prostaglandin endoperoxide receptor, on blood vessels

193

European Journal of Pharmacology, 168 (1989) 193-200 Elsevier

WP 50948

Effects of ONO-3708, an antagonist of the thromboxane A ,/prostaglandin endoperoxide receptor, on blood vessels Kigen Kondo,

Rumi Seo, Nagashige Omawari, Haruo Imawaka, Korekiyo Tadao Okegawa and Akiyoshi Kawasaki

Wakitani,

Heizo Kit-a,

Minase Research Instriute, Ono Pharmaceutical Co., Ltd., Shwnamoto-rho, Mishima-gun, Osaka 618, Japan Received

2 May 1989, accepted

20 June 1989

The pharmacological properties of a novel thromboxane A,/prostaglandin endoperoxide receptor antagonist, ONO-3708, on blood vessels were examined in vitro and in vivo. ONO-3708, 10 FM, inhibited the rabbit aorta H, (U-46619) or contractions induced by thromboxane A,, prostaglandin H,, 11,9-epoxymethano-prostaglandin prostaglandin F,,without affecting the contractions induced by angiotensin II, serotonin or norepinephrine. ONO-3708, at a concentration of 1 to 100 nM, appeared to be a competitive inhibitor of the contractile responses of the canine basilar artery to 9,11-epithio-11,12-methano-thromboxane A, (STA,), U-46619 and PGF,,, and a non-competitive inhibitor of the contractile responses to 15-hydroperoxy-eicosatetraenoic acid (15-HPETE). In in vivo studies, ONO-3708 (10 and 100 pg/kg per min i.v.) ameliorated the decrease in diameter of the basilar artery induced by the i.v. infusion of STA, (0.1 pg/kg per min) in cats. Furthermore, infusion of ONO-3708 (10 and 30 pg/kg per min i.v.) prevented the cerebral vasospasm in an experimental subarachnoid hemorrhage model in dogs. These results indicate that ONO-3708 is a potent antagonist of the thromboxane A,/prostaglandin endoperoxide receptor in vitro and in vivo and may be of therapeutic use in preventing cerebral vasospasm. Thromboxane A,/prostaglandin endoperoxide receptor; Cerebral vasospasm; Thromboxane A ,/prostaglandin endoperoxide receptor antagonists

1. Introduction

Thromboxane A 2 (TXA 2) and prostaglandin (PG) endoperoxides are potent substances for contracting blood vessels and aggregating platelets (Needleman et al., 1976). The participation of TXA, and PG endoperoxides in vascular diseases, cerebral vasospasm in dogs (Sasaki et al., 1982) and coronary spasm in humans (Tada and Kuzuya, 1985) has been suggested. However, the roles of the TXA,/PG endoperoxide receptor have not

Correspondence Pharmaceutical 618. Japan.

to: R. Seo, Minase Research Institute, Ono Co., Ltd., Shimamoto-cho, Mishima-gun, Osaka

0014-2999/89/$03.50

0 1989 Elsevier Science

Publishers

been fully elucidated because of the short half-life of TXA *, 0.5 min at 37 ’ C, and of PG endoperoxides, 5 min at 37 o C (Hamberg and Samuelsson, 1974; Hamberg et al., 1975). Especially, in in vivo studies, it is almost impossible to examine their sustained actions on the vascular system. We recently developed 9,11-epithio-11,12-methanoTXA, (STA,) as a stable TXA z agonist (Katsura et al., 1983: Mais et al., 1985) and 7-[2a,4cy-(dimethylmethano)-6P-(2-cyclopentyl-2fi-hydroxyacacid etamido)-icy-cycle-hexyll-5(Z)-heptenoic (ONO-3708) as a stable TXA, antagonist. It has been reported that ONO-3708 shows preventive effects on platelet aggregation and thrombosis and that these effects might be based on antagonism to the TXA-JPG endoperoxide receptor (Kondo et al., 1989).

