High Dose N--Acetylcysteine in Patients With Exacerbations of Chronic Obstructive Pulmonary Disease

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1 High Dose N--Acetylcysteine in Patients With Exacerbations of Chronic Obstructive Pulmonary Disease R. Zuin; A. Palamidese; R. Negrin; L. Catozzo; A. Scarda; M. Balbinot Abstract Objective: To investigate the efficacy and tolerability of high-dose N-acetylcysteine (NAC) in the treatment of patients with exacerbations of chronic obstructive pulmonary disease (COPD). Design and Patients: Randomised, double-blind, double-dummy, placebo-controlled study in 123 patients experiencing an acute exacerbation of COPD. Interventions: NAC 1200 mg/day, 600 mg/day or placebo administered once daily for 10 days. Main Outcome Measures: The primary objective was to assess the proportion of patients with normalised C- reactive protein (CRP) levels. Also assessed were effects on interleukin (IL)-8 levels, lung function and symptoms. Results: Both NAC 600 and 1200 mg/day were associated with a significantly higher proportion of patients achieving normalised CRP levels compared with placebo (52% and 90% vs 19% of patients; p = 0.01); however, NAC 1200 mg/day was superior to NAC 600 mg/day (p = 0.002). Furthermore, treatment with NAC 1200 mg/day was more efficacious than NAC 600 mg/day in reducing IL-8 levels and difficulty of expectoration, while the two active regimens had similar beneficial effects on lung function and other clinical outcomes (cough intensity and frequency, and lung auscultation). Treatments were well tolerated with one adverse event reported in NAC 1200 mg/day recipients and two reported in placebo recipients. Conclusion: Treatment with NAC 1200 mg/day improved biological markers and clinical outcomes in patients with COPD exacerbations. It is speculated that the effect of NAC on inflammatory markers may be due to both mucolytic and antioxidant properties. Introduction Chronic obstructive pulmonary disease (COPD) is characterised by irreversible or only partially reversible airway obstruction with episodes of symptom exacerbation that are believed to contribute to the progression of the disease and progressive loss of lung function. [1,2] Bacterial or viral infections appear to be responsible for exacerbations by causing an influx of inflammatory cells, such as activated neutrophils and macrophages responsible for the release of elastase and myeloperoxidase, [3] in the bronchial mucosa. [4] Release of elastase and mye- loperoxidase may lead to production of oxygen free radicals, [3] which have pro-inflammatory properties [5] and inhibit a-1 antitrypsin, the most important inhibitor of elastase. [6] A role for oxygen free radicals in the pathogenesis of COPD has been confirmed in a recent study showing a large increase in exhaled hydrogen peroxide during exacerbations. [5] Thus, it seems reasonable to assume that treatment of exacerbations of COPD with drugs possessing antioxidant properties may lead to reductions in markers of airway inflammation and improve clinical outcomes. Data suggest that N-acetylcysteine (NAC) at dosages of mg/day may reduce symptoms, exacerbation rates and lung function decline in COPD patients, although not all studies have yielded consistent results. [7-9] These effects were originally attributed to the ability of NAC to reduce mucus viscosity and facilitate expectoration. However, other studies have shown that NAC has anti-inflammatory and antioxidant properties. [10-13] In vitro, NAC inhibits neutrophil chemotaxis, interleukin (IL)-8 secretion, and other proinflammatory mediators such as the transcription nuclear factor (NF)-?B, which is directly correlated with the production of the systemic inflammatory marker C-reactive protein (CRP). [14]

