Fluticasone

A Randomized Controlled Study to Compare Effects of Inhaled
Fluticasone and Low Dose Deriphyllin® Combination with Inhaled
Fluticasone Alone in Patients with Moderate to Severe
Chronic Obstructive Pulmonary Disease
A DISSERTATION
SUBMITTED TO THE TAMIL NADU Dr. M.G.R. MEDICAL UNIVERSITY,
CHENNAI,
IN PARTIAL FULFILLMENT OF THE REGULATIONS FOR THE AWARD OF
M. D. DEGREE IN PHARMACOLOGY (BRANCH VI) EXAMINATION TO BE
HELD IN MARCH 2009
DEPARTMENT OF PHARMACOLOGY AND CLINICAL PHARMACOLOGY,

CHRISTIAN MEDICAL COLLEGE, VELLORE – 632002.
DECLARATION
I, Dr. Linda Jose P, hereby declare that this dissertation titled “A Randomized
Controlled Study to Compare Effects of Inhaled Fluticasone and Low Dose
Deriphyllin® Combination with Inhaled Fluticasone Alone in Patients with
Moderate to Severe Chronic Obstructive Pulmonary Disease” has been
prepared by me under direct guidance of Dr. Kalpana Ernest, Professor and Head,
Department of pharmacology, and co-guidance of Dr. D. J. Christopher, Professor
and Head, Department of Pulmonary Medicine and Dr. B. S. Ramakrishna,
Professor and Head, Department of Gastrointestinal Sciences, Christian Medical
College, Vellore, in partial fulfillment of university regulations for award of M.D.
Degree in Pharmacology. I have not submitted this dissertation in any part or full to
any other university or towards any other degree.
Place:
Date: Dr. Linda Jose P

Dr Kalpana Ernest M.D.
Professor and Head,
Department of Pharmacology & Clinical Pharmacology
Christian Medical College,
CERTIFICATE
Certified that this dissertation entitled “A Randomized Controlled Study
to Compare Effects Of Inhaled Fluticasone and Low Dose Deriphyllin®
Combination with Inhaled Fluticasone Alone in Patients with Moderate to
Severe Chronic Obstructive Pulmonary Disease” submitted by Dr. Linda Jose P
in partial fulfillment for the award of M.D. degree in Pharmacology, The Tamil
Nadu Dr. M. G. R. Medical University, Chennai, Tamil Nadu, is a bonafide work
done under my guidance and supervision and completed to my satisfaction.
Place:
Date:
Dr Kalpana Ernest M.D.
Dr D J Christopher DTCD,
DNB, FCCP, FRCP.
Professor and Head, .
Department of Pulmonary Medicine,
Christian Medical College.
CERTIFICATE
Certified that that this dissertation entitled “A Randomized Controlled
Study to Compare Effects of Inhaled Fluticasone and Low Dose Deriphyllin®
Nadu Dr M G R Medical University, Chennai, Tamil Nadu, is a bonafide work done
under my co-guidance and supervision and completed to my satisfaction.
Place:
Date: Dr D J Christopher
Dr B.S. Ramakrishna MD, DM,
PhD, FAMS
Professor and Head, .
Department of Gastroenterology &
Hepatology,
Christian Medical College.
CERTIFICATE
Certified that that this dissertation entitled “A Randomized Controlled
Study to Compare Effects of Inhaled Fluticasone and Low Dose Deriphyllin®
Nadu Dr M G R Medical University, Chennai, Tamil Nadu, is a bonafide work done
under my co-guidance and supervision and completed to my satisfaction.
Place: Dr B.S. Ramakrishna

Date:
Acknowledgements
The work carried out in this thesis was a collaborative effort of Pharmacology
Department with Departments of Pulmonary Medicine, Clinical pathology and Wellcome
Research Lab, at Christian Medical College, Vellore. Several people who have helped,
supported or contributed to this thesis deserve acknowledgment at this place.
I express my wholehearted thanks to Dr Kalpana Ernest, Professor and Head,
Department of Pharmacology and my guide, for always being supportive and
encouraging throughout my candidature. I am also thankful to her for allowing me to
pursue my interests in the area of pulmonary pharmacology.
Dr. D J Christopher, Professor and Head, Department of Pulmonary Medicine
and my co-guide - I acknowledge you Sir, with gratitude for allowing me to be a part of
pulmonary research team. I am very thankful to your ideas, suggestions, encouragement
and support throughout my course of study. I am indebted to you for motivating me to
initiate this project and helping me in every possible way throughout my dissertation.
I was fortunate to have the support and guidance of Dr. B.S. Ramakrishna,
Professor and Head, Department of Gastrointestinal Sciences, throughout my work. He
has been a great source of inspiration through his enormous knowledge and willingness
to support scientific research. I wish to express my deep gratitude to him for the
encouragement and invaluable support rendered to me in the fulfillment of this work.
I express my heartfelt thanks to Dr. Sukesh C Nair, Professor, Department of
Clinical Pathology and my co-investigator, for his support throughout this thesis work.
I particularly thank him for the excellent environment of scientific research I enjoyed in
his laboratory.
Dr Jacob Peedicayil, your easy accessibility and constructive critical review of
my efforts was most appreciated. I am grateful to you for sharing your insights, during
our discussions on epigenetics and the innumerable suggestions you gave during the
preparation of the manuscript. I would like to express my sincere gratitude to Dr. Sujith
Chandy, for always being supportive and inculcating interest in research. I am
particularly grateful to him for the time he freely made available from his busy schedule
for discussion and for the valuable inputs he gave to this study. I thank Dr. Anna
Mathew for all her encouragement and support and Dr. Manoj Tyagi, for stimulating
my interest in basic research.
I am overwhelmed with gratitude towards Dr. Denise, Dr. Binu, Mrs Anitha
and other staff in Clinical Pharmacology Unit for teaching me the intricacies of HPLC
and the inputs they provided to my study. To Dr. Gautham T.P, I would like to express
my deep gratitude for his inspiring enthusiasm and generous help in the fulfillment of this
work. I can honestly say that I would still be working, if it were not for your help. I thank
Dr. Joe Varghese, Mr. Kalyan, and Mr. Jayakumar for their guidance. My heart felt
thanks are also to Ms. Abitha for her generous help. I express my gratitude towards Mr.
Jayakanthan for the support provided in the performance of ELISA tests.

I thank all my friends that I had the pleasure to work with: Dr. Bhagwat Gunale
for being a great friend, Dr. Ratna, my colleague for always being supportive to me, Dr.
Sonish, for being there always to help me through my difficult times. I thank Dr.
Dhanasekar, Dr. Babu, Dr. Mannvizhi for their support. I also thank Dr. Meeta, Mrs.
Deepa, Ms Jisha, Ms Divya and other staff in Department of Pulmonary Medicine
for their enthusiasm and active interest in this work. I thank Mr. David Imbakumar
and Mr. Kamal for their assistance in sample processing and analysis. I appreciate their
genuine dedication and enthusiasm to help me at times of need.
I express my heartfelt thanks to Dr. Yasuyuki Nasuhara, Hokkaido University
School of Medicine, Japan, for sending me a soft copy of his work on low dose
theophylline and inspiring my thoughts. My sincere acknowledgments are offered to the
Research committee of the Christian Medical College for approving and facilitating
my study through funding and also to the patients for their enthusiasm to take part in this
study.
There are many others who deserve more than thanks during the time that I was
working for this thesis; I express my gratitude to each one as without them this thesis
would never have been completed
Finally I thank my family, for their love and understanding helped me to undertake
my course of study.
CONTENTS
Topic Page
I. Introduction 1
II. Aim and Objectives 4
III. Review of Literature
Chronic Obstructive Pulmonary Disease 5
Chronic Airway Inflammation in COPD 7
Oxidative stress in COPD 13
Anti-Inflammatory Effects of Corticosteroids 18
Corticosteroid Resistance in COPD 21
Effects of Theophylline in COPD 23
Clinical Implications of the Study 26
IV. Materials and Methods
Study Subjects 27
Study Design 28
Pulmonary Function Test 29
Six Minute Walk Test and Borg Dyspnea Score 30
Sputum Induction 30
Sputum Processing and Cell Count 31
Estimation of IL8 Level in Induced Sputum 32

Estimation of MPO Activity in Induced Sputum 34
Estimation of Plasma Theophylline Concentration by HPLC 35
Sample Size and Sampling 37
Randomization and Blinding 37
Statistical Analysis 37
V. Results
Background and Run-In Period 38
Effect of Treatment on Sputum Inflammatory Variables 40
Effect of Treatment on Lung Function Indices 43
VI. Discussion 47
VII. Conclusions 51
VIII. Summary 52
IX. Bibliography
X. Annexure
List of Tables
List of Figures
Abbreviations
Patient Information Sheet & Informed Consent Form
Case Record Form
Research Committee Approval
Ethics Committee Approval

Introduction …
Introduction:
Chronic Obstructive Pulmonary Disease (COPD) is a preventable and
treatable lung disease with significant extrapulmonary effects. It is a major cause of
chronic morbidity and mortality, being the fourth leading cause of death in the world
and continues to increase in its prevalence (Lopez et al., 2006). Tobacco smoking
continues to be a major cause of COPD (Lange et al., 1992). However, COPD may
also be caused by other environmental factors such as cooking fumes, pollutants,
passive smoking, or other inhaled toxins.
COPD is characterized by progressive airflow limitation manifesting
clinically with shortness of breath, chronic cough and sputum production. The airflow
limitation is associated with an abnormal inflammatory response of the lung to
noxious particles or gases (Rabe et al., 2007). A wide variety of inflammatory
mediators have been shown to be released from activated macrophages and
neutrophils in response to cigarette smoke and other inhaled particles (Barnes, 1999).
These inflammatory mediators attract inflammatory cells from the circulation and
amplify oxidative stress, which enhances the expression of inflammatory genes and
induces structural changes in the airway (Barnes, 2004a). Stimuli that switch on
inflammatory genes do so by changing the chromatin structure of the gene (Adcock et
al., 2005). Chromatin is made up of DNA wound almost twice around histone
proteins. Remodeling of this chromatin structure is through acetylation of the core

histone proteins which allows the chromatin structure to transform from the resting
closed conformation to an activated open form which initiates gene transcription
(Barnes, 2006c). This process of histone acetylation and gene transcription is
reversible with deacetylation of acetylated histones by histone deacetylases (HDACs)
resulting in gene silencing (Barnes et al., 2005).
Inhaled or oral corticosteroids are the most commonly used anti-
inflammatory drugs in COPD (Rabe et al., 2007). Fluticasone, Beclomethasone,
Budesonide and Ciclesonide are the widely prescribed inhaled corticosteroids in our
country. Corticosteroids exerts their anti-inflammatory effects mainly by recruiting
HDACs to the activated transcriptional complex, resulting in deacetylation of
histones, and, thus, a decrease in inflammatory gene transcription (Ito et al., 2005)
However, large controlled trials of inhaled corticosteroids in COPD (Keatings et al.,
1997b, Culpitt et al., 1999, Loppow et al., 2001) showed no reduction in disease
progression, as they did not show significant effect on neutrophil counts, granule
proteins, inflammatory markers or proteases in induced sputum. This may be due to
an active resistance to corticosteroids caused by the oxidative and nitrative stress in
COPD specifically impairing HDACs. Thus new types of non-steroidal anti-
inflammatory treatment are in need. These novel anti inflammatory drugs which
include phosphodiesterase inhibitors, inhibitors of nuclear factor-kappa beta (NF-kB),
inhibitors of p38 mitogen-activated protein kinase, and interleukin- 10 are under
clinical trials (Barnes, 1999).

