Effects of the Toll-like receptor 7 (TLR7) agonist, AZD8848, on allergen-induced responses in patients with mild asthma: a double-blind, randomised, parallel-group study.

BACKGROUND: Although allergic asthma is a complex area with many interacting factors involved, the 'hygiene hypothesis' proposes that a lack of exposure to infection during childhood may polarise the immune system towards allergen-reactive Th2-type responses in genetically susceptible individuals. Toll-like receptors (TLRs) play a key role within the innate immune system and TLR7 agonists have previously been shown to up-regulate Th1 responses and down-regulate Th2 responses to allergens in murine models of allergic or chronic asthma. This study aimed to examine the efficacy and safety of the novel TRL7 agonist AZD8848, which has been developed as an antedrug. METHODS: In this double-blind, randomised, parallel-group study, AZD8848 60 µg or placebo was administered intranasally once-weekly for 8 weeks in patients with mild-to-moderate allergic asthma (NCT00999466). Efficacy assessments were performed at 1 and 4 weeks after the last dose. The primary outcome was the late asthmatic response (LAR) fall in forced expiratory volume in 1 s (FEV1) after allergen challenge at 1-week post-treatment. RESULTS: AZD8848 significantly reduced average LAR fall in FEV1 by 27% vs. placebo at 1 week after treatment (p = 0.035). This effect was sustained at 4 weeks post-treatment; however, it did not reach clinical significance. AZD8848 reduced post-allergen challenge methacholine-induced airway hyper-responsiveness (AHR) vs. placebo at 1 week post-dosing (treatment ratio: 2.20, p = 0.024), with no effect at 4 weeks. There was no significant difference between the two groups in plasma cytokine, sputum Th2 cytokine or eosinophil responses post-allergen challenge at 1 week after treatment. The incidence of adverse events was similar in the two groups. AZD8848 was generally well tolerated. CONCLUSIONS AND CLINICAL RELEVANCE: In patients with allergic asthma, TLR7 agonists could potentially reduce allergen responsiveness by stimulating Type 1 interferon responses to down-regulate the dominant Th2 responses. TRIAL REGISTRATION: clinicaltrials.gov identifier NCT00999466.

Evaluation of systemic absorption and bronchodilator effect of glycopyrronium bromide delivered by nebulizer or a dry powder inhaler in subjects with chronic obstructive pulmonary disease.

BACKGROUND: Effective bronchodilator therapy depends upon adequate drug deposition in the lung. COPD patients who are unable to administer medications efficiently with conventional inhalers may benefit from the use of a nebulizer device. The aim of this study was to evaluate the systemic bioavailability and bronchodilator response of glycopyrronium bromide (GLY) administered by a novel nebulizer (eFlow® closed system [CS] vibrating membrane nebulizer) or dry powder inhaler (DPI) in subjects with moderate-to-severe chronic obstructive pulmonary disease (COPD). METHODS: In this randomized, open-label, single-dose, five-way crossover study, subjects received a sequence of either 50 µg GLY delivered by eFlow CS nebulizer (GLY/eFlow) or 63 µg GLY delivered by DPI (GLY/DPI), with and without activated charcoal, followed by intravenous infusion of 50 µg GLY with a washout period of 7 days between doses. Endpoints included plasma pharmacokinetics, safety and efficacy. RESULTS: The mean (± SD) baseline predicted forced expiratory volume in 1 s (FEV1) of the 30 subjects who completed the study was 51 ± 15%, with a FEV1/forced vital capacity ratio of 50 ± 11%. Without charcoal, the absolute systemic bioavailability of GLY/eFlow and GLY/DPI were approximately 15 and 22%, respectively. Changes from baseline in FEV1 at 60 min post-dose, without administration of charcoal, were 0.180 L and 0.220 L for GLY/eFlow and GLY/DPI, respectively; FEV1 improvements were similar when charcoal was administered (0.220 L for both GLY/eFlow and GLY/DPI). There were no significant differences in spirometry between the two devices. Fewer subjects administered GLY/eFlow reported adverse events (n = 15) than GLY/DPI (n = 18). CONCLUSIONS: After single doses, GLY/DPI delivered numerically higher peak and steady state levels of drug than did GLY/eFlow. Nebulized GLY produced similar bronchodilation but lower systemic levels of drug than GLY/DPI. Slightly higher number of subjects reported adverse events with GLY/DPI than with GLY/eFlow. Nebulized GLY may offer an effective alternative to patients with COPD not adequately treated with other devices. TRIAL REGISTRATION: NCT02512302 (ClinicalTrials.gov). Registered 28 May 2015.

