In vitro and in vivo anti-parasitic activity of biogenic antimony sulfide nanoparticles on Leishmania major (MRHO/IR/75/ER).

The aims of this study were to produce biogenic antimony sulfide nanoparticles (NPs) using Serratia marcescens (S. marcescens) and investigate the potential anti-leishmanial effects of these NPs on Leishmania major (L. major) (MRHO/IR/75/ER) in both in vitro and in vivo experiments. Biogenic antimony sulfide NPs were synthesized through intracellular biological methods using S. marcescens. The efficiency of various concentrations of antimony sulfide NPs was assessed using in vitro experiments on amastigotes of L. major at various times post-infection. In vivo experiments were carried out in BALB/c mice inoculated subcutaneously with 2 × 106L. major promastigotes (MHROM/IR/75/ER) and treated with antimony sulfide NPs (70 µg/mL, tropically), meglumine antimoniate (glucantime) as positive control and sterile phosphate-buffered saline (PBS, pH 7.4) as vehicle control. Results of in vitro experiments revealed that the anti-leishmanial activity increased when the antimony sulfide NPs concentration increased. The IC50 (50% inhibitory concentration) of antimony sulfide NPs against amastigotes was calculated as 62.5 µg/mL. In in vivo experiments, the average size of lesions significantly decreased to 8.6 ± 2.7 mm2 in mice inoculated with L. major promastigotes and treated with antimony sulfide NPs, compared with that in the negative control group (P = 0.015). Furthermore, results showed that antimony sulfide NPs significantly decreased the parasite load in the test group, compared with the negative control group (P = 0.001). Various concentrations of antimony sulfide NPs showed a great anti-leishmanial efficiency against L. major (MRHO/IR/75/ER), with the greatest efficiency shown by a concentration of 62.5 µg/mL in in vitro and in vivo experiments.

Combination oral therapy against Leishmania amazonensis infection in BALB/c mice using nanoassemblies made from amphiphilic antimony(V) complex incorporating miltefosine.

Clinically available drugs for mucocutaneous and cutaneous leishmaniases (CL) include mainly pentavalent antimony (Sb(V)) complexes, liposomal amphotericin B, and miltefosine (HePC). However, they present at least one of the following limitations: long-term parenteral administration through repeated doses, severe side effects, drug resistance, and high cost. HePC is the only oral drug available, but the appearance of resistance has resulted in changes of its use from monotherapy to combination therapy. Amphiphilic Sb(V) complexes, such as SbL8 obtained from reaction of Sb(V) with N-octanoyl-N-methylglucamide, were recently found to be orally active against experimental CL. The property of SbL8 to self-assemble in aqueous solution, forming nanostructures, led us to investigate the incorporation of HePC into SbL8 nanoassemblies and the therapeutic efficacy of SbL8/HePC nanoformulation by oral route in a murine model of CL. HePC incorporation into the SbL8 nanosystem was evidenced by using a fluorescent analog of HePC. The antileishmanial activity of SbL8/HePC nanoassemblies was evaluated after daily oral administration for 30 days in Leishmania amazonensis-infected BALB/c mice, in comparison with monotherapies (SbL8 or HePC) and saline control. All the treatments resulted in significant reduction in the lesion size growth, when compared with control. Strikingly, only SbL8/HePC nanoassemblies promoted a significant decrease of the parasite burden in the lesion. This work establishes the therapeutic benefit of SbL8/HePC association by oral route in a CL model and constitutes an important step towards the development of new orally active drug combination.

Early Cutaneous Leishmaniasis Patients Infected With Leishmania braziliensis Express Increased Inflammatory Responses After Antimony Therapy.

