Characterization of drugs as antioxidant prophylactics

Free Radical Biology & Medicine, Vol. 20, No. 5, pp. 675-705, 1996 Copyright 0 1996Elsevier Science Inc. Printed in the USA. All rights reserved 0891.5849/96$15.00 + .OO ELSEVIER

-+

0891~5849(95)02110-8

Review Article CHARACTERIZATION

OF DRUGS

AS ANTIOXIDANT

PROPHYLACTICS

OKEZIE I. ARUOMA Pharmacology Group, University of London King’s College, London, UK (Received 24 May 1995; Revised 28 August 1995; Accepted 31 August 1995)

Abstract-There is growing interest in the evaluation of drugs (prescription only medicines and over-the-counter medicines) as antioxidant prophylactics. Although free radical mechanism in human degenerative diseases is now generally recognised, the mechanisms of tissue injury in humans are very complex and it may not be possible to clearly identify the role played by free radicals in the process. This review examines the current evidence to support the notion that drugs for a particular therapeutic category might possess useful antioxidant capacity hence rninimising tissue injury due to free radicals. Keywords-Free

radicals, Oxidative stress, Human diseases, Antioxidants,

Drug design

independent existence) possessing one or more unpaired electrons, an unpaired electron being one that is alone in an orbital. Electrons are more stable when paired together in orbitals: the two electrons in a pair have different directions of spin. Hence, radicals are generally less stable than nonradicals, although their reactivities vary (see Table 1) , Once radicals form they can either react with another radical or with another molecule by various interactions.3 The rate and selectivity of reactions of this type occurring depends, on high concentrations of the radicals, delocalization of the single electron of the radical (thus increasing its life time), and on the absence of weak bonds in any other molecules present with which the radical could interact (further comments may be found in refs. 17, 162, 206, 231, 394396). Gerschman et a1.7 proposed “that oxygen poisoning and radiation injury have at least one common basis of action, possibly the formation of oxidizing free radicals.” This pioneering idea soon began to capture the imaginations of scientists. In the early 196Os, superoxide was found to be associated with a number bf enzymes, including xanthine oxidase (the reader is referred to the history of SOD told by McCord and Fridovich384). However, it was believed that this free radical was “bound” to the enzyme. In 1968, Joe McCord and Irwin Fridovich385 discovered that superoxide was secreted into solution, allowing superoxide

INTRODUCTION

Fundamental interest in free radicals began with the work of Moses Gomberg, ’who, in 1900, demonstrated the existence of the triphenylmethyl radical (PhsC’) . A free radical is any chemical species (capable of Okezie I. Aruoma received his B.Sc. in Biochemistry from the University of Sussex, UK, in 1981. Following an M.Sc. course in Biopharmacy at the University of London Chelsea College in 1984, he joined the research group of Professor Barry Halliwell, University of London King’s College in 1985. He was awarded a Ph.D. in Biochemistry in 1988. After a successful year at the U.S. Department of Commerce National Institute of Standards and Technology (Gaithersburg, MD) working as a guest scientist with Professor Miral Dizdaroglu, he returned to King’s College to continue his research work in free radical biochemistry. Okezie was awarded the higher doctorate degree, Doctor of Science (D.Sc.) by the University of London in 1994, in recognition of his eminence in the field of free radical reactions in biological systems. In the same year he was elected Fellow of the Royal Society of Chemistry (C.Chem. FRSC). Okezie has published over 75 scientific papers in major international ioumals and has edited three books: Free Radicals and Food Addikves (Halliwell as coeditor) published by Taylor & Francis, London, in 1991; DNA & Free Radicals (Halliwell as coeditor) published by Ellis Horwood, London, in 1993, and Free Radicals in Tropical Diseases, published by Harwood Academic Publishers. London. in 1993. Okezie holds the degree of Master of Business Administration (MBA) awarded in 1995 by the Warwick Business School, University of Warwick, UK. His project, entitled the Competitiveness of the PharmaceuticaZ Industry, inspired this review article. Dr. Aruoma has a particular interest in the role of free radicals and antioxidants in foods and in human health. Address correspondence to: 0. I. Aruoma, Pharmacology Group, University of London King’s College, Mam-esa Road, London SW3 6LX, UK. 675

0. I. ARUOMA

676

Table 1. Chemical Reactions of Free Radicals* Unimolecular Radical Reactions Reactions result from the instability of the first formed radical. The radicals may completely decompose or rearrange before reaction with other molecules or radicals present. Decomposition: reaction in which the radical decomposes to give a stable molecule and a new radical Rearrangement 1. breaking of an adjacent C-C bond in a cyclic system with concomitant formation of a new bond, usually carbonyl and a new isomeric radical 2. migration of an atom, via intramolecular abstraction by the radical center, thus creating a new, isomeric radical. Radical-Molecule Interactions Addition to unsaturated systems: 1. Addition of a radical to an olefinic double bond to give a new saturated, radical. Typical reaction is the radical induced polymerization of olefins. 2. Addition of a radical to an aromatic double bond. This intermediate step is widespread in free radical chemistry, e.g., in the radical substitution of aromatic compounds (homolytic aromatic substitution). The net overall reaction is displacement of an aromatic substituent by a radical: AR-X+Y’+AR-Y+X Abstraction or displacement: SH2 reactions+ - biomolecular reaction involving homolytic attack of a radical on a molecule. The radical attacks a univalent atom, usually a terminal halogen or hydrogen in an abstraction reaction to give rise to a new radical, e.g. Ph’ + CBrC& --t ‘CCL, + PhBr -

Homolytic substitution at multivalent atoms also occurs but both do not normally occur at saturated carbon centers. Reaction with oxidizing agents Radicals readily undergo l-electron oxidations with oxidizing reagents of suitable redox potential to give positive ions. Example is the Meerwein reaction, which involves the oxidation of cinnamyl derived radicals by cupric ions: PhCHCHRC02Et + Cu*+ -+ PhC+HCHRC02Et + Cu+ Radical-Radical Interactions Dimerization or radical coupling Localized radicals (methyl, phenyl radicals) react readily with little chance of dimerization. Only delocalized radicals have a high probability of dimerization in solution. Thus, R” + R”’--t R’ - R” When R’ = R”, the reaction is dimerization and when R’ f R” the reaction is radical coupling or combination. Radical disproportionation Involves collision of the radicals resulting in the abstraction of an atom, usually hydrogen, by one radical from the other. This leads to me formation of two stable molecules, with the atom abstracted being p to the radical center? e.g., the disproportionation of two phenylethyl radicals to give styrene and ethylbenzene * The reader is referred to Cadogat? for an extensive overview of the reaction sequences highlighted in this table and an examination of the complex chemistry of organic radicals. A recent paperback by Perkh@ on Radical Chemistry is worth perusing. + Sn2 stands for substitution homolytic biomolecular. * The disproportionation reaction derives its driving force from the formation of two new strong bonds and from the fact that the P--CH bonds in radicals are usually weak.

mediate cellular toxicity. With the discovery that an enzyme, SOD8 reacts with superoxide at diffusion controlled rates, thereby controlling cellular levels of this radical, the field of free radicals in biology and medicine was ushered in (for reviews and extensive discussions, the following references 8- 19, 384-391, are illustrative). The role of free radical reactions in human disease, biology, toxicology, and the deterioration of food has become an area of intense interest. One of the classic demonstrations of the role of free radicals in humans

to

elucidation of the mechanism of carbon tetrachloride (CC&) toxicity. Ingestion of this compound produces severe hepatic damage but it has been well established that the damage is not done by Ccl, itself, but a free radical produced by its metabolism in the liver.20*21 Reactive oxygen species (ROS) (Table 2) are constantly formed in the human body. Many of them serve useful physiological functions, but they can be toxic when generated in excess or in inappropriate environments, and this toxicity is often aggravated by the pres-

is the

Drugs as antioxidant prophylactics

ence of ions of such transition metals as iron or copper. Excess generation of reactive oxygen species within tissues can damage DNA, lipids, proteins, and carbohydrates. Which of these is the most important target of damage depends upon the cell type subjected to the oxidative stress and upon how it is imposed. Free radical mechanisms have been implicated in numerous clinical conditions.‘0~‘2V14~392*393~404*4~ Illustrative examples include ischemia-reperfusion injury ( stroke/myocardial infarction and organ transplantation) , cancer, aging, alcoholism, red blood cell defects (favism, malaria, sickle cell anemia, Fanconi’s anemia, protoporphyrin photo-oxidation), iron overload (nutritional deficiencies, Kwashiorkor, thalassemia, dietary iron overload, idiopathic hemochromatosis) , kidney (metal ion-mediated nephrotoxicity, aminoglycoside nephrotoxicity, autoimmune nephrotic syndromes ) , gastrointestinal tract (e.g., oral iron poisoning, endotoxin liver injury, free fatty acid-induced pancreatitis, nonsteroidal antiinflammatory drug induced gastrointestinal tract lesions, diabetogenic actions of alloxan) , inflammatory-immune injury [e.g., rheumatoid artluitis, glomerulonephritis, autoimmune diseases, vasculitis (hepatitis B virus)], brain (e.g., Parkinson’s disease, neurotoxins, allergic encephalomyelitis, potentiation of traumatic injury, hypertensive cerebrovascular injury, vitamin E deficiency), heart and cardiovascular system [e.g., atherosclerosis, adriamycin cardiotoxicity, Keshan disease (selenium deficiency), alcohol cardiomyopathy ] , eye (e.g., photic retinopathy, ocular hemorrhage, cataractogenesis, degenerative retinal damage), amyotrophic lateral sclerosis, and age-related macular degeneration. HUMAN ANTIOXIDANT DEFENSES

The toxicity of 02’- and Hz02 in living organisms is due to their conversion into OH’ and into reactive radical metal complexes. These processes are often referred to as either the iron catalyzed Haber-Weiss reaction22 (Eq. 1) or the superoxide-driven Fenton reaction 13V23~24 (Eq. 2). The reader is also referred to the “Centennial of Fenton Reaction” review by Koppeno1.25 Haber-Weiss: FelCu

Hz02 + 02’-

+

02 + OH’ + OH-

(1)

catalyst

Fenton: Fe2+ + Hz02 + intermediate + Fe3+ + OH’ + OH-

(2)

