Conditions: Asthma
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 23
Healthy volunteers: no

Brief summary:
This is a prospective, active-controlled, randomized, open label, single-center, multiple dose, two-period crossover clinical trial to assess the efficacy, safety and pharmacokinetics of AQ001S compared to a budesonide inhalation suspension (comparator) in adults with mild asthma.

Both treatments will be administered by nebulization.

Detailed description:
Adults patients with mild asthma (18 to 65 years old), who are 'inhaled corticosteroid (ICS)'-naïve for minimum 60 days at Screening Visit will be enrolled in the study. The patients will be treated for 28 days. The primary endpoint will be assessed by a provocative concentration of methacholine that results in a 20% drop (PC20) in the first second forced expiratory volume (FEV1) as determined by methacholine challenge test. The pharmacokinetics (PK) endpoint, i.e. PK profile of budesonide in plasma, and pharmacodynamics (PD) endpoints, i.e. recording of symptom scores, use of reliever drugs as needed, biomarkers of airway inflammation and pulmonary function tests will be assessed during the study.

Arms:
arm 1: AQ001S 0.125 mg/ml is a budesonide inhalation solution administered by nebulization once daily. arm 2: Budesonide 0.125 mg/ml is a budesonide inhalation suspension administered by nebulization once daily.

Interventions: intervention 1: AQ001S 0.125 mg/ml intervention 2: Budesonide 0.125 mg/ml inhalation suspension
Intervention descriptions: intervention 1: administered by nebulization once daily intervention 2: administered by nebulization once daily

Protocol document:
Prot_000.pdf: 






 
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2. SYNOPSIS 
 
Clinical trial code 
BOREAS  
Sponsor 
Aquilon Pharmaceuticals S.A.  
Investigational medicinal 
product (IMP) under 
investigation 
AQ001S 0.125 mg/ml (budesonide 0.125 mg/ml inhalation solution) 
Title 
A prospective, active-controlled, randomized, open label, single-center, multiple 
dose, two-period crossover clinical trial to assess the efficacy, safety and 
pharmacokinetics of a budesonide inhalation solution (AQ001S) compared to a 
budesonide inhalation suspension (comparator) in adults with mild asthma. 
Phase  
Phase 1/2a 
First-In-Human 
Proof-Of-Concept 
Primary Objective - 
Efficacy  
To compare the efficacy, i.e. the bronchoprotection of AQ001S 0.125 mg/ml with 
the comparator. 
Safety Objective  
To assess the safety of AQ001S 0.125 mg/ml.  
Secondary Objectives  
1. To compare the pharmacokinetics (PK) of AQ001S 0.125 mg/ml with the 
comparator 
2. To compare the secondary Efficacy/Pharmacodynamics (PD) of AQ001S 
0.125 mg/ml with the comparator. 
Trial design 
Prospective, active-controlled, randomized, open label, single-center, multiple 
dose, two-period crossover clinical trial 
Trial population 
Adults with mild asthma (18 to 65 years old), who are ‘inhaled corticosteroid 
(ICS)’-naïve for minimum 60 days at Screening Visit. 
A total of 24 subjects will be recruited into the clinical trial. Considering potential 
dropouts, a total of 20 subjects are anticipated to complete the clinical trial. 
Trial location 
This clinical trial will be conducted in 1 trial site in Belgium. 
Main Inclusion Criteria 
 
1. Reliable subjects who are willing to be available for the duration of the 
clinical trial and willing to comply with clinical trial procedures. 
2. Subjects who have the ability to understand the requirements of the 
clinical trial.  
3. Subjects who have given written informed consent. 
4. Subjects aged between 18 and 65 years, inclusive. 
5. Body mass index between 18.5 and 29 kg/m2. 
6. Documented clinical diagnosis of stable, persistent, asthma for at least 3 
months, i.e.: 
• 
for whom forced expiratory volume in one second (FEV1) ≥ 
70% of predicted, and 
• 
treated with as-needed reliever medication (short-acting beta2-
agonist-containing medication) only.  
7. Subjects who are ICS-naïve for minimum 60 days at Screening Visit.  
8. Positive methacholine (MCh) challenge test (concentration of MCh 
provoking an FEV1 fall of 20% [PC20] < 8 mg/ml or dose of MCh 
provoking an FEV1 fall of 20% [PD20] < 0.2 mg) in the last year. 
9. Post-bronchodilator FEV1 at least 80% of the predicted, documented in 
the last year.  
10. Clinical laboratory test results, 12-lead electrocardiogram (ECG), blood 
pressure and heart rate (supine) within normal reference range or judged 
to be not clinically significant by the Investigator. 
11. Female subjects of childbearing potential should have a negative 
pregnancy test at Screening Visit. 
12. Female subjects of childbearing potential using a highly effective method 
of contraception (i.e., pregnancy rate of < 1% per year) on a stable 
regimen, for at least 28 days, and pursuing this contraception during the 
trial and for 28 days after the last administration of the IMP. The highly 
effective methods of contraception must be one of the following: 


 
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combined estrogen and progestogen hormonal contraception with 
inhibition of ovulation, progestogen-only hormonal contraception 
associated with inhibition of ovulation, intrauterine device, intrauterine 
hormone-releasing system, bilateral tubal occlusion, vasectomized 
partner, or agreement on continuous abstinence from heterosexual 
intercourse. 
Main Exclusion criteria 
 
1. Current smokers or recent (< 8 weeks) ex-smokers or ex-smokers if > 10 
pack-years. 
2. Pregnant or breastfeeding female subjects.  
3. Inability to carry out pulmonary function testing.  
4. FEV1 < 70%. 
5. History of near-fatal asthma and/or intensive care unit admission for 
asthma symptoms. 
6. Exacerbations of asthma requiring oral steroids, hospitalization or change 
in asthma treatment in the previous three months.  
7. Evidence of symptomatic chronic or acute respiratory infection in the 
previous 8 weeks. 
8. Diagnosis of chronic obstructive pulmonary disease (COPD) or 
bronchiectasis. 
9. Pulmonary malformations, tuberculosis, cystic fibrosis. 
10. History of hypersensitivity or existing contraindication to budesonide or 
any other IMP ingredients. 
11. Untreated oral candidiasis.  
12. Immunosuppressive treatment, including systemic corticosteroids (e.g., 
oral, parenteral, ocular, nasal), within 28 days before Screening Visit. 
13. Use of ICS within 60 days before Screening Visit. 
14. Use of anti-leukotrienes, immunoglobulins, beta-blockers, digitalis, 
amiodarone, antifungals, macrolides, antidepressants, monoamine 
oxidase inhibitors, antiretroviral drugs, cholinesterase inhibitors, 
histamine, theophylline, non-steroidal anti-inflammatory drugs, 
anticholinergic drugs, neuroleptics, curariform drugs, antihistaminic 
(anti-H1) drugs, calcium channel blockers, long acting beta2-antagonists, 
mast cell stabilizers (e.g. natrium cromoglycate). 
15. History of alcohol or drug abuse. 
16. Unstable or life-threatening cardiac disease: subjects with any of the 
following would be excluded: 
• 
Myocardial infarction or unstable angina in the 6 months before 
Screening Visit, 
• 
Unstable or life-threatening cardiac arrythmia requiring 
intervention in the 3 months prior to Screening Visit, 
• 
New York Heart Association class IV Heart failure. 
17. History or presence of prolonged QT interval (> 470 ms), or any other 
clinically significant ECG abnormalities as judged by the Investigator 
based on 12-lead ECG recordings at Screening Visit. 
18. Diabetes mellitus. 
19. Neuropsychiatric diseases. 
20. Clinically relevant laboratory abnormalities at Screening Visit.  
21. Blood or plasma donation within 30 days prior to Screening Visit. 
22. History or presence of malignancy of any system organ class (other than 
localized basal cell carcinoma of the skin), treated or untreated, within the 
past 5 years prior to Screening Visit, regardless of whether there is 
evidence of local recurrence or metastases. 
23. Any surgical or medical condition which might significantly alter the 
absorption, distribution, metabolism, or excretion of drugs, or which may 
jeopardize the subject in case of participation in the clinical trial.   
24. History or presence of any other clinically relevant disease of any major 
system organ class (e.g. cardiovascular, pulmonary, renal, hepatic, 




 
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difference of the parameter between AQ001S 0.125 mg/ml and comparator. If this 
p-value is significant and the difference prefers AQ001S 0.125 mg/ml, superiority 
is shown. 
Additionally, an adjusted 95%-confidence interval for the mean difference between 
AQ001S 0.125 mg/ml and the comparator will be obtained by the ANCOVA. 
For the safety analysis, the treatment groups will be compared in a descriptive way. 
Tabulations of frequencies/proportions will be used for the evaluation of categorical 
(qualitative) data, and tabulations of descriptive statistics (at least number of 
observations, mean, standard deviation, median, minimum and maximum) will be 
used to analyze continuous (quantitative) data. 
 






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Procedures 
Periods 
Screenin2 
Treatment period 1 (TPl) 
Treatment period 2 (TP2) 
Visit 1 
Visit 4 
Visits1 
Pre-Tt·ial 
Screening 
(Randomi-
Visit 2 
Washout 
Visit 3 
(End-of-
Visit 
Visit 
zation and 
Baseline) 
Trial}2 
Timing of visits 
BetweenD-
Bet:weenD-7 
TPl DO and 
TPl D28 
TP2 DO and 
TP2D28 
67 (-2) and 
14 (+2) days 
Dl 
(treatment day) 
D-53 
and D-5 
Dl 
(+2) 
(+2) 
PK sampling 
x16 
x11 
x16 
x11 
Ovemight stay of subiect at trial site 
X 
X 
IMP dispensing 
X 
X 
Retum of IMP and dmg accountabilitv 
X 
X 
Subiect diary dispensing18 
X 
Subiect diary retum and review 
X 
X 
X 
X 
(Serious) Adverse Events19 assessment 
X 
X 
X 
X 
Local tolerabilitv20 
X 
X 
X 
X 
Subseauent theraov counselling 
X 
16 Sixteen (16) blood samples over 24 hours (sampling at the clinical site), including I pre-dose before IMP administration and 15 post-dose at     ,     ,    , 20, 30, 45, 60, 90,
120, 180, 240, 360 minutes and IO, 18, 24 hours after IMP administration (full single 24-hour PK). 
17 Seven (7) blood samples taken over 6 hours (sampling at the clinical site on D28[+2]), including I pre-dose before IMP administration and 6 post-dose at     ,     ,    , 120,
240 and 360 minutes after IMP administration (abridged PK). 
18 Trial subject diaries include daily day- and night-time asthma symptom questionnaires, weekly ACQ-5 and monthly ACT questionnaires, information about as-needed
reliever medication (SABA) and other concomitant medication use. 
19 
Whenever a (serious) adverse event takes place. 
20 
Through physical and clinical examination: increased bronchial irritability, paradoxical bronchospasm, oropharyngeal examination by the Investigator (physician) ( e.g. 
vocal cord myopathy, oral fungal infection). 
ACQ = asthma control questionnaire; ACT = asthma control test; D = treatment day, DO being the first IMP administration of the treatment period; ECG = electrocardiogram; 
FeNO = fractional concentration of exhaled nitric oxide; FEV1= forced expiratory volume at 1 second; FIVC = forced inspiratory vital capacity; FVC = forced vital capacity; 
ICS = inhaled corticosteroid; ID = identification (nUlllber); IMP = investigational medicinal product; MCh = methacholine (chloride); PC20 = concentration ofMCh provoking 
an FEV1 fall of20%; PD = pharmacodynamics; PD20 = dose ofMCh provoking an FEV1 fall of20%; PK = pha1macokinetics; SABA = sho1t-acting beta2-agonist 
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4. TABLE OF CONTENTS, FIGURES AND TABLES 
CONTENTS 
1. 
CLINICAL TRIAL SUMMARY INFORMATION .............................................................. 2 
2. 
SYNOPSIS ................................................................................................................................. 4 
3. 
FLOW CHART OF TRIAL PROCEDURES ......................................................................... 8 
4. 
TABLE OF CONTENTS, FIGURES AND TABLES .......................................................... 11 
5. 
ACRONYMS AND ABBREVIATIONS ............................................................................... 16 
6. 
INTRODUCTION ................................................................................................................... 18 
6.1. 
BACKGROUND INFORMATION ...................................................................................... 18 
6.2. 
RATIONALE FOR CONDUCTING THE CLINICAL TRIAL .................................................. 20 
6.3. 
BENEFIT-RISK STATEMENT ......................................................................................... 21 
 
Benefits and Risks Related to the Investigational Medicinal Products
 
21 
 
Benefits and Risks Related to the Trial Procedures ........................... 22 
 
Benefit-Risk Conclusion ..................................................................... 22 
7. 
CLINICAL TRIAL PURPOSE AND OBJECTIVES .......................................................... 23 
7.1. 
PURPOSE OF THE CLINICAL TRIAL ............................................................................... 23 
7.2. 
PRIMARY OBJECTIVES ................................................................................................. 23 
7.3. 
SECONDARY OBJECTIVES ............................................................................................ 23 
8. 
INVESTIGATIONAL PLAN ................................................................................................. 23 
8.1. 
PRIMARY AND SECONDARY PARAMETERS .................................................................. 23 
 
Primary Efficacy Parameter .............................................................. 23 
 
Safety Parameters .............................................................................. 23 
 
Pharmacokinetic Parameters ............................................................. 24 
 
Secondary Pharmacodynamic/Efficacy Parameters .......................... 24 
8.2. 
OVERALL CLINICAL TRIAL DESIGN AND PLAN ............................................................ 24 
8.3. 
DISCUSSION ON CLINICAL TRIAL DESIGN AND CHOICE OF CONTROL GROUP(S) ....... 25 
 
Justification of Clinical Trial Design ................................................. 25 
 
Choice of Control Group(s) ............................................................... 26 
 
Choice of Clinical Trial Parameters .................................................. 26 
 Primary Efficacy Parameter ........................................................... 26 
 Safety Parameters ........................................................................... 27 
 Pharmacokinetic Parameters .......................................................... 27 
 Secondary Pharmacodynamic/Efficacy Parameters and Techniques
 
28 
8.4. 
CLINICAL TRIAL POPULATION ..................................................................................... 28 
 
Population Base ................................................................................. 28 
 
Inclusion Criteria ............................................................................... 28 
 
Exclusion Criteria .............................................................................. 29 
 
Screening Failures ............................................................................. 30 
 
Subject Identification ......................................................................... 30 
8.5. 
INVESTIGATIONAL MEDICINAL PRODUCTS ................................................................. 30 
 
Treatments .......................................................................................... 30 
 
Name and Description of Investigational Medicinal Products .......... 31 


 
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Route of Administration ..................................................................... 31 
 
Packaging and Labelling ................................................................... 31 
 
Conditions for Storage and Use ......................................................... 32 
 
Dosage and Dosage Regimen ............................................................ 32 
 
Preparation and Method of Administration ....................................... 32 
8.6. 
AUXILIARY MEDICINAL PRODUCT USED IN THE CLINICAL TRIAL .............................. 33 
 
Name and Description of Auxiliary Medicinal Product ..................... 33 
 
Labelling of Auxiliary Medicinal Product ......................................... 33 
 
Traceability and Destruction of Auxiliary Medicinal Product .......... 33 
8.7. 
MEDICAL DEVICE USED IN THE CLINICAL TRIAL ........................................................ 33 
8.8. 
PRIOR TREATMENTS AND CONCOMITANT MEDICATIONS ........................................... 33 
 
Prior and Concomitant Medication ................................................... 33 
 
Forbidden and As-Needed Reliever Medication ................................ 34 
8.9. 
ASSIGNMENT OF SUBJECTS TO TREATMENT GROUPS.................................................. 34 
8.10. 
BLINDING AND EMERGENCY CARDS ........................................................................... 34 
8.11. 
TREATMENT COMPLIANCE........................................................................................... 35 
8.12. 
DRUG DISPENSING AND ACCOUNTABILITY ................................................................. 35 
9. 
CLINICAL TRIAL CONDUCT ............................................................................................ 35 
9.1. 
PROCEDURES BY VISIT ................................................................................................. 36 
 
Pre-Trial Visit .................................................................................... 36 
 
Screening Visit ................................................................................... 36 
 
Visit 1 – Randomization and Baseline ............................................... 37 
 
Visit 2 ................................................................................................. 39 
 
Visit 3 ................................................................................................. 40 
 
Visit 4 – End-of-Trial ......................................................................... 41 
 
Early Termination Visit ...................................................................... 41 
 
Unscheduled Visit .............................................................................. 42 
 
Follow-up Contact ............................................................................. 42 
9.2. 
ASSESSMENTS AND THEIR APPROPRIATENESS ............................................................ 42 
 
Safety Assessments ............................................................................. 42 
 Assessments for Safety Events ......................................................... 42 
9.2.1.1.1. Adverse Events and Adverse Reactions........................................ 42 
9.2.1.1.2. Serious Adverse Events ................................................................ 45 
9.2.1.1.3. Other Relevant Safety Information .............................................. 46 
 Clinical Laboratory Tests ............................................................... 47 
9.2.1.2.1. Hematology, Biochemistry, Urinalysis and Pregnancy Tests ...... 47 
9.2.1.2.2. Amounts and Combination of Blood and Urine Collections ........ 47 
9.2.1.2.3. Assessment of Hypothalamic Pituitary Adrenocortical Axis 
Function 
48 
 Physical and Clinical Examination................................................. 48 
 Vital Signs ....................................................................................... 49 
 Twelve-Lead Electrocardiogram .................................................... 49 
 Local Tolerability............................................................................ 49 
 Spirometry ....................................................................................... 50 
 Administration of As-Needed Reliever Medication ......................... 50 
 
Efficacy and Pharmacodynamic Assessments .................................... 50 
 Methacholine Challenge Test and PC20 ........................................ 50 
 Subject Diaries and Questionnaires ............................................... 52 
 Spirometry ....................................................................................... 52 
 Fractional Concentration of Exhaled Nitric Oxide ........................ 52 
 Blood Eosinophil Count .................................................................. 53 
 
Pharmacokinetic Assessments ............................................................ 53 


 
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 Single Dose 24-Hour PK Sampling ................................................ 53 
 Abridged PK Sampling ................................................................... 53 
 Processing and Bioanalysis of the PK Samples .............................. 54 
 Pharmacokinetic Parameters .......................................................... 54 
 
Other Assessments .............................................................................. 55 
 Demographics ................................................................................. 55 
 Medical and Surgical History ......................................................... 55 
 Respiratory and Asthma History ..................................................... 55 
 Prior/Concomitant Medications ..................................................... 55 
9.2.4.4.1. Medication History – Prior Medication Use ............................... 55 
9.2.4.4.2. Concomitant Medication Use ....................................................... 56 
 Subsequent Therapy Counseling ..................................................... 56 
10. 
TRIAL MANAGEMENT ....................................................................................................... 56 
10.1. 
RESPONSIBILITIES ........................................................................................................ 56 
 
Investigator(s) .................................................................................... 56 
 
Information to Investigator(s) ............................................................ 56 
10.2. 
DURATION OF THE CLINICAL TRIAL ............................................................................ 57 
 
Planned Duration for an Individual Subject ...................................... 57 
 
Time Windows Used in this Trial Including Tolerances .................... 57 
 
Planned duration for the clinical trial as a whole ............................. 58 
10.3. 
PROTOCOL AMENDMENTS ........................................................................................... 58 
10.4. 
PROTOCOL DEVIATIONS............................................................................................... 58 
10.5. 
WITHDRAWAL AND REPLACEMENT OF SUBJECTS ....................................................... 58 
 
Premature Subject Withdrawal .......................................................... 58 
 
Subject Replacement Policy ............................................................... 59 
10.6. 
TERMINATION OF THE CLINICAL TRIAL ....................................................................... 59 
 
Premature Termination of the Clinical Trial ..................................... 59 
 Early Termination of the Clinical Trial ........................................ 59 
 Early Termination of Trial Site ..................................................... 59 
10.7. 
DATA HANDLING AND RECORD KEEPING ........................................................ 60 
 
Source Data and Records .................................................................. 60 
 
Case Report Forms ............................................................................ 60 
 
Investigator’s Site File ....................................................................... 61 
 
Provision of Additional Information .................................................. 61 
 
