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ALS and other motor neuron disorders 2000 (Suppl 4), 5–15 © 2000 ALS and other motor neuron disorders. All rights reserved. ISSN 1466-0822
Supplement
Antioxidant therapy in ALS Erik P Pioro
Oxidative stress in ALS Free radicals and reactive oxygen species Oxidative metabolism in the mitochondrial respiratory chain supplies almost all the energy required by the human brain and spinal cord. During this process, oxygen is reduced to water by cytochrome oxidase, and ATP is generated. The resultant high-energy electrons move along the electron transport chain, and of these, a small percentage (< 5%) normally ‘leak’ onto oxygen to form free radicals.1–4 Free radicals are oxygen-containing molecules with one or more unpaired electrons, represented in chemical formulas by a dot (). Such molecules can exist independently, hence the term ‘free’. Free radicals are also known as reactive oxygen species. Free radicals have been reported to be involved in a variety of neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and ALS.1,5,6 Free radicals can be generated through either oxidative or excitoxic processes – the former via the electron transport chain as described above, the latter via activation of xanthine oxidase, nitric oxide synthase, and the production of arachidonic acid by phospholipase A2. Reactive oxygen species can damage cells at a variety of levels, including DNA (resulting in apoptosis), protein (enzyme inactivation, protein degradation), and lipids (loss of membrane potential and integrity, increased permeability to calcium and metal ions, formation of toxic aldehydes).
Superoxide dismutase (SOD) protection against reactive oxygen species One of the main forms of defense of eukaryotic cells against free radical damage is the enzyme superoxide dismutase (SOD).7 Three forms of SOD have been identied, each with a distinct distribution and coded by a separate gene: cytosolic copper, zinc-superoxide dismutase (SOD1), mitochondrial manganese-superoxide dismutase (SOD2), and an extracellular superoxide dismutase (SOD3). SOD1, which has a copper and zinc at the active site, is of particular interest in ALS pathogenesis because SOD1 gene mutaCorrespondence: Erik P Pioro MD PhD, Director, Center for ALS and Related Disorders, Section of Neuromuscular Diseases and EMG, Department of Neurology, S90, The Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, OH 44195, USA (Fax: +1 216 445 4653; e-mail:
[email protected]).
tions have been identied in approximately 20% of patients with inherited or familial ALS (fALS).8 In the presence of proton (H1 ), SOD scavenges the superoxide radical (O22 ) to form hydrogen peroxide (H2O2) and oxygen (Equation 1). In the normal state, hydrogen peroxide is then metabolized to water and oxygen by the enzymes glutathione peroxidase (GPx) and catalase (Cat) (Equation 2). 2O22 1
SOD
2H1 ¾ ¾ ® GPx, Cat
2H2O2 ¾ ¾ ®
O2 1
H2O2 1 2H2O
O2
(1) (2)
Cascade formation of free radicals The superoxide radical (O22 ) normally generated through oxidative metabolism is relatively unreactive per se, but it can initiate a cascade formation of other, more reactive oxygen species.3,5 By donating or accepting electrons, O22 can convert nonradical molecules into radicals. SOD normally maintains very low levels of the superoxide radical, but when SOD activity is signicantly reduced (as has been noted in some patients with fALS who have the SOD1 gene mutation,9–11) superoxide radicals can accumulate and a cascade of reactive species formation can ensue. Major reactive species generated and their intracellular reactants are listed in Table 1. Reactive species have been shown to directly cause neuronal death (e.g. peroxynitrite12,13). They also disrupt critical cell function such as maintenance of intact cell membranes (e.g. hydroxyl radical – via oxidation of polyunsaturated fatty acids) and are postulated to interfere with signal transduction and neurolament processing via SOD1-dependent protein nitration causing resistance to phosphorylation. In addition to causing direct damage,
Reactive species
Superoxide anion (O22 ) Nitric oxide radical (NO) Peroxynitrite (ONOO2 ) Nitronium ion (NOO1 ) Hydroxyl radical (OH)
Reactants
Nitric oxide (NO) Hydrogen peroxide (H2O2) Transition metals (Cu21 , Fe31 )
Table 1 Selected reactive species and intracellular reactants
6
Erik P Pioro
Supplement free radicals can react with intracellular reactants to trigger further formation of radicals. Free radicals can reduce transition metals by donating an electron to the metal, converting the metal from its stable oxidized state (e.g. Cu21 ) to an unstable reduced state (Cu1 ). The unstable metal can then donate its electron to an intermediate, normally unreactive compound such as hydrogen peroxide, triggering formation of the highly toxic hydroxyl radical (OH) (Equation 3- Fenton or Haber-Weiss reaction). Such reactions are normally kept in check by extracellular metal-binding proteins (e.g. transferrin, ferritin) that keep copper and other transition metals in an unreactive state. Most intracellular components are highly sensitive to damage by hydroxyl radicals (Table 2). In the normal state, hydrogen peroxide is maintained at low intracellular levels (Equation 2) and its reduction by SOD is not detrimental when the copper ion is in a stable oxidized state (Cu21 ) (Equation 4). However the mutant SOD1 enzyme is thought to have abnormal folding which exposes the copper ion core, resulting in a higher propensity for the copper to be reduced by a neighboring free radical and subsequent initiation of the radical cascade.9,14 SOD2 Cu1 1 SOD2 Cu21 1
H2O2 ¾ ® H2O2 ¾ ®
SOD2 Cu21 1
OH 1
SOD2 Cu1 1
O22 1
OH2 2H1
(3) (4)
SOD1 gene mutations Although mutations in the SOD1 gene account for about 20% of fALS, or only 1–2% of all ALS, this discovery has been a major advance in our understanding of the neurodegenerative mechanisms in ALS for two reasons: 1. there is little difference clinically and pathologically between fALS and sporadic ALS (sALS), so ndings related to the SOD1 mutation may be relevant to sALS, and 2. transgenic mice overexpressing high levels of the mutated human SOD1 gene develop a progressive MND, primarily of the hindlimbs, resembling human ALS. This animal
model has stimulated numerous studies into the cause and mechanisms of the disease as well as providing a means for preclinical screening of neuroprotective therapies.15
Role of antioxidants in ALS Therapies suppressing oxidative damage As discussed above, increasing evidence supports the hypothesis that oxidative damage contributes to motor neuron degeneration in ALS. Therefore, inhibition or suppression of neuronal oxidation may slow or even stop disease progression. Testing of candidate antioxidant drugs (Table 3) has generally been limited to in vitro studies from cell cultures and in vivo studies in mouse models of ALS (transgenic mouse model, wobbler mouse model). Efforts to investigate these drugs in human ALS have been limited.
