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Recent Patents on Anti-Infective Drug Discovery, 2006, 1, 17-31

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1574-891X/06 $100.00+.00

© 2006 Bentham Science Publishers Ltd.

Recent Progress in Pharmacological Research of Antioxidants in

Pathological Conditions: Cardiovascular Health

Victor M. Victor*

,#

, Kenneth J. McCreath and Milagros Rocha

a,#

#

Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) Unidad Mixta CNIC-Universidad

de Valencia (CNIC-UVEG), Departamento de Farmacologia, Facultad de Medicina, Avda. Blasco Ibañez 15, 46010

Valencia, Spain;

a

Departamento de Fisiologia (Fisiologia Animal II), Facultad de Ciencias Biologicas, Universidad

Complutense, 28040 Madrid, Spain

Received: July 20, 2005; Accepted: August 31, 2005; Revised: September 08, 2005

Abstract: Antioxidants are essential, and are involved in several important biological processes such as immunity,

protection against tissue damage, reproduction, growth and development. Antioxidants preserve adequate function of cells

against homeostatic disturbances such as those caused by septic shock, aging and, in general, processes involving

oxidative stress. Each year, many scientific articles are published describing the pharmacological and biological properties

of antioxidants. This review article compiles recent findings on these properties, focusing mainly on the anti-inflammatory

properties of antioxidants in different pathological areas, such as cardiovascular damage and sepsis. In relation to this

process, this review focuses on the involvement of reactive oxygen and nitrogen species. Finally, the protective role of

antioxidants against homeostatic disturbances such as those caused by endotoxin toxicity and cardiovascular damage, their

potential clinical use, and the effects on the redox state of immune cells are discussed.

Keywords: Antioxidant, cardiovascular dysfunction, endotoxin, flavonoids, immune system, ischemia, mitochondria, N-acetyl-

cysteine, NF-κB, nitric oxide, patent, peroxynitrite, reactive oxygen species, septic shock, vitamin.

INTRODUCTION

The presence of free radicals in biological materials was

discovered about 50 years ago [1]. Today, there is a large

body of evidence indicating that patients in hospital intensive

care units (ICUs) are exposed to excessive free radicals from

drugs and other substances that alter cellular reduction -

oxidation (redox) balance, and disrupt normal biological

functions [2-3]. However, low levels of free radicals are also

vital for many cell signaling events and are essential for

proper cell function.

Excess free radicals can result from a variety of

conditions such as tissue damage and hypoxia (limiting

oxygen levels), overexposure to environmental factors

(tobacco smoke, ultraviolet radiation, and pollutants), a lack

of antioxidants, or destruction of free radical scavengers.

When the production of damaging free radicals exceeds the

capacity of the body's antioxidant defenses to detoxify them,

a condition known as oxidative stress occurs. The resultant

cellular injury caused by oxidative stress has been linked to

over 200 clinical disorders, many of which are seen in ICU

patients units [4].

FREE RADICALS AND ANTIOXIDANTS: AN

OVERVIEW

A free radical can be described as any atom or a group of

atoms or molecules in which there is at least one unpaired

electron in the outermost shell [5]. These free radicals are

*Address correspondence to this author at the (VM Victor) at Centro

Nacional de Investigaciones Cardiovasculares (CNIC), Universidad de

Valencia, Departamento de Farmacologia, Facultad de Medicina, Avda.

Blasco Ibañez 15, 46010 Valencia, Spain; Tel: 34 963864630; Fax: 34

963864625; E-mail: vmvictor@cnic.es

very reactive with adjacent molecules such as lipids,proteins,

and carbohydrates and can cause cellular damage [6].

Paradoxically, free radicals can also be produced by many

cells as a protective mechanism, for example neutrophils

produce free radicals to attack and destroy pathogens, while

the liver uses free radicals for detoxification [7]. However,

the presence of free radicals within the body can also have a

significant role in the development and progression of many

disease processes for example heart disease, hypertension,

cerebrovascular accidents, and diabetic complications.

Any free radical involving O

2

is referred to as a reactive

oxygen species (ROS) [8]. Normal cellular metabolism

involves the production of ROS, and in humans, superoxide

(O

2

-

) is the most commonly produced free radical.

Phagocytic cells such as macrophages and neutrophils are

prominent sources of O

2

-

. During an inflammatory response,

these cells generate free radicals that attack invading

pathogens such as bacteria and, because of this, the

production of O

2

-

by activated phagocytic cells in response to

inflammation is one of the most studied free radical-

producing systems [5].

O

2

-

produced from a one-electron reduction of O

2

can

undergo either spontaneous or enzyme-catalyzed dismutation

to hydrogen peroxide (H

2

O

2

). H

2

O

2

, although not technically

considered an oxygen free radical, is a member of the ROS

family and may selectively participate in free radical

generation. The majority of the H

2

O

2

is broken down to O

2

and water by the antioxidant enzyme catalase. In addition to

catalase, glutathione peroxidase can also break down H

2

O

2

and also any peroxides that form on lipids within the body

[5]. When O

2

-

reacts with nitric oxide (NO), the toxic product

peroxynitrite (ONOO

-

) is formed. Although it is true that

18 Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1

Victor et al.

most ROS originate from O

2

-

generated as a by-product of

oxidative phosphorylation (mitochondrial respiration), they

differ in their mechanism of production, necessary cofactors,

diffusion range, hydrophobicity, biological targets,

detoxification pathways and breakdown products. O

-

2

is

membrane impermeant and its damaging reactions largely

involve disassembly of iron-sulphur clusters in proteins. In

fact, studies in rat liver lysates have shown that H

2

O

2

or O

-

2

alone lacked reactivity toward iron regulatory protein-1

(IRP-1), but a combined action of the two species induced

reversible inactivation of IRP-1. Such an effect was

attributed to direct interactions of O

-

2

and H

2

O

2

with a

preformed pool of IRP-1, resulting in reversible

modifications of -SH residues; in fact, its action would be

limited to removing only iron atoms, an effect sufficient to

abolish enzyme activity.

The hydroxyl radical (

.

OH) is the most reactive of the

free radical molecules [1].

.

OH damages cell membranes and

lipoproteins by a process termed lipid peroxidation. In fact,

lipid peroxidation can be defined as the process whereby free

radicals “steal” electrons from the lipids in our cell

membranes, resulting in cell damage and increased

production of ROS. This process takes place in 3 stages:

1. Initiation: In a peroxide-free lipid system, the initiation of

a peroxidation sequence refers to the attack of an ROS (with

sufficient reactivity) able to abstract a hydrogen (H) atom

from a methylene group (- CH

2

-), as these hydrogens have

very high mobility. This attack easily generates free radicals

from polyunsaturated fatty acids.

.

OH is the most efficient

ROS for this attack, whereas O

-

2

is insufficiently reactive.

2. Propagation: A peroxyl radical is able to abstract H from

another lipid molecule (adjacent fatty acid), especially in the

presence of metals such as copper or iron, thus causing an

autocatalytic chain reaction. The peroxyl radical combines

with H to give a lipid hydroperoxide (or peroxide). This

reaction characterizes the propagation stage.

3. Termination: formation of a hydroperoxide.

Lipid peroxidative damage to lipids in low-density

lipoprotein (LDL) plays an important role in atherosclerosis

[9].

