Tiotropium/Olodaterol treatment reduces cigarette smoke extract-induced cell death in BEAS-2B bronchial epithelial cells.

BACKGROUND: Cigarette smoking is a critical risk factor for the destruction of lung parenchyma or the development of emphysema, which is characteristic of COPD. Disruption of epithelial layer integrity may contribute to lung injury following cigarette smoke extract (CSE) exposure. Tiotropium/olodaterol acts as a bronchodilator for COPD treatment; however, the effect of dual bronchodilators on epithelial cell injury and its underlying mechanism remain unclear. In this study, we evaluated the effect of tiotropium/olodaterol on CSE-mediated cell death and the underlying mechanisms. METHODS: Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis, necrosis, and autophagy were evaluated using flow cytometry. Autophagy-related protein, phosphorylated ERK, expression was determined using Western blotting. RESULTS: Tiotropium/olodaterol significantly inhibited CSE-induced cell death, mitochondria dysfunction, and autophagy, which had no significant effect on apoptosis or necrosis in BEAS-2B human bronchial epithelial cells. Moreover, tiotropium/olodaterol attenuated CSE-induced upregulation of JNK. CONCLUSIONS: CSE induced cell death and caused consistent patterns of autophagy and JNK activation in BEAS-2B human bronchial epithelial cells. Tiotropium/olodaterol treatment protected bronchial epithelial cells from CSE-induced injury and inhibited activation of autophagy and upregulation of JNK phosphorylation. These results indicate that tiotropium/olodaterol may protect epithelial cells from the deleterious effects of CSE exposure, which is associated with the regulation of autophagy and JNK activation.

Tiotropium and Fluticasone Inhibit Rhinovirus-Induced Mucin Production via Multiple Mechanisms in Differentiated Airway Epithelial Cells.

Human rhinoviruses (HRVs) are associated with acute exacerbations in patients with chronic obstructive pulmonary disease (COPD) and asthma, which are accompanied by mucus hypersecretion. Whereas, various studies have shown that HRVs increase epithelial mucin production and thus may directly contribute to mucus hypersecretion. The effects of drugs used in the treatment of COPD and asthma on HRV-induced mucin production in epithelial cell cultures have not been studied. In the present study, we assessed effects of HRVs on mucin production and secretion in well-differentiated primary human bronchial epithelial cells (PBEC) and studied the effect of the inhaled corticosteroid fluticasone propionate and the long-acting muscarinic antagonist tiotropium bromide on this process. Differentiated PBEC that were cultured at the air-liquid interface (ALI-PBEC) were infected with HRV-A16 and HRV-1B. Quantitative PCR, immunofluorescence staining, ELISA, periodic acid-Schiff (PAS) staining and immunostaining assays were used to assess the effects of HRV infection. Here we demonstrate that both HRV-A16 and HRV-1B increased mucin (MUC5AC and MUC5B) gene expression and protein release. When exploring this in more detail in HRV-A16-infected epithelial cells, mucin expression was found to be accompanied by increases in expression of SAM-pointed domain-containing Ets-like factor (SPDEF) and SPDEF-regulated genes known to be involved in the regulation of mucin production. We also found that pre-treatment with the purinergic P2R antagonist suramin inhibits HRV-enhanced MUC5AC expression and protein release, implicating involvement of purinergic signaling by extracellular ATP. We furthermore found that both fluticasone and tiotropium decreased HRV-induced mucin production without affecting viral replication, and obtained evidence to suggest that the inhibitory effect of fluticasone involved modulation of SPDEF-regulated genes and extracellular ATP release. These data show that both tiotropium and fluticasone inhibit HRV-induced epithelial mucin production independent of viral clearance, and thus provide insight into the mechanisms underlying beneficial effects of tiotropium and fluticasone in the treatment of COPD, asthma and accompanying exacerbations in these patients. Furthermore, our findings provide additional insight into the mechanisms by which HRV increases epithelial mucin production.

Functional analysis of OCTN2 and ATB0,+ in normal human airway epithelial cells.

In human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by ß-oxidation. Aim of the present study was to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI for different times (8d or 21d of culture) were used as comparison. OCTN2 transporter was equally expressed in both models and functional at the basolateral side. ATB0,+ was, instead, highly expressed and active on the apical membrane of EpiAirway™ and only in early-cultures of Calu-3 (8d but not 21d ALI). In both cell models, L-carnitine uptake on the apical side was significantly inhibited by the bronchodilators glycopyrrolate and tiotropium, that hence can be considered substrates of ATB0,+; ipratropium was instead effective on the basolateral side, indicating its interaction with OCTN2. Inflammatory stimuli, such as LPS or TNFα, caused an induction of SLC6A14/ATB0,+ expression in Calu-3 cells, along with a 2-fold increase of L-carnitine uptake only at the apical side; on the contrary SLC22A5/OCTN2 was not affected. As both OCTN2 and ATB0,+, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the study of drug inhalation and pulmonary delivery.

