ABSTRACT
Despite efforts, available alternatives for the treatment of leishmaniasis are still scarce. In this work we tested a class of 15 quinolinylhydrazone analogues and presented data that support the use of the most active compound in cutaneous leishmaniasis caused by Leishmania amazonensis. In general, the compounds showed activity at low concentrations for both parasitic forms (5.33-37.04 µM to promastigotes, and 14.31-61.98 µM to amastigotes). In addition, the best compound (MHZ15) is highly selective for the parasite. Biochemical studies indicate that the treatment of promastigotes with MHZ15 leads the loss of mitochondrial potential and increase in ROS levels as the primary effects, which triggers accumulation of lipid droplets, loss of plasma membrane integrity and apoptosis hallmarks, including DNA fragmentation and phosphatidylserine exposure. These effects were similar in the intracellular form of the parasite. However, in this parasitic form there is no change in plasma membrane integrity in the observed treatment time, which can be attributed to metabolic differences and the resilience of the amastigote. Also, ultrastructural changes such as vacuolization suggesting autophagy were observed. The in vivo effectiveness of MHZ15 in the experimental model of cutaneous leishmaniasis was carried out in mice of the BALB/c strain infected with L. amazonensis. The treatment by intralesional route showed that MHZ15 acted with great efficiency with significantly reduction in the parasite load in the injured paws and draining lymph nodes, without clinical signs of distress or compromise of animal welfare. In vivo toxicity was also evaluated and null alterations in the levels of hepatic enzymes aspartate aminotransferase, and alanine aminotransferase was observed. The data presented herein demonstrates that MHZ15 exhibits a range of favorable characteristics conducive to the development of an antileishmanial agent.
Subject(s)
Apoptosis , Hydrazones , Leishmaniasis, Cutaneous , Mice, Inbred BALB C , Mitochondria , Animals , Apoptosis/drug effects , Mice , Mitochondria/drug effects , Mitochondria/metabolism , Hydrazones/pharmacology , Hydrazones/chemistry , Leishmaniasis, Cutaneous/drug therapy , Leishmaniasis, Cutaneous/parasitology , Antiprotozoal Agents/pharmacology , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/therapeutic use , Leishmania/drug effects , Reactive Oxygen Species/metabolism , Female , Leishmania mexicana/drug effects , Membrane Potential, Mitochondrial/drug effectsABSTRACT
Leishmaniasis is a group of infectious diseases caused by protozoan parasites that belong to the genus Leishmania. Currently, there is no human vaccine, and the available treatments are associated with toxicity, high cost, and the emergence of resistant strains. These factors highlight the need to identify new antileishmanial candidates. In this study, we synthesized twenty-four methoxylated cinnamides containing 1,2,3-triazole fragments and evaluated their antileishmanial activity against the Leishmania braziliensis species, which is the main etiological agent responsible for American Tegumentary Leishmaniasis (ATL). The cinnamides were synthetically prepared using nucleophilic acyl substitution and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions. The compounds were characterized using infrared, nuclear magnetic resonance, and high-resolution mass spectrometry techniques. We performed preliminary studies to evaluate the biological activity of these compounds against L. braziliensis promastigotes and axenic amastigotes. Compound 28, N-((1-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)-1H-1,2,3-triazole-4-yl) methyl)-3,4-dimethoxy cinnamide, demonstrated relevant antileishmanial activity with low toxicity in murine cells. The selectivity index values for this compound were superior compared with data obtained using amphotericin B. Furthermore, this cinnamide derivative reduced the infection percentage and number of recovered amastigotes in L. braziliensis-infected macrophages. It also induced an increase in reactive oxygen species production, depolarization of the mitochondrial potential, and disruption of the parasite membrane. Taken together, these findings suggest that this synthetic compound holds potential as an antileishmanial candidate and should be considered for future studies in the treatment of ATL.
