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.

Synthetic catalytic scavengers of reactive oxygen species (ROS) may have broad clinical applicability. In previous papers, two salen-manganese complexes, EUK-8 and EUK-134, had superoxide dismutase (SOD) and catalase activities and prevented ROS-associated tissue injury. This study describes two series of salen-manganese complexes, comparing catalytic ROS scavenging properties and cytoprotective activities. The compounds vary widely in ability to scavenge hydrogen peroxide, with this activity most influenced by salen ring alkoxy substitution and aromatic bridge modifications. In contrast, all compounds show comparable SOD activities. The most active alkoxy-substituted catalase mimetics protected cultured cells from hydrogen peroxide, and a subset of these were also neuroprotective in a rodent stroke model. Thus, structural modification of the prototype EUK-8 yields compounds with enhanced catalase activity and, in turn, biological effectiveness. This supports the concept that salen-manganese complexes represent a class of SOD and, in particular, catalase mimetics potentially useful against ROS-associated diseases.

There is both theoretical and therapeutic interest in establishing whether the signals conveyed by the enkephalins are turned off under the action of a specific peptidase which might, in this case, represent a target for a new class of psychoactive agents. Enkephalinase, a dipeptidyl carboxypeptidase cleaving the Gly3-Phe4 bond of enkephalins and distinct fropm angiotensin coverting enzyme (ACE), might be selectively involved in enkephalinergic transmission. It is a membrane-bound enzyme whose localization in the vicinity of opiate receptors in the central nervous system is suggested by parallel regional and subcellular distributions as well as by the effects of lesions. Such a role is further supported by the ontogenetic development of enkephalinase, its substrate specificity accounting for the increased biological activity of several enkephalin analogues and its adaptive increase following chronic treatment with morphine. To investigate the functional role of this enzyme further, we have designed a potent and specific enkephalinase inhibitor. We report here that this compound, thiorphan [(DL-3-mercapto-2-benzylpropanoyl)-glycine; patent no. 8008601] protects the enkephalins from the action of enkephalinase in vitro in nanomolar concentration and in vivo after either intracerebroventricular or systemic administration. In addition, thiorphan itself displays antinociceptive activity which is blocked by naloxone, an antagonist of opiate receptors.

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.

Delivery of nonlipophilic drugs to the brain is hindered by the tightly apposed capillary endothelial cells that make up the blood-brain barrier. We have examined the ability of a monoclonal antibody (OX-26), which recognizes the rat transferrin receptor, to function as a carrier for the delivery of drugs across the blood-brain barrier. This antibody, which was previously shown to bind preferentially to capillary endothelial cells in the brain after intravenous administration (Jefferies, W. A., Brandon, M. R., Hunt, S. V., Williams, A. F., Gatter, K. C. & Mason, D. Y. (1984) Nature (London) 312, 162-163), labels the entire cerebrovascular bed in a dose-dependent manner. The initially uniform labeling ofbrain capillaries becomes extremely punctate =4 hr after injection, suggesting a time-dependent sequestering of the antibody. Capillary-depletion experiments, in which the brain is separated into capillary and parenchymal fractions, show a timedependent migration of radiolabeled antibody from the capillaries into the brain parenchyma, which is consistent with the transcytosis of compounds across the blood-brain barrier.Antibody-methotrexate conjugates were tested in vivo to assess the carrier ability of this antibody. Immunohistochemical staining for either component of an OX-26-methotrexate conjugate revealed patterns of cerebrovascular labeling identical to those observed with the unaltered antibody. Accumulation of radiolabelod methotrexate in the brain parenchyma is greatly enhanced when the drug is conjugated to OX-26.The levels of various substances in the blood, such as hormones, amino acids, and ions, undergo frequent small fluctuations that can be brought about by activities such as eating and exercise (1). If the brain were not protected from these variations in serum composition, the result could be uncontrolled neural activity. The blood-brain barrier (BBB) functions to ensure that the homeostasis of the brain is maintained. Specialized characteristics of the endothelial cells that form brain capillaries are responsible for this barrier (1, 2). Brain capillary endothelial cells are joined together by tight intercellular junctions that form a continuous wall against the passive movement of substances from the blood to the brain (3, 4). These cells lack continuous gaps or channels connecting the luminal and abluminal membranes, which, in other endothelial cells, allow relatively unrestricted passage of blood-borne molecules into tissue.The isolation of the brain from the bloodstream is not complete; were this the case, the brain would be unable to function properly due to a lack ofnutrients and because ofthe need to exchange hormones and other compounds with the rest of the body. The presence of specific transport systems within the capillary endothelial cells, such as those for amino acids, transferrin, glucose, and insulin (2,(5)(6)(7)(8) A problem posed by the BBB is that, in the process of protecting the brain, it also excludes many potentially useful therapeutic agents. Currently, only sub...

Oxidative stress has been implicated in cognitive impairment in both old experimental animals and aged humans. This implication has led to the notion that antioxidant defense mechanisms in the brain are not sufficient to prevent age-related increase in oxidative damage and that dietary intake of a variety of antioxidants might be beneficial for preserving brain function. Here we report a dramatic loss of learning and memory function from 8 to 11 months of age in mice, associated with marked increases in several markers of brain oxidative stress. Chronic systemic administration of two synthetic catalytic scavengers of reactive oxygen species, Eukarion experimental compounds EUK-189 and EUK-207, from 8 to 11 months almost completely reversed cognitive deficits and increase in oxidative stress taking place during this time period in brain. In particular, increase in protein oxidation was completely prevented, whereas increase in lipid peroxidation was decreased by Ϸ50%. In addition, we observed a significant negative correlation between contextual fear learning and levels of protein oxidation in brain. These results further support the role of reactive oxygen species in age-related learning impairment and suggest potential clinical applications for synthetic catalytic scavengers of reactive oxygen species.A ging in humans, as well as in experimental animals, is associated with a slow deterioration of cognitive performance and, in particular, of learning and memory (1-5). In humans, recent studies (6) have indicated the importance of impaired learning and memory processes, because 12% of humans with mild cognitive impairment will develop Alzheimer's disease as opposed to 2% of the general population. Oxidative damage has long been proposed to be critically involved in several pathological manifestations of aging (7,8). Numerous studies (9 -12) have indeed reported increases in protein oxidation and lipid peroxidation in various regions of aged mammalian brains. These findings have led to the notion that antioxidant defense mechanisms in the brain are not sufficient to prevent age-related increase in oxidative damage and that dietary intake of a variety of antioxidants might be beneficial for preserving brain function. In agreement with this idea, synthetic catalytic molecules that exhibit both superoxide dismutase (SOD) and catalase activity significantly increase the mean and maximum lifespan in Caenorhabditis elegans (13) and alleviate lethal oxidative pathologies in mice with genetically deleted . The present studies tested, in wild-type mice, the effects of such molecules on age-related learning and memory impairment, and on markers of oxidative damage in the brain. We found that C57BL͞6N Sim mice exhibit a dramatic decrease in learning and memory function between 8 and 11 months of age, accompanied by a marked increase in brain oxidative damage. Chronic treatment of mice with two recently developed SOD͞catalase mimetics over a 3-month period almost completely reversed the age-related decline in cognitive functi...

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