GSK Clinical Study Register

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1 In February 2013, GlaxoSmithKline (GSK) announced a commitment to further clinical transparency through the public disclosure of GSK Clinical Study Reports (CSRs) on the GSK Clinical Study Register. The following guiding principles have been applied to the disclosure: Information will be excluded in order to protect the privacy of patients and all named persons associated with the study Patient data listings will be completely removed* to protect patient privacy. Anonymized data from each patient may be made available subject to an approved research proposal. For further information please see the Patient Level Data section of the GSK Clinical Study Register. Aggregate data will be included; with any direct reference to individual patients excluded *Complete removal of patient data listings may mean that page numbers are no longer consecutively numbered

2 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 BL2001/00008/00 Study No. Clinical Study Report for Study (Phase II) Document Number: BL2001/00008/00 A single centre, randomised, double-blind, placebo-controlled, crossover, pilot study to determine the efficacy and safety of 400 µg and 200 µg doses of Ventolin inhaler versus placebo for the treatment of smoking related cough. Initiation Date: 20 February 2001 Completion Date: 24 May 2001 Early Termination Date: N/A Date of Report: Sept 2001 Sponsor Signatory: (and Medical Officer) Dr Medical Director, SWITCH and Innovations Group GSK Consumer Health This study was performed in compliance with Good Clinical Practices and Glaxo Wellcome Standard Operating Procedures for all processes involved, including the archiving of essential documents. Copyright 2001 the GlaxoSmithKline group of companies. All rights reserved. Unauthorised copying or use of this information is prohibited. 1 1

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5 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 BL2001/00008/00 Study No. Synopsis Document Number: BL2001/00008/00 Study Number: NAME OF COMPANY: Glaxo Wellcome NAME OF FINISHED PRODUCT: Ventolin MDI NAME OF ACTIVE INGREDIENT(S): Salbutamol INDIVIDUAL STUDY TABLE REFERRING TO PART IV OF DOSSIER Volume: Page: xx xx (FOR NATIONAL AUTHORITY USE ONLY) Title: A single centre, randomised, double-blind, placebo-controlled, crossover, pilot study to determine the efficacy and safety of 400 µg and 200 µg doses of Ventolin inhaler versus placebo for the treatment of smoking related cough. Investigator: Professor Study center: Publication(s): None at the time of this report Study period: 08 Mar May 2001 Clinical phase: ІІ Objectives: Efficacy data derived from the study was used to compare the effectiveness of (1) 400µg and 200µg of Ventolin, delivered via an MDI, compared to (2) placebo for the treatment of smokers cough, as evidenced by changes in objective and subjective measurements of cough. Safety was determined by comparing the proportion of subjects that experienced adverse events Methodology: A single centre, randomised, double-blind, placebo controlled, cross-over, proof of concept pilot study involving patients suffering from smokers cough. Subjects who met screening requirements reported to the investigational site. Subjects read a subject information sheet and provided written informed consent. The clinician assessed their suitability, and baseline objective and subjective measurements of cough were recorded. A diagnosis of asthma was excluded using the methacholine challenge test. Patients were then discharged home with a screening diary, cough counter and min peak flow meter, following instruction in the use of these items. The screening period lasted for 5 days. On return to the unit, the screening diary was assessed for completeness, and suitable subjects were then allocated to one of the two treatment sequences according to a randomised list. Following an over night stay, during which subjects abstained from smoking, they were administered a dose of 400µg of Ventolin or placebo, and were allowed their first cigarette after 20 minutes. Citric acid challenge was then performed at 1, 2 and 4 hours post dose. Subjects took their next dose (200µg or placebo) of medication in the clinic at on day 1, after which they were discharged along with study medication, a diary card, a cough counter and peakflow 4

6 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 BL2001/00008/00 Study No. NAME OF COMPANY: Glaxo Wellcome NAME OF FINISHED PRODUCT: Ventolin MDI NAME OF ACTIVE INGREDIENT(S): Salbutamol INDIVIDUAL STUDY TABLE REFERRING TO PART IV OF DOSSIER Volume: Page: xx xx (FOR NATIONAL AUTHORITY USE ONLY) Title: A single centre, randomised, double-blind, placebo-controlled, crossover, pilot study to determine the efficacy and safety of 400 µg and 200 µg doses of Ventolin inhaler versus placebo for the treatment of smoking related cough. meter as before. They took a further 1 dose on day 1, and 3 doses on day 2. Subjects recorded subjective measurements of their cough in their diaries. On days 3 to 5, subjects took the assigned study medication as required up to 3 times/day (every 6 hours). Subjects then returned to the centre with their diaries and any remaining medication, where a compliance check and AE determination was performed. This was repeated for a second treatment phase, where they received the opposite medication (Ventolin or placebo) to the first treatment phase. A follow up visit was performed in order to check for adverse events and patient preference. Number of subjects: A total of 44 patients were randomised, from 71 patients screened. Of the 44 patients who were both randomised and treated, n received Ventolin in the first treatment period, followed by placebo (sequence A), and N received placebo followed by Ventolin (sequence B). Diagnosis and criteria for inclusion: Subjects were generally healthy male and females, > 18 years of age but < 65, who had a persistant cough associated with a cigarette smoking, a pack history of greater than 5 years and willing to give written informed consent. Test product, dose and mode of administration, batch number(s): Ventolin (Salbutamol) MDI 100µg per actuation. Batch number A, Expiry date Duration of treatment: Two treatment periods, during each of which subjects received over a period of 5 days, 6 doses of study medication over the first 2 days, thereafter taking up to 3 doses a day, as required, until day 5. Reference therapy, dose and mode of administration, batch number(s): Placebo to match Salbutamol MDI E00B215/D015987, Expiry date Criteria for evaluation: Cough Frequency during the first hour following waking on day 1 of each treatment period was used to determine treatment efficacy in this study. They were asked, on waking, to record their nocturnal disturbance caused by cough. During the study, subjects were also asked to complete the subjective assessments in diary cards. Subjects were asked to describe the severity of various cough symptoms using subjective 10 point box scales daily, and to record the amount of phlegm expectorated. Objective measures included the number of cough episodes counted, as well as to record, on waking, at midday and at 10pm, the highest of 3 peak flow 5

7 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 BL2001/00008/00 Study No. NAME OF COMPANY: Glaxo Wellcome NAME OF FINISHED PRODUCT: Ventolin MDI NAME OF ACTIVE INGREDIENT(S): Salbutamol INDIVIDUAL STUDY TABLE REFERRING TO PART IV OF DOSSIER Volume: Page: xx xx (FOR NATIONAL AUTHORITY USE ONLY) Title: A single centre, randomised, double-blind, placebo-controlled, crossover, pilot study to determine the efficacy and safety of 400 µg and 200 µg doses of Ventolin inhaler versus placebo for the treatment of smoking related cough. recordings. On days 1 to 5 of each treatment period, subjects were asked to document their medication usage in their diary cards. At the end of the study, subjects were asked to record their overall treatment preference. The primary endpoint was reduction in cough frequency over the first hour following waking, on day 1 of each treatment period. The secondary endpoints were nocturnal disturbance, change in cough symptoms (severity, chest tightness, ease of expectoration, global symptom score and expectoration volume), overall treatment preference, lung function and cough threshold elevation at 1,2 and 4 hours following waking. Statistical methods: As this was a pilot study, there was no information currently available on which to base a sample size calculation. It was anticipated that 40 patients should be a sufficient number to show trends in treatment efficacy. This was also a reasonable number of patients that one center could expect to recruit. Approximately 100 subjects were planned to be screened in order to obtain 40 randomised patients. In all analysis, the null hypothesis is that of no treatment difference. The alternative hypothesis asserts a difference between treatments. This was tested using a two-sided significance level of α=0.05. P-values of <=0.05 were considered significant. Confidence intervals for the difference used a confidence level of 95%. As this is an exploratory study, no adjustments were made for multiple comparisons. The primary population for all efficacy analyses is the ITT population. The primary efficacy endpoint(s) were analysed using both the ITT and the PP populations. The secondary efficacy endpoint(s) were analysed using the ITT population. If a difference was demonstrated between the ITT and the PP analysis carried out on the primary efficacy endpoint(s) then the secondary efficacy endpoint(s) would also be analysed using the PP population. It was assumed that there is a sufficient wash-out period between treatment periods (2 7 days). Therefore no carry-over effect was investigated. 6

8 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 BL2001/00008/00 Study No. NAME OF COMPANY: Glaxo Wellcome NAME OF FINISHED PRODUCT: Ventolin MDI NAME OF ACTIVE INGREDIENT(S): Salbutamol INDIVIDUAL STUDY TABLE REFERRING TO PART IV OF DOSSIER Volume: Page: xx xx (FOR NATIONAL AUTHORITY USE ONLY) Title: A single centre, randomised, double-blind, placebo-controlled, crossover, pilot study to determine the efficacy and safety of 400 µg and 200 µg doses of Ventolin inhaler versus placebo for the treatment of smoking related cough. Summary and Conclusions: The primary endpoint of the difference in cough frequency during the first hour on Treatment Day 1 was not found to be statistically significant. {[Difference 1.4, 95% CI 3.7, 0.9; p=0.216]} The secondary efficacy endpoint of cough threshold evaluation measured as response to citric acid challenge was found to be statistically significant at 1 hour: {[Difference 107.1, 95% CI 17.5, 196.7; p=0.020]} The secondary efficacy endpoint of Evening PEF was found to statistically significant, giving confidence that patient compliance was good: {[Difference 9.3, 95% CI 2.5, 16.1; p=0.009]} Analysis of all other secondary endpoints failed to show any statistically significant differences between the active group and placebo at all time points. Adverse events in total reported against placebo and Ventolin MDI were 13 (30%) and 20 (45%), whilst drug related adverse events were 7 (16%) and 12 (27%) respectively. Conclusion: The results show that the principle of efficacy of Ventolin MDI in the treatment of smoking related cough was not proven, hence Ventolin MDI, in its current formulation, warrants no further development as a cough medicine for smoking related cough. Date of Report: 27 November

9 CONFIDENTIAL BL2001/00008/00 Table of Contents Page 1. INTRODUCTION STUDY OBJECTIVE(S) AND ENDPOINT(S) Study Objective(s) Study Endpoint(s) INVESTIGATIONAL PLAN Study Design Control Group(s) Protocol Amendment(s) Study Population Inclusion Criteria Exclusion Criteria Criteria for Premature Study Drug and/or Study Discontinuation Study Treatments Study Drugs Treatment Assignment Treatment Administration Blinding of Study Drug and Blinding of Treatment Assignment Treatment Compliance Concurrent Medications and Non-Drug Therapies Medical Devices MEASUREMENTS AND EVALUATIONS Demographic and Baseline Characteristics Study Drugs Efficacy Measures Primary Efficacy Measure(s) Secondary Efficacy Measure(s) Safety Measures Adverse Events Medical Examination Medical Devices DATA ANALYSIS METHODS Data Quality assurance Quality Control Measures Sample Size Considerations Interim Analyses and Data Monitoring General Considerations for Data Analyses Derived and Transformed Data Composite Global Symptom Score Nocturnal Disturbance and PEF

10 CONFIDENTIAL BL2001/00008/ Examination of subgroups Changes from the Data Analysis Plan Study Population Subject Accountability Protocol Deviations Treatment Compliance Other Descriptions of Study Population Efficacy Analyses Primary Efficacy Measure(s) Secondary Efficacy Measure(s) Other Efficacy Measures Safety Analyses Extent of Exposure Adverse Events Deaths and Serious Adverse Events Adverse Events Leading to Discontinuation of Study Drug and/or Withdrawal from the Study and Other Significant Adverse Events Pregnancies STUDY POPULATION RESULTS Subject Accountability Protocol Deviations Populations Analyzed Demographic and Baseline Characteristics Demographics Medical Conditions Concurrent Therapy Treatment Compliance EFFICACY RESULTS Primary Efficacy Measure - Cough Frequency Secondary Efficacy Measure(s) Cough Frequency Nocturnal Disturbance Cough Severity Chest Tightness Volume of Expectoration Composite Global Symptom Score Treatment Preference Morning and Evening PEF D2 and D5 (Mm) Efficacy Conclusions SAFETY RESULTS Extent of Exposure Adverse Events

11 CONFIDENTIAL BL2001/00008/ Deaths Serious Adverse Events Adverse Events Leading to Discontinuation of Study Drug and/or Study Withdrawal Other Significant Adverse Events Pregnancies Medical Device Incidents, Near-Incidents, Malfunctions and Remedial Action Safety Conclusions DISCUSSION CONCLUSIONS REFERENCES TABLES Table 1 Summary of Subjects Screened Table 2 Summary of Subject Accountability Table 3 Summary of Subject Accountability - End of Study Record Table 4 Summary of End of Study Drug Record Table 5 Summary of Major Protocol Violations Table 6 Summary of Analysis Populations Table 7 Summary of Demographic and Screening Characteristics Table 8 Summary of Smoking History Table 9 Summary of Child-Bearing Potential Table 10 Summary of Current Medical Conditions Table 11 Summary of Baseline Vital Signs Table 12 Summary of Concurrent Medications Started and Stopped Prior to Study Treatment Table 12 Summary of Concurrent Medications Started Prior to Study Treatment and Ongoing During Study Table 12 Summary of Concurrent Medications Started During Study Treatment Table 13 Summary Statistics of Cough Frequency Table 14 Analysis of Cough Frequency Table 15 Summary Statistics of Cough Frequency Table 16 Analysis of Cough Frequency Table 17 Summary of Mean Nocturnal Disturbance Table 18 Analysis of Mean Nocturnal Disturbance Table 19 Summary Statistics of Cough Severity Table 20 Analysis of Cough Severity Table 21 Summary Statistics of Chest Tightness Table 22 Analysis of Chest Tightness Table 23 Summary of Volume of Expectoration Table 24 Summary of Composite Global Symptom Score [1] Table 26 Summary of Morning PEF Table 27 Analysis of Morning PEF

12 CONFIDENTIAL BL2001/00008/00 Table 28 Summary of Evening PEF Table 29 Analysis of Evening PEF Table 30 Summary of D2 Concentrations Table 31 Analysis of D2 Concentrations Table 32 Summary of D5 Concentrations Table 33 Analysis of D5 Concentrations Table 34 Summary of Study Drug Exposure Table 35 Summary of All Adverse Events Started Pre-Treatment Table 35 Summary of All Adverse Events Started During Treatment. 126 Table 35 Summary of All Adverse Events Started Post-Treatment Table 36 Summary of Drug-Related Adverse Events Table 37 Summary of Serious Adverse Events Table 38 Summary of Treatment-Limiting Adverse Events LISTINGS Listing 1 Listing of Populations Listing 2 Listing of Randomisation Code Listing 3 Listing of Inclusion/Exclusion Criteria Listing 4 Listing of Subjects Who Did Not Satisfy Inclusion/Exclusion Criteria Listing 5 Listing of Subjects For Whom the Treatment Blind was Broken During the Study Listing 6 Listing of Subjects with Protocol Violations Listing 7 Listing of Subject Accountability - End of Study Record Listing 8 Listing of Demographic Characteristics Listing 9 Listing of Child-Bearing Potential Status and Types of Contraception Used Listing 10 Listing of Current Medical Conditions Listing 11 Relationship between Previous and Concurrent Medication Group Terms and Preferred Terms Listing 12 Listing of Concurrent Medications Treatment Sequence : AB Listing 13 Listing of Vital Signs (Screening) Listing 14 Listing of FEV1 (Screening) Listing 15 Listing of ECG Data (Screening) Listing 16 Listing of Diagnosis of Asthma and ability to Cough (Screening) Listing 17 Listing of Smoking History Listing 18 Listing of Subjective Cough Assessments (Screening) Listing 19 Listing of Number of Cough Episodes (Screening) Listing 20 Listing of Number(teaspoons) of Cough Expectorations (Screening) Listing 21 Listing of Peak Flow (L/min) (Screening) Listing 22 Listing of Nocturnal Disturbance (Screening) Listing 23 Listing of Carbon Monoxide Monitoring/Peak Expiratory Flow

13 CONFIDENTIAL BL2001/00008/00 Listing 24 Listing of Dosing with Ventolin/Placebo/Time of First Cigarette Listing 25 Listing of Cough Frequency Listing 26 Listing of Citric Acid Challenge Listing 27 Listing of Subjective Cough Assessments Listing 28 Listing of Number of Cough Episodes Listing 29 Listing of Number(teaspoons) of Cough Expectorations Listing 30 Listing of Peak Flow (L/min) Listing 31 Listing of Nocturnal Disturbance Listing 32 Listing of Dosing Times Listing 33 Listing of Exposure to Study Drug Listing 34 Listing of Treatment Preference Listing 35 Relationship of Adverse Event Body Systems, Grouped Terms and Verbatim Text Listing 36 Listing of Subject Numbers for Individual Adverse Events Listing 37 Listing of All Adverse Events Treatment Sequence: AB

14 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 BL2001/00008/00 Study No. Abbreviations AE ANOVA BP CI CIB CRF CTU DAP DRC ECG FEV1 GCP GW ICF ICH IEC ITT IUD MIDAS PACT PP PV SD SAE SPC WPSP Adverse Event Analysis of Variance Blood Pressure Confidence Interval Clinical Investigator s Brochure Case Report Form Clinical Trials Unit Data Analysis Plan Daily Record Card Electrocardiogram Forced Expiratory Volume in 1 Second Good Clinical Practice Glaxo Wellcome Informed Consent Form International Conference on Harmonization Independent Ethics Committee Intent-to-Treat Intra-Uterine Device Medicines, Indications, Diagnoses, Adverse Events and Symptoms (Glaxo Wellcome Data Coding System for Adverse Events) Patient Allocation Clinical Trials Per Protocol Protocol Violator Standard Deviation Serious Adverse Event Summary of Product Characteristics Worldwide Product Safety and Pharmacovigilance 13 8

15 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 BL2001/00008/00 Study No. Ethics Independent Ethics Committee (IEC) or Institutional Review Board (IRB) The study protocol and any amendments and other information that required pre-approval were reviewed by an investigational center ethics committee: Ethical Conduct of the Study The study was conducted in accordance with "good clinical practice" (GCP) and all applicable regulations, including, where applicable, the Declaration of Helsinki. Subject Information and Consent The investigator or his designee informed the subject of all aspects pertaining to the subject s participation in the study. The process for obtaining subject informed consent was in accordance with all applicable regulatory requirements. The investigator or his designee and the subject both signed and dated the ICF before the subject participated in the study. The subject received a copy of the signed and dated form and the original was retained in the site study records. The decision regarding subject participation in the study, that was made by the subject, was entirely voluntary. The investigator or his designee emphasized to the subject that consent regarding study participation may be withdrawn at any time without penalty or loss of benefits to which the subject is otherwise entitled. The ICF had not been amended during the study and it accurately reflected the study protocol, was clearly identified by version number and date, and was approved by the The investigator had followed all applicable regulatory requirements pertaining to approval of the ICF by the IEC. 14 9

16 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 BL2001/00008/00 Study No. Investigators and Study Administration Professor Head of Department Tel : Fax : Tel : Fax :

17 1. INTRODUCTION CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. Many habituated smokers have a chronic cough, for which there is currently no therapy proven to be effective [1]. Smoking may cause increased reactivity of the airways, resulting in bronchospasm of the lower airway. Ventolin contains the β 2 -agonist Salbutamol, an effective bronchodilator, and may therefore have antitussive properties in smoking related cough [2,3]. 2. STUDY OBJECTIVE(S) AND ENDPOINT(S) 2.1. Study Objective(s) The aim of the study was to determine the efficacy and safety of 400 µg and 200 µg Ventolin (Salbutamol) inhaler compared with placebo on natural (i.e. on waking, before contact with potential triggers) and evoked (following the first cigarette) cough in habituated smokers, as evidenced by changes in objective and subjective measurements of cough [4] Study Endpoint(s) The primary endpoint was the cough frequency during the 1st hour on treatment day 1. The secondary endpoints were cough frequency on treatment day 1 (after the 1st hour) and on treatment days 2 to 5, nocturnal disturbance due to cough (over treatment days 1 to 5), change in cough symptoms (recorded at 12 noon and 10pm on Days 1 to 5), treatment preference, lung function (peak flow) and cough threshold evaluation measured as response to citric acid challenge. 3. INVESTIGATIONAL PLAN 3.1. Study Design This proof of principle pilot study was a single-centre, randomised, double-blind, placebo-controlled, crossover study, consisting of: a 5 day screening phase, during which baseline cough measurements were recorded; a first treatment phase of five days; a washout phase of between two to seven days; a second treatment phase of five days; and a follow up visit, as shown below. Screening (optional interval) Treatment 1 Washout Treatment 2 Follow up (5 days) (max 2 days) (5 days) (2-7 days) (5 days) (single visit) Visit Visit Visit 3 Visit

18 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. During the screening period, subjects were screened for eligibility, and if eligible, were asked to record baseline cough frequency, nocturnal disturbance, cough symptoms and peak flow. Subjects were asked to return to the Clinical Trials Unit the evening before each treatment period. Prior to treatment period one, each subject had their eligibility confirmed and, if eligible, were randomised to receive either active medication during treatment period 1 and placebo during treatment period 2, or the reverse. On waking on the morning of the first day of both treatment periods, while under supervision in the clinical trials unit, subjects received an initial dose of 400 µg Ventolin inhaler or placebo. Twenty minutes after receiving study medication, subjects smoked their first cigarette of the day which was expected to provoke a cough reflex. One hour after drug administration subjects underwent a cough challenge test with nebulised citric acid. They were then discharged home at noon on the first treatment day. All subsequent doses of Ventolin inhaler were 200 µg or matched placebo. During the 5 day treatment period, subjects were asked to complete their diary cards as accurately as possible, to take their study medication at the pre-determined times. For days 1 and 2 of each treatment period, subjects were asked to use their study medication 3 times per day, at 08.00, and For days 3-5, they could take study medication up to 3 times per day as required Control Group(s) A placebo control, identical in presentation to the study medication, was used in this study. All subjects received Ventolin during one treatment period, and placebo during the other Protocol Amendment(s) There were no amendments to the protocol Study Population Observations, assessments and investigations were made in every subject who volunteered for the study on the morning or afternoon preceding inclusion, i.e. prior to entry into the screening phase of the study. The results of these observations, in addition to the written informed consent of each subject, enabled the investigator to make a decision regarding inclusion or exclusion of the subject. At the end of the screening phase, subjects were again assessed for suitability, as evidenced by diary card compliance and absence of illness during the screening phase. If judged suitable, subjects were then randomised to treatment. All subjects included in the study responded to advertisements posted in and around the complex. They had been selected on a walk-in basis after a careful clinical examination carried out by one of the Clinical Trials Unit Physicians. Excluding smokers cough, all subjects were defined as healthy individuals if they were free from significant cardiac, pulmonary, gastrointestinal, hepatic, renal, haematological, neurological and psychiatric disease as determined by medical history and physical examination

19 Inclusion Criteria CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. A subject was eligible for inclusion in this study only if all of the following criteria applied: Men or women, aged 18 to 65 years, suffering from smoking related cough but otherwise healthy. A female was eligible to enter and participate in this study if she was of: a non-childbearing potential (i.e., physiologically incapable of becoming pregnant, including any female who is post-menopausal); or, b child-bearing potential, had a negative pregnancy test (urine) at screen, and agreed to one of the following: -Complete abstinence from intercourse throughout the study -Implants of levonorgestrel (except Norplant system); or, -Injectable progestogen; or, -Oral contraceptive (combined or progestogen only); or, -Any intrauterine device (IUD) with published data showing that the lowest expected failure rate is less than 1% per year; or, -Any other methods with published data showing that the lowest expected failure rate for that method is less than 1% per year; or, -Barrier method only if used in combination with any of the above acceptable methods; or, The subject was sterilised. Healthy subjects as determined by medical history, physical examination, vital signs, and ECG. FEV 1 > 80% of predicted Subjects smoking greater than 15 cigarettes a day and unwilling give up. Subjects who had a greater than 5 pack year smoking history (1 pack year = 20 cigarettes per day for 1 year) Subjects recorded morning cough on all five days of screening diary record card assessment. History of persistent daily morning cough for a minimum of 3 months prior to entry. Subject had the ability to read comprehend and record information required in the study. Subject had signed and dated written informed consent prior to participating in the study

