MD Dieter Ukena
email: dieter.ukena@klinikum-bremen-ost.de

Christian Biberger

Volker Steinijans

Volker Von Behren

Ronitta Malek

Hans H Weber

Ekkehard Beck

Anneliese Linnhoff

Ciclesonide is more effective than budesonide

Methods: A total of 399 patients with asthma were randomised to receive once-daily ciclesonide 320 µg ex-actuator (equivalent to 400 µg ex-valve) or once-daily budesonide 400 µg for 12 weeks. The primary endpoint was forced expiratory volume in 1 second (FEV 1 ). Additional efficacy variables included forced vital capacity (FVC), peak expiratory flow (PEF), asthma symptoms, use of rescue medication and time to onset of effect. Adverse events were monitored throughout the study.

Results: Both ciclesonide and budesonide significantly increased FEV 1 from baseline (416 and 321 ml, respectively; p<0.0001). The increase in FEV 1 was significantly greater in ciclesonide-treated patients (95% confidence interval: 0.016 to 0.174; p=0.019 versus budesonide). Similarly, ciclesonide and budesonide significantly improved FVC and PEF from baseline (p<0.0001), and significantly greater increases occurred with ciclesonide (p=0.034 and 0.019 versus budesonide, respectively). Analysis of morning PEF revealed an earlier onset of action for ciclesonide versus budesonide; a significant improvement was seen by Day 2 (p=0.039 versus baseline) with ciclesonide compared with Day 7 for budesonide (p=0.047 versus baseline). Adverse events occurred with a similar incidence in both treatment groups. Neither treatment caused significant changes in urinary cortisol levels.

Conclusion:

Once-daily ciclesonide was more effective than once-daily budesonide in improving FEV 1 , FVC and PEF. Ciclesonide also had an earlier onset of action than budesonide in patients with persistent asthma. Both ciclesonide and budesonide had good safety and tolerability profiles. Inhaled corticosteroids (ICS) are the most effective anti-inflammatory therapy for the long-term control of persistent asthma. [START_REF] Georgitis | The 1997 Asthma Management Guidelines and therapeutic issues relating to the treatment of asthma[END_REF][START_REF]Global strategy for asthma management and prevention[END_REF] However, poor adherence with prescribed ICS medication may be a major factor contributing to the suboptimal control of persistent asthma. Therefore, ongoing efforts are aimed at improving the level of treatment adherence in patients with asthma, including the development of ICS that are effective, have a rapid onset of action and are suitable for once-daily dosing. [START_REF] Boulet | Once-daily inhaled corticosteroids for the treatment of asthma[END_REF][START_REF] Guest | Switching asthma patients to a once-daily inhaled steroid improves compliance and reduces healthcare costs[END_REF] Ciclesonide is delivered as a solution to the lung via a hydrofluoroalkane-propelled metered-dose inhaler (HFA-MDI). [5] Pulmonary esterases convert ciclesonide into the active metabolite, desisobutyryl-ciclesonide (des-CIC). [START_REF] Mealy | Ciclesonide: treatment of allergic rhinitis antiallergy/antiasthmatic[END_REF][START_REF] Nave | Formation of fatty acid conjugates of ciclesonide active metabolite in the rat lung after 4-week inhalation of ciclesonide[END_REF] Subsequently, des-CIC undergoes reversible esterification to form fatty acid conjugates in the lung that serve as a depot for the slow release of the active drug. [START_REF] Nave | In vitro metabolism of ciclesonide in human lung and liver precision-cut tissue slices[END_REF] Finally, des-CIC is converted into a series of inactive metabolites that are eliminated from the body. [START_REF] Peet | Identification of enzymes involved in phase I metabolism of ciclesonide by human liver microsomes[END_REF] Several clinical studies have reported the efficacy and safety profiles of ciclesonide in patients with persistent asthma over a range of doses (80-640 µg).[10-12] For example, ciclesonide 160 and 640 µg once daily has been shown to maintain pulmonary function with a side-effect profile comparable to that of placebo. [START_REF] Chapman | Maintenance of asthma control by once-daily inhaled ciclesonide in adults with persistent asthma[END_REF] In addition, although the pathophysiology of asthma is responsive to circadian changes, [START_REF] Pincus | Chronobiology and chronotherapy of asthma[END_REF] it has been demonstrated that ciclesonide 160 µg can be administered once daily either in the morning or evening with comparable improvements in lung function and asthma symptom control.

[10] Budesonide is approved for use either once or twice daily in patients with asthma, providing similar improvements in lung function in asthma patients when administered as a once-daily 400-µg dose or a twice-daily 200-µg dose. [START_REF] Jones | Pulmicort Turbohaler once daily as initial prophylactic therapy for asthma[END_REF] In a comparative study, ciclesonide 160 µg administered once daily, either in the morning or evening, was shown to be comparable to budesonide 200 µg twice daily (400 µg per day) in maintaining lung function, asthma symptoms and rescue-medication use. [START_REF] Niphadkar | Comparison of the efficacy of ciclesonide 160 µg QD and budesonide 200 µg BID in adults with persistent asthma: a phase III, randomized, double-dummy, and openlabel study[END_REF] A further study compared the effects of a similar dose of ciclesonide (320 µg) and budesonide (400 µg) once-daily. Due to the circadian fluctuations in asthma, it is important to determine the effects of ICS at different times of the day. When administered in the A c c e p t e d m a n u s c r i p t morning, ciclesonide was at least as effective as budesonide in patients with persistent asthma. [START_REF] Boulet | Comparative efficacy of once-daily ciclesonide and budesonide in the treatment of persistent asthma[END_REF] However, the corresponding comparison of ciclesonide versus budesonide using a similar dose and once-daily dosing in the evening has not been performed.

Therefore, the aim of the current study is to determine the effects of once-daily ciclesonide 320 µg (ex-actuator dose), administered via HFA-MDI in the evening, and once-daily budesonide 400 µg, administered via dry powder inhaler (DPI;

Turbohaler TM ; AstraZeneca GmbH, Wedel, Germany) in the evening, in patients with moderate, persistent asthma.

METHODS

Patients

Patients (aged 12-75 years), with at least 6 months' history of asthma as defined by American Thoracic Society criteria [START_REF] Ats | Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease and asthma[END_REF], were recruited at 43 study centres. Pretreatment with ICS (≤500 µg/day beclomethasone dipropionate or equivalent) and/or other asthma medications (cromones, leukotriene antagonists or theophylline) was permitted with no change in treatment during the 4 weeks immediately before enrollment. Patients who used only rescue medication were also allowed to participate in this study. Patients with an asthma exacerbation or lower respiratory tract infection in the 4 weeks immediately before enrollment were excluded.

Additional exclusion criteria were use of systemic steroids during the 4 weeks before enrollment or more than three times in the previous 6 months, use of injectable steroids for 6 weeks before enrollment, or use of rescue medication other than shortacting β 2 -agonists. Pregnancy, current smoking or previous smoking (history of ≥10 pack-years), chronic obstructive pulmonary disease or hypersensitivity to ICS also resulted in patient exclusion. Upon study entry, patients discontinued their current therapy except for the use of short-acting β 2 -agonists, as needed. After a run-in period of up to 4 weeks, patients were eligible for randomisation if forced expiratory volume in 1 second (FEV 1 ) was 50%-90% predicted (or if FEV 1 was >90% of predicted, the FEV 1 :forced vital capacity (FVC) ratio was ≤0.7). Patients treated with ICS before the run-in period had to exhibit a decrease in FEV 1 of ≥10% during the

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run-in period to demonstrate their need for ICS. In addition, reversible bronchial obstruction (FEV 1 increase of ≥15% of initial value after application of salbutamol or a ≥15% variation in the diurnal peak expiratory flow [PEF]) (≥3 of the last 7 days of the run-in period) was required.

