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Magnus Nilsson,1 Magdalena Rhedin,2 Ramon Hendrickx,3 Susanne Berglund,1 Antonio Piras,2 Parmis Blomgran,2 Anders Cavallin,2 Mia Collins,2 Göran Dahl,4 Bilel Dekkak,5 Therese Ericsson,3 Niklas Hagberg,6 Ann Aurell Holmberg,3 Agnes Leffler,2 Anders J Lundqvist,3 Thomais Markou,5,7 James Pinkerton,5,7 Lars Rönnblom,6 Stacey Siu,8 Vanessa Taylor,8 Tiiu Wennberg,2 Dimitrios Zervas,5,7 Arian D J Laurence,9 Suman Mitra,2 Maria G Belvisi,5,7 Mark Birrell,5,7 Annika Borde2 1Medicinal Chemistry, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 2Bioscience, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 3DMPK, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 4Discovery Science, R&D, AstraZeneca, Gothenburg, Sweden; 5Respiratory Pharmacology Group, Division of Airway Disease, National Heart and Lung Institute, Imperial College London, London, UK; 6Rheumatology and Science for Life Laboratories, Department of Medical Sciences, Uppsala University, Uppsala, Sweden; 7Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden; 8Rigel Pharmaceuticals, South San Francisco, CA, USA; 9Department of Haematology, University College London Hospitals NHS Foundation Trust, London, UKCorrespondence: Magnus Nilsson, Medicinal Chemistry, Research and Early Development, Respiratory & Immunology, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, SE-431 83, Sweden, Tel +46722237222, Email Magnus.Nilsson@astrazeneca.comPurpose: Janus kinase 1 (JAK1) is implicated in multiple inflammatory pathways that are critical for the pathogenesis of asthma, including the interleukin (IL)-4, IL-5, IL-13, and thymic stromal lymphopoietin cytokine signaling pathways, which have previously been targeted to treat allergic asthma. Here, we describe the development of AZD0449 and AZD4604, two novel and highly selective JAK1 inhibitors with promising properties for inhalation.Methods: The effects of AZD0449 and AZD4604 in JAK1 signaling pathways were assessed by measuring phosphorylation of signal transducer and activator of transcription (STAT) proteins and chemokine release using immunoassays of whole blood from healthy human volunteers and rats. Pharmacokinetic studies performed on rats evaluated AZD0449 at a lung deposited dose of 52 μg/kg and AZD4604 at 30 μg/kg. The efficacy of AZD0449 and AZD4604 was assessed by evaluating lung inflammation (cell count and cytokine levels) and the late asthmatic response (average enhanced pause [Penh]).Results: Both compounds inhibited JAK1-dependent cytokine signaling pathways in a dose-dependent manner in human and rat leukocytes. After intratracheal administration in rats, both compounds exhibited low systemic exposures and medium-to-long terminal lung half-lives (AZD0449, 34 hours; AZD4604, 5 hours). Both compounds inhibited STAT3 and STAT5 phosphorylation in lung tissue from ovalbumin (OVA)-challenged rats. AZD0449 and AZD4604 also inhibited eosinophilia in the lung and reduced the late asthmatic response, measured as Penh in the OVA rat model.Conclusion: AZD0449 and AZD4604 show potential as inhibitors of signaling pathways involved in asthmatic immune responses, with target engagement demonstrated locally in the lung. These findings support the clinical development of AZD0449 and AZD4604 for the treatment of patients with asthma.Keywords: AZD0449, AZD4604, JAK, STAT

