Drug Antioxidant Effects a Basis for Drug Selection?

REVIEW ARTI LE

Drug Antioxidant Effects A Basis for Drug Selection?

Barry Halliwell Pulmonary Medicine, UC Davis Medical Center, Sacramento , California, USA

Contents 570 Summary 570 I. What is a Free Radical? 570 1.1 Definition of a Free Radical 57/ 1.2 Hydroxyl Radical 572 1.3 Formation of Oxygen Radicals In Vivo 574 1.4 Reactive Oxygen Species 575 1.5 Transition Metal Ions and Free Radical Reactions 575 1.6 Antioxidant Defence In Vivo: Intracellular 576 1.7 Antioxidant Defence In Vivo: Extracellular 579 2. Free Radical Damage to Cells and the Protective Ability of Antioxidants 579 2.1 Mechanisms of Cell Damage: The Molecular Targets 58/ 2.2 How to Characterise an Antioxidant 5 2 3. The Role of Free Radicals in Human Disease 582 3.1 Causation of Disease 582 3.2 A Significant Consequence of Disease Pathology 584 3.3 A General Consequence of Tissue Injury 585 3.4 What Can We Expect from Antioxidant Therapy? 586 586 4. Antioxidants in Disease Therapy 586 4.1 Use of Naturally Occurring Antioxidants and Derived Compounds 587 4.1.1 Superoxide Dismutase 587 4.1.2 Superoxide Dismutase 'Mimics' 588 4.1.3 a-Tocopherol 589 4.1.4 Ascorbic Acid (Vitamin C) 589 4.1.5 Adenosine 589 4.1.6 Lactoferrin 590 4.1.7 Nicotinamide 59/ 4.1.8 Glutathione and its Precursors 59/ 4.1.9 Carotenoids 59/ 4.2 'Synthetic' Antioxidants 59/ 4.2.1 Mercaptopropionylglycine and Other Thiols 592 4.2.2 Ebselen: A Glutathione Peroxidase Mimic? 593 4.2.3 Allopurinol, Oxypurinol and Other Xanth ine Oxidase Inhibitors 593 4.2.4 Inhibitors of the Generation of Reactive Oxygen Species by Phagocytes 595 4.2.5 Chelating Agents: Deferoxamine 595 4.2.6 Other Iron Chelators: Hydroxypyridones 596 4.2.7 Other Iron Chelators: ICRF-187 and the 'Lazaroids' 4.2.8 Probucol and Other Chain-Breaking Antioxidants 570 Drugs 42 (4) 1991

596 5. Antioxidant Properties of Drugs Developed for Other Purposes 597 6. Conclusion

Summary A free radical is any species capable of independent existence that contains one or more unpaired electrons. Free radical reactions have been implicated in the pathology of more than 50 human diseases. Radicals and otherreactive oxygen species areformed constantly in the human body, both by deliberate synthesis (e.g. by activated phagocytes) and by chemical side-reactions. They are removed by enzymic and nonenzymic antioxidant defence systems. Oxidative stress, occurring when antioxidant defences are inadequate, can damage lipids, proteins, carbohydrates and DNA. Afew clinical conditions arecaused by oxidative stress, but moreoften thestress results from the disease. Sometimes it then makes a significant contribution to thedisease pathology, and sometimes it does not. Several antioxidants are available for therapeutic use. They include mo lecules naturally present in the body[superoxide dismutase (SOD), a-tocopherol, glutathione and its precursors, ascorbic acid, adenosine, lactoferrin and carotenoidsJ as well as synthetic antioxi dants{such as thiols, ebselen (PZ51), xanthineoxidaseinhibitors, inhibitors ofphagocyte function, iron ion chelators and probucol]. The therapeutic efficacy of SOD, a-tocopherol and ascorbic acid in the treatment ofhuman disease is generally unimpressive to date although dietary deficiencies ofthelasttwomolecules shouldcertainly beavoided. Xanthine oxidase inhibitors may beoflimited relevance as antioxidants for human use. Excitingpreliminary results with probucol (antiather osclerosis), ebse1en (anti-inflammatory), and iron ion chelators (in thalassaemia, leukaemia, ma laria, stroke, traumatic brain injuryand haemorrhagic shock) needto be confirmed by controlled clinical trials. Clinical testing ofN-acetylcysteine in HIV-1-positive subjects may alsobe merited. A few drugs already in clinicaluse may havesome antioxidant properties, but this ability is not widespread and drug-derived radicals may occasionally cause significant damage.

Free radicals and their relationship to human possible spin states. Indeed, the technique of elec disease is an important research area at the mo tron spin resonance detects radicals because it ment, especially since it has been reported that free measures the energy changes that occur as elec radical reactions are implicated in the pathology of trons change their spin state. Because electrons are over 50 human diseases (listed in Halliwell & Gut more stable when paired together in orbitals, rad teridge 1989). icals are in general more reactive than nonradical species, although there is a considerable variation 1. What is a Free Radical? in their individual reactivity (reviewed by Halli 1.1 Definition well & Gutteridge 1989). Radicals can react with other molecules in a Electrons in atoms occupy regions of space number of ways. Thus, if 2 radicals meet, they can known as orbitals. Each orbital can hold a maxi combine their unpaired electrons (symbolised by -) mum of 2 electrons, spinning in opposite direc and join to form a covalent bond (a shared pair of tions. A free radical can be simply defined as any electrons): species capable of independent existence (for how A· + A· -+ A-A (Eq. I) ever brief a time) that contains one or more un paired electrons, an unpaired electron being one A radical might donate its unpaired electron to that is alone in an orbital. Most biological mole another molecule (a reducing radical) or it might cules are nonradicals, containing only paired elec take an electron from another molecule in order to trons (Halliwell & Gutteridge 1989). pair (an oxidising radical). However, if a radical An electron occupying an orbital by itself has 2 gives one electron to, takes one electron from or Drug Antioxidants and Disease 571

simply joins on by addition to a nonradical, that 1.2 Hydroxyl Radical nonradical becomes a radical. Thus, a feature of the reactions of free radicals with nonradicals The most reactive free radical known is the hy (which includes most biological molecules) is that droxyl radical (·OH), whose formation plays a key they tend to proceed as chain reactions: one radical role in the damage suffered by tissues exposed to begets another and so on. Only when 2 free radicals high-energy radiation (Von Sonntag 1987). When meet can these chain reactions be terminated (re tissues are exposed to, for example, 'Y-radiation, action 1). most of the energy they take up is absorbed by water, largelybecause there is more water in tissues than any other molecule. The radiation causes one

Removal of H· Initiation (can occur at several 1places in the chain)

Minor reactions Attack on membrane proteins; 1:=',m ---c..~ crosslinking if 2 radicals meet

~ A

Major reaction l~

Attack on membrane proteins; ~B)___.. reaction of 2 peroxyl radicals to 02' cause singlet oxygen (102) formation Lipid peroxyl radical ~CH02' +)CH02'- )C=O +~C-OH + 102 H· abstraction 1 from adjacent membrane lipid \F\=J\=/\ c 02H Lipid hydroperoxide

Fig. 1. The free radical chain reaction of lipid peroxidation. Attack of a reactive species such as ·OH upon the side-chains of unsaturated fatty acids causes hydrogen atom abstraction . The consequences of such attack upon a fatty acid side-chain containing 3 double bonds are shown. Hydrogen can be abstracted at different points in the side-chain, so that several isomeric lipid hydroperoxides can result. Cyclic peroxides can also be formed. A, Band C have conjugated diene structures (carbon atoms linked by a double, then a single, then another double bond). 572 Drugs 42 (4) 1991

ofthe oxygen-hydrogen covalent bonds in water to bility. However, under physiologicalconditions, the split, leaving a singleelectron on the hydrogen atom most likely fate of carbon-centered radicals is to and one on the oxygen atom and creating two rad combine with oxygen, creating yet another type of icals, H· and ·OH. Since it is so reactive, 'OH does radical, the peroxyl radical (sometimes abbreviated not persist for even a microsecond before combin to 'peroxy radical'): ing with a molecule in its immediate vicinity. Be II cause it is a radical, however, its reactions leave - C· + Oz -+ -C-Oz' (Eq. 3) behind a legacy in the cell in the form of propa II gating chain reactions. Thus, if 'OH attacks DNA, Peroxyl radicals can combine with each other free radical chain reactions occur and cause strand or attack membrane proteins (fig. 1), but they are breakage, deoxyribose fragmentation and extensive also reactive enough to attack adjacent fatty acid chemical alteration of the purine and pyrimidine side-chains, abstracting hydrogen: bases (Von Sonntag 1987).The latter pattern of base changes in DNA is apparently diagnostic for attack i III - C- Oz· + - CH -+ - C-OzH + -C' (Eq. 4) by 'OH (Aruoma et al. 1989a; Halliwell & Aruoma IIII 1991). Imperfect repair of DNA damage caused by lipid 'OH can result in proto-oncogene activation and hydroperoxide carcinogenesis. Hence, high-energy radiation can lead to cancer, which implies that 'OH is a com Another carbon-centered radical is generated, and plete carcinogen (reviewed by Breimer 1988). so the chain reaction (reactions 3 and 4) can con Perhaps the best characterised biological dam tinue. Hence, one 'OH can result in the conversion age caused by 'OH (although not necessarily the of several hundred fatty acid side-chains into lipid most important: see Halliwell 1987) is its ability to hydroperoxides (Halliwell & Gutteridge 1989). Ac initiate the free radical chain reaction known as cumulation oflipid hydroperoxides in a membrane lipid peroxidation (fig. 1). This occurs when the disrupts its function and can cause it to collapse. 'OR is generated close to or within membranes and In addition, lipid hydroperoxides can decompose attacks the fatty acid side-chains of the membrane to yield a wide range of highly cytotoxic products, phospholipids. It preferentially attacks side-chains the most studied'of which are aldehydes (reviewed derived from fatty acids with several double bonds, by Esterbauer et al. 1988). Most attention has been such as arachidonic acid.The 'OH abstracts an atom paid in the literature to malonaldehyde (sometimes of hydrogen from one of the carbon atoms in the called malondialdehyde) but this is relatively un side-chain and combines with it to form water: reactive compared with such noxious products as I I 4-hydroxynonenal (Curzio 1988; Esterbauer et al. - C- H + ·OH -+ -C' + HzO (Eq. 2) 1988). Peroxyl radicals and reactive aldehydes can I I also cause severe damage to membrane proteins, Reaction 2 removes the 'OH, but leaves behind inactivating receptors and membrane-bound en a carbon-centered radical (Co) in the membrane. zymes (e.g. Dean et al. 1986; Roubal & Tappel Carbon-centered radicals formed from polyunsa 1966). turated fatty acid side-chains usually undergo mo lecular rearrangement to give conjugated diene 1.3 Formation of Oxygen Radicals In Vivo structures, which can have various fates. Thus, if 2 such radicals collided in the membrane, cross Interest in the role of free radicals in human linking of fatty acid side-chains could occur as the metabolism was raised by the discovery in 1968 of 2 electrons join to form a covalent bond (fig. 1). an enzyme specific for a free radical substrate, su Reaction with membrane proteins is also a possi- peroxide dismutase (SOD). This enzyme activity is Drug Antioxidants and Disease 573