Free radical scavengers Vitamin E Vitamin E (a -tocopherol) has been recognized as an antioxidant since the 1930s and was among the medications given to Lou Gehrig.16 One of the body’s most important antioxidants, it is a lipophilic molecule that diffuses into cell membranes and protects polyunsaturated fatty acids against damage caused by lipid peroxidation,17 thus maintaining cell membrane integrity. Tocopherol donates a hydrogen atom (with its single electron) to harmful lipid peroxide radicals (OO2 L) to form a neutral lipid hydroperoxide (HOO2 L) and a relatively unreactive vitamin E radical (O2 tocopherol) (Equation 5). This reaction occurs faster than the lipid peroxide radical can react with adjacent fatty acid chains or membrane proteins. The resultant lipid hydroperoxides are then processed by glutathione peroxidase. Some of the vitamin E radical is regenerated by vitamin C (ascorbate) as reduced tocopherol (Equation 6). Vitamins E and C may thus have complementary antioxidant functions.5
Cellular Component
Type of damage
Consequence
DNA
Base modication Strand breakage
Apoptosis2
Lipid
Peroxidation of polyunsaturated fatty acids3
Formation of toxic aldehydes Permeability to Ca21 , metal ions Loss of membrane integrity, potential
Protein
Cross-linking Fragmentation Site-specic lesion
Protein degradation Protein inactivation (e.g. glutamine synthetase)66
Table 2 Hydroxyl radical-mediated damage to cellular components
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Antioxidant therapy in ALS
Supplement Free radical scavengers
Antioxidants
Metal chelators
‘Cocktail’ therapies
Aminosyn (28 amino acids) Alsamin (Selenium, antioxidants, amino acids, calcium channel blocker) Alsemet (L-methionine, vit E, selenium)
Vitamin E Vitamin C SOD N-acetylcysteine 21-aminosteroids (Lazaroids) Nitrone derivatives (spin traps) DMPO Gingko biloba
Catalase (substrate of peroxidase) Estrogen Glutathione a -Lipoic acid Selegiline Allopurinol Oligomeric proanthocyanidins (OPCs) Carotene (a , b ) Retinol Selenium
Desferrioxamine EDTA 21-aminosteroids L-, D-penicillamine Trientene
Table 3 Potential antioxidant therapies in ALS
HO-tocopherol1
OO2 L ¾ ®
O2 tocopherol 1
O-tocopherol 1
ascorbate ¾ ®
semihydroascorbate 1 (6) HO-tocopherol
HOO-L (5)
In vitro evidence indicating a potential role for vitamin E in the treatment of ALS includes the following: 1. Rat cortical cell cultures exposed to CSF from ALS patients show a decreased death rate in the presence of vitamin E.18 Of note, this decrease is even greater when vitamin E is combined with allopurinol; 2. Decreased vitamin E levels are found in the spinal cord of transgenic SOD1 mice19; 3. Mutant SOD1 mouse spinal cord cultures are protected from glutamate toxicity by pretreatment with vitamin E20; 4. Rat neuronal cells expressing mutant SOD1 are partially protected from death by the addition of vitamin E21; 5. Mutant SOD1 transgenic mice given daily oral vitamin E (200 IU/d, although doses may have been higher) and selenium (8 mg/kg) do not show any increase in survival, but demonstrated a delay in disease onset by 12 to 15 days, a 14% improvement over controls.22 In addition, dietary supplementation with vitamin E and selenium slightly improves the spinal cord vitamin E content of transgenic mice, thus appearing to restore some of the normal age-dependent increase in vitamin E observed in wild-type mice. Vitamin E was rst chemically synthesized in 1938 and was immediately used in humans. Because rats decient in vitamin E developed encephalomalacia and muscle weakness, it was thought to have a benecial effect on the nervous system. Anecdotal case reports in the 1940s and later, reported occasional benet of vitamin E in ALS.23–27 Uncontrolled studies showed lack of effectiveness of oral vitamin E in large (200–2000 IU/d) and massive (up to 20,000 IU/day for 6 months, n 5 20) doses.25 Poor absorption was suspected in megadose therapy because of frequent reports of intact vitamin E capsules in patients’
stools. Intramuscular administration of vitamin E (200 IU/d) also showed no benet and was complicated by subcutaneous necrosis at injection sites.25 In 1969, a small trial of vitamin E in 12 patients with sALS failed to show any detectable benet.28 In spite of these negative results, the nding that disease onset was delayed in mutant SOD1 transgenic mice given vitamin E22 suggests that a -tocopherol may benet patients with fALS caused by SOD1 mutations. Presymptomatic treatment may be needed to affect survival rather than disease onset alone. A clinical trial of vitamin E in ALS is currently underway in Europe. Vitamin C Vitamin C (L-ascorbate) can attain concentrations in the central nervous system (CNS) tenfold higher than in plasma, and even higher levels in cells because of active transport mechanisms.29 At high concentrations, vitamin C (and other avonoids) acts as a powerful antioxidant, although it can also act as a pro-oxidant and stimulate production of hydroxyl radicals. This generally occurs when vitamin C is present in low concentrations or in the presence of excess amounts of transition metals (e.g. catalytic iron, Fe31 ).3,30 Because SOD1 mutations may make the internal copper ion more accessible, such excess metals may be present in fALS.9,14 Therefore administration of vitamin C may actually be detrimental to patients with fALS and caution is required when using antioxidant avonoid therapies to treat ALS in general. One recent study31 examined the effect of administering high dose vitamin C and the metal chelator trientine to mutant SOD1 transgenic mice. A delayed onset of symptoms by 16 days (~8% improvement over controls) was reported. The relative effects of vitamin C and trientine were difcult to determine since both were given together. Past studies have shown improvement from administering metal chelators (see below) but no study has specically evaluated the effect of vitamin C alone in ALS.
