Dahlén et al. Page 1
File: ERJ-00955-2008 Online Depository
Revised 081104
Online Depository for:
EFFECT OF FORMOTEROL WITH OR WITHOUT
BUDESONIDE IN REPEATED LOW-DOSE ALLERGEN
CHALLENGE
Barbro Dahlén MD PhD1,5, Ann-Sofie Lantz RN1,5, Elisabeth Ihre MD PhD2, Maria
Skedinger MD PhD1,5, Elisabeth Henriksson BMA1,5, Leif Jörgensen MSci3, Tommy
Ekström MD PhD3, Sven-Erik Dahlén MD PhD4,5, Kjell Larsson MD PhD4,5
1
Division of Respiratory Medicine and Allergy, Department of Medicine at Karolinska
University Hospital Huddinge, 2Cityakuten, Stockholm, 3AstraZeneca Sweden, 4Division
of Physiology, The National Institute of Environmental Medicine and 5Centre for Allergy
Research at Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Corresponding author:
Dr Barbro Dahlén MD, PhD
Lung and allergy clinic, M53
Karolinska University Hospital Huddinge
SE-141 86 Stockholm, Sweden
Tel: +468 5858 1833
Fax: +468 711 7306
E-mail: barbro.dahlen@ki.se
Dahlén et al. Page 2
METHODS
Subjects
Fifteen nonsmoking subjects with stable intermittent [1] allergic asthma treated only with
a short-acting beta2-agonist as needed participated (Table 1 in the main text). All had a
post-bronchodilator FEV1 greater than 80 per cent of predicted normal value and airway
hyperresponsiveness to methacholine with a PD20 FEV1 being less than 7256 µg
(provocative dose causing a 20% fall in FEV1). Exclusion criteria were significant
allergen exposure, COPD or any significant respiratory disease other than asthma, a
respiratory tract infection within four weeks and use of glucocorticosteroids within eight
weeks prior to the study.
The Ethics Committee at The Karolinska University Hospital approved the study
(Dnr 04-470/1-4) and the subjects gave written informed consent.
Study design
The study (NCT00288379) was a three-period, cross-over, double-blind, double dummy
comparison between formoterol Turbuhaler™ 4.5µg, budesonide 160µg /formoterol
4.5µg Turbuhaler™, and placebo (AstraZeneca, Lund, Sweden) on airway functional and
inflammatory changes and symptoms, induced by repeated low-dose allergen exposure
(Figure 1 in the main text). The study medication was taken under observation in the
clinic as two puffs 30 minutes after allergen inhalation on every low-dose challenge day
(i.e. visit day day 2 to 8 in each period).
The subjects participated in two screening visits prior to randomization. At the first
visit, inclusion and exclusion criteria were examined, a skin prick test was performed (cat
dander, dog dander, timothy, birch, Dermatophagoides Pteronyssinus) as well as pre-
Dahlén et al. Page 3
study measurements of lung function, exhaled nitric oxide concentrations and a
methacholine challenge. At a second screening visit a cumulative, high-dose allergen
inhalation challenge (vide infra) was undertaken to establish current sensitivity, expressed
as allergen PD20FEV1 [2]. At this visit a short-acting beta2-agonist (terbutalin, Bricanyl
Turbuhaler™ 0.25 mg) was provided for use as as-needed medication. The patients were
instructed to refrain from its use 8 hours before the next visit as well as before every
clinic visit in the study.
The study proper started after a two-week wash-out, with each period consisting of
nine clinic visits, always in the morning, and with methacholine challenges and induced
sputum collection (vide infra) on visit days 1 and 9, i.e. pre and post the repeated allergen
exposure period. Exhaled NO (NIOX™, Aerocrine AB, Stockholm, Sweden) and FEV1
(Jaeger MasterScope, IntraMedic Inc, Sweden) were measured daily according to current
recommendations [3,4] and the values obtained before methacholine challenge on visit
day 1 were taken as pre-allergen exposure, pre-treatment baseline in the respective
period.
Allergen (Aquagen™, ALK Laboratories, Copenhagen, Denmark) was inhaled as a
single dose on seven consecutive week days, i.e Monday-Friday one week and Mon/Tues
next week (visit days 2-8) (Figure 1 in the main text). The allergen dose selected as the
low dose, was calculated from the screening allergen challenge (vide infra) as the
cumulative dose causing a fall in FEV1 of approximately 5% (PD5FEV1) from postdiluent value (Table 1 in the main text). Spirometry was obtained before and 10, 20 and
30 minutes after allergen inhalation on each occasion. The randomized study treatment
was then inhaled under observation before the subject was allowed to leave the clinic.
