Tmp 3466

1. Introduction
2. Physiology of penile erection
3. Role of L-arginine and erectile
function
4. Literature of ED
5. Current standard conservative
treatment for ED
6. Novel therapies for ED
7. Role of arginase
8. Conclusion
9. Expert opinion
Review
New approaches to the design
and discovery of therapies to
prevent erectile dysfunction
Haroldo A Toque
†
& Robert William Caldwell
Georgia Regents University, Augusta, Medical College of Georgia, Department of Pharmacology and
Toxicology, Augusta, GA, USA
Introduction: Nitric oxide (NO) is critically involved in erectile function. Since
NO synthase (NOS) and arginase compete for the same substrate L-arginine,
limiting arginase activity may provide more NO and thus be a beneficial ther-
apeutic approach to erectile dysfunction (ED). In the corpora cavernosa, exces-
sive arginase activity/expression has been implicated through studies of
preclinical and clinical models of ED. Further, the inhibition of arginase has
shown to increase vascular system relaxation and enhance blood flow in
penile circulation. Further studies, therefore, looking at therapies targeting
arginase could prove to be clinically useful.
Areas covered: The authors review gene- and cell-based therapies, the
involvement of RhoA/Rho-kinase (ROCK), MAPK and arginase in ED.
Expert opinion: Extensive literature supports the view that upregulated argi-
nase activity in cavernosal tissue can reduce NOS function and NO production.
Excessive arginase activity has been shown to contribute to the progression of
aging-, hypertension- and diabetes-induced vascular dysfunction as well as
ED. Earlier studies have shown that RhoA/ROCK and subsequent activation
of p38 MAPK mediate elevation of arginase expression/activity in diabetic
and hypertensive mice. Reducing corporal arginase activity by gene-based or
pharmacological therapy and/or inhibition of upstream regulators of arginase
expression may provide novel therapeutic approaches in the management
of ED.
Keywords: arginase, cavernosal smooth muscle relaxation, erectile dysfunction, nitric oxide,
penile erection
Expert Opin. Drug Discov. [Early Online]
1. Introduction
Erectile function is an important component of quality of life in men. Impairment
of erectile function can affect total health, relationships, overall quality of life and
potentially procreative advancement [1]. Over the past 20 years, erectile dysfunction
(ED) has been extensively studied because over 30 million men suffer with this dis-
order in the USA and the number is expected to rise considerably over the next
25 years, impacting > 300 million men by 2025 [2]. The fact that ED often coexists
with obesity, hypertension, cigarette smoking, aging and diabetes has led to the con-
clusion that ED is predominantly a disease of vascular origin [3]. Beyond its associ-
ation with vascular risk factors, ED is a strong predictor for coronary artery disease
mainly in men > 40 years [4].
Nitric oxide (NO), synthesized from L-arginine by NO synthase (NOS), is the
key physiological mediator of penile erection [1]. Given that NOS and arginase share
and compete for their common substrate L-arginine, NO bioavailability or biosyn-
thesis is likely linked to the regulation of arginase activity. Arginase is highly
expressed in corpus cavernosum (CC) and is implicated in ED [3]. Pharmacological
10.1517/17460441.2014.949234 © 2014 Informa UK, Ltd. ISSN 1746-0441, e-ISSN 1746-045X 1
All rights reserved: reproduction in whole or in part not permitted
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therapies have focused essentially in NO/cGMP signaling
pathway in the treatment of ED. The oral PDE5 inhibitors
have become the first-line treatment for ED [5]. However,
the efficacy of PDE5 inhibitors would be reduced when NO
bioavailability is decreased. Inhibition of PDE5 has limita-
tions in men with impaired penile vascular flow due to
advanced diabetes or cardiovascular disease (CVD). Thus,
either significant endothelial dysfunction or autonomic neu-
ropathy may limit the efficacy of a PDE5 inhibitor. New ther-
apeutic strategies including melanocortin receptor (MCR)
targets, shockwave therapy, combined therapy of testosterone
and a PDE5 inhibitor, supplementation of L-citrulline or
L-arginine, gene and cell-based therapy, soluble guanylyl
cyclase (sGC) stimulators and inhibition of arginase may pro-
vide better overall treatment for ED.
2. Physiology of penile erection
Penile erection involves interaction between the central neural
and peripheral pathways. Tumescence is a neurovascular event
modulated by sensory stimulation (such as visual, tactile,
olfactory and imaginative stimuli), psychological and hor-
monal factors. On sexual arousal, neurotransmitters are
released from the cavernous nerve terminals and the penile
endothelial cells, relaxing the cavernosal arteries and arterioles
and increasing the penile blood flow. Evidences indicate that
pudendal artery is the main supplier of blood (70%) to the
CC of the penis [6]. Concomitantly, relaxation of the trabecu-
lar smooth muscle increases the compliance of the sinusoids,
resulting in an engorgement of the penis with blood.
Penile blood flowis mediated by the autonomic nervous sys-
tem, which includes nonadrenergic noncholinergic (NANC)
nerves. These nerves are responsible for the release of NO to
the trabecular smooth muscle, which results in relaxation.
Because the CC is surrounded by the tunica albuginea, a tissue
that does not distend easily, the increased blood flow to the
penis enlarges the CC and enhances the intracavernosal pres-
sure (ICP). This change causes mechanical compression of
the emissary veins, which voids their ability to drain blood
and thereby results in penile rigidity. In the flaccid state in
which CC tissue is contracted, blood flow is limited and is
used only for nutritional purposes. In this state, blood pressure
within the CC is low. However, on arousal and erection, ICP
increases to about 100 mmHg [7]. Therefore, penile erection
includes sinusoidal relaxation, arterial dilation and venous
compression [8].
Central neurotransmitters such as dopamine and oxytocin
are also involved in a facilitatory effect on penile erection [9].
Increasing levels of dopamine in patients with Parkinson’s dis-
ease have shown to improve libido. Although both D1 and
D2 dopamine receptors have been implicated in central erec-
tile functions, the D2 receptor seems to have the predominant
role [10]. Research on several compounds designed to act selec-
tively on D2 receptors (ABT 724, PD 168077 and PIP 3EA)
have been found to induce erectile activity when given system-
ically or into the lateral ventricles [11]. The mechanism and
potency of these agents are similar to those observed for
apomorphine [11,12].
Penile detumescence begins with cessation of neurotrans-
mitter release, the breakdown of second messengers or activa-
tion of the a-adrenergic receptors on the cavernous arteries
and trabecular smooth muscle, leading to a reduction in arte-
rial inflow and a reduction of lacunar spaces. Contraction of
the trabecular smooth muscle restores the venous outflow
and the trapped blood is expelled and flaccidity returns [9].
In the pathophysiology context, several factors can disrupt
the normal physiologic mechanisms involved in penile erec-
tion (Table 1). A mix of two or more factors, including those
psychogenic and organic, is usually observed in patients with
ED. The most important therapeutic targets in the patho-
physiology of ED are addressed in Figure 1.
3. Role of L-arginine and erectile function
3.1 NO and erectile function
It is well documented that NO is the main factor mediating
penile erection. It is released from both endothelium and
NANC nerves. When released, NO diffuses into adjacent
smooth muscle cells of the CC, binds to sGC and converts
GTP to 3¢,5¢- cGMP. In turns, cGMP activates protein kinase
G (PKG) and exerts actions on ion channels, contractile reg-
ulatory proteins and PDE, which results in smooth muscle
relaxation (Figure 2) [13-15]. Thus, at the onset of sexual stimu-
lation, neuronal NOS (nNOS) induced by neuronal depolar-
ization and endothelial NOS (eNOS) induced in response to
shear forces brought on by dilation of arterial vessels and
increased blood flow into the sinuses of the CC serve as the
Article highlights.
. New therapies are needed for erectile dysfunction (ED).
.
An overview of currently used and available conservative
therapy options for ED is provided. In particular,
pharmacotherapies such as melanocortin receptor
agonist, testosterone replacement, supplementation of
L-citrulline or L-arginine or their combination and
stimulators of soluble guanylyl cyclase are described.
. Treatments that are not just palliative but restore
erectile function, such as shockwave, gene and stem cell
therapies are also addressed.
.
Enhanced arginase activity has been strongly implicated
in the pathogenesis of endothelial dysfunction and
impaired erectile function.
.
In models of ED such as diabetes, hypertension and
aging, inhibition or deletion of arginase gene has shown
to increase nitric oxide bioavailability and cGMP levels
accompanied with enhanced erectile function.
.
Strategies for limiting arginase activity and expression
and/or inhibition of its upstream regulators promise new
therapy for ED.
This box summarizes key points contained in the article.
H. A. Toque & R. W. Caldwell
2 Expert Opin. Drug Discov. (2014) 9(12)
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initiating factor in the erectile function process. PDE5 selec-
tively degrades cGMP and attenuates the erectile process.
This enzyme is highly expressed in the CC and controls the
cGMP accumulation caused by NO signaling and conse-
quently limits its relaxant actions [5]. Possible key causes of
reduced formation of NO are: i) limitation of the substrate
L-arginine; ii) increased levels of endogenous inhibitors of
NOS in plasma and tissues; and iii) upregulation of vascular
arginase levels.
3.2 Endogenous NOS inhibitors and erectile function
NO deficiency contributes to ED and is associated with
reduced endothelial and nitrergic nerve functions. Elevated
levels of endogenous NOS inhibitors such as asymmetric
dimethylarginine (ADMA), symmetric dimethylarginine
(SDMA), or N(G)-monomethyl-L-arginine have been
reported in patients with adverse cardiovascular events for
which endothelial dysfunction has been observed as a com-
mon etiology [16]. Increased ADMA has also been associated
with impaired NO-mediated urethral, trigonal and cavernosal
relaxation by pelvic ischemia [17].
ADMA is formed in endothelial cells by catabolism of
proteins containing methylated arginine residues. ADMA is
metabolized by dimethylarginine dimethylaminohydrolase
(DDAH), and reduced activity/expression of DDAH is
reported to be responsible for elevation of ADMA [18-21].
Studies have indicated that increased ADMA levels are
involved in reduced NOS activity and the pathogenesis of
ED in animal and human models [21]. Concentrations of
ADMA and SDMA are higher in those patients with arterio-
genic ED compared with those of nonarteriogenic ED and
controls. The authors concluded that increased ADMA
and SDMA concentrations contribute to NO deficiency and
impaired NO-mediated relaxation in the lower urinary tracts
and CC.
4. Literature of ED
ED is a common complaint in men over 40 years of age and
becomes more frequent in those over the age of 60 [22-24].
A well-accepted definition of ED is the consistent inability
to achieve or maintain an erection sufficient for satisfactory
sexual performance [14]. The Massachusetts Male Aging Study
predicts that 300 million men in the USA will suffer from ED
by 2025 [2,25]. Clearly, ED is now considered as a major health
problem for the increasing healthy aging population. Physical,
emotional and medical factors contribute to ED, and this
condition also is usually associated with vascular risk factors.
However, increased observation of young patients seeking
clinical help for problems with erection has been reported.
A recent study states that one of four men < 40 years have
ED [26]. Although the sample number is limited, the authors
suggest that lifestyle changes such as chronic use of cigarettes,
alcohol or consumption of illicit drugs, which is more fre-
quently observed in younger than older population, may
influence the onset of ED in young men. Further studies
with larger population samples are needed to confirm this
report and to characterize the potential role of ED severity
as a harbinger of medical disorders in men below the age of
40 years.
Over the past 20 years, several studies have revealed the
molecular mechanisms involved in the pathogenesis of ED
and have provided convincing evidence that ED is a disease
of psychogenic and organic etiologies. Organic causes include
neurogenic, endocrinologic, and vasculogenic damage as well
as drug-induced ED, which in total constitutes now > 80%
of clinical presentations. Psychogenic causes are mental prob-
lems, traumatic past experiences, relationship problems and
family or social pressures [4].
It is widely recognized that ED is associated with diseases
related to decreased NO bioavailability such as arterial
Table 1. List of gene therapy in different models of ED.
Model of ED Vector used Gene target Cell/tissue target Functional outcome Ref.
Aged rats eNOS Adenovirus Endothelial Improved ICP and cGMP [56]
Diabetic rats eNOS Adenovirus Endothelial Improved ICP [58]
Diabetic rats VEGF and Ang-1 Adenovirus Endothelial Improved ICP, cGMP, and eNOS levels [80]
Diabetic rats EC-SOD Adenovirus Endothelial Improved ICP and cGMP [72]
Aged rats EC-SOD Adenovirus Endothelial Reduced O
2
-
formation and
restored erection
[71]
Aged rat nNOS Adenovirus Neuronal Improved ICP [54]
Aged rat PIN Cytomegalovirus Neuronal Improved ICP and cGMP [55]
Aged mice Anti-arginase Adenovirus Endothelial Improved ICP and cGMP [88]
Castrated VEGF Adenovirus Penile Preserved erectile function [77]
Hypercholesterolemic
rats
VEGF Adenovirus Penile Enhanced CC angiogenesis and
erectile function
[80]
Aged rats iNOS Adenovirus Penile Ameliorated erectile function [61]
Diabetic rats PKG1-a Adenovirus Penile Restored PKG activity and improved ICP [83]
Ang-1: Angiopoietin-1; CC: Corpus cavernosum; EC-SOD: Extracellular superoxide dismutase; ED: Erectile dysfunction; eNOS: Endothelial nitric oxide synthase;
ICP: Intracavernosal pressure; iNOS: Inducible nitric oxide synthase; nNOS: Neuronal nitric oxide synthase; O
2
-
: Superoxide anion; PIN: Protein inhibitor of neuronal
nitric oxide synthase; PKG1: cGMP-dependent protein kinase 1.
New approaches to the design and discovery of therapies to prevent erectile dysfunction
Expert Opin. Drug Discov. (2014) 9(12) 3
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hypertension, hypercholesterolemia and diabetes. Impaired
NO/cGMP signaling pathways in the penile vasculature result
in reduced cavernosal smooth muscle relaxation and/or
increased smooth muscle contraction and ED. In 1998, the
treatment of ED was revolutionized by effective oral therapy
with PDE5 inhibitors such as sildenafil, tadalafil and vardena-
fil. Clinical efficacy of oral agents marked the beginning of
noninvasive pharmacological treatment for ED. Although
PDE5 inhibitors are effective in most of ED cases, these
agents have been shown to be less effective in certain disease
states, such as diabetes, post-radical prostatectomy, and severe
veno-occlusive dysfunction [27].
Additionally, recent reports indicate that ED is an early
marker of coronary artery disease, stroke and other adverse
cardiac events, because it precedes by 2 -- 3 years in advance
to the occurrence of a cardiovascular event [28,29]. Both cavern-
ous and coronary arteries share the common property of being
‘end’ arteries without collateral circulation. However, penile
arteries have a smaller diameter (1 -- 2 mm) than coronary ves-
sels (3 -- 4 mm), thus making them more prone to fail to ini-
tiate erection, even with a minimal narrowing [28]. The
importance of examining the relationship of ED to the onset
of CVD has key relevance in public health and clinical issues
as it may assist in reducing CVD complications [30].
6
7
4
3
2
1
Peripheral
nerves
Central nervous system
Sensory stimuli
(visual, erotic thought, tactile,
olfactory and imaginative)
Neurotransmitter release
(serotonin, dopamine, NO)
NANC nerves
Parasympathetic
nerves
Endothelial cell
NO
eNOS L-arginine arginase
(L-citruline)
Urea +
L-ornithine
cGMP
cGMP
inactive
PDE5
PKG Lower Ca
2+
PGE1
Relaxation
Cavernosal smooth muscle cell
sGC
[ ]
Ca
2+
NO
VIP
AC
ATP
cAMP
Ameliorates ED
Arginase
inhibitors
Endogenous
NOS inhibitors
Dopamine agonists
(apomorphine, ABT 724, PD168077)
sGC
activators
[


