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Dl-3-n-Butylphthalide (NBP):A Promising Therapeutic Agent for Ischemic Stroke

Authors:

Abstract

Background and objective: Stroke is a leading cause of morbidity and mortality in both developed and developing countries all over the world. The only drug for ischemic stroke approved by FDA is recombinant tissue plasminogen activator (rtPA). However, only 2-5% stroke patients receive rtPAs treatment due to its strict therapeutic time window. As ischemic stroke is a complex disease involving multiple mechanisms, medications with multi-targets may be more powerful compared with single-target drugs. Dl-3-n-Butylphthalide (NBP) is a synthetic compound based on l-3-n- Butylphthalide that is isolated from seeds of Apium graveolens. The racemic 3-n-butylphthalide (dl- NBP) was approved by Food and Drug Administration of China for the treatment of ischemic stroke in 2002. A number of clinical studies indicated that NBP not only improved the symptoms of ischemic stroke, but also contributed to the long-term recovery. The potential mechanisms of NBP for ischemic stroke treatment may target different pathophysiological processes, including anti-oxidant, antiinflammation, anti-apoptosis, anti-thrombosis, and protection of mitochondria et al. Conclusion: In this review, we have summarized the research progress of NBP for the treatment of ischemic stroke during the past two decades.
CNS & Neurological Disorders - Drug Targets
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REVIEW ARTICLE
Dl-3-n-Butylphthalide (NBP): A Promising Therapeutic Agent for
Ischemic Stroke
Shan Wang1,#, Fei Ma1,#, Longjian Huang1, Yong Zhang1, Yuchen Peng1, Changhong Xing2,
Yipu Feng1, Xiaoliang Wang1,* and Ying Peng1,*
1State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chi-
nese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China; 2Departments of Radiol-
ogy and Neurology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA
A R T I C L E H I S T O R Y
Received: April 02, 2018
Revised: May 27, 2018
Accepted: June 08 , 2018
DOI:
10.2174/1871527317666180612125843
Abstract: Background and Objective: Stroke is a leading cause of morbidity and mortality in both
developed and developing countries all over the world. The only drug for ischemic stroke approved by
FDA is recombinant tissue plasminogen activator (rtPA). However, only 2-5% stroke patients receive
rtPAs treatment due to its strict therapeutic time window. As ischemic stroke is a complex disease in-
volving multiple mechanisms, medications with multi-targets may be more powerful compared with
single-target drugs. Dl-3-n-Butylphthalide (NBP) is a synthetic compound based on l-3-n-
Butylphthalide that is isolated from seeds of Apium graveolens. The racemic 3-n-butylphthalide (dl-
NBP) was approved by Food and Drug Administration of China for the treatment of ischemic stroke in
2002. A number of clinical studies indicated that NBP not only improved the symptoms of ischemic
stroke, but also contributed to the long-term recovery. Th e potential mech anisms of NBP fo r ischemic
stroke treatment may target different pathophysiological processes, including anti-oxidant, anti-
inflammation, anti-apoptosis, anti-thrombosis, and protection of mitochondria et al.
Conclusion: In this review, we have summarized the research progress of NBP for the treatment of
ischemic stroke during the past two decades.
Keywords: Dl-3-n-butylphthalide, ischemic stroke, cerebral microcirculation, neuroprotection, mitochondria, apoptosis, oxida-
tive stress.
1. INTRODUCTION
As one of the leading causes of morbidity and mortality,
stroke places a great deal of economic burden on patients,
their relatives as well as the entire society [1]. In China, ac-
cording to the World Bank Data, there will be about 31.77
million stroke patients by 2030 with the cost of as much as
$40.0 billion per year [2]. Approximately 87% of strokes are
ischemic in nature [3]. Recombinant tissue plasminogen
activator (rtPA), a thrombolytic that restores blood flow to
the ischemic brain, remains to be the only specific medica-
tion approved by the FDA for clinical management in acute
ischemic stroke (AIS). Unfortunately, due to its limited
*Address correspondence to this author at the Pharmacology Department,
Institute of Materia Medica, Chinese Academy of Medical Sciences & Pe-
king Union Medical College, No.1, Xiannongtan Street, Xicheng District,
Beijing 100050, China; Tel: +86-10-63165173; Fax: +86-10-63017757;
E-mail: ypeng@imm.ac.cn and Tel: +86-10-63165330; Fax: +86-10-
63165330; E-mail: wangxl@imm.ac.cn
#Shan Wang and Fei Ma contributed equally to this work.
therapeutic time window, rtPA is only provided to a minority
of patients (2%-5%) [4]. Although scientists attempted to
develop novel medications for ischemic stroke, a large num-
ber of clinical trials failed, including thrombolytic agents,
antiplatelet agents, anticoagulants, modulators of excitatory
amino acids, modulators of calcium influx, metabolic activa-
tors, anti-edema agents, inhibitors of leukocyte adhesion,
free radical scavengers, promoters of membrane repair and
so on [2, 5].
