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 interleukin6 (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 IL6 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 microorganisms 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 postbronchodilator peak expiratory flow (PEF) (Mini-Wright Clement Clark International
Ltd, Harlow, UK). Patients recorded a change in their symptoms using a letterannotated 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 prespecified 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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