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provided by Elsevier - Publisher Connector Vol. 95 (2001) 734^739

Budesonide but not nedocromil sodium reduces exhaled nitric oxide levels in asthmatic children S. Carra' *,L.Gagliardi{, S. Zanconato*,M.Scollo*,N.Azzolin*, F. Zacchello* and E. Baraldi*

*Department of Paediatrics,Unit of Allergy and Respiratory Medicine,University of Padova, Padova and {Department of Neonatology, ICP, Milano, Italy

Abstract Exhaled nitric oxide (ENO) has been proposed as a marker of airwayin£ammation in and could be usefulto evaluate the response to anti-in£ammatory treatment.Weinvestigated the e¡ect of and nedocro- mil sodium on ENO levels and lung function in asthmatic children.Twenty stable steroid-na|«ve asthmatic children were randomizedin a single blind, cross-over study to receive inhaled budesonide (group A) or nedocromilsodium (group B) for 6 weeks.ENO wasmeasured with a chemiluminescence analyser at baseline and atthe end of eachtreatmentperiod. Repeated-measures ANOVAwas carried out.In asthmatic baseline ENOlevels [mean 32?5 ppb,95% con¢dence interval (CI) 26?4to38?7] were signi¢cantly higher compared to reference values (8?7ppb,95%CI8?1to9?2, P50?001).There were no treatment-order e¡ect, no carry-over e¡ect and in both groups the response pattern was the same: budeso- nide signi¢cantly lowered ENO levels from 41?0ppbto22?8 ppb in group A (mean, P50?001) and from 22?6ppbto 13 ?0 ppb in group B, (mean, P50?05), while nedocromil did not reduce ENO values (from 24?4ppbto22?6ppbingroup Bandfrom22?8 ppb to 38?0 ppb in group A, mean, P ¼NS and P50?01respectively). After budesonide treatment ENO values of asthmatics were stillsigni¢cantly higher thanin healthy children.The baseline values of FEV1and FEF25^75 were normal in both groups and no signi¢cant changes were observed during the study.In conclusion, our study shows that budesonide, but not nedocromilsodium, signi¢cantlyreduces ENOlevelsin stable asthmatic children evenin absence of changes in the lung function. c 2001Harcourt Publishers Ltd doi:10.1053/rmed.2001.1130, available online at http://www.idealibrary.com on

Keywords asthma, budesonide, nedocromil sodium, exhaled nitric oxide, children.

INTRODUCTION reasons, a recent international consensus on the man- agement of childhood asthma continues to recommend Inhaled (ICS) and are, for the chromones as the ¢rst-line prophylactic for time being, the main long-term-control for children (5). Some studies have supported the clinical childhood asthma and are recommended as the ¢rst-line e⁄cacy of chromones treatment for childhood asthma prophylactic treatment (1,2). (7^10) and nedocromil sodium has been shown to inhibit ICS are highly e¡ective anti-in£ammatory agents and bronchoconstriction by an e¡ect on both the early and recently their early introduction has been proposed for the late asthmatic response (11). Unfortunately, speci¢c school-aged children requiring continuous prophylactic studies to measure the anti-in£ammatory e¡ects of ne- treatment (3,4).This strategy, however, is not universally docromil sodium are very limited and recently the e¡ec- accepted (5). It has been shown that they may improve tiveness of chromones as maintenance therapy in symptoms, lung function and prevent complications, children with asthma has been questioned (12). such as irreversible airway damage or exacerbations (6). Recent evidence suggests that exhaled nitric oxide Although ICS have a much lower potential for systemic (ENO) represents a sensitive, non-invasive marker for as- e¡ects than oral steroids, there remains the possibility sessing the degree of ongoing airway in£ammation and the of side-e¡ects from a low level of systemic activity in pa- response to therapy in patients with asthma and other air- tientsreceiving long-term therapy even if itis notclear at way diseases (13^17). Nitric oxide (NO) is produced by what level they become clinically signi¢cant. For these many cells within the respiratory tract and can be easily detected in the exhaled air (14,15). The concentration of Received 28 December 2000 and accepted in revised form14 May 2001. Correspondence should be addressed to: Dr Eugenio Baraldi, ENO is increased in patients with airway in£ammation such Department of Paediatrics,Via Giustiniani 3, 35128 Padova, Italy. as asthma (16,18)and a rapidreduction of ENOlevels occurs Fax: +39-049-8213502; E-mail: [email protected] in asthmatics after a course of oral steroid therapy (16,19). BUDESONIDE, NEDOCROMIL AND EXHALED NO 735

