Pyrazinoindolone inhibitors of MAPKAP-K2.

Optimization of pyrazinoindolone inhibitors of MAPKAP-K2 (MK2) provides a reasonable balance of cellular potency and physicochemical properties. Mechanistic studies support the inhibition of MK2 which is responsible for the sub-micromolar cellular efficacy.

Reduction of antigen-induced airway hyperreactivity and eosinophilia in ICAM-1-deficient mice.

A murine model of asthma is described in which we examined the role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of airway reactivity, pulmonary eosinophilia, and inflammation. We sensitized wild-type control [C57BL/6J, (+/+)] and ICAM-1 knockout [C57BL/6J-ICAM-1, (-/-)] mice to ovalbumin (OVA), and challenged them with OVA delivered by aerosol (OVA-OVA) to induce a phenotype consistent with an asthmatic response. Bronchial responsiveness to methacholine and counts of cell numbers and measurements of eosinophil content and cytokine levels in bronchoalveolar lavage fluid (BALF) were significantly attenuated in ICAM-1(-/-) mice as compared with (+/+) mice. We also showed that the absence of ICAM-1 had no significant affects on the production of serum IgE antibody, but did have an effect on ex vivo lymphocyte proliferation. Additionally, immunohistochemistry: (1) revealed increased staining for vascular cell adhesion molecule-1 (VCAM-1) after antigen challenge in the ICAM-1(-/-) mice but not in the ICAM-1(+/+) controls; and (2) confirmed the presence of alternatively spliced forms of ICAM-1 in the lungs of ICAM-1(-/-) mice. Thus, despite the availability of alternate adhesion pathways in ICAM-1(-/-) mice, the absence of ICAM-1 prevented eosinophils from entering the airways. In summary, we found that the ICAM-1 knockout mice exhibited a significantly inhibited response to aerosol antigen challenge for most of the parameters examined, and conclude that ICAM-1 is an important ligand mediating T-cell proliferation in response to antigen, eosinophil migration into the airways, and the development of airway hyperreactivity (AHR) in allergen-sensitized and -challenged mice.

The role of 5-lipoxygenase products in preclinical models of asthma.

BACKGROUND: The action of 5-lipoxygenase on arachidonic acid generates potent inflammatory mediators that may contribute to the pathophysiology of asthma. METHODS: Using the potent and selective 5-lipoxygenase inhibitor BI-L-239, we have examined the role of 5-lipoxygenase products in three animal models of asthma. RESULTS: In vitro BI-L-239 inhibited 5-lipoxygenase product generation from human lung mast cells, alveolar macrophages, and peripheral blood leukocytes with a concentration that would provide 50% inhibition values of 28 to 340 nmol/L. A 36-fold selectivity for immunoreactive leukotriene C4 versus immunoreactive prostaglandin D2 inhibition was demonstrated in mast cells. In anesthetized cynomolgus monkeys, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced immunoreactive leukotriene C4 release (maximum, 73%; bronchoalveolar lavage [BAL], 20 minutes), late-phase bronchoconstriction (maximum, 41%; +6 to 8 hours), and neutrophil infiltration (maximum, 63%; BAL, +8 hours). In conscious sheep, inhaled BI-L-239 provided dose-dependent inhibition of the inhaled Ascaris-induced late-phase bronchoconstriction (maximum, 66%; +6 to 8 hours) and increase in airway responsiveness (maximum, 82%; carbachol, +24 hours). The acute bronchoconstriction was shortened, and neutrophil infiltration diminished (maximum, 61%; BAL, +8 hours) in this model. Finally in conscious actively sensitized guinea pigs pretreated with pyrilamine and indomethacin, inhaled BI-L-239 attenuated acute bronchoconstriction (maximum, 80%; +5 to 15 minutes), leukocyte infiltration (58%; BAL, +3 days) and increase in airway responsiveness (100%; methacholine, +3 days) induced by three alternate-day ovalbumin inhalations. CONCLUSIONS: In conclusion, results in these three animal models indicate that 5-lipoxygenase products may be major contributors to the bronchoconstriction (especially late phase), leukocyte infiltration, and airway hyperresponsiveness that characterize asthma.

The role of intercellular adhesion molecule-1 in chronic airway inflammation.

