Potent Inhibitor Of Lipid Peroxidation Research Articles

Novel membrane localized iron chelators as inhibitors of iron-dependent lipid peroxidation

Attachment of various iron chelating moieties to hydrophobic steroids greatly enhanced their abilities to inhibit iron-dependent lipid.peroxidation. Using whole rat brain homogenates, lipid peroxidation initiated by the addition of 200 μM Fe 2+ was assessed by the formation of thiobarbituric acid reactive products (TBAR). Under these conditions, 50% inhibitory concentrations of Fe 3+ chelators such as desferrioxamine or N 1, N 8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide (compound II) were around 170 and 50 μM respectively. Coupling desferrioxamine or compound II to a steroid at the D ring increased their potency in lipid peroxidation assays by 5- to 10-fold. Evidence that inhibition of lipid peroxidation by the steroid-chelator adducts was due to iron chelation was suggested by the fact that methylation of the catechol oxygens of compound II, which are essential for chelation, completely eliminated activity of the steroid adduct. A series of 21-aminosteroids which complex Fe 2+ iron and potently inhibit iron-dependent lipid peroxidation has also been synthesized. Coupling Fe 2+ chelators to hydrophobic steroids increased their inhibitory potencies by as much as 10- to 100-fold. Some steroid-based Fe 2+ chelators stimulated lipid peroxidation at low concentrations in the presence of Fe 3+. The degree of stimulation was related to the affinity of a compound for Fe 2+ with the stronger chelators causing greater stimulation. The most potent inhibitors of lipid peroxidation in the 21-aminosteroid series were found to be those compounds forming the weakest Fe 2+ complexes. The findings suggest that it is iron at or near the membrane that is responsible for the catalysis of lipid peroxidation. The compounds described should provide useful tools for studies of the involvement of iron in the lipid peroxidation process.

Effects of treatment with U-74006F on neurological outcome following experimental spinal cord injury.

The compound U-74006F is one of a series of 21-aminosteroids that lack glucocorticoid or mineralocorticoid activity. These potent inhibitors of lipid peroxidation have been specifically developed for the acute treatment of central nervous system trauma and ischemia. This study evaluated the dose-response characteristics and capability of U-74006F to promote functional recovery in cats subjected to compression trauma of the upper lumbar (L-2) spinal cord. Thirty minutes following injury, randomized and investigator-blinded treatment was initiated with the intravenous administration of either vehicle (citrate-buffered saline) or one of eight doses of U-74006F. Initial doses of U-74006F ranged from 0.01 to 30 mg/kg. Subsequent doses consisted of intravenous bolus injections followed by a continuous 42-hour intravenous infusion. Over the 48-hour treatment period, cats received total U-74006F doses ranging from 0.048 to 160 mg/kg. The animals were evaluated weekly for neurological recovery based upon an 11-point behavioral scale. With the exception of two cats in one group, the animals receiving accumulated doses of U-74006F (ranging from 1.6 to 160.0 mg/kg/48 hrs) exhibited nearly 75% of normal neurological function by 4 weeks after injury. Lower total doses of 0.16 and 0.48 mg/kg/48 hrs were associated with approximately 50% return of normal function, which was not significantly better than the recovery in the vehicle-treated control group. The lowest total dose tested (0.048 mg/kg/48 hrs) gave results indistinguishable from those in vehicle-treated cats, which had recovered only 20% of their preinjury neurological function by 4 weeks. These findings demonstrate that over a 100-fold range of doses, U-74006F has a remarkable capacity to promote functional recovery in spinal cord-injured cats.

21-Aminosteroid reduces ion shifts and edema in the rat middle cerebral artery occlusion model of regional ischemia.