The neurovascular unit is not only an anatomical con-
struct but also serves as a functional unit for the interactions
between neurons, glial cells and blood vessels [6]. The con-
cept of neurovascular unit has been used not only in the in-
vestigation of acute stroke pathophysiology, but also ex-
tended to dissect the delayed phases of stroke recovery. In
addition, this conception emphasized that the brain injury of
ischemic stroke should be regarded as an integral structure to
be protected, and neuroprotectants with multi-targets might
be a promising option for ischemic stroke. In this context, an
ideal therapeutic target for stroke would be one that rescues
1996-3181/18 $58.00+.00 © 2018 Bentham Science Publishers
NBP, A Promising Therapeutic Agent for Ischemic Stroke CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 339
neurovascular signaling and is active in multiple brain cell
types. However, some researchers believe that the first step
of neuroprotective strategy should be revascularization, in
which case, neuroprotectants could enter the salvageable
tissues and then exert their neuroprotective effects more effi-
ciently. Briefly, for the treatment of ischemic stroke,
neuroprotective strategy should be comprehensive which
includes revascularization, using neuroprotectants, and the
long-term recovery of neurofunction [7]. Dl-3-n-
Butylphthalide (NBP) may be one of these candidates.
NBP is a synthetic chiral compound based on l-3-n-
butylphthalide which is originally isolated from seeds of
Apium graveolens. Its chemical name is ()3-butyl-3H-2-
benzonfuran-1-one (chemical formula: C12H14O2), and mo-
lecular weight is 190.24 (Fig. 1). NBP is a colorless or light
yellow viscous liquid with a celery odor. In 1995, NBP was
first time used to treat ischemic stroke by Feng et al. [8]. In
the preclinical studies, NBP showed protective effects
against ischemic stroke in various animal models [9-15].
Treatment with NBP (20 mg·kg-1, i.p.) following 15 minutes
of middle cerebral artery occlusion (MCAO) in rat models
significantly reduced infarct volume by 87% below the con-
trol value and improved neurological deficits [13]. Similar
effects were also obtained even administrating with NBP (80
mg·kg-1, p.o., b.i.d.) after 2 hours of MCAO in mouse mod-
els as well as stroke-prone spontaneously hypertensive rat
models [10, 14]. The underlying mechanisms of protective
action of NBP might be involved in increasing regional
blood flow [9, 16], ameliorating brain edema and blood-
brain barrier damage [11], reducing the chance of thrombosis
[17, 18], improving microcirculation in arterioles [10],
improving energy metabolism [8], decreasing oxidative
damage [19], preventing the inflammatory response [20], and
reducing neuronal apoptosis [21, 22]. In this paper, the
pharmacokinetics, clinical trial and pharmacological
mechanisms of NBP on ischemic stroke treatment would be
reviewed.
CH2CH2CH2CH3
O
dl-NBP
O
Fig. (1). Structure of dl-NBP.
2. PHARMACOKINETICS AND CLINICAL TRAILS
Pharmacokinetics studies in human revealed that the peak
plasma concentration of NBP was achieved at approximately
0.75 hours after an oral administration of 200 mg NBP
(t.i.d.) soft capsules, with mean value of 514 ng·ml-1. In addi-
tion, the average AUC0-∞ value of NBP was 864 ng·h·ml-1,
and the mean t1/2 is 5.33 hours [23]. After being absorbed,
NBP primarily undergoes hydroxylation on the n-butyl side
chain and C-3, and forms four principal metabolites, includ-
ing 10-keto-NBP (M2), 3-hydroxy-NBP (M3-1), 10-
hydroxy-NBP (M3-2), and NBP-11-oic acid (M5-2). No
parent drug of NBP was detected in the urine or feces, and
the urinary metabolites accounted for about 81.6% of the
administrated dosage [23-25]. Actually, while NBP is effec-
tive, out of hydrophobicity, its bioavailability being as low
as 15%, which to some extent, limits the application of NBP
capsule [26]. Given this problem, potassium 2-(1-
hydroxypentyl)-benzoate (PHPB), a pro-drug of NBP, which
could entirely change into NBP in the body, has been devel-
oped. With extremely high solubility in water, PHPB has
better bioavailability than NBP [27]. Currently, PHPB is in
phase II-III clinical studies in China, and its clinical applica-
tion might be achieved in the near future. Safety studies in
human showed that the incidence of serious and non-serious
adverse events was similar between the NBP (25 mg, i.v.,
b.i.d. or, 200 mg, p.o., b.i.d.) treated group and placebo
group [24, 26] except that NBP had mild hepatotoxicity [23].
In different stages of clinical trials (II, III, IV), the incidence
of the elevated alanine aminotransferase of NBP in patients
ranged from 3.0% to 17.5% [26, 28, 29]. However, this ef-
fect was recoverable and it has been suggested that the ele-
vated alanine aminotransferase might be dose-related [26].
As a result, during the administration period of NBP, it is
necessary to regularly monitor the liver function.