Effect of Tiotropium Bromide on Airway Inflammation and Programmed Cell Death 5 in a Mouse Model of Ovalbumin-Induced Allergic Asthma.

Rationale: We previously demonstrated increased expression of programmed cell death 5 (PDCD5) in asthmatic patients and ovalbumin-induced allergic asthma. International guidelines (GINA 2019) have included the use of tiotropium bromide for chronic treatment of the most severe and frequently exacerbated asthma in patients ≥6 years old, who do not have good response to inhaled corticosteroids. Objective: To explore the role of tiotropium and its effect on PDCD5 level in a mouse model of chronic asthma. Methods: We divided 12 female mice into 2 groups: untreated asthma (n = 6) and tiotropium-treated asthma (n = 6). The impact of tiotropium was assessed by histology of lung tissue and morphometry. Pulmonary function was tested by using pressure sensors. The number of cells in bronchoalveolar lavage fluid (BALF) was detected. Levels of PDCD5, active caspase-3, and muscarinic acetylcholine receptors M2 (ChRM2) and M3 (ChRM3) were examined. Results: Tiotropium treatment significantly reduced airway inflammation and remodeling in asthmatic mice and intensified the lung function. PDCD5 level was reduced with tiotropium (p < 0.05). Moreover, active caspase-3 level was decreased with tiotropium (p < 0.001), and ChRM3 level was increased. Conclusions: Tiotropium treatment may alleviate the pathological changes with asthma by regulating apoptosis.

Tiotropium inhibits proinflammatory microparticle generation by human bronchial and endothelial cells.

Tiotropium is a muscarinic antagonist that reduces the risk of acute exacerbations of chronic obstructive pulmonary disease, possibly through an as yet incompletely characterized anti-inflammatory activity. We hypothesized that muscarinic activation of bronchial epithelial cells and endothelial cells causes the release of proinflammatory microparticles and that tiotropium inhibits the phenomenon. Microparticle generation was assessed by a functional assay, by flow cytometry and by NanoSight technology. Immortalized bronchial epithelial cells (16HBE) and umbilical vein endothelial cells were treated with acetylcholine in the presence of varying concentrations of tiotropium. Intracellular calcium concentration, extracellular regulated kinase phosphorylation and chemokine content in the conditioned media were assessed by commercial kits. Acetylcholine causes microparticle generation that is completely inhibited by tiotropium (50 pM). Microparticles generated by acetylcholine-stimulated cells increase the synthesis of proinflammatory mediators in an autocrine fashion. Acetylcholine-induced upregulation of microparticle generation is inhibited by an inhibitor of extracellular regulated kinase phosphorylation and by a phospholipase C inhibitor. Tiotropium blocks both extracellular regulated kinase phosphorylation and calcium mobilization, consistent with the hypothesis that the drug prevents microparticle generation through inhibition of these critical pathways. These results might contribute to explain the effect of tiotropium in reducing acute exacerbations of chronic obstructive pulmonary disease.

Pharmacological characterization of the interaction between tiotropium bromide and olodaterol on human bronchi and small airways.

Combining a long-acting ß2-agonist (LABA) with a long-acting muscarinic antagonist (LAMA) is the cornerstone to treat patients with chronic obstructive pulmonary disease (COPD). In this study we have characterized the interaction between the LAMA tiotropium bromide, and the LABA olodaterol, on the contractile tone of human medium bronchi and small airways. The response to a combination of tiotropium bromide and olodaterol was assessed at sub-maximal contractile tone induced by carbachol. The duration of action was studied in tissue contracted by transmural stimulation. Relaxation of bronchial tone was expressed as % of maximal response to papaverine. Drug interactions were analyzed by the Bliss Independence method and Unified Theory. Tiotropium bromide/olodaterol combination induced a significant synergistic relaxant response (P < 0.05 vs. expected additive effect) in medium bronchi and small airways pre-contracted by carbachol, by enhancing relaxation +22.13 ± 4.42% and +26.31 ± 12.39%, respectively. The combination of tiotropium bromide and olodaterol also reduced the airway smooth muscle contractility elicited by transmural stimulation by 73.60 ± 3.10%. The extent of synergy was strong to very strong, and was supported by the release of neuronal acetylcholine, cyclic adenosine monophosphate levels, and activation of iberiotoxin-sensitive KCa++ channels. Conversely, the interaction between tiotropium bromide and olodaterl was independent of the activity at M2 muscarinic receptors. These results indicate that tiotropium bromide/olodaterol combination leads to a potent and durable synergistic relaxation of human medium bronchi and small airways. Further pharmacological studies are needed to confirm these results in clinical settings.

