12: 1-13. 325 http://dx.doi:10.1016/0166-6851(84)90039-2. 326 23. Hart DT, Lauwers WJ, Willemsens G, Vandenbossche H, Opperdoes FR. 1989. 327 Perturbation of sterol biosynthesis by itraconazole and ketoconazole in Leishmania-mexicana-328 mexicana infected macrophages. Mol Biochem Parasitol. 33: 123-134. http://dx.doi:10.1016/0166-329 6851(89)90026-1. 330 24. Park S.W. 2013. Intestinal and hepatic Niemann Pick C1-Like 1. Diabetes Metab J. 37:240-8. 331 http://dx.doi:10.4093/dmj.2013.37.4.240. 332 25. Beach DH, Goad LJ, Holz GG. 1988. Effects of antimycotic azoles on growth and sterol 333 biosynthesis of Leishmania promastigotes. Mol Biochem Parasitol. 31:149-162. 334 http://dx.doi:10.1016/0166-6851(88)90166-1 335 26. Daly H, De Lima H, Kato H, Sordillo EM, Convit J, Reyes-Jaimes O, Zerpa O, Paniz 336 Mondolfi AE. 2014. Intermediate cutaneous leishmaniasis caused by Leishmania (Viannia) 337 braziliensis successfully treated with fluconazole. Clin Exp Dermatol 39(6): 708-12. 338 http://dx.doi:10.1111/ced.12359. 339 27. Rodrigues JC, Attias M, Rodriguez C, Urbina JA, Souza Wd. 2002. Ultrastructural and 340 biochemical alterations induced by 22,26-azasterol, a delta(24(25))-sterol methyltransferase 341 inhibitor, on promastigote and amastigote forms of Leishmania amazonensis. Antimicrob Agents 342 Chemother. 46(2):487-99. http://dx.doi:10.1111/j.1574-6968.2010.02178.x. 343 28. Macedo-Silva ST, Urbina JA, de Souza W, Rodrigues JC. 2013. In Vitro Activity of the 344 Antifungal Azoles Itraconazole and Posaconazole against Leishmania amazonensis. PLos One 345 2013, 8(12):e83247. http://dx.doi:10.1371/journal.pone.0083247 346 29. Kulkarni MM, Reddy N, Gude T, McGwire BS. 2013. Voriconazole suppresses the growth 347 of Leishmania species in vitro. Parasitol Res. 112(5):2095-9. http://dx.doi:10.1007/s00436-013-348 3274-x. 349

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30. Haughan, P.A., Chance, M.L., Goad, L.J. (1992). 1992. Synergism in vitro of lovastatin and 350 miconazole as antileishmanial agents. Biochem Pharmacol. 44:2199-2206. http://dx. 351 doi:10.1016/0006-2952(92)90347-l. 352 31. de Macedo-Silva ST, Visbal G, Urbina JA, de Souza W, Rodrigues JC. 2015. Potent in vitro 353 antiproliferative synergism of combinations of ergosterol biosynthesis inhibitors against Leishmania 354 amazonensis. Antimicrob Agents Chemother. 59(10):6402-18. http://dx. doi: 10.1128/AAC.01150- 355 32. Handler MZ, Patel PA, Kapila R, Al-Qubati Y, Schwartz RA. 2015. Cutaneous and 356 mucocutaneous leishmaniasis: Differential diagnosis, diagnosis, histopathology, and management. J 357 Am Acad Dermatol. 73(6):911-26. http://dx.doi:10.1016/j.jaad.2014.09.014. 358 33. Salmanpour R, Handjani F, Nouhpisheh MK. 2001. Comparative study of the efficacy of 359 oral ketoconazole with intra-lesional meglumine antimoniate (Glucantime) for the treatment of 360 cutaneous leishmaniasis. J Dermatolog Treat. 2001. 12(3):159-62. 361 http://dx.doi:10.1080/09546630152607899. 362 34. Gangneux JP, Dullin M, Sulahian A, Garin YJ, Derouin F. 1999. Experimental evaluation of 363 second-line oral treatments of visceral leishmaniasis caused by Leishmania infantum. Antimicrob 364 Agents Chemother.43(1):172-4. 365 366

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Drug Promastigotes IC50 (μM)

Intracellular amastigotes IC50 (μM)

Indication Chemical Class Mode of Action

Amoxycillin >100 >25 Antibiotic Aminopenicillin Binds to and inactivates penicillin-binding proteins on the inner bacterial cell wall.

Atenolol >100 >25 Antianginal/antihypertensive

Beta-adrenergic blocker

Inhibits stimulation of beta-receptor sites.

