Beneficial effect of CV-4151 (isbogrel), a thromboxane A2 synthase inhibitor, in a rat middle cerebral artery thrombosis model

Thrombosis

Pergamon

Research, Vol. 79, No. 1, pp. 95-107, 1995 Copyright 0 1995 Elseviet Science Ltd Printed in the USA. All rights resewed 0049-3tw3/95 $9.50 + Do

0049-3848(95)ooo94-1

BENEFICIAL EFFECT OF CV-4151 (ISBOGREL), A THROMBOXANE AZ SYNTHASE INHIBITOR, IN A RAT MIDDLE CEREBRAL ARTERY THROMBOSIS MODEL Yoshimi Imura, Yoshihiro Kiyota, Yasuo Nagai, Kohei Nishikawa* and Zen-i&i Terashita Pharmaceutical Research Laboratories I, Pharmaceutical Research Division*, Takeda Chemical Industries, Ltd. 17-85 Juso-Homnachi 2-Chome, Yodogawa-Ku, Osaka, 532, Japan

(Received 23 February 1995 by Editor K. Suzuki; revised/accepted

18 April 1995)

Abstract Effects of thromboxane & (TXA2.) synthase inhibitors (CV-4151 and ozagrel) on cerebral thrombosis and cerebral damage were examined in a rat middle cerebral artery (MCA) thrombosis model and their potencies were compared with the conventional antithrombotic agents, aspirin and ticlopidine. CV-4151 significantly inhibited photochemically induced MCA thrombosis by oral (1 and 10 mgikg) and intravenous (1 mg/ kg) administration. Ozagrel(l0 mg/kg, p.o.) also inhibited it. The potency of CV-4151 was about 10 times stronger than that of ozagrel, being comparable with the inhibition of blood TXA2 generation. Aspirin (100 mg/kg, p.o.) and ticlopidine (300 mg/kg, p.o.) showed an inhibitory tendency on MCA thrombosis. Twenty-four h after photochemical stimulation, cerebral edema and cerebral infarction were observed, and the lactate content in the brain increased. CV-4151 and ozagrel prevented this edema, and the antiedema effects of the drugs were correlated with the antithrombotic effect on thrombotic MCA occlusion. CV-4151 (10 mgkg, p.o.), furthermore, significantly reduced the infarct size and inhibited the increase in lactate content. These results indicate that TX42 synthase inhibitors inhibit cerebral damage by inhibition of MCA occlusion with thrombosis, probably resulting from the inhibition of TXA2 generation, and their effects are superior to those of aspirin and ticlopidine. TXA2 might play an important role in cerebral damage in the MCA thrombosis model. CV-4151 might be a useful drug for the treatment of cerebral thrombosis and for the prevention of cerebral infarction.

Thromboxane & @X42), a cyclooxygenase (COX) metabolite of arachidonic acid formed mainly by platelets, is a potent inducer of platelet aggregation and vasoconstrictor (1,2). Prostacyclin (PG12), another COX metabolite, formed mainly by endothelial cells, is a strong inhibitor of platelet aggregation and vasodilator. An imbalance of the TXA2-PGI2 system is thought to be associated with the pathogenesis of various thrombotic and ischemic disorders (3,4). Enhanced TXA2 production has been reported in patients with ischemic heart diseases, arteriosclerosis, diabetic disorders, and cerebral thrombosis (4). Therefore, inhibition of TXA2 generation or Key words: CV-4151, Isbogrel, Thromboxane &, Cerebral thrombosis model Corresponding author: Yoshimi Imura, Ph. D. Pharmaceutical Research Laboratories I, Takeda Chemical Industries, Ltd., 17-85 Juso Homnachi 2-Chome, Osaka, 532, Japan 95

