Clinical trial NCT02054156
Conditions: Cystic Fibrosis
Keywords: Cystic fibrosis (CF) Azithromycin Tobramycin solution for inhalation (TIS) Pseudomonas aeruginosa (Pa) Early Pseudomonas aeruginosa infection Pulmonary exacerbation Standardized anti-pseudomonal therapy
Sponsor: Bonnie Ramsey
Start date: 2014-06-01; Completion date: 2018-08-23
Enrollment: 221 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this trial is to compare the effects of treatment with tobramycin solution for inhalation (TIS) with and without azithromycin in people with cystic fibrosis (CF) age 6 months to 18 years who have early isolation of Pseudomonas aeruginosa (Pa) from a respiratory culture. Specimens of blood and sputum or throat swabs will be taken during the study along with pulmonary function testing. Participants will receive initial treatment with TIS followed additional treatment with TIS if quarterly respiratory cultures are positive for Pa in addition to either azithromycin or placebo for 18 months.

Detailed description:
Cystic fibrosis (CF) lung disease begins in the first few months of life and follows a course of recurrent lower airway bacterial infection and inflammation and progression of disease over years and decades at a variable pace. With the development of chronic lung infection, obstructive disease progressively worsens, ultimately leading to respiratory failure. Pseudomonas aeruginosa (Pa) is the most important pathogen infecting the CF lower airways, and its acquisition early in life is associated with a pro-inflammatory effect, lower lung function, poor nutritional outcomes, and decreased survival.

Pseudomonas aeruginosa (Pa) infection of the cystic fibrosis (CF) airway typically proceeds from early infection to chronic infection. Although some studies have shown that a minority of individuals with CF spontaneously clear early Pseudomonas aeruginosa (Pa) infection, data from multiple studies suggest that antibiotics are superior to no treatment in clearing Pseudomonas aeruginosa (Pa) from respiratory cultures. Understanding the transition period from early to chronic Pseudomonas aeruginosa (Pa) infection is thus of critical importance in identifying strategies to prevent this progression.

The study will assess the clinical and microbiologic efficacy and safety of azithromycin given three times weekly in combination with standardized tobramycin solution for inhalation (TIS) therapy among children with early Pseudomonas aeruginosa (Pa). TIS therapy is defined as an initial eradication treatment with 1-2 courses of 28 days TIS and subsequent 28 day treatments only at times a quarterly respiratory culture is positive for Pseudomonas aeruginosa (Pa). Eligible participants will be randomized within one month of their Pseudomonas aeruginosa (Pa) positive culture to receive one of the following two treatment strategies for 18 months: (1) oral placebo in addition to standardized TIS therapy, or (2) oral azithromycin in addition to standardized TIS therapy.

At the first study visit, participants will undergo a physical examination and a review of their medical history. Lung function will be measured via spirometry (in children greater than four years of age who are able to perform spirometry), electrocardiogram (ECG) testing will be conducted, and hearing ability will be measured via audiometry. Blood will be drawn for laboratory tests and a specimen will be obtained for a respiratory culture before randomization and study drug dispensing occurs. Subsequent study visits will take place at Day 21, Weeks 13, 26, 39, 52, 65, and 78. At each visit, participants will undergo a physical examination, a spirometry test (as appropriate), a respiratory specimen for Pseudomonas aeruginosa (Pa) culture will be collected and study drug will be dispensed (except at Week 78). Participants will complete self-report or parent-completed respiratory symptom questionnaires and signs and symptoms evaluations will be performed at all visits. Repeat hearing and laboratory tests will be performed at Weeks 39 and 78 and ECG testing will be repeated at Day 21 and Week 78. Participants will be required to maintain a medication diary throughout the study.

Arms: arm 1: azithromycin and tobramycin solution for inhalation (TIS) Azithromycin 3 times weekly, oral suspension, 10 mg/kg/dose up to 500 mg, for 18 months Tobramycin solution for inhalation (TIS), 300 mg, twice daily for 28 days when respiratory cultures are found positive for Pa at study visits for 18 months arm 2: placebo and tobramycin solution for inhalation (TIS) Placebo 3 times weekly, oral suspension, volume-matched to azithromycin, for 18 months Tobramycin solution for inhalation (TIS), 300 mg, twice daily for 28 days when respiratory cultures are found positive for Pa at study visits for 18 months
Interventions: intervention 1: azithromycin intervention 2: placebo intervention 3: Tobramycin solution for inhalation
Intervention descriptions: intervention 1: 3 times weekly, oral suspension, 10 mg/kg/dose up to 500 mg, for 18 months intervention 2: 3 times weekly, oral suspension, volume-matched to azithromycin, for 18 months intervention 3: 300 mg, twice daily for 28 days when respiratory cultures are found positive for Pa at study visits for 18 months

Protocol documents:
Prot_001.pdf: 


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PROTOCOL AGREEMENT 
 
I have read the protocol specified below.  In my formal capacity as Investigator, my duties include 
ensuring the safety of the study participants enrolled under my supervision and providing the Sponsor 
with complete and timely information, as outlined in the protocol.  It is understood that all information 
pertaining to the study will be held strictly confidential and that this confidentiality requirement applies 
to all study staff at this site.  Furthermore, on behalf of the study staff and myself, I agree to maintain the 
procedures required to carry out the study in accordance with accepted GCP principles and to abide by 
the terms of this protocol. 
 
Protocol Number:  OPTIMIZE-IP-12 
 
Protocol Title:  OPTIMIZing Treatment for Early Pseudomonas aeruginosa Infection in Cystic 
Fibrosis: The OPTIMIZE Multicenter, Placebo-Controlled, Double-Blind, Randomized Trial 
 
Protocol Date:  22 August 2016 
 
 
 
 
Investigator Signature  
 
Date 
 
Print Name and Title 
Site # 
 
 
Site Name 
 
Address 
 
 
 
 
 
 
 
Phone Number 
 


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TABLE OF CONTENTS 
 
1 
BACKGROUND AND RATIONALE .................................................................................................................... 18 
1.1 
RISK / BENEFIT ASSESSMENT ....................................................................................................................................... 21 
2 
STUDY OBJECTIVES ........................................................................................................................................... 22 
2.1 
PRIMARY OBJECTIVE .................................................................................................................................................... 22 
2.2 
SECONDARY OBJECTIVES ............................................................................................................................................. 22 
3 
STUDY DESIGN ..................................................................................................................................................... 23 
3.1 
STUDY OVERVIEW ....................................................................................................................................................... 23 
4 
CRITERIA FOR EVALUATION .......................................................................................................................... 24 
4.1 
PRIMARY EFFICACY ENDPOINT .................................................................................................................................... 24 
4.2 
SECONDARY ENDPOINTS .............................................................................................................................................. 24 
5 
PARTICIPANT SELECTION ................................................................................................................................ 24 
5.1 
STUDY POPULATION ..................................................................................................................................................... 24 
5.2 
INCLUSION CRITERIA ................................................................................................................................................... 25 
5.3 
EXCLUSION CRITERIA ......................................................................................................................................................... 25 
5.4 
STUDY SPECIFIC TOLERANCE FOR INCLUSION/EXCLUSION CRITERIA .......................................................................... 26 
5.5 
SCREEN FAIL CRITERIA ................................................................................................................................................ 26 
6 
CONCURRENT MEDICATIONS ......................................................................................................................... 26 
6.1 
ALLOWED MEDICATIONS AND TREATMENTS ............................................................................................................... 26 
6.2 
PROHIBITED MEDICATIONS AND TREATMENTS ............................................................................................................ 27 
7 
STUDY TREATMENTS ......................................................................................................................................... 27 
7.1 
METHOD OF ASSIGNING PARTICIPANTS TO TREATMENT GROUPS ................................................................................ 27 
7.2 
BLINDING ..................................................................................................................................................................... 28 
7.3 
FORMULATION OF TEST AND CONTROL PRODUCTS ...................................................................................................... 28 
7.3.1 
FORMULATION OF TEST PRODUCT ........................................................................................................................... 28 
7.3.2 
FORMULATION OF CONTROL PRODUCT ................................................................................................................... 29 
7.3.3 
PACKAGING AND LABELING .................................................................................................................................... 29 
7.4 
SUPPLY OF STUDY DRUG TO THE SITE .......................................................................................................................... 29 
7.4.1 
DOSAGE/DOSAGE REGIMEN .................................................................................................................................... 29 
7.4.2 
DISPENSING ............................................................................................................................................................. 29 
7.4.3 
ADMINISTRATION INSTRUCTIONS ............................................................................................................................ 29 
7.4.4 
STORAGE ................................................................................................................................................................. 30 
7.5 
DOSE MODIFICATION ................................................................................................................................................... 30 
7.5.1 
DOSE MODIFICATION DUE TO STUDY DRUG INTOLERANCE .................................................................................... 30 
7.5.2 
MISSED DOSAGES .................................................................................................................................................... 30 
7.6 
STUDY DRUG ACCOUNTABILITY .................................................................................................................................. 30 
7.7 
MEASURES OF STUDY DRUG TREATMENT COMPLIANCE .............................................................................................. 31 
7.8 
TOBRAMYCIN INHALATION SOLUTION (TIS) ................................................................................................................ 31 
7.8.1 
USE OF TOBI PODHALER AND OTHER NEBULIZED TOBRAMYCIN PRODUCTS ......................................................... 32 
7.8.2 
TIS DOSE MODIFICATION DUE TO PERSISTENT INFECTION ..................................................................................... 32 
7.8.3 
TIS ACCOUNTABILITY ............................................................................................................................................. 32 
7.8.4 
MEASURES OF TIS TREATMENT COMPLIANCE ........................................................................................................ 32 


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STUDY PROCEDURES AND GUIDELINES ....................................................................................................... 32 
8.1 
CLINICAL ASSESSMENTS .............................................................................................................................................. 32 
8.1.1 
CONCOMITANT MEDICATIONS ................................................................................................................................. 33 
8.1.2 
DEMOGRAPHICS ...................................................................................................................................................... 33 
8.1.3 
MEDICAL HISTORY .................................................................................................................................................. 33 
8.1.4 
MICROBIOLOGY HISTORY........................................................................................................................................ 33 
8.1.5 
RECORDING OF CLINICAL CARE MICROBIOLOGY RESULTS ..................................................................................... 33 
8.1.6 
PHYSICAL EXAMINATION ........................................................................................................................................ 33 
8.1.7 
WEIGHT AND HEIGHT/LENGTH ................................................................................................................................ 33 
8.1.8 
VITAL SIGNS............................................................................................................................................................ 34 
8.1.9 
OXIMETRY ............................................................................................................................................................... 34 
8.1.10 
SPIROMETRY ....................................................................................................................................................... 34 
8.1.11 
AUDIOMETRY ..................................................................................................................................................... 34 
8.1.12 
ELECTROCARDIOGRAM (ECG) ........................................................................................................................... 35 
8.1.13 
SIGNS AND SYMPTOMS EVALUATION FOR PULMONARY EXACERBATIONS ......................................................... 36 
8.1.14 
CYSTIC FIBROSIS RESPIRATORY QUESTIONNAIRES ............................................................................................. 36 
8.1.15 
ADVERSE EVENTS ............................................................................................................................................... 36 
8.1.16 
HOSPITALIZATION .............................................................................................................................................. 36 
8.1.17 
HEMATOLOGY .................................................................................................................................................... 37 
8.1.18 
BLOOD CHEMISTRY PROFILE .............................................................................................................................. 37 
8.1.19 
PREGNANCY TEST ............................................................................................................................................... 37 
8.1.20 
RESPIRATORY CULTURES FOR CF PATHOGENS INCLUDING P. AERUGINOSA AND S. AUREUS AND NON-
TUBERCULOUS MYCOBACTERIA (NTM) ................................................................................................................................. 37 
8.1.21 
SUBJECT DIARY .................................................................................................................................................. 38 
8.2 
RESEARCH LABORATORY ASSESSMENTS ..................................................................................................................... 38 
8.2.1 
PLASMA FOR INFLAMMATORY BIOMARKER ANALYSIS AND BANKING .................................................................... 38 
8.2.2 
OP SWABS FOR MICROBIOME ANALYSIS ................................................................................................................. 38 
9 
EVALUATIONS BY VISIT .................................................................................................................................... 38 
9.1 
BASELINE VISIT 1 – SCREENING AND RANDOMIZATION (DAY 0) ................................................................................. 38 
9.2 
VISIT 2 (WEEK 3, DAY 21 ± 2 DAYS) ............................................................................................................................ 39 
9.3 
VISIT 3 (WEEK 13 ± 2 WEEKS) ...................................................................................................................................... 40 
9.4 
VISIT 4 (WEEK 26 ± 2 WEEKS) ...................................................................................................................................... 41 
9.5 
VISIT 5 (WEEK 39 ± 2 WEEKS) ...................................................................................................................................... 41 
9.6 
VISIT 6 (WEEK 52 ± 2 WEEKS) ...................................................................................................................................... 42 
9.7 
VISIT 7 (WEEK 65 ± 2 WEEKS) ...................................................................................................................................... 43 
9.8 
VISIT 8 - END OF STUDY (WEEK 78 ± 2 WEEKS) ........................................................................................................... 43 
9.9 
UNSCHEDULED ASSESSMENT – CHANGE IN ANTIBIOTICS OR AS DETERMINED BY INVESTIGATOR ............................... 44 
9.10 
EARLY WITHDRAWAL FROM STUDY ........................................................................................................................ 44 
10 
ADVERSE EXPERIENCE REPORTING AND DOCUMENTATION ................................................................ 45 
10.1 
ADVERSE EVENTS ................................................................................................................................................... 45 
10.2 
SERIOUS ADVERSE EXPERIENCES (SAE) ................................................................................................................. 46 
10.3 
SERIOUS ADVERSE EXPERIENCE REPORTING........................................................................................................... 47 
10.4 
MEDICAL MONITORING ........................................................................................................................................... 47 
11 
DISCONTINUATION AND REPLACEMENT OF PARTICIPANTS ................................................................. 47 
11.1 
PERMANENT DISCONTINUATION OF STUDY DRUG................................................................................................... 47 
11.1.1 
PERMANENT DISCONTINUATION OF STUDY DRUG DUE TO PERSISTENT PA INFECTION ...................................... 48 
11.2 
TEMPORARY DISCONTINUATION OF STUDY DRUG .................................................................................................. 48 


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11.3 
DISCONTINUATION OF TIS ....................................................................................................................................... 48 
11.4 
WITHDRAWAL OF PARTICIPANTS FROM THE STUDY ................................................................................................ 49 
11.5 
REPLACEMENT OF PARTICIPANTS ............................................................................................................................ 49 
12 
PROTOCOL VIOLATIONS .................................................................................................................................. 49 
13 
DATA SAFETY MONITORING ........................................................................................................................... 49 
14 
STATISTICAL METHODS AND CONSIDERATIONS ....................................................................................... 50 
14.1 
DATA SETS ANALYZED ........................................................................................................................................... 50 
14.2 
DEMOGRAPHIC AND BASELINE CHARACTERISTICS .................................................................................................. 50 
14.3 
ANALYSIS OF PRIMARY ENDPOINT .......................................................................................................................... 50 
14.4 
ANALYSIS OF SECONDARY ENDPOINTS ................................................................................................................... 50 
14.5 
MISSING DATA ........................................................................................................................................................ 54 
14.6 
INTERIM ANALYSIS ................................................................................................................................................. 54 
14.7 
SAMPLE SIZE ........................................................................................................................................................... 54 
15 
DATA COLLECTION, RETENTION AND MONITORING ............................................................................... 55 
15.1 
DATA COLLECTION INSTRUMENTS .......................................................................................................................... 55 
15.2 
DATA MANAGEMENT PROCEDURES ........................................................................................................................ 55 
15.3 
DATA QUALITY CONTROL AND REPORTING ............................................................................................................ 56 
15.4 
SECURITY AND ARCHIVAL OF DATA ........................................................................................................................ 56 
15.5 
AVAILABILITY AND RETENTION OF INVESTIGATIONAL RECORDS ........................................................................... 56 
15.6 
MONITORING ........................................................................................................................................................... 57 
15.7 
PARTICIPANT CONFIDENTIALITY ............................................................................................................................. 57 
16 
ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS ........................................................... 57 
16.1 
PROTOCOL AMENDMENTS ....................................................................................................................................... 57 
16.2 
INSTITUTIONAL REVIEW BOARDS ............................................................................................................................ 57 
16.3 
INFORMED CONSENT FORM ..................................................................................................................................... 58 
16.3.1 
CONSENT FOR COLLECTION AND USE OF CFF REGISTRY ID NUMBER ............................................................... 59 
16.4 
PUBLICATIONS ......................................................................................................................................................... 59 
16.5 
INVESTIGATOR RESPONSIBILITIES ........................................................................................................................... 59 
17 
LITERATURE CITED ........................................................................................................................................... 60 
18 
APPENDIX I: SCHEDULE OF EVENTS .............................................................................................................. 68 
19 
APPENDIX II:  DEFINITION OF PULMONARY EXACERBATION ............................................................... 70 
20 
APPENDIX III: COMPOSITE LIST OF DRUGS THAT PROLONG QT AND/OR CAUSE TORSADES DE 
POINTES (TDP) .............................................................................................................................................................. 71 
 


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LIST OF ABBREVIATIONS 
AE 
adverse event 
AFB 
acid fast bacilli 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
BAL 
bronchoalveolar lavage 
BUN 
blood urea nitrogen 
CCC 
Clinical Coordinating Center 
CF 
cystic fibrosis 
CFF 
Cystic Fibrosis Foundation 
CFR 
Code of Federal Regulations 
CFRSD 
Cystic Fibrosis Respiratory Symptoms (or Signs) Diary 
CFRSD-CRISS 
CFRSD-Chronic Respiratory Infection Symptom Score  
CFTR 
cystic fibrosis transmembrane conductance regulator 
CRF 
case report form 
CRP 
C-reactive protein 
CT 
Computed tomography 
CTCAE 
Common Terminology Criterion for Adverse Events 
dBHL 
Decibels Hearing Level 
DCC 
Data Coordinating Center 
DMC 
Data Monitoring Committee 
DSMB 
Data Safety Monitoring Board 
ECG 
electrocardiogram 
eCRF 
electronic case report from 
EDC 
electronic data capture 
ESR 
erythrocyte sedimentation rate 
FDA 
Food and Drug Administration 
FEF25%-75% 
forced expiratory flow 
FEV1 
forced expiratory volume over one second 
FVC 
forced vital capacity 
GCP 
Good Clinical Practice 
GGT 
gamma-glutamyl transferase 
HIPAA 
Health Insurance Portability and Accountability Act of 1996 
ICF 
informed consent form 


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ICH 
International Conference on Harmonisation 
IGF 
insulin growth factor 
IL-8 
Interleukin-8 
IM 
IRB 
Inflammatory Biomarkers 
Institutional Review Board 
IRT  
immunoreactive trypsinogen 
IV 
intravenous 
LDH 
lactate dehydrogenase 
MD 
Medical Doctor 
MOA 
mechanism of action 
msec 
millisecond 
NHLBI 
National Institutes of Health/National Heart, Lung, and Blood 
Institute  
NIDDK 
National Institute of Diabetes and Digestive and Kidney 
Diseases 
NP 
Nurse Practitioner 
NTM 
Non-tuberculous mycobacteria 
OAE 
Otoacoustic emissions 
Pa 
Pseudomonas aeruginosa 
PA 
Physician Assistant 
PFT 
pulmonary function test 
PI 
Principal Investigator 
PR 
Measure from beginning of P wave to onset of ventricular 
depolarization (R) - ECG measure 
QRS 
QRS interval; duration of the QRS complex in milliseconds – 
ECG measure 
QT 
QT interval; duration between start of Q wave and end of T 
wave in milliseconds - ECG measure 
QTc 
QT interval corrected 
RC 
Research Coordinator 
RN 
Registered Nurse 
Sa 
Staphylococcus aureus 
SAE 
serious adverse experience 
SGOT 
serum glutamic oxaloacetic transaminase 
SGPT 
serum glutamate pyruvate transaminase 


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TCH CTRC 
The Children’s Hospital Clinical and Translational Research 
Center  in Denver 
TDNCC 
Therapeutics Development Network Coordinating Center 
TIS 
tobramycin inhalation solution 
VRA 
Visual reinforcement audiometry  
 
 


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PROTOCOL SYNOPSIS 
TITLE 
OPTIMIZing Treatment for Early Pseudomonas aeruginosa 
Infection in Cystic Fibrosis: The OPTIMIZE Multicenter, 
Placebo-Controlled,  Double-Blind, Randomized Trial 
SPONSOR 
Bonnie Ramsey, M.D. 
FUNDING 
ORGANIZATION 
National Institutes of Health/National Heart, Lung, and Blood 
Institute (NHLBI)  
Study Drug provided by Pfizer, Inc  
Study Drug provided by Novartis Pharmaceuticals Corp  
Study Compressors & Nebulizers provided by PARI 
Respiratory Equip, Inc  
NUMBER OF SITES 
Approximately forty-five (45)  
RATIONALE 
Cystic fibrosis (CF) is the most common autosomal recessively 
inherited disease among Caucasians, affecting approximately 
25,000 to 30,000 individuals in the United States(1). Although 
CF is a complex multi-system disease, 85% of the mortality 
results from lung disease, characterized by persistent infection 
and inflammation leading to progressive airway obstruction and 
bronchiectasis, and ultimately to respiratory failure(2). 
Pseudomonas aeruginosa (Pa) is the most important pathogen 
and is acquired early in life, with an age specific prevalence of 
30% by 10 years of age and ~ 70% by age 18(3, 4). We have 
recently completed two multicenter trials demonstrating that 
chronic therapies in children with CF and mild disease may 
positively affect disease progression by different, potentially 
complementary mechanisms. First, the NIH-funded EPIC trial 
focused on identifying the optimal treatment strategy for early 
Pa infection in young children with CF(5).  The trial 
demonstrated that monotherapy with tobramycin inhalation 
solution (TIS) given only when quarterly cultures were Pa 
positive (termed “culture-based”) was as efficacious as more 
intensive antibiotic regimens in terms of clinical and 
microbiologic efficacy, including pulmonary exacerbations and 
initial Pa eradication(5). Yet, nearly 34% of EPIC trial 
participants still experienced Pa recurrence during the 18 
month study and 50% experienced an exacerbation, one of the 
key signs of acute clinical worsening associated with a shorter 
time to recurrence of Pa infection(6) and poorer long-term 
clinical outcomes(7, 8). Second, we conducted a randomized, 
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CF uninfected with Pa that demonstrated a 50% reduction in 
pulmonary exacerbations in parallel with an anti-inflammatory 
effect among those treated with azithromycin(9, 10), similar to 
results in trials of azithromycin in older individuals with CF 
chronically infected with Pa(11-13). Additional findings from the 
EPIC trial have led us to hypothesize that combining TIS and 
oral azithromycin, given their complementary routes and 
mechanisms of action (MOA), will be additive in their impact 
on early CF lung infection with Pa. First, among EPIC 
participants failing initial Pa eradication therapy, those with 
more frequent exacerbations were more likely to have chronic 
Pa infection(6). In addition, persistent Pa strains had a 
phenotype consistent with early infection (highly antibiotic 
susceptible and non-mucoid), suggesting little exposure to the 
inhaled antibiotic, possibly due to inhibition of penetration by 
airway mucus, edema and inflammation. This theory is 
supported by the Australian longitudinal bronchoalveolar 
studies in infants with CF which demonstrated a significant 
inflammatory response in the first months of life and that Pa is 
the most pro-inflammatory of bacterial pathogens isolated(14, 15). 
Based upon these findings, we hypothesize that a therapeutic 
approach that reduces the frequency of exacerbations and 
reduces airway inflammation may prolong the time to Pa 
recurrence.  Specifically, we hypothesize that chronic oral 
azithromycin will provide added clinical benefit to 
standardized anti-pseudomonal therapy with TIS among 
children with early Pa and will decrease the risk of 
pulmonary exacerbations, reduce inflammation, and reduce 
the rate of Pa recurrence.   
STUDY DESIGN 
This is a multicenter, double-blind, randomized, placebo-
controlled clinical trial in 274 children with CF ages 6 mos -18 
years with early Pa, defined as either a first lifetime 
documented Pa culture or a Pa positive culture after at least 
two years of negative cultures.  The study will assess the 
clinical and microbiologic efficacy and safety of azithromycin 
given three times weekly in combination with standardized TIS 
therapy among children with early Pa.  TIS therapy is defined 
as an initial eradication treatment with 1-2 courses of 28 days 
TIS and subsequent 28 day treatments only at times a quarterly 
oropharyngeal culture is positive for Pa.  Eligible participants 
will be randomized within 40 days from the date of collection 
of their Pa positive culture. Participants initiating TIS more 
than 14 days prior to the baseline visit for treatment of their Pa 
positive culture will be excluded.  Participants will be 


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randomized in a 1:1 fashion to receive one of the following two 
treatment strategies for 18 months: (1) oral placebo in addition 
to standardized TIS therapy, or (2) oral azithromycin in 
addition to standardized TIS therapy.   
Three times weekly administration of oral azithromycin or 
matching placebo will be continued throughout the 18-month 
trial.  All participants will receive a 28-day course of TIS 
therapy during the first quarter of the trial and have a follow-up 
culture obtained to document Pa clearance. Participants who 
remain Pa positive after the first 28-day course of TIS will 
receive a second 28-day course of TIS.  Subsequently, 
participants will receive TIS therapy (administered as a single 
28-day course) only if their quarterly cultures are Pa positive. 
Cultures will be obtained at the start of each quarter for the 
duration of the 18-month study.   
Clinical and microbiologic outcomes will be assessed 
throughout the 18-month trial, including the occurrence of 
pulmonary exacerbations and Pa recurrence.   
PRIMARY 
OBJECTIVE 
To compare the time to pulmonary exacerbation between 
participants randomized to oral placebo and culture-based TIS 
versus oral azithromycin and culture-based TIS  
SECONDARY 
OBJECTIVES 
 Compare Pseudomonas aeruginosa (Pa) recurrence 
between treatment groups 
 Evaluate the safety profile of azithromycin in combination 
with culture-based TIS therapy as compared to culture-
based TIS therapy alone, including the incidence of 
treatment emergent pathogens    
 Compare changes in plasma inflammatory markers between 
treatment groups   
SECONDARY 
EXPLORATORY 
OBJECTIVES 
 Compare hospitalization rates and other indices of clinical 
efficacy, including changes in height, weight, pulmonary 
function (in participants ≥4 years old) and respiratory 
symptoms, between treatment groups  
 Compare changes in the respiratory microbiome between 
treatment groups  
 Develop a comprehensive baseline predictive model of poor 
microbiologic or clinical response to therapy, including 
respiratory microbiome, inflammatory biomarkers, 
demographic and clinical characteristics in order to identify 
high risk children for future studies of alternative 
therapeutic approaches 
 Establish a linked data and specimen repository for future 