Tiotropium inhibits methacholine-induced extracellular matrix production via ß-catenin signaling in human airway smooth muscle cells.

Background: Airway remodeling is an important feature of chronic obstructive pulmonary disease (COPD) that is associated with disease severity and irreversible airflow limitation. An extensive alteration of the extracellular matrix (ECM) surrounding the airway smooth muscle (ASM) bundle is one of the pathological manifestations of airway remodeling, which contributes to the decline in lung function. Tiotropium, a long-acting inhaled muscarinic receptor antagonist, has been confirmed to play a role in preventing airway remodeling including ECM deposition beyond bronchodilation in vivo, but the relationship between ASM cell (ASMC) relaxation and ECM production remains unclear. Purpose: In this study, we attempted to investigate the influence of tiotropium on ECM production by ASMCs and the underlying mechanism. Methods: Tiotropium was added 30 minutes before the addition of methacholine to primary cultured human ASMCs. Protein expression was analylized by Western Blot and mRNA abundance was determined by real-time PCR. Results: We found that tiotropium reduced collagen I protein expression, and the mRNA abundance of collagen I, fibronectin, and versican. ß-catenin signaling was inactivated by inhibiting glycogen synthase kinase 3ß (GSK3ß) phosphorylation in this process. Tiotropum inhibited the amount of active ß-catenin and its transcription activity. Furthermore, overexpression of active ß-catenin by adenoviruses carrying the S33Y mutant resisted the suppressive effect of tiotropium on collagen I protein expression. However, silencing ß-catenin by specific small interfering RNA enhanced the negative effect of tiotropium. Conclusion: These findings suggest that relaxation of ASMCs by tiotropium can prevent ECM production through ß-catenin signaling.

Protective effects of tiotropium alone or combined with budesonide against cadmium inhalation induced acute neutrophilic pulmonary inflammation in rats.

As a potent bronchodilator, the anti-inflammatory effects of tiotropium and its interaction with budesonide against cadmium-induced acute pulmonary inflammation were investigated. Compared to values obtained in rats exposed to cadmium, cytological analysis indicated a significant decrease of total cell and neutrophil counts and protein concentration in bronchoalveolar lavage fluid (BALF) in rats pretreated with tiotropium (70µg/15ml or 350µg/15ml). Zymographic tests showed a decrease of MMP-2 activity in BALF in rats pretreated only with high concentration of tiotropium. Histological examination revealed a significant decrease of the severity and extent of inflammatory lung injuries in rats pretreated with both tested concentrations of tiotropium. Though tiotropium (70µg/15ml) or budesonide (250µg/15ml) could not reduce cadmium-induced bronchial hyper-responsiveness, their combination significantly decreased bronchial contractile response to methacholine. These two drugs separately decreased the neutrophil number and protein concentration in BALF but no significant interaction was observed when both drugs were combined. Although no inhibitory effects on MMP-2 and MMP-9 was observed in rats pretreated with budesonide alone, the combination with the ineffective dose of tiotropium induced a significant reduction on these parameters. The inhibitory effect of tiotropium on lung injuries was not influenced by budesonide which alone induced a limited action on the severity and extent of inflammatory sites. Our findings show that tiotropium exerts anti-inflammatory effects on cadmium-induced acute neutrophilic pulmonary inflammation. The combination of tiotropium with budesonide inhibits cadmium-induced inflammatory injuries with a synergistic interaction on MMP-2 and MMP-9 activity and airway hyper-responsiveness.

Molecular Basis of Ligand Dissociation from G Protein-Coupled Receptors and Predicting Residence Time.

