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The purpose of this study was to explore whether cyclooxygenase products derived from endothelium or vascular muscle participate in the response of guinea-pig uterine arterial rings to prostaglandin F2 alpha (PGF2 alpha). Contraction to PGF2 alpha (0.1-30 microM) occurred with and without endothelium at similar potency and efficacy (pEC50 (-log EC50) values respectively 5.87 +/- 0.06 and 5.97 +/- 0.07; maximal response respectively 78.1 +/- 1.3% and 76.9 +/- 1.5% of contraction induced by 126 mM KCl). Indomethacin (3-30 microM) suppressed the maximum response to PGF2 alpha and induced a rightward shift of concentration-response curves, regardless of the presence of endothelium. pIC50 values for indomethacin were 4.67 and 4.74 for vessels with and without endothelium, respectively. In contrast, the thromboxane synthesis inhibitor OKY-046 (10 and 100 microM) did not affect the response to PGF2 alpha. We conclude that the PGF2 alpha-induced contraction in guinea-pig uterine artery is mediated, at least in part, through constrictor non-thromboxane prostanoid(s) of vascular muscle origin.

The mechanism responsible for the rapid increase of thromboxane A2 synthesis by cells of the human monoblastoid cell line U937, which were differentiated with 12-O-tetradecanoyl-phorbol-13-acetate, induced by lipopolysaccharide (LPS) was studied. Both RNA blot and immunoblot analyses showed that LPS increased the levels of prostaglandin endoperoxide synthase-1 (PES-1) and -2 (PES-2) in a time-dependent manner, and the modes of induction of the two isozymes differed. The maximum PES-1 mRNA level was 1.6 times higher 36 h after than before stimulation by LPS, and that of PES-2 mRNA was elevated about 20-fold at its peak at 12 h after stimulation. Consequently, the immunoreactive PES-1 and PES-2 protein levels also increased time-dependently after LPS stimulation. However, the effects of LPS on the thromboxane synthase mRNA and protein levels were much less marked. These results indicate that LPS-induced thromboxane synthesis by the differentiated cells was regulated at the levels of the two PES isozymes, predominantly at the PES-2 level.

The production of 14CO2 from labelled glucose by isolated uterine strips from ovariectomized-diabetic rats has been studied. U46619, an analogue of TXA2 did not affect basal glucose metabolism; however, insulin-induced increment in CO2 production was completely blocked, both in ovariectomized (OVD) or ovariectomized-estrogenized (OVED) diabetic uterus. OKY064 as well as UK38485, both inhibitors of TXA2 synthesis, stimulated glucose metabolism (p0.05) similar to that of insulin in uterine tissue from OVD and OVED rats. Inhibition in the synthesis and release of TXB2 was detected (p0.01) by uterine radioconversion of 14C-arachidonic acid when adding OKY38485 to the incubation medium, and the production of other prostanoids such as 6-keto-PGF1 alpha, PGF2 alpha and PGE2 was enhanced. In summary, TXA2 inhibited insulin-induced glucose metabolism in diabetic animals.

The effects of ridogrel (a thromboxane synthetase inhibitor / endoperoxide receptor antagonist) were assessed in an ovine model of pregnancy-induced hypertension. Maternal serum prostacyclin and thromboxane levels were quanitiated using RIA, and maternal and neonatal coagulation status was assessed. Pregnancy and neonatal outcome were recorded. Ridogrel,(E)-5-[[[3-pyridinyl)[3-(tri-fluoromethyl)phenyl]methylen]amin]oxy]pentanoic acid, was administered in one bolus dose at 0.1 or 1.0mg/kg IV, three hours following the onset of a 27 hour magnesium sulfate infusion given hypertensive ewes to prevent maternal seizures. At both doses, ridogrel improved neonatal outcome (0% neonatal mortality in each ridogrel group versus 67% neonatal mortality in the magnesium sulfate group), and ridogrel at 0.1mg/kg IV normalized birth weights. Abnormalities of maternal platelet function (abnormal or no response to collagen), occurring during the ovine syndrome, resolved following ridogrel treatment. Ridogrel's effects on maternal and neonatal coagulation were more dramatic at the 0.1mg/kg IV dose. Ridogrel appeared to be beneficial in this model of pregnancy-induced hypertension.

Tumor necrosis factor alpha (TNF alpha) and thromboxane A2 (TXA2) are major products of the activated alveolar macrophage and serve as key mediators of lung injury. In order to determine if the synthesis of TXA2 and the release of TNF alpha are associated, the production of these inflammatory agents by the human alveolar macrophage (AM), as a result of activation by lipopolysaccharide (LPS), was assessed in the absence and presence of the thromboxane synthase inhibitors UK 38,485 (Dazmegrel) and OKY 046. UK 38,485 and OKY 046 inhibited both LPS-stimulated TXA2 production and TNF alpha release in a dose-dependent manner. Prostaglandin E2 (PGE2) production was not increased by UK 38,485 or OKY 046. Neither LPS nor UK 38,485 had any effect on LTB4 production by AM. Neither UK 38,485 or OKY 046 had any effect on LPS-stimulated interleukin-1 beta release. However, the TXA2 mimetic, U46619, did not stimulate TNF alpha release by AM either in the absence or presence of UK 38,485. These findings suggest that 1) UK 38,485 and OKY 046 are inhibitors of both TXA2 production and TNF alpha release by activated human AM, 2) UK 38,485 probably does not exert its inhibitory action on TNF alpha release through effects on eicosanoid production and 3) the possibility that TNF alpha- and TXA2-induced lung injury may be subject to amelioration by imidazole-based compounds should be further evaluated.

It has been proposed that thromboxane synthetase inhibitors may be of use in the treatment of hypertensive disorders of pregnancy. A patient in whom aspirin did not prevent the development of pre-eclampsia in a previous pregnancy was treated with a thromboxane synthetase inhibitor (dazmegrel, Pfizer) in addition to low-dose aspirin. Increased urinary levels of 6-keto-prostaglandin Flα were found throughout pregnancy, which is consistent with the mode of action. At 17–18 weeks of gestation urinary prostaglandin E2 and FDα levels were increased compared with control pregnancies. These increases in PGE2 and PGF2α production were associated with mid-trimester abortion. In vitro studies were carried out to determine the effects of dazmegrel on platelet eicosanoid production. In whole blood from non-pregnant female volunteers this compound inhibited thromboxane B2 production and significantly enhanced prostaglandin E2 production and slightly increased prostacyclin production, demonstrating a redirection of prostaglandin endoperoxides. This suggested that similar changes in arachidonic acid metabolite production may occur in vivo and in vitro , and that thromboxane synthetase inhibitors should not be used during early pregnancy, since increased production of prostaglandins E2 and F2α may result in preterm labour or abortion.

