Pathophysiology and Pharmacology of Reactive Oxygen Species (ROS)

V. Bauer, V. Bauer, Š. Mátyás, S. Štolc, R. Sotníková, Š. Mátyás, S. Štolc, R. Sotníková, V. NosáľováV. Nosáľová

Reactive Oxygen Species as Mediators of Tissue Injury, Diseases and their Pharmacology

Institute of Experimental Pharmacology, Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, SKSlovak Academy of Sciences, Bratislava, SK

The beginningsThe beginningsThe beginningsThe beginnings

1775 - Priestley: discovery of O2 observation of toxic effect of O2

1900 - Gomberg: discovery of triphenylmethyl radical Until 1950/60: minimal attention was given to biological actions of free radicals and reactive oxygen species

Evidence on the existence of ROSEvidence on the existence of ROSEvidence on the existence of ROSEvidence on the existence of ROS

1954 - Gerschman et al. : Recognition of similarities between radiation and oxygen toxicity

1969 - McKord and Fridovich: Discovery of superoxide dismutase and suggestion of the existence of endogenous superoxide

1973 - Babior et al.: Recognition of the relationship between superoxide production and bactericidal activity of neutrophils

1981 - Granger et al.: recognition of the relationship between local ROS production and ischemia/reperfusion induced gut injury

Free radicals have one or more unpaired electrons in their outer orbital, indicated in formulas as []. As a consequence they have an increased reactivity with other molecules. This reactivity is determined by the ease with which a species can accept or donate electrons.

The prevalence of oxygen in biological systems means that oxygen centered radicals are the most common type found.

O2 acts in a process that is central to metabolism in aerobic life, as a terminal electron acceptor, being reduced to water. Transfer of electron to oxygen yields the reactive intermediates.

The term reactive oxygen species (ROS) rather than oxygen radicals is now generally preferred because singlet oxygen (its one form), hydrogen peroxide, hypochlorous acid, peroxide, hydroperoxide and epoxide metabolites of endogenous lipids and xenobiotics have chemically reactive oxygen containing functional groups, but are not radicals and do not necessarily interact with biological tissues via radical reactions.

Molecular oxygen is a biradical, having two unpaired electrons of parallel spin. As it is a terminal electron acceptor being reduced to water, oxygen acts in processes that are central to metabolism in aerobic life.

4O2 + 4H+ + 4e- 2H2O + 3O2 4O2 + 16H+ + 16e- 8H2O (yields to production of ATP)

4O2

4e-

4O2-

4e-

4O22-

4e-

4O23-

4e-

4O24-

16H+

8H2O

4O2

4e-

4O2.-

2H2O2 + 2O2

2H2O + O2

cyto

chro

me

c-ox

idas

eXOat presence of NADPHO

Reactive Oxygen Metabolite Cytochrome Cascade Cascade (ROM Cycle)

SOD

CAT

Half-life of Half-life of some rsome reactive eactive sspeciespeciesHalf-life of Half-life of some rsome reactive eactive sspeciespecies

RReactive specieseactive species Half-lifeHalf-life (s)(s) Half-lifeHalf-life (s)(s)

Hydroxyl radical (Hydroxyl radical (OH)OH)Alcoxyl radical (ROAlcoxyl radical (RO))Singlet oxygen (Singlet oxygen (11OO22))Peroxynitrite anion (ONOOPeroxynitrite anion (ONOO--))Peroxyl radical (ROOPeroxyl radical (ROO))Nitric oxide (Nitric oxide (NO)NO)Semiquinone radicalSemiquinone radicalHydrogen peroxide (HHydrogen peroxide (H22OO22))

Superoxide anion (OSuperoxide anion (O22--))

HypochloHypochlorous acidrous acid (HOCl) (HOCl)

Hydroxyl radical (Hydroxyl radical (OH)OH)Alcoxyl radical (ROAlcoxyl radical (RO))Singlet oxygen (Singlet oxygen (11OO22))Peroxynitrite anion (ONOOPeroxynitrite anion (ONOO--))Peroxyl radical (ROOPeroxyl radical (ROO))Nitric oxide (Nitric oxide (NO)NO)Semiquinone radicalSemiquinone radicalHydrogen peroxide (HHydrogen peroxide (H22OO22))

Superoxide anion (OSuperoxide anion (O22--))

HypochloHypochlorous acidrous acid (HOCl) (HOCl)

1010-9-9

1010-6-6

1010-5-5

00..05 – 105 – 1..0077

1 - 101 - 10minutes/hoursminutes/hours

sspontpontan. hours/an. hours/daysdays((accelerated by accelerated by enzymeenzymess))

sspontpontan. hours/an. hours/daysdays((by SOD accel.by SOD accel. to to 1010-6-6))dep. on substratedep. on substrate

1010-9-9

1010-6-6

1010-5-5

00..05 – 105 – 1..0077

1 - 101 - 10minutes/hoursminutes/hours

sspontpontan. hours/an. hours/daysdays((accelerated by accelerated by enzymeenzymess))

sspontpontan. hours/an. hours/daysdays((by SOD accel.by SOD accel. to to 1010-6-6))dep. on substratedep. on substrate

Physiol conc.Physiol conc. ((mol/lmol/l))

Physiol conc.Physiol conc. ((mol/lmol/l))

1010-9-9

1010-9-9 - 10- 10--77

1010--12 12 - 10- 10--1111

ROS present in mammalian tissues have both endogenous and exogenous origin. Their production is essential to normal function or metabolism of most mammalian cells.

