Phase I Assessment of Hypertonic Saline in Moderate to Severe Asthmatics
Phase: NA
Conditions: Moderate to Severe Asthma
Sponsor: University of North Carolina, Chapel Hill

 
 
 
 
 
Master Protocol 
 
 
Phase I Assessment of Hypertonic Saline in Moderate to Severe Asthmatics 
 
 
 
 
 
 
NCT number NCT03556683 
 
 
 
 
 
 
Document Date 06/08/2023 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Phase I Assessment of Hypertonic Saline in Moderate to Severe Asthmatics 
David B. Peden, MD  
Version 06/08/2023 
 
2 
 
 
 
 
 
 
 
 
 
Protocol: 
 
Complete Title:  
Phase I Assessment of Hypertonic Saline in Moderate to Severe 
Asthmatics 
 
 
Drug Name:  
Sodium Chloride 7% solution for inhalation (Hypertonic Saline); albuterol  
Protocol Date:  
June 8, 2023 
 
 
Study Principal Investigator: David B. Peden, MD, MS 
UNC Center for Environmental Medicine, Asthma and Lung Biology 
104 Mason Farm Road, CB 7310 
Chapel Hill NC 27599-7310 
Phone 919-966-0768 
email: david_peden@med.unc.edu 
 
 
 
 
 
 
 
 
 
 
 

Phase I Assessment of Hypertonic Saline in Moderate to Severe Asthmatics 
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ABBREVIATIONS AND DEFINITIONS OF TERMS 
 
Abbreviation 
Definition 
AE 
Adverse Events 
AED 
Automated External Defibrillator  
ATS 
American Thoracic Society 
BP 
Blood Pressure 
CEMALB 
Center for Environmental Medicine, Asthma and Lung Biology 
CF 
Cystic Fibrosis 
Co57 
Cobalt 57 
COPD 
Chronic Obstructive Pulmonary Disease 
Covid 
Sars CoV 2 virus/infection  
CTCAE 
Common Terminology Criteria for Adverse Events 
EMS 
Emergency Medical Services 
FVC 
Forced Vital Capacity 
FEF25-75 
Maximal Mid-Expiratory Flow Rate 
FEV1 
Forced Vital Capacity in one second 
IUD 
Intra Uterine Device 
HR 
Heart Rate 
HS 
Hypertonic Saline 
KeV 
Kilo electron volts 
LABA 
Long Acting Bronchodilator Agent 
LAIV 
Live Attenuated Influenza Vaccine 
MMAD 
Mass Mean Aerodynamic Diameter 
MCC 
Mucociliary Clearance 
mCi 
Millicurie 
NaCl 
Sodium Chloride 
NHLBI 
National Heart, Lung and Blood Institute 
NIH 
National Institute of Health 
NSAIDS 
Non-steroidal anti-inflammatory drugs 
PE 
Physical Examination 
PEF 
Peak Expiratory Flow 
PHI 
Personal Health Information 
PPE 
Personal Protective Equipment 
RR 
Respiratory Rate 
Tc99m-SC 
Sulfur colloid 
uCi 
Microcurie  
UP 
Unanticipated Problems 

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PROTOCOL SYNOPSIS 
Study Title 
Phase I Assessment of Hypertonic Saline in Moderate to Severe Asthmatics 
Funder 
NIH 
Clinical Phase 
Phase I 
Study Rationale 
Mucociliary clearance (MCC) is the process by which mucus is propelled from 
the lung toward the upper airway.  Studies at our Center and others have 
demonstrated that hypertonic saline (HS) increases the volume of airway 
surface liquid, improves rheologic properties of mucus, and accelerates 
mucus transport rates in vitro. Inhaled 7% HS enhances MCC in healthy 
volunteers as well as those with muco-obstructive diseases like cystic fibrosis 
and chronic bronchitis.  Improvements in MCC were also associated with 
improved lung function in CF. Use of HS as a treatment for asthma has been 
limited. Airway mucus plugging is a key feature of asthma and is responsible 
for much of the morbidity and mortality associated with the disease. We will 
explore the efficacy of albuterol alone and 7% HS with albuterol, compared to 
baseline for improving MCC in moderate to severe asthmatics.  
Study 
Objective(s) 
Primary Objective 
To determine the efficacy of HS for accelerating MCC acutely (immediately 
following HS) compared to baseline in well-controlled moderate to severe 
asthmatics. 
Secondary Objectives 
- 
To compare the efficacy of  albuterol alone vs 7% HS with albuteol 
pretreatment for accelerating MCC compared to baseline 
- 
To assess the safety of HS in moderate to severe asthmatics by 
measuring spirometry, vital signs, and symptom questionnaire scores. 
- 
Induced sputum samples after HS to assess for mucins and inflammatory 
endpoints 
Test Article(s) 
Sodium Chloride 7% solution for inhalation (Hereafter referred to as 
Hypertonic Saline or 7% HS) and albuterol for oral inhalation  
Study Design 
 
This is an open label proof-of-concept study.  Participants will have a baseline 
MCC assessment, followed by sputum induction.  They will return for another 
MCC assessment which will include treatment with 4 puffs of albuterol,  and 
followed by a sputum induction . For the HS visit, in order to assess for acute 
effects of HS on MCC, subjects will be treated with 4 puffs of albuterol, and 
then will receive 7% HS inhalation immediately after start of the MCC 
measurement.  Subjects will undergo sputum induction procedure after 
completion of MCC. All subjects will return the day following the HS visits for 

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medical assessment and to obtain lung images to approximate retention of 
radiolabeled particles at 24 hours.  
Subject 
Population - 
Criteria 
for 
Inclusion 
and 
Exclusion: 
Inclusion Criteria 
1. Age 18-60. 
2. Moderate to severe asthma at baseline (determined by Step 3 therapy 
or greater or by degree of impairment secondary to asthma).  Subjects 
will be Well Controlled asthma at the time of enrollment, or Not Well 
Controlled as determined by National Heart, Lung and Blood Insistute 
(NHLBI) Expert Panel Report 3 guidelines for diagnosis and treatment 
of asthma.  Subjects who meet the Not Well Controlled criteria must 
be free of wheezing, and must be at their baseline prior to any 
hypertonic saline inhalation. 
3. Subjects of childbearing potential must be non-pregnant, non-
lactating, and using an acceptable method of birth control (an IUD  
with a failure rate of <1%, hormonal contraceptives, barrier method, or 
abstinence) for the duration of participation study; if practicing 
abstinence, the subject must agree to use one of the acceptable 
methods of contraception if she becomes sexually active. 
 
Exclusion Criteria 
1. Current tobacco smokers (or >10 pack years of smoking) or e-cigarette 
users (including use within 1 year of the study) 
2. Other chronic illness, which could impact safety or study results 
3. Radiation exposure within the past year that would cause them to 
exceed Federal radiation safety regulations. 
Study Duration 
Each subject’s participation will last up to 4.5 months. 
The entire study is expected to last up to 6 years. 
Study Phases 
Screening 
Study 
Treatment 
Follow-Up   
(1) Baseline MCC:  Informed consent obtained, medical history including 
radiation exposure, vital signs and lung function assessed with 
spirometry. FEV1 must be ≥ 70% of predicted. A baseline MCC scan will 
be performed over 2 hours.  
(2) Baseline MCC with 4 puffs of albuterol:  Subjects will return at least 1 
week later for a MCC scan with 4 puffs of albuterol immediately after the 
start of the MCC measurement.  
 (3) Acute effects of HS on MCC: At least 1 week after albuterol visit, subjects 
will receive 4 puffs of albuterol and a dose of 7% HS immediately after the 
start of MCC measurement over a 2 hour period to assess the acute effects 
of HS on MCC.    Subjects will undergo sputum induction after each MCC visit.  
(4) Follow-up: Medical screening for safety endpoints, spirometry, and lung 
retention imaging will be performed the day following each treatment visit, and 
a discontinuation visit will be performed 5 to 10 days after the last study visit. 
Efficacy 
Evaluations 
To determine the efficacy of albuterol and HS with albuterol for acutely 
accelerating MCC in adult patients with moderate to severe asthma. We will 
prioritize comparison of acute effects of HS on MCC to baseline MCC 
measurements.    