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TXAz action might be one approach for clinical intervention in these diseases. Aspirin, a COX inhibitor, has antithrombotic and beneficial effects in patients with atherosclerotic disease, myocardial infarction, and transient ischemic attack (5-9). However, in addition to inhibition of platelet COX, the simultaneous inhibition of endothelial COX by aspirin limits its antithrombotic activity, as it would also inhibit the endothelial production of PGI2. Over the last decade, a considerable effort has been made in the search for, and development of selective TXA2 synthase inhibitors. Such inhibitors might have antithrombotic effects superior to those of aspirin, as they would selectively inhibit TXAz generation, and simultaneously increase the production of PGIz as a consequence of transfer of accumulated platelet prostaglandin (PG) endoperoxide, the common precursor of TXA2 and PG12, to endothelial cells (10-14). (Y-4151 (isbogrel), (E)-7-phenyl-7-(3-pyridyl)-6-heptenoic acid, is a potent and selective TXA2 synthase inhibitor (15, 16). CV-4151 inhibits carotid artery thrombosis in rabbits and femoral vein platelet-rich thrombosis in rats by inhibiting TXA2 generation and increasing PGI2 production (17, 18). Furthermore, CV-4151 improves neuronal deficiency and cerebral hypoperfusion in the dog cerebral ischemia-reperfusion injury model possibly by inhibiting thrombus formation and cerebral blood vessel spasm after reperfusion (19). To evaluate further the antithrombotic effect of CV4151 in a cerebral thrombosis model, and to compare the effects of TXA2 synthase inhibitors with those of aspirin and ticlopidine, an antiplatelet agent (20, 21), we investigated the antithrombotic and antiedema effect of these agents in the well established rat MCA thrombosis model induced by photochemical reaction (22, 23). Since 24 h after thrombotic occlusion of the MCA, cerebral edema is observed in this model, the antiedema effects of these drugs were also examined. MATERW

AND METHODS

Effect on blood TXAz generation in vitro

Blood was collected by aortic puncture from anesthetized rats and guinea-pigs, by cardiac puncture from rabbits, by cephalic vein puncture from dogs and by cubital vein puncture from healthy human volunteers who had not ingested any drugs for 2 weeks previously. The blood (950 ~1) was incubated with 50 ~1 drug solution or vehicle (saline) at 25 “C (for rat, human, dog and guinea-pig) or at 37 “C (for rabbit ) for 90 min, and then centrifuged at 8,000g for 3 min. The obtained serum was stored at -70 “C until used for the measurement of TXB2, a stable metabolite of TXA2. The level of TXB:! in serum was measured by radioimmunoassay (RIA) (16). Crossreactivity of the TXB2 antiserum used in this study with other prostaglandins was less than 0.1 % for PGEl and PGE2, 0.6% for PGFzo 0.1% for 6-keto PGFl oand 1.1% for PGD2. The detection limit of TXBz was 10 pg/50 pi/tube. ex vivo Two h after oral administration or before and 1 and 60 mm after intravenous (i.v.) bolus injection of agents, 1 ml of blood was withdrawn from the abdominal aorta of orally treated rats, or 0.4 ml of blood was taken via a right carotid artery camrula for i.v.-injected rats. The blood was incubated at 25 “C for 90 min to produce TXA2 and then centrifuged at 8,000g for 3 min. Serum TXB2 was measured by RIA as described above.

Effect on blood TXA2 generation in rats

MCA thrombosis model

The rat MCA thrombosis model was produced according to the method of Umemura et al. (23). Male Sprague-Dawley rats (300-400 g) were anesthetized with pentobarbital(50 mg/kg, i.p.), and the body temperature was maintained at 37 “C with a heating pad. The left femoral vein was camrulated for administration of rose bengal and drugs. The scalp and temporalis muscle were reflected, and a subtemporal craniotomy was performed using a dental drill under an operating microscope (SMZ-2T Nikon, Japan). Then, a circular bone window about 3 mm diameter was opened without cutting the dura mater, and revealing the main trunk of the left MCA. Rats with the MCA 74 - 134 pm in diameter were selected for the experiment. MCA thrombosis was triggered by vessel wall damage with a photochemical reaction. Photo-irradiation with green light was performed using a xenon lamp (L4997: Hamamatsu Photonics, Japan) with a heat absorption filter and a green filter. A 3-mm-diameter photofiber mounted on a micromanipulator was placed 5 mm