Study design and treatment

This was a double-blind, double-dummy, randomised, parallel-group study. After the run-in period, patients were randomised (at baseline) to receive either ciclesonide 320 µg (HFA-MDI, 160 µg/puff) or budesonide 400 µg (Turbohaler

Patient assessments

Patients were evaluated during the run-in period and during treatment visits (Weeks 1, 2, 4, 8, and 12) and at follow-up (ongoing adverse events only). At each study visit, FEV 1 , PEF and FVC were measured by spirometry. Diary data (morning and evening PEF, daytime and nighttime asthma symptom scores and number of puffs of rescue medication) were recorded electronically using an electronic asthma monitor (AM2, VIASYS; Höchberg, Germany) that captured the time and date of data entry.

Asthma scores were recorded on 5-point scales as follows: daytime asthma scores were recorded on a scale from 0 (very well, no symptoms) to 4 (asthma very bad, unable to carry out daily activities as usual); and nighttime asthma scores ranged from 0 (no symptoms, slept through the night) to 4 (bad night, awake most of the night because of asthma).

The onset of effect of ciclesonide and budesonide treatment was determined by comparing values for morning PEF, sum of asthma symptom scores and rescuemedication use on a daily basis during the first week and on a weekly basis thereafter compared with the week before randomization (baseline). Patients exhibiting an asthma exacerbation, i.e. deterioration in asthma (e.g. increase in asthma symptoms, distinct drop in lung function) requiring treatment with oral

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steroids were withdrawn from the study. Safety was evaluated by monitoring 24-hour urinary free cortisol levels, adverse events, vital signs and laboratory parameters.

Statistical analysis

Statistical analysis of each study endpoint was conducted in the full analysis set (intention to treat; patients who received at least 1 dose of study medication) and valid cases set (per-protocol; patients who completed the study without any major protocol violations). Results are reported for the full analysis set only unless there were differences in statistical conclusions between the full analysis set and valid cases set. For the primary variable, FEV 1 , treatment comparisons were based on differences between the end of treatment and the baseline value at the time of randomisation (last value analysis). An analysis of covariance was performed with the value at baseline and age as covariates, and sex and study centre as factors.

Initially, a test of non-inferiority was performed based on the per-protocol population and a non-inferiority acceptance limit of -200 ml. If non-inferiority was demonstrated, a subsequent test of superiority of ciclesonide over budesonide was performed using the full analysis set. Two-sided p-values are presented, with least-squares means and two-sided 95% confidence intervals (CI) calculated for treatment differences. A sample size of 200 patients per treatment group was required to ensure a power of 90% to demonstrate superiority of ciclesonide versus budesonide (α=0.025, one sided; expected difference in means=150 ml; common standard deviation=450 ml).

Secondary variables -FVC, PEF by spirometry, and morning and evening PEF from diarieswere analysed analogously to FEV 1 (non-inferiority limits were as follows: significant changes. These comparisons were conducted by means of a step-down procedure using sequentially earlier and shorter time periods until differences were no longer significant. Changes in urinary cortisol levels were analysed by paired t tests (within groups) and independent t tests (between groups).

RESULTS

Study population

A total of 437 patients were enrolled in this study, and 399 patients were randomised to either ciclesonide 320 µg once daily (n=198) or budesonide 400 µg once daily (n=201; full analysis set). Patient demographics and baseline characteristics were similar in both treatment groups (Table 1). Based on % predicted FEV 1 , the majority of patients had moderate, persistent asthma. Forty-one percent of patients in the ciclesonide group and 35% in the budesonide group had received previous ICS treatment.

During the study, 10 patients withdrew from the ciclesonide group and 18 patients withdrew from the budesonide group. Reasons for study discontinuation were asthma exacerbations (ciclesonide, two patients; budesonide, one patient), other adverse events (ciclesonide, three patients; budesonide, two patients) and nonmedical reasons (ciclesonide, five patients; budesonide, 15 patients). All cases of study discontinuation due to adverse events were assessed as unrelated to study medication. A total of 54 patients had protocol violations during the study; 30 patients in the ciclesonide group and 24 patients in the budesonide group. The majority of these violations related to randomisation criteria (8% of patients) and use of prohibited concomitant medications (3% of patients). Therefore, 168 ciclesonide patients and 177 budesonide patients were included in the valid cases set.

[Table 1 near here]

Efficacy

Lung function variables

During the first 7 days of treatment, FEV 1 rapidly increased from baseline in patients

A c c e p t e d m a n u s c r i p t treated with either ciclesonide or budesonide (Figure 1). In both treatment groups, FEV 1 reached a plateau after 2 weeks and remained at constant levels until the end of the study. After 12 weeks of treatment (last value analysis), FEV 1 increased by 416 ml in the ciclesonide group and by 321 ml in the budesonide group (p<0.0001 versus baseline for both; Figure 2, Table 2), resulting in a between-treatment difference of 95 ml. As the lower limit of the 95% CI (16 to 174 ml) for the difference in FEV 1 improvements between ciclesonide and budesonide exceeded not only the non-inferiority margin of -200 ml, but also 0, superiority of ciclesonide over budesonide was demonstrated (p=0.019, full analysis set). The significantly greater improvement in FEV 1 for ciclesonide compared with budesonide was even more pronounced in the subgroup of patients with a history of smoking (171 ml; p=0.033).

In the subgroup of non-smokers, ciclesonide was numerically better than budesonide (35 ml), but not statistically superior (p=0.498).

Ciclesonide-and budesonide-treated patients also experienced significant improvements in FVC and PEF by spirometry after 12 weeks of treatment (p<0.0001 versus baseline for both) (Table 2). In addition, patients treated with ciclesonide achieved a significantly greater increase in FVC (0.46 versus 0.35 l; p=0.034) and PEF by spirometry (80 versus 61 l/min; p=0.019) compared with budesonide.

[Figures 1 and2, and Table 2 near here]

Asthma symptoms and rescue-medication use

Statistically significant increases in asthma symptom scores (sum of daytime and nighttime symptoms) and decreases in use of rescue medication were observed after 12 weeks of treatment (last value analysis) with either ciclesonide or budesonide (Figure 3; Table 3). Ciclesonide significantly improved asthma symptom scores from baseline (change -0.62 units; p<0.0001). A similar improvement from baseline was observed in patients receiving budesonide (change -0.74 units; p<0.0001). The difference in the sum of asthma symptom scores between ciclesonide and budesonide treatment was not significant (p=0.863). Furthermore, ciclesonide and budesonide improved rescue-medication use relative to baseline (p<0.0001) (Figure 3; Table 3). The reduction in rescue-medication use was

A c c e p t e d m a n u s c r i p t

comparable in the ciclesonide and budesonide treatment groups (-0.42 versus -0.57 puffs/day, respectively; p=0.687).

[Figure 3 and Table 3 near here]

Onset of treatment effect

The time required to detect a change from baseline in morning PEF, asthma symptom scores and rescue-medication use was assessed to determine the onset of effect of ciclesonide and budesonide treatment. Ciclesonide treatment achieved a significant improvement in morning PEF by Day 2 (p=0.039 versus baseline) compared with Day 7 for budesonide (p=0.047 versus baseline; full analysis set) (Figure 4). In patients who completed the study according to the protocol (valid cases set), ciclesonide achieved a significant improvement in morning PEF by Day 3 (p=0.004 versus baseline) compared with Week 2 for budesonide (p<0.001 versus baseline). Both treatments significantly improved asthma symptom scores (p<0.0001 ciclesonide; p<0.001 budesonide) and decreased rescue-medication use (p<0.0001 ciclesonide; p=0.001 budesonide) on the first treatment day.

[Figure 4 near here]

Safety

Adverse events occurred with a similar incidence in both treatment groups. Seventythree adverse events occurred in 28% of patients treated with ciclesonide and 71 adverse events occurred in 27% of patients treated with budesonide. The most frequent adverse events were related to the respiratory system, in particular upper respiratory tract infection, rhinitis and bronchitis, which occurred in ≥3% of patients in both treatment groups. Most adverse events were of mild or moderate intensity and were assessed as unrelated to the study drug. There were four adverse events (dyspnoea, voice alteration, cough and headache) potentially related to ciclesonide and none in the budesonide group.