The effects of ridogrel, a thromboxane synthetase inhibitor and endoperoxide receptor antagonist, were studied in twelve pregnant, nulliparous Rhesus monkeys (Macaca mulatta) during the last trimester of pregnancy. Ridogrel was administered intravenously in two groups of animals (n = 6), at either 0.1 mg/kg or 1.0 mg/kg. Ultrasonic assessment of the fetuses during and after the infusion period revealed no obvious changes in fetal condition. Both dosages reduced serum thromboxane levels 30 minutes after administration, 96.6% and 99.6% suppression, 0.1 mg/kg and 1.0 mg/kg, respectively (P < 0.0001), while the 1.0 mg/kg dose produced continued suppression for 24 hours (78% suppression, P < 0.0001) and was lower than the 0.1 mg/kg 24 hour value (P < 0.008). Prostacyclin levels increased to 340% and 472% of baseline values, 0.1 mg/kg and 1.0 mg/kg, respectively at 30 minutes and to 928% and 255% of baseline at 24 and 48 hours after treatment in the 1.0 mg/kg group (P < 0.0003). Ridogrel caused no changes in maternal or neonatal outcome. The potential for the use of this compound for the treatment of preeclampsia is discussed.

The role of the eicosanoids in the pathophysiology of preeclampsia is reviewed, and the results of animal model and human studies with thromboxane synthetase inhibitors in preeclampsia are described. Potential benefits and limitations of therapy are discussed.

The influence of a transient asphyxia on cochlear potentials, i.e. endolymphatic potential (EP), summating potential (SP) and cochlear microphonics (CM) was investigated in guinea pigs when pretreating the animals with various substances interacting with the arachidonic acid (AA) cascade at the level of thromboxane. The controls showed the well-known decline of the EP, CM and the SP increase. When infusing a thromboxane synthetase inhibitor (dazoxiben) or two different thromboxane receptor blockers (daltroban or sulotraban) before the 3-minutes' period of asphyxia was started, the electrophysiological responses of the inner ear (cochlea) could significantly be influenced. The results indicate that a shift of the thromboxane (TXA2)/prostacyclin (PGI2)-balance in favour of the last improve the metabolic conditions for a survival of the cochlea when it is challenged.

Thromboxane A2 (TxA2) augments hypothalamus-pituitary-adrenal axis activity in both fetal and adult animals. We have proposed that TxA2 acts as a neuromodulator within the brain to stimulate the release of corticotropin releasing hormone (CRH) or arginine vasopressin (AVP) into the hypophyseal-portal blood. We performed the present experiments to identify immunoreactive thromboxane synthase (TxS) within fetal brain regions and to quantify developmental changes in the TxS immunoreactivity measurable within those regions. We found that immunoreactive TxS was present in fetal hypothalamus, pituitary, brainstem, and lung. In fetal hypothalamus, we found immunoreactive TxS in three identifiable molecular weights, approximately 65, 42, and 35 kD. In fetal pituitary and lung, we found the 65 and 35 kD forms, and in the brainstem we found only the 35 kD form. In fetal pituitary, there was a clear ontogenetic change in TxS immunoreactivity. The 42 kD TxS immunoreactivity was not present in the youngest fetal sheep studied (86-90 days' gestation), but was expressed in the other age groups (125-128, 135-139, 141-term, and postnatal ages). The other molecular weight forms appeared to increase in the older fetuses, but the changes were not significant. In the hypothalamus, all three forms of TxS were measurable at all ages, and there was no significant change in relative abundance. We conclude that immunoreactive TxS is present in the fetal brain throughout the last half of fetal gestation, but that the significance of multiple molecular weight forms is not clear.

We isolated a rat homolog of thromboxane (TX) synthase cDNA (-1.8 kb) from the kidney with a fragment of human TX synthase cDNA amplified by polymerase chain reaction with placenta cDNA as a template. Northern blot analysis has shown that rat TX synthase gene is expressed abundantly in lung, liver, and uterus; moderately in kidney. TX synthase mRNA expression was up-regulated in hydronephrotic kidney made by ureter ligation. In conclusion, we have revealed the structure of rat kidney TX synthase. Up-regulation of renal TX synthase, which may cause stimulation of TX synthesis, is possibly implicated in the tissue injury in hydronephrotic kidney.

The acute effects of a newly synthesized thromboxane dual blocker (KDI-792), a combined thromboxane synthase inhibitor and receptor antagonist, on lower limb circulation were examined using two-dimensional color and pulse Doppler ultrasonography and laser Doppler flowmetry. A randomized single-masked, placebo-controlled trial was performed on 36 type 2 diabetic patients with minimally impaired baseline flow. The anatomical cross-sectional area (CSA), maximum flow velocity (MFV) and flow volume index (FVI) in the right dorsal pedis artery (DPA) and right femoral artery (FA) were determined by Doppler ultrasonography before and 45 and 90 minutes after the administration of either 100 or 200 mg of KDI-792 to the dose groups or placebo to the control group. Periflux blood flow (PBF) in the right foot was determined simultaneously by laser Doppler flowmetry. Both CSA and MFV in the dose groups were significantly increased in both the FA and DPA. FVI was markedly increased from 21.4 +/- 3.7 to 68.3 +/- 26.8 in the DPA (M +/- SD, P0.01) and from 365.4 +/- 35.3 to 771.7 +/- 75.7 in the FA (P0.01) in the 200 mg dose group. In the 100 mg dose group, FVI was markedly increased from 20.0 +/- 8.7 to 68.3 +/- 26.8 (P0.01) in the DPA and from 372.5 +/- 130.0 to 677.5 +/- 187.8 (P0.01) in the FA. PBF was also increased in both dose groups (from 4.15 +/- 1.4 to 7.0 +/- 4.0 ml/min/100 g tissue in the 200 mg dose group, P0.01), whereas there were no significant changes in either measurement in the control group. There were no significant changes in pulse rate or blood pressure after administration in either the dosage group or the placebo group. These and previous findings indicate that a single administration of KDI-792 markedly increases lower limb blood flow and might have a more potent vasodilating effect than that of prostaglandin I2 derivatives.

The modulation of the production of prostacyclin and thromboxane from rat and cat aortic tissue slices by different vasoactive agents has been studied in order to reveal whether the release of these main two vasoactive prostanoids goes in parallel or may be controlled independently. Norepinephrine, isoproterenol, phentolamine, propranolol, angiotensin II, vasopressin, bradykinin, thrombin, verapamil, gallopamil, dopamine or methionin enkephalin were added to the incubation medium and 6-keto-PGF 1α (the stable metabolite of prostacyclin) and TXB 2 (the stable metabolite of thromboxane) were determined in the supernatant by radioimmunoassay. The ratio of the release of prostacyclin and thromboxane was computed. Norepinephrine increased both prostacyclin and thromboxane release. Isoproterenol increased the ratio of prostacyclin and thromboxane released in cat aortic tissue slices. Phentolamine and propranolol had no effects. Angiotensin II induced a slight but statistically insignificant increase in the ratio of the two prostanoids released. Vasopressin increased thromboxane release only. Bradykinin stimulated the prostacyclin while thrombin stimulated the thromboxane release. Verapamil decreased both prostacyclin and thromboxane production. Gallopamil decreased prostacyclin release and increased thromboxane release from vessel wall slices in a certain concentration range causing a characteristic dose dependent minimum in the ratio of prostacyclin and thromboxane release. Dopamine separately increased prostacyclin release while enkephalin had no significant effect. The data obtained show that in vascular tissue some unidentified yet cytophysiological mechanisms might exist which specifically control the activities of the prostacyclin synthase and thromboxane synthase enzymes.

