TNF-α-induces airway hyperresponsiveness to cholinergic stimulation in guinea pig airways.

BACKGROUND AND PURPOSE: TNF-α is an inflammatory cytokine implicated in the pathogenesis of asthma and it causes airway inflammation, bronchoconstriction and airway hyperresponsiveness to a number of spasmogens following inhalation. EXPERIMENTAL APPROACH: We compared contractions of guinea pig isolated trachea incubated with saline or TNF-α for 1, 2 or 4 days to electrical field stimulation (EFS), 5-HT or methacholine. In addition, we compared bronchoconstriction in anaesthetized guinea pigs 6 h after intratracheal instillation of saline or TNF-α to vagal nerve stimulation, i.v. 5-HT or methacholine. Differential counts were performed on the bronchoalvelolar lavage fluid (BALF). KEY RESULTS: Maximum contractions to methacholine, 5-HT and EFS were not different between freshly prepared and saline-incubated tissues. Exposure to TNF-α concentration-dependently potentiated contractions to 5-HT and EFS, but not methacholine. All contractions were atropine-sensitive, but not hexamethonium-sensitive. 5-HT-evoked contractions were inhibited by ketanserin or epithelial denudation. Only EFS-evoked contractions were tetrodotoxin-sensitive. Vagal stimulation, i.v. 5-HT or MCh caused a significant atropine-sensitive, frequency- and dose-dependent bronchoconstriction and decreased blood pressure similarly in both saline and TNF-α pre-treated animals. TNF-α potentiated the bronchoconstriction to vagal stimulation and 5-HT, but not MCh. The BALF from saline-treated animals contained predominantly macrophages, whereas that from TNF-α-treated animals contained neutrophils. CONCLUSIONS AND IMPLICATIONS: TNF-α caused airway hyperresponsiveness to nerve stimulation in vivo and increased contractility in vitro. However, responsiveness to MCh was unchanged, suggesting a pre-synaptic action of TNF-α on parasympathetic nerves. TNF-α-induced airway hyperresponsiveness to 5-HT suggested an increased 5-HT(2A) receptor-mediated acetylcholine release from epithelial cells.

In vitro and in vivo characterisation of anti-murine IL-13 antibodies recognising distinct functional epitopes.

Interleukin-13 (IL-13) sequentially binds to IL-13Ralpha1 and IL-4Ralpha forming a high affinity signalling complex. This receptor complex is expressed on multiple cell types in the airway and signals through signal transducer and activator of transcription factor-6 (STAT-6) to stimulate the production of chemokines, cytokines and mucus. Antibodies have been generated, using the UCB Selected Lymphocyte Antibody Method (UCB SLAM), that block either binding of murine IL-13 (mIL-13) to mIL-13Ralpha1 and mIL-13Ralpha2, or block recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. Monoclonal antibody (mAb) A was shown to bind to mIL-13 with high affinity (K(D) 11 pM) and prevent binding of mIL-13 to mIL-13Ralpha1. MAb B, that also bound mIL-13 with high affinity (K(D) 8 pM), was shown to prevent recruitment of mIL-4Ralpha to the mIL-13/mIL-13Ralpha1 complex. In vitro, mAbs A and B similarly neutralised mIL-13-stimulated STAT-6 activation and TF-1 cell proliferation. In vivo, mAbs A and B demonstrated equipotent, dose-dependent inhibition of eotaxin generation in mice stimulated by intraperitoneal administration of recombinant mIL-13. In an allergic lung inflammation model in mice, mAbs A and B equipotently inhibited muc5ac mucin mRNA upregulation in lung tissue measured two days after intranasal allergen challenge. These data support the design of therapeutics for the treatment of allergic airway disease that inhibits assembly of the high affinity IL-13 receptor signalling complex, by blocking the binding of IL-13 to IL-13Ralpha1 and IL-13Ralpha2, or the subsequent recruitment of IL-4Ralpha.

The effect of anti-integrin monoclonal antibodies on antigen-induced pulmonary inflammation in allergic rabbits.