G protein-coupled receptors (GPCRs) are integral membrane proteins and represent the largest class of drug targets. During the past decades progress in structural biology has enabled the crystallographic elucidation of the architecture of these important macromolecules. It also provided atomic-level visualization of ligand-receptor interactions, dramatically boosting the impact of structure-based approaches in drug discovery. However, knowledge obtained through crystallography is limited to static structural information. Less information is available showing how a ligand associates with or dissociates from a given receptor, whose importance is in fact increasingly recognized by the drug research community. Owing to recent advances in computer power and algorithms, molecular dynamics stimulations have become feasible that help in analyzing the kinetics of the ligand binding process. Here, we review what is currently known about the dynamics of GPCRs in the context of ligand association and dissociation, as determined through both crystallography and computer simulations. We particularly focus on the molecular basis of ligand dissociation from GPCRs and provide case studies that predict ligand dissociation pathways and residence time.

Tiotropium inhibits mucin production stimulated by neutrophil elastase but not by IL-13.

Tiotropium, a muscarinic antagonist, is approved for the treatment of chronic obstructive pulmonary disease and poorly controlled asthma. Because mucus hypersecretion is characteristic of both of these diseases, and muscarinic agonists stimulate mucus secretion, we hypothesized that tiotropium would attenuate airway MUC5AC expression. We grew normal human bronchial epithelial (NHBE) cells to a goblet cell phenotype with 1 or 5 ng/mL of IL-13 and exposed these cells to 10 nM tiotropium or excipient for the full 14 days. Normally differentiated NHBE cells (without IL-13) were exposed to neutrophil elastase (NE) 1 × 10-7 or 5 × 10-7 M for 1 h. MUC5AC was measured by quantitative PCR and ELISA. Acetylcholine production by the epithelium was evaluated by quantitative PCR and by choline/acetylcholine quantification. Tiotropium had no effect on IL-13-stimulated MUC5AC, but attenuated MUC5AC stimulated by NE (p = 0.007 at 5 × 10-7 M). IL-13 increased CarAT mRNA (p < 0.001 at 5 ng/mL) and acetylcholine concentration in the medium (p = 0.018 at 5 ng/mL), while NE had no effect. Tiotropium had no direct effect on IL-13 or NE-induced CarAT or acetylcholine concentration. Tiotropium decreased MUC5AC stimulated by NE, but had no effect on MUC5AC stimulated by IL-13. These results may be due to IL-13, but not NE, increasing acetylcholine production.

Ipratropium is 'luminally recycled' by an inter-play between apical uptake and efflux transporters in Calu-3 bronchial epithelial cell layers.

The mechanism by which quaternized anticholinergic bronchodilators permeate the airway epithelium remains controversial to date. In order to elucidate the role of drug transporters, ipratropium bidirectional transport as well as accumulation and release studies were performed in layers of the broncho-epithelial cell line Calu-3 grown at an air-liquid interface, in presence or absence of a range of transporter inhibitors. Unexpectedly, a higher transepithelial permeability was observed in the secretory direction, with an apparent efflux ratio of > 4. Concentration-dependent and inhibitor studies demonstrated the drug intracellular uptake was carrier-mediated. Interestingly, monitoring drug release post cell loading revealed the presence of an efficient efflux system on the apical side of the cell layers. Acting in concert, apical transporters seem to promote the 'luminal recycling' of the drug and hence, limit its transcellular transport. The data are in agreement with an apical Organic Cation Transporter (OCT) being involved in this process but also suggest the participation of unknown uptake and efflux transporters sensitive to probenecid. This study suggests the absorption of ipratropium across the pulmonary barrier is primarily governed by paracellular passive diffusion but transporters might play a significant role in controlling the drug local concentrations in the lungs.

The effect of tiotropium in combination with olodaterol on house dust mite-induced allergic airway disease.

One of the major goals of asthma therapy is to maintain asthma control and prevent acute exacerbations. Long-acting bronchodilators are regularly used for the treatment of asthma patients and in clinical studies the anti-cholinergic tiotropium has recently been shown to reduce exacerbations in patients with asthma. So far it is unclear how tiotropium exerts this effect. For this purpose, we designed an allergen-driven rechallenge model of allergic airway inflammation in mice, to assess the effectiveness of tiotropium and the long-acting ß-2 adrenoceptor agonist olodaterol on allergen-induced exacerbations of airway disease. Female C57BL/6J mice were sensitized intranasally (i.n.) with 1 µg of house dust mite (HDM) extract followed by a challenge regime (5 consecutive days 10 µg HDM extract i.n.) after one week. Mice were exposed to a secondary challenge five weeks after sensitization and were treated i.n. with different concentrations of tiotropium or olodaterol (1, 10 and 100 µg/kg) or a combination thereof (10 µg/kg each) prior to and during the secondary challenge period. Three days after the last challenge, bronchoalveolar lavage (BAL) fluid and lung tissue were collected for flow cytometry and histologic analysis, respectively. Secondary challenge with HDM extract strongly induced allergic airway disease reflected by inflammatory cell infiltration and goblet cell metaplasia. Treatment with tiotropium, but not with olodaterol reduced tissue inflammation and goblet cell metaplasia in a dose-dependent manner. The combination of tiotropium and olodaterol was more effective in significantly reducing tissue inflammation compared to tiotropium treatment alone, and also led to a decrease in BAL cell counts. These data suggest that in a model of relapsing allergic airway disease tiotropium directly prevents exacerbations by reducing inflammation and mucus production in the airways. In addition, the combination of tiotropium and olodaterol exerts synergistic effects.