PAF was administered as an intravenous bolus (0.1 μg/kg) to eight chronically instrumented awake sheep. The effects of pretreatment with an inhibitor of cyclooxygenase (meclofenamate) on PAF-induced changes in lung function were compared to those observed with a specific inhibitor of thromboxane synthase (DP1904). Each animal was studied four times in varied order: PAF alone, PAF + DP1904, PAF + meclofenamate, and DP1904 alone. Saline alone (control), DP1904 alone, and meclofenamate alone did not cause changes in any of the measured variables. DP1904 and meclofenamate significantly attenuated the PAF-induced fall in lung compliance, elevation in peak pulmonary artery pressure, and increased lung lymph flow. Both drugs abolished the PAF-induced increases in lung lymph thromboxane B 2 concentrations. Meclofenamate, but not DP1904, blocked the rise in lymph 6-keto-PGF 1α . Although meclofenamate blocked the rise in lymph PGE 2 , DP1904 resulted in levels 2.7 times higher than PAF alone. We conclude that: (1) inhibition of thromboxane synthase is as effective as inhibition of cyclooxygenase in attenuating PAF-induced changes in lung function, and (2) thromboxane synthase inhibition results in augmented production of PGE 2 following PAF administration in vivo .

Cyclosporine A (CyA) nephrotoxicity is associated with impaired renal hemodynamic function and increased production of the vasoconstrictor eicosanoid thromboxane A2 (TxA2). In CyA toxic rats, renal dysfunction can be partially reversed by inhibitors of thromboxane synthase. However, interpretation of these results is complicated since inhibition of thromboxane synthase may cause accumulation of prostaglandin endoperoxides that can act as partial agonists at the TxA2 receptor and may blunt the efficacy of treatment. Furthermore, these endoperoxides may be used as substrate for production of vasodilator prostaglandins causing beneficial effects on hemodynamics which are independent of thromboxane inhibition. To more specifically examine the role of TxA2 in CyA toxicity, we investigated the effects of the thromboxane receptor antagonist GR32191 on renal hemodynamics in a rat model of CyA nephrotoxicity. In this model, administration of CyA resulted in a significant decrease in glomerular filtration rate (GFR) (2.85 +/- 0.26 [CyA] vs 6.82 +/- 0.96 ml/min/kg [vehicle]; p less than 0.0005) and renal blood flow (RBF) (21.65 +/- 2.31 [CyA] vs 31.87 +/- 3.60 ml/min/kg [vehicle]; p less than 0.025). Renal vascular resistance (RVR) was significantly higher in rats given CyA compared to animals treated with CyA vehicle (5.32 +/- 0.55 [CyA] vs. 3.54 +/- 0.24 mm Hg/min/ml/kg [vehicle]; p less than 0.05). These renal hemodynamic alterations were associated with a significant increase in urinary excretion of unmetabolized, "native" thromboxane B2 (TxB2) (103 +/- 18 [CyA] vs 60 +/- 16 pg/hour [vehicle]; p less than 0.05). Only minimal histomorphologic changes were apparent by light microscopic examination of kidneys from both CyA and vehicle treated animals. However, with immunoperoxidase staining, a significantly greater number of cells expressing the rat common leukocyte antigen was found in the renal interstitium of rats given CyA. There was no detectable increase in monocytes/macrophages in the kidneys of CyA toxic animals. In rats treated with CyA, intraarterial infusion of GR32191 at maximally tolerated doses significantly increased GFR and RBF, and decreased RVR. Although both RBF and RVR were restored to levels not different from controls, GFR remained significantly reduced following administration of GR32191. These data suggest that the potent vasoconstrictor TxA2 plays an important role in mediating renal dysfunction in CyA nephrotoxicity. However, other factors may be important in producing nephrotoxicity associated with CyA.

The effects of the thromboxane synthase inhibitor CGS13080 on the in vivo synthesis of thromboxane and prostacyclin were determined in six healthy volunteers. Two different doses (0.08 and 0.25 mg/kg × h) were infused for six hours under strictly controlled conditions and 2,3-dinor-TxB2 and 2,3-dinor-6-keto-PGF1α were measured in urine using gaschromatography - mass spectrometry. The in vivo synthesis of thromboxane was inhibited by 80-75% while there was no effect on the in vivo prostacyclin synthesis.

The endoperoxide PGH 2 serves as a common intermediate for the enzymatic production of prostaglandins (PGEs and PGFs), thromboxanes (Tx) and prostacyclin (PGI 2 ). These compounds have quite different physiological activities and apparently perform important regulatory functions in various tissues and organs. We have obtained information on the distribution of individual enzymes responsible for the bioconversion of PGH 2 into these compounds in various tissue preparations. [1-C 14 ] PGH 2 was incubated with a membrane fraction from each tissue homogenate. The products were isolated and identified by radiometric TLC and gas chromatography-mass spectrometry. Short life intermediates were detected by their specific biological activities. With this approach, we have demonstrated the formation of thromboxanes in rhesus monkey platelets, spleen and bone marrow, guinea pig lung and spleen, rabbit lung, human platelets and thioglycollate stimulated peritoneal macrophage from rat. On the other hand, the membrane preparation of bovine and mare corpus luteum, uteri from rabbit, monkey and human, rat stomach and small intestine, and rabbit lung produced predominantly prostacyclin. In addition, a PGH 2 to PGD 2 isomerase was found in the homogenate of rat brain and polymorphonuclear leukocytes. In those tissues which possess more than one enzyme catalyzing the metabolism of prostaglandin endoperoxide, substrate availability appeared to be one factor controlling the metabolic fate of the endoperoxide. The wide occurrence of thromboxane and prostacyclin synthetases suggests that their biological roles are not limited to the cardiovascular system.

The prostaglandin endoperoxide PGH2, HHT, HETE, thromboxane A2, and thromboxane B2, which are all products of arachidonic acid metabolites of human platelets, were tested for their ability to modulate platelet cyclic nucleotide levels. None of the compounds tested altered the basal level of cAMP or cGMP, and only PGH2 and thromboxane A2 inhibited PGE1-stimulated cAMP accumulation. Thromboxane A2 was found to be a more potent inhibitor of PGE1-stimulated cAMP accumulation and inducer of platelet aggregation thatn PHG2.

