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Dehydropeptidase 1 (DPEP1) is a zinc-dependent metalloproteinase that is expressed aberrantly in several cancers. The role of DPEP1 in cancer remain controversial. In this study, we demonstrate that DPEP1 functions as a positive regulator for colon cancer cell metastasis. The expression of DPEP1 mRNA and proteins were upregulated in colon cancer tissues compared to normal mucosa. Gain-of-function and loss-of-function approaches were used to examine the malignant phenotype of DPEP1-expressing or DPEP1-depleted cells. DPEP1 expression caused a significant increase in colon cancer cell adhesion and invasion in vitro, and metastasis in vivo. In contrast, DPEP1 depletion induced opposite effects. Furthermore, cilastatin, a DPEP1 inhibitor, suppressed the invasion and metastasis of DPEP1-expressing cells. DPEP1 inhibited the leukotriene D4 signaling pathway and increased the expression of E-cadherin. We also show that DPEP1 mediates TGF-β-induced EMT. TGF-β transcriptionally repressed DPEP1 expression. TGF-β treatment decreased E-cadherin expression and promoted cell invasion in DPEP1-expressing colon cancer cell lines, whereas it did not affect these parameters in DPEP1-depleted cell lines. These results suggest that DPEP1 promotes cancer metastasis by regulating E-cadherin plasticity and that it might be a potential therapeutic target for preventing the progression of colon cancer.

Th17 cell trafficking in response to leukotriene signaling is poorly understood. Here we showed that Th17 cells express high levels of leukotriene B4 receptor 1 (LTB4R1) and cysteinyl leukotriene receptor 1 (CysLTR1). Th17 cells migrated under the guidance of leukotriene B4 and D4. The migration of Th17 cells was more efficient than that of Th1 and Th2 cells, and it was blocked by specific inhibitors of LTB4R1 or CysLTR1. Studies in an animal model of experimental autoimmune encephalomyelitis revealed that treatment with montelukast alleviated disease symptoms and inhibited the recruitment of Th17 cells to the central nervous system. Thus, leukotrienes may act as chemoattractants for Th17 cells.

Inflammation and altered immunity are recognized components of severe pulmonary arterial hypertension in human patients and in animal models of PAH. While eicosanoid metabolites of cyclooxygenase and lipoxygenase pathways have been identified in the lungs from pulmonary hypertensive animals their role in the pathogenesis of severe angioobliterative PAH has not been examined. Here we investigated whether a cyclooxygenase-2 (COX-2) inhibitor or diethylcarbamazine (DEC), that is known for its 5-lipoxygenase inhibiting and antioxidant actions, modify the development of PAH in the Sugen 5416/hypoxia (SuHx) rat model. The COX-2 inhibitor SC-58125 had little effect on the right ventricular pressure and did not prevent the development of pulmonary angioobliteration. In contrast, DEC blunted the muscularization of pulmonary arterioles and reduced the number of fully obliterated lung vessels. DEC treatment of SuHx rats, after the lung vascular disease had been established, reduced the degree of PAH, the number of obliterated arterioles and the degree of perivascular inflammation. We conclude that the non-specific anti-inflammatory drug DEC affects developing PAH and is partially effective once angioobliterative PAH has been established.

Purpose: Leukotriene D(4) (LTD(4)) is a central mediator in asthma inducing bronchoconstriction and profound disturbances in pulmonary gas exchange in asthmatic subjects. The aim of the study was to compare, for the first time, the influence of the bronchodilators salbutamol (400 μg) and ipratropium (80 μg) on lung function changes induced by inhaled LTD(4)., Methods: Treatments were evaluated in a randomized, three-period, double-blind, placebo-controlled, cross-over study where spirometric and pulmonary gas exchange indices were followed in 12 subjects with mild asthma before and after LTD(4) challenge., Results: Compared with placebo, salbutamol provided significant protection against the fall in FEV(1) (forced expiratory volume in 1 s) after LTD(4) challenge. Salbutamol also abolished the LTD(4)-induced gas exchange disturbances [decreased arterial oxygen tension (PaO(2)) and increased alveolar-arterial oxygen tension difference (AaPO(2))]. Ipratropium provided significant but less marked attenuation of the changes in FEV(1) and arterial oxygenation induced by LTD(4)., Conclusion: Despite the equal bronchodilatory effects of salbutamol and ipratropium before the challenge with LTD(4), salbutamol was superior to ipratropium in preventing spirometric and gas exchange abnormalities. This result indicates a broader action of salbutamol on several of the disturbances that contribute to airway obstruction including, for example, exudation of plasma in the airway mucosa. The clinical implication of this new finding is that in this model of acute asthmatic airway obstruction, salbutamol was more effective than ipratropium.

Cysteinyl leukotrienes (CysLTs) are potent inflammatory mediators that predominantly exert their effects by binding to cysteinyl leukotriene receptors of the G protein-coupled receptor family. CysLT receptor 2 (CysLT(2)R), expressed in endothelial cells of some vascular beds, has been implicated in a variety of cardiovascular functions. Endothelium-specific overexpression of human CysLT(2)R in transgenic mice (hEC-CysLT(2)R) greatly increases myocardial infarction damage. Investigation of this receptor, however, has been hindered by the lack of selective pharmacological antagonists. Here, we describe the characterization of 3-(((3-carboxycyclohexyl)amino)carbonyl)-4-(3-(4-(4-phenoxybutoxy)phenyl)-propoxy)benzoic acid (BayCysLT(2)) and explore the selective effects of this compound in attenuating myocardial ischemia/reperfusion damage and vascular leakage. Using a recently developed β-galactosidase-β-arrestin complementation assay for CysLT(2)R activity (Mol Pharmacol 79:270-278, 2011), we determined BayCysLT(2) to be ∼20-fold more potent than the nonselective dual CysLT receptor 1 (CysLT(1)R)/CysLT(2)R antagonist 4-(((1R,2E,4E,6Z,9Z)-1-((1S)-4-carboxy-1-hydroxybutyl)-2,4,6,9-pentadecatetraen-1-yl)thio)benzoic acid (Bay-u9773) (IC(50) 274 nM versus 4.6 μM, respectively). Intracellular calcium mobilization in response to cysteinyl leukotriene administration showed that BayCysLT(2) was >500-fold more selective for CysLT(2)R compared with CysLT(1)R. Intraperitoneal injection of BayCysLT(2) in mice significantly attenuated leukotriene D(4)-induced Evans blue dye leakage in the murine ear vasculature. BayCysLT(2) administration either before or after ischemia/reperfusion attenuated the aforementioned increased myocardial infarction damage in hEC-CysLT(2)R mice. Finally, decreased neutrophil infiltration and leukocyte adhesion molecule mRNA expression were observed in mice treated with antagonist compared with untreated controls. In conclusion, we present the characterization of a potent and selective antagonist for CysLT(2)R that is useful for discerning biological activities of this receptor.

