Prostaglandins Leukotrienes and Medicine 21: 197-206, 1986

EFFECT OF THROMBOXANE SYNTHETASE INHIBITORS (0~y-046, OKY-1580) 0~ EXPERIMENTALLY

INDUCED AIR EMBOLISM IN ANESTHETIZED DOGS

Yoko Ishihara? Yuko Uchida and Satoshi Kitamura

Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan * Division of Cell Biology, National Institute of Health, Bethesda, MD 20205 (reprint requests to SK)

ABSTRACT

We investigated the effects of OKY-046 and OKY-1580, thromboxane A2(TxA2) synthetase inhibitors, on plasma levels of 6-keto PGFld and TxB

E in anesthetized dogs with experi-

mentally induced air em olism. The percentage increase of tracheal pressure induced

by room air infusion was suppressed significantly by premed- ication with OKY-046. The percentage increase of pulmonary artery blood pressure was suppressed significantly by pre- medication with OKY-046 compared to that in control group. By room air infusion after the premeditation with OKY-046 and OKY-1580, systemic artery blood pressure did not show any significant changes. Plasma 6-keto PGFlo( level showed a marked increase by an intravenous infusion of room air, and such an increase became more predominant by the premed- ication with OKY-046 and OKY-1'580. On the other hand,plasma TxB2 level showed a marked increase by an intravenous infusion of room air, and such an increase became less predominant by the premeditation with OKY-046 and OKY-1580. These results may suggest that OKY-046 and OKY-1580 are not only TxA2 synthetase inhibitors but also PG12 synthetase accelerators and are useful drugs for treatment and preven- tion of thromboembolism.

INTRODUCTION

Prostaglandin 12(PGI2) and thromboxane A2(TxA2) are produced from PGG2 and PGH2 by the actions of TxA2 and PG12 synthetase (1.). PG12 is synthetized mainly in the vascular

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endothelial cells (2) and TxA2 in platelets (3). PG12 has a relaxation effect on the vascular and the tracheobronchial smooth muscle (4) and has an anti-platelet aggregation effect (5), whereas TxA2 has a contraction effect on the vascular and tracheobronchial smooth muscle (6) and has an accelerating effect on platelet aggregation (7).

The lung synthetizes, releases or inactivates various vasoactive substances, and its vascular beds are so vast as compared to those in other organs that various prosta- glandins including PG12 and TxA2 are metabolized efficiently. However, from the fact that the lung has the vast capillary beds comparing with those in other organs, the lung is also well known as the site of the microthrombus formation.

Recently, roles of PG12 and TxA2 in pathophysiological aspects of pulmonary thromboembolism and myocardial infar- ction have been investigated. Thus, the development of drugs which decrease serum TxA2 level and/or increase the PG12 level are expected as therapeutic drugs for pulmonary thromboembolism and myocardial infarction. In the present study we used OKY-046 and OKY-1580 which were developed as TxA2 synthetase inhibitors to demonstrate the effect of these drugs on plasma levels of 6-keto PGFlo( and TxB2 in anesthetized dogs with experimentally induced air embolism.

MATERIALS AND METHODS

The animals used in this study were young mongrel dogs aged less than 1 year weighing from 8 to 10 kg. Dogs were anesthetized with sodium pentobarbital (25 mg/kg), placed in dorsal position, a tracheal incision was perfomed, and a tracheal cannula was inserted. Then, a catheter was intro- duced into the left femoral artery to measure the systemic blood pressure and an another catheter into the left femoral vein to infuse Locke-Ringer's solution and drugs. Immediately after the intravenous injection of 2% succinyl- choline chloride, an artificial ventilation was started by a volume type respirator (Harvard Apparatus, U.S.A.) using room air. Thoracotomy was performed at the left 4th inter- costal region and a catheter was introduced into the pulmonary arterial trunk through an arterial branch of the left upper lung lobe to measure the pulmonary arterial pressure. The tracheal pressure was measured at the inlet of the tracheal cannula. The experiment was started 30 min after the thoracotomy. Experimental air embolism was con- ducted by an injection of 15 ml of room air into the femoral vein. Three hours after the injection of room air and various parameters were returned to the control level, OKY- 046 or OKY-1580 was injected into the femoral vein, and 15 min after the injection room air injection was repeated.

