Effect of an interleukin-4 variant on late phase asthmatic response to allergen challenge in asthmatic patients: results of two phase 2a studies

Article (PDF Available)inThe Lancet 370(9596):1422-31 · November 2007with280 Reads
DOI: 10.1016/S0140-6736(07)61600-6 · Source: PubMed
Abstract
Increases in T helper (Th) 2 cytokine concentrations have been seen in atopic asthma, with interleukin 4 and interleukin 13 thought to have a role in the physiological response to allergen challenge. Our aim was to assess the therapeutic effect of pitrakinra, an interleukin-4 variant that targets allergic Th2 inflammation by potently inhibiting the binding of interleukin 4 and interleukin 13 to interleukin-4Ralpha receptor complexes. In two independent randomised, double-blind, placebo-controlled, parallel group phase 2a clinical trials, patients with atopic asthma were treated with pitrakinra or placebo via two routes. In study 1, patients were randomly assigned to receive either 25 mg pitrakinra (n=12) or placebo (n=12) by subcutaneous injection once daily. In study 2, patients were randomly assigned to receive either 60 mg pitrakinra (n=16) or placebo (n=16) by nebulisation twice daily. Inhaled allergen challenge was done before and after 4 weeks of treatment. The primary endpoint for study 1 was maximum percentage decrease in forced expiratory volume in 1 s (FEV1) over 4-10 h after allergen challenge, whereas that in study 2 was average percentage decrease in FEV(1) over 4-10 h after allergen challenge. All patients except those with baseline data only were included in our analyses. These trials are registered with ClinicalTrials.gov, numbers NCT00535028 and NCT00535031. No patients dropped out or were lost to follow-up in study 1; in study 2, two patients in the placebo group and one in the pitrakinra group dropped out or were lost to follow-up. These individuals had baseline data only, and were excluded from the analyses. In study 1, there was a 17.1% maximum percentage decrease in FEV1 in the pitrakinra group; by contrast, the maximum decrease was 23.1% in the placebo group (difference 6%, 95% CI -4.37 to 16.32; p=0.243). In study 2, there was a 4.4% average percentage decrease in FEV1 in the pitrakinra group; by contrast, the average percentage decrease was 15.9% in the placebo group (3.7 [95% CI 2.08-6.25] times lower in the pitrakinra group; p=0.0001). There were fewer asthma-related adverse events (p=0.069) and fewer adverse events requiring beta-agonist rescue (p=0.031) after subcutaneous administration of pitrakinra than with placebo. There were too few asthma-related adverse events in study 2 to assess the effect of inhalation of pitrakinra on adverse events. Local treatment, targeted at inhibition of interleukins 4 and 13 in the lung, could substantially diminish the symptoms of asthma.

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Articles
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Eff ect of an interleukin-4 variant on late phase asthmatic
response to allergen challenge in asthmatic patients: results
of two phase 2a studies
Sally Wenzel, Darren Wilbraham, Rick Fuller, Elise Burmeister Getz, Malinda Longphre
Summary
Background Increases in T helper (Th) 2 cytokine concentrations have been seen in atopic asthma, with interleukin 4
and interleukin 13 thought to have a role in the physiological response to allergen challenge. Our aim was to assess
the therapeutic eff ect of pitrakinra, an interleukin-4 variant that targets allergic Th2 infl ammation by potently
inhibiting the binding of interleukin 4 and interleukin 13 to interleukin-4Rα receptor complexes.
Methods In two independent randomised, double-blind, placebo-controlled, parallel group phase 2a clinical trials,
patients with atopic asthma were treated with pitrakinra or placebo via two routes. In study 1, patients were randomly
assigned to receive either 25 mg pitrakinra (n=12) or placebo (n=12) by subcutaneous injection once daily. In study 2,
patients were randomly assigned to receive either 60 mg pitrakinra (n=16) or placebo (n=16) by nebulisation twice daily.
Inhaled allergen challenge was done before and after 4 weeks of treatment. The primary endpoint for study 1 was
maximum percentage decrease in forced expiratory volume in 1 s (FEV
1
) over 4–10 h after allergen challenge, whereas
that in study 2 was average percentage decrease in FEV
1
over 4–10 h after allergen challenge. All patients except those
with baseline data only were included in our analyses. These trials are registered with ClinicalTrials.gov, numbers
NCT00535028 and NCT00535031.
Findings No patients dropped out or were lost to follow-up in study 1; in study 2, two patients in the placebo group and
one in the pitrakinra group dropped out or were lost to follow-up. These individuals had baseline data only, and were
excluded from the analyses. In study 1, there was a 17·1% maximum percentage decrease in FEV
1
in the pitrakinra
group; by contrast, the maximum decrease was 23·1% in the placebo group (diff erence 6%, 95% CI –4·37 to 16·32;
p=0·243). In study 2, there was a 4·4% average percentage decrease in FEV
1
in the pitrakinra group; by contrast, the
average percentage decrease was 15·9% in the placebo group (3·7 [95% CI 2·08–6·25] times lower in the pitrakinra
group; p=0·0001). There were fewer asthma-related adverse events (p=0·069) and fewer adverse events requiring
β-agonist rescue (p=0·031) after subcutaneous administration of pitrakinra than with placebo. There were too few
asthma-related adverse events in study 2 to assess the eff ect of inhalation of pitrakinra on adverse events.
Interpretation Local treatment, targeted at inhibition of interleukins 4 and 13 in the lung, could substantially diminish
the symptoms of asthma.
Introduction
The concept that upregulation of T helper (Th) 2 cytokines
is critical for the allergic infl ammation associated with
asthma is nearly 20 years old.
1
However, confi rmatory
evidence that Th2 cytokines such as interleukin 4 or
interleukin 13 have a critical role in the onset and
development of clinical asthma has, until now, been
lacking. Asthma has been reported to be characterised by
infi ltration of activated T lymphocytes and eosinophils
into the bronchial mucosa.
2
These cells, along with
resident mast cells, secrete soluble growth factors and
infl ammatory mediators (including interleukin 4 and
interleukin 13) that could directly and indirectly modify
the mucosal surface. In murine models, this Th2-type
immune response leads to a bronchoconstrictive and
infl ammatory response to allergens which enhances
non-specifi c bronchial hyper-responsiveness.