Effect of a single exacerbation on decline in lung function in COPD.

BACKGROUND: COPD exacerbations are associated with accelerated lung function decline, but whether they are causal is unknown. We evaluated the effect of a single exacerbation on rate of lung function change using data from the 4-year Understanding Potential Long-term Impacts on Function with Tiotropium (UPLIFT®) trial. METHODS: Retrospective analysis of annual rates of decline in FEV1 and FVC before and after a single (and the only) moderate-to-severe exacerbation in patients during UPLIFT® (exacerbator subgroup), compared with changes between the first and second half of the study in a non-exacerbator subgroup. A sensitivity analysis examined annual rates of decline in matched pairs of exacerbators and non-exacerbators. RESULTS: Following the single moderate-to-severe exacerbation, mean annual decline in post-bronchodilator lung function increased compared with the rate of decline before the exacerbation (FEV1 76.5 vs. 39.1 mL/year, p = 0.003; FVC 106.5 vs. 34.7 mL/year, p = 0.011). In non-exacerbators, there were no differences in rates of decline between the first and second halves of the study (post-bronchodilator FEV1 38.2 vs. 41.8 mL/year, FVC 45.3 vs. 43.9 mL/year. Before the single (moderate-to-severe) exacerbation in the exacerbator subgroup, declines in post-bronchodilator FEV1 or FVC were similar to non-exacerbators in the first half of the study; after the single exacerbation they were significantly higher than for non-exacerbators in the second half of the study. The sensitivity analysis showed similar results. CONCLUSION: A single COPD exacerbation may result in significant increase in the rate of decline in lung function.

Efficacy and safety of indacaterol in patients with chronic obstructive pulmonary disease aged over 65 years: A pooled analysis.

BACKGROUND: Although the prevalence of chronic obstructive pulmonary disease (COPD) increases with age, no specific therapeutic approaches are available till date for the elderly population. AIM: To assess the efficacy and safety of once-daily indacaterol 150 and 300 µg in elderly patients with moderate to severe COPD. METHODS: Data were pooled from 11 randomized, double-blind, placebo- and active-controlled studies (8445 patients with COPD). The patient population was stratified into age groups: young (≥40-<65 years; 52.3%), elderly (≥65-<75 years; 36.4%), and very elderly (≥75 years; 11.4%). The efficacy outcomes included improvements in trough forced expiratory volume in 1 s (FEV1), transition dyspnea index (TDI), and health status (St. George's Respiratory Questionnaire [SGRQ]); safety was also assessed at 12 weeks. RESULTS: At Week 12, the mean improvement in FEV1 with indacaterol 150 µg versus placebo was comparable in the elderly (150 mL), very elderly (160 mL), and young (170 mL) groups (p < 0.001 for all comparisons). Similar improvement in FEV1 was observed with indacaterol 300 µg versus placebo in each group (p < 0.001). This improvement was also significantly higher with indacaterol than formoterol, salmeterol, and tiotropium in all groups (p < 0.01). Both TDI and SGRQ scores significantly improved with indacaterol versus placebo across age groups (p < 0.001) and were significantly higher than that for tiotropium (p < 0.001). Incidences of adverse events among indacaterol- or placebo-treated patients were similar, regardless of the age group. CONCLUSIONS: This pooled analysis suggests that the efficacy and safety of indacaterol treatment is similar between elderly and younger patients with COPD.

Long-term safety and efficacy of glycopyrrolate/formoterol metered dose inhaler using novel Co-Suspension™ Delivery Technology in patients with chronic obstructive pulmonary disease.

