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Astaxanthin: A Novel Potential Treatment for Oxidative Stressand Inflammation in Cardiovascular Disease

Fredric J. Pashkow, MD,a,b,* David G. Watumull,b and Charles L. Campbell, MDc

Oxidative stress and inflammation are implicated in several different manifestationsof cardiovascular disease (CVD). They are generated, in part, from the overproduc-tion of reactive oxygen species (ROS) and reactive nitrogen species (RNS) thatactivate transcriptional messengers, such as nuclear factor–�B, tangibly contributingto endothelial dysfunction, the initiation and progression of atherosclerosis, irrevers-ible damage after ischemic reperfusion, and even arrhythmia, such as atrial fibrilla-tion. Despite this connection between oxidative stress and CVD, there are currentlyno recognized therapeutic interventions to address this important unmet need. An-tioxidants that provide a broad, “upstream” approach via ROS/RNS quenching orfree radical chain breaking seem an appropriate therapeutic option based on epide-miologic, dietary, and in vivo animal model data. However, human clinical trials withseveral different well-known agents, such as vitamin E and �-carotene, have beendisappointing. Does this mean antioxidants as a class are ineffective, or rather thatthe “right” compound(s) have yet to be found, their mechanisms of action under-stood, and their appropriate targeting and dosages determined? A large class ofpotent naturally-occurring antioxidants exploited by nature—the oxygenated caro-tenoids (xanthophylls)— have demonstrated utility in their natural form but haveeluded development as successful targeted therapeutic agents up to the present time.This article characterizes the mechanism by which this novel group of antioxidantsfunction and reviews their preclinical development. Results from multiple speciessupport the antioxidant/anti-inflammatory properties of the prototype compound,astaxanthin, establishing it as an appropriate candidate for development as a thera-peutic agent for cardiovascular oxidative stress and inflammation. © 2008 Elsevier
Inc. All rights reserved. (Am J Cardiol 2008;101[suppl]:58D– 68D)
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therosclerosis is an inflammatory disease of the arterialall that remains a principal cause of death and disability,espite the application of statin and antiplatelet therapies.he severe clinical manifestations of the disease—myocar-ial infarction (MI) and stroke—are mainly caused by thebrupt obstruction of the vessel lumen by a thrombus trig-ered by the rupture or erosion of an atheroscleroticlaque.1 Existing data strongly suggest that immunoinflam-atory–related mechanisms are the major determinants of

hese atherothrombotic plaque sequelae.2 Thus, most of themportant advances in the comprehension of the mecha-isms of atherothrombosis come from studies of the criticalomponents involved in the modulation of the immunoin-ammatory balance within the plaque. Despite an increas-

ng understanding of these processes, there have been nopproved therapeutic interventions in vascular biology that

aJohn A. Burns School of Medicine, University of Hawaii, Honolulu,awaii, USA; bCardax Pharmaceuticals, Inc., Aiea, Hawaii, USA; and

Gill Heart Institute, University of Kentucky, Lexington, Kentucky, USA.Statement of author disclosure: Please see the Author Disclosures

ection at the end of this article.*Address for reprints: Fredric J. Pashkow, MD, 99-193 Aiea Heights

rive, Suites 400, Aiea, Hawaii 96701.
lE-mail address: [email protected].
002-9149/08/$ – see front matter © 2008 Elsevier Inc. All rights reserved.oi:10.1016/j.amjcard.2008.02.010

ully incorporate the current understanding of oxidativetress and inflammation.3

ole of Reactive Oxygen and Nitrogen Speciesn Cardiovascular Inflammation

eactive oxygen species (ROS) and reactive nitrogen spe-ies (RNS) are well recognized for functioning as bothotentially harmful and beneficial cell-signaling molecules.ormally generated by tightly regulated enzymes, such asicotinamide adenine dinucleotide phosphate oxidaseNADPH) and nitric oxide synthase (NOS), excessivend/or chronic overproduction of ROS/RNS either from theitochondrial electron transport chain or various otherOS/RNS– generating enzymes, NADPH or NOS, results inxidative stress, a harmful process that can be an importantource of damage to cellular components, including lipids,roteins, and DNA. In contrast, beneficial effects of ROS/NS (eg, superoxide radical and nitric oxide) occur tran-

iently at low-to-moderate concentrations and mediatehysiologic roles in cellular responses to oxygen depriva-ion: defense against infectious agents, modulation of cel-ular signaling pathways, and the induction of cellular pro-
iferation. Paradoxically, various ROS-mediated actions, in
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59DPashkow et al/Astaxanthin: A Novel Potential Treatment for Oxidative Stress and Inflammation in Cardiovascular Disease

act, may protect cells against ROS-induced oxidative stressnd reestablish or maintain “redox balance” or “redox ho-eostasis.”4

In postischemic myocardium, elevated levels of exoge-ous ROS are generated in cardiomyocytes, endothelialells, and infiltrating neutrophils that can lead to cellularysfunction and necrosis. ROS most likely contribute to theathogenesis of MI and serve as mediators of the reversibleentricular dysfunction (stunning) that often accompanieseperfusion of ischemic myocardium.