ABSTRACT
Quinoline and 1,2,3-triazoles are well-known nitrogen-based heterocycles presenting diverse pharmacological properties, although their antileishmanial activity is still poorly exploited. As an effort to contribute with studies involving these interesting chemical groups, in the present study, a series of compounds derived from 4-aminoquinoline and 1,2,3-triazole were synthetized and biological studies using L. amazonensis species were performed. The results pointed that the derivative 4, a hybrid of 4-aminoquinoline/1,2,3-triazole exhibited the best antileishmanial action, with inhibitory concentration (IC50) values of ~1 µM against intramacrophage amastigotes of L. amazonensis , and being 16-fold more active to parasites than to the host cell. The mechanism of action of derivative 4 suggest a multi-target action on Leishmania parasites, since the treatment of L. amazonensis promastigotes caused mitochondrial membrane depolarization, accumulation of ROS products, plasma membrane permeabilization, increase in neutral lipids, exposure of phosphatidylserine to the cell surface, changes in the cell cycle and DNA fragmentation. The results suggest that the antileishmanial effect of this compound is primarily altering critical biochemical processes for the correct functioning of organelles and macromolecules of parasites, with consequent cell death by processes related to apoptosis-like and necrosis. No up-regulation of reactive oxygen and nitrogen intermediates was promoted by derivative 4 on L. amazonensis -infected macrophages, suggesting a mechanism of action independent from the activation of the host cell. In conclusion, data suggest that derivative 4 presents selective antileishmanial effect, which is associated with multi-target action, and can be considered for future studies for the treatment against disease.
Subject(s)
Aminoquinolines/pharmacology , Antiprotozoal Agents/pharmacology , Leishmania mexicana/drug effects , Triazoles/pharmacology , Aminoquinolines/chemical synthesis , Animals , Antiprotozoal Agents/chemistry , Apoptosis/drug effects , Cell Cycle/drug effects , Cell Membrane/chemistry , Cell Membrane/metabolism , DNA Fragmentation/drug effects , Female , Lipid Metabolism/drug effects , Macrophages/parasitology , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred BALB C , Organelles/drug effects , Phosphatidylserines/pharmacology , Reactive Oxygen Species/metabolism , Triazoles/chemical synthesisABSTRACT
BACKGROUND: Despite high seroprevalence of asymptomatic infection in humans, toxoplasmosis can manifest as a severe systemic disease, as occurs in the congenital infection. Here we evaluate the seroprevalence of Toxoplasma infection among pregnant women in a highly urbanized area of Brazil. METHODS: A robust seroepidemiological study was conducted using laboratory databases of anti-Toxoplasma gondii serological results together with information on age, month/year of diagnosis and place of residence of pregnant women in the public health system of the city of Juiz de Fora, Brazil. RESULTS: Of 5895 pregnant women analysed, 54.7% showed seronegativity and 44.4% showed seropositivity for immunoglobulin G (IgG) antibodies against Toxoplasma gondii. This seropositivity rate increased to 68.3% when only considering participants from rural areas. Multivariate analysis revealed higher odds of being seropositive associated with age (odds ratio [OR] 1.06 [confidence interval {CI} 1.05 to 1.07]) and with living in rural areas (OR 2.96 [CI 1.64 to 5.36]). The spatial distribution of IgG seropositivity indicated a higher prevalence concentrated in rural and peripheral neighbourhoods. CONCLUSIONS: This is the first report to use spatial analysis to show a cluster of Toxoplasma infection in rural and peripheral neighbourhoods of a highly urbanized municipality, which highlights the need for adequate healthcare actions to be implemented for women living in these areas.
