Randomized Placebo-controlled Study Comparing a Leukotriene ...

Randomized Placebo-controlled Study Comparing a Leukotriene Receptor Antagonist and a Nasal Glucocorticoid in Seasonal Allergic Rhinitis TEET PULLERITS, LEA PRAKS, BENGT-ERIC SKOOGH, RAIVO ANI, and JAN LÖTVALL Lung Pharmacology Group, Department of Respiratory Medicine and Allergology, Institute of Heart and Lung Diseases, Göteborg University, Gothenburg, Sweden; Lung and Otorhinolaryngology Clinic, University of Tartu, Tartu, Estonia

Allergic rhinitis is an inflammatory disorder associated with local leukotriene release during periods of symptoms. Therefore, it has been suggested that antileukotrienes may be beneficial in the treatment of this disease. Leukotriene receptor antagonists have recently become available for asthma treatment, but little is known of their effects on allergic rhinitis. We have evaluated the effects of the leukotriene receptor antagonist zafirlukast versus placebo in patients with allergic rhinitis during the grass pollen season, using the nasal glucocorticoid beclomethasone dipropionate (BDP) as a positive treatment control. Thirty-three patients with seasonal allergic rhinitis were in a double-blind, doubledummy fashion randomized to treatments with oral zafirlukast (20 mg twice a day), intranasal beclomethasone dipropionate (200 mg twice a day), or placebo. The treatment was initiated 3 wk prior to the expected beginning of the grass pollen season. Patients completed a daily symptom-score list for sneezing, rhinorrhea, nasal itch, and nasal blockage during the 50-d treatment period. Nasal biopsies for quantification of local tissue eosinophilia (immunohistochemistry; EG2) were taken 1 mo before initiation of treatment and immediately after the peak of grass pollen season. Patients receiving treatment with zafirlukast had degrees of nasal symptoms similar to those in the placebo group, whereas the BDP group had significantly less symptoms compared with both treatments (p 5 0.01 and p 5 0.005, respectively). The numbers of activated eosinophils in the nasal tissue increased significantly during the pollen season in both the zafirlukast and the placebo groups, but not in the BDP group. These results obtained with a limited number of patients do not support any clinical efficacy of regular treatment with an oral antileukotriene in seasonal allergic rhinitis but rather favor the use of a nasal glucocorticoid. Pullerits T, Praks L, Skoogh B-E, Ani R, Lötvall J. Randomized placebocontrolled study comparing a leukotriene receptor antagonist and a nasal glucocorticoid in seasonal allergic rhinitis. AM J RESPIR CRIT CARE MED 1999;159:1814–1818.

Allergic rhinitis is a common disease characterized by infiltration of inflammatory cells into the nasal mucosa (1–3). In parallel with the cellular inflammation, which is dominated by eosinophils, numerous mediators are released locally during symptomatic allergic rhinitis. Two of the most abundant and potent mediators released are histamine and cysteinyl leukotrienes (4, 5). Exogenous administration of cysteinyl leukotrienes into the nose increases nasal resistance and local blood flow and causes local exudation of plasma (6), suggesting that this group of mediators is not only released in allergic rhinitis but may also cause some of the pathophysiologic changes observed in the (Received in original form October 5, 1998 and in revised form January 12, 1999 ) Supported by the Vårdal Foundation. No financial support was received from the pharmaceutical industry for this study. Dr. Pullerits is the recipient of a grant from the King Oscar Foundation. Dr. Lötvall is the recipient of a grant from Herman Krefting’s Foundation. Correspondence and requests for reprints should be addressed to Jan Lötvall, Lung Pharmacology Group, Department of Respiratory Medicine and Allergology, Göteborg University, Guldhedsgatan 10A, S-413 46 Gothenburg, Sweden. Am J Respir Crit Care Med Vol 159. pp 1814–1818, 1999 Internet address: www.atsjournals.org

