Synthesis and structure-activity relationships of novel 5-(hydroxamic acid)methyl oxazolidinone derivatives as 5-lipoxygenase inhibitors.

Oxazolidinone hydroxamic acid derivatives were synthesised and evaluated for inhibitory activity against leukotriene (LT) biosynthesis in three in vitro cell-based test systems and on direct inhibition of recombinant human 5-lipoxygenase (5-LO). Thirteen of the 19 compounds synthesised were considered active ((50% inhibitory concentration (IC50) ≤ 10 µM in two or more test systems)). Increasing alkyl chain length on the hydroxamic acid moiety enhanced activity and morpholinyl-containing derivatives were more active than N-acetyl-piperizinyl derivatives. The IC50 values in cell-based assay systems were comparable to those obtained by direct inhibition of 5-LO activity, confirming that the compounds are direct inhibitors of 5-LO. Particularly, compounds PH-249 and PH-251 had outstanding potencies (IC50 < 1 µM), comparable to that of the prototype 5-LO inhibitor, zileuton. Pronounced in vivo activity was demonstrated in zymosan-induced peritonitis in mice. These novel oxazolidinone hydroxamic acid derivatives are, therefore, potent 5-LO inhibitors with potential application as anti-allergic and anti-inflammatory agents.

Antiallergic and antiasthmatic effects of a novel enhydrazinone ester (CEE-1): inhibition of activation of both mast cells and eosinophils.

Activation of mast cells and eosinophils is a fundamental process in the pathophysiology of allergic diseases. We have previously reported that the novel enhydrazinone ester CEE-1 (ethyl 4-phenylhydrazinocyclohex-3-en-2-oxo-6-phenyl-1-oate) possesses potent anti-inflammatory activity. We have now tested whether the compound also possesses antiallergic and antiasthmatic effects in vitro and in vivo. The compound significantly inhibited degranulation and leukotriene C4 (LTC4) release from activated human eosinophils, as well as IgE-dependent degranulation and LTC4 release from passively sensitized rat basophilic leukemia cells and bone marrow-derived mouse mast cells. In human eosinophils, the drug was more potent in inhibiting degranulation than LTC4 release {IC50 = 0.4 µM [confidence interval (CI): 0.1-0.9] versus 3.8 µM (CI: 0.9-8.3)}, whereas in mast cells the reverse was essentially the case. The drug did not affect stimulus-induced calcium transients in eosinophils but significantly inhibited early phosphorylation of extracellular signal-regulated kinases 1/2 and p38-mitogen-activated protein kinases (MAPK). In vivo, topical application of 4.5-15 mg/kg of the compound significantly inhibited allergen-induced passive cutaneous anaphylaxis in mice. Similarly, in the mouse asthma model, the intranasal administration of 6.5-12.5 mg/kg of the compound significantly inhibited bronchial inflammation and eosinophil accumulation in bronchial lavage fluid, as well as abolishing airway hyper-responsiveness to methacholine. These results show that CEE-1 inhibits the activation of both mast cells and eosinophils in vitro, probably by blocking MAPK-activation pathways, and that these effects are translated into antiallergic and antiasthmatic effects in vivo. The compound, therefore, has potential application in the treatment of asthma and other allergic diseases.

Anti-allergic, anti-asthmatic and anti-inflammatory effects of an oxazolidinone hydroxamic acid derivative (PH-251) - A novel dual inhibitor of 5-lipoxygenase and mast cell degranulation.

