The Effects of Fluticasone With or Without Salmeterol On Systemic

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1 The Effects of Fluticasone With or Without Salmeterol On Systemic Biomarkers of Inflammation in COPD Don D. Sin, S. F. Paul Man, Darcy D. Marciniuk, Gordon Ford, Mark FitzGerald, Eric Wong, Ernest York, Rajesh R. Mainra, Warren Ramesh, Lyle S. Melenka, Eric Wilde, Robert L. Cowie; Dave Williams; Wen Q. Gan, Roxanne Rousseau on behalf of the ABC (Advair, Biomarkers in COPD) Investigators 1

2 Methods ONLINE DATA SUPPLEMENT Rationale For The Measurements The details of the trial design are described elsewhere (E1). In brief, this trial was a double blind randomized controlled trial (RCT) comparing the effects of inhaled fluticasone alone or in combination with salmeterol against placebo on three circulating biomarkers. The primary end point was C-reactive protein (CRP). Secondary molecules of interest were interleukin-6 (IL-6) and surfactant protein D (SPD). We limited our initial assessment to CRP, IL-6 and SPD in order to mitigate Type 1 error. Other secondary molecules such as IL-8, tumor necrosis factor-α, monocyte chemoattractant protein and other molecules will be measured at a later date as part of a post hoc exploratory work. CRP was chosen because it is a robust (but non-specific) marker of systemic inflammation and has been associated with clinical outcomes such as hospitalization and mortality in stable COPD patients (E2, E3). IL-6 was chosen because it has been associated with adverse clinical outcomes in the general community (E4) and is the main cytokine regulator of CRP production in the liver (E5). Although both CRP and IL-6 are generally accepted biomarkers of systemic inflammation, their use in COPD may be limited because they are produced predominantly in non-pulmonary organs and therefore lack specificity for COPD outcomes. SPD, on the other hand, is mainly secreted by alveolar type II pneumocytes and its circulating levels increase with lung injury (E6). The SPD measurements were added later to the protocol owing to the growing recognition of this molecule in the pathogenesis of COPD (E7) and its lungspecificity of expression and synthesis (E8). Elevated circulating levels of SPD have also been associated with poor outcomes including death in patients with lung injury (E9). Thus, SPD was chosen to evaluate the effects of the drugs on lung inflammation and injury. Other secondary measures included health status as assessed by the St. 2

3 George s Respiratory Questionnaire (SGRQ), and forced expiratory volume in one second (FEV 1 ). Study Participants All participants had a clinical diagnosis of COPD as defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines (E10). Spirometric criteria included a forced expiratory volume in one second (FEV 1 ) of less than 80% of predicted with an FEV 1 to forced vital capacity (FVC) ratio of less than 0.70 (post-bronchodilator values). Additional inclusion criteria were a cigarette smoking history of more than 10 pack-years, clinical stability as defined by the absence of exacerbations for at least 4 weeks, age 40 years and an absence of known chronic systemic infections or inflammatory conditions (e.g. rheumatoid arthritis, systemic lupus erythematosis, active sarcoidosis). Overview of the Trial Design Regardless of their prior medication history, all study participants underwent a run-in phase during which they received fluticasone (500 mcg bid; Flovent Diskus, GlaxoSmithKline Canada, Mississauga, ON) for 4 weeks. This was followed by a medication withdrawal phase wherein inhaled corticosteroids, long-acting β 2 - adrenoceptor agonists and theophylline products were withdrawn for 4 weeks. All other medications including short-acting β 2 -adrenoceptor agonists, anticholinergics and tiotropium were permitted during all phases of the study. After the completion of the withdrawal phase, participants were randomly assigned to one of three arms: placebo (placebo discus), inhaled fluticasone (500 mcg bid; Flovent Diskus, GlaxoSmithKline Canada, Mississauga, ON) or inhaled fluticasone/salmeterol combination (500/50 mcg bid; Advair Diskus, GlaxoSmithKline Canada, Mississauga, ON). Randomization was carried out centrally according to a computer-generated sequence stratified according to current smoking status with allocation concealment in a 1 (placebo arm) to 2 (fluticasone 3

