Montelukast and bronchial inflammation in asthma: A randomised, double-blind placebo-controlled trial

Respiratory Medicine (2009) 103, 995e1003

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/rmed

Montelukast and bronchial inflammation in asthma: A randomised, double-blind placebo-controlled trial C.F. Ramsay a, P. Sullivan b, M. Gizycki b, D. Wang c, A.S. Swern d, N.C. Barnes a, T.F. Reiss d, P.K. Jeffery b,* a

Department of Respiratory Medicine, Royal London Hospital, London, UK Department of Gene Therapy, Imperial College London, Royal Brompton Hospital, London, UK c Medical Statistics Unit, London School of Hygiene and Tropical Medicine, London, UK d Merck Research Laboratories, Rahway, NJ, USA b

Received 6 November 2008; accepted 19 January 2009 Available online 26 February 2009

KEYWORDS Asthma; Eosinophils; Inflammation; Leukotrienes; Receptors

Summary Background: Examination of bronchoalveolar lavage, induced sputum, and peripheral blood indicate that cysteinyl leukotriene receptor blockers decrease inflammatory cells in asthma but these do not examine airway tissue per se. Objectives: Our objective was to determine the effect of montelukast, a leukotriene receptor antagonist, on airway tissue inflammatory cells by direct bronchoscopic examination of the bronchial mucosa. Methods: Adult subjects with mild asthma (pre-bronchodilator FEV1  70% predicted; PC20 of 4 mg/mL) were given 10 mg/day oral montelukast (N Z 38) or placebo (N Z 37) for 6 weeks. Bronchial mucosal eosinophils and mast cells were identified and counted. Results: Change from baseline in numbers of biopsy EG2þ (‘‘activated’’) eosinophils was the primary endpoint; numbers of total (chromotrope 2Rþ) eosinophils and (tryptaseþ) mast cells were secondary. Unexpectedly, there were many patients with zero EG2þ eosinophils at baseline. There was a within-group decrease in EG2þ cells, from 13.54 cells/mm (at baseline) to 0.79 cells/mm at 6 weeks in the montelukast group (LS mean change; 95% confidence interval Z 13.59 [25.45, 1.74] cells/mm; P < 0.05), a change not observed in the placebo group (1.17 [13.26, 10.91] cells/mm; NS). The zero-inflated Poisson statistical model demonstrated that montelukast significantly reduced post-treatment EG2þ cells by 80% compared with placebo (95% CI [70.6e86.8%]; P < 0.0001). The data for total eosinophils showed similar changes. The reduction in mast cell numbers was 12% (95% CI [7.9, 16.0]; P < 0.0001).

* Corresponding author. Lung Pathology, Royal Brompton Hospital, Sydney Street, London SW3 6NP, UK. Tel.: þ44 0 207 351 8422; fax: þ44 0 207 351 8435. E-mail address: [email protected] (P.K. Jeffery). 0954-6111/$ - see front matter ª 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2009.01.019

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C.F. Ramsay et al. Conclusion: Direct examination of airway tissue confirms that montelukast decreases the number of eosinophils and mast cells in asthma. ª 2009 Elsevier Ltd. All rights reserved.

