Effects of tetrandrine on smooth muscle contraction induced by mediators in pulmonary hypertension.

AIM: In attempt to characterize tetrandrine on pulmonary hypertension, biological activities induced by a range of mediators implicated in the pathogenesis of pulmonary hypertension were investigated. METHODS: Pulmonary artery rings and tracheal segments were contracted with couples of bioactive substances in which a series experiments including effects of tetrandrine on calcium agonist, endothelin, thromboxane A2, angiotensin II, neuropeptide Y, histamine, 5-methyl furmethide were performed, the influences of tetrandrine in the concentration of 1 to 30 micromol/L were investigated. RESULTS: Tetrandrine inhibited calcium agonist BayK8644, endothelin-1 and thromboxane A2 mimetic U46619, angiotensin II- and neuropeptide Y-induced contractile responses with depression of the maximal contraction of pulmonary artery rings in a varying extent. Tetrandrine inhibited leukotriene E4-induced concentration-response curve in a competitive antagonist manner with a pKB of (5.29+/-0.11) without any influence leukotriene C4, leukotriene D4, histamine, and 5-methyl furmethide induced contractile responses of guinea pig trachea. CONCLUSION: Tetrandrine may produce multiple pharmacological effects against calcium channel antagonist, U46619, endothelin-1,angiotension II, and neuropeptide Y induced vasoconstriction in rat pulmonary arteries in varying extent and inhibition of leukotriene E4 rather than C4, D4, histamine, and 5-methyl furmethide induced contractile responses on rat tracheal segments. These pharmacological characteristics are considered to contribute to its antihypertensive action during pulmonary hypertension.

Leukotriene modifiers: novel therapeutic opportunities in asthma.

Cysteinyl leukotrienes (Cys-LT) are powerful proinflammatory autacoids that cause long-lasting bronchoconstriction, plasma leakage, increased mucus production; their biological activity suggests a prominent role in the etiopathology of asthma and several Cys-LT receptor antagonists and synthetase inhibitors have been developed as new antiasthmatic drugs. Zafirlukast was discovered by a mechanism-based approach to drug discovery; early structure-activity relationship analyses of the prototype SRS-A antagonist FPL-55712, lead to the identification of an indole-containing lead compound that was more specific than FPL-55712. Modifications were made on the lipid-like tail, indole backbone and acidic head region of this lead compound, resulting in potent and selective leukotriene receptor antagonists such as ICI-198615 and 204219 (zafirlukast). On the basis of successful results in preclinical asthma models, zafirlukast was recommended for clinical development and became the first leukotriene-modifier to be approved for the treatment of asthma. Leukotriene biosynthesis inhibitors (LSI) also represent a promising approach to the treatment of asthma and may theoretically provide a broader protection than Cys-LT receptor antagonists by inhibition of the synthesis of the two major leukotrienes, the Cys-LT and the chemotactic LTB4. The LSI BAY X-1005 is the result of a broad chemistry program that identified 15-HETE as an endogenous inhibitor of leukotriene synthesis and REV 5901 as a lead prototypic quinoline-based 5-lipoxygenase (5-LO) inhibitor. Clinical studies demonstrated the effectiveness of BAY X-1005 in experimental conditions such as allergen provocation and cold-air induced asthma. However, no consistent treatment effect in the overall asthma population (mild to moderately severe asthmatics) lead to discontinuation of its development.

Studies on 5-lipoxygenase inhibitors. II. Discovery, optical resolution and enantioselective synthesis of FR110302, a highly potent non-redox type 5-lipoxygenase inhibitor.