Dahlén et al. Page 4
Diary cards were administered on day 1 in each period and the subjects were asked
to record their symptom score (0-10) and their use of short-acting beta2-agonist every
evening covering the previous 24 hours. In addition, evening measurements of FEV1
were recorded at home using a pocket spirometer (Spirobank™, IntraMedic Inc,
Sweden).
The three periods were separated by a 15 day wash-out, which was extended to a
maximum eight weeks in the case of remaining asthma deterioration after the previous
exposure period, such as a difference in baseline FEV1 by >15% from pre study baseline
or if methacholine PD20 had not returned to within 2.5 doubling doses. Likewise an
interfering respiratory tract infection or a short, maximum four-day, course of inhaled or
oral corticosteroids given to reverse excessive deterioration after an allergen exposure
period, would necessitate a minimum four week extension of the wash-out.
Challenge procedures
Cumulative allergen inhalation challenge
Allergen was administered for inhalation after nebulisation by a dosimeter-controlled jetnebulizer (Spira Elektro 2™, Respiratory Care Center, Hameenlinna, Finland) set to
produce an output of 7.1 µL per breath (Table 1). As indicated in Table 2, by the use of
three different concentrations and by variations in the number of tidal breaths (1 to 7),
step-wise half-log increments of the cumulative dose of allergen were created.
Allergen solution:
Three concentrations of allergen extract (1000, 10.000 and 100.000 SQ/mL) were
prepared from powder dissolved in diluent (Aquagen™, ALK Laboratories, Copenhagen,
Denmark). A minimum of 1 mL of solution is required in the holding vessel.
Dahlén et al. Page 5
Determination of baseline FEV1:
Baseline FEV1 and PEFR were measured as the best of three reproducible efforts [4].
Provided baseline FEV1 was >70% of predicted, the test was performed.
Determination of diluent response and post-diluent baseline:
The diluent (7 breaths) was inhaled and FEV1 recorded after 15 min. If stable (i.e. not
deviating by more than 10% from the pre-diluent value), this value was used as postdiluent baseline to calculate the percent drop in FEV1 during the challenge. If a
significant response to the diluent occurred, the diluent inhalation was repeated. If a
significant response remained, the subject’s asthma was considered not stable enough to
pursue the methacholine challenge on that day.
Inhalation of allergen:
The challenge always started by inhalation of the lowest dose of allergen, and
incremental doses were administered every 20 minutes according to Table 2. Spirometry
was obtained as a single technically acceptable measurement at 18 minutes after each
dose increment. The provocation was terminated when FEV1 had fallen at least 20%
from the post-diluent baseline, or the maximum cumulative dose of allergen had been
reached (7100 SQ). If FEV1 was reduced between 15 and 20%, a further assessment was
made after 10 minutes and if still a less than 20% fall, a decision was made by the
investigator whether to administer a repeat or a higher dose of allergen. After a
significant 20% fall was obtained, FEV1 and PEFR were followed every 15 minutes
during the first hour, thereafter at hourly intervals until bedtime in order to detect a
potential late asthmatic reaction (LAR).
Dahlén et al. Page 6
The patient was always observed in the clinic until FEV1 was back within 90% of
baseline. Before leaving the clinic, it was written on the PEFR-chart at which predefined
level of drop in PEFR or in FEV1 the subject should use rescue medication and/or contact
the hospital. Contact details to the responsible investigator were provided on the chart.
The patients were told not to undertake physical exercise and not to expose themselves to
allergen in the same day (and night) as the challenge test. In addition to their short-acting
β-agonist, the patients were provided with oral corticosteroids for use in the case of a
severe LAR.
The allergen PD20FEV1 was derived by linear interpolation from the log-cumulated
dose response curves.
Methacholine inhalation challenge
Methacholine was administered for inhalation after nebulisation by a dosimetercontrolled jet-nebulizer (Spira Elektro 2™, Respiratory Care Center Ltd, Hameenlinna,
Finland). The protocol is modified after Nieminen MM et al. [5]. With the nebulizer set
to give an output of 7.1 µL per breath (Table 1), the use of increasing methacholine
concentrations and by increasing the number of breaths, doubling incremental doses were
administered (Table 3).
Methacholine solutions:
Three concentrations of methacholine chloride (1, 8 and 64 mg/mL) prepared at
Norrlands University Hospital Pharmacy and 2, 4 and 8 breaths were used according to
the protocol in Table 3.
