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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

Idiopathic pulmonary fibrosis is a fatal interstitial lung disease with limited therapeutic options. Current evidence suggests that IPF may be initiated by repeated epithelial injury in the distal lung followed by abnormal wound healing responses which occur due to intrinsic and extrinsic factors. Mechanisms contributing to chronic damage of the alveolar epithelium in IPF include dysregulated cellular processes such as apoptosis, senescence, abnormal activation of developmental pathways, aging, as well as genetic mutations. Therefore, targeting the regenerative capacity of the lung epithelium is an attractive approach in the development of novel therapies for IPF. Endogenous lung regeneration is a complex process involving coordinated cross-talk between multiple cell types and re-establishment of a normal extracellular matrix environment. This review will describe the current knowledge of reparative epithelial progenitor cells in the alveolar region of the lung and discuss potential novel therapeutic approaches for IPF focusing on endogenous alveolar repair. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Transient receptor potential (TRP) channels are multifunctional signalling molecules with many roles in sensory perception and cellular physiology. Therefore, it is not surprising that TRP channels have been implicated in numerous diseases, including hereditary disorders caused by defects in genes encoding TRP channels (TRP channelopathies). Most TRP channels are located at the cell surface, which makes them generally accessible drug targets. Early drug discovery efforts to target TRP channels focused on pain, but as our knowledge of TRP channels and their role in health and disease has grown, these efforts have expanded into new clinical indications, ranging from respiratory disorders through neurological and psychiatric diseases to diabetes and cancer. In this Review, we discuss recent findings in TRP channel structural biology that can affect both drug development and clinical indications. We also discuss the clinical promise of novel TRP channel modulators, aimed at both established and emerging targets. Last, we address the challenges that these compounds may face in clinical practice, including the need for carefully targeted approaches to minimize potential side-effects due to the multifunctional roles of TRP channels., This Review discusses recent findings in transient receptor potential (TRP) channel structural biology and the impact of these findings in drug development and clinical indications. It also addresses the challenges of modulating TRP channels and the need for targeted approaches to minimize potential side-effects due to the multifunctional roles of TRP channels.

With prevalent global air pollution, individuals with certain genetic predispositions and sensitivities are at of higher risk of developing respiratory symptoms including chronic cough. Studies to date have relied on patient-filled questionnaires in epidemiological studies to evaluate the gene-by-environment interactions. In a controlled human exposure study, we evaluated whether genetic risk score (GRS) based on cough-related single-nucleotide polymorphisms (SNPs) are associated with a cough count over 24 h post-exposure to diesel exhaust (DE), a model for traffic-related air pollution. DE is a mixture of several known air pollutants including PM2.5, CO, NO, NO2, and volatile organic compounds. Under closely observed circumstances, we determined that GRS constructed from 7 SNPs related to TRPA1, TRPV1, and NK-2R were correlated with cough count. Selection of channels were based on prior knowledge that SNPs in these channels lead to acute airway inflammation as a result of their increased sensitivity to particulate matter. We performed a linear regression analysis and found a significant, positive correlation between GRS and cough count following DE exposure (p = 0.002, R2 = 0.61) and filtered air (FA) exposure (p = 0.028, R2 = 0.37). Although that correlation was stronger for DE than for FA, we found no significant exposure-by-GRS interaction. In summary, cough-relevant GRS was associated with a higher 24 h cough count in a controlled setting, suggesting that individuals with a high GRS may be more susceptible to developing cough regardless of their exposure. The trend towards this susceptibility being more prominent in the context of traffic-related air pollution remains to be confirmed.Trial registration: ClinicalTrial.gov NCT02236039; NCT0223603. Registered on August 11, 2014, https://clinicaltrials.gov/ct2/show/NCT02236039.

Aims This 3-part, randomised, phase 1 first-in-human study (NCT03436316) investigated the safety, tolerability and pharmacokinetics (PK) of AZD8154, a dual phosphoinositide 3-kinase (PI3K) γδ inhibitor developed as a novel inhaled anti-inflammatory treatment for respiratory disease. Methods Healthy men, and women of nonchildbearing potential, were enrolled to receive single and multiple ascending inhaled doses of AZD8154 in parts 1 and 3 of the study, respectively, while part 2 characterised the systemic PK after a single intravenous (IV) dose. In part 1, participants received 0.1-7.7 mg AZD8154 in 6 cohorts. In part 2, participants were given 0.15 mg AZD8154 as an IV infusion. In part 3, AZD8154 was given in 3 cohorts of 0.6, 1.8 and 3.1 mg, with a single dose on Day 1 followed by repeated once-daily doses on Days 4-12. Results In total, 78 volunteers were randomised. All single inhaled, single IV and multiple inhaled doses were shown to be well tolerated without any safety concerns. A population PK model, using nonlinear mixed-effect modelling, was developed to describe the PK of AZD8154. The terminal mean half-life of AZD8154 was 18.0-32.0 hours. The geometric mean of the absolute pulmonary bioavailability of AZD8154 via the inhaled route was 94.1%. Conclusion AZD8154 demonstrated an acceptable safety profile, with no reports of serious adverse events and no clinically significant drug-associated safety concerns reported in healthy volunteers. AZD8154 demonstrated prolonged lung retention and a half-life supporting once-daily dosing.

