Inhaled and systemic

corticosteroid response in
severe asthma assessed by
alveolar nitric oxide: a
randomized crossover pilot
study of add-on therapy
Peter A. Williamson, Philip M. Short, Sriram Vaidyanathan &
Brian J. Lipworth
Asthma and Allergy Research Group, Division of Medical Sciences, University of Dundee, Dundee,
Scotland, UK
Correspondence
Professor Brian J. Lipworth MRCP, Asthma
and Allergy Research Group, Division of
Medical Sciences, University of Dundee,
Dundee, DD1 9SY, Scotland, UK.
Tel.: +44 13 8263 2983 (direct);
+44 13 8263 2588 (secretary)
Fax: +44 13 8264 4972
E-mail: brianlipworth@gmail.com
----------------------------------------------------------------------
Keywords
alveolar nitric oxide, asthma
----------------------------------------------------------------------
Received
10 November 2011
Accepted
30 April 2012
Accepted Article
Published Online
9 May 2012
WHAT IS ALREADY KNOWN ABOUT
THIS SUBJECT
Extra ne inhaled corticosteroid
formulations such as HFA beclomethasone
may be used to target the small airways in
asthma. However, it is unclear which
outcome measures may detect such small
airways effects in patients with severe
asthma.
WHAT THIS STUDY ADDS
The alveolar fraction of exhaled nitric oxide
is not a sensitive outcome measure for
following effects of extra ne particle
inhaled beclomethasone in severe asthma.
However there were signicant effects on
nitric oxide fractions measured at 50 ml s
-1
and bronchial ux with the extra ne
formulation, which did not produce
signicant cortisol suppression.
AIMS
Alveolar nitric oxide (CA
NO
) is a potential biomarker of small airway
inammation. We investigated effects on CA
NO
of the addition of coarse and ne
particle inhaled corticosteroids to standard therapy in severe asthma.
METHODS
Severe asthmatics taking 1600 mg day
-1
budesonide or equivalent performed
a randomized open-label crossover study. Subjects with FEV
1
< 80%, gas
trapping and CA
NO
2 ppb entered a 6 week dose-ramp run-in of
uticasone/salmeterol(FPSM) 250/50 mg twice daily for 3 weeks, then 500/50 mg
twice daily for 3 weeks. Patients then received additional HFA-beclomethasone
diproprionate (BDP) 200 mg twice daily or FP 250 mg twice daily for 3 weeks in a
crossover. Participants then received prednisolone(PRED) 25 mg day
-1
for
1 week. Nitric oxide, lung function, mannitol challenge, systemic inammatory
markers and urinary cortisol were measured.
RESULTS
Fifteen completed per protocol: mean (SD) age 51 (12) years, FEV
1
58 (13)%
predicted, residual volume 193 (100)% predicted and mannitol
PD10
177 (2.8) mg.
There was no signicant difference between FPSM and add-on therapy for CA
NO
.
FPSM/BDP and FPSM/PRED suppressed broncial ux (Jaw
NO
) and FE
NO
compared
with FPSM alone, but there was no signicant difference between FPSM/BDP
and FPSM/FP. ECP, e-selectin and ICAM-1 were suppressed by FPSM/PRED
compared with FPSM and FPSM/FP but not FPSM/BDP. Plasma cortisol was
signicantly suppressed by FPSM/PRED.
CONCLUSION
In severe asthma, CA
NO
is insensitive to changes in dose and delivery of inhaled
corticosteroids and is not suppressed by systemic corticosteroids. Additional
inhaled HFA-BDP reduced FE
NO
and Jaw
NO
without adrenal suppression. There
was a trend to reduction in FE
NO
and Jaw
NO
with additional FP but this did not
reach statistical signicance. PRED reduced FE
NO
and Jaw
NO
with suppression of
systemic inammatory markers and urinary cortisol.
British Journal of Clinical
Pharmacology
DOI:10.1111/j.1365-2125.2012.04319.x
Br J Clin Pharmacol / 75:1 / 93102 / 93 2012 The Authors
British Journal of Clinical Pharmacology 2012 The British Pharmacological Society
Introduction
Asthma is characterized by inammation of the airways
associated with smooth muscle hypertrophy, bronchial
hyper-reactivity and airway remodelling [1, 2]. Whilst
inhaled corticosteroids (ICS) are effective for the treatment
for most asthmatics, 515% of patients are poorly con-
trolled despite combination therapy, or require high dose
therapy to control their disease [3]. Pathology series
suggest that in severe asthma, disease may be more dis-
