Duration of effect of single-dose inhaled fluticasone propionate on AMP-induced bronchoconstriction

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1 Eur Respir J 2004; 23: DOI: / Prited i UK all rights reserved Copyright #ERS Jourals Ltd 2004 Europea Respiratory Joural ISSN Duratio of effect of sigle-dose ihaled fluticasoe propioate o AMP-iduced brochocostrictio B. Luijk*, R.D. Kempsford #, A.M. Wright #, P. Zae*, J-W.J. Lammers* Duratio of effect of sigle-dose ihaled fluticasoe propioate o AMP-iduced brochocostrictio. B. Luijk, R.D. Kempsford, A.M. Wright, P. Zae, J-W.J. Lammers. #ERS Jourals Ltd ABSTRACT: Airway hyperresposiveess iduced by adeosie-59-moophosphate (AMP) is regarded as a reliable model for allergic asthma ad for the evaluatio of ati-asthmatic drugs. Sigle-dose ihaled corticosteroids (ICS) are kow to be protective i this model, but the duratio of actio of these drugs i this model has ever bee studied. The duratio of ICS protectio was determied by admiistratio of sigle-dose fluticasoe propioate (FP; 1,000 mg) up to 26 h before AMP challege. A radomised, double-blid, placebo-cotrolled, four-way crossover study was performed i 13 mild asthmatics (mea SD predicted forced expiratory volume i oe secod (FEV1) 98 7%). Each subject received placebo ad FP (at 26, 14 or 2 h prior to the AMP challege). Furthermore, the marker exhaled itric oxide (eno) was studied after admiistratio at these time poits to ivestigate whether eno also demostrates the duratio of actio of ICS. The doublig cocetratios differece (DCD) of AMP causig a 20% fall i FEV1, whe FP was admiistered 26, 14 or 2 h prior to challege, was sigificatly icreased as compared with placebo: DCD (95% cofidece iterval) at 26 h, 0.73 ( ), p=0.008; 14 h, 1.50 ( ), pv0.001; ad 2 h, 2.89 ( ), pv However, eno was ot sigificatly affected at these time poits. I coclusio, a sigle dose of 1,000 mg ihaled fluticasoe propioate protects agaist adeosie-59-moophosphate airway hyperresposiveess up to 26 h after dosig. This study suggests that adeosie-59-moophosphate challege ca be used as a sesitive marker to study the duratio of actio of ihaled corticosteroids. Eur Respir J 2004; 23: *Dept of Pulmoary Diseases, Heart Lug Ceter Utrecht, Uiversity Medical Ceter Utrecht, the Netherlads. # GlaxoSmithKlie, Research ad Developmet, Greeford, UK. Correspodece: J-W.J. Lammers Dept of Pulmoary Diseases Heart-Lug Ceter Utrecht Uiversity Medical Ceter Utrecht Heidelberglaa CX Utrecht The Netherlads Fax: j.w.j.lammers@hli.azu.l Keywords: Adeosie-59-moophosphate airway hyperresposiveess asthma exhaled itric oxide fluticasoe propioate Received: April Accepted after revisio: November This study was fuded by GlaxoSmithKlie, UK. Ihaled corticosteroids (ICS) are commoly used as protective ati-iflammatory drugs i the treatmet of asthma [1]. The ati-iflammatory effects of ICS ca be show by demostratig their ifluece o airway hyperresposiveess (AHR), which is oe of the characteristics of chroic airway iflammatio i asthma [2]. AHR ca be measured usig ihalatio of direct brochocostrictors (e.g. histamie or methacholie) or idirect agets such as adeosie-59-moophosphate (AMP) [3]. Ihaled AMP fuctios, at least i part, through activatio of mast cells that release the secodary brochocostrictig mediators leukotriees ad histamie [4, 5]. These idirect airway challeges appear to be a better surrogate marker of the ICS-iduced ati-iflammatory effects tha direct challeges [3, 6, 7]. Recetly, two studies demostrated a rapidly iduced protective effect whe a sigle dose of a ICS was admiistered prior to a idirect challege. A protective effect of fluticasoe propioate (FP) agaist AMP challege was observed withi 2 h of admiistratio ad a protective effect of budesoide agaist hypertoic salie challege was foud withi 6 h of admiistratio [8, 9]. Iterestigly, the study with sigle-dose ihaled FP demostrated protectio towards AMP-AHR but ot after direct challege with histamie. Therefore, AMPiduced brochocostrictio appears to be a rapid model to evaluate the ati-iflammatory effect, the direct potecy ad thus efficacy of sigle-dose ICS i asthmatics. However, curretly o iformatio exists o the duratio of effect of a sigle ihaled dose of FP o AHR. This duratio