Antioxidant Therapy in Neurologic Disease FREE

The potential involvement of free radical or oxidative damage in the pathogenesis of human disease has received an enormous amount of study in the last decade.^{1 - 4} Free radicals are atoms or molecules with unpaired electrons in their outer orbits, making them highly reactive with macromolecular structures, leading to cellular injury and homeostatic disruption. Free radicals are produced as a byproduct of normal metabolism, and endogenous mechanisms exist to reduce their formation or enhance their inactivation.^{5 - 6} Disruption of the pro-oxidant and antioxidant balance in favor of the former may be a potential fundamental mechanism of human disease. A large body of evidence supports the concept that increased production of free radicals causes or accentuates neuronal injury and leads to disease, and this evidence has recently been reviewed by ourselves and others.^{7 - 9} Therapy aimed at boosting antioxidant defenses or reducing pro-oxidant production with free radical scavengers or antioxidants may be efficacious in preventing, ameliorating, or arresting many neurologic diseases. This approach is receiving increasing attention in clinical neurology both in large randomized controlled trials in common disorders, such as stroke and Parkinson disease, and in individual patients with rarer conditions, such as mitochondrial disorders. Moreover, widespread use of over-the-counter antioxidants and dietary supplements with presumed antioxidant ingredients has placed increasing pressure on physicians to be aware of data regarding the use of antioxidants as therapeutic agents. This review will summarize the main trials of antioxidant therapies in neurologic disorders and discuss methodological issues pertaining to these and future studies.

Antioxidants are endogenous or exogenous compounds that either reduce the formation of free radicals or react with and neutralize them, thus potentially protecting the cell from oxidative injury. Because the biochemistry of free radical injury is complex, many substances may act as potential antioxidants and thus provide protection against disease or limit its consequences. The classification of antioxidants presented in Table 1 is based on whether compounds are primarily endogenous or exogenous and their underlying mechanisms of action. In order for a particular antioxidant compound to enter the brain parenchyma, it must penetrate the blood-brain barrier (BBB) to allow for a critical therapeutic concentration within the central nervous system (CNS). Antioxidants may be lipid soluble (eg, vitamin E) or water soluble (eg, vitamin C) and possess varying degrees of BBB penetrance. Antioxidants that readily pass through the BBB are good therapeutic candidates for use in neurologic disorders. The pyrrolopyrimidines, a novel class of antioxidants that inhibit lipid peroxidation, have excellent BBB penetrance and are neuroprotective in animal models of both focal and generalized cerebral ischemia.^{10 - 11} These compounds have superior efficacy in such models compared with the 21-aminosteroids (lazaroids), a class with lower BBB penetrance.¹⁰ Coenzyme Q₁₀(ubiquinone) is a lipid-soluble mitochondrial antioxidant cofactor that readily crosses the BBB and that has been shown to be neuroprotective in several animal models of neurodegenerative diseases.¹²

Individual antioxidants may have differential effects in protecting nucleic acids, proteins, and lipids from free radical damage, and some compounds may be preferentially localized within specific organelles. Thus, rational combination therapy using different and potentially synergistic free radical scavengers could be superior to single agents, although this approach has rarely been used in trials of antioxidant therapy. For example, the use of vitamin E and vitamin C together may be superior to either agent alone,^{13 - 14} and the use of catalase may augment the potential beneficial effects of exogenously administered superoxide dismutase.¹⁵ Similarly, the combined use of a mitochondrial antioxidant (eg, coenzyme Q₁₀), an inhibitor of lipid peroxidation (eg, a pyrrolopyrimidine), and an iron-binding chelator (eg, deferoxamine) may be a rational therapeutic approach in some situations, eg, in early Parkinson disease. Novel derivative compounds are also being developed, eg, OPC-14117, an analogue of vitamin E.¹⁶ An intravenous formulation of vitamin E has also been studied,¹⁷ and chimeric compounds (eg, ascorbyl gamma–linolenic acid¹⁸ ) are also under development.

Although this article will focus on individual antioxidants in specific clinical trials, a number of studies using epidemiologic dietary evidence also suggest that naturally occurring antioxidants in food may protect against a number of neurologic disorders (although this type of evidence may often be confounded by lifestyle factors).⁹ Thus, dietary intervention may ameliorate free radical–mediated injury, and large-scale population educational campaigns could help reduce the burden of neurologic disease in the future. Increased dietary intake of tea (containing flavonoid compounds¹⁹ ) and tomatoes (rich in the non–vitamin A carotenoid lycopene²⁰ ) are examples of possible population-based antioxidant strategies.

