Identification of a selective nonpeptide antagonist of the anaphylatoxin C3a receptor that demonstrates antiinflammatory activity in animal models.

The anaphylatoxin C3a is a potent chemotactic peptide and inflammatory mediator released during complement activation which binds to and activates a G-protein-coupled receptor. Molecular cloning of the C3aR has facilitated studies to identify nonpeptide antagonists of the C3aR. A chemical lead that selectively inhibited the C3aR in a high throughput screen was identified and chemically optimized. The resulting antagonist, N(2)-[(2,2-diphenylethoxy)acetyl]-L-arginine (SB 290157), functioned as a competitive antagonist of (125)I-C3a radioligand binding to rat basophilic leukemia (RBL)-2H3 cells expressing the human C3aR (RBL-C3aR), with an IC(50) of 200 nM. SB 290157 was a functional antagonist, blocking C3a-induced C3aR internalization in a concentration-dependent manner and C3a-induced Ca(2+) mobilization in RBL-C3aR cells and human neutrophils with IC(50)s of 27.7 and 28 nM, respectively. SB 290157 was selective for the C3aR in that it did not antagonize the C5aR or six other chemotactic G protein-coupled receptors. Functional antagonism was not solely limited to the human C3aR; SB 290157 also inhibited C3a-induced Ca(2+) mobilization of RBL-2H3 cells expressing the mouse and guinea pig C3aRS: It potently inhibited C3a-mediated ATP release from guinea pig platelets and inhibited C3a-induced potentiation of the contractile response to field stimulation of perfused rat caudal artery. Furthermore, in animal models, SB 290157, inhibited neutrophil recruitment in a guinea pig LPS-induced airway neutrophilia model and decreased paw edema in a rat adjuvant-induced arthritis model. This selective antagonist may be useful to define the physiological and pathophysiological roles of the C3aR.

SB 239063, a p38 MAPK inhibitor, reduces neutrophilia, inflammatory cytokines, MMP-9, and fibrosis in lung.

The effects of a second generation p38 mitogen-activated protein kinase (MAPK) inhibitor, SB 239063 [trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridim idi n-4-yl)imidazole; IC(50) = 44 nM vs. p38 alpha], were assessed in models that represent different pathological aspects of chronic obstructive pulmonary disease (COPD) [airway neutrophilia, enhanced cytokine formation and increased matrix metalloproteinase (MMP)-9 activity] and in a model of lung fibrosis. Airway neutrophil infiltration and interleukin (IL)-6 levels, assessed by bronchoalveolar lavage 48 h after lipopolysaccharide (LPS) inhalation, were inhibited dose dependently by 3-30 mg/kg of SB 239063 given orally twice a day. In addition, SB 239063 (30 mg/kg orally) attenuated IL-6 bronchoalveolar lavage fluid concentrations (>90% inhibition) and MMP-9 activity (64% inhibition) assessed 6 h after LPS exposure. In guinea pig cultured alveolar macrophages, SB 239063 inhibited LPS-induced IL-6 production (IC(50) of 362 nM). In a bleomycin-induced pulmonary fibrosis model in rats, treatment with SB 239063 (2.4 or 4.8 mg/day via osmotic pump) significantly inhibited bleomycin-induced right ventricular hypertrophy (indicative of secondary pulmonary hypertension) and increases in lung hydroxyproline synthesis (indicative of collagen synthesis and fibrosis). Therefore, SB 239063 demonstrates activity against a range of sequelae commonly associated with COPD and fibrosis, supporting the therapeutic potential of p38 MAPK inhibitors such as SB 239063 in chronic airway disease.

Inhibition of p38 MAP kinase as a therapeutic strategy.

