Reactive oxygen species and inhibitors of inflammatory enzymes, NADPH oxidase, and iNOS in experimental models of Parkinson's disease.

Abstract

Reactive oxygen species (ROSs) are emerging as important players in the etiology of neurodegenerative disorders including Parkinson's disease (PD). Out of several ROS-generating systems, the inflammatory enzymes nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and inducible nitric oxide synthase (iNOS) were believed to play major roles. Mounting evidence suggests that activation of NADPH oxidase and the expression of iNOS are directly linked to the generation of highly reactive ROS which affects various cellular components and preferentially damage midbrain dopaminergic neurons in PD. Therefore, appropriate management or inhibition of ROS generated by these enzymes may represent a therapeutic target to reduce neuronal degeneration seen in PD. Here, we have summarized recently developed agents and patents claimed as inhibitors of NADPH oxidase and iNOS enzymes in experimental models of PD.

Full text

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Hindawi Publishing Corporation
Mediators of Inflammation
Volume 2012, Article ID 823902, 16 pages
doi:10.1155/2012/823902
Review Article
Reactive OxygenSpeciesandInhibitorsof Inflammatory
Enzymes,NADPH Oxidase,and iNOSinExperimentalModelsof
Parkinson’s Disease
SushrutaKoppula, HemantKumar, InSuKim, and Dong-KugChoi
Department of Biotechnology, Research Institute of Inflammatory Diseases, Konkuk University, Chungju 380-701, Republic of Korea
Correspondence should be addressed to Dong-Kug Choi, choidk@kku.ac.kr
Received 3 November 2011; Revised 23 December 2011; Accepted 9 January 2012
Academic Editor: Luc Valli` eres
Copyright © 2012 Sushruta Koppula et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Reactiveoxygenspecies(ROSs)areemergingasimportantplayersintheetiologyofneurodegenerativedisordersincludingParkin-
son’s disease (PD). Out of several ROS-generating systems, the inflammatory enzymes nicotinamide adenine dinucleotide phos-
phate(NADPH)oxidaseandinduciblenitricoxidesynthase(iNOS)werebelievedtoplaymajorroles.Mountingevidence suggests
that activation of NADPH oxidase and the expression of iNOS are directly linked to the generation of highly reactive ROS which
affectsvariouscellularcomponentsandpreferentiallydamagemidbraindopaminergicneuronsinPD.Therefore,appropriateman-
agement or inhibition of ROS generated by these enzymes may represent a therapeutic target to reduce neuronal degeneration
seen in PD. Here, we have summarized recently developed agents and patents claimed as inhibitors of NADPH oxidase and iNOS
enzymes in experimental models of PD.
1.Introduction
Brain inflammation may contribute to a wide variety of neu-
rodegenerative pathologies. Major regulators of brain in-
flammation that may exert a direct effect on neurons are
reactiveoxygenspecies(ROSs).ROSsareemergingasimpor-
tant players in the etiology of neurodegenerative disorders
[1]. Due to the reduced capacity of neuronal regeneration
and high metabolic rate, the brain is believed to be pro-
foundly liable to the damaging effects of ROS and the dopa-
minergic neurons in the substantia nigra of Parkinson’
disease (PD) patients are undoubtedly susceptible. Different
data sets suggest that oxidative stress is at the center of
various neurodegenerative diseases. Postmortem brain tis-
suesfrompatientswithneurodegenerativediseasesincluding
Alzheimer’s disease (AD), PD, Huntington’s disease (HD),
and amyotrophic lateral sclerosis (ALS) clearly show increa-
sedlevelsofROSinaffectedbrainregions[2–4].Thoughone
cannot assume that only ROSs are the major cause of these
disease states, it is necessary to question what is responsible
for this increased ROS generation.
ROSsareawiderangeofsmallsignalingmoleculeswhich
are highly reactive unpaired valence electrons. ROSs include
superoxide (O2−), hydrogen peroxide (H2O2), hydroxyl rad-
ical (OH•), and peroxynitrite (ONOO−) [5]. Although ROSs
have some biological advantages, excessive generation may
lead to threatened homeostasis of the biological system [6–
9]. ROSs are constantly generated through a variety of path-
ways,includingbothenzyme-catalyzedreactionsandnonen-
zyme reactions [10]. Whenever the balance between ROS
generationandthenaturalantioxidant defensesystemis lost,
a series of events may occur deregulating cellular functions
whichmayleadtovariouspathologicalconditionsforalmost
allvitalorgans[11].ROScanreactwithvitalcellcomponents
and alter intrinsic membrane properties like fluidity, ion
transport, loss of enzyme activity, protein cross-linking,
inhibition of protein synthesis, and DNA damage ultimately
resulting in cell death [12].
A more direct effect on neurons is the ROS produced
by the activation of the several inflammatory enzymes such
as nicotinamide adenine dinucleotide phosphate (NADPH)
oxidase, the expression of the inducible nitric oxide synthase