Within this context, it is also imperative that the neurologic scientific community closely examine compounds with putative antioxidant properties. Although potentially beneficial, the widespread uncontrolled use of compounds with supposed antioxidant properties is cause for concern and should encourage the study of safety issues in trials of novel antioxidant compounds. The dietary antioxidants vitamin E and vitamin C (ascorbic acid) appear to be safe and free of serious adverse effects when used at high doses in adults.²¹ Although there has been some concern that vitamin E may enhance the effect of warfarin, a double-blind study failed to demonstrate a significant effect on the international normalized ratio at doses of vitamin E up to 1200 IU/d.²² However, the use of high doses of nutritional antioxidant compounds should not be assumed to be safe in all clinical situations. For example, in the landmark Finnish Smokers' Study, mortality was greater in those smokers randomized to receive beta carotene.²³ As the number of clinical trials of novel putative antioxidants will likely continue to increase, continued vigilance will be required to detect potential harmful effects.

Although many animal studies^{24 - 37} (Table 2) support the importance of oxidative mechanisms in neurologic disorders, potential publication bias may favor positive studies, a problem less likely to occur with human studies. Randomized controlled clinical trials of compounds with antioxidant properties have yielded positive, negative, marginal, or conflicting results, both in neurologic and nonneurologic disorders. Compounds tested in neurologic disease include vitamin E, tirilazad, N-acetylcysteine, ebselen, selegiline, idebenone, and extract of Gingko biloba. Diseases studied include Parkinson disease, Alzheimer disease, multi-infarct dementia, amyotrophic lateral sclerosis (ALS), Huntington disease, acute ischemic stroke, subarachnoid hemorrhage, head and spinal cord injury, and intractable childhood epilepsy. Unfortunately, none of the clinical trials performed to date has measured markers of oxidative injury as a surrogate marker of drug efficacy, either in pretrial dose-finding studies or in subgroups of patients during the trials. This is important because some of the compounds used may not be effective antioxidants in vivo in humans, or alternatively the compounds may be effective but have been given in suboptimal doses. Some compounds (eg, vitamin E, probucol) may have antioxidant or pro-oxidant effects depending on dosage, concomitant treatments, and study models.^{38 - 39} These considerations underscore the importance of pretrial dose-finding studies using novel markers of free radical injury now available, such as isoprostanes,^{40 - 41} 8-hydroxy-2-deoxyguanosine,⁴² or 3-nitrotyrosine,⁴³ reliable markers of lipid, DNA, and protein oxidation, respectively. Measurement of potential reduction of biochemical indices of free radical injury should be made in subgroups of patients as a surrogate marker of antioxidant effectiveness in future controlled trials.⁴⁴ The objective measurement of antioxidant effectiveness could also provide critical information in individual treatment trials of putative antioxidants in rare neurologic disorders in which large prospective studies are not possible.

Vitamin E is regarded as the prototypic antioxidant vitamin by both physicians and patients and is the one most extensively studied in neurology, particularly in the area of chronic neurodegenerative disease.

In the Parkinson Study Group trial of 800 patients with otherwise untreated early Parkinson disease, treatment with vitamin E at 2000 IU/d had no effect in delaying need for levodopa therapy in patients who were followed up over a mean period of 14 months.⁴⁵ In this study, treatment with 10 mg of selegiline per day delayed the need for levodopa by a median time of about 9 months. Although selegiline is thought to exert its therapeutic effect by selectively inhibiting the enzyme monoamine oxidase B and thus increasing brain dopamine levels, experimental evidence also supports its role as a neuroprotective antioxidant.⁴⁶ Selegiline may in part protect against MPTP (N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) toxicity by scavenging the highly toxic radical MPP+ (1-methyl-4-phenyl-pyridinium).⁴⁷ A recent clinical trial from Scandinavia of 157 patients with newly diagnosed Parkinson disease confirmed that early treatment with selegiline delays the need for levodopa, and moreover, there was no significant symptomatic deterioration during a 2-month washout period of selegiline withdrawal prior to initiation of levodopa therapy, lending support to the concept that selegiline is indeed neuroprotective in human Parkinson disease.⁴⁸

This apparent lack of efficacy of vitamin E in Parkinson disease underscores the fact that pretrial considerations of likely underlying disease mechanisms may be critical in choosing the most appropriate antioxidant compound or compounds for evaluation. A specific antioxidant may not reach the critical concentration in the cellular compartment important in the pathophysiology of a particular disease, and thus an inappropriate antioxidant may be tested, eg, an inhibitor of lipid peroxidation in a disease characterized mainly by cytoplasmic oxidative stress. Increasing evidence indicates that Parkinson disease is primarily a mitochondrial disorder,⁴⁹ and thus the use of compounds with good mitochondrial penetrance, such as coenzyme Q₁₀,¹² could potentially be of benefit in this disease. Some investigators have concluded that, because treatment with one compound with antioxidant properties fails to show benefit in a particular disease, "antioxidant therapy does not benefit this disease," or that oxidative mechanisms are not important in pathophysiology.⁵⁰ This may be an oversimplified view of oxidative pathobiology.