20 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. Subject was willing to comply with the study requirements (including abstinence from cough treatments, abstinence from any other medications containing codeine, diary card completion at 10pm) Exclusion Criteria A subject was not be eligible for inclusion in this study if any of the following criteria applied: Inability to co-operate with study protocol. Chronic obstructive pulmonary disease - (emphysema + bronchitis + asthma) or asthma alone; all subjects were required to undergo a Methacholine challenge, a positive response was considered diagnostic for asthma and resulted in exclusion from the study. Other significant concurrent illness (clinical laboratory investigations were conducted if, in the opinion of the investigator, they were required in order to exclude the possibility of significant concurrent illness). Any concurrent medication that the study clinician considered contra-indicated to the study e.g. antitussives or mucolytics within 24 hours or anti-histamines within 72 hours before commencement of the study. Participation in another clinical study in which the subject was exposed to an investigational or non-investigational drug or device in the previous month. Known sensitivity to Salbutamol. History of drug or alcohol abuse (no more than 4 units per day, 1 unit being ½ pint of beer, or 1 glass of wine, or measure of spirit). Recent (within 14 days) respiratory tract infection. Pregnancy or lactation. Norplant system as a method of contraception Criteria for Premature Study Drug and/or Study Discontinuation Patients who met any of the following criteria were discontinued from the study: Presence of a medically relevant AE or intercurrent medical illness such that continued participation in the study posed a serious medical risk. Significant non-compliance with the study procedures as determined by the investigator and/or GW monitors. Inability to tolerate the protocol specified dose of the study medication. Decision on the part of the investigator or the patient to discontinue treatment for any reason. Use of any other treatment for their cough than the study medication that the investigator felt seriously compromised data quality 14 19

21 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. A patient could voluntarily discontinue participation in this study at any time. The investigator could also, at his discretion, discontinue the patient from participating in this study at any time Study Treatments Study Drugs GlaxoWellcome Research and development supplied Salbutamol inhalers (Ventolin MDI) and Salbutamol inhaler placebo. Tested product : Salbutamol MDI Batch Number A Placebo : Placebo MDI identical in appearance, smell and taste. Batch Number E00B215 or D Dosing methods used in this study corresponded to normal use conditions Dose Rationale The doses selected are based on the effective doses used in asthma. 400 µg Ventolin is expected to have the greater therapeutic effect, 200 µg Ventolin is expected to be better tolerated Dosages and Dosing Ventolin was available as a 100µg per actuation metered dose inhaler. To ensure blinding, placebo inhalers were manufactured using exactly the same process and propellants as the Ventolin MDI, with the exception of the active ingredient. Subjects received both Ventolin and placebo, one each during each of the treatment periods. The order was determined according to a randomised list Overdose and toxicity management No specific treatment was recommended, and the investigator was expected to use clinical judgement in treating overdose. The preferred antidote for overdose with Salbutamol is a cardioselective β-blocking agent. Beta-blocking drugs were to be used with caution in patients with history of bronchospasm. As hypokalaemia may occur following overdose with Salbutamol, serum potassium levels were to be monitored Treatment Assignment Subjects were assigned to study treatment in accordance with the randomisation schedule. Glaxo Wellcome, using a computer system PACT designed to create random schemes, generated the randomisation code prior to the study start

22 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. A sealed codebreak envelope for each treatment number identifying whether the subject has been assigned to sequence 1 Ventolin MDI during treatment period 1 and placebo during period 2 or sequence 1 placebo during treatment period 1and Ventolin MDI during period 2, was sent by Glaxo Wellcome to a nominated individual at the study site (centre) Each subject who satisfied the criteria for randomisation was assigned the next sequential treatment number at the centre. Once a treatment number had been assigned to a subject, it could not be assigned to any other subject. In the event that the treatment blind was broken for a subject, then this subject was to be withdrawn from the study if the study clinician considered continued participation to be contra-indicated Treatment Administration Subjects received a first dose of 400 µg Ventolin or placebo, and a second dose of 200 µg Ventolin or placebo while under supervision in the CTU. All subsequent doses were administered without supervision. During days 1 and 2 of each treatment period subjects received study medication three times a day (tid), during days 3, 4 and 5 of each treatment period subjects were allowed to take medication as required (prn), but not more than three times in 24 hours. Subjects were required to record the dosing times on their diary cards, and to record their subjective cough assessments 2 hours post dosing Blinding of Study Drug and Blinding of Treatment Assignment Study medication was blinded according to the randomisation procedures described above, such that the identity of the medication in each inhaler was unknown to the investigator and the subject. All medicines were supplied in inhalers contained in identical packaging. Only in the case of an emergency, when knowledge of the study drug was essential for the clinical management or welfare of the subject, the investigator could have unblinded a subject s treatment assignment. Had the blind been broken for any reason, the investigator was to notify the appropriate GW representative immediately. If the investigator broke the blind for an individual subject, the date and reason was to be recorded on the Status of Treatment Blind CRF. The investigator was not to reveal the blind to the monitor. Individual codebreak envelopes allowed the code to be unblinded for one subject whilst information for all other subjects would remain blinded. Treatment blind was not broken in the conduct of this study

23 Treatment Compliance CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. Patient s daily records of all medication taken during the study, including the exact time of administration of the inhaler, served as an estimate of compliance with therapy. The investigator maintained a drug accountability list, which included the start and stop dates of study medication and reconciliation of drug dispensed and returned for the home phase Concurrent Medications and Non-Drug Therapies As noted in the exclusion criteria, there were mandatory requirements to avoid concomitant therapies that might affect the results. During the treatment periods, subjects were asked to refrain from taking any other medication that might affect the results of the study, however use of non-opiate analgesics was allowed. One subject took a concomitant therapy during the study that could have interfered with the interpretation of the study results. The patient was not withdrawn from the study. However, if the investigator had felt that the study results were seriously compromised, the subject could have been withdrawn from the study at the discretion of the investigator. Any therapy that was taken during the study was reported on the diary card and appropriate section of the CRF Medical Devices GW provided, for use in the study, the following: Mini Wright peak flow meters Volumetric spacers Portable counters for recording the number of daily cough episodes The investigator provided, for use in the study, the following medical devices: Methacholine and Citric acid challenge testing equipment 4. MEASUREMENTS AND EVALUATIONS 4.1. Demographic and Baseline Characteristics Subjects who had signed the informed consent document confirming their agreement to take part in the study were assessed by the study clinician to determine their suitability. The study clinician recorded their gender, height, weight and measured their vital signs i.e. temperature, pulse and blood pressure. This information was recorded directly into the subject s Case Report Forms (CRF). The clinician took medical history and performed a urine pregnancy test (if female) and a brief medical examination with emphasis on the respiratory system. This information was recorded in the subjects Case Report Form. ECG and lung function (FEV1) data was collected. A Methacholine 17 22

24 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. challenge was performed in order to detect previously undiagnosed asthma, and a citric acid challenge was performed in order to assess the subjects ability to cough. A smoking history was obtained, including the number of pack years for that subject. Blood and urine samples will be collected and x-rays will be taken for clinical investigations if considered necessary by the investigator to exclude diagnosis of significant concurrent illness. Pregnancy testing will also be conducted using urine samples. Following successful completion of the initial screening visit, subjects entered a 5 day screening period. During the screening period subjects were provided with a cough counter and peak flow meter, and asked to complete a screening diary card to record baseline values for cough frequency, nocturnal disturbance, cough symptoms, and peak flow. Subjects were required to abstain from using any cough treatments and any products containing codeine from visit 1 until completion of the second treatment period. An interval of up to two days could be observed between completion of the screening period and the start of treatment period Study Drugs The investigator maintained a drug accountability list, which included the start and stop dates of study medication and reconciliation of drug dispensed and returned for the home phase. The doses of Ventolin syrup used in this study were 400µg for the first dose of each treatment period and 200µg for all subsequent doses. The route of administration was by inhalation. No treatment blind was broken during the study. Patients received a minimum of 6 doses of study medication, but were able to take treatment up to a maximum 5 days if required Efficacy Measures On the night before treatment day one, subjects were admitted to the Clinical Trials Unit at to ensure compliance with study procedures, and to confirm eligibility following clinical laboratory investigation results and review of the screening diary card (visit 2). If subjects were eligible to enter, they were randomised and told to continue smoking as normal until midnight when they were requested to abstain from cigarettes. Overnight smoking abstinence was confirmed by the use of carbon monoxide monitoring (Smokealyser). On waking the following morning (study day one) subjects recorded peak flow. They then received, in a blinded randomised fashion, 400 µg of Salbutamol or matched placebo via metered dose inhaler (MDI) fitted with a spacer. Drug administration was followed by a one hour observation period when both morning cough and plasma Salbutamol should have been at maximum 6 Cough frequency was recorded by voice activated analogue tape recorder and microphone during the 0-10 minute and minute intervals following drug administration. Twenty minutes after drug administration subjects entered the designated smoking room and were asked to have their first cigarette (own brand) of the day. Subjects were 18 23

25 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. observed for the number of coughs produced by this manoeuvre, coughs being counted for 20 minutes after the cigarette and a subsequent 20 minute period (i.e minutes post drug administration and 40 minutes - 1 hour post drug administration). Subjects then (i.e. 1 hour after drug administration) underwent cough challenge with nebulised citric acid according to the established protocol 7 (see appendix 1 for SOP). Briefly, they received incremental doses of citric acid interspaced at random intervals with nebulised saline. The sensitivity of the cough reflex was expressed as D2 and D5 measurements. These are the doses of citric acid causing at least two or five coughs per inhalation. Subjects were then allowed to smoke ad libitum but with the number and time of cigarettes consumed recorded on the diary record card. Cough challenge was repeated at two hours and four hours. Subjects received their second dose of study medication and were discharged from the CTU with instructions to continue their allotted medication and to complete the treatment period 1 diary card. Morning and afternoon/evening cough frequency, symptoms, expectoration; nocturnal disturbance; twice daily peak flow measurements; and any adverse events were recorded as separate items on the diary card. Investigators questioned subjects regarding any adverse events (see section 7) Primary Efficacy Measure(s) The primary efficacy endpoint was reduction in cough frequency over the first hour following waking on treatment day 1. Cough frequency at all other times during the treatment periods was a secondary endpoint Secondary Efficacy Measure(s) The secondary endpoints were: Nocturnal disturbance due to cough (5 point scaled answer); Daily recording of change in cough symptoms: severity, chest tightness, ease of expectoration, global symptom score (10 point scaled answers) and expectoration volume (approximate number of teaspoons); Overall treatment preference (treatment 1, treatment 2, or no preference); Lung function on waking and at 10pm (peak flow); and Cough threshold elevation 1,2 and 4 hours following waking on treatment day 1 only (D2 and D5 values in response to citric acid challenge) Safety Measures Vital signs (heart rate, blood pressure) and temperature were recorded. Safety was evaluated by adverse event reporting. Adverse events were coded and grouped by body 19 24

26 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. systems using the GW MIDAS system. A comparison of treatment safety as evidenced by adverse event reporting was summarized and compared between treatment groups Adverse Events The investigator was responsible for the detection and documentation of events meeting the definition of an AE or SAE as defined below. During each treatment period, when there was a safety evaluation, the investigator or study site personnel were responsible for detecting AEs and SAEs. Adverse event data was collected by questioning subjects on their well-being and any medication taken in addition to the study drug and also in conjunction with their diary card entries. In order to fulfil international safety reporting obligations, the investigator included in his assessment any SAEs resulting from study participation (e.g., complications from taking a blood sample). An AE is any untoward medical occurrence in a Subject or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with this treatment. An AE can therefore be any unfavourable and unintended sign (that could include a clinically significant abnormal laboratory finding), symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. An AE does include a/an: exacerbation of a pre-existing illness. increase in frequency or intensity of a pre-existing episodic event or condition. condition detected or diagnosed after study drug administration even though it may have been present prior to the start of the study. continuous persistent disease or symptoms present at baseline that worsen following the start of the study. An AE does not include a/an: medical or surgical procedure (e.g., surgery, endoscopy, tooth extraction, transfusion); the condition that leads to the procedure is an AE. pre-existing disease or conditions present or detected at the start of the study that do not worsen. situations where an untoward medical occurrence has not occurred (e.g., hospitalisations for cosmetic elective surgery, social and/or convenience admissions). the disease or disorder being studied or sign or symptom associated with the disease or disorder unless more severe than expected for the subject s condition. overdose of either study drug or concurrent medication without any signs or symptoms

27 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. An SAE is any adverse event occurring at any dose that results in any of the following outcomes: a b c d e f Death A life-threatening adverse event Inpatient hospitalisation or prolongation of existing hospitalisation A disability/incapacity A congenital anomaly in the offspring of a subject who received drug Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse event when, based upon appropriate medical judgement, they may jeopardise the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in Subject hospitalization, or the development of drug dependency or drug abuse Medical Examination Medical examination was performed within one hour prior to first drug treatment and involved medical history and brief medical examination with emphasis on the respiratory system. Results were reported in the CRF Vital Signs Blood pressure and pulse were recorded before entering the study. Also, tympanic temperature was taken and data were recorded in the CRF Pregnancy Urine pregnancy testing was performed on females of child-bearing potential at screening and at the end of the treatment period (post treatment visit day 7). Information on whether pregnancy occurred was collected from accepting participation in the study by signing a written informed consent until the end of the study treatment period (day 7 or day of withdrawal, whichever came first.) No subjects became pregnant during the course of the study Medical Devices GW provided medical devices for use in this study. In order to fulfil international reporting obligations the investigator was responsible for the detection and documentation of events meeting the definitions of incident, near-incident, or 21 26

28 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. malfunction that occurred during the study with such devices, whether the devices are manufactured by GW or by another company. GW provided, for use in the study, the following: Mini Wright peak flow meters Volumetric spacers Portable counters for recording the number of daily cough episodes The investigator provided, for use in the study, the following medical devices: Methacholine and Citric acid challenge testing equipment Medical Device Any instrument, apparatus, appliance, material or other article, the principal intended action of which is typically fulfilled by physical means (including mechanical action, physical barrier, replacement of, or support to, organs or body functions). Examples of medical devices include measuring spoons, syringes, springloaded auto-injector, infusion pumps, and endotracheal tubes. The detection and documentation procedures described in the protocol apply to all medical devices provided by GW for use in the study, whether such devices are manufactured by GW or by another company. Incident Any medical occurrence that occurs with a medical device, provided by GW for use in the study, which results in death or a serious deterioration in the state of health whether or not due to a malfunction of the device. Incidents include for example: inhalation of an object that has accidentally entered a spacer device and consequent tracheal obstruction. Incidents do not include for example: medical occurrences associated with metered-dose inhalers that do not fulfil the definition of a medical device (such events should be reported as medicinal product AEs) non-serious medical occurrences which have no further safety implications for the subject or the device Near-incident Any potential incident occurring with a medical device provided by GW for use in the study. Near-incidents are events that could have jeopardized the study subject, causing death or a serious deterioration of health, if medical intervention or other fortunate circumstances had not occurred. This would include deficiencies or inaccuracies in the instructions for use of the device and/or malfunctions

29 Near-incidents include for example: CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. discovery of caustic damage to the plastic parts of a device caused by contamination with an ointment, where no actual injury to a subject occurred but the reporter recognised that such an event could happen in normal medical use of the device and could kill or seriously harm a subject. Malfunction A failure of a device to perform in accordance with its intended purpose when used in accordance with the manufacturer s instructions. Remedial Action Any action other than routine maintenance or servicing of a device where such action is necessary to prevent recurrence of an incident or near-incident. This includes any amendment to the design to prevent recurrence. 5. DATA ANALYSIS METHODS 5.1. Data Quality assurance The investigator or designee recorded all required subject data using the previously specified data collection method defined by Glaxo Wellcome. An explanation was documented for any missing data. The investigator signed and dated a declaration on the CRF attesting to his responsibility for the quality of all data recorded and that the data represented a complete and accurate record of each subject s participation in the study. All data retrieved from the site was entered onto a quality controlled database. These data were subsequently analysed according to the methods outlined below. The methods for analysis and presentation of results were detailed prior to unblinding of treatments and the start of analysis in a Data Analysis Plan (DAP), dated 19 July Apart from minor modifications to the layout of some tables and listings and production of additional summaries, the analysis was performed according to the DAP. Principal changes from analyses planned in the protocol and DAP are described in Section The statistical analysis was performed using SAS version 6.12 systems and procedures Quality Control Measures All CRF and diary data were independently double-data-entered into SAS PC version Data were compared using programs developed by GSK and any discrepancies were resolved. The database was locked on 25 July Sample Size Considerations This was a pilot study and no data on the variability in spontaneous cough following cigarettes was available. Therefore it was not possible to base the power calculations around the primary endpoint. However, the change in cough sensitivity with citric acid (main secondary endpoint) is well described. A sample size of 35 subjects was required to have an 80% chance of detecting a change of one cough per cough challenge at the 5% level

30 CONFIDENTIAL CONFIDENTIAL 5.4. Interim Analyses and Data Monitoring BL2001/00008/00 Study BL2001/00008/00 No. No interim analyses were planned or performed for this study. In accordance with applicable regulations, GCP, ICH and GW procedures, monitors periodically contacted the site, including conducting on-site visits. The extent, nature and frequency of on-site visits were based on such considerations as the study objectives and/or endpoints, the purpose of the study, study design complexity and enrollment rate. During the contacts the monitor: Checked and assessed the progress of the study Reviewed study data collected Conducted Source Data Verification Identified any issues and addressed their resolution This was done in order to verify that: Data was authentic, accurate and complete Safety and rights of subjects were being protected Study was conducted in accordance with the currently approved protocol, GCP and all applicable regulatory requirements The investigator agreed to allow the monitor direct access to all relevant documents and to allocate his time and the time of his staff to the monitor to discuss findings and any relevant issues. In addition to contacts during the study, the monitor contacted the site prior to the start of the study to discuss the protocol and data collection procedures with site personnel General Considerations for Data Analyses Except where stated otherwise, all analyses requiring significance testing used a twosided test at the 5% significance level, and all 95% confidence intervals (CIs) are shown Derived and Transformed Data The cough severity and chest tightness profiles {(R i, t i ), i = 0,, k} for each subject are summarised using weighted mean responses calculated as the areas under the responsetime profiles using trapezoidal integration, divided by the duration of the specified observation period: Weighted mean response = ½ (t i - t i-1 ) (R i-1 + R i ) / (t k t 0 ), summing over i = 1,, k. Actual times {t i } were used for the derivation of weighted mean responses, if an actual time was missing the expected time was used

31 CONFIDENTIAL CONFIDENTIAL Composite Global Symptom Score BL2001/00008/00 Study BL2001/00008/00 No. The composite global symptom score was derived as the sum of the cough severity score and the chest tightness score. For the purposes of this pilot study, individual symptom scores were given equal weight in the derivation of the global symptom score Nocturnal Disturbance and PEF The mean nocturnal disturbance and mean am PEF for each subject was calculated using all available data from Days 2 to 6 for the screening period and each treatment period. The mean pm PEF for each subject was calculated using all available data from Days 1 to 5 for the screening period and each treatment period Examination of subgroups The study was not powered to detect interactions. Therefore, tests for two-factor interactions between treatment and sex, age and baseline (where appropriate) were to be tested for statistical significance at the 0.10 level. However as the data was normally distributed and the trial was a cross over trial this was not performed Changes from the Data Analysis Plan The examination of subgroups was not performed. The protocol states that Prescotts test will be used for the statistical analysis, however as the data was normally distributed an ANCOVA was performed unless the assumptions were not satisfied Study Population Subject membership of all analysis populations was determined blind to study treatment. The following three analysis populations were used: Intent-to-Treat population (ITT) - all subjects who were randomised to treatment and received at least one dose of trial medication and had at least one post-treatment efficacy assessment. Per Protocol Population (PP) The PP population consists of subjects in the ITT population but excluded any subjects who had a major protocol violation during the study. Major violations leading to the exclusion of a subject from the PP population were detailed and confirmed prior to breaking the blind. Safety population - consists of all subjects randomised to study treatment who received at least one dose of trial medication. This population was to be used if it differed from the ITT population for the safety summaries. Subjects were only excluded from the ITT and Safety populations if there was documented proof that they received no study medication

32 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. Before the randomisation code was broken, full and precise agreement regarding subject membership of all analysis populations were reached, in line with International Conference on Harmonisation (ICH) guidelines Subject Accountability The number of subjects randomised into the study and the number contained within each analysis population (i.e. intent-to-treat, per-protocol and safety) have been listed and summarised. The number of subjects who completed the study or who discontinued prematurely was also listed and summarised by reason Protocol Deviations Subject data was examined for evidence of protocol violations in order to assess how well the protocol was followed, any such violations have been summarised and listed. Inclusion and exclusion criteria are detailed in sections and of the protocol. Violators of these criteria were summarised and listed. They were not excluded from any ITT population analyses Major Protocol Violations Subjects who committed major protocol violations were excluded from the Per Protocol Population. Subjects are either major or partial major protocol violators. A major protocol violator was completely excluded from the Per Protocol population whilst a partial major protocol violator had some data excluded from the data the violation occurred. The major protocol violations are detailed below. Violators of inclusion criteria Males or females <18 years or >65 years old FEV1 80 % of predicted Subjects smoking 15 or less cigarettes a day Subjects with 5 pack years or less smoking history Females of child-bearing potential not following one of the contraceptive methods detailed in the protocol and/or no negative pregnancy test performed Subjects who did not provided informed consent prior to participation in the study Subjects not recording morning cough on all five days of screening diary record card assessment Subjects without a history of persistent daily morning cough for a minimum of 3 months prior to entry Subjects not able to read comprehend and record information required in the study 26 31

33 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. Subjects not willing to comply with the study requirements (including abstinence from cough treatments, abstinence from any other medications containing codeine, diary card completion at required times) Subjects not using GW Acceptable Contraceptive Methods. Violators of exclusion criteria Subject had asthma (Confirmed by post study reversibility testing and diagnosed by principal investigator) Subjects with chronic obstructive pulmonary disease - (emphysema + bronchitis + asthma); or emphysema alone; or asthma alone; all subjects will be required to undergo a methacholine challenge, a positive response would be considered diagnostic for asthma and resulted in exclusion from the study. Subjects with mild chronic bronchitis alone were not excluded from the study provided they fulfiled all of the inclusion criteria and none of the exclusion criteria. Subject with a clinically significant illness or disease e.g. cardiovascular, gastrointestinal, endocrinological, neurological diseases as determined by their medical history and physical examination Subject had taken any medication that the study clinician considers contra-indicated to the study e.g. antitussives or mucolytics within 24 hours or anti-histamines within 72 hours before commencement of the study Subject has a history of drug and/or alcohol abuse Subject was concurrently participating or has participated in another clinical study in which the subject was exposed to an investigational or non-investigational drug or device in the previous month Subject had taken a product containing menthol within the hour before commencement of the study Subject had a known sensitivity to salbutamol Subjects with a recent (within 14 days) respiratory tract infection Subjects using a Norplant system as a method of contraception Subject unable to cooperate with study protocol Minor Protocol Violations All the protocol violations in section 6.2 are major, and caused the subject to be excluded from the Per Protocol Population. In addition the listing identify the following violations, but the subject was not excluded from the Per Protocol Population. Subjects who became pregnant or who were lactating Subjects whose visits fell outside the visit windows specified in the protocol Subjects for whom the treatment blind was broken 27 32

34 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No Treatment Compliance A listing of subjects for whom the treatment blind was broken during the study is presented. Treatment compliance was also listed by treatment period Other Descriptions of Study Population The following characteristics were used to describe the study population. Supporting by-subject listings of this information are presented in each case Demographic and baseline characteristics Age, height, weight,number of pack years and FEV 1 were summarised using descriptive statistics (number of subjects [n], mean, SD, median, minimum and maximum) and the distribution (n, %) of sex, race and smoking status are presented. The distribution (n, %) of child-bearing potential status and contraception method used are presented for female subjects. Baseline vital sign data (systolic and diastolic blood pressure, pulse and tympanic temperature) was also summarised using descriptive statistics. Concurrent medical conditions were summarised (n, %) for each category. No statistical testing of the baseline characteristics was performed due to the ambiguity of the interpretation of baseline hypothesis testing Concurrent medications Concurrent medications were identified and summarised (n, %) by group term and preferred term, where terms are sorted in descending order of total incidence by group term, and by preferred term within group term. Supporting by-subject listings of this information are presented. All medication verbatim text were coded using the THERAPY dictionary to obtain the appropriate therapy group and preferred term. These summaries are split according to whether the medications were stopped prior to randomisation, started prior to randomisation and continued after randomisation or started/changed after randomisation. In addition, a listing summarising the relationship between group terms and preferred terms was produced for all concurrent medications Efficacy Analyses In all analyses described below, the null hypothesis is that of no treatment difference. The alternative hypothesis asserts a difference between treatments. This will be tested using a two-sided significance level of α=0.05. P-values of <= 0.05 will be considered 28 33