3–7
However,
recent clinical failure of drugs targeting the Th2 process
(ie, anti-interleukin 5 and interleukin-4-specifi c antag-
onists) has lessened enthusiasm for this pathway’s
singular importance. Despite these failures, concern has
remained that targeting of interleukin 4, to the exclusion
of interleukin 13, might have been too selective and that
studies that inhibit both of these cytokines are needed.
8,9
One possible approach to inhibiting both interleukin 4
and interleukin 13 is through inhibition or antagonism
of interleukin 4Rα, the signalling component of the
heterodimeric receptor complex for both interleukin 4
and interleukin 13.
10
Interleukin 4Rα forms a complex
with the common γ receptor on T cells, where it
specifi cally binds interleukin 4. Interleukin 4Rα can also
dimerise with interleukin 13Rα1 on other cell types to
bind both interleukin 4 and interleukin 13.
11
We developed
pitrakinra (Aerovant), a recombinant human interleukin-4
variant that competitively inhibits the interleukin-4Rα
receptor complex to interfere with the actions of both
interleukin 4 and interleukin 13. This drug, administered
either subcutaneously or via nebulisation, protected
allergic cynomolgus monkeys from allergen-induced
airways hyper-responsiveness and lung eosiniophilia in
Lancet 2007; 370: 1422–31
See Comment page 1396
University of Pittsburgh,
Division of Pulmonary, Allergy,
and Critical Care Medicine,
Pittsburgh, PA, USA
(S Wenzel MD); Guys Drug
Research Unit, Quintiles Ltd,
London, UK
(D Wilbraham MBBS); and
Aerovance Inc, Berkeley, CA,
USA (R Fuller FRCP,
E Burmeister Getz PhD,
M Longphre PhD)
Correspondence to:
Dr Malinda Longphre,
Aerovance Inc, 2929 7th Street,
Berkeley, CA 94710, USA
malinda.longphre@aerovance.
com
Articles
www.thelancet.com Vol 370 October 20, 2007
1423
both prophylactic and therapeutic model settings.
12,13
Subcutaneous administration in monkeys for 6 weeks or
more also reduced the cutaneous wheal response and
circulating concentrations of allergen-specifi c IgE.
14
In
human beings, pitrakinra decreased the eczema clinical
score and circulating IgE concentrations, and normalised
T-cell subsets in patients with severe atopic eczema after
4 weeks of subcutaneous administration.
15
Our aim was
to do two phase IIa trials of pitrakinra to investigate
whether Th2 immunity was important in a clinical setting
where asthmatics are experimentally challenged with
aerosolised allergen.
Methods
Patients
For both studies, patients with atopic asthma
(ages >18 years) were recruited to the Guy’s Drug
Research Unit, London, UK. Patients with asthma were
included if they had a baseline forced expiratory volume
in 1 s (FEV
1
) of 70% or more of predicted, needed regular
or as required use of β-agonists, and showed a late phase
response (≥15% drop in FEV
1
between 4–10 h) to allergen
challenge at screening. They must have been on a stable
regimen of medications for asthma for 1 month or more,
and could not have had systemic immunosuppressive
therapy within 1 month of screening. Additionally,
patients were screened for airways reactivity. In study 1,
patients were required to have a PC
20
(provocative
concentration that causes a 20% fall in FEV
1
from the
saline alone value) to methacholine of less than 8 mg/mL
and in study 2, participants were required to have a PC
20
to adenosine monophosphate of more than 3·125 mg/mL.
Individuals were excluded if they had any medical
condition that would preclude allergen challenge, had a
greater than 10 pack-year smoking history, or had smoked
in the 3 months before screening. Patients were also
excluded if they had received any corticosteroid
medications (systemic or inhaled) in the month before
screening. Participants were to continue their
non-steroidal concomitant treatments without change
during the study. No participants used leukotriene-
receptor antagonists while on study.
Both studies were done under a clinical trials
authorisation from the UK Medicines and Healthcare
products Regulatory Agency after review by the Guy’s
research ethics committee (London, UK). The studies
were done according to the principles of good clinical
practice and applicable laws and regulations and the
Declaration of Helsinki. All participants gave written
informed consent before any study-specifi c procedure.
Procedures
Both studies were double-blind, placebo-controlled,
parallel group in design. Patients were screened and
randomised within 1 month of dosing. Three screening
visits consisted of assessment of medical history, allergen
skin-prick test, lung function testing, determination of
lung function response to inhaled allergen, inhaled
methacholine, or inhaled adenosine monophosphate,
and basic safety screening. In study 1, participants were
randomly assigned to receive pitrakinra or placebo
subcutaneously once a day; whereas in study 2 participants
were randomly assigned to receive pitrakinra or placebo
via nebulisation twice a day. Allocation to treatment was
according to a predetermined random order in block
sizes of four. The randomisation list was generated with
SAS version 8.2 by the Guy’s Drug Research Unit.
Participants were randomised to active treatment or
placeo in a 1 to 1 ratio. Both pitrakinra and placebo were
dispensed from the same nebuliser pots to maintain
blinding.
Randomised participants returned to the clinic
once daily for 28 days to receive study medication. Final
follow-up visits were held 5–10 days after the fi nal dose
for both studies (fi gure 1).
Pitrakinra is a recombinant form of the wild-type
human interleukin 4 containing two functional mutations
at positions 121 (arginine to aspartic acid) and 124
(tyrosine to aspartic acid). For these studies, the drug was
manufactured by Bayer Ag (Wuppertal, Germany). The
placebo drug product was sterile saline for injection or
nebulisation. In study 1, 25 mg pitrakinra or placebo
(1·5 mL) was injected subcutaneously once daily in the
clinic. In study 2, 60 mg (nominal) of pitrakinra or
matched volume placebo was nebulised twice daily with
a Pari LC Plus nebuliser (Starnberg, Germany), with the
morning dose given in the clinic and the evening dose
self-administered at home. The nebuliser was expected
to deliver about 10 mg pitrakinra to the lung, in view of
its e ciency of delivery.