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Pharmacokineti
c Evaluations 
None 
Safety 
Evaluations 
Vital signs, symptom questionnaire scores, spirometry 
Statistical 
And 
Analytic Plan 
Primary Endpoint: difference between MCC measured at baseline and MCC 
measured with albuterol compared to MCC measured during and immediately 
after 7%HS inhalation, with MCC reported as the average percent retention of 
radiolabeled particles over 120 minutes of acquiring images. We will perform 
descriptive analysis of this difference; all point estimates will be tabulated 
along with their 95% confidence intervals. As we have assumed normality of 
MCC measurements, we will test the null hypothesis of no difference between 
baseline MCC and MCC immediately post-HS, against the alternative 
hypothesis of some difference, using a paired two-sided one-sample t-test 
with a 5% type I error rate. If the observed p-value is above 0.05, the test will 
be interpreted as being inconclusive. Following this test, we will perform 
sensitivity analyses to assess how realistic the assumption of normality was, 
and how much it may have impacted our conclusions. In particular, we will 
examine normal q-q plots and other descriptive tabulations, and we will 
examine the results of a Wilcoxon Signed Rank test. Should extreme or 
questionable observations arise in the study, we will also examine regression 
diagnostics to assess the influence of how those data were handled.  
 
Secondary Endpoints: difference between albuterol  and 7% HS effect on 
MCC and mucins/neutrophils in induced sputum post treatment.   Finally, we 
will report the proportion of enrolled patients who do not tolerate HS at 
baseline, along with the 95% confidence interval of this proportion.  
DATA 
AND 
SAFETY 
MONITORING 
PLAN 
A study coordinator will enter data into REDCap, and then a second person 
will verify the data.  Subjects will be monitored in real time for safety by study 
staff. A study physician will be immediately available during inhalation 
procedures and will determine the severity and relatedness of any adverse 
events (AEs). The NC TraCS Data Safety Monitoring Board (DSMB) will be 
chartered to set data monitoring and reporting requirements, in collaboration 
with the PI and study biostatistician. The DSMB will address issues regarding 
safety concerns, efficacy concerns, termination of the trial due to pre-specified 
stopping criteria, and ethical concerns. The DSMB will be furnished with 
relevant information by the Principal Investigator to make these decisions. The 
DSMB will review after 5 subjects have completed study procedures or at 6 
months after enrollment begins, whichever is soonest, and then every 6 
months thereafter. 
 
1 
BACKGROUND AND RATIONALE 
 
1.1 
Introduction 
Asthma is a chronic, inflammatory disease of the lungs.  It is the most commonly 
encountered respiratory disease in children and adults in the United States, and is a 
leading cause of morbidity worldwide. Asthma exacerbations account for a significant 
proportion of healthcare costs associated with asthma. Viral infection, allergen, and 
pollutant exposures are the most common triggers for asthma exacerbation, which is 
characterized by airway inflammation, mucosal edema, contraction of airway smooth 

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muscle, and increased production of airway mucus. Airway dysfunction leads to reduced 
mucociliary clearance (MCC), which contributes to airway mucus plugging and further 
deterioration of lung function. Mucus plugging is a feature associated with fatal asthma (1-
3).  
 
Pulmonary MCC is dependent on airway secretory cells and submucosal glands that 
produce a mucin-rich fluid layer on the airway surface and ciliated cells that hydrate and 
propel mucus out of the lung and into the upper airway. Rates of MCC are dependent on 
ciliary beat frequency, hydration, and the rheologic properties of mucus. In vitro studies 
have demonstrated that HS, through an osmotic effect on airway surfaces, improved 
hydration and mucus rheologic properties, and accelerated mucus transport rates. Inhaled 
HS has also been shown to produce an acute acceleration of MCC (4-6), suggesting 
potential benefit in the context of increased mucus production and plug formation 
associated with asthma.  
 
MCC is estimated in vivo through the use of gamma scintigraphy. A gamma camera is an 
imaging technique used to map functions and processes within the body by detecting 
radiation from a tracer introduced into the volunteer’s body. The most commonly used 
tracer is technetium-99m (Tc99m). Briefly, volunteers inhale aerosolized radiolabeled 
sulfur colloid (Tc99m-SC) then sit in front of a gamma camera, which detects emitted 
radiation, for a period of 120 minutes. During this time, technicians measure an average 
percent retention of radiolabeled particles in the lung. This is used to estimate the 
proportion of particles that are cleared from the lung per unit time, also referred to as MCC. 
 
1.2 
Name and Description of Investigational Product or Intervention  
• 
Albuterol sulfate for oral  inhalation  
• 
Sodium Chloride (NaCl) 7% solution for inhalation (Hypersal®, PARI Respiratory 
Equipment, Inc, Midlothian, VA). 
 
1.3 
Non-Clinical and Clinical Study Findings 
Studies at our Center and others have demonstrated that hypertonic saline (HS) increases 
the volume of airway surface liquid, improves rheologic properties of mucus, and 
accelerates mucus transport rates in vitro, and that inhaled HS enhances mucocilliary 
clearance (MCC) in healthy subjects with and without asthma (4-6) as well as patients with 
cystic fibrosis (7, 8, 9). The improvement in MCC with HS in CF appears dose-dependent 
based on the findings of one study, showing increasingly greater improvements in MCC 
from NaCl 0.9% up to NaCl 12% (10). In a study examining the effect of a single dose of 
nebulized HS 7% vs amiloride, HS 7% + amiloride, or NaCl 0.9%,  HS 7% alone was 
superior for increasing MCC in cystic fibrosis patients (11).   
 
1.4 
Relevant Literature and Data 
Much of the published data relevant to this proposal has been generated by investigators 
in the UNC Center for Environmental Medicine, Asthma and Lung Biology (CEMALB); 
these are highlighted below. 
 
The following is a short list of articles that provide support for our rationale and objectives. 
1. Kuyper LM, Pare PD, Hogg JC, Lambert RK, Ionescu D, Woods R, Bai TR. 
Characterization of airway plugging in fatal asthma. Am J Med 2003; 115: 6-11. 
2. Carroll NG, Mutavdzic S, James AL. Increased mast cells and neutrophils in 

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submucosal mucous glands and mucus plugging in patients with asthma. Thorax 2002; 
57: 677-682. 
3. Green FH, Williams DJ, James A, McPhee LJ, Mitchell I, Mauad T. Increased 
myoepithelial cells of bronchial submucosal glands in fatal asthma. Thorax 2010; 65: 32-
38. 
4. Alexis NE, Bennett W, Peden DB. Safety and benefits of inhaled hypertonic saline 
following airway challenges with endotoxin and allergen in asthmatics. J Asthma 2017; 54: 
957-960. 
5. Bennett WD, Wu J, Fuller F, Balcazar JR, Zeman KL, Duckworth H, Donn KH, 
O'Riordan TG, Boucher RC, Donaldson SH. Duration of action of hypertonic saline on 
mucociliary clearance in the normal lung. J Appl Physiol (1985) 2015; 118: 1483-1490. 
6. Daviskas E, Anderson SD, Gonda I, Eberl S, Meikle S, Seale JP, Bautovich G. 
Inhalation of hypertonic saline aerosol enhances mucociliary clearance in asthmatic and 
healthy subjects. Eur Respir J 1996; 9: 725-732. 
7. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. Mucus 
clearance and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006; 
354: 241-250. 
8. Elkins MR, Robinson M, Rose BR, Harbour C, Moriarty CP, Marks GB, Belousova EG, 
Xuan W, Bye PT, National Hypertonic Saline in Cystic Fibrosis Study G. A controlled trial 
of long-term inhaled hypertonic saline in patients with cystic fibrosis. N Engl J Med 2006; 
354: 229-240. 
9. Forouzan A, Masoumi K, Delirrooyfard A, Asgari Darian A, Mokhtar Gandomani L. 
Effect of Nebulized 3% Hypertonic Saline with Salbutamol on Management of Acute 
Asthma in Outpatient Adults: A Double-blind, Randomized Clinical Trial in Emergency 
Department. Iran J Allergy Asthma Immunol 2017; 16: 370-377. 
10. Robinson M, Hemming AL, Regnis JA, Wong AG, Bailey DL, Bautovich GJ, King M, 
Bye PT. Effect of increasing doses of hypertonic saline on mucociliary clearance in 
patients with cystic fibrosis. Thorax 1997; 52: 900-903. 
11. Robinson M, Regnis JA, Bailey DL, King M, Bautovich GJ, Bye PT. Effect of hypertonic 
saline, amiloride, and cough on mucociliary clearance in patients with cystic fibrosis. Am 
J Respir Crit Care Med 1996; 153: 1503-1509. 
12. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis CW, Boucher RC. 
Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the 
pathogenesis of cystic fibrosis airways disease.  Cell 95: 1005–1015, 1998. 
13. Alexis NE, Hu SC, Zeman K, Alter T, Bennett WD. Induced sputum derives from the 
central airways: confirmation using a radiolabeled aerosol bolus delivery technique. Am J 
Resp Crit Care Med 164: 1964–1970, 2001 
14. Sood N, Bennett WD, Zeman K, et al. Increasing concentration of inhaled saline with 
or without amiloride: effect on mucociliary clearance in normal subjects. Am J Respir Crit 
Care Med 167: 158–163, 2003. 
15. Button B, Cai LH, Ehre C, Kesimer M, Hill DB, Sheehan JK,, Boucher RC, Rubinstein 
M.  A periciliary brush promotes the lung health by separating the mucus layer from airway 
epithelia. Science 24: 937–941, 2012. 
 