The integrin adhesion molecules are involved in the recruitment and activation of inflammatory cells at sites of inflammation in a variety of diseases. In the present study, we have investigated the effects of blocking monoclonal antibodies (mAbs) directed against CD49d (alpha(4) integrin), CD18 (beta(2) integrin) and the alpha sub-units of beta(2) integrin CD11a (LFA-1 integrin) and CD11b (Mac-1 integrin), on antigen (Ag)-induced acute bronchoconstriction and cellular recruitment in allergic rabbits in vivo. Inhaled Ag (Alternaria tenuis) challenge of neonatally sensitised rabbits caused an acute bronchoconstriction demonstrated by an increase in lung resistance (R(L)) and decrease in dynamic compliance (C(dyn)) and pulmonary inflammation characterised by an increase in bronchoalveolar lavage (BAL) inflammatory cells, particularly eosinophils, 24 h after challenge. Pre-treatment with the anti-CD49d mAb (Max-68P), significantly inhibited the Ag-induced acute bronchoconstriction in terms of R(L) and (C(dyn)). Treatment with the other anti-integrin mAbs had no effect on the acute bronchoconstriction after inhaled Ag challenge.Pre-treatment with the anti-integrin mAbs had differential effects in blocking the recruitment of inflammatory cells 24 h after inhaled Ag in the allergic rabbits. The data show that in the allergic rabbit model of asthma, VLA-4 (CD49d/CD29) only, is involved in the acute bronchoconstriction, suggesting an involvement of mast cell degranulation. Furthermore, eosinophil recruitment and activation appears to be mediated by a combination of VLA-4 (CD49d/CD29) and LFA-1 (CD18/CD11a). However in contrast, lymphocyte recruitment appears to be mediated by a combination of LFA-1 (CD18/CD11a) and Mac-1 (CD18/CD11b).

CDP840. A prototype of a novel class of orally active anti-inflammatory phosphodiesterase 4 inhibitors.

The discovery, synthesis and biological activity of a series of triarylethane phosphodiesterase 4 inhibitors is described. Structure-activity relationship studies are presented for CDP840 (29), a potent, chiral, selective inhibitor of PDE 4 (IC(50) 4nM). CDP840 is non-emetic in the ferret at 30mgkg(-1) (po), active in models of inflammation and reverses ozone-induced bronchial hyperreactivity in the guinea pig.

Discovery and evaluation of potent, tyrosine-based alpha4beta1 integrin antagonists.

Using disulphide cysteine-based inhibitors as lead structures, this communication describes our strategy for identifying more stable, potent antagonists of the alpha4beta1 integrin. These studies ultimately discovered potent, low molecular weight inhibitors based on D-thioproline-L-tyrosine.

Inhibition of bronchospasm and ozone-induced airway hyperresponsiveness in the guinea-pig by CDP840, a novel phosphodiesterase type 4 inhibitor.