of effect could the be used i comparative studies with other ICS. Furthermore, the curret authors were iterested i whether the AMP challege is solely affected by a sigle dose of ICS or whether aother surrogate marker of airway iflammatio is as sesitive as AMP-AHR to show this rapid ati-iflammatory respose. This was the ratioale for studyig eno, which has also bee show to be a sesitive marker of ICS-iduced ati-iflammatory effects [10]. Therefore, the aim of this study was to ivestigate the duratio of effect of a sigle dose of ihaled FP by usig the AMP-challege model ad to compare AMP-AHR with eno as surrogate markers of airway iflammatio. A relatively high optimal dose of 1,000 mg FP, as idicated by the results of KETCHELL et al. [8], was studied to determie the duratio of protectio after a sigle dose of FP. Subjects Materials ad methods Thirtee osmokig, atopic subjects with itermittet or mild persistet asthma, accordig to the GINA (Global Iitiative o Asthma) guidelies [11], were icluded i the

2 560 B. LUIJK ET AL. study. Presece of atopy was cofirmed by at least oe positive ski-prick test. At screeig ad durig treatmet periods, all subjects9 asthma was stable ad demostrated a provocatio cocetratio of AMP causig a 20% fall i forced expiratory volume i oe secod (FEV1) (AMP-PC20) ofv50 mg?ml -1 at screeig. After a ru-i period of 7 days, a secod AMP challege was performed to cofirm stability of the AMP-PC20. If the AMP-PC20 was ot withi 1.5 doublig cocetratios from the screeig value the subject was excluded from further participatio i the study. Every patiet had a baselie FEV1 of w70% predicted accordig to Europea Respiratory Society values [12]. ICS ad other atiasthmatic medicatios were ot allowed 4 weeks before the screeig visit ad throughout the study, except for shortactig b 2 -agoists, which had to be withheld 12 h before each visit. Subjects who used oral corticosteroids withi 8 weeks before eterig the study ad throughout the study were also excluded. Caffeie-cotaiig food ad beverages ad alcohol were ot allowed 8 h before ad durig each treatmet period. The study was approved by the medical ethics committee of the Uiversity Medical Ceter Utrecht (Utrecht, the Netherlads) ad all patiets gave their writte iformed coset. Study desig All subjects uderwet four treatmet periods accordig to a radomised, double-blid, crossover, placebo-cotrolled desig. The duratio ad efficacy of a sigle-dose ICS was determied by admiistratio of 1,000 mg FP from a four blister diskhaler device (GlaxoSmithKlie, Steveage, UK) i each treatmet period. There were four, 2-day treatmet periods. Each subject received four differet treatmets: three active (FP) ad oe with placebo oly. For the active treatmets, sigle-dose FP 1,000 mg was admiistered 26, 14 or 2 h prior to AMP challege. For each active treatmet period, matchig placebo (lactose powder) was admiistered at the other time poits to maitai the treatmet blidig. The placebo-aloe treatmet was lactose powder at 26, 14 ad 2 h pre-amp challege. There was a washout period of at least 1 week betwee each treatmet. Durig the study, oly salbutamol was permitted to ihale as rescue medicatio if eeded. Seve to 14 days after the fial treatmet period, subjects retured to the hospital for a post-study fial visit for safety blood tests ad to establish stable asthmatic symptoms. AMP challege AMP was ihaled i doublig cocetratios at 5-mi itervals i the rage mg?ml -1, accordig to a stadardised challege protocol to determie AMP-PC20 as previously described [13, 14]. FEV1 was measured i duplicate at 30 ad 90 s after 2-mi tidal breathig from a calibrated (0.13 ml?mi -1 ) ebuliser (model 646; Devilbiss Ic., Somerset, PA, USA) while the ose was clipped. Durig the doubleblid period, ihaled AMP was give util the FEV1 fell by o20% from baselie or whe the maximum cocetratio of AMP was ihaled. AMP (Sigma-Aldrich, St. Louis, MO, USA) was dissolved i salie (0.9%) solutio to produce the doublig cocetratios. Forced expiratory volume i oe secod measuremets FEV1 measuremets were recorded with the same mobile bi-directioal digital spirometer (Sesorloop; SesorMedics Corp, Yorba Lida, CA, USA) durig treatmet periods at pre-dose at day 1 (i.e. before the first of the three