Pulmonary vascular response to thrombin: Effects of thromboxane synthetase inhibition with OKY-046 and OKY-1581

We examined the effects of thromboxane synthetase inhibition with OKY-1581 and OKY-046 on pulmonary hemodynamics and lung fluid balance after thrombin-induced intravascular coagulation. Studies were made in anesthetized sheep prepared with lyng lymph fistulas. Pulmonary intravascular coagulation was induced by i.v. infusion of α-thrombin over a 15 min period. Thrombin infusion in control sheep resulted in immediate increases in pulmonary artery pressure (P ) and pulmonary vascular resistance (PVR), which associated with rapid 3-fold increase in pulmonary lymph flow (Q̇lym) and a delayed increase in lymph-to-plasma protein concentration (L/P) ratio, indicating an increase in the pulmonary microvascular permeability to proteins. Thrombin-induced intravascular coagulation alos increased arterial thromboxane B₂ (a metabolite of thromboxane A₂) and 6-keto-PGF_1α concentrations (a metabolite of prostacyclin). Both OKY-1581 and OKY-046 prevented thromboxane B₂ and 6-keto-PGF_1α generation. The initial increments in P and PVR were attenuated in both treated groups. The increases in Q̇lym were gradual in the treated groups but attained the same levels as in control group. However, the increases in Q̇lym were associated with decreases in L/P ratio. In both treated groups, the leukocyte count decreased after thrombin infusion but then increased steadily above the baseline value, whereas the leukocyte count remained depressed in the control group after thrombin. These studies indicate that a part of the initial pulmonary vasoconstrictor response to thrombin-induced intravascular coagulation is mediated by thromboxane generation. In addition, thromboxane may also contribute to the increase in lung vascular permeability to proteins that occurs after intravascular coagulation and this effect may be mediated by a thromboxane-neutrophil interaction.     

Pulmonary vascular response to thrombin: effects of thromboxane synthetase inhibition with OKY-046 and OKY-1581

We examined the effects of thromboxane synthetase inhibition with OKY-1581 and OKY-046 on pulmonary hemodynamics and lung fluid balance after thrombin-induced intravascular coagulation. Studies were made in anesthetized sheep prepared with lung lymph fistulas. Pulmonary intravascular coagulation was induced by i.v. infusion of alpha-thrombin over a 15 min period. Thrombin infusion in control sheep resulted in immediate increases in pulmonary artery pressure (Ppa) and pulmonary vascular resistance (PVR), which were associated with rapid 3-fold increase in pulmonary lymph flow (Qlym) and a delayed increase in lymph-to-plasma protein concentration (L/P) ratio, indicating an increase in the pulmonary microvascular permeability to proteins. Thrombin-induced intravascular coagulation also increased arterial thromboxane B2 (a metabolite of thromboxane A2) and 6-keto-PGF1 alpha concentrations (a metabolite of prostacyclin). Both OKY-1581 and OKY-046 prevented thromboxane B2 and 6-keto-PGF1 alpha generation. The initial increments in Ppa and PVR were attenuated in both treated groups. The increases in Qlym were gradual in the treated groups but attained the same levels as in control group. However, the increases in Qlym were associated with decreases in L/P ratio. In both treated groups, the leukocyte count decreased after thrombin infusion but then increased steadily above the baseline value, whereas the leukocyte count remained depressed in the control group after thrombin. These studies indicate that a part of the initial pulmonary vasoconstrictor response to thrombin-induced intravascular coagulation is mediated by thromboxane generation. In addition, thromboxane may also contribute to the increase in lung vascular permeability to proteins that occurs after intravascular coagulation and this effect may be mediated by a thromboxane-neutrophil interaction.     

Effects of OKY-046, a selective thromboxane synthetase inhibitor, on blood pressure and thromboxane synthesis in spontaneously hypertensive rats