We have recently reported the discovery of a series of oxazolidinone hydroxamic acid derivatives that are potent inhibitors of 5-lipoxygenase (5-LO) [arachidonate 5-lipoxygenase; EC 1.13.11.34]. We now report that one of the most active members of this series, compound PH-251, [(R)-N-((3-(3-fluoro-4-morpholinophenyl)-2-oxooxazolidin-5-yl) methyl)-N-hydroxyoctanamide], also possesses a unique and strong ability to concurrently inhibit mast cell degranulation. PH-251 inhibited the biosynthesis of leukotriene C4 (LTC4), as well as degranulation of IgE/allergen-activated bone marrow-derived mouse mast cells (BMMC) in vitro. In contrast, zileuton (the prototype 5-LO inhibitor) inhibited leukotriene generation, but not degranulation. Consistent with its dual activity, compound PH-251 also significantly inhibited both the early and the late anaphylactic contractions of guinea pig lung parenchymal strip, whereas zileuton inhibited only the late (leukotriene-dependent) contractions. Comparative structure-activity analysis of PH-251 and its structural analogues showed that the anti-degranulation effect appeared to be dependent on the length of the straight-chain hydrocarbon substitution on the hydroxamic acid moiety. In the in vivo studies, PH-251 (3-30 mg/kg s.c.) strongly inhibited various components of zymosan-induced peritonitis - a typical non-allergic LT-dependent animal model of inflammation. In the mouse allergic asthma model, the compound significantly inhibited allergen-induced bronchial eosinophilic inflammation and airway hyper-responsiveness to inhaled methacholine. These results show that PH-251 is a unique dual inhibitor of 5-LO and mast cell degranulation, with in vivo activity in animal models of disease and may therefore offer potential advantages over single-target drugs in the treatment of asthma and other allergic and inflammatory diseases.

Interactions between theophylline and salbutamol on cytokine release in human monocytes.

The combination of beta(2)-adrenoceptor agonists (beta(2)-agonists) with inhaled steroids has become the standard treatment for mild to moderate asthma. Theophylline has also been combined successfully with inhaled steroids. However, the possible interaction between theophylline and beta(2)-agonists, with regard to their anti-inflammatory effects, has not been clarified. The aim of this study was to investigate the in vitro interaction between theophylline and salbutamol on cytokine generation from human monocytes and compare it with a similar interaction between dexamethasone and salbutamol. Purified monocytes from normal donors were pretreated with the drugs (alone or in combination) and stimulated with lipopolysaccharide for 24 h. Released tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), and their corresponding mRNA expressions, were determined and analyzed. Salbutamol (>or= 0.1 microM) significantly inhibited the release of TNF-alpha, but also significantly enhanced that of IL-6. In contrast, theophylline (50 microM) and dexamethasone (0.1 microM) strongly inhibited the generation of both cytokines. It is noteworthy that when the drugs were used in combination the effects of theophylline and salbutamol were additive in inhibiting TNF-alpha release, but theophylline blocked the IL-6-enhancing effect of salbutamol. A similar effect was seen when dexamethasone was combined with salbutamol. These results show that beta(2)-agonists have opposing effects on the generation of TNF-alpha and IL-6, but that when they were combined with clinically relevant concentrations of theophylline, theophylline, like dexamethasone, was capable of augmenting the anti-inflammatory effects of the beta(2)-agonists while at the same time preventing their proinflammatory effect. Thus, theophylline may have a potentially useful steroid-sparing effect.

Ang-(1-7)/ MAS1 receptor axis inhibits allergic airway inflammation via blockade of Src-mediated EGFR transactivation in a murine model of asthma.

The angiotensin-(1-7) [Ang-(1-7)]/MAS1 receptor signaling axis is a key endogenous anti-inflammatory signaling pathway. However, the mechanisms by which its mediates the anti-inflammatory effects are not completely understood. Using an allergic murine model of asthma, we investigated whether Ang-1(1-7)/MAS1 receptor axis a): inhibits allergic inflammation via modulation of Src-dependent transactivation of the epidermal growth factor receptor (EGFR) and downstream signaling effectors such as ERK1/2, and b): directly inhibits neutrophil and/or eosinophil chemotaxis ex vivo. Ovalbumin (OVA)-induced allergic inflammation resulted in increased phosphorylation of Src kinase, EGFR, and ERK1/2. In addition, OVA challenge increased airway cellular influx, perivascular and peribronchial inflammation, fibrosis, goblet cell hyper/metaplasia and airway hyperresponsiveness (AHR). Treatment with Ang-(1-7) inhibited phosphorylation of Src kinase, EGFR, ERK1/2, the cellular and histopathological changes and AHR. Ang-(1-7) treatment also inhibited neutrophil and eosinophil chemotaxis ex vivo. These changes were reversed following pre-treatment with A779. These data show that the anti-inflammatory actions of Ang-(1-7)/ MAS1 receptor axis are mediated, at least in part, via inhibition of Src-dependent transactivation of EGFR and downstream signaling molecules such as ERK1/2. This study therefore shows that inhibition of the Src/EGRF/ERK1/2 dependent signaling pathway is one of the mechanisms by which the Ang-(1-7)/ MAS1 receptor axis mediates it anti-inflammatory effects in diseases such as asthma.

