Tiotropium bromide exerts anti-inflammatory activity in a cigarette smoke mouse model of COPD

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Abstract

Tiotropium bromide is a long acting muscarinic antagonist (LAMA), marketed under the brand name Spiriva®, for the treatment of chronic obstructive pulmonary disease (COPD). Besides its proven direct bronchodilatory activity, recent clinical studies demonstrated that tiotropium is able to reduce the exacerbation rate and impact the clinical course of COPD. One significant pathological feature believed to be causative for the progressive nature of COPD is chronic pulmonary inflammation. The aim of the present study was to investigate the anti-inflammatory activity of tiotropium on cigarette smoke-induced pulmonary inflammation in mice. C57Bl/6 mice were exposed to cigarette smoke (CS) for four days with increasing exposure time for up to 6 h per day to elicit pulmonary inflammation and mediator release. One hour before smoke exposure, animals were treated with tiotropium by inhalation (0.01–0.3 mg/mL) for 5 min; 18 h after the last CS exposure a bronchoalveolar lavage was performed. Tiotropium concentration-dependently inhibited pulmonary neutrophilic inflammation with an IC50 of 0.058 mg/mL and a maximum inhibition of 60% at 0.3 mg/mL. Furthermore, the CS-induced pulmonary release of leukotriene B4, interleukin-6, keratinocyte-derived chemokine, monocyte chemotactic protein-1, macrophage inflammatory protein-1alpha and −2, and tumor necrosis factor alpha was dose-dependently reduced. The bronchodilatory activity of tiotropium against acetycholine-induced bronchoconstriction was found to be in the same dose range as the anti-inflammatory activity with an IC50 of 0.045 mg/mL and a maximum bronchodilation of 90% at 0.3 mg/mL. Our data suggest that the beneficial effects of tiotropium on the course of COPD shown in patients may be associated with an anti-inflammatory activity.

Introduction

Chronic obstructive pulmonary disease (COPD) is currently ranked as the fifth leading course of death in the world with tobacco smoke inhalation representing the most common cause of COPD and is predicted to be the 3rd leading cause of death in 2020 (WHO). The disease is characterised by pulmonary inflammation and airflow obstruction that progressively deteriorates over time and that has limited reversibility. However, increased cholinergic tone is the major reversible component of the disease [1], [2].

According to the present guidelines, bronchodilators represent the first line therapy in COPD (www.GOLDCOPD.org). Tiotropium bromide, the active ingredient of Spiriva®, is the only long acting muscarinic antagonist (LAMA) available for the treatment of patients with COPD. Tiotropium blocks the binding of acetylcholine to muscarinic receptors with a subnanomolar affinity. The receptor binding half-life is prolonged at the muscarinic M3 receptor subtype, the particular receptor inducing bronchoconstriction [3].

The efficacy as well as the long duration of action of tiotropium bromide has been confirmed in numerous clinical studies showing beneficial effects on lung function, COPD exacerbations and quality of life of COPD patients [4], [5].

A recent large 4-year trial in 5993 COPD patients (UPLIFT® trial) confirmed the favourable safety profile of tiotropium bromide and showed sustained improvements in lung function, quality of life and COPD exacerbations [6]. Viral and/or bacterial infections, with subsequent pulmonary inflammation, are believed to be the main cause of exacerbations. Although the decline in lung function across all COPD groups over 4 years was not influenced, subgroup analysis revealed a significant reduction in FEV1 decline in COPD/GOLD stage I/II patients [7]. Together with the reduction of exacerbations this analysis supports the concept that tiotropium might exert anti-inflammatory or anti-remodeling effects. A detailed review describing alternative mechanisms for tiotropium besides its well known bronchodilatory activity was recently published [8].

Acetylcholine, its synthesizing enzyme choline acetyl transferase (ChAT) and muscarinic receptors have been shown to be expressed not only in the parasympathetic nervous system but in nearly any cell types present in the airways [9], [10]. Acetylcholine was shown to be released from bronchial epithelium as well as from inflammatory cells [9] and mediates the release of the neutrophil chemotactic factor interleukin-8 (IL-8) by human bronchial epithelial cells [11]. A recent publication showed that muscarinic M3 receptor activation increases cigarette smoke (CS)-induced Interleukin-8 (IL-8) secretion by human airway smooth muscle cells [12]. Tiotropium has been tested for anti-inflammatory properties in various preclinical settings. The drug showed anti-inflammatory effects both in in-vitro experiments as well as in animal studies. Tiotropium suppressed acetycholine-induced release of the neutrophil chemotactic factor leukotriene B4 (LTB4) [13] and attenuated lipopolysaccharide (LPS)-induced IL-8 release from human airway epithelial cells. Tiotropium was shown to inhibit allergen-induced airway remodelling in a guinea pig model of ongoing asthma [14]. McQueen et al. [15] explored the role of muscarinic receptors in a diesel particle-induced rat model of pulmonary neutrophilic inflammation. In this study bilateral vagotomy as well as atropine pre-treatment reduced neutrophilic lung inflammation. Ipratropium bromide was shown to reduce cadmium-induced pulmonary inflammation in rats [16]. However, the role of muscarinic receptors in CS-induced pulmonary inflammation is presently not known. The aim of this study was to investigate the effect of muscarinic receptor inhibition by tiotropium in a CS-induced mouse model of pulmonary neutrophilic inflammation.

Section snippets

Animals

Female C57Bl/6 mice (18–23 g) were obtained from Charles River (Kisslegg, Germany). Animals were housed in isolated ventilated cages under a 12-h light-dark cycle and received food and water ad libitum. All animal experimentation was conducted in accordance with German national guidelines and legal regulations.

Inhalative administration of tiotropium

Aqueous solutions of tiotropium bromide (Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany) at concentrations of 0.01, 0.03, 0.1, and 0.3 mg/mL were aerosolized with a jet

Inhibition of cigarette smoke-induced pulmonary inflammation by tiotropium

To explore the possible anti-inflammatory effects of tiotropium a short-term mouse model of CS-induced pulmonary inflammation was utilized. Three independent experiments were performed with n = 5–10 animals per group, each (Table 1). Differential cell counts were determined in the bronchoalveolar lavage fluid (BALF). After 4 days of CS exposure pulmonary inflammation was characterized by a prominent statistically significant (p < 0.001) neutrophilic cell accumulation in all experiments. After CS

Discussion

In this study we show for the first time that tiotropium exerts prominent anti-inflammatory activity in an in vivo model of COPD. Tiotropium dose-dependently inhibited severe CS-induced pulmonary neutrophilia and increased levels of inflammatory mediators. The extent of the anti-inflammatory effect of tiotropium seems to be dependent on the stimulus used to exert the pulmonary inflammation. In our less severe LPS-induced pulmonary inflammation experiments tiotropium pre-treatment caused only a

Acknowledgements

The excellent technical assistance of Sylvia A. Blum, Martina Hagel, Martina Keck, and Eva Zeller is gratefully acknowledged.

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