Study of The Discovery and Development of Specific Inhibitors of Phosphodiesterase Type 5

STU
UDY OF THE DISC

COVERY AND
A
DEV
VELOPM
MENT OF
F SPECIFIIC
INHIIBITORS OF PHO
OSPHODIIESTERASE TYPE 5 (PDE5)) AND TH
HE
CLIN
NICAL PR
ROBLEM
MS ASSOC
CIATED W
WITH TH
HOSE INH
HIBITOR
RS
A Disssertation iss submitted for the parttial fulfillment of the coourse of Pharmaceuticcal
Resea
arch (PHRM
M 404) of th
he Departm
ment of Pharrmacy, East West Univeersity for th
he
degree of Bachelor of Ph
harmacy.
Supe
ervised b
by
Hossain
Dr.
D Chowd
dhury Faiz H
Professor
P
&
Ch
hairperson
Departmeent of Pharrmacy
East West
W Univerrsity
Sub
bmitted B
By
Deb
basree Paull
ID: 20
010-3-70-0036
Departme
D
ent Of Phaarmacy
East West
W Univerrsity
Dedication
This Research Paper is dedicated to
My beloved parents,
Who are my biggest Inspirations
DECLARATION BY THE CANDIDATE

I, Debasree Paul, hereby declare that this dissertation, entitled Study of the discovery and
development of specific inhibitors of phosphodiesterase type 5 (PDE5) and the clinical
problems associated with those inhibitors submitted to the Department of Pharmacy, East
West University, in the partial fulfillment of the requirement for the degree of Bachelor of
Pharmacy (Honors) is a genuine & authentic research work carried out by me under the guidance
of Dr. Chowdhury Faiz Hossain, Professor & Chairperson, Department of Pharmacy, East West
University, Dhaka. The contents of this dissertation, in full or in parts, have not been submitted
to any other Institute or University for the award of any Degree or Diploma of Fellowship.
---------------------------------Debasree Paul
ID: 2010-3-70-036
Department of Pharmacy
East West University
Aftabnagar, Dhaka
CERTIFICATION BY THE SUPERVISOR
This is to certify that the dissertation, entitled Study of the discovery and development of
specific inhibitors of phosphodiesterase type 5 (PDE5) and the clinical problems associated
with those inhibitors is an original literature research work done, under our guidance and
supervision by Debasree Paul (ID: 2010-3-70-036), in partial fulfillment of the requirement for
the degree of Bachelor of Pharmacy.
--------------------------------------Dr. Chowdhury Faiz Hossain

Professor & Chairperson
Department of Pharmacy
East West University
Aftabnagar, Dhaka
ACKNOWLEDGEMENTS
It is my pleasure and proud privilege to express my heartiest regards and gratitude to my
respected teacher and supervisor to Dr. Chowdhury Faiz Hossain, Professor and Chairperson,
Department of Pharmacy, East West University, for his expert supervision, constructive
criticism, valuable advice, optimistic counseling, constant support and continuous backup and
encouragement throughout every phase of the project as well as to prepare this dissertation.
I would also like to put forward my most sincere regards and profound gratitude to him for
giving me the opportunity to conduct such an interesting project and for facilitating a smooth
conduction of my study.
Last but not the least, I would like to thank my family, and friends for their care and
encouragement during my research work.
CONTENTS
1. Introduction
1.1 About phosphodiesterase enzyme
1-5
1
1.1.1 Nucleotide and phosphate bond linkage
1.1.2 cAMP and cGMP
1.1.3 Cyclic nucleotide phosphodiesterases
1.2 Studies on PDE
1.3 Multiple forms of phosphodiesterases
1.4 Phosphodiesterases as drug targets
2. Phosphodiesterase Family Classification

2.1 Phosphodiesterase classification
7-16
7
2.1.1 About PDE1
2.1.2 About PDE2
2.1.3 About PDE3
2.1.4 About PDE4
2.1.5 About PDE5
2.1.6 About PDE6
2.1.7 About PDE7
2.1.8 About PDE8
10
2.1.9 About PDE9
10
2.1.10 About PDE10
10
2.1.11 About PDE11
11
2.2 Overview of PDE isoform localization
11
2.3 Function of phosphodiesterase enzymes
15
2.4 PDE inhibitors
16
3. PDE5 Inhibitors
18-50
3.1 Cyclic guanosine monophosphate (cGMP)
18
3.2 Pathological importance of PDE5 inhibitor
18
3.2.1 Erectile dysfunction

3.3 Some PDE5 inhibitors and their mechanism of action
18
19
3.3.1 Treatment of erectile dysfunction
19
3.3.2 Treatment of pulmonary hypertension
21
3.4 Development and discovery of Sildenafil, Vardenafil and Tadalafil
21
3.5 Sildenafil dosing
26
3.6 Sildenafil usage
27
3.6.1 Pediatric usage
27
3.6.2 Geriatric usage
28
3.6.3 Pregnancy usage
28
3.6.4 Breastfeeding
28
3.7 Molecular structure of Sildenafil
28
3.8 Drug-drug interactions
31
3.9 Vardenafil brand names and indications
36
3.10 Vardenafil dosage
36
3.11 Vardenafil metabolism, elimination and usage
37
37
37
37
3.11.4 Breastfeeding
38
3.12 Drug interactions
38
3.13 Tadalafil brand names and indications
42
3.14 Tadalafil potency
43
3.15 Tadalafil dosage
43
3.16 Tadalafil usage
44
44
44
3.16.3 Pregnancy category
44
45
45
3.18 Pharmacokinetics of the three PDE5 inhibitors
46
3.19 Side-effects of PDE5 inhibitors (Viagra, Cialis, Levitra)
46
3.20 Precautions of PDE5 inhibitors (Viagra, Cialis, Levitra)
46
3.21 Selectivity compared among the three PDE5 inhibitors
49
3.22 Herbal erectile dysfunction treatments
49
3.23 Adulteration of herbal products with synthetic PDE5 inhibitors
50
4. Conclusion
52-56
4.1 Conclusion
52
4.1.1 Therapeutic agent for parasitic disease
52
4.1.2 Better enhanced erectile function
52
4.1.3 PDE5 inhibitors for treatment of Alzheimers disease
53
4.1.4 Development of acute and chronic cardioprotective therapy
53
4.1.5 PDE5 inhibitors for the treatment altitude pulmonary edema
53
4.1.6 Treatment of benign prostatic hyperplasia
54
4.1.7 Antidote to psychotropic-induced sexual dysfunction in women
54
4.1.8 Improve uterine artery blood flow and endometrial development

in IVF
55
4.1.9 Enhances Vasodilatation in Fetal Growth Restriction
56
4.1.10 Therapy for severe early-onset intrauterine growth restriction
56
4.1.11 Increased focus on development of selective PDE inhibitors
56
5. Reference
58-66
LIST OF FIGURES
Figure 1: Structure of cyclic cGMP
Figure 2: Structure of cAMP and cGMP
Figure 3: Phosphodiesterases hydrolyze the 3 cyclic phosphate bond.
Figure 4: Cyclic nucleotide signaling and regulation
Figure 5: Schematic diagram showing the inhibitory effect of Viagra (PDE5

inhibitor)
Figure 6: Effect of PDE 5 inhibitors on blood vessels
20
21
Figure 7: Development of sildenafil for erectile dysfunction and pulmonary

hypertension
25
Figure 8: Chemical structure of Sildenafil, Vardenafil and Tadalafil
26
Figure 9: Sildenafil
29
Figure 10: Sildenafil shown in the binding pocket of PDE5. The essential
interactions occur with Gln817 and Phe820.
30
Figure 11: H-loop (red) section of PDE5 protein (purple) closes off binding
pocket after sildenafil binds.
30
Figure 12: Close-up of sildenafil in the binding pocket. H-loop (red),

magnesium (orange), zinc (light blue) and Gln659 (white)
31
LIST OF TABLES
Table 1: PDE isoform localization
11
Table 2: Overall function of different types of phosphodiesterase enzymes

and their subclass
15
Table 3: Different types of PDE inhibitors
16
Table 4: The main stages in the development of Viagra
23
ABSTRACT
Cyclic nucleotide phosphodiesterases (PDEs) are enzymes that regulate the cellular levels of the
second messengers, cAMP and cGMP, by controlling their rates of degradation. There are 11
different PDE families, with each family typically having several different isoforms and splice
variants. These unique PDEs differ in their three-dimensional structure, kinetic properties, modes
of regulation, intracellular localization, cellular expression, and inhibitor sensitivities. Current
data suggest that individual isozymes modulate distinct regulatory pathways in the cell. These
properties, therefore, offer the opportunity for selectively targeting specific PDEs for treatment
of specific disease states. The feasibility of these enzymes as drug targets is exemplified by the
commercial and clinical successes of the erectile dysfunction drugs, sildenafil (Viagra), tadalafil
(Cialis), and vardenafil (Levitra). PDE inhibitors are also currently available or in development
for treatment of a variety of other pathological conditions. In this review the basic biochemical
properties, cellular regulation, expression patterns, and physiological functions of the different
PDE isoforms is discussed based on original research papers. How these properties relate to the
current and future development of PDE inhibitors as pharmacological agents is especially
considered. PDEs hold great promise as drug targets and recent research advances make this an
exciting time for the field of PDE research.
Keywords: Cyclic nucleotide phosphodiesterases, cAMP, cGMP, PDE inhibitors, sildenafil,
tadalafil, vardenafil.
Chapter One
INTRODUCTION
1.1 Abou
ut phospho
odiesterase enzyme
e
A phosphodiesterasee is an enzy
yme that cattalyzes the hhydrolysis oof phosphoddiester bondss, for
m
off cyclic AMP
P or cyclic G
GMP(Lomass and Zaccollo, 2014). It plays
instance a bond in a molecule
a role in signal transsduction by regulating the
t intracelluular concenttration of cyyclic nucleotides.
This phosphodiesterrase catalyzzes the speecific hydroolysis of ccGMP to 55'-GMP. Huuman
phosphod
diesterase 5 is mainly responsible for the deggradation off cyclic GM
MP in the coorpus
cavernosum (Georgee, 2014; Jayaashankar, 200
07).
The phossphodiesteraases that cleaave cyclic nu
ucleotides, thhat are impoortant for traansmitting siignals
within th
he cell, are known
k
as cy
yclic nucleo
otide phosphhodiesterasess (PDEs). P
Phosphodiestterase
inhibitorss can be used as drugs, and
a are used
d commerciaally to treat m
male erectilee dysfunctionn and
other con
nditions (Geo
orge, 2014).
1.1.1 Nu
ucleotide an
nd phosphate bond link
kage
A nucleo
otide is a com
mpound thatt has an arom
matic base ccontaining nnitrogen, a suugar that is eeither
ribose orr deoxyribosse, and a ph
hosphate gro
oup. DNA annd RNA aree long strandds of nucleootides
with each
h nucleotide linked sequ
uentially to th
he next and are polymerrs. A phosphhate bond linnkage
is an imp
portant part of
o the polym
merization off these nucleootide chains (Lehninger,, 1975; Albeerts et
al., 2002).
The cycllic portion reefers to the two single bonds
b
betweeen the phosphate groupp and the riibose.
This mak
kes the comp
pound cyclicc, enabling itt to bind protteins differently (Lehninnger, 1975).
Fiigure 1: Stru
ucture of cycclic cGMP
1.1.2 cA
AMP and cG
GMP
A cyclic nucleotide is
i a single-ph
hosphate nuccleotide withh a cyclic boond arrangem
ment betweeen the
sugar and phosphatee groups. Like other nucleotides, cyyclic nucleootides are coomposed of three
functionaal groups: a sugar, a niitrogenous base,
b
and a ssingle phospphate groupp. The two ccyclic
nucleotid
des in all ceells are cycllic AMP (cA
AMP) and ccyclic GMP
P (cGMP) (L
Lehninger, 11975;
Alberts et
e al., 2002).
Figu
ure 2: Structture of cAMP
P and cGMP
P
As can be seen in
n the cycliic adenosinee monophoosphate (cA
AMP) and ccyclic guannosine
monopho
osphate (cG
GMP) imagees, the 'cycclic' portionn consists oof two bonnds betweenn the
phosphatte group and
d the 3' and 5' hydroxyl groups of thhe sugar, veery often a riibose. cAMP
P and
cGMP haave a single base of aden
nine and guaanine, respecctively (Franncis and Corbbin, 1999).
These co
ompounds react
r
in man
ny differentt cellular prrocesses, annd are know
wn as seconndary
messengeers. The firsst signal is relayed
r
from
m outside of the cell by the binding of a hormone or
neurotran
nsmitter. Th
his binding then triggerrs an increaase in cAM
MP or cGMP
P concentrattions,
which grreatly ampliffies the magn
nitude of thee original siggnal (Lehninnger, 1975).
Numerou
us cellular fu
unctions are regulated by
y these seconnd messengeers, cAMP aand cGMP. IIn the
cardiovasscular system, blood prressure is reegulated byy contractionn and relaxaation of vasscular
smooth muscle
m
in asssociation witth vascular endothelial
e
ffunctions. Beeating of carrdiac myocyytes is
accuratelly controlled
d to pump blood
b
out off the heart tto other parrts of the boody accordinng to
environm
mental cond
ditions. Theese events in hemodyynamics aree ingeniously regulatedd by
extracellu
ular stimulaation through
h alteration of intracelluular cyclic nucleotide llevels, which are
determin
ned by a ballance betweeen their pro
oduction andd degradatioon by 3,5-ccyclic nucleeotide
phosphod
diesterases (PDEs) (Albeerts et al., 20
002; Omori and Kotera, 2006).
1.1.3 Cy
yclic nucleottide phosph
hodiesterasses
Cyclic nucleotide
n
ph
hosphodiesteerases degraade the cycclic nucleotidde by cleavving a phospphate
bond thaat keeps thee nucleotide cyclic. Thiis is knownn as phosphoodiester bonnd cleavage, and
causes deegradation of the cyclic nucleotide
n
(G
George, 20114).
Figure 3: Phosphodiesterases hydrolyze