]
Ca
2+
Ca
2+
nNOS
5. PDE5 inhibitors: sildenafil, tadalafil, vardenafil
1. Arginaseinhibitors : BEC, ABH
2. Endogenous NOS inhibitors: ADMA, SDMA,
3. Oral supplement: L-arginine, L-citrulline, L-argilin
4. sGC activators: (BAY 41-2272; BAY 60-4552;
BAY 63-2521, etc.)
7. Prostaglandin E1 stimulator: alprostadil
PDE5
Oral
supplement
6. Vasoactive intestinal peptide
G-protein
5
GTP
inhibitors
Figure 1. Established and new therapeutic targets for the pathophysiology of ED are shown. CNS coordinates sensory stimuli
froma variety of sources. Release of neurotransmitter such as dopamine also contributes to sexual stimuli. Peripheral excitation
evokes stimulation of the NANC and parasympathetic nerves, increases Ca
2+
influxes and promotes eNOS and nNOS activation.
All these events enhance NO formation in the penis. NO binds to sGC and catalyzes conversion of GTP to cGMP in the smooth
muscle cell. Arginase enzyme that competes with eNOS for the substrate L-arginine. Release of VIP from the NANC nerve or
administration of PGE1 increases cAMP levels, whichtogether withcGMP causes vasodilation of arteries andsinusoidal spaces of
the CC. Reduction of intracellular CC calciumlevels by protein kinase Aand PKGleads to penile erection. Therapeutic targets for
the treatment of ED are addressed in this review such as, endogenous NOS inhibitors, arginase inhibitors, sGC activators, oral
supplementation of both L-citrulline and L-arginine or their combination. Increased reaction of 1 and 2 impairs cavernosal
relaxation and penile erection. Agents 3, 4 and 5 enhances NO/sGC/cGMP pathway which contributes to cavernosal smooth
muscle relaxation. Agents 6 and 7 enhance AC/cAMP pathway and contribute to cavernosal relaxation.
ABH: 2(S)-amino-6-boronohexanoic acid; AC: Adenylate cyclase; BEC: S-(2-boronoethyl)-L-cysteine; CC: Corpus cavernosum; ED: Erectile dysfunction; eNOS: endo-
thelial nitric oxide synthase; NANC: Nonadrenergic noncholinergic; nNOS: Neuronal nitric oxide synthase; NO: Nitric oxide; NOS: Nitric oxide synthase; PGE1: Pros-
taglandin E1; PKG: Protein kinase G; sGC: Soluble guanylate cyclase; VIP: Vasoactive intestinal peptide.
H. A. Toque & R. W. Caldwell
4 Expert Opin. Drug Discov. (2014) 9(12)
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5. Current standard conservative treatment
for ED
Despite the broad efficacy, tolerability and safety of PDE5
inhibitors for treatment of ED, an array of other treatments
and combination therapies have been examined and devel-
oped. A wide variety of ED treatments exist and are publicized
in the market. Each patient may choose a treatment according
to his attitude and experience and the treatment’s cost
or availability.
Porst et al. [31] have proposed five levels for ED treatment.
Level 1 includes PDE5 inhibitors, intracavernosal injection of
prostaglandin E1 (PGE1, alprostadil) or vasoactive intestinal
peptide/phentolamine and transurethral PGE1 therapy.
Also, treatment of hypogonadism with testosterone treatment,
adopting an active lifestyle with resultant weight loss and opti-
mal treatment of concomitant CVDs can either improve ED
or add to the efficacy of PDE5 inhibitors. Level 2 includes
vacuum-erection therapy, oral L-arginine, topical PGE1,
intracavernosal injection therapy with papaverine/phentol-
amine (Bimix) or papaverine/phentolamine/PGE1 (Trimix)
combination mixtures. Level 3 includes oral treatment with
yohimbine in non-organic ED, and combination therapies
of PDE5 inhibitors and either transurethral or intracavernosal
injection which generate better efficacy rates than either ther-
apy alone. Level 4 includes the combination of vacuum-
erection therapy and either a PDE5 inhibitor or transurethral
PGE1 or intracavernosal injection therapy. Level 5 includes
combination therapy of PDE5 inhibitors and L-arginine or
daily dosing of tadalafil and short-acting PDE5 inhibitors.
Mechanical therapy as vacuum-erection devices (VED) can
be successfully applied nearly in all etiologies of ED. Vacuum
therapy can also be used in combination with other therapies
such as PDE5 inhibitors or others [32]. However, some contra-
indications of VEDs are reported in patients with prolonged
erections, in patients with severe penile curvature, and those
with severe bleeding disorders [33]. It is documented that about
30% do not respond to either monotherapy. Combined medi-
cal therapies have been reported for patients who fail monother-
apy to produce satisfactory erection. Thus, basic research and
medical findings together with sexual medical experts are still
needed to obtain a higher efficacy for the management of ED.
Shear force
ACh
[Ca
+2
]
Arginase
L-arginine
eNOS
NO
Urea
L-ornithine
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
NO
sGC
cGMP
GTP
5′GMP
PKG
PDE5
PDE5
inhibitor
K
–
Ca
+2
Smooth muscle
relaxation
NO
nNOS
Nitrergic
nerve
NO
NO
NO
NO
Stimulation
Inhibition
Corpora cavernosa
smooth muscle
Penile erection
Endothelial
cells
Figure 2. Regulation of cavernosal smooth muscle relaxation and penile erection is shown. Central or local excitation evoked
by endothelial cells and nitrergic nerves in the penis causes calcium [Ca
2+
] influxes and promotes eNOS and nNOS activation,
which in turn increases NO production. When released, NO diffuses into cavernosal smooth muscle cells, binds to sGC and
catalyzes the conversion of GTP to cGMP. In turns, cGMP activates PKG and exerts actions on calcium and potassium ion
channels, which results in smooth muscle relaxation and penile erection. PDE5 selectively degrades cGMP and attenuates
erectile process. Inhibition of PDE5 enhances cGMP levels and cavernosal relaxation.
Ach: Acetylcholine; eNOS: Endothelial nitric oxide synthase; NO: Nitric oxide; NOS: Nitric oxide synthase; nNOS: Neuronal nitric oxide synthase; PKG: Protein kinase G;
sGC: Soluble guanylate cyclase.
New approaches to the design and discovery of therapies to prevent erectile dysfunction
Expert Opin. Drug Discov. (2014) 9(12) 5
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6. Novel therapies for ED
Current advances in sexual medicine fieldhave led to an increased
understanding of the underlying molecular mechanisms involved
in erectile function. Overall, PDE5 inhibitors represent the treat-
ment of choice in ED. However, targeting MCRs, shockwave
therapy and testosterone replacement in combination with
PDE5 inhibitors have been reported to be alternative therapies
for ED [27,34-36]. Additionally, gene- and cell-based therapies
seem to act at the cellular level to improve specific cellular and
enzymatic functions which delay/reverse ED. Further, since
NOS and arginase share the same substrate L-arginine, limiting
arginase activity may provide a beneficial therapeutic approach
for preventing impaired erectile function. The current review
addresses the major cellular and molecular targets of biological
systems responsible for erectile function and the new therapeutic
approaches in the treatment of ED. Current literature and exper-
imental data support the prospect of enhanced arginase activity
playing a key role in endothelium impairment and ED.
6.1 MCR agonist therapy
Melanocortins, peptides derived from a larger precursor mole-
cule known as the pro-opiomelanocortin protein, have been
shown to cause penile erection following intracerebroventricu-
lar administration in animal models. This includes a-melano-
cyte-stimulating hormone (a-MSH) and adrenocorticotropic
hormone (ACTH) [37]. The authors concluded that the proer-
ectile effects of a-MSH and ACTH were mediated by cAMP
and occur only in the presence of testosterone. However, the
specific MCR subtype(s) through which these peptides
produce erectogenic effects is not known [38].
Targeting MCR is an alternative therapy for ED. Adminis-
tration of PT-141, a nonselective MCR agonist, has been
shown to initiate erectile responses in a dose-dependent man-
ner in men with ED [39]. However, side effects such as flush-
ing and nausea, but not hypotension, were observed.
6.2 Shockwave therapy
Low-energy shockwave therapy (LESWT) was initially used to
breakup/dissolve and treat renal calculi without open sur-
gery [40]. More recently, the technology has been applied for
treatment of gall stones, pancreatic stones, parotid gland
stones and pseudarthrosis [41-43]. However, the mechanism
underlying the effects of LESWT is not well understood. Pre-
vious reports have indicated that LESWT may exert its effect
by increasing angiogenic factors and promoting neovasculari-
zation, thus improving blood supply [43]. Additionally,
LESWT is reported to improve recruitment of circulating
endothelial progenitor cells in patients with chronic ischemic
disease [44]. At present, this technique is widely used for treat-
ing myocardial ischemia, diabetic foot, wound healing,
Peyronie’s disease, weight loss and ED [45].
Shockwave therapy is a transmitted sonic wave with fre-
quencies around 16 to 20 MHz, duration < 10 µs and is often
applied into the target tissues or organs, creating a focal zone
during its treatment. The goal of LESWT in ED treatment is
to reverse the pathological changes in CC to regain spontane-
ous sexual activity with few side effects [35,46]. Recent studies
have reported that LESWT treatment improves erectile func-
tion of diabetic rats by increasing the expression levels of a-
smooth muscle actin, von willebrand factor, nNOS and
VEGF, and by reducing receptor of advanced glycation end-
products levels in CC tissues [47,48]. Additionally, LESWT
has been shown to improve erectile responses in patients
with ED [49]. Also, clinical studies have reported that LESWT
markedly enhances the duration of erection, penile rigidity
and penile endothelial function in patients who did not
respond to oral treatment of PDE5 inhibitors [50]. Further,
combined treatment of tadalafil and LESWT markedly
increases erectile responses in patients with Peyronie’s disease
and ED [51]. These findings suggest that LESWT could repre-
sent an effective approach for patients with severe ED who
respond poorly to PDE5 inhibitors therapy. However, further
studies are necessary to support these findings.
6.3 Gene therapy
Gene therapy involves introduction of new genetic material
into an appropriate cell type in vivo or in vitro to produce a pos-
itive effect by restoring or correcting a defective or deficient
gene in the nucleus of the cell. This therapy may provide an
alternate approach to that of PDE5 inhibitors which treat
only the symptoms but do not aim to cure the underlying con-
dition [52]. Because the penis is one of the few organs with easy
access and homogeneous parenchymatous content that pro-
vides an ideal location for gene delivery, this approach offers
the possibility in theory of curing ED after a single injection
of the cDNA construct into the penis. Alternatively, the com-
bination of local gene therapy with any of the currently avail-
able oral medications (such as PDE5 inhibitors) could
synergistically improve the efficacy of both therapies. Gene
delivery for treatment of ED represents a novel and promising
approach. Figure 3 shows the scheme of gene-based therapy.
The sequential order of gene therapeutic approach include:
i) administration of the vector to the specific cell/organ;
ii) cellular uptake; iii) nuclear translocation; iv) chromosomal
integration; and v) transcriptional activity and expression of
the transgene product [53]. Several gene therapy strategies
have investigated the improvement of ED in animal models
(19). Since NO pathway regulates corporal cavernosal
smooth muscle relaxation, resulting in penile erection, gene
therapeutic approach to restore NO production is highly con-
sidered in patients with ED. Since nNOS may initiate caver-
nosal tissue relaxation, whereas activated eNOS may facilitate
attainment and maintenance of penile erection, gene delivery
involved with NO production in CC has been examined for
potential therapies of ED. The following section summarizes
the available findings of preclinical animal studies for gene
therapy of ED in NO/cGMP/PKG signaling pathway.
H. A. Toque & R. W. Caldwell
6 Expert Opin. Drug Discov. (2014) 9(12)
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6.3.1 NOS gene therapy
6.3.1.1 nNOS gene therapy
Adenovirus (AdV) vector-mediated overexpression of nNOS
has been examined in aged rats [54]. When recombinant
nNOS AdV was injected in the CC of aged rats, penile
nNOS was effective in ameliorating age-related ED at
18 days after administration by electroporation, without
inducing the expression of cytotoxic genes [54]. The authors
suggest that a significant portion of the nNOS gene is taken
up by the CC and corpus spongiosum smooth muscles, and
to a much lesser degree by the cavernosal sinusoids space
and vascular endothelium, with only a minor fraction going
into the sparse nerve terminals of the penis. Additionally,
the role of protein inhibitor of NOS (PIN), a physiological
inhibitor of nNOS and nitrergic neurotransmission in the
penis was investigated [55]. Treatment with short hairpin
RNA construct targeting PIN in the aged rat penis signifi-
cantly decreased PIN messenger RNA and protein levels,
increased nNOS activity and ameliorated aging-related
ED [55]. Although PIN inhibition restores erectile function
in aged rats, further studies are necessary to determine the
effect of PIN ablation in molecular and cellular targets before
it would be applied in human gene therapy.
6.3.1.2 eNOS gene therapy
The role of eNOS has been examined by gene therapy
approach in vascular pathologies leading to ED with
promising findings [56-59]. Adenoviral vector-mediated overex-
pression of eNOS has been reported in age- and diabetic-
related rat models of ED [56,58]. After delivery of recombinant
AdV containing eNOS gene into the CC of aged rats,
increased expression of eNOS transgene accompanied with
enhancement of cGMP levels and ICP were observed in penile
tissue compared to control rats. Similarly, in a rat model of
diabetes-induced ED, intracavernous transduction of eNOS
enhanced erectile responses to cavernous nerve stimulation
and increased corporal NO bioavailability [58]. Additionally,
combination therapy of AdV-mediated eNOS gene and acute
systemic administration of PDE5 inhibitor sildenafil in strep-
tozotocin (STZ)-induced diabetic rats resulted in a synergistic
erectile function accompanied with enhanced cGMP levels
that was greater than that of either therapy alone [60]. Delivery
of eNOS gene to the penis may offer therapeutic utility by
restoring regulatory balance associated with NO signaling of
penile erection. Although most studies have examined vascular
disease models and implicated eNOS gene therapy with prom-
ising results, the next goal is to translate the benefit of NOS
gene therapy in animal models into clinical practice.
6.3.1.3 Inducible NOS gene therapy
In addition to nNOS and eNOS gene therapies, plasmid
DNA containing inducible NOS (iNOS) has been shown to
increase ICP by cavernosal nerve stimulation and enhance
NOS activity in a rat model of age-induced ED [61]. Further,
p p
p
p p
p p
p p
p
p p
Transcription
Translation
Gene
DNA vector
Viral vector
Nucleus
Target
cell
Protein
Cytoplasm
Promoter
Figure 3. Schematic representation of gene-based therapy. Therapeutic gene of interest is placed in viral vector that enters
into the nucleus of a targeted cell to alter its function. This process of transcription occurs only in the nucleus and requires a
promoter sequence of DNA to drive the process. The mRNA is then translated, which results in the new protein. The
translation process occurs only within the cytoplasm of the cell.
New approaches to the design and discovery of therapies to prevent erectile dysfunction
Expert Opin. Drug Discov. (2014) 9(12) 7
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modest delivery of iNOS gene into the CC of aged rats was
compared using three different techniques: AdV, AdV-
transduced myoblast cells or plasmid vector alone [62].
Myoblast-mediated gene therapy markedly increased erectile
responses to cavernosal nerve stimulation over that of AdV
or plasmid injection. Transduction of iNOS gene into the
penis in low abundance may provide important advantages
over eNOS and nNOS as a therapeutic tool for ED from
neurogenic and vascular causes.
6.4 Additional gene therapies
6.4.1 Superoxide dismutase gene therapy
Superoxide dismutase (SOD) is an endogenous antioxidant,
which catalyzes conversion of super oxide to hydrogen perox-
ide. Several studies have considered the therapeutic effect of
AdV gene delivery of SOD in models of heart failure, hyper-
cholesterolemia, hypertension and aging [63,64]. Gene therapy
of SOD represents a potential target in the treatment of ED
because it is the major endogenous antioxidant that protects
against upregulation of the reactive oxygen species (ROS) as
superoxide (O
2
-
). Vascular diseases, including hyperten-
sion [65], hypercholesterolemia [66], atherosclerosis [67], aging
and diabetes mellitus [68] can interfere with the intricate vascu-
lar mechanisms underlying normal erection, and they are
often associated with increased O
2
-
formation in the penile
vasculature. O
2
-
reacts with NO to form peroxynitrite, result-
ing in a diminished NO bioavailability [69]. SOD catalyzes the
dismutation of the O
2
-
into hydrogen peroxide and water and
is expressed in a number of cells including the vascular endo-
thelium. Three SOD isozymes have been identified: mito-
chondrial MnSOD, cytosolic CuZn-SOD and extracellular
(EC)-SOD (EC-SOD) [70].
In a model of age-related ED in rats, AdV gene transfer of
EC-SOD into the penis resulted in enhanced SOD activity,
cGMP levels and higher expression of EC-SOD mRNAs and
proteins. These alterations were accompanied with increased
erectile response to cavernosal nerve stimulation similar to those
of young rats [71]. Further, transduction of AdV EC-SOD into
the penis of aged rats decreased oxidative stress as measured by