In a double-blind placebo-controlled randomized trial of 341 community-dwelling patients with moderately severe Alzheimer disease, when the analysis was adjusted for the baseline score on the Mini-Mental State Examination (scores were higher in the placebo group), treatment with vitamin E at 2000 IU/d was associated with an average 7.4-month delay to reach one of the following markers of disease progression: death, institutionalization, loss of ability to perform activities of daily living, or progression to severe dementia during the 2-year treatment period.⁵¹ Although less well publicized, patients in the trial who were randomized to receive selegiline, 10 mg/d, had a similar beneficial effect. Combination therapy with vitamin E and selegiline did not provide an additional benefit compared with either alone.

In a small trial of 73 patients with mild to moderate Huntington disease, treatment with vitamin E, 3000 IU/d, for 1 year failed to provide benefit, although there was a trend toward some clinical benefit in those patients with early disease.⁵² However, this study was confounded by the administration of 1 g of vitamin C and 25,000 IU of beta carotene given daily to both the placebo group and the active treatment group. In another small trial in Huntington disease using a different antioxidant, treatment of 48 patients with idebenone at 90 mg three times per day for 1 year failed to provide obvious clinical benefit compared with 43 patients treated with placebo.⁵³ However, neither of the above trials was powered to detect a less dramatic but still clinically important therapeutic effect.

Apart from trial size, there are several other reasons why trials with antioxidants in neurodegenerative diseases such as Huntington disease would not appear to demonstrate a positive outcome, even if oxidative damage plays a role in the pathophysiology of the disease. If the mechanisms underlying the disease are multiple and redundant, and neuronal injury occurs as a consequence of a number of concurrent or sequential processes, antioxidant therapy alone may not be effective. Protection against one pathway of cell damage may not be enough to halt disease progression. Multiple modes of therapy may be needed for an appreciable clinical effect in complex neurological disorders, eg, the combined use of potent antioxidants, N-methyl D-aspartate (NMDA) and non-NMDA receptor antagonists, inhibitors of apoptosis, and centrally acting calcium channel blockers. Furthermore, multimodal therapy may need to be delivered in a specific temporal sequence depending on the time course of various pathogenetic mechanisms of cellular injury, eg, following head injury. In addition, therapy may start too late in the course of an established disease process to demonstrate a clinical effect, as the pathologic effects of many neurodegenerative disorders are well advanced at the time of clinical presentation.

In a small trial using vitamin E in 24 children aged 5 to 18 years with refractory epilepsy, treatment of 12 children with vitamin E, 400 IU/d, over 3 months improved seizure control in 10 children, indicated by a reduction in seizure frequency of greater than 60% compared with a 3-month run-in baseline period.⁵⁴ Six of these 10 children showed a 90% to 100% reduction in seizure frequency. The two children who did not respond were identified as noncompliant with therapy, and this was confirmed by the measurement of plasma vitamin E levels. None of the 12 children given placebo showed any improvement in seizure frequency. To our knowledge, the encouraging results of this trial have not been replicated in a larger study.

Tirilazad mesylate, a 21-aminosteroid that inhibits lipid peroxidation,⁵⁵ has been studied in 4 acute neurologic disorders, with mixed results.

In acute ischemic stroke, 276 patients treated within 6 hours of stroke onset with tirilazad mesylate at a dosage of 6 mg/kg per day for 3 days fared no better than 280 patients treated with placebo.⁵⁶

Interestingly and somewhat surprisingly, two very similar large trials of tirilazad in subarachnoid hemorrhage, one in Europe, Australia, and New Zealand and the other in North America, have provided conflicting results.^{57 - 58} In the former trial,⁵⁷ patients treated with tirilazad mesylate, 6 mg/kg per day, for 10 days had reduced mortality and a better 3-month outcome compared with those given a vehicle alone, an effect that was more pronounced in men. In the North American study,⁵⁸ no difference was detected between those given the same dose of tirilazad and placebo. Although the reason for the different results of these 2 trials is unclear, it may be related to the fact that significantly more patients in the North American trial were treated with phenytoin, which is known to decrease the bioavailability of tirilazad.⁵⁸ A follow-up study using a higher dose of tirilazad is ongoing.⁵⁸

In acute spinal cord injury, treatment with tirilazad mesylate, 2.5 mg/kg every 6 hours, for 48 hours following an initial bolus of methylprednisolone appeared to have equal efficacy (but no obvious advantage) compared with a continuous 24-hour infusion of methylprednisolone.⁵⁹ Two large multicenter trials of tirilazad in moderate and severe head injury failed to show any clear difference in outcome between the treated and placebo groups.^{60 - 61} A novel group of compounds, the pyrrolopyrimidines, have been shown to be as effective as the 21-aminosteroids as inhibitors of lipid peroxidation while having a much greater BBB penetrance.²⁸ These compounds may replace the 21-aminosteroids as candidates in clinical trials in the future and may demonstrate more consistent efficacy.