The randomized, double-blinded, placebo-control, multi-
center clinical trials suggested that NBP might be a safe and
effective treatment for ischemic stroke [24, 26, 30]. In a
clinical trial, 573 patients were administrated with NBP (25
mg, i.v., b.i.d. or 200 mg, p.o., t.i.d.) within 48 hours of onset
of ischemic stroke for 90 days. According to the measure-
ments of modified Rankin scale, NBP might be more effec-
tive and safer than thromboxane A2 (TXA2) synthase inhibi-
tor ozagrel and aspirin, especially for those patients with
moderate severity of ischemic stroke [26]. In another trial
with an enrollment of 60 patients within 12 h of AIS, a sig-
nificant improvement of National Institutes of Health Stroke
Scale (NIHSS) scores at 21-day after stroke onset was ob-
served in NBP-treated group compared with Cerebrolysin
[24]. NBP (200 mg, p.o., t.i.d.) also elevated the number of
circulating endothelial progenitor cells in a study including
170 patients with AIS [30]. Furthermore, NBP seemed to
improve the long-term recovery of stroke-related disability,
such as walking, limb motor, language, sensor, thinking and
memory [26]. With the accumulating evidences, NBP has
been approved as a novel drug for ischemic stroke by the State
Food and Drug Administration of China since 2002. Up to
now, several clinical trials of NBP are ongoing on Cere-
brovascular Occlusion (NCT02594995), AIS (NCT02905565
and NCT02149875), Vascular Cognitive Impairment no De-
mentia (NCT02993367), AD (NCT02711683), and Restenosis
(NCT01405248). The phase II clinical trial of NBP softgel
capsules for ischemic stroke patients began in the United
State (NCT02905565) in 2017.
3. IMPROVE CEREBRAL MICROCIRCULATION
The cerebral circulatory system plays a vital part in keep-
ing an optimal cerebral blood flow (CBF) for the brain’s needs
of nutrients and oxygen [31]. Ischemic stroke is characterized
by luminal obstruction and reduced blood flow to the brain
[32]. Hence, restoring reperfusion via thrombolysis, intravas-
cular clot removal, or vasodilation is associated with an im-
proved outcome. NBP might be an effective drug to increase
CBF.
340 CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 Wang et al.
3.1. Improve Vasodilation
Administration with NBP (10 mg·kg-1, i.p.) after 10 min-
utes of MCAO in rat models, the regional CBF of NBP
groups were significantly increased by 107%, 211% and
370% above the control value respectively after 60, 120 and
180 minutes of MCAO [33]. The action of NBP on regional
CBF was mainly related to the vasodilation induced by ele-
vation of nitric oxide (NO), a strong vasodilator generated by
nitric oxide synthase (NOS) in endothelial cells, neurons and
glial cells. NBP (1 or 10 μmolL-1) significantly increased
the activity of NOS and the production of extracellular NO
in bovine aortic endothelial cells and bovine cerebral endo-
thelial cells, which might improve the cerebral microcircula-
tion and restore the supply of oxygen and nutrients to
ischemic hemisphere [34].
3.2. Inhibit Platelet Aggregation and Thrombosis Forma-
tion
After ischemia, a great deal of arachidonic acid (AA) is
produced by phospholipid decomposing by PLA2 and me-
tabolized into Prostaglandin I2 (PGI2) and TXA2 by cy-
clooxygenase. PGI2 is a vasodilator with the effect of anti-
platelet aggregation, while TXA2 can activate the aggrega-
tion of platelet and induce vasoconstriction. The disturbance
of the balance between PGI2 and TXA2 is related to some
pathological process, such as thrombogenesis and angio-
spasm [35]. After 5 minutes and 120 minutes of MCAO in
rats, treatment with NBP (10 or 20 mg·kg-1, i.p.) could down-
regulate the expression of PLA2 and depress the release of
AA in the cerebral cortex by about 60% below the control
value [36]. Moreover, post-treatment with NBP (10 or 20
mg·kg-1, i.p.) markedly elevated the ratio of PGI2/TXA2 after
reperfusion in rats, which might have beneficial effects on
the impaired microcirculation in post-ischemic brain tissues
[18]. In addition, NBP was a potent antiplatelet drug. Pre-
incubated with 300 μmolL-1 NBP could prevent the aggre-
gation of the platelets via inhibiting TXA2 synthesis and in-
creasing the level of cAMP in a dose-dependent manner in
human platelets [17]. Apart from that, experiments in rats
also confirmed the antithrombotic and antiplatelet activity of
NBP, and the decrease of 5-HT release from platelets might
be involved in this effect [37].
3.3. Regulate Angiogenesis
Vascular endothelial growth factor (VEGF) is the most
potent factor that stimulates angiogenesis and vasculogenesis
[38]. Besides VEGF, the basic fibroblast growth factor
(bFGF) and transforming growth factor-β1 (TGF-β1) are
also important angiogenic factors [39-41]. In a cold-induced
ischemic stroke model of stroke-prone renovascular hyper-
tensive rats, pretreatment with NBP (80 mg·kg-1, p.o.) mark-
edly reduced the attack incidence and the infarct volume and
increased the perfused microvessels [10]. NBP significantly
up-regulated the expressions of VEGF and bFGF in the hip-
pocampus andVEGF and TGF-β1 levels in the peri-
infarcted area of different models [42-44]. Lu et al. found
that NBP-induced in vitro angiogenesis was mediated by the
up-regulation of FGF-2 expression and the activations of
both ERK1/2 and PI3K/Akt-eNOS signaling pathways [45].