611

Some 6 years before Gomberg’ s work, Fentonz3 reported that ferrous iron promoted the oxidation of tartaric acid by H202. This led to the proposal of Eq. 2. About 40 years later, Haber and Weiss suggested that the OH’ formed in Eq. 2 actually arose by the reaction (Eq. 1) . However, from a thermodynamic standpoint, the second order rate constant of the reaction (Eq. 1) is so low that it could not occur at the low steady-state concentrations of 02’- and H202 that exist in vivo. Thus, the OH’ formation can be explained, because the Haber-Weiss reaction is catalyzed by traces of transition metals. On production in vivo, OH’ reacts at its site of formation. OH’ has an estimated half-life in cells of only lop9 s. Thus, in the case of OH’ generation by Fenton-type chemistry, the extent of OH’ formation is largely determined by the availability and location of the metal ion catalyst. Iron salts have been widely studied as catalysts of free radical reactions in vivo, but copper ions may also be important if one extrapolates in vitro results. For example, in an in vitro study in which the technique of gas chromatography with selected ion monitoring (GCNS/SIM) was used to compare the role of copper and iron ions in promoting damage to DNA by H202, ‘78X2112 added copper ions were significantly more reactive in causing DNA damage in the presence of H202 than were equimolar iron ions. This catalytic ability suggests that the availability of “free” iron and copper in vivo should be controlled. This is, indeed, the case. Iron ions are absorbed from the gut and transported to iron requiring cells by the protein transfer-i-in. Iron specifically bound to transferrin does not participate in free radical reactions.400.40’ Excess iron is stored as ferritin and hemosiderin in an attempt to keep the iron pool as small as possible. Most of or all of the plasma copper in humans is attached to the protein ceruloplasmin, which will not stimulate free radical reactions4” under normal conditions. Evidence in the literature suggests that OH’ generation can take place when the hemostasis is altered. Chevion et al.4o2have shown that copper and iron ions released into perfusates can cause ischemia/reperfusion injury. Ramos et al.,4o3 using the ESR spin trapping system detected OH’ as the cw-hydroxyethyl spin trapped adduct of 4-pyridyl l-oxide N-tert-butylnitrone formed from phorbol 12-myristate 13-acetate-stimulated human neutrophils and monocytes without the addition of supplemental iron. In systems that lacked myeloperoxidase, it as necessary to add iron to detect the OH’ adduct. This observation demonstrates that human neutrophils and monocytes can generate OH’ through a myeloperoxidase-dependent mechanism, 299 which could have microbicidal implications. Tissue injury can itself cause ROS generation (e.g.,

678

0. I. ARUOMA Table 2. Examples of Reaction Oxygen Species (ROS)

The Radicals Superoxide

02-

Hydroxyl

OH

Peroxyl, alkoxyl

ROT’, RO

Oxides of nitrogen

NO’, NO?’

The Nonradicals Hydrogen peroxide

Hypochlorous acid

HOC1

Ozone

03

Singlet oxygen

‘02

Oxygen-centered radical with selective reactivity. This species is produced by a number of enzyme systems, by autooxidation reactions and by nonenzymatic electron transfers that univalently reduce molecular oxygen. In aqueous solution, OZ.- can oxidize ascorbic acid. It can also reduce certain iron complexes such as cytochrome c and ferric-ethylenediamine-tetraacetic acid (Fe3+ EDTA). SOD accelerates the dismutation of OZ.-, converting it to hydrogen peroxide (H202) and oxygen (0,). A highly reactive oxygen-centered radical that attacks all molecules in the human body including tissues such as may occur in the myocardium. Typically, organic radicals often encountered as intermediates during the breakdown of peroxides of lipids in the free radical reaction of peroxidation. CCljOI’ has been used extensively to study potential antioxidant action of biomolecules. Nitric oxide is formed in vivo from the amino acid L-arginine. Nitrogen dioxide is formed when NO reacts with Oz and is found in polluted air and smoke such as from cigarettes. Formed in vivo when 02’- dismutates and also by many oxidase enzymes. H202 at low levels also appears poorly reactive. However, higher levels of H202 can attack several cellular energyproducing systems; for example, it inactivates the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. HzOz also forms OH’ in the presence of transition metal ions and OZ.- can facilitate this reaction A powerful oxidant often present in household bleaches but formed in the human neutrophils at sites of inflammation by action of the enzyme myeloperoxidase. May also react with 02’- to generate OH’ in neutrophils. Formed in the environment habited by humans. This noxious gas has been shown to deplete plasma antioxidants vitamin D, vitamin E, and uric acid. Here the spin of one of the electrons of the two outer orbitals is inverted, removing the quantum mechanical spin restrictions of molecular oxygen. Of the types of singlet oxygens, the delta singlet oxygen (‘ga02) is more biologically important because of its longer lifespan compared with the sigma singlet oxygen ‘Zg+.

by causing activation of phagocytes or releasing transition metal ions from damaged cells), which may (or may not, depending on the situation) contribute to a worsening of the injury. For example, many areas of the human brain, such as the substantia nigra, are particularly rich in iron. When the brain is injured by trauma or by ischemia (as happens in stroke), cell death tends to occur and those iron ions could be released. The iron ions thus released may then stimulate free radical reactions in the surrounding area and cause a spreading of damage. The sequelae of traumatic brain injury and stroke may involve a postinjury stimulation of iron ion-dependent free radical reactions. Parkinson’s disease is caused by death of cells in the substantia nigra. Lysis of dead cells could cause iron ion release. Indeed, evidence exists that patients with Parkinson’s disease are under oxidative stress and that free radical reactions are contributing to the degeneration of the substantia nigra (for reviews and further discussion, perusal of refs. 398, 399, and 405-412 may be worthwhile). The consequences of uncontrolled iron availability can also be seen in “iron-overload” diseases, such as idiopathic hemochromatosis (IH). In this disease, there is a progressive increase in total body iron stores with deposition of iron in the parenchymal cells of the liver, pancreas, heart, and endocrine organs. Plasma samples from iron-overloaded patients with IH contain

nontransferrin bound iron in a form that can catalyse free radical reactions that are suspected to be of importance in the pathology of iron overload.4’3V414,424 Iron overload causes severe tissue damage in which the liver, heart, joints, and pancreatic p-cells are especially affected. Thus, the nature of the damage done by excess formation of H202 and 02’- will be affected by the location and concentration of metal ion catalysts of reaction with the cells. It also follows that if no catalytic metal ions are available 02’- and H202 will have limited, if any, damaging effects. These fundamental principles underlie the importance of examining the availability and distribution of “catalytic“ metal ions in explaining oxidative damage to cells, and the abilities of antioxidants to minimise such damage. It is also worth pointing out that all organisms suffer some exposure to OH’ because it is generated in vivo by the homolytic fission of oxygen-hydrogen bonds in water driven by continuous exposure to background ionizing radiation.4’5 Halpern et a1.,4’6using spin-trapping/low-frequency EPR method demonstrated in situ detection of OH’ produced from ionizing radiation in the tumor of a living mouse. The interrelationship between ROS and antioxidants in humans is very complex indeed. The protective mechanisms do not act independently of one another, but rather they tend to function cooperatively in the form of a cascade (see Fig. 1) . Much has been written

Drugs as

679

antioxidantprophylactics

about antioxidants and disease prevention 26-453392 and their role in food preservation.37346-65 In humans, antioxidant defenses to remove 02’and H202 exist. Superoxide dismutases ( SODS)*,~‘~ remove O,‘- by greatly accelerating its conversion to H202. Human cells have a SOD enzyme-containing manganese at its active site (MnSOD) in the mitochondria. An SOD with copper and zinc at the active site (Cu,Zn-SOD) is also present, but largely in the cytosol.~~‘OCatalases in the peroxisomes convert H202 into water and O2 and help to dispose of H202 generated by the action of oxidase enzymes located in these organelles. However, the most important H202-removing enzymes in human cells are glutathione peroxidases (GSHPX) ,422enzymes that require selenium (as selenocysteine at the active site) for their action. GSHPX enzymes remove H202 by using it to oxidize

reduced glutathione (GSH) to oxidized glutathione (GSSG) . Glutathione reductase, an FAD-containing enzyme, regenerates GSH from GSSG, with NADPH as a source of reducing power (Fig. 1) . The scavenging enzymes and antioxidants can inhibit free radical production by chelating the transition metal catalysts, breaking chain reactions, reducing concentrations of ROS, and by scavenging initiating radicals. Cytoprotective enzymes are located within both the hydrophilic and hydrophobic compartments of the cell, while antioxidants are both intra- and extracellular. The tissue damage alluded to arises as a consequence of oxidative stress.16 Oxidative stress may be mediated by increased activity of the radical generating enzymes (e.g., xanthine oxidase) and/or their substrates (e.g., hypoxanthine), activation of phagocytes, activation of phospholipases, cyclooxygenases,

Tissue damage

Hypochlorous acid NADP

02*- + 02”

SOD

+ 2H+

L

‘4202 \

Glulrtbione pcroxidasc

,$ !.,

w “20

/J OH’ -

Polyunsaturated _ fatty acids

RO.

Lipid bydropcroxidcs

RO,’ -

Dcbydrckcorbic acid t

Ascorbate /

Fig. 1. Some interrelationships betweenfree radicals,reactiveoxygen species,and antioxidants.P-Caroteneand other plantderived antioxidants may also react with the radical intermediates, protecting tissues depicts a rationale for evaluating the role of antioxidants and drugs in biological implicated. From the standpoint of antioxidant supplementation to minimize tissue index of such injury and then develop universal methods for measuring free radical

against lipid peroxidation. The figure also systems where oxidative stress has been injury, it is necessary to first validate the generation in humans.

680

0. I. ARUOMA

and lipoxygenases, dilution and destruction of antioxidants, release of “free” metal ions from sequestered sites and/or muscle, release of heme proteins (hemoglobin, myoglobin) , and disruption of electron transport chains and increased electron leakage for o*.-. 9,10,16,19,66 Natural sources of vitamins and antioxidants are normally fruits and vegetables. It is important to understand that antioxidant vitamins as naturally present in food are in balanced biochemical equilibrium. In addition to antioxidant defense enzymes, living organisms contain a variety of radical-scavenging antioxidants, including GSH, uric acid, a-tocopherol, and ascorbic acid. cY-Tocopherol delays lipid peroxidation by reacting with chain-propagating peroxyl radicals faster than these radical can react with proteins or fatty acid side chains.26,66-7’ Burkart et a1.430 have shown that a-tocopherol also constitutes part of eukaryotic cells’ defense against NO toxicity. Muller397 have reviewed the role of vitamin E in neurological function. It is widely believed, but not yet rigorously proven, that ascorbic acid (and possibly GSH) can reduce this radical back to a-tocopherol, as suggested in Fig. 1. Ubiquinol (reduced coenzyme Q) might also regenerate a-tocopherol in membranes and lipoproteins.72X383.4s6 Ascorbate (vitamin C) is often claimed to be an important antioxidant in vivo. Its ability to show antioxidant properties is related to the fact that the semidehydroascorbate radical is much less reactive than are many of the radicals that can be scavenged by ascorbate. 73,74,4’7Enzymic systems exist in vivo to reduce this radical back to ascorbate using NADH (the NADH-semidehydroascorbate reductase enzyme) or GSH (the dehydroascorbate reductase enzyme) as sources of reducing power. However, these enzymes seem to be largely intracellular, and so ascorbic acid is rapidly depleted in human extracellular fluids under conditions of oxidative stress.26,44@‘X70 Free radical generation occurs normally in the human body, and rates of free radical generation are probably increased in most diseases. Their importance as a mechanism of tissue injury is still uncertain. However, the development of new assays applicable to humans should allow rapid evaluation of the role of free radicals in disease pathology and provide a logical basis for the therapeutic use of antioxidants.3”37’75-78X4’6 (see Figs. 2 and 4). It is worthwhile commenting on the therapeutic categories relevant to this review before turning to the issue of pro-oxidant and antioxidant characterization. I will take a nonscientific look at some of the therapeutic categories primarily to establish the view point of this review. However, informa-

Lntioxidants that occur

Administration of synthetic

laturally a-tocopherol,

antioxidants such as probucol,

glutathione, SOD,

lipoic acid, dihydrolipoic

flavonoids and

acid, or chelating agent,

polyphenols.

xanthine oxidase inhibitors, Zl-aminosteroids

""MAN CONDITION IN WHICH A ROLE HAS BEEN IMPLICATED

>

I

"se of drugs developed to protect against other mechanisms of tissue injury and human diseases, probably because they have antioxidant properties e.g. tamoxifen, captopril, and other ACE inhibitors, carvedilal and other P-blockers, Ca2+ antagonists, Nacetylcysteine, anti-inflammatory drugs, sulphasalazine and metabolites.

