Parasitological and immunological evaluation of a quinoline derivative salt incorporated into a polymeric micelle formulation against Leishmania infantum infection.

Leishmaniasis is a parasitic disease caused by Leishmania protozoa, which presents a large spectrum of clinical manifestations. In the present study, a quinoline derivative salt named N-(2-((7-chloroquinolin-4-yl)amino)ethyl)-N-(prop-2-yn-1-yl)prop-2-yn-1-aminium chloride or QDS3 was in vitro and in vivo tested against L. infantum by means of its incorporation in Poloxamer 407-based polymeric micelles (QDS3/M). The in vitro antileishmanial activity of QDS3 and QDS3/M was investigated in L. infantum promastigotes, axenic amastigotes and infected macrophages. BALB/c mice were infected with L. infantum, and parasitological parameters were evaluated 1 and 15 days post-treatment by determining the parasite load by a limiting dilution assay, besides a quantitative PCR (qPCR) method. Immunological response was assessed based on production of cellular cytokines, as well as by quantification of nitrite levels and specific antibodies. In vitro results showed that QDS3 free or in micelles presented effective antileishmanial action against both parasite stages, being more effective in amastigotes. In vivo data showed that treatment using QDS3 or QDS3/M reduced the parasite load in the livers, spleens, draining lymph nodes (dLN) and bone marrows of the treated animals, 1 and 15 days after treatment, when compared to values found in the control groups. Additionally, treated mice developed a polarized Th1-type immune response, with higher levels of IL-12, IFN-γ, GM-CSF and nitrite, besides high production of specific IgG2a antibodies, when compared to the controls. Parasitological and immunological data obtained using the micellar composition were better than the others. In conclusion, QDS3, mainly when applied in a delivery adjuvant system, could be considered for future studies as therapeutic candidate against VL.

Flau-A, a naphthoquinone derivative, is a promising therapeutic candidate against visceral leishmaniasis: A preliminary study.

Visceral leishmaniasis (VL) is a neglected tropical disease found in tropical and subtropical regions in the world. The therapeutics used for the treatment against disease presents problems, mainly related to drug toxicity, route of administration, high cost and/or by emergence of resistant strains. In this context, the search for alternative antileishmanial candidates is desirable. Recently, a naphthoquinone derivative namely 2-(2,3,4-tri-O-acetyl-6-deoxy-ß-L-galactopyranosyloxy)-1,4-naphthoquinone or Flau-A showed an effective in vitro biological action against Leishmania infantum. In the present study, the efficacy of this naphthoquinone derivative was evaluated in an in vivo infection model. BALB/c mice (n = 12 per group) were infected and later received saline or were treated with empty micelles (B/Mic), free Flau-A or it incorporated in Poloxamer 407-based micelles (Flau-A/Mic). The products were administered subcutaneously in the infected animals, which were then euthanized one (n = 6 per group) and 15 (n = 6 per group) days post-therapy, when immunological and parasitological evaluations were performed. Results showed that animals treated with Flau-A or Flau-A/Mic produced significantly higher levels of antileishmanial IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibody, when compared to data found in the control (saline and B/Mic) groups; which showed significantly higher levels of parasite-specific IL-4, IL-10 and IgG1 antibody. In addition, animals receiving free Flau-A or Flau-A/Mic presented also significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, when compared to the controls. A low hepatic and renal toxicity was also found. Overall, Flau-A/Mic showed better immunological and parasitological results, when compared to the use of free molecule. In conclusion, preliminary data suggest that this composition could be considered in future studies as promising therapeutic candidate against VL.

ChimLeish, a new recombinant chimeric protein evaluated as a diagnostic and prognostic marker for visceral leishmaniasis and human immunodeficiency virus coinfection.

Visceral leishmaniasis (VL) is a neglected tropical disease of global importance caused by parasites of the genus Leishmania, and coinfection with human immunodeficiency virus (HIV) is common in countries where both diseases are endemic. In particular, widely used immunological tests for VL diagnosis have impaired sensitivity (Se) and specificity (Sp) in VL/HIV coinfected patients and there is also cross-reactivity with other endemic diseases, e.g., Chagas disease, malaria, and tuberculosis. To develop new antigens to improve the diagnosis of VL and VL/HIV coinfection, we predicted eight specific B-cell epitopes of four Leishmania infantum antigens and constructed a recombinant polypeptide chimera antigen called ChimLeish. A serological panel of 195 serum samples was used to compare the diagnostic capabilities of ChimLeish alongside the individual synthetic peptides. ChimLeish reacted with sera from all VL and VL/HIV coinfected patients [Se = 100%; Sp = 100%; area under the curve (AUC) = 1.0]. Peptides showed lower reactivities (Se = 76.8 to 99.2%; Sp = 67.1 to 95.7%; AUC between 0.87 and 0.98) as did a L. infantum antigenic preparation used as an antigen control (Se = 56.8%; Sp = 69.5%: AUC = 0.45). Notably, ChimLeish demonstrated a significant reduction (p < 0.05) of anti-ChimLeish antibodies after treatment and cure of a small number of patients. Although only a limited serological panel was tested, preliminary data suggest that ChimLeish should be evaluated in larger sample studies for the diagnosis of VL and VL/HIV coinfection.

