Pharmacology & Pharmacy, 2011, 2, 306-314
doi:10.4236/pp.2011.24039 Published Online October 2011 (http://www.SciRP.org/journal/pp)
Neuroprotective Effect of a Prostacyclin Agonist
(ONO-1301) with Thromboxane Synthase
Inhibitory Activity in Rats Subjected to
Cerebral Ischemia
Mai Hazekawa1, Yoshiki Sakai2, Miyako Yoshida1, Tamami Haraguchi1, Takahiro Uchida1
1Department of Clinical Pharmaceutics, Faculty of Pharmaceutical Sciences, Mukogawa Women’s University, Hyogo, Japan; 2Ono
Pharmaceutical Co., Ltd., Research Headquarters, Osaka, Japan.
Received June 18th, 2011; revised July 28th, 2011; accepted August 6th, 2011.
ABSTRACT
ONO-1301 has been developed as a novel long-acting prostacyclin agonist with thromboxane synthase inhibitory activ-
ity. In the present study, we investigated the cerebroprotective effect of ONO-1301 on post-ischemic injury induced by
cerebral ischemia in rats. ONO-1301 (1 and 10 mg/kg) was administrated orally at reperfusion and then twice a day for
42 days. The cell damage induced by cerebral ischemia in the h ippocampal CA1 was evaluated using both Nissl stain-
ing and p roliferating cell nuclear antigen (PCNA) staining on the 42 days after cerebral ischemia. Activated astrocytes
were evaluated using immunofluorescence staining with GFAP on the 42 days after cerebral isch emia. Spatial learning
was assessed using a Morris water maze (MWM) task on the 56 days (i.e. after a 14 days washout period). ONO-1301-
treated rats (1 and 10 mg/kg) significantly improved cell death in the h ippocampal CA1, the number of PCNA-positive
cells and astrocyte activation . The spatial learning of ONO-1301-trea ted rats compared with vehicle-trea ted rats in the
MWM task. These results suggest that repeated treatment with oral ONO-1301 could prevent or limit post-ischemic
brain damage. In particular, treatment with ONO-1301 within 7 days after ischemia is most effective to imp rove ische-
mic damage.
Keywords: ONO-1301, Prostacyclin Agonist, Neuroprotection, Cerebral Ischemia, Astrocytes
1. Introduction
ONO-1301 is a novel long-acting prostacyclin agonist
with thromboxane synthase inhibitory activity. Unlike
prostacyclin, ONO-1301 does not possess a five-mem-
bered ring or allylic alcohol in its molecular structure,
making it more biologically and chemically stable. Its
inhibitory effect on thromboxane synthetase is mediated
by binding of thromboxane synthase to the 3-pyridine
moiety and a carboxylic acid group in ONO-1301 [1].
Prostacyclin (PGI2), produced in the vascular endothe-
lium, is a potent vasodilator and a strong inhibitor of
platelet aggregation [2-4]. Thromboxane A2 (TXA2) on
the other hand, although also a powerful vasodilator,
promotes platelet aggregation [3]. An imbalance of PGI2
and TXA2 may therefore result in vasospasm [5]. Ex-
perimental and clinical data have shown that PGI2 may
reduce vasoconstriction elicited by subarachnoid hemor-
rhage [6-9], and beneficial effects of low-dose prostacy-
clin infusion (0.5 ng/kg/min) have been reported in se-
vere traumatic brain injury [10,11].
A prostaglandin analogue, beraprost sodium, is known
to significantly increase tail nerve conduction velocity
(NCV) in rats with streptozotocin (STZ)-induced diabe-
tes [12]. In a previous study, we reported that ONO-
1301-loaded PLGA microspheres improved delayed
NCV in the lower limb of STZ-induced diabetic rats [13],
while in other studies, it has been reported that ONO-
1301 improves pulmonary hypertension [14,15], and
attenuates pulmonary fibrosis [16]. However, the neuro-
protective effect of ONO-1301 has not been clarified.