A novel series of 2,2-dialkyl-5-(2-quinolylmethoxy)-1,2,3, 4-tetrahydro-1-naphthols was synthesized and evaluated as 5-lipoxygenase (5-LO) inhibitors. Systematic optimization led to identification of several highly potent non-redox type 5-LO inhibitors with nanomolar IC50s as racemic mixtures. Optical resolution of racemate 50 indicated that its 5-LO inhibitory activity was enantiospecific and due to the (+)-enantiomer. An efficient synthetic route to the (+)-enantiomers via asymmetric reduction of tetralone intermediates was established. The best compound, (+)-2,2-dibutyl-5-(2-quinolylmethoxy)-1,2,3,4-tetrahydro-1-naphtho l (FR110302, (+)-50), showed potent inhibitory activity against leukotriene (LT) biosynthesis by intact neutrophiles in rats (IC50 4.9 nM) and in humans (IC50 40 nM). Furthermore oral administration of FR110302 significantly inhibited neutrophil migration in the rat air pouch model at 1 mg/kg.

Leukotrienes antagonist-inhibitor and the bronchial response to inhaled antigen in actively sensitized guinea-pigs.

The aim of the study was to estimate an eventual contribution of leukotrienes in respiratory mechanic changes observed in guinea-pig during bronchial anaphylaxis. Twenty-eight guinea-pigs, actively sensitized to ovalbumin, were anesthetized and curarized before being challenged, during controlled ventilation, with antigen administered as an aerosol. Antigen challenge was performed before and after pretreatment with nebulized FPL55712 and BW755C, respectively used as an antagonist of leukotrienes and as a cyclo-oxygenase/lipoxygenase inhibitor. The experiments were carried out during continuous recording of tracheal pressure (Ptr), airflow (V) and tidal volume (VT) signals variations evidencing respiratory asynchronism (AS) and allowing measurements of the changes in airway resistance (AR) and dynamic compliance (Cdyn) during all the challenge. Administration of nebulized ovalbumin was stopped at the onset of AS appearance chosen as the threshold of the antigen-induced bronchoconstriction. The results showed that separate or combined pretreatment with FPL55712 and BW755C did not significantly modify the threshold of the ovalbumin-induced bronchoconstriction in guinea-pigs. Nevertheless pretreatment with nebulized FPL55712 reduced significantly the intensity and the duration of the response of these animals to inhaled leukotriene D4 (LTD4). Moreover the response of guinea-pigs to inhaled LTD4, characterized by a starting decrease in Cdyn, appeared quite different from the response to the antigen starting by an abrupt rise in RA induced by a sudden bronchoconstriction. From these results, we concluded that leukotrienes seem not to be the main mediator of the bronchial anaphylaxis in guinea-pigs.

Effects of a specific cysteinyl leukotriene antagonist, pranlukast, on antigen-induced cysteinyl leukotriene-mediated rhinitis in guinea pigs.

To examine the effects of a specific cysteinyl leukotriene (cysLT) antagonist, pranlukast, on allergic rhinitis, antigen-induced rhinitis in guinea pigs was modified by pretreatment with an cyclooxygenase inhibitor (indomethacin) followed by an H1-blocker (pyrilamine). Intranasal ovalbumin (OVA) administration in actively sensitized guinea pigs resulted in concentration-dependent increases in nasal permeability and nasal airway resistance (NAR). Although pyrilamine (1 mg/kg, i.v.) abolished these antigen-induced changes, pretreatment with indomethacin (5 mg/kg, i.v.) followed by pyrilamine enhanced these responses to a degree similar to that observed with OVA challenge alone. Analyses of nasal perfusate in indomethacin/pyrilamine-pretreated animals showed that cysLTs increased by 270.8%, whereas thromboxane B2 decreased by 88.3% as compared with those on challenged with OVA alone. Oral administration of pranlukast (1-10 mg/kg) dose-dependently prevented increases in nasal permeability and NAR of indomethacin/pyrilamine-pretreated animals. However, an anti-allergic agent, azelastine, did not affect these responses. These results indicate that pranlukast suppresses antigen-induced cysLT-mediated responses of allergic rhinitis in actively sensitized guinea pigs. A cysLT antagonist, pranlukast, may thus prevent cysLT-mediated symptoms of allergic rhinitis.