present in all human cells and functions to remove phagocytes to kill some of the bacterial strains that superoxide radical, the I-electron reduction prod they can engulf. This can be illustrated by exam uct of oxygen: ining patients with chronic granulomatous disease, 02 + e- -- Oi- (Eq. 5) a collective name given to several conditions in which the membrane-bound NADPH oxidase sys Superoxide dismutase removes Oi- by catalys tem in phagocytes that makes the Oi- does not ing a dismutation reaction: one is oxidised to Oi- work (Curnutte & Babior 1987). Such patients have 02 and the other reduced to H202: phagocytes that engulf and process bacteria per (Eq. 6) fectly normally, but several bacterial strains are not killed and are released in viable form when the In the absence of SOD, dismutation of Oi- oc phagocytes die. Thus, patients suffer severe, per curs nonenzymically but at a rate approximately 4 sistent, and multiple infections with several organ orders of magnitude less at physiological pH. isms, especially Staphylococcus aureus. The discovery of SOD led to the realisation that Another killing mechanism used by neutrophils Oi- is formed in vivo in living organisms, and SOD (but not by macrophages) is the enzyme myelo functions to remove it. Formation of some in Oi- peroxidase (Weiss 1989). It uses H202 produced vivo appears to be a chemical accident (Fridovich by dismutation of Oi- to oxidise chloride ions into 1983; Imlay & Fridovich 1991). For example, when hypochlorous acid, HOCI, a powerful antibacterial mitochondria are functioning, some of the elec agent (many household bleach products contain the trons passing through the respiratory chain leak sodium salt of hypochlorous acid): from the electron carriers and pass directly onto oxygen, reducing it to Oi- (Fridovich 1983). Many (Eq. 7) molecules oxidise on contact with oxygen, such as More recently discovered examples of physio when an adrenalin solution left to stand on a la logical roles for 0i- include its possible involve boratory bench 'goes off and eventually forms a ment in fibroblast proliferation (Meier et al. 1990; pink product. The first stage in this oxidation is Murrell et al. 1990) and in vasoregulation (Halli transfer of an electron from the adrenalin onto 02, well 1989a). It is now widely thought (reviewed by giving Oi-. Such oxidations undoubtedly proceed Marietta 1989), but not yet completely certain in vivo as well (reviewed in Halliwell & Gutteridge 1989). For example, several sugars, including glu (Myers et al. 1990) that endothelium-derived re cose, react with proteins to produce oxygen radi laxing factor (EDRF), a humoral agent, produced cals. Thus, it has been suggested that years of ex by vascular endothelium, which is an important posure ofbody tissues to elevated blood glucose in mediator ofvasodilator responses induced by sev diabetic patients can result in them suffering an eral pharmacological agents (including acetylchol 'oxidative stress' that may contribute to the side ine and bradykinin), is identical with nitric oxide. effects of hyperglycaemia (discussed by Wolff & Nitric oxide (NO) has one unpaired electron and Dean 1987). Elevated blood glucose leads to a non thus qualifies as a free radical. Vascular endothel enzymatic glycosylation of proteins, to which free ium may also produce small amounts of Oi- which radical generation might contribute (Wolff & Dean can react with NO. Both NO itself (if generated in 1987). excess) and some of the products of its reaction Although some of the Oi- production in vivo with Oi- may be highly cytotoxic (Beckman 1990; may be accidental, much is functional. Thus, ac Beckman et al. 1990; Saran et al. 1990), although tivated phagocytic cells generate Oi- as has been others have disputed this and argued that the inter shown for monocytes, neutrophils, eosinophils and action of NO and Oi- may have a beneficial role most types of macrophage (Curnutte & Babior in regulating vascular tone (Halliwell 1989a; Saran 1987). Radical production is important in allowing et al. 1990). 574 Drugs 42 (4) 1991

1.4 Reactive Oxygen Species Oi- production by phagocytes (Curnutte & Babior 1987):

Superoxide dismutases remove Oi- by con cyt c (Fe+++) + Oi 7 -+ 02 + cyt c (Fer") (Eq, 9) verting it into 0i- and H202. They work in con junction with two enzymes that remove H202 in Another reactive oxygen species (but not a rad l ~g, human cells: catalase and glutathione peroxidase ical) is singlet oxygen a form of oxygen that (reviewed by Halliwell & Gutteridge 1989). The has undergone an electronic rearrangement that al study ofinborn errors of metabolism strongly sug lows it to react much faster With biological mole gests that glutathione peroxidase is the more im cules than does 'normal' (ground-state) oxygen. For portant in removing H202 in human tissues, prob example, singlet 02 reacts directly with polyunsa ably because it is located in the same subcellular turated fatty acid side-chains in membrane lipids compartments (mostly cytosol and some in mito to give lipid peroxides. Singlet 02 is produced when chondria) as SOD. Glutathione peroxidase is the several pigments are illuminated: light is absorbed only human enzyme known so far that requires the by the pigment, which enters a higher electronic element selenium for its activity: a selonocysteine excitation state. As this returns to the ground state, residue (side-chain -SeH instead of -SH, as in energy is transferred onto oxygen, to make singlet normal cysteine) is present at the enzyme's active 02. Several compounds found in plants, some cos site. It is unlikely, however, that the sole function metics and some drugs (e.g. tetracyclines) can cause of selenium in humans is to act as a cofactor for singlet 02 formation in the presence of light (re glutathione peroxidase (reviewed by Levander viewed by Epstein 1982; Kanofsky 1989). 1987). Glutathione peroxidase removes H202 by Usually such 'photosensitisation' reactions are using it to oxidise reduced glutathione (GSH) into a nuisance. For example, many of the side effects oxidised glutathione (GSSG): of the nonsteroidal anti-inflammatory drug ben oxaprofen could have been related to its ability to 2GSH + H202 -+ GSSG + 2H20 (Eq. 8) act as a photosensitiser (Artuso et al. 1990; Sik et H202 is a fairly unreactive molecule that can al. 1983), and patients with certain forms of por cross cell membranes easily. Like Oi-, it may per phyria become light-sensitive because photosensi form some physiological roles, e.g. control ofplate tising porphyrins accumulate in their skin (Ma let adherence (Salvemini & Botting 1990). Since thews-Roth 1987). However, photosensitisation can the H202 molecule has no unpaired electrons, be made use ofin medicine. Components ofa por H202 cannot be described as a radical. Hence, the phyrin derivative called haematoporphyrin deriv term 'reactive oxygen species' has been introduced ative (HPD) are taken up by tumour tissues . Ir in the biomedical literature to describe collectively radiation of the tumour with light at a wavelength not only Oi- and ·OH (radicals) but also H202 absorbed by HPD can then damage or kill the tum (nonradical). Hypochlorous acid (HOCl) produced our: this method has been applied in the treatment by myeloperoxidase can also be included under this of some types of lung cancer (reviewed by Ash & heading: it is a powerful oxidising agent but not a Brown 1989), although its real clinical benefit re radical, having no unpaired electrons. H202, Oi-, mains to be fully evaluated. ·OH and HOCI are sometimes collectively called Small amounts of singlet 02 can be generated 'oxidants'. This is a valid description for H202, in peroxidising lipid systems (fig. 1; Wefers 1987) ·OH and HOCl, which are oxidising agents. How but, apart from photosensitisation reactions, sin ever, Oi- has both oxidising and reducing prop glet 02 does not seem to be a major damaging spe erties. Indeed, the latter property is made use ofin cies in the human body. However, it can be pro a popular assay for 0i-, the SOD-inhibitable re duced by activated eosinophils under certain duction ofcytochrome c, often applied to measure circumstances (Kanofsky 1989). Drug Antioxidants and Disease 575

1.5 Transition Metal Ions and Free ticipate in the formation ofspecies able to abstract Radical Reactions hydrogen. There is considerable debate (e.g. see Ar uoma et al. 1989b; Minotti & Aust 1987) about Most transition metals have variable oxidation which species initiate peroxidation in biological numbers, e.g, iron as Fe++ or Fe+++and copper as lipid systems (such as lipoproteins and mem Cu" or Cur". Changing between oxidation states branes); candidates include ferryl, perferryl, Fe++ involves accepting and donating single electrons, Fe+++-02 complexes and ·OH. However, there is e.g.: general agreement that metal ions are needed for Fe+++ + e" ~ Fe?" (Eq. 10) formation ofinitiating species, except in organisms exposed to high doses of ionising radiation. Sec (Eq. II) ond, transition metal ions decompose lipid hydro Thus, most transition metal ions are remarka peroxides into peroxyl and alkoxyl (lipid-O) rad bly good promoters of free radical reactions (Hill icals that can abstract hydrogen and perpetuate the 1981). For example, Fe++ ions react with H202 to chain reaction of lipid peroxidation. This may be form a number of highly-reactive species, one of represented, in a simplified form, by the equations which is ·OH (reviewed by Halliwell & Gutteridge below, in which L· symbolises a carbon-centered I 990a): radical (fig. 2): Fe++ + H202 --. intermediate oxo-iron ion LOOH + Fe++ (Cu") --. LO· + Fe+++ (Cur") + OH complexes (e.g. ferryl) alkoxyl i radical (Eq. 14) Fe+++ + ·OH + OH- (Eq. 12) LOOH + Fe+++ --. LOO· + Fe++ + H+ peroxyl Superoxide can re-reduce Fe+++ to Fe?": radical (Eq. 15)

Fe+++ +Oi- --. intermediate oxo-iron ion complex (perferryl) LO· + LH --. LOH + L· (Eq. 16) ~ Fe++ + 02 (Eq. 13) LOO· + LH --. LOOH + L· (Eq. 17) L· + 02 --. LOO· (Eq. 18) Copper ions can also convert H202 into ·OH (Aruoma et al. 199Ia). Generation of ·OH and other Reducing agents, such as ascorbic acid or Oi-, reactive species appears to account for some or all can accelerate these metal ion-dependent peroxi of the toxicity observed when Oi- and H202 are dation reactions because Cut and Fe?" ions seem generated in excess in vivo (reviewed in Halliwell to decompose peroxides faster than do Cur" and & Aruoma 1991; Halliwell & Gutteridge I990a; Fe+++, respectively. The end-products of these Imlay & Linn 1988). There are very few significant complex metal ion-catalysed breakdowns of lipid cellular targets in mammals that can be attacked hydro peroxides include the cytotoxic aldehydes by Oi- or H202 generated at physiological rates. mentioned previously, as well as hydrocarbon gases It has also been suggested that the peroxynitrite such as ethane and pentane (reviewed in Halliwell species generated by reaction ofOi- with NO may & Gutteridge 1989). decompose to produce ·OH under physiological 1.6 Antioxidant Defence In Vivo: Intracellular conditions (Beckman 1990) but doubt has been ex pressed about this (Saran et al. 1990). The word 'antioxidant' can be defined in vari Iron and copper ions also accelerate lipid per ous ways. Often the term is implicitly restricted to oxidation, for at least 2 reasons. First, they can par- chain-breaking antioxidant inhibitors of lipid per- 576 Drugs 42 (4) 1991

oxidation, such as a-tocopherol (see below). How tocopherol help to minimise the consequences of ever, the author prefers a broader definition - an lipid peroxidation in membranes, should this pro antioxidant is any substance that, when present at cess begin. low concentrations compared to those of an oxi The terms 'a-tocopherol' and 'vitamin E' are often disable substrate, significantly delays or prevents used synonymously. This is not quite correct: vi oxidation of that substrate (Halliwell & Gutteridge tamin E is defined nutritionally as a factor needed 1989). The term 'oxidisable substrate' includes al in the diet of rats for normal reproduction (Di most everything found in living cells, including plock 1985), and compounds other than a-toco proteins, lipids, carbohydrates and DNA. pherol (e.g. (3-, 'Y- and a-tocopherols) have some ef The major intracellular antioxidants in the hu fect in this assay. However, a-tocopherol is the most man body are probably the enzymes superoxide effective lipid-soluble antioxidant in animals . dismutase, catalase and glutathione peroxidase. Two isoenzymes of SOD exist in human cells: an 1.7 Antioxidant Defence In Vivo: Extracellular enzyme containing copper and zinc ions (largely located in the cytosol) and a mitochondrial SOD Antioxidant defence in the extracellular com containing manganese ions. Both catalyse the same partments of the human body relies largely on dif reaction (reaction 6; Fridovich 1983). In addition, ferent strategies than does intracellular defence. a membranes contain the chain-breaking antioxidant Tocopherol is still important. Thus, the content of a-tocopherol, a lipid-soluble molecule that is lo a-tocopherol in plasma lipoproteins helps to de cated in the interior of biological membranes. a termine their resistance to lipid peroxidation, but Tocopherol is the most important chain-breaking it is not the only antioxidant present. This is rel antioxidant present in human membranes (Ingold evant to the development of atherosclerosis, since et al. 1986) but not the only one (Esterbauer et al. peroxidation oflow-density lipoproteins appears to 1989). Attached to the hydrophobic structure of a contribute to the progression of atherosclerosis (Es tocopherol is an -OH group whose hydrogen atom terbauer et al. 1989). Indeed, low plasma concen is very easy to remove. Thus, when peroxyl and trations of a-tocopherol and vitamin C seem to be alkoxyl radicals are generated during lipid peroxi associated with an increased incidence of myo dation, they combine preferentially with the an cardial infarction and of some types of cancer (Gey tioxidant, e.g.: et al. 1987; Gey 1990). However, it could be argued that these low levels are merely indicative of a poor LOO + tocopherol-OH --+ LOOH + tocopherol-Or (Eq. 19) diet, which predisposes to most diseases. Thus, al though one could assume that taking commercial instead of with an adjacent fatty acid. This ter vitamin pills would protect against atherosclerosis , minates the chain reaction, hence the name chain improving one's diet is a more logical recommen breaking antioxidant. It also converts the a-toco dation. If low density lipoproteins isolated from pherol into a new radical, tocopherol-O. This rad human plasma are placed under oxidative stress, ical is poorly reactive, being unable to attack ad peroxidation does not begin until tocopherols, {3 jacent fatty acid side-chains, so the chain reaction carotene, Iycopene and phytofluene have been ox is stopped. Evidence exists (Esterbauer et al. 1989; idised (Esterbauer et al. 1989). McCay 1985) that the tocopherol radical can mi By contrast with the intracellular environment, grate to the surface of membranes and lipoproteins enzymic antioxidant defence enzymes are much less and be converted back to a-tocopherol by reactions prominent in extracellular fluids. Blood plasma, with ascorbic acid (vitamin C) and possibly with tissue fluid, cerebrospinal fluid, synovial fluid and GSH, although it is not yet absolutely certain that seminal plasma contain little or no catalase activ this happens in vivo (Burton et al. 1990). However, ity, and only low activities of superoxide dismutase it is widely believed that both vitamin C and a- and selenium-containing glutathione peroxidase can Drug Antioxidants and Disease 577

