Effect of the Addition of Montelukast to Inhaled Fluticasone Propionate ...

Effect of the Addition of Montelukast to Inhaled Fluticasone Propionate on Airway Inflammation Siobha´n O’ Sullivan, Martijn Akveld, Conor M. Burke, and Leonard W. Poulter Department of Clinical Immunology, Royal Free and University College School of Medicine, London; R&D GlaxoSmithKline, Uxbridge, United Kingdom; and Department of Respiratory Medicine, James Connolly Memorial Hospital, Dublin, Ireland

The aim of the study was to investigate the effect of addition of montelukast to inhaled fluticasone propionate (FP) therapy, compared with FP therapy alone (100 ␮g twice a day) on airway immunopathology in individuals with mild asthma. Twenty-eight subjects received FP (100 ␮g twice a day) or FP (100 ␮g twice a day) plus montelukast (10 mg at night) for 8 weeks and were then crossed over to the alternate treatment for a further 8 weeks. Physiological measurements and bronchial biopsies were obtained at ⫾ 2 days before treatment and ⫾ 2 days at the end of each treatment period. A two-period crossover analysis was performed and the mean and SE were calculated. There was no significant difference in percent predicted FEV1 (p ⫽ 0.51) or PC20 mg/ml (p ⫽ 0.81) between the two treatment regimes after 8 weeks of therapy. There was no difference in the efficacy of either treatment in decreasing T cell (p ⫽ 0.97), CD45RO⫹ (p ⫽ 0.37), mast cell (p ⫽ 0.37), or activated eosinophils (p ⫽ 0.55) numbers in bronchial biopsies. There was no significant difference in the percentage area stained for IFN-␥ (p ⫽ 0.76) or interleukin-4 (p ⫽ 0.61) between treatments. Reduction of inflammatory cell numbers in the bronchial mucosa achieved with FP plus montelukast was not significantly different from the reduction observed with FP alone in individuals with mild asthma. Keywords: fluticasone; montelukast; inflammation; asthma

Airway inflammation is considered to be an integral part of the pathogenesis of asthma. A significant inflammatory component has been observed even in patients with mild to moderate disease (1). Corticosteroids are the most effective antiinflammatory agents currently available for the treatment of asthma (2). Several clinical studies have clearly demonstrated that inhaled corticosteroid (ICS) therapy improves lung function, reduces airway hyperreactivity (3), and mediates a marked reduction in the number of mast cells, macrophages, T lymphocytes, and eosinophils in the bronchial epithelium and submucosa (4). Hence, corticosteroids form the cornerstone of asthma treatment and are recommended as first-line therapy in mild, moderate, and severe persistent asthma (2). Leukotriene receptor antagonists (LTRAs) have been suggested both as suitable monotherapy and add-on therapy to ICS for the treatment of asthma (5, 6). The cysteinyl leukotrienes (LTC4/D4/E4) induce bronchoconstriction, mucus hypersecretion, mucosal edema, enhance airway hyperreactivity, and act as chemoattractants for eosinophils in the airway (7–9). Therefore, it is not surprising that LTRAs improve lung function, attenuate bronchial hyperresponsiveness, and reduce the number of exacerbations in patients with mild to

moderate asthma (10). Moreover, addition of LTRAs to ICS results in better control of asthma (11, 12) and can decrease the requirement for ICS (13, 14). Effects of LTRAs on inflammatory markers are less certain. Treatment with LTRA montelukast has resulted in a significant decrease in serum eosinophil cationic protein and both sputum (15) and peripheral blood eosinophils (16). Calhoun and coworkers have reported reductions in histamine and tumor necrosis factor-␣ concentrations in bronchoalveolar lavage fluid after segmental allergen challenge after treatment with zafirlukast (17). Furthermore, the LTRA montelukast has been shown to reduce nitric oxide in exhaled air, a noninvasive marker of inflammation (18). However, there is a paucity of biopsy data relating to possible antiinflammatory effects of LTRAs in the asthmatic airway. Nakamura and coworkers obtained bronchial biopsies before and after 4 weeks of treatment with pranlukast (225 mg twice a day). There was a reduction in activated eosinophils, T cells, and mast cells compared with the placebo-treated group (19). Whether there is any additional antiinflammatory effect of the LTRAs above and beyond that achieved by ICS in the asthmatic airway is unclear. In this study, we evaluate any additional effect on inflammatory cells in bronchial biopsies and soluble inflammatory mediators of the LTRA montelukast in individuals with mild asthma receiving the ICS fluticasone propionate (FP). METHODS Subjects Thirty-four (18 male/16 female, mean age 27.7 ⫾ 1.1, range 19–50 years) subjects with atopy, mild asthma, and who were nonsmokers were included in the study. Atopy was defined as a positive skin prick test to house dust mite or two other commonly inhaled allergens. Subjects were required to have an FEV1 of 60% or more of the predicted value, a ⌬FEV1 of 12% or more to salbutamol, and a provocative concentration of histamine to cause a 20% drop in FEV1 (PC20) of 4 mg/ml or less. All but one of the subjects had never received corticosteroids before the study, whereas all of them used short-acting ␤-agonists when necessary. Subjects had a pre-existing history of asthma and had been referred to a specialist clinic by their general practitioner due to persistence or worsening of their symptoms. One subject had previously received inhaled corticosteroids; however, this was not within 6 weeks of the commencement of the study. The Ethics Committee of James Connolly Memorial Hospital and The Irish Medicines Board approved the study. The use of a placebo was explained to subjects both orally and in the patient information leaflet. All subjects gave written informed consent before participation.

