Published ahead of print on October 18, 2007, doi:10.1164/rccm.200707-1134OC
© 2008 American Thoracic Society doi: 10.1164/rccm.200707-1134OC
Clarithromycin Targets Neutrophilic Airway Inflammation in Refractory Asthma1 NHMRC Centre for Respiratory and Sleep Medicine, School of Medicine and Public Health, University of Newcastle, Callaghan, Australia; 2 Department of Respiratory and Sleep Medicine, and 3 Department of Immunology and Infectious Diseases, Hunter Medical Research Institute, John Hunter Hospital, New Lambton, Australia; and 4 Department of Medical Genetics, School of Biomedical Science, Hunter Medical Research Institute, University of Newcastle, Callaghan, Australia Correspondence and requests for reprints should be addressed to Peter G. Gibson, M.B.B.S. F.R.A.C.P., Level 3, HMRI, John Hunter Hospital, Locked Bag 1, Hunter Region Mail Centre, Newcastle, NSW 2310, Australia. E-mail: peter.gibson{at}hnehealth.nsw.gov.au
Rationale: Patients with refractory asthma have persistent symptoms despite maximal treatment with inhaled corticosteroids and long-acting bronchodilators. The availability of add-on therapies is limited, and effective add-on therapies that target noneosinophilic airway inflammation are needed. Macrolide antibiotics, such as clarithromycin, have in vitro efficacy against IL-8 and neutrophils, key inflammatory mediators in noneosinophilic asthma. Objectives: To determine the efficacy of clarithromycin in patients with severe refractory asthma and specifically in a subgroup of patients with noneosinophilic asthma. Methods: Subjects with severe refractory asthma (n = 45) were randomized to receive clarithromycin (500 mg twice daily) or placebo for 8 weeks. Measurements and Main Results: The primary outcome for this study was sputum IL-8 concentration. Other inflammatory outcomes assessed included sputum neutrophil numbers and concentrations of neutrophil elastase and matrix metalloproteinase (MMP)-9. Clinical outcomes were also assessed, including lung function, airway hyperresponsiveness to hypertonic saline, asthma control, quality of life, and symptoms. Clarithromycin therapy significantly reduced airway concentrations of IL-8 and neutrophil numbers and improved quality-of-life scores compared with placebo. Reductions in neutrophil elastase and MMP-9 concentrations were also observed. These reductions in inflammation were most marked in those with refractory noneosinophilic asthma. Conclusions: Clarithromycin therapy can modulate IL-8 levels and neutrophil accumulation and activation in the airways of patients with refractory asthma. Macrolide therapy may be an important additional therapy that could be used to reduce noneosinophilic airway inflammation, particularly neutrophilic inflammation, in asthma. Clinical trial registered with the Australian Clinical Trials Registry www.actr.org.au (No. 12605000318684).
Key Words: refractory asthma • macrolides • induced sputum • IL-8 • quality of life
Refractory asthma is a term applied to patients with asthma who require high levels of medication to maintain good disease control or to those who experience persisting symptoms despite high levels of medication (1). Although affecting only a small proportion of all asthma sufferers, patients with refractory asthma have a disproportionate impact on health care costs. Current guidelines suggest a stepwise approach to asthma treatment, adding therapy until asthma control is achieved (2). For those with refractory asthma who are taking high doses of inhaled corticosteroids with long-acting β-agonists, add-on therapies are limited to oral corticosteroids and anti–IgE antibody therapy. These therapies act specifically on eosinophilic inflammation and allergic IgE pathways and offer little or no relief to neutrophilic airway inflammation, a recognized feature of severe asthma (3). Noneosinophilic forms of asthma are common (4) and are difficult to distinguish from eosinophilic asthma using typical clinical tools such as spirometry and airway challenges (5). Patients with noneosinophilic asthma (NEA) have increased neutrophils and IL-8 levels in the airways (6), and conventional therapies such as inhaled corticosteroids, although very effective in those with eosinophilic inflammation, have limited efficacy when eosinophils are within normal range (7). Thus, there is a need for effective antiinflammatory therapies for patients suffering from NEA. Macrolide antibiotics, such as clarithromycin, have separate and distinct antibiotic and antiinflammatory actions. Macrolides have been widely used in the treatment of infections caused by bacteria such as Chlamydia pneumoniae and Mycoplasma pneumoniae. There is extensive in vitro and growing in vivo evidence of antiinflammatory activity of macrolides. Reductions in IL-8 and neutrophil numbers have been observed after macrolide therapy in diffuse panbronchiolitis (8) and chronic airway disease (9). Recent case reports have shown that clarithromycin is an effective add-on therapy in prednisone-dependent asthma (10). We therefore hypothesized that macrolide antibiotics would be an effective add-on therapy in refractory NEA. Given the limited availability of treatment options for refractory asthma, this study aimed to determine the efficacy of 500 mg clarithromycin twice daily for 8 weeks in patients with refractory asthma, and specifically the effects in a subgroup of patients with NEA. Some of the results of this study have been previously reported in the form of abstracts (11, 12).
