Contraction of guinea pig inferior vena cava by eicosanoids

Summary Guinea pig inferior vena cava contracted in response to leukotriene (LT)C₄, LTD₄, LTE₄ U46619, phenylephrine, histamine, and KCl. Although LTC₄, LTD₄, and U46619 were the most potent agonists, active tension generated by these eicosanoids was only about half that of histamine or KCl. LTE₄ and phenylephrine were marginally active. Biochemical analysis showed vena cava able to convert about 23% LTC₄ to LTD₄ and LTE₄ in 45 min. Pretreatment with acivicin prevented this by abrogating conversion of LTC₄ to LTD₄. A subthreshold concentration of LTE₄ reduced responses to LTC₄ and LTD₄. LY171883 and WY-48252 competitively antagonized LTD₄-induced contractions of vena cava. In contrast, these antagonists blocked contractions to LTC₄ in a biphasic manner. Lower segments of the LTC₄ concentration-response curves were less affected than the upper portion suggesting the possibility of 2 LTC₄ receptor subtypes. Our results indicate that LTE₄ is a weak or partial agonist in this tissue and furthermore they suggest a lack of high affinity receptors for LTE₄ favoring LTC₄ and LTD₄. Indomethacin did not influence contractions to the leukotrienes or histamine. However, the response to U46619 was greatly enhanced suggesting release of a vasodilator prostaglandin as part of the overall response of the vena cava to the thromboxane A2 mimetic.Presented in part at a meeting of the American Society for Pharmacology and Experimental Therapeutics, Salt Lake City, Utah Correspondence to: J. H. Fleisch at the above address     

Pharmacologic analysis of LY188695 (KB-2413), 1-(2-ethoxyethyl)-2-(4-methyl-1-homopiperazinyl)-benzimidazole difumarate, a potent histamine1 receptor antagonist

LY188695 was evaluated both in vitro and in vivo in the guinea pig to determine its pharmacologic profile. The compound antagonized histamine-induced contractions of ileum, aorta, and trachea with pKB values of 9.9, 9.9, and 9.2 respectively. In the lung parenchymal strip, LY188695 caused a rightward shift of the histamine concentration-response curve with a reduction in the maximal response at all antagonist concentrations tested. The reason for this effect is unknown, but it was not due to a nonspecific depressant action of the compound on the parenchyma. Selectivity was shown by its inactivity against leukotriene D4, bradykinin, prostaglandin F2 alpha, acetylcholine, norepinephrine, and serotonin on various guinea pig and rat smooth muscles. Similarly, H2 receptor-mediated relaxation of the rat uterus was unaltered by LY188695. Increases in total pulmonary impedance caused by i.v. histamine to anesthetized guinea pigs were reduced by as little as 3 micrograms/kg given orally 1 hour prior to histamine challenge. In this system, LY188695 was 15 times more potent than chlorpheniramine and 100 times more potent than terfenadine. Similar responses elicited by acetylcholine were not antagonized by LY188695. A duration of action greater than 4 hours was observed in this model. Ovalbumin given i.v. to sensitized guinea pigs increased total pulmonary impedance which was markedly decreased after oral administration of 30 or 100 micrograms/kg LY188695. These results indicate that LY188695 is a very potent antagonist of H1-mediated responses and suggest that this agent might be useful in disease states characterized by an overproduction of histamine.     

Potentiation of leukotriene formation in pulmonary and vascular tissue

Summary Leukotriene (LT) release from vascular and pulmonary tissue was assessed by a radioimmunoassay for peptide leukotrienes (i.e., LTC₄, LTD₄ and LTE₄). The calcium ionophore A-23187 at 1–3 g/ml and platelet activating factor (PAF) at 10 g/ml produced marked formation of peptide leukotrienes in minced cat pulmonary tissue. This was also confirmed by bioassay of the incubates in isolated perfused cat coronary arteries. Rat pulmonary tissue was comparable to cat with regard to LT production, but guineapig lung produced about 30–50% less on a weight basis. In addition, aortic and coronary artery vessel walls produced significant amounts of LTs. The time course for maximal leukotriene production occurred at 45–60 min of incubation at 37°C in both the radioimmunoassay and the bioassay. Cat coronary artery constricted markedly to LTC₄ or LTD₄ (30–40 mm Hg) and to the lung or blood vessel incubate. This constriction was virtually totally blocked by the leukotriene antagonist FPL-55712, but not by the thromboxane receptor antagonist, pinane thromboxane A₂, the -adrenergic receptor antagonist, phenoxybenzamine, or the angiotensin receptor antagonist, saralasin. Thus, pulmonary and vascular tissue produce leukotrienes that appear to exert coronary constrictor effects on specific leukotriene receptors. These results indicate that the ischemia of shock and anaphylaxis may be accentuated by the release of peptide leukotrienes.Supported in part by Research Grant No. HL-25575 of the National Heart, Lung and Blood Institute of the NIH     