BACKGROUND: The long-term safety and efficacy of a novel Co-Suspension™ Delivery Technology glycopyrrolate (GP)/formoterol fumarate (FF) 18/9.6 µg fixed-dose combination metered dose inhaler (GFF MDI) were investigated in a 28-week safety extension study (PINNACLE-3, NCT01970878) of two randomized controlled Phase III trials (PINNACLE-1 and -2; NCT01854645 and NCT01854658) in subjects with moderate-to-very severe chronic obstructive pulmonary disease (COPD). METHODS: Subjects completing 24 weeks' treatment with GFF MDI, GP MDI, FF MDI (all twice-daily) or open-label tiotropium 18 µg (once-daily) in PINNACLE-1 or -2 were randomly selected to continue treatment for 28 weeks. The target enrollment for PINNACLE-3 was 850 subjects. Safety and efficacy were evaluated over 52 weeks. RESULTS: Of 3274 subjects randomized to active treatment in PINNACLE-1 or -2, 892 entered PINNACLE-3. Incidences of adverse events, serious adverse events and major adverse cardiovascular events were similar across treatment groups with no unexpected safety findings. For change from baseline in morning pre-dose trough forced expiratory volume in 1 s (FEV1), treatment differences for GFF MDI versus GP MDI, FF MDI and open-label tiotropium over 52 weeks were 57, 65 and 25 mL, respectively (p ≤ 0.0117). Average daily rescue medication use was significantly reduced for GFF MDI versus GP MDI and open-label tiotropium (p ≤ 0.0002). Statistically significant improvements were observed with GFF MDI versus monocomponents in Self-Administered Computerized Transition Dyspnea Index focal score, and in St George's Respiratory Questionnaire total score versus GP MDI. CONCLUSIONS: Results confirmed the long-term safety and tolerability of GFF MDI 18/9.6 µg twice-daily in subjects with moderate-to-very severe COPD. Improvements in efficacy endpoints were also sustained over 52 weeks.

Consistent improvement in health-related quality of life with tiotropium in patients with chronic obstructive pulmonary disease: Novel and conventional responder analyses.

INTRODUCTION: Improving health-related quality of life (HRQoL) in COPD patients is an important pharmacotherapeutic objective. This study investigated the extent, consistency, and durability of tiotropium maintenance therapy impact on HRQoL in moderate-to-very severe COPD. METHODS: Patients received once-daily tiotropium 18 µg (n = 5244) or placebo (n = 4799) via HandiHaler® (10 trials), or once-daily tiotropium 5 µg (n = 2622) or placebo (n = 2618) via Respimat® inhaler (3 trials). St George's Respiratory Questionnaire (SGRQ) total scores were measured at baseline, and 6 months (13 trials) and 1 year (9 trials) from treatment start. Adjusted mean differences between treatments for change from baseline in total scores were calculated at each time-point for each trial. Responder and deteriorator rates (decrease or increase in score ≥4 units from baseline, respectively), net benefit (responder rate increase plus deteriorator rate decrease), and cumulative improvement and deterioration were determined. RESULTS: Adjusted mean total score differences between treatments for change from baseline were significant (p < 0.05) in favor of tiotropium in 10/13 trials at 6 months and in 8/9 trials at 1 year. In all trials, estimated differences in responder rates between treatments favored tiotropium (significant [p < 0.05]: 5/13 trials at 6 months; 8/9 trials at 1 year). Net benefit favored tiotropium and cumulative improvement rates were consistently greater and deterioration rates consistently lower for tiotropium versus placebo. CONCLUSIONS: Tiotropium maintenance therapy significantly and consistently improved HRQoL in moderate-to-very severe COPD patients in a durable manner. These results may provide a benchmark for assessing benefits on HRQoL of other COPD treatments.

Effects of indacaterol versus tiotropium on exercise tolerance in patients with moderate COPD: a pilot randomized crossover study / Efeitos do indacaterol versus tiotrópio na tolerância ao exercício em pacientes com DPOC moderada: estudo cruzado randomizado piloto

