Antimonial drugs entrapped into phosphatidylserine liposomes: physicochemical evaluation and antileishmanial activity

Abstract: INTRODUCTION: Leishmaniasis is a disease caused by the protozoan Leishmania that resides mainly in mononuclear phagocytic system tissues. Pentavalent antimonials are the main treatment option, although these drugs have toxic side effects and high resistance rates. A potentially alternative and more effective therapeutic strategy is to use liposomes as carriers of the antileishmanial agents. The aims of this study were to develop antimonial drugs entrapped into phosphatidylserine liposomes and to analyze their biological and physicochemical characteristics. METHODS: Liposomes containing meglumine antimoniate (MA) or pentavalent antimony salt (Sb) were obtained through filter extrusion (FEL) and characterized by transmission electron microscopy. Promastigotes of Leishmania infantum were incubated with the drugs and the viability was determined with a tetrazolium dye (MTT assay). The effects of these drugs against intracellular amastigotes were also evaluated by optical microscopy, and mammalian cytotoxicity was determined by an MTT assay. RESULTS: Liposomes had an average diameter of 162nm. MA-FEL showed inhibitory activity against intracellular L. infantum amastigotes, with a 50% inhibitory concentration (IC50) of 0.9μg/mL, whereas that of MA was 60μg/mL. Sb-FEL showed an IC50 value of 0.2μg/mL, whereas that of free Sb was 9μg/mL. MA-FEL and Sb-FEL had strong in vitro activity that was 63-fold and 39-fold more effective than their respective free drugs. MA-FEL tested at a ten-times higher concentration than Sb-FEL did not show cytotoxicity to mammalian cells, resulting in a higher selectivity index. CONCLUSIONS: Antimonial drug-containing liposomes are more effective against Leishmania-infected macrophages than the non-liposomal drugs.

Expresión diferencial de proteínas en Leishmania (Viannia) panamensis asociadas con mecanismos de resistencia a antimoniato de meglumina / Differential expression of proteins in Leishmania (Viannia) panamensis associated with mechanisms of resistance to meglumine antimoniate.

Introducción. Los mecanismos de resistencia al antimonio pentavalente conocidos hasta el momento, se han descrito ampliamente en cepas del subgénero Leishmania, pero poco se sabe sobre las proteínas involucradas en los mecanismos de resistencia presentes en cepas del subgénero Viannia, como Leishmania panamensis. Objetivo. Identificar proteínas diferencialmente expresadas entre las cepas de L. panamensis (UA140), sensible y resistente al antimonio pentavalente, y analizar el posible papel de estas proteínas en mecanismos de resistencia. Materiales y métodos. Las proteínas de las cepas, sensible y resistente al antimonio pentavalente, se compararon usando electroforesis bidimensional. Las proteínas con aumento de la expresión fueron aisladas e identificadas por espectrometría de masas mediante MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). La expresión del ARNm de cinco de estas proteínas se cuantificó mediante PCR en tiempo real. Resultados. Los geles bidimensionales de las cepas sensible y resistente detectaron 532±39 y 541±43 manchas proteicas. Se encontraron 10 manchas con aumento de la expresión en la cepa resistente, identificadas como proteínas de choque térmico (Hsp60 mitocondrial, Hsp70 mitocondrial y citosólica), isomerasa de disulfuro, proteasa de cisteína, enolasa, factor de elongación 5-α, la subunidad 5-α del proteasoma y dos proteínas hipotéticas nombradas como Sp(2) y Sp(25). Conclusión. Este es el primer estudio llevado a cabo con una cepa resistente al antimonio pentavalente en L. panamensis, en el cual se han identificado proteínas que están relacionadas con el mecanismo de resistencia del parásito frente al medicamento, abriendo el camino para futuros estudios de estas proteínas como blancos terapéuticos.

