Original Paper Received: July 10, 2014 Accepted after revision: November 18, 2014 Published online: January 13, 2015
Pharmacology 2015;95:36–41 DOI: 10.1159/000369977
Novel Antitussive Effect of Suplatast Tosilate in Guinea Pigs Jian-Rong Zhou a Ryo-ichi Syono b Syu-ichi Fukumi b Kenji Kimoto b Tetsuya Shirasaki b Fumio Soeda b Kazuo Takahama b
a
Laboratory of Presymptomatic Medical Pharmacology, Faculty of Pharmaceutical Sciences, Sojo University, and Department of Environmental and Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan
Key Words Codeine-resistant coughs · Suplatast tosilate · Bronchitis model · Vagal afferent nerve discharge · Antitussive effects
Abstract We studied the antitussive effects of suplatast, a Th2 cytokine inhibitor, and compared them with the effects of codeine using an experimental cough model in guinea pigs. Suplatast and codeine dose-dependently inhibited cough caused by mechanical stimulation of the larynx, but they did not inhibit cough caused by mechanical stimulation of the bifurcation of the trachea. In guinea pigs with bronchitis, suplatast had an antitussive effect on cough caused by stimulation of the larynx, whereas codeine did not inhibit such cough. In SO2-exposed guinea pigs, suplatast tended to inhibit cough caused by mechanical stimulation of the tracheal bifurcation. Further, suplatast inhibited citric acid-induced cough augmented by pretreatment with an angiotensinconverting enzyme inhibitor, whereas codeine did not inhibit such cough. Suplatast also inhibited bradykinin-induced discharges of airway vagal afferent nerves and significantly inhibited 4-aminopyridine-induced discharges of airway vagal afferent nerves. These findings indicate that the antitussive effects of suplatast are mediated by a novel mechanism involving the peripheral nervous system. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0031–7012/15/0952–0036$39.50/0 E-Mail [email protected] www.karger.com/pha
Introduction
Although many antitussives are available, there is still a need for more effective ones for clinical applications. Chronic cough, which occurs in patients with various respiratory diseases such as cough variant asthma (CVA), atopic cough, chronic bronchitis, and laryngeal allergy, is often resistant to antitussive treatment [1–3]. CVA, the major cause of chronic cough, is characterized only by chronic dry cough [4] and is not relieved by an inhaled codeine [2]. It has been reported that suplatast [suplatast tosilate, ±-[2-[4-(3-ethoxy-2-hydroxypropoxy)phenylcarbamoyl]ethyl]dimethylsulfonium p-toluenesulfonate] improves the cough score and the cough threshold for capsaicin in patients with atopic cough and CVA [5, 6]. Suplatast, an anti-allergic drug, is a selective Th2 cytokine inhibitor [7] that suppresses the synthesis of IL-4 and IL-5 in vitro [8, 9] as well as allergen-induced increases in peritoneal eosinophils in mice [8]. It is unknown whether these effects of suplatast contribute to the mechanisms of its antitussive effects in atopic cough and CVA. An animal model of CVA has not been developed. We have previously found that in guinea pigs with bronchitis, cough caused by mechanical stimulation of the airway is not inhibited by codeine but by bakumondoto, a Chinese herbal medicine [10]. Other scientists have found that in guinea pigs treated with angiotensin-conJian-Rong Zhou, PhD Laboratory of Presymptomatic Medical Pharmacology Faculty of Pharmaceutical Sciences, Sojo University 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082 (Japan) E-Mail zhoujr @ ph.sojo-u.ac.jp
Downloaded by: University of Tokyo 157.82.153.40 - 6/3/2015 1:34:43 PM
b
Methods Animals This study was approved by the Committee of Animal Welfare and Control of the Faculty of Pharmaceutical Sciences, Kumamoto University, and was conducted according to the guidelines for care and use of experimental animals approved by the Council of the Pharmacological Society of Japan. Male Hartley guinea pigs (5–7 weeks old; Kyudo Pharm, Kumamoto, Japan) were housed at a room temperature of 22 ± 2 ° C. Guinea pigs with subacute bronchitis were prepared by exposure to 200 ppm SO2 gas for 2 h/day for 7 days, as described elsewhere [10]. The guinea pigs exposed to SO2 under these conditions exhibited bronchitis characterized by an increase in the number of inflammatory cells in the bronchoalveolar lavage fluid, an increase in the sensitivity of the bronchial smooth muscle to acetylcholine and histamine, and a reduction of neutral endopeptidase activity in the trachea [14]. Some of these bronchitic animals coughed during SO2 exposure but did not cough spontaneously after the exposure to SO2 was terminated. To facilitate the detection of the cough potentiation by ACE inhibitors, the normal and bronchitic animals that were used in the experiments were those in which the frequency of cough induced by cough stimulants was 1–4 coughs every 15 min. This cough frequency was designated as the preadministration value of the cough response. A group of 7–20 animals was used for each dose of each drug.
