INTRODUCTION

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Aspirin-induced asthma (AIA) is a syndrome of life threatening bronchoconstriction, nasal congestion, and flushing which is precipitated in approximately 10% of adult asthmatics by small amounts of aspirin and other cyclooxygenase (COX) inhibitors (1). Treatment of patients with this syndrome is often challenging; half of them require maintenance therapy with systemic corticosteroids in addition to topical steroids for satisfactory control of asthma symptoms. In a recent large survey 25% of asthmatic patients requiring emergency mechanical ventilation were found to be aspirin-intolerant (7).

In AIA, inhibition of COX is associated with an increased production of cysteinyl-leukotrienes (cys-LT). Leukotrienes have been found in increased amounts in urine (8), the nasal cavity (9, 10), and bronchi after challenge with aspirin (11). The essential enzyme for cys-LT synthesis, LTC₄ synthase, is overexpressed in bronchial biopsies of patients with AIA (12). The degree of LTC₄ synthase expression correlates with the release of cys-LT into BAL fluid and with bronchial hyperresponsiveness to inhaled aspirin. A common polymorphism of the LTC₄ synthase promoter is overrepresented in patients with AIA (13). Evidence for the critical role of cys-LTs has been provided by several recent studies employing drugs that interfere with cys-LTs action or formation. These compounds, which are available for clinical use, attenuate aspirin-precipitated bronchoconstriction (14) and are useful in chronic treatment of AIA (15).

Recently, we suggested (16) that the protective effect of salbutamol against aspirin-induced bronchoconstriction may not rely solely on the bronchodilator action of the drug. In this study we wondered if salmeterol, a new long-acting ₂-agonist (17), could be beneficial in AIA through a mechanism involving inhibition of the release of proinflammatory and bronchoconstrictor eicosanoids. Because of the essential role of cys-LTs and the specific response of prostanoids to aspirin in AIA (11), we measured the urinary excretion of LTE₄, believed to reflect the global production of cys-LTs (18), and PGD-M, the major urinary metabolite of PGD₂ (19, 20).

	METHODS

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Subjects

Studies were performed in 10 stable, nonsmoking asthmatics with aspirin sensitivity confirmed by oral and/or inhalation provocation test. The patients characteristics are presented in Table 1. The subjects were instructed to withhold inhaled adrenergic agents for 8 h, aminophylline for 24 h, antihistamines for 48 h, and inhaled steroids for 48 h prior to aspirin challenge. None of them was treated with long-acting ₂-agonists. Their baseline FEV₁ was > 60% of the predicted on each study day.

Study Design

The study was performed in a double-blind, placebo-controlled crossover fashion. Each study day began at 8:00 A.M. with pulmonary function testing (MasterLab; Jaeger, Würzburg, Germany), followed by an inhalation of seven breaths of nebulized saline. The study was continued if the fall in FEV₁ was smaller than 10% of the initial value at 10 and 20 min after inhalation of saline.

Part I. Fifteen minutes after administration of 50 µg of salmeterol or placebo via Rotadisk (Serevent; Glaxo, UK), patients underwent bronchial challenge test with increasing doses of L-ASA (see ASPIRIN PROVOCATION TEST below for details), and the dose of L-ASA that caused a fall in the FEV₁ of 20% was determined for each arm of the study.

Two weeks before Part II of the study (see below) standard provocations with increasing doses of aspirin were repeated in an open, single-blind fashion, and the values of aspirin PD₂₀ were calculated. The purpose of the challenges was to establish the PD₂₀ doses of L-ASA that were used during Part II of the study and to determine the repeatability of the challenge procedure. One patient was excluded from further study in Part II because of asthma exacerbation.

Part II. Nine subjects were randomized to receive either 50 µg of salmeterol or placebo 15 min before inhalation of a single PD₂₀ dose of L-ASA (PD₂₀ aspirin defined as described above). FEV₁ was measured at baseline, then every 30 min for 8 h after aspirin inhalation. Urine for analysis of LTE₄ and PGD-M, and blood for determination of eosinophils were collected at baseline (from 6:00 A.M to 8:00 A.M.), and every 2 h for 8 h after L-ASA inhalation.