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above the window, then the trunk of the MCA was irradiated with green light (wavelength 540 nm, 500,000 lux) through the window for 4 min. One min after the irradiation, rose bengal (10 mg/kg) was injected for 1 min through femoral vein cannula. Thrombus formation was monitored continuously over 60 min under the operating microscope, with videotape recording using a beam splitter, a black-and-white video camera (CS3440 Type TV 5747A6 Tokyo Electric Industry, Japan), and a Hi-Fi videorecorder (Hi-Fi MULTIPLEX SVHS Panasonic video, video-monitor WV-5410, Matsushita Electric Company, Japan). The total magnification of this system was x58 using this video-recording system, blood flow in the MCA is visualized as gray, whereas thrombus is visualized as white. The thrombus and complete thrombotic occlusion in the MCA can be observed under the microscope. The effect of drugs was evaluated in terms of prevention of the incidence of complete thrombotic occlusion 60 min after irradiation. In a sham-operation group, irradiation without rose bengal was performed. Drugs were suspended in a gum arabic solution and administered orally 90 min (CV-4151, ozagrel hydrochloride and aspirin) or 180 min (ticlopidine) before the experiment. In post-treatment experiments, CV-4151 with equimolar sodium hydrochloride, sodium ozagrel (Cataclot@ Ono Pharmaceuticals, Japan) and lysine aspirin (Wenopyrin@ Green cross, Japan) were solved in physiological saline, and then injected through the femoral vein cannula 1 min after the completion of irradiation. In the control group, gum arabic suspension or saline was administered as a vehicle. Measurement of water and electrolyte content in the cerebral cortex

The incision made in treated rats was closed quickly, and the rats were anesthetized 24 h after the completion of irradiation. Blood was collected by aortic puncture to examine blood TXA2 generation, and the cerebrum was removed. Then, the left (photo-irradiated) and right (contralateral) cortex were isolated from the cerebrum for measurement of water and electrolyte contents. Isolated cerebral cortices in preweighed vials were weighed to obtain the wet weight (W), then dried in an electric drying oven (SF32D, Advantec) at 95 “C for 72 h and reweighed to obtain the dry weight (D). The water content (%) was calculated as (W-D)/W x 100. The dried tissues were digested in 2.5 ml of 5 N nitric acid for 1 week and the electrolyte content in the digest was measured by atomic absorption spectroscopy (Flame Photometer, Corning). Electrolyte content was expressed as mEq/kg tissue dry weight. Drugs were given by oral administration 90 min before the operation. Measurement of relative change in lactate content by IH-magnetic

resonance spectroscopy

Relative change in lactate content in the brain was determined using lH-magnetic resonance spectroscopy (rH-MRS). ‘H-MRS was performed for sham, control and CV-4151 (10 mg/kg, p.o.)-treated rats using a 4.7 Tesla NMR spectrometer (CSI-II Omega System, Bruker, USA) 24 h after the irradiation. Skin and bilateral temporal and occipital muscle were retracted to eliminate contamination of the signal arising from muscles around the head, and each animal was placed in a specially designed Plexiglas holder. A surface coil tuned for a ‘H resonance frequency of 81.042 MHz was positioned centrally over the cranium. After positioning the animal in the center of the magnet bore and optimizing magnetic field homogeneity by shimming on the lH resonance of water, ‘H-MRS was carried out using 1.1-2.2 spin echo water suppression technique (echo time was 136 m seconds). The pre-delay time was 1,000 m seconds, and 90 degree pulse width was 20 p seconds. The accumulated free induction delays of 120 scans (spectra width 4,000 Hz, 2048 data points) were analyzed following zero filling, application of a 5 Hz Gaussian filter, and Fourier transformation. The relative lactate level was calculated from the peak ratio using Nacetylaspartate as an internal standard. Rats were kept anesthetized during measurement by inhalation of 1.5-2.0% halothane in @. (Y-4151 was given orally 90 min before the operation. Measurement of cerebral infarct volume by magnetic resonance imaging

Magnetic resonance imaging (MRI) of rat brain was performed under halothane anesthesia using a NMR spectrometer (CSI-II OMEGA system 4.7T/33 cm, Broker) with a superconducting magnet and an 8.5-cm inner-diameter, low-pass birdcage proton imaging coil. T2-weighted spin-echo imaging (TR 2000 ms, TE 90 ms, Slice thickness 2.0 mm, slice separation 2.0 mm) of 4 multislices were obtained with a field of view (Fov) of 100 mm in which two scans were averaged for each one of the 256 phase-encoding steps resulting in a total acquisition time of about

