Introduction: Goblet cell hyperplasia (GCH) within the epithelium of the conducting airways is a common feature of human respiratory diseases, including asthma and chronic obstructive pulmonary disease. GCH can be initiated or exacerbated by environmental agents, including air pollution, viral infection, and tobacco smoke. Experimental model systems for investigating causes of GCH and possible remedies are therefore of high interest. Materials and Methods: The current article utilized commercially available in vitro organotypic models of human mucociliary airway epithelium (EpiAirway™) and epithelial/stromal cocultures (EpiAirway-FT™) to characterize and compare aspects of GCH induced by Th2 cytokines, double-stranded RNA [Poly(I:C), a mimetic of viral infection], and whole cigarette smoke. Results and Discussion: Induction of GCH by interleukin (IL)-13, IL-4, Poly(I:C), and cigarette smoke was demonstrated in both the EpiAirway and EpiAirway-FT in vitro human airway models. Morphological induction of GCH was most prominent in EpiAirway-FT, while the response in EpiAirway revealed GCH overlapping with additional squamous changes and occasional appearance of epithelial voids. The results suggest that the fibroblast containing stromal component of EpiAirway-FT may play a role in modulating the outcome of epithelial exposures to these agents. Gene expression changes confirmed the induction of MUC5AC by Th2 cytokines, double-stranded RNA, and whole tobacco smoke in both EpiAirway and EpiAirway-FT. However, gene expression patterns also reveal noteworthy differences in the response to these agents. Azithromycin (AZ), a therapeutic drug that has been shown to improve clinical symptoms of pathogen and disease-associated mucus production in vivo, effectively reduced mucin protein and gene expression induction by all three agents. Conclusion: Taken together, the data demonstrate that organotypic in vitro human airway models are useful tools for evaluation of GCH and other clinically relevant responses to environmental agents.

(R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl] carbamoyl}phenyl)sulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide trifluoroacetic acid (GS-5759) is a bifunctional ligand composed of a quinolinone-containing pharmacophore [β2-adrenoceptor agonist orthostere (β2A)] found in several β2-adrenoceptor agonists, including indacaterol, linked covalently to a phosphodiesterase 4 (PDE4) inhibitor related to 6-[3-(dimethylcarbamoyl)benzenesulphonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GSK 256066) by a pent-1-yn-1-ylbenzene spacer. GS-5759 had a similar affinity for PDE4B1 and the native β2-adrenoceptor expressed on BEAS-2B human airway epithelial cells. However, compared with the monofunctional parent compound, β2A, the KA of GS-5759 for the β2-adrenoceptor was 35-fold lower. Schild analysis determined that the affinities of the β-adrenoceptor antagonists, (2R,3R)-1-[(2,3-dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1-methylethyl) amino]-2-butanol (ICI 118551) and propranolol, were agonist-dependent, being significantly lower for GS-5759 than β2A. Collectively, these data can be explained by “forced proximity,” bivalent binding where the pharmacophore in GS-5759 responsible for PDE4 inhibition also interacts with a nonallosteric domain within the β2-adrenoceptor that enhances the affinity of β2A for the orthosteric site. Microarray analyses revealed that, after 2-hour exposure, GS-5759 increased the expression of >3500 genes in BEAS-2B cells that were highly rank-order correlated with gene expression changes produced by indacaterol and GSK 256066 in combination (Ind/GSK). Moreover, the line of regression began close to the origin with a slope of 0.88, indicating that the magnitude of most gene expression changes produced by Ind/GSK was quantitatively replicated by GS-5759. Thus, GS-5759 is a novel compound exhibiting dual β2-adrenoceptor agonism and PDE4 inhibition with potential to interact on target tissues in a synergistic manner. Such polypharmacological behavior may be particularly effective in chronic obstructive pulmonary disease and other complex disorders where multiple processes interact to promote disease pathogenesis and progression.