Safety Monitoring ............................................................................... 61 
11. 
STATISTICAL METHODS AND SAMPLE SIZE ............................................................. 61 
11.1. 
DETERMINATION OF SAMPLE SIZE............................................................................... 61 
 
Calculation Option 1 (Lipworth) ....................................................... 62 
 
Calculation Option 2 (Kraan) ............................................................ 62 
 
Sample Size Results ............................................................................ 64 
11.2. 
RANDOMIZATION, STRATIFICATION AND CODE RELEASE .......................................... 64 
11.3. 
STATISTICAL ANALYSIS ............................................................................................... 64 
 
Populations for Analysis .................................................................... 65 
 
Primary Efficacy Endpoint and Analysis Plan ................................... 65 
 
Safety Endpoints and Analysis Plan ................................................... 65 
 
Pharmacokinetic Endpoints and Analysis Plan ................................. 66 
 
Secondary Pharmacodynamic/Efficacy Endpoints and Analysis Plan
 
66 
 
Handling of Data and Specific Analyses ............................................ 66 
 Handling of Dropouts or Missing Data ........................................ 66 
 Adjustments for Covariates ........................................................... 67 




 
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APPENDICES 
 
APPENDIX 1: PARI LC SPRINT NEBULIZER – INSTRUCTIONS FOR USE .................. 73 
APPENDIX 2: PARI TURBOBOY SX COMPRESSOR – INSTRUCTIONS FOR USE ..... 74 
APPENDIX 3: LABORATORY TESTS (INCLUDING OPTIONAL URINALYSIS AND 
URINARY CORTISOL/CREATININE RATIO) ................................................................... 75 
 
 


 
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5. ACRONYMS AND ABBREVIATIONS 
ACT 
Asthma Control Test 
ACQ 
Asthma Control Questionnaire 
AE  
Adverse Event 
ANCOVA 
Analysis of Covariance 
AR  
Adverse Reaction 
AxMP 
Auxiliary Medicinal Product 
BE 
Belgium 
COPD 
Chronic Obstructive Pulmonary Disease 
CRO  
Contract Research Organization 
(e)CRF  
(electronic) Case Report Form 
ECG 
Electrocardiogram 
EDC  
Electronic Data Capture 
EFF 
Efficacy population 
FeNO 
Fractional exhaled Nitric Oxide 
FEV1 
Forced Expiratory Volume in 1 second 
FIH 
First-In-Human 
FIVC 
Forced Inspiratory Vital Capacity 
FVC 
Forced Vital Capacity 
GCP  
Good Clinical Practice 
GDPR 
Global Data Protection Regulation 
GINA 
Global Initiative for Asthma 
HIV 
Human Immunodeficiency Virus 
HPA 
Hypothalamic Pituitary Adrenocortical 
HP-Betadex 
2-hydroxypropyl-β-cyclodextrin 
IB 
Investigator’s Brochure 
ICS 
Inhaled Corticosteroid 
ID 
Identification (number) 
IEC  
Independent Ethics Committee 
IFU 
Instructions For Use 
IMP  
Investigational Medicinal Product 
IRB  
Institutional Review Board 
LABA 
Long-Acting Beta2-Agonist 
LAMA 
Long-Acting Muscarinic Antagonist 
MCh 
Methacholine (chloride) 
ms 
Millisecond(s) 
PC20 
Concentration of methacholine provoking an FEV1 fall of 
20% 
PD 
Pharmacodynamics 
PD20 
Dose of methacholine provoking an FEV1 fall of 20% 
pH 
hydrogen Potential 
PK  
Pharmacokinetics 


 
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POC 
Proof-Of-Concept 
QD 
quaque die (once daily) 
SABA 
Short-Acting Beta2-Agonist 
SAE  
Serious Adverse Event 
SAF 
Safety population 
SAP 
Statistical Analysis Plan 
SARS-CoV-2 
Severe acute respiratory syndrome coronavirus 2 
SD 
Standard Deviation 
SDV 
Source Data Verification 
SmPC 
Summary of Product Characteristics 
SUSAR  
Suspected Unexpected Serious Adverse Reaction 
US(A) 
United States (of America) 
 
 
 


 
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6. INTRODUCTION 
6.1. Background Information 
Asthma is a chronic inflammation of the airways that causes coughing, chest tightness, wheezing or 
shortness of breath. The disease is mainly due to genetic (inherited) and/or environmental factors such 
as allergens, air pollutants and irritants like smoke. Respiratory infections and physical activity can also 
trigger asthma [1]. 
 
To improve asthma prevention and management, the “Global Initiative for Asthma” (GINA), a network 
of public health officials and health care professionals created by the American National Lung Institute 
and the World Health Organization, have released the GINA strategy, i.e. a global consensus on how to 
treat asthma (last version: 2019) [2]. Every practitioner worldwide is invited to follow the recommended 
step-up approaches (summarized in Figure 1) to select the most appropriate treatment according to 
patient’s disease severity, disease control and age category. In brief, asthma maintenance treatment is 
mainly based on lung inflammation control by anti-inflammatory drugs, in particular inhaled 
corticosteroids (ICS), and on airway opening with bronchodilator drugs (mainly long-acting beta2-
agonists [LABAs]) from low to high doses according to the stage of the disease. In case of acute crisis, 
low dose ICS and LABA combinations are now the preferred as-needed reliever medication beside the 
traditional short-acting beta2-agonist (SABA) bronchodilators. When the disease is very severe, other 
types of maintenance treatment should be added, i.e. long-acting muscarinic antagonists (LAMAs) 
bronchodilators or oral corticosteroids. 
 
Figure 1 - Summary of recommended treatment approaches for asthma  
 
Source: GINA report 2019 [2] 
BDP: beclometasone dipropionate; bud: budesonide; FEV1= forced expiratory volume at 1 second; form: 
formoterol; GINA: Global Initiative for Asthma; HDM SLIT: house dust mite sublingual immunotherapy; ICS: 
inhaled corticosteroid; IgE: immunoglobulin E; anti-IL(R): anti-interleukin (receptor); LABA: long-acting beta2-
agonist; LTRA: leukotriene receptor antagonist; OCS: oral corticosteroid; SABA: short-acting beta2-agonist. 
 
 
The majority of these treatments are administered by inhalation. Despite the established efficacy of ICS 
in asthma treatment, the lung deposition of currently marketed reference therapies is low and 


 
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heterogeneous. In case of administration by nebulization, on average, only 10% of the dose released 
from the traditional jet nebulizers will reach the site of action, i.e. deep lung areas [3–5]. This leads to 
decreased treatment efficacy and higher ICS doses are usually prescribed to compensate these 
drawbacks. These higher ICS doses increase the risk of local and systemic dose-dependent side effects. 
Side effects of ICS, in particular local ones such as hoarseness, throat irritation and oral fungal infection, 
are common in clinical practice due to the usual chronic administration of ICS [2,6,7].  
Budesonide is an ICS approved since 1981 [8] and marketed in liquid and powder formulations intended 
for inhalation to treat asthma (Pulmicort Respules®, Turbohaler®, Flexhaler® and generics) [9–15]. 
Budesonide is recommended by the international and national regulatory authorities as a first-line 
maintenance therapy for asthma management in adult and pediatric patients and its safety is well 
established [16–20]. 
Currently, the only budesonide liquid formulation for nebulization available on the market is an aqueous 
suspension (Pulmicort Respules® and generics), i.e. a heterogenous mixture containing solid particles, 
because of budesonide’s lipophilic properties and low solubility in water [10,12,14]. Suspensions can 
only be nebulized with traditional jet nebulizers [10,11,14,21–23]. The more recent nebulizers, 
especially the vibrating mesh nebulizers, which offer several benefits compared to the jet ones (e.g. 
hand-held, battery-driven, shorter time of administration) are not compatible with suspensions because 
of their specific technologies [24–26] and can only be used with aqueous solutions, i.e. homogenous 
mixtures where particles have been totally dissolved. Hence patients treated with suspension 
formulations, e.g. budesonide suspensions, cannot benefit from the advantages offered by more recent 
nebulizers. 
The low solubility of budesonide can therefore create technical difficulties and, consequently, jeopardize 
treatment efficacy and safety, which could have been improved through the use of these more recent 
nebulizers. 
 
Aquilon uses 2-hydroxypropyl-β-cyclodextrin (HP-Betadex) as a new excipient for inhaled drugs to 
solubilize budesonide, as both molecules form together a molecular inclusion complex. The resulting 
formulation is a homogenous budesonide inhalation solution (AQ001S) [27–29]. HP-Betadex is a cyclic 
oligosaccharide able to form water-soluble inclusion complexes of lipophilic water-insoluble drugs. HP-
Betadex is used to this intent in marketed oral, ocular, parenteral and rectal products in Europe and in 
the US (on the market: Sporanox®, Indocollyre®; withdrawn: Propulsid®, Mitozytrex®). Moreover, 
an orphan drug designation has been issued since 2009 for the compassionate use of HP-Betadex at high 
dosages (intrathecal and intracerebroventricular administration) [30–34]. However, the use of HP-
Betadex in inhaled drugs is new. To assess its safety and potential activity when administered by 
inhalation, HP-Betadex has been tested in 2 clinical trials (phase 1 and phase 2) by inhalation. This study 
showed that HP-Betadex administered by inhalation (nebulization) in multiple doses is proved to be safe 
and well-tolerated [35,36]. Preclinical toxicology studies on HP-Betadex and AQ001S in rats did not 
point out any safety concerns for the intended clinical dose strengths (also further described and 
summarized in AQ001S’ Investigator’s Brochure [IB]). 
 
In vivo Proof-Of-Concept (POC) studies performed by Aquilon in experimental murine asthma models 
have shown that intranasal administration of AQ001S has higher efficacy in reducing peribronchial 
allergen-induced inflammation than a budesonide suspension given intranasally at the same dose 
[28,37]. Indeed, AQ001S showed the same efficacy against several asthma outcomes as Pulmicort 
Respules®, but at lower budesonide doses. At the same budesonide doses, AQ001S efficacy against 
asthma symptoms was higher than Pulmicort Respules®. Hence, budesonide complexation by 
HP-Betadex increases budesonide efficacy in these animal models of asthma. This could lead, in 
humans, to a reduction of the budesonide dose required to obtain efficacy against asthma symptoms, 
and therefore to a decrease of patient exposure to ICS. 
 
These promising data open new therapeutic perspectives in asthma management, i.e.: 
- 
the possibility to use AQ001S with any nebulizer device in contrast to budesonide suspensions,  






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The benefits of AQ001S versus the currently marketed budesonide liquid formulations, that may rely 
on the use of the HP-Betadex excipient, are described in Section 6.1, i.e. the possibility to use AQ001S 
with any nebulizer device and a decrease of patient exposure to ICS with a reduction of ICS-related 
AEs. The use of HP-Betadex in inhaled drugs is new. The nonclinical, clinical and toxicology studies 
conducted with inhaled HP-Betadex and AQ001S do not point out any safety concerns for the intended 
clinical dose strengths (also further described and summarized in AQ001S’ IB). 
However, safety is an endpoint of the present clinical trial and the low dose chosen is a risk minimization 
measure. 
The potential benefits and risks of AQ001S are further described and summarized in AQ001S’ IB The 
potential benefits and risks of Budesonide                        inhalation suspension (comparator) are 
reviewed in its summary of product characteristics (SmPC) 
The dose strengths of both IMPs that will be administered during this clinical trial are low and chosen 
on purpose to demonstrate AQ001S’ superiority versus the comparator. Even if 0.125 mg QD 
budesonide inhalation suspension, i.e. the comparator, is not efficient, this should have no negative 
impact on the asthma control of the mild asthmatic subjects that will be selected to participate in this 
clinical trial. Moreover, the clinical trial allows the subjects to continue their usual treatment with as-
needed reliever medication (SABA only). 
 Benefits and Risks Related to the Trial Procedures 
Methacholine (MCh) challenge testing will be performed in this clinical trial to determine the primary 
efficacy parameter PC20 (concentration of MCh provoking a fall of 20% of the forced expiratory volume 
in 1 second [FEV1]), with the use of MCh as a provocative agent (Section 9.2.2.1).  
Methacholine is a classical provocative agent used in asthma diagnosis and the related safety measures 
are well described [41] and part of the routine procedures of the trial site. The trial subject will always 
be overseen by qualified site staff, under the responsibility of the Principal Investigator. After each 
challenge, spirometry assessments are scheduled (Section 9.2.1.7) to measure FEV1 and, along with the 
presence of asthma-related clinical symptoms (Section 9.2.1.6), to determine the need to administer as-
needed reliever medication (SABA) to the subject (if FEV1 < 70%; Section 9.2.1.8). In that case and at 
any time during the clinical trial, as-needed reliever medications will be available at trial site. 
The potential benefits and risks of the MCh (Provocholine®) used in this clinical trial are summarized 
in its technical information sheet. 
For the PK analyses, 16 blood samples will be taken at Visits 1 and 3, and 7 blood samples at Visits 2 
and 4. The total blood volumes, combined with those necessary for the clinical laboratory tests (Section 
9.2.1.2) and blood eosinophil count (Section 9.2.2.5), are mentioned in Section 9.2.1.2.2, i.e.: 
-
a maximum of 72.5 ml of blood taken per visit.
-
an estimated total maximum blood volume needed for a subject during the trial (when
completing the trial up until the last visit) of 260.5 ml, over 77 to 85 days.
These blood volumes are below the maximum allowable blood draws as recommended by: 
-
the Duke University Health System - Human Research Protection Program [42], i.e. for an
adult, not more than 5 ml/kg in any one 24-hour period, and 7 ml/kg in any 8-week period,
and
-
the Dana Farber Harvard Cancer Center [43], i.e. not more than 275 ml over a 28-day period.
The IMPs will be administered by nebulization with a Pari Boy Classic compressor combined with Pari 
LC Sprint nebulizer. The warnings and precautions of use related to these devices are described in their 
instructions for use (IFU) (Appendix 1 and Appendix 2). 
 Benefit-Risk Conclusion 




 
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• 
Adverse events (AEs) and serious adverse events (SAEs), including asthma exacerbations 
• 
General tolerability: vital signs, 12-lead electrocardiogram (ECG), physical examination 
• 
Laboratory parameters: hematology, biochemistry and urinalysis  
• 
Local tolerability:  
o Increased bronchial irritability  
o Paradoxical bronchospasm 
o Oropharyngeal examination (e.g. vocal cord myopathy, fungal infection) 
• 
Forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC), measured by 
spirometry  
• 
Assessment of hypothalamic pituitary adrenocortical (HPA) axis function: urinary 
cortisol/creatinine ratio in first waking urine. 
 
 Pharmacokinetic Parameters 
The PK profile of budesonide in plasma will be evaluated for each treatment period at: 
• 
Start of treatment period: 
o single dose 24-hour PK parameters at the time of the first scheduled IMP administration 
(Section 9.2.3.1 and 9.2.3.4). 
• 
End of treatment period: 
o abridged PK at the time of the last scheduled IMP administration (Section 9.2.3.1 and 
9.2.3.4). 
 Secondary Pharmacodynamic/Efficacy Parameters 
The secondary efficacy/PD will be evaluated using the following parameters: 
• 
Symptom scores will be recorded using day- and night-time asthma symptom scoring and 
validated asthma-specific Asthma Control Questionnaire (ACQ)-5 and Asthma Control Test 
(ACT) questionnaires (included in trial subject diary). 
• 
Use of as-needed reliever medication (reported in trial subject diary). 
• 
Airway inflammation PD biomarkers: fractional concentration of exhaled nitric oxide (FeNO) 
and blood eosinophil count. 
• 
FEV1 and forced inspiratory vital capacity (FIVC), measured by spirometry. 
 
8.2. Overall Clinical trial Design and Plan 
This is a prospective, active-controlled, randomized, open label, single-center, multiple dose, two-period 
crossover clinical trial in adults with mild asthma. This clinical trial is conducted in a single trial site in 
Belgium. Twenty-four (24) ICS-naïve for minimum 60 days, mild asthmatic adults (18 to 65 years old) 
will be recruited. Considering potential dropouts, a total of 20 subjects are anticipated to complete the 
clinical trial. 
 
Eligible subjects, i.e. fulfilling the criteria defined in Section 8.4 and having given their informed 
consent, will be randomized at Visit 1 in a 1 to 1 ratio to receive, for a first treatment period, one of the 
following IMPs: 
- 
AQ001S 0.125 mg/ml or 
- 
The comparator (0.125 mg/ml). 
All subjects will receive the randomly allocated IMP for 29 (+2) days. After a 14 (+2) days washout 
period, the subjects will receive the other IMP for a second treatment period of 29 (+2) days (crossover 
design).  
 
For each subject, the clinical trial will last 77 to 85 days from Screening Visit, including two treatment 
periods of 29 (+2) days and one washout period of 14 (+2) days, between the treatment periods.  








 
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  Secondary Efficacy/Pharmacodynamic Parameters and Techniques 
Besides the primary efficacy parameter, classical asthma efficacy/PD parameters will be evaluated in 
the BOREAS clinical trial: 
- 
Symptom scores will be recorded through the use of day- and night-time asthma 
symptom scoring and validated asthma-specific ACQ-5 and ACT questionnaires 
(included in trial subject diary) [45]. 
- 
Use of as-needed reliever medication (reported in trial subject diary) [45]. 
- 
Airway inflammation PD biomarkers: FeNO [45] and blood eosinophil count. 
- 
FEV1 and FIVC, measured by spirometry [45]. 
The blood eosinophil count (Section 9.2.2.5) is a surrogate biomarker of eosinophilic airway 
inflammation, a common feature of specific asthma phenotypes. Although less sensitive, it has been 
preferred to the sputum eosinophil count [45] as it has been reported to be less variable and less labor 
intensive for the subject than the sputum collection procedure [50]. 
 
8.4. Clinical Trial Population 
 Population Base 
Adults with mild asthma (18 to 65 years old), who are ICS-naïve for minimum 60 days at Screening 
Visit. 
 
 Inclusion Criteria 
Potential trial subjects may be entered in the clinical trial if they meet all of the following criteria: 
1. Reliable subjects who are willing to be available for the duration of the clinical trial and willing 
to comply with clinical trial procedures. 
2. Subjects who have the ability to understand the requirements of the clinical trial.  
3. Subjects who have given written informed consent. 
4. Subjects aged between 18 and 65 years, inclusive. 
5. Body mass index between 18.5 and 29 kg/m2. 
6. Documented clinical diagnosis of stable, persistent, asthma for at least 3 months, i.e.  
• for whom FEV1 ≥ 70% of predicted, and 
• treated with as-needed reliever medication (SABA-containing medication) only.  
7. Subjects who are ICS-naïve for minimum 60 days at Screening Visit.  
8. Positive MCh challenge test (PC20 < 8 mg/ml or PD20 < 0.2 mg) in the last year. 
9. Post-bronchodilator FEV1 at least 80% of the predicted documented in the last year. 
10. Clinical laboratory test results, 12-lead ECG, blood pressure and heart rate (supine) within 
normal reference range or judged to be not clinically significant by the Investigator. 
11. Female subjects of childbearing potential should have a negative pregnancy test at Screening 
Visit. 
12. Female subjects of childbearing potential using a highly effective method of contraception 
(i.e., pregnancy rate of < 1% per year) on a stable regimen, for at least 28 days, and pursuing 
this contraception during the trial and for 28 days after the last administration of the IMP. The 
highly effective methods of contraception must be one of the following: combined estrogen 
and progestogen hormonal contraception with inhibition of ovulation, progestogen-only 
hormonal contraception associated with inhibition of ovulation, intrauterine device, 
intrauterine hormone-releasing system, bilateral tubal occlusion, vasectomized partner, or 
agreement on continuous abstinence from heterosexual intercourse. 
 