Subject(s)
Toxoplasma , Antibodies, Protozoan , Brazil/epidemiology , Cities , Cross-Sectional Studies , Female , Humans , Immunoglobulin M , Pregnancy , Pregnant Women , Risk Factors , Seroepidemiologic StudiesABSTRACT
INTRODUCTION: The drugs currently available for leishmaniasis treatment have major limitations. METHODS: In vitro and in vivo studies were performed to evaluate the effect of a quinoline derivative, Hydraqui (7-chloro-4-(3-hydroxy-benzilidenehydrazo)quinoline, against Leishmania amazonensis. In silico analyses of absorption, distribution, metabolism, excretion, and toxicity (ADMET) parameters were performed. RESULTS: Hydraqui showed significant in vitro anti-amastigote activity. Also, Hydraqui-treated mice exhibited high efficacy in lesion size (48.3%) and parasitic load (93.8%) reduction, did not cause hepatic and renal toxicity, and showed appropriate ADMET properties. CONCLUSIONS: Hydraqui presents a set of satisfactory criteria for its application as an antileishmanial agent.
Subject(s)
Antiprotozoal Agents/therapeutic use , Leishmania mexicana/drug effects , Leishmaniasis, Cutaneous/drug therapy , Quinolines/therapeutic use , Animals , Disease Models, Animal , Female , Leishmaniasis, Cutaneous/parasitology , Mice , Mice, Inbred BALB C , Parasite Load , Quinolines/chemistryABSTRACT
Abstract INTRODUCTION: The drugs currently available for leishmaniasis treatment have major limitations. METHODS: In vitro and in vivo studies were performed to evaluate the effect of a quinoline derivative, Hydraqui (7-chloro-4-(3-hydroxy-benzilidenehydrazo)quinoline, against Leishmania amazonensis. In silico analyses of absorption, distribution, metabolism, excretion, and toxicity (ADMET) parameters were performed. RESULTS: Hydraqui showed significant in vitro anti-amastigote activity. Also, Hydraqui-treated mice exhibited high efficacy in lesion size (48.3%) and parasitic load (93.8%) reduction, did not cause hepatic and renal toxicity, and showed appropriate ADMET properties. CONCLUSIONS: Hydraqui presents a set of satisfactory criteria for its application as an antileishmanial agent.
Subject(s)
Animals , Female , Quinolines/therapeutic use , Leishmania mexicana/drug effects , Leishmaniasis, Cutaneous/drug therapy , Antiprotozoal Agents/therapeutic use , Quinolines/chemistry , Leishmaniasis, Cutaneous/parasitology , Disease Models, Animal , Parasite Load , Mice , Mice, Inbred BALB CABSTRACT
The alkylaminoalkanethiosulfuric acids (AAATs) are amphipathic compounds effective against experimental schistosomiasis, of low toxicity, elevated bioavailability after a single oral dose and prompt tissue absorption. OBJECTIVES: To explore the in-vitro antileishmanial potential of AAATs using five compounds of this series against Leishmania (Viannia) braziliensis. METHODS: Their effects on promastigotes and axenic amastigotes, and cytotoxicity to macrophages were tested by the MTT method, and on Leishmania-infected macrophages by Giemsa stain. Effects on the mitochondrial membrane potential of promastigotes and axenic amastigotes and DNA of intracellular amastigotes were tested using JC-1 and TUNEL assays, respectively. KEY FINDINGS: The 2-(isopropylamino)-1-octanethiosulfuric acid (I) and 2-(sec-butylamino)-1-octanethiosulfuric acid (II) exhibit activity against both promastigotes and intracellular amastigotes (IC50 25-35 µm), being more toxic to intracellular parasites than to the host cell. Compound I induced a loss of viability of axenic amastigotes, significantly reduced (30%) the mitochondrial membrane potential of both promastigotes and axenic amastigotes and promoted selective DNA fragmentation of the nucleus and kinetoplast of intracellular amastigotes. CONCLUSIONS: In this previously unpublished study of trypanosomatids, it is shown that AAATs could also exhibit selective antileishmanial activity, a new possibility to be investigated in oral treatment of leishmaniasis.
