Cumulative Neuroprotection by a Combination of Ozagrel Sodium

● Original
Cumulative Neuroprotection by a Combination of
Ozagrel Sodium and Edaravone against
Photochemical Thrombotic Ischemia in Rats
Tatsushi Kamiya, Chikako Nito, Masayuki Ueda, Toshiki Inaba,
Simon Amemiya, Kengo Kato, Yasuhiro Nishiyama,
Satoshi Suda and Yasuo Katayama
Abstract
A selective thromboxane A2 synthetase inhibitor ozagrel sodium and a free radical scavenger edaravone
are clinically available therapeutic agents for ischemic stroke in Japan. The present study sought to investigate whether such clinically available combination would yield further improvements in ischemic brain injury,
using a rat photochemical thrombotic model. Experimental ischemia was induced by irradiating the brain with
green light together with injecting a photosensitizing rose bengal dye in combination with common carotid artery occlusion. Animals were treated with intravenous injection of 10.0 mg!
kg of ozagrel sodium, 3.0 mg!
kg of
edaravone or combination of both. Vehicle-treated animals underwent physiological saline injection, instead of
the therapeutics. The combined therapy, but not each monotherapy, significantly improved neurological outcome and reduced infarct and edema volumes, which were assessed 72 hours after irradiation, compared to
the vehicle-treatment. In conclusion, a clinically available combination with ozagrel sodium and edaravone at
clinically relevant dosages shows cumulative neuroprotection against photochemical thrombotic ischemia in
rats. The present data provide an experimental basis for such combination that can be chosen in the clinical
field.
（Cerebral Blood Flow and Metabolism 17 : 233―240, 2005）
Key words : ozagrel sodium, edaravone, combined therapy, photochemical thrombotic ischemia, neuroprotection
mental ischemic brain injury9,3）. In addition to altera-
Introduction
tion in TXA2 synthesis, oxidative stress via reactive
oxygen species is also involved in ischemic brain
Arachidonic acid（AA）cascade plays an important
4）
27）
damage6,5）. Edaravone（MCI-186）
, a free radical
role in mediating ischemic brain injury . AA me-
scavenger, theoretically reduces
tabolite thromboxane A2（TXA2）
, which has strong
stress, and also displays protective effects against
effects on platelet aggregation and vascular constric-
experimental cerebral ischemia1,25,13,2）. Following pre-
tion, is increased under cerebral ischemia, and ag-
marketing trials17,18）, ozagrel and edaravone has been
gravates infarct and edema formations7）. Thus oza-
clinically available in Japan since 1992 and 2001, re-
14）
grel sodium（ozagrel ; OKY-046）
, a selective TXA2
spectively. Because various factors are associated
synthetase inhibitor, is theoretically one of the fun-
with deteriorating ischemic brain injury, combined
damental therapeutics for cerebral ischemia, and it
therapy with several therapeutic agents or strate-
indeed shows therapeutic benefits against experi-
gies may be more effective than each monotherapy,
Division of Neurology, Second Department of Internal
Medicine, Nippon Medical School, 1―1―5 Sendagi, Bunkyoku, Tokyo 113―8603, JAPAN
such
oxidative
as described in recent publications24,21,12,15,28,16）. Interestingly, superoxide anion is generated during AA
metabolic process11）, and edaravone is known to re― 233 ―
脳循環代謝
第 17 巻
第4号
duce AA-induced brain edema26）. These suggest a
ological saline to the concentration of 20 mg!
ml, was
close interaction between free radical production
intravenously infused over 1 minute, starting at 1
and AA metabolism, and expect a possible cumula-
minute after irradiation. The ipsilateral CCA was
tive effect by scavenging free radicals combined
doubly ligated just after irradiation, and then the
with inhibiting TXA2 production on ischemic brain
contralateral CCA was transiently occluded using an
injury. However, any experimental evidences for
aneurysmal clip for 1 hour. Rectal temperature was
neuroprotection by combined therapy with ozagrel
maintained at 37±0.5℃ using a heating lamp during
sodium and edaravone against cerebral ischemia
the
surgical
procedures.
Physiological
variables
have not been documented. The present study was,
（mean blood pressure, pH, pO2, pCO2）were meas-
therefore, to investigate whether such clinically
ured before, just after and 60 minutes after the irra-
available combination would yield further improve-
diation.
ments in ischemic brain injury and neurological out-
Animals were randomly divided into vehicle（VE）,
come following photochemically-induced thrombosis
ozagrel sodium（OZG）
, edaravone（ED）and ozagrel
in rats.
sodium & edarabone（OZG!
