Conditions: Emphysema
Keywords: Emphysema Prostaglandin E Bronchoalveolar lavage
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 118
Healthy volunteers: yes

Brief summary:
The Prostaglandin Inhibition for Emphysema (PIE) study will determine if a currently available therapy, ibuprofen 600 mg three times daily, can block PGE production in the lower respiratory tract and if this results in improvement in measures of lung repair function.

Detailed description:
The Prostaglandin Inhibition for Emphysema (PIE) study will determine if a currently available therapy, ibuprofen 600 mg three times daily, can block PGE production in the lower respiratory tract and if this results in improvement in measures of lung repair function. This is a proof-of-concept study. The PIE study will set the stage for novel therapy to modify the course of chronic obstructive pulmonary disease (COPD).

COPD is the third leading cause of death in the United States. No currently available treatment can meaningfully restore lung function that is lost in this disease. Emphysema is a major component of COPD and results when lung damage exceeds the ability of the lung to repair. Recent evidence indicates that the repair processes present in the normal lung are deficient in patients with emphysema and that this is due, in part, to suppression of repair by an inflammatory mediator: prostaglandin E (PGE). Currently available therapies can block PGE production, but whether this can be achieved in the lung in COPD is unknown. The PIE study will answer that question.

This will be accomplished by performing a randomized, double blind, placebo-controlled, parallel group study that will compare a widely used and well-tolerated non-steroidal anti-inflammatory drug, ibuprofen 600 mg three times daily, with placebo. PGE will be measured directly in the lower respiratory tract by sampling the lung with the technique of bronchoalveolar lavage. Secondary measures will be made, quantifying PGE in induced sputum and quantifying PGE metabolites in blood and urine. In addition, the current proposal will determine if biochemical measures of lung repair are restored by treatments that block PGE production. Additional outcomes will also be assessed including the effect of treatment on PGD and other eicosanoids and assessing IL-8 and neutrophils in sputum and BAL fluid and selected inflammatory biomarkers present in serum that may be associated with lung function decline. Finally, in an accompanying Ancillary Study, the current proposal will determine if alveolar macrophages over-produce PGE and/or PGD in COPD and will determine if the microRNA miR-146a modulates the production of these prostaglandins, as we have demonstrated for lung fibroblasts. The Ancillary Study will also determine if genetic variation in a miR-146a is related to differential expression.

The proposed research will, therefore, determine if inhibition of PGE production can be achieved in the lung, if this appears to restore lung repair mechanisms and will help determine who would benefit from such a therapeutic approach. This is a highly novel approach to the treatment of emphysema and has the potential to restore lost lung function, a crucial unmet medical need for a major public health problem.

Arms:
arm 1: Placebo three times daily arm 2: Control subjects, no intervention arm 3: 600 mg ibuprofen three times daily for 48 weeks

Interventions: intervention 1: 600 mg ibuprofen three times daily for 48 weeks intervention 2: Placebo three times daily
Intervention descriptions: intervention 1: None intervention 2: None

Protocol document:
Prot_SAP_000.pdf: 
 
 
Rennard, Stephen I. 
 1
 
Appendix I – Rennard RFA-HL-12-22 
 
Prostaglandin Inhibition for Emphysema 
(PIE) 
 
 
 
 
 
Revised 06/05/2015 
 
 
NCT02006576 
 
 


 
 
Rennard, Stephen I. 
 2
       Abstract  
 
 
 
 
 
 
 
 
 
 4 
 
 
1. Background and rationale  
 
 
 
 
 
 
 5 
 
1.1 COPD and emphysema 
 
 
 
 
 
 
 
 
5 
1.2 Tissue repair and emphysema 
 
 
 
 
 
 
 
6 
1.3 Prostaglandin E, tissue repair and emphysema 
1.4 NSAIDs, PGE inhibition and emphysema, observational                           
 
8         studies 
1.5 In vivo assessment of lung fibroblast repair activity 
 
 
 
10 
1.6 PGE inflammation and COPD 
 
 
 
 
 
12 
1.7 Choice of interventional drug  
 
 
 
 
 
13 
1.8 Subject identification and recruitment 
 
 
 
 
 
15 
1.9 Summary of background and preliminary data                   
 
16                            
supporting the concept of the current proposal 
 
2. Objectives and hypotheses 
 
 
 
 
 
 
 
18 
2.1  Primary clinical objective 
 
 
 
 
 
 
18 
2.2  Clinical study hypotheses 
 
 
 
 
 
 
18 
2.3  Ancillary study objectives 
 
 
 
 
 
 
20 
2.4  Ancillary study hypotheses 
 
 
 
 
 
 
20 
 
3. Study design  
 
 
 
 
 
 
 
 
21 
3.1  Overview 
 
 
 
 
 
 
 
 
 
21 
3.2  Enrollment criteria, emphysema subjects  
 
 
 
22 
3.3  Study visits, emphysema subjects  
 
 
 
 
23 
3.4  Enrollment criteria, smoking and ex-smoking control                                         
subjects 
 
 
 
 
 
 
 
 
 
25 
3.5  Enrollment criteria non-smoking control subjects  
 
 
26 
3.6  Study visits, control subjects  
 
 
 
 
 
26 
 
4. Statistical considerations  
 
 
 
 
 
 
28 
4.1  Study design 
 
 
 
 
 
 
 
 
28 
4.2  Sample size considerations  
 
 
 
 
 
28 
4.3  Interim monitoring and stopping rules 
 
 
 
 
30 
4.4  Analysis plans  
 
 
 
 
 
 
 
31 
 
5. Study organization and conduct  
 
 
 
 
 
34 
5.1  Data Coordinating Center 
 
 
 
 
 
 
34 
5.2  Clinical Sites 
 
 
 
 
 
 
 
 
34 
5.3  Radiology Reading Center 
 
 
 
 
 
 
34 


 
 
Rennard, Stephen I. 
 3
5.4  Study procedures manual and data forms  
 
 
 
34 
5.5  Study drug 
 
 
 
 
 
 
 
 
34 
5.6  Study materials  
 
 
 
 
 
 
 
34 
5.7  Quality control measures 
 
 
 
 
 
 
35 
5.8  Subject recruitment 
 
 
 
 
 
 
 
35 
 
6. Regulatory issues  
 
 
 
 
 
 
 
36 
6.1  IRB approvals  
 
 
 
 
 
 
 
36 
6.2  FDA approval  
 
 
 
 
 
 
 
36 
6.3  Study registration 
 
 
 
 
 
 
 
36 
 
7. References  
 
 
 
 
 
 
 
 
37 
 
 
 
 
  
 
 
 
 
 
 
 
 
 


 
 
Rennard, Stephen I. 
 4
Abstract 
 
The Prostaglandin Inhibition for Emphysema (PIE) study will determine if a 
currently available therapy, ibuprofen 600 mg three times daily, can block PGE 
production in the lower respiratory tract and if this results in improvement in measures 
of lung repair function.   This is a proof-of-concept study.  The PIE study will set the 
stage for novel therapy to modify the course of chronic obstructive pulmonary disease 
(COPD).   
COPD is the third leading cause of death in the United States.  No currently 
available treatment can meaningfully restore lung function that is lost in this disease.  
Emphysema is a major component to COPD and results when lung damage exceeds 
the ability of the lung to repair.  Recent evidence indicates that the repair processes 
present in the normal lung are deficient in patients with emphysema and that this is due, 
in part, to suppression of repair by an inflammatory mediator: prostaglandin E (PGE).  
Currently available therapies can block PGE production, but whether this can be 
achieved in the lung in COPD is unknown.  The PIE study will answer that question.   
This will be accomplished by performing a randomized, double blind, placebo-
controlled, parallel group study that will compare a widely used and well-tolerated non-
steroidal anti-inflammatory drug, ibuprofen 600 mg three times daily, with placebo.  PGE 
will be measured directly in the lower respiratory tract by sampling the lung with the 
technique of bronchoalveolar lavage.  Secondary measures will be made, quantifying 
PGE in induced sputum and quantifying PGE metabolites in blood and urine.  In 
addition, the current proposal will determine if biochemical measures of lung repair are 
restored by treatments that block PGE production.  Additional outcomes will also be 
assessed including the effect of treatment on PGD and other eicosanoids and 
assessing IL-8 and neutrophils in sputum and BAL fluid and selected inflammatory 
biomarkers present in serum that may be associated with lung function decline.  Finally, 
in an accompanying Ancillary Study, the current proposal will determine if alveolar 
macrophages over-produce PGE and/or PGD in COPD and will determine if the 
microRNA miR-146a plays modulates the production of these prostaglandins, as we 
have demonstrated for lung fibroblasts.  The Ancillary Study will also determine if 
genetic variation in a miR-146a is related to differential expression.   
The proposed research will, therefore, determine if inhibition of PGE production 
can be achieved in the lung, if this appears to restore lung repair mechanisms and will 
help determine who would benefit from such a therapeutic approach.  This is a highly 
novel approach to the treatment of emphysema and has the potential to restore lost 
lung function, a crucial unmet medical need for a major public health problem. 
 
 
 
 
 
 
 
 
 
 


 
 
Rennard, Stephen I. 
 5
1.  Background and Rationale 
 
1.1 COPD and emphysema.  COPD is currently the third leading cause of death 
in the United States1.   It is characterized by progressive loss of lung function that can 
result either from small airways disease or from emphysema.  Current therapy is largely 
symptomatic. To date, medical treatments have been without meaningful effect in 
slowing lung function loss2-4. 
Smoking is the most important etiologic factor leading to COPD.  Smoking 
cessation improves symptoms, particularly those of bronchitis.  If cessation is achieved 
early enough, disease progression is slowed5. However, once disease is sufficiently 
advanced, progression occurs despite cessation6,7.  This may be particularly true for 
emphysema7,8, which leads to the concept that, in susceptible individuals, smoking 
initiates a process leading to the development of emphysema and that the process 
remains active despite subsequent cessation. 
Abundant animal and human data support the concept that inflammation, which 
is induced by smoking, leads to lung damage and the formation of emphysema3,9.  To 
have tissue loss and emphysema, however, damage must exceed the capacity of the 
lung to repair.   
Several lines of evidence support the concept that compromised tissue repair 
also contributes to the pathogenesis of emphysema10.   
 
1.2 Tissue repair and emphysema.  Inadequate tissue maintenance due to loss 
of growth factors has been demonstrated to lead to emphysema in animal models11.  
Similarly, severe calorie restriction, sufficient to induce weight loss, can cause 
emphysema in animals7,12,13.  Emphysema has been reported in young non-smokers 
with bulimia7,14 and in young individuals who died from starvation7,15.  Finally, in animal 
models, inhibition of repair mechanisms greatly worsens emphysema16-20.  Thus, it is 
likely that impaired repair can contribute to the development of emphysema, both as a 
sole risk factor and as a co-factor potentiating the effects consequent to tissue damage. 
In addition, lung repair following the development of emphysema can be induced 
in animal models.  Retinoic acid, which drives the formation of alveolar septa during 
neonatal development, can induce formation of new alveolar wall in emphysema models 
in the mouse and rat 21,22.  In addition, growth factors, including hepatocyte growth 
factor (HGF) 23,24, granulocyte colony stimulating factor (G-CSF) 25, KGF 26 and 
adrenomedulin 27,28 have all been reported to stimulate new alveolar wall formation 
following the development of emphysema.  
Augmentation of repair mechanisms in the lung, therefore, has the potential to 
both slow the development of emphysema and restore lost function.  Improved repair 
within the lung is a reasonable target, as recent evidence had demonstrated that lung 
repair mechanisms are actively INHIBITED by a pharmacologically targetable 
mechanism.  Specifically, repair processes in fibroblasts, the major lung mesenchymal 
cells responsible for the production and maintenance of extracellular matrix, are 
inhibited by the inflammatory mediator prostaglandin E (PGE), which is produced in 
greater amounts by lung fibroblasts and is present in the lower respiratory tract at higher 
concentrations than in controls.  Inhibition of PGE production, therefore, has the 
potential to restore lung fibroblast repair functions.  This, in turn, could improve the 


 
 
Rennard, Stephen I. 
 6
balance of destruction and repair, thus slowing lung function loss and potentially 
restoring lost function. 
Several lines of investigation have demonstrated reduced repair functions in cells 
cultured from the lungs of patients with emphysema.  Compared to fibroblasts from 
controls, fibroblasts cultured from the lungs of patients with emphysema proliferate 
more slowly29,30, are more sensitive to inhibition by smoke31 and fail to express elastin 
mRNA32 and the growth factors HGF and KGF when stimulated33. We have 
demonstrated that COPD fibroblasts are deficient in their chemotactic response and in 
their ability to contract collagen matrices34. These differences persist with passage in 
culture, consistent with an alteration in cellular phenotype in COPD.  
 
1.3  Prostaglandins, tissue repair and emphysema.  In vitro human cell 
culture studies.  We have also demonstrated a key role for PGE in mediating the 
reduced repair function in fibroblasts from emphysema patients.  PGE is an 
inflammatory mediator that is a potent inhibitor of fibroblast-mediated repair responses35.  
A role for PGE in the modulation of tissue remodeling in the lung has been suggested in 
idiopathic pulmonary fibrosis36,37.  In this condition, PGE levels are reduced in the lower 
respiratory tract, and this has been suggested to contribute to excessive fibroblast-
mediated repair and fibrosis37,38.  The converse may contribute to emphysema.  As 
detailed below, PGE levels are increased in the lungs of COPD patients39-41, which 
would be expected to inhibit fibroblast repair functions.  In addition, we have shown that 
fibroblasts from COPD patients over-produce PGE and that inhibition of fibroblast 
production of PGE can partially correct their repair defects in vitro34.   
Animal studies.  Animal studies also support the potential for inhibition of PGE 
production as a therapy for emphysema.  COX inhibition can reduce smoke-induced 
inflammation in the mouse42.  More importantly, inhibition of PGE production in smoke-
exposed rats results in reduced severity of emphysema43. 
Human studies.  PGE levels.  Although published data are limited, three studies 
have demonstrated increased PGE levels in the lower respiratory tract in COPD. 
Monuschi et al. reported increased PGE levels in exhaled breath condensate of 
subjects with COPD39.  Chen et al.40 and Profita et al.41 have reported increased PGE 
levels in induced sputum from patients with COPD.  Chen also reported a significant 
relationship between PGE levels and reduced FEV1, although the number of subjects 
evaluated, 45, was relatively small, resulting in large confidence intervals in the 
estimated relationship.  While these studies demonstrate increased PGE in the lung, 
they do not obviously assess the alveolar compartment.  However, we have utilized the 
technique of bronchoalveolar lavage (BAL), which does sample the alveolar space.  
Moreover, we have used the method of fractional BAL processing 44.  When 20 ml 
aliquots are sequentially infused and immediately aspirated, the first aliquot is relatively 
enriched for bronchial material, while the subsequent are enriched for alveolar material.  
By separately processing these samples, partial separation of alveolar and bronchial 
samples can be obtained.  With this method, we demonstrated that “bronchial” samples 
have higher PGE concentrations than “alveolar” but that for both, COPD patients have 
increased PGE compared to normals (Figure 1).  This study thus confirms the published 
results and specifically demonstrates that alveolar PGE concentrations are increased 


 
 
Rennard, Stephen I. 
 7
compared to control.  In addition, even though the number of subjects in our study was 
also limited, we also confirmed the association of PGE level with FEV1 (p=0.0285). 
 
