INTRODUCTION

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Propranolol, a prototype -blocker used mainly for the treatment of cardiovascular diseases, causes bronchoconstriction in asthmatics when administered orally, intravenously, or by inhalation. After propranolol challenge of asthmatic patients bronchoconstriction occurs usually within 10 min (1, 2) and this reaction can be severe, prolonged, and difficult to reverse. However, this agent does not produce bronchoconstriction or increase bronchial responsiveness in normal subjects (3, 4).

It has been proposed that cholinergic nerve activity plays a role in the pathophysiology of propranolol-induced bronchoconstriction (PIB) (5). On the other hand, it is well known that mast cells in human lung possess -receptors on their surface and -adrenoreceptor agonists have been shown to inhibit anaphylactic release of mediators, including histamine, cysteinyl leukotrienes (cLTs) (LTC4, LTD4, and LTE4), and thromboxane A2 (TXA2) (6), suggesting that the PIB may be caused based on release of chemical mediators. Indeed, histamine was found to be involved in the PIB (7). However, to our knowledge, involvement of other important lipid mediators such as cLTs and TXA2 in the PIB has not been reported.

We have shown that propranolol causes bronchoconstriction when it is inhaled 20 min after an antigen challenge in guinea pigs (8) and cLT and TXA2 antagonists inhibit the bronchoconstriction (8, 9), suggesting the involvement of lipid mediators in the PIB of asthmatics. Thus, we conducted this randomized, double-blinded, placebo-controlled, crossover study to elucidate the role of cLTs and TXA2 in the PIB using a cLT antagonist, pranlukast, and a TXA2 antagonist, seratrodast.

	METHODS

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Subjects

Informed consent was obtained from all subjects. This study was approved by the ethics committee of our university hospital based on the recommendations by the Declaration of Helsinki.

Nine patients with mild to moderate stable asthma, in whom inhalation of 20 mg/ml or less propranolol caused a 20% or more fall in FEV₁ before the study (run-in), participated in this study, with a mean age of 56.7 ± 19.8 SD (range, 22 to 76 yr) (Table 1). Three men and two women had extrinsic asthma, and three men and one women had intrinsic asthma. The study was performed when their symptoms were mild and stable while they were receiving oral theophylline (Theo-Dur), oral and/or aerosol ₂-agonists, aerosol antimuscarinic agents, and/or inhaled corticosteroids. None of these patients had experienced viral infection for at least 4 wk prior to the study. All of them were nonsmokers. Concomitant medication was maintained without any modification during the study period, although all the medication was stopped during the washout period of more than 24 h from 8:00 A.M. on the previous day to 11.00 A.M. on the day of propranolol challenging, and until the end of the propranolol challenge test.

Measurement of Propranolol-induced Bronchoconstriction

DL-propranolol hydrochloride (Wako Pure Chemical Ind., Ltd., Osaka, Japan) was dissolved in physiologic saline to make propranolol solutions of 0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10, 20, and 40 mg/ml. Saline and propranolol were inhaled from a Devilbiss 646 nebulizer (Devilbiss Co, Somerset, PA) operated by compressed air at 5 L/min. The nebulizer output was 0.14 ml/ min. Saline was inhaled first for 1 min and FEV₁ was measured on a dry wedge spirometer (Transfer Test; P.K. Morgan Ltd., Chatham, Kent, UK). After confirming that the change in FEV₁ from the baseline after inhalation of saline was 10% or less in all subjects, inhalation of propranolol was started. Propranolol was inhaled for 1 min by tidal mouth breathing wearing a noseclip, and 5 min later spirometry was performed. Propranolol of increasing concentrations was successively inhaled until a fall of 20% or more in the FEV₁ occurred. The measured values were plotted on semilogarithmic graph paper, and the provocative concentration of propranolol producing a 20% fall in the FEV₁ (PC₂₀) was calculated.