ED）groups（n＝7, each）.
Ozagrel sodium（Kissei Pharmaceutical Co. Ltd., Na-
Materials and Methods
gano, Japan）was dissolved in physiological saline,
and was prepared at the concentration of 10.0 mg!
All experimental protocols were approved by our
ml. Edaravone（Mitsubishi Pharma Corporation, To-
institutional animal care committee in accordance
kyo, Japan）was dissolved in 1N NaOH and titered to
with the NIH guidelines for laboratory animal use
pH 7.4 using 1N HCl, and was diluted with physi-
and care. Twenty-eight male Sprague-Dawley rats
ological saline to the concentration of 3.0 mg!
ml.
（Sankyo Laboratories, Tokyo, Japan）
, weighing 250-
Rats were treated with intravenous injection of eda-
300 g, were used in the present study. Animals were
ravone（3.0 mg!
kg）
, ozagrel sodium（10.0 mg!
kg）or
initially anesthetized with 4％ halothane, and then
both. Edaravone and!
or ozagrel sodium were in-
maintained with 1％ halothane in a mixture of 70％
jected at 1 minute and!
or 30 minutes after the irra-
N2O and 30％ O2 following an overnight fast. The
diation, respectively. Vehicle-treated animals re-
caudal artery and the left femoral vein were cannu-
ceived intravenous injection of physiological saline,
lated for monitoring of arterial blood pressure, analy-
instead of these therapeutics. Animals were allowed
sis of arterial blood gases and blood glucose levels,
free access to food and water after recovery from
and for intravenous injection of photosensitizing dye
anesthesia for 72 hours. An experimental protocol is
and therapeutics.
presented in Fig. 1.
Experimental cerebral ischemia was induced using
Neurological symptoms in each rat were evaluated
a photochemical thrombotic technique, according to
72 hours after ischemia in a blind fashion using a
the previous protocol3） with several modifications,
neurological deficit score based on the detection of
which were necessary for introducing more severe
hemiparesis and abnormal posture, as described in
and consistent ischemia. Briefly, both common ca-
details previously16,2）. Briefly, flexor response in the
rotid arteries（CCAs）were carefully exposed through
right hindlimb of each rat was evaluated after ex-
a ventral midline neck incision. A small craniotomy
tending the limb using round tipped forceps to
of 4 mm in diameter was made at 3 mm posterior to
evaluate hemiparesis, and was graded as 0 ; normal,
the bregma and 5 mm lateral to the midline in the
1 ; slight deficit, 2 ; moderate deficit, and 3 ; severe
left skull. An optic fiber bundle was connected to a
deficit. Forelimb flexion and body twisting of each
xenon lamp light source system L4887（Hamamatsu
rat were evaluated after suspending by the tail to
Photonics, Shizuoka, Japan）, fitted with infrared and
assess abnormal posture, and were scored as 0 ; nor-
540 nm band pass filters, was placed at the craniot-
mal, 1 ; slight twisting, 2 ; marked twisting, and 3 ;
omy. Green light（wave length, 540 nm）was irradi-
marked twisting & forelimb flexion.
ated for 10 min, and 20 mg!