FEV
1
was assessed in accordance with American
Thoracic Society (ATS)/European Respiratory Society
(ERS) guidelines
16,17
with a MasterScope spirometer
25 mg pitrakinra subcutaneously/placebo once daily
60 mg pitrakinra nebulised/placebo twice daily
S1 S2
Screening visits
Study 2
On study days
On study days
Follow-up
Follow-up
Allergen
challenge
Allergen
challenge
Allergen
challenge
Methacholine Methacholine
Adenosine
monophosphate
Allergen
challenge
Adenosine
monophosphate
Screening visits
Study 1
S3 1 7 14 21 27 28 +5–10
S1 S2 S3 1 7 14 21 28 29 +5–10
Figure 1: Treatment time line
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(software version JLAB 4.61.0.1, Jaeger, Hoechberg,
Germany). Allergen inhalation challenge was done after
determination of general sensitivity by standard
skin-prick testing with cat, grass, and house dust mite
allergens. All allergens were sourced from ALK-Abelló
(Round Rock, TX, USA) and were nebulised with a Pari
LC Plus nebuliser. In study 2, grass pollen skin-prick
responders were excluded during hay fever season.
Administration of allergens was done according to the
ve-breath dosimeter method by use of a calibrated
Electro II inhalation dosimeter (Spira, Hämeenlinna,
Finland), under the supervision of experienced respiratory
clinical physiologists. The concentration of aerosolised
allergen (starting at 250 standardised quality units per
mL; SQU/mL) was doubled every 10 min until a decline
in lung function of about 20% was achieved (maximum
dose of 32 000 SQU/mL). This dose of allergen was then
set as the target for the post-treatment challenge.
However, for safety reasons, if a fall of more than 15%
was achieved before reaching the target dose, the allergen
challenge was terminated early. FEV
1
was measured after
allergen inhalation challenge at 20 min, 30 min, 45 min,
1 h, and then every 30 min from 1 to 10 h. After the
10-h post-allergen pulmonary function test, the patient
was given at least two pu s of β-agonist to relieve
bronchoconstriction as part of standard operating
procedure.
In study 1, 24 h after allergen challenge, doubling doses
of nebulised methacholine (Provocholine, Methapharm
Inc, Brantford, ON, Canada) were administered with a
Pari LC Plus and Electro II inhalation dosimeter every
5 min starting with 0·03125 mg/mL and continuing to
32 mg/mL or until a 20% fall in FEV
1
was achieved. This
approach was based on ATS guidelines.
18
In study 2,
adenosine monophosphate (Sigma Aldrich, St Louis,
MO, USA) was nebulised initially at 3·125 mg/mL and
increased to 400 mg/mL or until a 20% fall in FEV
1
was
achieved.
19
For each stimulus, the PC
20
was determined.
In study 2, fractional expiratory nitric oxide (F
E
NO) was
measured with a NIOX system (Aerocrine, Solna, Sweden)
according to ATS F
E
NO guidelines.
20
F
E
NO was measured
at the second screening and at day 27 (pre-allergen
challenge), and at the third screening and day 28 (24 h
post-allergen challenge). Total IgE was also measured in
patients in study 2 from blood taken pre-dose on day 1
and day 28 by ImmunoCap1000 assay (Phadia, Uppsala,
Sweden) at Laboratoire Marcel Merieux (Lyon, France).
Sputum induced with hypertonic saline was collected
from participants in study 1 (but not study 2) on screening
visit three and day 29 (24 h after allergen challenge and
1 h after methacholine challenge). Sputum cytospins
were fi xed within 2 h of collection and sent to Laboratory
Marcel Merieux (Lyon, France) for cytology analysis.
Eosinophils were reported as a proportion of
non-squamous cells in the sample.
In addition to being done at screening, allergen
skin-prick tests were done at day 28 in study 1. The
cutaneous allergen response was determined at screening
only in study 2, to guide the choice of allergen for
respiratory challenge.
Clinical safety endpoints, including haematology,
serum biochemistry, urinalysis, vital signs, and ECG,
were assessed. Extra lung function measurements for
safety were assessed in study 2 because this was the fi rst
time pitrakinra had been administered directly to the
lung. Adverse events were reported in conventional
format that refl ect International Conference on
118 patients screened
94 excluded
39 methacholine challenge
14 skin-prick test
14 allergen challenge
3 medical history
3 reversible FEV
1
3 drug screen
1 laboratory
1 physical examination
16 other
24 patients
randomised
12 received placebo
(volume-matched once daily)
12 received pitrakinra
(25 mg subcutaneously
once daily)
0 withdrew
A
B
0 withdrew
12 reached primary endpoint
194 patients screened
162 excluded
48 skin-prick test
36 allergen challenge
27 adenosine
monophosphate challenge
18 medical history
12 reversible FEV
1
7 baseline FEV
1
1 drug screen
13 other
32 patients
randomised
16 received placebo
(volume-matched twice daily)
2 withdrew
(adverse event)
1 withdrew
(non-compliance)
14 reached primary endpoint
15 reached primary endpoint
16 received pitrakinra
(60 mg nebulised twice daily)
12 reached primary endpoint
Figure 2: Trial profi les
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1425
Harmonisation/WHO Good Clinical Practice require-
ments (coding with MedDRA version 8.0). IgG antibody
levels against pitrakinra were also measured in blood
samples taken pre-dose on day 1 and either 5–10 days
after the last dose (study 1) or pre-dose on day 28 (study 2)
and analysed by ELISA at Aerovance Inc. 96-well plates
were coated with 1 g/mL pitrakinra overnight, washed
and blocked with casein (Pierce, Rockford, IL, USA). The
samples were diluted (minimum dilution 1:20) and
incubated on the plates. After washing, horseradish
peroxidase-conjugated goat anti-human IgG Fc antibody
(Jackson ImmunoResearch, West Grove, PA, USA)
number 109-036-098, 1:10 000) and 3,3,5,5
tetra-methylbenzidine (TMB) were used as the detection
reagents. The neutralisation potential of samples that
were positive for antibodies against pitrakinra was also
assessed with an ELISA-based binding assay to detect
the ability of such antibodies to block the binding of
pitrakinra to interleukin 4Rα. Serum samples confi rmed
positive for IgG antibodies against pitrakinra were
incubated with 5 g/mL pitrakinra to allow formation of
the antibody-analyte complex. Interleukin 4Rα (R&D
Systems, Minneapolis, MN, USA) was immobilised on
96-well plates, then incubated with the antibody-analyte
complex. Pitrakinra bound to interleukin 4Rα was
detected with biotinylated rat antibody against the drug,
followed by horseradish peroxidase-conjugated
streptavidin and the TMB substrate. Cynomolgus
monkey IgG against pitrakinra was used as a positive
control in both assays. The assay formats and detection
thresholds were designed in accordance with current
guidelines.