Dahlén et al. Page 7
Determination of baseline FEV1 and diluent response:
Baseline pulmonary function and assessment of diluent response were performed as
described for the allergen challenge protocol above.
Inhalation of methacholine:
The methacholine solution was inhaled every three minutes according to Table 3. FEV1
was obtained as a single technically acceptable measurement at 2.5 minutes after each
dose increment. The provocation was terminated when FEV1 had fallen at least 20%
from the post-diluent baseline, or the maximum cumulative dose of methacholine had
been reached (7256 g). After the challenge the patient was observed until FEV1 had
returned to within 90% of baseline. The methacholine PD20FEV1 values were calculated
from the log-cumulated dose response curves by linear interpolation.
Sputum induction
Sputum induction was performed according to ERS guidelines [6]. The subjects were
pre-treated with 250µg inhaled terbutaline to inhibit excessive airway constriction. The
subjects were instructed to expectorate sputum into a sterile tube after inhaling an aerosol
of hypertonic saline administered from an ultrasonic nebuliser (Ultra Neb 2000;
DeVilbiss Health Care, Somerset, PA) with an output of 1 mL·min-1. A fixed
concentration of 4.5% sterile saline solution was used and induction was performed for 1,
4 and 5 minutes followed by three further 5-minute periods. Spirometry was obtained at
the end of each induction interval and the induction was stopped if there was a fall in
FEV1 of 20% or more compared with the postbronchodilator value, or if symptoms
occurred. The subjects were instructed to cough deeply and spit after 5, 10, 15 and 20
Dahlén et al. Page 8
min of induction, or whenever they got the urge to do so, and to blow their nose and to
rinse their mouth with water prior to delivering the sputum sample.
Sputum processing
Whole sputum was processed according to the ERS guidelines [7]. Dithiothreitol (DTT;
Sputolysin® Reagent, EMD Biosciences, Inc., San Diego, CA) 0.1% was added to the
sample equivalent to 4x sputum weight and rocked for 15 minutes. Phosphate-buffered
saline was added to an equal volume of DTT and the sample was rocked for an additional
5 minutes. The sample was filtered through a 70 µm cell strainer (BD Falcon™, BD
Biosciences, San Jose, CA) and centrifuged at 400 x g, 10 minutes. The supernatant was
stored at -70ºC until assayed for PGD2. Cytospins were prepared from the cell pellet and
stained with May-Grunwald Giemsa (Sigma-Aldrich Co.).
Sputum cell counting
Cell differential was performed by counting 500 cells in random fields. Each cytospin
preparation was counted twice by each of two independent observers. The results of the
cell differentials are expressed as a percentage of the total number of non-squamous cells,
except for the squamous cells that are expressed as the percentage of the total cell
number.
Measurement of PGD2
Analyses of PGD2 in sputum supernatant was performed in serially diluted aliquots of by
enzyme immunoassay using the PGD2-MOX assay (Cayman Chemical Co., Ann Arbor,
MI) with detection limit 7.8 pg/mL. The rabbit polyclonal antiserum (anti-PGD2-MOX
rabbit IgG) cross reacts with un-methoxylated PGD2 by 0.2%, for all other primary
prostanoids the cross-reactivity is less than 0.01%.
Dahlén et al. Page 9
Statistical analysis
Within period changes and treatment differences in log-transformed PD20, in FEV1,
FENO values and diary card data using period mean values, were analyzed using a
repeated measures analysis of covariance (ANCOVA) model, with subject, period and
treatment as factors, and with baseline pre-allergen, pre-treatment values in each period
as covariate (Table 4). The mean asthma symptom score was calculated for each
treatment period and compared between treatments by an analysis of co variance
(ANCOVA).
Data are presented as adjusted least square means (LS)means (or geometric means for
PD20) and 95% CI. Period and carry-over effects of the drug treatments were analyzed by
substituting treatment with period and calculating trends throughout the study. The
sample size was calculated to be 12 completed patients assuming a standard deviation of
log10-transformed PD20 to be 0.23, a significance level of 5%, a 80% power, a two-sided
alternative hypothesis, and a between-treatment difference (increase) of 83% in PD20.
nQuery from Statistical Solutions, Stonehill Corporate Center, Suite 104, 999 Broadway,
Saugus, MA 01906, USA and SAS Version 8.02 from SAS Institute Inc., Cary, NC 27513
were used for statistical calculations.
Differences were considered significant if p<0.05.