 
 
 


 
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 Exclusion Criteria 
Potential trial subjects will not be entered into the clinical trial if any of the following apply: 
1. Current smokers or recent (< 8 weeks) ex-smokers or ex-smokers if > 10 pack-years. 
2. Pregnant or breastfeeding female subjects.  
3. Inability to carry out pulmonary function testing.  
4. FEV1 < 70%. 
5. History of near-fatal asthma and/or intensive care unit admission for asthma symptoms. 
6. Exacerbations of asthma requiring oral steroids, hospitalization or change in asthma treatment 
in the previous three months.  
7. Evidence of symptomatic chronic or acute respiratory infection in the previous 8 weeks. 
8. Diagnosis of chronic obstructive pulmonary disease (COPD) or bronchiectasis. 
9. Pulmonary malformations, tuberculosis, cystic fibrosis. 
10. History of hypersensitivity or existing contraindication to budesonide or any other IMP 
ingredients. 
11. Untreated oral candidiasis. 
12. Immunosuppressive treatment, including systemic corticosteroids (e.g., oral, parenteral, ocular, 
nasal), within 28 days before Screening Visit. 
13. Use of ICS within 60 days before Screening Visit. 
14. Use of anti-leukotrienes, immunoglobulins, beta-blockers, digitalis, amiodarone, antifungals, 
macrolides, antidepressants, monoamine oxidase inhibitors, antiretroviral drugs, cholinesterase 
inhibitors, histamine, theophylline, non-steroidal anti-inflammatory drugs, anticholinergic 
drugs, neuroleptics, curariform drugs, antihistaminic (anti-H1) drugs, calcium channel blockers, 
LABAs, mast cell stabilizers (e.g. natrium cromoglycate). 
15. History of alcohol or drug abuse. 
16. Unstable or life-threatening cardiac disease: subjects with any of the following would be 
excluded: 
• Myocardial infarction or unstable angina in the 6 months before Screening Visit, 
• Unstable or life-threatening cardiac arrythmia requiring intervention in the 3 months 
prior to Screening Visit, 
• New York Heart Association class IV Heart failure. 
17. History or presence of prolonged QT interval (> 470 ms), or any other clinically significant 
ECG abnormalities as judged by the Investigator based on 12-lead ECG recordings at Screening 
Visit. 
18. Diabetes mellitus. 
19. Neuropsychiatric diseases. 
20. Clinically relevant laboratory abnormalities at Screening Visit.  
21. Blood or plasma donation within 30 days prior to Screening Visit. 
22. History or presence of malignancy of any system organ class (other than localized basal cell 
carcinoma of the skin), treated or untreated, within the past 5 years prior to Screening Visit, 
regardless of whether there is evidence of local recurrence or metastases. 
23. Any surgical or medical condition which might significantly alter the absorption, distribution, 
metabolism, or excretion of drugs, or which may jeopardize the subject in case of participation 
in the clinical trial. 
24. History or presence of any other clinically relevant disease of any major system organ class (e.g. 
cardiovascular, pulmonary, renal, hepatic, gastrointestinal, reproductive, endocrinological, 
neurological, psychiatric or orthopedic disease) as judged by the Investigator. 
25. Human immunodeficiency virus (HIV) and SARS-CoV-2 infections. 
26. Subjects who participated in an investigational trial within the 12 weeks prior to the start of the 
trial. 
 
 
 










 
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The Investigator will evaluate the medication information collected in view of the inclusion and 
exclusion criteria to ensure that the list of medications is not indicative of a pathology listed in the 
exclusion criteria. In case the medication history demonstrates that one or more of the 
inclusion/exclusion criteria are not met, the subject will be withdrawn before randomization. This 
subject will be replaced and considered as a screening failure (Section 8.4.4). 
 
 Forbidden and As-Needed Reliever Medication 
Any type of asthma-related medication is forbidden during the clinical trial except the use of a SABA 
as as-needed reliever medication in case of asthma crisis necessitating treatment. In case the subject 
takes another asthma-related medication than the authorized as-needed medication (SABA only), the 
Investigator will document it and consider subject withdrawal after discussion with Sponsor’s medical 
monitor (Section 10.5.1). 
 
Other forbidden concomitant medications are: 
• 
Immunosuppressive treatment, including systemic corticosteroids (e.g., oral, parenteral, 
ocular, nasal). 
• 
ICS  
• 
Anti-leukotrienes, immunoglobulins, beta-blockers, digitalis, amiodarone, antifungals, 
macrolides, antidepressants, monoamine oxidase inhibitors, antiretroviral drugs, cholinesterase 
inhibitors, histamine, theophylline, non-steroidal anti-inflammatory drugs, anticholinergic 
drugs, neuroleptics, curariform drugs, antihistaminic (anti-H1) drugs, calcium channel 
blockers, long-acting beta2-antagonists, mast cell stabilizers (e.g. natrium cromoglycate). 
 
The Investigator and the subject’s treating physician should however always consider the interest of the 
subject as the priority. Therefore, in case any forbidden concomitant medication is needed by the subject, 
they will consider the subject’s interest rather than the clinical trial continuation. 
 
Any concomitant medications will be recorded according to Section 9.2.4.4 and the subjects will record 
the use of as-needed reliever medications (SABA only) in their diaries (Section 9.2.2.2). 
 
8.9. Assignment of Subjects to Treatment Groups 
Eligible subjects, i.e. fulfilling the criteria defined in Section 8.4 and having given their informed 
consent, will be randomized in a 1 to 1 ratio to one of the following treatment sequences at Visit 1 
(crossover design): 
 
Treatment sequence A 
Treatment sequence B 
Treatment period 1: AQ001S 0.125 mg/ml for 29 (+2) 
days 
Treatment period 1: comparator 0.125 mg/ml for 29 
(+2) days  
Washout for 14 (+2) days  
Washout for 14 (+2) days  
Treatment period 2: comparator 0.125 mg/ml for 29 
(+2) days  
Treatment period 2: AQ001S 0.125 mg/ml for 29 (+2) 
days  
 
The randomization of the subjects will be performed according to Section 11.2. 
The Investigator will inform the monitor of new randomized subjects. 
8.10. Blinding and Emergency Cards 
This is an open label clinical trial.  
The subject emergency card will mention his/her participation in the clinical trial as well as emergency 
phone numbers of the Principal Investigator. 
 


 
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8.11. Treatment Compliance 
AQ001S 0.125 mg/ml and comparator will be administered to the subjects both at the trial site (PK, first 
dose and last dose of each treatment) and at the subject’s home where the IMP will be prepared by the 
study nurse (for the whole treatment period except first and last dose). For each administration, trial staff 
and study nurses will fill in the nebulizer device with either of the IMPs as described in Section 8.5.7 
and will initiate the inhalation for the subject. For that purpose, the study nurses will receive training on 
IMP administration and handling prior to any subject visits. They will document the treatment 
procedures in a log, including IMP identification (Randomization ID, treatment group and treatment 
period), date and time of administration, allowing the monitoring of treatment compliance.  
 
At the first home visit, the study nurse will bring the nebulizer to the subject’s home. The nebulizer will 
then stay at subject’s home until the last home IMP administration of the clinical trial. After this last 
home administration, the nebulizer will be brought back to the trial site.  
8.12. Drug Dispensing and Accountability 
Both AQ001S 0.125 mg/ml and comparator batches will be shipped to the trial site after site initiation 
visit and stored according to recommended storage conditions. 
 
The IMPs will be dispensed to the subjects by the Investigator or authorized trial staff at the time 
points/visits indicated in the flowchart in Section 3. 
 
Any IMPs provided to the site will be accounted for. This includes IMPs received at the site, IMPs 
dispensed to the subjects, and unused and partially used IMPs returned by the study nurse. 
 
A drug inventory and dispensing log will be kept current by the Investigator or authorized trial staff, 
detailing quantities of IMPs received (including information about time and date), as well as quantities 
dispensed to each subject (including information about time and date) and the remaining quantity. The 
drug inventory and dispensing log will be available to the monitor to verify drug accountability during 
the clinical trial. 
 
At the subject’s home, the study nurse will collect the used and partially used IMPs in a disposal bin 
and will return them to the trial site as soon as feasible. At Visits 2 and 4, i.e. after the last home 
administration of each treatment period, the subjects will bring back the unused IMPs. 
The drug inventory and dispensing log will be updated accordingly by the Investigator and authorized 
trial staff to ensure the drug accountability. 
 
For every subject, the drug accountability of his/her first treatment period should be closed during visit 
2 at trial site before starting his/her second treatment period, so to avoid that first treatment doses are 
still in circulation when starting the second treatment period. 
 
At the end of the clinical trial, unused and partially used IMPs must be returned to the Sponsor or 
Sponsor’s designated subcontractor for destruction. 
 
Destruction can be initiated only after accountability has been verified and fully reconciled by the 
monitor and after the Sponsor has granted written approval of destruction. 
 
9. CLINICAL TRIAL CONDUCT 
The flow chart of assessments planned for each trial visit is given in Section 3. The clinical trial will 
include: 
- 
one Pre-Trial Visit,  
- 
one Screening Visit,  
- 
one Randomization and Baseline Visit (Visit 1), with the start of the first treatment period 


 
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- 
three intermediate visits (Visits 2 to 4), with a washout period of 14 (+2) days between 
Visit 2 and Visit 3.  
- 
The Visit 4 is also the End-of-Trial visit. 
Visit 1 and Visit 2 correspond to the beginning and the end of the first treatment period (TP1), 
respectively. 
Visit 3 and Visit 4 correspond to the beginning and the end of the second treatment period (TP2), 
respectively. 
9.1. Procedures by Visit 
The procedures described in this section are either closely linked or dependent on the outcome(s) of the 
previous procedure(s). Moreover, some procedures require physical efforts that could possibly interfere 
with other procedures planned during the same visit. Therefore, the sequence of the procedures as 
described for each visit needs to be strictly followed. 
 Pre-Trial Visit 
The Pre-Trial Visit aims at informing the subject appropriately on the clinical trial rationale, objectives 
and constraints and on his/her rights when accepting to participate into the clinical trial (GCP 
requirements). Subject will also give his/her explicit consent to allow to use his/her data after being 
informed on the identity of the controller, the goals of the data treatment, the fair processing of his/her 
data, and on his/her data subjects rights (EU General Data Protection Regulation [GDPR] requirements). 
It is the Investigator’s responsibility to ensure that each potential subject has the ability to provide 
adequate and informed consent, understands the nature and the scope of the clinical trial, and has signed 
informed consent prior to any participation in any phase of this clinical trial, including screening 
evaluations. The Investigator will also verify that an appropriate washout period has been respected for 
medication that the subject is using prior to trial participation, if applicable. 
 
At Pre-Trial Visit, the following trial procedures will be performed: 
1. Inform the subject, orally and in writing, about the purpose, procedures, and general risks of 
participating in the clinical trial. Answer his/her questions until he/she is satisfied with the 
information.  
2. Obtain his/her informed consent and GDPR explicit consent in writing. 
3. Give a copy of subject information and signed informed consent to the subject. 
 
Only after having obtained the subject’s informed consent in writing, the next assessments can be 
performed: 
4. Assign subject Screening ID (Section 8.4.5).  
5. Check the washout period for ICS*. 
6. Schedule the Screening Visit*. 
7. Instruct the subject to come at Screening Visit fasted (for at least 8 hours)*.   
8. Instruct the subject to come at Screening Visit without having taken any as-needed 
medications during the 4 hours before the visit. If this administration is necessary for the 
subject, he/she should inform the trial site and the visit should be rescheduled accordingly. 
 
*Pre-Trial Visit might be performed at the same time as the Screening Visit if the ICS washout period 
is ≥ 60 days and if the subject has been fasting for at least the last 8 hours. Otherwise, the Screening 
Visit should be planned as soon as all these conditions are met, so at maximum 60 (+2) days after the 
Pre-Trial Visit. The subject should then be instructed to come at Screening Visit fasted (for at least 8 
hours). 
 Screening Visit 
The procedures of this Screening Visit should be performed at least 4 hours after the subject has taken 
any as-needed reliever medications and the subject should come fasted (for at least 8 hours). If this is 
not the case, the visit should be rescheduled accordingly. 


 
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At Screening Visit, the following trial procedures will be performed: 
1. Review and document inclusion and exclusion criteria (Section 8.4). 
2. Record demographic information (Section 9.2.4.1). 
3. Check the fasting status of the subject. 
4. Obtain the medical and surgical history (Section 9.2.4.2). 
5. Obtain the respiratory and asthma history (Section 9.2.4.3). 
6. Obtain the medication history (prior medication), including respiratory medication and 
especially the use of SABA (as-needed reliever medication) and any other asthma medications 
(Section 9.2.4.4.1, and Section 8.8.2 for forbidden medications). 
7. Perform a physical and clinical examination (Section 9.2.1.3), including COVID-19 symptoms 
assessment21. 
8. Perform the following assessments needed to collect all missing inclusion and exclusion criteria 
information (perform these investigations in the mentioned order and do not proceed with the 
next one if subject’s exclusion is decided based on the previous one): 
a. Measure vital signs (Section 9.2.1.4). 
b. Perform 12-lead ECG (Section 9.2.1.5). 
c. Perform spirometry (Section 9.2.1.7) and measure FEV1.  
d. Collect fasting (8-hour) blood samples for hematology, biochemistry and pregnancy 
test, and urine samples for urinalysis (Section 9.2.1.2.1). 
e. Perform MCh challenge test if not performed in the last year and if FEV1 ≥ 70% 
(Section 9.2.2.1). 
f. Perform spirometry (Section 9.2.1.7): 
i. Measure FEV1. 
ii. If FEV1 < 70% or presence of asthma-related clinical symptoms (Section 
9.2.1.6), administer as-needed reliever medication (SABA) (Section 9.2.1.8). 
g. Perform a PCR nasopharyngeal swab test for SARS-cov-2 detection 
9. Dispense subject diary and give the subject the instructions to complete it over the whole 
clinical trial (Section 9.2.2.2), including the questionnaire about day- and night-time asthma 
symptoms, the ACQ-5 and ACT questionnaires to complete in the evening before Visit 1. 
10. Schedule Visit 1.  
11. Instruct the subject to collect his/her first waking urine between 6 and 9 AM in the morning of 
the Visit 1 and to write down the collection time in the subject diary and dispense the urine 
collection bottle.  
12. Instruct the subject to come to Visit 1 fasted (for at least 8 hours). 
13. Instruct the subject to come to Visit 1 without having taken any as-needed medications during 
the 4 hours before the visit. If this administration is necessary for the subject, he/she should 
inform the trial site and the visit should be rescheduled accordingly. 
 
 Visit 1 – Randomization and Baseline 
Visit 1 will take place 5 to maximum 7 days after the Screening Visit, when all laboratory results of the 
subject have been received. 
The Investigator will complete the eligibility assessment of the subject on the basis of the laboratory 
results (Section 8.4). 
The procedures of Visit 1 should be performed at least 4 hours after the subject has taken any as-needed 
reliever medications. 
 
At Visit 1, the following trial procedures will be performed: 
 
21 A PCR nasopharyngeal swab test must be performed at screening  


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1.
Obtain prior medication use information and especially the use of SABA (as-needed reliever 
medication) and any other asthma medications (Section 9.2.4.4.1, and Section 8.8.2 for 
forbidden medications).
2.
Perform a physical and clinical examination (Section 9.2.1.3), including local tolerability
(Section 9.2.1.6) and COVID-19 symptoms   assessments22.
3.
Measure vital signs (Section 9.2.1.4).
4.
For female subjects, perform a urine pregnancy test (Section 9.2.1.2.1).
5.
If subject’s eligibility is confirmed, randomize the subject to assign him/her to a treatment 
group (Section 8.9).
6.
Allocate subject’s Randomization ID accordingly (Section 8.4.5).
7.
Query the subject as to the occurrence of any safety events observed from Screening Visit to 
Visit 1 and record any safety events observed or spontaneously volunteered by the subject
(Section 9.2.1.1).
8.
Collect the first waking urine sample for urinary cortisol/creatinine ratio determination
(Section 9.2.1.2.3) and urinalysis (Section 9.2.1.2.1).
9.
Collect the diary pages containing the baseline symptom scoring information, i.e. 
questionnaire about day- and night-time asthma symptoms, the ACQ-5 and ACT 
questionnaires, the timing of urine collection and check their completion (Section 9.2.2.2).
10. Insert the catheter and collect the blood samples for hematology and  biochemistry (Section 
9.2.1.2.1), single dose 24-hour PK pre-dose time point (Section 9.2.3.1) and baseline blood 
eosinophil count (Section 9.2.2.5).
11. Perform FeNO (Section 9.2.2.4).
12. Perform spirometry (Section 9.2.1.7) and measure FEV1, FVC and FIVC.
13. Perform MCh challenge test (determine baseline PC20) (Section 9.2.2.1).
14. Perform spirometry (Section 9.2.1.7):
a.
Measure FEV1, FVC and FIVC.
b.
If FEV1 < 70% or presence of asthma-related clinical symptoms (Section 9.2.1.6), 
administer as-needed reliever medication (SABA) (Section 9.2.1.8).
15. Administer the D0 (single dose 24-hour PK) dose of IMP of treatment period 1 (2 ml, 
corresponding to 0.250 mg budesonide) (Section 8.5.7).
16. Admit the subject at the trial site for overnight stay.
17. Perform the PK sampling procedure (first post-dose time point =        minutes after first dose 
nebulization, in total 15 blood samples after first IMP dose, over 24 hours; Section 9.2.3.1).
18. Record safety events during the whole stay at the clinical site if occurring and/or reported by 
the subject (Section 9.2.1.1).
19. At the end of the single dose 24-hour PK sampling, administer the second dose of IMP of 
treatment period 1 (1 ml, corresponding to 0.125 mg budesonide) (Section 8.5.7).
20. Obtain concomitant medication use since the first IMP administration, i.e. during the whole 
stay at the trial site, and especially the use of SABA (as-needed reliever medication) and any 
other asthma medications (Section 9.2.4.4.2).
21. Dispense IMPs needed for treatment period 1 to the subject (Section 8.12).
22. Schedule or confirm the next trial visit (Visit 2).
23. Instruct the subject to use his/her SABA medication as needed and to record the use in his/her 
subject diary (Section 8.8.2).
24. Instruct the subject to complete his/her diary and to bring it to the next visit (Section 9.2.2.2).
25. Instruct the subject to collect his/her first waking urine between 6 and 9 AM in the morning of 
the next visit and to write down the collection time in the subject diary and dispense the urine 
collection bottle.
26. Instruct the subject to come to Visit 2 fasted (for at least 8 hours).
22 A PCR nasopharyngeal swab test may be performed during the study at the investigator’s discretion 








 
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site staff to contact the subject and ascertain the reason(s) for not returning. As a minimum, the site staff 
will contact the subject twice by phone and, if no response, will send a letter by registered mail. 
 
If a subject does not complete the clinical trial for any reason (including at Investigator’s discretion), 
the reason and circumstances for the subject’s early termination must be fully documented. If possible, 
the trial procedures specified for the Visit 4 should be performed. 
 
The Sponsor or designee must be informed in a timely manner if any subject withdraws from the trial, 
regardless of the cause.  
 
 Unscheduled Visit 
If deemed necessary for the patient’s safety and well-being, the Investigator can initiate an unscheduled 
visit. All such visits will be documented in the eCRF with any additional required documentation based 
on the nature of the unscheduled visit. 
 
 Follow-up Contact 
In order to collect any post-trial SAEs, the Investigator will contact each subject 30 (+2) days after 
his/her last visit, regardless of whether the subject has completed the clinical trial or has been withdrawn 
before the end of the trial.  
In case a post-trial SAE is identified, it should be entered into the eCRF within 24 hours after becoming 
aware of the information. In case the eCRF is not available, the safety information should be entered in 
the SAE Report Form and submitted to Clinfidence pharmacovigilance department within 24 hours after 
becoming aware of the information (Section 9.2.1.1.2). 
 
9.2. Assessments and their Appropriateness 
 Safety Assessments 
 Assessments for Safety Events 
All of the following safety information (safety events) will be collected: 
• 
AEs, SAEs, and post-trial SAEs  
• 
Pregnancies, IMP overdose, drug interaction, medication error. 
All safety events observed during the trial from the time the subject signs the informed consent until 
the last contact will be collected. 
 
9.2.1.1.1. Adverse Events and Adverse Reactions 
Definitions 
 
Adverse event (AE): An AE is any untoward medical occurrence in a trial subject receiving an IMP and 
which does not necessarily have a causal relationship with this treatment. An AE can therefore be any 
unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease 
temporally associated with the use of an IMP, whether related to the IMP or not. 
Adverse reaction (AR): ARs are all untoward and unintended responses to an investigational product 
related to any dose administered.  
Other significant AEs: Any marked laboratory abnormalities or any AEs that lead to an intervention, 
including withdrawal of IMP treatment, dose reduction, or significant additional concomitant therapy. 