All these findings demonstrated that NBP might improve
angiogenesis after cerebral ischemia.
4. PROTECT BLOOD BRAIN BARRIER
The blood brain barrier (BBB) is an essential factor in the
homeostatic regulation of the brain microenvironment [46].
Structurally, the BBB comprises endothelium and surrounding
astrocytes, pericytes and perivascular microglia [47]. BBB is
a highly selective permeability barrier which controls the
flux of some molecules between the blood and central nerve
system. After cerebral ischemia, the function of all
constituents of the BBB, including endothelial cells, astrocytes
and pericytes, was influenced [48]. The pinocytotic vesicles in
the endothelial cells were increased and the tight junction
proteins were deranged, which enhanced the permeability of
BBB and damaged its integrity [49].
NBP reduced IgG extravasation after focal cerebral
ischemia in a dose-dependent manner. Furthermore, ultra-
structure damage of capillaries and brain edema were re-
markably alleviated after post-treatment with NBP in a
reperfusion-induced BBB damage in rats (10 or 20 mg·kg-1,
i.p.) [11]. Additionally, NBP (10 μmolL-1) could protect
endothelial cells against oxidative/nitrosative stress, mito-
chondrial damage and subsequent cell death triggered by
oxygen glucose deprivation (OGD) [50]. It has been well
demonstrated that activation of Rho A led to the increase of
BBB permeability and cerebral edema [51]. NBP (40 mg kg-1,
p.o.) inhibited Rho A protein expression in the cerebral
cortex around focal cerebral infarction, consequently reduced
BBB damage and cerebral edema in rats after focal cerebral
infarction [52]. One latest study revealed the protective
effect of post-treatment with NBP (60 mg kg-1, p.o.) on the
structural integrity and the functional stability of BBB in rats
exposed to CO through the increases of Zonula occludens-1
(ZO-1) and claudin-5, two important molecules in tight
junction complexes [53].
5. IMPROVE MITOCHONDRIAL FUNCTIONS
The primary physiological function of mitochondria is to
generate adenosine triphosphate (ATP) through oxidative
phosphorylation by the electron transport chain [54], so mi-
tochondria is a major target in hypoxic or ischemic injury
[55]. NBP has been shown to improve mitochondrial func-
tions through several different aspects.
5.1. Increase Mitochondrial Complexes
Most cellular energy is produced through oxidative phos-
phorylation, a process catalyzed by a series of respiratory
enzyme complexes (complexes I–V) located in the inner
membrane of mitochondria. Xiong et al. found that complex
IV suffered a sharp decrease from 0.167 mmolkg-1min-1 to
0.09 mmolkg-1min-1 following one hour of MCAO in rats
and came back to the normal level after reperfusion for three
hours. The reduction of complex IV triggered by ischemia
could be markedly attenuated in the presence of NBP (5 mg
kg-1 or 10 mg kg-1, i.p., 10 minutes before ischemia) [56]. In
primary cultured neurons, exposure to 1 μmolL-1 NBP could
completely avert the disruption of complex IV induced by 6-
hour hypoglycemia and hypoxia [56]. In addition, the levels
NBP, A Promising Therapeutic Agent for Ischemic Stroke CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 341
of phosphocreatine and ATP were efficiently elevated by
1.5- and 2-fold after NBP (150 mg kg-1 or 200 mg kg-1, s.c.)
treatment in complete brain ischemia mouse model [8].
Hence, we inferred that NBP might directly act on mito-
chondria to upregulate the activity of respiratory enzyme
complexes and the synthesis of intracellular energy.
5.2. Protect the Structure and Function of Mitochondrial
Membrane
Two major factors in the physiologic function of mito-
chondriamitochondrial membrane potential (MMP) and
mitochondrial membrane fluidity (MMfu), were impaired by
oxygen-glucose deprivation in both human umbilical vein
endothelial cells and primary cultured neurons. However, 10
μmolL-1 NBP effectively suppressed OGD-induced HIF-1α
enhancement [50]. Xiong et al. had also demonstrated that
NBP (5 mg kg-1 or 10 mg kg-1, i.p., 10 minutes before
ischemia) could inhibit the decrease of MMfu in MCAO rats,
and NBP reversed the augment of microviscosity of mito-
chondrial membrane. Additionally, NBP could also prevent
mitochondria from swelling, fracturing as well as vacuolat-
ing [57].
5.3. Elevate Mitochondrial ATPase Activity
ATPase plays an important role in intracellular ionic ho-
meostasis and would be strongly perturbed by ischemia. The
activities of mitochondrial Ca2+-ATPase, Na+, K+-ATPase
and Mg2+-ATPase were sharply cut down due to cerebral
ischemia in rats, while NBP (5 mg kg-1 or 10 mg kg-1 or 20
mg kg-1, i.p., 10 minutes before ischemia) effectively ele-
vated the activities of Ca2+-ATPase and Na+, K+-ATPase
compared to the vehicle group respectively [19]. This effect
was confirmed by in vitro study that NBP (0.1 μmolL-1)
could maintain the level of ATPase in primary cultured neu-
rons subjected to OGD [57].
