“NITRIC OXIDE AS THERAPEUTIC

AGENT”
A
PROJECT REPORT
FOR ELECTIVE SUBJECT
SUBMITTED
TO
HEMCHANDRACHARYA NORTH GUJARAT UNIVERSITY, PATAN.

IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR THE DEGREE

CERTIFICATE OF BACHELOR OF PHARMACY
YEAR: 2006-2007

SUBMITTED BY

DUSHYANT B. PATEL
SHREE S.K.PATEL COLLEGE OF PHARMACEUTICAL
EDUCATION & RESEARCH,
GANPAT VIDYANAGAR, KHERVA-382711
NORTH GUJARAT
 CERTIFICATE

This is to certify that the project report for the elective

subject entitled “NITRIC OXIDE AS THERAPEUTIC
AGENT” is the bonafide work of DUSHYANT B. PATEL
satisfactorily carried under my guidance and supervision in the
Department Of Pharmacology of Shree S.K.Patel College of
Pharmaceutical Education and Research, Ganpat Vidyanagar,
during the academic year 2006-2007 sincerely and methodically.
This work is upto my satisfaction.

Guide:
Dr. N.J. PATEL
(M.Pharm, Ph.D.)
Head Of Department
Department of pharmacology,
Shree S.K. Patel College of Pharmaceutical Education & Research,
Ganpat Vidyanagar, Kherva

Principal:
Dr.MADHUBHAI M.PATEL
M.Pharm, PhD, LLB, FIC.
Department of Pharmaceutical Technology
Shree S.K. Patel College of Pharmaceutical Education & Research,
Ganpat Vidyanagar.

Date:
Place:

ACKNOWLEDGEMENT

The successful completion of a project “NITRIC OXIDE AS

THERAPEUTIC AGENT ’’ is generally not an individual effort. It is an

outcome of the cumulative effort of a number of persons, each having his
own importance to the objective. This session is a vote of thanks and
gratitude towards all those persons who have directly or indirectly
contributed in their own special way towards the completion of this
project.
First of all I would like to thank my guide respected Dr.N.J.Patel for
giving me guidance and also for supporting me in all the way to complete
this project.
I also like to thank our respected faculty members Mr. R.J.Patel,
Mr. R.K.Patel,Mr.N.J.Patel for their valued guidance and encouragement
towards the completion of our project.
I am also very much thankful to our honorable principal Dr.M.M.Patel for
providing infrastructure and research facilities at college.
And I am really grateful to all those, whose ideas have been helpful in
preparing this report.
I am also thankful to our librarian Mr.P.I.Patel, Mahadevbhai,
Mukeshbhai for providing me books & reference.
I heartily thankful to Chaula madam for her great help to me in computer
laboratory.
I am thankful to all my friends for always help me in my all difficulty.
At last but not at least I express thanks to my dearest brother HASMUKH
for his support and constant encouragement for successful completion of
my project work.
“May the candle be lightened forever, the joy is not of light alone, but of
presence of those, who played the role behind the curtain.”

GANPAT VIDYANAGAR
DUSHYANT B. PATEL
2006-07
FOURTH B.PHARM
 Dedicated to
God, My Family &
Teachers
 INDEX

SR. NO. CONTENT PAGE

NO.

1. INTRODUCTION AND HISTORY OF NITRIC OXIDE

2. CHEMISTRY AND PROPERTIES OF NITRIC OXIDE
3. 3.1 BIOSYNTHESIS OF NITRIC OXIDE
3.2 DIFFERENT FORMS OF NITRIC OXIDE SYNTHASE
3.3 DEGRADATION AND CARRIAGE OF NITRIC OXIDE
4. PHYSIOLOGICAL AND PHARMACOLOGICAL EFFECT OF
NITRIC OXIDE
5. 5.1 PHARMACOKINETICS
5.2 INDICATIONS
5.3 CONTRAINDICATION OF NITRIC OXIDE
6. DRUGS FOR MODULATING THE NO AND NOS ACTIVITY
7. REFERENCES
NITRIC OXIDE AS THERAPEUTIC AGENT

DISCOVERY OF NITRIC OXIDE

 Robert Furchgott of the State University of New York (SUNY) in Brooklyn set
out to solve in the 1950s, how blood vessel dilation works on the molecular level.
Furchgott's starting point was the neurotransmitter acetylcholine, which was
known to make blood vessels dilate when injected into animals. Presumably the
acetylcholine was instructing the muscle cells that surround blood vessels to relax,
thus increasing the diameter of the blood vessels.
 To get at the steps between acetylcholine and dilation, Furchgott tried to replicate
the acetylcholine response in the laboratory, using isolated strips of blood vessels
and the muscles surrounding them. A lengthening of the strips would indicate that
the muscles were relaxing and, by inference, that the blood vessel was dilating.
But with acetylcholine he saw the strips shorten (muscle contraction instead of
relaxation) every time. This was a puzzle that Furchgott temporarily set to one
side.
 Many years later Furchgott planned an experiment to determine the relative
potencies of several chemicals as relaxing agents of blood vessels. Carbachol, a
chemical relative of acetylcholine (which Furchgott's puzzling earlier experiments
had shown was a contracting agent), was to be used to contract the blood vessel
preparations so that Furchgott could then observe the relaxing effects of the three
agents he was investigating.
 Furchgott laid out a detailed protocol for his technician, David Davidson, which
began with tests to see that the tissue was reacting correctly. First came a test
contraction with the neurotransmitter norepinephrine, then a wash with fresh
saline solution to remove the norepinephrine, and then a test contraction with
carbachol. After another wash to remove the carbachol, the actual experiment
would commence. The experiment was planned for May 5, 1978. As it happened,
Davidson forgot the first wash. To the blood vessel preparation, still contracted by
the norepinephrine, he added the carbachol. But rather than contracting further,
the vessel relaxed.
 Furchgott had added acetylcholine or carbachol to vessels treated with various
chemicals many times before and seen only contraction. The only difference in
experimental procedure was that this time he was using rings of blood vessels
instead of strips. In further experiments Furchgott took rings, all of which had
relaxed in preliminary tests with acetylcholine, cut them into strips, and retested
the strips with acetylcholine. Infuriatingly, some of these strips continued to relax,
but some of them now contracted. He noticed that the ones that contracted had
curled up when they were cut earlier and had required some manipulation.
Perhaps the manipulation had done some damage.
 Sure enough, rubbing any of the strips on their inner surface took away their
ability to relax when acetylcholine was added. Furchgott recognized that the
methodical way that he had prepared the strips--always pulling the cut surface
over his finger to keep it out of the way--had wiped off something crucial.
 Furchgott demonstrated experimentally in 1980 that the missing something was
endothelial cells, which form the lining of blood vessels. When he made a
sandwich of two blood vessels--one with endothelial cells and one without--both
strips would relax in response to acetylcholine. The acetylcholine seemed to
instruct the endothelial cells to make a second messenger--which Furchgott
dubbed endothelium-derived relaxing factor, or EDRF. The EDRF then directed
the relaxation of the surrounding muscle cells in both strips.
 Although Furchgott knew that EDRF existed, he could not isolate and identify it.
For the moment it was simply defined as the substance produced by acetylcholine
treatment of endothelial cells.
EDRF AND NITRIC OXIDE ARE SAME