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studies of disease mechanisms, potential novel therapeutic 
approaches and predicting response to therapy  
NUMBER OF 
SUBJECTS 
Approximately two-hundred seventy-four (274) 
SUBJECT 
SELECTION 
CRITERIA 
Inclusion Criteria: 
1. Age  ≥ 6 months to ≤ 18 years 
2. Documentation of a CF diagnosis as evidenced by one or 
more clinical features consistent with the CF phenotype or 
positive CF Newborn Screening result for IRT/DNA or 
IRT/IRT and one or more of the following criteria: 
a. sweat chloride ≥ 60 mEq/liter by quantitative 
pilocarpine iontophoresis test (QPIT)  
b. two well-characterized mutations in the cystic 
fibrosis transmembrane conductive regulator 
(CFTR) gene 
c. Abnormal nasal potential difference (change in NPD 
in response to a low chloride solution and 
isoproteronol of less than -5 mV)  
3. Documented new positive oropharyngeal, sputum or lower 
respiratory tract culture for Pa obtained within 40 days of 
the Baseline Visit (Visit 1), defined as: a) first lifetime 
documented Pa positive culture; or b) Pa recovered after at 
least a two-year history of Pa negative respiratory cultures 
( 1 culture/ year) 
4. Clinically stable with no evidence of any significant 
respiratory symptoms at the Baseline Visit that would 
require administration of intravenous anti-pseudomonal 
antibiotics, oxygen supplementation, and/or hospitalization 
as determined by the study physician 
5. Written informed consent obtained from participant or 
participant’s legal representative (and assent when 
applicable) and ability for participant to comply with the 
requirements of the study  
Exclusion Criteria: 
1. Macrolide antibiotic use within 30 days of the Baseline 
Visit  
2. Initiation of current course of treatment with TIS >14 days 
prior to Baseline Visit 


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3. Weight <6.0 kg at the Baseline Visit 
4. History of aminoglycoside hypersensitivity or adverse 
reaction to inhaled aminoglycoside 
5. History of azithromycin hypersensitivity or adverse reaction 
to azithromycin or allergy to macrolide antibiotics  
6. History of positive respiratory culture for NTM or B. 
cepacia complex within 2 years of the Baseline Visit 
7. History of unresolved, abnormal renal function (defined as 
serum creatinine greater than 1.5 times the upper limit of 
normal for age).   
8. History of unresolved,  abnormal liver function tests 
(defined as ALT and/or AST greater than 4 times the upper 
limit of normal range) or history of portal hypertension   
9. History of unresolved, abnormal neutropenia (ANC ≤ 1000)  
10. Abnormal ECG test at the Baseline Visit defined as a QTc 
(B) of ≥460 msec or history of ventricular arrhythmia 
11. History of abnormal hearing sensitivity defined as  hearing 
threshold levels >25 dB HL (decibels Hearing Level) for 
VRA (visual reinforcement audiometry) at any frequency 
(500-4000Hz) or >20 dB HL for play or standard 
audiometry at any two frequencies (500-8000Hz)  in either 
ear, not associated with middle ear disease (including 
infection) or a flat (Type B) tympanogram   
12. New initiation of chronic therapy (greater than 21 days) 
with drugs known to prolong QT interval (refer to 
Appendix III) within 30 days prior to the Baseline Visit or 
co-administration of nelfinavir or oral anticoagulants 
13. Positive serum or urine pregnancy test at the Baseline Visit 
(to be performed on all females of child-bearing potential) 
or for females of child bearing potential: pregnant, 
breastfeeding, or unwilling to use barrier contraception 
during participation in the study 
14. Administration of any investigational drug within 30 days 
prior to the Baseline Visit 
15. Presence of a condition or abnormality (e.g., pre-existing 
heart disease) that in the opinion of the site investigator 
would compromise the safety of the participant or the 
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TEST PRODUCT, 
DOSE, AND ROUTE 
OF 
ADMINISTRATION 
Active treatment: azithromycin provided as 900 mg oral 
suspension in bottles.  Product is reconstituted with 12 mL 
water.  Final concentration = 200mg/5mL (final volume = 22.5 
mL) 
Participant dose: Approximately 10 mg/kg up to a maximum of 
500 mg per dose orally three times weekly  
CONTROL 
PRODUCT, DOSE 
AND ROUTE OF 
ADMINISTRATION 
Placebo: vehicle suspension.  Product is reconstituted with 12 
mL water.  Final volume = 22.5 mL 
Participant dose:  Equivalent volume to match test product dose 
of approximately 10 mg/kg up to a maximum of 500 mg per 
dose orally three times weekly 
STANDARD OF 
CARE 
TREATMENT:  
TOBRAMYCIN 
INHALATION 
SOLUTION 
Participants receive TIS (TOBI®) 300 mg BID.  Each treatment 
course will be administered for 28 consecutive days by 
inhalation using the PARI LC PLUS™ Nebulizer.   
The initial two treatment courses of TIS will be provided as 
part of the study, unless TIS was prescribed and administered 
in the 14 days prior study enrollment.  Pa positive cultures at 
subsequent study visits will be treated with TIS as standard of 
care and is required per protocol.   
DURATION OF 
SUBJECT 
PARTICIPATION 
AND DURATION 
OF STUDY 
Participants will be on study for 18 months 
Screening:  Eligible participants will be scheduled for 
Baseline Visit 1 within 40 days of their Pa positive culture 
and prior to receiving any anti-pseudomonal therapy other 
than TIS and ≤14 days from initiating TIS 
Treatment:  18 months (78 weeks ±2 weeks) 
Follow-up:  Final Visit at Week 78  
The total duration of the study is expected to be forty-nine (49) 
months:  thirty-one (31) months for participant recruitment and 
eighteen (18) for final participant follow-up. 
CONCOMITANT 
MEDICATIONS 
 
 
Allowed: Standard therapy for CF is allowed except for 
treatments noted in the exclusion criteria and the prohibited 
medications section below.  Ongoing chronic treatment (>30 
days prior to the Baseline Visit) with inhaled dornase alpha, 
high dose ibuprofen, hypertonic saline, inhaled or systemic 
steroids, short and long-acting bronchodilators and airway 
clearance, and FDA-approved CFTR modulators is 
allowed.  Acceptable chronic therapies initiated after study 
enrollment if clinically indicated include: inhaled dornase 
alpha, hypertonic saline, short/long acting bronchodilators, 
airway clearance, and FDA-approved CFTR modulators.  


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Acute treatment with drugs categorized as known to prolong 
QT interval is allowed according to the following:  
 
For drugs commonly used to treat CF patients (e.g. 
ciprofloxacin, levofloxacin, moxifloxacin, fluconazole, 
propofol, sevoflurane, ondansetron), study drug does not 
need to be stopped.   
 
For all other drugs categorized as known to prolong QT 
interval, acute use is allowed, but study drug (azithromycin 
or placebo) must be temporarily stopped (Refer to Section 
11.2).   After acute treatment is completed, participants 
should re-start study drug on the next scheduled treatment 
day. Drugs known to prolong QT interval are described as 
follows: “Substantial evidence supports the conclusion that 
these drugs prolong the QT interval AND are clearly 
associated with a risk of torsades de pointes (TdP), even 
when taken as directed in official labeling” and listed on the 
CredibleMeds® Website (Composite List of All QT Drugs 
under the category “Known Risk of TdP”).  
Treatment for pulmonary exacerbations and as required for 
acute care is allowed except for treatments noted in the 
prohibited medications section below.  Physicians are 
encouraged to prescribe acute antibiotic therapy only in the 
presence of symptoms.   
Prohibited: The following medications are prohibited until the 
end of study (Visit 8, Week 78):  
 
Investigational therapies 
  Chronic inhaled antibiotics (e.g., Cayston®, colistin). 
Chronic treatment is defined as more than 1 course of 
therapy within a 6-month period.  
 Initiation of chronic treatment (defined as >21 days of 
treatment) with high dose ibuprofen and systemic steroids 
The following medications are prohibited when the participant 
is concurrently receiving study drug.  Therefore, study drug 
must be stopped during concurrent administration of these 
medications.  
 
Macrolides (other than study drug): Acute treatment with 
macrolides, including azithromycin is allowed however no 
acute macrolides may be prescribed without stopping study 
drug (azithromycin or placebo). After acute treatment is 
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next scheduled treatment day. 
 
Drugs known to prolong QT interval (Composite List of All 
QT Drugs under the category “Known Risk of TdP”).  
(Refer to Appendix III and Section 6.1).   
 
Nelfinavir or oral anticoagulants 
 
 
EVALUATIONS 
 
PRIMARY 
ENDPOINT 
Difference between treatment groups in the time to a protocol-
defined pulmonary exacerbation.   
Pulmonary exacerbations will be defined according to an a 
priori sign and symptom-based definition.   
SECONDARY 
ENDPOINTS 
1. Time to Pa recurrence over the 18-month study period 
2. Safety as measured by the incidence of adverse events and 
laboratory abnormalities over the 18-month study period  
3. Changes from baseline in plasma inflammatory markers  
 
Secondary Exploratory Endpoints: 
4. Proportion of participants with initial Pa eradication 
success, defined as a Pa negative respiratory culture at 
Week 13 
5. Proportion of all cultures obtained that are positive for Pa 
during the 18-month study period 
6. Prevalence of treatment-emergent Staphylococcus aureus 
(methicillin susceptible and resistant), Haemophilus 
influenzae, Burkholderia cepacia complex, 
Stenotrophomonas maltophilia, and Alcaligenes 
(Achromobacter) xylosoxidans (from oropharyngeal and 
sputum cultures), and non-tuberculous mycobacteria (in 
sputum only) during the 18-month period  
7. Proportion of participants hospitalized over the 18-month 
study 
8. Proportion of participants prescribed acute oral, inhaled, 
and IV acute antibiotics over the 18-month study 
9. Change from baseline to the end of the 18-month study in 
pulmonary function (FVC, FEV1, FEF25-75%) in those old 
enough to perform spirometry (≥4 years of age) 
10. Change from baseline to the end of the 18-month study in 
weight and height 
11. Change from baseline to the end of the 18-month study in  
the self-report and parent-completed Cystic Fibrosis 
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12. Changes from baseline in the respiratory microbiome 
13. Association of baseline inflammatory marker values, 
microbial diversity measures and other baseline clinical and 
demographic characteristics with clinical and microbiologic 
outcomes over the 18-month study 
PLANNED 
INTERIM 
ANALYSES  
An independent data safety monitoring board (DSMB) will 
review comprehensive interim reports on a semi-annual basis.  
A priori interim stopping rules for efficacy and safety of the 
primary endpoint will be guided as outlined in the DSMB 
charter.  Serious adverse events will be monitored by the 
committee on an ongoing basis throughout the study. 
STATISTICS 
Primary Analysis 
Plan 
An intent to treat analysis will be used for the primary analysis, 
including all participants who were randomized.  The primary 
endpoint, time to pulmonary exacerbation, will be compared 
between treatment groups using Cox proportional hazards 
regression adjusting for randomization strata (age 6 mos-3 
years, >3-6 years, >6-12 years, >12-18 years).  Hazard ratios 
and 95% confidence intervals will be used to describe the 
treatment effect and p-values will be evaluated against a two-
sided 0.05 level of significance.   
Rationale for 
Number of Subjects 
Assuming a two-sided 0.05 level type I error and a sample size 
of approximately 137 children randomized to each treatment 
group (culture-based TIS with oral placebo or culture-based 
TIS with oral azithromycin), the study has 90% power to detect 
a hazard ratio for the time to exacerbation of 0.53 or smaller (a 
47% or greater reduction) between treatment groups and 80% 
power to detect a hazard ratio of 0.58 or smaller (a 42% or 
greater reduction).  These estimates account for six planned 
interim analyses and one final analyses of the primary endpoint.  
With a maximum projected withdrawal rate of at most 9% over 
the course of the 18 month study based on the prior EPIC trial 
with similar duration of follow-up, it is conservatively 
estimated that the study still has at minimum 80% power to 
detect a hazard ratio of 0.56 or smaller between treatment 
groups (a 44% or greater reduction).   
 


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1 
BACKGROUND AND RATIONALE 
CF lung disease begins in the first few months of life and follows a course of recurrent lower 
airway bacterial infection and inflammation(16-18), early gas trapping and bronchiectasis(19), and 
progression of disease over years and decades at a variable pace. With the development of chronic lung 
infection, obstructive disease progressively worsens, ultimately leading to respiratory failure(3, 4, 20, 21). 
Pseudomonas aeruginosa (Pa) is the most important pathogen infecting the CF lower airways(17, 22), and 
its acquisition early in life is associated with a pro-inflammatory effect(14), lower lung function, poor 
nutritional outcomes, and decreased survival(21).  Greater morbidity and cost of care is associated with 
chronic Pa infection including increased rate of hospitalizations and greater need for antibiotic therapy(2, 
23). In spite of aggressive therapy, more rapid decline in lung function and higher mortality rates ensue(24-
31). 
Pa infection of the CF airway typically proceeds from early infection to chronic infection, 
defined as >50% of cultures positive for Pa in a 12 month period(3, 32). Early Pa strains have distinct 
characteristics more likely to respond to antibiotic therapy, including non-mucoid phenotype, broad 
antimicrobial susceptibility and significantly lower colony density (16). Over time, Pa forms biofilms 
within the CF airway environment(33) and develops a mucoid phenotype (31) making it more refractory to 
treatment.   Although some studies have shown that a minority of individuals with CF spontaneously 
clear early Pa infection(3, 34), data from multiple studies suggest that antibiotics are superior to no 
treatment in clearing Pa from respiratory cultures(3, 34-39). Understanding the transition period from early 
to chronic Pa infection is thus of critical importance in identifying strategies to prevent this progression.  
Therapeutic approaches to treatment of early Pa infection have varied by antibiotic type, route, 
duration of treatment, and use of single or multiple courses, making interpretation challenging for 
clinicians eager to adopt these treatment strategies(3, 34-39). The focus of these trials was primarily 
towards the microbiologic endpoint of clearance of Pa from either the upper(34, 35, 37) or lower (40, 
41)airway rather than clinical endpoints. Several studies examined the durability of microbiologic effect 
(i.e., time to Pa recurrence)(36, 37, 39, 40).  One of the largest studies was the ELITE trial(39), which 
compared the efficacy of tobramycin inhalation solution (TIS, TOBI®) given for 28 days as compared to 
56 days for individuals with CF >6 months of age (90% were under 18 years of age) with early Pa 
infection and demonstrated that time to Pa recurrence did not differ by duration of initial therapy, with 
an average of 33% of individuals having Pa recurrence within the 27 months of follow-up. Two 
intervention studies of early Pa infection have compared multiple anti-pseudomonal treatment strategies, 
one focused on Pa eradication after 28 days of  therapy(42) and the other on clinical outcomes, in 
addition to microbiologic endpoints, over an 18 month period(5). The 28-day study was a randomized, 
open label trial which compared inhaled colistin with oral ciprofloxacin (the CC group, n=105) versus 
TIS and oral ciprofloxacin (the TC group, n=118) at the time of initial Pa infection. At 28 days, the 
eradication rate was not significantly different between the 2 regimens (CC: 62.8% vs. TC: 65.2%). The 
second study  was the EPIC clinical trial supported by NHLBI, NIDDK and CFF (Grant U01-
HL080310)(5) in which 306 children with early Pa were randomized to TIS given at quarterly intervals 
vs. TIS given only when quarterly cultures were positive for Pa (culture-based therapy). The primary 
clinical outcome was time to pulmonary exacerbation. All children enrolled in the trial received an 
initial course of TIS (with or without oral ciprofloxacin) to induce eradication before proceeding to their 
randomized treatment arms. There were several key findings from this study. First, there was no 
evidence to suggest that more intensive quarterly treatment with TIS (with or without ciprofloxacin) was 


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more effective than therapy given only when cultures were positive for Pa, both in terms of time to 
pulmonary exacerbation and Pa recurrence(5). Second, the initial TIS treatment course successfully 
resulted in negative Pa cultures in 85% of children although by the end of the study 34% had at least one 
Pa recurrence. The addition of ciprofloxacin to the TIS therapy conferred no additional benefit 
throughout the 18 month study. Third, 50% of all participants experienced a pulmonary exacerbation, 
and the subset of children failing initial eradication were at the highest risk for exacerbations and 
progression to chronic Pa infection(6). 
Based upon the accumulative published data from the studies outlined above, a working group of 
European and North American CF clinicians met in April 2012 to make recommendations for early Pa 
eradication therapy. The consensus of the group was that antibiotic treatment for first or recent Pa 
acquisition is indicated (consistent with a recent Cochrane Review in 2009(38)) and that either TIS 
monotherapy(5, 39)  or inhaled colistin with oral ciprofloxacin are efficacious(42) and should be considered 
standard of care(43). In general, inhaled colistin with oral ciprofloxacin is used at European sites and TIS 
is primarily used in the US. Among a sub-cohort of children enrolled in the U.S. Cystic Fibrosis 
Foundation (CFF) National Patient Registry(44) with first or recent acquisition of Pa in 2010, 82% were 
treated with TIS.  By contrast, no clinical trials have demonstrated azithromycin to be bactericidal for Pa 
as evidenced by a decrease in bacterial load (13). For this reason, azithromycin has not been considered as 
a potential therapy for eradication of early Pa infection and its use in this population is low. The 2010 
Cystic Fibrosis Foundation (CFF) national patient registry reported only 27% of patients with first or 
recent Pa had been prescribed chronic azithromycin therapy (personal communication, Bruce Marshall, 
MD, CFF). 
There have been, however, multiple trials assessing the clinical, rather than microbiologic impact 
of azithromycin for established Pa infection(11-13) with clear evidence of impact on clinical endpoints 
including significant reductions in exacerbations, improvement in pulmonary function, and weight gain 
as compared to placebo(11-13).  Part of this clinical impact may be due to an anti-inflammatory effect(10, 11, 
45), as demonstrated by a reduction in a range of serum inflammatory markers including calprotectin, 
serum amyloid antigen, absolute neutrophil count and C-reactive protein (CRP) at both 28 and 168 days 
of chronic therapy(10).  Azithromycin has been studied in younger, healthy children with CF without 
chronic Pa(9) showing a 50% reduction in the rate of pulmonary exacerbations among those treated with 
azithromycin and improved body weight(9). These are encouraging findings but no studies of macrolide 
antibiotics such as azithromycin have been focused on the question of enhancing eradication of early Pa 
infection even though the complementary mechanisms of action of azithromycin could enhance the 
efficacy of proven anti-pseudomonal therapies, such as TIS. 
It is very encouraging that the majority of children with early Pa infection will rapidly clear the 
pathogen from both upper(5, 39) and lower(40) airway secretions. Challenges, however, remain. First and 
foremost, Pa recurrence may occur within months(5, 39).   In both the EPIC and ELITE trials, one-third of 
participants had recurrence of Pa during the 18 month and 27 month follow-up periods, respectively. In 
the EPIC Observational study, an ongoing prospective study of risk factors for Pa acquisition in young 
Pa-negative children with CF(46), the rate of initial Pa acquisition among 889 children with a mean age 
of 4.5 (SD 3.5) years was 18 cases per 100 person-years (95% confidence interval [CI] 17, 20), while the 
rate of subsequent Pa acquisition was slightly higher at 23 cases (95% CI: 21, 25) per 100 person-years. 
These data suggest that children with a history of Pa, even following successful eradication, are a 
higher-risk population requiring close monitoring. There are likely multiple factors associated with re-
infection. In the EPIC Clinical trial, a significant association was observed between the occurrence of an 


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exacerbation and Pa recurrence(6).  Notably since 50% of the children in the EPIC trial experienced an 
exacerbation requiring acute antibiotic intervention (in addition to the study drug) during the follow up 
across treatment arms, a therapeutic approach that reduces the frequency of exacerbations may prolong 
the time to re-infection and potentially delay chronic Pa infection (Figure 1).   
 
Figure 1. Hypothetical Benefits of the Proposed Intervention.  We hypothesize that the addition of 
azithromycin to TIS in children and adolescents with CF and early Pa will decrease exacerbation 
frequency, resulting in a delay in time to Pa recurrence. Through this process it is hoped that 
progression to chronic infection will also be delayed, although the time course for progression to 
chronic infection is beyond the duration of this study. 
 
The current proposal is a multicenter, randomized, placebo-controlled trial designed to evaluate 
the efficacy and safety of combining two therapeutic mechanisms of action to address early Pa infection 
in CF. Participants will be randomized to either oral azithromycin or oral placebo, in addition to 
receiving a standardized anti-pseudomonal treatment regimen with TIS. For the purposes of this 
proposal, TIS therapy is defined as an initial eradication treatment with 1-2 courses of 28 days TIS and 
subsequent 28 day treatments only at times a quarterly oropharyngeal or sputum culture is positive for 
Pa. TIS, an aminoglycoside, is a well established bactericidal antibiotic in vitro(47) and in vivo(48) in CF 
respiratory secretions. Azithromycin, a macrolide antibiotic, has no demonstrated antimicrobial efficacy 
in vivo(13)or in vitro against Pa growing in culture in logarithmic phase(49), but may be effective in vivo 
at killing Pa in biofilms(50, 51).  In addition, it has a demonstrated anti-inflammatory effect(10, 11, 13, 45). 
These two approaches are complementary and should be additive. It is additionally possible that the two 
antibiotic approaches may be synergistic in vivo because macrolides may reduce quorum-sensing, 
motility and biofilm formation, all critical to Pa virulence and its ability to evade killing by 
aminoglycosides(52-55). A modest degree of in vitro synergy has also been demonstrated against Pa in CF 
(56). Thus, the combined therapy, as compared to TIS monotherapy, is hypothesized to demonstrate 
improved efficacy both for clinical outcomes including decreasing the risk of pulmonary exacerbation, 
and microbiologic endpoints including delaying Pa recurrence (Figure 1).  The primary goal of this 
proposed study is to evaluate the effectiveness of adjunctive therapy with azithromycin to improve 
clinical outcome.  Based upon the findings of these 2 trials and our current understanding of the drugs' 
mechanisms of action, we hypothesize that the use of chronic oral azithromycin will provide added 
clinical benefit to the use of standardized anti-pseudomonal therapy with TIS among children 


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with early Pa and decrease the risk of pulmonary exacerbation, reduce inflammation, and reduce 
the rate of Pa recurrence.  
1.1 
Risk / Benefit Assessment 
Risks of Study Drug:  While the safety and efficacy of azithromycin treatment given for 6 months has 
been well studied, there is no published safety and efficacy data for azithromycin for longer periods in 
the context of a placebo-controlled clinical trial or among children with CF less than 6 years of age(57). 
Overall, gastrointestinal side effects have been comparable in individuals with CF to those described in 
other populations(58). One study reported 33% of azithromycin vs.16% of placebo participants had 
nausea and 23% of azithromycin vs. 8% of placebo participants had diarrhea(13) and a subsequent study 
performed in children and adolescents with CF, demonstrated comparable gastrointestinal side effects 
between azithromycin and placebo groups(9). Hepatotoxicity associated with azithromycin has not been 
detected as measured by elevations in AST, ALT, or GGT (9, 13).  No increase in subjective hearing loss 
or tinnitus has been noted among azithromycin participants in the CF studies (9, 12, 13).  However, hearing 
loss has been reported in adults with chronic obstructive lung disease(59) and cardiovascular (CV) 
disease(60) who received chronic azithromycin therapy for one year. Hearing loss and/or tinnitus are 
noted under post-marketing experience in the drug label although causal relationship to drug product is 
not possible to ascertain.  A report(61) of a retrospective analysis of Tennessee Medicaid records  
identified a small increase risk ( HR 2.88,95%CI 1.79,4.63, p<0.001) for CV sudden death in older 
adults (mean age > 60 years) with CV risk factors. By contrast, 2 large randomized controlled trials(60, 62) 
of adults with known CV disease receiving chronic azithromycin therapy did not observe an increase in 
CV death and a large retrospective Danish study(63) found the risk of CV death comparable to penicillin, 
an antibiotic not known to have CV effects. Because of the concern raised by the Tennessee study( 63) 
that azithromycin may have arrhythmogenic potential in patients with increased CV risk (i.e., history of 
documented prolonged QT interval, hypokalemia, hypomagnesemia, bradycardia, or co-administration 
of other known arrhythmogenic drugs), in 2013, the FDA revised the azithromycin label to reflect this 
information(64).  The eligible population for this proposed CF study, children with no known CV risk 
factors, should not be at increased risk for macrolide associated CV sudden death. An increased risk of 
treatment-emergent macrolide-resistant S. aureus and H. influenzae has been observed (9) but there are 
no data to suggest an impact on clinical outcome. Although a recent report presents data from 
experiments using an animal model and clinical data from one site about a potential link between 
macrolides and increased risk of M. abscessus(65), no increased risk of treatment-emergent non-
tuberculous mycobacteria (NTM) has been observed in any controlled clinical trials(9, 13, 66).  A large 
epidemiologic study conducted using the CFF National patient registry found that patients with the 
greatest number of years of chronic macrolide usage were the least likely to develop incident NTM(67). 
 