Approximately, 90% of all oxygen consumed by mammalian cells is catalytically reduced by four electrons to yield two molecules of water. It is now clear that oxygen may also be reduced by less than four electrons in enzymatic and nonenzymatic reactions.

ROS are, however, also destructive unless tightly controlled. Mammalian cells have developed a battery of defenses to prevent and repair the injuries caused by oxidative stress.

Origin of ROS

HH++

..NO OONONO OONO-- HOONO HOONO NONO22

..

ee-- e e-- e e-- e e--

OO22 OO22..--

HH22OO2 2 ..OHOH HH22OO

OO22..--

OO2 2 FeFe2+2+ Fe Fe3+3+ H H++

ClCl--

Myelo-Myelo- peroxidaseperoxidase

HH22OO

HOCl HOCl 11OO22 + Cl + Cl--

HH22OO22 HH22OO

Generation in mammalian organism

Sources Sources endogenousendogenous exogenousexogenous

prostaglandin synth.prostaglandin synth. radiation, ultrasound radiation, ultrasound respiratory chainrespiratory chain cigarette smoke cigarette smokeautooxidationautooxidation drugs drugs

FREE RADICAL SFREE RADICAL S

phagocytes phagocytes heatheat oxyhemoglobinoxyhemoglobin pesticidespesticides oxidative enzymesoxidative enzymes infectionsinfections accumul reduced.metab.accumul reduced.metab. hyperoxia, exercise hyperoxia, exercise

air pollution (NOair pollution (NOxx, O, O33))

Enzymatic sources of ROS

Xanthine oxidaseXanthine oxidase

Hypoxanthine + 2OHypoxanthine + 2O2 2 Xanthine + Xanthine + OO22.-.- + + HH22OO22

NADPH oxidaseNADPH oxidase

NADPH + ONADPH + O2 2 NADPNADP+ + + + OO22.-.-

Amine oxidasesAmine oxidases R-CHR-CH22-NH-NH2 2 + H+ H22OO + O+ O2 2 R-CHO + NHR-CHO + NH3 3 + + HH22OO22

Myeloperoxidase Myeloperoxidase Hypohalous acid formation Hypohalous acid formation

HH22OO22 + X+ X- - + H+ H+ + HOX HOX + H+ H22OONADH oxidase reactionNADH oxidase reaction Hb(Mb)-FeHb(Mb)-Fe3+ 3+ + ROOH + ROOH Compound I + ROHCompound I + ROH Compound I + NADPH Compound I + NADPH NADNAD+ Compound II + Compound II Compound II + NADH Compound II + NADH NADNAD+ E-Fe+ E-Fe3+ 3+

NADNAD+ O+ O2 2 NADNAD+ + + + OO22.-.-

Aldehyde oxidaseAldehyde oxidase

2R-CHO + 2O2R-CHO + 2O22 2R-COOH + 2R-COOH + OO22.-.-

Dihydroorotate dehydrogenaseDihydroorotate dehydrogenase

Dihydroorotate + NADDihydroorotate + NAD+ O+ O2 2 NADH + NADH + OO22.-.- + Orotic acid + Orotic acid

0 100 200 300 400 500 600 700 800 900

-0.02

0.00

0.02

0.04

0.06

XO 2 mU/ml

X100mol/l

+ SOD 200 U/ml

no SOD

time (s)

Ab

sorb

ance

55

0 n

mEffect of superoxide dismutase (SOD) on reduction of cytochrome c by O2

.- which is produced by xanthine oxidase (XO) in the presence of xanthine (X)

Institute of Experimental Pharmacology, SASc, Bratislava, SK

Nonenzymatic sources of ROS and autooxidation reactions

FeFe2+ 2+ + O+ O2 2 FeFe3+3++ + OO22.-.-

Hb(Mb)-FeHb(Mb)-Fe2+ 2+ + O+ O2 2 Hb(Mb)-Fe Hb(Mb)-Fe3+3+++ OO22.- .-

Catecholamines + OCatecholamines + O2 2 Melanin + Melanin + OO22.-.-

Reduced flavinReduced flavin

Leukoflavin + OLeukoflavin + O2 2 Flavin semiquinone + Flavin semiquinone + OO22.-.-

CoenzymeCoenzyme

Q-hydroquinone + OQ-hydroquinone + O2 2 Coenzyme Q (ubiquinone) + Coenzyme Q (ubiquinone) + OO2 2 .-.-

Tetrahydropterin + 2 OTetrahydropterin + 2 O2 2 Dihydropterin + 2 Dihydropterin + 2 OO22.-.-

UntilUntil the 1960s,the 1960s, free radicalsfree radicals were not considered were not considered particularly relevant for mammalian physiology and particularly relevant for mammalian physiology and pathology. pathology.