 The discovery of EDRF caused an explosion in research, with many different

groups around the world making important contributions to the search for its
identity. But the realization that EDRF and NO were one and the same substance
took six years of intense research. From 1980 to 1986 reports of similarities
between the two gradually mounted. In hindsight this may seem to have been an
inevitable accumulation of data, but at the time the picture was quite confusing.
 NO is an extremely reactive free radical--a result of the molecule's unpaired
electron in its outer electron "shell." This high reactivity meant that scientists
trying to zero in on EDRF often unwittingly perturbed NO levels when they were
trying to shut off other molecular pathways, leading the researchers to the
erroneous conclusion that these other pathways were responsible for producing
EDRF. Moreover, NO it self seemed a poor candidate for cellular messenger. Its
reaction with oxygen, for example, leads to the formation of the corrosive gas
nitrogen dioxide (NO2), which is readily converted to nitric acid. NO previously
known biological signaling molecule was a free radical, let alone a radical that
was a poisonous gas.
 But then there were the mounting coincidences. For starters, EDRF and NO both
caused blood vessel dilation, and both did so by turning on GC. This and other
evidence led Ferid Murad to propose in 1986 that EDRF could be considered an
"Endogenous Nitrate."
 The decisive experiments that identified EDRF as NO were performed
independently by Ignarro at Tulane and the University of California, Los Angeles,
by Furchgott at SUNY, and by Salvador Moncada at the Wellcome Research
Laboratories in Beckenham, England. All three researchers found that NO and
EDRF both decayed in a matter of seconds, were stabilized by the same
conditions, and were turned off by the same battery of chemical treatments. In
addition, Ignarro found that NO and EDRF underwent identical reactions with a
complex chemical--an unlikely occurrence unless NO and EDRF were identical.
Thus, EDRF was chemically identified to be NO.
 Ignarro and Furchgott presented their results to a skeptical audience at a
conference at the Mayo Clinic, in Rochester, Minnesota, in July 1986. Ignarro felt
 that not "a single person" in the audience believed them, but when the data were
published in both 1987 and 1988, opinion swung their way. Moncada clinched the
argument in an important 1987 paper. In this widely cited article he
unambiguously showed that NO was made by endothelial cells. First, he
measured the amount of NO produced by a known relaxant (bradykinin) acting on
cultured endothelial cells. Then he added exactly that amount of NO to a blood
vessel and showed that the added NO could cause a full relaxant response. Thus,
the actions of NO could explain the actions of EDRF. A commentary
accompanying Moncada's paper described these findings as "the climax of one of
the most exciting sagas in vascular physiology and pharmacology."
 The molecule initially considered toxic and now is found to have many beneficial
physiological properties. The properties were diverse and interesting and so it was
chosen as the molecule of the year 1992 by the editors of Science. Drs. Furchgott,
Ignarro and Murad were awarded Nobel Prize in Medicine and Physiology in the
year 1998 for their pioneering work on NO. Now it has become a wonder
molecule with diverse physiological properties.
 NO is the endogenous activator of soluble guanylate cyclase, leading to the
formation of cyclic GMP (cGMP), which functions as a second messenger in
many cells including nerves, smooth muscle, monocytes and platelets. Nitrogen
and oxygen are neighbors in periodic table and NO shares several properties with
O2, in particular a high affinity for haem and other iron-sulfur groups.
 Nitric oxide (NO) is a multifunctional biological mediator that is being
investigated as an agent for improving oxygenation in patients with impaired
pulmonary circulation. Despite the recent explosion of interest in the biological
role of NO, there are considerable gaps in current knowledge of its medicinal
uses.
 NO serves as an intracellular and intercellular messenger molecule with both
autocrine and paracrine functions, and its role in cardiorespiratory diseases, CNS
disorders, inflammation and immunity, and as endogenous activator of the soluble
form of guanyl cyclase is widely documented.
CHEMISTRY AND PROPERTIES OF NITRIC OXIDE

 Nitric oxide is a diatomic molecule. It is a colorless, tasteless and volatile gas,

moderately soluble in water 3mM at saturation.
 Molecular formula : NO
 Molecular weight : 30 gm/mol
 Solubility : Its solubility in water is 1 in 5×10-7
 It is the second (Nitrogen oxidation state +2) in the redox sequence of oxy
compounds of nitrogen that includes NO- (oxidation state +1) and NO+ (oxidation
state +3).
 The characteristics chemistry of NO is largely the result of the arrangement of the
eleven electrons in the valence shell; nitrogen and oxygen combine to form four
bonding and four antibonding orbitals, which are sequentially filled by eight
bonding and three antibonding electrons, giving a bond order of 2.5 and a bond
length intermediate between double and triple bonding (1.150 A°).
 The single unpaired electron in the π2p* electron shell defines the molecule as a
free radical (the presence of this unpaired electron is implicit in the formula NO,
this is sometimes emphasized by writing the formula as ⋅NO).
 It exists in different oxidized forms exerting sometimes contrasting
pharmacological actions.
o NO radical
o NO- nitroxide anion (by addition of one electron)
o NO+ nitrosonium cation (removal of one electron)
o Can be oxidized to NO2- nitrite ion and NO3- nitrate ion
 o NO + O2 →NO2→Stable Nitrites and Nitrates
o NO can react with superoxide forming peroxynitrate ONOO- and OH
radical causing the undesired effect.
 The unpaired electron is antibonding and is easily detached to allow NO+ to be
formed (ionization potential 9.25 eV), although the lifetime of NO+ in aqueous
media has been calculated to be very short: about 3×10-10 s.
 Nitric oxide has a boiling point of –151.7 °C and melting point of –163.6 °C. It is
noncombustible but supports combustion. NO does not exhibit the high reactivity
characteristic of most free radicals and shows little tendency to dimerize.
 It is thermodynamically unstable (∆G°formation=86.32kJ), although as decomposition
is kinetically hindered the gas can be stored indefinitely at room temperature and
1 atmosphere pressure. At elevated pressure, disproportionation of NO to N 2O and
NO2 can occur by a reaction that obeys third order kinetics with a rate constant of
k=2.6×10-5 M-5h-1 at 30 °C. Therefore, for example at 30 °C and 200 atmosphere
pressure, 40% of NO initially present will be converted into N2O and NO2 after
300 h.
 NO does not interact with water and is not affected by light. It reacts with oxygen
at room temperature. In aqueous solutions NO exhibits solubility and diffusibility
similar to those of other diatomic gases (solubility: 1.7×10-3 molL-1 at 25 °C and 1
atmosphere pressure; diffusibility : 4.8×10-5 cm2s-1 at 37 °C).
 NO can participate in a variety of chemical reactions with metals, thiols, and other
reactive oxygen species.the probable targets in the cells are metals, reduced thiols,
molecular superoxide. Reaction with superoxide is very fast, forms peroxynitrile-
ONOO- a powerful oxidant that can modify proteins and lipids by nitration. It is
highly toxic reactive nitrogen species.
BIOSYNTHESIS OF NITRIC OXIDE

 Nitric oxide is synthesized by a family of enzymes that are collectively called as

