Correlative alteration of thromboxane A2 with antigen-induced bronchoconstriction and the role of platelets as a source of TXA2 synthesis in guinea pigs: effect of DP-1904, an inhibitor of thromboxane synthetase

Pulmonary mast cells (PMC) are important components of the inflammatory process in equine allergic lung diseases such as heaves. Very little, however, is known of the degranulation kinetics of these cells and thus, their pathophysiologic role remains largely speculative. The purpose of this study was to develop a repeatable protocol for in vitro equine PMC degranulation. Five mature horses (sex: 2 M, 3 F; age: 8.8 +/- 6.5 y), historically free of pulmonary disease and normal on clinical respiratory examination, arterial blood gas analysis, pulmonary mechanics testing and histamine inhalation challenge, were studied. Bronchoalveolar lavage was performed on 4 separate occasions, at least 2 d apart, in a different lung lobe on each occasion. The lavage fluid was concentrated by centrifugation. Cells were resuspended in modified HEPES/Tyrode, assessed for viability by Trypan blue exclusion, and PMC concentration determined. Cell inocula containing 30,000 PMC were incubated with 10(-8) to 6 x 10(-5) M A23187. Cells were then separated by centrifugation and histamine release (HR) was determined by fluorometric assay. The procedure was readily performed and yielded sufficient PMC for 30 to 60 inocula per lavage. Maximal HR (34.4 +/- 16.1%) was obtained with 10(-5) M A23187. The degranulation process was largely complete by 20 min but cell lysis was negligible. The challenge was repeatable within horse and produced a mean coefficient of variability of 23.0% following 20 min incubation with 10(-5) M A23187. We conclude that equine PMC degranulation can be repeatably performed in vitro and speculate that this protocol may be useful in further studies on the pathophysiology and treatment of equine allergic lung diseases.     

ZD1542, a potent thromboxane A2 synthase inhibitor and receptor antagonist in vitro

1. The thromboxane A2 synthase (TXS) inhibitory activity and the thromboxane A2 (TP)-receptor blocking action of ZD1542 (4(Z)-6-[2S,4S,5R)-2-[1-methyl-1-(2-nitro-4-tolyloxy)ethyl]-4-(3- pyridyl)-1,3-dioxan-5-yl]hex-4-enoic acid) has been evaluated in vitro on platelets and whole blood from a range of species including man. Antagonist activity has also been investigated in vascular and pulmonary smooth muscle preparations in vitro. 2. ZD1542 caused concentration-dependent inhibition of human platelet microsomal thromboxane B2 (TXB2) production in vitro (IC50 = 0.016 microM); this inhibition was associated with an increase in prostaglandin E2 (PGE2) and PGF2 alpha formation. 3. ZD1542 also inhibited collagen-stimulated TXS in human, rat and dog whole blood giving IC50 values of 0.018, 0.009 and 0.049 microM respectively. The drug did not modify platelet cyclo-oxygenase activity as inhibition of TXB2 formation was associated with a concomitant increase in the levels of PGD2, PGE2 and PGF2 alpha. ZD1542 had little if any effect against cultured human umbilical vein endothelial cell (HUVEC) cyclo-oxygenase (IC50 > 100 microM) and prostacyclin (PGI2) synthase (IC50 = 18.0 +/- 8.6 microM). 4. ZD1542 caused concentration-dependent inhibition of U46619-induced aggregation responses of human, rat and dog platelets yielding apparent pA2 values of 8.3, 8.5 and 9.1 respectively. The drug was selective as, at concentrations up to 100 microM, it did not modify 5-hydroxytryptamine (5-HT) or the primary phases of adenosine diphosphate (ADP) and adrenaline-induced aggregation. Furthermore, ZD1542 (100 microM) modified only weakly the platelet effects of PGD2, PGE1 and PGI2. 5. ZD1542 also caused concentration-dependent inhibition of U46619-mediated contractions of rat thoracic aorta, guinea-pig trachea and lung parenchyma preparations giving apparent pA2 values of 8.6,8.3 and 8.5 respectively. At concentrations approaching three orders of magnitude greater than those required to block U46619-mediated contractions, the drug did not affect the actions of non-prostanoid agonists or exhibit agonist activity in any of the smooth muscle preparations employed; neither did it interact at EP- or FP-receptors.6. In conclusion, the present study demonstrates that ZD1542 is a drug that exhibits both potent,selective TXS inhibition and TXA2 receptor antagonism.     