During the follow-up period, one patient in the budesonide group reported oral candidiasis, which was assessed as "likely related" to study medication. Three

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serious adverse events were reported in the ciclesonide group (surgery, stroke, dyspnoea/heart failure), all of them being assessed as unrelated to the study medication. There were no statistically significant or clinically relevant differences from baseline or between the two treatment groups in 24-hour urinary free cortisol or standard laboratory variables.

DISCUSSION

This study demonstrated that once-daily evening administration of ciclesonide (320 µg ex-actuator; HFA-MDI) achieved significantly greater improvements in spirometric lung function measurements (FEV 1 , FVC and PEF) compared with once-daily evening administration of budesonide (400 µg; Turbohaler TM ) in patients with persistent asthma. Comparable improvements in PEF by diary, asthma symptom scores and rescue-medication use were observed in ciclesonide-and budesonidetreated patients. Treatment adherence was not measured in this study because of technical reasons. However, due to the applied administration pattern (2 puffs of active treatment and placebo device each, given in the evening) it is unlikely that differences in lung function between ciclesonide-and budesonide-treated patients were related to differences in adherence rates.

In a previous study, ciclesonide 160 µg administered once daily was shown to be comparable to budesonide 200 µg twice daily (400 µg per day) in maintaining lung function, asthma symptoms and rescue medication. [START_REF] Niphadkar | Comparison of the efficacy of ciclesonide 160 µg QD and budesonide 200 µg BID in adults with persistent asthma: a phase III, randomized, double-dummy, and openlabel study[END_REF] In addition, a study comparing the effects of ciclesonide 320 µg and budesonide 400 µg once daily when administered in the morning showed that ciclesonide was at least as effective as budesonide in patients with persistent asthma. [START_REF] Boulet | Comparative efficacy of once-daily ciclesonide and budesonide in the treatment of persistent asthma[END_REF] Ciclesonide was non-inferior to budesonide for FEV 1 , and statistically superior to budesonide (p=0.010) for FVC. In addition, asthma symptom scores were comparable and median percentage of symptom-free days was significantly higher for ciclesonide versus budesonide (p=0.017). [START_REF] Boulet | Comparative efficacy of once-daily ciclesonide and budesonide in the treatment of persistent asthma[END_REF] Therefore, due to the circadian fluctuations in asthma, [START_REF] Pincus | Chronobiology and chronotherapy of asthma[END_REF] the current study compared the same once-daily doses of ciclesonide and budesonide administered in the evening. Importantly, the current study provided similar findings to those of the previous study administering both treatments in the morning, [START_REF] Boulet | Comparative efficacy of once-daily ciclesonide and budesonide in the treatment of persistent asthma[END_REF] A c c e p t e d m a n u s c r i p t providing further support for the similar efficacies of ciclesonide and budesonide, regardless of timing of administration.

The onset of effect of ciclesonide treatment appeared to be more rapid compared with budesonide in the current study; improvements in morning PEF were evident by Day 2 in the ciclesonide group, whereas budesonide did not significantly increase morning PEF until Day 7. In a recent study, budesonide 1,600 µg/day increased FEV 1 and morning PEF after the first week of treatment, [START_REF] Phillips | Time course of action of two inhaled corticosteroids, fluticasone propionate and budesonide[END_REF] which is in line with the results seen in the current study. Asthma symptom scores and rescue-medication use improved in both groups after the first dose. It is important to note that electronic diaries were used in this study to record assessments suitable to determine the onset of treatment effect. Compared with traditional patient diaries, electronic diaries are a more reliable tool for data collection because they capture the date and time at which assessments were recorded and, thereby, confirm the validity of the study data. The rapid onset of effect of ciclesonide treatment compares favourably with that of budesonide.

Previous studies have reported that ciclesonide and budesonide undergo fatty acid esterification in the lung. [START_REF] Nave | In vitro metabolism of ciclesonide in human lung and liver precision-cut tissue slices[END_REF][START_REF] Miller-Larsson | Reversible fatty acid conjugation of budesonide. Novel mechanism for prolonged retention of topically applied steroid in airway tissue[END_REF][START_REF] Tunek | Reversible formation of fatty acid esters of budesonide, an antiasthma glucocorticoid, in human lung and liver microsomes[END_REF] Ciclesonide fatty acid conjugates are five-fold more lipophilic than budesonide fatty acid conjugates. [START_REF] Nave | Formation of fatty acid conjugates of ciclesonide active metabolite in the rat lung after 4-week inhalation of ciclesonide[END_REF] Increased lipophilicity and the ability to form fatty acid conjugates may increase ciclesonide retention in the lung [START_REF] Nave | Formation of fatty acid conjugates of ciclesonide active metabolite in the rat lung after 4-week inhalation of ciclesonide[END_REF][START_REF] Nave | In vitro metabolism of ciclesonide in human lung and liver precision-cut tissue slices[END_REF] and, thereby, may contribute to the clinical efficacy of once-daily ciclesonide observed in this study. In addition, the high pulmonary deposition of ciclesonide and the high affinity of the active metabolite for the glucocorticoid receptor potentially contribute to the improvements in lung function and asthma symptoms. [START_REF] Newman | High lung deposition of 99m Tclabeled ciclesonide administered via HFA-MDI to patients with asthma[END_REF][START_REF] Rohatagi | Risk-benefit value of inhaled glucocorticoids: a pharmacokinetic/pharmacodynamic perspective[END_REF] Approximately 52% of the ciclesonide dose, administered via HFA-MDI, is delivered to the lungs, with the highest deposition (55% of the deposited dose) in the peripheral regions of the lung. [START_REF] Newman | High lung deposition of 99m Tclabeled ciclesonide administered via HFA-MDI to patients with asthma[END_REF] In contrast, pulmonary deposition of budesonide via dry powder inhaler is lower (15%-28% of the administered dose) and deposition is localised primarily to the central region of the lung. [START_REF] Borgström | Lung deposition[END_REF][START_REF] Hirst | A comparison of the lung deposition of budesonide from Easyhaler, Turbuhaler and pMDI plus spacer in asthmatic patients[END_REF] Furthermore, the active metabolite of ciclesonide (des-CIC) has a high relative glucocorticoid receptor affinity (1200 versus 900 for budesonide; dexamethasone reference is 100) and possesses substantial anti-inflammatory activity. [START_REF] Rohatagi | Risk-benefit value of inhaled glucocorticoids: a pharmacokinetic/pharmacodynamic perspective[END_REF][START_REF] Stoeck | In vitro and in vivo anti-inflammatory activity of the new glucocorticoid ciclesonide[END_REF] A c c e p t e d m a n u s c r i p t

The most common side effects in both treatment groups were related to the respiratory system. However, most were of mild or moderate intensity and did not result in treatment discontinuation. No serious adverse events were assessed as related to the study drug. Furthermore, neither ciclesonide nor budesonide demonstrated any significant or clinically relevant changes in urinary cortisol measurements. Previous studies have shown that ciclesonide does not induce cortisol suppression at daily doses as high as 1280 µg. [START_REF] Szefler | Ciclesonide, a novel inhaled steroid, does not affect hypothalamic-pituitary-adrenal axis function in patients with moderate-to-severe persistent asthma[END_REF][START_REF] Derom | Effects of inhaled ciclesonide and fluticasone propionate on cortisol secretion and airway responsiveness to adenosine 5'monophosphate in asthmatic patients[END_REF] Similarly, budesonide 400 µg once daily did not demonstrate significant cortisol suppression in healthy subjects or patients with asthma. [START_REF] Wilson | Adrenocortical activity with repeated administration of one-daily inhaled fluticasone propionate and budesonide in asthmatic adults[END_REF][START_REF] Andersson | A randomized controlled assessment of the effects of different dosing regimens of budesonide on the HPA-axis in healthy subjects[END_REF] In another study, twice-daily budesonide 200 µg (400 µg per day) was shown to significantly suppress urinary cortisol compared with once-daily ciclesonide 320 µg (p<0.05). [START_REF] Hansel | Ciclesonide 80 µg or 320 µg once daily achieves lung function improvement comparable with budesonide 200 µg twice daily in patients with persistent asthma[END_REF] In conclusion, this study demonstrated that once-daily ciclesonide achieved a significantly greater improvement in lung function (FEV Ciclesonide 320 µg is an ex-actuator dose, equivalent to 400 µg ex-valve.