kg of rose bengal dye
To assess infarct and edema volumes, rats were
（Sigma Chemical Co., MO, USA）
, dissolved in physi-
deeply anesthetized again with 4％ halothane, and
― 234 ―
combination of ozagrel sodium and edaravone
Table 1. Physiological parameters before and during ischemia
pH
pO2
（mm Hg）
pCO2
（mm Hg）
MABP
（mm Hg）
BG
（mg/dl）
TT
（℃）
Vehcle
7.338 ± 0.032
115.5 ± 23.6
39.1 ± 4.9
97.4 ± 15.6
95.7 ± 20.1
37.1 ± 0.3
Ozagrel-Na
7.379 ± 0.034
113.7 ± 10.8
34.6 ± 3.9
92.4 ± 21.5
88.3 ± 16.9
37.2 ± 0.6
Edaravone
7.337 ± 0.038
115.9 ± 7.6
41.1 ± 7.3
101.0 ± 13.8
88.9 ± 15.2
36.9 ± 0.5
Ozagrel-Na ＋ Edaravone
7.326 ± 0.041
117.3 ± 25.5
42.0 ± 8.2
83.6 ± 10.6
84.1 ± 7.7
36.7 ± 0.6
Vehcle
7.352 ± 0.042
101.2 ± 13.6
36.1 ± 9.1
114.6 ± 34.2
85.1 ± 11.8
37.0 ± 0.5
Ozagrel-Na
Edaravone
7.385 ± 0.027
7.388 ± 0.048
112.0 ± 9.7
111.7 ± 14.7
34.5 ± 3.6
36.8 ± 8.6
126.1 ± 37.4
129.5 ± 16.1
87.7 ± 13.9
84.3 ± 12.2
37.0 ± 0.6
37.2 ± 0.4
7.357 ± 0.02
106.7 ± 16.1
40.2 ± 5.7
109.9 ± 27.8
90.4 ± 13.5
36.7 ± 0.4
Vehcle
Ozagrel-Na
7.367 ± 0.037
7.373 ± 0.039
95.5 ± 14.8
99.2 ± 17.7
34.2 ± 4.7
33.1 ± 5.5
103.7 ± 25.5
91.4 ± 23.9
88.9 ± 11.6
80.7 ± 16.0
37.3 ± 0.3
37.5 ± 0.3
Edaravone
Ozagrel-Na ＋ Edaravone
7.393 ± 0.024
7.343 ± 0.071
106.4 ± 16.9
99.4 ± 13.9
36.5 ± 7.0
37.8 ± 9.8
102.9 ± 16.3
90.9 ± 22.0
79.5 ± 10.2
88.1 ± 11.2
37.2 ± 0.2
37.2 ± 0.3
Before ischemia
Immediately after ischemia
Ozagrel-Na ＋ Edaravone
1 hour after ischemia
There were no significant differences in any parameters between the groups（ANOVA followed by Bonferroni’s test）
Values are mean ± S.D.. MABP : mean arterial blood pressure ; BG : blood glucose level ; TT : temporal muscle temperature.
were decapitated 72 hours after ischemia. The brain
Results
was removed quickly, and was cut into seven 2 mmthick coronal sections using a rat brain matrix. Sections at a 2 mm-interval were stained with 2％ 2, 3,
1. Physiological parameters
5-triphenyltetrazolium chloride（TTC）solution（Wako
Table 1 shows physiological variables before and
Pure Chemical Industries, Ltd., Osaka, Japan）. Bilat-
just after and 60 minutes after focal ischemia in the
eral hemispheric areas and infarct area of each sec-
four groups. Physiological variables belonged to nor-
tion were traced using an image analyzing software
mal ranges without any statistically significant dif-
Mac Scope 2.55（Mitani Corporation, Fukui, Japan）on
ferences among the groups. There were no statisti-
a Macintosh computer, and were measured using
cally significant differences in the physiological pa-
NIH Image 1.62（National Institute of Health, MD,
rameters between the experimental groups.
USA）in a blind fashion. These areas from each TTCstained section were separately summed and multi-
2. Infarct and edema volumes
plied by the interval thickness to yield infarct,
ischemic hemispheric and non-ischemic hemispheric
Representative coronal rat brain sections are
shown in Fig. 2.
volumes. Edema volume was determined by sub-
Fig. 3 shows the infarct（A）and edema volumes
tracting the non-ischemic hemispheric volume from
（B）in the groups. The infarct volume in the VE,
the ischemic hemispheric volume.
OZG, ED and OZG!
ED group was 195.2±59.6,
Statistical analyses were performed using Stat-
119.6±33.2, 108.4±24.4 and 45.1±12.5 mm3, respec-
view 5.0（SAS Institute Inc., NC, USA）on a Macin-
tively. The edema volume in the VE, OZG, ED and
tosh computer. Neurological scores were compared
OZG!
ED group was 57.4±17.5, 31.3±9.0, 31.6±8.3
using a Kruskal-Wallis method followed by a Mann-
and 12.0±4.1 mm3, respectively. The infarct and
Whitney U test with Bonferroni correction. A multi-
edema volumes were significantly reduced in the
group
variance
OZG!
ED group compared to the VE group（p＜0.05）.
