Search tips
Search criteria 


Logo of aacPermissionsJournals.ASM.orgJournalAAC ArticleJournal InfoAuthorsReviewers
Antimicrob Agents Chemother. 2016 November; 60(11): 6844–6852.
Published online 2016 October 21. Prepublished online 2016 September 6. doi:  10.1128/AAC.01545-16
PMCID: PMC5075094

Antileishmanial Activity of Ezetimibe: Inhibition of Sterol Biosynthesis, In Vitro Synergy with Azoles, and Efficacy in Experimental Cutaneous Leishmaniasis


Leishmaniasis affects mainly low-income populations in tropical regions. Radical innovation in drug discovery is time-consuming and expensive, imposing severe restrictions on the ability to launch new chemical entities for the treatment of neglected diseases. Drug repositioning is an attractive strategy for addressing a specific demand more easily. In this project, we have evaluated the antileishmanial activities of 30 drugs currently in clinical use for various morbidities. Ezetimibe, clinically used to reduce intestinal cholesterol absorption in dyslipidemic patients, killed Leishmania amazonensis promastigotes with a 50% inhibitory concentration (IC50) of 30 μM. Morphological analysis revealed that ezetimibe caused the parasites to become rounded, with multiple nuclei and flagella. Analysis by gas chromatography (GC)-mass spectrometry (MS) showed that promastigotes treated with ezetimibe had smaller amounts of C-14-demethylated sterols, and accumulated more cholesterol and lanosterol, than untreated promastigotes. We then evaluated the combination of ezetimibe with well-known antileishmanial azoles. The fractional inhibitory concentration index (FICI) indicated synergy when ezetimibe was combined with ketoconazole or miconazole. The activity of ezetimibe against intracellular amastigotes was confirmed, with an IC50 of 20 μM, and ezetimibe reduced the IC90s of ketoconazole and miconazole from 11.3 and 11.5 μM to 4.14 and 8.25 μM, respectively. Subsequently, we confirmed the activity of ezetimibe in vivo, showing that it decreased lesion development and parasite loads in murine cutaneous leishmaniasis. We concluded that ezetimibe has promising antileishmanial activity and should be considered in combination with azoles in further preclinical and clinical studies.


Leishmaniasis is a noncontagious infectious disease caused by parasites of the genus Leishmania and is usually transmitted by sandflies belonging to the genus Phlebotomus or Lutzomyia. Depending on the species of Leishmania involved, the infection can affect the skin and mucous membranes (cutaneous leishmaniasis [CL]) or the internal organs (visceral leishmaniasis [VL]) (1, 2). Official estimates point to an annual incidence of approximately 300,000 VL cases and 1.0 million CL cases worldwide (3). Chemotherapy of leishmaniasis has its origin in the 1912 work of Vianna, who successfully treated a patient with emetic tartar (a trivalent antimonial), a formulation widely used at that time to treat infectious diseases (4). From this empirical approach, pentavalent antimonials were developed in the 1940s and have saved thousands of lives for several decades. Other drugs, such as amphotericin B, pentamidine, and, more recently, miltefosine, have been repositioned to treat leishmaniasis. Unfortunately, the therapeutic arsenal for leishmaniasis has become quite limited and outdated. Antimonials have many toxic effects, including cardiac, hepatic, pancreatic, and renal toxicity. They should be used with caution, and clinical and laboratory monitoring should be performed for patients with heart or liver disease (5). Miltefosine, the only oral drug licensed, represents the most important advance in the treatment of leishmaniasis in recent years. Currently, it is the treatment of choice in the program for the elimination of visceral leishmaniasis in India, and it was recently approved by the U.S. FDA for all forms of leishmaniasis, but cases of resistance, with increased treatment failures, have been reported (6,9).

This scenario reflects the difficulty of launching new chemical entities as drugs for this disease. Radical innovation in drug discovery is a time-consuming and costly process. Drug repositioning is an approach much used in the past that can be useful nowadays for addressing specific demands in public health areas such as neglected and orphan diseases (10,12). This strategy offers the advantage of working with compounds that have already been proved to be safe and to have favorable pharmacokinetic profiles in humans. Therefore, we selected a small set of drugs currently in clinical use for several morbidities and tested them against Leishmania amazonensis, the causal agent of diffuse cutaneous leishmaniasis.