tally distributed compared with mild-to-moderate disease
[2, 4, 5].
Fractional exhaled nitric oxide (NO) measured at a ow
rate of 50 ml s
-1
(FE
NO
) is correlated with airway eosinophila
and has been used as a surrogate of steroid responsiveness
[6]. In recent years extended exhaled NO techniques have
been developed which calculate values representative of
NO derived from the large conducting airways and the
small airways and alveoli, respectively, the so-called two
compartment model of NO production [7]. The technique
involves the measurement of exhaled NO at several ow
rates from which alveolar NO (CA
NO
) and bronchial ux
(Jaw
NO
) can be derived. The majority of traditional FE
NO
is
derived from Jaw
NO
[8].
Early papers reported that CA
NO
was elevated in severe
asthmatics suggesting that it could be a useful biomarker
for the monitoring of small airway disease in severe
asthma [9, 10]. It is now recognized that the two compart-
ment model was an over-simplication in that it does not
account for NO diffusion between compartments. Hence a
proportion of NO produced proximally (Jaw
NO
) will diffuse
back into the alveolar compartment resulting in false
elevation of CA
NO
andvice versa. Mathematical adjustments
are now used to correct for this [11, 12]. This correction is
especially important when Jaw
NO
(and hence FE
NO
) are
elevated as is often the case in severe asthma. Hence in
severe asthma, the response of CA
NO
to different prepara-
tions of corticosteroids has not been studied in a con-
trolled trial with correction for back-diffusion.
The aim of the present study was to investigate the
effects on CA
NO
of the addition of coarse or ne particle ICS
to standard therapy in severe asthma, using oral predniso-
lone (PRED) as a positive control. Other surrogates of
airway and systemic inammation were assessed for
comparison.
Methods
Study design
An open-label randomized crossover study was performed
(Figure 1). Participants were never-smokers or ex-smokers
with <10 pack years, aged 1665 years, a respiratory physi-
cian diagnosis of asthma, prescribed 1000 mg day
-1
uti-
casone or 1600 mg day
-1
budesonide in the previous year.
Subjects required evidence of persistent airow obstruc-
tion (post-bronchodilator FEV
1
: FVCratio < 0.7, FEV
1
< 80%)
and gas trapping (total lung capacity and residual volume
> 100%). Exclusion criteria included diagnosis of bron-
chiectasis or allergic broncho-pulmonary aspergillosis and
long term oral corticosteroid use. Subjects were also
required to have an elevated uncorrected alveolar NO
(2 ppb) following a 6 week dose-ramp run-in of
uticasone/salmeterol (FPSM) 500 mg day
-1
for 3 weeks,
then 1000 mg day
-1
for 3 weeks via dry powder inhaler
(Seretide Accuhaler, Allen & Hanbury).
Screen
3 weeks 3 weeks 3 weeks 3 weeks 3 weeks 1 week
Dose ramp
Step 1
Seretide
Accuhaler
250/50 mg
1 puff
twice daily
Seretide
Accuhaler
500/50 mg
1 puff
twice daily
Seretide Accuhaler 500/50 mg 1 puff twice daily throughout study period
Dose ramp
Step 2
Run-out
Additive
therapy :
HFA-BDP
100 mg 2 puff
twice daily
OR
Fluticasone
Accuhaler
250 mg
twice daily
Additive
therapy :
Fluticasone
Accuhaler
250 mg
twice daily
OR
HFA-BDP
100 mg 2 puff
twice daily
Additive
therapy :
Prednisolone
25 mg
once daily
Figure 1
Study diagram
P. A. Williamson et al.
94 / 75:1 / Br J Clin Pharmacol
For the treatment periods, participants continued
FPSM 1000 mg day
-1
and were additionally randomized to
extra-ne HFA-beclomethasone diproprionate (BDP)
400 mg day
-1
(BDP) (Qvar easibreathe, Teva Pharmaceuti-
cals) or uticasone propionate 500 mg day
-1
(FP) (Flixotide
Accuhaler, Allen & Hanbury) for 3 weeks in a crossover
fashion. Following a 3 week run-out all participants were
allocated PRED 25 mg day
-1
(PRED) for 1 week. Inhaler
technique was assessed and corrected at every visit using
an In-check dial (Clement Clarke International Limited,
Essex, UK). Compliance was re-enforced at each visit and
dose counters checked for DPI treatments. Computer gen-
erated simple randomization was performed to allocate
subjects to cross-over sequence.
At baseline and following each treatment the following