21
The primary endpoint of both studies was change in
FEV
1
over 4–10 h post-allergen challenge (ie, during late
asthmatic response). Study 1 defi ned the primary endpoint
as the maximum percentage fall in FEV
1
, while study 2
defi ned it as the average percentage fall in FEV
1
. Secondary
endpoints included F
E
NO (study 2 only), PC
20
, cutaneous
allergen response (study 1 only), sputum eosinophils
(study 1 only), and blood total IgE (study 2 only).
Statistical analysis
The primary endpoint of each study was analysed
independently by Aptuit (Edinburgh, Scotland).
Additional analyses of safety endpoint data were done at
Guy’s Drug Research Unit and of e cacy endpoint data
at Aerovance Inc.
E cacy endpoints were interpreted with analysis of
covariance (ANCOVA), with treatment group as the main
variable of interest and a covariate for the baseline
(screening) value. All patients, except those with baseline
data only, were included in our analyses. The single
exception was sputum eosinophils, for which the day 29
values were analysed non-parametrically (Kruskal-Wallis
test) because of violation of residual normality
assumptions not remedied by data transformation and
poor within-patient correlation between baseline and
day 29 values for the few participants (four receiving
study medication, seven receiving placebo) with paired
(baseline and day 29) results.
To facilitate comparison between studies, FEV
1
in late
asthmatic response was expressed in three ways for both
studies: maximum percentage fall, average percentage
fall, and area under the FEV
1
-versus-time curve (AUC).
The average percentage fall in FEV
1
was calculated by
dividing the area under the curve of the percentage fall in
FEV
1
by the duration of the response period. Maximum
percentage fall in FEV
1
and AUC were analysed on the
original scale. Estimated treatment arithmetic means
and an estimate of treatment di erence with 95% CI and
associated two-sided p value were determined for
maximum percentage fall in FEV
1
and AUC. Average
percentage fall in FEV
1
was log transformed before
analysis. After a back-transformation to the original scale,
the estimated treatment geometric means and an
estimate of treatment ratio with 95% CI and associated
p value were determined for average percentage fall in
FEV
1
. Three of the participants who received pitrakinra
and one of the placebo recipients in study 1, and two of
those who received pitrakinra and none of the placebo
recipients in study 2, had a negative average percentage
fall in FEV
1
at the end of treatment. These negative values
were truncated at zero to allow log transformation of the
time-normalised areas. AUC and area under the curve of
the percentage fall in FEV
1
were also calculated during
the early asthmatic response period (defi ned as 0–2 h
post-allergen).
F
E
NO data were analysed on the original scale. PC
20
,
IgE, and cutaneous allergen response data were
log-transformed before analysis. Blood eosinophil
percentages were square-root transformed before
analysis. Sputum eosinophil percentages were analysed
non-parametrically on the original scale.
Adverse event data were analysed with a Pearson’s χ
test, in which the number of days on which subjects had
an asthma-related adverse event, or required a β-agonist,
were assessed in the context of the number of days available
for adverse event observations, or β-agonist treatment.
All data were analysed with SAS version 8.2 or Minitab
release 14.13.
Study 1 Study 2
Pitrakinra Placebo Pitrakinra Placebo
Sex (male/female) 5/7 7/5 12/3 7/8
Ethnic origin (black/white/other) 2/10/0 2/9/1 0/12/3 1/13/1
Age (years) 31 (10) 30 (9) 25 (5) 29 (8)
PC
20
* (mg/mL) 1·37 (1·51) 1·84 (1·92) 16·6 (13·6) 34·5 (44·3)
FEV
1
(L) 3·72 (1·01) 3·70 (0·77) 4·09 (1·02) 3·52 (1·05)
FEV
1
(percentage predicted) 102% (13) 100% (20) 99% (15) 96% (18)
Total IgE (kU/L) .. .. 315 (303) 459 (399)
Data are n or mean (SD). *Study 1 for methacholine; study 2 for adenosine monophosphate.
Table 1: Demographic and baseline characteristics of study participants
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These trials are registered with ClinicalTrials.gov,
numbers NCT00535028 and NCT00535031.
Role of the funding source
The study sponsor, the principal investigator, and the
contracted research organisation had primary
responsibility for the study design, conduct, data
collection, and analysis. All authors had full access to the
data, and were responsible for the decision to submit
for publication.
Results
The trial profi les are shown in fi gure 2. There were no
study failures or withdrawals in study 1; by contrast, two
patients in the placebo group of study 2 withdrew because
of adverse events, one classifi ed as serious (both were
Hours
B
A
Placebo Pitrakinra subcutaneously
Placebo
Pitrakinra inhaled
Hours
Proportion of baseline FEV
1
(%)Proportion of baseline FEV
1
(%)
Hours
Hours
0246810
0
70
80
90
100
110
0246810
0
70
80
90
100
110
0246810
0
70
80
90
100
110
0246810
0
70
80
90
100
110
After 4 weeks treatment
Pre-dose at screeening
Placebo Pitrakinra Comparison Diff erence or ratio (95% CI) p value
Study 1 (pitrakinra group n=12, placebo group n=12)
Average percentage fall in FEV
1
9·4% 3·1% Placebo/pitrakinra 3·0 (0·91 to 10·12) 0·068
AUC (L·h) 17·6 19·2 Placebo–pitrakinra –1·6 (–3·25 to 0·03) 0·054
Maximum percentage fall in FEV
1
23·1% 17·1% Placebo–pitrakinra 6·0% (–4·37 to 16·32) 0·243
Study 2 (pitrakinra group n=15, placebo group n=14)
Average percentage fall in FEV
1
15·9% 4·4% Placebo/pitrakinra 3·7 (2·08 to 6·25) 0·0001
AUC (L·h) 18·4 21·6 Placebo–pitrakinra –3·1 (–4·63 to –1·65) 0·0002
Maximum percentage fall in FEV
1
27·6% 16·1% Placebo–pitrakinra 11·5% (5·21 to 17·85) 0·0009
Treatment means (or geometric mean, for the analysis of average percentage fall in FEV
1
after log transformation) were determined from the ANCOVA model.
Table 2: Allergen-induced FEV
1
response during the late phase
Figure 3: Average lung function after 4 weeks of treatment
Average lung function (FEV
1
) response to allergen challenge shown for all patients in (A) study 1 and (B) study 2 by treatment group pre-dose at screening (red) and
after 4 weeks of treatment (green). Error bars are SE.