disease. However, to definitely determine whether a mediator is involved in a disease, it is important to evaluate the effects of receptor antagonists or synthesis inhibitors. Three studies using antileukotriene drugs in rhinitis have been presented. In an allergen challenge study a 5-lipoxygenase inhibitor attenuated nasal congestion, but not sneezing (7). A placebo-controlled parallel group study in patients with ragweed-induced nasal symptoms showed that a single dose per day of 20 or 40 mg, but not 100 mg, of the leukotriene receptor antagonist zafirlukast slightly improved allergen-induced symptoms of nasal congestion compared with placebo, and symptoms of sneezing and rhinorrhea were also significantly reduced (8). The third study suggested that 4 wk of treatment with the leukotriene receptor antagonist pranlukast improved symptoms by only 24% compared with placebo on Week 4 of treatment (9). However, one allergen provocation study could not confirm any effect of a leukotriene antagonist in early allergic reactions in the nose (10). Thus, only a limited number of placebo-controlled studies suggest that cysteinyl leukotriene antagonists may have a small beneficial effect on allergic rhinitis. Importantly, however, there is no information about how this group of compounds compares with the most efficacious and safe treatment available, which is locally administered glucocorticoids (11).

Pullerits, Praks, Skoogh, et al.: Leukotriene Antagonist in Allergic Rhinitis

It has been suggested that treatment with an oral leukotriene antagonist may have the capacity to attenuate the symptoms of allergic rhinitis during the pollen season, but also that any such effect may be associated with decreased local eosinophilic inflammation since cysteinyl leukotrienes have been shown to be chemotactic for eosinophils (12). To test this theory, we randomized patients allergic to grass in a double-blind fashion to treatment with either oral zafirlukast (20 mg twice a day) or placebo during the pollen season. A third group was treated with an intranasal glucocorticoid because this treatment has previously been shown to be highly effective in attenuating both symptoms of allergic rhinitis as well as the local eosinophilic inflammation (13–15).

METHODS The study was approved by the Ethics Review Committee of Clinical Research Studies of the University of Tartu, and all patients gave their written informed consent.

Patients and Study Design Thirty-three patients (11 women and 22 men 16 to 45 yr of age; mean age, 27 yr) with a known history of allergic rhinitis during the grass pollen season the two previous years were recruited into the study. Allergy to grass pollen was confirmed by a positive skin prick test to three common grass pollen extracts (Phleum praténse, Lólium perénne, and Festúca praténsis) (ALK, Hørsholm, Denmark). Exclusion criteria were presence of any significant concurrent illness, symptoms of perennial rhinitis, or positive skin prick test against tree pollens appearing earlier in the year. Also, patients were excluded if they had a history of acute respiratory tract infections or had used glucocorticoids in any form within 2 wk prior to the inclusion visit. One month prior to the start of treatment, nasal biopsies were taken with cup and ring biopsy forceps (Entermed B.V., Woerden, Holland) from the lower border of the inferior turbinate after anesthesia with 10% lidocaine spray and subsequent cotton sponge with epinephrine (1 mg/ml) to reduce bleeding. After biopsies had been taken, the patients were randomized in a double-blind, doubledummy fashion to receive treatment with beclomethasone dipropionate nasal spray (BDP; 50 mg per dose) (Beclonase; Glaxo/Wellcome, Mölndal, Sweden) twice in each nostril twice a day (a total dose of 400 mg daily), the leukotriene receptor antagonist zafirlukast (Accolate 20 mg; Zeneca Pharmaceuticals, Wilmington, DE) purchased from IDIS Limited (Kingston upon Thames, UK) in capsules taken twice a day, or matching placebo treatments for both nasal spray and capsules (produced by Apoteksbolaget, Göteborg, Sweden) (n 5 11 in each group). The efficacy of 400 mg of BDP and 40 mg of zafirlukast daily has been previously demonstrated in seasonal allergic rhinitis (16) and asthma (17), respectively. Treatment was started 3 wk prior to the expected beginning of the grass pollen season, and patients were instructed to administer all drugs 1 h before or 2 h after meals. During the whole treatment period, patients recorded their daily symptoms for sneezing, rhinorrhea, nasal itch, and blockage using a score from 0 to 4, where 0 5 symptom not present, 1 5 symptom present, no discomfort, 2 5 some discomfort, 3 5 marked discomfort, no interference with activities, 4 5 interference with daily activities. Patients were allowed to use a short-acting antihistamine as a symptom-relieving drug and cromolyn eye drops for controlling eye symptoms. Use of antihistamine was, however, recorded as three points on the symptom score scale. This scoring system results in a maximum of 19 symptom points per day (18). During the late part of the grass pollen season, 6 wk after start of the treatment, the second nasal biopsy samples were taken as described above. Patients continued to use the prescribed treatment for one additional week until the end of the grass pollen season, resulting in a total of 50 d of treatment.