measurements were performed: extended exhaled NO,
spirometry, impulse oscillometry, body plethysmography,
asthma quality of life questionnaire (AQLQ), asthma
control questionnaire (ACQ), mannitol challenge and
blood for ECP, ICAM-1 and E-selectin. Subjects took study
medication on the morning of visits, but withheld as
required medications. Leukotriene receptor antagonists
were not permitted during the study. Alveolar NO was the
primary outcome and other measurements secondary. All
measurements were performed at the Asthma and Allergy
Research Group at Ninewells Hospital, Dundee and Perth
Royal Inrmary, Perth. The study was approved by the
Tayside Committee on Medical Research Ethics and regis-
tered on clinicaltrials.gov (NCT00829257).
Measurements
Lung function Spirometry was performed in accordance
with guidelines [13] using a SuperSpiro spirometer (Micro
Medical Ltd, Chatham, Kent, UK). AJaeger Masterscreen (Erich
Jaeger, Hoechberg, Germany) was used for impulse oscillom-
etry (IOS) andfull body plethysmography. IOS was performed
as previously published [14]. If FEV
1
> 60% the provocative
dose of mannitol required for a 10% fall in FEV
1
(mannitol
PD
10
) was performed as previously published [15].
Extended exhaled nitric oxide An on-line chemilumines-
cence analyzer (Niox, Aerocrine AB, Solna, Sweden) was
used to quantify exhaled NO at multiple ow rates,
50 ml s
-1
, 100 ml s
-1
and 200 ml s
-1
. Online graphs of NO
exhalation manoeuvres were examined to ensure that an
adequate plateau had been achieved. Measurements were
repeated as necessary to ensure a minimum of two satis-
factory readings at each ow rate with <10% variation.
CA
NO
and Jaw
NO
were derived using the two-compartment
models described by Tsoukias & George and Tsoukias et al.
[7, 16] with correction for axial diffusion as described by
Kerckx et al. [12].
Laboratory analysis Urine was collected overnight for
10 h prior to study visits. Urinary creatinine was deter-
mined using a Roche Cobas analyzer (Roche, Burgess Hill,
UK; coefcient of variance (CV) 4.9%) and cortisol by radio-
immunoassay (Diasorin, Bracknell, UK; CV 8.6%). ECP was
determined using the UNICAP method (Phadia Ltd, Milton
Keynes, UK; CV 4.0%). E-selectin and ICAM-1 were mea-
sured through use of enzyme immunoassays (Diaclone,
Besancon, France; CV 1.5% and 2.8%).
Statistical analysis
Data were assessed for normality using the Shapiro-Wilk
test and inspection of boxplots. Non-Gaussian data were
log-transformed prior to analysis. Values for baseline
(FPSM), treatment limbs (FPSM/FP and FPSM/FP) and posi-
tive control limb (FPSM/PRED) were compared using an
ANOVA of repeated measures followed by Bonferroni cor-
rection for multiple comparisons. A priori power calcula-
tions predicted that 14 participants in crossover design
ensured 90% power, with an a error of 0.05, to detect a
0.5 ppb change in CA
NO
, assuming a within patient stan-
dard deviation of 0.37 ppb based on previous studies in
asthmatics [9, 17]. Analyses were performed using SPSS
version 17.0 (Chicago, Illinois, USA) or GraphPad PRISM
version 5.01 (San Diego, California, USA).
Results
Fifty-two screens were performed of which 26 were suit-
able to enter the 6 week dose ramp run-in. Following
run-in 17 met entry criteria for randomization to treat-
ments. Two subjects were withdrawn post randomization
due to exacerbation requiring systemic steroids (one on
each treatment) (see gure 2). Demographic data and
medication at screen are shown in Table 1. Mean (SD) age
and FEV
1
were 51 (12) years and 58 (13)% predicted
respectively. All were current non-smokers and 13/15
were never smokers.
There was no signicant difference between FPSM
and any add-on therapy for corrected CA
NO
. Mean (95%
CI) values were 1.39 ppb (0.48, 2.31) for FPSM, 1.79 ppb
(0.58, 3.00) for FPSM/FP, 1.71 ppb (0.69, 2.73) for FPSM/
BDP and 1.29 ppb (0.62, 1.96) for FPSM/PRED (Figure 3,
52 screened 26 screen fail
2 withdrawn from ramp
2 fail post ramp criteria
2 withdrawn post randomization
26 enter dose ramp
17 randomized
15 complete per protocol
Figure 2
Consort diagram
Effects on alveolar NO in severe asthma
