Mitigating effect of EUK-207 on radiation-induced cognitive impairments.

The brain could be exposed to irradiation as part of a nuclear accident, radiological terrorism (dirty bomb scenario) or a medical radiological procedure. In the context of accidents or terrorism, there is considerable interest in compounds that can mitigate radiation-induced injury when treatment is initiated a day or more after the radiation exposure. As it will be challenging to determine the radiation exposure an individual has received within a relatively short time frame, it is also critical that the mitigating agent does not negatively affect individuals, including emergency workers, who might be treated, but who were not exposed. Alterations in hippocampus-dependent cognition often characterize radiation-induced cognitive injury. The catalytic ROS scavenger EUK-207 is a member of the class of metal-containing salen manganese (Mn) complexes that suppress oxidative stress, including in the mitochondria, and have been shown to mitigate radiation dermatitis, promote wound healing in irradiated skin, and mitigate vascular injuries in irradiated lungs. As the effects of EUK-207 against radiation injury in the brain are not known, we assessed the effects of EUK-207 on sham-irradiated animals and the ability of EUK-207 to mitigate radiation-induced cognitive injury. The day following irradiation or sham-irradiation, the mice started to receive EUK-207 and were cognitively tested 3 months following exposure. Mice irradiated at a dose of 15Gy showed cognitive impairments in the water maze probe trial. EUK-207 mitigated these impairments while not affecting cognitive performance of sham-irradiated mice in the water maze probe trial. Thus, EUK-207 has attractive properties and should be considered an ideal candidate to mitigate radiation-induced cognitive injury.

Mitigation of radiation-induced lung injury with EUK-207 and genistein: effects in adolescent rats.

Exposure of civilian populations to radiation due to accident, war or terrorist act is an increasing concern. The lung is one of the more radiosensitive organs that may be affected in people receiving partial-body irradiation and radiation injury in lung is thought to be associated with the development of a prolonged inflammatory response. Here we examined how effectively damage to the lung can be mitigated by administration of drugs initiated at different times after radiation exposure and examined response in adolescent animals for comparison with the young adult animals that we had studied previously. We studied the mitigation efficacy of the isoflavone genistein (50 mg/kg) and the salen-Mn superoxide dismutase-catalase mimetic EUK-207 (8 mg/kg), both of which have been reported to scavenge reactive oxygen species and reduce activity of the NFkB pathway. The drugs were given by subcutaneous injection to 6- to 7-week-old Fisher rats daily starting either immediately or 2 weeks after irradiation with 12 Gy to the whole thorax. The treatment was stopped at 28 weeks post irradiation and the animals were assessed for levels of inflammatory cytokines, activated macrophages, oxidative damage and fibrosis at 48 weeks post irradiation. We demonstrated that both genistein and EUK-207 delayed and suppressed the increased breathing rate associated with pneumonitis. These agents also reduced levels of oxidative damage (50-100%), levels of TGF-ß1 expression (75-100%), activated macrophages (20-60%) and fibrosis (60-80%). The adolescent rats developed pneumonitis earlier following irradiation of the lung than did the adult rats leading to greater severe morbidity requiring euthanasia (∼37% in adolescents vs. ∼10% in young adults) but the extent of the mitigation of the damage was similar or slightly greater.

Mitigation of lung injury after accidental exposure to radiation.

There is a serious need to develop effective mitigators against accidental radiation exposures. In radiation accidents, many people may receive nonuniform whole-body or partial-body irradiation. The lung is one of the more radiosensitive organs, demonstrating pneumonitis and fibrosis that are believed to develop at least partially because of radiation-induced chronic inflammation. Here we addressed the crucial questions of how damage to the lung can be mitigated and whether the response is affected by irradiation to the rest of the body. We examined the widely used dietary supplement genistein given at two dietary levels (750 or 3750 mg/kg) to Fischer rats irradiated with 12 Gy to the lung or 8 Gy to the lung + 4 Gy to the whole body excluding the head and tail (whole torso). We found that genistein had promising mitigating effects on oxidative damage, pneumonitis and fibrosis even at late times (36 weeks) when drug treatment was initiated 1 week after irradiation and stopped at 28 weeks postirradiation. The higher dose of genistein showed no greater beneficial effect. Combined lung and whole-torso irradiation caused more lung-related severe morbidity resulting in euthanasia of the animals than lung irradiation alone.