be measured. There is also very little reduced glu dant defence of human plasma is to prevent Oi tathione (GSH) in most extracellular fluids - about and H202 from reacting to form dangerous species 2 JLmoljL in human plasma (Svardal et al. 1990). such as ·OH, by binding transition metal ions in The selenium-containing glutathione peroxidase forms that will not stimulate free radical reactions, enzyme has been purified from human plasma, and or by otherwise preventing the metal ions from shown to be different from the intracellular en participating in such reactions. Thus, safe seques zyme. The functional role of this enzyme is some tration of iron and copper ions into forms that will thing of an anomaly, since its Km (substrate con not catalyse free radical reactions is an important centration for half-maximal velocity) for GSH is antioxidant strategy in the human body (Halliwell in the millimolar range and so it would be unlikely & Gutteridge I990a,b). to function effectively at the micromolar levels of How is this sequestration achieved? The iron GSH found in plasma (reviewed in Halliwell & transport protein transferrin in plasma from healthy Gutteridge 1990b). Although much higher (~ 100 humans is only 20 to 30%loaded with iron, so that JLmoljL) concentrations of GSH are present in hu the content of free ionic iron in plasma is effec man lung lining fluid, its content of glutathione tively nil. Iron, bound to transferrins will not par peroxidase is low (Cantin et ai, 1987). ticipate in ·OH radical formation or lipid peroxi 'Extracellular' superoxide dismutase (EC-SOD) dation (Aruoma & Halliwell 1987;Gutteridge et al. enzymes have also been described. Although EC 1981). The physiological importance of this se SODs contain copper and zinc, they are very dif questration of iron ions into 'safe' forms is clearly ferent from intracellular CuZnSOD in that EC illustrated by an inspection of the multiorgan dam SODs have a much higher relative molecular mass age seen in patients with iron-overload disease (about 135000), and possess attached carbohy (McLaren et al. 1983), in whom low-molecular-mass drate. The biological role of EC-SODs is unclear; iron ion complexes capable of stimulating lipid such low activities are present in plasma and in peroxidation and ·OH generation (Gutteridge et al. other extracellular fluids that bulk-scavenging of 1985) circulate in the plasma. Oi- in extracellular fluids has always seemed (to Haemoglobin can be liberated into plasma from this author) to be an unlikely physiological func disrupted erythrocytes, and myoglobin can be re tion for them. Marklund has shown that some EC leased from muscles after injury (e.g, myocardial SODs bind to heparin and has proposed that, in infarction, or skeletal muscle damage during stren vivo, they may be associated with endothelial cell uous exercise). Both of these haem-containing pro surfaces as a protective antioxidant 'layer' over the teins are potentially dangerous in that they can ac cells (Marklund, 1990). Trace amounts of EC-SODs celerate peroxidation of lipids (Galaris et al. 1988; are found attached to cells in various tissues, so Kanner & Harel 1985), including plasma lipopro the term 'extracellular' does not really mean a lo teins (Bruckdorfer et al. 1990), in the presence of cation within extracellular fluids. H202. They do this by two mechanisms. First, re Thus, enzymic removal of Oi- and H202 con action of the protein with equimolar concentra tributes little to the antioxidant activity of extra tions of H202 produces an oxo-haem species, cellular fluids. Indeed, Oi- and/or H202 gener probably ferryl, whose formation is associated with ated extracellularly in small amounts by production of a reactive oxygen species [possibly a endothelium (Arroyo et al. 1990), lymphocytes tyrosine peroxyl radical (Davies 1990)] that can (Maly 1990),platelets (Salvemini & Botting, 1990), stimulate lipid peroxidation and damage other fibroblasts (Murrell et al. 1990) and other cells biomolecules (Davies 1990; Galaris et al. 1988; (Burdon & Rice-Evans 1989) may play important Puppo & Halliwell, 1988a). Second, excess H202 physiological roles and thus should not be scav can cause degradation of the haem rings of myog enged with 100%efficiency. Halliwell and Gutter lobin and haemoglobin, releasing from the proteins idge (l990a,b) have argued that a major antioxi- iron ions that are capable of stimulating ·OH pro- 578 Drugs 42 (4) 1991

duction and lipid peroxidation (Gutteridge 1986; thelial cells or of erythrocytes, where binding of Puppo & Halliwell 1988a,b). Haem, released from Cut" ions could lead to oxidative damage. Binding damaged haem proteins, is also well known to be to albumin might also help to stop copper ions from a powerful stimulator of lipid peroxidation (e.g. accelerating the peroxidation of low-density lipo Gutteridge & Smith 1988). proteins and promoting atherosclerosis (Esterbauer Because myoglobin and haemoglobin can be lib et al. 1989). The copper ion-albumin complex might erated into body fluids after muscle injury (e.g, in be a safe 'transit form' of copper, that can be re the 'crush' syndrome) plasma contains the haemo moved from the circulation by the liver (Halliwell globin-binding proteins known as haptoglobins, as 1988). well as a haem-binding protein (haemopexin). Albumin transports fatty acids in the blood, and Binding of haemoglobin to haptoglobins, or of haem the bile pigment bilirubin is bound to it. It has been to haemopexin, diminishes their effectiveness in claimed that bilirubin acts as an antioxidant in stimulating lipid peroxidation (Gutteridge & Smith hibitor oflipid peroxidation in vitro (Stocker 1990). 1988). The haemoglobin-haptoglobin or haem-hae Perhaps bilirubin protects albumin-bound fatty mopexin complexes are rapidly cleared from the acids against peroxidation in vivo. Indeed, Stocker circulation. (1990) has argued that induction of the enzyme The plasma copper-containing protein caerulo haem oxygenase (shown to occur in cells exposed plasmin is thought to play an essential role in iron to oxidative stress) serves not only to remove a metabolism, but it also has antioxidant properties pro-oxidant (haem) but also to increase antioxi (Gutteridge Stocks 1981). First, caeruloplasmin & dants (e.g. bilirubin). However, the prevention of has a ferroxidase activity - it oxidises Fe++to Fe+++ copper ion-mediated damage by albumin is prob whilst reducing oxygen to water. However, unlike ably a property of the protein itself. Albumin is the nonenzymatic oxidation of Fe":" ions by 02, also a powerful scavenger of HOCI in plasma (Wasil this caeruloplasmin-catalysed oxidation of Fe":" et al. 1987). does not release any damaging oxygen radicals: they Ascorbic acid, present in plasma from healthy are kept on the active site of the protein. The fer humans at concentrations of50 to 200 jlmolfL, has roxidase activity ofcaeruloplasmin allows it to in multiple antioxidant properties (listed in Halliwell hibit iron ion-dependent lipid peroxidation and 1990), probably including the ability to regenerate ·OH formation from H202 under most conditions, and this is probably the major antioxidant activity a-tocopherol by reducing o-tocopheryl radicals at of the protein (Halliwell & Gutteridge 1990b). the surface of lipoproteins and membranes. High Albumin can also bind copper ions, and it usu concentrations of ascorbic acid are also present ally inhibits copper ion-dependent lipid peroxi intracellularly, especially in brain and lung cells. dation and ·OH radical formation (reviewed in Mixtures of ascorbic acid and H202 with iron or Halliwell 1988). In fact, the reactions often con copper ions have powerful pro-oxidant properties, tinue at the sites of metal ion binding and damage however, because they can form ·OH and other re the protein, but the high concentration of albumin active species. This emphasises the need for careful in plasma and the rapid turnover of this protein sequestration of 'free' transition metal ions in the mean that such damage is probably biologically in human body in order for ascorbic acid to exert its significant. Thus, binding to albumin of any cop antioxidant action. The fact that large amounts of per ions released into plasma may lead to damage ascorbic acid can safely be given to healthy hu to the protein if Oi- and H202 are generated in mans illustrates how effective this sequestration of plasma. However, albumin may well prevent the iron and copper ions is, and means that ascorbic copper ions from attaching to more important tar acid is probably an important antioxidant in healthy gets, such as -SH groups essential to membrane humans. The administration of ascorbic acid to function, e.g. on the membrane proteins of endo- patients with iron overload can lead to serious con- Drug Antioxidants and Disease 579

sequences, however, unless desferrioxamine is given creased production of 0i- and HzOz can occur as simultaneously (Nienhuis 1981). a result of: It thus seems that a major antioxidant action of I. Raised Oz concentrations (sometimes inad blood plasma is the prevention of transition metal vertantl y; culture of most mammalian cells under ions from accelerating damaging free-radical reac 95% Oz or even under air exposes them to higher tions such as lipid peroxidation. By contrast, the Oz concentrations than are present in vivo). metal ion-binding antioxidant defences of some 2. Adding certain toxins (such as alloxan, pa other extracellular fluids may be much weaker. raquat or doxorubicin (adriamycin)] that increase Human cerebrospinal fluid (reviewed by Halliwell intracellular formation of reactive oxygen species. 1989b) contains little transferrin , albumin or ca 3. Activating a large number of phagocytes at a eruloplasmin, but has high concentrations of as localised site (Oi - and HzOzare produced by ac corbic acid (about 10 times those in plasma) and tivated phagocytes and are essential for the killing also contains uric acid, which has some antioxi of many bacterial strains, but they 'can do tissue dant properties (Ames et al. 1981). Synovial fluid damage when generated in excess). has lower concentrations of albumin, transferrin One early event frequently observed in mam and caeruloplasmin than does plasma (Gutteridge malian cells subjected to oxidative stress is DNA 1987), whereas the fluid that lines the alveoli of strand breakage, which leads to activation of the lung also has a low protein content but a very poly(ADP-ribose) synthetase, an enzyme that high concentration of ascorbic acid (reviewed by cleaves NAD+ to give ADP-ribose, which is then Heffner & Repine 1989). It has also been shown polymerised. Excess activation of poly(ADP-ri that human alveolar lining fluid, unlike other hu bose) synthetase can lead to large falls in cellular man extracellular fluids, contains significant con NAD+ concentrations, so interfering with ATP centrations of glutathione (Buhl et al. 1989). Sem synthesis (Schraufstatter et al. 1987). Oxidative inal plasma may have poor antioxidant capacity, stress can also lead to rises in intracellular 'free' since spermatozoa seem to peroxidise readily (Ait Cat" within the cell by inactivating enzyme sys ken 1989),but more studies on this fluid are needed. tems in the endoplasmic reticulum and mitochon dria that sequester free Car", and possibly also by 2. Free Radical Damage to Cells and the damaging transport systems in the plasma mem Protective Ability ofAntioxidants brane that eject Ca?" from the cell (discussed by 2.1 Mechanisms of Ceil Damage: The Albano et al. 1991 ; Orrenius et al. 1989). If free Molecular Targets intracellular Car" becomes too high, proteases can be activated that cleave the cytoskeleton. Calcium There is still a general tendency in the literature could also activate endonucleases that produce to equate 'oxidative damage' with 'lipid peroxi DNA strand breaks (Orrenius et al. 1989). dation', so that antioxidants are often characterised In addition, cells contain a small low-molecu only by their ability to inhibit lipid peroxidation. lar-mass iron pool, which supplies iron for the syn However, membrane lipids are only one possible thesis of ferroproteins. The exact intracellular lo target of oxidative damage (fig. 2). Indeed, very few cation of this pool is not clear, but it may be largely toxins that injure cells by generating excess reac compartmentalised into organelles such as mito tive oxygen species seem to cause damage by ac chondria. Its existence may explain why superox celerating the bulk peroxidation of membrane lip ide dismutase and HzOz-removing enzymes are ids (discussed by Kappus 1987 and by Halliwell & such important intracellular antioxidants; it is vital Gutteridge 1984, 1990a). to remove as much Oi- and HzOz as possible be Oxidative stress in cells and tissues results from fore they come into contact with this low molec increased generation of reactive oxygen species and/ ular mass iron pool and generate damaging species or from decreases in antioxidant defences. In- such as ·OH (Halliwell & Gutteridge 1990b). Thus, 580 Drugs 42 (4) 1991

Increased lipid peroxidation

PolyAOPribose synthetase actiVation

Metal Ion release Into surrounding tissues.Injury to adjacentcells

Fig. 2. Schematic illustrating the multiple derangements of cell metabolism that can be brought about by oxidative stress. Direct damage to DNA, proteins and/or lipids is possible. Secondary damage can arise when oxidative stress produces rises in 'free' intracellular metal ions, such as Ca++and possibly Fer" , Ca++can stimulate proteases and nucleases, damaging both DNA and the cytoskeleton. Membrane 'blebs' are 'blow-outs' of the plasma membrane characteristic of cells under stress; the rupture of large blebs may lead to cell death (Orrenius et al. 1989).

