Tiotropium Bromide Improves Neutrophilic Asthma by Recovering Histone Deacetylase 2 Activity.

BACKGROUND: The value of tiotropium bromide (TIO) in neutrophilic asthma was meaningful in previous study. We hypothesized that TIO's mechanism of action is associated with histone deacetylase 2 (HDAC2) activity, which is key for controlling the transcription of inflammatory cytokines and usually downregulated in neutrophilic asthma. METHODS: The effects of TIO and dexamethasone (DEX) on HDAC2 activity, nuclear factor kappa B (NF-κB), and C-X-C motif chemokine ligand 1 (CXCL1) were evaluated in neutrophilic asthma mouse model (C57BL, 6-week-old). An in-vitro study was conducted using primary human bronchial/tracheal epithelial (HBE) cells from asthma patients. Western blot analyses were performed for phospho-phospholipase Cγ-1 (PLCγ-1) and inositol trisphosphate (IP3) receptors (IP3R) with treating lipopolysaccharide (LPS) and TIO. RESULTS: Ovalbumin was used to induce eosinophilic inflammation in this study. After neutrophilic asthma was induced by LPS (O+L group), HDAC2 activity was diminished with increased NF-κB activity and CXCL1 compared to the control group. TIO significantly improved NF-κB activity, CXCL1, and HDAC2 activity compared with the O+L group in in-vivo study (P < 0.05, each). Western blot analyses showed that LPS treated HBE cells from asthma patients increased PLCγ-1 and diminished IP3 receptor levels. After TIO treatment, recovery of IP3R and improved PLCγ-1 levels were observed. CONCLUSION: These results support the hypothesis that TIO modulates inflammation by recovering HDAC2 activity from the acetylcholine-stimulated inflammation cascade in neutrophilic asthma. The detailed inflammation cascade of recovering HDAC2 activity by TIO might be associated with PLCγ-1-IP3-IP3R mediated intracellular calcium ion pathway.

Effect of daily dosing with tiotropium against methacholine induced bronchoconstriction in asthmatics.

INTRODUCTION: Loss of bronchoprotection against direct and indirect acting stimuli following regular use of inhaled beta2-agonists occurs with both short and long-acting formulations. Comparatively little is known about the development of tolerance following regular use of inhaled muscarinic receptor antagonists. Two investigations with the short-acting muscarinic receptor antagonist ipratropium bromide have reported no tolerance after regular use against inhaled methacholine. To our knowledge, there are no data regarding loss of bronchoprotection following regular use of long-acting muscarinic receptor antagonist. We therefore looked at the effect of daily dosing with tiotropium on methacholine induced bronchoconstriction in a population of mild asthmatics. METHODS: We performed a randomized, double-blind, placebo-controlled cross-over study comparing tiotropium Respimat® 5 µg to placebo in adult asthmatics. Each treatment arm began with baseline methacholine challenge followed immediately by treatment administration. One hour later a post treatment methacholine challenge was performed. Participants dosed daily (two puffs) at home for the next six days and returned to the lab on Day 8 for a final dose of treatment 1 h prior to methacholine challenge. RESULTS: The average doubling dose increase in methacholine PD20 following a single dose of tiotropium was 3.9 doubling doses whereas that following placebo was 0.93 (p = 0.003). After regular use, methacholine PD20 was further increased to 6.4 doubling doses following tiotropium whereas that following placebo decreased by 0.57 doubling doses (p < 0.001). CONCLUSION: LAMA are indicated for use as add-on monotherapy or in triple therapy combination for poorly controlled asthma. It may be reassuring to know therefore, that regular use does not result in loss of bronchoprotection like that which occurs with beta2-agonist bronchodilators.

Crystal structures of the M1 and M4 muscarinic acetylcholine receptors.

Muscarinic M1-M5 acetylcholine receptors are G-protein-coupled receptors that regulate many vital functions of the central and peripheral nervous systems. In particular, the M1 and M4 receptor subtypes have emerged as attractive drug targets for treatments of neurological disorders, such as Alzheimer's disease and schizophrenia, but the high conservation of the acetylcholine-binding pocket has spurred current research into targeting allosteric sites on these receptors. Here we report the crystal structures of the M1 and M4 muscarinic receptors bound to the inverse agonist, tiotropium. Comparison of these structures with each other, as well as with the previously reported M2 and M3 receptor structures, reveals differences in the orthosteric and allosteric binding sites that contribute to a role in drug selectivity at this important receptor family. We also report identification of a cluster of residues that form a network linking the orthosteric and allosteric sites of the M4 receptor, which provides new insight into how allosteric modulation may be transmitted between the two spatially distinct domains.