Idiopathic pulmonary fibrosis (IPF) is a fatal disorder characterised by progressive, destructive lung scarring. Despite substantial progress, the genetic determinants of this disease remain incompletely defined. Using whole genome and whole exome sequencing data from 752 individuals with sporadic IPF and 119,055 UK Biobank controls, we performed a variant-level exome-wide association study (ExWAS) and gene-level collapsing analyses. Our variant-level analysis revealed a novel association between a rare missense variant in SPDL1 and IPF (NM_017785.5:g.169588475 G > A p.Arg20Gln; p = 2.4 × 10−7, odds ratio = 2.87, 95% confidence interval: 2.03–4.07). This signal was independently replicated in the FinnGen cohort, which contains 1028 cases and 196,986 controls (combined p = 2.2 × 10−20), firmly associating this variant as an IPF risk allele. SPDL1 encodes Spindly, a protein involved in mitotic checkpoint signalling during cell division that has not been previously described in fibrosis. To the best of our knowledge, these results highlight a novel mechanism underlying IPF, providing the potential for new therapeutic discoveries in a disease of great unmet need., Ryan Dhindsa et al. conducted an exome-wide association study to identify a rare variant in SPDL1 as a risk factor for idiopathic pulmonary fibrosis (IPF). Their findings implicate mitotic checkpoint signalling as a new mechanism underlying IPF.

COPD and idiopathic pulmonary fibrosis (IPF) together represent a considerable unmet medical need, and advances in their treatment lag well behind those of other chronic conditions. Both diseases involve maladaptive repair mechanisms leading to progressive and irreversible damage. However, our understanding of the complex underlying disease mechanisms is incomplete; with current diagnostic approaches, COPD and IPF are often discovered at an advanced stage and existing definitions of COPD and IPF can be misleading. To halt or reverse disease progression and achieve lung regeneration, there is a need for earlier identification and treatment of these diseases. A precision medicine approach to treatment is also important, involving the recognition of disease subtypes, or endotypes, according to underlying disease mechanisms, rather than the current “one-size-fits-all” approach. This review is based on discussions at a meeting involving 38 leading global experts in chronic lung disease mechanisms, and describes advances in the understanding of the pathology and molecular mechanisms of COPD and IPF to identify potential targets for reversing disease degeneration and promoting tissue repair and lung regeneration. We also discuss limitations of existing disease measures, technical advances in understanding disease pathology, and novel methods for targeted drug delivery.

The interplay of type-2 inflammation and antiviral immunity underpins asthma exacerbation pathogenesis. Virus infection induces type-2 inflammation–promoting chemokines CCL17 and CCL22 in asthma; however, mechanisms regulating induction are poorly understood. By using a human rhinovirus (RV) challenge model in human airway epithelial cells in vitro and mice in vivo, we assessed mechanisms regulating CCL17 and CCL22 expression. Subjects with mild to moderate asthma and healthy volunteers were experimentally infected with RV and airway CCL17 and CCL22 protein quantified. In vitro airway epithelial cell- and mouse-RV infection models were then used to define STAT6- and NF-κB–mediated regulation of CCL17 and CCL22 expression. Following RV infection, CCL17 and CCL22 expression was higher in asthma, which differentially correlated with clinical and immunological parameters. Air–liquid interface–differentiated primary epithelial cells from donors with asthma also expressed higher levels of RV-induced CCL22. RV infection boosted type-2 cytokine–induced STAT6 activation. In epithelial cells, type-2 cytokines and STAT6 activation had differential effects on chemokine expression, increasing CCL17 and suppressing CCL22, whereas NF-κB promoted expression of both chemokines. In mice, RV infection activated pulmonary STAT6, which was required for CCL17 but not CCL22 expression. STAT6-knockout mice infected with RV expressed increased levels of NF-κB–regulated chemokines, which was associated with rapid viral clearance. Therefore, RV-induced upregulation of CCL17 and CCL22 was mediated by NF-κB activation, whereas expression was differentially regulated by STAT6. Together, these findings suggest that therapeutic targeting of type-2 STAT6 activation alone will not block all inflammatory pathways during RV infection in asthma.

