Antioxidants: The good, the bad and the ugly (original) (raw)

Can J Cardiol. 2006 Feb; 22(Suppl B): 61B–65B.

Montreal Heart Institute, Montreal, Québec

Correspondence: Dr Jean-Claude Tardif, Director, Montreal Health Institute Research Centre, 5000 Belanger Street, Montreal, Québec H1T 1C8. Telephone 514-376-3330 ext 3612, fax 514-593-2500, e-mailgro.ihm-mci@fidrat.edualc-naej

Abstract

Inflammation has a fundamental role in mediating all stages of atherosclerotic disease. The key role of oxidation in linking lipids and inflammation to atherosclerosis is compelling and is supported by experimental evidence. However, the relevance of the antioxidant hypothesis for the treatment of patients with atherosclerosis has not been definitively proven. Results of randomized trials with ‘antioxidant’ vitamins have been disappointing, and there are potentially important problems associated with their use, including their potential pro-oxidant effects. Probucol has reduced postpercutaneous coronary intervention (PCI)-restenosis and progression of carotid atherosclerosis in clinical trials. The antioxidant vascular protectant AGI-1067 has also been effective at preventing atherosclerosis in all tested animal models. The nonintervened reference coronary segments of the PCI vessel demonstrated improvements with AGI-1067 in the Canadian Antioxidant Restenosis Trial-1 (CART-1), evidence supportive of a clinical effect on slowing atherosclerosis progression. Two trials test the antioxidant/anti-inflammatory hypothesis with AGI-1067; CART-2 assesses its value for the reduction of both atherosclerosis progression and post-PCI restenosis, and Aggressive Reduction of Inflammation Stops Events (ARISE), which is evaluating its effects on hard cardiovascular outcomes.

Keywords: Antioxidants, Atherosclerosis, Inflammation, Oxidative stress

Résumé

L’inflammation joue un rôle fondamental de médiation à tous les stades de l’athérosclérose. Le rôle central de l’oxydation dans les phénomènes qui relient les lipides et l’inflammation à l’athérosclérose n’est plus à démontrer et s’appuie sur des preuves expérimentales. Par contre, la pertinence de l’hypothèse antioxydante pour le traitement des patients qui souffrent d’athérosclérose n’a pas encore été prouvée hors de tout doute. Les résultats d’essais randomisés menés sur des vitamines « antioxydantes » ont donné des résultats décevants et leur utilisation est associée à des problèmes potentiellement importants, notamment à un risque d’effets pro-oxydants. Le probucol a réduit la resténose consécutive aux angioplasties coronariennes percutanées (ACP) et a ralenti la progression de l’athérosclérose carotidienne lors d’essais cliniques. L’AGI 1067, un antioxydant vasoprotecteur, s’est également révélé efficace à prévenir l’athérosclérose dans tous les modèles animaux testés. Les segments coronariens de référence sur lesquels les chercheurs ne sont pas intervenus lors d’ACP ont montré des signes d’amélioration avec l’AGI-1067 dans le cadre de l’étude CART-1 (pour Canadian Antioxidant Restenosis Trial-1); ces signes en confirment l’effet clinique sur le ralentissement de l’athérosclérose. Deux essais vérifient par ailleurs l’hypothèse de l’effet antioxydant/anti-inflammatoire de l’AGI 1067 : l’essai CART-2 évalue son utilité dans le ralentissement de la progression de l’athérosclérose et la réduction de la resténose post-ACP et l’essai ARISE (pour Aggressive Reduction of Inflammation Stops Events) mesure ses effets sur les paramètres cardiovasculaires définitifs.

