RESUMEN
Treatment against leishmaniasis presents problems, mainly due to the toxicity of the drugs, high cost, and the emergence of resistant strains. A previous study showed that two vanillin-derived synthetic molecules, 3s [4-(2-hydroxy-3-(4-octyl-1H-1,2,3-triazol-1-yl)propoxy)-3-methoxybenzaldehyde] and 3t [4-(3-(4-decyl-1H-1,2,3-triazol-1-yl)-2-hydroxypropoxy)-3-methoxybenzaldehyde], presented antileishmanial activity against Leishmania infantum, L. amazonensis, and L. braziliensis species. In the present work, 3s and 3t were evaluated to treat L. amazonensis-infected mice. Molecules were used pure or incorporated into Poloxamer 407-based micelles. In addition, amphotericin B (AmpB) and its liposomal formulation, Ambisome®, were used as control. Animals received the treatment and, one and 30 days after, they were euthanized to evaluate immunological, parasitological, and biochemical parameters. Results showed that the micellar compositions (3s/Mic and 3t/Mic) induced significant reductions in the lesion mean diameter and parasite load in the infected tissue and distinct organs, as well as a specific and significant antileishmanial Th1-type immune response, which was based on significantly higher levels of IFN-γ, IL-12, nitrite, and IgG2a isotype antibodies. Drug controls showed also antileishmanial action; although 3s/Mic and 3t/Mic have presented better and more significant parasitological and immunological data, which were based on significantly higher IFN-γ production and lower parasite burden in treated animals. In addition, significantly lower levels of urea, creatinine, alanine transaminase, and aspartate transaminase were found in mice treated with 3s/Mic and 3t/Mic, when compared to the others. In conclusion, results suggest that 3s/Mic and 3t/Mic could be considered as therapeutic candidates to treat against L. amazonensis infection.
Asunto(s)
Antiprotozoarios , Benzaldehídos , Leishmania mexicana , Ratones Endogámicos BALB C , Micelas , Animales , Ratones , Benzaldehídos/farmacología , Benzaldehídos/química , Leishmania mexicana/efectos de los fármacos , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Antiprotozoarios/química , Leishmaniasis Cutánea/tratamiento farmacológico , Femenino , Anfotericina B/farmacología , Anfotericina B/uso terapéutico , Poloxámero/química , Poloxámero/farmacología , Masculino , Bazo/parasitologíaRESUMEN
Leishmania virulence proteins should be considered as vaccine candidates against disease, since they are involved in developing infection in mammalian hosts. In a previous study, a Leishmania guanosine-5'-triphosphate (GTP)-binding protein was identified as a potential parasite virulence factor. In the present work, the gene encoding GTP was cloned and the recombinant protein (rGTP) was evaluated as a vaccine candidate against Leishmania infantum infection. The protein was associated with saponin (rGTP/Sap) or Poloxamer 407-based micelles (rGTP/Mic) as adjuvants, and protective efficacy was investigated in BALB/c mice after parasite challenge. Both rGTP/Sap and rGTP/Mic compositions induced a Th1-type immune response in vaccinated animals, with significantly higher levels of IFN-γ, IL-12, IL-2, TNF-α, GM-CSF, nitrite, specific IgG2a isotype antibody and positive lymphoproliferation, when compared to the control groups. This response was accompanied by significantly lower parasite load in the spleens, livers, bone marrows and draining lymph nodes of the animals. Immunological and parasitological evaluations indicated that rGTP/Mic induced a more polarized Th1-type response and higher reduction in the organ parasitism, and with lower hepatotoxicity, when compared to the use of rGTP/Sap. In conclusion, our preliminary data suggest that rGTP could be considered for further development as a vaccine candidate to protect against VL.
Asunto(s)
Leishmania infantum , Leishmaniasis Visceral , Leishmaniasis , Animales , Antígenos de Protozoos , Proteínas Portadoras , Guanosina , Guanosina Trifosfato , Mamíferos , Ratones , Ratones Endogámicos BALB C , Micelas , Poloxámero , Polifosfatos , Proteínas RecombinantesRESUMEN
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.