Fig. 2. Human antioxidant strategies. Although the drugs in question have been designed for a specific purpose, a number of important questions must be addressed when formulating an antioxidant cocktail for therapeutic use. An inhibitor of lipid peroxidation is unlikely to be useful if it mediates oxidative attack on proteins or DNA. So, what biomolecule is the compound supposed to protect? Will the compound be present in vivo at or near the biomolecule in sufficient concentration? How does the compound protect: by scavenging radicals, by preventing their formation, or by repairing damage? If the antioxidant acts by scavenging radicals, can the resulting antioxidant-derived radicals do biological damage? Can the antioxidant cause damage in biological systems that differ from those in which it exerts protection. Several inhibitors of lipid peroxidation have the potential to accelerate free radical damage to other biomolecules.

tion on some of the drugs may be found in established texts. 418-421

PHARMACEUTICAL

THERAPEUTIC

CATEGORIES

The key therapeutic categories are cardiovascular, antiinfective, gastrointestinal, central nervous system, respiratory, musculoskeletal, and joint disorders (see Fig. 3)) cancer, and immunology. Typical drugs in each of the categories are identified in Table 3. The eight therapeutic categories mentioned account for about 45% of the total pharmaceutical market. The pharmaceutical business environment is undergoing radical change, partly due to government health care

Drugs as

reforms that are occurring in the countries to the pharmaceutical industry.79X80

681

antioxidant prophylactics

important

calcium antagonists (Table cardiovascular drugs.

3 ) are the two top selling

Antiinfective

Cardiovascular Cardiovascular disease is a heterogeneous group of disorders that affects the heart and blood vessels. The diseases are characterized by angina, hypertension, congestive heart failure, acute myocardial infarction (heart attacks), stroke, and arrhythmia. Strokes are the third most common form of death in the western economy and are the most common contributor to neurological disability. The major risk factor of stroke is age, heart disease, obesity, diabetes, hypertension, and smoking. The majority of strokes are caused from a blockage in the blood flow in the brain due to blood clot fragments from a diseased heart. The largest segment is for antihypertensive drugs. ACE inhibitors and

The four basic classifications of drugs to treat infectious diseases are antibacterials, antivirals, antifungals, and antiprotozoals. Antibacterial drugs are used to treat infections resulting from bacterial organisms. Although infections can be easy to treat, complications arise because of the development of multiple resistance to antibiotics by many bacterial strains. There are few players in the antibacterial market, the entry barrier being created by the lack of expertise in traditional fermenting techniques required for the manufacture of antibiotics such as penicillin, tetracyclines, cephalosporins, and erythromycin. Nevertheless, synthetic quinolines (quinoline Ciprobay ) are becoming a major drug in the antibiotic market. The antiviral market is relatively new and is dominated by the drugs Retrovir for the treatment of AIDS and Zovirax for the treatment of herpes/shingles virus. Treatment of viral infections are often complicated by the fact that a virus is able to undergo mutations, making it difficult to eradicate. The classic example is the flu virus where the vaccine must be changed each season due to the changing virus. Another more sinister factor is that the virus is often bound up and genetically integrated with the cells, making it difficult to target drugs that will kill the virus without injuring or killing the infected cell.

Central nervous a

system (CNS)

The CNS drugs market consists of drugs for a wide range of disorders. The major disorders are anxiety (the largest segment), depression, insomnia, dementia (senile dementia such as Alzheimer’s), epilepsy, psychosis, and Parkinson’s disease. There is now the drug dependency segment, for example the smoking cessation treatments. Prozac is a selective serotonin reuptake inhibitor that has less potential for abuse in comparison with older generation antidepressants. The smoking cessation products were the CNS success stories of the 90s.

Cancer b

Fig. 3. Typical

hand deformities of rheumatoid arthritis showing ulnar deviations of the fingers and swelling of the metacarpophalangeal and proximal interphalangeal joints. (A) Early changes; (B) advanced disease state with gross destruction and ulnar drift. (Courtesy of Professor David Blake of the Royal London Hospital, UK.)

The prevalence of cancer has continued to grow in the world. This disease is responsible for one in four deaths in the developed world. Cancer is a heterogeneous disease with a variety of causes (aging, smoking, oxidative stress, etc. . . ), but the longer the exposure to a carcinogen, the greater the likelihood of the dis-

682

0. I. ARUOMA Table 3. Typical Drugs in the Pharmaceutical Therapeutic Categories

Central NervousSystem Drugs

Respiratory Drugs

Gastrointestinal Drugs

Antiinfective Drugs

Brand

Generic

Brand

Generic

Brand

Generic

Brand

Generic

Prozac XaIlaX Nicotine11 ‘ITS Ativan Tegretol

fluoxetine aIprazolam nicotine patch lorazepam carbamazepine

Ventolin range Seldane Becotide Proventil Intal

zantac Tagamet Losec Pepcid Gaster

ranitidine cimetidine omeprazole famotidine famotidine

Zovirax Augmentin Ceclor Rocephin Ciprobay

acyclovir co-amoxiclav ceflacor ceftriaxone ciprofloxacin

Parlodel AVaIl Dilantin Nicoderm

bromocriptine idebenone phenytoin nicotine patch

Pulmicort Zaditen Mucosolvan Atrovent

Prilosec Axid Prepulsid Selbex

omeprazole nizatidine cisaptide teprenone

Primaxin Diflucan Fortum Zinnat

imipenem/cilastin fluconazole ceftazidime cefuroxime axetil

BuSpar

buspirone

Hismanal

salbutamol terfanadine beclomethasone alputerol sodium cromoglycate budesonide ketotifen ambroxol ipatropium bromide astemizole

Maalox

Amoxil

amoxycillin

Sinemet Pamelor

co-careldopa nortriptyline

Beconase Seldane D

beclometbasone terfanidine

Carafate Buscopan

Retrovir Claforan

zidovudine cefotaxime

sertraline

Bricanyl

terbutaline

s0stri1

Mg and Al hydroxides sucralfate hyoscine butylbromide ranitidine

Valium

diazepam

Triludan

terfanadine

Neuer

Rivotril Leponex/ c102ari1

clonazepam

Claritin

loratadine

clozapine

Berodual

Hydergin

codergrocine

Nimotop Anafranil Sermion

nimodipine clomipramine nicergoline

all penicillins Erythromycin

erytbromycin

Cytotec

ceetraxate hycrochloride misoprostol

Tarivid

ofloxacin

Gastrosepin

perenzipine

Cleocin

lincomycin

Bemtec

iprat/fenot. comb. fenoterol

Imodium

Asmacort Intal Azeptin

triamcinoline cromoglycate azelastine

Dulco-Lax

loperamide hydrochloride bisacodyl

Duricef Nizoral Minocin

ampicillin/ sublactam cefadroxil ketoconazole minocylline

Musculoskeletal and joint disorder drugs

Cardiovascular Drugs

Immunology Drugs

Brand

Generic

Brand

Generic

Brand

Vasotec Capoten Mevacor Procardia family Cardizem family Tenormin Mevalotin Adalat Lopid

enalapril captopril lovastatin nifedipine

diclofenac naproxen piroxicam etodolac nabumetone methylprednisolone ketoprofen loxoprofen flurbiprofen

Sandimmun Intron A Sumifereon Roferon-A

atenolol pravastatin nifedipine gemfibrozil

Voltaren Naprosyn Feldene Lodine Relifex Medrol Orudis Loxonin Ansaid

Zestril

lisnopril

Zyloric

allopurinol

Zocor Calan/Calan SR

simvastatin verapamil

Tilcotil Brufen

tenoxicam ibuprofen

Orthoclone 0KT3 Mochida Interferon-p

Trental Herbesser Pravachol Nitroderm TTS Lopressor

pentoxyfyline diltiazem pravastatin nitroglycerin metoprolol

Surgam Orudis Dolobid Indocin Inteban

tiapofenic acid ketoprofen diflunisal indomethacin indomethacin

Perdipine Prinivil Lasix

nicardipine lisnopril frusemide

Salazopyrin Froben Tolectin

azulfidine flurbiprofen tolmetin sodium

Timunox Ceredase Krestin Feron

cyclosporin interferon-alpha interferon-alpha interferon-alpha azathioprine thymopentin glucocerebrocidase krestin interferon-b (natural) muroMAbCD3

Cancer Drugs Brand

Generic

Neupogen Nolvadex Zoferan Lupron VePesid Paraplatin Provera Zoladex Eulexin

G-CSF tamoxifen ondansetron leuprorelin etoposide carboplatin medroxy-progesterone goserilin flutamide

Pharmorubicin

epirubicin

interferon beta

Gran L-eucovorin

Wellferon Canferon A Picibanil Frone Leucomax

interferon-alpha interferon-alpha picihanil &interferon GM-CSF

Platinol Adriamycin Sandostatin 5-Fu Androcur

Imunace Proleukin Biogamma

Interleukin-2 Interleukin-2 interferon-gamma

Novantrone Suprecur Ifex

G-CSF leucovorin calcium Cisplatin doxorubicin octreotide fluorouracil cyproterone acetate mitozantrone buserelin ifosphamide

This table is for illustrative purpose alone and does not include drugs that are about to be launched in the market. Brand and generic names are identified. For more information, see ref. 79.

ease. The treatment of cancer is also heterogeneous, with a large number of different types of drugs each having specific application to a specific form of the diseases. Many of the drugs are cytotoxic, and affect healthy and malignant cells. Drugs that would mitigate

the worst effects (e.g., vomiting and white blood cell death) of cancer chemotherapy have been developed. Adjunct therapy drugs such as Zofran (antiemetic) and Neupogen (used to increase white blood cell count during chemotherapy) are earmarked to stimulate re-

Drugs as antioxidant prophylactics

naissance in chemotherapy, as patients could then tolerate larger and more effective forms of chemotherapy.