Nitric oxide synthase (NOS). NOS has three isoforms. These isoformsare heme-
containing flavoproteins employing L-arginine as substrate and requiring
NADPH, flavin adenine dinucleotide and tetrahydrobiopterin as cofactors.
 Formation of nitric oxide from L-arginine and several nitric oxide donors is
shown in figure. Activation of NOS by the influx of extracellular calcium and
binding of calmodulin, as in the case of the constitutive enzymes or following the
activation of the inducible NOS by cytokines, results in the metabolism of L-
arginine to nitric oxide and L-citrulline is inhibited by several arginine
competitors such as N-monomethyl-L-arginine.
 Some nitric oxide donors eg. Oxygenated nitroprusside, spontaneously generate
nitric oxide in aqueous solutions whereas others, such as furoxans and organic
nitrates and nitrites such as nitroglycerin, require the presence of a thiol
compound such as cysteine.
 Once generated , NO interacts with the heme moiety of the soluble guanylyl
cyclase in the cytoplasm of the cells. This results in allosteric transformation and
activation of the enzyme and leads to the formation of 3’,5’-cGMP from GTP.
Activation of soluble guanylyl cyclase by NO can be inhibited by methylene blue.
 The affinity of NO for iron is also responsible for its inhibitory effect on several
enzymes by interacting with iron-sulfur centers of these enzymes such as
cytochrome P450 by NO is major problem in inflammatory liver disease and can be
reversed by NOS inhibitors. Carbon monoxide and other gaseous compound
 produced endogenously from the catabolism of heme, shares many of the
properties of NO such as activation of soluble guanylyl cyclase.

FIG. Scheme of the formation of NO from L-arginine and its regulatory factors. cNOS,
constitutive NOS; AS, argininosuccinate synthetase; AL, argininosuccinate lyase; OTC,
ornithine transcarbamylase; CAT, cationic amino acid transporter in the cell membrane.
 EMBED Word.Picture.8

.The heme moiety of the enzyme is colored.

In the NOS reaction the guanidino nitrogen of Arg undergoes a five-electron oxidation
via an NOHLA (N-ω-hydroxy-L-arginine) intermediate to yield NO.

DIFFERENT FORMS OF NITRIC OXIDE SYNTHASE (NOS)

1) NEURONAL NITRIC OXIDE SYNTHASE OR nNOS OR NOS-I :

 • It has molecular weight of 168 kDa
• This is constitutive calcium dependent enzyme. It was first identified in neurons.
It is highly expresses in skeletal muscles and in the peripheral nerves. It regulates
muscle contraction, exercise-induced glucose uptake.
• It regulates the relaxation of vascular and non-vascular smooth muscles, mediates
relaxation of corpus cavernosum-penile erection. Its inhibition causes (as studied
by knock out mice) dilated bladder, increase in urinary frequency, pyloric stenosis
and dilated stomach.
• Excess of NO produced by this enzyme results in neurological disorders leading
to neuronal injury in central and peripheral nervous system. It is stimulated by
glutamate, HIV gp-120 and b-amyloid peptide.

2) INDUCIBLE NITRIC OXIDE SYNTHASE OR iNOS OR NOS-II :

• It has molecular weight of 130 kDa.
• It was first identified in macrophages as a mechanism for cytotoxicity. This is
considered as harmful NOS. iNOS is tightly bound to calmodulin and so is not
dependent on calcium ion levels for activation.
• It is activated by inflammatory stimuli like cytokines, interferon γ, oxidized LDL
and bacterial lipopolysaccharide. Inhibition of iNOS plays a major role in the
treatment of multiple sclerosis, septic shock and decrease of auto immune system.

3) ENDOTHELIAL NITRIC OXIDE SYNTHASE OR eNOS OR NOS-III :

• It has molecular eight of 135 kDa.
• They are subjected to regulation by calcium ion and calcium binding protein-
calmodulin (CAM). Increase in local or intracellular Ca2+ levels result in eNOS
activation through a Ca2+/CAM complex. It maintains vasculation in a relaxed
state. It inhibits platelet adhesion, suppresses replication of smooth muscle cells.
• Prolonged inhibition of this enzyme is detrimental. Pharmacological inhibition
causes vasoconstriction, hypertension enhanced platelet activity, increase
artherogenesis in animal models.
• It is stimulated by thrombin, acetylcholine, shear stress and bradykinin.
The isoenzymes of NOS
Features Neuronal NOS Constitutive NOS Inducible NOS
Alternative Type I Type III Type II
description nNOS cNOS iNOS
cnNOS eNOS macNOS
bNOS ecNOS
Molecular Weight 168 kDa 135 kDa 130 kDa
Human 12 7 17
chromosome
Calcium Yes Yes No
dependency
Subcellular Binds to a Targets to the Golgi and -
localization specific proteins to calveoli via N-
via an N-terminal terminal myristoylation
PDZ domain and palmitoylation
Tissue expression NANC neurons Endothelial cells Macrophages
Skeletal muscle Epithelial cells Neutrophills
White blood cells Cardiomyocytes Hepatocytes
Pancreatic islet Astrocytes
cells Fibroblasts
Respiratory and Chondrocytes
GIT epithelium Smooth muscle cells
Epithelial cells
Stimulus for Glutamate Acetylcholine Lipopolysaccharides
activation/induction HIV gp-120 Bradykinin Interferon γ
b-amyloid peptide Thrombin Cytokines
Shear stress Oxidized LDL
Amount released small, pulses Small,pulses Large, continuous
Function Regulatory Regulatory Host defence
CONTROL OF CONSTITUTIVE NOS (cNOS)
 The activity of constitutive isoforms of NOS is controlled by intracellular
calcium-calmodulin. Control is exerted in two ways:
1. Many endothelium-dependent agonists (e.g. acetylcholine, bradykinin,
substance-P) increase the cytoplasmic concentration of calcium ions,
[Ca2+]i ; the consequent increase in calcium-calmodulin activates eNOS or
nNOS.
2. phosphorylation of specific residues on eNOS renders it more active at a
given concentration of calcium-calmodulin. This can increase NO
synthesis in the absence of any change in [Ca2+]i.
 The main physiological stimulus controlling endothelial NO synthesis in
resistance vessels is probably shear stress. This is sensed by endothelial
mechanoreceptors and transduced via a serine-threonine protein kinase called Akt
or protein kinase B. Agonists that increase cAMP in endothelial cells (e.g.β2
agonists) also influence the phosphorylation of eNOS.
 In contrast to constitutive NOS isoforms, the activity of iNOS is independent of
[Ca2+]i. Though iNOS contains a binding site for calcium-calmodulin, the very
high affinity of this site for its ligand means that iNOS is activated even at the low
values of [Ca2+]i present under resting conditions.
 The enzyme is induced by bacterial lipopolysaccharide (LPS) and/or cytokines
synthesized in response to LPS, notably interferon γ the antiviral effect of which
can be explained by this action. Tumour necrosis factor-α and interleukin-1 are
not effective in inducing iNOS in their own right, but they each synergise with
interferon γ in this regard.
 Induction of iNOS is inhibited by glucocorticoids and by several cytokines,
including transforming growth factor-. There are important species differences in
the inducibility of iNOS, which despite the importance of iNOS in humans, is less
readily induced in humans than in mouse cells.
Receptors
(acetylcholine,bradykinin,substance P etc.) Mechanical shear stress

↑ [Ca+2]i
+
Akt
+
Calmodulin Ca+2-calmodulin
+
ENDOTHELIAL NOS NOS
CELL (active) (inactive)