Immunohistochemistry Nasal biopsy samples were immediately embedded in Tissue-Tek O.C.T. Compound (Miles Inc., Elkhart, IN) and rapidly frozen in a 2-methylbutane (Sigma Chemical Company, St. Louis, MO) container

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placed in liquid nitrogen. Frozen samples were stored at 2808 C until cut into 5-mm-thick sections using a cryostat Leitz 1720 digital (Ernst Leitz Wetzlar GmbH, Wetzlar, Germany). Sections were fixed in 4% formaldehyde for 15 min and pretreated with a solution of phosphatebuffered saline (PBS) containing 0.0064% sodium azide, 0.18% glucose, and 1:1,000 glucose oxidase for 15 min to inhibit endogenous peroxidase. For detection of the form of eosinophil cationic protein (ECP) that is suggested to be secreted by activated eosinophils (19), sections were incubated for 1 h with mouse antihuman EG2 antibody (1:500; Kabi Pharmacia Diagnostics AB, Uppsala, Sweden), followed by incubation with horseradish-peroxidase-conjugated sheep F(ab9)2 antimouse immunoglobulins (1:100; Amersham International plc., Little Chalfont, UK). Bound antibodies were visualized using the 3,39-diaminobenzidine (DAB) chromogen system (Dako Corporation, Carpinteria, CA), giving a brown reaction product of positive staining. Finally, the sections were counterstained with Mayer’s hematoxylin. As controls, species- and isotype-matched immunoglobulins (mouse IgG1; Sigma) were used, and for negative control staining the primary antibody was omitted. Stained sections were assessed in a blinded fashion using a light microscope (Olympus BH-2; Olympus Optical Co., Tokyo, Japan) at a magnification 3400. Quantification of EG2-positive cells was performed using a grid attached to the eyepiece of the microscope along the whole epithelial layer and within 200 mm of subepithelium, and the number of EG2-positive cells per mm2 was calculated. Data were expressed as the change of EG2-positive cells from the preseason samples to the peak grass pollen season samples (delta).

Pollen Counts Measurement Grass pollen counts in Tartu during the season of 1997 were measured daily with a Burkard volumetric pollen trap (Burkard Manufacturing Co. Ltd., Rickmansworth, UK).

Data Analysis Nasal symptoms during the period of increased grass pollen in the air, defined from the pollen count data, were denoted to be the main endpoint for this study. For statistical evaluation of this variable, a threestep approach was used. Initially, symptom score data were transformed by square rooting (log-transformation could not be performed because several data points were zero). On this transformed data, a paired two-way t test was performed within each group, evaluating whether the pollen season changed symptoms in any of the studied group versus the mean of the first week after randomization. Secondly, analysis of variance of the square-rooted total symptom score during the whole high-pollen season was performed to evaluate whether the overall change in symptoms during the season showed variance among the groups. Only if these two analyses were found to be significantly different (p , 0.05) did we proceed to the third step, comparing individual groups using Fisher’s PLSD multiple t test. Any difference between the individual groups was considered to be significant if the p value was found to be less than 0.05. Effects on local eosinophilic inflammation, which were denoted as a secondary endpoint, were evaluated by counting the number of tissue EG21 eosinophils per mm2 of a transsection of the nasal mucosal biopsy. Firstly, Wilcoxon’s signed rank test was performed to evaluate whether the number of eosinophils increased in any individual group between the preseason and peak-season biopsy samples. If such a difference was observed, a Kruskal-Wallis test was performed on the change of eosinophils per area (calculated as the difference in number of cells between baseline to peak pollen season) to evaluate whether there was any significant variance among the three groups, and if this was seen, a Mann-Whitney U test was performed to evaluate whether any difference between individual groups could be detected. Data were analyzed using Statview standard software for Macintosh.