Cough Experiments Mechanical Stimulation Two methods of mechanical stimulation were used in the cough experiments. The first method is one that we have previously reported [11]. Briefly, one group of 5–9 animals was used for each dose of suplatast or vehicle. The guinea pigs were anesthetized with urethane (1.1–1.2 g/kg i.p.). The animals were then placed on their backs, and a small hole (3 × 4 mm) was made in the ventral wall of the trachea about 3.5 cm caudal to the cricoid cartilage. The animals thus anesthetized coughed stably in response to the mechanical stimulation of the larynx or the bifurcation of the trachea for about 120 min after the beginning of the experiment. To pro-
Antitussive of Suplatast
duce cough responses, mechanical stimulation was applied to the larynx or the bifurcation of the trachea using a rabbit whisker with a 70-μm diameter tip. The coughing was monitored and recorded as changes in air flow within a tube connected to a pneumograph positioned at the caudal end of the breast using a polygraph system (RM 6100; Nihon Kohden, Japan) and a flowmeter (TUR-3200; Nihon Kohden). The mechanical stimulation was applied at 20min intervals before drug administration. The mean amplitude of the cough response before drug administration was used as a preadministration control value. Then, suplatast (diluted in saline) or codeine (diluted in saline) or vehicle (saline) was administered intravenously. The mechanical stimulation was applied 15, 30, 45 and 60 min after the administration of suplatast or vehicle, and the amplitude of the response to each stimulation was recorded. The lowest amplitude after the administration was considered the postadministration value and was expressed as the percentage of the preadministration value. The mean postadministration value of the suplatast group was compared to those of the codeine- and vehicle-treated groups. Chemical Stimulation The inhalation of chemicals is known to stimulate bronchopulmonary C fibers. Unlike mechanical stimulation of the extrapulmonary airways, selective C-fiber stimulants are less effective or ineffective at evoking cough in anesthetized animals [15]. Nonanesthetized guinea pigs were placed individually in a plethysmograph and forced to inhale a 0.1 mol/l citric acid solution for 2 min using an ultrasonic nebulizer (Tur-3200; Nihon-Kohden) that was connected to the chamber and adjusted to an output volume of 2.2 ml/min. The coughing was monitored and recorded as changes in air flow within a tube connected to the plethysmograph using the polygraph system and flowmeter described above. The sound of the coughing was also recorded on a tape recorder using a small microphone positioned within the plethysmograph. The animals were continuously observed during the experiment by a trained and uninformed observer. Coughing could be distinguished from sneezing, because the two responses differed in terms of sound and the behavior of the animals. The number of coughs during the 2-min inhalation period and the subsequent 13 min was counted and statistically compared between suplatast and vehicle groups. The ACE inhibitor enalapril was administered at 1 mg/kg s.c. 30 min before the inhalation of citric acid. Suplatast, codeine or vehicle was intravenously administered 10 min before the citric acid challenge. Airway Vagal Afferent Discharge Experiment Airway vagal afferent discharges were recorded using a method described elsewhere [10]. Briefly, guinea pigs were anesthetized with urethane (1.5 g/kg i.p.). The fine nerve fascicle (diameter, approx. 50 μm) that responded to the inhaled citric acid solution was detached from the right vagus. The nerve fascicle was drawn onto a small vinyl sheet containing 40 μl of Tyrode’s solution, in which voltage was recorded using a platinum-iridium filament electrode (diameter, 120 μm). The discharges were conventionally processed by a biophysical amplifier (AVB-10; Nihon Kohden), monitored on an oscilloscope (VC-10; Nihon Kohden), and recorded on a computer (Mac lab/200; AD instrument) for further analysis. For a quantitative analysis of the data, the firing rate of discharges before and after drug administration was determined using the computer. Suplatast or vehicle was injected into the vein. Then, 30 min
Pharmacology 2015;95:36–41 DOI: 10.1159/000369977
37
Downloaded by: University of Tokyo 157.82.153.40 - 6/3/2015 1:34:43 PM
verting enzyme (ACE) inhibitors, citric acid-induced cough is not inhibited by codeine but by bakumondo-to [11]. It has been reported that bakumondo-to is effective for the treatment of intractable coughs that are resistant to treatment with other antitussives [12, 13]. Thus, there is a precedent for novel antitussives inhibiting cough in codeine-resistant cough models. These codeine-resistant coughs in animal models should be useful to develop new antitussive drugs for the treatment of chronic cough. In the present study, we examined the antitussive effects of suplatast using various guinea pig cough models. We also examined the effects of suplatast on vagal afferent discharges of the lower airway in guinea pigs to compare the effects of suplatast with those of other peripherally acting antitussives.
