Evaluation of Liposomal and Conventional Amphotericin B in Experimental Fungal Keratitis Rabbit Model.

PURPOSE: We evaluate the efficacy of liposomal amphotericin (Fungisome) compared to conventional amphotericin (AMB) for the treatment of fungal keratitis (FK) in an experimental rabbit model. METHODS: FK was induced in 48 New Zealand White rabbits using Aspergillus flavus and Candida albicans (24 rabbits each). Rabbits were divided into four groups: 0.1% and 0.05% Fungisome-, and 0.1% AMB-treated groups, and one untreated control group. Clinical scores were recorded throughout the study while fungal burden was estimated by corneal button culture on day 19 (study endpoint). RESULTS: A statistically significant improvement in clinical score was seen on day 11 in the 0.1% and 0.05% Fungisome versus untreated groups (13.91 and 14.4 vs. 19.3; P < 0.001) in the A. flavus model, and on day 9 in the 0.1% Fungisome-treated versus untreated groups (12.96 vs. 14.2; P = 0.006) in the C. albicans model. At endpoint, the mean clinical scores of the untreated controls, and the 0.1% and 0.05% Fungisome-, and 0.1% AMB-treated groups were 20 ± 1.4, 5.33 ± 1.85, 9.66 ± 2.41, and 8.16 ± 1.95, respectively, in the A. flavus model and 15.85 ± 1.87, 3.08 ± 1.31, 4.21 ± 1.370, and 4.13 ± 1.38, respectively, in the C. albicans model. Conjunctival hyperemia score was higher in the 0.1% AMB- versus 0.1% Fungisome-treated groups (1.33 vs. 0.5, P = 0.452). Lowest fungal burden in both models was seen in the 0.1% Fungisome-treated groups. CONCLUSIONS: Clinical improvement was observed with Fungisome relative to untreated controls. However, no statistically significant differences in outcomes were observed between animals treated with Fungisome and AMB. Although the results are encouraging, future studies in humans are warranted. TRANSLATIONAL RELEVANCE: FK is a leading cause of corneal blindness and is on the rise especially in developing countries. Despite the availability of various antifungal agents, heterogeneous treatment outcomes are seen due to lack of a standardized treatment regimen for FK. Although the use of liposomal AMB has been substantiated by clinical evidence in systemic infections, to our knowledge there are no in vivo studies evaluating the role of topical liposomal versus conventional formulation in FK. Our study investigated the efficacy and toxicity profile of liposomal versus conventional formulation of AMB in an experimental rabbit FK model.

In vitro antifungal activity of Indian liposomal amphotericin B against clinical isolates of emerging species of yeast and moulds, and its comparison with amphotericin B deoxycholate, voriconazole, itraconazole and fluconazole.

Fungisome(TM) is a liposomal preparation of amphotericin B (AMB), already marketed in India. However, its antifungal activity has not been evaluated against a wide range of fungal pathogens. The study was planned to elucidate the in vitro antifungal activity of Fungisome(TM) against wide range of fungi and compare it with AMB deoxycholate (AMB-d), voriconazole (VOR), itraconazole (ITR) and fluconazole (FLU). Minimum inhibitory concentrations (MICs) of the drugs were determined for 262 clinical fungal isolates, including yeast, dimorphic and filamentous fungi, by broth microdilution method approved by Clinical and Laboratory Standards Institute, USA (yeast, M27-A3; filamentous fungi, M38-A2). The MIC(90s) of Fungisome(TM) were 0.125, 0.5 and 0.25 mg l(-1) against yeast, filamentous and dimorphic fungi respectively. In comparison, MIC(90s) of AMB-d, FLU, ITR and VOR were 1, 1 and 1 mg l(-1) (AMB-d), 4, 64 and 64 mg l(-1) (FLU), 1, 16 and 16 mg l(-1) (ITR) and 0.5, 4 and 16 mg l(-1) (VOR) against yeast, filamentous and dimorphic fungi respectively. The MIC of Fungisome(TM) was two to 16-fold lower than AMB-d. These results reveal an efficient in vitro activity of Fungisome(TM).

Amphotericin-B-loaded polymersomes formulation (PAMBO) based on (PEG)3-PLA copolymers: an in vivo evaluation in a murine model.