nitrotyrosine formation. Additionally, in a STZ-diabetic rat
model, protein levels of EC-SOD were markedly decreased,
contributing to elevation of ROS formation. Further, delivery
of AdV EC-SOD into the penis of STZ-diabetic rats decreased
O
2
-
levels and increased NO bioavailability and cGMP lev-
els [72]. These studies suggest that transfer of AdV EC-SOD
into the penis represent a viable gene therapeutic target in dia-
betic- and age-associated ED involving increased ROS forma-
tion into the penile endothelium.
6.4.2 VEGF gene therapy
Growth factor therapy has emerged as a novel therapeutic tool
for the treatment of ED because it mediates endothelial and
smooth muscle physiology and regulates biological process
of erectile function. VEGF stimulates angiogenic activity
in vivo and in vitro, and its mRNA isoforms are extensively
expressed in CC of humans and rats [73]. Decreased VEGF
has been associated with a number of pathophysiological
changes in the penis [74].
Several studies have indicated the potential benefits of
VEGF in the treatment of ED. In a model of
atherosclerosis-associated ED in rabbits, intracavernosal injec-
tion of VEGF prevented impairment of endothelial-
dependent CC smooth muscle relaxation [75]. Similarly,
VEGF and adeno-associated virus (AAV) carrying brain-
derived neurotrophic factor seemed to alleviate the neurogenic
and vasculogenic ED associated with hypercholesterol-
emia [74,76]. Additionally, intracavernosal VEGF injection
and AAV-mediated VEGF gene therapy prevented and
reversed venogenic ED in castrated rats [77]. Further studies
have shown a protective effect of intracavernosal VEGF injec-
tion in traumatic arteriogenic and diabetic models [78]. Results
of these studies suggested that VEGF protein exerts its effect
on vascular endothelium by causing hyperplasia and hypertro-
phy of endothelial cells which may counteract the endothelial
apoptosis common to some manifestation of ED [79].
In a model of diabetes-induced ED in rats, combination of
an AdV-VEGF construct and injection of angiogenic factor-1
(Ang-1) into the CC prevented ED accompanied with
increased cavernous angiogenesis, eNOS phosphorylation and
cGMP levels, whereas either AdV-VEGF or Ang-1 alone eli-
cited partial improvement [80]. All these basic research studies
have clarified the role of VEGF and its effect on vascular endo-
thelium in the penis. Translation of these findings into clinical
treatments of vasculogenic ED needs to be explored. Future
clinical trials will promise whether this form of therapy will be
useful in restoring normal erectile function in patients with ED.
6.4.3 cGMP-dependent PKG1 gene therapy
When NO is released from the endothelium or nitrergic
nerves on to the smooth muscle cells, it activates sGC and
consequently cGMP levels, which results in cavernosal smooth
muscle relaxation. PKG1 is one of the most important effector
targets for cGMP to increase vascular compliance in the erec-
tile process [81]. It is reported that PKG1 also activates large
conductance Ca
2+
-activated potassium channels [82], which
hyperpolarize smooth muscle cell membranes, causing muscle
relaxation. Mice lacking PKG1 have been shown to exhibit
ED [81]. Reduced levels of cavernosal cGMP have been
observed in models of ED [82,83]. In a model of STZ-diabetic
rat, reduced activity of PKG1-a and -b has been observed in
cavernosal tissues. However, adenoviral-mediated gene deliv-
ery of PKG1-a into the penis of diabetic rats restores PKG
activity to levels similar to those observed in control rats
accompanied with improved erectile function during caver-
nosal nerve stimulation [83].
6.4.4 Anti-arginase gene therapy
Recent evidence indicates that elevated arginase activity con-
tributes to impaired nitrergic nerve and endothelium-
mediated relaxation of cavernosal smooth muscle in aging,
H. A. Toque & R. W. Caldwell
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hypertension and diabetes [65,68,84]. Given that NOS and argi-
nase share L-arginine as their common substrate, elevation of
arginase activity can limit availability of L-arginine for NOS,
thereby reducing NO production and impairing vascular
function. Previous studies have shown that human diabetic
CC with ED exhibits higher arginase activity and diminished
NO synthesis with reduced cavernosal relaxation [85]. Supple-
mentation of the amino acid L-arginine, either through the
diet or direct infusion, has been shown to increase NO forma-
tion in the vasculature and restore endothelium-dependent
vasorelaxation in the penis of aged rats [3]. Additionally, in
vascular tissues, inhibition of arginase has been shown to
enhance NO production and reduce vascular dysfunction in
hypertensive, high-fat diet and diabetic states [86,87].
Considering that increased arginase impairs vascular func-
tion, Bivalacqua et al. examined the physiological role of argi-
nase in modulating erectile function in vivo using AAV gene
transfer of anti-arginase-1 into the penis of aged mouse [88].
The data showed that AAV anti-arginase-1: i) decreases
arginase-1 protein and mRNA; ii) restores endothelial and erec-
tile function in vivo; iii) increases NOS activity; and iv) elevates
penile cGMP levels in aged mice penis. These findings in vivo
demonstrated that arginase is involved in ED through attenua-
tion of endothelial-derived NO in the aged mice penis.
6.5 Considerations of gene therapy in ED
Although delivery of viral genes into the penis has shown
beneficial effects to restore erectile function, a number of
potential issues need to be considered. Viral vectors for local
delivery could enter the systemic circulation, causing random
transgene expression in undesired tissues or organs. Risk of
immune responses induced by viral vectors also represents
another concern, as this could hinder use of the therapy in
either acute or repeated clinical treatments. To limit unde-
sired immune and inflammatory responses, it would be bene-
ficial to decrease the virus load required to transduce cells in
the penis. The possibility of increasing efficiency and specific-
ity of viral vectors to target specific cells could allow for injec-
tion systemically rather than intracavernously. Experimental
techniques to increase the specificity of viral vectors have
been examined such as transcriptional and transductional tar-
geting. Transcriptional targeting works through a specific cell
promoter, whereas transductional targeting utilizes cell-
specific membrane markers to induce delivery of the viral
gene. Several potential risks of viral vectors have limited the
use in humans, such as endogenous viral recombination, can-
cer development and immunological reactions [89]. Although
preclinical animal findings of gene therapy are highly promis-
ing for treatment of ED, attention is needed in regard to spec-
ificity and long-term safety of viral therapy before the
translation to clinical application is attempted.
6.6 Stem cell therapy
Therapy with stem cells constitutes a novel approach for the
treatment of ED because of their ability to self-renew and
differentiate into several different types of cells. As a result
of degenerative diseases such as diabetes and heart failure, cells
become dysfunctional or succumb to apoptosis or necrosis [90].
Stem cells have the capacity for functional regeneration of
damaged tissues, depending on the stimuli or signals that
they received. Differing from other pharmacotherapies, the
strategy of stem cells in the field of ED is to replace the lost
or damaged cells, providing normal penile tissue and
function.
Three types of stem cells have been reported: totipotent,
pluripotent and multipotent [91]. Totipotent cells are formed
from the fusion of an egg and sperm cell [91], and they can
turn into any cell in the body but do not reproduce them-
selves indefinitely. Pluripotent stem cells are derived from
the inner cell mass of the embryo and can form any of
the > 200 different cell types in the human body. Embryonic
stem cell isolated from the inner cell mass of blastocysts is an
example of pluripotent cells [92]. Multipotent stem cells are
derived from fetal tissue, cord blood and adult tissues. These
cells are capable of self-renewal and can form all cell types
within the embryonic lineage to which these cells belong. Sev-
eral laboratories have induced some multipotent cells to
reprogram and become pluripotent cells. These cells are called
induced pluripotent stem cells. Since endothelial dysfunction
is one of the primary causes of ED, transplanted stem cell
approaches to restore the penile endothelium and cavernosal
smooth muscle cell function are highly needed for patients
with ED. Wessells and Williams [93] were the first to demon-
strate endothelial cell transplantation into the rat CC for
cell-based therapy.
Among the various types of adult stem cells, bone marrow-
derived stem cells (BM-SCs), adipose-derived stem cells
(ADSC) and umbilical cord blood stem cells represent the
most attractive adult stem cells for their use in practical, clin-
ical and cell-based therapeutic purposes. BM-SCs have been
evaluated for their potential to treat vasculogenic ED [92].
Umbilical cord blood stem cells is also an attractive stem
cell type because they can be readily and noninvasively col-
lected from donors, and their collection and use raise no prob-
lematic ethical implications. The principal routes of stem cell
transplant are intravascular infusion, via lumbar puncture,
and during surgery when it is used to treat cardiovascular or
vascular lesions, internal organ lesions and nervous lesions,
including lesions in the brain or spinal cord.
6.6.1 Mesenchymal stem cells therapy
Mesenchymal stem cells (MSCs) are multipotent adult stem
cells formed in the BM with the capacity to differentiate
into endothelial and smooth muscle cells in vivo, making
them a viable option for the treatment of vasculogenic
ED [94]. Transplantation of MSCs showed effective therapy
against vascular diseases in vivo, suggesting the possibility
that this stem cell population may be effective in replacing
or rejuvenating the dysfunctional analogous tissues within
the penis [95]. In a age-associated ED rat model, transplanted
New approaches to the design and discovery of therapies to prevent erectile dysfunction
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MSCs alone or eNOS-enhanced MSCs into the penis demon-
strated improvement of erectile function through mechanisms
involving enhanced endothelium-derived NO biosynthe-
sis [3,96]. Similarly, transplantation of MSCs into the penis
markedly increased dilation of sinusoidal vascular spaces in
the CC and restored erectile function in age-associated
ED [97].
In addition, the efficacy of MSCs alone or in combination
with VEGF gene therapy is observed in a model of diabetes-
associated ED [98]. While the survival of engrafted stem cells
in the target tissue is a concern during the treatment, MSCs
survival increased when combined with VEGF treatment, as
noted by increased pro-survival factors -- phosphorylated Akt
and Bcl-xL. Further, combination of MSCs and VEGF gene
therapy in the rat penis increased endothelial and smooth
muscle cells and enhanced erectile responses compared with
those of MSCs alone [98]. These findings suggest that MSCs
are cell-based therapy of ED, especially when combined
with gene therapeutic treatments. Similarly, administration
of MSCs into major pelvic ganglion showed the preservation
of erectile function after bilateral cavernous nerve injury
(CNI). However, the use of collagen-based biocompatible
polymer matrix enhanced the implantation of MSCs and
improved the therapeutic effect of MSCs against CNI [95].
This latter study suggested that use of polymers may promote
the retention of stem cells in the target tissues as observed
previously [99].
In patients with type 2 diabetes-associated ED, stem cell
therapy produced improvements in the libido, erectile func-
tion and blood glucose levels after intracavernous transplant
of human umbilical cord blood stem cells without immune
suppression [100]. Despite the positive effects on diabetic
ED, the exact mechanisms underlying these effects are
missing.
6.6.2 ADSC therapy
ADSCs are found in fat tissue and have the ability of self-
renewal and differentiation into multiple cell phenotypes. In
terms of differentiation and therapeutic potentials, ADSCs
are similar to MSCs but are easier and safer to harvest in large
quantities. ADSCs have been shown to be vascular precursor
stem cells, making them an appropriate choice for stem cell
therapy of ED. Earlier studies demonstrated that ADSCs
can differentiate into endothelial cells in many tissues [101]
including the penis. Fibroblast growth factor 2 is reported
to promote ADSCs differentiation into endothelial cell, and
intracavernous injection of ADSCs can improve erectile func-
tion in rats with ED [102].
In a rat model of bilateral CNI-induced ED, intracaver-
nous injection of ADSCs restored erectile function by repopu-
lating endothelial and smooth muscle cells in the penis [103].
Also, intracavernosal injection of ADSCs showed enhanced
erectile function in obese type 2 diabetic zucker diet fat
(ZDF) rats compared with untreated animals [104]. The
increased erectile response by ADSCs observed in ZDF rats
resulted from an increased population of endothelial cells,
elevated nNOS protein levels and decreased intracorporal tis-
sue apoptosis compared with untreated rats. These findings
indicate that there may have been a restorative impact on
nitrergic neuron axons and ganglia leading to improved erec-
tile function. However, it is also possible that this increase in
nNOS was a result of increased oxygenated blood flow sup-
plying more dorsal nerve nNOS following erectile function
improvement.
VEGF is a cytokine with strong angiogenic properties that
improves survival of transplanted MSCs in a myocardial
infarction model [105]. Application of stem cell together with
VEGF gene therapy would result in higher stem cell survival
and increased efficiency for tissue repair [106]. Cell-based ther-
apy with ADSCs expressing VEGF accelerates the recovery of
erectile function by increasing endothelial function. It also
improves smooth muscle growth through its paracrine effects
and VEGF expression, compared with ADSCs injection alone
in STZ-diabetic rats [107]. Further, in a rat model of CNI ED,
combined therapeutic effect of PDE5 inhibitor and ADSCs
expressing brain-derived neurotrophic factor-membrane sys-
tem protected cavernous nerve and improved angiogenesis in
the penis compared to untreated controls [108].
In conclusion, MSCs and ADSCs are creating an impres-
sive record of high efficacy in various preclinical disease mod-
els. Their success in clinical trials of various CVDs promises
extension of the applicability of these cells for other vascular
diseases such as ED. Currently, many studies have demon-
strated that transplantation of either MSCs or ADSCs sources
results in improvement of erectile function in models of ED.
We believe that stem cell therapy for ED has unveiled new
horizons for the future of medicine and clinical treatments.
The use of gene and stem cell therapies alone or in combina-
tion with each other and other treatments such as pharmaco-
logical therapy is highly promising for the management of
patients concerned with ED.
6.7 Testosterone replacement therapy
Androgens play a key role in male sexual function and the
physiological mechanisms of penile erection [109]. Evidence
suggests that testosterone deficiency is an essential factor
observed in hypogonadism- and aging-induced ED [110]. Tes-
tosterone plays a critical role in maintaining the structure and
function of the vascular smooth muscle components of the
penis and mediates the penile vasculature tone [111]. It has
been shown that testosterone regulates not only cGMP forma-
tion, through NOS stimulation, but also its catabolism,
through PDE5 activity [112]. Deficiency of testosterone levels
may have an indirect effect to elevate arginase. However, it
is currently unknown.
It has been shown that hypogonadism reduces number and
quality of erections, and reports indicate that testosterone
replacement therapy improve erectile function in > 50% of
men with ED [113,114]. Testosterone replacement therapy in
hypogonadal men with sexual disorders has been
H. A. Toque & R. W. Caldwell
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recommended as an initial treatment by several investigators,
followed by specific ED treatment such as PDE5 inhibi-
tors [115,116]. The prevalence of hypogonadism is higher in
men with diabetes, and low testosterone is associated with sex-
ual dysfunction and reduced response to oral therapy for
ED [117]. In human studies, long-acting testosterone undeca-
noate therapy improved sexual function and quality of life
but not the metabolic disorders in men with type 2
diabetes [118].
Recent studies have proposed the syndrome of late-onset
hypogonadism as a clinical and biochemical state in men
with advancing age. It is characterized by both low testoster-
one levels and sexual symptoms (as decreased sexual interest
and reduced erections) independently. Indication of low
testosterone in men having sexual symptoms offers an oppor-
tunity to identify a small subgroup of aging men at particu-
larly high risk of dying [119].
6.8 L-arginine and L-citrulline supplemental therapy
L-arginine is the substrate required by eNOS to produce NO,
and adequate supply of L-arginine is essential for normal vas-
cular function. Reduced levels of L-arginine have been
reported in diabetic animals and patients [120,121]. Oral admin-
istration of L-arginine has been reported to prevent
impairment of endothelium-dependent vasorelaxation in
CVD models [122,123]. However, several studies in experimen-
tal animal models and in human clinical trials have reported
no beneficial effect on chronic administration of L-argi-
nine [124]. It is because a large portion of L-arginine passes