h
thhe 3 cyclic pphosphate boond.
This deg
gradation regulates the duration, lo
ocalization, and amplituude of the signaling oof the
compoun
nd(Lugnier, 2006).
Figure 4: Cyclic nucleeotide signalling and reguulation
Localization of fundamental molecules involved in cAMP and cGMP signaling is illustrated.

Effector molecules of cAMP and cGMP are indicated by arrows from each cyclic nucleotide.
Phosphorylation of PDEs by PKA and PKG is demonstrated by dotted arrows. Modulation of
PDE activity by cGMP is shown by a thick arrow. Cellular and physiological outputs of cyclic
nucleotide signaling are shown in gray-colored boxes. NPs indicate natriuretic peptides; NO,
nitric oxide; NPRs, natriuretic peptide receptors; sGCs, soluble guanylyl cyclase; AC, adenylyl
cyclase; Gs, GTP-binding protein subunit; GPCRs, G proteincoupled receptors; Epac,
exchange protein directly activated by cAMP; CNG-channel, cyclic nucleotide-gated channel
(Omori and Kotera, 2006; Kobiaka and Gorczyca, 2000).
Production of cAMP by adenylyl cyclase (AC) can be regulated by GPCR and PKA. cAMP
signaling can lead to the activation of PKA, Epac and ion channels. Cyclic AMP can also be
degraded by PDEs (PDE1, 2, 3, 4, 7, 8, 10, or 11) and cGMP can inhibit selective PDE cAMP
degrading activity. Production of cGMP by guanylyl cyclase (GC) can be regulated by nitric
oxide (NO) and PKG. cGMP signaling can lead to the regulation of other PDEs, PKG and ion
channels. Cyclic GMP can also be degraded by PDEs (PDE1, 2, 3, 5, 6, 9, 10, or 11) (Omori and
Kotera, 2006; Taskn and Aandahl, 2004).
1.2 Studies on PDE

Almost immediately after the discovery of cAMP by Sutherland and colleagues, cyclic
nucleotide PDE activity was described (Butcher and Sutherland, 1962). With the subsequent
discovery of cGMP, it was found that both cAMP and cGMP could be hydrolyzed by the same
type of activity, i.e., hydrolysis of the 3 cyclic phosphate bond. On the basis of substrate
competition studies, it was clear that at least some of these activities must have the same
catalytic site. In fact, many of the early studies on cyclic nucleotides were directed toward
understanding PDE activity since at that time it was much easier to measure PDE activity than
either cAMP or cGMP themselves or the enzymes that catalyzed their synthesis. With the advent
of assays using radioactive substrate, it became clear that there were likely to be multiple forms
of PDEs with different kinetic and regulatory properties (Thompson et al., 1979; Beavo et al.,
1982). However, it was not until higher resolution fractionation techniques, monoclonal
antibodies, and molecular cloning and sequencing procedures were applied to the PDEs that the
truly large number of different gene products was fully appreciated (Lugnier, 2006).
1.3 Multiple forms of phosphodiesterases

One of the most important reasons that the PDEs are recognized as being good drug targets is the
fact that there are so many different isoforms. Only since the completion of the human and
mouse genome projects has the extent of this enzyme diversity begun to be fully appreciated.
Originally, PDEs were classified on the basis of their substrate specificity, modes of regulation,
and elution order from ion exchange columns. As soon as primary amino acid and nucleotide
sequences started to become available, they were further classified according to family
relationships based on homologies in primary sequence. Currently, it is widely accepted that
there are 11 different families of PDE comprising 21 different gene products (Bender and Beavo,
2006).
1.4 Phosphodiesterases as drug targets

Immediately after the discovery of PDE activity, it was found that caffeine was an effective
inhibitor of PDE activity and a number of nonselective PDE inhibitors including the caffeine
analog, theophylline, have been in use as therapeutic agents for many years now. Thus, the
principle that inhibition of PDE activity could be a valid therapeutic target is now well accepted.
However, most of the early PDE inhibitors had a very narrow therapeutic index, due at least in
part to the fact that nearly all of the early inhibitors would inhibit most, if not all, PDE activity in
every tissue (Bender and Beavo, 2006).
One important general reason that PDEs have been pursued as therapeutic targets is related to the
basic pharmacological principle that regulation of degradation of any ligand or second messenger
can often make a more rapid and larger percentage change in concentration than comparable
regulation of the rates of synthesis. This is true for either pharmacokinetic changes in drug levels
or changes in amounts of an endogenous cellular regulatory molecule or metabolite (Bender and
Beavo, 2006).
It has been apparent for many years now that there are a rather extraordinarily large number of
different forms of PDEs expressed in mammalian tissues, each of which can have a unique
architecture at the active site. Moreover, there is increasing evidence that many of these PDEs
are tightly connected to different physiological functions in the body and by inference also to
different pathological conditions. Therefore, it has been widely believed that it should be
possible to develop isoform selective inhibitors that can target specific functions and
pathological conditions without a high likelihood of causing nonspecific side effects. The recent
therapeutic and commercial success of agents such as sildenafil (Viagra), a selective PDE5
inhibitor, has validated the concept (Bender and Beavo, 2006).
Another reason PDEs are likely to be good drug targets relates to the concentrations of their
substrates in the cell. It is commonly accepted that the levels of cAMP and cGMP in most cells
are typically <1 to 10 M. This means that a competitive inhibitor would not need to compete
with very high levels of endogenous substrate to be effective. This fact has, for example,
hindered the development of most protein kinase inhibitors, as they need to have high enough
affinity to displace millimolar concentrations of ATP. At the same time, such an inhibitor must
be selective among thousands of other enzymes that use ATP. However, the challenging
development of protein kinase inhibitors is not impossible as selective inhibitors are beginning to
appear. So the fact that PDEs are relatively unique in their substrate binding requirements and
also that they use a substrate that is 100 to 1000 times lower than ATP makes them an
intrinsically more attractive pharmacological target than many other enzymes that use more
abundant substrates (Bender and Beavo, 2006).
Chapter Two
PHOSPHODIESTERASE
FAMILY CLASSIFICATION
2.1 Phosphodiesterase classification

There are many types of this class of phosphodiesterase, specialized for different functions.
Eleven families of PDEs with varying selectivities for cAMP or cGMP have been identified in
mammalian tissues. Within these families, multiple isoforms are expressed either as products of
different genes or as products of the same gene through alternative splicing. Regulation of PDEs
is important for controlling myriad physiological functions, including the visual response,
smooth muscle relaxation, platelet aggregation, fluid homeostasis, immune responses, and
cardiac contractility(Francis et al., 2001).
The PDEs differ in many aspects, including their biochemical properties and which cyclic
nucleotide they can act upon. PDEs are critically involved in feedback control of cellular cAMP
and cGMP levels. These are part of the criteria used to assign them to families. Some degrade
only cAMP, while others only affect cGMP. Other PDEs can degrade both of these cyclic
nucleotides (Jeon et al., 2005).
Activities of the various PDEs are highly regulated by panoply of processes, including
phosphorylation events, interaction with small molecules such as cGMP or phosphatidic acid,
subcellular localization, and association with specific protein partners. The PDE superfamily
continues to be a major target for pharmacological intervention in a number of medically
important maladies (Francis et al., 2001).
2.1.1 About PDE1

Descriptive Name: Calmodulin-dependent PDE
Regulators: Ca2+/Calmodulin
Substrate Specificity: cAMP or cGMP
Inhibitors: Vinpocetine
Major Tissue Expression: Brain, heart, smooth muscle, olfactory cilia
Physiological
Function:
Sperm
development
and
maturation;
Monocyte/macrophage
differentiation; Olfactory neuron regulation; Neuronal regulation

Disease Relevance: Fertility; Inflammation; Olfaction (O'Donnell and Zhang, 2004; Bender and
Beavo, 2006; Omori and Kotera, 2006).
2.1.2 About PDE2

Descriptive Name: cGMP-stimulated PDE
Regulators: cGMP
Inhibitors: EHNA (Erythro-9-(2-hydroxy-3-nonyl)adenine)
Major Tissue Expression: Adrenal cortex; Brain; Heart
Physiological Function: 2/3 regulation of cardiac myocytes; Endothelial cell function
Disease Relevance: Heart disease; Anti-angiogenic (Page and Spina, 2012; Zhang et al., 2004;
Bender and Beavo, 2006; Omori and Kotera, 2006)
2.1.3 About PDE3

Descriptive Name: cGMP-inhibited PDE
Regulators: cGMP; Insulin; Leptin
Inhibitors: Cilostamide; Enoximone; Imazodan; Trequinsin; Milrinone
Major Tissue Expression: Heart; Adipose; Pancreas; Platelets
Physiological Function: Platelet function; Adipocyte function
Disease Relevance: Intermittent claudication peripheral arterial occlusive disease; Restenosis;
Obesity; Type-2 diabetes (Bender and Beavo, 2006; Omori and Kotera, 2006).
2.1.4 About PDE4

Descriptive Name: cAMP-specific PDE
Regulators: PKA (cAMP-dependent protein kinase); ERK; Phosphatidic acid
Substrate Specificity: cAMP
Inhibitors: Rolipram
Major Tissue Expression: Many tissues
Physiological Function: Regulates monocyte, macrophage T-cell, eosinophil, neutrophil
function; Regulates neuronal function and differentiation; Regulates functions of inflammatory
cells and vascular smooth muscle cells; Pro-apoptotic; Anti-apoptotic; Endothelial cell function;
Inhibition of bone loss (Zhang et al., 2004; Bender and Beavo, 2006; Omori and Kotera, 2006;
Omori and Kotera, 2006).
Disease Relevance: Airway inflammation (asthma, COPD); Rheumatoid arthritis; Crohn's

disease; Learning; Memory; Schizophrenia; Spinal cord injury; Stroke; Restenosis; Chronic Bcell lymphocytic leukemia; Spinal cord injury; Parkinson's Disease; Anti-angiogenic; Osteopenia
(including osteoporosis) (Page and Spina, 2012; Bender and Beavo, 2006; Zhang et al., 2004;
Omori and Kotera, 2006).
2.1.5 About PDE5

