Effects of alpha tocopherol and probucol supplements on allergen-induced airway inflammation and hyperresponsiveness in a mouse model of allergic asthma.

OBJECTIVE: We investigated the role of antioxidants in airway hyperresponsiveness to acetylcholine using young asthma model mice, which were sensitized and stimulated with ovalbumin. METHODS: The mice had been fed either a normal diet, an alpha-tocopherol-supplemented diet or a probucol-supplemented diet 14 days before the first sensitization. They were immunized with antigen at intervals of 12 days and, starting from 10 days after the second immunization, they were exposed to antigen 3 times every 4th day using an ultrasonic nebulizer. Twenty-four hours after the last antigen inhalation, airway responsiveness to acetylcholine was measured and bronchoalveolar lavage fluid (BALF) was collected. A blood and lung tissue study was also carried out. RESULTS: Twenty-four hours after the last antigen challenge, both IL-4 and IL-5 in the BALF of alpha-tocopherol-supplemented mice were significantly decreased. The IL-5 level in probucol-supplemented mice was also decreased, but there was no difference in IL-4 levels. The serum IgE level was decreased in probucol-supplemented mice. Differential cell rates of the fluid revealed a significant decrease in eosinophils due to antioxidant supplementation. Airway hyperresponsiveness to acetylcholine was also repressed in antioxidant-supplemented mice. In histological sections of lung tissue, inflammatory cells and mucus secretion were markedly reduced in antioxidant-supplemented mice. We investigated the antioxidant effect on our model mice by examining 8-isoprostane in BALF and lung tissue, and acrolein in BALF; however, our experiment gave us no evidence of the antioxidant properties of either alpha-tocopherol or probucol contributing to the reduction of airway inflammation. CONCLUSION: These findings indicate that alpha-tocopherol and probucol suppress allergic responses in asthma model mice, although these two drugs cause suppression in different ways that are unrelated to antioxidation.

Evaluation of pharmacokinetic interaction between cyclosporin A and probucol in rats.

PURPOSE: The purpose of this study was to clarify the mechanism of pharmacokinetic interaction between cyclosporin A and probucol in clinical cases. METHODS: The whole blood concentration of cyclosporin A was measured after oral administration of cyclosporin A with or without probucol in rats. Cyclosporin A was administered as three types of solutions: the contents of the conventional formulation (Sandimmun capsule) diluted with corn oil and the contents of the new microemulsion preconcentrate formulation (Neoral capsule) diluted with saline or corn oil. The solubility of cyclosporin A and another lipophilic agent tacrolimus in water with or without probucol was also measured. RESULTS: The area under the blood concentration-time curve (AUC) after the administration of Sandimmun (corn oil) and Neoral (corn oil) was significantly decreased to 26% and 41% of the control by coadministration of probucol. However in the case of Neoral (saline), it was unchanged. The terminal elimination rate constant was not affected by probucol in any type of cyclosporin A solution. The solubility of cyclosporin A or tacrolimus in water dropped to 49% or 16% of the respective control in the presence of probucol. CONCLUSION: The interaction between cyclosporin A and probucol is caused by the decreased absorption of cyclosporin A partly based on the lowered solubility in the presence of probucol.

Inhibition of VCAM-1 expression in the arterial wall is shared by structurally different antioxidants that reduce early atherosclerosis in NZW rabbits.

We previously established that probucol decreases basal expression of VCAM-1 in the aorta of WHHL rabbits and inhibits the up-regulation of VCAM-1 expression that normally accompanies atherogenesis. To determine whether this effect is shared by other antioxidants in vivo, we now investigated whether a structurally unrelated antioxidant, vitamin E, also inhibits arterial VCAM-1 expression and whether the degree of VCAM-1 inhibition correlates with the reduction of atherosclerosis or the antioxidant protection of LDL. Atherogenesis and VCAM-1 mRNA and protein were determined in four groups of NZW rabbits (n = 6;-8) fed 0.5% cholesterol alone or supplemented with 0.1% vitamin E, a low dose (0.04;-0.075%) of probucol yielding the same degree of antioxidant protection of plasma LDL as vitamin E, or a high dose (0.5%) of probucol, and in normocholesterolemic rabbits. After 81 days, extensive atherosclerosis and a greater than 4-fold up-regulation of VCAM-1 mRNA was seen in rabbits on high cholesterol diet, mostly in the intima. Treatment with vitamin E, high-dose probucol, and low-dose probucol significantly decreased VCAM-1 mRNA by 49.0, 74.9, and 57. 5%, respectively, and reduced atherosclerosis in adjacent segments of the thoracic aorta to a similar degree as reported by previous studies. Immunocytochemistry confirmed that lesions of antioxidant-treated animals also contained less VCAM-1 protein. Neither the degree of VCAM-1 inhibition nor the extent of atherosclerosis correlated with the degree of antioxidant protection of plasma LDL.In summary, treatment with structurally unrelated antioxidants conveyed different degrees of antioxidant protection to plasma LDL but significantly reduced VCAM-1 expression in vivo and inhibited atherogenesis. This is consistent with the assumption that antiatherogenic effects of antioxidants may in part be mediated by interference with oxidation-dependent intracellular signaling.