 
2. 
STUDY OBJECTIVES  
2.1  
Primary Objective 
This study will assess the efficacy of inhaled HS for accelerating MCC acutely in well-
controlled moderate to severe asthmatics. 
2.2 
Secondary Objectives 

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To determine if albuterol is as effective as 7% HS in accelerating MCC. We will also 
evaluate the safety of HS in moderate to severe asthmatics by determining the proportion 
who are ‘intolerant’ to HS treatment (see Section 5.3). 
 
3.       INVESTIGATIONAL PLAN (brief overview) 
3.1      Study Design 
Type of design: Prospective open label study 
Provide brief overview of the study phases:  
• 
Visit 1 Baseline MCC: Participants will inhale Tc99m-SC, assessment of MCC over 
a 2-hour period to define baseline MCC.  Participants will then perform spirometry, 
followed by sputum induction.   
• 
Visit 2 Follow-up Baseline Visit:  Participants will return the following morning for a 
query of symptoms overnight. Spirometry will be performed. Participants will sit in front 
of the gamma camera for 30 minutes to estimate retention of Tc99m-SC in the airways.  
• 
Visit 3 Albuterol Treatment  visit:  Between 1-6   weeks of the baseline visit, 
participants will return to the lab to complete spirometry and the symptom 
questionnaire.  They will then inhale Tc99m-SC and immediately be scanned by 
gamma scintigraphy to measure deposition of the radiolabeled particles in the airways 
(4 minute scan). While they remain seated in front of the gamma camera they will then 
inhale 4 puffs of albuterol.  MCC measurement will be performed for a 2-hour period 
post-radioaerosol inhalation  to measure acute effects of albuterol on MCC.  Subjects 
will undergo sputum induction at the completion of the MCC scan. 
• 
Visit 4 Follow-up Albuterol Treatment Visit:  Participants will return the following 
morning for a query of symptoms overnight. Spirometry will be performed.  Participants 
will sit in front of the gamma camera for 30 minutes to estimate retention of Tc99m-
SC in the airways.  
• 
Visit 5: 7% HS concentration comparison:  Participants will return to the lab  
• 
Between 1-6   weeks of the albuterol visit, participants will return to the lab to complete 
spirometry and the symptom questionnaire.  They will then inhale Tc99m-SC and 
immediately be scanned by gamma scintigraphy to measure deposition of the 
radiolabeled particles in the airways (4 minute scan). While they remain seated in front 
of the gamma camera they will then inhale 4 puffs of albuterol followed by 4 mL of 
nebulized 7% HS.  MCC measurement will be performed for a 2-hour period post-
radioaerosol inhalation (which includes the period of HS inhalation) to measure acute 
effects of HS on MCC.  Subjects will undero sputum induction at the completion of the 
MCC scan. 
• 
Visit 6 Follow-up HS Treatment Visit:  Participants will return the following morning 
for a query of symptoms overnight. Spirometry will be performed. Participants will sit 
in front of the gamma camera for 30 minutes to estimate retention of Tc99m-SC in the 
airways. 
• 
Visit 7 Study Discontinuation: The subject will return to the research lab 5-10 days 
after the completion of the treatment visits for AE inquiry, vital signs and spirometry.  
• 
Unscheduled Visits: There is no expectation for unplanned visits.  Subjects will 
simply be rescheduled if they have a change in health status.  
 
3.2  
Allocation to Treatment Groups and Blinding (if applicable): N/A 
 
3.3  
Study Duration, Enrollment and Number of Subjects:  All subjects will be involved in 
the study for seven study visits. The Treatment Visits will occur within 1-6 weeks of the 

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screening visit. The total commitment for each subject will be a maximum of 14 weeks; 
however if a subject becomes ill we will delay visits until they are well.  This may extend 
the total time for an individual.  A total of 28 subjects will be enrolled to complete the study.  
The entire study should be completed in about 4I years.   
 
3.4 Study Population 
Inclusion Criteria  
1. Age 18-60 of both genders 
2. Moderate to severe asthma at baseline (determined by Step 3 therapy or greater 
or by asthma impairment) and be well-controlled at the time of enrollment, as 
determined by NHLBI Expert Panel Report 3 guidelines for diagnosis and 
treatment of asthma.  Or  
3. Moderate to severe asthma at baseline (determined by Step 3 therapy or greater 
or by asthma impairment) and not be well-controlled at the time of enrollment, as 
determined by NHLBI Expert Panel Report 3 guidelines for diagnosis and 
treatment of asthma 
4. Negative pregnancy test for females who are not s/p hysterectomy with 
oopherectomy 
5. FEV1 of at least 70% of predicted for age, sex, height, and race/ethnicity (without 
use of bronchodilating medications for 12 hours or long acting beta agonists for 24 
hours).   
6. Documented Covid 19 vaccination 
 
 
 
 
Exclusion Criteria  
 
Subjects who meet any of these criteria are not eligible for enrollment as study 
participants:  
 
 
      1. Clinical Contraindications:  
a) Any chronic medical condition considered by the PI as a contraindication to the 
study including significant cardiovascular disease, diabetes, chronic renal 
disease, 
chronic 
thyroid 
disease, 
history 
of 
chronic 
infections/immunodeficiency, or history of tuberculosis  
b) Any acute infection requiring antibiotics within 4 weeks of study. 
c) Mental illness or history of drug or alcohol abuse that, in the opinion of the 
investigator, would interfere with the participant’s ability to comply with study 
requirements. 
d) Medications which may impact the results of the study treatment, or may 
interfere with any other medications potentially used in the study (to include 
systemic steroids, beta antagonists, non-steroidal anti-inflammatory agents) 
e) Active smoking to include e-cigarettes within 1 year of the study, or lifetime of 
> 10 pack years of smoking 
f) Allergy/sensitivity to study drugs, or their formulations. 
g) History of intubation for asthma 
h) Unwillingness to use reliable contraception if sexually active (birth control 
pills/patch, condoms).  
i) Viral upper respiratory tract infection within 4 weeks of challenge. 
j) Radiation exposure history in the past year that would cause the participant to 
exceed Federal radiation safety guidelines.  
k) Positive Covid test in the past 90 days  

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2.  Pregnant women and children (< 18 years as this is age of majority in NC) will 
also be excluded since the risks associated with hypertonic saline inhalation to the 
fetus or child and the risk of radiation are unknown and cannot be justified.  
Breastfeeding women will be excluded from this protocol. 
 
 
         3.  Use of the following medications:  
a. 
Systemic corticosteroids; subjects with systemic corticosteroid-dependent 
asthma will be excluded.  All use of systemic steroids in the last year will be 
reviewed by a study physician. 
b. 
Use of daily theophylline within the past month 
 
c.   Use of beta blocking medications 
 
e. 
Receipt of LAIV (Live Attenuated Influenza Vaccine), also known as FluMist®, 
within the prior 30 days, or any vaccine within the prior 5 days 
 
f. 
Multivitamins, Vitamin C or E or herbal medications in the 4 days prior to the 
treatment visit 
 
g. 
Non-steroidal anti-inflammatory drugs in the 4 days prior to the treatment visit  
 
 
 
4.  Allergy/sensitivity to study drugs or their formulations: 
Known 
IgE-mediated 
hypersensitivity 
to 
albuterol, 
diphenhydramine 
or 
corticosteroids.  
 
