Experimental validation of biocompatible nanostructured lipid carriers of sophorolipid: Optimization, characterization and in-vitro evaluation.

To date, the potential of sophorolipids (an important class of glycolipids) has been exploited solely as amphipathic molecules but their ability to formulate lipid nanoparticles has never been explored. In this report, for the first time, lipid nanostructures coated with polysorbates (Tweens) were formulated by a hot dispersion method. By varying the amount of lipid, type of surfactant, and alcohol, dilution ratio etc., the formulation was optimized with respect to its stability, which is a central aspect of their potential applications. Their comprehensive physicochemical characterization was done using static and dynamic light scattering (SLS, DLS), small angle neutron scattering (SANS), zeta-potential, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and atomic force microscopy (AFM) techniques. Further hemolysis study was conducted to understand the in-vitro cytotoxicity levels of the lipidic nanoparticles prior to its application as a potent drug delivery device for countermanding the problems associated with challenging tuberculosis and leprosy drug-Rifampicin. Attaining high entrapment efficiency and sustained release from the developed carrier, further interaction with bovine serum albumin was investigated, to understand the in-vivo behavior of the nanostructured lipid carriers (NLCs).

A study of the free radical scavenging effects of Piper betle leaf extract in patients with vitiligo.

BACKGROUND: Vitiligo is an idiopathic skin disease manifested by depigmented macules. It is characterised by melanocyte destruction, and redox imbalance is proposed to play a contributory role. AIM: The aim of this study was to analyze the effects of an ethanolic extract of Piper betle leaves on the generation of reactive oxygen species in erythrocytes sourced from vitiligo patients. METHODS: The effect of Piper betle on the generation of reactive oxygen species in erythrocytes was measured by flow cytometry in patients with active and stable vitiligo versus healthy controls, using 5-(and-6)-chloromethyl-2'-7'-dichlorodihydrofluorescein diacetate. RESULTS: The generation of reactive oxygen species in erythrocytes was higher in patients with vitiligo (n = 23) compared to healthy controls (n = 18). The geometrical mean fluorescence channel was 23.05 ± 2.11 in patients versus 17.77 ± 1.79 in controls, P = 0.039. The levels of reactive oxygen species were higher in patients with active vitiligo. Treatment of erythrocytes with Piper betle in concentrations of 0.5 and 1.0 µg/ml significantly decreased the baseline levels of reactive oxygen species by 31.7% in healthy controls, and 47.6% and 44.3% in patients with active vitiligo, respectively. Piper betle effectively scavenged hydrogen peroxide, which was evident by a decrease in the geometrical mean fluorescence channel by 52.4% and 62.9% in healthy controls, and 45.0% and 57.0% in patients with active vitiligo. LIMITATIONS: The study had a small sample size. Future studies should focus on evaluation of the antioxidant role of Piper betle at the lesional site. CONCLUSION: This pilot study indicates that patients with active vitiligo demonstrate enhanced generation of reactive oxygen species in erythrocytes, which was significantly reduced following ex vivo treatment with Piper betle.

Clofazimine Inhibits the Growth of Babesia and Theileria Parasites In Vitro and In Vivo.

The present study evaluated the growth-inhibitory effects of clofazimine, currently used for treating leprosy, against Babesia bovis, B. bigemina, B. caballi, and Theileria equi in in vitro culture and against Babesia microti in mice. The 50% inhibitory concentrations (IC50s) of clofazimine against the in vitro growth of B. bovis, B. bigemina, B. caballi, and T. equi were 4.5, 3, 4.3, and 0.29 µM, respectively. In mice infected with B. microti, treatment with 20 mg/kg of body weight of clofazimine administered orally resulted in a significantly lower peak parasitemia (5.3%) than that in the control group (45.9%), which was comparable to the subcutaneous administration of 25 mg/kg diminazene aceturate, the most widely used treatment for animal piroplasmosis. Although slight anemia was observed in both clofazimine- and diminazene aceturate-treated infected mice, the level and duration of anemia were lower and shorter, respectively, than those in untreated infected mice. Using blood transfusions and PCR, we also examined whether clofazimine completely killed B. microti On day 40 postinfection, when blood analysis was performed, parasites were not found in blood smears; however, the DNA of B. microti was detected in the blood of clofazimine-treated animals and in several tissues of clofazimine- and diminazene aceturate-treated mice by PCR. The growth of parasites was observed in mice after blood transfusions from clofazimine-treated mice. In conclusion, clofazimine showed excellent inhibitory effects against Babesia and Theileria in vitro and in vivo, and further study on clofazimine is required for the future development of a novel chemotherapy with high efficacy and safety against animal piroplasmosis and, possibly, human babesiosis.

Erythrocyte superoxide dismutase, catalase activities and hydrogen peroxide induced lipid peroxidation in leprosy.