Introduction: Asthma is a chronic inflammatory disease of the airways with typical symptoms manifesting in coughing, wheezing and dyspnoea. Whilst current treatments can be effective, some patients are not properly controlled, and the medications are not ideal and are associated with unwanted side effects. Thus, new therapies are urgently required. Typically, the chronic inflammation endotype is associated with increased levels of type 2 cytokine such as IL-4, IL-5, IL-9 & IL-13. These pro-inflammatory signalling pathways critically depend on Janus Kinase (JAK) I or II signal transduction, therefore the inhibition of JAK pathways represents a promising therapeutic target in asthma. Aims and Objectives: To assess the potential benefit of JAK inhibition using two structurally distinct inhibitors in a in vivo model system. Methods: Two JAK inhibitors, AZD4604 & AZD0449, were intratracheally dosed into sensitised Brown Norway rats one hour prior to aerosolised allergen (OVA) challenge. In one set of animals we assessed target engagement (TE) in the lung tissue by measuring levels of pSTATs. In a parallel set, we measured the Late Asthmatic Response (LAR) and lung inflammation. Results: Administration of both AZD4604 and AZD0449 resulted in dose related reduction of pSTAT3 and pSTAT5, indicating TE in the lung. Furthermore, AZD4604 & AZD0449 administration suppressed LAR and airway inflammation to a similar level to that achieved with the ICS comparator, budesonide. Conclusions: These data indicate that JAK inhibition may be of benefit to patients suffering from allergic asthma.

Background Asthmatics that are exposed to inhaled pollutants such as cigarette smoke (CS) have increased symptom severity. Approximately 25% of adult asthmatics are thought to be active smokers and many sufferers, especially in the third world, are exposed to high levels of inhaled pollutants. The mechanism by which CS or other airborne pollutants alter the disease phenotype and the effectiveness of treatment in asthma is not known. The aim of this study was to determine the impact of CS exposure on the phenotype and treatment sensitivity of rodent models of allergic asthma. Methods Models of allergic asthma were configured that mimicked aspects of the asthma phenotype and the effect of CS exposure investigated. In some experiments, treatment with gold standard asthma therapies was investigated and end-points such as airway cellular burden, late asthmatic response (LAR) and airway hyper-Reactivity (AHR) assessed. Results CS co-exposure caused an increase in the LAR but interestingly attenuated the AHR. The effectiveness of LABA, LAMA and glucocorticoid treatment on LAR appeared to be retained in the CS-exposed model system. The eosinophilia or lymphocyte burden was not altered by CS co-exposure, nor did CS appear to alter the effectiveness of glucocorticoid treatment. Steroids, however failed to reduce the neutrophilic inflammation in sensitized mice exposed to CS. Conclusions These model data have certain parallels with clinical findings in asthmatics, where CS exposure did not impact the anti-inflammatory efficacy of steroids but attenuated AHR and enhanced symptoms such as the bronchospasm associated with the LAR. These model systems may be utilised to investigate how CS and other airborne pollutants impact the asthma phenotype; providing the opportunity to identify novel targets.

Introduction: Asthma prevalence has increased world-wide especially in children; thus there is a need to develop new therapies that are safe and effective especially for patients with severe/refractory asthma. CD4 + T cells are thought to play a central role in disease pathogenesis and associated symptoms. Recently, TRPV4 has been demonstrated to regulate the activation and inflammatory properties of CD4 + cells. The aim of these experiments was to determine the role of TRPV4 in an asthma model using a TRPV4 inhibitor and genetically modified (GM) animals. Methods: Mice (wild type or TRPV4 -/- ) and rats were sensitised with antigen (HDM or OVA) and subsequently topically challenged with the same antigen. Key features associated with an allergic asthma type phenotype were measured: lung function (Late asthmatic response [LAR] in the rat model and airway hyperreactivity [AHR] in the mouse model), allergic status (IgE levels) and airway inflammation. Results : Targeting TRPV4 using either GM mice or a pharmacological inhibitor (dosed after sensitisation but before challenge) abolished IgE levels in the mouse model (100% inhibition of the HDM response) but had no impact in the rat. Airway inflammation was significantly decreased in both models (Rat BALF eosinophilia: 92.5% inhibition of the OVA response, mouse BALF eosinophilia: 88.5% inhibition of the HDM response). This was associated with suppression of antigen triggered changes in lung function (Rat LAR: 74.9% inhibition of the signal; mouse AHR: 92.4% inhibition of the response to 3 mg/ml of aerosolised 5-HT). Conclusion : These data would support the evaluation of TRPV4 inhibitors in allergen challenge models in proof of concept clinical trials.