（ANOVA）or one-way ANOVA followed by Bonfer-
The infarct and edema volumes in the OZG and ED
roni’
s test was used to compare physiological vari-
groups were relatively smaller than those in the VE
ables and infarct and edema volumes. Data were
group, although these differences did not show sta-
presented as mean±SE, and p＜0.05 was considered
tistical significance.
statistically significant.
3. Neurological scores
repeated-measure
analysis
of
Fig. 4 demonstrates the neurological scores re― 235 ―
脳循環代謝
第 17 巻
第4号
Fig. 1. A experimental protocol in this study
Fig. 2. Representative coronal rat brain sections 72 hours after ischemia
The infarct and edema volumes were obviously improved by the combined therapy with ozagrel
sodium and edaravone compared to the vehicle-treatment, while ozagrel sodium and edaravone
monotherapies showed little improvement in either infarct or edema volume.
― 236 ―
combination of ozagrel sodium and edaravone
Fig. 4. Neurological scores regarding hemiparesis（A）
and abnormal posture（B）in the experimental groups.
The hemiparesis score, but not abnormal posture
score, was significantly improved by the combined
therapy with ozagrel sodium and edaravone compared
to the vehicle-treatment. Ozagrel sodium and edaravone monotherapies did not show significant improvements in the neurological scores.
Data were presented as mean±SE, and ＊ indicates significant difference from vehicle-treated group by a
Kruskal-Wallis method followed by a Mann-Whitney U
test with Bonferroni correction（p＜0.05）.
Abbreviations are same as indicated in Fig. 3.
Fig. 3. Infarct（A）and edema（B）volumes in the experimental groups.
The infarct and edema volumes were significantly reduced by the combined therapy with ozagrel sodium
and edaravone compared to the vehicle-treatment.
Ozagrel sodium and edaravone monotherapies did not
demonstrate significant reduction in neither infarct nor
edema volume.
Data were presented as mean±SE, and ＊ indicates significant difference from vehicle-treated group by oneway analysis of variance followed by Bonferroni’
s test
（p＜0.05）.
VE, vehicle-treatment ; OZG, ozagrel-monotherapy ; ED,
edaravone-monotherapy ; OZG!
ED, combined therapy
with ozagrel and edaravone
groups were also relatively smaller than those in the
VE group, however these did not show statistically
garding hemiparesis（A）and abnormal posture（B）
significant differences.
in the groups. The hemiparesis score in the VE,
Discussion
OZG, ED and OZG!
ED group was 2.0±0.2, 1.6±0.2,
1.4±0.2 and 1.1±0.1, respectively. The abnormal
posture score in the VE, OZG, ED and OZG!
ED
The present study clearly demonstrated that the
group was 1.7±0.2, 1.4±0.2, 1.4±0.2 and 1.1±0.1, re-
combination with ozagrel sodium and edaravone pro-
spectively. The OZG!
ED-treatment significantly im-
vided excellent neuroprotection together with better
proved the hemiparesis score compared to the VE-
neurological outcome, but that each monotheraphy
treatment（p＜0.05）
. The abnormal posture score in
was not enough to improve ischemic injury in the
the OZG!
ED group was relatively lower than that in
present experimental condition.
the VE group, although the difference was not statis-
Ozagrel sodium monotherapy improved neither
tically significant. Both scores in the OZG and ED
neurological outcome nor ischemic injury in the pre-
― 237 ―
脳循環代謝
第 17 巻
第4号
sent study. We have previously revealed that imme-
strategy does not have enough power for neuropro-
diate, but not delayed, injection of ozagrel sodium at
tection. Ozagrel sodium, a potent cerebral circulation
a dose of 10.0 mg!
kg reduced infarct and edema vol-
activating drug10）, reportedly recovers post-ischemic
umes in a rat photochemical thrombotic model3）.
decrease in cortical PO2 after focal ischemia9）, and
However cortical irradiation was performed through
maintains post-ischemic adenosine triphosphate level
the intact skull bone without CCA occlusion in the
following global ischemia20）. On the other hand, eda-
previous protocol3）, which might be less invasive
ravone reportedly inhibits mitochondrial permeabil-
than the present procedure. In addition, ozagrel so-
ity transition pore19）, and activates Bax!