Immunology Human diseases such as rheumatoid arthritis (see Fig. 3)) psoriasis, and type II diabetes have an immunological basis. Palliative measures are often used to treat these diseases because immunological drugs do not have the inherent sophistication to treat specific disorders. Immunosuppressants are used to prevent rejection after an organ transplant. The active drug and market leader here is Sandimmun, whose use is being extended to the treatment of psoriasis and rheumatoid arthritis. Interferons are the largest segment of the cytokines market. Cytokines are natural human proteins involved in the control and coordination of the immune system. Their usage extends to treatment of certain cancers (hairy cell leukemia) and certain viral infections such as hepatitis B. For example, Orthoclone OK3 is a monoclonal antibody product used to prevent transplant rejection, and Ceredase is a human enzyme used to treat the rare genetic disorder, Gaucher’s disease.

CHARACTERIZATION

OF ANTIOXIDANTS

Before a widescale use of natural and synthetic compounds as antioxidant prophylactics can be suggested, it is necessary to establish the properties of such molecules. The author’s laboratory and others have developed a series of assays for characterizing the antioxidant/pro-oxidant action of such molecules 37.81-95.461 (Fig. 4). Such research is critical in strategic formulation of antioxidant supplementation regimens and also in fortification practice during certain types of food processing. Extrapolation of in vitro data allows one to predict in vivo profiles of antioxidants. Nevertheless, several important questions that should be addressed when formulating an antioxidant cocktail designed either for therapeutic use or for addition to food have been suggested.3’X37’8’Zg0 In designing antioxidant drugs and/or supplements for therapeutic purposes, it is important to consider the physiological state of the subject consuming the antioxidant. The bioavailability of drugs is determined by their pharmacokinetics, which in turn, is determined by the degree of absorption, metabolism, distribution, and subsequent elimination. The drugs must, however, become available at a physiologically relevant concentration for the intended target. How much of the consumed antioxidant drugs and/or supplement becomes available to the patient on consumption, depends on the chemical nature of the molecule, the effect of nutrient

683

components, and other drugs. The physiological state of the patient is equally important. The premise in this review is that drugs designed for a specific therapeutic efficacy might in addition be evaluated for their ability to minimise oxidative damage in humans (see Figs. 2 and 4). Comments on assays of lipid peroxidation, reactions of peroxyl radicals, reactions with hypochlorous acid, pro-oxidant assays involving DNA damage, and the application of the deoxyribose assay are worthwhile before discussing recent work on some of the drugs in their respective therapeutic categories (Table 3). Extensive details and practical information on these assays may be found in refs. 81-84, 90, and 184, and the citations therein. Thus, brief commentaries are hereby presented. The concept of phospholipid and/or rat liver microsome peroxidation. An antioxidant may be defined as a substance that when present at low concentrations compared with that of an oxidizable substrate significantly delays or prevents the oxidation of that substrate. Food manufacturers have continued to use food grade antioxidants during food processing. The basic goal is to maintain the shelf life and nutritional quality of the product over a defined period. In the laboratory, rat liver or cardiac microsomes, ox brain phospholipid liposomes, and arachidonic acid are often used as lipid model systems in peroxidation studies. Antioxidants act at different levels in the oxidative sequence involving lipids. They may act by decreasing localized oxygen concentration, preventing first-chain initiation by scavenging initial radicals such as hydroxyl radicals, binding metal ions in forms that will not generate the lipid peroxidation initiating species, decomposing peroxides by converting them to nonradical products such as alcohols, and chain breaking, whereby intermediate radicals such as peroxyl and alkoxyl radicals are scavenged to prevent continued hydrogen abstraction. The extent to which oxidation of fatty acids and their esters occurs depends on the chemical structure of the fatty acid. Reactions with trichloromethyl peroxyl radicals. The CC1302’ is an organic peroxyl radical that has been adopted as a model for the peroxyl radical intermediate encountered during the process of lipid oxidation. The mechanism of free radical oxidation of unsaturated lipids reviewed by Porter et a1.470is recommended to the reader. Delineation of lipid peroxidation as a major pathway in most degenerative diseases (e.g., cancer, atherosclerosis, and neurodegenerative diseases such as Parkinson diseases), depends on adequate standardiza-

684

0.1.

DNA damage: HPLC and GUMS

measurement of

ARuoMA

Reperfusion

HPLC based assays for markers of oxidative damage e.g.

experiments

-

levels of prostaglandin F2a (ref. 96,464,465)

(refs. 105-109)

-

levels of thiol specific antioxidants (ref. 97)

xidized bases, Comet - measurement of level of protein tyrosines (ref. 9_',9?) -

aromatic hydroxylation (ref. 100-104)

HPLC mea*lreiren:*Of OXIDATIVE

uric acid/allantoin

DAMAGE

IN

HUMANS

level*, vitamin*, j3-

Assessment af protein

1

damage Irefs. 110-120,

4

>

472).

carotene levels,

Assessment of lipid damage Measurement of levels of

-

see also Table 4

antioxidant enzymes - SOD,

-

isoprostane measurements

catalases, glutathione

-

hydroxynonenals

peroxidases, glutathione

-

TBA reactive material by HPLC

reductases (refs 86,153-161,468)

lrefs. 121-140.467)

Fig. 4. Potential indices for evaluating the ability of drugs to modulate oxidative damage in humans. Although the references cited do not necessarily use such drugs as suggested in Table 3, the abilities of such drugs to modulate free radical reaction could be investigated using the methods cited. The references quoted are representative of the vast literature in the measurement of free radical damage in biological systems.

tion and control of measurement conditions. The link between DNA damage, faulty repair of DNA, protooncogene activation, and the ability of some of the end products of lipid peroxidation to act as promoters of carcinogenesis has continued to be a major research focus of many laboratories.

Reactions with nitric oxide NO98,'85-187,427-429,431-436,447

reacts rapidly with lipid derived peroxyl radicals to form products with sufficient stability to inhibit LDL oxidation.188,437 The antioxidant vs. pro-oxidant outcomes of reactions involving NO is dependent on relative ratios of the reactants. According to Rubbo et a1.,437“pro-oxidant reactions of NO’ will occur after O,‘- reaction with NO’ to yield ONOO- and the antioxidant effects of NO’ residues on the direct reaction with alkoxyl and peroxyl radical intermediate during lipid peroxidation.” This reaction may be highly relevant to the pathogenesis of atherosclerosis and other cardiovascular dysfunctions. The high reactivity of nitric oxide with ROz’, and the stability of the product may help protect the arterial wall. The rate constant for the reac-

tion of NO with R02’ to be akin to that of a good antioxidant should be between 106-lo9 M-‘s-l. Indeed, Huie and Padmajalg9 quoted the value to be l3 X 10’ M-‘s-l (this was obtained by flash photolysis of air-saturated nitrite solutions containing alcohols). For comparison, a-tocopherol, Lovastatin, and ascorbic acid react with CC1302’(generated by pulse radiolysis of Ccl4 and propan-2-01) with rate constants of 4.9 X log, 5.2 x log, and 1.2 X lo8 M-‘s-l, respectively. A role for NO has also been demonstrated in such human diseases as malaria where NO appears to be partially involved in resistance to malaria infection, in cardiovascular disease, acute inflammation, cancer, neurodegenerative diseases, and diabetes. Sessa et al. 190recently advocated that in dogs, “chronic exercise presumably by increasing endothelial shear stress, increases EDRF/NO production and endothelial cell NO synthase gene expression, and may contribute to the beneficial effects (cf. antihypertensive) of exercise on the cardiovascular effort.” de Rojas-Walker et al.38o have shown that the reaction between NO’ and 02’leads to DNA oxidative damage due to the formation of peroxynitrite, which may have OH--like potential. OH’ produces extensive DNA damage. 171-‘73Peroxynitrite has the propensity to damage supercoiled DNA

Drugs as antioxidant

in plasmid ~BR322.~~ White et al.438 have also suggested that the reaction of 02’- with NO’ is involved in the development of atherosclerotic disease and in limiting NO’ stimulation of vascular smooth muscle guanylate cyclase activity by virtue of the product ONOO-. Interestingly, Tarpey et al.439 reported that “vascular exposure to peroxynitrite potentiates endothelial-dependent activation of guanylate cyclase in a thiol-dependent fashion.” Peroxynitrite mediates a host of chemical reactions (reviewed in ref. 448) ; these include bactericidal activity,499 oxidation of proconverting LDL to a form recognized by teins 440,44’,450 the macrophage scavenger receptor,442,45’ being associated with antioxidant depletion in human plasma,423 and trypanocidal activity.471 Methods for direct measurement of NO in humans have been reported.443-445 Using an electrochemical microsensor inserted into a hand vein, Vallence et al. 443 measured NO release (from the L-arginine/nitric oxide pathway) after infusion of bradykinin and acetylcholine. Pro-oxidant assays. Antioxidants that protect lipids against free radical damage may actually accelerate damage to other molecules such as DNA, carbohydrates, and proteins under certain conditions. It is, therefore, important to examine suggested antioxidant activity or the activity of components with a proposed “antioxidant cocktail” using assays involving DNA, proteins, and carbohydrates. The deoxyribose assay allows determination of rate constants of reactions with OH’ radicals, assessment of abilities to exert pro-oxidant action, and assessment of abilities to chelate metal iron. The net outcome can be further investigated using assays involving DNA damage to assess pro-oxidant actions. These assays have unique features. The positive pro-oxidant actions in the deoxyribose system rely on the ability of the compounds to promote reduction of Fe3+ to Fe2+ chelates and, hence, OH’ formation in the presence of H202. Assays involving DNA rely on ability to reduce either the iron-bleomycin-DNA or copper- l,lO,phenanthroline -DNA complex. If the compound under test while inhibiting lipid peroxidation promotes the two reactions described, it possesses a pro-oxidant property and has to be subjected to a more careful evaluation. A compound might be prooxidant in the deoxyribose system and/or the DNA systems but sometimes not in both.463 Fortunately, organic solvents do not affect the outcome of DNAdependent assays. Thus, where the deoxyribose assay (described below) cannot be performed due to solubility restriction, the copper-phenanthroline assay would suffice. Both assays involve mechanisms whereby OH’ is produced.