U74006F is a member of a new family of steroid drugs called 21-aminosteroids, which are potent inhibitors of lipid peroxidation with little or no glucocorticoid or mineralocorticoid activity. We investigated the effects of U74006F on the early ionic edema produced by middle cerebral artery occlusion in rats. Intravenous doses of 3 mg/kg U74006F were given 10 minutes and 3 hours after occlusion. Tissue concentrations of Na+, K+, and water at and around the infarct site were measured by atomic absorption spectroscopy and by wet-dry weight measurements 24 hours after occlusion. Compared with vehicle treatment, U74006F treatment reduced brain water entry, Na+ accumulation, K+ loss, and net ion shift by 25-50% in most brain areas sampled in the frontal and parietal cortex. However, reductions of ionic edema were most prominent and reached significance (p less than 0.005, unpaired two-tailed t test) mostly in the frontoparietal and parietal cortex areas adjacent to the infarct site. Our findings suggest that a steroid drug without glucocorticoid or mineralocorticoid activity can reduce edema in cerebral ischemia but that the effects are largely limited to tissues in which collateral blood flow may be present.

Role of transferrin and ceruloplasmin in antioxidant activity of lung epithelial lining fluid.

Lung epithelial lining fluid (ELF) is a thin layer of plasma ultrafiltrate and locally secreted substances that may provide antioxidant protection and serve as a "front-line" defense for the lower respiratory tract epithelium. To characterize the antioxidant properties of ELF, young, healthy, nonsmoking volunteers underwent bronchoalveolar lavage with determination of ELF volumes and ELF proteins. ELF (greater than 0.4 ml) is a potent inhibitor of lipid peroxidation as measured by malondialdehyde (MDA) production in an in vitro iron-dependent assay system. Two serum proteins, transferrin and ceruloplasmin, were quantitated in ELF and found to be potent inhibitors of lipid peroxidation. Other ELF components, including vitamin E, vitamin C, and albumin, did not function as antioxidants in this system. Several experimental observations suggest that ELF transferrin was more important than ceruloplasmin in inhibiting lipid peroxidation: 1) ELF concentrations of transferrin were 20-fold higher than those for ceruloplasmin; 2) ELF antioxidant activity was abolished by preincubation with Fe3+; 3) ELF antioxidant activity was minimally affected by sodium azide, which is known to inhibit ceruloplasmin ferroxidase activity; and 4) ELF ceruloplasmin ferroxidase activity was virtually nondetectable. ELF possesses a significant antioxidant activity that may be important in vivo in protecting the lung from oxidant injury.

Effects of the 21-aminosteroid U74006F on experimental head injury in mice.

The ability of a novel non-glucocorticoid 21-aminosteroid U74006F to inhibit lipid peroxidation of central nervous system tissue in vitro and to enhance the early neurological recovery and survival of mice after a severe concussive head injury is described. In the in vitro studies, U74006F was found to be an extremely potent inhibitor of lipid peroxidation in an assay system where the glucocorticoid steroid methylprednisolone and the non-glucocortoid steroids U72099E and U75718A were almost completely ineffective. In the head-injury studies, unanesthetized male CF-1 mice were subjected to a 900 gm-cm closed head injury produced by a 50-gm weight being dropped 18 cm. This concussive injury resulted in immediate unconsciousness (loss of righting reflex) in all animals and death in approximately 30%. Survivors received a tail vein injection of either vehicle or U74006F (0.001 to 30 mg/kg) within 5 minutes postinjury. Their neurological status was evaluated 1 hour later using a grip test. The grip-test score indicated that intravenous administration of a single dose of U74006F resulted in a significant improvement by as much as 168.6% in the neurological status 1 hour postinjury over a broad dose range (0.003 to 30 mg/kg). A 1-mg/kg dose given intravenously within 5 minutes and again at 1 1/2 hours after a severe injury, in addition to improving early recovery, also increased the 1-week survival rate to 78.6% compared to 27.3% in vehicle-treated mice (p less than 0.02). The compound was also effective in enhancing early recovery after a more moderate injury. This study demonstrates that early treatment after severe concussive head injury with a potent inhibitor of iron-dependent lipid peroxidation can significantly benefit the injured brain in mice and promote both early neurological recovery and long-term survival.