Introduction: Chronic cough is a key symptom that plagues the lives of IPF patients, indeed, it is often the reason the disease is initially diagnosed. The aim of this project was to develop a pre-clinical model of IPF-associated cough and compare the mediator profiles with clinical samples, to aid in the search for effective treatments. Methods: Male guinea pigs (around 325g) were intratracheally dosed with vehicle (saline) or bleomycin (6 USP/kg) and coughs were quantified through manual observation of in-cage video footage, by watching 4.5 hours a day, for 3 weeks. Lung tissue, bronchoalveolar lavage fluid (BALF) and blood was harvested from a parallel group of animals. These were used to assess the extent of fibrosis and measure mediator production (e.g. a marker of oxidative stress: 8-isoprostane). Human BALF was obtained from IPF patients and healthy controls. Results: Spontaneous coughing was recorded in guinea pigs given bleomycin, which was associated with an increase in lung fibrosis and BALF 8-isoprostane (a marker of oxidative stress). We noted similar increases in BALF 8-isoprostane levels in the clinical samples. Conclusions: We have developed a preclinical model to aid in the search for a treatment for IPF-associated coughing. Furthermore, initial data suggests that an increase in oxidative burden (a known trigger of coughing) could be an attractive target.

Rationale: Effective cough treatments are a significant unmet need in patients with lung cancer. Aprepitant is a licensed treatment for nausea and vomiting, which blocks substance P activation of NK-1 (neurokinin 1) receptors, a mechanism also implicated in cough. Objectives: To assess aprepitant in patients with lung cancer with cough and evaluate mechanisms in vagal nerve tissue. Methods: Randomized double-blind crossover trial of patients with lung cancer and bothersome cough. They received 3 days of aprepitant or matched placebo; after a 3-day washout, patients crossed to the alternative treatment. The primary endpoint was awake cough frequency measured at screening and Day 3 of each treatment; secondary endpoints included patient-reported outcomes. In vitro, the depolarization of isolated guinea pig and human vagus nerve sections in grease-gap recording chambers, indicative of sensory nerve activation, was measured to evaluate the mechanism. Measurements and Main Results: Twenty patients with lung cancer enrolled, with a mean age 66 years (67.7); 60% were female and 80% had non–small cell cancer, 50% had advanced stage, and 55% had World Health Organization performance status 1. Cough frequency improved with aprepitant, reducing by 22.2% (95% confidence interval [CI], 2.8–37.7%) over placebo while awake (P = 0.03), 30.3% (95% CI, 12.7–44.3) over 24 hours (P = 0.002), and 59.8% (95% CI, 15.1–86.0) during sleep (P = 0.081). Patient-reported outcomes all significantly improved. Substance P depolarized both guinea pig and human vagus nerve. Aprepitant significantly inhibited substance P–induced depolarization by 78% in guinea pig (P = 0.0145) and 94% in human vagus (P = 0.0145). Conclusions: Substance P activation of NK-1 receptors appears to be an important mechanism driving cough in lung cancer, and NK-1 antagonists show promise as antitussive therapies.

Chronic cough is a burden of many respiratory diseases with no effective & safe treatment. The prostaglandin PGE2 acts on EP receptors (EP1-4) with beneficial & adverse effects in the lungs. We have shown EP4 agonism inhibits activation of airway sensory nerves, blocking cough1. We aimed to determine the signalling downstream of EP4 in airway sensory nerves, to better understand its anti-tussive effect. We isolated guinea pig (GP) vagal ganglia neurons, using retrograde tracer dye to identify airway neurons in vitro. We assessed intracellular calcium & cAMP in these neurons using chemical & virally transfected fluorophores. We also used a grease-gap recording technique to pharmacologically examine EP4 signalling in GP & human vagus nerves in vitro. We have shown that EP4 agonist inhibits capsaicin-induced activation of GP airway neurons. EP4 is known to be a Gαs-coupled GPCR, so we imaged cAMP, observing that EP4 agonism causes cAMP elevation. Dual-fluorophore imaging showed cAMP elevation in neurons where EP4 agonism had inhibitory effects. From previous work we know EP4 agonist inhibits capsaicin-induced GP & human vagus nerve activation. We looked whether the main cAMP effectors (PKA & EPAC) were involved. EPAC (but not PKA) blockers (ESI-09 & KT5720) abolished the inhibitory effect of EP4 agonism. EPAC activator (8-CPT-2Me-cAMP) mimicked the effect of EP4 agonism; this was blocked by EPAC inhibitor. These data reveal a role for cAMP & EPAC in the inhibitory & anti-tussive effects of EP4 agonism on airway sensory nerves. Understanding how EP4 exerts its beneficial effects on airway sensory nerves is important to better inform drug discovery efforts for anti-tussive therapies. 1.Wortley(2019)AJRCCM;199:A2862

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.

Research using animal models of asthma is currently dominated by mouse models. This has been driven by the comprehensive knowledge on inflammatory and immune reactions in mice, as well as tools to produce genetically modified mice. Many of the identified therapeutic targets influencing airway hyper-responsiveness and inflammation in mouse models, have however been disappointing when tested clinically in asthma. It is therefore a great need for new animal models that more closely resemble human asthma. The guinea pig has for decades been used in asthma research and a comprehensive table of different protocols for asthma models is presented. The studies have primarily been focused on the pharmacological aspects of the disease, where the guinea pig undoubtedly is superior to mice. Further reasons are the anatomical and physiological similarities between human and guinea pig airways compared with that of the mouse, especially with respect to airway branching, neurophysiology, pulmonary circulation and smooth muscle distribution, as well as mast cell localization and mediator secretion. Lack of reagents and specific molecular tools to study inflammatory and immunological reactions in the guinea pig has however greatly diminished its use in asthma research. The aim in this position paper is to review and summarize what we know about different aspects of the use of guinea pig in vivo models for asthma research. The associated aim is to highlight the unmet needs that have to be addressed in the future.