RESULTS The patients in the different treatment groups resembled each other regarding atopy (Kruskal-Wallis test among study groups for the diameter of skin prick test against grass pollens, p 5 0.3; for total IgE, p 5 0.5; for anti-grass pollen IgE, p .

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Figure 1. Study design and the 1997 grass pollen counts in Tartu during the course of the study.

0.9); age, p 5 0.3; and duration of disease, p 5 0.8. All 33 patients completed the treatment protocol. However, one patient from the placebo group was excluded from further analysis before performing data evaluation and unblinding of the code because he developed severe rhinitis prior to the pollen season because of an unrelated allergen or local infection. The second biopsy sample from another patient in the placebo group was not acquired because of personal reasons, and this patient is therefore not included in the analysis of EG21 staining but is included in symptom-score analysis. The grass pollen counts in Tartu during the season of 1997 rose as expected and are presented in Figure 1. Symptoms

In both the placebo and zafirlukast groups, the mean daily symptom score increased above the mean daily symptom score of the first treatment week starting from the twentieth day of the treatment (Figure 2). Square-rooted total symptom score during the whole high-pollen season was 8.70 6 0.96 for

Figure 2. Mean daily symptom score of patients with allergic rhinitis induced by grass pollen who were treated for 50 d with oral zafirlukast 20 mg twice a day, intranasal beclomethasone 200 mg twice a day, or matching placebos.

placebo, 8.21 6 1.09 for zafirlukast, and 3.90 6 1.24 for the BDP-treated group. Analysis of variance of this square-roottransformed data showed highly significant variance of symptoms among the groups (p 5 0.008). Multiple t tests showed that there were significant differences between both the placebo and the zafirlukast-treated groups versus the BDP group (p 5 0.005 and 0.01, respectively) but no significant difference between placebo and zafirlukast-treated groups (p 5 0.8). Local Inflammation

There was no significant variance in the number of EG21 cells between the different groups in the preseason samples (Figure 3). The number of EG21 cells in nasal biopsies taken immediately after the peak season was significantly increased compared with the preseason values in the placebo group (from 25.6 6 17.7 to 216.1 6 77.4 EG21 cells/mm2; Wilcoxon’s signed rank test, p 5 0.01) and in the group treated

Figure 3. Number of EG21 cells prior to the grass pollen season, and during the peak of the grass pollen season in nasal biopsies of patients with allergic rhinitis induced by grass pollen who were treated with oral zafirlukast, intranasal beclomethasone, or matching placebos.

Pullerits, Praks, Skoogh, et al.: Leukotriene Antagonist in Allergic Rhinitis

with zafirlukast (from 8.4 6 4.7 to 124.2 6 56.5; p 5 0.01), but not in the BDP-treated group (from 30.8 6 12.8 to 54.0 6 36.7; p 5 0.9) (Figure 3). Mean increase in the number of EG21 cells from pre- to peak grass pollen season was found to be significantly different between the groups, using the KruskalWallis test (p 5 0.04). The increase in EG21 cells was significantly lower for the BDP-treated group than for the placebo group (delta change, 23.2 6 39.4 versus 190.5 6 75.1; p 5 0.015). The increase in EG21 cells was not significantly different between the placebo and the zafirlukast groups (190.5 6 75.1 versus 115.8 6 52.6; p 5 0.4) or between the BDP and zafirlukast groups (p 5 0.08).