Doubling doses were calculated according to the formula: (Log10 PD20 post - Log10 PD20
pre )/Log10(2) for within period analyses, and the corresponding difference for Log10
shifts in PD20 used for the comparison between treatments.
Dahlén et al. Page 10
RESULTS
Power calculations for PD20, FEV1 and FENO
Power calculations for methacholine PD20, FEV1 and FENO have been performed based
on the results in the present study. Thus, a sample size of 8 in each parallel group will
have 80% power to detect a difference in geometric means of 359 in PD20 comparing
budesonide/formoterol with placebo assuming that the common standard deviation is 233
using a two group t-test with a 0.050 two-sided significance level.
A sample size of 11 in each parallel group will have 80% power to detect a difference in
means of 0.225 in FEV1 in % of predicted, assuming that the common standard deviation
is 0.175 using a two group t-test with a 0.050 two-sided significance level. For FENO a
sample size of 32 in each parallel group will have 80% power to detect a difference in
means of 18.0 ppb, assuming that the common standard deviation is 25.0 using a two
group t-test with a 0.050 two-sided significance level. The Standard Error of the Mean
(SEM) for FENO was approximately 10 ppb (Table 5).
Analyses of carry over effects
Tables 6, 7 and 8 show small changes between the study periods for PD20, FENO and
FEV1. The change from the start of one treatment period to the start of the next period
was calculated, and tested for significance using a t-test. Analyses showed no carry over
effects for the primary variable PD20 (Table 9), nor for FENO being the most sensitive
measure in the study (Table 10).
Dahlén et al. Page 11
REFERENCES
1.
Global Initiative for Asthma. Global Strategy for asthma management and
prevention: NHLBI/WHO workshop report. Bethesda, MD:National Heart Lung
and Blood Institute, 2006. (www.ginasthma.org).
2.
Roquet A, Dahlén B, Kumlin M, Ihre E, Anstrén G, Binks G et al. Combined
antagonism of leukotrienes and histamine produces predominant inhibition of
allergen-induced early and late phase airway obstruction in asthmatics. Am J
Respir Crit Care Med 1997; 155:1856-1863.
3.
Recommendations for standardized procedures for the on-line and off-line
measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in
adults and children. Am J Respir Crit Care Med 1999;160:2104– 2117.
4.
American Thoracic Society. Standardization of Spirometry, 1994 update. Am J
Respir Crit Care Med 1995;152:1107-1136.
5.
Nieminen MM, Lahdensuo A, Kellomaeki L, Karvonen J, Muittari A.
Methacholine bronchial challenge using a dosimeter with controlled tidal breathing.
Thorax 1988;43:896-900.
6.
Paggiaro PL, Chanez P, Holz O, Ind PW, Djukanovic R, Maestrelli P, Sterk PJ.
Sputum induction. Eur Respir J Suppl 2002;37:3s-8s.
7.
Efthimiadis A, Spanevello A, Hamid Q, Kelly MM, Linden M, Louis R, Pizzichini
MM, Pizzichini E, Ronchi C, Van Overvel F, Djukanovic R. Methods of sputum
processing for cell counts, immunocytochemistry and in situ hybridisation. Eur
Respir J Suppl 2002;37:19s-23s.
Dahlén et al. Page 12
Table 1. Nebulizer settings
Inspiratory flow rate:
0.5 L/s
Starting volume:
0.1 L
Tidal volume:
0.5 - 0.6 L
Duration of nebulization:
0.5 seconds
Table 2. Protocol for dosing of allergen
Allergen
concentration
SQ/mL
No of breaths
1000
1000
1000
10000
10000
100000
100000
Dose
SQ units
1
2
7
2
7
2
7
Cumulated dose
SQ units
7
14
50
142
497
1420
4970
7
21
71
213
710
2130
7100
Table 3. Protocol for dosing of methacholine
Methacholine
concentration
mg/mL
No of breaths
Dose
g
1
1
1
8
8
8
64
64
64
2
4
8
2
4
8
2
4
8
14.2
28.4
56.7
114
227
454
909
1818
3635
Cumulated dose
g
14.2
42.6
99.3
213
440
894
1803
3621
7256
Dahlén et al. Page 13
Table 4. Assessments during each study period
Assessment
Fr
FENO
FEV1
Sa
Su
Mo
Tu
We
Th
Fr
X
(x)
X
X
X
X
(x)
X
X
X
X
X
FEV1 10, 20, 30 min
post allergen
Sa
Su
Mo
Tu
We
X
X
X
X
X
X
X
X
X
X
X
X
X
MethacholinePD20
X
X
Induced sputum
X
X
Diary; FEV1
X
X
X
X
X
X
X
X
X
X
X
X
X
Diary; beta2-agonist
doses
X
X
X
(x)
X
X
X
X
X
X
X
X
X
Diary; symptoms
X
X
X
(x)
X
X
X
X
X
X
X
X
X
X=values used as baseline
X=values used in calculations of changes
(x)=values not used
Dahlén et al. Page 14
Table 5. Group mean (SEM) for FENO for each treatment and each timepoint.