Idiopathic pulmonary fibrosis (IPF) is a fatal disorder characterised by progressive, destructive lung scarring. Despite significant progress, the genetic determinants of this disease remain incompletely defined. Using next generation sequencing data from 752 individuals with sporadic IPF and 119,055 controls, we performed both variant- and gene-level analyses to identify novel IPF genetic risk factors. Our variant-level analysis revealed a novel rare missense variant in SPDL1 (NM_017785.5 p.Arg20Gln; p = 2.4 × 10−7, odds ratio = 2.87). This signal was independently replicated in the FinnGen cohort (combined p = 2.2 × 10−20), firmly associating this variant as a novel IPF risk allele. SPDL1 encodes Spindly, a protein involved in mitotic checkpoint signalling during cell division that has not been previously described in fibrosis. Our results highlight a novel mechanism underlying IPF, providing the potential for new therapeutic discoveries in a disease of great unmet need.

Background:Oral corticosteroids such as prednisolone are potent anti-inflammatory drugs but their use is limited by side effects caused by unwanted actions on the glucocorticoid receptor (GR), such as increased insulin resistance, and off-target actions on the mineralocorticoid receptor (MR) that disrupt electrolyte balance and increase water retention. AZD9567 is an oral, selective, non-steroidal glucocorticoid receptor modulator being developed to treat inflammatory diseases. Pre-clinical and phase 1 clinical data indicate that AZD9567 is the first GR modulator with an improved efficacy–dysglycaemic side effect profile versus prednisolone.Objectives:To compare the efficacy, safety and tolerability of AZD9567 with prednisolone in patients with active rheumatoid arthritis (RA), at doses with predicted equivalent anti-inflammatory activity.Methods:In this phase 2a, randomised, double-blind, parallel-group, multicentre study in RA patients with DAS28-CRP ≥ 3.2 despite stable treatment with conventional disease-modifying anti-rheumatic therapies (NCT03368235), patients were randomised 1:1 to AZD9567 40 mg or prednisolone 20 mg orally once daily for 14 days. The primary endpoint was change from baseline in DAS28-CRP at day 15. Secondary outcomes included components of DAS28-CRP, TJC68, SJC66, ACR response (ACR20, ACR50, ACR70) and safety outcomes, including serum electrolytes.Results:All 21 randomised patients (AZD9567, n = 11; prednisolone, n = 10) completed the study. There was a slight imbalance between the treatment groups at baseline, with higher age (mean ± SD: 64.5 ± 8.4 vs 55.5 ± 13.6 years), more women (8 vs 5) and greater disease severity (DAS28-CRP, mean ± SD: 5.26 ± 0.98 vs 4.90 ± 0.74) in the AZD9567 group versus the prednisolone group. There was no statistically significant or clinically meaningful (i.e. > 1.2) difference in change from baseline to day 15 in DAS28-CRP between AZD9567 and prednisolone, although this was numerically lower with AZD9567 (Table 1). Similar results were observed for TJC68, SJC66, CRP and GH (Table 1). The proportions of patients achieving ACR20, 50 and 70 response criteria were similar in both groups, albeit numerically lower with AZD9567. Similar numbers of patients in each group reported treatment-emergent adverse events (AZD9567, n = 10, prednisolone, n = 9); most were mild in severity. One serious adverse event, suicidal depression, was reported after completing AZD9567 treatment. Morning fasting serum sodium/potassium ratio at day 15 was not altered with AZD9567 but was increased from baseline with prednisolone (Figure 1).Conclusion:AZD9567 40 mg had a similar efficacy profile to prednisolone 20 mg in patients with active RA. Both drugs were well tolerated, with no new safety signals. Unlike prednisolone, AZD9567 had no effect on serum sodium/potassium ratio, suggesting selectivity of AZD9567 for the GR over the MR. These results support further trials of AZD9567 in patients with inflammatory disease.Table 1.Change from baseline to day 15 in clinical disease activity measures.AZD9567 (n = 11)Prednisolone (n = 10)Comparison(AZD9567 – prednisolone)LSM CFB (SE)95% CILSM CFB (SE)95% CILSMD (SE)95% CIp valueDAS28−CRP score−1.93 (0.35)−2.66, −1.21−2.40 (0.34)−3.11, −1.700.47 (0.46)−0.49, 1.430.315TJC28 score−6.12 (1.25)−8.76, −3.49−6.07 (1.21)−8.61, −3.52−0.05 (1.60)−3.43, 3.320.973SJC28 score−5.14 (0.65)−6.51, −3.76−5.40 (0.63)−6.73, −4.080.26 (0.84)−1.50, 2.030.757GH score−27.7 (7.3)−42.8, −12.5−37.4 (7.1)−52.3, −22.69.8 (9.7)−10.5, 30.10.325CRP, mg/L−10.8 (2.4)−15.9, −5.8−15.6 (2.5)−20.9, −10.34.8 (3.5)−2.5, 12.00.187TJC68 score−9.02 (2.46)−14.21, −3.82−7.90 (2.36)−12.88, −2.91−1.12 (3.12)−7.69, 5.460.724SJC66 score−6.24 (0.89)−8.13, −4.36−6.66 (0.86)−8.48, −4.850.42 (1.14)−1.98, 2.810.717CFB, change from baseline; CRP, C-reactive protein; DAS28, 28-joint disease activity score; GH, global health; LSM(D), least-squares mean (difference); SJC, swollen joint count; TJC, tender joint count.Acknowledgements:Medical writing support was provided by Richard Claes PhD of PharmaGenesis London, London, UK, funded by AstraZeneca, Gothenburg, Sweden in accordance with Good Publication Practice 3 (GPP3) guidelines (http://www.ismpp.org/gpp3).This study was funded by AstraZeneca. AZD9567 is an investigational medical product with no approved indication.Disclosure of Interests:Jacob M. van Laar Consultant of: Honoraria from - Abbvie, Arxx Tx, Galapagos, Gesyntha, Leadiant, Magenta, Roche, Sanofi Genzyme, Grant/research support from: AstraZeneca, Pfizer, Roche, Alejhandra Lei Shareholder of: AstraZeneca., Grant/research support from: Boehringer Ingelheim in 1998, Bristol-Myers Squibb in 1999, Employee of: AstraZeneca. Past employee of Almirall, Grünenthal, Boehringer Ingelheim, CESIF Pharma, Mary Safy-Khan Grant/research support from: Student grant from AstraZeneca 2015-2018., Joachim Almquist Shareholder of: AstraZeneca, Consultant of: AstraZeneca., Employee of: AstraZeneca., Carol Astbury Shareholder of: AstraZeneca., Employee of: AstraZeneca., Maria Belvisi Shareholder of: AstraZeneca., Grant/research support from: AstraZeneca and Chiesi, Employee of: AstraZeneca., Adam Platt Shareholder of: AstraZeneca., Employee of: AstraZeneca., Susanne Prothon Shareholder of: AstraZeneca., Employee of: AstaZeneca., Sara Samuelsson Shareholder of: AstraZeneca, Employee of: AstraZeneca, Petter Svanberg Employee of: AstraZeneca, Christina Keen Shareholder of: AstraZeneca., Employee of: AstraZeneca.