Bronchodilators are a central therapy for symptom relief in respiratory diseases such as chronic obstructive pulmonary disease ( COPD ) and asthma, with inhaled β 2 ‐adrenoceptor agonists and anticholinergics being the primary treatments available. The present studies evaluated the in vivo pharmacology of (R)‐6‐[[3‐[[4‐[5‐[[2‐Hydroxy‐2‐(8‐hydroxy‐2‐oxo‐1,2‐dihydroquinolin‐5‐yl)ethyl]amino]pent‐1‐ynyl]phenyl]carbamoyl]phenyl]sulfonyl]‐4‐[(3‐methoxyphenyl)amino]‐8‐methylquinoline‐3‐carboxamide ( GS ‐5759), a novel bifunctional compound with both phosphodiesterase 4 ( PDE 4) inhibitor and long‐acting β 2 ‐adrenoceptor agonist ( LABA ) activity, which has been optimized for inhalation delivery. GS ‐5759 dose‐dependently inhibited pulmonary neutrophilia in a lipopolysaccharide ( LPS ) aerosol challenge model of inflammation in rats with an ED 50 ≤ 10 μ g/kg. GS ‐5759 was also a potent bronchodilator with an ED 50 of 0.09 μ g/kg in guinea pigs and 3.4 μ g/kg in dogs after methylcholine ( MC h) and ragweed challenges respectively. In cynomolgus monkeys, GS ‐5759 was dosed as a fine‐particle dry powder and was efficacious in the same dose range in both MC h and LPS challenge models, with an ED 50 = 70 μ g/kg for bronchodilation and ED 50 = 4.9 μ g/kg for inhibition of LPS ‐induced pulmonary neutrophilia. In models to determine therapeutic index (T.I.), efficacy for bronchodilation was evaluated against increased heart rate and GS ‐5759 had a T.I. of 700 in guinea pigs and >31 in dogs. In a ferret model of emesis, no emesis was seen at doses several orders of magnitude greater than the ED 50 observed in the rat LPS inflammation model. GS ‐5759 is a bifunctional molecule developed for the treatment of COPD , which has both bronchodilator and anti‐inflammatory activity and has the potential for combination as a triple therapy with a second compound, within a single inhalation device.

Inhaled long-acting β2-adrenoceptor agonists (LABA) that act as bronchodilators and the oral anti-inflammatory phosphodiesterase 4 (PDE4) inhibitor roflumilast are both approved therapies for chronic obstructive pulmonary disease (COPD). Here we describe the activity of a novel, inhaled, bifunctional, small molecule (R)-6-[(3-{[4-(5-{[2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl]amino}pent-1-yn-1-yl)phenyl]carbamoyl}phenyl)sulfonyl]-4-[(3-methoxyphenyl)amino]-8-methylquinoline-3-carboxamide (GS-5759), which has specific β2 agonist and PDE4 inhibitory activity. GS-5759 demonstrated potent and full agonist activity at β2 adrenoceptors (EC50 = 8 ± 4 nM) and is a potent inhibitor of the PDE4 enzyme (IC50 = 5 ± 3 nM). In cell assays, GS-5759 inhibited lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNFα) production in human peripheral mononuclear cells (PBMC) with an IC50 = 0.3 nM [confidence interval (CI) 0.1–0.6] and in human neutrophils formyl-methionyl-leucyl-phenylalanine (fMLP)-induced super oxide anion production with an IC50 = 3 nM (CI 0.8–8). The addition of the β2 antagonist ICI 118551 shifted the IC50 in these cell assays to 4 and 38 nM, respectively, demonstrating the contribution of both β2 agonist and PDE4 inhibitory activity to GS-5759. GS-5759 was also a potent inhibitor of profibrotic and proinflammatory mediator release from human lung fibroblasts. GS-5759 relaxed guinea pig airway smooth muscle strips precontracted with carbachol in a concentration-dependent manner with an EC50 = 0.5 µM (CI 0.2–2) and had slow dissociation kinetics with an Off T1/2 > 720 minutes at an EC80 concentration of 3 µM. GS-5759 is a novel bifunctional molecule with both potent β2 agonist and PDE4 inhibitor activity that could provide inhaled bronchodilator and anti-inflammatory therapy for COPD.

Mucus clearance is an important component of the lung's innate defense system. A failure of this system brought on by mucus dehydration is common to both cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD). Mucus clearance rates are regulated by the volume of airway surface liquid (ASL) and by ciliary beat frequency (CBF). Chronic treatment with macrolide antibiotics is known to be beneficial to both CF and COPD patients. However, chronic macrolide usage may induce bacterial resistance. We have developed a novel macrolide, 2'-desoxy-9-(S)-erythromycylamine (GS-459755), that has significantly diminished antibiotic activity against Staphylococcus aureus, Streptococcus pneumonia, Moraxella catarrhalis, and Haemophilus influenzae. Since neutrophilia frequently occurs in chronic lung disease and human neutrophil elastase (HNE) induces mucus stasis by activating the epithelial sodium channel (ENaC), we tested the ability of GS-459755 to protect against HNE-induced mucus stasis. GS-459755 had no effect on HNE activity. However, GS-459755 pretreatment protected against HNE-induced ASL volume depletion in human bronchial epithelial cells (HBECs). The effect of GS-459755 on ASL volume was dose dependent (IC₅₀ ~3.9 μM) and comparable to the antibacterial macrolide azithromycin (IC₅₀ ~2.4 μM). Macrolides had no significant effect on CBF or on transepithelial water permeability. However, the amiloride-sensitive transepithelial voltage, a marker of ENaC activity, was diminished by macrolide pretreatment. We conclude that GS-459755 may limit HNE-induced activation of ENaC and may be useful for the treatment of mucus dehydration in CF and COPD without inducing bacterial resistance.