(Received in original form August 1, 2002; accepted in final form December 4, 2002)

Study Design

Supported by GlaxoSmithKline R&D, UK (Protocol FMS40242).

The study was a double-blind, randomized, crossover study with a 2-week placebo run-in period to familiarize subjects with study procedures. During the run-in period subjects were allocated a placebo Diskus inhaler matched to FP that they received on a twice daily basis and a placebo capsule matched to montelukast (10 mg) that they received at night. At the end of the run-in period, subjects were randomly assigned to receive FP (100 ␮g twice a day) by Diskus inhaler and a placebo

Correspondence and requests for reprints should be addressed to Siobha´n O’ Sullivan, Ph.D., Department of Clinical Immunology, Royal Free and University College Hospital Medical School, London NW3 2QG, UK. E-mail: [email protected] Am J Respir Crit Care Med Vol 167. pp 745–750, 2003 Originally Published in Press as DOI: 10.1164/rccm.200208-783OC on December 12, 2002 Internet address: www.atsjournals.org

746

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 167 2003

montelukast capsule or FP (100 ␮g twice a day) plus a montelukast capsule (10 mg at night) for 8 weeks. Subjects were then crossed over to the alternate treatment for a further 8 weeks. Subjects were also provided with a salbutamol Diskus (200 ␮g/puff) rescue inhaler for use when necessary during the run-in and treatment period for symptomatic relief. Bronchoscopy, pulmonary function, and histamine provocations were performed on the last day of the run-in period and ⫾ 2 days after the 8-week treatment periods. On the same occasion serum and urine samples were collected. When measurements were made 2 days after the 8-week treatment period, medication was continued for the additional 2 days.

Pulmonary Function Tests Pulmonary function tests were performed at the same time in the morning, using a V-Max 22D (Sensor Medics, Yorba Linda, CA) computerized system, and the best of three valid attempts were recorded. Bronchodilators were withheld for 8 hours before testing. Spirometery and histamine provocation were performed as described previously (20). Peak flow measurements were made on a twice daily basis after instruction by the supervising physician, with subjects recording the best of three attempts before taking any inhaled medication.

Bronchial Biopsies Endobronchial biopsies (3⫻) were obtained from the second to fourth generation bronchi on the right side under general anesthesia as described previously (20). Biopsy material was immediately snap frozen in iso-Pentane (BDH, Poole, UK), cooled to ⫺80⬚C, and then stored in liquid nitrogen. Sections, 6 ␮m in thickness, were subsequently cut from the specimens onto poly-l-lysine–coated glass slides (BDH). The integrity and architecture of the tissue was confirmed by staining with toludine blue dye. Sections were then air dried for 1 hour, fixed in chloroform:acetone 1:1 solution for 10 minutes, and were air dried again for a further 20 minutes before storage at ⫺20⬚C.