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18 min. Area of the infarct region was measured as the high intensity region using an MCID (Micro Computer Imaging Device, Neuroscience), and infarct volume was calculated by multiplying the infarct area and slice thickness. Total infarct volume was calculated by summation of the infarct volume from slice No. 1 to No. 4, which were coordinated with the stereotaxic atlas of the rat brain described by Pellegrino and Cushman (slice No. 1 anterior 1.0-3.0; slice No. 2 anterior 3.0-5.0; slice No.3 anterior 5.0-7.0; slice No. 4 anterior 7.0-9.0). Materials

CV-4151, (E)-7-phenyl-7-(3-pyridyl)-6-heptenoic acid, TXBz and ozagrel (OKY-046), (E)-3-[P(lH-imidazol-1-ylmethyl) phenyll-2-propenoic acid, were synthesized at Takeda Chemical Ind. Ltd. Ticlopidine, 5-[(2-chlorophenyl) methyl]-4,5,6,7-tetrahydrothieno [3,Zc]pyridine hydrochloride, was extracted from Panaldinea tablets (Daiichi Seiyaku, Japan). The antibody against TXBz was prepared at Takeda Chemical Ind. Ltd. Rose bengal (Wake Pure Chemicals, Japan), aspirin (Sigma, U.S.A.), and [3H]TXB2 (150 Ci/mmol, New England Nuclear, U.S.A.) were purchased commercially. Statistical analysis

Data are shown as mean + SEM. The significance of drug effects versus the control group was determined by x 2-test for evaluation of antithrombotic effects in the MCA thrombosis model, and Dunnett’s test and Student’s t-test for other experiments. RESULTS Znhibitory effect of drugs on blood TXA2 generation in vitro

To select a suitable animal for examining the antithrombotic effect of CV-4151 in vivo, the potency of its inhibitory action on TXA2 generation in blood was studied in rats, rabbits, humans, dogs and guinea-pigs. (X-4151 inhibited TXA2 generation in rat, rabbit and human blood with ICso values of 0.09 (95% confidence limits: 0.08-O.lO), 0.05 (0.02-0.14) and 0.27 (0.12-0.60) PM, respectively (FIG. 1A). In these three species, CV-4151 inhibited blood TXA2 generation in a same range of concentration. In dog and guinea-pig blood, inhibitory effect of CV-4151 was weaker than that in rat, rabbit and human blood, with ICso values of 1.09 (0.36-3.59) and 11.3 (8.6-14.9) PM, respectively. Gzagrel and aspirin inhibited TXA2 generation in rat blood with ICso values of 0.26 (0.13-0.53) and 11.9 (6.81-21.5)pM, respectively (FIG.lB).

f

‘T

[

?

. P . lL!z!

m 60’ 2 40’ F z 20’ c 0’ gO = r

n

:Rat : Rabbit : Human :Dog : Guinea -pig

: cv-4151 A:oza rel n :AspnnPl

.

.

-2o-

6

Concentration of

W-4151

7

Concmttation

(-IogM)

FIG.

6

of drug

6

4

(-IogM)

1

Inhibitory effect of (X-4151 on TX42 generation in rat, rabbit, human! *dog and guinea-pig blood (A), and inhibitory effects of CV-4151, ozagrel and aspnm on rat blood (B) in vitro. The concentrations (@ml) of TXB2 in controls were 91kll for rat, 74232 for rabbit, 150*30 for human, 595254 for dog and 194*6 for guinea-pig. Data shown are mean &EM. n=3-7.

CV-4151 AND MCA THROMBOSIS

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99

effects of drugs on the MCA thrombosis model in rats

Irradiation with green light alone failed to produce thrombus formation in the MCA. In irradiated rats that received rose bengal, a thrombus was formed immediately, sometimes flowed upstream, and finally produced occlusion in 6 of 7 rats, with a mean time to occlusion of 6.021.0 min. Pretreatment with (X-4151 (1 and 10 mgkg, p.o.) and ozagrel (10 mgkg, p.o.) significantly inhibited thrombotic occlusion (FIG. 2A). Aspirin (100 mg/kg, p.o.) and ticlopidine (300 mg/kg, p.o.) showed an inhibitory tendency. In post-treatment experiments, drugs were injected intravenously 1 min after the completion of irradiation, when the thrombus had already been formed, but had not produced occlusion. CV-4151 (0.1 and 1 mg/kg, i.v.) dose-dependently inhibited the thrombotic occlusion (FIG.2B), ozagrel (1 mg/kg, i.v.) also showed inhibition. Aspirin at 3 mgkg showed inhibitory tendency, but at a higher dose, 30 mg/kg, it had no effect. The antithrombotic potency of CV-4151 was about 10 times stronger than that of ozagrel in both the pre- and post-treatment experiments. A 100‘