Citrulline + NO Arginine

GC GC SMOOTH
(basal) (activated) MUSCLE
CELL

cGMP GTP

RELAXATION

CONTROL OF cNOS BY Ca+2-CALMODULIN

DEGRADATION AND CARRIAGE OF NITRIC OXIDE

 NO reacts with oxygen to form N2O4, which combines with water to produce a
mixture of nitric and nitrous acids. Nitrite ions are oxidized to nitrate by
oxyhaemoglobin. These reactions are summarized:
2NO + O2 → N2O4
N2O4 + H2O → NO3- + NO2- + 2H+
NO2- + HbO → NO3- + Hb
 Low concentration of NO are relatively stable in air because above first reaction
is a second-order reaction. Consequently, small amounts of NO produced in the
lung escape degradation and can be detected in exhaled air. In contrast, NO reacts
very rapidly with even low concentrations of superoxide anion (O2-) to produce
peroxynitrate anion (ONOO-), which responsible for some of its toxic effects.
 Haem has an affinity for NO > 10000 times greater than for oxygen. In the
absence of oxygen, NO bound to haem is relatively stable but in the presence of
oxygen, NO is converted to nitrate and the haem iron oxidized to
methaemoglobin.
 Distinct from this inactivation reaction, evidence is accumulating that the globin
part of haemoglobin carries NO under normal physiological conditions,
optimizing oxygen delivery in the periphery and contributing to the control of
respiration.
 NO binds reversibly to a specific cysteine residue in globin. The resulting S-
nitrosylated haemoglobin is believed to be involved in various NO-related
activities including the control of vascular resistance, blood pressure and
respiration. This is still controversial, particularly as regards quantitative aspects,
but key features include:
1. Nitrosylation of haemoglobin is reversible.
2. It depends on the state (R or T) of the haemoglobin, which consequently
takes up NO in the lungs and releases it in tissues, including the
respiratory center in the brain, in concert with release of oxygen.
3. NO is released not in to the cytoplasm of erythrocytes ,but is transported
out of the red cells via cysteine residues in the haemoglobin-binding
cytoplasmic domain of an anion exchanger called AE1.(AE1 is responsible
for the exchange of chloride and bicarbonate ions across the cell
membrane, the ‘Hamburger shift’ beloved of red cell physiologists. It is
the most abundant protein in red cell membrane).
PHYSIOLOGICAL AND PHARMACOLOGICAL EFFECTS OF NITRIC OXIDE

 NO activates guanylate cyclase by combining with its haem group and the
physiological effects of low concentrations of NO produce under normal
conditions by the constitutive enzymes are made by cGMP. These effects are
prevented by inhibitors of guanylate cyclase (e.g. ODQ), which are useful
investigational tools in this regard.
 NO also combines with the haem groups in other biologically important proteins
(e.g. cytochrome c oxidase where it may compete with oxygen, thus contributing
to the control of cellular respiration), thereby influencing their function. Cytotoxic
and/or cytoprotective effects of higher concentrations of NO relate to its
chemistry as a free radical.

VASCULAR EFFECTS
 Nitric oxide has a significant effect on vascular smooth muscle tone and blood
pressure. It is released by acetylcholine and other endothelium dependent
vasodilators. It may play a role in the normal regulation of vascular tone. It
increases vascular tone and elevates mean arterial pressure.
 The effects of vasopressor drugs are increased by inhibition of NOS. Increased
cGMP synthesis by guanylyl cyclase results in smooth muscle relaxation. Apart
from being a vasodilator, nitric oxide is also a potent inhibitor of neutrophil
adhesion to the vascular endothelium. This is due to the inhibitory effect of nitric
oxide on the expression of adhesion molecules on the endothelial surface.

RESPIRATORY EFFECTS
 Nitric oxide inhalation decreases pulmonary arterial pressure and improves blood
oxygenation. Thus when pulmonary resistance is elevated, it is possible to exploit
the vasodilator properties of nitric oxide by administering it via inhalation of a
few parts per million.
 Nitric oxide may have an additional role in relaxing airway smooth muscle and
thus act as bronchodilator. For these reasons, nitric oxide inhalation therapy is
 being widely tested in both infants and adults with acute respiratory distress
syndrome.

ATHEROSCLEROSIS
 Vascular plaque formation in hypercholesterolemia leads to reduced nitric oxide
formation and endothelium-dependent vasodilator responses. In addition, nitric
oxide may act as an antioxidant, blocking the oxidation of low-density
lipoproteins (LDL) and thus preventing the formation of foam cells in the
vascular wall.

PLATELETS
 Nitric oxide is a potent inhibitor of platelet adhesion and aggregation. Thus,
endothelial dysfunction and the associated decrease in nitric oxide generation may
result in abnormal platelet function. Nitric oxide may have an additional
beneficial effect on blood coagulation by enhancing fibrinolysis via an effect on
plasminogen.

ORGAN TRANSPLANTATION
 By reducing free radical toxicity during organ transplantation, nitric oxide may
act as a cytoprotective agent, inhibiting platelet and neutrophils aggregation and
adhesion to the vascular wall.

THE CENTRAL NERVOUS SYSTEM

 Nitric oxide a major role in the central nervous system- as a neurotransmitter, as a
modulator of ligand-gated receptors, or both. In addition, nitric oxide probably
plays a role in neuronal degeneration in some conditions.
 The likely cellular targets of NO in the central nervous system include presynaptic
and postsynaptic nerve terminals. Nitric oxide modifies neurotransmitter release
in different areas of the brain. Postsynaptic release of nitric oxide following
activation of the NMDA receptor may initiate presynaptic transmitter release of
glutamate, i.e., nitric oxide may function as a retrograde messenger that is
 synthesized in postsynaptic sites following opening of the Ca 2+ channels and
activation of NOS.
 Nitric oxide (like other substances) may have role in short and long term
potentiating effects on excitatory amino acids in brain development and learning.
Inhibition of calcium ATPase by peroxynitrite may in turn lead to enhanced Ca +2
accumulation and associated neurodegeneration. NOS-2 has been implicated in
several other degenerative neurologic conditions, eg., Alzheimer’s disease,
multiple sclerosis and Huntington’s disease.
 High levels of nitric oxide causes destruction of photoreceptor cells in the retina.
This is due to a prolonged increase in cGMP formation. Finally, nitric oxide and
cGMP also have a role in epileptic seizures.

INFLAMMATION
 Nitric oxide has role in both acute and chronic inflammation. eNOS is involved in
the vasodilation associated with acute inflammation. In acute inflammation,
inhibitors of eNOS have a dose-dependent protective effect, suggesting that the
nitric oxide promotes edema and vascular permeability. NO has a detrimental
effect in arthritis, dietary L-arginine supplementation exacerbates arthritis.
 Synovial fluid from patients with arthritis contains increased oxidation products
of nitric oxide, particularly peroxynitrite. Nitric oxide stimulates the synthesis of
inflammatory prostaglandins by activating COX-2. Thus inhibition the nitric
oxide pathway may have a beneficial effect on inflammatory diseases. Nitric
oxide is also required for maintaining COX-2 gene expression.
 Nitric oxide also plays important protective role in the body via immune cell
function. When challenged with foreign antigens, TH1 cells respond by
synthesizing nitric oxide.