Risks of Study Procedures: All study procedures performed during the study are consistent with standard 
of clinical care and there are no perceived additional risks in regard to physical well-being. During the 
study, there will be additional blood draws and respiratory culture collections.  The risk of collecting 
blood includes soreness, bruising, mild pain, mild bleeding or infection at the site. There is also risk of 
fainting or light-headedness. Topical application of a numbing agent to reduce the pain from the blood 
draws will be allowed. This may cause mild skin discoloration or swelling that resolves in 1 to 2 hours. 
The collection of the respiratory samples from oropharyngeal swab may cause minor brief discomfort, 
gagging or coughing. There is a small risk of wheezing, shortness of breath, and increased cough when 


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performing spirometry and redness of the skin or minor skin irritation may occur when performing ECG 
testing and oximetry.   
Risks to Confidentiality: As with any research study, there is a potential risk of breach of confidentiality. 
Study personnel at each site will enter data from source documents into a protocol-specific electronic 
Case Report Form (eCRF). Study participants will not be identified by name in the study database or on 
any data capture screens, but will be identified by initials and a subject identification number unique to 
this study. Only authorized individuals will be able to link the study ID to the subject’s name (site 
personnel at the individual sites and DCC clinical site monitors or auditors). All investigators and staff 
involved in the proposed research will have completed human subjects’ protection training and be bound 
by the agreement of confidentiality.  
Possible Benefit: The combined therapy of TIS and azithromycin, as compared to TIS monotherapy, is 
hypothesized to demonstrate improved efficacy both for clinical outcomes including decreasing the risk 
of pulmonary exacerbation and for microbiologic endpoints including delaying Pa recurrence. 
2 
STUDY OBJECTIVES 
2.1 
Primary Objective 
The primary objective of this study is to compare the time to pulmonary exacerbation between 
participants randomized to oral placebo with TIS versus oral azithromycin with TIS.    
2.2 
Secondary Objectives 
The secondary objectives of this study are to: 
1. Compare Pseudomonas aeruginosa (Pa) recurrence between treatment groups 
2. Evaluate the safety profile of azithromycin in combination with culture-based TIS therapy as 
compared to culture-based TIS therapy alone, including the incidence of treatment emergent 
pathogens     
3. Compare changes in plasma inflammatory markers between treatment groups  
Secondary Exploratory Objectives 
4. Compare hospitalization rates and other indices of clinical efficacy, including changes in height, 
weight, pulmonary function (in participants ≥4 years old) and respiratory symptoms, between 
treatment groups  
5. Compare changes in the respiratory microbiome between treatment groups  
6. Develop a comprehensive baseline predictive model of poor microbiologic or clinical response to 
therapy, including respiratory microbiome, inflammatory biomarkers, demographic and clinical 
characteristics in order to identify high risk children for future studies of alternative therapeutic 
approaches 
7. Establish a linked data and specimen repository for future studies of disease mechanisms, potential 
novel therapeutic approaches and predicting response to therapy 


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3 
STUDY DESIGN 
3.1 
Study Overview 
This is a multicenter, double-blind, randomized, placebo-controlled clinical trial in 274 children with CF 
ages 6 mos -18 years with early Pa, defined as either a first lifetime documented Pa culture or a Pa 
positive culture after at least two years of negative cultures.  The study will assess the clinical and 
microbiologic efficacy and safety of azithromycin given three times weekly in combination with 
standardized TIS therapy among children with early Pa.  TIS therapy is defined as an initial eradication 
treatment with 1-2 courses of 28 days TIS and subsequent 28-day treatments only at times a quarterly 
oropharyngeal culture is positive for Pa.  Eligible participants will be randomized within 40 days from 
the date of collection of their Pa positive culture. Participants initiating TIS more than 14 days prior to 
the baseline visit for treatment of their Pa positive culture will be excluded.  Participants will be 
randomized in a 1:1 fashion to receive one of the following two treatment strategies for 18 months: (1) 
oral placebo in addition to standardized TIS therapy, or (2) oral azithromycin in addition to standardized 
TIS therapy.   
Three times weekly administration of oral azithromycin or matching placebo will be continued 
throughout the 18 month trial.   All participants will receive a 28-day course of TIS therapy during the 
first quarter of the trial and have a follow-up culture obtained to document Pa clearance. Participants 
who remain Pa positive after the first 28-day course of TIS will receive a second 28-day course of TIS.  
Subsequently, participants will receive TIS therapy (administered as a single 28-day course) only if their 
quarterly cultures are Pa positive. Cultures will be obtained at the start of each quarter for the duration 
of the 18 month study (see Figure 2).   
Clinical and microbiologic outcomes will be assessed throughout the 18-month trial, including the 
occurrence of pulmonary exacerbations and Pa recurrence.   
Figure 2 
 


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The total duration of the study is expected to be forty-nine (49) months: thirty-one (31) months for 
participant recruitment and eighteen (18) for final participant follow-up. 
4 
CRITERIA FOR EVALUATION 
4.1 
Primary Efficacy Endpoint 
The primary endpoint in the study is the difference between treatment groups in the time to a protocol-
defined pulmonary exacerbation, comparing oral placebo with TIS versus oral azithromycin with TIS.  
Pulmonary exacerbations will be defined according to an a priori sign and symptom-based definition 
(Appendix II).   
4.2 
Secondary Endpoints 
1. Time to Pa recurrence over the 18-month study period 
2. Safety as measured by the incidence of adverse events and laboratory abnormalities over the 18-
month study period  
3. Changes from baseline in plasma inflammatory markers  
Secondary Exploratory Endpoints 
4. Proportion of participants with initial Pa eradication success, defined as a Pa negative respiratory 
culture at Week 13 
5. Proportion of all cultures obtained that are positive for Pa during the 18-month study period 
6. Prevalence of treatment-emergent Staphylococcus aureus (methicillin susceptible and resistant), 
Haemophilus influenzae, Burkholderia cepacia complex, Stenotrophomonas maltophilia, and 
Alcaligenes (Achromobacter) xylosoxidans (from oropharyngeal and sputum cultures), and non-
tuberculous mycobacteria (in sputum only) during the 18-month period 
7. Proportion of participants hospitalized over the 18-month study 
8. Proportion of participants prescribed acute oral, inhaled, and IV acute antibiotics over the 18-month 
study 
9. Change from baseline to the end of the 18-month study in pulmonary function (FVC, FEV1, FEF25-
75%) in those old enough to perform spirometry (≥4 years of age) 
10. Change from baseline to the end of the 18-month study in weight and height 
11. Change from baseline to the end of the 18-month study in the self-report and parent-completed 
Cystic Fibrosis Respiratory Diary scores 
12. Changes from baseline in the respiratory microbiome 
13. Association of baseline inflammatory marker values, microbial diversity measures and other 
baseline clinical and demographic characteristics with clinical and microbiologic outcomes over the 
18-month study 
5 
PARTICIPANT SELECTION 
5.1 
Study Population 
Children and adolescents with a diagnosis of CF who meet the inclusion and exclusion criteria will be 
eligible for participation in this study.   


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5.2 
Inclusion Criteria 
1. Age  ≥ 6 months to ≤ 18 years  
2. Documentation of a CF diagnosis as evidenced by one or more clinical features consistent with the 
CF phenotype or positive CF Newborn Screening result for IRT/DNA or IRT/IRT and one or more 
of the following criteria: 
a. 
sweat chloride ≥ 60 mEq/liter by quantitative pilocarpine iontophoresis test (QPIT)  
b. 
two well-characterized mutations in the cystic fibrosis transmembrane conductive 
regulator (CFTR) gene 
c. 
Abnormal nasal potential difference (change in NPD in response to a low chloride 
solution and isoproteronol of less than -5 mV)  
3. Documented new positive oropharyngeal, sputum or lower respiratory tract culture for Pa obtained 
within 40 days of the Baseline Visit (Visit 1), defined as: a) first lifetime documented Pa positive 
culture; or b) Pa recovered after at least a two-year history of Pa negative respiratory cultures ( 1 
culture/ year) 
4. Clinically stable with no evidence of any significant respiratory symptoms at the Baseline Visit that 
would require administration of intravenous anti-pseudomonal antibiotics, oxygen supplementation, 
and/or hospitalization as determined by the study physician 
5. Written informed consent obtained from participant or participant’s legal representative (and assent 
when applicable) and ability for participant to comply with the requirements of the study  
5.3 
Exclusion Criteria 
1. Macrolide antibiotic use within 30 days of the Baseline Visit  
2. Initiation of current course of treatment with TIS >14 days prior to Baseline Visit 
3. Weight <6.0 kg at the Baseline Visit 
4. History of aminoglycoside hypersensitivity or adverse reaction to inhaled aminoglycoside 
5. History of azithromycin hypersensitivity or adverse reaction to azithromycin or allergy to macrolide 
antibiotics  
6. History of positive respiratory culture for NTM or B. cepacia complex within 2 years of the Baseline 
Visit 
7. History of unresolved, abnormal renal function (defined as serum creatinine greater than 1.5 times 
the upper limit of normal for age).   
8. History of unresolved, abnormal liver function tests (defined as ALT and/or AST greater than 4 
times the upper limit of normal range) or history of portal hypertension  
9. History of unresolved, abnormal neutropenia (ANC ≤ 1000)   
10. Abnormal ECG test at the Baseline Visit defined as a QTc (B) of ≥460 msec or history of ventricular 
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11. History of abnormal hearing sensitivity defined as hearing threshold >25 dB HL (decibels Hearing 
Level) for VRA (visual reinforcement audiometry) at any frequency (500-4000Hz) or  >20 dB HL 
for play or standard audiometry at any two frequencies (500-8000Hz)  in either ear, not associated 
with middle ear disease (including infection) or a flat (Type B) tympanogram  
12. New initiation of chronic therapy (greater than 21 days) with drugs known to prolong QT interval 
(refer to Appendix III) within 30 days prior to the Baseline Visit or co-administration of nelfinavir or 
oral anticoagulants 
13. Positive serum or urine pregnancy test at the Baseline Visit (to be performed on all females of child-
bearing potential) or for females of child bearing potential: pregnant, breastfeeding, or unwilling to 
use barrier contraception during participation in the study 
14. Administration of any investigational drug within 30 days prior to the Baseline Visit 
15. Presence of a condition or abnormality (e.g., pre-existing heart disease) that in the opinion of the site 
investigator would compromise the safety of the participant or the quality of the data 
5.4 
Study Specific Tolerance for Inclusion/Exclusion Criteria 
Participants who fail to meet all the inclusion criteria or who meet any of the exclusion criteria will not 
be enrolled in this study.  Waivers of any of the above study entry criteria will not be granted. 
5.5 
Screen Fail Criteria 
Any consented participant who is excluded from the study before randomization is considered a screen 
failure.  All screen failures must be documented with the reason for the screen failure adequately stated.  
Participants who fail screening can be rescreened once if the site investigator feels they meet eligibility 
criteria and following confirmation from the study monitor.  Rescreened participants will complete all 
screening procedures (e.g., results from previous screening tests cannot be used). 
6 
CONCURRENT MEDICATIONS 
All participants should be maintained on the same medications throughout the entire study period, 
without introduction of new chronic therapies, unless medically indicated. 
6.1 
Allowed Medications and Treatments 
Standard therapy for CF is allowed except for treatments noted in the prohibited medications section 
below.   
Ongoing chronic treatment (>30 days prior to the Baseline Visit) with inhaled dornase alpha, high dose 
ibuprofen, hypertonic saline, inhaled or systemic steroids, short and long-acting bronchodilators, airway 
clearance, and FDA-approved CFTR modulators is allowed.  Acceptable chronic therapies initiated after 
study enrollment if clinically indicated include: inhaled dornase alpha, hypertonic saline, short/long 
acting bronchodilators, airway clearance, and FDA-approved CFTR modulators.  
Acute treatment with  drugs categorized as known to prolong QT interval is allowed according to the 
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 
For drugs commonly used to treat CF patients (e.g. ciprofloxacin, levofloxacin, moxifloxacin, 
fluconazole, propofol, sevoflurane, ondansetron), study drug does not need to be stopped.   
 
For all other drugs categorized as known to prolong QT interval, acute use is allowed, but study drug 
(azithromycin or placebo) must be temporarily stopped (Refer to Section 11.2).   After acute 
treatment is completed, participants should re-start study drug on the next scheduled treatment day.  
Drugs known to prolong QT interval are described as follows: “Substantial evidence supports the 
conclusion that these drugs prolong the QT interval AND are clearly associated with a risk of torsades 
de pointes (TdP), even when taken as directed in official labeling” and listed on the CredibleMeds® 
Website (Composite List of All QT Drugs under the category “Known Risk of TdP”).  
Treatment for pulmonary exacerbations and as required for acute care is allowed except for treatments 
noted in the prohibited medications section below.  Physicians are encouraged to prescribe acute 
antibiotic therapy only in the presence of symptoms.   
6.2 
Prohibited Medications and Treatments 
The following medications are prohibited until the end of study (Visit 8, Week 78):  
 Investigational therapies  
 Chronic inhaled antibiotics (e.g., Cayston®, colistin). Chronic treatment is defined as more than 1 
course of therapy within a 6-month period.  
 Initiation of chronic treatment (defined as >21 days of treatment) with high dose ibuprofen and 
systemic steroids 
The following medications are prohibited when the participant is concurrently receiving study 
drug.  Therefore, study drug must be stopped during concurrent administration of these 
medications.  
 Macrolides (other than study drug): Acute treatment with macrolides, including azithromycin is 
allowed however no acute macrolides may be prescribed without temporarily stopping study drug 
(azithromycin or placebo). After acute treatment is completed, participants should re-start study drug 
on the next scheduled treatment day.  
 Drugs known to prolong QT interval (Composite List of All QT Drugs under the category “Known 
Risk of TdP”).  (Refer to Appendix III and Section 6.1) 
 Nelfinavir or oral anticoagulants 
7 
STUDY TREATMENTS 
7.1 
Method of Assigning Participants to Treatment Groups 
Study personnel at the investigative site will use the Medidata Rave® and Balance™ systems to 
randomize each participant.  An adaptive randomization (dynamic allocation based on minimization(68)) 
will be employed with the goal of ensuring equal representation in each study arm for each age strata. 
Participants will be randomly assigned in a 1:1 ratio to one of two arms: 1) oral placebo with TIS 
therapy, or 2) oral azithromycin with TIS therapy.  The adaptive randomization algorithm will include 


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baseline age group (age 6 mos-3 years, >3-6 years, >6-12 years, >12-18 years).  The dynamic allocation 
algorithm seeks to optimize randomization balance by minimizing a weighted average of the marginal 
imbalance(69) of  treatment allocation for each factor and for the study overall.  A random element is 
added to the otherwise deterministic minimization algorithm to reduce allocation predictability by using 
a biased coin(70) to include a chance of allocation to a treatment arm other than the arm that optimizes 
balance.  After baseline data collection, participants will be randomized to one of the two study groups.  
To randomize a new participant, study personnel at the investigative site will complete the electronic 
case report form (eCRF) in the Medidata Rave system required to provide the stratification information 
and the participant will then be assigned to a study arm. 
7.2 
Blinding 
Due to the objectives of the study, the identity of azithromycin and placebo treatments will not be 
known to participants, parents/legal guardians of the participants, and site research staff.  The following 
study procedures will be in place to ensure double-blind administration of test and control 
(azithromycin/placebo) study treatments: 
 Randomization assignments will be stored in a secure database and appropriately protected and 
backed up 
 Access to the randomization code will be strictly controlled and limited to select members of the 
Data Coordinating Center (DCC) who are involved in the generation of interim reports for the 
DSMB   
 Azithromycin and placebo will be manufactured to be identical in appearance and taste-matched 
 Packaging and labeling of treatments will be identical 
During the study, the blind may be broken for individual participants in emergencies when knowledge of 
the participant’s treatment group is necessary for further patient management or when the event meets 
the FDA expedited reporting requirements as a suspected adverse reaction that is serious and unexpected 
(21 CFR 312.32(c)(1)(i)).  In those cases, knowledge of the treatment received is necessary for 
interpreting the event.  If the blind is broken and the subject was receiving placebo, an IND safety report 
would not be submitted.  However, if an IND safety report is submitted to FDA, all participating 
investigators would be notified.  Emergency unblinding procedures are provided in the Study Procedures 
Manual (Refer to Protocol Section 10.3 for Serious Adverse Experience reporting).    
The study blind will be broken on completion of the clinical study and after the study database has been 
locked.  The site investigators will be provided with each participant’s treatment assignment after the 
statistical report is finalized.   
7.3 
Formulation of Test and Control Products 
7.3.1 
Formulation of Test Product 
Active treatment: Azithromycin, 900 mg oral suspension will be provided by Pfizer, Inc.  The product 
must be reconstituted with 12 mL water before use.  Final concentration = 200mg/5mL (final volume = 
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7.3.2 
Formulation of Control Product 
Placebo: Vehicle suspension, taste- matched to the azithromycin will be provided by Pfizer, Inc.  The 
placebo is reconstituted with 12 mL water before use.  Final volume = 22.5 mL 
7.3.3 
Packaging and Labeling 
Azithromycin and Placebo- Product will be provided as 900 mg bottles by the manufacturer.  Bottles 
will be labeled and assembled into kits containing 30 bottles each.  Kits will be labeled as blinded study 
drug.  A 30-bottle kit will provide sufficient study drug for 12 weeks + 3 weeks.   
7.4 
Supply of Study Drug to the Site 
The Investigational Drug Service at Seattle Children’s Hospital (Central Pharmacy) will ship study drug 
to the investigational sites.  The initial study drug shipment will be shipped after site activation (i.e., all 
required regulatory documentation has been received by the Sponsor and a contract has been executed).  
Subsequent study drug shipments will be made as needed. 
7.4.1 
Dosage/Dosage Regimen 
Azithromycin or Placebo – All participants receive azithromycin or placebo for the 18 months of study 
enrollment.  Each participant administers a dose of approximately 10mg/kg/dose up to maximum of 500 
mg per dose orally three times weekly (e.g. Monday-Wednesday-Friday) per the Table 1 below.  
Table 1 
Weight (kg)  
Dose volume 
Dose (mg)  
5.0-7.9 
1.5 mL 
60 mg 
8.0-9.9 
2.5 mL 
100 mg 
10.0-14.9 
3 mL 
120 mg 
15.0-27.9 
5 mL  
200 mg  
28.0-39.9 
7.5 mL    
300 mg  
40.0-49.9 
10 mL  
400 mg 
≥ 50.0 kg  
12.5 mL  
500 mg  
7.4.2 
Dispensing 
Azithromycin or Placebo - Kits of a 12 (+3)-week supply will be dispensed to the participant by the site 
pharmacist (or designee) at enrollment and every 3-month visit for a total of 6 visits.  Kits will be 
dispensed with a measuring syringe and/or cup to help with reconstitution of the suspension and with 
dosing. 
7.4.3 
Administration Instructions  
Azithromycin and placebo: Bottles of study drug are reconstituted with water weekly by the participant 
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The correct amount of reconstituted study drug, based on the participant’s weight, should be taken 3 
times weekly (refer to Table 1).    This study medication may be administered without regard to meals 
(on either a full or empty stomach).    
Further information is provided in the Pharmacy Manual.  
7.4.4 
Storage 
Azithromycin and placebo: Oral powder for suspension is stored at room temperature 15 - 30°C (59 - 
86°F).  Reconstituted suspension is stored between 5º to 30ºC (41-86ºF) and must be used within 10 days 
of mixing.  The bottle should be kept tightly closed. 
All study drug should be stored by the study site at controlled temperatures in a secure area under 
restricted access according to local regulations.  If the temperature of study drug storage in the 
clinic/pharmacy exceeds or falls below this range, this should be reported to the Sponsor or designee.  
Participants will be instructed to store study drug in the original packaging according to the instructions 
outlined above. 
7.5 
Dose Modification 
Additional guidelines for study drug dose modifications are provided in the Study Procedures Manual.  
7.5.1 
Dose Modification Due to Study Drug Intolerance 
If a study participant has moderate to severe intolerance (refer to Study Procedures Manual) the 
following schedule for dosage adjustment can be made at the discretion of the site investigator: 
 If a study participant receiving study drug TIW (e.g. Monday-Wednesday-Friday) experiences 
drug-related intolerance, the dosing INTERVAL of the study drug will be extended to BIW (e.g. 
Monday and Thursday). 
 If a study participant does not tolerate receiving study drug BIW, the dosing INTERVAL will be 
extended to once weekly (e.g. Monday).  
 If a dose modification other than an interval change is needed, the modification should be 
discussed with the Medical Monitor or Sponsor. 
Refer to Section 11 for permanent and temporary discontinuation of study drug, including guidelines for 
permanent study drug discontinuation for chronic treatment with azithromycin for persistent Pa 
infection. 
7.5.2 
Missed Dosages 
Missed doses may be made up as long as they are not taken within 24 hours of the next scheduled dose 
(refer to Study Procedures Manual). 
7.6 
Study Drug Accountability 
An accurate and current accounting of the dispensing and return of study drug for each participant will 
be maintained on an ongoing basis by a member of the study site staff.  The number of bottles dispensed 


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will be recorded on the Investigational Drug Accountability Record.  The study monitor will verify these 
documents throughout the course of the study. 
7.7 
Measures of Study Drug Treatment Compliance 
Participants will be asked to return all unused study drug and both used and unused study drug bottles.  
Treatment compliance will be based on the number of doses reported taken by the participant. 
Further information is provided in the Study Procedures Manual and Pharmacy Manual. 
7.8 
Tobramycin Inhalation Solution (TIS) 
All participants receive a 28-day course of TIS therapy (300 mg/5 mL) administered twice daily, with 
the initial course starting no more than 14 days prior to the Baseline Visit.  
Participants who remain Pa positive after the first 28-day course of TIS will receive a second contiguous 
28-day course of twice daily TIS (56 continuous days of treatment).  For participants who received TIS 
in the 14 days prior to enrollment, Visit 2 should be conducted within the protocol-specified windows.  
However, if those participants require a second course of TIS, the administration may not be continuous 
but should be within 7-14 days of the last dose of the previous TIS 28-day course.   
After the first quarter, participants will continue to receive culture-based TIS therapy (one 28-day, twice 
daily administration) according to Pa culture results available at the start of each quarter and for the 
duration of the 18 month study.   
The initial two treatment courses of TIS will be provided as part of the study, unless TIS was prescribed 
and administered in the 14 days prior to study enrollment. Participants with Pa positive cultures at 
subsequent study visits (Visits 3 through 7) will be treated with TIS as standard of care.  TIS will not be 
provided at these subsequent visits as part of the study.   
TIS, 300 mg/5mL (TOBI®, Novartis Pharmaceutical Corp) is commercially packaged in open-label 
cartons containing 56 single dose ampules containing 300 mg tobramycin.  The ampules are packaged in 
sets of 4 enclosed within a laminated foil pouch.  Fourteen pouches will be contained in each carton. 
Each TIS dose will be administered using the PARI LC PLUS™ Nebulizer and a suitable compressor.  
The nebulizers will be provided by the study. Compressors will be provided if the participant does not 
already have one.  
Young children will be evaluated at the study site to determine if a PARI baby mask is required for 
dosing and to fit the child with an appropriate mask (size 1, 2, or 3) as needed.  
The first dose of TIS may be administered at the study site if deemed appropriate by the site 
investigator.  
Instructions for administration and for cleaning of the nebulizer and compressor will be reviewed at the 
first visit and copies of the instructions will be provided to the participant or parent.  
TIS should be stored by the study site at controlled temperatures (2-8ºC or 36-46ºF) in a secure area 
under restricted access according to local regulations.  If the temperature of study drug storage in the 
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Participants will be instructed to store TIS in the original packaging in the refrigerator.  However, 
ampules may be stored at room temperature (25ºC/77ºF) for up to 28 days. 
7.8.1 
Use of TOBI Podhaler and other nebulized tobramycin products 
Use of both the TOBI Podhaler and other commercially available nebulized tobramycin formulations 
prescribed by the participant’s clinician is allowed in place of the TIS described above.  Only TIS is 
provided as part of the study.  Participants will be required to use the study-provided TIS (TOBI) for 
the first two cycles (if applicable) of dosing unless they were prescribed another formulation in the 2 
weeks prior to enrollment.  If a participant enrolls while on another inhaled tobramycin formulation for 
less than 14 days, they will be allowed to complete that 28 day course but will be asked to use TIS for 
further therapy. All participants will be encouraged to use TIS throughout the study period.   
7.8.2 
TIS Dose Modification Due to Persistent Infection  
At the discretion of the primary CF care clinician, treatment with TIS may be revised to a 28-day on/ 28-
day off regimen if required for treatment of persistent Pa infection.  Persistent infection is defined in this 
study as any two respiratory cultures collected at study visits, positive for Pa from study Qtr 2 (Visit 3) 
onward.   If the positive Pa culture occurs outside of a study visit, site investigators will be asked to 
consult with the Medical Monitor or study investigator-sponsors.  The participants will be encouraged to 
continue in the study and complete all remaining scheduled visits and procedures.  Refer to section 11.3 
for guidelines for permanent TIS discontinuation. 
Refer to section 11.1.1 for guidelines for permanent study drug discontinuation for chronic treatment 
with azithromycin for persistent Pa infection. 
7.8.3 
TIS Accountability 
Drug accountability for TIS will only be performed for TIS provided as part of the study.  The number 
of kits and ampules dispensed will be recorded on the Investigational Drug Accountability Record.  The 
study monitor will verify these documents throughout the course of the study.  
7.8.4 
Measures of TIS Treatment Compliance 
Participants will be asked to return all used and unused TIS ampules provided as part of the study.   
8 
STUDY PROCEDURES AND GUIDELINES 
8.1 
Clinical Assessments 
All assessments will be performed as shown in the Schedule of Events, Appendix I.   
Prior to conducting any study-related activities, written informed consent and the Health 
Insurance Portability and Accountability Act (HIPAA) authorization must be signed and dated by the 
participant or the participant’s legal representative.  If appropriate, assent must also be obtained prior to 
conducting any study-related activities. 