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2 Mediators of Inflammation
(iNOS), myeloperoxidase, lipoxygenase, and cyclooxygenase
(COX).Theseenzymescontributetothepathogenesisofvar-
ious neurodegenerative diseases including PD. Earlier stud-
ies postulated that NADPH oxidase and iNOS are not exp-
ressed in normal CNS conditions, but in PD patients and
in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)
intoxicated mice. Both are clearly expressed and activated in
glial cells in the ventral midbrain. These two major inflam-
matory enzymes that produce ROS may have a pathogenic
role in PD, because when they are lacking in mutant mice,
they show less loss of dopaminergic neurons [13–15].
In this paper, we discuss recent inhibitors of ROS-gene-
rating inflammatory oxidative enzymes, in particular the
NADPH oxidase and iNOS as a therapeutic strategy for the
treatmentofPD.Wealsosummarizeclaimsbyrecentpatents
for several agents as potential NADPH oxidase and iNOS
inhibitors.
2.Reactive OxygenSpeciesand PD
PD, the second most major neurodegenerative disease after
AD, is a pathological condition characterized by the degener-
ation of dopaminergic neurons in the substantia nigra pars
compacta and loss of striatal dopamine content [16, 17].
Although several pathogenic hypotheses have been proposed
for PD, oxidative stress via the generation of ROS is consi-
dered one of the major contributors. Evidence for the role of
ROS was first observed in human PD brains showing mito-
chondrial dysfunction and oxidative damage in the degene-
rating areas including the substantia nigra [18, 19]. Dopa-
mine is a relatively unstable molecule subject to hydroxyl
radical attack that can induce ROS damage both from within
and outside the cell [20]. ROS generated from mitochondrial
and/or extramitochondrial sources appear to be the main
contributor of oxidative stress-mediated neurodegeneration
in PD models [16, 21–27]. Importantly, generation of ROS
from toxicity induced by accumulation of MPP+in the inner
mitochondrial membrane, disruption of complex I in the
electron transport chain, and interaction of MPP+with iron
stores within the pigmented substantia nigra cells are the
known sources of oxidative stress [28–31].
Increasing evidence indicates that inflammatory-activa-
ted microglia and astrocytes are considered to be a consequ-
ence of neuronal cell death in AD, PD, HD ALS, and mul-
tiple sclerosis [32–36]. Activated microglia highly expresses
several enzymes including iNOS, NADPH oxidase, COX-2,
andmyeloperoxidaseresponsibleforinflammatoryprocesses
mediated by oxidative stress. These enzymes contribute to
the pathogenesis of various neurodegenerative diseases [13,
37–39]. ROS-generating multimeric enzymes are indispens-
able for protecting the host against infections and injuries.
Inappropriate activation of these enzymes may be harmful
in noninfectious neurodegenerative disorders [40, 41]. Thus,
the discovery of various novel agents that inhibit the activa-
tion of these enzymes may prove to be a therapeutic strategy
inamelioratingvariousneurodegenerativediseasesincluding
PD.
3.Role of NADPH-Oxidase-DerivedROS
SignalinginPD
Epidemiological studies suggest that inflammation increases
the risk of developing PD [42]. Several studies revealed that
a significant source of ROS generated during inflammation
was by NADPH oxidase. Nervous system cells contain the
NADPH oxidase complex, which, when assembled and acti-
vated, produces free radicals in abundance that can lead to
tissue damage [43]. NADPH oxidase which is composed of
gp91phox, p67phox, p47phox, p40phox, and p22phoxsubunits was
first studied because of its essential role in host defense
[44]. In resting brain cells, NADPH oxidase is inactive be-
cause p47phox, p67phox, and p40phox, which are present in
the cytosol as a complex, are separated from gp91phoxand
p22phox, which are transmembrane proteins. Upon activa-
tion, the p47phoxsubunit gets phosphorylated and translo-
cates to the membrane as a complex to assemble with
gp91phoxand p22phoxto form an active NADPH oxidase cap-
able of reducing oxygen to a superoxide radical (O2−) to
generate microglial and/or extramitochondrial-derived ROS
[45–48].
Brain regions that are rich in catecholamines, such as
adrenaline, noradrenaline, and dopamine, are also exceptio-
nally vulnerable to free radical generation. Catecholamines
can spontaneously break down to free radicals or be meta-
bolized to free radicals by endogenous enzymes such as
monoamine oxidases. Activated microglia also contribute
to the degeneration of dopaminergic neurons by releasing
neurotoxic factors such as NADPH-oxidase-derived super-
oxide and cytokines [49]. Activated microglia can produce
a host of toxic molecules including reactive nitrogen species
and ROS. Microglia in the vicinity of dopaminergic neurons
in disease appears to have an upregulated capacity for
ROS production due to increased expression of NADPH
oxidase. Release of aggregated and nitrated α-synuclein
from dying or damaged dopaminergic neurons in the SN
is thought to contribute, in part, to their activation [50,
51].
Additionally, neurons in the vicinity of activated micro-
glia may thus be exposed to NADPH-oxidase-derived O2−
and other secondary oxidants, such as H2O2. NADPH oxi-
dase can be quickly activated to elevate the level of ROS
within a few minutes after stimulation by a variety of growth
factors, such as cytokines and hormones including inter-
leukin (IL)-1 [52], platelet-derived growth factor (PDGF)
[53], or nerve growth factor (NGF) [54].
Several studies indicated that NADPH oxidase has been
linked to microglia-derived oxidative stress from a variety
of neurotoxic insults, such as rotenone [55], diesel exhaust
particles [56], α-synuclein [50], amyloid beta [57], para-
quat [58], dopamine neuronal injury [13, 59], and cerebral
ischemia-reperfusion injury [60], indicating that microglial
NADPH oxidase activation may also be a common denom-
inator of microglial activation associated with neurotoxi-
city.