B.V. (Biomedical

Division)

194

We now describe the effects of ONO-3708 on the contractile responses of the isolated rabbit aorta and canine basilar artery, and the efficacy of ONO-3708 to prevent cerebral vasospasm in two in vivo animal models: the cerebral vasoconstriction induced by i.v. infusion of STA, in cats and the cerebral vasospasm after experimental subarachnoid hemorrhage in dogs.

2. Materials and methods 2.1. In vitro studies 2.1.1. Chemicals The following drugs were used: STA,, PGF,,, 11,9-epoxymethano-PGH, (U-46619) and ONO3708 from Ono Pharmaceutical Co., Ltd.; angiotensin II from Peptide Research Foundation; dlnorepinephrine from Sankyo Co., Ltd.; serotonincreatinine sulfate from Nakarai Chemicals; atropine sulfate from Takeda Chemical Industries Co., Ltd.; propranolol hydrochloride from Sumitomo Chemical Industries Co., Ltd.; mepyramine maleate from May & Baker; methysergide bimaleate from Sandoz; phenoxybenzamine hydrochloride from Tokyo Chemical Industries Co., Ltd.; indomethacin and sodium aracbidonate from Sigma; [l-‘4C]arachidonic acid from New England Nuclear. PGH, and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) were prepared according to a previously reported method (Ohki et al., 1979). TXA, was prepared as described previously (Needleman et al., 1976) with the modification that rabbit platelet microsomes were used as the enzyme source. The test solution of ONO3708 was prepared by dissolving it in physiological saline in the presence of L-arginine (molar ratio, 1 : 1.2). 2.1.2. Contractile responses of the isolated rabbit aorta and canine basilar artety to various substances Male albino rabbits weighing 2.0 to 3.0 kg were killed by rapid exsanguination. The thoracic aorta was excised and cut helically (3.0 mm in width and 50 mm in length) in Krebs-Henseleit medium (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO,. 7H,O, 1.2 mM KH,PO,, 2.5 mM CaCl, .2H,O,

25 mM NaHCO, and 5.8 mM glucose) aerated with 95% 0, and 5% CO,. The helical strips were superfused at a rate of 5 ml per n-tin with the Krebs-Henseleit medium. The resting tension was adjusted to 1.0 g. In the experiment on TXA,-induced contraction, a modified Krebs-Henseleit medium which contained atropine sulfate (0.1 pg/ml), propranolol hydrochloride (2 pg/ml), methysergide bimaleate (0.2 pg/ml), mepyramine maleate (0.1 pg/ml) and phenoxybenzamine hydrochloride (0.1 pg/ml) was used to block the actions of vasoactive substances in platelet microsomes. The contractions induced by vasoactive substances (injection volume; 50 ~1) were measured by means of a force-displacement transducer (Nihon Kohden; SB-1T) equipped with a carrier amplifier (Nihon Kohden; PR-5A) and were recorded on a recticorder (Nihon Kohden; RJG4128). ONO-3708 was infused into the stream of medium at a rate of 0.05 ml/mm (X 100 dilution). The contractile responses of the isolated canine basilar artery to vasoactive substances were studied according to the method of Koide et al. (Koide et al., 1982). Briefly, mongrel dogs of either sex weighing 6 to 13 kg were anesthetized with pentobarbital sodium (Pitman-Moore Inc.; 30 mg/kg i.v.) and were killed by rapid exsanguination. After isolation of the brain, the basilar artery was excised and cut into a cylindrical ring of 3 mm length. The ring was bathed in a chamber containing 10 ml of modified Krebs-Ringer medium (120 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl,. 2H,O, 1.0 mM MgSO,. 7H,O, 1.0 mM KH,PO,, 27 mM NaHCO,, 10 mM glucose and 0.01 mM EDTA-2Na) and gassed with 95% 0, and 5% CO,. The resting tension was adjusted to 3.0 g. The contractions of the preparation were measured as described above. The pA, values were determined from Schild plot analysis (Tallarida et al., 1979). 2.1.3. Cyclic GMP content in rabbit aorta The rabbit thoracic aorta was isolated and cut into cylindrical rings of 1 mm length. Two rings were immersed in Krebs-Henseleit medium aerated with 95% 0, and 5% CO,. Drugs were added after 2-min preincubation at 37” C and the reaction mixture was incubated further for 100 s. The

