METHODS

Subjects

Eighteen subjects (seven men) of mean (SD) age 26.1 (8.4) years participated in the study (table 1). They were non-smokers with mild to moderate persistent asthma and were atopic as defined by positive skin prick tests (>3 mm wheal response) to one or more common aeroallergens (grass pollen, tree pollen, cat fur, dog hair, and Dermatophagoides pteronyssinus (Bayer Corporation, Elkhart, USA). Subjects had a baseline forced expiratory volume in 1 second (FEV₁) ≥70% of their predicted values (or >1.5 l) and were only using short acting inhaled β₂ adrenoreceptor agonists. None of the subjects was taking oral or inhaled corticosteroids or theophylline. Bronchodilators were withheld for 8 hours before each visit to the laboratory. No subject was studied within 4 weeks of an upper respiratory tract infection or exacerbation of asthma.

Subjects gave written informed consent and the study was approved by the Southampton and South West Hampshire local research ethics committee.

Study design

The study was of a randomised, double blind, placebo controlled, crossover design. With the sample size of 18, it was designed to have 80% power (p=0.05, two sided test) to detect a threefold increase in the provocative concentration of inhaled AMP needed to decrease FEV₁ by 20% of baseline (PC₂₀AMP). This was based on the PC₂₀ being log₁₀ normally distributed and within subject variance in log PC₂₀ of 0.56.¹³

Each subject attended the laboratory on five occasions over a period of 4 weeks. At the first visit a medical and asthma history was taken, subjects underwent a full physical examination, and an electrocardiogram was performed. Before enrolment, a histamine bronchial challenge test was performed as a standard research screening procedure to establish baseline bronchial reactivity and therefore eligibility into the study. Subjects were included with a histamine PC₂₀ of ≤ 16 mg/ml. Within 1 week of the screening visit they attended the department for an AMP inhalation bronchial provocation test, and were required to demonstrate a 20% fall in FEV₁ after an AMP challenge to be eligible for the study. Urine was collected 4 hours after the AMP challenge. At the end of visit 2, subjects were randomly allocated the first 10 mg dose of montelukast (Singulair; Merck Sharp & Dohme Ltd, Hertfordshire, UK) or matched placebo orally. Visit 3 took place on the following day when the subject receiving the second dose of montelukast (10 mg) or placebo in the morning. An AMP challenge was then performed 4 hours after dosing and urine was collected 4 hours later. Serial FEV₁ measurements were measured every 15 minutes for 1 hour after the AMP challenge. Subjects were crossed over after a washout period of 14 days and received either montelukast or placebo. All tests performed initially (AMP challenge, urinary LTE₄ assay) were repeated.

Bronchial provocation

Pulmonary function was measured before and during the provocation using a dry wedge spirometer (Vitalograph Ltd, Buckinghamshire, UK). On each challenge day histamine acid phosphate (BCN Chemicals Inc, Beaconsfield, Quebec, Canada) and AMP (Sigma Chemical Co, Poole, Dorset, UK ) were made up freshly in 0.9% (w/v) sodium chloride solution to produce a range of increasing doubling concentrations from 0.03 to 32 mg/ml (0.1–104 mmol/l) and from 0.39 to 400 mg/ml (4.48–1151.6 mmol/l), respectively. The solutions were administered using a dosimeter (SPIRA Electro 2 inhalation dosimeter; Spira Resp Care Center Ltd, Helsinki, Finland). Wearing a nose clip, subjects inhaled the aerosolised solutions in five breaths from end tidal volume to full inspiratory capacity via a mouthpiece. Subjects were trained to take 3 seconds to reach full inspiratory capacity. All subjects were challenged using the same spirometer and dosimeter, which was calibrated before each patient use.

Baseline FEV₁ was performed at the beginning of each challenge. After inhalation of 0.9% sodium chloride, repeated measurements of FEV₁ at 1 and 3 minutes were noted, the higher value being recorded. If FEV₁ did not fall by >10% of the baseline value, the bronchoprovocation challenge with histamine (screening) or AMP (study) was performed. Increasing doubling concentrations of the stimulant (histamine or AMP) were inhaled at 5 minute intervals until FEV₁ had fallen by >20% of the post saline value or the highest concentration of the stimulant had been administered.

Urine collection for LTE₄ quantification

Urine was collected in separate containers without preservatives 4 hours after the AMP challenge at visit 2 and 4 hours after the AMP challenge (8 hours after dosing) at visit 3. The same intervals were used during the crossover phase. Aliquots (30 ml) of each sample were stored at –20°C and assayed after a 5 month period. Urinary LTE₄ levels were measured using a Biotrak leukotriene C₄/D₄/E₄ enzyme immunoassay (EIA) system (Amersham Pharmacia Biotech UK Ltd, Amersham, Buckinghamshire, UK) as described and validated by Kumlin et al.¹⁴ Creatinine was measured in all urinary samples and the results expressed as pmol excreted LTE₄/mmol creatinine.

