INTRODUCTION

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Inhaled ₂-adrenoceptor agonists are the most effective bronchodilators available for the treatment of acute symptoms in asthma (1). The introduction of the long-acting ₂-agonists salmeterol and formoterol allowed these drugs to be prescribed as regular treatment in subjects who continue to be symptomatic despite the use of inhaled corticosteroids (2). In addition to their bronchodilator effects, inhaled ₂-agonists also protect against a variety of direct and indirect bronchoconstrictor stimuli (3). Short-acting ₂-agonists provide greater bronchoprotection against the indirect stimulator adenosine 5'-monophosphate (AMP) than against direct airway smooth muscle stimulators, such as methacholine or histamine, in subjects with mild asthma (4, 7). Because AMP stimulates the release of histamine and other preformed spasmogens from mast cells (8, 9), the differential bronchoprotection by ₂-agonists has been interpreted as mast cell stabilization. However, this differential bronchoprotective effect was not seen with salmeterol at either 2 h (10) or 14 h after a single dose (11). This lack of mast cell-stabilizing effect of salmeterol may reflect the fact that it is a partial agonist compared with albuterol or terbutaline.

We hypothesized that formoterol, which is a virtually full agonist, should have a greater protective effect on mast cell mediator release than the partial agonist salmeterol. We investigated the mast cell-stabilizing effects of formoterol 30 min after a single dose in subjects with mild asthma. We hypothesized that formoterol would provide greater protection against the mast cell stimulus AMP than against a direct challenge with histamine. We also studied the protective effect against AMP or histamine of a single dose of albuterol (given via the same inhaler device) as a control.

	METHODS

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Subjects

Sixteen nonsmoking subjects meeting the American Thoracic Society criteria for asthma (12) (9 females and 7 males; mean age, 29.1 ± 1.5 yr; Table 1) took part. All had mild asthma (FEV₁ > 70% predicted), and a positive skin prick test to common aeroallergens (Dermatophagoides pteronyssinus, grass pollen, Aspergillus fumigatus, or cat). Subjects demonstrated sensitivity to inhaled histamine (PC₂₀ [provocative concentration of histamine or AMP causing a 20% drop in FEV₁] < 1 mg/ml) and AMP (PC₂₀ < 20 mg/ml) in the 2 wk before entry. None had an exacerbation of asthma or an upper respiratory tract infection in the previous 6 wk. Subjects were steroid free for at least 2 mo before entry and took no regular treatment for their asthma other than intermittent short-acting ₂-agonists. Written informed consent was obtained from each subject and the study was approved by the Ethics Committee of the Royal Brompton Hospital and the National Heart and Lung Institute (London, UK).

Study Protocol

The trial was double-blind, placebo-controlled, randomized, and cross-over. Subjects attended two screening visits to determine bronchial reactivity to AMP and histamine. At the initial screening visit subjects were converted to ipratropium bromide as a rescue inhaler for the duration of the study. After screening, subjects attended for six treatment visits with an identical protocol: baseline spirometry was followed by drug administration. Subjects inhaled either 12 µg of formoterol by Turbuhaler (Astra Draco AB, Lund, Sweden) (2 × 6 µg) plus two puffs of placebo, or 200 µg of albuterol by Turbuhaler (2 × 100 µg) plus two puffs of placebo or two matched placebo inhalers (two puffs of each). Thirty minutes later, spirometry was performed again and the subject was challenged with either histamine or AMP. The sequence of challenges was randomized such that one challenge agent was administered on the first three visits and the other on the final three visits. A "washout" period (minimum, 72 h; maximum, 2 wk) separated each visit. Subjects were asked to refrain from taking rescue medication or caffeinated beverages for at least 12 h before each visit and attended at a similar time of day for each visit.