685

prophylactics

The deoxyribose assay. Fenton reactions can be accelerated by the addition of reducing agents, hence causing more damage to the biological molecule. For example, the superoxide radical, ascorbate, and paraquat radical can accelerate the iron-catalysed Haber-Weiss reaction by generating the reduced form of the iron complex, for example: Fe3+-chelate

Fe3+ -chelate

+ 02’- -+ Fe*+-chelate

+ ascorbate Fe*+ -chelate

+ 0,

(3)

radical,

(4)

-+ + ascorbate

which will decompose hydrogen peroxide to generate the damaging species, hydroxyl radical. The hydroxyl radical formed in the reaction between Fe3+-EDTA and H202 in the presence of ascorbic acid attacks the sugar deoxyribose to form products that, on heating with tbiobarbituric acid (TBA) at low pH, yield a pink chromogen. OH’ radicals are involved in the reaction involving Fe3+/EDTA (as the chelate)/H,O,/ascorbate system. Studies using gas chromatography-mass spectrometry with selected ion monitoring (GCA4S / SIM) on DNA show that the pattern of the base damaged by ionizing radiation, where the base modification has been shown to be unequivocally due to OH’ generated in free solution, is similar to that produced by the Fe3+ -EDTA/ascorbate/H202 system. The hydroxyl radical that escapes scavenging by the EDTA will become available to attack the deoxyribose molecule, and any compound present in the reaction mixture will compete for the hydroxyl radical. The rate at which hydroxyl radical reacts with the target depends on the rate constants for that reaction and on the concentrations of molecules present as targets. In proteins and in the DNA, the constituent amino acids and bases will, respectively, have different rate constants for reaction with the hydroxyl radical. Another feature of the deoxyribose method is that it is possible to obtain information on the likelihood that the compounds under test could chelate iron in a way that prevents that iron from catalysing OH’ formation. Iron ions bound to the deoxyribose molecule will stimulate damage in the presence of ascorbate and H,O,. Compounds able to chelate the catalytic iron ions array from the deoxyribose, will prevent the ‘site specific’ reaction only if they inhibit the reaction of Fe with ascorbate or H202.234 A number of comments have been expressed in the literature concerning the deoxyribose method.452-4”” It has been suggested that iron ions might play a role in the deoxyribose assay via formation of other species

686

0. I.

such as the ferry1 or perferryl radical. Although no concrete evidence exists for these in the deoxyribose assay, the author maintains that the mechanism by which the oxidation of deoxyribose occurs in the present discussions remain valid if the assay conditions as described in 82384 and more recently in 453,454,473 are applied. There is very little doubt that use of Fe(II1) EDTA in the presence of a reducing agent such as ascorbate and H202 or the superoxide radical from the xanthine/oxidase-hypoxanthine system produces The author accepts that the chemistry of OH W.171,2423330 the reactions in the assay is very complicated. In tests for the ability of a compound to exert prooxidant action in vitro, it is the ability of the compound to mediate reaction similar to that of ascorbate that constitutes the basis of the evaluation. The Fe( III) EDTA complex has a tested propensity to be reduced by the pro-oxidant (if, indeed, it is able to do so). The redox potentials of other metal complexes (which may be physiologically relevant) would vary. In the absence of EDTA, iron ions are equally available to both the deoxyribose and the compound under test. Thus compounds that are able to preferentially chelate iron and present the resulting metal-complex in a less redoxactive form compared with EDTA-metal complex, will protect deoxyribose against damage in the presence of ascorbate and H202. Substances that inhibit in the assay are also those that are able to bind iron ions strongly enough to remove them from deoxyribose. Thus, EDTA removes iron ions from deoxyribose, but iron-EDTA chelates are very effective in generating OH’ so that the deoxyribose is still degraded-this time by OH’ in “free” solution, rather than by OH’ formed on the deoxyribose molecule. If a compound inhibits site-specific radical damage to deoxyribose by chelating iron ions and rendering them less active in producing OH’, two possibilities can account for the latter property. First, the inhibitormetal ion complex may be incapable of reacting with 02’- or H202, so blocking OH’ formation. Secondly, it may be that the inhibitor-metal ion complex still undergoes redox reactions, but that the OH’ is largely intercepted by the inhibitor and is not allowed to escape into free solution (i.e., damage is directed onto the inhibitor and away from the deoxyribose) . To distinguish between these mechanisms, one can examine the fate of the inhibitor in the reaction mixture by chemical analysis (i.e., does the inhibitor undergo chemical modification as the reaction proceeds?)907453 The deoxyribose method remains an easy to use laboratory tool. 81-84,90,94,213,216,248,268,454. 459,473 with hypochlorous acid. Hypochlorous acid, HOCl, is produced by the neutrophil-derived enzyme

Reactions

ARUOMA

myeloperoxidase at sites of inflammation and when activated neutrophils infiltrate reoxygenated tissue.“’ The enzyme oxidizes chloride ions, Cl-, in the presence of Hz02. A total of 5 X 106-activated human neutrophils in 1 ml produce 88 pM HOC1 in 2 h at 22”c.19* HOC1 can attack many biological molecules. For example, the proteolytic inhibitor alAP is the major inhibitor in human plasma of proteolytic enzymes such as elastase. alAP accounts for about 90% of the elastase-inhibitory capacity of the human serum.193 Thus, its inactivation by HOC1 might greatly potentiate tissue damage, because elastase is also released from activated neutrophils. Ramos et a1.2W,403 has demonstrated that HOC1 can also react with 02’- to generate HO’ in an iron-independent mechanism. Pertinent to this review, phenolic compounds react effectively with HOC1 and can protect susceptible targets against oxidation by this molecule.37 This may have physiological significance, given the interest in the use of dietary natural antioxidants to manipulate disease states. Of the assays to assess the ability of compounds to react with HOCl, one is illustrated in Fig. 5. The ability of a compound to protect alAP against inactivation by HOC1 in a physiologically meaningful way is a good test of whether the compound might be capable of scavenging HOC1 at a biologically significant rate. In Fig. 5 elastase hydrolyzes the substrate N-succinyl-trialayl-p-nitroanilide to yield a yellow colour that can be monitored at 410 nm (Fig.

r

Time Fig. 5. Assay profile for assessing reactions with hypochlorous acid. Hypothetical diagram illustrating the principle of the elastase assay. Line A, elastase alone; line B, elastase plus a,-antiproteinase (alAP); line C, elastase plus (HOC1 plus alAP); line D, elastase plus HOC1 (from refs. 184 and 194).

687

Drugs as antioxidant prophylactics

5A) ; a’ AP inhibits this reaction (Fig. 5B ) . In the presence of HOCl, the activity of elastase is restored, since a’AP is inactivated by the HOC1 present (Fig. 5C). In performing the assay, the concentration of HOC1 used (in principle close to the physiological levels of 88 PM) should not inactivate the elastase (Fig. 5D). Thus, the window between lines B and C provide the basis for conducting tests on compounds to see if they could react with HOC1 and thereby protect a’AP. Assays involving reactions with hydrogen peroxide (H,O,) , reaction with superoxide radicals (reduction of cytochrome c and nitroblue tetrazolium) and iron availability assays provide additional information on ability of the compounds to scavenge ROS. It is important that the compounds under test do not interfere with the assays.8’-84*g0*‘84 The tests outlined above enable one to examine the possibility that a given drug could act as an antioxidant in one or more ways in vivo. The tests may clearly show that an antioxidant role is unlikely. Alternatively, they could show that an antioxidant action is feasible, in that the drug shows protective action at concentrations within the range present in vivo. One can then select the drug for further investigation. The concepts will now be illustrated by examining research that has employed the ideas advocated. pBlockers/ Ca2+ antagonists, anticancer drugs, nonsteroidal antiinflammatory drugs, antiatherogenic drugs, and antiinfective drugs that cover most of the therapeutic categories in Table 3 are considered.

includes: increased lipid peroxidation products in serum and synovial fluid, 3’oX3” depletion and oxidation of ascorbate in serum and synovial fluid,3’2,3’3increased exhalation of pentane, 3’4 increased concentrations of products of uric acid oxidation, ‘41~‘42 formation of fluorescent proteins 3’5and increased levels of protein carbonyls, 316increased urinary excretion of S-hydroxydeoxyguanosine, 3’7 and formation of 2,3dihydroxybenzoate from salicylate.“’ Beggot et a130gsuggested that the peak plasma concentrations (mmol/L) following a daily dose (g) of a number of antiinflammatory drugs are Piroxicam (0.02 mM, 0.02 g); Hydroxychloroquine (0.001 mM, 0.4 g); Penicillamine (0.2 mM, 1.5 g; Phenylbutazone (0.5 mM, 0.6 g) ; Aspirin (2 mM as salicylate, 5-8 g) ; Mefanamic acid (0.08 mM, l-2 g); Ibuprofen (0.4 n&I, l-3 g), Naproxen (0.4 mM, 0.5-1.0 g); Indomethacin (0.02 mM, 0.150.2 g), Sulindac (0.02 n&I, 0.3-0.4 g); Sulphasalazine (0.05 mM, 3-4 g), and Salicylate (2 mM, 5-8 g) .309For a molecule to efficiently scavenge ROS, its concentration and perhaps the ability to chelate iron (where this is the inherent mechanism) and present the iron-scavenger complex in the form that is unable to favour oxidant formation is critical. Reaction of antiin$ammatoly

drugs with HOC1

Neutrophils contain myeloperoxidase, which catalyzes the oxidation of chloride ions (Cl-) by H202 to a highly reactive oxidant HOCl. HOC1 is a selective oxidant and reacts quickly with methionine and with the thiol functions in proteins.2m’220Studies of the reacANTIINFLAMMATORY DRUGS AS ANTIOXIDANT tion between antiinflammatory drugs and HOC1 show PROPHYLACTICS that most of the drugs are able to react with HOC1 Drugs in the therapeutic category of musculoskeleand protect biological important targets such as cr,tal and joint disorder are shown in Table 3. The nonsteantiproteinase (a’AP) .22’,222Thus, phenylbutazone, roidal antiinflammatory drugs (NSAIDs) are among penicillamine, and thiomalic acid (the thiol component the most frequently prescribed drugs that benefit paof gold sodium thiomalate) were shown to protect tients with rheumatoid arthritis (Fig. 6)) osteoarthritis, a’AP against inactivation by HOC1.222 Chadwick et and related conditions. NSAIDs share a common a1.223have shown that the amount of (r’AP in human mechanism in that they interfere with the activity of synovial fluids appears to be decreased presumably due cyclooxygenase reducing prostaglandin formation. The to oxidative attack upon this protein. However, the slow-acting antirheumatic drugs (SAARDs) on the scavenging of HOC1 by antiinflammatory drugs will other hand, affect the cellular aspects of inflammation. only be physiologically significant if at the concentraExtensive reviews may be found elsewhere.‘g5-203~425*426 tion of drug 309that can be achieved in vivo its reaction Oxidants such as 02’-, H202, ‘OH, and HOC1 are with HOC1 is fast enough to protect the biological formed at sites of inflammation, and appear to contribtarget from attack.222,224 ute to the tissue damage in some acute and chronic The author was among the first to suggest that inflammatory diseases.2”-207 It has been suggested that part of the mechanism of the antiinflammatory drug many antiinflammatory drugs might exert part of their sulphasalazine with respect to antioxidant action was action by scavenging oxidants, and decreasing formadue to ability to scavenge HOC1.224Sulphasalazine is tion of ROS by activated phagocytes.208-2’8~222~224~2ggeffective in the treatment and prophylactics of ulceraThe occurrence of oxidative stress in RA patients has tive colitis.225-227The drug is cleaved into sulphapyribeen reviewed recently by Halliwell.2’g The evidence dine and 5-aminosalicylate (SASA) by the gut flora,

0. I. ARUOMA

688

Central CC peripheral

Activities

pain relief Paracetamol,

include inhibition

of eicosanoid

Codeine

cytokine

synthesis,

synthesis

Prednisolone,

etc.