xidative Stress and Endothelial Dysfunction Areommon among Risk Factors and All Manifestationsf Cardiovascular Disease

uring the past 25 years, the literature has established theivotal role of the endothelium in preserving vascular ho-eostasis. Nitric oxide produced in endothelial cells and plate-

ets is believed to be the main factor responsible for endo-helial functional integrity (ie, vasorelaxation). Reduceditric oxide bioavailability causes so-called “endothelialysfunction,” which constitutes the molecular dysfunctionommon to both the “stable” lesions of atherosclerotic nar-owing and acute plaque rupture leading to unstable anginar MI. Substantial evidence is accumulating on the role ofxidative stress in reducing nitric oxide bioavailability andubsequent increased endothelial dysfunction as well aslterations of cell signaling pathways. The role of endothe-ial cell apoptosis as a possible end stage of endothelialysfunction and plaque disruption has also been proposed.n vitro and in vivo evidence suggest a role of oxidativetress as a putative mechanism, ultimately leading to plaquerosion and activation through increased endothelial cellpoptosis. Thus, oxidative stress, regardless of the stage oftherosclerotic disease, likely represents a key mechanismn vascular disease progression and a possible clinical targetor prevention.5–7

mportance of the Nuclear Factor–�Branscription Family

he nuclear factor–�B (NF-�B)/Rel pathway has beenhown to be critical to the inflammatory process and worthyf targeting for medicinal intervention. When activated byignals sourced from pathogens, oxidative stress, or otherhysiologic stresses, the NF-�B/Rel family of effector pro-eins translocate to the nucleus, resulting in the increasedranscription of proinflammatory genes.8 As a molecularexus point in the inflammatory process, NF-�B is fre-uently found to be activated at sites of inflammation iniverse diseases and is particularly important in cardiovas-ular disease (CVD), where increased transcription of proin-ammatory cytokines, chemokines, cell adhesion mole-
ules, matrix metalloproteinases, cyclooxygenase-2, and t
nducible nitric oxide synthase play a critical role in theisease etiology.9

Important activators of NF-�B and oxidative stress arelosely associated with many of the various risk factors fortherosclerosis: hypercholesterolemia, diabetes mellitus andts associated advanced glycation end products, uncon-rolled hypertension, and elevated plasma homocysteineevels. Activated NF-�B has been identified in the smooth

uscle cells, macrophages, and endothelial cells of humantherosclerotic lesions. Additionally, enhanced activation ofhis transcription factor has been shown to occur very earlyfter administration of a high-fat diet in the low-densityipoprotein (LDL) receptor–deficient atherosclerotic mouseodel.10 In patients with unstable angina, enhanced levels

f oxidized LDL increased NF-�B activation, suggestingies between oxidative stress and inflammation in this path-ay.11

otential Impact of Upstream Reduction innflammatory Activity and the Potential Significancef Balanced Reactive Oxygen Species

nzymatic antioxidants (eg, superoxide dismutase and glu-athione peroxidase, and chemical antioxidants, such asarotenoids, vitamin E, etc.) scavenge or quench excessipid and aqueous phase ROS/RNS, allowing signaling mol-cules and cellular pathways, such as NF-�B, to operateormally in homeostasis with the associated downstreamroducts from these pathways, which results in a balancedOS/RNS redox environment. However, in response toommonly encountered inflammatory stimuli (eg, cyto-ines, pathogens, etc.), ROS/RNS production transientlyncreases dramatically, temporarily overwhelming normalellular enzymatic and chemical antioxidants. Cell signalingathways are now activated, which in the case of NF-�Besults in large increases in the previously normal levels ofownstream inflammatory mediators, enzymes, and cyto-ines, such as cyclooxygenase-2, prostaglandin E2, and tu-or necrosis factor–� (TNF-�), which at their higher levels

ow drive potentially curative inflammation (Figure 1).owever, in the case of many prolonged disease patholo-ies, including many forms of CVD, ROS/RNS levels arehronically elevated, resulting in a state of prolonged oxi-ative stress and inflammation because of the depletion ofatural and dietary antioxidants, ultimately leading to aersistently malevolent redox homeostasis.

onnexins, Gap Junction Communication, andardiovascular Disease

ap junction proteins called connexins mediate gap junc-ion intercellular communication (GJC) and are a group of

20 highly conserved proteins with developmental and
issue-specific expression patterns. GJC allows the direct

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60D The American Journal of Cardiology (www.AJConline.org) Vol 101 (10A) May 22, 2008

ransmission among neighboring cells of ions, small hydro-hilic metabolites, and messengers �1–2 kDa in size. GJClays an important role in normal development and physi-logy, with a loss of function implicated in various humaniseases and pathologies.

In the heart, electrical activation of the myocardium isecessary to produce effective pumping of blood and de-ends on the orderly coordinated spatial and temporal trans-er of current (ions) from one cell to another. In normalentricular myocytes, this is accomplished through exten-ive cell–cell coupling via gap junctions. Cells have theapacity to rapidly regulate the function (posttranslationalegulation), quantity (transcriptional regulation), and com-osition (assembly regulation) of connexins within the gapunctional structures to accommodate physiologic demandsor altered cell–cell communication. Specifically, connexin3 is the most ubiquitously expressed connexin in tissuesnd is essential for normal cardiac function and contrac-ion. Connexin 40, a related protein, is primarily local-

igure 1. Balanced reactive oxygen species (ROS) environment and nopregulate the production of downstream inflammatory mediators, includyclooxygenase-2 (COX-2), tumor necrosis factor–� (TNF-�), interleukin-1f appropriate amounts of signaling molecules to operate normally alonnvironment and normal cellular health. (Courtesy of Cardax Pharmaceut

zed in the atria and exhibits altered levels and localiza- t

ion in cardiac tissue after atrial fibrillation. Figure 2A 12

hows a schematic of the hexameric oligomerizationf connexins to assemble a connexon (gap junctionemichannel) that when paired with a connexon from andjoining cell, forms a water-filled channel allowingransfer of ions and molecules via GJC. Figure 2B depictshe transmembrane organization of individual connexinroteins in the plasma membrane.