Epithelium selectively controls hypersensitization of the response of smooth muscle to leukotriene D4 by endogenous prostanoid(s) in guinea-pig trachea

Summary The effect of epithelium removal on the sensitivity of smooth muscle to carbachol and leukotriene D₄ (LTD₄) was investigated in guinea-pig isolated tracheal preparations untreated and treated with a cyclooxygenase inhibitor, flurbiprofen (1 ol/1). The pD₂ value of carbachol was not changed by epithelium removal or by flurbiprofen-treatment, alone or in combination. On the other hand, the pD₂ of LTD₄ was higher in the tracheal strips without epithelium than in the intact preparations. In preparations devoid of epithelium, the pD₂ value of LTD₄ was decreased by treatment with flurbiprofen, suggesting that the sensitivity of the smooth muscle (namely, epithelium-free preparations) to LTD₄ is enhanced by intramurally produced excitatory prostaglandin(s) [PG(s)]. However, in intact preparations with epithelium, no such sensitized response to LTD₄ was observed. After flurbiprofen-treatment, in the continued presence of prostaglandin F₂ (PGF₂, 200 nmol/l), the sensitivity to LTD₄ was partially recovered in the epithelium-free preparations, but not in the intact ones. These results suggest that epithelium diminishes the sensitizing effect of intramural excitatory PG(s) on responsiveness of tracheal smooth muscle to LTD4, possible via a non-prostanoid substance(s).Send offprint requests to I. Takayanagi     

Effect of Nedocromil Sodium on Sulfidopeptide Leukotrienes-stimulated Human Alveolar Macrophages in Asthma

Summary: Alveolar macrophages (AM) may take part in the amplification of the inflammatory mechanism involved in asthma. During an asthma attack, mast cells and eosinophils release arachidonic acid derivative mediators of inflammation such as sulfidopeptide leukotrienes. Among them, LTC₄ has been shown to be present in bronchoalveolar fluid. In asthmatic patients, we showed that the ability of AM to transform LTC₄ into its derivatives LTD₄ and LTE₄ was related to the intensity of the local inflammation observed during endoscopy. AM from asthmatics incubated in the presence of LTC₄ or LTE₄, generated LTB₄ and 5-HETE, which are potent chemoattractants. Nedocromil sodium (10^-4 M) decreased LTB₄ releasability and intracellular 5-HETE concentrations in zymosan-stimulated AM from asthmatic patients, and was shown to decrease the LTC₄ or LTE₄-promoted formation of LTB₄ and 5-HETE.     

Modulation of the contractile activity of the guinea-pig lung parenchymal strip by exogenous 5,8,11,14,17-eicosapentaenoic acid

Summary Exogenous eicosapentaenoic acid (EPA, 16.5 mol/l or 33 mol/l) inhibited dose-dependently the anaphylactic contractile response of guinea-pig lung parenchymal strips suspended in an organ bath. As determined by radioimmunoassay, EPA inhibited in a dose-dependent manner the anaphylactic release of the cyclooxygenase products thromboxane (TX) B₂ and 6-keto-prostaglandin (PG) F₁ but simultaneously enhanced the release of sulfidopeptide (SP)-leukotrienes (LT). Indomethacin (2.8 mol/1) abolished the release of cyclooxygenase products but potentiated the release of SP-LT. However, indomethacin treatment did not affect the inhibitory action of EPA on the contractile response of the anaphylactic lung strips. The lipoxygenase inhibitor, esculetin (50 mol/1), inhibited the release of SP-LT and also that of cyclooxygenase products of polyunsaturated fatty acid metabolism. The combination of esculetin and EPA resulted in enhanced inhibition of the anaphylactic contractile response as compared to EPA alone. By reversed phase high pressure liquid chromatography (HPLC), SP-LT from anaphylactic lung parenchymal strips was shown to consist of LTD₄ and LTE₄. EPA-pretreated lung strips released upon immunologic challenge additional immunoreactivity comigrating with authentic LTC₄, LTC₅, LTD₅ and LTE₅. While anaphylactic control strips also released LTB₄, in the bath fluid of EPA-treated strips, an additional immunoreactive compound migrating with the retention time of LTB₅ was observed. In non-sensitized guinea-pig lung parenchymal strips EPA inhibited the myotropic activity of exogenous mediators such as histamine (9 gmol/1), LTC₄ (16 nmol/1) and the TX mimetic U 46619 (28.4 nmol/1), an effect which was neither affected by indomethacin (2.8 gmol/1) nor by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 gmol/1). We conclude that exogenous EPA is able to inhibit the myotropic activity of endogenous and exogenous mediators of the anaphylactic reaction in the guinea-pig lung parenchymal strip. Thus, EPA itself and not a metabolite of the cyclooxygenase or lipoxygenase pathway of polyunsaturated fatty acid metabolism seems to act as a functional antagonist to various anaphylactic mediators in lung tissue. Send offprint requests to Th. Simmet at the above address     