 
 
 
Figure 1.   Bronchoalveolar lavage was performed as described44,45 with five serial 
20 ml aliquots, which allows partial separation of the “alveolar” and “bronchial” 
contents.  Levels were four- to five-fold higher in the bronchial fractions.  BAL 
PGE concentration was greater in both alveolar and bronchial fractions in 
patients with COPD.  Note that the vertical axis is a log scale.  *p<0.05.  
 
The cellular source(s) of the increased PGE in COPD is(are) undetermined.  We 
have demonstrated that fibroblasts cultured from the lungs of patients with COPD 
produce more PGE than do fibroblasts from control subjects34,46.  In addition, PGE 
production is inversely related to airflow and to the diffusion capacity, which is a 
measure of the severity of emphysema. Importantly, inhibition of PGE production in the 
COPD fibroblasts partially restores repair functions34, confirming the role of PGE and 
suggesting that improving repair functions is a reasonable therapeutic goal.   
We also demonstrated that COPD fibroblasts under-express the microRNA miR-
146a46.  This microRNA targets the PGE biosynthetic enzyme cyclo-oxygenase-2 (COX-
2).  We demonstrated that under-expression of miR-146a leads to over-expression of 
COX-2 and contributes to over-production of PGE in COPD fibroblasts, thus 
establishing its mechanistic role46.  Detailed sequencing of the miR-146a gene from 40 
fibroblast strains (17 controls and 23 COPD patients), including 5000 bp of the 5’ 
regulatory region, exons 1 and 2 and 2000 bp of the 3’ untranslated region revealed that 
polymorphisms in the gene were relatively common. We found 24 polymorphisms, 23 of 
which were novel, which had a minor allele frequency greater than 5%. Importantly, 


 
 
Rennard, Stephen I. 
 8
fibroblasts with the promoter polymorphism miR146a/-3147AG/GG under-expressed 
miR-146a compared to cells with the AA genotype.  In a population of 280 COPD 
subjects and controls, this same promoter polymorphism was found to be associated 
with a decreased FEV1/FVC ratio (p=0.01).  Interestingly, this polymorphism affected a 
putative AP1 transcription factor binding site at miR146a/-3147A that was disrupted with 
risk allele G.  These pilot data, therefore, strongly support the concept that 
polymorphisms in the miR-146a gene may be related to COPD 47.  
Both fibroblasts and alveolar macrophages are major sources of PGE in the 
lung35,48,49.  Whether macrophages contribute to the increased production of PGE in 
COPD patients will be addressed in the Ancillary Study (see Ancillary Study).  In 
addition, whether miR-146a modulates PGE production in alveolar macrophages and 
whether polymorphisms in the miR-146a gene predict PGE levels in the lung and 
production by alveolar macrophages will also be assessed.  Thus, the Ancillary Study 
will advance understanding of the mechanisms that underlie increased PGE levels in 
the lower respiratory tract in COPD.  In addition, the Ancillary Study will explore a 
potentially diagnostic biomarker (miR-146a gene polymorphisms) that has the potential 
to identify individuals at risk for increased PGE levels.  
 
1.4  NSAIDs, PGE inhibition and emphysema, observational studies.  
Currently available medications can inhibit PGE production.  PGE is derived from 
membrane phospholipids in a three-step process50.  First, a phospholipase A2 cleaves 
arachidonic acid from phospholipid.  Second, arachidonic acid is oxidized to form PGG 
then PGH by a cyclo-oxygenase (COX).  Finally, PGH is converted to PGE by a 
prostaglandin E synthase (PGES).  Several distinct enzymes can mediate each step.  In 
addition, at each step, other enzymes can produce alternate products.  
The most commonly used inhibitors of PGE production are COX inhibitors, 
sometimes termed non-steroidal anti-inflammatory drugs (NSAIDs)50.    Non-selective 
agents that inhibit both COX enzymes, COX-1 and COX-2, and agents that are 
selective for COX-2 are in use clinically.  They differ in their adverse side effects, which 
are mediated by several mechanisms.  These include inhibition of COX in cells other 
than the desired target cell, effects due to inhibition of COX- derived mediators other 
than PGE and, possibly, to shunting of substrate to alternate products.   More selective 
inhibition of PGE production is possible as specific inhibitors of PGE synthases have 
been developed, although none are available clinically.  Which PGE synthase(s) are 
most important in COPD, however, is as yet undetermined.  Thus, it is possible that a 
selective PGE synthase inhibitor may be of use clinically.  However, non-selective 
inhibition of PGE production provides a more robust test for the proof-of-concept that 
PGE can be targeted to treat emphysema.  Interestingly, while limited, several 
observational studies have related NSAID use to features of COPD, and these studies 
provide observations supporting the concept that NSAIDs may reduce the severity of 
COPD in general and emphysema specifically. 
McKeever and colleagues reviewed the NHANES dataset and observed that 
users of the non-selective NSAID ibuprofen had significantly better lung function, i.e. 
higher FEV1, than did non-users51.  Improved lung function was observed among 
occasional users of ibuprofen.  No clear dose response effect was observed in this trial, 
in part because there were relatively few daily users.  Observational studies with a 


 
 
Rennard, Stephen I. 
 9
therapy will be substantially confounded by selection bias: those using NSAIDs on a 
regular basis are likely to differ in a number of respects from those who use 
occasionally or not at all. Nevertheless, the observation that ibuprofen use is associated 
with better lung function provides suggestive information supporting the premise of the 
current proposal.  
The improved lung function associated with ibuprofen observed by McKeever 
and colleagues was not observed with acetaminophen51.   While acetaminophen is also 
a non-selective COX inhibitor, it does not have a marked anti-inflammatory effect.  This 
is thought to be due to blockade of its COX inhibitory effect by high concentrations of 
peroxides present in an inflammatory milieu50.  Since persistent inflammation is a 
characteristic feature of the lung in COPD, it is likely that acetaminophen would be less 
effective than ibuprofen.  
The ECLIPSE study is an observational study that included 2165 COPD subjects 
who underwent spirometry of whom 1729 had CT scans52.  Many reported NSAID use 
on entry.  Consistent with the results reported by McKeever51, NSAID use was 
marginally associated with better lung function assessed as post-bronchodilator FEV1 
(p=0.09).  Because CT scans were done, it was possible to assess emphysema.  
Patients with COPD had greater emphysema than controls, as reflected by lower lung 
density (data not shown).  Emphysema in COPD patients was more marked in the 
upper lobes, as would be expected (Figure 2).  Data from ECLIPSE also suggest that 
NSAID use may ameliorate the development of emphysema.  Specifically, NSAID users 
had significantly greater lung density suggesting less emphysema bilaterally in the lower 
lobes (Figure 2).   
 
Figure 2.  Use of NSAIDs is associated with reduced lower lobe emphysema.  
Data were evaluated from the ECLIPSE study52.  Quantitative CT scan was 
0.12 
0.13 
0.14 
0.15 
0.16 
Mean Lung Density 
Users!
Non-Users!
Upper!
Lower!
Middle!
Right Lung Lobes!
Upper!
Lower!
Left Lung Lobes!
*!
*!


 
 
Rennard, Stephen I. 
 10 
performed on 1729 subjects enrolled in the trial.  At the time of entry, 427 
reported using NSAIDs on a regular basis.  CT scan density was quantified as a 
measure of emphysema53-55.  Those who reported using NSAIDs (clear bars) had 
significantly greater density (less emphysema) in the lower lobes than those not 
using NSAIDs (grey bars).  * p < 0.05. 
 
The mean age of COPD subjects of all severities in ECLIPSE was about 63 
years.  While it is likely that NSAID use began several years before enrollment, it is 
highly likely that cigarette smoking and the development of emphysema preceded the 
use of NSAIDs by several decades.  Thus, an effect of NSAIDs on the lower lobes, 
where emphysema is less severe, is consistent with an effect in mitigating an early 
stage in the disease pathogenesis.  This might be the expected effect of improving 
repair, which may be able to sustain or repair lung structure in the face of mild injury, 
but may be much less effective in the presence of severe lung destruction.  
 
1.5  In vivo assessment of lung fibroblast repair activity.  The studies noted 
above demonstrate that PGE levels are increased in COPD, that fibroblast repair 
processes are inhibited by COPD, that inhibition of PGE production can improve 
fibroblast repair functions in vitro and can mitigate emphysema in animal models.  The 
current proposal is a proof-of-concept study designed to determine if inhibition of PGE 
can be achieved in the lung of patients with emphysema as a means to restore repair 
functions.  This concept can be tested by quantification of PGE levels following use of 
treatments that can inhibit PGE production and that are available for clinical use.  It is 
also possible, in this proof-of-concept study, to obtain information that inhibition of PGE 
production can restore repair functions. 
Collagen is a major component of extracellular matrix, and fibroblasts are the 
major source56,57.  Collagen is synthesized as a precursor, procollagen, that has 
terminal non-triple helical regions.  These must be cleaved so that the triple helical 
portion can form mature collagen fibers, a process that takes place after collagen is 
secreted into the extracellular space.  Because of this, the procollagen terminal peptides 
can serve as a marker of collagen synthesis.  This biomarker has proved very useful in 
gauging bone synthesis58-60.  It has also proved useful as a measure of collagen 
production in the lung.  A number of investigators61-63, including ourselves64,65, have 
demonstrated increases in procollagen peptide associated with interstitial lung disease, 
which is believed to reflect increased collagen production.  Similar increases have been 
reported following the development of ARDS66-68.  
Measurement of procollagen peptide, therefore, can provide a measure of 
fibroblast activity.  Inhibition of PGE production would be predicted to increase collagen 
production and generation of procollagen peptides.  To demonstrate the feasibility of 
this approach, we quantified one of the procollagen peptides, pro-collagen I C-terminal 
peptide in the alveolar fraction of BAL from COPD patients and controls.  This study not 
only confirms the ability to quantify pro-collagen peptide in BAL fluid of COPD patients, 
but levels in COPD patients were significantly reduced compared to controls, consistent 
with the concept of inhibited repair (Figure 3). 
 
 


 
 
Rennard, Stephen I. 
 11 
 
 
 
 
 
 
 
 
 
 
 
Figure 3.  Pro-collagen I C-terminal peptide levels in COPD.  BAL samples 
(alveolar fraction) from COPD subjects and normal controls were evaluated for 
pro-collagen I C-terminal peptide by ELISA.  Levels were significantly lower 
(Mann-Whitney, two-tailed, p=0.012) in the COPD subject BAL fluids compared to 
control, although there was considerable overlap.  Values were undetectable in a 
substantial number of subjects (this does not affect the rank order non-
parametric statistical test used to compare the groups). 
 
 
 
 
 
 
 
 
 
 


 
 
Rennard, Stephen I. 
 12 
 
 
 
 
1.6  PGE and inflammation in COPD.  COPD is an inflammatory disease 
characterized by increased numbers of macrophages and neutrophils in the lower 
respiratory tract3.  Associated with these cells, several chemotactic factors, including 
interleukin-8 (IL-8), are also increased in COPD69,70. PGE is an inflammatory mediator 
that is increased in COPD.  Interestingly, we have observed that PGE levels are 
significantly related to IL-8 levels in both the bronchial and alveolar portions of BAL fluid 
(Figure 4).   
 
Figure 4.  Bronchoalveolar lavage PGE and IL-8 levels.  BAL was performed in 
patients with COPD with 5 X 20 ml aliquots as described44,45 and the first aliquot, 
enriched for bronchial material, processed separately from the subsequent four 
aliquots, enriched for alveolar material.  PGE and IL-8 levels are higher in the 
bronchial portion than the alveolar, but correlate significantly in both portions. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


 
 
Rennard, Stephen I. 
 13 
 
 
One interesting possibility is that increased PGE levels in COPD may contribute, in part, 
to driving the increased levels of IL-8.  In support of this concept, we have demonstrated 
that PGE can stimulate IL-8 production from fibroblasts (Figure 5).   
 
Figure 5.  PGE stimulates IL-8 production by human lung fibroblasts.  Human 
lung fibroblasts (HFL-1) were cultured to confluence in vitro and stimulated with 
PGE for 24 hours.  PGE stimulation resulted in a concentration-dependent 
induction of IL-8 production.  Similar results were obtained with adult lung 
fibroblasts.  *p<0.05; **p<0.01.   
 
While the current study will focus on restoration of repair functions, reduction in 
inflammation could also be beneficial in COPD, and its demonstration would be a 
measure of a pharmacodynamic drug effect.  Moreover, reduced inflammation can 
provide an independent measure of proof-of-concept that PGE inhibition could be 
beneficial in emphysema. 
1.7  Other prostaglandins.  Prostaglandins other that PGE may also play a role 
in COPD.  Prostaglandin D2 (PGD) is present in the lower respiratory tract and has 
been related to recruitment of inflammatory cells including lymphocytes and 
eosinophils71,72.  IBlockade of the D-prostaglandin (DP) receptor reduced inflammation 
in smoke exposed mice73.  PGD also has the potential to modulate fibroblast-mediated 
repair responses 74,75.  Whether PGD or other prostanoids might also contribute to 
altered repair in emphysema is unknown. Montuschi et al. observed no difference in 
PGD levels in exhaled breath condensate in contrast to the increased levels of PGE that 
were observed 76.  The current study will determine if PGD and other prostanoid levels 
are altered in the lungs of COPD patients using BAL and induced sputum. 
 


 
 
Rennard, Stephen I. 
 14 
 
 
1.8  Choice of interventional drug.  Drug choice.  Several considerations have 
influenced the choice of interventional drug for the current proof-of-concept study, which 
is designed to determine if PGE production can be inhibited effectively in the lower 
respiratory tract and if this is associated with biomarkers suggestive of improved tissue 
repair and reduced inflammation.  To this end, a non-selective COX inhibitor is more 
likely to be effective, as both COX enzymes may play a role.  While subsequent studies 
may demonstrate advantages for more selective approaches, the approach used is 
appropriate for the initial proof-of-concept study.  In this regard, a variety of NSAIDs that 
vary in their selectivity for COX-1 or COX-2 are in clinical use50.  COX-2 selective 
agents have reduced risk of gastrointestinal side effects, but are associated with an 
increase in cardiac events.    
Ibuprofen is a widely used, non-selective COX inhibitor.  It is not associated with 
an increased risk for cardiac events77,78, although risks may be greater among 
individuals with a history of myocardial infarction79.  Ibuprofen can cause gastritis, but 
monitoring for this adverse event is possible by following symptoms, stool blood and 
hemoglobin levels.  Naproxen is also a non-selective COX inhibitor that is slightly more 
selective for COX-1.  It has a reduced cardiovascular risk compared to COX-2 selective 
agents, but an increased risk of gastrointestinal side effects79,80. It has a much longer 
half-life than ibuprofen and is used clinically on a twice-daily regimen.  In contrast, 
ibuprofen is often dosed four times daily.  However, twice daily dosing with ibuprofen 
has been used in cystic fibrosis and has resulted in significant lowering of inflammatory 
measures81 and slowed deterioration in lung function82,83.  In addition, the effect of 
conventional doses of ibuprofen has been assessed on BAL levels of PGE; in contrast, 
to our knowledge, there are no data on the effect of naproxen in the lung.  For these 
reasons, ibuprofen has been selected for use in the current short-term, proof-of-concept 
trial. 
 