The present study has been designed to pharmacologically investigate the effect of Montelukast sodium, a leukotriene D(4) receptor antagonist, and 1,2,3,4, tetrahydroisoquinoline, a leukotriene D(4) synthetic pathway inhibitor, on the pathophysiological progression of seizures using mouse models of kindled epilepsy and status epilepticus induced spontaneous recurrent seizures. Pentylenetetrazole (40 mg kg(-1)) (PTZ) administration every second day for a period of 15 d was used to elicit chemically induced kindled seizure activity in mice. In a separate set of groups, fifty consecutive electroshocks were delivered to mice using corneal electrodes with continuously increasing intensity with an inter-shock interval of 40s. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score (KSSS). Pilocarpine (100 mg kg(-1)) was injected every twenty minutes until the onset of status epilepticus. A spontaneous recurrent seizure severity score (SRSSS) was recorded as a measure of quantitative assessment of the progressive development of spontaneous recurrent seizures induced after pilocarpine status epilepticus. Sub-acute PTZ administration and electroshock induced the development of severe form of kindled seizures in mice. Severity of kindled seizures was assessed in terms of a composite kindled seizure severity score. Further, pharmacological status epilepticus elicited a progressive evolution of spontaneous recurrent seizures in the animals. However, Montelukast sodium, a leukotriene D(4) receptor antagonist, as well as 1,2,3,4, tetrahydroisoquinoline, a leukotriene D(4) synthetic pathway inhibitor, markedly and dose dependently suppressed the development of kindled seizures as well as pilocarpine induced spontaneous recurrent seizures. Therefore, leukotriene D(4) may be implicated in the pathogenesis of seizures., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The conjunctiva is a mucous membrane that covers the sclera and lines the inside of the eyelids. Throughout the conjunctiva are goblet cells that secrete mucins to protect the eye. Chronic inflammatory diseases such as allergic conjunctivitis and early dry eye lead to increased goblet cell mucin secretion into tears and ocular surface disease. The purpose of this study was to determine the actions of the inflammatory mediators, the leukotrienes and the proresolution resolvins, on secretion from cultured rat and human conjunctival goblet cells. We found that both cysteinyl leukotriene (CysLT) receptors, CysLT(1) and CysLT(2,) were present in rat conjunctiva and in rat and human cultured conjunctival goblet cells. All leukotrienes LTB(4), LTC(4), LTD(4), and LTE(4), as well as PGD(2), stimulated goblet cell secretion in rat goblet cells. LTD(4) and LTE(4) increased the intracellular Ca(2+) concentration ([Ca(2+)](i)), and LTD(4) activated ERK1/2. The CysLT(1) receptor antagonist MK571 significantly decreased LTD(4)-stimulated rat goblet cell secretion and the increase in [Ca(2+)](i). Resolvins D1 (RvD1) and E1 (RvE1) completely reduced LTD(4)-stimulated goblet cell secretion in cultured rat goblet cells. LTD(4)-induced secretion from human goblet cells was blocked by RvD1. RvD1 and RvE1 prevented LTD(4)- and LTE(4)-stimulated increases in [Ca(2+)](i), as well as LTD(4) activation of ERK1/2. We conclude that cysteinyl leukotrienes stimulate conjunctival goblet cell mucous secretion with LTD(4) using the CysLT(1) receptor. Stimulated secretion is terminated by preventing the increase in [Ca(2+)](i) and activation of ERK1/2 by RvD1 and RvE1.

Background: Histamine and cysteinyl leukotrienes are pivotal mast cell mediators which contribute considerably and likely complementary to the symptoms of allergic rhinitis. Currently, we sought to explore the direct actions of histamine and leukotriene D(4) (LTD(4)), a cysteinyl leukotriene, on porcine nasal arteries and veins. We also studied combined blocks of histamine and cysteinyl leukotrienes using loratadine and montelukast in an in vivo model of allergy-mediated nasal inflammation., Methods: For the evaluation of the action of histamine and LTD(4) on arteries and veins, porcine nasal mucosa was isolated and cut into slices (100-300 microm thick). Real-time images of the nasal arteries and veins were recorded and vessel activities estimated by changes in cross-sectional area before and after the tested drugs. For the in vivo studies, the effect of loratadine and montelukast given alone and in combination was examined on upper airway inflammation in ovalbumin-sensitized and -challenged Brown Norway rats., Results: Both histamine (0.001-10 micromol/l) and LTD(4) (0.001-10 micromol/l) produced a concentration-dependent increase in the lumen area of nasal mucosa arteries and veins. Histamine (0.01 micromol/l) alone produced a 24 and 12% increase in cross-sectional areas of arteries and veins, respectively. LTD(4) (0.001 micromol/l) alone increased artery and vein dilation by about 17 and 9%, respectively. Combination treatment with histamine (0.01 micromol/l) and LTD(4) (0.001 micromol/l) increased vessel dilation by 65% (arteries) and 26% (veins). In our in vivo Brown Norway rat studies, oral loratadine (0.01-10 mg/kg) and montelukast (0.01-10 mg/kg) significantly reduced antigen-induced total nasal inflammatory cell infiltration in a dose-dependent manner. The antiinflammatory dose-response curve of loratadine was shifted to the left when studied in combination with montelukast (0.01 mg/kg). Similarly, the dose-response characteristics of montelukast (0.01-10 mg/kg) was shifted in the presence of loratadine (0.01 mg/kg)., Conclusion: Our studies support the position that histamine and cysteinyl leukotrienes may act collaboratively to elicit allergic nasal pathologies such as upper airway inflammation and nasal vessel dilation (which may translate into increased nasal mucosal engorgement). Furthermore, the current results are supportive of the hypothesis that combined treatment of allergic rhinitis with an H(1) receptor antagonist and a CysLT(1) receptor antagonist may have greater benefit than sole treatment with these agents alone., (Copyright 2010 S. Karger AG, Basel.)

TLRs sense microbial products and initiate adaptive immune responses by activating dendritic cells (DCs). DCs have been shown to produce leukotrienes and, conversely, leukotrienes are known to modulate several DC functions. In this study, we examined the modulation of expression and function of cysteinyl-leukotriene receptor type 1 (CysLT1) on human monocyte-derived DCs during their differentiation and subsequent maturation with zymosan, a TLR2 agonist. Maturation of DCs with zymosan reduced CysLT1 mRNA levels and protein expression in a time-dependent fashion and was associated with a diminution of functional responsiveness to leukotriene D(4) as assessed by intracellular calcium mobilization, CCL2 and CCL3 production, and chemotaxis. The effect of zymosan was mediated by both TLR2 and dectin-1 activation. Zymosan also induced a rapid expression of cyclooxygenase-2 and the production of PGE(2) and IL-10. Addition of an anti-IL-10 neutralizing Ab or inhibitors of cyclooxygenase greatly reduced the ability of zymosan to down-regulate CysLT1 expression. Down-regulation of CysLT1 expression by zymosan could be reproduced by a combination of IL-10 and PGE(2), and was dependent on MAPK activation. Taken together, our findings indicate that zymosan down-regulates CysLT1 expression in DCs with consequently reduced functional responsiveness of the cells to leukotriene D(4) stimulation. This effect is partially dependent on an endogenous production of PGs and IL-10 by DCs.

The intracellular parent of the cysteinyl leukotrienes (cysLTs), leukotriene (LT) C(4), is formed by conjugation of LTA(4) and reduced glutathione by LTC(4) synthase in mast cells, eosinophils, basophils, and macrophages. After extracellular export, LTC(4) is converted to LTD(4) and LTE(4) through sequential enzymatic removal of glutamic acid and then glycine. Only LTE(4) is sufficiently stable to be prominent in biologic fluids, such as urine or bronchoalveolar lavage fluid, of asthmatic individuals and at sites of inflammation in animal models. LTE(4) has received little attention because it binds poorly to the classical type 1 and 2 cysLT receptors and is much less active on normal airways than LTC(4) or LTD(4). However, early studies indicated that LTE(4) caused skin swelling in human subjects as potently as LTC(4) and LTD(4), that airways of asthmatic subjects (particularly those that were aspirin sensitive) were selectively hyperresponsive to LTE(4), and that a potential distinct LTE(4) receptor was present in guinea pig trachea. Recent studies have begun to uncover receptors selective for LTE(4): P2Y(12), an adenosine diphosphate receptor, and CysLT(E)R, which was observed functionally in the skin of mice lacking the type 1 and 2 cysLT receptors. These findings prompt a renewed focus on LTE(4) receptors as therapeutic targets that are not currently addressed by available receptor antagonists.