Study Protocol

This study was performed in a double-blinded, placebo-controlled, crossover fashion. The three treatment regimens studied were (1) pranlukast 225 mg twice a day, (2) seratrodast 80 mg once a day, and (3) placebo two tablets twice a day during a 2-wk period. The order of the three treatment regimens was randomized. Pranlukast or placebo was orally administered at 8:00 A.M. and 8:00 P.M., and seratrodast was given at 8:00 P.M. On the day of propranolol challenging each drug was given at 8:00 A.M. Each subject attended at 10:30 A.M. on three separate days at intervals of 14 d. After resting for 15 min or more, propranolol provocation was started at 11:00 A.M.

Data Analysis

In the three test conditions (pranlukast, seratrodast, and placebo), baseline pulmonary function and bronchial responsiveness to propranolol were compared. Propranolol PC₂₀ values were presented as geometric means with the geometric standard error of the mean (GSEM), and those for baseline FVC, FEV₁, and FEV₁/FVC ratio were presented as arithmetic means and standard errors of the mean (SEM) in the text.

When a 20% or greater fall in FEV₁ was not obtained by the final concentration of propranolol (40 mg/ml) the PC₂₀ value was assumed be 80 mg/ml for statistical analysis.

Geometric mean PC₂₀ values and values for FVC, FEV₁, and FEV₁/FVC ratio with pranlukast, seratrodast, and placebo were compared by Student's paired t test. Results were also presented as means and the 95% confidence interval (95% CI) of the ratio of PC₂₀ with pranlukast or seratrodast to PC₂₀ with placebo. In assessing correlation of changes in PC₂₀ value to changes in FEV₁ by pranlukast or seratrodast, simple regression analysis was employed using log (PC₂₀ with pranlukast or seratrodast/PC₂₀ with placebo) and FEV₁ with pranlukast or seratrodast/FEV₁ with placebo. The difference with a p value of 0.05 or less was considered statistically significant.

	RESULTS

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Mean baseline values (± SEM) for FVC, FEV₁, FEV₁/FVC ratio, and GSEM for propranolol PC₂₀ on the first, the second, and the third test day are shown in Table 2. These values were not significantly different between the three test days.

Although there were no significant differences in the FVC, FEV₁, or FEV₁/FVC ratio between treatment with pranlukast, seratrodast, and placebo, the FEV₁ value tended to be greater with pranlukast than with placebo (p = 0.0543) (Table 3).

Individual PC₂₀ values on the treatment with pranlukast, seratrodast, and placebo and in run-in period are shown in Figure 1. The geometric mean value for PC₂₀ was not significantly altered with pranlukast (11.4 [GSEM, 1.35] mg/ml, ratio of 1.01 with 95% CI of 0.76 to 1.36) or seratrodast (10.7 [GSEM, 1.29] mg/ml, ratio of 0.95 with 95% CI of 0.81 to 1.12) compared with placebo (11.2 [GSEM, 1.28] mg/ml).

View larger version (19K): [in this window] [in a new window]   Figure 1.   Effect of 2-wk administration of seratrodast (80 mg/d) and pranlukast (450 mg/d) on aerosolized propranolol-induced bronchoconstriction in nine patients with stable asthma. Each thick horizontal bar represents geometric mean value of PC20. PC20 was defined as a propranolol concentration causing a 20% fall in FEV1.

There was no significant correlation between increase in PC₂₀ and increase in FEV₁ by pranlukast (r = 0.24, p = 0.55) or seratrodast (r = 0.01, p = 0.97). Methacholine concentration causing a 20% fall in FEV₁ did not correlate with PC₂₀ in the run-in period (r = 0.403, p = 0.28).

Any adverse effects were not recognized for each tested drug.

