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RECENT TRENDS
IN
PHARMACOLOGY OFNITRIC OXIDE
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OVERVIEW
Nitric oxide(NO)/ Endothelium Derived RelaxingFactor (EDRF)-Properties
Synthesis, Mechanism of action and NO Signalling
Physiological Role- In Body systems
Role in diseases/pathophysiological states, cancer
Pharmacology : NO- related drugs NOS modulating drugs activators and inhibitors
NO Donors : Classification, clinical potential
NO and gene therapy, stem cell-based therapy
and nutraceuticals Future prospects and Conclusion
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Background Information
Prior to 1990: An air pollutant Named Molecule of the Year by Science magazine in 1992
Robert Furchgott, Louis J Ignore, Ferid Murad:
Nobel Prize 1998
Properties of NO:
Small water and lipid soluble gas
Gaseous free radical
Three interchangeable forms:NO: Nitric Oxide
NO+: Nitrosonium cation
NO- : Nitroxyl Radical
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EDRF/NO!
EDRF was claimed to be NO by (Ignarro,
1989, Furchgott, 1990 and Skvaril,
2000,.others).
EDFR/NO system presented in manytissues, is a regulatory system likely to be
important physiologically.
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NOSs, is a family of related enzymes encoded
by separate genes. It is one of the most
regulated enzymes in biology.
Three known isoforms, two are constitutive(cNOS) and the third is inducible (iNOS)
(NOS2).
Nitric Oxide Synthases
Source: (Majano et al., 1998) (Tylor et al., 1997) (Freid Murad.1998 )
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NOSs
Constitutive
Neuornal
NOS1
bcNOS
Endothelial
NOS3
ecNOS
Inducible
NOS2
iNOS
Nitric Oxide
Synthases
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Nitric oxide synthesis involves
arginine, oxygen, and nicotinamideadenine dinucleotide phosphate
(NADPH).
Nitric Oxide Biosynthesis
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L-arginine
NOSsL-
Citrulline
Nitric Oxide Biosynthesis
Nitrite (NO2-)Nitrate (NO3-)
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Synthesis of Nitric Acid
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Nitric Oxide Synthase oxidizes the quanidine
group of L-arginine in a process that consumes
five electrons and results in the formation of NO
with stoichiometric formation of L-citrulline.
The process involves the oxidation of NADPHand the reduction of molecular oxygen.
The transformation occurs at a catalytic siteadjacent to a specific binding site of L-arginine.
Nitric Oxide Synthesis
Source: (Ignarro, 2001)
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Activation of NOS
Glutamate neurotransmitter binds to NMDA receptors
Ca++ channels open causing Ca influx into cell
Activation of calmodulin, which activates NOS
Mechanism for start of synthesis dependent on body
system
NO synthesis takes place in endothelial cells, lung cells,
and neuronal cells
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Http://www.kumc.edu/research/medicine/biochemistry/bioc800/sig02-06.
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Types of NOS
NOS I
Central and peripheral neuronal cells
Ca+2 dependent, used for neuronal communication
NOS II
Most nucleated cells, particularly macrophages
Independent of intracellular Ca+2
Inducible in presence of inflammatory cytokines
NOS III Vascular endothelial cells
Ca+2 dependent
Vascular regulation
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Constitutive Inducible
Cytosolic
NADPH dependent
Dioxygenase
Inhibited by L-arginineanalogues
Ca2+/calmodulin dependent
Picomoles NO released
Short-lasting release
Unaffected by glucocorticoids
Cytosolic
NADPH dependent
Dioxygenase
Inhibited by L-arginineanalogues
Ca2+/calmodulin independent
Nanomoles NO released
Long-lasting release
Induction inhibited by
glucocorticoids
Source: (Moncada et al., 1991)
cNOS/iNOS
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Neuronal NOS
(nNOS or NOS1)
Inducible NOS
(iNOS or NOS2)
Endothelial NOS
(eNOS or NOS3)
Originally cloned
from
Neuronal Cell Macrophage Endothelial Cell
Chromosome
localization
NOS1,
Chromosome 12
12q24.2
NOS2,
Chromosome 17
17 cen-q11.2
NOS3, Chromosome
7
7q35-7q36
Ca dependent Ca dependent
(Ca-dystrophin)
Ca-Calmodulin
independent
Ca dependent
(Ca-Calmodulin)
Ca independent
eNOS/nNOS/iNOS
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Nitric Oxide Signaling
Enzyme Gene No. of
exons
No. of
residues
Subcellular
location
Regulation
nNOS NOS1 29 1429-
1433
Mainly
soluble(brain);
Ca2+/CaM
iNOS NOS2 27 1144-
1153
Mainly
soluble
Ca2+
independent
eNOS NOS3 26 1203-
1205
Mainly
particulate
Ca2+/CaM
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Neuronal Nitric Oxide Synthase (nNOS)
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Epithelial Nitric Oxide Synthase (eNOS)
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Inducible Nitric Oxide Synthase (iNOS)
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Mechanism of Action of NO
Various stimuli5
- HTAcetylcholineThrombinCalcium ionophore A23187Arachidonic AcidChanges in AP &ES
NO Release
Platelet antiaggregation &Vasorelaxation effect
Prostacyclin
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Contd NO
NO bind to Fe 2+haem groupof Guanylyl Cyclase
Active Guanylate Cyclase
Increased cGMP
Increased intracellular Ca 2+
Relaxes muscle
Dilating the vessel &
lowering B.P.