BackgroundNavafenterol (AZD8871) belongs to a new class of bronchodilator, the single-molecule muscarinic antagonist and β-agonist, developed for the treatment of COPD. This study aimed to evaluate the efficacy, pharmacokinetics and safety of navafenterol versus placebo and an active comparator treatment for moderate-to-severe COPD.MethodsThis phase 2a, randomised, multicentre (Germany and UK), double-blind, double-dummy, three-way complete crossover study (ClinicalTrials.gov identifier: NCT03645434) compared 2 weeks’ treatment of once-daily navafenterol 600 µg via inhalation with placebo and a fixed-dose combination bronchodilator (umeclidinium/vilanterol (UMEC/VI); 62.5 µg/25 µg) in participants with moderate-to-severe COPD. The primary outcome was change from baseline in trough forced expiratory volume in 1 s (FEV1) on day 15. Secondary end-points included change from baseline in peak FEV1; change from baseline in Breathlessness, Cough and Sputum Scale (BCSS); change from baseline in COPD Assessment Tool (CAT); adverse events; and pharmacokinetics.Results73 participants were randomised. After 14 days, trough FEV1 was significantly improved with navafenterol compared with placebo (least-squares (LS) mean difference 0.202 L; p1 between navafenterol and UMEC/VI (LS mean difference −0.046 L; p=0.075). COPD symptoms (CAT and BCSS) showed significantly greater improvements with both active treatments versus placebo (all pConclusionOnce-daily navafenterol was well tolerated, improved lung function and reduced COPD-related symptoms, similar to an established once-daily fixed-dose combination bronchodilator.

Cough is the most common reason for patients to visit a primary care physician, yet it remains an unmet medical need. It can be idiopathic in nature but can also be a troublesome symptom across chronic lung diseases such as asthma, COPD and idiopathic pulmonary fibrosis (IPF). Chronic cough affects up to 12% of the population and yet there are no safe and effective therapies. The cough reflex is regulated by vagal, sensory afferent nerves which innervate the airway. The Transient Receptor Potential (TRP) family of ion channels are expressed on sensory nerve terminals, and when activated can evoke cough. This review focuses on the role of 4 TRP channels; TRP Vannilloid 1 (TRPV1), TRP Ankyrin 1 (TRPA1), TRP Vannilloid 4 (TRPV4) and TRP Melastatin 8 (TRPM8) and the purinergic P2X3 receptor and their possible role in chronic cough. We conclude that these ion channels, given their expression profile and their role in the activation of sensory afferents and the cough reflex, may represent excellent therapeutic targets for the treatment of respiratory symptoms in chronic lung disease.