Monoclonal anti-idiotypic antibody to a potent thromboxane A2 receptor antagonist and its interaction with thromboxane A2 receptor

A monoclonal anti-idiotypic antibody that interacts with thromboxane A2 receptor was generated using an anti-idiotypic approach. Idiotypic antibodies against a potent receptor antagonist, HS-145, were generated in rabbit. The idiotypic antibodies were then selected by an affinity procedure using SQ29,548-Affi-Gel-102 matrix. The selected idiotypic antibodies were used as surrogate receptor for anti-idiotypic antibody generation. A mouse monoclonal antibody, 3D-9E-12, was generated. It was shown to displace 125I-HS-145 from affinity-purified idiotypic antibodies. It also inhibits 125I-IS-145 binding to thromboxane A2 receptor in human platelet membranes in a dose-dependent manner. Furthermore, it attenuated U46,619-induced increase in [35S]guanosine 5'-O-(thiotriphosphate) binding and GTPase activity in human platelet membranes. Finally, it inhibited U46,619- but not PAF-induced platelet aggregation. These results indicate that 3D-9E-12 acts as a specific antagonist in the thromboxane A2 receptor.     

Inhaled ICI 204,219 blocks antigen-induced bronchoconstriction in subjects with bronchial asthma

Three inhalation formulations of ICI 204,219 were compared for antagonism of antigen-induced bronchoconstriction in 16 subjects with asthma who demonstrated reproducible hypersensitivity to allergen during screening challenges. Each subject received a single 0.2-mg dose of each formulation and was challenged with ragweed 30 min after administration of ICI 204,219 until the forced expiratory volume in 1 s (FEV1) decreased by 20 percent or the maximum allergen concentration (100 micrograms/ml) was reached. The majority of subjects tolerated 100 micrograms/ml of allergen without a 20 percent decrease in FEV1. Inhalation formulations of ICI 204,219 successfully inhibited bronchoconstriction in subjects with reproducible sensitivity to ragweed challenges.     

Effects of aerosol administration of a thromboxane receptor antagonist (S-1452) on experimental asthma in guinea pigs

The importance of thromboxane A2 (TXA2), one of the arachidonate metabolites, in the pathogenesis of bronchial asthma has been emphasized recently. Because aerosolized administration of antiasthmatic drugs is effective and safe, this study examined the effect of aerosolized TXA2 receptor antagonist (S-1452) on allergic bronchoconstriction in passively sensitized and mechanically ventilated guinea pigs. Under the cover of antihistamine, antigen-induced bronchoconstriction was markedly inhibited by pretreatment with aerosolized S-1452 inhalation in a dose-dependent manner. Although aerosolized S-1452 itself provoked weak bronchoconstriction for its partial agonist effect, bronchial responsiveness to inhaled histamine did not change 10 min after S-1452 inhalation. These results indicate that aerosolized S-1452 may be useful in treating bronchial asthma.     

A thromboxane A2 synthetase inhibitor as an anticoagulant for left ventricular assist devices

A comparative study to establish more adequate anticoagulation therapy for left ventricular assist devices was done by administering various anticoagulants: heparin, argatroban (a pure thrombin inhibitor), a thromboxane A2 synthetase inhibitor, and protease inhibitor. Results of the investigation revealed that use of no anticoagulation activates the intrinsic pathway of blood coagulation and causes severe coagulopathy, heparin or argatroban causes bleeding tendency, and a thromboxane A2 synthetase inhibitor can maintain blood coagulation within the acceptable range, but not completely. Combined administration of a thromboxane A2 synthetase inhibitor and protease inhibitor was found to be the best anticoagulation therapy in this study.     