THE PERIPHERAL NERVOUS SYSTEM

 Nonadrenergic, noncholinergig (NANC) neurons are widely distributed in
peripheral tissues, especially the gastrointestinal and reproductive tracts.
 Considerable evidence implicates nitric oxide as a mediator of certain NANC
actions, and some NANC neurons appear to release nitric oxide.

FIG. Interaction between nitrergic, cholinergic and adrenergic nerves in pre- and
postjunctional sites. Minus denotes "inhibition". NE, norepinephrine; L-Arg, L-arginine; L-
Citrus, L-citrulline; M2, M2 muscarinic receptor; 1 and 2, 1- and 2-adrenoceptors,
respectively; GC, guanylyl cyclase; DG, diacyl glycerol.
FIG. Schematic presentation of the diameter of cerebral and peripheral arteries as affected
by NO (basal and stimulated) released from the nitrergic nerve and endothelium and by
sympathetic nerve activation (only for peripheral artery). Basal diameter, the diameter
without tonic sympathetic discharge and basal and stimulated release of NO; resting
diameter, the diameter under influences of basal release of NO from the nerve and
endothelium on the basal diameter; NO-induced vasodilatation, the diameter increased
from the resting diameter by stimulated release of NO.

SEXUAL EFFECTS
 Penile erection is caused by the release of nitric oxide from NANC neurons,
Nitric oxide promotes relaxation of the smooth muscle in the corpora carvenosa-
the inhibiting factor in penile erection. Thus, impotence is a possible clinical
indication for the use of a nitric oxide donors.
Postulated roles of endogenous Nitric oxide
System Physiological role Pathological role
Excess Inadequate
production production/action
Cardiovascular
Endothelium/ Control of blood pressure and Hypotension Artherogenesis,
vascular smooth regional blood flow (septic shock) thrombosis,(e.g. in
muscle Maintenance of vascular hypercholesterolaemia,
integrity diabetes mellitus)
Platelets Limitation of adhesion or - -
aggregation
Host defence
Macrophages, Defence against viruses, - -
neutrophils, bacteria, fungi, protozoa,
leucocytes parasites,non-specific
immunity, Apoptosis,
tumorocidal effects
Nervous system
Central Neurotransmission; long term Excitotoxicity -
potentiation; plasticity (e.g. ischaemic
(memory,appetite,nociception) stroke,
Huntington’s
disease, AIDS
dementia)
Peripheral Neurotransmission (e.g. gastric - Hypertrophic pyloric
emptying, penile erection) stenosis, erectile
dysfunction

PHARMACOKINETICS
 Several methods for measuring the concentration of inhaled NO concentration are
available. Electrochemical systems rely on the oxidation of NO:
NO + H2O → HNO3 + 3e- +3 H+
 To exclude other oxidized species, the electrode is surrounded by a selectively
permeable chloroprene rubber membrane. Despite some concerns about the
specificity of this technique, many workers have used it for clinical measurement,
finding that in a clinical setting, and with humidified gases, repeatability is 2%.
An electrochemical technique based on a nickel-porphyrin electrode has been
designed which has a claimed detection limit of 10 nM.
 Measurement of NO concentration by chemiluminescence relies on oxidizing NO
with ozone to give NO2 in a state of electron excitation; light is emmited on the
return to the ground state. Chemiluminescence has an absolute sensitivity of about
10-13 M, and has been effective in many clinical studies.
 Nitric oxide is administered by inhalation. Inhaled NO is taken up into the
precapillary airspaces and alveoli at a rate many times faster than inhaled oxygen
and the diffusing capacity of the lung is 4.5 times faster than for carbon
monoxide.
 The anatomic proximity of the airspaces to muscular arterioles allows diffusing
NO to come in contact with the abluminal surface of these vessels. The
concentration of endogenous NO in the lung has been estimated at ~8ppb, while
that of free NO in plasma is ~3nM.
 Because of the rapid conversion of NO to nitrite by reaction with haemoglobin,
the effects of NO inhalation are generally considered to be localized to the lung
tissue, with an effective half-life of 2-6 s. the half-life of potential ‘stabilized ’
forms of NO (S-nitrosothiols) is longer and these compounds can be found in the
plasma of normal individuals.
 Inhaled NO undergoes rapid metabolism in the lungs and it is probable that no
unchanged drug enters the circulation. The metabolites of NO, nitrate and nitrite
are cleared from the body by the kidneys within 5-8 h.
 There have been no reports of any effects of concurrent illness or old age on the
disposition of NO. It is unlikely that any NO following its inhalation will be
excreted in breast milk or cross the placenta, but specific information is lacking.
INDICATIONS

 A considerable body of experimental evidence suggests that inhaled NO may be

beneficial in disease states characterized by pulmonary vasoconstriction and
ventilation perfusion mismatch. However, most available reports have studied
small numbers of patients, and there is as yet insufficient evidence to conclude
that NO is a life-saving therapy.
 Currently, the best indication for NO administration is a part of a clinical trial
during intensive therapy for life-threatening hypoxia associated with one of the
causes of pulmonary vasoconstriction.

1) PERSISTENT PULMONARY HYPERTENSION OF THE NEWBORN

 Infants present soon after birth with cyanosis resulting from right-to-left shunting
of blood through the ductus arteriosus and foramen ovale. The condition can be
idiopathic, or secondary to perinatal hypoxia, sepsis, meconium aspiration,
diaphragmatic hernia, congestive heart failure, severe respiratory distress
syndrome or polycythemia.
 Nitric oxide seems to be effective in reversing the hyposemia resulting from
persistent pulmonary hypertension of the newborn. In one study, inhaled NO at a
dose of 80 ppm raised the mean postductal oxygen tension to normal levels within
10 min, while in another, successful therapy was achieved with 6 ppm No.
 In one patient, 30 min of treatment produced resolution of the pulmonary
hypertension and in another prolonged NO therapy for 23 days (median
concentration 20 ppm) allowed a sustained improvement in oxygenation. An open
multicenter trial involving over 100 term and preterm infants has reported similar
results with 10-80 ppm NO.

2) CONGENITAL HEART DISEASE

  Congenital heart disease is frequently complicated by both pulmonary vascular
disease and pulmonary hypertensive crises. The conditions are difficult to reverse,
and contribute substantially to postoperative morbidity and mortality.
 In infants with congenital heart disease complicated by pulmonary hypertension,
inhaled NO at 20-80 ppm effectively reduced the pulmonary arterial pressure and
NO at doses of as little as 1 ppm was successful in treating pulmonary
hypertensive crises. Inhaled NO appears to produce more potent and selective
pulmonary vasodilation than administration of prostacyclin.

3) RESPIRATORY DISTRESS SYNDROME OF THE NEWBORN

 Pulmonary vascular resistance is increased in preterm neonates with respiratory
distress syndrome (RDS). In premature infants with RDS who required in an
inspired oxygen fraction of greater than 0.6, brief exposure to NO at 5-40 ppm
produced significant improvements in oxygenation. It is possible that early
treatment with inhaled NO in severe RDS may allow reductions in ventilation
pressures and FiO2, potentially reducing the injury caused by barotraumas and
oxygen toxicity in the susceptible premature lung.