Buflomedil >100 >25 Vasoactive agent/Non-selective, competitive α-adrenoceptor antagonist

Pyrrolidinyl-trimethoxylphenyl

Non-selective competitive inhibition of α-adrenoceptors in vascular smooth muscle.

Cefalexin >100 >25 Antibiotic cephalosporin Binds to and inactivates penicillin-binding proteins on the inner bacterial cell wall.

Cetirizine 100±10.5 >25 Antihistamine Piperazine Potent H1-receptor antagonist. Chlorthalidone >100 >25 Antihypertensive/diuretic Phthalimidine

derivative of benzenesulfonamide

Promotes sodium, chloride, and water excretion by inhibiting sodium reabsorption in the distal tubules of the kidneys.

Clarithromycin >100 >25 Antibiotic Macrolide derivative Inhibits RNA-dependent protein synthesis in many types of aerobic, anaerobic, gram-positive, and gram-negative bacteria.

Diclofenac sodium >100 >25 Non-steroidal anti-inflammatory

Phenylacetic acid derivate

Inhibitor of cyclooxygenase

Domperidone 51±0.6 >25 Antiemetic/antimuscarinic Phenothiazine Specific blocker of dopamine receptors. Enalapril >100 >25 Antihypertensive Dicarbocyl-

containing ACE inhibitor

Reduce blood pressure by affecting the renin-angiotensin-aldosterone system.

Ezetimibe 30±1.2 20±1.6 Antihypercholesterolemic Azetidinone Reduces blood cholesterol by inhibiting its absorption through the small intestine.

Famotidine >100 >25 Antiulcer agent/gastric acid secretion inhibitor

Thiazole derivative H2-receptor antagonist.

Gabapentin >100 >25 Anticonvulsant Cyclohexane-acetic acid derivative

Structural analog of gamma-aminobutyric acid (GABA).

Gemfibrozil >100 >25 Antihyperlipidemic Fibric Acid derivative

Reduces triglycerides and increases cholesterol carried in high-density lipoprotein (HDL) in the blood.

Glimepiride 54±1.5 >25 Antidiabetic Sulfonylurea Stimulates insulin release from beta cells in pancreas.

Hydrochlorothiazide >100 >25 Diuretic Benzothiadiazide Promotes movement of sodium, chloride and water from blood in peritubular capillaries into nephron’s distal convolutes tubule.

Ibuprofen >100 >25 Analgesic, anti-inflammatory, antipyretic

Propioninc acid derivative

Inhibitor of cyclooxygenase.

Lisinopril >100 >25 Antihypertensive/vasodilator

Lysine ester of enalapril

Reduce blood pressure by affecting the renin-angiotensin-aldosterone system.

Losartan >100 >25 Antihypertensive Angiotensin II receptor antagonist

Blocks binding of angiotensin II to receptor sites.

Meloxicam >100 >25 Anti-inflammatory Oxicam derivative Inhibitor of cyclooxygenase. Mesalazine >100 >25 Anti-inflammatory Aminosalicylic acid Their mode of action is unclear but a local

effect on epithelial cells by a variety of mechanisms to moderate the release of lipid mediators, cytokines and reactive oxygen species is proposed.

Metoprolol >100 >25 Antianginal/antihypertensive

Beta-adrenergic antagonist

Inhibits stimulation of beta-receptors.

Nimesulide >100 >25 non-steroidal anti-inflammatory

Phenoxymethanesulfonamilide

Inhibitor of cyclooxygenase.

Oxacillin >100 >25 Antibiotic Penicillin Inhibits bacterial cell wall synthesis. Pantoprazole >100 >25 Antiulcer Substituted

benzimidazole Inhibiting proton pump in gastric parietal cells.

Prednisolone >100 >25 Anti-inflammatory/immunosuppressant

Glicocorticoid Binds to intracellular glucocorticoid receptors.

Ranitidine >100 >25 Antiulcer agent, gastric acid secretion inhibitor

Aminoalkyl-substituted furan derivative

Competitive blockade of histaminergic H2 receptors

Scopolamine >100 >25 Anticholinergic Belladonna alkaloid, tertiary amine

Competitively inhibits acetylcholine at autonomic postganglionic cholinergic receptors

Secnidazole >100 >25 Antimicrobial Nitroimidazole DNA damage Tenoxicam >100 >25 Non-steroidal anti-

inflammatory Thienothiazine Inhibitor of cyclooxygenase

367 368

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369 370 Table 2: Sterol composition of L. amazonensis promastigotes under effect of ezetimibe. 371 Compound MW Relative amount (%)

Control Miconazole Ezetimibe 8 μM 10 μM 20 μM 40 μM

(1) Cholesterol 386 1.58 31.15 16.91 19.30 43.28 (2)14α-methylergosta- 8,24(28)-dien-3β-ol