Human cortical hydronephrotic microsomes converted [14C] arachidonic acid to [14C] thromboxane B2 as the major metabolic product. Using [14C] PGH2 as substrate, similar enzymatic conversions were noted with HHT greater than TXB2 less than 6KPGF1 alpha greater than PGE2 greater than PGE2 alpha as the major products. Inhibition of thromboxane synthetase with imidazole 5 mM reduced thromboxane B2 production by 60% and the major product then was 6 keto PGF1 alpha. After addition of imidazole, the metabolic profile showed PKPGF1 alpha greater than PGE2 greater than HHT greater than PGF 2 alpha. Control experiments were carried out using normal cortical tissue obtained from kidneys removed surgically for carcinoma of kidney and rejected for transplantation secondary to fracture as a consequence of blunt trauma. These control kidneys, while they demonstrated an ability to generate thromboxane B2 in vitro, had much less activity than hydronephrotic kidneys and with PGH2 as substrate PGE2 greater than TxB2. In addition, inhibition with imidazole produced mainly PGE2. Thus, like the rabbit and rat, there is enhanced thromboxane and prostacyclin synthesis in human ureteral obstruction and are, therefore, potential vasoactive compounds which may in part be responsible for the hemodynamic alterations occurring in human obstructive uropathy.

We investigated the effects of a new pyridoquinazoline thromboxane synthetase inhibitor infused before administering Escherichia Coli endotoxin into 18 anesthetized sheep with lung lymph fistulas. In normal sheep increasing plasma Ro 23-3423 concentrations were associated with increased plasma levels of 6-keto-PGF1 alpha, a reduced systemic vascular resistance (SVR, r = -0.80) and systemic arterial pressure (SAP, r = -0.92), the mean SAP falling from 80 to 50 mm Hg at the 20 and 30 mg/kg doses. Endotoxin infused into normal sheep caused transient pulmonary vasoconstriction associated with increased TxB2 and 6-keto-PGF1 alpha levels while vasoconstriction and TxB2 increase were significantly inhibited by pretreatment with Ro 23-3423 in a dose-dependent manner. When compared to controls, plasma and lymph levels of 6-keto-PGF1 alpha, PGF2 alpha and PGE2 after endotoxin infusion were increased several-fold by administering Ro 23-3423 up to plasma levels of 10 micrograms/ml. Doses over 30 mg/kg with blood levels above 10 micrograms/ml reduced plasma and lymph levels of 6-keto-PGF1 alpha, PGF2 alpha and PGE2, suggesting cyclooxygenase blockade at this dose. The peak 6-keto-PGF1 alpha levels at 60 min after endotoxin infusion in sheep with Ro-23-3423 levels below 10 micrograms/ml were associated with the greatest systemic hypotension due to a reduced SVR (r = -0.86). After endotoxin infusion the leukotrienes B4, C4, D4 and E4 in lung lymph were assayed by radioimmunoassay and high pressure liquid chromatography and remained at baseline values.

Dog platelets challenged with arachidonic acid fail to aggregate but synthesize a substance which aggregates rabbit and human platelets, this aggregation being suppressed by dibutyryl cyclic AMP. The aggregating substance contracts strips of rabbit aorta and of coeliac and mesenteric arteries, is soluble in diethyl ether, has a half-life of about 40 seconds at 37 degrees C and of 100 seconds at 22 degrees C. Its generation is blocked by various inhibitors of prostaglandin biosynthesis. The thromboxane A2 synthetase inhibitor imidazole and its analogue benzimidazolamine also suppress generation of vessel contracting activity in incubates of dog platelets and prostaglandin H2. Since dog platelets also transform prostaglandin H2 into thromboxane A2 their failure to aggregate, when stimulated by arachidonic acid or by prostaglandin H2, is not due to lack of thromboxane synthesizing ability.

Liberation and metabolism of arachidonic acid may be the common final pathway of different stimuli on the pulmunary vascular bed. In a model of isolated, ventilated rabbit lungs, perfused with krebs Henseleit albumin buffer in a recirculating system, changes of pulmonary vascular resistance and of vascular permeability are monitored continously. The addition of free arachidonic acid or of the Ca-ionophore A 23187 to the perfusion fluid consistently evokes a biphasic increases in vascular resistance as well as an initially reversible increase in vascular permeability, followed by pulmonary edema. Both phases of increased vascular resistance are completely suppressed by inhibition of the cyclooxygenase, decreased to a large degree by inhibitors of thromnoxane synthetase, and markedly augmented by short preincubation of arachidonic acid with ram seminal vescular microsomes and by sulfhydryl reagents. The increased pulmonary vascular permeability is augmented by inhibition of cyclooxygenase and reduced by simulteneous lipoxygenase inhibition. Antagonists of histamine, serotonin and sympathic or parasympathic activity do not have any influence. PG F 2α , TxB E 2 and PG I 2 alter the pulmonary vascular resistance, but do not increase vascular permeability. In inclusion, increased availability of free arachidonic acid evokes a rise in pulmonary vascular resistance, which can be ascribed to cyclooxygenase products, especially to thromboxane, and causes a rise in vascular permeability which can be ascribed to lipoxygenase products. The findings may be related to acute pulmonary lesions with increase in vascular resistance and with vascular leakage.

Thromboxane A 2 was generated by incubation of arachidonic acid with a suspension of human platelets. The filtrate contained 266 ± 46 ng/ml (n=10) of thromboxane A 2 and 25 ng/ml or less of prostaglandin endoperoxides (prostaglandins G 2 +H 2 ). Thromboxane A 2 was 2–10 times more potent than prostaglandin H 2 and 9–102 times and 26–308 times more potent than prostaglandins E 2 and F 2 α, respectively, in causing contractions of the superfused swine coronary artery.

In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 μM, respectively. Further examination of the effect of AHR-5333 on individual reaction of the 5-lipoxygenase pathway (i.e. conversion of LTA4, to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 expoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 μM) to 5-HETE was half maximally inhibited by 20 μM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformationof exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparation of sheep vesicular glands.

7-Oxabicyclo[2.2.1]heptane analogs of prostaglandin (PG) H2 can act as thromboxane (Tx) A2 receptor antagonists or agonists, PGI2 and/rr PGD2 receptor agonists, or exhibit a mixture of the above activities. SQ 28,852, a new analog with a hexyloxymethyl omega side chain, is a potent inhibitor of PG synthesis. SQ 28,852 inhibited collagen and arachidonic acid (AA)-induced platelet aggregation and TxB2 and PGE2 formation, but did not block platelet aggregation induced by ADP or the TxA2 mimics, 9,11-azoPGH2, SQ 26,655, and U-46,619. It also blocked conversion of AA to TxB2, PGE2, and 6-ketoPGF1α by microsomal preparations of human platelets, bovine seminal vesicles, and bovine aortas, respectively, but did not inhibit the conversion of PGH2 to TxA2 by the platelet microsomal preparation. SQ 28,852 (p.o.) protected mice against the lethal effects of AA (75 mg/kg, i.v.). The I50 values for SQ 28,852, indomethacin and aspirin were 0.025, 0.05 and 15 mg/kg, respectively. Neither SQ 28.852 nor indomethacin protected mice from death caused by 9,11-azoPGH2. SQ 28,852 (0.01 to 1 mg/kg, i.v.) inhibited AA-induced bronchoconstriction in anesthetized guinea pigs for at least 60 min. As an inhibitor of AA-induced bronchoconstriction, SQ 28,852 was 16- and 45-times more potent than indomethacin at 3 and 60 min after i.v. administration, respectively. SQ 28,852 did not inhibit brochoconstriction induced by histamine or 9.11-azoPGH2, indicating its specificity of action in vivo . SQ 28,852 is the first example of a new class of cyclooxygenase inhibitors whose structure is similar to that of the naturally occurring endoperoxide, PGH2.