Introduction

Methods

Asthma is a chronic inflammatory disorder of the airways associated with increased numbers of mucosal eosinophils, mast cells and T lymphocytes.1,2 These can be reduced, at least in part, by treatment with either inhaled or oral corticosteroids.3e6. However, there is evidence that corticosteroids do not treat all of the component inflammatory mediators of asthma. Among these, lipid-derived mediators, the cysteinyl leukotrienes (cysLTs) LTC4, LTD4 and LTE4, have potent biologic effects, including recruitment of eosinophils to the bronchial mucosa,7 increased vascular permeability, mucus hypersecretion, hyperresponsiveness and bronchoconstriction.8e11 In addition, cysLTs have the capacity in animal models of asthma to induce chronic remodelling of the airway wall.12 There are distinct approaches to reducing the actions of cysLTs: inhibition of biosynthesis (e.g., inhibitors of 5-lipoxygenase (5-LO) and 5-LO-activating protein) or blockade of cysLT receptors using leukotriene receptor antagonists (LTRAs).13 CysLT receptors have been cloned and characterised and identified in lung tissue.14,15 Moreover, a recent study of endobronchial biopsies has demonstrated the presence of a number of distinct cysLT receptor1-bearing cells in the airway mucosa of normal healthy humans and shown that their numbers are increased in stable asthma and further increased in patients experiencing severe exacerbations of asthma.16 Thus, targeting cysLT receptor1-bearing cells for the purpose of attenuating the many actions of cysLTs is a valid approach to anti-inflammatory therapy in asthma. In support of this, drugs that block either the production or site of action of the cysteinyl leukotrienes have shown clinical benefit in the treatment of chronic asthma in both adults and children.17,18 There are indications from studies of bronchoalveolar lavage (BAL),19,20 induced sputum21 and peripheral blood17,22 that cysteinyl leukotriene receptor blockers decrease the numbers of inflammatory cells in asthma, particularly eosinophils. For example, the LTRA montelukast reduces sputum eosinophils both in the asthmatic adult and child.21,23 Additionally, montelukast reduces eosinophil products and LT concentrations in nasal washes taken from children with asthma.24 Pre-treatment with zafirlukast significantly reduces the allergen-induced increase of BAL lymphocytes and basophils seen at 48 h after segmental allergen challenge.20 The only interventional study with an LTRA examining human bronchial tissue was that of Nakamura et al.,25 who reported that pranlukast given for 4 weeks reduced the numbers of eosinophils, mast cells and T-lymphocytes. The present multicentre study set out to determine whether treatment with the potent and specific LTRA montelukast in a broader population of mild asthmatic patients would reduce the numbers of eosinophils and mast cells detected in bronchial biopsies of individuals with mild persistent asthma.

Subjects Our entry criteria were based on GINA guidelines and a physician assessment of a clinical diagnosis of asthma backed up by a histamine challenge test confirming bronchial hyperreactivity. Patients between the ages of 18 and 45 years with a clinical history of mild asthma of 1 year duration and a prebronchodilator FEV1 of 70% predicted (with b2-agonist treatment withheld for 6 h) participated in the study. The 70% cut off was designed to exclude patients whose asthma was of a severity that it would be clinically inappropriate not to start them on inhaled steroids. Our subjects could be classified either as ‘‘intermittent’’ or ‘‘mild persistent’’ depending upon their reported symptoms or documented PEF variability. Subjects’ symptoms were controlled by ‘‘as required’’ short-acting b2-agonist use. None had received regular treatment with inhaled, oral or injected steroids or inhaled cromolyn, nedocromil or ketotifen 3 months (or any single dose for 6 weeks), nor taken oral theophylline within 2 weeks or a long-acting b2-agonist within 1 week of starting the study. All subjects were current non-smokers, had not smoked for at least a year prior to the study, and had a history equal to or less than 10 pack-years. Patients were in good health and had no other significant disease or medication requirements before or during the study.

Study design This was a randomised, double-blind, placebo-controlled, parallel group study performed at five clinical centres worldwide (UK, Finland, Chile, Peru and Guatemala). The protocol was approved by the local institutional review boards and in keeping with the Declaration of Helsinki. All patients gave written informed consent to participate in the study before any study-related procedures were performed. Patients underwent a pre-study screening visit where a full clinical assessment including baseline spirometry, chest X-ray, electrocardiogram, skin-prick testing to common allergens and histamine challenge testing were performed. Qualified patients were randomly assigned to receive either montelukast or placebo according to a computer-generated, randomised allocation schedule with a blocking factor of four. Patients subsequently underwent two bronchoscopies: the first prior to and the second after 6 weeks of treatment with either montelukast or placebo. All bronchoscopies, biopsy procedures, and specimen processing were performed according to a protocol standardised across the study centres. The primary endpoint was the change from baseline in the number of EG2þ (‘‘activated’’) eosinophils per unit area of bronchial biopsy in patients who had received montelukast for 6 weeks compared with those who had received placebo. Secondary endpoints comprised the number of

Anti-inflammatory effects of montelukast total eosinophils (chromotope 2R) and the number of mast cells (AA1 tryptase) in biopsy specimens.