Mechanisms for anti-inflammatory effects of 1-[15(S)-hydroxyeicosapentaenoyl] lysophosphatidylcholine, administered intraperitoneally, in zymosan A-induced peritonitis

BACKGROUND AND PURPOSE

Lysophosphatidylcholines (lysoPCs) with polyunsaturated acyl chains are known to exert anti-inflammatory actions. 15-Lipoxygeanation is crucial for anti-inflammatory action of polyunsaturated acylated lysoPCs. Here, the anti-inflammatory actions of 1-(15-hydroxyeicosapentaenoyl)-lysoPC (15-HEPE-lysoPC) and its derivatives were examined in a mechanistic analysis.

EXPERIMENTAL APPROACH

Anti-inflammatory actions of 15-HEPE-lysoPC in zymosan A-induced peritonitis of mice were examined by measuring plasma leakage and leucocyte infiltration, and determining levels of lipid mediators or cytokines.

KEY RESULTS

When each lysoPC, administered i.v., was assessed for its ability to suppress zymosan A-induced plasma leakage, 15-HEPE-lysoPC was found to be more potent than 1-(15-hydroperoxyeicosapentaenoyl)-lysoPC or 1-eicosapentaenoyl-lysoPC. Separately, i.p. administration of 15-HEPE-lysoPC markedly inhibited plasma leakage, in contrast to 15-HEPE, which had only a small effect. 15-HEPE-lysoPC also decreased leucocyte infiltration. Moreover, it reduced the formation of LTC₄ and LTB₄, 5-lipoxygenation products, as well as the levels of pro-inflammatory cytokines. The time-course study indicated that 15-HEPE-lysoPC might participate in both the early inflammatory phase and resolution phase. Additionally, 15-HEPE-lysoPC administration caused a partial suppression of LTC₄-induced plasma leakage and LTB₄-induced leucocyte infiltration. In the metabolism study, peritoneal exudate was shown to contain lysoPC-hydrolysing activity, crucial for anti-inflammatory activity, and a system capable of generating lipoxin A from 15-hydroxy eicosanoid precursor.

CONCLUSIONS AND IMPLICATIONS

15-HEPE-lysoPC, a precursor for 15-HEPE in target cells, induced anti-inflammatory actions by inhibiting the formation of pro-inflammatory leukotrienes and cytokines, and by enhancing the formation of lipoxin A. 15-HEPE-lysoPC might be one of many potent anti-inflammatory lipids in vivo.     

Pharmacologic and pharmacokinetic profile of SK&F S-106203, a potent,orally active peptidoleukotriene receptor antagonist,in guinea-pig