35 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. statistically significant. Confidence intervals for the difference used a confidence level of 95%. As this is an exploratory study, no adjustments were made for multiple comparisons. The primary population for all efficacy analyses is the ITT population. The primary efficacy endpoint(s) were analysed using both the ITT and the PP populations. The secondary efficacy endpoint(s) were analysed using the ITT population. If a difference was demonstrated between the ITT and the PP analysis carried out on the primary efficacy endpoint(s) then the secondary efficacy endpoint(s) would also be analysed using the PP population. It was assumed that there is a sufficient wash-out period between treatment periods (2 7 days). Therefore no carry-over effect was investigated. The protocol states that Prescotts test will be used for the statistical analysis, however ANCOVA was performed unless the assumptions were not satisfied Primary Efficacy Measure(s) The primary efficacy endpoint was the reduction in cough frequency after waking over the following timepoints on Treatment Day 1: 0-10 mins, mins, mins and 40mins - 1 hour This data was summarised and listed for each timepoint. The cough frequency for each time point will be calculated and analysed using ANCOVA containing terms for sequence, subject(sequence), period and treatment. No formal statistical testing for carryover was performed. The model fitted will be: Cough Frequency = sequence + subject(sequence) + period + treatment Adjusted means were presented, along with the estimated treatment difference and the associated 95% confidence intervals Secondary Efficacy Measure(s) Cough Frequency These data were listed and summarised by treatment group. Cough Frequency was also measured between 1 hour after treatment to 12:00 noon and 12:00 noon to 10pm on Treatment Day 1 and also from waking to 12:00 noon and 12:00 noon - 10pm on Treatment Days 2-5. The data collected on treatment Day 1 was summarised only. The data collected on Treatment Days 2-5 was analysed as Section

36 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No Nocturnal Disturbance These data were listed and summarised by treatment group. The mean nocturnal disturbance derived for each period (as defined in section 5.7) will be analysed using ANCOVA containing terms for sequence, subject(sequence), period and treatment. No formal statistical testing for carryover will be performed Cough Severity The analysis of cough severity will be performed using the weighted mean (derived as defined in section 5.5.1) using an Analysis of Covariance (ANCOVA) as in section 5.7. Adjusted means were presented, along with the estimated treatment difference and the associated 95% confidence intervals Chest Tightness Chest tightness was analysed as in section Volume of expectoration These data were summarised and listed by treatment period Composite global symptom score These data were summarised and listed by treatment group. No formal analysis was performed. The global symptom score was summarised by treatment period Treatment Preference Treatment preference was analysed using McNemar s test[2] Morning and Evening PEF Peak flow measurements were collected on waking and at 10pm on all screening and treatment days. The mean of morning and evening PEF was calculated for each subject and treatment period using all data available from days 1 to 5 (as defined in section 5.5.1). The means were analysed using ANCOVA as in section D2 and D5 The concentrations required to produce 2 and 5 coughs per citric acid challenge for each subject were analysed using ANCOVA as in section

37 Other Efficacy Measures CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. No other efficacy endpoints were recorded Safety Analyses Safety was assessed through the monitoring of AEs throughout the subject s participation in the study. AEs were coded and the frequency of subjects reporting each AE was summarised by treatment group. Safety analysis was based on the ITT Population Extent of Exposure A summary of study drug exposure was produced. Descriptive statistics (n, mean, SD, median, minimum, maximum) for the distribution of number of study drug applications are presented, along with the distribution (n, %) of number of applications Adverse Events Adverse events were coded and grouped by body system. Adverse events occurring pretreatment and during the active treatment period (during treatment) are tabulated separately. Treatment limiting AE s (i.e. leading to withdrawals) and drug related AE s was also tabulated. Adverse event codes were translated using MIDAS. In addition, a listing summarising the relationship between body system, group terms and verbatim text was produced Deaths and Serious Adverse Events If any non-fatal serious adverse events were reported a table would be produced. If any fatal adverse events or treatment limiting adverse events were reported a table would be produced Adverse Events Leading to Discontinuation of Study Drug and/or Withdrawal from the Study and Other Significant Adverse Events A summary table and a listing of treatment-limiting AEs were produced. For the purposes of this study, it was assumed that a treatment-limiting AE was one for which the CRF showed that the action taken with study drug as a result of the AE was one of the following: (1) dose adjusted, (2) temporarily interrupted or (3) permanently discontinued. All events not coded in this way were regarded as non-treatment-limiting Pregnancies Pregnancies occurring during the study were to be summarised in case narratives written by GW International Product Safety and Pharmacovigilance (IPSP) personnel

38 CONFIDENTIAL CONFIDENTIAL 6. STUDY POPULATION RESULTS BL2001/00008/00 Study BL2001/00008/00 No Subject Accountability One UK centre screened 71 subjects and randomised a total of 44 subjects into the study (Table 1). All randomised subjects received both Placebo and Ventolin Protocol Deviations There were a total of four major protocol violations for three subjects (Table 5 and Listing 6). There were 32 subjects who were partial major protocol violators (Table 5). In line with ICH guidelines, decisions that these violations constituted major protocol violations were taken prior to breaking the randomisation code Populations Analyzed All major protocol violators were completely excluded from the PP Population, and the partial major protocol violators were excluded from the point at which they started taking the prohibited medication. The ITT Population consisted of 44 subjects, and the PP Population consisted of 41 subjects (Table 6)

39 CONFIDENTIAL CONFIDENTIAL 6.4. Demographic and Baseline Characteristics BL2001/00008/00 Study BL2001/00008/00 No Demographics Demographic characteristics are summarised below. OVERVIEW OF SUBJECT DISPOSITION (ITT) Characteristic Total (N=44) Mean/n Range or % Age (years) Gender Male 31 70% Female 13 30% Race White % Height (cm) Weight (kg) FEV Source Table 7. Demographic characteristics are summarised in Table 7 and Smoking history in Table 8. Child-bearing potential status is summarised in Table 9. Of the 13 female subjects randomised, 11 (85%) were potentially able to bear children Medical Conditions Current medical conditions at baseline are presented by subject in listing 10 and summarised in Table 10. In the ITT population 31 had a current medical condition. Allergies were the most common current medical condition, with 11 (25%) subjects having allergies Concurrent Therapy There were 12 subjects who took concomitant medications that started and stopped prior to study treatment seven subjects (16%) taking paracetamol (Table 12). There were 16 subjects who took a concomitant medication prior to study treatment and continued during the study. There were 14 subjects who took concomitant medications during the study treatment, nine subjects (20%) taking Paracetamol

40 6.7. Treatment Compliance CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. All subjects took both medications for at least 2 days. The mean length of exposure was 5.0 days in the Placebo group and 5.0 days in the Ventolin (Table 34)

41 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. 7. EFFICACY RESULTS 7.1. Primary Efficacy Measure Cough Frequency Tables 13,14 and Listing 25 address the primary endpoint for the ITT Population. Table 14 shows the results of the ANCOVA fitted to the primary endpoint for the ITT Population. There is a significantly smaller cough frequency in the first ten minutes in the Ventolin group in comparison to the placebo group, statistical significance was not found at any other timepoints. Mean Cough Frequency ITT population Placebo (N=44) Ventolin (N=44) Adjusted mean 0-10 minutes (SE) 3.2 (0.3) 2.3 (0.3) Adjusted mean minutes (SE) 2.8 (0.3) 2.3 (0.3) Adjusted mean minutes (SE) 3.9 (0.3) 3.9 (0.3) Adjusted mean minutes (SE) 2.5 (0.3) 2.6 (0.3) Active minus Placebo (0-10 minutes) Difference P-value 95% CI Active minus Placebo (10-20 minutes) Difference P-value 95% CI Active minus Placebo (20-40 minutes) Difference P-value 95% CI Active minus Placebo (40-60 minutes) Difference P-value 95% CI Source: Table , , , ,

42 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. The above summaries and analyses were repeated for the PP Population and are presented in Tables 15,16. The results from the PP analysis confirm those found in the ITT Population Secondary Efficacy Measure(s) Cough Frequency The mean cough frequency for the non primary endpoints during the study is summarised in Table 13 and presented by-subject in listing 25. The statistical analysis of this parameter is summarised in Table 14. There were no statistical difference at any timepoints Nocturnal Disturbance The mean nocturnal disturbance during the study is summarised in Table 17 and presented by-subject in listing 31. The statistical analysis of this parameter is summarised in Table 18. The weighted mean score was 0.3 in both the Placebo and Ventolin groups. The adjusted mean treatment difference between Ventolin and Placebo was 0 and this was not statistically significant (p=0.780, 95% CI -0.1, 0.1) Cough Severity The mean cough severity during the study is summarised in Table 19 and presented bysubject in listing 27. The statistical analysis of this parameter is summarised in Table 20. The weighted mean score was 2.1 and 2.2 in the Placebo and Ventolin groups respectively. The adjusted adjusted mean treatment difference between Ventolin and Placebo was 0 and this was not statistically significant (p=0.947, 95% CI -0.3, 0.4) Chest Tightness The mean chest tightness during the study is summarised in Table 21 and presented bysubject in listing 27. The statistical analysis of this parameter is summarised in Table 22. The weighted mean score was 1.5 and 1.6 in the Placebo and Ventolin groups respectively. The adjusted mean treatment difference between Ventolin and Placebo was 0.1 but this was not statistically significant (p=0.359, 95% CI -0.1, 0.4) Volume of Expectoration The volume of expectoration during the study is summarised in Table 23 and presented by-subject in listing 29. The mean expectoration was 2.57 and 2.71 in the Placebo and Ventolin groups respectively 36 41

43 CONFIDENTIAL CONFIDENTIAL Composite Global Symptom Score BL2001/00008/00 Study BL2001/00008/00 No. The composite global symptom score during the study is summarised in Table 24 and presented by-subject in listing 27. The mean score was 3.58 and 3.67 in the Placebo and Ventolin groups respectively Treatment Preference There was no difference in treatment preference recorded by the subjects Morning and Evening PEF The mean morning PEF during the study is summarised in Table 26 and presented bysubject in listing 30. The statistical analysis of this parameter is summarised in Table 27. The adjusted mean change was 10.1 and 14.5 in the Placebo and Ventolin groups respectively. The adjusted mean treatment difference between Ventolin and Placebo was 4.4 but this was not statistically significant (p=0.193, 95% CI -2.3, 11.2). The mean evening during the study is summarised in Table 28 and presented by-subject in listing 30. The statistical analysis of this parameter is summarised in Table 29. The adjusted mean change was 8.2 and 17.5 in the Placebo and Ventolin groups respectively. The adjusted mean treatment difference between Ventolin and Placebo was 9.3 this was statistically significant (p=0.009, 95% CI 2.5, 16.1) D2 and D5 (Mm) The mean D2 concentrations at 1 hour, 2 hour and 4 hour are summarised in Table 30 and presented by-subject in listing 26. The statistical analysis of this parameter is summarised in Table 31. Concentrations recorded as > 1000 where corrected to 1001, therefore the precision in these results has been reduced. After 1 hour the mean D2 concentration was and in the Placebo and Ventolin groups respectively. The adjusted mean treatment difference between Ventolin and Placebo was this was statistically significant (p=0.020, 95% CI 17.5, 196.7). After 2 hours the mean D2 concentration was and in the Placebo and Ventolin groups respectively. The adjusted mean treatment difference between Ventolin and Placebo was 34.0 but this was not statistically significant (p=0.440, 95% CI 54.0, 121.9). After 4 hours the mean D2 concentration was and in the Placebo and Ventolin groups respectively. The adjusted mean treatment difference between Ventolin and Placebo was 34.3 but this was not statistically significant (p=0.501, 95% CI 67.5, 136.0)

44 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. The mean D5 concentrations at 1 hour, 2 hour and 4 hour are summarised in Table 32 and presented by-subject in listing 26. The statistical analysis of this parameter is summarised in Table 33. After 1 hour the mean D5 concentration was and in the Placebo and Ventolin groups respectively. The adjusted mean treatment difference between Ventolin and Placebo was 11.5 but this was not statistically significant (p=0.786, 95% CI 73.8, 96.8). After 2 hours the mean D5 concentration was and in the Placebo and Ventolin groups respectively. The adjusted mean treatment difference between Ventolin and Placebo was 49.0 but this was not statistically significant (p=0.196, 95% CI 26.3, 124.3). After 4 hours the mean D5 concentration was and in the Placebo and Ventolin groups respectively. The adjusted mean treatment difference between Ventolin and Placebo was 35.3 but this was not statistically significant (p=0.285, 95% CI 30.4, 101.0) Efficacy Conclusions The primary endpoint of the reduction in cough frequency after waking over the time point on treatment day 1, 0-10 mins showed a statistically significant reduction in cough. This was not felt to be clinically significant. All other time points for the primary endpoint were not shown to be statistically significant. The secondary endpoint of difference in morning and evening peak flow was shown to be statistically significant, giving confidence that subject compliance had been good. Similarly, the secondary endpoint of mean D2 concentration necessary to elicit cough was statistically significant at one hour. Analyses of all other secondary endpoints were not statistically significant

45 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. 8. SAFETY RESULTS 8.1. Extent of Exposure Exposure to study drug is summarised in the table below. OVERVIEW OF NUMBER OF DAYS OF EXPOSURE No. of Days exposure Placebo Ventolin (N=44) (N=44) n % N % <= 1 day and Source: Table 34 Exposure to study drug is summarised in Table 34. A total of 44 subjects received study drug; 44 received Placebo and 44 received Ventolin Adverse Events AE data are presented in Tables and Listings There were no deaths or SAEs during the study. Nineteen subjects (23%) reported at least one AE, which started during pre-treatment. Thirty-three subjects (75%) reported at least one AE, 13 (30%) whilst receiving placebo and 20 subjects (45%) whilst receiving Ventolin. The most common AE was headache, reported by seven subject (16%) whilst receiving placeco and 12 subjects (27%) whilst receiving Ventolin (Table 35). Thirteen subjects (23%) reported at least one drug-related AE, which started during treatment: seven subjects (16%) whilst receiving placebo group and six subjects (14%) whilst receiving Ventolin. The most common drug-related AE was headache, reported by seven subject (16%) whilst receiving placeco and five subjects (11%) whilst receiving Ventolin (Table 36 and Listing 35)

46 8.3. Deaths CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. There were no deaths during this study Serious Adverse Events There were no SAEs during this study Adverse Events Leading to Discontinuation of Study Drug and/or Study Withdrawal There were no AEs Leading to Discontinuation of Study Drug and/or Study Withdrawal 8.6. Other Significant Adverse Events There were no treatment-limiting AES Pregnancies There were no pregnancies reported during this study Medical Device Incidents, Near-Incidents, Malfunctions and Remedial Action No incidents, near-incidents or malfunctions were reported with the use of the medical device(s) manufactured or marketed by Glaxo Wellcome for this study Safety Conclusions There were no safety issues raised during this study

47 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. 9. DISCUSSION This study was performed to test the hypothesis that the beta agonist salbutamol would cause diminution in a naturalistic model of cough chronic cigarette smoking. Since salbutamol is an effective bronchodilator a possible mode of action would have been to remove bronchoconstriction secondary to airway inflammation and thus diminish the increased afferent stimulation on the cough reflex seen with bronchoconstriction [5]. The study was adequately powered to detect a change similar to that seen with commonly used antitussive medications on citric acid cough challenge, and indeed, this was an important secondary end point to the study [6]. The primary end point however had little data with which to calculate degree of confidence, were the study to prove negative. However, to have an entirely negative study would rule out a major clinical effect of salbutamol in smoking related cough. With regard to the major end point. This study was indeed negative in that there was no difference in cough frequency during the first hour of treatment day one. In retrospect this may have not been an entirely fair comparison since the first hour contained two different type of cough data. The first twenty minute period was pre the initial cigarette of the day. The second forty minutes of the hour was taken up with the number of coughs after cigarette smoke exposure. When cough counts were broken down into smaller epochs there was a significant reduction in cough count associated with salbutamol exposure in the time period of 0-10 minutes. One possible interpretation of this is that salbutamol is ineffective against cough produced by cigarette smoke but may be helpful in protecting against cough caused by previous smoking. In support of salbutamol s action on the cough reflex one of the main secondary end points, the citric acid challenge, was also significantly improved one hour post salbutamol. The cough challenge result should be regarded as similar to a methacholine challenge result with bronchodilators, which show a greater sensitivity than measurements such as the FEV 1 [7]. The other major secondary end point which altered was the evening peak flow. All of the subjects were screened with methacholine challenge and salbutamol reversibility in order to eliminate a possible diagnosis of asthma. This bronchodilation is an interesting observation, which not only confirms the patient compliance but would be surprising for an entirely normal group of subjects. This result is a pointer to the validity of the original hypothesis that smoking related cough may be associated with a degree of bronchoconstriction. The results of this study clearly demonstrate that salbutamol by inhalation is unlikely to be effective in cough induced by smoking. But, there was some evidence of efficacy before cigarette exposure which may indicate that bronchodilator therapy, particularly with a drug of more sustained activity, may be efficacious in cough associated with smoking cessation [8]

48 10. CONCLUSIONS CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. This study failed to support the clinical effectiveness of salbutamol in smoking related cough. Sub analysis of the primary end point and evaluation of some secondary end points indicated that salbutamol did have a small but clinically unimportant activity on the cough reflex. More effective and longer lasting bronchodilators coupled with a different therapeutic target smoking cessation cough may prove fruitful

49 CONFIDENTIAL CONFIDENTIAL BL2001/00008/00 Study BL2001/00008/00 No. 11. REFERENCES 1. Smith CA, Adamson DL, Choudry NB, Fuller RW. The effect of altering airway tone on the sensitivity of the cough reflex in normal volunteers. Eur.Respir.J. 1991; 4: Pounsford JC, Birch MJ, Saunders KB. Effect of bronchodilators on the cough response to inhaled citric acid in normal and asthmatic subjects. Thorax 1985; 40: Taylor IK, OShaughnessy KM, Choudry NB, Adachi M, Palmer JBD, Fuller RW. A comparative study in atopic subjects with asthma of the effects of salmeterol and salbutamol on allergen-induced bronchoconstriction, increase in airway reactivity, and increase in urinary leukotriene E4 excretion. J.Allergy Clin.Immunol. 1992; 89: OConnell F, Thomas VE, Studham JM, Pride NB, Fuller RW. Capsaicin cough sensitivity increases during upper respiratory infection. Resp.Med. 1996; 90: Rennard SI, Daughton D, Fujita J, et al. Short-term smoking reduction is associated with reduction in measures of lower respiratory tract inflammation in heavy smokers. Eur.Respir.J. 1990; 3: Goldstein DA, Tan YK, Soldin SJ. Pharmacokinetics and absolute bioavailability of salbutamol in healthy adult volunteers. Eur.J.Clin.Pharmacol. 1987; 32: Wong, C. H. and Morice, A. H. Cough threshold in patients with chronic obstructive pulmonary disease. Thorax 54, Jones, B and Kenward, MG. Design and analysis of cross-over trials, Chapman and Hall

50 12. TABLES CONFIDENTIAL BL2001/00008/00 49

51 Protocol: Population: All Subjects Table 1 Summary of Subjects Screened Number of Subject Screened Number of Subject Randomised Page 1 of 1 CONFIDENTIAL BL2001/00008/00

52 51 Protocol: Population: All Subjects Table 2 Summary of Subject Accountability Total (N=71) Subjects Entered into Period 1 44 (62%) Subjects Completing Period 1 44 (62%) Subjects Entered into Period 2 44 (62%) Subjects Completing Period 2 44 (62%) Subjects Attending the Follow-Up Visit 44 (62%) Page 1 of 1 CONFIDENTIAL BL2001/00008/00

53 52 Protocol: Population: All Subjects Table 3 Summary of Subject Accountability - End of Study Record Total (N=71) Completed 43 (61%) Prematurely Discontinued 28 (39%) Reason for Premature Discontinuation [1]: Adverse Event 3 (4%) Consent Withdrawn 6 (8%) Lost to Follow-Up 0 Protocol Violation 1 (1%) Other 18 (25%) Pregnancy [2] 0 Death [3] 0 [1] Primary reason for which the subject discontinued [2] Denominator is based on the number of female subjects (see Table 7) [3] Death may also be included in the adverse event category Page 1 of 1 CONFIDENTIAL BL2001/00008/00

54 53 Protocol: Population: All Subjects Table 4 Summary of End of Study Drug Record Placebo Ventolin Total (N=44) (N=44) (N=44) Discontinued Study Drug 0 1 (2%) 1 (2%) Prematurely Adverse Event 0 1 (2%) 1 (2%) Consent Withdrawn Lost to Follow-Up Protocol Violation Other Page 1 of 1 CONFIDENTIAL BL2001/00008/00

55 54 Protocol: Population: Intent-to-Treat Table 5 Summary of Major Protocol Violations Total (N=44) Number of Subjects with any 3 (7%) Major Protocol Violation[1] Number of Subjects with a Major Protocol Violation Invalid Medication 2 (5%) Did Not Fulfil Inclusion Criteria 2 (5%) Number of Subjects with a Partial 32 (73%) Major Protocol Violation[2] Invalid Medication 1 (2%) Did Not Fulfil Inclusion Criteria 2 (5%) Non Compliant to Visit Windows 31 (70%) [1] Subjects whose data are totally excluded from analysis using Per Protocol Analysis Population [2] Subjects who provide post-baseline efficacy assessments prior to the protocol violation in the Per Protocol Analysis Population Note:Subjects can violate for more than one reason Page 1 of 1 CONFIDENTIAL BL2001/00008/00

56 55 Protocol: Population: All Subjects Table 6 Summary of Analysis Populations Analysis Population Total All Subject Screened 71 Intent-to-treat 44 (62%) Population [1] Per Protocol Analysis 41 (58%) Population [2] [1] Subjects who were randomised and received at least one dose of study treatment [2] Subjects in the Intent-to-Treat Population who were not excluded as major protocol violators. Note that subjects who were partial major protocol violators are included in this summary. Only subjects who are fully excluded from the Per Protocol Analysis Population are excluded here Page 1 of 1 CONFIDENTIAL BL2001/00008/00

57 56 Protocol: Population: Intent-to-Treat Table 7 Summary of Demographic and Screening Characteristics Total (N=44) Age (years) n 44 Mean 33.9 SD 9.5 Median 34.0 Min. 20 Max. 61 Sex n 44 Male 31 (70%) Female 13 (30%) Race n 44 White 44 (100%) Black 0 Asian 0 American Hispanic 0 Other 0 Page 1 of 2 CONFIDENTIAL BL2001/00008/00

58 57 Protocol: Population: Intent-to-Treat Table 7 Summary of Demographic and Screening Characteristics Total (N=44) Height (cm) n 44 Mean SD 9.0 Median Min. 156 Max. 196 Weight (kg) n 44 Mean SD Median Min Max Fev1 (L) n 44 Mean 4.10 SD 0.89 Median 3.83 Min. 2.8 Max. 6.0 Page 2 of 2 CONFIDENTIAL BL2001/00008/00

59 58 Protocol: Population: Intent-to-Treat Table 8 Summary of Smoking History Total (N=44) Smoking Status n 44 Never Smoked 0 Current Smoker 44 (100%) Former Smoker 0 Number of Pack Years n 44 Mean 20.4 SD 15.6 Median 18.0 Min. 5 Max. 100 Page 1 of 1 CONFIDENTIAL BL2001/00008/00

60 59 Protocol: Population: Intent-to-Treat Table 9 Summary of Child-Bearing Potential Total (N=44) Child-Bearing Potential n 13 Potentially Able 11 (85%) Premenarche 0 Post menopause 1 (8%) Sterile (of child-bearing age) 1 (8%) Contraception Method Used [1] n 11 Oral Contraceptive 4 (36%) Intrauterine Device 0 Depot Contraceptive 2 (18%) Physical Barrier 1 (9%) Abstinence 2 (18%) Sterilization of male partner 2 (18%) Other 0 [1] Subjects can use more than one method Page 1 of 1 CONFIDENTIAL BL2001/00008/00

61 Protocol: Population: Intent-to-Treat Table 10 Summary of Current Medical Conditions Condition Total Body System Present? (N=44) 60 Number of Subjects reporting abnormalities 31 Ear, Nose & Throat n 44 Yes 3 (7%) No 41 (93%) Eyes n 44 Yes 5 (11%) No 39 (89%) Respiratory n 44 Yes 1 (2%) No 43 (98%) Cardiovascular n 44 Yes 0 No 43 (98%) Missing 1 (2%) Gastrointestinal n 44 Yes 2 (5%) No 42 (95%) Page 1 of 3 CONFIDENTIAL BL2001/00008/00

62 61 Protocol: Population: Intent-to-Treat Table 10 Summary of Current Medical Conditions Condition Total Body System Present? (N=44) Hepatobiliary and pancreas n 44 Yes 0 No 43 (98%) Missing 1 (2%) Urinary System n 44 Yes 1 (2%) No 43 (98%) Reproductive System n 44 Yes 0 No 44 (100%) Neurology n 44 Yes 4 (9%) No 40 (91%) Blood and lymphatic n 44 Yes 0 No 44 (100%) Page 2 of 3 CONFIDENTIAL BL2001/00008/00