8
Erik P Pioro
Supplement Superoxide dismutase (SOD) Superoxide dismutase (SOD), as previously discussed, acts as a free radical scavenger (Equation 1). When administered exogenously, it may compensate for diminished endogenous enzyme activity and may help prevent ALS progression. Delivery of the enzyme can be difcult because of poor penetration of SOD into the CNS. In vitro and in vivo studies have suggested a benecial effect of SOD in models of ALS. Interpretation of these data however must be tempered by the fact that SOD1 enzyme expression levels in transgenic mice are manyfold higher than in human fALS and that results in mice may therefore be difcult to extrapolate to humans. Studies of SOD have shown that SOD mimics EUK-8 and EUK-134 partially protect rat neuronal cells expressing mutant SOD1 from death.21 In another study, lecithinized SOD given subcutaneously to wobbler mice (a model of motor neuron disease) signicantly retarded disease progression.32 A case report has been published of SOD use in one patient.33 The patient, a 47 year old respirator-dependent, quadriparetic fALS patient received intrathecal, then intraventricular bovine SOD (bSOD) for 5 months as bolus then continuous infusions at doses of 25–50 mg/d. It was found that high concentrations of bSOD in the lateral ventricle could only be obtained with intraventricular (rather than intrathecal) administration. Muscle strength was measured twice over 6 weeks during the bolus administration period, and both times found to be approximately 2/5 (MRC scale) as determined by a physical therapist’s assessment. Muscle strength then dropped rapidly to just over 0/5 during a period when no bSOD was received because of catheter complications. The bSOD was well tolerated by the patient. There have been no other published reports of SOD use in humans. N-acetylcysteine N-acetylcysteine (NAC) is an attractive antioxidant for use in humans. It is commonly used as an antidote to acetaminophen toxicity, has few if any side effects, and crosses the blood-brain barrier, especially in its procysteine form. It is a free-radical scavenger and promotes the removal of the hydroxyl radical and hydrogen peroxide.34 It is also a precursor of glutathione, an important intracellular antioxidant.35 In addition, it may act as a chelator. Animal studies of NAC have shown 1. a mild although nonsignicant decrease in motoneuron degeneration in wobbler mice;36 2. no signicant change in symptom onset or survival in transgenic SOD mice;37 3. complete prevention of motor neuron loss in transgenic SOD mouse spinal cord organotypic cultures.38 A randomized, double-blind, placebo-controlled trial of N-acetylcysteine (50 mg/kg/d sc for 12 months) in 110 patients with ALS showed no change in disease progression but resulted in an overall mortality risk reduction of 26% at one year.39 This decrease however was not statistically signicant. Subgroup analyses revealed a surprising divergence of results in patients with spinal-onset ALS compared to bulbar-onset ALS. Limb-onset ALS patients
had a 50% decrease in mortality risk compared to placebo, and bulbar onset ALS patients had a 52–66% increase in mortality risk compared to controls. Neither of these results however were statistically signicant. Hypotheses set forth to possibly explain this difference included the following: 1. Bulbar-onset ALS generally shows a more rapid clinical deterioration and may have required longer periods of treatment than were given; 2. A benecial effect of NAC in the bulbar-onset group may have been masked by good supportive care including the use of gastrotomy; 3. NAC may have caused liquication of mucus which was more difcult for dysphagic patients with bulbar-onset ALS to accommodate; 4. Bulbar-onset ALS may differ from spinal-onset ALS in its response to treatment. Finally it should be noted that baseline forced vital capacity was better at baseline in the spinal-onset group than in the bulbar-onset group. In interpreting the results of this study, it is important to recall that none of the results shown were statistically signicant, and that wide ranges of uncertainty (condence intervals) were observed. 21-aminosteroids (Lazaroids) Lazaroids are glucocorticoid-derived 21-aminosteroids with antioxidant properties but without glucocorticoid activity. Aminosteroids have been shown in vitro to decrease neuronal nitric oxide synthase and behave as free radical scavengers preventing membrane lipid peroxidation.19 In addition, certain aminosteroids (e.g. U74006F) have been found to have chelating properties, as discussed below.40 The aminosteroid U-74389F has been found to decrease expression of GAP-43 mRNA in wobbler mice spinal motoneurons. GAP43 is a neuronal growth-associated protein preferentially expressed during embryogenesis and after CNS injury. It is hypothesized that the attenuation of GAP-43 mRNA expression with U-74389F may represent reversal of motoneuron degeneration or aberrant sprouting. Of note, a similar effect was achieved with natural corticosterone.41 There have been no trials of aminosteroids in humans.