To protect against oxidative damage, organisms have

developed a variety of antioxidant defenses that include

proteins, compounds such as vitamins, and specialized

antioxidant enzymes. Antioxidants can be described as

substances capable of counteracting the damaging effects of

oxidation in body tissues. Antioxidants are divided in

general into two classes based on mechanisms of action:

1. chain-breaking antioxidants, such as Vitamin E and beta-

carotene, “break the chain” of free radical formation by

donating an electron to stabilize an existing free radical. In

general, they act by reacting with peroxyl radicals.

2. preventive antioxidants are enzymes that scavenge

initiating radicals before they start an oxidation chain.

Chain-breaking antioxidants are found in the blood and

the fluids of the extracellular space, where preventive

antioxidant enzymes are absent or present in very small

quantities [8]. These small-molecule antioxidants include

both water and lipid-soluble varieties. The lipid-soluble

antioxidants are located in the cellular membranes and

lipoproteins, whereas the water-soluble antioxidants are

present in the aqueous environments, such as fluids inside

cells and in the blood.

Preventative antioxidant enzymes inside the cell are an

important defense against free radicals. The main enzymatic

“scavengers” responsible for the prevention of ROS

formation and oxidation are superoxide dismutase (SOD),

catalase, and glutathione peroxidase. SOD is found in

virtually every oxygen-based organism, and its major

function is to catalyze the dismutation of O

2

-

to H

2

O

2

. This

reaction is generally considered to be the body's primary

antioxidant defense because it prevents further generation of

free radicals. In humans, the highest levels of SOD are found

in the liver, adrenal gland, kidney, and spleen [10].

Catalase and glutathione peroxidase both work to

detoxify O

2

-reactive radicals by catalyzing the formation of

H

2

O

2

derived from O

2

-

. The liver, kidney, and red blood cells

possess high levels of catalase, which helps to detoxify

chemicals in the body. The water-soluble tripeptide-thiol

glutathione also plays an important role in a variety of

detoxification processes. Glutathione is found in millimolar

concentrations in the cell cytosol and other aqueous phases,

and readily interacts with free radicals, especially the

hydroxyl radical, by donating a hydrogen atom. This reaction

provides protection by neutralizing reactive hydroxyl

radicals that are thought to be a major source of free radical

pathology, including cancer [11]. Glutathione is also an

essential co-factor for antioxidant enzymes.

Below, the data is summarized regarding some of the

most frequently antioxidants in pathologies which implicate

free radicals production such as sepsis and cardiovascular

dysfunction. These pathogenesis generally involve

impairment in endothelial function.

SEPSIS AND SIGNALING PATHWAYS

Serious infections trigger systemic inflammatory

response and can result in sepsis. It is believed that sepsis

and therefore septic shock are due to the inappropiate

increase in the innate immune response via circulating and

tissue inflammatory cells, such as monocytes/macrophages

and neutrophils. These cells normally exist in a non-

activated state but are rapidly activated in response to

bacteria, their products or inflammatory mediators, to

become highly active phagocytes. In doing so, they can

contribute to the development of injuries by producing

mediators such as cytokines and ROS. In fact, sepsis induces

a dysfunction in immune cells [12-14].

As most infections occur primarily in the tissue and not

in the blood stream, extravasation of leukocytes is essential

to bring inflammatory cells and foreign pathogens into

contact. This requires both a chemotactic gradient and

coordinated up-regulation of endothelial and inflammatory

cell adhesion molecule expression (ICAM). Leukocytes have

only a finite life-span at the inflammation site. Neutrophils

rapidly undergo apoptosis to be cleared by inflammatory

macrophages, which themselves emigrate from the inflamed

site during the resolution phase. Thus, a successful

inflammatory event requires not only appropriate activation

of cells and mediators with subsequent phagocytosis and

Antioxidants and Pathological Conditions

Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1 19

removal of the exciting stimulus, but also a consequent

elimination of the inflammatory cells and debris to allow the

tissues to reform a normal architecture and function.

LIPOPOLYSACCHARIDE (LPS) SIGNALING

The molecular mechanisms by which LPS induces gene

activation, and hence inflammatory mediator expression,

have been reviewed recently [15]. LPS of Gram-negative

organisms induces macrophages to secrete cytokines, which

in turn activate T, and B cells to upregulate the adaptive

immune responses. Toll-like receptor 4 (TLR4) is the LPS

receptor and its stimulation induces nuclear factor κB (NF-

κB) activation. The activation of NF-κB involves phos-

phorylation and degradation of IκB, an inhibitor of NF-κB,

which allows the translocation of NF-κB heterodimers to the

nucleous to bring about its action. The NF-κB/IκB system

exerts transcriptional regulation on proinflammatory genes

encoded for various adhesion molecules and cytokines. Most

of these genes have functional NF-κB binding elements in

their promoter regions. Activation of NF-κB leads to the

induction of NF-κB binding elements in their promoter

regions. Activation of NF-κB leads to the induction of NF-

κB dependent effector genes such as TNFα, IL-1,

chemokines, adhesion molecules and also ROS production.

All these biologically active molecules/factors produce

modifications in blood flow, and aggregation of neutrophils,

and platelets. This results in damaged endothelium and also

coagulation abnormalities often seen in patients with sepsis

and septic shock. Therefore, NF-κB is reported to be an O

2

sensor in LPS-induced endotoxemia. A schematic

representation of the LPS/NFκB pathway is illustrated in

(Fig. 1).

Accordingly, there is a need to improve our

understanding of IκB degradation and for the identification

of modulators of this degradation process for use in treating

diseases associated with activation of NF-κB. Some patent

compounds satisfy these needs and further provide other

related advantages by using modulating agents that comprise

a recognition domain for E3 ubiquitin ligase, a necessary

factor for ubiquitin-controlled IκB degradation [16,17].

In addition, some patents have been developed in order to

counteract the effect of other important mediator in sepsis,

TNFα. For example, [18] shows that anti-TNF antibodies,

fragments and regions thereof, which are specific for human

TNFα, are useful for in vivo diagnosis and therapy of a

number of TNFα-mediated pathologies and conditions.

Further, polynucleotides coding for murine and chimeric

antibodies, methods of producing the antibody, methods of

use of the anti-TNF antibody, or fragment, region or

derivative thereof, in immunoassays and immunotherapeutic

approaches are described.

FREE RADICALS AND ANTIOXIDANTS IN SEPSIS

The pathogenetic sequence of reactions mediated by

endotoxin (LPS) leading to the development of sepsis

involves ROS [12-13,19]. Among ROS,

.

OH either singly or

in combination with ONOO

-

, leads to tissue damage often

observed during septic injury. Inactivation of these damaging

radicals by antioxidants or NO inhibitor(s) may be helpful

Fig. (1). Schematic diagram of LPS-induced signalling pathway of

the host inflammatory response in monocytes, macrophages and

neutrophils. For a detailed explanation of this figure see text. NF-

κB, nuclear factor κB; LPS, lipopolysaccharide; LBP, LPS-binding

protein; CD14, CD14 surface receptor; TLR4, Toll like receptor 4;

IKK-NF-κB, IkB kinase-NFκB pathway; NF-κB-IκB, NF-kB-

inhibitory protein IκB; complex MAPK, mitogen-activated protein

kinase pathway.

for protecting sepsis mediated derangements, but the

application of these agents as drugs in humans has not been

fully successful.

The sources of ROS during sepsis are:

(1) the mitochondrial respiratory chain.

(2) the metabolic cascade of arachidonic acid.

(3) the protease-mediated enzyme xanthine oxidase.

(4) granulocytes and other phagocytes activated by

complement, bacteria, endotoxin, lysosomal enzymes,

etc.

(5) Other oxidases mainly NADPH oxidase.