2 CRP levels are elevated during exacerbations of COPD and significantly reduced with treatment; thus, they can be used to monitor clinical improvement. [15,16] IL-8 has also been shown to be significantly elevated during exacerbations of COPD. [17] The aim of this study was to evaluate the efficacy of high-dose NAC (1200 mg/day) in patients with an acute exacerbation of COPD. Since the degree of anti-inflammatory effect of NAC is associated directly with CRP and IL-8 secretion, the anti-inflammatory effect of NAC was inferred from changes in CRP and IL-8, while clinical efficacy was assessed by changes in lung function, symptoms and physical examination. Tolerability was also evaluated by standard clinical and laboratory tests. Patients One-hundred and twenty-three adult outpatients participated in the study. They were required to have a history of COPD with at least two exacerbations in the previous 2 years, to have a post-bronchodilator forced expiratory volume in 1 second (FEV 1 ) between 40% and 70% of predicted, and to be currently experiencing an acute exacerbation of COPD characterised by one or more of the following symptoms: dyspnoea, wheezing, chest tightness, mucus production and fever. Patients with serious concomitant diseases (cardiac, hepatic, renal or cancer) or a-1-antitrypsin deficiency were not included. The protocol was approved by the local Ethics Committees and patients gave their written informed consent. Study Design This was a double-blind, double-dummy, placebo-controlled study. The participating subjects were randomly assigned, using a randomisation list for six-patient blocks, to one of three treatment groups: (a) a 10-day course of once-daily treatment with NAC 1200 mg/day (NAC1200; two 600mg tablets; n = 41); (b) NAC 600 mg/day (NAC600; one 600mg tablet plus a placebo tablet; n = 39); or (c) placebo (two tablets; n = 42). Concomitant use of inhaled corticosteroids, theophylline, anticholinergics and ß 2 -adrenoceptor agonists was permitted during the study, while the use of mucus-active, antitussive and systemic corticosteroids was prohibited to avoid any potential confounding effect on clinical endpoints. A total daily water intake of about 1L was recommended throughout the study. After the screening visit (T0), randomised patients attended the clinic after five (T5) and ten (T10) days of treatment. Assessment of Efficacy The primary efficacy endpoint was anti-inflammatory effect assessed by normalisation of serum CRP (<0.5 mg/dl). Secondary endpoints included serum IL-8 levels and clinical efficacy measures: FEV 1 before and after administration of salbutamol 200µg; symptoms (frequency and intensity of cough, difficulty of expectoration); and auscultation data. A Sensor Medics Model 922 spirometer was used to measure FEV 1 and forced vital capacity (FVC). The FEV 1 /FVC ratio was then calculated. Symptoms were recorded daily by patients on diary cards. Signs and symptoms were scored on a 4-point scale. For frequency of cough: 0 = no cough; 1 = rare cough; 2 = once or more per hour; 3 = cough or stimulus always present. For intensity of cough: 0 = no cough; 1 = mostly diurnal not interfering with activities; 2 = diurnal and nocturnal slightly interfering with activities and sleep; 3 = diurnal and nocturnal interfering with activities and sleep, may be associated with headache or thoracic/abdominal pain. For difficulty of expectoration: 0 = no difficulty; 1 = easy, requiring one or two efforts; 2 = moderately difficult, requiring repeated efforts; 3 = very difficult, requiring several efforts. For auscultation: 0 = normal breath sound; 1 =

3 coarse breath sound with wheeze; 2 = coarse breath sound with occasional or localised wheeze and crackles; 3 = coarse breath sound with constant or diffuse wheeze and crackles. Assessment of Tolerability Vital signs (heart rate, systolic and diastolic blood pressure, and breathing frequency) and whole blood cell count were determined at each visit. Urinalysis was performed at T0 and T5, and blood chemistry was assessed at T0 and T10. Statistics The sample size was determined in order to test the hypothesis that normalisation of CRP occurred at the end of the treatment period in 20% of patients receiving placebo and in 70% of patients receiving NAC 1200 mg/day, with a = 0.05 for a two-tailed test and a power of 80%. Standard deviations were used to show the character of the populations. Normally distributed variables were analysed by the paired t-test and analysis of variance (ANOVA) between and within groups. Non-normally distributed variables were compared within groups by signed-rank test and between groups by Kruskal-Wallis tests. Categorical and dichotomic variables were analysed by the chi-square (X 2 ) or Fisher's Exact test, as appropriate. As the primary variable was CRP normalisation, only those patients with abnormal CRP at baseline were analysed for this outcome (per-protocol