Protein kinase C- and reactive oxygen species-dependent stimulation of intracellular cAMP in human eosinophils. The role of extracellular signal-regulated protein kinases.

OBJECTIVE: The objective of this study was to investigate the role of the extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the signalling pathway of the novel protein kinase C (PKC)- and reactive oxygen species (ROS)-dependent stimulation of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) production in human eosinophils. MATERIALS AND METHODS: Immunomagnetically purified human eosinophils were stimulated in vitro with a PKC activator, phorbol myristate acetate (PMA), and the cAMP response in the presence of a phosphodiesterase inhibitor, rolipram, was determined. The role of ERK1/2 phosphorylation was investigated using specific inhibitors and Western blot analysis. RESULTS: The PMA-stimulated eosinophils responded with a profound increase in intracellular levels of cAMP that was dependent on both PKC and ROS, as confirmed by the use of specific inhibitors: Ro 31-8220 for PKC and diphenyleneiodonium (DPI) for the ROS-generating enzyme NADPH oxidase. Pre-treatment of cells with the ERK1/2 inhibitor PD 98059, but not the p38-MAPK inhibitor SB203580, nor the PI3 kinase inhibitor, wortmannin, abolished the response. PMA treatment induced the phosphorylation of ERK1/2 with a time course that is consistent with a role in the cAMP response. The ERK1/2 phosphorylation was abolished by the ERK1/2 inhibitor PD 98059 and the PKC inhibitor Ro 31-8220, but not the NADPH oxidase inhibitor DPI. CONCLUSION: These results reveal the involvement of ERK1/2 in the signalling mechanism of PMA-stimulated, PKC- and ROS-dependent stimulation of cAMP production in human eosinophils, and show that ERK1/2 phosphorylation is upstream of ROS production in the signalling pathway.

Reactive oxygen species mediate phorbol ester-stimulated cAMP response in human eosinophils.

Recently, we showed that phorbol 12-myristate 13-acetate (PMA) can cause a direct, PKC-dependent, stimulation of intracellular cAMP in human eosinophils. Since PMA also stimulates the release of reactive oxygen species in these cells, we have investigated whether reactive oxygen species are involved in the cAMP response. Provided eosinophils were incubated for <20 min at 37 degrees C before stimulation, PMA potently stimulated cAMP generation that surpassed that of histamine. Pre-treatment of the cells with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI) and apocynin, strongly inhibited the cAMP production induced by PMA, but not that induced by histamine. This treatment also strongly inhibited the release of superoxide anions (O(2)(-)). The cAMP response was also inhibited by pre-treatment with the specific peroxide scavenger, ebselen, but not superoxide dismutase, or NG-nitro-l-arginine methyl ester (L-NAME), thus, suggesting the possible involvement of a peroxide rather than O(2)(-) or nitric oxide (NO). These results reveal a novel involvement of intracellular reactive oxygen species in protein kinase C (PKC)-dependent stimulation of cAMP production in human eosinophils.

Effect of cyclosporin and tacrolimus (FK506) on the antigen-induced mediator release, membrane potential and 86Rb+/K+ and Ca2+ fluxes in the RBL-2H3 cell line.