6. INHIBIT OXIDATIVE STRESS
Reactive oxygen species (ROS) are essential signaling
molecules that play a pivotal role in maintaining physiologi-
cal cell functions. ROS can regulate the activities of protein
kinase pathways including receptor tyrosine kinases, protein
kinase C (PKC), and mitogen-activated protein kinases
(MAPK) as well as key transcription factors such as activator
protein-1 (AP-1) and nuclear factor-kappa-B (NF-kB) [58].
In the pathophysiology of cerebral ischemia, oxidative stress
initiates a series of complicated biochemical cascades and
finally accelerates the malignant progression of ischemic
stroke [59]. ROS are excessively produced during cerebral
ischemia or reperfusion, and oxidize cellular components
like lipids, proteins and DNA resulting in cellular damage
and death [60, 61]. Increased production of ROS results in
dysfunction of mitochondria, including the breakup of mito-
chondrial inner membrane integrity, the mitochondrial depo-
larization, the inhibition of mitochondrial electron transfer
chain, the opening of the mitochondrial permeability transi-
tion pore, and disruption of the intracellular calcium homeo-
stasis [62-66]. Besides, ROS itself also triggers the apoptotic
signaling pathway following ischemic damage [67, 68]. Mul-
tiple antioxidant effects of NBP after ischemia have been
found.
6.1. Enhance Anti-oxidant Enzymes Activities
Physiologically, ROS are continuously developed, but
are balanced by endogenous antioxidant defense mecha-
nisms. The homeostasis would be ruined by brain ischemia.
The primary sources of ROS in the setting of cerebral ische-
mia/reperfusion are the mitochondrial respiratory chain,
NAPDH oxidases, and xanthine oxidase (XO) [69]. In a
four-vessel occlusion model of rats, NBP (20 or 40 mg.kg-1,
i.p., 20 min before ischemia) dose-dependently inhibited the
accumulation of purine metabolites including hypoxanthine,
the substrates of XO, which showed the most dramatic de-
creasing from the peak 5.41 to 1.24 nmolml-1 [70]. Consis-
tently, NBP (0.1 μmolL-1) significantly inhibited the activity
of XO and decreased the formation of superoxide anion
compared with the vehicle group in vitro [71]. In addition,
NBP (20 mg.kg-1, i.p.) enhanced the activities of two en-
dogenous antioxidants L-Glutathione (GSH) and superoxide
dismutase (SOD), and decreased the content of malondialde-
hyde (MDA) in MCAO rats [19, 71].
6.2. Increase the Expression of Nrf2
The nuclear factor erythroid 2-related factor 2 (Nrf2) is a
key regulator of antioxidative defense responses and an im-
portant protective-survival factor in central nervous system
[72, 73]. Under normal condition, Nrf2 binds the cytosolic
protein Keap1 in an inactive state. After activated by redox
stimulation, Nrf2 translocates to the nucleus and binds anti-
oxidant response elements (ARE) [74]. Nrf2-ARE binding
can initiate transcription of hundreds of protective genes,
including SOD, thioredoxin (TRX), glutathione peroxidase
(GPX), NAD(P)H quinine oxidoreductase (NQO1), and
heme oxygenase (HO-1) [75]. The activation of Nrf2 can
exert neuroprotective effects against permanent cerebral
ischemia [76], whereas the lack of Nrf2 results in cortical
astrocytes and neurons more susceptible to oxidative stress
[77]. Compared with ischemia/reperfusion (I/R) group in
rats, the expression of Nrf2 in the cortex nuclear fraction was
significantly higher in the NBP (60 mg kg-1, p.o.) group,
demonstrating that NBP treatment induced Nrf2 activation
and subsequent nuclear localization [78]. In a carbon monox-
ide (CO)-induced brain damage rat model, the expressions of
Keap1, Nrf-2, and NQO-1 were up-regulated after 1, 3 and 7
day of NBP (60 mg kg-1, p.o.) administration compared with
the CO poisoning group [79].
7. INHIBIT APOPTOSIS AND AUTOPHAGY
After cerebral ischemia, apoptosis is triggered through
different signal pathways. The extrinsic pathway is triggered
by the involvement of death receptor, which initiates a sig-
naling cascade regulated by caspase-8 activation. By con-
trast, the intrinsic pathway is engaged when various apop-
totic stimuli activate the release of cytochrome c from mito-
chondria. Both pathways eventually lead to the activation of
caspase-3, degrading cellular proteins that are essential to
maintain cell survival and integrity [80]. In the penumbra
region of MCAO mice, NBP (100 mg kg-1, i.p., two hours
before and immediately after ischemia) treatment remarkably
decreased the release of apoptosis inducing factor (AIF) and
cytochrome c from cytosol to the mitochondria, and down-
regulated caspase-9 and caspase-3 by 33% and 43% respec-
342 CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 Wang et al.
tively [21]. In addition, NBP (40 mg kg-1, i.p., immediately
after I/R) markedly increased the ratio of Bcl-2 (anti-
apoptosis)/Bax (pro-apoptosis) in the hippocampus of Mon-
golian gerbil after global cerebral ischemia and reperfusion
damage [81]. These data illustrated that NBP treatment
might inhibit mitochondria-dependent cellular apoptotic cas-
cade after ischemia and reperfusion.