Blood samples collected from femoral vein were put into ice-cooled tubes containing EDTA and indomethacin and were centrifuged for 10 min at 3000 r.p.m., and plasma samples were stored at -20°C until their assay.

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OKY -046 OKY - 1580

(E) -3-(4-t 1-Imidazolylmethyl)

phenyl]-2-propenoic acid

hydrochloride

(E)-3-[4-(3-Pyridylmethyl)

phenyl)-2-methyl-2-propenoic

acid

Fig.1 Structural formula of OKY-046 and OKY-1580

Fig.2 Effect of OKY-046 on PTR, PPA and PSYST increase induced by intravenous infusion of room air in an anesthetized dog

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Plasma 6-keto PGFld and TxB2 levels were determined by radioimmunoassay kit(NEN). Results were analyzed statis- tically using Student's paired t-test with ~40.05 taken as showing a significant difference. The structural formula of OKY-046 and OKY-1580 are shown in Fig.1.

RESULTS

Fig.2 is the original charts showing the effect of OKY-046 on various parameters changes induced by air embo- lism in an anesthetized dog. By injecting 15ml of room air, tracheal pressure (PTR) and pulmonary arterial blood pressure (PPA) showed a marked increase, while systemic blood pressure (P YST) showed a decrease (Fig.2 left). Fifteen minutes a ter the intravenous infusion of OKY-046 ? (100mg) room air injection was repeated. Then the degrees of PTR and PPA increase and PSYST decrease were markedly suppressed (Fig.2 right).

Fig.3 summarizes the results obtained from the experi- ments mentioned above. This figure shows percentage change of PTR induced by room air infusion. The ordinate repre- sents the percentage change of PTR. The left figure shows the effect of OKY-046, and the right figure shows the effect of OKY-1580. The percentage increase of P R was suppressed significantly by premeditation with OKY-04 , while there ; was no significant suppression in OKY-1580 group.

Fig.4 summarizes the percentage change of PpA. As the dose of heparin was limited as much as possible in this experiment, a catheter inserted into pulmonary artery was blocked very often with clotted mass, there were small number of dogs from which information on PpA could be ob- tained. However, PPA level was decreased significantly in OKY-046 group compared to that in control group.

On the other hand, 3 of 5 dogs in OKY-1580 group showed an increase in PpA, although there was no significant dif- ference between control and OKY-1580 groups. Fig.5 summa- rizes the effects of OKY-046 and OKY-1580 on percentage change of systemic arterial blood pressure (PS

W By room

air infusion after the premeditation with OKY- and OKY- 1580, PSyST did not show any significant changes.

Fig.6 summarizes the change of plasma levels of 6-keto PGFlo( after intravenous infusion of room air. The upper figure (A) shows the effect of OKY-046 and the lower (B) OKY-1580 on plasma level of 6-keto PGFTo( after intravenous infusion of room air in anesthetized dogs. Plasma 6-keto- PGFlo( level showed a marked increase by an intravenous infusion of room air, and such an increase became more predominant by premeditation with OKY-046 and OKY-1580.

Fig.7 summarizes the change of plasma levels of TxB2 after intravenous infusion of room air. Plasma Tx82 levels showed a marked increase by an intravenous infusion of room air, and such an increase became less predominant by the premeditation with OKY-046 and OKY-1580.Plasma 6-keto PGFlo(

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I P<O.O2 ) I P<O.5

control OKY-046 Control OKY-1660 (lOOm6.i.v.) (I OC+W.v.)