Immunohistochemistry The number of primed T cells, eosinophils, mast cells, and macrophages per unit area in the lamina propria was determined with an indirect immunoperoxidase method (20). The total T cell concentration was determined by the use of a cocktail of mouse anti-human CD3, CD5, and CD8 (monoclonal antibody raised in the Royal Free and University College School of Medicine) at dilutions of 1 in 5 in phosphate-buffered saline (PBS) pH 7.2. The concentration of primed or memory T cells was determined by the use of mouse anti-human (IgG2␣) anti-CD45RO (University College Hospital, London) at a dilution of 1 in 5 in PBS pH 7.2. Activated eosinophils were determined by a mouse anti-human IgG anti-EG2 (Pharmacia, Uppsala, Sweden) again at a dilution of 1 in 5, whereas total eosinophils were determined by the use of mouse antihuman IgG anti-EG1 (Pharmacia) also at a dilution of 1 in 5 in PBS pH 7.2. Mast cells were determined by a mouse anti-human IgG AA1 antibody at a dilution of 1 in 50 in PBS pH 7.2. Macrophage concentrations were determined by the use of mouse anti-human (IgG) antiCD68 (DAKO, A/S, Denmark) at a dilution of 1 in 10 in PBS pH 7.2. The CD4:CD8 ratio was determined within the lamina propria using an indirect double immunofluorescence technique (20). Mouse antihuman CD4 IgG1 was used at a concentration of 1 in 10 in PBS pH 7.2 in conjunction with mouse anti-human anti-CD8 (DAKO, A/S). The percentage area stained for interleukin-4 and IFN-␥ was assessed by a biotin–alkaline phosphatase–streptavidin technique. Briefly, mouse anti-human IFN-␥ IgG2␣ (R&D Systems Europe, Abingdon, Oxon, UK) and mouse anti-human interleukin-4 IgG1 (Pharmingen, San Diego, CA) were diluted 1 in 100 and 1 in 10, respectively, in PBS⫹ 1% bovine serum albumin (BSA) and incubated overnight. An IgG affinity-purified horse anti-mouse biotinylated second layer (Vector Laboratories, Peterborough, UK) diluted 1/100 in PBS–BSA was added for 1 hour at room temperature. After rinsing in fresh Tris-buffered saline, sections were then incubated with streptavidin–alkaline phosphatase (Vector Laboratories) diluted 1/100 in PBS–BSA for 1 hour at room temperature. Sections were rinsed in fresh Tris-buffered saline, and the reaction was developed by a 15-minute application of a substrate solution (5 mg naphthol phosphate, 10 ml Tris–hydrogen chloride [pH 8.2], 200 ␮l dimethylformamide, 10 mg Fast Red, and 10 drops of levamisole added just before use). Sections were then washed in tap water, distilled

water, and counterstained with Mayers hematoxylin before mounting in PBS:glycerol (9:1). Negative controls were included for each section by omitting primary layer reagents, and positive controls were performed on sections of human palatine tonsil. All slides were coded and counted in a blind fashion by two observers. Positively stained cells in a minimum of three to five framed areas in the lamina propria (15-cell depth) were quantified using an image analysis system (Axiocam; Zeiss, Jena, Germany). The number of cells counted was divided by the area of the frame, and this reduced each framed area to unity. Results are expressed as cells/104 ␮m2. To determine the CD4:CD8 ratio, the total number of CD4⫹ and CD8⫹ cells were determined in five or more randomly selected areas as described previously and expressed as a ratio.

Analysis of Cellular and Soluble Markers Blood sampling and urine collection were performed on the same day the bronchial biopsies were obtained. Subjects provided a 3-hour urinary collection in a 24-hour urinary container without any addition of preservatives. A 50-ml urinary aliquot and serum samples were stored at ⫺20⬚C until analysis. Serum myeloperoxidase was quantified by a sandwich ELISA (Bioxytech-Oxis International, Portland, OR). To obtain a concentration within the assay range, serum samples were diluted at least 10-fold. The lower limit of detection for myeloperoxidase was 1.6 ng/ml. Enzyme immunoassay analysis of urinary 9␣,11␤-PGF2 and LTE4 were performed with polyclonal antisera and acetylcholinesteraselinked tracers (Cayman Chemical Co., Ann Arbor, MI). The detection limit for both assays was 7.8 pg/ml. Urinary eosinophil protein X was quantified with a double antibody radioimmunoassy (Pharmacia and UpJohn, Uppsala, Sweden). The lower limit of detection for eosinophil protein X was 3 ␮g/L. Creatinine was measured in all urine samples by a colorimetric assay, using an alkaline picrate method. Hence, urinary data are expressed as nanograms compound per millimoles filtered creatinine to correct for possible variations in diuresis.

Statistical Analysis Calculations of geometric mean PC20 values were performed on logtransformed data. The study was not powered on the basis of any one inflammatory marker or cell, as different inflammatory processes may be driving the pathology in particular individuals with asthma. The study has the power to detect at least one doubling difference in cell number per 0.1 mm2 (21). End-of-treatment values were analyzed as a two-period crossover using analysis of covariance (ANCOVA), with covariates subject, period, and treatment. The adjusted (least-squares) means from the general linear model, SE, adjusted mean differences (FP-adjusted mean-FP plus montelukast-adjusted mean), and p value were calculated. A p value of less than 0.05 was considered significant. A second confirmatory method was used to analyze the first treatment period change from baseline values using ANCOVA, allowing for effects due to treatment, age, sex, and baseline value. This allowed for consideration of the possible carry-over effect in the second period, due to the lack of a washout period. Analyses with both methods resulted in corresponding data; therefore, the two-period crossover analyses will be presented in this paper. There is a danger with multiple measures that spurious p values may be generated. As there were no significant differences between the two treatment regimes for the majority of outcome measures, this is not a significant concern in this study. Within treatment changes from baseline at the end of treatment were performed using analysis of variance, adjusted for period. Analyses are based on the 28 subjects who completed the study.

