Adaptive study design to assess effect of TRPV4 inhibition in patients with chronic cough.

OBJECTIVE: Airway sensory nerves involved in the cough reflex are activated by adenosine triphosphate (ATP) agonism of P2X purinoceptor 3 (P2X3) receptors. Transient receptor potential vanilloid 4 (TRPV4) channel activation causes ATP release from airway cells, and it is hypothesised that a TRPV4-ATP-P2X3 axis contributes to chronic cough. An adaptive study was run to determine if TRPV4 inhibition, using the selective TRPV4 channel blocker GSK2798745, was effective in reducing cough. METHODS: A two-period randomised, double blinded, placebo-controlled crossover study was designed with interim analyses for futility and sample size adjustment. Refractory chronic cough patients received either GSK2798745 or placebo once daily for 7 days with a washout between treatments. Pharmacokinetic samples were collected for analysis of GSK2798745 at end of study. The primary end-point was total cough counts assessed objectively during day-time hours (10 h) following 7 days of dosing. RESULTS: Interim analysis was performed after 12 participants completed both treatment periods. This showed a 32% increase in cough counts on Day 7 for GSK2798745 compared to placebo; the pre-defined negative criteria for the study were met and the study was stopped. At this point 17 participants had been enrolled (mean 61 years; 88% female), and 15 had completed the study. Final study results for posterior median cough counts showed a 34% (90% credible interval: -3%, +85%) numerical increase for GSK2798745 compared to placebo. CONCLUSION: There was no evidence of an anti-tussive effect of GSK2798745. The study design allowed the decision on lack of efficacy to be made with minimal participant exposure to the investigational drug.

Biological effects of p38 MAPK inhibitor losmapimod does not translate to clinical benefits in COPD.

RATIONALE: p38 mitogen-activated protein kinase (MAPK) expression is increased in chronic inflammatory disease. Losmapimod, a p38 MAPK inhibitor, has been developed as a potential anti-inflammatory therapy in COPD. OBJECTIVES: To evaluate the effect of losmapimod in reducing exacerbations in subjects with moderate-to-severe COPD. METHODS: In this double-blind, parallel-group study, subjects at risk of COPD exacerbations and ?2% blood eosinophils at screening, were randomized 1:1 to losmapimod 15 mg or placebo (variable treatment duration: 26-52 weeks). The primary endpoint was the annualized rate of moderate/severe exacerbations. Using a Bayesian framework, treatment success was defined as >90% posterior probability that the true ratio of the losmapimod/placebo exacerbation rate was <1. Lung function and health status (St George's Respiratory Questionnaire (SGRQ)) were also assessed. RESULTS: A planned interim analysis resulted in early study termination due to the low probability of a successful study outcome; a total of 94 subjects were randomized to placebo and 90 to losmapimod 15 mg, and 14 and 10 subjects respectively completed the study. Losmapimod treatment was not associated with an improvement in the adjusted posterior median annualized exacerbation rate (losmapimod/placebo ratio: 1.04 (95% Cr I: 0.63, 1.73)). The posterior probability for the losmapimod/placebo annualized rate ratio being <1 was 0.44 (success criterion: >0.90). A statistically significant improvement in post-bronchodilator forced expiratory volume in 1 s was seen at Week 26, at the 5% significance level, with losmapimod treatment versus placebo (p = 0.007). Changes from baseline in SGRQ total score were similar in both groups. No new risks or safety signals were identified with losmapimod treatment. CONCLUSIONS: Losmapimod treatment did not reduce the rate of exacerbations in, subjects with COPD at high risk of exacerbation and ?2% blood eosinophils. These data do not support its use as a therapy in COPD in addition to standard of care.

Effects of a novel sodium channel blocker, GSK2339345, in patients with refractory chronic cough
.