Reactive oxygen species (ROS) include both free radicals, which typically have an oxygen- or nitrogen-based unpaired electron in their outer orbits, and other species, such as hydrogen peroxide, that act as oxidants. The mitochondria, the cellular membrane oxidases, such as NADPH oxidase, nitric oxide synthase and myeloperoxidase are major sources of ROS. The byproducts associated with the metabolism of arachidonic acid by cyclooxygenase, lipoxygenase and cytochrome P450 monooxygenase also result in the production of ROS. The targets for damage from ROS are not only lipids, such as polyunsaturated fatty acids in cellular membranes and low density lipoproteins (LDL), but also proteins and nucleic acids.

Lipid peroxidation products react strongly with various biological substrates leading to cellular injury. Oxidative damage to polyunsaturated fatty acids of membrane phospholipids leads to a destabilization in their intermolecular packing characteristics and subsequent loss in membrane integrity. Lipid peroxides break down into smaller molecules that either remain covalently linked to the phospholipid glycerol backbone or are released into the cytosol. In addition, the oxidized LDL particle does not effectively bind to the high-affinity LDL receptor, thus extending its circulation and enhancing its uptake into macrophages. Besides lipids, other constituents of the cell are vulnerable to ROS-induced damage, especially nucleic acids associated with nuclear and mitochondrial DNA, and oxidizable proteins. Damage to nucleic acids can be broadly categorized as either strand breaks or base modifications. Oxidizable amino acids associated with cellular proteins are also vulnerable to ROS-induced damage, leading to a loss in normal function. The effects of protein oxidation products such as residue-specific changes, fragmentation products and cross-linked reaction products with other intracellular molecules can be highly deleterious to the cell (for example, disrupting proteins involved in transport or cell regulation).

OXIDATIVE STRESS AND ATHEROSCLEROSIS

Atherosclerosis is now understood to be a chronic inflammatory disease characterized by the excess accumulation of monocytederived macrophages within the arterial wall (1). Compelling evidence points to oxidative stress as an important trigger in the complex chain of events leading to atherosclerosis (2). The expression of chemotactic factors, such as monocyte chemotactic protein-1, and of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), are enhanced by oxidative stress and oxidized LDL (3,4). The release of macrophage colony-stimulating factor is also stimulated by modified LDL (5). Expression of these factors results in the attraction and adhesion of monocytes to the arterial wall, and the promotion of their differentiation into tissue macrophages. Exposure to the superoxide ion activates the nuclear factor-kappa B regulatory complex and triggers the transcription of several atherosclerosis-related genes (6). This series of events leads to the accumulation of macrophages in the arterial wall, which incorporate oxidized LDL to form foam cells. Oxidized LDL, in turn, stimulates the release of interleukin-1 from macrophages (7). The activity of matrix metalloproteinases, also regulated by oxidative stress, appears to be closely coupled with smooth muscle cell activation and migration, and has also been implicated in the pathophysiology of plaque rupture (8). Furthermore, reactive oxygen species can lead to platelet activation and thrombus formation (9). Therefore, oxidative stress appears to be important in both the early and the later stages of the atherosclerotic process.

There is also evidence that oxidative stress occurs early after angioplasty (10,11). Reactive intermediates can be generated by damaged endothelium, activated platelets and neutrophils at the angioplasty site. These oxidizing metabolites can induce chain reactions that result in endothelial dysfunction, macrophage activation, smooth muscle cell migration and proliferation, and matrix remodelling (12,13).