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Under normal basal conditions in blood vessels,NO is continually being produced by cNOS but the
activity of iNOS is very low.
During inflammation the amount of NO producedby iNOS may be a 1,000-fold greater than that
produced by cNOS. The activity of iNOS isstimulated during inflammation by bacterial
endotoxins (e.g., lipopolysaccharide) and cytokines
such as tumor necrosis factor (TNF) and
interleukins. Source: (Archer, 1993) (Davies et al., 1995)
Nitric Oxide Synthesis
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The chemical effect of NO n biologicalsystems is
extensive and complex, it is divided into twomajor categories:
Direct Those reactions fast enough to occur between NO and
specific biological molecules.
Indirect Do not involve NO, but rather are mediated by reactive
nitrogen oxide species (RNOS) formed from the reactionof NO either with oxygen or superoxide. RNOS formedfrom NO can mediate either nitrosative or oxidative
stress. Source: (Wink and Mitchell, 1998)
Nitric Oxide
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Oxidation of iron containing proteins such as
ribonucleotide reductase and aconitase,
Activation of the soluble guanylate cyclase,
ADP ribosylation of proteins,
Protein sulphhydryl group nitrosylation, and iron
regulatory factor activation (Shami et al., 1995).
.
Mechanism of Action
(Source: Shami et al., 1995)
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NO acts through the stimulation of thesoluble guanylate cyclase which is a
heterodimeric enzyme withsubsequent formation of cyclic GMP.
Cyclic GMP activates protein kinasesand leads ultimately to the
dephosphorylation of the myosine light
chain. Source: (Denninger and Marletta, 1999)
Mechanism of Action
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Intracellular calciumNitric Oxide SynthaseNitric OxideGuanylate cyclaseCyclic GMPProtein kinase GProtein phosphatase
Phosphodiestrase
NO/cGMP signaling processes (Davies et al., 1997)
Nitric Oxide
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NO is an important messenger moleculeinvolved in many physiological and
pathological processes within themammalian body both beneficial and
detrimental.
Source: (Kane et al., 1997)
Nitric Oxide
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Functions as a signaling molecule thattells the body to make blood vessels
relax and widen. This physiologicalreaction is important when the bodyneeds more blood.
Works as a signaling molecule in thecardiovascular system, the nervoussystem, and in other tissues,..
Nitric Oxide: Signaling Molecules
Source: (Kuwana, 1998)
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Appropriate levels of NO production are
important in protecting an organ such as theliver from ischemic damage.
Sustained levels of NO production result indirect tissue toxicity and contribute to thevascular collapse associated with septicshock.
Chronic expression of NO is associatedwith various carcinomas and inflammatoryconditions, including juvenile diabetes,multiple sclerosis, arthritis and ulcerativecolitis,.. Source: (Tylor etal., 1997)
Nitric Oxide
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The synthesis of NO by vascular endothelium is responsiblefor the vasodilator tone that is essential for the regulation ofblood pressure.
NO also contributes to the control of platelet aggregation and
the regulation of cardiac contractility. It is now established that NO is the physiological mediator ofpenile erection.
In the central nervous system, NO is a neurotransmitter thatunderlines several functions including the formation ofmemory.
In the periphery, there is a widespread network of nerves,previously recognized as nonadrenergic and noncholinergic,that operate through a NO dependent mechanism to mediatesome forms of neurogenic vasodilatation and regulate variousgastrointestinal, respiratory, and genitourinary tract functions.
Physiological Role
Source:(Moncada and Higgs, 1993)
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Nitric Oxide cGMP Pathway
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Physiology of Nitric oxide
NO play important role :
Penile erection
Lung vasodilatation
Physiological stimuli for generation of NO are not fullyunderstood, but pulsatile flow and shear forces may be themain determinant.
In biological system NO is not stored and diffuses freely to its
site of action where it bind covalently to its effectors (t1/2=3-5second)
In coronary artery disease, basal level of NO as well asstimulated release of NO reduced
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What is the role of Nitric Oxide in
the human body? Nitric Oxide in the human body has
many uses which are best
summarized under five categories.
NO in the nervous system NO in the circulatory system
NO in the muscular system
NO in the immune system NO in the digestive system
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Nitric Oxide in the Nervous
System
Nitric oxide as a neurotransmitter
NO is a signaling molecule, but not necessarily a neurotransmitter
NO signals inhibition of smooth muscle contraction, adaptive
relaxation, and localized vasodilation
Nitric oxide believed to play a role in long term memory
Memory mechanism proposed is a retrograde messenger that
facilitates long term potentiation of neurons (memory)
Synthesis mechanism involving Ca/Calmodulin activates NOS-I
NO travels from postsynaptic neuron back to presynaptic neuronwhich activates guanylyl cyclase, the enzyme that catalyzes cGMP
production
This starts a cycle of nerve action potentials driven by NO
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Is Nitric Oxide a
neurotransmitter?
