Antileishmanial Activity of Niosomal Combination Forms of Tioxolone along with Benzoxonium Chloride against Leishmania tropica.

In this study, we carried out extensive in vitro studies on various concentrations of tioxolone along with benzoxonium chloride and their niosomal forms against Leishmania tropica. Niosomes were prepared by the hydration method and were evaluated for morphology, size, release study, and encapsulation efficiency. This study measured leishmanicidal activity against promastigote and amastigote, apoptosis and gene expression levels of free solution and niosomal-encapsulated tioxolone along with benzoxonium chloride. Span/Tween 60 niosome had good physical stability and high encapsulation efficiency (more than 97%). The release profile of the entrapped compound showed that a gradual release rate. The combination of niosomal forms on promastigote and amastigote were more effective than glucantime. Also, the niosomal form of this compound was significantly less toxic than glucantime (P≤0.05). The flowcytometric analysis on niosomal form of drugs showed that higher number of early apoptotic event as the principal mode of action (89.13% in 200 µg/ml). Also, the niosomal compound increased the expression level of IL-12 and metacaspase genes and decreased the expression level of the IL-10 gene, which further confirming the immunomodulatory role as the mechanism of action. We observed the synergistic effects of these 2 drugs that induced the apoptotic pathways and also up regulation of an immunomodulatory role against as the main mode of action. Also, niosomal form of this combination was safe and demonstrated strong anti-leishmaniasis effects highlights further therapeutic approaches against anthroponotic cutaneous leishmaniasis in future planning.

Niosomal formulation of amphotericin B alone and in combination with glucantime: In vitro and in vivo leishmanicidal effects.

This study aimed to evaluate the efficacy of glucantime and amphotericin B (AmB) encapsulated in niosome against cutaneous leishmaniasis (CL) using in vitro and in vivo models. The niosomal formulations of the drugs alone and in combination were prepared and characterized. Subsequent to the examination of their cytotoxicity, their efficacy was evaluated using an in vitro MTT assay, macrophage model, flow cytometry, and gene expression profiling. For evaluation of therapeutic effect of niosomal combination on the lesion induced by Leishmania major in inbred BALB/c mice, the size of lesions and number of parasites in spleen was assessed. The niosomal formulations demonstrated significantly greater inhibitory effects compared with the non-niosomal forms when the IC50 was considered. The niosomal combination showed an increase in the apoptotic values and gene expression levels of IL-12 and metacaspase and a decrease in the levels of IL-10 with a dose-response effect. The niosomal combination was also effective in reducing the lesion size and splenic parasite burden in mice. Our findings indicated that there is a synergistic effect between AmB and glucantime in niosomal form in the inhibition of intracellular and extracellular forms of L. tropica. Additionally, the in vivo results on L. major suggest that topical niosomal formulation could be useful in the treatment of CL.

A Novel Niosomal Combination of Selenium Coupled with Glucantime against Leishmania tropica.

There is no effective treatment modality available against different forms of leishmaniasis. Therefore, the aim of this study was to improve the penetration and efficacy of selenium and glucantime coupled with niosomes and compared them with their simple forms alone on in vitro susceptibility assays. In this study, the niosomal formulations of selenium and in combination with glucantime were prepared. The size and morphology of the niosomal formulations were characterized and the effectivity of the new formulation was also evaluated using in vitro MTT assay, intra-macrophage model, and gene expression profile. From the results obtained, no cytotoxicity effect was observed for niosomal and simple forms of drugs, as alone or in combination. Niosomal formulations of the drugs significantly showed more inhibitory effects (P ≤ 0.001) than the simple drugs when the selectivity index was considered. The gene expression levels of Interleukin (IL-10) significantly decreased, while the level of IL-12 and metacaspase significantly increased (P ≤ 0.001). The results of the present study showed that selenium plus glucantime niosome possess a potent anti-leishmanial effect and enhanced their lethal activity as evidenced by the in vitro experiments.

Host's immune response in unresponsive and responsive patients with anthroponotic cutaneous leishmaniasis treated by meglumine antimoniate: A case-control study of Th1 and Th2 pathways.