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8.1.1 
Concomitant Medications 
All concomitant medication and concurrent therapies ongoing and started at the Baseline Visit and 
through Visit 8 will be documented.  Dose, route, unit, frequency of administration, indication for 
administration and dates of medication will be captured.  History of antibiotic use in the year prior to 
enrollment and any chronic macrolide use will also be collected for the study.   
8.1.2 
Demographics  
Demographic information (date of birth, sex, race) will be recorded at the Baseline Visit. 
8.1.3 
Medical History  
Relevant medical history, including history of current disease, other pertinent respiratory history, and 
information regarding underlying diseases will be recorded at the Baseline Visit. 
8.1.4 
Microbiology History 
Results of the study-qualifying Pa positive respiratory culture will be entered into the study database.  
Additional historic culture results will be recorded for study purposes as specified in the Study 
Procedures Manual.  
8.1.5 
Recording of Clinical Care Microbiology Results 
Information regarding the microbiology results from cultures that are collected during unscheduled 
assessments (refer to Appendix I Schedule of Events) will be extracted from the clinical record and 
entered into the study database for the duration of the study.   
8.1.6 
Physical Examination 
Physical examinations (complete and abbreviated) will be performed by either the site investigator or 
investigator-designated qualified staff (MD, NP, RN, PA).  A complete physical exam will be performed 
at the Baseline Visit, and the End of Study Visit (or Early Withdrawal), if applicable.  An abbreviated 
physical exam will be performed at all other study visits. The abbreviated exam includes respiratory, 
cardiovascular, and abdominal assessments.  After the Baseline Visit, new clinically significant 
abnormal physical exam findings must be documented and will be followed by a physician or other 
qualified staff at the next scheduled visit.  
8.1.7 
Weight and Height/Length   
Height/Length and Weight measurements will be obtained at all study visits. 
Height/Length 
Recumbent length should be measured on all participants approximately  18 months of age.  Height 
should be measured on all participants approximately > 18 months of age.  Consistent equipment for 
each type of measurement (standing or recumbent) should be used.  
Weight 
Participants who are approximately  18 months of age should be weighed without any clothing or 
diapers.  Participants who are approximately > 18 months of age can be weighed in street clothing, with 


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a dry diaper (if applicable), and no shoes.  Weight may be measured using either a standing or sitting 
scale (as appropriate) and should be obtained using consistent equipment for each type of measurement 
(standing or sitting).  
8.1.8 
Vital Signs 
Resting (minimum of 5 minutes) measurements of pulse and respirations will be performed and recorded 
at all study visits.  Body temperature and blood pressure will be performed as clinically indicated.   
8.1.9 
Oximetry 
A resting (minimum of 5 minutes) measurement of oximetry will be measured on room air and recorded 
at all study visits. 
8.1.10 Spirometry 
Spirometry will be performed at all study visits by all participants 4 years of age and older at the time of 
the visit who are able to reproducibly perform spirometry(71).  Procedures will be performed in 
accordance with the current American Thoracic Society recommendations for the performance and 
interpretation of tests at all study visits.  For preschool age participants performing spirometry, a single 
satisfactory maneuver is acceptable.   
The same spirometry equipment should be used for the duration of the study whenever possible.  Raw 
lung function numbers will be recorded along with percent of predicted calculated centrally.  
Participants who routinely use bronchodilators should use them prior to spirometry as noted below:  
 
Participants who routinely use short acting inhaled bronchodilators should use them 15 minutes 
to 2 hours prior to PFTs during study visits. 
 
Participants who routinely use long acting bronchodilator agents should use them 15 minutes to 6 
hours prior to PFTs during study visits. 
8.1.11 Audiometry 
Hearing testing will be performed at the Baseline Visit, Visit 5, and the End of Study Visit (or at Early 
Withdrawal).  Testing will be conducted at the site or at a qualified testing laboratory identified by the 
study site by a licensed audiologist with experience testing children.  Because scheduling of audiology 
testing may be difficult at some sites, testing can be conducted up to 2 weeks after the Baseline Visit and 
up to 2 weeks before or after Visit 5, and up to 2 weeks before the End of Study Visit (or at Early 
Withdrawal).  
Participants a) unable to complete audiology; b) with acute middle ear disease; c) with a flat (Type B) 
tympanogram; or d) with an abnormal audiogram at Visit 1 or 5, can remain in the study on study drug if 
they meet all other eligibility and protocol criteria.  
Testing will include visual reinforcement audiometry  (VRA) using sound field testing or earphones 
(supraural or insert) for participants approximately 6 to 30 months old, play audiometry with earphones 
for participants approximately 30 months to 5 years (developmental age), and standard audiometry for 
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to the discretion of the audiologist. For young infants and for special populations where behavioral 
audiometry cannot be completed, otoacoustic emissions testing will be performed.    
Audiometric responses will be measured from 500 – 6000 Hz for VRA and 250 to 8000 Hz for play and 
standard audiometry. When performing VRA, it is not necessary to test below 15dBHL. Bone 
conduction testing shall be performed if air conduction test results are elevated.  Tympanometry will be 
performed with all audiometry testing. 
Abnormal hearing sensitivity will be defined as hearing threshold levels >25 dB HL for VRA (visual 
reinforcement audiometry) at any one frequency (500-6000Hz) and >20 dB HL for play or standard 
audiometry at any two frequencies (250-8000Hz) in either ear.   
All abnormal test results, as reported by the site audiologist, should be forwarded in real time to an 
independent audiologist to be over-read.  If a determination of sensorineural hearing loss is made, the 
site will be notified and the study drug must be stopped for the duration of the study.  These participants 
will be encouraged to continue in the study and complete all study visits.  Treatment and follow-up will 
be determined at the discretion of the primary CF care clinician. Instructions for the over-reading 
reporting process are included in the Study Procedures Manual.   
Subjective hearing loss, tinnitus, and vertigo as reported by the participant or parent, should be recorded 
as adverse events and managed at the discretion of the primary CF care clinician.    
Sensorineural hearing loss, confirmed by the over-reader, will be reported as Serious Adverse 
Events. (Refer to Section 10.2) 
8.1.12 Electrocardiogram (ECG)  
Standard 12-lead ECG tracing will be obtained at the Baseline Visit, Visit 2, and the End of Study Visit 
(or Early Withdrawal).  Testing will be conducted and reviewed at the site by trained personnel with 
experience interpreting electrocardiograms in children.  Any test results of clinical concern, should be 
reported to the study PI to determine follow up actions as described below.   
Testing will be performed using an ECG machine that automatically calculates the heart rate and 
measures PR, QRS, QT, and QTc intervals.  All tests will be measured in Lead II using Bazett correction 
factor and QTc(B) will be reported.   
Abnormal baseline measurement for QTc(B) is defined as ≥ 460 msec.  Abnormal results at the time of 
follow-up testing are defined as QTc(B) >500 msec, an absolute QT >500 msec, or  an increase in 
QTc(B) of >30 msec.  
Abnormal test results, as defined in the protocol, will be over-read by an independent cardiologist and 
should be forwarded in real time as instructed in the Study Procedures Manual.  
Participants with abnormal ECG findings of QTc >500msec or QTc change of >60msec at V2 and V8 
will be instructed to temporarily stop study drug (at V2) and the PI will continue to observe the 
participant for clinical signs of arrhythmia (specifically torsades de pointes) while the tracings are 
reviewed by the over-reader.  Refer to the Study Manual for additional instructions on follow up and 
requirements for permanent discontinuation of study drug.   
Abnormal test results, as defined by the protocol and confirmed by over-reading, will be reported as 
Serious Adverse Events. (Refer to Section 10.2) 


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The ECG tracing may be collected for additional safety review.   
8.1.13 Signs and Symptoms Evaluation for Pulmonary Exacerbations 
Key changes in signs and symptoms will be reviewed and documented at all visits and when 
unscheduled assessments occur throughout the duration of the study.  Unscheduled assessments will be 
conducted when a participant initiates a new antibiotic therapy, has a change in antibiotic therapy, or as 
determined by the site investigator to assess possible symptoms of a pulmonary exacerbation.   
The review of pulmonary signs and symptoms will be standardized for assessment of acute pulmonary 
exacerbations and will be used to derive a protocol defined acute pulmonary exacerbation. Criteria for 
the protocol defined acute pulmonary exacerbation were based on the definition used in both the Early 
Pseudomonas Infection Control (EPIC001) clinical trial (NIH U01HL080310)(25) and recent multicenter 
randomized trial of azithromycin in children and adolescents uninfected with Pa(9)  Refer to Appendix II 
for the criteria for a pulmonary exacerbation. 
8.1.14 Cystic Fibrosis Respiratory Questionnaires 
The self-report Cystic Fibrosis Respiratory Symptom Diary (CFRSD) - Chronic Respiratory Infection 
Symptom Score (CRISS©) and the parent-completed Cystic Fibrosis Respiratory Symptom Diary 
(CFRSD) will be completed at each study visit and the responses will be entered into the electronic case 
report forms.   Both questionnaires take less than 5 minutes to complete.  The CFRSD-CRISS has now 
been used in over 700 patients with CF. 
The CFRSD-CRISS contains 8 questions and the CFRSD includes 16 questions (8 symptom items, 4 
emotional impact items, and 4 activity impact items) for parent symptom reporting(72-74).  The 
parent/guardian will complete the CFRSD for participants ≤ 11 years of age and participants ≥ 12 years 
of age will complete the CFRSD-CRISS self-report questionnaire.    
Additionally, parent/guardians will be asked to complete a new version of the questionnaire which is 
currently being evaluated.  The Cystic Fibrosis Respiratory Signs Diary for ages 0-6 years (CFRSD 0-6© 
- 18 questions) or ages 7-11 (CFRSD 7-11© - 15 questions).   
8.1.15  Adverse Events 
Information regarding occurrence of adverse events will be captured throughout the study.  Duration 
(start and stop dates), severity, outcome, treatment and relation to study drug will be recorded on the 
case report form (CRF).  If an adverse event occurs, at the discretion of the site investigator, the study 
drug can be stopped. 
If a participant has study drug discontinued because of an adverse event, the participant will be followed 
and treated by the site investigator until the abnormal parameter or symptom has resolved or stabilized.  
Participants who discontinue study drug early will be encouraged to complete all remaining scheduled 
and follow-up visits and procedures.   
8.1.16 Hospitalization 
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8.1.17 Hematology 
At the Baseline Visit, Visit 5 (Week 39), and the End of Study Visit (or Early Withdrawal, if 
applicable), blood will be obtained and sent to each site’s clinical hematology lab for a complete blood 
count (hemoglobin, hematocrit, red blood cell count, white blood cell count, white blood cell 
differential, and platelet count). 
8.1.18 Blood Chemistry Profile 
At the Baseline Visit, Visit 5 (Week 39), and the End of Study Visit (or Early Withdrawal, if 
applicable), blood will be obtained and sent to each site’s clinical chemistry lab for determination of 
serum sodium, potassium, chloride, bicarbonate, glucose, BUN, creatinine, aspartate aminotransferase 
(AST/SGOT), alanine aminotransferase (ALT/SGPT), alkaline phosphatase, total bilirubin, direct 
bilirubin, gamma-glutamyl transferase (GGT), albumin and LDH. 
8.1.19 Pregnancy Test 
At the Baseline Visit and the End of Study Visit (or Early Withdrawal), a urine or serum pregnancy test 
will be performed and the results reported for female participants who are of childbearing potential. 
8.1.20 Respiratory Cultures for CF Pathogens including P. aeruginosa and S. aureus and non-tuberculous 
mycobacteria (NTM) 
Respiratory specimens will be obtained at all study visits and cultured qualitatively at the site clinical 
laboratory for identification of Pa and Sa (methicillin susceptible and resistant), Haemophilus 
influenzae, Burkholderia species, Stenotrophomonas maltophilia, and Alcaligenes (Achromobacter) 
xylosoxidans (from oropharyngeal swabs or sputum, expectorated or induced, or BAL, if available), and 
non-tuberculous mycobacteria (for sputum-producers only). The respiratory specimen will be collected 
no sooner than 24 hours after the last dose of TIS.   
Collection: Oropharyngeal specimens will be collected with a swab from the posterior oropharyngeal 
wall and tonsillar pillars.  Established standardized procedures for collection of OP swabs developed by 
the CCC will be utilized.  Participants will be encouraged to cough prior to collection of the OP 
specimen.  If a participant is able to produce sputum or induced sputum is collected at any visit, 
specimens will also be cultured for non-tuberculous mycobacteria (NTM).  
Detailed instructions for specimen collection, processing, storage and shipping of samples intended for 
research laboratory analysis will be provided in the Study Laboratory Manual.  
If B. cepacia complex is isolated from respiratory cultures during the study, the participant is allowed to 
continue use of study drug.   
If NTM is isolated from sputum cultures  during the study, the participant must permanently discontinue 
study drug, but should be encouraged to continue with all remaining study visits.  If the AFB smear is 
positive from sputum cultures during the study, the subject will temporarily discontinue study drug 
pending the culture results.  If NTM is isolated, the participant must permanently discontinue study 
drug.  If the culture is negative, study drug may be restarted on the next scheduled treatment day.  


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8.1.21 Subject Diary 
Participants will be requested to complete a diary to document each dose of study drug and TIS taken 
and to record changes in concomitant medications and symptoms from Visit 1 through the End of Study 
Visit (or Early Withdrawal).  A diary will be dispensed at the Baseline Visit and again at Visits 3 
through 7 and reviewed at every study visit after the Baseline Visit.  Adverse events will be recorded in 
the CRF, as applicable. 
8.2 
Research Laboratory Assessments 
8.2.1 
Plasma for Inflammatory Biomarker Analysis and Banking 
Blood Collection and Processing:  At the Baseline Visit, Visit 5 and End of Study Visit (or Early 
Withdrawal), approximately 4mL blood will be collected for plasma samples.  Blood processing 
instructions will be provided in the Study Laboratory Manual.  At the end of the processing, the sample 
will be split into aliquots, which will be placed immediately into a –70oC freezer for storage until 
shipment. 
Specimen Shipment:  At the end of the study or upon request, all samples will be shipped on dry ice via 
overnight courier to The Children’s Hospital Clinical and Translational Research Center (TCH CTRC) 
in Denver for analysis and banking. 
Inflammatory Biomarker Analysis: Plasma collected from participants will be used for measurement of 
C-reactive protein (CRP), myeloperoxidase (MPO), and calprotectin. 
Banking: Plasma collected from participants will also be stored for indefinite banking.  Separate 
informed consent regarding this process will be obtained from each study participant.  
8.2.2 
OP swabs for Microbiome Analysis 
When specimens for respiratory culture are collected at all study visits, an additional oropharyngeal 
swab will be collected for microbiome analysis, using the same collection procedure.  Swabs will be 
placed into a –70oC freezer for storage until shipment. 
Specimen Shipment:  At the end of the study or upon request, all swabs will be shipped on dry ice via 
overnight courier to The Children’s Hospital Clinical and Translational Research Center (TCH CTRC) 
in Denver for analysis and banking. 
Banking: A suspension from the swabs collected from participants will be stored for indefinite banking.  
Separate informed consent regarding this process will be obtained from each study participant.  
9 
EVALUATIONS BY VISIT 
Underlined procedures are performed as part of standard of care at the quarterly clinical care visit.   
9.1 
Baseline Visit 1 – Screening and Randomization (Day 0) 
1. Review the study with the participant (participant’s legal representative) and obtain written 
informed consent and HIPAA authorization and assent, if appropriate.  Ensure participant has 
opted either in or out of CFF Registry ID collection and long-term specimen banking.  
2. Assign the participant a unique participant number. 


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3. Record demographics data. 
4. Record medical history, including antibiotic use and CF diagnosis and date 
5. Record microbiology history.  
6. Review and record concomitant medications. 
7. Complete CF Respiratory Diaries (participant or parent). 
8. Review Pulmonary Symptoms.   
9. Perform a complete physical examination.  
10. Measure and record height/length and weight. 
11. Obtain and record vital signs. 
12. Obtain and record oximetry. 
13. Collect blood for clinical laboratory testing (chemistry and hematology) at site lab.    
14. Collect blood or urine for pregnancy test (if female of child-bearing potential).  
15. Obtain results from chemistry and hematology drawn at this visit. 
16. Collect plasma for inflammatory marker analysis and banking (if banking consent was obtained). 
17. Perform and record spirometry (if participant is ≥ age 4 years) 
18. Perform audiometry at or within 2 weeks after visit.  Obtain signed copies of testing and 
evaluation. 
19. Perform ECG testing. Obtain signed copies of testing and evaluation. 
20. Obtain respiratory culture for processing at site microbiology lab (OP swab or sputum). 
21. Collect additional OP swab for microbiome.  
22. Randomize participant to azithromycin or placebo (blinded). 
23. Dispense azithromycin/placebo and dosing instructions. 
24. Dispense TIS study medication, nebulizer cleaning instructions and dosing instructions, unless 
first course was taken by the participant prior to enrollment in the study.  
25. Administer first dose of TIS in the clinic, unless first course was taken by the participant prior to 
enrollment in the study. (The first dose of TIS may be administered at the study site if deemed 
appropriate by the site investigator.)  
26. Dispense nebulizer (and masks if applicable) and compressor for TIS as needed. 
27. Dispense Subject Diary and review completion instructions. 
28. Schedule participant to return in 3 weeks (19-23 days after Screening Visit).   
9.2 
Visit 2 (Week 3, Day 21 ± 2 days) 
1. Complete CF Respiratory Diaries (participant or parent). 
2. Review Pulmonary Symptoms.   
3. Review Subject Diary. 
4. Record any adverse events. 
5. Record any hospitalizations.  
6. Record changes to concomitant medications. 


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7. Perform an abbreviated physical exam. 
8. Measure and record height/length and weight. 
9. Obtain and record vital signs. 
10. Obtain and record oximetry. 
11. Perform and record spirometry (if participant is ≥ age 4 years). 
12. Perform ECG testing. Obtain signed copies of testing and evaluation. 
13. Obtain respiratory culture for processing at site microbiology lab (OP swab or sputum). 
14. Collect additional OP swab for microbiome. 
15. Schedule participant to return in 11-15 weeks (Week 13 Visit) 
16. Advise the participant that RC will contact them if their respiratory culture results are positive 
for Pa and ask them to begin treatment without interruption of their TIS if possible. 
17. Following Visit 2: For participants with Visit 2 respiratory cultures positive for Pa, prescribe 
TIS within one week of visit. 
9.3 
Visit 3 (Week 13 ± 2 weeks) 
Underlined procedures are performed as part of standard of care at the quarterly clinical care visit. 
1. Complete CF Respiratory Diaries (participant or parent). 
2. Review Pulmonary Symptoms.  
3. Review Subject Diary. 
4. Perform study drug and study-dispensed TIS accountability. 
5. Record any adverse events.  
6. Record any hospitalizations.  
7. Record changes to concomitant medications. 
8. Perform abbreviated physical examination. 
9. Measure and record height/length and weight. 
10. Obtain and record vital signs. 
11. Obtain and record oximetry. 
12. Perform and record spirometry (if participant is ≥ age 4 years). 
13. Obtain respiratory culture for processing at site microbiology lab (OP swab or sputum). 
14. Collect additional OP swab for microbiome. 
15. Dispense azithromycin/placebo. 
16. Dispense Subject Diary. 
17. Schedule participant to return in 11-15 weeks for Visit 4 (Week 26) 
18. Advise the participant that RC will contact them if their respiratory culture results are positive 
for Pa and ask them to begin treatment within one week of visit. 
19. Following Visit 3: For participants with Visit 3 respiratory cultures positive for Pa: prescribe 
TIS within one week of visit. 


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9.4 
Visit 4 (Week 26 ± 2 weeks) 
Underlined procedures are performed as part of standard of care at the quarterly clinical care visit. 
1. Complete CF Respiratory Diaries (participant or parent). 
2. Review Pulmonary Symptoms.  
3. Review Subject Diary. 
4. Perform study drug accountability. 
5. Record any adverse events. 
6. Record any hospitalizations.  
7. Record changes to concomitant medications. 
8. Perform abbreviated physical examination. 
9. Measure and record height/length and weight. 
10. Obtain and record vital signs. 
11. Obtain and record oximetry. 
12. Perform and record spirometry (if participant is ≥ age 4 years). 
13. Obtain respiratory culture for processing at site microbiology lab (OP swab or sputum). 
14. Collect additional OP swab for microbiome. 
15. Dispense azithromycin/placebo.  
16. Dispense Subject Diary. 
17. Schedule participant to return in 11-15 weeks for Visit 5(Week 39). 
18. Advise the participant that RC will contact them if their respiratory culture results are positive 
for Pa and ask them to begin treatment within one week of visit. 
19. Following Visit 4: For participants with Visit 4 respiratory cultures positive for Pa, prescribe 
TIS within one week of visit. 
9.5 
Visit 5 (Week 39 ± 2 weeks) 
Underlined procedures are performed as part of standard of care at the quarterly clinical care visit. 
1. Complete CF Respiratory Diaries (participant or parent). 
2. Review Pulmonary Symptoms.  
3. Review Subject Diary. 
4. Perform study drug accountability. 
5. Record any adverse events. 
6. Record any hospitalizations.  
7. Record changes to concomitant medications. 
8. Perform abbreviated physical examination. 
9. Measure and record height/length and weight. 
10. Obtain and record vital signs. 
11. Obtain and record oximetry. 


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12. Collect blood for clinical laboratory testing (chemistry and hematology) at site lab.    
13. Collect plasma for inflammatory marker analysis and banking (if banking consent was obtained). 
14. Perform and record spirometry (if participant is ≥ age 4 years). 
15. Perform audiometry at or within 2 weeks of visit.  Obtain signed copies of testing and evaluation. 
16. Obtain respiratory culture for processing at site microbiology lab (OP swab or sputum). 
17. Collect additional OP swab for microbiome.  
18. Dispense azithromycin/placebo.   
19. Dispense Subject Diary. 
20. Schedule participant to return in 11-15 weeks for Visit 6 (Week 52). 
21. Advise the participant that RC will contact them if their respiratory culture results are positive 
for Pa and ask them to begin treatment within one week of visit.   
22. Following Visit 5: For participants with Visit 5 respiratory cultures positive for Pa, prescribe 
TIS within one week of visit. 
9.6 
Visit 6 (Week 52 ± 2 weeks) 
Underlined procedures are performed as part of standard of care at the quarterly clinical care visit. 
1. Complete CF Respiratory Diaries (participant or parent). 
2. Review Pulmonary Symptoms.  
3. Review Subject Diary. 
4. Perform study drug accountability. 
5. Record any adverse events. 
6. Record any hospitalizations.  
7. Record changes to concomitant medications. 
8. Perform abbreviated physical examination. 
9. Measure and record height/length and weight. 
10. Obtain and record vital signs. 
11. Obtain and record oximetry. 
12. Perform and record spirometry (if participant is ≥ age 4 years). 
13. Obtain respiratory culture for processing at site microbiology lab (OP swab or sputum). 
14. Collect additional OP swab for microbiome. 
15. Dispense azithromycin/placebo.  
16. Dispense Subject Diary. 
17. Schedule participant to return in 11-15 weeks for Visit 7(Week 65). 
18. Advise the participant that RC will contact them if their respiratory culture results are positive 
for Pa and ask them to begin treatment within one week of visit. 
19. Following Visit 6: For participants with Visit 6 respiratory cultures positive for Pa, prescribe 
TIS within one week of visit. 


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9.7 
Visit 7 (Week 65 ± 2 weeks) 
Underlined procedures are performed as part of standard of care at the quarterly clinical care visit. 
1. Complete CF Respiratory Diaries (participant or parent). 
2. Review Pulmonary Symptoms.  
3. Review Subject Diary. 
4. Perform study drug accountability. 
5. Record any adverse events. 
6. Record any hospitalizations.  
7. Record changes to concomitant medications. 
8. Perform abbreviated physical examination. 
9. Measure and record height/length and weight. 
10. Obtain and record vital signs. 
11. Obtain and record oximetry. 
12. Perform and record spirometry (if participant is ≥ age 4 years). 
13. Obtain respiratory culture for processing at site microbiology lab (OP swab or sputum). 
14. Collect additional OP swab for microbiome.  
15. Dispense azithromycin/placebo. 
16. Dispense Subject Diary. 
17. Schedule participant to return in 11-15 weeks for Visit 8 (Week 78). 
18. Advise the participant that RC will contact them if their respiratory culture results are positive 
for Pa and ask them to begin treatment within one week of visit.. 
19. Following Visit 7: For participants with Visit 7 respiratory cultures positive for Pa, prescribe 
TIS within one week of visit. 
9.8 
Visit 8 - End of Study (Week 78 ± 2 weeks)  
Underlined procedures are performed as part of standard of care at the quarterly clinical care visit. 
1. Complete CF Respiratory Diaries (participant or parent). 
2. Review Pulmonary Symptoms.  
3. Review Subject Diary. 
4. Perform final study drug accountability. 
5. Record any adverse events. 
6. Record any hospitalizations.  
7. Record changes to concomitant medications. 
8. Perform a complete physical examination. 
9. Measure and record height/length and weight. 
10. Obtain and record vital signs. 
11. Obtain and record oximetry. 


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12. Perform audiometry at or within 2 weeks of visit.  Obtain signed copies of testing and evaluation. 
13. Perform ECG testing. Obtain signed copies of testing and evaluation. 
14. Collect blood for clinical laboratory testing (chemistry and hematology) at site lab.    
15. Collect blood or urine for pregnancy test (if female of child-bearing potential). 
16. Collect plasma for inflammatory marker analysis and banking (if banking consent was obtained). 
17. Perform and record spirometry (if participant is ≥ age 4 years). 
18. Obtain respiratory culture for processing at site microbiology lab (OP swab or sputum). 
19. Collect additional OP swab for microbiome.  
20. If the participant has any unresolved adverse events at this visit, advise them that RC will contact 
them for additional follow-up.  
9.9 
Unscheduled Assessment – Change in Antibiotics or as determined by Investigator 
If the participant notifies the study site of a change in antibiotic therapy, the study site should collect the 
following information.  
1. Review Pulmonary Symptoms.  
2. Record any adverse events. 
3. Record changes to concomitant medications. 
4. Obtain clinical records of any respiratory cultures collected as part of the assessment. 
9.10 Early Withdrawal from Study 
If the participant withdraws early from the study and attends a final visit, the following procedures 
should be completed if possible: 
1. Complete CF Respiratory Diaries (participant or parent). 
2. Review Pulmonary Symptoms.  
3. Review Subject Diary.  
4. Record any adverse events. 
5. Perform final study drug accountability. 
6. Record any hospitalizations.  
7. Record changes to concomitant medications. 
8. Perform complete physical examination. 
9. Measure and record height/length and weight. 
10. Obtain and record vital signs. 
11. Obtain and record oximetry. 
12. Collect blood for clinical laboratory testing (chemistry and hematology) at site lab.    
13. Collect blood or urine for pregnancy test (if female of child-bearing potential). 
14. Collect plasma for inflammatory marker analysis and banking (if banking consent was obtained). 
15. Perform and record spirometry (if participant is ≥ age 4 years). 