JNKs, members of MAPK family, can be activated by
many insults including ischemia [82]. Once activated, JNKs
phosphorylate a variety of transcription factors and cellular
proteins that are related to apoptosis including Bcl-2, P53,
and others [83]. Wen et al. found that NBP (15 mgkg-1, p.o.,
t.i.d., 20 min after I/R) decreased the apoptosis of CA1 py-
ramidal neurons in the I/R-induced brain injury in rats by
preventing phosphorylation of JNK and the activations of its
two important downstream proteins, FasL and c-jun [84].
NBP (75 mgkg-1, q.d., p.o.) treatment also down-regulated
pro-apoptotic signaling mediated by phospho-JNK and p38
in I/R injury of rats [85].
The activations of calcineurin and calpain changed the
structure of chromosome, activated the endonuclease, and
induced DNA fragmentation and the apoptosis of ischemic
neurons [86]. Interestingly, NBP (20 mg.kg-1, i.p., 10 min
before ischemia) inhibited the activities of calcineurin and
calpain in focal cerebral ischemia rats [87], and NBP (10
μmolL-1) prevented DNA fragmentation and attenuated
morphological changes in rat cortical neurons after hypoxia
and hypoglycemia [88].
Fig. (2). Possible targets of NBP on multiple factors that might be involved in stroke. NBP can improve rCBF through the effect of vasodila-
tion with the elevation of NO, PGI2 and the decrease of TXA2 which could also prevent the platelet aggregation. NBP is also beneficial for
cerebral circulation mainly by up-regulating VEGF, bFGF, TGF-β1 with the effect of angiogenesis, these effects could in turn protect BBB.
During ischemic stroke, mitochondria is being damaged, NBP can restore it through improving the activity of respiratory enzyme complexes,
protecting the structure and function of mitochondrial membrane, as well as maintaining the level of ATPase. Ischemic stroke could induce
oxidative stress by increasing ROS, RNS and MDA levels while decreasing SOD and NOS activities, leading to apoptosis as well as inflam-
mation. However, NBP can modulate these processes by decreasing oxidative stress, up-regulating anti-apoptotic protein Bcl-2, down-
regulating pro-apoptotic proteins Bax, Bad, and Bid expression, inhibiting levels of TNF-α, IL-1β, IL-6, and Nrf2, NF-κB, JNK might also
be involved in these effects. In addition, NBP could decrease the releases of glutamate and intracellular calcium. Up arrows denote enhance-
ment, down arrows denote inhibition. BBB, blood-brain barrier; ROS, reactive oxygen species; bFGF, basic fibroblast growth factor; MMP,
mitochondrial membrane potential; MMfu, mitochondrial membrane fluidity; PLA2, phospholipase A2; TXA2, thromboxane A2; VEGF,
vascular endothelial growth factor; HGF, hepatocyte-growth factor; SOD, superoxide dismutase; MDA, malondialdehyde; GSH,
L-Glutathione; NO, nitric oxide; Nrf2, the nuclear factor erythroid 2-related factor 2; ARE, antioxidant response elements; TGF-β1, trans-
forming growth factor-β1.
glutamate
EAAT
Membrane
ADP
Ca
2
-ATPase
ATP
Ca
2
lactate
Na+-K+-ATPase
ETC complex IV
Cyt c
ATP
MMP
MMfu
+++++++
Mitochondrial
PLA
2
AA
COX
PGG
2
\PGH
2
PGI
2
TXA
2
neurongenesis
Vasodilation
platelet aggregation
angiogenesis
cGMP GSH
Microcirculation
ROS/RNS damage
ATP Ade
Ino Hyp Xan
XO
O
2
O
2
H
2
O
2
H
2
O
ONOO
-
-
NO
NOS SOD
L-Arginine
peroxidation
MDA
Endoplasmic reticulum
Ca
2+
release
Ca
2+
inflammation
TNF-aIL-1bIL-6
HO-1
SOD
Caspase 9, 3
Apoptosis
Apoptosome
P38
JNK
SOD2
Bd-XL
Bd-2 Cyt c Apaf-1
Bid
Bax
Bad
IV
e-
Ke ap1
Nrf-2
Nrf-2
Nrf-2
ARE
NF-
K
BHIF1 STAT3
Nuclear
gliogenesis
vEGF
FGF
bFGF
TGF-
b1
disruption
BBB
ATP
ADP
Na
+
stroke brain
NBP, A Promising Therapeutic Agent for Ischemic Stroke CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 343
Recently, autophagy has been found to be crucial in the
process of cellular apoptosis as it can degrade and clean up
damaged mitochondria [89, 90]. NBP (10 μmolL-1, begin-
ning of the reperfusion) could inhibit ischemia-induced neu-
roamyloidogenesis by down-regulating autophagy and sup-
pressing the activation of NF-kB pathway in neuroblastoma
2a/amyloid precursor protein 695 cells by 6 h OGD/12 h
reperfusion, indicating that autophagy might be one of the
potential mechanisms of the protective effects of NBP [91].