The aim of our work is to evaluate the e¡ect of 6 dium for 6 weeks. At the screening visit, patient weeks of treatment with budesonide and nedocromil admission details and medical histories were recorded sodium on ENO levels and lung function in steroid-na|«ve and each subject underwent ENO measurement and 1 asthmatic children.To our knowledge, no similar studies spirometry.The dosage of budesonide (Pulmaxan As- have previously addressed this issue in children. tra) was 400 mgday71 (200 mg twice daily) under the age of 9 years and 600 mgday71 (400 mginthemorning and 200 mg in the evening) after the age of 9 years. The METHODS 1 dosage of nedocromil sodium (Tilade Rhone-Poulenc) Patients was 4 mg three times daily. Budesonide was given by a 1 dry powder device (Turbohaler Astra) and ne- Twenty asthmatic children (13 males, seven females) ran- docromil sodium by MDI through a large spacer (Volu- ging in age from 5 to14 years (9?9+0?5years,mean+SEM) 1 matic Glaxo). Children and parents were instructed were recruited.The patients attended the Pulmonology/ for the use of the two inhalation devices prior to entry Allergy Outpatient Clinic of the Department of Paedia- into the study.The ¢rst study treatment was given for 6 trics, University of Padova. All children had mild or mod- weeks (t ), then the patients were switched to the other erate asthma (1) for at least 1year.Children were eligible 1 treatment for a further 6 weeks (t ).There was no wash- for our study if (a) asthma was stable, (b) they had not 2 out between the study periods. received any form of regular therapy (ICS, chromones, Patients performed ENO measurement and spirome- , long-acting b -agonists) for at least 2 2 try in three occasions: at baseline, at the end of the ¢rst months prior to the study.Only inhaled albuterol was al- treatment (after 6 weeks, t ), and then at the end of the lowed when needed. Stable asthma was de¢ned as having 1 second treatment (after other 6 weeks, t ). Measure- a forced expiratory volume in 1sec (FEV )480% pre- 2 1 ments were always done in the afternoon by the same dicted, symptom-free interval, no oral steroid use and investigator who was blinded to the treatment.ENO va- no history of asthma exacerbation within the previous 2 lues of asthmatics were compared to normative data months. Patients were excluded if they had su¡ered from previously obtained using the same method (20). The a respiratory tract infection in the month before ENO protocol was approved by our institutional review measurements. The diagnosis of asthma was based on board, and informed consent was obtained from all par- the clinical history, examination, and pulmonary function ents and patients. tests. All the subjects underwent skin-prick testing by use of a panel of common inhalant : mixed grass Measurement of exhaled NO pollen, Parietaria, Artemisia Vulgaris, Dermatophagoides pteronyssinus and farinae, Alternaria and cat (Bayropharm, ENO was measured by an online tidal breathing method Bayer, Milano, Italy). Sixteen patients were shown to be (14,15) using a chemiluminescence analyser.The details of positive to common allergens. To avoid potential con- the method have been previously described (16). Brie£y, founding e¡ect by pollens we designed the time of the the child was asked to breathe at tidal volume, through study to be outside the grass pollen season. a mouthpiece directly connected by aTe£on tube to the analyser via a two-way valve to avoid rebreathing. To ex- Controls clude the impact of environmental NO, the child inhaled NO free-air accumulated in a collapsible reservoir.ENO As reference values for ENO we used the data previously levels were continuously analysed by the chemilumines- obtained in159 healthy children (71males and 88 females, cence analyser (CLD 700 Al-Med, Ecophysics, Switzer- age range 6^15 years) using the same tidal breathing land) sampling at a constant £ow of 0?7lmin71.NO method (20). All children were caucasian, within the nor- levels were recorded when a steady state was reached. mal range for height and weight. There was no history In order to keep the soft palate closed (14,15), the breath- for allergy,respiratory diseases and smoking experience. ing circuit was provided with an internal restrictor that No children were taking medication and/or had had re- allows the exhalation with a low resistance providing a cent respiratory tract infections (20). pressure of 3^ 4 cm H2O at the mouthpiece.Before each study, the chemiluminescence analyser was calibrated Study design with a certi¢ed calibration mixture of NO in nitrogen (NO ¼ 300 ppb, NO ¼ 308 ppb) (SIAD, Bergamo, Italy) This study was a prospective, cross-over,self-controlled, x with guaranteed stability.ENO concentrations were re- single blind (blind to the investigator who performed ported in parts per billion (ppb). ENO measurements), randomized trial to evaluate the e¡ects of inhaled budesonide and nedocromil sodium on Spirometry ENO and lung function in asthmatic children. Following the screening visit, included patients were randomly allo- Pulmonary function parameters were measured by cated to receive either budesonide or nedocromil so- means of a 10-l bell spirometer (Biomedin, Padua, Italy), 736 RESPIRATORY MEDICINE and the best of three manoeuvres was expressed as a pliance with the therapy.Overall, six patients in group A percentage of predicted values according to Polgar and completed the sequence budesonide ^nedocromil and Promadhat (21). All patients abstained from using albu- eight patients in group B completed the sequence nedo- terol for at least12h before the test. cromil ^ budesonide.