This paper deals with in vivo evaluation of a new amphotericin-B-loaded polymersomes (PAMBO) formulation in terms of pharmacokinetics, toxicity, tissue distribution profile, and its efficacy in a murine model of disseminated candidiasis. Pharmacokinetic and tissue distribution studies of the PAMBO showed sustained levels of the drug in plasma as well as in target organs which harbor fungal and leishmanial infection. PAMBO was found to be much less toxic than Fungizone. It was observed that 700% increment in the dose is tolerated without observable toxicity which is supported by survival, biochemical, and histopathological results. PAMBO showed a significant improvement in the survival rate of immunosuppressed mice infected with Candida albicans as compared to control. It also showed better dose to dose (1 mg/kg) efficacy as compared to Fungizone and a significant improvement in the life expectancy at 3 and 5 mg/kg dose levels in the animals. Colony forming unit (CFU) counts in the target organs revealed significant reduction in Candida burden with PAMBO treatment. Kidney, spleen, and lung were cleared of infection, although liver was carrying a very low level of infection. Overall, PAMBO formulation is found to be more efficacious and less toxic in a fungal mice model.

Inhibition of NF-kappaB activation by 5-lipoxygenase inhibitors protects brain against injury in a rat model of focal cerebral ischemia.

BACKGROUND: Stroke is one of the leading causes of death worldwide and a major cause of morbidity and mortality in the United States of America. Brain ischemia-reperfusion (IR) triggers a complex series of biochemical events including inflammation. Leukotrienes derived from 5-lipoxygenase (5-LOX) cause inflammation and are thus involved in the pathobiology of stroke injury. METHODS: To test the neuroprotective efficacy of 5-LOX inhibition in a rat model of focal cerebral IR, ischemic animals were either pre- or post-treated with a potent selective 5-LOX inhibitor, (N- [3-[3-(-fluorophenoxy) phenyl]-1-methyl-2-propenyl]-N-hydroxyurea (BW-B 70C). They were evaluated at 24 h after reperfusion for brain infarction, neurological deficit score, and the expression of 5-LOX. Furthermore, the mechanism and the anti-inflammatory potential of BW-B 70C in the regulation of nuclear factor kappa B (NF-kappaB) and inflammatory inducible nitric oxide synthase (iNOS) were investigated both in vivo and in vitro. RESULTS AND DISCUSSION: Both pre- and post-treatment with BW-B 70C reduced infarctions and improved neurological deficit scores. Immunohistochemical study of brain sections showed IR-mediated increased expression of 5-LOX in the neurons and microglia. BW-B 70C down-regulated 5-LOX and inhibited iNOS expression by preventing NF-kappaB activation. Two other structurally different 5-LOX inhibitors were also administered post IR: caffeic acid and 2,3,5-trimethyl-6-[12-hydroxy-5,10-dodecadiynyl]-1,4-benzoquinone (AA-861). As with BW-B 70C, they provided remarkable neuroprotection. Furthermore, in vitro, BW-B 70C inhibited lipopolysaccharide (LPS) mediated nitric oxide production, iNOS induction and NF-kappaB activation in the BV2 microglial cell line. Treating rat primary microglia with BW-B70C confirmed blockage of LPS-mediated translocation of the p65 subunit of NF-kappaB from cytosol to nucleus. CONCLUSION: The study demonstrates the neuroprotective potential of 5-LOX inhibition through down-regulation of NF-kappaB in a rat model of experimental stroke.

Combination of systemic hypothermia and N-acetylcysteine attenuates hypoxic-ischemic brain injury in neonatal rats.