Under normal physiological conditions, the majority of

ROS are formed during cellular respiration and by activated

phagocytic cells, including neutrophils, involved in the

inflammatory response. ROS have physiologically essential

roles in mitochondrial respiration, prostaglandin production

pathways and host defense [20]. The electron reduction of O

2

occurs in the mitochondrial electron transport system of all

aerobically respiring cells. The enzyme catalyzing this

20 Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1

Victor et al.

reaction (cytochrome c oxidase or Complex IV) contains the

transition metals iron and copper in its active site. These ions

can be paramagnetic and contain stable unpaired electrons.

By using the unpaired electrons in these transition metals to

control the O

2

reactions, mitochondria prevent the unwanted

release of ROS.

In sepsis, there are several potential sources of ROS,

including the mitochondrial respiratory electron transport

chain, xanthine oxidase activation as a result of ischemia and

reperfusion, the respiratory burst associated with immune

cell activation, arachidonic acid metabolism and NADPH

oxidase. In fact, activated immune cells produce O

2

-

as a

cytotoxic agent as part of the respiratory burst via the action

of membrane-bound NADPH oxidase on O

2

.

The increase of ROS after LPS challenge has been

demonstrated in different models of septic shock in

peritoneal macrophages and lymphocytes [12-13,19]. This

disturbance in the balance between pro-oxidants (ROS) and

antioxidants in favor of the former is characteristic of

oxidative stress in immune cells in response to endotoxin. In

this context, a typical behavior of these cells under an

oxidative stress situation implies changes in different

immune functions such as an increase in adherence and

phagocytosis and a decrease in chemotaxis [12-15,19].

Neutrophils play a crucial role in the primary immune

defense against infectious agents. They have several well-

established functions, which include phagocytosis and the

production of ROS. However, in addition to the microbicidal

activity, neutrophils can also damage the host by disrupting

tissue integrity and function. In fact, neutrophils are

activated and increased in number during sepsis as a means

to control infection, but may, also be destructive to the

tissue. Because of this dual role, targeting therapies for

sepsis may involve inhibiting or enhancing neutrophil

function.

Neutrophils and macrophages also produce NO, which

can react with O

-

2

to produce peroxynitrite (ONOO

-

), itself a

powerful oxidant, which may decompose to form HO

-

.

Under ischemic conditions followed by subsequent reperfu-

sion, the enzyme xanthine oxidase catalyzes the formation of

uric acid with the co-production of O

-

2

, whose release results

in the recruitment and activation of neutrophils and their

adherence to endothelial cells.

During oxidative stress, damage mediated by ROS can

occur. Oxidation of DNA and proteins takes place, along

with membrane damage due to lipid peroxidation, leading to

alterations in membrane permeability, modification of

protein structure and functional changes [21].

ANTIOXIDANT DEFENSES

Antioxidants are central to the redox balance in the

human body. They do not act in isolation, but synergistically

with other classes of molecules. Primary antioxidants

prevent oxygen radical formation, by either removing free

radical precursors or by inhibiting catalysis, e.g. the enzymes

glutathione peroxidase and catalase. Secondary antioxidants

react with ROS which have already been formed, either to

remove or inhibit them, e.g. vitamins C and E. Endogenous

antioxidant defenses exist in a number of locations, namely

intracellularly, on the cell membrane and extracellularly [5].

The immune system is highly reliant on accurate cell-cell

communication for optimal function, and any damage to the

signaling systems involved will result in an impaired

immune responsiveness. Oxidant-mediated tissue injury is a

particular hazard to the immune system, since phagocyte

cells produce ROS as part of the defense against infection.

Therefore, adequate amounts of neutralizing antioxidants are

required to prevent damage to the immune cells themselves.

For example, vitamin A deficiency can affect the function of

different cells in the immune system. Different studies have

reported defects in phagocytic activity (defect in chemotaxis,

adhesion and the ability to generate ROS in neutrophils) and

impairment of T and B cell function. In general,

improvement of immune function and increased resistance to

infection is observed in vitamin A-deficient hosts after

supplementation [22].

Several patents have described antioxidant treatment in

order to improve immune function by diet before or during

oxidative stress situations such as:

U.S. Pat. No. 4,981,844 [23] discloses a method of imp-

roving the immune response in patients comprising the

ingestion of a diet that provides 20-60 kilo calories per kg of

patient body weight and wherein 20-80% of the calories are

derived from linoleic acid. The Alexander et al. patent also

teaches the consumption of from 100-1000 IU per day of

vitamin E.

U.S. Pat. No. 5,556,644 [24] discloses a multi-nutrient

nutritional supplement designed to be effective in increasing

immunity and decreasing the instances and severity of

infection among the elderly. This patent specifically teaches

the consumption of a nutritional supplement having recited

levels of various vitamins and minerals. The patent more

specifically teaches the consumption of the nutritional

supplement by the elderly to improve their immunological

status.

U.S. Pat. No. 5,444,054 [25] discloses a nutritional

product for patients suffering from ulcerative colitis or

inflammation of the colon. The nutritional product utilizes an

oil blend containing specified fatty acids and a source of

indigestible carbohydrate. The indigestible carbohydrate is

disclosed as being metabolized to short-chain fatty acids by

microorganisms present in the human colon.

U.S. Pat. No. 5,223,285 [26] discloses a liquid nutritional

product that contains a specific lipid blend for pulmonary

patients. This patent discloses that the lipid should have a

particular ratio of n-6 to n-3 fatty acids. Further, this

reference describes a nutritional product containing

quantities of nutrients having antioxidative properties in

vivo. Examples of such antioxidative nutrients include β-

carotene, vitamin E, vitamin C, selenium and taurine.

U.S. Pat. No. 4,871,768 [27] describes a dietary supple-

ment that contains a structured glyceride comprising n-3

fatty acids and medium chain fatty acids. This patent

describes synthetic triglycerides or structured lipids that

provide a high energy fat source and fatty acids that assist in

fighting infection. This patent also describes a method of

minimizing the effects of infection and minimizing the

Antioxidants and Pathological Conditions

Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1 21

effects of subsequent infection by administering a diet

containing 10-80% by weight of an oil-based fraction

comprising glycerol, fatty acids and combinations thereof,

wherein 50-90% of the fatty acids are caprylic acid, capric

acid or mixtures thereof and 10-50% by weight of n-3 fatty

acids. This reference teaches that the dietary supplement will

not prevent the onset of infections, however, it will promote

survival of infected patients. This patent fails to suggest that

stress-induced down regulation of the immune system can be

lessened by a nutritional composition that comprises (1) a

structured glyceride; and (2) an antioxidant system

comprising of at least vitamin E, vitamin C, selenium and β-

carotene. Other patents have included similar compounds in

order to treat diabetes or other pathologies [28].

U.S. Pat. No. 4,607,052 [29] describes triglycerides

wherein specific polyunsaturated acyl fragments are present

at the sn-2 position of the glycerol molecule. The structured

lipids of Mendy et al. are described as being useful for the

treatment of lipid digestion problems, metabolic diseases,

nutritional deficiencies, hypertension and in conditions

where immune modulation is desired. There is neither

teaching nor suggestion in the Mendy et al. patent that a

structured glyceride, when combined with a specific

antioxidant system, would be effective in reducing the

immunosuppression typically seen in animal models

subjected to stress.