Combination of serine protease inhibitor FUT-175 and thromboxane synthetase inhibitor OKY-046 decreases cerebral vasospasm in patients with subarachnoid hemorrhage

The preventive effect of the serine protease inhibitor FUT-175 (nafamostat mesilate), a potent inhibitor of the complement system, against vasospasm was evaluated in 34 high risk patients with thick and diffuse subarachnoid hemorrhage (SAH) demonstrated by computed tomography corresponding to Fisher group 3. All patients underwent surgery within 96 hours following SAH and received the thromboxane A2 synthetase inhibitor, OKY-046, as part of standard care. FUT-175 (40-160 mg/day) was administered during the initial 4 days following surgery. 455 patients treated without FUT-175 in the Nagasaki SAH Data Bank (non-FUT group) formed the control group. FUT-175 significantly decreased the incidence of symptomatic vasospasm in patients with severe neurological grade (Hunt and Hess grade 3, p < 0.02; Hunt and Hess grade 4, p < 0.02). The incidence of favorable outcome was 76.5% in the FUT group and 60.4% in the non-FUT group, but not statistically different. However, when patients of Hunt and Hess grade 5 were excluded, the FUT group had a significantly improved outcome (p < 0.05). This study suggests that FUT-175 has an additive effect to OKY-046 in preventing vasospasm in high risk patients with severe SAH.     

Effects of inhaled KAA-276, a selective histamine H1 receptor antagonist, on antigen- and histamine-induced bronchoconstriction in animals

The antiasthmatic profile of KAA-276 (1-[1-(4-fluorophenylmethyl)-1H-benzimidazole-2-yl]-5-[2-[4-(2- carboxethyl) phenyl]ethyl]-1,5-diazacyclooctane sulfate, CAS 167264-26-8), a newly synthesized histamine H1 receptor antagonist, given by inhalation as an aerosol was investigated and compared with the profiles obtained using other routes of administration. When given by inhalation, or by intravenous or oral routes, KAA-276 inhibited antigen-induced bronchoconstriction in rats with ID50 (a dose to inhibit the antigen-induced response by 50%) values of 0.054%, 1 mg/kg, and 51.2 mg/kg, respectively. KAA-276 prevented the histamine-induced wheal reaction in rats dose-dependently with ID50 values of 0.22% by inhalation, 0.18 mg/kg by the intravenous route, and 2.3 mg/kg by the oral route. To judge from these results, inhaled KAA-276, unlike intravenous or oral KAA-276, had no inhibitory effect on the histamine-induced wheal reaction at a dose (0.054%) that is effective against the antigen-induced airway asthmatic response. Inhaled KAA-276 suppressed antigen-induced bronchoconstriction in actively sensitized guinea pigs, and histamine-induced bronchoconstriction in monkeys. These results suggest that inhalation of KAA-276 would benefit patients with bronchial asthma without inducing unwanted systemic effects.     

HN-11500--a novel thromboxane A2 receptor antagonist with antithrombotic activity in humans at arterial blood flow conditions

Effect of dietary rapeseed oil from 00-varieties of rapeseed (0, 1.5% or 3% respectively in the wet compounded diets) on plasma thyroxine (T4), reproductive performance and kit weight gain during lactation was investigated with 3 groups of each 20 mink females. Plasma T4, which has not previously been reported for female mink, was significantly lower in lactating than in non-pregnant females. Unlike in an earlier experiment with growing male mink, it was not affected by dietary rapeseed oil. Reproductive performance, female weight development, feed consumption, and kit weight gain was normal in all treatment groups and there were no significant effects of the experimental treatment.     

Protective effect of thromboxane synthetase inhibitor on hypertensive renal damage in Dahl salt-sensitive rats

1. The effects of OKY-046, a specific thromboxane (TX) synthetase inhibitor, on blood pressure, urinary excretion of TX and its release from blood platelets and renal papilla, and pathological change of glomeruli were evaluated in Dahl salt-sensitive rats. 2. Average daily intakes of OKY-046-treated rats were 0.93 mg/kg (low dose), 9.8 mg/kg (moderate dose), and 88 mg/kg (high dose). 3. Systolic blood pressure tended to decrease by 6.3, 11.4, and 10.9% in three OKY-treated groups. 4. OKY-046 suppressed the release of TX from platelets in a dose-dependent fashion. Both TX in urine and released from renal papilla decreased in OKY-treated groups with moderate and high dose. OKY-046 resulted no change in urinary excretion or release from renal papilla of prostaglandin E2 or 6-keto-prostaglandin F1alpha. 5. Glomerular sclerosis score decreased significantly in both groups treated with moderate and high doses of OKY-046. 6. An inhibition of renal TX synthesis by TX synthetase inhibitor has a protective effect on the development of hypertensive renal damage with minor antihypertensive effect in Dahl salt-sensitive rats.     