Participants Nonsmoking adults with symptomatic refractory asthma (n = 79), according to GINA (Global Initiative for Asthma) guidelines (2), with demonstrated airway hyperresponsiveness to hypertonic saline (13) were studied. Participants were recruited from the Ambulatory Care Service of the Department of Respiratory and Sleep Medicine at the John Hunter Hospital (New Lambton, Australia). During the study, participants continued with their baseline medications as prescribed by their physician. At screening, participants were educated about the correct use of their baseline medication devices and any errors rectified. Participants were excluded if they had smoked more than 5 pack-years or if they had any known sensitivity to macrolide antibiotics. Antihistamine therapies were ceased for the duration of the study. The Hunter Area Health Service and University of Newcastle Research Ethics Committees approved this study.
Study Design
Assessments
Adverse events. The primary outcome was IL-8 levels in sputum supernatant, with secondary outcomes of sputum neutrophil numbers, neutrophil elastase (NE), and matrix metalloproteinase (MMP)-9 levels. Clinical outcomes were reported, including FEV1% predicted, dose–response slope to hypertonic saline, symptom severity, asthma control score, and asthma quality-of-life questionnaire score.
Sputum induction and saline challenge.
Sputum analysis. IL-8, NE, and total MMP-9 levels were determined by ELISA (R&D Systems, Minneapolis MN, and Calbiochem, San Diego, CA) (19), and IL-8 gene expression was determined using real-time polymerase chain reaction (20).
Statistical Analysis
Seventy-nine patients were screened for the study and 46 were eligible and allocated to treatment (23 to clarithromycin and 23 to placebo). One participant was randomized but did not complete the first week of treatment and was withdrawn. Analyses were performed on 45 participants (22 male) who completed the study (Figure 1). The duration of sputum induction was not different between the treatment groups at any visit. A sputum sample was collected on 216 of 225 occasions, giving a success rate of 96%. There were no significant differences between treatment groups at baseline with respect to age, gender, atopy, lung function, asthma control score, or daily inhaled corticosteroid dose (Table 1).
The 45 participants who completed the study had a mean age of 58 years and had moderate airflow obstruction with a mean FEV1% predicted of 71% (SD, 17%). The group had poor asthma control (mean [SD] asthma control score. 1.5 [0.8]) and were receiving high daily maintenance doses of inhaled corticosteroids (median [IQR] dose, 2,000 [1,000–2,000] µg). Thirty-nine (87%) participants were receiving long-acting bronchodilators, the majority (92%) in the form of combination therapies. In the past year, 20 (44%) participants had received a course of oral steroids, 3 had been hospitalized, and a further 10 (22%) had visited their general medical practitioner due to asthma exacerbation. Allergy history and rhinitis symptoms can be found in Table E1 of the online supplement. Clarithromycin treatment significantly reduced airway IL-8 levels from a median (IQR) of 6.6 (2.7–11.8) ng/ml before treatment to 3.9 (1.8–5.4) ng/ml after treatment (Figure 2A). There was also a significant reduction in neutrophil numbers and reductions in both IL-8 gene expression and neutrophil activation as assessed by concentrations of NE. Total MMP-9 levels were lower after clarithromycin; however, the differences were not significant (Table 3). There was no change in the number or proportion of eosinophils with clarithromycin treatment (data not shown).
Total quality-of-life score significantly improved with clarithromycin treatment (Table 2). Nine (39%) participants had a clinically significant improvement in their score of 0.5 or higher. The number-needed-to-treat (NNT) was 6. Within individual domains, there were significant improvements in both the activities and environmental stimuli domains (Figure 4A). In particular, the greatest improvement was found in the environmental stimuli domain where 15 (65%) participants had an improvement in their score of 0.5 or greater (Figure 4C) (NNT = 3). There was no change in the symptom score or levels of exhaled nitric oxide with clarithromycin treatment (see the online supplement).