RESULTS Twenty-eight of the thirty-four subjects entered completed the study. One of the subjects experienced an exacerbation of asthma after the baseline bronchocopsy that required treatment with oral corticosteroids. Four subjects (two of whom were taking FP plus montelukast and two taking FP only) were lost to follow up (failed to attend for all three bronchoscopies), and one subject

O’ Sullivan, Akveld, Burke, et al.: Addition of Montelukast to Fluticasone

747

TABLE 1. TWO-PERIOD CROSSOVER ANALYSIS OF LUNG FUNCTION PARAMETERS AFTER TREATMENT WITH FLUTICASONE PROPIONATE AND FLUTICASONE PROPIONATE PLUS MONTELUKAST

FEV1, % predicted PC20, mg/ml‡ PEF morning, L/min PEF evening, L/min

Baseline*

FP*

FP ⫹ Montelukast*

p Value†

90.7 ⫾ 3.4 0.43 (0.05–16.65) 441 ⫾ 15.6 453 ⫾ 15.8

93.7 ⫾ 3.3 1.47 (0.05–3.95)§ 463 ⫾ 16.3 473 ⫾ 15.5

92.6 ⫾ 2.8 1.59 (0.06–16)§ 463 ⫾ 15.5 472 ⫾ 14.8

0.51 0.81 0.46 0.19

Definition of abbreviations: FP ⫽ fluticasone propionate; PC20 ⫽ provocative concentration of histamine causing a 20% drop in FEV1. * Mean ⫾ SE. † Comparison of FP and FP plus montelukast groups. ‡ Geometric mean and range. § Within group mean change from baseline p ⬍ 0.05.

was withdrawn when found to be noncompliant with medication during the FP plus montelukast treatment period. No other adverse events were reported.

or increase in percentage IFN-␥ staining (p ⫽ 0.76) compared with treatment with FP (Figures 2A and 2B).

Pulmonary Function Tests

There was a statistically significant decrease from baseline in blood eosinophils in the combination group (p ⫽ 0.02) not seen in the group treated with FP only (Table 2). The percentage blood eosinophils in the FP plus montelukast group were significantly lower than in the group treated with FP (p ⫽ 0.005). Urinary eosinophil protein X (␮g/L) and LTE4 (ng/mmol creatinine) were decreased from baseline after treatment with both FP plus montelukast and FP alone; however, there was no difference between treatments (Table 2). Serum myeloperoxidase (ng/ml) remained unchanged from baseline levels in both treatment groups, with no difference observed between treatments. In contrast, levels of the mast cell marker 9␣,11␤-PGF2 (ng/mmol cr) increased from baseline after treatment with FP plus montelukast, whereas treatment with FP resulted in a slight decrease. Hence, there was a statistically significant difference between the two treatments with respect to this PGD2 metabolite (p ⫽ 0.043) (Table 2).

Over the 8-week period no significant change in FEV1 (% predicted) was recorded after treatment with FP or FP plus montelukast, and there was no difference between the two treatments (Table 1). Treatment with both regimes resulted in a statistically significant increase from baseline in PC20 (mg/ml); however, improvements in the FP plus montelukast group were not different from that observed in the FP group (Table 1). Morning and evening PEF measurements increased from baseline after treatment with FP and with FP plus montelukast; however, this increase did not reach statistical significance in either group, and there was no difference between the treatment groups (Table 1). Bronchial Biopsies

