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.

Recombinant human enkephalinase (neutral endopeptidase) prevents cough induced by tachykinins in awake guinea pigs.

To determine whether recombinant enkephalinase (neutral endopeptidase, EC 3.4.24.11) prevents cough induced by exogenously applied and endogenously released neuropeptides, we measured cough responses to aerosolized solutions of substance P or of capsaicin for 2 min in random-source guinea pigs before or after exposing them to aerosolized recombinant human enkephalinase. Substance P (10(-16) M) increased coughing compared with its vehicle. Enkephalinase (120 micrograms) inhibited cough induced by subsequent exposure to substance P compared with the response to substance P alone, but after further exposure to the enkephalinase inhibitor leucine-thiorphan (10(-5) M), substance P increased cough significantly. Similar results were obtained for capsaicin-induced cough. In pathogen-free guinea pigs, after they inhaled inactive recombinant enkephalinase (33 micrograms), capsaicin (10(-13) M) increased cough significantly. In contrast, after they inhaled active recombinant enkephalinase (33 micrograms), capsaicin increased cough only slightly. These results suggest that aerosolized enkephalinase reaches the sites of release or actions of endogenous neuropeptides and, by degrading them, prevents cough induced by their release. Furthermore, these studies suggest that recombinant enkephalinase might be useful in the treatment of cough and other symptoms of diseases involving peptides cleaved by this enzyme.

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.

Free radicals in reperfusion-induced arrhythmias: study with EUK 8, a novel nonprotein catalytic antioxidant.

Oxyradicals have been implicated as a possible cause of postischemic reperfusion arrhythmias (RA). However, the ability of enzymatic scavengers such as superoxide dismutase and/or catalase to reduce RA remains controversial. The purpose of the present work was to determine whether a nonprotein catalytic antioxidant, EUK 8, may limit RA in isolated heart preparations. The catalytic dismutation of H2O2 by EUK 8 was demonstrated using a Clark electrode. EUK 8's ability to scavenge oxyradicals was studied in vitro by electron spin resonance (ESR) in presence of superoxide-anion generating system. ESR concentration-effect curves obtained led us to use EUK 8 at 50 mumol/l in isolated heart preparations. Isolated rat hearts were submitted to 10 min regional ischemia induced by left coronary artery ligation. Reperfusion was achieved by releasing the coronary ligation, and the incidence and duration of early ventricular arrhythmias were then investigated. In the treated-group, EUK 8 was added to the perfusion fluid (50 mumol/l) 90 s before reperfusion. Our results show that EUK 8 significantly reduced the severity of RA as assessed by the arrhythmia score measurement (control: 3.46 +/- 0.21 vs. EUK 8: 2.73 +/- 0.27, p < .05). In conclusion, EUK 8 is able to limit RA in our experimental model. This effect might be related to the catalytic antioxidant properties of this complex.

Molecular cloning and amino acid sequence of human enkephalinase (neutral endopeptidase).

We have isolated a cDNA clone encoding human enkephalinase (neutral endopeptidase, EC 3.4.24.11) in a lambda gt10 library from human placenta, and present the complete 742 amino acid sequence of human enkephalinase. The human enzyme displays a high homology with rat and rabbit enkephalinase. Like the rat and rabbit enzyme, human enkephalinase contains a single N-terminal transmembrane region and is likely to be inserted through cell membranes with the majority of protein, including its carboxy-terminus, located extracellularly.

Brain-specific gene expression.

The brain of an adult rat expresses approximately 30,000 different brain-specific mRNAs. To investigate their encoded proteins, we have selected cDNA clones corresponding to mRNAs expressed exclusively in rat brain, determined their nucleotide sequences and generated antisera against synthetic peptides mimicking short regions of the deduced protein sequences. The clone plB236 encodes a protein that defines a widely distributed neuronal system and may be the precursor for a family of novel neuropeptides. A second clone, plB208, encodes rat brain proteolipid protein, the major protein component of central nervous system myelin. These studies have also identified an 82 nucleotide genetic element called an ID (identifier) sequence that may be involved in the regulation of transcription of brain-specific genes.

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.

Effects of EUK-8, a synthetic catalytic superoxide scavenger, on hypoxia- and acidosis-induced damage in hippocampal slices.