Cough response (% of preadministration)
100
(n = 20)
80
Larynx
Bifurcation 100
(n = 7) (n = 14)
(n = 14)
(n = 7)
* ***
60 40
***
20 0
Saline 100 μl
10 mg/kg 30 mg/kg
1 mg/kg
Suplatast
3 mg/kg
Codeine
Cough response (% of preadministration)
Larynx
(n = 20)
Bifurcation
(n = 8) (n = 12)
80
(n = 14)
(n = 8)
##
***
60
(n = 8)
***
40
***
20 0
Normal
SO2
Normal
Saline 100 μl
SO2
Normal
Suplatast 10 mg/kg
SO2
Codeine 1 mg/kg
Fig. 1. Effect of suplatast and codeine on coughs caused by mechanical stimulation of the larynx and the bifurcation of the trachea in guinea pigs. Suplatast suppressed cough caused by mechanical stimulation of the larynx, but did not suppress cough caused by mechanical stimulation of the tracheal bifurcation. Codeine also strongly suppressed cough caused by mechanical stimulation of the larynx. *** p < 0.001, * p < 0.05, significantly different from the saline group.
Fig. 2. Effect of suplatast on coughs caused by mechanical stimulation of the larynx and the bifurcation of the trachea in SO2-exposed guinea pigs. In SO2-treated guinea pigs, codeine did not suppress coughs caused by mechanical stimulation of the larynx or the tracheal bifurcation. Suplatast significantly suppressed coughs caused by mechanical stimulation of the larynx in SO2-treated guinea pigs. *** p < 0.001, significantly different from the saline group. ## p < 0.01, significantly different from the SO2-treated saline group.
later, the stimulant [bradykinin (BK) or the voltage-gated potassium channel blocker 4-aminopyridine (4-AP)] was administered by a close arterial injection into the lower airway in a volume of 10 μl/100 g body weight with an injection speed of 20 μl/s. The distribution of the injected stimulant in the lower airway was confirmed by an injection of Evans blue via the same route. The firing rate before the administration of BK or 4-AP was designated as 100%. The effect of suplatast on BK- or 4-AP-induced increases in the firing rate was determined and compared with that of the vehicle control group.
dose of 10 or 30 mg/kg inhibited an antigen-induced cough hypersensitivity in guinea pigs [16]. Suplatast at a dose of 10 mg/kg significantly inhibited cough caused by stimulation of the larynx, reducing the cough response to 58.4 ± 7.9% compared to the preadministration control value. Higher doses of suplatast also significantly inhibited cough caused by stimulation of the larynx. However, both suplatast and codeine had no significant effect on cough caused by stimulation of the tracheal bifurcation. In bronchitic guinea pigs, codeine had no antitussive effect (fig. 2), which is consistent with previous findings [17]. In contrast, suplatast significantly inhibited cough caused by larynx stimulation in both normal and bronchitic guinea pigs. Moreover, suplatast tended to inhibit cough caused by stimulation of the tracheal bifurcation in bronchitic guinea pigs, but the inhibition was not statistically significant.