We have investigated the time course of formation of thromboxane A2, thromboxane B2, and the C-17 hydroxy fatty acid, HHT, from arachidonic acid in a washed human platelet suspension. Our results indicate that HHT is not a breakdown product of thromboxane A2, but rather thromboxane A2 decomposes exclusively into thromboxane B2. The kinetics of formation of thromboxane B2 from the endoperoxide prostaglandin H2 in human platelet microsomes was examined. Our data suggest that a bimolecular reaction is involved in the formation of thromboxane A2 from prostaglandin H2 and that thromboxane synthase is not an isomerase, but may be acting via a dismutase-type reaction. One possibility is that thromboxane and HHT are produced simultaneously from two molecules of prostaglandin H2.

Piroxicam is a potent inhibitor of prostaglandin biosynthesis. Experiments utilizing cell culture and microsomes derived from various sources have demonstrated that piroxicam is a selective inhibitor of the cyclooxygenase step of arachidonic acid metabolism. Little blocking activity is observed at the phospholipase, thromboxane or prostacyclin synthetase, and arachidonic acid lipoxygenase steps.

The comparative effects of three so called “thromboxane-synthetase- inhibitors” (imisazole, N-0.164, and U-51065) on arachidonate metabolism and on platelet aggregation were studied. All three compounds blocked platelet microsomal thromboxane synthesis from prostaglandin endoperoxides without affecting platelet adenyl cyclase. Imidazole, blocked thromboxane synthesis in intact platelets either from arachidonic acid or PGH2, without affecting aggregation. U-51605 simultaneously inhibited thromboxane synthesis and platelet suspension aggregation. N-0164 inhibited aggregation probably at extracellular sites, at concentrations that did not alter arachidonate or PGH2 metabolism. High concentrations of N-0164 simultaneously inhibited PG cyclo-oxygenase and thromboxane synthetase. The lack of specificity of these compounds requires that other actions of these compound must be considered when they are used as pharmacological tools to inhibit thromboxane synthetase.

Injection of γ-carrageenin into t he pleural cavity of rats caused the accumulation of the pleural exudate. When levels of prostaglandins (PGs) and thromboxane (TX) B 2 were quantified by gas chromatography-mass spectrometry as their methyl ester (ME)-dimethyllisopropylsilyl (DMiPS) ether or ME-methoxine-DMiPS ether derivatives, 6-keto-PGF 1α reached the maximum at 1 hr after carrageenin, then PGE 2 and TXB 2 showed peaks at 3 hr and waned off before 9 hr. he PGF 2α level was kept low, but PGD 2 , PGE 1 and PGF 1α were not detected. Aspirin (100 mg/kg, i.p.) significantly decreased the PG and TXB 2 levels and suppressed the rate of plasma exudation until 5 hr, but did not at 7 hr, when it was measured by the amount of exuded pontamine sky blue injected intravenously. OKY-025 (300 mg/kg, i.p.), a selective TXA synthetase inhibitor, and tranylcypromine (20 mg/kg, i.p.), a PGI synthetase inhibitor, could not extensively inhibit the accumulation of the exudate. These results suggest that the cyclooxygenase products of arachidonic acid, particularly PGE 2 , definitely play an important role in the exudation during the first 5 hr.

The role of thromboxane A2 (TxA2) in CCl4-induced liver disease was investigated in mice. Significant elevation of TxB2 in the liver was observed 6 hours after the injection of CCl4. Administration of OKY-046, a selective TxA2 synthetase inhibitor (10 and 50 mg/kg) and ONO-3708, a TxA2 receptor antagonist, (0.5, 1 and 2 mg/Kg) suppressed the elevation of serum GOT and GPT levels and histopathological changes of the liver. In addition, OKY-046 inhibited the elevation of TxB2 in the liver. When U-46619, a stable TxA2 mimetic was injected i.v. into the mice, clear elevation of serum GOT and GPT levels and histopathological score of the liver were observed. These results suggest that TxA2 play a role for the onset of CCl4-induced liver injury in mice.

It has been proposed that thromboxane synthase inhibition (TXSI) may be a useful form of anti-thrombotic therapy and that this is due, in part, to redirection of PGH2 metabolism in favour of PGI2, a potent vasodilator and anti-platelet agent. While redirection has been observed ex vivo there are conflicting reports of its occurrence in vivo . We now describe the characterisation of an acute intravenous challenge model using thrombin, collagen, arachidonic acid (AA) and PGH2 for the study of PGH2 metabolism. Following challenge, plasma concentrations of TXB2, 6-oxo-PGF1α, alleged metabolites of PGI2 (PGI2m) and PGE2 were measured by radioimmunoassay (RIA). Thrombin and collagen challenge resulted in a dose-related increase in plasma TXB2 while AA and PGH2, in addition, elevated 6-oxo-PGF1α and PGI2m. Injection of PGH2 elevated 6-oxo-PGF1α, PGI2m, TXB2 and PGE2 levels. Experimental conditions were defined such that challenge with thrombin (40 NIH units kg−1), collagen (100 kg−1), AA (1mg kg−1) and PGH2 (5μg kg−1) and measurement of eicosanoids 0.5min following challenge (5μg kg−1) and measurement of eicosanoids 0.5min following challenge were optimal for detection of redirection of PGH2 metabolism in vivo . The identity of immunoreactive TXB2 and 6-oxo-PGF1α was further supported by experiments in which the extracted immunoreactive eicosanoids co-eluted with authentic [3H]standards when subject to reverse phase high performance liquid chromatography (RPHPLC). Evidence is also presented that the levels of plasma eicosanoids measured in this model reflect in vivo biosynthesis.