Anoxia produces deleterious effects on synaptic transmission in the hippocampal slice preparation. A proposed source of damage is the superoxide radical (.O2-) produced during the earlier period of reoxygenation. The present study tested the effects of a synthetic, catalytic superoxide radical scavenger (EUK-8) on CA1 pyramidal cell responses elicited by electrical stimulation of the Schaffer-commissural pathway after severe anoxic episodes. Following reoxygenation, slices incubated with EUK-8 (50 microM) exhibited significantly better recovery of excitatory postsynaptic potentials (EPSPs) than control slices. In addition, repeated episodes of anoxia produced irreversible loss of synaptic transmission in the majority of control slices (93 +/- 7%, n = 15), compared to a small fraction in EUK-8-incubated slices (27 +/- 12%, n = 15). A thiobarbituric acid (TBA) test was used to assess the effect of EUK-8 on lipid peroxidation elicited in hippocampal slices by acidosis and lactic acid (pH 5.0 and 30 mM lactic acid). Incubation in the presence of EUK-8 totally prevented the increase in lipid peroxidation produced by acidosis and lactic acid in both the incubation medium and the slice homogenates. These results indicate that a superoxide scavenger like EUK-8 prevents damage produced by acidosis and anoxia in hippocampal slices and suggest the possibility of using this type of molecule under various pathological conditions.

EUK-134, a synthetic superoxide dismutase and catalase mimetic, protects rat kidneys from ischemia-reperfusion-induced damage.

The effect of a new synthetic superoxide dismutase and catalase mimetic was investigated on renal ischemia-reperfusion syndrome in rats. Synthetic salen-manganese complexes have characteristics that might facilitate their potential usefulness as therapeutic agents: (1) unlike proteinaceous antioxidant enzymes, synthetic complexes, due to their low molecular weight, have a better stability and bioavailability; (2) they have a catalytic activity enhancing their efficiency over noncatalytic reactive oxygen metabolite scavengers; and finally, (3) exhibiting combined superoxide dismutase and catalase activity, they destroy both superoxide anions and hydrogen peroxides, thereby enhancing their protective effect on ischemically injured tissues. One such compound, EUK-134, was tested in uninephrectomized rats that underwent a left renal artery clamping. After a 75-min left renal artery clamping, a single intravenous injection of EUK-134 at 0.2 mg/kg, just before unclamping, provided significantly better renal function recovery during the week after the ischemic insult compared with recovery of untreated animals. Two hours after several periods of renal ischemia (30, 45, 60, and 75 min of left renal artery clamping), EUK-134 given at a similar dose significantly improved the glomerular filtration rate after an acute ischemia of 30 and 45 min, as assessed by EDTA 51Cr. Overall, these results show that synthetic superoxide dismutase-catalase mimetics such as EUK-134 can protect ischemically injured rat kidneys from ischemia-reperfusion syndrome when administered just before reperfusion.

Characterization of endopeptidase 3.4.24.11 ("enkephalinase") activity in human plasma and cerebrospinal fluid.

The presence of the neutral metallo-endopeptidase 3.4.24.11 ("enkephalinase") activity was investigated by fluorimetric assay in human body fluids. Although the enzyme was previously known to occur exclusively in membrane bound form in the human or animal central nervous system, its activity was detected in human cerebrospinal fluid (CSF), plasma and amniotic fluid. Although the endopeptidase 3.4.24.11 activity found in human body fluids has properties closely related to the membrane bound enzyme such as affinity constant for the inhibitors, optimal pH and Km for the substrate, the Vmax values were in CSF, plasma and amniotic fluid, respectively, 2 x 10(3), 10(2), 10 lower than mouse brain homogenate.

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.

Role of oxidant stress in the adult respiratory distress syndrome: evaluation of a novel antioxidant strategy in a porcine model of endotoxin-induced acute lung injury.

Reactive oxygen metabolites (ROMs) are thought to play a key role in the pathogenesis of the adult respiratory distress syndrome (ARDS). Accordingly, the use of ROM scavengers, such as N-acetyl-cysteine or dimethylthiourea, as therapeutic adjuncts to prevent oxidant-mediated damage to the lung have been evaluated extensively in animal models of ARDS. Results with this approach have been quite variable among studies. Another strategy that has been examined in animal models of ARDS is the administration of various enzymes, particularly superoxide dismutase (SOD) or catalase (CAT), in an effort to promote the conversion of ROMs to inactive metabolites. In theory, this strategy should be more effective than the use of ROM scavengers since a single molecule of a catalytically active molecule can neutralize a large number of molecules of a reactive species, whereas most scavengers act in a stoichiometric fashion to neutralize radicals on a mole-for-mole basis. This notion is supported by studies showing that prophylactic treatment with CAT provides impressive protection against acute lung injury induced in experimental animals by the administration of lipopolysaccharide (LPS). Results with SOD have been more variable. Recently, we have utilized a porcine model of LPS-induced ARDS to investigate the therapeutic potential of EUK-8, a novel, synthetic, low molecular salen-manganese complex that exhibits both SOD-like and CAT-like activities in vitro. Using both pre- and post-treatment designs, we have documented that treatment with EUK-8 significantly attenuates many of the features of LPS-induced acute lung injury, including arterial hypoxemia, pulmonary hypertension, decreased dynamic pulmonary compliance, and pulmonary edema. These findings support the view that salen-manganese complexes warrant further evaluation as therapeutic agents for treatment or prevention of sepsis-related ARDS in humans.