population). Changes in CRP values from baseline and all other variables were analysed on the intention-to-treat population. Results Baseline Characteristics Patients' disease characteristics and demographic data at screening (T0) are reported in Table 1. At the screening visit (T0), CRP levels were higher than normal (=0.5 mg/dl) in 81 (66.4%) patients ( Table 1 ). These patients comprised the per-protocol population. The proportion of patients with specific causes of exacerbation in each treatment arm are shown in Table 1. Efficacy In patients with abnormal CRP at baseline, normalisation of CRP was achieved in 27 (90%) patients in the NAC1200 group, 13 (52%) in the NAC600 group and five (19%) in the placebo group, with statistically significant differences between groups (figure 1). Figure 1. C-reactive protein (CRP) normalisation rates in 81 patients with exacerbations of chronic obstructive pulmonary disease and baseline CRP levels =0.5 mg/dl treated for 10 days with N- acetylcysteine (NAC) 600 or 1200 mg/day or placebo; p = for NAC 600 vs placebo; p = for NAC 1200 vs NAC 600. These categorical data were confirmed by the progressive reduction in CRP concentrations seen over the treatment period in the intention-to-treat population (figure 2) and the per-protocol population (data not shown). Compared with T0, CRP was significantly decreased at T5 and T10 with NAC600 and NAC1200 (p < for

4 all comparisons) but not with placebo. Moreover, the reduction of CRP at T10 was significantly greater in the NAC1200 group than in the NAC600 group (p < 0.002). Figure 2. Mean changes in C-reactive protein (CRP) levels over a 10-day treatment period in patients with exacerbations of chronic obstructive pulmonary disease receiving N-acetylcysteine (NAC) 600 or 1200 mg/day or placebo (intention-to-treat population). Data bars are standard deviations. T0 = screening visit; T5 = intermediate visit after 5 days of treatment; T10 = final visit after 10 days of treatment. * p < vs T0 and placebo, p < vs NAC 600mg. IL-8 (figure 3) was significantly decreased at T10 compared with T0 in the NAC1200 group (p < 0.001). It was unchanged in the NAC600 group and significantly increased in the placebo group (p < 0.001). Figure 3. Mean changes in interleukin (IL)-8 levels in patients with exacerbations of chronic obstructive pulmonary disease treated for 10 days with N-acetylcysteine (NAC) 600 or 1200 mg/day or placebo. Data bars are standard deviations. T0 = screening visit; T5 = intermediate visit after 5 days of treatment; T10 = final visit after 10 days of treatment. * p < vs T0. Changes in FEV 1 were small when measured both before (figure 4) and after (data not shown) salbutamol use. However, significant increments compared with T0 were observed at T5 with NAC1200 (p < 0.001) and at T10 with both NAC600 (p = 0.004) and NAC1200 (p < 0.001). No significant changes were observed in the placebo group or between groups. Figure 4. Mean changes in pre-bronchodilator forced expiratory volume in 1 second (FEV1) in patients with exacerbations of chronic obstructive pulmonary disease treated for 10 days with N-acetylcysteine (NAC) 600 or 1200 mg/day or placebo. Data bars are standard deviations. T0 = screening visit; T5 = intermediate visit after 5 days of treatment; T10 = final visit after 10 days of treatment. * p < vs T0 (NAC 1200mg), p = vs T0 (NAC 600mg). Clinical outcomes were improved significantly more frequently in patients treated with NAC than placebo (figure 5). At T10, the proportion of patients with improvement in cough frequency and intensity tended to be higher in the NAC600 group than in the placebo group (p = and p = 0.066, respectively), and was significantly higher in the NAC1200 group than in the placebo group (p = and p = 0.002, respectively). There were no significant differences between the NAC600 and NAC1200 groups in terms of improvement in cough frequency and intensity. The proportion of patients with decreased difficulty of expectoration was significantly higher in both the NAC600 and NAC1200 groups than in the placebo group (p = and p < 0.001, respectively) and significantly higher with NAC1200 than with NAC600 (p = 0.05). The proportion of patients with improved lung auscultation was significantly greater in the NAC1200 and NAC600 groups than in those receiving placebo (both p = 0.05).