63 62 Protocol: Population: Intent-to-Treat Table 10 Summary of Current Medical Conditions Condition Total Body System Present? (N=44) Endocrine and metabolic n 44 Yes 1 (2%) No 43 (98%) Musculoskeletal n 44 Yes 8 (18%) No 36 (82%) Skin n 44 Yes 6 (14%) No 38 (86%) Psychiatry n 44 Yes 5 (11%) No 39 (89%) Allergies n 44 Yes 11 (25%) No 33 (75%) Page 3 of 3 CONFIDENTIAL BL2001/00008/00

64 63 Protocol: Population: Intent-to-Treat Table 11 Summary of Baseline Vital Signs Total (N=44) Systolic Blood Pressure (mmhg) n 44 Mean SD 16.1 Median Min. 96 Max. 163 Diastolic Blood Pressure (mmhg) n 44 Mean 79.1 SD 10.4 Median 77.5 Min. 52 Max. 105 Page 1 of 2 CONFIDENTIAL BL2001/00008/00

65 64 Protocol: Population: Intent-to-Treat Table 11 Summary of Baseline Vital Signs Total (N=44) Heart Rate (beats/min) n 44 Mean 74.5 SD 11.8 Median 73.0 Min. 53 Max. 106 Temperature (C) n 44 Mean SD 0.42 Median Min Max Page 2 of 2 CONFIDENTIAL BL2001/00008/00

66 Protocol: Population: Intent-to-Treat Table 12 Summary of Concurrent Medications Started and Stopped Prior to Study Treatment Group Term Total Preferred Term (N=44) 65 Any Medication 12 (27%) Miscellaneous analgesics Any Medication 7 (16%) Paracetamol 7 (16%) Salicylates Any Medication 2 (5%) Anadin 1 (2%) Aspirin 1 (2%) Anti-tussives,expectorants + mucolytics Any Medication 1 (2%) Lockets 1 (2%) Local anesthetics Any Medication 1 (2%) Local anesthetic 1 (2%) NSAIDs Any Medication 1 (2%) Ibuprofen 1 (2%) Narcotic + other analgesics combined Any Medication 1 (2%) Kapake 1 (2%) Page 1 of 1 CONFIDENTIAL BL2001/00008/00

67 Protocol: Population: Intent-to-Treat Table 12 Summary of Concurrent Medications Started Prior to Study Treatment and Ongoing During Study Group Term Total Preferred Term (N=44) 66 Any Medication 16 (36%) Estrogens + progestogens combined Any Medication 4 (9%) Brevinor 1 (2%) Loestrin 1 (2%) Logynon 1 (2%) Minulet 1 (2%) Other anti-depressants Any Medication 4 (9%) Fluoxetine hydrochloride 1 (2%) Paroxetine hydrochloride 2 (5%) Venlafaxine hydrochloride 1 (2%) Anti-cholinergics Any Medication 1 (2%) Cyclopentolate hydrochloride 1 (2%) Corticosteroids Any Medication 1 (2%) Hydrocortisone 1 (2%) Corticosteroids + anti-infectives combin Any Medication 1 (2%) Otomize 1 (2%) Page 1 of 2 CONFIDENTIAL BL2001/00008/00

68 67 Protocol: Population: Intent-to-Treat Table 12 Summary of Concurrent Medications Started Prior to Study Treatment and Ongoing During Study Group Term Total Preferred Term (N=44) Insulins Any Medication 1 (2%) Human intermediate/long-acting insulin 1 (2%) NSAIDs Any Medication 1 (2%) Ibuprofen 1 (2%) Progestogens Any Medication 1 (2%) Medroxyprogesterone acetate 1 (2%) Proton pump inhibitors Any Medication 1 (2%) Lansoprazole 1 (2%) Thyroid preparations Any Medication 1 (2%) Thyroxine sodium 1 (2%) Page 2 of 2 CONFIDENTIAL BL2001/00008/00

69 Protocol: Population: Intent-to-Treat Table 12 Summary of Concurrent Medications Started During Study Treatment Group Term Total Preferred Term (N=44) 68 Any Medication 14 (32%) Miscellaneous analgesics Any Medication 9 (20%) Paracetamol 9 (20%) Anti-migraine preparations Any Medication 2 (5%) Naratriptan hydrochloride 1 (2%) Paramax 1 (2%) Salicylates Any Medication 2 (5%) Anadin 2 (5%) Macrolides Any Medication 1 (2%) Erythromycin 1 (2%) NSAIDs Any Medication 1 (2%) Ibuprofen 1 (2%) Narcotic analgesics Any Medication 1 (2%) Codeine phosphate 1 (2%) Page 1 of 2 CONFIDENTIAL BL2001/00008/00

70 Protocol: Population: Intent-to-Treat Table 12 Summary of Concurrent Medications Started During Study Treatment Group Term Total Preferred Term (N=44) Penicillins Any Medication 1 (2%) Amoxycillin 1 (2%) 69 Page 2 of 2 CONFIDENTIAL BL2001/00008/00

71 70 Protocol: Population: Intent-to-Treat Table 13 Summary Statistics of Cough Frequency Placebo Ventolin (N=44) (N=44) Day1,0-10 minutes n Mean SD Median Min. 0 0 Max Day1,10-20 minutes n Mean SD Median Min. 0 0 Max Day1,20-40 minutes n Mean SD Median Min. 0 0 Max Page 1 of 5 CONFIDENTIAL BL2001/00008/00

72 71 Protocol: Population: Intent-to-Treat Table 13 Summary Statistics of Cough Frequency Placebo Ventolin (N=44) (N=44) Day1,40-60 minutes n Mean SD Median Min. 0 0 Max Day1,1hr-12pm n Mean SD Median Min. 2 1 Max Day1,12pm-10pm n Mean SD Median Min. 4 1 Max Page 2 of 5 CONFIDENTIAL BL2001/00008/00

73 72 Protocol: Population: Intent-to-Treat Table 13 Summary Statistics of Cough Frequency Placebo Ventolin (N=44) (N=44) Day2,waking-12pm n Mean SD Median Min. 2 1 Max Day2,12pm-10pm n Mean SD Median Min. 2 1 Max Day3,waking-12pm n Mean SD Median Min. 2 1 Max Page 3 of 5 CONFIDENTIAL BL2001/00008/00

74 73 Protocol: Population: Intent-to-Treat Table 13 Summary Statistics of Cough Frequency Placebo Ventolin (N=44) (N=44) Day3,12pm-10pm n Mean SD Median Min. 0 0 Max Day4,waking-12pm n Mean SD Median Min. 2 1 Max Day4,12pm-10pm n Mean SD Median Min. 1 0 Max Page 4 of 5 CONFIDENTIAL BL2001/00008/00

75 74 Protocol: Population: Intent-to-Treat Table 13 Summary Statistics of Cough Frequency Placebo Ventolin (N=44) (N=44) Day5,waking-12pm n Mean SD Median Min. 2 0 Max Day5,12pm-10pm n Mean SD Median Min. 1 1 Max Page 5 of 5 CONFIDENTIAL BL2001/00008/00

76 75 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 1,0-10 minutes (N=44) (N=44) Raw Mean at 0-10 minutes 3.2 (3.1) 2.3 (2.1) Adjusted Mean(se) 3.2 (0.3) 2.3 (0.3) Column trt. minus Placebo Difference (se) -0.9 (0.4) 95% Confidence Interval -1.7, -0.1 p-value Page 1 of 12 CONFIDENTIAL BL2001/00008/00

77 76 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 1,10-20 minutes (N=44) (N=44) Raw Mean at minutes 2.8 (2.8) 2.3 (2.1) Adjusted Mean(se) 2.8 (0.3) 2.3 (0.3) Column trt. minus Placebo Difference (se) -0.5 (0.4) 95% Confidence Interval -1.3, 0.2 p-value Page 2 of 12 CONFIDENTIAL BL2001/00008/00

78 77 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 1,20-40 minutes (N=44) (N=44) Raw Mean at minutes 3.9 (3.5) 3.9 (3.6) Adjusted Mean(se) 3.9 (0.3) 3.9 (0.3) Column trt. minus Placebo Difference (se) -0.0 (0.4) 95% Confidence Interval -0.9, 0.8 p-value Page 3 of 12 CONFIDENTIAL BL2001/00008/00

79 78 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 1,40-60 minutes (N=44) (N=44) Raw Mean at minutes 2.5 (3.0) 2.6 (2.2) Adjusted Mean(se) 2.5 (0.3) 2.6 (0.3) Column trt. minus Placebo Difference (se) 0.1 (0.4) 95% Confidence Interval -0.8, 0.9 p-value Page 4 of 12 CONFIDENTIAL BL2001/00008/00

80 79 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 2,waking-12pm (N=44) (N=44) Raw Mean at waking-12pm 10.3 (6.8) 10.8 (8.5) Adjusted Mean(se) 10.3 (0.8) 10.7 (0.8) Column trt. minus Placebo Difference (se) 0.5 (1.1) 95% Confidence Interval -1.8, 2.8 p-value Page 5 of 12 CONFIDENTIAL BL2001/00008/00

81 80 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 2,12pm-10pm (N=44) (N=44) Raw Mean at 12pm-10pm 12.6 (11.5) 11.8 (9.5) Adjusted Mean(se) 12.6 (1.0) 11.7 (1.0) Column trt. minus Placebo Difference (se) -0.9 (1.5) 95% Confidence Interval -3.8, 2.1 p-value Page 6 of 12 CONFIDENTIAL BL2001/00008/00

82 81 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 3,waking-12pm (N=44) (N=44) Raw Mean at waking-12pm 12.0 (11.6) 12.0 (8.8) Adjusted Mean(se) 12.3 (1.2) 11.7 (1.2) Column trt. minus Placebo Difference (se) -0.6 (1.7) 95% Confidence Interval -4.1, 2.9 p-value Page 7 of 12 CONFIDENTIAL BL2001/00008/00

83 82 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 3,12pm-10pm (N=44) (N=44) Raw Mean at 12pm-10pm 13.0 (11.6) 13.1 (9.8) Adjusted Mean(se) 13.0 (1.0) 12.9 (1.0) Column trt. minus Placebo Difference (se) -0.0 (1.4) 95% Confidence Interval -2.9, 2.9 p-value Page 8 of 12 CONFIDENTIAL BL2001/00008/00

84 83 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 4,waking-12pm (N=44) (N=44) Raw Mean at waking-12pm 11.3 (9.0) 12.8 (10.7) Adjusted Mean(se) 11.3 (0.7) 12.6 (0.7) Column trt. minus Placebo Difference (se) 1.4 (1.0) 95% Confidence Interval -0.6, 3.4 p-value Page 9 of 12 CONFIDENTIAL BL2001/00008/00

85 84 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 4,12pm-10pm (N=44) (N=44) Raw Mean at 12pm-10pm 11.9 (9.4) 12.7 (11.1) Adjusted Mean(se) 11.9 (0.8) 12.5 (0.8) Column trt. minus Placebo Difference (se) 0.7 (1.2) 95% Confidence Interval -1.7, 3.0 p-value Page 10 of 12 CONFIDENTIAL BL2001/00008/00

86 85 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day 5,waking-12pm (N=44) (N=44) Raw Mean at waking-12pm 11.3 (9.1) 10.5 (7.9) Adjusted Mean(se) 11.1 (1.1) 10.6 (1.1) Column trt. minus Placebo Difference (se) -0.5 (1.6) 95% Confidence Interval -3.7, 2.6 p-value Page 11 of 12 CONFIDENTIAL BL2001/00008/00

87 86 Protocol: Population: Intent-to-Treat Table 14 Analysis of Cough Frequency Placebo Ventolin Day5,12pm-10pm (N=44) (N=44) Raw Mean at 12pm-10pm 13.0 (12.3) 12.8 (11.1) Adjusted Mean(se) 13.0 (0.9) 12.7 (0.9) Column trt. minus Placebo Difference (se) -0.4 (1.2) 95% Confidence Interval -2.9, 2.1 p-value Page 12 of 12 CONFIDENTIAL BL2001/00008/00

88 87 Protocol: Population: Per Protocol Table 15 Summary Statistics of Cough Frequency Placebo Ventolin (N=41) (N=41) Day1,0-10 minutes n Mean SD Median Min. 0 0 Max Day1,10-20 minutes n Mean SD Median Min. 0 0 Max Day1,20-40 minutes n Mean SD Median Min. 0 0 Max Page 1 of 5 CONFIDENTIAL BL2001/00008/00

89 88 Protocol: Population: Per Protocol Table 15 Summary Statistics of Cough Frequency Placebo Ventolin (N=41) (N=41) Day1,40-60 minutes n Mean SD Median Min. 0 0 Max Day1,1hr-12pm n Mean SD Median Min. 2 1 Max Day1,12pm-10pm n Mean SD Median Min. 4 1 Max Page 2 of 5 CONFIDENTIAL BL2001/00008/00

90 89 Protocol: Population: Per Protocol Table 15 Summary Statistics of Cough Frequency Placebo Ventolin (N=41) (N=41) Day2,waking-12pm n Mean SD Median Min. 2 1 Max Day2,12pm-10pm n Mean SD Median Min. 2 1 Max Day3,waking-12pm n Mean SD Median Min. 2 1 Max Page 3 of 5 CONFIDENTIAL BL2001/00008/00

91 90 Protocol: Population: Per Protocol Table 15 Summary Statistics of Cough Frequency Placebo Ventolin (N=41) (N=41) Day3,12pm-10pm n Mean SD Median Min. 0 0 Max Day4,waking-12pm n Mean SD Median Min. 2 1 Max Day4,12pm-10pm n Mean SD Median Min. 1 0 Max Page 4 of 5 CONFIDENTIAL BL2001/00008/00

92 91 Protocol: Population: Per Protocol Table 15 Summary Statistics of Cough Frequency Placebo Ventolin (N=41) (N=41) Day5,waking-12pm n Mean SD Median Min. 2 0 Max Day5,12pm-10pm n Mean SD Median Min. 1 1 Max Page 5 of 5 CONFIDENTIAL BL2001/00008/00

93 92 Protocol: Population: Per Protocol Table 16 Analysis of Cough Frequency Placebo Ventolin Day 1,0-10 minutes (N=41) (N=41) Raw Mean at 0-10 minutes 3.0 (2.8) 2.2 (2.1) Adjusted Mean(se) 2.9 (0.3) 2.2 (0.3) Column trt. minus Placebo Difference (se) -0.7 (0.4) 95% Confidence Interval -1.4, 0.0 p-value Page 1 of 4 CONFIDENTIAL BL2001/00008/00

94 93 Protocol: Population: Per Protocol Table 16 Analysis of Cough Frequency Placebo Ventolin Day 1,10-20 minutes (N=41) (N=41) Raw Mean at minutes 2.6 (2.7) 2.2 (2.2) Adjusted Mean(se) 2.5 (0.3) 2.2 (0.3) Column trt. minus Placebo Difference (se) -0.3 (0.4) 95% Confidence Interval -1.1, 0.5 p-value Page 2 of 4 CONFIDENTIAL BL2001/00008/00

95 94 Protocol: Population: Per Protocol Table 16 Analysis of Cough Frequency Placebo Ventolin Day 1,20-40 minutes (N=41) (N=41) Raw Mean at minutes 3.8 (3.6) 3.8 (3.7) Adjusted Mean(se) 3.7 (0.3) 3.8 (0.3) Column trt. minus Placebo Difference (se) 0.1 (0.5) 95% Confidence Interval -0.8, 1.1 p-value Page 3 of 4 CONFIDENTIAL BL2001/00008/00

96 95 Protocol: Population: Per Protocol Table 16 Analysis of Cough Frequency Placebo Ventolin Day 1,40-60 minutes (N=41) (N=41) Raw Mean at minutes 2.6 (3.1) 2.6 (2.2) Adjusted Mean(se) 2.5 (0.3) 2.6 (0.3) Column trt. minus Placebo Difference (se) 0.0 (0.4) 95% Confidence Interval -0.8, 0.9 p-value Page 4 of 4 CONFIDENTIAL BL2001/00008/00

97 96 Protocol: Population: Intent-to-Treat Table 17 Summary of Mean Nocturnal Disturbance Placebo Ventolin Total (N=44) (N=44) (N=44) Screening (Day 2 to 6) n 44 Mean 0.53 SD 0.52 Median 0.40 Min. 0.0 Max. 2.2 Period 1 (Day 2 to 6) n Mean SD Median Min Max Period 2 (Day 2 to 6) n Mean SD Median Min Max Page 1 of 1 CONFIDENTIAL BL2001/00008/00

98 97 Protocol: Population: Intent-to-Treat Table 18 Analysis of Mean Nocturnal Disturbance Placebo Ventolin (N=44) (N=44) Raw Mean at Baseline (sd) 0.5 (0.5) 0.5 (0.5) Raw Mean Day 2 to 6 (sd) 0.3 (0.5) 0.3 (0.5) Adjusted Mean(se) 0.3 (0.0) 0.3 (0.0) Adjusted Mean change(se) -0.2 (0.0) -0.2 (0.0) Column trt. minus Placebo Difference (se) 0.0 (0.1) 95% Confidence Interval -0.1, 0.1 p-value Page 1 of 1 CONFIDENTIAL BL2001/00008/00

99 98 Protocol: Population: Intent-to-Treat Table 19 Summary Statistics of Cough Severity Placebo Ventolin (N=44) (N=44) Raw Change Raw Change Baseline [1] n Mean SD Median Min Max Day 1,12pm n Mean SD Median Min Max Day 1,10pm n Mean SD Median Min Max [1] Mean of all baseline time points [2] Weighted Mean Score calculated using all available data Page 1 of 4 CONFIDENTIAL BL2001/00008/00

100 99 Protocol: Population: Intent-to-Treat Table 19 Summary Statistics of Cough Severity Placebo Ventolin (N=44) (N=44) Raw Change Raw Change Day 2,12pm n Mean SD Median Min Max Day 2,10pm n Mean SD Median Min Max Day 3,12pm n Mean SD Median Min Max [1] Mean of all baseline time points [2] Weighted Mean Score calculated using all available data Page 2 of 4 CONFIDENTIAL BL2001/00008/00

101 100 Protocol: Population: Intent-to-Treat Table 19 Summary Statistics of Cough Severity Placebo Ventolin (N=44) (N=44) Raw Change Raw Change Day 3,10pm n Mean SD Median Min Max Day 4,12pm n Mean SD Median Min Max Day 4,10pm n Mean SD Median Min Max [1] Mean of all baseline time points [2] Weighted Mean Score calculated using all available data Page 3 of 4 CONFIDENTIAL BL2001/00008/00

102 101 Protocol: Population: Intent-to-Treat Table 19 Summary Statistics of Cough Severity Placebo Ventolin (N=44) (N=44) Raw Change Raw Change Day 5,12pm n Mean SD Median Min Max Day 5,10pm n Mean SD Median Min Max Weighted Mean Score [2] n Mean SD Median Min Max [1] Mean of all baseline time points [2] Weighted Mean Score calculated using all available data Page 4 of 4 CONFIDENTIAL BL2001/00008/00

103 102 Protocol: Population: Intent-to-Treat Table 20 Analysis of Cough Severity Placebo Ventolin (N=44) (N=44) Raw Mean of Weighted Mean Score at 2.7 (1.5) 2.7 (1.5) Baseline (sd) Raw Mean of Weighted Mean Score (sd) 2.1 (1.3) 2.2 (1.6) Adjusted Mean(se) 2.1 (0.1) 2.2 (0.1) Adjusted Mean change(se) -0.6 (0.1) -0.6 (0.1) Column trt. minus Placebo Difference (se) 0.0 (0.2) 95% Confidence Interval -0.3, 0.4 p-value Page 1 of 1 CONFIDENTIAL BL2001/00008/00

104 103 Protocol: Population: Intent-to-Treat Table 21 Summary Statistics of Chest Tightness Placebo Ventolin (N=44) (N=44) Raw Change Raw Change Baseline [1] n Mean SD Median Min Max Day 1,12pm n Mean SD Median Min Max Day 1,10pm n Mean SD Median Min Max [1] Mean of all baseline time points [2] Weighted Mean Score calculated using all available data Page 1 of 4 CONFIDENTIAL BL2001/00008/00

105 104 Protocol: Population: Intent-to-Treat Table 21 Summary Statistics of Chest Tightness Placebo Ventolin (N=44) (N=44) Raw Change Raw Change Day 2,12pm n Mean SD Median Min Max Day 2,10pm n Mean SD Median Min Max Day 3,12pm n Mean SD Median Min Max [1] Mean of all baseline time points [2] Weighted Mean Score calculated using all available data Page 2 of 4 CONFIDENTIAL BL2001/00008/00

106 105 Protocol: Population: Intent-to-Treat Table 21 Summary Statistics of Chest Tightness Placebo Ventolin (N=44) (N=44) Raw Change Raw Change Day 3,10pm n Mean SD Median Min Max Day 4,12pm n Mean SD Median Min Max Day 4,10pm n Mean SD Median Min Max [1] Mean of all baseline time points [2] Weighted Mean Score calculated using all available data Page 3 of 4 CONFIDENTIAL BL2001/00008/00

107 106 Protocol: Population: Intent-to-Treat Table 21 Summary Statistics of Chest Tightness Placebo Ventolin (N=44) (N=44) Raw Change Raw Change Day 5,12pm n Mean SD Median Min Max Day 5,10pm n Mean SD Median Min Max Weighted Mean Score [2] n Mean SD Median Min Max [1] Mean of all baseline time points [2] Weighted Mean Score calculated using all available data Page 4 of 4 CONFIDENTIAL BL2001/00008/00

108 107 Protocol: Population: Intent-to-Treat Table 22 Analysis of Chest Tightness Placebo Ventolin (N=44) (N=44) Raw Mean of Weighted Mean Score at 2.2 (1.7) 2.2 (1.7) Baseline (sd) Raw Mean of Weighted Mean Score (sd) 1.5 (1.5) 1.6 (1.6) Adjusted Mean(se) 1.5 (0.1) 1.6 (0.1) Adjusted Mean change(se) -0.8 (0.1) -0.7 (0.1) Column trt. minus Placebo Difference (se) 0.1 (0.1) 95% Confidence Interval -0.1, 0.4 p-value Page 1 of 1 CONFIDENTIAL BL2001/00008/00

109 108 Protocol: Population: Intent-to-Treat Table 23 Summary of Volume of Expectoration Placebo Ventolin Total (N=44) (N=44) (N=44) Screening (Day 1 to 5) n 44 Mean 3.03 SD 3.15 Median 1.89 Min. 0.0 Max Period 1 (Day 1 to 5) n Mean SD Median Min Max Period 2 (Day 1 to 5) n Mean SD Median Min Max Page 1 of 2 CONFIDENTIAL BL2001/00008/00

110 109 Protocol: Population: Intent-to-Treat Table 23 Summary of Volume of Expectoration Placebo Ventolin Total (N=44) (N=44) (N=44) Combined n Mean SD Median Min Max Page 2 of 2 CONFIDENTIAL BL2001/00008/00

111 110 Protocol: Population: Intent-to-Treat Table 24 Summary of Composite Global Symptom Score [1] Placebo Ventolin Total (N=44) (N=44) (N=44) Screening (Day 1 to 5) n 44 Mean 4.82 SD 2.93 Median 3.56 Min. 0.4 Max Period 1 (Day 1 to 5) n Mean SD Median Min Max Period 2 (Day 1 to 5) n Mean SD Median Min Max [1] The sum of the scores of Chest Tightness and Cough Severity Page 1 of 2 CONFIDENTIAL BL2001/00008/00

112 111 Protocol: Population: Intent-to-Treat Table 24 Summary of Composite Global Symptom Score [1] Placebo Ventolin Total (N=44) (N=44) (N=44) Combined n Mean SD Median Min Max [1] The sum of the scores of Chest Tightness and Cough Severity Page 2 of 2 CONFIDENTIAL BL2001/00008/00

113 112 Protocol: Population: Intent-to-Treat Table 26 Summary of Morning PEF Placebo Ventolin Total (N=44) (N=44) (N=44) Screening n 44 Mean SD Median Min Max Period 1 n Mean SD Median Min Max Period 2 n Mean SD Median Min Max Page 1 of 1 CONFIDENTIAL BL2001/00008/00

114 113 Protocol: Population: Intent-to-Treat Table 27 Analysis of Morning PEF Placebo Ventolin (N=44) (N=44) Raw Mean at Baseline (sd) (76.9) (76.9) Raw Mean of PEF (sd) (75.6) (74.8) Adjusted Mean(se) (2.4) (2.4) Adjusted Mean change(se) 10.1 (2.4) 14.5 (2.4) Column trt. minus Placebo Difference (se) 4.4 (3.4) 95% Confidence Interval -2.3, 11.2 p-value Page 1 of 1 CONFIDENTIAL BL2001/00008/00