Proatherogenic and antiatherogenic effects of probucol and phytosterols in apolipoprotein E-deficient mice: possible mechanisms of action.

BACKGROUND: The effects of probucol and a phytosterol mixture (FCP-3PI) on atherosclerotic lesion formation, plasma lipoproteins, hepatic and lipoprotein lipase activities, antioxidant enzyme activities, and plasma fibrinogen were investigated in apolipoprotein E-knockout (apoE-KO) mice. METHODS AND RESULTS: Three groups of 8 mice were fed a diet containing 9% (wt/wt) fat (controls) or the foregoing diet supplemented with either 1% (wt/wt) probucol (the probucol group) or 2% (wt/wt) FCP-3PI (the FCP-3PI group) for 20 weeks. Compared with controls, atherosclerotic lesion size was 3 times greater in the probucol group, whereas it was decreased by half in the FCP-3PI group. Probucol treatment resulted in high plasma probucol concentrations, which correlated (r=0.69) with the lesion area. HDL cholesterol was reduced (>75%) in the probucol group and slightly increased (14%) in the FCP-3PI-treated group. Postheparin lipoprotein lipase (LPL) activity was significantly reduced in both treatment groups, but only FCP-3PI significantly decreased hepatic lipase activity. Plasma fibrinogen was increased 42% by probucol and decreased 19% by FCP-3PI relative to controls. Probucol significantly increased plasma glutathione reductase, glutathione peroxidase, and superoxide dismutase activities (P<0.05). In contrast to findings in apoE-KO mice, there was no probucol-induced atherosclerosis in their wild-type counterparts fed the same dose for the same period of time. CONCLUSIONS: Antiatherogenic activity of FCP-3PI in apoE-KO mice is associated with an increase in HDL cholesterol concentration along with decreases in hepatic lipase activity and plasma fibrinogen concentrations. Proatherogenic effects of probucol may be related to increased plasma fibrinogen, decreased HDL cholesterol concentrations along with decreased LPL activity, or its direct "toxicity" due to very high plasma concentration. Our studies demonstrate that the antioxidant and cholesterol-lowering properties of probucol do not prevent atherogenesis in this particular animal model.

Measurement of reactive oxygen species by chemiluminescence in diet-induced atherosclerosis: protective roles of vitamin E and probucol on different radical species.

We have investigated the effects of a high-cholesterol diet on the production of different reactive oxygen species in rabbit aortic rings and evaluated the protective effects of vitamin E and probucol in preventing peroxidative changes. Twenty-five male albino rabbits were divided into five groups. Control rabbits were fed a vitamin E-poor rabbit chow. Rabbits in the second group were given a vitamin E-poor diet supplemented with 2% cholesterol. Other groups received either 50 mg/kg vitamin E, 1% probucol, or both, in addition to 2% cholesterol for 4 weeks. Reactive oxygen species formation in aortic rings was measured by enhanced chemiluminescence using luminol and lucigenin. (The results were given as cpm/mg wet weight.) Further differentiation of radical species involved in luminol-enhanced chemiluminescence was performed using sodium azide and L-nitroarginine, a selective inhibitor of nitric oxide production. Our results indicated that cholesterol feeding increased lucigenin and luminol chemiluminescence, where the contribution of free radicals inhibited by sodium azide (radicals originating from endothelial cells or from phagocytes) were 53% and peroxynitrite 24%. Both vitamin E and probucol were effective as scavengers of free radicals, but the effect of vitamin E was more pronounced. In conclusion, the present study demonstrated excessive generation of reactive oxygen species within the atherosclerotic vessel. Peroxidative changes could be prevented by vitamin E and probucol treatment, but vitamin E seemed to be more efficient.

Effect of probucol on LDL oxidation and atherosclerosis in LDL receptor-deficient mice.