doses) ad pre-dose at day 2 (i.e. before the last of the three doses, FEV1 oly), pre-challege (i.e. 2 h after the last admiistered dose), ad regular time poits durig AMP-challege protocol. Exhaled itric oxide Exhaled itric oxide (eno) was measured i parts per billio, accordig to the America Thoracic Society stadards, with a calibrated chemilumiescece aalyser (type CLD 77 AM; Eco Physics, Duerte, Switzerlad) with a flow rate of 250 ml?s -1 [15]. Exhalatios ito a NO reader were repeated three times ad the the overall mea was calculated. eno was measured at similar time poits to FEV1, except the pre-dose value at day 2. The eno recordigs were always obtaied before the FEV1 values. Measuremet of plasma fluticasoe propioate levels Blood samples were take for determiatio of plasma FP cocetratios to demostrate the absece of circulatig FP prior to dosig ad to determie the plasma FP cocetratios at AMP challege. Blood samples (5 ml) were collected before the first dose ad immediately pre-amp challege (2 h after the last dose was ihaled), ad placed immediately o ice ad cetrifuged at 1,0006g for 10 mi at 4uC. The plasma superatat was stored i tubes at -70uC util further processig. FP was the quatified i plasma by automated solid-phase extractio followed by liquid chromatography tadem mass spectrometry (Applied Biosystems API3000; Applied Biosystems, Warrigto, UK). The limit of quatificatio was 10 pg?ml -1. Data aalysis The plaed sample size of 12 subjects was predicted to have 80% power to detect a ratio for AMP-PC20 of 2.4 betwee ay two of the treatmet groups, assumig a withisubject SD o the log e scale of 0.77 ad a sigificace level of 5%. This ratio is equivalet to a differece of 1.26 doublig cocetratios differece (DCD) betwee treatmet groups. The AMP-PC20 ad eno measuremets were aalysed usig aalysis of variace. The followig factors were icluded i the models: subject, period ad treatmet group. The data were log trasformed prior to aalyses. For the PC20 data, differeces ad 95% cofidece itervals (CI) betwee treatmet groups were preseted i terms of DCDs. For the NO data, the comparisos were calculated i terms of a ratio (eno ratio) ad 95% CI (FP/placebo). A ratio of 1 would idicate o effect of FP compared with placebo. All other data were expressed as (geometric) meas (95% CI) uless otherwise stated. Differeces were cosidered to be statistically sigificat if the p-level was v0.05. Subjects Results Subject characteristics are listed i table 1. The AMP-AHR of the icluded subjects was stable before startig the four treatmet periods (idividual AMP-PC20 values were withi the above-metioed 1.5 DCD). The mea AMP-PC20 differece betwee screeig ad ru-i period expressed i DCD SD was

3 ICS AND AMP-INDUCED BRONCHOCONSTRICTION 561 Table 1. Subject characteristics of icluded mild asthmatic patiets Subject Sex Age yrs Atopy # FEV1 % pred eno ppb AMP-PC20 mg?ml -1 Screeig Ru-i 1 M 22 H M 19 H, C M 24 H,G,C F 21 H,G F 20 H,G,C F 22 H, C M 22 H M 24 H,G M 20 H,G,C F 23 H,G F 21 H,C F 24 H,G,C M 22 H,C Mea SD } (50) 6.72(94) 5.56(129) M: male; F: female; FEV1: forced expiratory volume i oe secod; eno: exhaled itric oxide; AMP-PC20: provocatio cocetratio of AMP causig a 20% fall i FEV1. # : atopy defied as positive ski-prick tests for house dust mite (H), grass (G) ad cat (C); } : geometric mea (coefficiet of variatio %). Due to icorrect diskhaler use, oe subject received the same treatmet twice (FP 14 h before the challege). Although this subject completed the trial, a additioal subject was recruited. Data from all 13 subjects were used i the aalyses. Effect of sigle-dose ihaled fluticasoe propioate o forced expiratory volume i oe secod Durig the four treatmet periods with either active FP at the three differet FP admiistratio time poits or the placebo treatmet, o differeces were foud i pre-dose ad post-dose FEV1 values (FP at 26 h: 3.88 L ( ); FP at 14 h: 3.93 L ( ); FP at 2 h: 3.83 L ( ); ad durig placebo: 3.76 L ( )). Furthermore, o differeces i baselie FEV1 values were observed betwee the four treatmet periods durig the study. differet from placebo treatmet at all three time poits. The DCD (95% CI; FP to placebo) were as follows: 0.73 ( ), p=0.008; 1.50 ( ), pv0.001; ad 2.89 ( ), pv0.001, for the 26 h, 14 h ad 2 h groups, respectively, as show i figure 1a ad table 3. The AMP- PC20 measured at 2 h after FP ihalatio was also sigificatly higher tha the AMP-PC20 values at 26 ad 14 h, respectively (DCD 2 26 h: 2.16 ( ), pv0.001; 2 14 h: 1.39 ( ), pv0.001), ad a sigificat differece betwee 26 ad 14 h time poits of FP admiistratio (DCD: 0.77 ( ), p=0.005) was observed. Although there is oe clear upper outlier i the AMP-PC20 dataset at 26 h FP admiistratio, this outlier did ot have a large ifluece o the results. A aalysis was performed excludig this subject ad the differece at 26 h was still statistically sigificat. Effect of sigle-dose ihaled fluticasoe propioate o AMP-PC20 The idividual AMP-PC20 data are give i table 2. The AMP-PC20 after FP admiistratio was statistically sigificatly The effect of sigle-dose fluticasoe propioate o exhaled itric oxide I table 4, the geometric meas ad ratios for the eno values for the four treatmet groups are preseted. Table 2. The idividual provocatio cocetratio of AMP causig a 20% fall i forced expiratory volume i oe secod values durig the four treatmet periods (placebo, 26, 14 ad 2 h) i 13 asthmatics Subject Placebo 26 h 14 h 2 h Geomea (95% CI) 7.4 ( ) 12.3 ( ) 21.0 ( ) 54.9 ( ) CI: cofidece iterval.

4 562 B. LUIJK ET AL. a) DCD active FP placebo b) h 14 h 2 h Table 4. Geometric meas ad ratio of the pre-challege exhaled itric oxide (eno) cocetratios durig placebo ad fluticasoe propioate (FP) admiistered 26, 14 ad 2 h before AMP challege Treatmet group # eno ppb } Ratio z (CI) p-value Placebo 18.7 FP 1,000 mg 26 h ( ) h ( ) h ( ) 0.75 CI: cofidece iterval. # : for the active treatmet groups, the legth of time betwee the dose ad AMP challege is idicated; } : geometric meas i parts per billio (ppb) adjusted for period effects; z : betwee active treatmet group ad placebo; : for pairwise comparisos with placebo. at 26 h FP admiistratio compared to placebo (table 4 ad fig. 1b). eno ratio active FP placebo h 14 h 2 h Time of FP admiistratio Fig. 1. a) Doublig cocetratios differece (DCD) ad 95% cofidece itervals (CI) of the provocative cocetratio of AMP causig a 20% fall i the forced expiratory volume i oe secod (AMP-PC20) with the admiistered active fluticasoe propioate (FP) treatmet compared with placebo at the idicated time poits. b) Ratio ad 95% CI of exhaled itric oxide (eno) with the admiistered active FP treatmet compared with placebo at the idicated time poits. There was a osigificat decrease i pre-challege eno values whe FP was admiistered 26 ad 14 h prior to the AMP challege, with a lowest ratio of 0.83 ( , p=0.06) Table 3. Geometric meas ad doublig cocetratio differeces (DCD) of the provocatio cocetratio of AMP causig a 20% fall i forced expiratory volume i oe secod (AMP-PC20) values durig placebo ad fluticasoe propioate (FP) admiistered 26, 14 ad 2 h before AMP challege Treatmet group # AMP-PC20 DCD (CI) z p-value mg?ml -1} Placebo 7.4 FP 1,000 mg 26 h ( ) h ( ) v h ( ) v0.001 CI: cofidece iterval; # : for the active treatmet groups, the legth of time betwee the dose ad AMP challege is idicated; } : adjusted for period effects; z : betwee active treatmet group ad placebo expressed i terms of doublig cocetratios of AMP (see fig. 1a); : for pairwise comparisos with placebo. Plasma fluticasoe propioate levels Pre-challege plasma FP cocetratios were below the limit of quatificatio i the placebo treatmet period ad i all the pre-treatmet samples. Plasma FP levels immediately prior to AMP challege (i.e. 2 h post-dose) were detectable i all subjects whe FP was give 14 h (media 32.8 pg?ml -1 (rage )) ad 2 h (147.1 pg?ml -1 ( )) before the AMP challege. Whe FP was admiistered 26 h prior to the AMP challege, plasma FP levels were below the limit of quatificatio i half the subjects, whereas the other subjects demostrated a media of 15.3 pg?ml -1 ( ). There was o correlatio betwee DCD AMP-PC20 improvemet ad the 2-h post-dose plasma FP levels at the time poits studied. Discussio I this study, the authors foud that a sigle dose of 1,000 mg ihaled FP, admiistered 26, 14 or 2 h prior to challege, protected agaist AMP-iduced brochocostrictio. A sigle dose of 1,000 mg FP give at these time poits did ot appear to