The phosphodiesterase 4 inhibitor (PDE4i) roflumilast has been approved in the US and EU for treatment of GOLD stage 3 and 4 chronic obstructive pulmonary disease (COPD). Inhaled β2 adrenoceptor agonist bronchodilators and anti-inflammatory glucocorticosteroids are also used as standard of care in COPD. We investigated the anti-inflammatory interaction of roflumilast in combination with long-acting β2 agonists (LABA), salmeterol or formoterol, or a glucocorticosteroid, dexamethasone, on cytokine production from LPS-stimulated human primary peripheral blood mononuclear cells (PBMC). Salmeterol or formoterol caused a concentration-dependent inhibition of tumor necrosis factor-α (TNFα) secretion with an IC50 of 0.33 pM (C.I. 0.006-19) and 34 pM (C.I. 13-87), respectively. When roflumilast was evaluated, the addition of salmeterol (1 nM) to roflumilast caused the IC50 for roflumilast to shift from 1.8 nM (C.I. 0.8-4) to 4.1 pM (C.I.0.3-69) (p < 0.01), and maximal inhibition increased from 72.5 ± 3.2% to 90.9 ± 3.1%. Addition of formoterol to roflumilast also produced an increased TNFα inhibition more than either drug alone (p < 0.05). The inhibition of TNFα production with salmeterol was both β2 adrenoceptor- and protein kinase A-dependent. Addition of roflumilast (10 nM) in the presence of dexamethasone increased the inhibition of LPS-induced TNFα and CCL3. Roflumilast in combination with salmeterol, formoterol, or dexamethasone increased the inhibition of LPS-induced TNFα from human PBMC, in an additive manner. Addition of roflumilast to either a β2 adrenoceptor agonist or a glucocorticosteroid may provide superior anti-inflammatory activity and greater efficacy in COPD patients and be dose sparing.

Background: Small airway narrowing is an important pathology which impacts lung function in chronic obstructive pulmonary disease (COPD).The accumulation of fibroblasts and myofibroblasts contribute to inflammation, remodeling and fibrosis by production and release of mediators such as cytokines, profibrotic factors and extracellular matrix proteins.This study investigated the effects of the phosphodiesterase 4 inhibitor roflumilast, combined with the long acting β 2 adrenergic agonist indacaterol, both approved therapeutics for COPD, on fibroblast functions that contribute to inflammation and airway fibrosis.Methods: The effects of roflumilast and indacaterol treatment were characterized on transforming growth factor β1 (TGFβ1)-treated normal human lung fibroblasts (NHLF).NHLF were evaluated for expression of the profibrotic mediators endothelin-1 (ET-1) and connective tissue growth factor (CTGF), expression of the myofibroblast marker alpha smooth muscle actin, and fibronectin (FN) secretion.Tumor necrosis factor-α (TNF-α) was used to induce secretion of chemokine C-X-C motif ligand 10 (CXCL10), chemokine C-C motif ligand 5 (CCL5) and granulocyte macrophage colony-stimulating factor (GM-CSF) from NHLF and drug inhibition was assessed.Results: Evaluation of roflumilast (1-10 μM) showed no significant inhibition alone on TGFβ1-induced ET-1 and CTGF mRNA transcripts, ET-1 and FN protein production, alpha smooth muscle expression, or TNF-α-induced secretion of CXCL10, CCL5 and GM-CSF.A concentration-dependent inhibition of ET-1 and CTGF was shown with indacaterol treatment, and a submaximal concentration was chosen for combination studies.When indacaterol (0.1 nM) was added to roflumilast, significant inhibition was seen on all inflammatory and fibrotic mediators evaluated, which was superior to the inhibition seen with either drug alone.Roflumilast plus indacaterol combination treatment resulted in significantly elevated phosphorylation of the transcription factor cAMP response elementbinding protein (CREB), an effect that was protein kinase A-dependent.Inhibition of protein kinase A was also found to reverse the inhibition of indacaterol and roflumilast on CTGF.Conclusions: These results demonstrate that addition of roflumilast to a LABA inhibits primary fibroblast/ myofibroblast function and therapeutically this may impact lung fibroblast proinflammatory and profibrotic mediator release which contributes to small airway remodeling and airway obstruction in COPD.