Dexamethasone

1 Treatmentof >

rheumatoid arthritis

may

f-

act

es

immunoauppressants. Gold,

Penicillamine,

I

NON

Activities resulting

ANTI-INFL.ue%ToRYDRUGS

STEROIDAL

include inhibition in inhibition

of cycle-oxygenase

of prostanoid

several other cellular

processes

synthesis,

including

activity modulates

decreases in

lymphocyte responses and cartilage Aspirin,

Indomethacin,

Xetoprcfen,

metabolism Pluroprofen,

Modulation of inrmunesystem by cytotoxic

action OT by

stimulation.

neutrophil and monocyte migration and phagocytosis, suppression of bradykinin production and alteration

~I+KlNOt4OD~TORs

of

Azathioprine,

Levanisole,

Chloramhucil, Cyclophosphamide

Phenylbutazone

Fig. 6. Classes of drugs used in the treatment of inflammatory disorder rheumatoid arthritis.

and it is thought that SASA is the true antiinflammatory agent.225~227-229 Reaction with hydroxyl radicals al-Antiproteinase (a,AP) has been used to assess the potential sensitivity of biomolecules to OH’ relevant to inflamation.230 In in vitro systems, a,AP can be inactivated by exposure to OH’ generated by pulse radiolysis and/or by an Fe3+-EDTA/H202/ascorbic acid 230, and this can be inhibited by uric acidlM and some antiinflammatory drugszz4 (Table 5). The significance of these observations is not very clear. For example, activated neutrophils produce little OH’ unless transition metal ions are available to convert H202 into this radical, but such metal ions do appear to be present in the fluid of at least some patients with RA. The inactivation of the antiproteinase by OH’ generated under anoxic condition was decreased by adding a range of antiinflammatory drugs to the reaction mixture. The thiol compound penicillamine promoted damage to the protein under conditions that favor the formation of oxysulphur radicals.215X230 This raises the possibility that certain sulphur-containing compounds might react in such a way as to produce secondary radicals that could contribute to the side effects of such compounds. 86,215,245,246,326-329

The drug phenylbutazone was one of the first nonsteroidal antiinflammatory drugs to be used in the treatment of rheumatoid arthritis.‘959196The drug is effective against ankylosing spondylitis but it has a number of side effects. As such, it has not been recommended for use in the treatment of osteoarthritis or soft-tissue rheumatism. Reaction of phenylbutazone with ROS may not necessarily be a good thing. Phenylbutazone, acetaminophen, and penicillamine reacts with ROS to produce drug-derived radicals 230*232,246 that could potentially oxidize molecules and cause further tissue damage in vivo. Interestingly, both compounds react with OH’ with rate constant (M-‘s-l) of 4.4-6.5 X lo9 for penicillamine and 1.3-1.7 x 10” (1.1-1.5 x 10” by pulse radiolysis) for phenylbutazone. It has recently been shown that phenylbutazone and a series of fenamic acids could cause damage to alAP and accelerate the peroxidation of arachidonic acid in the presence of myoglobin and H202 .247Phenylbutazone could also cause inactivation of alAP in the presence of cytochrome c and H202. Grisham et a1.233have shown that SASA could exert pro-oxidant action in vitro, by promoting deoxyribose degradation due to OH’. Some OH’ scavengers have a degree of metal-binding capacity, and can pull metal ions away from sensitive targets. If the metal ion-scavenger complex is still

Drugs as antioxidant Table 4. Measurement

and Detection

689

prophylactics of Biological

Lipid Peroxidation”’

What is Measured

Method

Remarks

Loss of unsaturated fatty acids Lipid peroxides

Analysis of fatty acids by GLC or HPLC Iodine liberation

Lipid peroxides

Heme degradation of peroxides (often first separated by HPLC)

Lipid peroxides

Glutatbione (GSPase)

Lipid peroxides

Cyclooxygenase

Aldehydes

Fluorescence

TBA-reactive material (TBARS)

TBA test

Cytotoxic aldehydes

GClHPLClantibody

Diene-conjugated structures

Diene conjugation

Pentane

Hydrocarbon

Very useful for assessing lipid peroxidation stimulated by different metal complexes that give different product distributions. Lipid peroxides oxidize I- to I2 for titration with thiosulphate. Useful for bulk lipids, e.g., foodstuffs. H202 also oxidizes I- to IZ. Method can be applied to extracts of biological samples if other oxidizing agents are absent. Heme moiety of proteins can decompose lipid peroxides with formation of reactive intermediates. Microperoxidase is particularly effective. Radicals produced can be reacted with isoluminol to produce light, giving a sensitivity of lo- 12 mol peroxide. Diode array detectors may be used. Linked to a redox dye, a sensitivity of lo-‘* mol hydroperoxide can be achieved. GSPase reacts with HzOz and hydroperoxides, oxidizing GSH to GSSG. Addition of glutathione reductase and NADPH to reduce GSSG back to GSH results in consumption of NADPH which can be related to peroxide content. Sensitivity 3 nmol ml-’ peroxide. Cannot measure peroxides within membranes: they must first be cleaved out by phospholipases. (Availability of phospholipid hydroperoxide GSPase may simplify this problem.) Stimulation of cyclooxygenase activity can be used to measure trace amounts of peroxide in biological fluids. Sensitivity picomoles of peroxide. This assay cannot be used to identify specific peroxides present, but it is potentially interesting because it relates the presence of peroxides to one of their potential biological actions, i.e., stimulation of eicosanoid synthesis. The assay has not been widely used to date. Aldehydes such as malondialdehyde (MDA) can react with amino groups to form Shiff bases (at acid pH only). At neutral pH fluorescent dihydropyridines may be formed. Aldehydes can also polymerize to produce fluorescent products in the absence of amino groups. Formation of fluorescent products is a minor reaction pathway and has very complex chemistry, but is a highly sensitive method. It should never be assumed, without detailed characterization, that fluorescent products accumulating in vivo are end products of lipid peroxidation. The test material is heated at low pH with thiobarbituric acid (TBA) and the resulting pink chromogen is measured by absorbance at 532 nm or by fluorescence at 553 nm. The chromogen can be extracted into butan-l-01. Most of the aldehydes that react with TBA are derived from peroxides and unsaturated fatty acids during the test procedure. Simple and nonspecific assay. rigorous controls required. Hydroxyalkenals such as 4-hydroxynonenal are products of lipid peroxidation that are cytotoxic at nanomolar concentrations. They can be measured by HPLC or GC. Several techniques have been developed which use antibodies to detect proteins modified by lipid peroxidation products, e.g., proteins modified by reaction with unsaturated aldehydes. Oxidation of unsaturated fatty acids is accompanied by an increase in UV absorbance at 230-235 nm. Useful for bulk lipids (e.g., LDL). Requires extraction or separation techniques for use on biological fluids or tissues. Greater sensitivity and specificity can be gained by measuring second-derivative spectra. Serious problems arise when used on human (and some animal) body fluids, GC measurement of gases formed during lipid peroxide decomposition. Only a minor reaction pathway but can be used as a noninvasive in vivo measure of peroxidation. Results in practice have been variable: some authors have found the technique to work well and others have abandoned it. Rigorous controls are required as hydrocarbon gases are produced by bacteria and are air pollutants (thus partitioning into body tissues). Gas production is also affected by O2 concentration in vivo and by the metabolism of pentane to pentanol. Hydrocarbon gas production depends on the presence of metal ions to decompose lipid peroxides and so may not give an adequate index of the overall peroxidation process if such ions are only available in limited amounts, as is often the case in viva.

and ethane

peroxidase

Measurement of lipid peroxidation carries misleading artefacts.

techniques

gases

remains

problematic

in that of all the assays available

redox active, some or all of the OH’ radical generated by it will be directed towards the scavenger, and the target will be protected. However, would antiinflammatory drugs be able to interfere with site-specific OH’ radical formation caused by iron ions bound to critical

each one measures

something

different

and each

sites? In the case of Fenton-type chemistry, the site of hydroxyl radical generation is often determined by the availability and location of the metal ion catalyst. The damage caused by the OH’ depends on where it is formed.” If antiinflammatory drugs were to protect

0. I. ARUOMA

690

Table 5. Protection of cY,-Antiproteinase Against Hydroxyl Radical Attack by Antiinflammatory Drugs

Drug Added to Reaction Mixture None None (unirradiated (Yjantiproteinase) 5-Aminosalicylate 120 PM 240 PM Piroxicam 120 /.LM 240 /LM DL-Penicillamine disulphide 120 PM 240 PM DL-Penicillamine 120 ,LLM 240 /.LM Diclofenac sodium 120 PM 249 ,uM Chloroquine 120 PM 240 I.LM Sulphapyridine 120 /.LM 240 PM Hydroxychloroquinie 120 /IM 240 I.LM Indometbacin 120 PM 240 PM

Elastase Activity (AA x lO?s) 13.1

% Protection of LV,APby Drug (as % Decrease in Elastase Activity)

-

attack deoxyribose. Once sufficient scavenger has been added to convert all available iron into an iron-scavenger complex, the remaining deoxyribose degradation is caused by OH’ generated in free solution by that iron-scavenger complex.213,234*235 Metal complexes of antiinjlammatory drugs