Antioxidants Catalase Catalase (CAT) is an enzyme which scavenges and detoxies hydrogen peroxide produced by the action of SOD (Equation 2) and inhibits the formation of nitric oxide. Putrescine-modied catalase (PUT-CAT) has a 2.4 fold increase in blood-brain-barrier permeability. PUT-CAT was administered to transgenic SOD1 mice with both high expression (He) and low expression (Le) of the mutant gene, at a dose of 720–900 U/d subcutaneous for He and 60,000 U/kg intraperitoneal twice per week for Le.42 Both PUT-CAT and CAT delayed the onset of clinical disease and weakness in He and Le groups, with PUT-CAT having a more marked effect (Table 4). Survival analyses were generally not signicant except for a trend to increased survival in a subgroup analysis of Le mice given PUT-CAT (10 days increased survival compared to controls; p < 0.05).
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Antioxidant therapy in ALS
Supplement Symptom onset
Weakness
Putrescine-modied catalase High-expressor mice Low-expressor mice Controls
¯ 26% (p < 0.001) ¯ 5% (p < 0.001) —
¯ 26% (p < 0.001) ¯ 9% (p < 0.001) —
Catalase High-expressor mice Low-expressor mice Controls
¯ 12% (p < 0.01) ¯ 7% (p < 0.01) —
¯ 14% (p < 0.001) ¯ 6% (p < 0.05) —
Table 4 Effect of catalase administration to high and low transgenic mouse expressors of mutant SOD142
Estrogen Although it has not been used in the treatment of human ALS, estrogen has demonstrated antioxidant properties43,44 and estrogen’s reported benet in Alzheimer’s disease is hypothesized to be due in part to its antioxidant properties.45,46 In vitro studies have shown decreased lipid peroxidation of rat cortical cultures and human brain homogenates with estrogen hormones.44 Moreover, mutant SOD1 mouse spinal cord motor neuron cultures are protected from glutamate toxicity by pretreatment with estrogen.20 a -Lipoic acid The metabolic antioxidant a -lipoate (thioctic acid, 1,2dithiolane-3-pentanoic acid) is a low molecular weight substance that easily crosses the blood-brain-barrier and has very low toxicity.47 After being taken up into cells and tissue, it is reduced to dihydrolipoate, which is even a more potent antioxidant. Being a thiol, like glutathione, a lipoate and dihydrolipoate are central to the body’s antioxidant defense system. Among its many actions, a -lipoate raises intracellular glutathione levels (by 30–70%), scavenges hydroxyl radicals, peroxynitrite, nitric oxide, hydrogen peroxide, and oxygen free-radicals. It also chelates transition metals like iron and copper and appears to protect against glutamate excitotoxicity. An oral dose of 10 mg/kg (radiolabeled) reached peak levels in rat cortex, peripheral nerve and spinal cord within 30 minutes of administration. It has a relatively short halflife with levels dropping to 5% of peak doses at 24 hours. Cultured rat hippocampal neurons exposed to 1 µM a lipoate 2 hours after plating and with every media change were signicantly protected against glutamate toxicity (1 mM) with 92% viability, compared to non-lipoate treated cells with 9% viability.48 Lipoic acid has been used in Germany for the treatment of diabetic neuropathy and clinical trials for its use in this condition are underway in the United States. Selegiline Selegiline (Deprenyl®) is a monoamine oxidase B (MAOB) inhibitor with antioxiant properties which has some benet in Parkinson’s disease. Three randomized, placebo-
controlled trials using selegiline 10 mg PO qd have failed to show any benet in disease progression or survival. An open randomized, placebo-controlled trial of selegiline in 53 patients for 6 months revealed no difference in MRC score, Norris score, or functional vital capacity.49 A randomized, double-blind, placebo-controlled trial using a 3 month crossover design in 10 patients showed no difference in Norris or spinal bulbar scores.50 Finally, another randomized, double-blind, placebo-controlled trial of 133 patients over 6 months showed no difference in Appel score.51
Metal chelators Metal chelators prevent reactive iron and other transition metals from participating in reactions producing hydroxyl radicals and lipid peroxidation. Desferrioxamine, a well characterized chelator, prevents most iron-dependent radical reactions and has successfully decreased oxidative damage in animal models of human disease.3 However, desferrioxamine and most antioxidants do not cross the blood-brain barrier; therefore other chelating agents with antioxidant activity have been developed, including a group of 21-aminosteroids containing tirilazad mesylate (U74006F).40 Mutations of SOD114 are thought to expose the normally shielded copper core active site of the enzyme. Normally, the copper ion is in a stable oxidized state (Cu21 ) (Equation 4). However exposure of the copper core increases the likelihood for the copper to be reduced by free radicals and trigger a cascade of further radical generation. In vitro studies have suggested benet from the use of metal chelators: 1. d,l-penicillamine and ethylenediaminetetraacetic acid (EDTA) minimize the production of free radicals by the mutant SOD1 enzyme;52 2. the copper chelator TEPA (tetraethylene-pentamine) partially protects SOD1 transfected rat neurons from death.21 In vivo studies have further suggested benet in animal models of ALS: 1. Oral d-penicillamine given to SOD1 transgenic mice (0.1 g/kg gavage or in chow) resulted in a delay of symptom onset of 10 days in gavaged animals (7.5% improvement over controls; p 5 0.0015) and delayed mortality by 11 and 10 days respectively in
