Methods
Study Design
An international, 16-week, randomized, double-blind, placebo-controlled, parallel group study (Figure 1) investigating the effect of roflumilast 500 μg once daily versus placebo on inflammation parameters in bronchial biopsy tissue specimens, sputum and blood serum (clinical trial identifier: NCT01509677). Patients will be equally randomized to either roflumilast treatment or placebo in a 1:1 ratio by means of a computerized central randomization system IVRS/IWRS. The system will assign one or two appropriate trial treatment kit(s) from the stock available at the site for each patient. The primary endpoint of the study will be the number of CD8+ cells (cell counts per mm) in bronchial biopsy tissue specimens (sub-mucosa) evaluated from randomization to the end of the intervention period. The key secondary endpoint will be the number of CD68+ cells (cell counts per mm), assessed over the same timeframe, but a host of other secondary outcomes will also be assessed (Table 1). The study will be conducted at 11 European sites specializing in lung diseases.
(Enlarge Image)
Figure 1.
Schematic design of the clinical study.
Ethical Considerations
This trial will be conducted in accordance with the Declaration of Helsinki, Good Clinical Practice (GCP) guidelines and any additional local regulations. Ethical approval has been gained from the NRES Committee East of England - Cambridge South, UK; Regionala etikprövningsnämnden i Lund, Sweden; Komisja Bioetyczna UJ, Poland; Ethik-Kommission der Ärztekammer Schleswig-Holstein (Ethik-Kommission I), Germany as well as the UK, Swedish, Polish and German regulatory agencies.
Patient Population
Patients with a history of COPD for at least 12 months, associated with chronic productive cough for at least three months, in each of the two years prior to baseline visit will be recruited into the study. Patients with moderate-to-severe COPD (stages II and III) will be enrolled (according to GOLD 2009). Patients with a history of a recent exacerbation (within six months prior to baseline) will be excluded as will patients who have had a respiratory tract infection, which has not resolved at least four weeks before baseline. Standard bronchodilator therapy use will be permitted throughout the study. However, concomitant medications, including glucocorticoids (inhaled corticosteroids [ICSs]), oral steroids), long-acting β2-agonist (LABA)/ICS combinations, theophylline, lipoxygenase inhibitors, anti-platelet therapy and leukotriene antagonists will not be permitted throughout the trial and will be withdrawn at the start of the run-in period. Bronchodilators starting at least 6 weeks prior to run-in will be allowed, although these treatments must remain stable throughout the study. Other drugs for the treatment of concurrent disease will be permitted, but their doses must also be kept constant throughout study (including the run-in period). In addition to meeting the admission criteria (Table 2), patients must also satisfy the following conditions in order to be eligible for randomization into the double-blind treatment period:
No COPD exacerbation between baseline and randomization (as defined by treatment and/or hospitalization)
Tablet compliance ≥ 80% and ≤ 125%
Information regarding patients' COPD severity (stage II versus stage III), smoking status, concomitant LABA use and previous inhaled corticosteroid usage will be recorded for the purpose of sub-group analyses and stratification of the most important confounding variables. The number of enrolled patients will be capped at 150 in total.
Roflumilast and placebo tablets will be of identical appearance, shape and colour and will have identical labelling and packaging.
Sample Size
The sample size of 150 has been calculated from the available information on the primary endpoint and has been kept as small as possible. Previous trials have shown that drop-out rates in bronchial biopsy studies may be as high as 30% from enrolled patients; therefore, if a conservative estimate of 30% is applied, the present study may have 105 patients who complete the trial. Drop-outs are difficult to handle in bronchial biopsy studies. If drop-outs are excluded this could unfairly bias the trial, however, if drop-outs continue in the study after having treatment with antibiotics and systemic corticosteroids, this could also bias the results. It is therefore important to choose a group of patients who are unlikely to exacerbate or drop out during the course of the trial, which is the case in our study design. A universally recognized level of clinical relevance regarding the primary endpoint (sub-mucosal CD8+ cells) has not yet been agreed on within the scientific community, but a 30% improvement using roflumilast treatment over placebo may be of clinical relevance. We have calculated that with a dispersion of 25, tissue area of 0.3 mm and further assumptions (1:1 randomization, two-sided α = 0.05, power = 0.90, event rate on roflumilast = 200 cells/mm, event rate on placebo = 285 cells/mm), the trial would have a high power (approximately 90%) to detect treatment differences. However, the study will not be statistically powered to investigate any outcomes with regards to the effectiveness of COPD treatment.
Technical Aspects
Bronchoscopy. Bronchoscopies will be performed in line with the American Thoracic Society (ATS) guidelines, Endobronchial Biopsy Workshop, modified protocol of O'Shaughnessy et al. and according to local clinical standards of care. Endobronchial biopsies will be taken from each lobar and sub-segmental carina using Olympus EndoJaw single-patient use biopsy cut forceps. In order to take into account both inter and intra-patient variability, 2–3 biopsies will be taken from the lobar bronchus and 2–3 from the sub-segmental airways, at each bronchoscopy session (randomization and end of treatment period). The left and right lobes will be alternated between subjects, but all biopsies will be harvested from the same lung during any given session. The second set of biopsies (after the treatment period) will be taken from the same airway level, but from a different specific site.