Abstract Objective: To compare a once-daily long-acting β2 agonist (indacaterol 150 µg) with a once-daily long-acting anticholinergic (tiotropium 5 µg) in terms of their effects on exercise endurance (limit of tolerance, Tlim) in patients with moderate COPD. Secondary endpoints were their effects on lung hyperinflation, exercise-related dyspnea, and daily-life dyspnea. Methods: This was a randomized, single-blind, crossover pilot study involving 20 patients (mean age, 60.9 ± 10.0 years; mean FEV1, 69 ± 7% of predicted). Spirometric parameters, Transition Dyspnea Index scores, Tlim, and exertional dyspnea were compared after three weeks of each treatment (with a one-week washout period between treatments). Results: Nineteen patients completed the study (one having been excluded because of COPD exacerbation). Improvement in Tlim from baseline tended to be greater after treatment with tiotropium than after treatment with indacaterol (96 ± 163 s vs. 8 ± 82 s; p = 0.06). Tlim significantly improved from baseline after treatment with tiotropium (having increased from 396 ± 319 s to 493 ± 347 s; p = 0.010) but not after treatment with indacaterol (having increased from 393 ± 246 to 401 ± 254 s; p = 0.678). There were no differences between the two treatments regarding improvements in Borg dyspnea scores and lung hyperinflation at "isotime" and peak exercise. There were also no significant differences between treatments regarding Transition Dyspnea Index scores (1.5 ± 2.1 vs. 0.9 ± 2.3; p = 0.39). Conclusions: In patients with moderate COPD, tiotropium tends to improve Tlim in comparison with indacaterol. No significant differences were observed between the two treatments regarding their effects on lung hyperinflation, exercise-related dyspnea, and daily-life dyspnea. Future studies, including a larger number of patients, are required in order to confirm our findings and explore mechanistic explanations. (ClinicalTrials.gov identifier: ...

RESUMO Objetivo: Comparar um β2-agonista de longa duração administrado uma vez por dia (indacaterol 150 µg) a um anticolinérgico de longa duração administrado uma vez por dia (tiotrópio 5 µg) quanto a seus efeitos na resistência ao exercício (limite de tolerância, Tlim) em pacientes com DPOC moderada. Os desfechos secundários foram seus efeitos na hiperinsuflação pulmonar, na dispneia causada pelo exercício e na dispneia na vida diária. Métodos: Estudo piloto randomizado cruzado e simples cego com 20 pacientes (média de idade: 60,9 ± 10,0 anos; média do VEF1: 69 ± 7% do previsto). Parâmetros espirométricos, pontuação no Transition Dyspnea Index, Tlim e dispneia aos esforços foram comparados após três semanas de cada tratamento (com uma semana de intervalo entre os tratamentos). Resultados: Dezenove pacientes completaram o estudo - um foi excluído por causa de exacerbação da DPOC. A melhora no Tlim tendeu a ser maior com tiotrópio do que com indacaterol (96 ± 163 s vs. 8 ± 82 s; p = 0,06). Em comparação com os valores basais, o Tlim melhorou significativamente com tiotrópio (aumentando de 396 ± 319 s para 493 ± 347 s; p = 0,010), mas não com indacaterol (aumentando de 393 ± 246 para 401 ± 254 s; p = 0,678). Não houve diferença entre os tratamentos quanto à melhora na pontuação na escala de dispneia de Borg e na insuflação pulmonar no "isotempo" e no pico do exercício. Também não houve diferenças significativas entre os tratamentos quanto à pontuação no Transition Dyspnea Index (1,5 ± 2,1 vs. 0,9 ± 2,3; p = 0,39). Conclusões: Em pacientes com DPOC moderada, o tiotrópio tende a melhorar o Tlim em comparação com o indacaterol. Não houve diferenças significativas entre os tratamentos quanto a seus efeitos na insuflação pulmonar, na dispneia durante o exercício e na dispneia na vida diária. São necessários mais estudos, com um número maior de pacientes, para confirmar nossos achados e explorar explicações mecanicistas. (ClinicalTrials.gov ...

Tiotropium improves lung function, exacerbation rate, and asthma control, independent of baseline characteristics including age, degree of airway obstruction, and allergic status.

BACKGROUND: Many patients with asthma remain symptomatic despite treatment with inhaled corticosteroids (ICS) with or without long-acting ß2-agonists (LABAs). Tiotropium add-on to ICS plus a LABA has been shown to improve lung function and reduce exacerbation risk in patients with symptomatic asthma. OBJECTIVE: To determine whether the efficacy of tiotropium add-on therapy is dependent on patients' baseline characteristics. METHODS: Two randomized, double-blind, parallel-group, twin trials (NCT00772538 and NCT00776984) of once-daily tiotropium Respimat(®) 5 µg add-on to ICS plus a LABA were performed in parallel in patients with severe symptomatic asthma. Exploratory subgroup analyses of peak forced expiratory volume in 1 s (FEV1), trough FEV1, time to first severe exacerbation, time to first episode of asthma worsening, and seven-question Asthma Control Questionnaire responder rate were performed to determine whether results were influenced by baseline characteristics. RESULTS: 912 patients were randomized: 456 received tiotropium and 456 received placebo. Tiotropium improved lung function, reduced the risk of asthma exacerbations and asthma worsening, and improved asthma symptom control, compared with placebo, independent of baseline characteristics including gender, age, body mass index, disease duration, age at asthma onset, and FEV1 % predicted at screening and reversibility. CONCLUSION: Once-daily tiotropium 5 µg compared with placebo improved lung function, reduced the risk of asthma exacerbations and asthma worsening, and improved asthma symptom control, independent of a broad range of baseline characteristics, as add-on to ICS plus LABAs in patients with severe symptomatic asthma. TRIAL REGISTRY: ClinicalTrials.gov; numbers NCT00772538 and NCT00776984 URL: www.clinicaltrials.gov.

