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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

Airway epithelial damage is a common feature in respiratory diseases such as COPD and has been suggested to drive inflammation and progression of disease. These features manifest as remodeling and destruction of lung epithelial characteristics including loss of small airways which contributes to chronic airway inflammation. Histone deacetylase 6 (HDAC6) has been shown to play a role in epithelial function and dysregulation, such as in cilia disassembly, epithelial to mesenchymal transition (EMT) and oxidative stress responses, and has been implicated in several diseases. We thus used ACY-1083, an inhibitor with high selectivity for HDAC6, and characterized its effects on epithelial function including epithelial disruption, cytokine production, remodeling, mucociliary clearance and cell characteristics.Primary lung epithelial air-liquid interface cultures from COPD patients were used and the impacts of TNF, TGF-β, cigarette smoke and bacterial challenges on epithelial function in the presence and absence of ACY-1083 were tested. Each challenge increased the permeability of the epithelial barrier whilst ACY-1083 blocked this effect and even decreased permeability in the absence of challenge. TNF was also shown to increase production of cytokines and mucins, with ACY-1083 reducing the effect. We observed that COPD-relevant stimulations created damage to the epithelium as seen on immunohistochemistry sections and that treatment with ACY-1083 maintained an intact cell layer and preserved mucociliary function. Interestingly, there was no direct effect on ciliary beat frequency or tight junction proteins indicating other mechanisms for the protected epithelium.In summary, ACY-1083 shows protection of the respiratory epithelium during COPD-relevant challenges which indicates a future potential to restore epithelial structure and function to halt disease progression in clinical practice.

Inverse agonists of the nuclear receptor RORC2 have been widely pursued as a potential treatment for a variety of autoimmune diseases. We have discovered a novel series of isoindoline-based inverse agonists of the nuclear receptor RORC2, derived from our recently disclosed RORC2 inverse agonist 2. Extensive structure-activity relationship (SAR) studies resulted in AZD0284 (20), which combined potent inhibition of IL-17A secretion from primary human TH17 cells with excellent metabolic stability and good PK in preclinical species. In two preclinical in vivo studies, compound 20 reduced thymocyte numbers in mice and showed dose-dependent reduction of IL-17A containing γδ-T cells and of IL-17A and IL-22 RNA in the imiquimod induced inflammation model. Based on these data and a favorable safety profile, 20 was progressed to phase 1 clinical studies.