Hypoxic ischemic (HI) injury in neonates may have devastating, long-term consequences. Recently completed clinical trials in HI neonates indicate that hypothermia within 6 h of birth results in modest improvement in the combined outcome of death or severe disability. The aim of this study was to investigate the effects of combining hypothermia and N-acetylcysteine (NAC) on brain injury, neonatal reflexes and myelination after neonatal HI. Seven-day-old rats were subjected to right common carotid artery ligation and hypoxia (8% oxygen) for 2 h. Systemic hypothermia (30 + 0.5 degrees C) was induced immediately after the period of HI and was maintained for 2 h. NAC (50 mg/kg) was administered by intraperitoneal injection daily until sacrifice. Brain infarct volumes were significantly reduced at 48 h post-HI in the hypothermia plus NAC group (21.5 +/- 3.84 mm3) compared with vehicle (240.85 +/- 4.08 mm3). Neonatal reflexes were also significantly improved by combination therapy at days 1 and 7. There was a significant loss of right hemispheric brain volume in the untreated group at 2 and 4 wk after HI insult. Brain volumes were preserved in hypothermia plus NAC group and were not significantly different when compared with the sham group. Similarly, increased myelin expression was seen in brain sections from hypothermia plus NAC group, when stained for Luxol Fast Blue (LFB), Myelin Basic Protein (MBP) and Proteolipid protein (PLP). These results indicate that hypothermia plus NAC combination therapy improves infarct volume, myelin expression and functional outcomes after focal HI injury.

Cerebrovascular protection by various nitric oxide donors in rats after experimental stroke.

The efficacy of nitric oxide (NO) treatment in ischemic stroke, though well recognized, is yet to be tested in clinic. NO donors used to treat ischemic injury are structurally diverse compounds. We have shown that treatment of S-nitrosoglutathione (GSNO) protects the brain against injury and inflammation in rats after experimental stroke [M. Khan, B. Sekhon, S. Giri, M. Jatana, A. G. Gilg, K. Ayasolla, C. Elango, A. K. Singh, I. Singh, S-Nitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke, J. Cereb. Blood Flow Metab. 25 (2005) 177-192.]. In this study, we tested structurally different NO donors including GSNO, S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitroprusside (SNP), methylamine hexamethylene methylamine NONOate (MAHMA), propylamine propylamine NONOate (PAPA), 3-morpholinosydnonimine (SIN-1) and compared their neuroprotective efficacy and antioxidant property in rats after ischemia/reperfusion (I/R). GSNO, in addition to neuroprotection, decreased nitrotyrosine formation and lipid peroxidation in blood and increased the ratio of reduced versus oxidized glutathione (GSH/GSSG) in brain as compared to untreated animals. GSNO also prevented the I/R-induced increase in mRNA expression of ICAM-1 and E-Selectin. SNAP and SNP extended limited neuroprotection, reduced nitrotyrosine formation in blood and blocked increase in mRNA expression of ICAM-1 and E-Selectin in brain tissue. PAPA, MAHMA, and SIN-1 neither protected the brain nor reduced oxidative stress. We conclude that neuroprotective action of NO donors in experimental stroke depends on their ability to reduce oxidative stress both in brain and blood.

S-Nitrosoglutathione reduces inflammation and protects brain against focal cerebral ischemia in a rat model of experimental stroke.

Preservation of endothelial functions with low-dose nitric oxide (NO) and inhibition of excessive production of NO from inducible NO synthase (iNOS) is a potential therapeutic approach for acute stroke. Based on this hypothesis, an NO modulator, S-nitrosoglutathione (GSNO) was used, which provided neuroprotection in a rat model of focal cerebral ischemia. Administration of GSNO after the onset of ischemia reduced infarction and improved cerebral blood flow. To understand the mechanism of protection, the involvement of inflammation in ischemic brain injury was examined. Treatment with GSNO reduced the expression of tumor necrosis factor-alpha, interleukin-1beta, and iNOS; inhibited the activation of microglia/macrophage (ED1, CD11-b); and downregulated the expression of leukocyte function-associated antigen-1 and intercellular adhesion molecule-1 in the ischemic brain. The number of apoptotic cells (including neurons) and the activity of caspase-3 were also decreased after GSNO treatment. Further, the antiinflammatory effect of GSNO on expression of iNOS and activation of NF-kappaB machinery in rat primary astrocytes and in the murine microglial cell line BV2 was tested. Cytokine-mediated expression of iNOS and activation of NF-kappaB were inhibited by GSNO treatment. That GSNO protects the brain against ischemia/reperfusion injury by modulating NO systems, resulting in a reduction in inflammation and neuronal cell death was documented by the results.