The immunosuppressants cyclosporin A (CsA) and tacrolimus (FK506) inhibit the activation by antigen of T-lymphocytes as well as mast cells. The mechanism of their action on mast cells has yet to be elucidated. We, therefore, assessed their effect on antigen-induced histamine and beta-hexosaminidase release, membrane potential changes (bis-oxonol fluorescent probe), 86RB+ (marker for K+)-efflux, the intracellular free calcium concentration ([Ca2+]i in single cells) and 45Ca2+ uptake (CsA only) in RBL-2H3 cells, a mucosal-type mast cell line, passively sensitized with monoclonal mouse IgE antibody. Antigen addition induced depolarization within 1-2 min, followed by slower repolarization, reaching a steady state (approximately 90% repolarization) after 7-9 min. CsA and FK506 each dose-dependently inhibited antigen-induced histamine and beta-hexosaminidase secretion and the membrane repolarization phase, with similar IC50s for both actions, approximately 20 nM for CsA and approximately 2 nM for FK506. Antigen-induced 86Rb+-efflux was also significantly inhibited. Antigen-evoked increase in [Ca2+]i (area under the curve, AUC) was reduced by 35% and 52% in the presence of CsA or FK506 (1 microM each), respectively. However, 45Ca2+-uptake was not inhibited by CsA. These results suggest that both CsA and FK506 may inhibit mediator release from mast cells via blocking two interrelated processes, which are involved in the secretory process: 1. Membrane repolarization phase, which is essential for optimal mediator secretion and is mediated by a Ca2+-sensitive K+-efflux, yet to be further characterized, and (2) Increase in [Ca2+]i, probably via reduction of Ca(+2)-release from intracellular stores, [Ca2+]s.

Protein kinase C activation inhibits eosinophil degranulation through stimulation of intracellular cAMP production.

The mechanism of inhibition of eosinophil degranulation by protein kinase C (PKC) was investigated in complement C5a (C5a)-stimulated degranulation of highly purified human eosinophils using the specific PKC activator - phorbol 12-myristate 13-acetate (PMA). C5a-induced release of eosinophil peroxidase and eosinophil cationic protein was potently inhibited in a concentration-dependent manner by PMA (IC(50): 3 and 5 nM, respectively). The inhibition by PMA, but not histamine, was significantly reversed by the specific, but isoform nonselective, PKC inhibitor Ro 31-8220 (1 microM). In the presence of phosphodiesterase inhibitor rolipram (5 microM), PMA stimulated a pronounced concentration-dependent increase in intracellular cAMP, with a potency 400 times that of histamine (EC(50): 55 nM vs 22.5 microM). The inactive PMA analogue, 4alpha-PMA, had no such effect. The cAMP production by PMA, but not histamine, was significantly reversed by Ro 31-8220 (1 microM) and the selective inhibitor of the novel PKCdelta, rottlerin (1-3 microM), but not the selective inhibitor of the classical PKC isoforms, Gö 6976 (0.01-0.1 microM). Western blot analysis revealed the presence of six PKC isoforms (alpha, betaI, betaII, delta, iota and zeta) in isolated eosinophils. Chelation of internal or external calcium had no effect on PMA-induced cAMP response, but abolished that induced by histamine. There was a good correlation between increase in intracellular cAMP and inhibition of degranulation. These results show, for the first time, that in human eosinophils, PMA, via activation of PKCdelta isoform, can stimulate cAMP production, and that this may be the basis for its potent anti-degranulatory effect.

Loss of surface beta-2 adrenoceptors accounts for the insensitivity of cultured human monocytes to beta-2 adrenoceptor agonists.