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Mediators of Inflammation3
4.NADPH OxidaseInhibitorsand
ExperimentalModels of PD
It is well documented that NADPH oxidase is upregulated
in PD [13]. Reports reveal that NADPH-oxidase activation
plays a critical role in the degeneration of dopaminergic
neurons and inactivation of this enzyme may be a promising
target for PD treatment [13, 55, 61]. Reports also suggest
that microglial toxin, LPS-induced loss of nigral dopami-
nergic neurons in vivo and in vitro, was significantly less pro-
nouncedinNADPH-oxidase-deficientmice,whencompared
to normal control mice [62]. In the normal CNS, NADPH
oxidase is quiescent, but, in patients with PD and in MPTP-
intoxicated mice, NADPH oxidase is clearly expressed and
activated in glial cells in the ventral midbrain. Thus, agents
that inhibit NADPH oxidase activation may be ideal ther-
apeutic agents for the management of PD. Here, we sum-
marize a list of recently published and patented compounds
which act as NADPH-oxidase-derived ROS inhibitors in
experimental animal models focusing on PD pathology.
Ethyl pyruvate (EP, Figure 1(a)) is an effective scavenger
of ROS, especially hydrogen peroxide, by virtue of its non-
enzymatic oxidative decarboxylation reaction. EP has been
reported to exert pharmacological effects, such as scavenging
of ROS, suppression of inflammation, inhibition of apopto-
sis, and support of cellular ATP synthesis. Recently, EP has
been shown to rescue nigrostriatal dopaminergic neurons
by regulating glial activation in an MPTP intoxicated mouse
model of PD. A single injection of EP (10, 25, and
50mg/kgbodyweight) per day administered to mice into the
peritoneum at 12h after the last MPTP injection exerted
neuroprotection which was associated with suppression
of NADPH-oxidase-derived ROS production by activated
microglia [63].
Aminoethyl-benzenesulfonylfluoride
1(b)), an NADPH oxidase inhibitor, was reported to be use-
ful in ameliorating oxidative stress and apoptosis in mesen-
cephalic dopaminergic neuronal cells. AEBSF at 300μM
significantly blocked 1-methyl-4-phenylpyridinium ion-
(MPP+-) induced ROS production for over 45min in N27
cells in a dose-dependent manner and rescued the cells from
apoptotic cell death. The study supported that NADPH
oxidase play a critical role in the oxidative damage in PD
and inhibiting this enzyme activation by AEBSF may lead to
novel therapy for PD [61].
Apocynin (4-hydroxy-3-methoxyacetophenone, Figure
1(c)), a selective inhibitor of NADPH-oxidase, can block the
production of superoxide and oxygen-free radicals that typ-
ically accompany inflammation. Apocynin is a compound
originally isolated from the medicinal plant Picrorhiza kur-
roa, which inhibited both intracellular and extracellular ROS
production by interfering with the phagosomal association
of the cytosolic protein p47phox. Apocynin has been used to
prevent oxidative-stress-mediated cell damage in several dis-
ease models. Recently, apocynin has been proved to improve
neurological function and can be used as a neuroprotective
agent [64–68]. In the presence of microglial NADPH oxidase
activation in vitro, apocynin was able to reduce extracellular
ROS and cellular damage. Apocynin pretreatment (0.5mM
(AEBSF,Figure
for 30min) also attenuated the rotenone-induced release of
superoxides from activated microglia mediated by NADPH
oxidase [55].
In another study, exposure to paraquat (50μM), a herbi-
cide with a structure similar to the neurotoxin MPP+, has
been shown to produce PD-like symptoms and generated
ROS (including superoxide anions) in BV-2 cells. Paraquat-
inducedcytotoxicityinBV-2cellswasaccompaniedbytrans-
location of the p67phoxcytosolic subunit of NADPH oxidase
to the membrane. Paraquat-induced ROS production was
significantly inhibited by apocynin (1mM) [69]. Data from
the studies above indicated that apocynin may inhibit the
release of microglial NADPH-oxidase-mediated superoxide
in microglia-enhanced degeneration of dopaminergic neu-
rons.
Diphenyliodonium (DPI, Figure 1(d)) is a widely used
selective NADPH oxidase inhibitor which was documented
to act as a neuroprotective agent due to its potent anti-in-
flammatory properties mediated through the inhibition of
ROS. At very low concentrations, DPI inhibited both extra-
cellularsuperoxideandintracellularROSproductionindop-
aminergic neurons from LPS-induced degeneration and in
LPS-treated microglial activation [70]. In a report by Ma
andZhou,DPIat3–30μMconcentrationsinhibitedintrinsic
NADPH oxidase activity in N27 cells. Furthermore, DPI
blockedMPP+-inducedROSproduction.DPIalsopromoted
the survival of primary striatal neurons [71] and protected
against glutamate-induced apoptosis in dopaminergic SH-
SY5Y cells [72]. In addition, several in vivo studies demon-
strated that DPI delivered protection against global cerebral
ischemia [64], rotenone- [55], paraquat- [73], 6-OHDA-
[74],MPTP-[59],andIFN-γ/LPS-[75]induced dopaminer-
gic degeneration. In a recent study, Gao et al. [76] developed
a two-hit (neuroinflammation and mutant α-synuclein (α-
syn) overexpression) animal model to investigate mecha-
nisms through which mutant α-syn and inflammation work
inconcerttomediatechronicPDneurodegeneration.Results
revealed that LPS stimulation within the brain to transgenic
mice over expressing human A53T mutant α-syn developed
persistent neuroinflammation, chronic progressive degener-
ation of the nigrostriatal dopamine pathway, accumulation
of aggregated, nitrated α-syn, and formation of Lewy body-
like inclusions in nigral neurons. Continuous inhibition of
NADPH oxidase by 4-week infusion of DPI (5μg/kg/h)
blocked α-syn pathology and nigral neurodegeneration.
ThesestudiesprovideapotentialroleofDPIinthetreatment
of PD.
Dextromethorphan (DM, Figure 1(e)) is neuroprotective
through inhibition of microglial activation and NADPH
oxidase activation. Earlier reports reveal that DM protects
dopaminergic neurons against inflammation-mediated deg-
eneration induced by LPS, through inhibition of superoxide
radicals[77].Furthermore,Lietal.[78]reportedthatfemto-
(10−13and 10−14M) and micromolar (10−5to 10−7M) con-
centrations of DM (both pre- and posttreatment) showed
equal efficacy in protecting LPS-induced dopaminergic neu-
ron death in midbrain neuron-glia cultures. These stud-
ies indicated that the neuroprotective effect elicited by
femtomolar concentrations of DM is mediated through