The protocol was approved by the Jagiellonian University Ethical Committee, and informed consent was obtained from each patient.

Aspirin Provocation Test

An aspirin provocation test was performed using a dosimeter-controlled jet nebulizer, driven by compressed air (Spiro Electro 2; Hengityshoitokeskus Co., Hameenlinna, Finland). Crystalline lysine aspirin (Aspisol; Bayer AG, Levercusen, Germany) was dissolved freshly each day in saline to produce a 1 M (0.1 M = 18 mg/ml of acetylsalicylic acid) stock solution and a tenfold dilution. The challenge was started with inhalation of saline, and FEV₁ measurements were made at 10 and 20 min thereafter. If the fall in FEV₁ was less then 10%, the provocation was continued with increasing concentrations of L-ASA aerosol given every 30 min beginning with a dose of 0.18 mg/ml to maximum of 115.2 mg/ml of aspirin (the dose increments of aspirin were 0.18, 0.36, 0.90, 2.34, 7.2, 16.2, 39.6, and 115.2 mg). FEV₁ was measured at 10, 20, and 30 min after each dose. The provocation was stopped when FEV₁ had fallen 20% or greater from baseline value (which was equal to the FEV₁ obtained at 20 min after saline inhalation), or when the maximal dose of aspirin had been reached (the maximum cumulative dose of aspirin was 182.0 mg). Dose-response relations for aspirin were constructed and used to calculate the PD₂₀ by linear interpolation. In some patients pretreated with salmeterol, FEV₁ failed to drop by 20% at the highest dose of aspirin. For statistical evaluation their PD₂₀ values were set as equal to the highest cumulative dose of acetylsalicylic acid given.

Measurement of Urinary LTE₄

LTE₄ was measured in unpurified urine samples by direct enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) as previously reported (21).

Measurement of Urinary PGD-M

PGD-M (9, 11-dihydroxy-15-oxo-2,3,18,19-tetranorprost-5-ene-1,20-dioic acid) was quantified by gas chromatography/negative ion chemical ionization-mass spectrometry as previously described (22). Briefly, to 1 ml of urine [¹⁸O₄]PGD-M internal standard was added. Then the sample was acidified to pH 3 with HCl and left to stand at room temperature for 30 min to allow quantitative cyclization of the lower side chain to a hemiketal lactone. After extraction with a C-18 Sep-Pak cartridge (Waters Chromatography Div., Millipore, Milford, MA), methylation of the upper carboxyl, and methoxymation of the keto group at C-15, borate buffer (pH, 9.1) was added and neutral lipids were extracted with ethyl acetate. The aqueous layer was then acidified to pH 3 with HCl, and PGD-M was extracted with methylene chloride. The lower carboxyl was then converted to a pentafluorobenzyl ester, and the partially derivatized PGD-M was purified on thin-layer chromatography. After conversion to a trimethylsilyl ether derivative, quantification of PGD-M was accomplished by selected ion monitoring: mass-to-charge ratio was 514 for endogenous PGD-M and 522 for the internal standard.

Eosinophil Count

Eosinophils were counted using a light microscope, after dissolving the whole blood sample in Pilot's solution (23).

Statistical Evaluation

Statistical evaluation was performed using PC and Statsoft-Statistica^TM software. All PD₂₀ and IgE values were log transformed before analysis, and descriptive statistics were expressed as geometric mean (GM) and 95% confidence interval (95% CI). All other summary statistics were expressed as mean (M) and standard deviation (SD). Because of the non-normality of our data as determined by the Shapiro-Wilk W Test, a global parametric ANOVA was avoided. Instead, a Mann-Whitney U Rank Sum Test was employed to compare two independent groups; Wilcoxon's Signed Rank Test for matched pair studies, and a Kruskal-Wallis test and Friedman's Rank ANOVA for comparison of several groups were used. The relationship between variables was determined with Sperman's r Correlation Coefficient. The repeatability of the challenge procedure was expressed in terms of fold differences according to the equation of Peat and colleagues (24), and was illustrated according to Bland and Altman (25). In all procedures the influence of outliers on statistical conclusions was checked by performing all tests with and without the observation suspected to be an outlier.