NO serves in the body as a neurotransmitter, but there
are definite differences between other neurotransmitters
used commonly in the body
NO is synthesized on demand vs. constant synthesis
NO diffuses out of the cells making it vs. storage in vesicles and release
by exocytosis
NO does not bind to surface receptors, but instead exits cytoplasm,
enters the target cell, and binds with intracellular guanylyl cyclase
Similarities to normal NTs Present in presynaptic terminal
Natural removal from synaptic junction
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Nitric Oxide in the Circulatory
System
NO serves as a vasodilator
Released in response to high blood flow rate and signaling
molecules (Ach and bradykinin)
Highly localized and effects are brief
If NO synthesis is inhibited, blood pressure skyrockets (Diagram of vasodilation mechanism after muscular system)
NO aids in gas exchange between hemoglobin and cells
Hemoglobin is a vasoconstrictor, Fe scavenges NO
NO is protected by cysteine group when O2 binds to hemoglobin
During O2 delivery, NO locally dilates blood vessels to aid in gas
exchange
Excess NO is picked up by HGB with CO2
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Nitric Oxide in the Muscular
System NO was orginally called EDRF (endothelium
derived relaxation factor)
NO signals inhibition of smooth muscle contraction
Ca+2 is released from the vascular lumen activating NOS
NO is synthesized from NOS III in vascular endothelialcells
This causes guanylyl cyclase to produce cGMP
A rise in cGMP causes Ca+2 pumps to be activated, thus
reducing Ca+2 concentration in the cell
This causes muscle relaxation
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Http://www.kumc.edu/research/medicine/biochemistry/bioc800/sig02-1
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Nitric Oxide in the Immune
System
NOS II catalyzes synthesis of NO used in host defense
reactions
Activation of NOS II is independent of Ca+2 in the cell
Synthesis of NO happens in most nucleated cells,particularly macrophages
NO is a potent inhibitor of viral replication
NO is a bactericidal agent
NO is created from the nitrates extracted from food near thegums
This kills bacteria in the mouth that may be harmful to the
body
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Nitric Oxide in the Digestive
System
NO is used in adaptive relaxation
NO promotes the stretching of the stomach inresponse to filling.
When the stomach gets full, stretch receptors
trigger smooth muscle relaxation through NO
releasing neurons
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New research ideas involving
Nitric Oxide
The role NO might play in neuronal
development
The mechanism of NO inhibiting the different
forms of NOS
Diazeniumdiolates as NO releasing drugs
Excessive NO release as the cause of most
brain damage after stroke
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Cond.
NO inhibitor of platelet activation
Alteration in formation of NO
Vasoconstriction, Platelet adhesion and Aggregation
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Contd
Isosorbide dinitrate
NITRIC OXIDE
Reduced Platelet deposition & Increased survival timein patients with peripheral vascular disease
Nit i O id d Di
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NO
HypertensionAtherosclerosis
Diabetes
NANCdysfunction
Inflammati
on
Coagulopathies
Organdysfunction
Vascularinjury
Ischemia
reperfusioninjury
Vasospasm
Microcirculatory
dysfunction
Neuronalfunction
Nitric Oxide and Diseases
++
+
+
+
+
+++
+
+
+
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Vascular actions of NO
Direct vasodilation (flow dependent and receptormediated)
Indirect vasodilation by inhibiting vasoconstrictor
influences (e.g., inhibits angiotensin
IIand sympathetic vasoconstriction) Anti-thrombotic effect - inhibits platelet adhesion to
the vascular endothelium
Anti-inflammatory effect - inhibits leukocyte
adhesion to vascular endothelium; scavengessuperoxide anion
Anti-proliferative effect - inhibits smooth muscle
hyperplasia
NO when its production is C
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NO, when its production is
impaired or its bioavailability is
reduced, the following can
result:
Vasoconstriction (e.g.,coronary vasospasm, elevated
systemic vascular resistance,
hypertension)
Thrombosis due to platelet
aggregation and adhesion tovascular endothelium
Inflammation due to
upregulation of leukocyte and
endothelial adhesion
molecules Vascular hypertrophy and
stenosis
Conditions Associated with
Abnormal NO Production
and Bioavailability
Hypertension
Obesity
Dyslipidemias (particularly
hypercholesterolemia and
hypertriglyceridemia)
Diabetes (both type I andII)
Heart failure
Atherosclerosis
Aging
Cigarette smoking
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NO, plays a variety of roles, which are at timescontradictory.
NO, has a dual complex action, at least dual, ontumor growth that may depend on the local
concentration of NO.
Additional factors such as the presence of ROS,and the type of tumor and its susceptibility to NO.
Nitric Oxide and Cancer
Source: (Jenkins et al., 1995) (Wink et al., 1998)
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Low NO concentration
Pro-angiogenic and protumor growth
Higher NO concentration Inhibit mitochondrial respiration, the citric acid cycle
glycolysis, and DNA replication. Locally high levels of
reactive oxygen species (ROS), insufficient oxygen supply
to the tumor tissue, may exacerbate these toxic effects by
generating even more reactive compounds, such asperoxynitrite (ONOO-). The latter compound arises from
the diffusion limited interaction of NO and O2 and is even
more reactive than NO.
Source: (Vamvakas and Schmidt, 1997 and Masuda et al., 2002)
Nitric Oxide
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Nitric Oxide
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Inhalation of 25 ppm of mixed nitrogenoxide, the recommended threshold
limit, may cause pulmonary irritation.
Higher doses of NO may cause
minimal irritation initially but result inhemorrhagic pulmonary edema
several days later.
Toxicology of NO
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Individuals exposed to nitrogen oxide
should be carefully monitored and receive
supportive care, including supplemental O2,
morphine and steroid therapy.
NO must be handled with extreme caution.