Deriphyllin Retard is a sustained release formulation that contains etofylline
and theophylline anhydrous. Theophylline has been used as a bronchodilator in the
treatment of COPD for over 70 years, but has lost popularity as better tolerated and
more effective bronchodilators have been introduced. However, new insights into the
molecular action of theophylline have shown that theophylline activates HDAC
activity and therefore suppress the expression of inflammatory genes (Adcock et al.,
2005). Moreover, theophylline significantly reduces the concentrations of 3-
nitrotyrosine and thus the oxidative stress (Hirano et al., 2006). Thus theophylline
would have an anti-inflammatory action in its own right and, of more importance,
may reverse the resistance to the anti-inflammatory effects of corticosteroids.
Moreover, this anti-inflammatory effect is seen at low plasma concentrations of
theophylline (5 to 10 μg/ml) which avoids side effects and drug interactions; making
it an attractive and safe treatment (Culpitt et al., 2002)
This study aims to evaluate the effects of inhaled fluticasone and low
dose Deriphyllin® combination, as compared to inhaled fluticasone monotherapy in
patients with stable moderate to severe COPD. If proven to be effective, it may raise
the possibility that this relatively old and inexpensive medicine may come back into
favour as an anti-inflammatory treatment and may even reverse steroid resistance in
COPD.
Aim & Objectives …
Aim of the study
To compare the effects of inhaled fluticasone and low dose Deriphyllin®
combination against inhaled fluticasone alone in patients with stable, moderate to
severe COPD.
Primary objective: To compare the anti-inflammatory effects of inhaled
fluticasone and low dose Deriphyllin® combination against inhaled fluticasone
monotherapy, in patients with stable moderate to severe COPD, as measured by
changes in
1. Sputum total white cell count
2. Concentration of interleukin8 (IL8) in induced sputum
3. Myeloperoxidase (MPO) activity in induced sputum
Secondary objectives: To compare the improvement in lung function
indices with inhaled fluticasone and low dose Deriphyllin® combination against
inhaled fluticasone monotherapy, in patients with stable, moderate to severe COPD,
as measured by changes in
1. Forced Expiratory Volume in 1 second (FEV1)
2. Dyspnea as measured by Borg dyspnea score(BDS)
3. Exercise tolerance as measured by 6 minute walk test (6 MWT)

Literature review …
Review of Literature:
1. Chronic obstructive pulmonary disease
Chronic obstructive pulmonary disease (COPD) is a chronic
inflammatory disease of the airways with some significant extrapulmonary effects
that may contribute to its severity. Its pulmonary component is characterized by
airflow limitation that is not fully reversible but usually progressive. Weight loss,
nutritional abnormalities and skeletal muscle dysfunction are well-recognized
extrapulmonary effects of COPD.
The chronic airflow limitation characteristic of COPD is
associated with an abnormal inflammatory response of the lung to noxious particles
or gases. This abnormal inflammatory response may induce parenchymal tissue
destruction (resulting in emphysema), and disrupt normal repair and defense
mechanisms (resulting in small airway fibrosis). Thus a new definition has recently
been adopted by the Global Initiative on Obstructive Lung Disease (GOLD 2007)
(Rabe et al., 2007) to encompass the role of chronic inflammation in COPD: COPD is
“a disease state characterized by airflow limitation that is not fully reversible. The
airflow limitation is usually progressive and associated with an abnormal
inflammatory response of the lungs to noxious particles and gases”.
Risk factors for COPD

Cigarette smoking is by far the most commonly encountered risk factor for
COPD (Lange et al., 1992). Other types of tobacco smoking popular in our country
(beedies) are also risk factors for COPD (Jindal et al., 2006), although their risk
relative to cigarette smoking has not been reported. The risk for COPD in smokers is
dose-related (Burrows et al., 1977) depending on the age at starting to smoke, total
pack-years smoked, and current smoking status. Occupational dusts and chemicals
(Hnizdo et al., 2002) are known to cause COPD on their own. Biomass fuels like
wood, animal dung, crop residues, and coal, typically burned in open fires or poorly
functioning stoves, may lead to very high levels of air pollution (Shrestha et al., 2005)
Infections (viral and bacterial) may contribute to the pathogenesis and
progression of COPD, and the bacterial colonization associated with airway
inflammation, may also play a significant role in COPD exacerbations (Seemungal et
al., 2001). Malnutrition and weight loss can reduce respiratory muscle strength and
endurance, apparently by reducing respiratory muscle mass and thus, contribute to the
cachexia seen among COPD patients (Casaburi, 2001).
COPD is a polygenic disease and a classic example of gene-environment
interaction (Silverman et al., 2002). Thus, of two people with the same smoking
history, only one may develop COPD due to differences in genetic predisposition to
the disease, or on how long they live (Molfino, 2007).

2. Chronic airway inflammation in COPD
Pathological changes characteristic of COPD include chronic airway
inflammation causing (a) mucus hypersecretion, (b) chronic obstructive bronchitis
with fibrosis and obstruction of small airways and (c) emphysema with enlargement
of airspaces and destruction of lung parenchyma (Jeffery, 2000).
The chronic airway inflammation characteristic of COPD appears to be an
abnormal inflammatory response of the respiratory tract to irritants such as cigarette
smoke. In COPD patients, lung inflammation is amplified by oxidative stress and an
excess of proteinases, leading to the characteristic pathological changes.
COPD is characterized by a specific pattern of inflammation involving
neutrophils (Stockley, 2002), macrophages (Tetley, 2002), and CD8+lymphocytes
(Saetta et al., 1998).These cells release inflammatory mediators and interact with
structural cells in the airways and lung parenchyma (Barnes, 2004a).
2.1 Inflammatory Cells in COPD
 Neutrophils: Increased neutrophils are found in sputum of normal
smokers. Few neutrophils are seen in lung tissue. It is further increased in
COPD in proportion to disease severity (Williams et al., 2001).They may be
important in mucus hypersecretion and also secrete serine proteases like
neutrophil elastase (NE), cathepsin G, and proteinase-3 which may contribute
to alveolar destruction (Stockley, 2002).

 Macrophages: Increased numbers are seen in airway lumen, lung
parenchyma, and bronchoalveolar lavage fluid (Tetley, 2002). They produce
increased inflammatory mediators including tumor necrosis factor (TNF)–α,
IL-8, LTB4 and proteases (Grashoff et al., 1997) in COPD patients in
response to cigarette smoke and may show defective phagocytosis (Churg et
al., 2003).
 T lymphocytes: Both CD4+ and CD8+ cells are increased in the
airway wall and lung parenchyma, with increase in CD8+:CD4+ ratio.
Increased CD8+ T cells (Tc1) secrete interferon-γ and express the chemokine
receptor CXCR3 (Cosio et al., 2002). CD8+ cells may be cytotoxic to alveolar
cells, contributing to their destruction (Saetta et al., 1998).
 B lymphocytes: Increased in peripheral airways and within lymphoid
follicles, possibly as a response to chronic colonization and infection of the
airways (Hogg et al., 2004).
 Eosinophils: Increased eosinophils are seen in the airway (Gompertz
et al., 2000) and increased eosinophil proteins are present in induced sputum
during exacerbations (Tetley, 2005).
 Epithelial cells: May be activated by cigarette smoke to produce
inflammatory mediators. They are an important source of transforming growth
factor (TGF)-α, which induces local fibrosis (Takizawa, 2003).

2.2 Inflammatory Mediators Involved in COPD
A wide variety of inflammatory mediators have been shown to be
increased in COPD patients which attract inflammatory cells from the circulation
(chemotactic factors), amplify the inflammatory process (pro-inflammatory
cytokines), and induce structural changes (growth factors).
Chemotactic factors:
 Lipid mediators: e.g., Leukotriene B4 (LTB4) is a potent chemo-attractant of
neutrophils and T lymphocytes (Woolhouse et al., 2002).
 Chemokines: e.g., IL-8 is selectively chemo-attractant to neutrophils (Beeh et al.,
2003). It signals via two receptors: a low-affinity CXCR (chemokine receptor)1 and a
high affinity CXCR2 which may also be increased in COPD (Qiu et al., 2003).
Pro-inflammatory cytokines: e.g., Tumor necrosis factor-α (TNF- α), IL-1β,
and IL-6 amplify the inflammatory process of COPD (Keatings et al., 1996). TNF- α
activates NF-kB, which switches on the transcription of inflammatory genes, in
epithelial cells and macrophages and thus contribute to the cachexia of severe COPD
(Yagi et al., 2006).
Growth factors: The elevated concentrations of G-CSF in COPD increase
neutrophil survival and may enhance neutrophilic inflammation (Barnes, 2004a).
Small airway and alveolar epithelial cells of COPD patients show increased
expression of transforming growth factor-ß (TGF-ß) which might play a role in the
characteristic fibrosis in small airways (Ogawa et al., 2007)
.
2.3 Inflammatory Mechanisms in COPD
There is probably a complex interaction between cells and mediators
in COPD, resulting in progressive obstructive changes in small airways and
destruction of lung parenchyma.
Neutrophils secrete serine proteases like neutrophil elastase (NE),
proteinase-3, and cathepsin-G. These are potent stimulants of mucus secretion and
may play an important role in causing emphysema (Stockley, 2002). Macrophages
and neutrophils secrete proteinases, mainly matrix metalloproteinases (MMPs)
(Belvisi et al., 2003). Increased levels of collagenase (MMP-1) and gelatinase B
(MMP-9) have been detected in bronchial lavage fluid of patients with emphysema
(Muroski et al., 2008). Alveolar macrophages also express a unique MMP,
macrophage metalloelastase (MMP-12) (Babusyte et al., 2007). Moreover, cathepsins
K, L, and S which are released from macrophages also have potent elastolytic activity
(Barnes et al., 2003).
Normally, proteolytic enzymes are counteracted by an excess of
antiproteases. The major inhibitors of serine proteases are alpha-1 antitrypsin (α1-AT)
in lung parenchyma (Ying et al., 2002) and airway epithelium–derived secretory
leukoprotease inhibitor (SLPI) (Weldon et al., 2007) in the airways. At least four
tissue inhibitors of MMPs (TIMP1-4) counteract MMPs (Xu et al., 2003, Russell et
al., 2002). In smokers who develop COPD, the production of antiproteases may be
inadequate to neutralize the effects of multiple proteases resulting in amplification of
inflammatory response to irritants (Stockley, 2001, Piccioni et al., 1992)

Table 1: Protease-Antiprotease Imbalance in COPD
Proteases and Antiproteases Involved in COPD
Increased Proteases Decreased Antiproteases
Serine proteases
Neutrophil elastase alpha-1 antitrypsin
Cathepsin G alpha-1 antichymotrypsin
Proteinase 3 Secretory leukoprotease inhibitor
Cysteine proteinases
Cathepsins K, L, S
Matrix metalloproteinases Tissue inhibitors of MMP

(MMPs) (TIMPs)
MMP-8, MMP-9, MMP-12 TIMP1-4
Source : Global Strategy for the Diagnosis, Management and Prevention of COPD,
Global Initiative for Chronic Obstructive Lung Disease (GOLD) 2007. page 26
Available from: http://www.goldcopd.org .
Figure 1: Inflammatory Mechanisms in Chronic
Obstructive Pulmonary Disease
.
Cigarette smoke and other irritants activate alveolar macrophages and airway
epithelial cells which release neutrophil chemotactic factors, including interleukin-8
and leukotriene B4. Neutrophils and macrophages release proteases that break down
connective tissue in the lung parenchyma, resulting in emphysema, and also stimulate
mucus hypersecretion. Proteases are normally counteracted by protease inhibitors.
Cytotoxic T cells (CD8+ lymphocytes) may also be involved in the inflammatory
cascade. MCP-1 denotes monocyte chemotactic protein 1, which is released by and
affects macrophages.
Source: Chest / 117 / 2 / February, 2000 Supplement
3 Oxidative Stress in COPD
Oxidants like reactive oxygen species (ROS) are generated from
activated cells such as macrophages and neutrophils on exposure to cigarette smoke
and other inhaled particulates (MacNee, 2001). ROS rapidly combine with nitric
oxide to form the potent radical peroxynitrite (ONOO -), which itself generates
hydroxy radical (OH-) (MacNee, 2001).
ROS are normally counteracted by antioxidants, both endogenous
(glutathione, uric acid, bilirubin) and exogenous (vitamin C and vitamin E from diet)
(MacNee, 2000)There is evidence for a reduction in antioxidant defenses in patients
with COPD, which may further enhance oxidative stress. Biomarkers like hydrogen
peroxide and 8-isoprostane are increased in the sputum, and exhaled breath of COPD
patients (Montuschi et al., 2000) marking increased oxidative stress in COPD.
Peroxynitrite reacts with tyrosine residues in certain proteins to cause several
deleterious effects (Kanazawa et al., 2003).
 oxidants activate nuclear factor–·B (NF-κB), a transcription factor that orchestrates
the expression of multiple inflammatory genes, including IL-8, TNF-α, and
MMP-9 (Yagi et al., 2006).
 oxidants inactivate antiproteases resulting in protease-antiprotease imbalance
(MacNee, 2005).
 H2O2 directly constricts airway smooth muscle in vitro, and hydroxyl radicals (OH–
) potently induce plasma exudation in airways (Barnes, 2004b).