De Michele et al. [30] describes a patent directed to the

discovery of immunonutritional products that are useful in

reducing the immunological system suppression that results

from stress. The stress may be in the form of physical

exertion, mental exhaustion, disease states and the like. In

one embodiment, the invention relates to a nutritional

composition comprising a structured glyceride component

and an antioxidant system. This nutritional composition has

been shown to be highly effective in reducing immune

system downregulation or dysregulation as a result of stress.

As previously described, the SOD enzymes are a family

of metalloenzymes which rapidly promote the conversion of

O

2

-

to H

2

O

2

. Three forms of SOD are recognized to be

important: copper-zinc SOD (cytoplasmic-located), manga-

nese SOD (mitochondrial-located) and extracellular SOD

(extracellular matrix-located). Catalase and glutathione

peroxidase, a selenium containig enzyme which requires the

presence of reduced GSH for its action, both catalyze the

conversion of H

2

O

2

to H

2

O. GSH also has direct antioxidant

activity, through donation of hydrogen ions, to repair

damaged DNA. Oxidative stress and modulation on

GSH/GSSG (GSSG=oxidized GSH) levels also up-regulate

gene expression of several other antioxidant proteins, such as

manganese SOD, glutathione peroxidase, thioredoxin (Trx)

and metallothionein.

The hydrophobic lipid interior of membranes require a

different spectrum of antioxidants. Fat-soluble vitamin E (α-

tocopherol) is the most important antioxidant in this

environment. β-carotene, lycopene and co-enzyme Q have

also been implicated as membrane antioxidants. Lipid-

soluble antioxidants are important in preventing membrane

polyunsaturated fatty acids from undergoing lipid

peroxidation, which leads to loss of membrane integrity.

ROS may also be present in the extracellular

compartment, especially as a result of neutrophil activation.

The plasma and red cell components of blood both act as

antioxidants; red cells have a copper-zinc SOD-dependent

pathway for the inactivation of O

2

-

, and catalase and

glutathione peroxidase for dealing with H

2

O

2

. A number of

metal-binding plasma proteins function as valuable antioxi-

dants in addition to their transport roles, including

apotransferrin, lactoferrin and caeruloplasmin. Albumin is

also effective via its oxidizable thiol group, which permits

radical scavenging, and the binding of reactive transition

metal ions. A number of important smaller molecules are

present in the plasma, which act as secondary antioxidants.

These include vitamin E, vitamin C, uric acid and bilirubin.

Vitamin C interacts with O

-

2

to form dehydroascorbic acid.

Vitamin C may also reduce Fe

3+

to Fe

2+

, which can then be

involved in iron-catalyzed hydroxyl generation, thereby

implicating vitamin C as both pro-oxidant and an

antioxidant. Vitamin C exerts anti-inflammatory effects in

human and animal studies. Vitamin E is an important

nutrient for maintaining the immune system, and plays a role

in the differentiation of immature T cells in the thymus.

Many groups have investigated the possibilities of

quenching ROS production in different pathologies which

involve oxidative stress. In this regard, there are several

approximations to modulate arachidonic acid metabolism.

For example, the compounds heterocyclo-alkylsulfonyl

pyrazoles and sulfonyl heteroaryl triazoles have demonstra-

ted anti-inflammatory/analgesic properties [31]. Both

compounds are reported to control cycloxygenase (COX) in

different pathogenic situations.

Other approximations to control free radical production,

include compounds such as hydroxyguanidines which

control the enzyme xanthine oxidase in different pathogenic

situations that include sepsis as well as ischemic diseases,

hypoxia or arrhytmias [32].

In reference to another important source of free radicals,

NADPH oxidase, there are several patents which have

demonstrated excellent results to modulate this enzyme such

as o-methoxycatechol (apocynin) [33], as well as in another

method [34], another patent has demonstrated that

compositions derived from low molecular weight GTP-

binding proteins (LMWG), mastaparan, GAP proteins, and

related peptides can regulate the oxidative burst [35]. This

method contemplates compositions useful in inhibiting

activation of NADPH oxidase or in promoting GDP/GTP

exchange and therefore inhibits O

-

2

production in phagocytic

cells, reducing the severity of inflammation and

inflammatory disorders.

EFFECTS OF NITRIC OXIDE

NO is synthesized from L-arginine by different

isoenzymes of (NOS) [36], and is implicated in a wide range

of disease processes, exerting both detrimental and beneficial

effects at the cellular and vascular levels. To date, three main

isoforms of NOS are known: neuronal NOS (NOS-1 or

nNOS), inducible NOS (NOS-2 or iNOS), and endothelial

NOS (NOS-3 or eNOS).

NO has been shown to play a key role in the pathogenesis

of septic shock [37]. Hyperproduction of NO induces

22 Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1

Victor et al.

excessive vasodilation, changes in vascular permeability, and

inhibition of noradrenergic nerve transmission, all charac-

teristics of human septic shock.

The recogniton of NO production by activated

macrophages as part of the inflammatory process was an

important milestone for assesing both the biological

production of NO and the phenomenon of induction of NOS

activity [38]. The observation has been extended to

neutrophils [39], lymphocytes [40], and other cell types. The

role of NO in the pathophysiology of endotoxic shock was

advanced by Thiemermann and Vane [41], who observed

that administration of the specific NOS inhibitor N-methyl-

L-arginine (L-NMMA) decreased the severe hypotension

produced by administration of LPS. Other groups

simultaneously reported similar results indicating that

endotoxin increases NO production [42,43] and prompted

the idea that pharmacological inhibition of NOS may be

useful in the treatment of inflammation and septic shock

[44]. However, clinical trials using L-NMMA failed to show

a beneficial effect in septic shock patients [45]. The major

limitation for the use of NOS inhibitors in clinical studies is

the development of pulmonary hypertension as a side effect

of NOS blockade, which can be alleviated by the use of

inhaled NO. However, several compounds which modulate

NO synthesis have been patented in recent years, such as

various inflammatory mediators that have been implicated in

the induction and activation of iNOS, particularly IFNγ,

TNFα, IL-1β, and platelet-activating factor (PAF) alone or

synergistically [46,47]. In addition to the activation of iNOS,

cytokines and endotoxin may increase NO release by

increasing arginine availability through the opening of the

specific y

+

channels and the expression of the cationic

aminoacid transporter (CAT), or by increasing tetrahyd-

robiopterin levels, a key cofactor in NO synthesis. Several

experimental studies have demonstrated a decrease in NOS

activity resulting in an impairment in endothelial-dependent

relaxation during endotoxemia and experimental sepsis [48],

possibly as the result of a cytokine-or hypoxia-induced

shortened half-life of NOS mRNA [49], or of altered calcium

mobilization [50]. Other investigators have shown increased

endothelial NO release immediately after endotoxin

administration either directly [51] or indirectly as suggested

by the lack of effect of a specific iNOS inhibitor on the

initial hypotension after endotoxin administration.

NO exerts in vitro toxic effects including nuclear

damage, protein and membrane phospholipid alterations, and

the inhibition of mitochondrial respiration in several cell

types. Mitochondrial impairment could also be considered as

an adaptive phenomenon, decreasing cellular metabolism

when the energy supply is limited. The relevance of

mitochondrial impairment is, however, questionable, as

administration of the NO donor SIN-1 in a canine model of

endotoxic shock increased O

2

extraction capabilities [52].

The toxicity of NO itself may be enhanced by the formation

of ONOO

-

from the reaction of NO with O

-

2

[53]. Therefore,

the multiple organ failure syndrome (MOFS) that often

accompanies severe sepsis may be related to the cellular

effects of excess NO or ONOO

-

. In contrast, NO may protect

cells from oxidative damage by scavenging ROS [54].