The effects of the selective muscarinic M3 receptor antagonist darifenacin, and of hyoscine (scopolamine), on motion sickness, skin conductance & cognitive function.

AIMS: The aim of this study was to compare the effects of the selective M3 muscarinic acetylcholine receptor antagonist darifenacin, oral hyoscine hydrobromide and placebo on motion sickness induced by cross-coupled stimulation. METHODS: The effects of darifenacin 10 mg or 20 mg, hyoscine hydrobromide 0.6 mg and placebo were assessed in a randomized, double-blind, four-way cross over trial of 16 healthy subjects. Motion sickness, skin conductance (a measure of sweating) and psychomotor cognitive function tests were investigated. RESULTS: Hyoscine hydrobromide produced significantly increased tolerance to motion versus placebo (P < 0.05 to P < 0.01). The motion protection effect of darifenacin (10 or 20 mg) was approximately one third that of hyoscine hydrobromide but was not significant versus placebo. Darifenacin and hyoscine hydrobromide both significantly reduced skin conductance versus placebo. Darifenacin produced either no effect or an enhanced effect on cognitive function in contrast to hyoscine hydrobromide, where there was significant impairment of psychomotor performance. CONCLUSION: The results suggest that selective antagonism of the M3 receptor may not be important in the prevention of motion sickness. However, selective M3 antagonism does not impair cognitive function. These observations may be important given that long-term treatment with non-selective anti-muscarinic agents such as oxybutynin may lead to an increased incidence of dementia.

Efficacy and safety of nebulized glycopyrrolate for administration using a high efficiency nebulizer in patients with chronic obstructive pulmonary disease.

AIMS: To establish the dose-response for pharmacodynamics (bronchodilatation), safety and pharmacokinetics for a nebulized formulation of the long acting muscarinic antagonist glycopyrrolate (EP-101) with a high efficiency nebulizer in patients with chronic obstructive pulmonary disease (COPD). METHODS: Patients with moderate to severe COPD (GOLD II/III), with reversible lung function, were enrolled into this randomized, double-blind, placebo-controlled, six period crossover study (n = 42). Patients received single doses of EP-101 (12.5-400 µg) and placebo via a high efficiency nebulizer (eFlow® PARI nebulizer), with washout between treatments. Plasma pharmacokinetics were assessed in a subset of patients (n = 11). RESULTS: All treatments were well tolerated with similar adverse event rates reported with placebo and at all doses. There were no clinically relevant changes in heart rate, systolic and diastolic blood pressure or in ECG parameters including QTc interval. Following treatment with EP-101 at all doses there was a rapid bronchodilator response within 5 min. Significant improvements in mean change from baseline FEV1 at 24 h were reported at doses ≥ 50 µg compared with placebo, with a clear dose-response relationship. Mean changes in FEV1 were 0.10 l (95% CI 0.06, 0.14) and 0.12 l (95% CI 0.08, 0.16) for 100 µg and 200 µg, respectively. CONCLUSION: Single doses of EP-101 ranging from 12.5 µg to 400 µg were well tolerated. EP-101 delivered by high efficiency nebulizer device produced a rapid onset of bronchodilatation with clinically meaningful improvements in lung function maintained over a 24 h period at all doses >50 µg.

Using magnetic resonance imaging to quantify the inflammatory response following allergen challenge in allergic rhinitis.

Current rhinometric and flow assessments measure nasal patency and are often poorly correlated with rhinitis symptoms. To evaluate magnetic resonance imaging (MRI) as a new method to measure inflammatory changes in nasal and sinus mucosa following nasal allergen challenge. A pilot study (n = 6) determined the optimal technical settings for MRI to measure inflammatory change which were then adopted for the main study. This study was a single blind, placebo-controlled, three-way crossover trial in 14 subjects with seasonal allergic rhinitis. Effects of cetirizine, cetirizine and pseudoephedrine (Cet+PE), or placebo on total nasal symptom scores (TNSS), peak nasal inspiratory flow (PNIF), nasal nitric oxide (nNO), acoustic rhinometry, and MRI end points following nasal intranasal allergen challenge were measured. There were significant changes in all parameters after allergen challenge (P < 0.01), except for nNO. MRI end points were less variable and more consistent than PNIF and acoustic rhinometry in detecting changes after allergen challenge. Total nasal airspace volume was the most sensitive and reproducible MRI measurement, with a mean reduction from -5.37 cm(3) (95%CI -7.35, -3.38; P < 0.001), which was maximal 60 min after allergen challenge. A change of one in TNSS corresponded to a change in MRI volume of -0.57 cm(3). There was an improvement in all parameters (except nNO) in subjects taking Cet+PE compared with placebo, however this did not achieve significance probably because of the small study size (overall analysis P > 0.07; comparison of active versus placebo P > 0.09). MRI provides novel insights into the anatomical inflammatory changes post allergen challenge and provides a new method for assessment of nasal patency and objective measurement of inflammatory responses.