Substance P contracts the human iris sphincter: possible modulation by endogenous enkephalinase.

Substance P-immunoreactive neurons have been found in the irides of many species including humans. In several species, substance P has been shown to induce contraction of the sphincter muscle but this action of substance P has not been previously demonstrated in the human eye. Using an eye cup model in which the sensitivity of the iris muscle to substance P is increased compared to the isolated sphincter muscle, we have observed that nanomolar amounts of substance P induced contraction of the sphincter in the human iris. This contractile response was enhanced in eyes pretreated with thiorphan, an enkephalinase inhibitor, suggesting that endogenous enkephalinase (E.C. 3.4.24.11) may modulate the substance P contraction in the human iris. Further support for this hypothesis was the finding of enkephalinase-like immunoreactivity and enzyme activity in the human iris sphincter muscle.

Localization of enkephalinase mRNA in rat brain by in situ hybridization: comparison with immunohistochemical localization of the protein.

The messenger RNA (mRNA) encoding enkephalinase (EC. 3.4.24.11; neutral endopeptidase) has been localized in rat brain by in situ hybridization using 35S- or 32P-labelled cRNA probes. Hybridization was observed only in few brain areas, and was particularly strong in the striatum, olfactory bulb and pontine nuclei. The enkephalinase protein was also localized in brain sections using a radiolabelled monoclonal antibody. While some brain regions contained both the mRNA and its translation product, others, including in particular the substantia nigra, were rich in enkephalinase but did not contain any detectable amount of enkephalinase mRNA. Enkephalinase mRNA-containing cells could be identified in regions containing neurons known to project to the substantia nigra. The discrepancy between the mRNA and the protein labelling is likely to reflect the fact that the mRNA is exclusively located within the soma of the cells while the translated protein may be found anywhere along the axonal processes.

Regional distribution of a high-affinity enkephalin-degrading peptidase ('enkephalinase') and effects of lesions suggest localization in the vicinity of opiate receptors in brain.

The distribution of a high affinity enkephalin-dipeptidylcarboxypeptidase between regions of mouse brain is markedly heterogenous and parallels that of opiate receptors. Furthermore intrastriatal administration of kainic acid as well as interruption of the nigrostriatal dopaminergic pathway by 6-hydroxydopamine (6-OHDA) lead to similar decreases in this peptidase activity and in the number of opiate receptors. On the contrary, no correlation was found between low affinity enkephalin degrading enzymes and opiate receptors.

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.

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.

Protective effect of the superoxide scavenger EUK8 against ultrastructural alterations induced by ischemia and reperfusion in isolated rat hearts.

We have recently shown that iron overload may increase the susceptibility of isolated rat hearts to ischemia and reperfusion, an effect that could be related to an iron-dependent activation of the Fenton reaction, thus producing cytotoxic hydroxyl radicals. The purpose of our study was to investigate the influence of EUK8, a new catalytic scavenger of superoxide anions that is similar to superoxide dismutase, on cardiac ultrastructural alterations associated with reperfusion. Experiments were carried out ex vivo in iron-overloaded rat hearts perfused at a constant flow rate (11 ml/min) with (EUK8) or without (control) EUK8 (50 mumol/L) throughout the perfusion. Hearts were submitted to 15 min global normothermic no-flow ischemia and then reperfused for 15 min before examination. For each heart, six micrographs at a magnification of x 11,300 were used to quantitate ultrastructural alterations. In our experimental model, EUK8 prevented some of the ultrastructural modifications associated with ischemia and reperfusion in iron-overloaded rat hearts. This beneficial effect could be related to the limitation of reperfusion-induced oxygen free radical production under the effect of EUK8.

Recombinant enkephalinase effectively inhibits substance P-induced miosis in the rabbit eye cup model.

Enkephalinase (EC 3.4.24.11) is a naturally occurring, membrane-bound peptidase that degrades substance P in vivo and in vitro. Addition of this neutral endopeptidase to a rabbit eye cup model partially inhibits substance P-induced contraction of the iris sphincter muscle. Inactivation of substance P is reversed by thiorphan, a specific inhibitor of enkephalinase. These results show that enkephalinase degradation of substance P produces metabolites that are physiologically inactive in iris contraction. We also observed that atropine acts synergistically with enkephalinase to completely abolish substance P-induced iris contraction suggesting that the action of substance P on the iris contains an acetylcholine-stimulatory effect which is not lost by enkephalinase treatment.