10
Erik P Pioro
Supplement gavaged and chow animals (gavage 7.2% improvement; p 5 0.001; chow 6.4% improvement; p 5 0.001). 53 There was no effect on the overall duration of disease; 2. Coadministration of oral high dose vitamin C and the copper chelator trientine (125–170 mg/kg/d) to mutant SOD1 transgenic mice results in a delay of symptom onset by 16 days (7.7% improvement over controls; p 5 0.03), as well as a delayed endpoint (total paralysis of hindlimbs) by 25 days (9.4% improvement over controls; p 5 0.045). 31 As previously mentioned, the relative effects of vitamin C and trientine are difcult to determine since both were given together. Trientene is used in the treatment of Wilson’s disease (hepatolenticular degeneration), which results from abnormalities in copper metabolism. Most reports of metal chelator use in human ALS are anecdotal.54,55 Small series of patients in the 1970s and 1980s (5–10 patients per study) reported no clinical benet in the majority of patients using doses of penicillamine ranging from 300 mg po BID-TID for 3 months to 1 year. This nding may, in part, be due to poor penetration of chelating agents into the CNS. A few reports have described improvement in patients with known exposure to heavy metals.56–58
‘Cocktail’ Therapies Empirical combinations of amino acids, vitamins, antioxidants, and metal chelators have been used by ALS patients and physicians since at least the 1980s. Few of these combination therapies have been reported or carefully evaluated. Reported studies are difcult to interpret because they are often brief, usually retrospective, and involve relatively few patients. Moreover, it is of course difcult to isolate which, if any, component may be more benecial than the others. One such study reported the use of Aminosyn® (Abbott Pharmaceuticals), an intravenous solution of 18 amino acids (including leucine, isoleucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine). Aminosyn was given intravenously to 12 patients with ALS twice weekly for one month.25 Continued progression of ALS was noted. In another study, 36 patients given various combinations of N-acetylcysteine, vitamin C, vitamin E, Nacetylmethionine, dithiothreitol, dimercaptosuccinic acid (DMSA) failed to show any increase in survival when compared to historical controls.54 Alsamin, a combination of selenium, antioxidants, amino acids, and the calciumchanel blocker nimodipine was reported to increase glutathione activity and vitamin E serum levels in 35 sALS patients after 9 weeks.59 Finally, a double-blind, pacebo-controlled trial of Alsemet (a combination of L-methionine (2 g), antioxidants (not specied), vitamin E (400 IU), and selenium [3 3 102 5 g]) in 32 patients with ALS resulted in increased serum glutathione activity and vitamin E levels. In addition, slower deterioration in muscle testing (p < 0.025) and bulbar function scores (p 5 0.05), but no difference in limb function scores were noted. Survival at 1 year was
85% for the Alsemet group but only 50% for the placebo group (possibly a statistically signicant difference, although calculations not reported).60
Anecdotal reports Nutritional/herbal supplements Anecdotal reports of improvement of ALS with nutritional supplements and herbs have been reported since the 1960s, with few if any hard data to justify such use. Administration of ‘organ extracts and other nutrients’ was reported in the 1960s without any measurable benet. Octacosanols, long-chain alcohols present in the CNS but absent from a normal diet have been felt by some to be of potential benet in CNS disorders. Such long-chain alcohols can be obtained from wheat germ oil. A 1987 placebo-controlled, crossover, double-blind trial of oral octacosanol in 11 patients with ALS showed no change in disease course.25 Informal discussion with patients and examination of ALS-related Internet websites indicate that oligomeric proanthocyanidins (OPCs) are currently thought to be of benet. OCPs, found in the bark of french pine (Pinus Maritima; pycnogenol) and in grape seed extract are said to have an antioxidant potency up to 50fold greater than vitamin E and 20-fold greater than vitamin C. However, no peer-reviewed data are available to support such statements. Finally, many ALS patients are taking other substances with known antioxidant properties which have not been tested either in vitro or in vivo for use in ALS. These include a - and b -carotene, retinol, selenium (a cofactor for glutathione synthetase), and gingko biloba (a free radical scavenger).
