Eva Wex

Mode of action of nintedanib in the treatment of idiopathic pulmonary fibrosis

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. Nintedanib interferes with processes active in fibrosis, e.g. fibroblast proliferation, migration and differentiation http://ow.ly/Iae9z

Induced Syk deletion leads to suppressed allergic responses but has no effect on neutrophil or monocyte migration in vivo.

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.

Syk and Lyn mediate distinct Syk phosphorylation events in FcɛRI-signal transduction: Implications for regulation of IgE-mediated degranulation

Spleen tyrosine kinase (Syk) is a key regulatory factor in the IgE-mediated allergic signal transduction pathway in mast cells and basophils. Syk is phosphorylated on a number of tyrosines following the binding of IgE/allergen complexes to FcɛRI receptors leading to initiation of inflammatory signaling via downstream enzymes and scaffolding proteins. We examined the kinases responsible for the phosphorylation of key Syk tyrosines in rat RBL-2H3 basophilic cells and bone marrow-derived mast cells (BMMCs). The phosphorylation of Syk tyrosine 346 was completely blocked by the novel Src family kinase inhibitor BIRA766, suggesting this tyrosine is a pure substrate for Src family kinases. This was supported by the findings that kinase-dead (KD) Syk was efficiently phosphorylated on this tyrosine and that a specific Syk inhibitor BAY61-3606 was without effect. The phosphorylation of other Syk tyrosines 317, 342, 519 and 520 was reduced by Syk and Src family inhibitors, suggesting a role for auto- and trans-phosphorylation. Lyn was the predominant Src family kinase expressed and activated in RBL-2H3 cells, meanwhile Lyn knockdown with a specific siRNA interfered with the phosphorylation of all Syk tyrosines and the Syk substrates SLP-76 and LAT. Pharmacological inhibition of Syk completely blocked the degranulation of RBL-2H3 and BMMCs. However, Lyn knockdown sensitized RBL-2H3 cells to FcɛRI-induced degranulation. We showed that whilst interference with Lyn expression disrupts FcɛRI proximal signaling via Syk and its direct substrates including SLP-76 and LAT, distal activation of downstream proteins including Erk is enhanced. This study identifies the responsible kinases for the phosphorylation of key Syk tyrosines and the propagation of FcɛRI receptor mediated signal transduction in allergic responses.

A Novel Model of IgE-Mediated Passive Pulmonary Anaphylaxis in Rats

Mast cells are central effector cells in allergic asthma and are augmented in the airways of asthma patients. Attenuating mast cell degranulation and with it the early asthmatic response is an important intervention point to inhibit bronchoconstriction, plasma exudation and tissue oedema formation. To validate the efficacy of novel pharmacological interventions, appropriate and practicable in vivo models reflecting mast cell-dependent mechanisms in the lung, are missing. Thus, we developed a novel model of passive pulmonary anaphylaxis in rats. Rats were passively sensitized by concurrent intratracheal and intradermal (ear) application of an anti-DNP IgE antibody. Intravenous application of the antigen, DNP-BSA in combination with Evans blue dye, led to mast cell degranulation in both tissues. Quantification of mast cell degranulation in the lung was determined by (1) mediator release into bronchoalveolar lavage, (2) extravasation of Evans blue dye into tracheal and bronchial lung tissue and (3) invasive measurement of antigen-induced bronchoconstriction. Quantification of mast cell degranulation in the ear was determined by extravasation of Evans blue dye into ear tissue. We pharmacologically validated our model using the SYK inhibitor Fostamatinib, the H1-receptor antagonist Desloratadine, the mast cell stabilizer disodium cromoglycate (DSCG) and the β2-adrenergic receptor agonist Formoterol. Fostamatinib was equally efficacious in lung and ear. Desloratadine effectively inhibited bronchoconstriction and ear vascular leakage, but was less effective against pulmonary vascular leakage, perhaps reflecting the differing roles for histamine receptor sub-types. DSCG attenuated both vascular leakage in the lung and bronchoconstriction, but with a very short duration of action. As an inhaled approach, Formoterol was more effective in the lung than in the ear. This model of passive pulmonary anaphylaxis provides a tissue relevant readout of early mast cell activity and pharmacological benchmarking broadly reflects responses observed in patients with asthma.

Effects of conventional tobacco smoke and nicotine‐free cigarette smoke on airway inflammation, airway remodelling and lung function in a triple allergen model of severe asthma

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.

