Cigarette Smoke Nasal and Whole Blood Challenge in Patients With Chronic Obstructive Pulmonary Disease (COPD)
Cigarette Smoke Nasal and Whole Blood Challenge in Patients With Chronic Obstructive Pulmonary Disease (COPD)
This is a clinical research study to assess whether after exhaling a single cigarette smoke
 through the nose there are changes in the inflammatory cells and proteins of nasal
 secretions.

 A single blood sample from each subject will be stimulated with cigarette smoke in the
 laboratory to see the effects on inflammatory blood cells.

 Comparison of findings between smokers with COPD and "Healthy" smokers will be carried out.

 We hypothesize that some subjects have amplified inflammatory response to a single cigarette,
 and these will be those subjects who develop chronic obstructive pulmonary disease (COPD)
 after decades of smoking. We hope to develop an acute challenge model that relates to the
 causation of COPD. When studying the effects of new drugs, these may be detected in small
 numbers of patients in a challenge situation, when we would need to study many more
 unchallenged patients to demonstrate drug effects. In clinical research on asthma and
 allergy, the nasal allergen challenge has been a very successful model, and we hope to
 validate a comparable model for COPD.
Introduction Chronic obstructive pulmonary disease (COPD) is a common respiratory disease
 that causes widespread morbidity and mortality, the Global Burden of Disease Study estimating
 COPD to be currently the sixth most common cause of death worldwide. Decades of cigarette
 smoking is by far the major contributing factor, and this causes a range of respiratory
 disorders; including chronic bronchitis, obstructive bronchiolitis, emphysema, and pulmonary
 vascular disease. Medical treatment of COPD is largely palliative; involving smoking
 cessation, use of inhaled bronchodilators, and long term oxygen therapy for respiratory
 failure. Recently, it has been recognised that those smokers who develop COPD have
 amplification of inflammation compared with "healthy smokers", and that there is the urgent
 need to develop new challenge models to assess why COPD develops in some heavy smokers.

 Cigarette Smoke Nasal Challenge Model The nose is an accessible part of the respiratory
 system, from which it is possible to obtain tissue exudates and cell brushings in a
 non-invasive manner. A comprehensive review of the extensive clinical research experience
 with these nasal methodologies has recently been published. There is also the insight that
 patients with COPD commonly have inflammation of both the airways and nasal passages, with a
 similar type of inflammatory infiltrate in lower and upper airways. In COPD the inflammation
 is predominantly involving neutrophils and macrophages, as opposed to the increase in mast
 cells and eosinophils characteristic of asthma.

 It has long been recognised that there is a strong functional and immunological relationship
 between the nose and bronchi, especially in terms of infiltrating leukocytes and inflammatory
 mediators when comparing allergic rhinitis and allergic asthma. The upper and lower airways
 have related respiratory epithelium and similar responses to allergen challenge.

 The nasal and bronchial mucosa of smokers with COPD have been demonstrated to be infiltrated
 with CD8+ T lymphocytes and neutrophils. This corresponds to the presence of nasal symptoms
 during exacerbations of COPD, and sputum and nasal levels of IL-8 are correlated in COPD.

 In order to sample nasal exudates for allergic inflammatory mediators, the classical methods
 of nasal lavage are those described by Naclerio et al., the nasal pool method of Greiff et
 al., and the use of a Foley's catheter by Grünberg and colleagues. Lavage is performed with
 saline at volumes of between 1 and 10ml. The repeatability and validity of different nasal
 lavage methods have been compared.

 Tommy Sim and colleagues have developed the use of filter paper strips, that are placed on
 the turbinates to absorb nasal secretions. The nasal filter paper method has the advantage of
 directly sampling nasal secretions which are less diluted and can therefore pick up protein
 signals which are below the detection limits of nasal lavage. The matrix or filter paper
 method has been used to measure chemokines and cytokines after NAC.