Histamine and AMP Challenges

Solutions of histamine and AMP (Sigma, Poole, UK) were made up immediately before challenge in 0.9% saline in concentrations of 0.0625-32 mg/ml for histamine and of 0.39-800 mg/ml for AMP. For AMP, the 800-mg/ml dose was kept at ~ 30° C to keep the AMP in solution. Doses were inhaled via a nebulizer attached to a breath-activated dosimeter (Mefar, Brescia, Italy), which delivered particles with an aerodynamic mass median diameter of 3.5-4.0 µm at an output of 9 µl per breath. A standard procedure was used for all provocation tests. Baseline FEV₁ was measured using a dry wedge spirometer (Vitalograph, Buckingham, UK). Three values of FEV₁ were recorded at 1-min intervals and the best taken as baseline. Subjects inhaled five breaths of normal saline as a control, each breath taking 1 s with a 6-s breath-hold. FEV₁ was measured 2 min later. The subject then inhaled a series of doubling concentrations of histamine or AMP, starting with the lowest dose, at 3-min intervals with FEV₁ measured 2 min after each dose. Challenges were terminated when the FEV₁ fell by > 20% from the post-saline value or the top dose was reached. A log dose-response curve was constructed and the concentration causing a 20% fall in FEV₁ from the post-saline value (PC₂₀) was calculated by linear interpolation and expressed in noncumulative units. If a subject did not respond to the highest concentration, a linear extrapolation was made using the final two doses. If this extrapolation gave a value between the highest concentration and double the highest concentration, the extrapolated value was used for analysis. If the value was greater than the highest concentration doubled, the highest concentration doubled was used.

Data Analysis

Results are presented as means ± 1 standard error of the mean (SEM) unless otherwise stated. PC₂₀ values were log-transformed for analysis and geometric means calculated. The protective effect of formoterol or albuterol against AMP or histamine was calculated by comparing in each subject the difference in PC₂₀ after inhaling the respective active treatment with that achieved after inhaling placebo. The effect is expressed as doubling doses, using the formula:

To determine the validity of comparing the effects of the drugs on different challenges, dose-response curves were constructed for each spasmogen after each treatment. FEV₁ values after inhalation of the final four concentrations of spasmogen were calculated as a percentage of the post-saline value and compared by multifactorial analysis of variance (ANOVA). Values were included in this analysis only if a 20% fall in FEV₁ was achieved. Serial measurements within groups (baseline FEV₁, change from baseline FEV₁ with treatment) were analyzed by repeated measures ANOVA, followed by pairwise comparisons using the Bonferroni correction (13). Paired data (log PC₂₀ values, doubling dose changes in log PC₂₀) and unpaired data between groups (screening data, doubling dose changes in log PC₂₀) were compared using the appropriate two-tailed t test. Statistical significance was taken as p < 0.05.

	RESULTS

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Bronchodilator Effect

Baseline FEV₁ values did not differ between screening visits or any of the six study visits (AMP challenges: placebo, 3.23 ± 0.14; formoterol, 3.22 ± 0.12; albuterol, 3.12 ± 0.15; histamine challenges: placebo, 3.15 ± 0.12; formoterol, 3.22 ± 0.13; albuterol, 3.24 ± 0.14). Both formoterol and albuterol caused significant increases in FEV₁ before AMP and histamine challenge (Figure 1). There was no significant difference in the size of the bronchodilator response to inhaled formoterol or albuterol (Figure 1).

View larger version (19K): [in this window] [in a new window]   Figure 1.   Bronchodilator response 30 min after inhalation of placebo (open bars), 12 µg of formoterol (closed bars), or 200 µg of albuterol (hatched bars). Values shown represent the increase in forced expiratory volume in 1 s (FEV1) in liters before challenge with adenosine 5'-monophosphate (AMP) or histamine. Data are means and 1 SEM for 16 subjects. ***p < 0.001 versus placebo; NS = nonsignificant.