through the gastrointestinal tract and hepatic system where
it is catabolized by arginase I to ornithine and urea [125].
Chronic L-arginine treatment may produce undesirable effects
on cardiovascular function by enhancing arginase expression.
L-citrulline supplementation may become an important sub-
stitute for L-arginine supply where it is decreased under
pathologic conditions.
Supplementation of L-citrulline, a production of NOS and
the precursor of L-arginine, can also increase formation of
NO. L-citrulline is recycled into L-arginine sequentially by
argininosuccinate synthetase and argininosuccinate lyase [126].
Unlike L-arginine, L-citrulline is not largely affected by hepatic
metabolism and is not a substrate for arginase. Systemic
administration of L-citrulline appears to be a more efficient
way to increase blood NO levels than L-arginine [127]. Previous
studies have revealed that supplemental L-citrulline does not
increase arginase activity in blood vessels or liver and is able
to enhance endothelium-dependent NO production and
hypotension in rabbits [126]. Interestingly, the relevance of L-
citrulline on ED models has also been reported. Studies
have revealed that oral administration of L-citrulline improves
erectile function in arteriogenic and castrated ED rat model
by increasing blood NO levels and by attenuating damage
of cavernosal smooth muscle cells [128].
Additionally, oral L-citrulline supplementation for 1 month
increased erection hardness enough to restore normal erectile
function in patients with mild ED [129]. Although less effective
than PDE5 inhibitors, the authors concluded that in the short
term, L-citrulline treatment is safe and well accepted and may
become an alternative and less-expensive treatment for mild-
to-moderate ED patients. We believe that long-term oral sup-
plemental therapy of L-citrulline may be effective in supplying
L-arginine and NO formation in the cavernosal vasculature
with enhancement of endothelial and nitrergic function in
patients with ED. Combination of L-arginine and L-citrulline,
named Argilinin in the European market, has shown protec-
tive effect in CVDs. However, its efficacy for ED is still
unknown and further studies are needed.
6.9 sGC stimulator therapy
On its release, NO diffuses into adjacent cavernosal smooth
muscle cells and binds with its physiologic receptor, sGC.
NO promotes enzyme sGC activation to generate cGMP,
which in turn promotes cavernosal relaxation leading to penile
erection [14,15]. Decreased formation of NO is observed in
patients with diabetes-induced ED [85]. In support of this,
STZ-induced diabetes or spontaneously diabetic mice have
reduced endothelial and nitrergic responses [65,130]. Pharmaco-
logical agents that inhibit cGMP degradation such as the PDE
inhibitors and/or agents that enhance cGMP levels have
attracted much interest as potential therapeutic strategy in
the management of ED. However, studies reported that
> 30% of patients with ED do not respond to treatment
with PDE5 inhibitors, indicating that endogenous levels of
NO are reduced to such extent that PDE5 inhibitors can no
longer increase cGMP levels to a sufficient extent [131]. Phar-
macological stimulators of sGC may be beneficial for those
patients suffering with ED.
The benzylindazole derivate YC-1 was identified to be the
first NO-independent sGC stimulator to cause erectile
responses [132]. This pharmacological agent formed a lead
structure for the development of optimized sGC stimulators
with improved potency and specificity for heme-dependent
sGC stimulators (including CFM-1571, BAY 41-2272, BAY
41-8543, BAY 60-4552 and BAY 63-2521) and heme-
independent sGC activators (e.g., BAY 58-2667, BAY
60-2770, A-778935) [133]. Results from animal and human
studies have reported that BAY 41-2272 relaxes CC and
causes penile erection in vivo [134,135]. The relaxing effect of
BAY 41-2272 is uniquely mediated by sGC stimulation and
not by PDE5 inhibition [136]. Additionally, BAY
41-2272 has been shown to act in synergy with the NO
donor, sodium nitroprusside, to produce penile erection in
rabbits, implying that these compounds will enhance the
response to endogenous NO released during sexual stimula-
tion and thus facilitate a natural penile erection [134]. Further,
BAY 41-2272 also reduces O
2
-
formation and NADPH oxi-
dase expression in the CC, thereby increasing the bioavailabil-
ity of NO [135,137].
Recent studies have also observed synergistic effects of
combined administration of a sGC stimulator, BAY
New approaches to the design and discovery of therapies to prevent erectile dysfunction
Expert Opin. Drug Discov. (2014) 9(12) 11
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60-4552 and a PDE5 inhibitor, vardenafil, in CC relaxation
with increased proerectile effect in a CNI model [138]. Addi-
tionally, BAY 60-2770 has been shown to increase intracaver-
nosal responses under cavernosal nerve injury [139]. Thus, the
development of pharmacological agents capable of directly
stimulating the NO receptor, sGC, may represent a potential
therapeutic strategy in the management of ED.
6.10 RhoA/Rho-kinase pathway and ED
Rho-kinase (ROCK), a serine/threonine protein kinase, has
been identified as the major downstream effector of RhoA
which mediates calcium (Ca
2+
) sensitization [140]. The
ROCK isoforms found in the vasculature are ROCK-a/
ROCK2 and ROCK-b/ROCK1. ROCK is necessary for
diverse functions such as blood flow, arterial blood pressure,
airway resistance and intestinal peristalsis. ROCK activation
permits actin--myosin interactions and smooth muscle cells
contraction by maintaining the activity of myosin light-chain
kinase, independently of the free cytosolic Ca
2+
levels. Acti-
vated RhoA/ROCK pathway has been implicated in many
pathological conditions including arterial hypertension [141],
atherosclerosis [142], heart attack [143], stroke [144], coronary
vasospasm [145], myocardial hypertrophy [146], myocardial
ischemia reperfusion injury [147], aging [148], diabetes [149]
and ED [150]. Past studies have indicated that the use of
ROCK inhibitors like Y-27632, H1152 and fasudil offers
clinical benefits regarding the treatment of these diseases, as
well as contributes pharmacological tools for vascular stud-
ies [151]. The effect of ROCK inhibitors in preclinical models
of ED will be described below.
The release of NO from eNOS and nNOS into the penile
vasculature is fundamental for cavernosal smooth muscle
relaxation and normal erectile function [15]. Since the main
function of ROCK is the regulation of smooth muscle tone,
the upregulation of the ROCK pathway increases cavernosal
smooth muscle contraction, leading to ED [140,148]. One of
the first studies demonstrated that the ROCK inhibitor
Y-27632 caused a dramatic increase in CC pressure and erec-
tion independent of NO in an in vivo rat model [152]. Also,
higher expression of active RhoA contributes to flaccid state
of CC, and oral treatment with ROCK inhibitor reversed
impaired ED [153]. Additionally, a study combined an NO
donor and a ROCK inhibitor in the rat CC and observed a
synergistic effect of the erectile response to electrical field
stimulation compared to each therapy alone [152]. This study
demonstrated that inhibition of the RhoA/ROCK pathway
enhances the action of NO. In experimental preclinical mod-
els of ED, several studies have reported elevations in ROCK
activity/expression in association with impaired erectile func-
tion [141,150,154]. The interaction of NO and ROCK pathway
has been studied in the vasculature, focusing on disease states.
Accumulating evidence indicates that eNOS is regulated by
the ROCK pathway [155]. Inhibition of ROCK upregulates
eNOS in vivo via mechanisms that involve stabilization of
eNOS mRNA and increase in NO synthesis. Inhibition of
RhoA in CC of STZ-induced diabetic rats has been shown
to increase eNOS activity/expression, restoring erectile func-
tion [155]. These observations suggest that RhoA/ROCK
signaling suppresses NO synthesis.
Delivery of gene AAV encoding dominant negative RhoA
into the penis of aged and young rats for 7 days reduced
ROCK activity and markedly improved erectile function in
aged rats when compared with those of young rats [148]. Sim-
ilarly, in age-associated ED, ROCK inhibitor Y-27632 signif-
icantly improved erectile function compared to younger
rats [156]. Also, decreased expression of nNOS accompanied
with impaired erectile function in aged rats is reversed by
treatment with ROCK inhibitor, restoring the imbalance
between nNOS and ROCK activity [157]. Further, chronic
administration of fasudil, an oral ROCK inhibitor, prevented
the development of both vasculogenic ED and pelvic
atherosclerosis [158].
6.11 Implication of RhoA/ROCK and arginase in ED
Growing evidence indicate that enhanced RhoA/ROCK path-
way function is linked to upregulation of arginase in endothe-
lial cells exposed to oxidative species [159], angiotensin II [160],
thrombin [161] and in inflammatory bowel disease [162]. Two
isoforms of ROCK (1 and 2) have been identified in mamma-
lian tissues and both isoforms are expressed in vascular
smooth muscle and endothelial cells. To circumvent potential
nonspecific effects of ROCK inhibitors and their identical
actions on the two ROCK isoforms, haploinsufficient mouse
models with partial deletion of ROCK 1
+/-
and ROCK 2
+/-
genes have been used to address the specific role of each
ROCK isoform in activation of corporal arginase.
Recently, our study in CC tissues confirmed that diabetes
increases ROCK activity, protein expression of ROCK 2,
active p38 MAPK, arginase 2 (ARG2) and vascular arginase
activity as well as decreases endothelial and nitrergic nerve-
dependent relaxation responses. Diabetic heterozygous
ROCK 2
+/-
knockout mice evoked less ROCK activity, lower
levels of CC arginase activity/expression, less p38 MAPK acti-
vation and less impairment of endothelium-dependent and
nitrergic nerve-mediated relaxation than diabetic wild-type
mice [150]. Thus, our findings indicate that increased corporal
arginase activity caused by diabetes involves the ROCK and
p38 MAPK pathways, which contribute to penile vascular
dysfunction.
6.12 MAPKs
MAPK constitute a class of serine/threonine protein kinases
that evoke an intracellular signaling cascade in response to
EC stimuli such as proliferation, stresses, proinflammatory
cytokines, ROS, growth factors and apoptosis [163]. Several
groups of MAPK have been so far identified including EC
signal-regulated kinases (ERK1/2), p38 MAPK, c-Jun
N-terminal kinases (JNKs), ERK5 (MAPK7), ERK3
(MAPK6), ERK4 (MAPK4) and ERK7/8. The major
MAPK signaling pathways associated with various vascular
H. A. Toque & R. W. Caldwell
12 Expert Opin. Drug Discov. (2014) 9(12)
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diseases including hypertension, diabetes cancer, inflamma-
tion and neurodegeneration are p38 MAPK, ERK1/2 and
the JNK. However, inhibitors of p38 MAPK and
ERK1/2 are been tested in preclinical models of ED, and their
outcomes will be discussed in this review.
6.12.1 p38 MAPK and ED
Activation of p38 MAPK is a stress-sensitive mechanism trig-
gered by angiotensin II, diabetes, hyperglycemia and oxidative
stress that has been linked to the pathogenesis of vasculopathy
via increased endothelial cell proliferation with lesion forma-
tion and impairment of endothelial function [160,164]. At the
present time, few published studies are known to evaluate
the role of p38 MAPK on ED. In the first of these studies,
Nangle et al. [164] examined the effects of the p38 MAPK
inhibitor, LY2161793, on penile neurovascular function in
STZ-induced diabetic mice. This study demonstrated that
inhibition of p38 MAPK corrects nitrergic neurovascular dys-
function in diabetic mice CC. Subsequently, Toque et al. [65]
showed that p38 MAPK is involved in increased arginase
activity and contributes to endothelial dysfunction in CC of
mice treated for 2 weeks with angiotensin II. This study
showed that in vivo treatment with the p38 MAPK inhibitor,
SB 203580, attenuates angiotensin II-induced activation of
p38 MAPK, suppresses arginase activity and arginase II
expression and blocks angiotensin II-induced endothelial dys-
function in mice CC. Also, decrease in eNOS phosphoryla-
tion at Ser1177 observed in CC of angiotensin II-treated
mice is prevented by p38 inhibitor.
Additionally, Lysiak et al. [165] reported that ED and cell
death observed after bilateral cavernous nerve resection in
the penile tissue is due to an increase in apoptotic endothelial
cells and cavernous smooth muscle accompanied with
increased activation of p38 MAPK and JNK. Evidence indi-
cates that RhoA/ROCK pathway is an upstream regulator of
MAPK activity such as p38 MAPK [160]. Recently,
Toque et al. [150] demonstrated that p38 MAPK activation is
a downstream target of Rho/ROCK pathway, which is
involved in increased arginase activity and impaired endothe-
lial and nitrergic relaxation in diabetic mice CC.
6.12.2 ERK1/2 and ED
ERK1/2 can be triggered by cellular stresses such as oxidative
stress and hyperglycemia, which play an important role in the
development of diabetic complications, a disease associated
with ED. Currently few studies have focused on the role of
ERK1/2 in ED. In the first of these studies, Sommer et al.
[166] demonstrated that ERK1/2 is present and active in
human CC, and the expression of ERK1/2 is more pro-
nounced in endothelial rather than smooth muscle cells.
Higher levels of active ERK1/2 are also observed in diabetic
patients with ED. Recently, Nunes et al. [167] showed that
inhibition of ERK1/2 corrects penile arginase activity and
protects against ED caused by STZ-induced diabetic mice.
It seems that the MAPKs are indirectly associated with NOS
regulation, which affects NO availability. Earlier study
showed that ERK plays a key role in eNOS regulation. Phos-
phorylation of eNOS catalyzed by ERK can lead to a reduc-
tion in enzyme activity [168].
Additionally, increased activation of RhoA/ROCK and
ERK1/2 are associated with mineralocorticoid hypertension-
induced ED [169]. Although further studies are needed to bet-
ter clarify the exact role of both p38 MAPK and
ERK1/2 pathway in ED, new insights point to these pathways
as novel therapeutic targets worthy of consideration for
clinical trials.
7. Role of arginase
Arginase is the hydrolytic enzyme that catalyzes the conver-
sion of L-arginine into urea and L-ornithine. Arginase com-
petes with NOS for their common substrate L-arginine and
can alter NOS function when elevated during disease or
aging [125]. When the supply of L-arginine is insufficient,
NOS produce less NO and becomes ‘uncoupled’ and uses
more molecular oxygen as substrate to produce O
2
-
which
reacts rapidly with any available NO to generate the toxic oxi-
dant peroxynitrite. Competition between NOS and arginase
for L-arginine within the cell to produce either NO or orni-
thine and urea is quite feasible given their individual enzy-
matic properties. Although the affinity of L-arginine is much
higher for NOS (K
m
~ 6 µM) than for arginase
(K
m
~ 5 mM), the maximum activity (V
max
) of arginase
is > 1000 times than that of NOS, indicating similar rates
of substrate utilization at physiological L-arginine levels [170].
Two isoforms of arginase exist: the cytosolic isozyme argi-
nase I that is mainly expressed in the liver where it is a central
player in the urea cycle, and the mitochondrial isozyme argi-
nase II that is highly expressed in the kidney. Each is encoded
by a separate gene and is found in vascular tissues, endothelial
and smooth muscle cells, but their distribution appears to be
vessel- and species-dependent [171]. Both arginase isoforms
have been identified in vascular tissue and endothelial cells
and have important roles in endothelial dysfunction in diabe-
tes and other diseases. Arginase provides substrate for the
ornithine decarboxylase pathway, producing polyamines.
Arginase also plays a role in the production of proline, a crit-
ical component of collagen, through the ornithine amino-
transferase/pyrroline-5-carboxylate reductase pathway [125].
Therefore, increased arginase activity is associated with cell
growth and collagen formation and fibrosis.
7.1 Arginase and ED
Enhanced arginase activity has been strongly implicated in
the pathogenesis of vascular inflammatory reactions [172]
and endothelial and ED [85,87]. Penile erection and flaccidity
are regulated mainly by neurophysiological process involving
the relaxation and contraction of cavernosal smooth muscle.
Strong evidence indicates NO as the principal mediator of
penile erection [14]. NO released from sinusoidal endothelial
New approaches to the design and discovery of therapies to prevent erectile dysfunction
Expert Opin. Drug Discov. (2014) 9(12) 13
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cells (eNOS) or from nitrergic nerves (nNOS) causes CC
smooth muscle relaxation [14,15]. The pathological process
is characterized by impaired eNOS and nNOS relaxation
due to decreased production and bioavailability of NO.
Reduced availability of the substrate L-arginine to eNOS
has been implicated in vascular dysfunction in diabetes and
a variety of other disease conditions (Figure 4). Cavernosal
tissues from human diabetic patients with ED exhibit ele-
vated arginase activity and expression, diminished NO syn-
thesis and reduced cavernosal relaxation [85]. Conversely,
inhibition of arginase with 2(S)-amino-6-boronohexanoic
acid (ABH) or S-(2-boronoethyl)-L-cysteine maintains cellu-
lar L-arginine concentrations, which in turn enhance NOS
activity and NO-dependent cavernosal smooth muscle relax-
ation in human and rabbit penile CC and penile erection in
live rabbits. Additionally, deletion of arginase II gene in
mouse can enhance CC relaxation and prevent vascular dys-
function in diabetic mice [68].
The most common factors/pathologies involved with