In this report the pharmacologic and pharmacokinetic profile of the leukotriene receptor antagonist 3(S)-1(2-carboxyethyl)thio]-3-12-(8-phenyloctyl)phenyl] propanoic acid (SK&F S-106203) in guinea-pigs is described. In isolated guinea-pig tracheae SK&F S-106203 was a potent, competitive antagonist of leukotriene (LT) D₄-induced contractions (pA₂ = 7.6). SK&F S-106203 was also a potent antagonist of LTE₄-induced contractions (pK_B = 7.3), but had little effect on those elicited by LTC₄ (pK_B = 5.5). SK&F S-106203 (10 μM) had no effect on contractions produced by histamine, carbachol, KCI, U-44069, PGF_2α or PGD₂. In addition, SK&F S-106203 (10 μM) did not inhibit cyclic nucleotide phosphodiesterase (PDE) activity of several PDE isozymes. In guinea-pig lung membrane preparations, SK&F S-106203 was a potent antagonist of ³H-LTD₄ binding with a K_i = 19.4 ± 2.1 nM (n = 5). The pharmacokinetic profile of SK&F S-106203 was determined in unanesthetized guinea-pigs. Following an i.v. (bolus) dose (25 mg/kg), SK&F S-106203 disappeared from plasma in a biphasic fashion with half-lives of 0.1 h (50% of the area under the plasma concentration-time curve, AUC) and 11 h. The AUC obtained for SK&F S-106203 following i.v. administration was 87.3 ± 7.5 μg-h/ml. Following an oral dose of SK&F S-106203 (100 mg/kg), the maximal plasma concentration (C_max) and the time C_max was achieved (T_max were 21.62 ± 2.26 kg/ml and 4 ± 1 h, respectively; the AUC was 279.9 ± 41.8 μg-h/ml. Studies examining the effects of i.v. infusion of SK&F S-106203 revealed that marked inhibition of LTD₄-induced bronchospasm was produced with steady-state plasma levels of SK&F S-106203 < 1 μg/ml (< 2 μM). Oral (p.o.) pretreatment with 100 μmol/kg SK&F S-106203 for up to 24 h essentially abolished LTD₄-induced bronchospasm; this correlated with sustained plasma concentrations of > 2 μg/ml. The results indicate that in guinea-pig airways, SK&F S-106203 is a potent and selective LT receptor antagonist that is active via aerosol, oral and i.v. routes of administration. When given orally, SK&F S-106203 is highly bioavailable and has a very long duration of action which correlates with the pharmacokinetic profile of the compound. SK&F S-106203 may be useful therapy in asthma and other disorders in which the Us are thought to play a prominent pathophysiological role.     

Effects of KP-496, a novel dual antagonist at the cysteinyl leukotriene receptor 1 and the thromboxane A(2) receptor, on airway obstruction in guinea pigs

BACKGROUND AND PURPOSE: KP-496 is a novel dual antagonist for cysteinyl leukotriene receptor 1 (CysLT(1)) and thromboxane A(2) (TXA(2)) receptor (TP). The aim of this study was to evaluate the pharmacological profile of inhaled KP-496 and its effects on airway obstruction.EXPERIMENTAL APPROACH: Antagonist activities of inhaled KP-496 were investigated using bronchoconstriction induced in guinea pigs by LTD(4) or U46619, a stable TXA(2) mimetic. Guinea pigs sensitized with injections of ovalbumin were used to assess the effects of inhaled KP-496 on bronchoconstriction induced by antigen (i.v.). Another set of guinea pigs were sensitized and challenged with ovalbumin by inhalation and the effects of inhaled KP-496 on immediate and late airway responses and airway hyperresponsiveness were investigated.KEY RESULTS: KP-496 significantly inhibited LTD(4)- and U46619-induced bronchoconstriction in a dose-dependent manner. The inhibitory effects of KP-496 (1%) were comparable to those of montelukast (a CysLT(1) antagonist, p.o., 0.3 mg kg(-1)) or seratrodast (a TP antagonist, p.o., 3 mg kg(-1)). KP-496 (1%) and oral co-administration of montelukast (10 mg kg(-1)) and seratrodast (20 mg kg(-1)) significantly inhibited antigen-induced bronchoconstriction, whereas administration of montelukast or seratrodast separately did not inhibit antigen-induced bronchoconstriction. KP-496 exhibited dose-dependent and significant inhibitory effects on the immediate and late airway responses and airway hyperresponsiveness following antigen challenge.CONCLUSIONS AND IMPLICATIONS: KP-496 exerts effects in guinea pigs which could be beneficial in asthma. These effects of KP-496 were greater than those of a CysLT(1) antagonist or a TP antagonist, in preventing antigen-induced airway obstruction.     

Co-operative signalling through DP(1) and DP(2) prostanoid receptors is required to enhance leukotriene C(4) synthesis induced by prostaglandin D(2) in eosinophils

BACKGROUND AND PURPOSE

Prostaglandin (PG) D₂ has emerged as a key mediator of allergic inflammatory pathologies and, particularly, PGD₂ induces leukotriene (LT) C₄ secretion from eosinophils. Here, we have characterized how PGD₂ signals to induce LTC₄ synthesis in eosinophils.

EXPERIMENTAL APPROACH

Antagonists and agonists of DP₁ and DP₂ prostanoid receptors were used in a model of PGD₂-induced eosinophilic inflammation in vivo and with PGD₂-stimulated human eosinophils in vitro, to identify PGD₂ receptor(s) mediating LTC₄ secretion. The signalling pathways involved were also investigated.