The effects of OKY-046, a specific thromboxane (TX) synthetase inhibitor, on blood pressure, urinary TX excretion, TX synthesis in blood platelets, kidney slices and aortic strips, were evaluated in adult spontaneously hypertensive rats (SHR). OKY-046 was dissolved in drinking water at concentrations of 1, 10, 100 mg/dl. The average intakes of OKY-046 were 1.4 +/- 0.1, 13.0 +/- 1.1, and 147 +/- 12 mg/kg/day, in rats who took 1, 10, 100 mg/dl of OKY-046 solutions for drinking water, respectively. The systolic blood pressure was significantly decreased by 34 mmHg only with the high dose of OKY-046 (147 mg/kg/day). OKY-046 suppressed the platelet aggregability to ADP and the release of TX B2, a stable metabolite of TX A2, from blood platelets in a dose-dependent fashion. Urinary excretion of TX B2 decreased significantly in both groups treated with moderate (13.0 mg/kg/day) and high doses of OKY-046 (147 mg/kg/day). The release of TX B2 from kidney slices was decreased only by the high dose of OKY-046, while the release of TX B2 from aortic strips was not changed even by the high dose of OKY-046. OKY-046 had no effect on urinary excretion of 6-keto-prostaglandin F1 alpha, a stable metabolite of prostacyclin, or, on its release from the kidney slices and aortic strips. These results suggest that the effect of OKY-046 on TX synthesis has organ specificity and that the antihypertensive effect of this drug in SHR is related to reduced renal TX synthesis.     

A study on the effectiveness of a thromboxane synthetase inhibitor (OKY-046) in increasing the survival length of skin flaps

In an experimental study to test the thromboxane (TX) synthetase inhibitor OKY-046, two random-pattern skin flaps, each measuring 15.5 x 2 cm, and caudally based, were elevated on the backs of rabbits, and the effect of the test drug on their survival length was evaluated. The results indicated that the survival length of the skin flaps was 4.5 +/- 0.2 cm in the control group and 6.8 +/- 0.3 cm in the OKY-046-treated group, hence exceeding the control value by more than 50 percent, which was statistically significant. A laser speckle flow-meter showed that the OKY-046-treated flaps had significantly greater blood flow as compared with the control group both at 1 and 48 hours after operation. Whereas the blood flow values were significantly lower at 48 hours than at 1 hour after operation in the control group, no such reduction was noted in the OKY-046-treated group. On the other hand, while plasma TXB2 was found elevated at 1 hour postoperatively in the control group, such a response to the surgical intervention was blocked and the plasma TXB2/6-keto prostaglandin (PG) F1a ratio was decreased in the OKY-046-treated group. These results clearly indicated that OKY-046 suppressed a plasma thromboxane elevation induced by surgery, it augmented the flap blood flow, and it thereby increased flap survival length, suggesting that the drug might be helpful clinically and that further investigation must be carried out concerning its application.     

Role of thromboxane (Tx) A2 in guinea pig forssman shock and the effect of OKY-046, TX A2 synthetase inhibitor

To study the role of thromboxane (Tx) A₂ in Forssman systemic shock (FSS) in guinea pig, the effect of (E)-3-[p(1H-Imidazol-1-ylmethyl) phenyl]-2-propenoic acid hydrochloride (OKY-046), a specific Tx A₂ synthetase inhibitor, was studied. OKY-046 administered intravenously clearly prolonged survival time and protected against fatal shock. In shocked animals, definite decreases in serum complement hemolytic activity (CH50), leucocyte counts and platelet counts and an increase in lactate dehydrogenase (LDH) activity were observed. In addition, a significant increase of Tx B₂ and incoagulability of blood were observed after shock. Whereas OKY-046 had no effect on the decreases in CH₅₀, platelet counts and leucocyte counts, it inhibited the increase of Tx B₂ and increased the amount of 6-keto PG F_1α. When Forssman antibody (half a lethal dose) was injected, a diphasic increase in airway resistance was observed. OKY-046 inhibited this diphasic increase in airway resistance. These data suggest a pathophysiological role for Tx A₂ in FSS. OKY-046 inhibited the Forssman antibody induced respiratory disorders probably due to the inhibition of Tx A₂ synthesis after shock.     

Protective effect of a thromboxane synthetase inhibitor,OKY-1581, on increased lung vascular permeability in pulmonary microembolization in dogs

To evaluate the potential beneficial effect of a thromboxane synthetase inhibitor, OKY-1581, on increased pulmonary vascular permeability in pulmonary microembolization, we have measured the filtration coefficient in the nonembolized lung after unilateral microembolization in dogs. The unilateral microembolization caused marked elevations in pulmonary artery pressure and blood flow to the nonembolized lung, while pulmonary venous pressure in nonembolized lung did not change. The pulmonary vascular resistance in nonembolized lung did not increase significantly. The filtration coefficient (K_f) in nonembolized lung increased to 0.14 ± 0.02 from the baseline value of 0.07 ± 0.01 ml/min/mmHg/100g at 30 min after microembolization when the initial hemodynamic changes reduced toward the baseline value. In OKY-1581 treated dogs, similar hemodynamic changes did not result in the increase in the filtration coefficient in nonembolized lung. Platelet aggregation was also inhibited after microembolization in OKY-1581 treated dogs. Based on these results, we could conclude that OKY-1581 could prevent the increase in pulmonary vascular permeability following pulmonary microembolization by inhibiting platelet aggregation and possibly by preventing the release of thromboxane A₂.     

