Antagonizing the human M3 muscarinic receptor (hM3R) over a long time is a key feature of modern bronchodilating COPD drugs aiming at symptom relief. The long duration of action of the antimuscarinic drug tiotropium and its kinetic subtype selectivity over hM2R are investigated by kinetic mapping of the binding site and the exit channel of hM3R. Hence, dissociation experiments have been performed with a set of molecular matched pairs of tiotropium on a large variety of mutated variants of hM3R. The exceedingly long half-life of tiotropium (of more than 24 h) is attributed to interactions in the binding site; particularly a highly directed interaction of the ligands' hydroxy group with an asparagine (N508(6.52)) prevents rapid dissociation via a snap-lock mechanism. The kinetic selectivity over hM2R, however, is caused by differences in the electrostatics and in the flexibility of the extracellular vestibule. Extensive molecular dynamics simulations (several microseconds) support experimental results.
Background: A major cause for severe asthma exacerbations are viral infections. Asthma exacerbations are characterized by a sudden decrease in lung function associated with increased inflammation in the lung. In severe cases exacerbations cannot be sufficiently controlled by using inhaled or oral corticosteroids. Aim: Investigate the anti-inflammatory and broncho-protective effects of the selective PDE4 inhibitor Roflumilast and the corticosteroids Dexamethasone and Fluticasone in a murine influenza-induced asthma exacerbation model. Methods: BALB/c mice were sensitized with ovalbumin on day 0, 14 and 21 followed by two challenges with ovalbumin aerosol on day 26 and 27. The infection with Influenza A virus was performed intranasally on day 25. Roflumilast, Dexamethasone or Fluticasone were given either orally or intratracheally on day 25, 26 and 27. Mice were analysed on day 28. Results: All three treatments significantly reduced the numbers of macrophages and eosinophils and Th2 cytokine levels in the airways. However, only Roflumilast attenuated pulmonary neutrophilic cell influx, KC concentrations and airway hyperresponsiveness (AHR). Conclusion: In this murine model of an acute asthma exacerbation, steroid treatment had no impact on AHR or neutrophil numbers, parameters believed to be important in human asthma exacerbations. In contrast, Roflumilast reduced both parameters. These pre-clinical data suggest that in patients where asthma exacerbations cannot be controlled by an inhaled or oral corticosteroid, a PDE4 inhibitor like Roflumilast may help to reduce symptoms and the frequency of exacerbations.
Abstract The spleen tyrosine kinase (Syk) is a key mediator of immunoreceptor signaling in immune cells. Thus, interfering with the function of Syk by genetic deletion or pharmacological inhibition might influence a variety of allergic and autoimmune processes. Since conventional Syk knockout mice are not viable, studies addressing the effect of Syk deletion in adult animals have been limited. To further explore functions of Syk in animal models of allergy and to shed light on the role of Syk in the in vivo migration of neutrophils and monocytes, we generated inducible Syk knockout mice. These mice harbor a floxed Syk gene and a tamoxifen‐inducible Cre recombinase under the control of the ubiquitously active Rosa26 ‐promoter. Thus, treatment of mice with tamoxifen leads to the deletion of Syk in all organs. Syk‐deleted mice were analyzed in mast cell‐dependent models and in models focusing on neutrophil and monocyte migration. We show that Syk deletion in adult mice reduces inflammatory responses in mast cell‐driven animal models of allergy and asthma but has no effect on the migration of neutrophils and monocytes. Therefore, the inducible Syk knockout mice presented here provide a valuable tool to further explore the role of Syk in disease‐related animal models.
Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease characterised by fibrosis of the lung parenchyma and loss of lung function. Although the pathogenic pathways involved in IPF have not been fully elucidated, IPF is believed to be caused by repetitive alveolar epithelial cell injury and dysregulated repair, in which there is uncontrolled proliferation of lung fibroblasts and differentiation of fibroblasts into myofibroblasts, which excessively deposit extracellular matrix (ECM) proteins in the interstitial space. A number of profibrotic mediators including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and transforming growth factor-β are believed to play important roles in the pathogenesis of IPF. Nintedanib is a potent small molecule inhibitor of the receptor tyrosine kinases PDGF receptor, FGF receptor and vascular endothelial growth factor receptor. Data from in vitro studies have shown that nintedanib interferes with processes active in fibrosis such as fibroblast proliferation, migration and differentiation, and the secretion of ECM. In addition, nintedanib has shown consistent anti-fibrotic and anti-inflammatory activity in animal models of lung fibrosis. These data provide a strong rationale for the clinical efficacy of nintedanib in patients with IPF, which has recently been demonstrated in phase III clinical trials.