The discoveries of the existence ofThe discoveries of the existence of superoxide superoxide dismutase dismutase (SOD)(SOD) activity in mammalian cells activity in mammalian cells inin 1969 1969 by McCord and Fridovichby McCord and Fridovich and association of and association of bactericidal activity of neutrophils with production bactericidal activity of neutrophils with production of theof the superoxide superoxide radical (Oradical (O22

.-.-)) by Babior and by Babior and coworkers in 1973, coworkers in 1973, linked linked free radicalsfree radicals to numerous to numerous physiological and pathophysiological processes.physiological and pathophysiological processes.

One decade later,One decade later, in 1981, Granger and coworkers in 1981, Granger and coworkers established a hypothesis on the role of these reactive established a hypothesis on the role of these reactive species in thespecies in the reperfusion injuryreperfusion injury after intestinal after intestinal ischemia. ischemia.

ROS are tightly controlled resulting in a physiological balance between their

production and elimination

ROS are tightly controlled resulting in a physiological balance between their

production and elimination

c-cytosolic, m-mitochondrial, p-peroxisomal

ROS:ROS: OO22

, H, H22OO2 2 , , 11OO2 2

OH, HOClOH, HOCl

Enzymes:Enzymes: SOD (SOD (c, mc, m) , ) , GPX (GPX (c, mc, m), CAT (), CAT (c, pc, p))

Non-enzyme antioxidants:Non-enzyme antioxidants: vitamines (E,A,C), thiols, vitamines (E,A,C), thiols, phenols, ceruloplasmin, phenols, ceruloplasmin,

transferrin, uric acid,transferrin, uric acid, albumin, etc.albumin, etc.

Biological antioxidant defense mechanisms

Defense mechanisms in the organisma. Catalytic free radical removal O2

.- - spontaneous dismutation - superoxide dismutase (SOD) - ceruloplasmin H2O2 - glutathion peroxidase (GTPx) - catalase (CAT) Organic hydroperoxides - GTPx Disulphide - GTPx Oxidised ascorbate - GTPx b. Free radical scavengers (antioxidants) Vitamin E (-tocopherol) O2

.-, .OH, LPO Reduced ascorbic acid in high concentrations of O2

.-, .OH, LPO Low m.w. thiols (e.g. cystein) Large m.w. thiols (e.g. albumin)c. Removal of Fe and Cu Ferritin, transferrin, lactoferrin (Fe) Ceruloplasmin (Cu, Fe) Serum albumin (Cu)

H2O2

H2O + O2 H2O

O2.-

.OH

LPO

Supe

roxi

de d

ism

utas

es

(C

u/Z

n-M

n)

Cat

alas

e

GSH-Peroxidase

GSH

GSSG

Fe2+ Fe3+

Ferritin

Vita

min

E

Under pathological condition the physiological balance is lostUnder pathological condition the physiological balance is lost

Consequences are shown in the next panels

ROS:ROS: OO22

, H, H22OO2 2 ,, 1 1OO22, , OH, HOClOH, HOCl

Enzymes:Enzymes: SOD,GPX,CATSOD,GPX,CATNon-enzyme antioxidants:Non-enzyme antioxidants: vitamines (E,A,C), thiols, vitamines (E,A,C), thiols, uric acid, ceruloplasmin, uric acid, ceruloplasmin,

transferrin, phenols, transferrin, phenols, albumin, etc.albumin, etc.

Disbalance between production and elimination of ROS develops during inflammation, ischemia/reperfusion, altered metabolism, action of drugs, pollutants, etc.

Such disbalance causes pathology of brain, heart, vessels, gut, airways, muscle, parenchy- matous organs (liver, kidney, pancreas), eye, skin, joints, etc.

Exposure of the tissues to ROS in a variety of biological systems has documented their ability to damage lipids, proteins and DNA. The resulting damage potentiated by increased free intracellular Ca2+ causes activation/deacti-vation of various enzyme systems and cell injury or death.

Mechanisms of ROS induced cell injuryMechanisms of ROS induced cell injury

Lipid Oxidation of thiols DNA damage Schiff basesLipid Oxidation of thiols DNA damage Schiff bases peroxidation Carbonyl formationperoxidation Carbonyl formation Damage to CaDamage to Ca2+2+ and Poly ADP Altered gene and Poly ADP Altered gene other ion transport ribosylation expressionother ion transport ribosylation expression systemssystems Amadori productsAmadori products

Membrane Instability to maintain Depletion of ATPMembrane Instability to maintain Depletion of ATP damage normal ion gradients and NAD(P)(H)damage normal ion gradients and NAD(P)(H)

Activation/Deactivation of AGEsActivation/Deactivation of AGEs various enzyme systems various enzyme systems ( (Advanced glycation Advanced glycation end products)end products)