Dose.  Like most NSAIDs, ibuprofen is a competitive inhibitor of arachidonic acid 
oxidation50.  As such, at clinical doses, NSAIDs do not completely prevent the formation 
of eicosanoids, and their degree of inhibition is concentration dependent.  After single 
doses of 400 and 600 mg, ibuprofen resulted in a 10 and 15% reduction in the blood 
level of the PGE metabolite 13,14-dihydro-15-keto-PGE 2 (PGEM)84.  The effects may 
be greater with chronic dosing or with assessments over longer time frames.  McAnulty 
and colleagues assessed marathon runners who were chronic users of ibuprofen 
compared to non-users85.  Although chronic dosing was not controlled, users took 600 
mg the evening before and 200 mg the morning of a race and then 200 mg every four 
hours.   Urinary levels of PGEM were about 50% of the levels observed in non-users 
both before and after running (treatment effect p=0.016)85.   
Pulmonary effects.  Effects of ibuprofen may be greater in the lung.  Hazucha 
and colleagues measured PGE levels in bronchoalveolar lavage (BAL) fluid before and 
after exercise in normal men while breathing ozone86.  Pretreatment with 800 mg 
ibuprofen followed by 200 mg during the exercise reduced PGE levels by 60% 
(p<0.05)86.   Similarly, in a COPD population, Montuschi et al. reported a 77% reduction 
(p<0.0001) in exhaled breath condensate PGE following treatment with 400 mg 
ibuprofen every six hours for two days87.    


 
 
Rennard, Stephen I. 
 15 
 
Because of its anti-inflammatory effect, ibuprofen has been tested in patients with 
cystic fibrosis.  Reduction in the rate of decline of FEV1 has been observed in two 
placebo-controlled trials82,83.  Both studies employed relatively high doses of ibuprofen 
(20-30 mg/kg), but the drug was administered twice daily.  Konstan and colleagues 
have also demonstrated a relationship between neutrophil recruitment in vivo and blood 
levels81.  With twice daily administration, reduction in neutrophil recruitment was 
observed in both normal volunteers and cystic fibrosis patients, but only when the Cmax 
exceeded 50 ug/ml.  None of these studies quantified eicosanoid production. 
 
Rationale for current study design.  The current study is a proof-of-concept 
study to demonstrate inhibition of PGE production and associated biological effects in 
the lower respiratory tract.  Because ibuprofen is a competitive inhibitor, at conventional 
doses, it only partially inhibits PGE production.  Nevertheless, at doses that are within 
usual clinical practice, reduction in lower respiratory tract PGE levels by greater than 
50% seems a reasonable target85-87.  In addition, at these doses, ibuprofen has been 
observed to reduce the rate of lung function loss in cystic fibrosis.  It is likely that the 
effect in cystic fibrosis is due to an effect on airways inflammation.  While not the 
primary goal of the present trial, this provides evidence that is helpful in confirming the 
choice of dose and in establishing tolerability for treatment in patients with lung disease 
and provides support for an important secondary outcome measure.   
Based on this prior experience, the current trial will use ibuprofen at a dose of 
600 mg three times daily vs. placebo. The primary analysis will be by intention-to-treat.  
However, blood levels will be determined after four weeks of use one to two hours after 
dosing.  This will not be used as a measure of compliance, as ibuprofen has a short 
half-life.  However, it will be used in a secondary analysis to evaluate variable 
absorption or metabolism, which may be particularly important in a proof-of-concept trial.  
Because of the logistical complexity that would require dummy adjustments in the 
placebo and the generally good response to clinical doses of ibuprofen, a dose 
adjustment strategy was not felt warranted. 
 
1.9  Subject identification and recruitment. A major limitation to the study of 
interventions affecting repair in emphysema has been access to subjects with relatively 
limited disease.  Because COPD is diagnoses relatively late in the course, and because 
emphysema is not specifically detected by spirometry, individuals with mild disease are 
most often undiagnosed and, in most COPD patients, the extent of emphysema is 
unknown.  The current study will specifically enroll subjects with emphysema who do 
not have severe or very severe COPD.  This has several important theoretical and 
practical advantages.  First, it is likely that improvement in repair can mitigate 
emphysema progression more if the disease is not markedly advanced.  In addition, 
restoration of lost structure and function may be more readily achieved if abnormalities 
are not severe.  That is, it may be easier to fix a mild anatomic defect rather than a 
severe one. The current study is possible because of the opportunity of accessing a 
previously identified cohort of appropriate study subjects from the COPDGene Study.  


 
 
Rennard, Stephen I. 
 16 
COPDGene (NCT00608764) is an NIH-funded study of more than 10,000 
subjects who have undergone lung function measurement, CT scanning and collection 
of blood for DNA analysis88.  Subjects have provided consent to be recontacted, and 
several studies have been initiated that utilize this cohort (e.g. NCT01253473).  The four 
clinical centers participating in the current proposal are all COPDGene centers.  The 
anticipated number of subjects at each center with emphysema (>5% of voxels <-950 
Hounsfield Units) for each level of COPD severity is shown in Table 1. A telephone 
screen of 10 subjects was able to contact nine, and seven of these expressed interest in 
participation.  Thus, the available population of 919 potential subjects should represent 
an adequate pool of potential subjects for the current proposal.  If needed, it would be 
possible to augment these numbers with GOLD 3 subjects. 
 
Another advantage of recruiting COPDGene subjects is that, when possible, 
study protocols will use COPDGene methods.  This will greatly simplify training and 
quality control, which have already been implemented at each study site as part of 
COPDGene. 
 
 
 
1.10  Summary of background and preliminary data supporting the concept 
of the current proposal.  COPD is the third leading cause of death in the United States.   
It progresses despite smoking cessation, and this appears to be particularly true for 
emphysema.  No currently available therapy has been demonstrated to meaningfully 
slow disease progression or to restore lost lung function.  The destruction of lung tissue 
that characterizes emphysema results from tissue damage that exceeds the capacity of 
tissue repair.  Both play roles in emphysema.  Importantly, impaired mesenchymal cell, 
i.e. fibroblast, repair functions result, in part, from over-production of PGE.  In vitro and 
animal studies suggest that inhibition of PGE can restore repair functions to COPD 
fibroblasts from human patients and can ameliorate the development of emphysema in 
animal models.  In addition, data from human observational studies suggest that the use 
of NSAIDs, which can inhibit PGE production, is associated with reduced severity of 
emphysema.  PGE, moreover, may contribute to the chronic inflammation that 
characterizes COPD, the inhibition of which could be of therapeutic benefit.  Moreover, 


 
 
Rennard, Stephen I. 
 17 
pro-collagen peptide, a biochemical measure that reflects tissue repair, has been used 
in several clinical settings, including lung disease, and its use to gauge fibroblast-
mediated repair activity in emphysema is practicable.  Finally, the appropriate 
population for a proof-of-concept study, individuals with definite emphysema but with 
mild or moderate COPD, has been identified. 
Taken together, these observations suggest that inhibition of PGE production has 
the potential to slow the development of emphysema and potentially may allow tissue 
repair to correct anatomic defects and that a proof-of-concept study is readily feasible.    
 


 
 
Rennard, Stephen I. 
 18 
2.  Objectives and hypotheses 
 
 
2.1 Primary clinical objective.  Previous studies support the concept that 
impaired lung repair contributes to the pathogenesis of emphysema and that PGE 
present in the lower respiratory tract inhibits repair function of lung mesenchymal cells, 
i.e. fibroblasts.  The primary goal of the current proposal is to test, as a proof-of-concept, 
that inhibition of PGE production will reduce PGE levels in the lower respiratory tract of 
patients with emphysema and will this lead to increased biochemical markers of tissue 
repair.  This will be tested by intention-to-treat comparing the ibuprofen-treated subjects 
with placebo treated subjects and is essential to demonstrate prior use of PGE inhibition 
strategies as therapy to slow disease progression or to restore lung function in 
emphysema.  Several secondary questions will also be pursued. 
 
2.2  Clinical study hypotheses 
 
Primary hypothesis:  
H1: Will ibuprofen, 600 mg three times daily, decrease PGE concentrations in the 
alveolar portion of BAL fluid in subjects with emphysema in comparison to placebo? 
 
H2: Will ibuprofen, 600 mg three times daily, increase pro-collagen peptide 
fragment concentrations in the alveolar portion of BAL fluid in subjects with emphysema 
in comparison to placebo? 
 
 
Other hypotheses: 
 
Effects on PGE 
 
H3: Will ibuprofen, 600 mg three times daily, reduce prostaglandin E 
concentrations in the alveolar portion of BAL fluid in subjects with emphysema to those 
present in smoking, former smoking and healthy non-smoking controls? 
 
H4:  Will ibuprofen 600 mg three times daily, reduce prostaglandin E 
concentrations in the bronchial portion of BAL fluid in subjects with emphysema 
compared to placebo?   
H4.1  Will levels reduce to those present in former smoking and healthy 
non-smoking controls? 
 
H5:  Will ibuprofen 600 mg three times daily, reduce prostaglandin E 
concentrations in induced sputum from subjects with emphysema compared to 
placebo?   
H5.1  Will levels reduce to those present in former smoking and healthy 
non-smoking controls? 
 
H6: Will ibuprofen 600 mg three times daily, reduce the PGE metabolite PGEM in 
the peripheral blood of subjects with emphysema compared to placebo?   


 
 
Rennard, Stephen I. 
 19 
H6.1  Will levels reduce to those present in former smoking and healthy 
non-smoking controls? 
 
H7: Will ibuprofen 600 mg three times daily, reduce the PGE metabolite PGEM in 
the urine of subjects with emphysema compared to placebo?   
H7.1  Will levels reduce to those present in former smoking and healthy 
non-smoking controls? 
 
 
Effects on biomarkers of repair 
 
H8: Will ibuprofen, 600 mg three times daily, increase pro-collagen peptide 
fragment concentrations in the alveolar portion of BAL fluid in subjects to the range 
observed in normal individuals or to higher levels? 
 
H9: Will ibuprofen 600 mg three times daily, increase the procollagen III amino 
peptide excreted into the urine of subjects with emphysema compared to placebo? 
 
 
Effects on inflammation 
H10: Will ibuprofen, 600 mg three times daily, decrease IL-8 concentrations in 
the alveolar portion of BAL fluid in subjects with emphysema in comparison to placebo?  
 
 
H10.1:  Will levels be reduced to those observed in former smoking and 
healthy non-smoking controls? 
 
H11: Will ibuprofen, 600 mg three times daily, decrease neutrophil 
concentrations in the alveolar portion of BAL fluid in subjects with emphysema in 
comparison to placebo?   
H11.1  Will levels be reduced to those observed in former smoking and 
healthy non-smoking controls? 
 
H12: Will ibuprofen 600 mg three times daily, reduce IL-8 concentrations in the 
bronchial portion of BAL fluid in subjects with emphysema compared to placebo?   
H12.1  Will levels reduce to those present in former smoking and healthy 
non-smoking controls? 
 
H13: Will ibuprofen 600 mg three times daily, reduce IL-8 concentrations in 
induced sputum from subjects with emphysema compared to placebo?   
H13.1  Will levels reduce to those present in former smoking and healthy 
non-smoking controls? 
 
H14: Will ibuprofen 600 mg three times daily, reduce PMN concentrations in the 
bronchial portion of BAL fluid in subjects with emphysema compared to placebo?   
H14.1  Will levels reduce to those present in former smoking and healthy 
non-smoking controls? 
 


 
 
Rennard, Stephen I. 
 20 
H15: Will ibuprofen 600 mg three times daily, reduce PMN concentrations in 
induced sputum from subjects with emphysema compared to placebo?   
H12.1  Will levels reduce to those present in former smoking and healthy 
non-smoking controls? 
 
 
 
H16:  Will ibuprofen 600 mg three times daily, alter LTB4 concentrations in 
subjects with emphysema compared to placebo? 
 
 
H16.1:  Alveolar portion of BAL 
 
 
H16.2:  Bronchial portion of BAL 
 
 
H16.3   Induced sputum 
 
 
H17: Will ibuprofen 600 mg three times daily, reduce concentrations of CC-16 in 
serum from subjects with emphysema compared to placebo? 
 
 
H18: Will ibuprofen 600 mg three times daily, reduce concentrations of CRP in 
serum from subjects with emphysema compared to placebo? 
 
H19: Will ibuprofen 600 mg three times daily, reduce concentrations of SP-d in 
serum from subjects with emphysema compared to placebo? 
 
 
Other prostanoids  
 
H20:  Are levels of PGD increased in the alveolar component of BAL fluid in 
patients with COPD. 
 
 
H20.1  Are levels of PGD in alveolar lavage fluid related to FEV1 
 
 
H20.2  Are levels of PGD in alveolar lavage fluid related to severity 
of emphysema 
 
 
H 21. Are levels of TXB2 (the primary metabolite of thromboxane) increased in 
the alveolar component of BAL fluid in patients with COPD. 
H21.1  Are levels of TXB2 (the primary metabolite of thromboxane) in 
alveolar lavage fluid related to FEV1 
H21.2  Are levels of TXB2 (the primary metabolite of thromboxane) in 
alveolar lavage fluid related to severity of emphysema 
 
H22:  Are levels of 6kPGF1a (the primary metabolite of prostacyclin) increased in 
the alveolar component of BAL fluid in patients with COPD. 
H22.1  Are levels of 6kPGF1a (the primary metabolite of prostacyclin) in 
alveolar lavage fluid related to FEV1 
H22.2  Are levels of 6kPGF1a (the primary metabolite of prostacyclin) in 
alveolar lavage fluid related to severity of emphysema 
 
H23: Will ibuprofen 600 mg three times daily, reduce the eicosanoids other than 
PGE in subjects with emphysema compared to placebo in BAL and induced sputum?   
H23.1 PGD 
H23.2 TXB2 (the primary metabolite of thromboxane) 


 
 
Rennard, Stephen I. 
 21 
H23.3 6kPGF1a (the primary metabolite of prostacyclin) and LTB4 in the  
bronchial and alveolar portions of BAL  
 
 
Exploratory clinical measures 
 
H24: Will ibuprofen 600 mg three times daily alter the rate of change of 
emphysema progression as assessed by CT scan? 
 
 
H25: Will ibuprofen 600 mg three times daily alter the rate of change of DLCO? 
 
H26: Will ibuprofen 600 mg three times daily alter the rate of change of FEV1? 
 
H27:  Will ibuprofen 600 mg three times daily alter the rate of COPD 
exacerbations? 
 