Statistical analysis Exhaled NO The primary endpoint of the study was to evaluate the Baseline ENO values were signi¢cantly higher in asth- changes in ENO through the treatments.To test the hy- matics than in healthy children (41?0ppb,95%CI30?2to pothesis that treatments modi¢ed the parameters 51 ?7groupA;24?4 ppb, 95% CI 15?8to33?0groupB; (ENO levels, FEV ,FEF ), multivariate repeated-mea- 1 25^75 8?7 ppb, 95% CI 8?1to9?2 healthy controls, P50?001). sures analysis of variance (RMANOVA) was applied (22), Randomization notwithstanding, the baseline ENO va- treatment being the within-subjects factor. The treat- lues of the two groups of asthmatic patients were di¡er- ment order (nedocromil/budesonide vs. budesonide/ne- ent, group A having a higher value. Values of ENO docromil) was inserted in the model as a between- decreased after budesonide, but not after nedocromil subject factor (Fig. 1). After cross-over, the pattern was similar. RMA- to test the treatment-order e¡ect. If the result of NOVA showed no signi¢cant treatment-order e¡ects RMANOVA was signi¢cant, orthogonal contrast com- nor interactions. As the sequence of treatments did not parisons were applied. All analyses were performed with the SPSS/PC+ statistical package (SPSS Inc, Chicago, IL, U.S.A.).The results are expressed as mean with 95% con- ¢dence intervals (CI). AP-value lower than 0?05 was con- sidered as signi¢cant.

RESULTS Subjects demographics Twenty asthmatic children were recruited. Patients’ characteristics at baseline are presented inTable1. Eleven children were randomized to receive budesonide in the ¢rst 6 weeks and then switched to nedocromil sodium for the other 6 weeks (group A).The other nine children (group B) received nedocromil sodium for the ¢rst 6 weeks of the study and then switched to budesonide for the other 6 weeks. Of the 20 patients who entered the treatmentperiod,sixwerewithdrawnduringthelast6 weeks of treatment, ¢ve from group A and one from group B.Five patients of group Awere withdrawn during the treatment with nedocromil sodium for inadequate asthma control. The patient in group B was withdrawn during the treatment with budesonide due to low com-