Acarbose presents in vitro and in vivo antileishmanial activity against Leishmania infantum and is a promising therapeutic candidate against visceral leishmaniasis.

Treatment against visceral leishmaniasis (VL) is mainly hampered by drug toxicity, long treatment regimens and/or high costs. Thus, the identification of novel and low-cost antileishmanial agents is urgent. Acarbose (ACA) is a specific inhibitor of glucosidase-like proteins, which has been used for treating diabetes. In the present study, we show that this molecule also presents in vitro and in vivo specific antileishmanial activity against Leishmania infantum. Results showed an in vitro direct action against L. infantum promastigotes and amastigotes, and low toxicity to mammalian cells. In addition, in vivo experiments performed using free ACA or incorporated in a Pluronic® F127-based polymeric micelle system called ACA/Mic proved effective for the treatment of L. infantum-infected BALB/c mice. Treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes when compared to the controls, as well as the development of antileishmanial Th1-type humoral and cellular responses based on high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and IgG2a isotype antibodies. In addition, ACA or ACA-treated animals suffered from low organ toxicity. Treatment with ACA/Mic outperformed treatments using either Miltefosine or free ACA based on parasitological and immunological evaluations performed one and 15 days post-therapy. In conclusion, data suggest that the ACA/Mic is a potential therapeutic agent against L. infantum and merits further consideration for VL treatment.

In vitro and in vivo antileishmanial activity of ß-acetyl-digitoxin, a cardenolide of Digitalis lanata potentially useful to treat visceral leishmaniasis.

Current treatments of visceral leishmaniasis face limitations due to drug side effects and/or high cost, along with the emergence of parasite resistance. Novel and low-cost antileishmanial agents are therefore required. We report herein the antileishmanial activity of ß-acetyl-digitoxin (b-AD), a cardenolide isolated from Digitalis lanata leaves, assayed in vitro and in vivo against Leishmania infantum. Results showed direct action of b-AD against parasites, as well as efficacy for the treatment of Leishmania-infected macrophages. In vivo experiments using b-AD-containing Pluronic® F127 polymeric micelles (b-AD/Mic) to treat L. infantum-infected mice showed that this composition reduced the parasite load in distinct organs in more significant levels. It also induced the development of anti-parasite Th1-type immunity, attested by high levels of IFN-γ, IL-12, TNF-α, GM-CSF, nitrite and specific IgG2a antibodies, in addition to low IL-4 and IL-10 contents, along with higher IFN-γ-producing CD4+ and CD8+ T-cell frequency. Furthermore, low toxicity was found in the organs of the treated animals. Comparing the therapeutic effect between the treatments, b-AD/Mic was the most effective in protecting animals against infection, when compared to the other groups including miltefosine used as a drug control. Data found 15 days after treatment were similar to those obtained one day post-therapy. In conclusion, the results obtained suggest that b-AD/Mic is a promising antileishmanial agent and deserves further studies to investigate its potential to treat visceral leishmaniasis.