195

reaction was terminated by the addition of 6% trichloroacetic acid. The cyclic GMP content was determined by the method described previously (Honma et al., 1977) after the removal of trichloroacetic acid with water-saturated diethyl ether. 2.2. In vivo studies 2.2.1. Cerebral vasospasm induced by STA, infusion in cats Adult cats of either sex weighing 2.6 to 4.0 kg were anesthetized with ketamine hydrochloride (Sankyo Co., Ltd.,; 20 mg/kg i.m.) and the head was placed in a stereotaxic instrument. After endotracheal intubation, the cats were paralyzed with gallamine triethiodide (Sigma; 10 mg/kg i.v.) and lightly anesthetized with 0.5% halothane (Hoechst). Catheters were introduced into the femoral vein and artery. Arterial blood gases were measured frequently (Radiometer; ABL 30) and were maintained within the following ranges: pH 7.3 to 7.5; PaO, 80 to 120 mmHg; PaCO, 30 to 40 mmHg with a respirator (Shinano; SN-480-6). The basilar artery was exposed by a transcervico-transclival approach (Echlin, 1965) under an operating microscope (Olympus OME). Regional cerebral blood flow was determined by a standard inhalation hydrogen clearance method (Tamura et al., 1980). Two to four urethane-coated platinum electrodes were positioned into the piriform cortex around the basilar artery at a depth of 1 mm, and the regional cerebral blood flow was measured with a UH-meter (Unique Medical; MHG-DI). Photographs of the basilar artery were taken and were magnified (X 20) to measure the diameter of the artery. STA, (0.1 pg/kg per min i.v.) was administered for 2 h, and ONO-3708 (10 and 100 pg/kg per min i.v.) was infused continuously from 1 h after the start of STA, infusion. 2.2.2. Cerebral vasospasm in an experimental subarachnoid hemorrhage model in dogs Mongrel dogs of either sex weighing 8 to 12 kg were anesthetized with pentobarbital sodium (30 mg/kg i.v.). The left vertebral artery was cannulated for angiography and the femoral vein and artery were cannulated for drug infusion and monitoring arterial blood gases, respectively. The

head was placed in a stereotaxic instrument and arterial blood gases were maintained within the physiological conditions as previously described above. Experimental subarachnoid hemorrhage was induced by the intracisternal injection of fresh arterial blood; a 22-G needle was inserted into cisterna magna and cerebrospinal fluid (7 ml) was replaced with an equivolume of autologous fresh arterial blood within 60 s. The dogs were tilted with tail up for 30 min to facilitate settling of the blood around the basilar artery. ONO-3708 was infused (i.v.) for 3 days from 2 h after the arterial blood injection. Angiograms were taken [Toshiba Medical; KXO-12( M)] with urografin (Schering AG; 4 ml) and were magnified photographically (x2). The diameter of the basilar artery was measured at three selected points with the help of a magnifying lens ( X 10). 2.3. Statistics Statistical comparisons were made using the analysis of variance of the software package SAS (Statistical Analysis System, Cary, USA) followed by Tukey’s comparison or Student’s t-test. P values of 0.05 or less were considered statistically significant. All data are presented as means + S.E.M.

3. Results 3.1. In vitro studies ONO-3708 at a concentration of 10 PM significantly inhibited the contractile responses of isolated superfused preparations of rabbit aorta to PGH,, U-46619 and PGF,, (table 1). TXA,, However, it did not affect the contractions induced by angiotensin II, norepinephrine and serotonin. Cumulative concentration-response curves were plotted for the effect of STA,, U46619, PGF,, and 15-HPETE on canine basilar arteries. The negative logarithms of the ECSo (M) values for STA,, U-46619, PGFz, and 15-HPETE were 9.10 + 0.05 (n = 36) 8.55 k 0.06 (n = 27) 6.28 f 0.08 (n = 34) and 6.47 f 0.09 (n = 37) respectively. ONO-3708 (1 to 100 nM) shifted the