Figure 2: Oxidative Stress in Chronic Obstructive
Pulmonary Disease
Reactive oxygen species from cigarette smoke or from inflammatory cells have
several damaging effects, including (a)decreased antiprotease defenses; (b) activation
of nuclear factor-kB, resulting in inflammatory gene amplification causing increased
production of the cytokines ( interleukin-8,tumor necrosis factor α )and isoprostanes;
and (c) direct effects on airway functions. O2- denotes super-oxide anion, H2O2:
hydrogen peroxide, OH: hydroxyl radical, and ONOO: peroxynitrite.
Source: NEJM 2000; Volume 343 Number 4 275

3.1 Inflammatory Gene Amplification
Activated macrophages and neutrophils in the airways of COPD
patients, release multiple inflammatory mediators such as nuclear factor-kB (NF-kB)
and activator protein-1 (AP-1) (Barnes, 2004a) which interact and activate structural
changes at the site of inflammation (Barnes 2004b). The increased expression of most
of these inflammatory proteins is regulated at the level of gene transcription by
remodeling the chromatin structure of the gene (Adcock et al., 2005).
Chromatin remodeling and gene expression
Chromatin is composed of DNA and histones, which are basic proteins
that provide the structural backbone of the chromosome. In the resting cell, DNA is
wound tightly around core histones, excluding the binding of the enzyme RNA
polymerase II, which activates gene transcription and the formation of messenger
RNA. This conformation of the chromatin structure is described as closed and is
associated with suppression of gene expression (Barnes, 2006c)
Each core histone has a long N-terminal tail that is rich in lysine residues,
which may become acetylated, thus reducing their charge which allows the chromatin
structure to transform from the resting closed conformation to an activated open form
(Roth et al., 2001) opening the chromatin structure, with unwinding of DNA so that .
RNA polymerase II and basal transcription complexes can now bind to DNA to
initiate gene transcription.(Barnes, 2006c)

Histone acetyl transferases and co-activators
Inflammatory signals such as IL-1b, tumor necrosis factor-α (TNF- α)
or endotoxin, result in acetylation of histones. This results in unwinding of DNA,
binding of TATA box binding protein (TBP), TBP-associated factors and RNA
polymerase II, which then initiates gene transcription (Shapiro, 2005). This requires
the engagement of co-activator molecules such as CREB binding proteins (CBP)
which interact with transcription factors such as CREB (cAMP response element
binding protein), AP-1 and NF-κB and act as the molecular switches that control gene
transcription and all have intrinsic histone acetyl transferases (HAT) activity.(Barnes,
2006c)
Histone deacetylase and co repressors

The histone acetylation and thus gene transcription, is reversible by
histone deacetylases (HDACs). HDACs act as co-repressors along with other co-
repressor proteins, such as nuclear receptor co-repressor (NCoR) and silencing
mediator of retinoid and thyroid hormone receptors (SMRT), (Adcock et al., 2005)
forming a co-repressor complex that deacetylate histones and thus silence gene
expression
Impaired HDAC in COPD
In COPD, increased oxidative stress and cigarette smoke increase
histone acetylation as well as impair the function of HDAC resulting in increased
expression of inflammatory genes (Barnes, 2006d). However, the mechanisms hereby
oxidative stress leads to impairment in activity of HDAC remain to be determined.

Figure 3: Gene regulation by histone acetylation
Co-activator molecules such as CBP interact with transcription factors such as CREB,
AP-1 and NF-κB, resulting in activation of their intrinsic HAT activity. This results in
acetylation (Ac) of core histones, opening up the chromatin structure to allow binding
on RNA polymerase II, which initiates gene transcription.
Source: British Journal of Pharmacology (2006) 148, 247

4. Anti-inflammatory effects of corticosteroids
The current recognition of COPD as a chronic airway inflammation
makes anti-inflammatory therapy both a logical and attractive approach to managing
this condition. Regular treatment with inhaled glucocorticosteroids has been shown to
reduce the frequency of exacerbations and thus improve health status for symptomatic
COPD patients. Current guidelines suggest that inhaled corticosteroids should be
considered in patients with a FEV1 < 50% predicted (Stage III: Severe COPD and
Stage IV: Very Severe COPD) and repeated exacerbations (who have at least one
exacerbation per year) (Rabe et al., 2007).
Mechanism of Action
The anti-inflammatory effects of glucocorticoids include modulation
of cytokine and chemokine production; inhibition of eicosanoid synthesis; marked
inhibition of accumulation of leukocytes in lung tissue; and decreased vascular
permeability (Barnes, 2006b).
Glucocorticoid receptors
Corticosteroids diffuse readily across cell membranes and bind to
glucocorticoid receptors (GR) in the cytoplasm. This results in rapid transport of the
activated GR–corticosteroid complex into the nucleus, where two GR molecules bind
together as a homodimer and binds to DNA at specific sequences in the promoter
region of corticosteroid-responsive genes known as glucocorticoid response elements
(GRE) and modulate gene transcription (Barnes, 2006a)

Anti-inflammatory gene activation by corticosteroids
Corticosteroids switch on several genes that have anti-inflammatory
effects, including annexin-1 (lipocortin-1), SLPI, interleukin-10 (IL-10) and the
inhibitor of NF-kB (IkB-a) (Barnes, 2006c).Corticosteroids also switch on the
synthesis of two proteins that affect inflammatory signal transduction pathways,
glucocorticoid-induced leucine zipper protein (GILZ), which inhibits both NF-kB and
AP-1 (Mittelstadt et al., 2001) and MAP kinase phosphatase-1 (MKP-1), which
inhibits p38 MAP kinase (Barnes, 2006a)
Repression of inflammatory genes by corticosteroids
The major mechanism by which corticosteroids exerts their anti-
inflammatory effects is by inhibiting the synthesis of multiple inflammatory proteins
through suppression of the genes that encode them. This effect occurs through
reversal of the histone acetylation either by binding to CBP or other co-activators and
directly inhibiting their HAT activity (Ito et al., 2000) or more importantly, recruiting
HDAC2 to the activated transcriptional complex, resulting in deacetylation of
histones, and, thus, a decrease in inflammatory gene transcription (Ito et al., 2000)
Non-genomic inhibitory effects on human neutrophil

degranulation
Glucocorticoids have a rapid anti-secretory effect as. it has demonstrated at low
concentration , dexamethasone decreases Ca2+-dependent Chloride secretion in
human bronchial epithelial cells (Urbach et al., 2002)

.
Figure 4: Anti-inflammatory effects of corticosteroids
Interaction of GR with GRE classically leads to an increase in gene transcription
(trans-activation), but negative GRE sites have also been described where binding of
GR leads to gene suppression (cis-repression) Corticosteroids inhibit the effects of
pro inflammatory transcription factors, such as AP-1 and NF-kB, that regulate the
expression of genes that code for many inflammatory proteins like cytokines,
inflammatory enzymes, adhesion molecules and inflammatory receptors (trans-
repression).
Source: Eur Respir J 2006; 27: 415

5. Corticosteroid resistance in COPD
Inhaled corticosteroids provide relatively little therapeutic benefit in
COPD, as there was no reduction in inflammatory cells, cytokines or proteases in
induced sputum even with high doses of inhaled and oral corticosteroids (Keatings et
al., 1997b);(Culpitt et al., 1999);(Loppow et al., 2001). In vitro studies show that
cytokine release from alveolar macrophages is markedly resistant to the anti-
inflammatory effects of corticosteroids in COPD patients , compared to cells from
normal smokers (Culpitt et al., 2003).
Mechanism of corticosteroid resistance
The lack of response to corticosteroids in COPD may be explained, at
least in part, by the effect of cigarette smoking and oxidative stress on HDAC
function (Ito et al., 2005)). Even patients who have stopped smoking continue to have
oxidative stress (Montuschi et al., 2000). Oxidative stress in the presence of increased
nitric oxide production results in the formation of peroxynitrite, which may then
nitrate HDAC and impairs its catalytic effect (Barnes et al., 2005). The reduction in
HDAC also prevents deacetylation of acetylated GR so that corticosteroids are no
longer able to repress their receptor (Barnes, 2006c). Thus it is likely that oxidative
and nitrative stress in COPD specifically impairs HDAC resulting in corticosteroid
resistance
Figure 5: Mechanism of corticosteroid resistance in
COPD
Stimulation of alveolar macrophages activates NF-κB and other transcription factors

to switch on HAT leading to histone acetylation and subsequently to transcription of
genes encoding inflammatory proteins, such as TNF-α, IL-8 and G-CSF.
Corticosteroids reverse this by binding to GR and recruiting HDAC. This reverses the
histone acetylation induced by NF-κB and switches off the activated inflammatory
genes. In COPD patients, cigarette smoke generates oxidative stress (acting through
the formation of peroxynitrite) to impair the activity of HDAC. This reduces the anti-
inflammatory effect of corticosteroids, as HDAC is now unable to reverse histone
acetylation.
Source: Eur Respir J 2006; 27: 422

Reversal of corticosteroid resistance
As oxidative stress and peroxynitrite appear to inhibit HDAC activity
and mimic the defect in HDAC seen in COPD patients, antioxidants (MacNee, 2000),
inhibitors of inducible nitric oxide synthase (iNOS) (MacNee, 2000) or peroxynitrite
scavengers, might be expected to be effective in restoring corticosteroid
responsiveness.
6. Effects of theophylline in COPD

Bronchodilatory effects
At high concentrations, theophylline inhibits cyclic nucleotide
phosphodiesterase (PDEs), thereby preventing breakdown of cyclic AMP and cyclic
GMP to 5¢-AMP and 5¢-GMP, respectively. This will lead to an accumulation of
cyclic AMP and cyclic GP, thereby increasing signal transduction through these
pathways (Rabe et al., 1995).
Another proposed mechanism is the inhibition of cell surface receptors
for adenosine. These receptors modulate adenylyl cyclase activity, and adenosine has
been shown to cause contraction of isolated airway smooth muscle and to provoke
histamine release from airway mast cells (Ramsdell, 1995).
Anti inflammatory effects
Theophylline directly activates HDAC activity and therefore suppresses the
expression of inflammatory genes (Adcock et al., 2005). This effect is seen at
therapeutic concentrations of theophylline (10_6/10_5 M) and is lost at higher

concentrations (10_4 M) (Barnes, 2006c). This is in contrast with corticosteroids, the
effects of which are due to recruitment of HDAC2 to the active transcription site
without direct effect on HDAC activation. Moreover, theophylline significantly
reduces the concentrations of 3-nitrotyrosine, the stable product of peroxynitrite,
which is raised in sputum cells of COPD patients (Hirano et al., 2006) and markedly
reduce the damage of HDAC. This suggests that theophylline might restore HDAC
activity in two ways: by activation of the HDAC and by reducing oxidative stress in
COPD patients.
Potentiation of the anti-inflammatory actions of

corticosteroids
Theophylline alone has a relatively weak anti-inflammatory action as
HDAC are not effective in switching off inflammatory genes unless recruited to the
active inflammatory site by activated glucocorticoid receptors. However low dose
theophylline can restore HDAC activity, and thus may potentiate the anti-
inflammatory actions of corticosteroids. This underlies the benefit of low-dose
theophylline added to low or high doses of inhaled corticosteroids seen in clinical
studies of patients with asthma (Evans et al., 1997). This suggests that theophylline
may “unlock” the resistance to corticosteroids that is seen in patients with COPD and
where impaired responsiveness to corticosteroids therapy is also seen.

Figure 6: Anti-inflammatory effects of theophylline
Corticosteroids activates the glucocorticoid receptors and cause recruitment of
HDACs to the activated transcriptional complex via (GR), resulting in suppression of
inflammatory genes. Theophylline directly activates histone deacetylases
(HDACs),which deacetylate core histones that have been acetylated by the histone
acetyl transferase (HAT) activity of co-activators. This predicts that theophylline and
corticosteroids may have a synergistic effect in repressing inflammatory gene
expression.
Source: Am J Respir Crit Care Med Vol 167. p 815, 2003

Safety of low dose theophylline
A major limitation in the use of theophylline is the frequency of
adverse effects. The most common side effects are headache, nausea and vomiting,
abdominal discomfort, and restlessness.(Sessler, 1990) At high concentrations,
convulsions, cardiac arrhythmias, and death may occur(Shannon, 1999). Some of the
adverse effects of theophylline (central stimulation, gastric secretion, diuresis, and
arrhythmias) may be due to adenosine receptor antagonism while nausea,
gastrointestinal symptoms, and headache are due to inhibition of certain PDEs
(Howell et al., 1990). Moreover theophylline is metabolized in the liver by the P-450
isoenzyme CYP1A2 (Robson et al., 1987) Thus, drugs that inhibit CYP1A2, such as
macrolide and quinolone antibiotics, may increase plasma theophylline
concentrations to levels that produce side effects. These adverse effects of
theophylline tend to occur at plasma concentrations of 10 to 20 mg/L.(Mitenko et al.,
1973) However, the anti-inflammatory effects are seen at low concentrations of
theophylline (5 to 10 mg/L)(Culpitt et al., 2002) (Kobayashi et al., 2004) which
avoids side effects and drug interactions and makes it an attractive safe treatment.
7. Clinical implications of the study
Low dose theophylline may have a unique effect in the treatment of
COPD by restoring reduced HDAC activity to normal levels, thus suppressing
inflammation but also potentially making the patients responsive to orticosteroids.
This treatment would be a relatively cheap and safe approach to the global epidemic
of COPD.
Materials & Methods
…
Materials and Methods:
The study protocol was approved by Institutional Review Board (Research and Ethics
Committees), Christian Medical College, Vellore, India. The study was conducted in
pulmonary function laboratory in the Department of Pulmonary Medicine, Christian
Medical College, and Vellore. The study was carried out as per the rules of
declaration of Helsinki Patients were recruited for the study from December 2007 and
followed up till May 2008.
1. Study Subjects:
Inclusion Criteria:
1. Adult patients aged more than 40 years, having moderate to severe COPD
2. Smokers or Ex-smokers with a history of 10 or more pack-years
3. FEV1 less than 80% and FEV1/FVC < 70% of predicted
4. Post bronchodilator reversibility less than 12 % and 400 ml
Exclusion Criteria:
1. History suggestive of asthma
2. Any significant uncontrolled medical condition other than COPD
3. Recent episode of acute myocardial infarction, cardiac failure or cardiac
arrhythmias within the last 6 months
4. Exacerbation of COPD or respiratory tract infection 6 weeks before screening
5. Home oxygen therapy in last 6 weeks before screening

