Pharmacological studies on the TXA2 synthetase inhibitor (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid (OKY-046)

The effects of (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid (OKY-046) on thromboxane A2 (TXA2) synthetase in vitro and on experimental animal models of sudden death and cerebral infarction were studied. IC50 values of OKY-046 for the TXA2 synthetase of human, rabbit, dog and guinea pig washed platelets were 0.004, 0.004, 0.26 and 2.4 microM, respectively. OKY-046 at concentrations up to 1 mM, however, did not inhibit prostacyclin (PGI2) synthetase from bovine aorta microsomes or cyclooxygenase and PGE2 isomerase from sheep seminal vesicle microsomes. Similarly, platelet 12-lipoxygenase was not affected by OKY-046. Evidence for a re-direction of arachidonate metabolism from thromboxane synthesis toward PGI2 synthesis was obtained using rat peritoneal cells. Namely, OKY-046 increased PGI2 production accompanied by an inhibition of TXA2 production at a concentration of more than 1 microM. OKY-046 at a dose of 0.1 mg/kg (i.v.) in dogs inhibited the aortic and mesenteric arterial contraction of rabbit induced by the addition of arachidonate to extracorporated blood of the dogs. OKY-046 at a dose of 0.3 mg/kg (i.v.) prevented the arachidonate-induced sudden death and also decreased the incidence of cerebral infarction induced by injection of arachidonate into the internal carotid artery in rabbits. Aspirin also decreased the incidence of cerebral infarction at a dose of 30 mg/kg (i.v.). These results suggest that OKY-046 may be valuable for the treatment of cerebrovascular and cardiovascular diseases associated with vasoconstriction and thrombosis due to TXA2.     

Bradykinin-induced airway responses in guinea pig: effects of inhibition of cyclooxygenase and thromboxane synthetase.

We studied the effects of indomethacin (10 mg/kg i.v.), a cyclooxygenase inhibitor, and OKY-046 (1, 10 and 30 mg/kg i.v.), a selective thromboxane synthetase inhibitor, on airflow obstruction and airway plasma exudation induced by bradykinin (150 nmol) instilled by the airway route to anesthetized guinea pigs. To do this, we studied changes in lung resistance (RL) and extravasation of Evans Blue dye respectively. Instilled bradykinin produced an immediate and marked increase in RL which peaked at approximately 30 s. We also observed a delayed increase in RL, reaching a second peak at approximately 3 min. Bradykinin produced airway plasma exudation at all airway levels, measured as extravasation of Evans Blue dye. Indomethacin significantly inhibited both the immediate and the delayed increase in RL after bradykinin. OKY-046 had a similar significant and dose-dependent inhibitory effect on these responses. In addition, both drugs inhibited bradykinin-induced Evans blue dye extravasation in intrapulmonary airways. Bradykinin instilled by the airway route significantly decreased systemic blood pressure but this effect was not altered in animals pretreated with either indomethacin or OKY-046. We conclude that the bronchoconstrictor response and airway plasma exudation induced by instilled-bradykinin may be mediated in part via thromboxane A2 generation.     

Pharmacological profile of 48740 R.P., a PAF-acether antagonist

The pyrrolo-thiazole derivative 48740 R.P. inhibited the platelet-activating factor (PAF-acether)-induced aggregation of human and rabbit platelets and was poorly effective against ADP- and arachidonic acid-induced platelet aggregation. 48740 R.P. prevented the activation of guinea-pig alveolar macrophages by PAF-acether, and the PAF-acether-induced thromboxane B₂ production from guinea-pig lungs. 48740 R.P. (3 mg/kg i.v.) antagonized selectively in anaesthetized guinea-pigs the bronchoconstriction due to PAF-acether without affecting that due to acetylcholine, histamine, serotonin, thromboxane A₂ analogue U-46,619 and arachidonic acid. A higher dose of 48740 R.P. (10 mg/kg i.v.) was required to block the thrombocytopenia and the leucopenia induced by PAF-acether in the propranolol-treated guinea-pigs. 48740 R.P. (30 mg/kg i.v.) antagonized the PAF-acether effects when bronchoconstriction was induced by aerosolized PAF-acether. 48740 R.P. is a selective antagonist of PAF-acether under in vitro and in vivo conditions.     