In selected patients, three protected brush specimens will be collected during the bronchoscopy procedures. If performed, these specimens will be collected prior to the bronchial biopsy procedure at the same part of the lower lobe bronchus. The specimens will be used to evaluate longitudinal changes in COPD airway microbiota in placebo-treated patients and to define the effect of roflumilast therapy on the airway microbiome.
Biopsy Sample Processing, Cell Quantification and Biopsy Quality. Biopsies will be gently extracted and sent to the site's laboratory for further processing (fixation and paraffin wax embedding). Immunostaining and quantification of inflammatory cells will be performed according to standard procedures. Briefly, inflammatory cells will be identified using indirect immunohistochemistry (using the peroxidase-antiperoxidase complex-PAP- and diaminobenzidine as substrate or the alkaline phosphatase-anti-alkaline phosphatase complex; APAAP and Fast Red). For each antibody, the total number of positively stained cells will be counted to a depth of 100 μm below the epithelial basement membrane using a computerized image analysis.
To ensure adequate quality and consistency between investigators at different sites, centralized training, covering all aspects of material collection, handling and processing, will be provided. The quality of biopsy material will also be validated for each site, by requesting that sites provide pseudonymized samples from the first three enrolled patients. In order to be considered a good quality sample, the biopsied tissue area must be ≥0.1 mm, contain ≥1 mm of basement membrane and be ≥100 μm deep. Only after the biopsy samples are considered to be of sufficiently good quality will the site be allowed to recruit further patients into the trial.
Inflammatory Biomarkers in Induced Sputum. Sputum will be induced, collected and initially processed at investigational sites. The quality of sputum samples will also be estimated by investigators on a scale from one to six. Total and differential cell counts of neutrophils, macrophages, eosinophils and lymphocytes will be performed and inflammatory biomarkers will be analyzed using the 46-biomarker Multi-Analyte Profiling (MAP) technology (Human InflammationMAP® v 1.0; Myriad RBM). This tool contains quantitative, multiplexed immunoassays for 46 biomarkers, but the ones of primary interest with regards to this study and roflumilast will be: alpha-2 macroglobulin, interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), matrix metalloproteinase type 9 (MMP-9), tissue inhibitor of metalloproteinase (TIMP) and vascular endothelial growth factor (VEGF). The remaining 40 biomarkers will only be analyzed exploratively (Table 1). To ensure adequate quality and consistency of samples, centralized hands-on training will be provided and samples will be assessed on an ongoing basis.
Inflammatory Biomarkers in Blood Serum. Blood withdrawal for the measurement of inflammatory biomarkers will be performed at approximately the same time each day (±2 hours), but no later than 10:00 am at each respective visit. Inflammatory biomarkers will be quantified using MAP technology (Human InflammationMAP® v 1.0; Myriad RBM).
Blood serum, sputum and biopsy samples will be collected and appropriately stored for up to three years after the end of the study to allow for future analyses of biomarkers of scientific interest.
Pulmonary Function Tests. Spirometry will be performed according to the recommendation of the ATS – European Respiratory Society (ATS/ERS) consensus guidelines on pulmonary function testing. Sites will use their own devices, performing maintenance and calibration of instruments according to usual standards of practice. FEV1 (absolute and percentage predicted values), forced vital capacity (FVC) (absolute values) and the ratio of FEV1/FVC will also be recorded.
Safety. Following bronchoscopy with bronchial biopsy, patients will be closely monitored for at least two hours and will only be discharged when the effects of sedation and local anaesthesia disappear as judged by the investigator. All patients will be provided with a 24-hour emergency contact number and a safety follow-up visit will be performed within two weeks after each bronchoscopy session.
Statistical and Analytical Plans. The intention-to-treat (ITT) analysis will be based on the full analysis set (FAS). It will be the primary analysis for this study and will be performed for all primary and secondary endpoints. The primary endpoint relates to pulmonary inflammation expressed as CD8+ cell counts per mm in sub-mucosal bronchial biopsy tissue specimens measured before and after the double-blind treatment period. The key secondary endpoint relates to CD68+ cell counts per mm in sub-mucosal bronchial biopsy tissue specimens. Roflumilast/placebo comparisons for these endpoints will be performed via a multiple test procedure such that the family-wise error rate of 5% is controlled in the strong sense. The two null hypotheses are: equal CD8+ counts/mm and equal CD68+ counts/mm for roflumilast and placebo. These null hypotheses will be ordered, so that the CD8+ comparison comes first and the CD68+ comparison comes second. If the comparison with respect to CD8+ is significant at the nominal level α = 5%, the corresponding null hypothesis will be rejected and the CD68+ comparison will be performed in a confirmatory way (otherwise confirmatory testing stops). This will again be done at nominal level α = 5%. If significant (after a significant result in the first comparison), the corresponding null hypothesis will be rejected. If the first comparison is not significant at 5% level, then neither null hypothesis must be rejected. The component tests of the multiple test on CD8+ and CD68+ will be based on Poisson regression models with CD8+ (CD68+) at end of treatment period as a dependent variable and treatment and baseline values of the respective dependent variable as covariates. A dispersion parameter and an offset (equivalent to the bronchoscopy sampling area) will be taken into account.
Analyses of the other secondary endpoints will be descriptive on treatment and visit, by ANCOVA on absolute change from baseline to last available measurement during double-blind treatment for continuous variables, or Poisson regression for count data. In addition, analyses will be performed in subgroups stratified by COPD stage, smoking status, concomitant LABA and former ICS use.