Subject(s)
Antiprotozoal Agents/pharmacology , Leishmania braziliensis/isolation & purification , Leishmaniasis/drug therapy , Sulfuric Acids/pharmacology , Administration, Oral , Animals , Antiprotozoal Agents/administration & dosage , Antiprotozoal Agents/chemistry , Inhibitory Concentration 50 , Leishmania braziliensis/drug effects , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred BALB C , Structure-Activity Relationship , Sulfuric Acids/administration & dosage , Sulfuric Acids/chemistryABSTRACT
OBJECTIVES: The in vitro antileishmanial effect of analogues of resveratrol (AR) present in the N-aryl imines and N-aryl hydrazones series was investigated. In addition, possible parasite targets were evaluated. METHODS: Antipromastigote activity of Leishmania amazonensis, L. braziliensis and L. infantum, as well as the cytotoxicity on macrophages was determined by MTT assay and L. braziliensis-infected macrophages effect by Giemsa stain. After staining, effects on the parasite targets were analysed by flow cytometry or by fluorescence microscopy. KEY-FINDINGS: Among the tested compounds, the derivative AR26 showed the best effect against promastigotes of all Leishmania species (IC50 < 3.0 µg/ml), being more active than miltefosine, the control drug. AR26 was also effective against amastigotes of L. braziliensis (IC50 = 15.9 µg/ml), with low toxicity to mammalian cells. The evaluation of mechanism of action of AR26 on L. braziliensis promastigotes indicates mitochondrial potential depolarization, plasma membrane permeabilization, interference in the progression of the cell cycle and accumulation of autophagic vacuoles. In addition, any increase of the reactive oxygen species levels was detected in the treated L. braziliensis-macrophages. CONCLUSIONS: Data indicate that the antileishmanial activity of AR26 is related to multitarget action, and the resveratrol analogues could be used in future studies as antileishmanial agent.
Subject(s)
Antiprotozoal Agents/pharmacology , Leishmania/drug effects , Leishmaniasis/drug therapy , Resveratrol/pharmacology , Animals , Antiprotozoal Agents/administration & dosage , Antiprotozoal Agents/chemistry , Disease Models, Animal , Female , Hydrazones/administration & dosage , Hydrazones/chemistry , Hydrazones/pharmacology , Imines/administration & dosage , Imines/chemistry , Imines/pharmacology , Inhibitory Concentration 50 , Leishmaniasis/parasitology , Macrophages/drug effects , Mice , Mice, Inbred BALB C , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/pharmacology , Reactive Oxygen Species/metabolism , Resveratrol/administration & dosage , Resveratrol/analogs & derivativesABSTRACT
A nucleoside triphosphate diphosphohydrolase-1 (NTPDase 1) was identified on the surface, flagellum and kinetoplast from L. infantum promastigotes by immunocytochemistry and confocal laser scanning microscopy, using immune sera that recognized specifically the B domain of NTPDase 1 and produced against synthetic peptides (LbB1LJ and LbB2LJ) derived from this domain. The polyclonal antibodies had effective antileishmanial effect, reducing significantly in vitro promastigotes growth (21-25%), an antiproliferative effect also demonstrated by immune sera produced against recombinant r-pot B domain, and two other synthetic peptides (potB1LJ and potB2LJ). In addition, using these biomolecules in ELISA technique, IgG1 and IgG2 subclasses reactivities of either healthy dogs or infected by L. infantum and classified clinically as asymptomatic, oligosymptomatic and symptomatic were tested. Analysis of distinct IgG1 and IgG2 seropositivities patterns suggested antibody subclasses binding epitopes along B domain for protection against infection, indicating this domain as a new tool for prophylactic and immunotherapeutic investigations.