The purpose of this study was to investigate the neu-
roprotective effect of ONO-1301 in rats subjected to
cerebral ischemia. We used a modification of the stroke
model developed by Pulsinelli and Brierley [17], termed
cerebral ischemia model. Firstly, plasma level of radio-

Neuroprotective Effect of a Prostacyclin Agonist (ONO-1301) with Thromboxane Synthase 307
Inhibitory Activity in Rats Subjected to Cerebral Ischemia
activity derived from 14C-ONO-1301 was measured in
rats treated orally (p.o.) with 1 and 10 mg/kg 14C-ONO-
1301, in order to determine the pharmacokinetics of
ONO-1301 in vivo. Then we examined the effect of 42
days post-ischemic ONO-1301 treatment on neuronal
damage, expression of PCNA-positive cells and activated
astrocytes in the hippocampal CA1 region using cerebral
ischemia model. In order to investigate the protective
effect of ONO-1301 on physiological function, spatial
learning was assessed using the Morris water maze
(MWM) task.
2. Materials and Methods
2.1. Animals
Male Wistar rats weighing 250 - 300 g were obtained
from Charles River Japan Inc. (Hino, Japan), and were
housed in groups of 4/5 animals per cage in a tempera-
ture-controlled room (23˚C ± 2˚C) with a relative humid-
ity of 60% ± 10%. The lights were on from 7:00 to 19:00.
The animals had free access to food (CRF-1, Oriental
Yeast Co., Ltd., Tokyo, Japan) and water. Experiments
were conducted between 9:00 and 17:00. All procedures
regarding animal care and use were carried out according
to the regulations of the Experimental Animal Care and
Use Committee of Mukogawa Women’s University. All
experiments conformed to the guidelines on the ethical
use of animals of the Japanese Government Notification,
and all efforts were made to minimize both the number
of animals used and their suffering.
2.2. Cerebral Ischemia
Animals were subjected to cerebral ischemia according
to a previously published method [18]. Rats were anaes-
thetized with intraperitoneal (i.p.) sodium pentobarbital
(35 mg/kg) and immobilized in a stereotaxic apparatus
(Type SR-6; Narishige Scientific Instrument Laboratories,
Tokyo, Japan). The bilateral arteries beneath the alar
foramina of the first vertebra were electrocoagulated us-
ing a bipolar coagulator (MICRO-3D; Mizuho Industrial
Co., Tokyo, Japan). The following day the common ca-
rotid arteries were compressed using a Schwartz vessel
clip (1.7 × 8 mm jaw; World Precision Instrument, Sara-
sota, FL, USA) to each artery, and cerebral circulation
was interrupted for 10 min. This was repeated once after
a 1-min interval. Rats that did not demonstrate loss of
their righting reflex during arterial occlusion were ex-
cluded from subsequent experiments.
Animals were randomly divided into seven groups: a
sham-ischemia group treated with 0.5 w/v% carboxy-
methyl cellulose solution for 8 days (8d Vehicle treat-
ment group); 8 days post-ischemic ONO-1301 groups
treated with 10 mg/kg p.o. (8d ONO-1301 10 mg/kg
treatment group); a sham-ischemia group treated with 0.5
w/v% carboxymethyl cellulose solution for 42 days (42d
Sham treatment group); two 42 days post-ischemic
ONO-1301 groups treated with 1 or 10 mg/kg p.o. (42d
ONO-1301 1 mg/kg, 10 mg/kg treatment group); a group
treated post-ischemia with vehicle p.o. for 8 days and
ONO-1301 10 mg/kg p.o. for 34 days (34d ONO-1301
10 mg/kg treatment group); and a vehicle-ischemia group
treated with 0.5 w/v% carboxymethyl cellulose solution
for 42 days post-ischemic treatment (42d Vehicle treat-
ment group). ONO-1301 or vehicle was administered
once directly after reperfusion and twice a day thereafter.