damage to proteins and to DNA can occur not only & Linn 1988; Orrenius et al. 1989; Schraufstatter as a result of activation of hydrolytic enzymes, but et al. 1987; Starke & Farber 1985). In addition, ox also by generation of·OH. The relative significance idative stress can create more metal ion promoters of these 2 mechanisms is an area of current debate of free radical reactions. Thus, Oi- can mobilise (Halliwell 1987; Halliwell & Aruoma 1991 ; Imlay a small percentage of the iron ions stored within Drug Antioxidants and Disease 581

the protein ferritin (Biemond et al. 1984; Bolann Table I. Questions to ask when evaluating the proposed role & Ulvik 1990) and may also be able to release iron of an 'antioxidant' in vivo ions from the active sites of some iron-sulphur pro 1. What biomolecule is the compound supposed to protect? teins (Flint & Emptage 1991). Similarly, HzOz in For example. an inhibitor of lipid peroxidation is unlikely to excess can 'degrade haem proteins to liberate iron be useful if the oxidative damage is mediated by an attack (Gutteridge 1986; Puppo & Halliwell I988a,b). upon proteins or DNA. 2. Is the compound present in vivo at or near that 2.2 How to Characterise an Antioxidant biomolecule at sufficient concentration? For example, many compounds have been suggested to act as 'OH scavengers in vivo. In order to compete with biological In evaluating the likelihood of a proposal that molecules for 'OH, a scavenger has to be present at at a compound already present in vivo or admini least millimolar concentrations in vivo. Most drugs never stered as a drug is acting as an antioxidant in vivo, achieve this sort of concentration. it is important to ask the right questions; as sum 3. How will the compound protect - by scavenging ROS, by preventing their formation or by repairing damage? marised in table I (Halliwell 1990). Antioxidants 4. For naturally occurring antioxidants, is antioxidant can act by scavenging reactive oxygen species (e.g. protection the primary biological role of the molecule or a SOD removing Or), by inhibiting their formation secondary one? For example. SOD has probably evolved (e.g. by blocking activation of phagocytes), by as an antioxidant enzyme. By contrast, transferrin has probably evolved as an iron transport protein, although the binding transition metal ions and preventing for binding of iron ions to transferrin stops them accelerating mation of ·OH and/or decomposition of lipid hy radical reactions, giving this protein an important droperoxides, by repairing damage (e.g. a-toco secondary role in extracellular antioxidant defence. pherol repairing peroxyl radicals and so terminating 5. If the antioxidant acts by scavenging a ROS, can the the chain reaction oflipid peroxidation) or by any antioxidant-derived radicals themselves do biological damage? combination of the above. 6. Can the antioxidant cause damage in biological systems In testing putative antioxidant activity, it is im different from those in which it exerts protection? portant to use biologically relevant reactive oxygen Abbreviation: ROS = reactive oxygen species; SOD = superoxide species. Fairly simple methods exist for measuring dismutase. the rate of reaction ofa compound with Oi-, HzOz, HOCI, ·OH, peroxyl radicals, singlet Oz and oxi dants generated when haem proteins react with HzOz. Methods also exist to measure the ability of can sometimes do damage in biological systems. compounds to suppress metal ion-dependent ·OH This may occasionally be because reactive oxygen generation, production of reactive oxygen species species have useful physiologicalfunctions and thus by phagocytes or inhibition of the peroxidation of too much antioxidant activity can be counter-pro fatty acids or of membranes (reviewed by Halliwell ductive. Hence, intravenous administration of ex 1990). In all cases, it is essential to test the com cess a-tocopherol to premature babies has been pound at the concentrations that are actually pre claimed to lead to an increase in serious infections, sent in vivo. which are often too low for direct presumably by suppression of bacterial killing by scavenging of reactive oxygen species to be feasi phagocytes (discussed by Ehrenkranz 1989). In ad ble. For example, although many drugs react with dition , many lipid-soluble chain-breaking antioxi ·OH in vitro, it is unlikely that this is often a sig dant inhibitors of lipid peroxidation can have pro nificant mechanism of antioxidant action in vivo oxidant properties under certain circumstances, simply because the drugs are usually present at often because they can bind Fe+++ ions and reduce concentrations that are too low to compete with them to Fe++ (Laughton et al. 1989). Even a-to biological targets for any ·OH generated (explained copherol can be made to exert pro-oxidant effects in table I). in vitro, although these almost certainly have no It must also be borne in mind that antioxidants physiological relevance (discussed by Halliwell 582 Drugs 42 (4) 1991

1990). Propyl gallate, an antioxidant sometimes 3. Role ofFree Radicals in used in the food industry, has limited solubility in Human Disease water, but this is enough to allow it to accelerate both 'OH formation from H202 by Fenton chem 3.1 Causation of Disease istry and DNA damage by the antibiotic bleomy cin, in both cases by its ability to reduce Fe+++ to Does increased formation of free radicals and Fer" (Aruoma et al. 1990; Gutteridge & Xaio other reactive oxygen species cause any human dis Chang 1981). Many phenolic compounds found in ease? Radiation-induced cancer may be initiated plants (especially flavonoids) have been styled as by radicals, as discussed previously. The signs pro duced by chronic dietary deficiencies of selenium 'antioxidants' because they inhibit lipid peroxida (Keshan disease) or of vitamin E (neurological dis tion - hence the appearance of 'bioflavonoids' (plus orders seen in patients with inborn errors in the or minus vitamin C) on the shelves of health-food mechanism ofintestinal fat absorption) might also stores. However, several plant phenolics can ac be mediated by reactive oxygen species (reviewed celerate oxidative damage to nonlipid biomole by Levander 1987; Howard 1990). In the prema cules such as DNA (e.g, Laughton et al. 1989).They ture infant, exposure of the incompletely vascular can do this by reducing Fe+++ ions to Fe?" and/ ised retina to elevated concentrations of oxygencan or by oxidising to produce Oi- and H202. Thus, lead to retinopathy of prematurity, which in its an antioxidant in one system is not an antioxidant most severe forms leads to blindness. Several con in all systems, and this must be borne in mind. trolled clinical trials have documented the effi A similar principle might be applicable to as ciency of a-tocopherol in minimising the severity corbic acid. Tissue injury can result in release of of the retinopathy, suggesting (but by no means intracellular metal ions and a consequent stimu proving) a role for lipid peroxidation (Howard lation offree radical reactions (section 3.2). Whereas 1990). Vitamin E supplementation may also di ascorbic acid is a good antioxidant in the absence minish the incidence of intraventricular haemor of transition metal ions, in the presence of such rhage (Chiswick et al. 1991). ions it can accelerate oxidative damage. It thus fol lows that the large doses (often > 10 g/day) of as 3.2 A Significant Consequence of corbic acid sometimes recommended for con Disease Pathology sumption might be harmless to healthy subjects, but not necessarily harmless in certain human dis For most human diseases, the excess formation eases. In sickle-celldisease (Jain & Williams 1985), of reactive oxygen species is not a cause of the dis ease, but is secondary to the primary disease pro rheumatoid arthritis (Lunec & Blake 1985), adult cess. For example, activated neutrophils produce respiratory distress syndrome (ARDS) [Cross et al. Oi-, H202 and HOCI as part of the mechanism 1990] and iron overload disease (Nienhuis 1981), of killing bacteria. However, if large numbers of ascorbic acid concentrations in body fluids are phagocytes become activated in a localised area, markedly subnormal. In rheumatoid arthritis and they can produce tissue damage. For example, the ARDS, this may be because of the rapid uptake synovial fluid in the swollen.joints of rheumatoid and oxidation of ascorbate by activated neutro patients contains many activated neutrophils and phils as well as by the reaction of ascorbate with there is reasonable evidence that reactive oxygen 0i- and HOCl. However, replacement of this as species and other products derived from neutro corbate might not necessarily be totally beneficial. phils and macrophages are contributing to the joint It might even be argued that this loss of ascorbic injury (reviewed by Halliwell et al. 1988a).In some acid is beneficial, if tissue injury is making metal forms of ARDS the lung damage is thought to be ions available (Halliwell & Gutteridge 1990b). mediated by an influx of neutrophils into the lung, Drug Antioxidants and Disease 583

where they become activated to produce prosta cells and macrophages have been shown to be ca glandins, leukotrienes, proteolytic enzymes, such pable of oxidising LOL in vitro so that macro as elastase, and reactive oxygen species (Cross et phages will internalise it faster. The modification al. 1990). Among other effects, the reactive oxygen process that allows recognition by the scavenger re species can inactivate the proteins (such as aq ceptors involves the derivatisation of lysine resi antiproteinase) within the lung that normally in dues of the apoprotein B moiety of LOL by lipid hibit the action of serine proteinases, such as elas peroxidation products, such as the cytotoxic alde tase, and prevent them from attacking lung elastic hyde 4-hydroxynonenal (Steinberg et al. 1989).Hu fibres. The precise contribution that generation of man atherosclerotic lesions accumulate the pig reactive oxygen species makes to lung damage in ment ceroid, which is widely thought to be an end AROS is unknown, but deserves more investiga product of peroxidation (Ball et al. 1987). More tion in view of the high mortality rate of AROS rigorous evidence for the presence of peroxidised (Cross et al. 1990). lipid in atherosclerotic plaques is provided by the Another example of secondary oxidative dam use of antibodies. For example, antibodies directed age is provided by the work of Akaike et al. (1990). against LOL that has undergone peroxidation or They found that superoxide dismutase protects has been treated with 4-hydroxynonenal, bind to mice against the lethality of an influenza virus in rabbit aortic atherosclerotic lesions. In addition, fection, apparently by protecting the lungs against LOL eluted from such lesions can bind to antibody excess reactive oxygen species generated by acti specific for malondialdehyde-treated LOL. Plasma vated phagocytes and by the enzyme xanthine ox samples from hypercholesterolaemic rabbits and idase. Similar oxidative stress may occur in some from several human subjects have been found to other viral infections (Peterhans et al. 1988) and contain antibodies that will react with peroxidised during malarial infections (Clark et al. 1986; Hunt LOL, suggesting that peroxidation does indeed take & Stocker 1990). place in vivo (Steinberg et al. 1989). Reactive oxygen species seem also to play a role Peroxidation ofLOL within the vesselwallcould in the formation of foam cells in atherosclerotic have a number of deleterious effects. It has been plaques (reviewed by Steinberg et al. 1989). Inter suggested that products formed in peroxidised LOL, est in the role of lipid peroxidation in atheroge such as lysophosphatidyl choline, might act as nesis was raised when it was found that the com chemotactic factors for blood monocytes, encour pound probucol has a marked antiatherogenic effect aging their recruitment into an atherosclerotic le in hyperlipidaemic rabbits (Carew et al. 1987). sion and development into macrophages (Stein Probucol, like a-tocopherol, has easily donatable berg et al. 1989). In addition, low (submicromolar) hydrogen atoms and is a powerful chain-breaking concentrations of lipid hydroperoxides might ac inhibitor of lipid peroxidation. That does not mean, celerate cyclooxygenase- and lipoxygenase-cata ofcourse, that any protective action of probucol in lysed reactions in endothelium and in any platelets vivo is due to inhibition of lipid peroxidation. present, leading to enhanced formation of prosta Macrophages possess receptors for low-density glandins and leukotrienes (Warso & Lands 1983). lipoprotein (LOL) but if LOL has become perox Oxidised LOL might also stimulate the production idised it is recognised by a separate class ofmacro of cytokines and growth factors by macrophages, phage receptors known as the scavenger receptors. which are complex multifunctional cells (Yokode There may be more than one type of scavenger re et al. 1988). There have been several speculations ceptor. LOL bound to these receptors is taken up that oxidation products of cholesterol might also with enhanced efficiency, so that cholesterol rap be involved in atherogenesis; cholesterol is oxi idly accumulates within the macrophage and may dised into a wide variety of products in peroxidis convert it into a foam cell (Mitchinson & Ball ing lipid systems (Sevanian & Peterson 1986;Smith 1987). Arterial endothelial cells, smooth muscle 1987) and oxidised cholesterol has been reported 584 Drugs 42 (4) 1991