This communication infrastructure is involved in theathophysiology of multiple cardiovascular disorders.13–15

onnexin levels and localization during cardiac tissue re-odeling after atrial fibrillation has been a focus of recent

nterest and thus constitutes a plausible therapeutic targetor the treatment of dysrhythmias.16,17 In mice, genetic de-letion of connexin 43 localized in both mitochondrial andlasma membranes abolishes the protection of ischemia andiazoxide-induced preconditioning.

Mechanistically, this tissue protective effect stems fromOS formation in response to the transport of diazoxide to

llular health. Transcription factors, such as nuclear factor–�B (NF-�B)ucible nitric oxide synthase (iNOS), matrix metalloproteinases (MMPs),�), and others. During normal cellular function, ROS allow the productionthe downstream products of these pathways, creating a balanced ROSc.)

rmal ceing ind� (IL-1g withicals, In

he inner mitochondrial membrane.18 Mitochondrial con-

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exin 43 content is also decreased in aged mouse hearts, andeduced levels of connexin 43 may contribute to the loss ofardioprotection.19 Several carotenoids have been shown topregulate connexin 43 protein levels and GJC.20

ingle Pathway/Enzyme Drug Targeting versusroader Upstream Therapeutic Approaches

espite the association among oxidative stress, inflam-ation, and CVD, no therapies have been successfully

erified through randomized clinical trials.21 Individuallock and key” therapies targeted to specific pathwaysnd proteins, such as kinases, although mechanisticallyttractive, may produce off-target side effects or, whenhe obstruction of a pathway is complete, potentiallyevere drug-related adverse events. Free radical–scav-nging antioxidants may reduce the overall burden ofOS/RNS and a broad upstream downregulation of oxi-ative stress, but studies examining the pharmacologicotential of antioxidants that show promise in epidemi-logy and in vivo animal studies have failed in prospec-ive human trials. A better understanding of therapeuticode of action, uptake, distribution, pharmacokinetics,

nd metabolism of all proposed antioxidants is a prereq-isite to the successful discovery and development of

igure 2. Organization of connexins in the plasma membrane. ConA) Diagrammatic cross-section through an area of cell–cell contact cohospholipid bilayer in the plasma membrane (blue). Each cell contributesection (foreground). (B) A single connexin molecule traverses the plasmassemble to form a connexon by forming 3 sulfhydryl bonds between thonnexon is bound by 18 sulfhydryl bonds to produce a tight seal blockedicine.12)

herapeutic agents combating oxidative stress. d

embrane Alignment of Antioxidant Compoundsredicts Activity

ecent work by McNulty et al22 suggests that conforma-ional differences in membrane alignment of antioxidantsay help explain their biologic activity and disparate study

esults. In particular, the antioxidant, anti-inflammatory ac-ivity of these molecules appears, in part, to be a conse-uence of their precise transmembrane alignment in theipid bilayer of cellular membranes. In addition to prevent-ng lipid-based oxidation, this alignment provides a varia-ion in the ability of the hydrophilic biophores in the endieces to be adequately exposed to interact with reactivepecies in the aqueous environment, potentially facilitatingOS shuttling along the double bonds of the carbon back-one of the compound (Figure 3). The transmembranelignment of the molecule also likely provides proximity toofactors, such as vitamin C, which serve as a sink forccepting the radical cations, effectively recharging thelectron transfer capacity.23

he Xanthophyll Carotenoid Astaxanthin Targetsxidative Stress

he most effective compound in these membrane positionaltudies is the xanthophyll carotenoid astaxanthin. A red-

roteins organize into connexon cylinders in the plasma membrane.g gap junctions. Connexin proteins are shown (yellow) traversing theexins to form a cylinder enclosing a central water-filled pore seen in crossne 4 times with both N- and C-terminal ends in the cytoplasm. Connexinsconserved cysteine residues present in each opposing loop. Thus, eachentry of extracellular ions, such as Ca2�. (Adapted from Science and

nexin pntainin6 connmembrae highlying the

ish-colored C-40 compound, astaxanthin is a powerful

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road-ranging antioxidant that occurs naturally in a wideariety of living organisms, such as microalgae, fungi, com-lex plants, and crustaceans.24 It is a quencher of ROS/RNSingle- and 2-electron oxidants as well as a chain-breakingcavenger of free radicals. The potent antioxidant activity ofstaxanthin has been observed to modulate biologic func-ions ranging from lipid peroxidation to tissue protectiongainst light damage.22,25 A water-dispersible astaxanthinerivative has demonstrated efficacy in both in vitro and inivo experimental animal models.26 After an extensive USood and Drug Administration (FDA) review of its safetyrofile, a synthetic, racemic form of astaxanthin was ap-roved as an animal food additive in 1987 (Roche Vitamins;SM, Heerlen, Netherlands). Natural microalga or shrimp

hell extracts containing esterified astaxanthin compoundsave been approved by the FDA as nutraceuticals.27 Forarious biologic, strategic, and business-related reasons,ncluding bioavailability, solubility, and manufacturingtandards, among others, neither the synthetic, racemicstaxanthin nor the nutraceutical extracts are likely to beeveloped or commercialized as pharmaceutical products.n contrast, biocompatible and bioavailable derivatives of

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igure 3. Transmembrane orientation of polar carotenoids facilitates electroembranes is likely responsible for their antioxidant properties and their b

hydrophilic) ends of the molecule to the internal cytoplasm and to thentermembrane space of mitochondria), potentially facilitating electronntramembranous alignment of the molecule also likely provides proximityffectively “recharging” the capacity of the degraded carotenoid. RNS �

staxanthin are being developed for pharmaceutical appli- o

ations in human health where oxidative stress-mediatednflammation may be important, including CVD, prostatend other cancers, hepatitis, Alzheimer disease, and age-elated macular degeneration.24

afety and Metabolism of theanthophyll Carotenoids

n general, xanthophyll carotenoids are highly lipophilicnd water insoluble. However, several of the xanthophyllarotenoid derivatives under development demonstrate sig-ificantly improved solubility and oral bioavailability.iven orally, these compounds enter the circulation via the

ymphatic system, are processed in the liver, and are thenistributed to targeted tissues via plasma lipids.28 Therefore,he strong safety profile of the un-derivatized molecules isoth relevant and important.