115 114 Protocol: Population: Intent-to-Treat Table 28 Summary of Evening PEF Placebo Ventolin Total (N=44) (N=44) (N=44) Screening n 44 Mean SD Median Min Max Period 1 n Mean SD Median Min Max Period 2 n Mean SD Median Min Max Page 1 of 1 CONFIDENTIAL BL2001/00008/00

116 115 Protocol: Population: Intent-to-Treat Table 29 Analysis of Evening PEF Placebo Ventolin (N=44) (N=44) Raw Mean at Baseline (sd) (80.3) (80.3) Raw Mean of PEF (sd) (80.4) (77.2) Adjusted Mean(se) (2.4) (2.4) Adjusted Mean change(se) 8.2 (2.4) 17.5 (2.4) Column trt. minus Placebo Difference (se) 9.3 (3.4) 95% Confidence Interval 2.5, 16.1 p-value Page 1 of 1 CONFIDENTIAL BL2001/00008/00

117 116 Protocol: Population: Intent-to-Treat Table 30 Summary of D2 Concentrations Placebo Ventolin (N=44) (N=44) 1 hr n Mean SD Median Min Max hr n Mean SD Median Min Max hr n Mean SD Median Min Max Note values recorded as >1000 as summarised as 1001 Page 1 of 1 CONFIDENTIAL BL2001/00008/00

118 117 Protocol: Population: Intent-to-Treat Table 31 Analysis of D2 Concentrations Placebo Ventolin 1hr (N=44) (N=44) Raw Mean at 1hr (251.7) (330.9) Adjusted Mean(se) (31.4) (31.4) Column trt. minus Placebo Difference (se) (44.4) 95% Confidence Interval 17.5, p-value Note values recorded as >1000 as analysed as 1001 Page 1 of 3 CONFIDENTIAL BL2001/00008/00

119 118 Protocol: Population: Intent-to-Treat Table 31 Analysis of D2 Concentrations Placebo Ventolin 2hr (N=44) (N=44) Raw Mean at 2hr (299.1) (318.7) Adjusted Mean(se) (30.8) (30.8) Column trt. minus Placebo Difference (se) 34.0 (43.6) 95% Confidence Interval -54.0, p-value Note values recorded as >1000 as analysed as 1001 Page 2 of 3 CONFIDENTIAL BL2001/00008/00

120 119 Protocol: Population: Intent-to-Treat Table 31 Analysis of D2 Concentrations Placebo Ventolin 4hr (N=44) (N=44) Raw Mean at 4hr (325.1) (335.4) Adjusted Mean(se) (35.6) (35.6) Column trt. minus Placebo Difference (se) 34.3 (50.4) 95% Confidence Interval -67.5, p-value Note values recorded as >1000 as analysed as 1001 Page 3 of 3 CONFIDENTIAL BL2001/00008/00

121 120 Protocol: Population: Intent-to-Treat Table 32 Summary of D5 Concentrations Placebo Ventolin (N=44) (N=44) 1 hr n Mean SD Median Min Max hr n Mean SD Median Min Max hr n Mean SD Median Min Max Note values recorded as >1000 as summarised as 1001 Page 1 of 1 CONFIDENTIAL BL2001/00008/00

122 121 Protocol: Population: Intent-to-Treat Table 33 Analysis of D5 Concentrations Placebo Ventolin 1hr (N=44) (N=44) Raw Mean at 1hr (231.4) (230.5) Adjusted Mean(se) (29.5) (30.2) Column trt. minus Placebo Difference (se) 11.5 (42.2) 95% Confidence Interval -73.8, 96.8 p-value Note values recorded as >1000 as analysed as 1001 Page 1 of 3 CONFIDENTIAL BL2001/00008/00

123 122 Protocol: Population: Intent-to-Treat Table 33 Analysis of D5 Concentrations Placebo Ventolin 2hr (N=44) (N=44) Raw Mean at 2hr (261.7) (214.9) Adjusted Mean(se) (26.1) (26.7) Column trt. minus Placebo Difference (se) 49.0 (37.3) 95% Confidence Interval -26.3, p-value Note values recorded as >1000 as analysed as 1001 Page 2 of 3 CONFIDENTIAL BL2001/00008/00

124 123 Protocol: Population: Intent-to-Treat Table 33 Analysis of D5 Concentrations Placebo Ventolin 4hr (N=44) (N=44) Raw Mean at 4hr (268.4) (247.4) Adjusted Mean(se) (22.7) (23.3) Column trt. minus Placebo Difference (se) 35.3 (32.5) 95% Confidence Interval -30.4, p-value Note values recorded as >1000 as analysed as 1001 Page 3 of 3 CONFIDENTIAL BL2001/00008/00

125 124 Protocol: Population: Intent-to-Treat Table 34 Summary of Study Drug Exposure Placebo Ventolin (N=44) (N=44) Number of Subjects receiving Medication 44 (100%) 44 (100%) Number of days of exposure n Mean SD Median Min. 3 3 Max. 6 5 <= 1 day exposure 0 0 >= 2 days and <= 4 days exposure 1 (2%) 1 (2%) >= 5 days exposure 43 (98%) 43 (98%) Page 1 of 1 CONFIDENTIAL BL2001/00008/00

126 125 Protocol: Population: Intent-to-Treat Started Pre-Treatment Table 35 Summary of All Adverse Events BODY SYSTEM Placebo Ventolin Event (N=44) (N=44) ANY EVENT 11 (25%) 8 (18%) NEUROLOGY Any Event 7 (16%) 5 (11%) Headaches 6 (14%) 5 (11%) Compressed nerve syndromes 1 (2%) 0 Migraines 1 (2%) 0 MUSCULOSKELETAL Any Event 2 (5%) 2 (5%) Musculoskeletal pain 2 (5%) 1 (2%) Arthritis 0 1 (2%) EAR NOSE & THROAT Any Event 2 (5%) 1 (2%) Throat irritation 2 (5%) 1 (2%) NON-SITE SPECIFIC Any Event 3 (7%) 0 Chest symptoms 2 (5%) 0 Malaise & fatigue 1 (2%) 0 GASTROINTESTINAL Any Event 1 (2%) 1 (2%) Dental operations 1 (2%) 0 Nausea & vomiting 0 1 (2%) Page 1 of 1 CONFIDENTIAL BL2001/00008/00

127 126 Protocol: Population: Intent-to-Treat Started During Treatment Table 35 Summary of All Adverse Events BODY SYSTEM Placebo Ventolin Event (N=44) (N=44) ANY EVENT 13 (30%) 20 (45%) NEUROLOGY Any Event 8 (18%) 14 (32%) Headaches 7 (16%) 12 (27%) Dizziness 1 (2%) 2 (5%) EAR NOSE & THROAT Any Event 3 (7%) 6 (14%) Throat irritation 2 (5%) 5 (11%) Ear signs & symptoms 1 (2%) 1 (2%) Nasal sinus disorders 0 1 (2%) Rhinorrhea/post nasal drip 1 (2%) 0 Sneezing 1 (2%) 0 GASTROINTESTINAL Any Event 4 (9%) 4 (9%) Nausea & vomiting 3 (7%) 3 (7%) Dental discomfort & pain 1 (2%) 1 (2%) LOWER RESPIRATORY Any Event 2 (5%) 2 (5%) Cough 1 (2%) 1 (2%) Breathing disorders 1 (2%) 0 Sputum abnormalities 0 1 (2%) Page 1 of 2 CONFIDENTIAL BL2001/00008/00

128 127 Protocol: Population: Intent-to-Treat Started During Treatment Table 35 Summary of All Adverse Events BODY SYSTEM Placebo Ventolin Event (N=44) (N=44) MUSCULOSKELETAL Any Event 2 (5%) 2 (5%) Musculoskeletal pain 1 (2%) 2 (5%) Bone & skeletal pain 0 1 (2%) Muscle pain 1 (2%) 0 NON-SITE SPECIFIC Any Event 2 (5%) 2 (5%) Chest symptoms 2 (5%) 1 (2%) Malaise & fatigue 0 1 (2%) PSYCHIATRY Any Event 0 1 (2%) Aggression & hostility 0 1 (2%) SKIN Any Event 1 (2%) 0 Sweating 1 (2%) 0 Page 2 of 2 CONFIDENTIAL BL2001/00008/00

129 Protocol: Population: Intent-to-Treat Started Post-Treatment Table 35 Summary of All Adverse Events There were no such events 128 Page 1 of 1 CONFIDENTIAL BL2001/00008/00

130 Protocol: Population: Intent-to-Treat Table 36 Summary of Drug-Related Adverse Events 129 BODY SYSTEM Placebo Ventolin Event (N=44) (N=44) ANY EVENT 7 (16%) 6 (14%) NEUROLOGY Any Event 7 (16%) 5 (11%) Headaches 7 (16%) 5 (11%) LOWER RESPIRATORY Any Event 0 1 (2%) Sputum abnormalities 0 1 (2%) Page 1 of 1 CONFIDENTIAL BL2001/00008/00

131 Protocol: Population: Intent-to-Treat Table 37 Summary of Serious Adverse Events There were no such events 130 Page 1 of 1 CONFIDENTIAL BL2001/00008/00

132 Protocol: Population: Intent-to-Treat Table 38 Summary of Treatment-Limiting Adverse Events There were no such events 131 Page 1 of 1 CONFIDENTIAL BL2001/00008/00

133 13. LISTINGS CONFIDENTIAL BL2001/00008/00 132

134 This section contained data from each individual patient, rather than in aggregate. They have been excluded to protect patient privacy. Anonymized data from each patient may be made available subject to an approved research proposal. For further information please see the Patient Level Data section of the GSK Clinical Study Register.

135 CONFIDENTIAL Glaxo Wellcome Research and Development BL2000/00007/00 Division: Self-Medication Document Number: BL2000/00007/00 Directorate Document Type: Concept Protocol Study Number: Site of Issue: Stockley Park, UK Classification: Level 2 Document Date: 12 December 2000 Title: A single centre, randomised, double-blind, placebo-controlled, crossover, pilot study to determine the efficacy and safety of 400µg and 200µg doses of Ventolin inhaler versus placebo for the treatment of smoking related cough. Abstract: (For Internal Use Only) Ventolin inhaler contains the β 2 -agonist salbutamol, an effective bronchodilator. Smoking may cause increased reactivity of the airways resulting in bronchospasm in the lower airway. This single centre, randomised, double-blind, placebo-controlled, crossover, pilot study investigates whether or not Ventolin inhaler is effective in treating smoking related cough. The study involves 40 subjects, men or women, aged 18 to 65 years, suffering from smoking related cough but otherwise healthy. The study consists of: a 5 day screening phase during which baseline cough measurements will be recorded; a first treatment phase of five days; a washout phase of between two to seven days; a second treatment phase of five days; and a follow up visit. Subjects will receive active medication during one treatment phase and matched placebo during the other, the order in which they receive active treatment and placebo will be randomised and double-blinded. On waking on the first day of each treatment phase, while under supervision in the clinical trials unit, subjects will receive an initial dose of 400 µg Ventolin inhaler or placebo. Twenty minutes after receiving study medication, subjects will smoke a cigarette which is likely to provoke a cough reflex. Subjects will undergo a citric acid challenge test and be discharged home at noon on the first treatment day, all subsequent doses of Ventolin inhaler will be 200 µg or matched placebo. Study endpoints include: cough frequency; nocturnal disturbance; change in cough symptoms; treatment preference; lung function (peak flow); and response to citric acid challenge. Safety and tolerability will be monitored via adverse event reporting. The study will be conducted at the UK. Authors: Prof. MA MD FRCP, BSc, PhD Glaxo Wellcome Compound Numbers/Keywords (if applicable): Salbutamol (Ventolin )

136 CONFIDENTIAL Glaxo Wellcome Research and Development BL2000/00007/00 Distribution: *denotes summary only, **denotes partial copy only For European and Japanese Authors: Records Management Group, Information Services, UK (2 copies, unbound, plain paper, single-sided) For documents with US or Canadian site/business prefix Document Numbers: US Internal Databases, RTP J (1 copy, unbound, plain paper, single-sided). For all clinical documents from the UK or Rest of World: UK INDI Team, Bldg. 20E, Greenford (2 copies, unbound, plain paper, single-sided). Dr Dr Dr Dr Study Team Leader, Self- Medication Directorate (SMD), Clinical Research Medical Director, SMD Clinical Strategy, SMD Regulatory Affairs, SMD Worldwide Product Surveillance and Pharmacovigilance (WPSP) Senior Statistician, Medical Data Sciences (MDS) International CRF Design and Development European Data Management, (MDS) International Clinical Supplies Medical Affairs, SMD Category Manager, SMD

137 CONFIDENTIAL Glaxo Wellcome Research and Development BL2000/00007/00 Title: A single centre, randomised, double-blind, placebo-controlled, crossover, pilot study to determine the efficacy and safety of 400 µg and 200 µg doses of Ventolin inhaler versus placebo for the treatment of smoking related cough. Author(s): BL2000/00007/00 Study No: Document Date: 12 December 2000 Study Team Leader: Signature: Date: Senior Clinical Programme Head Prof. Principal Investigator Sponsor Signatory: Signature: Date: Dr. Head of Clinical Strategy Self Medication Directorate Senior Statistician Medical Data Sciences-Respiratory Statistics Section Head MDS Stats-Respiratory Revision Chronology: BL2000/00007/00 Copyright Glaxo Wellcome Research and Development All rights reserved. Unauthorised copying or use of this information is prohibited.

138 CONFIDENTIAL BL2000/00007/00 SPONSOR INFORMATION PAGE Title: Study Number: A single centre, randomised, double-blind, placebo-controlled, crossover, pilot study to determine the efficacy and safety of 400 µg and 200 µg doses of Ventolin inhaler versus placebo for the treatment of smoking related cough. Study No: Glaxo Wellcome plc Stockley Park West Uxbridge, Middlesex, UB11 1BT, UK Telephone: Medical Contact: MBBCh, BSc, FFPM Glaxo Wellcome plc, Stockley Park West, Uxbridge, Middlesex, UB11 1BT, UK Telephone: Fascimile: CONTACTS Clinical Trial Serious Adverse Events must be reported to: Study Team Leader: Glaxo Wellcome plc Stockley Park West, Uxbridge, Middlesex, UB11 1BT, UK Telephone: Fascimile: Or in his absence: Dr Glaxo Wellcome plc Stockley Park, Middlesex, UB11 1BT, UK Telephone: Fascimile: i

139 CONFIDENTIAL BL2000/00007/00 INVESTIGATOR PROTOCOL AGREEMENT PAGE I agree: To assume responsibility for the proper conduct of the study at this site. To conduct the study in compliance with this protocol, any future amendments, and with any other study conduct procedures provided by Glaxo Wellcome (GW). Not to implement any deviations from or changes to the protocol without agreement from the sponsor and prior review and written approval from the Institutional Review Board (IRB) or Independent Ethics Committee (IEC), except where necessary to eliminate an immediate hazard to the subjects, or for administrative aspects of the study (where permitted by all applicable regulatory requirements). That I am thoroughly familiar with the appropriate use of the investigational drug(s), as described in this protocol, and any other information provided by the sponsor including, but not limited to the following: the current Clinical Investigator s Brochure (CIB) or equivalent document, CIB supplement (if applicable), and approved product label (if the product is marketed in this country and the label is not already provided as an equivalent to a CIB). That I am aware of, and will comply with, good clinical practices (GCP) and all applicable regulatory requirements. To ensure that all persons assisting me with the study are adequately informed about the investigational drug(s) and of their study- related duties and functions as described in the protocol. That I have been informed that certain regulatory authorities require the Sponsor to obtain and supply details about the investigator s ownership interest in the Sponsor or the study drug, and more generally about his/her financial ties with the Sponsor. GW will use and disclose the information solely for the purpose of complying with regulatory requirements. Hence I: Agree to supply GW with any information regarding ownership interest and financial ties (including those of my spouse and dependent children); Agree to promptly update this information if any relevant changes occur during the course of the study and for 1 year following completion of the study; and Agree that GW may disclose this information about such ownership interests and financial ties to regulatory authorities. ii

140 CONFIDENTIAL BL2000/00007/00 Investigator Name: Investigator Signature Date The following co-signature is required only when the investigator is not a physician. Physician Name: Physician Signature Date iii

141 CONFIDENTIAL BL2000/00007/00 TABLE OF CONTENTS PAGE ABBREVIATIONS...VII PROTOCOL SUMMARY...VIII 1. INTRODUCTION Background Study Rationale STUDY OBJECTIVE(S) AND ENDPOINT(S) Study Objectives Study Endpoints Primary Efficacy Endpoint Secondary Efficacy Endpoints Safety and Tolerability Endpoints INVESTIGATIONAL PLAN Study Design Screening Treatment Period Washout Treatment Period Follow-up Visit Medication Schedule Study Population Inclusion Criteria Exclusion Criteria Other Study Eligibility Criteria Considerations Treatment During Study Study Drugs and Dosages Study Treatment Assignment Concurrent Medications and Non-Drug Therapies STUDY DRUG MANAGEMENT Study Drug Packaging and Labeling Study Drug Handling Study Drug Accountability Procedures MEASUREMENTS AND EVALUATIONS Time and Events Schedule Demographic and Baseline Characteristics Study Drugs Efficacy Safety Pregnancy Premature Discontinuation Premature Discontinuation from the Study Premature Discontinuation of the Study Drug iv

142 CONFIDENTIAL BL2000/00007/00 6. DATA ANALYSIS METHODS Sample Size Determination General Considerations Analysis Populations Other Issues Efficacy Primary Efficacy Measure Secondary Efficacy Measures Safety AEs AND SAEs Definition of an AE Definition of a SAE Lack of Efficacy as an AE or SAE Clinical Laboratory Abnormalities and Other Abnormal Assessments as AEs and SAEs Method, Frequency, and Time Period for Detecting AEs and SAEs Documenting AEs and SAEs Follow-up of AEs and SAEs Prompt Reporting of SAEs to GW Timeframes for Submitting SAE Reports to GW Transmission of the SAE Reports Regulatory Reporting Requirements For SAEs Post-study AEs and SAEs SAEs Related to Study Participation SAEs Involving a Non-GW Product MEDICAL DEVICES INCIDENTS AND NEAR-INCIDENTS (INCLUDING MALFUNCTIONS) Definitions of a Medical Device, Incident, Near-Incident, Malfunction, and Remedial Action Time Period for Detecting Medical Device Incidents and Near- Incidents Documenting Medical Device Incidents and Near-Incidents Follow-up of Medical Device Incidents and Near-Incidents Prompt Reporting of Medical Device Incidents and Near-Incidents to GW Timeframes for Submitting Medical Device Incident and Near-Incident Reports to GW Reports Regulatory Reporting Requirements For Medical Devices Post-Study Medical Device Incidents and Near-Incidents STUDY ADMINISTRATION Data Management Subject Tracking Data Collection Database Processing Regulatory and Ethical Considerations Regulatory Authority Approval Notification of Primary Care Physician Ethical Conduct of the Study and Ethics Approval v

143 CONFIDENTIAL BL2000/00007/ Subject Informed Consent Investigator Reporting Requirements Study Monitoring Quality Assurance Study and Site Closure Records Retention Investigator Access to Data and Provision of Study Results Information Disclosure and Inventions REFERENCES TABLES Time and Events Schedule FIGURES Figure 1. Study Overview APPENDICES Appendix 1 - Summary of Product Characteristics Appendix 2 - Standard Operating Procedure for Cough Challenge with Citric Acid Appendix 3 - Compensation and for Patients and Indemnity Appendix 4 Declaration of Helsinki vi

144 CONFIDENTIAL BL2000/00007/00 ABBREVIATIONS CRF AE SAE ICF IRB IEC GCP GW ITT PP SCA SPC URTI AUC CIB WPSP ANCOVA CTU Case Report Form Adverse Event Serious Adverse Event Informed Consent Form Independent Review Board Independent Ethics Committee Good Clinical Practice Glaxo Wellcome Intend To Treat (population) Per Protocol Standard Clarification Agreement Summary of Product Characteristics Upper Respiratory Tract Infection Area Under Curve Clinical Investigator Brochure World Product Safety and Pharmacovigilance Analysis of Covariance Clinical Trials Unit vii

145 CONFIDENTIAL BL2000/00007/00 PROTOCOL SUMMARY Study Rationale Many habituated smokers have a chronic cough, for which there is currently no therapy proven to be effective. Smoking may cause increased reactivity of the airways, resulting in bronchospasm of the lower airway. Ventolin contains the β 2 -agonist salbutamol, an effective bronchodilator, and may therefore have antitussive properties in smoking related cough. Study Objective(s) The primary objective is to determine the efficacy of 400 µg and 200 µg Ventolin (salbutamol) inhaler versus placebo on natural (i.e. on waking, before contact with potential triggers) and evoked (following the first cigarette) cough in habituated smokers. The secondary objective is to investigate the safety of 400 µg and 200 µg Ventolin (salbutamol) inhaler for the treatment of cough in habituated smokers. Study Endpoint(s) Primary Efficacy Endpoint Cough Frequency during the 1st hour on Treatment Day 1 Secondary Efficacy Endpoints Cough Frequency on Treatment Day 1 (after the 1st hour) and on Treatment Days 2 to 5 Nocturnal Disturbance due to Cough (over Treatment Days 1 to 5) Change in Cough Symptoms (recorded at 12 noon and 10pm on Days 1 to 5) Treatment Preference Lung Function (Peak Flow) Cough threshold evaluation measured as response to citric acid challenge Safety Assessments Adverse Event Reporting Study Design The study is a single-centre, randomised, double-blind, placebo-controlled, crossover pilot study, consisting of: a 5 day screening phase, during which baseline cough measurements will be recorded; a first treatment phase of five days; a washout phase of viii

146 CONFIDENTIAL BL2000/00007/00 between two to seven days; a second treatment phase of five days; and a follow up visit, as shown below. During the screening period, subjects will be screened for eligibility, and if eligible, will be asked to record baseline cough frequency, nocturnal disturbance, cough symptoms and peak flow. Screening (optional interval) Treatment 1 Washout Treatment 2 Follow up (5 days) (max 2 days) (5 days) (2-7 days) (5 days) (single visit) Visit Visit Visit 3 Visit 4 Subjects will be asked to return to the Clinical Trials Unit the evening before each treatment period. Prior to treatment period one, each subject will have their eligibility confirmed and, if eligible, will be randomised to receive either active medication during treatment period 1 and placebo during treatment period 2, or the reverse. On waking on the morning of the first day of both treatment periods, while under supervision in the clinical trials unit, subjects will receive an initial dose of 400 µg Ventolin inhaler or placebo. Twenty minutes after receiving study medication, subjects will smoke their first cigarette of the day which is expected to provoke a cough reflex. One hour after drug administration subjects will undergo a cough challenge test with nebulised citric acid. They will then be discharged home at noon on the first treatment day, all subsequent doses of Ventolin inhaler will be 200 µg or matched placebo. Planned Sample Size There will be 40 subjects randomised; 20 to receive Ventolin (salbutamol) in treatment period 1 and placebo in treatment period 2 and 20 to receive placebo in treatment period 1 and Ventolin (salbutamol) in treatment period 2. Study Population The study population will be subjects: Men or women, aged 18 to 65 years, suffering from smoking related cough but otherwise healthy. A female is eligible to enter and participate in this study if she is of: a b non-childbearing potential (i.e., physiologically incapable of becoming pregnant, including any female who is post-menopausal); or, child-bearing potential, has a negative pregnancy test (urine) at screen, and agrees to one of the following: ix

147 CONFIDENTIAL BL2000/00007/00 Complete abstinence from intercourse throughout the study Implants of levonorgestrel (except Norplant system); or, Injectable progestogen; or, Oral contraceptive (combined or progestogen only); or, Any intrauterine device (IUD) with published data showing that the lowest expected failure rate is less than 1% per year; or, Any other methods with published data showing that the lowest expected failure rate for that method is less than 1% per year; or, Barrier method only if used in combination with any of the above acceptable methods; or, The subject is sterilised. Healthy subjects as determined by medical history, physical examination, vital signs, and ECG. FEV 1 > 80% of predicted Subjects smoking greater than 15 cigarettes a day and unwilling to give up. Subjects who have a greater than 5 pack year smoking history (1 pack year = 20 cigarettes per day for 1 year) Subjects recording morning cough on all five days of screening diary record card assessment. History of persistent daily morning cough for a minimum of 3 months prior to entry. Subject has the ability to read comprehend and record information required in the study. Subject has signed and dated written informed consent prior to participating in the study. Willing to comply with the study requirements (including abstinence from cough treatments, abstinence from any other medications containing codeine, diary card completion at required times). Study Drugs and Dosages Subjects will receive active medication during one treatment phase and matched placebo during the other, the order in which they receive active treatment and placebo will be randomised and double-blinded. Medication will be administered via metered dose inhaler (MDI) fitted with a spacer (Volumatic Spacer Device provided by Allen & Hanburys Ltd). The first two doses of each treatment phase will be taken while subjects are under supervision in the clinical trials unit. The first dose will be 400 µg Ventolin inhaler or placebo, all subsequent doses will be of 200 µg Ventolin inhaler or placebo. Days 1 and 2 (3 doses, tid): First dose in the morning on waking, second dose at mid-day, third dose in the evening. x