NO has effects in vascular tone. During sepsis and

endotoxemia, convincing data suggest a pivotal role for NO

in the endotoxin-induced [51] vasodilation and vascular

hyporeactivity to vasoconstrictors. Both pharmacological

inhibition and iNOS gene deficiency are associated with a

loss of endotoxin-induced vasodilation [55]. The increased

NO release has also been implicated in the diminished

response to vasoconstrictors [56].

Some patents have been designed in order to modulate

the levels of NO and pro-inflammatory cytokines, such as

described by Singh et al. [57]. This invention first provides a

method for suppressing the induction of iNOS and/or

proinflammatory cytokines in a cell comprising contacting

said cell with an effective amount of at least one induction

suppressor and/or inhibitor of iNOS. Preferred cells

throughout the various embodiments of the invention are

lymphocytes, macrophages, endothelial cells, astrocytes,

masengial cells, myocytes, Kuffer cells, epithelial cells,

microglia, oligodendrocytes and neurons. Proinflammatory

cytokines that are preferred include TNF-alpha, IL-1β, IL-2,

IL-6, IL-8 and IFNγ. Inhibition of NO cytotoxicity includes

inhibition of iNOS activity, production of iNOS protein,

production or translation of iNOS mRNA, and inhibition of

LPS- or cytokine-induced NF-κΒ activation in a cell. In

preferred aspects of the invention, the induction suppressor

and/or inhibitor of iNOS and/or proinflammatory cytokines

may be selected from the group including, but not limited to,

lovastatin, mevastatin, FPT inhibitor II, forskolin, rolipram,

phenylacetate (NaPA), N-acetyl cysteine (NAC), pyrolidine

dithiocarbamate (PDTC), 4-phenylbutyrate (4PBA), 5-

aminoimidazole-4-carboxamide ribonucleoside (AICAR),

theophylline, papaverine, cAMP, 8-bromo-cAMP, (S)-

cAMP, and salts, analogs, or derivatives thereof.

Other patents have described amidino compounds useful

as NOS inhibitors in inflammation disorders [47]. Apart

from this, the following individual publications disclose

compounds that inhibit NO synthesis and preferentially

inhibit the iNOS [58-64].

INVOLVEMENT OF NITROGEN SPECIES

ROS and reactive nitrogen species (RNS) play an

important role in inflammatory processes as mediators of

injury. RNS is a collective name that includes NO, ONOO

-

,

nitrogen dioxide radical (NO

2

.

), other oxides of nitrogen and

products arising when NO reacts with O

-

2

, RO

.

and RO

2

. NO

reacts rapidly with ferrous iron, and at physiological

concentrations, NO also binds to soluble guanylate cyclase

and to another hemoprotein, cytochrome c oxidase (Complex

IV), the terminal enzyme of the mitochondrial respiratory

chain. NO can therefore control cellular functions via the

reversible inhibition of respiration [65]. There are a number

of reactive NO species, such as N

2

O

3

and ONOO

-

that can

also alter critical cellular components.

During the first hours after injury, iNOS-mediated NO

production is upregulated, producing a burst of NO that far

exceeds basal levels. This overabundance of NO produces

significant cellular injury via several mechanisms. First NO

may directly promote overwhelming peripheral vasodilation,

resulting in vascular decomposition; second, NO may

upregulate the transcription NF-κB initiating an inflamma-

Antioxidants and Pathological Conditions

Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1 23

tory signaling pathway that, in turn, triggers numerous

inflammatory cytokines [66]. NO also interacts with the O

-

2

to yield ONOO

-

, a highly reactive compound that

exacerbates the injury produced by either O

-

2

alone or NO

alone [67]. These investigators have proposed that the

ONOO

-

generation which occurs during fluid resuscitation in

the injured subject produces cellular death by enhancing

DNA single strand breakage, activating the nuclear enzyme

polyADP ribose synthetase (PARS), which in turn leads to

cellular energy depletion and cellular necrosis [68]. The

detrimental effects of ONOO

-

in shock and resuscitation

have been attributed to oxidation of sulfhydryl groups, the

nitration of tyrosine, tryptophane, and guanine, as well as

inhibition of the membrane sodium-potassium adenosine

triphosphatase. In addition, PARS activation depletes NAD

and thus alters electron transport, ATP synthesis, and

glycolysis; more recently, studies have shown that PARS

activates the caspase cascade, which results in DNA

fragmentation and cellular apoptosis [66,69].

The activation of monocytes, macrophages and

endothelial cells by LPS results in the expression of iNOS,

and consequently increases the transformation of L-arginine

to NO, which can combine with O

2

-

to form ONOO

-

, causing

tissue injury during shock, inflammation and ischemia-

reperfusion [70]. NO also stimulates H

2

O

2

and O

-

2

production by mitochondria [71], possibly by inhibiting

COX, thus increasing leakage of electrons from the

respiratory chain. H

2

O

2

, in turn, participates in the upregul-

ation of iNOS expression via NFκB activation [72]. Cultured

astrocytes and macrophages expressing iNOS produce up to

1 µM NO, and so it is possible that catalase in these cells

could be inhibited. Indeed, iNOS expression in activated

astrocytes leads to increased H

2

O

2

levels [73], as well as

inhibition of cellular respiration at the level of COX [74].

ONOO

-

has also been shown to stimulate H

2

O

2

production

by isolated mitochondria [75]. Paradoxically, NO can also

decrease ROS-produced damage that occurs at physiological

levels of NO [76]. The high reactivity of NO with radicals

might be beneficial in vivo, for example, by scavenging

peroxyl radicals and inhibiting peroxidation. Additionally, it

has been suggested that ONOO

-

may also be a signal

transmitter and can mediate vasorelaxation, similarly to NO

[77]. Apart from ONOO

-

, other RNS such as nitrogen oxides

and nitryl chloride are believed to cause several different

pathophysiological events, including inflammation [78].

Inflammatory reactions play an important role in

endotoxin-induced tissue injury. The latter is mediated by

adhesion and migration of leukocytes through the

endothelium, generation of ROS and RNS [79], and release

of several proinflammatory cytokines by monocytes/

macrophages. Local generation of RNS contributes to tissue

injury. Recent studies have demonstrated that activation of

the nuclear enzyme poly(ADP-ribose) polymerase-1 by

RNS-mediated DNA damage is an important pathway of

tissue injury in conditions associated with oxidative stress

[80]. Increased formation of RNS in response to endotoxin

challenge is organ specific [81].

Some authors have described different methods to avoid

the harmful effects of ONOO-. For example Szabo et al. [82]

have shown that mercaptoethylguanidine and guanidine

inhibitors of NOS react with ONOO and protect against

ONOO-induced oxidative damage.

CARDIOVASCULAR DYSFUNCTION

There is mounting evidence for involvement of oxidative

stress in the mechanism of cardiovascular disease (stroke,

congestive heart failure, hypertension and coronary artery

disease). Among the most numerous causes of mortality is

cardiac arrest. In relation to the heart, a principal deleterious

influence is arrhythmia, resulting from toxic action on

membranes that are concerned with cardiac conduction.