We assessed the effect of a specific thromboxane synthetase inhibitor (an imidazole derivative) on pulmonary hemodynamics and the concentrations of TxB 2 (TxA 2 ), 6-keto-PGF 1α (PGI 2 ), and PGF 2α in pulmonary lymph and transpulmonary blood samples following intravenous administration of E. coli endotoxin (1 μg/kg) in sheep. In control animals the rise in pulmonary artery pressure correlated with increases in plasma and lymph TxB 2 concentrations and large transpulmonary concentration gradients of this metabolite were measured. In imidazle treated animals both pulmonary hypertension as well as increases in plasma and lymph TxB 2 concentrations were substantially reduced. In contrast, peak concentrations of 6-keto-PGF 1α (PGI 2 ) and PGF 2α were severalfold higher than those measured in control animals. This suggests a shunting of endoperoxide metabolism towards prostacyclin and primary prostaglandins and documents the specificity of the thromboxane synthetase inhibitor. Out study provides evidence that endotoxin-induced pulmonary hypertension is mediated by pulmonary synthesis of TxA 2 .

The pharmacokinetics of a new, selective thromboxane synthase inhibitor, sodium 5-(3'-pyridinylmethyl)benzofuran-2-carboxylate were determined for single dose, bolus intravenous injections (1, 3, and 10 mg/kg); for continuous 24 hr infusions (10 and 30 micrograms/kg/min); and for oral doses of gelatin encapsulated powdered drug (3, 10, and 30 mg/kg). Drug disappeared biexponentially after intravenous administration, and plasma concentrations were proportional to the dose. Absorption of drug occurred rapidly after its oral administration; peak plasma levels occurred 1-2 hours after ingestion, and circulating drug was detectable within 30 minutes. For all experiments, inhibition of cellular thromboxane B2 production, ex situ, corresponded with plasma drug levels and its reactivation corresponded with disappearance of the drug indicating that it was not accumulated by platelets.

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 pa ) and pulmonary vascular resistance (PVR), which 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 alos increased arterial thromboxane B2 (a metabolite of thromboxane A2) and 6-keto-PGF1α concentrations (a metabolite of prostacyclin). Both OKY-1581 and OKY-046 prevented thromboxane B2 and 6-keto-PGF1α generation. The initial increments in P pa 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.

PAF-receptor antagonists are known to inhibit gastrointestinal damage induced by endotoxin. In the present study, the interaction between the biosynthesis of PAF and thromboxane (TX) A2, as putative mediators of the acute intestinal damage induced by endotoxin, has been investigated in the anaesthetised rat. Bolus intravenous administration of lipopolysaccharide from E. coli (5-50 mg/kg) induced dose-related jejunal damage, assessed using both macroscopic and histological techniques. This damage was accompanied by significant increases in the jejunal formation of PAF determined by bioassay, and of TXB2, determined by radioimmunoassay. Pretreatment with the structurally-unrelated thromboxane synthase inhibitors, 1-benzyl imidazole (10-50 mg/kg) or OKY 1581 (25 mg/kg) substantially reduced both jejunal damage and TXB2 formation, but did not inhibit PAF formation. Likewise, pretreatment with indomethacin (5 mg/kg) or BW 755C (50 mg/kg) reduced jejunal damage and TXB2 formation but did not affect PAF formation. Pretreatment (2h) with dexamethasone (4 mg/kg) reduced jejunal damage and the formation of both TXB2 and PAF. Intravenous infusion of PAF (100 ng/kg/min for 10 min) induced jejunal damage and significantly increased the formation of TXB2, whereas non-specific jejunal damage induced by oral administration of ethanol did not augment PAF formation. The present findings that inhibition of jejunal thromboxane formation is associated with a substantial reduction in jejunal damage, with no corresponding inhibition in PAF formation, therefore suggests a complex interaction or sequential release of these tissue destructive mediators underlying the intestinal damage induced by endotoxin.

Addition of the one-, two- or three- series endoperoxide to human platelet-rich plasma tend to supress aggregation, through the action of their respective non-enzymatic breakdown products PGE 1 , PGD 2 , or PGD 3 all of which elevate cyclic AMP levels. On the other hand, these stable primary products do not arise in appreciable amounts from intrinsic endoperoxides generated from either endogenous or exogenous free fatty acids. 5,8,11,14,17-Eicosapentaenoic acid (EPA) suppresses arachidonic acid (5,8,11,14-eicosatetraenoic acid) conversion by cycloogygenase (as well as lipoxygenase) to aggregatory metabolites in platelets. Exogenously added EPA was capable of inhibiting PRP aggregation induced either by exogenous or endogenous (released by ADP or collagen) arachidonate. The hypothetical combination of an EPA-rich diet and a thromboxane synthetase inhibitor might abolish production of the pro-aggregatory species, thromboxane A 2 , and enhance formation of the anti-aggregatory metabolite, prostacyclin. Whereas EPA is not detectably metabolized by platelets, dihomo-γ-linolenic acid (8,11,14,-eicosatrienoic acid) is primariley converted by cyclooxygenase and thromboxane synthetase into the inactive metabolite, 12-hydroxyheptadecadienoic (HHD) acid. Pretreatment of human platelet suspensions with the thromboxane synthetase inhibitor imidazole unmasks the aggregatory property of PGH 1 and DLL which was partially compromised by the PGE 1 formed. The combination of the thromboxane synthetase inhibitor and an adenylate cyclase inhibitor unmasks a complete irreversible aggregation by DLL or PGH 1 . The basis of a dietary strategy that replaces AA with DLL must rely on the production by the platelet of an inactive metabolite (HHD) rather than thromboxane A 2 .

Furosemide increases the synthesis of two major renal eicosanoids, prostacylin (PGI 2 ) and thromboxane A 2 (TXA 2 ), by stimulating the release of arachidonic acid which in turn is metabolized to PGG 2 /PGH 2 , then to PGI 2 and TXA 2 . PGI 2 may mediate, in part, the early increment in plasma renin activity (PRA) after furosemide. We hypothesized that thromboxane synthetase inhibition should direct prostaglandin endoperoxide metabolism toward PGI 2 , thereby enhancing the effects of furosemide on renin release. Furosemide (2.0 mg.kg −1 i.v.) was injected into Sprague-Dawley rats pretreated either with vehicle or with U-63, 557A (a thromboxane synthetase inhibitor, 2 mg/kg −1 followed by 2 mg/kg −1 .hr −1 ). Urinary 6ketoPGF 1 α and thromboxane B 2 (TXB 2 ), reflecting renal synthesis of PGI 2 and TXA 2 , as well as PRA and serum TXB 2 , were measured. Serum TXB 2 was reduced by 96% after U-63, 557A. U-63, 557A did not affect the basal PRA. Furosemide increased PRA in both vehicle and U63, 557A treated rats. However, the PRA-increment at 10, 20 and 40 min following furosemide administration was greater in U-63, 557A-treated rats than in vehicle-treated rats and urine 6ketoPGF 1 α excretion rates were increased. These effects of thromboxane synthesis inhibition are consistent with a redirection of renal PG synthesis toward PGI 2 and further suggest that such redirection can be physiologically relevant.