Background: Early cutaneous leishmaniasis (ECL) is characterized by a nonulcerated papular lesion and illness duration less than 30 days. Approximately 4 weeks later, the cutaneous leishmaniasis (CL) ulcers appear. We were surprised to find that failure after antimony therapy (Sb5) is higher in ECL than CL. We hypothesize that the inflammatory response in ECL patients may increase during Sb5 therapy, which leads to treatment failure. Methods: A cohort of 44 ECL patients infected by Leishmania braziliensis was established to evaluate the response to Sb5 and to compare immunologic responses in ECL patients with CL and healthy subjects. Results: A hierarchical clustering based on cytokine levels showed a weak positive correlation between proinflammatory cytokine levels and those patients that failed Sb5 treatment. Although Sb5 therapy decreased interferon-γ and tumor necrosis factor levels in CL patients, we were surprised to find that an increase in these cytokines was observed in ECL patients. Moreover, interleukin (IL)-10 was less able to down-modulate immune responses in ECL. Conclusions: The enhanced production of proinflammatory cytokines, due in part to the decreased ability of IL-10 to down-modulate immune response during therapy in ECL, promotes the development and persistence of leishmania ulcer despite antimony therapy.

Fluconazole in the Treatment of Cutaneous Leishmaniasis Caused by Leishmania braziliensis: A Randomized Controlled Trial.

BACKGROUND: The treatment of cutaneous leishmaniasis (CL) caused by Leishmania braziliensis in Brazil with pentavalent antimony (Sbv) is associated with a high rate of failure, up to 45% of cases. In addition, Sbv can only administered parenterally and has important toxic effect. An effective, safe, and oral treatment for CL is required. METHODS: A randomized controlled clinical trial was conducted to compare the efficacy and safety of high-dosage oral fluconazole (6.5-8.0 mg/kg/d for 28 days) versus a standard Sbv protocol (20 mg/kg/d for 20 days) for the treatment of CL in Bahia, Brazil. RESULTS: A total of 53 subjects were included in the trial; 26 were treated with Sbv, and 27 with fluconazole. Intention-to-treat analysis showed initial cure rates (2 months after treatment) of 22.2% (6 of 27) in the fluconazole and 53.8% (14 of 26) in the Sbv group (P = .04). Six months after treatment, the final cure rate remained the same in both groups, without any relapses. The frequencies of adverse effects in the Sbv and fluconazole groups were similar, 34.6% versus 37% respectively. One patient treated with fluconazole discontinued treatment owing to malaise, headache, and moderate dizziness (Common Terminology Criteria for Adverse Events grade 2). CONCLUSIONS: Oral fluconazole at a dosage of 6.5-8 mg/kg/d for 28 days should not be considered an effective treatment for CL caused by L. braziliensisClinical Trials Registration. NCT01953744.

Low concentrations of antimony impair DNA damage signaling and the repair of radiation-induced DSB in HeLa S3 cells.

Antimony is utilized in a large variety of industrial applications, leading to significant environmental and occupational exposure. Mainly based on animal experiments, the IARC and MAK Commission have classified antimony and its inorganic compounds as Group 2B or 2 carcinogens, respectively. However, the underlying mode(s) of action are still largely unknown. In the present study, we investigated the impact of non-cytotoxic up to cytotoxic concentrations of SbCl3 on DNA DSB repair and cell cycle control in HeLa S3 cells. We induced DSB by γ-irradiation and analyzed inhibitory actions of antimony on potential molecular targets of the DSB repair machinery. Antimony disturbed cell cycle control, affecting phosphorylation of Chk1. Furthermore, the repair of DSB was impaired in the presence of antimony, as monitored by pulsed-field gel electrophoresis and γH2AX foci formation of cells in G1 and G2 phase. Specifically, BRCA1 and RAD51 were identified as molecular targets. Our results point towards an interference with both non-homologous end-joining (NHEJ) and homologous recombination (HR), and inhibitory effects may be explained by interactions with critical cysteine groups; this needs to be further investigated. Altogether, the results provide further evidence for the impairment of DNA repair processes as one underlying mechanism in antimony-induced carcinogenicity.

Mixed antimony(V) complexes with different sugars to modulate the oral bioavailability of pentavalent antimonial drugs.