KEY RESULTS

In vivo and in vitro assays with receptor antagonists showed that PGD₂-triggered cysteinyl-LT (cysLT) secretion depends on the activation of both DP₁ and DP₂ receptors. DP₁ and DP₂ receptor agonists elicited cysLTs production only after simultaneous activation of both receptors. In eosinophils, LTC₄ synthesis, but not LTC₄ transport/export, was activated by PGD₂ receptor stimulation, and lipid bodies (lipid droplets) were the intracellular compartments of DP₁/DP₂ receptor-driven LTC₄ synthesis. Although not sufficient to trigger LTC₄ synthesis by itself, DP₁ receptor activation, signalling through protein kinase A, did activate the biogenesis of eosinophil lipid bodies, a process crucial for PGD₂-induced LTC₄ synthesis. Similarly, concurrent DP₂ receptor activation used Pertussis toxin-sensitive and calcium-dependent signalling pathways to achieve effective PGD₂-induced LTC₄ synthesis.

CONCLUSIONS AND IMPLICATIONS

Based on pivotal roles of cysLTs in allergic inflammatory pathogenesis and the collaborative interaction between PGD₂ receptors described here, our data suggest that both DP₁ and DP₂ receptor antagonists might be attractive candidates for anti-allergic therapies.

LINKED ARTICLE

This article is commented on by Mackay and Stewart, pp. 1671–1673 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01236.x     

Novel leukotriene biosynthesis inhibitors (2012-2016) as anti-inflammatory agents

Introduction: Leukotrienes (LTs) are lipid mediators produced from arachidonic acid with a broad variety of bioactivities in allergy and inflammation. The biosynthesis of LTs mainly involves 5-lipoxygenase (5-LO) and its 5-lipoxygenase-activating protein (FLAP), LTA₄ hydrolase and LTC₄ synthase that all may represent potential targets for LT biosynthesis inhibitors.

Areas covered: We introduce the LT biosynthetic pathway and its cellular regulation, the diverse biological actions of LTs and their receptors, and we briefly describe the pharmacological strategies for suppression of LT formation as well as the classes of current LT biosynthesis inhibitors. The main focus is placed on the comprehensive discussion of recently reported inhibitors of 5-LO, FLAP, LTA₄ hydrolase and LTC₄ synthase, based on literature search (PubMed and Thomson Innovation Patents Searches), covering 2012–2016.

Expert opinion: Although many new series of 5-LO inhibitors have been presented without patenting, essentially by academia, novel FLAP inhibitors (many patented) are most advanced in clinical development and are apparently the focus of pharmaceutical companies. Only few novel inhibitors of LTA₄ hydrolase and LTC₄ synthase were reported. Major issues in the development of LT synthesis inhibitors are related to loss of potency in biological relevant environment, poor pharmacokinetics, lack of oral efficacy, and side effects.     

Pharmacological characterization of the first potent and selective antagonist at the cysteinyl leukotriene 2 (CysLT2) receptor

Background and purpose:

Cysteinyl leukotrienes (CysLTs) have been implicated in the pathophysiology of inflammatory and cardiovascular disorders. Their actions are mediated by CysLT₁ and CysLT₂ receptors. Here we report the discovery of 3-({[(1S,3S)-3-carboxycyclohexyl]amino}carbonyl)-4-(3-{4-[4-(cyclo-hexyloxy)butoxy]phenyl}propoxy) benzoic acid (HAMI3379), the first potent and selective CysLT₂ receptor antagonist.

Experimental approach:

Pharmacological characterization of HAMI3379 was performed using stably transfected CysLT₁ and CysLT₂ receptor cell lines, and isolated, Langendorff-perfused, guinea pig hearts.

Key results:

In a CysLT₂ receptor reporter cell line, HAMI3379 antagonized leukotriene D₄- (LTD₄-) and leukotriene C₄- (LTC₄-) induced intracellular calcium mobilization with IC₅₀ values of 3.8 nM and 4.4 nM respectively. In contrast, HAMI3379 exhibited very low potency on a recombinant CysLT₁ receptor cell line (IC₅₀ > 10 000 nM). In addition, HAMI3379 did not exhibit any agonistic activity on both CysLT receptor cell lines. In binding studies using membranes from the CysLT₂ and CysLT₁ receptor cell lines, HAMI3379 inhibited [³H]-LTD₄ binding with IC₅₀ values of 38 nM and >10 000 nM respectively. In isolated Langendorff-perfused guinea pig hearts HAMI3379 concentration-dependently inhibited and reversed the LTC₄-induced perfusion pressure increase and contractility decrease. The selective CysLT₁ receptor antagonist zafirlukast was found to be inactive in this experimental setting.