Anti-allergic effects of (E)-3-[p-(1H-imidazol-1-ylmethyl)phenyl]-2-propenoic acid (OKY-046), a specific thromboxane (TX) A2 synthetase inhibitor: effect of OKY-046 on II-IV type allergic reactions

We studied the effects of OKY-046 on types II, III and IV allergic reactions, as classified by Coombs and Gell. In Type II, OKY-046 at 30-100 mg/kg intraduodenally (i.d.) and at 1-30 mg/kg intravenously (i.v.) inhibited the bronchoconstriction in a dose-dependent manner after Forssman antigen injection. Aspirin (3 mg/kg, i.v.) also suppressed it. OKY-046 (30-100 mg/kg, i.d.) suppressed the increase of TXB2 level in the plasma in a dose-dependent manner. However, there was no effect of OKY-046 and aspirin on the decrease in complement activity (CH50), platelets and leukocytes. Additionally, OKY-046 (300 mg/kg, p.o.) prolonged the survival time following Forssman antigen injection. However, the immune hemolysis reaction was not prevented by OKY-046 (10(-6)-10(-3) M). FUT-175 protected against the Forssman shock at 1 mg/kg, i.v. and the in vitro immune hemolysis reaction at 10(-5) M. In Type III, OKY-046 (300 mg/kg, p.o.) significantly suppressed the direct passive Arthus reaction and immune complex nephritis in rats. There was no effect of OKY-046 on the delayed-type hypersensitive response to picryl chloride in mice. We think that OKY-046 should be a beneficial drug for the treatment of types II and III allergic reactions.     

Effect of thromboxane synthetase inhibitor on cerebral circulation and metabolism during experimental cerebral ischemia in spontaneously hypertensive rats

The protective effect of thromboxane synthetase inhibitor, OKY-046, on brain ischemia was studied in spontaneously hypertensive rats. Cerebral ischemia was developed by bilateral carotid artery ligation (BCL) for 1 or 3 h and thereafter, circulation was restored for 15 min. OKY-046, 5 or 30 mg/kg, or saline as control was administered i.v. before BCL. Neither blood pressure nor blood gases were altered by OKY-046 or saline injection. During BCL, cerebral cortical blood flow was reduced to 25 and 15% of the resting value at 30 and 60 min, respectively, and these changes were not different among the groups. In rats with ischemia longer than 1 h, the blood flow was well preserved by OKY-046, 30 mg/kg, to 10-17% of the resting level, thus significantly higher than that (less than 5%) in non-treated rats. After 15 min recirculation, the supratentorial lactate level was lower and adenosine triphosphate (ATP) was higher in OKY-046-treated rats than in the saline-treated ischemic rats. Plasma thromboxane B2 was increased markedly in 1 h ischemic-reperfused rats without treatment and the increase was almost completely inhibited by OKY-046. In contrast, 6-keto-prostaglandin F1 alpha was increased 8.5-fold after ischemia and the increase was not affected by the treatment. OKY-046 seems to have an antiischemic effect on acutely induced cerebral ischemia. Selective inhibition of thromboxane A2 production and an inversely high level of prostaglandin I2 may be an important contribution to protection of the microcirculation during ischemia and preservation of ischemic cerebral metabolism.     