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16. Perform audiometry at or within 2 weeks of visit.  Obtain signed copies of testing and evaluation. 
17. Perform ECG testing. Obtain signed copies of testing and evaluation. 
18. Obtain respiratory culture for processing at site microbiology lab (OP swab or sputum). 
19. Collect additional OP swab for microbiome. 
20. If the participant has any unresolved adverse events at this visit, advise them that RC will contact 
them for additional follow-up.  
10 ADVERSE EXPERIENCE REPORTING AND DOCUMENTATION 
10.1 Adverse Events 
An adverse event (AE) is any untoward medical occurrence in a clinical investigation of a patient 
administered a pharmaceutical product and that does not necessarily have a causal relationship with the 
treatment.  An AE is therefore any unfavorable and unintended sign (including an abnormal laboratory 
finding), symptom or disease temporally associated with the administration of an investigational 
product, whether or not related to that investigational product.  An unexpected AE is one of a type not 
identified in nature, severity, or frequency in the current Prescribing or Product Information of greater 
severity or frequency than expected based on the information in the Prescribing or Product Information. 
The Investigator will probe, via discussion with the participant, for the occurrence of AEs during each 
participant visit and record the information in the site’s source documents.  Adverse events will be 
recorded in the patient CRF.  Adverse events will be described by duration (start and stop dates and 
times), severity, outcome, treatment and relation to study drug. 
AE Severity 
The National Cancer Institute’s Common Terminology Criteria for Adverse Events (CTCAE) Version 
4.0, as modified for cystic fibrosis, should be used to assess and grade AE severity, including laboratory 
abnormalities judged to be clinically significant.  The modified criteria can be found in the study 
manual.  If the experience is not covered in the modified criteria, the guidelines shown in Table 1 below 
should be used to grade severity.  It should be pointed out that the term “severe” is a measure of 
intensity and that a severe AE is not necessarily serious. 
Table 1.  AE Severity Grading 
Severity (Toxicity Grade) 
Description 
Mild (1) 
Mild; asymptomatic or mild symptoms; clinical or diagnostic 
observations only; intervention not indicated  
Moderate (2) 
Moderate; minimal, local or noninvasive intervention indicated; 
limiting age-appropriate Instrumental activities of daily living (e.g., 
preparing meals, using the telephone, managing money) 
Severe (3) 
Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; 
limiting self care activities of daily living (e.g., bathing, dressing, 
feeding self, using toilet, taking medications) 
Life-threatening (4) 
Life-threatening consequences; urgent intervention indicated 


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Death (5) 
Death related to AE 
 
AE Relationship to Study Drug 
The relationship of an AE to the study drug should be assessed using the following the guidelines in 
Table 2. 
Table 2.  AE Relationship to Study Drug 
Relationship 
to Drug 
Comment 
Definitely 
Previously known toxicity of agent; or an event that follows a reasonable temporal 
sequence from administration of the drug; that follows a known or expected response 
pattern to the suspected drug; that is confirmed by stopping or reducing the dosage of 
the drug; and that is not explained by any other reasonable hypothesis. 
Probably 
An event that follows a reasonable temporal sequence from administration of the 
drug; that follows a known or expected response pattern to the suspected drug; that is 
confirmed by stopping or reducing the dosage of the drug; and that is unlikely to be 
explained by the known characteristics of the subject’s clinical state or by other 
interventions. 
Possibly 
An event that follows a reasonable temporal sequence from administration of the 
drug; that follows a known or expected response pattern to that suspected drug; but 
that could readily have been produced by a number of other factors. 
Unrelated 
An event that can be determined with certainty to have no relationship to the study 
drug. 
10.2 Serious Adverse Experiences (SAE) 
An SAE is defined as any AE occurring at any dose that results in any of the following outcomes: 
 
death 
 
a life-threatening adverse experience 
 
inpatient hospitalization or prolongation of existing hospitalization 
 
a persistent or significant disability/incapacity 
 
a congenital anomaly/birth defect  
Other important medical events may also be considered an SAE when, based on appropriate medical 
judgment, they jeopardize the participant or require intervention to prevent one of the outcomes listed.  
For this study, important medical events that will be reported as an SAE include: 
 
Sensorineural hearing loss, determined by audiometry and confirmed by the over-reader  
 
ECG testing results of  QTc(B) >500 msec, an absolute QT >500 msec, or an increase in QTc(B) 
of >30 msec, that has been confirmed by over-reading (Refer to Section 8.1.12). 


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10.3 Serious Adverse Experience Reporting 
Study sites will document all SAEs that occur (whether or not related to study drug) on an SAE Report 
Form.  The collection period for all SAEs will begin after informed consent is obtained and end after 
procedures for the final study visit have been completed. 
All SAE Report Forms will be reviewed by the site investigator and sent to the DCC within one business 
day of the site learning of the event.  Sites will scan and email or fax the SAE report to: 
Email address:  cfsaesfacsys@seattlechildrens.org 
Direct dial fax number:  206-985-3278 
The site will notify the DCC of additional information or follow-up to an initial SAE Report as soon as 
relevant information is available.  Follow-up information is reported on an SAE Report Form. 
In accordance with the standard operating procedures and policies of the local Institutional Review 
Board (IRB) the site investigator will report SAEs to the IRB.   
10.4 Medical Monitoring 
The Medical Monitor for the Data Coordinating Center should be contacted directly at this number to 
report medical concerns or questions regarding safety. 
Pager: (800) 341-0961 
11 DISCONTINUATION AND REPLACEMENT OF PARTICIPANTS 
A participant may be discontinued from study drug (azithromycin or placebo) at any time if the 
participant, the site investigator, or the Sponsor feels that it is not in the participant’s best interest to 
continue.   
11.1 Permanent Discontinuation of Study Drug  
A participant must be permanently discontinued from study drug (azithromycin or placebo) for the 
following reasons:    
 
Pregnancy 
 
Isolation of NTM from a respiratory culture during the study 
 
Determination of sensorineural hearing loss (determined by audiometry and confirmed by the 
over-reader).   Participants will be encouraged to continue in the study and complete all 
remaining scheduled visits and procedures (See Section 8.1.11). 
 
Abnormal ECG findings at a study visit after Visit 1, defined as a QTc(B) >500 msec, an 
absolute QT >500 msec, or  an increase in QTc(B) of >30 msec that has been confirmed by over-
reading. 
Additional reasons for study drug discontinuation are listed below.  Participants should continue with all 
remaining study visits if possible.   
 
Abnormal clinically significant laboratory safety test results, as determined by the site 
investigator 


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 
Adverse event, as determined by the investigator*  
 
Subject decision 
 
Protocol violation, as determined by the investigator or Sponsor 
 
Death 
*If a participant is discontinued from study drug due to an adverse event, the participant will be 
followed and treated by the Investigator until the abnormal parameter or symptom has resolved or 
stabilized.  All dose modifications should be recorded appropriately in the source documents and CRFs. 
11.1.1 Permanent Discontinuation of Study Drug Due to Persistent Pa Infection 
At the discretion of the primary CF care clinician and in consultation with the participant, treatment with 
study drug (blinded placebo or azithromycin) can be discontinued and chronic azithromycin can be 
initiated if required for treatment of persistent Pa infection.  Persistent infection is defined in this study 
as any two respiratory cultures collected at study visits, positive for Pa from study Qtr 2 (Visit 3) 
onward.  The participants will be encouraged to continue in the study and complete all remaining 
scheduled visits and procedures. 
If one or more of the positive Pa cultures occurs outside of a study visit, investigators will be asked to 
consult with the Medical Monitor or study investigator-sponsors.   
Refer to section 7.8.2 for guidelines for TIS dose modifications due to persistent Pa infection. 
11.2 Temporary Discontinuation of Study Drug  
Study drug (azithromycin or placebo) must be temporarily stopped if acute treatment with azithromycin 
or any macrolide is required.  After acute treatment is completed, participants are allowed to re-start 
study drug (azithromycin or placebo) on the next scheduled treatment day. (See Section 6.2) 
Study drug must be temporarily stopped if a sputum AFB smear result is AFB positive.  If the culture is 
negative, study drug may be restarted on the next scheduled treatment day. (See Section 8.1.20) 
Study drug must be temporarily stopped if acute treatment with any drug known to prolong QT interval 
is required (refer to Appendix III).  After acute treatment is completed, participants are allowed to re-
start study drug on the next scheduled treatment day.  
Study drug may also be temporarily stopped or dose-modified due to an abnormal clinically significant 
laboratory safety test results, as determined by the site investigator.  Refer to protocol Section 7.5 for 
dose modification information.   
11.3 Discontinuation of TIS 
Participants who are discontinued from use of TIS during the study may continue use of study drug 
(azithromycin or placebo).  All participants who permanently discontinue use of TIS should be 
encouraged to complete all remaining scheduled visits and procedures. 
If a participant is discontinued from TIS use due to an adverse event, the participant will be followed 
and treated by the Investigator until the abnormal parameter or symptom has resolved or stabilized.  All 
dose modifications should be recorded appropriately in the source documents and CRFs.  
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11.4 Withdrawal of Participants from the Study 
All participants are free to withdraw from participation at any time, for any reason, specified or 
unspecified, and without prejudice.  This may include participants who withdraw from study treatment 
early and who decline to continue to come in for the remaining study visits.   
Reasonable attempts will be made by the site investigator to provide a reason for participant 
withdrawals.  The reason for the participant’s withdrawal from the study will be specified in the 
participant’s source documents.  Participants who withdraw from the study should be encouraged to 
come in for a final visit (Section 9.10: Early Withdrawal from Study).   
11.5 Replacement of Participants 
Participants who withdraw from the study treatment or from the study will not be replaced.   
12 PROTOCOL VIOLATIONS 
A protocol violation occurs when the participant, site investigator, or Sponsor fails to adhere to 
significant protocol requirements affecting the inclusion, exclusion, participant safety and primary 
endpoint criteria.  Protocol violations for this study include, but are not limited to, the following: 
 Failure to meet inclusion/exclusion criteria 
 Use of a prohibited concomitant medication  
 Inappropriate administration of study medication that impacts the safety of the participant or 
inappropriate dispensing of study drug (e.g., wrong container # dispensed) 
 
Failure to comply with Good Clinical Practice (GCP) guidelines will also result in a protocol violation.   
When a protocol violation occurs, it will be discussed with the site investigator and a Protocol Violation 
Form detailing the violation will be generated.  This form will be signed by a Sponsor representative and 
the site investigator.  A copy of the form will be filed in the site’s regulatory binder and in the Sponsor’s 
files.  The site will report the violation to their IRB in accordance with their IRB reporting requirements. 
13 DATA SAFETY MONITORING  
An independent data safety monitoring board (DSMB) appointed by the NHLBI will review 
comprehensive interim enrollment, safety, and efficacy data reports on a semi-annual basis.  A priori 
interim stopping rules for efficacy and safety with respect to the primary endpoint will be in place and 
evaluated as outlined in the DSMB charter, with approximately 6 planned interim analyses and a final 
analyses.  Serious adverse events will be monitored by the committee on an ongoing basis throughout 
the study. Further details regarding the timing and content of the interim reviews is included in the 
statistical section below and in the DSMB charter. 


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14 STATISTICAL METHODS AND CONSIDERATIONS 
A detailed statistical analysis plan will be written that will describe all analyses, tables, figures, and data 
listings that will be generated for this study.  All analyses will be performed using SAS® (SAS Institute, 
Inc., Cary, NC, USA) or R (R Foundation for Statistical Computing, Vienna, Austria).  
14.1 Data Sets Analyzed 
All analyses will be performed using an intent-to-treat (ITT) population, which is defined as all 
randomized participants.  The primary efficacy analyses will be repeated in the per-protocol population, 
which is defined as participants having completed ≥80% of doses (azithromycin or placebo). 
Participants who are discontinued from study drug are encouraged to complete all remaining study visits 
and will remain in the analyses population according to ITT. 
14.2 Demographic and Baseline Characteristics 
Treatment groups will be described and compared with respect to baseline demographic and clinical 
characteristics including age, gender, CFTR genotype, race, height, weight, randomization strata (6 mos-
3 years, >3-6 years, ≥6-12 yrs, >12-18 yrs), lifetime history of Pa positivity (ever versus never positive 
greater than 2 years prior to enrollment),  presence of Sa and/ or other gram-negative bacteria, FEV1% 
predicted among those able to perform spirometry, and use of chronic medications (inhaled dornase alfa 
and hypertonic saline). For all analyses, baseline will be defined as the measurement prior and closest to 
the administration of the first dose of study drug (azithromycin or placebo).   
14.3 Analysis of Primary Endpoint 
The primary endpoint will be time to a protocol-defined pulmonary exacerbation, comparing the two 
treatment groups:  oral placebo and culture-based TIS treatment versus oral azithromycin and culture-
based TIS treatment.  Follow-up time will begin at first dose of study drug (Day 0) and end after 
completion of the final study visit (at approximately 18 months).  Time to exacerbation will be presented 
using Kaplan-Meier survival estimation and compared between treatment groups using a Cox 
proportional hazards model with effects for treatment group and randomization strata (age group), with a 
test of the treatment effect at a two-sided significance level of 0.05.  The hazard ratio and its 95% 
confidence interval will be presented.  Additional secondary analysis may employ a stratified log-rank 
test of treatment arm adjusting for the randomization strata.  The proportion having an event at each 
study visit will be presented by treatment group using Kaplan-Meier estimation.   
Estimates of the hazard ratio and associated 95% confidence interval for the primary endpoint will also 
be reported by subgroups defined by demographic and disease specific baseline characteristics of 
interest.  No adjustments for multiple comparisons will be made.  
14.4 Analysis of Secondary Endpoints 
Descriptive analyses and graphical displays will be used to summarize all secondary endpoints, 
including changes from baseline in continuous endpoints and rates over the follow-up period for event 
based endpoints.  The ITT population will be used for all secondary analyses. 
 
 


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Safety  
All reported SAEs and AEs will be coded using MedDRA and grouped by body system.  (S)AEs will be 
tabulated by treatment group using standard coding terms sorted by body system.  The incidence of AEs 
in each treatment arm will be tabulated by seriousness, severity, and relationship to study drug.  If an 
adverse event is reported more than once during the study period for a given participant, the greatest 
severity and the worst-case relationship will be presented in tables.  The number of (S)AEs will be 
summarized for each treatment group as follows: (i) The proportion of participants with at least one 
(S)AE, (ii) The average number of (S)AEs per patient, and (iii) The rate of (S)AEs per patient month of 
follow-up.  Histograms showing the frequency of the number of (S)AEs in each treatment group will be 
included.  Rates of (S)AEs by System Organ Class (SOC) will be presented by treatment group.  Poisson 
regression modeling will be used to derive rate ratios and 95% confidence intervals for each SOC.  The 
rate ratios will be compared using a two-sided 0.05 level test for Poisson count data. 
Clinical laboratory data at each study visit and changes from baseline will be summarized by treatment 
group.  In addition, the following clinical laboratory summaries will be presented by treatment group: (i) 
the incidence of clinically significant abnormalities at each study visit; and (ii) tables summarizing the 
frequencies of participants below, within, and above the normal reference ranges at baseline and end of 
study; (iii) tables displaying baseline to end of study shifts in each laboratory value (shifts between 
below, within or above normal range).  
The proportion of participants permanently or temporarily discontinuing study drug will be tabulated by 
treatment group.  Drug discontinuation events will be categorized as:  (1) Permanently discontinued 
study drug, (2) Permanently discontinued study drug and withdrew from study, and (3) Temporarily 
discontinued study drug.  Reason for permanent drug discontinuation will be summarized.  Compliance 
measures will be computed separately for the inhaled tobramycin and oral azithromycin therapies.  
Those patients with less than 80% average compliance for either azithromycin or placebo will be 
excluded from the per protocol population.   
Microbiology 
Descriptive analyses and graphical displays will be used to summarize the emergence and disappearance 
of microbiologic organisms identified throughout the study.  In the event that both an OP culture and an 
expectorated sputum culture are available from a participant at the same visit and produce discordant 
results, the positive result will be used in the analysis for this visit.  The proportion of participants with 
Pa positive cultures after the first quarter of treatment will also be summarized for each treatment group 
with differences between treatment groups tested using a 0.05 level of significance Chi-square test. 
Time to Pa recurrence from weeks 13 through 78 will be presented using Kaplan-Meier survival 
estimation and compared between treatment groups using a Cox proportional hazards model with effects 
for treatment arm and randomization strata, with a test of the treatment effect at a two-sided significance 
level of 0.05.  The hazard ratio and its 95% confidence interval will be presented.  The proportion of Pa-
positive respiratory cultures among all respiratory cultures taken from weeks 13 through 78 will also be 
modeled.  The response will be binary (positive or negative culture).  The proportion of Pa-positive 
respiratory cultures at each visit will be graphically displayed by treatment group.  A generalized 
estimating equation (GEE) model using a logit link will be used to model this data with robust variance 
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randomization strata(75).  The significance of the treatment group variable will be tested using a two-
sided 0.05 level of significance.  The treatment associated odds ratio from this model and corresponding 
95% confidence interval will be the measure of treatment effect.  The estimated treatment effect will be 
interpreted as the marginal odds ratio of Pa positive respiratory cultures during the 18 months.    
Inflammatory Markers 
Inflammatory markers will be appropriately transformed (e.g. log10) and will be descriptively 
summarized and graphically displayed for each visit and for changes from baseline.  Differences 
between treatment groups in the 39 and 78 week change from baseline will be estimated with 
corresponding 95% confidence intervals and tested using a two-sided 0.05 level two-sample t-test.  
Several exploratory regression models will be developed to investigate the association between the 
inflammatory markers and clinical outcomes including time to pulmonary exacerbation.  Baseline 
inflammatory markers in addition to other baseline characteristics will be included in a Cox proportional 
hazards model for time to exacerbation with a goal of identifying a set of baseline inflammatory markers 
predictive of clinical outcome.  Separate models may be generated for each treatment group to reduce 
the number of potential interactions with treatment.  Additional models exploring specific microbiologic 
outcomes including time to Pa recurrence will be similarly developed.  Repeated measures regression 
models will also be generated investigating the association between baseline inflammatory markers 
(adjusting for other baseline predictors) and changes from baseline in continuous outcomes such as 
spirometry, anthropometric measures.   
Hospitalization and Acute Antibiotic Usage 
Endpoints for hospitalization, intravenous antibiotic usage, inhaled antibiotic usage, and oral antibiotic 
usage will be descriptively summarized.  Graphical displays for the proportion of participants with each 
event and corresponding event rates will also be provided.  For each endpoint, the proportion of 
participants experiencing the event will be compared between treatment groups using a logistic 
regression model including effects for treatment group and randomization strata.  Odds ratios and 95% 
confidence intervals will be used to describe the treatment effect and the significance of the treatment 
group variable will be tested using a two-sided 0.05 level of significance.  
 
Pulmonary Function 
Baseline clinical and demographic characteristics will be summarized among all participants with 
spirometry values available at the baseline visit.  Spirometry analyses will only include the subset of 
participants who are 4 years of age or greater and for whom a spirometry measure at baseline is 
available. FEV1, FEV1 % predicted, FVC, and FEF25%-75% will be descriptively summarized and 
graphically displayed for each visit and for changes from baseline.  Predicted values at each visit will be 
calculated centrally.  For each spirometry measure, a repeated measures linear regression model will be 
used to model the change in the measure over time (in days).  Robust variance estimation of the 
coefficients will be implemented within the generalized estimating equation framework with an 
independence working correlation matrix.  The models will include effects for treatment and 
randomization strata.  The difference between treatment groups in the 18-month change from baseline in 
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confidence interval.  The models will include effects for treatment and randomization strata.  The 
significance of the treatment group variable will be tested using a two-sided 0.05 level of significance.  
Anthropometric Measures 
Height/length, height/length percentile, weight, and weight percentile will be descriptively summarized 
and graphically displayed for each visit and for changes from baseline.  For each anthropometric 
measure, a repeated measures linear regression model will be used to model the change in the measure 
over time (in days).  Robust variance estimation of the coefficients will be implemented within the 
generalized estimating equation framework with an independence working correlation matrix.  The 
models will include effects for treatment and randomization strata.  The difference between treatment 
groups in the 18 month change from baseline in each anthropometric variable will be estimated via the 
repeated measures model with corresponding 95% confidence interval.  The models will include effects 
for treatment and randomization strata.  The significance of the treatment group variable will be tested 
using a two-sided 0.05 level of significance.  
Respiratory Symptoms 
The CFRSD and CFRSD-CRISS will be analyzed as composite scores calculated based on “yes” 
responses to a specified set of the CFRSD and CFRSD-CRISS questions.  Component responses and 
composite scores will be descriptively summarized at baseline in addition to changes from baseline.  
Change from baseline in each composite score will be analyzed using a repeated measures linear 
regression model with time from baseline (in days) included in the model.  Robust variance estimation 
of the coefficients will be implemented within the generalized estimating equation framework with an 
independence working correlation matrix.  The models will include effects for treatment and 
randomization strata.  The difference in the scores between treatment groups in the 18 month change 
from baseline will be estimated via the repeated measures model with corresponding 95% confidence 
interval.  The significance of the treatment group variable will be tested using a two-sided 0.05 level of 
significance.  
Respiratory Microbiome  
Respiratory microbial communities are complex multivariate systems and will be descriptively 
summarized at both baseline and end of study using OP swab samples.   Total bacterial load (16S rDNA 
copy per OP swab) will be calculated for each sample and averaged.  Sequencing and phylogenetic-
based clustering techniques classify the bacteria into operational taxonomic units (OTUs, e.g. genus) and 
their relative proportion of the entire community (in a given sample) can be calculated(76). True diversity 
of varying orders will calculated for each sample and graphically investigated to evaluate the evenness 
and richness across the treatment groups(77, 78).  Shannon diversity, or true diversity of order 1, will be 
used for statistical comparisons across treatment groups and over time using a two-sample t-test and 
paired t-test, respectively. Microbial community data contain only relative information due to the fact 
that they are constrained to a unit sum. Therefore, we propose to use an isometric log ratio 
transformation (ilr) applied to the relative abundance measures prior to statistical analysis(79, 80).  
Relative abundance of each OTU will be summarized via graphical displays, which will include 
principal component analysis and biplots. Change from baseline in the dominant OTUs will be 
summarized by treatment group and compared using bootstrap methodology(81). Both baseline and 


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change in the respiratory microbiome will be correlated with clinical and microbiologic outcomes using 
exploratory regression models to estimate the association between microbiome characteristics in the 
respiratory tract and both the risk of a pulmonary exacerbation and risk of Pa recurrence. 
Comprehensive Baseline Predictive Model of Eradication Failure 
 A multivariable logistic regression model will be developed to identify key baseline predictors 
associated with initial eradication failure, defined by the Pa status at Week 13.  Baseline demographic, 
clinical, microbiologic, inflammatory, and respiratory microbiome variables will all be assessed for 
inclusion in the model.  Stepwise regression methods will be used for variable selection.  The positive 
predictive value, negative predictive value, sensitivity, and specificity based on the model will be 
evaluated and corresponding ROC curves generated.  The ultimate goal is to develop a robust predictive 
model to identify children with early Pa who are at high risk for eradication failure.  This model could 
then be used in future studies to identify this high risk subset and evaluate alternative first line treatment 
strategies to promote Pa eradication.   
14.5 Missing Data 
With respect to the primary endpoint, missing data may arise from missing exacerbation symptom 
assessment data or due to early withdrawal.  Site training, automatic queries in the electronic data 
capture system, and thorough clinical site monitoring will be performed in order to minimize the 
occurrence of missing data.  Participants who discontinue treatment will be encouraged to remain in the 
study while completing all study procedures.  Participants who discontinue study will be censored at the 
date of last contact.  The impact of missing data on the primary endpoint will be assessed through 
sensitivity analysis.  A sensitivity analysis of the primary endpoint using only participants with complete 
follow up data will be performed.  Furthermore, the potential influence of acute respiratory events not 
meeting the primary endpoint definition will also be assessed by censoring participants with acute 
antibiotic usage prior to meeting the primary endpoint. Further details regarding the handling of missing 
data will be provided in the statistical analysis plan. 
14.6 Interim Analysis 
An independent data safety monitoring board (DSMB) appointed by the NHLBI will review 
comprehensive interim enrollment, safety, and efficacy data reports on a semi-annual basis. A priori 
stopping rules for efficacy and safety with respect to the primary endpoint will be guided as outlined in 
the DSMB charter.  Serious adverse events will be monitored by the committee on an ongoing basis 
throughout the study.  All study personnel and investigators will remain blinded to results of the interim 
analyses, with the exception of designated Data Coordinating Center (DCC) personnel who will prepare 
the interim reports.  In addition to interim DSMB reports, semi-annual reports will also be generated for 
the Steering Committee to the study.  This Committee will remain blinded, and will only be provided 
enrollment and site performance data.  
14.7 Sample Size 
Results from a recently completed randomized trial in children with CF uninfected with Pa 
demonstrated a 51% reduction in exacerbations in the group receiving azithromycin as compared to 
those receiving placebo( 9).  Further, results from a randomized trial in children and adults chronically 