Patients with a history of smoking were defined as individuals having a history of <10 pack years or ≥3 months of smoking abstinence.

  Key words: Ciclesonide, budesonide, asthma, inhaled corticosteroids, cortisol,

FVC - 0

 0 .20 l; PEF -25 l/min). Differences in the sum of asthma symptom scores and the use of rescue medication were analysed by non-parametric tests using the modification of Wilcoxon's signed-rank test according to Pratt (within groups) and the Mann-Whitney U test (between groups). Between-treatment comparisons for asthma symptom-free days and rescue-medication-free days were analysed using Mann-Whitney U tests. The time to onset of treatment effect was determined by comparing pre-treatment and post-treatment values of each of the relevant efficacy variables (morning PEF, sum of asthma symptom scores and rescue-medication use) for A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

  Biberger and Volker Steinijans are full time employees of ALTANA Pharma AG, Konstanz, Germany. Volker von Behren, Ronitta Malek, Hans H. Weber, Ekkehard Beck and Anneliese Linnhoff have no conflicts of interest to disclose.

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

Figure 1 .FEV 1 =

 11 Figure 1. Change in forced expiratory volume in 1 second (FEV 1 ) during 12 weeks of treatment (time course). Full analysis set. Values are presented as least squares means ± standard error of the least squares mean. Ciclesonide 320 µg is an ex-actuator dose, equivalent to 400 µg ex-valve. FEV 1 = Forced expiratory volume in 1 second; CIC = Ciclesonide; BUD = Budesonide.

Figure 2 .

 2 Figure 2. Change in forced expiratory volume in 1 second (FEV 1 ) after 12 weeks of treatment (last value analysis). Full analysis set. Values are presented as least squares means ± standard errors of the least squares mean. Ciclesonide 320 µg is an ex-actuator dose, equivalent to 400 µg ex-valve. *p<0.0001 vs baseline; † ciclesonide vs budesonide. FEV 1 = Forced expiratory volume in 1 second; CIC = Ciclesonide; BUD = Budesonide.

Figure 3 .

 3 Figure 3. Asthma symptom scores and rescue-medication use after 12 weeks of treatment. Full analysis set. Data are presented as non-parametric point estimates. Asthma symptom scores are sum of morning and evening scores. Ciclesonide 320 µg is an ex-actuator dose, equivalent to 400 µg ex-valve. *p<0.0001 vs baseline; † ciclesonide vs budesonide (last value analysis). CIC = Ciclesonide; BUD = Budesonide.

Figure 4 .A c c e p t e d m a n u s c r i p t

 4 Figure 4. Onset of action: change in morning peak expiratory flow (PEF) during first 4 weeks of treatment. Improvements with ciclesonide and budesonide therapy compared with baseline first reached statistical significance on day 2 (*p=0.039) and day 7 ( ‡ p=0.047), respectively (dashed lines).Full analysis set. Data reported as least squares mean ± standard error of the least squares mean. † p<0.0001 vs baseline. Ciclesonide 320 µg is an ex-actuator dose, equivalent to 400 µg ex-valve. PEF = Peak expiratory flow; CIC = Ciclesonide; BUD = Budesonide.

Figure 1 :A c c e p t e d m a n u s c r i p t

 1 Figure 1:

Figure 2 :

 2 Figure 2:

Figure 3 :

 3 Figure 3:

Figure 4 :

 4 Figure 4:

Table 1 . Demographic and baseline characteristics Characteristics Ciclesonide 320 µg once daily Budesonide 400 µg once daily

 1

	TABLES
	Patients, n	198	201
	Sex (male/female), %	43/57	49/51
	Median age, years (range)	44 (12-74)	46 (13-75)
	Patients without/with history of smoking,	64/36	67/33
	%
	ICS not pre-treated/ICS pre-treated, %	59/41	65/35
	Asthma severity, %
	Mild (FEV 1 ≥80% predicted)	18	24
	Moderate (FEV 1 >60% but
	<80% predicted)	70	61
	Severe (FEV 1 ≤60% predicted)	12	15
	FEV 1 , ml ± SD	2323 ± 632	2337 ± 641
	FEV 1 , % predicted ± SD	71 ± 11	72 ± 11
	Reversibility: change in FEV 1 , % ± SD	24 ± 11	24 ± 11
	Morning PEF (diary) l/min ± SD	365 ± 117	352 ± 113
	Morning PEF (diary), % predicted ± SD	79 ± 20	76 ± 21
	PEF variability, % ± SD	12 ± 7	12 ± 6
	ICS = Inhaled corticosteroid; FEV 1 = Forced expiratory volume in 1 second; SD = Standard
	deviation; PEF = Peak expiratory flow.
	Full analysis set.

ACKNOWLEDGEMENTS

This study was funded and sponsored by ALTANA Pharma. The authors would like to thank ProEd and Medicus International for editorial assistance. Editorial support was funded by ALTANA Pharma. The following investigators participated in the study: Germany: Dr. Volker Aldinger, Haßloch; Dr. Ekkehard Beck, Rüdersdorf; Dr. Albrecht Bezler, Aalen; Dr. Mike Bucher, Frankenthal; Dr. Klaus Colberg, Bad Segeberg; Dr. Wolfgang Daut, Kallstadt; Dr. Vera Grimm-Sachs, Bruchsal; Dr. Gerrit Hoppe, Berlin; Dr. Siegfried Ilg, Königslutter; Dr. Jakob Junggeburth, Bad Wörishofen; Dr. Christiane Klein, Künzing; Dr. Jürgen Krehbiel, Rödersheim-Gronau; Dr. Birgit Kreische, Kamenz; Dr. Ludger Lindemann, Gelsenkirchen; Dr. Anneliese Linnhoff, Berlin; Dr. Ronitta Malek, Frankfurt; Dr. Dieter Müller, Tostedt; Dr. Ingomar Naudts, Rodgau; Dr. Gerhard Neumann, Delitzsch; Dr. Joachim Pettenkofer, Berlin; Dr. Herbert Querfurt, Bochum; Dr. Werner Reimann, Bensheim; Dr. Habib Samiri, Frankfurt; Dr. Björn Schmorell, Forchheim; Dr. Renita Schnorr, Berlin; Dr. Thomas Schultz, Berlin; Dr. Reinhard Storch, Neulußheim; Dr. Jürgen Thomas, Wiesbaden; Dr. Lutz Volgmann, Hannover; Dr. Volker von Behren, Wiesbaden; Dr. Hans Gert Weber, Borna; Dr. Hans H. Weber, Hannover. The Netherlands: Dr. Bonne Biesma, Hertogenbosch; Dr. David Cheung, Schiedam; Dr. Wim Evers, Sneek; Dr. Aloysius Greefhorst, Hengelo; Dr. Noel Schlösser, Utrecht. Switzerland: Dr. Hans Bettschen, Spiez; Dr. Werner Graf, Bern; Dr. Maurus Pfister, Rorschach; Dr. Tamás Töth, Thun; Dr. Heini Wacker, Allschwil; Dr. Andreas Züllig, Wädenswil.

This study was supported by a grant from

CONFLICT OF INTEREST STATEMENT

Dieter Ukena has participated in advisory board meetings at ALTANA Pharma AG and AstraZeneca, and received lecture fees from AstraZeneca, Boehringer, Pfizer, ALTANA Pharma AG, Merck Sharp & Dohme and GlaxoSmithKline. Christian Data are presented for the full analysis set.

P values are two sided at the 5% level for test of differences.

Ciclesonide 320 µg is an ex-actuator dose, equivalent to ciclesonide 400 µg ex-valve. Data are presented as medians (change from baseline) and non-parametric point estimates (PE, changes vs BUD).