   5.  Physical/laboratory indications:  
a. 
Abnormalities on lung auscultation 
b. 
Temperature > 37.8 
c. 
Systolic BP >150 mm hg or < 90 mm Hg or diastolic BP> 90 mm Hg or < 50 
d.    Oxygen saturation of < 93% 
 
   6.   Inability or unwillingness of a participant to give written informed consent. 
 
 
4  STUDY PROCEDURES  (See Appendix for Table of Study Procedures)  
 
4.1 Visit 1 Baseline MCC:  Subjects will be phone screened for potential eligibly. All subjects will 
be verbally screened for Covid 19 infection or exposure following current guidelines.  The study, 
including risks and benefits, and what is expected of the subject will be explained and subjects 
will be given time to ask any questions.  Once consent is obtained, a medical history, including all 
medications, will be collected.  Subjects will be queried about any radiation exposure in the past 
year.  A negative urine pregnancy test will be required of all women.  Vitals signs, including 
Baseline
MCC 
1. Inhale 7% HS 
2. Immediate 
MCC 
1.Albuterol 
treatment.         
2.Immediate
MCC 
Final 
study 
visit 
(N=28). 

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temperature, respiratory rate (RR), heart rate (HR), oxygen saturation, blood pressure (BP), and 
symptom questionnaires will be collected.  Fractional exhaled Nitric Oxice (FeNO) will be 
measured in exhaled breath (used to classify asthma as being predominated by eosinophilic vs 
non-eosinophilic airway inflammation). Spirometry will be performed to establish that day’s 
baseline values. FEV1 must be ≥ 70% before albuterol. Participants will inhale Tc99m-SC and 
immediately be scanned by gamma scintigraphy to measure deposition of the radiolabeled 
particles in the airways (4 minute scan). MCC will be assessed over a 2-hour period to define 
baseline MCC. A baseline symptom questionnaire will be obtained.  Subjects will then undergo a 
sputum induction procedure 
 
Any subject who experiences a > 10% reduction in FEV1 from that day’s baseline at the 
spirometry measure, or who increases HR, BP or RR more than 30% from baseline, or with 
audible wheezing on physical exam, or participants who report any symptom as severe (score of 
3) on the symptom questionnaire will be considered a intolerant to HS and will not be included in 
further study.  Oxygen saturation will be monitored, and a transient decrease is an expected 
finding when using mucolytic agents.  Sustained (< 90% for more than 1 hour) decrease in oxygen 
saturation will be considered intolerance.  Testing will be stopped at any point the volunteer 
requests that it stop.  Any subject who does not tolerate HS will be monitored until the vital signs 
return to normal limits, and the symptoms resolve.  Subjects will be discharged to home with with 
contact information for a study physician, an asthma action plan reviewed by the study 
coordinator, and emergency medications if needed overnight, including an albuterol MDI with 
spacer and 60 mg of prednisone.   Subjects will be instructed to call the physician on call if they 
require prednisone treatment. 
 
4.2 Visit 2 Follow-up Baseline Visit: Participants will return the following morning for vital signs, 
symptom questionnaire, 24-hour lung retention imaging, FeNO, and spirometry. Subjects who 
tolerate the HS dose and do not meet stopping criteria will be invited back for the remaining study 
visits. 
 
4.3 Visit 3 effect of albuterol inhalation on MCC:  At least 1 week but no greater than 6 weeks 
from Visit 2, subjects will return to the lab.    This visit may be delayed longer if the subject has an 
asthma related exacerbation to ensure good health for a total of 4 weeks before the visit. Vital 
signs, symptom and radiation exposure questionnaire are collected, and FeNO and spirometry 
performed.  A negative urine pregnancy test will be required of all female participants. Medical 
history will be updated, including any new medication.  Subjects will undergo spirometry and  
FEV1 must be at least > 70% of predicted for age, height, sex, and race/ethnicity. Participants will 
then inhale Tc99m-SC and immediately be scanned by gamma scintigraphy to measure 
deposition of the radiolabeled particles in the airways (4 minute scan). While they remain seated 
in front of the gamma camera, they will be provided with 4 puffs of albuterol with spacer, followed 
by a 15-minute wait period.  .  MCC measurement will be performed for a 2-hour period post-
radioaerosol inhalation to measure acute effects of albuterol on MCC.  Vital signs, symptom 
questionnaire, spirometry and discharge procedures will be identical to those described for visit 
1.  
 
Subjects will undergo sputum induction following the 2 hour MCC scan.  
 
4.4 Visit 4 Follow-up Albuterol Treatment Visit: Participants will return the following morning 
for vital signs, symptom questionnaire, 24-hour lung retention imaging, FeNO, and spirometry. 
Subjects who complete the acute treatment visit and do not meet stopping criteria will be invited 
back for the remaining study visits. 

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4.5 Visit 5  Treatment (effect of 7% HS on MCC): Subjects will return at least 1 week, but no 
more than 6 weeks later to the lab.  This visit may be delayed longer if the subject has an asthma 
related exacerbation to ensure good health for a total of 4 weeks before the visit. Vital signs, 
symptom and radiation exposure questionnaire are collected, and FeNO and spirometry 
performed. A negative urine pregnancy test will be required of all female participants. Medical 
history will be updated, including any new medication. FEV1 must be at least 10% of baseline 
(established at screening visit) and > 70% of predicted for age, height, sex, and race/ethnicity.  
Subjects will undergo spirometry and  FEV1 must be at least > 70% of predicted for age, height, 
sex, and race/ethnicity. Participants will then inhale. Tc99m-SC and immediately be scanned by 
gamma scintigraphy to measure deposition of the radiolabeled particles in the airways (4 minute 
scan). While they remain seated in front of the gamma camera, they will be provided with 4 puffs 
of albuterol with spacer, followed by a 15-minute wait period  They will then inhale 4 mL of 
nebulized 7% HS.  MCC measurement will be performed for a 2-hour period post-radioaerosol 
inhalation (which includes the period of HS inhalation) to measure acute effects of HS on MCC.  
Vital signs and symptom questionnaire will be performed at 5 and 30 minutes after HS inhalation 
while sitting in front of the gamma camera. Vital signs, symptom questionnaire, spirometry and 
discharge procedures will be identical to those described for visit 1. 
 
Subjects will undergo sputum induction following the MCC scan.  
 
4.6 Visit 6 Follow-up HS treatment  Visit: Participants will return the following morning for vital 
signs, symptom questionnaire, 24-hour lung retention imaging, FeNO, and spirometry.  
 
4.7 Visit 7 Study Discontinuation: The subject will return to the research lab 5-10 days after the 
completion of the treatment visits for AE inquiry, vital signs and spirometry. 
 
4.8 Unscheduled visits:  Any subject who has an unexpected problem as a result of the study 
treatment will have access to a study physician.  Subjects will be invited back to the lab for vital 
signs collection, physical exam, and spirometry.  If the subject has any adverse event not 
associated with the study, he/she will not complete the study until the AE resolves such that it 
does not interfere with the study or with safety. 
 
4.9 Concomitant Medication documentation:  Concomitant medications are collected at the 
screening visit and throughout the study visits.  Subjects will use previously prescribed medication 
for their asthma.  Subjects will not be asked to withhold any LABA/steroid combination medication 
on the morning of study visits. The study will provide all subjects with an albuterol MDI with spacer, 
and with 60 mg of prednisone as part of the asthma action plan at discharge.  Subjects will be 
allowed to keep albuterol MDI with spacers for their personal use.  Subjects will be asked to return 
unused prednisone tablets to the study staff during the follow-up visits.   
 
4.10 Rescue medication administration: As with any testing in a population of subjects with 
asthma, albuterol (Ventolin® HFA) will always be available for rescue.  There is also a code cart 
immediately available with epinephrine autoinjectors.  Oxygen is available for supplementation if 
required. 
 
4.11 Subject Completion/ Withdrawal procedures: Subjects are considered completed when 
the final 24-hour imaging, symptom questionnaire, vital signs and spirometry are collected.  
Subjects may withdraw at any time.  Subjects will be asked to return to the lab for a final visit if 
they withdraw from the study prior to completion of all study procedures to monitor safety.   

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Subjects may be withdrawn at any time for safety concerns.  Subjects who have any unanticipated 
problems (UPs) with the study procedures will be monitored until resolution of the symptoms.  
 
Criteria for safety prior to HS inhalation: 
(Subjects not meeting these criteria will not proceed with HS inhalation): 
1. FEV1 of at least 70% of predicted  
2. FEV1 within 10% of baseline value established at the screening visit. 
2. Baseline oxygen saturation of at least 93%. 
3. No history of viral respiratory tract symptoms within 4 weeks of challenge. 
4. Vital signs within the previously defined normal parameters. 
5. Symptom questionnaire score ≤ 2, , and no more than mild symptoms (ranked as “1”) 
reported for any category (see Section 5.3). 
 