to be toxic to many cell types, including arterial been used include thiol compounds (e.g. mercapto smooth muscle cells and endothelial cells. It seems propionylglycine), catalase, recombinant human likely that cell breakdown in the advanced athero superoxide dismutase and desferrioxamine, a che sclerotic plaque can lead not only to activation of lator that binds iron ions and usually stops them lipoxygenases, but also to release of transition metal from accelerating free radical reactions (Gutteridge ions that can stimulate free radical reactions such et aI. 1979). The use of antioxidants in the treat as lipid peroxidation (Halliwell I989c). ment of shock and in the preservation of organs The clinical consequences ofsevere atheroscler for tranplantation is also receiving attention (Bolli osis (stroke and myocardial infarction) may also 1988; Currin et aI. 1990; Fuller et aI. 1988; Sanan involve reactive oxygen species. One of the most et aI. 1989), as discussed in section 4. exciting areas of basic research in recent years has Superoxide dismutase and catalase have been extensively explored as protective agents when been that of ischaemia/reperfusion injury (Me added to reperfusates in many types of experiment. Cord, 1985), although it has yet to yield clinically In some models significant protection is observed useful developments. When tissues are deprived of whilst in others the enzymes are without effect.The 02 they are injured and after a period the injury author's general impression is that SOD is often becomes irreversible and the tissue will die. The very protective against myocardial arrhythmias and length of this critical period depends on the extent sometimes protective against stunning, whereas its of02 deprivation (whether it is total ischaemia or ability to diminish infarct size is less well estab merely hypoxia) and the tissue in question: skeletal lished (Bolli 1991;Cohen 1989; Downey et aI. 1991; muscle can be rendered bloodless for hours with Engler & Gilpin 1989). Protection by some antiox out much injury, whereas in the brain the period idants is often only observed when the antioxidant is much shorter. For the heart, up to about 60 min is added before ischaemia or at the start of reper utes seems to be a tolerable period. Thus, ischae fusion: addition after reperfusion has started (even mia injures cells and will eventually kill them, and after only 60 seconds) gives no protective effect. the aim should be to restore blood flow as soon as This phenomenon has been demonstrated with possible: hence, the use of thrombolytic agents in desferrioxamine and with mercaptopropionylgly the treatment of myocardial infarction (reviewed cine (Bolli et aI. 1989) in an open-chest dog model by Bolli 1988). of myocardial stunning after reperfusion. However, studies on isolated organs and in ani Why should ischaemia lead to increased gen mals have shown that, provided the period of is eration of reactive oxygen species when tissues are chaemia does not itselfdo irreversible damage, tis reoxygenated? The answer is not yet clear: possi sue function is better preserved upon reoxygenation bilities include release of iron ions, catalytic for free if certain antioxidants are included in the reper radical reactions, from their normal storage sites fusion medium (Bolli 1988; McCord 1985; Simp within the cell (Gower et aI. 1989a; Halliwell 1989a; son & Lucchesi 1987). Hence, restoration of the Sanan et aI. 1989),disruption of mitochondrial res oxygen supply, although obviously beneficial over piratory chains so that more electrons leak to oxy all, causes increased formation of reactive oxygen gen to form Oi- and increases in the activities of species in the damaged tissue and temporarily certain enzymes, such as xanthine oxidase, that worsens the injury. For example, depending on the generate Oi- (McCord 1985). However, there is model system studied, the heart can respond to re considerable doubt about therole of xanthine ox perfusion by showing several dysfunctions , such as idase in human myocardial injury (discussed by Kehrer 1989). arrhythmias, delayed return of contractile function ('stunning') and , it has been claimed, an increase 3.3 A General Consequence of Tissue Injury in infarct size. Thus, inclusion of antioxidants when Mechanical (e.g. crushing), chemical or isch blood flow is restored offers protective effects in aemic injury to tissues may cause cells to rupture many animal experiments: antioxidants that have and release their contents into the surrounding area Drug Antioxidants and Disease 585

(fig. 3). These contents will include transition metal ions into the surrounding area. These ions can fa ions, which are potentially capable of increasing free cilitate further damage to the surrounding areas by radical reactions in the surrounding area. For ex accelerating free radical reactions (fig. 3). This pro ample, administration of cytotoxic drugs to patients posal has been given some support from animal with acute myeloid leukaemia has been shown to studies, using agents (such as amino steroids) that create a temporary 'iron overload' state, probably interfere with iron-dependent radical reactions. One due to extensive drug-induced lysis of the leu such aminosteroid, U74006F, has been observed, kaemic cells. This increased iron availability could in animal experiments, to decrease reperfusion in contribute to the side effects of cytotoxic chemo jury, post-traumatic spinal cord degeneration and therapy (Halliwell et al. 1988b). Thus, as figure 3 neurological damage after head injury (reviewed by shows, any disease that increases cell death could Hall & Braughler 1989). conceivably result in an increased rate of free rad ical reactions. This may be completely trivial, i.e. 3.4 What Can We Expect from the increased rates of free radical reactions may Antioxidant Therapy? make no significant contribution to the disease pathology. An example of this may be the in Tissue destruction and degeneration can result creased amounts of lipid hydroperoxides measured in increased oxidative damage, by such processes in dystroph ic muscle, even though there is no evi as metal ion release, lipoxygenase activation (fig. dence that inhibitors ofIipid peroxidation or other 3), phagocyte stimulation and disruption of mito antioxidants are therapeutically useful in muscular chondrial electron transport chains (so that more dystrophy (Davison et al. 1988; Stern et al. 1982). electrons 'escape' to oxygen to form 02"-). Hence, Sometimes, however, this injury-related damage almost any disease is likely to be accompanied by may be important: perhaps the greatest interest in increased formation of reactive oxygen species, and this area lies in the sequelae of traumatic or isch it is not surprising that the list of diseases in which aemic injury to the brain. Some areas of the human their formation has been implicated is long and brain are rich in iron but cerebrospinal fluid has grows longer (Halliwell 1987). For atherosclerosis, no significant iron-binding capacity, since its con rheumatoid arthritis, some forms of ARDS, reoxy tent of transferrin is low. Thus, it has been pro genation injury, and traumatic or ischaemic dam posed (Halliwell & Gutteridge 1985) that injury to age to the central nervous system, there is reason the brain by mechanical means (trauma) or by oxy able evidence to suggset that free radical reactions gen deprivation (stroke) can result in release of iron make a significant contribution to the disease path-

Tissue Injury Cell death. Releaseof ... membrane .... 'catalytic' transition Heat ... rupture metal ions Mechanical (trauma) Infections t--- Radiation ,. Activation of Ultrasound Increased free phospholipases, ...... radical reactions Toxins cyclo-oxygenases .. in surrounding tissue Oxygendeprivation (ischaemia) and Iipoxygenases

Fig. 3. Diagram showing how cell injury can predispose to increased free radical reactions in the surrounding tissue. Cell injury can activate synthesis of prostaglandins and leukotrienes. It can also release transition metal ions that can accelerate free radical reactions. This stimulation of free radical reactions as a result of injury can sometimes result in a significant spreading of the injury. In other situations, it may be of no biological consequence. 586 Drugs 42 (4) 1991

ology. As has been stressed previously, however crease the number of neutrophils entering sites of (Halliwell & Gutteridge 1984), it is equally likely inflammation. It also diminishes their adherence that in some (perhaps most) diseases, the increased to endothelium (Suzuki et al. 1989). However, SOD formation of reactive oxygen species is an epi enzymes of equal catalytic activity do not suppress phenomenon that is of little clinical relevance and acute inflammation to the same extent in rats, and antioxidants are unlikely to be of therapeutic bene it has been suggested that SOD must bind to spe fit. This is by no means a new ' situation. Tissue cific sites to exert its anti-inflammatory activity. injury usually increases synthesis of prostaglan Hence, the surface charge distribution on the SOD dins, yet cyclooxygenase inhibitors are of little protein is an important factor (Jadot et al. 1986; benefit in many human diseases. Thus, in rheu Schalkwijk et al. 1985). Similar results have been matoid arthritis, they decrease pain and swelling obtained in studies of the ability of SOD to sup of the joints, but fail to halt the joint destruction press adjuvant-induced polyarthritis in rats (Vaille processes. . et al. 1990). These general principles can help us to interpret There is a considerable amount of older litera the results of antioxidant therapy in various hu ture claiming that injection of SOD into the knee man diseases, and to make proposals for the fu joints of patients with osteoarthritis or rheumatoid ture. arthritis produces a beneficial effect, but these data have been severely criticised (reviewed by Green 4. Antioxidants in Disease Therapy wald 1990). Even the early proponents of the use of SOD in humans now admit that SOD is not, Attempts to use antioxidants in the treatment and will probably never become, a drug of choice of human disease can be divided into 3 main areas: in the- treatment of rheumatoid arthritis (Flohe 1. Administration ofantioxidants that occur na 1988). The author has been unable to find any re turally in the human body, such as a-tocopherol, port of a well-controlled clinical trial of the effect glutathione or superoxide dismutase (SOD). of SOD in inflammatory joint disease or autoim 2. Administration of synthetic antioxidants, such mune disease in a high-quality journal, although as probuco1 or che1ating agents that suppress iron briefreports of uncontrolled studies continue to be ion-dependent free radical reactions . published. Now that recombinant human copper 3. The possibility that drugs developed to pro zinc-containing (Ambrosio et al. 1986) and man tect against-other mechanisms of tissue injury might ganese-containing SODs (Beck et al. 1988) are have additional physiological action because they available, such well-controlled studies should be have antioxidant properties; for example, the . performed to settle the question as to whether SOD angiotensin converting enzyme inhibitor captopril will be of any use as an anti-inflammatory agent. has been suggested to function additionally as an A wide variety of SOD conjugates are available, antioxidant in vivo (Bagchi et al. 1989). However, including polyethyleneglycol (PEG)-SOD, cation a detailed evaluation of these proposals has shown ised SOD, genetically engineered SOD polymers, that the antioxidant activity of captopril at the con pyran SOD arid SOD-albumin complexes (re centrations actually achieved in vivo is likely to be viewed by Greenwald 1990). These have longer cir very limited (Aruoma et al. 1991b). culating half-lives than the unconjugated SOD mo 4.1 Use of Naturally Occurring Antioxidants lecules. and Derived Compounds There has also been intense interest in the use of SOD in protection against reoxygenation injury. 4.1.1 Superoxide Dismutase The reported effects of SOD in decreasing infarct Copper-zinc containing SOD has a well-estab size after prolonged myocardial ischaemia seem in lished anti-inflammatory effect in animal models creasingly questionable (Bolli et al. 1991; Downey of acute inflammation, in part because it can de- 1991; section 3.2) and so hope is being concen- Drug Antioxidants and Disease 587

trated in 2 areas: does a combination of SOD and (EC-SOD; section 1.7) has been cloned and ex a thrombolytic agent produce increased functional pressed (Tibell et al. 1987). Since reoxygenation in activity and decreased mortality compared with a jury to tissues probably involves endothelial dam thrombolytic agent alone in cases of recent myo age (Forman et al. 1989; Halliwell 1989a; Tsao & cardial infarction , and would SOD be helpful in Lefer 1990)and some EC-SODsbind to endothelia extending the preservation time of organs for (Marklund 1990), it has been proposed by Mark transplantation? An answer to the first question lund's group that EC-SODs might be especially ef must await the results of controlled clinical trials ficient as protective agents. To the author's know currently in progress, although Petch (1990) and ledge, this proposal has not yet been tested in Editorial (1989a) have reported that reperfusion in humans by controlled clinical trial. Inoue et al. jury does not seem to be a significant problem in (1990) constructed and expressed in yeast a fusion the clinical use of thrombolytic agents. One reason gene consisting of cDNA encoding human Cu for this could be that reperfusion in most animal ZnSOD and heparin-binding peptide. The result model systems is sudden, whereas thrombolytic re ing enzyme retained activity and was able to bind perfusion in humans is slow, and slow reoxygen to and protect endothelial cells. ationmay minimise the injury (e.g. Perry & Wadhwa 1988). Although markers of oxidative re 4.1.2 Superoxide Dismutase 'Mimics' perfusion injury can be detected during postis Several low-molecular-mass compounds that chaemic reperfusion of the hearts of patients after react with 0 2'- have been described. They usually open-heart surgery (del Nido et al. 1987; Ferrari et contain transition metal ions. Examples include al. 1990), the clinical significanceof this injury may iron porphyrins (Nagano et al. 1989), a complex of be questioned (Bell et al. 1990; Hermens et al. manganese ions with the chelating agent desfer 1988). Even if human myocardial reoxygenation rioxamine (Beyer& Fridovich 1989) or copper ions injury were clinically significant, SOD need not be chelated to amino acids or to anti-inflammatory the ideal protective agent. For example, SOD failed drugs (Sorenson 1984; Weser et al. 1980). Super to protect against myocardial stunning in open-chest oxide scavengers that do not involve metal ions, dogs, although the combination of SOD and cat such as nitroxides, have also been described alase was protective (Patel et al. 1990)., Further (Mitchell et al. 1990). Apart from the various cop comments about this are made in section 5. per complexes (Sorenson 1984) little information Treatment of human kidney transplant recipi is available on the toxicity of SOD mimics (Stein ents with bovine SOD produced no significant de kuhler et al. 1990). Indeed, the potential use of these crease in rates of acute renal failure, but there was compounds in treatment of human disease is un a suggestion of benefit that merits further study certain in view of the availability of recombinant (Schneeburger et al. 1989). The reported ability of human SOD enzymes of very low toxicity. SOD in some animal experiments to offer.protec tion against endotoxic or hypovolaemic shock 4.1.3 a-Tocopherol (Carden et al. 1989; Schneider 1988), pancreatitis Prolonged deficiency of a-tocopherol, as in fat (Sanfey et al. 1984), gastrointestinal ischaemia malabsorption syndromes, produces severe neu (McCord 1985; Vohra et al. 1989)and preservation rological damage (reviewed by Bieri et al. 1983). of organs other than kidney (Fuller et al. 1988; The antioxidant action of a-tocopherol in plasma Huang et al. 1987; Weiss et al. 1989) does lnot yet lipoproteins might help to protect against athero appear to have been assessed critically in humans. sclerosis, as suggested by epidemiological studies Even in animal experiments, there is considerable (Gey et al. 1987; Gey 1990). Hence, there are good variability in results between different laboratories reasons for maintaining an adequate vitamin E in (e.g. McCord 1985). take by eating a healthy diet and for giving sup The gene encoding human extracellular SOD plementary vitamin E to patients with diseases af- 588 Drugs 42 (4) 1991