Bronchoconstriction is a key feature of asthma but the disease drivers and signalling mechanisms involved are not fully understood. TRPM3, a member of the Transient Receptor family of ion channels, has recently been shown to activate airway sensory nerves in both animal and human tissue (Bonvini et al 2018, AJRCCM:197:A7411). However, its role in direct bronchoconstriction is currently untested. We therefore aimed to investigate the role of TRPM3 in contraction of airway smooth muscle. The TRPM3 agonist CIM0216 was shown to cause bronchospasm in vivo in the anaesthetised guinea pig (GP), independent of sensory nerve activation. Tissue bath assays indicated that CIM0216 caused a concentration dependent contraction of GP trachea, which was inhibited by the TRPM3 antagonist Primidone. Further investigation revealed that both removal of the epithelium and addition of the selective NK2 antagonist MEN10376 significantly inhibited the contraction induced by CIM0216. RTPCR studies revealed that the TRPM3 gene was expressed on GP epithelial cells, and the TACR2 gene, which is the receptor for NK2, was expressed on GP ASM. These data suggest that activation of TRPM3 on epithelial cells leads to the release of tachykinins which activate the NK2 receptor on airway smooth muscle to cause bronchoconstriction. Tachykinins such as NKA have been well documented to cause contraction in the asthmatic airway. Although further work is required, this implicates the TRPM3 receptor on epithelial cells as a potential non-neuronal source of tachykinin release and subsequent bronchoconstriction, and also highlights it as a possible novel therapeutic target.

Over interpretation of cough challenge data only adds to the confusion in our understanding of the mechanisms underlying chronic coughhttp://bit.ly/2X52CQt

The past 3 years have seen expansion of ERS fellowship programmes, driven by the 2013–2016 ERS fellowship strategyhttp://ow.ly/6r9D30279J7

Most airway diseases, including chronic obstructive pulmonary disease (COPD), are associated with excessive coughing. The extent to which this may be a consequence of increased activation of vagal afferents by pathology in the airways (e.g., inflammatory mediators, excessive mucus) or an altered neuronal phenotype is unknown. Understanding whether respiratory diseases are associated with dysfunction of airway sensory nerves has the potential to identify novel therapeutic targets.To assess the changes in cough responses to a range of inhaled irritants in COPD and model these in animals to investigate the underlying mechanisms.Cough responses to inhaled stimuli in patients with COPD, healthy smokers, refractory chronic cough, asthma, and healthy volunteers were assessed and compared with vagus/airway nerve and cough responses in a cigarette smoke (CS) exposure guinea pig model.Patients with COPD had heightened cough responses to capsaicin but reduced responses to prostaglandin E2 compared with healthy volunteers. Furthermore, the different patient groups all exhibited different patterns of modulation of cough responses. Consistent with these findings, capsaicin caused a greater number of coughs in CS-exposed guinea pigs than in control animals; similar increased responses were observed in ex vivo vagus nerve and neuron cell bodies in the vagal ganglia. However, responses to prostaglandin E2 were decreased by CS exposure.CS exposure is capable of inducing responses consistent with phenotypic switching in airway sensory nerves comparable with the cough responses observed in patients with COPD. Moreover, the differing profiles of cough responses support the concept of disease-specific neurophenotypes in airway disease. Clinical trial registered with www.clinicaltrials.gov (NCT 01297790).

Background and Purpose Airway microvascular leak (MVL) involves the extravasation of proteins from post-capillary venules into surrounding tissue. MVL is a cardinal sign of inflammation and an important feature of airway inflammatory diseases such as asthma. Prostaglandin E2 (PGE2), a product of cyclooxygenase mediated metabolism of arachidonic acid, binds to 4 receptors, termed EP1-4. PGE2 has a wide variety of effects within the airway, including modulation of inflammation, sensory nerve activation and airway tone. However, the effect of PGE2 on airway MVL and the receptor/s that mediate this have not been described. Experimental Approach Evans Blue dye was used as a marker of airway MVL and selective EP receptor agonists and antagonists were used alongside EP receptor deficient mice to define the receptor subtype involved. Key Results PGE2 induced significant airway MVL in mice and guinea pigs. A significant reduction in PGE2-induced MVL was demonstrated in Ptger2-/- and Ptger4-/- mice and in wild type mice pre-treated simultaneously with EP2 (PF-04418948) and the EP4 (ER-819762) receptor antagonists. In a model of allergic asthma, an increase in airway levels of PGE2 was associated with a rise in MVL; this change was absent in Ptger2-/- and Ptger4-/- mice. Conclusions and Implications PGE2 is a key mediator produced by the lung and has widespread effects according to the EP receptor activated. Airway MVL represents a response to injury and under ‘disease’ conditions is a prominent feature of airway inflammation. The data presented highlight a key role for EP2 and EP4 receptors in MVL induced by PGE2. This article is protected by copyright. All rights reserved.

