ABSTRACT
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.
Subject(s)
Azoles/pharmacology , Ezetimibe/pharmacology , Leishmania mexicana/drug effects , Leishmaniasis, Cutaneous/drug therapy , Trypanocidal Agents/pharmacology , Animals , Disease Models, Animal , Drug Evaluation, Preclinical/methods , Drug Synergism , Inhibitory Concentration 50 , Leishmania mexicana/metabolism , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/parasitology , Mice, Inbred BALB C , Sterols/biosynthesisABSTRACT
Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is the second major cause of death by parasites, after malaria. The arsenal of drugs against leishmaniasis is small, and each has a disadvantage in terms of toxicity, efficacy, price, or treatment regimen. Our group has focused on studying new drug candidates as alternatives to current treatments. The pterocarpanquinone LQB-118 was designed and synthesized based on molecular hybridization, and it exhibited antiprotozoal and anti-leukemic cell line activities. Our previous work demonstrated that LQB-118 was an effective treatment for experimental cutaneous leishmaniasis. In this study, we observed that treatment with 10 mg/kg of body weight/day LQB-118 orally inhibited the development of hepatosplenomegaly with a 99% reduction in parasite load. An in vivo toxicological analysis showed no change in the clinical, biochemical, or hematological parameters. Histologically, all of the analyzed organs were normal, with the exception of the liver, where focal points of necrosis with leukocytic infiltration were observed at treatment doses 5 times higher than the therapeutic dose; however, these changes were not accompanied by an increase in transaminases. Our findings indicate that LQB-118 is effective at treating different clinical forms of leishmaniasis and presents no relevant signs of toxicity at therapeutic doses; thus, this framework is demonstrated suitable for developing promising drug candidates for the oral treatment of leishmaniasis.
Subject(s)
Antiprotozoal Agents/pharmacology , Hepatomegaly/prevention & control , Leishmania infantum/drug effects , Leishmaniasis, Visceral/drug therapy , Naphthoquinones/pharmacology , Parasitemia/prevention & control , Pterocarpans/pharmacology , Splenomegaly/prevention & control , Animals , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Gastric Absorption , Humans , Inhibitory Concentration 50 , Intubation, Gastrointestinal , Leishmania infantum/growth & development , Leishmaniasis, Visceral/parasitology , Leishmaniasis, Visceral/pathology , Mice , Mice, Inbred BALB C , Organ Specificity , Toxicity Tests, SubacuteABSTRACT
An orally delivered, safe and effective treatment for leishmaniasis is an unmet medical need. Azoles and the pyrazolylpyrimidine allopurinol present leishmanicidal activity, but their clinical efficacies are variable. Here, we describe the activity of the new pyrazolyltetrazole hybrid, 5-[5-amino-1-(4'-methoxyphenyl)1H-pyrazole-4-yl]1H-tetrazole (MSN20). MSN20 showed a 50% inhibitory concentration (IC50) of 22.3 µM against amastigotes of Leishmania amazonensis and reduced significantly the parasite load in infected mice, suggesting its utility as a lead compound for the development of an oral treatment for leishmaniasis.