Cell injuryCell injury

LIPIDS PROTEINS DNA SUGARS

Involvement of ROS in APOPTOSIS Involvement of ROS in APOPTOSIS

NOXANOXA

(trauma, hypoxia (trauma, hypoxia under homeostaticunder homeostatic

metabolic insufficiency metabolic insufficiency control to a certaincontrol to a certain

activation of excitatory receptors)activation of excitatory receptors) limit limit

Ion disbalanceIon disbalance caspase/calpainecaspase/calpaine ROS generationROS generation Mitochondrial failureMitochondrial failure activation activation

Bcl-2 / Bax disbalanceBcl-2 / Bax disbalance

CELL DEATHCELL DEATH (necrosis / apoptosis)(necrosis / apoptosis)

Currently it is believed that free radicals are definitely paticipating in several health disorders.

There are different pathologic conditions where extracellular, intracellular or both ROS act at least in part.

However, in spite of the extensive studies our knowledge concerning the role and action of free radicals and ROS is still incomplete and changing.

Pathological conditions that may have a free radical component and sites of ROS actions

Atherosclerosis

Smoking, air pollutants & drug induced reactions

intracellular extracellularIntracellular & extracellular

Hypo-, hyper-oxygenation & Reperfusion after ischemia

Cataractogenesis Immunereactions

Parkinsonism

Diabetes

Iron, drug& chemicaltoxicity

Chemical cancerogenesis Radiation injury

Ageing & senile dementia

FREE RADICALS

Cancer

Inflammatory reactions

ROS generation during ischemia and reperfusion ATP

I S AMP Xanthine dehydrogenase C H Adenosine E Ca2+ proteases M Inosine I

A Hypoxanthine+Xanthine oxidase O2.- activated chemoattractants

REOXYGENATION

Cl- H2O2 O2.-

Extravasated Circulating neutrophils neutrophils MPO Fe2+ Fe3+

Neutrophil

HOCl .OH activators Chemoattractants

Tissue damageTissue damage

ROS in the sequence of events in ROS in the sequence of events in STROKE STROKE

HYPOXIAHYPOXIA ATP depletionATP depletion Cell depolarizationCell depolarization (( Mg block of NMDA rec.) Mg block of NMDA rec.) Excitatory aminoacid releaseExcitatory aminoacid release CaCa2+2+ influx into the cellsinflux into the cells Slow accumul. Ca Slow accumul. Ca2+2+ in mitochondria in mitochondria Activation of phosholipases, MPT pore opening in mitochondriaActivation of phosholipases, MPT pore opening in mitochondria proteinkinases, proteases, Hproteinkinases, proteases, H+ + gradient collapse in mitochondriagradient collapse in mitochondria endonucl., phosphatases etc.endonucl., phosphatases etc. ROS generationROS generation ONOOONOO- - generationgeneration

Devastatory effect in cellsDevastatory effect in cells

NEURONAL DEATHNEURONAL DEATH Therapeutic interventionsTherapeutic interventions:: cyklosporine (specific MPT pore inhibitor), cyklosporine (specific MPT pore inhibitor), antioxidants (lazaroidsantioxidants (lazaroids, deferoxamine, SOD in liposomes, allopurinol, deferoxamine, SOD in liposomes, allopurinol))

Frequent targets of ROS Frequent targets of ROS

OO22--

HH22OO22

HOClHOClOOHH

gutgut

heart & heart & vesselsvessels

airwaysairways

brain &brain &nervesnerves

ROS affect different tissues and tissue components.

They affect e.g. not only the smooth muscle cells, but also their epithelium, endothelium, innervation, membrane lipids, receptors, transmitter systems, prostanoid production, Ca2+ homeo- stasis, etc.)

Effects of H2O2 on guinea pig ileum

wh

ole

ile

um

lon

git

ud

inal

mu

scle

wh

ole

ileu

m untreated atropine & guanethidine

treated

1mol/l Hi5 min

10 mN

0.5 mmol/l H2O2

Institute of Experimental Pharmacology, SASc, Bratislava, SK

2 min

Effects of various ROS on guinea pig trachea

H2O2

OH (H2O2 + FeSO4)

OH (FeSO4 + Ascorbic acid)

O2 - (Xanthine + Xanthine oxidase)

1 mN

Institute of Experimental Pharmacology, SASc, Bratislava, SK

Proposed mechanisms of ROS actions in airways

No changes

Dominantcontraction

Dominantrelaxation

Dia

met

ero

f tr

ach

ea

Restingtone

Initial changeof tone

after ROS

Latechangeof tone

after ROS

Dominantcontraction

Long-lastingcontraction

Intensivecontraction

Dia

met

ero

f tr

ach

ea

Restingtone

Initial changeof tone

after ROS

Late changeof tone

after ROS

In physiological conditions In pathological conditions

O2.-

H2O2

.OH

O2.-

H2O2

.OH

epithelium

smooth muscleSOD- superoxide dismutase; Cat – catalase; LMWAO – low molecular weight antioxidants Institute of Experimental Pharmacology, SASc, Bratislava, SK

NO reacting with O2-- gives rise to

unstable peroxynitrite, which decom-poses also to the most toxic OH.

