Twenty-four nonsmoking patients (10 men) with moderate, persistent asthma participated in the study. The inclusion criteria were age 18 to 60 years of age and a history of stable asthma as defined by the American Thoracic Society. Patient characteristics are summarized in Table 1. All patients had a baseline FEVj > 70% of predicted, FEV1 reversibility > 15%, and required regular treatment with moderate doses of inhaled corticosteroids (beclomethasone dipropionate, 800 ^g/d or equivalent) for > 3 months. All patients were hyperresponsive as measured by a provocative concentration of methacholine causing a 20% fall in FEV1 (PC20) < 8 mg/mL. None of the patients had a history of respiratory disease other than asthma, and none required oral glucocorticoid treatment within 3 months before study entry and used any other medication except study medication during the trial. The patients were included during a clinically well-controlled period without symptoms of an upper respiratory tract infection for 4 weeks prior to the study. The study was approved by the Royal Brompton and Harefield NHS Hospital Trust Ethics Committee, and informed consent was given by all patients. All patients suffering from asthma may order asthma inhalers online to treat asthma attacks.
In order to determine the onset of asthma exacerbation after corticosteroid withdrawal, the study was conducted in a randomized, double-blind, placebo-controlled manner by comparing the effects of 10 weeks of treatment with budesonide (400 μg bid or placebo via Turbuhaler; AstraZeneca; Lund, Sweden). The study periods consisted of a 2-week run in phase and a 10-week exacerbation-induction phase. Before the start of the study, patients were screened on 2 separate days. Long-acting P2-agonists and theophylline preparations were discontinued at least 72 h before the first screening visit. On the first day, FEV1 was measured before and after inhalation of 400 μg of albuterol by metered-dose inhaler with a large-volume spacer device, and on the second visit a methacholine challenge test was performed. The patients stopped regular asthma medication for 2 weeks. Thereafter, patients were randomized to receive either matched placebo or budesonide, 400 μg bid, via Turbuhaler. Patients who had exacerbations during the run-in period were excluded from the study. Patients visited the hospital at the end of the run-in period, at 2-week intervals during the exacerbation-induction period, and at the time of an exacerbation to undertake lung function testing and sputum induction. During the study, the patients kept a diary to record morning and evening peak expiratory flow measurements.
During the 2-week run-in phase, all patients had to have stable disease without lower respiratory tract infection. Throughout the study, only terbutaline via Turbuhaler was permitted on an on-demand basis as a rescue inhaler. A study physician was accessible by telephone 24 h/d.
Induction of Exacerbation
Steroid withdrawal was performed by inhalation of placebo, and the study was discontinued when an exacerbation developed or when no exacerbation of asthma occurred within the 10-week study period. Order ventolin and treat asthma attacks. Patients undergoing exacerbation during treatment period were withdrawn from the study. Thereafter, inhaled budesonide was increased to 800 |j,g bid, and systemic corticosteroids (prednisolone 30 mg/d) were administered.
An exacerbation was defined as at least one of three crite-ria: (1) a drop in morning peak flow > 20% below baseline (mean of the last 7 days run-in period) on 2 consecutive days; (2) wakening due to asthma on 2 consecutive nights, and requiring rescue medication; (3) > 50% increase in 24-h rescue medication use on at least 2 consecutive days compared to mean use during the last 7 days of the run-in period, which also exceeded the equivalent of four puffs of terbutaline. FEV1 was then measured within 24 h of the exacerbation.
Lung Function Measurements and Methacholine Challenge Tests
FEV1 and FVC were measured using a dry wedge spirometer (Vitalograph; Buckingham, UK). Values are expressed as percentage of predicted. Baseline values were measured after 15 min of rest and taken as the highest of three readings. Single readings only were taken at other times. Airway hyperresponsiveness to methachoHne was assessed using the method of Sterk and colleagues. Methacholine was inhaled by tidal breathing in doubling concentrations (0.015 to 32 mg/mL) for 2 min at 5-min intervals. Measurements of FEV1 were made at baseline and after each dose. The challenge test was discontinued if FEV1 dropped > 20% from baseline. PC20 was calculated by linear interpolation of the log-dose response curves.