Descriptive Name: cGMP-binding PDE
Regulators: PKG (cGMP-dependent protein kinase)
Substrate Specificity: cGMP
Inhibitors: Sildenafil; Vardenafil; Dipyridamole; Zaprinast
Major Tissue Expression: Lung; Platelets; Smooth muscle; Corpus collusum
Physiological Function: Vascular smooth muscle cell relaxation
Disease Relevance: Penile erectile dysfunction; Asthma; COPD; Pulmonary hypertension;
Migraine (Zhang et al., 2004; O'Donnell and Zhang, 2004; Bender and Beavo, 2006; Omori and
Kotera, 2006)
2.1.6 About PDE6

Descriptive Name: Photoreceptor PDE
Regulators: Light
Inhibitors: Zaprinast; Dipyridamole
Major Tissue Expression: Rod and cone; Photoreceptor; Outer segments
Physiological Function: Visual signal transduction
Disease Relevance: Retinitis pigmentosa (Keravis and Lugnier, 2012; Bender and Beavo, 2006;
Omori and Kotera, 2006)
2.1.7 About PDE7

Descriptive Name: High affinity cAMP-specific PDE
Regulators: Not Known
Inhibitors: BRL50481
Major Tissue Expression: Skeletal muscle; T-cells (Keravis and Lugnier, 2012; Bender and
Beavo, 2006; Omori and Kotera, 2006)
Physiological Function: Not Known
2.1.8 About PDE8

Descriptive Name: cAMP-specific PDE
Inhibitors: Dipyridamole
Major Tissue Expression: Testis; Liver; Thyroid
Physiological Function: Thyroid function; T-cell activation
Disease Relevance: Hyperthyroidism; Metabolic bone disease (Bender and Beavo, 2006; Omori
and Kotera, 2006)
2.1.9 About PDE9

Descriptive Name: High affinity cGMP-specific PDE
Inhibitors: SCH-51866 which is cis-5,6a,7,8,9,9a-Hehahydro-2-[4-(trifluoromethyl)phenylme
thyl]-5-methyl-cyclopent[4,5]imidazo[2,1-b]purin-4(3H)-one
Major Tissue Expression: Kidney
Physiological Function: Not Known
Disease Relevance: Fertility (Keravis and Lugnier, 2012; Zhang et al., 2004; Bender and Beavo,
2006; Omori and Kotera, 2006)
2.1.10 About PDE10

Descriptive Name: Dual specificity PDE
Regulators: PKA
10
Major Tissue Expression: Testis; Brain

Physiological Function: Striatal neuron function
Disease Relevance: Parkinsonism; Schizophrenia; Obsessive compulsive disorders; Addictions
(Page and Spina, 2012; Bender and Beavo, 2006; Omori and Kotera, 2006)
2.1.11 About PDE11

Descriptive Name: Dual specificity PDE
Major Tissue Expression: Skeletal muscle; Prostate
Physiological Function: Sperm function (motility, number)
Disease Relevance: Fertility (Page and Spina, 2012; Bender and Beavo, 2006)
2.2 Overview of PDE isoform localization

Table 1: PDE isoform localization (Bender and Beavo, 2006; Jayashankar, 2007)
Isoform
Localization
Tissue/Cellular
PDE1A
Intracellular
Smooth muscle, heart, lung, brain, Predominantly cytosolic

sperm; PDE1A1 in lung and heart,
PDE1A2 in brain
PDE1B
PDE1B1 in neurons, lymphocytes, Cytosolic

and smooth muscle; PDE1B2 in
macrophages and lymphocytes
PDE1C
Brain, proliferating human smooth Cytosolic

muscle,
spermatids;
PDE1C2
in
olfactory epithelium
PDE2A
Adrenal medulla, brain, heart, platelet, PDE2A3
and
macrophage subsets, endothelial cell membrane-bound,
PDE2A2
whereas
variants
PDE2A1
are
is
subsets; PDE2 is highly localized to cytosolic
11
Isoform
Localization
unique neuronal populations and brain
regions
PDE3A
Heart,
vascular
smooth
muscle, Can be either membrane-associated or
platelets, oocyte, kidney; PDE3A cytosolic, depending on the variant and the
variants have differential expression cell type it is expressed in
in cardiovascular tissues
PDE3B
Vascular smooth muscle, adipocytes, Predominantly

hepatocytes,
kidney,
membrane-associated;
cells, Localized to endoplasmic reticulum and
developing sperm, T lymphocytes, microsomal fractions

macrophages
PDE4A
Widely expressed with mRNA found PDE4A5 is localized to membrane ruffles

in many tissues; olfactory system, through its Src homology domain; the
immune cells, and testis; high levels supershort variant PDE4A1 is entirely
of
several
of
the
variants
are membrane-associated; PDE4A4 associates
distributed throughout the brain
with Src family kinases; PDE4A5 is

localized by AKAP binding; PDE4s can be
recruited to interact with -arrestin; PDE4A
is recruited to a lipid raft fraction in
activated T cells
PDE4B
High levels of mRNA detected in a PDE4s can be recruited to interact with variety of tissues; notable expression arrestin; is recruited to a lipid raft fraction in
in immune cells and the brain
PDE4C
activated T cells
More restricted expression compared Predominantly cytosolic; PDE4s can be

with other PDE4 isoforms; mRNA recruited to interact with -arrestin
found in lung, testis, and several cell
lines mainly of neuronal origin
PDE4D
mRNA widely distributed and found Depending on the identity of the variant, can
in a variety of tissues; protein levels be
found
in
cytosolic
or
particulate
high in the brain and in several other fractions; PDE4D3 is localized by binding
tissues as well; expression of variants to mAKAP and AKAP450; PDE4D3 is a
12
Isoform
Localization
seems to be localized to specific part of the cardiac RyR2 channel complex;
tissues and regions; variants are found PDE4D5 interacts with RACK1; can be
in many commonly used cell lines recruited to interact with -arrestin; is
(HEK293, COS) and in inflammatory recruited to a lipid raft fraction in activated
cells
PDE5A
T cells
Platelets, vascular smooth muscle, Cytosolic

brain, lung, heart, kidney, skeletal
muscle; PDE5A1 and PDE5A2 are
widely expressed, whereas PDE5A3
is specific to vascular smooth muscle
PDE6A/
High expression in rod cells of the Targeted
PDE6B
photoreceptor layer of retina; also isoprenylation;

found in pineal gland
PDE6C
to
the
membrane
association
with
by
the
subunit results in cytosolic localization
High expression in cone cells of the Cytosolic by virtue of its association with
photoreceptor layer of retina; also the subunit
found in pineal gland
PDE7A
Immune cells, heart, skeletal muscle, Cytosolic; localizes with AKAP MTG in
endothelial cells; PDE7A1 protein lymphocytes
detected in a variety of immune cells,
whereas PDE7A2 protein was found
only in cardiac tissue
PDE7B
mRNA found in brain, heart, liver, Cytosolic

skeletal muscle, pancreas, testis; in
rat, PDE7B3 expression was restricted
to the heart, whereas PDE7B2 was
only found in testis; PDE7B1 is
expressed in multiple tissues
PDE8A
mRNA found in many tissues but Found in both cytosolic and particulate
highest
in
testis,
spleen,
small fractions
intestine, ovary, colon, and kidney;
13
Isoform
Localization
PDE8A2-5 is expressed in much
lower
abundance
than
PDE8A1;
differential localization of PDE8A1

and PDE8A2 mRNA
PDE8B
Brain and thyroid; variant expression Found in both cytosolic and particulate
is differential as PDE8B1 is expressed fractions
only in thyroid, whereas PDE8B3 is
expressed equally in brain and thyroid
PDE9A
mRNA for most variants has been PDE9A5 protein has been shown to be
detected in nearly every tissue tested cytosolic, whereas PDE9A1 is localized to
with highest levels in kidney, brain, the nucleus
spleen,
various
tissues,
and
gastrointestinal
prostate;
variants
expressed differentially
PDE10A
Highest expression in brain, testis, PDE10A1 and PDE10A3 variants are

heart, and thyroid; also reported in cytosolic, whereas PDE10A2 is particulate;
pituitary gland and in striated and PDE10A2
phosphorylation
triggers
cardiac muscle; PDE10A2 mRNA translocation of the enzyme from the Golgi
expression seems to be higher than to the cytosol
that for PDE10A1 in most tissues
PDE11A
mRNA found in skeletal muscle, Cytosolic

prostate, testis, salivary gland, thyroid
gland, and liver; PDE11A1 is most
prominent
PDE11A3
in
is
skeletal
specific
muscle;
to
testis;
PDE11A4 is highest in prostate
14
2.3 Function of phosphodiesterase enzymes

Table 2: Overall function of different types of phosphodiesterase enzymes and their subclass
(Bender and Beavo, 2006)
PDE Family
PDE1
Function(s)
PDE1A probably serves to regulate vascular smooth muscle contraction and
may play a role in sperm function; PDE1B is involved in dopaminergic
signaling as well as immune cell activation and survival; PDE1C is required
for vascular smooth muscle cell proliferation and may also regulate sperm
function and neuronal signaling
PDE2
PDE2 frequently mediates cross-talk between cGMP and cAMP pathways; it

regulates aldosterone secretion from the adrenal gland, cAMP and PKA
phosphorylation of Ca+2 channels in the heart, cGMP in neurons, long-term
memory, and barrier function of endothelial cells under inflammatory
conditions
PDE3
PDE3A regulates cardiac contractility, platelet aggregation, vascular smooth

muscle contraction, oocyte maturation, and regulation of renin release;
PDE3B mediates insulin signaling, especially its antilipolytic effects;
PDE3B also regulates cell cycle/proliferation and mediates the inhibitory
effects of leptin and other signals on insulin secretion and renin release
PDE4
At least one form is expressed in most cells, and PDE4s play roles in a wide
array of processes, including brain function, monocyte and macrophage
activation, neutrophil infiltration, vascular smooth muscle proliferation,
fertility, vasodilation, and cardiac contractility
PDE5
PDE5 is a well-documented regulator of vascular smooth muscle

contraction, especially in penis and lung; it is involved in NO-cGMP
signaling in platelets to control aggregation and may also play a role in
regulation of cGMP signaling in the brain
PDE6
PDE6 is involved in signal transduction of the photoresponse in the eye; it

may also regulate melatonin release from the pineal gland
15
PDE Family
PDE7
Function(s)
PDE7 is implicated to play a role in T-cell activation and activation of other
inflammatory cells
PDE8
PDE8 may play a role in T cell activation, sperm, or leydig cell function
PDE9
The function of PDE9 is currently unknown, but it has been postulated to

regulate NO-cGMP signaling in the brain
PDE10
PDE10A is thought to be a regulator of cGMP in the brain and may play a

role in learning and memory
PDE11
PDE11 possibly has a role in sperm development and function
2.4 PDE inhibitors