6. Patients currently using inhaled corticosteroids, long acting β 2 agonist
7. Patients who had prednisolone or other oral corticosteroids more than 10 mg/d
during the past 4 weeks
Informed Consent Procedure:
Information sheet and consent form were explained to patients in the
language they understood and adequate time was given for them to make a decision
as to whether to take part in the study or not. They were also informed of their right to
withdraw from the study at anytime without giving any reasons. Written informed
consent was obtained before commencing the study.
2. Study Design:
It was a randomized, single blind, two arm comparative study. Subjects were
recruited from the outpatient department (OPD) of Pulmonary Medicine, Christian
Medical College and Hospital, Vellore. India
Screening visit
After taking informed consent, a detailed clinical proforma of patient’s history
and physical examination was recorded. Screening spirometry, ECG and 6 MWT (6
minute walk test) were performed. If they were using long acting β2 agonists, inhaled
corticosteroids and/or theophylline, these drugs were discontinued for 1week (run in
period). Subjects were allowed use of rescue medication (inhaled salbutamol with or
without short acting anticholinergics) as and when needed through out the study.
Baseline visit
Patients who met the inclusion criteria and were stable during the run in
period were randomized to the study. Baseline spirometry and 6 minute walk test with
Borg Dyspnea score were performed and blood samples for total and differential
white cell count and induced sputum were collected. The patients were randomized to
one of the two arms of study.
Table 2: Treatment arms of the study
A Inhaled Fluticasone 500 μg + Deriphyllin 150 mg twice a

day
B Inhaled Fluticasone 500 μg twice a day
End of treatment visit
At the end of 4 weeks of treatment, follow up spirometry and 6 minute walk
test with Borg Dyspnea score were performed and blood samples for total and
differential white cell count and induced sputum were collected.
3. Pulmonary Function Tests
Spirometry was done between 7 AM and 10 AM on all the study days. Inhaled
salbutamol and ipratropium were withheld for an eight hour period prior to the test.
spirometry was done with the pneumotachometer type MultiSpiro device. The
spirometer was calibrated every morning before doing any tests. The principal
investigator was trained in effectively performing spirometry before starting the
study. Patients were instructed and trained adequately to perform spirometry with
maximal effort so as to avoid variability due to patient effort. Spirometry was done
according to American Thoracic Society (ATS) guidelines (Brusasco et al., 2005).
4. Six minute walk test and Borg Dyspnea Score
6 MWT was done according to ATS Guidelines (Brooks et al., 2003). Borg’s
10-point scale for dyspnea was administered before and after 6 MWT. A Thirty meter
long straight corridor was used for 6 MWT which was marked at 1 meter intervals.
The corridor had adequate light and ventilation. Patients were asked to walk at their
own pace and as much distance they could cover in 6 minutes. Pulse rate and oxygen
saturation (SpO2) were monitored with a portable pulse oximeter (PALCO,
MEDIAID mIUel no. 340, USA).
5. Sputum Induction
Sputum induction was performed as per the guidelines of European
Respiratory Society (ERS) (Paggiaro et al., 2002). Patients were given detailed and
clear instructions prior to the procedure. Pre bronchodilator FEV 1 was measured
followed by administration of 200 μg inhaled salbutamol. After 10 min, post
bronchodilator FEV1 was measured. Hypertonic saline (3.0%) was aerosolized by
ultrasonic nebuliser ( itama, Japan), with output set at < 1 mL/min. Induction was
performed at 5 minutes intervals for 20 minutes. Patients were asked to cough up
sputum at 5 minute intervals for 20 minutes, and in addition whenever they had the
urge to do so. They were asked to spit saliva into a cup before coughing sputum into
another, to avoid salivary contamination. Expectorated sputum was collected in a
sterile cup, which was immediately placed on ice until processing. Induction was
stopped if symptoms aggravated or if patients encountered significant discomfort.
Patients were monitored in the laboratory until their forced expiratory volume in one
second or peak expiratory flow had returned to within 5% of the baseline value.
5.1 Sputum Processing and Cell count
Sputum processing was performed as per ERS guidelines (Paggiaro et
al., 2002). The collected sputum samples were examined as early as possible or
within two to four hours. The entire expectorate was transferred into a pre-weighed
polystyrene tube and weighed again to estimate the weight of the sputum. It was
mixed with equal volume of 0.1% dithiothreitol (DTT) to break the disulphide bonds
in mucin molecules allowing cells to be released. After mixing well for 10 seconds,
samples were vortexed for 30 minutes at room temperature and then incubated in a
water bath at 370C for 10 minutes. Samples were then filtered through a 100 micron
nylon mesh to remove debris. Total count was measured in an automated
haemocytometer. The cell suspension was then centrifuged at 2000 rpm for 5 minutes
at 40C. Supernatants were removed and stored at -700C until analysis. Cell pellets ere
resuspended in phosphate-buffered saline (PBS) and stored at -70 0C for further
analysis.
6. Estimation of IL8 level in induced sputum (Brightling et al.,
2000)
The samples to be tested for antigen A are coated onto the surface of plastic wells.
Residual sticky sites on the plastic are blocked by adding irrelevant proteins. The
labeled antibody is then added to the wells under conditions, so that only binding to
antigen A causes the labeled antibody to be retained on the surface. Unbound labeled
antibody is removed from all wells by washing, and bound antibody is detected by an
enzyme-dependent color-change reaction.
Materials used ( BD Biosciences Pharminogen)
1. Capture antibody : Anti-human IL-8 monoclonal antibody
2. Detection Antibody : Biotinylated antihuman IL-8 monoclonal antibody
3. Enzyme Reagent : Streptavidin- horseradish peroxidase conjugate (SAv-HRP)
4. Standards : Recombinant human IL-8, lyophilized
5. 96 well ELISA plates
6. Distilled water
7. Microplate reader capable of measuring absorbance at 450 nm
Sputum Sample preparation
1 : 10,000 dilution of sputum supernatants were prepared in PBS.
Standards preparation
Calibrators were prepared by sequential dilutions to obtain concentrations of 200
pg/ml, 100 pg/ml , 50 pg/ml , 25 pg/ml, 12.5 pg/ml , 6.25 pg/ml and 3.125 pg/ml.
Assay procedure
 100 μl of diluted capture antibody was added to each well and incubated
overnight at 4 C.
 Wells were aspirated and washed 3 times.
 Wells were blocked with 200 μl of Assay Diluent per well and incubated at
room temperature (RT) for 1 hour.
 Wells were aspirated and washed 3 times.
 100 μl of standard , sample and control were added to each well and incubated
2 hours in RT.
 Wells were then aspirated and washed 5 times.
 100 μl working detector ( Detection Ab + SAv-HRP) were added to each well
and incubated for 1 hour RT.
 Wells were then aspirated and washed 7 times.
 100 μl of substrate solution were added to each well and incubated for 30
minutes at RT in dark.
 50 μl of stop solution were added to each wells. Absorbance read at 450 nm
Calculations
A standard curve was constructed from the absorbance of the standards on y axis and
IL-8 concentration on the x axis. The best fit curve had a regression coefficient (r) of
0.996.The intercept (b) and slope (m) of the standard curve was calculated.
Concentration of the samples were calculated as per linear regression, y = m.x + b
where b = intercept, m = slope, y = absorbance and x = concentration of the sample

7. Estimation of MPO activity in induced sputum (Llewellyn-Jones et al.,
1996).
Principle:
MPO is an intracellular enzyme localized to neutrophil granules. MPO catalyzes the
breakdown of its substrate, H2O2 to release nascent oxygen and water. Nascent
oxygen oxidizes o-dianisidine to give a yellow coloured product which is measured
by spectrophotometer at 450nm. MPO activity in the sample was expressed as IU/mg
of sputum cells.
Materials used:
1. Cetrimide : Hexadecyl trimethyl ammonium bromide
2. 50mM Phosphate buffer saline (PBS) (pH 6.0)
3. O- dianisidine dihydrochloride (10mg / dL buffer)
4. 1% H2O2 (0.1µ L H2O2 )
5. 96 well Microtiter plates
6. Microplate reader capable of measuring absorbance at 450 nm
Sample preparation
1 ml of cell suspensions were centrifuged at 2000 rpm for 5 minutes
and PBS were removed. The cell pellets were weighed and mixed with phosphate
buffer containing Cetrimide (0.5%) as 1ml to every 50mg sputum cells. Cell
suspensions were freeze thawed for 5 cycles and centrifuged at 2000 rpm for 4
minutes. Supernatants were alliquoted for MPO estimation.

Assay Procedure
 200 μl of phosphate buffer containing O-dianisidine was added to each well.
 50 μl of sample was added to each well in duplicate except for blank.
 50 μl of fresh H2O2 was added to each well simultaneously.
 The absorbance was read at 460nm at an interval of 10 sec for 1 minute.
 Sequential samples for each patient were assayed on the same microtitre plate.
Calculations
A = ε x c x l. Thus, c = A/ ε.l where A = absobance, c = concentration, l = path
length, ε = molar absorbance coefficient of O-d for 1 cm path length = 11.3/
mmol/cm. MPO activity IU / mg sputum cells = ΔA 460 / min /11.3 * 1 / Volume
taken *1 / mg sputum cells.
8. Estimation of plasma theophylline concentration by high

performance liquid chromatography (HPLC)
Chromatographic conditions followed
Waters isocratic discovery column, Temperature: 30ºC, UV detection: 275nm.
Mobile phase: 5 mM acetate buffer (pH- 4.0) and Acetonitrile (ACN) at the ratio of
0.7: 0.3, at the flow rate of 1 ml/mt.
Therapeutic range of theophylline: 10 -20 µg/ml.
Lower limit of quantification: 2µg/ml.

Preparation of standards and samples
Working standards of 2μg/ml, 12µg/ml, and 30µg/ml were prepared from a stock of
20 mg/ 100ml. 16µg/ml was used as quality control (QC)). 5ml of venous blood was
collected from each patient into a test tube containing EDTA and was mixed well. It
was then centrifuged at 1500 rpm and plasma were separated into eppendorf tubes
and stored at -200C.
HPLC setup
The pumps were purged to remove the existing solvent and set up with the mobile
phase at a flow rate of 1ml/minute.
Procedure
200µl of plasma (standards, QC and patients) were pipetted into eppendorfs and
vortexed for 30 seconds. 400µl of ACN was added into each eppendorf to precipitate
the proteins. The eppendorfs were then vortexed for 1 minute in high speed and then
centrifuged at 13,000 rpm for 8 minutes. The supernatant was separated and injected
to HPLC system for analysis. The retention time of the peak was 3 minutes.
Calculation
A standard curve was constructed from the area under curve of the peaks on y axis
and theophylline concentration of the standards on the x axis. The best fit curve had a
regression coefficient (r) of 0.999. The intercept (b) and slope (m) of the standard
curve was calculated. Theophylline concentration in the patient samples were
calculated using the equation y = m.x + b, where b = intercept, m = slope,
y = area under curve of the sample and x = concentration of the sample.