Physiologic measurements All spirometric measurements were made at 9 am  2 h on a spirometer calibrated weekly and conforming to accepted American Thoracic Society criteria. Patients demonstrated airway hyperresponsiveness to histamine with a PC20 of 4 mg/mL. Histamine challenge testing was performed with a Devilbiss 646 nebuliser calibrated to an output of 0.13 mL/min. Doubling concentrations of histamine starting with 0.03 mg/mL were administered for 2 min at 5-min intervals until a decrease in FEV1 of 20% was achieved. The histamine PC20 was interpolated from the last two data points on a plot of the log concentration of histamine against percent decrease in FEV1.

Bronchoscopy All bronchoscopies, biopsy procedures, and specimen processing were performed according to a protocol standardised across the study centres. Prior to each bronchoscopy at 10 am  2 h, all patients received 2.5 mg nebulised salbutamol, 600 mg intravenous atropine, 4e10 mg intravenous midazolam and topical xylocaine spray to the nose and throat. During the procedure, patients received topical lignocaine (4% and 2%) to the vocal cords and airways and also nasal oxygen supplementation at 2e4 L/min. The bronchoscopies were performed by one of two designated persons at each site. At each bronchoscopy, six endobronchial biopsies were taken from the sub-carinae of the right lung. The biopsies were taken with fenestrated cup biopsy forceps (Pentax KW2411S) that were replaced at least every four bronchoscopies. A note was made of the sub-carinae from which the biopsies were taken, and at the second bronchoscopy, the same airway generations but different microanatomical sites of the right lung were sampled.

Biopsy handling Three biopsies were fixed in 10% buffered formaldehyde for between 1 and 4 days, when they were processed to paraffin wax. Five-micrometre-thick sections were stained with an antibody directed against EG2 (to identify ‘‘activated’’ eosinophils) or chromotrope (2R) (a marker for total eosinophils), and AA1 tryptase (mast cells). Chromotrope2Rþ and immuno-positive cells were counted in an area of subepithelium to a pre-defined depth of 100 mm that excluded mucus-secreting glands and bronchial smooth muscle, and their numbers were expressed per millimetre length reticular basement membrane (RBM). Although the study was not designed nor powered to examine changes in RBM thickness, this was recorded in the biopsies of 28 montelukast-treated and 26 placebo-treated subjects. Total thickness of the RBM was assessed by a single observer by light microscopy using haematoxylin and eosin-stained tissue sections and image analysis. Repeat measurements were taken at 20-mm intervals and, where adequate tissue was available, the geometric mean of 30 measurements was taken for each subject, as previously recommended.26