The present study was designed to investigate the effect of montelukast sodium, a leukotriene D(4) receptor antagonist, and 1,2,3,4,tetrahydroisoquinoline, a leukotriene D(4) synthetic pathway inhibitor, on the development of morphine dependence in a mouse model of naloxone-induced opioid withdrawal syndrome. Morphine (5 mg/kg, i.p.) was administered twice daily for a period of 5 days following which a single injection of naloxone (8 mg/kg, i.p.) precipitated the opioid withdrawal syndrome in mice. Behavioral observations were made for a period of 30 min immediately after naloxone treatment. The withdrawal syndrome was quantitatively assessed in terms of withdrawal severity score and the frequency of jumping, rearing, fore paw licking and circling. Montelukast sodium as well as 1,2,3,4,tetrahydroisoquinoline, markedly and dose dependently (p<0.01) attenuated the morphine-naloxone-induced opioid withdrawal syndrome in mice. However, administration of montelukast sodium or 1,2,3,4,tetrahydroisoquinoline did not alter the activity of the central nervous system, assessed in terms of locomotor activity count thus ruling out any per se sedative action of montelukast sodium. Further, pretreatment with montelukast sodium or 1,2,3,4,tetrahydroisoquinoline did not alter the acute analgesic effect of morphine. Thus, leukotriene D(4) may be involved in the development of opioid dependence and the precipitation of its withdrawal syndrome and thus may serve as a viable pharmacological target to tackle the problem of opioid addiction.

The bronchoconstriction caused by inhaled neurokinin A (NKA) in patients with asthma is indirect. The mediators involved in NKA-induced bronchoconstriction are unknown. Studies with various H1 receptor antagonists were negative, making an important contribution of histamine unlikely. To study the role of cysteinyl leukotrienes in neurokinin A-induced bronchoconstriction, we performed a randomised, double-blind, cross-over, placebo controlled trial in 12 patients with mild to moderate asthma. Zafirlukast and matching placebo were given orally, 40 mg the evening before and 40 mg the morning of assessment. In one period NKA was administered, in the other period leukotriene D4 (LTD4). Increasing concentrations of NKA and LTD4 were inhaled from a 30 L bag, after nebulization via a Mallinckrodt nebuliser. The difference between log10PC20LTD4 after treatment with placebo or zafirlukast was highly significant (p<0.0001). A trend was observed towards a difference between log10PC20 neurokinin A after treatment with placebo or zafirlukast (p=0.0741). The dose ratio for the neurokinin A provocation was 4.4 and for the LTD4 provocation 67.7. In conclusion, zafirlukast had a large inhibitory effect on LTD4-induced bronchoconstriction, but offered only limited protective effect against neurokinin A-induced bronchoconstriction. We suggest that leukotrienes play a limited role in the bronchoconstrictor effect of neurokinin A in patients with asthma.

Leukotrienes (LTs) are lipid mediators implicated in asthma and other inflammatory diseases. LTB(4) and LTD(4) also participate in antimicrobial defense by stimulating phagocyte functions via ligation of B leukotriene type 1 (BLT1) receptor and cysteinyl LT type 1 (cysLT1) receptor, respectively. Although both Galpha(i) and Galpha(q) proteins have been shown to be coupled to both BLT1 and cysLT1 receptors in transfected cell systems, there is little known about specific G protein subunit coupling to LT receptors, or to other G protein-coupled receptors, in primary cells. In this study we sought to define the role of specific G proteins in pulmonary alveolar macrophage (AM) innate immune responses to LTB(4) and LTD(4). LTB(4) but not LTD(4) reduced cAMP levels in rat AM by a pertussis toxin (PTX)-sensitive mechanism. Enhancement of FcgammaR-mediated phagocytosis and bacterial killing by LTB(4) was also PTX-sensitive, whereas that induced by LTD(4) was not. LTD(4) and LTB(4) induced Ca(2+) and intracellular inositol monophosphate accumulation, respectively, highlighting the role of Galpha(q) protein in mediating PTX-insensitive LTD(4) enhancement of phagocytosis and microbicidal activity. Studies with liposome-delivered G protein blocking Abs indicated a dependency on specific Galpha(q/11) and Galpha(i3) subunits, but not Galpha(i2) or G(beta)gamma, in LTB(4)-enhanced phagocytosis. The selective importance of Galpha(q/11) protein was also demonstrated in LTD(4)-enhanced phagocytosis. The present investigation identifies differences in specific G protein subunit coupling to LT receptors in antimicrobial responses and highlights the importance of defining the specific G proteins coupled to heptahelical receptors in primary cells, rather than simply using heterologous expression systems.

Background: A dry powder inhaler of KP-496 is currently in clinical development in Japan as an anti-asthmatic agent. The aim of this study was to evaluate the in vitro pharmacological profile of KP-496., Methods: The antagonistic activities of KP-496 for leukotriene (LT) D(4) and thromboxane (TX) A(2) receptors were examined using the LTD(4)- and U46619-induced contractions of the isolated guinea pig trachea. The selectivity of KP-496 was examined using various agonist-induced contractions in the isolated guinea pig trachea., Results: KP-496 produced parallel rightward shifts of the LTD(4) and U46619 concentration-response curves in a concentration-dependent manner. Schild plot analyses of the antagonistic activities of KP-496 demonstrated that it is a competitive antagonist for LTD(4) and TXA(2) receptors with pA(2) values of 8.64 and 8.23, respectively. The LTD(4) antagonistic activity of KP-496 was comparable to that of pranlukast and zafirlukast but was more potent than that of montelukast. The TXA(2) antagonistic activity of KP-496 was comparable to that of seratrodast. KP-496 and seratrodast also inhibited the prostaglandin (PG) D(2)- and PGF(2alpha)-induced contractions of the isolated guinea pig trachea. KP-496 had no effect on the histamine-, acetylcholine-, serotonin- and substance P-induced contractions of the isolated guinea pig trachea., Conclusions: These results indicate that KP-496 is a selective dual antagonist for LTD(4) and TXA(2) receptors. LTD(4) and TXA(2) play important roles in asthma, and antagonists for these mediators are being used for the treatment of asthma. Thus, KP-496 is expected to become a novel potent therapeutic agent for asthma.

Twenty five years after the structure elucidation of slow reacting substance of anaphylaxis, antileukotrienes are established as a new therapeutic modality in asthma. The chapter reviews the biochemistry and pharmacology of leukotrienes and antileukotrienes with particular focus on the different usage of antileukotrienes for treatment of asthma and rhinitis in Europe and the US. Further research needs and new areas for leukotriene involvement in respiratory diseases are also discussed.