148 CONFIDENTIAL BL2000/00007/00 Days 3, 4, 5 (prn): medication to be taken as required (but not more than 3 doses in any 24 hour period). Measurements and Evaluations Cough frequency: over the following time periods following waking: Treatment day 1: 0-10 mins, mins, mins, 40 mins - 1 hour, 1 hour - 12:00 noon, and 12:00 noon - 10pm. Treatment days 2-5 and screening days: waking to 12:00 noon, 12:00 noon to 10pm Nocturnal disturbance due to cough Scaled answer: 0 (no symptoms during the night) to 4 (symptoms so severe that I did not sleep at all) on nights following days 1-5 of each treatment period and screening days. Change in cough symptoms Scaled answers, recorded at 12:00 noon for the period from waking until 12:00 noon, and at 10pm for the period from 12:00 noon until 10pm on all: i. Morning and afternoon/evening cough severity (including questions relating to ease of expectoration) ii. Morning and afternoon/evening chest tightness iii. Morning and afternoon/evening expectoration volume iv. Morning and afternoon global symptom score Treatment preference Scaled answer: preference for treatment 1, no preference, preference for treatment 2, following administration of both treatments. Lung function Peak flow measurements on waking and at 10pm on all screening and treatment days. Cough threshold: Response to citric acid challenge 1, 2 and 4 hours after drug administration, on treatment day 1 of each treatment period. Safety and tolerability: Adverse event reporting Data Analysis Methods The primary population for all efficacy analyses will be the ITT population. Pairwise comparisons will be made between Ventolin and Placebo. As the study is a pilot study, no adjustment will be made for multiplicity. All confidence intervals calculated in the study analyses will be symmetric and of size 95% and all hypotheses tests will be 2-sided and conducted using a 5% significance level. The distribution of the reduction in cough frequency will be analysed using Prescott s test. xi

149 CONFIDENTIAL BL2000/00007/00 1. INTRODUCTION Despite the best efforts of both health care professionals and subjects themselves, many smokers find that they are unable to give up the habit. Unsurprisingly many of these habituated smokers have a chronic cough productive of small quantities of sputum, typically most severe early in the morning. There is currently no therapy for smoking related cough that is proven to be effective. In this proposal we wish to study the effect of inhaled salbutamol on cough in habituated smokers Background The effect of salbutamol on the cough reflex has been the subject of a number of studies. In healthy normal volunteers salbutamol appears to have little effect on the cough reflex 1, however, in disease states such as asthma when inflammation and bronchoconstriction are present then salbutamol has measurable antitussive activity 2. In these circumstances it is likely that the inflammation leads to some degree of bronchoconstriction which causes heightening of the cough reflex through slowly adapting receptors. Salbutamol, by relaxing bronchial smooth muscle, inhibits these reflex responses and restores the cough reflex towards normal Study Rationale A number of studies have shown that the cough reflex, as measured by cough challenge, becomes abnormal in lung disease such as viral respiratory tract infection 4. There is increasing evidence that smoking causes airway inflammation 5. We plan to use these abnormalities of the cough reflex in order to test the hypothesis that salbutamol has antitussive properties in smoking related cough. 2. STUDY OBJECTIVE(S) AND ENDPOINT(S) 2.1. Study Objectives The primary objective is to determine the efficacy of 400 µg and 200 µg Ventolin (salbutamol) inhaler versus placebo on natural (i.e. on waking, before contact with potential triggers) and evoked (following the first cigarette) cough in habituated smokers. The secondary objective is to investigate the safety of 400 µg and 200 µg Ventolin (salbutamol) inhaler for the treatment of cough in habituated smokers Study Endpoints Full details of the endpoints described below can be found in Section

150 CONFIDENTIAL BL2000/00007/ Primary Efficacy Endpoint Reduction in cough frequency over the following time periods after waking on treatment day 1 (both treatment periods): 0-10 min, min, min, 40 min - 1 hour Secondary Efficacy Endpoints 1. Reduction in cough frequency over the following time periods: Treatment day 1: 1 hour after waking 12 noon, 12:00 noon - 10pm Treatment days 2-5: waking to 12:00 noon, 12:00 noon to 10pm 2. Nocturnal disturbance due to cough 3. Change in cough symptoms (severity [including ease of expectoration], chest tightness, expectoration volume, global symptom score) 4. Treatment preference 5. Lung function recorded by peak flow measurements 6. Cough threshold elevation measured as response to citric acid challenge Safety and Tolerability Endpoints Adverse event reporting 3. INVESTIGATIONAL PLAN 3.1. Study Design The study will be a randomised placebo controlled crossover. Each limb of the crossover will be separated by at least two days Screening Subjects will attend for an initial screening visit (Visit 1) during which they will be asked for their informed consent to take part in the study. Demographics, medical history, physical examination, vital signs, ECG and lung function (FEV1) data will be collected. Subjects will be assessed for compliance with the inclusion/exclusion criteria, including methacholine challenge to detect previously undiagnosed asthma and citric acid challenge to confirm an ability to cough due to this test. Blood and urine samples will be collected and x-rays will be taken for clinical investigations if considered necessary by the investigator to exclude diagnosis of significant concurrent illness. Pregnancy testing will also be conducted using urine samples. Following successful completion of the initial screening visit, subjects will enter a 5 day screening period. During the screening period subjects will be provided with a cough counter and peak flow meter, and asked to complete a screening diary card to record baseline values for cough frequency, nocturnal disturbance, cough symptoms, and peak flow. 2

151 CONFIDENTIAL BL2000/00007/00 Subjects will be required to abstain from using any cough treatments and any products containing codeine from Visit 1 until completion of the second treatment period. Any concomitant medications taken during the trial will be recorded. An interval of up to two days may be observed between completion of the screening period and the start of treatment period Treatment Period 1 On the night before treatment day one, subjects will be admitted to the Clinical Trials Unit (CTU) at to ensure compliance with study procedures, and to confirm eligibility following clinical laboratory investigation results and review of the screening diary card (Visit 2). If subjects are eligible to enter, they will be randomised and told to continue smoking as normal until midnight when they will abstain from cigarettes. Overnight smoking abstinence will be confirmed by the use of carbon monoxide monitoring (Smokealyser). On waking the following morning (study day one) subjects will record peak flow. They ZLOOWKHQUHFHLYHLQDEOLQGHGUDQGRPLVHGIDVKLRQ JRIVDOEXWDPRORUPDWFKHG placebo via metered dose inhaler (MDI) fitted with a spacer (volumatic). Drug administration will be followed by a one hour observation period when both morning cough and plasma salbutamol should be at maximum. Cough frequency will be recorded by voice activated analogue tape recorder and microphone during the 0-10 minute and minute intervals following drug administration. Twenty minutes after drug administration subjects will enter the designated smoking room and be asked to have their first cigarette (own brand) of the day. Subjects will be observed for the number of coughs produced by this manoeuvre, coughs being counted for 20 minutes after the cigarette and a subsequent 20 minute period (i.e minutes post drug administration and 40 minutes - 1 hour post drug administration). Subjects will then (i.e. 1 hour after drug administration) undergo cough challenge with nebulised citric acid according to the established protocol 6 (see appendix 1 for SOP). Briefly, they will receive incremental doses of citric acid interspaced at random intervals with nebulised saline. The sensitivity of the cough reflex will be expressed as D2 and D5 measurements. These are the doses of citric acid causing at least two or five coughs per inhalation. Subjects will then be allowed to smoke ad libitum but with the number and time of cigarettes consumed recorded. Cough challenge will be repeated at two hours and four hours. Subjects will receive their second dose of study medication and will be discharged from the CTU with instructions to continue their allotted medication and to complete the treatment period 1 diary card. Morning and afternoon/evening cough frequency, symptoms, expectoration; nocturnal disturbance; twice daily peak flow measurements; and any adverse events will be 3

152 CONFIDENTIAL BL2000/00007/00 recorded as separate items on the diary card. Investigators will question subjects regarding any adverse events (see Section 7) Washout Subjects will then enter the wash out period for a minimum of two days and maximum of seven days, returning for the second limb of the study the evening before the start of the second treatment period, when the diary card for the first treatment period will be returned Treatment Period 2 All procedures for treatment period 2 will be carried out as for treatment period 1. Subjects will be asked to smoke with the same frequency and at the same time points as they did during study day one of treatment period 1, during the time they are in the CTU (Visit 3). Following discharge from the CTU, subjects will be allowed to smoke ad libitum. Subjects will be issued with a diary card identical to the one used for treatment period 1, except that at the end of the second treatment period they will be asked to record any treatment preference Follow-up Visit Subjects will return for a follow-up visit (Visit 4) within one week of completing treatment period 2 to return the diary card. Any adverse events ongoing at the follow-up visit will be followed according to standard clinical practice until resolved or until not requiring further medical treatment in the opinion of the investigator Medication Schedule On day 1 of each treatment period (while resident in the CTU), the first dose will be 400 JVDOEXWDPRORUPDWFKHGSODFHERYLD0',ILWWHGZLWKDVSDFHU$OOVXEVHTXHQWGRVHV ZLOOEH JVDOEXWDPRORUPDWFKHGSODFHERYLDWKHVDPHGHYLFH7KHVHFRQGGRVHZLOO be at mid-day, also while under supervision in the CTU. Subjects will then be discharged from the CTU with instructions to take their remaining doses in the same manner. Days 1 and 2 (3 doses, tid): First dose in the morning on waking, second dose at mid-day, third dose in the evening Days 3, 4, 5 (prn): medication to be taken as required (but not more than 3 doses in any 24 hour period) 3.2. Study Population Volunteers with chronic troublesome cough but without significant systemic illness will be recruited for this study by external advertisement. Subjects will be required to have a history of greater than five pack years smoking and to be unwilling to give up cigarettes. 4

153 CONFIDENTIAL BL2000/00007/ Inclusion Criteria A subject will be eligible for inclusion in this study only if all of the following criteria apply: Men and women aged 18 to 65 years Healthy subjects as determined by medical history, physical examination, vital signs, and ECG FEV 1 > 80% of predicted Subjects smoking greater than 15 cigarettes a day and unwilling to give up Subjects who have a greater than 5 pack year smoking history (1 pack year = 20 cigarettes per day for 1 year). Subjects recording morning cough on all five days of screening diary record card assessment History of persistent daily morning cough for a minimum of 3 months prior to entry Subject has the ability to read comprehend and record information required in the study Subject has signed and dated written informed consent prior to participating in the study Willing to comply with the study requirements (including abstinence from cough treatments, abstinence from any other medications containing codeine, diary card completion at required times) Healthy subjects are defined as individuals who are free from clinically significant illness or disease as determined by their medical history (including family), physical examination, laboratory studies, and other tests GW Acceptable Contraceptive Methods, when used consistently and in accordance with both the product label and the instructions of a physician, are as follows: Complete abstinence from intercourse throughout the study Female sterilization Sterilization of male partner Implants of levonorgestrel - (Except Norplant system) Injectable progestogen Oral contraceptive (combined or progestogen only) Any intrauterine device (IUD) with published data showing that the lowest expected failure rate is less than 1% per year Any other methods with published data showing that the lowest expected failure rate for birth control is less than 1% per year 5

154 CONFIDENTIAL BL2000/00007/00 Barrier method, only if used in combination with any of the above acceptable methods Exclusion Criteria A subject will not be eligible for inclusion in this study if any of the following criteria apply: Inability to cooperate with study protocol Chronic obstructive pulmonary disease - (emphysema + bronchitis + asthma); or emphysema alone; or asthma alone; all subjects will be required to undergo a methacholine challenge, a positive response will be considered diagnostic for asthma and will result in exclusion from the study. Subjects with mild chronic bronchitis alone will not be excluded from the study provided they fulfil all of the inclusion criteria and none of the exclusion criteria. Other significant concurrent illness (clinical laboratory investigations may be conducted if, in the opinion of the investigator, they are required in order to exclude the possibility of significant concurrent illness) Any concurrent medication that the study clinician considers contra-indicated to the study e.g. antitussives or mucolytics within 24 hours or anti-histamines within 72 hours before commencement of the study Participation in another clinical study in which the subject was exposed to an investigational or non-investigational drug or device in the previous month Known sensitivity to salbutamol History of drug or alcohol abuse (no more than 4 units per day, 1 unit being ½ pint of beer, or 1 glass of wine, or measure of spirit) Recent (within 14 days) respiratory tract infection Pregnancy or lactation Norplant system as a method of contraception Inability to cough due to citric acid challenge Other Study Eligibility Criteria Considerations In order to assess any potential impact on subject eligibility with regard to safety, the investigator must refer to the following document(s) for detailed information regarding warnings, precautions, contraindications, adverse events, and other significant data pertaining to the study drug(s) being used in this study: relevant documents including, but not limited to, CIB/equivalent document, CIB supplement (if applicable), and approved product label (if applicable). 6

155 CONFIDENTIAL BL2000/00007/ Treatment During Study Study Drugs and Dosages Study drugs Ventolin (salbutamol) inhaler or matched placebo Dose rationale Doses selected are based on the effective doses used in asthma. 400 µg Ventolin is expected to have the greater therapeutic effect, 200 µg Ventolin is expected to be better tolerated Dosages and dosing Subjects will receive a first dose of 400 µg Ventolin or placebo, and a second dose of 200 µg Ventolin or placebo while under supervision in the CTU. All subsequent doses will be administered without supervision. During days 1 and 2 of each treatment period subjects will receive study medication three times a day (tid), during days 3, 4 and 5 of each treatment period subjects will be allowed to take medication as required (prn), but not more than three times in 24 hours. Subjects will be required to record the dosing times on their diary cards Overdose and toxicity management No specific treatment is recommended, and the investigator should use clinical judgement in treating the overdose. The preferred antidote for overdose with salbutamol is a cardioselective beta-blocking agent. Beta-blocking drugs should be used with caution in patients with history of bronchospasm. Hypokalaemia may occur following overdose with salbutamol, serum potassium levels should be monitored Study Treatment Assignment Subjects will be assigned to study treatment in accordance with the randomisation schedule. Glaxo Wellcome, using a computer system PACT designed to create random schemes will generate the randomisation code prior to the study start. A sealed codebreak envelope for each treatment number identifying whether the subject has been assigned to Sequence 1 (AB) or Sequence 2 (BA), where A is Ventolin and B is Placebo, will be sent by Glaxo Wellcome to a nominated individual at the study site (centre). Each subject who satisfies the criteria for randomisation will be assigned the next sequential treatment number at the centre. Once a treatment number has been assigned to a subject, it cannot be assigned to any other subject. 7

156 CONFIDENTIAL BL2000/00007/00 Study medication will be blinded according to the randomisation procedures described above, such that the identity of the medication is unknown to the investigator and the subject. All medicines will be supplied in identical packaging. Only in the case of an emergency, when knowledge of the study drug is essential for the clinical management or welfare of the subject, the investigator may unblind a subject s treatment assignment. If the blind is broken for any reason, the investigator must notify the appropriate GW representative immediately. If the investigator breaks the blind for an individual subject, the date and reason must be recorded on the Status of Treatment Blind CRF. The investigator must not reveal the blind to the monitor. Individual codebreak envelopes allow the code to be unblinded for one subject whilst information for all other subjects remains blinded. If a serious adverse event (SAE; as defined in Section 7.2, Definition of an SAE ) is reported to Glaxo Wellcome (GW), Worldwide Product Safety & Pharmacovigilance (WPSP) staff will unblind the treatment assignment for the individual subject. If an expedited regulatory report to one or more regulatory agencies is required, the report will identify the subject s treatment assignment. When applicable, a copy of the regulatory report may be sent to investigators in accordance with relevant regulations, GW policy, or both. In the event that the treatment blind is broken for a subject, then this subject will be withdrawn from the study if the study clinician considers continued participation to be contra-indicated Concurrent Medications and Non-Drug Therapies During the laboratory phase the following are specifically prohibited - any other medication - lozenges or medicated sweets or chewing gum During the home phase subjects will be asked to refrain from taking any other medication, taking lozenges, medicated sweets and chewing gum, however use of non-opiate analgesics will be allowed. 4. STUDY DRUG MANAGEMENT 4.1. Study Drug Packaging and Labeling The contents of the label will be in accordance with all applicable regulatory requirements. 8

157 CONFIDENTIAL BL2000/00007/ Study Drug Handling Study drug will be dispatched to a site only after receipt of required documents in accordance with all applicable regulatory requirements and GW procedures. The study medication will be allocated, dispensed and administered by the study site clinician or his designees. Study drug must be dispensed or administered according to procedures described herein. Only subjects enrolled in the study may receive study drug, in accordance with all applicable regulatory requirements. Only authorized site personnel may supply or administer study drug. All study drugs must be stored in a secure area with access limited to the investigator and authorized study site personnel and under physical conditions that are consistent with study drug-specific requirements. At the end of the study the investigator will ensure that all unused medication will be disposed in accordance with the centre s standard operating procedures Study Drug Accountability Procedures The investigator is responsible for study drug accountability, reconciliation, and record maintenance. In accordance with all applicable regulatory requirements, the investigator or designated study site personnel must maintain study drug accountability records throughout the course of the study. This person(s) will document the amount of study drug received from GW, the amount supplied and/or administered to and returned by subjects, if applicable. 5. MEASUREMENTS AND EVALUATIONS 5.1. Time and Events Schedule Please see Table 1 and Figure 1 for Time and Events schedule. The study will start in February 2001 and is planned to be complete by May Forty subjects will be recruited in total, randomised to receive either active study medication in treatment period 1 and placebo in treatment period 2, or the reverse. Subjects will stay in the CTU in groups of 4, each group will be solely men or women, since the sleeping area in the CTU is communal. Two blocks of 4 subjects can be studied per week. Two months will be allowed for recruitment, therefore total study duration from first patient first visit (FPFV) to last patient last visit (LPLV) will be a maximum of 2 months and 3 weeks. 9

158 CONFIDENTIAL BL2000/00007/ Demographic and Baseline Characteristics Subjects who have signed the informed consent document confirming their agreement to take part in the study will be assessed by the study clinician to determine their suitability. The study clinician will record their gender, height, weight and will measure their vital signs i.e. temperature, pulse and blood pressure. This information will be recorded directly into the subjects Case Report Forms (CRF). The clinician will take a medical history and perform a urine pregnancy test and a brief medical examination with emphasis on the respiratory system. This information will be recorded in the subjects Case Report Form Study Drugs The investigator will maintain a drug accountability list, which will include the start and stop dates of study medication and reconciliation of drug dispensed and returned for the home phase Efficacy Subjective and objective methods of cough recording will be used to determine treatment efficacy in this study. A wide range of measures will be used in order to capture as many potential treatment effects as possible, whether a reduction in cough frequency, a reduction in cough severity, a change in the nature of the cough, or an initial productive cough with subsequent relief. The primary efficacy endpoint is reduction in cough frequency over the first hour following waking on treatment day 1. Cough frequency at all other times during the treatment periods is a secondary endpoint. Other secondary endpoints are: Nocturnal disturbance due to cough (5 point scaled answer); Daily recording of change in cough symptoms: severity and chest tightness (10 point scaled answers) global symptom score (composite score) and expectoration volume (approximate number of teaspoons); Overall treatment preference (treatment 1, treatment 2, or no preference); Lung function on waking and at 10pm (peak flow); and Cough threshold elevation 1,2 and 4 hours following waking on treatment day 1 only (D2 and D5 values in response to citric acid challenge) Safety The following safety data will be recorded: Vital signs (heart rate, blood pressure) at screening. 12-lead ECG at screening. 10

159 CONFIDENTIAL BL2000/00007/00 Adverse Events AEs will be collected from the time a subject consents to participate in the study, prior to inhaling any study medication until the end of the study. Serious adverse events All visits except 1 st visit (screening visit) Pregnancy Pregnancy testing Urine pregnancy testing will be performed on females of child-bearing potential at screening and at the end of the treatment period (Visit 4) or study discontinuation visit which ever comes first Time period for collecting pregnancy information Information on pregnancy will be collected at screening through to follow-up (or withdrawal) after acceptance to participate in the study by signing a written informed consent Occurrence of pregnancy Any female subject or female partner of a male subject who becomes pregnant while participating in this study will be followed to determine the outcome of the pregnancy. Generally, follow-up will be no longer than 6 to 8 weeks following the estimated delivery date. The investigator, or his or her designee, will collect pregnancy information on the appropriate GW form and submit it to GW within 2 weeks of learning of the subject s pregnancy. While pregnancy itself is not considered to be an adverse event (AE) or SAE, any pregnancy complication or elective termination of a pregnancy for medical reasons will be recorded as an AE or a SAE, as described in Section 7.6, Documenting AEs and SAEs and will be followed-up as described in Section 7.7, Follow-up of AEs and SAEs. A spontaneous abortion is always considered to be a SAE. Furthermore, any SAE occurring as a result of a post-study pregnancy and is reasonably related to the study drug will be reported to GW as described in Section 7.10, Post-study AEs and SAEs, if the investigator learns of this SAE through spontaneous reporting Premature Discontinuation Premature Discontinuation from the Study A subject may voluntarily discontinue participation in this study at any time. The investigator may also, at his or her discretion, discontinue the subject from participating in this study at any time. If a subject is prematurely discontinued from participation in the study for any reason, the investigator must make every effort to perform the following evaluations: Check for adverse events, collect home phase diary, collect returned 11

160 CONFIDENTIAL BL2000/00007/00 medication and conduct peak flow measurements inclusive of nocturnal cough count and pregnancy tests for female subjects. These data will be recorded, as they comprise an essential evaluation that needs to be done prior to discharging any subject from the study. In the event that a subject is prematurely discontinued from the study at any time due to an AE (as defined in Section 7.1, Definition of an AE ) or SAE (as defined in Section 7.2, Definition of a SAE ), the procedures stated in Section 7, ( AEs and SAEs ) must be followed. Subjects are considered to have completed the study when they return to the Centre for the follow-up visit (Visit 4). Subjects will be withdrawn from the study if protocol violations will ensue which would compromise their safety. Subjects prematurely withdrawn from the study will not be replaced Premature Discontinuation of the Study Drug The study drug may be prematurely discontinued for a subject if the clinician considers that this is in subject s best interest. A subject from whom the study drug is prematurely discontinued will not be replaced. 6. DATA ANALYSIS METHODS 6.1. Sample Size Determination This is a pilot study and no data on the variability in spontaneous cough following cigarettes is available. Therefore it has not been possible to base the power calculations around the primary endpoint. However, the change in cough sensitivity with citric acid (main secondary endpoint) is well described. A sample size of 35 subjects will be required to have an 80% chance of detecting a change of one cough per cough challenge at the 5% level General Considerations Analysis Populations The Intent-to-Treat (ITT) population will comprise all subjects randomised to treatment who have received at least one dose of trial medication, have baseline efficacy assessments and have at least one post-treatment efficacy assessment. The Per Protocol (PP) population will comprise all subjects in the ITT population with no major protocol violations. Major violations leading to the exclusion of a subject from the PP population will be detailed and confirmed prior to breaking the blind. 12

161 CONFIDENTIAL BL2000/00007/00 The Safety population will comprise all patients randomised to study treatment who received at least one dose of trial medication. Subjects will only be excluded from the ITT and Safety populations if there is proof that they received no study medication Other Issues The analysis methodology for this study is described in the sections below. If at any time after protocol finalisation, a change to the planned analysis is considered necessary, this will be documented and justified in the Data Analysis Plan (if the change is made prior to database authorisation). Similarly, if a change is made after the final statistical analysis has been performed, this will be documented and justified in the Final Study Report. All confidence intervals calculated in the study analyses will be symmetric and of size 95% and all hypotheses tests will be 2-sided and conducted using a 5% significance level. Pairwise comparisons will be made between Ventolin and placebo. As the study is a pilot study, no adjustment will be made for multiplicity. For a within subject comparison, subjects will need to have available data from both treatment periods Efficacy The primary population for all efficacy analyses will be the ITT population. The primary efficacy endpoint(s) will be analysed using both the ITT and the PP populations. The secondary efficacy endpoint(s) will be analysed using the ITT population. If a difference is demonstrated between the ITT and the PP analysis carried out on the primary efficacy endpoint(s) then the secondary efficacy endpoint(s) will also be analysed using the PP population. If examination of residual plots for parameters being analysed under the assumption of normality suggest marked deviations from normality or variance homogeneity, a log-transformation will be applied to the data, which will then be analysed as described. It is assumed that there is a sufficient wash-out period between treatment periods (2-7 days). Therefore no carry-over effect will be investigated Primary Efficacy Measure The primary efficacy endpoint is the reduction in cough frequency after waking over the following timepoints on Treatment Day 1: 0-10 mins, mins, mins and 40 mins - 1 hour 13