Administration of N-acetylcysteine after focal cerebral ischemia protects brain and reduces inflammation in a rat model of experimental stroke.

Free radicals and inflammatory mediators are involved in transient focal cerebral ischemia (FCI). Preadministration of N-acetylcysteine (NAC) has been found to attenuate the cerebral ischemia-reperfusion injury in a rat model of experimental stroke. This study was undertaken to investigate the neuroprotective potential of NAC administered after ischemic events in experimental stroke. FCI was induced for 30 min by occluding the middle cerebral artery (MCA). NAC (150 mg/kg) was administered intraperitoneally at the time of reperfusion followed by another dose 6 hr later. Animals were sacrificed after 24 hr of reperfusion. The cerebral infarct consistently involved the cortex and striatum. Infarction was assessed by staining the brain sections with 2,3,5-triphenyltetrazolium chloride. Animals treated with NAC showed a significant reduction in infarct area and infarct volume and an improvement in neurologic scores and glutathione level. Reduction in infarction was significant even when a single dose of NAC was administered at 6 hr of reperfusion. Immunohistochemical and quantitative real-time PCR studies demonstrated a reduction in the expression of proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha) and interleukin 1beta (IL-1beta) and inducible nitric oxide synthase (iNOS) in NAC compared to that in vehicle-treated animals. The expression of activated macrophage/microglia (ED1) and apoptotic cell death in ischemic brain was also reduced by NAC treatment. These results indicate that in a rat model of experimental stroke, administration of NAC even after ischemia onset protected the brain from free radical injury, apoptosis, and inflammation, with a wide treatment window.

Apoptotic positive cells in Krabbe brain and induction of apoptosis in rat C6 glial cells by psychosine.

Globoid cell leukodystrophy (GLD) is an autosomal recessive disorder of infants, caused by deficient activity of cerebroside-beta-galactosidase resulting in loss of myelin accompanied by loss of oligodendrocytes. The loss of oligodendrocyte population is accompanied by accumulation of psychosine, which is considered as the molecule responsible for the observed pathophysiology of GLD. We were able to detect apoptotic cells by terminal dUTP nick-end labeling assay and nuclear localization of p53 in postmortem brain tissue of Krabbe's disease patients, which were not detected in the control brain. To study the role of psychosine in cell death, we investigated the effect of psychosine on C6 glial cell survival by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Similar to ceramide (43.8% loss) the galactopsychosine and glucopsychosine treatment killed up to 46.3 and 48.75% of cells, respectively. On the other hand, sphingosine had no effect. DNA laddering assay confirmed these results. Moreover, psychosine-induced detection of annexin-V positive cells supports a role for psychosine in C6 glial cell death via the apoptotic pathway. These results indicate that psychosine may play a role in apoptotic cell loss observed in GLD brain.

Galactosylsphingosine (psychosine)-induced expression of cytokine-mediated inducible nitric oxide synthases via AP-1 and C/EBP: implications for Krabbe disease.

Globoid cell leukodystrophy (Krabbe disease) is characterized by the accumulation of a toxic metabolite, psychosine (galactosylsphingosine), which is a substrate for the deficient enzyme (galactocerebroside beta-galactosidase). This study underscores the possible role of psychosine in the effect of inducible nitric oxide synthase (iNOS) -derived NO in the pathophysiology of this demyelinating disease. For the first time, we provide evidence of the expression of iNOS in CNS of Krabbe patient and show that the iNOS-expressing cells in the CNS were astrocytes. Psychosine potentiated the LPS-induced production of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) in primary rat astrocytes and regulated the cytokine-mediated production of NO in C6 glioma and primary rat astrocyte. Psychosine induced cytokine-mediated nuclear translocation of AP-1 and C/EBP by potentiating the expression of Fra-1 and C/EBP-delta proteins. This suggests that psychosine maintained or sustained the cytokine-primed expression of iNOS by further potentiating the nuclear translocation of AP-1 and C/EBP without modulating the cytokine-mediated transcription activity of NF-kappaB. This study hypothesizes that accumulated psychosine leads to production of cytokines and iNOS expression. The ensuing excessive production of NO and ONOO- may play a role in pathogenesis of Krabbe disease.