excessive arginase-associated ED are described below.
7.1.1 Arginase and aging
Evidences from epidemiological studies confirm that age is
the primary risk factor for ED, and the prevalence and severity
of ED augments with age. A recent summary reported by the
International Consultation Committee for Sexual Medicine
reported that 39% of men have some degree of ED by the
age of 40 years, reaching 67% by the age of 70 years [173].
These findings are in agreement with large-scale studies as
reported previously [174]. Aging is associated with marked
changes in the penile endothelium and smooth muscle cells.
Therefore, age-related vascular endothelial dysfunction has
been associated with increased arginase activity/expression
[175], reduced expression and activity of eNOS and increased
oxidative stress [176]. Moreover, aging contributes to the devel-
opment of ED through the occurrence of endothelial dysfunc-
tion [3]. Because arginase participates in the regulation of NO
production by modulating the availability of L-arginine for
NOS, inhibition of arginase can have a potential therapeutic
effect in treating impaired NO-dependent cavernosal smooth
muscle relaxation in ED.
In the past years, few in vitro and in vivo studies have
reported the use of arginase inhibitors in the treatment of
age-associated ED. Bivalacqua et al. [88] demonstrated that
penile endothelial cells isolated from the aged mouse penis
over expressed arginase, which results in decreased eNOS
activity and impaired vascular function. The use of an argi-
nase inhibitor, ABH, or delivery of AVV anti-arginase gene
into the penis of aged mice increased the penile eNOS activity
and cGMP levels, restoring endothelial-derived NO vasodila-
tation and erectile function. Additionally, attenuated nNOS
protein levels and increased arginase activity contributed to
impaired neurogenic relaxation in aged rabbit CC [177]. Fur-
ther, oral inhibition of arginase with ABH results in improved
erectile function in aged rats [178]. All these findings support
that inhibition of arginase may represent a novel molecular
therapeutic target for the treatment of age-associated
vasculogenic ED.
Vascular endothelial
dysfunction
Hypertension Angiotensin II
Diabetes
Aging Atherosclerosis
Smoking
Arginase
(activity/expression)
NO
L-citrulline
NOS
L-arginine
Urea
L-ornithine
Arginase
O
2
–
ONOO
–
Erectile
dysfunction
Figure 4. Enhanced arginase activity and expression is linked to many cardiovascular risk factors. Loss of functional
endothelium and subsequent endothelial dysfunction play a key role in the occurrence of erectile dysfunction. Increased
arginase competes with NOS for their common substrate L-arginine and can alter NOS function during disease. When
L-arginine is insufficient, NOS produces less NO and becomes uncoupled and uses molecular oxygen to produce superoxide,
which reacts rapidly with any available NO to generate peroxynitrate.
NO: Nitric oxide; NOS: Nitric oxide synthase.
H. A. Toque & R. W. Caldwell
14 Expert Opin. Drug Discov. (2014) 9(12)
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7.1.2 Arginase and diabetes
Both type 1 and type 2 diabetes represent a common risk
factor of ED, which in turns develops in 50 -- 75% of male
diabetics [173,179]. ED also occurs three times more frequently
in diabetics than non-diabetics patients [180]. ED was the first
sign of diabetes mellitus in 12% of patients [181]. Vascular
changes such as endothelial impairment, reduced nitrergic
innervation, smooth muscle changes and reduced corporal
compliance are observed in diabetes [173]. Decreases in plasma
L-arginine have been reported in diabetic patients [120,121] and
in vascular tissue of STZ-diabetic rats [120]. Increased arginase
activity seems to be involved in these conditions.
Our group has demonstrated that increases in arginase
activity and arginase I expression in diabetes and high
glucose-induced endothelial dysfunction of aorta, coronary
and retinal arteries [182,183]. Researchers in the sexual medicine
field have examined effects of arginase inhibitor administra-
tion on these pathophysiological disturbances. Cox et al.
[184] showed arginase activity in human CC, and that inhibi-
tion of arginase significantly enhanced nitrergic nerve-
mediated relaxation of cavernosal smooth muscle cells.
Increased expression of arginase II in human diabetic CC
accompanied with diminished NO production contributes
to ED [85]. Additionally, enhanced arginase activity is
observed in the reproductive system of diabetic dogs, which
contributes to ED and low fertility in diabetics [185]. Further,
in a genetic spontaneously non-obese type 1 diabetes mouse
model, cavernosal tissues exhibited increased arginase activ-
ity/expression, contributing to impaired endothelial and
nitrergic function and reduced NO production [130].
Inhibitors of arginase are not isoform-selective and their
specificity may be species-dependent [125]. Thus, it is not pos-
sible to identify the role and function of a specific arginase iso-
form using pharmacological tools. Development of transgenic
mice lacking specific arginase isoforms has provided a more
complete and precise view of arginase function in a living sys-
tem. Our group has recently reported that deletion of arginase
II in mice markedly attenuated endothelial-dependent and
nitrergic nerve relaxation responses in CC of diabetic
mice [68]. Arginase II appears to modulate decreased NO pro-
duction in the cavernosal vasculature in diabetes. Therefore,
arginase is a potential target for therapeutic intervention in
the treatment of ED.
7.1.3 Arginase and hypertension
Hypertension is a major risk factor of CVDs by decreasing
NO bioavailability, increasing O
2
-
production and decreas-
ing endothelial levels of eNOS cofactor tetrahydrobiopterin
or substrate L-arginine [186-188]. Acute administration of
L-arginine has been shown to restore endothelium-
dependent vasodilator function in patients with essential
hypertension [189]. Recent clinical and basic science findings
have confirmed a higher incidence and prevalence of ED in
patients with hypertension (> 68%) in which endothelial
dysfunction is the major contributing factor of penile vascu-
lar pathology [190-192].
Elevated arginase activity has been reported in aorta, heart
and lung of spontaneously hypertensive rats (SHR). Treat-
ment with the arginase inhibitor N
w
-hydroxy-nor-L-arginine
reduced systemic blood pressure and cardiac fibrosis and
improved vascular function in SHR [84]. In deoxycorticoster-
one acetate (DOCA)-salt hypertensive rats, expression and
activity of arginase I protein in the aorta are elevated accom-
panied with increased blood pressure. These findings suggest
that arginase is involved in the pathophysiology of arterial
hypertension [193]. Additionally, the detrimental role of argi-
nase I in mediating elevation of blood pressure and endothe-
lial dysfunction is reported in the DOCA-salt hypertensive
mice [194]. Our group has determined that elevated angioten-
sin II is a key component of endothelial dysfunction in CVDs,
including hypertension and diabetes and has been linked to
upregulation of arginase activity in mice [65,160]. Angiotensin
II, which is found in human CC endothelial and smooth
muscle cells [195], appears to play a significant role in the reg-
ulation of the erection process since higher concentrations of
it are associated with penile detumescence [196]. Our group
has demonstrated that angiotensin II-treated mice have
increased systolic blood pressure accompanied with elevation
of aortic and cavernosal arginase activity/expression and
impaired vascular and CC endothelial function [65]. These
findings suggest that increased arginase activity and decreased
phospho-eNOS expression in the positive regulatory site may
contribute to impaired vascular function in hypertension-
associated ED.
7.1.4 Arginase and smoking
Cigarette smoking is an independent risk factor of vasculo-
genic ED. When adults rabbits are given subcutaneous injec-
tion of cigarette smoke extract daily for 5 weeks, a marked
increase in cavernosal arginase activity/expression (arginase I)
has been observed. Additionally, increased levels of endoge-
nous NOS inhibitors (monomethylarginine and ADMA)
decreased nNOS expression, attenuated NOS activity and
reduced cGMP levels were observed. These results suggest
that impaired NO production would result from blunted
NOS activity, which is possibly brought by the downregula-
tion of nNOS protein, accumulation of endogenous NOS
inhibitors and enhanced arginase activity together with upre-
gulation of arginase I protein in cavernous tissue [197].
8. Conclusion
Study findings that inhibition of arginase or deletion of argi-
nase gene enhances NO production and reduces vascular
endothelial dysfunction in CVD models, including ED
appear to be highly relevant for future management of ED.
Elevation of arginase activity can limit availability of L-argi-
nine for NOS, resulting in reduced NO production accompa-
nied by impaired CC smooth muscle relaxation. Evidence
New approaches to the design and discovery of therapies to prevent erectile dysfunction
Expert Opin. Drug Discov. (2014) 9(12) 15
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that increased vascular system relaxation and enhanced blood
flow in the penile circulation through inhibition of arginase
advances the possibility that therapies targeting arginase could
indeed be clinically useful. Identification of signal transduc-
tion steps involved in enhancing arginase expression and
activity could provide novel therapeutic targets for develop-
ment of pharmacological treatments that limit its activities
in pathological conditions.
9. Expert opinion
Over the past several years, there have been a large number of
preclinical and clinical studies reporting significant advances
in our understanding of the physiology and the pathophysiol-
ogy of penile erection. The exact etiological mechanisms
responsible for impaired erectile function have not yet been
determined. The main target in the mechanisms associated
with erectile function is still NO, and the deep understanding
of NO/cGMP pathway has added significantly to the treat-
ment of ED. Decreased NO bioavailability in the penile
endothelium observed in preclinical models of ED may be
caused by lack of substrate (L-arginine), reduction of eNOS,
diminished cofactors of eNOS, uncoupled NOS or increased
reaction of NO with O
2
-
anion. Extensive literature supports
the concept that upregulated arginase activity in cavernosal
tissue can alter NOS function, producing less NO. Excessive
arginase activity has been shown to contribute to the progres-
sion of aging-, hypertension- and diabetes-induced vascular
dysfunction and ED [65,68,85,88].
Additionally, significant advances in basic research have
shown the usefulness of pharmacotherapy such as supplemen-
tation of L-citrulline, MCR agonists, testosterone replace-
ment, sGC stimulators and shockwave, stem cell and gene
therapies in the treatment of ED. Administration of anti-
arginase genes into the penis of aged mice has been shown
to restore endothelial function and to enhance cGMP levels
followed by improved erectile function. Upstream mediators
of arginase activation are being investigated in various disease
conditions. Studies by our group and others have shown that
RhoA/ROCK and subsequent activation of p38 MAPK medi-
ate elevation of arginase expression and activity in STZ-
diabetic mice [150], angiotensin II-induced hypertensive
mice [65,160] and endothelial cell treated with thrombin [161]
or ROS [159]. Activated RhoA/ROCK pathway has been
shown to be involved in penile detumescence and ED, and
partial deletion of ROCK II gene in diabetic mice evoked
less ROCK activity, lower levels of CC arginase activity, less
p38 MAPK activity and less impairment of endothelium-
dependent and nitrergic nerve-mediated relaxation than dia-
betic control mice [150]. Our findings indicate that increased
p38 MAPK
Arginase
Impaired cavernosal
relaxation
G-protein
Extracellular
Intracellular
Diabetes
BEC
SB-203580
RhoA
Rho-kinase
Inhibition
Stimulation
p38 MAPK inhibitor: SB203580
Arginase inhibitor: BEC
Figure 5. Excessive arginase activity by diabetes-induced RhoA/Rho-kinase and p38 MAPK activation leads to impaired
cavernosal relaxation.
BEC: S-(2-boronoethyl)-L-cysteine.
H. A. Toque & R. W. Caldwell
16 Expert Opin. Drug Discov. (2014) 9(12)
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corporal arginase activity caused by diabetes involves the
ROCK and p38 MAPK pathways, which contribute to
impaired CC relaxation and ED. Thus, reduction of corporal
arginase activity and/or inhibition of upstream regulators of
arginase expression represent important targets for the treat-
ment of ED (Figure 5).
Despite the efficacy of PDE5 inhibitors and their position
as the primary choice for treating ED, the search for new
drugs for treatment of ED has been extensive. The efficacy
of sildenafil in the relaxation of the CC is decreased when
NOS is blocked [198]. Moderate reduction of arginase function
represents a promising new strategy since it can drive greater
NO production at the endothelial levels and can enhance
the effectiveness of PDE5 inhibitors in patients with ED.
Indeed, targeting arginase by gene-based or pharmacological
therapy may provide a novel therapeutic approach in the
management of patients who do not respond to conventional
treatment of ED.
Declaration of interest
HA Toque is funded by a Scientist Development Grant
(13SDG17410007) from the American Heart Association
(National Affiliate) and by a research grant from Sexual Med-
icine Society of North America. The authors have no other
relevant affiliations or financial involvement with any organi-
zation or entity with a financial interest in or financial conflict
with the subject matter or materials discussed in the manu-
script apart from those disclosed.
Bibliography
Papers of special note have been highlighted as
either of interest () or of considerable interest
() to readers.
1. Albersen M, Mwamukonda KB,
Shindel AW, et al. Evaluation and
treatment of erectile dysfunction.
Med Clin North Am 2011;95(1):201--12
2. Ayta IA, McKinlay JB, Krane RJ. The
likely worldwide increase in erectile
dysfunction between 1995 and 2025 and
some possible policy consequences.
BJU Int 1999;84(1):50--6
3. Bivalacqua TJ, Usta MF, Champion HC,
et al. Endothelial dysfunction in erectile
dysfunction: role of the endothelium in
erectile physiology and disease. J Androl
2003;24(6 Suppl):S17--37
4. Shamloul R, Ghanem H. Erectile
dysfunction. Lancet
2013;381(9861):153--65
5. Carson CC, Lue TF. Phosphodiesterase
type 5 inhibitors for erectile dysfunction.
BJU Int 2005;96(3):257--80
6. Manabe K, Heaton JP, Morales A, et al.
Pre-penile arteries are dominant in the
regulation of penile vascular resistance in
the rat. Int J Impot Res
2000;12(3):183--9
7. Sattar AA, Salpigides G,
Vanderhaeghen JJ, et al. Cavernous
oxygen tension and smooth muscle
fibers: relation and function. J Urol
1995;154(5):1736--9
8. Hafez ES, Hafez SD. Erectile
dysfunction: anatomical parameters,
etiology, diagnosis, and therapy.
Arch Androl 2005;51(1):15--31
9. Giuliano F, Rampin O. Neural control
of erection. Physiol Behav
2004;83(2):189--201
10. Padma-Nathan H, Christ G, Adaikan G,
et al. Pharmacotherapy for erectile
dysfunction. J Sex Med
2004;1(2):128--40
11. Melis MR, Succu S, Sanna F, et al.
PIP3EA and PD-168077, two selective
dopamine D4 receptor agonists, induce
penile erection in male rats: site and
mechanism of action in the brain.
Eur J Neurosci 2006;24(7):2021--30
12. Cowart M, Latshaw SP, Bhatia P, et al.
Discovery of 2-(4-pyridin-2-ylpiperazin-
1-ylmethyl)-1H-benzimidazole (ABT-
724), a dopaminergic agent with a novel
mode of action for the potential
treatment of erectile dysfunction.
J Med Chem 2004;47(15):3853--64
13. Andersson KE. Pharmacology of penile
erection. Pharmacol Rev
2001;53(3):417--50
14. Dean RC, Lue TF. Physiology of penile
erection and pathophysiology of erectile
dysfunction. Urol Clin North Am
2005;32(4):379--95, v
15. Toda N, Ayajiki K, Okamura T. Nitric
oxide and penile erectile function.
Pharmacol Ther 2005;106(2):233--66
16. Beltowski J, Wojcicka G,
Jamroz-Wisniewska A. Role of nitric
oxide and endothelium-derived
hyperpolarizing factor (EDHF) in the
regulation of blood pressure by leptin in
lean and obese rats. Life Sci
2006;79(1):63--71
17. Masuda H. Significance of nitric oxide
and its modulation mechanisms by
endogenous nitric oxide synthase
inhibitors and arginase in the micturition
disorders and erectile dysfunction.
Int J Urol 2008;15(2):128--34
18. Jiang DJ, Jia SJ, Yan J, et al.
Involvement of DDAH/ADMA/NOS
pathway in nicotine-induced endothelial
dysfunction. Biochem Biophys
Res Commun 2006;349(2):683--93
19. Lin KY, Ito A, Asagami T, et al.
Impaired nitric oxide synthase pathway
in diabetes mellitus: role of asymmetric
dimethylarginine and dimethylarginine
dimethylaminohydrolase. Circulation
2002;106(8):987--92
20. Masuda H, Azuma H. [Biological and
pathophysiological roles of endogenous
methylarginines as inhibitors of nitric
oxide synthase].
Nihon Yakurigaku Zasshi
2002;119(1):29--35
21. Park K, Lee DG, Kim SW, et al.
Dimethylarginine
dimethylaminohydrolase in rat penile
tissue: reduced enzyme activity is
responsible for erectile dysfunction in a
rat model of atherosclerosis. Int J
Impot Res 2009;21(4):228--34
22. Zhang X, Gao J, Liu J, et al.
Distribution and factors associated with
four premature ejaculation syndromes in
outpatients complaining of ejaculating
prematurely. J Sex Med
2013;10(6):1603--11
23. Nicolosi A, Glasser DB, Kim SC, et al.
Sexual behavior and dysfunction and
help-seeking patterns in adults aged 40-
80 years in the urban population of
Asian countries. BJU Int
2005;95(4):609--14
New approaches to the design and discovery of therapies to prevent erectile dysfunction
Expert Opin. Drug Discov. (2014) 9(12) 17
E
x
p
e
r
t