CR3465 (L-Tyrosine, N-[(2-quinolinyl)carbonyl]-O-(7-fluoro-2-quinolinylmethyl) sodium salt) is a potent antagonist of [3H]leukotriene D4 ([3H]LTD4) binding to guinea pig lung preparations, its Ki (4.7+/-0.7 nM) being comparable with that of montelukast (5.6+/-0.6 nM). In tracheal strips from standard or ovalbumin-sensitized guinea pigs, CR3465 caused parallel rightward shifts in the concentration-response curves obtained with either LTD4 or antigen (pA(2), 8.74 and 8.15). Intravenous (i.v.) administration of the agent both antagonized (ED50, 9.9+/-1.9 microg/kg) and reverted LTD4 -induced bronchoconstriction of anesthetized guinea pigs. CR3465 reduced inflammatory infiltrates in the bronchoalveolar lavage fluid after antigen challenge of sensitized animals, and proved also active in inhibiting phosphodiesterase 3 (PDE3) and phosphodiesterase 4 (PDE4) activities exhibited by human platelets and neutrophils (IC50, 2.01+/-0.07 and 4.7+/-0.5 microM). In line with properties shown by phosphodiesterase inhibitors, CR3465 reduced the contractile response of guinea pig airways to histamine and decreased N-formyl-Met-Leu-Phe (fMLP)-induced degranulation of human neutrophils (IC50, 13.8 microM). Oral administration (20 mg/kg) of the compound in rats produced a significant (37%) ex vivo inhibition of tumor necrosis factor-alpha (TNF-alpha) release from lipopolysaccharide-stimulated whole blood. Pharmacokinetic data in the rat demonstrated approximately 100% bioavailability of the agent. We conclude that CR3465 represents a potent leukotriene CysLT1 receptor antagonist with enhanced effects, being also useful for counteracting spasmogenic and inflammatory stimuli other than those elicited by cysteinyl-leukotrienes (Cys-LTs).

In the present study, we examined the inhibitory effects of the beta2-adrenoceptor agonists isoproterenol, salbutamol, fenoterol, and clenbuterol, on the release of chemical mediators from cultured human mast cells after prolonged treatment with the agonists. Although preincubation of sensitized mast cells for 10 min with beta2-adrenoceptor agonists potently inhibited mediator release, prolongation of the preincubation period up to 240 min attenuated the inhibition. The attenuation of histamine release inhibition was potent when compared with that of prostaglandin D2 (PGD2) and cysteinyl leukotriene (LT) release inhibition. In contrast, forskolin inhibited mediator release and the inhibition increased gradually in proportion to the preincubation period. The reduced inhibition by the beta2-adrenoceptor agonists was compensated for by simultaneous treatment with cholera toxin. The beta2-adrenoceptor agonists elevated intracellular cAMP levels after 10-min incubation and the elevated levels were almost comparable to those after 240-min incubation. Forskolin elevated the intracellular cAMP levels more potently after incubation for 240 min than after 10 min. When mast cells were incubated for 3 d with the beta2-adrenoceptor agonists, similar attenuation of mediator release inhibition was observed. Elevation of intracellular cAMP levels was also attenuated, although beta2-adrenoceptor mRNA expression was potentiated. The present results collectively indicate that the attenuation of mediator release inhibition by beta2-adrenoceptor agonists under the present experimental conditions involves uncoupling between beta2-adrenoceptors and Gs proteins. Furthermore, the beta2-adrenoceptor desensitization causes differential attenuating effects on the inhibition of histamine, PGD2, and LT release, suggesting that downstream events involved in each inhibitory pathway have different sensitivity to receptor desensitization.

Addition of LTD4 (10 nM) to Xenopus laevis oocytes expressing the mCysLT1 receptor together with hBK or hIK channels resulted in the activation of both channels secondary to an LTD4-induced increase in [Ca2+]i. In addition, the hIK channel is activated by low concentrations of LTD4 (<0.1 nM), which did not result in any increase in [Ca2+]i. Even though activation of hIK by low concentrations of LTD4 was independent of an increase in [Ca2+]i, a certain "permissive" level of [Ca2+]i was required for its activation, since buffering of intracellular Ca2+ by EGTA completely abolished the response to LTD4. Neither hTBAK1 nor hTASK2 was activated following stimulations with LTD4 (0.1 and 100 nM).

Background: Evidence suggests that small airways contribute to clinically significant processes in asthma. Cysteinyl leukotrienes (CysLTs) are considered to be pivotal mediators in the pathogenesis of asthma. Montelukast (MK), a specific CysLT1 receptor antagonist, is metabolized in two main hydroxylated metabolites (termed M5 and M6, respectively)., Objectives: The aims of this study were to compare the responsiveness of small and large human bronchi to the three CysLTs, to evaluate the antagonist activity of MK, M5 and M6 in these preparations of human bronchi, and to characterize the CysLT receptors involved in the contractile response., Methods and Results: In isolated small bronchus (i.d. 0.5-2 mm), the potencies (-log molar EC50) of LTC4, LTD4 and LTE4 were 9.3 (n=11), 9.1 (n=30) and 8.4 (n=14), respectively. The three CysLTs were about 30-fold more potent in small bronchi than in larger bronchi (i.d. 4-6 mm). In small bronchi, MK significantly shifted to the right the CysLT concentration-effect curves with pA2 values against LTC4, LTD4 and LTE4 of 9.1 (n=3), 9.0 (n=11) and 8.7 (n=5), respectively. The antagonist potencies of M6 and M5 were similar to MK and fivefold lower, respectively. A similar activity of MK against the three CysLTs suggested that CysLT1 receptors are involved in the contraction of human bronchus. Analysis by RT-PCR also indicated that human bronchus mainly expressed CysLT1 receptors., Conclusion: MK exerts a potent antagonist activity against the particularly potent constricting effects of CysLTs in isolated human small bronchi, which only expressed the CysLT1 receptor subtype. The metabolites of MK are also potent in vitro antagonists, but may not participate in the therapeutic activity of MK due to their low plasma concentrations in patients treated with the recommended dose of MK.

We investigated signal transduction pathways for LTD4 in the human promonocytic cell line U937 known, upon differentiation, to express CysLT1 receptors. We confirmed the presence of high-affinity binding sites for 3H-LTD4, which, in functional studies, displayed the features of CysLT1 receptor. In fact, three potent and selective CysLT1 receptor antagonists were able to completely inhibit LTD4-induced response. In turn, cytosolic Ca2+ ([Ca2+]i) increase (EC50 = 3.4 nM +/- 27% CV) was only partially sensitive to pertussis toxin (PTx) as well as to the prenylation inhibitor fluvastatin and to the specific geranylgeranylation and farnesylation inhibitors BAL 9504 and FPT II. Finally, Clostridium sordellii lethal toxin, inhibitor of the Ras family of GTPases, and FTS, a potent methyltransferase inhibitor, were both able to partially inhibit LTD4-induced [Ca2+] increase, suggesting a role for a Ras family member in [Ca2+]i regulation. In conclusion, in dU937 LTD4 signal transduction involves: (a) at least two pathways, one sensitive and one insensitive to PTx; (b) isoprenylated proteins, such as betagamma subunits and, possibly, a small G protein of the Ras family.

Background: Tachykinins are potent contractors of human airways producing a dose-related bronchoconstriction when administered by means of inhalation to asthmatic subjects., Objective: The aim of this study was to examine the effective role played by leukotrienes (LTs) in neurokinin A (NKA)-induced bronchoconstriction in asthmatic patients., Methods: To address this question, we investigated the protective effect of a selective cysteinyl LT receptor antagonist, montelukast, against inhaled NKA and determined LTE(4) excretion in the urine., Results: Inhaled NKA in the absence of any drug treatment produced a concentration-related bronchospasm with a geometric mean provocative concentration required to produce a 15% decrease in FEV(1) from the postsaline baseline value (PC(15)) value of 290.9 microg/mL (+SE, 407.1 microg/mL; -SE, 207.84 microg/mL). Montelukast pretreatment significantly increased (P <.01) the PC(15) NKA value (708.8 microg/mL; +SE, 890.47 microg/mL; -SE, 564.15 microg/mL) in comparison with placebo (394.4 microg/mL; +SE, 491.88 microg/mL; -SE, 248.16 microg/mL) and produced a shift of the NKA concentration-response curve to the right in all the subjects studied. When compared with placebo, montelukast did not have a significant protective effect against methacholine challenge; the geometric mean PC(15) values obtained were 0.87 and 0.96 mg/mL with placebo and montelukast, respectively. Although we have not observed any increase in urinary LTE(4) excretion after NKA inhalation, we have shown that pretreatment of asthmatic subjects with montelukast elicits a significant protection against NKA-induced bronchoconstriction., Conclusion: In asthmatic subjects NKA-induced bronchoconstriction is indirectly caused by the release of LTs, and this mechanism could explain some of the antiasthmatic and anti-inflammatory effects of LT antagonists.