Subject(s)
Antibodies, Protozoan/immunology , Dog Diseases/immunology , Immunoglobulin G/immunology , Leishmania infantum/enzymology , Leishmania infantum/immunology , Leishmaniasis, Visceral/veterinary , Nucleoside-Triphosphatase/immunology , Animals , Antibodies, Protozoan/metabolism , Dog Diseases/parasitology , Dogs , Leishmaniasis, Visceral/immunology , Leishmaniasis, Visceral/parasitology , Protein Domains/immunologyABSTRACT
Currently, available treatment options for leishmaniasis are limited and unsatisfactory. In a previous study, a quinoline derivative (AMQ-j), exhibited a strong effect against Leishmania amazonensis and its antileishmanial activity was preliminarily associated with mitochondrial dysfunction. The present study further explores the antileishmanial effect of this compound against L. amazonensis, as well as determines the main cellular processes involved in the death of the parasite. Moreover, this study evaluated the in vivo effect of the AMQ-j in BALB/c mice experimentally infected by L. amazonensis. The results showed that the compound AMQ-j induces a set of morphological and biochemical features that could correlate with both autophagy-related and apoptosis-like processes, indicating intense mitochondrial swelling, a collapse of the mitochondrial membrane potential, an abnormal chromatin condensation, an externalization of phosphatidylserine, an accumulation of lipid bodies, a disorganization of cell cycle, a formation of autophagic vacuoles, and an increase of acidic compartments. Treatment with AMQ-j through an intralesional route was effective in reducing the parasite burden and size of the lesion. No significant increase in the serum levels of hepatic or renal damage toxicity markers was observed. These findings contribute to the understanding of the mode of action of quinoline derivatives involved in the death of Leishmania parasites and encourage new studies in other experimental models of Leishmania infection.
Subject(s)
Aminoquinolines/pharmacology , Antiprotozoal Agents/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Leishmania mexicana/drug effects , Leishmaniasis, Cutaneous/drug therapy , Aminoquinolines/therapeutic use , Aminoquinolines/toxicity , Animals , Antiprotozoal Agents/therapeutic use , Antiprotozoal Agents/toxicity , Cell Cycle/drug effects , Chlorocebus aethiops , Creatinine/metabolism , Ear, External/parasitology , Ear, External/pathology , Female , Inhibitory Concentration 50 , Kidney/drug effects , Leishmania mexicana/cytology , Leishmania mexicana/growth & development , Leishmania mexicana/ultrastructure , Liver/drug effects , Liver/enzymology , Mice , Mice, Inbred BALB C , Vero CellsABSTRACT
Parasitic diseases, such as malaria and leishmaniasis, are relevant public health problems worldwide. For both diseases, the alarming number of clinical cases and deaths reported annually has justified the incentives directed to better understanding of host's factors associated with susceptibility to infection or protection. In this context, over recent years, some studies have given special attention to B lymphocytes with a regulator phenotype, known as Breg cells. Essentially important in the maintenance of immunological tolerance, especially in autoimmune disease models such as rheumatoid arthritis and experimentally induced autoimmune encephalomyelitis, the function of these lymphocytes has so far been poorly explored during the course of diseases caused by parasites. As the activation of Breg cells has been proposed as a possible therapeutic or vaccine strategy against several diseases, here we reviewed studies focused on understanding the relation of parasite and Breg cells in malaria and leishmaniasis, and the possible implications of these strategies in the course of both infections.
Subject(s)
B-Lymphocytes, Regulatory/immunology , Leishmania/immunology , Leishmaniasis/immunology , Malaria/immunology , Plasmodium/immunology , B-Lymphocytes, Regulatory/parasitology , Humans , Immune Tolerance , Leishmaniasis/parasitology , Malaria/parasitologyABSTRACT
In this study, we have investigated the antileishmanial activity of ten 7-chloro-4-quinolinylhydrazone derivatives. Among the compounds tested, compounds 2a and 2j presented activity against promastigotes (IC50 values of 52.5 and 21.1 µM, respectively) and compounds 2a and 2c were active against intracellular amastigotes (IC50 of 8.1 and 15.6 µM, respectively) of Leishmania amazonensis. The majority of compounds did not show toxicity against murine macrophages. Compound 2a exhibited low cytotoxicity to human erythrocytes and induced an oxidative imbalance in promastigote forms, reflected by an increase in the formation of reactive oxygen species (ROS) and a reduction of mitochondrial membrane potential. No alteration in the plasma membrane integrity of parasites was observed. Taken together, these results suggest that compound 2a is a selective antileishmanial agent, and preliminary observations suggest that its effects appear to be mediated by mitochondrial dysfunction.