T cells were significantly reduced from baseline after 8 weeks of treatment with FP plus montelukast (5.49 ⫾ 0.5 vs. 3.83 ⫾ 0.26; p ⫽ 0.01), but not after treatment with FP (3.85 ⫾ 0.46; p ⫽ 0.2). (Figure 1A). However, the reduction in T cells (p ⫽ 0.97) and CD45RO cells (p ⫽ 0.37) (Figure 1B) between treatment groups was not different. Treatment with both the combination (2.07 ⫾ 0.2 vs. 0.86 ⫾ 0.12; p ⫽ 0.001) and with FP alone (1.09 ⫾ 0.12; p ⫽ 0.046) decreased the number of eosinophils (EG1) from baseline (Figure 1C). Similarly, the number of activated eosinophils (EG2) was also decreased from baseline when subjects were treated with FP plus montelukast (1.02 ⫾ 0.11 vs. 0.30 ⫾ 0.07; p ⫽ 0.002) and FP (0.40 ⫾ 0.1; p ⫽ 0.04). (Figure 1D). There was, however, no difference in the number of eosinophils (p ⫽ 0.25) or activated eosinophils (p ⫽ 0.55) between subjects treated with FP alone and FP plus montelukast. Mast cell numbers were significantly decreased from baseline in the group treated with FP (2.86 ⫾ 0.33 vs. 1.33 ⫾ 0.19; p ⫽ 0.03). There was also a reduction in mast cells in the group treated with FP plus montelukast (1.50 ⫾ 0.24; p ⫽ 0.04); however, there was no difference between the two treatment groups (p ⫽ 0.37) (Figure 1E). The number of macrophages did not significantly decrease from baseline after treatment with FP or FP plus montelukast. After 8 weeks of therapy there was no difference between the treatment groups (p ⫽ 0.24) (Figure 1F). After treatment with FP for 8 weeks, the percentage interleukin-4 staining significantly decreased from baseline (23.3 ⫾ 1.5 vs. 18.8 ⫾ 0.8; p ⫽ 0.03); however, this was not seen in the FP plus montelukast group (p ⫽ 0.29). Percentage IFN-␥ staining did not increase significantly from baseline in either treatment group. Treatment with FP plus montelukast did not result in a significantly greater decrease in percentage interleukin-4 (p ⫽ 0.61)

Cellular and Soluble Markers

DISCUSSION Despite treatment with ICS, patients with incompletely controlled asthma who require additional therapy still remain, and there are now a number of studies in the literature investigating the effect of LTRAs in patients taking steroids (11–14). However, the remit of these studies has been largely confined to the additional benefits of LTRAs on lung function and symptoms. To the best of our knowledge this is the first study that examines the potential benefit of the addition of a LTRA to an ICS with respect to modulating inflammation in the asthmatic lung. We could not document any significant difference in the number of T cells, mast cells, eosinophils, or macrophages in the bronchial muscosa between individuals with asthma treated with FP or the combination FP plus montelukast. The reduction in inflammatory cells in the bronchial mucosa of individuals with mild to moderate asthma after treatment with FP is well documented (22, 23). Nakamura and coworkers have reported a reduction in the number of T cells, mast cells, and activated eosinophils in the bronchial mucosa of 17 individuals with mild to moderate asthma, after 4 weeks of treatment with the LTRA pranlukast in a double blind, placebo-controlled study (19). Furthermore, Pizzichini and coworkers demonstrated that 4 weeks of treatment with montelukast resulted in a decrease in both sputum and peripheral blood eosinophils (16). It would therefore appear that the LTRAs do have antiinflammatory effects both peripherally and in lung. However, the results of the current

748


Figure 1. Mean ⫾ SE of A, T cells, B, CD45RO⫹ cells, C, eosinophils, D, activated eosinophils, E, mast cells F, and macrophages in bronchial biopsies of subjects with asthma before treatment (open bar), after 8 weeks of treatment with FP (closed bar), and after 8 weeks of treatment with FP plus montelukast (hatched bar). Treatment with FP plus montelukast did not result in a significantly greater decrease in any of the cells compared with treatment with FP.

investigation would suggest that the suppression of inflammatory cells by FP in the bronchial mucosa is so profound in individuals with mild asthma that little is gained by the addition of a LTRA such as montelukast. It could be argued that there was insufficient residual inflammation after low-dose ICS treatment to see any effect of the LTRA. On the basis of FEV1 values, the subjects in this study would be classified as ones with mild asthma. However, the mean PC20, arguably a better gauge of inflammation, would place the subjects in the mild to moderate category. Data from pretreatment biopsies clearly indicated that the subjects had an active inflammatory process in their airways, and subjects were treated with the lowest dose of FP ethically permissible. We have demonstrated previously in a group of individuals with asthma with similar characteristics as those in the present study, that 500 mg/ day is the optimal dose for decreasing inflammatory cell numbers in bronchial biopsies (24). Furthermore, Pauwels and coworkers have demonstrated residual inflammation after treatment of up to 1 year with higher does of ICS (25).