An analysis of the X-ray structure of cilastatin bound to membrane dipeptidase, together with docking studies, is presented here to reveal how a simple amide may act as a high-affinity, reversible, amidase inhibitor. Cilastatin binds as a normal substrate and is orientated in a perfect near-attack conformer for formation of a tetrahedral intermediate with the zinc-bound water/hydroxide. This intermediate is fated, however, only to revert to its starting components as scission of the amide bond is prevented by the precise fit of cilastatin within the active site. The cilastatin alkyl end groups that are tightly buttressed against amino acid residues on opposite sides of the active site, are aligned along the C-N reaction coordinate axis thereby preventing collapse of the intermediate via rupture of the C-N bond. Such a feature could have more general applicability in the explicit design of substrate variants as selective, tight-binding, and reversible inhibitors.

Mesangial cells (MC) serve a number of functions in the renal glomerular capillary including structural support of the capillary tuft, modulation of glomerular hemodynamics, and a phagocytic function allowing removal of macromolecules and immune complexes. The proliferation of MC is a prominent feature of glomerular disease including IgA nephropathy, membranoproliferative glomerulonephritis, lupus nephritis, and diabetic nephropathy. In experimental animal models of nephritis, MC proliferation frequently precedes and is linked to the increase of extracellular matrix in the mesangium and glomerulosclerosis. Reduction of MC proliferation in glomerular disease models by treatment with heparin, low-protein diet, or antibodies to platelet-derived growth factor (PDGF), have been shown to reduce extracellular matrix expansion and glomerulosclerotic changes. Therefore, MC proliferation inhibitors may offer therapeutic opportunities for the treatment of proliferative glomerular disease. It is also known that the MC proliferation is inhibited by many kinds of pharmacological drugs, for example, angiotensin converting enzyme (ACE) inhibitors, leukotriene D(4) (LTD(4)) antagonists, PDGF inhibitors, matrix metalloproteinases (MMP) inhibitors, 3-hydroxy-3 methyl glutaryl-coenzymeA (HMG-CoA) inhibitors, cyclin-dependent kinases (CDK) inhibitors, and others. This review summarizes the recently reported MC proliferation inhibitors with their pharmacological properties on the basis of their chemical structures., (Copyright 2002 Wiley Periodicals, Inc.)

Stem cell factor (SCF) is directly involved in the induction of airway hyperreactivity during allergen-induced pulmonary responses in mouse models. In these studies, we examined the specific mediators and mechanisms by which SCF can directly induce airway hyperreactivity via mast cell activation. Initial in vitro studies with bone marrow-derived mast cells indicated that SCF was able to induce the production of bronchospastic leukotrienes, LTC(4) and LTE(4). Subsequently, when SCF was instilled in the airways of naive mice, we were able to observe a similar induction of LTC(4) and LTE(4) in the bronchoalveolar lavage (BAL) fluid and lungs of treated mice. These in vivo studies clearly suggested that the previously observed SCF-induced airway hyperreactivity may be related to the leukotriene production after SCF stimulation. To further investigate whether the released leukotrienes were the mediators of the SCF-induced airway hyperreactivity, an inhibitor of 5-lipoxygenase (5-LO) binding to the 5-LO activating protein (FLAP) was utilized. The FLAP inhibitor MK-886, given to the animals before intratracheal SCF administration, significantly inhibited the release of LTC(4) and LTE(4) into the BAL fluid. More importantly, use of the FLAP inhibitor nearly abrogated the SCF-induced airway hyperreactivity. In addition, blocking the LTD(4)/E(4), but not LTB(4), receptor attenuated the SCF-induced airway hyperreactivity. In addition, the FLAP inhibitor reduced other mast-derived mediators, including histamine and tumor necrosis factor. Altogether, these studies indicate that SCF-induced airway hyperreactivity is dependent upon leukotriene-mediated pathways.

Mechanisms governing the release of drugs from controlled delivery systems are mainly diffusion, osmosis and erosion. For poorly soluble drugs, the existing mechanisms are limited to osmosis and matrix erosion, that are commonly observed in single unit matrix dosage forms. This study reports formulation and dissolution performance of Eudragit L 100 55 and Eudragit S 100 based multi-unit controlled release system of a poorly soluble thiazole based leukotriene D(4) antagonist, that was obtained by an extrusion/spheronization technique. Effect of triethyl citrate, that was incorporated in the matrix, on the dissolution performance of the drug was also evaluated. In vitro matrix erosion and drug release from the pellets were determined by the use of USP Dissolution Apparatus I, pH 6.8 phosphate buffer, gravimetry and UV spectrophotometry, respectively. Results obtained demonstrated that matrix erosion and drug release occurred simultaneously from the pellets. Pellets eroded with a consequent reduction in size without any change in the pellet geometry for over 12 h. Matrix erosion and drug release followed zero order kinetics. Data obtained strongly suggested a polymer controlled, surface erosion mechanism.

Galphimia glauca is used for the treatment of asthma and allergies in Latin American traditional medicine. The ethylacetate fraction from its aerial parts was assayed for bronchoconstriction induced by antigen and several agonists in guinea pig tracheae. The organic fraction significantly inhibited the contractile response to ovalbumin in tracheae from sensitized guinea pigs, and significantly and selectively inhibited the bronchoconstriction induced by leukotriene D(4) (LTD(4)). The relative potency of the ethylacetate fraction of G. glauca to produce a concentration-dependent rightward shift of LTD(4) concentration-response curve was similar to that reported for SK&F 104353, a well-known competitive LTD(4)-antagonist.

To investigate the effect of pranlukast on leukotriene- induced airway mucosal epithelial dysfunction, samples of human nasal mucosa obtained during surgery for facial trauma were exposed to leukotriene C4 and/or D4 and observed on a TV screen magnified x 2,500. Leukotriene C4- and D4-induced ciliary inhibition and delayed mucosal surface alterations appeared several hours later. Pranlukast prevented both the mucosal epithelial cell dysfunction and the delayed epithelial cell alteration.

The lipoxygenase-derived eicosanoids leukotrienes and lipoxins are well defined regulators of hemeodynamics and leukocyte recruitment in inflammatory conditions. Here, we describe a novel bioaction of lipoxin A(4) (LXA(4)), namely inhibition of leukotriene D(4) (LTD(4))-induced human renal mesangial cell proliferation, and investigate the signal transduction mechanisms involved. LXA(4) blocked LTD(4)-stimulated phosphatidylinositol 3-kinase (PI 3-kinase) activity in parallel to inhibition of LTD(4)-induced mesangial cell proliferation. Screening of a human mesangial cell cDNA library revealed expression of the recently described cys-leukotriene(1)/LTD(4) receptor. LTD(4)-induced mesangial cell proliferation required both extracellular-related signal regulated kinase (erk) and PI 3-kinase activation and may involve platelet-derived growth factor receptor transactivation. LTD(4)-stimulated the MAP kinases erk and p38 via a pertussis toxin (PTX)-sensitive pathway dependent on PI 3-kinase and protein kinase C activation. On screening a cDNA library, mesangial cells were found to express the previously described LXA(4) receptor. In contrast to LTD(4), LXA(4) showed differential activation of erk and p38. LXA(4) activation of erk was insensitive to PTX and PI 3-kinase inhibition, whereas LXA(4) activation of p38 was sensitive to PTX and could be blocked by the LTD(4) receptor antagonist SKF 104353. These data suggest that LXA(4) stimulation of the MAP kinase superfamily involves two distinct receptors: one shared with LTD(4) and coupled to a PTX-sensitive G protein (G(i)) and a second coupled via an alternative G protein, such as G(q) or G(12), to erk activation. These data expand on the spectrum of LXA(4) bioactions within an inflammatory milieu.