Emerging muscarinic receptor antagonists for the treatment of asthma.

INTRODUCTION: The increased acetylcholine signaling in asthma pathophysiology offers the rationale for the use of LAMAs in the treatment of asthmatic patients. Tiotropium is still the only LAMA approved for use in asthma but there is a real interest in developing novel LAMAs for the treatment of asthma, or at least to extend this indication to other LAMAs already on the market. AREAS COVERED: We examined and discussed trials and research that have studied or are evaluating the role of LAMAs already on the market in asthma and possible novel muscarinic acetylcholine receptor antagonists. EXPERT OPINION: Glycopyrronium and umeclidinium will soon be included in the GINA strategy with the same current indications of tiotropium. It is likely that the choice of the LAMA will be influenced not so much by its pharmacological profile as by the type of triple therapy chosen. It is extremely difficult to identify a new LAMA that is more effective than tiotropium, but is it plausible that new technologies that will allow delivering the drug in a more targeted way and with a lower risk of adverse effects may represent the real progress in the use of LAMAs in asthma in the coming years.

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.

Effects of tiotropium bromide on airway hyperresponsiveness and inflammation in mice exposed to organic dust.

INTRODUCTION: Acute exposure to organic dust (OD) in pig barns induces intense airway inflammation with neutrophilia and hyperresponsiveness. This reaction is likely associated with increased cholinergic activity. Therefore, the involvement of cholinergic mechanisms in the reaction to acute exposure of OD was investigated in mice using the long-acting muscarinic antagonist tiotropium. METHODS: BALB/c mice received tiotropium (2-200 ng) intranasally on day 1 of the study. On days 2-4, mice received vehicle or OD (25 µg) intranasally. Airway hyperresponsiveness to methacholine was assessed 24 h following the last OD exposure. Bronchoalveolar lavage (BAL) fluid, lung tissue and blood were collected for analyses. RESULTS: Organic dust elevated airway responsiveness to methacholine compared with controls (PBS) assessed as Newtonian resistance (1.5 ±â€¯0.1 vs 0.9 ±â€¯0.1 cm H2O x s/mL), tissue damping (12.4 ±â€¯1.4 vs 8.9 ±â€¯0.9 cm H2O∙s/mL) and tissue elastance (41.1 ±â€¯5.3 vs 27.2 ±â€¯2.5 cm H2O∙s/mL). Tiotropium (200 ng) decreased the Newtonian resistance and tissue damping after exposure to PBS or OD. Organic dust exposure increased inflammatory cells in BAL fluid by almost 400%, mainly due to neutrophil influx, which was unaffected by tiotropium. Organic dust increased levels of mainly Th1 mediators. Tiotropium treatment attenuated OD-induced release of IL-2, IL-4 and IL-6. CONCLUSIONS: Tiotropium decreased the OD-induced increase of specific cytokines without influencing the OD-induced increase of airway responsiveness and neutrophil infiltration into the lungs. We conclude that the cholinergic pathway contributes to the pro-inflammatory effects caused by inhalation of OD from pig barns.

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.

Combining long-acting bronchodilators with different mechanisms of action: A pharmacological approach to optimize bronchodilation of equine airways.

The ultra long-acting ß2 -adrenoceptor agonist olodaterol plus the ultra long-acting muscarinic antagonist tiotropium bromide are known to relax equine airways. In human bronchi combining these drugs elicits a positive interaction, thus we aimed to characterize this information further in equine isolated airways stimulated by electrical field stimulation (EFS) and using the Concentration-Reduction Index (CRI) and Combination Index (CI) equations. The drugs were administered alone and together by reproducing ex vivo the concentration-ratio delivered by the currently available fixed-dose combination (1:1). The single agents elicited a significant (p < .05) concentration-dependent reduction in the EFS-induced contractility, that was synergistically improved (CI 0.18) when administered in combination (0.9 logarithms more potent, 24% more effective than the monocomponents). The drugs mixture allowed a reduction in the concentration of olodaterol from ≃1 to ≃2.3 logarithms. A favorable CRI was detected also for tiotropium bromide, whose concentration can be reduced ≃1 logarithm at medium effect levels, remaining positive up to submaximal relaxant effect in the presence of olodaterol. The combination of tiotropium bromide/olodaterol allows the reduction in the concentration of the monocomponents to achieve airway smooth muscle relaxation, thus potentially decreases the risk of adverse events when these drugs are used to treat severe asthmatic horses.