The effect of BAY u 3405, a thromboxane receptor antagonist, on prostaglandin D2-induced nasal blockage

BACKGROUND: Nasal lavage and challenge studies in allergic rhinitis implicate prostaglandin (PG) D2 in the genesis of nasal blockage. PG D2 is known to act via at least two receptors, the thromboxane prostanoid receptor and the PG D2 prostanoid (DP) receptor; the lower airway effects are mediated chiefly by the TP receptor. The receptor involved in the genesis of PG D2-induced nasal blockage is unknown. BAY u 3405 is a potent selective competitive TP receptor antagonist, which inhibits the lower airway response to PG D2, and shifts the dose-response curve to the right by up to 16-fold. METHODS: The efficacy of a single oral dose of 20 mg of BAY u 3405 was examined in comparison with PG D2 nasal insufflation in a randomized, double-blind, placebo-controlled crossover study, with objective measurement of nasal resistance by active posterior rhinomanometry. RESULTS: BAY u 3405 afforded no protection against PG D2-induced nasal blockage. CONCLUSIONS: This suggests that PG D2-induced nasal blockage may be mediated by the DP receptor rather than the TP receptor and that TP receptor antagonists are unlikely to be of benefit in the treatment of allergic rhinitis. In vivo investigation with specific potent DP receptor antagonists is awaited.     

The influence of misoprostol on post-aspirin bronchoconstriction in patients with aspirin sensitive asthma

It is believed that aspirin (ASA) and other nonsteroidal antiinflammatory drugs elicit dysponea in ASA sensitive asthmatics by blocking the cyclooxygenase. It is unclear whether this bronchospasm is due to shunting of arachidonic acid into the lipooxygenase pathway or removal of cyclooxygenase product which prevent bronchospasm. Diminished tissue concentration of PGE may cause bronchoconstriction. PGE play also modulatory function to mast call decreasing the release of mediators of anaphylaxis. There are some evidences concerning the mast cell degranulation in postaspirin reaction in ASA sensitive asthmatics. The authors investigated the influence of synthetic analogue of PGE1--misoprostol (Cototec, Searle) on the postaspirin bronchoconstriction in seven ASA sensitive asthmatics aged 33-62. Aspirin threshold doses ranged from 10 to 150 mg. Postaspirin bronchoconstriction begun usually within 1-2 hrs after digestion of ASA and 200 micrograms were additionally given 2 h later. Seven days later misoprostol (400 micrograms) was administered together with previously determined dose of ASA. One the other day the bronchodilating effect of misoprostol alone was examined. In all but one patients we observed the protective influence of misoprostol on ASA induced bronchoconstriction. Max. fall in FEV1 in % after ASA in each of the patients was 40, 25, 24, 33, 47 and 54, and after ASA with misoprostol, respectively 10, 9, 4, (+8), 10, (+2) and 45. Misoprostol given together with ASA attenuated aspirin-induced bronchoconstriction reaching statistical significance at 3 and 3.5 h, and also diminished extrapulmonary symptoms. The authors discuss the possible mechanism of protective influence of misoprostol.     

Effects of uroguanylin and guanylin against antigen-induced bronchoconstriction and airway microvascular leakage in sensitized guinea-pigs