Criteria for safety of a given individual following HS inhalation which would suspend 
the individual from further participation in the study will include: 
1. A 10% or greater decline in FEV1 (from that day’s baseline) at the 30-minute spirometry 
assessment.  
2. Continued Symptoms of asthma exacerbation such as wheezing or respiratory distress. 
3. Heart rate, blood pressure, or respiratory rate increase of more than 30% from baseline 
values (established on the same day). 
4. Oxygen saturation < 90% for > 1 hour. 
5. ANY reported shortness of breath, and any symptom ranked as severe (“3”). 
6. Requirement of oral corticosteroid treatment for exacerbation of asthma associated with 
the HS inhalation. 
 
Safety criteria for iniating suspension of further study until consultation with the 
DSMB and IRB will include the following: 
 
1.  The occurrence of any Serious Adverse Event. 
2. If 3 of the first 10 participants fail the individual safety criteria outlined above.  
Subjects will be under direct supervision of a study physician throughout the HS inhalation 
and MCC measurement, and will have contact information for after-hours access to a study 
physician.  An asthma action plan with emergency medications will be provided to each 
subject after the HS visits in the event of an unexpected event, such as wheezing or 
bronchospasm overnight.  
 
4.12 Screen failure procedures.  For subjects who do not meet enrollment criteria, all study 
related activity will stop once it is determined that the subject does not meet criteria. 
 
 
5.  STUDY EVALUATIONS AND MEASUREMENTS  
List variables that will be abstracted from medical charts: none  
Describe screening evaluation: Vital signs, including heart rate, respiratory rate, temperature 
and blood pressure will be collected.  Oxygen saturation levels will be noted, as well as breath 
sounds.  A brief physical examination, including but not limited to the cervical lymph nodes, eyes, 
ears, nose, throat, cardiovascular and respiratory systems.   
Describe how measurements will be taken: 

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Covid: 
All University, National and State guidelines will be followed during the Covid 19 pandemic, and 
the protocol will make changes as required to be in compliance.  There will only be 1 subject at 
any given time in the study area, and areas will be cleaned between subjects.  As many of the 
procedures for this study are considered to be aerosol generating, all subjects will be verbally 
screened for signs or symptoms of Covid, or recent potential exposure.  Staff will be encouraged 
to receive vaccination.  Study particiants will be masked when it does not interfere with 
measurements.  Study staff will wear appropriate PPE during visits.  Spirometry and aerosol 
treatments will be done in rooms with either > 6 air exchanges per hour or with running HEPA 
filters. Any subject will be deferred for at least 90 days after a positive Covid test, and volunteers 
will be required to show proof of up to date vaccination.  
 
Spirometry:  
This test measures the volume of air that can be exhaled and the rate of airflow during exhalation 
after a maximal inhalation, and is performed to American Thoracic Society (ATS) guidelines. 
Subjects will inhale as deeply as possible, then exhale as rapidly and completely as possible into 
the spirometer.  Measurements obtained from each maneuver include the forced vital capacity 
(FVC), the forced expiratory volume in the first second (FEV1), the maximal mid-expiratory flow 
rate (FEF 25-75%) and the peak expiratory flow (PEF).  The largest FVC and FEV1, from at least 
3 acceptable trials, are selected for analysis; the flow rates are selected from the trial with the 
largest sum of FVC and FEV1. 
 
FeNO:  
Fractional Exhaled nitric oxide (FeNO) is measured using the Niox MINO (Aerocrine Inc) device. 
The subject inhales through the mouth to total lung capacity (TLC) and then exhales immediately. 
The NO analyzer samples the expirate continuously. Since exhaled NO plateau values can vary 
with exhalation flow rate, subjects must achieve a standardized expiratory flow rate for the results 
to be acceptable. eNO is measured in parts per billion (ppb). The procedure is performed in 
accordance with ATS guidelines. 
 
HS Inhalation: 
The subject will be instructed on the proper use of the PARI LC Star Nebulizer System, an 
approved device for aerosol inhalation.  The subject will use the nebulizer until the HS dose is 
completed.  All subjects in this study will be pretreated with albuterol prior to HS inhalation. 
 
MCC procedures:  
Prior to each MCC study, a transmission Co57 scan will be performed to define the lung 
boundaries, to assign regions of interest, and to normalize these regions for lung volume 
differences (Fig 2). A rectangular phantom containing the radioisotope Co57 (< 25 mCi) will be 
placed in front (5cm) of the subject sitting with his/her back to the gamma camera for 30 seconds. 
The transmission scan has been used by us (e.g. 05-2358, 08-0795, 06-1016, 13-1605, 15-938) 
and others to provide a delineation of lung boundaries for assessing regional deposition 
/clearance of the inhaled radioaerosol.  Prior to the transmission scan on each study day we will 
place 2 spot markers of Americium241 (0.9 microcurie (uCi) each, gamma 66 KeV) on the upper 
and lower back of each subject during scanning (both Tc99m-SC deposition/retention and Co57 
transmission). With dual isotope imaging, these spot markers will allow alignment of images for 
more accurate determination of regional deposition/retention. These very low radiation sources 
have been obtained from commercially available home smoke alarms. The placement of these 
markers will be determined to be outside the lung field during the transmission scan. Their location 

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will be marked in semi-permanent ink for later placement during Tc99m deposition/retention 
scans. The shielded side of this source will be placed/taped onto the subject’s skin. 
 
Radiolabeled Tc99m-sulfur colloid will be delivered using a modified Pari-LL nebulizer (MMAD 
9.5 um).  This is a closed delivery system that produces 80 ml/sec air flow, and therefore limits 
the inspiratory flow rate to this value.  While seated in front of a gamma camera subjects will 
perform single inhalations lasting ~10 seconds each from the delivery system, and will exhale at 
500 ml/sec (using feedback from a flow meter in the breathing circuit).  Approximately 5 of these 
inhalation maneuvers will be required to deposit an 
adequate isotope dose to the lung.  Subjects will be 
allowed to breathe normally (off the nebulizer) in 
between each inspiratory maneuver. Each volunteer will 
practice these maneuvers prior to the actual radioaerosol 
inhalation to guarantee his/her proficiency.   The activity 
of Tc99m-SC loaded in the nebulizer will be adjusted to 
provide an estimated 40 uCi deposited in the lung for 
each MCC scan. A single crystal detector will be placed 
at the subject’s back during inhalation to monitor dose to 
the lung. Total inhalation time should be less than 5 
minutes in all cases. Immediately following isotope 
inhalation, the subject will gargle and drink water to clear 
activity that deposited in the mouth into the stomach. The 
subject will then (within a minute of final inhalation 
maneuver) be seated in front of a large-field-of-view 
gamma camera to begin acquiring particle retention 
images.  
 
For the baseline MCC measurement, the gamma image 
capture will begin immediately and will capture 
continuously for the first 34 minutes. Thereafter, 2 
consecutive 2-minute images will be obtained at the start 
of every 10-minute period until 2 hours have passed. For 
visit 3/5, participants will first inhale Tc99m-SC as described above then sit in front of the gamma 
camera for 4 minutes during which images of the lung will be continuously captured. While still 
sitting in front of the gamma camera, they will inhale HS. Image capture will be continuous during 
HS inhalation and for the next 34 minutes, after which 2 consecutive 2-minute images will be 
obtained at the start of every 10-minute period until 2 hours have passed. The subject will also 
return the day (visit 4/6) following each MCC measurement to sit in front of the gamma camera 
for 30 minutes to obtain images of the lungs to visualize retention of Tc99m-SC particles. No 
Tc99m-SC will be administered at this visit. Spirometry, symptom questionnaire, and vital signs 
will be collected prior to discharge on the follow up day. 
 