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1427
asthma attacks precipitated by the allergen challenge
protocol). These individuals provided no FEV
1
data after
1 h post allergen dose, and so did not contribute to the
statistical analysis of the late asthmatic response or PC
20
data. Another patient in the pitrakinra group of study 2
withdrew because of poor clinic attendance and missed
doses. This individual was also excluded from our
analyses. Demographic and baseline data for the enrolled
participants who completed the study are shown in
table 1.
The decrease in FEV
1
in response to allergen challenge
was attenuated after 4 weeks of treatment with pitrakinra,
compared with placebo, irrespective of route of
administration (fi gure 3 and table 2). After subcutaneous
administration, pitrakinra conferred a 3·0 (95% CI
0·91–10·12, p=0·068) times reduction in the average
allergen-induced percentage fall in FEV
1
from pre-challenge
baseline during the late phase; by contrast, administration
by inhalation conferred a 3·7 (2·1–6·3, p=0·0001) times
reduction. The AUC, as well as the maximum percentage
fall in late asthmatic response FEV
1
from pre-challenge
baseline, were also improved after 4 weeks of subcutaneous
or inhaled administration of pitrakinra (table 2), although
treatment-related improvements were only signifi cant
after administration by inhalation. No signifi cant di erence
between pitrakinra and placebo was seen on the early
asthmatic response with either method of administration
(table 3).
Some participants received a lower dose of allergen on
day 28 (study 1) or 27 (study 2) relative to the screening
dose. The lower end-of-study allergen dose a ected more
placebo recipients than those who received pitrakinra,
with similar changes in allergen dose across groups
(study 1: four of 12 in the placebo group, mean change of
1·25 doubling doses, two of 12 in the pitrakinra group,
mean change of 1·0 doubling dose; study 2: seven of 14
in the placebo group, mean change of 1·43 doubling
doses, four of 15 in the pitrakinra group, mean change of
1·75 doubling doses). Despite the lower dose of allergen
in some participants in the post-treatment challenge, the
magnitude of the early response was not di erent from
that at screening. Additionally, the e ect of inhaled
pitrakinra on late asthmatic response FEV
1
relative to
placebo remained signifi cant when the subset of
participants with matching allergen dose at screening
and study end (seven in placebo group, 11 in the pitrakinra
group; study 2) was analysed (ANCOVA-adjusted ratio of
average percentage fall in FEV
1
3·3, 95% CI 1·4–7·6;
p=0·009).
No signifi cant di erence between pitrakinra and
placebo was seen on airway hyper-responsiveness to
methacholine (study 1) or adenosine monophosphate
(study 2). After subcutaneous administration, individuals
receiving pitrakinra tolerated 0·84 doubling doses more
methacholine before reaching a 20% fall in FEV
1
than did
placebo recipients, although this increase was not
signifi cant: ANCOVA-adjusted geometric mean PC
20
was
1·61 mg/mL in the pitrakinra group versus 0·90 mg/mL
in the placebo group (pitrakinra/placebo ratio 1·79,
95% CI 0·67–4·83; p=0·234). Likewise, after inhaled
administration, participants receiving pitrakinra tolerated
0·82 doubling doses more adenosine monophos phate
before reaching a 20% fall in FEV
1
than did those in the
placebo group; ANCOVA-adjusted geometric mean PC
20
was 34·6 mg/mL in the pitrakinra group versus
19·6 mg/mL in the placebo group (1·76, 0·84–3·70;
p=0·128).
Baseline (pre-allergen, predose, screening 2) F
E
NO was
high (63·2 [SD 39·2] ppb, n=22, fi gure 4; normal range
0–25 ppb), as might be expected for the asthmatic
population.
20
Resting (pre-allergen) F
E
NO was signifi cantly
lower after 4 weeks of inhaled pitrakinra than with
placebo (fi gure 4; ANCOVA adjusted mean 34·9 ppb with
pitrakinra, 63·1 ppb with placebo; treatment di erence
28·3 ppb, 95% CI 9·4–47·2; p=0·005).
24 h after allergen challenge (at screening 3), F
E
NO was
signifi cantly increased in all patients pre-randomisation
to 111·7 (SD 47·9) ppb (p<0·0001, relative to screening 2).
A signifi cant e ect of allergen was also seen at study end
across all participants (pre-allergen day 27 vs post-allergen
day 28; p<0·0001; data not shown). There was a signifi cant
linear relationship between the change in post-allergen
F
E
NO (day 28–screening 3) and the change in resting
F
E
NO (day 27–screening 2; p<0·002, Pearson correlation
coe cient 0·64, R² 0·41) in all participants in study 2.
At baseline in study 1, skin-prick area measurements
ranged from 31·0 to 176·6 mm (geometric mean
55·4 mm [CV%
15%] in the pitrakinra group; 76·2 mm
[19%] in the placebo group). At screening in study 2, areas
ranged from 21·7 to 108·5 mm (geometric mean
63·1 mm [15%] in the pitrakinra group; 56·3 mm [19%]
in the placebo group). At day 28 in study 1, there was no
signifi cant di erence in the cutaneous allergen response
between treatment groups (ANCOVA, 1·01, 95% CI
0·61–1·63; p=0·983).
At baseline, sputum eosinophils as a percentage of
non-squamous cells ranged from 2 to 52%, with median
percentages of 10% (n=9, placebo) and 11% (n=7,
pitrakinra). There was no signifi cant di erence in
sputum eosinophils at day 29 (p=0·965; data not shown),
although this assessment was limited by the high
proportion (54%) of participants without paired (baseline
and day 29) data, due mainly to insu cient sputum
volume.
Placebo Pitrakinra Placebo/pitrakinra
ratio (95% CI)
p value
Study 1 (pitrakinra group n=12, placebo group n=12)
Average percentage fall in FEV
1
6·5% 2·8% 2·3 (0·57–9·0) 0·56
Study 2 (pitrakinra group n=15, placebo group n=14)
Average percentage fall in FEV
1
4·2% 4·4% 0·97 (0·43–2·20) 0·94
Table 3: Allergen-induced FEV
1
response during the early phase
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At baseline, blood eosinophils as a proportion of white
blood cells were within the normal range of 0–6%
for 18 of 24 (study 1) and for 25 of 30 (study 2) participants
(data not shown). There was no signifi cant di erence in
blood eosinophils between the two treatment groups in
either study after 4 weeks of treatment (p=0·313, study 1;
p=0·715, study 2; data not shown).
