IL-13 and IL-4, but not IL-5 nor IL-17A, induce hyperresponsiveness in isolated human small airways.

BACKGROUND: Specific inflammatory pathways are indicated to contribute to severe asthma, but their individual involvement in the development of airway hyperresponsiveness remains unexplored. OBJECTIVE: This experimental study in human small bronchi aimed to provide insight into which of the type 2 and type 17 cytokines cause hyperresponsiveness of airway smooth muscle. METHODS: Explanted small bronchi isolated from human lung tissue and human airway smooth muscle cells were treated for 2 and 1 day(s), respectively, with 100 ng/mL of IL-4, IL-5, IL-13, or IL-17A, and contractile responses, Ca2+ mobilization, and receptor expression were assessed. RESULTS: Treatment with IL-13 increased the potency of histamine, carbachol, and leukotriene D4 as contractile agonists. IL-4, but not IL-5 or IL-17A, also increased the potency of histamine. In human airway smooth muscle cells, IL-13 and IL-4, but not IL-5 and IL-17A, enhanced the histamine-induced Ca2+ mobilization that was accompanied with increased mRNA expression of histamine H1 and cysteinyl leukotriene CysLT1 receptors. RNA sequencing of isolated bronchi confirmed the IL-13-mediated upregulation of H1 and CysLT1 receptors, without showing an alteration of muscarinic M3 receptors. Dexamethasone had no effects on IL-13-induced hyperresponsiveness in human bronchi, the increased Ca2+ mobilization, or the enhanced receptor expression. In contrast, antagonism of the common receptor for IL-13 and IL-4 by the biologic dupilumab prevented the effects of both IL-13 and IL-4 in human bronchi and human airway smooth muscle cells. CONCLUSIONS: The glucocorticoid-insensitive hyperrresponsiveness in isolated human airways induced by IL-13 and IL-4 provides further evidence that the IL-4Rα pathway should be targeted as a new strategy for the treatment of airway hyperresponsiveness in asthma.

Long-Acting ß2-Adrenoceptor Agonists Enhance Glucocorticoid Receptor (GR)-Mediated Transcription by Gene-Specific Mechanisms Rather Than Generic Effects via GR.

In asthma, the clinical efficacy of inhaled corticosteroids (ICSs) is enhanced by long-acting ß2-adrenoceptor agonists (LABAs). ICSs, or more accurately, glucocorticoids, promote therapeutically relevant changes in gene expression, and, in primary human bronchial epithelial cells (pHBECs) and airway smooth muscle cells, this genomic effect can be enhanced by a LABA. Modeling this interaction in human bronchial airway epithelial BEAS-2B cells transfected with a 2× glucocorticoid response element (2×GRE)-driven luciferase reporter showed glucocorticoid-induced transcription to be enhanced 2- to 3-fold by LABA. This glucocorticoid receptor (GR; NR3C1)-dependent effect occurred rapidly, was insensitive to protein synthesis inhibition, and was maximal when glucocorticoid and LABA were added concurrently. The ability of LABA to enhance GR-mediated transcription was not associated with changes in GR expression, serine (Ser203, Ser211, Ser226) phosphorylation, ligand affinity, or nuclear translocation. Chromatin immunoprecipitation demonstrated that glucocorticoid-induced recruitment of GR to the integrated 2×GRE reporter and multiple gene loci, whose mRNAs were unaffected or enhanced by LABA, was also unchanged by LABA. Transcriptomic analysis revealed glucocorticoid-induced mRNAs were variably enhanced, unaffected, or repressed by LABA. Thus, events leading to GR binding at target genes are not the primary explanation for how LABAs modulate GR-mediated transcription. As many glucocorticoid-induced genes are independently induced by LABA, gene-specific control by GR- and LABA-activated transcription factors may explain these observations. Because LABAs promote similar effects in pHBECs, therapeutic relevance is likely. These data illustrate the need to understand gene function(s), and the mechanisms leading to gene-specific induction, if existing ICS/LABA combination therapies are to be improved.

Selective Nonsteroidal Glucocorticoid Receptor Modulators for the Inhaled Treatment of Pulmonary Diseases.

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate 1b (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, 15m (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid 3 (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment.

Benchmarking of Human Dose Prediction for Inhaled Medicines from Preclinical In Vivo Data.