1. The activity of CDP840, a novel, potent and selective cyclic nucleotide phosphodiesterase type 4 (PDE 4) inhibitor, was evaluated in guinea-pig models (in vitro and in vivo) of bronchospasm, ozone-induced airway hyperresponsiveness (AHR) and non-cholinergic bronchoconstriction. Comparisons were made with (i) other PDE 4 inhibitors: CT1731 (S-enantiomer of CDP840), rolipram, RP73401 and (ii) the clinically used agents salbutamol and theophylline. 2. CDP840 relaxed isolated trachea, under basal tone (EC50 4.5 +/- 1.1 microM) being 17 fold less potent than rolipram (EC50 0.26 +/- 0.13 microM) but attaining the same Emax (83 +/- 6% of the response to 300 microM papaverine). 3. CDP840 relaxed tracheae pre-contracted with carbachol (IC25 39 +/- 9 microM) and histamine (IC25 4 +/- 1 microM) producing monophasic curves. Stereoselectivity was not observed with CT1731 against either carbachol (IC25 33 +/- 11 microM) or histamine (IC25 17 +/- 10 microM). Aminophylline was 1.6 fold (carbachol) and 11 fold (histamine) less potent than CDP840. Rolipram and RP73401 produced tri-phasic relaxation curves but were of similar potency (at the IC25 level) to CDP840 against carbachol (rolipram 18 +/- 5 microM, RP73401 39 +/- 1 microM) whereas against histamine they were approximately 20 fold more potent (rolipram 0.2 +/- 0.1 microM, RP73401 0.2 +/- 0.1 microM). In producing > 30% (carbachol) and > 60% (histamine) relaxation these inhibitors had similar potency and were poor compared to salbutamol. 4. Pre-incubation with CDP840 (10 microM) did not antagonize histamine-induced contraction of isolated trachea; however, it did cause a slight potentiation of the subsequent relaxation to salbutamol (IC50 23 +/- 1 to 15 +/- 2 nM). 5. Pretreatment (1 h) with either CDP840 (1 mg kg-1, i.p. or 3 mg kg-1, i.v.) or rolipram (1 mg kg-1, i.p.) did not bronchodilate or antagonize bronchospasm due to inhaled histamine in anaesthetized, ventilated guinea-pigs. Salbutamol (1 mg kg-1, i.p.) did not bronchodilate but caused a parallel 7 fold rightward shift in the histamine dose-response curve. 6. Stimulation of the vagus nerve in the presence of atropine resulted in a frequency-related bronchoconstriction. CDP840 and rolipram (i.v.) inhibited the response being approximately equipotent (EC50 approximately 10 micrograms kg-1). Neither drug inhibited bronchospasm to inhaled substance P. 7. CDP840 (1-10 micrograms kg-1 i.p.) dose relatedly inhibited ozone-induced bronchoconstriction. CT1731 (1 mg kg-1), rolipram (1 mg kg-1), RP73401 (10 micrograms kg-1) and aminophylline (10 mg kg-1) had no effect. Ozone-induced AHR to inhaled histamine was inhibited by CDP840 in a dose-related manner, 10 micrograms kg-1 abolishing the AHR. This effect was stereoselective as CT1731 was approximately 30 fold less potent than CDP840. Rolipram was approximately 100 fold less potent and RP73401 and aminophylline had no effect. CDP840 was orally active being approximately 10 fold less potent compared to i.p. administration. 8. CDP840 is a poor spasmolytic and anti-spasmogenic agent in response to exogenous mediators; however, it potently inhibits vagally mediated non-cholinergic bronchoconstriction and ozone-induced AHR to histamine. It is possible that regulation of cyclic AMP by PDE 4 contributes to neuronal sensitivity in the airways. Furthermore, CDP840 may suppress AHR without being an overt bronchodilator. Such a profile of activity may have therapeutic benefit in airways diseases such as asthma.

Effect of the glucocorticosteroid budesonide and a novel phosphodiesterase type 4 inhibitor CDP840 on antigen-induced airway responses in neonatally immunised rabbits.

1. The effects of the inhaled corticosteroid budesonide and a novel PDE 4 inhibitor CDP840 given systematically, were evaluated in a model of antigen-induced airway inflammation in the rabbit. 2. Adult litter-matched NZW rabbits (2.4-3.5 kg) immunised within 24 h of birth with Alternaria tenuis antigen were pretreated with budesonide (total dose 100 micrograms, inhaled over 2 days) or CDP840 (total dose 7 mg kg-1, i.p. over 3 days), before antigen challenge. For each drug-treated group a parallel group of rabbits was pretreated with the appropriate vehicle. In all groups airway responsiveness to inhaled histamine was assessed and bronchoalveolar lavage (BAL) performed 24 h before and after antigen challenge. 3. Basal lung function in terms of total lung resistance (RL; cmH2O l 1s-1) and dynamic compliance (Cdyn; ml cmH2O-1) were unaltered by pretreatment with budesonide or CDP840 compared to their respective vehicles 24 h before or after antigen challenge. 4. The RL component of the acute bronchoconstriction induced by inhaled Alternaria tenuis aerosol was unaffected by pretreatment with budesonide. However, budesonide prevented the fall in Cdyn due to antigen. Treatment with CDP840 significantly reduced antigen-induced acute bronchoconstriction in terms of both RL and Cdyn. 5. Airway hyperresponsiveness (AHR) to inhaled histamine was indicated by reduced RL PC50 (2.4-4.5 fold) and Cdyn PC35 (2.1-3.9 fold) values 24 h after antigen challenge. Treatment with either budesonide or CDP840 abolished the antigen-induced increase in responsiveness to inhaled histamine. 6. Total cells recovered per ml of BAL fluid increased 24 h after antigen challenge. Antigen-induced pulmonary eosinophilia was reduced (93%) in budesonide and (85%) in CDP840 treated rabbits. Antigen-induced increases in neutrophil numbers were reduced (76%) with budesonide but not CDP840 pretreatment. 7. Inhalation of Alternaria tenuis aerosol elicited an acute bronchoconstriction, followed 24 hours later by an increased responsiveness to inhaled histamine and pulmonary neutrophil and eosinophil recruitment. CDP840 was more effective than budesonide in preventing the antigen-induced increase in total lung resistance (RL); however, both drugs prevented the antigen-induced reduction in dynamic compliance (Cdyn). CDP840 and budesonide also prevented antigen-induced AHR and eosinophilia in the immunised rabbit.