On contact with air NO interacts with O2,producing a dimeric form of nitrogen
dioxide, a reddish- brown gas (Archer.,
1993).Source: (Archer., 1993)
Nitric Oxide
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More about NO
Nitric oxide and hypertension (Leclercq et al., 2002) Nitric oxide and pulmonary system ( Persson et al.,1994)
Nitric oxide and the nervous system (Kuwana, 1998) Nitric oxide and gastrointestinal tract (Miller and
Sandoval, 1999) Nitric oxide in liver failure (Tomas et al., 1992) Nitric oxide levels in patients after trauma and during
sepsis (Tylor et al., 1997).
Nitric oxide as a cytostatic and cytotoxic agent(Moncada and Higgs, 1993).
Nitric oxide in immunity and inflammation (Hadas et al.,2002)
Nitric oxide and cancer. ..
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Nitric Oxide Signaling
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Arg NO
GTP cGMP
5) NO binds to Guanyly l c yclase
Relaxationof smooth muscle
NO
Smooth muscle cellblood vessel wall
4)NO diffuses
across membranes
2) AChbinds to receptorson endothelial cells
3)Activate NO syn thase
1)Stimulated nerve releases
Acety lchol ine(ACh) atNerve
terminal
Nitric Oxide Signaling
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NO in Ischemic myocardium:
ACE inhibitor
Inhibition in degradation of Bradykinin
Accumulation of Bradykinin and NO
Prevent coronary Vasoconstriction
Increase in coronary blood flow
1.Stimulation of Bradykinin
Receptor
2. Inhibit Kininase II
Reduced Degdn. OfBradykinin
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Contd
Kitakaze et al.(2000) ACE I attenuate both reversible andirreversible myocardial cellular injury via bradykinin/ NO- dependent
manner
ACE I, enalaprilat, improves transmural myocardial perfusion at rest
and after stress and restore impaired sub endothelial coronary flow
and vasodilator reserve .
The effect of Enalaprilat is bradykinin and NO dependent.
ACE I increase Bradykinin and NO:
Potent cardio protection
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Role of NO inHypertension
In hypertension, morphological vascular alterationaffecting
Endothelium
Intima Vascular smooth muscle
Abnormalities of endothelial cells-- vascular resistanceincrease in Arterial Pressure.
Endothelium produce contracting substances: O 2-
Thromboxane A2
Endothelin-1(Peptide)
Endothelin-1: Potent vasoconstrictor
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Contd
Increase in endothelin plasma conc. observed in patients with
primary hypertension compared to normal.
Mitogenic activation described in hypertension is induced by :
Increase in sympathetic activity
Release of vasoactive agents such as endothelin,
Angiotensin II, PG
Basal formation of NO decreased in Hypertension.
Recently Das,U. N. (2004), the overall role of NO and O2 (superoxide anion ) in hypertension
Patient with hypertension have elevated conc. Of super oxideanion , H2O2 ,Lipid peroxides, endothelin, with simultaneousdecrease in eNO, SOD, Vit E and LCPUFAs.
Nitroglycerine was used for many years to treat
" i " ( h i ) d d d bl d fl
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"angina" (chest pain) due to reduced blood flow
in heart arteries without any knowledge of mechanism
Lumen diameter increasesand resistance to blood
flow decreases
Heart
("coronary")
artery
NO
N
O O
O
N
O O
O
N
O O
O
C C C
H H H H H H
N-ONitro
glycerine
We now know nitroglycerine does notact directly but is degraded to NO
NO
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Nitric oxide (NO) is a gaseous signaling
molecule that readily diffuses across cellmembranes and regulates a wide range of
physiologic and pathophysiologic processes
including cardiovascular, inflammation,
immune, and neuronal functions.
Nitric oxide should not be confused withnitrous oxide (N2O), an anesthetic gas.
DISCOVERY OF ENDOGENOUSLY GENERATED NITRIC
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DISCOVERY OF ENDOGENOUSLY GENERATED NITRIC
OXIDE
The first observations of the biologic role ofendogenously generated NO -in rodent macrophagesand neutrophils:
In vitro exposure of these cells to endotoxinlipopolysaccharide resulted in the accumulation ofsignificant amounts of nitrite and nitrate in the cellculture medium.
Furthermore, injection of endotoxin in animalselevated urinary nitrite and nitrate, the two oxidationproducts of NO.
The second observation was made by investigators in
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The second observation was made by investigators in1980 who found that the ability of acetylcholine toelicit relaxation of isolated strips of rabbit aorta was
entirely dependent on the presence of theendothelium.
If the endothelium was removed, the vessel stillexhibited normal relaxation responses to
nitroglycerin, but not to acetylcholine or carbachol. They discovered that following stimulation with
acetylcholine or carbachol, the endothelium releaseda short-lived molecule that resulted in relaxation and
dilation of surrounding vascular smooth muscle. The synthesis of this factor was not affected by
cyclooxygenase inhibitors, indicating that it wasdistinct from endothelium-derived prostacyclin..
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Synthesis
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Synthesis
NO, written as NO to indicate an unpaired electron in itschemical structure, or simply NO, is a highly reactive signaling
molecule that is made in a wide variety of cells, mostprominently neurons, skeletal muscle, endothelial cells, and
certain immune system cells.
In these cells, NO is synthesized by one or more of three
closely related NO synthase (NOS) isoenzymes, each of whichis encoded by a separate gene and named for the initial cell
type in which it was isolated
These enzymes, neuronal NOS (nNOS or NOS-1),
macrophage or inducible NOS (iNOS or NOS-2), andendothelial NOS (eNOS or NOS-3), despite their names, areeach expressed in a wide variety of cell types, often with anoverlapping distribution.