Probucol is a powerful inhibitor of atherosclerosis in a number of animal models. However, it is unknown whether this is due to the strong antioxidant protection of low density lipoprotein (LDL), to antioxidant effects in the artery wall, or to cellular effects not shared by other antioxidants. To investigate whether murine models are suitable to study the antiatherogenic mechanisms of probucol, three experiments following different protocols were carried out in 135 male and female LDL receptor-deficient (LDLR-/-) mice. Treatment groups received a high (0.5%) or low (0.025%) dose of probucol, or low-dose probucol plus a high dose (0.1%) of vitamin E for periods ranging from 6 to 26 weeks. In all experiments, probucol strongly protected LDL against ex vivo oxidation (lag times exceeding 1400 min in 0.5% probucol-treated mice). Treatment with 0.5% probucol significantly lowered both HDL-cholesterol and plasma apolipoprotein (apo)A-I concentrations. In all three experiments, treatment with 0.5% probucol consistently increased the size of lesions in the aortic origin, from 1.3-fold (n.s.) to 2.9-fold (P < 0.05) in female mice and from 3.6- to 3.7-fold in males (P < 0.001). Even treatment with 0.025% probucol increased atherosclerosis 1.6-fold in male mice (P < 0.01). Addition of the high dose of vitamin E did not attenuate the pro-atherogenic effect of 0.025% probucol. In conclusion, probucol not only failed to decrease but actively increased atherogenesis in LDLR-/- mice in a dose-dependent manner, even though it provided a very strong antioxidant protection of LDL. This suggests that the reduction of atherosclerosis observed in other animal models is due to intracellular effects of probucol not found in mice, to differences in the metabolism of probucol, and/or to an overriding atherogenic effect of the decrease in HDL in murine models.

Endothelial dysfunction in streptozotocin-diabetic rats is not reversed by dietary probucol or simvastatin supplementation.

Oxidative stress and dyslipidaemia are key features of diabetes mellitus and may be involved in mediating the vascular endothelial dysfunction associated with this disease. The aim of this study was to examine the effect of dietary lipid-lowering and antioxidant agents on vascular endothelial function and oxidative stress. Diabetic male Sprague-Dawley rats (i.v. streptozotocin, 45 mg/kg) were fed for 4 weeks on a standard diet or on a diet supplemented with either the lipid-lowering antioxidant probucol (1% w/w in diet) or the 3-hydroxy 3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitor simvastatin (0.01% w/w in diet). Responses to noradrenaline, acetylcholine, and sodium nitroprusside were assessed in small mesenteric arteries (mean internal diameter 300+/-5 microm, n = 80) mounted on a small vessel myograph. Plasma concentrations of total cholesterol and triglycerides were significantly raised in standard-fed diabetic rats and significantly reduced in probucol and simvastatin-fed diabetic rats 8-epi-prostaglandin (PG)F2alpha, an indicator of oxidative stress, was raised in liver and aorta from diabetic rats compared to controls. Probucol supplementation reduced 8-epi-PGF2alpha in aorta and liver of diabetic rats but increased 8-epi-PGF2alpha content in plasma and aorta from control animals. The abnormal relaxation to acetylcholine in arteries from the diabetic rats (pEC550 diabetic 6.763+/-0.172 vs control 7.541+/-0.175; p < 0.05) was not improved by probucol or simvastatin. These data, therefore, do not support a role for oxidative stress or dyslipidaemia in mediating the impaired ACh-induced endothelium-dependent relaxation of small mesenteric arteries from the streptozotocin-diabetic rat.

Comparative study on the effect of low-dose vitamin E and probucol on the susceptibility of LDL to oxidation and the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbits.

The diet of Watanabe heritable hyperlipidemic (WHHL) rabbits was supplemented with a low dose (0.025% [wt/wt]) of the antioxidant vitamin E or probucol. The effect of 6 months of treatment on the susceptibility of low-density lipoproteins (LDLs) to oxidative modification and on established atherosclerotic lesions was studied. Vitamin E administration had no effect on plasma lipid levels; probucol supplementation decreased plasma total cholesterol. Vitamin E levels in plasma and LDL increased threefold in the course of treatment with this antioxidant. Six months of treatment with vitamin E and probucol increased the lag time of conjugated-diene formation of LDL subjected to in vitro oxidation by 54% (P < .001) and 51% (P = .019), respectively. In this LDL-oxidizability assay, only vitamin E reduced the maximal rate of conjugated-diene production (-24%, P < .001). Neither vitamin E treatment nor probucol therapy reduced the amount of thiobarbituric acid-reactive substances in plasma. Vitamin E treatment reduced the specific LDL apolipoprotein B-100 fluorescence (-10%, P = .035) compared with controls. Probucol was without effect on this index of in vivo LDL oxidation. At the end of the 6-month study, the mean +/- SD percentage area of aorta covered with plaques was 58.7 +/- 10.1% in control animals, 62.7 +/- 12.0% in the probucol-treated animals, and 48.9 +/- 13.8% in the animals treated with vitamin E; these differences were not significant. This study demonstrates that at this low dosage, vitamin E is a more effective antioxidant than probucol.