Asunto(s)
Antiprotozoarios/uso terapéutico , Digitoxigenina/uso terapéutico , Reposicionamiento de Medicamentos/métodos , Leishmania infantum/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Poloxámero/uso terapéutico , Anfotericina B/uso terapéutico , Animales , Ácido Desoxicólico/uso terapéutico , Combinación de Medicamentos , Femenino , Hígado/parasitología , Macrófagos/efectos de los fármacos , Macrófagos/parasitología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Micelas , Carga de Parásitos , Especies Reactivas de Oxígeno , Bazo/parasitologíaRESUMEN
Leishmania proteins have been evaluated as vaccine candidates against leishmaniasis; however, most antigens present low immunogenicity and need to be added with immune adjuvants. A low number of licensed adjuvants exist on the market today; therefore, research conducted to produce new products is desirable. The present study sought to evaluate the immunogenicity and protective efficacy of a recombinant Leishmania hypothetical protein, namely LiHyR, administered with saponin or liposomes in BALB/c mice. Immunological and parasitological parameters were evaluated, and results showed significant protection against Leishmania infantum infection produced by both compositions in the immunized animals; however, this was not identified when the antigen was used alone. In addition, the liposomal formulation was more effective in inducing a polarized Th1 response in the vaccinated animals, which was maintained after challenge and reflected by lower parasitism found in all evaluated organs when the limiting dilution technique and RT-PCR assay were employed. The protected animals showed higher levels of protein and parasite-specific IFN-γ IL-2, IL-12, GM-CSF, and TNF-α, which were evaluated by capture ELISA and flow cytometry, in addition to a higher production of anti-protein and anti-parasite IgG2a antibodies, both before and after challenge. The Lip/rLiHyR combination induced higher IFN-γ production through both CD4+ and CD8+ T cell subtypes. Results indicate the possibility of using the LiHyR, containing a liposomal formulation, as a vaccine candidate against visceral leishmaniasis.
Asunto(s)
Citocinas/inmunología , Inmunogenicidad Vacunal , Leishmania infantum/inmunología , Vacunas contra la Leishmaniasis/farmacología , Leishmaniasis Visceral/prevención & control , Proteínas Protozoarias/farmacología , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Vacunas contra la Leishmaniasis/inmunología , Leishmaniasis Visceral/inmunología , Leishmaniasis Visceral/patología , Liposomas , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/inmunologíaRESUMEN
In the present study, a Poloxamer 407-based amphotericin B (AmpB)-containing polymeric micelles system (AmpB/M) was employed in the treatment of Leishmania amazonensis-infected BALB/c mice. Initially, the in vitro antileishmanial activity (IC50 value) of AmpB/M and B-AmpB/M (empty micelles) against stationary promastigotes and amastigotes-like forms of the parasites was determined, and results were of 1.83 ± 0.4 and 22.1 ± 0.7 µM, respectively, for the promastigotes, and of 2.27 ± 0.5 and 33.98 ± 2.6 µM, respectively, for the amastigotes-like. The cytotoxic concentration (CC50) values of these products were also evaluated, and we found the results of 119.5 ± 9.6 and 134.7 ± 10.3 µM, respectively. With these values, the selectivity index (SI) was calculated and results were of 65.3 and 5.4, respectively, for the promastigotes, and of 59.3 and 3.96, respectively, for the amastigotes-like of the parasites. Free AmpB showed IC50 values of 1.2 ± 0.3 and 2.5 ± 0.5 µM for the promastigotes and amastigotes-like, respectively, whereas the CC50 value was of 9.5 ± 0.4 µM. The SI values of this drug were of 7.9 and 3.8, respectively, for the promastigote and amastigote-like stages of the parasites. After, animals were infected and received saline or were treated subcutaneously with free AmpB, AmpB/M or B-AmpB/M. In the results, free AmpB-treated and infected mice showed reductions in their body weight, which were associated with hepatic and renal damage; however, no organic alteration was observed in the AmpB/M-treated animals. In addition, these animals showed significant reductions in their lesion average size and in the parasite burden in all evaluated infected tissue and organs, when compared to the other groups; as well as significantly higher levels of antileishmanial IFN-γ, IL-12, GM-CSF and nitrite, which were associated with low production of IL-4, IL-10 and IgG1 isotype antibodies. In conclusion, this AmpB/M system could be considered as an alternative for future studies in the treatment of tegumentary leishmaniasis.
Asunto(s)
Anfotericina B/administración & dosificación , Antiprotozoarios/administración & dosificación , Excipientes , Leishmania mexicana/efectos de los fármacos , Leishmaniasis Cutánea/tratamiento farmacológico , Poloxámero , Anfotericina B/farmacología , Anfotericina B/uso terapéutico , Anfotericina B/toxicidad , Animales , Antiprotozoarios/farmacología , Antiprotozoarios/uso terapéutico , Antiprotozoarios/toxicidad , Supervivencia Celular/efectos de los fármacos , Citocinas/biosíntesis , Relación Dosis-Respuesta a Droga , Femenino , Inmunoglobulina G/biosíntesis , Concentración 50 Inhibidora , Leishmania mexicana/crecimiento & desarrollo , Leishmania mexicana/inmunología , Leishmaniasis Cutánea/inmunología , Leishmaniasis Cutánea/parasitología , Leishmaniasis Cutánea/patología , Hígado/parasitología , Ganglios Linfáticos/parasitología , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Micelas , Polímeros , Ratas , Bazo/citología , Bazo/inmunología , Bazo/parasitologíaRESUMEN
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.