Using superoxide dismutase/catalase mimetics to manipulate the redox environment of neural precursor cells.

Past work has shown that neural precursor cells are predisposed to redox sensitive changes, and that oxidative stress plays a critical role in the acute and persistent changes that occur within the irradiated CNS. Irradiation leads to a marked rise in reactive oxygen species (ROS) that correlates with oxidative endpoints in vivo and reductions in neurogenesis. To better understand the impact of oxidative stress on neural precursor cells, and to determine if radiation-induced oxidative damage and precursor cell loss after irradiation could be reduced, a series of antioxidant compounds (EUK-134, EUK-163, EUK-172, EUK-189) were tested, three of which possess both superoxide dismutase (SOD) and catalase activities and one (EUK-163) whose only significant activity is SOD. Our results show that these SOD/catalase mimetics apparently increase the oxidation of a ROS-sensitive fluorescent indicator dye, particularly after short (12 h) treatments, but that longer treatments (24 h) decrease oxidation attributable to radiation-induced ROS. Similarly, other studies found that cells incubated with CuZnSOD showed some increase in intracellular ROS levels. Subsequent data suggested that the dye-oxidising capabilities of the EUK compounds were linked to differences in their catalase activity and, most likely, their ability to catalyse peroxidative pathways. In unirradiated mice, the EUK-134 analogue induced some decrease of proliferating precursor cells and immature neurons 48 h after radiation, an effect that may be attributable to cytotoxicity and/or inhibition of precursor proliferation. In irradiated mice, a single injection of EUK-134 was not found to be an effective radioprotector at acute times (48 h). The present results support continued development of our in vitro model as a tool for predicting certain in vivo responses, and suggest that in some biological systems the capability to scavenge superoxide but produce excess H(2)O(2), as is known for CuZnSOD, may be potentially deleterious. Our results also show that the ability of catalase mimetics, like true catalases, to catalyse peroxidase reactions can complicate the interpretation of data obtained with certain fluorescent ROS-indicator dyes.

Lifespan extension and rescue of spongiform encephalopathy in superoxide dismutase 2 nullizygous mice treated with superoxide dismutase-catalase mimetics.

Superoxide is produced as a result of normal energy metabolism within the mitochondria and is scavenged by the mitochondrial form of superoxide dismutase (sod2). Mice with inactivated SOD2 (sod2 nullizygous mice) die prematurely, exhibiting several metabolic and mitochondrial defects and severe tissue pathologies, including a lethal spongiform neurodegenerative disorder (Li et al., 1995; Melov et al., 1998, 1999). We show that treatment of sod2 nullizygous mice with synthetic superoxide dismutase (SOD)-catalase mimetics extends their lifespan by threefold, rescues the spongiform encephalopathy, and attenuates mitochondrial defects. This class of antioxidant compounds has been shown previously to extend lifespan in the nematode Caenorhabditis elegans (Melov et al., 2000). These new findings in mice suggest novel therapeutic approaches to neurodegenerative diseases associated with oxidative stress such as Friedreich ataxia, spongiform encephalopathies, and Alzheimer's and Parkinson's diseases, in which chronic oxidative damage to the brain has been implicated.

Attenuation of staurosporine-induced apoptosis, oxidative stress, and mitochondrial dysfunction by synthetic superoxide dismutase and catalase mimetics, in cultured cortical neurons.