Effect of the selective thromboxane A2 receptor antagonist, S-1452, on antigen-induced sustained bronchial hyperresponsiveness

Long-lasting bronchial hyperresponsiveness to i.v. acetylcholine was observed in actively sensitized guinea-pigs after aerosol ovalbium exposure. The response became significant at 7 h post-challenge and persisted for at least 120 h compared to the response of unsensitized animals. Pretreatment of animals with the specific thromboxane A₂ receptor antagonist, S-1452 (calcium (1R, 2S, 3S, 4S)-(5Z)-7-(((phenylsulfonyl)amino)bicyclo[2.2.1]hept-2-yl) hept-5-enoate dihydrate), almost completely inhibited the onset of bronchial hyperresponsiveness, as assessed at 24 and 120 h post-challenge. However, it was ineffective when administered at 1 h post-challenge or 2 h before assessment of bronchial responsiveness. Lung vascular injury occured transiently immediately after antigen challenge, the kinetics of injury being associated with those for the production of thromboxane B₂ in bronchoalveolar lavage fluid. The vascular injury was dramatically suppressed by pretreatment with S-1452. These findings suggest that acutely generated thromboxane A₂ plays an important role in the pathogenesis of antigen-induced long-lasting bronchial hyperresponsiveness, probably by producing vascular damage in the lungs.     

Effect of DP-1904, a thromboxane synthetase inhibitor, on antigen- and spasmogen-induced bronchoconstriction in rodents

The effect of DP-1904 [6-(1-imidazolylmethyl)-5,6,7,8-tetra-hydronaphthalene-2-car boxylic acid hydrochloride], a selective thromboxane synthetase inhibitor, was examined on antigen- and spasmogen-induced bronchoconstriction in rodents. Oral administration of DP-1904 (1, 3, 10 mg/kg) as well as OKY-046 (sodium (E)-3[4-(1-imidazolylmethyl)-phenyl]-2-propanoate, 100 mg/kg), significantly inhibited immunoglobulin G-mediated bronchoconstriction in actively sensitized guinea pigs. Immunoglobulin E-mediated bronchoconstriction in actively sensitized rats was also inhibited by both DP-1904 (1, 10 mg/kg) and OKY-046 (100 mg/kg). DP-1904 (3-30 mg/kg) and OKY-046 (30 mg/kg) suppressed leukotriene D4-induced bronchoconstriction in guinea pigs. In these models, the endogenous levels of thromboxanes significantly increased following the stimulus (antigen and leukotriene D4). DP-1904 (10 mg/kg) inhibited the increase in thromboxane level in both plasma and bronchial alveolar lavage fluid. These actions of DP-1904 persisted for more than 12 h, indicating a long-lasting effect of DP-1904 on bronchoconstriction. The results showed that the biological activity of DP-1904 in our rodents models is more potent than that of OKY-046 (Ozagrel), which is available as an anti-asthma agent in Japan.     

Effect of imidazole on prostaglandin and thromboxane accumulation in urate arthritis

High concentrations of prostaglandins E₂ and F₂ and much lower concentrations of thromboxane B₂ occur in joint washes of chicken 1–3 h after intra-articular injection of urate crystals. Pretreatment with 200 mg/kg imidazole i.v. reduced the concentration of prostaglandons and of thromboxane B₂ in the joint washes significantly. Simultaneously, leucocyte invasion was delayed and development of oedema was inhibited. The results suggest that in urate crystal arthritis the effect on prostaglandin and thromboxane accumulation at the site of inflammation contributes to the anti-inflammatory activity of imidazole.     

Desensitization of substance P-induced K+ release in rat parotid

The angiotensin converting enzyme inhibitor enalapril (0.5 mg/kg i.v.) potentiated significantly the inhibitor effect of the thromboxane A₂ antagonist L-640,035 (1 mg/kg i.v.) on electrically induced platelet accumulation in the rabbit in vivo. Enalapril had no effect upon platelet accumulation when given alone. The hypotensive effects of enalapril did not account for the potentiation because a combination of hexamethonium (5 mg/kg i.v.) and hydralazine (1 mg/kg i.v.), which decreased blood pressure similarly to enalapril, did not augment the effect of L-640,035. Determination by radioimmunoassay of plasma levels of immunoreactive 6-keto-PGF₁, suggested that increases in PGI₂ levels after combined administration of enalapril and L-640,035 could explain the observed potentiation.     