Cysteinyl leukotrienes are potent inflammatory molecules playing a major role in asthma. The involvement of these mediators in hypersensitivity in mice is not well known. This study aimed at elucidating their implication by using MK-571, a cysLT(1)receptor antagonist. Mice were sensitized with a suspension of ovalbumin (8 microg) adsorbed to alum (2 mg) and were challenged with an aerosolized ovalbumin solution (0.5%). Inflammatory cell infiltration in the bronchoalveolar lavage (mostly eosinophils) following antigen challenge was inhibited by dexamethasone (0.1, 1 and 5 mg kg(-1)s.c.) and MK-571 (1, 10, 100 mg kg(-1)i.v.) in a dose-dependent manner. Maximal inhibition was 95% with 5 mg kg(-1)dexamethasone and 90% with 100 mg kg(-1)MK-571. When injected together they showed an additive inhibitory effect on eosinophil infiltration. Bronchial hyperreactivity, measured by the increased pulmonary insufflation pressure to carbachol injections, was also inhibited dose-dependently by MK-571. The EC(50)values for carbachol were of 22.39+/-1.12 microg kg(-1)in sensitized and challenged animals that did not receive MK-571 and increased to 43.65+/-1.10, 50.12+/-1.15 and 83.18+/-1.16 microg kg(-1)in animals treated with 1, 10 and 100 mg kg(-1)MK-571 respectively. Lung microvascular leakage (as measured by Evans blue extravasation) induced by antigen bronchoprovocation was reduced by 22% after treatment with 10 mg kg(-1)MK-571. All these inhibitory effects of MK-571 suggest a role for leukotriene D(4)in this animal model of allergic asthma., (Copyright 2000 Harcourt Publishers Ltd.)

The antagonistic activity of oral YM158 (3-[(4-tert-butylthiazol-2-yl)methoxy]-5'-[3-(4-chlorobenzenesu lfonyl)propyl]-2'-(1H-tetrazol-5-ylmethoxy)benzanilide monosodium salt monohydrate), a new dual antagonist for leukotriene (LT) D4 and thromboxane (TX) A2 receptors, was investigated. Oral YM158 caused dose-dependent inhibition of LTD4-induced increases in plasma leakage and LTD4- or U46619-induced increases in airway resistance, with ED50 values of 6.6, 8.6 and 14 mg/kg, respectively. The dose-range of YM158's inhibitions was almost the same for both LTD4 and TXA2 receptors, and repeated oral doses did not affect its efficacy. Furthermore, oral YM158 inhibited antigen-induced bronchoconstriction. Although the potency of pranlukast for LTD4 receptor antagonism (ED50 = 0.34 mg/kg) is greater than that of YM158 (ED50 = 8.6 mg/kg), the doses of both pranlukast and YM158 for significant inhibition of the antigen-evoked airway response were the same, indicating that the TXA2 receptor antagonism of YM158 plays an important role in its anti-asthmatic effects. In conclusion, YM158 promises to be a novel agent for treating bronchial asthma.

A group of new 4-[2-(7-heterocyclemethoxynaftalen-2-ylmethoxy)ethyl]benzoic acids have been synthesized and pharmacologically evaluated as LTD(4)-antagonists. Thiazole derivatives, especially 4-[2-[7-(4-cyclobutylthiazole-2-ylmethoxyl)naphthalen- 2-ylmetho-xy]et hyl]benzoic acid, present considerable activity and improved pharmacokinetic profiles in comparison with our quinoline containing lead molecule confirming the interest of our compounds as potentially oral antiasthmatics and that the 4-alkylthiazole system can be considered as bioisosteric of the quinoline ring at least in our series of compounds.

The effect of drug loading, water required for granulation and spheronization time on porosity parameters (intrusion-extrusion isotherms, pore size distribution, total pore surface area, mean pore diameter, shape and morphology of pores) and drug release rates from pellets of a poorly soluble drug was investigated. Porosity parameters were determined by mercury intrusion porosimetry. The drug loading was found to have a profound effect on the porosity parameters. Pellets with low drug loading showed increased pore surface area with small mean pore diameters and an increased number of total pores. On the other hand, pellets with high drug loading had decreased pore surface areas with larger mean pore diameters and a reduction in the total number of pores. With high drug loading, the drug release rate decreased. Water required for granulation had a direct effect on the total porosity of the pellets. Spheronization time from 2 to 10 min had a pronounced effect on porosity parameters and release rates. No changes in porosity parameters and release rates were observed from 10 to 20 min of spheronization time. It was shown that each porosity parameter investigated was well correlated with drug release rates and thus it is important to study the effect of porosity parameters in evaluating the in vitro performance of the multi-unit erosion matrix for the controlled release of a poorly soluble drug.

Leukotrienes can be generated from a wide variety of cells including mast cells and eosinophils. The biological properties of these products include bronchial smooth muscle contraction, stimulation of mucous production, enhancement of vascular permeability, and recruitment of eosinophils. These properties can contribute significantly to the pathobiology of asthma. Recently, zafirlukast and montelukast, and zileuton, leukotriene D4 receptor antagonists and 5-lipoxygenase inhibitors, respectively, have been developed and are available for treating asthma. Studies have found these compounds modify bronchospasm with exercise, the pulmonary reaction to aspirin in sensitive subjects, and the airway response to inhaled antigen. Furthermore, in patients with chronic asthma, leukotriene modifiers improve airflow obstruction, decrease the need for rescue medication, and diminish symptoms. Moreover, these drugs can prevent asthma exacerbations. However, there is little evidence that these medications have potent anti-inflammatory activity. Nonetheless, leukotriene modifiers represent new, and effective, therapeutics in the treatment of asthma; at present, the positioning of these products in relationship to inhaled corticosteroids, for example, in the treatment of asthma has not been fully defined but will emerge with further study and use in the clinic setting.

Purpose: The aim of this study was to construct a reliable dry powder inhalation (DPI) testing system for use in guinea pigs. Using this system, we were able to demonstrate the superiority of pulmonary administration of hydrophilically surface-modified pranlukast hydrate powder (SM-DP) over IV and PO administration as reflected in improved pharmacological action. Our ultimate aim is the development of an ideal treatment system for bronchial asthma involving topical administration to the lung., Methods: The reliability of the present DPI system was validated by continuously monitoring the concentration and particle size distribution of aerosols generated with an ambient particulate monitor and an Andersen air sampler, respectively. The pharmacological effect of SM-DP intratracheally administered to guinea pig was investigated by measuring the degree of bronchoconstriction and microvascular leakage induced by leukotriene D4., Results: The mass concentration of aerosols generated by the DPI system was stable and the mass median aerodynamic diameter of aerosols insufflated from the respirator of the DPI system ranged from 1.4 to 1.7 microm, within respirable limits. Inhibition of bronchoconstriction and airway microvascular leakage induced by leukotriene D4 was achieved successfully with a dramatically lower dose of DP, or a further lower dose of SM-DP, comparable with that of the drug solution injected intravenously. The plasma pranlukast hydrate level with SM-DP at 50% inhibition of bronchoconstriction and airway microvascular leakage was reduced to 1/10 or less that following IV and PO administration., Conclusions: The hydrophilically surface-modified pranlukast hydrate powders were ideally aerosolized by the present DPI system, and were uniformly deposited in the lung lobes after inhalation. The pulmonary administration system with SM-DP is strongly recommended as an ideal system for the treatment of bronchial asthma in order to avoid systemic side-effects due to a dramatically reduced ED50, comparable with or lower than IV, and the low plasma concentration of drug, 1/12 or less than that following IV and PO administration.