Neuronal apoptosis induced by staurosporine (STS) involves multiple cellular and molecular events, such as the production of reactive oxygen species (ROS). In this study, we tested the efficacy of two synthetic superoxide dismutase/catalase mimetics (EUK-134 and EUK-189) on neuronal apoptosis, oxidative stress, and mitochondrial dysfunction produced by STS in primary cortical neuronal cultures. Exposure of cultures to STS for 24 h increased lactate dehydrogenase (LDH) release, the number of apoptotic cells, and decreased trypan blue exclusion. Pretreatment with 20 microM EUK-134 or 0.5 microM EUK-189 significantly attenuated STS-induced neurotoxicity, as did pretreatment with the caspase-1 inhibitor, Ac-YVAD-CHO, but not the caspase-3 inhibitor, Ac-DEVD-CHO. Posttreatment (1-3 h following STS exposure) with 20 microM EUK-134 or 0.5 microM EUK-189 significantly reduced STS-induced LDH release, in a time-dependent manner. Exposure of cultures to STS for 1 h produced an elevation of ROS, as determined by increased levels of 2,7-dichlorofluorescein (DCF). This rapid elevation of ROS was followed by an increase in lipid peroxidation, and both the increase in DCF fluorescence and in lipid peroxidation were significantly blocked by pretreatment with EUK-134. STS treatment for 3-6 h increased cytochrome c release from mitochondria into the cytosol, an effect also blocked by pretreatment with EUK-134. These results indicate that intracellular oxidative stress and mitochondrial dysfunction are critically involved in STS-induced neurotoxicity. However, there are additional cellular responses to STS, which are insensitive to treatment with radical scavengers that also contribute to its neurotoxicity.

Oxidative signalling and inflammatory pathways in Alzheimer's disease.

It is well established that inflammation and oxidative stress are key components of the pathology of Alzheimer's disease (AD), but how early in the pathological cascade these processes are involved or which specific molecular components are key, has not been fully elucidated. This paper describes the pharmacological approach to understand the molecular components of inflammation and oxidative stress on the activation of microglial cells and neuronal cell viability. We have shown that activation of microglia with the 42-amino-acid form of the beta-amyloid peptide (A beta 42) activates the production of cyclooxygenase-2, the inducible form of nitric oxide synthase and tumour necrosis factor-alpha and there appears to be little interactive feedback between these three mediators. Moreover, we explore the effects of a series of salen-manganese complexes, EUK-8, -134 and -189, which are known to possess both superoxide and catalase activity. These compounds are able to protect cells from insults produced by hydrogen peroxide or peroxynitrite. Moreover, EUK-134 was also able to limit the output of prostaglandin E2 from activated microglial cells. The mechanisms underlying these effects are discussed. Together, these data support a pivotal role for oxidative stress and inflammation as key mediators of the pathological cascade in AD and provide some ideas about possible therapeutic targets.

Synthetic superoxide dismutase/catalase mimetics reduce oxidative stress and prolong survival in a mouse amyotrophic lateral sclerosis model.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that causes motoneuron degeneration, paralysis and death. Mutations in Cu, Zn superoxide dismutase (SOD1) are one cause of this disease. It is widely suspected that increased reactive oxidative species (ROS) is involved in motoneuron degeneration but whether such an involvement plays a role in ALS progression in vivo is uncertain. We treated mice expressing human mutant SOD1 G93A with EUK-8 and EUK-134, two synthetic SOD/catalase mimetics that have shown efficacy in several animal models of human diseases. These treatments reduced levels of oxidative stress and prolonged survival. The results suggest that oxidative stress plays an active role in ALS and illustrate the potential for treatment strategies aimed specifically against ROS.

6,6'-Bis(2-hydroxyphenyl)-2,2'-bipyridine manganese(III) complexes: a novel series of superoxide dismutase and catalase mimetics.

A series of novel manganese(III) complexes is described based on a 6,6'-bis(2-hydroxyphenyl)-2,2'-bipyridine template. These complexes show superoxide dismutase and catalase activity. The effect of the aromatic substitution pattern on the SAR is described.

Prevention of 1-methyl-4-phenylpyridinium- and 6-hydroxydopamine-induced nitration of tyrosine hydroxylase and neurotoxicity by EUK-134, a superoxide dismutase and catalase mimetic, in cultured dopaminergic neurons.