196 TABLE

1

Effect of ONO-3708 (10 PM) on vasocontractile responses of isolated rabbit aorta to various agents used in the superfusion method. TXA, was generated by the incubation (0 o C, 30 s) of PGH, (1 ug) with indomethacin-treated rabbit platelet microsomes (0.5 to 1 mg) in 0.1 ml of M/15 phosphate buffer pH 7.4. Aliquots (50 ~1) of the reaction mixture were applied just above the vessels. ’ P < 0.05 and h P < 0.01 (Student’s paired t-test). n

Agent (dose)

TXA 2 (0.5 pg) PGH, (3 /tg) U-46619 (0.1 ug) PGF,, (10 pg) Angiotensin II (0.1 ng) Norepinephrine (1 ag) Serotonin (1 pg)

3 3 6 6 5 5 3

Tension

(g) During treatment

0.720 0.602 0.497 0.489 0.765 1.038 0.921

0.255 + 0.040 0.322 f 0.027 0.171 +0.080 0.109*0.051 0.702 k 0.092 1.085 f 0.063 0.952 f 0.105

+ f + + i + f

0.030 0.017 0.161 0.121 0.123 0.088 0.059

concentration-response curves for STA,, U-46619 and PGF,, but not those for 15-HPETE to the right in parallel (fig. 1). Figure 2 shows Schild

30-

Inhibition

Pretreatment

’ ’ ’ h

(R)

64.7, 4.6 46.7* 3.5 76.Okll.l 83.5+ 6.0 5.7k 5.2 -6.Ok 4.7 -3.1 * 7.5

plots of the inhibition by ONO-3708 of the contractile responses to STA2, U-46619 and PGF,, based on the data of fig. 1. The pA, values of

U-46619

STA2

50 -

STA2 (Id)

U-46619

PGF2a ( M )

15-HPETE

(M 1

(bl)

Fig. 1. Effects of ONO-3708 on contractile responses of the isolated canine basilar artery to STA,, U-46619, PGF,, and 15-HPETE. Each compound was added cumulatively 5 min after the addition of ONO-3708. Each point represents the mean f S.E.M. of at least 4 determinations.

197 U-46619

STA, 2.0 S&e

= 1.19 (0.51-1.67

pA2 = 6.44 I e d $

)

PGF2a

93pe = 0.94

( 0.62-126 )

)

sbpe = 0.95 (0.67-1.23

( 7.91-897 1

1.0

B E 1

0 7

9

8

-Log C ONO-3706

Fig. 2. Schild plot of the inhibition

7

1 Mdar

8

9

-Log C ONO-3708

I

I

T

7

6

9

by ONO-3708 of the contractile response of canine basilar artery Each value in parentheses indicates the 95% confidence limit.

ONO-3708 were 8.44, 9.01 and 8.96 for STA,, U-46619 and PGF,,, respectively, with slope values approximated to - 1. To examine the possibility of ONO-3708 acting as a non-specific vasodilator, its effects on the cyclic GMP content in rabbit aorta were examined. ONO-3708 did not affect the cyclic GMP content in rabbit aorta, as the cyclic GMP levels were 6.3 + 0.9 and 6.3 L- 0.7 pmol/mg protein per 100 s with and without ONO-3708 (1 PM), respectively.

1 Molar

-Log C ONO-3708

1 Motar

to STA,.

U-46619

-

and PGF2,.

ONO-3708

(A)

STA2

0 Ivgikglrmn

100 pglkgimln(N=5) 10 pglkglmm

Pre

30

60

90

(N=5)

120

Time (mid (6)

Before

During

inlusion

of

STAz

(O.lpglkglmin)

100

)rg/kg/ml” (N=lO)

10

,,gikg,mm (N=13)

“ehlcle (N=l

I

1)

1

Pre

30

60

90

120

Time (min)

Fig. 3. Vasoconstriction of the infusion in cats. Left: before during i.v. infusion of STA, Arrows indicate the points

basilar artery induced by STA 2 administration of STA,. Bight: (0.1 pg/kg per min for 1 h). for diameter measurement.