Statistical Analysis Each value represents the mean ± SEM for 7–20 mice. Comparisons between the two groups were carried out using unpaired Student’s t test. Multiple comparisons were performed using a Dunnett test. p values
Pharmacology 2015;95:36–41 DOI: 10.1159/000369977
Novel Antitussive Effect of Suplatast Tosilate in Guinea Pigs Jian-Rong Zhou a Ryo-ichi Syono b Syu-ichi Fukumi b Kenji Kimoto b Tetsuya Shirasaki b Fumio Soeda b Kazuo Takahama b
a
Laboratory of Presymptomatic Medical Pharmacology, Faculty of Pharmaceutical Sciences, Sojo University, and Department of Environmental and Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan
Key Words Codeine-resistant coughs · Suplatast tosilate · Bronchitis model · Vagal afferent nerve discharge · Antitussive effects
Abstract We studied the antitussive effects of suplatast, a Th2 cytokine inhibitor, and compared them with the effects of codeine using an experimental cough model in guinea pigs. Suplatast and codeine dose-dependently inhibited cough caused by mechanical stimulation of the larynx, but they did not inhibit cough caused by mechanical stimulation of the bifurcation of the trachea. In guinea pigs with bronchitis, suplatast had an antitussive effect on cough caused by stimulation of the larynx, whereas codeine did not inhibit such cough. In SO2-exposed guinea pigs, suplatast tended to inhibit cough caused by mechanical stimulation of the tracheal bifurcation. Further, suplatast inhibited citric acid-induced cough augmented by pretreatment with an angiotensinconverting enzyme inhibitor, whereas codeine did not inhibit such cough. Suplatast also inhibited bradykinin-induced discharges of airway vagal afferent nerves and significantly inhibited 4-aminopyridine-induced discharges of airway vagal afferent nerves. These findings indicate that the antitussive effects of suplatast are mediated by a novel mechanism involving the peripheral nervous system. © 2015 S. Karger AG, Basel
© 2015 S. Karger AG, Basel 0031–7012/15/0952–0036$39.50/0 E-Mail [email protected] www.karger.com/pha
Introduction
Although many antitussives are available, there is still a need for more effective ones for clinical applications. Chronic cough, which occurs in patients with various respiratory diseases such as cough variant asthma (CVA), atopic cough, chronic bronchitis, and laryngeal allergy, is often resistant to antitussive treatment [1–3]. CVA, the major cause of chronic cough, is characterized only by chronic dry cough [4] and is not relieved by an inhaled codeine [2]. It has been reported that suplatast [suplatast tosilate, ±-[2-[4-(3-ethoxy-2-hydroxypropoxy)phenylcarbamoyl]ethyl]dimethylsulfonium p-toluenesulfonate] improves the cough score and the cough threshold for capsaicin in patients with atopic cough and CVA [5, 6]. Suplatast, an anti-allergic drug, is a selective Th2 cytokine inhibitor [7] that suppresses the synthesis of IL-4 and IL-5 in vitro [8, 9] as well as allergen-induced increases in peritoneal eosinophils in mice [8]. It is unknown whether these effects of suplatast contribute to the mechanisms of its antitussive effects in atopic cough and CVA. An animal model of CVA has not been developed. We have previously found that in guinea pigs with bronchitis, cough caused by mechanical stimulation of the airway is not inhibited by codeine but by bakumondoto, a Chinese herbal medicine [10]. Other scientists have found that in guinea pigs treated with angiotensin-conJian-Rong Zhou, PhD Laboratory of Presymptomatic Medical Pharmacology Faculty of Pharmaceutical Sciences, Sojo University 4-22-1 Ikeda, Nishi-ku, Kumamoto 860-0082 (Japan) E-Mail zhoujr @ ph.sojo-u.ac.jp
Downloaded by: University of Tokyo 157.82.153.40 - 6/3/2015 1:34:43 PM
b
Methods Animals This study was approved by the Committee of Animal Welfare and Control of the Faculty of Pharmaceutical Sciences, Kumamoto University, and was conducted according to the guidelines for care and use of experimental animals approved by the Council of the Pharmacological Society of Japan. Male Hartley guinea pigs (5–7 weeks old; Kyudo Pharm, Kumamoto, Japan) were housed at a room temperature of 22 ± 2 ° C. Guinea pigs with subacute bronchitis were prepared by exposure to 200 ppm SO2 gas for 2 h/day for 7 days, as described elsewhere [10]. The guinea pigs exposed to SO2 under these conditions exhibited bronchitis characterized by an increase in the number of inflammatory cells in the bronchoalveolar lavage fluid, an increase in the sensitivity of the bronchial smooth muscle to acetylcholine and histamine, and a reduction of neutral endopeptidase activity in the trachea [14]. Some of these bronchitic animals coughed during SO2 exposure but did not cough spontaneously after the exposure to SO2 was terminated. To facilitate the detection of the cough potentiation by ACE inhibitors, the normal and bronchitic animals that were used in the experiments were those in which the frequency of cough induced by cough stimulants was 1–4 coughs every 15 min. This cough frequency was designated as the preadministration value of the cough response. A group of 7–20 animals was used for each dose of each drug.