 
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Withdrawal due to AE/AR: AE/AR leading to discontinuation of treatment with IMP. Any such events 
will be followed up by the Investigator until the event is resolved or until the medical condition of the 
subject is stable. All follow-up information collected will be made available to the Sponsor. 
 
Collection of AEs 
 
The condition of the subject will be monitored throughout the clinical trial. At each visit, whether 
scheduled or unscheduled, AEs will be elicited using a standard nonleading question such as "How have 
you been since the last visit/during the previous trial period?" In addition, the Investigator will check 
the subject diaries for any documented event. 
 
Any AE or AR, which occurs during the clinical trial, at trial site or reported to the home-visiting study 
nurses, will be noted in detail in the eCRF. If the subject reports several signs or symptoms representing 
a single syndrome or diagnosis, the diagnosis should be recorded in the eCRF. The Investigator will 
grade the severity of all AEs or ARs (mild, moderate, or severe), the seriousness (nonserious or serious), 
and the causality (probable, possible, unlikely, not related or unclassified).  
 
In the event of clinically significant abnormal laboratory findings, the test results will be confirmed, and 
the subject followed up until the laboratory values have returned to normal and/or an adequate 
explanation for the abnormality has become available. 
 
Diseases, signs and symptoms, and/or laboratory abnormalities already present before the first 
administration of IMP will not be considered AEs unless an exacerbation in intensity or frequency 
(worsening) occurs. 
 
The Investigator will provide detailed information about any abnormalities and about the nature of and 
reasons for any action taken as well as any other observations or comments that may be useful for the 
interpretation and understanding of an AE or AR. 
 
Asthma exacerbations 
 
Asthma exacerbations will be considered as adverse if they reflect, to the opinion of the Investigator, a 
worsening of the subject's disease. To allow this evaluation, all asthma exacerbations will be reported 
as AEs, as well as the treatment (concomitant medication) administered and a report, issued by the data 
management department or the pharmacovigilance department, will be sent to the Investigator on a 
weekly basis, according to the instructions described in the safety management plan. 
 
Severity of AEs 
 
The intensity/severity of AEs will be graded as follows: 
 
• 
Mild: an AE, usually transient, which causes discomfort but does not interfere with the subject’s 
routine activities, 
• 
Moderate: an AE which is sufficiently discomforting to interfere with the subject’s routine 
activities, 
• 
Severe: an AE which is incapacitating and prevents the pursuit of the subject's routine activities. 
 
The grading of an AE is up to the medical judgement of the Investigator and will be decided on a case-
by-case basis. 
 
Causality of AEs 


 
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The relationship of AEs to the administered IMP will be assessed by the Investigator: 
 
• 
Probable: reports including good reasons and sufficient documentation to assume a causal 
relationship, in the sense of plausible, conceivable, likely, but not necessarily highly probable. 
A reaction that follows a reasonable temporal sequence from administration of the IMP; or that 
follows a known or expected response pattern to the suspected medicine; or that is confirmed 
by stopping or reducing the dosage of the medicine and that could not reasonably be explained 
by known characteristics of the subject’s clinical state, 
• 
Possible: reports containing sufficient information to accept the possibility of a causal 
relationship, in the sense of not impossible and not unlikely, although the connection is uncertain 
or doubtful, for example because of missing data or insufficient evidence. A reaction that 
follows a reasonable temporal sequence from administration of the IMP; that follows a known 
or expected response pattern to the suspected medicine; but that could readily have been 
produced by a number of other factors, 
• 
Unlikely: reports not following a reasonable temporal sequence from IMP administration. An 
event which may have been produced by the subject's clinical state or by environmental factors 
or other therapies administered, 
• 
Not related (unrelated): events for which sufficient information exists to conclude that the 
etiology is unrelated to the IMP,  
• 
Unclassified: reports which for one reason or another are not yet assessable, e.g., because of 
outstanding information (can only be a temporary assessment). 
 
Product-related causality (ARs) will be defined as events that are probably related or possibly related to 
the IMP. Events will be categorized as unrelated to IMP if they are reported as unlikely, unclassified or 
not related. 
 
Outcome of AEs 
 
The outcome of all reported AEs has to be documented as follows: 
 
1. Recovered, resolved 
2. Recovering, resolving 
3. Not recovered, not resolved 
4. Recovered, resolved with sequelae 
5. Fatal 
6. Unknown 
 
A subject's death per se is not an event, but an outcome. The event which resulted in the subject's death 
must be fully documented and reported, even in case the death occurs within 30 days after IMP treatment 
end and regardless of whether or not it is considered treatment related. 
 
Action(s) taken with subject 
 
AEs requiring action or therapy will be treated with recognized standards of medical care to protect the 
health and well-being of the subject. Appropriate resuscitation equipment and medicines will be 
available to ensure the best possible treatment in case of an emergency. 
The actions taken with each subject by the Investigator will be documented. 
The Investigator will follow up on each AE until it has resolved or until the medical condition of the 
subject has stabilized. Any follow-up information will be reported to the Sponsor. 
The Investigator can withdraw the subject from the clinical trial for safety reasons (Section 10.5.1). 
 


 
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Action(s) taken with IMP under investigation 
 
As described in Section 10.1.2, Sponsor is responsible for the ongoing safety evaluation of the IMP 
under investigation and will expedite the notification to the Principal Investigator and regulatory 
authorities of findings that are both serious and unexpected (Section 9.2.1.1.2) and/or that could 
adversely affect the safety of subjects, the conduct of the clinical trial or alter the Independent Ethics 
Committee (IEC)/Institutional Review Board (IRB)’s approval to continue the clinical trial. 
Significant worsening of the benefit/risk ratio of AQ001S 0.125 mg/ml will lead to reevaluate the 
appropriateness to continue, hold or stop the clinical trial (Section 10.6.1.1).  
 
 
Documentation of AEs 
 
All AEs that occur during the trial are to be recorded in the eCRF. The Investigator should complete all 
the details requested, including dates of onset, time of onset, stop date (when applicable), stop time 
(when applicable), severity, action taken, outcome, and relationship to IMP and to trial procedures. 
 
9.2.1.1.2. Serious Adverse Events 
A serious adverse event (SAE) is any untoward medical occurrence that at any dose: 
 
• 
Results in death, 
• 
Is life-threatening (see below), 
• 
Requires hospitalization or prolongation of existing hospitalization, 
• 
Results in persistent or significant disability/incapacity, 
• 
Is a congenital anomaly/birth defect. 
Any other important medical event that may not result in death, be life-threatening or require 
hospitalization may be considered an SAE when, based upon appropriate medical judgement by the 
Principal Investigator, the event may jeopardize the participant or may require an intervention to prevent 
one of the other serious outcomes listed above. 
NOTE: The term 'life-threatening' refers to an event in which the subject was, in the view of the reporting 
Investigator, at immediate risk of death at the time of the event; it does not refer to an event which may 
hypothetically have caused death had it been more severe. 
 
Suspected Unexpected Serious Adverse Reactions (SUSARs) 
Unexpected ARs are SUSARs if the following three conditions are met:  
1. The event must be serious,  
2. There must be a certain degree of probability that the event is a harmful and an undesirable 
reaction to the IMP, regardless of the administered dose,  
3. The AR must be unexpected, that is to say, the nature and severity of the AR are not in 
agreement with the product information as recorded in the IB (Section 8) or other reference 
safety information of the IMP. 
Should a SUSAR occur, it will be reported to the IEC/IRB and to the applicable authorities in accordance 
with Directive 2001/20/EC. 
SAE Reporting Timelines 




 
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reported as an AE. In case of suspected overdose, the patient should be treated according to standard 
medical practice based on the Investigator’s judgment. 
 
Drug interaction 
 
A drug interaction is a situation in which a substance or medicinal product affects the activity of an IMP, 
i.e., increases or decreases its effects, or produces an effect that none of the products would exhibit on 
its own. The reaction of a suspected drug interaction must be clearly identified and reported as an AE. 
 
Medication error 
 
A medication error involves the inadvertent administration or unintended use of a medicinal product 
which may be caused by the naming, presentation of pharmaceutical form/packaging or IFU/labelling. 
The reaction must be clearly identified as a medication error. 
 
 Clinical Laboratory Tests 
9.2.1.2.1. Hematology, Biochemistry, Urinalysis and Pregnancy Tests 
Clinical laboratory tests include blood analysis (hematology and biochemistry) and urinalysis. 
Subject should be fasting for at least the last 8 hours before blood collection, particularly for the 
Screening Visit to assess eligibility of the subject (Section 9.1.2). 
Clinical laboratory tests will be performed at the time points/visits indicated in the flowchart in Section 
3. On dosing days at trial site, the assessments will be done according to the sequence described in the 
visit procedures (Section 9.1). 
 
A local laboratory will perform the classical clinical laboratory tests on blood samples, including serum 
pregnancy test for female subjects of childbearing potential allowing to exclude pregnant subjects from 
entering the clinical trial. 
 
For urinalysis, test strip analyses will be performed on the first waking urine samples at the trial site at 
the time of sample collection. If a test result is suspicious/unsure, a urine sample will be sent to the local 
laboratory for in-depth urinalysis with the optional tests listed in Appendix 3. 
At Visit 1, a confirmatory urine pregnancy test will be performed at the trial site for female subjects of 
childbearing potential allowing to exclude pregnant subjects from receiving the IMPs. 
 
The appended table (Appendix 3) lists all laboratory tests to be performed, normal values and reasons 
to test.  
Blood and urine samples will be prepared, and shipments made to the laboratory, according to their 
recommendations, and sample storage conditions will be documented. Only sample collection date and 
time must be entered in the eCRF. 
 
The methods of laboratory test determination will be provided in the clinical study report. These methods 
are validated methods used in clinical routine by the laboratory. 
 
All out-of-range laboratory values that are considered by the Investigator to be clinically significant will 
be recorded as AEs (Section 9.2.1.1.1). 
 
The laboratory that will perform the sample analyses is specified in Section 1. 
9.2.1.2.2. Amounts and Combination of Blood and Urine Collections 


 
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The estimated amount of blood to be collected per visit, including all blood/plasma assessments is, for 
each subject:  
• 
A total of 24.5 ml of blood at Screening Visit (for eligibility criteria), 
• 
A total of 72.5 ml of blood at Visit 1 (24.5 ml for hematology and biochemistry, including blood 
eosinophil count, and 45 ml for full single 24-hour PK), 
• 
A total of 45.5 ml of blood at Visit 2 (24.5 ml for hematology and biochemistry, including blood 
eosinophil count, and 21 ml for abridged PK), 
• 
A total of 72.5 ml of blood at Visit 3 (24.5 ml for hematology and biochemistry, and 45 ml for 
full single 24-hour PK), 
• 
A total of 45.5 ml of blood at Visit 4 (24.5 ml for hematology and biochemistry, including blood 
eosinophil count, and 21 ml for abridged PK). 
 
The estimated total maximum blood volume needed per subject during the trial (when completing the 
trial until the last visit) is 260.5 ml, over 77 to 85 days, from Screening Visit until Visit 4 (End-of-Trial). 
 
At Screening Visit, blood and urine samples will be respectively collected with classical venipuncture 
and urine collection materials. 
At Visits 1, 2, 3 and 4, the blood and urine collections for hematology, biochemistry and urinalysis will 
be combined with those necessary for the blood PK sampling (Section 9.2.3) and eosinophil count 
(Section 9.2.2.5), i.e. through a catheter, and for the urinary cortisol/creatinine ratio, i.e. in the first 
waking urine (Section 9.2.1.2.3).  
9.2.1.2.3. Assessment of Hypothalamic Pituitary Adrenocortical Axis Function  
Hypothalamic pituitary adrenocortical (HPA) axis function will be assessed through specific urinalysis 
at the time points/visits indicated in the flowchart in Section 3. On dosing days at trial site, the 
assessments will be done according to the sequence described in the visit procedures (Section 9.1). 
For that purpose, the urinary cortisol/creatinine ratio will be determined in first waking urine collected 
by the subject between 6 and 9 AM. 
The local laboratory that will determine the cortisol/creatinine ratio analysis in the urinary samples is 
specified in Section 1. 
Population reference values for the urinary cortisol/creatinine ratio in first waking urine for urine 
collection between 6 and 9 AM have been established by the laboratory prior to the clinical trial 
(Appendix 3). The methods of cortisol/creatinine ratio determination will be provided in the clinical 
study report. Urine samples will be prepared, and shipments made to the laboratory in charge of the 
cortisol/creatinine ratio determination according to their recommendations and the storage conditions 
will be documented. The timing of first waking urine collection will be recorded by the subjects in their 
diary (Section 9.2.2.2). 
 
All out-of-range laboratory values that are considered by the Investigator to be clinically significant will 
be recorded as AEs (Section 9.2.1.1). 
 Physical and Clinical Examination 
The physical and clinical examination include height (only measured at Screening Visit), weight, body 
mass index (calculated), oropharyngeal examination and covid-19 symptoms assessments by the 
Investigator (physician), and will be recorded at the time points/visits indicated in the flowchart in 
Section 3. On dosing days at trial site, the examinations will be done according to the sequence described 
in the visit procedures (Section 9.1). 
The oropharyngeal examination will include the investigation of specific ICS-related signs, such as 
vocal cord myopathy and fungal infection. 
 
All abnormal findings in physical and clinical examination that are considered by the Investigator to be 
clinically significant will be recorded as AEs (Section 9.2.1.1). 


 
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 Vital Signs 
Vital signs will be measured at the time points/visits indicated in the flowchart in Section 3. On dosing 
days at trial site, the measurements will be done according to the sequence described in the visit 
procedures (Section 9.1). 
Vital signs will include records of blood pressure (seated), body temperature, heart rate, and blood 
oxygen saturation. Vital sign measurements will be taken while the subject is seated after at least 5 
minutes at rest. 
Systolic and diastolic blood pressure measurements will be made using a sphygmomanometer with a 
cuff size appropriate for each individual subject.  
Heart rate and blood oxygen saturation will be measured with a pulse oximeter.  
Temperature will be measured orally. 
 
All abnormal findings in vital signs that are considered by the Investigator to be clinically significant 
will be recorded as AEs (Section 9.2.1.1). 
 Twelve-Lead Electrocardiogram 
Single 12-lead paper ECG will be obtained at the time points/visits indicated in the flowchart in Section 
3. On dosing days at trial site, the measurements will be done according to the sequence described in 
the visit procedures (Section 9.1). 
The single 12-lead ECG will be recorded while subject is in resting position (supine) for at least 5 
minutes. For each ECG, the following parameters will be calculated and recorded into the eCRF: 
• 
Heart rate, 
• 
QT interval duration, 
• 
corrected QT interval (QTc), estimating the QT interval at a standard heart rate of 60 beat per 
minute. 
Twelve-lead ECGs will be interpreted by the Investigator at the site as soon as possible after the time of 
ECG collection, and ideally while the subject is still present at trial site, for immediate subject 
management. 
All abnormal findings in ECGs that are considered by the Investigator to be clinically significant will 
be recorded as AEs (Section 9.2.1.1). 
 Local Tolerability  
Local tolerability will be assessed at the time points/visits indicated in the flowchart in Section 3 through 
physical and clinical examination (Section 9.2.1.3). On dosing days at trial site, the measurements will 
be done according to the sequence described in the visit procedures (Section 9.1). 
 
In particular, the following specific parameters will be reported: 
- 
Increased bronchial irritability, 
- 
Paradoxical bronchospasm, 
- 
Oropharyngeal examination (e.g. vocal cord myopathy, fungal infection). 
 
The presence of asthma-related clinical symptoms (e.g. dyspnea, cough, wheezing, bronchospasm, etc.) 
after MCh challenges, along with the FEV1 spirometry measurements at Visits 1, 2 and 4, will determine 
the need to administer as-needed reliever medication (SABA) to the subject (if FEV1 < 70%; Section 
9.2.1.8).  
 
 


 
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 Spirometry 
Spirometry, a common pulmonary function test, will be performed according to the American Thoracic 
Society/European Respiratory Society standardization of spirometry [51]. The following parameters will 
be measured at the time points/visits indicated in the flowchart in Section 3: 
- 
Forced expiratory volume at 1 second (FEV1) to check eligibility, as safety parameter and as 
efficacy parameter, 
- 
Forced vital capacity (FVC) as safety parameter, 
- 
Forced inspiratory vital capacity (FIVC) as efficacy parameter. 
FEV1 and FIVC are indeed meant to be improved by an asthma treatment. 
 
Therefore, spirometry will be performed at the indicated visits in order to: 
- 
Measure FEV1 to check inclusion criteria: Screening Visit, 
- 
Measure FEV1, FVC and FIVC before and after MCh challenge test for safety and efficacy 
assessments (baseline values): Visit 1, 
- 
Measure FEV1, FVC and FIVC before and after MCh challenge test for safety and efficacy 
assessments (end-of-treatment period values): Visits 2 and 4, 
- 
Measure FEV1 and FVC for safety assessments: Visit 3. 
 
On dosing days at trial site, the measurements will be done according to the sequence described in the 
visit procedures (Section 9.1). 
 
The FEV1 measurements before and after MCh challenge tests allow to determine the PC20 as described 
in Section 9.2.2.1 and according to the guidelines of the American Thoracic Society [41]. 
 
The FEV1 measurements after MCh challenge test will determine, along with the presence of asthma-
related clinical symptoms (Section 9.2.1.6), the need to administer as-needed reliever medication 
(SABA) to the subject (if FEV1 < 70%; Section 9.2.1.8).  
 Administration of As-Needed Reliever Medication 
The administration of as-needed reliever medication (SABA) at any time of the clinical trial will be 
recorded, including by the subject in his/her diary on a daily basis.  
 
At Visits 1, 2 and 4, the FEV1 measurements after MCh challenge tests (Section 9.2.1.7) and the 
recording of asthma-related clinical symptoms (Section 9.2.1.6) will determine the need to administer 
as-needed reliever medication (SABA) to the subject (if FEV1 < 70% or presence of asthma clinical 
symptoms).  
 
 Efficacy and Pharmacodynamic Assessments 
 Methacholine Challenge Test and PC20 
 
In the present clinical trial, MCh challenge tests will be performed at the time points/visits indicated in 
the flowchart in Section 3, using a marketed MCh powder (Section 8.6) as a provocative agent (i.e. the 
auxiliary medicinal product [AxMP] Provocholine®). On dosing days at trial site, the measurements 
will be done according to the sequence described in the visit procedures (Section 9.1). 
 
The following protocol, procedures and sequence described in the American Thoracic Society guidelines 
[41] will be followed to perform this MCh challenge test.  
 
Protocol:  
A continuous 2-minute tidal breathing dosing protocol will be used in the present clinical trial. 