CONCERNS ASSOCIATED WITH THE USE OF ‘ANTIOXIDANT’ VITAMINS

The results of prospective epidemiological studies have supported a protective role for antioxidant vitamins in cardiovascular diseases (14). Such observational studies are limited by their inability to control for the effects of unknown or unmeasured confounders. In contrast, the results of randomized clinical trials with antioxidant vitamins have been disappointing. An excess risk for cancer and cardiovascular mortality was observed with beta-carotene in the large Alpha-Tocopherol/Beta-Carotene (ATBC) (15) and Carotene And Retinol Efficacy Trial (CARET) (16) studies. In terms of the results with vitamin E, except for the Cambridge Heart Antioxidant Study (CHAOS) (17), most secondary prevention trials have not shown protective effects (18–20). The Gruppo Italiano per lo Studio della Sopravivenza nell’Infarcto miocardico (GISSI) prevention trial was an open-label, randomized trial assessing dietary supplements of vitamin E (300 mg daily) and n-3 polyunsaturated fatty acids in 11,324 patients who had had a recent myocardial infarction (18). The Heart Outcomes Prevention Evaluation (HOPE) study tested an angiotensin-converting enzyme inhibitor and vitamin E (400 U daily) in a factorial design for the prevention of cardiovascular morbidity and mortality (19). The study population included more than 9000 patients who were at high risk of developing a major cardiovascular event. The Heart Protection Study (HPS) recruited more than 20,000 patients at high risk of atherosclerosis-related events because of a past history of myocardial infarction, or other evidence of atherosclerosis, diabetes mellitus or hypertension (20). The patients were randomly assigned in a two by two factorial design to receive simvastatin alone, antioxidant vitamins (600 mg vitamin E, 250 mg vitamin C and 20 mg beta-carotene daily), a combination of simvastatin and vitamins, or placebo. Vitamin E did not offer cardiovascular protection in the GISSI, HOPE and HPS trials (18–20).

There are potentially important problems associated with the use of vitamins, which include their potential pro-oxidant effects (21,22). Alpha-tocopherol itself becomes a radical when it scavenges a free electron. Because the tocopherol radical is relatively unstable, it can become a donor of free radicals or act as a pro-oxidant. Bowry et al (21) showed that, under high concentrations of vitamin E, lipid peroxidation is actually faster in the presence of alpha-tocopherol, and lipid peroxidation is propagated within LDL particles by reaction with the tocopherol radical. This may explain why a high dose of alpha-tocopherol worsens endothelial-dependent vasodilation, whereas a low dose improved it in cholesterol-fed rabbits (22). These problems associated with vitamin E supplementation may explain the negative results of the vitamin arms of the GISSI, HOPE, HPS, Women’s Angiographic Vitamin and Estrogen (WAVE) and MultiVitamins and Probucol (MVP) trials (18–20,23,24). Moreover, antioxidant vitamins negated in part the beneficial effects of lipid-lowering agents in patients with coronary artery disease in the recently published High density lipoprotein Atherosclerosis Treatment Study (HATS) (25). This phenomenon had also been previously observed in the MVP trial (24,26,27).

RESULTS WITH POWERFUL SYNTHETIC ANTIOXIDANTS

Animal studies have shown a beneficial effect of antioxidants on both neointimal formation and arterial remodelling after balloon angioplasty (28–32). More recently, the antioxidant probucol was shown to promote the regeneration of functional endothelium in balloon-injured rabbit aortas (33). Small clinical studies, along with the MVP and the Probucol Angioplasty Restenosis Trial (PART), have shown that probucol started before balloon angioplasty reduces coronary restenosis (13,24,26,27,34–38). However, prolongation of the QT interval and lowering of high density lipoprotein cholesterol with probucol remain potential long-term safety concerns. An agent that would offer antioxidant properties similar to probucol but with more favourable safety and pharmacokinetic profiles would have significant potential for the prevention of both restenosis and atherosclerosis progression. The recently developed antioxidant and vascular protectant AGI-1067 appears to fulfill these criteria and was therefore assessed in the Canadian Antioxidant Restenosis Trial-1 (CART-1) (39).

AGI-1067, the monosuccinic acid ester of probucol, is a phenolic antioxidant member of a novel class of agents termed vascular protectants (40). It has strong antioxidant properties, equipotent to those of probucol, and anti-inflammatory properties (4,41). AGI-1067 also exhibits greater water solubility and cell permeability than probucol. AGI-1067 has the ability to selectively block the expression of oxidation-sensitive inflammatory genes that code for VCAM-1 and monocyte chemotactic protein-1 (42). Although AGI-1067 and probucol are equipotent antioxidants, they produce different effects on VCAM-1 expression in human aortic endothelial cells. AGI-1067 produces a concentration-related decrease in tumour necrosis factor-alpha-stimulated VCAM-1 expression in the concentration range of 2.5 μmol/L to 10 μmol/L, while probucol was shown to have no effect at concentrations as high as 100 μmol/L (41,43). AGI-1067 has also been a powerful inhibitor of smooth muscle cell proliferation in experimental studies.