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4 Mediators of Inflammation
the inhibition of LPS-induced proinflammatory mediators,
especially superoxide. In another study, DM was reported
to elicit neuroprotective effects in the MPTP-intoxicated PD
model in mice. DM significantly reduced the MPTP-induced
production of both extracellular superoxide free radicals and
intracellular ROS in both in vitro and in vivo experiments
[79].
Recently, Ramanathan et al. [80] revealed that DM in-
creased superoxide dismutase (SOD) and catalase, reduced
thiobarbituric acid reactive substances in the hippocampal
and striatal regions of monosodium glutamate-induced neu-
rodegeneration in rats, and improved neuroprotection based
on its antioxidant properties. Another recent report also
indicated that increased ROS production in activated BV-2
microglial cells by LPS was associated with increased exp-
ression of NADPH oxidase (NOX)-2, a subunit component
of NADPH oxidase and DM significantly suppressed the
upregulation of NOX-2 as well as subsequent ROS pro-
duction in activated BV-2 cells [81]. In light of this, DM
may form the potential therapeutic strategy for the treat-
ment of PD. The metabolite of DM, 3-hydroxymorphinan
(Figure 2(a)),alsoprotectedthenigrostriatalpathwayagainst
MPTP-elicited damage both in vivo and in vitro by reducing
MPTP-elicited reactive microgliosis as evidenced by the
decreased production of ROS due to its potent neuroprotec-
tion in PD [82].
Resveratrol(3,4,5-trihydroxy-trans-stilbene,Figure 2(b)),
a nonflavonoid polyphenol naturally found in red wine
and grapes, has been found to possess antioxidant, anti-
cancer, and anti-inflammatory properties. Recent results
from Zhang et al. [83] clearly demonstrated that resveratrol
pretreatment (15–60μM) for 30min stimulated with LPS
(10ng/mL) protected dopaminergic neurons against LPS-
induced neurotoxicity in concentration and time-dependent
manners through the inhibition of microglial activation and
the subsequent reduction in release of proinflammatory fac-
tors. The authors showed that resveratrol reduced NADPH-
oxidase-mediated generation of ROS and inhibited the LPS-
induced translocation of NADPH oxidase cytosolic subunit
p47phoxto the cell membrane. The most important finding is
that resveratrol failed to exhibit neuroprotection in cultures
from NADPH-oxidase-deficient mice. This data indicates
that NADPH oxidase may be a major player in resveratrol-
mediated neuroprotection in the models of PD.
The epigallocatechin (EGCG, Figure 2(c)), a catechin
polyphenol, was reported to reduce neuronal NADPH exp-
ression in rats exposed to acute hypoxia [84]. EGCG was
alsoreportedtoinhibitcytosolicsubunitsofNADPHoxidase
from translocating into the membrane, suggesting that inhi-
bition of NADPH oxidase activity may prevent oxidative
stress. In a recent report, the effects of EGCG on dichlorodi-
phenyltrichloroethane (DDT), a pesticide which is believed
to play a causative role in the etiology of PD, was studied.
It was found that EGCG concentration dependently (1μM,
3μM, and 10μM) reduced DDT-induced cell death in dopa-
minergic SH-SY5Y cells. Reports also indicated that EGCG
was capable of reducing dopaminergic neurotoxin 6-hyd-
roxydopamine-(6-OHDA-) induced cell death in SH-SY5Y
cells[85]andMPTP-inducedneurotoxicityinmice[86].The
O
O
O
H3C
CH2CH3
(a)
CH2 CH2 NH2
–
F
S
O
O
–
(b)
O
OCH3
OH
(c)
+
(d)
OCH3
H
N
H3C
(e)
Figure 1: Chemical structure of ethyl pyruvate (a), aminoethyl-
benzenesulfonylfluoride (b), apocynin (c), diphenyliodonium (d)
and dextromethorphan (e).
authors suggest that consumption of green tea, which con-
tains high concentrations of EGCG, may provide potential
prophylactic effects in reducing the risk of developing PD.
Few neuropeptides such as pituitary adenylate cyclase-
activating polypeptide (PACAP) 38, PACAP 27, and its inter-
nal peptide, Gly-Ile-Phe (GIF), were reported to be neuro-
protective at 10−13M against LPS-induced dopaminergic
neurotoxicity. PACAP is widely distributed in the peripheral
and central nervous system, where PACAP release is reported
to serve as a neuronal survival factor [87, 88]. PACAP is re-
ported to have diverse functions and has been shown to act
as a neurotransmitter/neuromodulator [89] and a neuro-
protectant [90, 91]. PACAP 38 and GIF also protected
against MPTP-induced neurotoxicity in animal models. The
polypeptides significantly ameliorated the production of
microglia-derived ROS, where both LPS- and phorbol 12-
myristate 13-acetate-induced superoxide and intracellular
ROS were inhibited. The study showed that PACAP38 and
GIF were neuroprotective only in normal cultures and not
in NADPH oxidase deficient cultures, proving the impor-
tant role of NADPH oxidase for GIF and PACAP 38s neuro-
protection [92].
The steroid hormone, 17β-estradiol (E2, Figure 2(d)), is
released into the blood where it can exert trophic or reg-
ulatory effects on many different target tissues such as the
breast, ovary, uterus, bone, and brain. Reports revealed that
E2 treatment (0.025mg; 14–21 day release via minipumps)
strongly attenuated the elevation of NADPH oxidase activity

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Mediators of Inflammation5
O
N
HO
(a)
HO
OH
OH
(b)
HO
O
OH
OH
HO
OH
OH
(c)
H
H
H
HO
OH
(d)
H3C
N
H
OCH3
O
OH
OCH3
(e)
Figure 2: Chemical structure of 3-hydroxymorphinan (a), resveratrol (b), epigallocatechin (c), estradiol 17-β (d), and sinomenine (e).
in the hippocampal CA1 region following cerebral ischemia
in brain, which correlated with its suppression of O2−levels
and its neuroprotective effect [93]. Moreover, E2 inhibited
activation of the GTPase, Rac1, in an Akt-dependent manner
following cerebral ischemia, which is critical for NOX-2 acti-
vation. Due to its neuroprotective effect and potent role in
inhibiting NADPH oxidase expression, E2 may be further
developed for the treatment of PD [94].
Transforming growth factor (TGF)-β1 is a pleiotropic
cytokinethatplaysacriticalroleinthecontrolofcellgrowth,
differentiation, inflammation, cell chemotaxis, apoptosis,
andhematopoiesis.StudieshaveshownthatTGF-β1canpro-
tectneuronsfromcelldeathinducedbyoxidativeinjury[95].
A recent report by Qian and Flood [96] revealed that the
neuroprotectiveeffectsofTGF-β1aremainlyattributedtoits
ability to inhibit the production of ROS from microglia dur-
ing their activation or reactivation. TGF-β1 inhibited LPS-
induced NADPH oxidase subunit p47phoxtranslocation from
the cytosol to the membrane in microglia, thereby exerting
potent anti-inflammatory and neuroprotective properties.
Sinomenine (Figure 2(e)), a natural dextrorotatory mor-
phinan analog, was reported to possess anti-inflammatory
and neuroprotective properties by the inhibition of micro-
glial NADPH oxidase. Sinomenine pretreatment for 30min
at micromolar (10−6–10−5M) and subpicomolar concentra-
tions (10−14–10−13M) showed equivalent efficacy in protec-
ting against dopaminergic neuron death in rat midbrain
neuron-glial cultures. Furthermore, sinomenine suppressed
LPS-induced extracellular ROSproduction via the inhibition
of NADPH cytosolic subunit p47phoxtranslocation to the
cell membrane. These findings strongly suggest that the
protective effects of sinomenine are most likely mediated
throughtheinhibition of microglial NADPHoxidase activity
[70].
N-[2-(4-hydroxy-phenyl)-ethyl]-2-(2,5-dimethoxy-phe-
nyl)-3-(3-methoxy-4-hydroxy-phenyl)-acrylamide
Figure 3(a)) is a squamosamide derivative reported to medi-
ate anti-inflammatory and neuroprotective effects in both
LPS and MPTP-intoxicated models of PD [97]. For in vivo
studies, FLZ (75mg/kg, p.o.) was administered 30min
before every MPTP injection (15mg/kg, s.c.) for 6 conse-
cutive days. For LPS (2ng/mL) stimulation, 10μM of FLZ
was pretreated for 1h. The neuroprotective effect of FLZ
was attributed to a reduction in LPS-induced microglial
production of proinflammatory factors such as superoxide,
tumor necrosis factor-α (TNF-α), nitric oxide (NO), and
prostaglandin E2 (PGE2). Findings from this study revealed
that the anti-inflammatory properties of FLZ were mediated
through inhibition of NADPH oxidase, the key microglial
superoxide-producing enzyme [97].
Phycocyanobilin (PCB, Figure 3(b)), a chromophore de-
rived from biliverdin, plays an essential light-harvesting role
in many blue-green algae and cyanobacteria. It constitutes
up to 1% of the dry weight of spirulina. Recently, it was
reported that C-phycocyanin administered orally (the spiru-
lina holoprotein that includes PCB) suppresses the neuro-
toxic impact of the excitotoxin kainite in rats, and a diet high
in spirulina ameliorates the loss of dopaminergic neurons
in the MPTP-induced Parkinsonian syndrome in mice. The
central physiological effects of PCB may also reflect inhibi-
tion of neuronal NADPH oxidase, which is known to have
a modulatory impact on neuron function, and can mediate
neurotoxicity in certain neurodegenerative diseases [98].
PCB has been shown to be a potent inhibitor of NADPH
(FLZ,