2.3. Measurement of Radioactivity
14C-ONO-1301 was synthesized at SEKISUI Medical
Co., Ltd. (Tokyo, Japan). The specific activity was 1.028
MBq/mg. Plasma levels of radioactivity derived from
14C-ONO-1301 were measured after a single p.o. admini-
stration of 1 or 10 mg/kg 14C-ONO-1301. Blood was
drawn from the inferior vena cava of four rats 0.16, 0.50,
1, 2, 3, 4, 6, 8, 10, 24, 36, 72 h after a single p.o. admini-
stration. Plasma was directly analyzed by liquid scintilla-
tion counting (LSC). Radioactivity for all in a Tri-Carb
460 C liquid scintillation counter obtained from Packard
Instrument Company (USA) for at least 5 min or 100,000
counts. This specification was met for the sample ali-
quots that had radioactivity greater than 100 dpm. Scin-
tillation counting data (cpm) were automatically cor-
rected for counting efficiency using the external stan-
dardization technique and an instrument-stored quench
curve generated from a series of sealed quenched stan-
dards.
2.4. Fixation and Processing of Tissue for
Histology
Rats were killed by deep anaesthetization with pentobar-
bital (35 mg/kg, i.p.) and transcardial perfusion with cold
heparinized saline, followed by perfusion of 4% para-
formaldehyde. The brain was then removed from the
skull and post-fixed overnight in paraformaldehyde, be-
fore being dehydrated and embedded in paraffin. Coronal
sections 10-µm thick were made using a rotary micro-
tome. For hippocampal morphology, a 3.3-mm section
was taken from the bregma [19].
2.5. Histological Examination
In order to assess cell damage in the hippocampus after
cerebral ischemia, the number of surviving cells was
counted in Nissl-stained hipocampal sections. Other rep-
resentative coronal sections were stained with proliferat-
ing cell nuclear antigen (PCNA), or glial fibrillary acidic

Neuroprotective Effect of a Prostacyclin Agonist (ONO-1301) with Thromboxane Synthase 308
Inhibitory Activity in Rats Subjected to Cerebral Ischemia
protein (GFAP). These sections were examined by light
microscopy, and the neuronal density of CA1 neurons
(per mm2) in the stratum pyramidale measured.
Immunohistochemical detection (PCNA and GFAP) of
cell damage in paraffin-embedded brain sections was
performed by the streptavidin-avitin-biotin-immunop-ero-
xidase complex method [20]. Briefly, 10-µm thin PCNA-
stained sections on poly-L-lysine-coated slides were de-
paraffinized and rehydrated. Endogenous peroxidase acti-
vity was blocked with 1% H2O2 in 0.1 mol/L Tris-NaCl
(pH 7.4) for 30 min. After incubation with 5% normal
bovine serum albumin (BSA; Serologicals, GA, USA)
for 1 h at 37˚C, sections were incubated overnight at
40˚C with primary antibody mouse anti-rat PCNA (Dako
Cytmation, Glostrup, Denmark) in 1% BSA using a 1:50
dilution. Sections were then incubated with a biotinylated
secondary antibody goat anti-mouse IgG (Dako Cytma-
tion, Glostrup, Denmark) for 30 min at 37˚C at a 1:200
dilution.
This was followed by incubation with streptavidin
peroxidase (1:100) for 1 h and subsequent chromogen
development with 0.5% 3,3’-diaminobenzedrine tetrahy-
drochloride (DAB) and 0.33% H2O2 in 0.5 mol/L Tris-
NaCl as substrate. The sections were counterstained with
Harris Haematoxylin (H&E), then dehydrated, mounted,
and used as a positive control.
In the GFAP staining, rabbit anti-rat GFAP (Dako Cyt-
mation, Glostrup, Denmark) was used as primary antibody.
A biotinylated goat anti-rabbit IgG (Dako Cytmation,
Glostrup, Denmark) was used as secondary antibody.
Damage was scored in the CA1 according to the
method of Freund et al. [21]: normal (score 0). No posi-
tive cells detected; mild (score 1), <20% positive cells;
moderate (score 2), 20% - 50% positive cells; severe
(score 3), 50% - 70% positive cells; very severe (score 4),
>70% positive cells. The damage score (from 0 to 4) for
each subfield was calculated as a mean of the damage
score in all sections in which the subfield was present.
