ß-Arrestin-2 mediates the proinflammatory effects of proteinase-activated receptor-2 in the airway.

Proteinase-Activated receptor-2 (PAR(2)), a G-protein-coupled Receptor, activated by serine proteinases, is reported to have both protective and proinflammatory effects in the airway. Given these opposing actions, both inhibitors and activators of PAR(2) have been proposed for treating asthma. PAR(2) can signal through two independent pathways: a ß-arrestin-dependent one that promotes leukocyte migration, and a G-protein/Ca(2+) one that is required for prostaglandin E(2) (PGE(2)) production and bronchiolar smooth muscle relaxation. We hypothesized that the proinflammatory responses to PAR(2) activation are mediated by ß-arrestins, whereas the protective effects are not. Using a mouse ovalbumin model for PAR(2)-modulated airway inflammation, we observed decreased leukocyte recruitment, cytokine production, and mucin production in ß-arrestin-2(-/-) mice. In contrast, PAR(2)-mediated PGE(2) production, smooth muscle relaxation, and decreased baseline airway resistance (measures of putative PAR(2) "protective" effects) were independent of ß-arrestin-2. Flow cytometry and cytospins reveal that lung eosinophil and CD4 T-cell infiltration, and production of IL-4, IL-6, IL-13, and TNFα, were enhanced in wild-type but not ß-arrestin-2(-/-) mice. Using the forced oscillation technique to measure airway resistance reveals that PAR(2) activation protects against airway hyperresponsiveness by an unknown mechanism, possibly involving smooth muscle relaxation. Our data suggest that the PAR(2)-enhanced inflammatory process is ß-arrestin-2 dependent, whereas the protective anticonstrictor effect of bronchial epithelial PAR(2) may be ß-arrestin independent.

Nebulized perflubron and carbon dioxide rapidly dilate constricted airways in an ovine model of allergic asthma.

BACKGROUND: The low toxicity of perfluorocarbons (PFCs), their high affinity for respiratory gases and their compatibility with lung surfactant have made them useful candidates for treating respiratory diseases such as adult respiratory distress syndrome. We report results for treating acute allergic and non-allergic bronchoconstriction in sheep using S-1226 (a gas mixture containing carbon dioxide and small volumes of nebulized perflubron). The carbon dioxide, which is highly soluble in perflubron, was used to relax airway smooth muscle. METHODS: Sheep previously sensitized to house dust mite (HDM) were challenged with HDM aerosols to induce early asthmatic responses. At the maximal responses (characterised by an increase in lung resistance), the sheep were either not treated or treated with one of the following; nebulized S-1226 (perflubron + 12% CO2), nebulized perflubron + medical air, 12% CO2, salbutamol or medical air. Lung resistance was monitored for up to 20 minutes after cessation of treatment. RESULTS: Treatment with S-1226 for 2 minutes following HDM challenge resulted in a more rapid, more profound and more prolonged decline in lung resistance compared with the other treatment interventions. Video bronchoscopy showed an immediate and complete (within 5 seconds) re-opening of MCh-constricted airways following treatment with S-1226. CONCLUSIONS: S-1226 is a potent and rapid formulation for re-opening constricted airways. Its mechanism(s) of action are unknown. The formulation has potential as a rescue treatment for acute severe asthma.

Mucosal allergic sensitization to cockroach allergens is dependent on proteinase activity and proteinase-activated receptor-2 activation.

We have shown that proteinase-activated receptor-2 (PAR(2)) activation in the airways leads to allergic sensitization to concomitantly inhaled Ags, thus implicating PAR(2) in the pathogenesis of asthma. Many aeroallergens with proteinase activity activate PAR(2). To study the role of PAR(2) in allergic sensitization to aeroallergens, we developed a murine model of mucosal sensitization to cockroach proteins. We hypothesized that PAR(2) activation in the airways by natural allergens with serine proteinase activity plays an important role in allergic sensitization. Cockroach extract (CE) was administered to BALB/c mice intranasally on five consecutive days (sensitization phase) and a week later for four more days (challenge phase). Airway hyperresponsiveness (AHR) and allergic airway inflammation were assessed after the last challenge. To study the role of PAR(2), mice were exposed intranasally to a receptor-blocking anti-PAR(2) Ab before each administration of CE during the sensitization phase. Mucosal exposure to CE induced eosinophilic airway inflammation, AHR, and cockroach-specific IgG1. Heat-inactivated or soybean trypsin inhibitor-treated CE failed to induce these effects, indicating that proteinase activity plays an important role. The use of an anti-PAR(2) blocking Ab during the sensitization phase completely inhibited airway inflammation and also decreased AHR and the production of cockroach-specific IgG1. PAR(2) activation by CE acts as an adjuvant for allergic sensitization even in the absence of functional TLR4. We conclude that CE induces PAR(2)-dependent allergic airway sensitization in a mouse model of allergic airway inflammation. PAR(2) activation may be a general mechanism used by aeroallergens to induce allergic sensitization.

Carbon dioxide enhances substance P-induced epithelium-dependent bronchial smooth muscle relaxation in Sprague-Dawley rats.