Hypertonic Saline Induced Sputum procedure:  
Prior to sputum induction, subjects will have spirometry measured for FEV1 and FVC values. 
Next, an ultrasonic nebulizer filled with 20 cc of 3% hypertonic saline (inhalation grade for 
respiratory use only, 3% NaCl) will be set to the maximum output setting and turned on. The 
subject will be instructed to latch his/her mouth onto the nebulizer mouthpiece and breathe 
normally (i.e., tidal breaths) for 7 minutes as the saline is nebulized through the mouthpiece in a 
jet stream and inhaled. The nose will not be occluded for this procedure. The subject will be 
encouraged to come off the mouthpiece at any time to cough if a sputum sample from the lower 

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airways (i.e. not from the back of the throat) is ready for expectoration. Prior to expectoration, 
subjects will be asked to blow their nose, rinse their mouth with water, and clear their throat to 
avoid the inclusion of non airway fluid samples. The sample will be expectorated into a sterile 
specimen jar and capped.  
 
Following the measurement of FEV1 after the first 7 minute inhalation period, the concentration 
of saline will be increased from 3% to 4%, provided the FEV1 decrement is < 10% from the post 
bronchodilator value. If the FEV1 falls between 10 20% of the post bronchodilator value, the test 
will proceed but the concentration of saline will remain the same.  If the FEV1 falls by > 20% or if 
troublesome symptoms occur, the nebulization will be discontinued, and albuterol will be 
immediately available if necessary to relieve symptoms. The same procedure will be followed for 
the final 7 minute inhalation period using 5% hypertonic saline provided the FEVl safety 
parameters described above have been met. The nebulization is stopped after a total of 21 
minutes or earlier if a sputum sample of good quality is obtained (i.e. visible sputum plugs).  
Subjects may request early termination at any time the procedure becomes intolerable.  
 
5.1 Efficacy Evaluation (if applicable) 
To determine the efficacy of HS for accelerating MCC associated with  3% vs. 7% HS treatment 
as compared to baseline MCC in adult patients with moderate to severe asthma. MCC is 
measured as the average % retention of radiolabeled particles in the lung over a 120 minute 
measurement period.  
 
5.2 Pharmacokinetic Evaluation (if applicable) 
None 
 
5.3 Safety Evaluations: The primary safety criteria will be change in FEV1 from baseline 
measures obtained on the same day.   A drop in FEV1 by 10% at the 30-minute mark after HS 
inhalation will be considered an adverse event for patient safety and will result in withdrawal of 
the subject from further participation in the study.   
 
During visit 1, subjects will be monitored for 1 hour after HS inhalation.  If a subject’s FEV1 is 
reduced by ≥ 10% from that day’s baseline at the 5-minute mark, spirometry will be repeated 
every 5 minutes up to 30 minutes.  If at the 30-minute mark FEV1 is still reduced by ≥ 10%, 4 
puffs of albuterol will be administered. Albuterol 4 puffs with spacer will be repeated if the 
participant has symptoms, or if FEV1 does not spontaneously return to within 5% of baseline 
values at the 1-hour mark.  
 
Oxygen saturation will be monitored during the HS inhalation through the rest of the day’s study 
procedures.  Several studies have shown that the oxygen saturation drops transiently in patients 
with CF and COPD when they inhale a mucolytic agent.  It is anticipated that asthmatic subjects 
will have a similar response.   
 
Vital signs are monitored, including HR, RR, and BP.  Auscultation is performed to determine if 
wheezing is present. 
 
We will assess symptoms at baseline, prior to HS, and following HS whenever vital signs are 
measured. We will use the following symptom score questionnaire: 
Symptom score (scale 0-none, 1-mild, 2-moderate, 3-severe):  
Malaise____, Fatigue____, Shortness of Breath_____, Cough_____, Myalgia_____, 
Chills_____, Fever_____, Headache______ 

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Mild: Symptom minimally noticeable but would not ordinarily cause you to stop normal activities 
such as going to work or school and would not keep you from undertaking physical exercise. 
 
Moderate:  Symptom present but would not ordinarily cause you to stop normal activities such as 
going to work or school, although it might stop you from doing physical exercise (“working out”) 
 
Severe: Symptom clearly present and would cause you to consider not going to work or school 
or seek medical attention (such as calling a health care provider, going to student health, going 
to the emergency room, or taking medications for these symptoms). 
 
HS treatment will be deferred if baseline total symptom score > 2, OR any shortness of breath or 
fever are reported, OR if any symptom rated as moderate (2) or severe (3). For symptom scores 
obtained after HS treatment, subjects will be assessed by a study physician if total symptom score 
>2, if ANY shortness of breath is reported, or if any symptom is rated as moderate (2) or severe 
(3). If following HS treatment, the participant ranks any symptom as severe (3), that participant 
will be excluded from further study.  
 
6 STATISTICAL CONSIDERATION   
 
6.1. Primary Endpoint:  
change in MCC from baseline immediately after 7% HS inhalation 
 
6.2 Secondary Endpoints 
a. change in MCC from baseline immediately after  albuterol inhalation. This endpoint is 
of exploratory interest. 
b. Safety measures for HS treatment in moderate to severe asthmatics will include change 
in the following from baseline: 
 
Vital signs (HR, RR, BP and oxygen saturation) 
 
Spirometry (FEV1, FVC) 
 
Symptom Questionnaires 
 
Physical exam (wheezing on chest auscultation, for example) 
We will also assess: 
 
Adverse events and Serious Adverse events 
 
Proportion of participants who are deemed ‘intolerant’ to HS (i.e. those who 
experience a persistent reduction in FEV1 of ≥ 10% from that day’s 
baseline at 30 minutes post-HS, those who report shortness of breath, 
those who report a score of “3” for any individual symptom criterion, or 
those who experience oxygen saturation levels below 90% for > 1 hour).  
  
Safety endpoints will be assessed continuously during the study period for monitoring of adverse 
events and individual stopping criteria. All adverse events whether or not listed in the NCI-CTCAE 
(see Section 9) or whether they are or are not related to study participation will be graded on a 
scale from 1 to 5 according to the standards in the NCI-CTCAE manual.  Adverse events will be 
reported to the UNC IRB, the UNC DSMB, and the NHLBI project officer. The DSMB will review 
the protocol after 5 subjects have completed study procedures or at 6 months after enrollment 
begins, whichever is soonest, and then every 6 months thereafter. 
 
Exploratory endpoints will also be assessed. These will focus on sputum cellularity, mediators in 
sputum (e.g. T1 cytokines: IL-1β, IL-6, IL-8, TNFα, T2 cytokines, IL-4, IL-5, IL-13), eicosanoids, 

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and mucins (total mucins, MUC5AC, MUC5B).  
 
 
6.3 Statistical Methods 
Our analytical plan was developed in collaboration with Dr. Haibo Zhou, the biostatistician 
for the CEMALB who will oversee all statistical analyses. The primary analysis of interest 
pertains to the difference between MCC measured at baseline and MCC measured during 
and after 7% HS challenge. The MCC measurements that define this difference are 
defined in section 5, and this difference has a unit of absolute percentage points. We will 
perform descriptive analysis of this difference; all point estimates will be tabulated along 
with their 95% confidence intervals. As we have assumed the sufficient underlying 
normality of MCC measurements, we will test the null hypothesis of no difference between 
baseline MCC and MCC immediately during and after 7% HS, against the alternative 
hypothesis of some difference, using a paired two-sided one-sample t-test with a 5% type 
I error rate. That is, if we let µΔ3% denote the mean difference between MCC measured at 
baseline and MCC measured during and immediately after the albuterol treatment, i.e. we 
will test the following hypothesis:  
H0: µΔalbuterol=0 vs. H1: µΔalbuterol≠0. 
 
If the observed p-value is above 0.05, the test will be interpreted as being inconclusive. 
Following this test, we will perform sensitivity analyses to assess how realistic the 
assumption of normality was, and how much it may have impacted our conclusions. In 
particular, we will examine normal q-q plots and other descriptive tabulations, and we will 
examine the results of a Wilcoxon Signed Rank test. Should extreme or questionable 
observations arise in the study, we will also examine regression diagnostics to assess the 
influence of how those data were handled. The secondary analysis, which is of exploratory 
interest, pertains to the difference between MCC measured at baseline and MCC 
measured during and immediately after the 7% HS challenge. We will analyze this 
difference in a similar manner, i.e we will test the hypothesis  
H0: µΔ7%=0 vs. H1: µΔ7%≠0. 
We will report the proportion of enrolled patients who do not tolerate HS at baseline, along 
with the 95% confidence interval of this proportion.  
 
The effect of albuterol treatment on MCC will be assessed as a secondary endpoint, using 
the same techniques as described above for studies of 7% HS. We will also compare 
results following treatment with  albuterol to those observed after 7% saline as an 
exploratory outcome. To do this analysis, we will assess the difference of the outcomes of 
these treatments within each individual and conduct appropriate paired t-tests. If these 
differences are not normally distributed, we will do proper transformation of the data to 
achieve a normal distribution. We will also conduct Wilcoxon signed rank tests in when 
data are not normally distributed.  
 