Ezetimibe, the drug found to be most active in the initial screening, was then evaluated for its mode of action and its effectiveness in combination with well-known leishmanicidal azoles in vitro and in vivo.



Ezetimibe was extracted from 60 10-mg Ezetrol tablets (Merck Sharp & Dohme Corp., Kenilworth, NJ, USA) by use of dichloromethane (1:0.3, wt/vol ezetimibe/dichloromethane). The mixture was incubated for 30 min under agitation and was filtered afterwards. The solvent was evaporated, yielding 200 mg. The extracted product was dissolved in deuterated chloroform and was analyzed in a 400-MHz 1H nuclear magnetic resonance (NMR) spectrometer (see Fig. S1 in the supplemental material). All other drugs were provided by the Sigma-Aldrich Corp. (St. Louis, MO, USA). The drugs were dissolved in a 10 mM stock of dimethyl sulfoxide (DMSO) or phosphate-buffered saline (PBS) and were stored at −20°C.

Maintenance and cultivation of parasites.

Leishmania amazonensis promastigotes (strain MHOM/BR/77/LTB 0016) were maintained at 26°C in RPMI medium (Sigma-Aldrich Corp., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml streptomycin, 100 U/ml penicillin, and 5 mg/ml hemin. Subcultures were performed twice a week until the 10th passage. Afterwards, old cultures were discarded, and fresh parasites were obtained from infected BALB/c mice.

Promastigote morphology.

L. amazonensis promastigotes (1.0 × 106/ml) were grown with 10, 20, or 40 μM ezetimibe. After 72 h, the parasites were washed twice with PBS, fixed, and stained using the Instant Prov hematological dye system (Newprov, Curitiba, Brazil).

Antipromastigote activity.

Assays were performed with L. amazonensis promastigotes at 26°C in RPMI medium without phenol red (Sigma-Aldrich Corp., St. Louis, MO, USA), supplemented as described above. The tests were performed in 96-well plates, with an initial inoculum of 1.0 × 106 cells/ml and compound concentrations ranging as high as 24 μM for ketoconazole and miconazole and as high as 100 μM for all other drugs. In combination experiments, ezetimibe concentrations were 2.5, 5, and 10 μM. Plates were incubated at 26°C for 72 h. After this period, parasite growth was evaluated by adding 10% MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] (5 mg/ml) per well. The plates were incubated at 26°C for a further 1 h, and formazan crystals were dissolved by adding 80 μl DMSO to each well. The reaction was analyzed in a spectrophotometer at a wavelength of 570 nm. Fifty percent inhibitory concentrations (IC50s) were obtained by nonlinear regression using GraphPad Prism software (version 6; GraphPad Software, Inc., La Jolla, CA, USA). The fractional inhibitory concentration index (FICI) for the analysis of synergy was calculated as [(IC50 of drug A in combination)/(IC50 of drug A alone)] + [(IC50 of drug B in combination)/(IC50 of drug B alone)]. According to published guidelines (13), a FICI of ≤0.5 was interpreted as indicating synergy, a FICI of >4.0 as indicating antagonism, and a FICI of >0.5 to 4.0 as indicating “no interaction.”

Toxicity to macrophages.

Mouse peritoneal macrophages in 96-well plates were treated with ezetimibe, ketoconazole, or miconazole alone or in combination for 72 h at 37°C. After this period, cell viability was evaluated by adding 10% tetrazolium salt (MTT) (5 mg/ml) to each well. The plates were incubated at 37°C for another 4 h, and the resulting formazan crystals were dissolved by adding 80 μl DMSO to each well. The plates were then read at 570 nm. The number of viable treated macrophages was expressed as a percentage of the number of viable untreated-control macrophages (taken as 100%).

Antiamastigote activity.