Bronchoprotective Effect

After formoterol treatment, the geometric mean PC₂₀ for AMP was 263.0 mg/ml, compared with 4.3 mg/ml after placebo (p < 0.0001), and after albuterol it was 72.4 mg/ml (p < 0.0001 versus placebo) (Table 2). The geometric mean PC₂₀ for histamine after formoterol treatment was 6.8 mg/ml, compared with 0.4 mg/ml after placebo (p < 0.0001), and 4.1 mg/ml after albuterol (p < 0.0001 versus placebo) (Table 2). One subject reached the top dose during histamine challenge after both formoterol and albuterol treatment. The top dose of AMP was reached in seven subjects after formoterol treatment and in three subjects after albuterol treatment. The protective effect of albuterol against AMP and histamine did not differ significantly: 4.1 ± 0.5 and 3.5 ± 0.4 doubling doses, respectively (Figure 2). However, the protective effect of formoterol was significantly greater against AMP (6.0 ± 0.8 doubling doses) than against histamine (4.2 ± 0.4 doubling doses, p < 0.05) (Figure 2). The dose-response curves to AMP and histamine were similar in shape after placebo treatment and were not significantly altered by either formoterol or albuterol treatment (Figure 3).

View larger version (16K): [in this window] [in a new window]   Figure 2.   The protective effect of formoterol (12 µg) or albuterol (200 µg) on airway responsiveness to adenosine 5'-monophosphate (AMP) (closed bars) or histamine (open bars) determined at 30 min. Data are means and 1 SEM for n = 16 subjects. *p < 0.05.

View larger version (16K): [in this window] [in a new window]   Figure 3.   Dose-response curves for bronchial provocation challenge after inhalation of placebo, formoterol (12 µg), or albuterol (200 µg). Forced expiratory volume in 1 s (FEV1) values are expressed as a percentage of the post-saline value obtained after inhaling the final four doubling increments of AMP (A) or histamine (B) required to reach a 20% fall in FEV1 from the post-saline value (PC20). X denotes the final concentration, X/2 the penultimate concentration, and so on.

	DISCUSSION

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The present study has shown that a single dose of inhaled formoterol reduced airway responsiveness to histamine by 4.2 doubling doses but caused a greater reduction in airway responsiveness to AMP (6.0 doubling doses). In contrast, a single dose of inhaled albuterol decreased airway responsiveness to AMP and histamine to a similar extent (4.1 and 3.5 doubling doses, respectively). This greater protective effect of formoterol against AMP suggests a mast cell-stabilizing effect for formoterol.

The greater protective effect of formoterol against AMP compared with histamine cannot be explained by functional antagonism alone, and may be due to nonsmooth muscle activity. AMP indirectly stimulates bronchoconstriction in asthma, and is thought to activate airway mast cells to release bronchospastic mediators, including histamine (14, 15). ₂-Agonists inhibit release of histamine from human mast cells in vitro, an effect demonstrated for both albuterol (16) and long-acting ₂-agonists salmeterol and formoterol (17, 18). Albuterol also inhibits the increase in plasma histamine after allergen challenge in vivo (19), an action likely to be mediated by mast cells. These findings suggest that the greater protective effect of ₂-agonists against AMP is due to a protective effect on airway mast cells.

We included in the analysis extrapolated PC₂₀ data on seven subjects in whom we were unable to calculate a true PC₂₀ value after treatment because of profound bronchoprotection (three with AMP and one with histamine after albuterol; seven with AMP and one with histamine after formoterol). These data may lead to an underestimation of the protective effect of the drugs, or to an overestimation of the effect if the dose-response curve to the next dose suddenly steepens. Overestimation is unlikely to account for the differential protective effect of formoterol against AMP compared with histamine seen in the present study. For the seven subjects who reached the top dose of AMP after formoterol treatment, all estimates lay on the flat part of the dose-response curve (FEV₁ values of 3.3 ± 0.2, 3.2 ± 0.2, 3.2 ± 0.2, and 3.2 ± 0.2 L/min, respectively, after the final four doses of AMP challenge).