TABLE 1. Baseline patients’characteristics FIG.1. (a) Meanlevel of FEV1% predicted (95% CI) ateach per- iodof assessment.No signi¢cantdi¡erenceswere observeddur- Group A Group B ing the study.(b) Mean levels of ENO (95% CI) at each period of Number 11 9 assessment during the study.Data are from subjects that com- Age (years) 11 ?2+0?58?3+0?6 pleted the study.Subjects of group A (&) were ¢rsttreatedwith mean+ SEM budesonide for 6 weeks and then switched to nedocromil so- Range 8^14 5^11 dium.Group B (*) received the treatmentsinthe reverse order. Sex (F/M) 8/3 5/4 In both groups budesonide treatment was followed by a signi¢- Atopic 11 5 cant reduction of ENO values. Reference values for ENO (95% Non-atopic 0 4 CI) are from 159 healthy children (20). *P50?05, **P50?01, ***P50?001. P-values represent comparison between the end and the startof eachtreatment period. BUDESONIDE, NEDOCROMIL AND EXHALED NO 737

TABLE 2. ENO values of the two treatment pooled together in14 children completing the study

Baseline Budesonide Di¡erence (Group A+B) (Group At1+Bt2) NO (ppb) 31?5(24to39) 17?2(12?5to21?9)* 714 ?3(721 ?4to77?2) Baseline Nedocromil Di¡erence (Group A+B) (Group At2+Bt1) NO (ppb) 31?5(24to39) 29?2(23?0to35?3) { 72?3(77?7to3?1) P-values representcomparison between baseline and the end of eachtreatment. t1,t2 see methods. ENO values are expressedin ppb, as meanwith 95% CI. *P50?001, {P¼NS.