Asunto(s)
Clioquinol/uso terapéutico , Leishmaniasis Visceral/tratamiento farmacológico , Micelas , Poloxámero/química , Animales , Anticuerpos Antiprotozoarios/sangre , Clioquinol/química , Citocinas/inmunología , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Femenino , Leishmania infantum , Ratones , Ratones Endogámicos BALB C , Carga de Parásitos , Poloxámero/uso terapéutico , Células TH1/inmunologíaRESUMEN
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.
Asunto(s)
Antiprotozoarios/uso terapéutico , Leishmania/efectos de los fármacos , Leishmaniasis/tratamiento farmacológico , Micelas , Naftoquinonas/uso terapéutico , Poloxámero/uso terapéutico , Animales , Antiprotozoarios/química , Antiprotozoarios/farmacocinética , Excipientes/química , Excipientes/farmacocinética , Excipientes/uso terapéutico , Femenino , Leishmania/metabolismo , Leishmaniasis/metabolismo , Ratones , Ratones Endogámicos BALB C , Naftoquinonas/química , Naftoquinonas/farmacocinética , Poloxámero/química , Poloxámero/farmacocinética , Resultado del TratamientoRESUMEN
Clioquinol (5-chloro-7-iodoquinolin-8-ol or ICHQ) was recently showed to presents an in vitro effective antileishmanial action, causing changes in membrane permeability, mitochondrial functionality, and parasite morphology. In the present study, ICHQ was incorporated into a Poloxamer 407-based polymeric micelles system (ICHQ/M), and its antileishmanial activity was in vivo evaluated in L. amazonensis-infected BALB/c mice. Amphotericin B (AmpB) and its liposomal formulation (Ambisome®) were used as controls. Parasitological and immunological evaluations were performed 30â¯days after the treatment. Results indicated more significant reductions in the average lesion diameter and parasite burden in ICHQ or ICHQ/M-treated mice, which were associated with the development of a polarized Th1 immune response, based on production of high levels of IFN-γ, IL-12, TNF-α, GM-CSF, and antileishmanial IgG2a antibody. Control groups´ mice produced high levels of IL-4, IL-10, and IgG1 isotype antibody. No organic toxicity was found by using ICHQ or ICHQ/M to treat the animals, although those receiving AmpB and Ambisome® have presented higher levels of renal and hepatic damage markers. In conclusion, results suggested that the ICHQ/M composition can be considered as an antileishmanial candidate to be tested against human leishmaniasis.
Asunto(s)
Antiprotozoarios/inmunología , Antiprotozoarios/uso terapéutico , Clioquinol/inmunología , Clioquinol/uso terapéutico , Leishmania mexicana/efectos de los fármacos , Leishmaniasis Visceral/tratamiento farmacológico , Poloxámero/administración & dosificación , Anfotericina B/administración & dosificación , Anfotericina B/uso terapéutico , Anfotericina B/toxicidad , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/inmunología , Antígenos de Protozoos/uso terapéutico , Antiprotozoarios/administración & dosificación , Antiprotozoarios/toxicidad , Clioquinol/administración & dosificación , Citocinas/biosíntesis , Citocinas/inmunología , Sistemas de Liberación de Medicamentos/métodos , Humanos , Inmunoglobulina G/sangre , Interferón gamma/biosíntesis , Interferón gamma/inmunología , Leishmania mexicana/crecimiento & desarrollo , Leishmaniasis Visceral/inmunología , Ratones , Ratones Endogámicos BALB C , Micelas , Carga de Parásitos , Poloxámero/química , Células TH1RESUMEN
Human tegumentary leishmaniasis (HTL), characterized by skin ulcers that may spread and cause dreadful and massive tissue destruction of the nose and mouth, is considered a neglected tropical disease, and it is a serious threat to global health due to its continuous expansion, favored by the lifecycle of its causative organism that is maintained in domestic animal reservoirs and anthropophilic sand fly species. Serodiagnosis of HTL is a great challenge due to many biological factors, including hampered specificity and/or sensitivity. This investigation addresses the unmet need for new diagnostic markers of HTL, and describes a simple platform to improve the serodiagnosis. A constrained conformational phage display random peptide library combined with a magnetic microsphere-based subtraction strategy was used to identify ligands with potential diagnostic applications. Six clones were selected against IgG antibodies from HTL patients, characterized by sequencing and confirmed by a phage-ELISA using sera from patients developing visceral leishmaniasis (n=20), Chagas disease (n=10), mucosal (n=30) and cutaneous (n=20) leishmaniasis; as well as from healthy subjects living in endemic (n=20) and non-endemic (n=30) areas of leishmaniasis. A wild-type M13-phage clone and a soluble Leishmania antigenic extract were used as negative and positive controls, respectively. Three clones reached 100% sensitivity and specificity, without any cross-reactivity with sera from patients with leishmaniasis-related diseases. Briefly, we describe for the first time a set of serological markers based on three immunodominant mimotopes that showed 100% accuracy, and that could be used in a phage-ELISA assay for the HTL serodiagnosis.