fecting fat absorption. Therapeutic administration effects' of the succinate ester itself (Carini et al. of vitamin E to premature babies has been re 1990; Fariss 1990; Fariss et al. 1989). ported to diminish the severity of retrolental fibro Several structural analogues of a-tocopherol with plasia (although there is some controversy about improved antioxidant activity (as assayed in vitro this: discussed in Ehrenkranz 1989) and the inci and/or tested in animals) have been described (In dence of both haemolytic syndrome of prematurity gold et al. 1986, 1990; Mukai et al. 1989). A water and intraventricular haemorrhage (Chiswick et al. soluble analogue of a-tocopherol, 'Trolox C', also 1991; Sinha et al. 1987). Vitamin E supplementa exists (Thomas & Bielski 1987)but caution should tion has also been recommended as beneficial in be employed in using it upon humans because it patients suffering from haemolytic syndromes has been shown to be capable of accelerating oxi caused by an inborn lack of glutathione synthetase dative damage to substrates other than lipids in or glucose-6-phosphate dehydrogenase in erythro vitro (Aruoma et al. 1990). In general, the author cytes (e.g. Corash et al. 1980). However, it has been feels that antioxidant inhibitors of lipid peroxida reported that high doses of intravenous vitamin E tion are not likely to be of general use in treating can produce an increase in the infection rate in pre human disease, because lipid peroxidation is often mature babies, probably by depressing phagocyte a late stage in the oxidative damage mechanism function (Engle et al. 1988). Depression of phago (section 2.1). However, they may have great bene cyte function can also be demonstrated in cells iso fit as protective agents in some cases, e.g. in pro lated from adults taking oral vitamin E but this tecting against the development of atherosclerosis, effect does not seem to be clinically significant provided that they can enter and remain in LDL (Bendich & Machlin 1988). (Esterbauer et al. 1989). The results of a clinical However, attempts to use vitamin E to treat hu trial (Deprenyl and Tocopherol Antioxidative man diseases in which oxidative stress is thought Therapy of Parkinsonism, or DATATOP) in which to be important, such as anthracycline-induced the combination of vitamin E with an inhibitor of cardiac damage (reviewed by Powis 1989), have monoamine oxidase B (deprenyl) is being evalu been largely disappointing. One reason for this ated in the treatment of Parkinson's disease are could be that it takes a considerable time to raise awaited with interest (Parkinson Study Group the vitamin E content of many tissues, especially 1989). So far, however, the beneficial effect of de that of brain, whereas oxidative damage can often prenyl has been much more obvious than that of be very fast (e.g. within minutes in the reoxygen a-tocopherol. ation injury experiments described by Bolli et al. 1989). In addition, oxidative damage frequently 4.1.4 Ascorbic Acid (Vitamin C) occurs by mechanisms other than lipid peroxida Vitamin C is essential in the human diet as a tion (Powis 1989; section 2.1). cofactor for several hydroxylase enzymes (re Pharmacological doses of vitamin E have been viewed by Levine 1986)and deficiency ofit should claimed to be beneficial in thrombotic vascular be avoided, especially as deficiency might also pre disease (reviewed by Bieri et al. 1983), but even if dispose to atherosclerosis, as is suggested by .epi true this might be due to effects other than an demiological studies (Gey et al. 1987).It is possible tioxidant action, such as partial inhibition of li that smokers need a higher vitamin C intake than poxygenase (Reddanna et al. 1985) or a weak nonsmokers, since smoking appears to lead to in suppression of platelet aggregation (Jandak et al. creased vitamin C consumption (e.g. Chow et al. 1989). It is also interesting to note that the reported 1986)and vitamin C is thought to be an important protective effects of o-tocopheryl succinate against antioxidant in the lung (Bendich et al. 1986; Cross oxidative damage in isolatedhepatocytes could not et al. 1990). It may also protect against the carcin be explained simply by its hydrolysis to free to ogenicity of nitrosamines (Shamberger 1984; Tan copherol and may have involved 'cytoprotective nenbaum et al. 1991). In the absence of metal ions, Drug Antioxidants and Disease 589

ascorbate is a powerful antioxidant in vitro (Frei et protective against ischaemic injury (Engler 1987). al. 1989), but in their presence, it can be dangerous, Compounds acting in a comparable way to aden as discussed in section 1.7. osine might be very useful as anti-inflammatory Administration of very high doses (up to 20g agents and in protection against reoxygenation in daily in some cases) of vitamin C to healthy hu jury (e.g. Kaminski & Proctor 1990), to the extent mans appears to do no significant harm (Bendich that this phenomenon is clinically important (sec et al. 1986) but, to the author's knowledge, no tion 4.1.1). clinical benefit has yet been demonstrated in well controlled clinical trials in any disease: indeed, high 4.1.6 Lactoferrin dose vitamin C appeared to lead to lethal hae Lactoferrin is an iron ion-binding protein, sim morrhagic tumour necrosis in 4 patients with ad ilar to transferrin but with a higher affinity for iron vanced cancer (Cameron & Campbell 1974). Ces ions (especially at acidic pH values). It is present sation of megadose therapy may lead to rebound in human secretions (milk, saliva, tear fluid, nasal low levels of vitamin C in body fluids (e.g. Tsao secretions) and released by activated neutrophils. & Salimi 1984). Iron ions bound to lactoferrin are unable to cata A number oflipophilic ascorbate esters (e.g. as lyse free radical reactions (Aruoma & Halliwell corbyl palmitate) have also been described (e.g. 1987; Gutteridge et al. 1981). Indeed, it has been Baader et al. 1988), but reports of properly con argued that the release of lactoferrin by activated trolled trials of their use in the treatment of human neutrophils is a protective mechanism that helps disease have not yet appeared. to diminish iron-dependent free radical reactions in the surrounding area (Halliwell et al. 1981). For 4.1.5 Adenosine example, Ward et al. (1983) found that lactoferrin Under certain circumstances, neutrophils can was able to protect rats against lung injury me potentiate injury to tissues (section 3.2). For ex diated by reactive oxygen species. Lactoferrin has ample, antibodies preventing neutrophil adherence many other effects on phagocyte function, however decreased infarct size in a dog model of cardiac (Birgens 1984). Several groups are cloning and ex reoxygenation injury after ischaemia (Werns & pressing the gene for human lactoferrin (e.g. Stow Lucchesi 1990). The first step in neutrophil accu ell et al. 1991) and it will be interesting to see if mulation in several tissues involves adherence to the human protein has any therapeutic uses. the endothelium, a process that is regulated by many substances produced by endothelial cells. For 4.1.7 Nicotinamide example, prostacyclin decreases adherence, whereas Subjecting cells to oxidative stress frequently Oi- increases it (Suzuki et al. 1989). leads to depletion of intracellular nicotinamide nu The nucleoside adenosine (adenine-ribose) af cleotides by activation of poly(ADP-ribose) syn fects the function of both neutrophils (e.g. it de thetase (section 2.1). Several authors have reported creases Oi- production) and endothelium and it protection against cell killing under these circum has been shown to have protective effects in some stances by inhibitors of poly(ADP-ribose) synthe animal models of reoxygenation injury (Forman et tase, such as aminobenzamide, or by supplemen al. 1989; Grisham et al. 1989; Pitarys et al. 1991). tation of the cell culture medium with nicotinamide Indeed, it has been argued that adenosine is a nat or nicotinic acid (niacin) [e.g. Schraufstatter et al. ural anti-inflammatory agent during transient in 1987; Weitberg 1989]. Weitberg (1989) found that jury, helping to prevent premature activation of the the prolonged ingestion of supplementary nicotinic inflammatory response (Cronstein et al. 1990; En acid by human volunteers led to an increase in the gler 1987). For example, 5-amino-4-imidazole car NAD+ concentrations in their circulating lympho boxamide-riboside accelerates adenosine release cytes, and that lymphocytes isolated from these from ischaemic cells, and has been reported to be volunteers were more resistant to oxidative dam- 590 Drugs 42 (4) 1991

age. Wang et al. (1990) found that pretreatment of GSH itself is not rapidly taken up by cells, but hamsters with nicotinamide decreased the lung fi several approaches can be used to overcome this. brosis observed after subsequent exposure to the Methyl and ethyl esters of glutathione have been antitumour antibiotic bleomycin. described, which can apparently cross cell mem Whether these various observations will tum out branes and be hydrolysed to GSH within the cell to have therapeutic relevance to humans is un (Anderson & Meister 1989a). However, there have clear. A potential problem with the use of ADP been some reports of toxicity of GSH esters, pos ribose synthetase inhibitors is that blocking the ac sibly due to contaminating compounds arising dur tion of this enzyme may decrease DNA repair, so ing their preparation (Thomalley 1991). The com increasing the risk ofcarcinogenesis: this effect has pound L-2-oxathiazolidine-4-carboxylate has been been demonstrated in mice whose pancreatic ,B-cells reported to be hydrolysed to cysteine in vivo,which were damaged by treatment with streptozotocin, may lead to an increased synthesis of GSH (Meis leading to diabetes. The animals could be protected ter 1988; Porta et al. 1991). N-Acetylcysteine has against the diabetogenic action of streptozotocin been used as an antioxidant in a wide variety of by administration of ADP-ribose synthetase inhib experiments (Moldeus et al. 1986) and is very ef itors, but a significant number of animals then de fective in the treatment of paracetamol (acetami veloped insulinomas (Okamoto 1986). The results nophen) overdosage in humans (Smilkstein et al. with nicotinamide again illustrate the point that a 1988). Paracetamol is metabolised by the hepatic healthy diet may be a good protection against tis cytochrome P450 system to a reactive quinone sue injury due to oxidative damage. imine that can combine with GSH, so causing sev ere GSH depletion in the liver. N-Acetylcysteine 4.1 .8 Glutathione and its Precursors may protect by entering cells and being hydrolysed Glutathione has multiple functions in human to cysteine, which stimulates GSH synthesis . Ad metabolism, including the detoxification of xeno ditionally, N-acetylcysteine has also been widely biotics. It is also important in antioxidant defence, used in humans for treatment of various respira e.g. by helping to protect DNA against damage by tory disorders; it has very limited toxicity but its ionising radiation and by acting as a substrate for therapeutic benefits in these conditions have been glutathione peroxidases and transferases (Held questioned. 1988; Ross 1988). There have therefore been many Mixtures of thiols with transition metal ions can suggestions that reduced glutathione (GSH) may be cytotoxic because ofreactions that produce both be therapeutically useful as an antioxidant and as oxygen radicals (02'- and 'OH) and sulphur-con a general cytoprotective agent, e.g. in the preser taining radicals such as RS·, RSO· and RSOz· vation of organs for transplantation and in pro (Rowley & Halliwell 1982; Sevilla et al. 1989). GSH tecting against tissue damage by cytotoxic drugs is unstable in solution, particularly if iron or cop such as cyclophosphamide (Currin et al. 1990; per ions are present, and the oxidation products Friedman et al. 1990; Menasche et al. 1986). Use can cause damage. This must be borne in mind if of aerosolised GSH solutions has been suggested solutions containing GSH or other thiols are to be as a means ofdiminishing lung damage by reactive stored for any length of time, e.g. in cell culture oxygen species generated extracellularly from ac media or in fluids for organ preservation (e.g. tivated phagocytes in such diseases as cystic fibro Boudjema et al. 1990; Currin et al. 1990). Cysteine sis (Buhl et al. 1990). There is also evidence for seems to be more toxic than GSH in such systems abnormalities ofGSH metabolism in HIV-infected (Saez et al. 1982), perhaps because it oxidises faster cells and the use of GSH or its precursors in and because the cysteine-So radical can abstract hy asymptomatic HIV-positive and AIDS patients has drogen atoms from fatty acids and initiate lipid been suggested (reviewed by Halliwell & Cross peroxidation (Schoneich et al. 1989). Thus, cys 1991). teine accelerates iron ion-dependent peroxidation Drug Antioxidants and Disease 591