As part of an FDA feed additive petition filed in 1987,oche Vitamins selected the xanthophyll carotenoid astax-nthin to conduct both good laboratory practices and non–ood laboratory practices safety pharmacology and toxicol-

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ling. Specific physicochemical interactions of antioxidant compounds withbenefits. The transmembranous alignment provides exposure of the polar

s environment external to the cell (or the mitochondrial matrix and ther via the double bonds of the carbon scaffold of the compound. The

in C, which serves as a “sink” for accepting the radical cations generatednitrogen species; ROS � reactive oxygen species.

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igure 4. Oxidative stress is the prime, common mediator of endothelial dysfunction. When triggered by risk factors, such as hypertension, dyslipidemia,iabetes mellitus, and obesity, increased oxidative stress results in decreased bioavailability of nitric oxide, increased production of endothelin-1 (ET-1),ncreased angiotensin-converting enzyme (ACE), and increased local tissue generation of angiotensin II, which in turn, further increases oxidative stress.CAM � intracellular adhesion molecule; MMPs � matrix metalloproteinases; NO � nitric oxide; PAI-1 � plasminogen activator inhibitor–1; VCAM �
ascular cell adhesion molecule. (Adapted from Circulation.35)
igure 5. Astaxanthin delays formation of oxidized low-density lipoprotein (LDL). Conjugated dienes are formed in the LDL fraction in the presence of 10g/mL of various carotenoids. Oxidation of LDL (35 �g/mL protein) catalyzed by the addition of V-70 (final 200 �mol/L) was monitored continuously by

he diene formation at 234 nm. Astaxanthin clearly delays the formation of conjugated dienes as well as the amount of conjugated dienes formed, when
ompared with active controls, including �-tocopherol. (Reprinted with permission from J Atheroscler Thromb.36)

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tudies (oral and parenteral routes) in rats (up to 1.2 g/kg peray for 91 days) and dogs (up to 162 mg/kg per day for 91ays) demonstrated no effects in dogs at the highest dose,ith only minor findings in rats. Ames and micronucleus

ssays also demonstrated no mutagenicity profile at theoses tested. Reproductive toxicology (teratology and em-ryotoxicity) was evaluated in both rats and rabbits; again,o effects were seen with astaxanthin doses of up to 1,000g/kg.29 Since the approval by the FDA of this feed addi-

ive petition in 1987, astaxanthin has been used in thenimal feed market (primarily salmon), with no evidence ofoxicity. The safety of astaxanthin in humans is supportedy published human clinical trials using astaxanthin-con-aining dietary supplements.30

staxanthin for the Potential Treatment of Reactivexygen Species–Mediated Inflammation andxidation in Cardiovascular Disease

xidative stress and inflammation play an important role innumber of aspects of CVD, including endothelial dysfunc-

ion,31 functional lipid disorders,32 periprocedural myocar-ial damage,33 and atrial fibrillation.34

When triggered by risk factors, such as hypertension, dys-ipidemia, diabetes, and obesity, increased oxidative stress re-ults in decreased bioavailability of nitric oxide, increasedroduction of endothelin-1, increased angiotensin-convertingnzyme, and increased local tissue generation of angioten-in II, which, in turn, further increases oxidative stress and

igure 6. Astaxanthin derivative reduces mean infarct size. Mean infarct sizeCON) received vehicle for 4 days before ischemia and reperfusion. Acutealt (ADD) 2 hours before ischemia-reperfusion. Chronically treated animalsp �0.01; ��p �0.001. (Reprinted with permission from Mol Life Sci.38

ndothelial dysfunction (Figure 4).35 R

Astaxanthin reduced measurements of LDL oxidation inumans in contrast to other antioxidants, such as vitamin E�-tocopherol) and lutein, compared with controls (Figure 5).36

n a separate study conducted by Mason and colleagues,37

staxanthin eliminated the lipid peroxidation caused by ro-ecoxib in cellular membrane models. The ability of astax-nthin to protect LDL from oxidation was further supportedy ex vivo analysis of LDL particles from humans fedstaxanthin. In this study, astaxanthin conferred increasedesistance to copper-induced oxidation seen as increasedDL oxidation lag times.36

tudies of Ischemia-Reperfusion Injury with anstaxanthin Derivative

schemia-reperfusion injury follows the restoration of bloodow to ischemic tissue and is mediated by ROS/RNS.ithin the myocardium, this occurs with mechanical or

harmacologic reperfusion in the setting of MI or at the timef flow restoration in coronary artery bypass grafting oreart transplantation. Reperfusion, after a period of isch-mia, is thought to result in part from a brisk inflammatoryesponse and the massive production of ROS from variousources, especially neutrophils. Studies in animal modelsndicate that redox-sensitive transcription factors are acti-ated, including NF-�B and the mitogen-activated proteininases. In response, there are numerous antioxidant sys-ems produced by the cell that attenuate the damage done by

sed as a percentage of infarct size/area at risk (IS/AAR%). Control animalsed animals (C-A) received 50 mg/kg of astaxanthin disuccinate disodiumreceived ADD 50 mg/kg for 4 days before ischemia-reperfusion on day 5.