Previous studies have shown that the association of the drug meglumine antimoniate (MA) with ß-cyclodextrin can improve its bioavailability by the oral route. In this work, ribose and maltose were investigated for their ability to form mixed or association complexes with MA, release MA and modulate the serum levels of Sb after oral administration in mice. Analysis of the MA/ribose composition by high performance liquid chromatography coupled to mass spectrometry (LCMS-IT-TOF) revealed the presence of mixed meglumine-Sb-ribose and Sb-ribose complexes. Analysis of the MA/maltose composition suggested the formation of MA-maltose association compounds. Circular dichroism characterization of these compositions following dilution in water at 37 °C suggested a partial and slow dissociation of the association compounds. When the MA/ribose composition was administered orally and compared to MA, the serum concentration of Sb was significantly lower after 1 h and greater after 3 h. On the other hand, the MA/maltose composition showed similar serum Sb concentration after 1 h and higher level of Sb after 3 h, when compared to MA. In conclusion, the present study has demonstrated the formation of mixed or association complexes of MA with sugars, such as maltose and ribose, which promoted sustained serum level of Sb after oral administration.

Intralesional antimony for single lesions of bolivian cutaneous leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis is an ultimately self-curing disease for which systemic therapy with pentavalent antimony (Sb) is effective but with side effects. We evaluated 2 local treatments, intralesional (IL) Sb and cryotherapy, for single lesions due to Bolivian Leishmania (v.) braziliensis in a placebo-controlled study. METHODS: Patients were randomized between IL Sb (650 µg/mm(2) of lesion area on days 1, 3, and 5), cryotherapy (days 1 and 14), and placebo cream (daily for 20 days) in a 3:2:3 allocation. Lesion area was measured prior to therapy, and at 1, 3, and 6 months after therapy. The criteria for lesion cure were as follows: not doubling in size at 1 month, at least 50% diminution in size at 3 months, and complete reepithelialization at 6 months. Local adverse effects were recorded. RESULTS: Cure rates were 21 of 30 (70%; 95% confidence interval [CI], 52%-83%) for IL Sb, 4 of 20 (20%; 95% CI, 8%-42%) for cryotherapy, and 5 of 30 (17%; 95% CI, 7%-34%) for placebo cream (P < .001 for IL Sb vs each other group). IL Sb adverse events were limited to injection site pain, with a mean value of 1.0 (mild). CONCLUSIONS: The comparative cure rate, small amount of drug administered, and tolerance data for IL Sb suggest that if local therapy for single L. braziliensis lesions is chosen, this treatment is attractive. Given the difficulties of performing placebo-controlled trials in the New World, the combined placebo and cryotherapy cure rate (18%; 95% CI, 10%-31%) is likely to become the standard against which future interventions for L. braziliensis are compared. CLINICAL TRIALS REGISTRATION: NCT01300975.

A pilot study comparing low-dose liposomal amphotericin B with N-methyl glucamine for the treatment of American cutaneous leishmaniasis.

BACKGROUND: Cutaneous leishmaniasis is an infectious re-emerging disease that has increased in incidence worldwide. Antimony, a highly toxic drug, remains the first choice therapy to treat it. Liposomal amphotericin B is active against Leishmania and is less toxic than antimony. OBJECTIVE: To compare low-dose liposomal amphotericin B with N-methyl glucamine for the treatment of American cutaneous leishmaniasis. PATIENTS/METHODS: In a controlled open-label trial 35 patients with a localized form of American cutaneous leishmaniasis were included. They were allocated to a first group treated with 1.5 mg/kg/day of liposomal amphotericin B for 5 days, or to a second one treated with 20 mgSbV/kg/day of N-methyl glucamine for 20 days. RESULTS: In the first group, 50% and 81% of patients experienced a clinical cure and clinical improvement respectively. There was a 100% clinical cure in the second group. CONCLUSION: Liposomal amphotericin B seems to be promising and safe for the treatment of American cutaneous leishmaniasis.

Activity of Free and Liposomal Antimony Trioxide in the Acute Promyelocytic Leukemia Cell Line NB4.