PURPOSE: A scientifically robust prediction of human dose is important in determining whether to progress a candidate drug into clinical development. A particular challenge for inhaled medicines is that unbound drug concentrations at the pharmacological target site cannot be easily measured or predicted. In the absence of such data, alternative empirical methods can be useful. This work is a post hoc analysis based on preclinical in vivo pharmacokinetic/pharmacodynamic (PK/PD) data with the aim to evaluate such approaches and provide guidance on clinically effective dose prediction for inhaled medicines. METHODS: Five empirically based methodologies were applied on a diverse set of marketed inhaled therapeutics (inhaled corticosteroids and bronchodilators). The approaches include scaling of dose based on body weight or body surface area and variants of PK/PD approaches aiming to predict the therapeutic dose based on having efficacious concentrations of drug in the lung over the dosing interval. RESULTS: The most robust predictions of dose were made by body weight adjustment (90% within 3-fold) and by a specific PK/PD approach aiming for an average predicted 75% effect level during the dosing interval (80% within 3-fold). Scaling of dose based on body surface area consistently under predicted the therapeutic dose. CONCLUSIONS: Preclinical in vivo data and empirical scaling to man can be used as a baseline method for clinical dose predictions of inhaled medicines. The development of more sophisticated translational models utilizing free drug concentration and target engagement data is a desirable build.

Neutrophils in ulcerative colitis: a review of selected biomarkers and their potential therapeutic implications.

OBJECTIVES: This review article describes the role of neutrophils in mucosal injury and the resulting crypt abscesses characteristic of ulcerative colitis. We also review selected biomarkers for monitoring neutrophil presence and activity in the mucosa as well as their potential as therapeutic targets. MATERIAL: We have collated and selectively reviewed data on the most prominent well-established and emerging neutrophil-related biomarkers and potential therapeutic targets (calprotectin, lactoferrin, CXCR1, CXCR2, MMP-9, NGAL, elafin, HNE, pANCAs, MPO, CD16, CD177, CD64, HNPs, SLPI and PTX3) in ulcerative colitis. RESULTS: Systemic and intestinal neutrophil activity increases substantially in active ulcerative colitis, driving tissue damage and extra-intestinal manifestations. Calprotectin is a robust neutrophil and disease biomarker, and a few neutrophil-related targets are being clinically explored as therapeutic targets. CONCLUSION: We propose that targeting neutrophils and their inflammatory mediators per se is an opportunity that should be explored to identify new effective medical therapies. The overall clinical goal for neutrophil-targeted therapy will be to modulate, but not completely silence, neutrophil activity, thereby abolishing the destructive inflammation with associated acute and chronic tissue damage without compromising host-defense.

Effect of oral administration of AZD8309, a CXCR2 antagonist, on the severity of experimental pancreatitis.

INTRODUCTION: Acute pancreatitis is a common gastrointestinal disorder burdened with a high mortality. Two pathophysiological events during experimental pancreatitis are thought to determine the clinical course: premature digestive protease activation and tissue infiltration by inflammatory cells. We have investigated the effect of AZD8309, a potent and orally bioavailable antagonist of the chemokine receptor CXCR2, which has been proposed to regulate the transmigration of neutrophils. METHODS: Male C57BL6 mice (25-30 g) received gavage feeding of AZD8309 (50 mg/kg/BW) or mannitol (controls) twice daily starting 3 h prior to pancreatitis induction. Mild pancreatitis was induced by i.p. caerulein administration (50 µg/kg BW), severe pancreatitis by intraductal taurocholate (2%). Pancreas, lung, and serum was harvested up to 48 h after pancreatitis induction and used for histopathology, amylase, lipase, cathepsin B, trypsin, and elastase activity measurements, myeloperoxidase (MPO) content and cytokine concentrations. RESULTS: Oral administration of AZD8309 significantly reduced MPO in the pancreas and lungs (8 h & 24 h) and reduced intrapancreatic trypsin and elastase activity (8 h) in caerulein-pancreatitis. In taurocholate-pancreatitis AZD8309 reduced cathepsin B activity and MPO. Serum cytokine levels were reduced by AZD8309 as well as histopathological damage. CONCLUSION: The CXCR2 antagonist AZD8309 reduced the transmigration of neutrophils as well as intrapancreatic protease activation in experimental pancreatitis. This effect was sufficient to reduce the overall severity of the disease. CXCR2 may therefore be a viable therapeutic target and AZD8309 a suitable agent for the treatment of acute pancreatitis.