Iron-initiated tissue oxidation: lipid peroxidation, vitamin E destruction and protein thiol oxidation. Inhibition by a novel antioxidant, U-78517F.

Oxidative injury was initiated by addition of ferrous ammonium sulfate (FAS) to a suspension of whole rat brain homogenate in Krebs buffer. After FAS addition, tissue vitamin E dropped sharply over a 30-sec interval and then recovered marginally for 5 min. After 5 min, vitamin E levels dropped to a low and constant level. Also after 5 min, TBARS (thiobarbituric acid reactive substances, a color test for lipid peroxidation) showed a statistically significant (P < or = 0.05) increase that continued through the remainder of the 30-min experiment. Reduced protein thiols decreased significantly (P < or = 0.05) at 15 min post FAS addition. This suggests that, in this model of iron-initiated lipid peroxidation (LP), the endogenous antioxidant vitamin E is first depleted before membrane lipids and membrane bound proteins show evidence of oxidative injury. A novel antioxidant, U-78517F, inhibited the destruction of vitamin E, LP and protein thiol oxidation in this model. The efficacy of the compound after different times of addition is described.

Comparison of the efficacy of mechanistically different antioxidants in the rat hemorrhagic shock model.

Four pharmacological mechanisms for antagonizing free radical generation or reactions were compared in terms of their efficacy in attenuating hemorrhagic shock in rats. These included opposing superoxide generation by xanthine oxidase (e.g., oxypurinol), inhibiting arachidonic acid oxidation by cyclooxygenase (e.g., ibuprofen), chelating iron (e.g., desferal), and inhibiting lipid peroxidation (e.g., tirilazad mesylate [U-74006F] and U-78517G). Animals were hemorrhaged to a mean arterial pressure (MAP) of 43-45 mmHg where they were held for 2 hr. Five minutes prior to the end of the hemorrhage period, either vehicle, U-74006F (10 mg/kg), U-78517G (10 mg/kg), oxypurinol (10 or 25 mg/kg), desferal (10 or 25 mg/kg), or ibuprofen (10 mg/kg) was administered i.v., followed by the reinfusion of shed blood. In vehicle-treated animals, MAP declined progressively over the 2 hr post-reinfusion. Ibuprofen, desferal, and oxypurinol treatments each failed to attenuate this decline. In contrast, both U-74006F and U-78517G resulted in a significantly improved maintenance of MAP. Evidence of shock-induced lipid peroxidation was observed in terms of a 73.8% depletion in liver vitamin E content at 2 hr post-reinfusion in vehicle-treated rats. This decrease was prevented by both U-74006F and U-78517G. Inhibition of free radical-induced lipid peroxidation appears more effective for attenuating free radical pathophysiology in hemorrhagic shock that attempting to inhibit specific pathways of oxygen radical generation.

U-78517F: a potent inhibitor of lipid peroxidation with activity in experimental brain injury and ischemia.

U-78517F (2-[4-[2,6-di-(1-pyrrolidinyl)-4-pyridinyl)-1-piperazinyl] methyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol, dihydrochloride), which combines the antioxidant ring portion of alpha-tocopherol together with the amine of the previously described 21-aminosteroids (e.g., U-74006F), is a novel inhibitor of iron-catalyzed lipid peroxidation. U-78517F was found to have a 50% inhibitory concentration (IC50) of 0.6 microM against 200 microM ferrous chloride-initiated lipid peroxidation in rat brain homogenates, compared to 8 microM for U-74006F, 28 microM for alpha-tocopherol and 43 microM for the ring portion of alpha-tocopherol (i.e., trolox). Both stereoisomers of the racemic U-78517F proved to be equally active antioxidants. Against lipid peroxidation initiated by xanthine/xanthine oxidase, U-78517F was even more potent, with an IC50 of 0.01 microM. U-78517F was also observed to protect cultured mouse spinal neurons against iron-induced damage, with an IC50 of approximately 0.5 microM. When administered to male CF-1 mice i.v. at 5 min after a severe concussive head injury. U-78517F produced a dose-related improvement in the 1-hr neurological recovery. The minimum effective i.v. dose was 1.0 micrograms/kg. Measurement of U-78517 concentrations in the brains of mice after administration of a 10-mg/kg i.v. dose revealed effective antioxidant levels for as long as 2 hr. Evidence of an in vivo antioxidant action was provided by the attenuation of iron-induced blood-brain barrier disruption (i.e., Evans' blue extravasation) in rats pretreated with U-78517F.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in postischemic neuronal necrosis in gerbils.