TITLE: Activité antileishmaniale in vitro et in vivo de la ß-acétyl-digitoxine, un cardénolide de Digitalis lanata potentiellement utile pour traiter la leishmaniose viscérale. ABSTRACT: Les traitements actuels de la leishmaniose viscérale font face à des limitations dues aux effets secondaires des médicaments et/ou à leur coût élevé, ainsi qu'à l'émergence d'une résistance parasitaire. Des agents antileishmaniaux nouveaux et peu coûteux sont donc nécessaires. Nous rapportons ici l'activité antileishmaniale de la ß-acétyl-digitoxine (b-AD), un cardénolide isolé à partir de feuilles de Digitalis lanata, mesurée in vitro et in vivo contre Leishmania infantum. Les résultats ont montré une action directe de la b-AD contre les parasites, ainsi qu'une efficacité pour le traitement des macrophages infectés par Leishmania. Des expériences in vivo utilisant des micelles polymériques Pluronic® F127 contenant de la b-AD (b-AD/Mic) pour traiter des souris infectées par L. infantum ont montré que cette composition réduisait à des niveaux plus significatifs la charge parasitaire dans différents organes, ainsi que le développement d'une immunité antiparasitaire de type Th1, attestée par les taux élevés d'IFN-γ, d'IL-12, de TNF-α, de GM-CSF, de nitrite et d'anticorps IgG2a spécifiques, en plus des faibles taux d'IL-4 et IL-10, ainsi qu'une fréquence plus élevée des cellules T CD4+ and CD8+ productrices d' IFN-γ. De plus, une faible toxicité a été trouvée dans les organes des animaux traités. En comparant l'effet thérapeutique des traitements, b-AD/Mic était le plus efficace pour protéger les animaux contre l'infection, par rapport aux autres groupes comprenant la miltefosine utilisée comme contrôle médicamenteux. Les données trouvées 15 jours après le traitement étaient similaires à celles obtenues un jour après le traitement. En conclusion, les résultats obtenus suggèrent que b-AD/Mic est un agent antileishmanial prometteur et mérite des études supplémentaires pour étudier son potentiel à traiter la leishmaniose viscérale.

Leishmania eukaryotic elongation Factor-1 beta protein is immunogenic and induces parasitological protection in mice against Leishmania infantum infection.

Treatment for visceral leishmaniasis (VL) is hampered mainly by the toxicity and/or high cost of antileishmanial drugs. What is more, variability on sensitivity and/or specificity of diagnostic tests hinders effective disease management. In this context, prophylactic vaccination should be considered as a strategy to prevent disease. In the present study, immunogenicity of the Leishmania eukaryotic Elongation Factor-1 beta (EF1b) protein, classified as a Leishmania virulence factor, was evaluated in vitro and in vivo and tested, for the first time, as a vaccine candidate against Leishmania infantum infection. The antigen was administered as DNA vaccine or as recombinant protein (rEF1b) delivered in saponin. BALB/c mice immunization with a DNA plasmid and recombinant protein plus saponin induced development of specific Th1-type immunity, characterized by high levels of IFN-γ, IL-12, GM-CSF, both T cell subtypes and antileishmanial IgG2a isotype antibodies, before and after infection. This immunological response to the vaccines was corroborated further by parasitological analysis of the vaccinated and then challenged mice, which showed significant reductions in the parasite load in their liver, spleen, bone marrow and draining lymph nodes, when compared to the controls. Vaccination using rEF1b/saponin induced a more robust Th1 response and parasitological protection when compared to the DNA vaccine. Furthermore, in vitro analysis of lymphoproliferation, IFN-γ and IL-10 levels in human PBMC cultures showed as well development of a specific Th1-type response. In conclusion, data suggest that EF1b could be a promising vaccine candidate to protect against L. infantum infection.

Ivermectin presents effective and selective antileishmanial activity in vitro and in vivo against Leishmania infantum and is therapeutic against visceral leishmaniasis.

Treatment for visceral leishmaniasis (VL) is hindered mainly by the toxicity and/or high cost of therapeutic drugs. In addition, parasite resistance has been registered. Thus, there is an urgent need for the identification of novel, effective and low-cost antileishmanial agents. Since drug discovery is a long and expensive process, drug repositioning for treatment of leishmaniasis should be considered. In the present study, Ivermectin (IVE), a broad-spectrum drug used for treatment of parasitic diseases, was evaluated in vitro and in vivo against Leishmania infantum species. Results in vitro showed that IVE presented 50% Leishmania and macrophage inhibitory concentrations (IC50 and CC50, respectively) of 3.64 ± 0.48 µM and 427.50 ± 17.60 µM, respectively, with a selectivity index (SI) of 117.45; whereas Amphotericin B (AmpB), which was used as control, showed IC50 and CC50 values of 0.12 ± 0.05 µM and 1.06 ± 0.23 µM, respectively, with a corresponding SI of 8.90. Treatment with IVE effectively reduced the infection percentage and parasite burden in infected and treated macrophages and displayed a prophylactic activity by inhibiting macrophage infection with pre-treated parasites. Furthermore, preliminary studies suggested that IVE targets the parasite's mitochondria. Activity of IVE in its free format or incorporated into Pluronic® F127-based polymeric micelles (IVE/Mic) was also evaluated in vivo as a treating drug for L. infantum-infected BALB/c mice. Miltefosine was used as a control. Results showed that Miltefosine, IVE and IVE/Mic-treated animals presented significant reductions in the parasite load in their spleens, livers, bone marrows and draining lymph nodes, as well as development of an antileishmanial Th1-type immune response one and 15 days after treatment. Notably, IVE/Mic showed a better parasitological and immunological response in comparison to other alternative treatments. In conclusion, results suggest that IVE/Mic could be considered in future studies as a therapeutic alternative to treat VL.