Impaired myocardial contractile function is a well-

documented feature in both experimental and human septic

shock [83] that greatly contributes to the mortality associated

with this pathological condition [84]. Several hypotheses

have been proposed to explain endotoxin-induced cardiovas-

cular failure, including microvascular dysfunction, the

presence of activated leukocytes, and the effects of various

circulating and/or locally produced proinflammatory

cytokines, such as TNFα and IL-1β [85]. Most of the

deleterious effects of proinflammatory cytokine release have

been attributed to the production of large amounts of NO,

which may exert detrimental effects on the myocardium in

animal models, isolated hearts, and isolated cardiac

myocytes [86]. Deleterious effects of NO have been mainly

reported in biological conditions in which ROS generation is

also enhanced [87]. The relevance of these biological events

in mediating myocardial dysfunction is further illustrated by

the finding that antioxidant therapy [79] and inhibition of

NO synthase activity can improve endotoxin and cytokine-

induced contractile dysfunction [79,88].

In the cardiovascular system, endothelium, which

separates the vascular wall from the circulation and the blood

components, plays a key role, because it regulates vascular

homeostasis by elaborating a variety of factors that act

locally in the blood vessel wall and lumen, such as NO,

prostacyclin and endothelin. Endothelium, in fact plays

important roles in maintaining a normal vascular tone and

blood fluidity, reduced platelet activity and leukocyte

adhesion, and limiting vascular inflammatory reactions.

In sepsis, NO may exert direct and indirect effects on

cardiac function. Sustained generation of NO occurs in

systemic inflammatory reactions, such as septic shock with

involvement in circulatory failure. In fact, myocardial iNOS

activity has been reported in response to endotoxin and

cytokines and inversely correlated with myocardial

performance [89].

Recently, several studies have added to the confusion

surrounding the role of NO by demonstrating no effect of

NO or NOS inhibition on the myocardium or on β-

adrenergic responsiveness [90]. Nevertheless, in most

studies, low-to-moderate doses of iNOS inhibitors restore

myocardial contractility in hearts exposed to proinflamma-

tory cytokines, whereas at higher doses, the effects are

reversed. This finding may indicate that small amounts of

NO produced by iNOS may be necessary to maintain

contractility [90] and can be cardio-protective in

experimental sepsis [91].

24 Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1

Victor et al.

Systemic sepsis is associated with cardiovascular dysfun-

ction through its effects on the myocardium, endothelium

and vascular smooth muscle [92]. Moreover, during septic

shock, ventricular function is altered despite normal or

increased cardiac output.

The number of endogenously produced inflammatory

mediators which can potentially contribute to septic

myocardial depression is extensive. The list includes

arachidonic acid metabolites, PAF, histamine, and endorp-

hins. Some evidence also suggests that ROS may increase

the left ventricular diameter and decrease left ventricular

contractility [93].

The most proposed mechanism implicated in the

pathogenesis of endothelial dysfunctions is related to an

increased production of ROS. This would deplete the

bioavailability of NO and exacerbate local oxidative stress

by directly reacting with NO to form ONOO

-

, which in turn,

would further sustain an oxidative injury to the endothelium.

In human neutrophils, ONOO

-

triggers the down-

regulation of L-selectin expression, and upregulation of

CD11/CD18 expression [94]. These effects are likely to be

mediated by the ability of ONOO

-

to trigger nuclear factor

NF-κB activation [95]. The development of ONOO

-

neutralizers has provided a more direct approach to assess

the role of ONOO

-

in organ injury in a variety of

inflammation states [82]. For example, mercaptoethylguani-

dine (MEG), a ONOO

-

scavenger and iNOS inhibitor [82]

and 5,10,15,20-tetrakis(4-sulfonatophenyl)-porphyrinato iron

(III) (FeTPPS), which catalyzes the isomerization of ONOO

-

to nitrate anion [96], may decrease the generation of highly

reactive intermediates such as nitrogen dioxide and OH

radicals. Besides, ONOO

-

neutralizers may attenuate inflam-

matory processes associated with ischemia reperfusion [97],

and infection [98]. Future investigations are necessary to see

the

effects

in

endotoxin-induced

cardiovascular

inflammation.

ROLE OF OXIDATIVE STRESS IN HEART FAILURE

The pathological mechanism of heart failure is complex

and involves the activation of numerous secondary pathways

(cytokines, oxidative stress and nitrosative stress) (Fig. 2),

which leads to: (i) abnormalities in various signaling

processes, cardiac receptors and Ca

2+

homeostasis; (ii)

contractile protein desensitization; and (iii) endothelial

dysfunction.

Experimental and clinical studies demostrate increased

production of ROS in the pathogenesis of acute heart failure.

In fact, malondyaldehyde (a marker of lipid peroxidation), is

increased in patients with ischemic cardiomyopathy.

The generation of ROS in the myocardium is triggered by

ischemia and reperfusion and by exposure of the heart to

inflammatory cytokines. Source of ROS in failing

myocardium includes, among others, xanthine and NADPH

oxidoreductases, the mitochondrial electron transport chain

and activated neutrophils [99].

The generation of ONOO

-

has been demonstrated in

acute heart failure. In fact, the pathogenesis of endothelial

dysfunction associated with acute heart failure involves

enhanced oxidative stress from various local sources. A

potential additional source of ROS is iNOS, which,

paradoxically, can produce O

2

-

.

Fig. (2). Progression of heart failure and the role of oxidative and nitrosative stress.

Antioxidants and Pathological Conditions

Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1 25

There are several mechanisms of ONOO

-

-induced cardiac

and vascular dysfunction (Fig. 3). For example, ONOO

-

-

induced tyrosine nitration can lead to dysfunctional nitrated

proteins, whereas ONOO

-

-induced oxidation of sulfydryl

groups can inhibit important enzymes in the mitochondrial

respiratory chain. Furthermore, ONOO-modified cellular

proteins are subject to accelerated degradation via the

proteasome and ONOO

-

inhibits a variety of ion pumps

[100]. ONOO

-

also potently oxidizes various molecules

including BH

4

, a crucial cofactor of NOS, to quinoid 5,6-

dihydropterin. The generation of ONOO

-

also decreases the

availability of NO for G-protein stimulation and vasodila-

tion, thus further contributing to endothelial dysfunction.

In addition, ONOO

-

can inhibit SOD, glutaredoxin and

other antioxidant molecules and systems, which leads to

positive feedback cycles of intracellular oxidant generation

and oxidative injury [100]. ONOO

-

can also activate matrix

metalloproteinases (MMPs) [101] and the nuclear enzyme

poly(ADP-ribose) polymerase (PARP) [102]. ONOO

-

also

triggers the expression of P-selectin, ICAM-1 and Mac-1,

and mediates the cytokine-induced expression of IL-8 in

human leukocytes via enhancement of NF-kB activation

[100], thereby promoting proinflammatory responses.

Some patents have been designed in order to modulate

cardiovascular damage, by supplementing with antioxidants.

For example, Zeng et al. (2004) [103], describe an invention

with antioxidant composition comprising the constituents of

α-thioctacid and vitamin E, which prevents cardiovascular

and cerebrovascular diseasess. Other patents describe

inventions which include an antioxidant cocktail designated

to treat and prevent heart disease and stroke [104-106].

Besides heart failure, ROS and vascular oxidative stress

have been involved in hypertension and atherogenesis.

Compelling experimental evidence indicates that ROS play

an important pathophysiological role in the development in

atherogenesis and therefore in hypertension. This is due, in

large, to oxidative stress and decreased NO bioavailability in

the vasculature and to ROS-mediated cardiovascular

remodeling.

Atherosclerosis, a chronic inflammatory disease of the

arterial wall, is a major cause of morbidity and mortality

from cardiovascular disease in much of the world’s

population. For this reason, we would like to mention some

important patents in the treatment of these pathologies. For

example, Morawalla et al. [107] describes the medicinal

properties of Terminalia arjuna in alleviation of various

health related disorders, showing cardio-protective

principles. In other patent, De Simone [108] describes an

antioxidant combination composition to protect from

oxidative stress in cardiovascular damage. The composition

includes: L-carnitine inner salt, acetyl L-carnitine inner salt,

α-lipoic acid, coenzyme Q10, vitamin E and

selenomethionine.