6.4 Sample Size and Power 
The primary endpoint of this study is the difference between baseline MCC and MCC 
immediately after HS administration. In reviewing MCC data under baseline conditions in 
patients who would fit criteria for moderate asthma, Burbank et al. found a mean average  % 
retention of 21% with a standard deviation of 9% (16). We wish to detect a difference of mean 
MCC equal to 7%. Given the paired nature of the study, we believe 10% to be a reasonable 
upper bound of the standard deviation of this difference in means. Assuming β=0.9 and 
α=0.05, a sample size of n=21 would be adequate. We will inflate this by 30% to account for 

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attrition with multiple study visits, taking the recruited sample to n=28. 
 
6.5 Interim Analysis 
 
Interim analyses of efficacy of 7% HS will be performed.  
 
7. STUDY INTERVENTION (drug, device or other intervention details) 
• 
Description: Sodium chloride 7% solution, which is commercially available as Hypersal® 
(PARI Respiratory Equipment, Inc, Midlothian, VA).  
• 
Receipt/Storage: HS will be delivered and stored within the CEMALB. 
• 
Packaging/Labeling: This is an open label study. 
• 
Dosing: 4mL vial of HS is nebulized and inhaled until the dose is complete, typically 15 
minutes. 
• 
Treatment compliance and Adherence: Subjects are observed during all dosing periods. 
• 
Drug Return/Destruction: NA 
• 
Drug Accountability: NA 
 
8. STUDY INTERVENTION ADMINISTRATION (if applicable)  
• Randomization procedures:  N/A  
• Blinding procedures:  This is an open label study. 
 
9. SAFETY MANAGEMENT 
• 
Definition of Adverse Event (AE) and Serious Adverse Event (SAE) 
o An adverse event for a given volunteer will be defined as failure of any of the safety criteria 
outlined above. Any decrease in lung function as outlined above will be considered an 
adverse event. Any symptoms that induce a volunteer to seek medical attention from any 
provider within 96 hours of a study visit will be considered an adverse event.  A serious 
adverse event will be defined as any event that requires hospitalization or results in life 
threatening illness or injury, permanent (or likely to be permanent) illness or injury, or death 
if these events occur within 96 hours of a study visit (or if the clinical scenario leading up to 
hospitalization, illness, injury or death begins within 96 hours of a study treatment visit).   
Grading criteria 
 
In addition to determining whether an adverse event fulfills criteria for a serious adverse 
event or not, the severity of adverse events experienced by study participants will be 
graded according to the criteria set forth in the National Cancer Institute’s Common 
Terminology Criteria for Adverse Events Version 4.0. This document (referred to herein 
as the NCI-CTCAE manual) provides a common language to describe levels of severity, 
to analyze and interpret data, and to articulate the clinical significance of all adverse 
events.   
 
All adverse events whether or not listed in the NCI-CTCAE will be graded on a scale from 
1 to 5 according to the following standards in the NCI-CTCAE manual (A semi-colon 
indicates ‘or’ within the description of the grade.): 
 
Grade 1 = Mild; asymptomatic or mild symptoms; clinical or diagnostic observations only; 
intervention not indicated. 
 
Grade 2 = Moderate; minimal, local or noninvasive intervention indicated; limiting age-
appropriate instrumental ADL (preparing meals, shopping for groceries or clothes, using 
the telephone, managing money, etc). 
 
Grade 3 = Severe or medically significant but not immediately life-threatening; 
hospitalization or prolongation of hospitalization indicated; disabling; limiting self care ADL 
(bathing, dressing and undressing, feeding self, using he toilet, taking medications, and 

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not bedridden).  
 
Grade 4 = Life-threatening consequences; or urgent intervention indicated. 
 
Grade 5 = Death related to AE. 
• 
Adverse Event/Serious Adverse Event reporting procedures: AE’s and UP’s will be 
reported to the IRB.  The IRB, DSMB, and funding agency will be notified of any SAEs.  The 
DSMB will review progress after the first 5 participants have completed the protocol or at 6 
months after enrollment begins, whichever is soonest, and then every 6 months thereafter. 
• 
Medical Emergency procedures: There is a study physician available for all inhalation 
procedures.  A code cart and Automated External Defibrillator (AED) are available in the 
event of a cardiopulmonary event.  Albuterol, both inhalers and nebulizers, are available in 
the event of bronchospasm. If a subject has an event that is does not immediately respond 
to care in the research lab, he/she will be transported via EMS to UNC Healthcare for 
treatment. 
• 
Data Safety Monitoring Plan: Data is initially recorded on paper documents, and the 
documents are maintained in a binder which is kept in a locked office within the EPA Human 
Studies facility. The EPA HSF is a secure facility with a guarded entrance and requires 
identification for entry. This data is then entered into REDCap by an initial data entry person, 
and confirmed by a second user. 
• Potential Risks: 
 
Potential Risks of Hypertonic Saline Inhalation: 
Inhalation of hypertonic saline carries a small risk of bronchospasm, which may lead to the 
onset of asthma symptoms.   
 
Potential Risks of Spirometry: 
Potential risks include possible lightheadedness or wheezing. 
 
Potential Risks of Mucociliary clearance scan: 
The radiation risks for the MCC scan, including the Cobalt 57 transmission scan and the 
Americium 241 disks, which are used as fiducial markers is approximately 133 mRems.   
Adults in the New York City area receive about 300mRems per year in natural radiation 
exposure. 
 
Potenial Risks of Sputum Induction: 
• 
Sputum induction may induce cough, chest tightness or bronchospasm Protections Against 
Risk: 
 
Protections to Minimize Risk of Hypertonic Saline Inhalation: 
A CEMALB physician is present in the building at all times during HS inhalation. All asthmatic 
subjects are pretreated with albuterol inhalation prior to hypertonic saline administration. 
Additional albuterol will be given after HS inhalation if FEV declines by ≥ 10% from that day’s 
baseline at the 30 minute spirometry measurement, if the participant develops symptoms of 
an asthma attack such as wheezing, or if the participant requests albuterol treatment.   
 
Protections to Minimize Risk of Spirometry: 
Subjects will be seated in a non-rolling chair when spirometry is performed and standard 
methodology conforming to the American Thoracic Society guidelines for measurement of 
spirometry will be used.  Subjects will be instructed to notify the study staff if they feel 
lightheaded, and albuterol will be available in the event the subject experiences any 
unanticipated bronchoconstriction. 
 
Protections to Minimize Risk of Mucociliary clearance scan: 
Radiation history is collected.   Any subject who will exceed safe annual exposure limits will 
not be enrolled.  
 
Protections to minimize Risk of Sputum Induction: 
Subjects will have been given 4 puffs of albuerol in the 2-3 hour time period prior to sputum 
induction.  In the event of bronchospasm associated with the induction, albuterol will be 

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immediately available for rescue.   
 
 
10. DATA COLLECTION AND MANAGMENT  
• 
Monitoring Plan: We will use RedCap for data management.  The data will be entered 
by one person (typically the coordinator or, for lab analysis, a lab staff member) and 
then checked by a second person.  The data is not marked "complete" until the 2nd 
person verifies the entry.  REDCap creates a data dictionary when the database is 
established, this is used as the codebook.  
• 
Database documentation: All databases using equipment generated have a date and 
user attached to the electronic file. Data will be linked with a codebook (i.e. sample ID) 
using Excel. A second entry is done manually in a lab notebook, which includes date, 
samples assayed, assay used, any issues (like missing samples/data), reference to 
where data is stored, and a printout of the data (all as hardcopies in a lab notebook).  
Adherence to the codebook is ensured by having a second individual to check the 
entries. The PI takes responsibility for data management computations. 
• Case report forms: Case report forms will be developed by the study team, using 
templates from previous studies. These forms are maintained by study coordinators, and 
reviewed by investigators as needed.  
• How will confidentiality be maintained: Subjects will be issued a subject number when 
they enroll into the study, and this number will only be used to label samples.  All PHI will 
be maintained by the study coordinator or the investigators and will be kept in locked 
areas when not in use.  The identifiers that go into REDCap will only be accessible to 
those who need them for their job, specifically study physicians, investigators and 
coordinators.  Subjects sometimes undergo procedures – such as sitting in front of the 
gamma camera – at the same time, however other than basic introductions, nothing about 
one subject is disclosed to the other subject. 
  