Bcl-2 depend-
dium was injected 30 min after the irradiation in this
ent anti-apoptotic mechanisms without affecting cor-
present study. The differences between the proto-
tical cerebral blood flow2）. The cumulative neuropro-
cols may interpret the insufficient protection by oza-
tection observed in the present study might result
grel sodium monotherapy observed in the present
from an interaction between such different protec-
study. Indeed, ozagrel sodium is reportedly ineffec-
tive mechanisms of both drugs.
tive under severe ischemia in a rat photochemical
Clinical daily dosages were 160.0 mg for ozagrel
thrombotic model requiring craniotomy and CCA
sodium17） and 60.0 mg for edaravone8）. Each dose
clipping23）, which mimics the present model.
（mg!
kg）injected in the present study was approxi-
Edaravone monotherapy also reduced neither neu-
mately 3-folds amount of the clinically approved dos-
rological deficit nor ischemic injury in the present
age of each agent. Moreover edaravone was well tol-
study, although neuroprotective effects of edaravone
erated even at a dose of 2.0 mg!
kg in a phase I clini-
against ischemic damage have been variously dem-
cal trial22）. Thus the present protocol for the combi-
onstrated1,25,26,13,2）. We have previously shown that 3.0
nation with ozagrel sodium and edaravone was con-
mg!
kg of edaravone injected twice after reperfusion
sidered clinically relevant.
In conclusion, a combination with ozagrel sodium
reduced infarct and edema volumes in rat transient
2）
focal ischemia . Kawai, et al. also reported that eda-
and edaravone shows strongly cumulative neuropro-
ravone with the same injection protocol improved in-
tection against photochemical thrombotic ischemia in
farct formation in rat photochemically-induced focal
rats. Since the present protocol is clinically available
13）
ischemia . In contrast, edaravone was administered
combination at clinically relevant dosages, the pre-
just once in the present study, suggesting that sin-
sent data provide an experimental basis for such
gle injection of edaravone might not be enough for
combination that can be chosen in the clinical field.
neuroprotection in the present model.
Regardless of the negative results in both mono-
Acknowledgments
therapies, the combined therapy demonstrated cu-
Ozagrel sodium was generously supplied from Kissei Phar-
mulative neuroprotection in the present study. We
maceutical Co. Ltd.（Nagano, Japan）, and edaravone was
have previously demonstrated that mild hypothermia at 35℃ enhanced neuroprotective effects of a se-
kindly provided by Mitsubishi Pharma Corporation（Tokyo, Japan）.
lective thrombin inhibitor argatroban12） and edaravone15） on focal ischemic injury. Furthermore, we
have also shown that immunosuppressant FK506
combined with such mild hypothermia provided excellent neuroprotection against focal ischemia under
a condition that each monotherapy was not effective16）. In addition, various therapeutic agents or
strategies, regardless of whether clinically available,
have been tested as the means for combined therapies24,21,28）. These observations suggest synergistic effect of a combination with several therapeutic strategies in different mechanisms, even though each
― 238 ―
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脳循環代謝
第 17 巻
第4号
ラット脳血栓モデルにおけるエダラボンとオザグレルナトリウムの
併用療法による脳保護効果増強作用
神谷
達司，仁藤智香子，上田
加藤
健吾，西山
雅之，稲葉
康裕，須田
俊東，雨宮
智，片山
志門
泰朗
日本医科大学第二内科
脳保護薬エダラボン（ED）はフリーラジカル消去作用をもち，細胞膜脂質の過酸化を抑制することにより脳
保護作用を示し，トロンボキサン合成酵素阻害薬オザグレルナトリウム（OZG）は，微少循環改善や血小板凝
集抑制により脳保護作用を示す．今回我々は，ラット光感受性脳血栓モデルの虚血 72 時間後における ED の神
経保護作用を検討し，さらに OZG を併用することで，その脳保護効果が増強するかどうかを検討した．実験動
物は，I：対照群，II：OZG 群，III：ED 群，IV：OZG!
ED 群の 4 群に分類した．脳梗塞体積の検討では，ED
群と OZG 群は対照群に比し共に縮小傾向を認めたが，有意差は認めなかった．しかし，OZG!
ED 群では，対照
群に比し脳梗塞の有意な縮小を認め，脳浮腫体積や神経症候も有意に改善した（p＜0.05）
．ED はラット脳血栓
モデルにおいて脳梗塞抑制効果を示し，さらに OZG を併用することにより ED の脳保護効果が増強する可能性
が示唆され，この併用療法の臨床応用の有用性が示唆された．
キーワード：オザグレルナトリウム，エダラボン，併用療法，光感受性脳血栓モデル，脳保護療法
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