Levels of both serum eosinophil cationic protein and urinary LTE4 decreased with treatment; however, there was no difference between treatment groups. Levels of myeloperoxidase were unchanged after either treatment. Airway neutrophilia remains apparent in patients with severe asthma, even after treatment with high doses of corticosteroids (26). Interestingly, levels of the mast cell marker, 9␣,11␤-PGF2 decreased after FP treatment but increased when montelukast was added to the treatment regime. Mast cells numbers in the bronchial mucosa were decreased after treatment with both FP and FP plus montelukast. Thus, the effect of montelukast does not seem to be on the mast cells themselves. On a speculative note, montelukast may be indirectly affecting cyclooxygenase or 11-ketoreductase resulting in increased quantities of PGD2, the parent compound, or 9␣,11␤-PGF2, respectively. Prostaglandin E2 levels in rats pretreated for 12 days intracolonically with montelukast were increased 12 hours after trinitrobenzene sulphonic acid–induced colitis (27). It should be noted that in the case of both eosinophils and mast cells, indirect markers did not correlate well with the

Figure 2. Mean ⫾ SE percentage area stained for IFN-␥ and interleukin-4 before (open bar) and after treatment with FP (closed bar) and FP plus montelukast (hatched bar).

O’ Sullivan, Akveld, Burke, et al.: Addition of Montelukast to Fluticasone

749

TABLE 2. TWO-PERIOD CROSSOVER ANALYSIS OF INFLAMMATORY CELL/MEDIATORS AFTER TREATMENT WITH FLUTICASONE PROPIONATE AND FLUTICASONE PROPIONATE PLUS MONTELUKAST Baseline* Blood eosinophils, % Serum MPO, ng/ml Urinary EPX, ␮g/L Urinary LTE4, ng/mmol cr Urinary 9␣,11␤-PGF2, ng/mmol cr

5.97 80.1 173.4 22.2 70.5

⫾ ⫾ ⫾ ⫾ ⫾

0.95 8.7 67.7 3.7 10

FP* 3.96 79.8 137.2 16.2 64.1

⫾ ⫾ ⫾ ⫾ ⫾

0.56 7.9 28.6 2.0 8.1

FP ⫹ Montelukast*

p Value†

⫾ ⫾ ⫾ ⫾ ⫾

0.005 0.67 0.70 0.34 0.04

2.88 83.0 119.8 19.1 108

0.35‡ 8.1 15.7 3.8 22.2

Definition of abbreviations: EPX ⫽ eosinophil protein X; FP ⫽ fluticasone propionate; MPO ⫽ myeloperoxidase. * Mean ⫾ SE. † Comparison of FP and FP plus montelukast groups. ‡ Within-group mean change from baseline, p ⬍ 0.05.

cell data from the biopsies. Thus, when direct measures of markers of inflammation are inaccessible, consideration should be given to using combinations of inflammatory mediators, e.g., in the case of the mast cell use of histamine and 9␣,11␤-PGF2 may prove useful. In the current investigation, we did not document any significant benefit in terms of FEV1 with the addition of montelukast to FP. With use of montelukast in conjunction with 400 ␮g/day of beclomethasone dipropionate, Laviolette and coworkers could demonstrate significant additional benefit in terms of improving FEV1 in individuals with mild to moderate asthma (6). However, the percent predicted FEV1 of these subjects at baseline was markedly less than that of the current subjects, potentially allowing more room for improvement. Pranlukast and montelukast have been shown to significantly improve adenosine monophosphate and methacholine PC20 respectively, when compared with placebo in individuals with mild to moderate asthma (15, 28). In the current study, the geometric mean histamine PC20 significantly increased from baseline in both treatment groups, however, there was no difference between the groups. Dempsey and coworkers have recently reported that the methacholine PD20 (provocative dose to cause a 20% drop in FEV) was significantly greater in a group of 24 individuals with mild to moderate asthma receiving 100 ␮g of beclomethasone, plus the LTRA, zafirlukast, compared with 100 ␮g of beclomethasone alone (29). The disparity between this and the current study is more than likely due to the more severe nature of asthma of the subjects in the aforementioned study. Primarily for logistic reasons a washout period was not included in the study design. It was believed that an 18-week clinical trial with three bronchoscopies was sufficiently demanding to warrant exclusion of a washout period. Previous crossover studies with montelukast have included washout periods of up to 4 weeks (30–32). To validate our assumption of no carry-over effect, the first treatment period change from baseline values was analyzed using ANCOVA, allowing for effects due to treatment, age, sex, and baseline value. Results obtained were in accordance with those generated using the two-period crossover analyses, making a carryover effect highly unlikely. The position of LTRAs in asthma treatment regimes has not yet achieved consensus. The lack of a dose–response for ICS treatment in asthma validates the preferential use of add-on therapy over increasing the dose of ICS (24). Lofdahl and coworkers have demonstrated the steroid-sparing effects of montelukast in patients requiring moderate to high doses of ICS (13). The findings from the current study would suggest however, that there is very little antiinflammatory benefit to be gained from the addition of montelukast to low-dose FP in individuals with mild asthma. However, in patients with more severe disease (12) and aspirin-intolerant asthma (33), addition of LTRAs has been