We have examined the role of intercellular adhesion molecule-1 (ICAM-1) in chronic airway inflammation and airway hyperresponsiveness in a primate model of asthma. Airway cellular composition was assessed by bronchoalveolar lavage (BAL) and airway responsiveness was measured as the bronchoconstrictor response to inhaled methacholine. In animals with chronic airway inflammation (increased BAL eosinophils) and sustained airway hyperresponsiveness, a 7 day dosing scheme with a murine anti-human ICAM-1 monoclonal antibody (R6.5, 2 mg/kg/day; i.v.) did not reduce the existing airway inflammation or airway hyperresponsiveness. In contrast, a similar dosing scheme with dexamethasone (0.2 mg/kg/day, i.m.) was found to significantly reduce both the airway eosinophilia and hyperresponsiveness. However, one week after cessation of dexamethasone treatment, the airway inflammation and hyperresponsiveness returned to pre-treatment levels. In further experiments where animals were first treated with dexamethasone (7 days) followed by a 7 day treatment with R6.5, the reoccurrence of airway inflammation and subsequent increase in airway responsiveness was prevented. We conclude that the efficacy of ICAM-1 is primarily associated with inhibition of the influx of inflammatory cells into the airways and subsequent reduction in airway responsiveness. These data suggest that in lungs with pre-existing inflammation the modulation of ICAM-1 following treatment with glucocorticoids may be a novel and more selective long-term treatment for control of the chronic airway inflammation and hyperresponsiveness associated with bronchial asthma.

Effects of single and multiple inhalations of platelet-activating factor on airway cell composition and responsiveness in monkeys.

Platelet-activating factor (PAF) is a potent pro-inflammatory mediator that may play a role in the pathogenesis of airway hyper-responsiveness and asthma. In man, a single inhalation of PAF induces a small but prolonged increase in airway responsiveness in some individuals. The purpose of this study was to determine the effects of single and multiple inhalations of PAF on airway cell composition and responsiveness in monkeys. Anaesthetized and intubated adult male cynomolgus monkeys were studied. Airway cell composition was measured by bronchoalveolar lavage (BAL). Airway responsiveness was measured by determining the concentration (PC100) of inhaled methacholine that caused a 100% increase in respiratory system resistance (Rrs). Airway cell composition (BAL) and responsiveness (PC100) were determined 1 day before and 20 hr after a single inhalation of PAF (approximately 200 micrograms) or 3 days before (Day 0) and 3 days after (Day 10) 3-alternate-day (Days 3, 5 and 7) inhalations of PAF (each approximately 600 micrograms). The single inhalation of PAF (n = 8) caused an acute increase in Rrs (147 +/- 69%), an increase in BAL granulocytes, and a decrease in PC100 in four of eight animals that was moderate (greater than eight fold) in only one animal. The mean +/- s.e. change in log PC100 was -0.29 +/- 0.18. The multiple inhalations of PAF (n = 8) caused acute increases in Rrs (143 +/- 38%, 175 +/- 44% and 156 +/- 39%, respectively), an increase in BAL granulocytes, and a decrease in PC100 in four of eight animals that was moderate in two animals. The mean +/- s.e. change in log PC100 was -0.43 +/- 0.22.

Endothelial leukocyte adhesion molecule-1 mediates antigen-induced acute airway inflammation and late-phase airway obstruction in monkeys.

This study examines the role of endothelial leukocyte adhesion molecule-1 (ELAM-1) in the development of the acute airway inflammation (cell influx) and late-phase airway obstruction in a primate model of extrinsic asthma. In animals sensitive to antigen, a single inhalation exposure induced the rapid expression of ELAM-1 (6 h) exclusively on vascular endothelium that correlated with the influx of neutrophils into the lungs and the onset of late-phase airway obstruction. In contrast, basal levels of ICAM-1 was constitutively expressed on vascular endothelium and airway epithelium before antigen challenge. After the single antigen exposure, changes in ICAM-1 expression did not correlate with neutrophil influx or the change in airway caliber. This was confirmed by showing that pretreatment with a monoclonal antibody to ICAM-1 did not inhibit the acute influx of neutrophils associated with late-phase airway obstruction, whereas a monoclonal antibody to ELAM-1 blocked both the influx of neutrophils and the late-phase airway obstruction. This study demonstrates a functional role for ELAM-1 in the development of acute airway inflammation in vivo. We conclude that, in primates, the late-phase response is the result of an ELAM-1 dependent influx of neutrophils. Therefore, the regulation of ELAM-1 expression may provide a novel approach to controlling the acute inflammatory response, and thereby, affecting airway function associated with inflammatory disorders, including asthma.