In study 2, baseline total IgE in peripheral blood was
slightly above (geometric mean 277 kU/L [CV% 36%]) the
normal range of less than 150 kU/L. There was no
signifi cant di erence in blood IgE between the
two treatment groups after 4 weeks of treatment
(ANCOVA-adjusted geometric mean 312 kU/L in
pitrakinra group vs 284 kU/L in placebo group; treatment
ratio 0·91, 95% CI 0·81–1·02, p=0·096).
There were no signifi cant changes in haematology,
serum biochemistry, urinalysis, vital signs, or ECG tests
over time after administration of pitrakinra by either
route; there were also no signifi cant di erences in safety
outcomes between those who received pitrakinra and
those who received placebo. Pitrakinra had no negative
e ects on pulmonary function. A summary of adverse
events for both studies is shown in table 4.
There were more general disorders reported with
subcutaneously administered pitrakinra than with
placebo, mainly due to injection site reactions or
discomfort. Of the 672 injections administered, there
were 39 injection site-related adverse events reported
across both groups (35 events reported by eight of
12 patients receiving pitrakinra and four events by two
of 12 placebo recipients). Fewer asthma-related adverse
events requiring β-agonist rescue occurred in the
pitrakinra group than in the placebo group in study 1
(three events in three participants in the pitrakinra
group compared with 11 events in fi ve of the placebo
recipients; p=0·031). Participants who received
subcutaneous pitrakinra reported fewer adverse events
related to asthma (wheezing, dyspnoea, and chest
discomfort) than did those who received placebo (six
events were reported by four participants in the
160
60
30
0
–30
–60
–90
140
120
100
80
60
40
20
0
Resting F
E
NO at baseline (ppb)
Change in resting F
E
NO (%)
Pitrakinra inhaled Placebo
AB
Pitrakinra
Placebo
Figure 4: Pre-allergen baseline F
E
NO at screening 2 (A) and after 4 weeks of treatment (B)
Darker dots in (B) represent mean for each treatment group, with error bars representing 95% CI. Lighter dots are individual measurements.
Study 1, pitrakinra (n=12) Study 1, placebo (n=12) Study 2, pitrakinra (n=16) Study 2, placebo (n=16)
Participants Events Participants Events Participants Events Participants Events
Number of participants with an
adverse event
12 73 11 41 12 45 15 44
Nervous system disorders* 4 (33%) 9 5 (45%) 5 6 (55%) 8 6 (42%) 7
General disorders and
administration site conditions†
8 (67%) 40 4 (36%) 11 7 (63%) 7 3 (21%) 4
Skin disorders‡ 3 (25%) 4 3 (27%) 4 1 (9%) 1 2 (14%) 2
Gastrointestinal disorders§ 3 (25%) 4 1 (9%) 1 4 (36%) 7 4 (29%) 6
Respiratory, thoracic disorders¶ 5 (42%) 8 7 (63%) 14 10 (90%) 15 11 (78%) 22
Ear, labrynth|| 2 (17%) 2 0 (0%) 0 0 0 0 0
Musculoskeletal disorders** 2 (17%) 3 3 (27%) 4 3(27%) 3 2 (14%) 2
Infections†† 1 (8%) 1 3 (27%) 3 0 0 0 0
Reproductive disorders‡‡ 2 (17%) 2 0 (0%) 0 0 0 0 0
*For example, headaches, somnolence, dizziness. †For example, injection site discomfort, reaction, increased energy. ‡For example, rash, dry skin, pruritus. §For example,
nausea, abdominal discomfort, diarrhoea. ¶For example, wheezing, chest discomfort, dyspnoea, nasal congestion. ||For example, ear ache. **For example, muscle soreness,
back pain. ††For example, upper respiratory infection. ‡‡For example, mennoraghia, dysmenorraghia.
Table 4: Treatment-emergent adverse events by system organ class (>15% of participants)
Articles
www.thelancet.com Vol 370 October 20, 2007
1429
pitrakinra group vs 14 events in seven placebo recipients;
p=0·069). There were fewer overall asthma-related
adverse events requiring β-agonist rescue outside the
allergen challenge protocol in study 2, with only
two participants receiving pitrakinra and fi ve of those
in the placebo group a ected.
IgG antibodies against pitrakinra were detected in
follow-up blood samples from three of ten participants
tested after subcutaneous administration (titres of 1:40,
1:80, and 1:80) and from three of 15 participants after
inhalation (titres of 1:30, 1:60, and 1:480). All samples
confi rmed to contain IgG antibodies against pitrakinra
were not able to block binding of the drug to interleukin
4Rα—ie, they were non-neutralising antibodies.
Discussion
Our data show that, compared with placebo, decreases in
FEV
1
after allergen challenge were signifi cantly attenuated
after 4 weeks of inhalation of pitrakinra, lending support
to the hypothesis that dual inhibition of interleukin 4 and
interleukin 13 can a ect the course of the late asthmatic
response after experimental allergen challenge. The
frequency of spontaneous asthma attacks requiring
rescue medication use was also diminished in the fi rst
study, suggesting improved control over asthma
symptoms after treatment with pitrakinra.
Animal models and human studies suggest an
important role of Th2 immune responses in atopic
asthma,
1,3–5
and interference with the Th2 immune
pathway improves asthma symptoms in animal
models.
22–24
However, clinical data to support this
hypothesis has been lacking. Experimental treatments
that target interleukin 4 or interleukin 5 alone have not
been especially e ective, presumably because of
redundancies with interleukin 13 in many allergic
processes.
8
Animal studies suggest that interleukin 4 is
more important in the initiation of an immune response
and interleukin 13 is more involved in the amplifi cation
or expansion of immune responses.
5,8,25,26
Our data bolster
support for the immunological mechanisms previously
established in preclinical model systems that emphasise
a cooperative or redundant role for interleukin 4 and
interleukin 13 in allergic disease.
In addition to improvements in the late asthmatic
response, the resting infl ammatory status of the lungs
(as measured by F
E
NO) was signifi cantly attenuated after
inhalation of pitrakinra for 4 weeks, compared with
placebo,
27
in line with observations that interleukin 4 and
other pro-infl ammatory mediators induce nitric oxide
synthase (iNOS) through STAT1 and STAT6 in epithelial
cells.