Perturbations in oxidative metabolism and increased oxidative stress may underlie neuronal degeneration in ALS.^{62 - 63} It is now well established that mutations in the copper-zinc superoxide dismutase gene underlie the pathogenesis of some forms of familial ALS.^{9 ,62} Remarkably, only one small randomized controlled trial of antioxidant therapy in ALS has been reported.⁶⁴ Treatment of 55 patients of N-acetylcysteine, 50 mg/kg per day, over 1 year showed no benefit compared with 56 patients given placebo as measured by mortality, disability, decline in muscle strength, and pulmonary and bulbar function.

Extract of the plant Gingko biloba is a popular complementary remedy marketed for use for a variety of disorders, including supposed enhancing effects on cognition. Its beneficial effects are believed to derive in part from its high antioxidant flavonoid and terpenoid content.⁶⁵ In a welcome recent clinical trial of this "neutraceutical,"⁶⁶ treatment with Gingko biloba extract, 120 mg/d, in 96 patients with moderate to severe dementia secondary to either Alzheimer disease or multi-infarct dementia appeared to stabilize or improve cognition and social functioning (as assessed by the patients' caregivers) during 1 year of intervention compared with 104 patients taking a placebo with a similar taste and smell.⁶⁷ Also in Alzheimer disease, a recent report from Germany indicated a dose-dependent beneficial effect of idebenone in a total of 450 patients with mild to moderate dementia that was sustained over a 2-year follow-up period.⁶⁸

Two trials from Japan have recently been reported using the novel antioxidant ebselen, a compound with an action similar to that of glutathione peroxidase.^{69 - 70} In 300 patients with acute ischemic stroke, ebselen therapy commenced within 48 hours of stroke onset at a dosage of 150 mg twice per day for 2 weeks significantly improved outcome as measured by the Glasgow Outcome Scale at 1 month.⁶⁹ Improvement was maintained at 3 months, although this failed to reach statistical significance. Post hoc analysis showed that the improvement occurred especially in those in whom treatment was initiated within 24 hours. In 286 patients with subarachnoid hemorrhage, treatment with ebselen initiated within 96 hours using a dose schedule similar to that in the ischemic stroke trial improved the outcome at 3 months in those patients (58 in the placebo group, 52 in the ebselen group) who suffered neurologic deficit secondary to vasospasm, and this improvement correlated with findings on computed tomography.⁷⁰ There was no difference in outcome between the treatment and placebo groups in those patients without clinically obvious vasospasm.

Two small studies of antioxidant therapy in patients with human immunodeficiency virus–associated cognitive impairment have been published.^{16 ,71} However, both of these trials were short tolerability studies (10-12 weeks) and were not powered to assess efficacy. The vitamin E analogue OPC-14117 was as well tolerated as placebo in a study of 30 patients, and despite the trial limitations, there was a trend toward improved cognitive test scores in the treated group.¹⁶ In the other trial by the same group in a total of 36 patients, both α-lipoic acid and selegiline were well tolerated, and patients treated with selegiline showed marked improvement on tests of verbal memory after 10 weeks of treatment.⁷¹

A large body of evidence suggests that oxidative injury is important in either the primary or downstream secondary pathophysiological mechanisms underlying many neurologic disorders, and that therapy with appropriate antioxidants may be beneficial. However, the clinical trials performed to date provide conflicting data. There are several potential reasons for this, including suboptimal dose schedules, the inappropriate use of a particular antioxidant for a given disease, and the redundancy of disease pathophysiology. Measurement of novel indices of free radical injury now available should be used to provide biochemical evidence of antioxidant effectiveness and to provide surrogate markers of efficacy in clinical trials. To date, trial planners may not have adequately considered the complex biochemistry of free radical biology. In the future, the availability of potential screening strategies to identify subjects at risk for some disorders may compel us to study the potential utility of predisease antioxidant therapy. Identification of high-risk individuals is now possible for Huntington disease and forms of Alzheimer disease due to autosomal dominant gene mutations. Advances in molecular neurogenetics may soon allow us to identify many diseases of the nervous system at the preclinical stage. The ethics of such selective screening and the design of testable early treatment strategies in asymptomatic individuals, including antioxidant therapies, is one of the important issues facing neurology in this new millenium.

Accepted for publication September 24, 1999.

We thank Arthur Asbury, MD, Christopher Clark, MD, and Garret FitzGerald, MD, for reviewing the manuscript and for their helpful comments.

Corresponding author: Norman Delanty, Department of Clinical Neurological Sciences, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland (e-mail: normandelanty@eircom.net).