The total hippocampal damage score was calculated as
the sum of subfield damage scores.
2.6. MWM Task for Spatial Learning
The swimming pool (Neuroscience Inc.) was a circular
water tank, 148 cm in diameter and 44 cm deep, which
was modified according to Morris [22]. It was filled to a
depth of 32 cm with clear water at a temperature of 23˚C
± 2˚C. The test was performed with the illumination of a
100-W bulb. A platform, 12 cm in diameter and 30 cm in
height, was present inside the tank, its top surface being
0.5 cm below the surface of the water. The pool was lo-
cated in a large test room, and surrounded by many cues
external to the maze (e.g. the experimenter, ceiling lights,
racks, etc.), which were visible from within the pool and
could be used by the rat for spatial location. The posi-
tions of the cues were unchanged throughout the test pe-
riod. The CCD camera, equipped with a personal com-
puter, was used for behavioral analysis (AXIS-30, Neu-
roscience Inc).
Each rat received two trials daily for four consecutive
days. A trial consisted of placing the rat by hand into the
water facing the wall of the pool, at one of three starting
positions (excluding the quadrant containing the plat-
form). The pool was divided into sections (north: N,
south: S, east: E, or west: W). The platform was located
in a constant position in the middle of one quadrant and
its location was the same for all rats. During each block
of three trials, each rat started once at each of the three
starting positions, but the sequence of the positions was
selected at random. In each trial, the time taken to swim
to the hidden platform was recorded; the cut-off time was
90 s. If a rat found the platform, it was permitted to re-
main there for 30 s. If a rat failed to find the platform
within 90 s, the rat was forced to remain on the platform
for 30 s. At the end of a trial, the rat was returned to its
cage. The inter-trial interval time was approximately 30
min. The performance of the test animal in each trial was
assessed by two parameters: swimming time and swim-
ming distance, using the personal computer. Swimming
speed was calculated by Swimming length (m) per Goal
latency (s).
2.7. Statistical Analysis
Data of Figures 1-3 are expressed as mean ± S.D. Data
of Figures 4-6 are expressed as mean ± S.E.M. Data on
the number of CA1 pyramidal neurons and the amount of
cell damage were analyzed by Bonferroni/Dunnett’s test
after one-way (repeated measures) analysis of variance
(ANOVA). The scores of expressed PCNA-positive cells
and activated astrocytes (GFAP-positive cells) were ana-
lyzed for statistical significance using the cumulative
chi-square test. Goal latency and swimming length in the
MWM task was evaluated by one-way ANOVA. SAS
software (ReL.8.2 TS020.SAS Version 5.0; SAS Institute
Inc.) and the Yukms statistics library (Yukms statistics
library Version 5.0, Yukms Co. Ltd.) were used; P-val-
ues of less than 0.05 were considered to be statistically
significant.
3. Results
3.1. Pharmacokinetic Study of ONO-1301 in
Intact Rats
14C-ONO-1301-derived radioactivity was detected at all
the time points through 72 h postdose. As shown in Fig-
ure 1, the concentration of plasma AUC0-∞ and Cmax of

Neuroprotective Effect of a Prostacyclin Agonist (ONO-1301) with Thromboxane Synthase 309
Inhibitory Activity in Rats Subjected to Cerebral Ischemia
10 mg/kg group were approximately 10-fold greater than
the plasma those of 1 mg/kg group (Table 1). The time at
which the maximal concentration occurred (Tmax) was
similar between 1 mg/kg and 10 mg/kg group. The
elimination T1/2 of 1 mg/kg group (8.9 ± 4.1 h) was
greater than that of 10 mg/kg group (5.1 ± 1.7 h) (Table
1; Figure 1).