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These isoforms generate NO from the amino acid L-
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garginine in an O2- and NADPH-dependent reaction
This enzymatic reaction involves enzyme-bound
cofactors, including heme, tetrahydrobiopterin, andflavin adenine dinucleotide.
In the case of nNOS and eNOS, NO synthesis is evoked byagents and processes that increase cytosolic calcium
concentrations. Binding of calcium-calmodulin complexes to eNOS and
nNOS leads to enzyme activation.
On the other hand, iNOS is not regulated by calcium, but
is inducible. In macrophages and several other cell types,
inflammatory mediators induce the transcriptionalactivation of the iNOSgene, resulting in accumulation of
iNOS and generation of increased quantities of NO.
Nitric oxide generation from L-arginine and nitric oxide donors and the
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formation of cGMP.
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Signaling Mechanismsof NO
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NO mediates its effects by covalent modification of proteins.
There are three major effector targets of NO :
Metalloproteins
Thiols
Tyrosine Nitration
Metalloproteins NO interacts with metals, especially iron in heme.
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, p y
Soluble guanylyl cyclase (sGC), an enzyme that generatescyclic GMP from guanosine triphosphate (GTP), containsheme, which binds readily to NO.
NO binding to heme results in activation of sGC andelevation in intracellular cGMP levels.
cGMP activates protein kinase G (PKG), whichphosphorylates specific proteins.
Effects ,mediated :
Vasodilatory effects, which are largely mediated by NO-dependent elevations in cGMP and PKG activity.
Inhibitory effect on enzymes that contain iron-sulfurclusters such as the tricarboxylic acid cycle enzymeaconitase.
Inhibits mitochondrial respiration by inhibition ofcytochrome oxidase.
Inhibition of the heme-containing cytochrome P450enzymes - major pathogenic mechanism in inflammatoryliver disease.
Thiols
NO t ith thi l ( d t i i th SH )
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NO reacts with thiols (compounds containing the SH group)
to form nitrosothiols.
In proteins, the thiol moiety is found in amino acid cysteine.
Upon exposure to NO, certain proteins are found toaccumulate nitrosothiols, which can activate or inhibit the
activity of these proteins.
This posttranslational modification, termed S-nitrosylation,
is reversed by chemical reduction by intracellular reducingagents.
The formation of nitrosothiols is not mediated by directreaction of NO with thiols, but rather requires either metals
or oxygen to catalyze the formation of this adduct. NO undergoes both oxidative and reductive reactions,
resulting in the formation of a variety of oxides of nitrogen
that can nitrosylate thiols, nitrate tyrosines,are stable
oxidation products
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Glutathione a major intracellular sulfhydryl-
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Glutathione, a major intracellular sulfhydrylcontaining compound, also interacts with NOunder physiologic conditions to generate S-
nitrosoglutathione, a more stable form of NO.
Nitrosoglutathione may serve as an endogenouslong-lived adduct or carrier of NO.
Vascular glutathione is decreased in diabetesmellitus and atherosclerosis, and this mayaccount for the increased incidence of
cardiovascular complications in theseconditions.
Tyrosine Nitration
NO ffi i l i h id f i i
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NO reacts very efficiently with superoxide to form peroxynitrite(ONOO), a powerful oxidant that leads to DNA damage,irreversible nitration of tyrosine, and oxidation of cysteine todisulfides or to various oxides (SOX).
In several diseases, cellular degeneration, due to apoptoticmechanisms or due to ischemia, leads to excess superoxideproduction, and a consequent increase in peroxynitrite levels.
Numerous proteins have been found to contain nitrotyrosines, andthis modification can be associated with either activation or
inhibition of protein function. However, it is not yet clear whether tyrosine nitration has essential
roles in either physiologic signaling or in the pathology of anydisease.
Protein tyrosine nitration is also used as a marker for the presenceof oxidative and nitrosative stress.
Peroxynitrite-mediated protein modification is regulated by thecellular content of glutathione, which can protect against tissuedamage by scavenging peroxynitrite.
Factors that regulate the biosynthesis and decomposition ofglutathione may have important consequences on the toxicity of
NO.
Inactivation
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Inactivation
The lability of NO is related to its rapid reactionswith metals and reactive oxygen species.
Thus, NO reacts with heme and hemoproteins,including oxyhemoglobin, which catalyzes NOoxidation to nitrate.
NO reactions with hemoglobin may also result inpartial S-nitrosylation of hemoglobin, resultingin transport of NO throughout the vasculature.
NO is also inactivated by superoxide, and
scavengers of superoxide anion such assuperoxide dismutase may protect NO,enhancing its potency and prolonging itsduration of action.
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VASCULAR EFFECTS
Apart from being a vasodilator, NO protects against thrombosis andatherogenesis through several mechanisms
The antithrombotic effects of NO are also mediated by NO-dependent inhibitionof platelet aggregation. Both endothelial cells and platelets themselves contain
eNOS, which acts to regulate thrombus formation
Thus, endothelial dysfunction and the associated decrease in NO generation mayresult in abnormal platelet function. As in vascular smooth muscle, cGMP
mediates the effect of NO in platelets.
an additional inhibitory effect on blood coagulation by enhancing fibrinolysis viaan effect on plasminogen.
reduces endothelial adhesion of monocytes and leukocytes, key features of the
early development of atheromatous plaques.
may act as an antioxidant, blocking the oxidation of low-density lipoproteins andthus preventing or reducing the formation of foam cells in the vascular wall.
. Atherosclerosis risk factors, such as smoking, hyperlipidemia, diabetes, andhypertension, are associated with decreased endothelial NO production, and thusenhance atherogenesis.