H28:  Will ibuprofen 600 mg three times daily, alter health status assessed by the 
SGRQ or COPD status assessed by the CAT in subjects with emphysema  
 
 
Emphysema phenotype 
 
H29:  Are there differences between subjects with upper lobe vs. lower lobe 
emphysema 
 
 
H29.1  Do PGE levels in BAL fluid, sputum  and metabolites in blood and 
urine differ between subjects with upper lobe vs. lower lobe emphysema 
 
 
H29.2 Do procollagen peptides differ in BAL fluid between subjects with 
upper lobe vs. lower lobe emphysema 
 
 
H29.3  Do their eicosanoids (PGD, 6kPGF1a, TXB2) urine differ between 
subjects with upper lobe vs. lower lobe emphysema 
 
 
H29.4  Do measures of inflammation differ between subjects with upper vs. 
lower lobe emphysema 
 
 
H29.5  Is the response to 600 mg ibuprofen three times daily different in 
subjects with upper lobe vs. lower lobe emphysema 
 
 
 
H29.5.1 Response of PGE 
 
 
 
H29.5.2 Response of measures of repair 
 
 
 
H29.5.3 Response of other eicosanoids 
 
 
 
H29.5.4 Response of measures of inflammation 
 
H30:  Are there differences between subjects with clustered vs. diffuse 
emphysema 
 
 
H30.1  Do PGE levels in BAL fluid, sputum  and metabolites in blood and 
urine differ between subjects with clustered vs. diffuse emphysema 
 
 
H30.2 Do procollagen peptides differ in BAL fluid between subjects with 
clustered vs. diffuse emphysema 
 
 
H30.3  Do the eicosanoids (PGD, 6kPGF1a, TXB2) urine differ between 
subjects with clustered vs. diffuse emphysema 


 
 
Rennard, Stephen I. 
 22 
 
 
H30.4  Do measures of inflammation differ between subjects with upper vs. 
lower lobe emphysema 
 
 
H30.5  Is the response to 600 mg ibuprofen three times daily different in 
subjects with clustered vs. diffuse emphysema 
 
 
 
H30.5.1 Response of PGE 
 
 
 
H30.5.2 Response of measures of repair 
 
 
 
H30.5.3 Response of other eicosanoids 
 
 
 
H30.5.4 Response of measures of inflammation 
 
 
Note: for all questions evaluating the effect of ibuprofen, a secondary analysis will be 
performed to determine the relationship with individual subject ibuprofen levels. 
 
Progression of COPD 
H31:  Will BAL PGE levels assessed at study initiation be related to the rate of 
emphysema progression determined by the change in CT quantified lung density from 
the time of assessment in COPDGene to the current study (estimated 3-6 years)? 
 
H32: Will BAL PGE levels assessed at study initiation be related to the rate of 
emphysema progression determined by the change in FEV1 from the time of 
assessment in COPDGene to the current study (estimated 3-6 years)? 
 
 
H33: Will measures of prostanoids other than PGE assessed at study initiation 
be related to the rate of emphysema progression determined by the change in CT 
quantified lung density from the time of assessment in COPDGene to the current study 
(estimated 3-6 years)? 
 
H34: Will measures of inflammation assessed at study initiation be related to the 
rate of emphysema progression determined by the change in FEV1 from the time of 
assessment in COPDGene to the current study (estimated 3-6 years)? 
 
 
 
2.3  Ancillary Study Objectives. Prior studies have shown that fibroblasts from 
patients with COPD over-produce PGE, that this is due to under-expression of the 
microRNA miR-146a and that genetic polymorphisms may contribute to miR-146a 
under-expression.  The Ancillary Study will determine if alveolar macrophages 
contribute to PGE over-production in emphysema and will determine if miR-146a plays 
a role in these cells.  We will also determine if the other major prostanoid produced by 
alveolar macrophages, PGD, is over-produced in COPD and if miR-146a modulates its 
production. 
 
Ancillary Study Hypotheses 
 
 
Primary Hypothesis 


 
 
Rennard, Stephen I. 
 23 
AH1:  Do alveolar macrophages from patients with emphysema over-produce 
PGE compared to alveolar macrophages from controls? 
 
Secondary Hypotheses 
AH2:  Do alveolar macrophages from patients with emphysema over-produce 
PGD compared to alveolar macrophages from controls? 
 
AH3:  Does miR-146a expression differ in alveolar macrophages from 
emphysema subjects compared to controls?   
AH3.1 Are alveolar macrophage miR-146a levels related to macrophage 
PGE production?   
AH3.2  Are alveolar macrophage miR-146a levels related to macrophage 
COX-2 mRNA and protein expression levels? 
 
AH4:  Does miR-146a modulate COX-2 mRNA and protein expression and PGE 
production in alveolar macrophages? 
 
AH5:  Does miR-146a modulate COX-2 mRNA and protein expression and PGE 
production in alveolar macrophages? 
 
AH6:  Are polymorphisms in the miR-146a gene, including nearby regulatory 
sequences and the large intron, related to miR-146a expression in alveolar 
macrophages?   
 
AH7:  Are the polymorphisms related to COX-2 mRNA and protein expression?   
 
AH8:  Are the polymorphisms related to PGE production? 
 


 
 
Rennard, Stephen I. 
 24 
3.  Study design 
 
3.1 Overview.  The Prostaglandin Inhibition for Emphysema (PIE) trial is a proof-
of-concept trial designed to determine if a conventional dose of a well-tolerated NSAID 
can reduce PGE levels in the lower respiratory tract of subjects with emphysema.  The 
study is a randomized, placebo controlled, parallel group study that will compare 
ibuprofen, 600 mg three times daily, with placebo, administered for 12 months.  The 
primary outcome will be PGE levels quantified in the alveolar portion of BAL fluid.  The 
primary comparison will be intention-to-treat comparing the difference between pre- and 
post-treatment in the treated group to pre- and post-treatment in the placebo group.   
One hundred forty emphysema subjects will be randomized at a 1:1 ratio (70 
subjects in each group).  This based on a sample size estimate based on preliminary 
data from our center and from the literature that 52 subjects with paired BAL samples in 
each group would provide 90% power to detect a 50% reduction in PGE levels at a 
significance level of 0.05, estimates that we believe are conservative.  To have 52 
evaluable subjects with paired BAL samples in each group, 70 randomized subjects in 
each group, 140 total subjects, will be required if there is a 25% drop-out rate, an 
estimate we believe conservative. 
The secondary objective is to determine if ibuprofen 600 mg three times daily 
compared to placebo increases lower respiratory tract pro-collagen peptide levels. 
Conditional power analyses suggest our sample size will have a 69% power to detect a 
standardized effect size of 0.485 (which was the difference observed in pilot studies), 
comparing the effect of treatment to that of placebo and a 92% power to detect a 
difference of treatment before to after within the treated group.    
Additional objectives are to determine if treatment with ibuprofen alters PGE 
concentration in induced sputum, if there are changes in other eicosanoids, in particular 
PGD, and eicosanoid metabolites in BAL, sputum, blood and urine and, by using 
previously collected data required for subject enrollment, if PGE, other eicosanoid and 
measures of inflammation levels are related to the rate of emphysema progression.  
Control subjects will also be assessed on one occasion so that levels in the emphysema 
subjects before and after treatment can be compared to control.  Control subjects will 
not receive treatment with ibuprofen.   Finally, the primary goals will be achieved by 
assessing subjects with bronchoscopy and BAL after 3 months of treatment.  Subjects 
will continue to receive randomized treatment for a total of 48 months.  At the end of 
that time, emphysema assessed by CT-scan, lung function and health status will be 
assessed to provide an estimate of effect size that can help with the design of Phase 3 
efficacy studies. 
The study will be conducted at least three clinical centers including: Temple 
University Hospital, Philadelphia, National Jewish Health, Denver and Harbor/UCLA 
Hospital, Los Angeles.  All three centers were participants in COPDGene.  The 
University of Nebraska Medical Center (UNMC) will serve as the Data Coordinating 
Center (DCC).  Samples will be shipped to the DCC where they will be banked and 
where the biochemical and cellular study-specific analyses will be performed.  Clinical 
tests required for subject enrollment and to assure safety for bronchoscopy will be 
performed at the study sites.  National Jewish Health (NJH), which served as the 
radiology reading center for COPDGene, will serve as the radiology reading center.  


 
 
Rennard, Stephen I. 
 25 
Annonomized CDs with the CT data will be shipped to NJH.  Where possible, 
standardized procedures adopted by COPDGene will be used. 
 All emphysema subjects will be prior COPDGene participants or other subjects 
who have had CT scans demonstrating emphysema as determined by the radiology 
center at NJH.   Smoking and former control subjects will also be COPDGene 
participants or subjects who have had a CT scan reviewed and accepted by the 
radiology center at NJH.  No non-smokers were enrolled in COPDGene.  For this 
reason, 20 healthy non-smoking non-COPDGene subjects will also be recruited. 
After randomization, subjects will be treated for 48 weeks. following which they 
will be reassessed. 
Because of the invasive nature of the BAL, an interim analysis with a formal 
futility stopping rule is include to minimize unnecessary exposure of study subjects to 
risk.  
 
 
 Figure 6. Schematic time line of key study events. 
 
The study will include 18 visits (Figure 6) 
Visit 1: Screening will include a history and physical, demographics, stool for 
occult blood, blood chemistry, CBC, spirometry, DLCO, SGRG and CAT. 
Visit 2 (within two weeks of screening): CT scan, induced sputum 


 
 
Rennard, Stephen I. 
 26 
Visit 3 (2-3 weeks after visit 2): Bronchoscopy, blood and urine for biomarkers; 
randomize to ibuprofen or placebo 
Visit 4 (phone call): follow-up after one week of medication 
Visit 5* (4 weeks after randomization): follow-up including stool for occult blood 
Visit 6* (8 weeks after randomization): follow-up including stool for occult blood 
Visit 7* (10 weeks after randomization): sputum 
Visit 8* (12 weeks after randomization, at least 2 weeks after visit 7): 
Bronchoscopy, blood and urine for biomarkers 
Visit 9* (1 week after bronchoscopy phone call): follow up 
Visit 10*-17* (every 4 weeks from week 16 to week 44 after randomization) 
follow-up including stool for occult blood 
Visit 18* (48 weeks after randomization): CT-scan, blood and urine for 
biomarkers, spirometry, DLCO, SGRG and CAT. 
 
*Emphysema subjects only. 
 
 
3.2  Enrollment criteria, emphysema subjects.  At each study site, 
emphysema subjects will be recruited from prior COPDGene subjects or from other 
subjects who have had CT scans demonstrating emphysema.  All must have consented 
to be recontacted for additional studies.  All must meet the following criteria  
 
Age > 45 years 
 
Emphysema (>5% of voxels <-950 Hounsfield Units determined on a CT scan 
quantified at the COPDGene radiology center, i.e. NJH) 
 
Post-bronchodilator FEV1 > 35% predicted 
 
Smoker or ex-smoker (10 pack years minimum) 
 
Subjects who meet the entry criteria will be contacted by phone.  If interested in 
participating, subjects will be informed of the following exclusion criteria by phone: 
 
Contraindication to bronchoscopy or other study procedures 
 
Pregnancy or plans to become pregnant within six months 
 
Aspirin-sensitive asthma 
 
Regular use of systemic glucocorticoid 
 
Regular use of an NSAID   
 
Unstable medical condition  
 
History of myocardial infarction or unstable angina within six months 
 
Allergy to or history of adverse effect from ibuprofen or other NSAID 
 
History of gastrointestinal bleeding within one year 
 
Any condition that, in the opinion of the investigator, places the subject at 
untoward risk 
 
Inability to provide informed consent 
 
Subjects who meet the entry criteria and who believe they do not have any exclusions 
and who are interested in participating will be invited to the study site. 
 
3.3 Study visits, emphysema subjects 


 
 
Rennard, Stephen I. 
 27 
 
Visit 1.  Day 0. 
Informed consent.  Consent will be obtained by a study physician who is qualified to 
explain all study procedures including bronchoscopy. 
Medical history 
Physical examination 
Clinical laboratory 
 
Blood chemistry panel including Na, K, Cl, HCO3, albumin, glucose, creatinine, 
BUN 
 
CBC 
 
Urine pregnancy test (women only) to be repeated in serum if positive 
 
Stool occult blood assessment 
 
Spirometry, pre- and post-bronchodilator 
 
DLCO 
 
SGRQ  
 
CAT 
 
Smoking history 
 
 
Confirmation that entry criteria are met and that no exclusions are present; 
confirm that subject continues to meet study criteria by spirometry 
 
Schedule CT Scan, induced sputum and bronchoscopy 
 
Visit 2. (can be two separate days, but CT scan must precede sputum.  To be 
performed 0-14 days after screening) 
 
CT scan:  this will not be repeated is an acceptable CT scan has been performed 
within 1 year.  A CT scan will be deemed acceptable if the radiology center confirms 
that emphysema can be quantified. 
 
Induced sputum 
 
Visit 3. (two to three weeks after visit 2) 
 
Subjects will be called within one week of visit to review schedule and 
instructions for bronchoscopy. 
 
Review of study and procedure and consent for bronchoscopy and BAL 
 
Limited interval history and physical 
 
Collect 3 tubes of blood for research study.  Tube 1 (DNA); Tube 2 (serum); Tube 
3 (plasma) 
 
Urine for biomarkers 
 
Bronchoscopy and BAL 
 
Distribute study medication* 
 
Call one day after procedure to assure post bronchoscopy recovery 
 
*Subject randomization will be determined by the DCC.  This will be done in blocks for 
each site.  Study medication will be prepared by a vendor (tentatively the Temple 
University Pharmacy) and shipped to study sites with a subject number-specific code.  
Subjects will be given acetaminophen for PRN use. 
 


 
 
Rennard, Stephen I. 
 28 
 
Visit 4. (one week +/- 4 days after randomization) 
 
Phone call to check use of study medication, potential problems 
 
Visit 5. (four weeks +/- one week after randomization) 
 
Call one week and one day prior to visit to remind about appointment 
 
Visit to occur 1-2 hours after morning dose of study medication 
 
Interval history 
 
Stool for occult blood 
 
Distribute study medication 
 
Collect 1 tube of blood for ibuprofen level 
 
Visit 6. (eight weeks +/- one week after randomization) 
 
Call one week 
 
Interval history 
 
Stool for occult blood 
 
Distribute study medication 
 
Schedule induced sputum 
 
Visit 7. (10 weeks +/- four days after randomization) 
Induced sputum 
 
Visit 8. (12 weeks +/- 1 week after randomization, at least 2 weeks after visit 7) 
 
Subjects will be called within one week of visit to review schedule and 
instructions for bronchoscopy 
 
Review of study and procedure and consent for bronchoscopy and BAL 
 
Limited interval history and physical 
 
Urine pregnancy test (women only) to be repeated in serum if positive 
 
Collect 2 tubes of blood for research study.  Tube 1 (serum); Tube 2 (plasma).   
 
Urine for biomarkers 
 
Bronchoscopy and BAL 
 
Call one day after procedure to assure post-bronchoscopy recovery 
 
Visit 9.  (one week +/- 4 days after visit 8) 
 
Phone call follow-up 
 
Visit 10-17.  (Every 4 from week 16 until week 44 after randomization). 
 