162 CONFIDENTIAL BL2000/00007/00 The distribution of the reduction in cough frequency for each time point will be calculated and analysed using Prescott s test Secondary Efficacy Measures Summary statistics will be provided for all secondary endpoints. Inference analysis will be performed wherever specified Cough Frequency (CF) Cough Frequency is also measured between 1 hour after treatment to 12:00 noon and 12:00 noon to 10pm on Treatment Day 1 and also from waking to 12:00 noon and 12:00 noon - 10pm on Treatment Days 2-5. The data collected on treatment Day 1 will be summarised only. The data collected on Treatment Days 2-5 will be analysed as Section Nocturnal Disturbance The mean nocturnal disturbance over each set of 5 days will be calculated for each subject. These values will be analysed as in Section Cough Severity and Chest Tightness For each measure, the analysis will be performed using the weighted mean of the score. The weighted mean responses will be calculated as the area under the response-time profile using trapezoidal integration, divided by the duration of the observation period. Actual times will be used for the derivation of the weighted mean responses. An Analysis of Covariance (ANCOVA) will be performed on the weighted means, including terms for subject, period and treatment. Adjusted means will be presented, along with the estimated treatment difference and the associated 95% confidence intervals Volume of expectoration The volume of expectoration data will be summarised by treatment period Composite global symptom score The composite global symptom score will be derived as the sum of the cough severity score and the chest tightness score. For the purposes of this pilot study, individual symptom scores will be given equal weight in the derivation of the global symptom score. The global symptom score will be summarised by treatment period. 14

163 CONFIDENTIAL BL2000/00007/ Treatment preference Treatment preference will be analysed using McNemar s test Morning and evening PEF Peak flow measurements will be collected on waking and at 10pm on all screening and treatment days. The mean of morning and evening PEF will be calculated for each subject and treatment period using all data available from days 1 to 5. The means will be analysed using analysis of covariance including terms for subject, period and treatment D2 and D5 The concentrations required to produce 2 and 5 coughs per citric acid challenge for each subject will be analysed using ANCOVA including terms for subject, treatment and period Safety Adverse Events will be coded and grouped by body systems using the GW MIDAS system. A comparison of treatment safety as evidence by adverse event reporting will be summarised and compared between treatment groups. 7. AEs AND SAEs The investigator is responsible for the detection and documentation of events meeting the definition of an AE or SAE as provided in this protocol. During each treatment period, when there is a safety evaluation, the investigator or study site personnel will be responsible for detecting AEs and SAEs, as detailed in this section of the protocol. In order to fulfill international safety reporting obligations, the investigator will include in his or her assessment any SAEs resulting from study participation (e.g., complications resulting from the taking of a blood sample) Definition of an AE An AE is any untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical product and which does not necessarily have a causal relationship with this treatment. An AE can therefore be any unfavorable and unintended sign (that could include a clinically significant abnormal laboratory finding), symptom, or disease temporally 15

164 CONFIDENTIAL BL2000/00007/00 associated with the use of a medicinal product, whether or not considered related to the medicinal product. An AE does include a/an: exacerbation of a pre-existing illness. increase in frequency or severity of a pre-existing episodic event or condition. condition detected or diagnosed after study drug administration even though it may have been present prior to the start of the study. continuous persistent disease or symptoms present at baseline that worsen following the start of the study. An AE does not include a/an: medical or surgical procedure (e.g., surgery, endoscopy, tooth extraction, transfusion); the condition that leads to the procedure is an AE. pre-existing disease or conditions present or detected at the start of the study that do not worsen. situations where an untoward medical occurrence has not occurred (e.g., hospitalizations for cosmetic elective surgery, social and/or convenience admissions). the disease or disorder being studied or sign or symptom associated with the disease or disorder unless more severe than expected for the subject s condition. overdose of either study drug or concurrent medication without any signs or symptoms. For GW clinical trials, AEs may include pre- or post-treatment events that occur as a result of protocol-mandated procedures (i.e., invasive procedures, modification of subject s previous therapeutic regimen). Information on managing an overdose, including drug and non-drug therapies, is described in Section , Overdose and Toxicity Management Definition of a SAE An SAE is any adverse event occurring at any dose that results in any of the following outcomes: a b c d e Death A life-threatening adverse event Inpatient hospitalization or prolongation of existing hospitalization A disability/incapacity A congenital anomaly in the offspring of a subject who received drug 16

165 CONFIDENTIAL BL2000/00007/00 f Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a serious adverse event when, based upon appropriate medical judgment, they may jeopardize the patient or subject and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse. Clarifications: Occurring at any dose does not imply that the subject is receiving study drug. Life-threatening means that the subject was, in the view of the investigator, at immediate risk of death from the event as it occurred. This definition does not include an event that, had it occurred in a more severe form, might have caused death. Hospitalization for elective treatment of a pre-existing condition that did not worsen during the study is not considered an AE. Complications that occur during hospitalization are AEs. If a complication prolongs hospitalization, the event is an SAE. Inpatient hospitalization means the subject has been formally admitted to a hospital for medical reasons. This may or may not be overnight. It does not include presentation at a casualty or emergency room. With regard to criteria f above, medical and scientific judgment should be used in deciding whether prompt reporting is appropriate in this situation Lack of Efficacy as an AE or SAE Lack of efficacy per se will not be reported as an AE. The signs and symptoms or clinical sequelae resulting from lack of efficacy will be reported if they fulfill the AE or SAE definition (including clarifications) Clinical Laboratory Abnormalities and Other Abnormal Assessments as AEs and SAEs Abnormal laboratory findings (e.g., clinical chemistry, hematology, urinalysis) or other abnormal assessments (e.g ECGs, X-rays, vital signs) that are judged by the investigator as clinically significant must be recorded as AEs or SAEs if they meet the definition of an AE, as defined in Section 7.1, ( Definition of an AE ), or SAE, as defined in Section 7.2, ( Definition of a SAE ). Clinically significant abnormal laboratory findings or other abnormal assessments that are detected after study drug administration or that are present at baseline and worsen following the start of the study are included as AEs or SAEs. However, clinically significant abnormal laboratory findings or other abnormal assessments that are associated with the disease being studied, unless judged by the investigator as more severe than expected for the subject s condition, or that are present or detected at the start of the study that do not worsen, are not included as AEs or SAEs. 17

166 CONFIDENTIAL BL2000/00007/00 The investigator will exercise his or her medical and scientific judgement in deciding whether an abnormal laboratory finding or other abnormal assessment is clinically significant. As defined by protocol in Section 7.2, all Grade 4 laboratory abnormalities will be reported as SAEs Method, Frequency, and Time Period for Detecting AEs and SAEs AE and SAE evaluation will be performed at each clinic visit during the study and will be captured from the time the patient signs a written informed consent until the follow-up visit. In the Informed Consent form, patients will be instructed to report any unusual or unexpected symptoms to the study staff immediately. At the end of the laboratory phase of the study, once the patient has an opportunity to spontaneously report any adverse event, the investigator or designee will ask How do you feel? During the home phase subjects will be asked to report any unusual or unexpected symptoms in their home phase diaries and on returning to the Centre after each treatment period, subjects will again be asked (1) Have you had any medical problems since the last visit? (2) Have you taken any new medicines, other than those given to you in this study, since your last visit? A subject who withdraws or is prematurely dropped out of the study will also be asked the same question to check for adverse events, if possible Documenting AEs and SAEs When an AE/SAE occurs, it is the responsibility of the investigator to review all documentation (e.g., hospital progress notes, laboratory, and diagnostic reports) relative to the event. The investigator must then record all relevant information regarding an AE/SAE on the CRF. Any AE occurring during the study must be documented in the subject s medical records, in accordance with the investigator s normal clinical practice, and on the AE page of the CRF. SAEs that occur during the study must be documented in the subject s medical record and on the SAE page of the CRF. A separate set of SAE pages should be used for each SAE. However, if at the time of initial reporting, multiple SAEs are present that are temporally and/or clinically related, they may be reported on the same SAE page. The investigator should attempt to establish a diagnosis of the event based on signs, symptoms, and/or other clinical information. In such cases, the diagnosis should be documented as the AE and/or SAE and not the individual signs/symptoms. If a clinically significant abnormal laboratory finding or other abnormal assessment meets the definition of an AE or SAE, then the AE CRF page or SAE CRF page must be completed, as appropriate. A diagnosis, if known, or clinical signs and symptoms if diagnosis is unknown, rather than the clinically significant abnormal laboratory finding, should be completed on AE or SAE CRF page. If no diagnosis is known and clinical 18

167 CONFIDENTIAL BL2000/00007/00 signs and symptoms are not present, then the abnormal finding should be recorded. The laboratory data should either be recorded in Section 10 of the SAE form with the reference range and baseline value(s) or copies of the laboratory reports and reference ranges should be sent with the SAE CRF pages. The SAE pages of the CRF should be completed as thoroughly as possible and signed by the investigator or his/her designee before transmittal to GW. It is very important that the investigator provide his/her assessment of causality to study drug at the time of the initial SAE report Follow-up of AEs and SAEs All AEs and SAEs must be followed until resolution, until the condition stabilizes, until the event is otherwise explained, or the subject is lost to follow-up. The investigator is responsible to ensure that follow-up includes any supplemental investigations as may be indicated to elucidate as completely as practical the nature and/or causality of the AE or SAE. This may include additional laboratory tests or investigations, histopathological examinations, or consultation with other health care professionals. GW may request that the investigator perform or arrange for the conduct of supplemental measurements and/or evaluations. If a subject dies during participation in the study or during a recognized follow-up period, GW will be provided with a copy of any post-mortem findings, including histopathology. New or updated information will be recorded on the originally completed SAE CRF with all changes signed and dated by the investigator Prompt Reporting of SAEs to GW SAEs must be reported promptly to GW as described in the following table once the investigator determines that the event meets the protocol definition of an SAE Timeframes for Submitting SAE Reports to GW Initial SAE Reports Additional Information on a Previously Reported SAE Type of SAE Time Frame Documents Time Frame Documents Death or Life- Threatening Event a 24/48 hrs a SAE CRF pages Other SAEs 48 hrs SAE CRF pages 48 hrs Updated SAE CRF pages 48 hrs Updated SAE CRF pages Initial notification should be sent to GW within 24 hours of the investigator learning of the death or life-threatening event. Fully completed documents ( SAE CRF pages) should be sent to GW within 48 hours. 19

168 CONFIDENTIAL BL2000/00007/ Transmission of the SAE Reports Facsimile transmission of the SAE CRF is the preferred method to transmit this information to the project contact for SAE receipt. In the absence of facsimile equipment, notification by telephone is acceptable for deaths and life-threatening events, with a copy of the SAE CRF sent by overnight mail. For SAEs that are not deaths or life-threatening events, telephone notification, in the absence of facsimile equipment, is not acceptable. Instead, a copy of the SAE CRF will be sent by overnight mail. GW will provide separately a list of project contacts for SAE receipt, fax numbers, and mailing addresses. (See CONTACTS on Page i) Regulatory Reporting Requirements For SAEs The investigator must promptly report all SAEs to GW in accordance with the procedures detailed in Section 7.8, Prompt Reporting of SAEs to GW. GW has a legal responsibility to notify both the local regulatory authority and other regulatory agencies about the safety of a drug under clinical investigation. Prompt notification of SAEs by the investigator to the appropriate project contact for SAE receipt is essential so that legal obligations and ethical responsibilities towards the safety of other subjects are met. The investigator, or responsible person according to local requirements, must comply with the applicable local regulatory requirements related to the reporting of SAEs to regulatory authorities and the Institutional Review Board (IRB)/Independent Ethics Committee (IEC) Post-study AEs and SAEs Investigators are not obligated to actively seek AEs or SAEs in former study participants. However, if the investigator learns of any SAE at any time after a subject has been discharged from the study, and such event(s) is(are) reasonably related to the study drug, the investigator will promptly notify GW SAEs Related to Study Participation An SAE considered related to study participation (e.g., procedures, invasive tests), even if it occurs during the pre- or post-treatment period, will be reported promptly to GW (see Section 7.8, Prompt Reporting of SAEs to GW ) SAEs Involving a Non-GW Product In those instances where an SAE has occurred in a subject receiving a non-gw product as a comparator or concurrent medication, the report must be sent to the appropriate project contact for SAE receipt in the same time frames as if it were a GW product (see Section 7.8, Prompt Reporting of SAEs to GW ). 20

169 CONFIDENTIAL BL2000/00007/00 8. MEDICAL DEVICES INCIDENTS AND NEAR-INCIDENTS (INCLUDING MALFUNCTIONS) GW medical devices are being provided for use in this study. In order to fulfil international reporting obligations the investigator is responsible for the detection and documentation of events meeting the definitions of incident, near-incident, or malfunction that occur during the study with such devices, whether the devices are manufactured by GW or by another company. GW will be providing, for use in the study, the following medical devices: Salbutamol Metered Dose Inhalers Volumatic Spacer Devices (provided by Allen & Hanburys Ltd) 8.1. Definitions of a Medical Device, Incident, Near-Incident, Malfunction, and Remedial Action Medical Device Any instrument, apparatus, appliance, material or other article, the principal intended action of which is typically fulfilled by physical means (including mechanical action, physical barrier, replacement of, or support to, organs or body functions). Examples of medical devices include measuring spoons, syringes, spacers and spring-loaded auto-injectors. The detection and documentation procedures described in this protocol apply to all GW medical devices provided for use in the study. Incident Any medical occurrence that occurs with a GW medical device, provided for use in the study, which results in death or a serious deterioration in the state of health whether or not due to a malfunction of the device. Incidents include for example: inhalation of an object that has accidentally entered a spacer device and consequent tracheal obstruction. Incidents do not include for example: medical occurrences associated with metered-dose inhalers that do not fulfil the definition of a medical device (such events will be reported as medicinal product AEs) non-serious medical occurrences which have no further safety implications for the subject or the device Near-incident Any potential incident occurring with a GW medical device provided for use in the study. Near-incidents are events that could have jeopardized the study subject, causing death or a serious deterioration of health, if medical intervention or other fortunate circumstances had not occurred. This would include deficiencies or inaccuracies in the instructions for use of the device and/or malfunctions. 21

170 CONFIDENTIAL BL2000/00007/00 Near-incidents include for example: discovery of caustic damage to the plastic parts of a device caused by contamination with an ointment, where no actual injury to a subject occurred but the reporter recognized that such an event could happen in normal medical use of the device and could kill or seriously harm a subject. Malfunction A failure of a device to perform in accordance with its intended purpose when used in accordance with the manufacturer s instructions. Remedial Action Any action other than routine maintenance or servicing of a device where such action is necessary to prevent recurrence of an incident or near-incident. This includes any amendment to the design to prevent recurrence Time Period for Detecting Medical Device Incidents and Near-Incidents Medical device incidents, near-incidents or malfunctions will be detected, documented and reported during all study periods in which the GW medical devices that are provided are available for use Documenting Medical Device Incidents and Near-Incidents Any medical device incident or near-incident occurring during the study must be documented in the subject s medical records, in accordance with the investigator s normal clinical practice, and on the "Medical Device Incident Report Form". In addition, for incidents and near-incidents fulfilling the definition of an AE or an SAE, the appropriate pages of the CRF must be completed as described in Section 7.6. The "Medical Device Incident Report Form" will be completed as thoroughly as possible and signed by the investigator or his/her designee before transmittal to Glaxo Wellcome. It is very important that the investigator provides his/her assessment of causality to the medical device provided by GW at the time of the initial report, and describes any corrective or remedial actions taken to prevent recurrence of the incident Follow-up of Medical Device Incidents and Near-Incidents All medical device incidents, and near-incidents involving an AE, must be followed until resolution of the event, until the condition stabilizes, until the condition is otherwise explained, or until the subject is lost to follow-up. The investigator is responsible for ensuring that follow-up includes any supplemental investigations as may be indicated to elucidate as completely as practical the nature and/or causality of the incident. New or updated information will be recorded on the originally completed "Medical Device Incident Report Form", with all changes signed and dated by the investigator. 22

171 CONFIDENTIAL BL2000/00007/ Prompt Reporting of Medical Device Incidents and Near-Incidents to GW Medical device incidents and near-incidents must be reported promptly to GW as described below once the investigator determines that the event meets the protocol definition of a medical device incident or near-incident Timeframes for Submitting Medical Device Incident and Near-Incident Reports to GW Initial Medical Device Incident/Near-Incident Reports Additional Information on a Previously Reported Medical Device Incident/Near-Incident Type of Event Time Frame Documents Time Frame Documents Incident 24 hours "Medical Device Incident Report Form" Near-Incident 48 hours "Medical Device Incident Report Form" 48 hours Updated "Medical Device Incident Report Form" 48 hours Updated "Medical Device Incident Report Form" Reports Facsimile transmission of the "Medical Device Incident Report Form" is the preferred method to transmit this information to the GW project contact. The same individual will be the GW project contact for receipt of medical device reports and SAEs. In the absence of facsimile equipment, notification by telephone is acceptable for incidents, with a copy of the "Medical Device Incident Report Form" sent by overnight mail. For near-incidents, telephone notification, in the absence of facsimile equipment, is not acceptable. Instead, a copy of the "Medical Device Incident Report Form" will be sent by overnight mail Regulatory Reporting Requirements For Medical Devices The investigator must promptly report all incidents and near-incidents occurring with any GW medical device provided for use in the study, in accordance with the procedures detailed in Section 8.5, Prompt Reporting of Incidents and Near-Incidents to GW. GW has a legal responsibility to notify appropriate regulatory bodies and other entities about certain safety information relating to medical devices being used in clinical studies. Prompt notification of incidents and near-incidents by the investigator to the appropriate project contact is essential in order to meet legal obligations and ethical responsibility towards the safety of other subjects. 23

172 CONFIDENTIAL BL2000/00007/00 The investigator, or responsible person according to local requirements, must comply with the applicable local regulatory requirements relating to the reporting of incidents and near-incidents to regulatory bodies, and the IRB/IEC Post-Study Medical Device Incidents and Near-Incidents Investigators are not obligated to actively seek reports of medical device incidents or near-incidents in former study participants. However, if the investigator learns of any incident or near-incident at any time after a subject has been discharged from the study, and such incident/near-incident is reasonably related to a GW medical device provided for the study, the investigator will promptly notify GW. 9. STUDY ADMINISTRATION 9.1. Data Management Data Management will be performed in accordance with all applicable GW standards and procedures Subject Tracking Subject tracking will be conducted by staff at the Clinical Trials Unit (CTU) monitoring visits to the unit. and by GW Data Collection Subject will complete a centre Source Data Form. This will document the following: subjects initials, address, date of birth, sex, telephone number, address, name and address of subjects general practitioner, study protocol number and sponsor, screening number, inclusion/exclusion status, treatment number (if applicable), visit dates, current medical condition, previous medical condition and/or surgery, current medication or any in the previous month, history of allergy, smoking status, participation in another clinical study in the previous month, clinician s diagnosis of current medical indication, pregnancy test result. This Source Data Form will be used as source data for the study. A screening log will be maintained for the study to track the study status. Apart from the information available from the Source Data Form all other data for this study will be recorded directly into subjects CRFs and home phase diaries. The investigator or designee must record all required subject data using the previously specified data collection method defined by Glaxo Wellcome. An explanation must be documented for any missing data. The investigator must sign and date a declaration on the CRF attesting to his/her responsibility for the quality of all data recorded and that the data represents a complete and accurate record of each subject s participation in the study. 24

173 CONFIDENTIAL BL2000/00007/00 All data retrieved from the site will be entered into a quality controlled database. The data will subsequently be analyzed according to the methods outlined in Section 6, Data Analysis Methods Database Processing CRF data will be converted to electronic form via a data entry application and loaded into a quality controlled database. Data will be reviewed and validated. Data clarifications will be requested of investigators or their designees, and the database will be edited appropriately. Selected variables will be coded. The database will be authorized for release when all data management quality control procedures are completed. The data will subsequently be analyzed according to the methods outlined in Section 6, "Data Analysis Methods." 9.2. Regulatory and Ethical Considerations Regulatory Authority Approval GW will obtain approval to conduct the study from the appropriate regulatory agency Medicines Control Agency (MCA) in accordance with applicable country-specific regulations prior to the site initiating the study. This study will be conducted in accordance with good clinical practice (GCP) and all applicable regulations, including, where applicable, the Declaration of Helsinki, June 1964, as modified by the 48th World Medical Association, Republic of South Africa, October Notification of Primary Care Physician If agreed by the subject, the investigator should notify the subject s primary care physician (if applicable) of the subject s participation in the study. The primary care physician may contact the investigator for any further information regarding the subject s participation in the study. This requirement will also be stated in the Patient Informed Consent Ethical Conduct of the Study and Ethics Approval It is the investigator s responsibility to ensure that this protocol is reviewed and approved by the appropriate IEC or IRB. The IEC or IRB must also review and approve the site s informed consent form (ICF) and any other written information provided to the subject prior to any enrolment of subjects, and any advertisement that will be used for subject recruitment. The investigator or his/her designee must forward to GW copies of the IEC or IRB approval and the approved informed consent materials, that GW must receive prior to the start of the study. 25

174 CONFIDENTIAL BL2000/00007/00 If, during the study, it is necessary to amend either the protocol or the informed consent form, the investigator will be responsible for ensuring the IEC or IRB reviews and approves these amended documents. IEC or IRB approval of the amended ICF must be obtained before new subjects consent to take part in the study using this version of the form. Copies of the IEC or IRB approval of the amended ICF and the approved amended ICF must be forwarded to GW as soon as available Subject Informed Consent The investigator or his designee will inform the subject or, where applicable, the subject s legally authorized representative (e.g., a parent, guardian, next of kin, other individual or other body with appropriate jurisdiction) of all aspects pertaining to the subject s participation in the study. The process for obtaining subject informed consent will be in accordance with all applicable regulatory requirements. The investigator or his/her designee and the subject or the subject s legally authorized representative must both sign and date the ICF before the subject can participate in the study. The subject or subject s legally acceptable representative will receive a copy of the signed and dated form and the original will be retained in the site study records. The decision regarding subject participation in the study, that is made either by the subject or the subject s legally acceptable representative, is entirely voluntary. The investigator or his designee must emphasize to the subject or the subject s legally acceptable representative that consent regarding study participation may be withdrawn at any time without penalty or loss of benefits to which the subject is otherwise entitled. If the ICF is amended during the study, the investigator must follow all applicable regulatory requirements pertaining to approval of the amended ICF by the IEC or IRB and use of the amended form (including for ongoing subjects) Investigator Reporting Requirements In accordance with applicable local regulatory requirements, the investigator may be obligated to provide periodic safety updates on the conduct of the study at his or her site and notification of study closure to the IRB or IEC. Such periodic safety updates and notifications are the responsibility of the investigator and not of Glaxo Wellcome Study Monitoring In accordance with applicable regulations, GCP, and GW procedures, monitors will periodically contact the site, including conducting on-site visits. The extent, nature and frequency of on-site visits will be based on such considerations as the study objective and/or endpoints, the purpose of the study, study design complexity, and enrollment rate. During these contacts, the monitor will: check and assess the progress of the study 26

175 CONFIDENTIAL BL2000/00007/00 review study data collected conduct Source Document Verification identify any issues and address their resolution This will be done in order to verify that the: data are authentic, accurate, and complete safety and rights of subjects are being protected study is conducted in accordance with the currently approved protocol (and any amendments), GCP, and all applicable regulatory requirements. The investigator agrees to allow the monitor direct access to all relevant documents and to allocate his time and the time of his staff to the monitor to discuss findings and any relevant issues. In addition to contacts during the study, the monitor will also contact the site prior to the start of the study to discuss the protocol and data collection procedures with site personnel. At study closure, monitors will also conduct all activities as indicated in Section 9.5, Study and Site Closure Quality Assurance At its discretion, GW may conduct a quality assurance audit of this study. If such an audit occurs, the investigator agrees to allow the auditor direct access to all relevant documents and to allocate his time and the time of his staff to the auditor to discuss findings and any relevant issues. In addition, regulatory agencies may conduct a regulatory inspection of this study. If such an inspection occurs, the investigator agrees to allow the inspector direct access to all relevant documents and to allocate his time and the time of his staff to the inspector to discuss findings and any relevant issues Study and Site Closure Upon completion of the study, the monitor will conduct the following activities in conjunction with the investigator, as appropriate: Return of all study data to Glaxo Wellcome Data clarifications and/or resolutions Accountability, reconciliation, and arrangements for unused study drugs Review of site study records for completeness Return of treatment codes to Glaxo Wellcome 27