Conclusions and implications:

HAMI3379 was identified as a potent and selective CysLT₂ receptor antagonist, which was devoid of CysLT receptor agonism. Using this compound, we showed that the cardiac effects of CysLTs are predominantly mediated by the CysLT₂ receptor.     

Curcumin Inhibits the Activation of Immunoglobulin E-Mediated Mast Cells and Passive Systemic Anaphylaxis in Mice by Reducing Serum Eicosanoid and Histamine Levels

Curcumin is naturally occurring polyphenolic compound found in turmeric and has many pharmacological activities. The present study was undertaken to evaluate anti-allergic inflammatory activity of curcumin, and to investigate its inhibitory mechanisms in immunoglobulin E (IgE)/Ag-induced mouse bone marrow-derived mast cells (BMMCs) and in a mouse model of IgE/Ag-mediated passive systemic anaphylaxis (PSA). Curcumin inhibited cyclooxygenase-2 (COX-2) dependent prostaglandin D₂ (PGD₂) and 5-lipoxygenase (5-LO) dependent leukotriene C₄ (LTC₄) generation dose-dependently in BMMCs. To probe the mechanism involved, we assessed the effects of curcumin on the phosphorylation of Syk and its downstream signal molecules. Curcumin inhibited intracellular Ca²⁺ influx via phospholipase Cγ1 (PLCγ1) activation and the phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor-κB (NF-κB) pathway. Furthermore, the oral administration of curcumin significantly attenuated IgE/Ag-induced PSA, as determined by serum LTC₄, PGD₂, and histamine levels. Taken together, this study shows that curcumin offers a basis for drug development for the treatment of allergic inflammatory diseases.     

A histological method for studying the effects of drugs on mediator-induced airway microvascular leakage in rodents

Methods are described for studying the effects of drugs on increases in tracheobronchial microvascular permeability (leakage) induced by inflammatory mediators. A model in conscious guinea pigs (in which the leakage effects resulted from circulating mediator) and a model in anesthetized rats (in which the leakage effects resulted from application of the mediator to the airway lumen) are described. Inhibitor drugs were given i.v. 2 min before the mediator. In guinea pigs either histamine or leukotriene D₄ (LTD₄) and colloidal carbon (C, the tracer molecule for leakage) were administered together iv.; in rats 5-hydroxytryptamine (5-HT, 100 μg) was injected intratracheally followed by i.v. colloidal carbon. Tracheal and bronchial tissues were removed 15 min later from the killed animals and prepared for histology, and the number of C-labeled microvessels in the mucosal/submucosal region of 7-μm sections was counted. In guinea pigs, leakage produced by LTD₄ or histamine was related to the dose administered and the relative potency of LTD₄: histamine was approximately 123:1, on a molar basis. Leakage caused by histamine, but not by LTD₄, was prevented by mepyramine (1 mg/kg) and LTD₄-induced leakage was prevented by FPL 55712 (1 mg/kg). Terbutaline (1 mg/kg) attenuated leakage to both mediators but never abolished it. In rats, leakage was also seen to 5-HT, which was prevented by methysergide (1 mg/kg) and markedly attenuated by ketanserin (1 mg/kg) or by terbutaline (1 mg/kg). It is suggested that the colloidal carbon tracer technique has application to pharmacological studies designed to examine the effects of drugs on mediator-induced permeability to macromolecules in the tracheobronchial microcirculation.     

Effect of BAY x7195, an oral receptor antagonist of cysteinyl-leukotrienes,on leukotriene D4-induced bronchoconstriction in normal volunteers

Leukotrienes (LT) have been proposed to play an important role in the pathogenesis of asthma. This paper reports the results of two studies investigating the effect of BAY x 7195, a new oral receptor antagonist of cysteinyl-leukotrienes, on LTD₄-induced bronchoconstriction in healthy male volunteers. Using a double-blind, placebo-controlled, crossover design, volunteers received 250 mg (n=6; study 1) and 100 and 500 mg (n=6; study 2) of BAY x 7195. Bronchoprovocation with nebulized LTD₄ was performed 2 (250 mg) and 2 and 8 (100 and 500 mg) hp.a. The specific airway's conductance (SGaw) was used to assess the airway's response. Blood samples to determine plasma concentrations of BAY x 7195 were taken at the end of bronchoprovocation. BAY x 7195 showed no effect on baseline lung function. Compared to placebo, the different doses of BAY x 7195 increased the concentration of LTD₄ needed to produce a 35% decrease in SGaw 2h p.a. between 1- and 23-fold. Eight hours p.a., 100 and 500 mg caused shifts in the concentration-response curve of between 1- and 13-fold. There was no predictive relationship between plasma concentrations of BAY x 7195 and the response to LTD₄ challenge. However, there was a relationship between dose and effect. No relevant adverse effects were reported. In conclusion, the present results suggest that BAY x 7195 is an effective LTD₄-receptor antagonist in man.     