Clinical assessment 
 
Stool for occult blood 
 
Distribute study medication 
 
Visit 18 (48 weeks after randomization) 
 
Clinical assessment 
 
Stool for occult blood 
 
CT-scan 
 
Spirometry (pre and post bronchodilator) 


 
 
Rennard, Stephen I. 
 29 
 
DLCO 
 
SGRQ 
 
CAT 
 
Blood (serum and plasma) for biomarkers 
 
Urine for biomarkers 
 
 
At each visit, all study information will be recorded in the study binder.  All fields in the 
study report forms must be completed for each visit.  Sample inventory forms must be 
completed when samples are initially stored.  Sample shipping forms must be 
completed when samples are shipped, which will be batchwise. 
 
 
 
3.4  Enrollment criteria smoking and ex-smoking control subjects.  At each 
study site, smoking and ex-smoking control subjects will be recruited from prior 
COPDGene subjects or other subjects that have had a CT scan reviewed and accepted 
by the radiology center at NJH.  All must have consented to be recontacted for 
additional studies.  All must meet the following: 
 
Age > 45 years 
 
No emphysema (<3% of voxels <-950 Hounsfield Units determined on a CT scan 
quantified at the COPDGene radiology center, i.e. NJH) 
 
Normal lung function  (pre bronchodilator FEV1/FVC > 0.7 and FEV1 > 80% 
predicted) 
 
Smoker or ex-smoker (10 pack years minimum). 
 
Subjects who meet the entry criteria will be contacted by phone.  If interested in 
participating, subjects will be informed of the following exclusion criteria by phone: 
 
Contraindication to bronchoscopy or other study procedures 
 
Pregnancy or plans to become pregnant within six months 
 
Aspirin-sensitive asthma 
 
Regular use of systemic glucocorticoid 
 
Regular use of an NSAID   
 
Unstable medical condition  
 
History of myocardial infarction or unstable angina within six months 
 
Allergy to or history of adverse effect from ibuprofen or other NSAID 
 
History of gastrointestinal bleeding within one year 
 
Any condition that, in the opinion of the investigator, places the subject at 
untoward risk 
 
Inability to provide informed consent 
 
Subjects who meet the entry criteria and who believe they do have any exclusions and 
who are interested in participating will be invited to the study site. 
 
3.5 Entry criteria healthy non-smoker controls.  Because COPDGene does 
not include healthy non-smokers, these subjects will be recruited de novo.  Subjects 
must be healthy without a chronic medical problem that requires treatment and without 


 
 
Rennard, Stephen I. 
 30 
having had an acute medical problem requiring treatment within six months.  In addition, 
all must be non-smokers (less than 100 cigarettes per lifetime) and must meet the 
following criteria: 
 
Age > 45 years 
Normal lung function (pre bronchodilator FEV1/FVC > 0.7 and FEV1 > 80% 
predicted); lung function criteria will be established at the screening visit 
 
and have none of the exclusions that apply to other subjects: 
 
Contraindication to bronchoscopy or other study procedures 
 
Pregnancy or plans to become pregnant within six months 
 
Aspirin-sensitive asthma 
 
Regular use of systemic glucocorticoid 
 
Regular use of an NSAID   
 
Unstable medical condition  
 
History of myocardial infarction or unstable angina within six months 
 
Allergy to or history of adverse effect from ibuprofen or other NSAID 
 
History of gastrointestinal bleeding within one year 
 
Any condition that, in the opinion of the investigator, places the subject at 
untoward risk 
 
Inability to provide informed consent 
 
CT scan must show no emphysema (<5% Housfield units).  If emphysema is 
present, a subject will be excluded as a normal non-smoking control.  
 
3.6  Study visits, control subjects 
 
Visit 1.  Day 0 
Informed consent.  Consent will be obtained by a study physician who is qualified 
to explain all study procedures including bronchoscopy. 
Medical history 
Physical examination 
Clinical laboratory 
 
Blood chemistry panel including Na, K, Cl, HCO3, albumin, glucose, creatinine, 
BUN 
 
Urine pregnancy test (women only); followed by serum pregnancy test if positive 
 
CBC 
 
Stool occult blood assessment 
 
Spirometry, pre- and post-bronchodilator 
 
EKG 
 
DLCO 
 
SGRQ  
 
CAT 
 
Smoking history 
 


 
 
Rennard, Stephen I. 
 31 
 
Confirmation that entry criteria are met and that no exclusions are present; 
confirm that subject continues to meet study criteria by spirometry 
 
Schedule CT Scan, induced sputum and bronchoscopy 
 
Visit 2. (can be two separate days, but CT scan must precede sputum.  To be 
performed 0-14 days after screening.) 
 
CT scan 
 
Induced sputum 
 
Visit 3. (2-3 weeks after visit 2) 
 
Subjects will be called within one week of visit to review schedule and 
instructions for bronchoscopy. 
 
Review of study and procedure and consent for bronchoscopy and BAL 
 
Limited interval history and physical 
 
Collect 3 tubes of blood for research study.  Tube 1 (DNA);  Tube 2 (serum); 
Tube 3 (plasma). 
 
Urine for biomarkers 
 
Bronchoscopy and BAL 
 
 
Call one day after procedure to assure post-bronchoscopy recovery 
 
Visit 4.  (one week +/- 4 days after visit 3) 
 
Phone call follow-up 
 
 
At each visit, all study information will be recorded in the study binder.  All fields in the 
study report forms must be completed for each visit.  Sample inventory forms must be 
completed when samples are initially stored.  Sample shipping forms must be 
completed when samples are shipped, which will be batchwise. 
 
 


 
 
Rennard, Stephen I. 
 32 
4.  Statistical considerations 
 
 
 
4.1 Study design.  The PIE is a double blind, placebo controlled, parallel group, 
multi-center study designed to determine if ibuprofen 600 mg three times daily can 
reduce lower respiratory tract PGE levels compared to placebo.  The primary outcome 
will be PGE levels in the alveolar portion of BAL fluid.   
The primary measure will be difference from beginning to end of treatment 
comparing treated vs. placebo groups on an intention-to-treat basis.  The secondary 
outcome will be procollagen peptides measured in the alveolar portion of the BAL 
analyzed by the same plan. All three pro-collagen peptides will be assessed and 
evaluated separately. Levels in BAL fluid will be compared to levels determined in a set 
of smoking, ex-smoking and non-smoking controls.   
Additional analyses will include PGE in the bronchial portion of the BAL fluid and 
induced sputum, other eicosanoids and metabolites in  BAL fluid, blood and urine and 
inflammatory markers, including IL-8 and neutrophils measured in BAL and sputum.  
Ibuprofen levels will be measured, and a secondary analysis based on blood levels will 
be performed.   
An exploratory analysis relating the change in emphysema over time to BAL 
levels of PGE will be performed.  This will be accomplished by using historical data 
collected in the COPDGene study. 
 
4.2  Sample size considerations. The primary question is:  Will ibuprofen 
treatment reduce lower respiratory tract PGE levels?  The question will be tested by 
comparing the difference in PGE concentration in the alveolar BAL fraction in the 
treated group to the difference in the placebo group after three months of treatment.  
Based on preliminary data, the observed standardized effect size for the difference in 
PGE concentration between COPD and controls was 1.3 (COPD mean=13.13, SD=9.2; 
Control mean=4.39, SD=1.9). Using a 0.05 level two-sided independent sample t-test, a 
sample size of 52 per group will have a 90% power to detect an effect half this size 
(0.65). This difference in effect size equates to a 33% reduction in mean PGE (13.13 to 
8.76).  Based on the literature, we believe this is a conservative effect size, as inhibition 
of PGE of 60% or greater has been reported.  In prior studies, we have had less than 
10% drop-out in studies requiring multiple bronchoscopies over intervals of three 
months or less 89-92.  Using a conservative estimate of 25% drop-outs, we intend to 
enroll a total of 140 subjects, 70 in each group.   
The study has been powered on the primary outcome, change in PGE level 
compared to placebo.  We have, however, estimated the power to detect a difference in 
the secondary outcome, pro-collagen peptide.  Based on our pilot studies with pro-
collagen I N-terminal peptide, our study with an anticipated 52 subjects will have 69% 
power to detect a standardized effect size of 0.485 (as observed in pilot studies) for the 
difference between groups using a 0.05 level two-sided independent sample t-test if 
inhibition of PGE corrects the pro-collagen I peptide levels to that of normal subjects.  
There is over 92% power to detect an improvement of this size within the treatment 
group using a 0.05 level two-sided paired t-test. With 52 subjects per group, we will 
have 80% power to detect an effect of 0.55 (slightly larger than in pilot studies) between 
groups and 0.396 (slightly smaller than in pilot studies) within group at the 0.05 level. 


 
 
Rennard, Stephen I. 
 33 
The same power estimates described above apply to the analyses of PGE in 
sputum samples.  However, we anticipate obtaining satisfactory sputum samples in 
75% (39) of subjects.  As a result, we will have 80% power to detect the same effect 
size in the sputum specimens, as there will be fewer subjects with paired sputums than 
with paired BALs.  However, we feel our estimates are conservative, as they are based 
on a 50% reduction in PGE concentration, which was readily achieved in prior studies. 
Preliminary data do not exist to estimate the rate of change of emphysema 
quantified by emphysema.  As a result, we do not have a basis to estimate the 
conditional power for the exploratory analysis.  The analysis planned, however, should 
provide a basis for estimating sample sizes for follow-on studies that would use CT-
quantified emphysema as an outcome measure. 
For the Ancillary Study, the primary outcome is macrophage PGE production by 
COPD macrophages compared to controls. Data from all evaluable subjects will be 
used.  Each subject will be considered as a single data point.  The number of data 
points, therefore, is determined by the sample size considerations of the primary aim of 
the clinical trial.  We anticipate obtaining samples from 140 COPD subjects and 60 
controls.  Based on the mean (SD) from preliminary studies for PGE production by 
fibroblasts, which is 641 (409) ng/105 cells/day for COPD subjects and 260 (161) for 
controls, and estimating adequate samples from 80% of subjects, a conservative 
estimate, and assuming the variance is the same and the difference in production of 
PGE is half that we observed in COPD fibroblasts, we will have greater than 93% power 
to detect a difference at the 0.05 level between COPD and control groups. 
For the secondary aim of the Ancillary Study evaluating miR-146a levels in 
COPD subjects vs. controls, again using as pilot data that obtained from fibroblasts that 
showed the mean(SD) of miR-146a expression following stimulation to be 392(177) for 
COPD and 681(456) for controls, we will have 80% power to detect a standardized 
effect size of 0.486 for the difference in miR-146a expression in COPD macrophages 
compared to controls, at the 0.05 level.  This effect is reasonable to assume, as the 
observed effect size in the pilot data with fibroblasts was 0.868.  For assessing the 
functional role of miR-146a in modulating macrophage COX-2 mRNA, we used a similar 
analysis based on prior results with fibroblasts that shows the observed means(SD) are 
for COX-2 mRNA expression in fibroblasts following an miR-146a mimic were 60(25) 
treated and 130(25) untreated. With the conservative estimate that the role of miR-146a 
in alveolar macrophages will be 50% that in fibroblasts, samples from eight subjects will 
have 90% power to detect differences in COX-2 expression in response to miRNA-146a 
mimic at a 0.05 level.  However, because of COPD heterogeneity, sample sizes of less 
than 10-15 are generally regarded skeptically.  Thus, we plan to assess cells from 20 
COPD subjects and 20 control smoker, ex-smoker and non-smoker subjects, which we 
believe will be adequate to assess the functional role of miR-146a. 
 
 
 
 
 
 
 


 
 
Rennard, Stephen I. 
 34 
 
The final aim of the Ancillary Study is to relate genetic variants in the miR-146a 
gene to COPD.  Conditional power for this aim was estimated for 200 subjects (140 
COPD, 60 controls) based on the binomial distribution. Table 2 shows excellent power 
to detect alleles present in at least twice in the study sample (frequency ≥ 0.5%). 
Power to detect association between SNPs of allele frequency ≥1% and miR-146a 
expression levels was calculated using Quanto software93, at α=0.05 (no multiple testing 
correction) and at α=0.001 to adjust for 50 SNPs tested (Table 3).   
 
 
 
 
4.3  Interim monitoring and stopping rules.  A Data and Safety Monitoring 
Board (DSMB) will be constituted.  It will consist of four members, two pulmonologists, 
one cardiologist and a statistician, who will review the progress of the study on a 
quarterly basis.  The status of each subject after bronchoscopy and the results of the 
monthly gastritis monitoring will be reported monthly in summary reports to the DSMB 
members.  In addition, the DSMB will receive reports of all Serious Adverse Events 
within 24 hours of their being reported to the DCC.  
Because this trial involves an invasive procedure (bronchoscopy), formal interim 
analyses and formal stopping rules were believed to be required to help prevent subject 
exposure to unnecessary risks. Formal interim analysis of outcomes will be performed 
once, when approximately 50% of patients have completed the post-treatment 
bronchoscopy. We will use an O’Brien-Fleming monitoring boundary 94 (truncated at 
three standard deviations) to assess whether the interim results are sufficient to 
conclude that ibuprofen is effective at reducing PGE. We will also apply a futility 
Table 2.  Power to detect 
variants by sequencing 
Allele 
frequency 
power 
0.25% 
0.63 
0.50% 
0.87 
1% 
0.98 
2% 
1 
5% 
1 
Table 3.  Power to detect association between SNPs (allele frequency 
! 1%) and miR-146a expression levels 
α=0.05 
α=0.001 
Percent of 
variation 
explained 
COPD cases 
All subjects 
COPD cases 
All subjects 
5% 
0.76 
0.89 
0.27 
0.47 
10% 
0.97 
1 
0.71 
0.9 
15% 
1 
1 
0.93 
0.99 


 
 
Rennard, Stephen I. 
 35 
monitoring rule. The monitoring boundary nominal significance level associated with the 
interim analysis using the O’Brien-Fleming spending function will be 0.003 and 0.012, 
with the final analysis being done with =0.047. Should there be any meaningful 
prevalence of concerning adverse events, e.g. myocardial infarctions, the DSMB can 
ask for an unblinded set of data to determine if the effect is related to the intervention.  
The DSMB is empowered to stop the study should concerns regarding subject safety 
warrant. 
 