Protection against carbon tetrachloride hepatotoxicity by 5,10-dihydroindeno[1,2-b]indole, a potent inhibitor of lipid peroxidation

The influence of 5,10-dihydroindeno[1,2-b]indole (indenoindole) on carbon tetrachloride (CCl 4)-mediated hepatotoxicity and lipid peroxidation were examined. Indenoindole (25 mg/kg body weight) ameliorated the increase in liver enzymes appearing in the plasma 24 hr after CCl 4 administration, with about a 63% reduction for alanine transaminase, 56% for ornithine transcarbamylase and 84% for alkaline phosphatase. Indenoindole also partially prevented, in a dose-dependent fashion, the decrease in hepatic cytochromes P-450, total tissue reducing equivalents and hepatic ascorbate levels resulting 4 hr after CCl 4 administration. In a homogeneous chemical system consisting of purified soybean phospholipid substrate in chlorobenzene, azobisisobutyronitrile-initiated lipid peroxidation was inhibited by indenoindole, with 50% inhibition occurring at about 17 μ m. Inhibition by indenoindole of iron-ascorbate-initiated lipid peroxidation in aqueous buffer containing phospholipid vesicles was about tenfold more efficient, with 50% inhibition occurring at about 1.5 μ m. Presumably, this was due to the increased concentration of indenoindole in the membrane of the phospholipid vesicle. The efficiency of inhibition of lipid peroxidation was in the order of indenoindole = butylated hydroxytoluene (BHT) > α-tocopherol ⪢ indole > indene. These 50% inhibition values of lipid peroxidation for these compounds were similar in an assay system composed of NADPH-fortified mouse-liver musomes initiated with CCl 4. For indenoindole, the 50% inhibition value (1.3 μ m) was more than two orders of magnitude less than the spectral binding constant for indenoindole to mouse-liver cytochrome P-450 ( K s = 236 μ m ), implying that the partial inhibition of metabolic activation of CCl 4 was not responsible for the inhibition of lipid peroxidation observed with indenoindole in this system. It appears that indenoindole may trap reactive radicals and inhibit lipid peroxidation in vitro. Regardless of whether inhibition is at the level of scavenging CCl 4 metabolite radicals, or lipid radicals in membranes, radical trapping provides a plausible mechanism by which this compound inhibited CCl 4 hepatotoxicity.

Novel 21-amino steroids as potent inhibitors of iron-dependent lipid peroxidation.

Two representative compounds from a novel chemical series of potent inhibitors of lipid peroxidation are described. The compounds 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-16 alpha-methylpregna-1,4,9(11)-triene-3,20-dione monomethane sulfonate (U74006F) and 21-[4-(3,6-bis(diethylamino)-2-pyridinyl)-1-piperazinyl]-16 alpha-methylpregna-1,4,9(11)triene-3,20-dione hydrochloride (U74500A) inhibited lipid peroxidation in brain homogenates and purified brain synaptosomes under a variety of conditions involving iron. With IC50 values ranging from 2 to 60 microM, U74006F and U74500A were comparable in potency to alpha-tocopherol or butylated hydroxytoluene and were nearly 100 times as potent as desferrioxamine. Some specificity for intact phospholipid membranes is suggested since the ability of U74006F or U74500A to inhibit lipid peroxidation was greatly reduced in methanol solutions of arachidonic acid. Despite close similarities in their structures, their response to increasing concentrations of Fe2+ in lipid peroxidation assays differed qualitatively. One of the compounds, U74500A, may act as a membrane localized chelator of iron.

Effects of anthrapyrazole antineoplastic agents on lipid peroxidation

The effects of three anthrapyrazoles and an aminoacridine derivative on doxorubincin- and iron-stimulated lipid peroxidation in rabbit hepatic microsomes have been characterized. Two anthrapyrazoles, CI-937 and CI-942, were potent inhibitors of lipid peroxidation with 15 μM drug inhibiting the rate of peroxidation 70 to 90%. In contrast CI-941 was relatively ineffective in inhibiting lipid peroxidation with only 35% inhibition occurring at 100 μM drug. CI-921, an aminoacridine derivative, diminished lipid peroxidation by 65% at 15 μM. All four drugs failed to decrease the rate of doxorubicin-stimulated NADPH oxidation at concentrations <50 μM, suggesting that inhibition of lipid peroxidation was not the result of diminished enzyme activity. CI-937 formed a 2:1 complex with ferric ion, K D = 47 μM, which was reversible with EDTA.

Toxic and protective effects of antioxidants in biomembranes.