Peritoneal macrophages from BALB/c mice were infected with L. amazonensis promastigotes (stationary-growth phase) at a 3:1 parasite-to-macrophage ratio, incubated for 4 h in Lab-Tek chambers (Nunc, Roskilde, Denmark), and kept at 37°C. After 4 h, the chambers were washed, and the cultures were treated with azole derivatives alone or combined with ezetimibe in supplemented RPMI medium for 72 h. For the initial screening, the concentration of drugs was fixed at 25 μM, and a threshold of at least 50% inhibition was established. A drug-activity curve was made with ezetimibe, the only drug that reached the threshold, with concentrations ranging as high as 40 μM. In the combination assays, the concentration of ezetimibe was fixed at 20 μM, while the concentration of ketoconazole or miconazole ranged as high as 16 or 32 μM, respectively. After incubation, the slides were stained, and the infection rate was determined by counting under a light microscope. The infection rate was calculated using the following formula: (percentage of infected macrophages × number of amastigotes)/(total number of macrophages).

Extraction of lipids.

Lipids were extracted from L. amazonensis promastigotes by use of the method published by Bligh and Dyer in 1959 (14). Briefly, samples were pelleted, and a solution of methanol, chloroform, and water (2:1:0.5, vol/vol) was added. After the mixture was stirred for 1 h, the samples were centrifuged for 20 min at 3,000 rpm, and the supernatant, containing the lipids, was separated from the precipitate. The precipitate was subjected to a second extraction under the same conditions. The supernatants were combined, and a mixture of chloroform and water (1:1, vol/vol) was added. After 40 s of stirring, the material was centrifuged (at 3,000 rpm for 30 min) again. The lower layer (organic) containing the lipids was then separated with the aid of a glass syringe and was transferred to a 1.5-ml tube resistant to organic solvents (Axygen Scientific, Inc., Union City, CA, USA). The solvent was evaporated under a N2 flux, and the lipids were analyzed by gas chromatography coupled with mass spectrometry (GC-MS), as described below.

Analysis of the sterol profile by GC-MS.

L. amazonensis promastigotes were cultured with 10, 20, or 40 μM ezetimibe or in culture medium alone. After 72 h, 1 × 108 parasites of each culture were washed three times in cold PBS (pH 7.5), and the sterols were extracted as described above. The samples were injected into a GCMS-QP2010 Ultra machine (Shimadzu Scientific Instruments, Tokyo, Japan). After injection, the column temperature was maintained at 50°C for 1 min and was then increased, first to 270°C at a rate 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 constant at 1.1 ml/min. The temperatures of the injector and detector were 250°C and 280°C, respectively (15).

Ethics statement.

Studies with L. amazonensis-infected BALB/c mice were performed in accordance with protocols approved by the Ethics Committee for Animal Use of the Instituto Oswaldo Cruz (L026/2015).

In vivo assay.

To assess the in vivo activity of the combination of ezetimibe and ketoconazole, BALB/c mice (9 animals per group) were infected in the right ear with 2 × 106 L. amazonensis promastigotes in stationary phase. The treatment started 10 days after infection. The animals were treated by the oral route with either ezetimibe (10 mg/kg of body weight/day), ketoconazole (100 mg/kg/day), miltefosine (20 mg/kg/day), or the combination of ezetimibe at 10 mg/kg/day and ketoconazole at 100 mg/kg/day. The animals were treated 5 days per week in a total of 20 doses. Negative controls were also similarly treated with PBS. The thickness of ears was recorded once a week. After the treatment period, the animals were euthanized for the determination of parasite loads and toxicological analysis. Levels of urea, albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, total bilirubin, creatine kinase, and cholesterol in serum were measured in a Vitros 250 system (Ortho Clinical; Johnson & Johnson) using dry chemistry methodology.


Antileishmanial screening.

The complete list of drugs tested and their IC50s for promastigotes and intracellular amastigotes of L. amazonensis can be found in Table 1. Among all compounds tested, only glimepiride, domperidone, and ezetimibe showed activity against promastigotes, with IC50s of 54, 51, and 30 μM, respectively. For intracellular amastigotes, the clinically relevant stage of the parasite, only ezetimibe showed an IC50 lower than the arbitrated threshold of 25 μM, at 20 μM. Ezetimibe also drew attention by inducing striking morphological changes in L. amazonensis promastigotes. We observed that the parasites became rounded and displayed multiple nuclei and flagella; this effect was concentration dependent and occurred mainly at 40 μM (Fig. 1).