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Preparation of the MCh concentrations: 
The following 4 concentrations of MCh in sterile vials using a sterile solution of NaCl 0,9% as diluent 
must be prepared, placed in a holder, and stored in a refrigerator (2 to 8°C). These solutions can be 
stored for not more than 2 weeks in a refrigerator.  
Diluent: sterile NaCl 0.9% solution for injection  
MCh concentrations: 0.25, 1, 4 and 16 mg/ml (see Table 2) 
Table 2 - Provocholine® powder dilutions (one 100-mg vial of methacholine chloride) 
with 0.9% NaCl injection in sterile vials 
Label 
strength 
Take 
Add NaCl 
0.9% 
(diluent) 
Total 
volume 
Dilution 
factor 
MCh 
dilution X 
MCh dilution 
concentration 
100 mg 
100 mg 
6.25 mL 
6.25 mL 
N/A 
Dilution A 
16 mg/mL 
3 mL of Dilution A 
9 mL 
12 mL 
1:4 
Dilution B 
4 mg/mL 
3 mL of Dilution B 
9 mL 
12 mL 
1:4 
Dilution C 
1 mg/mL 
3 mL of Dilution C 
9 mL 
12 mL 
1:4 
Dilution D 
0.25 mg/mL 
Rationale for the concentration’s selection: 
The above-mentioned MCh concentrations were selected for the present clinical trial because of 
concerns about the safety of the 10-fold changes in dilution strengths described in the protocol of 
Provocholine® IFU. Quadrupling concentration increments are recommended by the guidelines of the 
American Thoracic Society [41].  
Preparation of the subject for MCh challenge test: 
-
The test must be explained to the subject. The subjects should be told they may experience some
minor symptoms, such as cough or chest tightness, but that most subjects have no symptoms.
They should be warned that occasional severe symptoms may occur.
-
The subject should be asked if they would like to urinate before the test (stress incontinence
could be precipitated, especially in older women).
-
The subjects should be evaluated for contraindications and medication use should be reviewed.
Dosage and administration: 
-
The MCh dilution vials should be removed from the refrigerator 30 min before testing, so that
the mixture warms to room temperature before use.
-
Before MCh challenge test is begun, baseline pulmonary function tests must be performed
inserting 3 mL of diluent (i.e., sterile solution of NaCl 0.9% for injection) into the nebulizer,
using a sterile syringe. A subject to be challenged must have an FEV1 of at least 70% of the
predicted value.
-
The MCh challenge test is performed by giving the subject ascending serial concentrations of
Provocholine® (i.e., starting from dilution D [0.25 mg/mL], dilution C [1 mg/mL], dilution B
[4 mg/mL] and ending by dilution A [16 mg/mL] subsequently).
-
During the MCh challenge test, the subject should be instructed to relax and breathe quietly
(tidal breathing) for 2 min. The timer must be set for 2 min. A noseclip may be applied.
-
3 ml of the appropriate dilution must be inserted into the nebulizer, using a sterile syringe.
-
FEV1 must be measured about 30 and 90 s after the nebulization of each serial concentration is
completed. An acceptable-quality FEV1 must be obtained at each time point. To keep the


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cumulative effect of MCh relatively constant, the time interval between the commencement of 
two subsequent concentrations should be kept to 5 min. 
-
If the FEV1 falls less than 20%, empty the nebulizer (any remaining solution should be
discarded) and repeat the nebulization with the inhalation of 3 ml of the next highest MCh
concentration.
-
If the FEV1 falls more than 20% from baseline (or the highest MCh concentration [i.e., dilution
A] has been given), no further MCh must be given. The signs and symptoms experienced by the
subject must be noted. Inhaled as-needed reliever medication (SABA) must be administered.
Spirometry must be repeated after 10 minutes.
-
The PC20 FEV1 must be calculated by linear interpolation based on the antepenultimate
Provocholine® dilution and on the last Provocholine® dilution that induced a FEV1 that fell
more than 20% from baseline.
-
The PC20 must be recorded in the eCRF.
To ensure the quality of the tests, subjects must not ingest caffeine-containing products and must be 
under fasting conditions at the visits during which these tests are performed. 
At Visit 1 (baseline), Visit 2 and Visit 4, a MCh challenge test will be performed, i.e. up to the 
administration of a concentration of MCh provoking an FEV1 fall of 20% (PC20). 
Subject Diaries and Questionnaires 
Subject diaries will be given to the participating subjects (in their native language) at the time 
points/visits indicated in the flowchart in Section 3. Subjects will be instructed on how to complete their 
diary by the Investigator or designee.  
The following information will be recorded by the subject in his/her diary: 
•
Symptom questionnaires and scores will be answered by the trial subjects:
a.
Daily questionnaire: day- and night-time asthma symptom scoring,
b.
Weekly Asthma Control Questionnaire™ (ACQ-5) questionnaire,
c.
Monthly Asthma Control Test™ (ACT) questionnaire.
•
Use of as-needed reliever medication (daily),
•
Use of concomitant medications (weekly),
•
Time of first waking urine collection in the morning of Visits 1 to 4.
The daily questionnaire is based on questions published in the literature [52]. 
The ACQ-5 is a 5-item, subject-based simple questionnaire to measure the adequacy of asthma control 
and change in asthma control which occurs either spontaneously or as a result of treatment. It is a 
clinically validated assessment of asthma control [53].  
The ACT is a brief, 5-item subject-based assessment designed to measure dimensions of asthma control 
outlined in the current asthma management guidelines as defined by the National Heart, Lung, and Blood 
Institute: asthma symptoms, use of as-needed reliever medications, and the impact of asthma on 
everyday functioning. It is suitable for use with or without lung-function testing [54].  
At the time points/visits indicated in the flowchart in Section 3, the completed subject diary pages will 
be collected and reviewed by the Investigator.  
 Spirometry 
See Section 9.2.1.7. 
Fractional Concentration of Exhaled Nitric Oxide 






 
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• 
λz: apparent first-order rate constant associated with the terminal slope of the semi-
logarithmic budesonide concentration-time curve, 
• 
t1/2z: terminal elimination half-life associated with the terminal slope (λz) of the semi-
logarithmic budesonide concentration-time curve, 
• 
AUC0-6h: area under the plasma concentration-time curve from time 0 to 6-hours post 
administration (ng.h/ml), 
• 
AUC0-last: area under the plasma concentration-time curve from time 0 to the last quantifiable 
concentration (ng.h/ml), 
• 
AUC0-inf: area under the plasma concentration-time curve from time 0 to infinity (ng.h/ml), 
• 
CL/F: apparent clearance following budesonide inhalation (l/h), 
• 
V/F: apparent volume of distribution following budesonide inhalation (l), 
• 
Ctrough: plasma budesonide concentration at the end of a dosing interval, immediately before 
next IMP administration (ng/ml). 
 
 Other Assessments 
 Demographics  
The demographic and baseline characteristics include sex and age and will be recorded at Screening 
Visit. 
 Medical and Surgical History  
Medical and surgical history of the subject will be obtained by interviewing the subject. Clinically 
significant findings as well as pre-existing conditions present prior to screening will be recorded at 
Screening Visit (see also inclusion/exclusion criteria in Section 8.4). 
This includes a review of: 
- 
All body systems, 
- 
Previous surgical intervention(s), including indication, date and outcome. 
 Respiratory and Asthma History 
Respiratory and asthma history, obtained by interviewing the subject, will be recorded at the time 
points/visits indicated in the flowchart in Section 3 (see also inclusion/exclusion criteria in Section 8.4). 
This includes: 
- 
Date of asthma diagnosis, including date of appearance of first symptoms, 
- 
Asthma exacerbations in the last year, 
- 
Other respiratory history: COPD, bronchiectasis, pulmonary malformations, tuberculosis, cystic 
fibrosis.  
 Prior/Concomitant Medications 
Information about prior and concomitant medications will be obtained by interviewing the subject and 
recorded weekly by the subject in the subject diary (Section 9.2.2.2).  
 
Medications with a start date occurring before the first IMP dose date will be identified as prior 
medications.  
The following medications will be identified as concomitant medications : 
- 
Medications with a start date occurring on or after the first IMP dose date,  
- 
Prior medications that continue or ends on or after the first IMP dose date.  
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Subjects will be queried about their prior medication use at the time points/visits indicated in the 
flowchart in Section 3 (see Section 8.8.2 for forbidden medications). Prior medications used by the 
subjects will be documented including the name of the drug, the pharmaceutical form, the strength, the 
dose regimen, the frequency, the route of administration, the date of initiation and discontinuation, and 
the reason for use. 
Any medications taken in the 3 months prior to Screening Visit will be checked and recorded for 
subject’s inclusion, in particular the use of ICS (Section 8.4). 
Any medications taken in the 3 months prior to Screening Visit, including respiratory medications and 
especially the use of as-needed reliever medication (SABA-containing medication) and any other asthma 
medications, will be recorded at Screening Visit (see Section 8.8.2 for forbidden medications). 
9.2.4.4.2. Concomitant Medication Use 
Subjects will be queried about their concomitant medication use and their diaries checked for correct 
completion (Section 9.2.2.2) at the time points/visits indicated in the flowchart in Section 3 (see Section 
8.8.2 for forbidden medications). Concomitant medications taken by the subjects will be documented 
including the name of the drug, the pharmaceutical form, the strength, the dose regimen, the frequency, 
the route of administration, the date of initiation and discontinuation, and the reason for use. 
The reason for use or change of strength or dose regimen of a concomitant therapy may need to be 
reported as an AE (Section 9.2.1.1). 
 
 Subsequent Therapy Counseling 
At the end of the clinical trial (Visit 4, End-of-Trial Visit) or in case of subject’s withdrawal, the 
subsequent therapy will be at the discretion of the subject’s treating physician. 
 
10. TRIAL MANAGEMENT 
10.1. Responsibilities 
 
Investigator(s) 
The Principal Investigator is accountable for the conduct of the clinical trial. Responsibilities may be 
delegated to appropriately qualified persons (designees). 
 
A delegation of authority log will be filled in and signed by the Principal Investigator. In accordance 
with this authority log, trial site staff (e.g., sub-investigators, nurses) is authorized to perform tasks 
relating to the clinical trial. 
 
Information to Investigator(s) 
IMP under investigation: AQ001S 0.125 mg/ml 
An Investigator's Brochure (IB) will be handed out to the Investigator before the start of the clinical 
trial. The IB contains all information in the Sponsor's possession necessary for the Investigator to be 
fully and accurately informed about the safety of AQ001S 0.125 mg/ml and the respective benefit/risk 
ratio. 
The IB will be updated by the Sponsor at regular intervals and whenever relevant new information 
concerning AQ001S 0.125 mg/ml becomes available. 
The Investigator will be informed about the methods for rating relevant clinical trial outcomes and for 
completing eCRFs. 
The Investigator will be kept informed of important data that relate to the safe use of AQ001S 0.125 
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Planned duration for the clinical trial as a whole 
The clinical trial is planned to last approximately 6 months, including a recruitment period of 3 months.  
The clinical trial will be considered completed when all subjects have completed the End-of-Trial Visit 
(Visit 4) except in case of unresolved safety issues. 
10.3. Protocol Amendments 
Any prospective change to the protocol will be agreed between the Principal Investigator and the 
Sponsor prior to its implementation. 
Any such amendments will be submitted to the competent IEC/IRB and/or competent authority as 
required by applicable regulations. 
IEC/IRB approval will, at a minimum, be requested for any change to this protocol which could affect 
the safety of the subjects, the objective/design of the clinical trial, any increase in dosage or duration of 
exposure to the IMP, an increase in the number of subjects treated, the addition of a new test or 
procedure, or the dropping of a test intended to monitor safety. 
 
10.4. Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol or GCP requirements. The 
noncompliance may be either on the part of the subject, the Investigator, or the trial staff. 
 
An Investigator must not make any changes or deviate from this protocol, except to protect the rights, 
safety and well-being of a trial subject in case of an emergency. 
It is the responsibility of the Investigator to use continuous vigilance to identify and report deviations as 
soon after becoming aware of the deviation. As a result of deviations, corrective actions are to be 
developed by the site and implemented promptly. 
 
All deviations from the protocol, with the reason for the deviation and the date of occurrence, must be 
documented and reported to the Sponsor. Sites may also be required to report deviations to the IEC/IRB, 
per local guidelines and government regulations. 
 
Deviations will be reviewed and evaluated on an ongoing basis and, as necessary, appropriate corrective 
and/or preventive actions will be implemented by the Sponsor. 
 
10.5. Withdrawal and Replacement of Subjects 
 
Premature Subject Withdrawal 
Subjects have the right to withdraw from the clinical trial at any time for any reasons, without the need 
to justify their decision. The Investigator also has the right to withdraw subjects in case of AEs, poor 
compliance, or other reasons. Since an excessive rate of withdrawals can render the clinical trial 
noninterpretable, any unnecessary withdrawal of subject should be avoided. 
 
The Investigator can withdraw the subject from the clinical trial for safety reasons, including pregnancy 
(Section 9.2.1.1). In this case, the Investigator will immediately inform the Sponsor and will fill in an 
(S)AE form in the eCRF. If subject’s withdrawal is due to safety reason, for example an (S)AE, the 
subject will be observed until complete resolution of the (S)AE. Any sequel will have to be reported. 
Subjects will be contacted 1 month after the complete resolution of the case before closing the subject’s 
eCRF. Report of this contact will be recorded in the eCRF. 
 
Investigator will withdraw the subject from the clinical trial in case of pregnancy (Section 9.2.1.1.3). In 
this case, the Investigator will immediately inform the Sponsor and will fill in a pregnancy notification 
form in the eCRF. If subject’s withdrawal is due to pregnancy occurring during the clinical trial, both 


 
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mother and fetus will be observed until childbirth, upon subject’s consent. The Investigator will collect 
all follow-up information on the outcome and send it to a Sponsor representative. 
 
Subject’s withdrawal will have to be considered by the Investigator in case of non-compliance to the 
clinical trial procedures, in case of smoking start and in case of use of forbidden concomitant 
medications. In this case, the Investigator will inform the Sponsor of this deviation to the protocol; the 
Sponsor will take the final decision to withdraw or not the subject from the clinical trial. If the subject 
is withdrawn, no new data will be collected from the trial subject, but data already collected will be 
cleaned before subject’s eCRF closure. 
 
For any withdrawals after clinical trial entry, i.e. from Visit 1 randomization, the Investigator will obtain 
all the required details and document the reason(s) for discontinuation. In addition, the Investigator will 
schedule, if possible, an early termination visit (Section 9.1.7).  
 
If the reason for withdrawal of a subject is an AE, the event or laboratory test leading to the subject’s 
withdrawal will be recorded. The Investigator will make thorough efforts to clearly document the 
outcome. 
 
 
Subject Replacement Policy 
Randomized subjects will be replaced only in case of a withdrawal proportion in excess of 15%.  
Any replacement subject will be assigned to the same treatment sequence as the withdrawn subject. 
Subjects withdrawn from the clinical trial for safety reasons will not be replaced. 
 
10.6. Termination of the Clinical trial 
If the clinical trial is terminated prematurely or suspended, the appropriate IEC/IRB and regulatory 
authority(ies) will be promptly informed of the termination or suspension and will be provided the 
reason(s) for the termination or suspension. All obligations and responsibilities of the Sponsor and the 
Investigator following GCP, national regulations and the Declaration of Helsinki will remain in force if 
the clinical trial is terminated prematurely. 
 
 
Premature Termination of the Clinical Trial 
Both the Investigator and the Sponsor reserve the right to terminate the clinical trial at any time. In this 
event, any necessary procedures will be arranged on an individual basis after review and consultation 
by both parties. In terminating the clinical trial, the Sponsor and the Investigator will ensure that 
adequate consideration is given to the protection of the subjects' interests. 
Early termination of the clinical trial as a whole or by trial site may apply for the reasons detailed in the 
following subsections: 
 
 Early Termination of the Clinical Trial 
At any time, the clinical trial as a whole will be terminated prematurely if new toxicological or 
pharmacological findings or safety reports invalidate the earlier positive benefit-risk assessment.  
Significant worsening of the benefit/risk ratio of AQ001S 0.125 mg/ml will lead to reevaluate the 
appropriateness to continue, hold or stop the clinical trial.  
 
 Early Termination of Trial Site 
At any time, the clinical trial can be terminated at the trial site if: 
• 
The trial site cannot comply with the requirements of the protocol, 


 
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• 
The trial site cannot comply with GCP standards, 
• 
The trial site cannot comply with applicable Good Manufacturing and Distribution Practice, 
• 
The trial site's first subject is not recruited within 2 weeks after initiation of the trial site, 
• 
The required recruitment rate is not met. 
 
Should the clinical trial be prematurely terminated, all trial materials (completed, partially completed, 
and blank eCRFs, IMPs, etc.) must be returned to the Sponsor. 
 
10.7. DATA HANDLING AND RECORD KEEPING 
 
Source Data and Records 
Source data are defined as all information related to clinical findings, observations, or other activities in 
the clinical trial, written down in original records or certified copies of original records, allowing 
reconstruction and evaluation of the clinical trial. 
The Investigator will maintain adequate source records (e.g., case histories or subject files for each 
subject enrolled). Source records should be preserved for the maximum period of time required by local 
regulations. 
For each subject enrolled, the Investigator will indicate in the source record(s) that the subject 
participates in this clinical trial. 
All data entered in the eCRF must be supported by source data in the subject records. 
The Investigator will permit trial-related monitoring, audit(s), IEC/IRB review(s), and regulatory 
inspection(s), by providing direct access to the source data records. 
The Investigator may authorize site staff (e.g., sub-Investigators, nurses) to enter clinical trial data into 
the eCRF. This must be documented in the delegation of authority log signed by the Investigator. 
 
 
Case Report Forms 
For each subject enrolled, an electronic case report form (eCRF) will be completed within the electronic 
data capture (EDC) system and approved by the Investigator or an authorized sub-Investigator. 
 
Trial site staff (e.g., study nurse) will be responsible for entering subject data into the validated EDC 
system. All site staff will be trained on the EDC system and trial specific eCRFs prior to receiving access 
to the live database for data entry. 
 
The site is also provided with the approved eCRF completion guidelines which will assist in data entry 
and data issues/questions. The site will be notified once the database is active to begin data entry. 
Additional site training may be provided as refreshers throughout the clinical trial, if needed. All people 
allowed to enter or change eCRF data must be listed in the delegation of authority log. 
 
Changes to Case Report Form Data 
Monitors will perform source data verification (SDV) as defined for the clinical trial. 
If any errors or discrepancies in the eCRFs are found during data entry or review, discrepancies will be 
generated automatically within the EDC system, and 'manual' queries will be created by either a monitor 
or Data Management. 
Discrepancies and queries can only be corrected by the Investigator(s) or other authorized site staff. An 
audit trail will document all changes to the data over the entire clinical trial period. If the reason for a 
change is not obvious, a comment must be supplied in the query's response, stating the reason for the 
change, prior to closing. The clinical trial monitor should provide guidance to Investigator(s) and the 
Investigator(s)' designated representatives on making such corrections. 


 
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Once queries have been resolved by the site staff, the resolutions are assessed by Data Management. If 
the query response provided confirms the data as correct, the discrepancy will be closed. If the response 
does not adequately address the question raised, a new query will be issued for further clarification. 
Manual checks are performed, and programs are run throughout the clinical trial until the data is clean 
and the database is ready for lock. All discrepancies will be resolved prior to database lock. There will 
be a final run of the programmed checks to ensure all discrepancies are closed out, SDV will be 
confirmed as complete by the monitor, and all eCRFs will be approved by the Investigator prior to 
database lock. 
 
 
Investigator’s Site File 
The Investigator is responsible for maintaining all records to enable the conduct of the clinical trial to 
be fully documented. Essential documents as required by GCP guidelines and regulations (e.g., copies 
of the protocol, trial approval letters, all original informed consent forms, site copies of all eCRFs, IMP 
dispensing and accountability logs, correspondence pertaining to the clinical trial, etc.) should be filed 
accurately and kept by the Investigator for the maximum period of time required by local regulations. 
The Investigator is responsible for maintaining a confidential subject identification code list, which 
provides the unique link between named source records and eCRF data for the Sponsor. The Investigator 
must arrange for the retention of this confidential list for the maximum period of time required by local 
regulations. 
No clinical trial document should be destroyed without prior written agreement between the Investigator 
and the Sponsor. Should the Investigator elect to assign the trial documents to another party, or move 
them to another location, the Sponsor must be notified in writing and will have to give its formal written 
agreement prior to implementing such change. 
 
Provision of Additional Information 
On request, the Investigator will supply the Sponsor with additional data relating to the clinical trial, or 
copies of relevant source records, ensuring that the subject's confidentiality is maintained. In case of 
particular issues or governmental queries, it is also necessary to have access to the entire clinical trial 
records, provided that the subject's confidentiality is protected in accordance with applicable regulations. 
 
 
Safety Monitoring  
The Sponsor Pharmacovigilance Manager, the Safety Monitor and the Investigator will review relevant 
data periodically during the clinical trial and will give advice on the continuation, modification, or 
termination of the clinical trial. The benefit/risk of AQ001S 0.125 mg/ml (Section 6.3.1 and described 
in more details in the AQ001S’ IB) will be reevaluated by the Sponsor Pharmacovigilance Manager at 
each SAE report. His/her evaluation and clinical justification will be entered in the eCRF in a text section 
of the AE reporting form. In case his/her evaluation leads to a modification of the previous benefit/risk, 
the Sponsor and Investigator will decide together of the corrective actions that need to be implemented.  
An independent data monitoring committee will not be established by the Sponsor for the clinical trial.  
A written clinical trial-specific safety plan will define in detail the responsibilities and procedures used 
for recording, reporting and assessing AE and any safety information during the clinical trial conduct.  
 
11. STATISTICAL METHODS AND SAMPLE SIZE 
11.1. Determination of Sample Size 
A total of 24 subjects will be recruited into the clinical trial. Considering potential dropouts, a total of 
20 subjects are anticipated to complete the clinical trial. 
 