Br J Clin Pharmacol / 75:1 / 95
Table 2). The FPSM/BDP and FPSM/PRED suppressed
Jaw
NO
and FE
NO
compared with FPSM alone (Figure 3,
Table 2, Table 4). For FE
NO
values were 22.5 ppb (17.0, 27.9)
and 21.4 ppb (16.4, 26.4) for FPSM/BDP and FPSM/PRED,
respectively compared with 30.7 ppb (21.1, 40.4) for FPSM
alone. There was a numerical trend for a reduction in FE
NO
and Jaw
NO
with FPSM/FP, but this did not reach statistical
signicance (P = 0.09 for both). There was no signicant
difference between FPSM/FP and FPSM/BDP (Figure 3,
Table 2, Table 4).
There was no statistically signicant difference
between treatments for any measure of pulmonary func-
tion including spirometry, IOS, body plethysmography and
bronchial challenge, although there was a numerical trend
for improvement in body plethysmography with FPSM/
PRED (Tables 2 and 3).
Plasma ECP, e-selectin and ICAM-1 were signicantly
suppressed by FPSM/PRED compared wit FPSM and
FPSM/FP but not FPSM/BDP. However FPSM/BDP was not
signicantly reduced compared with FPSM/FP or FPSM
(Figure 4, Table 2).
Plasma cortisol was signicantly suppressed by FPSM/
PRED compared with all other limbs (Figure 4, Table 2).
There was no difference in overnight urinary cortisol :
creatinine ratio (OUCC) for inhaled treatment (FPSM,
FPSM/FP or FPSM/BDP). OUCC was not tested in the FPSM/
PRED limb due to cross-reactivity of the assay to urinary
metabolites.
ACQ and AQLQ values are shown (Figure 5). There
was no signicant difference in symptoms scores for any
treatment.
Discussion
This is the rst study to examine the potential role of CA
NO
for assessing therapies in severe asthma. Whilst early
studies suggested CA
NO
could be an attractive biomarker
to assess the small airways we have demonstrated it is
insufciently sensitive to detect a dose response to inhaled
corticosteroids or a response to systemic therapy.
Secondary outcomes in the present study showed that
inhaled HFA-BDP produced signicant reductions in FE
NO
and Jaw
NO
when added to a maximal dose of FPSM,
without causing adrenal suppression. Similar effects on
FE
NO
and Jaw
NO
were observed with oral PRED, but were
associated with signicant cortisol suppression. The addi-
tion of high dose FP showed a numerical trend for reduc-
tion which did not reach statistical signicance (Table 2,
Table 4). There was no statistically signicant difference
between FPSM/BDP and FPSM/FP.
Severe or refractory asthma is difcult to characterize
for research purposes and recommendations have been
reviewed several times in recent years [3, 1821]. The joint
AmericanThoracic Society statement on refractory asthma
suggests that criteria should include measures of airway
obstruction and level of therapy a patient requires for sta-
bility [3]. We therefore selected participants on the basis of
lung function as well as the dose of inhaled corticosteroids.
In order to include a positive control limb of systemic ste-
roids and to select a group stable enough to tolerate
changes to medication we did not include subjects on
long term oral corticosteroid therapy. In this regard our
patients may not be considered as severe as cohorts in
Table 1
Demographics at screen
Screen
number
Age(years)/
Gender
BDP
(mg)
LABA (L), Nasal
steroid (N), LAMA(T),
Antihistamine (A)
FEV1
(%)
Residual
volume
(%)
Resistance
at 5 Hz
(R5%)
Mannitol
PD10
1 50/M 1600 L 77 159 202 95.53
2 52/F 2000 L 66 456 115 30.39
6 52/M 1000 LNA 67 83 109 139.73
3 63/M 1600 LN 54 142 215 170.37
10 37 M 2000 LA 71 170 129 13.28
23 65F 1000 L 49 216 192 337.32
27 48 M 2000 L 67 130 130 192.50
31 40/M 2000 LA 44 224 213 NR
32 28/M 1000 L 64 140 214 96.94
36 43 M 2000 L 70 162 286 455.89
37 65/M 1600 69 147 151 129.27
38 60/M 2000 57 128 153 443.57
39 61/F 2000 N 48 185 142 NR
40 52/M 2000 L 63 153 200 121.69
41 52/M 2000 LN 38 188 306 NR
Mean (SD) 51 (10) 1635 (448) 58 (13) 193 (100) 184 (56) 177 (2.81)
Challenge not performed as FEV1 < 50% predicted. BDP, beclomethasone diproprionate equivalent; LABA, long acting beta-agonist; LAMA, long acting anti-muscarinic.
P. A. Williamson et al.
96 / 75:1 / Br J Clin Pharmacol
FPSM
0
10
20
30
40
50
FPSM/FP
Treatment
F
E
N
O

