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 collageninduced 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.

In animal models of asthma, ovalbumin (OVA) aerosol results in bronchoconstriction characterized by a histamine-related early asthmatic response (EAR) followed by a neuropeptide-related late asthmatic response (LAR) which can be modulated by transient receptor potential (TRP) channel A1 antagonists (Thorax. 2012, 67, 19-25). The aim of the study was to assess the potential of two TRPA1 antagonists on the EAR and LAR in OVA-induced bronchoconstriction in anaesthetized guinea pigs. Bronchoconstriction was induced by intra-tracheal administration of a single dose of OVA (50 µg/kg) and lung resistance recorded for 30 minutes. The animals were pre-treated with pyrilamine (2 mg/kg i.v.) or its vehicle (saline) 10 min before OVA. The TRPA1 antagonists HC-030031 and A-967079 or their vehicle (0.5 % methylcellulose) were administered i.p. 1 h before OVA. Without pyrilamine pre-treatment, OVA induced a fast increase in lung resistance (max. 60 ± 13 ml overflow after 1 min) which was not reduced by the TRPA1 antagonists. Under pyrilamine pre-treatment, OVA induced a slow increase in lung resistance (max. 33 ± 10 ml overflow at the end of the recording). HC-030031 (1 µg/kg – 1 mg/kg) dose-dependently inhibited the non-histamine-related OVA- induced bronchoconstriction (ED 50 = 0.01 mg/kg) with a maximum bronchoprotection of 76 % at 0.03 mg/kg (p 50 = 0.01 mg/kg) with a maximum bronchoprotection of 78 % at 0.03 mg/kg (p This study shows that the EAR is histamine related, while the LAR is modulated by the TRPA1 channel in OVA-induced bronchoconstriction in anaesthetized guinea pigs.

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.

G protein coupled receptors (GPCRs) exhibit a spectrum of functional behaviours in response to natural and synthetic ligands. Recent crystal structures provide insights into inactive states of several GPCRs. Efforts to obtain an agonist-bound active-state GPCR structure have proven difficult due to the inherent instability of this state in the absence of a G protein. We generated a camelid antibody fragment (nanobody) to the human β2 adrenergic receptor (β2AR) that exhibits G protein-like behaviour, and obtained an agonist-bound, active-state crystal structure of the receptor-nanobody complex. Comparison with the inactive β2AR structure reveals subtle changes in the binding pocket; however, these small changes are associated with an 11 Å outward movement of the cytoplasmic end of transmembrane segment 6, and rearrangements of transmembrane segments 5 and 7 that are remarkably similar to those observed in opsin, an active form of rhodopsin. This structure provides insights into the process of agonist binding and activation. Two papers by Brian Kobilka and colleagues describe the X-ray crystal structure of the human β2 adrenergic receptor (β2AR) bound to various agonists. β2AR is a member of the G protein coupled receptor (GPCR) family of membrane-spanning receptors that sense molecules outside the cell and activate internal signalling pathways. With a ubiquitous role in human physiology, GPCRs are prime targets for drug discovery. A third paper by Christopher Tate and his team describes crystal structures of a similar GPCR, the turkey β1-adrenergic receptor (β1AR), bound to full and partial agonists. Together, these new structures reveal the subtle structural changes that accompany agonist binding, showing how binding events inside and outside the cell membrane stabilize the receptor's active state. Agonist binding to β1AR is shown to induce a contraction of the catecholamine-binding pocket relative to the antagonist-bound receptor, and molecular-dynamics simulations of the β2AR agonist complex suggest that the agonist-bound active state spontaneously relaxes to an inactive-like state in the absence of a G protein. The X-ray crystal structure of the human β2 adrenergic receptor, a G-protein-coupled receptor, in an agonist-bound 'active' state is solved. Comparison of this structure with a previously published structure of the same GPCR in an inactive state indicates that minor changes in the binding pocket of the protein lead to major changes elsewhere — there is a large outward movement of the cytoplasmic end of one of the transmembrane segments and rearrangements of two other transmembrane segments. This structure provides insights into the process of agonist binding and activation.