Since the discovery of p38 MAP kinase in 1994, our understanding of its biology has progressed dramatically. The key advances include (1) identification of p38 MAP kinase homologs and protein kinases that act upstream and downstream from p38 MAP kinase, (2) identification of interesting and potentially important substrates, (3) elucidation of the role of p38 MAP kinase in cellular processes and (4) the establishment of the mechanism by which the pyridinylimidazole p38 MAP kinase inhibitors inhibit enzyme activity. It is now known that there are four members of the p38 MAP kinase family. They differ in their tissue distribution, regulation of kinase activation and subsequent phosphorylation of downstream substrates. They also differ in terms of their sensitivities toward the p38 MAP kinase inhibitors. The best-studied isoform is p38 alpha, whose activation has been observed in many hematopoietic and non-hematopoietic cell types upon treatment with appropriate stimuli. The pyridinylimidazole compounds, exemplified by SB 203580, were originally prepared as inflammatory cytokine synthesis inhibitors that subsequently were found to be selective inhibitors of p38 MAP kinase. SB 203580 inhibits the catalytic activity of p38 MAP kinase by competitive binding in the ATP pocket. X-ray crystallographic studies of the target enzyme complexed with inhibitor reinforce the observations made from site-directed mutagenesis studies, thereby providing a molecular basis for understanding the kinase selectivity of these inhibitors. The p38 MAP kinase inhibitors are efficacious in several disease models, including inflammation, arthritis and other joint diseases, septic shock, and myocardial injury. In all cases, p38 activation in key cell types correlated with disease initiation and progression. Treatment with p38 MAP kinase inhibitors attenuated both p38 activation and disease severity. Structurally diverse p38 MAP kinase inhibitors have been tested extensively in preclinical studies.

SB 239063, a potent p38 MAP kinase inhibitor, reduces inflammatory cytokine production, airways eosinophil infiltration, and persistence.

The anti-inflammatory/antiallergic activity of a novel second-generation p38 mitogen-activated protein kinase inhibitor, SB 239063[trans-1-(4-hydroxycyclohexyl) -4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)imidazole], was investigated in vivo and in vitro. SB 239063 had an IC(50) of 44 nM for inhibition of recombinant purified human p38alpha. In lipopolysaccharide-stimulated human peripheral blood monocytes, SB 239063 inhibited interleukin-1 and tumor necrosis factor-alpha production (IC(50) values = 0.12 and 0.35 microM, respectively). A role for p38 kinase in cytokine-associated inflammation in the mouse was shown by p38 activation in the lung and inhibition of lipopolysaccharide-induced tumor necrosis factor-alpha production by SB 239063 (ED(50) = 5.8 mg/kg p.o.). Antiallergic activity was demonstrated by essential abolition (approximately 93% inhibition) of inhaled ovalbumin (OA)-induced airway eosinophilia by SB 239063 (12 mg/kg p.o.), measured by bronchoalveolar lavage (BAL) in OA-sensitized mice. In addition, p38 kinase was found by Western analysis to be activated in guinea pig lung. Administration of SB 239063 (10 or 30 mg/kg p.o.) in conscious guinea pigs markedly reduced ( approximately 50% inhibition) OA-induced pulmonary eosinophil influx, measured by BAL 24 h after antigen. SB 239063 (10 mg/kg b.i.d. p.o.) administered after leukotriene D(4) inhalation, reduced by 60% the persistent airway eosinophilia seen at 4 days. Apoptosis of cultured eosinophils isolated from guinea pig BAL was increased by SB 239063 (1-10 microM) in the presence of interleukin-5. These results indicate that SB 239063 is a potent inhibitor of inflammatory cytokine production, inhibits eosinophil recruitment, in addition to enhancing apoptosis of these cells. Collectively, the results support the potential utility of p38 kinase inhibitors, such as SB 239063, for the treatment of asthma and other inflammatory disorders.

1,4-Cyclohexanecarboxylates: potent and selective inhibitors of phosophodiesterase 4 for the treatment of asthma.

Evaluation of a variety of PDE4 inhibitors in a series of cellular and in vivo assays suggested a strategy to improve the therapeutic index of PDE4 inhibitors by increasing their selectivity for the ability to inhibit PDE4 catalytic activity versus the ability to compete for high affinity [3H]rolipram-binding sites in the central nervous system. Use of this strategy led ultimately to the identification of cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxyl ic acid (1, SB 207499, Ariflo), a potent second-generation inhibitor of PDE4 with a decreased potential for side effects versus the archetypic first generation inhibitor, (R)-rolipram.