Introduction. The well-known drug resistance mechanisms to pentavalent antimony have been widely described in strains of the Leishmania subgenus, but little is known about the mechanisms of resistance and the proteins associated with it in strains of the Viannia subgenus such as Leishmania panamensis. Objective. Differentially expressed proteins were identified between pentavalent antimonial sensitive and resistant L. panamensis (UA140) strains, and the role of these proteins was analyzed as possible resistance mechanisms. Materials and methods. The protein lysates of pentavalent antimony sensitive and resistant strains were separated by two-dimensional gel electrophoresis,and the protein patterns compared. The proteins identified as overexpressed were separated and analyzed using MALDI-TOF/TOF (Matrix Assisted Laser Desorption Ionization/Time of Flight). The level of mRNA expression of five of these proteins was quantified using real-time PCR. Results. On the 2-dimensional gels, 532 ± 39 protein spots were identified for the sensitive strains, and 541 ± 43 spots for the resistant strains. Ten spots were overexpressed in the resistant strain and identified as heat shock protein (Hsp60 mitochondrial, Hsp70 cytosolic and mitochondrial), disulfide isomerase, cysteine protease, enolase, elongation factor 5-alpha, the proteasome alpha-5 subunit and two hypothetical proteins named as Sp(2) and Sp(25). Conclusion. This is the first proteomic study conducted with a L. panamensis resistant strain where several proteins were identified and related with the parasite resistance mechanism to pentavalent antimony. This opens the way for future studies aimed at modulating the drug resistance or at evaluating these proteins as therapeutic targets.

In vitro and in vivo activity of meglumine antimoniate produced at Farmanguinhos-Fiocruz, Brazil, against Leishmania (Leishmania) amazonensis, L (L) chagasi and L (Viannia) braziliensis

The leishmanicidal activity of four batches of meglumine antimoniate, produced in Farmanguinhos-Fiocruz, Brazil (TAMs), was assessed and compared to Glucantime®-Aventis Pharma Ltda. Using the amastigote-like in vitro model, the active concentrations of Sb v varied from 10µg/ml to 300 µg/ml for L. (L.) chagasi and from 50µg/ml to 300µg/ml for L. (L.) amazonensis, with no statistically significant differences among the four batches of TAMs and Glucantime®. The inhibitory concentrations (IC50) determined by the amastigote-infected macrophage model for TAM01/03 and Glucantime® were, respectively: 26.3µg/ml and 127.6µg/ml for L. chagasi, 15.4µg /ml and 22.9µg/ml for L. amazonensis, and 12.1µg/ml and 24.2µg/ml for L. (V.) braziliensis. The activities of the four batches of TAMs were confirmed in an in vivo model by assessing, during eight weeks skin lesions caused by L. braziliensis in hamster that were treated with 20mg Sb v/Kg/day for 30 consecutive days. The meglumine antimoniate produced by Farmanguinhos was as effective as the reference drug, Glucantime®-Aventis, against three species of Leishmania that are of medical importance in Brazil.

Combined effect of the essential oil from Chenopodium ambrosioides and antileishmanial drugs on promastigotes of Leishmania amazonensis

To date, there are no vaccines against Leishmania, and chemotherapy remains the mainstay for the control of leishmaniasis. The drugs of choice used for leishmaniasis therapy are significantly toxic, expensive and with a growing frequency of refractory infections. Because of these limitations, a combination therapy is the better hope. This work demonstrates that the essential oil from Chenopodium ambrosioides shows a synergic activity after incubation in conjunction with pentamidine against promastigotes of Leishmania amazonensis. However, an indifferent effect has been found for combinations of meglumine antimoniate or amphotericin B and the essential oil.

Até hoje não temos vacina contra a Leishmania e a quimioterapia é a indicação para o controle desta doença. Os remédios que hoje utilizamos são tóxicos e muito caros e além disso o resultado não é sempre o desejado. Por isso, uma terapia de combinação é a melhor opção. Este trabalho mostra que o óleo de essência de C. ambrosioides tem atividade sinérgica junto com a pentamidina sobre os promastigotas de L. amazonensis, diferente do resultado da combinação de antimônio de meglumine e anfotericina B e o óleo de essência.

Effects of combined therapy with thalidomide and glucantime on leishmaniasis induced by Leishmania major in BALB/c mice

For treating Leishmania major infection in BALB/c mice, we used thalidomide in conjunction with glucantime. Groups of mice were challenged with 5 x 10(3) metacyclic promastigotes of L. major subcutaneously. A week after the challenge, drug treatment was started and continued for 12 days. Thalidomide was orally administrated 30 mg/kg/day and glucantime was administrated intraperitoneally (200 mg/kg/day). It was shown that the combined therapy is more effective than single therapies with each one of the drugs since the foot pad swelling in the group of mice received thalidomide and glucantime was significantly decreased (0.9 +/- 0.2 mm) compared to mice treated with either glucantime, thalidomide, or carrier alone (1.2 +/- 0.25, 1.4 +/- 0.3, and 1.7 +/- 0.27 mm, respectively). Cytokine study showed that the effect of thalidomide was not dependent on IL-12; however, it up-regulated IFN-gamma and down-regulated IL-10 production. Conclusively, thalidomide seems promising as a conjunctive therapy with antimony in murine model of visceral leishmaniasis.