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6 Mediators of Inflammation
H3CO
HO
H
H3CO
OCH3
O
HN
OH
(a)
O
N
NH
COOHCOOH
H
N
O
N
(b)
H3C
H3C
CH3
O
O
H
H
HO
H
O
O
CH3
H3C
(c)
O
OHO
OHOH
H2N
O
HH
N
N
OH
(d)
Figure 3: Chemical structure of FLZ (a), phycocyanobilin (b), simvastatin (c), and minocycline (d).
oxidaseactivityinvarioushumancellculturesatmicromolar
concentrations. PCB may thus have versatile potential for
preserving the healthy function of the CNS in advanced
old age patients suffering from neuroinflammatory diseases
including PD.
In a recent study, Santiago et al. [99] investigated the
effect of simvastatin (Figure 3(c)) a commonly used, choles-
terol-lowering drug, in LPS and MPTP neurodegenerative
models to identify its neuroprotective effects for PD. The
study suggested that simvastatin (5mg/kgbodyweight, i.p.)
could prevent neurotoxic damage by LPS stimulation in mic-
roglial cells. Studies by Brenneman et al. [90] also indicated
that simvastatin is associated with a reduced incidence of
dementia and PD in elderly patients. Simvastatin treatment
(10μM) blocked the rac1-dependent activation of NADPH
oxidase and O2−production and significantly diminished
microglialCCchemokineligand5(amajorchemoattractant
of inflammatory cells) expression induced by interferon-β
alone or by a combination of interferon-β/TNF-α, thereby
exerting its suppressive effects on inflammation in the CNS
[100]. Furthermore, simvastatin inhibited NADPH oxidase
andtheproductionofROSinmicroglia[101].Arecentstudy
showed that simvastatin protects dopaminergic neurodegen-
eration in in vivo parkinsonian models [102]. Further, sim-
vastatinwasalsoreportedtoattenuatesuperoxidegeneration
by NADPH oxidase activation, protecting the endothelial
cell barrier [103]. All these data suggest the protective effect
of simvastatin against the degeneration of dopaminergic
neurons and may be developed as a promising drug to pro-
vide neuroprotection in PD.
Minocycline (Figure 3(d)), a well-known semisynthetic
tetracycline derivative, is neuroprotective in several animal
models of neurodegeneration, including PD [104, 105]. Stu-
dies have demonstrated that the neuroprotective actions of
minocycline are attributable to inhibition of microglial acti-
vation accompanied by oxidative stress. Choi et al. [106] re-
ported that minocycline (25 or 50mg/kg) exerted neuropro-
tection by significantly attenuating thrombin-induced neu-
rotoxic effects through inhibition of NADPH oxidase activa-
tion and ROS production from activated microglia.
Several patents for various categories of compounds have
been claimed for selectively inhibiting NADPH oxidase by
proving to be useful in the treatment and/or prevention of
inflammatory conditions in neurodegenerative diseases. Pat-
ented compounds published over the last five years for selec-
tively inhibiting NADPH oxidase were collectively described
in our earlier review [107]. The most recent relevant patents
showing a possible role in ameliorating neurodegenerative
diseases such as PD include the pyrazolo pyridine derivatives
[108], tetrahydroindole derivatives [109], imipramine blue
analogs [110], quinolone derivatives [111], and hesperidin
and hesperetin analogs [112]. These compounds were shown
to selectively inhibit and downregulate the expression of
NADPH oxidase by suppressing ROS generation, conse-
quently proving their importance as novel therapies in ame-
liorating neuroinflammatory degenerative diseases including
PD.