sigificatly chage pre-amp challege FEV1 or eno levels. The fidigs of AMP-PC20 improvemet are i agreemet with previous studies demostratig similar improvemet after repeat-dose or sigle-dose ihalatio of ICS whe the last dose was admiistered shortly before challege [8, 9, 16, 17]. This early protective effect of FP agaist AMP challege is also cosistet with the recet fidigs of PROSPERINI et al. [7], who showed that AHR to ihaled AMP promptly detected iflammatory chages of the asthmatic airways as early as the first week of treatmet with budesoide ad that AMP-PC20 retured to ear baselie levels as early as the first week of treatmet [7]. I additio, a sigle dose of ICS admiistered just before a allerge challege ihibited the late asthmatic respose [18]. I the preset study, the duratio of ati-iflammatory effects of sigle-dose FP was directly assessed by alterig the FP-AMP challege iterval. ICS are kow for their topical ati-iflammatory effects ad ihaled FP i particular has a highly favourable topical efficacy compared with systemic safety [19]. This direct topical effect of ICS is supposed to be resposible for the acute cliical effect o FEV1 as soo as 2 h, as demostrated i a previous study i acute severe asthma [20]. This FEV1 improvemet was also show 6 h after sigle high-dose ICS

5 ICS AND AMP-INDUCED BRONCHOCONSTRICTION 563 treatmet with budesoide (2,400 ad 1,600 mg, respectively) [9, 21]. However, o effect was foud o the FEV1 2 h after admiistratio of a sigle dose of 1,000 mg FP i the study by KETCHELL et al. [8], which is comparable to the preset results at 2 h, or at the 14 ad 26 h time poits. This may be due to the curret mild stable disease of the asthmatics that were studied. Moreover, ICS iduce a acute ati-iflammatory effect o AHR measured by idirect challeges [8, 9]. The improvemet o AMP-AHR after FP ihalatio up to 26 h foud i this study may be due to local high affiity towards the glucocorticoid receptor of FP i the airways after ihalatio ad by the log half-life of the FP active steroid receptor complex of w10 h [22, 23]. The optimal FP protectio o AMP-iduced brochocostrictio was demostrated i a recet abstract, with the highest effect whe FP was admiistered 4 h prior to a AMP challege ad ot at the previously metioed 2 h [24]. I additio, the decreasig ati-iflammatory respose measured by AMP-AHR up to 26 h was associated with odetectable FP plasma levels i half of the subjects ad, without correlatio, with the doublig cocetratio of the AMP-PC20 differece at the three time poits, which further supports the otio that this duratio of actio is a sustaied topical FP effect rather tha a systemic ati-iflammatory effect [19]. Recetly, several mechaisms have bee proposed regardig the acute protective effect of sigle-dose ICS o AMPiduced brochocostrictio by ifluecig the adeosie receptors that may play a role i airway iflammatio [8]. The ihibitio of adeosie receptor activity by ICS after bidig to the glucocorticoid receptor o the membrae is suggested to be rapidly iduced by ogeomic atiiflammatory effects withi miutes ad by geomic atiiflammatory effects withi hours [8, 25]. It is likely that both the ogeomic ad geomic effects iteract with the adeosie receptor activity itracellularly or via a direct iteractio at the adeosie receptor site. This may cause the improvemet o AMP-AHR up to 26 h, with a effect that decreases i time, as foud for the decrease of protectio from 2 to 14 h, ad up to 26 h i this study. Furthermore, adeosie iduces plasma exudatio ad brochial blood flow icrease that are associated with the local vascular effects of microvascular leakage ad oedema of the airway wall that have bee described as factors of AHR [2, 26]. These vascular evets may be reduced by ICS, with FP, i particular, beig a potet aget to iduce a vasocostrictive effect [22, 25]. I additio, it has previously bee stated that FP decreases microvascular leakage ad airway mucosal blood flow [2, 27]. These effects together may cotribute to the prologed protectio by FP up to 26 h o AMP-iduced brochocostrictio i the preset study. However, the exact mechaisms behid this duratio of effect of acute ICS-iduced ihibitio o AMP-AHR eeds further study. This study may have some limitatios. First, the