The effect of glucantime™ on the labeling of blood constituents with technetium-99m

OBJETIVO: A marcação de hemácias sangüíneas (C) com 99mTc é muito utilizada nos procedimentos diagnósticos na medicina nuclear. Drogas podem alterar este método de marcação e modificar a biodisponibilidade de radiofármacos. Neste trabalho, foi avaliada a influência de glucantime na marcação de elementos sangüíneos com 99mTc. MÉTODOS: Sangue foi retirado de ratos e incubado com glucantime. Adicionou-se cloreto estanoso e 99mTc. Após centrifugação, plasma (P) e (C) foram isolados. Amostras de P e C foram precipitadas com TCA 5 por cento, centrifugadas e separadas em frações solúveis (FS) e insolúveis (FI). Os percentuais de atividade total injetada (por cento ATI) em C, FI-P e FI-C foram calculados (p<0,05). RESULTADOS: O %ATI em C diminuiu, em relação ao controle, nas seguintes concentrações de glucantime (6,25 por cento;12,5 por cento; 25 por cento; 50 por cento; 100por cento), respectivamente: 94,06±1,29 (controle) para 77,15±2,79; para 76,68±1,88; para 75,15±2,79; para 72,64±4,40 e para 63,05±3,84. Em FI-C, o %ATI diminuiu, em relação ao controle, em todas as concentrações de glucantime (3,125 por cento; 6,25 por cento; 12, 5 por cento; 25 por cento; 50 por cento; 100 por cento), respectivamente: 93,34±1,18 (controle) para 78,81±2,76; para 74,76±4,82; para 74,02±5,32; para 64,35±4,82; para 62,81±1,97 e para 54,55±3,58. CONCLUSÕES: Este efeito provavelmente foi devido a produtos presentes nesta droga que podem se complexar com íons (Sn+2 e 99mTcO-4) ou ter um efeito direto ou indireto na concentração intracelular do íon estanoso.

Novel methods for the encapsulation of meglumine antimoniate into liposomes

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58 percent) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

In vitro activity of meglumine antimoniate, a pentavalent antimonial drug, on Leishmania promastigotes

The activity of a pentavalent antimonial drug glucantime on the growth of promastigote forms of Leishmania strains involved in South American cutaneous and mucocutaneous leishmaniasis was investigated. A marked difference in susceptibility to glucantime among four different strains and cloned lines obtained from a single strain was observed. For the sensitive strains (L.braziliensis M2903 and L. guyanensis M1176), the cell growth was reduced in a dose-dependent manner for drug concentrations at a range of 0.23 to 23 mM. However, despite the relative sensitivity of the assay, no significant increase of effect was observed in the presence of higher drug concentrations. For the resistant strains (L. amazonensis M10996 and L. braziliensis LYB259) a dose-response line is obtained at a higher concentration range (20 mM to mM). The influence of the drug on surface properties, respiratory activity and incorporation of radiolabelled leucine by a sensitive strain - L-guyanensis M1176-was studied as an approach to its site of action. Despite the increased intensity of self-aglutination for cells growing in the presence of glucantime, no significant change was observed in electrophoretic mobility or Concanavian A reactivity. Since the oxygen uptake of glucose-stimulated promastigotes was only slightly reduced in the presence of 23 mM glucantime at 28-C, the reduction was not sufficient to explain the total growth inhibition observed. A significant decrease of 14C-leucine incorporation into the cold TCA-insoluble fraction of drug-treated cells was observed within the same concentration range that reduces promastigote growth

Efeitos da injeçäo intra-arterial de meios artificiais de contraste iodado: um estudo comparativo utilizando modelo experimental / Effects of intra-arterial injection of different contrast media

Este estudo foi concebido para que fossem avaliados os efeitos de injeçäo intra-arterial de diferentes meios artificiais de contrastes iodados, sobre o endotélio vascular e parênquima renal de ratos. Os resultados encontrados permitem-nos concluir que a toxicidade de tais soluçöes é nitidamente maior que a de soluçöes controle (soluçäo salina isotônica). As soluçöes de iodamida-meglumina-sódio parecem causar menos alteraçöes histológicas do que as soluçöes de diatrizoato de sódio a 50%