Page 7

Mediators of Inflammation7
H2N
COOH
S
N
H
NH
CH3
(a)
NH
H3C
H
N
H
NH2
COOH
(b)
NH
NH2
N
H
H2N
(c)
HO
OH
O
OH
OH
OH
O
(d)
Figure 4: Chemical structure of GW274150 (a), L-NIL (b), aminoguanidine (c), and quercetin (d).
5.Role of iNOS-DerivedROS SignalinginPD
Apart from the above-discussed NADPH-oxidase-derived
ROS systems, focus also points to nitrogen dioxide-derived
reactive species such as ONOO−, NO, and other unrecog-
nized potential reactive nitrogen species as the main culprits
[113]. Due to their highly unstable nature and reactivity,
biological molecules such as proteins, DNA, and lipids in
dopaminergic neurons in the brains of parkinsonian patients
could be targeted for oxidation resulting in extensive cellular
injury and cell death. Normally, inducible NO synthase
(iNOS) is not expressed in the brain, but in pathological
situations, especially those associated with gliosis, iNOS can
be induced.
In experimental PD models and MPTP neurotoxin-
induced models, induction of iNOS expression has been ob-
served[14,114].Earlierstudieshavealsosuggestedthatinhi-
bition of iNOS showed neuroprotection in the MPTP-indu-
ced PD model. In addition, inflammatory mediators such as
LPS and cytokines also cause an increase in iNOS expression
in microglia and astrocytes [115] and possibly in neurons
[116]. Once expressed, iNOS produces high levels of NO
continuously from microglia or astrocytes [117, 118].
Nitric oxide, a lipophilic diatomic molecule, can travel
several micrometers away from its site of production and
freely cross the plasma membrane to reach the intracellular
space of dopaminergic neurons. Also, the interaction of NO
and O2−will result in the formation of OONO−, a highly
reactive species. Peroxynitrite is a potent cytotoxic oxidant,
which in turn will inflict oxidative damage to biological tar-
gets such as inactivating ion channels, damaging DNA, and
nitrating tyrosine residues that can potentially inactivate
enzymes and disrupt signal transduction [119]. Therefore,
inhibition of glial activation-mediated oxidative stress by
reducing the iNOS may have therapeutic value in the treat-
ment of neuroinflammation related to PD. In the following
section, we describe recently available agents and patented
compounds that selectively inhibit iNOS activity and may
show a promising role in PD treatment.
6.InducibleNitric OxideSynthase Inhibitors
andExperimentalModels of PD
It is well documented that experimental PD models using
various toxins and MPTP-induced dopaminergic degenera-
tionexpresshighlevelsofiNOS[37,120].Recently,Broomet
al. [121] reported a selective iNOS inhibitor, GW274150 ([2-
[(1-iminoethyl) amino] ethyl]-L-homocysteine, Figure 4(a))
showing a potent role in the pathogenesis of PD. They indi-
cated that 6-OHDA administration produced an increased
number of cells expressing iNOS and was also associated
with increased microglial activation. GW274150 treatment
(3, 10, and 30mg/kg) orally twice daily for 7 consecutive
days leads to a suppression of iNOS expression and the
inflammatory response in the 6-OHDA model of PD and
was neuroprotective but at a narrow therapeutic range. The
findings from this paper support the concept that NO has
a detrimental effect on dopaminergic neurons and that
modulation of the inflammatory response may be a valid
neuroprotective therapeutic approach in treating PD.
Gahm et al. [122] reported that L-N-iminoethyl-lysine
(L-NIL, Figure 4(b)), a selective iNOS inhibitor, appeared
to protect the injured brain by limiting OONO−formation.
Their study analyzed a variety of parameters including neu-
ronal degeneration, survival, cellular apoptosis, and forma-
tion of nitrotyrosine following traumatic brain injury (TBI).
L-NIL significantly reduced iNOS activity in animals injured
by brain contusion. Moreover, neuronal degeneration and
NTimmunoreactivitysignificantlyreducedat24h.Neuronal
survival was unchanged at 24h but increased at 6 days in
L-NIL-treated animals. Cellular apoptosis of mononuclear
phagocytes (ED-1) and neuron (NeuN) positive cells were
significantly reduced following L-NIL treatment 6 days after

Page 8

8 Mediators of Inflammation
H3C
O
N
H
H2
C
OCH3
OH
CH2 CH CH2 OH
––
C
H2
7
–
(a)
H2N
N
NH2
NH2
(b)
HO
OCH3
OH
O
O
(c)
O
N
Se
(d)
Figure 5: Chemical structure of glyceryl nonivamide (a), agmatine (b), wogonin (c), and ebselen (d).
trauma. These findings also strongly support a putative
harmful role of iNOS induction early after TBI and the neu-
roprotectiveroleofL-NILinneurodegenerativediseasessuch
as PD.
The neuroprotective actions of aminoguanidine (AG,
Figure 4(c))havelongbeenstudied.Severalauthorsreported
that the selective inhibition of iNOS is one of the major
mechanisms by which AG exerts its neuroprotection [123–
125]. Cash et al. [126] reported the neuroprotective effects
of AG on transient focal ischemia in the rat brain. Lu et al.
[127] proposed the neuroprotective action of AG by com-
bined magnetic resonance imaging and histopathologic and
functional analysis after lateral fluid-percussive brain injury
in rats. The cerebroprotective effect of AG in a rodent model
of stroke was also studied [128]. In a recent report [129], the
effectiveness of AG was studied in modulating the toxicity
of aluminum chloride on the nitrite levels, malondialdehyde
concentration,reducedglutathionecontent,andcytochrome
c oxidase activity in Wistar rats and confirmed that the
inhibition of iNOS was responsible for this action. All these
studies have concluded that the potent inhibition of iNOS
activity was partly responsible for neuroprotection and AG
can be further developed in the prevention of various CNS
disorders including PD.
Quercetin (Figure 4(d)), a major flavonoid naturally
occurring in plants, deserves attention because of its benef-
icial effects observed in various in vitro and in vivo neural
damage models. Quercetin significantly exerted a neuropro-
tective effect through inhibition of the iNOS/NO system
and proinflammation gene expression in PC12 cells and in
Zebrafish [130]. The selective dopaminergic neurotoxin 6-
OHDA was used to induce neural damage in PC12 cells and
Zebrafish. Pretreatment with quercetin offered neuropro-
tection against 6-OHDA-induced PC12 apoptotic cell death
and dopaminergic neuronal loss in Zebrafish. A mechanis-
tic study revealed that quercetin could inhibit NO over-
production and iNOS overexpression in PC12 cells and
downregulates the overexpression of proinflammatory genes
suggesting that role of quercetin in neuroprotection leading
to its development as an effective therapeutic agent for the
treatment neurodegenerative diseases including PD.
The neuroprotective effects of glyceryl nonivamide
(GLNVA, Figure 5(a)), a vanilloid receptor (VR) agonist on
activated microglia and 6-OHDA-induced neurotoxicity in
dopaminergic SH-SY5Y cells were studied recently [131].
The authors revealed that GLNVA decreased LPS-activated
microglia-induced overexpression of neuronal nitric-oxide
synthase and gp91phoxon SH-SY5Y cells. GLNVA (1, 10,
100μM) for 24h diminished LPS-induced NO production,
overexpression of iNOS, and intracellular reactive oxygen
species in activated microglia. 6-OHDA-induced overexpres-
sionofnNOS,iNOS,COX-2,andgp91phoxwerealsoreduced
by GLNVA in SH-SY5Y cells. The neuroprotective effects
of GLNVA are mediated, at least in part, by decreasing
the inflammation- and oxidative-stress-associated factors
induced by microglia and 6-OHDA.
The neuroprotective effects of exogenous agmatine, a
guanidinium compound (Figure 5(b)), were investigated in
experimental spinal cord injury (SCI). Agmatine is a neuro-
transmitter—neuromodulator withboth N-methyl-d-aspar-
tate receptor (NMDAR)—antagonizing and NO synthase-
inhibiting activities. Agmatine administration following SCI
was shown to reduce NO levels significantly and suggested
that this drug may be helpful in the treatment of patients
with neurodegeneration especially in SCIs [132]. In a recent
study, the effects of agmatine on cell injury induced by
rotenone commonly used in establishing in vivo and in vitro
models of PD in a human-derived dopaminergic SH-SY5Y
celllinewereshown.Agmatinedose-dependentlysuppressed
rotenone-induced cellular injury through a reduction of oxi-
dative stress, by protecting dopaminergic neurons [133].
Wogonin(5,7-dihydroxy-8-methoxyflavone,Figure 5(c)),
an active component originated from the root of Scutel-
laria baicalensis, has been reported to possess antioxi-
dant and anti-inflammatory properties. Wogonin (5, 20,
50mM) inhibited inflammatory activation of cultured brain