Mast cell–airway smooth muscle (ASM) interactions play a major role in the immunoglobulin (Ig)E- dependent bronchoconstriction seen in asthma but less is known about IgE-independent mechanisms of mast cell activation. Transient receptor potential cation channel, subfamily V, member 4 (TRPV4) activation causes contraction of human ASMviathe release of cysteinyl leukotrienes (cysLTs) but the mechanism is unknown. The objective of the present study was to investigate a role for IgE-independent, mast cell–ASM interaction in TRPV4-induced bronchospasm.Bronchoconstriction was measured in anaesthetised guinea pigs and contraction of human and guinea-pig airway tissue assessed using isometric tension measurements. Increases in intracellular [Ca2+] were imaged using the Ca2+-sensitive dye FURA2, and time-lapse ptychography was utilised as a surrogate for contraction of ASM cells.The TRPV4 agonist GSK1016790A caused contractionin vivoin the guinea pig, and in human and guinea-pig tracheal tissue, which was inhibited by the TRPV4 antagonist GSK2193874. GSK1016790A increased [Ca2+]iand released ATP in human ASM cells without causing contraction. TRPV4 and ATP evoked contraction in isolated tracheal tissue but co-culture experiments indicated a requirement for human lung mast cells. Expression profiling and pharmacological studies demonstrated that mast cell activation was dependent upon ATP activating the P2X4 receptor. Trypsin was shown to evoke contraction of tracheal tissueviaactivation of PAR-2-TRPV4-ATP-cysLT axis indicating the potential disease relevance of this signalling pathway.TRPV4 activation increases [Ca2+]iand releases ATP from ASM cells triggering P2X4-dependent release of cysLTs from mast cells resulting in ASM contraction. This study delineates a novel mast cell–ASM interaction and TRPV4 as a driver of IgE-independent mast cell-dependent bronchospasm.

Introduction: Cough is an essential defensive reflex, however excessive/chronic cough is a problematic symptom of respiratory diseases. We have previously shown that inhaled LABA can attenuate cough responses by suppressing airway sensory nerves. As LABAs are commonly prescribed to asthmatic patients where coughing persists, we hypothesised that this beneficial property may be lost due to tachyphylaxis as seen in airway smooth muscle (ASM) (Finney et al, BJP, 2001). Methods: Guinea pigs were treated with inhaled vehicle/formoterol (3μg/ml, 10 min) b.i.d. for 7 days. On day 8 inhaled vehicle/formoterol was administered 1h prior to assessments. Formoterol ASM relaxation was assessed on tracheal segments. Formoterol anti-tussive activity was assessed on capsaicin-evoked cough in vivo, & capsaicin-induced vagus nerve depolarisation in vitro. Results: Formoterol relaxation of ASM was reduced in tissues from animals chronically dosed with LABA, indicating tachyphylaxis of the β2-adrenoreceptor (β2A) (Fig. 1a). Formoterol inhibition of cough and sensory nerve activation was not altered (Fig.1b/c), suggesting β2A on nerves was not desensitised. Conclusions: As reported by others in pre-clinical systems & observed clinically, prolonged LABA dosing can lead to β2A tachyphylaxis on ASM and reduced bronchodilator effectiveness. In this study, it appeared that the impact of LABAs on airway sensory nerves was not altered by chronic treatment.

Increasing concerns over the health effects of inhaling air pollutants such as diesel exhaust particles (DEP) has led to a worldwide effort to find alternative fuels such as biodiesel. Indeed, exposure to DEP has been associated with worsening of symptoms in asthmatics (Bernstein D.AAIR:2012,4,4,178-183). Recently, we reported that this could be due to the ability of DEP to activate TRPA1 ion channels on airway sensory nerves and subsequently trigger unwanted respiratory reflexes (Robinson R.JACI.2017.EPub). The aim of this study was to examine whether the exhaust particles from using rape methyl ester (RME) biodiesel shared the same impact as DEP on airway sensory nerves. We profiled the impact of exhaust extract from engines powered with petro-diesel and biodiesel on vagal sensory nerves in ex vivo and in vivo systems. Like DEP, aerosolised biodiesel extract was able to trigger firing of guinea pig pulmonary C-fibres. In the ex vivo system, both petro-diesel and biodiesel extract activated the vagus nerve. As reported with DEP, nerve activation by biodiesel was inhibited with an antioxidant and a TRPA1 antagonist (n-acetyl-cysteine 1mM: 0.11±0.08 – 0.0096±0.006mV and Jansenn-130 10µM: 0.093±0.003 mV – 0.006±0.0008mV). These data indicate that exhaust from an engine powered with biodiesel can activate airway sensory nerves through an oxidant-stress driven activation of TRPA1 (similar to petro-diesel). This suggests that exposure to it may also activate reflex events to produce or exacerbate respiratory symptoms.