Sample size and Sampling
As we could not find any study that had compared the effect of low dose
theophylline-d inhaled fluticasone combination with inhaled fluticasone alone in patients
with stable moderate COPD in Medline search we planned a pilot study. In consultation
with the statistician we decided to recruit 16 COPD subjects.
Randomization and Blinding

To achieve balance in treatment allocation, simple permuted block
randomization method was used. Pseudo random numbers were generated by computer n
blocks of 4 and 6 sizes. The group allocation was done by a pharmacist from the hospital
pharmacy who was not involved in recruiting patients for the study. Treatment allocation
ratio was 1:1 for the two treatment groups To avoid any observer bias, study drug
administration was done by a third person who was not involved in recruiting patients
and in assessing the bronchodilator response.
Statistical analysis
Baseline data was expressed as the mean + standard deviation (SD). Results were
expressed as the mean + standard error of the mean (SEM). We used SPSS.11 (Statistical
Package for the Social Science, version 11) for data analysis. A probability level less than
0.05 (p<0.05) was considered as statistically significant. The two groups were compared
with Mann Whitney U test. The changes in sputum inflammatory markers and lung
function indices before and after treatment in the groups were compared by Wilcoxson
Signed Ranks Tests. In all the figures in results, the error bars represent SEM.
Results …
Results:
1. Background and run-in period
Twenty-five patients were screened of whom 16 were eligible to be
included in the study. All the patients were males, older than 40 years and were either
current smokers or ex-smokers with more than 10 pack years of smoking. They had
moderate to severe COPD as per the GOLD guidelines (FEV1< 50% of the predicted).
Subjects in the combination group had a mean FEV 1 of 1.21 + 0.57 liters, mean SpO2
of 95.3 + 2.6 % and their mean dyspnea score was 1.5 + 1.5. In the monotherapy
group, mean FEV1 was 1.26 + 0.6 liters, mean SpO2 was 95.7 + 1.1 % and mean
dyspnea score was 0.57 + 0.7.
None of the subjects had a recent exacerbation or was using inhaled
corticosteroids or long acting β 2 agonist during the previous one month. Subjects
who were currently using oral theophylline were given a one week washout period
before being recruited into the study. Plasma theophylline levels were measured at the
baseline and at the end of the study by HPLC. There was one withdrawal from the
combination group as the plasma theophylline level was more than 10 μg/ml at the
baseline. The baseline mean plasma theophylline level was less than 2 μg/ml for the
rest of the study subjects. At the end of the study, it was 4.31 ± 0.1 μg/ml for those in
the combination group and less than 2 μg/ml for those in monotherapy group.
All patients were given salbutamol and ipratropium combination
(Duolin) as rescue medication. One subject in the combination group was excluded as
he contracted a coincidental airway infection during the study period. Eventually, 14
subjects (seven in each arm) completed the study.
Table 3: Baseline characteristics of the patients
(Data expressed as mean +/- standard deviation)

Fluticasone Fluticasone +
group Deriphyllin®
D group
Number of subjects 8 8
Dropouts 1 1
Mean age (years) 65.1 + 13.18 62 + 7.5
Current smoker / Ex smoker 5/2 6/1
Smoking pack years 24.29 + 7.61 30.03 + 14.65
Baseline FEV1(L) 1.26 + 0.6 1.21 + 0.57
D
Baseline FEV1( % of predicted ) 49.07 + 17.01 48.13 + 15.57
6 minute walk distance 315.8 + 51.14 301.1 + 53.9
Baseline Oxygen
95.71 + 1.1 95.29 + 2.6
Saturation(SPO2)
Baseline Borg Dyspnea Score 0.57 + 0.73 1.5 + 1.5
Baseline mean plasma

1.26 + 0.27 1.96 + 1.83
theophylline Level(mg/l)
End of therapy mean plasma
1.51 + 0.1 4.31 ± 0.1
theophylline level(mg/l)
Both groups were comparable. p value > 0.05

2. The effect of treatment on sputum inflammatory variables
Among the patients who received the combination of fluticasone 500 mcg
with Deriphyllin® 150 mg twice a day, MPO activity was significantly decreased at
the end of the treatment when compared to baseline. The MPO level at the end of
therapy was 1.78 + 0.9 IU per g sputum as compared to 4.06 + 0.9 IU per g sputum
at the baseline (p = 0.018) (Table 4; Figure 7). The IL- 8 levels in the sputum also
showed a decrease at the end of the treatment among those who received combination
therapy but the decrease was not statistically significant (26.24 + 12.3 ng/ml vs 15.13
+ 5.6 ng/ml at the baseline, p > 0.05) (Table 4; Figure 8).
In the monotherapy group, the MPO activity in the sputum showed a
minor decrease with the treatment. The MPO level at the end of the treatment was
1.93 + 0.6 IU per gm sputum as compared to 3.51 + 1 IU per gm sputum at the
baseline (p > 0.05) (Table 4; Figure 7). They showed a non significant increase in the
sputum IL-8 level at the end of the treatment (24.16 + 9.5 ng/ml) as compared to the
baseline (19.94 + 8.3 ng/ml),( p > 0.05) (Table 4; Figure 8).
We could not perform a differential cell count of the sputum samples
due to technical difficulties. However, the total cell count in the sputum showed a non
significant decrease with both the treatments. Among the subjects who received
combination therapy the total cell count decreased from 2.10 + 0.7 x 10 6 cells/ml at
baseline to 0.95 + 0.2 x 106 cells/ml at the end of study; while among those who
received monotherapy it decreased from 1.40 + 0.46 x 106 cells/ml to 0.95 + 0.2 x 106
cells/ml (Table 4; Figure 9).

Table 4: Effect of treatment on sputum inflammatory variables
Mean + SEM Pre treatment Post treatment p value
Fluticasone
Total cell count 2.10 + 0.7 0.95 + 0.2 NS

(x 106 cells/ml)
MPO 3.52 + 1 1.93 + 0.6 NS
(IU/g sputum)
IL-8 19.94 + 8.3 24.16 + 9.5 NS
(ng/ml)
Fluticasone with Deriphyllin®
Total cell count 1.40 + 0.46 0.85 + 0.2 NS

(x 106 cells/ml)
MPO 4.06 + 0.9 1.78 + 0.9 0.018
(IU/g sputum)
IL-8 26.24 + 12.37 15.13 + 5.6 NS
(ng/ml)
NS: p value not statistically significant
Figure 7: Effect on MPO activity in induced sputum
6.0
Fluticasone Fluticasone + Deriphyllin®

5.0
4.0
3.0
*
2.0
1.0
0.0
Pre treatment Post treatment Pre treatment Post treatment

(* p = 0.018)
Figure 8: Effect on IL-8 level in induced sputum
40.0

35.0
30.0
25.0
20.0
15.0
10.0
5.0
0.0
Figure 9: Effect on total cell count in induced sputum
3.00

2.50
2.00
1.50
1.00
0.50
0.00

3. The effect of treatment on lung function indices
Subjects who received the combination of fluticasone 500 μg with
Deriphyllin® 150 mg twice a day, showed a non significant increase in mean FEV 1 at
the end of the treatment when compared to baseline. The FEV1 at the end of therapy
was 1.37 + 0.2 l as compared to 1.21 + 0.2 l at the baseline (p = 0.06), (Table 5;
Figure 10). They showed a non significant change in the mean distance covered in 6
minutes (6MWD) at the end of the treatment (318.14 + 41.48 meters vs 301.14 + 53.9
meters at the baseline, p > 0.05) (Table 5; Figure 12). The Borg dyspnea score (BDS)
changed non-significantly among the subjects who received combination therapy
(1.57 + 0.5 vs 1 + 0.2 at the baseline, p > 0.05) (Table 5; Figure 11).
Among the subjects who received monotherapy with fluticasone 500 μg twice
daily, there was a non significant change in the FEV1 (1.26 + 0.23 l vs 1.33 + 0.23 l at
the baseline, p > 0.05) and in the mean 6MWD (318.14+ 41.48 m vs 301.14 + 53.9 m
at the baseline, p > 0.05) (Table 5; Figure 10 and 12). The BDS at the end of study
was 0.57 + 0.2 as compared to 0.5 + 0.2 at the baseline, p > 0.05 (Table 5; Figure11).
Oxygen saturation of the subjects (SpO2) did not show significant change
with either of the treatments. At the end of 4 weeks study, among those who received
combination treatment mean SpO2 was 94.71+1.1 % as compared to 95.2+ 0.9 % at
baseline while it changed from 95.57+ 1.1 % to 95.7+ 0.4 % among those who
received monotherapy (p>0.05) (Table 5; Figure 13).

Table 5: Effect of treatment on Lung function indices
Mean + SEM Pre treatment Post treatment p value
Fluticasone
1.26 + 0.2 1.33 + 0.2 NS

FEV1 (liters)
6 MWD 315.86 + 51.14 325.14 + 51.2 NS

(meters)
0.57 + 0.2 0.5 + 0.2 NS

BDS
95.7 + 0.4 95.57 + 1.1 NS

SpO2 (%)
Fluticasone with Deriphyllin®
1.21 + 0.2 1.37 + 0.2 NS

FEV1 (liters)
6 MWD 301.14 + 53.9 318.14 + 41.48 NS

(meters)
1.57 + 0.5 1 + 0.2 NS

BDS
95.2 + 0.9 94.71 + 1.1 NS

SpO2 (%)
NS: p value not statistically significant

Figure 10: Effect of treatment on FEV 1
1.8

1.6
1.4
1.2
1.0
0.8
0.6
0.4
Figure 11: Effect of treatment on Borg Dyspnea Score
2.00
1.80 Fluticasone Fluticasone + Deriphyllin®
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
0.00

Figure 12: Effects of treatment on 6 minute walk distance
400
350
300
250
200
150
Figure 13: Effect of treatment on SpO2
100
99
98
97
96
95
94
93
92
91
90

Discussion …
Discussion:
COPD is a disease of progressive airflow limitation, characterized by chronic
inflammation in the airways, parenchyma, and pulmonary vasculature. The
pathogenesis of airway obstruction in COPD has been attributed to recruitment of
inflammatory cells like macrophages and neutrophils into the lungs by inhaled
noxious particles, resulting in protease-antiprotease imbalance (Stockley, 1999) and
increased oxidative stress (Repine JE, 1997). These inflammatory cells which include
bronchial epithelial cells (Nakamura et al., 1991), alveolar macrophages and
neutrophils (Takahashi et al., 1993) secrete IL-8 and leukotriene B 4 (LTB 4), the
major chemo attractants which cause recruitment of more neutrophils to the airways
(Richman-Eisenstat et al., 1993). Moreover, the neutrophil activation markers
(constituents of neutrophil granules), myeloperoxidase and lactoferrin, are elevated in
the sputum supernatants of patients with COPD (Keatings et al., 1997a). This
neutrophilic inflammation seen in COPD is associated with rapid decline in forced
expiratory volume in 1 second (FEV1) (Stanescu et al., 1996) and leads to increased
risk of exacerbations and worsening of health status.
The objectives of this randomized, controlled study was to compare the
changes in sputum inflammatory indices and lung function parameters among stable,
moderate to severe COPD patients before and after treatment with fluticasone with
low dose Deriphyllin®, using the control group having fluticasone alone. We
analysed induced sputum to assess changes in inflammatory indices like total cell
count, myeloperoxidase and interleukin (IL)-8 levels caused by both the treatments.
The effect of treatment on lung function indices was measured as improvement in
FEV1, symptom score, 6 minute walk distance and O2 saturation.
The results of this study show that the combination of Deriphyllin®
150 mg-fluticasone 500 μg twice a day significantly decreased the levels of MPO in
sputum. This was in agreement with the results of the previous studies evaluating the
effect of low dose theophylline on myeloperoxidase. Culpitt et al (Culpitt et al., 2002)
who demonstrated in their study that low dose theophylline reduced MPO levels in
induced sputum in moderate to severe COPD patients after 4 weeks of treatment.
Kobayashi et al (Kobayashi et al., 2004) has shown the same result in mild to
moderate COPD.
The IL-8 level in sputum did not change significantly with either
treatment in the present study. This finding is in keeping with the results of the study
by Kobayashi et al (Kobayashi et al., 2004) who did not find a significant decrease in
IL-8 level after treatment with low dose theophylline in his series of patients and
with the results of the study by Culpitt et al (Culpitt et al., 1999) who showed that
fluticasone at a dose of 500 μg twice a day, had no effect on inflammatory indices
including IL-8 in the induced sputum of patients with COPD. On the contrary, Culpitt
et al (Culpitt et al., 2002) , in a separate study among COPD patients demonstrated a
significant decrease in IL-8 level with low dose theophylline at the end of 4 weeks of
treatment. This disparity in results may have been due to the higher mean serum
concentration of theophylline achieved by the patients in the study by Culpitt et al
study in comparison with the present study and that by Kobayashi et al. (The mean
serum concentration of theophylline: 9.5±0.3 μg/ml in the Culpitt et al study, 7.3 ±
2.1 μg/ml in Kobayashi et al study and 4.31 ± 0.1 μg/ml in the present study Thus the
effect of low dose theophylline and inhaled fluticasone treatment on the level of IL-8
in induced sputum is unclear and may need testing larger cohort of patients.
We could not analyze the differential cell count in the samples due to
technical difficulties; however the total cell count showed a trend to decrease with
both treatment arms in the current study. This correlates well with the finding in the
meta-analysis on the effects of inhaled corticosteroids on sputum cell counts in stable
COPD by Wen Qi Gan et al (Gan et al., 2005). They found that inhaled
corticosteroids, at a lower cumulative dose (< 60 mg) or shorter duration of therapy
(< 6 weeks) do not demonstrate a favorable effect on reducing sputum cell count in
stable chronic obstructive pulmonary disease.
Subjects in either group showed non-significant changes in mean
FEVI, the mean distance covered in 6 minutes and the mean dyspnea score (BDS) at
the end of the treatment when compared to baseline. Oxygen saturation (SpO 2) of the
subjects did not show any change with either of the treatments. This may be due to
the relatively shorter duration of our study as D.S Postma has stated in his review
(Postma et al., 1999) that inhaled corticosteroids administered over a period of 3–