Hypocapnia and hypercapnia constrict and relax airway smooth muscle, respectively, through pH- and calcium (Ca(2+))-mediated mechanisms. In this study we explore a potential role for the airway epithelium in these responses to carbon dioxide (CO(2)). Contractile and relaxant responses of isolated rat bronchial rings were measured under hypocapnic, eucapnic, and hypercapnic conditions. Substance P was added to methacholine precontracted bronchial rings with and without epithelium. The role of Ca(2+) was assessed using Ca(2+)-free solutions and a Ca(2+) channel blocker, nifedipine. The effects of pH were assessed in solutions with HEPES buffer. Hypocapnic challenge increased the organ bath's pH and increased bronchial smooth muscle resting tension. This effect was abolished with HEPES buffer and partially inhibited by nifedipine. Hypocapnic conditions suppressed substance P-induced epithelium-dependent relaxation, whereas hypercapnia augmented the response. The epithelial hypocapnic effect was pH dependent, whereas the hypercapnic effect was pH independent. CO(2) had no effect on the epithelial independent smooth muscle agonists methacholine and isoproterenol. In conclusion our data indicate that, in addition to the effects of pH and Ca(2+), CO(2) affects airway smooth muscle by a pH-independent, epithelium-mediated mechanism. These findings could potentially lead to new treatments for asthma involving CO(2)-sensing receptors in the airways.

A phase IIa proof-of-concept, placebo-controlled, randomized, double-blind, crossover, single-dose clinical trial of a new class of bronchodilator for acute asthma.

BACKGROUND: This study evaluates a novel bronchodilator, S1226, for its efficacy in reversing allergen-induced bronchoconstriction in subjects with mild, allergic asthma. S1226 is a new class of bronchodilator that is an aerosol/vapor/gas mixture combining pharmacological and biophysical principles for a novel mode of action. It contains a potent bronchodilator gas (carbon dioxide or CO2) and nebulized perflubron (a synthetic surfactant possessing mucolytic properties). It has demonstrated rapid reversal of allergen-induced bronchoconstriction in an ovine study model. METHODS: This was a phase IIa proof-of-concept, placebo-controlled, randomized, double-blind, crossover single-dose clinical trial to evaluate the safety, tolerability, and efficacy of S1226 (8% CO2) administered by nebulization following an allergen-induced early asthmatic response in 12 subjects with mild, allergic asthma. Primary safety endpoints were adverse events, vital signs, pulse oximetry, and spirometry. Efficacy endpoints included bronchodilator response (measured as the forced expiratory volume in 1 s or FEV1) over time, the area under the curve of FEV1 for the early asthmatic response over time, and achievement of responder status, defined as a 12% improvement after the allergen challenge. RESULTS: No significant safety issues were observed. All adverse events were non-serious, mild, and transient. There was a statistically significant decrease in peripheral blood oxygenation levels over time in the placebo group following allergen inhalation, whereas blood oxygenation was maintained at normal levels in the S1226-treated subjects (P = 0.028). This effect was greatest 5 min after start of treatment (P < 0.001). The recovery rate was faster but not significantly so (P = 0.272) for S1226 compared to the placebo at earlier time points (5, 10, and 15 min), as assessed by ≥12% reversal of FEV1. The recovery of FEV1 over time was significantly greater (P = 0.04) with S1226 compared to the placebo. CONCLUSIONS: S1226 was safe, tolerated well, and provided bronchodilation and improved blood oxygenation in subjects with mild atopic asthma following allergen-induced bronchoconstriction. Additional studies to optimize the therapeutic response are indicated. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02334553 . Registered on 12 November 2014.

A phase I, placebo-controlled, randomized, double-blind, single ascending dose-ranging study to evaluate the safety and tolerability of a novel biophysical bronchodilator (S-1226) administered by nebulization in healthy volunteers.

BACKGROUND: A major challenge in treating acute asthma exacerbations is the need to open constricted airways rapidly enough to reestablish ventilation and allow delivery of conventional medication to diseased airways. The solution requires a new approach that considers both biophysical and pharmacological aspects of treatments used in acute asthma. The result of testing several formulations was S-1226: carbon dioxide-enriched air delivered in nebulized perflubron, a synthetic surfactant. These agents act synergistically to rapidly reopen closed airways within seconds. The bronchodilator effect is independent of ß-adrenergic and cholinergic mediated-signaling pathways, offering a unique mechanism of action. S-1226 has a low toxicity profile and was effective in treating bronchoconstriction in animal models of asthma. The goal of the present study was to evaluate the safety and tolerability of S-1226 in healthy human subjects. METHODS: The phase I study was a single-center, randomized, double-blind, placebo-controlled, sequential, single-ascending-dose study conducted in Canada. Thirty-six subjects were distributed into three cohorts. Within each cohort, subjects were randomized to receive a single dose of S-1226 or a matching placebo administered over a 2-minute nebulization period. S-1226 was formulated with perflubron and 4 %, 8 %, or 12 % CO2. The dose of CO2 was sequentially escalated by cohort. The safety and tolerability of S-1226 were evaluated through assessment of adverse events, vital signs, 12-lead electrocardiograms, clinical laboratory parameters, and physical examinations. RESULTS: S-1226 was safe and well tolerated at all three CO2 levels (4 %, 8 %, and 12 %). A total of 28 adverse events were reported, and all were judged mild in severity. Twenty-four adverse events occurred in the S-1226 cohort, of which five were considered remotely related and six possibly related to S-1226. CONCLUSIONS: S-1226 is a novel drug being developed for the treatment of acute asthma exacerbations. It consists of CO2-enriched air and perflubron and has potential to offer rapid and potent bronchodilation. The results of the study indicate that S-1226 is safe and well tolerated. All adverse events were mild, reversible, and likely due to known side effects of CO2 inhalation. TRIAL REGISTRATION: ClinicalTrials.gov NCT02616770 . Registered on 25 November 2015.