(
p
p
b
)
FPSM/BDP
* *
FPSM/PRED FPSM
0
1000
2000
3000
4000
FPSM/FP
Treatment
J
a
w
N
O

(
n
l

s
-
1
)
FPSM/BDP
* *
FPSM/PRED
FPSM
0
2
4
NS
NS
6
FPSM/FP
Treatment
U
n
c
o
r
r
e
c
t
e
d

C
A
N
O

(
p
p
b
)
FPSM/BDP FPSM/PRED
FPSM
0
1
2
3
FPSM/FP
Treatment
C
A
N
O

(
p
p
b
)
FPSM/BDP FPSM/PRED
Figure 3
Extended nitric oxide measurements stratied by treatment group. Data displayed as mean with 95% condence intervals. * P < 0.05 compared with FPSM.
FP, uticasone; FPSM, uticasone/salmeterol; BDP, beclomethasone diproprionate; PRED, prednisolone
Table 2
Inammation and adrenal axis
Treatment FPSM FPSM/FP FPSM/BDP FPSM/PRED
Exhaled nitric oxide
FENO (ppb) 30.7 (21.140.4) 24.3 (18.929.7) 22.5 (17.027.9)* 21.4 (16.426.4)*
JawNO (nL.s
-1
) 2667 (17443590) 2008 (15172500) 1902 (13702434)* 1795 (13232266)*
CANO (ppb) 1.39 (0.482.31) 1.79 (0.583.00) 1.71 (0.692.73) 1.29 (0.621.96)
Uncorrected CANO (ppb) 3.74 (3.174.31) 3.59 (2.444.74) 3.37 (2.404.34) 2.90 (2.263.54)
Plasma ECP 33.3 (17.6 to 49.0) 31.2 (21.2 to 41.7) 23.1 (14.1 to 32.1) 15.9 (12.0 to 19.7)**
Plasma E-selectin 95.3 (82.2 to 108.3 103.7 (87.8 to 119.5) 94.0 (77.4 to 110.7) 78.0 (64.3 to 91.7)**
Plasma ICAM-1 546 (485 to 607) 540 (475 to 605) 513 (456 to 571) 479 (418 to 540)**
Mannitol PD10 (mg) 135 (57320) 133 (60294) 182 (72459) 119 (55260)
Adrenal axis
08.00 h plasma cortisol 394 (338 to 459) 419 (349 to 503) 407 (356 to 466) 61 (31 to 121)***
*P < 0.05 compared with FPSM. **P < 0.05 compared with FPSM and FPSM/FP. ***P < 0.05 compared with FPSM, FPSM/FP and FPSM/BDP. Presented as geometric mean and 95%
CI. FP, uticasone; FPSM, uticasone/salmeterol; BDP, beclomethasone diproprionate; PRED, prednisolone.
Effects on alveolar NO in severe asthma
Br J Clin Pharmacol / 75:1 / 97
other studies on refractory asthma [2224] but nonethe-
less represent a refractory group as evidenced by a lack of
improvement in lung function and symptoms even with
systemic therapy. To ensure enrolment of subjects with
airow obstruction due to asthma, we sought evidence of
gas trapping with body plethysmography (mean residual
volume 193% predicted), excluded current smokers or
ex-smokers with >10 pack year history and conrmed
bronchial hyper-reactivity in all subjects with sufcient res-
piratory reserve to tolerate a bronchial challenge (mean
mannitol PD
10
177 mg).
In order to select participants with evidence of small
airways inammation and room for further suppression of
CA
NO
, entry criteria also included a CA
NO
> 2 ppb following
the 6 week run-in period. This level was selected on the
basis of pilot data, but at the time of protocol design the
importance of correction for axial diffusion was not widely
recognized and therefore an uncorrected value was used
[25].
Whilst several studies have reported elevated levels of
CA
NO
in severe asthma, only one other study has assessed
its responsiveness to corticosteroids [9, 10, 17, 25]. A
number of studies have been performed in milder asthma
with regards to suppression of CA
NO
using ICS. However
methods and results are varied and results have been con-
icting [26]. In an uncontrolled study by Berry et al. CA
NO
was suppressed in severe asthmatics by oral PRED but not
by a doubling of inhaled FP [9]. The authors concluded that
systemic treatment may be required to target adequately
the small airways, in keeping with Bel et al. who have
Table 3
Lung function
Treatment FPSM FPSM/FP FPSM/BDP FPSM/PRED
Spirometry
FEV1 (l) 2.16 (1.81 to 2.51) 2.18 (1.82 to 2.54) 2.21 (1.81 to 2.61) 2.26 (1.89 to 2.63)
FEF2575 (l) 1.35 (0.98 to 1.71) 1.29 (0.92 to 1.66) 1.36 (0.95 to 1.77) 1.38 (1.00 to 1.76)
FVC (l) 3.42 (3.03 to 3.81) 3.40 (3.00 to 3.81) 3.45 (3.03 to 3.87) 3.49 (3.08 to 3.90)
RVC (l) 3.70 (3.25 to 4.16) 3.49 (3.03 to 3.94} 3.51 (3.06 to 3.96) 3.66 (3.26 to 4.06)
FEV1 (%) 63.4 (57.6 to 69.1) 64.2 (59.0 to 69.4) 64.6 (58.5 to 70.7) 65.5 (59.6 to 71.4)
FEF2575 (%) 34.8 (26.3 to 43.3) 34.0 (25.4 to 42.5) 35.0 (25.7 to 44.3) 35.6 (26.6 to 44.6)
FVC (%) 83.2 (78.5 to 88.0) 83.0 (80.4 to 85.6) 83.9 (81.2 to 86.7) 83.6 (78.5 to 88.7)
RVC (%0 88.0 (83.5 to 92.5) 82.5 (77.9 to 87.0) 82.6 (79.2 to 86.0) 84.8 (81.3 to 88.4)
Impulse oscillometry
R5 (kPa l
-1
s) 0.47 (0.39 to 0.56) 0.45 (0.40 to 0.59) 0.44 (0.41 to 0.66) 0.47 (0.37 to 0.68
R5-R20 0.13 (0.23 to 0.13) 0.11 (0.06 to 0.23) 0.11 (0.06 to 0.25) 0.09 (0.03 to 0.21)
X5 (kPa l
-1
s) -0.15 (-0.23 to -0.13) -0.17 (-0.24 to -0.13) -0.15 (-0.26 to -0.12) -0.15 (-0.33 to -0.12)
Body plethysmography
sRaw (kPa s
-1
) 1.87 (1.31 to 2.42) 1.94 (1.43 to 2.45) 2.02 (1.51 to 2.54) 1.57 (1.02 to 2.13)
sRaw (%) 166 (114 to 218) 175 (127 to 222) 180 (131 to 229) 142 (93 to 192)
RV (l) 3.28 (2.58 to 3.97) 3.16 (2.80 to 3.51) 3.44 (2.60 to 4.28) 2.76 (2.42 to 3.10)
RV (%) 159 (113 to 205) 144 (127 to 161) 170 (113 to 227) 130 (117 to 143)
RV/TLC 0.44 (0.38 to 0.51) 0.46 (0.41 to 0.50) 0.46 (0.41 to 0.52) 0.41 (0.36 to 0.46)
Presented as mean and (95% CI) except impulse oscillometry which is median (IQR). % shown as percent predicted for race, age and gender. FP, uticasone; FPSM, uticasone/
salmeterol; BDP, beclomethasone diproprionate; PRED, prednisolone.
FPSM
0
20
40
60
FPSM/
FP
Treatment
E
C
P