Variable

Ciclesonide 320 µg is an ex-actuator dose, equivalent to ciclesonide 400 µg ex-valve.


Stelmach Iwona

Grzelewski Tomasz

Bobrowska -Korzeniowska Monika

Stelmach Piotr

Kuna Piotr

I Stelmach

A randomized, double-blind trial of the effect of anti-asthma treatment on lung function in children with asthma

Keywords: Asthma, Children, Pulmonary Function, Budesonide, Montelukast, Formoterol

Background: Pulmonary function tests (PFTs) and especially spirometry measures are useful tools in evaluating early response to treatment of asthma in children mainly due to their worldwide availability. The aim of our study was to determine the effects of anti-asthma treatment in children, equally on FEV 1 , FEF25-75%, Rint and SRaw values. Methods: Children 6-18 years of age with moderate atopic asthma were randomized to 4week, placebo-controlled, double-blind trial. Patients were randomly allocated to receive 200 µg budesonide (B) (n=29), 5 or 10 mg (according to age) montelukast (M) (n = 29), 200 µg B + 5 or 10 mg M (n = 29), 200 µg B + 9 µg formoterol (F) (n=29) or placebo (n = 27). FEV 1, FEF25-75%, Rint, SRaw were measured before and after treatment.

Results: Rint, SRaw, FEV 1 improved significantly in all active treatment groups while FEF25-75% improved significantly only in BM group and M group. Combination therapy, showed significantly greater effects on Rint than monotherapy: BM group compared to B group (P =0.01) and M group (P =0.03) and BF group compared to B group (P =0.01) and M group (P =0.04).

Conclusion:

This study shows that using single parameter for monitoring asthma can be misleading. Using combination of lung function techniques provides better assessment of treatment. Results of our study confirm this hypothesis. The best effect on large and small airways was achieved with combined anti-inflammatory therapy.

Asthma is one of the most common chronic disease worldwide, imposing a substantial social burden on both children and adults [START_REF]Global Strategy for Asthma Management and Prevention[END_REF]. Normal lung function is one of the goals of asthma management [START_REF]Global Strategy for Asthma Management and Prevention[END_REF].

Symptom scores (SSc) and pulmonary function tests (PFTs) determine asthma severity and medication requirements [START_REF]Global Strategy for Asthma Management and Prevention[END_REF]. Exacerbation rates, frequency of daytime and nocturnal symptoms, and caregiver assessments of quality of life can be useful measures in evaluating outcomes in children with asthma [START_REF] Skoner | Outcome Measures in Childhood Asthma[END_REF][START_REF] Sharek | Agreement among measures of asthma status: a prospective study of low-income children with moderate to severe asthma[END_REF]. PFTs and especially spirometry measures are useful tools in evaluating early response to treatment of asthma in children mainly due to their worldwide availability. What needs to be kept in mind, especially in assessing early treatment effect of allergic inflammation in children with asthma, is that SSc and spirometry measures have also limitations, mainly their subjectivity (SSc), wide variabiliby, and lack of stability in short time period (SSc and FEV 1 ). Spirometry measures posess other disadvantages, like the influence of forced maneuvers on airway tone, requiring child cooperation and, even in symptomatic children, being often within the normal range [START_REF] Black | Assessment of airway function in young children with asthma: comparison of spirometry, interrupter technique, and tidal flow by inductance plethsmography[END_REF].

Additional PFTs such as resistance by the interrupter technique (Rint), plethysmography seem to be helpful additional tools evaluating early treatment effect of allergic inflammation in children with asthma. They assess airway resistances during quiet breathing, do not require active cooperation from children. It was shown that specific airway resistance (SRaw), Rint but not forced expiratory volume in one second (FEV 1 ) detected the subclinical increase in bronchial muscle tone in children [START_REF] Davis | Neonatal and pediatric respiratory diagnostics[END_REF]. It was revealed that, even in healthy children, salbutamol inhalation reduces the value of SRaw suggesting that plethysmography [START_REF] Hellinckx | Bronchodilator response in 3-6,5 yeras old healthy and stable asthmatic children[END_REF] can detect minimal changes in airway caliber [START_REF] Malmberg | Determinants of respiratory system input impedance and bronchodilator response in healthy Finnish preschool children[END_REF]. From the studies reported to date some authors

A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t [START_REF] Sly | Measurement of lung function in preschool children using the interrupter technique[END_REF][START_REF] Bisgaard | Lung function measurement in awake young children[END_REF] have confidence that Rint and SRaw are clinically useful, may precisely follow changes in lung function with growth and development and be used to manage lung disease in children, despite some reservations like questioning its repeatability. The interrupter technique has been shown to have a good short term repeatability (over a time span of minutes) in children [START_REF] Sly | Measurement of lung function in preschool children using the interrupter technique[END_REF]. Greater long term variability between measurements was found in children with persistent cough or previous wheeze [START_REF] Sly | Measurement of lung function in preschool children using the interrupter technique[END_REF], which suggests that the lower long term repeatability in symptomatic children might be due to the variability of the disease rather than the variability of the technique. On the other hand poor stability and wide variability of FEV 1 in many studies was observed [START_REF] Nakaie | A comparative study of clinical score and lung function tests in the classification of asthma by severity of disease[END_REF]11], suggesting the need of additional lung function tests usage that could accompany spirometry in studies of treatment monitoring in children with asthma.

Regular anti-asthma treatment with inhaled glucocorticosteroids improves lung function [START_REF] Bousquet | Comparison of roflumilast, an oral anti-inflammatory, with beclomethasone dipropionate in the treatment of persistent asthma[END_REF],

prevents bronchoconstriction, decreases symptoms, reduces asthma exacerbation [START_REF] Bjermer | Montelukast and fluticasone compared with salmeterol and fluticasone in protecting against asthma exacerbation in adults: one year, double blind, randomized, comparative trial[END_REF]. There is evidence that leukotriene modifiers used as add-on therapy reduce the dose of inhaled glucocorticosteroids in moderate to severe asthma, and may improve asthma control in patients whose asthma is not controlled with low or high doses of inhaled glucocorticosteroids alone [START_REF] Riccioni | Comparison of montelukast and budesonide on bronchial reactivity in subjects with mild-moderate persistent asthma[END_REF][START_REF] Virchow | Zafirlukast improves asthma control in patients receiving high-dose inhaled corticosteroids[END_REF][START_REF] Price | Clinical Outcomes with Montelukast as a Partner Agent to Corticosteroid Therapy (COMPACT) International Study Group. Randomised controlled trial of montelukast plus inhaled budesonide versus double dose inhaled budesonide in adult patients with asthma[END_REF][17]. Some authors demonstrate equivalent effects of long-acting inhaled β2agonists and leukotriene modifiers as add-on therapy on asthma exacerbations [START_REF] Bjermer | Montelukast and fluticasone compared with salmeterol and fluticasone in protecting against asthma exacerbation in adults: one year, double blind, randomized, comparative trial[END_REF]. However, recent findings of Bisgaard [START_REF] Bisgaard | Effect of long-acting beta2 agonists on exacerbation rates of asthma in children[END_REF] and Ni [START_REF] Ni | Addition of inhaled long-acting beta2-agonists to inhaled steroids as first line therapy for persistent asthma in steroid-naive adults[END_REF] showed the lack of evidence for the control of asthma exacerbations in children regularly using long-acting inhaled β2-agonists and one of these studies [START_REF] Bisgaard | Effect of long-acting beta2 agonists on exacerbation rates of asthma in children[END_REF] has brought into question its general use as add-on therapy in children.

Different surveys also revealed insufficient monitoring of asthma and its treatment [START_REF] Rabe | Clinical management of asthma in 1999: the Asthma Insights and Reality in Europe (AIRE) study[END_REF][START_REF] Van Den Berg | What general practitioners and paediatricians think about their patients' asthma[END_REF][START_REF] Hanania | Revisiting asthma control: How should it best be defined?[END_REF].