The diversity of reactions affected by these enzymes makes them promising targets for drug
therapy. Inhibition of the PDEs prolongs the reaction being mediated by the cyclic nucleotide.
Table 3: Different types of PDE inhibitors (Bender and Beavo, 2006; Jayashankar, 2007)
PDE
PDE1
Inhibitors
Vinpocetine
Usage
Only recently have selective PDE1 inhibitors been developed;
IC224 may be the most selective in intact cells
PDE2
EHNA (erythro- Inhibitors are being investigated for improving memory and
9-(2-hydroxy-3-
decreasing
nonyl)adenine);
conditions
endothelial
permeability
under
inflammatory
BAY 60-7550
PDE3
Cilostamide;
Milrinone is a currently approved treatment for short term
Milrinone;
congestive heart failure; cilastazol is a treatment for intermittent
Trequinsin;
claudication
Cilastazol
PDE4
Rolipram;
Multiple compounds have undergone trials for treatment of
Roflumilast;
chronic obstructive pulmonary disease but have experienced
Cilomilat
limited success because of side effects; compounds are also

under investigation for several other inflammatory conditions;
16
PDE
Inhibitors
Usage
interest exists in using PDE4 inhibitors for CNS disorders,
including depression and improvement of memory
PDE5
PDE6
Zaprinast;
Sildenafil, vardenafil, and tadalafil are in usage as erectile
Sildenafil;
dysfunction drugs; these compounds are in trials for other
Vardenafil;
indications such as pulmonary hypertension and benign prostatic
Tadalafil
hyperplasia
Sildenafil
Inhibition of PDE6 may be a source of sildenafil side effects on

vision; genetic mutations in PDE6 are the basis for several vision
related diseases, but PDE6 has not been investigated as a
therapeutic target
PDE7
BRL 50481
PDE7-selective inhibitors have been investigated as antiinflammatory agents in vitro but so far have shown limited utility
in vivo
PDE8
PDE9
No truly selective inhibitors are yet available

BAY 73-6691
BAY 73-6691 is in preclinical development for Alzheimer's

disease treatment
PDE10
No truly selective inhibitors are yet available
PDE11 Tadalafil
PDE11 has received pharmacological interest because it is also

inhibited by tadalafil and thus is a potential source for side
effects
17
Chapter Three
PDE5 INHIBITORS
3.1 Cyclic guanosine monophosphate (cGMP)

cGMP is a cyclic nucleotide (a second messenger) derived from guanosine triphosphate (GTP).
cGMP acts as a second messenger much like cyclic AMP, most notably by activating
intracellular protein kinases in response to the binding of membrane-impermeable peptide
hormones to the external cell surface. cGMP relaxes smooth muscle tissues. In blood vessels,
relaxation of vascular smooth muscles leads to vasodilation and increased blood flow. It also
regulates ion channel conductance, glycogenolysis, and cellular apoptosis(Waldman and Murad,
1987; Francis et al., 2001).
cGMP synthesis is catalyzed by guanylate cyclase (GC) which converts GTP to cGMP. Peptide
hormones such as the natriuretic factors activate membrane-bound GC, while nitric oxide
typically stimulates cGMP synthesis in soluble GC (Tremblay et al., 1988; Francis et al., 2001).
A phosphodiesterase type 5 inhibitor (PDE5 inhibitor) is a drug used to block the degradative
action of cGMP-specific phosphodiesterase type 5 (PDE5) on cyclic GMP in the smooth muscle
cells lining the blood vessels supplying the corpus cavernosum of the penis. PDE5 inhibitors are
used in the treatment of erectile dysfunction and were the first effective oral treatment available
for the condition (Eardley, 2010). Because PDE5 is also present in the arterial wall smooth
muscle within the lungs, PDE5 inhibitors have also been explored for the treatment of pulmonary
hypertension, a disease in which blood vessels in the lungs become overloaded with fluid,
usually as a result of failure of the right ventricle of the heart(Jayashankar, 2007).
3.2 Pathological importance ofPDE5 inhibitor

Phosphodiesterase 5 is responsible for the degradation of cGMP in the smooth muscle cells
lining the blood vessels supplying the corpus cavernosum of the penis, which leads to erectile
dysfunction (Aversa et al., 2006; Jayashankar, 2007).
3.2.1 Erectile dysfunction

Erectile dysfunction is defined as the persistent inability to attain or maintain penile erection
adequate for sexual intercourse. This condition, to some degree, affects more than one half of all
men over the age of 40, including 30 million men in the US and more than 150 million men
worldwide (Aversa et al., 2006). While it was thought for years that most cases of ED were of
18
psychogenic origin, current statistics suggest that more than half of the cases may be caused by
organic syndromes including,
Adverse drug reactions (diuretics and anti-hypertensives, antidepressants, antipsychotics,

anticonvulsants, and sedative/hypnotics),
Chronic disease (mainly diabetes but also heart, liver, and kidney disease)
Endocrine disorders (altered testosterone or prolactin levels)
Vascular disease (atherosclerosis)
Primary neurologic disorders (spinal cord injury, multiple sclerosis, Alzheimer's disease,
parkinsonism)
Surgical procedures that damage parasympathetic nerve fibers including prostatectomy,

cystectomy, and kidney transplantation (Jayashankar, 2007; Aversa et al., 2006).
3.3 Some PDE5 inhibitors and their mechanism of action

Phosphodiesterase type 5 inhibitors (PDE5) inhibitors include sildenafil (Viagra), vardenafil
(Levitra), and tadalafil (Cialis).Sildenafil (Viagra), tadalafil (Cialis), and vardenafil (Levitra) are
clinically indicated for the treatment of erectile dysfunction (Wang, 2010). Sildenafil and
tadalafil are also indicated for the treatment of pulmonary hypertension. Sildenafil, the
prototypical PDE5 inhibitor, was originally discovered during the search of a novel treatment for
angina. Studies in 2002 explored its potential for increasing neurogenesis after stroke (Zhang et
al., 2002).
3.3.1 Treatment of erectile dysfunction

Three medicines taken by mouth are approved for the treatment of erectile dysfunction are
Sildenafil (Viagra), Vardenafil (Levitra) and Tadalafil (Cialis)(Wang, 2010).
PDE-5 inhibitors can be used when an erection problem (erectile dysfunction) is caused by:
Diabetes.
High blood pressure.
Spinal cord injury or other problems affecting nerve function.
Prostate surgery
Side effects of medicines.
19
Em
motional or psychologica
p
al causes (Aversa et al., 2006)
Figure 5: Schem
matic diagraam showing the inhibitorry effect of V
Viagra (PDE
E5 inhibitor)
PDE-5 in
nhibitors wo
ork by block
king, or inh
hibiting, the action of P
PDE-5, preseent in the coorpus
cavernosum, the sp
pongy erecttile tissue of the pennis (Lin et al., 2000). Under noormal
circumstaances, sexuaal arousal in the male stim
mulates neuurons in the ccorpus caverrnosum to reelease
nitric ox
xide (NO), a chemicall compound
d that caus es the form
mation of ccyclic guannosine
monopho
osphate (cGM
MP). NO acctivates the enzyme
e
guannylate cyclase which ressults in increeased
levels off cyclic guaanosine mon
nophosphate (cGMP), lleading to ssmooth muscle relaxatioon in
blood veessels suppllying the co
orpus cavern
nosum, resuulting in inncreased bloood flow annd an
erection. PDE-5 breaaks down cG
GMP, and so
o the PDE-5 inhibitors, bby blocking the action oof the
enzyme, maintain hig
gher levels of
o cGMP, alllowing cGM
MP to accum
mulate and persist longerr, and
preserve a satisfactorry erection (A
Aversa et all., 2006; Jayaashankar, 20007).
20
3.3.2 Treatment of pulmonary

y hypertenssion
Phosphod
diesterase 5 (PDE 5) inh
hibitors are a type of taargeted theraapy used to treat peoplee with
pulmonarry hypertenssion (PH) (M
Maurice et all., 2003). Taargeted theraapies slow thhe progressiion of
PH and may
m even rev
verse some of
o the damag
ge to the heaart and lungs. There are ttwo types off PDE
5 inhibito
or currently used to treatt PH:
Silldenafil (Viaagra or Revaatio)
Taadalafil (Adccirca)
PDE 5 inhibitors
i
sto
op PDE5 en
nzymes, fou
und in bloodd vessel waalls, from w
working propperly.
These en
nzymes are involved
i
in controlling
c
blood
b
vessell constrictionn. PDE5 hellps control bblood
flow to the
t pulmonaary arteries. By stopping
g PDE5 froom working,, PDE 5 inhhibitors causse the
blood veessels to rellax. This in
ncreases blo
ood flow too the lungs and lowerss blood preessure
(Pulmonaary Hyperten
nsion Association UK, 2012).
2
E
of PD
DE 5 inhibitoors on blood vessels
Figure 6: Effect
3.4 Deveelopment an
nd discoverry of Silden
nafil, Vardeenafil and T
Tadalafil
Sildenafiil citrate, solld as Viagra, Revatio an
nd under variious other trrade names, is a drug ussed to
treat ereectile dysfunction and pulmonary
y arterial hhypertension (PAH). Itt was origiinally
discovereed by Pfizerr scientists Andrew
A
Bell, David Broown, and Niccholas Terreett (Boolell eet al.,
1996; Vaardi and Ninii, 2007).
21
The inventors of Viagra actually wanted to invent a medicine for the cure of cardio-vascular
diseases, but fortunately or unfortunately, they developed a medicine which could reverse
impotence or erectile dysfunction for the first time (Ghofrani et al., 2006)
In the year 1991, employees of Pfizer at Sandwich, it was discovered that chemical compounds
belonging to the pyrazolopyrimidinone class were useful in treating heart problems like angina
or chest pain. During clinical trials, it was found that the drug had little or insignificant effect on
angina. But during this phase, an unexpected side effect of this medicine was observed.
Sildenafil could improve and sustain a mans penile erection. Pfizer than stopped the research on
Sildenafil as heart medication and initiated investigation on it for penile erection (Trott, 2008).
Peter Dunn and Albert Wood are given the dues for the process that facilitated the invention of
Viagra by the British Press. The names of these two scientists appeared on Pfizers patent
application paper for the manufacturing process of Sildenafil Citrate. But it was only in the year
1991 that the potency of Sildenafil to cure angina was discovered by Andrew Bell, Dr David
Brown and Dr Nicholas Terrett. In fact Terrett was named in the 1991 patent for the use of
Sildenafil as a treatment for heart problems, and he is thereby recognized as the father of
Viagra (Trott, 2008).
And again it was Nicholas Terrett and his colleague Peter Ellis in 1994, who discovered that the
drug can be very effective in enhancing the flow of blood to the penile region in patients
suffering from erectile dysfunction while they were investigating on the utility of Sildenafil as a
heart medicine. Sildenafil was found to increase the muscle relaxing effects of nitric oxide, a
chemical that generally gets released when a person is sexually stimulated. The smooth
relaxation of the muscle in the penis facilitates higher rate of blood flow and helps in producing
an erection (Trott, 2008).
Dunn and Wood worked on the critical nine-step process to synthesize a Sildenafil compound
into a pill at that particular point of time. However, the patent name Viagra as the first
prescription pill for the treatment of impotence or erectile dysfunction was approved by the FDA
on March 27, 1998 (Trott, 2008).
22
Table 4: Th
he main stages in the devvelopment of Viagra
23
With the annual sales of Viagra hitting the $1 billion mark in 1999-2001, Viagra began to be
considered as one of the highest money grossing prescription drugs it has ever produced. In a
report published on July 10, 1998, in the New York Times, the sale of the erectile dysfunction
drug propelled in increasing the second-quarter profit margin of Pfizer by a whopping 38%
(Trott, 2008).
In 1998, ICOS Corporation and Eli Lilly and Company formed the Lilly ICOS, LLC, joint
venture company to further develop and commercialize tadalafil as a treatment for ED. Two
years later, Lilly ICOS, LLC, filed a new drug application with the FDA for compound IC351
(under the tadalafil generic name, and the Cialis brand name). In May 2002, Lilly ICOS reported
to the American Urological Association that clinical trial testing demonstrated that tadalafil was
effective for up to 36 hours, and one year later, the FDA approved tadalafil (Daugan et al.,
2003).
The FDA's approval of Viagra (Sildenafil) on March 27, 1998 was a ground-breaking
commercial event for the treatment of ED, with sales exceeding US$1 billion. Subsequently, the
FDA approved Levitra (vardenafil) on August 19, 2003, and Cialis (tadalafil) on November 21,
2003. Dunn and Wood worked on the critical nine-step process to synthesize a Sildenafil
compound into a pill at that particular point of time. However, the patent name Viagra as the first
prescription pill for the treatment of impotence or erectile dysfunction was approved by the FDA
on March 27, 1998 (Sildenafil aka Viagra, 2008; Daugan et al., 2003).
With the annual sales of Viagra hitting the $1 billion mark in 1999-2001, Viagra began to be
considered as one of the highest money grossing prescription drugs it has ever produced. In a
report published on July 10 1998 in the New York Times, the sale of the erectile dysfunction
drug propelled in increasing the second-quarter profit margin of Pfizer by a whopping 38%
(Sildenafil aka Viagra, 2008; Icos Corporation, 2014).
24
Figure 7: Development of sildenafil forr erectile dysfun