Uroguanylin and guanylin are isolated mainly from the gastrointestinal tract and are activators of guanylyl cyclase C receptor (GC-C), which mediates the production of intracellular cyclic guanosine 3',5'-monophosphate (cyclic GMP). The bronchodilator effects of agents that raise cyclic GMP levels, such as atrial natriuretic peptide, have been reported, and uroguanylin mRNA has recently been detected in extra-gastrointestinal tissues, including the lung, suggesting their role in pulmonary activity. In the first step of this study, we examined the relaxant effects of uroguanylin and guanylin on isolated tracheal smooth muscle of guinea-pigs, and measured tissue cyclic GMP levels by means of enzymeimmunoassay. Uroguanylin produced concentration-dependent relaxant effects on resting tone and significant elevated cyclic GMP levels. Guanylin produced the same, but less potent, effects. In this study, we first investigated the effects of uroguanylin and guanylin on antigen-induced bronchoconstriction and airway microvascular leakage in actively sensitized guinea-pigs. Anesthetized male guinea-pigs, ventilated via a tracheal cannula, were placed in a plethysmograph to measure pulmonary mechanics for 10 min after challenging with 1 mg/kg of ovalbumin. Evans blue dye was then extravasated into their airway tissues to measure microvascular leakage. Intravenous pretreatment with uroguanylin significantly inhibited ovalbumin-induced bronchoconstriction and microvascular leakage in a dose-dependent manner. These inhibitory effects were mimicked by 8-bromoguanosine 3', 5'-cyclic monophosphate. This study is the first to show that uroguanylin not only had a potent bronchodilatory effect but also inhibited microvascular leakage. These results encouraged us to continue the above experimental and clinical studies in bronchial asthma.     

Effects of FR173657, a non-peptide B2 antagonist, on kinin-induced hypotension, visceral and peripheral oedema formation and bronchoconstriction

The mutagenic and lethal effects of abasic sites in DNA are averted by repair initiated by 'class II' apurinic (AP) endonucleases, which cleave immediately 5'to abasic sites. We examined substrate binding by the human AP endonuclease, Ape protein (also called Hap1, Apex or Ref-1). In electrophoretic mobility-shift experiments, Ape bound synthetic DNA substrates containing single AP sites or tetrahydrofuran (F) residues. No complexes were detected with single-stranded substrates or unmodified duplex DNA. In EDTA, the concentration of Ape required to shift 50% of duplex F-DNA was approximately 50 nM, while the addition of 10 mM MgCl2 nearly eliminated detectable F-DNA@Ape complexes. Filter-binding studies demonstrated a half-life of approximately 50 s at 0 degrees C for F-DNA@Ape complexes in the presence of EDTA, and <15 s after the addition of Mg2+. The DNA recovered from F-DNA@Ape complexes was intact but was rapidly cleaved upon addition of Mg2+, which suggests that these protein-DNA complexes are on the catalytic pathway for incision. Methylation and ethylation interference experiments identified DNA contacts critical for Ape binding, and Cu-1, 10-phenanthroline footprinting suggested an Ape-induced structural distortion at the abasic site prior to cleavage.     

Effects of an AT1 receptor antagonist, an ACE inhibitor and a calcium channel antagonist on cardiac gene expressions in hypertensive rats

1. This study was undertaken to determine whether the AT1 receptor directly contributes to hypertension-induced cardiac hypertrophy and gene expressions. 2. Stroke-prone spontaneously hypertensive rats (SHRSP) were given orally an AT1, receptor antagonist (losartan, 30 mg kg-1 day-1), an angiotensin converting enzyme inhibitor (enalapril 10 mg kg-1 day-1), a dihydropyridine calcium channel antagonist (amlodipine, 5 mg kg-1 day-1), or vehicle (control), for 8 weeks (from 16 to 24 weeks of age). The effects of each drug were compared on ventricular weight and mRNA levels for myocardial phenotype- and fibrosis-related genes. 3. Left ventricular hypertrophy of SHRSP was accompanied by the increase in mRNA levels for two foetal phenotypes of contractile proteins (skeletal alpha-actin and beta-myosin heavy chain (beta-MHC)), atrial natriuretic polypeptide (ANP), transforming growth factor-beta-1 (TGF-beta 1) and collagen, and a decrease in mRNA levels for an adult phenotype of contractile protein (alpha-MHC). Thus, the left ventricle of SHRSP was characterized by myocardial transition from an adult to a foetal phenotype and interstitial fibrosis at the molecular level. 4. Although losartan, enalapril and amlodipine lowered blood pressure of SHRSP to a comparable degree throughout the treatment, losartan caused regression of left ventricular hypertrophy of SHRSP to a greater extent than amlodipine (P < 0.01). 5. Losartan significantly decreased mRNA levels for skeletal alpha-actin, ANP, TGF-beta 1 and collagen types I, III and IV and increased alpha-MHC mRNA in the left ventricle of SHRSP. Amlodipine did not alter left ventricular ANP, alpha-MHC and collagen types I and IV mRNA levels of SHRSP. 6. The effects of enalapril on left ventricular hypertrophy and gene expressions of SHRSP were similar to those of losartan, except for the lack of inhibition of collagen type I expression by enalapril. 7. Unlike the hypertrophied left ventricle, there was no significant difference between losartan and amlodipine in the effects on non-hypertrophied right ventricular gene expressions of SHRSP. 8. Our results show that hypertension causes not only left ventricular hypertrophy but also molecular transition of myocardium to a foetal phenotype and interstitial fibrosis-related molecular changes. These hypertension-induced left ventricular molecular changes may be at least in part mediated by the direct action of local angiotensin II via the AT1, receptor.     