0.1 Control

0’

1

1

cv-4151

0.01 Control

10

0.1 cv-4151

10

10

Aspirin

Ozagrel

0.1

1

Ozagrel

FIG.

100

1

100 300 (mg/kg) Ticlopidine

0.3

3

30

OWW

Aspirin

2

Antithrombotic effects of drugs by oral administration (A), and intravenous bolus injection (B) in the rat MCA thrombosis model. Drugs were given orally 2 h before irradiation, or intravenously 1 min after the completion of irradiation. Data shown are MCA occlusion rates. x 2-test was used for statistical analysis. *:p
100

CV-4151 AND MCA THROMBOSIS

Inhibitory

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effects of drugs on blood TXA2 generation ex vivo

Two h after oral administration, CV-4151 (0.01, 0.1, 1 and 10 mg/kg), ozagrel (0.1, 1 and 10 mg/kg) and aspirin (10 and 100 mg/kg) inhibited blood TXAz generation ex vivo in a dosedependent manner (TABLE I) (18). One min after intravenous injection, (X-4151 (0.1 and 1 mg/kg), ozagrel(O.1 and 1 mg/kg) and aspirin (3 and 30 mg/kg) markedly inhibited blood TxA;! generation, and their effects lasted for 60 min (TABLE II). TABLE

I

Inhibitory effects on blood TXA2 generation in rats 2 h after oral administration ex vivo

Dw cv-4151

Ozagrel Aspirin

Dose (mg/kg) 0.01 0.1 1 10 0.1 1 10 1 10 100

Number of experiments 10 20 13 14 5 10 5 5 5 13

30*4 6323

* **

9221 9520.4

** **

22*7 8324 98+0.5 9*12 7423 96+1

Data shown are mean&EM from reference (lS).*p:
Inhibition (%)

** ** ** **

**PC 0.01 vs control value.

II

Inhibitory effects of drugs on blood TXA2 generation in rats 1 and 60 min after bolus injection ex vivo

Dws Control (Y-4151 Ozagrel Aspirin

Dose@VW 0.01 0.1 A.1 1 0.3 3 30

Inhibition (%) 60 min after injection 1 min after injection -0.3*10 66+3 ** 9821 ** 100 ** 97cl * 100 ** 52*22 ** 8829 * lOO+O.l **

-19*17 31+1 ** 68*3 ** 9821 ** 45+6 ** 77*5 ** 42218 ** 86c8 * 100 *

Data shown are mean&EM from 3-8 experiments. *pcO.O5; **p< 0.01 vs pre-value. TXB2 concentrations of mean pre-value in each group were 120-176 &ml.

CV-4151 AND MCA THROMBOSIS

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101

effects of drugs on cerebral edema in the MCA thrombosis model in rats

In the sham group, 24 h after irradiation without rose bengal, the water and sodium contents in the irradiated left cerebral cortex were slightly but significantly increased in comparison with those in the contralateml right cerebral cortex (TABLE III). The potassium content did not change. The water and sodium contents in the irradiated cerebral cortex of control rats increased markedly, and the potassium content decreased in comparison with those in the irradiated cerebral cortex of shamtreated rats (TABLE III). Pre-treatment with (Y-4151 (0.1 - 10 mg/kg, p.o.) dose-dependently inhibited the changes in these parameters. Significant effects were observed at doses of 1 and 10 mg/kg CV-4151. Ozagrel (10 and 100 mg/kg, p.o.) and ticlopidine (300 mg/kg, p.o.) produced significant inhibition, and aspirin (100 mg/kg, p.o.) showed an inhibitory tendency. No significant difference in the water content on the contralateral cortex was observed among the groups. In this experiment, blood TXA2 generation 24 h after MCA injury was significantly inhibited in the CV4151 (1 and 10 mg/kg, p.o.)-, ozagrel (100 mg/kg, p.o.)- and aspirin (10 and 100 mg/kg, p.o.)treated groups (TABLE IV). TABLE