 
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Populations for Analysis 
Efficacy (EFF) population 
The EFF population encompasses all subjects that completed both treatment periods and for which the 
primary efficacy parameter PC20 is available at baseline (Visit 1) and after both treatment periods (Visit 
2 and Visit 4). 
The EFF population is a per-protocol set, in the sense that it is a subset of the full analysis set, where 
subjects with major protocol deviations which may have an impact on the evaluation of the primary 
clinical trial outcome parameter will be excluded. 
Examples of major and minor protocol deviations will be described in the SAP. 
A final decision about the classification of protocol deviations and their consequences regarding 
assignment of subjects to analysis sets will be made during the data review meeting. Decisions and 
outcome will be approved by the Sponsor. 
 
The safety (SAF) population will include all randomized subjects who received at least one dose of IMP 
(AQ001S 0.125 mg/ml or comparator).  
 
Pharmacodynamics (PK) population 
The PK population includes all subjects, which have two full evaluable PK profiles and did not have 
protocol deviations relevant to the evaluation of PK.  
 
 
Primary Efficacy Endpoint and Analysis Plan 
The primary efficacy analysis will be evaluated on the EFF population. The primary efficacy endpoint 
will be the change from baseline in PC20 after each treatment period (baseline is defined at Visit 1). 
 
For primary efficacy analysis, the mean PC20 changes from baseline between AQ001S 0.125 mg/ml 
and the comparator will be compared by analysis of covariance (ANCOVA) for crossover design. A p-
value will be calculated for the difference of the parameter between AQ001S 0.125 mg/ml and 
comparator. If this p-value is significant and the difference prefers AQ001S 0.125 mg/ml, superiority is 
shown. 
Additionally, an adjusted 95%-confidence interval for the mean difference between AQ001S 0.125 
mg/ml and the comparator will be obtained by the ANCOVA.  
 
 
 
Safety Endpoints and Analysis Plan 
The safety will be evaluated collecting the following information: 
• 
AEs, SAEs, including asthma exacerbations, 
• 
General tolerability: vital signs, ECG, physical examination, 
• 
Laboratory parameters: hematology, biochemistry and urinalysis,  
• 
Local tolerability:  
o Increased bronchial irritability,  
o Paradoxical bronchospasm, 
o Oropharyngeal examination (e.g. vocal cord myopathy, fungal infection), 
• 
FEV1 and FVC, measured by spirometry,  
• 
HPA axis function: assessment of urinary cortisol/creatinine ratio in first waking urine. 
 
The safety endpoints will be analyzed for the SAF population. Safety analyses will be descriptive in 
nature and no hypothesis testing is planned. 
 


 
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The treatment groups will be compared in a descriptive way. Tabulations of frequencies/proportions 
will be used for the evaluation of categorical (qualitative) data, and tabulations of descriptive statistics 
(at least number of observations, mean, standard deviation, median, minimum and maximum) will be 
used to analyze continuous (quantitative) data. 
 
Measurements (such as ECG, vital signs, or laboratory parameters) or AEs will be assigned to treatments 
based on the actual treatment at the planned time of the measurement or on the recorded time of AE 
onset (concept of treatment emergent AEs). 
 
AEs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA). Frequency, 
severity and causal relationship of AEs will be tabulated by treatment, system organ class and preferred 
term. Absolute and relative frequencies of subjects with at least one AE will be presented and the total 
number of AEs will be given by treatment group. SAEs and other significant AEs will be evaluated 
separately. 
 
 
Pharmacokinetic Endpoints and Analysis Plan 
The PK endpoint which is the PK profile of budesonide in plasma will be evaluated through the PK 
parameters listed in Section 9.2.3.4, calculated for each treatment period. 
 
The PK analysis will be performed based upon the PK set. The primary focus of the statistical analysis 
of this section is to describe and characterize the PK of AQ001S 0.125 mg/ml and the comparator. Thus, 
there are no formal hypotheses being tested. 
 
The individual plasma concentrations of budesonide will be listed and tabulated by treatment and time 
point. 
Descriptive statistics for the PK parameters will be calculated by treatment. The tables will include at 
least the number of observations, mean, standard deviation, median, minimum and maximum, and 
additionally the geometric mean and the geometric coefficient of variation (gCV). 
 
 
Secondary Efficacy/Pharmacodynamic Endpoints and Analysis 
Plan 
The secondary efficacy/PD endpoints will be the changes from baseline, after each treatment period, 
in: 
• 
Symptom scores,  
• 
Use of as-needed reliever medications,  
• 
FeNO, 
• 
Blood eosinophil count, 
• 
FEV1, 
• 
FIVC. 
 
The secondary efficacy and PD analysis will be performed on the SAF population. Those parameters 
will also be evaluated descriptively. Therefore, tabulations of frequencies/proportions will be used for 
the evaluation of categorical (qualitative) data, and tabulations of descriptive statistics (at least number 
of observations, mean, standard deviation, median, minimum and maximum) will be used to analyze 
continuous (quantitative) data. 
 
 
Handling of Data and Specific Analyses 
 Handling of Dropouts or Missing Data 


 
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In general, missing data will not be imputed. Calculations pertaining to person-year computations will 
be based on observed values only. Only in case of missing body weight will the last available weight 
measurement be used for calculating the dose per kg bodyweight (last observation carried forward). 
 
 Adjustments for Covariates 
The ANCOVA models used for primary analysis will be adjusted for the baseline value, treatment 
sequence, period and within-subject sequence.  
 Active Control Clinical Trials Intended to Show Non-Inferiority 
Not applicable. 
 
 Data Presentation 
Individual response data and other relevant clinical trial information will be presented in tables, with 
supporting individual data listings. 
 
 Interim Analysis 
No interim analysis will be performed in the framework of this clinical trial. AQ001S 0.125 mg/ml’s 
benefit/risk ratio will be evaluated on an ongoing basis by the Sponsor’s Pharmacovigilance Manager. 
Any modification of the benefit/risk ratio will be immediately communicated by the Sponsor’s 
Pharmacovigilance Manager to the Investigator. 
 
12. ETHICAL/REGULATORY LEGAL AND ADMINISTRATIVE ASPECTS 
12.1. Ethical/Regulatory Framework 
This clinical trial will be conducted in accordance with the ethical principles laid down in the Declaration 
of Helsinki. The clinical trial protocol and any subsequent amendment(s) will be submitted to an 
Independent Ethics Committee (IEC)/Institutional Review Board (IRB) and to the Regulatory Authority. 
The clinical trial will be conducted in compliance with the protocol, GCP guidelines, and applicable 
regulatory requirements. 
The regulatory application or submission for regulatory approval will be made by the Sponsor or 
designated third party (e.g., a contract research organization [CRO]) as required by national law. 
 
12.2. Approval of Clinical Trial Documents 
The clinical trial protocol, a sample of the subject information and informed consent form, any other 
materials provided to the subjects, and further requested information will be submitted by the Sponsor 
or the Investigator to the appropriate IEC/IRB and the Regulatory Authority. The clinical trial must be 
approved by the IEC/IRB and the Regulatory Authority before any IMP may be shipped to the trial sites 
and before any subject is exposed to a trial related procedure. 
The Sponsor, the Investigator, and any third party (e.g., CRO) involved in obtaining approval must 
inform each other in writing that all ethical and legal requirements have been met before the first subject 
is enrolled in the clinical trial. 
12.3. Subject Information and Informed Consent 
The Investigator will obtain freely given written consent from each subject after an appropriate 
explanation of the aims, methods, anticipated benefits, potential hazards, and any other aspect of the 
clinical trial which is relevant to the subject's decision to participate. 
 


 
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The informed consent form must be signed, with name, date and time noted by the subject, before the 
subject is exposed to any trial-related procedure, including screening tests for eligibility. 
 
The Investigator will explain that the subjects are completely free to refuse to enter the clinical trial or 
to withdraw from it at any time, without any consequences for their further care and without the need to 
justify. The Investigator will fill in the informed consent section of the eCRF for each subject enrolled. 
Each subject will be informed that his/her medical (source) records may be reviewed by the clinical trial 
monitor, a quality assurance auditor, or a health authority inspector, in accordance with applicable 
regulations, and that these persons are bound by confidentiality obligations. 
 
 
Subject’s Data Protection 
Besides the subject’s agreement to participate in the clinical trial, the informed consent form will also 
give to the subject detailed information on the intended treatment of his/her data. The EU GDPR is 
indeed applicable to any clinical trial. The subject’s consent will include both the consent to participate 
in the clinical trial and his/her explicit consent to the intended treatment of his/her data. 
In consequence, the Sponsor has the obligation to implement the appropriate technical and 
organizational measures to ensure and be able to demonstrate that the personal data are processed in 
accordance with the data protection rules (Article 24 of GDPR). The Sponsor will ensure compliance to 
all the data protection rules in GDPR, including ensuring respect of the data protection principles, 
appointing a responsible person for Data Protection, maintaining records of processing activities and 
facilitating the exercise of individuals’ rights. The name and contact details of the responsible person 
for Data Protection will be mentioned in the informed consent form. 
In addition, the subjects will be informed on the purposes of the processing as well as the legal bases of 
this data treatment (legal obligation of the sponsor to report safety & efficacy data to the competent 
authorities [Article 6(1)(c) of GDPR in conjunction with Article 9(2)(i)] and explicit consent of the 
subject [Article 6(1)(a) of GDPR in conjunction with Article 9(2)(a)] [56,57]), on the identity of the 
controller (sponsor), its contact details and, where applicable, those of the controller’s representative; 
the recipients or categories of recipients of the personal data, if any;  the finalities of the treatment of 
his/her data, the storage period (or if not possible, criteria used to determine that period), the clinical 
research purposes, the fair processing of his/her data, and on his/her data subjects rights (access; 
rectification; erasure; restriction on processing; objection to processing and data portability) as required 
by the EU GDPR. The subjects will be informed of their right to lodge a complaint with a supervisory 
authority. Subjects will also be informed that their data could be transferred to another party in another 
country. In case of such data transfer, the Sponsor will maintain high standards of confidentiality and 
protection of subject personal data and ensure that any data processor used is reliable and can make 
sufficient data protection guarantees (implementing appropriate technical and organizational measures). 
The Sponsor will establish Standard Contractual Clauses (SCC) on data transfer ensuring that all parties 
working with the sponsor are committed to protecting and keeping the subject’s personal data 
confidential. The subjects will also be informed that the treatment of his/her data will not give him/her 
any rights for any other compensation than the one linked to their participation to the clinical trial.  
Finally, as well as for GCP compliance, GDPR compliance is, in Belgium, in the framework of a clinical 
trial, the competence of the consulted IEC/IRB. The responsibility for the GDPR compliance check of 
the clinical trial dossier lies with the sponsor. 
12.4. Confidentiality of Subject Data 
The Investigator will ensure that the subject's confidentiality is preserved. Per GCP, on eCRFs or any 
other documents submitted to the Sponsor, the subjects will not be identified by their names, but by a 
unique subject identifier (data pseudonymization) (Section 8.4.5). Documents not intended for 


 
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CONFIDENTIAL 
submission to the Sponsor, i.e., the confidential subject identification code list, original consent forms, 
and source records, will be maintained by the Investigator in strict confidence. 
 
13. QUALITY CONTROL AND QUALITY ASSURANCE 
13.1. Periodic Monitoring 
The monitor will contact and visit the Investigator periodically to review all trial-related source 
data/records, verify the adherence to the protocol and the completeness, correctness and accuracy of all 
eCRF entries compared to source data. The Investigator will co-operate with the monitor to ensure that 
any discrepancies identified are resolved. 
 
A monitoring plan will be approved by the Sponsor before trial site initiation. 
 
The monitor must be given direct access to source documents (original documents, data and records). 
Direct access includes permission to examine, analyze, verify, and reproduce any records and reports 
that are important to the evaluation of the clinical trial. Source data will be available for all data in the 
eCRFs, including all laboratory results.  
 
13.2. Audit and Inspection 
The Investigator will make all trial-related source data and records available to a qualified quality 
assurance auditor mandated by the Sponsor, after reasonable notice, or to IEC/IRB/regulatory 
inspectors. The main purpose of an audit or inspection is to confirm that the rights and welfare of the 
subjects have been adequately protected, and that all data relevant for the assessment of safety and 
efficacy of AQ001S 0.125 mg/ml have been reported to the Sponsor. 
 
14. REPORTING AND PUBLICATION 
14.1. Clinical Study Report 
A clinical study report (in accordance with relevant guidelines and the Sponsor's procedures) will be 
prepared by the Sponsor after completion of the clinical trial. The Principal Investigator will approve 
the final clinical study report after review. 
 
14.2. Publication Policy 
The results of this clinical trial will be shared in the European Union Clinical Trials Register and in 
other mandatory registers as appropriate and according to the regulatory requirements. 
The results of this clinical trial may be published or presented at scientific meetings. 
If this is envisaged by the Investigator, the Investigator agrees to inform the Sponsor and to submit for 
review and approval all manuscripts or abstracts to the Sponsor prior to submission to an editorial board 
or scientific review committee. This will allow the Sponsor to protect proprietary information and to 
provide comments based on information that may not yet be available to the Investigator. 
Authorship will be determined by mutual agreement. 
The publication and disclosure policies are addressed in a separate agreement with the trial site. 
 
15. LIABILITIES AND INSURANCE 
In order to cover any potential damage or injury occurring to a subject in association with the IMPs or 
participation in the clinical trial, the Sponsor will contract insurance in accordance with local 
regulations. 
The Investigator is responsible for dispensing the IMPs according to this protocol and for the secure 
storage and safe handling of the IMPs throughout the clinical trial. 
The financing and insurance details are addressed in a separate agreement with the trial site.  
 
 


 
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16. REFERENCES 
 
1.  
Mayo Clinic. Asthma. Available at: https://www.mayoclinic.org/diseases-
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Global Initiative for Asthma (GINA). Global Strategy for Asthma Management and 
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Hou S, Wu J, Li X, Shu H. Practical, regulatory and clinical considerations for development of 
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Le Brun PP, de Boer AH, Heijerman HG, Frijlink HW. A review of the technical aspects of 
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Dahl R. Systemic side effects of inhaled corticosteroids in patients with asthma. Respir Med 
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AstraZeneca. PULMICORT FLEXHALERTM Prescribing Information. 2010.  
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AstraZeneca UK Limited. PULMICORT RESPULES nebuliser suspension. Summary of 
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Eurogenerics. Budesonide EG suspension pour inhalation par nébuliseur. Summary of Product 
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AstraZeneca. PULMICORT®, suspension pour inhalation par nébuliseur. Prescribing 
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AstraZeneca. PULMICORT TURBOHALER 200, 200 microgrammes/dose, poudre pour 
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BTS/SIGN, British Thoracic Society and Scottish Intercollegiate Guidelines Network. SIGN 
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National Asthma Council Australia. Australian Asthma Handbook: National guidelines for 
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NICE, National Institute for Health and Care Excellence advice. Key therapeutic topic: High-
dose inhaled corticosteroids in asthma (KTT5) - 15 January 2015. 2018;  
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Paterniti M. Evaluation of the Systemic Effects of Locally Acting Corticosteroids, US Food 
and Drug Administration (FDA) Public Workshop: Advancing the Development of Pediatric 
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GINA (Global Initiative for Asthma). Global Strategy for Asthma Management and 


 
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Prevention. 2020. Available at: https://ginasthma.org/gina-reports/. Accessed 15 January 2021. 
21.  
BUDESONIDE SANDOZ, suspension pour inhalation par nébuliseur en récipient unidose. 
Résumé des caractéristiques du Produit (SmPC). 2018. Available at: http://base-donnees-
publique.medicaments.gouv.fr/affichageDoc.php?specid=63036604&typedoc=R. Accessed 15 
January 2021. 
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AstraZeneca UK Limited. PULMICORT RESPULES nebuliser suspension SmPC. electronic 
Medicines Compendium (eMC). 2017.  
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Eurogenerics. Budesonide EG suspension pour inhalation par nébuliseur. SmPC. 2018.  
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Martin AR, Finlay WH. Nebulizers for drug delivery to the lungs. Expert Opin Drug Deliv 
2015; 12:889–900.  
25.  
Kesser KC, Geller DE. New Aerosol Delivery Devices for Cystic Fibrosis. Respir Care 2009; 
54:754–768.  
26.  
Najlah M, Parveen I, Alhnan MA, et al. The effects of suspension particle size on the 
performance of air-jet, ultrasonic and vibrating-mesh nebulisers. Int J Pharm 2014; 461:234–
241.  
27.  
Dufour G, Evrard B, de Tullio P. 2D-Cosy NMR Spectroscopy as a Quantitative Tool in 
Biological Matrix: Application to Cyclodextrins. AAPS J 2015; 17:1501–1510.  
28.  
Maes P, Cataldo D, Evrard B, Dufour G, De Tullio P. Cyclodextrins and budesonide derivative 
compositions and methods. World Intellectual Property Organization (WIPO)/Patent 
Cooperation Treaty (PCT), WO2015144938. 2015. Available at: 
https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2015144938&_cid=P20-K4Y083-
16767-1. Accessed 15 January 2021. 
29.  
Dufour G. Intérêts des cyclodextrines dans la formulation de préparations galéniques 
antiasthmatiques. 2016; :211.  
30.  
Hastings C. Request for intrathecal delivery of HPBCD for Niemann-Pick type C patients. C. 
Hastings, M.D. Principal Investigator Department of Pediatric Hematology Oncolog. 2010. 
Available at: http://addiandcassi.com/wordpress/wp-content/uploads/Hempel-Cyclodextrin-
Intrathecal-FDA-Filing-2010-Aug.pdf. Accessed 7 January 2020. 
31.  
European Medicines Agency (EMA), Inspections, Human Medicines Pharmacovigilance and 
Committees Division. PDCO monthly report of opinions on paediatric investigation plans and 
other activities. EMA/PDCO/853813/2016. Available at: 
https://www.ema.europa.eu/en/events/paediatric-committee-pdco-13-16-december-2016. 
Accessed 26 January 2021. 
32.  
Hempel C. FDA Approves Request for New Cyclodextrin Treatment For Niemann Pick Type 
C The Addi & Cassi Fund. 2010. Available at: http://addiandcassi.com/fda-approves-request-
cyclodextrin-treatment-niemann-pick-type/. Accessed 7 January 2020. 
33.  
Megías-Vericat JE, García-Robles A, Company-Albir MJ, et al. Early experience with 
compassionate use of 2 hydroxypropyl-beta-cyclodextrin for Niemann–Pick type C disease: 
review of initial published cases. Neurol Sci 2017; 38:727–743.  
34.  
Davis ME, Brewster ME. Cyclodextrin-based pharmaceutics: Past, present and future. Nat Rev 
Drug Discov 2004; 3:1023–1035.  
35.  
Cataldo D, Louis R. Tolérance de la bétacyclodextrine administrée par inhalation en dose 
unique et en doses répétées chez le volontaire sain. EudraCT 2005-005673-30. ULiège. Data on 
file. 2006;  
36.  
Cataldo D, Louis R. Étude d’efficacité de l’hydroxypropyl-béta-cyclodextrine administrée par 
inhalation en doses répétées chez l’asthmatique allergique. EudraCT 2005-005673-30. ULiège. 
Data on file. 2008.  
37.  
Dufour G, Bigazzi W, Wong N, et al. Interest of cyclodextrins in spray-dried microparticles 
formulation for sustained pulmonary delivery of budesonide. Int J Pharm 2015; 495:869–878.  
38.  
AstraZeneca. PULMICORT, suspension pour inhalation par nébuliseur. Prescribing 
information (Résumé des Caractéristiques du produit). FAMPH. 2018.  
39.  
Masoli M, Holt S, Weatherall M, Beasley R. Dose-response relationship of inhaled budesonide 
in adult asthma: a meta-analysis. Eur Respir J 2004; 23:552–558.  