3.2. Effect of Post-Ischemic ONO-1301
Treatment on Cell Death in the
Hippocampal CA1 Region Following
Cerebral Ischemia
Nissl staining revealed pyknosis, eosinophilia, karyrrhe-
xis, and chromosome condensation in the CA1 pyramidal
neurons in the vehicle-ischemia group compared with the
sham-ischemia group. Cerebral ischemia induced cell
death in hippocampal CA1 pyramidal neurons.
The 8 days ONO-1301 treatments of 10 mg/kg sig-
nificantly suppressed neuronal death compared with ve-
hicle (F = 21.89, P < 0.01) (Left: 8d Vehicle 11.0 ± 6.0, 8d
ONO-1301 10 mg/kg 90.9 ± 53.7; Right: 8d Vehicle 20.0
± 39.0, 8d ONO-1301 10 mg/kg 89.7 ± 51.8; Figure 2).
The 42 days ONO-1301 treatments (1 and 10 mg/kg)
significantly suppressed neuronal death (F = 24.32, P <
Table 1. The pharmacokinetics of ONO-1301 in rats follow-
ing a single oral dose of 1 or 10 mg/kg ONO-1301 (n = 4).
Dose
(mg/kg)
Cmax
(ng cq./ml) Tmax (h) AUC(0-∞) (ng cq.·h/ml) T1/2 (h)
1 354 ± 109 1.5 ± 0.62325 ± 604 8.9 ± 4.1
10 3981 ± 1569 1.5 ± 0.627586 ± 9230 5.1 ± 1.7
Figure 1. Plasma levels of radioactivity of derived from 14C-
ONO-1301 after administration of 14C-ONO-1301 in intact
rats. 14C-ONO-1301 was administered orally to intact rats;
plasma levels of radioactivity of derived from 14C-ONO-
1301 was detected in plasma for 72 h after a single injection
(○, 1 mg/kg; ●, 10 mg/kg). Values represent the mean ± S.D.
of 4 rats.
0.01, and P < 0.05 vs vehicle-ischemia group, respec-
tively; Figure 3), as did the 34 days ONO-1301 10 mg/
kg treatment (P < 0.05 vs vehicle-ischemia group; Fig-
ure 3). The neuroprotective effect of the 42 days ONO-
1301 treatment was, however, greater than the 34 days
ONO-1301 10 mg/kg treatment (Left: 42d Sham 163.1 ±
4.1, 42d Vehicle 24.0 ± 7.5, 42d ONO-1301 1 mg/kg
73.6 ± 15.5, 42d ONO-1301 10 mg/kg 91.2 ± 14.2, 34d
ONO-1301 10 mg/kg 48.3 ± 9.9; Right: 42d Sham 162.3
± 0.7, 42d Vehicle 19.6 ± 4.5, 42d ONO-1301 1 mg/kg
63.0 ± 5.1, 42d ONO-1301 10 mg/kg 76.4 ± 4.9, 34d
ONO-1301 10 mg/kg 43.2 ± 4.7; Figure 3).
3.3. Decrease of PCNA-Positive Cells Induced by
Cerebral Ischemia
There were significantly higher numbers of PCNA-posi-
tive cells in the vehicle-ischemia group compared with
the sham-ischemia group (P < 0.01). The 42 days post-
ischemic ONO-1301 treatment (1 mg/kg) significantly
inhibited the expression of PCNA-positive cells induced
by repeated cerebral ischemia (F = 6.24, P < 0.05 vs 42d
Vehicle group). The number of PCNA-positive cells in
(a)
(b)
Figure 2. Effect of post-ischemic ONO-1301 8 days treat-
ment on cell death in the hippocampal CA1 region following
cerebral ischemia. Animals received ONO-1301 10 mg/kg
orally for 8 days after cerebral ischemia. In this figure, 4VO
stands for four vessels occlusion. (a) Representative photo-
graphic results of surviving cells in the hippocampal CA1
region × 200, bar = 25 µm. (b) Surviving cells in the CA1
region of the hippocampus expressed as number of cells/
mm2. **P < 0.01 vs 8d Vehicle group (Bonferroni/Dunnett’s
test). The number of rats examined was 10/group.