III

inhibitory effects of drugs on cerebral edema in the rat MCA thrombosis model

Drug h&) Sham

Number 6

Cortex ‘Left 2)Right

Water content (%) 79.75kO.17 79.02 + 0.09

K content @wg)

Na content (mWW

1

**

**

I

Control 25 Left 81.48f0.13 cv-4151 0.1 8 Left 81.28 f0.28 1 8 Left 80.49*0.25** 10 Left 8 80.39f0.29** Ozagrel 1 Left 81.16kO.24 8 10 8 Left 80.68 *0.31* 100 Left 8 80.36*0.25** Aspirin 1 7 Left 81.38 zk 0.35 10 8 Left 81.13f0.25 100 9 Left 80.97 f 0.47 Ticlopidine 100 9 Left 81.27 f0.23 300 8 Left 80.74+0.34* Data shown are mean&EM. 1):Irradiated left cortex, 2): Contralateral right cortex. Dunnett’s test was used for statistical analysis. *:p
255hll 225f6

* 1

346+9

**

1

503+7 506+4 447+5

327+21 286f12** 290+14**

444+12 465f7 476+8*

325 f 14 296+16* 293 zk 14*

440f4 44959 443f8

350f21 330+.17 320f31

434213 456f3

335 * 13 289+20**

473+6* 472? ll*

471f6*

** 1

102

CV-4151 AND MCA THROMBOSIS

TABLE

IV

Inhibitory effects of drugs on blood TX& MCA occlusion Dose @g/kg) 0.1 1 10

Dw Sham Control Cv-415 1

Ozagrel

1 10 100 1 10 100

Aspirin

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generation in rats 24 h after

Number of experiments 6 25 8 8 8 8 8 8 7 8 9

Ticlopidine

100 9 300 8 Data shown are meamSEM. Dunnett’s test was used for statistical analysis. **:p
96fll 102f7 12s+20 46f13** 5+3** 131 ItI 17 119+20 35f6** 117+16 48+10** 12f3** 130f12 781+7

Eflect of CV-4151 on relative lactate content Following MCA occlusion, the relative lactate content was markedly increased in the control group in comparison with that in the sham-operated group. (X-4151 (10 mg/kg, p.o.) significantly inhibited the increase in the relative lactate content (FIG. 3).

FIG.3 Effect of (X-4151 on relative lactate content in the brain of rats with MCA occlusion. Data shown are mean c SEM for 3-8 experiments. Student’s ttest was used for statistical analysis. t:p
Sham

Control

cv-4151 10 mgikg, p.0.

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CV-4151 AND MCA THROMBOSIS

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Effect of CV-4151 on the volume of cerebral infarction

As shown in FIG. 4A, ‘ITweighted spin-echo MRI clearly demonstrated tissue injury 24 h after irradiation in the MCA regions. The anatomical distribution of increased signal intensity was observed mainly in the partial, sensory-motor and temporal cortice of the ipsilateral hemisphere of the irradiation and a small part of the parietal cortex of the contralateral hemisphere. In a more rostra1 part of the ipsilateml hemisphere, there was extensive cortical and subcortical involvement of gray matter. When (X-4151 (10 mg/kg, p.o.) was given 90 min before irradiation, the high intensity area was reduced (FIG. 4A). The amelioration was remarkable in the region between the parietal cortex and temporal cortex. The infarct volume was reduced by about 37 % by pretreatment with (Y-4151 (FIG. 4B).

A

Control

Control

CV-4151 (10 mg/kg, p.o.)

FIG.4 A typical magnetic resonance imaging in the brain of control and CV-4151 (10 mg/kg, p.o.)-treated rat (A), and ameliorative effect of CV-4151 on infarct volume in the brain bf iats with MCA’.&clusion (B). Data shown are mean*SEM for 5 experiments. Student’s t-test was used for statistical analysis. **:pcO.Ol.