Subject(s)
Antiprotozoal Agents/administration & dosage , Antiprotozoal Agents/pharmacology , Leishmaniasis, Cutaneous/drug therapy , Pyrazoles/chemistry , Tetrazoles/chemistry , Administration, Oral , Animals , Antiprotozoal Agents/chemistry , Inhibitory Concentration 50 , Mice , Structure-Activity RelationshipABSTRACT
Two series of N,N'-diphenyl-benzamidines were synthesized as part of a study to search potential new drugs with antileishmanial activity. These compounds were obtained by anilides in PCl5 halogenation reaction with generation in situ of the corresponding benzimidoyl chlorides, and subsequently treatment with adequate anilines. The series I showed expressive results of antileishmanial activity, highlighted the compounds 9a with IC50 = 81.28 µM (log IC50 = 1.91 µM) against Leishmania chagasi, 8e with IC50 = 26.30 (log IC50 = 1.52 µM) against Leishmania braziliensis. From the results obtained from SAR study (series I), the series II was planned from Craig 2-dimensional map, in which was possible the discovery of the potent compounds, 9v and 9j with IC50 = 12.60 µM (log IC50 = 1.10 µM) and 13.00 µM (log IC50 = 1.11 µM), respectively, against Leishmania amazonensis. The results obtained from the SAR and QSAR studies indicated the best results when electron-donor groups in the ring attached to amidinic carbon, unlike when electron-withdrawing groups at the phenyl-N ring showing inhibitory activity increased. Furthermore, the QSAR model obtained indicated the hydrophobicity as a fundamental property for antileishmanial activity presented by these series.
Subject(s)
Antiparasitic Agents/pharmacology , Benzamidines/pharmacology , Leishmania/drug effects , Antiparasitic Agents/chemical synthesis , Antiparasitic Agents/chemistry , Benzamidines/chemical synthesis , Benzamidines/chemistry , Dose-Response Relationship, Drug , Models, Molecular , Molecular Structure , Parasitic Sensitivity Tests , Structure-Activity RelationshipABSTRACT
OBJECTIVES: The pterocarpanquinone LQB-118, previously demonstrated to be effective in vivo via oral delivery, was investigated for its mechanism in selective parasite killing. METHODS: Oxidative stress in Leishmania amazonensis was analysed by evaluating reactive oxygen species (ROS) production (2',7'-dichlorodihydrofluorescein diacetate) and the loss of mitochondrial membrane potential (ΔΨm) using rhodamine, JC-1 and MitoCapture. Ultrastructural analysis was performed using transmission electron microscopy (TEM). DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). RESULTS: Treatment with LQB-118 induced ROS production in the promastigotes of L. amazonensis in a concentration-dependent manner for the first 4 h and was sustained for 24 h. TEM analysis revealed several alterations typical of apoptosis. Promastigotes presented a reduction of ΔΨm after 24 h of incubation with 2.5 µM (18.7%), 5 µM (63.7%) or 10 µM (70.7%) LQB-118. A sub-G0/G1 cell cycle phenotype was observed in 21%-83% of the promastigotes incubated with 1.25-10 µM LQB-118. Concentration-dependent DNA fragmentation was observed in promastigotes treated with 2.5-10 µM LQB-118, and selective DNA fragmentation was observed in intracellular amastigotes after 72 h with 2.5 µM treatment. CONCLUSIONS: Our results suggest that LQB-118 selectively induces ROS-triggered and mitochondria-dependent apoptosis in this parasite.
Subject(s)
Antiprotozoal Agents/pharmacology , Apoptosis , Leishmania/drug effects , Naphthoquinones/pharmacology , Oxidative Stress , Pterocarpans/pharmacology , DNA Fragmentation , In Situ Nick-End Labeling , Leishmania/physiology , Leishmania/ultrastructure , Membrane Potential, Mitochondrial/drug effects , Microscopy, Electron, Transmission , Reactive Oxygen Species/analysisABSTRACT
Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.
Subject(s)
Antiprotozoal Agents/pharmacology , Ketoconazole/pharmacology , Leishmania mexicana/drug effects , Leishmania mexicana/enzymology , /metabolism , Up-Regulation/drug effects , Parasitic Sensitivity Tests , Real-Time Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Protozoan/analysis , /geneticsABSTRACT
Ketoconazole is a clinically safe antifungal agent that also inhibits the growth of Leishmania spp. A study was undertaken to determine whether Leishmania parasites are prone to becoming resistant to ketoconazole by upregulating C14-demethylase after stepwise pharmacological pressure. Leishmania amazonensis promastigotes [inhibitory concentration (IC)50 = 2 µM] were subjected to stepwise selection with ketoconazole and two resistant lines were obtained, La8 (IC50 = 8 µM) and La10 (IC50 = 10 µM). As a result, we found that the resistance level was directly proportional to the C14-demethylase mRNA expression level; we also observed that expression levels were six and 12 times higher in La8 and La10, respectively. This is the first demonstration that L. amazonensis can up-regulate C14-demethylase in response to drug pressure and this report contributes to the understanding of the mechanisms of parasite resistance.