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Statistical methods Efficacy analyses were performed by an external consultant statistician (D. Wang); the sponsor statistician (A. Swern) confirmed the efficacy analyses and performed all additional analyses. A per-protocol analysis was performed for the analyses of the primary end point and other airway inflammatory cells in bronchial biopsies due to the scientific question being addressed. The intention-to-treat (allpatients-treated) principle, i.e., inclusion of all patients with a baseline and a post-baseline measurement, was also applied as a corroborative analysis for the bronchial biopsy end points. Change from baseline for the primary endpoint (EG2þ cells) and for the two secondary endpoints, chromotrope-positive cells and tryptase-positive mast cells, was analysed first using the a priori agreed ANOVA model that included factors for centre and treatment. If the 95% confidence interval for the mean change from baseline for a particular treatment group did not contain the value zero, then the within-group change is statistically significant (P < 0.05). After an initial review of the data when still blinded, it was initially thought that no transformation of these data would be required because of the robust nature of the ANOVA method. However, a review of the unblinded data demonstrated a significant number of samples with zero counts at both baseline and post-treatment, and it was agreed to conduct post-hoc analyses more appropriate to the distribution of these data. Models based on the normal distribution, such as the linear regression model, cannot be used even after a log-transformation of cell count data because of the excess zero counts. The Poisson regression model, ordinarily used for count data, is also not appropriate since each of the post-treatment means for each of the three post-treatment counts is much smaller than the corresponding variance, and a property of the Poisson distribution is that the mean and variance are equal. A more appropriate model for the post-treatment counts is the zero-inflated Poisson (ZIP) model, which incorporates an estimate for the excess of zeros over those allowed by the Poisson model.27e29 The baseline cell count is also included as a covariate in the ZIP model analysis to adjust for the baseline difference in the cell counts. The study was designed such that a sample size of N Z 24 in each group yielded 80% power to detect (at an a Z 0.05, two-tailed test) a 50% reduction from baseline in the number of EG2þ cells present in bronchial biopsy in the montelukast group compared with placebo. This statement was based on data from previous studies30,31 where the coefficient of variation was 86% (standard deviation [SD] for the log-transformed variable was 75%). Compared with placebo, changes to RBM thickness as the result of montelukast treatment were assessed by Mann Whitney U test.

Results Disposition of subjects Of the 88 patients randomised to the study [43 M, 45 P], 75 patients [85.2%; 38 M, 37 P] had evaluable biopsies and were included in the per-protocol dataset. One patient in the placebo group was discontinued from the study due to

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airway inflammation and pneumonia. Data from three patients taking montelukast and five patients taking placebo were invalid or were missing biopsies at either baseline or post-treatment. Data from one patient taking montelukast and one taking placebo were eliminated because their PC20 values were not in the protocol-specified range. Data from one patient taking montelukast and one taking placebo were removed from the per-protocol analysis dataset because the patients had not taken the study drug on one of two days before the biopsy (Fig. 1). Table 1 summarises baseline characteristics for the remaining 75 per-protocol patients. There were no clinically meaningful differences between the montelukast and placebo groups.

Counts of inflammatory cells Against expectation, eosinophils were found variably and infrequently and, when present, often clustered together. In contrast, mast cells were always present, frequent and evenly distributed throughout the tissue. The data for cell counts per unit area are summarised in Figs. 2ae4a and demonstrate the high proportion of sections in which EG2þ (48%) and chromotropeþ (24%) cells were undetected at baseline. There was no suggestion of an association between the presence or absence of biopsy eosinophils and a subject history of allergy. Individual patient data for each cell type are shown in Figs. 2be4b.

EG2-positive cells Using the ANOVA statistical model, there was a significant within-group decrease in the mean EG2þ cell count, from 13.54 cells/mm (at baseline) to 0.79 cells/mm in the

montelukast group (P < 0.05) with an LS mean change and associated 95% confidence interval of 13.59 [25.45, 1.74] cells/mm. By comparison, the change in the placebo group, from 6.70 to 6.34 cells/mm, was not statistically significant, with an LS mean change of 1.17 (95% CI [13.26, 10.91]) cells/mm. The difference between the montelukast and placebo groups in the change from baseline in EG2þ cells was not statistically significant (P Z 0.13). In contrast, the ZIP model, more appropriate to the observed non-normal distribution of these data, shows that compared with placebo and after controlling for the excess zeros in EG2þ cell counts at baseline and post-treatment, montelukast reduced the post-treatment EG2þ counts by 80.3% (95% CI [70.6%, 86.8%]) (Table 2).