After stimulation of the washed human blood platelets by arachidonic acid (AA), the concurrent evaluations for formed malondialdehyde (MDA) measured by the common photometrical thiobarbituric acid (TBA) method, and for thormboxane B2 (TXB2 measured by gas chromatography, revealed that the formed MDA exceeded the amount of TXB2 on a molar base. However, MDA and TXB2 originating from thromboxane synthesis activity should be produced in approximately equimolar amounts. By treatment of the stimulated platelet samples with stannous chloride it is possible to reduce all peroxidized products of AA which generate MDA otherwise during the TBA reaction and to estimate MDA and TXB2 in a ratio of nearly 1:1. the stannous chloride treatment does not destroy the MDA and does not influence the TBA reaction with MDA. Therefore the simple and quick TBA method can be used after stannous chloride treatment for estimation of thromboxane synthase activity in AA stimulated washed human platelets.

The pattern of prostaglandin synthetase products from human peripheral blood monocytes was examined. Thromboxane and prostaglandin E were found to be the major products released by monocytes/macrophages on day one of culture following cell adherence. If these cells were studied 24h after cell adherence had occurred, then thromboxane synthesis was noted to be markedly reduced and PGE was the major secretory product. A day one type pattern, i.e. high thromboxane, high PGE could be elicited from day two cultured cells if cell adherence was delayed until day two of culture. Inflammatory stimuli caused a consistent rise in PGE release from day one and day two cultures, no consistent change in thromboxane was observed. It is suggested that activation of the thromboxane synthetase pathway in monocytes and macrophages is primarily a consequence of cell adherence. Prostaglandin E and prostacyclin (PGI) appear to be the major products released in response to inflammatory stimuli. These data demonstrate that the pattern and sequence of prostaglandins synthesized are in part a function of the in vitro culture conditions, time in culture and the species studied. Further, these findings offer a possible explanation to the discrepant reports in the literature.

A newly synthesized 9 alpha-homo-9,11-epoxy-5,13-prostadienoic acid analogue, SQ 26, 536, (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z), 13(E)-15S-hydroxyprostadienoic acid) inhibited arachidonic acid (AA)-induced platelet aggregation with an I50 value of 1.7 microM. SQ 26,536 did not inhibit prostaglandin (PG) synthetase activity of bovine seminal vesicle microsomes or thromboxane (Tx) synthetase activity of lysed human blood platelets. SQ 26,536 also inhibited platelet aggregation induced by epinephrine (secondary phase), 9,11-azoPGH2 and collagen but did not inhibit the primary phase of epinephrine-induced aggregation or ADP-induced platelet aggregation. SQ 26,538 (8(R)9(S)11(R)12(S)-9 alpha-homo-9,11-epoxy-5(Z),13(E)-15R-hydroxyprostadienoic acid), a 15-epimer of SQ 26,536, induced platelet aggregation with an A50 value of 2.5 microM. SQ 26,536 competitively inhibited SQ 26,538-induced platelet aggregation with a Ki value of 3 microM. Neither indomethacin, a PG synthetase inhibitor, nor SQ 80,338 (1-(3-phenyl-2-propenyl)-1H-imidazole), a Tx synthetase inhibitor, inhibited SQ 26,538- or 9,11-azoPGH2-induced platelet aggregation. These data indicate that SQ 26,536 and SQ 26,538 are stable antagonist and agonist, respectively, of the human blood platelet thromboxane receptor.

Bovine gastric mucosal microsomes synthesize prostaglandins from arachidonic acid but thromboxane B2 is the principal product. Thromboxane B2 synthesis occurs at an appreciable rate from endogenous precursor but more rapidly with added arachidonate. Nonsteroidal antiinflammatory drugs inhibited synthesis of prostaglandins and thromboxanes with the following decreasing order of potency: indomethacin, fenoprofen, acetylsalicylic acid, phenylbutazone, sulfinpyrazone, and acetaminophen.

The TxA2 synthetase inhibitor, dazoxiben, and the TxA2 antagonist, +/- SQ 29,548, were examined for effects on release and vasoactivity of TxA2 and prostacyclin. Isolated perfused guinea pig lungs were used as the enzyme source from which TxA2 and prostacyclin were released in response to injections of arachidonic acid or bradykinin. Both dazoxiben and +/- SQ 29, 548 inhibited contraction of the superfused rat aorta and bovine coronary artery after arachidonic acid injection through the lung. +/- SQ 29,548 abolished contractions of the rat aorta, but significant aorta contracting activity persisted during dazoxiben treatment. Dazoxiben significantly inhibited arachidonate-induced release of TxA2 (immunoreactive TxB2) into the superfusate, but TxA2 release was significantly potentiated by +/- SQ 29,548. Thus, in the presence of enhanced TxA2 concentrations, +/- SQ 29,548 effectively antagonized the vasospastic effect of TxA2. Dazoxiben diverted a significantly greater amount of arachidonic acid into prostacyclin synthesis (immunoreactive 6-keto-PGF1 alpha), changing original coronary vasoconstriction into relaxation. +/- SQ 29,548 did not significantly modify lung prostacyclin synthesis. Moreover, with +/- SQ 29,548, the absence of TxA2-mediated coronary contraction unmasked active relaxation of the superfused bovine coronary artery, coincident with thromboxane and prostacyclin release. Dazoxiben consistently inhibited TxA2 synthesis and enhanced prostacyclin synthesis. +/- SQ 29,548 augmented TxB2 release in response to arachidonate, but not bradykinin, and did not significantly alter 6-keto-PGF1 alpha release in response to either arachidonate or bradykinin. In terms of vasoactivity measured in vitro, +/- SQ 29,548 and dazoxiben produced similar anti-vasospastic effects, although this was accomplished by completely different mechanisms.

In the preceding paper we described the characterisation of an acute intravenous challenge model for the evaluation of the effects of thromboxane synthase inhibition (TXSI) on eicosanoid metabolism. Herein we describe the biochemical pharmacology of two TXSI and aspirin in this model. Both TXSI caused significant inhibition of plasma TXB2 in vivo without elevation of 6-oxo-PGF1 alpha levels. Similar results were obtained when combined levels of 6-oxo-PGF1 alpha,13,14 dihydro 6-oxo-PGF1 alpha,13,14 dihydro 6,15-dioxo-PGF1 alpha and 6-oxo-PGE1 were measured as an index of PGI2 biosynthesis (PGI2m). Thus no evidence of in vivo redirection of PGH2 to PGI2 was found. Ex vivo experiments performed in serum gave an apparent stimulation of immunoreactive 6-oxo-PGF1 alpha following TXSI but RPHPLC analysis of extracted serum showed that this stimulation was accounted for by increase in a product co-eluting with [3H]PGF2 alpha. The implications of these findings in relation to TXSI and receptor antagonists are discussed.