8. PREV ENT INFLAMMATION
Inflammation plays an important role in the pathogenesis
of ischemic stroke. After ischemia onset, circulating leuko-
cytes adhere to vessel walls, migrate and accumulate into
ischemic brain tissue, subsequently release pro-inflammatory
mediators (e.g. TNF-α, IL-1), which will exacerbate cerebral
injury [92]. Cell adhesion molecules such as selectins and
intercellular adhesion molecule 1 (ICAM-1) play a vital role
in the infiltration of leukocytes [93-95]. The expressions of
ICAM-1, P-selectin and E-selectin were up-regulated on the
endothelial cell surface in ischemic brain [20]. And the level
of TNF-α in the ischemic cortex was also elevated in rat fo-
cal ischemic stroke models [96]. Administration of NBP (20
mgkg-1, i.p., 10 minutes and 60 minutes after MCAO) inhib-
ited the increases of both ICAM-1 and TNF-α in a MCAO
model, and NBP (0.01 μmolL-1) attenuated the neutrophil-
endothelial cells adhesion induced by TNF-α or IL-1 [20,
97]. TLR4/NF-κB is another important inflammation-related
signaling pathway in ischemic stroke. In a cerebral IR-
induced rat model, NBP (4.5 mgkg-1, i.p., q.d., after IR)
suppressed the activation of TLR4, the phosphorylation of
NF-kB, and subsequently the elevation of pro-inflammatory
cytokines, including TNF-a, IL-1, IL-6 and IL-18 [98].
9. DECREASE GLUTAMATE AND INTRACELLU-
LAR CALCIUM
Glutamate is an endogenous excitatory neurotransmitter
in central nervous system. During ischemia, the failure of
Na+, K+-ATPase resulting from ATP depletion leads to the
decrease of Na+ and K+ gradients and the depolarization of
presynaptic membranes. Sustained cell membrane depolari-
zation causes increased glutamate release from vesica and
decreased glutamate uptake by presynaptic membranes. Ac-
cumulation of glutamate in the synaptic cleft, ultimately,
results in neuronal swelling or necrosis [99]. 5-HT, an im-
portant vasoactive substance, modulates the post-synaptic
action of excitatory amino acids (EAA) by impacting the
activity of N-methyl-D-aspartate (NMDA) [100, 101]. After
global ischemia, the release of glutamate and 5-HT in hippo-
campus and striatum were markedly elevated [102]. NBP (10
μmolL-1) decreased the release of glutamate and 5-HT by
28.9% and 45.3% respectively in cultured cortical neurons
induced by hypoglycemia/hypoxia [103].
After ischemia, glutamate released from neurons and glia
activated the NMDA receptor and then induced the influx of
Ca2+, which is a key activator of ischemic cell death path-
ways [104-106]. Besides, Ca2+ reuptake into the endoplasmic
reticulum by the SERCA-ATPase is impaired due to the
scarce supplies of ATP, whereas Ca2+ release from intracel-
lular stores through the ryanodine receptor is enhanced
[107]. The elevation of intracellular Ca2+ causes the disor-
dered activation of a wide range of enzyme systems. These
enzymes and their metabolic products, such as oxygen free
radicals, cause mitochondrial damage, cytoskeleton break-
down as well as cell important components detrition, such as
proteins, lipids, and DNA [108-110]. NBP (10 μmolL-1)
attenuated the elevation of intracellular calcium induced by
thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-
ATPase in neurons. Nevertheless, NBP had no effect on in-
creased intracellular calcium induced by elevated K+, indi-
cating that NBP might only reduce calcium release from
intracellular stores [111]. Consistent with these results, NBP
(100 μmolL-1) was found to have inhibitory effect on the
release of intracellular calcium that was initiated by norepi-
nephrine in the rat isolated tail artery, but it exerted no effect
on the constriction when extracellular calcium was added
[112].
CONCLUSION
NBP has been widely used for treating acute ischemic
stroke in China and shows a good clinical effect. Moreover,
the phase II clinical trial of NBP softgel capsules in ischemic
stroke patients has started in United State in 2017. This will
be a randomized, double-blind, placebo-controlled, add-on to
standard-of-care study with a primary objective to assess the
safety of NBP treatment in patients with mild to moderate
acute ischemic stroke, and the results are looked forward to.