Oxidative stress has been implicated in the selective degeneration of dopaminergic (DAergic) neurons in Parkinson's disease (PD). In this study, we tested the efficacy of EUK-134, a superoxide dismutase (SOD) and catalase mimetic, on the nitration of tyrosine hydroxylase (TH), a marker of oxidative stress, and neurotoxicity produced by 1-methyl-4-phenylpyridinium (MPP(+)) and 6-hydroxydopamine (6-OHDA) in primary DAergic neuron cultures. Exposure of cultures to 10 microM MPP(+) reduced dopamine (DA) uptake and the number of tyrosine hydroxylase immunoreactive (THir) neurons to 56 and 52% of control, while exposure to 30 microM 6-OHDA reduced DA uptake and the number of THir neurons to 58 and 59% of control, respectively. Pretreatment of cultures with 0.5 microM EUK-134 completely protected DAergic neurons against MPP(+)- and 6-OHDA-induced neurotoxicity. Exposure of primary neuron cultures to either MPP(+) or 6-OHDA produced nitration of tyrosine residues in TH. Pretreatment of cultures with 0.5 microM EUK-134 completely prevented MPP(+)- or 6-OHDA-induced nitration of tyrosine residues in TH. Taken together, these results support the idea that reactive oxygen species (ROS) are critically involved in MPP(+)- and 6-OHDA-induced neurotoxicity and suggest a potential therapeutic role for synthetic catalytic scavengers of ROS, such as EUK-134, in the treatment of PD.

Extension of life-span with superoxide dismutase/catalase mimetics.

We tested the theory that reactive oxygen species cause aging. We augmented the natural antioxidant systems of Caenorhabditis elegans with small synthetic superoxide dismutase/catalase mimetics. Treatment of wild-type worms increased their mean life-span by a mean of 44 percent, and treatment of prematurely aging worms resulted in normalization of their life-span (a 67 percent increase). It appears that oxidative stress is a major determinant of life-span and that it can be counteracted by pharmacological intervention.

Oxidative neuropathology and putative chemical entities for Alzheimer's disease: neuroprotective effects of salen-manganese catalytic anti-oxidants.

Considerable evidence exists that the brains of individuals with Alzheimer's disease are subject to elevated levels of oxidative stress, particularly in regions exhibiting pathological damage. A major contributor to this oxidative stress appears to be the inflammatory process. Activation of rodent microglial cells by LPS or beta-amyloid peptide results in a marked up-regulation of inducible nitric oxide synthase (iNOS) and corresponding nitric oxide (NO) production. Elevated levels of iNOS are also observed in the brains of Alzheimer patients. The reaction of NO with superoxide leads to the generation of the highly reactive and damaging peroxynitrite free radical species. Peroxynitrite appears to play a key role in the generation of an oxidative stress in the Alzheimer brain as evidenced by widespread nitrotyrosine immunoreactivity. We have employed SIN-1 as a peroxynitrite generating system in cell cultures in order to characterize the effects of this free radical on neurons. SIN-1 treatment of primary rat hippocampal neurons in culture results in neurotoxicity by a necrosis mechanism according to electron microscopic criteria. One approach to limiting peroxynitrite mediated damage is to limit superoxide production. An approach we have evaluated is treatment with salen manganese compounds, a class of catalytic antioxidant compounds which behave as superoxide dismutase (SOD)/catalase mimetics to detoxify superoxide. A number of such salen manganese compounds, including EUK-8 and EUK-134, can markedly protect primary rat cortical neurons from hydrogen peroxide mediated oxidative stress. Such salen manganese compounds can similarly afford marked neuroprotection to an oxidative stress imposed by SIN-1, potentially attributable at least in part to their inherent SOD activity. The salen manganese SOD/catalase mimetics represent a promising class of catalytic antioxidant for attenuating oxidative stress.

EUK-134, a synthetic superoxide dismutase and catalase mimetic, prevents oxidative stress and attenuates kainate-induced neuropathology.

The present study tested the effects of EUK-134, a synthetic superoxide dismutase/catalase mimetic, on several indices of oxidative stress and neuropathology produced in the rat limbic system as a result of seizure activity elicited by systemic kainic acid (KA) administration. Pretreatment of rats with EUK-134 did not modify the latency for or duration of KA-induced seizure activity. It did produce a highly significant reduction in increased protein nitration, activator protein-1- and NF-kappaB-binding activity, and spectrin proteolysis as well as in neuronal damage resulting from seizure activity in limbic structures. These results support the hypothesis that kainate-induced excitotoxicity is caused, at least in part, by the action of reactive oxygen species. Furthermore, they suggest that synthetic superoxide dismutase/catalase mimetics such as EUK-134 might be used to prevent excitotoxic neuronal injury.