CART-1

CART-1 (39) was a double-blind, double-dummy, multicentre trial in which 305 patients scheduled to undergo elective percutaneous coronary intervention (PCI) with or without stent placement (85% were treated with stents) were randomly assigned to placebo, probucol 500 mg twice daily, or AGI-1067 70 mg, 140 mg or 280 mg once daily. Patients were treated for two weeks before and four weeks after PCI in this phase 2 trial. The minimal lumen area at the site of PCI on follow-up intravascular ultrasound (IVUS) was on average 2.66 mm2 in the placebo group, 3.69 mm2 in the probucol group, 2.75 mm2 in the AGI-1067 70 mg group, 3.17 mm2 in the AGI-1067 140 mg group and 3.36 mm2 in the AGI-1067 280 mg group (P<0.05 for AGI-1067 280 mg and probucol versus placebo) (39). There was also a significant dose-response relationship of AGI-1067 for the primary end point (P=0.02, Figure 1). The benefit of AGI-1067 and probucol was present immediately after PCI, raising the possibilities that countering oxidative stress may rapidly improve endothelial function (44) or that changes in plaque content (45) may have contributed to an optimal response to PCI.

Primary end point results in the Canadian Antioxidant Restenosis Trial-1 (CART-1). IVUS Intravascular ultrasound; PCI Percutaneous coronary intervention

Volumetric (three-dimensional) changes of nonintervened coronary reference segments, away from the PCI site, were also evaluated with IVUS in a post hoc analysis in CART-1 (39). The mean change in lumen volume (follow-up minus baseline) in the reference segments was −5.3 mm3 in the placebo group, −0.2 mm3 in the probucol group and −2.4 mm3 in the AGI-1067 70 mg group, but was +3.5 mm3 in the AGI-1067 140 mg group and +1.8 mm3 in the AGI-1067 280 mg group (P=0.05 for AGI-1067 140 mg versus placebo; P=0.077 for dose-response relationship). These findings may represent the first clinical evidence of vascular protection with AGI-1067 (46). This effect was due to trends for both the inhibition of negative remodelling and reduction of plaque burden. These clinical results are supported by demonstration of atherosclerosis prevention by AGI-1067 in all tested animal models (41), including in apolipoprotein E knockout and LDL receptor-deficient mice, and in hyperlipidemic primates.

The preclinical and clinical results obtained with AGI-1067 are also concordant with those in the Fukuoka AtheroSclerosis Trial (FAST) in which probucol induced regression of carotid atherosclerosis in 246 asymptomatic hypercholesterolemic patients (47). The incidence of major cardiac events was also significantly lower with probucol than in the control group in FAST (2.4% versus 13.6%; P<0.05). The results of the Probucol Quantitative Regression Swedish Trial (PQRST) appear to be discordant with those described here, but the design of that study raised several important issues (48). The primary end point of that study was lumen volume of femoral arteries using three-dimensional angiography, an approach rarely used in other clinical trials. The choice of the femoral location for assessment is also questionable in light of the preferential effect of probucol on younger lesions in the proximal thoracic aorta compared with the more advanced iliac lesions in experimental atherosclerosis in nonhuman primates (49). In addition, probucol was given to all patients (including those in the placebo group) for two months during the prerandomization phase, which represents another problematic design feature considering that probucol accumulates in tissues for prolonged periods.