Summary and recommendations for antioxidant therapy in ALS Because oxidant-mediated neurotoxicity has been convincingly demonstrated in numerous studies, the antioxidant therapeutic approach to the treatment of ALS has a solid theoretical basis. Although many compounds with antioxidant properties have been tested in vitro and in animal models of ALS, human data are scarce. One must however be cautious in extrapolating results from transgenic mice whose levels of human mutant SOD1 activity are usually manyfold greater than in humans. In addition, because these mice represent SOD1-mutated fALS, a rare defect in human ALS, a drug that is not effective in delaying disease onset or survival in transgenic mutant SOD1 mice may still be of value in sALS patients. Several studies suggest that a combination of antioxidants is likely to be more benecial than a single antioxidant, although which combination to use has not been systematically evaluated. Finally, as there is a substantial interaction between glutamate-mediated excitotoxicity and oxidative damage,61 the optimal treatment may be a combination of antioxidative
11
Antioxidant therapy in ALS
Supplement and antiexcitotoxic treatments, as well as neurotrophic factor-like agents.62–64 Based on the available data and on documented toxicities, the following recommendations for antioxidant therapy are suggested: 1. Avoid activities that reduce endogenous antioxidant levels, such as smoking which reduces serum vitamin C concentrations;65 2. High dose vitamin E (up to 2000 IU/d) has been shown to decrease lipid peroxidation (see Equation 5); 3. High dose vitamin C (500–1000 mg/d) can be added for synergy with vitamin E because vitamin C reduces the vitamin E radical (see Equation 6). 4. Based on animal model studies, the antioxidant enzyme (putrescine-modied) catalase, the copper chelator trientine (already used in treatment of Wilson’s disease), and the antioxidant lipoic acid, are promising compounds with low toxicity which warrant further evaluation. Continued research efforts are needed to investigate the therapeutic usefulness of antioxidants in the treatment of ALS.
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Acknowledgements Many thanks to Mathilde H Pioro for assistance in preparing the manuscript.
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49. Mazzini L, Testa D, Balzarini C, Mora G. An open randomized clinical trial of selegiline in amyotrophic lateral sclerosis. J Neurol 1994; 241: 223–227. 50. Jossan SS, Ekblom J, Gudjonsson J, Hagbarth KE, Aquilonius SM. Double blind crossover trial with deprenyl in amyotrophic lateral sclerosis. J Neural Transm 1994; 41: 237–241. 51. Lange DJ, Murphy PL, Diamond B, et al. Selegiline is ineffective in a collaborative double-blind, placebo-controlled trial for treatment of amyotrophic lateral sclerosis. Arch Neurol 1998; 55: 93–96. 52. Wiedau-Pazos M, Goto JJ, Rabizadeh S. Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis. Science 1996; 271: 515–518. 53. Hottinger AF, Fine EG, Gurney ME, Zum AD, Aebischer P. The copper chelator d-penicillamine delays onset of disease and extends survival in a transgenic mouse model of familial amyotrophic lateral sclerosis. European J Neurosci 1997; 9: 1548–1551. 54. Vyth A, Timmer JG, Bossuyt PM, Louwerse ES, deJong JM. Survival in patients with amyotrophic lateral sclerosis, treated with an array of antioxidants. J Neurol Sci 1996; 139 (Suppl): 99–103. 55. Conradi S, Ronnevi L-O, Nise G, Vesterberg O. Long-time penicillamine treatment in Amyotrophic Lateral Sclerosis with parallel determination of lead in blood, plasma and urine. Acta Neurol Scandinav 1982; 65: 203–211. 56. Bousser MG, Malier M. Penicillamine in amyotrophic lateral sclerosis. Lancet 1979; i: 168. 57. Justic A, Sostarko M. Improvement of spinal amyotrophy by penicillamine therapy. Lancet 1977; ii: 1034–1035. 58. House AO, Abbott RJ, Davidson DL. Response to penicillamine of lead concentrations in CSF and blood in patients with motor neuron disease. Br Med J 1978; ii: 1684. 59. Apostolski S, Marinkovicz Z, Nikolic A, Blagojevic D, Spasic MB, Michelson AB. Glutathione peroxidase in amyotrophic lateral sclerosis: the effects of selenium supplementation. J Environm Pathol Toxicol Oncol 1998; 17 (3–4): 325–329. 60. Stevic Z, Nikolic A, Blagojevic D, et al. A controlled trial of combination of methionine and antioxidants in amyotrophic lateral sclerosis. Proc ALS/MND International Alliance Meeting, Munich, Germany 1998; [Abstract]. 61. Mitsumoto H, Chad DA, Pioro EP. Excitotoxicity and oxidative damage in ALS pathogenesis. In: Amyotrophic Lateral Sclerosis. Philadelphia, PA: Oxford University Press; 1998: 197–225. 62. Rothstein JD. Therapeutic horizons for amyotrophic lateral sclerosis. Curr Opin Neurobiol 1996; 6: 679–687. 63. Louvel E, Hugon J, Doble A. Therapeutic advances in amyotrophic lateral sclerosis. Trends Pharmacol Sci 1997; 18: 196–203. 64. Eisen A, Weber M. Treatment of amyotrophic lateral sclerosis. Drugs Aging 1999; 14: 173–196. 65. Banerjee KK, Marimuthu P, Sarkar A, Chaudhuri RN. Inuence of cigarette smoking on vitamin C, glutathione and lipid peroxidation status. Indian J Public Health 1998; 42: 20–23. 66. Nakamura K, Stadtman ER. Oxidative inactivation of glutamine synthetase subunits. Proc Natl Acad Sci USA 1984; 81: 2011–2015.