Previous studies have demonstrated an increased bronchoprotective effect against AMP compared with histamine for both albuterol (4, 10) and terbutaline (7), suggesting that both drugs have mast cell-stabilizing properties. In contrast, in the present study, although the protective effect of albuterol was greater against AMP than against histamine (4.1 and 3.5 doubling doses, respectively), the difference was not significant. A possible explanation for the lack of difference is our inclusion of data on subjects demonstrating profound bronchoprotection after albuterol inhalation (two with AMP and one with both AMP and histamine). As discussed above, this may underestimate the protective effect of AMP to a greater extent than that of histamine. In support of this argument, the present study found a protective effect against histamine (3.5 doubling doses) similar to that reported previously (3.3 [4] and 3.8 [10] doubling doses). However, there was less protection against AMP (4.1 doubling doses) than in the latter two studies (4.7 and 5.1 doubling doses).

Second, a lower dose of albuterol (200 µg) was used herein, compared with 400 µg (10) and 2.5 mg (4) in previous studies. Although 200 µg of albuterol via Turbuhaler is comparable to 400 µg via Diskhaler in terms of bronchodilator effect (20), their bronchoprotective effects may not be comparable. The mast cell-stabilizing effect of albuterol in vitro is dose dependent, with albuterol being approximately 100 times less potent than formoterol (17). The protective effect of terbutaline against AMP in patients with asthma is also dose dependent (3). The dose of albuterol used in the present study may be too low for a mast cell effect to be demonstrated.

Finally, the short duration of action of albuterol may have contributed to its weaker protective effect compared with formoterol. The maximum possible time between inhalation of ₂-agonist and end of provocation challenge was ~ 69 min (30 min before the start of challenge plus up to ~ 39 min to administer all doses of spasmogen). However, the maximal bronchodilator effect of albuterol is maintained for at least 90 min (21). Consequently, loss of efficacy of albuterol over the time taken to achieve a response is unlikely to account for the weaker protective effect of albuterol compared with formoterol.

A comparison of the change in dose response with different challenges is valid only if the dose response to each challenge is similar, a unit shift in the dose-response curve for one stimulus being equivalent to the unit shift for another stimulus. To assess this, we compared dose-response curves after the final four doses of each spasmogen and found no differences with placebo or active treatments. Thus, both formoterol and albuterol caused parallel shifts in the AMP and histamine dose- response curves, confirming the validity of comparisons between AMP and histamine challenges in the present study. We did not include data from subjects in whom a PC₂₀ had not been achieved at the final dose of spasmogen, because they had not reached an equivalent part of the dose-response curve. Because the end point of the study was PC₂₀, we had insufficient points to calculate the true slope of the dose-response curves. Although we did not observe any steepening of the dose-response curve for either spasmogen after albuterol and formoterol treatment, it is possible this may become apparent with greater bronchoconstriction (22, 23).

Although mast cell stabilization has previously been demonstrated in vivo for short-acting ₂-agonists, this is the first study to demonstrate this effect with a long-acting ₂-agonist. Salmeterol has no in vivo mast cell-stabilizing properties either 2 h (10) or 14 h (11) after inhalation. A possible explanation for the lack of effect of salmeterol compared with formoterol is the time point studied. Protection in the present study was measured at 30 min rather than later, at 2 or 14 h (10, 11), which may allow time for receptor desensitization. However, desensitization after a single dose of salmeterol is unlikely to be apparent as early as 2 h after dosing. Another possibility is the partial agonist nature of salmeterol (24). In guinea pig eosinophils, formoterol inhibits release of mediators, whereas salmeterol not only is ineffective but also blocks the action of formoterol (25). It is not reported whether a similar phenomenon exists for human lung mast cells.

In conclusion, our results demonstrate that a single dose of formoterol not only causes inhibition of nonspecific bronchoconstriction but may have an additional inhibitory effect on bronchoconstrictor stimuli that involve mast cell activation. Thus, it may be that inhaled formoterol has an additional protective effect against stimuli thought to involve mast cell degranulation, such as exercise and allergen, in addition to that due to functional antagonism alone. Whether these beneficial actions of formoterol persist after prolonged therapy requires further study.