	RESULTS

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Part I. The mean PD₂₀ dose of aspirin was significantly higher after salmeterol than after placebo (68.5 versus 8.3 mg, respectively; p = 0.02, Wilcoxon's Matched Pairs Test) (Table 2). A protective effect of salmeterol against aspirin-induced bronchoconstriction occurred in nine of 10 patients. In six the maximal cumulative dose of L-ASA failed to produce a fall in FEV₁ of 20%.

Part II. After placebo, eight of nine subjects developed a fall in the FEV₁ of 20% or greater after provocation with a single dose of aspirin, which was determined in a screening study accomplished between Parts I and II of the study. In contrast, after salmeterol pretreatment none of the patients had a 20% drop in FEV₁ for 8 h after inhalation of the same single provocative dose of aspirin (Figure 1).

View larger version (17K): [in this window] [in a new window]   Figure 1.   FEV1 response to L-lysine aspirin (PD20) challenge preceded by salmeterol or placebo (Part II of the study).

The method of Bland and Altman (25) as extended by Peat and colleagues (24) was used to establish the agreement between the values of L-ASA PD₂₀ obtained after placebo inhalation in Part I and during screening provocation tests performed prior to Part II of the study. All repeatability computations were performed with (numbers in parentheses) and without (numbers in brackets) a subject suspected to be an outlier. Mean change in fold difference units (0.498) [0.743] with 95% CI: (0.169 to 1.462) [0.388 to 1.426] was not significant. The repeatability was independent of PD₂₀ aspirin dose (r = 0.30, p = 0.43), [r = 0.41, p = 0.32]. The measure of repeatability was expressed according to the equation of Peat and colleagues (24) as fold differences (0.10 to 9.83) [0.27 to 3.68] (Figure 2).

View larger version (9K): [in this window] [in a new window]   View larger version (10K): [in this window] [in a new window]   Figure 2.   Repeatability values for nine subjects presented in two ways. The top figure shows values with the line of identity. The bottom figure demonstrates value differences plotted against the mean with the line of no difference.

At baseline, mean LTE₄ levels did not differ on placebo and salmeterol days. After placebo pretreatment, provocation with L-ASA caused a significant rise in urinary LTE₄ at 2 h (p = 0.03), 4 h (p = 0.01), and 6 h (p = 0.05; Wilcoxon Matched Pairs Test, Table 3) after challenge. In contrast, after salmeterol pretreatment mean LTE₄ did not increase significantly at any time after aspirin inhalation (p > 0.05). The greatest mean LTE₄ level on placebo at 4 h after L-ASA challenge was significantly higher than the corresponding level on salmeterol day (Table 3). At 4 h the urinary LTE₄ levels correlated significantly with the dose of inhaled aspirin (p = 0.04, r = 0.68, Sperman's Correlation Coefficient) on placebo day. This correlation vanished in the group pretreated with salmeterol. In either group there was no correlation between LTE₄ levels and the fall in FEV₁.

In six of nine patients L-ASA inhalation preceded by placebo produced a rise in PGD-M by 7-580% at 2 and/or 4 h after the challenge. Two other patients responded by a fall in PGD-M of 50% and 60%, and in one patient PGD-M levels remained unchanged. The mean changes in PGD-M levels after provocation did not reach statistical significance (p = 0.05, Wilcoxon Matched Pairs Test, Table 3) compared to baseline values. Noticeably, pretreatment with salmeterol blocked the increase in the urinary PGD-M in all 6 patients who demonstrated such a response after placebo pretreatment. The mean PGD-M levels were significantly lower following salmeterol administration as compared to placebo at 2 h (p = 0.008), and 4 h (p = 0.05, Wilcoxon Test). The changes in PGD-M levels did not correlate with either the changes in FEV₁ or aspirin PD₂₀.

Eosinophil counts decreased on both salmeterol and placebo day. The decrease was most evident at 4 and 6 h after challenge (Table 3). On placebo, the decrease reached statistical significance at 6 h after aspirin in comparison to baseline (p = 0.04, Wilcoxon Test). No correlation was observed between the maximum fall in FEV₁ and the fall in eosinophil blood counts. There was no correlation between eosinophil count and PD₂₀ dose of aspirin in patients pretreated with either salmeterol or placebo.