4 arm) to 2 (fluticasone/salmeterol) distribution ratio. The study protocol was approved by the regional ethics board for each of the participating centers. Outcome measurements During every visit, venipuncture was performed using standard procedures and the blood samples were sent to a central laboratory in Vancouver for preparation, storage and analysis. From these samples, the participants' CRP, IL-6 and SPD levels were determined in triplicate using a commercially available high-sensitivity enzyme-linked immunosorbent assay (ELISA) kits (Alpha Diagnostics for CRP, San Antonio, Tx; R and D Systems, Minneapolis, MN for IL-6; Biovendor, Modrice, Czech Republic for SPD). The coefficient of variation for the CRP measurement was 5.4%, for the IL-6 measurement was 3.1% and for the SPD measurement was 2.6%. The lower limit of detection for the CRP assay was 0.35 ng/ml, for the IL-6 assay was pg/ml and for the SPD assay was 0.2 ng/ml. Spirometry was also performed at each visit in accordance with the guidelines from the American Thoracic Society (E11). Additionally, during each visit, the participants completed a disease-specific health status questionnaire, St. George s Respiratory Questionnaire (SGRQ) (E12). Information on exacerbations was captured throughout the study. A significant event was defined as worsening of COPD symptoms leading to hospitalization, a visit to the emergency room or to the use of an antimicrobial agent and/or systemic corticosteroids as an outpatient. Exacerbations were treated by the attending physician. For those who experienced a significant exacerbation, they were brought back to the study site for assessment at two weeks following full recovery of symptoms and discontinuation of all systemic corticosteroids and/or antibiotics. If patients were stable, they were re-initiated into the study beginning from the run-in period unless the exacerbation occurred during the treatment phase in which case they were re-initiated beginning at visit 4 (RCT phase). Statistical Analysis 4

5 Analyses were conducted based on an intention-to-treat principle and based on actual measurements: no data were imputed. The baseline characteristics of the study subjects were compared using a chi-square test with appropriate degrees of freedom for dichotomous variables and one-way analysis of variance (ANOVA) for continuous variables. Tukey s post hoc analysis was used to adjust for multiple (pairwise) comparisons. For non-normally distributed variables, Kruskal-Wallis test was used. Multiple Behrens-Fisher-Test (E13) was used to adjust for multiple (pairwise) comparisons of non-normally distributed variables. To determine which if any of the biomarkers were related to changes in clinical outcomes such as health status or FEV 1, we divided the subjects into quintiles of CRP, IL-6 or SPD and performed linear regressions for each quintile and used a chi-square trend test to test for the linearity of the relationship. All statistical tests were two-tailed in nature and p <0.05 was considered statistically significant. Normally distributed continuous variables are reported as mean±sd, while non-normally distributed variables are reported as median (interquartile range) unless otherwise indicated. 5

6 Results A total of 356 subjects were screened. Of these, 67 were excluded because they failed to meet the inclusion and exclusion criteria of the study, leaving 289 subjects for analysis. Between visits 1 and 2, 29 subjects dropped out of the study and 36 dropped out between visits 2 and 3 mostly because of worsening in their symptoms or heath status. This left 224 subjects, who were randomized at visit 3 into 3 arms of the trial: 45 to the placebo, 87 to fluticasone and 92 to fluticasone/salmeterol combination groups. A flow diagram summarizing the distribution of the subjects is shown in figure 1. The baseline characterizes of the study participants at each of the major visits is shown in table 1. Visit 1 (enrollment) The average age of the subjects was 68.8±9.0 years; 60.2% were males and 32.5% were current smokers. The average body mass index (BMI) was 27.7±6.0 kg/m 2 and the average FEV 1 was 1.36±0.54 L or 47.8±16.2% of predicted. 58.4% of the subjects were taking combination therapy, while 68.3% were taking an inhaled corticosteroid containing drug at the time of enrollment. The average total SGRQ score was 42.4±16.5 units. The median (interquartile range) SPD was ( ) ng/ml, CRP was 3.4 ( ) mg/l, and IL-6 was 2.2 ( ) pg/ml. Fifty-nine percent of the study participants were taking either an inhaled corticosteroid or an inhaled corticosteroid/laba combination at the time of enrollment (38% for inhaled corticosteroids and 21% for the combination products). Visit 2 (run-in phase; 1 month of fluticasone) The changes in the serum biomarkers across the various phases of the study relative to baseline are summarized in Table 2. After 1 month of fluticasone, the subjects CRP changed by a median (interquartile range) of 0.0 (-1.0 to 1.3) mg/l (p = 0.325); IL-6 changed by 0.0 (-0.6 to 0.6) pg/ml (p = 0.924) and SPD changed by 1.3 (-9.1 to 13.3) 6