Previous research indicates that lung function testing can provide a substantial insight into the outcome of asthma. However focusing on single parameter i.e. FEV 1 may underestimate bronchial obstruction [START_REF] Hanania | Revisiting asthma control: How should it best be defined?[END_REF], mainly because it reflects large and medium-size airway function

A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t [START_REF] Eid | Can peak expiratory flow predict airflow obstruction in children with asthma?[END_REF]. Even though there is no direct parameter capable of assessing small airways, it has been assumed that the forced expiratory flow at the 25 and 75% of the pulmonary volume (FEF 25-75) might be considered as a measure of the caliber concerning distal airways [START_REF] Bjermer | Past and future perspectives in the asthma treatment[END_REF][START_REF] Ferguson | Persisting airway obstruction in asymptomatic children with asthma with normal peak expiratory flow rates[END_REF][START_REF] Bye | The importance of spirometry in the assessment of childhod asthma[END_REF],

where airflow limitation is mainly dependent on asthmatic inflammatory process intensity, not due to bronchial muscle constriction. Rint has been shown to measure fall in resistive pressure across the airways [START_REF] Sly | Measurement of lung function in preschool children using the interrupter technique[END_REF]. SRaw is the pletysmographic parameter which also measures total airway resistance (sRaw=Thoracic gas volume x Absolute airway resistance) [START_REF] Olaguibel | Comparative analysis of the bronchodilator response measured by impulse oscillometry (IOS), spirometry and body plethysmography in asthmatic children[END_REF][START_REF] Larsen | Assessing respiratory function in young children:Developmental considerations[END_REF].

That is why Rint and SRaw measures are thought to precisely assess resistances across all the airways, where airflow limitation is dependent on all pathological features of asthma, including bronchial smooth muscle constriction and hypertrophy [START_REF]Global Strategy for Asthma Management and Prevention[END_REF][START_REF] Sly | Measurement of lung function in preschool children using the interrupter technique[END_REF][START_REF] Larsen | Assessing respiratory function in young children:Developmental considerations[END_REF].

Thus, measurement of all above parameters together, may reflect airway changes in asthmatic children in the best way and optimally assess the effect of treatment.

The aim of this study was to determine the effects of anti-asthma treatment on measurement of FEV 1 , FEF25-75%, Rint and SRaw values in children with moderate atopic asthma.

Patients and methods

Patients

Participants in the 8-week study comprised of 150 children with a typical history of moderate Diagnosis of asthma was established by symptoms of asthma and improvement in the prebronchodilator FEV 1 of ≥15% after administration of salbutamol (200µg). We defined moderate persistent asthma according to established guidelines [START_REF]Global Strategy for Asthma Management and Prevention[END_REF]. During the previous 6 months, before the study, all patients had been treated with inhaled steroids (budesonide, average dose 400 µg /day) and with long-acting bronchodilator (formoterol, 0.012 mg twice daily). Antihistamines (loratadine, 10 mg daily), nasal steroids (budesonide, 0.05 mg daily), and nasal nedocromil (nedocromil sodium, 2.6 mg four times daily) were also used. Asthma was stable in all patients, there had been no exacerbations of disease or need for other treatment for 6 months, and no hospitalisations due to asthma occured in the previous 6 months before the study.

For the purposes of the study skin prick tests (Nexter-Allergopharma, Germany) to standard allergen extracts were performed; a mean wheal diameter ≥3 mm at 15 min was defined as a positive response. Serum-specific IgE levels were measured by using a Pharmacia CAP kit (Uppsala, Sweden); >0.7 kU/L were defined as positive. Subjects were recruited from our Clinic Center.

Inclusion Criteria

Male and female outpatients, aged 6 to 18 with a clinical diagnosis of bronchial asthma with a duration of at least 6 months before the first visit and with current history of moderate persistent asthma, were enrolled. To become eligible for the active treatment period, patients and their parents were required to do reproducible spirometry, whole body pletysmography and interrupter technique.

Exclusion Criteria

Study exclusions included active upper respiratory tract infection within 3 weeks before the study and acute sinus disease requiring antibiotic treatment within 1 month before the study, previous intubation, or asthma hospitalisation during the 3 months before the first visit.

Additional criteria were other clinically significant pulmonary, hematologic, hepatic, gastrointestinal, renal, endocrine, neurologic, cardiovascular, and/or psychiatric diseases or malignancy that either put the patient at risk when participating in the study or could influence the results of the study or the patient's ability to participate in the study as judged by the

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

investigator. Excluded medications were β-blockers (eye drops included), astemizole within 3 months, or oral corticosteroids within 1 month before the first visit. Patients who were receiving immunotherapy were also excluded.

Study Design

A schema showing the flow of subjects through the trial is shown in Figure 1. The study was conducted fom April to October, when the exposure to dust mites was at the constant level.

This was a randomized, double-blinded, placebo-controlled trial comparing the effect of budesonide, montelukast, budesonide with montelukast, budesonide with formoterol and matching placebo. We measured PFTs such as FEF25-75%, Rint, SRaw and FEV 1 before and after treatment. Children 6-14 years received 5 mg of montelukast (Singulair, MSD, Whitehouse Station, NJ, USA) and children > 14 years old received 10mg oral tablet once daily at bedtime. Budesonide (Pulmicort turbuhaler, AstraZeneca, UK) was administered 200 µg per day, formoterol (Oxis turbuhaler, AstraZeneca, UK) was administered 9 µg per day.

Montelukast, placebo, turbuhalers containing drug or no drug were blinded by the hospital pharmacy.

There were three study visits. At the first visit, long acting β2-agonists, steroids, nasal cromolyn, and antihistamines were stopped, and patients were put on inhaled β2-agonist, (Ventolin, GlaxoSmithKline, USA) 100 µg/dose 'as needed' for symptomatic relief purposes.

They were informed of the aim of the study and were told how to use the inhalers. At the second visit, 4 weeks after the first visit, patients were randomized according to a computergenerated allocation schedule for anti-asthma treatment with:

• budesonide, montelukast placebo, formoterol placebo (29 patients) -budesonide group

• montelukast, formoterol placebo, budesonide placebo (29 patients) -montelukast group

• budesonide, montelukast, formoterol placebo (29 patients) -budesonide with montelukast performed. This always took place at the same time of day (11 a. m.) and on the same day of the week. Before lung function tesing all drugs were witheld for 24 hours. The measurements of FEF25-75%, FEV 1 , Rint and SRaw, were recorded at visit two and three.

Lung function measurements

Pulmonary function testing was done with a LUNGTEST 1000 unit (MES, Cracov, Poland).

and FEF25-75%

A spirometer was used to measure FEV 1 and FEF25-75%. The tests were performed according to American Thoracic Society standards [START_REF] Crapo | Standardization of Spirometry, 1994 Update. American Thoracic Society[END_REF]. The highest of three successful measurements was taken. The results were expressed as percentage of change from baseline.

SRaw

Measurements were made during tidal breathing. SRaw was measured in a constant-pressure, whole-body plethysmograph as the relationship between simultaneous variations in respiratory flow and maximum changes in plethysmographic pressure during inspiration and expiration. SRaw was calculated from the S-shaped resistance loops presented graphically by the computer connected with the plethysmograph. The relationship between flow and pressure was displayed on-line. The measurement was rejected if any alteration in the shape of the resistance loops was seen, since this could reflect face mask leakage, coughing, swallowing, or vocalization. Compensation for body temperature, barometric pressure, and water vaporsaturated (BTPS) conditions was done electronically [START_REF] Klug | Measurement of the specific airway resistance by plethysmography in young children accompanied by an adult[END_REF]. The respiratory rate was 30 to 45 breaths/min. The mean value of three sequential measurements of SRaw was used as the result. The results were expressed as percentage of change from baseline.

Rint

Measurements were carried out using the LUNGTEST 1000 Unit, device including a shutter owing to air leakage or altered ventilation pattern and were discarded as well [START_REF] Merkus | Interrupter resistance in preschool children: measurement characteristics and reference values[END_REF]. The mean value of five consecutive measurements was used as the result. The results were expressed as percentage of change from baseline.

Statistical methods.