Cysteinyl leukotrienes do not mediate lipopolysaccharide-induced airway hyperresponsiveness in guinea pigs.

Inhalation of bacterial lipopolysaccharide (LPS) by guinea pigs caused bronchial hyperreactivity to acetylcholine with a peak at 2 hr after exposure. Exposure to 0.01% LPS for 30 min resulted in an elevation of cysteinyl leukotrienes (cys-LTs) content in bronchoalveolar lavage fluid (BALF) which was obtained 1 hr after LPS exposure. The cys-LTs antagonist, ONO-1078 (10 mg/kg, p.o.), significantly inhibited LPS-induced bronchial hyperreactivity, but ICI-204,219 (10 mg/kg, p.o.), another cys-LT antagonist, did not. Each dose employed in the present study was sufficient to inhibit LTD4-induced broncho-constriction in guinea pigs. In order to investigate the inhibitory mechanism of ONO-1078, the effect on the LPS-induced production of tumor necrosis factor (TNF) was examined. The amount of TNF in BALF increased significantly 2 hr after exposure to LPS. The inhalation of murine recombinant TNF-alpha (5 x 10(4) u/ml) resulted in bronchial hyperreactivity in guinea pigs. ONO-1078 (10 mg/kg, p.o.) inhibited the increase of LPS-induced TNF in BALF, but ICI-204,219 (10 mg/kg, p.o.) had no effect. These results suggest that TNF plays an important role in the onset of LPS-induced bronchial hyper-reactivity, and that ONO-1078 inhibits the LPS-induced airway hyperreactivity probably due to the inhibition of TNF production.

SRS-A antagonist pyranoquinolone alkaloids from east African Fagara plants and their synthesis.

Three pyranoquinolone alkaloids isolated from two East African Fagara plants have been found to exhibit SRS-A antagonist action. Their synthesis has been accomplished, using a modified Coppola's method or a thermal cyclization followed by an electrocyclic ring closure.

Effects of ONO-1078, a leukotriene antagonist, on cardiovascular responses induced by vagal stimulation, capsaicin, and substance P in guinea pigs.

AIM: To determine the role of ONO-1078, 4-oxo-8 -[p-(4-phenylbutyloxy) benzoylamino]- 2-(tetrazol-5-yl) -4H-1-benzopyran hemihydrate, in cardiovascular responses induced by vagal stimulation, capsaicin, and substance P. METHODS: Evans blue extravasation in the atrium and ventricle, and mean arterial pressure (MAP) were observed. RESULTS: Electric stimulation of vagus (ESV, 10 Hz, 5 ms, 2 or 10 V, for 90 s) increased Evans blue extravasation in the hearts of atropine (1 mg.kg-1, i.v.)-pretreated guinea pigs. Capsaicin (0.05 mg.kg-1, i.v.) and substance P (1 microgram.kg-1, i.v.) enhanced the dye extravasation and elicited a drop in MAP. ONO-1078 (0.03 and 0.1 mg.kg-1, i.v.) inhibited ESV-induced response, especially at stimulation of 2 V. ONO-1078 (0.03 mg.kg-1) attenuated capsaicin-induced cardiac microvascular leakage and hypotensive response, but failed to inhibit substance P-induced responses. CONCLUSION: ONO-1078 can modulate the cardiovascular responses in neurogenic inflammation, possibly mediated by inhibiting sensory neuropeptide release.

Effects of the peptide leukotriene receptor antagonist ICI 198,615 on the in vivo and in vitro changes in guinea pig bladder function which occur after sensitization with ovalbumin.