5 Figure 5. Cough, expectoration and auscultation improvement rates in patients with exacerbations of chronic obstructive pulmonary disease treated for 10 days with N-acetylcysteine (NAC) 600 or 1200 mg/day or placebo. * p = 0.05 vs placebo, ** p < vs placebo, p = 0.05 vs NAC 600mg, 0.05 = p < 0.10 vs placebo; + p = vs placebo. Acute symptoms of COPD, dyspnoea, wheezing, chest tightness, mucus production and fever were treated as required during the study. The proportion of patients receiving concomitant medications in each treatment arm is shown in Table 2. Tolerability One adverse event was reported in the NAC1200 group. The event was mild gastric pain, which was judged as possibly related to treatment and disappeared after treatment discontinuation. Two adverse events occurred in the placebo group, one of which resulted in treatment discontinuation. No significant changes were observed in vital signs, blood chemistry, whole blood cell count or urinalysis. Discussion The major findings of this study of exacerbations of COPD can be summarised as follows: (a) among patients with elevated CRP, those receiving NAC were more likely to have this marker normalised than those receiving placebo, with NAC1200 being more efficacious in this regard than NAC600; (b) both dosages of NAC were superior to placebo in terms of improving lung function and clinical outcomes; (c) NAC1200 was superior to NAC600 in reducing IL-8 and difficulty of expectoration. COPD is a disease characterised by a progressive decline in lung function that is correlated with frequency of exacerbation. [1,2,18] Exacerbations occur in response to a series of events including mucus hypersecretion, reduced mucociliary clearance and viral or bacterial infections. [4,18] The ensuing influx of inflammatory cells, namely neutrophils, macrophages and T lymphocytes, may lead to damage of bronchial mucosa and lung parenchyma through the release of protease, myeloperoxidase and oxygen free radicals. [3,5] In addition to its well known mucolytic activity, [19] NAC has been shown to exert anti-inflammatory activity and antioxidant activity by inhibiting neutrophil chemotaxis [10] and promoting the synthesis of glutathione, which represents one of the most efficient antioxidant cellular systems. [11] The results of the present study, which is the first to demonstrate the effects of NAC on lung function, support a role for NAC as an anti-inflammatory agent in the treatment of COPD exacerbations. NAC reduced CRP and IL-8 in a dose-dependent manner, which was associated with a beneficial effect on clinical and functional outcomes. The data presented here do not allow us to ascertain whether NAC reduced CRP and IL-8 by a direct anti-inflammatory effect or by improving mucociliary clearance, as suggested by the much greater effect of NAC1200 than NAC600 on ease of expectoration. The mode of action of NAC in COPD has not been elucidated. If the effect is primarily a direct anti-inflammatory effect, the greatest treatment effect may be seen in the first few days after treatment initiation rather than at 5 or 10 days; however, this has not been studied to date and was not measured in this study. Further research is required to ascertain whether this is the case; such data would shed more light on the likely mechanism of effect of NAC. A possible confounding factor in any study of exacerbations of COPD is use of concomitant treatments, which cannot be avoided for ethical reasons. However, this does not appear to have been the case in the present