Characterization of LTC4 effects on rabbit Heal mucosa in vitro

Summary The effects of peptidoleukotriene C4 (LTC₄) on electrical properties and Na⁺ and Cl^– fluxes in the presence or absence of the LTD4/LTE4 antagonist, (2(S)-hydroxy3-(R)-carboxyethylthio)-3-[2-(8-phenyloctyl)phenyl] propanoic acid (SK&F 104353) were investigated in stripped ileal mucosa from rabbits placed in Ussing chambers. Results from this study demonstrate that serosal addition of LTC4 produces a dose-dependent increase in short-circuit current (I_sc) which is not affected by pretreatment of the tissue with SK&F 104353 (0.1 M). The concentration of LTC4 which produced an increase in I_sc of 4 µEq/h · cm² was 181 nM in the absence of SK&F 104353 and this value did not differ significantly from the value of 212 nM in the presence of SK&F 104353 (0.1 M). SK&F 104353 (0.1 M) reduced the increase in I_sc elicited by LTD4 and LTE4 by > 95%. Mucosal addition of LTC4 failed to alter I_sc. The time course of the increase in I_sc in response to LTC4 is qualitatively similar to that seen with both LTD4 and LTE4. Increases in I_sc produced by LTC4 are not inhibited by removal of Ca²⁺ from the serosal bathing solution but are inhibited by pretreatment of the tissue with indomethacin (1 µM). The histamine receptor antagonist, mepyramine, reduced the change in I_sc resulting from stimulation with LTC4 by 20% while the cholinergic antagonist, atropine, and the excitable tissue Na⁺ channel blocker, tetrodotoxin, were without effect. Measurement of unidirectional and net Na⁺ and Cl^– fluxes revealed that LTC4 reduces Na⁺ and Cl^– absorption and that subsequent addition of PGEI produced no further decrease. Thus, these results indicate that LTC4 as well as LTD₄ and LTE₄ can inhibit electrolyte absorption and that the receptors involved in stimulation by LTC₄ are distinct from those responsible for the effects of LTD₄ and LTE₄. Send offprint requests to P. L. Smith at the above address     

白三烯C4（LTC4）放射受体结合方法的建立及二苯乙烯低聚体和LTC4受体结合特性

目的：建立LTC₄放射受体结合实验方法,并比较二苯乙烯低聚体（Gn-3）和LTC₄受体的结合特性。方法：以豚鼠肺膜为实验材料,采用³H-LTC₄为放射配体,以FPL₅₅₇₁₂作阳性对照药物,Gn-3为实验药物,进行药物竞争结合实验。采用离体器官生物检测法鉴定Gn-3对LTC₄受体的拮抗作用。结果：3H-LTC₄与其相应受体呈现单一结合位点,Gn-3可明显取代3H-LTC₄与其受体结合。生物学检定法证实Gn-3可抑制LTC₄引起的生物学效应。 结论：豚鼠肺膜LTC₄受体为单一结合位点受体,Gn-3为高活性的LTC₄受体拮抗剂。     

Molsidomine on cardiovascular leukotriene D4 actions

Summary Anaesthetized open-chest dogs were used to study the effects of intracoronary leukotriene D₄ (LTD₄; 0.5 g/kg) on haemodynamics, the electrocardiogram (ECG), coronary blood flow in the left circumflex artery and coronary resistance in the absence or presence of the antianginal drug molsidomine (500 g/kg i.v.). LTD₄ increased left ventricular (LV) filling pressure from 6.5±3.8 to 14.7±3.2 mm Hg (P<0.01), decreased LV dP/dt _max from 2,500±200 to 1,240±205 mm Hg/s (P<0.05), elevated the S-T segment of the ECG from 0.3±0.2 to 2.4±0.6 mV (P<0.01), and coronary resistance from 3.4±0.9 to 33.7±4.8 mm Hg×min×ml^–1 (P<0.001). Coronary artery blood flow fell from 33.8±2.7 to 3±3 ml/min (P<0.001). Molsidomine treatment 15 min prior to repeated intracoronary LTD₄ application attenuated vasoconstrictor response to LTD₄ and the subsequent elevation in total coronary vascular resistance. The negative inotropic actions of the eicosanoid were counteracted by molsidomine. Filling pressure decreased and no temporary signs of ischaemia were noted in the ECG. Inhibition of the cyclooxygenase enzyme activity by i.v. indomethacin (5 mg/kg) had no effects on the LTD₄-induced haemodynamic alterations, the ECG, and coronary flow and resistance. The antagonistic molsidomine actions on haemodynamic and electrocardiographic LTD₄ effects were not influenced by previous indomethacin treatment. Thus, molsidomine actions on haemodynamics and assumed influences of the drug or its main metabolite on platelet function and platelet and coronary vascular eicosanoid formation may protect the heart against LTD₄-caused derangements.     