NITRIC OXIDE IN DISEASE
SEPTIC SHOCK
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SEPTIC SHOCK
Increased urinary excretion of nitrate, the oxidative productof NO, is a feature of gram-negative bacterial infection.
Lipopolysaccharide components from the bacterial wallinduce synthesis of iNOS, resulting in exaggeratedhypotension, shock, and, in some cases, death.
This hypotension is reversed by NOS inhibitors such as L-NMMA (in humans as well as in animal models.
A similar reversal of hypotension is produced by compoundsthat prevent the action of NO (such as methylene blue), aswell as by scavengers of NO (such as hemoglobin).
However, thus far there has been no correlation between thehemodynamic effects of relatively nonselective NOS
inhibitors and survival rate in gram-negative sepsis. The absence of benefit may reflect the inability of the NOS
inhibitors to differentiate between NOS isoforms or mayreflect concurrent inhibition of beneficial aspects of iNOSsignaling.
NITRIC OXIDE IN DISEASE
INFLAMMATION
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NO is an important microbicide and may have important roles in tissue adapting
to inflammatory states.
However, overproduction of NO may exacerbate tissue injury in both acute and
chronic inflammatory conditions. NO generated during inflammation is involvedin the vasodilation associated with acute inflammation and can interact withsuperoxide to generate peroxynitrite and subsequently modify proteins, lipids,
and nucleotides.
In experimental models of acute inflammation, inhibitors of iNOS have a dose-dependent protective effect, suggesting that NO promotes edema and vascular
permeability.
NO has a detrimental effect in chronic models of arthritis; dietary L-argininesupplementation exacerbates arthritis, whereas protection is seen with iNOS
inhibitors.
Recent studies have shown that NO stimulates the synthesis of inflammatory
prostaglandins by activating cyclooxygenase isoenzyme II (COX-2). Thus,inhibition of the NO pathway may have a beneficial effect on inflammatorydiseases, including joint diseases.
NITRIC OXIDE IN DISEASETHE CENTRAL NERVOUS SYSTEM
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THE CENTRAL NERVOUS SYSTEM NO has been proposed to have a major role in the central
nervous systemas a neurotransmitter, as a modulator of
ligand-gated receptors, or both. NO synthesis is induced at postsynaptic sites in neurons
upon activation of the NMDA subtype of glutamatereceptor, which results in calcium influx and activation ofnNOS.
In several neuronal subtypes, eNOS is also present andactivated by neurotransmitter pathways that lead tocalcium influx.
NO synthesized postsynaptically may function as aretrograde messenger and diffuse to the presynaptic
terminal to enhance the efficiency of neurotransmitterrelease through a cGMP or S-nitrosylation-dependentmechanism.
It has been suggested that a major role for NO is in theregulation of synaptic plasticity, the molecular process
that underlies learning and behavior
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THE PERIPHERAL NERVOUS SYSTEM
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THE PERIPHERAL NERVOUS SYSTEM
Nonadrenergic, noncholinergic (NANC) neurons are widelydistributed in peripheral tissues, especially the gastrointestinal andreproductive tracts
NO as a mediator of certain NANC actions, and some NANC neuronsappear to release NO.
Penile erection is thought to be caused by the release of NO fromNANC neurons; it is well documented that NO promotes relaxationof the smooth muscle in the corpora cavernosathe initiating factor
in penile erectionand inhibitors of NOS have been shown toprevent erection caused by pelvic nerve stimulation in the rat.
Thus, impotence is a possible clinical indication for the use of a NOdonor, and trials have been carried out with nitroglycerin ointmentand the nitroglycerin patch.
An established approach is to inhibit the breakdown of cGMP by thephosphodiesterase (PDE isoform 5) present in the smooth muscle ofthe corpora cavernosa with drugs such as sildenafil , tadanafil
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NITRIC OXIDE IN DISEASERESPIRATORY DISORDERS
NO has been shown to improve cardiopulmonaryfunction in adult patients with pulmonary arteryhypertension
It s administered by inhalation. It has also beenadministered by inhalation to newborns with
pulmonary hypertension and acute respiratorydistress syndrome.
NO may have an additional role in relaxing airwaysmooth muscle and thus acting as a bronchodilator.
For these reasons, NO inhalation therapy is beingwidely tested in both infants and adults with acuterespiratory distress syndrome. The adverse effects ofthis use of NO are being assessed.
PHARMACOLOGIC MANIPULATION OF
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NITRIC OXIDE
NITRIC OXIDE MODULATORS Inhibitors of Nitric Oxide Synthesis
Nitric Oxide Donors
Nitric oxide supplements
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Different Class of Nitric Oxide Donors
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SR Deshpandeet al, 2012
NITRIC OXIDE DONORS
NO donors which release NO or related NO species are
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NO donors, which release NO or related NO species, areused to elicit smooth muscle relaxation..
Organic Nitrates
Nitroglycerin,which dilates veins and coronary arteries, ismetabolized to NO by mitochondrial aldehyde reductase,an enzyme enriched in venous smooth muscle, accountingfor the potent venodilating activity of this molecule.
Other organic nitrates, such as isosorbide dinitrate aremetabolized to an NO-releasing species through acurrently unidentified enzyme.
Organic nitrates have less significant effects onaggregation of platelets, which appear to lack theenzymatic pathways necessary for rapid metabolicactivation.