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OS.4 Many pharmacologic interventions to limit ischemia-

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eperfusion injury have been evaluated, and this field con-inues to be an area of intense research interest.38,39

Significant cardioprotection has been demonstrated inultiple animal models (rat, rabbit, dog) of reperfusion-

schemia damage with a derivative, astaxanthin disuccinateisodium salt (ADD). These studies show up to 70% pro-ection from ischemic damage in dogs using standard mea-ures of myocardial salvage.40

When evaluated in a rat model of ischemia-reperfusion,arenteral administration of ADD was found to significantlyeduce infarct size compared with control animals. In thesexperiments, rats were treated with 1 of 3 doses of ADD fordays and then subjected to 30 minutes of ischemia and 2

ours of reperfusion. The animals that received ADD hadmaller infarctions in a dose-dependent manner than controlnimals. An inverse correlation was observed between in-arct size and the concentration of deesterified free astax-

igure 7. Effect of astaxanthin on reactive oxygen species (ROS) generaiNOS) expression. (A) RAW264.7 cells were treated with lipopolysacchariCFH2-DA (5 �mol/L) for an additional 30 minutes, and intracellular leve

B) RAW264.7 cells were treated with 1 �mol/L of hydrogen peroxide (H2

nalyzed by electrophoretic mobility shift assay (EMSA). Supershifting waacrophages were treated with H2O2 and/or interferon-� (10 units/mL) in

NOS were measured by Western blotting. (D) Nitrite was measured in thp �0.05. AX � astaxanthin. (Reprinted with permission from Mol Cells

nthin in plasma.41 Later, ADD was evaluated in a canine i

odel of ischemia-reperfusion. In these experiments, ADDas administered parenterally 2 hours before the ischemic

hallenge, or daily for 5 days before ischemia and comparedith control animals. In each case, 1 hour of ischemia was

ollowed by 2 hours of reperfusion. Animals treated for 2 hoursad significantly smaller infarcts than the control animals, andhe animals treated for 5 days had still smaller infarcts. Again,n inverse correlation between plasma levels of free astaxan-hin and tissue salvage was noted (Figure 6).40

ADD has been shown to reduce infarct size as well. Newealand white rabbits received ADD for 4 days before

nduction of ischemia for 30 minutes followed by 3 hours ofeperfusion. When evaluated histologically, animals treatedith ADD had significantly smaller infarcts than the control

nimals. There was also a strong trend of reduced serumroponin levels in the ADD-treated animals. Measurementsf tissue levels of ADD revealed the compound accumulates

clear factor–�B (NF-�B) activation, and inducible nitric oxide synthaseS) in the presence or absence of astaxanthin for 30 minutes, incubated withS were determined by fluorescence-activated cell sorter (FACS) analysis.

he presence or absence of astaxanthin for 2 hours, and NF-�B activity wasmed with antibody against the NF-�B p65 subunit (�-p65). (C) Peritonealsence or absence of astaxanthin (50 �mol/L) for 14 hours, and levels ofre medium after 24-hour culture. Data shown are means � SD (n � 3).

tion, nudes (LPls of ROO2) in ts perforthe pree cultu

n myocardial tissue. After 4 days of therapy, myocardial

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issue levels were orders of magnitude higher than plasmaevels at the time of the ischemia-reperfusion experiment.mmunofluorescence studies indicated the ADD treatmentesulted in less deposition of C-reactive protein and mem-rane attack complex in the ischemic tissue than in theontrols, indicating a less robust inflammatory response inhese treated animals.42

staxanthin Reduces Rethrombosis After Vascularhrombotic Occlusion

ntracellular generation of ROS is also thought to beequired for rethrombosis and platelet aggregation.43 Thestaxanthin derivative ADD has demonstrated efficacy incanine model of carotid artery rethrombosis and plateletggregation.44 After forming an occlusive thrombus inhe carotid artery, dogs were administered recombinantissue plasminogen activator intra-arterially to achievehrombolysis in the presence of either 0.9% sodium chlo-ide solution or ADD (10, 30, or 50 mg/kg via intrave-ous infusion). The results indicated a dose-dependenteduction in the incidence of carotid artery rethrombosis.n addition, ex vivo platelet aggregation and thrombuseights were dose-dependently inhibited by ADD. No

hange was recorded in bleeding time among the treat-ent groups. The data demonstrate that ADD signifi-

antly reduces the incidence of secondary arterial throm-osis in a clinically relevant model of thrombolysis,hile maintaining normal hemostasis.

staxanthin Modifies Oxidative Stress Messengersnd Inflammatory Mediators

s discussed earlier, the NF-�B inflammatory pathway haseen shown to be at least partially regulated by ROS and haseen implicated in various forms of CVD.45 Higuchi et al46

ave shown that TNF-�–induced cardiomyocyte hypertro-hy is mediated through ROS-induced NF-�B activation,ith oxidative species serving as a hypertrophic signaling

ransducer. This result is consistent with the observation ofolkentin and Dom47 of an emerging paradigm wherebyultiple signaling pathways operate together to orchestratehypertrophic response. The role of inflammatory media-

ors in the failing heart is evidenced by immunomodulationf cytokines in experimental models of heart failure.48 Theotential of NF-�B as a therapeutic target for treating car-iac hypertrophy has been suggested because experimentalnhibition of NF-�B signaling can attenuate cardiac hyper-rophy without sensitizing cardiomyocytes to apoptotic celleath.49