oflipids, whereas GSH and N-acetylcysteine do not sitising drugs (Mathews-Roth 1987).They may also (Haenen et al. 1989). Some thiols can also mobilise have an anticancer effect (Bendich & Olson 1989; iron ions from ferritin (e.g. Bonomi et al. 1989). Stahelin et al. 1991). It thus seems important to Indeed, cysteine is much more toxic when admin maintain an adequate dietary intake of vegetables. istered to animals that are N-acetylcysteine or GSH Carotenoids can also act as inhibitors oflipid per (Anderson & Meister 1989b). Similarly, high levels oxidation under certain circumstances, but their ofplasma homocysteine in homocystinuric patients actions are complex (discussed by Krinsky 1989). predispose to atherosclerosis (Ueland & Refsum Whether they will ever have a major therapeutic 1989). use remains unclear as yet. Sulphur-containing radicals can also be formed when thiols are oxidised by ·OH and possibly by 4.2 Synthetic Antioxidants other reactive oxygen species: 4.2.1 Mercaptopropionylglycine and RSH + ·OH --+ RS· + H20 (Eq. 20) Other Thiols Many thiols have been tested for their ability to RS· o~ RSO·, RS02· (Eq. 21) protect cells and small animals against ionizing ra As has been stressed previously (Halliwell 1990), diation. They include GSH and its precursors (cys when proposing the use of scavengers to remove teine, cysteamine), dimercaprol (British Anti-Lew reactive oxygen species, it is necessary to ask what isite) and mercaptopropionylglycine (von Sonntag happens to the radicals derived from the scaven 1987). In general, the author's impression is that ger. cysteamine (e.g. Isseis et al. 1987) and cysteine N-Acetylcysteine has been reported to have pro (section 4.1.8) are more toxic than the others, per tective effects in many animal models of lung in haps because they oxidise faster if transition metal jury. By contrast, it potentiated the lung damage ions are present and/or produce sulphur-contain produced by the anticancer drug bleomycin in ing radicals that are more damaging to biological hamsters (Giri et al. 1988). Bleomycin-dependent molecules. lung damage is thought to involve formation ofan The thiol compound mercaptopropionylglycine iron ion-bleomycin complex, which produces (MPG) has been shown to be protective against re strand breakage in DNA in the presence of certain perfusion injury after ischaemia in animal models reducing agents (reviewed by Halliwell & Gutter of myocardial stunning and infarction (discussed idge 1989). In vitro, N-acetylcysteine did not po by Bolli et al. 1989; Puppo et al. 1990). It may act tentiate iron ion-dependent damage to DNA by by its ability to scavenge reactive oxygen species, bleomycin, whereas cysteine did (Evans & Halli but which species it acts upon is as yet uncertain well, unpublished data) and so it is possible that (Puppo et .al. 1990). Several clinical studies have the N-acetylcysteine acted as a precursor of intra claimed that MPG is beneficial in the treatment of cellular cysteine, which could reduce the iron-bleo various diseases (listed in Bolli et al. 1989)but well mycin complex upon the nuclear DNA. Thus, in controlledtrials of its effects against reperfusion in discriminate use ofthiol compounds as therapeutic jury do not appear to have been published. agents cannot be recommended. 4.2.2 Ebselen: A Glutathione Peroxidase 4.1.9 Carotenoids Mimic? Carotenoid pigments, such as {3-carotene, are Ebselen (PZ51; 2-phenyl-I ,2-benzisoselenazo powerful quenchers of singlet 02 and have found lin-3(2H)-one) is an organoselenium compound that therapeutic use in protecting porphyria patients catalyses removal of peroxides by reaction with against photosensitisation damage to the skin, and GSH, and was developed as a glutathione peroxi in protecting animals against damage by photosen- dase 'mimic'. It has been shown to have anti-in- 592 Drugs 42 (4) 1991

flammatory effects in a number of small mammals sulphur, did not inhibit the enzyme any better in (Parnham et al. 1991). However, it is uncertain vivo than did oxypurinol (Spector et al. 1989), al whether these effects can be attributed to removal though it was a more powerful inhibitor in vitro. of H202, since ebselen also inhibits cyclooxygen Since allopurinol is widely used to treat hyper ase, lipoxygenase, the production of reactive oxy uricaemia in humans (Chalmers et al. 1968), its gen species by activated neutrophils and lipid per toxicological profile is well known and it might oxidation in membranes (Cotgreave et al. 1989; therefore seem to be the agent of choice in testing Hayaishi & Slater 1986;Leurs et al. 1989;Parnham for beneficial effects in diseases to which ischae et al. 1991). Ebselen does not appear to have been mia/reperfusion is thought to contribute: it has al effectively tested in controlled clinical trials for ways seemed to the author that preventing the for beneficial effects in humans to date, although its mation of excess reactive oxygen species is more metabolism in the human body has been described likely to be effective than attempting to scavenge (Fischer et al. 1988). One potentially worrying ob them once they have been formed. Unfortunately servation is that ebselen also inhibits elongation the evidence that xanthine oxidase makes any sig and desaturation of long-chain fatty acids in rat nificant contribution to the generation of reactive liver microsomes (Laguna et al. 1989), although the oxygen species in human heart and brain is very toxicity of the drug to animals has been reported weak (section 3.2; Wajner & Harkness 1989;Werns as very low (Parnham et al. 1991). & Lucchesi 1990). In addition, many studies in which protection has been shown have required 4.2.3 Allopurinol, Oxypurinol and Other pretreatment of animals with the drug, which is Xanthine Oxidase Inhibitors not usually possible in clinical cases of human is Allopurinol inhibits the production of Oi-, chaemia/reperfusion. However, xanthine oxidase H202 and uric acid by the enzyme xanthine oxi activity has been detected in human gut, in the pal dase. It inhibits by first acting as a substrate for mar fascia of patients with Dupuytren's contrac the enzyme, so producing the true inhibitor, oxy ture (Murrell et al. 1987), in the circulation of purinol (Spector 1988). Oxypurinol is also the ma patients after reperfusion of a tourniqued limb jor metabolite formed when allopurinol is ad (Friedl et al. 1990) and in the synovia of patients minstered to humans (Reiter et al. 1983). When it with rheumatoid arthritis (Merry et al. 1989). The was proposed that xanthine oxidase is the major therapeutic possibilities of treatment with allopu source of the reactive oxygen species generated rinol in these diseases have been discussed, but not when ischaemic tissues are reoxygenated (McCord yet evaluated clinically to date. Allopurinol and 1985), interest naturally arose in the use of allo oxypurinol could also be beneficial in enhanced purinol or oxypurinol as protectors against tissue preservation of organs for transplantation (Cham damage. Indeed, some animal experiments found bers et al. 1987). Salim (1990) has recently reported that allopurinol has protective effects against car that allopurinol was as effective as cimetidine in diac, cerebral or gastrointestinal reoxygenation in preventing the recurrence of duodenal ulcer in hu jury (Itoh et al. 1986; McCord 1985; Parks 1989) mans. as well as in some animal models of haemorrhagic As well as inhibiting xanthine oxidase, oxypu shock (Allan et al. 1986). It might be thought more rinol can also scavenge HOCI (Grootveld et al. logicalto use oxypurinol as a protective agent, since 1987), which might contribute to its protective ef it is the true inhibitor ofxanthine oxidase (Spector fects under certain circumstances. In vitro, both al 1988). However, allopurinol was reported to be lopurinol and oxypurinol can react with 'OH, but more effective than oxypurinol in reducing urinary the concentrations achieved in vivo are probably uric acid excretion in humans (Chalmers et al. too low for this to be a significant mechanism of 1968).Similarly B103U, an analogue of oxypurinol protection (discussed by Halliwell 1990). An ad in which the oxygen at position 6 is replaced by ditional mechanism by which allopurinol or oxy- Drug Antioxidants and Disease 593

purinol could protect tissues is by preventing ox chelator of iron ions that suppresses iron-depend idation of hypoxanthine, so enhancing its salvage ent free radical reactions. Based on this observa for reincorporation into adenine nucleotides when tion, the potential therapeutic use of inositol 1,2,6 the tissue is reoxygenated (Lasley et aI. 1988). triphosphate in human disease is being evaluated Other inhibitors of xanthine oxidase that have (Claxson et aI. 1990). been described in the literature include lodoxam The only reasonably specific iron ion chelator ide (White 1981), pterin-6-aldehyde (Nishino & approved for routine clinical use in humans is de Tsushima 1986) and amflutizole (Wems & Luc feroxamine (sometimes called desferrioxamine: ceshi 1990). commercial preparations of the drug are usually deferoxamine B methanesulphonate). Deferoxam 4.2.4 Inhibitors ofthe Generation of Reactive ine, which is produced by Streptomyces pilosus, is Oxygen Species by Phagocytes widely and effectively used for the prevention and Generation of reactive oxygen species by acti treatment of iron overload in patients who have vated phagocytes may contribute to inflammatory ingested toxic oral doses of iron salts, or who re and reoxygenation injury (section 3.2). Thus, it is quire multiple blood transfusions, e.g. for the treat possible that inhibitors of phagocyte recruitment, ment of thalassaemia (Modell et aI. 1982). Defer adherence to endothelium, or activation of the res oxamine also binds Al+++ ions, although with a piratory burst oxidase complex could be therapeu stability constant 6 orders of magnitude less than tically useful, as has already been discussed in the for its binding of Fe"?" ions, and has been used to case of adenosine (section 4.1.5). Several anti-in decrease body aluminium loads in renal dialysis flammatory drugs have been suggested to suppress patients (McCarthy et aI. 1990). It has recently been Oi- formation by phagocytes, but convincing evi claimed that deferoxamine has beneficial effects in dence for this mechanism of action in vivo has been the treatment of patients with Alzheimer's disease obtained only in a few cases (discussed by Halli (McLachlan et aI. 1991). well et aI. 1988a). Cross (1990) has reviewed the Deferoxamine is a powerful chelator of Fe"?". compounds that have been shown to inhibit phag In the presence ofphysiological buffer systems, de ocyte Oi- generation in vitro: none of them ap feroxamine inhibits iron ion-dependent lipid per pears suitable for therapeutic use as yet. Hart et aI. oxidation and the generation of highly-reactive ox (1990) reported that apocynin, a phenolic com idising species such as 'OH from Oi- and H202 pound of plant origin, is oxidised to a toxic prod in the presence of iron ions (reviewed by Halliwell uct by phagocytes containing peroxidase (such as I989d). Large doses of deferoxamine can be in neutrophils, which possess myeloperoxidase) and jected into animals or humans: at least 50 mg/kg thus exerted an anti-inflammatory effect in rats. bodyweight per day appears fairly safe in thalas Only activated phagocytes (producing the H202 saemic patients (Davis et aI. 1983) provided that necessary for peroxidase action) would be affected. they do not become iron-deficient (Porter et aI. 1989a). Hence, deferoxamine has been proposed 4.2.5 Chelating Agents: Deferoxamine for use as a 'probe' to study the importance of iron Chelating agents have been successfully used in dependent free radical reactions as mediators of the treatment of metal ion poisoning for many tissue injury (Gutteridge et aI. 1979). Indeed, de years, e.g. dimercaprol to treat arsenic poisoning, feroxamine has been used in this way in many ani penicillamine and trientine to treat Wilson's dis mal model systems (reviewed by Halliwell 1989d). ease and diethylenetriaminepentaacetic acid to re It is generally observed to be protective, probably move lead or plutonium (Jones 1991;Jones & Pratt by its iron-chelating actions in most cases, al 1976). Grafand Eaton (1990) have pointed out that though it has also been shown to interfere with the phytic acid (inositol hexaphosphate) , an insoluble uptake of paraquat by isolated lung cells (van der constituent of the human diet, is also a powerful Wal et aI. (990), which partially explains its pro- 594 Drugs 42 (4) 1991