The effects of thromboxane A2 inhibitors (OKY-046 and ONO-3708) and leukotriene inhibitors (AA-861 and LY-171883) on CCl4-induced chronic liver injury in mice

The effects of OKY-046, a selective thromboxane A2 (TxA2) synthetase inhibitor, ONO-3708, a novel TxA2 receptor antagonist, AA-861, a selective 5-lipoxygenase inhibitor and LY-171883, a peptide leukotrienes (p-LTs) receptor antagonist on the chronic liver injury were investigated in mice. The chronic liver injury was induced by the injection of carbon tetrachloride (CCl4) two times a week for twelve weeks in mice. In chronic liver injury models, significant histopathological changes in the liver and extensive elevation of glutamate transaminase (GOT and GPT) activity were observed. Administration of OKY-046, ONO-3708, AA-861 and LY-171883 for 12 weeks suppressed the elevation of serum GOT and GPT levels and histopathological changes in CCl4-induced chronic liver injury. These results suggest that TxA2 and LTs inhibitors are effective for the onset and development of chronic liver injury in mice.     

Effect of OKY-046, a selective thromboxane inhibitor,on prostaglandin production and function of pregnant rat uteri and platelets

The effect of OKY-046 (ONO, Japan), a selective TX inhibitor, was studied for its effect on uterine and platelet activity. On day 21 of pregnancy, rats were injected with either 0, 1, or 5 mg/kg OKY-046 via the tail vein. One hour following injections, in vitro activity of uteri and platelets was assessed. A decrease (P<.01) in uterine TXB₂ production (measured by RIA) occurred with increasing OKY-046 dose ( 104 ± 31 vs 44 ± 6 vs 24 ± 2 ng TXB₂/g tissue/45 min). OKY-046 treatment had no effect on other prostanoids. Contractile activity was not affected by OKY-046. The amount of TXB₂ produced in platelets from OKY-046 (5 mg/Kg) treated rats was 45.5% less than that from controls (P<.001). Likewise, arachidonate-induced aggregation of platelets from OKY-046 treated rats was 46.1% less (P<.05) than that of controls. In summary, in vivo administration of OKY-046 selectively reduced uterine TXB₂ without altering other prostanoids, or affecting uterine contractions. In contrast, both platelet TXB₂ production and platelet function (aggregation) was decreased.     

OKY-046 inhibits thromboxane synthesis with no effect on brain edema and neurological status in head traumatized rats

Head trauma (HT) was induced in the left hemisphere of rats by a weight drop device. Edema was maximal 24 h after HT in the injured zone, and PGE2, TXB2 and 6-keto-PGF1 alpha were elevated in both the injured and remote areas. The effect of a specific thromboxane synthetase inhibitor, OKY-046, on the outcome of HT was studied. OKY-046, 100 mg/kg, was given to rats immediately and 8 h after HT. The neurological severity score (NSS) was evaluated at 1 h after HT, and at 24 h, just prior to sacrifice. Specific gravity (SG) of both hemispheres was measured after decapitation. Prostaglandins (PGs) were extracted from the site of injury and from the frontal lobes, remote from the injury, and assayed by RIA. Basal levels of PGE2 and 6-keto-PGF1 alpha were not reduced by the drug while basal TXB2 levels were lowered. However, the increased production due to HT of all PGs, was inhibited by OKY-046, especially that of TXB2. The ratio of TXB2/6-keto-PGF1 alpha, known to affect vascular tone, was reduced by OKY-046 treatment as a result of TXA2 synthesis inhibition. Still, no effect was found on the neurological outcome (as evaluated by the NSS), or on edema formation (expressed by reduced SG). Thus, based on the present findings increased TXA2 synthesis cannot be implicated in the pathophysiology of cerebral edema or dysfunction following HT.     