Because of the large energy gain of the reduction of OH to H2O, this radical reacts instantaneously with any biological molecule in its immediate environment by abstracting hydrogen atom.

Production of ROS in endothelium and neutrophils

l-argininel-arginine NOSNOS

OO2 2 NADPH NADPH NONO

ATP AMP adenosineATP AMP adenosine

inosine hypoxanthineinosine hypoxanthine OO22

XDH XOXDH XO

uric acid uric acid OO22--

OH HOH H22OO22

OO22

HH22OO22

HOClHOCl

NADPHNADPH oxidaseoxidase

NADPHNADPH

MPOMPO

MPOMPO

NADPNADP++

eennddootthheelliiuumm

nneeuuttrroopphhiill

OHOH

NONO

OO22--

OONOOONO--OHOH

Institute of Experimental Pharmacology, SASc, Bratislava, SK

CAT – catalaseSOD – superoxide dismutase

Elimination by SOD with CAT of the effects of FMLP activated neutrophils (NEUT) generating O2

-- on noradrenaline (NA) precontracted rat aorta

Institute of Experimental Pharmacology, SASc, Bratislava, SK

Effects of ROS on the endothelium and development of atherosclerosis

atherosclerotic lesion cell proliferation release of growth factoratherosclerotic lesion cell proliferation release of growth factor

active oxygen,active oxygen, recruitment of collagenase, elastase, adherencerecruitment of collagenase, elastase, adherence macrophages lipases, proteases of plateletsmacrophages lipases, proteases of platelets

Plasma Plasma endothelial cellsendothelial cells

LDL LDL

Intima Intima activatedactivated oxygenoxygen Fatty Streak iron/copperFatty Streak iron/copper

Oxidatively modified LDLOxidatively modified LDL apoB-bound 4-hydoxynonenal, oxidized lipids,apoB-bound 4-hydoxynonenal, oxidized lipids, fatty acid hydroperoxidesfatty acid hydroperoxides

Monocyte

Monocyte

Tissuemacrophages

Foam cells

Membrane Membrane damagedamage

based on J.P. Kehrer (1993)

Diseases that may have ROS related pathogenesis

IAirways Normobaric hyperoxic injury Bronchopulmonary dysplasia Idiopathic pulmonary fibrosis Respiratory distress syndromes (ARDS, IRDS) Emphysema Chronic bronchitis & asthma bronchiale Asbestosis Inhaled pollutants, smoke, chemicals (e.g. paraquat,

bleomycin) & oxidants (e.g. SO2, NOx, O3)

Gut Ischemia/reperfusion Crohn’s disease Ulcerative colitis & necrotizing enterocolitis Gastric & intestinal ulcers Chemicals (e.g. NSAID)

IIHeart and vessels Ischemia/reperfusion (after infarction, transplantation) Chemicals (e.g. ethanol, doxorubicin) Atherosclerosis/hypertension Selenium deficiency Vasculitis

Brain and nerves Hyperbaric hyperoxic injury Parkinson's disease Alzheimer’s disease (details see in the next panel) Amyotrophic lateral scleroses Neuropathies (e.g. diabetic) Neurotoxins (e.g. 6-hydroxydopamine, MPTP) Vitamin E deficiency Neuronal ceroid lipofuscinoses Traumatic injury/hemorrhage/inflammation Ischemia/reperfusion HIV-dementia Multiple sclerosis

ALZHEIMER DISEASE ALZHEIMER DISEASE and oxidative stressand oxidative stress

Protein oxidation Protein oxidation (carbonyls) - „crosslinking“ (carbonyls) - „crosslinking“ Fe in neurons with fibrilary aggregates Fe in neurons with fibrilary aggregates ((-hyperphosphoryl.protein)-hyperphosphoryl.protein) Content of aluminium in neurons with fibrilllary aggregatesContent of aluminium in neurons with fibrilllary aggregates -amyloid generation -amyloid generation (direct cytotoxic action,(direct cytotoxic action, Ca Caii, generation of , generation of ROSROS

even in the absence of Meeven in the absence of Me2+2+)) Activity of microglia Activity of microglia (brain macrophages = (brain macrophages = ROSROS source)source) Activity of CAT without Activity of CAT without SOD activity resulting in SOD activity resulting in HH22OO2 2 andand OHOH Generation of lipid hydroperoxides and reactive cytotoxic aldehydes Generation of lipid hydroperoxides and reactive cytotoxic aldehydes

(e.g. HNE)(e.g. HNE)

Therapeutic interventionsTherapeutic interventions:: antioxidants and ROS scavengers antioxidants and ROS scavengers (e.g. U-74500A, U-78517F, U-83836E, vitamines E,C), (e.g. U-74500A, U-78517F, U-83836E, vitamines E,C), chelators, CAT, deprenylchelators, CAT, deprenyl