注射用重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白对活动性类风湿关节炎患者骨代谢的影响

目的:分析及评价注射用重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(etanercept)对活动性类风湿关节炎患者骨代谢的影响。方法:选取2015年在西南医科大学附属医院风湿免疫科确诊为类风湿关节炎(Rheumatoid Arthritis,RA)的100例骨代谢异常患者,年龄为40~59(51.3±7.6)岁,病程为12~41(29.5±14.1)个月,其中男性23例,女性77例,且所有患者DAS28评分>2.6,均通过双能X线骨密度检测仪及N-端前肽(PINP)、血清Ⅰ型胶原C-末端肽(CTX-I)评估骨代谢情况。根据初始骨密度检测结果,分为类风湿关节炎伴骨质疏松组(A组,58例)及类风湿关节炎伴骨量减少组(B组,42例),A、B组各随机分为两组,A组分为A_1组(29例)和A_2组(29例),B组分为B_1组(21例)和B2组(21例),其中A_1和B_1组给予注射用重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(25mg皮下注射biw×6个月,后改为25mg qw×6个月)+甲氨喋呤(15mg qw)+叶酸片(10mg qw)+硫酸羟氯喹(0.2bid)+钙尔奇D(600mg qd)+阿法骨化醇(0.5μg qd)治疗,A_2和B2组给予甲氨喋呤(15mg qw)+叶酸片(10mg qw)+硫酸羟氯喹(0.2bid)+钙尔奇D(600mg qd)+阿法骨化醇(0.5μg qd)治疗,A_1、B_1组为观察组,A_2、B2组为对照组,各组患者分别治疗前、治疗后6、12月检测股骨颈、颈椎、大转子、Ward三角骨密度值,同时检测PINP、CTX-I水平、并计算DAS28评分。结果:与A_2组比较,A_1组患者在性别构成比、DAS28评分、平均病程、平均发病年龄及初始PINP、CTX-I水平差异无显著性(P>0.05),大转子初始骨密度值明显低于A_2组,差异有显著性(P<0.05)。A_1组股骨颈、Ward三角在治疗6、12个月后骨密度值均高于同期A_2组,颈椎在治疗12个月后骨密度值高于A_2组,大转子在治疗6个月后骨密度值低于A_2组,差异均有显著性(P<0.05)。在治疗6、12个月后,A_1组PINP水平均低于同期A_2组,差异有显著性(P<0.05),A_1组CTX-I水平较同期A_2组降低,尤以12个月后更为显著(P<0.05);与B2组比较,B_1组在性别构成比、平均发病年龄及初始PINP、CTX-I水平差异无显著性(P>0.05),但B_1组DAS28评分较B2组高,平均病程较B2组短,大转子初始骨密度值低于B2组,差异有显著性(P<0.05)。B_1组股骨颈、颈椎、Ward三角在治疗12个月后骨密度值高于同期B2组,大转子在治疗6个月后骨密度值低于B2组,差异均有显著性(P<0.05)。在治疗6、12个月后,B_1组PINP水平均低于同期B2组,差异有显著性(P<0.05),B_1组CTX-I水平较同期B2组降低,尤以12个月后更为显著(P<0.05)。结论:注射用重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白可有效阻止活动性类风湿关节炎患者的骨量丢失,改善该类患者异常的骨代谢,随使用时间的延长,效果可能更加显著。     

In vivo enhancement of platelet activating factor-induced prostacyclin production by OKY-046, a selective inhibitor of thromboxane A2 synthase.