Apart from the potent pharmacological actions in ischemic
stroke, NBP also showed beneficial effects in multiple neu-
rodegenerative diseases, such as Alzheimer’s disease (AD)
[113-116], dementia [117-119], Parkinson’s disease [118,
120, 121], and Amyotrophic lateral sclerosis (ALS) [122]. A
number of studies demonstrated that NBP could affect the
two important pathophysiological aspects of ischemic stroke,
improving the condition of cerebral circulation and neuro-
protection by acting on multiple active targets (Fig. 2). Al-
though NBP has been considered as a multi-target drug, the
in-depth study of its target has been ongoing. The latest re-
search found NQO1, p53 and indoleamine 2,3-dioxygenase
(IDO) might be direct binding target of NBP [123]. The pre-
clinical and clinical studies of NBP could provide useful
clues for developing more potent and comprehensive thera-
peutic strategies for ischemic stroke. However, further study
will be needed to understand the precise molecular mecha-
nisms in the future.
LIST OF ABBREVIATIONS
AD = Alzheimer’s Disease
AIF = Apoptosis-Inducing Factor
AIS = Acute Ischemic Stroke
ALS = Amyotrophic Lateral Sclerosis
ARE = Antioxidant Response Elements
BBB = Blood-Brain Barrier
bFGF = Basic Fibroblast Growth Factor
CIR = Cerebral Ischemia-reperfusion
344 CNS & Neurological Disorders - Drug Targets, 2018, Vol. 17, No. 5 Wang et al.
CO = Carbon Monoxide
COPD = Chronic Obstructive Pulmonary Disease
EAA = Excitatory Amino Acids
HGF = Hepatocyte-Growth Factor
ICAM-1 = Intercellular Adhesion Molecule-1
I/R = Ischemia/Reperfusion
GSH = L-Glutathione
6-BBP = 6-Bromo-3-n-butylphthalide
LPS = Lipopolysaccharide
MCA = Middle Cerebral Artery
MCAO = Middle Cerebral Artery Occlusion
MDA = Malondialdehyde
MMfu = Mitochondrial Membrane Fluidity
MMP = Mitochondrial Membrane Potential
NBP = Dl-3-n-Butylphthalide
NIHSS = National Institutes of Health Stroke Scale
NMDA = N-methyl-D-aspartate
NOS = Nitric Oxide Synthase
NO = Nitric Oxide
Nrf2 = The Nuclear Factor Erythroid 2-related Factor 2
OGD = Oxygen-glucose Deprivation
PHPB = 2-(1-Hydroxypentyl)-benzoate
PLA2 = Phospholipase A2
PGI2 = Prostaglandin I2
ROS = Reactive Oxygen Species
rtPAs = Recombinant Tissue Plasminogen Activators
SOD = Superoxide Dismutase
TGF-β1 = Transforming Growth Factor-β1
TXA2 = Thromboxane A2
VEGF = Vascular Endothelial Growth Factor
CONSENT FOR PUBLICATION
The authors all agreed with the publication of this article.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
This project was supported by the grants from National
Natural Sciences Foundation of China (No. 81373387,
81473200 and 81673420), CAMS Innovation Fund for
Medical Sciences (No. 2017-I2M-2-004), and Beijing Key
Laboratory of New Drug Mechanisms and Pharmacological
Evaluation Study (BZ0150).
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PMID: 29895257
... Dl-3-n-butylphthalide (NBP) is a synthetic compound based on l-3-n-butylphthalide that is isolated from the seeds of Apium graveolens. NBP was approved by the Food and Drug Administration (FDA) of China for the treatment of ischemic stroke in 2002 (7). It has also been approved by the US FDA to undergo a phase II trial for the treatment of ischemic stroke (8). ...
... However, the continuous administration of 10 or 30 mg/kg NBP significantly reduced the levels of activated STEP 61 and increased those of p-ERK1/2 and p-CREB, but no significant differences were observed between the NBP groups (10 vs. 30 mg/kg; P>0.05). NBP concentrations were set to 10 and 30 mg/kg according to the published literature (7,31). In conclusion, NBP alleviated amyloid-induced learning and memory impairment, and decreased Aβ-induced activated STEP 61 levels and promote ERK1/2 and CREB phosphorylation. ...
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AIM: To study the effect of dl-3-n-butylphthalide (dl-NBP) on the function of mitochondrial respiratory chain and to elucidate the increasing effect of NBP on brain energy supply during cerebral ischemia. METHODS: Mitochondria were isolated from the brain of transient middle cerebral artery occluded (MCAO) rat and the activities of the four complexes of the respiratory chain were determined. RESULTS: The activity of complex IV was deeply decreased after 1 h-ischemia. It was back to normal level when treated with NBP (5 mg·kg -1 or 10 mg·kg -1 ip 10 min before ischemia). During the reperfusion period after ischemia, the activity of complex I was notably increased at 3 h, and that of complex II was decreased at 6 h. With NBP treatment, these altered activities also returned to normal level. In cultured neurons subjected to 6 h-hypoxia/hypoglycemia, the same increasing effect of NBP(d-, l-or dl-) on the activity of complex IV was also found, and d-NBP seemed to be more effective. CONCLUSION: NBP can act directly on complex IV to increase its activity. This action may play an important role in the increasing effect of NBP on brain energy supply during cerebral ischemia.
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