Chromotrope-positive cells Using the ANOVA statistical model, there was a significant (P < 0.05) within-group decrease after montelukast treatment in the mean chromotropeþ cell count from 3.89 cells/ mm (at baseline) to 1.13 cells/mm; the LS mean change was 2.65 (95% CI [4.69, 0.61]) cells/mm. By comparison, the placebo group showed a decrease from 2.80 to 2.17 cells/mm, which was not statistically significant; with the LS mean change of 0.42 (95% CI [2.50, 1.66]) cells/ mm. The difference between montelukast treatment and placebo in the change from baseline in chromotropeþ cells was not statistically significant (P Z 0.115). However, after applying the ZIP model, which took into account the excess zeroes in the distribution, the average reduction in chromotropeþ cells was 54.4% (95% CI [28.0, 71.1]; P < 0.001).

Randomized (88)

Montelukast Randomized (43)

Placebo Randomized (45) Discontinued (1) airway inflammation and pneumonia

Discontinued (4) invalid or missing biopsy data (3) biopsy not in correct range for PC20 hyperresponsiveness (1)

Discontinued (1)

Montelukast Completed study (43)

Montelukast Valid biopsy data (39)

Placebo Completed study (44)

Placebo Valid biopsy data (38)

Discontinued (6) invalid or missing biopsy data (5) biopsy not in correct range for PC20 hyperresponsiveness (1)

Discontinued (1) eliminated for not having taken study drug both days prior to biopsy

eliminated for not having taken study drug both days prior to biopsy

Montelukast Per protocol dataset (38)

Figure 1

Placebo Per protocol dataset (37)

Subject disposition.

Anti-inflammatory effects of montelukast Patient baseline characteristics.

Characteristic

Montelukast N

Sex Male Female History of allergic conjunctivitis Yes No History of allergic rhinitis Yes No History of allergic conjunctivitis or allergic rhinitis Yes No Positive skin test to at least one allergen Yes No

Age (years) Weight (kg) Height (cm) Duration of asthma (years) Smoking history (pack-years) FEV1 (percent predicted) FEV1 (percent reversibility) Blood eosinophils (103/mL) Histamine PC20 (mg/mL)

Placebo %

N

Total %

N

%

19 19

50.0 50.0

13 24

35.1 64.9

32 43

42.7 57.3

13 25

34.2 65.8

11 25

30.6 69.4

24 50

32.4 67.6

10 28

26.3 73.7

13 24

35.1 64.9

23 50

30.7 69.3

29 9

76.3 23.7

25 12

67.6 32.4

54 21

72.0 28.0

28 9

75.7 24.3

30 6

83.3 16.7

58 15

79.5 20.6

N Z 38 Mean (SD) 29.2 (7.7) 67.9 (10.6) 167.5 (8.6) 16.1 (10.7) 0.9 (2.2) 86.7 (15.8) 11.4 (8.2) 0.4 (0.3) 0.5 (0.7)

Median 27.0 65.8 167.0 16.2 0 85.0 10.3 0.2 0.2

Tryptase-positive mast cells By ANOVA, there was a significant (P < 0.05) within-group decrease in the mean tryptase-positive mast cell count from 164.64 cells/mm (at baseline) to 98.83 cells/mm in the montelukast group; the LS mean change was 68.00 (95% CI [103.91, 32.08]) cells/mm. The placebo group showed a within-group decrease from 129.57 to 101.35 cells/mm, which was not statistically significant; the LS mean change was 28.59 (95% CI [65.21, 8.03]) cells/mm. The difference between the placebo and montelukast groups in the change from baseline in mast cells was not statistically significant (P Z 0.114). After taking into account the excess zeros in the distribution, the average reduction in mast cell numbers was 12.1% (95% CI [7.9, 16.0]; P < 0.0001). There was no significant difference between the montelukast and placebo groups in epithelial RBM thickness. The median change was þ0.6 mm (95% CI [0.3, 1.7]) for montelukast and 0.5 (95% CI [1.2, 0.2]) for placebo.