7 ng/ml (p = 0.178); FEV 1 changed by -0.6 (-3.8 to 4.2) % of predicted (p = 0.779); and SGRQ changed by -1.7 (-6.7 to 4.5) units (p=0.012). Visit 3 (withdrawal phase) Withdrawal of fluticasone for one month led to the following changes: the subjects CRP changed by 0.0 (-1.3 to 1.3) mg/l (p = 0.829); IL-6 changed by -0.1 (-0.6 to 0.6) pg/ml (p = 0.728); SPD changed by 5.6 (-4.5 to 20.1) ng/ml (p < 0.001); FEV 1 changed by -1.0 (-5.4 to 2.2) % predicted (p < 0.001); and total SGRQ changed by 2.6 (-2.3 to 8.1) units (p < 0.001). The findings from the run-in and withdrawal phases are summarized in Table 2. Visit 4 (RCT phase) The clinical characteristics of the study subjects at the time of randomization are summarized in Table 4. The changes in biomarkers, health status and lung function between visit 3 and 4 are summarized in Table 3. Neither fluticasone nor the combination of fluticasone/salmeterol had any significant effect on CRP (primary end point) or IL-6 (secondary end point) levels. There was, however, a significant reduction in SPD levels with fluticasone and fluticasone/salmeterol compared with placebo (p=0.002). SGRQ also improved significantly in both the fluticasone and the fluticasone/salmeterol groups compared with placebo, driven mostly by improvements in the symptom scores. There were no significant differences between fluticasone and the fluticasone/salmeterol groups in any of these parameters. FEV 1 improved significantly in the fluticasone/salmeterol group compared with placebo. Changes in the circulating SPD levels were related to changes in SGRQ scores between visit 3 and 4 (figure 2). Subjects with the largest reduction in circulating SPD levels experienced the biggest improvements in health status as measured by the total SGRQ. Similarly, there was a significant relationship between circulating SPD and changes in FEV 1 between visit 3 and 4 (figure 3). Subjects with the largest reduction in SPD levels experienced the 7

8 biggest improvements in FEV 1. CRP changes were also associated with changes in total SGRQ scores (p<0.001) but not with changes in FEV 1 (p=0.339). There was no significant relationship between changes in IL-6 and total SGRQ (p = 0.847) or FEV 1 (p = 0.184). Exacerbation and Withdrawals There were significantly more exacerbations during the withdrawal than in the run-in phase. Moreover, subjects who received placebo diskus experienced more exacerbations and were more likely to drop out compared to subjects who received fluticasone or fluticasone/salmeterol (see table 5). All withdrawals occurred because of worsening of respiratory symptoms related to the participants COPD. There were no significant differences in exacerbations or drop-outs between the fluticasone and fluticasone/salmeterol groups. Other Adverse Events During the active treatment phase, 9% in the placebo, 6% in the fluticasone, and 10% in the combination arm complained of lower respiratory tract symptoms (which did not result in their withdrawal or any additional treatment) (p=0.620). Upper respiratory tract complaints defined as rhinorrhea, nasal congestion and/or headache were reported in 4% of placebo, 4% of fluticasone and 4% in the combination-treated groups (p=0.908). There were no reported cases of pneumonia. 8

9 References E1. Sin DD, Man SF, Marciniuk DD, Ford G, FitzGerald M, Wong E, York E, Mainra RR, Ramesh W, Melenka LS, et al. Can inhaled fluticasone alone or in combination with salmeterol reduce systemic inflammation in chronic obstructive pulmonary disease? Study protocol for a randomized controlled trial [nct ]. BMC Pulm Med 2006;6:3. E2. Dahl M, Vestbo J, Lange P, Bojesen SE, Tybjaerg-Hansen A, Nordestgaard BG. C-reactive protein as a predictor of prognosis in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2007;175: E3. Man SF, Connett JE, Anthonisen NR, Wise RA, Tashkin DP, Sin DD. C-reactive protein and mortality in mild to moderate chronic obstructive pulmonary disease. Thorax 2006;61: E4. Stork S, Feelders RA, van den Beld AW, Steyerberg EW, Savelkoul HF, Lamberts SW, Grobbee DE, Bots ML. Prediction of mortality risk in the elderly. Am J Med 2006;119: E5. Pepys MB, Hirschfield GM. C-reactive protein: A critical update. J Clin Invest 2003;111: E6. Griese M. Pulmonary surfactant in health and human lung diseases: State of the art. Eur Respir J 1999;13: E7. Hirama N, Shibata Y, Otake K, Machiya J, Wada T, Inoue S, Abe S, Takabatake N, Sata M, Kubota I. Increased surfactant protein-d and foamy macrophages in smokinginduced mouse emphysema. Respirology 2007;12: E8. Mori K, Kurihara N, Hayashida S, Tanaka M, Ikeda K. The intrauterine expression of surfactant protein d in the terminal airways of human fetuses compared with surfactant protein a. Eur J Pediatr 2002;161: E9. Eisner MD, Parsons P, Matthay MA, Ware L, Greene K. Plasma surfactant protein levels and clinical outcomes in patients with acute lung injury. Thorax 2003;58: E10. Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, Calverley P, Fukuchi Y, Jenkins C, Rodriguez-Roisin R, van Weel C, et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: Gold executive summary. Am J Respir Crit Care Med 2007;176: E11. Standardization of spirometry update. Statement of the american thoracic society. Am Rev Respir Dis 1987;136: E12. Jones PW, Quirk FH, Baveystock CM, Littlejohns P. A self-complete measure of health status for chronic airflow limitation. The st. George's respiratory questionnaire. Am Rev Respir Dis 1992;145:

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