12weeks generally do not change the level of airways obstruction, as assessed by
FEV1and peak expiratory flow (PEF). Moreover Kobayashi et al (Kobayashi et al.,
2004) has demonstrated minimal changes in lung function indices after 4 weeks
treatment with low dose theophylline.
There were few limitations in the present study. As the study was
planned as a pilot study we could recruit only 16 patients with moderate to severe
stable COPD and there were two dropouts in the study. We could not perform a
differential cell count of the sputum samples due to lack of a standardized technique.
However, our results are interesting and relevant for three reasons. First,
theophyllines are cheaper drugs compared to both long acting β 2 agonists and inhaled
corticosteroids. Second, systemic administration can make the drug available for the
periphery of the lungs where the concentration of inhaled drugs may be low. Third,
since the anti-inflammatory properties of theophylline are seen at concentrations
lower than those determining the bronchodilating effects, these drugs could be better
tolerated by COPD patients than in the past. Thus, low dose theophylline in
combination with corticosteroid inhalation may be an attractive option available for
controlling neutrophilic airway inflammation in COPD. However, further long term
studies are warranted in order to determine whether this anti-inflammatory effect
contributes to long-term clinical benefits in COPD patients

Conclusion …
Conclusions:
1. The myeloperoxidase level in sputum decreased significantly with the
combination of fluticasone 500 μg and Deriphyllin® 150 mg twice a day
while fluticasone 500 μg twice a day monotherapy caused only a minor
decrease in MPO level .The IL- 8 level showed changes with both therapies
when compared to baseline though the changes were not statistically
significant. Total cell count in the sputum did not change appreciably with
either of the treatments.
2. Subjects who received the combination therapy showed non-significant
increase in mean FEV1 and mean distance covered in 6 minutes with a
decrease in dyspnea score (BDS) at the end of the treatment. In the
monotherapy group also there was minor improvement in these lung function
indices at the end of the treatment. Oxygen saturation (SpO 2) of the subjects
did not show change with either of the treatments.
Thus low-dose Deriphyllin® and inhaled fluticasone combination
treatment for 4 weeks caused a better suppression of inflammatory markers in
moderate to severe COPD patients as compared to inhaled fluticasone monotherapy.
This suggests that low dose theophylline may potentiate the anti-inflammatory effect
of glucocorticoids in the treatment of COPD.