 Cells samples may be obtained from the nasal mucosal using small nylon dental flossing
 brushes which are gently rotated over the epithelium, and then the attached cells are
 dislodged in balanced salt solution. It has been demonstrated that nasal brushing can be used
 as an alternative to nasal biopsy [24], whilst nasal brush supernatants can be analysed for
 cytokine release [25]. An alternative method is to employ nasal mucosal scrapings using a
 plastic curette (Rhinoprobe, Arlington Scientific, Utah). Nasal brushing and scraping causes
 some discomfort but does not require local anaesthesia. Nasal biopsy is generally performed
 from the lower edge of the inferior turbinate by a specialist. This is a traumatic procedure
 that requires careful local anaesthesia, but a specimen of mucosal epithelium with basement
 membrane and submucosal tissue is obtained.

 Nasal allergen challenge (NAC) is of proven benefit to test novel anti-inflammatory therapy
 for allergic rhinitis and asthma.

 NAC has advantages over inhaled allergen challenge since it is easier to recruit subjects, it
 is less invasive, and repeated tissue samples are readily obtained. Small numbers of patients
 can be given even single doses of therapy, and the anti-inflammatory and clinical effects on
 NAC responses noted. Using these challenge methods in phase II of drug development for asthma
 and allergic rhinitis it is possible to provide an early and reliable "Go/No Go" decision
 before embarking on large-scale, long-term and costly phase III studies.

 We hope to develop an analogous challenge model for COPD by assessing effects of nasal
 challenge with cigarette smoke.

 A major challenge is to develop phase IIa clinical pharmacology designs that can be used to
 assess anti-inflammatory drugs being developed for COPD. Nasal epithelial and inflammatory
 responses to ozone exposure have been much studied [31-39]. Indeed COPD is characterised by
 chronic inflammation with an imbalance in oxidant/antioxidant levels, and systemic
 inflammation may trigger local exercise-induced oxidative stress.

 Three year clinical trials involving thousands of patients have found that inhaled
 corticosteroids do not affect the accelerated rate loss of lung function in COPD.
 Furthermore, ongoing phase III trials with oral phosphodiesterase type 4 (PDE4)inhibitors are
 also unconvincing, despite the initial smaller studies looking promising. There is clearly
 the compelling need to develop non-invasive biomarkers and clinical challenge models for use
 in phase II clinical pharmacology studies to enable reliable assessment of the effects of
 anti-inflammatory therapy in COPD.

 Use of nasal challenge models coupled to sensitive biomarkers and clinical endpoints could
 perhaps be used to establish clinical efficacy of new drugs for COPD in small scale studies.
 Novel therapies have the potential to selectively inhibit various cell types and particular
 mediators involved in diverse inflammatory diseases. We believe that nasal allergen challenge
 has advantages over inhaled challenge: safety, repeat non-invasive sampling.

 In the future this will ensure that nasal challenge will play a growing role in clinical
 pharmacology assessment of anti-inflammatory therapy. Major efforts are ongoing to develop
 nasal challenge models to mimic the inflammation found in COPD, that can be used in clinical
 pharmacology assessment of new anti-inflammatory drugs.

 Whole Blood Challenge in vitro with Cigarette Smoke We shall stimulate blood samples in the
 laboratory in an in vitro manner with a cigarette smoke extract. The cigarette smoke extract
 is produced in a fume cabinet ensuring the safety of laboratory personnel. Cigarette smoke
 contains many toxic compounds including free radicals and oxidants. Cigarette smoke
 conditioned medium (CSCM) is made by bubbling the smoke from burning cigarettes through
 buffered salt solution, and this can be used to stimulate cells in vitro. Cigarette smoke
 extract has been shown to be cytotoxic to an alveolar type II cell-derived line and this
 effect can be blocked by N-acetyl cysteine a scavenger of oxidants. {Hoshino, Mio, et al.
 2001 7463 /id}. We have developed methods of assessing neutrophil CD11b upregulation in
 response to stimulation with cytokines and chemokines. CD11b is a cell surface receptor on
 leukocytes that is mobilised from intracellular stores when cells are stimulated. Surface
 CD11b can be measured using fluorescent labelled antibodies and flow cytometry. We have
 developed a method of stimulating whole blood with CSCM which produces a 200% increase in
 CD11b upregulation after incubation for 20 minutes. This effect is further increased if
 plasma is removed from the whole blood prior to stimulation, and the effect can be partially
 inhibited by the presence of reduced glutathione. COPD patients and smokers have been shown
 to have decreased antioxidant capacity in their plasma. We aim to compare neutrophil
 responses to CSCM stimulation in whole blood, and washed blood where the plasma has been
 removed, to compare neutrophil responsiveness between COPD patients and smokers and possible
 anti-oxidant effects of plasma.