6 study as there were no significant differences in use of antibacterial or bronchodilator treatments between the groups. Furthermore, the number of patients receiving inhaled corticosteroids was small in all treatment groups. Therefore, the differences between treatments observed in the present study can be reasonably attributed to NAC and its dosage. In this study, patients with at least two exacerbations of COPD in the previous 2 years were included. It is likely that patients with fewer previous exacerbations may also benefit from this treatment. This is a matter for further study. Both NAC600 and NAC1200 were equally well tolerated, as shown by the constancy of vital signs and laboratory findings during the study, and also by patients' opinions on treatment acceptability, which was positive in >95% of patients for all groups. Conclusion This study provides evidence that a daily dose of NAC 1200mg, twice the currently recommended dosage, improves outcomes in patients with exacerbations of COPD. The beneficial effects of NAC are clinically apparent and are associated with reduction or normalisation of inflammatory markers. Further studies are necessary to ascertain whether the latter effects of NAC are directly related to its antioxidant activity or the result of improvements in mucus clearance. References 1. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. American Thoracic Society. Am J Respir Crit Care Med 1995; 152 (5 Pt 2): S Siafakas NM, Vermeire P, Pride NB, et al. Optimal assessment and management of chronic obstructive pulmonary disease (COPD). The European Respiratory Society Task Force. Eur Respir J 1995; 8 (8): Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989; 320 (6): Murphy TF, Sethi S. Bacterial infection in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992; 146 (4): Dekhuijzen PN, Aben KK, Dekker I, et al. Increased exhalation of hydrogen peroxide in patients with stable and unstable chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1996; 154 (3 Pt 1): Heffner JE, Repine JE. Pulmonary strategies of antioxidant defense. Am Rev Respir Dis 1989; 140 (2): Stey C, Steurer J, Bachmann S, et al. The effect of oral N-acetylcysteine in chronic bronchitis: a quantitative systematic review. Eur Resp J 2000; 16 (2): Grandjean EM, Berthet P, Ruffmann R, et al. Efficacy of oral long-term N-acetylcysteine in chronic bronchopulmonary disease: a meta-analysis of published double-blind, placebo-controlled clinical trials. Clin Ther 2000; 22 (2): Dekhuijzen PN. Antioxidant properties of N-acetylcysteine: their relevance in relation to chronic obstructive pulmonary disease. Eur Respir J 2004; 23 (4): Kharazmi A. The anti-inflammatory properties of N-acetylcysteine. Eur Respir Rev 1992; 2 (7): Aruoma OI, Halliwell B, Hoey BM, et al. The antioxidant action of N-acetylcysteine: its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid. Free Radic Biol Med 1989; 6 (6): De Benedetto F, Aceto A, Dragani B, et al. Long-term oral N-acetylcysteine reduces exhaled hydrogen peroxide in stable COPD. Pulm Pharmacol Ther 2005; 18 (1): Kasielski M, Nowak D. Long-term administration of N-acetylcysteine decreases hydrogen peroxide exhalation in subjects with chronic obstructive pulmonary disease. Respir Med 2001; 95 (6):

7 14. Pinkus R, Weiner LM, Daniel V. Role of oxidants and antioxidants in the induction of AP-1, NF-?B, and glutathione S-transferase gene expression. J Biol Chem 1996; 271 (23): van Beurden WJ, Smeenk FW, Harff GA, et al. Markers of inflammation and oxidative stress during lower respiratory tract infections in COPD patients. Monaldi Arch Chest Dis 2003; 59 (4): Malo O, Sauleda J, Busquets X, et al. Systemic inflammation during exacerbations of chronic obstructive pulmonary disease. Arch Bronconeumol 2002; 38 (4): Aaron SD, Angel JB, Lunau M, et al. Granulocyte inflammatory markers and airway infection during acute exacerbation of chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2001; 163 (2): Vestbo J, Prescott E, Lange P. Association of chronic mucous hypersecretion with FEV1 decline and chronic obstructive pulmonary disease morbidity. Copenhagen City Heart Study Group. Am J Respir Crit Care Med 1996; 153 (5): Allegra L, Blasi F, Capone P, et al. Proprietà mucoattive ed antiossidanti della NAC (N-acetilcisteina) ad alte dosi. G Ital Mal Torace 2002; 56 (3):