Summary …
Summary:
The efficacy of inhaled corticosteroids on neutrophilic airway
inflammation has not been demonstrated when treating COPD(Culpitt et al., 1999). A
new anti-inflammatory mechanism of action for theophylline has been proposed that
it induces histone deacetylase activity and decreases inflammatory gene expression
(Barnes, 2006c). This action of theophylline may make it possible to contribute to the
anti-inflammatory effects of inhaled corticosteroids in COPD. This is the first study
addressing the issue of low dose theophylline potentiating anti-inflammatory effects
of corticosteroids in COPD patients and thus its effects on lung function indices.
A total of 16 outpatients having stable, moderate to severe COPD were
enrolled in this randomized, controlled study, of whom 14 completed the study. The
study group was given Deriphyllin® 150 mg with fluticasone 500 µg twice a day
while the control group received fluticasone 500 µg twice a day alone. Induced
sputum was collected and analyzed at the baseline and at the end of treatment to
assess inflammatory markers like total cell count, myeloperoxidase and interleukin
(IL)-8 levels. The effect of treatment on lung function indices was measured as
improvement in FEV1, symptom score, 6 minute walk distance and O2 saturation
before and after treatment.
The patients who received the combination therapy, showed a
significant decrease in sputum MPO activity as compared to those in the monotherapy
group. IL-8 level and total cell count in the sputum did not change appreciably with
either of the treatments. They also showed a small but non significant improvement in
the FEV1, symptom score, 6 minute walk distance and O2 saturation after treatment as
compared to those who received monotherapy.
Thus combination of low-dose Deriphyllin® and inhaled fluticasone
given for 4 weeks caused a better suppression of inflammatory markers in moderate
to severe COPD patients as when compared with inhaled fluticasone monotherapy.
This suggests that low dose theophylline may potentiate the anti-inflammatory effect
of glucocorticoids and thus combination of fluticasone and Deriphyllin® might
provide a relatively inexpensive way of managing this common and globally
increasing disease.
Bibliography …
Bibliography:
ADCOCK, I. M., ITO, K. & BARNES, P. J. (2005) Histone deacetylation: an
important mechanism in inflammatory lung diseases. Copd, 2, 445-55.
BABUSYTE, A., STRAVINSKAITE, K., JEROCH, J., LOTVALL, J.,
SAKALAUSKAS, R. & SITKAUSKIENE, B. (2007) Patterns of airway
inflammation and MMP-12 expression in smokers and ex-smokers with
COPD. Respir Res, 8, 81.
BARNES, P. J. (1999) Novel approaches and targets for treatment of chronic
obstructive pulmonary disease. Am J Respir Crit Care Med, 160, S72-9.
BARNES, P. J. (2004a) Mediators of chronic obstructive pulmonary disease.
Pharmacol Rev, 56, 515-48.
BARNES, P. J. (2004b) Small airways in COPD. N Engl J Med, 350, 2635-7.
BARNES, P. J. (2006a) Corticosteroid effects on cell signalling. Eur Respir J,
27, 413-26.
BARNES, P. J. (2006b) Corticosteroids: the drugs to beat. Eur J Pharmacol,
533, 2-14.
BARNES, P. J. (2006c) How corticosteroids control inflammation: Quintiles
Prize Lecture 2005. Br J Pharmacol, 148, 245-54.
BARNES, P. J. (2006d) Reduced histone deacetylase in COPD: clinical
implications. Chest, 129, 151-5.
BARNES, P. J., ADCOCK, I. M. & ITO, K. (2005) Histone acetylation and
deacetylation: importance in inflammatory lung diseases. Eur Respir J, 25,
552-63.
BARNES, P. J., SHAPIRO, S. D. & PAUWELS, R. A. (2003) Chronic
obstructive pulmonary disease: molecular and cellular mechanisms. Eur
Respir J, 22, 672-88.
BEEH, K. M., KORNMANN, O., BUHL, R., CULPITT, S. V., GIEMBYCZ,
M. A. & BARNES, P. J. (2003) Neutrophil chemotactic activity of sputum
from patients with COPD: role of interleukin 8 and leukotriene B4. Chest, 123,
1240-7.
BELVISI, M. G. & BOTTOMLEY, K. M. (2003) The role of matrix
metalloproteinases (MMPs) in the pathophysiology of chronic obstructive
pulmonary disease (COPD): a therapeutic role for inhibitors of MMPs?
Inflamm Res, 52, 95-100.
BRIGHTLING, C. E., MONTEIRO, W., WARD, R., PARKER, D., MORGAN,
M. D., WARDLAW, A. J. & PAVORD, I. D. (2000) Sputum eosinophilia
and short-term response to prednisolone in chronic obstructive pulmonary
disease: a randomised controlled trial. Lancet, 356, 1480-5.
BROOKS, D., SOLWAY, S. & GIBBONS, W. J. (2003) ATS statement on six-
minute walk test. Am J Respir Crit Care Med, 167, 1287.
BRUSASCO, V., CRAPO, R. & VIEGI, G. (2005) Coming together: the
ATS/ERS consensus on clinical pulmonary function testing. Eur Respir J,
26, 1-2.
BURROWS, B., KNUDSON, R. J., CLINE, M. G. & LEBOWITZ, M. D.
(1977) Quantitative relationships between cigarette smoking and
ventilatory function. Am Rev Respir Dis, 115, 195-205.
CASABURI, R. (2001) Skeletal muscle dysfunction in chronic obstructive
pulmonary disease. Med Sci Sports Exerc, 33, S662-70.
CHURG, A., WANG, R. D., TAI, H., WANG, X., XIE, C., DAI, J., SHAPIRO,
S. D. & WRIGHT, J. L. (2003) Macrophage metalloelastase mediates
acute cigarette smoke-induced inflammation via tumor necrosis factor-
alpha release. Am J Respir Crit Care Med, 167, 1083-9.
COSIO, M. G., MAJO, J. & COSIO, M. G. (2002) Inflammation of the airways
and lung parenchyma in COPD: role of T cells. Chest, 121, 160S-165S.
CULPITT, S. V., DE MATOS, C., RUSSELL, R. E., DONNELLY, L. E.,
ROGERS, D. F. & BARNES, P. J. (2002) Effect of theophylline on
induced sputum inflammatory indices and neutrophil chemotaxis in
chronic obstructive pulmonary disease. Am J Respir Crit Care Med, 165,
1371-6.
CULPITT, S. V., MAZIAK, W., LOUKIDIS, S., NIGHTINGALE, J. A.,
MATTHEWS, J. L. & BARNES, P. J. (1999) Effect of high dose inhaled
steroid on cells, cytokines, and proteases in induced sputum in chronic
obstructive pulmonary disease. Am J Respir Crit Care Med, 160, 1635-9.
CULPITT, S. V., ROGERS, D. F., SHAH, P., DE MATOS, C., RUSSELL, R.
E., DONNELLY, L. E. & BARNES, P. J. (2003) Impaired inhibition by
dexamethasone
of cytokine release by alveolar macrophages from patients with chronic
obstructive pulmonary disease. Am J Respir Crit Care Med, 167, 24-31.
EVANS, D. J., TAYLOR, D. A., ZETTERSTROM, O., CHUNG, K. F.,
O'CONNOR, B. J. & BARNES, P. J. (1997) A comparison of low-dose
inhaled budesonide plus theophylline and high-dose inhaled budesonide
for moderate asthma. N Engl J Med, 337, 1412-8.
GAN, W. Q., MAN, S. F. & SIN, D. D. (2005) Effects of inhaled
corticosteroids on sputum cell counts in stable chronic obstructive
pulmonary disease: a systematic review and a meta-analysis. BMC Pulm
Med, 5, 3.
GOMPERTZ, S. & STOCKLEY, R. A. (2000) Inflammation--role of the
neutrophil and the eosinophil. Semin Respir Infect, 15, 14-23.
GRASHOFF, W. F., SONT, J. K., STERK, P. J., HIEMSTRA, P. S., DE
BOER, W. I., STOLK, J., HAN, J. & VAN KRIEKEN, J. M. (1997)
Chronic obstructive pulmonary disease: role of bronchiolar mast cells and
macrophages. Am J Pathol, 151, 1785-90.
HIRANO, T., YAMAGATA, T., GOHDA, M., YAMAGATA, Y.,
ICHIKAWA, T., YANAGISAWA, S., UESHIMA, K., AKAMATSU, K.,
NAKANISHI, M., MATSUNAGA, K., MINAKATA, Y. & ICHINOSE,
M. (2006) Inhibition of Reactive Nitrogen Species Production in Copd
Airways: Comparison between Inhaled Corticosteroid and Oral
Theophylline. Thorax.
HNIZDO, E., SULLIVAN, P. A., BANG, K. M. & WAGNER, G. (2002)
Association between chronic obstructive pulmonary disease and
employment by industry and occupation in the US population: a study of
data from the Third National Health and Nutrition Examination Survey.
Am J Epidemiol, 156, 738-46.
HOGG, J. C., CHU, F., UTOKAPARCH, S., WOODS, R., ELLIOTT, W. M.,
BUZATU, L., CHERNIACK, R. M., ROGERS, R. M., SCIURBA, F. C.,
COXSON, H. O. & PARE, P. D. (2004) The nature of small-airway
obstruction in chronic obstructive pulmonary disease. N Engl J Med, 350,
2645-53.
HOWELL, R. E., MUEHSAM, W. T. & KINNIER, W. J. (1990) Mechanism
for the emetic side effect of xanthine bronchodilators. Life Sci, 46, 563-8.
ITO, K., BARNES, P. J. & ADCOCK, I. M. (2000) Glucocorticoid receptor
recruitment of histone deacetylase 2 inhibits interleukin-1beta-induced
histone H4 acetylation on lysines 8 and 12. Mol Cell Biol, 20, 6891-903.
ITO, K., ITO, M., ELLIOTT, W. M., COSIO, B., CARAMORI, G., KON, O.
M., BARCZYK, A., HAYASHI, S., ADCOCK, I. M., HOGG, J. C. &
BARNES, P. J. (2005) Decreased histone deacetylase activity in chronic
obstructive pulmonary disease. N Engl J Med, 352, 1967-76.
JEFFERY, P. K. (2000) Comparison of the structural and inflammatory features
of COPD and asthma. Giles F. Filley Lecture. Chest, 117, 251S-60S.
JINDAL, S. K., AGGARWAL, A. N., CHAUDHRY, K., CHHABRA, S. K.,
D'SOUZA, G. A., GUPTA, D., KATIYAR, S. K., KUMAR, R., SHAH,
B. & VIJAYAN, V. K. (2006) A multicentric study on epidemiology of
chronic obstructive pulmonary disease and its relationship with tobacco
smoking and environmental tobacco smoke exposure. Indian J Chest Dis
Allied Sci, 48, 23-9.
KANAZAWA, H., SHIRAISHI, S., HIRATA, K. & YOSHIKAWA, J. (2003)
Imbalance between levels of nitrogen oxides and peroxynitrite inhibitory
activity in chronic obstructive pulmonary disease. Thorax, 58, 106-9.
KEATINGS, V. M. & BARNES, P. J. (1997a) Granulocyte activation markers
in induced sputum: comparison between chronic obstructive pulmonary
disease, asthma, and normal subjects. Am J Respir Crit Care Med, 155,
449-53.
KEATINGS, V. M., COLLINS, P. D., SCOTT, D. M. & BARNES, P. J. (1996)
Differences in interleukin-8 and tumor necrosis factor-alpha in induced
sputum from patients with chronic obstructive pulmonary disease or
asthma. Am J Respir Crit Care Med, 153, 530-4.
KEATINGS, V. M., JATAKANON, A., WORSDELL, Y. M. & BARNES, P. J.
(1997b) Effects of inhaled and oral glucocorticoids on inflammatory
indices in asthma and COPD. Am J Respir Crit Care Med, 155, 542-8.
KOBAYASHI, M., NASUHARA, Y., BETSUYAKU, T., SHIBUYA, E.,
TANINO, Y., TANINO, M., TAKAMURA, K., NAGAI, K.,
HOSOKAWA, T. & NISHIMURA, M. (2004) Effect of low-dose
theophylline on airway inflammation in COPD. Respirology, 9, 249-54.
LANGE, P., NYBOE, J., APPLEYARD, M., JENSEN, G. & SCHNOHR, P.
(1992) Relationship of the type of tobacco and inhalation pattern to
pulmonary and total mortality. Eur Respir J, 5, 1111-7.
LLEWELLYN-JONES, C. G., HARRIS, T. A. & STOCKLEY, R. A. (1996)
Effect of fluticasone propionate on sputum of patients with chronic
bronchitis and emphysema. Am J Respir Crit Care Med, 153, 616-21.
LOPEZ, A. D., SHIBUYA, K., RAO, C., MATHERS, C. D., HANSELL, A. L.,
HELD, L. S., SCHMID, V. & BUIST, S. (2006) Chronic obstructive
pulmonary disease: current burden and future projections. Eur Respir J,
27, 397-412.
LOPPOW, D., SCHLEISS, M. B., KANNIESS, F., TAUBE, C., JORRES, R.
A. & MAGNUSSEN, H. (2001) In patients with chronic bronchitis a four
week trial with inhaled steroids does not attenuate airway inflammation.
Respir Med, 95, 115-21.
MACNEE, W. (2000) Oxidants/antioxidants and COPD. Chest, 117, 303S-17S.
MACNEE, W. (2001) Oxidative stress and lung inflammation in airways
disease. Eur J Pharmacol, 429, 195-207.
MACNEE, W. (2005) Pathogenesis of chronic obstructive pulmonary disease.
Proc Am Thorac Soc, 2, 258-66; discussion 290-1.
MITENKO, P. A. & OGILVIE, R. I. (1973) Rational intravenous doses of
theophylline. N Engl J Med, 289, 600-3.
MITTELSTADT, P. R. & ASHWELL, J. D. (2001) Inhibition of AP-1 by the
glucocorticoid-inducible protein GILZ. J Biol Chem, 276, 29603-10.
MOLFINO, N. A. (2007) Genetic predisposition to accelerated decline of lung
function in COPD. Int J Chron Obstruct Pulmon Dis, 2, 117-9.
MONTUSCHI, P., KHARITONOV, S. A., CIABATTONI, G., CORRADI, M.,
VAN RENSEN, L., GEDDES, D. M., HODSON, M. E. & BARNES, P. J.
(2000) Exhaled 8-isoprostane as a new non-invasive biomarker of
oxidative stress in cystic fibrosis. Thorax, 55, 205-9.
MUROSKI, M. E., ROYCIK, M. D., NEWCOMER, R. G., VAN DEN STEEN,
P. E., OPDENAKKER, G., MONROE, H. R., SAHAB, Z. J. & SANG, Q.
X. (2008) Matrix metalloproteinase-9/gelatinase B is a putative
therapeutic target of chronic obstructive pulmonary disease and multiple
sclerosis. Curr Pharm Biotechnol, 9, 34-46.
NAKAMURA, H., YOSHIMURA, K., JAFFE, H. A. & CRYSTAL, R. G.
(1991) Interleukin-8 gene expression in human bronchial epithelial cells. J
Biol Chem, 266, 19611-7.
OGAWA, E., RUAN, J., CONNETT, J. E., ANTHONISEN, N. R., PARE, P. D.
& SANDFORD, A. J. (2007) Transforming growth factor-beta1
polymorphisms, airway responsiveness and lung function decline in
smokers. Respir Med, 101, 938-43.
PAGGIARO, P. L., CHANEZ, P., HOLZ, O., IND, P. W., DJUKANOVIC, R.,
MAESTRELLI, P. & STERK, P. J. (2002) Sputum induction. Eur Respir
J Suppl, 37, 3s-8s.
PICCIONI, P. D., KRAMPS, J. A., RUDOLPHUS, A., BULGHERONI, A. &
LUISETTI, M. (1992) Proteinase/proteinase inhibitor imbalance in
sputum sol phases from patients with chronic obstructive pulmonary
disease. Suggestions for a key role played by antileukoprotease. Chest,
102, 1470-6.
POSTMA, D. S. & KERSTJENS, H. A. (1999) Are inhaled glucocorticosteroids
effective in chronic obstructive pulmonary disease? Am J Respir Crit Care
Med, 160, S66-71.
QIU, Y., ZHU, J., BANDI, V., ATMAR, R. L., HATTOTUWA, K.,
GUNTUPALLI, K. K. & JEFFERY, P. K. (2003) Biopsy neutrophilia,
neutrophil chemokine and receptor gene expression in severe
exacerbations of chronic obstructive pulmonary disease. Am J Respir Crit
Care Med, 168, 968-75.
RABE, K. F., HURD, S., ANZUETO, A., BARNES, P. J., BUIST, S. A.,
CALVERLEY, P., FUKUCHI, Y., JENKINS, C., RODRIGUEZ-ROISIN,
R., VAN WEEL, C. & ZIELINSKI, J. (2007) Global strategy for the
diagnosis, management, and prevention of chronic obstructive pulmonary
disease: GOLD executive summary. Am J Respir Crit Care Med, 176,
532-55.
RABE, K. F., MAGNUSSEN, H. & DENT, G. (1995) Theophylline and
selective PDE inhibitors as bronchodilators and smooth muscle relaxants.
Eur Respir J, 8, 637-42.
RAMSDELL, J. (1995) Use of theophylline in the treatment of COPD. Chest,
107, 206S-209S.
REPINE JE, B. A., LANKHORST I. (1997) Oxidative stress in chronic
obstructive pulmonary disease Am. J. Resp. Crit. Care Med. , 156:341–57.
RICHMAN-EISENSTAT, J. B., JORENS, P. G., HEBERT, C. A., UEKI, I. &
NADEL, J. A. (1993) Interleukin-8: an important chemoattractant in
sputum of patients with chronic inflammatory airway diseases. Am J
Physiol, 264, L413-8.
ROBSON, R. A., MATTHEWS, A. P., MINERS, J. O., MCMANUS, M. E.,
MEYER, U. A., HALL, P. M. & BIRKETT, D. J. (1987) Characterisation
of theophylline metabolism in human liver microsomes. Br J Clin
Pharmacol, 24, 293-300.
RUSSELL, R. E., THORLEY, A., CULPITT, S. V., DODD, S., DONNELLY,
L. E., DEMATTOS, C., FITZGERALD, M. & BARNES, P. J. (2002)
Alveolar macrophage-mediated elastolysis: roles of matrix
metalloproteinases, cysteine, and serine proteases. Am J Physiol Lung Cell
Mol Physiol, 283, L867-73.
SAETTA, M., DI STEFANO, A., TURATO, G., FACCHINI, F. M.,
CORBINO, L., MAPP, C. E., MAESTRELLI, P., CIACCIA, A. &
FABBRI, L. M. (1998) CD8+ T-lymphocytes in peripheral airways of
smokers with chronic obstructive pulmonary disease. Am J Respir Crit
Care Med, 157, 822-6.
SEEMUNGAL, T., HARPER-OWEN, R., BHOWMIK, A., MORIC, I.,
SANDERSON, G., MESSAGE, S., MACCALLUM, P., MEADE, T. W.,
JEFFRIES, D. J., JOHNSTON, S. L. & WEDZICHA, J. A. (2001)
Respiratory viruses, symptoms, and inflammatory markers in acute
exacerbations and stable chronic obstructive pulmonary disease. Am J
Respir Crit Care Med, 164, 1618-23.
SESSLER, C. N. (1990) Theophylline toxicity: clinical features of 116
consecutive cases. Am J Med, 88, 567-76.
SHANNON, M. (1999) Life-threatening events after theophylline overdose: a
10-year prospective analysis. Arch Intern Med, 159, 989-94.
SHAPIRO, S. D. (2005) COPD unwound. N Engl J Med, 352, 2016-9.
SHRESTHA, I. L. & SHRESTHA, S. L. (2005) Indoor air pollution from
biomass fuels and respiratory health of the exposed population in Nepalese
households. Int J Occup Environ Health, 11, 150-60.
SILVERMAN, E. K., MOSLEY, J. D., PALMER, L. J., BARTH, M.,
SENTER, J. M., BROWN, A., DRAZEN, J. M., KWIATKOWSKI, D. J.,
CHAPMAN, H. A., CAMPBELL, E. J., PROVINCE, M. A., RAO, D. C.,
REILLY, J. J., GINNS, L. C., SPEIZER, F. E. & WEISS, S. T. (2002)
Genome-wide linkage analysis of severe, early-onset chronic obstructive
pulmonary disease: airflow obstruction and chronic bronchitis phenotypes.
Hum Mol Genet, 11, 623-32.
STANESCU, D., SANNA, A., VERITER, C., KOSTIANEV, S., CALCAGNI,
P. G., FABBRI, L. M. & MAESTRELLI, P. (1996) Airways obstruction,
chronic expectoration, and rapid decline of FEV1 in smokers are
associated with increased levels of sputum neutrophils. Thorax, 51, 267-
71.
STOCKLEY, R. A. (1999) Neutrophils and protease/antiprotease imbalance.
Am J Respir Crit Care Med, 160, S49-52.
STOCKLEY, R. A. (2001) Proteases and antiproteases. Novartis Found Symp,
234, 189-99; discussion 199-204.
STOCKLEY, R. A. (2002) Neutrophils and the pathogenesis of COPD. Chest,
121, 151S-155S.
TAKAHASHI, G. W., ANDREWS, D. F., 3RD, LILLY, M. B., SINGER, J. W.
& ALDERSON, M. R. (1993) Effect of granulocyte-macrophage colony-
stimulating factor and interleukin-3 on interleukin-8 production by human
neutrophils and monocytes. Blood, 81, 357-64.
TAKIZAWA, H. (2003) [Role of inflammatory cells in the development of
airway inflammation]. Nippon Rinsho, 61, 2107-12.
TETLEY, T. D. (2002) Macrophages and the pathogenesis of COPD. Chest,
121, 156S-159S.
TETLEY, T. D. (2005) Inflammatory cells and chronic obstructive pulmonary
disease. Curr Drug Targets Inflamm Allergy, 4, 607-18.
URBACH, V., WALSH, D. E., MAINPRICE, B., BOUSQUET, J. &
HARVEY, B. J. (2002) Rapid non-genomic inhibition of ATP-induced Cl-
secretion by dexamethasone in human bronchial epithelium. J Physiol,
545, 869-78.
WELDON, S., MCGARRY, N., TAGGART, C. C. & MCELVANEY, N. G.
(2007) The role of secretory leucoprotease inhibitor in the resolution of
inflammatory responses. Biochem Soc Trans, 35, 273-6.
WILLIAMS, T. J. & JOSE, P. J. (2001) Neutrophils in chronic obstructive
pulmonary disease. Novartis Found Symp, 234, 136-41; discussion 141-8.
WOOLHOUSE, I. S., BAYLEY, D. L. & STOCKLEY, R. A. (2002) Sputum
chemotactic activity in chronic obstructive pulmonary disease: effect of
alpha(1)-antitrypsin deficiency and the role of leukotriene B(4) and
interleukin 8. Thorax, 57, 709-14.
XU, X. M., SUN, T. Y., LIN, J. B. & ZHANG, H. S. (2003) [The role of matrix
metalloproteinases-9 and tissue inhibitor of metalloproteinaes-1 in chronic
obstructive pulmonary disease]. Zhonghua Yi Xue Za Zhi, 83, 1138-41.
YAGI, O., AOSHIBA, K. & NAGAI, A. (2006) Activation of nuclear factor-
kappaB in airway epithelial cells in patients with chronic obstructive
pulmonary disease. Respiration, 73, 610-6.
YING, Q. L. & SIMON, S. R. (2002) Elastolysis by proteinase 3 and its
inhibition by alpha(1)-proteinase inhibitor: a mechanism for the
incomplete inhibition of ongoing elastolysis. Am J Respir Cell Mol Biol,
26, 356-61.
Annexure …
List of Tables
Page
Table 1: Protease-Antiprotease Imbalance in COPD 11
Table 2: Treatment arms of the study 29
Table 3: Baseline characteristics of the patients 39
Table 4: Effect of treatment on sputum inflammatory variables 41
Table 5: Effect of treatment on Lung function indices 44