Background : Exacerbations of COPD are associated with increased risk of mortality. One major trigger of COPD exacerbations are influenza virus-induced respiratory tract infections. Aim : To compare the efficacy of the corticosteroid fluticasone to treatment with the PDE4 inhibitor roflumilast, and the long acting anticholinergic tiotropium in a COPD mouse model of acute virus-induced exacerbation. Methods : BALB/c mice were exposed to cigarette smoke (CS) for 2 weeks and infected with Influenza A H1N1 on day 8. In parallel to CS exposure, mice were treated with fluticasone, roflumilast or tiotropium. Mice were sacrificed on day 12 and pulmonary cell infiltration, cytokines in lung homogenate, lung function and viral replication were assessed. Results : Treatment with fluticasone showed that virus-induced neutrophilia was not only steroid insensitive, but steroid treatment lead to a worsening of health status, a more rapid and severe loss of body weight, an increase in pro-inflammatory cytokine release (IL-6, IFN-γ, IL-10 and KC), a decline in lung function and an increased viral load. Treatment with roflumilast reduced IFN-γ, IL-6, KC, and IL-10, but had only modest effects on cellular influx. Tiotropium was the only compound tested that did not only reduce IFN-γ, IL-6 and IL-10 but also significantly attenuated pulmonary cellular influx of neutrophils and macrophages. Conclusion : Our data demonstrate that in this in vivo model of Influenza-induced exacerbation fluticasone worsens health status. In contrast, treatment with roflumilast and tiotropium reduces release of pro-inflammatory cytokines. Furthermore, tiotropium efficiently reduces inflammatory cell infiltration.
β2 -adrenoceptor agonists are widely used in the management of obstructive airway diseases. Besides their bronchodilatory effect, several studies suggest inhibitory effects on various aspects of inflammation. The aim of our study was to determine the efficacy of the long-acting β2 -adrenoceptor agonist olodaterol to inhibit pulmonary inflammation and to elucidate mechanism(s) underlying its anti-inflammatory actions.Olodaterol was tested in murine and guinea pig models of cigarette smoke- and LPS-induced lung inflammation. Furthermore, effects of olodaterol on the LPS-induced pro-inflammatory mediator release from human parenchymal explants, CD11b adhesion molecule expression on human granulocytes TNF-α release from human whole blood and on the IL-8-induced migration of human peripheral blood neutrophils were investigated.Olodaterol dose-dependently attenuated cell influx and pro-inflammatory mediator release in murine and guinea pig models of pulmonary inflammation. These anti-inflammatory effects were observed at doses relevant to their bronchodilatory efficacy. Mechanistically, olodaterol attenuated pro-inflammatory mediator release from human parenchymal explants and whole blood and reduced expression of CD11b adhesion molecules on granulocytes, but without direct effects on IL-8-induced neutrophil transwell migration.This is the first evidence for the anti-inflammatory efficacy of a β2 -adrenoceptor agonist in models of lung inflammation induced by cigarette smoke. The long-acting β2 -adrenoceptor agonist olodaterol attenuated pulmonary inflammation through mechanisms that are separate from direct inhibition of bronchoconstriction. Furthermore, the in vivo data suggest that the anti-inflammatory properties of olodaterol are maintained after repeated dosing for 4 days.