Growth regulation of cultured endothelial cells by inflammatory cytokines: mitogenic, anti-proliferative and cytotoxic effects.

The inflammatory cytokines tumor necrosis factor-alpha (TNF alpha) and interleukin-1 alpha (IL-1 alpha) have angiogenic properties but generally inhibit cultured endothelial cell (EC) proliferation. Investigations into the growth-regulatory effects of these two agents on a variety of cultured EC types showed that they exert mitogenic, anti-proliferative or cytotoxic effects depending upon cell type and cytokine combinations. The anti-proliferative effect was distinct from cytotoxicity. Nitric oxide (NO) release from EC, examined as a potential mechanism underlying some of these effects, did not appear to mediate the anti-proliferative effects of these cytokines. However, NO also seemed to have a bimodal effect on EC proliferation depending upon whether the NO was endogenous or exogenous. These data underscore the diversity in cytokine and NO effects on cultured EC which, if reproducible in vivo, may be partly responsible for the variable and sometimes contradictory results obtained with regards to the role of inflammatory cytokines and NO on angiogenesis.

Synthetic combined superoxide dismutase/catalase mimetics are protective as a delayed treatment in a rat stroke model: a key role for reactive oxygen species in ischemic brain injury.

Stroke is a severe and prevalent syndrome for which there is a great need for treatment, including agents to block the cascade of brain injury that occurs in the hours after the onset of ischemia. Reactive oxygen species (ROS) have been implicated in this destructive process, but antioxidant enzymes such as superoxide dismutase (SOD) have been unsatisfactory in experimental stroke models. This study is an evaluation of the effectiveness of salen-manganese complexes, a class of synthetic SOD/catalase mimetics, in a rat focal ischemia model involving middle cerebral artery occlusion. We focus on EUK-134, a newly reported salen-manganese complex demonstrated here to have greater catalase and cytoprotective activities and equivalent SOD activity compared with the previously described prototype EUK-8. The administration of EUK-134 at 3 hr after middle cerebral artery occlusion significantly reduced brain infarct size, with the highest dose apparently preventing further infarct growth. EUK-8 was also protective but substantially less effective. These findings support a key role for ROS in the cascade of brain injury after stroke, even well after the onset of ischemia. The enhanced activity of EUK-134 suggests that, in particular, hydrogen peroxide contributes significantly to this injury. Overall, this study suggests that synthetic SOD/catalase mimetics might serve as novel, multifunctional therapeutic agents for stroke.

Role of oxidant stress and iron delocalization in acidosis-induced intestinal epithelial hyperpermeability.

Using Caco-2BBe monolayers as a model of the intestinal epithelium, we tested the hypothesis that reactive oxygen metabolites contribute to lactic acid-induced hyperpermeability. Compared to monolayers incubated at normal pH (i.e., 7.4) monolayers incubated in medium titrated to extracellular pH (pHo) 5.0 with 10 mM lactic acid demonstrated increased permeability to both fluorescein sulfonic acid (FS) and fluorescein isothiocyanate-dextran (average molecular mass = 4000 Da; FD4). Lactic acid-induced hyperpermeability to both FS and FD4 was reduced by adding either 30 microM EUK-8, a superoxide dismutase/catalase mimetic, or catalase (10(4) U/mL). Incubation of monolayers with lactic acid increased cellular malondialdehyde content, a measure of lipid peroxidation. EUK-8 (30 microM) completely abrogated this effect. Incubation with ferrous sulfate (100 microM) exacerbated both lactic acid-induced hyperpermeability to FS and lactic acid-induced lipid peroxidation. Iron chelation with 1 mM diethylene triamine pentaacetic acid (DTPA)-trisodium calcium salt attenuated lactic acid-induced hyperpermeability, whereas iron-loaded DTPA (1 mM) was not protective. Treatment with DTPA-trisodium calcium salt also ameliorated lactic acid-induced lipid peroxidation. Incubation with lactic acid (pHo 5.0) for 16 h increased the cellular content of low molecular weight iron species. Incubation with lactic acid (pHo 5.0) for 24 h significantly increased the percentage of oxidized protein-bound thiols in Caco-2BBe cells. We conclude that lactic acidosis induces hyperpermeability in Caco-2BBe monolayers, in part, via an iron-dependent increase in reactive oxygen metabolite-mediated damage.