N Z 37 Mean (SD) 30.6 (7.9) 69.4 (13.4) 166.5 (9.5) 14.6 (10.0) 1.6 (5.6) 89.3 (13.6) 11.3 (11.1) 0.3 (0.2) 0.4 (0.5)

Median 27.0 65.8 166.0 16.1 0 87.9 8.2 0.3 0.2

N Z 75 Mean (SD) 29.9 (7.8) 68.6 (12.0) 167.0 (9.0) 15.4 (10.4) 1.2 (4.3) 88.0 (14.7) 11.3 (9.7) 0.3 (0.2) 0.4 (0.6)

Median 27.0 65.8 166.0 16.2 0 87.2 9.4 0.3 0.2

change within the placebo group was not statistically significant. Application of the pre-specified ANOVA analysis demonstrated that the difference between montelukast and placebo in change of EG2þ cells was not statistically significant (P Z 0.13). However, there were a significant number of zero values in the data set, in excess of those anticipated. In order to take account of the excess of zero values in these non-normally distributed data, application 1000.0

EG2-positive Cells (cells/mm)

Table 1

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baseline visit 4

100.0

10.0

1.0

Discussion 0.1

The present study shows that 6 weeks of treatment with montelukast was associated with a significant within-group reduction of EG2þ cells, from 13 cells/mm at baseline to fewer than 1 cell/mm at the end of treatment, whereas the

Placebo (N = 37)

Montelukast (N = 38)

Figure 2 EG2-positive eosinophils. Graphical representation of the data for EG2-positive eosinophil counts per unit area (arrows Z median values).

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Chromotrope-positive Cells (cells/mm)

1000.0

baseline visit 4

Table 2 Results from the zero-inflated Poisson (ZIP) model analysis. Cell type

100.0

Treatment P-value effect on incidence rate

10.0

Post-EG2 0.1966 Post-mast 0.8795 Post-chromotrope 0.4558

1.0

95% confidence interval Lower limit

Upper limit

<0.0001 0.1312 <0.0001 0.8396 0.0010 0.2887

0.2944 0.9213 0.7198

Percent reduction expressed as (1.00  incidence rate)/ 1.00)  100. 0.1 Placebo (N = 37)

Montelukast (N = 38)

Figure 3 Chromotrope-positive eosinophils. Graphical representation of the data for chromotrope-positive eosinophil counts per unit area (arrows Z median values).

of the ZIP statistical model demonstrated that, compared with placebo, montelukast significantly reduced the number of post-treatment EG2þ cells by approximately 80% (P < 0.0001). The data for total eosinophils showed a similar pattern of change. In addition, there was a 12% reduction in mast cell numbers with montelukast treatment, also significant by ZIP analysis. The pro-inflammatory nature of LTs and anti-inflammatory effects of LTRAs have been reviewed.9e11 More recent studies of exhaled breath condensate in asthmatic patients treated with corticosteroids demonstrate that, in many, suppression of inflammation by corticosteroids is incomplete. In these patients, the LTRA montelukast, given for 4 weeks, reduces cysLT as well as LTB4 concentrations and improves symptoms.32 Montelukast reduces the number of sputum eosinophils in asthma,21 reduces eosinophil cationic protein (ECP) and LT concentrations in nasal washes, and improves lung function in children with persistent asthma.18,24 Moreover, patients with mild asthma challenged with allergen respond with an early-phase (0e3 h) and subsequent late-phase (3e8 h) reduction in lung function: both the early-phase and, importantly, the late-phase asthmatic responses are significantly attenuated by pre-

Tryptase-positive Cells (cells/mm )

1000

baseline visit 4

100

10

1 Placebo (N = 37)

Montelukast (N = 38)

Figure 4 Tryptase-positive mast cells. Graphical representation of the data for tryptase-positive mast cell counts per unit area (arrows Z median values).