BACKGROUND/AIM: Antimony is a chemical element used in the therapy of parasitic diseases with a promising anticancer potential. The aim of this study was to evaluate in vitro activity of free or liposomal vesicle-packed antimony trioxide (AT or LAT) in the t(15;17)(q22;q21) translocation-positive acute promyelocytic leukemia (APL) cell line NB4. MATERIALS AND METHODS: Cytotoxicity was analysed with trypan blue exclusion, the MTT assay and neutral red exclusion assay; cell proliferation with PicoGreen®; and reactive oxygen species (ROS) production with DCFDA. RESULTS: Liposomal particles did not change the pH of the cell culture medium and entered the cells. Both formulations resulted in a time- and concentration-dependent cytotoxicity and production of ROS. LAT showed higher toxicity at lower concentrations compared to AT. CONCLUSION: LAT may be used to decrease drug dosage and maintain high anti-tumoral effects on APL cells.

Dermal absorption of gallium antimonide in vitro and pro-inflammatory effects on human dermal fibroblasts.

Gallium antimonide (GaSb) is a group III-V compound semiconductor with a comparatively narrow band gap energy (0.73 eV at 300 K) that allows efficient operation in the near-infrared region. This property may be useful in developing new biomedical instruments such as epidermal optoelectronic devices. The present study investigated the absorption of GaSb in pig skin in vitro for 24 h using Franz cells. A donor solution was prepared by soaking GaSb thin films in synthetic sweat. The results showed that both gallium and antimony penetrated the skin, and permeation and resorption occurred for gallium. Histopathological findings showed no inflammatory responses in pig skin exposed to GaSb for 24 h. Cytotoxicity was significantly elevated after 3 and 7 days, and pro-inflammatory cytokines and IL-8 levels were low after 1 and 3 days but elevated 7 days following the direct culturing of human dermal fibroblasts (HDF) on GaSb thin films. These results demonstrate that the short-term cytotoxicity and pro-inflammatory effect of GaSb on HDF were relatively low.

The efficacy of Tc-99m sestamibi for sentinel node mapping in breast carcinomas: comparison with Tc-99m antimony sulphide colloid.

BACKGROUND: To study the value of periareolar intra-dermal injection of Tc-99m sestamibi (MIBI) for sentinel node mapping in breast carcinoma. MATERIAL AND METHODS: Fifty patients with early-stage breast cancer were included in our study. 17.5 MBq Tc-99m-MIBI was injected intradermally to 25 patients and the remainders were injected with the same dose of Tc-99m-antimony sulphide colloid. Anterior and lateral static images were taken at 2 minutes. If sentinel lymph node was not detected, delayed imaging by up to 180 minutes was carried out. The patients were operated on 2-4 hours post-injection. Sentinel lymph node biopsy was performed by the aid of gamma probe and blue dye during surgery. RESULTS: In the Tc-99m-MIBI group, 23 patients had lymph nodes on scintigraphy images, and sentinel nodes were detected during surgery in all 23 patients. In the Tc-99m-antimony sulphide colloid group, 24 patients had lymph nodes on scintigraphy images, and sentinel nodes were identified during surgery in 24 patients. CONCLUSIONS: We concluded that 99mTc-MIBI is a suitable radiopharmaceutical for sentinel node detection.

Factors affecting sentinel lymph node detection failure in breast cancer patients using intradermal injection of the tracer.

OBJECTIVE: The standard method for axillary lymph node staging in early breast cancer is sentinel lymph node biopsy. In some patients the sentinel lymph node can not be localized during surgery and these patients have to undergo standard axillary lymph node dissection. In this study we have evaluated the predictors of sentinel lymph node localization failure using (99m)Tc-antimony sulfide colloid and intradermal injection combined with blue dye technique. MATERIAL AND METHODS: 202 consecutive patients with early stage breast cancer (clinically stage I or II) were retrospectively evaluated. Patients whose sentinel lymph node was localized during surgery were compared to those with localization failure considering several variables. RESULTS: Sentinel lymph node was successfully located on the pre-operative lymphoscintigraphy images in 180 patients (89%). Both univariate and multivariate analyses showed that only sentinel lymph node non-visualization by pre-operative lymphoscintigraphy, experience of the surgeon, and axillary lymph node involvement are associated with sentinel node localization failure during surgery. CONCLUSIONS: This study shows the importance of pre-operative lymphoscintigraphy in order to identify the group of patients with possible localization failure during surgery and warning the surgeon beforehand. We also recommend that all surgeons pass the learning curve of sentinel lymph node biopsy before routinely performing this procedure.