nction and pulm
monary hypertension
25
In 1998, ICOS Corp

poration and
d Eli Lilly and Compaany formed the Lilly IC
COS, LLC, joint
c
to further dev
velop and co
ommercializze tadalafil aas a treatmeent for ED. Two
venture company
years lateer, Lilly ICO
OS, LLC, filed a new drug
d
applicattion with thee FDA for ccompound IC351
(under th
he tadalafil generic
g
namee, and the Ciialis brand nname). In Maay 2002, Lilly ICOS repported
to the Am
merican Uro
ological Asso
ociation thatt clinical triaal testing dem
monstrated tthat tadalafil was
effective for up to 36 hours, and
d one year laater, the FD
DA approvedd tadalafil (R
Revill, 2003;; Icos
Corporattion, 2014).
The FDA
A's approvaal of Viagrra (Sildenaffil) on Mar
arch 27, 19998 was a ground-breaaking
commerccial event for the treatmeent of ED, with
w sales exxceeding US
S$1 billion. S
Subsequentlyy, the
FDA app
proved Levittra (vardenafil) on Augu
ust 19, 20033, and Cialis (tadalafil) oon Novembeer 21,
2003 (Gaaines, 2004; Icos Corporration, 2014)).
Figuree 8: Chemiccal structure of Sildenafill, Vardenafill and Tadalaafil
3.5 Sildeenafil dosin

ng
Sildenafiil citrate is so
old as Viagrra and Revattio. Revatio iis only availlable as a rouund, white 220 mg
pill, to distinguish it from Viiagra, which
h is a bluee diamond-sshaped pill (Truven H
Health
Micromeedex, 2014).
26
The dose of this medicine will be different for different patients. The amount of medicine that is
taken depends on the strength of the medicine. Also, on the number of doses taken each day, the
time allowed between doses, and the length of time the medicine is taken depending on the
medical problem for which the medicine is used (Truven Health Micromedex, 2014; Loran et al.,
2009).
For treatment of erectile dysfunction: Oral dosage form (tablets):
Adults up to 65 years of age50 milligrams (mg) as a single dose no more than once a day,
1 hour before sexual intercourse. Alternatively, the medicine may be taken 30 minutes to 4
hours before sexual intercourse. Your doctor may adjust your dose if needed(Chamsi-Pasha,
2001; Loran et al., 2009).
Adults 65 years of age and older25 mg as a single dose no more than once a day, 1 hour
before sexual intercourse. Alternatively, the medicine may be taken 30 minutes to 4 hours
before sexual intercourse. Your doctor may adjust your dose if needed.
ChildrenUse is not recommended (Truven Health Micromedex, 2014).
For treatment of pulmonary arterial hypertension:Oral dosage forms (suspension or tablets):
Adults20 milligrams (mg) three times a day. Each dose should be taken about 4 to 6 hours
apart (Jackson et al., 2006).
ChildrenUse and dose must be determined by doctor (Truven Health Micromedex, 2014).
3.6 Sildenafil usage

In deciding to use a medicine, the risks of taking the medicine must be weighed against the good
it will do. For this medicine, the following should be considered:
3.6.1 Pediatricusage
Sildenafil should never be used in children for erectile dysfunction. In general, sildenafil should
not be used for pulmonary arterial hypertension in children, especially for chronic use (Truven
Health Micromedex, 2014).
27

Appropriate studies performed to date have not demonstrated geriatric-specific problems that
would limit the usefulness of sildenafil in the elderly. However, elderly patients are more likely
to have age-related liver, kidney, or heart problems, which may require an adjustment in the dose
for patients receiving sildenafil (Truven Health Micromedex, 2014).

Pregnancy Category is B. Animal studies have revealed no evidence of harm to the fetus,
however, there are no adequate studies in pregnant women OR animal studies have shown an
adverse effect, but adequate studies in pregnant women have failed to demonstrate a risk to the
fetus (Truven Health Micromedex, 2014).
3.6.4 Breastfeeding
There are no adequate studies in women for determining infant risk when using this medication
during breastfeeding. Weigh the potential benefits against the potential risks before taking this
medication while breastfeeding (Truven Health Micromedex, 2014).
3.7 Molecular structure of Sildenafil

Sildenafil is composed of three regions:
R1 pyrazolopyrimidinone group
R2 ethoxyphenyl group
R3 methylpiperazine(Wang et al., 2006)
28
Figure 9: Sildenaafil
The R1 group is reesponsible fo
or the bindiing affinity of sildenaffil to PDE5. Sildenafils R1
structure is similar to
o the purinee region on the
t cGMP m
molecule. Thhis region iss what allow
ws the
sildenafill ligand to biind into the active site in
n the PDE5 pprotein (Wanng et al., 20006).
Sildenafiil binds to PD
DE5 through
h many interractions:
Two
T
hydrogeen bonds bettween a nitro
ogen atom aand an oxygeen atom on Gln817 of P
PDE5
an
nd an oxygen atom and a nitrogen attom on the R
R1 segment oof sildenafil.
This
T
portion of the drug
g is also staabilized by the stackingg of a diffeerent amino acid,
Phe820.
All
A three segments of thee drug are involved
i
in V
Van der Waaals contactts and regioons of
hy
ydrophobic stability (Wang et al., 20
006).
29
Figuree 10: Sildenaafil shown in

n the binding
g pocket of P
PDE5. The eessential inteeractions occcur
with Gln
n817 and Phee820.
Sildenafiil works by triggering the
t H-Loop (red sectionn, in the figgure below) to shift oveer the
binding site
s renderin
ng the PDE5 protein inacctive, unablee to breakdoown cGMP. The H-loop R3
region in
nteraction is unknown; future
fu
work to
t study thiss region of ssildenafil is nneeded. Thee shift
can be ass great as 24 angstroms from
f
the unb
bound protei n (Wang et aal., 2006).
Figu
ure 11: H-loop (red) secttion of PDE5
5 protein (puurple) closess off bindingg pocket afteer
sildeenafil binds..
30
The amin
no acid Gln
n659 sits jusst before th
he H-loop annd likely accts as a hingge (in the ffigure
below). There
T
is rottation around some of the
t bonds inn sildenafil, leading to some flexibbility.
Evidencee suggests it can exist in
n three main positions. E
Each of thesse positions kkeeps R1 annd R2
constant,, with rotatio
on of the R3 region (Wan
ng et al., 20006).
Figure 12:
1 Close-up
p of sildenafi
fil in the bind
ding pocket. H-loop (redd), magnesiuum (orange), zinc
(light blue) and Gln6599 (white)
The R3 region
r
interacts with th
he H-loop. More
M
researcch is neededd to determiine three facctors,
discussed
d below, wh
hich will allo
ow for a mo
ore specific description of the moleecular interaaction
between sildenafil an
nd PDE5.
Establish
E
whiich of the thrree conformaations of silddenafil is acttive or most active.
Find out the position

p
of th
he H-loop within
w
the prootein.
Determine
D
ho
ow the silden
nafil ligand sits in the biinding pockket and whichh amino acids on
th
he H-loop th
he R3 region interacts wiith specificallly (Wang ett al., 2006).
3.8 Drug
g-drug interactions
Use of Sildenafil
S
cleearly contraiindicated wiith concurreent use of niitrates. List of representtative
organic nitrates
n
is as follows:
Niitroglycerin
31
Deponit
Minitran
Nitrok
Nitro-Bid
Nitrocine
Nitroderm
Nitro Disc
Nitro-Dur
Nitrogard
Nitroglycerin
Nitroglycerin T/R
Nitroglyn
Nitrol ointment
Nitrolingual spray
Nitrong
Nitro-Par
Nitropress
Nitro SA
Nitrospan
Nitro-Par
Nitropress
Nitro SA
Nitrospan
Nitrostat
Nitro-trans system
Nitro transdermal
Nitro-Time
Transiderm-Nitro
Tridil
Isosorbide Mononitrate
32
Imdur
ISMO
Isosorbide mononitrate
Monoket
Isosorbide Nitrate
Dilatrate-SR
Iso-Bid
Isordil
Isordil tembids
Isosorbide dinitrate
Isosorbide dinitrate LA
Sorbitrate
Sorbitrate SA
Pentaerythritol Tetranitrate
Peritrate
Peritrate SA
Erythrityl Tetranitrate
Cardilate
Isosorbide Dinitrate/Phenobarbital
Isordil w/PB
Illicit Substances Containing Organic Nitrates
Amyl nitrate or nitrite (It is known that amyl nitrate or nitrite is sometimes abused. In
abuse situations, amyl nitrate or nitrite may be known by various names, including
poppers.) (Truven Health Micromedex, 2014; Chamsi-Pasha, 2001).
Cardiovascular effects of Sildenafil may be potentially hazardous (use dependent on individual

clinical assessment) for
1) Patients with active coronary ischemia who are not taking nitrates (eg, positive exercise
test for ischemia)
33
2) Patients with congestive heart failure and borderline low blood pressure and borderline
low volume status
3) Patients on a complicated, multidrug, antihypertensive program
4) Patients taking drugs that can prolong the half-life of Viagra (Truven Health Micromedex,
2014; Jackson et al., 2005).
Sildenafil is broken down predominantly by an enzyme called CYP3A in the liver; therefore,
important interactions may occur with medications that affect this enzyme pathway. List of drugs
That Are Metabolized by or That Inhibit Cytochrome P450 3A4 are as follows:
Antibiotic/Antifungal:
Biaxin (clarithromycin)
Clotrimazole
Erythromycin
Diflucan
Sporanox
Ketoconazole
Miconazole
Noroxin
Troleandomycin (Truven Health Micromedex, 2014)
Cardiovascular:
Amiodarone
Norvast
Digitoxin
Diltiazem
Disopyramide
Plendil (felodipine)
DynaCirc (isradipine)
Cozaar (losartan)
Posicor (mibefradil)
Nifedipine
Quinidine
34
Verapamil (Truven Health Micromedex, 2014; Speakman and Kloner, 1999)
Statins (or HMG-CoA reductase inhibitors):
Lipitor (atorvastatin)
Baycol (cerivastatin)
Mevacor (lovastatin)
Zocor (simvastatin)
Central Nervous System:
Alprazolam
Carbamazepine
Prozac (fluoxetine)
Luvox (fluvoxamine)
Imipramine
Serzone (nefazodone)
Phenobarbital
Phenytoin
Zoloft
Triazolam (Truven Health Micromedex, 2014)
Others:
Acetaminophen
Hismanal (astemizole)
Tagamet (cimetidine)
Propulsid (cisapride)
Cyclosporine
Dexamethasone
Ethinyl estradiol
Naringenin (grapefruit juice)
Prilosec (omeprazole)
Rifampin
Tacrolimus
Seldane (terfenadine)
35
Theophylline
Rezulin (troglitazone)
Protease inhibitors: Crixivan (indinavir), Norvir (ritonavir), Viracept (nelfinavir), Invirase

(saquinavir) (Truven Health Micromedex, 2014)
Patients with human immunodeficiency virus (HIV or AIDS) who are taking medicines called
antiretroviral agents should not use a phosphodiesterase inhibitor such as sildenafil since it can
dramatically impair the efficacy of the antiretroviral. Use of sildenafil with epoprostenol may
reduce the blood level of sildenafil. Use of sildenafil with beta blockers (another type of heart or
blood pressure medicine) may increase the levels of sildenafil (Truven Health Micromedex,
2014).
3.9 Vardenafil brand names and indications