treatment with montelukast.33 This effect of montelukast is not attributable to direct bronchodilation, as baseline measures of lung function were unchanged: it likely reflects the suppression of smooth muscle constriction induced by the cysLTs released during the inflammatory response. There may also be chronic effects of LTs that affect the processes of airway smooth proliferation and remodelling. LTD4 potentiates epidermal growth factor-induced increases in myocyte DNA synthesis and cell number, and this effect can be abolished by LTRAs.34 In addition, montelukast can attenuate the increase of allergen challengeinduced myofibroblast formation and the in vitro migration of airway smooth muscle that occurs in response to platelet-derived growth factor; both are thought to play a role in the process of airway wall remodeling.35e37 In a placebo-controlled endobronchial biopsy study of asthmatics treated with the LTRA pranlukast for 4 weeks, immunohistologic analyses revealed that LTRA administration reduced the numbers of eosinophils, mast cells and Tcells in addition to improving patients’ asthma symptoms.25 In a double-blind parallel comparison of fluticasone and montelukast in bronchial biopsies obtained from 36 mild atopic asthmatics treated for 8 weeks, both treatments showed a similar non-significant trend to reduced numbers of tissue eosinophils, whereas the significant reduction of mast cells seen with fluticasone was not achieved following montelukast.38 Thus, on balance the evidence of the present study in combination with other published data suggests that treatment with the cysteinyl LTRA montelukast results in anti-inflammatory effects in patients with mild asthma. Moreover, these data in humans are supported by studies of animal models of asthma in which montelukast has been shown to block or reverse sub-acute allergen-induced eosinophilic inflammation and structural changes that include increases of smooth muscle mass and subepithelial fibrosis.39 In respect of the last-mentioned, the lack of effect on the RBM thickness in the present human study is likely a reflection of the relatively short duration of treatment. It has been shown previously that although antiinflammatory treatment with inhaled corticosteroids reduces inflammation within the first 3 months, reduction of RBM thickness in humans requires at least 1 to 2 years of treatment.5,6 Our study was neither designed nor powered to examine the effects of intervention on the remodelling process. Studies of longer duration will be needed to assess the effect of montelukast on airway RBM thickening and the

Anti-inflammatory effects of montelukast capacity of montelukast to further reduce the eosinophilia when added to optimal steroid therapy.

Variability of eosinophils At the time the study was designed and conducted (1995) it was commonly thought that inflammation was a cardinal feature of all asthma and that eosinophils would be present in the bronchial biopsies of all asthmatics. However, we observed a more erratic inter- and intra-biopsy distribution of eosinophils than expected, which challenged the assumption of broad eosinophilic inflammation in asthma. Thus, we were forced to reconsider the most appropriate analyses for these data. The observations of eosinophil scarcity in subsets of asthmatics have since been repeated and today there is formalised acceptance of asthma phenotypes, in part, based on the predominant pattern of inflammatory cells. One of these is characterised by eosinophil paucity and neutrophil predominance.40,41 Analyses made prior to and since the present study have shown that there is large variation in counts of inflammatory cells between patients, between airway generations, between biopsies, between tissue sections, around the internal circumference of the airway, and with time. Richmond et al.42 looked at variability within biopsies, lobes and patients in 12 mild asthmatic subjects biopsied 1 month apart, noting large intra-subject variations with time that outweighed intra-subject bronchoscopy differences. This and a previous study by Bentley and colleagues43 showed that using three or more step sections from the same biopsy gave relatively small coefficients of variability for a range of inflammatory cells. However, the study by Sullivan and co-workers26 found a high variance in bronchial biopsy inflammatory cell counts, noting that they were not normally distributed and that the cumulative weighted mean did not become stable until 5e10 mm of tissue underlying the RBM had been included. They calculated that the median number of sections required from a biopsy before the cumulative weighted mean was within 20% of the final result was two sections, but that the 75th percentile was six sections. Few studies have included such amounts of tissue in their analyses, including the present one. Since the present study was conducted, Gamble and colleagues44 have shown in subjects with chronic obstructive pulmonary disease (COPD) that the largest contribution to variability is time (39%), followed by airway generation (23%), biopsy (2.5%), zone within a tissue section (1.4%) and section (0.4%). In another study, the variability around the internal circumference of the airway of a CD8þ inflammatory cell (less variable than the eosinophil) was considerable, and the authors concluded that in order to achieve a representative count and to maximise statistical power to detect differences between study populations, biopsy tissue including a minimum of 5 mm of RBM should be examined.45 These observations of variability provide a possible explanation for the zero values for eosinophils that we observed in the present study. In addition, it must be recognised that the finding of a large number of zero values might represent a true biologic finding, given that most of the patients with zero values at baseline had zero values after treatment in either group. This finding could be consistent with the hypothesis that the eosinophil might be