The inhibition of antigen-induced eosinophilia and bronchoconstriction by CDP840, a novel stereo-selective inhibitor of phosphodiesterase type 4.

1. The novel tri-aryl ethane CDP840, is a potent and selective inhibitor of cyclic AMP phosphodiesterase type 4 (PDE 4) extracted from tissues or recombinant PDE 4 isoforms expressed in yeast (IC50S: 4-45 nM). CDP840 is stereo-selective since its S enantiomer (CT 1731) is 10-50 times less active against all forms of PDE 4 tested while both enantiomers are inactive (IC50S: > 100 microM) against PDE types 1, 2, 3 and 5. 2. Oral administration of CDP840 caused a dose-dependent reduction of interleukin-5 (IL-5)-induced pleural eosinophilia in rats (ED50 = 0.03 mg kg-1). The eosinophils in pleural exudates from CDP840-treated animals contained higher levels of eosinophil peroxidase (EPO) than cells from control animals, suggesting a stabilizing effect on eosinophil degranulation. CDP840 was approximately equi-active with the steroid dexamethasone in this model and was 10-100 times more potent than the known PDE 4-selective inhibitors rolipram and RP73401. The activity of CDP840 was not influenced by adrenalectomy, beta-sympathomimetics or beta-sympatholytics. 3. Antigen-induced pulmonary eosinophilia in sensitized guinea-pigs was reduced dose-dependently by CDP840 (0.01-1 mg kg-1, i.p.) and intracellular EPO levels were significantly higher. CDP840 was more potent in these activities than CT1731 or rolipram and comparable in potency to RP73401. 4. Rolipram or CDP840 were less active than dexamethasone in preventing neutrophil accumulation, or exudate formation in carrageenan-induced pleurisy in rats and thus do not exhibit general anti-inflammatory activity. 5. In sensitized guinea-pigs, aerosols of the antigen ovalbumin caused a dose-dependent bronchoconstriction demonstrated by an increase in pulmonary inflation pressure. Administration of CDP840 (0.001-1.0 mg kg-1, i.p.), 1 h before antigen challenge, resulted in dose-dependent reduction in response to antigen. This activity was not due to bronchodilatation since higher doses of CDP840 (3 mg kg-1) did not significantly change the bronchoconstrictor response to histamine. Rolipram was approximately 10 times less active than CDP840 in preventing antigen-induced bronchoconstriction. 6. These results confirm the observations that selective PDE 4 inhibitors reduce antigen-induced bronchoconstriction and pulmonary eosinophilic inflammation. CDP840 is more potent than rolipram in inhibiting native or recombinant PDE 4. Unlike the recently described potent PDE 4 inhibitor RP73401, CDP840 is more active than rolipram in the rat IL-5 model following oral administration. The novel series of tri-aryl ethanes, of which CDP840 is the lead compound, could be the basis of an orally active prophylactic treatment for human asthma.