412 - 41.18 4.44 6.21 2.43

(3) Ergosta-5,7,24-trien-3β-ol (dehydroepisterol)

396 93.32 - 62.18 23.70 4.58

(4) Ergosta-7,24-dien-3β-ol (Episterol)

398 5.10 - 12.20 9.83 1.72

(5) 4α-14α-dimethylergosta-8,24(28)-dien-3β-ol, (obtusifoliol)

426 - 33.60 - - -

(6) Unknown 424 - - - 3.71 8.75 (7) Lanosterol 426 - - 3.34 37.88 39.24 MW - Molecular Weight; Relative Amount (%) – Relative to total lipid content from each sample. 372 373 374 375

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Legends to Figures 376 Fig. 1. Micrographs of promastigotes of L. amazonensis treated with ezetimibe. L. amazonensis 377 promastigotes were grown at 10, 20 and 40 μM of ezetimibe to 72 hours. A control, B ezetimibe 10 378 μM, C ezetimibe 20 μM, D ezetimibe 40 μM. The slides were stained using Instant Prov 379 hematological dye system. 380 Fig. 2. Isobolographic analysis of the antileishmanial activity of association of ezetimibe and azoles. 381 L. amazonensis promastigotes were incubated with ezetimibe and the indicated azoles in different 382 concentrations for 72 hours at 26°C. Each plotted point in the isobolograms is the IC50 of the drugs 383 alone or in combination. The straight lines connecting the individual IC50s represent the theoretical 384 line of additivity for each combination. The experiments were performed in triplicate, n = 3. The 385 graphics are representative of a single experiment, with the standard deviation value. The graphics 386 and IC50 values were produced with GraphPad Prism 4 software. 387 Fig. 3 Antiamastigote activity of ezetimibe combined with azoles. Peritoneal macrophages infected 388 with L. amazonensis treated with ezetimibe and the indicated azoles were incubated for 72 hours at 389 37°C. After incubation, the cells were fixed and stained. The infection rate was calculated as 390 indicated in the methodology. The experiments were performed in triplicate, n = 3. The graphics are 391 representative of a single experiment, with the standard deviation value. * p < 0.05, ** p < 0.01, 392 *** p < 0.001. 393 Fig. 4 Toxicity to macrophages. Uninfected macrophages were incubated with ezetimibe, 394 miconazole, ketoconazole alone or in combination for 72 hours at 37 °C. After this period, the cells 395 were incubated with MTT for 1 hour at 37 °C. The absorbance was read in a spectrophotometer at 396 570 nm. (A) ezetimibe, (B), ketoconazole (C), miconazole (D) + Eze20 ketoconazole (E) Eze20 + 397 miconazole. The experiments were performed in triplicate, n = 3. *** p <0.001 compared to the 398 control. 399

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Fig. 5. Micrographs of L. amazonensis-infected macrophages treated with ezetimibe and azoles. 400 Peritoneal macrophages infected with L. amazonensis treated with ezetimibe and the indicated 401 azoles were incubated for 72 hours at 37°C. After incubation, the cells were fixed and stained. (A) 402 control, (B) ezetimibe 20 μM, (C) ketoconazole 8 μM, (D) ketoconazole 8 μM + ezetimibe 20 μM, 403 (E) miconazole 4 μM, (F) miconazole 4μM + ezetimibe 20 μM. 404 Fig. 6. In vivo activity of ezetimibe in combination with ketoconazole. BALB/c mice (9/group) 405 were infected on the right ear with 2 x 106 promastigotes of L. amazonensis stationary phase and 406 orally treated with ezetimibe (10 mg/kg/day) (Eze10), ketoconazole (100mg/ kg/day) (Keto100), 407 miltefosine (20 mg/kg/day) and ezetimibe + ketoconazole (10 mg/kg/day + 100 mg/kg/ day) (Eze10 408 + keto100). The treatment started ten days after infection. The animals were treated 5 days per 409 week, in a total of 20 doses. The euthanasia was proceeded after 35 days of infection. Negative 410 controls were also similarly treated with PBS. A - Lesion growth and parasite load. B-I - 411 Biochemical evaluation of toxicity of the treatment, according to the indicated parameters. Urea 412 (URE), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), 413 creatinine (CREA), total bilirubin (BIL), creatine kinase (CK) and cholesterol (CHO). 2way 414 ANOVA, * p <0.05, ** p <0.01 *** p <0.001 compared to the control group. # # p <0.01 compared 415 to the Eze10 group. π p <0.05, π π p <0.01 π π π p <0.001 compared to the Keto100 group. 416

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