Vardenafil (INN) is used for treating erectile dysfunction and pulmonary arterial hypertension
(PAH). It is sold under the trade names Levitra, Staxyn in India, and Vivanza in Italy. Vardenafil
was co-marketed by Bayer Pharmaceuticals, GlaxoSmithKline, and Schering-Plough under the
trade name Levitra. As of 2005, the co-promotion rights of GSK on Levitra have been returned
to Bayer in many markets outside the U.S. In Italy, Bayer sells vardenafil as Levitra and GSK
sells it as Vivanza. Thus, because of European Union trade rules, parallel imports might result in
Vivanza sold next to Levitra in the EU (Bayer Healthcare Pharmaceutical Inc., 2011; Thomson
Healthcare Inc., 2014).
An orally disintegrating form, marketed as Staxyn, has been gaining approvals in countries such
as the United States and Canada. Beyond its indications for erectile dysfunction, vardenafil may
be effective in the treatment of premature ejaculation, where it may significantly increase the
time from penetration to ejaculation (Thomson Healthcare Inc., 2014).
3.10 Vardenafil dosage

For most individuals, the recommended dose of vardenafil regular tablets is 10 mg per day taken
60 minutes before intercourse. If there is no response or side effects, the dose may be increased
to 20 mg or, if there are side effects, it may be reduced to 5 mg. Individuals 65 years of age or
older should begin therapy with 5 mg. Individuals who are taking medications that increase the
36
blood levels of vardenafil should start treatment with 2.5 to 5 mg of vardenafil (Bayer Healthcare
Pharmaceutical Inc., 2011).
Orally disintegrating tablets (ODT) are not interchangeable with regular vardenafil tablets
because they are better absorbed and produce higher blood levels than regular tablets. The
recommended dosing when using ODT is one tablet 60 minutes before intercourse. Only one
tablet should be used per day. It should be placed on the tongue until it disintegrates and should
not be swallowed with water (Bayer Healthcare Pharmaceutical Inc., 2011).
3.11 Vardenafil metabolism, elimination and usage

Vardenafil is metabolized predominantly by the hepatic enzyme CYP3A4, with contribution
from the CYP3A5 and CYP2C isoforms. The major circulating metabolite, M1, results from
desethylation at the piperazine moiety of vardenafil. M1 shows a phosphodiesterase selectivity
profile similar to that of vardenafil and an in vitro inhibitory potency for PDE5 28% of that of
vardenafil.
After oral administration, vardenafil is excreted as metabolites predominantly in the feces
(approximately 91-95% of administered oral dose) and to a lesser extent in the urine
(approximately 2-6% of administered oral dose).

Vardenafil is not indicated for use in children. Safety and efficacy have not been established
(Bayer Healthcare Pharmaceutical Inc., 2011).

would limit the usefulness of vardenafil in the elderly (Bayer Healthcare Pharmaceutical Inc.,
2011).

This drug is not indicated for use in female patients. The drugs FDA pregnancy category is B.
No evidence of specific potential for teratogenicity, embryotoxicity or fetotoxicity was observed
37
in rats and rabbits that received vardenafil at up to 18 mg/kg/day during organogenesis. There are
no studies of the use of this drug in pregnant women. Drugs which have been taken by only a
limited number of pregnant women and women of childbearing age, without an increase in the
frequency of malformation or other direct or indirect harmful effects on the human fetus having
been observed. Studies in animals have shown evidence of an increased occurrence of fetal
damage, the significance of which is considered uncertain in humans. Animal reproduction
studies have failed to demonstrate a risk to the fetus and there are no adequate and wellcontrolled studies in pregnant women (Bayer Healthcare Pharmaceutical Inc., 2011).
Following a single oral dose of 3 mg/kg, 3.3% of the administered dose was excreted into the
milk within 24 hours. There are no adequate studies in women for determining infant risk when
using this medication during breastfeeding. The potential benefits against the potential risks
should be weighed before taking this medication while breastfeeding (Bayer Healthcare
Pharmaceutical Inc., 2011).

Although certain medicines should not be used together at all, in other cases two different
medicines may be used together even if an interaction might occur. In these cases, doctor may
want to change the dose, or other precautions may be necessary. Using this medicine with any of
the following medicines is not recommended. The doctor may decide not to treat the patient with
this medication or change some of the other medicines, mentioned below, that the patient takes
(Bayer Healthcare Pharmaceutical Inc., 2011):
Amifampridine
Cisapride
Dronedarone
Erythrityl Tetranitrate
Fluconazole
Isosorbide Dinitrate
Isosorbide Mononitrate
38
Mesoridazine
Nelfinavir
Nitroglycerin
Pentaerythritol Tetranitrate
Pimozide
Piperaquine
Posaconazole
Riociguat
Sparfloxacin
Thioridazine
Using this medicine with any of the following medicines is usually not recommended, but may
be required in some cases. If both medicines are prescribed together, the doctor may change the
dose or how often the patient uses one or both of the medicines, which are mentioned below
(Bayer Healthcare Pharmaceutical Inc., 2011):
Acecainide
Amiodarone
Amitriptyline
Amoxapine
Apomorphine
Arsenic Trioxide
Asenapine
Astemizole
Azimilide
Azithromycin
Bretylium
Carbamazepine
Ceritinib
Chloroquine
Chlorpromazine
39
Ciprofloxacin
Citalopram
Clarithromycin
Clomipramine
Clozapine
Cobicistat
Crizotinib
Dabrafenib
Dasatinib
Delamanid
Desipramine
Disopyramide
Dofetilide
Dolasetron
Domperidone
Erythromycin
Escitalopram
Eslicarbazepine Acetate
Fingolimod
Flecainide
Fluoxetine
Gatifloxacin
Granisetron
Halofantrine
Haloperidol
Ibutilide
Iloperidone
Imipramine
Ivabradine
Ketoconazole
40
Lapatinib
Levofloxacin
Lopinavir
Lumefantrine
Mefloquine
Methadone
Mifepristone
Mitotane
Moricizine
Moxifloxacin
Nilotinib
Norfloxacin
Nortriptyline
Octreotide
Ofloxacin
Ondansetron
Paliperidone
Pazopanib
Perflutren Lipid Microsphere
Primidone
Procainamide
Prochlorperazine
Promethazine
Propafenone
Protriptyline
Quetiapine
Quinidine
Quinine
Ranolazine
Salmeterol
41
Saquinavir
Sematilide
Sevoflurane
Siltuximab
Simeprevir
Sodium Phosphate
Sodium Phosphate, Dibasic
Sodium Phosphate, Monobasic
Solifenacin
Sorafenib
Sotalol
Sunitinib
Tedisamil
Telaprevir
Telithromycin
Terfenadine
Tetrabenazine
Toremifene
Trazodone
Trifluoperazine
Trimipramine
Vandetanib
Vemurafenib
Vinflunine
Voriconazole
Ziprasidone (Bayer Healthcare Pharmaceutical Inc., 2011)
3.13 Tadalafil brand names and indications

Tadalafil relaxes muscles and increases blood flow to particular areas of the body. Tadalafil
under the name of Cialis is used to treat erectile dysfunction (impotence) and symptoms of
42
benign prostatic hypertrophy (enlarged prostate). Another brand of tadalafil is Adcirca, which is
used to treat pulmonary arterial hypertension and improve exercise capacity in men and women.
Tadalafil is used to treat men who have signs and symptoms of benign prostatic hyperplasia
(BPH). BPH is caused by an enlarged prostate. Men with BPH usually have difficulty urinating,
a decreased flow of urination, hesitation at the beginning of urination, and a need to get up at
night to urinate. Tadalafil will make these symptoms less severe and reduce the chance that
prostate surgery will be needed. This medicine is also used to treat erectile dysfunction and signs
and symptoms of BPH (Eli Lilly and Company, 2014).
Tadalafil is used in both men and women to treat the symptoms of pulmonary arterial
hypertension. This is high blood pressure that occurs in the main artery that carries blood from
the right side of the heart (the ventricle) to the lungs. When the smaller blood vessels in the lungs
become more resistant to blood flow, the right ventricle must work harder to pump enough blood
through the lungs. Tadalafil works on the PDE5 enzyme in the lungs to relax the blood vessels.
This will increase the supply of blood to the lungs and reduce the workload of the heart(Eli Lilly
and Company, 2014).
3.14 Tadalafil potency

Tadalafil is a highly potent and highly selective PDE5 inhibitor. Introduction of a 3,4methylenedioxy substitution on the phenyl ring in position 6 led to a potent PDE5 inhibitor with
increased cellular potency. Tadalafil has high selectivity for PDE5 vs PDE1-4 and PDE6. It
displays 85-fold greater selectivity vs PDE6 than sildenafil. It showed profound and long-lasting
blood pressure lowering activity (30 mmHg/>7 h) in the spontaneously hypertensive rat model
after oral administration (5 mg/kg)(Daugan et al., 2003).
3.15 Tadalafil dosage

For most individuals, the recommended starting dose of tadalafil is 10 mg per day taken before
sexual activity (tadalafil for use as needed). Depending on the adequacy of the response or side
effects, the dose may be increased to 20 mg or decreased to 5 mg a day. The effect of tadalafil
may last up to 36 hours. Individuals who are taking medications that increase the blood levels of
tadalafil should not exceed a total dose of 10 mg in 72 hours (See drug interactions). For once
43
daily use without regard to sexual activity the recommended dose is 2.5 to 5 mg daily. Tadalafil
should not be taken more than once daily (Eli Lilly and Company, 2014).
The recommended dose for BPH, or BPH and ED is 5 mg daily taken about the same time each
day. Tadalafil may be taken with or without food since food does not affect its absorption from
the intestine. The dose of tadalafil may require adjustment for patients with reduced kidney or
liver function (Eli Lilly and Company, 2014).
3.16 Tadalafil usage

In deciding to use a medicine, the risks of taking the medicine must be weighed against the good
it will do. This is a decision the patient and doctor will make. For this medicine, the following
should be considered:

Use of Cialis tablet is not indicated for use in the pediatric population. Safety and efficacy have
not been established. Appropriate studies have not been performed on the relationship of age to
the effects of Adcirca tablet in the pediatric population. Safety and efficacy have not been
established (Eli Lilly and Company, 2014).

would limit the usefulness of tadalafil in the elderly. However, elderly patients are more likely to
have age-related kidney problems, which may require caution and an adjustment in the dose for
patients receiving tadalafil (Eli Lilly and Company, 2014).
3.16.3 Pregnancy category

Tadalafil has been assigned to pregnancy category B by the FDA. Animal data have failed to
reveal evidence of teratogenicity or fetotoxicity. There are controlled data in human pregnancy.
Tadalafil is only recommended for use during pregnancy when benefit outweighs risk (Eli Lilly
and Company, 2014).
44
There are no data on the excretion of tadalafil in human milk. The manufacturer recommends
that caution be used when administering tadalafil to nursing women (Eli Lilly and Company,
2014).