The long‐acting β2‐adrenoceptor agonist olodaterol attenuates pulmonary inflammation

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 Attenuates Citric Acid-Induced Cough in Naïve and Ovalbumin-Sensitized and Challenged Guinea Pigs

Excessive coughing is a common feature of airway diseases. Different G-protein coupled receptors, including β2-adrenergic receptors (β2-AR), have been implicated in the molecular mechanisms underlying the cough reflex. However, the potential antitussive property of β2-AR agonists in patients with respiratory disease is a matter of ongoing debate. The aim of our study was to test the efficacy of the long-acting β2-AR agonist olodaterol with regard to its antitussive property in a pre-clinical model of citric acid-induced cough in guinea pigs and to compare the results to different clinically relevant β2-AR agonists. In our study β2-AR agonists were intratracheally administered, as dry powder, into the lungs of naïve or ovalbumin-sensitized guinea pigs 15 minutes prior to induction of cough by exposure to citric acid. Cough events were counted over 15 minutes during the citric acid exposure. Olodaterol dose-dependently inhibited the number of cough events in naïve and even more potently and with a greater maximal efficacy in ovalbumin-sensitized guinea pigs (p < 0.01). Formoterol and salmeterol showed a trend towards reducing cough. On the contrary, indacaterol demonstrated pro-tussive properties as it significantly increased the number of coughs, both in naïve and ovalbumin-sensitized animals (p < 0.001). In conclusion, olodaterol, at doses eliciting bronchodilation, showed antitussive properties in a model of citric acid-induced cough in naïve and ovalbumin-sensitized guinea pigs. This is in agreement with pre-clinical and clinical studies showing antitussive efficacy of β2-AR agonists. Indacaterol increased the number of coughs in this model, which concurs with clinical data where a transient cough has been observed after indacaterol inhalation. While the antitussive properties of β2-AR agonists can be explained by their ability to lead to the cAMP-induced hyperpolarization of the neuron membrane thereby inhibiting sensory nerve activation and the cough reflex, the mechanism underlying the pro-tussive property of indacaterol is not known.

BI 1002494, a Novel Potent and Selective Oral Spleen Tyrosine Kinase Inhibitor, Displays Differential Potency in Human Basophils and B Cells

BI 1002494 [(R)-4-{(R)-1-[7-(3,4,5-trimethoxy-phenyl)-[1,6]napthyridin-5-yloxy]-ethyl}pyrrolidin-2-one] is a novel, potent, and selective spleen tyrosine kinase (SYK) inhibitor with sustained plasma exposure after oral administration in rats, which qualifies this molecule as a good in vitro and in vivo tool compound. BI 1002494 exhibits higher potency in inhibiting high-affinity IgE receptor–mediated mast cell and basophil degranulation (IC50 = 115 nM) compared with B-cell receptor–mediated activation of B cells (IC50 = 810 nM). This may be explained by lower kinase potency when the physiologic ligand B-cell linker was used, suggesting that SYK inhibitors may exhibit differential potency depending on the cell type and the respective signal transduction ligand. A 3-fold decrease in potency was observed in rat basophils (IC50 = 323 nM) compared with human basophils, but a similar species potency shift was not observed in B cells. The lower potency in rat basophils was confirmed in both ex vivo inhibition of bronchoconstriction in precision-cut rat lung slices and in reversal of anaphylaxis-driven airway resistance in rats. The different cellular potencies translated into different in vivo efficacy; full efficacy in a rat ovalbumin model (that contains an element of mast cell dependence) was achieved with a trough plasma concentration of 340 nM, whereas full efficacy in a rat collagen-induced arthritis model (that contains an element of B-cell dependence) was achieved with a trough plasma concentration of 1400 nM. Taken together, these data provide a platform from which different estimates of human efficacious exposures can be made according to the relevant cell type for the indication intended to be treated.

Olodaterol shows anti‐fibrotic efficacy in in vitro and in vivo models of pulmonary fibrosis

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 β2‐adrenoceptors (β2‐AR) has been shown to inhibit pro‐fibrotic events primarily in cell lines, the role of β2‐adrenoceptor agonists has not yet been fully characterized. The aim of our study was to explore the anti‐fibrotic activity of the long‐acting β2‐adrenoceptor agonist olodaterol in primary human lung fibroblasts (HLF) and in murine models of pulmonary fibrosis.