Data analysis

Data were analysed using spss version 10 (SPSS, Chicago, IL, USA). The percentage decrease in FEV₁ was plotted against the cumulative concentration of AMP inhaled on a logarithmic scale and the concentration of agonist producing a 20% fall from the post saline value (PC₂₀) was determined by linear interpolation between the last two concentrations. Bronchoconstriction induced by AMP was expressed as concentration-response curves which were constructed as the logarithm of the concentration nebulised against the percentage change in FEV_1. Least squares regression was performed on % FEV₁ versus concentration, with treatment and concentration/treatment interaction terms. The interaction term was tested to assess parallelism of the response curves.

Both PC₂₀ and urinary LTE₄ levels were assumed to be log normally distributed. Analysis of covariance (ANCOVA), adjusting for subject, period and baseline, was used to estimate treatment effects on the log transformed data.¹⁵ The montelukast/placebo concentration ratio was calculated by dividing the PC₂₀AMP value obtained after administration of montelukast by that obtained after placebo. The geometric mean and 95% confidence intervals of the montelukast/placebo concentration ratio were calculated by transforming back to the original measurement scale.

RESULTS

There were no significant differences in baseline or post saline FEV₁ values on any of the study days. As part of the inclusion criteria, all subjects exhibited bronchial hyperreactivity to inhaled histamine with a baseline geometric mean PC₂₀ histamine of 2.0 mg/ml (range 0.29–8.82 mg/ml; table 1). All patients in the study showed bronchial reactivity to AMP at baseline. From the patients enrolled, the FEV₁ failed to fall by 20% after the placebo arm and after treatment with montelukast in one patient. This was different from this subject's baseline AMP challenge which showed a PC₂₀AMP. This difference compared with baseline can probably be explained by the expected variability of the bronchial challenge procedure. Two subjects failed to achieve a PC₂₀ after montelukast treatment. This differed from their baseline AMP challenge where a PC₂₀ was recorded, illustrating the protective effect of montelukast in some patients. Subjects who failed to show a 20% fall in FEV₁ were assigned a PC₂₀ of 1600 mg/ml (twice the maximum cumulative concentration).

The slopes of the concentration-response curves did not depart significantly from parallel (p>0.05). There was a displacement to the right of the AMP concentration-response curve in 13 of the 18 subjects (fig 1). The geometric mean (range) concentration of AMP required to produce a 20% decrease in FEV₁ PC₂₀ (PC₂₀AMP) was 51.6 mg/ml (range 8.42–1600) after placebo and 126.5 mg/ml (range 13.31–1600) after montelukast (fig 2A). Thirteen subjects had an increased PC₂₀AMP after montelukast (montelukast/placebo concentration ratio >1) while five subjects had little or no change in PC₂₀AMP. Adjusting for period and baseline effects, the treatment effect of montelukast remained statistically significant (p=0.006). The estimated geometric mean and 95% confidence interval for placebo and treatment were 52.6 mg/ml (35.2 to 78.7) and 123.9 mg/ml (83.0 to 185.0), respectively. The geometric mean of the montelukast/placebo concentration ratio was 2.4 (95% CI 1.3 to 4.2). The analysis was repeated omitting the three subjects who failed to show a decrease in FEV₁ of at least 20% from the post-saline FEV₁ after treatment with placebo or montelukast. Using ANCOVA, the treatment effect of montelukast remained statistically significant (p=0.004), with an estimated geometric mean of 42.7 mg/ml (95% CI 32.8 to 55.5) and 82.0 mg/ml (95% CI 60.4 to 111.2) for placebo and montelukast, respectively. The geometric mean of the montelukast/placebo concentration ratio was 1.9 (95% CI 1.3 to 2.9).

Complete data for evaluation of urinary LTE₄ levels were available in 14 of the 18 subjects. The geometric mean urinary LTE₄ level was 210.4 pmol/mmol creatinine (95% CI 168.9 to 268.4) after placebo and 230.9 pmol/mmol creatinine (95% CI 201.7 to 264.4) after montelukast (fig 2B).

The ratio of urinary LTE₄ levels after montelukast to after placebo was 0.9 (95% CI 0.7 to 1.2), p=0.566 (ANCOVA).

DISCUSSION

Consistent with previous studies, we have shown that inhaled AMP induces bronchoconstriction in patients with asthma.^16,17 We have further shown that the selective CysLT₁ receptor antagonist montelukast is a potent but partial antagonist of AMP induced bronchoconstriction, and that montelukast was not associated with a significant reduction in the mean post challenge urinary LTE₄ levels. The ability of montelukast to inhibit adenosine induced bronchoconstriction through CysLT₁ receptor blockade thus definitively establishes that cysteinyl leukotrienes contribute as mediators of adenosine induced bronchoconstriction.