The effect of the novel phosphodiesterase-4 inhibitor MEM 1414 on the allergen induced responses in mild asthma.

BACKGROUND: Inhaled allergen challenge is a standard method to study airway responses to inflammatory provocation and evaluate the therapeutic potential of novel anti-inflammatory compounds in asthma. MEM 1414 is a novel oral PDE4 inhibitor with high affinity and selectivity creating the potential for an improved side effect profile vs non-selective PDE inhibitors. We evaluated the tolerability and effect of MEM 1414 on airway responses in mild asthmatics. METHODS: A randomised double blind placebo controlled cross over study in two centres, in which sixteen steroid naïve atopic asthmatics were challenged with inhaled allergen. Subjects were dosed with MEM 1414 (600 mg) or placebo, twice daily orally for 7 days. Allergen challenge was performed on day 6 (2 hours post-dose), and methacholine responsiveness was measured 24 hours post allergen (day 7). Biomarkers of drug effects using ex vivo LPS stimulation of whole blood production of interleukin (IL)-6 and leukotriene (LT)-B4 and fractional exhaled nitric oxide (FeNO) were measured on day 6 (0, 2 and 8 hours post-dose). Plasma pharmacokinetics were measured on days 1, 6 and 7. The primary endpoint was the effect on late asthmatic response to allergen. RESULTS: Treatment with MEM 1414 abrogated the late phase response with a mean difference in FEV1 (LAR 3-10 hours) of 104 ml (25%) vs placebo (p < 0.005), with no effect on the early response. Biomarker responses were also attenuated with MEM 1414 treatment with reductions in LPS-stimulated whole blood assays for TNFα at 8 hours (p < 0.03) and LTB4 at 24 hours (p = 0.0808) with no change in the IL-6 response. The MEM 1414 treatment phase was associated with higher incidence of nausea (6/16 MEM 1414 vs 2/16 placebo) and vomiting (3/16 vs 0/16 placebo). CONCLUSIONS: Oral MEM 1414, a novel PDE4 inhibitor, significantly reduces the late response following inhaled allergen challenge. MEM 1414 also inhibited whole blood assays of cytokine production from inflammatory cells. MEM 1414 was associated with a typical adverse event profile of PDE4 inhibitors, namely nausea and vomiting although these were mild side effects. TRIAL REGISTRATION NUMBER: Current controlled trials ISRCTN48047493.

Inhibition of LPS-induced airway neutrophilic inflammation in healthy volunteers with an oral CXCR2 antagonist.

BACKGROUND: Inhaled lipopolysaccharide (LPS) induces a dose-dependent, acute neutrophilic response in the airways of healthy volunteers that can be quantified in induced sputum. Chemokines, such as CXCL1 and CXCL8, play an important role in neutrophilic inflammation in the lung through the activation of CXCR2 and small molecule antagonists of these receptors have now been developed. We investigated the effect of AZD8309, a CXCR2 antagonist, compared with placebo on LPS-induced inflammation measured in sputum of healthy volunteers. METHODS: Twenty healthy subjects were randomized in a double-blind placebo-controlled, cross-over study. AZD8309 (300 mg) or placebo was dosed twice daily orally for 3 days prior to challenge with inhaled LPS and induced sputum was collected 6 h later. RESULTS: Treatment with AZD8309 showed a mean 77% reduction in total sputum cells (p < 0.001) and 79% reduction in sputum neutrophils (p < 0.05) compared with placebo after LPS challenge. There was also a reduction in neutrophil elastase activity (p < 0.05) and CXCL1 (p < 0.05) and trends for reductions in sputum macrophages (47%), leukotriene B4 (39%) and CXCL8 (52%). CONCLUSIONS: AZD8309 inhibited LPS-induced inflammation measured in induced sputum of normal volunteers, indicating that this treatment may be useful in the treatment of neutrophilic diseases of the airways, such as COPD, severe asthma and cystic fibrosis. TRIAL REGISTRATION: NCT00860821.

Safety of regadenoson, an adenosine A2A receptor agonist for myocardial perfusion imaging, in mild asthma and moderate asthma patients: a randomized, double-blind, placebo-controlled trial.