OBJECTIVE: Voltage-gated sodium channels (VGSC) are important in the initiation and propagation of action potentials in afferent sensory nerve fibers responsible for evoking cough. This study investigated the efficacy of GSK2339345, a VGSC inhibitor, in the treatment of refractory chronic cough (RCC). METHODS: A three-part randomized, double-blind, placebo-controlled, cross-over study was conducted in the UK. In part A, patients with RCC received two inhaled doses of either GSK2339345 or placebo, 4 hours apart during three study periods. Patients were monitored for cough for 8 hours post-first dose using the VitaloJAK, ambulatory cough monitor. In parts B and C, patients underwent full dose-response cough challenges with capsaicin and citric acid respectively following single doses of randomly assigned GSK2339345 or placebo (4 study days). Part A was analyzed using a mixed effects model and parts B and C using population non-linear mixed effects models. RESULTS: Of 16 enrolled patients, 11 completed the study. 8-hour cough counts increased following GSK2339345 treatment compared with placebo (GSK2339345/placebo ratio of adjusted geometric means: 1.26 (90% credible interval 1.10, 1.44), associated with GSK2339345-evoked coughing, recorded during the 2 minutes post-dose. This was not observed with placebo. The effect of GSK2339345 on cough responses during cough challenges was inconclusive. GSK2339345 was well tolerated. CONCLUSIONS: While these data could not determine if GSK2339345 reached the target VGSC, they strongly suggest that GSK2339345 has no anti-tussive effect despite reaching airway sensory nerves as evidenced by the evoked transient cough.
.

A post-hoc subgroup analysis of data from a six month clinical trial comparing the efficacy and safety of losmapimod in moderate-severe COPD patients with ≤2% and >2% blood eosinophils.

BACKGROUND: A six month study of the p38 MAPK inhibitor, losmapimod, suggested a trend in reducing COPD exacerbations with the 15 mg twice daily dose. OBJECTIVE AND METHODS: Using data from this study which evaluated the efficacy of twice daily losmapimod, 2.5 mg, 7.5 mg, and 15 mg, versus placebo in patients with moderate-to-severe COPD, we analysed the effect of losmapimod in reducing the rate of moderate/severe exacerbations in patient subgroups with ≤2% and >2% blood eosinophils at baseline. Lung function, fibrinogen and hsCRP were also evaluated. RESULTS: In the ≤2% eosinophil subgroup, there was an exposure-related reduction in the rate of moderate/severe exacerbations with losmapimod relative to placebo (losmapimod 15 mg: 55% reduction; losmapimod 7.5 mg: 29%; losmapimod 2.5 mg: 10%); with the 15 mg dose reaching statistical significance (15 mg/placebo mean rate ratio [95% CI]: 0.45 [0.22; 0.90]). There was also an improvement in lung function with 15 mg losmapimod over Weeks 1-12. No improvement in the rate of moderate/severe exacerbations or post-bronchodilator FEV1 was observed for subjects treated with Losmapimod compared to placebo in the patient subgroup with blood eosinophils >2% at baseline. Transient reductions in fibrinogen and hsCRP were observed with losmapimod 7.5 mg and 15 mg in both eosinophil subgroups. CONCLUSIONS: These findings indicate eosinophil-related heterogeneity within COPD and suggest that losmapimod could be a potential therapy to reduce exacerbations in COPD patients with eosinophil levels ≤2%. This needs to be explored further in a prospectively designed study with pre-specified criteria for blood eosinophil subgroups in COPD patients.

Efficacy, safety and effect on biomarkers of AZD9668 in cystic fibrosis.