 
 
4.4  Analysis plans. The primary analysis will be by intention-to-treat. 
Descriptive statistics will be computed for all variables to ensure data quality and to 
evaluate the assumptions of statistical tests. Variable distributions will be described with 
box plots, histograms, measures of central tendency, measures of variation and 
frequencies. We will test the assumption of normality and equal variances for 
continuous outcome measures within each group and transform variables for which 
these assumptions are not met. Before testing the study hypotheses, we will examine 
the data for possible confounders. Potentially confounding variables, such as age, 
gender, and disease severity, will be controlled for by entering these variables as 
covariates. These variables will be entered as a covariate if the univariate two-sided test 
(either t-test or Chi-square test) comparing the two treatment groups is significant at the 
0.20 level. We will use a large alpha level here so that important potentially confounding 
effects are not overlooked. 
Linear mixed effects modeling will be used to determine the effect of ibuprofen on 
reducing PGE levels in COPD patients. Linear mixed effects modeling is a regression 
technique that accounts for the correlation due to multiple measurements on the same 
subject. This method will allow for the evaluation of differences within group (pre- to 
post-treatment change in PGE) as well as the difference between groups post treatment. 
The effects of treatment and time point, as well as any variables found to be significant 
in the univariate analysis, will be included in the model. Finally, a secondary analysis 
will be performed using blood levels of ibuprofen as a covariate.   
In order to place the magnitude of any reduction observed into perspective, BAL 
PGE levels in the COPD patients before and after treatment will be compared with the 
levels present in normals.  This comparison will be made estimating mean BAL PGE 
levels for each group and calculating their associated 95% confidence intervals. As the 
treatment is expected to lower PGE levels, the post-treatment is expected to be closer 
to controls.  
For the secondary outcome, change in alveolar fraction pro-collagen peptide 
concentration, we will use the same modeling and analysis approach described above.  
Pro-collagen peptides, pro-collagen I C-terminal and N-terminal peptides and pro-
collagen III N-terminal peptide will be quantified in BAL fluid. The change in each pro-
collagen peptide level following ibuprofen treatment will be compared to the change in 
placebo-treated subjects. 
 
A number of additional analyses are planned.  The alveolar fraction of the BAL 
will be assessed for COX-derived eicosanoids in addition to PGE: PGD, TXB2 (a 
metabolite of thromboxane A2) and 6kPGF1a (a metabolite of prostacyclin) by LC-
MS/MS. These metabolites will be quantified under the same chromatographic and 


 
 
Rennard, Stephen I. 
 36 
sample preparation conditions used for PGE analysis and using the same LC-MS/MS 
method. This will confirm the effect of COX inhibition of products other than PGE.  In 
addition, urea will be used to estimate concentrations of PGE in alveolar ELF.  Finally, 
we will assess PGE concentration in the bronchial portion of the BAL fluid and in 
induced sputum.  While these latter two measures will not assess alveolar PGE, they 
will provide independent measures of the effect of ibuprofen on PGE in the lung.  
Similarly, quantification of PGEM in blood and urine will provide an independent 
assessment of the effect on total body PGE production.  The same analysis plans 
described above for the analysis of PGE in the BAL fluid will be used for these 
secondary analyses. 
 
Use of legacy data from COPDGene will allow us to relate measures made to 
progression of COPD and emphysema.  To accomplish this, we will determine the 
difference between the legacy COPDGene CT-scan compared to that obtained upon 
PIE study entry.  Correction will be made for change in achieved lung volume95. Dividing 
by the time between the assessments (3-6 years) will estimate a rate of progression of 
emphysema.  The rate of change in emphysema for the entire population will then be 
related to PGE concentration in the BAL fluid on the initial (without treatment) BAL.  This 
will allow us to determine if PGE is related to the rate of progression of emphysema.  A 
similar analysis will be performed estimating the relationship with the rate of change in 
FEV1. 
 
We will also assess clinical measures after 48 weeks of treatment. We do 
not believe our study, which is powered to confirm lower respiratory tract inhibition of 
PGE, will be adequately powered to rigorously assess disease progression.  The 
sample size of 52 per group will provide 80% power to detect a standardized effect size 
for the mean change in lung volume of 0.57 between groups (a medium effect as 
defined by Cohen) and 0.4 within group (slightly smaller than a medium effect). The 
standardized effect sizes represent a difference between groups 57% as large as the 
standard deviation and change within group 40% as large as the standard deviation.  
We expect the change observed during this study to be smaller.  However, the results 
from this trial will be used as assessment of the likely effect size and variance and will 
be important is the design of future Phase 3 efficacy trials.  For this purpose, we will 
assess several outcome variables. 
At present the most accurate way of detecting emphysema severity is CT 
scanning. CT scan quantification of emphysema has been demonstrated to be sensitive 
to change over a one-year time frame and has been used as an outcome in several 
clinical trials95-97.  We will confirm the diagnosis and severity of emphysema at study 
entry by CT scan.  
Volumetric CT scans will be acquired at end inspiration using multi-detector 
scanners (≥ 64 detector rows) using the SPIROMICS CT protocol. Phantom scans will 
be acquired on a monthly basis at each site, to assure scanner stability. Phantom scans 
will be analyzed as in the COPDGene and SPIROMICS protocol. Any deviations from 
recommended phantom parameters will be communicated immediately to the sites, and 
further scans will be suspended until the deviation is corrected. To perform quantitative 
emphysema analysis, de-identified DICOM images will be segmented using the Apollo 
image analysis system (VIDA Diagnostics, http://www.vidadiagnostics.com)98. The 
software will divide each lung into lobes as well as three craniocaudal zones of equal z-


 
 
Rennard, Stephen I. 
 37 
axis lengths.  The following analyses will be performed on the segmented inspiratory 
lung images: total inspiratory lung volumes, mean lung attenuation, relative lung 
volumes (for the whole lung, each lung lobe, and apical and basal lung thirds) falling 
below attenuation coefficients of  -950, -910, and 856 HU. The 15th percentile point of 
the lung density histogram (PERC15) will also be calculated.  Additionally, to evaluate 
emphysematous “hole size”, the alpha value (the negative of the slope from the log-log 
relationship of hole size versus number of holes) will be measured for the entire lung 
and for the upper and lower lobes 99. 
We will also assess FEV1, DLCO and SGRQ as these additional clinical 
parameters that can also contribute to the design of phase 3 efficacy trials.   
Our assessment of CT scan will provide quantitative measures of two distinct 
patterns of emphysema, upper lobe vs. lower lobe  and clustered vs. diffuse.  Both of 
these distinct patterns have been associated with differences in natural history, clinical 
features and response to treatment.  Therefore, we will also conduct a secondary 
analysis based on these parameters. 
 
The first aim of the Ancillary Study is, do alveolar macrophages from COPD 
subjects produce more PGE than from controls?  Because the data distribution cannot 
be assumed to be normal, generalized linear modeling will be used.  Generalized linear 
modeling is a regression technique that allows for non-normally distributed outcome 
variables. In addition, a modeling approach will allow us to adjust for important 
covariates, as well as smoking status and disease severity. Secondary analyses will be 
performed similarly, evaluating current and former smoking COPD subjects separately.  
Each subject will be considered as a single data point.   
The second aim of the Ancillary Study will determine if miR-146a levels differ 
between COPD subjects and controls.  This will be approached with the same plan 
described above.  To determine if miR-146a levels are related to BAL fluid PGE and 
alveolar macrophage COX-2 mRNA, protein levels and PGE production, we will use 
Spearman’s correlation.  Similar analyses will be performed with macrophages following 
culture with and without IL-1ß/TNF- stimulation.  For analyses of functional 
macrophage studies using miR-146a mimics and inhibitors, each subject will be a single 
data point, and differences in the paired values will be analyzed using a non-parametric 
Wilcoxon signed ranks test.   
In the third aim of the Ancillary Study, we will test for association between miR-
146a polymorphisms and the BAL PGE levels (determined in the Clinical Study) and the 
immediate ex-vivo miR-146a and COX-2 expression in alveolar macrophages.  For 
common variants (allele frequency ≥ 1%), we will use linear regression in all subjects 
and in COPD cases only, adjusting for smoking status, if that is found to be relevant. 
For rare variants (<1%), we will employ one of several current methods that can be 
applied to quantitative traits, such as the Variable Threshold test100 or the SNP-set 
Kernel Association Test101.  We will adapt these methods for analysis of exome/genome 
sequencing data to the candidate gene analysis, by collapsing SNPs according to 
location within the gene (e.g. promoter, exon, 3’ UTR) and weighting by presumed 
functional effects, such as SNPs that alter transcription factor binding sites.  Methods for 
rare variant analysis are constantly evolving, and we will adopt best practices as they 
emerge.  In addition, we predict that polymorphisms in miR-146a related to AP-1 


 
 
Rennard, Stephen I. 
 38 
regulatory sites will specifically modulate response to IL-1ß/TNF-, and this will be 
ascertained by relating miR-146a polymorphisms to the results of the stimulation of 
PGE production by IL-1ß/TNF-, using the analysis methods above.   
In addition to PGE, alveolar macrophages produce PGD.  Our analytical method 
is able to quantify PGD and, but including an additional internal standard in the LC-
MS/MS runs, both PGE and PGD can be accurately determined with little increase in 
total cost 102. 
 
 


 
 
Rennard, Stephen I. 
 39 
5.  Study organization and conduct 
 
 
5.1  Data Coordinating Center.  The DCC will be located at the University of 
Nebraska Medical Center (UNMC).  The DCC will have the primary responsibility to 
assure that all study sites are fully equipped and trained to execute the study, will 
monitor the progress of the study and its compliance with protocol and regulatory 
requirements, will house all samples, will coordinate sample analyses, will have the 
responsibility for maintaining the study database and will perform statistical analyses.  In 
addition, the DCC will prepare the Study Procedures Manual and will oversee data entry 
and quality control.   
 
 
5.2  Clinical sites.  At least three clinical sites will participate: Temple University 
Hospital, National Jewish Health, and Harbor/UCLA Hospital.  All centers are previous 
COPDGene centers.  Study procedures will be harmonized with prior COPDGene 
procedures.  Each center will have a site Principal Investigator, a Study Coordinator and 
a Laboratory Technician, all of whom will complete formal training for study procedures.  
The study will be approved by the local Institutional Review Board and will comply with 
local HIPAA guidelines. 
 
 
5.3  Radiology reading center.  National Jewish Health (NJH) will serve as the 
Radiology Reading Center.  To perform quantitative emphysema analysis, de-identified 
DICOM images will be sent to NJH for analysis.  Data will then be transferred to the 
DCC. 
 
 
5.4  Study Procedures Manual, and Data Forms.  The Study Procedures 
Manual and Data Forms will be prepared by the DCC after study approval.  We expect 
this to be completed between notification and accomplished before the funding start 
date so that all study materials will be in place prior to the official funding date.  The 
Data Forms will be completed at the Study Sites, the Radiology Reading Center and at 
the analysis laboratories of the DCC.  In general, the same procedures used for 
COPDGene for the performance of CT Scans, for the transfer of CT data and for quality 
assurance of CT Scans, including staff training, data monitoring and scanning of 
phantoms will be used.  Other procedures, e.g. bronchoscopy and BAL and induced 
sputum, are currently based on standard UNMC procedures.  Should they become 
available prior to study initiation, these procedures will be harmonized with those of 
SPIROMICS as much as possible.  This will help with cross-study comparisons and will 
simplify staff training and quality control measures. 
 
 
5.5  Study drug.  Study drug, ibuprofen and matching placebo, will be prepared 
by an experienced vendor.  This is tentatively planned to be Temple University Hospital 
Pharmacy.  Medication will be packaged with a coded label and shipped to each study 
site.  Randomization of subjects at each site will be determined by the statistician at the 
DCC. 
 
 
5.6.  Study materials.  Study materials will be provided to the study sites by the 
DCC.  The materials provided will include data collection forms, sample tubes, coded 


 
 
Rennard, Stephen I. 
 40 
labels and consumables.  In addition, kits will be prepared for the processing of BAL 
and induced-sputum specimens.  These will include reagents needed for cell culture 
and harvest.  Each site is adequately equipped with the required equipment, e.g. 
spirometer, nebulizer, endoscopy suite, centrifuge, incubator, CT scanner, etc. 
 
 
5.7  Quality Control Measures.  Quality control measures will be established 
and included in the Study Procedures Manual.  These measures will include real time 
measures of quality for spirometry, CT Scanning, induced sputum and BAL.  The CT 
quality control measures will be the same as those implemented in COPDGene.  The 
BAL measures will include quantification of fluid return, cell return and cytology of 
bronchial and alveolar specimens.  In addition, although personnel at all study sites are 
highly experienced, the Study Principal Investigator will directly observe the initial 
bronchoscopy and BAL, including sample processing that will be performed at each site.  
Should it be necessary, subsequent procedures will also be directly observed. 
 
 
5.8  Subject recruitment.  COPD subjects and current and ex-smoking controls 
will be recruited at each study site from eligible participants in COPDGene and other 
subjects with qualifying CT scans.  The COPDGene Data Center (NJH) will be notified 
of subjects entered in the study using the COPDGene Study code, which is de-identified. 
The COPDGene Data Center will then transfer the data needed for the PIE study to the 
DCC at UNMC using only the COPDGene Study Code.  Neither the COPDGene Data 
Center nor the PIE DCC at UNMC will have access to subject identifiers.  Healthy non-
smoking control subjects will be recruited at the study sites and de-identified data sent 
to the DCC at UNMC. 
 
 


 
 
Rennard, Stephen I. 
 41 
6.  Regulatory Issues 
 
 
 
6.1  IRB approvals.  The study will be approved by the human studies 
committee (Institutional Review Board, IRB) at all study sites.  In addition, approval will 
be obtained from the IRBs at UNMC and at NJH (the COPDGene Data Center and the 
PIE Radiology Reading Center).  Final approval of the protocol will also be obtained 
from the COPDGene Steering Committee.  Preliminary interviews with the IRB at 
UNMC have indicated that the limited analysis of the miR-146a gene does not constitute 
genetic testing, and no separate genetics consent will be requested.  This question, 
however, will be revisited with all study site IRBs prior to study initiation.   
 
 
6.2  FDA approval.  Pre-IND consultation with the FDA will be requested.  The 
medication to be used, ibuprofen, is already approved and usage will be within currently 
approved dosing.  However, the indication in the current proposal is significantly 
different from approved indications, interstate transport of the drug and placebo will be 
required and the study involves more than minimal risk.  Thus an investigator 
Investigational New Drug (IND) Approval may be required.  If so, an investigator IND will 
be submitted to the FDA.  It is anticipated that this process will be completed prior to the 
scheduled date for funding approval. 
 