Subject(s)
Aminoquinolines/pharmacology , Erythrocytes/drug effects , Hydrazones/pharmacology , Leishmania mexicana/drug effects , Macrophages/drug effects , Aminoquinolines/chemistry , Aminoquinolines/toxicity , Animals , Erythrocytes/parasitology , Humans , Hydrazones/chemistry , Hydrazones/toxicity , Inhibitory Concentration 50 , Macrophages/parasitology , Membrane Potential, Mitochondrial/drug effects , Mice , Mitochondria/drug effects , Reactive Oxygen Species/metabolismABSTRACT
Leishmaniasis is a complex disease caused by protozoan parasite Leishmania and the treatment remains a serious problem since the available drugs exhibited high toxicity and side effects. Plant-derived natural products are promising leads for the development of novel chemotherapeutics. In this work the phytol-rich hexane fraction (PRF) from the leaves of Lacistema pubescens was obtained and identified by GC-MS analysis. When assayed for antileishmanial effects, PRF was active against promastigote and amastigote forms of Leishmania amazonensis (IC50 values of 44.0 and 25.8 µg/mL respectively). Furthermore, PRF did not show significant cytotoxicity on peritoneal macrophages being more destructive to the intracellular parasite than to mammalian cells. In addition, possible targets of PRF were investigated against L. amazonensis promastigotes. The results showed that PRF exerted its antipromastigote activity by marked depolarization of the mitochondrial membrane potential followed by the increase of ROS levels in L. amazonensis promastigotes. During these events, no rupture of the cell membrane integrity was observed. Our results indicated that PRF was effective and selective against L. amazonensis, and that this effect was mainly mediated by mitochondrial dysfunction associated to ROS production.
Subject(s)
Leishmania mexicana/drug effects , Magnoliopsida/chemistry , Phytol/pharmacology , Plant Extracts/pharmacology , Animals , Cell Membrane/drug effects , Dose-Response Relationship, Drug , Gas Chromatography-Mass Spectrometry , Inhibitory Concentration 50 , Leishmania mexicana/metabolism , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/parasitology , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred BALB C , Mitochondria/drug effects , Mitochondria/metabolism , Plant Leaves/chemistry , Reactive Oxygen Species/metabolismABSTRACT
Species of the genus Lacistema are traditionally used by Brazilian and Peruvian indigenous communities. The present study investigated the in vitro antileishmanial activity against several Leishmania species, cytotoxicity in murine peritoneal macrophages, antiproliferative activity against HL60 and Jurkat cells, and antibacterial activities against seven bacteria strains of the aerial parts of the methanolic crude extract and fractions of Lacistema pubescens. In addition, their chemical profile was also evaluated. Hexane fraction showed the most significant IC50 values against all promastigotes of Leishmania species tested, except for L. chagasi (IC50 = 4.2 µg/mL for L. major and IC50 = 3.5 µg/mL for L. amazonensis). This fraction also exhibited a strong activity against amastigotes of L. amazonensis (IC50 = 6.9 µg/mL). The antiproliferative activity was also observed for methanolic extract and hexane fraction with IC50 = 47.2 µg/mL and IC50 = 39.7 µg/mL for HL60, respectively. Regarding the antimicrobial activity, the overall antibacterial activity was not very significative. Phytol and sitosterol were identified in the methanolic extract. Additionally, previous studies also revealed the presence of those compounds in the hexane fraction. Among other compounds, phytol and sitosterol were probably involved in the antileishmanial and cytotoxicity activities observed in this study.