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Keywords:

  • airway inflammation;
  • asthma;
  • leukotriene antagonist;
  • montelukast;
  • nitric oxide;
  • steroid

Abstract

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References

Background:  Montelukast has been shown to be effective in controlling the increase in exhaled NO in asthmatic children re-exposed to house dust mite (HDM). This study compared the effect of low dose inhaled budesonide and oral montelukast in preventing the expected relapse of airway inflammation and reactivity in a group of 24 mild asthmatic children allergic to HDM after a brief period of exposure to relevant allergens.

Methods:  Lung function, bronchial hyperresponsiveness (BHR) to methacholine (PC20), fractional exhaled nitric oxide (FeNO) levels and sputum eosinophilia were evaluated.

Results:  Pulmonary function remained stable. The BHR was unchanged after exposure in the group treated with budesonide, whereas a significant increase (P = 0.028) was observed in the patients receiving montelukast. No significant difference was observed in FeNO levels after exposure to mite antigen in the two groups. In both the groups of asthmatic children we observed a significant increase in sputum eosinophil % after the exposure to mite antigen.

Conclusions:  The significant increase in BHR level observed in the group of children receiving montelukast suggests a more comprehensive effect as disease controller by inhaled steroids than by leukotriene antagonist in allergic asthmatic children re-exposed to relevant allergens.

Abbreviations:
BHR

bronchial hyperresponsiveness

FeNO

fractional exhaled nitric oxide

FEV1

forced expiratory volume in 1 s

FEF25−75

forced expiratory flow between 25 and 75% of the vital capacity

HDM

house dust mite

PC20

provocative concentration of methacholine causing a 20% fall in FEV1

Airway inflammation has a keyrole in the pathogenesis of asthma. Even mild asymptomatic asthmatic patients can still present a residual level of persistent airway inflammation and hyperreactivity, thus confirming the opportunity to consider treatment with anti-inflammatory agents also in mild patients (1).

It has been demonstrated that even brief periods of exposure to natural levels of house dust mite (HDM) can be responsible for an increase in fractional exhaled nitric oxide (FeNO) levels (1–3) and that inhaled steroids can prevent such increase (3). A study with an open design, has suggested that montelukast can be effective in controlling the increase of FeNO in asthmatic children re-exposed to HDM (4). More recently it has been observed that there is an adjunctive effect of the addition to montelukast in reducing the level of FeNO in asthmatic children treated with inhaled steroids (5–8).

In this study we aimed to compare the effect of low dose inhaled budesonide and oral montelukast in preventing the expected relapse of airway inflammation and reactivity in a group of mild asthmatic children allergic to HDM after a brief period of natural exposure to relevant allergens.

Subjects

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References

Twenty-four children (10 males, 14 females), ranging in age 6–13 years with a history of mild to moderate asthma according to the American Thoracic Society definition (9) and positive skin prick tests to HDM, were evaluated at the residential house ‘Istituto Pio XII’ (Misurina BL, Italy), situated at 1756 m in the Italian Alps. None of the patients had respiratory infections for at least 2 months before the beginning of the study and none of them was allergic to furred pets.

The children did not present any clinically evident asthma exacerbation and none of them had received corticosteroids for at least 2 months before the study.

The study was approved by the Istituto Pio XII Ethics Committee and both children and their parents gave informed consent.

Study design

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References

The children admitted to the study had been living at the Istituto Pio XII, an environment free of HDM allergen because of location at high altitude, for at least 3 months at the time of enrollment (T0). The first set of evaluations (T0) was performed immediately before the children left the Istituto Pio XII to spend Christmas holidays in their families’ homes at sea level. The second set of evaluations (T1) was performed at the time of re-admission to the Istituto Pio XII after the children had been re-exposed to HDM allergens for 2 weeks. This study was performed out of the pollen season in order to avoid interference of outdoor allergens for those patients who were also sensitized to pollens.

The evaluations at T0 and T1 consisted in the measurement of pulmonary function (FEV1, FEF25−75), airway reactivity to methacholine (PC20), NO exhalation levels and evaluation of cytology obtained by induced sputum.

Patients were randomly divided into two parallel groups of 12 subjects each, receiving either active budesonide (100 μg twice daily) and placebo montelukast, or placebo budesonide and active montelukast (5 mg administered once a day in the evening), starting the same day when they returned at home.

Budesonide placebo was prepared by emptying the Turbuhaler reservoir before the beginning of the study, whereas a nonmatching placebo for montelukast was obtained by selecting a small sweet cherry-tasting candy. The patients were selected from those who had never experienced before neither budesonide nor montelukast treatment. A doctor not participating to the following parts of the study prepared the study drugs and placebos, as well as the drug randomization table. The parents received sealed packages containing the study drugs when they were leaving the residential house in order to keep all the investigators blind about the treatment regimen for each single patient. A treatment randomization list was kept in a sealed envelope in the residential house in order to know the allocation of every patient in the case of an emergency.