The response of parenchymal strips from guinea-pig lungs to tracheobronchial lavage fluid (TBLF) collected from 8 normal horses and from 8 affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) was determined. TBLF was collected during the summer (July) and winter (February) seasons. The serum/TBLF urea nitrogen ratio was used to standardize the mediator concentration in the TBLF. Four strips were used from each guinea-pig. The first strip did not receive any antagonist and served as the control. The second, third and fourth strips received antagonists of PGE2, LTD4 and PAF, respectively at 10(-6) mol/L for 30 min. The tissues were then precontracted with a dose of histamine (10(-5) mol/L) and their responses to 1 ml of TBLF were determined. The study showed that TBLF obtained in the summer from unaffected horses produced a significantly greater relaxation than that from the affected horses, whereas TBLF obtained in the winter from unaffected or affected horses did not cause a significantly different degree of relaxation. Among the antagonist-treated strips, only those exposed to the PGE2 blocker showed a significant reduction in the relaxation caused by TBLF obtained in the summer from SPAOPD horses. This suggests that PGE2 is an important mediator present in the summer in the TBLF from horses affected with SPAOPD.

By addressing the issues of potency and metabolism in 3, a new series of LTD4 antagonists represented by (+)-26 was developed which is equipotent to clinical LTD4 antagonists Zafirlukast (1) and Pranlukast (2).

The in vivo model of nasal microvascular leakage was used for the nasal allergic challenge in ovalbumin (OA)-sensitized guinea pigs, or nasal stimulation with leukotriene D4 (LTD4) in non-sensitized animals. An intravenous injection of Evans blue dye was given as an index of nasal microvascular leakage. Following the nasal stimulation with LTD4, the concentration of dye in the nasal lavage fluid rapidly increased. Oral administration of ONO-1078 (pranlukast) (3-30 mg/kg) significantly inhibited the LTD4-induced nasal microvascular leakage. In OA-sensitized guinea pigs, the excretions of dye into nasal lavage fluid were recognized soon after the topical antigenic stimulation and continued for over 60 minutes. Oral administration of ONO-1078 (30 mg/kg) significantly inhibited the antigen-induced microvascular leakage. These results suggest that ONO-1078 may be of therapeutic use for nasal allergy.

Neurological transmitters including ACh, substance P (SP), and calcitonin gene-related peptide (CGRP) play an important role in regulating airway tone, and increased bronchial reactivity to cholinergic stimulation is a well-recognized phenomenon in patients with bronchial asthma. We postulated that ACh, SP, and CGRP might stimulate alveolar macrophages (AMs) to release neutrophil, monocyte, and eosinophil chemotactic activities. To test this hypothesis, bovine AMs were isolated by bronchoalveolar lavage and cultured. AMs released chemotactic activities in response to ACh in a dose- and time-dependent manner (P < 0.05). However, SP and CGRP did not stimulate bovine AMs. Checkerboard analysis revealed that these released activities were predominantly chemotactic. Partial characterization and molecular-sieve column chromatography revealed that low-molecular-weight lipid-soluble activity was predominant. Lipoxygenase inhibitors significantly blocked the release of chemotactic activities (P < 0.05). Leukotriene B4- and platelet-activating factor-receptor antagonists blocked the chemotactic activities. Immunoreactive leukotriene B4 significantly increased in supernatant fluids in response to ACh (P < 0.05), but platelet-activating factor did not. The receptor responsible for the release of the chemotactic activities was the muscarinic M3 receptor. These data demonstrate that ACh stimulates AMs to release lipoxygenase-derived chemotactic activities and plays a role in inflammatory cell recruitment into the airway.

The two geminal ethyl groups in the succinic acid moiety of CGP57698 (4-[3-(7-fluoro-2-quinolinyl-methoxy)phenyl-amino]-2,2-diethyl-4-oxo- butanoic acid) are responsible for the high in vitro and in vivo potency of this peptidoleukotriene antagonist of the quinoline type. The synthesis and structure activity relationships of CGP57698 and its analogs are described.

A set of 2-[4-(dialkylaminoalkoxy)phenyl]benzotriazoles and corresponding N-oxides was prepared. In a preliminary pharmacological investigation concerning some of these compounds, several in vitro and in vivo activities were shown. At concentrations in the range of 3-10 microM all tested compounds strongly inhibited (50-100%) the guinea pig ileum contractions induced either electrically or by means of several agonists; of particular interest was the antagonism to leukotriene D4. Compound 5b inhibited platelet aggregation induced by thromboxane A2, PAF and ADF (but not by arachidonic acid) and increased the bleeding time in mice. Compounds 5b and 6b protected mice from potassium cyanide hypoxia and exerted anti-hypercholesterolemic action; the first compound produced a ratio between HPL and total serum cholesterol concentrations below 0.92, thus indicating a potential anti-atherogenic activity.

1. Cysteinyl-leukotrienes (cysteinyl-LTs) are important mediators in the pathogenesis of asthma. They cause bronchoconstriction, mucus hypersecretion, increase in microvascular permeability, plasma extravasation and eosinophil recruitment. 2. We investigated the pharmacological profile of the cysteinyl-LT antagonists CGP 45715A (iralukast), a structural analogue of LTD4 and CGP 57698, a quinoline type antagonist, in human airways in vitro, by performing binding studies on human lung parenchyma membranes and functional studies on human isolated bronchial strips. 3. Competition curves vs [3H]-LTD4 on human lung parenchyma membranes demonstrated that: (a) both antagonists were able to compete for the two sites labelled by [3H]-LTD4; (b) as in all the G-protein coupled receptors, iralukast and CGP 57698 did not discriminate between the high and the low affinity states of the CysLT receptor labelled by LTD4 (Ki1=Ki2= 16.6 nM+/-36% CV and Ki1= Ki2 = 5.7 nM+/-19% CV, respectively); (c) iralukast, but not CGP 57698, displayed a slow binding kinetic, because preincubation (15 min) increased its antagonist potency. 4. In functional studies: (a) iralukast and CGP 57698 antagonized LTD4-induced contraction of human bronchi, with pA2 values of 7.77+/-4.3% CV and 8.51+/-1.6% CV, respectively, and slopes not significantly different from unity; (b) the maximal LTD4 response in the presence of CGP 57698 was actually increased, thus clearly deviating from apparent simple competition. 5. Both antagonists significantly inhibited antigen-induced contraction of human isolated bronchial strips in a concentration-dependent manner, lowering the upper plateau of the anti-IgE curves. 6. In conclusion, the results of the present in vitro investigation indicate that iralukast and CGP 57698 are potent antagonists of LTD4 in human airways, with affinities in the nanomolar range, similar to those obtained for ICI 204,219 and ONO 1078, two of the most clinically advanced CysLT receptor antagonists. Thus, these compounds might be useful drugs for the therapy of asthma and other allergic diseases.