Fig.3 Effect of OKY-046 and OKY-1580 on percentage change of tracheal pressure induced by room air infusion in anesthetized dbgs

I P<O.OZ

(56) 400

1 300

200

100

0 k

(%) 400

300

,200

100

0 tMl%Ot OIY-VN

~lom$.l.V.J

U.S.

(%f 400

300

200

100 b Fig.4 Effect of OKY-046 and OKY-1580 on percentage

change of pulmonary artery blood pressure induced by room air infusion in anesthetized dogs

201

(%J I (%) -80 -80

(So) -80 I (%I

-80

-70

-60

-70

-60

-70 -70

-60 -60

-50 -50

-40 -40

-30 -30

-20 -20

-10 -10

0 0 control OKY-046 control OKY-1560

(lOOm9,i.v.) (1 Ohn9,i.v.)

Fig.5 Effect of OKY-046 and OKY-1580 on percentage change of systemic artery blood pressure induced by room air infusion in anesthetized dogs

levels were significantly lower at 5 and 10 min after the intravenous infusion of room air in OKY-046 and OKY-1580 groups comparing with those in control group.

DISCUSSION

OKY-046 suppressed significantly the increase of tracheal pressure and pulmonary artery blood pressure induced by the intravenous infusion of room air, but did not affect in the fall of systemic artery blood pressure. On the other hand, OKY-1580 did not show any significant changes in tracheal pressure, pulmonary artery blood pres- sure and in systemic artery blood pressure increase induced by an intravenous infusion of room air. Although there were no significant differences between OKY-046 and OKY-1580 group and the control group, in plasma levels of 6-keto- PGFlti, plasma level of TxB2 showed a marked decrease by administration of these drugs.

It has been suggested that platelet aggregation plays an important role in pathophysiology of pulmonary thrombo- embolism induced by an intravenous injection of thrombin. In present investigation on pulmonary embolism we injected room air instead of thrombin in order to investigate the effects of OKY-046 and OKY-1580. In pulmonary air embolism, it seems that air embolism deposited in the pulmonary vascular beds will be rapidly absorbed into the circulating blood. From the results obtained in this experiment, it was demonstrated that plasma 6-keto PGFld and TxB2 levels showed marked increase by room air injection as well as by thrombin injection.

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(c13 contrd(n =4)

o---e OKY-046(n = 5) (lOOmg.i.v.)

600

500 iii f d___f____ ____---- -;

100 - i 1 O 0 5 10 20 30 (min)

Fig.6(A) Effect of OKY-046 on plasma level of 6-keto PGFlo( after the intravenous infusion of room air in anesthetized dogs

6ketoPGFI a be~ml) W control(n=4)

*---a OKY-158O(n=4) (1 OOrng, i.v.)

600 -

Fig.6(B) Effect of OKY-1580 on plasma level of 6-keto PGFlo( after the intravenous infusion of room air in anesthetized dogs

Because PGl2 and TxA2 are very unstable in circulating blood, we measured the plasma levels of 6-keto PGFlo( and TxB2 which were stable metabolites of PGI2 and TxA2, res- pectively. And from these values we could estimate those of PGI2 and TxA2.

IC50 of OKY-046 and OKY-1580 against TxA2 synthetase are 1.1 A10e8M and 2 X10-9 M, respectively, and these drugs showed marked inhibitory action on TxA2 synthetase. Although, these drugs did not show any inhibitory or accel- erating activities on cycle-oxygenase which synthetize

I prostaglandin endoperoxide at the concentration of 10‘ M

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or less (8,9). However, in our in vitro experiment using guinea pigs, these 2 drugs attenuated histamine-, serotonin-, acetylcholine-, bradykinin- and PGFzo(- induced contractile responses and potentiated isoproterenol-, salbutamol- and PGE2- induced relaxation in guinea pig tracheal strips dose- dependently. In addition, OKY-046 and OKY-1580 inhibited the biosynthesis of TxAz and accelerated the production of 6-keto PGFlo( from arachidonic acid in isolated perfused guinea pig lung lobes (10). These experimental results may suggest that OKY-046 and OKY-1580 have not only the inhibi- tory action on TxA2 synthetase but also the accelerating action on PGI2 synthetase.