Neuroprotective Effect of a Prostacyclin Agonist (ONO-1301) with Thromboxane Synthase 310
Inhibitory Activity in Rats Subjected to Cerebral Ischemia
(a)
(b)
Figure 3. Effect of post-ischemic ONO-1301 42 days treat-
ment on cell death in the hippocampal CA1 region following
cerebral ischemia. Animals received ONO-1301 1 or 10
mg/kg orally for 42 days after cerebral ischemia. (a) Repre-
sentative photographic results of surviving cells in the hip-
pocampal CA1 region × 200, bar = 25 µm; (b) Surviving
cells in the CA1 region of the hippocampus expressed as
number of cells/mm2. **P < 0.01 vs 42d Sham group; #P <
0.05, ##P < 0.01 vs 42d Vehicle group (Bonferroni/Dunnett’s
test). The number of rats examined was 10 - 12/group.
each group was as follows: 42d Sham 0.0 ± 0.0, 42d Ve-
hicle 2.0 ± 0.2, 42d ONO-1301 1 mg/kg 1.0 ± 0.3, 42d
ONO-1301 10 mg/kg 1.2 ± 0.3, 34d ONO-1301 10
mg/kg 1.5 ± 0.3; Figure 4).
3.4. Inhibition of Activated Astrocytes Induced
by Cerebral Ischemia
There were significantly higher numbers of GFAP- posi-
tive cells in the vehicle-ischemia group compared with
the sham-ischemia group (F = 12.51, P < 0.01). The 42
days post-ischemic ONO-1301 treatment (1 or 10 mg/kg)
significantly inhibited the expression of GFAP-positive
cells induced by repeated cerebral ischemia (P < 0.05 vs
vehicle group), whereas the 34 days ONO-1301 treat-
ment did not inhibit activated astrocytes (42d Sham 0.0 ±
0.0, 42d Vehicle 3.5 ± 0.1, 42d ONO-1301 1 mg/kg 1.9 ±
0.4, 42d ONO-1301 10 mg/kg 1.7 ± 0.4, 34d ONO-1301
10 mg/kg 2.5 ± 0.4; Figure 5).
(a)
(b)
Figure 4. Effect of post-ischemic ONO-1301 treatment on
presented PCNA-positive cells induced by cerebral ischemia.
Animals received ONO-1301 1 or 10 mg/kg orally for 42
days after repeated cerebral ischemia. (a) Representative
photographic results of PCNA-positive cell in the CA1 re-
gion of the hippocampus × 200, bar = 25 µm; (b) PCNA-
positive cell in the CA1 region of the hippocampus ex-
pressed as number of cells/mm2. **P < 0.01 vs 42d Sham
group; #P < 0.05 vs 42d Vehicle group (chi-square test). The
number of rats examined was 10 - 12/group.
3.5. Spatial Learning Using the MWM Task in
Post-Ischemic Rats
The MWM task was carried out on the 14 days after the
end of the 42 days ONO-1301 treatment period. The re-
peated measure one-way ANOVA for goal latency
showed a significantly delayed goal latency between the
sham-ischemic and vehicle-ischemic groups (F = 3.46, P
< 0.01). The goal latency of 42 days ONO-1301-treated
rats (10 mg/kg) was significantly improved compared
with the vehicle-ischemic group (P < 0.01) with an av-
erage goal latency in eight time trials of: 42d Sham 42.5
± 2.2 s, 42d Vehicle 58.4 ± 1.9 s, 42d ONO-1301 10
mg/kg 47.9 ± 2.3 s, and 34d ONO-1301 10 mg/kg 51.7 ±
2.2 s; Figure 6(a). Furthermore, there was a significant
difference between the swimming length of the sham-
ischemic and vehicle-ischemic groups (F = 4.05, P <
0.01). The swimming length of 42 days ONO-1301 treated

Neuroprotective Effect of a Prostacyclin Agonist (ONO-1301) with Thromboxane Synthase 311
Inhibitory Activity in Rats Subjected to Cerebral Ischemia
(a)
(b)
Figure 5. Effect of post-ischemic ONO-1301 treatment on
activated astrocytes induced by cerebral ischemia. Animals
received ONO-1301 1 or 10 mg/kg orally for 42 days after
repeated cerebral ischemia. (a) Representative photogra-
phic results of GFAP-positive cell in the CA1 region of the
hippocampus × 200, bar = 25 µm. (b) GFAP-positive cell in
the CA1 region of the hippocampus expressed as numbers
cells/mm2. **P < 0.01 vs 42d Sham group; ##P < 0.01 vs 42d
Vehicle group (cumulative chi-square test). The number of
rats examined was 10 - 12/group.