The short-acting beta-2 adrenoceptor agonists (ß(2)-agonists), such as salbutamol, are effective bronchodilators used to treat asthma. They lack significant anti-inflammatory effect in vivo as well as on isolated alveolar macrophages even though they exhibit this effect on freshly isolated monocytes. The purpose of this study was to determine if this observation is related to a change in the expression and/or function of surface ß(2)-receptors during the differentiation of these cells to macrophages. Purified monocytes, cultured for 1-48 h were pre-treated with the ß(2)-agonists (salbutamol or procaterol) or PGE(2) before being stimulated with bacterial lipopolysaccharide (LPS). Subsequently, the amount of TNF-α (a typical inflammatory mediator) released over 24 h, as well as agonist-stimulated cAMP, were determined by enzyme immunoassays. Western blotting techniques were used to study the expression of the membrane ß(2)-receptor protein. Results showed that in freshly isolated human monocytes, both the ß(2)-agonists and PGE(2) significantly inhibited LPS-induced TNF-α release as well as increased intracellular cAMP. After culturing adherent monocytes for 24-48 h, the ability of the ß(2)-agonists to produce both effects was completely lost, whereas that of PGE(2) was essentially intact. Western blotting data showed a near complete loss of surface expression of ß(2)-receptors in cells cultured for ≥24 h. These results show that as human monocytes adhere to surfaces to begin differentiation into macrophages, they selectively lose their surface ß(2)-receptors and hence become insensitive to the anti-inflammatory effect of ß(2)-agonists. This may explain why ß(2)-agonists lack significant anti-inflammatory effect on alveolar macrophages or in clinical asthma.

Anti-tussive and bronchodilator mechanisms of action for the enaminone E121.

AIMS: In this study, we investigated whether the enaminone, E121, has anti-tussive effects in a guinea pig model of cough, and if so, whether this effect is mediated via a central or peripheral site of action. We also assessed whether E121 has bronchodilator effects and the molecular mechanisms underlying any anti-tussive and/or bronchodilator effects. MAIN METHODS: Whole body plethysmography was used to assess both cough and airway obstruction. A stereotaxic apparatus was used to administer drugs intracerebroventricularly (i.c.v.). Effects of E121 were examined in vitro on contractile effects in guinea pig bronchioles. KEY FINDINGS: Pre-treatment of animals with E121 resulted in a significant inhibition in the citric acid-induced cough and airway obstruction compared to vehicle-pretreated animals. The K(ATP) antagonist, glibenclamide, significantly inhibited the anti-tussive and bronchoprotective effects of E121. Also, intra-tracheal administration of E121 resulted in a significant inhibition of both the citric acid-induced cough response and airway obstruction compared to vehicle-pretreated animals. By contrast, i.c.v. administration had no effect. Finally, E121 significantly inhibited carbachol-induced airway smooth muscle contractions, an effect that was reduced by both glibenclamide and propranolol. Interestingly, E121 enhanced histamine-induced cAMP release in human eosinophils although it did not directly elevate cAMP levels. SIGNIFICANCE: The enaminone, E121, has anti-tussive and bronchodilatory effects and is topically, but not centrally, active. The anti-tussive mechanism of action of E121 seems to be K(ATP) channel dependent, whereas its bronchodilatory effects appear to be mediated via activation of both K(ATP) channels and ß(2) receptors. Therefore, E121 may potentially represent a novel therapy for cough, particularly cough associated with airway obstruction.

In vitro and in vivo anti-inflammatory effects of andrographolide.

Andrographolide - the major active principle isolated from the plant Andrographis paniculata, has been shown to possess a strong anti-inflammatory activity. The possibility that the drug may affect asthmatic inflammation, through inhibition of the relevant inflammatory cytokines, has not been explored. The purpose of this study was, firstly, to investigate the ability of andrographolide to inhibit the release of inflammatory cytokines in vitro in a model of non-specific inflammation and subsequently to determine whether such effect can also be exerted in vivo in allergic lung inflammation. LPS-induced TNF-alpha and GM-CSF release from mouse peritoneal macrophages was inhibited by andrographolide in a concentration-dependent manner. The concentration of the drug producing 50% inhibition was 0.6 microM for TNF-alpha and 3.3 microM for GM-CSF. The maximal inhibition achieved (at 50 microM) was 77% and 94%, respectively, for the two cytokines. The drug was as efficacious as dexamethasone, but about 8-12 times less potent. The drug also suppressed LPS-induced expression of mRNA for the two cytokines, suggesting that this effect may contribute to the mechanism underlying its anti-inflammatory effects. In the in vivo study, intra-peritoneal treatment of ovalbumin-immunized and nasally-challenged mice with andrographolide significantly inhibited the elevation of bronchoalveolar fluid (BAF) levels of TNF-alpha and GM-CSF in a dose-dependent manner, with 30 mg/kg producing an inhibition of 92% and 65% of the cytokines, respectively) and almost completely abolishing the accumulation of lymphocytes and eosinophils. These results provide evidence that andrographolide is an effective anti-inflammatory drug that is active in vitro and in vivo, and affects both non-specific as well as antigen/antibody-dependent lung inflammation. Thus, andrographolide has the potential to be used in a variety of inflammatory conditions, including allergic lung inflammation.