	DISCUSSION

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The study showed that pretreatment with a single dose of 50 µg of salmeterol effectively attenuates aspirin-precipitated bronchoconstriction. The statistical approach used by us, and also by other investigators (26), underestimates the full protective potential of salmeterol because the highest cumulative dose of aspirin failed to produce a 20% fall in the FEV₁ in six of 10 salmeterol pretreated patients. For the purpose of statistical analysis, the PD₂₀ dose was set as 182 mg. In Part II of the study pretreatment with salmeterol before aspirin challenge with a single PD₂₀ aspirin dose: (1) significantly inhibited the increase in urinary LTE₄, (2) abolished the PGD-M rise in those patients who demonstrated it after placebo, and (3) prevented a fall in blood eosinophil count, a phenomenon that may result from eosinophil recruitment into the respiratory tract after aspirin provocation (27).

Analysis of urinary excretion of LTE₄ and PGD-M, the products of the two major pathways of arachidonic acid metabolism in vivo, offers new insights into the potential mechanism of the protective action of salmeterol in AIA. Urinary LTE₄ levels, believed to reflect global cys-LTs production (18), increased significantly after aspirin challenge. As described previously (27), the intensity of LTE₄ response depends on the dose of aspirin. On the other hand, there was no correlation between the levels of LTE₄ and the fall in FEV₁. PGD-M is the major urinary metabolite of PGD₂. The urinary excretion of PGD-M increases early after specific allergen challenge in atopic asthmatics. However, inhibition of PGD₂ synthesis and PGD-M release with indomethacin does not alter allergen provoked bronchoconstriction in atopic patients (19). Urinary PGD-M release after aspirin challenge has not been studied in AIA. O'Sullivan and colleagues (28) recently reported a small, though statistically significant, increase in urinary excretion of another PGD₂ metabolite, 9, 11-PGF₂, after aspirin challenge in aspirin-intolerant asthmatics. In our patients, the PGD-M increase after aspirin was not consistent: some patients showed an increase of varying magnitude, others a fall, reminiscent of the changes in PGD₂ measured in BAL fluid of patients with AIA shortly after intrabronchial instillation of lysine-aspirin (11). It may be that simultaneous activation of cellular synthesis of PGD₂ by aspirin and simultaneous inhibition of cyclooxygenase by aspirin leads to a variable release of PGD₂, depending on the extent of cyclooxygenase inhibition in the cell(s) producing PGD₂.

Two mechanisms might explain the bronchoprotective action of salmeterol. First, long-acting stimulation of ₂-receptor by the drug powerfully relaxes smooth muscles and makes the bronchi less responsive to the constrictor stimuli. Second, inhibition of the production of cys-LT and, at least in some patients, bronchoconstrictor prostanoids such as PGD₂ could ameliorate bronchoconstriction. Inhibition of cys-LTs metabolism by salmeterol might be unique in asthmatics with aspirin intolerance and suggests that different mechanisms are responsible for the generation of lipid mediators in aspirin- and allergen-induced responses. In the study by Taylor and colleagues (29), salmeterol pretreatment prevented allergen precipitated bronchoconstriction, but this effect was interpreted as caused solely by smooth-muscle relaxation since the drug did not inhibit urinary LTE₄ release. It has been suggested that salmeterol might have some antiinflammatory properties, but the evidence is incomplete. For example, salmeterol was a potent inhibitor of antigen-induced release of histamine, LTC₄/ D₄/E₄, and PGD₂ from passively sensitized fragments of human lung in vitro (30), and attenuated chemotactic responses of human eosinophils, although it did not inhibit LTC₄ production by activated human eosinophils (31). Moreover, pretreatment with salmeterol decreased the levels of potent inflammatory cytokines in the BAL fluid during late-phase response to segmental antigen challenge in human atopic asthmatics (32). The observation that salmeterol suppresses eicosanoid release during aspirin provoked asthmatic reaction may suggest that the drug possesses some antiinflammatory effects in aspirin-induced asthma, although this surmise will require further studies and confirmation.