Prevention and suppression of autoimmune encephalomyelitis by EUK-8, a synthetic catalytic scavenger of oxygen-reactive metabolites.

Breakdown of T cell tolerance to self-myelin basic protein induces an autoimmune process that leads to demyelination of the central nervous system (CNS) in multiple sclerosis (MS) patients. While the autoimmune disease is initiated by antigen-specific autoreactive T cells, there is accumulating evidence that CNS injury is essentially mediated by CNS-infiltrating inflammatory cells. In addition, it is established that activated macrophages and polymorphonuclear cells contribute to tissue damage in several inflammatory diseases by releasing highly reactive oxygen metabolites. It was therefore possible that demyelination associated with MS results from oxidative injury caused by a cascade of oxygen reactive metabolites produced by CNS-infiltrating activated macrophages and other inflammatory cells. To address this question, we tested the effect of a synthetic catalytic scavenger of oxygen radicals, EUK-8, on experimental allergic encephalomyelitis (EAE) in mice, the animal model for MS in humans. We observed that repeated injection of EUK-8 starting at the time of EAE induction delayed the onset and markedly reduced the severity of the disease. Strikingly, all EUK-8-treated mice completely recovered after 40 days. In addition, we showed that posttreatment with EUK-8 4 days after EAE induction also resulted in a significant amelioration of EAE disease. These results indicate that oxygen metabolites secreted by inflammatory cells at the site of tissue destruction play a major role in the induction and presumably the perpetuation of the autoimmune disease. This study also suggests that treatment with oxygen metabolites scavengers may represent a novel and promising strategy to prevent the onset and to block the course of ongoing autoimmune encephalomyelitis and other inflammatory autoimmune diseases.

A lipidated anti-Tat antibody enters living cells and blocks HIV-1 viral replication.

We have developed a chemical modification of antibodies, lipidation, which enables their intracellular delivery into living cells. Intracellular localization of lipidated antibodies was demonstrated by confocal microscopy and by measuring cellular uptake of 125I-labeled lipidated antibodies. Functionally, a lipidated monoclonal antibody directed against the Tat protein from human immunodeficiency virus type 1 (HIV-1) inhibited viral replication of several HIV-1 isolates by approximately 85% as shown by increased viability of infected cells and decreased reverse transcriptase activity. The antibody in its native form had no such effect. These data show that lipidated antibodies can reach and functionally inhibit intracellular targets. Lipidation may help to facilitate the development of intracellular immunotherapy for AIDS.

A competitive chemiluminescent enzyme-linked immunosorbent assay for the determination of RMP-7 in human blood.

RMP-7, a bradykinin agonist, is a synthetic nonapeptide designed to enhance the delivery of therapeutics to the central nervous system. A sensitive, competitive chemiluminescent enzyme-linked immunosorbent assay (ELISA) for quantifying RMP-7 in human blood samples has been developed. Rabbit antibodies against RMP-7 were produced using the conjugate of RMP-7 to keyhole limpet hemocyanin through glutaraldehyde. Biotinylated RMP-7, conjugated via N-hydroxysuccinimide ester, was used as the tracer. A premixed solution of biotinylated alkaline phosphatase and avidin was used to quantify the tracer, with a dioxetane-based compound as the chemiluminescent substrate. The method involves treating blood samples with organic solvents to precipitate proteins, evaporating the supernatants to dryness, reconstituting residues in PBS and assaying the buffer solutions with the ELISA. The assay, using 1.0 ml of whole blood, has precision and accuracy within +/- 20% over the concentration range 25-800 pg ml-1. There are no significant endogenous interferences. The assay has been successfully used to support clinical trials of RMP-7.