This drug should not be used with the following medications because very serious, possibly fatal
interactions may occur:
Drugs for heart conditions (nicorandil or nitrates)
Alpha blockers (used to treat high blood pressure, enlarged prostate or heart failure)
(Kloner et al., 2004).
If the patient is currently using any of these medications, the doctor or pharmacist should be
informed before starting tadalafil.
Before using this medication, the doctor or pharmacist should have the knowledge of all
prescription and non-prescription/herbal products the patient may use, especially of:
Antibiotics such as erythromycin, clarithromycin or rifampicin
Antifungals such as ketoconazole or itraconazole
Anticonvulsants such as phenobarbital, phenytoin and carbamazepine
Antivirals such as amprenavir, ritonavir or saquinavir (Eli Lilly and Company, 2014)
Therefore, before using tadalafil, the doctor or pharmacist should be informed of all the products
the patient uses.
Tadalafil can lower blood pressure, and combining it with ethanol may further increase this
effect. The patient may be more likely to experience symptoms such as dizziness,
lightheadedness, fainting, flushing, headache, and heart palpitations. The patient should avoid or
limit the use of alcohol while being treated with tadalafil, and use caution when getting up from a
sitting or lying position(Simon and Zieve, 2013;Gresser and Gleiter, 2002).
45
3.18 Pharmacokinetics of the three PDE5 inhibitors

As competitive inhibitors of PDE5, PDE5 inhibitors have structures derived from cGMP.
Sildenafil and vardenafil have very similar molecular structures. In contrast, tadalafil has a
different chemical structure from sildenafil and vardenafil. These structural differences lead to
differences in the pharmacokinetics of the three drugs (Rosen and Kostis, 2003).
The mean time to maximum plasma concentration of sildenafil and vardenafil is 1 h and for
tadalafil is 2 h, while the half-lives of sildenafil and vardenafil are 4 h and that of tadalafil is 17.5
h. Food high in fat delays and reduces the absorption of sildenafil and vardenafil, but does not
affect the rate or extent of absorption of tadalafil (Wright, 2006).
3.19 Side-effects of PDE5 inhibitors (Viagra, Cialis, Levitra)

Common side effects of Viagra, Levitra and Cialis are facial flushing and a blocked nose. They
can also cause headaches, muscle aches, and indigestion. Sometimes when taking Viagra, men
also experience brighter or a blue tinge to their vision (Gresser and Gleiter, 2002; Pfizer
Laboratories Div Pfizer Inc, 2014).
Most side-effects are not troublesome and only last an hour or so. Most men do not stop taking
these PDE5 inhibitors because of the side effects (Pfizer Laboratories Div Pfizer Inc, 2014).
3.20 Precautions of PDE5 inhibitors (Viagra, Cialis, Levitra)
PDE5 inhibitors produce a small effect on supine blood pressure in healthy individuals.
PDE5 inhibitors may induce a slight reduction in cardiac output
PDE5 inhibitors are not recommended or used only with caution for the following
populations:
patients with a myocardial infarction within the last 90 days
patients with unstable angina or angina occurring during sexual intercourse
patients with New York Heart Association Class 2 or greater heart failure in the last 6
months
patients with uncontrolled arrhythmia, hypotension (<90/50 mm Hg), or uncontrolled

hypertension (>170/100 mm Hg)
46
patients with a stroke within the last 6 months
patients with left ventricular outflow obstruction (Rnceus Interactive, 2012; Cheitlin et
al., 1999)
Anti-hypertension medications - can cause an additive effect when taken with PDE5
inhibitors. Concomitant use of organic nitrates present the most serious hypotensive risk but
alpha-adrenergic blocking agents may also cause significant vasodilation when taken with a
PDE5 inhibitor. Combining PDE5 inhibitors and a vasodilator can result in symptomatic
hypotension (e.g. syncope or dizziness). When PDE5 and antihypertension agents are taken
together the dosage of each drug may need to be adjusted. Intravascular volume and vascular
tone can also affect the cardiovascular response to these medications (Nehra, 2009).
Hepatic insufficiency- The cytochrome P450 enzyme CYP3A4 is the primary mechanism for
the metabolism and elimination of PDE5 medications from the system. Each passes through
the healthy liver and reduces the concentration of PDE5 inhibitors and active metabolites.
Therefore, disease or drugs that affect these enzymes will necessarily alter circulating time
and concentration of drugs cleared by this enzyme pathway (Rnceus Interactive, 2012).
Cimetidine is a non-specific CYP inhibitor that can cause a 56% increase in sildenafil
plasma concentration.
CYP3A4 inhibitors that reduce the clearance of PDE5 inhibitors include:

o erythromycin
o ketoconazole
o itraconazole
o ritonavir
o grapefruit
o starfruit
CYP3A4 inducers enhance the clearance of PDE5 inhibitors. They include:

o bosentan
o rifampin
Age - plasma levels of PDE5 inhibitors are increased in healthy patients >65 years. Lower
dosage may be considered
47
Renal insufficiency - plasma levels of PDE5 inhibitors are increased in patients with severe
renal insufficiency (e.g. creatinine clearance <30 mL/min)
Special Senses
Visiono Nonarteritic anterior ischemic optic neuropathy (NAION) is of unknown etiology.

NAION can cause temporary attenuation or permanent loss of vision, usually
unilaterally. NAION has been associated with PDE5 inhibitor use. Patients taking
PDE5 inhibitors for ED who experience visual disturbances should stop taking the
medication and seek medical advice.
o It is also recommended that sildenafil be administered with caution to patients with
retinitis pigmentosa. PDE-6 is known to be present in the photoreceptors of the
retina. It is presumed that sildenafil has some effect on PDE-6 which might explain
the color perception problems that occur at high doses.
o Dose related color discrimination impairment (blue/green tinge) is presumed to be a
result of the overlapping of effect on PDE6. Sildenafil seems to have a greater
crossover effect than the longer acting PDE5 inhibitors (Rnceus Interactive, 2012).
Hearing- Sudden attenuation, loss of hearing and tinnitus has been reported by people
taking PDE5 inhibitors. Patients taking PDE5 inhibitors for ED who experience auditory
disturbances should stop taking the medication and seek medical advice (Rnceus
Interactive, 2012).
Priapism - is a persistent erection of the penis. In most cases, the arterial supply is excessive,
the venous drainage is inadequate, or there is a combination of the two conditions. Sickle cell
anemia, leukemia, and multiple myeloma are conditions known to predispose patients to
priapism. It is recommended that PDE5 inhibitors be used with caution in patients that have
anatomical deformation of the penis and in patients who have conditions which may
predispose them to priapism (Burnett et al., 2006).
Combining treatment - Sildenafil is not recommended that it be taken in combination with

other ED drugs or treatment methods.
48
Coagulopathy - PDE5 is known to be present in platelets. Sildenafil should be administered

with caution in patients that have a bleeding disorder or peptic ulcer disease (Chughtai et al.,
2010).
3.21 Selectivity compared among the three PDE5 inhibitors

Tadalafil, sildenafil, and vardenafil all act by inhibiting the PDE5 enzyme. These drugs also
inhibit other PDE enzymes. Sildenafil and vardenafil inhibit PDE6, an enzyme found in the eye,
more than tadalafil. Some sildenafil users see a bluish tinge and have a heightened sensitivity to
light because of PDE6 inhibition. Sildenafil and vardenafil also inhibit PDE1 more than tadalafil.
PDE1 is found in the brain, heart, and vascular smooth muscle. It is thought that the inhibition of
PDE1 by sildenafil and vardenafil leads to vasodilation, flushing, and tachycardia. Tadalafil
inhibits PDE11 more than sildenafil or vardenafil. PDE11 is expressed in skeletal muscle, the
prostate, the liver, the kidney, the pituitary gland, and the testes. The effects on the body of
inhibiting PDE11 are not known (Bischoff, 2004).
3.22 Herbal erectile dysfunction treatments

When men are seeking an erectile dysfunction treatment in pill form, they have two options: a
natural erectile dysfunction treatment or PDE5 inhibitors. The most popular PDE5 inhibitors on
the market today to treat ED are Viagra, Cialis and Levitra. Millions of men seeking natural
erectile dysfunction treatments today, due to the unpleasant side effects PDE5 inhibitors present
(Sagan, 2012).
There are many herbs and herbal supplements which have been used by traditional medicine for
centuries to treat erectile dysfunction. The herb horny goat weed (botanical name epimedium) for
example, contains natural PDE 5 inhibitors (Wheeler, 2006). Below is a list of these herbs or
natural substances for effective impotence treatments:
Gingko biloba
Ginseng
Horny goat weed
Yohimbe
L-arginine (amino acid from chick peas and other foods)
49
Dehydroepiandrosterone or DHEA (soy or wild yam derived) (Wheeler, 2006)
The most popular natural erectile dysfunction ingredient that naturally boosts the production of
nitric oxide in the blood by herbal bio-active compound call icariin that have no known side
effects is epimedium extract. Unlike Viagra that is a temporary ED remedy, the natural
ingredient epimedium powder build up in your system and after 3-4 months, most men are able
to stop using epimedium powder and can achieve an erection naturally (Sagan, 2012).
3.23 Adulteration of herbal products with synthetic PDE5 inhibitors

Many products labeled "herbal" or "all natural" (herbal/natural) that claim to enhance sexual
performance and imply use for the treatment of erectile dysfunction (ED) are marketed as overthe-counter (OTC) dietary supplements. In a study by Pfizer Global Security, ninety-one samples
labeled as 58 distinct products and priced from $2.99 to $17.99 were evaluated. Although no
sample claimed to include synthetic substances, 74 (81%) contained PDE5-inhibitor
pharmaceutical ingredients, including tadalafil and/or sildenafil or PDE5-inhibitor analogs.
Because of the dangers of adulteration with synthetic PDE5 inhibitors, absent safety warnings,
and lack of quality or consistent manufacture, men with ED unknowingly risk their health by
using OTC herbal/natural products that claim to enhance sexual performance (Campbell et al.,
2013).
In another study seven herbal products (Biovigora; Super-X; Vip ViGa; Vuka Vuka; Herbal
Viaphrodisiac; Stamina-Rx; Herbal Erotica) marketed for the treatment of erectile
dysfunction were purchased via the Internet or at local health food stores. Specimens were for
contamination with PDE5 inhibitors. High performance liquid chromatography and mass
spectrometry were used to detect evidence of contamination with sildenafil, tadalafil or
vardenafil. Of the 7 tested products 2 contained pharmacological dosages of sildenafil and
tadalafil. Mean dosages of sildenafil and tadalafil were 30.2 and 19.7 mg, respectively (Fleshner
et al., 2005).
A significant proportion of natural products marketed for erectile dysfunction contain PDE5
inhibitors. Although marketed as natural products devoid of adverse effects, these agents are
50
known to have potentially fatal drug interactions with nitrates. Better regulation of the natural
health products industry is required to prevent such adulterations (Fleshner et al., 2005).
51
Chapter Four
CONCLUSION
4.1 Conclusion
The introduction of oral phosphodiesterase-5 inhibitors (PDE5Is) in the late 1990s and early
2000s revolutionized the field of sexual medicine and PDE5Is are currently first-line
monotherapy for erectile dysfunction (ED) (Dhir et al., 2011). In less than 20 years, the first
selective type 5 phosphodiesterase inhibitors have evolved from potential anti-angina drugs to
on-demand oral treatment for erectile dysfunction (Viagra), and more recently to a new orally
active treatment for pulmonary hypertension (Revatio) (Ghofrani et al., 2006). Research
continues at a substantial level to identify new, selective PDE5 inhibitors and to investigate their
usefulness and activity in other areas (Rotella, 2002). Below some recent clinical trials with
PDE5 inhibitors, advances in medicinal chemistry, and other activities and potential applications
of this class of compounds are summarized.
4.1.1 Therapeutic agent for parasitic disease

PDE5 has also been considered as a potential therapeutic agent for parasitic disease such as
African sleeping sickness. Strategic changes were made to the structure of sildenafil so the
molecule could project into a parasite-specific pocket (the p-pocket). Similar approach has been
used to design therapeutic agents Plasmodium falciparum (Wang et al., 2012).
4.1.2 Better enhanced erectile function

In a study it showed that vitamin E combined with sildenafil enhanced erectile function better
than either vitamin E or sildenafil alone in an animal model of diabetes (streptozotocin-induced
diabetes rats). The results of the study showed significant improvements in penile intracavernous
pressure and improved physiologic capacity (smooth muscle markers, endothelial cell and nNOS
staining) in the vitamin E plus sildenafil group. Early treatment with sildenafil plus vitamin E
demonstrated positive changes in intracavernous pressure, nNOS, smooth muscle alpha-actin
endothelial cell staining and an apparent decrease in PDE5 activity. Further work evaluating the
levels of free oxygen radicals and the impact of other scavenger molecules seems clinically
relevant for this large cohort of men who remain poorly responsive to current PDE5 inhibition
therapy (Young et al., 2003).
52
4.1.3 PDE5 inhibitors for treatment of Alzheimers disease