TXB, @c/ml) w control(n =4)

*---. OKY-046(n=5) (1oOmg.i.v.)

700

!J&&

0 5 10 20 30 Ml)

* o.os< P< 0.001

Fig.7(A) Effect of OKY-046 on plasma level of TxB2 after the intravenous infusion of room air in anesthetized dogs

TX& (w/ml)

- control(n=4)

*--* OKY-lseo(n=4) (loom09 i.v.1

300 200

W

+_f____________--l

100

Fig.7(B) Effect of OKY-1580 on plasma level of TxB2 after the intravenous infusion of room air in anesthetized dogs

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Mehta et al. demonstrated that PG12 production showed an increase in human umbilical vein in the presence of OKY- 1580 (11). In present investigaion plasma level of TxB showed a significant decrease, while plasma level of 6- 2 eto PGFlo( showed a tendency to increase compared to that in control group. Our previous study demonstrated that PG12 and its metabolite, 6-keto PGFld induced the relaxation response in bronchial smooth muscle (12). In present inves- tigation OKY-046 significantly inhibited an increase in tracheal pressure and pulmonary artery pressure as compared to OKY-1580. Although changes in plasma levels of 6-keto- PGFlo( and TxB2 after the intravenous infusion of OKY-046 and OKY-1580 were almost same. So, it seems to me that not only TxA2 but also various vasoactive substances such as arachidonic acid cascade metabolites, histamine, serotonin and bradykinin and vagus nerve stimulation might be involved in the rise of tracheal and pulmonary artery blood pressure in pulmonary air embolism.

Above results suggest that OKY-046 and OKY-1580 might be useful therapeutic agents for the treatment and preven- tion of pulmonary thromboembolism.

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REFERENCES

Ishihara Y., Kitamura S. Pulmonary circulation and pro- staglandins. Lung and Heart U:89, 1983. Moncada S., Vane, J.R. The discovery of prostaglandin (PGX); a fresh insight into arachidonic acid metabolism. In.Biochemical Aspects of Prostaglandins and Thrombo- xanes. ed. Kharasch N, Fried J. p.155 (London) Academic Press, 1977. Needleman P, Moncada S. Bunting S, Vane J.R. Hamberg M., Sammuelsson B. Identification of an enzyme in platelet microsome which generates thromboxane A2 from prosta- glandin endoperoxides. Nature 261:558, 1976. Kitamura, S., Ishihara, Y., Sugiyama, Y. et al. Effects of prostaglandins in airway system and pulmonary circulation systim in dogs. Jap. J. Thoracic. Dis. u:560, 1979 Moncada S., Gryglewski R.J. Bunting S., Vane J.R. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregaion. Nature &$:663, 1976. Svensson J., Strandberg K., Tuvemo T., Hamberg M. Throm- boxane A2 : Effects on airway and vascular smooth muscle. Prostaglandins U:425, 1977. Meyers K.M., Seachord C.L., Holmsten H., Smith J.B., Prieur D.J. A dominat role of thromboxane formaion in secondary aggregation of platelets. Nature m:331 1979. Iizuka K., Akahane K., Momose D. et al. Highly selective inhibitors of thromboxane synthetase 1. Imidatole deri- vatives. J.Med. Chem. &1139, 1981 Tanouchi T., Kawamura M., Ohyama I. et al. Highly select- ive inhibitors of thromboxane synthetase 2. Pyridine

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synthetase inhibitors (OKY-046, OKY-1580) on the action of bronchoactive agents in guinea pig tracheal strips and on arachidonate metabolism in guinea pig lung lobes. Prostaglandins, Leukotrienes and Medicine 14:341, 1984.

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12)Ishihara Y., Kitamura S., Kosaka K. Effects of 6-keto- prostaglandin F,H on bronchial smooth muscle. Jap. J. Thoracic. Dis. 20:274, 1982.

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