Digitoxigenin presents an effective and selective antileishmanial action against Leishmania infantum and is a potential therapeutic agent for visceral leishmaniasis.

Treatment for visceral leishmaniasis (VL) is hampered mainly by drug toxicity, their high cost, and parasite resistance. Drug development is a long and pricey process, and therefore, drug repositioning may be an alternative worth pursuing. Cardenolides are used to treat cardiac diseases, especially those obtained from Digitalis species. In the present study, cardenolide digitoxigenin (DIGI) obtained from a methanolic extract of Digitalis lanata leaves was tested for its antileishmanial activity against Leishmania infantum species. Results showed that 50% Leishmania and murine macrophage inhibitory concentrations (IC50 and CC50, respectively) were of 6.9 ± 1.5 and 295.3 ± 14.5 µg/mL, respectively. With amphotericin B (AmpB) deoxycholate, used as a control drug, values of 0.13 ± 0.02 and 0.79 ± 0.12 µg/mL, respectively, were observed. Selectivity index (SI) values were of 42.8 and 6.1 for DIGI and AmpB, respectively. Preliminary studies suggested that the mechanism of action for DIGI is to cause alterations in the mitochondrial membrane potential, to increase the levels of reactive oxygen species and induce accumulation of lipid bodies in the parasites. DIGI was incorporated into Pluronic® F127-based polymeric micelles, and the formula (DIGI/Mic) was used to treat L. infantum-infected mice. Miltefosine was used as a control drug. Results showed that animals treated with either miltefosine, DIGI, or DIGI/Mic presented significant reductions in the parasite load in their spleens, livers, bone marrows, and draining lymph nodes, as well as the development of a specific Th1-type response, when compared with the controls. Results obtained 1 day after treatment were corroborated with data corresponding to 15 days after therapy. Importantly, treatment with DIGI/Mic induced better parasitological and immunological responses when compared with miltefosine- and DIGI-treated mice. In conclusion, DIGI/Mic has the potential to be used as a therapeutic agent to protect against L. infantum infection, and it is therefore worth of consideration in future studies addressing VL treatment.

Parasitological and immunological evaluation of a novel chemotherapeutic agent against visceral leishmaniasis.

AIMS: Treatment for visceral leishmaniasis (VL) is hampered by the toxicity and/or high cost of drugs, as well as by emergence of parasite resistance. Therefore, there is an urgent need for new antileishmanial agents. METHODS AND RESULTS: In this study, the antileishmanial activity of a diprenylated flavonoid called 5,7,3,4'-tetrahydroxy-6,8-diprenylisoflavone (CMt) was tested against Leishmania infantum and L amazonensis species. Results showed that CMt presented selectivity index (SI) of 70.0 and 165.0 against L infantum and L amazonensis promastigotes, respectively, and of 181.9 and 397.8 against respective axenic amastigotes. Amphotericin B (AmpB) showed lower SI values of 9.1 and 11.1 against L infantum and L amazonensis promastigotes, respectively, and of 12.5 and 14.3 against amastigotes, respectively. CMt was effective in the treatment of infected macrophages and caused alterations in the parasite mitochondria. L infantum-infected mice treated with miltefosine, CMt alone or incorporated in polymeric micelles (CMt/Mic) presented significant reductions in the parasite load in distinct organs, when compared to the control groups. An antileishmanial Th1-type cellular and humoral immune response were developed one and 15 days after treatment, with CMt/Mic-treated mice presenting a better protective response. CONCLUSION: Our data suggest that CMt/Mic could be evaluated as a chemotherapeutic agent against VL.

A Leishmania amastigote-specific hypothetical protein evaluated as recombinant protein plus Th1 adjuvant or DNA plasmid-based vaccine to protect against visceral leishmaniasis.