PURPOSE: The present studies were designed to determine the effects of in vivo and in vitro administration of ICI 198,615 (ICI), a leukotriene receptor antagonist, on the inflammatory changes that occur in the bladder after sensitization with ovalbumin. MATERIALS AND METHODS: The effect of intravenous administration of ICI on urodynamic changes after instillation of ovalbumin to sensitized guinea pigs was evaluated by in vivo cystometry. Responses of in vitro bladder muscle strips to contractile stimuli and ovalbumin were also evaluated in the presence of ICI. RESULTS: In sensitized guinea pigs, in vivo cystometry with ovalbumin induced a marked decrease in bladder capacity and increase in intravesical pressure. Pretreatment with ICI prior to cystometry prevented the ovalbumin-induced changes in capacity and intravesical pressure. There were no significant differences between control and sensitized animals in the responses of in vitro bladder muscle strips to field stimulation or bethanechol. However, maximal contractile responses to ovalbumin were significantly greater in the strips from sensitized animals than in controls. Preincubation with ICI, indomethacin, or pyrilamine alone was unable to inhibit the contractile responses to ovalbumin. However, combined administration of ICI, indomethacin and pyrilamine completely blocked the responses. CONCLUSIONS: In vivo administration of the leukotriene receptor antagonist ICI 198,615 reversed the urodynamic changes induced by ovalbumin challenge in sensitized guinea pigs. These results indicate that leukotrienes are primarily responsible for the changes in in vivo bladder function associated with sensitization.

Differential activation of mitogen-activated protein kinases by H2O2 and O2- in vascular smooth muscle cells.

Increased generation of active oxygen species such as H2O2 and O2- may be important in vascular smooth muscle cell growth associated with atherosclerosis and restenosis. In previous work, we showed that H2O2 stimulated vascular smooth muscle cell growth and proto-oncogene expression. In the present study, we compared the effects of H2O2 and O2- on cultured rat aortic vascular smooth muscle cell growth and signal transduction. O2- was generated in a concentration-dependent manner by the naphthoquinolinedione LY83583. Vascular smooth muscle cell growth, as measured by [3H]thymidine incorporation, was stimulated by 200 mumol/L H2O2 (110% increase versus 0.1% serum) and 1 mumol/L LY83583 (175% increase) to levels comparable to 10 ng/mL platelet-derived growth factor (210% increase). Since activation of mitogen-activated protein kinase (MAP kinase) is one of the earliest growth factor signal events, the activity of MAP kinase was measured by changes in mobility on Western blot and by phosphorylation of myelin basic protein. There was a concentration-dependent increase in MAP kinase activity by LY83583 (maximum, 10 mumol/L) but not by H2O2. The time course for activation of MAP kinase by LY83583 showed a maximum at 5 to 10 minutes with return to baseline by 20 minutes. Activation of MAP kinase by LY83583 was protein kinase C dependent. Expression of MAP kinase phosphatase-1 (MKP-1), a transcriptionally regulated redox-sensitive protein tyrosine/threonine phosphatase, was also measured. Although H2O2 induced MKP-1 mRNA to a greater extent than did LY83583, the increased MKP-1 expression could not explain the inability of H2O2 to stimulate MAP kinase, because mRNA levels were not detected until 60 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-dependent mediation of leukotriene D4-induced airway microvascular leakage and bronchoconstriction in the guinea pig.