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Group discussion Ben Brooks, MD: If you look carefully at the Vitamin E papers, and they have taken a big hit over the years, you will see that they describe what we call in Wisconsin ‘The Minnesota Effect’. The Wexler Papers and all the papers from the 30’s and 40’s talk about the effect of Vitamin E on cramps, fasciculations. It is quite clear clinically that this is a (real) effect. You can ramp up the dose of vitamin E and show the effect on spontaneous and incident cramps. Usually spontaneous cramps are treated much more effectively at lower doses, still high dose by reconstitution or supplemental status. And then you have to go higher for the incident cramps, and so I think the Wexler Papers were correct but everyone was looking for the treatment effect on survival and didn’t really see what was also in those papers in terms of symptomatic improvement. When we look at our therapies we have to look at whether these therapies have symptomatic effects, effects on the propagating features of the disease or primary effect on the underlying pathology. If we keep that framework in mind when we evaluate all these therapies it will be helpful in putting together the combination therapies. Second thing, was the power of these studies, many of the studies that were termed ineffective in terms of the p-values, showed an effect size and I think if you had a drug that had a patent on it, you probably would take N-acetylcysteine and do another study with one thousand patients and show that the effect size would be consistent with the treatment effect. Third, procysteine is another model of a drug that ts into the category the N-acetylcysteine drugs. Merit Cudkowicz did a very nice Phase 1–2 study on that in terms of effect, and so I think when you look at whether you should raise glutathione, how should you do it? The issue is where’s lipoic acid in this group, the thionic acids? I think that we can learn a lot from the experimental diabetic neuropathy people because they have taken their animal models, put the combination therapies into the animal models and have tried to put back out in the clinic. Erik Pioro, PhD, MD: Alpha lipoate does have antioxidant properties and it may be another one to consider. There haven’t been any trials done in ALS with it. Procysteine to my understanding, unfortunately, was negative with the last studies that were done at MGH but that is also another drug that may be worth re-investigating in larger numbers. Really, we are faced with most of the antioxidant studies being small. If we had more patients and the power was increased we may be able to start seeing a benet but nothing was obvious with these at least. Your rst point about looking at symptoms and the benet is clearly important when we are dealing with treating this disease and all the aspects have to be looked at. The primary endpoint in a lot of these studies of course is death; secondary endpoints being progression of muscle weakness, but the quality of life and all of these issues are extremely important to look at. It is interesting that you mentioned the cramping being improved by Vitamin E. Peter Anderson described SOD1 mutations in the 90 position; these
patients complain of cramping which is very characteristic in the lower extremities. This seems to be immediately relieved by vitamin E. It is just miraculous in their eyes because it is very incapacitating. As you know, this particular mutation is very slow in its evolution, but they are really incapacitated by leg pain and cramping; Vitamin E takes it away immediately. Jeffrey Rosenfeld, PhD, MD: Dr Brooks’ comments about the studies being generally under-powered are very true. Most of the studies that we are going to hear about today, especially the investigator initiated studies, are largely under-powered. That is why I thought the result from the Alsemet Trial in Munich was so interesting. That study used a survival endpoint, and there is not usually a measurement error there. They demonstrated a signicant difference using that cocktail. That study showed an 85% survival using the cocktail vs. 50% survival in the placebo group. I thought it was an interesting and curious result in Munich and I think it is worth recognizing considering our conversation today. It hasn’t been formally published that I’m aware of. I think that is the most signicant antioxidant trial to date using the most basic dependent measure in 32 patients, which is an under-powered study looking at survival over that period of time. And they still saw what I thought to be a remarkable effect. I don’t know why we’re all not prescribing that cocktail? Wendy Johnston, MD: I think this a good time to make two comments. One is that I don’t think we are utilizing the families with ALS as experimental models. As you know there are a number of families with different mutations who have extremely stereotyped presentations including age of onset and course. And this is a very motivated group who would be very interested in pre-symptomatic therapy. So I think that certainly nationally and internationally we could get enough families to institute some of these in a prospective way taking onset and survival as endpoints. And it might be a study very amenable to an international effort using simple endpoints such as these. If we are going to give patient anecdotes, I have a patient with familial ALS. Her mother presented at the same age, same pattern – bulbar – and my patient is on estrogen replacement. She has had a very prolonged course compared to her mother. She is a physician. We’ve talked about whether estrogen is a factor. The second point is funding which is always an issue, the NIH does have a Complimentary and Alternative Medicine branch now. And I think again collectively we should be approaching them for funding of many of these trials. Stanley Appel, MD: Let me just make a couple of comments, one with respect to what we just heard. We have a patient who is 49 years of age, with a very strong A4V SOD mutation. He has a little bit of myotonia. Everyone in the family is strongly affected with an autosomal dominant pattern and is dead by this age. This man is still going on and he isn’t taking anything. So I think even