Persistent airway eosinophilia after leukotriene (LT) D4 administration in the guinea pig: modulation by the LTD4 receptor antagonist, pranlukast, or an interleukin-5 monoclonal antibody.

Aerosolized cysteinyl leukotrienes (CysLTs) elicit migration of eosinophils into guinea pig lungs and the airways of patients with asthma. The present studies were designed to analyze the concentration-response relationship, time course, and pharmacologic and histologic characteristics of leukotriene D4 (LTD4)-induced eosinophil influx into the airways of conscious guinea pigs. Animals were exposed to aerosols of 0.3 to 30 microg/ml LTD4 for 1 min, during which specific airway conductance (sGaw) was monitored. Bronchoalveolar lavages (BALs) of guinea pig airways were conducted at selected times from 4 h to 4 wk after LTD4 challenge. LTD4 produced maximal decreases in sGaw (70 to 90% reduction) at all concentrations tested and concentration-related increases in eosinophil levels in BALs, assessed 24 h after challenge. Increased numbers of eosinophils in the bronchial epithelium and subepithelium were confirmed histologically. Significant eosinophilia was maintained for up to 4 wk postchallenge. Pretreatment with the LTD4 receptor antagonist, pranlukast (ONO-1078, SB 205312) (20 mg/kg, intragastrically), significantly inhibited both the bronchoconstriction and the eosinophilia at 24 h, whereas the cyclooxygenase inhibitor, meclofenamic acid (5 mg/kg, intragastrically), had no effect on either parameter. Histologic observations were consistent with BAL results. Pretreatment with the rat anti-mouse antibody to interleukin-5 (IL-5), TRFK-5 (10-300 microg, intraperitoneally), produced dose-related inhibition of LTD4-induced eosinophilia, measured in 24 h or 3 wk BAL, but did not affect the acute bronchoconstriction. These results indicate that LTD4 elicits airway eosinophil influx in guinea pigs which persists as long as 4 wk after a single exposure, and provide the first evidence that IL-5 may have a role in LTD4-induced airways inflammation. This and other previously reported proinflammatory effects of LTD4 may contribute significantly to its overall influential role in the pathophysiology of asthma, and may underlie the therapeutic benefit of CysLT receptor antagonists, such as pranlukast, in this disorder.

Pharmacological characterization of SB 202235, a potent and selective 5-lipoxygenase inhibitor: effects in models of allergic asthma.

The peptidoleukotrienes and leukotriene B4, formed from arachidonic acid through the action of 5-lipoxygenase (5-LO), exert a spectrum of biological effects. It has been proposed that potent and selective 5-LO inhibitors will be effective therapy in diseases in which the peptidoleukotrienes and leukotriene B4 have been implicated, such as asthma and arthritis. The novel compound (S)-N-hydroxy-N-(2,3-dihydro-6-phenylmethoxy-3-benzyofuranyl )urea (SB 202235) was evaluated as a selective inhibitor of 5-LO in a cell-free system as well as in various cellular assays. In addition, the potential therapeutic value of SB 202235 was assessed in preclinical models of allergic asthma. The activity of the 5-LO enzyme isolated from rat basophilic leukemia-1 cells was inhibited by SB 202235 in a concentration-dependent manner with an IC50 value of 1.9 microM. Consistent with its ability to inhibit 5-LO, SB 202235 inhibited the production of leukotriene B4 by human monocytes and in human whole blood (IC50 values of 1.5 microM and 1.1 microM, respectively). The selectivity of SB 202235 was confirmed by its lack of effect against several other enzymes and receptors. SB 202235 potently and effectively inhibited the contraction produced by a single concentration of ovalbumin in guinea pig trachea (IC50 = 20 microM) and of anti-IgE in human bronchus (IC50 = 2 microM). SB 202235 (3-30 microM) also inhibited the contraction of guinea pig trachea in response to increasing concentration of ovalbumin. When administered orally (30 mg/kg) to conscious guinea pigs, SB 202235 attenuated antigen-induced broncho-constriction and the subsequent eosinophil influx.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphodiesterase IV inhibitors as therapy for eosinophil-induced lung injury in asthma.