The i.v. administration of leukotriene (LT)D4 to anesthetized guinea pigs produced dose-dependent increases in pulmonary microvascular permeability, as measured by extravasation of Evans blue dye into the trachea, main bronchi, and small airways, with an ED50 of approximately 0.05 microgram/kg. When LTD4 was administered at 0.3 microgram/kg, the resulting plasma extravasation into all three airway sections was markedly reduced by pretreatment with a cyclooxygenase inhibitor, meclofenamic acid (2.5 mg/kg, i.v.), a thromboxane (TX) receptor antagonist, SQ 29,548 (0.1 or 1 mg/kg, i.v.), or a peptidoleukotriene receptor antagonist, pranlukast (SB 205312) (0.1 or 1 mg/kg, i.v.), but not by the H1 histamine receptor antagonist, pyrilamine. When LTD4 was administered at 1.0 microgram/kg, meclofenamate (2.5 or 5 mg/kg, i.v.) or SQ 29,548 slightly attenuated plasma extravasation only in the small airway, whereas pranlukast was effective in all three airway segments. Administration of the 5-lipoxygenase inhibitor, zileuton (10 mg/kg, i.v.), or the PAF antagonist, L-659,989 (5 mg/kg, i.v.), did not affect the microvascular leakage response to 1.0 microgram/kg LTD4. In addition, i.v.-administered LTD4 (0.3 or 1.0 microgram/kg) or the prostaglandin (PG)/TXA2 receptor agonist, U-46619 (3.0 micrograms/kg), produced significant bronchoconstriction as measured by increases in pulmonary insufflation pressure. The bronchoconstrictor responses to LTD4 were markedly attenuated by the same inhibitors, namely meclofenamic acid, SQ 29,548, and pranlukast, that reduced the 0.3 microgram/kg LTD4-induced plasma extravasation throughout the airways and the 1.0 microgram/kg LTD4-induced extravasation into the small airways. U-46619-induced bronchoconstriction was blocked only by SQ 29,548.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium derived relaxing factor release from canine coronary artery by leukocytes.

Lectins, known to recognize endothelial cell adhesion molecules, have been shown to release endothelium-derived relaxing factor (EDRF) from blood vessels. We investigated the effects of different leukocyte-type cells to determine if these cells, by interacting with the endothelium, could release EDRF from the circumflex branch of the canine coronary artery. The following cells were investigated: human promyelocytic leukemia (HL-60), human monocyte (THP-1), and human Burkitt lymphoma (DAUDI). All of these cells produced a significant endothelium-dependent relaxation of the dog coronary artery in the presence of ibuprofen. The endothelium-dependent relaxations were reversed by hemoglobin (10 microM), methylene blue (3 microM), 6-anilino-5,8-quinolinedione (LY 83583, 30 microM), and NG-nitro-L-arginine methyl ester (L-NAME, 1 mM). HL-60 cells grown in the presence of 1 mM L-NAME retained their ability to cause endothelium-dependent relaxation of the canine coronary artery, suggesting that the source of the NO was the endothelium and not the HL-60 cells. The cell-induced vascular relaxation could be obtained in the absence of extracellular calcium. It is suggested that HL-60, THP-1, and DAUDI cells interact with a specific receptor on the endothelial cell and as a result of this interaction the endothelial cells are stimulated to release EDRF.

Cyclic GMP and guanylate cyclase mediate lipopolysaccharide-induced Kupffer cell tumor necrosis factor-alpha synthesis.

Tumor necrosis factor-alpha (TNF-alpha) is an important mediator in sepsis and septic shock. Kupffer cells (KCs) are the resident macrophages of the liver and are potent producers of TNF-alpha in response to inflammatory stimuli such as bacterial endotoxin or lipopolysaccharide (LPS). Although the effects of exogenous cytokines such as interferon-gamma on TNF-alpha production by macrophages have been fairly well studied, the intracellular pathways regulating KC TNF-alpha synthesis are largely unknown. We investigated the role of guanylate cyclase and cGMP in LPS-induced KC TNF-alpha synthesis. Exogenous 8-BrcGMP and dbcGMP increased LPS-stimulated TNF-alpha synthesis but had no effect on KC TNF-alpha in the absence of LPS. Sodium nitroprusside (SNP), a nitric oxide-releasing substance that stimulates guanylate cyclase, increased TNF-alpha synthesis in response to LPS, whereas methylene blue and LY83583, guanylate cyclase inhibitors, decreased KC TNF-alpha synthesis. The inhibitory effect of methylene blue could be overcome with exogenous dbcGMP or SNP. Our results demonstrate that guanylate cyclase and cGMP mediate LPS-induced KC TNF-alpha synthesis and suggest that agents that alter cyclic nucleotide metabolism in KCs may affect the response of these cells to inflammation and inflammatory stimuli.