O
p
i
n
.

D
r
u
g

D
i
s
c
o
v
.

D
o
w
n
l
o
a
d
e
d

f
r
o
m

i
n
f
o
r
m
a
h
e
a
l
t
h
c
a
r
e
.
c
o
m

b
y

M
e
d
i
c
a
l

C
o
l
l
e
g
e

o
f

G
e
o
r
g
i
a

o
n

0
9
/
1
0
/
1
4
F
o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.
24. Lochlainn MN, Kenny RA. Sexual
activity and aging. J Am Med Dir Assoc
2013;14(8):565--72
25. Stranne J, Malmsten UG, Areskoug B,
et al. Influence of age and changes over
time on erectile dysfunction: results from
two large cross-sectional surveys 11 years
apart. Scand J Urol 2013;47(3):198--205
26. Capogrosso P, Colicchia M,
Ventimiglia E, et al. One patient out of
four with newly diagnosed erectile
dysfunction is a young man--worrisome
picture from the everyday clinical
practice. J Sex Med 2013;10(7):1833--41
. Important exploratory analysis of
young men seeking first medical help
for onset of erectile dysfunction (ED)
as their primary sexual disorder.
27. Hakim L, Van der Aa F, Bivalacqua TJ,
et al. Emerging tools for erectile
dysfunction: a role for regenerative
medicine. Nat Rev Urol
2012;9(9):520--36
28. Montorsi P, Ravagnani PM, Galli S,
et al. The artery size hypothesis:
a macrovascular link between erectile
dysfunction and coronary artery disease.
Am J Cardiol 2005;96(12B):19M--23M
.
Information of the crosslink between
sexual dysfunction and cardiovascular
risk problems.
29. Miner M, Nehra A, Jackson G, et al. All
men with vasculogenic erectile
dysfunction require a cardiovascular
workup. Am J Med 2014;127(3):174--82
30. Montorsi F, Adaikan G, Becher E, et al.
Summary of the recommendations on
sexual dysfunctions in men. J Sex Med
2010;7(11):3572--88
31. Porst H, Burnett A, Brock G, et al. SOP
conservative (medical and mechanical)
treatment of erectile dysfunction.
J Sex Med 2013;10(1):130--71
32. Chen J, Sofer M, Kaver I, et al.
Concomitant use of sildenafil and a
vacuum entrapment device for the
treatment of erectile dysfunction. J Urol
2004;171(1):292--5
33. Limoge JP, Olins E, Henderson D, et al.
Minimally invasive therapies in the
treatment of erectile dysfunction in
anticoagulated cases: a study of
satisfaction and safety. J Urol
1996;155(4):1276--9
34. Hatzimouratidis K, Amar E, Eardley I,
et al. Guidelines on male sexual
dysfunction: erectile dysfunction and
premature ejaculation. Eur Urol
2010;57(5):804--14
35. Zhang X, Zang N, Wei Y, et al.
Testosterone regulates smooth muscle
contractile pathways in the rat prostate:
emphasis on PDE5 signaling. Am J
Physiol Endocrinol Metab
2012;302(2):E243--53
36. Buvat J, Maggi M, Guay A, et al.
Testosterone deficiency in men:
systematic review and standard operating
procedures for diagnosis and treatment.
J Sex Med 2013;10(1):245--84
37. Argiolas A, Melis MR, Murgia S, et al.
ACTH- and alpha-MSH-induced
grooming, stretching, yawning and penile
erection in male rats: site of action in the
brain and role of melanocortin receptors.
Brain Res Bull 2000;51(5):425--31
38. Lindblom J, Schioth HB, Larsson A,
et al. Autoradiographic discrimination of
melanocortin receptors indicates that the
MC3 subtype dominates in the medial
rat brain. Brain Res
1998;810(1-2):161--71
39. Rosen RC, Diamond LE, Earle DC,
et al. Evaluation of the safety,
pharmacokinetics and pharmacodynamic
effects of subcutaneously administered
PT-141, a melanocortin receptor agonist,
in healthy male subjects and in patients
with an inadequate response to Viagra.
Int J Impot Res 2004;16(2):135--42
40. Chaussy C, Brendel W, Schmiedt E.
Extracorporeally induced destruction of
kidney stones by shock waves. Lancet
1980;2(8207):1265--8
41. Bara T, Synder M. Nine-years experience
with the use of shock waves for
treatment of bone union disturbances.
Ortop Traumatol Rehabil
2007;9(3):254--8
42. Tandan M, Reddy DN, Santosh D, et al.
Extracorporeal shock wave lithotripsy of
large difficult common bile duct stones:
efficacy and analysis of factors that favor
stone fragmentation.
J Gastroenterol Hepatol
2009;24(8):1370--4
43. Wang CJ, Wang FS, Yang KD, et al.
Shock wave therapy induces
neovascularization at the tendon-bone
junction. A study in rabbits.
J Orthop Res 2003;21(6):984--9
44. Aicher A, Heeschen C, Sasaki K, et al.
Low-energy shock wave for enhancing
recruitment of endothelial progenitor
cells: a new modality to increase efficacy
of cell therapy in chronic hind limb
ischemia. Circulation
2006;114(25):2823--30
45. Vardi Y, Appel B, Jacob G, et al. Can
low-intensity extracorporeal shockwave
therapy improve erectile function? A 6-
month follow-up pilot study in patients
with organic erectile dysfunction.
Eur Urol 2010;58(2):243--8
46. Goertz O, Lauer H, Hirsch T, et al.
Extracorporeal shock waves improve
angiogenesis after full thickness burn.
Burns 2012;38(7):1010--18
47. Lei H, Liu J, Li H, et al. Low-intensity
shock wave therapy and its application to
erectile dysfunction. World J
Mens Health 2013;31(3):208--14
48. Liu J, Zhou F, Li GY, et al. Evaluation
of the effect of different doses of low
energy shock wave therapy on the erectile
function of streptozotocin (STZ)-induced
diabetic rats. Int J Mol Sci
2013;14(5):10661--73
49. Vardi Y, Appel B, Kilchevsky A, et al.
Does low intensity extracorporeal shock
wave therapy have a physiological effect
on erectile function? Short-term results of
a randomized, double-blind, sham
controlled study. J Urol
2012;187(5):1769--75
50. Gruenwald I, Appel B, Kitrey ND, et al.
Shockwave treatment of erectile
dysfunction. Ther Adv Urol
2012;5(2):95--9
51. Palmieri A, Imbimbo C, Creta M, et al.
Tadalafil once daily and extracorporeal
shock wave therapy in the management
of patients with Peyronie’s disease and
erectile dysfunction: results from a
prospective randomized trial.
Int J Androl 2012;35(2):190--5
52. Gonzalez-Cadavid NF, Rajfer J.
Molecular pathophysiology and gene
therapy of aging-related erectile
dysfunction. Exp Gerontol
2004;39(11-12):1705--12
53. Kay MA. State-of-the-art gene-based
therapies: the road ahead. Nat Rev Genet
2011;12(5):316--28
54. Magee TR, Ferrini M, Garban HJ, et al.
Gene therapy of erectile dysfunction in
the rat with penile neuronal nitric oxide
synthase. Biol Reprod
2002;67(3):1033--41
55. Magee TR, Kovanecz I, Davila HH,
et al. Antisense and short hairpin
H. A. Toque & R. W. Caldwell
18 Expert Opin. Drug Discov. (2014) 9(12)
E
x
p
e
r
t

O
p
i
n
.

D
r
u
g

D
i
s
c
o
v
.