Page 9

Mediators of Inflammation9
H3C
N
O
S
NH
O
O
(a)
O
O
O
H
H
N
H
F
(b)
H2N
N
A
N
N
H
R1
R11
(c)
X3
X4
X2
X5
A
R
O
O
X1
(d)
O
O
H
N
H
O
R1
OR2
H
O
O
OCH3
OH
(e)
Figure 6: Chemical structure of pioglitazone (a), paroxetine (b), imidazopyridine (c), coumarins (d), and theopederin derivatives (e).
microglia by diminishing LPS-induced NO production via
suppressing iNOS induction in microglia [134]. Further-
more, Chun et al. [135] reported the inhibitory activities of
wogoninderivativesonLPS-inducedNOproductioninBV-2
microglial cells and on H2O2-induced neuronal cell death in
SH-SY5Y cells. Wogonin and its derivatives ranging between
5, 10, 20, and 40μM concentration decreased the production
of NO and inflammatory cytokines owing to their potential
role in mitigating neuroinflammation seen in PD.
2-Phenyl-1,2-benzisoselenazole-3(2H)-one (ebselen), a
seleno organic compound and a strong ONOO−cleansing
agent (Figure 5(d)), possesses antioxidant and anti-inflam-
matory properties and is now under clinical trials for the
treatment of ischemic stroke. Earlier reports indicated that
ebselencanalsopreferentiallyinhibittheactivityofinducible
nitric oxide (NO) synthase within a certain concentration
range [136]. Ebselen prevented both neuronal loss and
clinical symptoms in a primate MPTP model of PD. Ebselen
(10μM) also prevented peroxide radical overproduction
induced by serum withdrawal in cultured PC12 cells and
hydroxyl radical generation induced by the mitochondrial
toxin, MPP+, in an in vivo system in the rat brain [137]. The
authors indicated that ebselen inhibited the free radical
production and may be useful as preventive treatment in
PD.
Pioglitazone, a PPARgamma agonist (Figure 6(a)), was
reported to protect mice from MPTP-induced dopaminergic
cell loss, glial activation, and loss of catecholamines in the
striatum. In addition, pioglitazone (10μM) provided neuro-
protective properties to substantia nigra dopaminergic
neurons in LPS-induced PD models both in vivo and
in vitro [138, 139]. In mice treated with pioglitazone, there
was reduced activation of microglia, reduced induction of
iNOS-positive cells and less glial fibrillary acidic protein
positive cells in both the striatum and substantia nigra pars
compacta. A comprehensive mechanistic study revealed that
pioglitazone-mediated neuroprotection involves inhibition
ofmicroglialactivationanddecreasedexpressionandactivity
of iNOS and may offer a treatment opportunity in PD to
slow the progression of disease that is mediated by neuro-
inflammation [138, 140].
Recently, the antidepressant paroxetine (Figure 6(b)) was
reported to promote the survival of nigrostriatal dopamin-
ergic neurons in the MPTP mouse model of PD. Treatment
with paroxetine (10mg/kgbodyweight, equivalent to 0.2–
0.25mg/day) into the peritoneum for 6 days, beginning at
12h after last MPTP injection, prevented the degeneration of
nigrostriatal DA neurons, increased striatal dopamine levels,
and improved motor function. The authors indicated that
the neuroprotection afforded by paroxetine may partly be
associated with the suppression of iNOS in activated mic-
roglia [141].
During the last five years, several novel compounds
that selectively inhibit iNOS expression have been patented.
Thomas et al. [142] claimed the usefulness of imidazopyri-
dinederivatives(Figure 6(c))asiNOSinhibitors.Theyfound
the novel 7-amino-3, 4, 5, 6, tetrahydro-2H-azepin-2-yl-
substituted imidazopyridine derivatives have valuable phar-
macological properties which make them commercially uti-
lizable. They are selective inhibitors of the enzyme iNOS. On
account of their iNOS-inhibiting properties, the compounds
according to the invention can be employed in human, vete-
rinary medicine, and therapeutics, where an excess of NO
or O2−is involved due to iNOS activation. They can be
used without limitation for the treatment and prophylaxis of
various neuroinflammatory and neurodegenerative diseases
including PD [142].