IL-17A induces chromatin remodeling promoting IL-8 release in bronchial epithelial cells: Effect of Tiotropium.

AIMS: IL-17A plays a key role in the persistence of airway inflammation, oxidative stress, and reduction of steroid-sensitivity in COPD. We studied the effect of IL-17A on chromatin remodeling and IL-8 production. MAIN METHODS: We measured the levels of IL-8 and IL-17A in induced sputum supernatants (ISS) from healthy controls (HCs), healthy smokers (HSs), and COPD patients by enzyme-linked immunosorbent assay (ELISA). A human bronchial epithelial cell line (16HBE) was stimulated with ISS from HCs, HSs, or COPD subjects. IL-8 was evaluated in 16HBE by Western blot and real-time polymerase chain reaction (PCR). Histone deacetylase 2 (HDAC2), acetyl histone H3 (Ac-His H3) (k9) and inhibitor kappa kinase alpha (IKKα) levels were evaluated in the nuclear extract by Western blot. Finally, we evaluated the effect of IL-17A depletion in ISS, the silencing of IKKα, and the anti-inflammatory effects of Tiotropium Spiriva® (100nM) on 16HBE. KEY FINDINGS: IL-8 and IL-17A levels were higher in ISS from COPD patients and HSs than from HCs. IL-8 protein and messenger RNA (mRNA) levels were increased in 16HBE stimulated with ISS from COPD patients compared with untreated cells. Furthermore, ISS from COPD patients reduced the nuclear levels of HDAC2 while increasing the activity of both Ac-His H3 (k9) and IKKα in stimulated 16HBE. IL-17A depletion in ISS and the IKKα silencing in 16HBE significantly increased the nuclear levels of HDAC2, reduced Ac-His H3 (k9), and promoted IL-8 synthesis in stimulated 16HBE. Tiotropium controls the proinflammatory activity generated by ISS from COPD patients in 16HBE. SIGNIFICANCE: IL-17A present in the airway of COPD patients, which induces chromatin remodeling, promotes the release of IL-8 in the bronchial epithelium. Tiotropium is able to control this proinflammatory activity.

Downregulation of the cough reflex by aclidinium and tiotropium in awake and anesthetized rabbits.

Long-acting muscarinic receptor antagonists (LAMAs) have been reported to attenuate cough in preclinical and clinical studies. The present study was performed on rabbits to compare aclidinium and tiotropium efficacy in the downregulation of the cough reflex. This reflex was evoked by citric acid inhalation in unanesthetized animals and by both citric acid inhalation and mechanical stimulation of the tracheobronchial tree in anesthetized animals 90 min following the inhalation of each drug (nebulizer output always at 1 mL/min). Aclidinium 4 mg/mL and tiotropium 200 µg/mL inhaled in 1 min proved to have similar protective effect on methacholine-induced bronchoconstriction in anesthetized animals. The total dosage employed for aclidinium and tiotropium was 4 mg and 200 µg, respectively. In awake animals, similar reductions in the cough number were observed following 10-min inhalation of each drug with a slight, not significant tendency to higher antitussive effects for aclidinium. In anesthetized animals, 1-min inhalation of each drug caused similar depressant effects on cough responses induced by both mechanical and chemical stimulation. A complete suppression of cough responses to mechanical stimuli was seen in some preparations. The results strongly suggest that the LAMA-induced downregulation of cough may be mediated not only by transient receptor potential vanilloid type 1 channels, as already reported, but also by acid-sensing ion channels and mechanoreceptors. The route of administration along with the more rapid hydrolysis of aclidinium into inactive metabolites minimize potential systemic side effects and give to this drug a very favorable safety profile.