The role of thromboxane A2 (TxA2) in allergic cutaneous reactions and the effect of (E)-3-[p-(1H-imidazol-1-ylmethyl) phenyl]-2-propenoic acid hydrochloride (OKY-046), a TxA2 synthetase inhibitor

To study the role of thromboxane A2 (TxA2) in cutaneous allergic reactions, the effect of (E)-3-[p-(1H-Imidazol-1-ylmethyl)phenyl]-2-propenoic acid hydrochloride (OKY-046), a selective TxA2 synthetase inhibitor, on cutaneous reactions in rats and mice was studied. Simultaneously, the effect of 9,11-methanoepoxy-prostaglandin H2 (U-46619), a stable analogue of TxA2, on capillary permeability in mouse and rat skin was investigated. Passive cutaneous anaphylaxis (PCA) in mouse ear was clearly inhibited by OKY-046 but not by indomethacin. The inhibitory action of OKY-046 was not influenced by pretreatment with indomethacin. Moreover, prostaglandin I2, which accumulated as a result of the inhibition of TxA2 synthetase, did not affect the PCA. But, the dye leakages caused by histamine, serotonin and leukotriene C4 in mouse ear were clearly inhibited by OKY-046. In addition, OKY-046 inhibited rat reversed cutaneous anaphylaxis, but its inhibitory action was not affected by pretreatment with indomethacin. Contrary to the above results, rat footpad passive Arthus reaction and mouse footpad tuberculin delayed hypersensitivity reaction were not affected by OKY-046. Additionally, U-46619 did not cause an increase of capillary permeability in either mouse and rat skin. These results suggest a slight role of TxA2 in cutaneous allergic reactions in mice and rats and the efficacy of OKY-046 on Type I and II reactions regardless of the inhibition of TxA2 synthetase activity.     

Effect of digitalis on the diaphragm in anesthetized dogs

We examined the effect of digitalis on diaphragmatic contractility and fatigability in 19 anesthetized mechanically ventilated dogs. The diaphragmatic force was assessed from transdiaphragmatic pressure (Pdi) developed at functional residual capacity against an occluded airway during cervical phrenic nerve stimulation. In a first group of five dogs, Pdi-stimulus frequency relationships were compared before and after administration of ouabain in doses of 0.01, 0.02, and 0.04 mg/kg. In a second group, diaphragmatic fatigue was produced by bilateral phrenic nerve stimulation at 30 Hz. Ten seconds of stimulation and 15 s of mechanical ventilation were repeated for 30 min. The rates of decrease in Pdi were compared between two groups, one of 0.05 mg/kg deslanoside-treated dogs (n = 7) and one of nontreated dogs (n = 7). After ouabain administration Pdi was significantly greater at each frequency in a dose-dependent manner. On the other hand, the rate of decrease in Pdi in the deslanoside group was significantly smaller than that in the nontreated group, whereas deslanoside did not greatly change the Pdi-frequency curves in fresh diaphragm. We conclude that ouabain improves contractility of the fresh diaphragm and that deslanoside has a protective effect against fatigability.     

Effect of glucagon-(1-21)-peptide on secretin-stimulated pancreatic exocrine secretion in anesthetized dogs

The effects of glucagon-(1-21)-peptide on pancreatic exocrine secretion and plasma glucose levels were studied and compared with those of native glucagon in anesthetized dogs. Intravenous bolus administration of 1 nmol or 10 nmol/kg of glucagon-(1-21)-peptide evoked a significant inhibition of secretin-stimulated pancreatic juice secretion and protein output in a dose-dependent manner, as equimolar doses of glucagon did. Native glucagon induced an immediate and transient increase in pancreatic juice volume, which was followed by a significant inhibition. However, glucagon-(1-21)-peptide showed only the inhibitory action. Glucagon-(1-21)-peptide had no effect on plasma glucose levels even when a dose of 10 nmol/kg was given. The results suggest that the N-terminal amino-acid residues of glucagon play an important role in the inhibition of pancreatic exocrine secretion.     

Effect of thromboxane antagonists on ozone-induced airway responses in dogs

Airway hyperresponsiveness after inhaled ozone in dogs may occur as a result of thromboxane release in the airway. In this study, two thromboxane receptor antagonists, L-655,240 and L-670,596, were used in doses that inhibit the response to an inhaled thromboxane mimetic, U-46619, to determine further the role of thromboxane in ozone-induced airway hyperresponsiveness. Dogs were studied on 2 days separated by 1 wk. On each day, the dogs inhaled ozone (3 ppm) for 30 min. On one randomly assigned day, 10 dogs received an infusion of L-655,240 (5 mg.kg-1.h-1) and 5 dogs received an infusion of L-670,596 (1 mg.kg-1.h-1); on the other day dogs received a control infusion. Airway responses to doubling doses of acetylcholine were measured before and after inhalation of ozone and were expressed as the concentration of acetylcholine giving a rise in resistance of 5 cmH2O.l-1.s from baseline (acetylcholine provocation concentration). The development of airway hyperresponsiveness after ozone was not inhibited by the thromboxane antagonists. The mean log difference in the acetylcholine provocative concentration before and after ozone on the L-655,240 treatment day was 0.62 +/- 0.12 (SE) and on the control day was 0.71 +/- 0.12 (P = 0.48); on the L-670,596 treatment day the mean log difference was 0.68 +/- 0.15 (SE) and on the control day it was 0.75 +/- 0.19 (P = 0.45). These results do not support an important role for thromboxane in causing ozone-induced airway hyperresponsiveness.     