Astaxanthin has been shown to inhibit inflammation-nduced nitric oxide production and inflammatory gene ex-ression by suppressing I-�B kinase–dependent NF-�B ac-
ivation.50 This inhibition of the expression or formation p
f proinflammatory mediators and cytokines was ob-erved in both a lipopolysaccharide–stimulated macro-hage cell line (RAW264.7) and isolated primary mac-ophages. Figure 7A shows the results of a cell culturexperiment quantifying ROS levels in RAW264.7 mac-ophages treated with lipopolysaccharide in the presence orbsence of various astaxanthin concentrations. Astaxanthinnhibited the intracellular accumulation of ROS in lipopo-ysaccharide–stimulated RAW264.7 cells in a dose-depen-ent manner, decreased hydrogen peroxide–induced NF-�Bctivation in RAW264.7 cells (Figure 7B), and loweredxpression of inducible nitric oxide synthase in primaryacrophages (Figure 7C/D). Moreover, astaxanthin blocked

uclear translocation of the activated NF-�B p65 subunitnd I-�B� degradation, which correlated with its inhibitoryffect on I-�B kinase activity. These results suggest thatstaxanthin, most likely because of its antioxidant activity,nhibits the production of inflammatory mediators by block-ng NF-�B pathway activation, possibly resulting from up-tream suppression of I-�B kinase activity, leading to de-reased I-�B� degradation.50 In the same study, astaxanthinas also shown to suppress serum levels of nitric oxide,rostaglandin E2, TNF-�, and interleukin 1-� in lipo-olysaccharide-treated mice. A separate study demonstratedhe ability of astaxanthin to suppress the development ofnflammation in an endotoxin-induced uveitis model.51

ere, they showed that in vivo astaxanthin (100 mg/kg) wass strong an anti-inflammatory as prednisolone (10 mg/kg),nd in RAW264.7 macrophage cells, again decreased nitricxide production, inducible nitric oxide synthase activity,nd prostaglandin E2 and TNF-� production.

ummary of Astaxanthin Preclinical Studies

reclinical studies of the xanthophyll carotenoid astaxanthinnd its derivatives demonstrate anti-inflammatory propertiesnd potential efficacy in the setting of ischemia-reperfusion,he reduction of lipid peroxidation, and the reduction ofethrombosis after thrombolysis. It remains to be seen if theesults of these ex vivo and animal studies can be repro-uced in human subjects. The long history of astaxanthinse in the aquaculture industry and the known safety profilef the nutraceutical compound make it an ideal choice forlinical study in settings such as elective percutaneous cor-nary interventions, acute MI, and coronary artery bypassrafting. Past experiences with the transition from animalodels of ischemia-reperfusion to human studies are

raught with disappointment likely because of difficulty inelivering compounds to acutely infarcting tissue and ineparating acute injury from irreversibly infarcted tissue.istribution and localization of astaxanthin in myocardium

learly support further development of the preclinical
rogram.

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onclusion

OS and RNS are produced as a consequence of normalellular metabolism. However, under certain conditions,xcessive amounts are produced, resulting in increased ox-dative stress. Excess ROS/RNS can activate numerousathways, leading to increased expression of proinflamma-ory genes and elevated production of proinflammatory cy-okines, mediators, and enzymes. The transcriptional acti-ator NF-�B—an important component of one of thesenflammatory pathways—directly upregulates the produc-ion of inflammatory mediators, including inducible nitricxide synthase, cyclooxygenase-1 and cyclooxygenase-2,NF-�, and interleukin 1-�. Although inhibition of this key

nflammatory factor may have therapeutic potential, at-empts to chronically inhibit NF-�B or other components ofhis pathway have led to undesirable side effects and/oro-called off-target effects. Elucidation of critical intercel-ular and intracellular signaling mechanisms can help in theetermination of mechanisms through which oxidativetress, inflammation, and pathologic precursors produce po-entially clinically relevant cardiovascular dysfunction andubsequent disease.

Evidence suggests that the xanthophyll carotenoids,hrough their unique physicochemical properties, may beble to dampen or modulate excessive ROS/RNS, with aonsequent impact on inflammatory processes in the ab-ence of the negative effects demonstrated with traditionalharmaceutical lock and key interventional strategies. Aasic understanding of the mode of action of astaxanthinnd its potential role in the regulation of important inflam-atory messengers as well as other modes of cellular com-unication is currently being investigated. Multiple ex vivo

nd in vivo studies have shown the effectiveness of astax-nthin in several potentially relevant cardiovascular pathol-gies driven by oxidative stress that results in inflammation.hus, there may be a potential therapeutic role for astaxan-

hin derivatives in the management of myocardial injury,xidized LDL, and rethrombosis after thrombolysis, as wells other cardiac diseases, such as atrial fibrillation. How-ver, the potential effect of the xanthophyll carotenoids,pecifically astaxanthin, on signal transduction pathways,ellular homeostasis, and biologic effects as potential clin-cal targets remains to be clarified.

cknowledgment

pecial thanks to Timothy J. King, PhD, for editorial assis-ance with this manuscript.

uthor Disclosures

he authors who contributed to this article have disclosed
he following industry relationships:
Fredric J. Pashkow, MD, is a salaried employee, cor-orate officer, and stockholder in Cardax Pharmaceuticals,nc., a company involved in the discovery and developmentf drugs targeted to treat oxidative stress and inflammation.ompounds in development by the company include deriv-tives of the antioxidant xanthophyll corotenoid family thatre discussed in this supplement.

David G. Watumull, is a salaried employee, corporatefficer, and stockholder in Cardax Pharmaceuticals, Inc., aompany involved in the discovery and development ofrugs targeted to treat oxidative stress and inflammation.ompounds in development by the company include deriv-tives of the antioxidant xanthophyll corotenoid family thatre discussed in this supplement.