Most studies evaluating vaccine candidates against visceral leishmaniasis (VL) have used parasite promastigote-expressed antigens; however, Leishmania proteins expressed in the amastigote forms should be considered, since few hours after infection this stage comes into contact with the host immune system and is responsible for the development of the disease. In this context, in the present study, a Leishmania amastigote-specific hypothetical protein, called LiHyJ, was evaluated as a recombinant protein plus saponin as an adjuvant or DNA vaccine to protect against VL. The vaccine effect was evaluated by means of the evaluation of immunological and parasitological analyses performed in BALB/c mice against Leishmania infantum infection. Results showed that rLiHyJ/saponin and DNA LiHyJ induced significantly higher levels of anti-protein and anti-parasite IFN-γ, IL-12, GM-CSF, and IgG2a isotype antibodies, which were associated with a low presence of IL-4 and IL-10. DNA vaccination induced higher IFN-γ production, mainly by CD8+ T cells, while rLiHyJ/saponin stimulated the production of this cytokine, mainly by CD4+ T cells. The parasite load evaluated in distinct organs showed that both immunization schedules significantly reduced organic parasitism, when compared to the controls. Similar results were found in the immunological and parasitological assays when using the recombinant protein or DNA, although the vaccination with rLiHyJ plus saponin induced a slightly higher Th1 response and lower parasite load, when compared to the use of DNA plasmid. The protein also proved to be immunogenic when peripheral blood mononuclear cells of treated VL patients and healthy subjects were in vitro stimulated, since higher IFN-γ and lower IL-4 and IL-10 levels were found in the culture supernatants. In conclusion, LiHyJ should be considered in future studies as a vaccine candidate to protect against VL.

A candidate vaccine for human visceral leishmaniasis based on a specific T cell epitope-containing chimeric protein protects mice against Leishmania infantum infection.

Leishmaniases are neglected diseases caused by infection with Leishmania parasites and there are currently no prophylactic vaccines. In this study, we designed in silico a synthetic recombinant vaccine against visceral leishmaniasis (VL) called ChimeraT, which contains specific T-cell epitopes from Leishmania Prohibitin, Eukaryotic Initiation Factor 5a and the hypothetical LiHyp1 and LiHyp2 proteins. Subcutaneous delivery of ChimeraT plus saponin stimulated a Th1 cell-mediated immune response and protected mice against L. infantum infection, significantly reducing the parasite load in distinct organs. ChimeraT/saponin vaccine stimulated significantly higher levels of IFN-γ, IL-12, and GM-CSF cytokines by both murine CD4+ and CD8+ T cells, with correspondingly low levels of IL-4 and IL-10. Induced antibodies were predominantly IgG2a isotype and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide. ChimeraT also induced lymphoproliferative responses in peripheral blood mononuclear cells from VL patients after treatment and healthy subjects, as well as higher IFN-γ and lower IL-10 secretion into cell supernatants. Thus, ChimeraT associated with a Th1 adjuvant could be considered as a potential vaccine candidate to protect against human disease.

Leishmania infantum pyridoxal kinase evaluated in a recombinant protein and DNA vaccine to protects against visceral leishmaniasis.

Leishmania infantum pyridoxal kinase (PK) protein was characterized after an immunoproteomics screening performed with the sera from patients suffering visceral leishmaniasis (VL). Since it was recognized by sera of mammalian hosts infected by a viscerotropic Leishmania species, PK could emerge as a new vaccine candidate against disease, due to its antigenicity and immunogenicity. In this context, in the present study, the effects of the immunization using PK were evaluated when administered as a DNA plasmid (pDNAA3/PK) or recombinant protein (rPK) plus saponin. The immune response elicited by both vaccination regimens reduced in significant levels the parasite load in spleen, liver, draining lymph nodes and bone marrow, being associated with the development of Th1-type immune response, which was characterized by high levels of IFN-γ, IL-12, GM-CSF, and specific IgG2a antibody, besides low production of IL-4, IL-10, and protein and parasite-specific IgG1 antibodies. CD8+ T cells were more important in the IFN-γ production in the pDNAA3/PK group, while CD4+ T cells contributed more significantly to production of this cytokine in the rPK/Saponin group. In addition, increased IFN-γ secretion, along with low levels of IL-10, were found when PBMCs from VL patients after treatment and healthy individuals were stimulated with the protein. In conclusion, when administered either as a DNA plasmid or recombinant protein plus adjuvant, PK can direct the immune response towards a Th1-type immune profile, protecting mice against L. infantum challenge; therefore, it can be seen as a promising immunogen against human VL.

Evaluation of the protective efficacy of a Leishmania protein associated with distinct adjuvants against visceral leishmaniasis and in vitro immunogenicity in human cells.