 
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40.  
Brand PLP. Inhaled corticosteroids reduce growth. Or do they? Eur Respir J 2001; 17:287–294.  
41.  
Crapo RO, Casaburi R, Coates AL, et al. Guidelines for Methacholine and Exercise Challenge 
Testing - 1999. Am J Respir Crit Care Med 2000; 161:309–329.  
42.  
Duke University Health System - Human Research Protection Program. Blood drawing for 
human subject research. 2012;  
43.  
Dana Farber / Harvard Cancer Center. Guidance on Maximum Blood Draw for Research 
Purposes. 2012;  
44.  
Lipworth BJ, Sims EJ, Das SK, Buck H, Paterson M. Dose-response comparison of budesonide 
dry powder inhalers using adenosine monophosphate bronchial challenge. Ann Allergy, 
Asthma Immunol 2005; 94:675–681.  
45.  
EMA. Guideline on the clinical investigation of medicinal products for the treatment of asthma. 
2015; :1–21.  
46.  
Dean BW, Birnie EE, Whitmore GA, et al. Between-visit variability in FEV1 as a diagnostic 
test for Asthma in adults. Ann Am Thorac Soc 2018; 15:1039–1046.  
47.  
EMA. Guideline on the Requirements for Clinical Documentation for OIP Including the 
Requirements for Demonstration of Therapeutic Equivalence Between Two Inhaled Products 
for Use in the Treatment of Asthma and COPD... 2009;  
48.  
McIntyre HD, Mitchell CA, Bowler SD, Armstrong JG, Wooler JA, Cowley DM. Measuring 
the systemic effects of inhaled beclomethasone: Timed morning urine collections compared 
with 24 hour specimens. Thorax 1995; 50:1280–1284.  
49.  
Lipworth BJ, Seckl JR. Measures for detecting systemic bioactivity with inhaled and intranasal 
corticosteroids. Thorax 1997; 52:476–482.  
50.  
Pavord ID, Agusti A. Blood eosinophil count: A biomarker of an important treatable trait in 
patients with airway disease. Eur Respir J 2016; 47:1299–1303.  
51.  
Graham BL, Steenbruggen I, Barjaktarevic IZ, et al. Standardization of spirometry 2019 update 
an official American Thoracic Society and European Respiratory Society technical statement. 
Am J Respir Crit Care Med 2019; 200:E70–E88.  
52.  
Santanello NC, Barber BL, Reiss TF, Friedman BS, Juniper EF, Zhang J. Measurement 
characteristics of two asthma symptom diary scales for use in clinical trials. Eur Respir J 1997; 
10:646–51.  
53.  
Juniper EF, O’Byrne PM, Guyatt GH, Ferrie PJ, King DR. Development and validation of a 
questionnaire to measure asthma control. Eur Respir J 1999; 14:902–907.  
54.  
Kosinski M, Bayliss MS, Sc M, Turner-bowker DM, Ph D. Asthma Control Test : A User ’ s 
Guide TM. 2008; 2008.  
55.  
Kraan J, Koëter GH, van der Mark TW, et al. Dosage and Time Effects of Inhaled Budesonide 
on Bronchial Hyperreactivity. Am Rev Respir Dis 1988; 137:44–48.  
56.  
European Commission Directorate-General for Health and Food Safety. Health systems and 
products, Medical products – quality, safety and innovation. Question and Answers on the 
interplay between the Clinical Trials Regulation and the General Data Protection Regulation, 
April 2019.  
57.  
European Data Protection Board (EDPB). Opinion 3/2019 concerning the Questions and 
Answers on the interplay between the Clinical Trials Regulation (CTR) and the General Data 
Protection regulation (GDPR) (art. 70.1.b)). Adopted on January 2019.  
 
 


 
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17. APPENDICES 
APPENDIX 1: PARI LC SPRINT NEBULIZER – INSTRUCTIONS FOR USE 
 
See Appended document. 
 
 


 
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APPENDIX 2: PARI BOY CLASSIC COMPRESSOR – INSTRUCTIONS FOR USE 
 
See Appended document. 
 
 
 








d£ 
AQ-PRO-005 
aquilon 
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18.PROTOCOL SIGNATURES
Signature of the Sponsor's Representatives 
This clinical trial is intended to be conducted in compliance with the protocol, Good Clinical 
Practice and applicable regulato1y requirements. 
 
Chief Executive Officer on behalf of the 
Signature 
Sponsor 
Clinical and Project Leader 
on behalf of the Sponsor 
Signature 
CONFIDENTIAL 
___ / ___ / __ _
Date (DD/MMM/YYYY) 
___ / ___ / __ _
Date (DD/MMM/YYYY) 


 
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Signature of the Principal Investigator 
 
 
I have read the protocol carefully and hereby confirm that it is scientifically and ethically sound. 
I agree to implement and conduct this clinical trial diligently, in strict compliance with the 
protocol, ICH (step 5) ‘Guidance on Good Clinical Practice [E6]’ and all applicable laws and 
regulations.  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
______/______/_______ 
                                             
Principal Investigator 
Signature 
Date (DD/MMM/YYYY) 
 
 
 




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19.
DOCUMENT HISTORY
VERSION 
HISTORY 
ISSUE DATE 
1.0 
Creation 
28/FEB/2020 
Review after IEC feedback (Comite 
d'Ethique Hospitalo-Facultaire 
Universitaire de Liege) dated 24 March 
2020: 
-
Changes in title
-
-
Emphasis on the primaiy efficacy
pai·aineter, e.g. PC20, explaining the
need for this assessment at Visits 1
(baseline), Visit 2 ( end of first
treatment period) and Visit 4 (end of
second treatment period)
-
Add of minimum BMI as inclusion
criterion (18.5 kg/m2)
-
Amendment of maximum BMI from
2.0 
30 to 29 kg/m2 as inclusion criterion
21/AUG/2020 
-
Addition of HIV and COVID-19 
infections as exclusion criterion
-
Add of assessment of COVID-19 
symptoms in physical and clinical 
exan1.ination
-
Ad of         timepoint in PK blood 
collection
-
Clarifications where needed
-
Change of compressor for the 
nebulizer used: Pai·i Boy Classic, 
which is compliant with the new 
Medical Device Regulation, instead 
of Pai·i Turbo Boy SX
CONFIDENTIAL 


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3.0 
Review before clinical trial start: 
-
Estimated clinical start postponed to
June 2021
-
Addition of the sponsor’s medical
advisor for the trial contacts
-
Amendment to the name of the
company doing the packaging and
labeling of IMPs
-
Change of Bioanalysis Laboratory
-
Clarification in the protocol of MCh
challenge test to ensure subject’s
safety during the test (PC20
endpoint)
-
Amendment of email address for
AEs reporting to be in line with
Safety Management Plan
-
Other clarifications where needed
15/JUN/2021 

SAP_001.pdf: 
Statistical Analysis Plan for Project BOREAS 
Protocol identification No.: 
Protocol version: 
Protocol date: 
EudraCT number: 
CRO study ID: 
Study title: 
lnvestigational product: 
Development phase: 
Sponsor: 
CRO: 
Statistical Analysis Plan 
AQ-PRO-005 
3.0 
15-JUN-2021
Principal investigator 
Author of SAP: 
SAP version / status: 
2019-002849-38 
BOREAS 
A prospective, active-controlled, randomized, open label, 
single-center, multiple dose, two-period crossover clinical 
trial to assess the efficacy, safety and pharmacokinetics 
of a budesonide inhalation solution (AQ001 S) compared 
to a budesonide inhalation suspension ( comparator) in 
adults with mild asthma. 
AQ001S 0.125 mg/ml (budesonide 0.125 mg/ml inhalation 
solution) 
1/2a 
Aquilon Pharmaceuticals S.A. (Aquilon) 
Quai de la Boverie, 59 
4020 Liege 
BELGIUM 
D r. 
Trial site name: 
Senior Biostatistician 
Final v2.0 
Page 
1 of 27 
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Statistical Analysis Plan for Project BOREAS 
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1 Version Control 
Version 
Date 
Section 
Description 
Final v1.0 
06-APR-2022
ALL 
Initial release 
Final v2.0 
29-SEP-2022
8 
Added log-transformation of the primary endpoint 


Statistical Analysis Plan for Project BOREAS 
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2 Statistical Analysis Plan Approval 
Author approval: 
Senior Biostatistician  
 
Date (dd/mmm/yyyy) 
Name 
Signature 
Statistics approval: 
Head of Biostatistics  
 
Date (dd/mmm/yyyy) 
Name 
Signature 
CRO approval: 
Clinical Research Professional 
 
Date (dd/mmm/yyyy) 
Name 
Signature 
Sponsor approval: 
Clinical and Project Leader 
Aquilon Pharmaceuticals S.A. 
Quai de la Boverie, 59 
4020 Liège 
Belgium  
 
Date (dd/mmm/yyyy) 
Name 
Signature 


Statistical Analysis Plan for Project BOREAS  
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3 Table of Contents 
1 
Version Control ................................................................................................................ 3 
2 
Statistical Analysis Plan Approval .................................................................................... 4 
3 
Table of Contents ............................................................................................................ 5 
4 
List of Abbreviations ........................................................................................................ 7 
5 
Introduction ..................................................................................................................... 9 
6 
Study Objectives and Endpoints .................................................................................... 10 
6.1 
Study Objectives .................................................................................................... 10 
6.1.1 
Primary objectives ........................................................................................... 10 
6.1.2 
Secondary objectives ...................................................................................... 10 
6.2 
Study overview ....................................................................................................... 10 
6.2.1 
Study design ................................................................................................... 10 
6.2.2 
Sample size ..................................................................................................... 12 
6.3 
Study Endpoints ..................................................................................................... 13 
6.3.1 
Primary Efficacy Endpoint ............................................................................... 13 
6.3.2 
Safety Endpoints ............................................................................................. 13 
6.3.3 
Pharmacokinetic Endpoints ............................................................................. 13 
6.3.4 
Secondary Efficacy/Pharmacodynamic Endpoints ........................................... 14 
7 
General Considerations Pertaining to the Statistical Analysis ........................................ 15 
7.1 
Definitions .............................................................................................................. 15 
7.2 
Analysis Sets .......................................................................................................... 16 
7.2.1 
Analysis Set for Efficacy Analyses ................................................................... 16 
7.2.2 
Analysis Set for Safety Analyses ..................................................................... 16 
7.2.3 
Analysis Set for Pharmacokinetic Analyses ..................................................... 16 
7.2.4 
Other Analysis Sets ......................................................................................... 16 
7.3 
DATA SCREENING AND ACCEPTANCE .............................................................. 17 
7.3.1 
Data Handling and Electronic Transfer of Data ................................................ 17 
7.3.2 
Handling of Missing Data ................................................................................. 17 
7.3.3 
Data review meeting ........................................................................................ 17 
8 
Statistical Analysis ......................................................................................................... 18 
8.1 
General principles .................................................................................................. 18 
8.2 
Specifications Related to the Whole Analysis ......................................................... 18 


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8.2.1 
Analysis Sets ................................................................................................... 18 
8.2.2 
Tabulation ....................................................................................................... 18 
8.3 
Subject Disposition ................................................................................................. 19 
8.4 
Demographics and Baseline Characteristics .......................................................... 19 
8.5 
Medical/Surgical History ......................................................................................... 19 
8.6 
Prior and Concomitant Medication .......................................................................... 20 
8.7 
Compliance ............................................................................................................ 20 
8.8 
Efficacy Analysis .................................................................................................... 20 
8.8.1 
Descriptive Analyses of Primary Efficacy Endpoint .......................................... 20 
8.8.2 
Primary Efficacy Analysis ................................................................................ 20 
8.8.3 
Secondary Efficacy/Pharmacodynamic analysis .............................................. 21 
8.8.4 
Subgroup Analyses ......................................................................................... 21 
8.9 
Safety Analysis ....................................................................................................... 21 
8.9.1 
Physical and Clinical Examination ................................................................... 21 
8.9.2 
Vital Signs ....................................................................................................... 22 
8.9.3 
ECG ................................................................................................................ 22 
8.9.4 
Laboratory measurements ............................................................................... 22 
8.9.5 
Adverse Events ............................................................................................... 22 
8.10 Pharmacokinetic analysis ....................................................................................... 22 
9 
Reporting Conventions .................................................................................................. 24 
10 Technical Details ........................................................................................................... 25 
11 List of Planned Contents of Tables, Figures and Listings .............................................. 26 
12 References .................................................................................................................... 27 


Statistical Analysis Plan for Project BOREAS 
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4 List of Abbreviations 
AE 
Adverse event 
av 
Airway volume 
ANCOVA 
Analysis of Covariance 
BMI 
Body mass index 
CRO 
Contract Research Organization 
CT 
Computed Tomography 
D 
Day 
ECG 
Electrocardiogram 
FeNO 
Fractional exhaled Nitric Oxide 
FEV1 
Forced Expiratory Volume in 1 second 
FIH 
First-In-Human 
FIVC 
Forced Inspiratory Vital Capacity 
FVC 
Forced Vital Capacity 
gCV 
Geometric Coefficient of Variation 
gMean 
Geometric Mean 
HPA 
Hypothalamic Pituitary Adrenocortical 
ICH 
International Conference on Harmonisation of Technical Requirements 
for Registration of Pharmaceuticals for Human Use 
ICS 
Inhaled Corticosteroid 
ID 
Identifier 
IMP 
Investigational medicinal product 


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lv 
Lobe volume 
MCh 
Methacholine (chloride) 
PC20 
Concentration of methacholine provoking an FEV1 fall of 20% 
PD 
Pharmacodynamics 
PK 
Pharmacokinetics 
POC 
Proof-Of-Concept 
RMSE 
Root Mean Squared Error 
SAE 
Serious Adverse Event 
SAP 
Statistical Analysis Plan 
TP 
Treatment Period 
V 
Visit 


Statistical Analysis Plan for Project BOREAS 
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5 Introduction 
The BOREAS study (Protocol AQ-PRO-005 version 3.0 from 15 June 2021, hereinafter 
referred to as “the protocol”) was designed to compare the efficacy, the safety and the 
pharmacokinetics of the budesonide inhalation solution AQ001S with those of the comparator 
Budesonide                 (budesonide inhalation suspension) in adults with mild asthma. It is a 
phase 1/2a 
First-In-Human 
(FIH) 
Proof-Of-Concept 
(POC) 
prospective, 
active-
controlled, randomized, open label, single-center, multiple dose, two-period crossover clinical 
trial. 
The purpose of this statistical analysis plan (SAP) is to provide details of the statistical 
analyses that have been outlined in the protocol of the BOREAS study AQ-PRO-005 and to 
ensure that the statistical methodologies that will be used, and the resulting data listings, 
summary tables, figures and report which will be produced are complete and appropriate 
to support valid conclusions regarding the study objectives of the clinical study.  
The SAP follows the principles of the Guidelines ICH (International Conference 
on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human 
Use) Topic E3 [1] and ICH Topic E9 [2].  
The statistical analysis of the data will be the responsibility of                    and will be 
performed by               . Of note, the statistical analysis of the PK data will be the 
responsibility of and will be performed by an external party (                             ). 


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6 Study Objectives and Endpoints 
6.1 Study Objectives 
6.1.1 Primary objectives 
The primary objectives of the clinical trial are: 
•
To compare the efficacy, i.e. the bronchoprotection, of AQ001S 0.125 mg/ml with the
comparator.
•
To assess the safety of AQ001S 0.125 mg/ml.
6.1.2 Secondary objectives 
The secondary objectives are: 
•
To compare the PK of AQ001S 0.125 mg/ml with the comparator
•
To compare the secondary efficacy/PD of AQ001S 0.125 mg/ml with the comparator.
6.2 Study overview 
6.2.1 Study design 
The present trial is a prospective, active-controlled, randomized, open label, single-center, 
multiple dose, two-period crossover clinical trial in mild asthmatic adults (18 to 65 years old), 
who are “inhaled corticosteroid (ICS)”-naïve for minimum 60 days at Screening Visit, 
comparing two IMPs: 
IMP under investigation: 
•
AQ001S 0.125 mg/ml is a budesonide inhalation solution administered by
nebulization once daily.
IMP used as a Comparator: 
•
Budesonide        0.125 mg/ml is a budesonide inhalation 
suspension administered by nebulization once daily.
For each subject, the clinical trial will last 77 to 85 days from Screening Visit, including two 
treatment periods of 29 (+2) days and one washout period of 14 (+2) days, between the 
treatment periods (Figure 1). In case the subject is taking or has taken ICS within the last 60 
days at the time of the Pre-Trial Visit (the visit before or combined to the Screening Visit), the 
total duration might be a maximum of 147 days from Pre-Trial Visit (Table 1.) 
Figure 1. Course of the BOREAS study 




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Table 2: Treatment sequences 
Treatment sequence A 
Treatment sequence B 
Treatment period 1: AQ001S 0.125 mg/ml for 
29 (+2) days 
Treatment period 1: comparator 0.125 mg/ml 
for 29 (+2) days  
Washout for 14 (+2) days 
Washout for 14 (+2) days 
Treatment period 2: comparator 0.125 mg/ml 
for 29 (+2) days  
Treatment period 2: AQ001S 0.125 mg/ml for 
29 (+2) days  
The randomization list will be generated using a pseudo random number generator and a seed 
number that ensures that the allocation is non-predictable and reproducible. The randomization 
list will contain a mirror list to allow for subject replacements. 
This is an open-label clinical trial so there is no blinding. 
This is a single-center study. Hence no pooling of different centers is necessary. 
6.2.2 Sample size 
A total of 24 subjects will be recruited into the trial. Considering potential dropouts, a total of 
20 subjects (10 per treatment sequence) are anticipated to complete the clinical trial. As 
discussed above, randomisation will be in a 1:1 ratio. 
The sample size was calculated considering PC20 as the primary efficacy endpoint, based on 
the internal animal efficacy data of the sponsor and on the work of Lipworth et al. (2005) [5] 
and of Kraan et al. (1988) [6]. Both Lipworth and Kraan studied the response of patients in 
terms of PC20 to various doses of inhaled budesonide, as detailed in the BOREAS protocol. 
The sample sizes obtained based on the dose-response between PC20 and inhaled 
budesonide dose observed by Lipworth et al. and Kraan et al. and using a power (1-beta 
probability) of 80% (0.8) are given in Table 3.  
Table 3 – Sample size calculation based on different assumptions of dose-response between 
PC20 and inhaled budesonide dose 
(power=0.8) 
Required sample size - number of subjects completing 
the trial to show superiority of AQ001S over 
comparator in terms of budesonide dose reduction of: 
-fold 
-fold 
Lipworth et al. 
 