The results were analyzed according to well known statistical methods by using StatSoft Statistica for Windows, release 6.0 (StatSoft, Inc., Tulsa, USA). Data was presented as mean with standard error (SEM) or standard deviation (SD) (for demographic data). Before analysis all parameters were logarytmically transformed to the normal distribution. To determined differences within groups Student's t-test was used. To compare changes with treatment between groups all parameters were analyzed using analysis of variance (ANOVA) for repeated measures. P-values < 0.05 were considered to be significant.

Ethics

This study was approved by the Medical Ethics Committee of the Medical University. All parents or guardians gave their written consent for participation in this study. The families

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

were fully informed that treatment withdrawal during wash-out period might result in a significant exacerbation of asthma and they were given a specific plan to manage any exacerbation that might occurre.

Results

One hundred and forty three subjects completed the study, [START_REF] Phh | Standardisation of lung function tests. 1993 Update. Report Working Party for the European Community for Steel and Coal[END_REF] Lung function was significantly improved after 4 weeks of treatment with budesonide, montelukast, budesonide with montelukast, and budesonide with formoterol and it was reflected by Rint, SRaw and FEV 1 within groups and compared to placebo at the endpoint after 4 weeks of treatment (Table 2).

Rint, SRaw and FEV 1 improved during active treatment period and deteriorated during administration of placebo (Table 2, Figure 2).

Additionally it was found that FEF25-75% values improved significantly after 4 weeks of

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

treatment with montelukast and budesonide with montelukast within groups and compared to placebo (Table 2, Figure 3).

Analysis of variance (ANOVA) showed no significant differences between active treatment groups in changes of SRaw and FEV 1 . However, we found significant difference between active treatment groups in Rint (Figure 2). Rint improved more significantly in budesonide with montelukast group compared to budesonide group (P=0.01) and compared to montelukast group (P=0.03). Also in budesonide with formoterol group compared to budesonide group (P=0.01) and compared to montelukast group (P=0.04), Rint improved more significantly.

Discussion

This is the first trial using measurement of FEV 1 , FEF25-75%, Rint and SRaw after the different therapeutic combinations for the management of childhood asthma. There have been many studies on this topic, however, none of them used the combination of various techniques to differentiate central and peripheral airway obstruction. In previous studies, rather single drug treatment effect on lung resistance parameters was assessed [START_REF] Bisgaard | Bronchoprotection with a leukotriene receptor antagonist in asthmatic preschool children[END_REF][START_REF] Von Berg | Formoterol and salbutamol metered aerosols: comparison of a new and an established beta-2-agonist for their bronchodilating efficacy in the treatment of childhood bronchial asthma[END_REF][START_REF] Kozlik-Feldmann | Long-term effects of formoterol and salbutamol on bronchial hyperreactivity and beta-adrenoceptor density on lymphocytes in children with bronchial asthma[END_REF][START_REF] Pao | Randomized controlled trial of fluticasone in preschool children with intermittent wheeze[END_REF][START_REF] Nielsen | Bronchodilation and bronchoprotection in asthmatic preschool children from formoterol administered by mechanically actuated dry-powder inhaler and spacer[END_REF].

Our study showed that budesonide, montelukast, budesonide with montelukast, and budesonide with formoterol significantly improved lung function based on FEV 1 , Rint and SRaw values after four weeks of treatment. We also showed that monotherapy with montelukast and montelukast combined with budesonide increased FEF25-75% confirming that montelukast improved the airflow in small airways in our patients. According to the Rint results, we demonstrated that combination therapy showed significantly greater effects than monotherapy across the airways (on large and small airways).

Although prior to the study all patients had been treated with budesonide and formoterol in doses higher than that of the study period, because of the fact that asthma was stable at least

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

from six months, we were able to modify treatment.

The present study was explorative and the low dose of budesonide was chosen with a view towards not overlooking any possible effect of other studied drugs on lung function. This low dose of budesonide was chosen to compare it favourably with the effects of montelukast. The justification for using montelukast alone for the therapy of moderate persistent asthma is to assess the single effect of montelukast on lung function in children.

There are many data showing significant improvement in lung function (based on FEV 1 or/and PEF results) in children with asthma treated with anti-leukotrienes [START_REF] Szefler | Characterization of within-subject responses to fluticasone and montelukast in childhood asthma[END_REF][START_REF] Stelmach | A randomized, double-blind trial of the effect of treatment with montelukast on bronchial hyperresponsiveness and serum eosinophilic cationic protein (ECP), soluble interleukin 2 receptor (sIL-2R), IL-4, and soluble intercellular adhesion molecule 1 (sICAM-1) in children with asthma[END_REF] inhaled steroids [START_REF] Szefler | Characterization of within-subject responses to fluticasone and montelukast in childhood asthma[END_REF][START_REF] O'byrne | Effects of montelukast (MK-0476); a potent cysteinyl leukotriene receptor antagonist, on bronchodilation in asthmatic subjects treated with and without inhaled corticosteroids[END_REF], anti-leukotrienes with inhaled steroids [42], long acting β2-mimetics with inhaled steroids [START_REF] Tal | Budesonide/formoterol in a single inhaler versus inhaled corticosteroids alone in the treatment of asthma[END_REF]. Nevertheless, there is limited data showing an efficacy of treatment based on FEF25-75%, SRaw and Rint measurements in children with asthma.

While currently glucocorticosteroids remains the reference drug as a controller in moderate persistent asthma, the study data demonstrated that administration of montelukast provided an important and additional effect on peripheral airways function in children. Moderate improvements in lung function, by means of spirometric measurements (in children 6 and older) and in asthma control (in children 2 and older) have been demonstrated with leukotriene receptor antagonist monotherapy in patients with severe and moderate disease [START_REF] Stelmach | A randomized, double-blind trial of the effect of treatment with montelukast on bronchial hyperresponsiveness and serum eosinophilic cationic protein (ECP), soluble interleukin 2 receptor (sIL-2R), IL-4, and soluble intercellular adhesion molecule 1 (sICAM-1) in children with asthma[END_REF][START_REF] Stelmach | The effect of treatment with montelukast on in vitro interleukin-10 production of mononuclear cells of children with asthma[END_REF][START_REF] Knorr | Montelukast for chronic asthma in 6-to 14-year-old children: a randomized, double-blind trial. Pediatric Montelukast Study Group[END_REF]. In his latest cross-over study Szefler et al. showed that phenotype of children with mild-to-moderate asthma, with normal pulmonary function and low levels of markers associated with allergic inflammation could receive montelukast as initial therapy with an assessment in response [START_REF] Szefler | Characterization of within-subject responses to fluticasone and montelukast in childhood asthma[END_REF]. However, some severe asthmatics could be particularly sensitive to the positive effects of montelukast [START_REF] Tonelli | Short-term effect of the addition of leukotriene receptor antagonists to the current therapy in severe asthmatics[END_REF].

In our study we revealed that children treated with montelukast, either in monotherapy or in small airways measured by impulse oscillometry in children but there was no effect on FEF25-75% parameter [START_REF] Nieto | Montelukast improves pulmonary function measured by impulse oscillometry in children with asthma (Mio study)[END_REF]. Only one study in adults with moderate persistent asthma revealed that montelukast in combination with salmeterol improved FEF25-75% after 2 weeks of treatment [START_REF] Currie | A proof of concept study to evaluate putative benefits of montelukast in moderate persistent asthmatics[END_REF]. Results of the two studies in children with asthma revealed that also budesonide monotherapy significantly improves FEF25-75% values [START_REF] Cengizlier | Circulating ICAM-1 levels in bronchial asthma and the effect of inhaled corticosteroids[END_REF][START_REF] Shapiro | Once-daily budesonide inhalation powder (Pulmicort Turbuhaler) maintains pulmonary function and symptoms of asthmatic children previously receiving inhaled corticosteroids[END_REF].