Lung function and FeNO measurement

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References

Lung function was measured by a Vitalograph Compact spirometer (Vitalograph Ltd, Buckingham, UK). FEV1, FEF25−75 were considered in the analysis of the results.

The FeNO was measured by chemiluminescence analyzer (Logan LR 2149; Rochester, Kent, UK) with a single breath on-line method according to ERS/ATS guidelines (10). FeNO was measured before methacholine challenge (11).

Methacholine provocation test

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References

Bronchial hyperresponsiveness (BHR) was evaluated by methacholine bronchial provocation (12, 13) performed by a MeFar dosimeter (MeFar, Brescia, Italy). Dosimeter and ampoules were calibrated in order to nebulize 0.01 ml of solution in 1.2 s, generating particle ranging from 0.5 to 3 μm in diameter. All inhalations were performed by slow inspiration starting from functional residual capacity without reaching the total lung capacity, followed by 5 s of breath holding. Before administering methacholine, five inhalations of buffer were given. One minute after the fifth inhalation of buffer solution, FEV1 was recorded again, and the test was to continue only if FEV1 difference has been <10%. The provocation was performed in each patient with increasing doses of methacholine. Data were plotted so that concentrations of inhaled methacholine were logarithmically reported on the abscissa, and percent decrease of FEV1, compared with FEV1 measured after buffer control nebulization, were arithmetically reported on the ordinate. The concentration of methacholine required to produce a FEV1 fall of 20%, defined as PC20 (mg/ml) was calculated (13).

Sputum induction

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References

Sputum was induced by inhalation of hypertonic saline solution, by a standardized method (14, 15). Briefly, 4 and 5% hypertonic saline solutions were nebulized using an ultrasonic nebulizer (UN202), for four periods of 5 min each, for a total time of 20 min, after premedication with inhaled salbutamol (two puffs, 200 μg). FEV1 was monitored before and 10 min after salbutamol and then every 5 min during the saline inhalation.

After each period of nebulization, subjects were instructed to rinse their mouth and then to cough sputum into a Petri dish. In the laboratory, plugs arising from lower respiratory tract were selected by visual inspection, suspended in dithiotreitrol for 30 min at 37°C and then, after centrifugation and washing, transferred onto slides by cytocentrifugation (500 rpm for 5 min) with CYTOSPIN 3 Shandon. Slides were stained with May–Grunwald–Giemsa and coded to be counted blind to the study design. Eosinophils and epithelial cells were expressed as mean percentages of at least 400 cells counted by two independent observers, excluding squamous epithelial cells to correct for salivary contamination.

Measurement of mite allergen levels

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References

Parents were asked to collect dust from the mattresses of the subjects participating the study in a standardized fashion, by vacuuming the mattress surface for an approximate area of 2 m2 for 5 min using a vacuum cleaner with a new bag on. Bags containing samples were stored in a sealed plastic bag at 4°C until the dust was processed for measurement of mite group I allergen levels, which was performed with MAbs in a two site immunometric enzyme-linked immunosorbent assay (16).

The results were expressed in micrograms of mite group I allergens for gram of dust (17).

Statistical analysis

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References

Nonparametric analysis was performed. The comparison within each group were performed by Wilkoxon test and inter-group differences were tested by Mann–Witney U-test. P-values <0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References

Of the 24 children who were admitted, 21 completed the study, three were not compliant with the scheduled regimen, as verified by checking the personal diary card and by evaluating the quantity of unused drug at the end of the study period. These subjects were excluded from the study and therefore the final analysis for the two groups excluded respectively one subject for budesonide and two for montelukast.

It was possible to obtain dust samples from 14 of the 21 children who completed the study. The mean level of mite allergen in samples collected in the residential house was <0.05 μg/g of dust. The mean level (SEM) of HDM group I allergens (Der pI and Der fI) were 43.8 ± 13.1 μg/g of dust in the budesonide group and 54.3 ± 13.8 μg/g of dust in the montelukast group (ns).

In these mild asthmatic patients, both in those treated with budesonide and in those receiving montelukast, no significant asthma symptom was reported during the study period and it was possible to gradually withheld medications within few days from the time of re-admission to the residential house.

Pulmonary function was comparable in terms of FEV1 and FEF25−75 at baseline determination and they remained stable after the 2-week period of exposure to HDM antigens (Table 1). PC20 was unchanged after exposure in the group treated with budesonide, whereas a significant increase (P = 0.028) was observed in the patients receiving montelukast (Fig. 1, panel A).