4) BRONCHOPULMONARY DYSPLASIA
 Bronchopulmonary dysplasia (BDP) is a common sequel to premature birth ,
characterized by marked maldistribution of ventilation to perfusion and
pulmonary hypertension. Inhaled NO at a dose of 3-40 ppm has been used
successfully in treating acute hypoxemic respiratory failure in infants with BPD.
in some infants with severe BPD who received prolonged inhalational NO therapy
could not be weaned from NO. Suppression of endogenous NO in these infants
may thus be a potential risk in this condition.

5) PRIMARY PULMONARY HYPERTENSION

 Administration of 40 ppm NO reduces pulmonary vascular resistance in adult
patients with pulmonary hypertension. The effect seems to be at least as effective
as intravenous prostacyclin, and does not lead to the decline in systemic vascular
resistance seen with vasodilator prostanoids.
6) ADULT RESPIRATORY DISTRESS SYNDROME
 NO at 18 ppm has been shown to reduce pulmonary vascular resistance, decrease
intrapulmonary shunting, and improve oxygenation in patients with adult
respiratory distress syndrome. In the same patients intravenous prostacyclin
reduced pulmonary artery pressure but increased ventilation: perfusion
mismatching, worsening oxygenation.
 Nitric oxide has been found to be useful in ameliorating pulmonary hypertension
and hypoxia in Acute Respiratory Distress Syndrome (ARDS). A sustained
improvement in arterial oxygenation along with a reduction in pulmonary arterial
pressure in response to NO in patients with ARDS is reported in a number of
studies since 1993.

Effects of Nitric Oxide on Pulmonary Circulation in ARDS

 Nitric oxide administered exogenously relaxes the pulmonary vascular smooth

muscle through an activation of the soluble guanylate cyclase. Contrary to the
other pulmonary vasodilators, inhaled NO has no systemic effect because it is
inactivated by fixation to hemoglobin as soon as it reaches the vascular lumen.
Nitric Oxide, administered exogenously by inhalational route has no effect on the
normal, nonconstricted pulmonary circulation.
 The pulmonary vasodilator effect of NO administered by inhalational route is
specific only to the pulmonary vessels of the ventilated part of the lung, as it does
not reach the vessels perfusing the non-ventilated zone. As a result, pulmonary
blood flow is preferentially redistributed towards the well-ventilated alveoli,
thereby decreasing the pulmonary shunt.
 During the early phases of ARDS, the only determinant of the decrease in
pulmonary vascular resistance index (PVRI) induced by inhaled NO is the basal
level of PVRI. The greater the baseline PVRI, the greater the inhaled NO-induced
decrease in PVRI. `NO-responders' are the patients with an increase in PVRI
related to pulmonary vasoconstriction (PVRI > 200 dynes.sec.cm-5.m2). `NO non-
responders' are either patients with normal pulmonary vascular tone or patients
 with an increase in PVRI mainly related to thrombosis or to anatomical
remodeling of the pulmonary vessels characterising late stages of ARDS.
 There is a poor correlation between the decrease in mean pulmonary artery
pressure (MPAP) in response to NO and the baseline MPAP. At least two reasons
can be advanced to explain this. Firstly, pulmonary hypertension can some times
be mainly related to an increase in the pulmonary blood flow, with a fairly normal
PVRI. In such an event, NO will not reduce MPAP eventhough the baseline
MPAP is elevated. Secondly, in the presence of right heart failure due to acute
pulmonary hypertension, inhalation of NO can indirectly increase cardiac output
by unloading the failing right ventricle. In this case, despite a decrease in PVRI,
MPAP may decrease only marginally.
 NO may cause a paradoxical increase in MPAP and pulmonary wedge pressure
leading to pulmonary edema in-patients with severe left ventricular failure
associated with pulmonary hypertension. In these patients the decrease in right
ventricular afterload induced by NO increases right ventricular output which, in
turn, increases venous return to the failing left ventricle.
 Inhaled NO are both an arterial and a venous vasodilator. Although it acts mainly
on the arterial side, there are experimental arguments in favor of inhaled NO-
induced venodilation. Abnormal proliferation of smooth muscle in non-muscular
pulmonary vessels is one of the features of ARDS and there is evidence that
Inhaled NO could prevent it.

Effects of Nitric Oxide on Gas Exchange in ARDS

 Inhaled nitric oxide influences oxygen transfer favourably across the alveolo-
capillary membrane. It also seems to affect the carbon dioxide elimination by
decreasing the alveolar dead space.

Effect on arterial oxygenation:

 Inhaled NO improves arterial oxygenation by diverting pulmonary blood flow

away from nonventilated towards ventilated lung areas, thereby, reducing lung
areas with low V/Q ratios. In contrast to intravenous vasodilators, inhaled NO do
 not inhibit hypoxic pulmonary vasoconstriction present in the nonventilated lung
areas.
 Inhaled NO-induced increase in PaO2 and decrease in pulmonary shunt correlate
well with the initial level of PVRI. The greater the baseline PVRI, the greater the
increase in PaO2 and decrease in pulmonary shunt. Application of PEEP
potentiates the improvement in arterial oxygenation with NO. This beneficial
effect of PEEP is observed only if PEEP induces alveolar recruitment. On the
other hand, When PEEP induces lung overdistention, the effect of inhaled NO on
arterial oxygenation does not differ between PEEP and ZEEP conditions.
Therefore, the effect of inhaled NO on arterial oxygenation should always be
tested after optimization of alveolar recruitment.
 Patients considered as `NO non-responders' under ZEEP might become `NO-
responders' after administration of PEEP. It is interesting to note that potentiation
of NO-induced increase in PaO2 by PEEP is not always associated with a
corresponding decrease in PVRI.
 A dramatic improvement in arterial oxygenation can be induced by inhaled NO in
ARDS patients with patent foramen ovale. In patients with patent foramen ovale,
pulmonary hypertension associated with ARDS may induce a right to left
intracardiac shunt which is partly responsible for hypoxia. By unloading the right
ventricle, NO causes a functional closure of the patent foramen ovale and
improvement in oxygenation. So, patent foramen ovale should be routinely looked
for in patients with ARDS with severe hypoxia. If present, low concentrations of
inhaled NO can be used to decrease the right to left intracardiac shunt. Similar
effects have been reported with NO in patients with pulmonary embolism having
a patent foramen ovale.

Effect on alveolar dead space:

 Most often, NO inhalation causes an increase in the end-tidal carbon dioxide

tension (PetCO2) independent of any modification in the minute ventilation and
cardiac output. This is generally associated with a small but significant reduction
in PaCO2 corresponding to a marked decrease in alveolar dead space. This
 phenomenon is most likely related to the re-opening of constricted pulmonary
arteries in ventilated lung regions.

Bronchial Effects

 Nitric oxide given by inhalational route inhibits methacholine-induced

bronchoconstriction in guinea pigs; this bronchodilating effect is dose-dependent
in the range of 5-15 ppm. But in patients with ARDS, it has not been shown to
decrease the respiratory resistance between 10 and 80 ppm. In spontaneously
breathing asthmatic patients, the bronchodilator effect of 80 ppm of NO is much
smaller than that of Beta2 agonists.