Platelet-activating factor (PAF), a likely mediator of endotoxin action, causes thromboxane A2 (TXA2) release and pulmonary hypertension in pigs. We examined the effect of selective TXA2 synthase inhibition with OKY-046 on cyclooxygenase metabolites during PAF-induced pulmonary hypertension. Six closed-chest pigs received PAF in escalating doses (0.1, 0.3, 1.0, and 3.0 nmol intravenously, i.v.) before and after (E)-3[4-(1-imidazolyl methyl) phenyl]-Z-propenoic acid hydrochloride monohydrate OKY-046, 10-mg/kg i.v. bolus plus 20-mg/kg/h infusion. Plasma samples at peak PAF effect had radioimmunoassay (RIA) for the stable metabolites of TXA2 (TXB2) and prostacyclin (6-keto-PGF1 alpha). Tachyphylaxis was not noted in 5 control pigs given sequential repeats of the PAF dosing series. Pulmonary vascular resistance (PVR) was 240 +/- 30 (SE) dyne s cm-5 at baseline and increased to 3,100 +/- 1,300 after 1.0 nmol PAF (p less than 0.05). When the same amount of PAF was given after OKY-046, PVR increased only to 820 +/- 280 dynes/s/cm-5. TXB2 was 34 +/- 7 pg/0.1 ml at baseline and increased to 70 +/- 4 pg/0.1 ml with PAF 1.0 nmol (p less than 0.001). TXB2 levels were unchanged from 34 +/- 4 pg/0.1 ml when PAF 1.0 nmol was administered after OKY-046 (NS vs. pre-OKY-046). In contrast, 6-keto-PGF1 alpha, 6 +/- 2 pg/0.1 ml at baseline, increased to 24 +/- 4 pg/0.1 ml after PAF 1.0 nmol and increased further to 50 +/- 8 pg/0.1 ml when PAF 1.0 nmol was given after OKY-046 (p less than 0.05 vs. pre-OKY-046).(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in arachidonic acid-induced respiratory distress in streptozotocin-diabetic mice]

Using streptozotocin (STZ)-diabetic mice, we examined the respiratory distress induced by arachidonic acid. Male ddY mice were made diabetic by injecting STZ (170 mg/kg, i.p.) 2 weeks prior to the experiment. Control mice received the vehicle (citrate buffer, pH 4.6). The duration of respiratory distress was observed by a slow i.v.-injection of sodium arachidonic acid (AA) into the caudal vein of mice at the dose of 50 mg/kg. Aspirin was i.p.-administered 30 min before AA. OKY-046 (specific thromboxane A2 (TXA2) synthetase inhibitor), OP-41483 (stable prostacyclin analog) and 9, 11 epithia-11, 12-methano-TXA2 (STA2, stable TXA2 analog) were i.v.-administered 30 min before AA. The duration of respiratory distress induced by AA was significantly reduced in STZ-diabetic mice. Aspirin (10-50 mg/kg) and OKY-046 (25-100 mg/kg) enhanced the AA-induced respiratory distress in STZ-diabetic mice. OP-41483 (1-100 micrograms/kg) reduced the AA-induced effect in both control and STZ-diabetic mice. STA2 (10 micrograms/kg) enhanced the AA-induced effect in both control and STZ-diabetic mice. ONO-1078 (1-10 mg/kg) did not affect the AA-induced effect in both control and STZ-diabetic mice. TMK-688 (0.01-1 mg/kg) reduced the AA-induced effect in the control mice, but not in the STZ-diabetic mice. These results suggest an involvement of leukotriene in the respiratory response to AA in diabetic mice, especially when cyclooxygenase and TXA2 synthetase are inhibited.     

Evidence for the participation of kinins in Freund's adjuvant-induced inflammatory and nociceptive responses in kinin B1 and B2 receptor knockout mice.