The PDE4 inhibitor CHF-6001 and LAMAs inhibit bronchoconstriction-induced remodeling in lung slices.

Combination therapy of PDE4 inhibitors and anticholinergics induces bronchoprotection in COPD. Mechanical forces that arise during bronchoconstriction may contribute to airway remodeling. Therefore, we investigated the impact of PDE4 inhibitors and anticholinergics on bronchoconstriction-induced remodeling. Because of the different mechanism of action of PDE4 inhibitors and anticholinergics, we hypothesized functional interactions of these two drug classes. Guinea pig precision-cut lung slices were preincubated with the PDE4 inhibitors CHF-6001 or roflumilast and/or the anticholinergics tiotropium or glycopyorrolate, followed by stimulation with methacholine (10 µM) or TGF-ß1 (2 ng/ml) for 48 h. The inhibitory effects on airway smooth muscle remodeling, airway contraction, and TGF-ß release were investigated. Methacholine-induced protein expression of smooth muscle-myosin was fully inhibited by CHF-6001 (0.3-100 nM), whereas roflumilast (1 µM) had smaller effects. Tiotropium and glycopyrrolate fully inhibited methacholine-induced airway remodeling (0.1-30 nM). The combination of CHF-6001 and tiotropium or glycopyrrolate, in concentrations partially effective by themselves, fully inhibited methacholine-induced remodeling in combination. CHF-6001 did not affect airway closure and had limited effects on TGF-ß1-induced remodeling, but rather, it inhibited methacholine-induced TGF-ß release. The PDE4 inhibitor CHF-6001, and to a lesser extent roflumilast, and the LAMAs tiotropium and glycopyrrolate inhibit bronchoconstriction-induced remodeling. The combination of CHF-6001 and anticholinergics was more effective than the individual compounds. This cooperativity might be explained by the distinct mechanisms of action inhibiting TGF-ß release and bronchoconstriction.

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.

Pharmacological characterization of the interaction between tiotropium and olodaterol administered at 5:5 concentration-ratio in equine bronchi.

Equine airways represent a suitable ex vivo model to study the functional impact of pharmacological treatments on human chronic obstructive pulmonary disorders, such as asthma and chronic obstructive pulmonary disease (COPD). We aimed to characterize the pharmacological interaction between the long-acting muscarinic antagonist (LAMA) tiotropium and the long-acting ß2-agonist (LABA) olodaterol in equine airways. The effect of tiotropium and olodaterol, administered alone and in combination at the ratio of concentrations reproducing ex vivo the concentration-ratio delivered by the currently available fixed-dose combination (FDC) (5:5), was investigated on the cholinergic contractile tone induced by the parasympathetic activation of equine isolated airways. The drug interaction was analysed by using the Bliss Independence and Unified Theory models. Both tiotropium and olodaterol induced a sub-maximal concentration-dependent inhibition of bronchial contractility (Emax: tiotropium 83.6 ± 14.8%, olodaterol 76.9 ± 17.9%; pEC50: tiotropium 8.2 ± 0.5; olodaterol 8.3 ± 0.6). When administered at 5:5 concentration-ratio, tiotropium plus olodaterol completely inhibited the bronchial contractility (Emax 102.7 ± 8.4%; pEC50 9.0 ± 0.7). Strong synergistic interaction was detected for tiotropium/olodaterol combination (combination index 0.011). When administered at low concentrations, the drug mixture elicited up to 94.6 ± 9.5% effect that was 36.0 ± 8.1% greater than the expected additive effect. The results of this study demonstrate that the co-administration of tiotropium plus olodaterol at 5:5 concentration-ratio leads to synergistic inhibition of equine bronchial contractility when compared with either drug administered alone. These findings suggest that the currently available LABA/LABA FDC may be effective in delivering tiotropium/olodaterol combination at equipotency concentrations of each monocomponent into the lung and, thus, inducing synergistic effect in the airways.

Tiotropium Attenuates Virus-Induced Pulmonary Inflammation in Cigarette Smoke-Exposed Mice.