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infected with Pa, the majority receiving concurrent TIS, demonstrated a 40% reduction in exacerbations 
in the group receiving azithromycin as compared to those receiving placebo(13).  It is felt that a 40-50% 
reduction in exacerbations is the minimal effect size that would need to be observed to justify the 
addition of a second antibiotic regimen for the treatment of children with CF and new onset Pa.  Data 
from the original EPIC trial indicates that approximately 50% of children with new onset Pa will 
experience a protocol-defined pulmonary exacerbation over the 18-month follow up period(5).  Thus, 
even in the presence of standard of care treatment with TIS, we expect a high rate of exacerbation 
among those randomized to receive oral placebo with TIS.  Assuming a two-sided 0.05 type I error and a 
sample size of approximately 137 children randomized to each treatment group (culture-based TIS with 
oral placebo or culture-based TIS with oral azithromycin), the study has 90% power to detect a hazard 
ratio for the time to exacerbation of 0.53 or smaller (a 47% or greater reduction) between treatment 
groups and 80% power to detect a hazard ratio of 0.58 or smaller (a 42% or greater reduction).  With a 
maximum projected withdrawal rate of at most 9% over the course of the 18-month study based on the 
prior EPIC trial with similar duration of follow-up, it is conservatively estimated that the study still has 
at minimum 80% power to detect a hazard ratio of 0.56 or smaller between treatment groups (a 44% or 
greater reduction).  These power estimates account for six planned interim analyses and one final 
analyses, assuming an O’Brien-Fleming type boundary with boundary parameter equal to 1.0(82).  
Further, it is anticipated that approximately 34% of the children in the placebo group will experience Pa 
recurrence over the 18-month study based on estimates from the EPIC trial (Section 2.2.2).   The study 
has 80% power to detect a hazard ratio of 0.52 or lower (48% or greater reduction) in the time to Pa 
recurrence between the azithromycin and placebo groups assuming a two-sided 0.05 type I error. 
15  
DATA COLLECTION, RETENTION AND MONITORING  
15.1 Data Collection Instruments 
An electronic data capture system, Medidata Rave, will be utilized for collection of study data.  The 
Investigator will prepare and maintain adequate and accurate source documents designed to record all 
observations and other pertinent data for each participant who signs informed consent.   
Study personnel at each site will enter data from source documents corresponding to a participant’s visit 
or assessment into the protocol-specific electronic Case Report Form (eCRF).  Participants will not be 
identified by name in the study database or on any study documents to be collected by the Sponsor (or 
designee), but will be identified by a site number, participant number and initials. 
If a correction is required for an eCRF, the time and date stamp will track the person entering or 
updating eCRF data and creates an electronic audit trail.   
The Investigator is responsible for all information collected on participants enrolled in this study.  All 
data collected during the course of this study must be reviewed and verified for completeness and 
accuracy by the Investigator.  A copy of the CRF will remain at the Investigator’s site at the completion 
of the study. 
15.2 Data Management Procedures 
The data will be entered into a validated database.  The DCC will be responsible for data processing, in 
accordance with procedural documentation.  Database lock will occur once quality assurance procedures 
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All procedures for the handling and analysis of data will be conducted using good computing practices 
for the handling and analysis of data for clinical trials. 
15.3 Data Quality Control and Reporting 
Once data have been entered into the study database, a system of computerized data validation checks 
will be implemented and applied to the database on a regular basis.  Queries are entered, tracked, and 
resolved through the EDC system directly.  The study database will be updated in accordance with the 
resolved queries.  All changes to the study database will be documented in an audit trail. 
15.4 Security and Archival of Data 
The EDC system is hosted by Medidata; the data are stored at Medidata’s primary data center in 
Houston, Texas, with fail-safe data centers in New Jersey.  Data are regularly backed up by Medidata 
and stored with Iron Mountain. 
Medidata maintains 21 CFR Part 11-compliant electronic systems, with procedures in place to safeguard 
against unauthorized acquisition of data.  Any authorized communication with the Medidata servers at 
the Houston Data Center is conducted via SSL (128-bit) encryption.  Robust password procedures, 
consistent with 21 Part 11, are in place.  Robust physical security procedures are in place at the Houston 
Data Center to prevent unauthorized personnel physical access to the server rooms.  EDC account access 
is maintained and monitored by the Data Coordinating Center. 
Other databases will be stored on Seattle Children’s servers and are safeguarded against unauthorized 
access by established security procedures.  Network accounts are password protected and maintained 
and monitored by the Seattle Children’s Information Services group.  Data is backed up regularly 
according to the Information Technology group’s procedures.  
 Note that there is an intention to make banked biospecimens and associated data available to 
investigators for future exploration. The biospecimens will be collected under Institutional Review 
Board (IRB)/Independent Ethics Committee (IEC) approval, processed according to a standard operating 
procedure and stored at a central facility, with appropriate procedures to enable long term, stable 
storage. Researchers may apply, via a standardized process, for use of de-identified data and specimens 
for research purposes. Applications will undergo a scientific review process. When applying for use of 
data or specimens, the applicant must agree to: (1) use the data and specimens only for research 
purposes and to not make any attempts to try to identify any individual subject; (2) securing the data and 
specimens using appropriate methods; and (3) destroy or return the data (and specimens) in accordance 
with the specimen/data use agreement after analyses are completed.  Before data or specimens will be 
released to an investigator, documentation of IRB exemption or approval from their institution must be 
provided. 
15.5 Availability and Retention of Investigational Records 
The Investigator must make study data accessible to the monitor, other authorized representatives of the 
Sponsor (or designee), IRB, and Regulatory Agency (e.g., FDA) inspectors upon request.  A file for 
each participant must be maintained that includes the signed Informed Consent, HIPAA Authorization 
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must ensure the reliability and availability of source documents from which the information on the CRF 
was derived. 
All study documents (e.g., patient files, signed informed consent forms, copies of CRFs, Study File 
Notebook, etc.) must be kept secured for a period of one year after database lock and accessible for 10 
years following database lock.  There may be other circumstances for which the Sponsor is required to 
maintain study records and, therefore, the Sponsor should be contacted prior to removing study records 
for any reason. 
15.6 Monitoring 
Monitoring visits will be conducted by representatives of the Sponsor according to the U.S. CFR Title 
21 Part 312 and ICH Guidelines for GCP (E6) and to ensure investigator compliance to 21 CFR Parts 
50, 56 and 312 and to GCP.  By signing this protocol, the Investigator grants permission to the Sponsor 
(or designee), and appropriate regulatory authorities to conduct on-site monitoring and/or auditing of all 
appropriate study documentation. 
15.7 Participant Confidentiality 
In order to maintain participant confidentiality, only a site number, participant number and participant 
initials will identify all study participants on CRFs and other documentation submitted to the Sponsor.  
If specific consent is given, the participant’s CFF patient registry number will also be collected.  
Additional participant confidentiality issues (if applicable) are covered in the Clinical Study Agreement. 
16 ADMINISTRATIVE, ETHICAL, REGULATORY CONSIDERATIONS 
The study will be conducted according to the Declaration of Helsinki, Protection of Human Volunteers 
(21 CFR 50), Institutional Review Boards (21 CFR 56), and Obligations of Clinical Investigators (21 
CFR 312). 
To maintain confidentiality, all laboratory specimens, evaluation forms, reports and other records will be 
identified by a coded number and initials only.  Clinical information will not be released without written 
permission of the participant, except as necessary for monitoring by the FDA.  The Investigator must 
also comply with all applicable privacy regulations (e.g., Health Insurance Portability and 
Accountability Act of 1996, EU Data Protection Directive 95/46/EC). 
16.1 Protocol Amendments 
Any amendment to the protocol will be written by the Sponsor.  Protocol amendments cannot be 
implemented without prior written IRB approval except as necessary to eliminate immediate safety 
hazards to participants.  A protocol amendment intended to eliminate an apparent immediate hazard to 
participants may be implemented immediately, provided the IRBs are notified within five working days. 
16.2 Institutional Review Boards  
The protocol and consent form will be reviewed and approved by the IRB of each participating center 
prior to study initiation.  Serious adverse experiences regardless of causality will be reported to the IRB 
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keep the IRB informed as to the progress of the study.  The Investigator will obtain assurance of IRB 
compliance with regulations. 
Any documents that the IRB may need to fulfill its responsibilities (such as protocol, protocol 
amendments, prescribing information, consent forms, information concerning patient recruitment, 
payment or compensation procedures, or other pertinent information) will be submitted to the IRB.  The 
IRB’s written unconditional approval of the study protocol and the informed consent form will be in the 
possession of the Investigator before the study is initiated.  The IRB’s unconditional approval statement 
will be transmitted by the Investigator to the Sponsor or designee prior to the shipment of study supplies 
to the site.  This approval must refer to the study by exact protocol title and number and should identify 
the documents reviewed and the date of review. 
Protocol and/or informed consent modifications or changes may not be initiated without prior written 
IRB approval except when necessary to eliminate immediate hazards to the participants or when the 
change(s) involves only logistical or administrative aspects of the study.  Such modifications will be 
submitted to the IRB and written verification that the modification was submitted and subsequently 
approved should be obtained.   
The IRB must be informed of revisions to other documents originally submitted for review; serious 
and/or unexpected adverse experiences occurring during the study in accordance with the standard 
operating procedures and policies of the IRB; new information that may affect adversely the safety of 
the participants or the conduct of the study; an annual update and/or request for re-approval; and when 
the study has been completed. 
16.3 Informed Consent Form  
Informed consent will be obtained in accordance with the Declaration of Helsinki, ICH GCP, US Code 
of Federal Regulations for Protection of Human Participants (21 CFR 50.25[a,b], CFR 50.27, and CFR 
Part 56, Subpart A), the Health Insurance Portability and Accountability Act (HIPAA, if applicable), and 
local regulations. 
The Investigator will prepare the informed consent form, assent and HIPAA authorization and provide 
the documents to the Sponsor or designee for approval prior to submission to the IRB.  The consent 
form generated by the Investigator must be acceptable to the Sponsor and be approved by the IRB.  The 
written consent document will embody the elements of informed consent as described in the 
International Conference on Harmonisation and will also comply with local regulations.  The 
Investigator will send an IRB-approved copy of the Informed Consent Form to the Sponsor (or 
designee) for the study file. 
A properly executed, written, informed consent will be obtained from each participant (or their legal 
representatives) prior to entering the participant into the trial.  Information should be given in both oral 
and written form and participants (or their legal representatives) must be given ample opportunity to 
inquire about details of the study.  If appropriate and required by the local IRB, assent from the 
participant will also be obtained.  If a participant is unable to sign the informed consent form (ICF) and 
the HIPAA authorization, a legal representative may sign for the participant.  A copy of the signed 
consent form (and assent) will be given to the participant or legal representative of the participant and 
the original will be maintained with the participant’s records. 


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During the course of the study, if modifications are made to the consent form that impact the participant, 
the participant will be re-consented as described above.  
16.3.1 Consent for Collection and Use of CFF Registry ID Number  
To facilitate possible future evaluation of retrospective and prospective information from all participants 
who screen for this study, consent will also be sought at the Baseline Visit to collect the participant’s 
CFF Registry ID number.  The CFF registry collects data on all patients with CF followed at CFF-
accredited care centers at each clinical encounter, at each hospitalization or course of antibiotics, and via 
a year-end survey.  Data include microbiology results, spirometry results, CF genotype and other 
information.   
16.4 Publications  
The preparation and submittal for publication of manuscripts containing the study results shall be in 
accordance with a process determined by mutual written agreement among the participating institutions.  
The publication or presentation of any study results shall comply with all applicable privacy laws, 
including, but not limited to, the Health Insurance Portability and Accountability Act of 1996.  
16.5 Investigator Responsibilities 
By signing the Agreement of Investigator form, the Investigator agrees to: 
1. Conduct the study in accordance with the protocol and only make changes after notifying the 
Sponsor (or designee), except when to protect the safety, rights or welfare of participants. 
2. Personally conduct or supervise the study (or investigation). 
3. Ensure that the requirements relating to obtaining informed consent and IRB review and 
approval meet federal guidelines, as stated in § 21 CFR, parts 50 and 56. 
4. Report to the Sponsor or designee any AEs that occur in the course of the study, in accordance 
with §21 CFR 312.64. 
5. Ensure that all associates, colleagues and employees assisting in the conduct of the study are 
informed about their obligations in meeting the above commitments. 
6. Maintain adequate and accurate records in accordance with §21 CFR 312.62 and to make those 
records available for inspection with the Sponsor (or designee). 
7. Ensure that an IRB that complies with the requirements of §21 CFR part 56 will be responsible 
for initial and continuing review and approval of the clinical study. 
8. Promptly report to the IRB and the Sponsor (or designee) all changes in the research activity 
and all unanticipated problems involving risks to participants or others (to include amendments 
and IND safety reports). 
9. Seek IRB approval before any changes are made in the research study, except when necessary to 
eliminate hazards to the patients/participants. 
10. Comply with all other requirements regarding the obligations of clinical investigators and all 
other pertinent requirements listed in § 21 CFR part 312. 


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17 LITERATURE CITED 
1. 
Welsh MJ, Ramsey BW, Accurso F, Cutting G. Cystic fibrosis. In: Scriver CR, Beaudet AL, Sly 
WS, Valle D, editors. The Metabolic and Molecular Basis of Inherited Diseases. 8th ed. New 
York: McGraw-Hill; 2001. p. 5121-88. 
2. 
Flume PA, Mogayzel PJ, Jr., Robinson KA, Rosenblatt RL, Quittell L, Marshall BC. Cystic 
fibrosis pulmonary guidelines: pulmonary complications: hemoptysis and pneumothorax. 
American Journal of Respiratory and Critical Care Medicine. 2010;182(3):298-306. 
3. 
Gibson RL, Burns JL, Ramsey BW. Pathophysiology and management of pulmonary infections in 
cystic fibrosis. American Journal of Respiratory and Critical Care Medicine. 2003;168(8):918-51. 
4. 
Hauser AR, Jain M, Bar-Meir M, McColley SA. Clinical significance of microbial infection and 
adaptation in cystic fibrosis. Clinical Microbiology Reviews. 2011;24(1):29-70. PMCID: 3021203. 
5. 
Treggiari MM, Retsch-Bogart G, Mayer-Hamblett N, Khan U, Kulich M, Kronmal R, Williams J, 
Ramsey BW, Investigators ftE. Comparative efficacy and safety of four randomized regimens to 
treat early Pseudomonas aeruginosa infection in children with cystic fibrosis. Archives of 
Pediatrics and Adolescent Medicine. 2011;165(9):847-56. 
6. 
Mayer-Hamblett N, Kronmal RA, Gibson RL, Rosenfeld M, Retsch-Bogart G, Treggiari MM, 
Burns JL, Khan U, Ramsey BW. Initial Pseudomonas aeruginosa treatment failure is associated 
with exacerbations in cystic fibrosis. Pediatric Pulmonology. 2012;47(2):125-34. PMCID: 
3214247. 
7. 
Sanders DB, Bittner RC, Rosenfeld M, Hoffman LR, Redding GJ, Goss CH. Failure to recover to 
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Respiratory and Critical Care Medicine. 2010;182(5):627-32. 
8. 
Sanders DB, Bittner RC, Rosenfeld M, Redding GJ, Goss CH. Pulmonary exacerbations are 
associated with subsequent FEV1 decline in both adults and children with cystic fibrosis. Pediatric 
Pulmonology. 2011;46(4):393-400. 
9. 
Saiman L, Anstead M, Mayer-Hamblett N, Lands LC, Kloster M, Hocevar-Trnka J, Goss CH, 
Rose LM, Burns JL, Marshall BC, Ratjen F. Effect of azithromycin on pulmonary function in 
patients with cystic fibrosis uninfected with Pseudomonas aeruginosa: a randomized controlled 
trial. JAMA. 2010;303(17):1707-15. 
10. Ratjen F, Saiman L, Mayer-Hamblett N, Lands LC, Kloster M, Thompson V, Emmett P, Marshall 
B, Accurso F, Sagel S, Anstead M. Effect of azithromycin on systemic markers of inflammation in 
cystic fibrosis patients uninfected with Pseudomonas aeruginosa. Chest. 2012, May 17;e-pub 
ahead of print. 


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6. 
12. Equi A, Balfour-Lynn IM, Bush A, Rosenthal M. Long term azithromycin in children with cystic 
fibrosis: a randomised, placebo-controlled crossover trial. Lancet. 2002;360(9338):978-84. 
13. Saiman L, Marshall BC, Mayer-Hamblett N, Burns JL, Quittner AL, Cibene DA, Coquillette S, 
Fieberg AY, Accurso FJ, Campbell PW, 3rd. Azithromycin in patients with cystic fibrosis 
chronically infected with Pseudomonas aeruginosa: a randomized controlled trial. JAMA. 
2003;290(13):1749-56. 
14. Gangell C, Gard S, Douglas T, Park J, de Klerk N, Keil T, Brennan S, Ranganathan S, Robins-
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15. Pillarisetti N, Williamson E, Linnane B, Skoric B, Robertson CF, Robinson P, Massie J, Hall GL, 
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16. Burns JL, Gibson RL, McNamara S, Yim D, Emerson J, Rosenfeld M, Hiatt P, McCoy K, Castile 
R, Smith AL, Ramsey BW. Longitudinal assessment of Pseudomonas aeruginosa in young 
children with cystic fibrosis. Journal of Infectious Diseases. 2001;183(3):444-52. 
17. Rosenfeld M, Gibson RL, McNamara S, Emerson J, Burns JL, Castile R, Hiatt P, McCoy K, 
Wilson CB, Inglis A, Smith A, Martin TR, Ramsey BW. Early pulmonary infection, inflammation, 
and clinical outcomes in infants with cystic fibrosis. Pediatric Pulmonology. 2001;32(5):356-66. 
18. Armstrong DS, Grimwood K, Carlin JB, Carzino R, Gutierrez JP, Hull J, Olinsky A, Phelan EM, 
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19. Stick SM, Brennan S, Murray C, Douglas T, von Ungern-Sternberg BS, Garratt LW, Gangell CL, 
De Klerk N, Linnane B, Ranganathan S, Robinson P, Robertson C, Sly PD. Bronchiectasis in 
infants and preschool children diagnosed with cystic fibrosis after newborn screening. Journal of 
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20. Parad RB, Gerard CJ, Zurakowski D, Nichols DP, Pier GB. Pulmonary outcome in cystic fibrosis 
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modestly by genotype. Infection and Immunity. 1999;67(9):4744-50. PMCID: 96804. 
21. Henry RL, Mellis CM, Petrovic L. Mucoid Pseudomonas aeruginosa is a marker of poor survival 
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23. Britto MT, Kotagal UR, Hornung RW, Atherton HD, Tsevat J, Wilmott RW. Impact of recent 
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24. Pamukcu A, Bush A, Buchdahl R. Effects of Pseudomonas aeruginosa colonization on lung 
function and anthropometric variables in children with cystic fibrosis. Pediatric Pulmonology. 
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25. Rabin HR, Butler SM, Wohl ME, Geller DE, Colin AA, Schidlow DV, Johnson CA, Konstan MW, 
Regelmann WE. Pulmonary exacerbations in cystic fibrosis. Pediatric Pulmonology. 
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26. Kerem E, Corey M, Gold R, Levison H. Pulmonary function and clinical course in patients with 
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1990;116(5):714-9. 
27. Konstan MW, Morgan WJ, Butler SM, Pasta DJ, Craib ML, Silva SJ, Stokes DC, Wohl ME, 
Wagener JS, Regelmann WE, Johnson CA. Risk factors for rate of decline in forced expiratory 
volume in one second in children and adolescents with cystic fibrosis. Journal of Pediatrics. 
2007;151(2):134-9, 9 e1. 
28. Kosorok MR, Zeng L, West SE, Rock MJ, Splaingard ML, Laxova A, Green CG, Collins J, Farrell 
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29. Nixon GM, Armstrong DS, Carzino R, Carlin JB, Olinsky A, Robertson CF, Grimwood K. 
Clinical outcome after early Pseudomonas aeruginosa infection in cystic fibrosis. Journal of 
Pediatrics. 2001;138(5):699-704. 
30. Emerson J, Rosenfeld M, McNamara S, Ramsey B, Gibson RL. Pseudomonas aeruginosa and 
other predictors of mortality and morbidity in young children with cystic fibrosis. Pediatric 
Pulmonology. 2002;34(2):91-100. 
31. Li Z, Kosorok MR, Farrell PM, Laxova A, West SE, Green CG, Collins J, Rock MJ, Splaingard 
ML. Longitudinal development of mucoid Pseudomonas aeruginosa infection and lung disease 
progression in children with cystic fibrosis. JAMA. 2005;293(5):581-8. 
32. Lee TW, Brownlee KG, Conway SP, Denton M, Littlewood JM. Evaluation of a new definition for 
chronic Pseudomonas aeruginosa infection in cystic fibrosis patients. J Cyst Fibros. 2003;2(1):29-
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33. Singh PK, Schaefer AL, Parsek MR, Moninger TO, Welsh MJ, Greenberg EP. Quorum-sensing 
signals indicate that cystic fibrosis lungs are infected with bacterial biofilms. Nature. 
2000;407(6805):762-4. 
34. Wiesemann HG, Steinkamp G, Ratjen F, Bauernfeind A, Przyklenk B, Doring G, von der Hardt H. 
Placebo-controlled, double-blind, randomized study of aerosolized tobramycin for early treatment 
of Pseudomonas aeruginosa colonization in cystic fibrosis. Pediatric Pulmonology. 1998;25(2):88-
92. 
35. Valerius NH, Koch C, Hoiby N. Prevention of chronic Pseudomonas aeruginosa colonisation in 
cystic fibrosis by early treatment. Lancet. 1991;338(8769):725-6. 
36. Munck A, Bonacorsi S, Mariani-Kurkdjian P, Lebourgeois M, Gerardin M, Brahimi N, Navarro J, 
Bingen E. Genotypic characterization of Pseudomonas aeruginosa strains recovered from patients 
with cystic fibrosis after initial and subsequent colonization. Pediatric Pulmonology. 
2001;32(4):288-92. 
37. Taccetti G, Campana S, Festini F, Mascherini M, Doring G. Early eradication therapy against 
Pseudomonas aeruginosa in cystic fibrosis patients. European Respiratory Journal. 
2005;26(3):458-61. 
38. Langton Hewer SC, Smyth AR. Antiobiotic strategies for eradicating Pseudomonas aeruginosa in 
people with cystic fibrosis. Cochrane Database of Systematic Reviews. 2009;CD004197. 
39. Ratjen F, Munck A, Kho P, Angyalosi G. Treatment of early Pseudomonas aeruginosa infection in 
patients with cystic fibrosis: the ELITE trial. Thorax. 2010;65(4):286-91. 
40. Gibson RL, Emerson J, Mayer-Hamblett N, Burns JL, McNamara S, Accurso FJ, Konstan MW, 
Chatfield BA, Retsch-Bogart G, Waltz DA, Acton J, Zeitlin P, Hiatt P, Moss R, Williams J, 
Ramsey BW. Duration of treatment effect after tobramycin solution for inhalation in young 
children with cystic fibrosis. Pediatric Pulmonology. 2007;42(7):610-23. 
41. Nixon GM, Armstrong DS, Carzino R, Carlin JB, Olinsky A, Robertson CF, Grimwood K, 
Wainwright C. Early airway infection, inflammation, and lung function in cystic fibrosis. Archives 
of Disease in Childhood. 2002;87(4):306-11. PMCID: 1763045. 
42. Taccetti G, Bianchini E, Cariani L, Buzzetti R, Costantini D, Trevisan F, Zavataro L, Campana S. 
Early antibiotic treatment for Pseudomonas aeruginosa eradication in patients with cystic fibrosis: 
a randomised multicentre study comparing two different protocols. Thorax. 2012;epub ahead of 
print Feb. 29. 
43. Doring G, Flume P, Heijerman H, Elborn JS. Treatment of lung infection in patients with cystic 
fibrosis: current and future strategies. J Cyst Fibros. 2012;11(6):461-79. 


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44. Treggiari MM, Rosenfeld M, Mayer-Hamblett N, Retsch-Bogart G, Gibson RL, Williams J, 
Emerson J, Kronmal RA, Ramsey BW. Early anti-pseudomonal acquisition in young patients with 
cystic fibrosis: rationale and design of the EPIC clinical trial and observational study'. Contemp 
Clin Trials. 2009;30(3):256-68. PMCID: 2783320. 
45. Steinkamp G, Schmitt-Grohe S, Doring G, Staab D, Pfrunder D, Beck G, Schubert R, Zielen S. 
Once-weekly azithromycin in cystic fibrosis with chronic Pseudomonas aeruginosa infection. 
Respiratory Medicine. 2008;102(11):1643-53. 
46. Rosenfeld M, Emerson J, McNamara S, Joubran K, Retsch-Bogart G, Graff GR, Gutierrez HH, 
Kanga JF, Lahiri T, Noyes B, Ramsey B, Ren CL, Schechter M, Morgan W, Gibson RL. Baseline 
characteristics and factors associated with nutritional and pulmonary status at enrollment in the 
cystic fibrosis EPIC observational cohort. Pediatric Pulmonology. 2010;45(9):934-44. 
47. Mendelman PM, Smith AL, Levy J, Weber A, Ramsey B, Davis RL. Aminoglycoside penetration, 
inactivation, and efficacy in cystic fibrosis sputum. American Review of Respiratory Disease. 
1985;132(4):761-5. 
48. Ramsey BW, Dorkin HL, Eisenberg JD, Gibson RL, Harwood IR, Kravitz RM, Schidlow DV, 
Wilmott RW, Astley SJ, McBurnie MA. Efficacy of aerosolized tobramycin in patients with cystic 
fibrosis. New England Journal of Medicine. 1993;328(24):1740-6. 
49. Baumann U, King M, App EM, Tai S, Konig A, Fischer JJ, Zimmermann T, Sextro W, von der 
Hardt H. Long term azithromycin therapy in cystic fibrosis patients: a study on drug levels and 
sputum properties. Canadian Respiratory Journal. 2004;11(2):151-5. 
50. Moskowitz SM, Foster JM, Emerson JC, Gibson RL, Burns JL. Use of Pseudomonas biofilm 
susceptibilities to assign simulated antibiotic regimens for cystic fibrosis airway infection. Journal 
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51. Moskowitz SM, Emerson JC, McNamara S, Shell RD, Orenstein DM, Rosenbluth D, Katz MF, 
Ahrens R, Hornick D, Joseph PM, Gibson RL, Aitken ML, Benton WW, Burns JL. Randomized 
trial of biofilm testing to select antibiotics for cystic fibrosis airway infection. Pediatric 
Pulmonology. 2011;46(2):184-92. 
52. Mizukane R, Hirakata Y, Kaku M, Ishii Y, Furuya N, Ishida K, Koga H, Kohno S, Yamaguchi K. 
Comparative in vitro exoenzyme-suppressing activities of azithromycin and other macrolide 
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1994;38(3):528-33. PMCID: 284493. 
53. Molinari G, Guzman CA, Pesce A, Schito GC. Inhibition of Pseudomonas aeruginosa virulence 
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54. Gillis RJ, Iglewski BH. Azithromycin retards Pseudomonas aeruginosa biofilm formation. Journal 
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55. Wagner T, Soong G, Sokol S, Saiman L, Prince A. Effects of azithromycin on clinical isolates of 
Pseudomonas aeruginosa from cystic fibrosis patients. Chest. 2005;128(2):912-9. 
56. Saiman L, Chen Y, Gabriel PS, Knirsch C. Synergistic activities of macrolide antibiotics against 
Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, and Alcaligenes 
xylosoxidans isolated from patients with cystic fibrosis. Antimicrobial Agents & Chemotherapy. 
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57. Southern KW, Barker PM, Solis A. Macrolide antibiotics for cystic fibrosis. Cochrane Database of 
Systematic Reviews. 2004(2):CD002203. 
58. Wilms EB, Touw DJ, Heijerman HG, van der Ent CK. Azithromycin maintenance therapy in 
patients with cystic fibrosis: A dose advice based on a review of pharmacokinetics, efficacy, and 
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59. Albert RK, Connett J, Bailey WC, Casaburi R, Cooper JA, Jr., Criner GJ, Curtis JL, Dransfield 
MT, Han MK, Lazarus SC, Make B, Marchetti N, Martinez FJ, Madinger NE, McEvoy C, 
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60. Grayston JT, Kronmal RA, Jackson LA, Parisi AF, Muhlestein JB, Cohen JD, Rogers WJ, Crouse 
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23. 
 