176 CONFIDENTIAL BL2000/00007/00 In addition, GW reserves the right to temporarily suspend or prematurely discontinue this study either at a single site or at all sites at any time and for reasons including, but are not limited to, safety or ethical issues or severe non-compliance. If such action is taken, GW will discuss this with the Investigator (including the reasons for taking such action) at that time. GW will promptly inform all other investigators and/or institutions conducting the study if the study is suspended or terminated for safety reasons, and will also inform the regulatory authorities of the suspension or termination of the study and the reason(s) for the action. If required by applicable regulations, the investigator must inform the IRB or IEC promptly and provide the reason for the suspension or termination. If the study is prematurely discontinued, all study data must be returned to GW. In addition, arrangements will be made for all unused study drugs in accordance with GW procedures for the study. Financial compensation to investigators and/or institutions will be in accordance with the agreement established between the investigator and GW Records Retention In accordance with applicable regulatory requirements, following closure of the study, the investigator will maintain a copy of all site study records in a safe and secure location. GW will inform the investigator of the time period for retaining these records in order to comply with applicable regulatory requirements Investigator Access to Data and Provision of Study Results GW will provide the investigator with a copy of the CRF data collected from the site. When a clinical study report is completed, GW will provide the major findings of the study to the investigator. In addition, details of the study treatment assignment will be provided to the investigator to enable him/her to review the data to determine the outcome of the study for his/her subject Information Disclosure and Inventions Ownership: All data and records provided by GW or generated during the study (other than a subject s medical records) and all inventions discovered in the course of conducting the study are the property of GW. If a written contract for the conduct of the study which includes ownership provisions inconsistent with this statement is executed, that contract s ownership provisions shall apply rather than this statement. Confidentiality: The investigator and other study site personnel will keep confidential any information provided by GW (including this protocol) related to this study and all data and records 28

177 CONFIDENTIAL BL2000/00007/00 generated in the course of conducting the study, and will not use the information, data, or records for any purpose other than conducting the study. These restrictions do not apply to: (1) information which becomes publicly available through no fault of the investigator or study site personnel; (2) information which it is necessary to disclose in confidence to an IEC or IRB solely for the evaluation of the study; (3) information which it is necessary to disclose in order to provide appropriate medical care to a study subject, or (4) study results which may be published as described in the next paragraph. If a written contract for the conduct of the study which includes confidentiality provisions inconsistent with this statement is executed, that contract s confidentiality provisions shall apply rather than this statement. Publication: The investigator shall inform GW of any publication plans. Prior to submitting for publication, presenting, using for instructional purposes or otherwise disclosing the results of the study, the investigator shall allow GW a period of at least thirty (30) days [or, for abstracts, at least five (5) working days] to review the proposed publication or disclosure prior to its submission for publication or other disclosure, but consent to publish would not be unreasonably withheld. Publications or disclosures of study results shall not include other, confidential information of Glaxo Wellcome s. If the proposed publication/disclosure risks GW s ability to patent any invention related to the study, the publication or disclosure will be modified or delayed, at the investigator s option, a sufficient time to allow GW to seek patent protection of the invention. This statement does not give GW any editorial rights over the content of a publication or disclosure, other than to restrict the disclosure of GW s confidential information. If a written contract for the conduct of the study, which includes publication provisions inconsistent with this statement is executed, that contract s publication provisions shall apply rather than this statement. GW policies regarding compensation for injury for subjects will apply and are described in full in these respective documents that are available upon request. A copy of the compensation information will be given to the subject upon request in accordance with country-specific requirements. GW policies regarding indemnification for investigators and institutions participating in the study will apply and are fully described in these respective documents which are available upon request Payments to Subjects Subjects will be paid for the inconvenience of participating in the study. The amount of payment is stated in the informed consent form. Subjects not completing the study for whatever reason will be paid at the discretion of the Investigator, generally on a pro rata basis. 29

178 CONFIDENTIAL BL2000/00007/ REFERENCES 1. Smith CA, Adamson DL, Choudry NB, Fuller RW. The effect of altering airway tone on the sensitivity of the cough reflex in normal volunteers. Eur.Respir.J. 1991; 4: Pounsford JC, Birch MJ, Saunders KB. Effect of bronchodilators on the cough response to inhaled citric acid in normal and asthmatic subjects. Thorax 1985; 40: Taylor IK, OShaughnessy KM, Choudry NB, Adachi M, Palmer JBD, Fuller RW. A comparative study in atopic subjects with asthma of the effects of salmeterol and salbutamol on allergen-induced bronchoconstriction, increase in airway reactivity, and increase in urinary leukotriene E4 excretion. J.Allergy Clin.Immunol. 1992; 89: OConnell F, Thomas VE, Studham JM, Pride NB, Fuller RW. Capsaicin cough sensitivity increases during upper respiratory infection. Resp.Med. 1996; 90: Rennard SI, Daughton D, Fujita J, et al. Short-term smoking reduction is associated with reduction in measures of lower respiratory tract inflammation in heavy smokers. Eur. Respir. J. 1990; 3: Wong, C. H. and Morice, A. H. Cough threshold in patients with chronic obstructive pulmonary disease. Thorax 1999; 54, Jones, B and Kenward, MG. Design and analysis of cross-over trials, Chapman and Hall. 30

179 CONFIDENTIAL BL2000/00007/ TABLES Time and Events Schedule SCREENING Day 1 (Visit 1) Days 2-5 Day 0 (Visit 2 a, 3 b ) TREATMENT PERIOD Day 1 Days 2-5 SCREENING Informed Consent Demographics and Medical History Physical examination, vital signs, ECG Lung function (FEV1) Methacholine/citric acid challenge Pregnancy test (if applicable) Screening Diary Card (Presence of morning cough; baseline efficacy measures: cough frequency, nocturnal disturbance, symptoms, peak flow; AE and conc. med. monitoring) x x x x x x x TREATMENT PERIOD Admit to clinical trials unit at approximately 8pm Adverse event detection by investigator Review screening a /treatment period 1 b diary card Randomisation Study medication: 400µg Ventolin/placebo 200µg Ventolin/placebo Cough frequency (0-10, 10-20, 20-40, 40min-1hr, 1hr-12 noon) First cigarette Citric acid challenge Adverse event detection by investigator Discharge from clinical trials unit x 31 x x x x On waking Xc waking onwards 20min after waking 1, 2 and 4 hrs After second dose x c

180 CONFIDENTIAL BL2000/00007/00 DIARY CARD Treatment diary card (Efficacy measures: cough frequency, nocturnal disturbance, symptoms, treatment preference b, peak flow; AE and conc. med. monitoring) SCREENING Day 1 (Visit 1) Days 2-5 Day 0 (Visit 2 a, 3 b ) TREATMENT PERIOD Day 1 a: treatment period 1 b: treatment period 2 c: Day 1: lunchtime and evening, Day 2:morning, lunchtime and evening, Days 3-5: as required. x Days 2-5 x FOLLOW-UP (Visit 4) Discontinuation Visit FOLLOW-UP Adverse event detection by investigator x x Review treatment period 2 diary card x Review AEs (if applicable) x x Pregnancy Test (if applicable) x x Review of preceding period diary card x 32

181 CONFIDENTIAL BL2000/00007/ FIGURES Figure 1. Study Overview Screening (optional Treatment 1 Washout Treatment 2 Follow up interval) (5 days) (max 2 days) (5 days) (2-7 days) (5 days) (single visit) Visit 1 Visit 2 Visit 3 Visit 4 screening Day 1 last screening day to treatment 1 day 1 last washout day to treatment 2 day 1 within 7 days of end of treatment 2 33

182 CONFIDENTIAL BL2000/00007/ APPENDICES Appendix 1 - Summary of Product Characteristics Product Summary 1. Trade Name of the Medicinal Product Ventolin Inhaler 2. Qualitative and Quantitative Composition Salbutamol BP 100 mcg per actuation. 3. Pharmaceutical Form Metered dose aerosol delivering 100 mcg salbutamol BP per actuation with a specially designed actuator. Clinical Particulars 4.1 Therapeutic Indications Salbutamol is a selective beta-2 adrenoceptor agonist. At therapeutic doses it acts on the beta-2 adrenoceptors of bronchial muscle, with little or no action on the beta-1 adrenoceptors of the heart. With its fast onset of action, it is particularly suitable for the relief of acute symptoms and the prevention of exercise-induced asthma. Salbutamol provides short-acting (4-6 hour) bronchodilation with a fast onset (within 5 minutes) in reversible airways obstruction. It is suitable for long term use in the relief and prevention of asthmatic symptoms. Ventolin should be used to relieve symptoms when they occur and to prevent them in those circumstances recognised by the patient to precipitate an asthmatic attack (e.g. before exercise or unavoidable allergen exposure). Ventolin is particularly valuable as rescue medication in mild, moderate or severe asthma, provided that reliance on it does not delay the introduction and use of regular inhaled corticosteroid therapy. 4.2 Posology and Method of Administration Route of administration: Oral inhalation 34

183 CONFIDENTIAL BL2000/00007/00 Salbutamol has a duration of action of 4 to 6 hours in most patients. A volumatic spacer device may be used in patients who find it difficult to synchronise aerosol actuation with inspiration of breath. 1. Adults For the relief of acute asthma symptoms on inhalation. May be administered as a single minimum starting dose. This may be increased to two inhalations if necessary. To prevent exercise induced symptoms, two inhalations should be taken minutes before exertion. 2. Children One of two inhalations for the relief of acute bronchospasm or before allergen exposure or exercise. The babyhaler spacer device may be used to facilitate administration to children under 5 years of age. The recommended dose for chronic therapy is two inhalations up to four times a day. 3. Elderly No specific dosage recommendations. On demand use of Ventolin should not exceed 8 inhalations in any 24 hours. Reliance on such supplementary use or a sudden increase in dose indicates poorly controlled or deteriorating asthma (see precautions). 4.3 Contra-indications Although intravenous salbutamol and occasionally salbutamol tablets are used in the management of premature labour, uncomplicated by conditions such as placenta praevia, ante-partum haemorrhage, or toxaemia of pregnancy, inhaled salbutamol preparations are not appropriate for managing premature labour. Salbutamol presentations should not be used for threatened abortion. Ventolin inhaler is contra-indicated in patients with a history of hypersensitivity to any of its components. 4.4 Special Warnings and Precautions for Use Bronchodilators should not be the only or main treatment in patients with severe or unstable asthma. Severe asthma requires regular medical assessment including lung function testing as patients are at risk of severe attacks and even death. Physicians should consider using oral corticosteroid therapy and/or the maximum recommended dose of inhaled corticosteroid in those patients. Increasing use of 35

184 CONFIDENTIAL BL2000/00007/00 bronchodilators in particular short-acting inhaled beta-2-agonists to relieve symptoms indicates deterioration of asthma control. If patients find that short acting relief bronchodilator treatment becomes less effective or they need more inhalations than usual, medical attention must be sought. In this situation patients should be reassessed and consideration given to the need for increased anti-inflammatory therapy (e.g. Higher doses of inhaled corticosteroids or a course of oral corticosteroids). Severe exacerbations of asthma must be treated in the normal way. In the event of a previously effective dose of inhaled Ventolin failing to give relief lasting at least three hours, the patient should be advised to seek medical advice in order that any necessary additional steps may be taken. Patients inhaler technique should be checked to make sure that aerosol actuation is synchronised with inspiration of breath for optimum delivery of the drug to the lungs. As there may be adverse effects associated with excessive dosing, the dosage or frequency of administration should only be increased on medical advice. Salbutamol should be administered cautiously to patients suffering from thyrotoxicosis. Salbutamol and not-selective beta-blocking drugs such as propranolol, should not usually be prescribed together. Potentially serious hypokalaemia may result from beta-2 agonists therapy mainly from parenteral and nebulised administration. Particular caution is advised in acute severe asthma as this effect may be potentiated by concomitant treatment with xanthine derivatives, steroids, diuretics and hypoxia. It is recommended that serum potassium levels are monitored in such situations. 4.5 Interactions with other Medicaments and other forms of Interaction None known. 4.6 Pregnancy and Lactation Administration of drugs during pregnancy should only be considered if the expected benefit to the mother is greater than any possible risk to the foetus. As with the majority of drugs, there is little published evidence of its safety in the early stages of human pregnancy, but in animal studies there was evidence of some harmful effects on the foetus at very high dose levels. As salbutamol is probably secreted in breast milk its use in nursing mothers requires careful consideration. It is not known whether salbutamol has a harmful effect on the neonate and so its use should be restricted to situations where it is felt that the expected benefit to the mother is likely to outweigh any potential risk to the neonate. 36

185 CONFIDENTIAL BL2000/00007/ Effects on Ability to Drive and Use Machines None known. 4.8 Undesirable Effects Ventolin inhaler may cause fine tremor of skeletal muscle, usually the hands are most obviously affected. This effect is dose related and is common to all beta adrenergic stimulants. Occasionally headaches have been reported. Tachycardia, with or without peripheral vasodilatation, may rarely occur. In FRPPRQZLWKRWKHU 2 agonists, cardiac arrhythmias (including artrial fibrillation, supraventricular tachycardia and extrasystoles) have been reported in association with the use of salbutamol, usually in susceptible patients. There have been very rare reports of muscle cramps. Hypersensitivity reactions including angioedema and urticaria, bronchospasm, hypotension and collapse have been reported vary rarely. Potentially serious hypokalaemia may result from beta-2 agonist therapy. As with other inhalation therapy, paradoxical bronchospasms may occur with an immediate increase in wheezing after dosing. This should be treated immediately with an alternative presentation or a fast-acting inhaled bronchodilator. Ventolin inhaler should be discontinued immediately, the patient assessed, and if necessary alternative therapy instituted. As with other beta-2 agonists hyperactivity in children has been reported rarely. Mouth and throat irritation may occur with inhaled salbutamol. 4.9 Overdose The preferred antidote for overdosage with Ventolin is a cardo-selective betablocking agent but beta-blocking drugs should be used with caution in patients with a history of bronchospasm. Hypokalaemia may occur following overdose with salbutamol. Serum potassium levels should be monitored. Pharmacological Properties 5.1 Pharmacodynamic Properties Salbutamol is a selective beta-2 adrenoceptor agonist. At therapeutic doses it acts on the beta-2 adrenoceptors of bronchial muscle. 37

186 CONFIDENTIAL BL2000/00007/ Pharmacokinetic Properties Salbutamol administered intravenously has half-life of 4 to 6 hours and is cleared partly renally and partly by metabolism to the inactive 4-0-sulphate (phenolic sulphate) which is also excreted primarily in the urine. The faeces are a minor route of excretion. The majority of a dose of salbutamol given intravenously, orally or by inhalation is excreted within 72 hours. Salbutamol is bound to plasma proteins to the extent of 10%. After administration by the inhaled route between 10 and 20% of the dose reaches the lower airways. The remainder is retained in the delivery system or is deposited in the oropharynx from where it is swallowed. The fraction deposited in the airways is absorbed into the pulmonary tissues and circulation but is not metabolised by the lung. On reaching the systemic circulation it becomes accessible to hepatic metabolism and is excreted, primarily in the urine, as unchanged drug and as the phenolic sulphate. The swallowed portion of an inhaled dose is absorbed from the gastrointestinal tract and undergoes considerable first-pass metabolism to the phenolic sulphate. Both unchanged drug and conjugate are excreted primarily in the urine. 5.3 Pre clinical Safety Data None stated. Pharmaceutical Particulars 6.1 List of Excipients Oleic acid Trichlorofluoromethane Dichlorodiflouromethane. 6.2 Incompatibilities None known. 6.3 Shelf Life 3 years. 6.4 Special Precautions for Storage Store below 30ºC protected from light and direct sunlight. The canister should not be broken, punctured or burnt, even when apparently empty. As with most 38

187 CONFIDENTIAL BL2000/00007/00 inhaled medications in aerosol canisters, the therapeutic effect of this medication may decrease when the canister is cold. 6.5 Nature and Contents of Container Aluminium can fitted with a metering valve and actuator. A dust cover fits over the mouthpiece of actuator. A volumatic spacer device is also available for use with Ventolin inhaler. 6.6 Instruction for Use/Handling None stated. Administrative Data 7. Marketing Authorisation Holder Allen & Hanburys Limited Horsenden House Oldfield Lane North Greenford Middlesex UB6 0HB 8. Marketing Authorisation Number PL 00045/5022R 9. Date of First Authorisation/Renewal of Authorisation MAA: 5 December 1984 Renewal: 26 June 1989 Renewal: 15 June Date of (Partial) Revision of the Text 9 February

188 CONFIDENTIAL BL2000/00007/ Appendix 2 - Standard Operating Procedure for Cough Challenge with Citric Acid Standard Operating Procedure Title: A dose-response cough challenge with citric acid using the Mefar dosimeter system SOP No: CTU70400 Approved By Professor SIGNED DATE 40

189 CONFIDENTIAL BL2000/00007/00 Evaluation of the Cough Challenge Methodology The protocol for the cough challenge methodology is reviewed on an annual basis. The date and the member of the Clinical Trials Staff, which reviewed this technique, are located below. DATE REVIEWED CHANGES MADE? NAME OF PERSONNEL INVOLVED SIGNATURE 41

190 CONFIDENTIAL BL2000/00007/00 Principle To measure the sensitivity of the cough reflex within: Healthy volunteers Chronic cough subjects Subjects with other respiratory disorders The method of administration of the citric acid is via a breath activated, 1-second nebulisation utilising a Mefar dosimeter. This standard operating procedure is intended for all appropriately qualified staff and physicians within the 42

191 CONFIDENTIAL BL2000/00007/00 INDEX 1. Things to consider before performing the citric acid cough challenge i. Personnel ii. Safety iii. Precautions for subject safety 2. Equipment and materials i. Solutions ii. Dilution Equipment iii. Storage iv. Equipment 3. Preparation for testing i. Subject preparation before testing ii. Calibration iii. Solution Preparation 4. Performing the test i. The subject ii. Test Sequence 5. Appendix Appendix 1 Masterscope Appendix 2 Exclusion Criteria 6. Signatories 43

192 CONFIDENTIAL BL2000/00007/00 1. Things to consider before performing the citric acid cough challenge i. Personnel Before performing the cough challenge on a subject you must fulfil the following criteria: 1. Be capable of managing the equipment including set-up, proper function, maintenance and cleaning. 2. Be proficient at spirometry. 3. Know the contra-indications (APPENDIX 2) to cough challenge testing. 4. Be familiar with safety and emergency procedures. 5. Know when to stop further testing. 6. Be proficient in the administration of inhaled bronchodilators and evaluation of the response to them. ii. Safety Inhaled citric acid may cause bronchoconstriction. Thus you should consider the safety of the subject. Precautions for subject safety A physician or other person appropriately trained to treat acute bronchospasm including appropriate use of resuscitation equipment should be close enough to respond to an emergency quickly. You should make sure that medications to treat severe bronchospasm are present within the testing area. These include epinephrine and atropine for subcutaneous injection and albuterol and ipratropium in metered dose inhalers or pre-mixed solutions for inhalation, oxygen must also be available. A small volume nebulizer should be readily available for the administration of bronchodilators. A stethoscope, sphygnomanometer, and pulse oximeter should also be available. 44

193 CONFIDENTIAL BL2000/00007/00 2. Equipment and materials 2.i. Solutions 10ml 10% 1M citric acid (prepared at Hallamshire Hospital, Sheffield) 0.98% (sterile) sodium chloride for irrigation (Baxter, U.K.) β adrenergic agonist nebule 2.5mg (Ventolin ) 2.ii. Dilution Equipment x1 5ml micropippete x16 5ml micropipette tips x9 sterile universal pots correctly labelled for each concentration of citric acid/saline x9 sterile dosimeter pots labelled so that only the operator can identify the concentration of citric acid within each pot gloves lab coat 2.iii. Storage Citric acid stored at room temperature in a tightly sealed container Saline diluent stored at room temperature β adrenergic agonist nebule (2.5mg) stored at room temperature 2.iv. Equipment A breath activated Mefar Dosimeter A sterile dosimeter mouth piece A Pariboy nebulizer with sterile chamber and mouth piece Masterscope system (Jaeger) with sterile mouth piece - Appendix 1 45

194 CONFIDENTIAL BL2000/00007/00 3. Preparation for testing 3.i. Subject preparation before testing a. Explain the test to the subject. Subjects should be told that they might suffer severe bouts of coughing and that they may experience some minor symptoms such as chest tightness or breathlessness. Care should be taken to ensure that the test description does not bias the result. b. Ask the subject if they would like to urinate before the test (stress incontinence could be precipitated, especially in older women). c. Evaluate the subject for contraindication (APPENDIX 2) and review medication use, list of medications affecting the cough challenge test are in APPENDIX 2. 3.ii. Calibration Before starting the test make sure both the Mefar Dosimeter system and the Jaeger Masterscope are calibrated to the standard procedures outlined in SOP no: CTU60100 and SOP no: CTU30200 respectively. 3.iii. Solution Preparation 1. Label 9 universal pots Follow the dilution sequence below, ensuring that each concentration of citric acid is in the correctly numbered universal pot and that a new pipette tip is used to make each concentration of citric acid. After addition of sodium chloride the lid is replaced and the solution is adequately mixed. 3. Using a sterile pipette tip for each citric acid solution remove 3ml of each citric acid solution into a sterile correctly numbered dosimeter pot. 4. Remove 3ml of 0.9% sodium chloride solution into pots 8 and 9 again using sterile pipette tips. 46

195 CONFIDENTIAL BL2000/00007/00 Dosimeter Citric Acid Vol. 0.9% NaCl [Citric Acid] Pot No. Solution Volume (ml) Generated used for dilution (ml) (mm) mM) (30mM) (100mM) (300mM) (1000mM) (1000mM) stock(1000mm)

196 CONFIDENTIAL BL2000/00007/00 4. Performing the test 4.i. The subject a. Subjects must be able to understand the procedure and perform reliable spirometric Manoeuvres b. Subjects should be seated comfortably throughout the test 4.ii. Test sequence 1. In the main menu click on the icon to allow you to input subject data 2. Enter relevant details of subject, include date of birth and a reference No: to specifically identify the subject. 3. Once entered subject data press F10 which will take you back to the main menu. 4. Now click the icon which allows you to perform a flow-volume measurement 5. Ask the subject to perform a flow volume loop- which will allow measure of both FEV1 and FVC (performing flow/volume loops SOP no: CTU20100). 6. The subject should perform at least two flow volume loops and the second loop FEV1 and FVC should be within 5% of the first recording, if not repeat flow-volume loops until achieve reproducibility but if over 8 attempts are taken do not proceed with test. 7. After each flow volume curve press the icon to end the test and calculate parameters and percentages 8. The Masterscope system will highlight the best performed FEV1 for you and this will act as the baseline measurement. 9. End flow-volume test by pressing key F Set the dosimeter at: 1.0 second inhalation time 30 seconds pause time 04 inhalations 48

197 CONFIDENTIAL BL2000/00007/00 Results Table Flow volume loop Box highlighted for best FEV1 Figure 1 - shows screen view when performing flow-volume loop 11. Place the sterilised mouthpiece on the dosimeter pot 8 and after attaching the air tubing to the bottom of the pot and the air sensor to the end of the mouthpiece and pass to the subject. 12. Instruct the subject to exhale to functional residual capacity (FRC) and then to inhale slowly for 1 second, through the mouthpiece until the dosimeter stops bleeping. 13. Count the number of coughs in the first ten seconds after inhalation of the dosimeter pot contents. 14. Wait the 30 seconds pause time 15. Repeat steps 12-14, 3 more times. 16. Repeat with each of the dosimeter pots starting with the lowest citric acid concentration (pot 1) and increasing to the highest (pot 7). 17. Randomly intersperse pot number 9 amongst the increasing doses of citric acid. 18. Log-dose response curves are used to obtain the D2 (concentration of citric acid causing 2 coughs/inhalation) and D5 (concentration of citric acid causing 5 coughs/inhalation). 19. Wait 5 minutes after completing the test and then repeat steps Now calculate whether this measure of FEV1 is within 5% of the reference FEV1 if so then the subject has completed the test and is safe to go home. If not follow the procedures outlined in SOP No: CTU

198 CONFIDENTIAL BL2000/00007/00 Masterscope This allows the measurement of important parameters for the determination of dynamic and static lung volumes. These are the parameters of slow (ERV, VCIN, VCEX) and forced FVC<FEV1, MEF50) breathing manoeuvres as well as the maximum voluntary ventilation (MVV). The standard measurement programs are Spirometry/Flow-volume (ERV, VCIN, FEV1, MEF50, MVV 50