BACKGROUND: Patients with reactive airways are at risk for adenosine-induced bronchoconstriction, mediated via A(2B) and/or A(3) adenosine receptors. METHODS AND RESULTS: To examine the effects of regadenoson, a selective adenosine A(2A) receptor agonist, on airway resistance, we conducted a randomized, double-blind, placebo-controlled crossover trial in asthmatic patients with a positive adenosine monophosphate challenge test. The mean ratio of the forced expiratory volume in 1 second (FEV(1)) at each tested time point relative to the baseline FEV(1) was significantly higher after treatment with regadenoson compared with placebo from 10 to 60 minutes after treatment. One patient had a substantial but asymptomatic FEV(1) reduction (-36.2%) after regadenoson that reversed spontaneously. The most common adverse events with regadenoson were tachycardia (66%), dizziness (53%), headache (45%), and dyspnea (34%). The mean heart rate significantly increased with regadenoson (maximum of +10.4 beats/min) versus placebo. CONCLUSIONS: In this pilot safety study of 48 patients with mild or moderate asthma who had bronchial reactivity to adenosine monophosphate, regadenoson was safe and well tolerated.

Optimized dialysis and protease inhibition of sputum dithiothreitol supernatants.

RATIONALE: Dithiothreitol (DTT) is commonly used to liquefy induced sputum samples before assessment of cytology, but causes reduction of disulfide bonds and denaturation of proteins. OBJECTIVES: To process sputum supernatants containing DTT to enable quantification of cytokines and chemokines. METHODS: A standard solution of 22 pooled chemokines and cytokines was incubated with DTT at the concentrations used during sputum liquefaction and then dialyzed under 20 different denaturant and redox conditions. MEASUREMENTS AND MAIN RESULTS: After incubation of the standard solution with DTT there was loss of detectable protein mediators on immunoassay, but optimized dialysis permitted recovery of chemokines to 96 +/- 4% and cytokines to 91 +/- 6%. Optimized dialysis of DTT supernatants from subjects with asthma covering a range of severities (n = 35) was performed in the presence of a cocktail of protease inhibitors and demonstrated significantly elevated levels of the chemokine CXCL10 (IFN-gamma-inducible protein-10), CXCL8 (IL-8), and CCL3 (macrophage inflammatory protein-1alpha); with lower but significantly elevated levels of CCL2 (monocyte chemotactic protein-1), CCL11 (eotaxin), and CCL5 (regulated on activation, normal T-cell expressed and secreted) in severe asthma. In sputum from subjects with severe asthma there were also significantly elevated levels of IL-4, IL-5, IL-13, tumor necrosis factor-alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and IL-12(p40). CONCLUSIONS: The technique of optimized dialysis and protease inhibition of sputum DTT supernatants aids the detection of chemokines and cytokines. The detection of elevated levels of particular sputum chemokines and cytokines in individual patients may provide a rationale for specific therapies.

The effects of a monoclonal antibody directed against tumor necrosis factor-alpha in asthma.

RATIONALE: Neutralization of tumor necrosis factor-alpha (TNF-alpha) is an effective antiinflammatory therapy for several chronic inflammatory diseases. METHODS AND OBJECTIVES: We undertook a double-blind, placebo-controlled, parallel-group design study in 38 patients with moderate asthma treated with inhaled corticosteroids but symptomatic during a run-in phase. Infliximab (5 mg/kg) or placebo was administered by intravenous infusion at Weeks 0, 2, and 6. We assessed clinical response by monitoring lung function, symptoms, and inhaled beta(2)-agonist usage using hand-held electronic devices. RESULTS: The primary endpoint, change in morning PEF at Days 50-56 compared with the last 7 d of the run-in, was not significantly different on treatment. However, infliximab was associated with a decrease in mean diurnal variation of PEF at Week 8 (p = 0.02; 95% confidence interval [CI], -8.1 to -0.72). Furthermore, there was a decrease in the number of patients with exacerbations of asthma (p = 0.01; 95% CI, 4.4 to 52.7) and an increased probability of freedom from exacerbation with time (p = 0.03) in patients on infliximab (n = 14) compared with placebo (n = 18). In addition, infliximab decreased levels of TNF-alpha (p = 0.01) and other cytokines in sputum supernatants. There were no serious adverse events related to the study agent. CONCLUSIONS: Treatment with infliximab was well tolerated and caused a decrease in the number of patients with exacerbations in symptomatic moderate asthma. The promising preliminary findings underscore the need to evaluate therapy directed against TNF-alpha in larger trials enrolling patients with more severe asthma.