β(2)-Adrenoceptor (β(2)-AR) agonists are powerful bronchodilators and play a pivotal role in the management of pulmonary obstructive diseases, such as asthma and chronic obstructive pulmonary disease. Although these agents first were used many years ago, progress in drug development has resulted in better tolerated, long-acting β(2)-AR agonists (LABAs), such as formoterol and salmeterol. Although LABAs have been on the market for several years, relatively little is known on the rationale(s) behind their long duration of action. In this study, we focused on olodaterol (previously known as BI1744CL), a novel inhaled LABA, which provides a bronchodilating effect lasting 24 h and is currently in Phase III clinical trials. To understand the rationale behind its long duration of action, different aspects of olodaterol were analyzed (i.e., its lipophilicity and propensity to accumulate in the lipid bilayer as well as its tight binding to the β(2)-AR). In line with its physicochemical properties, olodaterol associated moderately with lipid bilayers. Instead, kinetic as well as equilibrium binding studies indicated the presence of a stable [(3)H]olodaterol/β(2)-AR complex with a dissociation half-life of 17.8 h due to ternary complex formation. The tight binding of olodaterol to the human β(2)-AR and stabilization of the ternary complex were confirmed in functional experiments monitoring adenylyl cyclase activity after extensive washout. Taken together, binding, kinetic, and functional data support the existence of a stable complex with the β(2)-AR that, with a dissociation half-life >17 h, might indeed be a rationale for the 24-h duration of action of olodaterol.

Inhaled corticosteroids (ICS) and long-acting β 2 -agonists (LABAs) fixed dose combinations (FDCs) are widely used for the treatment of asthma while the long-acting muscarinic antagonist (LAMA) tiotropium is a standard therapy of chronic obstructive pulmonary disease (COPD). Additional improvement of lung function might be expected with the use of the triple therapy “ICS + LABA + LAMA”. The aim of this study was to assess potential synergistic bronchoprotection activity of olodaterol (LABA) combined with tiotropium (LAMA) and ciclesonide (steroid), against ovalbumin-induced bronchoconstriction in the anaesthetized guinea pig. Bronchoconstriction was induced by inhalation of a single dose of ovalbumin (50 μg/kg) and lung resistance was recorded for 10 minutes in anaesthetized guinea pigs. Compounds were given intra-tracheally 2 h before OVA challenge. Olodaterol dose-dependently inhibited the OVA-induced bronchoconstriction with an ED 50 of 0.2 μg/kg. Combination of olodaterol with either tiotropium or ciclesonide displayed bronchoprotection in a dose-dependent manner with ED 50 values 10–fold lower (0.02-0.03 μg/kg) than the ED 50 of olodaterol alone (p 50 of 0.003 μg/kg (p This study shows that olodaterol in combination with tiotropium and ciclesonide elicits potent synergistic bronchoprotective activity in the guinea pig. This effect is significantly higher than the summarized values of the respective monotherapies and supports the concept of the triple therapy.

Compound 4p was identified from a series of 6-hydroxy-4H-benzo[1,4]oxazin-3-ones as potent agonist of the human beta(2)-adrenoceptor with a high beta(1)/beta(2)-selectivity. A complete reversal of acetylcholine-induced bronchoconstriction which lasted over the whole study period of 5 h was demonstrated for 4p in a guinea pig in vivo model without any signs of cardiovascular effects up to 10-fold above the first dose reaching 100% bronchoprotection. The enantiomerically pure (R)-form of 4p exerted a bronchodilatory efficacy over 24 h in dogs and guinea pigs in the absence of systemic pharmacodynamic effects. Formoterol which was tested as comparator in the same in vivo models of acetylcholine-induced bronchoconstriction did not retain efficacy after 24 h. In summary, the preclinical pro. le of compound (R)-4p (olodaterol, also known as BI 1744 CL) suggests a potential for once-daily dosing in man accompanied with an improved safety pro. le. (C) 2010 Elsevier Ltd. All rights reserved.

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.