Subject(s)
Antiprotozoal Agents/pharmacology , Ketoconazole/pharmacology , Leishmania mexicana/drug effects , Leishmania mexicana/enzymology , Sterol 14-Demethylase/metabolism , Up-Regulation/drug effects , Inhibitory Concentration 50 , Parasitic Sensitivity Tests , RNA, Messenger/analysis , RNA, Protozoan/analysis , Real-Time Polymerase Chain Reaction , Sterol 14-Demethylase/geneticsABSTRACT
Leishmania parasites, despite being able to synthesize their own sterols, acquire and accumulate significant amounts of cholesterol through low density lipoprotein (LDL) particle endocytosis. The role of this system in Leishmania amazonensis promastigotes under pharmacological pressure by sterol biosynthesis inhibitors (SBIs) was investigated. First, thin layer chromatography demonstrated that L. amazonensis promastigotes, in response to ergosterol biosynthesis inhibition by treatment with 4.0 and 6.0 µM ketoconazole or miconazole, accumulate up to two times more cholesterol than controls. The treatment of promastigotes with ketoconazole and simvastatin, two SBIs with non-related mechanisms of action, showed that both drugs induce increases in (125)I-LDL endocytosis in a dose-dependent manner, indicating that the accumulation of exogenous cholesterol is due to the enhancement of LDL uptake. Finally, it was demonstrated that L. amazonensis promastigotes were rendered more susceptible to treatment with SBIs (ketoconazole, miconazole, simvastatin and terbinafine) in the absence of exogenous cholesterol sources, with a reduction of the IC50s of about 50% in three of the four tested drugs. These results show that the exogenous cholesterol uptake system in L. amazonensis plays a role as a compensatory mechanism in response to the presence of SBIs, suggesting that it may be a potential pharmacological target.
Subject(s)
Endocytosis , Leishmania/drug effects , Leishmania/metabolism , Lipoproteins, LDL/metabolism , Sterols/antagonists & inhibitors , Sterols/biosynthesis , Animals , Antiprotozoal Agents/metabolism , Inhibitory Concentration 50 , Ketoconazole/metabolism , Miconazole/metabolism , Naphthalenes/metabolism , Parasitic Sensitivity Tests , TerbinafineABSTRACT
OBJECTIVES: This paper describes the antileishmanial properties of LQB-118, a new compound designed by molecular hybridization, orally active in Leishmania amazonensis-infected BALB/c mice. METHODS: In vitro antileishmanial activity was determined in L. amazonensis-infected macrophages. For in vivo studies, LQB-118 was administered intralesionally (15 µg/kg/day, five times a week), intraperitoneally (4.5 mg/kg/day, five times a week) or orally (4.5 mg/kg/day, five times a week) to L. amazonensis-infected BALB/c mice throughout experiments lasting 85 or 105 days. At the end of the experiments, serum levels of alanine aminotransferase, aspartate aminotransferase and creatinine were measured as toxicological parameters. RESULTS: LQB-118 was active against intracellular amastigotes of L. amazonensis [50% inhibitory concentration (IC(50)) 1.4 µM] and significantly less so against macrophages (IC(50) 18.5 µM). LQB-118 administered intralesionally, intraperitoneally or orally was found to control both lesion and parasite growth in L. amazonensis-infected BALB/c mice, without altering serological markers of toxicity. CONCLUSIONS: These results demonstrate that the molecular hybridization of a naphthoquinone core to pterocarpan yielded a novel antileishmanial compound that was locally and orally active in an experimental cutaneous leishmaniasis model.