Certain metal complexes of antiinflammatory drugs have been suggested as effective agents against 11.9 9 For example, copper complexes of a number ROS. 236-240 8.7 34 of nonsteroidal antiinflammatory drugs are more potent 10.8 18 antiinflammatory agents and less ulcerogenic than their 7.6 42 parent antiinflammatory drugs.““~24’This was demonstrated for the copper complexes of penicillamine, flu12.2 8.8 33 fenamic acid, levamisole, aspirin and 2-amino-2-thiazoline.241 There was no obvious difference between the 11.7 11 activities of copper(I) and copper(I1) complexes, sug9.2 30 gesting the possibility of a common mechanism. 11.0 16 Upon administration, it is necessary for the copper 7.8 40 complexes to remain bound to minimize the possibility 10.3 21 of free copper ions becoming available at the site of 7.6 42 inflammation. Metal ions, especially those of copper and iron, are catalysts for free radical formation in biological 10.4 21 7.6 42 systems. Recent in vitro studies show that mixtures of copper ions with H202 and 02’- or ascorbate can do 8.7 34 considerable damage to DNA and proteins.242 Copper 7.5 43 ions mediate much more damage to DNA bases by H202 6.9 47 in the presence or absence of ascorbate than do iron 6.6 50 ions in comparable reaction mixture.“2 a,-Antiproteinase (1 mg/ml) was pulse-irradiated in N,O-satuUnder normal conditions, much less copper is presrated buffer over a period of approximately 6 min, to generate 180 ent in the human body than iron. For example, an adult pmol .01-I/dm3. Drugs were included in the reaction mixture at the body has on average 4.5 g of iron but only 0.08 g of final concentration stated. Elastase alone gave a A&i0 of 1.84 X lo-Vs. copper. Interestingly, proteins such as albumin are able From Aruoma ef ~1.‘~’ to inhibit formation of free radicals from free copper ions in free solution. Thus, much care is needed in evaluating the efficacy of the metal complexes of the against this type of damage, such action would depend antiinflammatory drugs. The integrity of the complexes on the effective concentration of the drugs at the site in vivo should be considered in view of the ability of of inflammation, the rate constant for reaction with free copper ions to form free radicals. ROS and the ability of the drugs to chelate iron (in Although it has been suggested that chelation of iron the case of iron-dependent reaction) ions.222*224Z234*235 and other metals may contribute to antiinflammatory Antiinflammatory drugs are able to inhibit the oxiaction, the use of iron chelator desferrioxamine (DFO) dation of deoxyribose in the presence of Fe-EDTA, *13 in humans has highlighted associated toxicity probindicating their ability to scavenge OH’. It is also possilems.243 Desferrioxamine causes reversible ocular abble that EDTA keeps the Fe bound to itself and does normalities that may be related to its high iron-binding not let it bind to the drugs. Antiinflammatory drugs capacity. However, the antiinflammatory drug D-myoalso inhibit oxidation of deoxyribose in reaction mixinositol-1,2,6-triphosphate, one of the isomers of inositures not containing EDTA (e.g., Fig. 7, line X), sugto1 triphosphate, *@has been shown to be able to chegesting that they have some degree of iron-binding late iron, decrease metal-induced lipid peroxidation, capacity. The initial phase of effective inhibition is due and afford protection against DFO ocular toxicity.W to a transfer of iron from the deoxyribose to the added Thus, in the event that free radicals and other reacscavenger, 234giving an iron-scavenger complex that is tive oxygen species are produced in free solution at poorly effective (for reasons of redox potential or of sites of inflammation, the ability of antiinflammatory self scavenging) in generating OH’ radical that can 0

-

Drugs as antioxidant

6

x

X

3

Y

I

I

0.70

o-35

0

CONCENTRATION

mM

Fig. 7. Inhibition of deoxyribose degradation by hydroxychloroquine. Details of the deoxyribose assay may be found in refs. 82, 84, and 213. The rate constant (k) for reaction with OH’ can be calculated from the equation (in the presence of EDTA and in the absence of EDTA for the apparent rate constants): k, = slope

x

kDR x [DR]

x

A”

where kDR is 3.1 x 10’ M-‘SK’. A” is the absorbance in the absence of added drug. [DR] is the concentration of deoxyribose in the reaction mixture. Line X, EDTA absent; Line Y, EDTA present. The calculated rate constant for hydroxychloroquine is ( 1.O- 1.3 ) X 1()10 M-Is-m

drugs to scavenge them would depend on the concentrations of the drugs at the site, the iron-binding properties of the drugs, and on the various rate constants of reaction of the drugs with ROS. One complication is that some of the antiinflammatory drugs might form secondary radicals that could, themselves, contribute to the resulting tissue damage. Perhaps the assessment of potential pro-oxidant activity could become part of toxicity testing of antiinflammatory agents using the Stanford Toxicity index.382

CALCIUM INHIBITORS,

ANTAGONISTS, AND

Hz-RECEPTOR

ANTIOXIDANT

P-BLOCKERS,

ACE

ANTAGONISTS

AS

PROPHYLACTICS?

Drugs considered here fall under the cardiovascular and gastrointestinal therapeutic categories (Table 3).

prophylactics

691

A number of P-blockers, ACE (angiotensin-converting enzyme) inhibitors, calcium antagonists, and H2-receptor antagonists either in clinical use or at certain stages of development have been reported to scavenge ROS, inhibit lipid peroxidation, and modulate oxidation of low density lipoprotein.248-268 Calcium ions are often involved in the injury to cells that can be produced by oxidative stress, and calcium may also play a direct role in reoxygenation injury. Thus, “calcium blockers” may improve myocardial function after reoxygenation.257~259Z270-272~279 Ca*+ channels play a critical role in regulating the activity of a variety of different excitable tissues. Indeed, the Ca*’ channel blockers, although a heterogenous group of compounds, fall into three main structural classes: benzothiazepines, aralkylamines, and dihydropyridines. Typical drugs in these classes are diltiazem, verapamil, and nitrendipine, respectively. Calcium antagonists are major therapeutic agents for hypertension and angina pectoris (often they are used in combination therapy with ACE inhibitors and pblockers)273 and have been tested for their effects on gastrointestinal motility.274 One mechanism via which oxidative stress can cause damage to DNA involves nuclease activation. Oxidative stress leads to the activation of Ca*+ binding by endoplasmic reticulum, inhibition of plasma membrane Ca*+ extrusion systems, and the release of Ca2+ from mitochondria. This sequence of events leads to increases in the levels of intracellular free calcium ions and subsequent DNA fragmentation without oxidative base modification often observed via the Fenton mechanism.275-277 Thus, from the standpoint that oxidative stress is associated with the disruption of the intracellular Ca2+ homeostasis leading to prolonged elevation of the cytosolic Ca*’ concentration and eventual cell death, evaluation of antioxidant/pro-oxidant actions of calcium channel antagonists seem logical enough. That /?-blockers can attenuate lipid peroxidation and oxidation of low-density lipoproteins249V278 is relevant to the management of cardiovascular disorders. Low-density lipoprotein (LDL) oxidation by endothelial cells, smooth muscle cells, or monocytes/macrophages is thought to be a key step in the progression of atherogenesis.280-283Specific antioxidant drugs, e.g., probucol and 21-aminosteroid, have been extensively investigated for their abilities to protect LDL and lipid membranes against oxidation282*2W-289 and, hence, contribute to the modulation of oxidative modification relevant to the atherogenesis hypothesis in humans. That P-blockers might provide additional antioxidant protection is supported by a body of evidence in the literature; 248~250~256~258~26’ however, ~278different workers appear to obtain different efficacies of the ability of P-blockers

692

0. I.

ARUOMA

to protect biological molecules against oxidation. The thesis advocated in this review should provide a comprehensive answer as to the potential antioxidant/pro-oxidant actions of new drugs in this area. The drug carvedi101is a third-generation ,&blocking agent with cardiovascular activity. Recent studies by the author have shown that carvedilol reacts rapidly with the model peroxyl radical CC1302’(calculated rate constant 1.54 2 0.08 X lo7 M-k-‘) generated by pulse radiolysis of a mixture of 1% v/v CC& and 49% v/v isopropyl alcohol in 50% 10 mM KHzP04-KOH buffer pH 7.0 at 25°C. Carvedilol was able to scavenge hypochlorous acid at a rate sufficient to protect a model molecule catalase231 against inactivation by HOCl. Histamine administration enhances positive inotropism (the force of ventricular contraction), increases positive chronotropism (the sinus rate), slows negative dromotropism (atrioventricular conduction), and causes positive bathmotropism (ventricular arrhythmias) . Histamine acts independently of the adrenergic system, and its effect is mediated by the H1 and Hz receptors. Extensive reviews may be found in refs. 290-296 and 428. The human heart is known to contain approximately 3 mg g-’ histamine.295 ROS plays a role in the pathophysiology of gastrointestinal disorder duodenal ulceration.297 Thus, Hz antagonists have been investigated for their abilities to scavenge ROS.253,29s Lapenna et a1.253suggested that famatidine, cimetidine, and ranitidine (Fig. 8) reacted rapidly with OH’ with rate constants 1.7 X 10” M-‘s-l, 1.6 X 10” M-‘s-‘, and 7.5 X lo9 M-‘s-l, respectively. van Zyl et a1.298 suggested that ranitidine and nizatidine but not famotidine were potent scavengers of HOCl. The potency of JB-9322 (N- [ 3 [ 3-(piperidin-l-ylmethyl)phenoxy ] propyll-N’-cyclopropylmethyl-2-nitrol,lethenediamine as a histamine H,-receptor antagonist has been described.381 It is hereby speculated that the cytoprotective properties of JB-9322 may also include ability to scavenge reactive oxygen species. The ACE inhibitors are used for the treatment of arterial hypertension and cardiac failure on patients with myocardial infarction. Extensive reviews of these class of pharmacological agents may be found in refs. 300-305. Captopril and other ACE inhibitors (such as Enalapril and Lisinopril) have beneficial effects on the reoxygenated myocardium. The effect has been suggested to be due to abilities to scavenge ROS 262,267,268,305-309

The sulf hydryl groups in captopril and related compounds have been suggested to be responsible for the observed ROS scavenging properties. Anticancer drugs as antioxidant prophylactics Carcinogenesis is the process that results in a cell growing abnormally and outside of the normal regula-

X-

CH3 CH2SCH$H2N=yNHCH3 HN-C=

N

HN,N

Cimetidine

CH2SCH2CH2NHiNHCH3 -

CHN02

\O d CbN(CH3)2 Ranitidine

CH2SCHzCH2;NHz -

NS02NHz

/i S ,N

Y

N=C(NH2)2 Famotidine

CH2SCH2CH2NH;NHCH3

,

-

CHN02

/-4 S /N

Y Ch$-W-ki)2 Nizatidine

Structural Fig.

formulae 8. Structural

of

formulae

H,-antagonists of H,-antagonists.

tory mechanisms of cell division. The initiation of tumor development probably involves interactions with environmental chemicals. Indeed, free radical intermediates of xenobiotics and ROS production by chemical carcinogens have been related to environmental carcinogenesis.6~ll,318-320,469