Organic nitrates are limited by the loss of therapeuticeffect during continuous administration. This nitratetolerance may derive from NO-mediated inhibition ofmitochondrial aldehyde reductase.
Organic Nitrites
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g
Organic nitrites, such as the volatile
antianginal isoamylnitrite, also requiremetabolic activation to elicit vasorelaxation,
although the responsible enzyme has not
been identified.
Nitrites are arterial vasodilators and do notexhibit the rapid tolerance seen with nitrates.
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NO Gas InhalationNO itself can be sed therape ticall
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NO itself can be used therapeutically. Inhalation of NO results in reduced pulmonary artery
pressure and improved perfusion of ventilated areas ofthe lung.
Inhaled NO has been used for acute respiratorydistress syndrome, acute hypoxemia, andcardiopulmonary resuscitation with evidence for
short-term improvements in pulmonary function.Alternate Strategies Another mechanism to enhance the activity of NO is
to enhance the downstream NO signaling pathway.
Sildenafil, an inhibitor of type 5 phosphodiesterase,results in prolongation of the duration of NO-inducedcGMP elevations in a variety of tissues pulmonaryhypertension in dogs..?
Different strategies to achieve NO-donating anti-
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Different strategies to achieve NO donating anti
inflammatory drugs (CINOD)
Ennio Ongini and Manlio Bolla, 2006
NO donor drugs
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g NO gas is notoriously difficult to handle on account of the
problems associated with complete exclusion of oxygen to
prevent oxidation to nitrogen dioxide. Nevertheless, the gas itself can be used therapeutically,
particularly in pulmonary hypertension (Griffiths and Evans,
2005) and in neonates (Greenough, 2000), where it is delivered
to the lungs via inhalation.
.
ORGANIC NITRATES
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Most commonly used NO donor drugs.
Glyceryl trinitrate (GTN; also known as nitroglycerin;) is the
best-studied nitrate, used mainly in acute relief of painassociated with angina, whereas other slower release
preparations, such as isosorbide mononitrate (ISMN), are
used for the treatment of chronic angina.
GTN ointments are also routinely used for the treatment of anal
fissure (Fenton et al., 2006), transdermal patches in heartfailure and chronic angina, whereas nebulized GTN may have
benefits in certain subgroups with pulmonary hypertension
Sodium nitroprusside (SNP).
Used to provide rapid lowering of blood pressure in
hypertensive crises. Relatively stable and does not release NO spontaneously in the
physiological environment; instead, NO generation requires
either light or a tissue-specific mode of release
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The toxicity of by-products needs to be more fully confirmed
(Lam et al., 2003), especially as subsequent reactions
between decomposition products could lead to the formation
of carcinogenic nitrosamines (Maragos et al., 1991).
Incorporation of NONOates into polymers may represent a
means of preventing the leaching of by-products (Mowery et
al., 2000).
At present, conjugated NONOates hold a great deal of
promise, especially for the treatment of certain cancers,
although further characterization of these drugs is essential
before they reach larger clinical trials. The potential for oral
preparations of NONOates
S-nitrosothiols contain a single chemical bond between a thiol (sulphydryl) group
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g ( p y y ) g p
(R-SH) and the NO moiety.
Biological activity of S-nitrosothiols is highly influenced by the
molecular environment of the parent thiol. That said, the complex chemistry of NO release from even the
most basic S-nitrosothiol gives these compounds several means
by which they can confer NO bioactivity.
For instance, S-nitrosothiols are considered to be NO+donors (see
below) and transfer of NO+ across the plasma membrane viaprotein disulphide isomerases (Zai et al., 1999) may allow even
large molecule weight S-nitrosothiols to transfer oxides of nitrogen
across cell membranes to subcellular targets.
A vast number of factors are capable of releasing NO from S-
nitrosothiols, including light, heat, transition metals, thiols,superoxide (Williams, 1999; Al-Sa'doni and Ferro, 2000; Megson
and Webb, 2002). and enzymes such as xanthine oxidase
(Trujillo et al., 1998), superoxide dismutase (Jourd'heuil et al.,
1999), protein disulphide isomerase (Ramachandran et al., 2001)
and various dehydrogenases (Liu et al., 2001).
S Nitrosothiols
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S-Nitrosothiols
Potential advantages over other classes of NO donor.
Firstly, some examples show tissue selectivity: S-nitroso-
glutathione (GSNO; ) is selective for arteries over veins,
giving them a different haemodynamic profile of action than
those of classical organic nitrates.
Additionally, S-nitrosothiols are potent antiplatelet agents,inhibiting aggregation at doses that do not influence vascular
tone (de Belder et al., 1994; Ramsay et al., 1995).
Furthermore, the ability of S-nitrosothiols to directly transfer
NO+ species allows biological activity to be passed on
through a chain of other thiols without the release of free NO. This mechanism of bioactivation may make S-nitrosothiols
less susceptible to conditions of oxidative stress by effectively
protecting the NO moiety from attack by oxygen-centred free
radicals.
GSNO has at least the potential to concurrently boost
NO hybrid drugs (Hybrid NO donor drugs)
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y g ( y g )
Represent a novel approach to the design of NO-releasing
compounds.
Structurally modified to incorporate NO-containing molecules.
The aim of this strategy is to synthesize drugs that retain the
pharmacological activity of the parent compound, but also
have the biological actions of NO. Importantly, the release of NO must be balanced to provide
sufficient activity within the concentration range of the parent
compound (Bandarage et al., 2000
NO-NSAIDs
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Low-dose aspirin is also routinely used prophylactically to
reduce the risk of thrombotic events associated with a wide
range of cardiovascular conditions. However, prolonged use ofaspirin leads to serious side effects in the gastrointestinal tract
that have been reported to cause 16 000 deaths each year in
the USA (Keeble and Moore, 2002).