Sputum Induction and Processing
Sputum was induced by inhalation for 15 min of 3.5% saline solution via an ultrasonic nebulizer (model 2000; DeVilbis; Heston, UK), as previously described. After each inhalation period, patients were asked to rinse their mouth and were encouraged to expectorate sputum. Briefly, the whole sputum sample was processed with dithiothreitol (Sigma Chemicals; Poole, UK). The homogenized sputum was centrifuged at 1,500g for 10 min. The supernatant was separated and frozen at — 70°C until further analysis. Total cell counts were made on a hemocy-tometer slide, using Kimura stain, and slides were prepared (Cytospin; Shandon; Runcorn, UK) and stained with May-Grun-wald-Giemsa stain. Differential cell counts were made by a blinded observer. Three hundred nonsquamous cells were counted on two slides for each sample in a blind way. Differential cell counts are expressed as percentages of nonsquamous cells. Two of the sputum samples from the same patient in budesonide treatment group at different visits were withdrawn from analysis because of containing > 80% squamous cells (Table 2).
The concentration of IL-8 in sputum supernatant was determined using commercially available enzyme-linked immunosorbent assay (R&D Systems; Minneapolis, MN) according to the manufacturer instructions.
Results are reported as mean ± SEM. We compared patients who lost control of asthma with those who did not after corticosteroid withdrawal. All statistical tests were two sided, and significance was accepted at the level of 95% and p < 0.05 using statistical software (GraphPad Prism; GraphPad Software; San Diego, CA). To determine whether changes in sputum neutrophil counts and sputum IL-8 levels accompanied asthma exacerbation, the differences of the last-visit measurements from baseline were used for analysis of correlation with Pearson product-moment technique. The data from subjects with and without exacerbation at each time point were categorized as a group before analysis. Sputum neutrophil counts, percentage of neutrophils, and IL-8 levels 2 weeks prior to an exacerbation were compared to those of the week-8 inflammatory markers in nonexacerbation group using the unpaired t test. The last-visit measurements of the exacerbation and nonexacerbation groups were also evaluated by the unpaired t test.
Table 1—Patient Characteristics
|Age, yr||36.9 ± 2.5||38.3 ± 3.1|
|Male/female gender, No.||4/8||5/7|
|Postbronchodilator FEVj, L||3.87 ± 0.17||3.75 ± 0.1|
|Reversibilty, %||20.4 ± 1.0||20.7 ± 1.2|
|PC20, mg/mL||0.98 ± 0.26||0.99 ± 0.27|
Table 2—Total and Specific Sputum Cell Counts After Budesonide
|Variables||Weeks of Budesonide Treatment|
|FEVj, % predicted Exacerbation, No.||72.3 ± 1.5||73.4 ± 2.1 1||71.8 ± 0.9||73.7 ± 0.6||73.2 ± 1.1||72.8 ± 1.7|
|Total cells, 106/mL||3.4 ± 0.3||3.2 ± 0.3||3.6 ± 0.2||3.2 ± 0.2||3.5 ± 0.4||3.6 ± 0.6|
|Neutrophils, %||33.9 ± 3.7||41.3 ± 4.1||38.2 ± 2.1||36.4 ± 5.2||37.7 ± 3.8||38.4 ± 4.5|
|Neutrophils, 106/mL||1.2 ± 0.6||1.3 ± 0.5||1.4 ± 0.4||1.2 ± 0.5||1.3 ± 0.6||1.3 ± 0.7|
|Eosinophils, %||4.8 ± 1.0||2.6 ± 0.8||1.8 ± 0.6||1.2 ± 0.5||0.45 ± 0.2||0.3 ± 0.1|
|Eosinophils, 106/mL||0.2 ± 0.03||0.09 ± 0.02||0.06 ± 0.03||0.04 ± 0.01||0.01 ± 0.004||0.02 ± 0.01|
|Macrophages, %||57.9 ± 3.4||53.3 ± 3.9||57.6 ± 2.7||60 ± 5.5||59.7 ± 4.8||59.5 ± 4.9|
|Macrophages, 106/mL||1.9 ± 0.2||1.7 ± 0.2||2.1 ± 0.2||2.0 ± 0.2||2.1 ± 0.9||2.2 ± 0.4|
|Lymphocytes, %||3.3 ± 0.9||2.8 ± 0.7||2.5 ± 0.7||2.4 ± 0.6||2.1 ± 1||2.0 ± 0.7|
|Lymphocytes, 106/mL||0.1 ± 0.01||0.1 ± 0.01||0.1 ± 0.02||0.07 ± 0.02||0.08 ± 0.03||0.1 ± 0.03|