Viral infections trigger exacerbations in chronic obstructive pulmonary disease (COPD), and tiotropium, a M3 receptor antagonist, reduces exacerbations in patients by unknown mechanisms. In this report, we investigated whether tiotropium has anti-inflammatory effects in mice exposed to cigarette smoke (CS) and infected with influenza virus A/PR/8/34 (H1N1) or respiratory syncytial virus (RSV) and compared these effects with those of steroid fluticasone and PDE4-inhibitor roflumilast. Mice were exposed to CS; infected with H1N1 or RSV; and treated with tiotropium, fluticasone, or roflumilast. The amount of cells and cytokine levels in the airways, lung function, and viral load was determined. NCI-H292 cells were infected with H1N1 or RSV and treated with the drugs. In CS/H1N1-exposed mice, tiotropium reduced neutrophil and macrophage numbers and levels of interleukin-6 (IL-6) and interferon-γ (IFN-γ) in the airways and improved lung function. In contrast, fluticasone increased the loss of body weight; failed to reduce neutrophil or macrophage numbers; increased IL-6, KC, and tumor necrosis factor-α (TNF-α) in the lungs; and worsened lung function. Treatment with roflumilast reduced macrophage numbers, IL-6, and KC in the lungs but had no effect on neutrophil numbers or lung function. In CS/RSV-exposed mice, treatment with tiotropium, but not fluticasone or roflumilast, reduced neutrophil numbers and IL-6 and TNF-α levels in the lungs. Viral load of H1N1 and RSV was significantly elevated in CS/virus-exposed mice and NCI-H292 cells after fluticasone treatment, whereas tiotropium and roflumilast had no effect. In conclusion, tiotropium has anti-inflammatory effects on CS/virus-induced inflammation in mice that are superior to the effects of roflumilast and fluticasone. This finding might help to explain the observed reduction of exacerbation rates in COPD patients.

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.

Effect of tiotropium on mucus hypersecretion and airway clearance in patients with COPD.

BACKGROUND: Increased sputum production is an important feature of COPD, in which a large amount of secretions stagnated in the respiratory lumen may aggravate airflow limitation, impair airway mucociliary transport, and cause recurrent respiratory infection and, hence, acute exacerbations of the diseases. There is evidence that airway mucus hypersecretion is associated with the severity and prognosis of COPD, but the symptoms are generally difficult to treat. METHODS: In an open, non-controlled study, we examined the effect of the anticholinergic agent tiotropium on airway mucus hypersecretion in 22 COPD patients. After a 4-week run-in period, the patients received 18 µg of tiotropium once daily delivered through the handihaler for 8 weeks, while symptoms and their impact associated with sputum were scored according to cough and sputum assessment questionnaire (CASA-Q). At week 0 and week 8, spirometry was performed before and 30 min after the administration of albuterol. To test the effect of tiotropium on airway mucociliary transport, nasal clearance time was measured. To evaluate airway mucus production, solid composition of the sputum (dry/wet weight ratio) was measured. RESULTS: Treatment with tiotropium increased both prebronchodilator FEV1 and postbronchodilator FEV1. Tiotropium decreased cough symptom scores and provided with favorable influences on sputum-related symptoms, and none of the patients complained of worsening of the symptoms judging from the CASA-Q score. Both solid composition of the sputum and mucin contents decreased and nasal clearance time was shortened from 29.4 ± 5.1 to 20.6 ± 4.1min (p < 0.05) during the 8-week treatment. CONCLUSIONS: Tiotropium decreases symptoms associated with sputum in COPD patients, an effect that may be related to the inhibition of airway mucus hypersecretion and improvement of airway mucociliary clearance.

Characterization of V0162, a new long-acting antagonist at human M3 muscarinic acetylcholine receptors.