Effect of ozagrel hydrochloride,a thromboxane synthetase inhibitor,on alcoholic beverage-induced bronchoconstriction in asthmatic patients

Acetaldehyde is thought to be a main factor of alcohol-induced asthma. The thromboxane (TX) synthetase inhibitor, ozagrel hydrochloride, inhibits acetaldehyde-induced bronchoconstriction in asthmatic patients. The present study evaluated the involvement of TXA(2) on alcoholic beverage-induced bronchoconstriction. Four patients with alcohol-induced asthma received ozagrel (400 mg for 4 days) or placebo using a single-blind, randomized, cross-over design. On two separate study days, each subject drank the same brand and volume of alcoholic beverage (beer or Japanese sake) and bronchoconstriction was assessed as the change in peak expiratory flow (PEF). The effect of ozagrel on the aerosolized challenge of acetaldehyde was investigated in the same subjects. Although aerosolized acetaldehyde-induced bronchoconstriction was significantly prevented by ozagrel, there were no differences in the time course of the decrease in PEF or the maximum fall in PEF after alcohol intake between placebo and ozagrel. We conclude that TXA(2) is not involved in alcoholic beverage-induced bronchoconstriction.     

Protective actions of a new thromboxane synthetase inhibitor in arachidonate induced sudden death

A new thromboxane synthetase inhibitor, OKY-046, at doses of 0.5 and 1.0 mg/kg prevented mortality induced by sodium arachidonate in 100% of the rabbits studied. Sodium arachidonate at a dose of 1.25 mg/kg uniformly decreased mean arterial blood pressure to values approximately 0 mm Hg, stopped respiration and produced sudden death within 3-5 minutes in all rabbits studied. OKY-046 prevented all these sequelae of the sodium arachidonate. Untreated rabbits challenged with sodium arachidonate develop large increases in circulating thromboxane B2 (TxB2) and 6-keto PGF1 alpha of about 12- to 18-fold. In contrast, OKY-046 prevented the increase in TxB2 concentrations and the pulmonary thrombosis, but did not block the rise in 6-keto PGF1 alpha following arachidonate injection. These results suggest that the protective mechanism of OKY-046 in arachidonate induced sudden death is via selective inhibition of thromboxane synthesis.     

Novel inhibitors of poly(ADP-ribose) synthetase.

In a search for new inhibitors of the nuclear enzyme poly(ADP-ribose) synthetase, it was found that various benzamides substituted in the 3-position were the most inhibitory compounds found to date. Two of the benzamides, 3-aminobenzamide and 3-methoxybenzamide, were found to be competitive inhibitors, with Ki values or less than 2 microM.     

Effect of cyclooxygenase and thromboxane synthetase inhibition on furosemide-stimulated plasma renin activity

We studied the effects of a specific thromboxane (TX) synthetase inhibitor (U-63,557A) and a cyclooxygenase inhibitor on furosemide-induced renin release. Furosemide (2.0 mg X kg-1) was injected into Sprague-Dawley rats pretreated with indomethacin (10 mg X kg-1, i.v.), U-63,557A (1.0-32.0 mg X kg-1, i.v.), or vehicle (Na2CO3 0.03 M). Plasma renin activity was measured in blood samples collected 0, 10, 20, and 40 min after the injection of furosemide. Blood was also collected after the administration of vehicle, indomethacin, or U-63,557A for serum TXB2, a measure of platelet TXA2 synthesis. The results demonstrated that plasma renin activity rose with time following furosemide in the various groups of rats; indomethacin suppressed the furosemide-induced increments in plasma renin activity, while U-63,557A at doses of 4-8 mg X kg-1 augmented it. At doses below 4 mg X kg-1 or above 8 mg X kg-1, U-63,557A did not augment renin secretion. Indomethacin and U-63,557A reduced serum thromboxane by 81 and 90%, respectively. Thus, these experiments suggest that thromboxane synthetase inhibition, within a narrow dosage range, potentiates furosemide-induced renin release while cyclooxygenase inhibition suppresses it.