Natural and synthetic antioxidants (AOs) are widely used as stabilizers of biomembranes against lipid peroxidation (LPO). Natural AOs (tocopherols, ubiquinols) containing hydrocarbon "tails" do not disturb the membrane lipid bilayer. Synthetic AOs devoid of hydrocarbon radicals may perturb the lipid bilayer. It was shown that AOs devoid of hydrocarbon tails (butylated hydroxytoluene, 2,2,5,7,8-pentamethyl-6-hydroxychromane) exerted toxic effects on erythrocyte membranes (induced hemolysis), on sarcoplasmic reticulum membranes (inhibited Ca2+-transport) and on platelet membranes (initiated Ca2+-dependent aggregation) in vitro. These AOs are the substrates of cytochrome P-450, and underwent oxidative hydroxylation. This suggests that they have short half-life times in biomembranes and in the organism. Antioxidants with hydrocarbon tails, are hydroxylated at very low rates and are slowly excreted. Antioxidants devoid of hydrocarbon tails, are 10-20 fold more potent LPO inhibitors than the corresponding AOs with hydrocarbon tails. The strategy of AOs application for long and short-term stabilization of biomembranes against LPO in vivo is discussed.

Inhibition of cephaloridine-induced lipid peroxidation

The present study was designed to elucidate whether cephaloridine-induced lipid peroxidation is inhibited by probenecid, cobalt chloride and antioxidants such as α-tocopherol and N, N′-diphenyl- p-phenylenediamine (DPPD). Kidney slices obtained from the renal cortex of male Wistar rats were incubated for 1 h in a cephaloridine or cefotaxime (1.25–10 mg/ml) containing medium. In another series of experiments, kidney slices were incubated with cephaloridine or cefotaxime (5 mg/ml) for different periods of time (30–120 min). Lipid peroxidation was monitored by measuring the production of malondialdehyde (MDA). Subsequently, kidney slices were incubated in both series of experiments, in a cephalosporin free medium containing tetraethyl-ammonium (TEA). Accumulation of TEA in renal cortical slices, expressed as slice to medium ratio (S/M), was used to measure changes in the transport capacity of the kidney cells. While cefotaxime had only a slight effect, cephaloridine induced a significant time- and concentration-dependent increase of MDA production and a significant time- and concentration-dependent decrease of TEA accumulation. Inhibition of the renal uptake of cephaloridine by probenecid induced a decrease in MDA production and complete recovery of TEA accumulation. The antioxidants DPPD and α-tocopherol inhibited cephaloridine-induced lipid peroxidation in a concentration-dependent manner. Recovery of TEA accumulation accompanied the decrease in lipid peroxidation. DPPD was a more potent inhibitor of lipid peroxidation than α-tocopherol. Cobalt chloride, known for its ability to decrease cellular concentration of cytocchrome P-450, effectively decreased cephaloridine-induced lipid peroxidation. Thus, these findings support the concept that lipid peroxidation has an important role in the development of cephaloridine-induced nephrotoxicity.

Effect of inhibitors of drug metabolizing enzymes on carbon tetrachloride hepatotoxicity

Several inhibitors of drug metabolizing enzymes were tested for their ability to prevent carbon tetrachloride-induced liver necrosis and fatty infiltration in the rat. CFT 1201, Sch 5705, Sch 5706, Sch 5712, and Lilly 18947 protected against necrosis to some extent. CFT 1201 and Sch 5706 were the most effective compounds. CFT 1201 also significantly prevented the fatty infiltration. DPEA and imipramine were unable to prevent necrosis. The preventive action of inhibitors of drug metabolizing enzymes against necrosis cannot be ascribed solely to their ability to inhibit either drug metabolism or lipid peroxidation, since imipramine was the most potent inhibitor of lipid peroxidation and it was not effective as a protector. Moreover, Sch 5706 was the most effective compound tested, and it neither inhibited carbon tetrachloride-induced lipid peroxidation nor prolonged the pentobarbital sleeping time of rats more than did the other compounds. The protective effect found by administration of a substrate of drug metabolizing enzymes such as aminopyrine suggests that part of the overall preventive effect of the inhibitors might be due to a substrate-like action competing with lipid peroxidation for TPNH and oxygen. Other factors like interference with gastrointestinal absorption or production of hypothermia could also be important.