The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a prophylactic vaccination is urgently required. In the present study, a Leishmania protein called LiHyE, which was suggested recently as an antigenic marker for canine and human VL, was evaluated regarding its immunogenicity and protective efficacy in BALB/c mice against Leishmania infantum infection. In addition, the protein was used to stimulate peripheral blood mononuclear cells (PBMCs) from VL patients before and after treatment, as well as from healthy subjects. Vaccination results showed that the recombinant (rLiHyE) protein associated with liposome or saponin induced effective protection in the mice, since significant reductions in the parasite load in spleen, liver, draining lymph nodes, and bone marrow were found. The parasitological protection was associated with Th1-type cell response, since high IFN-γ, IL-12, and GM-CSF levels, in addition to low IL-4 and IL-10 production, were found. Liposome induced a better parasitological and immunological protection than did saponin. Experiments using PBMCs showed rLiHyE-stimulated lymphoproliferation in treated patients' and healthy subjects' cells, as well as high IFN-γ levels in the cell supernatant. In conclusion, rLiHyE could be considered for future studies as a vaccine candidate against VL.

Liposomal Formulation of ChimeraT, a Multiple T-Cell Epitope-Containing Recombinant Protein, Is a Candidate Vaccine for Human Visceral Leishmaniasis.

Background: Leishmaniases are neglected diseases caused by infection with Leishmania parasites and there are no human vaccines in use routinely. The purpose of this study was to examine the immunogenicity of ChimeraT, a novel synthetic recombinant vaccine against visceral leishmaniasis (VL), incorporated into a human-compatible liposome formulation. Methods: BALB/c mice were immunized subcutaneously with ChimeraT/liposome vaccine, ChimeraT/saponin adjuvant, or ChimeraT/saline and immune responses examined in vitro and in vivo. Results: Immunization with the ChimeraT/liposome formulation induced a polarized Th1-type response and significant protection against L. infantum infection. ChimeraT/liposome vaccine stimulated significantly high levels of interferon (IFN)-γ, interleukin (IL)-12, and granulocyte macrophage-colony stimulating factor (GM-CSF) cytokines by both CD4 and CD8 T-cells, with correspondingly lower levels of IL-4 and IL-10 cytokines. Induced antibodies were predominantly IgG2a isotype, and homologous antigen-stimulated spleen cells produced significant nitrite as a proxy for nitric oxide (NO). Furthermore, we examined a small number of treated VL patients and found higher levels of circulating anti-ChimeraT protein IgG2 antibodies, compared to IgG1 levels. Conclusions: Overall, the liposomal formulation of ChimeraT induced a protective Th1-type immune response and thus could be considered in future studies as a vaccine candidate against human VL.

A clioquinol-containing Pluronic® F127 polymeric micelle system is effective in the treatment of visceral leishmaniasis in a murine model.

A clioquinol (ICHQ)-containing Pluronic® F127 polymeric micelle system (ICHQ/Mic) was recently shown to be effective against Leishmania amazonensis infection in a murine model. In the present study, ICHQ/Mic was tested against L. infantum infection. BALB/c mice (n = 12 per group) were infected with L. infantum stationary promastigotes through subcutaneous injection and, 45 days after challenge, received saline or were treated via the subcutaneous route with empty micelles, ICHQ or ICHQ/Mic. In addition, animals were treated with miltefosine by the oral route, as a drug control. Half of the animals were euthanized 1 and 15 days after treatment, aiming to evaluate two endpoints after therapy, when parasitological and immunological parameters were investigated. Results showed that the treatment using miltefosine, ICHQ or ICHQ/Mic induced significantly higher anti-parasite IFN-γ, IL-12, GM-CSF, nitrite and IgG2a isotype antibody levels, which were associated with low IL-4 and IL-10 production. In addition, a higher frequency of IFN-γ and TNF-α-producing CD4+ and CD8+ T-cells was found in these animals. The parasite load was evaluated in distinct organs, and results showed that the treatment using miltefosine, ICHQ or ICHQ/Mic induced significant reductions in organic parasitism in the treated and infected mice. A comparison between the treatments suggested that ICHQ/Mic was the most effective in inducing a highly polarized Th1-type response, as well as reducing the parasite load in significant levels in the treated and infected animals. Data obtained 15 days after treatment suggested maintenance of the immunological and parasitological responses. In conclusion, ICHQ/Mic could be considered in future studies for the treatment of visceral leishmaniasis.