Background Asthma prevalence has increased world-wide especially in children; thus there is a need to develop new therapies that are safe and effective especially for patients with severe/refractory asthma. CD4+ T cells are thought to play a central role in disease pathogenesis and associated symptoms. Recently, TRPV1 has been demonstrated to regulate the activation and inflammatory properties of CD4+ cells. The aim of these experiments was to demonstrate the importance of CD4+ T cells and the role of TRPV1 in an asthma model using a clinically ready TRPV1 inhibitor (XEN-D0501) and genetically modified (GM) animals. Methods Mice (wild type, CD4 −/− or TRPV1 −/−) and rats were sensitised with antigen (HDM or OVA) and subsequently topically challenged with the same antigen. Key features associated with an allergic asthma type phenotype were measured: lung function (airway hyperreactivity [AHR] and late asthmatic response [LAR]), allergic status (IgE levels) and airway inflammation. Results CD4+ T cells play a central role in both disease model systems with all the asthma-like features attenuated. Targeting TRPV1 using either GM mice or a pharmacological inhibitor tended to decrease IgE levels, airway inflammation and lung function changes. Conclusion Our data suggests the involvement of TRPV1 in allergic asthma and thus we feel this target merits further investigation.

Asthma is increasing globally and current treatments only manage a proportion of patients. There is an urgent need to develop new therapies. Lymphocytes are thought to play a central role in the pathophysiology of asthma through the production of inflammatory mediators. This is thought to be via the transcription factor NFAT which in turn can be activated through Ca(2+) release-activated Ca(2+) (CRAC) channels. The aim of this work was to investigate the role of CRAC in clinical and pre-clinical models of allergic asthma. Initial data demonstrated that the NFAT pathway is increased in stimulated lymphocytes from asthmatics. To confirm a role for the channel we showed that a selective inhibitor, Synta 66, blocked mediator production from lymphocytes. Synta 66 inhibited CD2/3/28 induced IL-2, IL-7, IL-13 & IFNΥ in a concentration-dependent manner in healthy and severe asthma donors, with over 60% inhibition observed for all cytokines. NFAT pathway was also increased in a pre-clinical asthma model. In this model we have demonstrated that CRAC played a central role in the airway inflammation and late asthmatic response (LAR). In conclusion, our data provides evidence that suggests targeting CRAC channels could be of therapeutic benefit for asthma sufferers.

BackgroundAsthma and chronic obstructive pulmonary disease (COPD) are chronic inflammatory diseases of the airway. Current treatment options (long acting β-adrenoceptor agonists and glucocorticosteroids) are not optimal as they are only effective in certain patient groups and safety concerns exist regarding both compound classes. Therefore, novel bronchodilator and anti-inflammatory strategies are being pursued. Prostaglandin E2 (PGE2) is an arachidonic acid-derived eicosanoid produced by the lung which acts on four different G-protein coupled receptors (EP1–4) to cause an array of beneficial and deleterious effects. The aim of this study was to identify the EP receptor mediating the anti-inflammatory actions of PGE2 in the lung using a range of cell-based assays and in vivo models.Methods and resultsIt was demonstrated in three distinct model systems (innate stimulus, lipopolysaccharide (LPS); allergic response, ovalbumin (OVA); inhaled pollutant, cigarette smoke) that mice missing functional EP4 (Ptger4−/−) receptors had higher levels of airway inflammation, suggesting that endogenous PGE2 was suppressing inflammation via EP4 receptor activation. Cell-based assay systems (murine and human monocytes/alveolar macrophages) demonstrated that PGE2 inhibited cytokine release from LPS-stimulated cells and that this was mimicked by an EP4 (but not EP1–3) receptor agonist and inhibited by an EP4 receptor antagonist. The anti-inflammatory effect occurred at the transcriptional level and was via the adenylyl cyclase/cAMP/ cAMP-dependent protein kinase (PKA) axis.ConclusionThis study demonstrates that EP4 receptor activation is responsible for the anti-inflammatory activity of PGE2 in a range of disease relevant models and, as such, could represent a novel therapeutic target for chronic airway inflammatory conditions.