Effect of leukotriene C4D4 antagonist on colonic damage induced by intracolonic administration of trinitrobenzene sulfonic acid in rats.

We examined the effects of eicosanoid antagonists on colonic damage induced by trinitrobenzene sulfonic acid (TNB) in a rat inflammatory bowel model. TNB (30 mg) dissolved in 0.25 ml of 50% ethanol, was given intrarectally. The appropriate doses of ONO-1078 (a leukotriene C4D4 antagonist), ONO-4057 (a leukotriene B4 antagonist), and OKY-046 (a thromboxane A2 synthetase inhibitor) were given to obtain the same blood level, either 4 h before (pre-treatment model) or 24 h after (the post-treatment model) the administration of TNB (n = 8 in all groups). Drugs were given once daily for 6 days through a gastric feeding tube. Autopsy was performed on the 7th day. Colonic damage was assessed in terms of colonic damage scores, and myeloperoxidase (MPO) activity and eicosanoid concentrations in colonic tissues were measured. Compared with the group given TNB alone, the colonic damage score was reduced to 10% in the pre-treatment model with ONO-1078, but the score was not reduced in other groups, MPO activity was not changed in any group. The concentration of leukotriene C4 was reduced with ONO-1078 treatment, in both pre- and post-treatment models. These results demonstrated that a leukotriene C4D4 antagonist reduced colonic inflammation; however, its anti-inflammatory effect was limited in this colitis model.

[Inhibition of chemically induced microvascular leakage by ONO-1078, a leukotriene antagonist, in rat skin].

This study is to determine whether ONO-1078, a potent leukotriene antagonist, influences chemically induced rat skin microvascular leakage which is considered to be, at least in part, due to stimulation of sensory nerve ending and release of sensory neuropeptides. Evans blue dye was used as a tracer for plasma leakage. Intradermal injections of chemical stimuli, histamine (10 micrograms), capsaicin (10 micrograms) and formalin (0.5 mg), evoked Evans blue dye extravasation in rat skin. Intraperitoneal ONO-1078 dose-dependently inhibited the dye extravasation induced by these stimuli, with ID5v values of 1.98 mg.kg-1 for histamine, 1.78 mg.kg-1 for capsaicin, and 2.23 mg.kg-1 for formalin. In contrast to chlorpheniramine, a H1 receptor antagonist, the inhibitory effect of ONO-1078 was weaker on histamine, but more potent on capsaicin and formalin. The inhibitory effect of dexamethasone was more potent than that of ONO-1078 on these stimuli. On the other hand, ONO-1078 inhibited the dye extravasation induced by leukotriene D4 (0.05 micrograms), but showed no effect on those induced by substance P (0.5 micrograms, a sensory neuropeptide), a larger dose of histamine (100 micrograms), and bradykinin (1 microgram). These results suggest that inhibition of chemically induced skin microvascular leakage by ONO-1078 may be mediated by inhibiting the release of sensory neuropeptides from capsaicin-sensitive sensory fibers.

[Effect of tremulacin on actions of SRS-A and histamine].

The effect of tremulacin (TRC) extracted from Mao Bai Yang (Folia Populus tomentosa Carr) on actions of SRS-A and histamine were investigated by using isolated guinea pig ileum and spectrofluorometric assay. TRC was found to inhibit the contraction of isolated guinea pig ileum induced by histamine and SRS-A, in a dose-dependent manner with IC50 of 1.78 x 10(-4) mol.L-1 and 2.51 x 10(-4) mol.L-1, respectively. TRC at the dose of 10(-4) mol.L-1 inhibited SRS-A release from sensitized isolated guinea pig lung. While at the dose of 10(-5) mol.L-1 inhibited histamine release from the peritoneal mast cells in sensitized rats. These results indicate that inhibition of the release of histamine and SRS-A may play an important role in the mechanism of antiinflammatory actions of TRC.