demonstrated to be effective, and it may be in these patients that LTRAs should be preferentially used as add-on therapy rather than in patients with mild to moderate asthma. Acknowledgment : The authors thank Ms. Huda Al Doujaily, Mr. Keith Berelowitz, Ms. Suzanne Doyle, and Sisters Anne Sullivan and Mary Toole for excellent technical assistance. They also thank Dr. Liam Cormican, Dr. Edward Moloney, and Dr. Mike McWeeney for clinical support. The authors gratefully acknowledge Ms. Deborah Tandy for her outstanding statistical work.

References 1. Vignola AM, Chanez P, Campbell AM, Souques F, Lebel B, Enander I, Bousquet J. Airway inflammation in mild intermittent and in persistent asthma. Am J Respir Crit Care Med 1998;157:403–409. 2. Global Strategy for Asthma Management and Prevention. National Heart, Lung and Blood Institute Report. National Institutes of Health; Revised 2002. 3. Barnes P. Current issues for establishing inhaled corticosteroids as the antiinflammatory agents of choice in asthma. J Allergy Clin Immunol 1998;101:S427–S433. 4. Djukanovic R, Wison J, Britten K, Wilson SJ, Walls AF, Roche WR, Howarth PH, Holgate ST. Effect of an inhaled corticosteroid on airway inflammation and symptoms in asthma. Am Rev Respir Dis 1992;145: 669–674. 5. Drazen JM, Isreal E. Should antileukotriene therapies be used instead of inhaled corticosteroids in asthma? Yes. Am J Respir Crit Care Med 1998;158:1699–1701. 6. Laviolette M, Malmstrom K, Lu S, Chervinsky P, Pujet JC, Peszek I, Zhang J, Reiss TF. Montelukast added to inhaled beclomethasone in treatment of asthma. Am J Respir Crit Care Med 1999;160:1862–1868. 7. Busse WW. Leukotrienes and inflammation. Am J Respir Crit Care Med 1998;157:S210–S213. 8. Laitinen LA, Laitinen A, Haahtela T, Vilkka V, Spur BW, Lee TH. Leukotriene E4 and granulocytic infiltration into asthmatic airways. Lancet 1993;341:989–990. 9. Gauvreau GM, Parameswaran KN, Watson RM, O’Byrne PM. Inhaled leukotriene E(4), but not leukotriene D(4), increased airway inflammatory cells in subjects with atopic asthma. Am J Respir Crit Care Med 2001;164:1495–1500. 10. Barnes NC. Effects of antileukotrienes in the treatment of asthma. Am J Respir Crit Care Med 2000;161:S73–S76. 11. Reiss TF, Chervinsky P, Dockhorn RJ, Shingo S, Seidenberg B, Edwards TB. Montelukast, a once-daily leukotriene receptor antagonist in the treatment of chronic asthma: a multicenter, randomized, double-blind trial. Arch Intern Med 1998;158:1213–1220. 12. Cristian-Virchow JC, Prasse A, Naya I, Summerton L, Harris A. Zafirlukast improves asthma control in patients receiving high-dose inhaled corticosteroids. Am J Respir Crit Care Med 2000;162:578–585. 13. Lofdahl CG, Reiss TF, Leff JA, Isreal E, Noonan MJ, Seidenberg AF, Capizzi T, Kundu S, Godard P. Randomised, placebo controlled trial of effect of a leukotriene receptor antagonist, montelukast, on tapering inhaled corticosteroids in asthmatic patients. BMJ 1999;319:87–90. 14. Tamaoki J, Kondo M, Sakai N, Nakata J, Takemura H, Nagai A, Takizawa T, Konno K. Leukotriene antagonist prevents exacerbation of asthma during reduction of high-dose inhaled corticosteroid: The Tokyo Joshi-Idai Asthma Research Group. Am J Respir Crit Care Med 1997;155:1235–1240.