Experiments were designed to investigate the role of kinin B₁ and B₂ receptors in Freund's adjuvant (CFA)-induced inflammation and nociception responses by the use of B₁ and B₂ null mutant mice. Intradermal (i.d.) injection of CFA produced time-dependent and marked hyperalgesic responses in both ipsilateral and contralateral paws of wild-type mice. Gene disruption of the kinin B₂ receptor did not interfere with CFA-induced hyperalgesia, but ablation of the gene of the B₁ receptor reduced the hyperalgesia in both ipsilateral (48±13%, at 12 h) and contralateral (91±22%, at 12 h) paws. Treatment of wild-type mice with the selective B₁ antagonist des-Arg⁹-[Leu⁸]-BK (150 nmol/kg, s.c.) reduced CFA-evoked thermal hyperalgesia, to an extent which was similar to that observed in mice lacking kinin B₁ receptor. I.d. injection of CFA produced a time-related and long-lasting (up to 72 h) increase in paw volume in wild-type mice. A similar effect was observed in B₁ knockout mice. In mice lacking B₂ receptor, the earlier stage of the CFA-induced paw oedema (6 h) was significantly greater compared with the wild-type animals, an effect which was almost completely reversed (76±5%) by des-Arg⁹-[Leu⁸]-BK. This data demonstrates that kinin B₁ receptor, but not B₂ receptor, exerts a critical role in controlling the persistent inflammatory hyperalgesia induced by CFA in mice, while B₂ receptor appears to have only a minor role in the amplification of the earlier stage of CFA-induced paw oedema formation. The results of the present study, taken together with those of previous studies, suggest that B₁ receptor antagonists represent a potential target for the development of new drugs to treat persistent inflammatory pain.     

Inhibitory action of OKY-O46.HCl, a specific TXA2 synthetase inhibitor, on platelet activating factor (PAF)-induced airway hyperresponsiveness of guinea pigs: role of TXA2 in development of PAF-induced nonspecific airway hyperresponsiveness

We studied a role of TXA2 in the development of PAF-induced nonspecific airway hyperresponsiveness in guinea pigs using a TXA2 synthetase inhibitor (OKY-O46.HCl) and a stable TXA2 mimetic agent (STA2). Inhalation of PAF (1 microgram/ml) and STA2 (1 or 10 ng/ml) increased the airway response to acetylcholine (ACh), histamine, leukotriene D4 and electrical vagal stimulation. Intraduodenal administration (i.d.) of OKY-O46.HCl (100 mg/kg) inhibited PAF-induced airway hyperresponsiveness. However, OKY-046.HCl (30 mg/kg, i.v.) did not suppress STA2-induced airway hyperresponsiveness. Neither hexamethonium (1 mg/kg, i.v.) nor hemicholinium-3 (10 mg/kg, i.v.) prevented the increase in the airway response to ACh after inhalation of PAF and STA2. In the presence of atropine (0.5 mg/kg, i.p.), PAF-induced airway hyperresponsiveness to histamine did not change. OKY-046.HCl (100 mg/kg, i.d.) inhibited the increase in ACh (10(-8) M)-induced 45Ca uptake into the lung tissue from PAF-inhalated guinea pigs. Inhalation of STA2 increased the number (Bmax) of muscarinic and H1-histaminergic receptors in the lung tissue from guinea pigs, but no changes were found on beta-adrenoceptors. These results suggest that TXA2 should act on the smooth muscle cells or alter functions of muscarinic and H1-histaminergic receptors, except beta-adrenoceptors, and then increase the membrane permeability to extracellular Ca2+. We also assume that OKY-046.HCl can inhibit PAF-induced nonspecific airway hyperresponsiveness by suppressing the generation of TXA2.     