Background: The Late Asthmatic Response (LAR) is a salient feature of asthma and is often used as a clinical endpoint when testing new medicines. Yet, it is poorly represented in preclinical studies and tends to rely on acute allergen challenge paradigms. These may not be predictive of therapeutic efficacy in asthma. The aim of this study was to characterise the impact of serial antigen challenges in a rodent model. Methods: Sensitised Brown Norway rats were challenged with aerosolised ovalbumin (OVA) once a week for up to five weeks. Lung function and airway inflammation were assessed after 1, 3 and 5 challenges to compare acute and chronic exposures. A time course of airway inflammation was further investigated 1, 2, 4, 6, 24 and 48 hours after a single or five challenges. The effect of the Gold standard asthma therapy, budesonide, was tested on the chronic model by dosing rats with vehicle or budesonide (0.03, 0.3, 3 or 30 mg/kg, p.o.) around the fifth challenge, and assessing lung function and airway inflammation. Results: Acute antigen challenge triggered a LAR followed by an increase in cellular burden in the lungs. After chronic exposure, the magnitude of the LAR remained whereas the cellular inflammation was attenuated. The time course of cellular inflammation appeared similar in profile after a single or multiple challenges. Treatment with budesonide was able to suppress both the LAR and the cellular inflammation. Conclusion: We have shown that the LAR signal is robust and sensitive to Gold standard asthma treatment after chronic antigen exposure. This model could be useful to study novel asthma therapies and to better understand the LAR.

Transient Receptor Potential (TRP) A1 channels are expressed on airway sensory nerves and are linked to the initiation of respiratory symptoms and disease pathogenesis. TRPA1 is believed to be an oxidative stress (OS) sensor and is triggered by a range of stimuli e.g. acrolein, PGE2 and bradykinin (BK), hydrogen peroxide (HP), diesel exhaust particles (DEP), hyper-osmolarity (HO) and low temperature (10°C). The aim was to investigate the mechanisms involved in TRPA1 activation on airway sensory nerves and study the role played by OS. We assessed activation of guinea-pig (gp), mice (wild-type versus TRPA1 KO) and human vagus nerve. We used a selective TRPA1 antagonist and three different antioxidants: N-acetyl-L-cysteine (NAC, an extracellular oxidative stress scavenger), Glutathione (GSH, an intracellular anti-oxidant) and VAS2780 (NADPH oxidase inhibitor). The results showed that the TRPA1 antagonist abolished responses to acrolein, HP, DEP, and 10°C and were lost in the tissue from TRPA1 KO mice, whereas responses to PGE2, BK and HO were partially suppressed (approx. 50%). NAC and GSH inhibited acrolein, HP and DEP activation. Whereas, PGE2, BK and HO induced activation appeared to be dependent on NADPH oxidase. Stimulation with 10°C was not sensitive to the anti-oxidants. We have shown a number of different modalities activate TRPA1 on sensory nerves and they, with the exception of 10°C, are mediated via an oxidative stress response. Interestingly, it appears that a number of stimuli are dependent on NADPH oxidase activity to activate TRPA1. This highlights the range of disease relevant mechanisms by which TRPA1 can be activated and implies that modulating the channel is a promising target for future drug discovery.

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.

Cellular senescence has been associated with the structural and functional decline observed during physiological lung aging and in chronic obstructive pulmonary disease (COPD). Airway epithelial cells are the first line of defense in the lungs and are important to COPD pathogenesis. However, the mechanisms underlying airway epithelial cell senescence, and particularly the role of telomere dysfunction in this process, are poorly understood. We aimed to investigate telomere dysfunction in airway epithelial cells from patients with COPD, in the aging murine lung and following cigarette smoke exposure. We evaluated colocalization of γ-histone protein 2A.X and telomeres and telomere length in small airway epithelial cells from patients with COPD, during murine lung aging, and following cigarette smoke exposure in vivo and in vitro. We found that telomere-associated DNA damage foci increase in small airway epithelial cells from patients with COPD, without significant telomere shortening detected. With age, telomere-associated foci increase in small airway epithelial cells of the murine lung, which is accelerated by cigarette smoke exposure. Moreover, telomere-associated foci predict age-dependent emphysema, and late-generation Terc null mice, which harbor dysfunctional telomeres, show early-onset emphysema. We found that cigarette smoke accelerates telomere dysfunction via reactive oxygen species in vitro and may be associated with ataxia telangiectasia mutated-dependent secretion of inflammatory cytokines interleukin-6 and -8. We propose that telomeres are highly sensitive to cigarette smoke-induced damage, and telomere dysfunction may underlie decline of lung function observed during aging and in COPD.