Fig. 4. Effects of i.v. infusion of ONO-3708 on vasoconstriction of the basilar artery (A) and decrease in regional cerebral blood flow (B) in cats. The number of cats (A) or electrodes (B) is shown in parentheses. r-CBF; regional cerebral blood flow. ONO-3708 at both doses significantly inhibited the changes in diameter from 60 to 120 min (P < 0.01. Tukey’s test), and at 100 pg/kg per min it significantly inhibited the decrease in regional cerebral blood flow (P < 0.05, Tukey’s test).

198 TABLE 2 Preventive effects of ONO-3708 on cerebral vasospasm after experimental subarachnoid hemorrhage in dogs. N u m b e r s in parentheses are the numbers of dogs used. SAH: Subarachnoid hemorrhage, a Time after SAH. h p < 0.05 (Tukey's test on day 3). Treatment

Vehicle ONO-3708 3/~g/kgpermin 1 0 / ~ g / k g per min 30/~g/kgpermin

% decrease in the diameter of basilar artery 1 day ~

2 days "

3 days a

16.6_+2.8(15)

19.1_+3.0(15)

25.9_+2.1

18.4_+6.7 (6) 12.4-+2.6 (7) 10.8_+5.8 (8)

23.1_+7.0 (6) 11.6_+3.4 (8) 10.3+_4.3 (8)

27.8_+9.1 (6) 8.2_+4.0 b (7) 13.8+_5.4 (7)

3.2. in vivo studies

STA 2 (0.1/~g/kg per min) elicited constrictions of the cat basilar artery (fig. 3). The arterial diameters were reduced by 28.1 to 37.1% in the 3 groups (fig. 4A) 60 min after STA 2 infusion. ONO-3708 (10 and 100 /~g/kg per min) significantly inhibited the changes in diameter of the basilar artery compared to those of the vehicle group from 60 to 120 min after the start of STA 2 infusion (P < 0.01, Tukey's test). There were no significant differences in the initial regional cerebral blood flow among the 3 groups; 47.8 _+ 4.6 (n = 11; where n represents the number of the electrodes), 48.8_+ 4.6 (n = 13) and 60.5 _+ 9.2 m l / 1 0 0 g per min (n = 10) in the vehicle, O N O 3708 10 and 100 t~g/kg per min treated group, respectively. The regional cerebral blood flow decreased by 23.5, to 31.1%, at 60 min after STA 2 infusion in the 3 groups (fig. 4B). ONO-3708 ameliorated the decreases in regional cerebral blood flow in a dose-dependent manner and there was a significant difference between the vehicle and ONO-3708 100 /~g/kg per min group (P < 0.05) at 60 to 120 min after the start of STA 2 infusion. These results suggest that ONO-3708 could have exerted antagonistic activities against the TXA J P G endoperoxide receptor even in vivo. Table 2 shows the effects of ONO-3708 on vasospasm in an experimental subarachnoid hemorrhage model in dogs. Injection of autologous arterial blood produced time-dependent vasospasm in vehicle-treated dogs. After the 3-day arterial blood injection, ONO-3708 ( 1 0 / ~ g / k g per min) reduced the cerebral vasospasm as compared

(14)

to that seen in the vehicle group (P < 0.05, Tukey's test).

4. Discussion

In in vitro studies, ONO-3708 inhibited the contractile responses of isolated vascular smooth muscle to T X A > S T A > PGH2, U-46619 and PGF2~ without affecting the contractions induced by angiotensin II, norepinephrine or serotonin. The vasodilating activity of PGE~ or P G I 2 was also not affected by ONO-3708 (data not shown). Furthermore, this compound did not affect the cyclic G M P content in rabbit aorta, indicating that ONO-3708 is neither an endothelium-dependent vasodilator nor a direct activating agent of guanylate cyclase in vascular smooth muscle. The results from the concentration-response relationship and Schild plots suggest strongly that ONO3708 might be a competitive antagonist of the contractile actions of T X A 2 / P G endoperoxides and PGF2~. The slope obtained from the Schild plot was close to - 1 . 0 in the presence of the compound. ONO-3708 inhibited PGF2~-induced contractions of both isolated rabbit aorta and canine basilar artery. These observations are consistent with the results obtained for other T X A z / P G endoperoxide receptor antagonists (Jones et al., 1982; Mais et al., 1985). Vasocontractile PGs and T X A 2 appear to share the same binding site responsible for vascular contraction. ONO-3708 also attenuated the contractile responses of the isolated canine basilar artery to 15-HPETE at concentrations similar to those in-

