Efficient prevention of membrane lipid peroxidation by vitamin E (alpha-tocopherol) may involve its regeneration by vitamin C (ascorbate). Conceivably, the efficacy of antioxidants designed as therapeutic agents could be enhanced if a similar regeneration were favorable; thus, a model membrane system was developed which allowed assessment of interaction of phenolic antioxidants with ascorbate and ascorbyl-6-palmitate. Ascorbate alone (50-200 microM) potentiated oxidation of soybean phosphatidylcholine liposomes by Fe2+/histidine-Fe3+, an effect which was temporally related to reduction of Fe3+ generated during oxidation. Addition of 200 microM ascorbate to alpha-tocopherol-containing liposomes (0.1 mol%) resulted in marked, synergistic protection. Accordingly, in the presence but not absence of ascorbate, alpha-tocopherol levels were maintained relatively constant during Fe2+/histidine-Fe3+ exposure. Probucol (4,4'-[(1-methylethylidine)bis(thio)]bis[2,6-bis(1,1- dimethylethyl)]phenol), an antioxidant which prevents oxidation of low density lipoproteins, and its analogues MDL 27,968 (4,4'-[(1-methylethylidene)bis(thio)]bis[2,6- dimethyl]phenol) and MDL 28,881 (2,6-bis(1,1-dimethylethyl)-4-[(3,7,11- trimethyldodecyl)thio]phenol) prevented oxidation but exhibited no synergy with ascorbate. Ascorbyl-6-palmitate itself was an effective antioxidant but did not interact synergistically with any of the phenolic antioxidants. Differential scanning calorimetry revealed significant differences among the antioxidants in their effect on the liquid-crystalline phase transition of dipalmitoyl phosphatidylcholine (DPPC) liposomes. Both alpha-tocopherol and MDL 27,968 significantly reduced the phase transition temperature and the enthalpy of the transition. MDL 28,881 had no effect while probucol was intermediate. The potential for ascorbate or its analogues to interact with phenolic antioxidants to provide a more effective antioxidant system appears to be dictated by structural features and by the location of the antioxidants in the membrane.

␣-Phenyl-tert-butyl nitrone (PBN) is a nitrone spin trap, which has shown efficacy in animal models of oxidative stress, including stroke, aging, sepsis, and myocardial ischemia/reperfusion injury. We have prepared a series of novel cyclic variants of PBN and evaluated them for radical trapping activity in vitro. The cyclic nitrones were shown to trap ⅐ OH with MDL 101,002 being 20 -25 times more active than PBN as assessed using 2-deoxyribose and p-nitrosodimethylaniline as substrates, respectively. Trapping of ⅐ OH by MDL 101,002 was also examined by using ESR spectroscopy. When Fenton's reagent was used, the ⅐ OH adduct of MDL 101,002 yielded a six-line spectrum with hyperfine coupling constants distinct from that of PBN. Importantly, the half-life of the adduct was nearly 5 min, while that of PBN is less than 1 min at physiologic pH. MDL 101,002 also trapped the O 2 . radical to yield a six-line spectrum with coupling constants very distinct from that of the ⅐ OH adduct. In mice, the cyclic nitrones ameliorated the damaging effects of oxidative stress induced by ferrous iron injection into brain tissue. Similar protection was not afforded by the lipid peroxidation inhibitor U74006F, thus implicating radical trapping as a unique feature in the prevention of cell injury. Together, the in vivo activity, the stability of the nitroxide adducts, and the ability to distinguish between trapping of ⅐ OH and O 2 . suggest the cyclic nitrones to be ideal reagents for the study of oxidative cell injury.

Oxidative damage in the CNS is proposed to play a role in many acute and chronic neurodegenerative disorders. Accordingly, the nitrone spin trap a-phenyl-Ntert-butylnitrone (PBN), which reacts covalently with free radicals, has shown efficacy in a variety of animal models of CNS injury. We have synthesized a number of cyclic variants of PBN and examined their activity as radical traps and protectants against oxidative damage in CNS tissue. By using electron spin resonance spectroscopy, the cyclic nitrones MDL 101,002 and MDL 102,832 were shown to trap radicals in a manner similar to that of PBN. All cyclic nitrones tested prevented hydroxyl radical-dependent degradation of 2-deoxyribose and peroxyl radical-dependent oxidation of synaptosomes more potently than PBN. The radical scavenging properties of the cyclic nitrones contributed to a three-to 25-fold increase in potency relative to PBN against oxidative damage and cytotoxicity in cerebellar granule cell cultures. Similar to the phenolic antioxidant MDL 74,722, the nitrones minimized seizures and delayed the time to death in mice following central injection of ferrous iron. Although ironinduced lipid peroxidation was inhibited by MDL 74,722, the nitrones had no effect on this biochemical end point, indicating that iron-induced mortality does not result solely from lipid peroxidation and suggesting additional neuroprotective properties for the nitrones. These results indicate that cyclic nitrones are more potent radical traps and inhibitors of lipid peroxidation in vitro than PBN, and their ability to delay significantly iron-induced mortality in vivo suggests they may be useful in the treatment of acute and chronic neurodegenerat~n. Furthermore, the stability of the spin trap adducts of the cyclic nitrones provides a new tool for the study of oxidative tissue injury.

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