Antinephritic effect of OKY-046, a thromboxane A synthetase inhibitor (1). Effects on crescentic-type anti-GBM nephritis in rats

To induce crescentic-type anti-glomerular basement membrane (anti-GBM) nephritis, male Sprague-Dawley rats were immunized with rabbit gamma-globulin in Freund's complete adjuvant following i.v. injection of anti-GBM serum. At the same time, original type anti-GBM nephritis was induced in other rats by anti-GBM serum only. The animals with crescentic-type anti-GBM nephritis showed significantly higher platelet aggregability than that in rats with original-type anti-GBM nephritis at days 5, 10 and 40 after anti-GBM serum administration, respectively. To estimate the antinephritic effect of OKY-046, a thromboxane A synthetase inhibitor, it was given orally to rats at doses of 0.5, 2.5 or 20 mg/kg for 39 days after anti-GBM serum. OKY-046 (20 mg/kg) significantly inhibited the increase in both urinary protein and plasma cholesterol levels (40% and 35% vs. control, respectively). Moreover, when examined by light microscopy, this drug remarkably prevented histological involvement of the glomeruli. OKY-046 at 20 mg/kg had suppressed the hyperaggregability of platelets by 77% by day 40 as compared with the control, but doses of 0.5 and 2.5 mg/kg did not. It is concluded from these data that OKY-046 has beneficial effects on crescentic-type anti-GBM nephritis and may act through inhibition of not only platelet TxA2 but also glomerular TxA2 in its action to prevent histological alterations.     

Preventive effect of the thromboxane A2 receptor antagonist S-1452 (d-s-145 Ca) on arachidonate-induced sudden death and cerebral infarction in rabbits

The thromboxane A₂ (TXA₂) receptor antagonist S-1452, d-S-145 Ca, calcium 5(Z)-1R,2S,3S,4S-7-[3-phenylsulfonylaminobicyclo[2.2.1]hept-2-yl]-5-heptenoate dihydrate, prevented sudden death induced by arachidonate or U46619 in rabbits when given in a dose range between 0.1 mg/kg and 1 mg/kg, p.o. OKY-046 and aspirin were also effective in this thromboembolism model, but 10 to 30 times larger doses were needed. Orally administered S-1452 successfully prevented the incidence of cerebral infarction in the rabbit, which was elicited by infusion of 10 mg/ml sodium arachidonate at a rate of 0.1 ml/min for 30 min via a left internal carotid artery. Significant reduction of the infarction grade was observed in dosing above 3 mg/kg of this compound 1 to 4 h before the arachidonate infusion. OKY-046 was effective similarly, but less active than S-1452 (the minimum effective dose: 10 mg/kg). Ticlopidine and aspirin were entirely inactive in this model even after much larger doses. S-1452 is a useful compound for prevention of TXA₂-mediated thrombosis in the brain. © 1993 wiley-Liss, Inc.     

Lipoxin A4 inhibits cholinergic neurotransmission through nitric oxide generation in the rabbit trachea

The effect of lipoxin A₄ and lipoxin B₄ on cholinergic neurotransmission in rabbit tracheal segments was studied under isometric conditions in vitro. Lipoxin A₄ attenuated the contractile responses to electrical field stimulation and caused a rightward shift of the frequency-response curves, so that the stimulus frequency required to produce a half-maximal effect (ES₅₀) increased from 8.1 ± 0.8 to 25.7 ± 1.9 Hz (P < 0.001), whereas lipoxin B₄ had no effect. In contrast, lipoxin A₄ did not alte contractile responses to acetylcholine. Pretreatment of tissues with N^G-nitro- -arginine methylester inhibited the effect of lipoxin A₄ on electrical field stimulation, but N^G-nitro- -arginine methylester did not. This inhibition by N^G-nitro- -arginine methylester was reversed by -arginine but not by -arginine. These results suggest that lipoxin A₄ prejunctionally reduces the vagal nerve-mediated contraction of airway smooth muscle, probably by inhibiting the release of acetylcholine, and that this effect may be exerted through stimulation of nitric oxide generation.     

