Combination therapy of tiotropium and ciclesonide attenuates airway inflammation and remodeling in a guinea pig model of chronic asthma

Abstract

Background: The long-acting anticholinergic tiotropium has recently been registered for the treatment of asthma, and its use is associated with a reduction in exacerbation frequency. Anti-inflammatory and anti-remodeling effects of tiotropium have been demonstrated in in vitro and in vivo models. Because tiotropium treatment is used in combination with inhaled corticosteroids, potential additive effects between the two would be clinically relevant. Therefore, the aim of this study was to investigate additive effects between tiotropium and ciclesonide on airway inflammation and remodeling in guinea pig models of asthma.

Methods: Guinea pigs (n = 3-8/group) were sensitized and challenged with ovalbumin in an acute (single challenge) and a chronic model (12 weekly challenges) of allergic asthma. Animals were treated with vehicle, nebulized tiotropium (0.01-0.3 mM) and/or intranasally instilled ciclesonide (0.001-1 mg/kg) before each challenge. Bronchoalveolar lavage fluid and lungs were collected for analysis of airway inflammation and remodeling.

Results: Tiotropium and ciclesonide treatment, alone or in combination, did not inhibit airway inflammation in the acute asthma model. In a dose-finding study, low doses of tiotropium and ciclesonide inhibited airway eosinophilia and airway smooth muscle thickening in the chronic asthma model. Threshold doses of 0.01 mM tiotropium (nebulizer concentration) and 0.01 mg/kg ciclesonide were selected to investigate potential additive effects between both drugs. At these doses, tiotropium and ciclesonide did not inhibit airway eosinophilia or airway smooth muscle thickening when administered alone, but significantly inhibited these allergen-induced responses when administered in combination.

Conclusions: Combined treatment with low doses of tiotropium and ciclesonide inhibits airway inflammation and remodeling in a guinea pig model of chronic asthma, suggesting that combined treatment with anticholinergics and corticosteroids may have anti-inflammatory and anti-remodeling activity in allergic airway diseases. Since tiotropium is registered as a therapy for asthma added on to corticosteroid treatment, these beneficial effects of the combination therapy may be clinically relevant.

Figures

Fig. 1

Experimental procedure. a acute protocol, b chronic protocol. Male Dunkin Hartley guinea pigs (n = 3–8 animals per group, see Additional file 1: Table S1-S3 for overview of groups) were sensitized to ovalbumin (OVA) by intraperitoneal injection of 0.5 ml allergen solution containing 100 μg/ml ovalbumin and 100 mg/ml Al(OH)3, and intracutaneous injection of 0.5 ml allergen solution. Subsequently, guinea pigs were challenged with OVA (0.05–0.1 %) via inhalation of aerosolized solution. Tiotropium (tio; 0.01–0.3 mM; 3 min inhalation time) was administered via aerosol inhalation and ciclesonide (cicl; 0.001–1 mg/kg) via intranasal instillation, 24 h and/or 1 h before every challenge. In the acute protocol, a bronchoalveolar lavage was performed 25 h after the ovalbumin challenge. In the chronic protocol, lungs were harvested for tissue sections 24 h after the last of 12 weekly OVA challenges

Fig. 2

Inflammatory cell numbers in bronchoalveolar lavage fluid in response to a single ovalbumin (OVA) challenge and treatment with saline (sal; control), tiotropium (tio; 0.1 mM; nebulizer concentration), and/or ciclesonide (cicl; 1 mg/kg). Guinea pigs were treated as described in Fig. 1a. A bronchoalveolar lavage was performed 25 h after OVA challenge and inflammatory cells were determined. a total cells, b eosinophils, c macrophages, d lymphocytes, e neutrophils. ** p < 0.01, *** p < 0.001. Data represent mean ± s.e.m. of 5–8 animals per group

Fig. 3

Airway eosinophilia in response to chronic ovalbumin (OVA) challenge and treatment with saline (sal; control), tiotropium (tio; 0.01 and 0.03 mM; nebulizer concencenrations) or ciclesonide (cicl; 0.01 and 0.1 mg/kg). Guinea pigs were treated as described in Fig. 1b. Lungs were collected 24 h after the last OVA challenge and eosinophil numbers were determined by H&E staining in the submucosa (a,c) and adventitia (b,d) of the non-cartilaginous (a,b) and cartilaginous airways (c,d). * p < 0.05, ** p < 0.01, *** p < 0.001. Data represent mean ± s.e.m. of 4 animals per group

Fig. 4

Airway smooth muscle mass thickening in response to chronic ovalbumin (OVA) challenge and treatment with saline (sal, control), tiotropium (tio; 0.01 and 0.03 mM; nebulizer concentration) or ciclesonide (cicl; 0.01 and 0.1 mg/kg). Guinea pigs were treated as described in Fig. 1b. Lungs were collected 24 h after the last OVA challenge and airway smooth muscle mass was determined by α-sm-myosin antibody staining of non-cartilaginous (a) and cartilaginous airways (b). * p < 0.05. Data represent mean ± s.e.m. of 4 animals per group

Fig. 5

Airway eosinophilia in response to chronic ovalbumin (OVA) challenge and treatment with saline (sal; control), tiotropium (tio; 0.01 mM; nebulizer concentration) and/or ciclesonide (cicl; 0.01 mg/kg). Guinea pigs were treated as described in Fig. 1b. Lungs were collected 24 h after the last OVA challenge and eosinophil numbers were determined by H&E staining in the submucosa (a, i) and adventitia (b,j) of non-cartilaginous (a,b) and cartilaginous (i,j) airways. Representative images are shown in panels c-h for non-cartilaginous airways (magnification 200x) and in panels K-P for cartilaginous airways (maginification 100x). * p < 0.05, ** p < 0.01. Data represent mean ± s.e.m. of 8 animals per group

Fig. 6

Airway smooth muscle mass thickening in response to chronic ovalbumin (OVA) challenge and treatment with saline (sal; control), tiotropium (tio; 0.01 mM; nebulizer concentration) and/or ciclesonide (cicl; 0.01 mg/kg). Guinea pigs were treated as described in Fig. 1b. Lungs were collected 24 h after the last OVA challenge and airway smooth muscle mass was determined by α-sm-myosin antibody staining. Quantification is shown in figure a for non-cartilaginous airways and representative images are shown in panels b-g (magnification 200x). * p < 0.05. Data represent mean ± s.e.m. of 8 animals per group