Charles L. Campbell, MD, is a member of the Speak-rs’ Bureau for Novartis Pharmaceuticals; and serves on thecientific Advisory Board for Cardax Pharmaceuticals, andedicure Pharmaceuticals, and sanofi-aventis.

1. Fuster V, Moreno PR, Fayad ZA, Corti R, Badimon JJ. Atherothrom-bosis and high-risk plaque. Part I: evolving concepts. J Am CollCardiol 2005;46:937–954.

2. Tedgui A, Mallat Z. Cytokines in atherosclerosis: pathogenic andregulatory pathways. Physiol Rev 2006;86:515–581.

3. Geisler T, Bhatt DL. The role of inflammation in atherothrombosis:current and future strategies of medical treatment. Med Sci Monit2004;10:RA308–RA316.

4. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Freeradicals and antioxidants in normal physiological functions and humandisease. Int J Biochem Cell Biol 2007;39:44–84.

5. Davidson SM, Duchen MR. Endothelial mitochondria: contributing tovascular function and disease. Circ Res 2007;100:1128–1141.

6. Muller G, Goettsch C, Morawietz H. Oxidative stress and endothelialdysfunction. Hamostaseologie 2007;27:5–12.

7. Schulz E, Anter E, Keaney JF Jr. Oxidative stress, antioxidants, andendothelial function. Curr Med Chem 2004;11:1093–1104.

8. Tak PP, Firestein GS. NF-�B: a key role in inflammatory diseases.J Clin Invest 2001;107:7–11.

9. Li Q, Verma IM. NF-�B regulation in the immune system. Nat RevImmunol 2002;2:725–734.

0. Cyrus T, Sung S, Zhao L, Funk CD, Tang S, Pratico D. Effect oflow-dose aspirin on vascular inflammation, plaque stability, andatherogenesis in low-density lipoprotein receptor-deficient mice. Cir-culation 2002;106:1282–1287.

1. Cominacini L, Anselmi M, Garbin U, Fratta Pasini A, Stranieri C,Fusaro M, Nava C, Agostoni P, Keta D, Zardini P, Sawamura T, LoCascio V. Enhanced plasma levels of oxidized low-density lipoproteinincrease circulating nuclear factor-�B activation in patients with un-stable angina. J Am Coll Cardiol 2005;46:799–806.

2. Bertram JS. Cellular communication via gap junctions. Sci Med(Phila) 2000;8:18–27.

3. Chadjichristos CE, Derouette JP, Kwak BR. Connexins in atheroscle-rosis. Adv Cardiol 2006;42:255–267.

4. Dhein S. Cardiac ischemia and uncoupling: gap junctions in ischemiaand infarction. Adv Cardiol 2006;42:198–212.

5. Severs NJ, Dupont E, Thomas N, Kaba R, Rothery S, Jain R, SharpeyK, Fry CH. Alterations in cardiac connexin expression in cardiomy-opathies. Adv Cardiol 2006;42:228–242.

6. Murray KT, Mace LC, Yang Z. Nonantiarrhythmic drug therapy foratrial fibrillation. Heart Rhythm 2007;4:S88–S90.

7. Dhein S. Role of connexins in atrial fibrillation. Adv Cardiol 2006;42:
161–174.

1

1

2

2

2

2

2

2

2

2

2

2

3

3

3

3

3

3

3

3

3

3

4

4

4

4

4

4

4

4

4

4

5

5


8. Rodriguez-Sinovas A, Boengler K, Cabestrero A, Gres P, Morente M,Ruiz-Meana M, Konietzka I, Miro E, Totzeck A, Heusch G, Schulz R,Garcia-Dorado D. Translocation of connexin 43 to the inner mitochon-drial membrane of cardiomyocytes through the heat shock protein90-dependent TOM pathway and its importance for cardioprotection.Circ Res 2006;99:93–101.

9. Boengler K, Konietzka I, Buechert A, Heinen Y, Garcia-Dorado D,Heusch G, Schulz R. Loss of ischemic preconditioning’s cardiopro-tection in aged mouse hearts is associated with reduced gap junctionaland mitochondrial levels of connexin 43. Am J Physiol Heart CircPhysiol 2007;292:H1764–H1769.

0. Hix LM, Lockwood SF, Bertram JS. Upregulation of connexin 43protein expression and increased gap junctional communication bywater soluble disodium disuccinate astaxanthin derivatives. CancerLett 2004;211:25–37.

1. Rodrigo R, Guichard C, Charles R. Clinical pharmacology and thera-peutic use of antioxidant vitamins. Fundam Clin Pharmacol 2007;21:111–127.

2. McNulty HP, Byun J, Lockwood SF, Jacob RF, Mason RP. Differen-tial effects of carotenoids on lipid peroxidation due to membraneinteractions: X-ray diffraction analysis. Biochim Biophys Acta 2007;1768:167–174.

3. May JM. Is ascorbic acid an antioxidant for the plasma membrane?FASEB J 1999;13:995–1006.

4. Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H. Astax-anthin, a carotenoid with potential in human health and nutrition. J NatProd 2006;69:443–449.

5. Santocono M, Zurria M, Berrettini M, Fedeli D, Falcioni G. Influenceof astaxanthin, zeaxanthin and lutein on DNA damage and repair inUVA-irradiated cells. J Photochem Photobiol B 2006;85:205–215.

6. Lockwood SF, Gross GJ. Disodium disuccinate astaxanthin (Cardax):antioxidant and antiinflammatory cardioprotection. Cardiovasc DrugRev 2005;23:199–216.