Fig (3). Role of oxidative stress in oxidant-induced cellular dysfunction and necrosis. Nitric oxide (NO) and superoxide (O

2

-

), induce cell

damage via lipid peroxidation, inactivation of enzymes by oxidation and nitration. Peroxynitrite (ONOO

-

) also acts on mitochondria

decreasing the membrane potential (Ψ), triggering the release of pro-apoptotic factors such as cytochrome c (Cyt c) and apoptosis-inducing

factor (AIF). These factors mediate caspase-dependent and caspase-independent apoptotic death pathways, respectively. Moreover,

peroxynitrite, causes strand breaks in DNA.

26 Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1

Victor et al.

ANTIOXIDANT PROTECTION IN SEPSIS

It has been reported that sepsis increases markers of

oxidative stress in different animal models and in humans.

This effect is correlated with an imbalance of antioxidant

amount [109]. The clear evidence for oxidative stress in

sepsis and the link with inflammatory gene expression has

provided a foundation for intervention to either reduce

oxidative stress or inhibit transcriptional activation. For this

reason, the protective role of antioxidants against endotoxin

toxicity and their potential clinical use is discussed.

Endogenous and synthetic antioxidants, such as iron-

chelating agents, N-acetyl-L-cysteine (NAC), apocynin,

probucol, and edaravone, are useful in preventing

cardiovascular injury and diseases. Endogenous antioxidants

in the myocardium include vitamins C and E, cysteine and

the enzymes, SOD, catalase and glutathione peroxidase. In

cases where events such as ischemia and hypoxia have

compromised the cardiac cell’s natural defense mechanisms,

synthetic antioxidants such as NAC, a thiol containing

compound, have proved successful in protection against

oxidative stress on reperfusion.

Secondary plant metabolites containing flavonoids and

coumaric acid derivatives from spinach are effective against

oxidative stress in the rat heart [79]. Targets for antioxidant

therapy include NADPH oxidase, a source of O

-

2

and

peroxides.

GSH comprises more than 90% of the nonprotein low

molecular weight reduced thiols in cells and is one of the

most important intracellular mechanisms protecting normal

cells from oxidative injury. GSH linked redox homeostasis

controls the levels of ROS that are constantly formed during

cellular metabolism. It is recognized that the antioxidant

GSH plays a very important role in several biological

processes, and is implicated in the modulation of immune

function and inflammatory response [110].

One of the treatments to enhance the GSH pool is NAC

supplementation, which, of course, also has direct

antioxidant activity [111]. NAC has been used safely to treat

acetaminophen overdose for two decades [112]. NAC shows

an in vitro and in vivo stimulatory effect on the phagocytic

process of macrophages, and controls the functions of

lymphocytes [113-116]. The regulation of redox state by

NAC in immune cells and in animals with endotoxic shock

[117], can lead to decreased NFκB activation [118], TNF-α

release and increased survival [119].

In the clinical setting, NAC alone or in combination with

other antioxidants has been shown to have variable results.

An early acute study, in which NAC was administered in

conjunction with vitamin C and α-tocopherol in patients with

septic shock, did not measure NFκB activation, and neither

total antioxidant capacity nor lipid peroxidation was changed

[120].

Victor et al. [117] demonstrated that NAC treatment in

animals with endotoxic shock decreased ROS, TNF, MDA

levels, the GSSG/GSH ratio and iNOS levels, and increased

the antioxidant defenses in peritoneal macrophages and

lymphocytes. Moreover, NAC treatment prevented the

activation of NF-κB, improved the redox state of these

immune cells and increased survival.

Byrne et al. [121] have demonstrated the therapeutic

antioxidant effects of glutamine and NAC composition.

Compositions comprising glutamine in combination with

other ingredients such as NAC are disclosed. Such

compositions can be administered to treat patients with

certain disease states. In fact, glutamine and other amino

acids have been given to patients with immune-compromised

states, both alone and in combination with an immuno-

potentiator such as levitinan, OK-432, or sizofiran [122].

Vitamin C is a powerful electron donor, reacting with both

O

-

2

and OH. Vitamin C plays an important role in the

defence against oxidative damage especially in leukocytes.

The main role of vitamin C in the organism is linked to its

function as a reductor, but it also participates in the

modulation of complex biochemical pathways which are an

essential part of the normal metabolism of immune cells.

This antioxidant also inhibits the activation of the nuclear

transcription factor NF-κB produced by endotoxin [123],

which could result in a decrease of TNFα. These results

suggest that early administration of vitamin C may be a

valuable adjunct treatment in sepsis. Although several

investigations revealed no association between vitamin C

intake and lower risk of coronary disease, high doses of this

supplement are reported to improve vascular function in

some cases.

Cardiovascular disease is the leading cause of death in

the United States and many other countries. Nutritional

factors are widely recognized as playing a role in preventing,

delaying the onset of and/or slowing the progression of

atherosclerosis and coronary heart disease. Attempts have

been made in the prior art to design multivitamin

supplements specifically for heart health. For example, U.S.

Pat. No. 5,770,215 [124] discloses, a multivitamin composi-

tion containing various vitamins, minerals, and acetyl-

salicylic acid. Boulos et al. [125] have recently paten-ted a

multivitamin/multimineral optimized for cardiovascular

health containing increased levels of Vitamin E and Folic

Acid, low levels of iron, as well as containing mixed

carotenoids.

There have been several studies which have reported low

circulating vitamin E (α-tocopherol) levels in sepsis.

Vitamin E has been shown to have several additional

biologically important effects apart from its role as an

antioxidant. These include the inhibition of arachidonic acid

oxidative metabolism and the inhibition of protein kinase C

(PKC) activity. A steady decrease in circulating α-

tocopherol levels in plasma was reported in a pig septic

shock model in both survivor and non-survivor animals. This

was accompanied by a simultaneous increase of markers of

oxidative injury in the animals which subsequently died

[126]. Vitamin E has been shown to be protective in

reducing the effects of oxidative stress in sepsis in a number

of animal studies [127]. Taken together, these studies reveal

vitamin E to be a potent immunomodulator [128] agent that

can interrupt the activation of inflammatory cells at the level

of signal transduction.

One strategy for preventing oxyradical-induced damage

is to inhibit the formation of oxyradicals such as O

-

2

. Iron

Antioxidants and Pathological Conditions

Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1 27

chelators, such as desferrioxamine (also called deferoxamine

or Desferol) and others, inhibit iron ion-dependent OH

generation and thus act as inhibitors of free radical formation

[129]. Amino-steroid-based antioxidants such as the 21-

aminosteroids termed "lazaroids" (e.g., U74006F) have also

been proposed as inhibitors of ROS formation. Desfer-

rioxamine, allopurinol, and other pyrazolopyrimidines such

as oxypurinol, have also been tested for preventing

oxyradical formation in a myocardial stunning model system

and following hemorrhagic and endotoxic shock [130].

However, each of these compounds has notable drawbacks

for therapeutic usage. For example, deferoxamine is not an

ideal iron chelator and its cellular penetration is quite

limited.