There was a significant reduction in the proportion of participants reporting wheeze in the clarithromycin group (86% reporting wheeze at visit 2, reduced to 50% at visit 4; P = 0.043). There were no changes in any symptoms in the placebo group. There was no change in FEV1% predicted, dose–response slope to hypertonic saline, or asthma control score after clarithromycin treatment (Table 3).
At the end of clarithromycin treatment, total MMP-9 levels were significantly lower in those who had received clarithromycin compared with placebo. Neutrophil numbers and concentrations of NE were also lower; however, these values were not statistically significant (Table 3). Participants were reassessed 4 weeks after withdrawal of therapy and a significant increase in the concentration of IL-8 and NE was observed in those participants who had taken clarithromycin (Figures 2A and 2B). Neutrophil numbers were increased and the quality-of-life score deteriorated after clarithromycin was withdrawn, but these changes were not statistically significant. The median (IQR) number of neutrophils had increased to 122.7 (23.6–196) x 104 cells/ml after treatment was withdrawn for 4 weeks, which was approaching the pretreatment numbers of 142.9 x 104 cells/ml. The median (IQR) total quality-of-life score had reduced from 6.2 (5.4–6.6) at the end of treatment to 5.8 (5.3–6.2). The concentration of total MMP-9 continued to remain low after the withdrawal of clarithromycin. The median (IQR) total MMP-9 concentration after clarithromycin was withdrawn was significantly lower than pretreatment concentrations (median [IQR]: 2,943 [1,900–8,609] vs. 9,111 [2,280–11,772] ng/ml; P = 0.04). These changes were not observed in the placebo-treated group.
NEA Subanalysis
At the end of treatment, levels of NE and MMP-9 were significantly lower in those who had received clarithromycin compared with placebo. Neutrophil numbers were also lower but these values were not significant (Table 4).
We report a double-blind, randomized, placebo-controlled trial of clarithromycin in severe refractory asthma and show that 8 weeks of clarithromycin therapy significantly reduces airway IL-8 and neutrophil numbers and improves quality of life. Concentrations of MMP-9 and NE were also reduced in those receiving clarithromycin, suggesting an overall down-regulation of neutrophil activation and mediator release. The antiinflammatory effect of clarithromycin treatment was most marked in those with refractory NEA and provides an opportunity to further investigate the potential of clarithromycin as an add-on therapy for refractory disease. Sputum IL-8 levels were significantly reduced after 8 weeks of clarithromycin therapy and returned to pretreatment levels when therapy was withdrawn. IL-8 is a potent neutrophil chemoattractant and activator of neutrophils and is produced by a number of airway inflammatory cells, including neutrophils (21). We have previously shown that levels of IL-8 are elevated in NEA (6), and in asthma are correlated with airflow obstruction (5), suggesting IL-8 may play a key inflammatory role in refractory NEA. IL-8 suppression with macrolide therapy has been reported in a number of other airway conditions, including bronchiectasis (22), bronchiolitis (23), and chronic sinusitis (14, 24). In chronic obstructive pulmonary disease (COPD), the evidence is conflicting with only two randomized controlled trials of clarithromycin treatment. In one study, there were significant reductions in sputum IL-8 levels (25), and in the other, no changes in sputum IL-8 levels were observed after 3 months of therapy with clarithromycin (26). A key difference between the two study designs is the dosing regime; in the first study, treatment was given twice daily and in the latter, once daily. It is possible that a single dose of clarithromycin may not be sufficient to induce and maintain the antiinflammatory actions of this treatment in the airways. Clarithromycin and other macrolides also inhibit IL-8 release from a number of other inflammatory cells, including monocytes (27), nasal epithelial cells (24), bronchial epithelial cells (28), and eosinophils (29). Treatment with clarithromycin significantly reduced the number of neutrophils in the airway lumen as well as the concentration of NE in the sputum supernatant, suggesting a reduction in both neutrophil accumulation and activation. Sputum IL-8 gene expression was reduced 2-fold and airway concentrations of MMP-9 reduced by 2.5-fold with clarithromycin therapy, consistent with a reduction in neutrophil activation. Reductions in neutrophils and NE have been reported in patients with chronic airway disease (diffuse panbronchiolitis and bronchiectasis) (9), and neutrophil proportions have been shown to be reduced in both bronchiectasis (22) and COPD (26). Resolution of neutrophilic inflammation in the airways is achieved by the removal of apoptotic neutrophils by alveolar macrophages (efferocytosis). One mechanism whereby clarithromycin may reduce neutrophil numbers is via improvements in phagocytosis of apoptotic cells. Alveolar macrophages treated with macrolide antibiotics have an increased phagocytic capacity for apoptotic neutrophils (30) and epithelial cells (31). Participants on clarithromycin therapy had a significant improvement in the Juniper asthma quality-of-life score, with better results in both the