Effects of theophylline and rolipram on antigen-induced airway responses in neonatally immunized rabbits.

1. The effects of the xanthine, theophylline, a non-selective phosphodiesterase (PDE) inhibitor, and the phosphodiesterase type 4 (PDE 4) inhibitor, rolipram, were evaluated in a model of antigen-induced airway responses in the allergic rabbit. 2. Adult litter-matched NZW rabbits (2.5-3.9 kg), immunized within 24 h of birth with Alternaria tenuis antigen, were pretreated twice daily for 3 days with theophylline (3 mg kg-1, i.p) or rolipram (1 mg kg-1, i.p) prior to antigen challenge (Alternaria tenuis). For each drug-treated group, a parallel group of rabbits were pretreated with the appropriate vehicle. In all groups airway responsiveness to inhaled histamine and bronchoalveolar lavage (BAL) was performed 24 h before and after antigen-challenge. 3. Basal lung function in terms of resistance (RL, cmH2O 1(-1)s-1) and dynamic compliance (Cdyn, ml cmH2O-1) were unaltered by pretreatment with theophylline or rolipram compared to their respective vehicles 24 h prior to or post antigen challenge. 4. The acute bronchoconstriction induced by inhaled Alternaria tenuis aerosol was unaffected by pretreatment with theophylline or rolipram. 5. Airway hyperresponsiveness to inhaled histamine was indicated by reduced RL PC50 (2.4-3.5 fold) and Cdyn PC35 (2.5-2.6 fold) values 24 h after antigen challenge. Treatment with rolipram, but not theophylline, prevented the increase in responsiveness to inhaled histamine 24 h after antigen challenge. 6. Total cells per ml of BAL fluid increased 24 h after antigen challenge due to the recruitment of neutrophils and eosinophils. Antigen-induced increases in pulmonary neutrophils were unaffected; however, eosinophils were reduced 57.5% in theophylline and 82% in rolipram-treated rabbits. 7. Inhalation of Alternaria tenuis aerosol elicits an acute bronchoconstriction, followed 24 h later by an increased responsiveness to inhaled histamine and pulmonary neutrophil and eosinophil recruitment in the immunized rabbit. With the dosing regimes used, both rolipram and theophylline inhibited eosinophil recruitment, whilst only rolipram prevented the development of airway hyperresponsiveness. Neither agent inhibited the acute bronchoconstriction due to inhaled antigen.

Capsaicin pre-treatment prevents the development of antigen-induced airway hyperresponsiveness in neonatally immunised rabbits.

The effect of a 3-day pre-treatment regime of capsaicin (8-methyl-N-vanillyl-6-nonenamide) (80 mg/kg s.c.) on airway changes induced by Alternaria tenuis aerosol challenge 3 days later was assessed in adult rabbits immunised from birth to the age of 3 months. Pre-treatment with capsaicin did not alter basal lung function or basal responsiveness to inhaled histamine. While capsaicin had no significant effect on the acute bronchoconstriction induced by antigen, this dose was sufficient to significantly inhibit the increase in airway responsiveness to inhaled histamine achieved 24 h following antigen challenge. The pulmonary recruitment of neutrophils and eosinophils induced by antigen was unaltered by prior treatment with capsaicin. In vitro contractile responsiveness to methacholine was not significantly different in bronchial tissues removed from capsaicin- and vehicle-pre-treated rabbits. In addition, there were no significant differences in responses to methacholine in preparations denuded of epithelium. Contraction of bronchial tissue induced by exogenously applied capsaicin in vitro, although modest, was significantly inhibited in capsaicin-pre-treated animals. In vehicle-pre-treated rabbits, contraction induced by a second challenge with capsaicin 45 min later was significantly reduced to a level that made responses not significantly different from those obtained in capsaicin-pre-treated tissues. The results of the present study demonstrate that antigen-induced airway hyperresponsiveness to inhaled histamine in immunised rabbits is inhibited by prior treatment with capsaicin. These findings suggest the involvement of capsaicin-sensitive nerves in antigen-induced airway hyperresponsiveness but not acute bronchospasm or cell infiltration induced by antigen.