DISCUSSION This randomized, placebo-controlled study has shown that a leukotriene receptor antagonist is less effective than a nasal glucocorticoid in improving local symptoms in patients with seasonal allergic rhinitis during a grass pollen season. Furthermore, the leukotriene receptor antagonist did not protect against the increase in local eosinophilic inflammation induced by the pollen season, whereas the nasal glucocorticoid was effective in this respect. The lack of significant improvement in nasal symptoms with zafirlukast 20 mg twice a day greatly discourages the use of leukotriene receptor antagonist as monotherapy in seasonal allergic rhinitis. However, our data do not exclude the possibility that a subgroup of patients with allergic rhinitis may benefit from this type of treatment. Furthermore, we acknowledge that our study did not have the power to detect small differences between the placebo and the zafirlukast groups. Two studies (8, 9) in fact suggest a small beneficial symptom-relieving effect (less than 30%) of leukotriene antagonists in allergic rhinitis, but the magnitude of the effect is much smaller than has previously been observed with local glucocorticoids (20, 21). The present study, for the first time directly comparing these two treatments, shows that a local glucocorticoid is more effective than a leukotriene receptor antagonist in seasonal allergic rhinitis. The grass pollen season during the study was of average severity for the study area, although it is generally recognized that grass pollen levels in this region are lower than those in countries with warmer climates such as in central Europe and Great Britain (22, 23). Influx of “activated” eosinophils as evaluated by the expression of EG2-positive cells into the inflamed mucosa is a well-known phenomenon in seasonal allergic rhinitis (24), as well as in asthma (25). Our data confirm the many previous findings showing attenuation of local eosinophilic inflammation by treatment with local glucocorticoids in patients with allergic rhinitis (13–15). It has been previously suggested that leukotrienes can contribute to the cellular inflammatory process in asthma (26, 27). However, our present study did not show statistical evidence of any reduction in the influx of the number of EG2-positive cells by regular treatment with zafirlukast, arguing against any pronounced inhibitory effect of a leukotriene receptor antagonist on the eosinophilic inflammation seen in seasonal allergic rhinitis. Leukotriene antagonists are to date available only for the treatment of asthma. However, discussion about the potentially beneficial role of this group of compounds in the treatment of both asthma and allergic rhinitis has emerged (28, 29) based on the data showing release of leukotrienes in both these diseases. Thus, granulocytes from patients with asthma and allergic rhinitis have been reported to release more LTC4 than those from normal subjects (30). Also, increased concentrations of LTC4 and LTD4 have been recovered from nasal

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secretions after allergen provocation in patients with atopic allergies (31), and release of LTC4, LTD4, and LTE4 has been found to correlate with clinical response in ragweed-sensitive subjects challenged intranasally with the antigen (5). However, it has been shown that nasal challenge with exogenous leukotrienes causes primarily nasal congestion, with very limited effects on itching, sneezing, or rhinorrhea (4, 6, 32). In the present study, treatment with zafirlukast did not have a statistically significant effect on any of the four recorded symptoms of allergic rhinitis. If treatment with a leukotriene receptor antagonist can provide improvement in nasal obstruction measurable with objective criteria such as rhinomanometry needs to be addressed in further studies. More pronounced effects of leukotrienes in lower compared with upper airways (33) may argue that these airway levels respond differently to leukotrienes, which may explain the lack of beneficial effect of a leukotriene receptor antagonist in our study. In conclusion, these results strongly favor the use of intranasal glucocorticoids over oral leukotriene receptor antagonists in the treatment of pollen-induced allergic rhinitis since BDP, but not zafirlukast, strongly attenuated both clinical symptoms as well as local allergic eosinophilic inflammation induced by natural pollen exposure. Acknowledgment : The writers are grateful to Dr. Anders Lindén for helpful discussion in the planning of this study, Professor Paul O’Byrne for valuable comments on the manuscript, and to Maret Saar for providing us with data on grass pollen counts.

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