The anticholinergic properties of the mequitazine enantiomer V0162 make it a drug candidate for the treatment of chronic obstructive airway diseases. Here, we compared V0162's in vitro pharmacological activity at recombinant human M3 muscarinic acetylcholine receptors (hM3Rs) with that of other anticholinergics, using (i) a radioligand binding assay, (ii) a functional reporter gene assay and (iii) a bronchoconstriction inhibition assay on human bronchial preparations. V0162 had high affinity for hM3Rs, with a pKi varying from 9.01 after a 2 h incubation to 9.21 after 23 h. The other mequitazine enantiomer (V0114) was less potent. V0162 displayed rapid off-kinetics and a biphasic time course of binding. V0162 was found to be an antagonist behaving as an inverse agonist for hM3R-mediated reporter gene activation, with much the same efficacy as atropine, ipratropium and tiotropium. However, in contrast to ipratropium and atropine, V0162's inhibitory potency was only slightly affected by compound washout. V0162 antagonized acetylcholine-mediated contractions in a human bronchial preparation; the pA2 values increased with the incubation time (up to 2 h). Moreover, there was a progressive increase in V0162's ability to inhibit electrically-induced contractions, which persisted after compound washout. In conclusion, V0162 is the most active mequitazine enantiomer at hM3Rs and shows a complex pattern of binding to the membrane compartment. These particular features may be of therapeutic value when persistent antagonism at hM3Rs is required.

Molecular hybridization yields triazole bronchodilators for the treatment of COPD.

A 1,2,4-triazole motif was employed as a bioisostere for the ester commonly used in muscarinic antagonists, and subsequent integrative conjugation to a ß2 agonist quinolinone furnished a new class of bifunctional MABAs for the treatment of COPD. Medicinal chemistry optimization using the principles of 'inhalation by design' furnished a clinical candidate with desirable pharmacological, pharmacokinetic and biopharmaceutical properties.

Cardiac safety of tiotropium in patients with COPD: a combined analysis of Holter-ECG data from four randomised clinical trials.

BACKGROUND: Tiotropium is generally well tolerated; however, there has been debate whether antimuscarinics, particularly tiotropium administered via Respimat(®) Soft Mist(™) Inhaler, may induce cardiac arrhythmias in a vulnerable subpopulation with cardiovascular comorbidity. The aim of this study was to provide evidence of the cardiac safety of tiotropium maintenance therapy. METHODS: Combined analysis of Holter electrocardiogram (ECG) data from clinical trials of tiotropium in chronic obstructive pulmonary disease (COPD). Trials in the Boehringer Ingelheim clinical trials database conducted between 2003 and 2012, involving tiotropium HandiHaler(®) 18 µg and/or tiotropium Respimat(®) (1.25-, 2.5-, 5.0- and 10-µg doses) were reviewed. All trials involving Holter-ECG monitoring during this period were included in the analysis. Men and women aged ≥ 40 years with a smoking history of ≥ 10 pack-years, and a clinical diagnosis of COPD were included. Holter ECGs were evaluated for heart rate (HR), supraventricular premature beats (SVPBs), ventricular premature beats (VPBs) and pauses. Quantitative and categorical end-points were derived for each of the Holter monitoring days. RESULTS: Four trials (n = 727) were included in the analysis. Respimat(®) (1.25-10 µg) or HandiHaler(®) (18 µg) was not associated with changes in HR, SVPBs, VPBs and pauses compared with placebo or the pretreatment baseline period. In terms of cardiac arrhythmia end-points, there was no evidence for an exposure-effect relationship. CONCLUSIONS: In this analysis, tiotropium maintenance therapy administered using Respimat(®) (1.25-10 µg) or HandiHaler(®) (18 µg) once daily for periods of up to 48 weeks was well tolerated with no increased risk of cardiac arrhythmia in patients with COPD.

Investigating the Unbinding of Muscarinic Antagonists from the Muscarinic 3 Receptor.

Patient symptom relief is often heavily influenced by the residence time of the inhibitor-target complex. For the human muscarinic receptor 3 (hMR3), tiotropium is a long-acting bronchodilator used in conditions such as asthma or chronic obstructive pulmonary disease (COPD). The mechanistic insights into this inhibitor remain unclear; specifically, the elucidation of the main factors determining the unbinding rates could help develop the next generation of antimuscarinic agents. Using our novel unbinding algorithm, we were able to investigate ligand dissociation from hMR3. The unbinding paths of tiotropium and two of its analogues, N-methylscopolamin and homatropine methylbromide, show a consistent qualitative mechanism and allow us to identify the structural bottleneck of the process. Furthermore, our machine learning-based analysis identified key roles of the ECL2/TM5 junction involved in the transition state. Additionally, our results point to relevant changes at the intracellular end of the TM6 helix leading to the ICL3 kinase domain, highlighting the closest residue L482. This residue is located right between two main protein binding sites involved in signal transduction for hMR3's activation and regulation. We also highlight key pharmacophores of tiotropium that play determining roles in the unbinding kinetics and could aid toward drug design and lead optimization.

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.