199

hibiting the contractions induced by STA,, U46619 and PGF,,. This effect does not seem to be competitive, because the shifts of the concentration-response curve were not parallel, suggesting that ONO-3708 could be a partial antagonist of 15-HPETE. It has been reported that isolated canine arteries release TXB, and that one of the endothelium-derived contracting factors is probably TXA, (Shirahase et al., 1987). Though indomethacin does not influence 15-HPETE-induced contractions (Koide et al., 1982) antagonism of endogenous contractile prostanoids cannot be excluded as mechanism for the inhibition of 15-HPETE-induced contraction by ONO-3708. In the in vivo studies, we first examined the efficacy of ONO-3708 to inhibit the basilar artery constrictions induced by the TXA,/PG endoperoxide receptor agonist. STA, exerted cardiovascular effects not only on the basilar artery but also on systemic blood pressure (increase in arterial blood pressure). The infusion (i.v.) of STA, decreased the diameter of the basilar artery and the regional cerebral blood flow by about 30%, indicating that STA, constricts the basilar artery through the TXA */PG endoperoxide receptor, even in vivo. Furthermore, significant inhibition of the basilar artery constriction by ONO-3708 would mean that it could interact with the TXA,/PG endoperoxide receptor in the in vivo vascular system. At a dose of 10 fig/kg per min, ONO-3708 inhibited the constriction of the basilar artery much more potently than the decrease in regional blood flow induced by STA, (fig. 4). This discrepancy might have been due to the method of measuring regional cerebral blood flow. Since we had inserted electrodes into the piriform cortex around the basilar artery, the regional cerebral blood flow measured might not have directly reflected the blood flow of the basilar artery itself. We further examined the effectiveness of ONO3708 on cerebral vasospasms induced by intracisternal injection of autologous arterial blood in dogs. Cerebral vasospasm following subarachnoid hemorrhage is due to the production of multiple vasoconstrictive substances in the subarachnoid space (Boullin et al., 1981, Shigeno, 1982 and Toda et al., 1980). These substances may include prostanoids and lipoxygenase products. We con-

firmed angiographically that the late spasm (3 days after blood injection) was prevented by the continuous i.v. infusion of ONO-3708. From a therapeutic point of view, it is more important to inhibit the late than the early spasm, because it has been shown that human subarachnoid hemorrhage lacks the early phase of vasospasm which corresponds to the early spasm in animal models (Wilkins, 1976). A recent report, based on the use of a specific TXAz synthetase inhibitor, OKY1581, suggests an important role for TXA, in the cerebral vasospasm in this canine model (Sasaki et al., 1982). Similar results were also observed in a rabbit model (Chan et al., 1984). The efficacy of OKY-1581 to prevent cerebral vasospasm is due not only to decreases in TXA, levels but also to increases in PGI, levels. Since ONO-3708 does not affect prostanoid synthesis in blood vessels or platelets (Kondo et al., 1989) the increase in PGI, could not have had a part in its efficacy. After subarachnoid hemorrhage, activated platelets and white blood cells may synthesize prostanoids and/or lipoxygenase products. Several reports indicate important roles for 15HPETE in vascular systems. An increase in lipid peroxides has been reported in patients with subarachnoid hemorrhage, and intracistemal injection of 15-HPETE in dogs causes cerebral vasospasm which is similar to that seen after the induction of subarachnoid hemorrhage in a canine model (Sasaki et al., 1981). Based on our experimental results and observations, ONO-3708 appeared to prevent cerebral vasospasm by inhibiting the constriction of the basilar artery induced by prostanoids and lipid peroxides, including 15-HPETE, and thus might be useful for the treatment of cerebral vasospasm.

References

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