Efficacy of EA575 as an Antitussive and Mucoactive Agent in Preclinical In Vivo Models

Abstract

Background: The efficacy of EA575 in the treatment of respiratory diseases is described in various clinical studies, improving patients' disease-related symptoms. However, mechanistic in vivo data proving its beneficial effects are limited. Methods: Focusing on the treatment of acute airway inflammation and accompanying cough, this study aimed to elucidate antitussive and mucoactive properties of EA575, applying two animal models. Animals were treated orally twice daily for 7 days, resulting in 43, 215.2, or 430.5 mg/kg bw/d of EA575. Antitussive effects were investigated within an acute lung inflammation model of bleomycin-treated guinea pigs after citric acid exposure. Hereby, the number of coughs, enhanced pause (penH), and bronchoalveolar lavage fluid (BALF) were investigated. Mucoactivity of EA575 was assessed within a murine model, determining phenol red concentration in BALF. Results: EA575 treatment within the acute lung inflammation model reduced cough events up to 56% while reducing inflammatory cell influx in BALF dose-dependently, e.g., reducing neutrophils in BALF up to 70.9%. This suggests a strong connection between anti-inflammatory and antitussive properties of EA575. Furthermore, penH decreased in a dose-dependent manner, suggesting an ease in respiration. Mucoactivity was shown by a dose-dependent increase in phenol red concentration in BALF up to 38.9%. Notably, EA575/salbutamol co-administration resulted in enhanced phenol red secretion compared to respective single administrations. Conclusions: These data highlight the benefits of EA575 in treating cough-related respiratory diseases, particularly when accompanied by sputum, as EA575 has been shown to obtain mucoactivity. Furthermore, the combinatory effect of EA575/salbutamol treatment provides a foundation for future research in the treatment of chronic respiratory diseases.

Keywords: EA575; antitussive; cough; ivy leaf dry extract; mucoactive agent; respiratory disease.

Figure 1

EA575 decreases cough events after citric acid exposure of bleomycin-challenged guinea pigs. The animals (n = 6 per group) were challenged once with phosphate-buffered saline (PBS) or bleomycin (BLM, 2.5 U/kg bw) and treated twice daily for seven days with vehicle, EA575, or dexamethasone. Cough events were recorded in a whole-body plethysmography system for 15 min starting with citric acid exposure. Comparisons between the BLM-only treated animals were made against each of the other groups using a one-way analysis of variance (ANOVA) followed by Dunnett’s test. * p ≤ 0.05, ** p ≤ 0.01. and *** p ≤ 0.001. bw: body weight.

Figure 2

EA575 decreases inflammatory cell influx in the BALF of bleomycin-challenged guinea pigs. The animals (n = 6 per group) were challenged once with phosphate-buffered saline (PBS) or bleomycin (BLM, 2.5 U/kg bw) and treated twice daily for seven days with vehicle, EA575, or dexamethasone. BALF was collected on Day 7 after the BLM challenge in all groups. Data for (A) total cell count, (B) neutrophils, (C) lymphocytes, (D) macrophages in BALF are shown as mean ± SD. Comparisons between the BLM-only treated animals were made against each of the other groups using a one-way analysis of variance (ANOVA), followed by Dunnett’s test. * p ≤ 0.05, ** p ≤ 0.01. and *** p ≤ 0.001. bw: body weight.

Figure 3

Correlation of cough events and neutrophil count for control and treated guinea pigs. Neutrophil count in BALF was chosen as a representative inflammation parameter and depicted against the number of cough events. A linear regression fit was performed to elucidate the correlation between these observations. bw: body weight.

Figure 4

EA575 attenuates enhanced pause increase after bleomycin administration in guinea pigs. The animals (n = 6 per group) were challenged once with phosphate-buffered saline (PBS) or bleomycin (BLM, 2.5 U/kg bw) and treated twice daily for seven days with vehicle, EA575, or dexamethasone. Inspiratory and expiratory flow, as well as the time between inspirations, were recorded using the whole-body plethysmography system. Comparisons between the BLM + vehicle-treated animals were made against each of the other groups using a one-way analysis of variance (ANOVA), followed by Dunnett’s test. * p ≤ 0.05, ** p ≤ 0.01. and *** p ≤ 0.001. bw: body weight.

Figure 5

EA575 induces phenol red secretion in BALF and enhances the effect of β₂ receptor agonist salbutamol. The animals (n = 8 per group) were treated twice daily for seven days with either vehicle or EA575. Treatment with salbutamol at 2 or 4 mg/kg bw occurred once, 30 min prior to phenol red administration. Phenol red (300 mg/kg bw) was applied intraperitoneally 30 min before the study. BALF was collected after study termination. Phenol red concentration was determined in alkaline BALF solution at 565 nm. Comparisons between the vehicle-treated animals were made against each of the other groups using a one-way analysis of variance (ANOVA), followed by Dunnett’s test. * p ≤ 0.05, ** p ≤ 0.01. and *** p ≤ 0.001. bw: body weight.