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DISCUSSION Thrombotic occlusion of the internal carotid artery (ICA) and the MCA is the most common cause of cerebral infarction. In the Japanese population, thrombotic MCA occlusion appears to have a higher incidence than ICA occlusion (24). In a previous study, we have found that CV-4151 inhibited rabbit carotid artery thrombosis induced by endothelial damage, and demonstrated that the antithrombotic efficacy of (X-4151 was significantly correlated with its inhibition of blood TX42 generation ex vivo (17). It was reported that thrombomodulin, a cofactor for protein C activation presented on endothelial cells, is not present in the brain (25). This finding indicates that cerebral arteries are relatively thrombogenic compared arteries in other tissues. To clarify whether CV-4151 has the same potent antithrombotic effect against cerebral artery thrombosis as it has against carotid artery thrombosis, we investigated the effect of (X-4151 in a cerebral artery thrombosis model. The photochemical reaction-induced rat MCA thrombosis model was used, since (X-4151 inhibited TXAz generation in human and rat blood in vitro with comparable efficacy (FIG. lA), and the photochemical reaction in blood vessels produces oxygen radicals which injure endothelial cell, resulting in reproducible platelet-rich thrombus formation (23). Oral administration of CV-4151 inhibited both thrombotic MCA occlusion and blood TXA2 generation with the same dose range (FIG. 2A and TABLE I). Ozagrel, a TXA2 synthase inhibitor with a different molecule as structure, also inhibited the occlusion. The antithrombotic potency of CV-4151 was about 10 times stronger than that of ozagrel which is in good agreement with the inhibition of blood TXA2 generation ex vivo (FIG. 2A, TABLE I). These findings indicate that the ratio of antithrombotic potency between CV-4151 and ozagrel is due to their inhibitory potency of TXA2 synthase ex vivo. Aspirin (100 mg/kg, p.o.), however, showed a moderate antithrombotic effect, although it inhibited blood TXA2 generation almost completely. These results support the previous hypothesis that in addition to the inhibition of TXA2 generation, increased synthesis of PGI2 by selective inhibition of TXA2 synthase might contribute to the antithrombotic effect of TXA2 synthase inhibitors (14, 17, 18). Although ticlopidine shows potent antiplatelet and antithrombotic effects upon repeated administration, its effects following a single dose were weaker than those of TXA2 synthase inhibitors (FIG.2A). Generally, TXA2 synthase inhibitors have weak inhibitory effects on platelet aggregation in vitro, because prostaglandin endoperoxide (PGHz), a precursor of both TXA2 and PGI2, accumulates as a result of the inhibition of TXA2 biosynthesis by TXA2 synthase inhibitors and acts as TXA2 agonist (26). However, in the presence of PGI2 synthase, which is the situation in the MCA thrombosis model, TXA2 synthase inhibitors might show potent antithrombotic effects by inhibiting TXA2 generation and redirecting the metabolism of the accumulating PGH2 to PGI2. In fact, (X-4151 markedly inhibits arachidonic acid-induced platelet aggregation in rabbit platelet-rich plasma in the presence of rat aortic rings, as a source of PGI2 synthase, while it moderately inhibits platelet aggregation without rat aortic rings (27). In the post-treatment experiments, established mural thrombi in the MCA, which were not occlusive, flowed upstream after i.v. injection of (X-4151 and ozagrel, and occlusive thrombi were not formed frequently. Aspirin (3 mg/kg, i.v.) tended to inhibit thrombotic occlusion, but at 30 m@g, i.v., which would have completely inhibited TXA2 generation, it had no effect. The apparent biphasic antithrombotic effects of aspirin might be “aspirin dilemma” (FIG.2). These findings coincided with those of our previous study using the rat femoral vein platelet-rich thrombosis model and a continuous and quantitative monitoring system (18, 28). In the femoral vein thrombosis model, a mural thrombus formed immediately after vessel wall injury, then sometimes some thrombus fragments flowed upstream, leading to final disappearance of the mural thrombus. Thrombus formation in wivo is dynamic, and regulated continuously by a balance of endogenous inducers, such as subendothelial collagen, tissue factor, thrombin, ADP and TXA2, and also by endogenous inhibitors, such as nitric oxide, PGI2, tissue type plasminogen activator and blood pressure (17, 29-32). Post-treatment with CV-4151 inhibited further thrombus formation and accelerated thrombus disappearance, due to the inhibition of TX42 generation and increase of PGI2 generation (16, 18). This might occur in the MCA thrombosis model, although there is a qualitative difference between arterial and venous thrombi. This evidence suggests that

