Article Figures & Data
Figures
- FIG 1
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. The slides were stained using the Instant Prov hematological dye system.
- FIG 2
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 point in the isobolograms is the IC50 of the drug alone or in combination. The straight dashed lines represent the theoretical line of additivity for each combination. The experiments were performed in triplicate (n = 3). The graphics are representative of the results of a single experiment. The graphics and IC50s were produced with GraphPad Prism software, version 4.
- FIG 3
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. The infection rate was calculated as described in Materials and Methods. The experiments were performed in triplicate (n = 3). The graphics are representative of a single experiment; error bars, standard deviations. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
- FIG 4
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 was read in a spectrophotometer at 570 nm. (A) Ezetimibe; (B) ketoconazole; (C) miconazole; (D) 20 μm ezetimibe (Eze) plus ketoconazole; (E) 20 μm Eze plus miconazole. The experiments were performed in triplicate (n = 3).
- FIG 5
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 and stained. (A) Control; (B) 20 μM ezetimibe; (C) 8 μM ketoconazole; (D) 8 μM ketoconazole plus 20 μM ezetimibe; (E) 4 μM miconazole; (F) 4 μM miconazole plus 20 μM ezetimibe.
- FIG 6
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), ketoconazole at 100 mg/kg/day (Keto100), miltefosine at 20 mg/kg/day, or Eze10 plus Keto100. The treatment started 10 days after infection. The animals were treated 5 days per week, in a total of 20 doses. They were euthanized after 35 days of infection. Negative controls were similarly treated with PBS. (A) Lesion growth. (Inset) Parasite load. Symbols indicate significant differences from the control group (*, P < 0.05; **, P < 0.01; ***, P < 0.001), the Eze10 group (##, P < 0.01), or the Keto100 group (π, P < 0.05; ππ, P < 0.01; πππ, P < 0.001) by two-way analysis of variance. (B) Biochemical evaluation of the toxicity of the treatment according to the parameters indicated: urea (URE), albumin (ALB), alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CREA), total bilirubin (BIL), creatine kinase (CK), and cholesterol (CHO).
Tables
- TABLE 1
Drugs tested for antileishmanial activity
Drug IC50 (μM) for: Indication Chemical classa Mode of actionb Promastigotes Intracellular amastigotes Amoxicillin >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/nonselective, competitive α-adrenoceptor antagonist Pyrrolidinyl-trimethoxyphenyl Nonselective competitive inhibition of α-adrenoceptors in vascular smooth muscle Cephalexin >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 Nonsteroidal anti-inflammatory Phenylacetic acid derivative Inhibitor of cyclooxygenase Domperidone 51 ± 0.6 >25 Antiemetic/antimuscarinic Phenothiazine Specific blocker of dopamine receptors Enalapril >100 >25 Antihypertensive Dicarbocyl-containing ACE inhibitor Reduces 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 GABA Gemfibrozil >100 >25 Antihyperlipidemic Fibric acid derivative Reduces triglycerides and increases cholesterol carried in 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 convoluted tubule Ibuprofen >100 >25 Analgesic, anti-inflammatory, antipyretic Propionic acid derivative Inhibitor of cyclooxygenase Lisinopril >100 >25 Antihypertensive/vasodilator Lysine ester of enalapril Reduces 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 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 Nonsteroidal anti-inflammatory Phenoxymethanesulfonamide Inhibitor of cyclooxygenase Oxacillin >100 >25 Antibiotic Penicillin Inhibits bacterial cell wall synthesis Pantoprazole >100 >25 Antiulcer Substituted benzimidazole Inhibits proton pump in gastric parietal cells Prednisolone >100 >25 Anti-inflammatory/immunosuppressant Glucocorticoid 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 Nonsteroidal anti-inflammatory Thienothiazine Inhibitor of cyclooxygenase - TABLE 2
Effect of ezetimibe on the sterol composition of L. amazonensis promastigotes
Compound Mol wt % of total lipid content of sample Control Miconazole (8 μM) Ezetimibe 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
Supplemental material
- Supplemental file 1 -
Supplemental Figure S1
PDF, 216K
- Supplemental file 1 -