TITLE: Un système à micelles polymériques Pluronic® F127 contenant du clioquinol est efficace pour le traitement de la leishmaniose viscérale dans un modèle murin. ABSTRACT: Un système à micelles polymériques Pluronic® F127 (ICHQ/Mic) contenant du clioquinol (ICHQ) s'est récemment révélé efficace contre l'infection à Leishmania amazonensis dans un modèle murin. Dans la présente étude, l'ICHQ/Mic a été testé contre l'infection à L. infantum. Les souris BALB/c (n = 12 par groupe) ont été infectées par des promastigotes stationnaires de L. infantum par injection sous-cutanée et ont reçu 45 jours après l'épreuve une solution saline ou ont été traitées par voie sous-cutanée avec des micelles vides, ICHQ ou ICHQ/Mic. De plus, les animaux ont été traités avec de la miltefosine par voie orale, comme contrôle médicamenteux. La moitié des animaux ont été euthanasiés 1 et 15 jours après le traitement, dans le but de mesurer deux critères d'évaluation après la thérapie, lorsque les paramètres parasitologiques et immunologiques ont été étudiés. Les résultats ont montré que le traitement par miltefosine, ICHQ ou ICHQ/Mic induisait des niveaux d'anticorps anti-parasite IFN-γ, IL-12, GM-CSF, nitrite et IgG2a significativement plus élevés, associés à de faibles productions d'IL-4 et IL-10. De plus, une fréquence plus élevée de cellules T CD4+ et CD8+ produisant de l'IFN-γ and TNF-α a été trouvée chez ces animaux. La charge parasitaire a été évaluée dans des organes distincts et les résultats ont montré que le traitement utilisant la miltefosine, ICHQ ou ICHQ/Mic induisait des réductions significatives du parasitisme des organes chez les souris traitées et infectées. Une comparaison entre les traitements a suggéré qu'ICHQ/Mic était le plus efficace pour induire une réponse de type Th1 polarisée, ainsi que pour réduire la charge parasitaire à des niveaux significatifs chez les animaux traités et infectés. Les données obtenues 15 jours après le traitement suggèrent le maintien des réponses immunologiques et parasitologiques. En conclusion, ICHQ/Mic pourrait être envisagé dans de futures études pour le traitement contre la leishmaniose viscérale.

Leishmania infantum amastin protein incorporated in distinct adjuvant systems induces protection against visceral leishmaniasis.

The control measures against visceral leishmaniasis (VL) include a precise diagnosis of disease, the treatment of human cases, and reservoir and vector controls. However, these are insufficient to avoid the spread of the disease in specific countries worldwide. As a consequence, prophylactic vaccination could be interesting, although no effective candidate against human disease is available. In the present study, the Leishmania infantum amastin protein was evaluated regarding its immunogenicity and protective efficacy against experimental VL. BALB/c mice immunized with subcutaneous injections of the recombinant protein with or without liposome/saponin (Lip/Sap) as an adjuvant. After immunization, half of the animals per group were euthanized and immunological evaluations were performed, while the others were challenged with L. infantum promastigotes. Forty-five days after infection, the animals were euthanized and parasitological and immunological evaluations were performed. Results showed the development of a Th1-type immune response in rAmastin-Lip and rAmastin-Sap/vaccinated mice, before and after infection, which was based on the production of protein and parasite-specific IFN-γ, IL-12, GM-CSF, and nitrite, as well as the IgG2a isotype antibody. CD4+ T cells were mainly responsible for IFN-γ production in vaccinated mice, which also presented significant reductions in parasitism in their liver, spleen, draining lymph nodes, and bone marrow. In addition, PBMC cultures of treated VL patients and healthy subjects stimulated with rAmastin showed lymphoproliferation and higher IFN-γ production. In conclusion, the present study shows the first case of an L. infantum amastin protein associated with distinct delivery systems inducing protection against L. infantum infection and demonstrates an immunogenic effect of this protein in human cells.

A chloroquinoline derivate presents effective in vitro and in vivo antileishmanial activity against Leishmania species that cause tegumentary and visceral leishmaniasis.