Asthma is a complex, multifactorial disease that is underpinned by airway inflammation. A variety of cytotoxic substances are released into the airway from infiltrating inflammatory cells, especially the eosinophil. These cytotoxic substances, including reactive oxygen metabolites, produce damage to the airway epithelium, a histologic feature of chronic asthma. Damage to the airway epithelium, in turn, is thought to be a major factor responsible for the development of airway hyperreactivity, a hallmark of asthma. One notable molecular target for novel antiasthmatic drugs is the cyclic AMP-specific phosphodiesterase (PDE) or PDE IV. This isozyme is the predominant form of cyclic nucleotide PDE activity in inflammatory cells. Thus, in view of the putative role of cyclic AMP as an inhibitory second messenger in these cells, PDE IV inhibitors have been shown to suppress inflammatory cell activity. The purpose of the present experiments was to examine the effect of the PDE IV inhibitor, R-rolipram, on three key functions of the guinea pig eosinophil: a) superoxide anion (O2-) production, b) adhesion to human umbilical vein endothelial cells (HUVECs), and c) infiltration into the airway. R-rolipram-elevated eosinophil cyclic AMP content (EC50 = 1.7 microM) and inhibited fMLP-induced O2- production in a concentration-dependent manner (IC50 = 0.3 microM). In contrast, neither siguazodan, a PDE III inhibitor, nor zaprinast, a PDE V inhibitor, had an appreciable effect. R-rolipram (30 microM) also reduced by 25 to 40% the adhesion of eosinophils to HUVECs stimulated with phorbol myristate acetate or tumor necrosis factor-alpha, particularly under conditions in which both cell types were simultaneously exposed to the PDE IV inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of antigen-induced bronchoconstriction and eosinophil infiltration in the guinea pig by the cyclic AMP-specific phosphodiesterase inhibitor, rolipram.

Selective inhibition of the low Km cyclic AMP-specific phosphodiesterase has been shown to inhibit inflammatory cell function and relax airway smooth muscle. These studies were conducted to characterize the bronchodilator and anti-inflammatory activity of rolipram, an archetypical cyclic AMP-specific phosphodiesterase inhibitor, in in vitro and in vivo guinea pig airway models. In isolated tracheal rings from ovalbumin (OA)-sensitive guinea pigs, both R- and S-enantiomers of rolipram (1 microM) significantly antagonized OA-induced contractions. In contrast, neither enantiomer at concentrations up to 1 microM significantly inhibited histamine- or LTD4-induced contractions. In superfusion and mediator release experiments, both enantiomers of rolipram significantly reduced antigen-induced prostaglandin D2 release, but had minimal effect on histamine release. In anesthetized, ventilated OA-sensitive guinea pigs, racemic rolipram or enantiomers reduced OA-induced bronchoconstriction with ID50 values of approximately 0.25 mg/kg i.v. Histamine- and leukotriene D4-induced bronchoconstriction were not affected by doses of rolipram which abolished the response to OA. Higher doses (3-10 mg/kg) reduced histamine-, but not the leukotriene D4-induced bronchoconstriction. In conscious OA-sensitive guinea pigs, intragastric pretreatment with rolipram dose-dependently reduced both the OA-induced decreases in specific conductance as well as the corresponding pulmonary eosinophil influx as assessed by both bronchoalveolar lavage and histological evaluation. Therefore, rolipram produces significant inhibition of antigen-induced bronchoconstrictor and inflammatory responses, thus providing strong evidence that this pharmacological approach may be of significant therapeutic value in allergic asthma.(ABSTRACT TRUNCATED AT 250 WORDS)