rats was significantly less than the vehicle-ischemic group
(P < 0.01) with average swimming times in eight time
trials of: 42d Sham 1068.9 ± 53.8 cm, 42d Vehicle
1500.7 ± 47.4 cm, 42d ONO-1301 10 mg/kg 1172.7 ±
57.9 cm, 34d ONO-1301 10 mg/kg 1267.1 ± 53.9 cm;
Figure 6(b). Swimming speed during 4 groups was no
significant differences (42d Sham 3.9 ± 0.1 m/s, 42d Ve-
hicle 3.9 ± 0.1 m/s, 42d ONO-1301 10 mg/kg 3.9 ± 0.1
m/s, 34d ONO-1301 10 mg/kg 3.9 ± 0.2 m/s).
4. Discussion
It has previously been reported that plasma ONO-1301
concentrations after a single subcutaneous administration
indicated that its activity would be sustained [14]. In or-
der to investigate the pharmacokinetics of ONO-1301
after oral administration, plasma level of radioactivity
derived from 14C-ONO-1301 were measured after single
oral doses of 1 and 10 mg/kg 14C-ONO-1301 in rats. In
(a)
(b)
Figure 6. Spatial learning in the MWM task in rats with
cerebral ischemia. The task was performed on the 14 days
after the final treatment with ONO-1301 (10 mg/kg). Two
trials were conducted per day for 4 days. (a) The data are
given as mean ± S.E.M. of goal latency time. (b) The data
are given as mean ± S.E.M. of swimming length. **P < 0.01
compared with 42d Sham group; #P < 0.05, ##P < 0.01 com-
pared with 42d Vehicle group (one-way ANOVA).
both 1- and 10-mg/kg treatment group, the concentration
of plasma AUC0-∞ and Cmax of 10 mg/kg group were ap-
proximately 10-fold greater than the plasma those of 1
mg/kg group. These results suggested that the release
profile of ONO-1301 showed a liner model. Liner model
is known to easy to repeated treatment. Furthermore,
plasma level of radioactivity of ONO-1301 is keep low
levels without being zero ng cq./ml 36 h after treatment
of ONO-1301. These finding suggested ONO-1301 has
possible to accumulate if ONO-1301 was repeated treat-
ment for long term. However, in order to exert maximum
neuroprotective effect, we decided ONO-1301 of treat-
ment time is twice a day in this experimental schedule
because T1/2 was about 5 - 8 h.
In this study, twice-daily treatment with ONO-1301

Neuroprotective Effect of a Prostacyclin Agonist (ONO-1301) with Thromboxane Synthase 312
Inhibitory Activity in Rats Subjected to Cerebral Ischemia
p.o. for 8 days after four-vessel occlusion (4VO) showed
a neuroprotective effect against cerebral ischemia. Twi-
ce-daily treatment with ONO-1301 p.o. for 42 days also
showed a neuroprotective effect against cerebral ische-
mia. However, twice-daily treatment with ONO-1301 p.o.
for 34 days was no significant in neuroprotective effect.