Drugs tested for antileishmanial activity
Micrographs of L. amazonensis promastigotes treated with ezetimibe. L. amazonensis promastigotes were grown with 10, 20, or 40 μM ezetimibe for 72 h. (A) Control; (B) 10 μM ezetimibe; (C) 20 μM ezetimibe; (D) 40 μM ezetimibe. ...

Ezetimibe interferes with sterol biosynthesis in L. amazonensis.

The morphological alterations seen in ezetimibe-treated promastigotes induced us to investigate whether this drug interferes with sterol biosynthesis in the parasite. Table 2 shows the results of analysis of the relative amount of each sterol by GC-MS. Promastigotes treated with miconazole, an inhibitor of C-14 demethylase, showed decreases in the amounts of the ergostane-derived sterols ergosta-5,7,24-trien-3β-ol (dehydroepisterol) (compound 3) and ergosta-7,24-dien-3β-ol (episterol) (compound 4). Miconazole also induced the accumulation of exogenous cholesterol collected from the culture medium and increases in the amounts of the methylated sterols 14α-methylergosta-8,24(28)-dien-3β-ol (compound 2) and 4α-14α-dimethylergosta-8,24(28)-dien-3β-ol (obtusifoliol) (compound 5). Treatment with ezetimibe also decreased the amounts of the C-14-demethylated sterols dehydroepisterol and episterol and promoted the accumulation of cholesterol (compound 1) and lanosterol (a C-14-methylated sterol) (compound 7). We also observed the accumulation of an unknown sterol (compound 6).

Effect of ezetimibe on the sterol composition of L. amazonensis promastigotes

Ezetimibe potentiates the effects of azoles.

Considering the significant alterations in the sterol patterns of the parasites after ezetimibe treatment, the antileishmanial activity of this drug against L. amazonensis promastigotes and amastigotes was also evaluated in combination with azoles (ketoconazole and miconazole). To graphically evaluate the interaction of ezetimibe with azoles against promastigotes, the IC50s of the drugs alone and in combination were plotted as isobolograms, and to estimate the type of interaction (synergy, antagonism, or neutral), the fractional inhibitory concentration index (FICI) was calculated. As expected, miconazole and ketoconazole were active against the promastigotes, with IC50 of 2.5 ± 0.1 and 2.7 ± 0.1 μM, respectively. The IC50s of the azoles decreased when they were combined with ezetimibe, generating fully concave isobolograms (Fig. 2). The calculation of FICI resulted in values of 0.4 for both combinations, confirming synergism. Due to the experimental difficulty of using a “checkerboard” approach to draw isobolograms for antiamastigote activity, we fixed the concentration of ezetimibe at the IC50 (20 μM) (Fig. 3A) and varied the concentrations of the azoles to calculate the IC90 (the concentration that inhibits 90% of the parasites) of each combination. When ketoconazole or miconazole was combined with ezetimibe, the IC90 was reduced from 11.3 ± 0.2 μM or 11.5 ± 0.1 μM to 4.14 ± 0.3 μM or 8.25 ± 0.2 μM, respectively (Fig. 3B and andC).C). Furthermore, ezetimibe, miconazole, and ketoconazole, alone or combined, showed no toxic effects on macrophages (Fig. 4A to toE).E). Figure 5 shows representative micrographs of infected macrophages treated with ezetimibe, alone or combined with ketoconazole or miconazole, that have normal morphology and reduced parasite loads (Fig. 5).