Subject(s)
Antiprotozoal Agents/administration & dosage , Leishmaniasis, Cutaneous/drug therapy , Administration, Oral , Administration, Topical , Alanine Transaminase/blood , Animals , Antiprotozoal Agents/adverse effects , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/pharmacology , Aspartate Aminotransferases/blood , Chemical and Drug Induced Liver Injury/diagnosis , Creatinine/blood , Disease Models, Animal , Inhibitory Concentration 50 , Leishmania mexicana/drug effects , Leishmaniasis, Cutaneous/parasitology , Liver/enzymology , Mice , Mice, Inbred BALB C , Naphthoquinones/administration & dosage , Naphthoquinones/adverse effects , Naphthoquinones/chemistry , Naphthoquinones/pharmacology , Pterocarpans/administration & dosage , Pterocarpans/adverse effects , Pterocarpans/chemistry , Pterocarpans/pharmacology , Rodent Diseases/drug therapy , Rodent Diseases/parasitology , Serum/chemistry , Treatment OutcomeABSTRACT
Leishmania parasites survive despite exposure to the toxic nitrosative oxidants during phagocytosis by the host cell. In this work, the authors investigated comparatively the resistance of Leishmania amazonensis promastigotes and axenic amastigotes to a relatively strong nitrosating agent that acts as a nitric oxide (NO) donor, sodium nitroprusside (SNP). Results demonstrate that SNP is able to decrease, in vitro, the number of L. amazonensis promastigotes and axenic amastigotes in a dose-dependent maner. Promastigotes, cultured in the presence of 0.25, 0.5, and 1 mmol L(-1) SNP for 24 h showed about 75% growth inhibition, and 97-100% when the cultures were treated with >2 mmol L(-1) SNP. In contrast, when axenic amastigotes were growing in the presence of 0.25-8 mM SNP added to the culture medium, 50% was the maximum of growth inhibition observed. Treated promastigotes presented reduced motility and became round in shape further confirming the leishmanicidal activity of SNP. On the other hand, axenic amastigotes, besides being much more resistant to SNP-mediated cytotoxicity, did not show marked morphological alteration when incubated for 24 h, until 8 mM concentrations of this nitrosating agent were used. The cytotoxicity toward L. amazonensis was attenuated by reduced glutathione (GSH), supporting the view that SNP-mediated toxicity triggered multiple oxidative mechanisms, including oxidation of thiols groups and metal-independent oxidation of biomolecules to free radical intermediates.
Subject(s)
Leishmania mexicana/drug effects , Life Cycle Stages/drug effects , Nitroprusside/toxicity , Animals , Arginase/metabolism , Culture Media, Conditioned/chemistry , Dose-Response Relationship, Drug , Glutathione/pharmacology , Leishmania mexicana/growth & development , Leishmania mexicana/metabolism , Nitric Oxide Donors/toxicity , Nitrites/analysis , Nitrites/metabolismABSTRACT
In a previous works searching for new drugs with high efficiency, we reported the in vitro and in vivo antileishmanial activity of a series of diarylheptanoid structurally related to curcumin against L. amazonensis. This work describes the in vitro antileishmanial activity of a new series of diarylheptanoids and diarylpentanoids derivatives. These drugs were assayed against Leishmania amazonensis, L. braziliensis and L. chagasi promastigotes containing a high percentage of metacyclic forms and the axenic amastigote form of L. amazonensis and using Pentamidine Isethionate as reference drug. Parasites in the log late phase culture were incubated with several concentrations of the drugs solubilized in dimethyl sulphoxide (DMSO) and then counted in a Neubauer's chamber. Controls without the drugs and with DMSO were done in parallel. The results showed that all diarylheptanoids and diarylpentanoids had a very good antileishmanial activity.