D
o
w
n
l
o
a
d
e
d

f
r
o
m

i
n
f
o
r
m
a
h
e
a
l
t
h
c
a
r
e
.
c
o
m

b
y

M
e
d
i
c
a
l

C
o
l
l
e
g
e

o
f

G
e
o
r
g
i
a

o
n

0
9
/
1
0
/
1
4
F
o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.
RNA (shRNA) constructs targeting PIN
(Protein Inhibitor of NOS) ameliorate
aging-related erectile dysfunction in the
rat. J Sex Med 2007;4(3):633--43
56. Bivalacqua TJ, Champion HC,
Mehta YS, et al. Adenoviral gene transfer
of endothelial nitric oxide synthase
(eNOS) to the penis improves age-related
erectile dysfunction in the rat. Int J
Impot Res 2000;12(Suppl 3):S8--17
57. Champion HC, Bivalacqua TJ,
Greenberg SS, et al. Adenoviral gene
transfer of endothelial nitric-oxide
synthase (eNOS) partially restores normal
pulmonary arterial pressure in eNOS-
deficient mice. Proc Natl Acad Sci USA
2002;99(20):13248--53
58. Bivalacqua TJ, Usta MF, Champion HC,
et al. Gene transfer of endothelial nitric
oxide synthase partially restores nitric
oxide synthesis and erectile function in
streptozotocin diabetic rats. J Urol
2003;169(5):1911--17
59. Cooney R, Hynes SO, Sharif F, et al.
Effect of gene delivery of NOS isoforms
on intimal hyperplasia and endothelial
regeneration after balloon injury.
Gene Ther 2007;14(5):396--404
60. Bivalacqua TJ, Usta MF, Champion HC,
et al. Effect of combination endothelial
nitric oxide synthase gene therapy and
sildenafil on erectile function in diabetic
rats. Int J Impot Res 2004;16(1):21--9
61. Garban H, Marquez D, Magee T, et al.
Cloning of rat and human inducible
penile nitric oxide synthase. Application
for gene therapy of erectile dysfunction.
Biol Reprod 1997;56(4):954--63
62. Chancellor MB, Tirney S, Mattes CE,
et al. Nitric oxide synthase gene transfer
for erectile dysfunction in a rat model.
BJU Int 2003;91(7):691--6
63. Fennell JP, Brosnan MJ, Frater AJ, et al.
Adenovirus-mediated overexpression of
extracellular superoxide dismutase
improves endothelial dysfunction in a rat
model of hypertension. Gene Ther
2002;9(2):110--17
64. Zanetti M, Sato J, Katusic ZS, et al.
Gene transfer of superoxide dismutase
isoforms reverses endothelial dysfunction
in diabetic rabbit aorta. Am J Physiol
Heart Circ Physiol
2001;280(6):H2516--23
65. Toque HA, Romero MJ, Tostes RC,
et al. p38 Mitogen-activated protein
kinase (MAPK) increases arginase activity
and contributes to endothelial
dysfunction in corpora cavernosa from
angiotensin-II-treated mice. J Sex Med
2010;7(12):3857--67
..
Important information in the
identification of novel signaling
pathway involved in hypertension-
induced ED.
66. Shukla N, Jones R, Persad R, et al.
Effect of sildenafil citrate and a nitric
oxide donating sildenafil derivative, NCX
911, on cavernosal relaxation and
superoxide formation in
hypercholesterolaemic rabbits.
Eur J Pharmacol 2005;517(3):224--31
67. Khan MA, Thompson CS, Emsley AM,
et al. The interaction of homocysteine
and copper markedly inhibits the
relaxation of rabbit corpus cavernosum:
new risk factors for angiopathic erectile
dysfunction? BJU Int 1999;84(6):720--4
68. Toque HA, Tostes RC, Yao L, et al.
Arginase II deletion increases corpora
cavernosa relaxation in diabetic mice.
J Sex Med 2011;8(3):722--33
. Valuable information on the role of
arginase in ED.
69. Li H, Horke S, Forstermann U.
Oxidative stress in vascular disease and
its pharmacological prevention.
Trends Pharmacol Sci
2013;34(6):313--19
70. Fukai T, Ushio-Fukai M. Superoxide
dismutases: role in redox signaling,
vascular function, and diseases.
Antioxid Redox Signal
2011;15(6):1583--606
71. Bivalacqua TJ, Armstrong JS,
Biggerstaff J, et al. Gene transfer of
extracellular SOD to the penis reduces
O2-* and improves erectile function in
aged rats. Am J Physiol
2003;284(4):H1408--21
72. Bivalacqua TJ, Usta MF, Kendirci M,
et al. Superoxide anion production in the
rat penis impairs erectile function in
diabetes: influence of in vivo extracellular
superoxide dismutase gene therapy.
J Sex Med
2005;2(2):187--97; discussion 97-8
73. Burchardt T, Burchardt M, Chen MW,
et al. Expression of VEGF splice variants
144/145 and 205/206 in adult male
tissues. IUBMB Life 1999;48(4):405--8
74. Lin CS, Lue TF. Growth factor therapy
and neuronal nitric oxide synthase. Int J
Impot Res 2004;16(Suppl 1):S38--9
75. Henry GD, Byrne R, Hunyh TT, et al.
Intracavernosal injections of vascular
endothelial gro h factor protects
endothelial dependent corpora cavernosal
smooth muscle relaxation in the
hypercholesterolemic rabbit:
a preliminary study. Int J Impot Res
2000;12(6):334--9
76. Gholami SS, Rogers R, Chang J, et al.
The effect of vascular endothelial growth
factor and adeno-associated virus
mediated brain derived neurotrophic
factor on neurogenic and vasculogenic
erectile dysfunction induced by
hyperlipidemia. J Urol
2003;169(4):1577--81
77. Rogers RS, Graziottin TM, Lin CS,
et al. Intracavernosal vascular endothelial
growth factor (VEGF) injection and
adeno-associated virus-mediated VEGF
gene therapy prevent and reverse
venogenic erectile dysfunction in rats.
Int J Impot Res 2003;15(1):26--37
78. Shirai M, Yamanaka M, Shiina H, et al.
Vascular endothelial growth factor
restores erectile function through
modulation of the insulin-like growth
factor system and sex hormone receptors
in diabetic rat. Biochem Biophys
Res Commun 2006;341(3):755--62
79. Yamanaka M, Shirai M, Shiina H, et al.
Vascular endothelial growth factor
restores erectile function through
inhibition of apoptosis in diabetic rat
penile crura. J Urol 2005;173(1):318--23
80. Ryu JK, Cho CH, Shin HY, et al.
Combined angiopoietin-1 and vascular
endothelial growth factor gene transfer
restores cavernous angiogenesis and
erectile function in a rat model of
hypercholesterolemia. Mol Ther
2006;13(4):705--15
81. Hedlund P, Aszodi A, Pfeifer A, et al.
Erectile dysfunction in cyclic GMP-
dependent kinase I-deficient mice.
Proc Natl Acad Sci USA
2000;97(5):2349--54
82. Chang S, Hypolite JA, Velez M, et al.
Downregulation of cGMP-dependent
protein kinase-1 activity in the corpus
cavernosum smooth muscle of diabetic
rabbits. Am J Physiol Regul Integr
Comp Physiol 2004;287(4):R950--60
83. Bivalacqua TJ, Kendirci M,
Champion HC, et al. Dysregulation of
cGMP-dependent protein kinase 1
(PKG-1) impairs erectile function in
diabetic rats: influence of in vivo gene
New approaches to the design and discovery of therapies to prevent erectile dysfunction
Expert Opin. Drug Discov. (2014) 9(12) 19
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o
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.

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o
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n
l
o
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d

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r
o
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i
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f
o
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a
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e
a
l
t
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c
a
r
e
.
c
o
m

b
y

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e
d
i
c
a
l

C
o
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e
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e

o
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o
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0
9
/
1
0
/
1
4
F
o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.
therapy of PKG1alpha. BJU Int
2007;99(6):1488--94
84. Bagnost T, Ma L, da Silva RF, et al.
Cardiovascular effects of arginase
inhibition in spontaneously hypertensive
rats with fully developed hypertension.
Cardiovasc Res 2010;87(3):569--77
85. Bivalacqua TJ, Hellstrom WJ,
Kadowitz PJ, et al. Increased expression
of arginase II in human diabetic corpus
cavernosum: in diabetic-associated erectile
dysfunction. Biochem Biophys
Res Commun 2001;283(4):923--7
.
Valuable information of excessive
arginase in diabetic cavernosal tissue
and its implication in ED.
86. Demougeot C, Prigent-Tessier A,
Bagnost T, et al. Time course of vascular
arginase expression and activity in
spontaneously hypertensive rats. Life Sci
2007;80(12):1128--34
87. Romero MJ, Platt DH, Tawfik HE,
et al. Diabetes-induced coronary vascular
dysfunction involves increased arginase
activity. Circ Res 2008;102(1):95--102
88. Bivalacqua TJ, Burnett AL,
Hellstrom WJ, et al. Overexpression of
arginase in the aged mouse penis impairs
erectile function and decreases eNOS
activity: influence of in vivo gene therapy
of anti-arginase. Am J Physiol Heart
Circ Physiol 2007;292(3):H1340--51
89. Gur S, Kadowitz PJ, Hellstrom WJ.
A review of current progress in gene and
stem cell therapy for erectile dysfunction.
Expert Opin Biol Ther
2008;8(10):1521--38
90. Grazette LP, Rosenzweig A. Role of
apoptosis in heart failure. Heart Fail Clin
2005;1(2):251--61
91. Vaegler M, Lenis AT, Daum L, et al.
Stem cell therapy for voiding and erectile
dysfunction. Nat Rev Urol
2012;Epub ahead of print
92. Strong TD, Gebska MA, Champion HC,
et al. Stem and endothelial progenitor
cells in erection biology. Int J Impot Res
2008;20(3):243--54
93. Wessells H, Williams SK. Endothelial
cell transplantation into the corpus
cavernosum: moving towards cell-based
gene therapy. J Urol 1999;162(6):2162--4
94. Song YS, Lee HJ, Park IH, et al.
Potential differentiation of human
mesenchymal stem cell transplanted in
rat corpus cavernosum toward endothelial
or smooth muscle cells. Int J Impot Res
2007;19(4):378--85
95. Kim SJ, Park SH, Sung YC, et al. Effect
of mesenchymal stem cells associated to
matrixen on the erectile function in the
rat model with bilateral cavernous nerve
crushing injury. Int Braz J Urol
2012;38(6):833--41
96. Deng W, Bivalacqua TJ,
Chattergoon NN, et al. Adenoviral gene
transfer of eNOS: high-level expression
in ex vivo expanded marrow stromal
cells. Am J Physiol Cell Physiol
2003;285(5):C1322--9
97. Abdel Aziz MT, El-Haggar S, Mostafa T,
et al. Effect of mesenchymal stem cell
penile transplantation on erectile
signaling of aged rats. Andrologia
2010;42(3):187--92
98. Qiu X, Sun C, Yu W, et al. Combined
strategy of mesenchymal stem cell
injection with vascular endothelial
growth factor gene therapy for the
treatment of diabetes-associated erectile
dysfunction. J Androl 2012;33(1):37--44
99. Canguven O, Burnett A. Cavernous
nerve injury using rodent animal models.
J Sex Med 2008;5(8):1776--85
100. Bahk JY, Jung JH, Han H, et al.
Treatment of diabetic impotence with
umbilical cord blood stem cell
intracavernosal transplant: preliminary
report of 7 cases. Exp Clin Transplant
2010;8(2):150--60
101. Cao Y, Sun Z, Liao L, et al. Human
adipose tissue-derived stem cells
differentiate into endothelial cells in vitro
and improve postnatal neovascularization
in vivo. Biochem Biophys Res Commun
2005;332(2):370--9
102. Ning H, Liu G, Lin G, et al. Fibroblast
growth factor 2 promotes endothelial
differentiation of adipose tissue-derived
stem cells. J Sex Med 2009;6(4):967--79
103. Bella AJ, Lin G, Cagiannos I, et al.
Emerging neuromodulatory molecules for
the treatment of neurogenic erectile
dysfunction caused by cavernous nerve
injury. Asian J Androl 2008;10(1):54--9
104. Garcia MM, Fandel TM, Lin G, et al.
Treatment of erectile dysfunction in the
obese type 2 diabetic ZDF rat with
adipose tissue-derived stem cells.
J Sex Med 2010;7(1 Pt 1):89--98
105. Zhou WW, Hu JG, Yang JF, et al.
Angiogenic effect of bone marrow
mesenchymal stem cells transfected with
human VEGF gene on myocardial
infarcts in rats. Zhong Nan Da Xue Xue
Bao Yi Xue Ban 2006;31(5):763--6; 71
106. Pons J, Huang Y, Arakawa-Hoyt J, et al.
VEGF improves survival of mesenchymal
stem cells in infarcted hearts.
Biochem Biophys Res Commun
2008;376(2):419--22
107. Liu G, Sun X, Bian J, et al. Correction
of diabetic erectile dysfunction with
adipose derived stem cells modified with
the vascular endothelial growth factor
gene in a rodent diabetic model.
PLoS One 2013;8(8):e72790
108. Jeong HH, Piao S, Ha JN, et al.
Combined therapeutic effect of udenafil
and adipose-derived stem cell (ADSC)/
brain-derived neurotrophic factor
(BDNF)-membrane system in a rat
model of cavernous nerve injury. Urology
2013;81(5):1108.e7--14
109. Traish AM, Goldstein I, Kim NN.
Testosterone and erectile function: from
basic research to a new clinical paradigm
for managing men with androgen
insufficiency and erectile dysfunction.
Eur Urol 2007;52(1):54--70
110. Bozkurt O, Bolat D, Demir O, et al.
Erectile function and late-onset
hypogonadism symptoms related to lower
urinary tract symptom severity in elderly
men. Asian J Androl 2013;15(6):785--9
111. Aversa A, Bruzziches R, Francomano D,
et al. Testosterone and phosphodiesterase
type-5 inhibitors: new strategy for
preventing endothelial damage in internal
and sexual medicine? Ther Adv Urol
2009;1(4):179--97
112. Filippi S, Vignozzi L, Morelli A, et al.
Testosterone partially ameliorates
metabolic profile and erectile
responsiveness to PDE5 inhibitors in an
animal model of male metabolic
syndrome. J Sex Med
2009;6(12):3274--88
113. Yassin AA, Haffejee M. Testosterone
depot injection in male hypogonadism:
a critical appraisal. Clin Interv Aging
2007;2(4):577--90
114. Jockenhovel F, Minnemann T,
Schubert M, et al. Comparison of long-
acting testosterone undecanoate
formulation versus testosterone enanthate
on sexual function and mood in
hypogonadal men. Eur J Endocrinol
2009;160(5):815--19
H. A. Toque & R. W. Caldwell
20 Expert Opin. Drug Discov. (2014) 9(12)
E
x
p
e
r
t

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p
i
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.

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r
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g

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i
s
c
o
v
.

D
o
w
n
l
o
a
d
e
d

f
r
o
m

i
n
f
o
r
m
a
h
e
a
l
t
h
c
a
r
e
.
c
o
m

b
y

M
e
d
i
c
a
l

C
o
l
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e
g
e

o
f

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o
r
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a

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0
9
/
1
0
/
1
4
F
o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.
115. Spitzer M, Bhasin S, Travison TG, et al.
Sildenafil increases serum testosterone
levels by a direct action on the testes.
Andrology 2013;1(6):913--18
116. Corona G, Maggi M. The role of
testosterone in erectile dysfunction.
Nat Rev Urol 2010;7(1):46--56
117. Hackett G, Cole N, Bhartia M, et al.
Testosterone replacement therapy with
long-acting testosterone undecanoate
improves sexual function and
quality-of-life parameters vs. placebo in a
population of men with type 2 diabetes.
J Sex Med 2013;10(6):1612--27
118. Hackett G, Cole N, Bhartia M, et al.
The response to testosterone undecanoate
in men with type 2 diabetes is dependent
on achieving threshold serum levels (the
BLAST study). Int J Clin Pract
2014;68(2):203--15
119. Pye SR, Huhtaniemi IT, Finn JD, et al.
Late-onset hypogonadism and mortality
in aging men. J Clin Endocrinol Metab
2014;99(4):1357--66
120. Pieper GM, Dondlinger LA. Plasma and
vascular tissue arginine are decreased in
diabetes: acute arginine supplementation
restores endothelium-dependent
relaxation by augmenting cGMP
production. J Pharmacol Exp Ther
1997;283(2):684--91
121. Hagenfeldt L, Dahlquist G, Persson B.
Plasma amino acids in relation to
metabolic control in insulin-dependent
diabetic children. Acta Paediatr Scand
1989;78(2):278--82
122. Jobgen W, Meininger CJ, Jobgen SC,
et al. Dietary L-arginine supplementation
reduces white fat gain and enhances
skeletal muscle and brown fat masses in
diet-induced obese rats. J Nutr
2009;139(2):230--7
123. Alam MA, Kauter K, Withers K, et al.
Chronic l-arginine treatment improves
metabolic, cardiovascular and liver
complications in diet-induced obesity in
rats. Food Funct 2013;4(1):83--91
124. Chin-Dusting JP, Willems L, Kaye DM.
L-arginine transporters in cardiovascular
disease: a novel therapeutic target.
Pharmacol Ther 2007;116(3):428--36
125. Morris SM Jr. Recent advances in
arginine metabolism: roles and regulation
of the arginases. Br J Pharmacol
2009;157(6):922--30
126. Romero MJ, Platt DH, Caldwell RB,
et al. Therapeutic use of citrulline in
cardiovascular disease.
Cardiovasc Drug Rev
2006;24(3-4):275--90
127. Schwedhelm E, Maas R, Freese R, et al.
Pharmacokinetic and pharmacodynamic
properties of oral L-citrulline and L-
arginine: impact on nitric oxide
metabolism. Br J Clin Pharmacol
2008;65(1):51--9
128. Hotta Y, Shiota A, Kataoka T, et al.
Oral l-citrulline supplementation
improves erectile function and penile
structure in castrated rats. Int J Urol
2014;21(6):608--12
129. Cormio L, De Siati M, Lorusso F, et al.
Oral L-citrulline supplementation
improves erection hardness in men with
mild erectile dysfunction. Urology
2011;77(1):119--22
130. Toque HA, Nunes KP, Yao L, et al.
Akita spontaneously type 1 diabetic mice
exhibit elevated vascular arginase and
impaired vascular endothelial and
nitrergic function. PLoS One
2013;8(8):e72277
131. Ghofrani HA, Osterloh IH,
Grimminger F. Sildenafil: from angina to
erectile dysfunction to pulmonary
hypertension and beyond. Nat Rev
Drug Discov 2006;5(8):689--702
132. Brioni JD, Nakane M, Hsieh GC, et al.
Activators of soluble guanylate cyclase for
the treatment of male erectile
dysfunction. Int J Impot Res
2002;14(1):8--14
133. Stasch JP, Hobbs AJ. NO-independent,
haem-dependent soluble guanylate cyclase
stimulators. Handb Exp pharmacol
2009(191):277--308
134. Bischoff E, Schramm M, Straub A, et al.
BAY 41-2272: a stimulator of soluble
guanylyl cyclase induces nitric oxide-
dependent penile erection in vivo.
Urology 2003;61(2):464--7
135. Claudino MA, da Silva FH, Monica FZ,
et al. Long-term oral treatment with BAY
41-2272 ameliorates impaired corpus
cavernosum relaxations in a nitric oxide-
deficient rat model. BJU Int
2011;108(1):116--22
136. Bischoff E, Stasch JP. Effects of the sGC
stimulator BAY 41-2272 are not
mediated by phosphodiesterase
5 inhibition. Circulation
2004;110(12):e320--1; author reply e-1
137. Teixeira CE, Priviero FB, Webb RC.
Effects of 5-cyclopropyl-2-[1-(2-fluoro-
benzyl)-1H-pyrazolo[3,4-b]pyridine-3-yl]
pyrimidin-4- ylamine (BAY 41-2272) on
smooth muscle tone, soluble guanylyl
cyclase activity, and NADPH oxidase
activity/expression in corpus cavernosum
from wild-type, neuronal, and
endothelial nitric-oxide synthase null
mice. J Pharmacol Exp Ther
2007;322(3):1093--102
138. Albersen M, Linsen L, Tinel H, et al.
Synergistic effects of BAY 60-4552 and
vardenafil on relaxation of corpus
cavernosum tissue of patients with
erectile dysfunction and clinical
phosphodiesterase type 5 inhibitor
failure. J Sex Med 2013;10(5):1268--77
139. Lasker GF, Pankey EA, Frink TJ, et al.
The sGC activator BAY 60-2770 has
potent erectile activity in the rat. Am J
Physiol Heart Circ Physiol
2013;304(12):H1670--9
140. Chitaley K, Webb RC, Mills TM.
RhoA/Rho-kinase: a novel player in the
regulation of penile erection. Int J
Impot Res 2001;13(2):67--72
141. Jin L, Ying Z, Hilgers RH, et al.
Increased RhoA/Rho-kinase signaling
mediates spontaneous tone in aorta from
angiotensin II-induced hypertensive rats.
J Pharmacol Exp Ther
2006;318(1):288--95
142. Zhou Q, Liao JK. Rho kinase:
an important mediator of atherosclerosis
and vascular disease. Curr Pharm Des
2009;15(27):3108--15
143. Hamid SA, Bower HS, Baxter GF. Rho
kinase activation plays a major role as a
mediator of irreversible injury in
reperfused myocardium. Am J Physiol
Heart Circ Physiol
2007;292(6):H2598--606
144. Rikitake Y, Kim HH, Huang Z, et al.
Inhibition of Rho kinase (ROCK) leads
to increased cerebral blood flow and
stroke protection. Stroke
2005;36(10):2251--7
145. Sato M, Tani E, Fujikawa H, et al.
Involvement of Rho-kinase-mediated
phosphorylation of myosin light chain in
enhancement of cerebral vasospasm.
Circ Res 2000;87(3):195--200
146. Higashi M, Shimokawa H, Hattori T,
et al. Long-term inhibition of Rho-kinase
suppresses angiotensin II-induced
cardiovascular hypertrophy in rats in
vivo: effect on endothelial NAD(P)H
oxidase system. Circ Res
2003;93(8):767--75
New approaches to the design and discovery of therapies to prevent erectile dysfunction
Expert Opin. Drug Discov. (2014) 9(12) 21
E
x
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i
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o
w
n
l
o
a
d
e
d