(

g

l

1
)
FPSM/
BDP
**
FPSM/
PRED
NS
FPSM
0
50
100
150
FPSM/
FP
Treatment
E
-
s
e
l
e
c
t
i
n

(
n
g

m
l

1
)
FPSM/
BDP
**
FPSM/
PRED
NS
FPSM
1
10
100
1000
FPSM/
FP
Treatment
C
o
r
t
i
s
o
l

(
n
m
o
l

l
-
1
)
FPSM/
BDP
***
FPSM/
PRED
NS
Figure 4
Plasma ECP, E-selectin and cortisol stratied by treatment group. Data displayed as mean and 95% condence intervals with exception of plasma cortisol,
displayed as geometric mean and 95% condence intervals. ** P < 0.05 compared with FPSM and FPSM/FP, *** P < 0.05 compared with FPSM, FPSM/ FP and
FPSM/BDP. FP, uticasone; FPSM, uticasone/salmeterol; BDP, beclomethasone diproprionate; PRED, prednisolone
P. A. Williamson et al.
98 / 75:1 / Br J Clin Pharmacol
shown benets of parenteral steroids in refractory asthma
[23]. However, in the paper by Berry et al. [9], no correction
for axial diffusion was applied and further inspection of the
data reveals that FE
NO
was signicantly suppressed with
PRED (32.4 vs. 17.8 ppb) but not by a doubling of ICS (41.6
vs. 41.3 ppb). Hence the apparent fall in uncorrected CA
NO
may represent a signicant reduction in back-diffusion
rather than a true reduction in alveolar NO. In the present
FPSM FPSM/FP FPSM/BDP FPSM/PRED
Treatment
FPSM FPSM/FP FPSM/BDP FPSM/PRED
Treatment
2
4
6
8
AQLQ
Optimal control
cut point
0
1
2
3
ACQ
Figure 5
Asthma control questionnaire (ACQ) and asthma quality of life questionnaire (AQLQ) stratied by treatment group. Data displayed as mean and 95%
condence intervals. FP, uticasone; FPSM, uticasone/salmeterol; BDP, beclomethasone diproprionate; PRED, prednisolone
Table 4
Difference in means and 95% condence intervals for extended nitric oxide measurements
Mean difference 95% CI Signicance
FENO (ppb)
FPSM vs. FPSM/FP 7.85 (1.10, 16.80) 0.094
vs. FPSM/BDP 10.41 (1.46, 19.36) 0.048
vs. FPSM/PRED 11.18 (2.23, 20.13) 0.041
FPSM/FP vs. FPSM/BDP 2.57 (6.38, 11.52) 0.485
vs. FPSM/PRED 3.33 (5.62, 12.28) 0.379
FPSM/BDP vs. FPSM/PRED 0.76 (8.19, 9.71) 0.829
JawNO (nl s
-1
)
FPSM vs. FPSM/FP 765.1 (103.5, 1634) 0.093
vs. FPSM/BDP 932.6 (63.96, 1801) 0.049
vs. FPSM/PRED 1027 (158.6, 1896) 0.047
FPSM/FP vs. FPSM/BDP 167.5 (701.1, 1036) 0.630
vs. FPSM/PRED 262.1 (606.5, 1131) 0.465
FPSM/BDP vs. FPSM/PRED 94.63 (774.0, 963.3) 0.783
CANO (ppb)
FPSM vs. FPSM/FP 0.56 (2.35, 1.24) 0.459
vs. FPSM/BDP 0.32 (2.12, 1.47) 0.651
vs. FPSM/PRED 0.15 (1.94, 1.64) 0.832
FPSM/FP vs. FPSM/BDP 0.23 (1.56, 2.03) 0.741
vs. FPSM/PRED 0.41 (1.39, 2.20) 0.631
FPSM/BDP vs. FPSM/PRED 0.17 (1.62, 1.97) 0.806
Uncorrected CANO (ppb)
FPSM vs. FPSM/FP 0.20 (1.33, 1.73) 0.740
vs. FPSM/BDP 0.60 (0.92, 2.13) 0.355
vs. FPSM/PRED 0.87 (0.66, 2.40) 0.213
FPSM/FP vs. FPSM/BDP 0.40 (1.13, 1.93) 0.520
vs. FPSM/PRED 0.67 (0.86, 2.19) 0.312
FPSM/BDP vs. FPSM/PRED 0.27 (1.26, 1.79) 0.660
FP, uticasone; FPSM, uticasone/salmeterol; BDP, beclomethasone diproprionate; PRED, prednisolone.
Effects on alveolar NO in severe asthma
Br J Clin Pharmacol / 75:1 / 99
study there was no signicant suppression of uncorrected
values, but the respective changes in FE
NO
and Jaw
NO
were
far more modest (Table 2). Our ndings are also consistent
with a more recent observational study of 15 asthmatics
demonstrating that CA
NO
is not elevated during acute
exacerbations and change in FE
NO
output was due to ux
rather than CA
NO
[27]. This paper also suggested that after
correction for Jaw
NO
, CA
NO
was not elevated in asthma com-
pared with age matched controls, in keeping with the lack
of response in the present paper and pilot work by our
department [25].
There are a number of possible reasons that CA
NO
was
not suppressed in the present study. The run-in of FPSM
may have maximally suppressed CA
NO
preventing any
further suppression. The run-in period included a dose-
ramp from FPSM 500 mg day
-1
to FPSM 1000 mg day
-1
to
assess if a doseresponse to CA
NO
could be demonstrated.
As in the main study, this showed a doseresponse in FE
NO
and Jaw
NO
but not corrected CA
NO
(data not shown). It has
been shown that for FE
NO