Isobolographic analysis of the antileishmanial activities of combinations of ezetimibe with azoles. L. amazonensis promastigotes were incubated with ezetimibe and the indicated azoles at different concentrations for 72 h at 26°C. Each plotted ...
Antiamastigote activity of ezetimibe combined with azoles. Peritoneal macrophages infected with L. amazonensis and treated with ezetimibe and the indicated azoles were incubated for 72 h at 37°C. After incubation, the cells were fixed and stained. ...
Toxicity to macrophages. Uninfected macrophages were incubated either with ezetimibe, miconazole, or ketoconazole alone or with a combination for 72 h at 37°C. After this period, the cells were incubated with MTT for 1 h at 37°C. The absorbance ...
Micrographs of L. amazonensis-infected macrophages treated with ezetimibe and azoles. Peritoneal macrophages infected with L. amazonensis and treated with ezetimibe and azoles were incubated for 72 h at 37°C. After incubation, the cells were fixed ...

For in vivo assays, BALB/c mice were infected with L. amazonensis and were treated with ezetimibe (10 mg/kg/day) and ketoconazole (100 mg/kg/day), alone or in combination, by the oral route. As can be seen in Fig. 6A, ketoconazole and ezetimibe were able individually to control lesion development. When the two drugs were combined, lesion growth was controlled more effectively. All treatments reduced parasite loads significantly (Fig. 6A, inset). Nonetheless, no significant differences in the levels of urea, albumin, ALT, AST, creatinine, bilirubin, or creatine kinase in serum were observed between treated and untreated animals, demonstrating that the treatment was not toxic to the mammalian host (Fig. 6B). Furthermore, no significant change in the cholesterol level in serum was observed (Fig. 6B).

In vivo activity of ezetimibe in combination with ketoconazole. BALB/c mice (9/group) were infected in the right ear with 2 × 106 stationary-phase L. amazonensis promastigotes and were orally treated with either ezetimibe at 10 mg/kg/day (Eze10), ...


Thirty drugs in clinical use were included in this study. Instead of focusing on antimicrobial agents, we selected compounds belonging to a wide spectrum of pharmacological classes (Table 1). There are no data in the literature about the antileishmanial activities of glimepiride and ezetimibe, but interestingly, domperidone has been used in clinical trials, aiming at veterinary use, with naturally infected dogs (16, 17). However, in those studies, no intrinsic antileishmanial activity was assigned to domperidone. The therapeutic effect of this drug in dogs has been attributed to its immunomodulatory activity (17).

Among the active drugs, ezetimibe drew attention due to its property of causing dramatic morphological alterations in promastigotes (Fig. 1). These morphological changes are consistent with those described previously as consequences of drug interference in the sterol metabolism of trypanosomatids (18,20). In fact, we observed that ezetimibe causes alterations in the sterol composition of the parasites that are compatible with C-14 demethylase inhibition (21,23). Curiously, ezetimibe is a potent inhibitor of intestinal cholesterol absorption mediated by the Niemann–Pick C1-Like 1 protein (NPC1L1) and is used clinically to reduce cholesterolemia (24). Nevertheless, no inhibition in the human sterol biosynthesis pathway has been reported for ezetimibe. The sterol pathway of the trypanosomatids, however, has diverged sufficiently to allow selective pharmacological inhibition. Thus, besides its effect on cholesterol transport in humans, it is possible that ezetimibe could also interfere with sterol biosynthesis in Leishmania parasites.

It is assumed that the sterol biosynthetic pathway is essential for Leishmania parasites, because inhibition of diverse enzymes of this pathway, such as 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), squalene epoxidase, C-14 demethylase, and C-24 methyltransferase, results in parasite death (25,30). Drug combination in the treatment of infectious diseases is an interesting approach. Drugs acting in the same pathway can be combined to improve their activities (31). Thus, we evaluated the activity of ezetimibe in combination with ketoconazole or miconazole. These combinations showed synergistic effects on promastigotes (Fig. 2) and increased the activities of these azoles against intracellular amastigotes of L. amazonensis (Fig. 3B and andCC).