Roles of endogenous prostacyclin and thromboxane A2 in the ischemic canine heart

The ability of the ischemic heart to release prostacyclin (PGI2) and thromboxane A2 (TXA2) was studied, together with the effects of these substances on the ischemic myocardium in open-chest dogs. We measured the plasma levels of 6-keto-PGF1 alpha and TXB2--which are stable metabolites of PGI2 and TXA2, respectively--as well as lactate and coronary venous blood flow. The dogs were divided into three groups of eight animals which received indomethacin (5 mg/kg), (E)-3-[4-l-imidazolylmethyl)phenyl]-2-propenoic acid hydrochloride monohydrate (OKY-046) (1 mg/kg), or the vehicle. A transient increase in 6-keto-PGF1 alpha was observed in the great cardiac vein 5 min after the ligation of the left anterior descending coronary artery (LAD). TXB2 and lactate increased 30 and 15 min, respectively, after the ligation. Indomethacin prevented significant increases in 6-keto-PGF1 alpha and TXB2, but accelerated the lactate release. OKY-046 prevented significant increases in TXB2 and lactate release, but did not counteract the increase in 6-keto-PGF1 alpha. Although coronary venous flow decreased significantly 5 min after the ligation in every group, the flow returned to the preligation level 15 min after the ligation in the OKY-046 and the vehicle groups. Thus, we have demonstrated the release of PGI2 and TXA2 from the ischemic heart and suggest beneficial effects of PGI2 and of a selective inhibitor of thromboxane synthetase on the ischemic myocardium.     

Effects of OKY-046, a selective thromboxane A2 synthetase inhibitor, on ventricular arrhythmias and prostaglandins during coronary artery ligation and reperfusion in anesthetized dogs

Effects of OKY-046, a selective thromboxane synthetase inhibitor, on ischemia-induced ventricular arrhythmias were investigated in anesthetized dogs. OKY-046 (30 mg/kg, intravenously) decreased ventricular arrhythmias significantly both during 30 min of coronary artery ligation and subsequent reperfusion, which corresponded with a significant reduction of thromboxane concentration, suggesting that thromboxane released during ligation is an important factor in the development of ventricular arrhythmias. Inhibition of thromboxane synthesis with OKY-046 might be useful for the prevention of ischemia-induced ventricular arrhythmias.     

Effects of verapamil on activation of arachidonic acid metabolism in guinea-pig lungs

The effects of (±)-verapamil and its optical isomers on the activation of arachidonic acid (AA) in guinea-pig isolated perfused lungs were investigated. The calcium ionophore A23187 (6–15 nmol), histamine (20–50 nmol) and leukotriene E₄ (1–2 nmol) induced the release of thromboxane A₂ (TxA₂), which was detected by bioassay and by radioimmunoassay of the stable metabolite TxB₂. Racemic (±)-verapamil (0.4–40 μM) caused concentration-dependent inhibition of A23187-induced release of TxA₂ without affecting the conversion of exogenous AA to TxA₂. Both (+)-verapamil and (−)-verapamil (1–10 μM) caused concentration-dependent inhibition of histamine-induced release of TxA₂. In contrast, racemic (±)-verapamil did not inhibit leukotriene E₄ (LTE₄)-induced release of TxB₂. Calcium depletion (with 2 mM ethylenediamine tetra acetate) significantly reduced both histamine-induced release of TxB₂ from 8.6 ± 2.6 to 1.8 ± 0.8 ng/min (P < 0.05) and LTE₄-induced release of TxB₂ from 4.9 ± 0.9 to 0.5 ± 0.2 ng/min (mean±S.E.M.)(P<0.05). Since histamine stimulates phospholipase A₂ in guinea-pig lungs, these results suggest that (±)-verapamil inhibits phospholipase A₂ and that A23187 activates AA metabolism by stimulating phospholipase A₂. Although all three agents activate AA metabolism by calcium-dependent processes, LTE₄ may stimulate calcium entry via separate mechanisms because it is not inhibited by (±)-verapamil.