there is a level of maximal sup-
pression for an individual which varies dependent on
factors such as age, gender, atopy and height [28]. Similarly,
it is plausible there is a level of maximal suppression for
CA
NO
. If so, inclusion of individuals with a CA
NO
>2 ppb on
FPSM 1000 mg day
-1
may have inadvertently selected indi-
viduals maximally suppressed at a high level, rather than
allowing further room for improvement as initially
hypothesized. Finally it is possible that the steroids simply
have no signicant effect on CA
NO
in severe asthma, as has
been suggested in COPD [29].
It has been suggested that small airway disease may be
an important target for asthma therapy. With the develop-
ment of extra-ne particle ICS it is now possible to achieve
greater small airways drug deposition [30]. This is particu-
larly pertinent in individuals with airow obstruction as
this leads to more central distribution of inhaled therapies
[31]. We had hypothesized that extra-ne HFA-BDP may
achieve additional suppression of CA
NO
compared with a
biological equivalent dose of coarse particle ICS. Whilst this
was not observed, extra-ne BDP achieved signicant sup-
pression of FE
NO
and Jaw
NO
compared with FPSM without
causing adrenal suppression. Similarly whilst FPSM/PRED
signicantly suppressed systemic markers of asthmatic
inammation (ECP, e-selectin and ICAM-1) compared with
FPSM and FPSM/FP, they were not signicantly lower than
extra-ne HFA-BDP (Table 2). The consistency of this trend
across ECP, e-selectin and ICAM-1 would suggest that a
larger study powered for these markers may have demon-
strated signicant suppression with FPSM/BDP compared
with FPSM and FPSM/FP, though this cannot be conrmed
with the present data. Alternatively, it is possible that sys-
temic markers of inammation can only be suppressed
with appreciable systemic bioavailability, which was dem-
onstrated for PREDbut not inhaled therapies. In this regard
only moderate doses of HFA-BDP were used as add-on
therapy. Hence suppression of inammatory markers and
cortisol may have been observed at higher doses. Whilst
the combined doses of inhaled therapy in the study would
be very high for most asthmatics, it should be recognized
that severe airow obstruction reduces the lung bioavail-
ability of inhaled steroid as has been shown previously
with FP [3235].
Although HFA-BDP showed additional suppression of
FE
NO
and ux, the clinical signicance of such changes in
steroid treated asthmatics is not established. Smith et al.
have examined personal best FE
NO
in 73 non-severe asth-
matics comparing FE
NO
at a threshold where control is lost,
optimized inhaled FP and oral corticosteroid. They also
found levels were signicantly lower on oral steroid com-
pared with optimized inhaled FP, but had no extra-ne
particle comparator [28]. Further adequately powered
studies would be required to establish whether extra-ne
particle ICS could achieve near personal best FE
NO
and
whether this would confer greater asthma control with
long term therapy.
We were also interested to see that there was a non-
signicant numerical trend towards improvement in
mannitol airway hyperreactivity with FPSM/BDP, though
this was less than the biological variability of one dou-
bling dilution. Again, larger studies would be warranted
to assess the relative benets of different particle size
over a wider dose range and sufciently powered to
assess effects on mannitol challenge, exacerbations and
systemic inammatory markers.
For lungfunction, whilst there was a numerical trendfor
improvement with FPSM/PRED, there were no statistically
signicant changes. These were secondary outcomes and
as such the study was not sufciently powered to detect
small changes. Furthermore, we felt it unethical to use our
standard medication withholding time of three half-lives
because of our cohorts relative instability. Insteadwe asked
participants to use their study medication on the morning
of the visit and as such measurements of lung function, IOS