 
6.3  Study registration.  The study will be registered with ClinicalTrials.gov. 
 
 


 
 
Rennard, Stephen I. 
 42 
7.  References 
 
 
 
1. 
Minino, A., Xu, J. & Kochanek, M. Deaths: Preliminary Data for 2008. National Vital 
Statistics Report 59, 1-71 (2010). 
2. 
Shapiro, S.D., Reilly, J.J.J. & Rennard, S.I. Chronic Bronchitis and Emphysema. in 
Textbook of Respiratory Medicine, Vol. I (ed. Robert J. Mason, V.C.B., Thomas R. 
Martin, Talmadge E. King, Jr., Dean E. Schraufnagel, John F. Murray and Jay A. Nadel) 
919-967 (Saunders Philadelphia, 2010). 
3. 
Global Strategy for Diagnosis, Management and Prevention of COPD. (2010). 
4. 
Rennard, S.I. Treatment of stable chronic obstructive pulmonary disease. Lancet 364, 
791-802 (2004). 
5. 
Anthonisen, N.R., Connett, J.E. & Murray, R.P. Smoking and lung function of Lung 
Health Study participants after 11 years. Am J Respir Crit Care Med 166, 675-9 
(2002). 
6. 
Kohansal, R., Martinez-Camblor, P., Agusti, A., Buist, A.S., Mannino, D.M. & Soriano, 
J.B. The natural history of chronic airflow obstruction revisited: an analysis of the 
Framingham offspring cohort. Am J Respir Crit Care Med 180, 3-10 (2009). 
7. 
Ohnishi, K., Takagi, M., Yoshimochi, K., Satomi, S. & Konttinen, Y.T. Matrix 
metalloproteinase mediated extracellular matrix protein degradation in human 
pulmonary emphysema. Lab Invest 78, 1077-1087 (1998). 
8. 
Patel, B.D., Coxson, H.O., Pillai, S.G., Agusti, A.G., Calverley, P.M., Donner, C.F., Make, 
B.J., Muller, N.L., Rennard, S.I., Vestbo, J., Wouters, E.F., Hiorns, M.P., Nakano, Y., 
Camp, P.G., Nasute Fauerbach, P.V., Screaton, N.J., Campbell, E.J., Anderson, W.H., 
Pare, P.D., Levy, R.D., Lake, S.L., Silverman, E.K. & Lomas, D.A. Airway wall thickening 
and emphysema show independent familial aggregation in chronic obstructive 
pulmonary disease. Am J Respir Crit Care Med 178, 500-5 (2008). 
9. 
Shapiro, S.D., Snider, G.L. & Rennard, S.I. Chronic Bronchitis and Emphysema. in 
Textbook of Respiratory Medicine, Vol. 1 (ed. Mason, R.J., Broadus, V.C.,  Murray, J.F., 
and Nadel, J.A.) 1115-1167 (Elsevier, Philadelphia, 2005). 
10. 
Rennard, S. Defective Repair in COPD: the American Hypothesis. in Long-Term 
Intervention in Chronic Obstructive Pulmonary Disease, Vol. 191 (eds. Pauwels, R.A. & 
Postma, D.S.) 165-200 (Marcel Dekker, New York, 2004). 
11. 
Kasahara, Y., Tuder, R.M., Taraseviciene-Stewart, L., Le Cras, T.D., Abman, S., Hirth, 
P.K., Waltenberger, J. & Voelkel, N.F. Inhibition of VEGF receptors causes lung cell 
apoptosis and emphysema. J Clin Invest 106, 1311-9. (2000). 
12. 
Sahebjami, H. Lung mechanics and ultrastructure in prolonged starvation. Am Rev 
Respir Dis 117, 77 (1978). 
13. 
Massaro, D., Massaro, G.D., Baras, A., Hoffman, E.P. & Clerch, L.B. Calorie-related 
rapid onset of alveolar loss, regeneration, and changes in mouse lung gene 
expression. Am J Physiol Lung Cell Mol Physiol 286, L896-906 (2004). 
14. 
Coxson, H.O., Chan, I.H., Mayo, J.R., Hlynsky, J., Nakano, Y. & Birmingham, C.L. Early 
emphysema in patients with anorexia nervosa. Am J Respir Crit Care Med 170, 748-
52 (2004). 


 
 
Rennard, Stephen I. 
 43 
15. 
Stein, J. & Fenigstein, H. Anatomie pathologique de la maladie de famine. in Maladie 
de famine. (ed. Apfelbaum, E.) 21-27 (American Joint Distribution Committee, 
Warsaw, Poland, 1946). 
16. 
Riley, D.J., Kramer, M.J., Kerr, J.S., Chae, C.U., Yu, S.Y. & Berg, R.A. Damage and repair 
of lung connective tissue in rats exposed to toxic levels of oxygen. Am Rev Respir Dis 
135, 441-447 (1987). 
17. 
Hoidal, J.R., Niewoehner, D.E., Rao, N.V. & Hibbs, M.S. The role of neutrophils in the 
development of cadmium chloride-induced emphysema in lathyrogen-fed hamsters. 
Am J Pathol 120, 22-29 (1985). 
18. 
Kuhn, C. & Starcher, B.C. The effect of lathyrogens on the evolution of elastase-
induced emphysema. Am Rev Respir Dis 122, 453-460 (1980). 
19. 
Hoidal, J.R. & Niewoehner, D.E. Cigarette smoke inhalation potentiates elastase-
induced emphysema in hamsters. Am. Rev. Respir. Dis. 127, 478-481 (1983). 
20. 
Cardoso, W.V., Sekhon, H.S., Hyde, D.M. & Thurlbeck, W.M. Collagen and elastin in 
human pulmonary emphysema. Am Rev Respir Dis 147, 975-981 (1993). 
21. 
Massaro, G.D. & Massaro, D. Postnatal treatment with retinoic acid increases the 
number of pulmonary alveoli in rats. Am J Physiol 270, L305-L3l0 (1996). 
22. 
Massaro, D. & Massaro, G.D. Pulmonary alveolus formation: critical period, retinoid 
regulation and plasticity. Novartis Found Symp 234, 229-36; discussion 236-41 
(2001). 
23. 
Hegab, A.E., Kubo, H., Yamaya, M., Asada, M., He, M., Fujino, N., Mizuno, S. & 
Nakamura, T. Intranasal HGF administration ameliorates the physiologic and 
morphologic changes in lung emphysema. Mol Ther 16, 1417-26 (2008). 
24. 
Shigemura, N., Sawa, Y., Mizuno, S., Ono, M., Ohta, M., Nakamura, T., Kaneda, Y. & 
Matsuda, H. Amelioration of pulmonary emphysema by in vivo gene transfection 
with hepatocyte growth factor in rats. Circulation 111, 1407-14 (2005). 
25. 
Ishizawa, K., Kubo, H., Yamada, M., Kobayashi, S., Numasaki, M., Ueda, S., Suzuki, T. & 
Sasaki, H. Bone marrow-derived cells contribute to lung regeneration after elastase-
induced pulmonary emphysema. FEBS Lett 556, 249-52 (2004). 
26. 
Yildirim, A.O., Muyal, V., John, G., Muller, B., Seifart, C., Kasper, M. & Fehrenbach, H. 
Palifermin induces alveolar maintenance programs in emphysematous mice. Am J 
Respir Crit Care Med 181, 705-17. 
27. 
Murakami, S., Nagaya, N., Itoh, T., Iwase, T., Fujisato, T., Nishioka, K., Hamada, K., 
Kangawa, K. & Kimura, H. Adrenomedullin regenerates alveoli and vasculature in 
elastase-induced pulmonary emphysema in mice. Am J Respir Crit Care Med 172, 
581-9 (2005). 
28. 
Vadivel, A., Abozaid, S., van Haaften, T., Sawicka, M., Eaton, F., Chen, M. & Thebaud, B. 
Adrenomedullin promotes lung angiogenesis, alveolar development, and repair. Am 
J Respir Cell Mol Biol 43, 152-60. 
29. 
Holz, O., Zuhlke, I., Jaksztat, E., Muller, K.C., Welker, L., Nakashima, M., Diemel, K.D., 
Branscheid, D., Magnussen, H. & Jorres, R.A. Lung fibroblasts from patients with 
emphysema show a reduced proliferation rate in culture. Eur Respir J 24, 575-9 
(2004). 
30. 
Noordhoek, J.A., Postma, D.S., Chong, L.L., Vos, J.T., Kauffman, H.F., Timens, W. & van 
Straaten, J.F. Different proliferative capacity of lung fibroblasts obtained from 
control subjects and patients with emphysema. Exp Lung Res 29, 291-302 (2003). 


 
 
Rennard, Stephen I. 
 44 
31. 
Nobukuni, S., Watanabe, K., Inoue, J., Wen, F.Q., Tamaru, N. & Yoshida, M. Cigarette 
smoke inhibits the growth of lung fibroblasts from patients with pulmonary 
emphysema. Respirology 7, 217-23 (2002). 
32. 
Plantier, L., Rochette-Egly, C., Goven, D., Boutten, A., Bonay, M., Leseche, G., Fournier, 
M., Crestani, B. & Boczkowski, J. Dysregulation of elastin expression by fibroblasts in 
pulmonary emphysema: role of cellular retinoic acid binding protein 2. Thorax 63, 
1012-7 (2008). 
33. 
Plantier, L., Marchand-Adam, S., Marchal-Somme, J., Leseche, G., Fournier, M., 
Dehoux, M., Aubier, M. & Crestani, B. Defect of hepatocyte growth factor production 
by fibroblasts in human pulmonary emphysema. Am J Physiol Lung Cell Mol Physiol 
288, L641-7 (2005). 
34. 
Togo, S., Holz, O., Liu, X., Sugiura, H., Kamio, K., Wang, X., Kawasaki, S., Ahn, Y., 
Fredriksson, K., Skold, C.M., Mueller, K.C., Branscheid, D., Welker, L., Watz, H., 
Magnussen, H. & Rennard, S.I. Lung fibroblast repair functions in patients with 
chronic obstructive pulmonary disease are altered by multiple mechanisms. Am J 
Respir Crit Care Med 178, 248-60 (2008). 
35. 
Bozyk, P.D. & Moore, B.B. Prostaglandin E2 and the Pathogenesis of Pulmonary 
Fibrosis. Am J Respir Cell Mol Biol. 
36. 
Prakash, U. Bronchoscopy. Vol. 1 617-650 (Elsevier, Philadelphia, 2005). 
37. 
Borok, Z., Gillissen, A., Buhl, R., Hoyt, R.F., Hubbard, R.C., Ozaki, T., Rennard, S.I. & 
Crystal, R.G. Augmentation of functional prostaglandin E levels on the respiratory 
epithelial surface by aerosol administratin of prostaglandin E. Am Rev Respir Dis 144, 
1080-1084 (1991). 
38. 
Maher, T.M., Evans, I.C., Bottoms, S.E., Mercer, P.F., Thorley, A.J., Nicholson, A.G., 
Laurent, G.J., Tetley, T.D., Chambers, R.C. & McAnulty, R.J. Diminished prostaglandin 
E2 contributes to the apoptosis paradox in idiopathic pulmonary fibrosis. Am J 
Respir Crit Care Med 182, 73-82. 
39. 
Montuschi, P., Kharitonov, S.A., Ciabattoni, G. & Barnes, P.J. Exhaled leukotrienes and 
prostaglandins in COPD. Thorax 58, 585-8 (2003). 
40. 
Chen, Y., Chen, P., Hanaoka, M., Droma, Y. & Kubo, K. Enhanced levels of 
prostaglandin E2 and matrix metalloproteinase-2 correlate with the severity of 
airflow limitation in stable COPD. Respirology 13, 1014-21 (2008). 
41. 
Profita, M., Sala, A., Bonanno, A., Riccobono, L., Ferraro, M., La Grutta, S., Albano, G.D., 
Montalbano, A.M. & Gjomarkaj, M. Chronic obstructive pulmonary disease and 
neutrophil infiltration: role of cigarette smoke and cyclooxygenase products. Am J 
Physiol Lung Cell Mol Physiol 298, L261-9. 
42. 
Castro, P., Nasser, H., Abrahao, A., Dos Reis, L.C., Rica, I., Valenca, S.S., Rezende, D.C., 
Quintas, L.E., Cavalcante, M.C., Porto, L.C. & Koatz, V.L. Aspirin and indomethacin 
reduce lung inflammation of mice exposed to cigarette smoke. Biochem Pharmacol 
77, 1029-39 (2009). 
43. 
Roh, G.S., Yi, C.O., Cho, Y.J., Jeon, B.T., Nizamudtinova, I.T., Kim, H.J., Kim, J.H., Oh, Y.M., 
Huh, J.W., Lee, J.H., Hwang, Y.S., Lee, S.D. & Lee, J.D. Anti-inflammatory effects of 
celecoxib in rat lungs with smoke-induced emphysema. Am J Physiol Lung Cell Mol 
Physiol 299, L184-91. 
44. 
Rennard, S.I., Ghafouri, M., Thompson, A.B., Linder, J., Vaughan, W., Jones, K., Ertl, R.F., 
Christensen, K., Prince, A., Stahl, M.G. & Robbins, R.A. Fractional processing of 


 
 
Rennard, Stephen I. 
 45 
sequential bronchoalveolar lavage to separate bronchial and alveolar samples. Am 
Rev Respir Dis 141, 208-217 (1990). 
45. 
Reynolds, h. Analysis of cellular and protein content of broncho-alveolar lavage fluid 
from patients wwith idiopathic pulmonary. J of Clin Invest (1977). 
46. 
Sato, T., Liu, X., Nelson, A., Nakanishi, M., Kanaji, N., Wang, X., Kim, M., Li, Y., Sun, J., 
Michalski, J., Patil, A., Basma, H., Holz, O., Magnussen, H. & Rennard, S.I. Reduced 
miR-146a increases prostaglandin E in chronic obstructive pulmonary disease 
fibroblasts. Am J Respir Crit Care Med 182, 1020-9. 
47. 
Weiler, Z., Sato, T., Nelson, A., Liu, X., Gunji, Y., Muller, K.-C., Magnussen, H., Rabe, K.F., 
Rennard, S. & Levan, T.D. Identification of Single Nucleotide Polymorphisms in 
MicroRNA 146a. Am. J. Respir. Crit. Care Med 183, A3526 (2011). 
48. 
Bitterman, P.B., Wewers, M.D., Rennard, S.I., Adelberg, S. & Crystal, R.G. Modulation 
of alveolar macrophage-driven fibroblast proliferation by alternative macrophage 
mediators. J Clin Invest 77, 700-708 (1986). 
49. 
Elias, J.A., Ferro, T.J. & Rossman, M.D. Differential prostaglandin production by 
unfractionated and density-fractionated human monocytes and alveolar 
macrophages. J Leukoc Bio, 114-121 (1987). 
50. 
Smyth, E.M., Burke, A. & FitzGerald, G.A. Lipid-derived autocoids: eicosanoids and 
platelet-activating factor. in Goodman and Gilman's Pharmacology (McGraw-Hill, 
2009). 
51. 
McKeever, T.M., Lewis, S.A., Smit, H.A., Burney, P., Britton, J.R. & Cassano, P.A. The 
association of acetaminophen, aspirin, and ibuprofen with respiratory disease and 
lung function. Am J Respir Crit Care Med 171, 966-71 (2005). 
52. 
Vestbo, J., Anderson, W., Coxson, H.O., Crim, C., Dawber, F., Edwards, L., Hagan, G., 
Knobil, K., Lomas, D.A., Macnee, W., Silverman, E.K. & Tal-Singer, R. Evaluation of 
COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE). Eur 
Respir J 31, 869-873 (2008). 
53. 
Kuwano, K., Matsuba, K., Ikeda, T., Murakami, J., Araki, A., Nishitani, H., Ishida, T., 
Yasumoto, K. & Shigematsu, N. The diagnosis of mild emphysema:  correlation of 
computed tomography and pathology scores. Am Rev Respir Dis 141, 169-178 
(1990). 
54. 
Newell, J.D., Jr. Quantitative computed tomography of lung parenchyma in chronic 
obstructive pulmonary disease: an overview. Proc Am Thorac Soc 5, 915-8 (2008). 
55. 
Coxson, H.O. Quantitative chest tomography in COPD research: chairman's summary. 
Proc Am Thorac Soc 5, 874-7 (2008). 
56. 
Chambers, R.C. & Laurent, G.J. Collagens. in The Lung: Scientific Foundations (eds. 
Crystal, R., West, J., Weibel, E. & Barnes, P.) 709-728 (Lippincott-Raven, Philadelphia, 
1997). 
57. 
Rennard, S.I. & Sharp, J.G. Mesenchymal Cells of the Lung. in Chronic Obstructive 
Pulmonary Disease (ed. Stockley, R., Rennard, S., Rabe, K., Celli, B) 308-318 
(Blackwell, Malden, 2007). 
58. 
Stoffel, K., Engler, H., Kuster, M. & Riesen, W. Changes in biochemical markers after 
lower limb fractures. Clin Chem 53, 131-4 (2007). 
59. 
Brown, J.E., McCloskey, E.V., Dewar, J.A., Body, J.J., Cameron, D.A., Harnett, A.N., 
Ruutu, M., Purohit, O.P., Tahtela, R. & Coleman, R.E. The use of bone markers in a 6-