OBJECTIVES: To assess erythrocyte superoxide dismutase (SOD) and catalase (CAT) activities and hydrogen peroxide induced lipid peroxidation in leprosy. DESIGN: One hundred leprosy patients and 50 normal healthy controls were studied for the parameters. The data was analysed by grouping the patients into Ridley-Jopling (RJ) types [Tuberculoid leprosy (TT, n = 22), Borderline tuberculoid leprosy (BT, n = 28), Borderline leprosy (BB, n = 13), Borderline lepromatous leprosy (BL, n = 16) and Lepromatous leprosy (LL, n = 21)] and into different levels of Bacteriological Index (BI) [bacteriologically negative (n = 32), BI = 0.1-1 (n = 22), BI = 1.1-2 (n = 16), BI = 2.1-3 (n = 14), BI = 3.1-4 (n = 10) and BI = 4.1-6 (n = 06)]. RESULTS: The induced peroxidation was significantly high and the enzyme activities were significantly low in leprosy (total patients) as compared to controls. A progressive increase in peroxidation was detected along the leprosy spectrum from TT to LL and the increase was significant in BB, BL and LL groups as compared to controls. Induced peroxidation in LL group as compared to TT, BT and BB and in the BL group as compared to TT and BT were significantly different. A concomitant progressive decline in enzyme activity was detected along the leprosy spectrum from TT to LL. The SOD activity in BB, BL and LL and the CAT activity in BL and LL were significantly low as compared to controls. SOD activity in BB, BL and LL groups as compared to TT and in the LL group as compared to BT were significantly different. A progressive trend of increasing peroxidation and decreasing SOD and CAT activity were also detected along the leprosy groups with advancing level of BI. Induced peroxidation and SOD activity were significantly different in bacteriologically positive groups as compared to controls and in the BI levels 1.1-2, 2.1-3, 3.1-4 and 4.1-6 as compared to bacteriologically negative group. The peroxidation was significantly different in BI levels 2.1-3, 3.1-4 and 4.1-6 as compared to BI level 0.1-1. The CAT activity was significantly different in BI levels 2.1-3, 3.1-4 and 4.1-6 as compared to controls. CONCLUSION: The study findings suggest oxidative stressful state associated with reduced antioxidant defence potential in erythrocytes of leprosy patients. The study implicates association of erythrocyte oxidative stress with bacterial load and type of leprosy.

Hemolytic drugs aniline and dapsone induce iron release in erythrocytes and increase the free iron pool in spleen and liver.

Incubation of rat erythrocytes with the hydroxylated metabolites of aniline and dapsone (4-4'-diaminodiphenylsulfone), phenylhydroxylamine and dapsone hydroxylamine, respectively, induced marked release of iron and methemoglobin formation. On the contrary, no release of iron nor methemoglobin formation was seen when the erythrocytes were incubated with the parent compounds (aniline and dapsone). The acute intoxication of rats with aniline or dapsone induced a marked increase in the erythrocyte content of free iron and methemoglobin, indicating that the xenobiotics are effective only after biotransformation to toxic metabolites in vivo. Prolonged administration of aniline or dapsone to rats produced continuous release of iron from erythrocytes. Marked iron overload was seen in the spleen and in the liver Kupffer cells, as detected histochemically. The spleen weight in these subchronically treated animals was significantly increased. The free iron pool was markedly increased in the spleen and to a lower extent in the liver. The possible relationships between iron release in erythrocytes, oxidative damage seen in senescent cells, hemolysis, overwhelmed capacity of spleen and liver to keep iron in storage forms and subsequent increase in low molecular weight, catalitically active iron is discussed.

Immunomodulatory assays to study structure-activity relationships of thalidomide.

Thalidomide, which has a long history of tragedy because of its ability to cause severe birth defects, is very effective in alleviating erythema nodosum leprosum in leprosy patients and aphthous ulcers in AIDS patients. The causes of these inflammatory diseases and the mechanism by which thalidomide diminishes them are unknown. It has been suggested that modulation of the immune response plays an important role. We found that thalidomide exerts immunomodulatory activity in three bioassays. It suppresses an IgM plaque forming cell response in mice injected with sheep erythrocytes: it inhibits TNF-alpha production by LPS stimulated human mononuclear cells: and it enhances IL-2 production by Con-A stimulated human mononuclear cells. We employed these bioassays to compare the activity of 15 analogs of thalidomide with thalidomide itself. Eight of the compounds were derivatives of the glutarimide moiety of thalidomide and the others were phthalimide or derivatives of the phthalimide moiety of thalidomide. N-hydroxyphthalimide, a simple derivative of phthalimide, was more effective than thalidomide and was also the most effective of the compounds assayed in suppressing the IgM plaque and TNF-alpha responses, but it did not enhance the IL-2 response, instead, it significantly suppressed it.

Dapsone-induced hemolytic anemia.