Page 10

10 Mediators of Inflammation
Dead neurons,
aggregated proteins,
inflammatory stimuli
(e.g LPS)
Activating factor from
apoptotic neuron
iNOS
L-NIL
GW
274150
AG
NO
Inflammatory
cytokine
(PGE2, IL-1β,
IFN-γ, TNF-α)
NADPH
oxidase
DPI
EGCG
DM
Activated microglia
NO
O2−
H2O2
ONOO−
H2O2
Protein
aggregation
NF-κB
Dead
genes
OH
Neuron
death
Neuron
NO
ONOO−
Protein adducts
Lipid preoxidation
DNA damage
ATP
depletion
Neurotoxin
e.g MPP+,
paraquat,
rotenone
ONOO−
Fe3+
Fe2+
O2−
Figure7:RoleofiNOSandNADPHoxidaseandtheirinhibitorsinoxidativestressandneuronaldamage.Reactiveoxygenspecies(ROS)can
be generated in distinct ways, for example, cell activation by neurotoxins (MPTP, paraquat, and rotenone), and mitochondrial dysfunction
andproteinaggregation.ROSshavearoleinoxidativestress,therebycausingneuronalinjuryorcelldeathwhichcanbeafactorformicroglial
activation. Several inhibitors of iNOS (L-N-iminoethyl-lysine (L-NIL), GW 274150, and aminoguanidine (AG)) and NADPH oxidase
inhibitors diphenyliodonium (DPI), epigallocatechin (EGCG), and dextromethorphan (DM) as shown inhibit the generation of nitric
oxide and superoxides, thereby inhibiting the formation of ROS and preventing neurodegeneration. Abbreviations: iNOS: inducible nitric
oxide synthase, LPS: lipopolysaccharides, NADPH oxidase: nicotinamide adenine dinucleotide phosphate-oxidase, NO: nitric oxide, O2
peroxy radical, ONOO−: peroxynitrite, NF-κB: nuclear factor kappa, H2O2: hydrogen peroxide, TNF-α: tumor necrosis factor-alpha, PGE2:
prostaglandin E2, IL-1β: interleukin-1 beta, and IFN-γ: interferon-gamma.
−:
A patent has also been obtained by Sharon et al. [143]
on a few novel coumarins (Figure 6(d)) for their use as
inhibitors of iNOS. Their study reveals that the compounds
synthesizedfromtheirinventioncanbeusedforvariousneu-
rodegenerative diseases including PD.
Recently, Tadayoshi et al. [144] filed a patent for a sense
oligonucleotide with sequence complementary to a single-
stranded RNA (antisense transcript) with sequence comple-
mentary to mRNA of the iNOS gene in order to control exp-
ression of iNOS. The authors reveal that the proposed inven-
tion can control the expression of iNOS and can be useful for
biological defense in ameliorating diseases related to exces-
sive production of NO, such as neuroinflammation.
Theopederin and derivatives (Figure 6(e)) are marine
natural substances isolated and purified from Porifera spe-
cies. In a recent patent, a pharmaceutical and health food
preparationcontainingtheopederinderivativesasactivecon-
stituentswasclaimedbyHeonjoongandHyun-Sil[145].The
authors claim that these derivatives selectively inhibit the
excessive generation of NO and iNOS activation and are use-
ful to treat and/or prevent various diseases including auto-
immune diseases, inflammatory diseases, multiple sclerosis,
and neurodegeneration.
There has also been a patent filed by Singh [146] on the
exemplary compound lovastatin, which is a sodium salt of
phenylacetic acid, FPT inhibitor II, N-acetyl cysteine, and
cyclic AMP, selectively inhibit the iNOS activation in LPS-
stimulated microglial cells, and can be used for the treatment
of various neuroinflammatory diseases.
The role of iNOS and NADPH oxidase in oxidative stress
and neuronal damage and the potential therapeutic strategy
of the discussed compounds were represented in Figure 7. In
view of the published reports and patents filed, inhibition of
iNOS and NADPH oxidase by various existing and emerging
molecules would be one of the ideal targets for the treatment
for PD.
7.Conclusion
The etiology of PD remains unknown, and the mechanisms
controlling the selective and progressive degeneration of the
nigrostriatal dopaminergic pathway are poorly understood.
Therapeutic intervention aimed at halting the degenerative
nature observed in PD is of prime importance and presents
some research opportunities. Studies of postmortem PD
brains and various cellular and animal models of PD in the
lasttwodecadesstronglysuggestthattheneuroinflammation
caused by oxidative stress is one of the major mechanisms in
PD pathology. It is believed that activated glial cells, which

Page 11

Mediators of Inflammation 11
comprisethemajorityofthisinflammatoryresponse,contri-
bute to neurodegeneration through the production of ROS.
Currently, ROS-generating oxidative enzymes are emerging
as major therapeutic targets to inhibit neurotoxicity seen in
PD.
A range of data during the past few years suggest that
anti-inflammatory agents with neuroprotective effects by
inhibiting inflammatory oxidative enzymes in experimental
models may be capable of preventing or reducing neuronal
degeneration and arrest the progression of PD. Although
several anti-inflammatory agents were known to inhibit
the ROS, none has been specific and the results achieved
are somewhat inconsistent and show limited mechanistic
relevance to PD. Thus, inhibitors of major ROS-generating
NADPH oxidase and iNOS that have a decent proven record
of safety and efficacy would be promising candidates. The
compounds discussed in this paper provide valuable infor-
mation in selectively inhibiting the ROS generated by oxida-
tive enzymes in in vitro and in vivo experimental models
of PD. These compounds must be proved for their safety,
selectivity, toxicity, bioavailability, therapeutic window, and
absenceofsignificant sideeffects.Furthermore,combination
therapy of selected agents in the correct time frame and dose
may also provide better results to achieve synergistic clinical
effects.
However, a complete understanding of the molecular
mechanisms of the specificities of ROS in PD, and larger
studies both epidemiologic and randomized clinical trials in
humans, as well as animal studies, are needed to validate
these findings in delivering beneficial effects in the treatment
of PD.
Acknowledgments
This work was financially supported by the Ministry of
Education, Science Technology (MEST) and Korea Indus-
trial Technology Foundation (KOTEF) through the Human
Resource Training Project for Regional Innovation and also
supported by Basic Science Research Program through the
National Research Foundation of Korea(NRF) funded by
the Ministry of Education, Science and Technology (2011-
0014923).
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