AGI-1067 was not different from placebo with respect to its effects on the QT interval after six weeks of therapy in CART-1, in contrast to probucol (46). This allows testing of the antioxidant/anti-inflammatory hypothesis with AGI-1067 in two other clinical trials.

RATIONALE AND DESIGN OF THE CART-2

AGI-1067 has been effective at preventing atherosclerosis in all tested animal models. The positive results observed with AGI-1067 in reference segments in CART-1 have also been described. Therefore, CART-2 assesses the value of AGI-1067 for the reduction of both post-PCI restenosis and atherosclerosis progression in non-PCI vessels after 12 months of treatment (measured with IVUS [50]). Based on preclinical and clinical data and also on its beneficial effects on prevention of LDL-lipid oxidation, blockage of monocyte adhesion and recruitment to inflamed endothelium through inhibition of endothelial adhesion molecule expression, it was hypothesized that AGI-1067 may significantly slow atherosclerosis progression or even induce regression in CART-2. Considering that oxidative stress and inflammation may persist for a prolonged period after stenting (51), treatment with AGI-1067 for the entire period of risk after PCI may result in enhanced protection against luminal renarrowing. This hypothesis is also tested in CART-2 (52).

RATIONALE AND DESIGN OF THE AGGRESSIVE REDUCTION OF INFLAMMATION STOPS EVENTS (ARISE) TRIAL

Despite improvements in imaging modalities, visualization of morphological details indicative of plaque stabilization is not yet possible with IVUS (50), and its predictive value for future clinical events is not yet definitely known. The greatest impact AGI-1067 may have clinically is the prevention of plaque rupture and subsequent cardiovascular morbidity and mortality. Modifications resulting from the administration of AGI-1067, which stabilize the plaque but do not result in overall plaque volume changes, are only evaluable through the conduct of a clinical events trial. Therefore, the ARISE trial is a necessary addition to the full evaluation of the potential utility of AGI-1067. Furthermore, ARISE represents a unique opportunity to test in a definitive fashion the antioxidant/anti-inflammatory hypothesis (53). The primary objective of ARISE is to determine whether long-term treatment with AGI-1067 will prevent major cardiovascular events in patients with coronary artery disease. Because the beneficial effects of AGI-1067 would likely be related to its potent antioxidant and anti-inflammatory properties, another key objective is to determine the effects of AGI-1067 on markers of inflammation and oxidation.

ARISE is a multicentre, double-blind, randomized, placebo-controlled trial involving more than 200 study sites (Figure 2). Patients with a recent diagnosis of coronary artery disease (unstable angina or myocardial infarction) and one additional risk factor are enrolled in the United States, Canada, Europe and South Africa. Patients are randomly assigned to AGI-1067 or placebo in a 1:1 ratio and treated for at least 12 months. The study will be complete when at least 990 patients have experienced a primary event. The primary study end point is the combined incidence of cardiovascular mortality, resuscitated cardiac arrest, nonfatal myocardial infarction, nonfatal stroke, need for coronary revascularization and urgent hospitalization for angina pectoris with objective evidence of ischemia.

Study design of the Aggressive Reduction of Inflammation Stops Events (ARISE) multinational, multicentre trial. CAD Coronary artery disease; MI Myocardial infarction; UA Unstable angina

CONCLUSION

Despite medical advancements made over the past decade, cardiovascular disease originating from atherosclerosis inflicts a large burden in terms of life expectancy, life quality and societal costs. Based on the aging of the population and trends toward environmental and lifestyle factors that increase the risk for atherosclerosis, it is anticipated that this burden will not dissipate in the near future. There is, therefore, a need for a pharmacological intervention that would provide further cardiovascular protection in patients with atherosclerosis over and above the protection offered by other medications available, including statins. In light of its favourable effects, ARISE is evaluating the value of AGI-1067 on clinical end points.

Acknowledgments

Dr Tardif holds the Canadian Institutes of Health Research (CIHR) and Pfizer Chair in Atherosclerosis.

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