Low-affinity IgE receptor (FcepsilonRII)-mediated activation of human monocytes by both monomeric IgE and IgE/anti-IgE immune complex.

Monocytes and macrophages of individuals with allergic diseases express increased levels of the low-affinity IgE receptors (FcepsilonRII or CD23) on their surfaces. The cross-linking of CD23-bound IgE antibody by allergen activates the cells to release inflammatory mediators. In mast cells, the binding of IgE to the high-affinity IgE receptors (FcepsilonRI) has recently been shown to activate these cells independent of allergen. It has not been determined if such is true of the binding of IgE to the low-affinity receptors. The purpose of this study was, therefore, to determine whether monomeric IgE alone can activate CD23-bearing human monocytes and how this may relate to the activation by IgE/anti-IgE immune complex. Purified monocytes, cultured for 48 h with IL-4 to up-regulate CD23 were sensitized with human myeloma IgE and further cultured for 24 h with or without anti-human IgE antibody. The release of cytokines TNF-alpha and MIP-1alpha (as an index of activation) was determined by enzyme immunoassay. Results showed that in IL-4-treated/CD23-bearing monocytes, sensitization with IgE alone caused a release of TNF-alpha and MIP-1alpha. The addition of anti-IgE antibody to cross-link the bound IgE resulted in the enhancement of the response. Such activation by monomeric IgE and IgE/anti-IgE immune complex was blocked with an anti-CD23 antibody, confirming the specific involvement of CD23 molecules. Neither of the activation modalities elevated intracellular cAMP, contrary to previous report. These results show for the first time, that in CD23-bearing monocytes, IgE sensitization alone can activate monocytes, and that ligation of such IgE by anti-IgE antibody only enhances the response. These observations have implications for the understanding of the pathophysiology of IgE-dependent inflammation accompanying many allergic diseases.

Positive coupling of atypical adenosine A3 receptors on human eosinophils to adenylyl cyclase.

Adenosine A(3) receptors are reported to couple negatively to adenylyl cyclase (AC) but their mediation of anti-inflammatory effects in human eosinophils prompted us to investigate their coupling to AC. The A(3)-selective agonists IB-MECA and Cl-IB-MECA evoked a concentration-dependent generation of cAMP (EC(50), 3.2 and 1.8 microM, respectively) and were more potent than the A(2A) agonist CGS 21680 (EC(50)=15.4 microM) and adenosine (EC(50)=19.2 microM). The cAMP response was additive to that produced by forskolin (10 microM). The effect of IB-MECA was insensitive to A(1) and A(2A) receptor antagonists, but was antagonized by the A(3)-selective antagonist MRS 1220 (0.1-2.5 microM) in a competitive manner. The estimated K(B) of 190 nM was, however, atypical. The cyclo-oxygenase inhibitor, indomethacin, had no effect on the cAMP response. A general inverse relationship between cAMP generation and inhibition of degranulation was seen. We conclude that in human eosinophils, an atypical form of A(3) receptors positively coupled to AC may exist. The resulting cAMP generation may underlie the anti-inflammatory actions of A(3) agonists in eosinophils.