Pharmacological characterization of GSK1004723, a novel, long-acting antagonist at histamine H(1) and H(3) receptors

BACKGROUND AND PURPOSE

Preclinical pharmacological characterization of GSK1004723, a novel, dual histamine H₁ and H₃ receptor antagonist.

EXPERIMENTAL APPROACH

GSK1004723 was characterized in vitro and in vivo using methods that included radioligand binding, intracellular calcium mobilization, cAMP production, GTPγS binding, superfused human bronchus and guinea pig whole body plethysmography.

KEY RESULTS

In cell membranes over-expressing human recombinant H₁ and H₃ receptors, GSK1004723 displayed high affinity, competitive binding (H₁ pKi = 10.2; H₃ pKi = 10.6). In addition, GSK1004723 demonstrated slow dissociation from both receptors with a t_1/2 of 1.2 and 1.5 h for H₁ and H₃ respectively. GSK1004723 specifically antagonized H₁ receptor mediated increases in intracellular calcium and H₃ receptor mediated increases in GTPγS binding. The antagonism exerted was retained after cell washing, consistent with slow dissociation from H₁ and H₃ receptors. Duration of action was further evaluated using superfused human bronchus preparations. GSK1004723 (100 nmol·L⁻¹) reversed an established contractile response to histamine. When GSK1004723 was removed from the perfusate, only 20% recovery of the histamine response was observed over 10 h. Moreover, 21 h post-exposure to GSK1004723 there remained almost complete antagonism of responses to histamine. In vivo pharmacology was studied in conscious guinea pigs in which nasal congestion induced by intranasal histamine was measured indirectly (plethysmography). GSK1004723 (0.1 and 1 mg·mL⁻¹ intranasal) antagonized the histamine-induced response with a duration of up to 72 h.

CONCLUSIONS AND IMPLICATIONS

GSK1004723 is a potent and selective histamine H₁ and H₃ receptor antagonist with a long duration of action and represents a potential novel therapy for allergic rhinitis.     

Comparative 5-HT4 receptor antagonist activity of LY353433 and its active hydroxylated metabolites

LY353433 is a selective, potent, and orally active 5-HT₄ receptor antagonist with a long duration of pharmacological activity following its oral administration to rats. After oral administration of LY353433 (100 mg/kg) to rats, peak plasma concentration of the parent material was approximately 25 ng/ml and rapidly declined such that 4 h after administration, plasma concentration of the parent material was barely detectable. However, two additional peaks (LY343031 and LY343032) were observed in plasma via HPLC and subsequently identified as hydroxylated metabolites of LY353433. Peak plasma concentrations of LY343031 (approximately 50 ng/ml) and of LY343032 (approximately 150 ng/ml) were achieved within 1 h after the oral administration of LY353433. Furthermore, plasma concentrations of these metabolites were maintained for several hours and declined more slowly than plasma concentrations of the parent material. Both metabolites inhibited esophageal 5-HT₄ receptors in a concentration range similar to that observed with LY353433. In addition, the receptor selectivity profiles for LY343031 and LY343032 were similar to that of LY353433 at α₁, α₂, β, Dopamine D₁, Dopamine D₂, benzodiazapine, histamine H₁, GABA_A, 5-HT₂, and muscarinic receptors. Thus, these hydroxylated derivatives of LY353433 were potent and selective 5-HT₄ receptor antagonists in vitro. Lastly, using ex vivo esophageal relaxation to serotonin to assess 5-HT₄ receptor antagonism, these compounds were less active after oral administration to rats than LY353433. Thus, the long duration of pharmacological activity observed after oral administration of LY353433 is likely related not only to the 5-HT₄ receptor antagonist activity of the parent molecule, but also to the 5-HT₄ receptor antagonist activity associated with its two hydroxylated metabolites. Drug Dev. Res. 43:193–199, 1998. © 1998 Wiley-Liss, Inc.