The aim of this study was to evaluate the safety and effect on clinical outcomes and biomarkers of inflammation and tissue damage of the neutrophil elastase inhibitor AZD9668 (60 mg twice daily orally for 4 weeks) in cystic fibrosis. This was a randomised, double-blind, placebo-controlled study. Primary outcome measures were sputum neutrophil count, lung function, 24-h sputum weight, BronkoTest® diary card data and health-related quality-of-life (revised cystic fibrosis quality-of-life questionnaire). Secondary end-points included sputum neutrophil elastase activity, inflammatory biomarkers in sputum and blood, urine and plasma desmosine (an elastin degradation marker), AZD9668 levels and safety parameters (adverse events, routine haematology, biochemistry, electrocardiogram and sputum bacteriology). 56 patients were randomised, of which 27 received AZD9668. There was no effect for AZD9668 on sputum neutrophil counts, neutrophil elastase activity, lung function or clinical outcomes, including quality of life. In the AZD9668 group, there was a trend towards reduction in sputum inflammatory biomarkers with statistically significant changes in interleukin-6, RANTES and urinary desmosine. The pattern of adverse events was similar between groups. Consistent reductions in sputum inflammatory biomarkers were seen in the AZD9668 group, and reduction in urinary desmosine suggests that AZD9668 impacts elastin cleavage by neutrophil elastase.

Stimulation of signal transducer and activator of transcription-1 (STAT1)-dependent gene transcription by lipopolysaccharide and interferon-gamma is regulated by mammalian target of rapamycin.

Mammalian target of rapamycin (mTOR) and phosphatidylinositol 3-kinase (PI3K) regulate cell growth, protein synthesis, and apoptosis in response to nutrients and mitogens. As an important source of nitric oxide during inflammation, human inducible nitric oxide synthase also plays a role in the regulation of cytokine-driven cell proliferation and apoptosis. The role of mTOR and PI3K in the activation of human inducible nitric oxide synthase transcription by cytokines and lipopolysaccharide (LPS) was investigated in lung epithelial adenocarcinoma (A549) cells. LY294002, a dual mTOR and PI3K inhibitor, blocked human inducible nitric oxide synthase (hiNOS) promoter activation and mRNA induction by cytokines and LPS in a PI3K-independent fashion. On gene expression analysis, LY294002 selectively blocked the induction of a subset of 14 LPS/interferon-gamma (IFN-gamma)-induced genes, previously characterized as signal transducer and activator of transcription-1 (STAT1)-dependent. LY294002, but not wortmannin, inhibited LPS/IFN-gamma-dependent STAT1 phosphorylation at Ser-727 and STAT1 activity. Consistent with dual inhibition of mTOR and PI3K by LY294002, dominant-negative mTOR, anti-mTOR small interfering RNA, or rapamycin each inhibited phosphorylation of STAT1 only in the presence of wortmannin. LPS/IFN-gamma led to the formation of a macromolecular complex containing mTOR, STAT1, as well as protein kinase C delta, a known STAT1alpha kinase. Thus, LPS and IFN-gamma activate the PI3K and mTOR pathways, which converge to regulate STAT1-dependent transcription of pro-apoptotic and pro-inflammatory genes in a rapamycin-insensitive manner.

STAT-1 and c-Fos interaction in nitric oxide synthase-2 gene activation.

Interferon-gamma (IFN-gamma) is required for induction of the human nitric oxide synthase-2 (NOS2) gene in lung epithelium. Although the human NOS2 promoter region contains many cytokine-responsive elements, the molecular basis of induction is only partially understood. Here, the major cis-regulatory elements that control IFN-gamma-inducible NOS2 gene transcription in human lung epithelial cells are identified as composite response elements that bind signal transducer and activator of transcription 1 (STAT-1) and activator protein 1 (AP-1), which is comprised of c-Fos, Fra-2, c-Jun, and JunD. Notably, IFN-gamma activation of the human NOS2 promoter is shown to require functional AP-1 regulatory region(s), suggesting a role for AP-1 activation/binding in the IFN-gamma induction of genes. We show that c-Fos interacts with STAT-1 after IFN-gamma activation and the c-Fos/STAT-1 complex binds to the gamma-activated site (GAS) element in close proximity to AP-1 sites located at 4.9 kb upstream of the transcription start site. Taken together, our findings support a model in which a physical interaction between c-Fos and STAT-1 participates in NOS2 gene transcriptional activation.