Effects on Platelet Function

 Activation of coagulation, mainly related to platelet aggregation, induces

microvascular thrombosis in the pulmonary circulation during ARDS.
Endogenous NO causes antiplatelet effects by a guanylate cyclase-dependent
mechanism. An antithrombotic effect and/or an increase in the bleeding risk
might be expected in patients receiving inhaled NO, if the antiplatelet effect is of
sufficient magnitude. In patients with ARDS without pre-existing coagulation
disorders, the beneficial effects of inhaled NO on arterial oxygenation and
pulmonary circulation are also associated with a significant inhibition of platelet
aggregation even at low NO concentrations (< 1ppm).This antithrombotic effect
is not associated with a significant prolongation of the bleeding time.

Other Effects of Potential Benefit

 Improvement in gas exchange with NO could also be explained by mechanisms

other than diversion of pulmonary blood flow. Because inhaled NO most likely
dilates both sides of the pulmonary circulation (arteries and veins), the reduction
in pulmonary arterial pressure may be associated with a reduction in pulmonary
capillary hydrostatic pressure, which in turn, could reduce the amount of
pulmonary edema. Inhaled NO could also limit the remodeling of the pulmonary
circulation due to smooth muscle proliferation, which occurs following acute lung
injury. Recent evidence also suggests an anti-inflammatory role for NO in ARDS.
7) CARDIOPULMONARY BYPASS
 Cardiopulmonary bypass can disrupt pulmonary endothelial function but the
smooth muscle activity can remain intact. Raised pulmonary vascular resistance
in this setting has been successfully treated in both adults and children by inhaled
NO at 20-40 ppm, even in whom a ventricular assist device was in situ.

8) ASTHMA/CHRONIC OBSTRUCTIVE PULMONARY DISEASE

 At present there is insufficient evidence to recommend inhaled NO for treatment
of either asthma or chronic obstructive pulmonary disease.

CONTRAINDICATIONS
1) METHEMOGLOBINEMIA
 Contraindications to NO therapy have not yet been defined; methemoglobinemia
(either congenital or secondary to malarial infection) is, however, a theoretical
contraindication.

ADVERSE REACTIONS
 Reports of adverse effects in clinical studies have been rare. Nevertheless, caution
is required as subtle but serious effects such as the exacerbation of inflammatory
lung diseases are theoretically possible, and the results of further studies are
awaited.
1) POTENTIALLY LIFE-THREATENING EFFECTS
 Paradoxical hypoxia during NO inhalation has been reported in one newborn
infant
2) ACUTE OVERDOSAGE
 Concentrations of 20000 ppm cause acute pulmonary edema and
methemoglobinemia. In a single case, acute overdosage with 600-800 ppm was
associated with death. Therapy is supportive.
3) OTHER EFFECTS
 Clinical studies have shown only mildly increased methemoglobin concentrations
in order of 1-2%. An increase in the bleeding time has been demonstrated in adult
 volunteers, although this does not seem to be a significant problem in clinical
practice

DRUGS FOR MODULATING THE NO AND NOS ACTIVITY

 There are many ways in which the NO can be modulated.

1. NO donor drugs where the NO is produced in the desired quantity for
eliciting their activity.
2. Indirect way of prolonging the effect of NO.
3. Substrates for NOS where some molecules having guanidine functions
will serve as substrates to result in NO production.
4. Inhibition of NOS so that undesirable effects of NO can be nullified.

1) NITRIC OXIDE DONORS :

A ) S-NITROSOTHIOLS
 Thiol groups of proteins and other biomolecules are nitrosated by either acidified
nitrite or aerobic NO. Resulting S-nitroso derivative serve as important storage
and transport forms. The limiting factor for use in clinical applications is the
spontaneous release of NO, highly dependent on trace metal contamination. But
still the above compound is used for dermal infection.
B ) DIAZENIUMDIOLATES
 Unlike the other NO drugs, anionic diazeniumdiolates spontaneously release NO.
NO generation is not affected by the presence of thiols or trace metal
contamination. They have different half lives, so a given amount of NO released
slowly over a long period of time has a less potent but more prolonged duration of
vasodilatory action than same amount released at once.

C ) NITRATE ESTERS – NITROGLYCERINE

 General information :
• It is an anti-anginal drug. It is Nitrate eater and relieves the angina by
generating enough NO on 3-electron metabolic reduction to dilate the
microvessels. On continuous use its efficiency decrease leading to Nitrate
tolerance. It is a function of dosage and frequency of administration.
• Tolerance may result from an inability of the vascular smooth muscle to
convert nitroglycerine to NO or to the activation of mechanism of extraneous
to vessel walls. The nitrites are isomylnitrite, isosorbide dinitrite, isosorbide
mononitrite, erythrytyl mononitrite.
 Mechanism of action :
• Nitrates cause direct, non-specific smooth muscle relaxation. This effect
occurs due to production of nitric oxide, which stimulates formation of cGMP
in the smooth muscle cells, which leads to dephosphorylation of myosin light
chain resulting in smooth muscle relaxation.
• Repeated frequent administration of high dose of organic nitrates leads to
decrease in magnitude of most of their pharmacological actions. They cause
more venodilatation than arteriolar dilatation, hence pre-load and diastolic
pressure is reduced which leads to decreased cardiac work.
 Pharmacokinetics:
• Administered by sublingual route, it is absorbed rapidly.
• On Set of Action
SL: 1-3 minutes; I.V.: 1-2 Minutes. SR: 20-45 minutes; topical ointment: 30 to
60 minutes.
 Adverse effects:
• Throbbing headache, weakness, sweating, flushing, dizziness, tachycardia,
palpitation, methaemoglobinaemia, fainting.
 Contraindications :
• Cerebral trauma, cerebral haemorrhage, hypertrophic cardiomyopathy.
 Special precautions :
• Pulmonary disease, cerebrovascular disease.
 Interactions :
 • Alcohol: Severe hypotension and cardiovascular collapse.
• Aspirin: Increase serum concentrations of nitrates and their actions.
• Antihypertensives, TCAs, Phenothiazine, Calcium channel blockers:
Marked symptomatic orthostatic hypotension.
• Lab Tests: May interfere with the Zlatkis-Zak colour reaction causing a false
report of decreased serum cholesterol.
 Indications:
• Tablets: Angina Pectoris.
• I.V.: Unstable angina, coronary vasospasm, LVF accompanying MI,
hypertension during cardiac surgery.
• Ointment & Transdermal patch: Prevention of angina pectoris, either
alone or in combination with other anti-anginal agents.
 Dosage :
• Adults: Tablet: Acute attacks 0.5mg every 3 minutes till cesation of pain or
limiting side effects appear.
• Prophylaxis: 0.5mg prior to activity. Absorbed through buccal mucosa &
hence chew & do not swallow.
• Inj.: Initially 5 mcg/minute through infusion. Increase with increments of 5
mcg/minute. SR/CR: 2.5-6.5 mg 8-12 hourly. Ointment: 1.5-3cm ribbon
thrice daily or more as required.
• Children: Not applicable.