Elevation of cGMP levels through inhibition of PDE5 provides a way of improving memory and
learning (Fiorito et al., 2013). Alzheimers disease (AD) is the most common form of dementia
among the elderly. In AD patients, memory loss is accompanied by the formation of betaamyloid plaques and the appearance of tau in a pathological form. Given the lack of effective
treatments for AD, the development of new management strategies for these patients is critical.
The continued failure to find effective therapies using molecules aimed at addressing the antibeta amyloid pathology has led researchers to focus on other non-amyloid-based approaches to
restore memory function. PDE5 inhibitors have also been shown to effectively restore memory
function and also improve memory performance in AD and non-AD animal models. So there has
been a great interest in PDE5 inhibitors as promising new therapeutic agents for treatment of
Alzheimers disease (Garca-Osta et al., 2012).
4.1.4 Development of acute and chronic cardioprotective therapy

In 2002, in a study a new and potentially important use for the male erectile dysfunction drugs
sildenafil (Viagra), vardenafil (Levitra) and tadalafil (Cialis) was discovered. Since these drugs
are potent inhibitors of phosphodiesterase-5 (PDE-5), an enzyme that regulates the levels of
intracellular messenger molecule, cGMP, the study showed that PDE-5 inhibitors have
significant protection against damage from ischemia/reperfusion injury in animals (Virginia
Commonwealth University, 2014).
In a major breakthrough, it was further validated that sildenafil protected the heart against
injuries caused by chemotherapeutic drug doxorubicin, both used to treat tumors and blood
cancers. This work may lead to future development of a new group of compounds that could be
evolved for both acute and chronic cardioprotective therapy against ischemia/reperfusion injury,
hypertrophy and heart failure (Virginia Commonwealth University, 2014).
4.1.5 PDE5 inhibitors for the treatment altitude pulmonary edema

The combination of a tendency to affect otherwise fit and healthy individuals, and a
characteristically rapid progression to death within hours of the first symptoms, renders high
altitude pulmonary edema (HAPE) a particularly devastating illness in travelers to high altitudes.
53
The alveolar edema and ventilation:perfusion mismatch initiate a catastrophic downward spiral
of worsening alveolar hypoxia (Bates et al., 2007). Many reviews discuss the rationale for the
use of phosphodiesterase (PDE5) inhibitors, like sildenafil and tadalafil, in HAPE, compare the
pharmacokinetic properties of the available agents, and appraise the relevant experimental
evidence (Maggiorini et al., 2006; Kleinsasser and Loeckinger, 2002). Although this class of
drugs show promise in high altitude medicine, further research is necessary to determine the
efficacy and safety of PDE5 inhibitors as a treatment for established (Bates et al., 2007).
4.1.6 Treatment of benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is prevalent in old men and often results in lower urinary
tract symptoms (LUTS). Phosphodiesterase-5 (PDE5) inhibitors increase intracellular
concentrations of cyclic guanosine monophosphate. PDE5 inhibitors (sildenafil, tadalafil,
vardenafil, etc.) are first-line treatments for erectile dysfunction. Recently, PDE5 inhibitors have
been found to regulate smooth muscle tone in human prostate. There has been increasing interest
in the use of PDE5 inhibitors to treat BPH/LUTS. Combination of PDE5 inhibitors and alpha1adrenergic blockers may have an additive beneficial effect on BPH/LUTS compared with
monotherapy. Mechanisms of action of nitric oxide/cyclic guanosine monophosphate/PDE5
pathway in the treatment of BPH/LUTS deserve further investigations. Larger-scale, well
designed clinical trials in future are needed to ascertain the safety, efficacy and cost-effectiveness
of PDE5 inhibitors in the treatment of LUTS secondary to BPH (Wang, 2010).
4.1.7 Antidote to psychotropic-induced sexual dysfunction in women

Sildenafil has been studied as an antidote to psychotropic-induced sexual dysfunction in women.
A recently published prospective, parallel-group, randomized, double-blind, placebo-controlled
clinical trial investigated the effect of sildenafil on pre-menopausal women whose major
depression was remitted by selective serotonin reuptake inhibitor antidepressants but who were
also experiencing sexual dysfunction. Primary and secondary outcome measures were
psychometrically validated questionnaires such as the Clinical Global Impression sexual function
scale, the Female Sexual Function Questionnaire, the Arizona Sexual Experience scale-female
version, the University of New Mexico Sexual Function Inventory-female version, and the
Hamilton Depression Rating scale. Testosterone blood levels were also assessed. Sildenafil
54
treatment was associated with a significant improvement in orgasmic function and the response
was best in women with the highest free testosterone values (Borg and Chavez, 2014).
In an open study, sildenafil (Viagra) was prescribed for nine women outpatients who reported
sexual dysfunction induced by antidepressant medication, primarily selective serotonin reuptake
inhibitors. The nine patients, all of whom had experienced either anorgasmia or delayed orgasm
with or without associated disturbances, reported significant reversal of sexual dysfunction,
usually with the first dose of 50 mg of sildenafil (Nurnberg et al., 1999).
In a case report, it was described that a woman with SSRI-induced anorgasmia and no other
sexual complaints had this side effect reversed by vardenafil. Larger-scale placebo-controlled
studies would be helpful in determining the effect size and whether other opportunities exist for
using vardenafil to reverse SSRI-induced sexual dysfunction (Ashton, 2004).
It is suggested that phosphodiesterase type 5 inhibitors may have clinical therapeutic benefit,
especially for selected female patients with sexual arousal and orgasm disorders with normal
values of testosterone. Further research is needed, but it is strongly recommended that the critical
influence of sex steroids on womens sexual responses be better appreciated and accounted for
(Reis and Abdo, 2014).
4.1.8 Improve uterine artery blood flow and endometrial development in IVF
Endometrial growth is thought to depend on uterine artery blood flow and the importance of
endometrial development on in-vitro fertilization (IVF) outcome has been previously reported.
Sildenafil citrate (Viagra), a type 5-specific phosphodiesterase inhibitor, augments the
vasodilatory effects of NO by preventing the degradation of cGMP. The combination of
sildenafil and oestradiol valerate improved blood flow and endometrial thickness in four patients
with prior failed assisted reproductive cycles due to poor endometrial response. Three of the four
patients conceived. Although greater numbers of patients and randomized evaluation are needed
to validate this treatment, vaginal sildenafil may be effective for improving uterine artery blood
flow and endometrial development in IVF patients with prior poor endometrial response (Sher
and Fisch, 2000).
55
4.1.9 Enhances Vasodilatation in Fetal Growth Restriction

Fetal growth restriction (FGR) affects up to 8% of all pregnancies and has massive short-term
(increased fetal morbidity and mortality) and long-term (increased incidence of cardiovascular
disease in adulthood) health implications. Increased myometrial small artery vasoconstriction
and decreased endothelium-dependent vasodilatation in vessels are seen in women whose
pregnancies were complicated by FGR. A study demonstrated that sildenafil citrate significantly
reduced vasoconstriction and significantly improved relaxation of FGR small arteries. So the use
of sildenafil citrate in vivo may potentially improve uteroplacental blood flow in pregnancies
complicated by FGR (Wareing et al., 2005).
4.1.10 Therapy for severe early-onset intrauterine growth restriction

Currently, there is no effective therapy for severe early-onset intrauterine growth restriction
(IUGR). Sildenafil citrate vasodilates the myometrial arteries isolated from women with IUGRcomplicated pregnancies. Women were offered Sildenafil (25 mg three times daily until
delivery) if their pregnancy was complicated by early-onset IUGR [abdominal circumference
(AC) < 5th percentile] and either the gestational age was <25+0 weeks or an estimate of the fetal
weight was <600 g (excluding known fetal anomaly/syndrome and/or planned termination).
Sildenafil treatment was associated with increased fetal AC growth [odds ratio, 12.9; 95%
confidence interval (CI), 1.3, 126; compared with institutional Sildenafil-naive early-onset IUGR
controls]. Randomised controlled trial data are required to determine whether Sildenafil
improves perinatal outcomes for early-onset IUGR-complicated pregnancies. (von Dadelszen et
al., 2011)
4.1.11 Increased focus on development of selective PDE inhibitors

Considerable attention has been given to the development of selective PDE inhibitors, especially
after the therapeutic success of PDE5 inhibitors in the treatment of erectile dysfunction. Several
associations between PDE genes and genetic diseases have been described, and more recently
PDE11A and PDE8B have been implicated in predisposition to tumor formation. This review
focuses on the possible function of PDEs in a variety of tumors, primarily in endocrine glands,
both in tumor predisposition and as potential therapeutic targets (Levy et al., 2011).
56
The effects of phosphodiesterase type 5 inhibitors on vasodilation mediated via nitric oxidecyclic guanosine monophosphate are well described but less is known about other mechanisms
through which phosphodiesterase type 5 inhibitors benefit endothelial function, including
normalization of serum biomarkers, increased levels of endothelial progenitor cells, ischemiareperfusion protection mechanisms, and other actions specific to patients with diabetes. Further
studies are carried out to understand their impact on several cardiovascular diseases, including
heart failure, high-altitude pulmonary edema, Raynaud's phenomenon, coronary artery disease,
diabetes, and atherosclerosis (Schwartz et al., 2013).
57
Chapter Five
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Study of The Discovery and Development of Specific Inhibitors of Phosphodiesterase Type 5

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Study of The Discovery and Development of Specific Inhibitors of Phosphodiesterase Type 5

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STU

UDY OF THE DISC

DECLARATION BY THE CANDIDATE

CERTIFICATION BY THE SUPERVISOR

--------------------------------------Dr. Chowdhury Faiz Hossain

1.1.1 Nucleotide and phosphate bond linkage

1.1.2 cAMP and cGMP

1.1.3 Cyclic nucleotide phosphodiesterases

1.2 Studies on PDE

1.3 Multiple forms of phosphodiesterases

1.4 Phosphodiesterases as drug targets

2. Phosphodiesterase Family Classification

2.1.1 About PDE1

2.1.2 About PDE2

2.1.3 About PDE3

2.1.4 About PDE4

2.1.5 About PDE5

2.1.6 About PDE6

2.1.7 About PDE7

2.1.8 About PDE8

2.1.9 About PDE9

2.1.10 About PDE10

2.1.11 About PDE11

2.2 Overview of PDE isoform localization

2.3 Function of phosphodiesterase enzymes

2.4 PDE inhibitors

3.1 Cyclic guanosine monophosphate (cGMP)

3.2 Pathological importance of PDE5 inhibitor

3.2.1 Erectile dysfunction

3.3.1 Treatment of erectile dysfunction

3.3.2 Treatment of pulmonary hypertension

3.4 Development and discovery of Sildenafil, Vardenafil and Tadalafil

3.5 Sildenafil dosing

3.6 Sildenafil usage

3.6.1 Pediatric usage

3.6.2 Geriatric usage

3.6.3 Pregnancy usage

3.7 Molecular structure of Sildenafil

3.8 Drug-drug interactions

3.9 Vardenafil brand names and indications

3.10 Vardenafil dosage

3.11 Vardenafil metabolism, elimination and usage

3.11.1 Pediatric usage

3.11.2 Geriatric usage

3.11.3 Pregnancy usage

3.12 Drug interactions

3.13 Tadalafil brand names and indications

3.14 Tadalafil potency

3.15 Tadalafil dosage

3.16 Tadalafil usage

3.16.1 Pediatric usage

3.16.2 Geriatric usage

3.16.3 Pregnancy category

3.17 Drug interactions

3.18 Pharmacokinetics of the three PDE5 inhibitors

3.19 Side-effects of PDE5 inhibitors (Viagra, Cialis, Levitra)

3.20 Precautions of PDE5 inhibitors (Viagra, Cialis, Levitra)

3.21 Selectivity compared among the three PDE5 inhibitors

3.22 Herbal erectile dysfunction treatments

3.23 Adulteration of herbal products with synthetic PDE5 inhibitors

4.1.1 Therapeutic agent for parasitic disease

4.1.2 Better enhanced erectile function

4.1.3 PDE5 inhibitors for treatment of Alzheimers disease