In order to find dual antagonists against both thromboxane A2 (TXA2) and leukotriene D4 (LTD4) receptors for a new antiasthmatic agent, various benzenesulfonamide derivatives were synthesized and evaluated for those pharmacological effects. TXA2 and LTD4 antagonistic activities in vitro were evaluated by the inhibitory effects on LTD4-induced and U-46619-induced contraction of guinea-pig trachea. Furthermore, TXA2 and LTD4 antagonistic activities in vivo were evaluated by the inhibitory effects on LTD4-induced and U-46619-induced bronchoconstriction of guinea-pig after oral administration of test compounds. It was found that 4-[5-[1-(4-chlorobenzenesulfonamido)-5-methylhexyl]-2-thi eny l]butyric acid and 4-[5-[1-(4-fluorobenzenesulfonamido)-5-methyl-hexyl]-2-thienyl]but yric acid possess good anti-LTD4 and anti-TXA2 activities by oral administration.

In order to find new antiasthmatic agents with dual antagonistic activity against thromboxane A2 (TXA2) and leukotriene D4 (LTD4) receptors, synthesis and pharmacological evaluation of various [4-[1-(benzenesulfonamido)-alkyl]phenyl]alkanoic acid derivatives were undertaken. TXA2 and LTD4 antagonistic activities in vitro were evaluated by measuring the inhibitory effects on L-46619-induced contraction of guinea-pig trachea and LTD4-induced contraction of guinea-pig ileum and trachea. Several compounds showed satisfactory dual antagonistic activities, and their effect (after oral administration) on LTD4-induced bronchoconstriction in guinea-pig in vivo was examined. The results demonstrated that both 4-[4-[1-(4-chlorobenzenesulfonamido)hexyl]phenyl]butyric acid (12e) and 4-[4-[1-(4-chlorobenzenesulfonamido)-5-methylhexyl]phenyl]bu tyric acid (12m) possessed good anti-LTD4 activities. Compounds 12e and 12m were then evaluated for other related pharmacological effects involving the arachidonic acid cascade. These compounds appear to be hybrid eicosanoids antagonists having antagonistic activity against contraction of guinea-pig trachea induced by prostaglandin D2 (PGD2) and PGF2 sigma, as well as TXA2 and LTD4 antagonistic activities.

Structure optimization of the leukotriene D4 antagonist Ro24-5913 was attempted by combinatorial chemistry. Three segments in its N-succinyl-3-(2-thiazolylethenyl)anilide skeleton, designated as A, B, and C coincided with the thiazolyl, aniline, and N-acyl moieties, respectively, and were selected for variations in a synthesis involving the sequences A + B-->AB and AB + C-->ABC to furnish the library (10A 7B 10C) containing 700 compounds. Lead candidates were identified by the LTD4-induced muscle-contraction assay. Assays of the C-partition 10(10A 7B C) of the set led to a subset of C elements associated with significant bioactivities, ic = {C1, C2, C3}, from which the preferred element C1 was selected. Incorporating this selection into the synthesis of the first reduced set gave the partition 7(10A B C1) whose assay revealed the set iB = {B1, B6} and hence the preferred B element B1. The second reduced set, 10(A B1 C1) incorporating the selected C1 and B1 moieties, revealed iA = {A1, A3, A4, A6, A7, A8, A9} In the resulting combinatiorial product iA x iB x iC, comprising 42 elements, A1 B1 C1 appears on top of the list. Thus, 4-[[3-[2-[4-(2,2-dimethyl ethyl)cyclobutyl-2-thiazolyl]ethenyl] phenyl]amino]-2,2-diethyl-4-oxobutanoic acid (Ro24-5913) was confirmed as the structure with the highest bioactivity. Analogues obtained by replacement of the cyclobutyl group in Ro24-5913 with 4-fluorophenyl and t-butyl were the runners-up. Of these, the former exhibited bioactivity comparable to that of Ro24-5913.

Human airways in vitro contract when stimulated by anti-IgE, whereas human pulmonary vessels relax. Leukotriene D4 (LTD4) induced a contractile response in the airways, while in pulmonary vessels both contractions and relaxations were observed. The LTD4 contractions in airways were blocked by cysLT1 receptor antagonists (MK 571, ICI 198615, and BAY x7195). In contrast none of the compounds affected the LTD4 contractions of pulmonary veins. These results suggest that the leukotrienes which are released during antigen challenge of airways and pulmonary vessels may be acting at distinct receptors in the human lung.

It has been reported that hyperpnea-induced bronchoconstriction in guinea pigs is a potential model for exercise-induced asthma in humans. We hypothesized that calcitonin gene-related peptide (CGRP) could modulate leukotriene D4 (LTD4)-induced responses and be involved in the pathophysiology in this asthma model. We measured tracheal (Ptr) and alveolar pressure (PA) using alveolar capsules in open-chested, mechanically ventilated (f = 1 Hz, VT = 9 ml/kg, PEEP = 4 cm H2O) guinea pigs. Animals were intravenously pretreated with saline (SAL), CGRP(8-37) (CGRP receptor antagonist), CGRP, MK-571 (LTD4 receptor antagonist), MK-886 (5-lipoxygenase inhibitor), or CGRP(8-37) + MK-571, and then underwent dry gas hyperpnea challenge (HC, 95% 02-5% CO2, 150 breaths/min, 7 min). We calculated resistance of lung (RL), tissue (Rti), and airway (Raw). HC increased RL, Rti, and Raw in SAL controls (322 +/- 27, 430 +/- 59, 299 +/- 23% baseline, respectively). MK-571, MK-886, and CGRP significantly reduced the responses to HC, while CGRP(8-37) enhanced HC-induced responses. Pretreatment with CGRP(8-37) and MK-571 in combination attenuated HC-induced constriction. In addition, pretreatment with CGRP reduced responses induced by intravenous administration of LTD4. These observations suggest that CGRP might be involved in the pathophysiology of hyperpnea-induced constriction in guinea pigs via modulation of LTD4-elicited responses.

Studies were performed in three models of acute gastric mucosal damage (induced by oral ethanol, aspirin and indomethacin) and a model of chronic gastritis (induced by 7 day treatment with iodoacetamide) in rats to establish the role of leukotrienes (LTs) in the pathogenesis of these lesions. The protective effects of highly selective 5-lipoxygenase (5-LO) inhibitors and leukotriene antagonists were thus examined in rats given these ulcerogens. Ethanol (1 mL, p.o.)-induced haemorrhagic lesions were significantly reduced by prior oral administration of the 5-LO inhibitor L-656224 (50 mg/kg), whereas lower doses of this drug were ineffective. Prior treatment with oral doses (5 and 10 mg/kg) of the 5-LO inhibitor L-655224 or the LT antagonists L-649923 or L-660711, failed to affect lesions induced by aspirin (100 mg/kg, p.o.) and indomethacin (400 mg/kg, s.c.), whereas higher doses of all three drugs (50 mg/kg) showed protective effects. Repeated prior dosing up to 5 h with the novel five lipoxygenase activating protein (FLAP) inhibitor, MK886 (50 and 100 mg/kg), reduced the lesions developed by indomethacin (30 mg/kg, s.c.). Twice daily dosing with the 5-LO inhibitor L-656224 (5 mg/kg) or the LT antagonist L-649923 (2 or 5 mg/kg) for 7 days significantly reduced the development of iodoacetamide-induced gastritis during the period of induction of this condition, but higher doses of these inhibitors were not protective. We conclude that 5-LO products partially mediate the production of gastric mucosal lesions induced by damaging agents, which varies according to the ulcer model employed; the limited protective effects of the respective 5-LO inhibitors and LT antagonists depend on their individual pharmacokinetics and their time of dosing.