	DISCUSSION

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In this study, we examined whether the lipid mediators cLTs and TXA2 are involved in the pathophysiology of propranolol-induced bronchoconstriction (PIB) in asthmatics. We showed that a 2-wk treatment with pranlukast or seratrodast did not influence PIB, whereas pranlukast, but not seratrodast, tended to reduce baseline bronchomotor tone. As the sample size of this study was small, the power was calculated retrospectively. The calculation showed that the sample size would give an 80% probability of detecting a true effect of pranlukast and seratrodast in the ratio of PC₂₀ with pranlukast and seratrodast to PC₂₀ with placebo (> 1.57 and > 1.28, respectively) when using a test at the 5% significance level and the standard deviation of the ratio investigated. Furthermore, the ratios with pranlukast and seratrodast were nearly zero, strongly suggesting that these drugs have no effects on PIB.

The important role of lipid mediators has been shown in bronchoconstrictor responses to some specific stimuli. The cLT antagonists have been shown to inhibit the antigen-induced immediate (IAR) and late (LAR) asthmatic response (10, 11), exercise-induced asthma (12), and cold air-induced bronchoconstriction (13). In addition, pranlukast has been proven to prevent aspirin-induced bronchoconstriction (14). A TXA2 synthesis inhibitor, ozagrel hydrochloride, has been shown to inhibit IAR, LAR, and exercise-induced bronchoconstriction (15, 16) and a TXA2 antagonist, GR32191, to reduce IAR (17), whereas the potency seems to be weaker than that of the cLT antagonists (18). Thus, many studies have shown the involvement of cLTs and TXA2 in specific bronchial responses in asthmatics, but the role of these lipid mediators in PIB has not been reported, leading us to design the present study.

Although a partial involvement of histamine in PIB has been proposed (7), it is obvious that only histamine cannot explain the exact mechanism. Other mediators are likely to be involved. To our knowledge, any mediator other than histamine has not been examined, and this is the first study to clarify the involvement of cLTs and TXA2 in the PIB.

Pranlukast, a selective LT receptor antagonist, and seratrodast, a selective TXA2 receptor antagonist, have been proven to be useful for management of asthma in Japan (19, 20), and these are now available as an asthma controller in Japan. The tested doses of pranlukast and seratrodast are the recommended doses for the treatment of asthma. Furthermore, a single administration of 225 mg pranlukast has been shown to strongly prevent aspirin-induced bronchoconstriction (14), and seratrodast has been reported to reduce bronchial hyperresponsiveness to methacholine when administered at a dose of 80 mg once a day for 4 wk (21). From these findings, the tested doses of both drugs are enough to assess the role of cLTs and TXA2 in humans. It has been shown that TXA2 antagonists antagonize action of not only TXA2 but also PGD2 (17).

cLTs, TXA2, and PGD2 are considered to be continuously released by ongoing inflammation in the airways of stable asthmatics. Biochemically, it has been shown that levels of cLTs, TXA2, and PGD2 are increased in bronchoalveolar lavaged fluid from stable asthmatics (22). Physiologically, cLT antagonists have been proven to have bronchodilator effect in asthmatics (25) as suggested in this study, indicating that a part of heightened bronchomotor tone of asthma is caused by cLTs. In addition, we previously reported that a 1-wk treatment with pranlukast in the same dose as tested in this study significantly reduced bronchial responsiveness to methacholine in stable asthmatics (26). These findings support the continuous release of cLTs in the asthmatic airways. On the other hand, it has been shown that selective TXA2 antagonists, seratrodast, BAYu 3405, and S-1452, significantly attenuated bronchial responsiveness to methacholine in stable asthmatics (21, 27). It has been shown that there is no correlation between nonspecific bronchial responsiveness and PIB in asthmatics (28) as shown in this study. Accordingly, although cLTs and TXA2 have been proposed to be involved in nonspecific bronchial responsiveness, these mediators may not have a role in PIB in asthmatics, indicating that our guinea pig model of PIB (8, 9) is not relevant to humans.

In conclusion, although histamine and cholinergic nerve innervation may be partially involved in the PIB of asthmatics (5, 7), the present study denied the role of cLTs or TXA2 in the PIB. As the entire mechanism has not been clearly understood up to now, further studies are necessary to elucidate the role of other mediators and autonomic nerve activities.