The objective of the present study was to compare the host's immune responses between unresponsive and responsive patients with anthroponotic cutaneous leishmaniasis (ACL) treated by meglumine antimoniate. A case-control study was carried out in an endemic focus in Iran. Blood samples were taken from patients and peripheral blood mononuclear cells (PBMCs) were isolated. Two wells were considered for each isolate of unresponsive and responsive patients; one was exposed to L. tropica (Lt-stimulated cells) and the other remained non-exposed (non-stimulated cells). After 24 h of incubation, whole RNA was extracted from each sample. Real-time quantitative PCR was carried out to confirm the differences in expression levels of IL-12 P40, IFN-γ, IL-1ß, IL-4 and IL-10 among isolates. Data were analyzed and P < 0.05 was considered to be statistically significant. In our study, Lt-stimulated cells and non-stimulated cells in unresponsive groups demonstrated significantly lower expression levels of IL-1ß, IL-12 P40 and IFN-γ genes and higher expression levels of IL-4 and IL-10 genes, compared to Lt-stimulated cells and non-stimulated cells in responsive groups. There was a negative correlation between IL-12 P40 with IL-10 and IL-1ß with IL-10 in ACL Lt-stimulated cells in unresponsive group, while a positive correlation between IL-12 P40 with IL-1ß and IL-12 P40 with IFN-γ in ACL Lt-stimulated cells in responsive group. Probably, different immune responses caused by various factors play a major role in the pathogenesis and development of unresponsiveness in ACL patients. The profile and timing of cytokine production correlated well with the treatment outcome of Leishmania infection.

A single-group trial of end-stage patients with anthroponotic cutaneous leishmaniasis: Levamisole in combination with Glucantime in field and laboratory models.

Currently, there is no satisfactory treatment modality available for cutaneous leishmaniasis (CL). The major objective of the present study was to explore the effect of immunomodulator-levamisole in combination with Glucantime in end-stage unresponsive patients with anthroponotic CL (ACL). Twenty end-stage unresponsive patients with ACL were identified for participation in this single-group trial study. Simultaneously, each patient was received a combination of levamisole pills along with Glucantime during the remedy course. Several in vitro complementary experiments were performed to evaluate the mode of action of levamisole and Glucantime alone and in combination using a macrophage model, in vitro MTT assay, flow cytometry and quantitative real time PCR (qPCR). Overall, 75% of the patients showed complete clinical cure, 10% partially improved and the remaining (15%) had underlying chronic diseases demonstrated no response to the treatment regimen. In in vitro studies, there was no cytotoxic effect associated with these drugs in the range of our experiments. The findings by the flow cytometric analysis represented that the highest apoptotic values corresponded to the drugs combination (32.23%) at 200 µg/ml concentration. Finally, the gene expression level of IL-12 p40, iNOS and TNF-α promoted while the level of IL-10 and TGF-ß genes reduced as anticipated. The findings clearly indicated that the combination of levamisole and Glucantime should be considered in end-stage unresponsive patients with ACL who have not responded to basic treatments. The immunomodulatory role of levamisole in mounting immune system as documented by the in vitro experiments and further substantiated by this single-group trail study was highlighted.

Emergence of a new focus of anthroponotic cutaneous leishmaniasis due to Leishmania tropica in rural communities of Bam district after the earthquake, Iran.

OBJECTIVES: To describe a new emerging focus of anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica in rural areas of Dehbakry county, south-eastern Iran, after the earthquake of 2003. METHODS: House-to-house survey of 3884 inhabitants for active leishmaniasis lesions or scars. The diagnosis was confirmed by smears, cultures and identification of the parasite by polymerase chain reaction (PCR). RESULTS: All age groups were affected, although patients ≤10 years of age showed the highest rate of infection (P = 0.0001). The overall prevalence rate was 5.3%; 6.3% in females and 4.3% in males. Of 204 cases, 1.8% had active sores and 3.5% had scars, with a significant difference between the sexes (P = 0.005). 47% of the lesions were on the face and 77.9% had one lesion. The incidence rose gradually 2004-2005, but grew exponentially 2006-2008. Electrophoresis of PCR products indicated that L. tropica was the causative agent. CONCLUSIONS: The current emergence was unexpected in this rural locality, where no previous history of CL was recorded. According to our knowledge this is the first report of a gradually establishing new ACL focus in rural communities after the 2003 earthquake.