Previously study was reported 4VO model rat was shown
two stage of neuronal cell death in ischemic damage
process. First stage is acute neuronal cell death in hippo-
campus during 7 days after ischemia. Second stage is de-
layed progressive neuronal cell death as chronic stage
[23-25]. In a previous study, it was reported that the
therapeutic window for cannabidiol treatment to delay
ischemic damage was determined by its inhibition of the
inflammatory reaction, which was divided into three
phases. Firstly, MPO-positive cells were expressed on 1
and 3 days after ischemia; secondly, microglia was acti-
vated on 1, 3, 7 and 14 days; and thirdly, astrocytes were
activated on 7 and 14 days [26]. That is why, rats were
treated vehicle for 8 days and ONO-1301 for 34 days. We
determined hirtological and behavior change about neu-
ron-protective effect of ONO-1301.
The histological changes taking place in the brain tis-
sue were investigated by assessment of PCNA- and
GFAP-positive cells. The 42 days ONO-1301 treatment
inhibited expression of both PCNA-positive cells and the
activated astrocyte reaction induced by repeated cerebral
ischemia, while the 34 days ONO-1301 treatment did not
inhibit the activated astrocyte reaction. These results
suggested that the first 8 days after ischemia-reperfusion
is the most important phase in which to inhibit activated
astrocytes.
Neutrophil elastase is known to be released from acti-
vated neutrophils during the process of inflammation,
elastase inhibitors exert a cardioprotective effect against
reperfusion injury, probably by inhibition of leukocyte
extravasation as indicated by the decrease in myeloper-
oxidase (MPO) activated within 7 days after ischemia
[27]. Neutrophil elastase decreases the endothelial pro-
duction of PGI2 through the inhibition of endothelial ni-
tric oxide synthase (NOS) activation and thereby con-
tributes to the development of ischemia-reperfusion- in-
duced liver injury [28]. In these reports, PGI2 production
is being regulated by two opposing effects after ische-
mia-reperfusion, being increased by noradrenaline and
decreased by neutrophil elastase. The results of this study
suggest that it is the PGI2-agonist effect of ONO-1301,
rather than the TXA2-inhibitory effect, which is respon-
sible for its neuroprotective effects. The neuroprotective
mechanism of ONO-1301 is probably inhibited infram-
mation reaction of PGI2 such as suppress the neutrophil
elastase activity in the first phase after ischemia-reper-
fusion, as the histological results showed the 34 days
ONO-1301 treatment group to have less effect than the
42 days ONO-1301 treatment group.
Cerebral ischemia induces cell death in the hippocam-
pal CA1 pyramidal neurons, so the number of surviving
CA1 pyramidal neurons gives a measure of the neuro-
protective effect of ONO-1301. Treatment with ONO-
1301 (1 and 10 mg/kg) for 42 days post-ischemia was
significantly suppressed neuronal death. We determined
behavior change as spatial learning using MWM task.
When spatial learning was assessed by the MWM task on
the 14 days after the end of the 42 days ONO-1301
treatment period, the goal latency of 42 days ONO-1301-
treated rats was significantly improved compared with
the vehicle-ischemia group. Furthermore, the swimming
time of the 42 days ONO-1301-treated rats was signifi-
cantly improved compared with the vehicle-ischemia
group. While the goal latency and swimming time of 34
days ONO-1301 10 mg/kg treatment group was also sig-
nificantly improved compared with the vehicle-ischemia
group. However, the improvement effect was less than
that of the 42 days ONO-1301 treatment group. These
results suggest that the critical phase for inhibition of the
progressive inflammatory reaction (i.e., the therapeutic
window) is less than 8 days after cerebral ischemia. Re-
peated treatment of ONO-1301 was indicated no affect to
swimming ability and safety because swimming speed
was no significant difference.
5. Conclusions
ONO-1301 has been developed as a novel long-acting
PGI2 agonist with thromboxane synthase inhibitory ac-
tivity. The primary finding of this study was ONO-1301
has neuroprotective effects when administered orally
after experimentally induced cerebral ischemia. Daily
treatment with oral ONO-1301 for 42 days post-ischemia
prevented brain damage by inhibiting activated astro-
cytes. It was necessary to commence treatment with
ONO-1301 within the 8 days period immediately fol-
lowing the ischemic insult. These findings suggest that
ONO-1301 may be a clinically useful treatment if used
soon after cerebrovascular accident.
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