Twenty-four hour postischemic neuronal necrosis was compared in male vs. female Mongolian gerbils subjected to a 3-h period of severe incomplete hemispheric ischemia produced by unilateral carotid occlusion. The incidence of stroke-prone males was 42.9% versus 26.7% for the females. Among the stroke-prone animals, the males displayed significantly greater neuronal necrosis at 24 h after ischemia compared to the females in the cerebral cortex and CA1 region of the hippocampus. In the CA1 region of the stroke-prone males, only 2.0% of the normal neuronal population remained by 24 h compared to 36.8% in the stroke-prone females (p less than 0.02). In the cerebral cortex, the males had only 19.9% of normal versus 58.2% in the females (p less than 0.05). In a second series of mechanistic experiments, no differences in cortical blood flow (CBF) were disclosed between preselected male and female stroke-prone animals before, during, or for 2 h after ischemia. As with the CBF, the extent of cortical extracellular hypocalcia during ischemia did not differ significantly. However, the degree of postischemic recovery of cortical extracellular calcium was significantly better in the females from 30 min to 2 h after reperfusion. In the same experiments, hemispheric vitamin E levels were measured at the 2 h time point as an index of postischemic brain lipid peroxidation. No difference in baseline vitamin E levels was observed between male and female sham-operated gerbils. In the males subjected to 3 h of ischemia plus 2 h of reperfusion, the hemispheric vitamin E decreased by 43.5% compared to the sham-operated males.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonsteroidal lazaroid U78517F in models of focal and global ischemia.

U78517F is a novel inhibitor of iron-catalyzed lipid peroxidation that combines the tetramethylchroman antioxidant ring portion of alpha-tocopherol with the amine of the previously described 21-aminosteroids (e.g., U74006F). U78517F inhibited 200 microM FeCl2-initiated lipid peroxidation in rat brain homogenates by 50% at a concentration of 0.6 microM compared with 8 microM for U74006F, 28 microM for alpha-tocopherol, and 43 microM for the ring portion of alpha-tocopherol (i.e., trolox). U78517F is devoid of hypothermic or antiexcitotoxic actions or interactions with known neurotransmitter receptors. When administered intraperitoneally to male gerbils at 10 minutes before and again at the end of a 3-hour period of unilateral carotid artery occlusion, U78517F decreased 24-hour postischemic cortical neuronal necrosis. Neuronal density in the medial portion of the cortex was increased from 34.2% of normal in vehicle-treated animals to 86.3% in the U78517F-treated animals. In the lateral cortical area, the vehicle group showed only 3.3% neuronal survival versus 48.2% in the drug-treated group. In a separate series of experiments with the same focal ischemia model, identical dosing with U78517F enhanced the postischemic recovery of cortical extracellular calcium without any effect on ischemic or postischemic cortical blood flow. The effect on calcium recovery was observed at intraperitoneal doses as low as 0.1 mg/kg. The compound also was effective in partially attenuating 1-week postischemic hippocampal CA1 neuronal loss in a gerbil global ischemia model involving brief (15-minute) bilateral carotid occlusion, but sustained dosing was required. These results document the anti-ischemic efficacy of a novel and potent inhibitor of iron-catalyzed lipid peroxidation and further support a key role of oxygen radicals in postischemic brain damage.