The next step was to evaluate the leishmanicidal activity of ezetimibe alone or combined with ketoconazole in vivo (Fig. 6A and andB).B). Ezetimibe or ketoconazole alone reduced lesion growth and parasite loads equally in a murine model of cutaneous leishmaniasis. The combination was more efficacious at controlling lesion development, but no difference was observed in parasite burdens. Ketoconazole was chosen to be evaluated in vivo, because when in combination with ezetimibe, it showed better results than the miconazole-ezetimibe combination against intracellular amastigotes (Fig. 3C). Moreover, clinical trials have demonstrated that ketoconazole could be advantageous for patients infected with New World species of Leishmania (1). Oral ketoconazole combined with intralesional injections of meglumine antimoniate (Glucantime) has been shown to be more effective than meglumine antimoniate alone in the treatment of localized cutaneous leishmaniasis (32). Furthermore, ketoconazole at 100 mg/kg/day reduced splenic parasite loads in a murine model of visceral leishmaniasis caused by Leishmania infantum; at the lower dose of 50 mg/kg/day, ketoconazole potentiated the effect of meglumine antimoniate (33).

In summary, our screening of FDA-approved drugs identified ezetimibe as a promising antileishmanial agent. It has antileishmanial activity in vitro and in vivo, presumably through its effect on parasite sterol biosynthesis, and acts synergistically with azoles. These data suggest that ezetimibe could be useful, alone or in combination with azoles, in further treatments for leishmaniasis.

Supplementary Material

Supplemental material:


We thank the Program for Technological Development in Tools for Health (PDTIS), FIOCRUZ, for evaluating the serum toxicological markers.

We report no conflict of interest.

Funding Statement

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), and the Programa Estratégico de Apoio à Pesquisa em Saúde (PAPES/FIOCRUZ; grant 407680/2012-8 to E.C.T.-S.).