750


15. Yoshida S, Ishizaki Y, Shoji T, Onuma K, Nakagawa H, Nakabayashi M, Akahori K, Hasegawa H, Amayasu H. Effect of pranlukast on bronchial inflammation in patients with asthma. Clin Exp Allergy 2000; 30:1008–1014. 16. Pizzichini E, Leff JA, Reiss TF, Hendeles L, Boulet L-P, Wei LX, Efthimiadis AE, Zhang J, Hargreave FE. Montelukast reduces airway eosinophilic inflammation in asthma: a randomized, controlled trial. Eur Respir J 1999;14:12–18. 17. Calhoun WJ, Lavins BJ, Minkwitz MC, Evans R, Gleich GJ, Cohn J. Effect of zafirlukast (Accolate) on cellular mediators of inflammation: bronchoalveolar lavage fluid findings after segmental allergen challenge. Am J Respir Crit Care Med 1998;157:1381–1389. 18. Bisgaard H, Loland L, Oj JA. NO in exhaled air of asthmatic children is reduced by the leukotriene receptor antagonist montelukast. Am J Respir Crit Care Med 1999;160:1227–1231. 19. Nakamura Y, Hoshino M, Sim JJ, Ishii K, Hosaka K, Sakamoto T. Effect of the leukotriene receptor antagonist pranlukast on cellular infiltration in the bronchial mucosa of patients with asthma. Thorax 1998;53:835–841. 20. Burke C, Sreenan S, Pathmakanthan S, Patterson J, Schmekel B, Poulter LW. Relative effects of inhaled corticosteroids on immunopathology and physiology in asthma: a controlled study. Thorax 1996;51:993–999. 21. Sont JK, Willems LN, Evertse CE, Hooijer R, Sterk PJ, van Krieken JH. Repeatability of measures of inflammatory cell number in bronchial biopsies in atopic asthma. Eur Respir J 1997;10:2602–2608. 22. Olivieri D, Chetta A, Del Donno M. Effect of short-term treatment with low-dose inhaled fluticasone propionate on airway inflammation and remodeling in mild asthma: a placebo controlled study. Am J Respir Crit Care Med 1997;155:1864–1871. 23. Faul J, Leonard C, Burke C, Poulter LW. Fluticasone propionate induced alterations to lung function and the immunopathology of asthma over time. Thorax 1998;53:753–761. 24. O’Sullivan S, Cormican L, Murphy M, Poulter LW, Burke CM. Effects of varying doses of inhaled corticosteroids on the physiology and

25.

26.

27.

28.

29.

30.

31.

32.

33.

bronchial wall immunopathology in mild to moderate asthma. Chest 2002;122:1966–1972. Pauwels RA, Lofdahl CG, Postma DS, Tattersfield AE, O’Byrne P, Barnes PJ, Ullman A. Effect of inhaled formoterol and budesonide on exacerbations of asthma: Formoterol and Corticosteroids Establishing Therapy (FACET) International Study Group. N Engl J Med 1997; 337:1405–1411. Louis R, Lau LC, Bron AO, Roldaan AC, Radermecker M, Djukanovic R. The relationship between airways inflammation and asthma severity. Am J Respir Crit Care Med 2000;161:9–16. Holma R, Salmenpera P, Riutta A, Virtanen I, Korpela R, Vapaatalo H. Acute effects of the cys-leukotriene-1 receptor antagonist, montelukast, on experimental colitis in rats. Eur J Pharmacol 2001;429:309–318. Dempsey OJ, Wilson AM, Sims EJ, Mistry C, Lipworth BJ. Additive bronchoprotective and bronchodilator effects with single doses of salmeterol and montelukast in asthmatic patients receiving inhaled corticosteroids. Chest 2000;117:950–953. Dempsey OJ, Fowler SJ, Wilson A, Kennedy G, Lipworth BJ. Effects of adding either a leukotriene receptor antagonist or low-dose theophylline to a low or medium dose of inhaled corticosteroid in patients with persistent asthma. Chest 2002;122:151–159. Minoguchi K, Kohno Y, Minoguchi H, Kihara N, Sano Y, Yasuhara H, Adachi M. Reduction of eosinophilic inflammation in the airways of patients with asthma using montelukast. Chest 2002;121:732–738. Wilson AM, Dempsey OJ, Sims EJ, Lipworth BJ. Evaluation of salmeterol or montelukast as second-line therapy for asthma not controlled with inhaled corticosteroids. Chest 2001;119:1021–1026. Vidal C, Fernandez-Ovide E, Pineiro J, Nunez R, Gonzalez-Quintela A. Comparison of montelukast versus budesonide in the treatment of exercise-induced bronchoconstriction. Ann Allergy Asthma Immunol 2001;86:655–658. Dahlen SE, Malmstrom K, Nizankowska E, Dahlen B, Kuna P, Kowalski M, Lumry WR, Picado C, Stevenson DD, Bousquet J, et al. Improvement of aspirin-intolerant asthma by montelukast, a leukotriene antagonist: a randomized, double-blind, placebo-controlled trial. Am J Respir Crit Care Med 2002;165:9–14.