 
 
Rennard, Stephen I. 
 46 
week study to assess the efficacy of oral clodronate in patients with metastatic bone 
disease. Calcif Tissue Int 81, 341-51 (2007). 
60. 
McClung, M.R., San Martin, J., Miller, P.D., Civitelli, R., Bandeira, F., Omizo, M., Donley, 
D.W., Dalsky, G.P. & Eriksen, E.F. Opposite bone remodeling effects of teriparatide 
and alendronate in increasing bone mass. Arch Intern Med 165, 1762-8 (2005). 
61. 
Blaschke, E., Eklund, A. & Hernbrand, R. Extracellular matrix components in 
bronchoalveolar lavage fluid in sarcoidosis and their relationship to signs of 
alveolitis. Am Rev Respir Dis 141, 1020-5 (1990). 
62. 
Luisetti, M., Bulgheroni, A., Bacchella, L., Pasturenzi, L. & Aprile, C. Elevated serum 
procollagen III aminopeptide levels in sarcoidosis. Chest 98, 1414-20 (1990). 
63. 
Harrison, N.K., McAnulty, R.J., Kimpton, W.G., Fraser, J.R., Laurent, T.C. & Laurent, G.J. 
Heterogeneity of type III procollagen N-terminal peptides in BAL fluid from normal 
and fibrotic lungs. Eur Respir J 6, 1443-8 (1993). 
64. 
Pohl, W.R., Thompson, A.B., Kohn, H., Losch, S., Umek, H., Legenstein, E., Kummer, F., 
Rennard, S.I. & Klech, H. Serum procollagen III peptide levels in subjects with 
sarcoidosis:  A five-year follow-up study. Am Rev Respir Dis 145, 412-417 (1992). 
65. 
Teschler, H., Thompson, A.B., Pohl, W.R., Konietzko, N., Rennard, S.I. & Costabel, U. 
Bronchoalveolar lavage procollagen-III-peptide in recent onset hypersensitivity 
pneumonitis: correlation with extracellular matrix components. Eur Respir J 6, 709-
14 (1993). 
66. 
Armstrong, L., Thickett, D.R., Mansell, J.P., Ionescu, M., Hoyle, E., Billinghurst, R.C., 
Poole, A.R. & Millar, A.B. Changes in collagen turnover in early acute respiratory 
distress syndrome. Am J Respir Crit Care Med 160, 1910-5 (1999). 
67. 
Marshall, R.P., Bellingan, G., Webb, S., Puddicombe, A., Goldsack, N., McAnulty, R.J. & 
Laurent, G.J. Fibroproliferation occurs early in the acute respiratory distress 
syndrome and impacts on outcome. Am J Respir Crit Care Med 162, 1783-8 (2000). 
68. 
Meduri, G.U., Tolley, E.A., Chinn, A., Stentz, F. & Postlethwaite, A. Procollagen types I 
and III aminoterminal propeptide levels during acute respiratory distress syndrome 
and in response to methylprednisolone treatment. Am J Respir Crit Care Med 158, 
1432-41 (1998). 
69. 
Mio, T., Romberger, D.J., Thompson, A.B., Robbins, R.A., Heires, A. & Rennard, S.I. 
Cigarette smoke induces interleukin-8 release from human bronchial epithelial cells. 
Am J Respir Crit Care Med 155, 1770-1776 (1997). 
70. 
Sethi, S., Maloney, J., Grove, L., Wrona, C. & Berenson, C.S. Airway inflammation and 
bronchial bacterial colonization in chronic obstructive pulmonary disease. Am J 
Respir Crit Care Med 173, 991-8 (2006). 
71. 
Matsuoka, T., Hirata, M., Tanaka, H., Takahashi, Y., Murata, T., Kabashima, K., 
Sugimoto, Y., Kobayashi, T., Ushikubi, F., Aze, Y., Eguchi, N., Urade, Y., Yoshida, N., 
Kimura, K., Mizoguchi, A., Honda, Y., Nagai, H. & Narumiya, S. Prostaglandin D2 as a 
mediator of allergic asthma. Science 287, 2013-7 (2000). 
72. 
Shiraishi, Y., Asano, K., Nakajima, T., Oguma, T., Suzuki, Y., Shiomi, T., Sayama, K., 
Niimi, K., Wakaki, M., Kagyo, J., Ikeda, E., Hirai, H., Yamaguchi, K. & Ishizaka, A. 
Prostaglandin D2-induced eosinophilic airway inflammation is mediated by CRTH2 
receptor. J Pharmacol Exp Ther 312, 954-60 (2005). 
73. 
Stebbins, K.J., Broadhead, A.R., Baccei, C.S., Scott, J.M., Truong, Y.P., Coate, H., Stock, 
N.S., Santini, A.M., Fagan, P., Prodanovich, P., Bain, G., Stearns, B.A., King, C.D., 


 
 
Rennard, Stephen I. 
 47 
Hutchinson, J.H., Prasit, P., Evans, J.F. & Lorrain, D.S. Pharmacological blockade of the 
DP2 receptor inhibits cigarette smoke-induced inflammation, mucus cell metaplasia, 
and epithelial hyperplasia in the mouse lung. J Pharmacol Exp Ther 332, 764-75. 
74. 
Kohyama, T., Liu, X.D., Wen, F.Q., Kim, H.J., Takizawa, H. & Rennard, S.I. 
Prostaglandin D2 inhibits fibroblast migration. Eur Respir J 19, 684-689 (2002). 
75. 
Kohyama, T., Wyatt, T.A., Liu, X., Wen, F.Q., Kobayashi, T., Fang, Q., Kim, H.J. & 
Rennard, S.I. PGD2 modulates fibroblast-mediated native collagen gel contraction. 
Am J Respir Cell Mol Biol 27, 375-381 (2002). 
76. 
Montuschi, P. Exhaled breath condensate analysis in patients with COPD. Clin Chim 
Acta 356, 22-34 (2005). 
77. 
Solomon, D.H., Avorn, J., Sturmer, T., Glynn, R.J., Mogun, H. & Schneeweiss, S. 
Cardiovascular outcomes in new users of coxibs and nonsteroidal antiinflammatory 
drugs: high-risk subgroups and time course of risk. Arthritis Rheum 54, 1378-89 
(2006). 
78. 
Solomon, D.H., Glynn, R.J., Rothman, K.J., Schneeweiss, S., Setoguchi, S., Mogun, H., 
Avorn, J. & Sturmer, T. Subgroup analyses to determine cardiovascular risk 
associated with nonsteroidal antiinflammatory drugs and coxibs in specific patient 
groups. Arthritis Rheum 59, 1097-104 (2008). 
79. 
Ray, W.A., Varas-Lorenzo, C., Chung, C.P., Castellsague, J., Murray, K.T., Stein, C.M., 
Daugherty, J.R., Arbogast, P.G. & Garcia-Rodriguez, L.A. Cardiovascular risks of 
nonsteroidal antiinflammatory drugs in patients after hospitalization for serious 
coronary heart disease. Circ Cardiovasc Qual Outcomes 2, 155-63 (2009). 
80. 
Trelle, S., Reichenbach, S., Wandel, S., Hildebrand, P., Tschannen, B., Villiger, P.M., 
Egger, M. & Juni, P. Cardiovascular safety of non-steroidal anti-inflammatory drugs: 
network meta-analysis. BMJ 342, c7086. 
81. 
Konstan, M.W., Krenicky, J.E., Finney, M.R., Kirchner, H.L., Hilliard, K.A., Hilliard, J.B., 
Davis, P.B. & Hoppel, C.L. Effect of ibuprofen on neutrophil migration in vivo in cystic 
fibrosis and healthy subjects. J Pharmacol Exp Ther 306, 1086-91 (2003). 
82. 
Konstan, M.W., Byard, P.J., Hoppel, C.L. & Davis, P.B. Effect of high-dose ibuprofen in 
patients with cystic fibrosis. N Engl J Med 332, 848-54 (1995). 
83. 
Lands, L.C., Milner, R., Cantin, A.M., Manson, D. & Corey, M. High-dose ibuprofen in 
cystic fibrosis: Canadian safety and effectiveness trial. J Pediatr 151, 249-54 (2007). 
84. 
Giagoudakis, G. & Markantonis, S.L. Relationships between the concentrations of 
prostaglandins and the nonsteroidal antiinflammatory drugs indomethacin, 
diclofenac, and ibuprofen. Pharmacotherapy 25, 18-25 (2005). 
85. 
McAnulty, S.R., Owens, J.T., McAnulty, L.S., Nieman, D.C., Morrow, J.D., Dumke, C.L. & 
Milne, G.L. Ibuprofen use during extreme exercise: effects on oxidative stress and 
PGE2. Med Sci Sports Exerc 39, 1075-9 (2007). 
86. 
Hazucha, M.J., Madden, M., Pape, G., Becker, S., Devlin, R., Koren, H.S., Kehrl, H. & 
Bromberg, P.A. Effects of cyclo-oxygenase inhibition on ozone-induced respiratory 
inflammation and lung function changes. Eur J Appl Physiol Occup Physiol 73, 17-27 
(1996). 
87. 
Montuschi, P., Macagno, F., Parente, P., Valente, S., Lauriola, L., Ciappi, G., Kharitonov, 
S.A., Barnes, P.J. & Ciabattoni, G. Effects of cyclo-oxygenase inhibition on exhaled 
eicosanoids in patients with COPD. Thorax 60, 827-33 (2005). 


 
 
Rennard, Stephen I. 
 48 
88. 
Regan, E.A., Hokanson, J.E., Murphy, J.R., Make, B., Lynch, D.A., Beaty, T.H., Curran-
Everett, D., Silverman, E.K. & Crapo, J.D. Genetic epidemiology of COPD (COPDGene) 
study design. COPD 7, 32-43. 
89. 
Millatmal, T., Daughton, D., Thompson, A.B., Floreani, A.A., Romberger, D., Epperson, 
K., Larson, L. & Rennard, S.I. Smoking reduction:  an alternative approach for 
smokers who cannot quit. Monaldi Arch Chest Dis 49, 421-424 (1994). 
90. 
Rennard, S.I., Daughton, D., Fujita, J., Oehlerking, M.B., Dobson, J.R., Stahl, M.G., 
Robbins, R.A. & Thompson, A.B. Short-term smoking reduction is associated with 
reduction in measures of lower respiratory tract inflammation in heavy smokers. 
Eur Respir J 3, 752-759 (1990). 
91. 
Thompson, A.B., Mueller, M., Heires, A.J., Bohling, T.L., Daughton, D., Yancey S, Sykes, 
S. & Rennard, S.I. Aerosolized beclomethasone in chronic bronchitis;  improved 
pulmonary function and diminished airway inflammation. Am. Rev. Respir. Dis. 146, 
389-395 (1992). 
92. 
Rennard, S.I., Umino, T., Millatmal, T., Daughton, D.M., Manouilova, L.S., Ullrich, F.A., 
Patil, K.D., Romberger, D.J., Floreani, A.A. & Anderson, J.R. Evaluation of subclinical 
respiratory tract inflammation in heavy smokers who switch to a cigarette-like 
nicotine delivery device that primarily heats tobacco. Nicotine Tob Res 4, 467-76 
(2002). 
93. 
Gauderman, W.J. Sample size requirements for matched case-control studies of 
gene-environment interaction. Stat Med 21, 35-50 (2002). 
94. 
O'Brien, P.C. & Fleming, T.R. A multiple testing procedure for clinical trials. 
Biometrics 35, 549-56 (1979). 
95. 
Dirksen, A., Piitulainen, E., Parr, D.G., Deng, C., Wencker, M., Shaker, S.B. & Stockley, 
R.A. Exploring the role of CT densitometry: a randomised study of augmentation 
therapy in alpha1-antitrypsin deficiency. Eur Respir J 33, 1345-53 (2009). 
96. 
Stolk, J., Putter, H., Bakker, E.M., Shaker, S.B., Parr, D.G., Piitulainen, E., Russi, E.W., 
Grebski, E., Dirksen, A., Stockley, R.A., Reiber, J.H. & Stoel, B.C. Progression 
parameters for emphysema: a clinical investigation. Respir Med 101, 1924-30 
(2007). 
97. 
Stockley, R.A., Parr, D.G., Piitulainen, E., Stolk, J., Stoel, B.C. & Dirksen, A. Therapeutic 
efficacy of alpha-1 antitrypsin augmentation therapy on the loss of lung tissue: an 
integrated analysis of 2 randomised clinical trials using computed tomography 
densitometry. Respir Res 11, 136. 
98. 
Hoffman, E.A., Simon, B.A. & McLennan, G. State of the Art. A structural and 
functional assessment of the lung via multidetector-row computed tomography: 
phenotyping chronic obstructive pulmonary disease. Proc Am Thorac Soc 3, 519-32 
(2006). 
99. 
Martinez, F.J., Foster, G., Curtis, J.L., Criner, G., Weinmann, G., Fishman, A., DeCamp, 
M.M., Benditt, J., Sciurba, F., Make, B., Mohsenifar, Z., Diaz, P., Hoffman, E. & Wise, R. 
Predictors of mortality in patients with emphysema and severe airflow obstruction. 
Am J Respir Crit Care Med 173, 1326-34 (2006). 
100. 
Price, A.L., Kryukov, G.V., de Bakker, P.I., Purcell, S.M., Staples, J., Wei, L.J. & Sunyaev, 
S.R. Pooled association tests for rare variants in exon-resequencing studies. Am J 
Hum Genet 86, 832-8. 


 
 
Rennard, Stephen I. 
 49 
101. 
Wu, M.C., Kraft, P., Epstein, M.P., Taylor, D.M., Chanock, S.J., Hunter, D.J. & Lin, X. 
Powerful SNP-set analysis for case-control genome-wide association studies. Am J 
Hum Genet 86, 929-42. 
102. 
Cao, H., Xiao, L., Park, G., Wang, X., Azim, A.C., Christman, J.W. & van Breemen, R.B. An 
improved LC-MS/MS method for the quantification of prostaglandins E(2) and D(2) 
production in biological fluids. Anal Biochem 372, 41-51 (2008).