 


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18 APPENDIX I: SCHEDULE OF EVENTS 
 
Quarter 1 
Quarter 2 
Quarter 3 
Quarter 4 
Quarter 5 
Quarter 6 
End of Study or  
Early Withdrawal 
 
 
Baseline 
Visit 1 
Screening  
(Day 0) 
Visit 2 
(Week 3  
± 2 days) 
Visit 3 
(Week 13  
± 2 weeks)  
Visit 4  
(Week 26 
± 2 weeks) 
Visit 5 
 (Week 39 
± 2 weeks) 
Visit 6 
(Week 52 
± 2 weeks) 
Visit 7 
(Week 65 
± 2 weeks) 
Visit 8 
(Week 78 
± 2 weeks)  
Unscheduled 
Assessment8 
Informed Consent 
X 
 
 
 
 
 
 
 
 
Consent for CF Registry ID & 
Banking 
X 
 
 
 
 
 
 
 
 
Respiratory Questionnaires 1 
X 
X 
X 
X 
X 
X 
X 
X 
 
Demographics & Medical 
History 
X 
 
 
 
 
 
 
 
 
Concomitant Medication  
X 
X 
X 
X 
X 
X 
X 
X 
X 
Microbiology History 
X 
 
 
 
 
 
 
 
 
Complete Physical Exam 
X 
 
 
 
 
 
 
X 
 
Abbreviated Physical Exam 
 
X 
X 
X 
X 
X 
X 
 
 
Height & Weight 
X 
X 
X 
X 
X 
X 
X 
X 
 
Vital Signs & Oximetry 
X 
X 
X 
X 
X 
X 
X 
X 
 
Spirometry2 
X 
X 
X 
X 
X 
X 
X 
X 
 
Collect plasma for IM and 
Banking3 
X 
 
 
 
X 
 
 
X 
 
Chemistry and Hematology 
performed at site lab 
X 
 
 
 
X 
 
 
X 
 
Audiometry4 
X 
 
 
 
X 
 
 
X 
 
12-lead ECG Testing  
X 
X 
 
 
 
 
 
X 
 
Pregnancy Test (urine or serum) 
X 
 
 
 
 
 
 
X 
 
Respiratory Culture (processed at 
site lab)5 
X 
X 
X 
X 
X 
X 
X 
X 
 
OP swab collection for 
X 
X 
X 
X 
X 
X 
X 
X 
 


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Quarter 1 
Quarter 2 
Quarter 3 
Quarter 4 
Quarter 5 
Quarter 6 
End of Study or  
Early Withdrawal 
 
 
Baseline 
Visit 1 
Screening  
(Day 0) 
Visit 2 
(Week 3  
± 2 days) 
Visit 3 
(Week 13  
± 2 weeks)  
Visit 4  
(Week 26 
± 2 weeks) 
Visit 5 
 (Week 39 
± 2 weeks) 
Visit 6 
(Week 52 
± 2 weeks) 
Visit 7 
(Week 65 
± 2 weeks) 
Visit 8 
(Week 78 
± 2 weeks)  
Unscheduled 
Assessment8 
microbiome sample3 
Randomization 
 to azithromycin 
or placebo 
X 
 
 
 
 
 
 
 
 
Dispense TIS  
X6 
[X] 6 
[X]7 
[X] 7 
[X] 7 
[X] 7 
[X] 7 
 
 
Dispense azithromycin/placebo 
X 
 
X 
X 
X 
X 
X 
 
 
Dispense Subject Diary 
X 
 
X 
X 
X 
X 
X 
 
 
Review Subject Diary 
 
X 
X 
X 
X 
X 
X 
X 
 
Drug Accountability 
 
 
X 
X 
X 
X 
X 
X 
 
Adverse Events Review 
 
X 
X 
X 
X 
X 
X 
X 
X 
Hospitalization Review 
 
X 
X 
X 
X 
X 
X 
X 
 
Pulmonary Symptom Review 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Clinical Care Micro Results 
(respiratory cultures) 
 
 
 
 
 
 
 
 
X 
1 Completed at each study visit  
2 Spirometry is only performed among participants 4 years of age or older able to reliably perform the procedure. 
3 Samples will be shipped at the end of study or upon request 
4 Audiometry can be performed up to 2 after weeks of Visit 1, up to 2 weeks before or after Visit 5, and up to 2 weeks before Visit 8. Testing will include visual reinforcement 
audiometry  (VRA) using sound field testing for participants approximately 6 to 30 months old, play audiometry with earphones for participants approximately 30 months to 5 
years (developmental age), and standard audiometry for those approximately 5 to 18 years of age. Tympanometry will be performed with all audiometry testing.  
5Respiratory cultures are processed at site microbiology laboratory.  Sputum will additionally be cultured for NTM at the site microbiology laboratory.  
6 All participants will receive a 28 day course of TIS at Visit 1, unless TIS was prescribed and administered in the 14 days prior study enrollment.  At Visit 2, participants whose 
respiratory culture are Pa + based on site microbiology lab results will receive a second 28-day TIS course during the first treatment quarter. 
7 At Visits 3, 4, 5, 6, and 7, participants whose respiratory culture is Pa + based on site microbiology lab results will receive a 28-day TIS course during the treatment quarter as 
standard of care.  
8 An unscheduled assessment will be conducted when a participant initiates a new antibiotic therapy, has a change in antibiotic therapy or as determined by the investigator to 
assess possible symptoms of a pulmonary exacerbation.


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19 APPENDIX II:  DEFINITION OF PULMONARY EXACERBATION  
 
The presence of a pulmonary exacerbation is established by the following: 
(1) 
One of the major criteria alone  
             or  
(2) 
Two of the minor signs/symptoms and fulfillment of symptom duration   
 
Major Criteria: (One finding alone establishes the presence of a pulmonary exacerbation) 
(1) Absolute decrease in FEV1 %predicted of 10%  
(2) Oxygen saturation <90% on room air or absolute decrease of  5%  
(3) New lobar infiltrate(s) or atelectasi(e)s on chest radiograph 
(4) Hemoptysis (more than streaks on more than one occasion in past week) 
 
Minor Signs/Symptoms: (Two minor signs/symptoms are required in the absence of major criteria. If at least 
2 minor signs/symptoms are present, at least one needs to be 3 or more days in duration to meet the PE 
definition) 
(1) Increased work of breathing or respiratory rate 
(2) New or increased adventitial sounds on lung exam 
(3) Weight loss 5% of body weight or decrease across 1 major percentile in weight percentile for age in 
past 6 months 
(4) Increased cough 
(5) Decreased exercise tolerance or level of activity 
(6) Increased chest congestion or change in sputum 
 
 
 
 


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20 APPENDIX III: COMPOSITE LIST OF DRUGS THAT PROLONG QT AND/OR CAUSE 
TORSADES DE POINTES (TDP)  
 
The list of drugs that prolong QT and/or cause torsades de pointes (TdP) may be updated during the study.   
Refer to the most recent version of the list at this website: Composite List of All QT Drugs under the 
category “Known Risk of TdP.”   
Drugs known to prolong QT interval are described as follows: “Substantial evidence supports the 
conclusion that these drugs prolong the QT interval AND are clearly associated with a risk of torsades de 
pointes (TdP), even when taken as directed in official labeling.”  
Instructions for searching for specific QT drugs will be posted on the OPTIMIZE study website. 

SAP_000.pdf: 
 
 
 
 
 Statistical Analysis Plan 
For the  
Final Statistical Report 
 
 
 
 
PROTOCOL NUMBER:   
OPTIMIZE-IP-12 
 
 
 
PROTOCOL TITLE: 
The OPTIMIZE Multicenter, Placebo-
Controlled, Double-Blind, Randomized 
Trial 
 
 
 
PRINCIPAL 
INVESTIGATORS:  
 
Nicole Mayer-Hamblett, PhD 
OPTIMIZE DCC 
 
Bonnie Ramsey, MD 
 
 
 
 
 
OPTIMIZE CCC 
 
 
FUNDING AGENCIES:   
National Institutes of Health/National 
Heart, Lung, and Blood Institute 
(NHLBI) 
 
 
PREPARED BY: 
 
 
Margaret Kloster, MS 
 
 
 
 
 
Shelly Ma, MS 
 
 
 
 
 
Nicole Hamblett, PhD 
 
 
 
 
RELEASE DATE:   
 
April 22, 2015 
 
  


 
 
PREFACE 
 
 
The Statistical Analysis Plan (SAP) as outlined in this document will be finalized prior to 
the completion of the first comprehensive DSMB interim report.  Any modifications to 
the SAP after finalization will be documented.  The SAP contains all modifications and 
updates to the planned analyses that were outlined in the original study protocol.  This 
plan details all a priori specified analyses that will be performed upon study completion 
and database lock, with detailed specifications for all tables, figures, and statistical 
models.   


 
 
Signature Page 
 
 
 
 
Analysis plan approved by: 
 
 
 
 
______________________________________________      ____________ 
Bonnie Ramsey, M.D. 
 
 
 
 
       
Date 
Principal Investigator 
OPTIMIZE CCC 
 
 
 
______________________________________________      ____________ 
Nicole Mayer Hamblett, PhD  
 
 
 
 
Date 
Principal Investigator 
OTPIMIZE DCC 
 
 
 
 
 
 
 
 
 
 
 
  
 


 
 
1.     Overview 
 
1.1 
Study Rationale and Design 
 
This is a multicenter, double-blind, randomized, placebo-controlled clinical trial in 274 
children with CF ages 6 mos -18 years with early Pa, defined as either a first lifetime 
documented Pa culture or a Pa positive culture after at least two years of negative 
cultures.  The study will assess the clinical and microbiologic efficacy and safety of 
azithromycin given three times weekly in combination with standardized TIS therapy 
among children with early Pa.  TIS therapy is defined as an initial eradication treatment 
with 1-2 courses of 28 days TIS and subsequent 28 day treatments only at times a 
quarterly oropharyngeal culture is positive for Pa.  Eligible participants will be 
randomized within one month of their Pa positive culture. Participants initiating TIS 
more than 14 days prior to the baseline visit for treatment of their Pa positive culture will 
be excluded.  Participants will be randomized in a 1:1 fashion to receive one of the 
following two treatment strategies for 18 months: (1) oral placebo in addition to 
standardized TIS therapy, or (2) oral azithromycin in addition to standardized TIS 
therapy.   
 
Three times weekly administration of oral azithromycin or matching placebo will be 
continued throughout the 18 month trial.   All participants will receive a 28-day course of 
TIS therapy during the first quarter of the trial and have a follow-up culture obtained to 
document Pa clearance. Participants who remain Pa positive after the first 28-day course 
of TIS will receive a second 28-day course of TIS.  Subsequently, participants will 
receive TIS therapy (administered as a single 28-day course) only if their quarterly 
cultures are Pa positive. Cultures will be obtained at the start of each quarter for the 
duration of the 18 month study (see Figure 1).   
 
Clinical and microbiologic outcomes will be assessed throughout the 18-month trial, 
including the occurrence of pulmonary exacerbations and Pa recurrence.   
 


 
 
 
The total duration of the study is expected to be forty-nine (49) months: thirty-one (31) 
months for participant recruitment and eighteen (18) for final participant follow-up. 
1.2 
Study Objectives and Endpoints 
 
The primary endpoint in the study is the difference between treatment groups in the time 
to a protocol-defined pulmonary exacerbation, comparing oral placebo with inhaled 
tobramycin versus oral azithromycin with inhaled tobramycin.  Pulmonary exacerbations 
will be defined according to an a priori sign and symptom-based definition (Appendix 
II).   
Secondary Endpoints 
1. Time to Pa recurrence over the 18-month study period 
2. Safety as measured by the incidence of adverse events and laboratory abnormalities 
over the 18-month study period  
3. Changes from baseline in plasma inflammatory markers  
Secondary Exploratory Endpoints 
4. Proportion of participants with initial Pa eradication success, defined as a Pa 
negative respiratory culture at week 13 
5. Proportion of all cultures obtained that are positive for Pa during the 18 month study 
period 
6. Prevalence of treatment-emergent Staphylococcus aureus (methicillin susceptible 
and resistant), Haemophilus influenzae, Burkholderia cepacia complex, 
Stenotrophomonas maltophilia, and Alcaligenes xylosoxidans (from oropharyngeal 


 
 
and sputum cultures), and non-tuberculous mycobacteria (in sputum only) during the 
18-month period 
7. Proportion of participants hospitalized over the 18-month study 
8. Proportion of participants prescribed acute oral, inhaled, and IV acute antibiotics 
over the 18-month study 
9. Change from baseline to the end of the 18-month study in pulmonary function (FVC, 
FEV1, FEF25-75%) in those old enough to perform spirometry (≥4 years of age) 
10. Change from baseline to the end of the 18-month study in weight and height 
11. Change from baseline to the end of the 18-month study in the self-report and parent-
completed Cystic Fibrosis Respiratory Diary scores 
12. Changes from baseline in the respiratory microbiome 
13. Association of baseline inflammatory marker values, microbial diversity measures 
and other baseline clinical and demographic characteristics with clinical and 
microbiologic outcomes over the 18-month study 
 
 
1.3 
Interim Data Monitoring Committee Reviews 
 
The DSMB will review quarterly abbreviated safety reports beginning 3 months after the 
first participant is randomized. Summaries of enrollment, drug discontinuations, and AEs 
and SAEs tabulated by treatment group will be included in the report.  
 
In addition to the quarterly safety reports, comprehensive interim reports will be 
generated and reviewed on a semi-annual basis beginning one year after the first 
participant enrolled. These report will include summaries of enrollment, randomization, 
demographic and baseline characteristics, safety (SAEs and AEs (including abnormal 
changes in audiology and electrocardiograms), hospitalizations, laboratory abnormalities, 
drug discontinuations), and key efficacy data (pulmonary exacerbations, microbiology, 
anthropometrics, spirometry, acute antibiotic usage).  
 
Formal stopping rules for efficacy, futility and safety and the timing of their evaluation 
are outlined in section 1.3. 
 
1.4 
Statistical Monitoring Guidelines and Stopping Rules for Planned Efficacy 
and Futility Analyses 
 
The DSMB will be guided by a formal stopping rule based on the relative risk of the 
primary endpoint, time to pulmonary exacerbation. With the sample size of 
approximately 137 per group and assuming a two-sided type 1 error of 0.05 and that 50% 
of participants will experience an exacerbation in the culture-based TIS with placebo 
group, the study has approximately 90% power to detect a 47% or greater reduction in 
exacerbations in the group receiving culture-based TIS with azithromycin (hazard ratio of 
0.53 or lower) and approximately 80% power to detect a 42% or greater reduction 
(hazard ratio of 0.58 or lower). These estimates take into account six planned interim 
analyses, which will be included in the comprehensive interim report, and one final 
analysis, included in the final report for the study. The first formal interim analysis will 


 
 
be described in the first comprehensive interim analysis generated one year after the first 
participant is randomized. 
 
Among the 137 participants enrolled in the culture-based TIS with placebo group, at a 
rate of 50%, we would expect approximately 69 primary endpoint events during the 18 
month randomized trial. Assuming that the addition of azithromycin conservatively 
results in a 40% reduction in exacerbations (relative risk ≤ 0.6), we would expect 
approximately 41 events among the 137 subjects enrolled in the azithromycin group for a 
total of 110 events during the study. The timing of the 7 analyses does not need to be 
equally spaced with respect to the accrual of PE events. However, if equally spaced 
analyses are performed, these would occur after 16, 31, 47, 63, 79, 94, and 110 events. 
Assuming an O’Brien-Fleming boundary1, the two-sided stopping rule would suggest 
that the trial be stopped early for safety if the observed hazard ratio were greater than or 
equal to 15.71, 3.96, 2.51, 1.88, 1.74, 1.58, and 1.48 at each analysis time (i.e. 
significantly greater number of events in the azithromycin group as compared to the 
placebo group) and the stopping rule for efficacy would suggest that the trial be stopped 
early if the observed hazard ratio were equal to or less than 0.06, 0.25, 0.39, 0.50, 0.58, 
0.63, and 0.68 at each analysis time (i.e. significantly less exacerbations in the 
azithromycin group as compared to placebo). With this example of equally spaced 
analyses, on the error spending function scale this corresponds to the following 
cumulative error spent: <0.00001, 0.023, 0.0333, 0.1384, 0.3396, 0.6323, and 1.0.  
The actual stopping boundaries will be computed at each analysis and in conjunction with 
comprehensive semi-annual report to the DSMB. Thus, the actual stopping boundaries 
may differ than those provided above depending on the number of events that have 
occurred. Splus SeqTrial software will be used to calculate the formal stopping boundary 
and adjust for this interim analysis in the final analysis.  
In addition to monitoring for the primary endpoint, the DSMB will also be guided by a 
safety threshold for which to monitor safety of the SAE defined as sensorineuronal 
hearing loss. Based on data from the EPIC clinical trial among children ages 1 – 12 years 
of age receiving TIS for newly acquired Pa, we expect approximately 8.6% of children to 
have an abnormal hearing test post-baseline (with a comparable definition to 
sensorineuronal hearing loss above) (17/197 children in EPIC, 95% CI 5.5%,13.4%). 
Based on this data, we recommend a stopping rule for the SAE sensorineuronal hearing 
loss as the following:  
 
(1) The proportion of patients with sensorineuronal hearing loss post-baseline is at 
least 8% greater in the azithromycin group as compared to placebo. This assumes, 
for example, at the end of the study that 12/137 participants (8.8%) of participants 
in the placebo group and 23/137 (16.8%) in the azithromycin group experience 
this SAE (an 8.0% difference, 95% CI: 0.06%, 16.1%)  
 
AND 
 
(2) The proportion of participants in the azithromycin group with sensorineural 
hearing loss is at least 13% or greater.  
 


 
 
 
2. Report Generation 
 
2.1 
Data Flow 
 
An electronic data capture system, Medidata Rave®, will be utilized for collection of 
study data. The Investigator will prepare and maintain adequate and accurate source 
documents designed to record all observations and other pertinent data for each 
participant who signs informed consent.  
 
Study personnel at each site will enter data from source documents corresponding to a 
participant’s visit or assessment into the protocol-specific electronic Case Report Form 
(eCRF). Participants will not be identified by name in the study database or on any study 
documents to be collected by the Sponsor (or designee), but will be identified by a site 
number, participant number and initials. If a correction is required for an eCRF, the time 
and date stamp will track the person entering or updating eCRF data and creates an 
electronic audit trail. The Investigator is responsible for all information collected on 
participants enrolled in this study. All data collected during the course of this study must 
be reviewed and verified for completeness and accuracy by the Investigator. A copy of 
the CRF will remain at the Investigator’s site at the completion of the study. 
 
 
2.2 
Report Generation 
 
The final statistical report will describe and justify any deviations from the original 
statistical plan described herein.  If necessary, such deviations will be treated as a 
protocol amendment. 
 
The final statistical report will be accompanied by a description of the data cleaning 
process and will provide summaries of key findings. All programs used to produce the 
statistical reports will be documented, validated, and archived. Statistical analyses will be 
generated by the OPTIMIZE DCC.     
 
The statistical reports will be prepared using current versions of SAS, R and/or S-PLUS 
statistical software.  All statistical tests are two-sided and will be evaluated at a 0.05 level 
of significance unless otherwise specified. 
 
2.3 
Definition of the Analysis Populations 
Intent-to-Treat Population 
The intent-to-treat population is defined as all randomized participants. Participants who 
are discontinued from study drug early are encouraged to complete all remaining study 
visits and will remain in the analysis population according to ITT. 
 
Per Protocol Population 
The per-protocol population is defined as all randomized participants who have 
completed ≥ 80% of expected doses of azithromycin or placebo, were not determined to 


 
 
be ineligible, did not incur any protocol violations, or who at any point in the study 
received IV anti-pseudomonal therapy in the absence of accompanying symptoms (as 
discouraged in the protocol).   Additionally, the per-protocol population will be analyzed 
according to the treatment they received (not how they were randomized).  
 
All analyses included in this report will be performed using the intent-to-treat (ITT) 
population unless otherwise specified. The primary efficacy analyses evaluating time to 
protocol-defined pulmonary exacerbation, and key secondary efficacy endpoints 
evaluating time to Pa recurrence and changes in inflammatory markers, will also be 
performed using the per-protocol population (see sections 4, 5 and 6, respectively).  
 
 
Missing Data  
Site training, automatic queries in the electronic data capture system, and thorough 
clinical site monitoring will be performed in order to minimize the occurrence of missing 
data. Participants who discontinue treatment will be encouraged to remain in the study 
while completing all study procedures. Details regarding the handling of missing data for 
analyses evaluating the primary efficacy endpoint, time to pulmonary exacerbation, and 
for analyses of the key secondary efficacy endpoints evaluating time to Pa recurrence and 
change in inflammatory markers will be provided in each of their respective sections of 
the statistical analysis plan (Section 4, 5 and 6).  Observed case analyses will be 
performed for all other secondary and exploratory endpoints. 
 
2.4 
Definitions 
 
Baseline – Baseline refers to the first study visit at which randomization occurs. 
 
Study drug – Study drug refers to the drug the participant was randomized to (either 
azithromycin or placebo) 


 
 
1.  Summary of Enrollment  
The cumulative enrollment of participants entering the study is graphically presented. 
The number of participants screened, eligible, randomized, treated, and withdrawn post-
Baseline are summarized by both treatment arm and site.  Reasons for screen failure 
are summarized. Screen failures are participants that signed informed consent but did 
not meet eligibility criteria or chose not to participate in the study. The number of 
participants who withdrew early from the study are tabulated by treatment arm and 
reason for withdrawal is summarized. 
2.  Summary of Participant Demographics and Baseline Characteristics 
Participant demographics and clinical characteristics at baseline are descriptively 
summarized by treatment arm and overall.   Characteristics summarized are gender, 
age, race, genotype (homozygous, heterozygous, other), FEV1, height, weight, and 
sweat chloride. P. aeruginosa (Pa) eligibility status (first lifetime Pa culture or history of 
Pa) as well as Pa and Staphylococcus aureus (SA) status at baseline are also 
summarized by treatment arm and overall. The number and percent of participants using 
either a nebulizer or a podhaler for administration of tobramycin at baseline is also 
summarized, in addition to season of enrollment (winter, spring, summer and fall). The 
number of participants using hypertonic saline and pulmozyme within thirty days of 
baseline is also summarized. Fisher Exact tests are used to test at a two-sided 0.05 
level of significance for differences in categorical variables across treatment arms. A 
two-sided t-test assuming unequal variance is used to test the difference between 
treatment arms for all continuous variables at a two-sided 0.05 level of significance. 
3.  Summary of Protocol Adherence and Per Protocol Population Definition 
The number and percentage of participants who completed each visit is summarized by 
treatment arm. A study visit is considered completed if a participant completed any study 
related procedures.  
Follow-up time is summarized as well as the average length of follow-up time for each 
participant. Follow-up time is defined as time between baseline and the last study visit 
(either end of study Visit 8 [Week 78] or withdrawal date).  
Study drug (azithromycin/placebo) could either be stopped permanently or temporarily at 
any time at the discretion of the subject, site investigator or the Sponsor. If a participant 
stopped study drug permanently, they were encouraged to remain in the study and 
complete all remaining study visits. If a participant experienced moderate to severe 
intolerance of study drug, dosage adjustments could be made at the discretion of the 
investigator. The number and proportion of participants temporarily or permanently 
stopping study drug at the discretion of the site investigator are summarized, as well as 
the reason for study drug stoppage. The number and proportion of participants modifying 
study drug dosage frequency, at the discretion of the site investigator, is also 
summarized as well as reasons. The total number and proportion of participants with 


 
 
physician initiated dose modifications is summarized and the difference in proportions 
between treatment arms is reported. A corresponding 95% confidence interval is 
calculated using Newcombe-Wilson method and p-values are from a Fisher’s exact test. 
Study drug compliance is measured based on participant self-report. Participants were 
instructed to maintain a daily diary in which they recorded doses taken. The diaries were 
reviewed at each study visit by the site Research Coordinator (RC) and any deviation 
from the expected dose will be discussed with the participant and reason for deviation 
noted. To determine compliance, the total number of expected doses for each participant 
in the study is calculated. Per protocol, 3 doses of study drug are to be taken each week 
until the end of the study. For participants who completed Visit 8, the number of 
expected doses is calculated as the number of full weeks between the first dose of study 
drug and Visit 8 multiplied by 3. For participants who withdrew from the study prior to 
Visit 8, the number of expected doses is based on the expected time on study per 
protocol (78 weeks); therefore the number of expected doses is 234. Study drug 
compliance is calculated as: (Number of doses reported taken / Number of expected 
doses) x 100. Compliance is not dependent on the number of bottles dispensed or 
returned. Study drug compliance is summarized by treatment arm and overall as well as 
the number and percentage of participants with ≥ 80% compliance.  
Adherence to the protocol as it pertains to inhaled tobramycin use throughout the trial is 
also summarized. All participants should have initiated at least a first course of inhaled 
tobramycin no more than fourteen days prior to Baseline. Subsequently, if a culture 
resulted in a Pa positive result at Visit 2 (Week 3) or any of Visits 3-8 (quarterly visits), 
an inhaled tobramycin course would be initiated. If the study visit culture resulted in a Pa 
negative result, no inhaled tobramycin would be initiated unless indicated clinically for 
symptoms. Additionally, at the discretion of the primary care clinician, treatment with TIS 
may be revised to a 28-day on/28-day off regimen if the Pa infection is persistent. A 
persistent Pa infection is defined as any two respiratory cultures collected at a study 
visits, positive for Pa from Visit 3 onward. Sites were encouraged to prescribe TIS, 
however, a participant could choose to use the TOBI podhaler (TIP) or other 
commercially available nebulized tobramycin formulations in place of TOBI. The total 
number and percent of participants initiating the first course of inhaled tobramycin and 
the time from Baseline to initiating of the first course is summarized. The total number of 
subsequent Pa positive quarters is summarized as well as the number of Pa positive 
quarters for which inhaled antibiotics were initiated and the type of inhaled antibiotic 
used (TIS, TIP, other inhaled tobramycin, other inhaled antibiotic). Finally, the number of 
subsequent Pa negative quarters is summarized as well as the number of Pa negative 
quarters for which inhaled antibiotics were initiated. A quarter is considered Pa positive 
when at least one Pa culture was positive at or between quarterly visits.  
A listing of all protocol violations is provided. The number of participants included in the 
per-protocol population is provided by treatment arm as well as reasons for exclusion. 
The per-protocol population is defined as all randomized participants who have 
completed ≥ 80% of expected doses of azithromycin or placebo, were not determined to 
be ineligible, did not incur any protocol violations, or who at any point in the study 


 
 
received IV anti-pseudomonal therapy in the absence of accompanying symptoms (as 
discouraged in the protocol).  The per-protocol population  will be analyzed according to 
the treatment they received (not how they were randomized). 
4.  Summary of Pulmonary Exacerbations 
The primary endpoint is time to a protocol-defined pulmonary exacerbation. In this study, 
we used an a priori definition for exacerbation based on signs and symptoms 
characterized as major criteria or minor criteria (protocol Appendix II).  Implementation of 
this definition was consistent with its definition in the randomized, placebo controlled trial 
of azithromycin in pediatric patients with CF culture negative for Pa (1, 2). At the 
initiation of each new antibiotic, site investigators completed an electronic case report 
form and indicated the presence or absence of signs and symptoms comprising the PE 
definition that prompted antibiotic treatment. However, the decision to utilize antibiotics 
was not dependent on whether or not the protocol definition for exacerbation was met.   
 