IIIBlood Chemicals (e.g. phenylhydrazine, primaquine,

sulphonamides, lead) Protoporphyrine photooxidation Malaria Anemias (sickle cell, favism)

Liver Ischemia/reperfusion Chemicals (e.g. halogenated hydrocarbons, quinones, ethanol, acetaminophen) Accumulation of iron or copper Endotoxin

Kidney Autoimmune nephrosis (inflammation, e.g. glomerulonephritis) Chemicals (e.g. aminoglycosides, heavy metals)

IVPancreas Acute & chronic pancreatitis Diabetes mellitus

Eye Retinopathy of prematurity Photic retinopathy Cataracts Laser photoablation

Skin Radiation (solar, ionising) Thermal injury Chemicals (photosensitizers, e.g. tetracyclines) Contact dermatitis Porphyria

VMuscle Muscular dystrophy Multiple sclerosis Exercise

Others Aging Pregnancy and newborn complications Radiation injury Cancer Chemicals (e.g. alloxan, iron overload, radiosensitizers) Autoimmune diseases (e.g. rheumatoid arthritis, lupus

erythematodes) Inflammation (in general)

Potential antioxidant therapy I Inhibitors of ROS synthesis NADPH-oxidase Inhibitors

Flavoprotein inhibitors (FAD analogs, antibodies of cytP450 reductase)

Agents forming complexes with Fe2+ in cyt b (butylisocyanide, imidazole, pyridine) Mg2+(enabling FAD binding),Fe2+ ,Cu2+ chelators (bathophenantroline, EDTA, EGTA, deferoxamine, bilirubin)

Thiol reagents (N-ethylmaleimide, 1-naphtol, 1,4- naphtoquinone)

NADPH analogs (NADPH 2,3-dialdehyde) Inhibitors of metabolism of AA and PLA2 IMAO (Deprenyl) Others (corticosteroids, diphenyliodonium) Inhibitors of xanthine oxidase (tungsten, oxypurinol, allopurinol, pterinaldehyde, folic acid) Antibodies against leukocytes

IIAgents supporting and complementing enzymatic protective systems Superoxide dismutase (SOD) SOD (Lip-SOD,PEG-SOD) Copper diisopropylsalicylate

SOD mimetics Catalase (Cat) Cat (Lip-CatTP, Peg-CatTP) Glutathionperoxidase (GTPx) GSH, GSH methylester, GSH diethylmaleate Low m.w. thiols (e.g. cystein) High m.w. thiols (e.g. albumin) L-2-oxothiazidolidine-4-carboxylate N-acetylcysteine Ebselen Selenium Lactoperoxidase & DT-diaphorase

III Drugs interfering with iron and copper metabolism

(deferoxamine, hemopexine, ferritin, transferrin, lactoferrin, ceruloplasmin, serum albumin)

Antioxidants Vitamins and their analogues (vitamin E, vitamin C, carotenoids, oxycarotenoids) Phenol derivatives (eugenol, guajacol, probucol, N,N-diphenyl- phenylendiamine) Flavone derivatives (flavonoids, isoflavonoids, allirazine, green tea) Indol derivatives (stobadine, carvedilol, melatonin, -carbolines) Xanthine derivatives (allopurinol, oxypurinol, uric acid) 21-amino steroids (lazaroids) Antiinflammatory drugs (piroxicam, flufenamic acid, mefenamic acid, hydroquinone, sulindac, fenylbutazone, indomethacin, ibuprofen, naproxen, levamisole, sulfasalazine, acetylsalicylic

acid) Hypolipidemics (lovastatin) Proteins (albumin)

IV

Agents containing sulfur (cysteine, cysteamine, GSH, dithiothreitol, N-acetylcysteine, ACE inhibitors, dimethylthiourea, thiourea, thiomalate, hypotaurine, taurine, penicillamine, 2-amino-2-thiazole, dihydrolipoate, -mercaptopropionyl glycine, N-2-mercaptopropionyl glycine, -mercaptoethanole, D,L-methionine, other low and high m.w. thiols) Nitroso compounds ( .NO, nitrosopine) Other drugs (-adrenolytics, H2-antihistaminics, calcium channel blockers, pentoxyphylline, carbanilates, urea, bilirubin, glucans,

manitol, glucose, 2-methylaminochromans, DMSO, BHT, BHA, 2-MEA, etoxiquin, -lipoic acid, Zn2+)

V

Inhibition of O2.- formation

Nonsteroid antiflogistics Antiasthmatics (adrenomimetics, corticoids, methylxanthines) Prostaglandins Flavonoids Antibiotics (e.g. minocycline) Antimalarics Inhibitors of ACE Dipyridamol

VI/aScavenging or removal of ROS Scavenging of generated O2

.-

Flavonoids & other natural products Vitamins E, C, A(-carotene)Synthetic analogs of PGB2DipyridamolPentoxiphyllineAntibiotics.NO donors

5-acetylsalicylic acid Uric acidScavenging HOCl

Uric acid Taurine, hypotaurineScavenging or quenching of 1O2

Silymarine -carotene Vitamin E Stobadine

VI/bScavenging or removal of ROS Removal of H2O2

Catalase (not working in the presence of .NO)

N-acetylcysteineElimination of OH.