Cough Experiments Mechanical Stimulation Two methods of mechanical stimulation were used in the cough experiments. The first method is one that we have previously reported [11]. Briefly, one group of 5–9 animals was used for each dose of suplatast or vehicle. The guinea pigs were anesthetized with urethane (1.1–1.2 g/kg i.p.). The animals were then placed on their backs, and a small hole (3 × 4 mm) was made in the ventral wall of the trachea about 3.5 cm caudal to the cricoid cartilage. The animals thus anesthetized coughed stably in response to the mechanical stimulation of the larynx or the bifurcation of the trachea for about 120 min after the beginning of the experiment. To pro-
Antitussive of Suplatast
duce cough responses, mechanical stimulation was applied to the larynx or the bifurcation of the trachea using a rabbit whisker with a 70-μm diameter tip. The coughing was monitored and recorded as changes in air flow within a tube connected to a pneumograph positioned at the caudal end of the breast using a polygraph system (RM 6100; Nihon Kohden, Japan) and a flowmeter (TUR-3200; Nihon Kohden). The mechanical stimulation was applied at 20min intervals before drug administration. The mean amplitude of the cough response before drug administration was used as a preadministration control value. Then, suplatast (diluted in saline) or codeine (diluted in saline) or vehicle (saline) was administered intravenously. The mechanical stimulation was applied 15, 30, 45 and 60 min after the administration of suplatast or vehicle, and the amplitude of the response to each stimulation was recorded. The lowest amplitude after the administration was considered the postadministration value and was expressed as the percentage of the preadministration value. The mean postadministration value of the suplatast group was compared to those of the codeine- and vehicle-treated groups. Chemical Stimulation The inhalation of chemicals is known to stimulate bronchopulmonary C fibers. Unlike mechanical stimulation of the extrapulmonary airways, selective C-fiber stimulants are less effective or ineffective at evoking cough in anesthetized animals [15]. Nonanesthetized guinea pigs were placed individually in a plethysmograph and forced to inhale a 0.1 mol/l citric acid solution for 2 min using an ultrasonic nebulizer (Tur-3200; Nihon-Kohden) that was connected to the chamber and adjusted to an output volume of 2.2 ml/min. The coughing was monitored and recorded as changes in air flow within a tube connected to the plethysmograph using the polygraph system and flowmeter described above. The sound of the coughing was also recorded on a tape recorder using a small microphone positioned within the plethysmograph. The animals were continuously observed during the experiment by a trained and uninformed observer. Coughing could be distinguished from sneezing, because the two responses differed in terms of sound and the behavior of the animals. The number of coughs during the 2-min inhalation period and the subsequent 13 min was counted and statistically compared between suplatast and vehicle groups. The ACE inhibitor enalapril was administered at 1 mg/kg s.c. 30 min before the inhalation of citric acid. Suplatast, codeine or vehicle was intravenously administered 10 min before the citric acid challenge. Airway Vagal Afferent Discharge Experiment Airway vagal afferent discharges were recorded using a method described elsewhere [10]. Briefly, guinea pigs were anesthetized with urethane (1.5 g/kg i.p.). The fine nerve fascicle (diameter, approx. 50 μm) that responded to the inhaled citric acid solution was detached from the right vagus. The nerve fascicle was drawn onto a small vinyl sheet containing 40 μl of Tyrode’s solution, in which voltage was recorded using a platinum-iridium filament electrode (diameter, 120 μm). The discharges were conventionally processed by a biophysical amplifier (AVB-10; Nihon Kohden), monitored on an oscilloscope (VC-10; Nihon Kohden), and recorded on a computer (Mac lab/200; AD instrument) for further analysis. For a quantitative analysis of the data, the firing rate of discharges before and after drug administration was determined using the computer. Suplatast or vehicle was injected into the vein. Then, 30 min
Pharmacology 2015;95:36–41 DOI: 10.1159/000369977
37
Downloaded by: University of Tokyo 157.82.153.40 - 6/3/2015 1:34:43 PM
verting enzyme (ACE) inhibitors, citric acid-induced cough is not inhibited by codeine but by bakumondo-to [11]. It has been reported that bakumondo-to is effective for the treatment of intractable coughs that are resistant to treatment with other antitussives [12, 13]. Thus, there is a precedent for novel antitussives inhibiting cough in codeine-resistant cough models. These codeine-resistant coughs in animal models should be useful to develop new antitussive drugs for the treatment of chronic cough. In the present study, we examined the antitussive effects of suplatast using various guinea pig cough models. We also examined the effects of suplatast on vagal afferent discharges of the lower airway in guinea pigs to compare the effects of suplatast with those of other peripherally acting antitussives.