SRaw and Rint values were used to assess efficacy of treatment with budesonide [START_REF] Nielsen | The effect of inhaled budesonide on symptoms, lung function, and cold air and methacholine responsiveness in 2-to 5-year-old asthmatic children[END_REF],

montelukast [START_REF] Bisgaard | Bronchoprotection with a leukotriene receptor antagonist in asthmatic preschool children[END_REF][START_REF] Spahn | Effect of montelukast on peripheral airflow obstruction in children with asthma[END_REF], formoterol [START_REF] Von Berg | Formoterol and salbutamol metered aerosols: comparison of a new and an established beta-2-agonist for their bronchodilating efficacy in the treatment of childhood bronchial asthma[END_REF][START_REF] Kozlik-Feldmann | Long-term effects of formoterol and salbutamol on bronchial hyperreactivity and beta-adrenoceptor density on lymphocytes in children with bronchial asthma[END_REF], salmeterol [START_REF] Tomac | Efficacy of salmeterol in the treatment of childhood asthma[END_REF], and fluticasone [START_REF] Pao | Randomized controlled trial of fluticasone in preschool children with intermittent wheeze[END_REF] in children with asthma. Study of Tomac et al. showed high efficacy of salmeterol administered for four weeks together with corticosteroid and disodium cromoglycate maintenance treatment by improving airway resistance and conductance [START_REF] Spahn | Effect of montelukast on peripheral airflow obstruction in children with asthma[END_REF]. In our study we also revealed significant decrease of SRaw together with Rint values within group of children treated with budesonide with formoterol. The effect of treatment on SRaw with one dose of formoterol was investigated and compared with one dose of salbutamol by Von Berg et al [START_REF] Von Berg | Formoterol and salbutamol metered aerosols: comparison of a new and an established beta-2-agonist for their bronchodilating efficacy in the treatment of childhood bronchial asthma[END_REF]. In this study formoterol produced a larger decrease in SRaw from baseline for longer periods after inhalation than did salbutamol, what was observed also in other study [START_REF] Nielsen | Bronchodilation and bronchoprotection in asthmatic preschool children from formoterol administered by mechanically actuated dry-powder inhaler and spacer[END_REF].

Study of Nielsen and Bisgaard revealed that Rint but not SRaw improved during treatment with budesonide in preschool children with asthma [START_REF] Nielsen | The effect of inhaled budesonide on symptoms, lung function, and cold air and methacholine responsiveness in 2-to 5-year-old asthmatic children[END_REF]. Also results of study of Pao and

McKenzie outline the improvement in Rint after treatment with fluticasone in younger wheezy children sensitized to common aeroallergens [START_REF] Pao | Randomized controlled trial of fluticasone in preschool children with intermittent wheeze[END_REF]. In our study we observed significant improvement of Rint and SRaw after treatment with 200 µg of budesonide. However we demonstrated that addition of montelukast or formoterol to budesonide therapy was associated with significantly greater improvements in Rint. Rint perform better than SRaw in terms of discriminating responses in the groups that received some form of chronic anti-inflammatory therapy. The basic assumption underlying the interrupter technique is that, following an instantaneous interruption of airflow at the airway opening (by closing a valve or shutter), there is an instantaneous equilibration of pressure between the alveoli and the airway opening (behind the occlusion) [START_REF] Sly | Measurement of lung function in preschool children using the interrupter technique[END_REF]. Rint has been shown to measure fall in resistive pressure across the airways, and includes also all Newtonian resistance from the respiratory system including components from the pulmonary tissues and chest wall [START_REF] Sly | Measurement of lung function in preschool children using the interrupter technique[END_REF], while SRaw concentrates specifically on airway resistance measurement [START_REF] Larsen | Assessing respiratory function in young children:Developmental considerations[END_REF]. We can speculate that the difference between the spectrum of these two measurements was responsible for greater significant improvement in Rint value in comparison with SRaw value after both combined therapies in our study.

In our study the lung function parameters in the active treatment groups improved compared to a deterioration in the placebo group. This certainly underscores the importance of objective monitoring of lung function in children with asthma. All patients from our study groups were able to easily perform all manouvers required for PFTs studied.

The aim of our study was to determine effect of anti-asthma treatment in children, equally on FEV 1 , FEF25-75%, Rint and SRaw values. There is no one single lung function parameter nor comparison as primary outcome in this study; there are four different treatment groups. In this case it did not allow us to adequately estimate power and sample size for our study.

This study shows that using single parameter for monitoring asthma can be misleading. Using combination of lung function techniques provides better assessment of treatment. Results of our study confirm this hypothesis. The best effect on large and small airways was achieved

  asthma who were sensitive to house dust mites as shown by positive skin-prick tests and by the presence of specific IgE to Dermatophagoides pteronyssimus or Dermatophagoides farinae (41 patients positive to Dermatophagoides pteronyssimus, 54 patients positive to Dermatophagoides farinae and 55 patients positive to both D. pteronyssimus and D. farinae).

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  s c r i p t group • budesonide, formoterol, montelukast placebo (29 patients) -budesonide with formoterol group • montelukast placebo, formoterol placebo, budesonide placebo (27 patients) -placebo group At visit two and three spirometry, whole body pletysmography and interrupter technique were

Flow

  and volume were measured with a pressure-screen-type pneumotachograph, calibrated daily. All measurements were performed by trained investigators. The sequence of measurements was Rint, whole body plethysmography and spirometry. Measurements were carried out in a familiar and quiet room. Standing height and weight were assessed: subjects were measured without shoes, wearing light summer clothing. During measurements, children were instructed to sit upright, and a nose clip and a noncompressible mouthpiece were used. When needed, an adult accompanied the subject during testing. Predicted values for all lung function variables were based on a previous study of healthy controls, provided by the lung function test equipment manufacturer [29, 30]. Mean relative change normalizes lung function improvement over a wide of baseline lung function and was defined as: PFT at the end of the study -PFT at the start PFT at the start x 100% PFT: pulmonary function test

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  and pneumotachograph, connected to computer with an online display showing mouth pressure, time of shutter closure and Rint values recorded during one session. Its software calculates Rint using the back extrapolation technique to t = 15 ms after shutter closure during 100 ms. Measurements were made during tidal breathing. The position of the pneumotachograph with Rint was adjusted on a support arm to facilitate unobstructed breathing. The functioning of the shutter was demonstrated once to make children familiar with the sound. A number of 5 correct tracings was then obtained for the expiratory maneuver. During this period the cheeks and chin were supported from behind by the observer, the head was positioned in slight extension. Shutter closure was programmed at maximal expiratory tidal flow. Thus, Rint values are obtained at or near midexpiration, minimizing the breath-to-breath variation in inflation level and hence on Raw. Timing of the shutter closure (at the peak of flow) can be checked on the display. Tracings were inspected immediately after the measurement in the presence of the child. Rejection criteria were: tachypnea, usage of the vocal cords, extreme neck flexion or extension, leakage of the mouthpiece. Tracings not showing the timing of the shutter closure were discarded; tracings with a horizontal or declining pressure signal after shutter closure were considered artifacts

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  combined therapy with budesonide demonstrate increase in FEF25-75% values after four weeks of treatment. This suggests that systemic drugs -like oral montelukast -can reach lower airways, whose inflammation plays a crucial role in the evolution of asthma, while inhaled drugs hardly reach them. Study of Nieto et al. showed good effect of montelukast on

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t 1 Flow

 1 Figure 1

Figure 2 ChangeFigure 3 A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

 23 Figure 2

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Figure 1 :

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Figure 2 :

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Figure 3 :

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Table 1

 1 Patients baseline characteristics. Data is presented as mean and standard error mean.

	Definition of abbreviations: NS= not statistically significant; %
	pred.=percentage of predicted value

Table 2

 2 Effect of treatment with budesonide, montelukast, budesonide with montelukast, budesonide with formoterol, placebo on FEV1, FEF25-75%, SRaw and Rint. Values are expressed as means with standard error mean (SEM).Definition of abbreviations: FEV 1 =forced expiratory volume in one second; FEF25-75%=forced expiratory flow between 25% and 75% of the vital capacity; SRaw=specific airway resistance; Rint=resistance by the interrupter technique; % pred.= percentage of predicted value.

This study was funded by grant 503-207-1 and 503-2056-