Supplemental material for this article may be found at


1. Handler MZ, Patel PA, Kapila R, Al-Qubati Y, Schwartz RA 2015. Cutaneous and mucocutaneous leishmaniasis: differential diagnosis, diagnosis, histopathology, and management. J Am Acad Dermatol 73:911–926. doi:.10.1016/j.jaad.2014.09.014 [PubMed] [Cross Ref]
2. Savoia D. 2015. Recent updates and perspectives on leishmaniasis. J Infect Dev Ctries 9:588–596. doi:.10.3855/jidc.6833 [PubMed] [Cross Ref]
3. Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M 2012. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7(5):e35671. doi:.10.1371/journal.pone.0035671 [PMC free article] [PubMed] [Cross Ref]
4. Vianna G. 1912. Comunicação à Sessão de 24 de abril de 1912 da Sociedade Brasileira de Dermatologia. Arch Bras Med 1:36–38.
5. Oliveira LF, Schubach AO, Martins MM, Passos SL, Oliveira RV, Marzochi MC, Andrade CA 2011. Systematic review of the adverse effects of cutaneous leishmaniasis treatment in the New World. Acta Trop 118:87–96. doi:.10.1016/j.actatropica.2011.02.007 [PubMed] [Cross Ref]
6. Kesselheim AS, Maggs LR, Sarpatwari A 2015. Experience with the priority review voucher program for drug development. JAMA 314:1687–1688. doi:.10.1001/jama.2015.11845 [PubMed] [Cross Ref]
7. Patra P, Guha SK, Maji AK, Saha P, Ganguly S, Chakraborty A, Kundu PK, Sarker S, Ray K 2012. Efficacy of oral miltefosine in visceral leishmaniasis in rural West Bengal, India. Indian J Pharmacol 44:500–503. doi:.10.4103/0253-7613.99326 [PMC free article] [PubMed] [Cross Ref]
8. Rijal S, Ostyn B, Uranw S, Rai K, Bhattarai NR, Dorlo TP, Beijnen JH, Vanaerschot M, Decuypere S, Dhakal SS, Das ML, Karki P, Singh R, Boelaert M, Dujardin JC 2013. Increasing failure of miltefosine in the treatment of kala-azar in Nepal and the potential role of parasite drug resistance, reinfection, or noncompliance. Clin Infect Dis 56:1530–1538. doi:.10.1093/cid/cit102 [PubMed] [Cross Ref]
9. Obonaga R, Fernández OL, Valderrama L, Rubiano LC, Castro MDM, Barrera MC, Gomez MA, Saravia NG 2014. Treatment failure and miltefosine susceptibility in dermal leishmaniasis caused by Leishmania subgenus Viannia species. Antimicrob Agents Chemother 5:144–152. doi:.10.1128/AAC.01023-13 [PMC free article] [PubMed] [Cross Ref]
10. Kaiser M, Mäser P, Tadoori LP, Ioset JR, Brun R 2015. Antiprotozoal activity profiling of approved drugs: a starting point toward drug repositioning. PLoS One 10(8):e0135556. doi:.10.1371/journal.pone.0135556 [PMC free article] [PubMed] [Cross Ref]
11. Sateriale A, Bessoff K, Sarkar IN, Huston CD 2014. Drug repurposing: mining protozoan proteomes for targets of known bioactive compounds. J Am Med Inform Assoc 21:238–244. doi:.10.1136/amiajnl-2013-001700 [PMC free article] [PubMed] [Cross Ref]
12. Andrews KT, Fisher G, Skinner-Adams TS 2014. Drug repurposing and human parasitic protozoan diseases. Int J Parasitol Drugs Drug Resist 4:95–111. doi:.10.1016/j.ijpddr.2014.02.002 [PMC free article] [PubMed] [Cross Ref]
13. Odds FC. 2003. Synergy, antagonism, and what the chequerboard puts between them. J Antimicrob Chemother 52:1. doi:.10.1093/jac/dkg301 [PubMed] [Cross Ref]
14. Bligh EG, Dyer WJ 1959. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917. doi:.10.1139/o59-099 [PubMed] [Cross Ref]
15. Torres-Santos EC, Sampaio-Santos MI, Buckner FS, Yokoyama K, Gelb M, Urbina JA, Rossi-Bergmann B 2009. Altered sterol profile induced in Leishmania amazonensis by a natural dihydroxymethoxylated chalcone. J Antimicrob Chemother 63:469–472. doi:.10.1093/jac/dkn546 [PMC free article] [PubMed] [Cross Ref]
16. Gómez-Ochoa P, Castillo JA, Gascón M, Zarate JJ, Alvarez F, Couto CG. 2009. Use of domperidone in the treatment of canine visceral leishmaniasis: a clinical trial. Vet J 179:259–263. doi:.10.1016/j.tvjl.2007.09.014 [PubMed] [Cross Ref]
17. Passos SR, Rodrigues TA, Madureira AP, Giunchetti RC, Zanini MS 2014. Clinical treatment of cutaneous leishmaniasis in dogs with furazolidone and domperidone. Int J Antimicrob Agents 44:463–465. doi:.10.1016/j.ijantimicag.2014.07.011 [PubMed] [Cross Ref]