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TXA2 synthase inhibitors may exert therapeutic effects at the early stage of thrombus formation in patients with cerebral thrombosis. It has been reported that MCA occlusion causes metabolic disturbances in the brain, resulting in edema (33). In the present study, 24 h after the MCA occlusion with a thrombus induced by irradiation with rose bengal, the water and sodium contents of the cerebral cortex increased significantly, the potassium content decreased, and proton NMR analysis showed a significantly increased lactate content. Boquillon et al, reported that in a photochemically induced cerebral infarction model in mice, the water and sodium contents of the cerebrum increased and the potassium content decreased in mice that received rose bengal, but not in those without rose bengal 24 h after irradiation through the intact skull surface (22). However, in our experiments, the water and sodium contents in the cortex of sham-treated rats were slightly but significantly increased (TABLE III). This seems to be the reason for the surgical damage. Umemura et al. observed cerebral infarction in this MCA thrombosis model 24 h after MCA occlusion with a thrombus and demonstrated that post-treatment with a thrombolytic agent reduced the infarct size by reopening the MCA (23). Since cerebral edema was also observed in this study, we examined the preventive effects of antiplatelet drugs on cerebral edema in the MCA thrombosis model. (Y-4151 inhibited the changes in the water, sodium and potassium contents at doses of 1 and 10 mgkg, which also inhibited blood TXAz generation by more than 50%, and at the latter dose inhibited the increase in lactate content. These results indicate that (Y-4151 inhibits cerebral edema through amelioration of metabolic disturbance, as a result of inhibition of MCA occlusion with thrombosis. Ozagrel (100 mg/kg, p.o.) inhibited the increase in water and sodium contents, but did not inhibit the decrease in potassium content. The reason for this result is not clear. Aspirin (100 mg/kg, p.o.) and ticlopidine (300 mg/kg, p.o.) moderately inhibited these changes, but their potency was weaker than those of TXA2 synthase inhibitors. The doses of both CV-4151 and ozagrel which show antiedema effect are comparable to those which show antithrombotic effects and inhibit blood TXA2 generation. Post-treatment with (X-4151 (10 mg/kg) after complete MCA occlusion did not have a significant antiedema effect in this MCA thrombosis model (unpublished data), indicating that TXA2 dose not directly contribute to cerebral edema. The main cause of cerebral edema in this model seems to be thrombotic MCA occlusion, which is caused by TX42 and not to be a direct effect of TXA2. This is also supported by the fact that ticlopidine at 300 mg/kg, a dose which dose not inhibit blood lXA2 generation, inhibits cerebral edema by the inhibition of MCA thrombosis. These pharmacological evidence indicates that TX& synthase inhibitors inhibit cerebral damage by inhibition of MCA occlusion with a thrombosis, probably resulting from the inhibition of TXA;! generation. TXA2 might play an important role in cerebral damage in the rat MCA thrombosis model. In conclusion, TX42 synthase inhibitors show antithrombotic and antiedema effects superior to those of the conventional antithrombotic drugs, aspirin and ticlopidine, in the rat MCA thrombosis model. The antithrombotic effect of (X-4151 was about 10 times more potent than that of ozagrel. CV-4151 might therefore be a useful drug for the treatment of cerebral thrombosis and for prevention of cerebral infarction. Acknowledgments The authors thank Mr. H. Nakagawa for excellent technical assistance, and also Dr. T. Naka for helpful discussion. REFERENCES 1. HAMBERG, M., SVENSSON, J. and SAMUELSSON. B. Thromboxanes: A new erouo of biologically active compounds derived from prostaglandin ‘endoperoxides. Proc Nat1 &ad’ Sci USA 72, 2994-2998,1975. 2. MONCADA, S. and VANE, J.R. Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane AZ and prostacyclin. Pharmacol Rev 30, 293-331, 1979. 3. WHITTLE, B. J.R. and MONCADA, S. Pharmacological interactions between prostacyclin and thromboxanes. Br Med Bull 39, 232-238, 1983.

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