 Overview of planned research

 Two characterised matched groups of smokers with COPD and "healthy" smokers that do not have
 lung disease will be enrolled for this study. Each group will comprise 8 subjects. Each
 subject will be required to make 2 visits to the NHLI Clinical Studies Unit as follows:

 Visit 1 : Screening During screening, candidates for the study will be fully informed about
 the nature of the study and possible AEs. Candidates must read the consent form and sign the
 document after the investigator has answered all questions to the candidate's satisfaction.
 Further procedures can begin only after the consent form has been signed. Two original
 informed consent form copies will be signed by the subject. An original signed consent form
 will be retained by the investigator and the second original will be given to the candidate
 to take home.

 Candidates will be evaluated for entry into the study according to the stated inclusion and
 exclusion criteria. The following procedures will be performed to establish each candidate's
 general health and qualifications for enrollment into the study:

 -  Obtain signed, written informed consent

 -  Record medication history over the past 30 days Record medical history, including
 smoking status

 -  Perform a physical examination of heart, lung and nose

 -  Pregnancy test for female subjects of child bearing age

 -  Conduct spirometry and test reversibility

 -  Review inclusion and exclusion criteria

 The investigator will evaluate the results of all examinations including clinical laboratory
 tests and will determine each candidate's suitability for the study. When the subject is
 suitable for the study, a blood sample (10mls) will be taken for cigarette smoke whole blood
 challenge.

 Visit 2: Nasal smoke challenge Each subject will be allowed to smoke at least 5 cigarettes
 between 9am to 9pm the day before attending the unit, and on the previous 7 days. On the
 study day, each patient will be asked to smoke a single cigarette but exhaling through the
 nose.

 Following this cigarette challenge, we will carry out the following procedures over a period
 of 8 hours (pre(x2), 1h, 2h, 3h, 4h,6h and 8h):

 1. Nasal lavage consists of washing the nose of 5ml (a teaspoon) of warm salt solution.
 This solution is gently passed from a syringe through a hollow "olive" into the end of
 the left nostril. The fluid does not enter the sinuses nor pass down the back of the
 nose. A total of 2 flushes will be carried out at each timepoints.

 2. Nasal filter paper consists of a 7mm x 30mm strips of paper that are gently place inside
 the end of the right nostril. They are left there for up to2 minutes until they are
 moist with nasal secretions.

 3. A cannula will be inserted to enable blood to be taken at the above time points for
 serum analysis of cytokines and chemokines.

 4. Pregnancy test for female subjects of child bearing age only.

 As part of the study, subjects will be informed of the harmful effects of cigarette smoking,
 and advised of the need to stop smoking. Subjects will be offered smoking classes to help
 them stop smoking. It is anticipated that the study day will be set as the quit date for
 subjects.
Inclusion Criteria (COPD Smokers):

 -  Smokers currently on at least 5 cigarettes per day, with a history of >10 pack years

 -  Post-bronchodilator FEV1 >30% of predicted and < 80% of predicted

 -  Pre-bronchodilator FEV1/FVC of <70%

 -  With or without chronic simple bronchitis

 Exclusion criteria (COPD Smokers):

 -  History of asthma, allergy (including rhinitis/eczema)

 -  Reversibility : an increase in FEV1 that is >400ml from the baseline pre-
 bronchodilator value (bronchodilate with salbutamol 400g delivered from a metered dose
 inhaler (MDI) into a spacer).

 Inclusion criteria (for "Healthy" Smokers):

 -  Smokers currently on at least 5 cigarettes per day, with a history of >10 pack years

 -  FEV1 >90% of predicted, FEV1/FVC of >70%

 -  Cannot have chronic simple bronchitis

 -  Age, sex, smoking history matched to COPD Smokers

 Exclusion criteria (for "Healthy" Smokers):

 -  History of asthma, allergy (including rhinitis/eczema

 -  Reversibility: an increase in FEV1 that is both >400ml from the baseline
 pre-bronchodilator value (bronchodilate with salbutamol 400mcg delivered from a
 metered dose inhaler (MDI) into a spacer)P

 -  Pregnancy