ManitolThiourea

Stobadine Melatonin Probucol 5-acetylsalicylic acid Lazaroids DMSO, DMTU, BHT Uric acid Glucose

Some positive results from preclinical and clinical studies with thepyridoindole STOBADINE, which possesses significant antioxidant, mainly hydroxyl radical scavenging, lipid oxidation chain breaking

and singlet oxygen quenching properties, as an example, are presented in the following panels.

VI/cScavenging or removal of ROS Lipid oxidation chain breaking antioxidants

(anti LO. and LOO.) Bilirubin Vitamins E Vitamin C -carotenoids and oxycarotenoids Stobadine Melatonin -lipoic acid Uric acid Lazaroids BHT, BHA Ehoxyquin 2-methylaminochroman

Protection by STOBADINE (STB) of the acetylcholine induced relaxation in rat aortic rings

caused by reversible occlusion of aorta in vivo (I/R)

05

1015

2025

3035

Sham I/R I/R + STB

% o

f M

ax. R

elax

. N

H3C

H

NCH3

H

H.2HCl

STOBADINEI – ischemia; R – reperfusion P<0.05 I/R vs Sham P<0.05 I/R+STB vs I/R

Institute of Experimental Pharmacology, SASc, Bratislava, SK

STOBADINE (STB) effect on experimetal myocardial infarction (MI) in dogs

3hr occlusion of the 3hr occlusion of the anterior descendent anterior descendent branch of the left branch of the left coronary arterycoronary artery

Stobadine (1 mg/kg iv) Stobadine (1 mg/kg iv) given 30 min after the given 30 min after the occlusionocclusion

Reduction of the Reduction of the infarction area by 28% infarction area by 28% (( PP < 0.05) < 0.05)0

5

10

15

20

25

C STB

MI

area

%

Institute of Experimental Pharmacology, SASc, Bratislava, SK

STOBADINE effect on transmission in rat hippocampal slices during hypoxia/reoxygenation

A-control , B-hypoxia, C- reoxygenation in untreated slices, D-reoxygenation in stobadine treated slices Institute of Experimental Pharmacology, SASc, Bratislava, SK

Effects of STOBADINE on acetic acid (AA)induced colitis in rats

Dose

mg/kg

Damage

score

Wet/dry

weight

ratio

Myelo-

peroxidase

unit/g ww

Residual

volume

µl/cm/h

GSH

nmol/g/

min

Evans blue

µg/g ww

Sham 0 4.3±0.3 8.6±1.9 140±11.5 399±23.4 6.9±0.62

AA+vehicle

4.3±0.3 5.3±0.2

33.9±5.6

213±11.3

171±14.5

86.8±14.5

AA+Stobadine 5

3.7±0.7 5.1±0.1

20.1±2.5

202±10.7

215±14.3

48.4±5.6

AA+Stobadine 10

2.1±.2

5.0±0.1

12.1±2.7

172±17 249±23.7

36.8±3.9

AA+Stobadine 20

1.2±0.2

4.8±0.1

8.2±3.3

138±7.2

302±21.8

29.5±2.7

P<0.05 AA+ vehicle or AA+ Stobadine vs Sham P<0.05 AA+Stobadine vs AA+vehicle Institute of Experimental Pharmacology, SASc, Bratislava, SK

Effects of drugs on hydrogen peroxide induced contractions of the guinea pig trachea

PO.O5

Institute of Experimental Pharmacology, SASc, Bratislava, SK

Therapeutic relevance of the use of antioxidantsI DISEASE ANTIOXIDANT THERAPEUTIC

SUCCESSCardiovascular Carotenoids

Ascorbic acidTocopherolsSelenProbucolFlavonoids

-++

Newborn hypoxia induced injuries

PenicillamineTocopherols

++

Ischemia/Reperfusion (of the heart, brain, gut, kidney)

SOD, SOD+CATLipoic acidAllopurinolTocopherolsDeferoxamine

++

Transplantation and tissue preservation

SOD, SOD+CATTocopherolsAscorbic acidCarotenoids

++++

II DISEASE ANTIOXIDANT THERAPEUTIC SUCCESS

Intravascular hemorrhage TocopherolsAscorbic acid

++

Platelets aggregation FlavonoidsStobadine

++

Hemochromatosis Deferoxamine ++Head trauma (details see in the next panel)

LazaroidsStobadine derivativesPhenyl-butyl-nitones

++

Subarrachnoidalhemorrhage

Lazaroids

Respiratory distress syndromes (IRDS, ARDS)

SODAllopurinolTocopherols

+++

Bronchial asthma SOD+CATThiolsTocopherols

- - -

Pulmonary injuries N-acetylcysteineThiols

++

ROS in the sequence of events in ROS in the sequence of events in NEUROTRAUMANEUROTRAUMA