in£uence the results, the two treatment sequences were and 97% (95% CI 62 to 132) in the group A and 97% (95% pooled together.The treatment e¡ect was the only fac- CI 90 to 105) and 109% (95% CI 75 to 137) in group B re- tor to explain the observed di¡erences, and was highly spectively, P ¼ NS]. During the study there were no sig- signi¢cant (P ¼ 0?002); contrast comparisons showed ni¢cant changes in the two groups at each period of that ENO values after nedocromil (At2+Bt1)werenot assessment (RMANOVA, P ¼ NS) (Fig.1). di¡erent from baseline (di¡erence 72?3 ppb, 95% CI 77?7to3?1, P ¼ 0?38), whereas ENO values after bude- sonide (At1+Bt2) were di¡erent from baseline (di¡erence DISCUSSION 714?3 ppb, 95% CI 721?4to77?2, P50?001) (Table 2). Given the di¡erence in baseline values between groups Recently, the measurement of ENO has been proposed A and B, a separate analysis was carried out within each as a sensitive, non invasive marker to assess the extent group. In both groups, RMANOVA showed that there of airway in£ammation in asthmatic patients and moni- were signi¢cant di¡erences between the three times tor the e¡ectiveness of anti-in£ammatory therapy (baseline, t1,t2).Values of ENO after nedocromil treatment (13,17).The results of this study show that, in stable ster- were never di¡erent from baseline (P ¼ 0?54 in group A, oid-na|«ve asthmatic children, 6 weeks of treatment with P¼0?57 in group B); values after budesonide treatment inhaled budesonide signi¢cantly reduce ENO levels, were signi¢cantly reduced compared to baseline (group A while nedocromil sodium does not signi¢cantly modify P50?001) and nedocromil treatment (group B P50?05). ENO production. However, the ¢nal levels of ENO after Moreover, even after budesonide treatment, both in group inhaled steroids were still increased compared to values A and group B ENO values of asthmatics were still signi¢- found in normal individuals. cantly higher than in healthy children ( P50?01). The ENOreduction found after budesonide treatment In order to verify a possible source of bias due to the con¢rms and extends the results of previous studies that presence of six drop-outs, we performed also an analysis showed a decrease in ENO levels in asthmatic patients of the data of the pre-cross-over period when all pa- treated with inhaled steroids (24^26). Recently, Jataka- tients were available.This amounts to carry out a paral- non et al. (17) assessed the e¡ect of inhaled budesonide lel-comparison design study, and allows to obtain on ENO and other in£ammatory parameters in asth- unbiased estimates of the e¡ects (23) (group A, n ¼11: matics. They found that, after inhaled steroids, there baseline ENO 38.6 ppb, 95% CI 30?2to47?1, post-treat- was a signi¢cant reduction in ENO, an improvement in ment ENO 25?8 ppb, 95% CI 17?1to34?5, P50?05; group methacholine PC20 and a reduction of sputum eosino- B, n ¼ 9: baseline ENO 25?1ppb, 95% CI17?5to32?6, post phils. Similar results have been obtained by van Rensen treatment ENO 22?1ppb,95% CI15?3to28?9, P ¼ NS with et al. after a 4-week treatment with £uticasone in asth- aStudent’st-test for paired data). Also with this analysis, matic adults (24). Taken together these ¢ndings support the results are similar to those already shown con¢rming the contention that ENO is a surrogate marker of airway RMANOVA results, i.e. that budesonide but not nedo- in£ammation. cromil sodium reduces ENO levels. In children it has been shown that during asthma ex- acerbation ENO is a more sensitive marker of asthma ac- tivity than serum markers (ECP and soluble interleukin), Spirometry and appears to be a more useful indicator of the bene¢- cial response to therapy (19). It could be At baseline there were no signi¢cant di¡erences in FEV1 speculated that the anti-in£ammatory e¡ect of steroids and FEF25^75 between the groups [95% (95% CI 83 to106) results in a down-regulation of the transcription of 738 RESPIRATORY MEDICINE inducible nitric oxide synthase (iNOS) with subsequent sonide treatment, ENO was still signi¢cantly increased reduced production of NO. It has been suggested that compared to healthy controls in both groups. This sug- these drugs inhibit the expression of iNOS probably by gests that normal values of ENO in asthmatics can prob- blocking the transcription factor nuclear factor-kappa B ably be reached only with higher dosages of steroids or that is critical for transcription of the iNOS gene (27). longer treatment periods. It is indeed known that treat- Furthermore, the e¡ect of steroids on epithelial expres- ment with inhaled steroids may provide only partial sup- sion of iNOS has been con¢rmedin vivo using endobron- pression of airway in£ammation (36) and that the e¡ect chial biopsies (28). of steroids on ENO is likely to be dose-dependent (17,25). We did not show a signi¢cant reduction of ENO after The inability to show an improvement in lung function nedocromil sodium therapy: in group A, ENO values in- following steroid treatment, despite the improvement creased, while in group B they remained unchanged. It of ENO, may be due to the normal baseline levels of needs to be highlighted that group A had lower baseline FEV1 (83^116% predicted), with little room for improve- ENO values because of previous budesonide treatment. ment. It is therefore likely that the suppression of airway This suggests that nedocromil sodium is unable to main- in£ammation induced by inhaled steroids in patients with tain the inhibition of iNOS expression reached with ster- stable asthma can not be objectively assessed by stan- oid treatment. These ¢ndings are similar to preliminary dard parameters of lung function. Similar results, i.e. a data reported by Al-Ali et al. in asthmatic adults (29). reduction of ENO without changes in lung function, have They found that ENO levels are reduced by budesonide been found by other studies (25,35). but not by nedocromil sodium or placebo. Similarly to In conclusion, our study shows that inhaled budeso- ours, this study included only steroid-na|«ve asthmatic nide, but not nedocromil sodium, signi¢cantly reduces patients. ENO levels in stable, steroid-na|«ve asthmatic children Nedocromil sodium is considered a safe alternative to even in absence of changes in lung function. 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