f
r
o
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i
n
f
o
r
m
a
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e
a
l
t
h
c
a
r
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.
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o
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b
y

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e
d
i
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C
o
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e
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o
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0
9
/
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0
/
1
4
F
o
r

p
e
r
s
o
n
a
l

u
s
e

o
n
l
y
.
147. Bao W, Hu E, Tao L, et al. Inhibition
of Rho-kinase protects the heart against
ischemia/reperfusion injury.
Cardiovasc Res 2004;61(3):548--58
148. Jin L, Liu T, Lagoda GA, et al. Elevated
RhoA/Rho-kinase activity in the aged rat
penis: mechanism for age-associated
erectile dysfunction. Faseb J
2006;20(3):536--8
149. Yao L, Chandra S, Toque HA, et al.
Prevention of diabetes-induced arginase
activation and vascular dysfunction by
Rho kinase (ROCK) knockout.
Cardiovasc Res 2013;97(3):509--19
150. Toque HA, Nunes KP, Yao L, et al.
Activated Rho kinase mediates diabetes-
induced elevation of vascular arginase
activation and contributes to impaired
corpora cavernosa relaxation: possible
involvement of p38 MAPK activation.
J Sex Med 2013;10(6):1502--15
..
Importance of Rho kinase and MAPK
signaling pathway in diabetes-
induced ED.
151. Rikitake Y, Liao JK. ROCKs as
therapeutic targets in cardiovascular
diseases. Expert Rev Cardiovasc Ther
2005;3(3):441--51
152. Mills TM, Chitaley K, Lewis RW, et al.
Nitric oxide inhibits RhoA/Rho-kinase
signaling to cause penile erection.
Eur J Pharmacol 2002;439(1-3):173--4
153. Wang H, Eto M, Steers WD, et al.
RhoA-mediated Ca2+ sensitization in
erectile function. J Biol Chem
2002;277(34):30614--21
154. Wingard CJ, Johnson JA, Holmes A,
et al. Improved erectile function after
Rho-kinase inhibition in a rat castrate
model of erectile dysfunction. Am J
Physiol Regul Integr Comp Physiol
2003;284(6):R1572--9
155. Bivalacqua TJ, Champion HC, Usta MF,
et al. RhoA/Rho-kinase suppresses
endothelial nitric oxide synthase in the
penis: a mechanism for diabetes-
associated erectile dysfunction. Proc Natl
Acad Sci USA 2004;101(24):9121--6
156. Rajasekaran M, White S, Baquir A, et al.
Rho-kinase inhibition improves erectile
function in aging male Brown-Norway
rats. J Androl 2005;26(2):182--8
157. Gao BH, Zhao ST, Meng FW, et al.
Y-27632 improves the erectile
dysfunction with ageing in SD rats
through adjusting the imbalance between
nNo and the Rho-kinase pathways.
Andrologia 2007;39(4):146--50
158. Park K, Kim SW, Rhu KS, et al.
Chronic administration of an oral Rho
kinase inhibitor prevents the
development of vasculogenic erectile
dysfunction in a rat model. J Sex Med
2006;3(6):996--1003
159. Chandra S, Romero MJ, Shatanawi A,
et al. Oxidative species increase arginase
activity in endothelial cells through the
RhoA/Rho kinase pathway.
Br J Pharmacol 2011;165(2):506--19
160. Shatanawi A, Romero MJ, Iddings JA,
et al. Angiotensin II-induced vascular
endothelial dysfunction through RhoA/
Rho kinase/p38 mitogen-activated
protein kinase/arginase pathway. Am J
Physiol Cell Physiol
2011;300(5):C1181--92
161. Ming XF, Barandier C,
Viswambharan H, et al. Thrombin
stimulates human endothelial arginase
enzymatic activity via RhoA/ROCK
pathway: implications for atherosclerotic
endothelial dysfunction. Circulation
2004;110(24):3708--14
162. Horowitz S, Binion DG, Nelson VM,
et al. Increased arginase activity and
endothelial dysfunction in human
inflammatory bowel disease. Am J
Physiol Gastrointest Liver Physiol
2007;292(5):G1323--36
163. Zhang J, Shen B, Lin A. Novel strategies
for inhibition of the p38 MAPK
pathway. Trends Pharmacol Sci
2007;28(6):286--95
164. Nangle MR, Cotter MA, Cameron NE.
Correction of nitrergic neurovascular
dysfunction in diabetic mouse corpus
cavernosum by p38 mitogen-activated
protein kinase inhibition. Int J
Impot Res 2006;18(3):258--63
165. Lysiak JJ, Nguyen QA, Kirby JL, et al.
Ischemia-reperfusion of the murine testis
stimulates the expression of
proinflammatory cytokines and activation
of c-jun N-terminal kinase in a pathway
to E-selectin expression. Biol Reprod
2003;69(1):202--10
166. Sommer F, Klotz T, Steinritz D, et al.
MAP kinase 1/2 (Erk 1/2) and serine/
threonine specific protein kinase Akt/
PKB expression and activity in the
human corpus cavernosum. Int J
Impot Res 2002;14(4):217--25
167. Nunes KP, Toque HA, Caldwell RB,
et al. Extracellular signal-regulated kinase
(ERK) inhibition decreases arginase
activity and improves corpora cavernosal
relaxation in streptozotocin (STZ)-
induced diabetic mice. J Sex Med
2011;8(12):3335--44
168. Bernier SG, Haldar S, Michel T.
Bradykinin-regulated interactions of the
mitogen-activated protein kinase pathway
with the endothelial nitric-oxide synthase.
J Biol Chem 2000;275(39):30707--15
169. Carneiro FS, Giachini FR, Lima VV,
et al. DOCA-salt treatment enhances
responses to endothelin-1 in murine
corpus cavernosum. Can J
Physiol Pharmacol 2008;86(6):320--8
170. Wu G, Morris SM Jr. Arginine
metabolism: nitric oxide and beyond.
Biochem J 1998;336(Pt 1):1--17
171. Bachetti T, Comini L, Francolini G,
et al. Arginase pathway in human
endothelial cells in pathophysiological
conditions. J Mol Cell Cardiol
2004;37(2):515--23
172. Mumenthaler SM, Yu H, Tze S, et al.
Expression of arginase II in prostate
cancer. Int J Oncol 2008;32(2):357--65
173. Lewis RW, Fugl-Meyer KS, Corona G,
et al. Definitions/epidemiology/risk
factors for sexual dysfunction. J Sex Med
2010;7(4 Pt 2):1598--607
174. El-Sakka AI. Erectile dysfunction,
depression, and ischemic heart disease:
does the existence of one component of
this triad necessitate inquiring the other
two? J Sex Med 2011;8(4):937--40
175. Berkowitz DE, White R, Li D, et al.
Arginase reciprocally regulates nitric
oxide synthase activity and contributes to
endothelial dysfunction in aging blood
vessels. Circulation 2003;108(16):2000--6
176. Kim JH, Bugaj LJ, Oh YJ, et al.
Arginase inhibition restores NOS
coupling and reverses endothelial
dysfunction and vascular stiffness in old
rats. J Appl Physiol (1985)
2009;107(4):1249--57
177. Numao N, Masuda H, Sakai Y, et al.
Roles of attenuated neuronal nitric-oxide
synthase protein expression and
accelerated arginase activity in impairing
neurogenic relaxation of corpus
cavernosum in aged rabbits. BJU Int
2007;99(6):1495--9
178. Segal R, Hannan JL, Liu X, et al.
Chronic oral administration of the
H. A. Toque & R. W. Caldwell
22 Expert Opin. Drug Discov. (2014) 9(12)
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y
.
arginase inhibitor 2(S)-amino-6-
boronohexanoic acid (ABH) improves
erectile function in aged rats. J Androl
2012;33(6):1169--75
179. Chitaley K. Type 1 and Type 2 diabetic-
erectile dysfunction: same diagnosis
(ICD-9), different disease? J Sex Med
2009;6(Suppl 3):262--8
180. Ponholzer A, Temml C, Mock K, et al.
Prevalence and risk factors for erectile
dysfunction in 2869 men using a
validated questionnaire. Eur Urol
2005;47(1):80--5; discussion 5-6
181. Gatti A, Mandosi E, Fallarino M, et al.
Metabolic syndrome and erectile
dysfunction among obese non-diabetic
subjects. J Endocrinol Invest
2009;32(6):542--5
182. Romero MJ, Iddings JA, Platt DH, et al.
Diabetes-induced vascular dysfunction
involves arginase I. Am J Physiol Heart
Circ Physiol 2012;302(1):H159--66
183. Elms SC, Toque HA, Rojas M, et al.
The role of arginase I in diabetes-
induced retinal vascular dysfunction in
mouse and rat models of diabetes.
Diabetologia 2013;56(3):654--62
184. Cox JD, Kim NN, Traish AM, et al.
Arginase-boronic acid complex highlights
a physiological role in erectile function.
Nat Struct Biol 1999;6(11):1043--7
185. Jelodar G, Razmi N, Gholampour V.
Arginase alteration in the reproductive
system of alloxan-diabetic dogs.
J Reprod Dev 2007;53(2):317--21
186. Luscher TF, Richard V, Tschudi M,
et al. Endothelial control of vascular tone
in large and small coronary arteries.
J Am Coll Cardiol 1990;15(3):519--27
187. Touyz RM, Schiffrin EL. Increased
generation of superoxide by angiotensin
II in smooth muscle cells from resistance
arteries of hypertensive patients: role of
phospholipase D-dependent NAD(P)H
oxidase-sensitive pathways. J Hypertens
2001;19(7):1245--54
188. Landmesser U, Dikalov S, Price SR,
et al. Oxidation of tetrahydrobiopterin
leads to uncoupling of endothelial cell
nitric oxide synthase in hypertension.
J Clin Invest 2003;111(8):1201--9
189. Lekakis JP, Papathanassiou S,
Papaioannou TG, et al. Oral L-arginine
improves endothelial dysfunction in
patients with essential hypertension.
Int J Cardiol 2002;86(2-3):317--23
190. Virag R, Bouilly P, Frydman D. Is
impotence an arterial disorder? A study
of arterial risk factors in 440 impotent
men. Lancet 1985;1(8422):181--4
191. Burchardt M, Burchardt T, Baer L, et al.
Hypertension is associated with severe
erectile dysfunction. J Urol
2000;164(4):1188--91
192. Mittawae B, El-Nashaar AR, Fouda A,
et al. Incidence of erectile dysfunction in
800 hypertensive patients: a multicenter
Egyptian national study. Urology
2006;67(3):575--8
193. Rodriguez S, Richert L, Berthelot A.
Increased arginase activity in aorta of
mineralocorticoid-salt hypertensive rats.
Clin Exp Hypertens 2000;22(1):75--85
194. Toque HA, Nunes KP, Rojas M, et al.
Arginase 1 mediates increased blood
pressure and contributes to vascular
endothelial dysfunction in
deoxycorticosterone acetate-salt
hypertension. Front Immunol
2013;4:219
195. Kifor I, Williams GH, Vickers MA, et al.
Tissue angiotensin II as a modulator of
erectile function. I. Angiotensin peptide
content, secretion and effects in the
corpus cavernosum. J Urol
1997;157(5):1920--5
196. Becker AJ, Uckert S, Stief CG, et al.
Possible role of bradykinin and
angiotensin II in the regulation of penile
erection and detumescence. Urology
2001;57(1):193--8
197. Imamura M, Waseda Y, Marinova GV,
et al. Alterations of NOS, arginase, and
DDAH protein expression in rabbit
cavernous tissue after administration of
cigarette smoke extract. Am J Physiol
Regul Integr Comp Physiol
2007;293(5):R2081--9
198. Kalsi JS, Ralph DJ, Thomas P, et al.
A nitric oxide-releasing PDE5 inhibitor
relaxes human corpus cavernosum in the
absence of endogenous nitric oxide.
J Sex Med 2005;2(1):53--7
Affiliation
Haroldo A Toque
†
PhD &
Robert William Caldwell
†
Author for correspondence
Georgia Regents University, Medical College of
Georgia, Department of Pharmacology and
Toxicology, 1459 Laney Walker Blvd, Augusta,
GA 30912-2308, USA
Tel: +1 706 721 6351;
Fax: +1 706 721 2347;
E-mail: [email protected]
New approaches to the design and discovery of therapies to prevent erectile dysfunction
Expert Opin. Drug Discov. (2014) 9(12) 23
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