Effect of DP-1904, a thromboxane A2 synthase inhibitor, on passive Heymann nephritis in rats

Despite the fact that aneuploidy is a major genetic cause of human morbidity and mortality, antimutagenicity studies have used predominantly short-term tests that detect gene mutations, chromosomal aberrations, and micronuclei. Therefore, the major deficiency in the use of short-term tests for antimutagenicity studies is those that detect chromosomal malsegregation leading to aneuploidy. Thus, we initiated a study on the utility of short-term tests for the detection of antianeugenic activity. We selected strain D61.M of Saccharomyces cerevisiae, nocodazole, and chlorophyllin as a model short-term test, aneugen, and antimutagen, respectively, for our initial study. Chlorophyllin strongly inhibited the aneugenic activity of nocodazole, but had no effect on the endpoints when tested alone, in strain D61.M. To our knowledge, this is the first report of an antianeugen. Furthermore, we conclude that strain D61.M can be used as a relatively simple, inexpensive, and rapid short-term test for the study of antianeugenicity.     

PGF2 alpha causes bronchoconstriction and pulmonary vasoconstriction via thromboxane receptors in rat lung

OBJECTIVE: The present study set out to determine general levels of distress, anxiety and depression in an obstetric-gynaecology inpatient population and to ascertain levels of patient satisfaction with services. METHOD: 200 English-speaking consecutive admissions to the hospital filled out a package of questionnaires consisting of a demographic data form, the 30-item General Health Questionnaire, the Speilberger State-Trait Anxiety Inventory, the Inventory to Diagnose Depression and a Patient Satisfaction with Services Questionnaire. 134 packages were fully completed. RESULTS: The most significant findings were: first, that high levels of anxiety prevailed, with approximately 1/3 of patients at or above the 75th percentile for anxiety levels; and second, that those patients born in non-English speaking countries were significantly more dissatisfied with their care. CONCLUSIONS: The results of this study suggest that there is a need for increased awareness of and further research into the psychological needs of obstetric-gynaecology patients.     

Effect of inhaled indomethacin on exercise-induced bronchoconstriction in children with asthma

We evaluated the effect of inhaled indomethacin, a nonsteroidal antiinflammatory drug (NSAID), on exercise-induced bronchoconstriction (EIB) in children with asthma. Nine asthmatic children (7 boys, 2 girls, with a mean +/- SEM age of 11.0 +/- 0.8 yr) with a history of EIB participated in this study. These subjects were pretreated with inhaled indomethacin (3 mg/m2 body surface area [BSA]) or placebo (0.9% saline) according to a double-blind, randomized, crossover design, and underwent an exercise challenge test 15 min after the pretreatment. Inhaled indomethacin significantly attenuated EIB. The mean maximal percent decrease in FEV1 following exercise was 36.1 +/- 5.7% after placebo and 18.0 +/- 4.6% after indomethacin pretreatment (p = 0.0310). Indomethacin also significantly reduced the mean maximal decrease in arterial oxygen saturation after exercise (p = 0.0378). The inhibition of local prostaglandin synthesis and/or ion transport in the airways may be a mechanism involved in the protective potency of inhaled indomethacin.