CpG ODN D35 improves the response to abbreviated low-dose pentavalent antimonial treatment in non-human primate model of cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) affects the lives of 0.7-1 million people every year causing lesions that take months to heal. These lesions can result in disfiguring scars with psychological, social and economic consequences. Antimonials are the first line of therapy for CL, however the treatment is lengthy and linked to significant toxicities; further, its efficacy is variable and resistant parasites are emerging. Shorter or lower dose antimonial treatment regimens, which would decrease the risk of adverse events and improve patient compliance, have shown reduced efficacy and further increase the risk emergence of antimonial-resistant strains. The progression of lesions in CL is partly determined by the immune response it elicits, and previous studies showed that administration of immunomodulatory type D CpG ODNs, magnifies the immune response to Leishmania and reduces lesion severity in nonhuman primates (NHP) challenged with Leishmania major or Leishmania amazonensis. Here we explored whether the addition of a single dose of immunomodulating CpG ODN D35 augments the efficacy of a short-course, low-dose pentavalent antimonial treatment regimen. Results show that macaques treated with D35 plus 5mg/kg sodium stibogluconate (SbV) for 10 days had smaller lesions and reduced time to re-epithelization after infection with Leishmania major. No toxicities were evident during the studies, even at doses of D35 10 times higher than those used in treatment. Critically, pentavalent antimonial treatment did not modify the ability of D35 to induce type I IFNs. The findings support the efficacy of D35 as adjuvant therapy for shorter, low dose pentavalent antimonial treatment.

[Effects of trivalent antimony on the rat hormonal status under experimental conditions].

The paper gives the data of experimental studies of oral trivalent antimony used at 1 and 20 maximum permissible concentrations (MPC) on the rat hormonal status. The findings are indicative of the specific features of changes in the hormonal status of experimental animals depending on the dose of trivalent antimony. The concentration of trivalent antimony at the level of 20 MPC is shown to act as a role of a reprotoxic agent that causes a change in the serum content of hormones, such as follicle-stimulating and luteinizing ones.

Topical photodynamic therapy with chloroaluminum phthalocyanine liposomes is as effective as systemic pentavalent antimony in the treatment of experimental cutaneous leishmaniasis.

BACKGROUND: In the Americas, one of the main causative species of cutaneous leishmaniasis is Leishmania (Leishmania) amazonensis. The systemic antimonials remain the most largely used option for disease control. However, this drug has significant toxicity. The development of new alternative therapies, including the identification of effective drugs for topical treatment of cutaneous leishmaniasis, is of utmost interest. In this sense, photodynamic therapy emerges as a new strategy. The aim of this study was to develop the chloroaluminum phthalocyanine-loaded liposome, characterize it, and evaluate its stability and efficacy in the topical treatment of cutaneous leishmaniasis caused by L. (L.) amazonensis. METHODS: Liposomes composed of egg phosphatidylcholine were prepared by Bangham's method. Storage stability of phthalocyanine-loaded liposomes was evaluated at 30 and 60 days after preparation. For the in vivo evaluation, the animals were infected with L. (L.) amazonensis and divided into groups: chloroaluminium phthalocyanine-loaded liposome, blank liposome, meglumine antimoniate (200 mgSb+5/Kg/day), and control. The lesion size was determined weekly after the beginning of the treatment. Upon completion, parasites were recovered from the skin lesion and spleen and evaluated by limiting dilution assay. RESULTS: Chloroaluminum phthalocyanine-loaded liposomes were stable and showed adequate characteristics for topical administration. The topical chloroaluminum phthalocyanine-loaded liposome was as effective as systemic pentavalent antimony in reducing the parasitic load in the lesion and spleen in infected animals. CONCLUSIONS: The present study showed that photodynamic therapy with chloroaluminum phthalocyanine-loaded liposomes is a promising strategy for the treatment of American cutaneous leishmaniasis caused by L. (L.) amazonensis.