TRAUMATRAUMA Excitatory aminoacid release (GLU)Excitatory aminoacid release (GLU)

CaCa2+ 2+ influxinflux into the cells into the cells Activ. of inflam. cascade Activ. of inflam. cascade Protease/lipase activation (PAF, eikosanoids,Protease/lipase activation (PAF, eikosanoids, Cell depolarizationCell depolarization cytokines, PMN activ.) cytokines, PMN activ.) ((Mg block of NMDA rec.) Mg block of NMDA rec.) ROS generationROS generation

NaNa++influxinflux cell devastation cell devastation EdemaEdema

NEURONAL DEATHNEURONAL DEATH

TRIAD : TRIAD : EXCITOTOXICITYEXCITOTOXICITY, , Ca-OVERLOADCa-OVERLOAD, , OXIDATIVE STRESSOXIDATIVE STRESS

Therapeutic interventionsTherapeutic interventions: -SH donors (N-acetylcysteine), lazaroids,: -SH donors (N-acetylcysteine), lazaroids, steroids, deferoxamine, SOD, vitamines A,E,C, pyridoindoles, steroids, deferoxamine, SOD, vitamines A,E,C, pyridoindoles,

stobadine, PBN, flavonoids (quercetine), PAF antag. (BN 520210) stobadine, PBN, flavonoids (quercetine), PAF antag. (BN 520210)

III DISEASE ANTIOXIDANT THERAPEUTIC SUCCESS

Flu (cold) Ascorbic acid Retrolental fibroplasia Tocopherols +Cataract Tocopherols +Inflammatory diseases of the gut (IBD)

5-aminosalicylatesSulfasalazineSulfapyridineSOD+CATGlucans

+++

Hepatopathies Lipoic acidSilymarinStobadine

+

Paracetamol intoxication N-acetylcysteine ++Chemical poisonings Glutathione

Deferoxamine++

Photosensibilization CarotenoidsTretionin

+++

UV irradiation Carotenoids ++

IV DISEASE ANTIOXIDANT THERAPEUTIC SUCCESS

Rheumatoid arthritis SODPenicillamineDeferoxamine

+

Parkinsomism Tocopherols

Wilson’s disease Penicillamine ++

Cerebro-vascular spasms CarotenoidsTocopherolsThiols

Cancer SOD+CATCarotenoidsAscorbic acidTocopherolsSelenFlavonoidsThiols

-

-

Antianginal effect of STOBADINE (STB) Phase II clinical study

Patients with angina Patients with angina pectoris (stable and effort) pectoris (stable and effort) (n = 13)(n = 13)

Effect of 4 week treatment Effect of 4 week treatment with with STOBADINESTOBADINE (up to (up to 100 mg/day p.o.)100 mg/day p.o.)

Significant decrease in the Significant decrease in the No. of anginal attacksNo. of anginal attacks

SignificantSignificant ( (* P P<0.05)<0.05) decrease in the No. of decrease in the No. of selfadministered selfadministered nitroglycerine tabletsnitroglycerine tablets

02468

-1 2 3 4 5w e e k s

Numb

er / w

eek

Attacks Nitroglycerine tbl.S T B

***

*

** *

*

Institute of Experimental Pharmacology, SASc, Bratislava, SK

increase of membrane lipid peroxidation increase of prostaglandin production increase of intracellular free calcium alteration of conductivity of ion channels alteration of enzyme activity alteration of release/action of neurotransmitters reduction of half-life of biologically active substances damage of proteins damage of DNA, genes and protein synthesis damage of carbohydrates

Conclusions IROS act by:Conclusions IROS act by:

Mechanisms of ROS action differ in various biological tissues.Their actions depend upon condition of the tissue itself, the

corresponding epithelium, endothelium, innervation, etc.

reduction of their generationreduction of their generation - elimination of undesirable physical and chemical influences - protection of tissues from chronic inflammation - protection of tissues from ischemia

their eliminationtheir elimination - substitution with antioxidant enzymes - substitution with non-enzyme antioxidants and scavengers

interaction with their effectsinteraction with their effects - protection of cells from intracellular free calcium accumulation and its effects

Conclusions IIThe effects of ROS could be prevented or

stopped by:

Conclusions IIITherapeutic success with the use of

antioxidants, quenchers and scavengers

There areThere are Promising clinical resultsPromising clinical results

e.g. in photosensibilization, paracetamol intoxication, e.g. in photosensibilization, paracetamol intoxication, hemochromatosishemochromatosis

Controversial clinical resultsControversial clinical resultse.g. in ischemia/reperfusion, subarachnoidal hemorrhage, e.g. in ischemia/reperfusion, subarachnoidal hemorrhage,

respiratory distress syndromesrespiratory distress syndromes Minimal therapeutic effects Minimal therapeutic effects

e.g. in asthma bronchiale, cancere.g. in asthma bronchiale, cancer