7. Guerin M, Huntley ME, Olaizola M. Haematococcus astaxanthin:applications for human health and nutrition. Trends Biotechnol 2003;21:210–216.

8. Wang XD, Krinsky NI, Marini RP, Tang G, Yu J, Hurley R, Fox JG,Russell RM. Intestinal uptake and lymphatic absorption of beta-caro-tene in ferrets: a model for human beta-carotene metabolism. Am JPhysiol 1992;263:G480–G486.

9. Astaxanthin as a pigmenter in salmon feed. Chemicals HVF.Hoffman-La Roche Inc., 1987.

0. Spiller GA, Dewell A. Safety of an astaxanthin-rich Haematococcuspluvialis algal extract: a randomized clinical trial. J Med Food 2003;6:51–56.

1. Félétou M, Vanhoutte PM. Endothelial dysfunction: a multifaceteddisorder (The Wiggers Award Lecture). Am J Physiol Heart CircPhysiol 2006;291:H985–H1002.

2. Nicholls SJ, Zheng L, Hazen SL. Formation of dysfunctional high-density lipoprotein by myeloperoxidase. Trends Cardiovasc Med2005;15:212–219.

3. Levitsky S. Protecting the myocardial cell during coronary revascu-larization: the William WL Glenn Lecture. Circulation 2006;114:I339–I343.

4. Van Wagoner DR. Recent insights into the pathophysiology of atrial
fibrillation. Semin Thorac Cardiovasc Surg 2007;19:9–15.
5. Dzau VJ, Antman EM, Black HR, Hayes DL, Manson JE, Plutzky J,Popma JJ, Stevenson W. The cardiovascular disease continuum vali-dated: clinical evidence of improved patient outcomes. Part I: patho-physiology and clinical trial evidence (risk factors through stablecoronary artery disease). Circulation 2006;114:2850–2870.

6. Iwamoto T, Hosoda K, Hirano R, Kurata H, Matsumoto A, Miki W,Kamiyama M, Itakura H, Yamamoto S, Kondo K. Inhibition of low-density lipoprotein oxidation by astaxanthin. J Atheroscler Thromb2000;7:216–222.

7. Mason RP, Walter MF, McNulty HP, Lockwood SF, Byun J, Day CA,Jacob RF. Rofecoxib increases susceptibility of human LDL andmembrane lipids to oxidative damage: a mechanism of cardiotoxicity.J Cardiovasc Pharmacol 2006;47(suppl 1):S7–S14.

8. Gross GJ, Auchampach JA. Reperfusion injury: does it exist? J MolCell Cardiol 2007;42:12–18.

9. Wang QD, Pernow J, Sjoquist PO, Ryden L. Pharmacological possi-bilities for protection against myocardial reperfusion injury. Cardio-vasc Res 2002;55:25–37.

0. Gross GJ, Lockwood SF. Acute and chronic administration of diso-dium disuccinate astaxanthin (Cardax) produces marked cardioprotec-tion in dog hearts. Mol Cell Biochem 2005;272:221–227.

1. Gross GJ, Lockwood SF. Cardioprotection and myocardial salvage bya disodium disuccinate astaxanthin derivative (Cardax). Life Sci 2004;75:215–224.

2. Gross GJ, Hazen SL, Lockwood SF. Seven day oral supplementationwith Cardax (disodium disuccinate astaxanthin) provides significantcardioprotection and reduces oxidative stress in rats. Mol Cell Biochem2006;283:23–30.

3. Krotz F, Sohn HY, Pohl U. Reactive oxygen species: players in theplatelet game. Arterioscler Thromb Vasc Biol 2004;24:1988–1996.

4. Lauver DA, Driscoll EM, Lucchesi BR. Disodium disuccinate astax-anthin prevents carotid rethrombosis and ex-vivo platelet activation.Pharmacology (in press).

5. Hall G, Hasday JD, Rogers TB. Regulating the regulator: NF-�Bsignaling in heart. J Mol Cell Cardiol 2006;41:580–591.

6. Higuchi Y, Otsu K, Nishida K, Hirotani S, Nakayama H, YamaguchiO, Matsumura Y, Ueno H, Tada M, Hori M. Involvement of reactiveoxygen species-mediated NF-�B activation in TNF-�-induced cardi-omyocyte hypertrophy. J Mol Cell Cardiol 2002;34:233–240.

7. Molkentin JD, Dorn IG II. Cytoplasmic signaling pathways that reg-ulate cardiac hypertrophy. Annu Rev Physiol 2001;63:391–426.

8. Matsumori A, Sasayama S. The role of inflammatory mediators in thefailing heart: immunomodulation of cytokines in experimental modelsof heart failure. Heart Fail Rev 2001;6:129–136.

9. Cook SA, Novikov MS, Ahn Y, Matsui T, Rosenzweig A. A20 isdynamically regulated in the heart and inhibits the hypertrophic re-sponse. Circulation 2003;108:664–667.

0. Lee SJ, Bai SK, Lee KS, Namkoong S, Na HJ, Ha KS, Han JA, YimSV, Chang K, Kwon YG, Lee SK, Kim YM. Astaxanthin inhibitsnitric oxide production and inflammatory gene expression by suppress-ing I(�)B kinase-dependent NF-�B activation. Mol Cells 2003;16:97–105.

1. Ohgami K, Shiratori K, Kotake S, Nishida T, Mizuki N, Yazawa K,Ohno S. Effects of astaxanthin on lipopolysaccharide-induced inflam-mation in vitro and in vivo. Invest Ophthalmol Vis Sci 2003;44:2694–
2701.

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