List of Figures
Page
Figure 1: Inflammatory Mechanisms in COPD 12
Figure 2: Oxidative Stress in COPD 14
Figure 3: Gene regulation by histone acetylation 17
Figure 4: Anti-inflammatory effects of corticosteroids 20
Figure 5: Mechanism of corticosteroid resistance in COPD 22
Figure 6: Anti-inflammatory effects of theophylline 25
Figure 7: Effect on MPO activity in induced sputum 41
Figure 8: Effect on total cell count in induced sputum 42
Figure 9: Effect on IL-8 level in induced sputum 42
Figure 10: Effect of treatment on FEV 1 45
Figure 11: Effect of treatment on Borg Dyspnea Score 45
Figure 12: Effects of treatment on 6 minute walk distance 46
Figure 13: Effect of treatment on SpO2 46

Abbreviations
GOLD – Global initiative for chronic Obstructive Lung Disease
COPD – Chronic Obstructive Pulmonary Disease
WHO – World Health Organization
ATS – American Thoracic Society
ERS – European Respiratory Society
LABA – Long Acting β2 Adrenergic Agonist
SABA – Short Acting β2 Adrenergic Agonist
SVN – Small Volume Nebulizer
FEV1 – Forced Expiratory Volume in 1 second
FVC – Forced Vital Capacity
l – Liters
6 MWT – 6 Minute Walk Test
cAMP – Cyclic Adenosine Monophosphate
SD – Standard Deviation
SEM – Standard Error of Mean

Patient information sheet
What is Chronic obstructive pulmonary disease?

COPD is a chronic inflammatory disease of the air ways characterized by worsening
airflow limitation with shortness of breath progressing to chronic cough and sputum
production. In patients with severe disease other features like cachexia, skeletal muscle
wasting, increased risk of cardiovascular disease, anemia, osteoporosis, and depression
are also seen.
What causes the COPD?

Worldwide, cigarette smoking is the most commonly encountered risk factor for
COPD, although in our country, air pollution resulting from the burning of wood and
other biomass fuels has also been identified as a COPD risk factor.
What are the treatment options available for COPD?
The treatment and management of COPD generally involves bronchodilators and anti
inflammatory agents mainly corticosteroids.
What is study medication?

The study involves two drugs, one is Fluticasone (Inhaled corticosteroid which is anti
inflammatory) and the other is Theophylline (Bronchodilator). Both these drugs are
approved and widely prescribed drugs for COPD. But in many COPD patients, inhaled
corticosteroids give less benefit due to inhibitory effect of smoking. Theophylline when
prescribed in low dose (plasma theophylline level < 10mg/ml ) is found to be able to
restore corticosteroid sensitivity in patients with COPD.
What is the purpose of the present study?

The purpose of the present study is to evaluate the efficacy of low dose theophylline
can improve inhaled corticosteroid responsiveness in patients with moderate COPD
What is the study procedure?
At the screening visit after taking your informed consent your history will recorded and
physical examination and a questionnaire regarding your wellbeing will be
administered. A screening spirometry and 6 MWT (6 minute walk test) will be
performed. If you are on inhaled corticosteroids and/or theophylline, you will be
discontinued for 1 week (run in period) but beta 2 agonist and anticholinergics will be
allowed to be taken throughout the study.
If you meet the study criteria and are stable during the run in period, you will be
randomized to the study. Baseline spirometry and 6 minute walk test with Borg
Dyspnea score will be performed , blood sample and induced sputum will be collected
and will be randomized to receive either Fluticasone 500 mcg 2 puffs + Sustained
release theophylline 150 mg twice a day or Fluticasone 500 mcg 2 puffs twice a day.
At the end of 4 weeks of treatment spirometry and 6 minute walk test with Borg
Dyspnea score will be performed, blood sample and induced sputum will be collected
again.
Who will do the study?

The study will be conducted by Dr.Linda Jose .P, Dept of Pharmacology, under the
supervision of Dr. D.J. Christopher and Dr. B.S. Ramakrishna.
Which is study site?

The study site is at Department of pulmonary medicine, Christian Medical College,
Vellore-1
What are the risks or discomforts of taking part in the study?

You will be required to undergo spirometry, 6 minute walk test and induced sputum
collection. Induction of sputum can cause some discomfort in few patients. If you
develop any such discomfort, rescue medications will be given immediately and
necessary care will be taken. Theophylline when given in low dose pose has less chance
of causing side effects like nausea, gastrointestinal symptoms, and headache.
What are the benefits of taking part in the study?

Both the study drug and the comparator drug are well established drugs for the
treatment of your condition. Apart from getting the drugs free, you may benefit from
the supportive care provided by the team of investigators; normally extended to study
patients .However you may not get any extraordinary benefits from participation.
What about the confidentiality?

Your identity will not be disclosed to anybody not directly connected with the study
except the ethics committee and/ or regulatory authorities during the course of the study
as well as after completion of the study including the publication of the data.
Will I be paid for participating in the study?

You will not be paid any monetary compensation for participating in the study.
However, in case of any study related injury you will be paid compensation.
Will I have to pay for any study related investigations / procedure / treatment?
You will not be required to pay for laboratory tests. The study medications used during
the study period will be free of cost.
Can I withdraw from the study once enrolled?

You can opt out of study at any time without giving any reasons for it and this will not
have any bearing on your subsequent treatment at the hospital.
Whom can I contact for further information?

You can contact the following person for further information:
Dr. Linda Jose .P
Department of Pharmacology
Christian medical college, Vellore
Informed Consent form
TITLE: A Randomized Controlled Study to Compare the Effects of Inhaled
Fluticasone and Low Dose Deriphyllin® Combination against Inhaled
Fluticasone Alone In Patients with Moderate to Severe Chronic Obstructive
Pulmonary Disease
Participant’s name:________________ Participant’s Initials:_______
Date of Birth / Age:_________________ Screening No:_______
(Please tick boxes)
1. I declare that I have read the information sheet provide

to me regarding this study and have clarified any
doubts that I had. [ ]
2. I also understand that my participation in this study is entirely

voluntary and that I am free to withdraw permission to
continue to participate at any time without affecting my
usual treatment or my legal rights. [ ]
3. I also understand that during the 4 weeks of the study,

the theophylline or fluticasone inhaler will be provided free,
but after this, I may have to pay for it. [ ]
4. I understand that I will receive free treatment for any study

related injury or adverse event but I will not receive and
other financial compensation. [ ]
5. I understand that the study staff and institutional ethics

committee members will not need my permission to look
at my health records even if I withdraw from the trial.
I agree to this access . [ ]
6. I understand that my identity will not be revealed in
any information released to third parties or published . [ ]
7. I voluntarily agree to take part in this study. [ ]
Name of the subject: ______________________

Signature of the subject : ______________________
Date: ______________________
Name of witness: _______________________

Relation to participant: _______________________
Date: _______________________
Name of the investigator: ______________________

Signature of the investigator: ______________________
Date: ______________________
Case Record Form
Screening Visit
Subject No _____ , Patient initial ____ , Age: ______ yrs

Weight: ______ Kg Height: ______ cm
Smoking history
(No. Of Cigarettes per day) _____ x ___ yrs /20 = ____ pack yrs
Current smoker : Yes / No
If No , Date of quitting: _________
COPD Status
Duration of COPD _________ yrs ______months
Date of last exacerbation __________
Current medications _____________________________

_______________________________
Pulse Rate _____ Blood Pressure _____

General Examination ___________________
Respiratory System _____________________________________
CVS ________________________________________________
GIT _______________________ Other systems _____________
Spirometry :
Post FEV1/FVC _________ Post FEV1 % pred _________
6- Minute Walk Test: _________
ECG: __________
Subject Randomized : Yes / No

Randomization No: _________
Baseline Visit
Subject Initial ______ Randomization No: ____

CVS ________________________________________________
Spirometry :
Blood Tests:
TC , DC , S. Potassium , Plasma theophylline level
Induced sputum collection
Time of collection: __________
Study Medication Supplied:
Rescue Medication Dispensed:

End of Treatment Visit
Subject Initial ______ Randomization No: ____

CVS ________________________________________________
Spirometry :
Blood Tests:
TC , DC , S. Potassium , Plasma theophylline level
Induced sputum collection
Time of collection: __________

Fluticasone

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Fluticasone

Uploaded by

Copyright:

Available Formats

A Randomized Controlled Study to Compare Effects of Inhaled

Fluticasone and Low Dose Deriphyllin® Combination with Inhaled

Fluticasone Alone in Patients with Moderate to Severe

Chronic Obstructive Pulmonary Disease

DEPARTMENT OF PHARMACOLOGY AND CLINICAL PHARMACOLOGY,

Controlled Study to Compare Effects of Inhaled Fluticasone and Low Dose

Deriphyllin® Combination with Inhaled Fluticasone Alone in Patients with

Moderate to Severe Chronic Obstructive Pulmonary Disease” has been

Department of pharmacology, and co-guidance of Dr. D. J. Christopher, Professor

and Head, Department of Pulmonary Medicine and Dr. B. S. Ramakrishna,

Professor and Head, Department of Gastrointestinal Sciences, Christian Medical

College, Vellore, in partial fulfillment of university regulations for award of M.D.

any other university or towards any other degree.

Date: Dr. Linda Jose P

Certified that this dissertation entitled “A Randomized Controlled Study

to Compare Effects Of Inhaled Fluticasone and Low Dose Deriphyllin®

Combination with Inhaled Fluticasone Alone in Patients with Moderate to

Severe Chronic Obstructive Pulmonary Disease” submitted by Dr. Linda Jose P

Nadu Dr. M. G. R. Medical University, Chennai, Tamil Nadu, is a bonafide work

done under my guidance and supervision and completed to my satisfaction.

Certified that that this dissertation entitled “A Randomized Controlled

Study to Compare Effects of Inhaled Fluticasone and Low Dose Deriphyllin®

Combination with Inhaled Fluticasone Alone in Patients with Moderate to

Severe Chronic Obstructive Pulmonary Disease” submitted by Dr. Linda Jose P

Nadu Dr M G R Medical University, Chennai, Tamil Nadu, is a bonafide work done

under my co-guidance and supervision and completed to my satisfaction.

Certified that that this dissertation entitled “A Randomized Controlled

Study to Compare Effects of Inhaled Fluticasone and Low Dose Deriphyllin®

Combination with Inhaled Fluticasone Alone in Patients with Moderate to

Severe Chronic Obstructive Pulmonary Disease” submitted by Dr. Linda Jose P

Nadu Dr M G R Medical University, Chennai, Tamil Nadu, is a bonafide work done

under my co-guidance and supervision and completed to my satisfaction.

Place: Dr B.S. Ramakrishna

Department with Departments of Pulmonary Medicine, Clinical pathology and Wellcome

supported or contributed to this thesis deserve acknowledgment at this place.

I express my wholehearted thanks to Dr Kalpana Ernest, Professor and Head,

Department of Pharmacology and my guide, for always being supportive and

encouraging throughout my candidature. I am also thankful to her for allowing me to

pursue my interests in the area of pulmonary pharmacology.

Dr. D J Christopher, Professor and Head, Department of Pulmonary Medicine

pulmonary research team. I am very thankful to your ideas, suggestions, encouragement

and support throughout my course of study. I am indebted to you for motivating me to

Professor and Head, Department of Gastrointestinal Sciences, throughout my work. He

encouragement and invaluable support rendered to me in the fulfillment of this work.

I express my heartfelt thanks to Dr. Sukesh C Nair, Professor, Department of

Dr Jacob Peedicayil, your easy accessibility and constructive critical review of

Chandy, for always being supportive and inculcating interest in research. I am

my interest in basic research.

Jayakanthan for the support provided in the performance of ELISA tests.

Deepa, Ms Jisha, Ms Divya and other staff in Department of Pulmonary Medicine

genuine dedication and enthusiasm to help me at times of need.

I express my heartfelt thanks to Dr. Yasuyuki Nasuhara, Hokkaido University

theophylline and inspiring my thoughts. My sincere acknowledgments are offered to the

would never have been completed

II. Aim and Objectives 4

III. Review of Literature

Chronic Obstructive Pulmonary Disease 5

Chronic Airway Inflammation in COPD 7

Oxidative stress in COPD 13

Anti-Inflammatory Effects of Corticosteroids 18

Corticosteroid Resistance in COPD 21

Effects of Theophylline in COPD 23

Clinical Implications of the Study 26