Another strategy for preventing oxyradical-induced

damage is to catalytically remove oxyradicals such as

superoxide once they have been formed. SOD and catalase

have been extensively explored, with some success, as

protective agents when added to reperfusates in many types

of experiments or when added pre-ischemia [131]. The

availability of recombinant SOD has allowed more extensive

evaluation of the effect of administering SOD in the

treatment or prevention of various medical conditions

including reperfusion injury of the brain and spinal cord

[132], endotoxemia [133], and myocardial infarction [134],

and for osteoarthritis and intestinal ischemia [135]. SOD also

has been reported to have positive effects in treating

systemic lupus erythematosus, Crohn's disease, gastric

ulcers, oxygen toxicity, burns patients, renal failure attendant

to transplantation, and herpes simplex infection.

An alternative strategy for preventing oxyradical-induced

damage is to scavenge oxyradicals such as O

2

-

once these

have been formed, typically by employing small molecule

scavengers which act stoichiometrically rather than

catalytically. Congeners of GSH have been used in various

animal models to attenuate oxyradical injury. For example,

N-2-mercaptopropionylglycine has been found to confer

protective effects in a canine model of myocardial ischemia

and reperfusion [136] and NAC ("Mucomyst") has been used

to treat endotoxin toxicity in sheep [137]. Dimethyl thiourea

(DMTU) and butyl-phenylnitrone (BPN) are believed to

scavenge OH, and have been shown to reduce ischemia-

reperfusion injury in rat myocardium and in rabbits [138].

Mannitol has also been used as a free radical scavenger to

reduce organ injury during reoxygenation [139]. Thus,

application of inhibitors of oxyradical formation and/or

enzymes that remove O

-

2

and H

2

O

2

and/or small molecule

oxyradical scavengers have all shown promise for preventing

reoxygenation damage present in a variety of ischemic

pathological states and for treating or preventing various

disease states associated with free radicals. However, each of

these categories contains several drawbacks. For example,

inhibitors of oxyradical formation typically chelate transition

metals which are used in essential enzymatic processes in

normal physiology and respiration; moreover, even at very

high doses, these inhibitors do not completely prevent

oxyradical formation. SOD and catalase are large

polypeptides which are expensive to manufacture, do not

penetrate cells or the blood-brain barrier, and generally

require parenteral routes of administration. Free radical

scavengers act stoichiometrically and are thus easily depleted

and must be administered in high dosages to be effective.

The complex formed between the chelator desfer-

roxamine and manganese has SOD activity and has shown

some activity in biological models, but the instability of the

metal ligand complex apparently precludes its pharmaceu-

tical use. Porphyrin-manganese complexes have been shown

to protect bacteria from paraquat toxicity and to promote the

aerobic survival of SOD-deficient E. coli mutants.

Based on the foregoing studies, it is clear that a need

exists for antioxidant agents which are efficient at removing

dangerous oxyradicals, particularly O

-

2

and H

2

O

2

, and which

are inexpensive to manufacture, stable, and possess

advantageous pharmacokinetic properties, such as the ability

to cross the blood-brain barrier and penetrate tissues. Such

versatile antioxidants would find use as pharmaceuticals,

chemoprotectants, and possibly as dietary supplements.

In fact, for example Malfroy-Camine et al. [140]

describes synthetic catalytic free radical scavengers useful as

antioxidants for prevention and therapy of disease. In this

patent, they provide antioxidant salen metal complexes,

compositions of such antioxidant salen-metal complexes

having O

-

2

activity, catalase activity, and/or peroxidase

activity, compositions of salen-metal complexes in a form

suitable for pharmaceutical administration to treat or prevent

a disease associated with cell or tissue damage produced by

free radicals such as O

-

2

, and cosmetic and free radical

quenching formulations of salen metal compounds.

Therefore, many groups of researchers have focused their

attention on the biological activity of antioxidants and its

active principles. Many scientific articles are published every

year in different international journals related to the

pharmacological properties of new antioxidants in order to

counteract the effect of ROS in different pathologies. For

example, Rao et al. have synthesized and used antioxidants

such as (-)-mesquitol from natural sources (Dichrostachys

cinerea), as well as (-)-Olivil from the plant Stereospermum

personatum. Both compounds have demonstrated antioxi-

dant, activity [141,142]. Other studies have implicated

mitochondria-targeted antioxidants which have also been

shown to modulate the role of mitochondrial ROS

production in redox signaling pathways [143].

CURRENT AND FUTURE DEVELOPMENTS

In an approach to design more optimally effective

antioxidant regimes by targeting cells rather normal systemic

route, Michael Murphy et al. have recently embarked on a

program of work to deliver antioxidant moieties (for

example vitamin E and ubiquinone) specifically to the

mitochondrion, the major site of oxidative stress [144]. By

using a lipophilic cationic group as a carrier, to facilitate

targeting to the highly charged mitochondrion, Murphy et al.

have shown that in principle, this method is very effective at

combating oxidative stress in a variety of in vitro scenarios

such as NFκB activation [145], Friedreich Ataxia

complications [146], and stress-related teleomere shortening

[147]. The first in vivo demonstration (proof-of-principle) of

mitochondrially-targeted antioxidants in the area of cardiac

ischemia reperfusion was recently reported [148]. These

successes now open the doorway for novel treatments for

28 Recent Patents on Anti-Infective Drug Discovery, 2006, Vol. 1, No. 1

Victor et al.

clinical conditions in which oxidative stress plays a major

role [149].

In this regard, the patent WO9926582A2 [150] relates to

antioxidants having a lipophilic cationic group and to uses of

these antioxidants, for example, as pharmaceuticals. This can

be to prevent the elevated mitochondrial oxidative stress

associated with particular diseases, or diseases associated

with mitochondrial DNA mutations. Accordingly, they have

the potential to be used as an adjunct to cell transplant

therapies for e.g. neurodegenerative diseases, as a means to

increase survival rates of the implanted cells. In addition,

these compounds could be used as prophylactics to protect

organs during transplantation, or ameliorate the ischemia-

reperfusion injury that occurs during surgery. The

compounds of the invention could also be used to reduce cell

damage following stroke and heart attack. As these therapies

use the selectivity of mitochondria, one can forsee that a

major advantage over current antioxidant therapies is that the

antioxidant is taken to the cellular compartment under the

greatest oxidative stress. It is thought that this should

increase the efficacy of the therapy.

In conclusion, antioxidants are involved in several

important biological processes such as immunity, protection

against tissue damage, reproduction, growth and develop-

ment. Antioxidants preserve adequate function of cells

against homeostatic disturbances such as processes involving

oxidative stress such as septic shock and cardiovascular

dysfunction. Many groups of researchers have focused their

attention on the biological activity of antioxidants and its

active principles. The protective role of antioxidants against

homeostatic disturbances such as those caused by endotoxin

toxicity and cardiovascular damage have been patented, their

potential clinical use and the effects on the redox state of

have been discussed.

Although there are a large number of patents which

describe beneficial effects of antioxidant treatment in several

oxidative stress disorders, in general, the results obtained so

far are slightly disappointing. From our point of view, the

use of novel targeted antioxidants, such as those described

by Murphy et al. appears to be an exciting new leap into the

use of antioxidant therapy.

ACKNOWLEDGEMENTS

This work was supported by grant CP03/00024 (FIS)

from Instituto Salud Carlos III, GV2004-A-147 from

Generalitat Valenciana and RECAVA network. Victor M

Victor is a recipient of a Contrato-Investigador FIS from

Instituto Salud Carlos III. KJ McCreath is a recipient of

Contrato Ramon y Cajal.

COMPETING INTERESTS STATEMENT

The authors declare that they have no competing

financial interests.

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