28–31
There have been some studies to suggest that
nitric oxide could have a benefi cial e ect in asthma
patients by enhancing bronchodilation.
32
However, our
data do not seem to support this hypothesis, since the fall
in F
E
NO was accompanied by, and even marginally
correlated with, improved lung function after allergen
challenge. The observation that resting F
E
NO was
signifi cantly decreased by pitrakinra, but post-challenge
F
E
NO was not, is interesting and could be caused by
di erences in pathways contributing to the increased
concentrations of nitric oxide. Basal F
E
NO could be more
dependent on up-regulation of iNOS by interleukin 4 and
interleukin 13, whereas the increase in F
E
NO after
allergen challenge could involve additional pathways in
epithelial cells and perhaps macrophages that are not
a ected by the inhibition of interleukin 4Rα. One could
postulate that pitrakinra down-regulates baseline Th2
infl ammation in the asthmatic lung while not interfering
with the lung’s natural defences in the face of large
amounts of foreign allergen.
Although F
E
NO is believed to give some indication of
the degree of eosinophilic infl ammation in the airways,
the relationship is not completely reliable. These
preliminary studies did not include any additional
measures of infl ammation, such as sputum or biopsy
studies. Because numerous animal and in-vitro studies
support the role of interleukin 4Rα in mucus production
from airway epithelial cells, it is certainly possible that
some of the e ect of pitrakinra is on epithelial cell
mucus production.
33
Subsequent studies will be needed
to determine the broader e ect of interleukin 4Rα
inhibition on eosinophils, mast cells, mucus and goblet
cell hyperplasia, and even smooth muscle hypertrophy
or hyperplasia.
34
However, as a number of genetic
studies have suggested that polymorphisms in the
interleukin 4/interleukin 13/interleukin 4Rα pathways
contribute to both lower lung function and severe
exacerbations in asthma, a potential for a long-term
e ect of this approach on disease outcomes clearly
exists.
35,36
Pitrakinra did not have any e ect on the early response
to allergen challenge, nor did we anticipate that it would
have such an e ect. The early response is largely believed
to be driven by a mast cell response secondary to
cross-linking of IgE receptors by allergen. We have no
reason to believe that a 1-month treatment would be
su cient to limit the activation of the mast cells.
Inhibition of the early response would require a reduction
in the numbers of mast cells present in the airways or in
the degree of binding of IgE to the mast cell. Neither of
these is likely to occur within the fi rst month of therapy,
but might be expected to occur after more prolonged
therapy. More and longer studies will be required to
determine whether inhibition of interleukin 4Rα has any
e ect on mast cells.
Treatment with pitrakinra did not signifi cantly a ect
airway hyper-responsiveness to either methacholine or
adenosine monophosphate measured 24 h after allergen
challenge. However, the di erence between active
treatment and placebo was close to one doubling dose,
which suggests that an improvement might have been
seen with greater numbers of participants. Furthermore,
variability of the measurement was high, so the lack of
statistical signifi cance could be due to a type 2 error
Articles
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rather than implying that airway hyper-responsiveness
was not a ected by pitrakinra. It is not uncommon for
the sample sizes used in these types of studies to be
associated with non-signifi cant changes, partly because
of the intrinsic variability of the test. Whereas some
laboratories have reported very consistent e ects of
allergen to increase airway hyper-responsiveness, others
have not. In fact, in study 1, an increase in airway
hyper-responsiveness was seen in only 13 of the
24 participants. When an increase in airway
hyper-responsiveness is not seen after allergen challenge,
it is much less likely that the compound of interest will
have a signifi cant e ect. Additionally, the drug was
administered for only 4 weeks. Even inhaled
corticosteroids have a modest e ect on airway
hyper-responsiveness after 4 weeks.
37
Our own animal
models and those from other laboratories support an
e ect of interleukin 4 and interleukin 13 on airway
hyper-responsiveness.
12,13
However, larger studies will be
needed to determine whether signifi cant improvements
in this endpoint will be seen in human beings.
Pitrakinra was associated with few adverse events in
the three phase 1 (unpublished data) and three phase 2
clinical studies to date, whether administered by
subcutaneous injection (up to 30 mg, for up to 13 weeks)
or by inhalation (up to 60 mg nebulised, for up to 4 weeks)
in participants with atopic asthma or atopic eczema
(133 participants in total). The most common adverse
event that we saw after subcutaneous administration was
injection site-related discomfort, a common event with
most injectable drugs. One should note that the events
were not associated with the development of antibodies,
nor were they associated with any discernible pattern (ie,
they were not more common at the end of the 4 weeks of
exposure). There were also fewer spontaneous asthma
attacks requiring rescue medication in participants who
received pitrakinra subcutaneously than in those in the
placebo group. There were also fewer respiratory-related
adverse events that required rescue medications in the
pitrakinra group than in the placebo group in study 2,
although there were too few events to determine
signifi cance.
The e ects of pitrakinra on late phase asthmatic
response are promising when compared with similar
studies with other successful anti-infl ammatory asthma
therapies, including anti-IgE,
38
leukotriene antagonists,
39
and inhaled corticosteroids.
40
No current class of asthma
drugs has failed in an allergen challenge model only to
succeed in a real world setting. Whether the e ect is due
to inhibition of interleukin 13 alone, or both interleukin 13
and interleukin 4, is not yet known. Future studies of this
drug, as well as molecules that specifi cally inhibit
interleukin 13, in asthmatic individuals of all levels of
severity over longer periods of time are clearly warranted.
Contributors
DW was the principal investigator. All authors contributed to discussions of
protocol design, data analysis, and interpretation of the results of the study.
Confl ict of interest statement
RF, EBG, and ML are all employed by Aerovance Inc. DW is employed by
Quintiles Ltd, who were contracted by Aerovance to do the clinical study.
SW is a consultant for Aerovance Inc and has been paid about US$1500 in
honoraria in the past 3 years for participation in advisory panel discussions.
Acknowledgments
We acknowledge the excellent technical and operational contributions of
everyone involved in these clinical studies, in particular Mahjabeen Lowe
and Noel Landsman. These trials were funded by Aerovance Inc, a
privately held biopharmaceutical company.