Patients with asthma who smoke have reduced lung function, increased exacerbation rates and increased steroid resistance compared to non-smoking asthmatics. In mice, cigarette smoke has been reported to have both pro- and anti-Th2 response effects.We hypothesized that combining tobacco cigarette smoke (tCS) with allergen exposure increases inflammation, airway remodelling and lung function in mice. To test this hypothesis, we combined a severe triple allergen model with tCS exposure and investigated whether effects were due to Toll-like receptor 4 signalling and/or nicotine and also observed when nicotine-free cigarettes were used.Mice were sensitized with ovalbumin, cockroach and house dust mite allergen in alum followed by intratracheal challenges with allergen twice a week for 6 weeks or additionally exposed to tCS during the allergen challenge period. Nicotine or nicotine-free herbal cigarette smoke was also applied to allergen challenged mice.tCS significantly reduced eosinophil numbers, IL-4 and IL-5 concentrations in the lung, total and allergen-specific IgE in serum, improved lung function and reduced collagen I levels. With the exception of collagen I all parameters reduced by tobacco cigarette smoke were also reduced in Toll-like receptor 4-deficient mice. Nicotine-free cigarette smoke also had significant anti-inflammatory effects on eosinophils, IL-4 and IL-5 concentrations in the lung and reduced airway hyperreactivity, albeit weaker than tobacco smoke. Applying nicotine alone also reduced Th2 cytokine levels and eosinophil numbers in the airways.Our experiments show that tCS exposure reduces allergen-induced Th2 response in the lung and associated collagen I production and development of airway hyperreactivity. With the exception on collagen I formation, these effects were not dependent on Toll-like receptor 4. The observed anti-Th2 effects of both nicotine and nicotine-free herbal cigarette smoke together suggests that tCS reduces the Th2 responses through nicotine and other products released by burning tobacco.
Background and Purpose Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease characterized by excessive fibroblast activation ultimately leading to scarring of the lungs. Although, the activation of β 2 ‐adrenoceptors (β 2 ‐AR) has been shown to inhibit pro‐fibrotic events primarily in cell lines, the role of β 2 ‐adrenoceptor agonists has not yet been fully characterized. The aim of our study was to explore the anti‐fibrotic activity of the long‐acting β 2 ‐adrenoceptor agonist olodaterol in primary human lung fibroblasts (HLF) and in murine models of pulmonary fibrosis. Experimental Approach We assessed the activity of olodaterol to inhibit various pro‐fibrotic mechanisms, induced by different pro‐fibrotic mediators, in primary HLF from control donors and patients with IPF (IPF‐LF). The in vivo anti‐fibrotic activity of olodaterol, given once daily by inhalation in either a preventive or therapeutic treatment regimen, was explored in murine models of lung fibrosis induced by either bleomycin or the overexpression of TGF‐β1. Key Results In both HLF and IPF‐LF, olodaterol attenuated TGF‐β‐induced expression of α‐smooth muscle actin, fibronectin and endothelin‐1 (ET‐1), FGF‐ and PDGF‐induced motility and proliferation and TGF‐β/ET‐1‐induced contraction. In vivo olodaterol significantly attenuated the bleomycin‐induced increase in lung weight, reduced bronchoalveolar lavage cell counts and inhibited release of pro‐fibrotic mediators (TGF‐ß, MMP‐9 and tissue inhibitor of metalloproteinase‐1). Forced vital capacity was increased only with the preventive treatment regimen. In the TGF‐β‐overexpressing model, olodaterol additionally reduced the Col3A1 mRNA expression. Conclusion and Implications Olodaterol showed anti‐fibrotic properties in primary HLF from control and IPF patients and in murine models of lung fibrosis.
Hintergrund: Nintedanib ist ein spezifischer Tyrosinkinaseinhibitor, der sich aktuell im Zulassungsprozess für die Behandlung der idiopathischen Lungenfibrose (IPF) befindet.
'%3e%3ccircle r='20' fill='%23008'/%3e%3ccircle r='17.5' fill='%23fff'/%3e%3ccircle r='3.5' fill='%23008'/%3e%3cg id='d'%3e%3cg id='c'%3e%3cg id='b'%3e%3cg id='a' fill='%23008'%3e%3ccircle r='.875' transform='rotate(7.5 -8.75 133.5)'/%3e%3cpath d='M0 17.5.6 7 0 2l-.6 5L0 17.5z'/%3e%3c/g%3e%3cuse xlink:href='%23a' transform='rotate(15)'/%3e%3c/g%3e%3cuse xlink:href='%23b' transform='rotate(30)'/%3e%3c/g%3e%3cuse xlink:href='%23c' transform='rotate(60)'/%3e%3c/g%3e%3cuse xlink:href='%23d' transform='rotate(120)'/%3e%3cuse xlink:href='%23d' transform='rotate(-120)'/%3e%3c/g%3e%3c/svg%3e)
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)