Hydroxyl radicals and other reactive oxygen species are implicated in molecular and tissue damage in human to DNA 170-173.330-331 ,& to cancer. 10,12,321-325 ~~~~~ lipids 333are the two potentially devastating effects of OH’. The link between DNA damage, faulty repair of DNA, proto-oncogene activation, in activation/loss of tumor suppressor genes and the ability of some end products of lipid peroxidation to act as promoters of carcinogenesis are much discussed.333,3?-337 Many carcinogens damage DNA or interfere with enzymes required for accurate DNA replication. It has been speculated that apoptosis (cell death) is an efficient method of preventing malignant transformations. Since apoptosis removes cells with genetic lesions, it has been argued that abnormal apoptosis can contribute

693

Drugs as antioxidant prophylactics

to cancer development by virtue of the accumulation of dividing cells and the inefficient removal of genetic variants with propensity to be malignant. Thus, the role of oncogenes, such as Bcl-2, the ~53 tumor suppressor genes and anticancer drugs in apoptosis and cancer, have been extensively discussed.338-344 The pro-oxidant actions of anticancer drugs (e.g., bleomycin, adriamycin, and the platinum antitumor drugs) and their potential cytotoxicity due to free radical mechanisms have been extensively reviewed.345-350 Thus, the adoption of a strategy for evaluating the potential pro-oxidant and antioxidant action of anticancer drugs in development and those in clinical use might contribute to a greater understanding of the pharmacoeconomics of such drugs with respect to antioxidant prophylactics. The drugs tamoxifen and etoposide (see Table 3) are illustrative. The anticancer drug tamoxifen and its metabolite 4-hydroxytamoxifen (Fig. 9) are widely used in che-

Tamoxifen 4-Hydroxytamoxifen

RI

Rl

H OH

OCHzCHzN(CH,)2 OCHzCHzN(CH,)z

motherapy. Trans-tamoxifen is used as the therapeutic agent of choice in the treatment of estrogen-dependent human breast tumors.351-354Tamoxifen has been reported to suppress the mitotic action of estrogen,355,356 exert antiproliferative effects on estrogen receptor-negative breast cancer cells,357,358suppress tumor promoter-induced hydrogen peroxide formation by human neutrophils, 35ginhibit lipid peroxidation in in vitro systems, 3M)and recently has been associated with increased risk of endometrial cancer.361 Tamoxifen and hydroxytamoxifen react rapidly with CC1302’with calculated rate constants of (1.88 2 0.09) X 10’ M-‘ss’ and (7.80 ? 0.39) X lo6 M-‘s-’ .37The possibility that the drugs could react with lipid oxidation intermediates (peroxyl radicals) contributes to the potential benefit of tamoxifen and its metabolite as antioxidant prophylactics in human breast cancer. The cytochrome P-450 catalyzed 4-hydroxylation has been suggested to generate the potent metabolite 4-hydroxytamoxifen in vivo.362-364Mani et a1.365served that tamoxifen-N-oxide can be reduced to tamoxifen hence the N-oxide may serve as a storage for tamoxifen in vivo. Etoposide (VP 16) (Fig. 9) is an antineoplastic agent that has been used in chemotherapy of several types of tumors.366,367Etoposide has been reported to inhibit anthracycline-induced lipid peroxidation, 368induce single-strand breaks in DNA in HeLa cells,369 and to cause concentration-dependent induction of apoptosis in immature thymocyte.370 The drug is a DNA topoisomerase II inhibitor.37’,372The interaction of etoposide phenoxyl radical with intracellular reductarns may explain the drug’s metabolic activation and cytotoxic effects.347 Like tamoxifen, etoposide reacted rapidly with peroxyl radicals with a calculated rate constant of (1.56 + 0.08) X 10’ M-‘SK’. The three drugs (see Table 6a,b) inhibited DNA damage in the bleomycin-Fe3+ system induced by propyl gallate, gallic acid, and/or Trolox C, but not in the damage mediated by ascorbate and/or the H202 system. The biological significance of this observation is being investigated. OTHER CONSIDERATIONS

Etoposide (v&‘-16)

Fig. 9. Structural formula of tamoxifen and etoposide.

Shahzeidi et a1.373have shown that oral N-acetyl cysteine reduced bleomycin-induced collagen deposition in the lungs of mice. N-Acetyl cysteine has been employed in the treatment of acute lung injury and is currently being evaluated in clinical trials as anti-AIDS drug. The antioxidant profile of N-acetyl cysteine is fully established.374-379It is interesting to speculate that drugs designed to incorporate features of “antioxidants” might present logical regime in chemotherapy.

694

0. I. ARUOMA

The principles have also been applied to study the potential antioxidant and pro-oxidant actions of neuroleptic drugs (chlorpromazine, prochlorperazine, metoclopramide, haloperidol, and methotrimeprazine)457 and to L-DOPA and dopamine.458 L-DOPA and dopamine have a mixture of pro- and antioxidant effects, which could contribute to tissue damage due to oxidative stress in Parkinson’s disease and other neurological disorders. The interfacial properties of the ‘antioxidant’ drugs should be a prime point for consideration in drug design. For example, can lipolytic drugs concentrate within hydrophobic regions such as the interior of membranes? Similarly, water-soluble antioxidants may not be of any use in protecting membrane lipids against oxidation if they are not able to become present. Frankel et al. have suggested that the “differences observed

Table 6A. Pro-Oxidant Actions of Gallic Acid, Trolox C, Ascorbate, and Propyl Gallate and Their Inhibition by Tamoxifen, 4-Hydroxytamoxifen, and Etoposide, Assessed by the Iron-Bleomycin-Dependent DNA Damage Extent of DNA Damage AS32

Systems tested 0

Ascorbate plus Tamoxifen plus 4-Hydroxytamoxifen plus Etoposide Gallic Acid plus Tamoxifen plus 4-Hydroxytamoxifen plus Etoposide Propyl gallate plus Tamoxifen plus 4-Hydroxytamoxifen plus Etoposide Trolox C plus Tamoxifen plus 4-Hydroxytamoxifen plus Etoposide

50 /.LM 50 /JM 100 /IM 50 PM 100 /LM 50 PM 100 /.LM 50 /.JM 50 /.LM 100 /LM 50 ,LLM 100 PM 50 /.LM 100 PM 50 /LM 50 /.LM 100 /LM 50 ELM 100 /IM 50 /LM 100 /LM 50 /.LM 50 PM 100 /.LM 50 /.LM 100 PM 50 ELM 100 PM

0.079 0.570 0.581 0.511 0.585 0.512 0.592 0.550 0.716 0.423 0.175 0.462 0.171 0.456 0.199 0.303 0.120 0.099 0.183 0.120 0.217 0.129 0.400 0.268 0.144 0.278 0.149 0.304 0.148

Percent Inhibition

0 10 0 10 0 4 41 76 35 76 36 72 60 67 40 60 28 57 33 64 31 63 24 63

* Values are the means from triplicate experimental points that varied by no more than 10%. Etoposide, tamoxifen and 4-hydroxytamoxifen were used at the final concentrations stated. Data illustrates ability to inhibit the pro-oxidant action of gallic acid, Trolox C, propyl galate, and ascorbate. Percent inhibitions were calculated from the individual control A532 for ascorbate, gallic acid, propyl gallate, and Trolox C, respectively.

Table 6B. DNA Damage by the Bleomycin-Fe’+ Complex and Its Inhibition With Catalase. Suueroxide Dismutase

Addition to Reaction Mixture/PM None Ascorbate + Catalase + SOD + Desferal + Tamoxifen + 4-OH Tamoxifen + Etoposide PG only + Catalase + SOD + Desferal + Tamoxifen + 4-OH Tamoxifen + Etoposide

200 /LM 1000 u/ml 500 U/ml 500 /LM 100 ELM 100 /LM 100 /LM 200 /JM 1000 u/ml 500 U/ml 500 ELM 100 /.LM 100 /.LM 100 /.LM

Extent of DNA Damage/A532 0.077 1.415 1.442 1.416 0.492 1.291 1.267 1.280 0.270 0.271 0.256 0.095 0.112 0.135 0.166

Percent Inhibition 0.0 0.0 65.0 9.0 11.0 10.0 0.0 0.0 65.0 59.0 50.0 40.0

Catalase and superoxide dismutase (SOD) were freshly diluted and units of activity were as described in the Sigma Co. catalog.

in the efficiency of antioxidants may be explained by their affinities towards bulk oils and emulsions and that the analytical method used to determine the extent and endpoint of oxidation is critical.“461 Similarly, Pryor et a1.462suggested that rate constants for antioxidants are sensitive to the system used for their measurement. Lipophilic drugs can be evaluated using the assays that rely on ability to reduce either the ironbleomycin-DNA or copper- 1,lO phenanthroline DNA complex.373833463 There are no solubility restrictions. If the drug compound under test whilst inhibiting lipid proxidation promotes the two reactions described, it possesses a pro-oxidant property and may, therefore, merit a more careful evaluation. CONCLUDINGCOMMENTS Free radical generation occurs normally in the human body, and rates of free radical generation are probably increased in most diseases. Their importance as a mechanism of tissue injury is still uncertain, but the development of new assays applicable to humans should allow rapid evaluation of the role of free radicals in disease pathology and provide a logical basis for the therapeutic use of drugs as antioxidant prophylactics. It is envisaged that drugs with multiple mechanisms of protective action, including antioxidant properties, may become available in attempts to minimize tissue injury in human disease. The subject of this review may help pharmaceutical R & D strategies. - I thank the UK Ministry of Agriculture Fisheries and Food, Nestec SA Switzerland, and tbe World Cancer Research Fund for research support. I respectfully acknowledge the

Acknowledgements

695

Drugs as antioxidant prophylactics role that my teacher and colleague Professor Barry Halliwell has played in my scientific career.

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ACE-angiotensin-converting enzyme AIDS-acquired immunodeficiency syndrome a,APa,-antiproteinase SASA-5-aminosalicylic acid CC1302’-trichloromethylperoxyl radical CCL-carbon tetrachloride CNS -central nervous system L-DOPA-L-3,4-dihydroxyphenylalanine DR-deoxyribose EDTA-ethylenediaminetetraacetic acid GC/hIS/SIM-gas chromatography, mass spectroscopy with selected ion monitoring GSH-glutathione GSHPX-glutathione peroxidase GSSG-reduced glutathione HOCl-hypochlorous acid LDL-low density lipoprotein NADPH-reduced nicotinamide adenine dinucleotide phosphate NSAIDs-bonsteroidal antiinflammatory drugs Ph&‘---triphenylmethyl radical ROS-reactive oxygen species SOD - superoxide dismutase TBA-thiobarbituric acid