A further increase in the risk of upper gastrointestinal bleeding
attributed to the co-administration of multiple antithrombotictherapies regularly prescribed for cardiovascular conditions
(Hallas et al., 2006).
NO has a number of effects in the gastrointestinal tract that
could counteract the loss of protective prostanoids caused by
aspirin. NO increases secretion of protective gastric mucus(Brown et al., 1993), increases blood flow to the gastric
mucosa, promoting repair and removal of toxins (Hallas et al.,
2006), decreases interaction of neutrophils with the gastric
microcirculation (Wallace, 1997) and may also promote the
healing of gastric ulcers (Ma and Wallace, 2000). Therefore,
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The first NO-NSAID compounds designed and releasedcommercially were the NicOx compounds, NCX4016 and
NCX4215.
Both are derivatives of aspirin (often referred to as
nitroaspirins')adapted to contain a nitrate group.
These compounds have been shown to retain the ability ofaspirin to inhibit inflammation and nociception without causing
gastric ulcers seen with equivalent concentrations of aspirin
(del Soldato et al., 1999; Fiorucci et al., 2003; Turnbull et al.,
2006b).
Also, several studies have shown that these compounds havecomparable or greater antiplatelet effects than the parent
NSAID, without causing excessive vasodilatation or
hypotension (Lechi et al., 1996; del Soldato et al.,
1999;Wallace et al., 1999a; Momi et al., 2000).
Emerging novel NO-NSAID compounds.
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In the past 5 years, there has been considerable investigation
into the mechanism that underpins the anti-inflammatory
properties of NO generated from nitroaspirins (Keeble and
Moore, 2002). Caspases are a family of proteases involved in
cytokine release and apoptosis.
NO from NCX4016 inhibits the action of capsase-1, and,
subsequently, the propagation of other cytokines such as IL-
1and IL-8 (Fiorucci et al., 2000).
NCX4016 induced inhibition of capsase-1 is mediated
through S-nitrosylation of a sulphydryl group (Dimmeler et al.,
1997), therefore, other NO-NSAIDs, such as S-nitroso-
diclofenac (see Figure 6a) may be more effective inhibitors of
capsase-1 through transnitrosation reactions.
Nitroaspirins also inhibit the release of TNF- from
lipopolysaccharide-stimulated macrophages (Minuz et al.,
2001), although it is difficult to determine whether this is a
direct effect of NO or throu h inhibition of other c tokines.
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NO-NSAIDs have attractive properties in a number of
cardiovascular conditions.
On top of the antiplatelet actions of NO-NSAIDs, the NO-
mediated anti-inflammatory properties would be useful in
vascular injury and atherosclerosis, given the central role of
inflammatory cells in the process (Ross, 1999).
NCX4016, but not aspirin, has been shown to reduce
experimental restenosis in hypercholesterolemic (Napoli et
al., 2001) and aged (Napoli et al., 2002) mice.
NCX4016 also reduces infarct size in several different models
of myocardial ischaemia-reperfusion injury (Rossoni et al.,2000,2001; Wainwright et al., 2002; Burke et al., 2006) and
has been shown to reduce plateletmonocyte interaction in
humans to a greater extent than aspirin alone (Fiorucci et al.,
2004).
Nitroaspirins
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Nitroaspirins
also show potential in cancer therapy. Upregulation of
cyclooxygenase-2 leading to enhanced prostaglandin output
is a feature of a number of cancers (Baron, 1995). Both the
gastro-sparing properties of nitroaspirins and the direct effect
of NO on cell proliferation could be beneficial, although
obtaining suitable balance between the different facets of a
nitroaspirin might prove difficult. That said, NCX4016 havebeen shown to be 2506000-fold more effective at inhibiting
the growth of a number of different cancer cell lines (Kashfi
and Rigas, 2005). Additionally, the compound also reduced
tumour growth in an in vivo rat model of colonic
adenocarcinoma to a greater extent than aspirin itself (Bak etal., 1998). Aside from cancer, NO-NSAIDs may have
applications in other areas of the body (Keeble and Moore,
2002). A NO-releasing derivative of flurbiprofen has been
shown to have beneficial actions in models of renal ablation
(Fujihara et al., 1998), bone degeneration (Armour et al.,'
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Given the tolerance issues associated with organic nitrates, it
is surprising that the majority of nitroaspirins investigated so
far exploit the same NO donor moiety, especially as it has
been shown for at least one example that the mechanism of
NO release is the same in hybrid drugs (Turnbull et al.,
2006a).
Many new NSAIDs/anti-inflammatory/analgesic agents with
nitrates groups
Paracetamol (Marshall et al., 2006), flurbiprofen (Fujihara et
al., 1998), naproxen (Young et al., 2005), mesalamine
(Wallace et al., 1999b), gabapentin (Wu et al., 2004),
predisolone and other steroids (Tallet et al., 2002). However,
the nitrate ester of nitroaspirin has been replaced with a
furoxan moiety (Cena et al., 2003; Turnbull et al., 2006a)
and S-nitroso- (Bandarage et al., 2000) and diazeniumdiolate
(Velazquez et al., 2005) forms of other NSAID drugs (e.g.
aspirin, diclofenac, indomethacin and ibuprofen) have also
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