tective effect against cytotoxicity of this herbicide. Because iron is essential for normal cell func Deferoxamine can also react directly with several tion, prolonged administration of any powerful reactive oxygen species, including R02", Oi-, "OH, iron-chelating agent to humans (other than patients HOC1, oxidants produced by mixing haem pro suffering from iron overload) will probably cause teins with H202, and peroxynitrite formed by re serious side effects. Hence, proposals to use long action of NO with Oi-. Most of these reactions term deferoxamine administration in the treat are of little or no significance in vivo, simply be ment of chronic disease .in humans, such as Alz cause the concentration of deferoxamine achieved heimer's disease or rheumatoid arthritis, must be during normal therapeutic use is too low for scav approached with caution , as must prolonged treat enging of reactive oxygen species to be a feasible ment of aluminium-overloaded patients with this mechanism of action (Halliwell 1989d). However, drug (Blake et a1. 1985; McCarthy et a1. · 1990). the ability of deferoxamine to scavenge peroxyni However, the side effects of chronic deferoxamine trite has been suggested to account for its ability administration should not preclude its acute use, to protect against reoxygenation injury in some e.g. in combination with thrombolytic agents in the myocardial systems (Beckman et a1. 1990), a pro treatment of myocardial reperfusion injury, in hae posal which merits further investigation. It has also morrhagic shock (Sanan et a1. 1989) or in amelio been shown (at high concentrations) to block the rating cell damage by toxins such as the anthra formation of xanthine oxidase activity in cultured cycline antitumour antibiotics (Halliwell et a1. bovine pulmonary endothelial cells ( Rinaldo & 1988b). Deferoxamine might be especially useful in Gorry 1990). Deferoxamine is very poor at re myocardial reperfusion systems (Menasche et a1. moving iron ions bound to transferrin or lactofer 1990). For example, it can sometimes protect rin, which may be advantageous since iron bound against myocardial stunning much more effectively to these proteins does not stimulate free radical re than does SOD (Bolli et a1. 1990). Thus, if clinical actions (Aruoma & Halliwell 1987; Gutteridge et trials (section 4.1.1) show that recombinant human a1. 1981). SOD has a beneficial effect when combined with Deferoxamine, at therapeutically relevant con thrombolytic agents in the treatment of recent centrations of 20 to 100 ,umoljL, can inhibit cell myocardial infarction, then the possibility of using proliferation in vitro, and in vivo (Estrov et al. deferoxamine as a much cheaper alternative is well 1988). It may do so by depriving the cells of iron worth exploring. Deferoxamine might also have and inhibiting the enzyme ribonucleoside diphos applications in the preservation of organs for trans phate reductase, but there is considerable debate plantation (Gower et al. 1989b; Yoon et al. 1989). as to whether or not this is the only cytostatic It is interesting to note that, despite the putative mechanism. Thus, when deferoxamine is admini iron ion dependency of the lung damage induced stered by continuous infusion over several days, as by the anticancer drug bleomycin in animals (sec in some studies of its effect upon chronic inflam tion 4.1.8) neither deferoxamine nor other iron mation or autoimmune disease in animals, it is chelators so far tested appear to offer any protec possible (but remains to be proved) that inhibition tive effects (Tryka 1989; Ward et a1. 1988). of the proliferation of inflammatory cells such as Hallaway et a1. (1989) have described high-mo lymphocytes could contribute to the protective ef lecular-mass derivatives of deferoxamine, made by fects observed. The cytostatic action of deferox attaching it to polymers such as dextran or hy amine or other iron ion- chelating agents might droxyethyl starch. Conjugation to these polymers conceivably be of therapeutic use in suppressing was reported not to diminish Fe+++-bindingactiv Iympocyte-mediated reactions (Bierer & Nathan ity, but to markedly raise the LDso and the cir 1990; Bradley et al. 1986), e.g. graft rejection or the culating plasma lifetime in animals. Whether these replication of HIV (Baruchel et al. 1991 ; Tabor et high-molecular-mass forms of deferoxamine will be a1. 1991). therapeutically useful remains to be seen. DrugAntioxidants and Disease 595

4.2.6 Other Iron Chelators: Hydroxypyridones tion of these compounds to humans, especially A major problem with deferoxamine is that it those not suffering from iron overload, without is not absorbed through the gut and has to be ad close medical supervision (Hershko 1988; Nathan ministered to thalassaemic patients by intravenous & Piomelli 1990). or subcutaneous infusion, leading to poor patient One aspect ofthe use of iron-chelating drugs that compliance. The drug is also expensive. There has deserves further attention is their antimalarial ac therefore been a search for orally active chelating tion . Deferoxamine (Hershko & Peto 1988; Pollack agents that are, hopefully, cheaper (Jones 1982). et al. 1987) inhibits the growth of malarial para Some work has been reported with pyridoxal iso sites in culture and in infected animals. Hydrox nicotinoyl hydrazone (discussed by Hershko 1988), ypyridones are also effective against parasites in deferrithiocin (Biener & Nathan 1990; Wolfe 1990) vitro (Heppner et al. 1988; Hershko et al. 1991). and 2,3-dihydroxybenzoate (Grady et al. 1978), but Similar suggestions have been made for use of che most attention is currently concentrated on the 3 lators against trypanosomal infections (Shapiro et hydroxypyrid-4-ones. For example, 1,2-dimethyl-3 al. 1982). Clark et al. (1986) have proposed that hydroxypyridin-4-one (LI) has undergone prelim reactive oxygen species are involved in the path inary tests in humans. It appears to be more toxic ology of malarial infections, and various antioxi than deferoxamine (Editorial I989b), and other hy dants have been found to be beneficial in malaria droxypyridones (such as the 1,2-diethyl com infected animals (e.g. Thumwood et al. 1989). By pound) are now undergoing evaluation (Porter et contrast, several antiparasitic drugs have been al. 1989b,c, 1990; Dobbin & Hider 1990; Hershko claimed to exert their action by increased genera et al. 1991). tion of reactive oxygen species in vivo (reviewed The increased toxicity of hydroxypyridones, by Docampo 1990). when compared with deferoxamine, might be due to features of their chemistry (Halliwell & Groot 4.2.7 Other Iron Chelators: ICRF-187 and veld 1987). Three moles of chelator are needed to the 'Lazaroids' complex I Fe+++ ion completely, whereas defer Another compound whose action in vivo may oxamine chelates Fe+++ at a I : I molar ratio: the relate to iron ion chelation is ICRF-187, which was deferoxamine molecule 'wraps around' the iron ion, originally synthesised as an anti-tumour agent and encasing it in an envelope of organic material. Upon extensively tested in animals and humans, but with dilution, 3 : I hydroxypyridone: iron ion com disappointing results. ICRF-187 is hydrolysed in plexes tend to dissociate into 2: I and I : I com vivo to produce a metal ion chelator structurally plexes, leaving available coordination sites on the similar to edetic acid (EDTA). ICRF-187 has been iron that could allow the complexes to catalyse free reported to ameliorate the cardiotoxicity of an radical reactions. Indeed, Kontoghiorghes et al. thracyclines in human breast cancer patients, while (1986) found that 3 : I chelator-iron complexes did not affecting the therapeutic efficiency of these not stimulate iron-dependent free radical reactions, drugs (Speyer et al. 1988). It has therefore been whereas 2 : I or I : I complexes seemed able to do suggested (Halliwell et al. 1988b; Halliwell & Bom so. In addition, hydroxypyridones can mobilise iron ford 1989) that careful use of low concentrations ions from transferrin (Kontoghiorghes 1986a) or of chelating agents may help to minimise anthra lactoferrin (Kontoghiorghes I986b). Thus, one can cycline-dependent cardiotoxicity in other cancers, envisage a scenario in which iron ions safely pro particularly the leukaemias, in which chemother tein-bound (i.e. unable to stimulate free radical re apy can cause transient iron overload (section 3.3). actions) are mobilised and transfemed to other sites ICRF-187 is the (+)-isomer of the racemic mixture in the body at which damage can be caused (Hal first described as ICRF-159 [I ,2-bis-(3,5-dioxopi liwell & Grootveld 1987). Caution therefore should perazine-I-yl)propane]. ICRF-187 is preferred for be employed in proposing long term administra- use because of its increased solubility in water. It 596 Drugs 42 (4) 1991

has also been reported to protect against the dia phenolic antioxidants has been described as 'not betogenic action of alloxan in mice, which is impressive' (Swingle et al. 1987). thought to be due to increased free radical for Many flavonoids and other phenols ofplant ori mation in the pancreatic islets (El-Hage et al. 1981). gin have been reported to have anti-inflammatory Since iron-dependent free radical reactions ap effects in some animal model systems (Lewis 1989; pear to make significant contributions to injury to Villar et al. 1984) and to inhibit lipid peroxidation the CNS after stroke or trauma (section 3.3), sev in vitro (reviewed by Larson 1988), but similar cau eral antioxidants (e.g. U74006F) have been devel tions apply (e.g. Donatus et al. 1990; Hodnick et oped that can cross the blood-brain barrier (Hall al. 1986;Laughton et al. 1989).Flavonoids are often & Braughler 1989). These compounds are 21-ami powerful inhibitors of lipoxygenase and cyclooxy nosteroids ('lazaroids') without glucocorticoid ac genase (e.g. Moroney et al. 1988) and they exert a tivity and they are powerful inhibitors ofiron ion wide range of other biochemical effects, so that it dependent lipid peroxidation in vitro. although is usually difficult to attribute any biological ac tions that they have to antioxidant activity. Several whether they act in vivo as iron ion chelators and/ plant phenolics can be oxidised by myeloperoxi or as chain-breaking antioxidants is unclear as yet. dase to give toxic products (Simons et al. 1990), They have been reported to diminish brain or spinal which may explain some of their reported effects cord injury resulting from stroke or trauma in ani in suppressing Oi- generation by activated neu mal experiments (Hall & Braughler 1989; Jacobsen trophils (Hart et al. 1990). Some phenols can also et al. 1990; Steinke et al. 1989). Results of clinical exert pro-oxidant effects in vitro (Laughton et al. trials in humans are awaited with interest. 1989). Interest in the role of lipid peroxidation in 4.2.8 Probucol and Other Chain-Breaking atherosclerosis (section 3.2) was raised when it was Antioxidants found that the phenolic compound probucol (4,4' Several phenolic chain-breaking antioxidants [isopropylidenedithio]bis [2,6-di-tert-butylphenol]) have been proposed for use in humans. They in has a marked antiatherogenic effect in hyperlipid clude MK-447 (2-aminomethyl-4-tert-butyl-6-io aemic rabbits (Carew et al. 1987). Probucol was dophenol), ONO-3144 (2-aminomethyl-4-tert-bu introduced as an agent that lowers blood choles tyl-6-propionylphenol) and R-830 (2,6-di-tert-butyl terol concentrations, but it is also a powerful chain 4-[2'-thenoyl]phenol). All have shown anti-inflam breaking inhibitor of lipid peroxidation (Parthas matory effects in animal models of acute inflam arathy et al. 1986) that does not appear to have mation (Swingle etal. 1987). R-830 is reported to pro-oxidant activity to other biomolecules (Aru be in early clinical trials as a topical preparation oma et al. 1990). The potential therapeutic use of for skin inflammation, but little information on probucol to diminish the development of athero human use of the other compounds has appeared. sclerotic lesions in coronary arteries after bypass It must be remembered that chain-breaking an surgery is currently being evaluated. There is no tioxidants can often show pro-oxidant properties direct proof as yet that any antiatherogenic effect towards nonlipid biomolecules (section 2.2), that of probucol is actually related to an inhibition of any anti-inflammatory action they have could be lipid peroxidation and not to other effects on mon related to interference with the synthesis of pros ocytes and macrophages within atherosclerotic le taglandins and/or leukotrienes (e.g. by inhibiting sions. lipoxygenase and/or cyclooxygenase) rather than 5. Antioxidant Properties ofDrugs by inhibiting lipid peroxidation, and that lipid per Developed for Other Purposes oxidation is often not a major mechanism of cell injury by oxidative stress (section 2.1). These rea Many drugs already used in humans have been sons may account for the observation that the anti discovered to be capable of acting as antioxidants inflammatory activity of commercially available in vitro in some assay systems. They include non- Drug Antioxidants and Disease 597

steroidal anti-inflammatory drugs (discussed by clinical use may well be doing several different Halliwell et al. 1988a), the angiotensin converting things. enzyme inhibitor captopril (Bagchi et al. 1989), the Drugs with multiple mechanisms of protective sulphasalazine metabolite 5-aminosalicylate (Aru action, including antioxidant properties, may be one oma et al. 1987) and several compounds acting as way forward to minimising tissue injury in human Car" antagonists (e.g. Janero et al. 1988). How disease. Certainly, the potential therapeutic effects ever, very few drugs are present in vivo at concen of N-acetylcysteine in HIV-I infection (Halliwell & trations that would allow them to compete with Cross 1991),probucol in atherosclerosis, ebselen in biological molecules for reaction with 'OH or HOCI chronic inflammation and iron chelators in Alz (table I): possible exceptions include salicylate, heimer's disease, leukaemia, organ preservation and penicillamine and gold thiols as used in the treat malaria merit urgent evaluation. Probably less at ment of rheumatoid arthritis (reviewed by Halli tention should be paid to SOD and vitamin E, well et al. 1988a) and sulfasalazine or aminosali which have been therapeutically disappointing (ex cylates as used in the treatment of inflammatory cept for vitamin E in ameliorating the conse bowel disease (Ahnfelt-Ronne et al. 1990). For drugs quences of prematurity and fat malabsorption syn that can act as radical scavengers in vivo, it is im dromes). portant to ensure that toxic drug-derived radicals are not produced during the reaction. For example, Acknowledgements sulphur-containing radicals derived from penicil lamine are capable of inactivating clq-antiprotein Theauthor thanks Dr JMC Gutteridge for his helpful comments. Research funding from the British Heart ase, the major inhibitor of serine proteases in hu Foundation and the Arthritis and Rheumatism Council man extracellular fluids (Aruoma et al. 1989c). (UK) is gratefully acknowledged.

6. Conclusion References

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9th International Symposium on Preparative and Industrial Chromatography - 'PREP 92' Date: 6-8 April 1992 Venue: Nancy, France

For further information, please contact: Congres ·PREp·92' Societe Francaise de Chimie 1. rue Grandville - B.P. 451 F-54001 Nancy Cedex FRANCE