Dapsone, an old drug introduced and used almost exclusively for the treatment of leprosy, is now utilized in an increasing number of therapeutic situations. However, its hemotoxicity is potentially severe and is often dose limiting. Effective countermeasures, based on resolution of the mechanisms underlying dapsone-induced hemotoxicity, could significantly enhance the therapeutic value of the drug. In studies on rat red cells, we have established that the N-hydroxy metabolites of dapsone, DDS-NOH and MADDS-NOH, are direct-acting hemolytic agents, that they are formed in amounts sufficient to account for the hemotoxicity of the parent drug, and that the action of these toxic metabolites in the red cell induces premature sequestration by the spleen. Incubation of rat red cells with hemolytic concentrations of arylhydroxylamines leads to the generation of hydroxyl, glutathiyl, and hemoglobinthiyl radicals, and the formation of protein-glutathione mixed disulfides. Disulfide-linked adducts are also formed between membrane skeletal proteins and hemoglobin monomers, as well as between the monomeric hemoglobin units forming dimers, trimers, tetramers, and pentamers. Profound morphological changes are seen with change from normal discoidocity to an extreme nonspherocytic enchinocyte shape. Parallel studies with human red cells indicate that the response of human cells is qualitatively similar but that there are notable differences in regard to skeletal membrane effects. A working hypothesis for the mechanism underlying dapsone hemolytic activity is proposed.

Stimulation of K-C1 cotransport in rat red cells by a hemolytic anemia-producing metabolite of dapsone.

Dapsone, a sulfone compound used in the treatment of leprosy and, more recently, Pneumocystis carinii pneumonia, produces as a major side effect a hemolytic anemia. This anemia is characterized by oxidation of hemoglobin to methemoglobin and increased splenic uptake of red blood cells. Using a rat model, Grossman and Jollow (J. Pharmacol. Exp. Ther. 244: 118-125, 1988) found that dapsone hydroxylamine (DDS-NOH), a dapsone metabolite, is responsible for its hemolytic effect in vivo. DDS-NOH also promotes hemoglobin binding to SH groups on rat red cell membrane proteins (Budinsky et al., FASEB J. 2: A801, 1988). Since the binding of hemoglobin and other reagents (e.g., N-ethylmaleimide) to membrane SH groups has been associated with increased K transport in red blood cells, we examined the effect of DDS-NOH on K efflux from rat red blood cells in vitro. Cells shrink when exposed to DDS-NOH (100 microM) in media with plasma-like ionic composition. This shrinkage is prevented if extracellular K is raised to 110 mM or if intra- and extracellular Cl are replaced by methylsulfate (MeSO4), suggesting involvement of a K-Cl cotransport pathway. Indeed, 100 microM DDS-NOH produces a 4- to 5-fold increase in K efflux in cells containing Cl but less than a 2-fold increase in cells containing MeSO4. This stimulatory effect is specific for K; Na efflux is slightly inhibited by 100 microM DDS-NOH. The concentrations of DDS-NOH required for half-maximal stimulation of Cl-dependent K efflux (53 microM) is similar to its half-maximal hemolytic concentration in rats (approximately 100 microM). Furthermore, the stimulation of Cl-dependent K efflux by DDS-NOH is greater than 80% reversed by subsequent treatment of the cells with dithiothreitol, suggesting involvement of SH groups. Our results indicate that DDS-NOH exposure stimulates an apparent K-Cl cotransport in rat red blood cells, resulting in cell shrinkage under physiological ionic conditions. Since shrinkage of red blood cells renders them less deformable (Mohandas et al., J. Clin. Invest. 66: 563-573, 1980), this suggests a pathophysiological mechanism whereby DDS-NOH exposure in vivo could promote increased splenic uptake of red blood cells and hemolytic anemia.

Cytogenetic effect of dapsone, an antileprotic drug, in the mouse in vivo system.

Potential genotoxicity of dapsone was evaluated in mice following in vivo cytogenetic assays. Adult male mice treated with different doses (20 mg, 40 mg, or 80 mg/kg/day for 4 weeks) and for different periods (40 mg/kg/day for 2, 4, or 8 weeks) provided bone marrow and testes for mitotic and meiotic chromosome analyses, respectively. A dose-response (20 mg, 40 mg, or 80 mg/kg/day for 2 weeks) analysis was done with a separate set of mice using a micronucleus test (MNT). Untreated mice served as controls. Both the metaphase analysis and MNT in bone-marrow cells revealed significantly higher incidences of clastogenicity for all of the dose levels and treatment periods. Chromosome aberrations, with and without gaps, in bone-marrow metaphases showed a positive correlation with the doses, but not with the treatment periods. Correlation was also lacking in the MNT. In the meiotic cells, the incidences of chromosome aberrations increased significantly with the highest dose and with the longest period of treatment.