Cough response (% of preadministration)
100
(n = 20)
80
Larynx
Bifurcation 100
(n = 7) (n = 14)
(n = 14)
(n = 7)
* ***
60 40
***
20 0
Saline 100 μl
10 mg/kg 30 mg/kg
1 mg/kg
Suplatast
3 mg/kg
Codeine
Cough response (% of preadministration)
Larynx
(n = 20)
Bifurcation
(n = 8) (n = 12)
80
(n = 14)
(n = 8)
##
***
60
(n = 8)
***
40
***
20 0
Normal
SO2
Normal
Saline 100 μl
SO2
Normal
Suplatast 10 mg/kg
SO2
Codeine 1 mg/kg
Fig. 1. Effect of suplatast and codeine on coughs caused by mechanical stimulation of the larynx and the bifurcation of the trachea in guinea pigs. Suplatast suppressed cough caused by mechanical stimulation of the larynx, but did not suppress cough caused by mechanical stimulation of the tracheal bifurcation. Codeine also strongly suppressed cough caused by mechanical stimulation of the larynx. *** p < 0.001, * p < 0.05, significantly different from the saline group.
Fig. 2. Effect of suplatast on coughs caused by mechanical stimulation of the larynx and the bifurcation of the trachea in SO2-exposed guinea pigs. In SO2-treated guinea pigs, codeine did not suppress coughs caused by mechanical stimulation of the larynx or the tracheal bifurcation. Suplatast significantly suppressed coughs caused by mechanical stimulation of the larynx in SO2-treated guinea pigs. *** p < 0.001, significantly different from the saline group. ## p < 0.01, significantly different from the SO2-treated saline group.
later, the stimulant [bradykinin (BK) or the voltage-gated potassium channel blocker 4-aminopyridine (4-AP)] was administered by a close arterial injection into the lower airway in a volume of 10 μl/100 g body weight with an injection speed of 20 μl/s. The distribution of the injected stimulant in the lower airway was confirmed by an injection of Evans blue via the same route. The firing rate before the administration of BK or 4-AP was designated as 100%. The effect of suplatast on BK- or 4-AP-induced increases in the firing rate was determined and compared with that of the vehicle control group.
dose of 10 or 30 mg/kg inhibited an antigen-induced cough hypersensitivity in guinea pigs [16]. Suplatast at a dose of 10 mg/kg significantly inhibited cough caused by stimulation of the larynx, reducing the cough response to 58.4 ± 7.9% compared to the preadministration control value. Higher doses of suplatast also significantly inhibited cough caused by stimulation of the larynx. However, both suplatast and codeine had no significant effect on cough caused by stimulation of the tracheal bifurcation. In bronchitic guinea pigs, codeine had no antitussive effect (fig. 2), which is consistent with previous findings [17]. In contrast, suplatast significantly inhibited cough caused by larynx stimulation in both normal and bronchitic guinea pigs. Moreover, suplatast tended to inhibit cough caused by stimulation of the tracheal bifurcation in bronchitic guinea pigs, but the inhibition was not statistically significant.
Statistical Analysis Each value represents the mean ± SEM for 7–20 mice. Comparisons between the two groups were carried out using unpaired Student’s t test. Multiple comparisons were performed using a Dunnett test. p values
![[Mutagenicity tests of suplatast tosilate (IPD-1151T)].](/assets/img/hold.png)