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    • "Activation of the type I complex leads to differentiation and proliferation of Th2 cells and activation of type II to develop pathological manifestations such as fibrosis, mucus production and epithelial hyperplasia and IgM /IgE switch (Mueller et al., 2002; LaPorte et al., 2008). As these pathological manifestation are hallmarks of asthma and atopic dermatitis IL-4R has become a therapeutic target and inhibition of IL-4R by the IL-4 variant pitrakinra or inhibitory monoclonal antibody dupilumab has shown efficacy in phase II clinical studies in asthma and atopic dermatitis, respectively (Wenzel et al., 2007; Beck et al., 2014). As UC is also thought to be a Th2 characterized inflammation albeit less clearly defined as in the two other diseases we thought it an attractive idea to use pitrakinra in order to simultaneously test a potential therapeutic benefit. "
    [Show abstract] [Hide abstract] ABSTRACT: Animal models reflective of ulcerative colitis (UC) remain a major challenge and yet are crucial to understand mechanisms underlying the onset of disease and inflammatory characteristics of relapses and remission. Mouse models in which colitis-like symptoms are induced by challenge with toxins such as oxazolone, DSS or TNBS have been instrumental in understanding inflammatory processes of UC, however, they neither reflect the heterogeneous patient population observed in UC nor can they be used when inhibitors require high homology between ligands and receptors. In an attempt to overcome these problems we have developed a mouse model which relies on NOD-SCID IL2rγ(null) mice reconstituted with peripheral blood mononuclear cells derived from patients suffering from UC. Upon challenge with ethanol mice developed colitis-like symptoms and changes of the colon architecture characterized by influx of inflammatory cells, edema, crypt loss, crypt abscesses and epithelial hyperplasia as previously observed in immune-competent mice. TARC, TGFß1 and HGF expression increased in distal parts of the colon. Analysis of human leucocytes isolated from mouse spleen revealed an increase in frequencies of CD1a+ -, CD64+ -, CD163+ -, and TSLPR+ CD14+ monocytes and antigen-experienced CD44+ CD4+ - and CD8+ T-cells in response to ethanol. Analysis of human leucocytes from colon of challenged mice identified CD14+ monocytes and CD11b+ monocytes as predominant populations. RTPCR analysis from distal parts of the colon indicated that IFNγ might be one cytokine driving inflammation. Treatment with infliximab ameliorated symptoms and pathological manifestations whereas pitrakinra had no therapeutic benefit. Thus, this model is partially reflective of the human disease and might help to increase translatability between animal- and clinical studies.
    Full-text · Article · Aug 2016
    • "Despite the positive effect of CHF6001 on the primary endpoint (LAR), no consistent effect on secondary endpoints, including induced sputum and methacholine challenge conducted after allergen challenge was observed. However, the study was not powered for these secondary endpoints, and many other previous allergen challenge studies of novel therapies have also shown positive efficacy on the primary LAR endpoints but failed to show efficacy on secondary endpoints such as methacholine challenge [8,10,17,18,23]. The FEV 1 measurements on days 4 and 9 (before allergen challenge ) showed no change compared to baseline. "
    [Show abstract] [Hide abstract] ABSTRACT: CHF6001 is an inhaled phosphodiesterase 4 (PDE4) inhibitor in development for the treatment of obstructive lung diseases. We investigated the efficacy and safety of CHF6001 using the allergen challenge model in a double blind, placebo controlled, 3-way cross-over study. Thirty six atopic asthmatics who were not taking inhaled corticosteroids and who demonstrated a late asthmatic response (LAR) to inhaled allergen at screening were randomised to receive CHF6001 400 μg or 1200 μg or placebo administered once a day using a dry powder inhaler. The three treatment periods were 9 days; allergen challenges were performed on day 9 and induced sputum was obtained after 10 h from challenge. Washout periods between treatments were up to 5 weeks. Both CHF6001 doses significantly attenuated the LAR; the primary endpoint analysis showed that CHF6001 400 μg and 1200 μg caused reductions of 19.7% (p = 0.015) and 28.2% (p < 0.001) respectively of the weighted FEV1 AUC4-10h compared with placebo. The difference between the CHF6001 doses was not statistically significant (p = 0.2). Compared with placebo, CHF 6001 caused greater reduction in sputum eosinophil counts, although these changes were not statistically significant. CHF6001 was well tolerated, with similar numbers of adverse events in each treatment period. This inhaled PDE4 inhibitor has the potential to provide clinical benefits in patients with atopic asthma.
    Full-text · Article · Jun 2016
    • "This IL-4DM modify the endogenous IL-4 (wild type) for interacting with receptor. It is synthesized as receptor antagonist and beneficial for treating eczema and eosinophillic asthma [144,145]. Viswandham with his colleagues developed IL-4DM by chemical mutation of hot spot residues which is highly potent for decreasing immunogenicity, and served an additional benefit of remarkable pharmacokinetics in-vivo [146]. e) Small molecular inhibitors As proteins have supremacy of evolution-selected specificity that causes little applicability of pharmaceutical agents. "
    [Show abstract] [Hide abstract] ABSTRACT: Studies on Interlukin-4 (IL-4) disclosed great deal of information about its various physiological and pathological roles. All these roles depend upon its interaction and signaling through either type-I (IL-4Rα/common γ-chain) or type-II (IL-4Rα/IL-13Rα) receptors. Another cytokine, IL-13, shares some of the functions of IL-4, because both cytokines use a common receptor subunit, IL-4Rα. Here in this review, we discuss the structural details of IL-4 and IL-4Rα subunit and the structural similarities between IL-4 and IL-13. We also describe detailed chemistry of type-I and type-II receptor complexes and their signaling pathways. Furthermore, we elaborate the strength of type-II hetero dimer signals in response to IL-4 and IL-13. These cytokines are prime players in pathogenesis of allergic asthma, allergic hypersensitivity, different cancers, and HIV infection. Recent advances in the structural and binding chemistry of these cytokines various types of inhibitors were designed to block the interaction of IL-4 and IL-13 with their receptor, including several IL-4 mutant analogs and IL-4 antagonistic antibodies. Moreover, different targeted immunotoxins, which is a fusion of cytokine protein with a toxin or suicidal gene, are the new class of inhibitors to prevent cancer progression. In addition few small molecular inhibitors such as flavonoids have also been developed which are capable of binding with high affinity to IL-4Rα and, therefore, can be very effective in blocking IL-4-mediated responses.
    Full-text · Article · Apr 2016
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