Introduction

Muscarinic antagonists, such as aclidinium, tiotropium and ipratropium, are known to exert their bronchodilator effects by blocking the actions of acetylcholine at M3 receptors on airway smooth muscle [1, 2]. However, interactions with muscarinic receptors outside of the respiratory tract confer a potential for unwanted systemic side effects. For example, blockade of the cardiac M2 receptors, the principal muscarinic receptor subtype expressed by cardiac myocytes [3–6], induces tachycardia, considered the most severe side effect associated with muscarinic antagonist use [7].

Here, we report the functional potency and duration of action of aclidinium bromide at M2 and M3 muscarinic receptors in isolated cardiac and airway tissues of human origin compared with tiotropium and ipratropium.

Methods

Human lung tissue was harvested from patients undergoing surgery for lung carcinoma and left atrial tissue from patients undergoing cardiac bypass surgery. None of the patients had a history of asthma. The protocol for the use of lung or cardiac tissues was approved by the Ethics Committee of University Clinic Hospital (Valencia, Spain), and informed consent was obtained from all patients.

Fresh bronchial strips were mounted in a superfusion chamber containing oxygenated Kreb’s solution (KHS) at 37 °C [8, 9]. Spontaneous tone was inhibited by zileuton 10 μM and fexofenadine 10 μM [10]. Electrical stimulation at 8 Hz, over 0.5 ms and 40–50 V was delivered as 10 s trains of square-wave pulses every 2 min. Once responses to electrical stimulation had stabilized, increasing and cumulative concentrations of aclidinium, ipratropium or tiotropium (0.03–10 nM) were added to the stimulated bronchial strips to measure potency at the M3 receptors.

To assess onset and offset of action, aclidinium, ipratropium or tiotropium (all 10 nM) were added to the bronchial strips to inhibit approximately 75% of the stable baseline contractions induced by electrical stimulation. After 30 min, the preparation was washed free of antagonist and recovery of tone was recorded for 14–15 h.

Macroscopically normal human left atrial tissue (3–4 mm long) was mounted in a superfusion chamber containing oxygenated KHS at 37 °C (pH 7.4). Each preparation (initial load, 2.0 g) was connected to a force displacement transducer, and changes in tension were recorded. Atrial contraction was induced by electrical stimulation at 1 Hz, over 5 ms and 2–5 V (20% higher than the threshold for contraction). Once responses to electrical stimulation had stabilized (60 min), carbachol 10 µM was added to the stimulated atria to inhibit electrically induced contractions.

To measure the duration of action at the M2 receptors, aclidinium, ipratropium or tiotropium were added to the carbachol (10 µM)-treated atria at a concentration that inhibited 70% of the maximum carbachol-induced relaxation (70, 80 and 50 nM, respectively). Once inhibition of tone was stable, the antagonists were washed out and the atria incubated with carbachol 10 µM for 240 min.

The rates of onset (t 1/2) and duration of action (offset) of the antagonists were defined as the time taken from antagonist addition to 50% inhibition of tone, and from antagonist washout to recover 50% (t 1/2) [8] or maximal recovery (t max) of the maximum carbachol-induced relaxation, respectively. The concentration of antagonist required for 50% inhibition of the electrically stimulated contraction (IC50) and the offset (t 1/2) was calculated using nonlinear regression analysis.

Statistical significance, set at the 0.05 level, was determined by parametric analysis of variance, using two-sided statistical tests, followed by Bonferroni’s multiple comparison test. All data analyses were performed using GraphPad Prism (San Diego, CA, USA).

Results

Aclidinium, ipratropium and tiotropium produced concentration-dependent relaxation in isolated human bronchial rings. All three muscarinic antagonists inhibited the contractile response induced by electrical stimulation with similar potency, as expressed by IC50 values (Table 1).

Table 1 Duration of action of muscarinic antagonists in isolated human bronchial rings and left atrial strips plus potency in human bronchial rings
Full size table

The duration of action (offset time; at a concentration that inhibited 75% of the electrically stimulated contraction) of aclidinium at the M3 receptor was significantly longer than that of ipratropium (Table 1; P < 0.05), whereas no recovery of tone was observed after washout of tiotropium for the duration of the study. The onset of action of aclidinium was similar to that of ipratropium and significantly faster than tiotropium (Table 1; P < 0.05).

The duration of action of aclidinium at the M2 receptor was significantly longer than that of ipratropium and shorter than that of tiotropium (Table 1; both P < 0.05); aclidinium inhibited the bradycardic effect of carbachol with a longer duration of action compared with ipratropium, but shorter than tiotropium (Table 1). Aclidinium had a shorter duration of action at the M2 receptor than at the M3 receptor (Table 1).

Discussion

Our results demonstrate that in human bronchial tissue, aclidinium had a similar potency at M3 receptors to that of tiotropium and ipratropium, although its onset of action was significantly faster than tiotropium and its duration of action was significantly longer than ipratropium. These results are consistent with previous preclinical studies in isolated guinea pig trachea [11, 12]. The faster onset of action of aclidinium compared with tiotropium and the long duration of action of both are consistent with the clinical profile of these compounds [13].

In human left atrial tissue, aclidinium had a shorter duration of action at M2 receptors than tiotropium, but longer than ipratropium. These results are also consistent with those previously described in isolated guinea pig left atria and in membranes expressing human M2 receptors [11, 12]. All three antagonists have a faster offset from human left atrial tissue compared with human bronchial tissue, indicating that in human tissue isolates, each compound is kinetically selective. Similar findings have been demonstrated in guinea pig tissue [12], indicating functional selectivity is maintained across species.

Conclusion

These results, together with the high plasma hydrolysis rate of aclidinium in comparison with tiotropium [14], may translate into a reduced propensity for systemic effects, particularly unwanted cardiovascular adverse events in the clinical setting, and there is increasing evidence to support this hypothesis [15–17].