Treatment
Budesonide 160µg /formoterol
4.5µg 2 puffs
Formoterol 4.5 µg 2 puffs
Placebo
Day in treatment
period
Mean
(ppb)
SEM
(ppb)
-3
47.4
7.4
0
1
2
3
4
7
8
9
-3
0
1
2
3
4
7
8
9
-3
0
1
2
3
4
7
8
9
54.1
53.8
54.5
53.5
51.9
51.7
54.9
53.0
47.3
53.4
62.0
68.1
74.1
76.2
61.2
71.7
74.6
45.6
50.0
61.9
52.5
80.6
79.9
64.2
76.2
73.3
8.7
8.6
9.6
10.3
9.5
9.0
10.8
10.1
7.0
8.9
9.9
11.0
12.5
13.0
8.4
12.4
13.1
8.0
8.0
9.9
12.4
14.8
14.0
10.4
12.6
11.7
Dahlén et al. Page 15
Tables 6-8 illustrate the absence of carry over effect.
Table 6. Descriptive statistics of PD20, study period 1, 2 and 3
All treatment groups
(N=15)
Study Period
Mean (SD)
Study Period 1
808.3 (1349)
Study Period 2
Study Period 3
Geometric mean (CV)
Median
(Min,Max)
407 (167)
448.5
(25 , 7379)
1078 (2109)
383 (196)
387.5
(7 , 11300)
736.4 (1214)
368 (165)
326.5
(49 , 6173)
Table 7. Descriptive statistics of FENO, study period 1, 2 and 3.
Study Period
All treatment groups
(N=15)
Mean (SD)
Median
(Min,Max)
Before randomisation
42.59 (30)
28.8
(10.1 , 98.2)
Study Period 1
61.57 (40)
52.0
(6.3 , 162)
Study Period 2
63.12 (44)
51.6
(6.3 , 190)
Study Period 3
60.63 (40)
55.9
(8 , 190)
Table 8. Descriptive statistics of FEV1 for study period 1, 2 and 3.
All treatment groups
(N=15)
Study Period
Mean (SD)
Median
(Min,Max)
Before randomisation
4.12 (0.77)
3.98
(3.14 , 5.28)
Study Period 1
3.98 (0.73)
3.76
(2.62 , 5.42)
Study Period 2
3.90 (0.69)
3.67
(2.68 , 5.21)
Study Period 3
3.89 (0.67)
3.75
(2.75 , 5.17)
Dahlén et al. Page 16
Table 9. Analyses of carry over effect for PD20.
bud/form
(N=15)
form
(N=15)
placebo
(N=15)
Start of
Start of
Start of
Start of
next
Start of
next
Start of
next
treatment treatment treatment treatment treatment treatment
N
11
11
10
10
9
9
Geometric mean
590
795
396
330
340
287
Median
566
642
430
373
247
253
Range
224, 1756 80, 6173
76, 3399
50, 1451
25, 2450
49, 1756
t-test vs baseline
0.24
0.65
0.49
Study period 3 has no baseline value for the next study period. The number of patients is therefore
lower than in the other analysis
Table 10. Analyses of carry over effect for FENO.
Bud/form
(N=15)
Start of
treatment
form
(N=15)
placebo
(N=15)
Start of
Start of
next
Start of
next
Start of
treatment treatment treatment treatment
Start of next
treatment
N
11
11
10
10
9
9
Mean (SD)
40.3 (23)
40.5 (26)
54.9 (34)
43.3 (24)
61.4 (39)
48.1 (30)
Median
36
38.1
37.7
46.1
74.5
51.2
Range
13.6 ,
84.6
8.6 , 89.0 17.1 , 122 8.1 , 96.7 13.6 , 121.5 11.8 , 96.5
t-test vs baseline
0.97
0.09
0.07
Study period 3 has no baseline value for the next study period. The number of patients is the refore
lower than in the other analysis