Antigen-induced mediator release in primates.

We examined the release of bronchoactive mediators into the airways of allergic primates during the acute response to specific antigen inhalation. Twelve adult male cynomolgus monkeys (Macaca fascicularis) with a naturally occurring respiratory sensitivity to inhaled Ascaris suum extract were anesthetized and intubated for each study. Respiratory system resistance (Rrs) and dynamic lung compliance (CLdyn) were measured before and after antigen inhalation, and the release of mediators into the airways was assessed by bronchoalveolar lavage (BAL). BAL samples were concentrated approximately 5-fold before quantitation of LTC4 and PGD2 by RP-HPLC and radioimmunoassay and histamine by a fluorometric assay. Antigen inhalation resulted in a 40-fold increase in BAL levels of i-LTC4 (1.5 +/- 0.7 to 41.6 +/- 12.7 ng, p less than 0.01), a 10-fold increase in i-PGD2 (2.4 +/- 0.9 to 25.9 +/- 5.5 ng, p less than 0.01), and a 20-fold increase in BAL histamine (1.0 +/- 1.5 to 21.4 +/- 2.3 micrograms, p less than 0.01). Dexamethasone (n = 7) inhibited the antigen-induced increase in BAL i-LTC4 (71 +/- 6%, p less than 0.01) and i-PGD2 (52 +/- 8%, p less than 0.05) while weakly inhibiting histamine release (43 +/- 10%). Indomethacin (n = 7) had a variable effect on i-LTC4 levels (6 +/- 51%), strongly inhibited i-PGD2 (88 +/- 9%, p less than 0.01), and had no effect on histamine release (25 +/- 8%). Pretreatment with iodoxamide tromethamine significantly blocked the release of each mediator, but mepyramine, an H1 antagonist, had no effect on mediator release.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of single and multiple inhalations of antigen on airway responsiveness in monkeys.

Airway hyperresponsiveness is an important and characteristic feature of asthma. In monkeys, we have reported that antigen (Ag) inhalation induces a prolonged airway eosinophilia, that chronic airway eosinophilia is associated with marked airway hyperresponsiveness, and that chronic Ag inhalations induce airway eosinophilia and hyperresponsiveness. In this study we have determined the effects of acute Ag inhalation(s) on airway responsiveness to obtain a protocol for the study of the mechanisms involved. Anesthetized and intubated adult male cynomolgus monkeys with a naturally occurring sensitivity to Ascaris suum extract were studied. Airway responsiveness (provocative concentration of nebulized and inhaled methacholine that induced a 100% increase in respiratory system resistance [Rrs] [PC100]; twofold decrease regarded as significant) and airway cell composition (bronchoalveolar lavage [BAL]) were determined 1 day before and 20 hours after a single inhalation of Ascaris extract, or 3 days before and 3 days after three alternate-day inhalations of Ascaris extract. The single inhalation of Ag (N = 7) caused an acute increase in Rrs (307% +/- 62%), an increase in BAL leukocytes, and a decrease in PC100 in three animals that was moderate (more than eightfold) in two animals. The mean +/- SE change in log PC100 was only -0.25 +/- 0.24. The multiple inhalations of Ag in the same animals caused acute increases in Rrs (178% +/- 48%, 380% +/- 83%, and 331% +/- 63%, respectively), an increase in BAL granulocytes, and a decrease in PC100 in six of seven animals (mean +/- SE change in log PC100 was -1.36 +/- 0.34) that was moderate in two and severe (more than 80-fold) in three animals.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of a 5-lipoxygenase inhibitor on antigen-induced mediator release, late-phase bronchoconstriction and cellular infiltrates in primates.

Inhaled BI-L-239 significantly inhibited i-LTC4 generation, late-phase bronchoconstriction and the influx of neutrophils into the lungs. We conclude that leukotriene generation and release within the lungs, following allergen exposure, in part mediate altered lung function and contribute to the development of airway inflammation. As such, treatment with a selective 5-lipoxygenase inhibitor may aid in the treatment of bronchial asthma and other allergic diseases.