Although the exact mechanism of bronchoconstriction produced by inhaled adenosine in asthma is not known, previous studies in vitro have shown that adenosine potentiates the release of both preformed and newly synthesised mediators of inflammation from human lung mast cells¹⁸ through stimulation of A2_B receptors and activation of the phosphatidylinositol cascade.^8,19 The contribution of histamine has been shown using the selective histamine H₁ receptor antagonists terfenadine, astemizole, and loratadine,²⁰ and strongly supports the idea that inhaled AMP potentiates the release of preformed histamine from immunologically primed mast cells in asthmatic airways.²¹

The contribution of cysteinyl leukotrienes to bronchoconstrictor responses of AMP is supported by the inhibitory effects of the cysteinyl leukotriene receptor antagonist ICI 198,615 and the FLAP (five lipoxygenase activating protein) inhibitor MK-886 against adenosine provoked contractile responses of asthmatic airways in vitro,¹¹ which suggests that adenosine acts indirectly by liberation of leukotrienes possibly from mast cells. A contribution of eicosanoids in vivo is indicated by the attenuation of adenosine induced bronchoconstriction in patients treated with selective inhibitors of cyclooxygenase²² and the experimental 5-LO inhibitor ABT-761.¹² Eicosanoids generated by human lung mast cells include PGD₂, LTB₄, and cysteinyl leukotrienes. The availability of potent and selective CysLT₁ antagonists has enabled us to define for the first time the specific contribution of cysteinyl leukotrienes to bronchoconstriction provoked by AMP.

In the present study montelukast produced a 2.9 fold increase in the concentration of AMP required for an equivalent fall in FEV₁ after placebo. Three of the 18 subjects failed to show a decrease of 20% in their FEV₁ from the post saline value after the maximum concentration of AMP was administered. Although a censored value of 1600 mg/ml was assigned to these subjects, when omitted from the analysis the treatment effect of montelukast still revealed a twofold protection against the bronchoconstrictor effect of AMP which remained highly significant (p=0.004).

The magnitude of the protection observed in vivo is considerably less than the 16-fold protection afforded by histamine H₁ receptor antagonism.²³ This difference in the relative inhibitory capacity of antagonistic inhibitors emphasises the central role that histamine plays in adenosine induced bronchoconstriction and emphasises that, while augmenting mast cell mediator release, stimulation of A2_B receptors preferentially augments degranulation with release of preformed mediators rather than stimulating the production of newly generated mediators.²¹ Although histamine would seem to be central to the adenosine induced bronchoconstriction response in most of our subjects, three demonstrated a predominantly leukotriene driven adenosine response as illustrated by a failure to achieve a PC₂₀ after the maximum concentration of AMP (400 mg/ml) was inhaled following montelukast (fig 1). This adds to the view that, within the asthmatic population, there is substantial heterogeneity among individuals in their therapeutic response to drugs that selectively interfere with mediator pathways. The reason(s) for such heterogeneity in the relative contribution of any one mediator to bronchoconstriction produced by an indirect stimulus is not known. In the case of leukotrienes, one possibility is promoter polymorphism as indicated in the 5-LO²⁴ and leukotriene C₄ synthase genes.²⁵

Urinary excretion of LTE₄ has often been used as a marker of cysteinyl leukotriene production in the airways. LTE4 concentrations in urine are significantly increased in response to allergen challenge,²⁶ aspirin challenge in aspirin sensitive asthmatic subjects,²⁷ and during spontaneous exacerbations of asthma.²⁶ In the present study pretreatment with montelukast was not associated with a significant reduction in the mean post challenge urinary LTE₄ levels when compared with placebo, in contrast to the effects of the 5-LO inhibitor ABT-761,¹² consistent with the different modes of action of these drugs.

In conclusion, our results confirm that selective CysLT₁ receptor antagonism produces a small but highly significant protection against AMP induced bronchoconstriction in subjects with atopic asthma, indicating that cysteinyl leukotrienes are generated by this indirect challenge probably from airway mast cells through preferential activation of their A2_B receptors. While the contribution of cysteinyl leukotrienes to the adenosine induced bronchoconstriction response is likely to be smaller than that of histamine overall, we have shown that cysteinyl leukotrienes play a markedly more pronounced role in a minority of subjects. These findings constitute the basis for further clinical studies to investigate whether the adenosine bronchoconstrictor response can be completely attenuated by a combination of histamine H₁ receptor and selective CysLT₁ receptor blockade as described in vitro.¹¹