11. RECRUITMENT STRATEGY  
Subjects will be recruited by posters and informational emails at UNC.  The study will be 
posted on the JOIN THE CONQUEST website, designed for recruiting subjects into 
research studies. “Dear Doctor” letters will be sent to pulmonary and family practices 
doctors who may have patients who are interested in volunteer to participate in the study. 
 
12. CONSENT PROCESS 
• 
Describe the procedure that will be used to obtain informed consent/HIPAA 
authorization and assent (if applicable): The study will be described in detail to the 
subject, including why the study is being conducted, the medication being studies, the 
risks and benefits, and what is expected of the subject.  The subject will be given 
adequate time to read the consent, and consent will be obtained prior to any study 
procedures.  Consent may take place on a separate day from the baseline procedures. 
• 
Who will obtain consent: Consent will be obtained by a study coordinator or a study 
physician.  
• 
Where will consent process take place: The consent will take place at the UNC 
CEMALB, located in the Human Studies Facility of the US EPA on Mason Farm Road 
in Chapel Hill. 
• 
How will investigator assure that subjects comprehend the nature of the study, 
procedures, the risks and benefits:  The subject will be encouraged to ask questions 

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regarding the study and procedures.  Open ended questions will be asked of the subject 
to solicit correct responses, to help ensure that the subject understands the study 
commitment, procedures and risks and benefits 
 
13. References 
 
1. Kuyper LM, Pare PD, Hogg JC, Lambert RK, Ionescu D, Woods R, Bai TR. Characterization of 
airway plugging in fatal asthma. Am J Med 2003; 115: 6-11. 
2. Carroll NG, Mutavdzic S, James AL. Increased mast cells and neutrophils in submucosal 
mucous glands and mucus plugging in patients with asthma. Thorax 2002; 57: 677-682. 
3. Green FH, Williams DJ, James A, McPhee LJ, Mitchell I, Mauad T. Increased myoepithelial 
cells of bronchial submucosal glands in fatal asthma. Thorax 2010; 65: 32-38. 
4. Alexis NE, Bennett W, Peden DB. Safety and benefits of inhaled hypertonic saline following 
airway challenges with endotoxin and allergen in asthmatics. J Asthma 2017; 54: 957-960. 
5. Bennett WD, Wu J, Fuller F, Balcazar JR, Zeman KL, Duckworth H, Donn KH, O'Riordan TG, 
Boucher RC, Donaldson SH. Duration of action of hypertonic saline on mucociliary 
clearance in the normal lung. J Appl Physiol (1985) 2015; 118: 1483-1490. 
6. Daviskas E, Anderson SD, Gonda I, Eberl S, Meikle S, Seale JP, Bautovich G. Inhalation of 
hypertonic saline aerosol enhances mucociliary clearance in asthmatic and healthy 
subjects. Eur Respir J 1996; 9: 725-732. 
7. Donaldson SH, Bennett WD, Zeman KL, Knowles MR, Tarran R, Boucher RC. Mucus clearance 
and lung function in cystic fibrosis with hypertonic saline. N Engl J Med 2006; 354: 241-
250. 
8. Elkins MR, Robinson M, Rose BR, Harbour C, Moriarty CP, Marks GB, Belousova EG, Xuan 
W, Bye PT, National Hypertonic Saline in Cystic Fibrosis Study G. A controlled trial of 
long-term inhaled hypertonic saline in patients with cystic fibrosis. N Engl J Med 2006; 
354: 229-240. 
9. Forouzan A, Masoumi K, Delirrooyfard A, Asgari Darian A, Mokhtar Gandomani L. Effect of 
Nebulized 3% Hypertonic Saline with Salbutamol on Management of Acute Asthma in 
Outpatient Adults: A Double-blind, Randomized Clinical Trial in Emergency Department. 
Iran J Allergy Asthma Immunol 2017; 16: 370-377. 
10. Robinson M, Hemming AL, Regnis JA, Wong AG, Bailey DL, Bautovich GJ, King M, Bye PT. 
Effect of increasing doses of hypertonic saline on mucociliary clearance in patients with 
cystic fibrosis. Thorax 1997; 52: 900-903. 
11. Robinson M, Regnis JA, Bailey DL, King M, Bautovich GJ, Bye PT. Effect of hypertonic saline, 
amiloride, and cough on mucociliary clearance in patients with cystic fibrosis. Am J Respir 
Crit Care Med 1996; 153: 1503-1509. 
12. O'Riordan TG, Zwang J, Smaldone GC. Mucociliary clearance in adult asthma. The American 
review of respiratory disease. 1992;146(3):598-603. doi: 10.1164/ajrccm/146.3.598. 
PubMed PMID: 1519834 
13. Shah RV, Amin M, Sangwan S, Smaldone GC. Steroid effects on mucociliary clearance in 
outpatient asthma. J Aerosol Med. 2006;19(2):208-20. doi: 10.1089/jam.2006.19.208. 
PubMed PMID: 16796545 
14. Sood N, Bennett WD, Zeman K, et al. Increasing concentration of inhaled saline with or without 
 
amiloride: effect on mucociliary clearance in normal subjects. Am J Respir Crit Care Med 
 
167: 158–163, 2003. 
15. Button B, Cai LH, Ehre C, Kesimer M, Hill DB, Sheehan JK,, Boucher RC, Rubinstein M.  A 
 
periciliary brush promotes the lung health by separating the mucus layer from airway 
 
epithelia. Science 24: 937–941, 2012. 
16. Burbank AJ, Duran CG, Pan Y, Burns P, Jones S, Jiang Q, Yang C, Jenkins S, Wells H, Alexis 

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N, Kesimer M, Bennett WD, Zhou H, Peden DB, Hernandez ML. Gamma tocopherol-enriched 
supplement reduces sputum eosinophilia and endotoxin-induced sputum neutrophilia in 
volunteers with asthma. J Allergy Clin Immunol 2017 Jun 20 (article in press). 
 
 
 
 
 
 
 
 
14. APPENDIX 
 
Study Personnel: 
 
Role in Project 
Name and Address 
Title 
Principal Investigator 
David B. Peden, MD, MS 
CEMALB, 104 Mason Farm Rd 
CB# 7310 
Chapel Hill, NC 27599-7310 
Professor of Pediatrics & 
Director, Center for 
Environmental Medicine, 
Asthma and Lung Biology 
Co-investigator 
Richard Boucher, MD 
7008 Marsico Hall 
Chapel Hill, NC 27599-7248 
James C. Moeser Eminent 
Professor of Medicine & 
Director of the Marsico 
Lung Institute 
Co-investigator 
William Bennett, PhD 
CEMALB, 104 Mason Farm Rd 
CB# 7310 
Chapel Hill, NC 27599-7310 
Professor of Medicine, 
Director of Mucociliary 
Clearance and Aerosol 
Research Lab, CEMALB 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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David B. Peden, MD  
Version 06/08/2023 
 
25 
 
 
 
 
 
 
Summary of Study Procedures: 
 
Study 
Procedures 
Visit 1: 
Baseline 
MCC Visit 
Visit 2: 
(24h f/u) 
 
Visit 3: 
Albuterol 
and MCC   
 
Visit 4: 
 (24h f/u) 
Visit 5: 
Acute 7% 
HS 
Visit 6 
(24h f/u)  
Study 
Completion 
visit 
Time between 
visits 
1-6 weeks 
1-6 weeks 
5-10 days 
Informed 
Consent 
X 
 
 
 
 
 
 
History, med 
review 
X 
 
X 
 
X 
 
X 
Review 
history, AEs 
X 
X 
X 
X 
X 
X 
X 
Vital Signs 
X 
X 
X 
X 
X 
X 
X 
Symptom 
Questionnaire 
X 
X 
X 
X 
X 
X 
X 
Physical exam 
X 
 
X 
 
X 
 
 
Urine 
pregnancy test 
X 
 
X 
 
X 
 
 
Spirometry 
X 
X 
X 
X 
X 
X 
X 
Albuterol  
X 
 
X 
 
X 
 
 
Nebulized HS 
 
 
 
 
X 
 
 
MCC 
Measurement 
X 
 
X 
 
X 
 
 
24h Lung 
Retention 
Images 
 
X 
 
X 
 
X 
 
Induced 
Sputum 
Collection  
X 
 
X 
 
X