Time to exacerbation is presented using Kaplan-Meier survival estimation and is 
compared between treatment groups using a Cox proportional hazards model with 
effects for treatment group and randomization strata (≥6 months – 3 years, ≥3 -6 years, 
≥6 – 12 years, ≥12 years), with a test of the treatment effect at a two-sided significance 
level of 0.05.  Participants who withdraw from the study early will be censored at the 
date of last contact. The results from the model, including the hazard ratio and its 95% 
confidence interval are presented using both the ITT population and the per-protocol 
population.  
 
Several sensitivity analyses will also be performed to assess the robustness of study 
results as outlined in the study protocol.  The first sensitivity analysis will utilize a 
composite endpoint of time to pulmonary exacerbation or repeated use of acute 
antibiotics (at least 2 acute antibiotic courses prescribed that do not meet the PE 
definition) (3). Furthermore, the potential influence of acute respiratory events not 
meeting the primary endpoint definition will also be assessed by censoring participants 
with acute antibiotic usage prior to meeting the primary endpoint. To investigate the 
impact of missing data, results from a model will be presented using only participants 
with complete follow up data. A model will also be presented assuming any censored 
participant experienced a pulmonary exacerbation at the time of withdrawal or loss to 
follow-up. Hazard ratios and 95% confidence intervals from each model adjusting for 
treatment group and randomization strata are presented.  
Exploratory subgroup analyses of the primary endpoint will also be performed.  
Estimates of the hazard ratio and associated 95% confidence interval along with p-
values for the primary endpoint will be reported by subgroups defined by pre-specified 
demographic and disease specific baseline characteristics of interest. No adjustments 
for multiple comparisons will be made. Additional exploratory analyses include 
summarizing the frequency of signs/symptoms presented during all pulmonary 
exacerbations events as well as comparing treatment groups with respect to the average 


 
 
number of PEs. The results from an Anderson-Gill Cox Model for the mean number of 
exacerbations with effects for randomization strata and treatment group is also 
presented with corresponding hazard ratio and 95% confidence interval. 
Additional exploratory analyses will be performed utilizing alternative definitions of the 
PE endpoint incorporating additional data collection during the study. In particular, 
additional signs and symptom data were collected at scheduled study visits independent 
of antibiotic use for exploratory purposes and will be used to investigate frequency of 
signs and symptoms consistent with the PE definition regardless of antibiotic initiation 
(defined a “significant respiratory event”) as well as the frequency of physician defined 
(rather than protocol defined) exacerbation events.   
 
 
References 
 
1. Saiman L, Anstead M, Mayer-Hamblett N, Lands LC, Kloster M, Hocevar-Trnka J, 
Goss CH, Rose LM, Burns JL, Marshall BC, Ratjen F; AZ0004 Azithromycin Study 
Group. Effect of azithromycin on pulmonary function in patients with cystic fibrosis 
uninfected with Pseudomonas aeruginosa: a randomized controlled trial. JAMA. 
2010 May 5;303(17):1707-15. 
2. Anstead M, Saiman L, Mayer-Hamblett N, Lands LC, Kloster M, Goss CH, Rose LM, 
Burns JL, Marshall BC, Ratjen F. Pulmonary Exacerbations in CF Patients with Early 
Lung Disease. Journal of Cystic Fibrosis. 2014 Jan;13(1):74-9. Epub 2013 Sep 10. 
PMID: 24029220. 
3. Mayer-Hamblett N, Saiman L, Lands LC, Rosenfeld M, Kloster M, Fisher L, Ratjen F.  
Impact of acute antibiotic therapy on the pulmonary exacerbation endpoint in cystic 
fibrosis clinical trials. Contemporary Clinical Trials.  2013 Sep;36(1):99-105. doi: 
10.1016/j.cct.2013.06.004. Epub 2013 Jun 14. PMID:23770109. 
5.  Summary of Pseudomonas aeruginosa (Pa) Endpoints 
Respiratory culture results are obtained at scheduled study visits with additional cultures 
obtained between visits only if clinically necessary. Analyses include only cultures 
obtained during scheduled study visits unless otherwise specified. Respiratory cultures 
could be collected via oropharyngeal (OP) swab, sputum (expectorated or induced) or 
bronchoalveolar lavage (BAL). In the event that more than one culture is obtained for the 
same visit from the same participant, a positive result for Pa in any of the specimens 
designates the participant positive for Pa at that visit. The number and percent of 
cultures obtained and by which method is summarized by visit and treatment arm. 
A key secondary efficacy endpoint is time to Pa recurrence after initial treatment with 
anti-pseudomonal therapy received during the first quarter of the study. Time to Pa 
recurrence from weeks 13 through 78 is presented using Kaplan-Meier survival 
estimation and compared between treatment arms using a Cox proportional hazards 
model with effects for treatment and age group (≥6 months – 3 years, ≥3 – 6 years, ≥6 – 
12 years, ≥12 years), with a test of the treatment effect at a two-sided significance level 


 
 
of 0.05. The results from the model, including hazard ratios and 95% confidence 
intervals, are presented. This main model will be performed for both the ITT population 
and the PP population (see Introduction). In the event that there are missing culture 
results at a scheduled study visit for a participant, the following imputation method will be 
used. If the participant has culture results available from the quarter directly following the 
missing culture quarter, the result of that culture will be used for the preceding missing 
quarter. If there are two or more missing quarters, the participant will be assumed to be 
a microbiologic “failure” and assumed positive for Pa. Further, a sensitivity model of time 
to Pa recurrence will be performed including culture results obtained both at acute study 
visits and scheduled study visits.  
Exploratory subgroup analyses of the time to Pa recurrence endpoint will also be 
performed. Estimates of the hazard ratio and associated 95% confidence interval along 
with p-values for the primary endpoint will be reported by subgroups defined by pre-
specified demographic and disease specific baseline characteristics of interest. No 
adjustments for multiple comparisons will be made.  
The proportion of Pa-positive respiratory cultures among all respiratory cultures taken at 
scheduled study visits from weeks 13 through 78 is also modeled. The response is 
binary (positive or negative culture) at each visit. A generalized estimating equation 
(GEE) model using a logit link is used to model this data with robust variance estimation 
and an independence working correlation matrix, including effects for both treatment and 
age group (≥6 months – 3 years, ≥3 – 6 years, ≥6 – 12 years, ≥12 years) in addition to 
the number of available cultures at scheduled study visits over the 18 month study 
period. The significance of the treatment arm variable is tested using a two-sided 0.05 
level of significance. The treatment associated odds ratio from this model and 
corresponding 95% confidence interval is the measure of treatment effect. The 
estimated treatment effect is interpreted as the marginal odds ratio of Pa positive 
respiratory cultures during the 18 months. A sensitivity model will also be performed 
including culture results obtained both at acute study visits and scheduled study visits.  
The proportion of participants with Pa positive cultures is summarized by visit and 
treatment. Differences and 95% confidence intervals are reported for each visit.  
The proportion of participants who achieved initial eradication at week 3 and after the 
first quarter of treatment at week 13 is summarized and compared between treatment 
groups, and additional summaries are provided for the proportion who were Pa positive 
at week 3 but were negative by week 13.The proportion of patients with at least one Pa 
positive culture result after the first quarter of treatment (on or after week 13) is also 
summarized for each treatment arm with the difference between treatment arms tested 
using a 0.05 level of significance Chi-square test. The number of participants with 
persistent Pa infection at some point during the trial is summarized. Persistence is 
defined in the protocol as any two respiratory cultures collected at study visits, positive 
for Pa from Week 13 onward. The difference between treatment arms is tested using a 
two-sided 0.05 level Chi-square test. Additionally, among those who turned persistent, 


 
 
the inhaled tobramycin formulation used and frequency of treatment (i.e. 28 days on/28 
days off, quarterly) after persistent infection is summarized. 
Time to persistent Pa infection is presented using Kaplan-Meier survival estimation and 
compared between treatment arms using a Cox proportional hazards model with effects 
for treatment and age group (≥6 months – 3 years, ≥3 – 6 years, ≥6 – 12 years, ≥12 
years), with a test of the treatment effect at a two-sided significance level of 0.05. The 
results from the model, including hazard ratios and 95% confidence intervals, are 
presented. 
Exploratory analyses of the frequency of mucoid Pa cultures were also performed. Pa 
mucoid status at each visit and the change for each visit from baseline is summarized by 
treatment arm. Pa resistance to aminoglycosides, quinolones and β-lactams were also 
tested when indicated clinically. The number and proportion of participants with at least 
one emergent drug-resistant Pa culture post-baseline is summarized by treatment arm 
for each drug class among those participants with at least one result available for 
testing. Differences and 95% confidence intervals are presented.  
A multivariable logistic regression model using stepwise regression is developed to 
identify key baseline predictors associated with initial eradication failure, determined 
using Pa status at Week 13. Baseline demographic, clinical, microbiologic and 
inflammatory variables will be assessed for inclusion of the model. Cross-validation 
techniques are used to determine a model. The model coefficients and standard errors 
(SE) are presented as well as corresponding 95% confidence intervals and the p-values. 
The positive predictive value, negative predictive value, sensitivity and specificity of the 
model are presented.Summary of Pseudomonas aeruginosa (Pa) Endpoints 
6.  Summary of Inflammatory Markers 
Serum inflammatory markers (myeloperoxidase (MPO), C-reactive Protein (CRP) and 
calprotectin) are transformed using log10 and summarized at each visit (Baseline, Week 
39 and Week 78) by treatment arm.  For values below the limit of detection, ½ the lower 
limit of detection is used. For values greater than the limit of detection, the upper limit of 
detection is used. 
Change from baseline to end of study in each inflammatory marker is a key secondary 
efficacy endpoint.  Linear regression will be used to estimate the difference between 
treatment groups in the 78 week change from baseline in each inflammatory marker, 
adjusted for randomization strata. These analyses will be performed on both the ITT and 
PP population. Additional sensitivity analyses will be performed to address missing data. 
Specifically, the least favorable treatment arm imputation method will be used which 
imputes that missing value with the mean change from the treatment arm with the worst 
change in the observed case analysis.   


 
 
Observed changes from baseline at Weeks 39 and 78 are also descriptively summarized 
by treatment arm. The change from baseline in inflammatory markers at Weeks 39 and 
78 are also graphed for each treatment arm along with 95% confidence intervals. 
7.  Summary of Adverse Events 
Treatment emergent adverse events (AEs) and serious adverse events (SAEs) will be 
comprehensively summarized. Treatment emergent adverse events are those events 
that occurred on or after Baseline as reported by the participant. The safety population is 
used in all summaries of SAEs and AEs. Note that all tables summarizing AEs also 
include SAEs (which are a subset of the AEs) unless otherwise specified.  
Included in the summaries are tables displaying: 1) the proportion of participants with at 
least one (S)AE, 2) the average number of (S)AEs per patient, and 3) the rate of (S)AEs 
per participant-month of follow-up. Poisson regression is used to compare the rate of 
(S)AEs by treatment arm. Rate ratios and corresponding 95% confidence intervals and 
p-values are reported. The difference in the proportion of participants experiencing at 
least one (S)AE between treatment arms is also reported, with  corresponding 95% 
confidence intervals  calculated using the Newcombe-Wilson method and p-values 
derived  from a Fisher’s exact test.  
A histogram showing the number of (S)AEs per participant in each treatment arm is 
displayed. Detailed summary tables of individual (S)AEs grouped by SOC will be 
included to show the number and percent of participants experiencing each (S)AE by 
treatment arm. The number and rate of events for each (S)AE is also reported by 
treatment arm. Poisson regression is used to derive rate ratios and corresponding 95% 
confidence intervals comparing treatment arms for each SOC. Treatment arms will be 
compared by plotting rates of (S)AEs and the proportion of participants experiencing 
(S)AEs by System Organ Class (SOC) as well as the rate ratio and difference in 
proportions with corresponding 95% confidence intervals to compare treatment arms. 
The most frequently occurring adverse events, defined as those events occurring in ≥ 
5% of participants, will be separately summarized by SOC and treatment arm.  
The severity of AEs will be tabulated by SOC and treatment arm according to whether 
the AE was mild, moderate, severe or life-threatening. The number and proportion of 
participants experiencing at least one AE classified as severe or worse is summarized 
by treatment arm. The difference in the proportion of participants experiencing at least 
one severe AE or worse between treatment arms is reported. Corresponding 95% 
confidence intervals calculated using Newcombe-Wilson method and p-values derived 
from a Fisher’s exact test. The proportion of participants experiencing AEs and rates of 
AEs will be summarized by the relation to study drug (unrelated, possibly, probably, 
definitely) by treatment arm.  Finally, the rate of AEs will be plotted by site.  
Both sensorineural hearing loss and protocol-defined abnormal ECG results, defined a 
priori as key safety endpoints, will be reported as SAEs and therefore will be included in 


 
 
SAE and AE tables. All SAE narratives will also be included in a listing appended to this 
report. 
8.  Safety Laboratory Parameters 
Blood was drawn for serum chemistry and hematology at Baseline, Visit 5 (Week 39) 
Visit 8 (Week 78) and at an Early Withdrawal Visit (if participant withdrew early). In the 
case that a participant had labs drawn at an Early Withdrawal Visit, those labs are 
included in the summaries for the visit that should have occurred closest to and after 
their early termination visit.  
Average lab values at each time point and average changes from Baseline are 
summarized by treatment arm. The difference between treatment arms in the mean 
change from Baseline to each visit are displayed with corresponding 95% confidence 
interval. If a participant did not have labs at a specified time point, they are excluded 
from that particular measure.  
The number and percent of participants with lab measures below (low), within (normal) 
and above (high) the normal range at each visit and for each lab are displayed by 
treatment arm. Each site used their own standard normal range, therefore, normal 
ranges for each measure may vary by site. For each abnormal lab value (low or high), 
the site PI determined if the abnormal lab was considered clinically significant or not. 
Thus, clinically significant lab values are a subset of the abnormal values. The number 
and percent of lab values deemed to be clinically significant are summarized by visit and 
treatment arm. Changes in abnormal and clinically significant lab values from Baseline at 
Visits 5 and 8 are summarized as emergent high or low. For example, a lab value is 
determined to be emergent low at a visit if the lab value was not low at Baseline but low 
at the visit . Emergent high values and emergent clinically significant low and high values 
are similarly categorized. The difference in proportions between treatment arms of 
participants with emergent clinically significant lab measures at Visit 5 and Visit 8 are 
displayed with corresponding 95% confidence interval. 
9.  Summary of Hospitalizations and Antibiotic Usage 
Descriptive statistics are used to summarize the percent of participants hospitalized (all 
cause) by treatment arm, including the difference between treatment groups with 
associated 95% confidence interval calculated using the Newcombe Wilson method. 
Differences between treatment groups with respect to the proportion of participants 
hospitalized is assessed using a logistic regression model adjusting for treatment and 
age category (≥6 months – 3 years, ≥3 – 6 years, ≥6 – 12 years, ≥12 years). The Odds 
ratio and 95% confidence interval from the model are presented along with a p-value. 
The rate of hospitalizations per month is also descriptively summarized and compared 
using Poisson regression, including follow-up time as an offset, with an associated rate 
ratio and 95% confidence interval 
 


 
 
Similarly, the percent of participants initiating oral, IV, and inhaled antibiotics after 
Baseline and over the follow up period and the number of days of oral and IV antibiotic 
use are descriptively summarized by treatment arm. Differences in proportions and the 
number of days of usage are summarized with associated 95% confidence intervals. The 
proportion of participants initiating oral, IV and inhaled antibiotics are compared using 
logistic regression modes adjusting for treatment arm and age category (≥6 months – 3 
years, ≥3 – 6 years, ≥6 – 12 years, ≥12 years ). The Odds ratios and 95% confidence 
intervals from the models are presented along with associated p-values. 
10.  Summary of Non-Pseudomonal Microbiologic Endpoints 
Respiratory culture results are obtained at each study visit with additional cultures 
obtained between study visits only if clinically necessary.  . Analyses include only 
cultures obtained at scheduled study visits. The respiratory culture could be collected via 
oropharyngeal (OP) swab, sputum (expectorated or induced) or bronchoalveolar lavage 
(BAL). In the event that more than one specimen is obtained for the same visit from the 
same participant, a positive result for an organism in any of the specimens designates 
the participant positive for that organism at that visit.  The number and percent of 
cultures obtained and by which method is summarized by visit and treatment arm. 
Non-pseudomonal microbiologic organism culture results (Staphylococcus aureus 
[methicillin susceptible and resistant], Haemophilus influenzae, Burkholderia cepacia 
complex, Stenotrophomonas maltophilia, and Alcaligenes (Achromobacter) 
xylosoxidans) are summarized for each visit and the change for each visit from baseline 
by treatment arm  Differences and 95% confidence intervals are presented. For each 
organism, the treatment emergent prevalence is summarized by treatment arm. 
Differences and 95% confidence intervals are presented as well as p-values from a Chi-
square test. NTM results are available only when sputum was produced at study visits in 
a quantity that allows for testing. The number and percent of participants who were able 
to produce sputum at any visit post-baseline is summarized as well as the number and 
percent of participants with an NTM positive result by treatment arm. The difference and 
95% confidence interval is presented Note that a positive NTM result triggered 
permanent study drug discontinuation for that particular participant although they were 
encouraged to stay in the study for full follow-up. 
11.  Spirometry Results 
Spirometry was to be performed at Baseline for all participants 4 years and older who 
could reproducibly perform spirometry. Baseline clinical and demographic characteristics 
are summarized among  the subset of participants with spirometry values available at 
the Baseline visit. All spirometry tables, figures and models include those participants 
with a Baseline measurement. FEV1 (liters and percent predicted), FVC and FEF25%-75% 
are descriptively summarized for each study visit and change (absolute and relative) 
from Baseline by treatment. The difference between treatment arms in the change from 
Baseline at each visit is presented along with 95% confidence intervals. FEV1 percent 


 
 
predicted is calculated using the Wang (1) equations (Female ≤ 16 years, Male ≤ 18 
years) or Hankinson (2)  equations (Female > 16 years, Male > 18 years) at each visit.   
Participants crossing age thresholds for a particular equation (e.g. from Wang to 
Hankinson) during the study remained on the same reference equations determined at 
Baseline to maintain continuity.  Relative change is defined as: [(Visit Measure – 
Baseline Measure) x100]/Baseline Measure. The mean absolute and relative change 
from Baseline are plotted by treatment for each spirometry measure with 95% 
confidence intervals at each time point.  
For each spirometry measure, a repeated measures linear regression model is used to 
model change over time among the subset of participants with spirometry at Baseline. 
The model adjusts for  age group (≥6 months – 3 years, ≥3 – 6 years, ≥6 – 12 years, ≥12 
years), time (days) and a time by treatment interaction. A generalized estimating 
equation framework with an independence working correlation matrix is utilized to 
calculate robust variance estimates. The coefficients of the model, along with 
corresponding standard error (SE), 95% confidence intervals (CI) and p-values are 
displayed. These models are utilized to estimate the 18-month change from Baseline 
with corresponding 95% CIs and p-values for each measure. 
References: 
 
1. Wang X, Dockery DW, Wypij D, Fay ME, Ferris BG, Jr. Pulmonary function 
between 6 and 18 years of age. Pediatr Pulmonol 1993; 15(2): 75-88. 
2. Hankinson JL, Odencrantz JR, Fedan KB. Spirometric reference values from a 
sample of the general U.S. population. Am J Respir Crit Care Med 1999; 159(1): 
179-87. 
12.  Anthropometric Results 
Height/length and weight were collected at each study visit. Height/length, height/length 
percentile, weight and weight percentile are descriptively summarized for each study 
visit and change (absolute and relative) from Baseline by treatment arm. WHO reference 
tables (1) were used to determine percentiles for participants < 2 years old at Baseline. 
CDC reference tables (2) were used to determine percentiles for participants ≥ 2 years 
old at Baseline. Participants crossing age thresholds for a particular equation (e.g. from 
WHO to CDC) during the study remained on the same reference equations determined 
at Baseline to maintain continuity. The difference between treatment arms in the change 
from Baseline at each visit is presented along with 95% confidence intervals. The mean 
absolute change from Baseline is plotted by treatment for each measure with 95% 
confidence intervals at each time point.  
For each anthropometric measure, a repeated measures linear regression is to model 
change over time among the subset of participants with spirometry at Baseline. The 
model adjusts for the age randomization factor (age group: ≤3 years, >3-6 years, ≥6-12 
years, >12-18 years), continuous time (in days) and a treatment by time interaction.  A 
generalized estimating equation framework with an independence working correlation 


 
 
matrix is utilized to calculate robust variance estimates. The coefficients of the model, 
along with corresponding standard error (SE), 95% confidence intervals (CI) and p-
values are displayed. These models are utilized to estimate the 18-month change from 
Baseline with corresponding 95% CIs and p-values for each measure. 
 
References 
 
1. WHO, Apr 2006. Child Growth Standards based on length/ehight,weight and age. 
Acta Paedreatr Suppl Apr 2006; 450:76-85 
2. Kuczmarski, Robert J., Cynthia L. Ogden, Laurence M. Grummer-Strawn, Katherine 
M. Flegal, Shumei S. Guo, Rong Wei, Zuguo Mei, Lester R. Curtin, Alex F. Roche, 
and Clifford L. Johnson. "CDC growth charts: United States." Advance data 314 
(2000): 1-27. 
13.  Summary of the Cystic Fibrosis Respiratory Symptoms Diary (CFRSD) 
The Cystic Fibrosis Respiratory Symptoms Diary (CFRSD) and the parent-completed 
CFRSD will be completed at each study visit for participants ≥12 years and <11 years, 
respectively.  
The CFRSD asks a participant (or parent) to state the extent of their 8 respiratory 
symptoms: difficulty breathing, felt feverish, felt tired, had chills or sweats, coughed, 
coughed up mucus, had tightness in the chest, or wheezed. If a symptom is present, 
they are asked to rate the severity of the symptom using a 4 point scale with possible 
answers that include “a little”, “somewhat”, “a good deal”, “a great deal” or “slightly”, 
“moderately”, “very”, “extremely”.  Each respiratory symptom is assigned a score from 0-
4 based on the response (0 in the absence of symptom). For each participant, a 
summed score is then calculated, ranging from 0 to 24. In turn, this score is converted to 
a 0 to 100 scale. This composite score is referred to as the CFRSD-CRISS.  
The CFRSD-CRISS is summarized at each visit and changes from Baseline by 
treatment. The difference between treatment arms in the change from Baseline for the 
CFRSD-CRISS at each visit are presented along with 95% confidence intervals. 
Summaries at each visit include scores from participants who responded to at least 7 of 
the respiratory questions. 
A repeated measures linear regression model was used to model changes in the 
CFRSD-CRISS over time. The model adjusts for  age group (≥6 months – 3 years, ≥3 – 
6 years, ≥6 – 12 years, ≥12 years), continuous time and a time by treatment interaction. 
A generalized estimating equation framework with an independence working correlation 
matrix is utilized to calculate robust variance estimates. The coefficients of the model, 
along with corresponding standard error (SE), 95% confidence intervals (CI) and p-
values are displayed. These models are utilized to estimate the 18-month change from 
Baseline with corresponding 95% CIs and p-values for the CFRSD-CRISS. 


 
 
The severity of symptoms from the CFRSD at Baseline and Visit 8 (Week 72) are 
summarized by treatment as well as change in severity of symptoms from Baseline. The 
proportion of participants experiencing symptoms at Baseline and Visit 8 are 
summarized by treatment arm as well as differences between treatment arms with 
corresponding 95% confidence intervals.  The proportion of participants with a decrease 
and increase in symptom severity from Baseline to Visit 8 is summarized by treatment 
arm as well as differences between treatment arms with corresponding 95% confidence 
intervals.