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.

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.

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)

2-nor-leukotriene analogs: antagonists of the airway and vascular smooth muscle effects of leukotriene C4, D4 and E4.

A structural analog of LTD4, 4R-hydroxy-5S-cysteinylglycyl-6Z-nonadecenoic acid (4R, 5S, 6Z-2-nor-LTD1) has been synthesized and pharmacologically characterized. It significantly antagonized the contractile action of LTD4, LTC4 and LTE4 in guinea pig airways. In addition, this compound antagonized the in vitro vasoconstrictive effects of LTD4 in the guinea pig pulmonary artery. The study of a series of structural analogs of 4R, 5S, 6Z-2-nor-LTD1 suggests that the spatial separation of the C-1 (eicosanoid) carboxyl relative to the hydroxyl is a critical determinant in LTD4 agonist/antagonist activity.

Antagonism of the pulmonary effects of the peptidoleukotrienes by a leukotriene D4 analog.

4R-hydroxy-5S-cysteinylglycine-6-Z-nonadecenoic acid (4R,5S, 6Z-2-nor-LTD1), a structural analog of leukotriene (LT) D4 (LTD4), significantly antagonized the pulmonary actions of LTD4 in several guinea pig models of LT-mediated bronchoconstriction and edema formation. In vitro, 4R,5S,6Z-2-nor-LTD1 (10(-5) and 10(-4) M) antagonized the LTD4-induced contraction of tracheal spirals and lung parenchymal strips. This antagonist action of 4R,5S,6Z-2-nor-LTD1 was specific for the LTs, in that LTC4- and LTE4-induced contractions of the trachea were also antagonized, whereas the contractions elicited by other spasmogens, e.g., histamine, carbachol, prostaglandin F2 alpha and KCl, were not antagonized. In vivo, the LTD4-induced bronchoconstriction in anesthetized, spontaneously breathing guinea pigs as reflected by decreases in dynamic lung compliance and airway conductance were attenuated significantly by a 1-min pretreatment with 4R,5S,6Z-2-nor-LTD1 at 5 mg/kg i.v. Similarly, the LTD4-induced increase in tracheal microvascular permeability, as assessed by extravasation of [125I]bovine serum albumin, was blocked by pretreatment with 4R,5S,6Z-2-nor-LTD1. These results provide the first demonstration that a structural analog of the peptidoleukotrienes can pharmacologically antagonize the potent actions of these LTs.