environmental stimuli and physical activity domains. More than 60% of participants had a clinically significant improvement in their environmental stimuli domain of 0.5 or greater. This domain asks the participant about his or her symptoms in the past month due to exposure to dusts, smoke, and other particulates. Such a marked improvement in this domain suggests that the immunologic response to these triggers may be reduced with clarithromycin therapy. Improvements in St. George's Respiratory Questionnaire scores have been observed when macrolides have been used to treat acute exacerbations of chronic bronchitis (32) and in the symptom score only for stable COPD (33). An improvement in the environmental stimuli domain may be explained by the IL-8–modifying effects of clarithromycin. By reducing the amount of IL-8 available, neutrophil activation responses are also modified, and symptoms that result from typical innate immune or nonallergic stimuli, such as particulates, smoke, and dust, may also diminish. We have previously demonstrated high levels of endotoxin and innate immune activation in the airways of patients with neutrophilic asthma and bronchiectasis (34), and macrolide therapy appears to be an effective neutrophil modifier in both diseases. Fewer participants reported wheeze after clarithromycin therapy, with a significant reduction of more than 30%. We observed no changes in airway hyperresponsiveness to hypertonic saline in the participants receiving clarithromycin. Although other studies have shown improvements in hyperresponsiveness after macrolide therapy (35–38), these have largely been on mild well-controlled asthma, which did not use hypertonic saline as the stimulus. Airway responsiveness to hypertonic saline may be a better marker of eosinophilic airway inflammation, and therefore relatively nonresponsive to therapies targeting noneosinophilic mechanisms in asthma. When patients with noneosinophilic refractory asthma were analyzed separately, 8 weeks of clarithromycin therapy significantly reduced IL-8 protein and gene expression. There was also a statistically significant reduction in sputum MMP-9 levels in the airways after clarithromycin therapy for those with NEA. Quality of life was also better with more than 75% of participants with refractory NEA having a clinically significant improvement in the environmental stimuli domain. There was a similar reduction in the frequency of participants who reported experienced wheezing in the previous month. This study was powered to detect differences in sputum IL-8 concentrations with clarithromycin treatment and not to investigate clinical markers of disease, such as airway hyperresponsiveness, asthma control, or quality of life. Despite this, we found evidence of improved quality of life with clarithromycin treatment in refractory asthma, specifically for those with NEA. It is possible that the other clinical markers chosen (e.g., presence of symptoms and asthma control score) may not be responsive measures of clinical improvement in NEA. Future larger studies are required to closely examine the effects of macrolide antibiotics on clinical asthma outcomes and should consider other clinical outcomes that may be more responsive to treatment, such as symptoms visual analog scale. The precise triggers of neutrophilic inflammation in NEA are unknown but may include colonization with typical or atypical bacterial pathogens. Both chlamydia and mycoplasma species have been found in the airways of patients with chronic asthma (39) with an increased frequency compared with healthy control subjects (40). Chlamydia pneumonia infection results in neutrophil accumulation and the bacteria are able to replicate within neutrophils, indicating that it may be an important trigger of neutrophilic inflammation (41). When patients with asthma and chlamydia or mycoplasma infection are treated with macrolide antibiotics, small improvements in lung function have been observed (42, 43). No study, however, has investigated alternations in airway IL-8 or neutrophil levels, and further research is required to examine atypical bacterial infection and macrolide therapy in patients with NEA. In conclusion, we have studied the antiinflammatory effect of clarithromycin in severe refractory asthma and provide data demonstrating significant reductions in levels of IL-8 and numbers of neutrophils in the airway lumen combined with a significant improvement in quality-of-life score and a reduction in self-reported wheeze. This approach confirms the potential of clarithromycin therapy in the treatment of airway inflammation, and more specifically in noneosinophilic refractory asthma where there are no proven treatment options.
The authors thank Ms. Noreen Bell, Ms. Rebecca Oldham, and Ms. Joanna Mimica for their technical assistance.
Supported by the National Health and Medical Research Council of Australia. J.L.S. was supported by the NHMRC Centre for Respiratory and Sleep Medicine; P.G.G. holds an NHMRC Practitioner Fellowship. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1164/rccm.200707-1134OC on October 18, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form July 31, 2007; accepted in final form October 18, 2007
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ABSTRACT
AT A GLANCE COMMENTARY
, 5%; power, 80%) based on previous studies in nasal polyposis (