The identification of new therapeutics to treat leishmaniasis is desirable, since available drugs are toxic and present high cost and/or poor availability. Therefore, the discovery of safer, more effective and selective pharmaceutical options is of utmost importance. Efforts towards the development of new candidates based on molecule analogs with known biological functions have been an interesting and cost-effective strategy. In this context, quinoline derivatives have proven to be effective biological activities against distinct diseases. In the present study, a new chloroquinoline derivate, AM1009, was in vitro tested against two Leishmania species that cause leishmaniasis. The present study analyzed the necessary inhibitory concentration to preclude 50% of the Leishmania promastigotes and axenic amastigotes (EC50 value), as well as the inhibitory concentrations to preclude 50% of the murine macrophages and human red blood cells (CC50 and RBC50 values, respectively). In addition, the treatment of infected macrophages and the inhibition of infection using pre-treated parasites were also investigated, as was the mechanism of action of the molecule in L. amazonensis. To investigate the in vivo therapeutic effect, BALB/c mice were infected with L. amazonensis and later treated with AM1009. Parasitological and immunological parameters were also evaluated. Clioquinol, a known antileishmanial quinoline derivate, and amphotericin B (AmpB), were used as molecule and drug controls, respectively. Results in both in vitro and in vivo experiments showed a better and more selective action of AM1009 to kill the in vitro parasites, as well as in treating infected mice, when compared to results obtained using clioquinol or AmpB. AM1009-treated animals presented significantly lower average lesion diameter and parasite burden in the infected tissue and organs evaluated in this study, as well as a more polarized antileishmanial Th1 immune response and low renal and hepatic toxicity. This result suggests that AM1009 should be considered a possible therapeutic target to be evaluated in future studies for treatment against leishmaniasis.

Evaluation of the in vitro and in vivo antileishmanial activity of a chloroquinolin derivative against Leishmania species capable of causing tegumentary and visceral leishmaniasis.

The treatment against leishmaniasis presents problems, since the currently used drugs are toxic and/or have high costs. In addition, parasite resistance has increased. As a consequence, in this study, a chloroquinolin derivative, namely 7-chloro-N,N-dimethylquinolin-4-amine or GF1059, was in vitro and in vivo tested against Leishmania parasites. Experiments were performed to evaluate in vitro antileishmanial activity and cytotoxicity, as well as the treatment of infected macrophages and the inhibition of infection using pre-treated parasites. This study also investigated the GF1059 mechanism of action in L. amazonensis. Results showed that the compound was highly effective against L. infantum and L. amazonensis, presenting a selectivity index of 154.6 and 86.4, respectively, against promastigotes and of 137.6 and 74.3, respectively, against amastigotes. GF1059 was also effective in the treatment of infected macrophages and inhibited the infection of these cells when parasites were pre-incubated with it. The molecule also induced changes in the parasites' mitochondrial membrane potential and cell integrity, and caused an increase in the reactive oxygen species production in L. amazonensis. Experiments performed in BALB/c mice, which had been previously infected with L. amazonensis promastigotes, and thus treated with GF1059, showed that these animals presented significant reductions in the parasite load when the infected tissue, spleen, liver, and draining lymph node were evaluated. GF1059-treated mice presented both lower parasitism and low levels of enzymatic markers, as compared to those receiving amphotericin B, which was used as control. In conclusion, data suggested that GF1059 can be considered a possible therapeutic target to be tested against leishmaniasis.

In vivo antileishmanial efficacy of a naphthoquinone derivate incorporated into a Pluronic® F127-based polymeric micelle system against Leishmania amazonensis infection.

New therapeutic strategies against leishmaniasis are desirable, since the treatment against disease presents problems, such as the toxicity, high cost and/or parasite resistance. As consequence, new antileishmanial compounds are necessary to be identified, as presenting high activity against Leishmania parasites, but low toxicity in mammalian hosts. Flau-A is a naphthoquinone derivative recently showed to presents an in vitro effective action against Leishmania amazonensis and L. infantum species. In the present work, the in vivo efficacy of Flau-A, which was incorporated into a Poloxamer 407-based micelle system, was evaluated in a murine model against L. amazonensis infection. Amphotericin B (AmB) and Ambisome® were used as controls. The animals were infected and later treated with the compounds. Thirty days after the treatment, parasitological and immunological parameters were evaluated. Results showed that AmB, Ambisome®, Flau-A or Flau-A/M-treated animals presented significantly lower average lesion diameter and parasite burden in tissue and organs evaluated, when compared to the control (saline and micelle) groups. Flau-A or Flau-A/M-treated mice were those presenting the most significant reductions in the parasite burden, when compared to the others. These animals developed also a more polarized antileishmanial Th1 immune response, which was based on significantly higher levels of IFN-γ, IL-12, TNF-α, GM-CSF, and parasite-specific IgG2a isotype; associated with low levels of IL-4, IL-10, and IgG1 antibody. The absence of toxicity was found in these animals, although mice receiving AmB have showed high levels of renal and hepatic damage markers. In conclusion, results suggested that the Flau-A/M compound may be considered as a possible therapeutic target to be evaluated against human leishmaniasis.