Effect of a thromboxane synthetase inhibitor (OKY-046) on bronchoconstriction induced by aerosol leukotriene C4 and histamine was studied in anesthetized, artificially ventilated guinea pigs in order to examine whether secondary release of thromboxane A2 is produced by aerosol leukotriene C4 or not. 0.01-1.0 micrograms/ml of leukotriene C4 and 12.5-400 micrograms/ml of histamine inhaled from ultrasonic nebulizer developed for small animals caused dose-dependent increase of pressure at airway opening (Pao) which is considered to be an index representing bronchial response. Pretreatment of the animals with intravenous OKY-046 (100mg/kg) significantly reduced the airway responses produced by inhalation of 0.1, 0.33 and 1.0 micrograms/ml of leukotriene C4, while the pretreatment did not affect the histamine dose-response curve. Based on these findings and previous reports (6,7), it is suggested that aerosol leukotriene C4 activates arachidonate cyclooxygenase pathway including thromboxane A2 synthesis and the released cyclooxygenase products have bronchodilating effect as a whole.

The conversion of (1- 14 C) PGH 2 was studied in human placental and fetal membrane cellular preparations (tissue fragments, homogenate, cytosol, microsomes). Placental and amnion homogenates convert labelled PGH 2 into PGE 2 through a very active PGE 2 isomerase. However isolated placental microsomes do not metabolise PGH 2 into PGE 2 but into T×A 2 (identified as T×B 2 by GC-MS) and presumably 12-HHT. This microsomal T×A 2 synthetase is not active in the whole tissue nor in the homogenate. Placental cytosol gives mainly PGD 2 . No conversion into PGI 2 (identofied as 6 keto PGF 1α ) nor PGF 2α was observed in any fraction. Some aspects of PG synthesis regulation by the placental cytosol were studied: the cytosol contains a heat-stable factor that inhibits T×A 2 synthesis and shifts PGH 2 placental microsome metabolism towards PGE 2 . In addition the placental cytosol inhibits human platelet-aggregation through a heat-labile factor which is not PGI 2 nor PGD 2 . A multiple step regulation of the various PG metabolites synthetised from arachidonic acid in the placenta can be outlined and its physiological implications are discussed.

L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) was found to inhibit several aspects of arachidonic acid (20:4) metabolism in human platelets; the primary effect being inhibition of thromboxane synthetase. Thromboxane B2 (TxB2) formation from exogenous 20:4 or PGH2, or from endogenous 20:4, was inhibited by TPCK at concentrations between 0.1 and 0.5 mM. Formation of malondialdehyde (MDA) and 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT), products which also arise from PGH2, was inhibited to a similar extent. Inhibition of formation from 20:4 of 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), the product of the lipoxygenase pathway, was observed; although the extent of this inhibition was less than that of TxB2 formation. A small inhibitory effect of TPCK on the release of 20:4 from platelet phospholipids was also observed. This evidence indicated that while a number of reactions are inhibited by TPCK, the primary effect appears to be inhibition of thromboxane synthetase.

Leukotriene D4 (LTD4) administered intravenously to anesthetized, spontaneously breathing guinea pigs elicited decreases in dynamic lung compliance (Cdyn) and airway conductance (GAW) with a maximal response achieved at 0.5 min. Simultaneously, plasma levels of the thromboxane metabolite, TxB2, and the prostacyclin metabolite, 6-keto-PGF1 alpha, increased 10-fold over pre-LTD4 levels. Pretreatment of the guinea pigs with meclofenamic acid delayed the onset of the LTD4-induced bronchoconstriction, antagonized the magnitude of the decreases in Cdyn and GAW, and blocked the increase in plasma TxB2 and 6-keto-PGF1 alpha levels. The thromboxane synthetase inhibitor, UK 37,248, suppressed the LTD4-induced bronchoconstriction, while it completely blocked TxB2 production without significantly affecting 6-keto-PGF1 alpha. The SRS-A end organ antagonist, FPL 55712, blocked both the LTD4-induced bronchoconstriction and the production of the arachidonic acid metabolites. These results suggest that thromboxane A2 plays an important role in mediating part of the bronchoconstriction elicited by intravenously administered LTD4 in the guinea pig.

It has been proposed that thromboxane synthase inhibition (TXSI) may be a useful form of anti-thrombotic therapy and that this is due, in part, to redirection of PGH2 metabolism in favour of PGI2, a potent vasodilator and anti-platelet agent. While redirection has been observed ex vivo there are conflicting reports of its occurrence in vivo. We now describe the characterisation of an acute intravenous challenge model using thrombin, collagen, arachidonic acid (AA) and PGH2 for the study of PGH2 metabolism. Following challenge, plasma concentrations of TXB2, 6-oxo-PGF1 alpha, alleged metabolites of PGI2 (PGI2m) and PGE2 were measured by radioimmunoassay (RIA). Thrombin and collagen challenge resulted in a dose-related increase in plasma TXB2 while AA and PGH2, in addition, elevated 6-oxo-PGF1 alpha and PGI2m. Injection of PGH2 elevated 6-oxo-PGF1 alpha, PGI2m, TXB2 and PGE2 levels. Experimental conditions were defined such that challenge with thrombin (40 NIH units kg-1), collagen (100 micrograms kg-1), AA (1 mg kg-1) and PGH2 (5 micrograms kg-1) and measurement of eicosanoids 0.5 min following challenge were optimal for detection of redirection of PGH2 metabolism in vivo. The identity of immunoreactive TXB2 and 6-oxo-PGF1 alpha was further supported by experiments in which the extracted immunoreactive eicosanoids co-eluted with authentic [3H]standards when subject to reverse phase high performance liquid chromatography (RPHPLC). Evidence is also presented that the levels of plasma eicosanoids measured in this model reflect in vivo biosynthesis.

This report outlines the activity of a new thromboxane synthase inhibitor sodium, 5-(3-pyridinylmethyl)-2-benzofurancarboxylate, (U-63557A). U-63557A is a potent inhibitor of the thromboxane synthase in human platelets in vitro, as well as in rhesus monkey platelets ex vivo. A single oral dose of 3.0 mg/kg U-63557A inhibits the platelet thromboxane synthase in rhesus monkeys approximately 80% for at least 12 hrs. U-63557A has been administered to monkeys twice a day, (10 mg/kg) for 14 days, without evidence of drug tachyphylaxis or rebound. U-63557A does not inhibit thrombin-stimulated PGI2 biosynthesis in human endothelial cells, the 5-lipoxygenase in human neutrophils, or the cyclo-oxygenase in a variety of test systems. In anesthetized dogs, U-63557A injected i.v. at 0.1 to 5 mg/kg prevented the blockage of stenosed coronary arteries caused platelet aggregation. Similar effects were obtained by oral administration of 1-5 mg/kg. The thromboxane synthase inhibitor was more efficacious than cyclooxygenase inhibitors and equal to PGI2 in efficacy. Under appropriate conditions the protective effects of U-63557A could be reversed by i.v. cyclooxygenase inhibitors suggesting that its efficacy depended in part on endogenous PGI2 formation. Due to its specificity, oral activity, and extended duration of action, U-63557A is a promising compound for the evaluation of the role of thromboxane synthase in a variety of pathophysiological states.