18. Vannier-Santos MA, Urbina JA, Martiny A, Neves A, de Souza W 1995. Alterations induced by the antifungal compounds ketoconazole and terbinafine in Leishmania. J Eukaryot Microbiol 42:337–346. doi:.10.1111/j.1550-7408.1995.tb01591.x [PubMed] [Cross Ref]
19. Dinesh N, Soumya N, Singh S 2015. Antileishmanial effect of mevastatin is due to interference with sterol metabolism. Parasitol Res 144:3873–3883. doi:.10.1007/s00436-015-4618-5 [PubMed] [Cross Ref]
20. Pérez-Moreno G, Sealey-Cardona M, Rodrigues-Poveda C, Gelb MH, Ruiz-Pérez LM, Castilho-Acosta V, Urbina JA, González-Pacanowska D 2012. Endogenous sterol biosynthesis is important for mitochondrial function and cell morphology in procyclic forms of Trypanosoma brucei. Int J Parasitol 42:975–989. doi:.10.1016/j.ijpara.2012.07.012 [PubMed] [Cross Ref]
21. Berman JD, Goad LJ, Beach DH, Holz GG 1986. Effects of ketoconazole on sterol biosynthesis by Leishmania mexicana mexicana amastigotes in murine macrophage tumor cells. Mol Biochem Parasitol 20:85–92. doi:.10.1016/0166-6851(86)90145-3 [PubMed] [Cross Ref]
22. Berman JD, Holz GG, Beach DH 1984. Effects of ketoconazole on growth and sterol biosynthesis of Leishmania mexicana promastigotes in culture. Mol Biochem Parasitol 12:1–13. doi:.10.1016/0166-6851(84)90039-2 [PubMed] [Cross Ref]
23. Hart DT, Lauwers WJ, Willemsens G, Vandenbossche H, Opperdoes FR 1989. Perturbation of sterol biosynthesis by itraconazole and ketoconazole in Leishmania mexicana mexicana infected macrophages. Mol Biochem Parasitol 33:123–134. doi:.10.1016/0166-6851(89)90026-1 [PubMed] [Cross Ref]
24. Park SW. 2013. Intestinal and hepatic Niemann Pick C1-Like 1. Diabetes Metab J 37:240–248. doi:.10.4093/dmj.2013.37.4.240 [PMC free article] [PubMed] [Cross Ref]
25. Beach DH, Goad LJ, Holz GG 1988. Effects of antimycotic azoles on growth and sterol biosynthesis of Leishmania promastigotes. Mol Biochem Parasitol 31:149–162. doi:.10.1016/0166-6851(88)90166-1 [PubMed] [Cross Ref]
26. Daly H, De Lima H, Kato H, Sordillo EM, Convit J, Reyes-Jaimes O, Zerpa O, Paniz Mondolfi AE 2014. Intermediate cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis successfully treated with fluconazole. Clin Exp Dermatol 39:708–712. doi:.10.1111/ced.12359 [PubMed] [Cross Ref]
27. Rodrigues JC, Attias M, Rodriguez C, Urbina JA, de Souza W 2002. Ultrastructural and biochemical alterations induced by 22,26-azasterol, a Δ24(25)-sterol methyltransferase inhibitor, on promastigote and amastigote forms of Leishmania amazonensis. Antimicrob Agents Chemother 46:487–499. doi:.10.1128/AAC.46.2.487-499.2002 [PMC free article] [PubMed] [Cross Ref]
28. de Macedo-Silva ST, Urbina JA, de Souza W, Rodrigues JC 2013. In vitro activity of the antifungal azoles itraconazole and posaconazole against Leishmania amazonensis. PLoS One 8(12):e83247. doi:.10.1371/journal.pone.0083247 [PMC free article] [PubMed] [Cross Ref]
29. Kulkarni MM, Reddy N, Gude T, McGwire BS 2013. Voriconazole suppresses the growth of Leishmania species in vitro. Parasitol Res 112:2095–2099. doi:.10.1007/s00436-013-3274-x [PubMed] [Cross Ref]
30. Haughan PA, Chance ML, Goad LJ (1992). 1992. Synergism in vitro of lovastatin and miconazole as antileishmanial agents. Biochem Pharmacol 44:2199–2206. doi:.10.1016/0006-2952(92)90347-L [PubMed] [Cross Ref]
31. de Macedo-Silva ST, Visbal G, Urbina JA, de Souza W, Rodrigues JC 2015. Potent in vitro antiproliferative synergism of combinations of ergosterol biosynthesis inhibitors against Leishmania amazonensis. Antimicrob Agents Chemother 59:6402–6418. doi:.10.1128/AAC.01150-15 [PMC free article] [PubMed] [Cross Ref]
32. Salmanpour R, Handjani F, Nouhpisheh MK 2001. Comparative study of the efficacy of oral ketoconazole with intra-lesional meglumine antimoniate (Glucantime) for the treatment of cutaneous leishmaniasis. J Dermatol Treat 12:159–162. doi:.10.1080/09546630152607899 [PubMed] [Cross Ref]
33. Gangneux JP, Dullin M, Sulahian A, Garin YJ, Derouin F 1999. Experimental evaluation of second-line oral treatments of visceral leishmaniasis caused by Leishmania infantum. Antimicrob Agents Chemother 43:172–174. [PMC free article] [PubMed]

Articles from Antimicrobial Agents and Chemotherapy are provided here courtesy of American Society for Microbiology (ASM)