and body plethysmography represent post bronchodilator
measurements. Any additional bronchodilator effect from
corticosteroids is therefore likely to be small and difcult to
detect even with techniques sensitive to changes in airway
obstruction undetectable by spirometry alone [36]. Simi-
larly, no signicant improvement was seen in symptom
scores. We would interpret the lack of improvement in
symptoms andlungfunction, evenonoral therapy, as good
evidence of the refractory nature of our cohorts asthma.
Lung function and symptoms were secondary outcomes
andas suchthestudy was not poweredfor theseoutcomes.
Large long term studies would be required to conrm or
refute whether changes in NO would translate to clinically
meaningful patient outcomes.
We must acknowledge that our study had no placebo
control arm which is always desirable in clinical trial
design. The severity of our patients meant that a true
placebo arm with no therapy was not possible. Instead a
positive control arm was used to characterize maximal
P. A. Williamson et al.
100 / 75:1 / Br J Clin Pharmacol
suppression. We believe this is still a clinically meaningful
comparison in individuals who, by denition, have treat-
ment resistant disease. We acknowledge that our cohort
was older than a general population of severe asthmatics,
perhaps reecting a population willing and able to engage
in active clinical research. We acknowledge several further
limitations to our study design which were required to
minimize risks of exacerbation in a severe cohort: a base-
line with a full washout would have been potentially haz-
ardous, full bronchial challenge in all patients would have
been dangerous due to low FEV
1
, as would inhalation of
hypertonic saline for induced sputum. The stringency of
the inclusion criteria meant that numbers were necessarily
limited, though a cross-over design was employed to maxi-
mize statistical power. Finally, some may feel that our study
design was contrived as addition of high dose ICS to com-
bination therapy is not a standard approach in refractory
disease and is not included in guidelines. Whilst we
acknowledge this, we feel there is a paucity of data to
inform on a pharmacological approach to refractory
asthma and much of the published work focuses on non-
pharmacological and co-morbid aspects of disease. We are
aware anecdotally of pulmonologists who may indeed add
high dose ICS to combination therapy rather than step-up
to maintenance dose oral corticosteroid. Therefore whilst
the study design is articial it is nonetheless of interest
pharmacologically.
In conclusion, in severe asthma, inhaled HFA-BDP pro-
duced additional reductions in FE
NO
and Jaw
NO
without
adrenal suppression. Oral PRED produced similar effects
but with additional suppression of systemic inammatory
markers and cortisol. There was a numerical trend for
reduction with FP but this did not reach statistical signi-
cance and no signicant difference between FPSM/BDP
and FPSM/FP. Alveolar NO is insensitive to changes in dose
and delivery of ICS and is not suppressed by systemic cor-
ticosteroids. This suggests that CA
NO
is either an insensitive
test of reversible small airway disease, or that this cohort of
patients require non-steroidal immunosuppressive treat-
ments. CA
NO
is therefore unlikely to be a useful tool in mea-
suring response to treatments targeted at small airway
disease in refractory asthma.
Competing Interests
BJL has received unrestricted educational grant support
from TEVA for the present study and has also received
funds for giving post graduate educational talks and
attending advisory boards for TEVA. There are no other
competing interests to declare.
Source of funding
Investigator led study supported by an unrestricted edu-
cational grant from TEVA Pharmaceuticals Inc. USA.
Contributions
Conception and design: PAW, SV, BJL. Analysis and interpre-
tation: PAW, PMS, BJL. Drafting of manuscript for important
intellectual content: PAW, PMS, SV, BJL.
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