2 ) SUBSTRATE INHIBITORS :
 Some of the guanidines, thioureas, bis thioureas have been used as substrate
analogues. These molecules have shown to inhibit the NOS enzymes. Drugs can
inhibit NO synthesis or action by several mechanisms. Currently, the most useful
drugs are arginine analogues, which compete with arginine for NOS and in some
cases also compete for the carrier that transports arginine into endothelial cells.
 Several such compounds, e.g. NG-monomethyl-L-arginine (L-NMMA) and NG-
nitro-L-arginine methyl ester (L-NAME), have proved of great value as
 experimental tools. One such compound, ADMA, is present in human urine and
its plasma concentration has recently been found to correlate strongly with
vascular mortality in patients receiving haemodialysis for chronic renal failure.
Consequently it appears likely that ADMA can influence the L-arginine/NO
pathway under pathological conditions in vivo and there is considerable interest
in the hydrolase enzymes that metabolise endogenous ADMA.
 Infusion of L-NMMA into the brachial artery causes vasoconstriction owing to
inhibition of the basal production of NO, without influencing blood pressure or
causing other systemic effects, whereas intravenous L-NMMA increases blood
pressure and causes vasoconstriction in renal, mesenteric, cerebral and striated
muscle resistance vessels.
 There is interest in selective inhibitors of different isoforms of NOS, although
high (>100-fold) selectivity has yet to be achieved. N-iminoethyl-L-lysine is a
selective inhibitor of iNOS. 7-nitroindazole inhibits mouse cerebellar NOS and
following intraperitoneal administration, inhibits nociception without altering
arterial blood pressure: this selectivity apparently results from an incompletely
understood pharmacokinetic effect related to access of the drug to NOS in brain.
 An endogenous protein inhibitor of nNOS (termed ‘PIN’) works by an entirely
different mechanism, namely destabilizing the NOS dimer.

3 ) POTENTIATION OF NITRIC OXIDE :

 Several means whereby the L-arginine/NO pathway could be enhanced are under
investigation. Some of these rely on existing drugs of proven value in other
contexts. The hope is that by potentiating NO they will prevent atherosclerosis or
its thrombotic complications or have other beneficial effects attributed to no.
possibilities include:
• Selective NO donors as ‘replacement’ therapy.
• Dietary supplementation with L-arginine.
• Antioxidants (to reduce superoxide anion formation and hence stabilize NO)
• Drugs that restore endothelial function in patients with metabolic risk factors for
vascular diseases (e.g. angiotensin converting enzyme inhinitors, statins, insulin,
oestrogens)
 • β2-adrenoceptor agonists and related drugs (e.g. nebivolol, a β1-adrenoceptor
antagonist that is metabolised to an active metabolite that activates the L-
arginine/NO-pathway)
• Sildenafil: It potentiates the action of NO a corpus carvenosa smooth muscle. It is
used to treat erectile dysfunction; other possible uses (e.g. in pulmonary
hypertension, gastric stasis) are being investigated.

A) SILDENAFIL
 General information :
• Sildenafil citrate is a potential oral therapy for treatment of erectile
dysfunction.
 Mechanism of action :
• Sildenafil induces erections in response to sexual stimulation by causing
smooth muscle relaxation in the corpus cavernosum through selective
inhibition of phosphodiesterase type 5 (PDE5). Since sildenafil only lowers
the inactivation of cyclic guanosine monophosphate (cGMP) and does not
enhance its production, penile erection due to sildenafil is achieved only in
response to sexual stimulation.
 Adverse effects:
• Headache, flushing, dyspepsia, nasal congestion, urinary tract infection,
abnormal vision, diarrhoea, dizziness, rash, back pain, arthralgia,
tachycardia, angina pectoris, hypotension, postural hypertension,
myocardial infarction, chest pain, allergic reaction.
 Contraindications :
• Hypersensitivity. Patients concurrently or intermittently using organic
nitrates in any form.
 Special precautions :
• Use with caution in patients with sever hepatic and renal impairment
(25mg is recommended).Avoid use in patients with cardiovascular diseases
in whom sexual activity is inadvisable.
 • Use with caution in patients with retinitis pigmentosa. Exercise caution in
treating erectile dysfunction in patients with anatomical deformation of the
penis (such as angulation, cavernosal fibrosis, or Peyronie’s disease) or in
patients who have conditions that may predispose them to priapism (such
as sickle cell anaemia, multiple myeloma, or leukaemia.)
 Interactions :
• Non-specific beta-blockers, loop and potassium-sparing diuretics enhance
efficacy of sildenafil. Concomitant administration of sildenafil 100mg and
amlodipine 5 or 10mg in hypertensive patients led to a mean additional
blood pressure reduction of 8mm Hg systolic and 7mm Hg diastolic.
Cytochrome P450 inhibitors like erythromycin, ketoconazole, itraconazole
are likely to reduce sildenafil clearance.
 Indications:
• Erectile dysfunction.
 Dosage :
• The usual recommended dose is 50mg taken as needed approximately 1
hour before sexual activity. Sildenafil may be taken anywhere from 4
hours to 30 minutes before sexual activity. Maximum recommended dose
is 100mg once daily.
• In patients >65 years, or those with hepatic or severe renal impairment, a
starting dose of 25mg may be considered.

B) NEBIVOLOL :
 General information :
• It is an antiadrenergic agent-beta adrenergic blocker.
 Mechanism of action :
• It is a competitive and highly selective beta-1 receptor antagonist with
nitric oxide (NO)-releasing properties.
 Pharmacokinetics :
• Absorption of nebivolol is rapid and not affected by food. It is extensively
metabolised, partly to active hydroxy-metabolites.
  Adverse effects :
• Headache, dizziness, tiredness and paraesthesia, nausea, diarrhoea,
constipation, visual disturbances, dyspnoea and oedema. Rarely raised
triglyceride levels have been reported.
 Contraindications :
• Hypersensitivity to nebivolol, asthma or history of obstructive airway
disease, hepatic impairment.
 Special precautions :
• Avoid abrupt withdrawal specially in angina, first-degree AV block. Renal
and hepatic insufficiency.
 Indications :
• Treatment of essential hypertension.
 Dosage :
• Adults: 5mg daily preferably at the same time of day.
• In elderly and in those with renal insufficiency the starting dose is 2.5mg
daily, which may be increased to 5mg, if necessary.

C). GINSENOSIDES

 Panax ginseng, one of the most famous Chinese herbs, has been used as an
aphrodisiac, hypotensive, cardiotonic, and sedative substance. It was noted that
the aqueous ginseng extract, mainly containing ginsenoside Rb1, augmented the
relaxation of isolated monkey cerebral arteries elicited by transmural electrical
stimulation or nicotine but did not affect the response to exogenous NO (Toda et
al., 2001). Ginsenosides also potentiate the NO-mediated, neurogenic relaxation
of isolated rabbit corpus cavernosum (Chen and Lee, 1995b) and have the ability
to liberate NO from the endothelium and scavenge oxygen free radicals,
including superoxide and hydroxyl radicals (Gillis, 1997). These actions of
ginsenosides may account for the aphrodisiac, hypotensive, and other
cardiovascular effects of Panax ginseng.
 In contrast to the potentiating action of PDE inhibitors, neurogenic responses of
the isolated monkey artery are prolonged by ginsenoside only in parallel with the
 increased magnitude of responses, and the response to exogenous NO is not
enhanced, suggesting that the PDE inhibitory action is not involved (Toda et al.,
2001). Since ginsenoside additionally increases the neurogenic response that has
already been augmented by treatment with atropine (Toda et al., 1997b), an
inhibition of prejunctional muscarinic receptors is not responsible for
potentiation by the Chinese herb. Mechanisms of the potentiating action must be
elucidated in future studies

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