Online Data Supplement
Effect of tiotropium on sputum and serum inflammatory markers and exacerbations in chronic obstructive pulmonary disease
Duncan J Powrie, Tom M Wilkinson, Gavin C Donaldson, Peter Jones, Kenneth Scrine, Klaus Viel, Steven Kesten, Jadwiga A Wedzicha
Methods
Study design
This was a one year, single centre, randomised, placebo controlled study to assess the effect of tiotropium on sputum inflammatory markers and exacerbation frequency. Ethics approval was obtained from East London and the City Research Ethics Committee, the trial was registered with ClinicalTrials.gov and all patients gave written informed consent. The primary endpoint was the concentration of interleukin-6 (IL-6) in sputum. Secondary end points included sputum interleukin-8 (IL-8) and myeloperoxidase (MPO), serum IL-6 and C-reactive protein (CRP), FEV1 and exacerbation frequency as assessed by our validated daily diary cards [1,2].
Patient selection
Patients aged ≥ 40 years with a diagnosis of COPD (FEV1 <80% predicted and FEV1 /FVC less than 70%) were recruited from primary care and the outpatients department of the London Chest Hospital between November 2002 and January 2004. Patients were required to be stable, that is, free from exacerbations in the preceding four weeks and have a minimum 10 pack year smoking history. Patients with a history of asthma or atopy were excluded, as were those on long term oxygen therapy or those with a clinically significant disease other than COPD (defined as one which in the opinion of the investigators may either put the patient at risk because of participation in the study or influence the results of the study or the patient’s ability to participate in the study). Patients on β-blockers were excluded as were those on oral β-agonists or nedocromil sodium. Anticholinergics other than the study drug were not allowed during the course of the study. Short and long acting inhaled β- agonists and inhaled steroids were permitted.
Study Protocol
Patients underwent a screening visit at which their eligibility was assessed. Physical examination was performed and concomitant drug therapy was documented. Smoking status was verified by urinary cotinine. Patients performed spirometry (Micro Medical, Chatham, UK) and provided sputum for assessment of sputum inflammatory markers and bacteriology. They also provided a serum sample for measurement of IL-6 and CRP. Following screening, eligible patients underwent a 2 week run-in period and following stratification according to smoking status and exacerbation history (<3 or >=3 exacerbations in the preceding 12 months) were randomised to tiotropium 18mcg once daily via the HandiHaler device or placebo. They were provided with a diary for recording of daily symptoms, morning peak flow and compliance with the study drug.
Patients were followed up for one year and seen at weeks 4, 16, 32 and 52 after randomisation by a doctor or nurse blinded to the intervention. Patients were seen in the morning prior to taking their inhaled medication. At each visit, patient diaries were examined to determine exacerbation frequency and adverse events were recorded. Patients produced a spontaneous or induced sputum sample for analysis and spirometry was carried out. Patients provided serum samples at weeks 32 and 52 and were asked to subjectively report any change in sputum volume on a simple scale of reduced, increased or unchanged. Patients experiencing an exacerbation in the preceding four weeks were not sampled and their visit deferred for four weeks post the onset of the exacerbation.
Sputum and Blood Sampling
Immediately following lung function measurement, stable patients were asked to spontaneously expectorate sputum into a sterile container. Patients unable to produce a sample of sputum spontaneously underwent sputum induction [3]. Once a sample was obtained sputum plugs were separated from saliva using sterile forceps and one third of the sputum taken and analyzed for quantitative bacterial culture [4,5], the remainder was homogenized, centrifuged, and aliquots of supernatant stored at -700C for later cytokine analysis [6]. Sputum samples containing <25 squamous epithelial cells per low powered field and >25 leukocytes per high powered were accepted for analysis. Sputum IL6, IL8 and MPO levels were measured using ELISA (R&D Systems Abingdon, UK). Contemporaneous blood samples were centrifuged at 40C and serum decanted and stored at -800C for subsequent analysis of IL6 levels using ELISA (R&D Systems Abingdon, UK). CRP assays were carried out by Pivotal Laboratories (York, UK).
Bacteriological analysis
For bacteriological analysis, tenfold serial dilutions of the homogenized sample were made in brain heart infusion broth and 100 µl aliquots were plated out onto the surface of a range of different media, incubated, counted and sub-cultured as previously described [4,5]. The number of colony forming units/ml sputum was calculated from the number of colonies obtained and the dilution of the sputum, expressed in colony forming units per ml (cfu ml-1). Bacteriology data are expressed as the total bacterial count in log base ten units. Potentially pathogenic micro-organisms are bacteria known to be common pathogens of the respiratory tract in subjects with COPD (Streptococcus pneumoniae, Haemophilus influenzae, Haemophilus parainfluenzae Moraxella catarhalis, Staphylococcus aureus, Pseudomonas aeruginosa and other gram negative enteric bacteria).
Exacerbations
At recruitment patients were taught how to record on diary cards each morning post- bronchodilator peak expiratory flow (PEF) (Mini-Wright Clement Clark International Ltd, Harlow, UK). Patients recorded a change in their symptoms using a letter-annotated system. When well or stable the patients were instructed not to record any of the symptom letters on the diary. However, when they perceived an increase over their normal, stable condition in symptoms; (major and minor, see below); they noted the corresponding symptom letter on their diary card. Therefore patients recorded symptom letters if a symptom was perceived as worse eg dyspnoea, or of new onset eg sore throat (as the latter is not usually present).
Patients were encouraged to report symptom changes to the study team; they were assessed within 24-48 hours in the study clinic by a respiratory physician prior to initiation of therapy for the exacerbation. The diagnosis of an exacerbation was based on symptomatic criteria previously validated by our group [1,2,7]. An exacerbation was defined as the presence for at least two consecutive days of increase in any two “major” symptoms (dyspnoea, sputum purulence, sputum amount) or increase in one “major” and one “minor” symptom (wheeze, sore throat, cough, symptoms of a common cold). Therapy was initiated by DP according to clinical circumstances and was usually amoxicillin or amoxicillin/clavulinic acid plus a one week course of prednisolone 30mgs.
Statistics
Analyses were carried out using the full analysis data set (all randomized, treated patients with efficacy data) using an analysis of covariance that adjusted for smoking status and exacerbation history over the previous year (<3 or >=3) recorded at recruitment. For sputum markers, the area under the curve (AUC) was calculated for each patient with missing data replaced by interpolation or the last observation carried forward. The model also included baseline inflammatory marker levels as a covariate. AUCs for IL-6 and MPO were skewed and therefore log10 transformed. For lung function, comparisons were between changes from start and end of the study. The pre-specified data analysis with AUCs was chosen for its ability to capture and combine any short or long-term effect of tiotropium. An additional analysis was also carried out using cross-sectional regression models (xtreg command in Stata 8). Systemic inflammatory markers were not sampled at weeks 4 and 16, so comparisons were made by Wilcoxon sign-rank test between changes from baseline to final sample.
The effect of tiotropium on individual annual rates (= the number of exacerbation divided by days on drug * 365) was tested using a Wilcoxon test. Differences in time to the next exacerbation were examined using a log-rank test.
Exacerbation recovery times were calculated as the time for a 3 day moving average of peak flow to return post-exacerbation to a baseline taken as the average on days 14 to 8 prior to exacerbation onset.
The statistical analysis was performed with SAS 8.2 and Stata 8.
References
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