Table 1.  Pulmonary function in the study groups (median, Q1; Q3)
 BudesonideMontelukast
T0T1T0T1
  1. FEV1%, forced expiratory volume in 1 s; FEF25−75%, forced expiratory flow between 25 and 75% of the vital capacity.

FEV1%108 (97; 119)99 (97; 117)99 (96; 112)100 (96; 107)
FEF25−75%112 (70; 116)103 (68; 119)87 (68; 105)82 (73; 94)
image

Figure 1. Median values of PC20 (panel A), FeNO levels (panel B) and sputum eosinophil cell count (%) (panel C) at T0 and T1, in two parallel groups: active budesonide —-bsl00001—- and active montelukast ⋯⋯•⋯⋯.

Download figure to PowerPoint

Both treatments have been demonstrated to be effective in preventing the expected (18) increase in FeNO after the 2-week period of exposure to mite antigen (Fig. 1, panel B).

In contrast, in both the groups of asthmatic children we observed a significant increase in sputum eosinophil % after the exposure to mite antigen (Fig. 1, panel C).

Discussion

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References

Leukotriene antagonist montelukast has been demonstrated to provide additional clinical benefits in asthmatic patients not satisfactorily treated with inhaled corticosteroids (5–8, 18, 19) and in those with exercise induced asthma (20).

Bisgaard et al. (18) have demonstrated that FeNO decreased by 20% in a group of asthmatic children after two weeks of treatment with montelukast, independently of concurrent steroid treatment. The authors concluded in favor of a potential anti-inflammatory role of leukotriene antagonist in asthma.

Ghiro et al. (5) showed that in asthmatic children receiving maintenance therapy with inhaled steroid there is a reduction in FeNO after montelukast treatment.

In this study we aimed to evaluate whether montelukast was effective in preventing the expected increase in airway inflammation in a group of children allergic to HDM. In this study no placebo group was included as the Ethic Committee objected that the previous data with similar experimental design had already confirmed that even a short period of antigen exposure was responsible for a prompt, significant, increase in airway inflammation in asthmatic children not receiving anti-inflammatory therapy (2, 21). In particular, in a recent study, it has been observed a significant increase in FeNO in children receiving placebo, but not in those treated with low dosage inhaled steroid after re-exposed to relevant allergens for 2 weeks, confirming the opportunity to consider the use of an anti-inflammatory treatment also in the case of brief period of allergen exposure (3). Therefore all the comparisons in the present study are considered with baseline for within group evaluations.

An increase in BHR to methacholine in the montelukast-treated group, but not in the budesonide-treated one, was the only significant difference we observed about the influence of the two treatments on the investigated parameters. Both treatments have been effective in preventing an increase in FeNO, and a worsening in lung function. In contrast, an increase in sputum eosinophil percentage has been observed in both the arms of the study. These results, therefore, confirm an effect of leukotriene antagonist in controlling the level of FeNO in asthmatic children, but they open new perspectives for discussion about the role of this class of drugs as controller in the treatment of asthma. In fact, the increase in BHR observed in the group receiving montelukast, but not in that treated with budesonide, further confirms the idea that airway inflammation and hyperresponsiveness may represent different features of the disease (22) thus suggesting that different classes of drugs may complement each other rather that to be considered as alternative.

On the contrary, both FeNO and sputum eosinophils are regarded as markers of airway inflammation in asthma and some relationship has been demonstrated between these parameters (23, 24). A correlation has been demonstrated to exist and to be stronger in steroid naïve patients, whereas steroid treatment seems to be more effective in reducing FeNO than sputum eosinophilia (24, 25). This observation suggests that FeNO may measure other aspects of airway inflammation in asthma aside from eosinophilia (1, 23, 24). Therefore, when interpretating the results of airway inflammation measurements in asthmatic patients both the potential different meaning of the different tests and the effect of steroid treatment has always to be kept into consideration.

As sputum eosinophilia precedes clinical deterioration (26) the observed increase in this parameter in both the arms of the study suggests that, despite a lack of deterioration in terms of FeNO levels, neither of the study treatments can be considered fully effective in preventing the relapse of airway inflammation when mite allergic asthmatic children are re-exposed to the relevant allergen.

In conclusion, the significant increase in BHR level observed in the group of children receiving montelukast suggests a more comprehensive effect as disease controller by inhaled steroids than by leukotriene antagonist in allergic asthmatic children re-exposed to relevant allergens.

References

  1. Top of page
  2. Abstract
  3. Material and methods
  4. Subjects
  5. Study design
  6. Lung function and FeNO measurement
  7. Methacholine provocation test
  8. Sputum induction
  9. Measurement of mite allergen levels
  10. Statistical analysis
  11. Results
  12. Discussion
  13. References
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