doserespose for the activity of FP admiistered up to 26 h prior to AMP challege was ot determied i this study ad therefore it is still uclear whether lower FP doses may cause similar improvemet i AMP-PC20. However, a recetly published study demostrated a dose-depedet effect after 2 h, usig FP at either 100, 250 or 1,000 mg [8]. Thus, the protectio by ihaled FP appears to be a topical effect rather tha a effect due to systemic exposure ad hece lower doses might also be effective 14 ad 26 h after ihalatio. Secodly, oly FP was studied. Other types of ICS could have bee used to demostrate the same duratio of effect. Although protectio o AMP-iduced brochocostrictio has bee show whe sigle doses of beclomethasoe 2,000 mg or budesoide 1,600 mg were admiistered 2 h prior to AMP challege, this may ot have similar results whe studyig duratio of actio i this model [28]. Thirdly, these sigle-dose effects of ICS towards AMP challege may be iflueced by the icreasig osmolarity of the ihaled doublig cocetratios of solutios, which has bee show to have a affect o the AHR i asthmatics [29]. Fially, the preset study shows a model to assess the duratio of effect of ICS for comparative studies o ewly developed types of ICS, ot for the purpose of dose regime for cliical beeficial effects of a prologed protectio by a sigle dose of 1,000 mg of FP. Despite the sigificat ati-iflammatory effect up to 26 h after FP admiistratio, this model caot be directly extrapolated to the cliical situatio where ICS are give o a log-term basis. I cotrast to AMP challege, the absece of a protective effect of FP o eno up to 26 h after FP admiistratio may idicate that these tests reflect differet aspects of airway iflammatio. Whether a ICS-iduced ati-iflammatory effect o eno release is dose-depedet, type of ICS depedet, time depedet or due to the mild disease of the icluded asthmatics has to be studied further. I additio, there is some evidece that it is dose depedet, as after 6 h, sigle high-dose ICS treatmet with ebulised budesoide (8 mg), the eno levels were reduced [10]. Furthermore, the percetage of sputum eosiophils may also be used to show a ICS-iduced ati-iflammatory respose, which was decreased 6 h after a sigle dose of budesoide [9]. These markers are all sesitive after hours of ICS treatmet, whereas direct challeges are ot [8]. These iflammatory markers ad FEV1 appear to be less sesitive to reflect the direct activities of ICS-iduced ati-iflammatory effects, as has bee show for AMP-AHR, which showed a directly proportioal effect to the dose or timig of ihaled ICS used. Thus, take together, these fidigs stregthe the view that AMP challege is exceptioally sesitive to the topical effects of ICS. I coclusio, a sigle dose of 1,000 mg ihaled fluticasoe propioate demostrated a duratio of actio of up to 26 h by protectio agaist adeosie-59-moophosphateiduced brochocostrictio, without a sigificat effect o exhaled itric oxide. This study further suggests that the ati-iflammatory respose measured by a adeosie-59- moophosphate challege is a sesitive tool for future comparative studies o duratio of actio of ewly developed ihaled corticosteroids. Refereces 1. Lipworth BJ. Fortightly review: moder drug treatmet of chroic asthma. BMJ 1999; 318: Bares PJ. Effect of corticosteroids o airway hyperresposiveess. Am Rev Resp Dis 1990; 141: S70 S Joos GF, O9Coor B, Aderso SD, et al. Idirect airway challeges. Eur Respir J 2003; 21: Polosa R. Adeosie-receptor subtypes: their relevace to adeosie-mediated resposes i asthma ad chroic obstructive pulmoary disease. Eur Respir J 2002; 20: Va Schoor J, Joos GF, Pauwels RA. Idirect brochial hyperresposiveess i asthma: mechaisms, pharmacology ad implicatios for cliical research. Eur Respir J 2000; 16: Va de Berge M, Meijer RJ, Kerstjes HA, et al. PC(20) adeosie 59-moophosphate is more closely associated with airway iflammatio i asthma tha PC(20) methacholie. Am J Respir Crit Care Med 2001; 163: Prosperii G, Rajakulasigam K, Cacciola RR, et al. Chages i sputum couts ad airway hyperresposiveess after budesoide: moitorig ati-iflammatory respose o the basis of surrogate markers of airway iflammatio. J Allergy Cli Immuol 2002; 110:

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