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Supplement there, there are going to be some constraints. I would just want to emphasize two points that Dr Pioro made in a slightly different way. You talked a little bit in your last slide about the importance of recognizing that SOD may not be quite the same as sporadic ALS. But there is another reason it may not be quite the same, in addition to the number of copies of the mutant SOD. In studies that Glen Smith and I have published and one in review, it’s quite clear that one of the measures of free radical stress, namely 4-hydroxy nonenal, which is a very potent and very toxic compound, is elevated dramatically in the CSF of patients with ALS. This turns out to also be in the blood. But it is only elevated in the sporadic ALS, it is not elevated in the familial ALS. So there may be something in addition that is very different which tells us that the pattern of free radical stress is very different in the SOD or in any of the familial cases because in our familial cases they were not elevated in the CSF or blood. Now if you go back and look at the data very carefully, you see there is some free radical stress within the cell detected immunohistochemically in the familial patients, but you have to look very, very hard and then maybe you’ll nd it. You don’t have to look hard at all in the sporadic patients. So there is something very different and therefore I’m not sure that familial is necessarily the best model for some of these free radical antioxidants that we are talking about. And then of course, the other issue which is far from settled is ‘why do motor neurons die in the SOD mutation?’ I know it is very easy for all of us to assume because SOD is a dismutase it should therefore affect free radicals. It is far from clear that we understand why they die or whether it is directly related to free radical stress. The recent notion that Don Cleveland put in the literature – a hypothesis that is going to be solved by a wonderful copper chaperone turns out not to be the case from what Phil Wong has said. So we are still up in the air as to the mechanism, and I think the emphasis ought to be on sporadic where it is clear that free radical stress is important. Erik Pioro, PhD, MD: Your point on the difference in the markers of the hydroxyl damage being higher in the sporadic ALS patients, rather than the familial is opposite of what I would have guessed based on these ndings and the mechanisms that copper may be more exposed in the mutated SOD-1. So it is a challenge to look more at the sporadic forms. At this point at least, the animal models that are available would not be able to address that. Your other comment about the patient with the A4V mutation is fascinating. Even though the genetics appear outwardly to be identical, there are clearly other factors at play i.e. modier genes, and such, that may also be of inuence. So when we are looking at results of clinical trials, especially when we are looking at a sporadic ALS background, we have to keep in mind that we are dealing with a myriad of potential gene interactions that are inuencing the behavior of the disease in that particular person. We are not dealing with a homogeneous patient population by any means, as we all know. But I think when it comes to interpreting results of trials, and this is where the power of the
study is so critical, you really need to have enough patients analyzed so that some of these conicting things will hopefully be averaged out. Jeffrey Rosenfeld, PhD, MD: For many reasons the familial ALS patients appear to be a group requiring separate study from the sporadic patients and for obvious reasons there is justication to think that this might be a group that theoretically would be selectively beneted by antioxidant therapy, although antioxidant enzyme activity is not the issue, it is rather the mutated form, the toxic form of the SOD. But we really haven’t addressed the question of a trial just for familial patients, who at least presumably have a more homogeneous pathophysiology. Erik Pioro, PhD, MD: If I can also add to Dr Johnston’s comment about using patients with familial mutations as test subjects, if you will, to study; patients that I have spoken with are extremely willing to participate and help in any way. It would be very benecial in patients who are presymptomatic to treat them prophylactically. The issue is that the disease has already begun in the sporadic ALS patients we see and it’s probably been going on for months or longer before they come to see us. We’re dealing with a disease where ‘the horse is out of the barn’ so to speak and so we are ghting an uphill battle. In a lot of the studies that I presented with the SOD1 transgenic mice the treatments were started pre-symptomatically. So that is also important to keep in mind. If these animals started treatment once the symptoms really began would we be seeing as much of a benet? But these are all issues we are dealing with, emphasizing that looking for markers to identify early disease is really important. John Wald, MD: The studies that you discussed on Vitamin E; do you know if they were alpha-tocopheral or were they mixed? Because the more recent evidence is that the mixed tocopherols are quite important to include. So if we are later on going to vote on what to include I think we want to make sure that we look harder at the beta and gamma tocopherols as well as just alpha. Erik Pioro, PhD, MD: What is your opinion on the importance of that? What is the evidence to support the mixed tocopherols as being more benecial? John Wald, MD: I’m not certain the evidence is very strong but the argument is that with excess alpha you are interfering with the effects of the beta and gamma and losing other important antioxidant properties that they potentially have in other mechanisms. But I wondered if you know whether it is all alpha in the studies? Erik Pioro, PhD, MD: The earlier studies did not specify but some indicated it was alpha. In the European Trial, I’m not sure. So that is a good point Dr Wald, and I think that we need to look at that more critically to see if using a mixture of vitamin E forms is going to make a difference.
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Supplement Stanley Appel, MD: It’s a very good point and I think it highlights how we should have half of the room lled with pharmacologists here because it goes back to the battle of natural versus synthetic as well and whether you get your vitamin E from your renery or your ‘natural’ sources. When you look at some of these things in terms of
the pharmacology, the racemic mixtures are sometimes better and the purer are not and the question is, is it a dose effect? When you look at the vitamin E studies in particular, making sure that you have equal amounts of alpha, or beta, or gamma is going to be one of the issues we are going to have to come up with.