1001 playing a supportive role rather than a primary role or that it is important in some patients and not in others.46 In addition, we noted that, unlike several of the cell types examined in the aforementioned studies, the eosinophils in our biopsies were not evenly distributed throughout the tissue (in contrast to mast cells), but rather were usually present as clusters, with large areas of tissue devoid of them. Such a sporadic distribution, together with the mild nature of the asthma in our subjects and an analysis of a single tissue section and biopsy from each subject likely all contribute to the excess of zero values seen in our primary endpoint. Yet despite these contributions to variability, there was still a significant reduction of eosinophils and mast cells afforded by montelukast treatment when the statistical model appropriate to the excess of zero values was applied. Further, this study highlights the critical importance of collaboration, standardisation, quality control, and the application of methodology appropriate and tailored to the study design and the question being asked.47

Conclusion Whilst there was a significant within-group reduction, the a priori agreed ANOVA did not demonstrate that montelukast given over 6 weeks significantly altered the change from baseline in the numbers of EG2þ bronchial mucosal eosinophils in comparison with placebo. However, the appropriateness of applying the pre-specified ANOVA analysis to the number of zero values observed after unblinding was questioned. Post-hoc analysis using the zero-inflated Poisson (ZIP) statistical model was more appropriate to these data as it controlled for the excess of zero values in the non-normally distributed baseline and post-treatment cell counts. Applying the ZIP model demonstrated that montelukast treatment significantly reduced the number of EG2þ (‘‘activated’’) eosinophils, chromotropeþ (total) eosinophils and mast cells.

Acknowledgements The study was sponsored and financed by Merck & Co., Inc., Rahway, NJ who were involved in its design, collection of study specimens, statistical analyses and decision to submit. Investigators for the protocol 059 study were: T. Haahtela, Finland; F. Galleguilos, Chile; R.J. Davies, UK; J. Martinez, Guatemala; and C. Villaran, Peru. We acknowledge the valued contributions of Drs D. Li, D. Wang, B. Knorr and J. Leff and Mr. T. Ansari and editorial assistance from Dr C. Hustad.

Conflict of interest statement CFR has received meeting sponsorship from Merck Inc., has attended Merck advisory boards and received fees for doing so and his department has been in receipt of a donation towards running costs for research work involving montelukast. During the conduct of the clinical trial reported above CR was in receipt of monies from Merck that contributed towards his salary, in part. NB has acted on Merck and

1002 GSK advisory boards and lectured at Merck-sponsored symposia for which he has received remuneration. PKJ has been reimbursed by GSK, AZ and Merck for attending international conferences and participating as a paid speaker, has served as a paid consultant to GSK, AZ and Argenta and received research grants from GSK, Argenta, AZ and Merck, including a grant to conduct the analyses of the study presented herein. TFR and ASS are employees of Merck Inc. and may own stock or stock options. DW, MG and PS have no declared or perceived conflicts of interest.

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15.

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Further reading Korn D, Van den Brande P, Potvin E, Dramaix M, Herbots E, Peche R. Efficacy of add-on montelukast in patients with non-controlled asthma: a Belgian open-label study. Curr Med Opinion 2009;25: 489e97.