 


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Kraan et al. 
The sample size calculations based on the PC20 as a primary endpoint in BOREAS indicate 
that a sample size of 20 subjects completing the clinical trial is enough to demonstrate an 
effect of AQ001S 0.125 mg/ml over the comparator with a     -fold factor (
        ) as well as with a       -fold factor between AQ001S 0.125 mg/ml and the comparator 
with a power of 80%. 
6.3  Study Endpoints 
6.3.1 Primary Efficacy Endpoint 
The primary efficacy endpoint will be the change from baseline in PC20 after each treatment 
period (baseline is defined at Visit 1). 
For PC20 evaluation, the subjects will perform a methacholine (MCh) challenge test. They 
will inhale sequentially increasing MCh concentrations (4) with a spirometry test 
(measurement of % fall in Forced Expiratory Volume in 1 second, i.e. FEV1) between each 
concentration. The test ends when MCh induces an FEV1 drop of more than 20% from 
baseline. The PC20 is then calculated by linear interpolation based on the antepenultimate 
MCh dilution and on the last MCh dilution that induced a FEV1 that fell more than 20% from 
baseline.  
6.3.2 Safety Endpoints 
The safety will be evaluated collecting the following information: 
•
AEs, SAEs, including asthma exacerbations,
•
General tolerability: vital signs, ECG, physical examination,
•
Laboratory parameters: hematology, biochemistry and urinalysis,
•
Local tolerability:
o
Increased bronchial irritability,
o
Paradoxical bronchospasm,
o
Oropharyngeal examination (e.g. vocal cord myopathy, fungal infection),
•
FEV1 and FVC, measured by spirometry,
•
HPA axis function: assessment of urinary cortisol/creatinine ratio in first waking urine.
6.3.3 Pharmacokinetic Endpoints
The PK endpoint which is the PK profile of budesonide in plasma will be evaluated through the 
PK parameters, calculated for each treatment period both at the start (24-hour PK analysis at 
Visit 1 and Visit 3) and at the end (abridged PK analysis over 6 hours at Visit 2 and Visit 4) of 
each TP. 
The following PK parameters will be calculated based on the budesonide plasma 
concentrations obtained from bioanalysis, for each treatment period: 


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•
Single dose 24-hour PK at the time of the first scheduled IMP administration of 0.250
mg budesonide (16 time points): Cmax, tmax, Clast, tlast, AUC0-inf, AUC0-last, AUC0-6h, CL/F,
V/F, λz and t1/2z,
•
Abridged PK at the time of the last scheduled IMP administration of 0.125 mg
budesonide (7 time points): Ctrough, Cmax, tmax, Clast, tlast, and AUC0-last, AUC0-6h.
PK parameter definitions: 
•
Cmax: maximum observed plasma budesonide concentration after dosing or during a
dosing interval (ng/ml),
•
tmax: time to reach the maximum observed plasma budesonide concentration (h),
•
Clast: last observed plasma budesonide concentration after dosing (ng/ml),
•
tlast: time to reach the last observed plasma budesonide concentration after dosing (h),
•
λz: apparent first-order rate constant associated with the terminal slope of the semi-
logarithmic budesonide concentration-time curve,
•
t1/2z: terminal elimination half-life associated with the terminal slope (λz) of the semi-
logarithmic budesonide concentration-time curve,
•
AUC0-6h: area under the plasma concentration-time curve from time 0 to 6-hours post
administration (ng.h/ml),
•
AUC0-last: area under the plasma concentration-time curve from time 0 to the last
quantifiable concentration (ng.h/ml),
•
AUC0-inf: area under the plasma concentration-time curve from time 0 to infinity
(ng.h/ml),
•
CL/F: apparent clearance following budesonide inhalation (l/h),
•
V/F: apparent volume of distribution following budesonide inhalation (l),
•
Ctrough: plasma budesonide concentration at the end of a dosing interval, immediately
before next IMP administration (ng/ml).
6.3.4 Secondary Efficacy/Pharmacodynamic Endpoints 
The secondary efficacy/PD endpoints will be the changes from baseline, after each treatment 
period, in: 
•
Symptom scores recorded using
o
daily day- and night-time asthma symptom scoring
o
Weekly validated Asthma Control Questionnaire™ (ACQ-5) questionnaire,
o
monthly validated Asthma Control Test (ACT) questionnaires,
•
Daily use of as-needed reliever medications,
•
fractional concentration of exhaled nitric oxide (FeNO) at Visit 1, 2 and 4
•
Blood eosinophil count at Visit 1, 2 and 4 (also measured at Visit 3 only for safety
purposes)
•
FEV1, at Visit 1, 2 and 4 (also measured at Visit 3 only for safety purposes)
•
FIVC. at Visit 1, 2 and 4 (also measured at Visit 3 only for safety purposes).


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7 General Considerations Pertaining to the Statistical Analysis 
7.1 Definitions 
Baseline 
Visit 1 is defined to be the Baseline timepoint for the evaluation of the primary efficacy endpoint. 
Unless otherwise specified, for all other measurements the baseline refers to the last 
measurement collected prior to the first dose of study medication. 
Baseline characteristics 
Baseline characteristics concern subjects data at Visit 1. They include all patient’s 
demographic, disease and treatment history and endpoint data recorded in the Registry prior 
to treatment start with AQ001S or Budesonide                       on Visit 1.  
Change from baseline 
Change from baseline refers to the endpoint values at Visit 2 (treatment period 1) or Visit 4 
(treatment period 2) minus the baseline value at Visit 1 for that parameter.  
Day 1 
Day 1 is defined for each subject as the day of first dose of AQ001S or Budesonide                 
for each treatment period (V1 or V3) receiving the double dose for 24h PK analysis (i.e. 0.250 
mg budesonide).  
Treatment sequence 
Treatment sequence defines the order of treatment (A or B) which was attributed to the subject 
by the randomization procedure. 
Treatment 
Treatment refers to AQ001S or Budesonide                . 
Treatment period 1 
Treatment period 1 refers to the first period (Day 1 to 28 from study start) the subject was under 
treatment (AQ001S or Budesonide              ). 
Treatment period 2 
Treatment period 2 refers to the second period (Day 1 to 28 from end of washout period) the 
subject was under treatment (AQ001S or Budesonide                 ). 
Assessment time points 
Expected assessment time points after initiation of AQ001S or Budesonide            
treatment are on Day 28 for each treatment period (Visits 2 and 4 for TP1 and TP2, 
respectively). 
End of treatment for an individual subject 
End of treatment for an individual subject is defined as the day when AQ001S or 
Budesonide           is stopped; the day of the last dose of AQ001S or Budesonide     (Day 28, 
29 or 30 


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of each treatment period) or the day the decision is made to permanently stop treatment, 
whichever occurs last. 
End of study for an individual subject 
End of study for an individual subject is defined as the day for withdrawal from study, follow-
up call or death. 
End of Study 
The study will conclude when all subjects will have completed the follow-up call, have 
withdrawn consent or died, whichever is sooner. 
7.2 Analysis Sets 
7.2.1 Analysis Set for Efficacy Analyses 
Efficacy Analysis set (EFF) 
The EFF population encompasses all subjects that completed both treatment periods and for 
which the primary efficacy parameter PC20 is available at baseline (Visit 1) and after both 
treatment periods (Visit 2 and Visit 4). 
The EFF population is a per-protocol set, in the sense that it is a subset of the full analysis set, 
where subjects with major protocol deviations which may have an impact on the evaluation of 
the primary clinical trial outcome parameter will be excluded. 
7.2.2 Analysis Set for Safety Analyses 
Safety Analysis set (SAF) 
The safety (SAF) population will include all randomized subjects who received at least one 
dose of IMP (AQ001S 0.125 mg/ml or comparator). 
7.2.3 Analysis Set for Pharmacokinetic Analyses 
Pharmacokinetics Analysis set (PK) 
The PK population includes all subjects, which have two full evaluable PK profiles and did not 
have protocol deviations relevant to the evaluation of PK. 
7.2.4 Other Analysis Sets 
Screened Analysis set (SCR) 
This set includes all subjects for whom the Screening Visit was performed. 
Randomized Analysis set (RAND) 
This set includes all randomized subjects. 


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7.3 DATA SCREENING AND ACCEPTANCE 
7.3.1 Data Handling and Electronic Transfer of Data  
All data will be captured in a web-based electronic CRF. Data will be downloaded in CSV 
format and transformed into SAS® data sets for the analyses. 
The PK data will be captured in an electronic database in the bioanalytical laboratory (, ). 
Data will be downloaded in CSV format by an external vendor (, ). The formal statistical 
analysis of the PK data is covered in the Analysis Plan of             and is not included in this 
document. 
7.3.2 Handling of Missing Data 
In general, missing data will not be imputed. Calculations pertaining to person-year 
computations will be based on observed values only. Only in case of missing body weight will 
the last available weight measurement be used for calculating the dose per kg bodyweight (last 
observation carried forward). 
7.3.3 Data review meeting 
In a blinded data review meeting prior to data base lock, assignment of subjects to the analysis 
sets will be confirmed and any necessary determinations regarding the relevance of protocol 
deviations will be made. These decisions will be based on study data listings (provided by data 
management) and other available pertinent documents such as site protocol deviation logs. 


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8 Statistical Analysis 
This section contains the detailed description of the statistical analysis of the data from the 
BOREAS clinical study. 
8.1 General principles 
The final statistical analysis will be performed after data base lock. 
In this BOREAS cross-over study, subjects will be analysed: 
•
according to their originally assigned treatment sequence (i.e., as randomized) in the
primary efficacy analyses
•
according to the treatment actually received (i.e., as treated) in all safety analyses and
other analyses.
A subject will be considered randomized as soon as a subject number has been assigned. 
General rules for handling missing data: In summary tables, the number of subjects with 
missing data will be presented when relevant but will be excluded from the calculation of 
percentages (numerator and denominator) unless otherwise specified.  
8.2 Specifications Related to the Whole Analysis 
8.2.1 Analysis Sets 
The primary efficacy analysis will be evaluated on the EFF set. 
The safety endpoints will be analysed for the SAF set. 
The PK analysis will be performed based upon the PK set. 
The secondary efficacy and PD analysis will be performed on the SAF set. 
8.2.2 Tabulation 
8.2.2.1 Listings 
Important Case Report Form data as well as all relevant generated and transformed variables 
together with the original data items will be listed. Unless otherwise specified, treatment and/or 
treatment sequence will always be included in listings, and listings will be sorted first by 
treatment if applicable, then by treatment sequence, subject identifier and finally, if applicable, 
by time point/visit number and/or a relevant date (e.g., date of onset of AE). 
The reference date for calculation of study days will be the day of first study medication (called 
Day 1). For subjects randomized but not treated the date of randomization will denote Day 1. 
All computed durations when expressed in number of days will be calculated by the difference 
of the two selected dates plus one day. 


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8.2.2.2 Tables 
Descriptive summary tables will be grouped by treatment and time point/visit as appropriate. 
For tables, which cannot be grouped by treatment, like e.g. subject disposition or demographics 
and baseline characteristics, the treatment sequence will be used as grouping variable. 
For quantitative data, number of subjects, number of observations, number of missing values, 
mean, standard deviation, minimum, 25%-percentile, median, 75%-percentile and maximum 
will be shown. Unless otherwise specified, confidence intervals will be calculated at the 95% 
level.  
Categorical data will be displayed in frequency tables showing number of missing values, 
absolute frequencies, and relative frequencies (in %). Presentation of these summary tables is 
referred to in the following as “X is/will be summarised”.  
8.3 Subject Disposition 
Subjects in the screened set will be listed with eligibility status, visit dates, study completion 
status, occurrence of temporary and/or permanent investigational product discontinuation, and 
reasons for study or product discontinuation (if applicable). Ineligible subjects will be listed with 
pertinent inclusion and/or exclusion criteria. 
Assignment of subjects in the randomized set to the sets EFF, SAF, and PK, and the reasons 
for exclusion from any of these analysis sets, if available, will be listed. Assignment of subjects 
in the randomized set to EFF, SAF, and PK will be summarised by treatment sequence. 
Visit completion status will be summarised by treatment and time point/visit for the EFF set. 
8.4 Demographics and Baseline Characteristics 
The demographic and baseline characteristics include sex and age recorded at Screening Visit 
will be listed and summarised by treatment sequence for the EFF set and for the SAF set, if 
the two sets differ. 
8.5 Medical/Surgical History 
Medical and surgical history items will be listed with indication, date of onset, date of resolution, 
treatment (if available), and outcome for the EFF set and for the SAF set, if the two sets differ. 
If a coding is available, then coded entities will each be summarised by treatment sequence 
for the EFF set and for the SAF set, if the two sets differ. 
Respiratory and asthma history of subjects will be listed with date of asthma diagnosis, date of 
appearance of first symptoms and asthma exacerbations in the last year for the EFF set and 
for the SAF set, if the two sets differ. Other respiratory history will also be listed additionally if 
available.  


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8.6 Prior and Concomitant Medication 
Prior and concomitant medication items will be listed with the name of the drug, the 
pharmaceutical form, the strength, the dose regimen, the frequency, the route of 
administration, the date of initiation and discontinuation, and the reason for use for the EFF set 
and for the SAF set, if the two sets differ. The coded prior and concomitant medication entities 
will be summarised by treatment sequence for the EFF set and for the SAF set, if the two sets 
differ. For coding the most recent WHODD version available at the time of database lock is 
used. 
8.7 Compliance 
Study product intake compliance with respect to the whole treatment phase will be listed and 
summarised by treatment sequence for the EFF set. 
8.8 Efficacy Analysis 
The primary efficacy analysis will be done using the EFF set. Statistical tests and confidence 
intervals will be two-sided at 5% significance level. The secondary efficacy and PD analysis 
will be performed on the SAF set. 
8.8.1 Descriptive Analyses of Primary Efficacy Endpoint 
For the parameter PC20 (concentration of MCh provoking an FEV1 fall of 20%), baseline 
measurements (Visit 1) and measurements at Visit 2 and Visit 4 will be listed by subject. 
Baseline measurements, measurements at Visit 2 and Visit 4, and changes from baseline will 
be summarised by treatment group. Line plots for baseline measurements and measurements 
at Visit 2 and Visit 4 will be produced by treatment sequence. 
8.8.2 Primary Efficacy Analysis 
For primary efficacy analysis, the mean PC20 changes from baseline between AQ001S 0.125 
mg/ml and the comparator will be compared by analysis of covariance (ANCOVA) for crossover 
design. A p-value will be calculated for the difference of the parameter between AQ001S 0.125 
mg/ml and comparator. If this p-value is significant and the difference prefers AQ001S 0.125 
mg/ml, superiority is shown. 
Additionally, an adjusted 95%-confidence interval for the mean difference between AQ001S 
0.125 mg/ml and the comparator will be obtained by the ANCOVA. 
As the PC20 values are expected to follow a log-normal distribution, a log10-transformation is 
done before performing the primary efficacy analysis. 
Analysis of Covariance (ANCOVA) 
The ANCOVA will be used to estimate the residual error, which is used to construct the 
confidence intervals, moreover to evaluate the presence of period or sequence effects. The 


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effects considered in the ANCOVA model will be: baseline value, treatment sequence, period, 
and within-subject sequence.  
8.8.3 Secondary Efficacy/Pharmacodynamic analysis 
The secondary efficacy/PD parameters mentioned in Section 6.2.4 will also be evaluated 
descriptively. The individual PD parameters will be listed by subject. Frequencies/proportions 
for categorical (qualitative) parameters and descriptive statistics for continuous (quantitative) 
parameters will be calculated and summarised by treatment.  
•
Symptom scores recorded using
o
Daily day- and night-time asthma symptom scoring
o
Weekly validated asthma questionnaires, i.e.  Asthma Control Questionnaire
(ACQ)-5
o
Monthly validated Asthma Control Test (ACT) questionnaires,
•
Use of as-needed reliever medications,
•
fractional concentration of exhaled nitric oxide (FeNO),
•
Blood eosinophil count,
•
FEV1,
•
FIVC.
8.8.4 Subgroup Analyses 
No subgroup analyses will be done. 
8.9 Safety Analysis 
All safety analyses will be done using the SAF set unless otherwise specified. Safety analyses 
will be descriptive in nature and no hypothesis testing is planned. 
Measurements (such as ECG, vital signs, or laboratory parameters) or AEs will be assigned to 
treatments based on the actual treatment at the planned time of the measurement or on the 
recorded time of AE onset (concept of treatment emergent AEs). 
8.9.1 Physical and Clinical Examination 
Physical examination results (normal, abnormal – not clinically significant, abnormal – clinically 
significant) and description of abnormality will be listed by organ system and time point/visit. 
Physical examination results will be summarised as appropriate, and shift in examination 
results categories will be presented by treatment group and time point/visit.  
Clinical examination (hight, weight, BMI) will be summarised by treatment group and visit and 
listed accordingly. 


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8.9.2 Vital Signs 
For each recorded vital sign (systolic & diastolic blood pressure, body temperature, heart rate, 
respiratory rate, and blood oxygen saturation), all available measurements, and changes from 
baseline to all post-randomisation time points/visits will be listed. Vital sign measurements, and 
changes from baseline to post-randomisation visits, will be summarised by treatment group 
and time point/visit. 
8.9.3 ECG 
ECG assessments will be listed with date-time, ECG parameters, interpretation, and 
abnormalities (if any). ECG interpretation (normal, abnormal – not clinically significant, 
abnormal – clinically significant) will be summarised by treatment group and time point/visit. 
8.9.4 Laboratory measurements 
Laboratory measurement results (normal, abnormal – not clinically significant, abnormal – 
clinically significant) and description of the abnormality will be listed by parameter and time 
point/visit. Laboratory data will be compared to their reference ranges. Values outside the 
reference range as well as possibly clinically significant values will be highlighted in the listings. 
For all recorded laboratory parameters, all available measurements will be listed. Changes 
from baseline to all post-randomisation time points/visits will be listed for quantitative 
parameters. Laboratory parameter measurements will be summarised by treatment group and 
time point/visit for all time points/visits. Changes from baseline to post-randomisation time 
points/visits will be summarised by treatment group for quantitative parameters. For laboratory 
parameters where normal ranges are available, shift from baseline will be tabulated by 
treatment group for all post-randomisation time points/visits. 
8.9.5 Adverse Events 
AEs will be assigned to treatments based on the recorded time of AE onset (concept of 
treatment emergent AEs). An overall listing of AEs and SAEs (including asthma exacerbations) 
by subject with information on reported term, system organ class, preferred term, date of onset, 
time of onset, stop date (when applicable), stop time (when applicable), actions taken, severity, 
seriousness, relationship to IMP and to trial procedures, and outcome will be produced.  
AEs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA). Frequency, 
severity, and causal relationship of AEs will be tabulated by treatment, system organ class and 
preferred term. Absolute and relative frequencies of subjects with at least one AE will be 
presented and the total number of AEs will be given by treatment group. SAEs and other 
significant AEs will be evaluated separately. 
8.10 Pharmacokinetic analysis 
The PK analysis will be performed on the PK set. The statistical analysis of this section will 
describe and characterize the PK of AQ001S 0.25 mg/ml and Budesonide 0.25 mg/ml for the 


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24-hour PK at the start of each TP, and the PK of AQ001S and Budesonide             0.125 mg/ml 
for the 6-hour abridged PK at the end of each TP. There are no formal hypotheses being tested.
The individual plasma concentrations of budesonide will be listed and tabulated by treatment 
and time point. 
Descriptive statistics for the PK parameters described in Section 6.3.3 will be calculated and 
summarised by treatment. This analysis is just mentioned here for completeness but will not 
be performed by                    . 


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9 Reporting Conventions 
P-values will be reported to three (3) decimal places; p-values less than 0.001 will be reported
as “<0.001”. Minimum and maximum will use the same number of decimal places as the
original data. All other statistics, including percentage values, will be reported to one (1)
decimal place greater than the original data.
Data in columns of a table will be formatted as follows: 
- Alphanumeric values are left-justified (in mixed upper- and lower-case)
- Integer numbers (e.g., counts) are center-justified
- Numbers containing fraction portions are decimal aligned
All fraction numeric values will be printed with a zero to the left of the decimal point (e.g., 0.12, 
0.3 etc.) 
Dates will be printed according to the alpha-numerical definition in the database. Missing 
portions of dates should be represented on patient listings as dashed (--JAN99). Dates that 
are missing because they are not applicable for the patient are output as “NA” (= not 
applicable), unless otherwise specified. 
In case that the definition of a date variable is numeric, dates will be printed in SAS® DATE9. 
Format (“DDMMMYYYY”: 01JAN2008). 
If measurements are repeated, the first one (scheduled one) will be used in the summary table, 
except for screening and Day -1: then the latest measurement (nearest to first dosing) will be 
used. All measurements including the repeated one will be presented in the individual listings. 


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10 Technical Details 
The 
analysis 
will 
be 
carried 
out 
according 
to 
standard 
operating 
procedure 
SOP001_PROGRAMMING [3]. The statistical analysis, including all tables, listings, figures of 
clinical and laboratory data will be performed using SAS® 9.4 or higher. 
SAS® programming will be performed according to         standards as defined in 
SOP001_PROGRAMMING [3] and related work instructions. Special attention will be paid to 
planning and performance of quality control measures as documented in the quality control 
plan for the analysis of this study (see also SOP002_PROGRAM_QC [4]). 


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11 List of Planned Contents of Tables, Figures and Listings 
Based on this SAP, an excel file is developed which covers all tables, figures and listings to be 
included in the clinical study report. This document is appended to this SAP. In accordance 
with ICH E3 [1] the statistical output is organised in Tables and Figures as follows: 
14 TABLES, FIGURES AND GRAPHS REFERRED TO BUT NOT INCLUDED IN THE TEXT 
14.1 Demographic data and baseline characteristics 
14.2 Efficacy data 
14.3 Safety data 
14.4 Pharmacokinetics data 
16 APPENDICES 
16.2 PATIENT DATA LISTINGS 


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12 References 
[1]
International Conference on Harmonisation. Note for Guidance on Structure and
Content of Clinical Study Reports (ICH E3). CPMP/ICH/137/95, 1996.
[2]
International Conference on Harmonisation. Note for Guidance on Statistical
Principles for Clinical Trials (ICH E9). CPMP/ICH/363/96, 1998.
[3]
SOP001_PROGRAMMING, “Standard Operating Procedure for Statistical
Programming”, current version
[4]
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