Gene expression analysis of antimony resistance in Leishmania tropica using quantitative real-time PCR focused on genes involved in trypanothione metabolism and drug transport.

Pentavalent antimonials remain the treatment of choice for all the clinical forms of leishmaniasis. The increasing rates of antimony resistance are becoming a serious health problem in treatment of anthroponotic cutaneous leishmaniasis (ACL). Accordingly, unraveling molecular markers is crucial for improving medication strategies and monitoring of drug-resistant parasites. Different studies have suggested the importance of genes involved in trypanothione metabolism and drug transport. In this regard, present study was designed to investigate the RNA expression level of five genes including γ-GCS, ODC, TRYR (involved in trypanothione metabolism), AQP1 (acts in drug uptake) and MRPA (involved in sequestration of drug) in sensitive and resistant Leishmania tropica isolates. Seven antimony-resistant and seven antimony-sensitive L. tropica clinical isolates were collected from ACL patients. Drug sensitivity test was performed on the samples as well as reference strains; afterwards, gene expression analysis was performed on clinical isolates by quantitative real-time PCR. The results revealed that the average expression level of AQP1 gene was decreased (0.47-fold) in resistant isolates compared to sensitive ones whereas MRPA (2.45), γ-GCS (2.1) and TRYR (1.97) was upregulated in resistant isolates. The average expression of ODC (1.24-fold) gene was not different significantly between sensitive and resistant isolates. Our findings suggest that AQP1, MRPA, GSH1 and TRYR can be considered as potential molecular markers for screening of antimony resistance in some L. tropica clinical isolates.

Characterizing molecular probes for diffusion measurements in the brain.

Brain diffusion properties are at present most commonly evaluated by magnetic resonance (MR) diffusion imaging. MR cannot easily distinguish between the extracellular and intracellular signal components, but the older technique of real-time iontophoresis (RTI) detects exclusively extracellular diffusion. Interpretation of the MR results would therefore benefit from auxiliary RTI measurements. This requires a molecular probe detectable by both techniques. Our aim was to specify a minimum set of requirements that such a diffusion probe should fulfill and apply it to two candidate probes: the cation tetramethylammonium (TMA(+)), used routinely in the RTI experiments, and the anion hexafluoroantimonate (SbF(6)(-)). Desirable characteristics of a molecular diffusion probe include predictable diffusion properties, stability, minimum interaction with cellular physiology, very slow penetration into the cells, and sufficiently strong and selective MR and RTI signals. These properties were evaluated using preparations of rat neocortical slices under normal and ischemic conditions, as well as solutions and agarose gel. While both molecules can be detected by MR and RTI, neither proved an ideal candidate. TMA(+) was very stable but it penetrated into the cells and accumulated there within tens of minutes. SbF(6)(-) did not enter the cells as readily but it was not stable, particularly in ischemic tissue and at higher temperatures. Its presence also resulted in a decreased extracellular volume. These probe properties help to interpret previously published MR data on TMA(+) diffusion and might play a role in other diffusion experiments obtained with them.

Colonoscopic preoperative localization using submucosal injection of radiolabelled colloid.

Malignant colonic polyps can be removed endoscopically but surgical resection is sometimes required. However, the polypectomy site can be difficult to locate. Current methods use various tattooing agents, with varying degrees of success. A new technique using preoperative injection of technetium-99m-labelled antimony colloid, with intraoperative localization using a handheld gamma probe, is described. Although unsuccessful in terms of localizing a previously partially resected polyp, the technique itself proved safe and simple, and has some advantages over other endoscopic approaches.