Evaluation of the Effect of a Nasal Allergen Challenge With Dermatophagoides Farinae Extract on Nasal Airway Inflammation in Allergic Individuals, Comparing E-cigarette Users to Cigarette Smokers and Non-smokers.
Phase: PHASE1
Conditions: Healthy Participants, Tobacco Smokers
Sponsor: University of North Carolina, Chapel Hill

 
 
 
 
 
February 20, 2017 version 
 
 
 
 
Evaluation of the effect of a nasal allergen challenge with 
Dermatophagoides farinae extract on nasal airway 
inflammation in allergic individuals, comparing e-cigarette 
users to cigarette smokers and non-smokers 
 
 
 
NCT number NCT03023397
    Document Date 02/20/2017
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
February 20, 2017 version 
 
 
6.1. Protocol 1: Evaluation of the effect of a nasal allergen challenge with 
Dermatophagoides farinae extract on nasal airway inflammation in allergic 
individuals, comparing e-cigarette users to cigarette smokers and non-smokers. 
 
a. Objectives and Purpose. The purpose of this pilot study is to evaluate allergen-
induced inflammation in nasal lavage samples following Dermatophagoides farinae 
intranasal challenge in e-cigarette users, cigarette smokers, and non-smokers. 
 
b. Patient inclusion and exclusion criteria. 
 
We will recruit allergic subjects with or without mild asthma, ages 18-50 years, including 
15 e-cigarette users, 15 tobacco cigarette smokers, and 15 non-smokers (classification 
described below). There will be no gender or ethnic restrictions. Allergy skin testing to 
D. farinae will be performed to confirm the presence of allergic sensitization.  Only 
individuals sensitive to house dust mite will be selected for participation.  Spirometry will 
be performed to determine the current level of lung function. Those with an FEV1 (forced 
expiratory volume in the first second) of at least 80% predicted for age, gender, race, and 
height and an FEV1 to FVC (forced vital capacity) ratio of at least .75will be eligible for 
the study. Subjects for this study will be both mild asthmatic and non-asthmatic healthy 
volunteers, ages 18-50years and to include both males and females.  Our recruitment goal 
includes 45 volunteers with at least 50% female subjects and at least a 30% minority 
representation. It is anticipated that the demographics of our patient population will 
reflect those of the local university community. Many determinations (e.g. spirometry, 
allergy skin testing) are made as a part of a separate screening protocol, IRB #98-0799, 
which is performed to identify subjects for potential participation is experimental 
challenge protocols such as this one.  
 
Inclusion criteria 
 
1. Specific allergy to house dust mite D. farinae confirmed by positive 
immediate skin test response. 
 
2. Subjects may be enrolled with mild asthma if an FEV1 of at least 80% of 
predicted and FEV1/FVC ratio of at least .75 (without use of bronchodilator 
medications for 12 hours), consistent with lung function of persons with mild 
episodic or mild persistent asthma is demonstrated. For the purpose of this 
protocol, an asthmatic individual will be defined as having a) positive 
methacholine challenge with a provocative concentration of methacholine 
producing a 20% fall in FEV1 (PC20 methacholine) with less than or equal to 
10 mg/ml; OR b) physician diagnosed asthma with symptoms and chronic 
daily therapy consistent with the mild asthma. 
 
3. Subjects will be classified as tobacco smokers, e-cigarette users, or non-
smokers according to the following guidelines: 

 
 
 
 
 
February 20, 2017 version 
 
 
 
Tobacco Smokers 
E-cigarette Users 
Non-smokers 
Average 
cigs/wk
>35 cigs/week 
<7 cigs/week 
none 
Average vapor 
puffs/day
<18 puffs/day 
≥18 puffs/day 
none 
Hookah use 
<30 minutes/week 
<30 minutes/week 
<30 minutes/week 
Duration of 
use 
If former heavy e-
cig user, must be 
<18 puffs/day for 
≥6 months 
E-cig user ≥6 months; 
if former heavy 
tobacco user, must be 
<7 cigs/week for ≥6 
months  
Must be abstinent 
from tobacco or e-cig 
products for ≥6 
months 
 
 
4. Ability to withhold antihistamine medications for one week prior to baseline 
and allergen challenge visits. 
 
5. Subjects must be able and willing to give informed consent. 
 
    
Exclusion criteria 
 
1. 
Any chronic medical condition considered by the PI as a contraindication 
to the allergen challenge study including significant cardiovascular 
disease, diabetes requiring medication, chronic renal disease, bleeding 
disorder, or chronic thyroid disease. 
 
2. 
Physician directed emergency treatment for an asthma exacerbation within 
the preceding 12 months. 
 
3. 
Use of systemic steroid therapy within the preceding 12 months for 
treatment of an asthma exacerbation. 
4. 
Use of inhaled or nasal steroids, cromolyn or leukotriene receptor 
antagonists (Montelukast or Zafirkulast) within the past month (except for 
use of  cromolyn exclusively prior to exercise). 
 
 
5.  
Subjects who smoke marijuana or use illicit drugs will be excluded.  
 
6. 
Use of daily theophylline within the past month. 
7. 
Use of nasal medications that might alter the response to nasal allergen 
challenge including anti-inflammatory and anti-histamine agents within 
one week of challenge. 
 

 
 
 
 
 
February 20, 2017 version 
 
8. 
Inability to withhold inhaled or oral bronchodilator medications for 12 
hours prior to allergen challenge. 
 
9. 
Pregnancy or nursing a baby. 
 
10.   
Women of child-bearing age who are not using dependable contraception 
(such as birth control pills, IUD, estrogen patches) or who are not 
completely abstinent.   
 
11.  
Nighttime symptoms of cough or wheeze greater than 1x/week at baseline 
(not during a clearly recognized viral induced asthma exacerbation) which 
would be characteristic of a person of moderate or severe persistent 
asthma as outlined in the current NHLBI guidelines for diagnosis and 
management of asthma. 
 
12.   
Exacerbation of asthma more than 2x/week which would be characteristic 
of a person with moderate or severe persistent asthma as outlined in the 
current NHLBI guidelines for diagnosis and management of asthma. 
 
13. 
 Daily requirement for albuterol due to asthma symptoms (cough, wheeze, 
chest tightness) which would be characteristic of a person of moderate or 
severe persistent asthma as outlined in the current NHLBI guidelines for 
diagnosis and management of asthma. (Not to include prophylactic use of 
albuterol prior to exercise). 
 
14.  
Viral upper respiratory tract infection or other acute inflammatory 
conditions of the nose or paranasal sinuses, such as sinusitis, within 4 
weeks of challenge. 
 
15.  
Any acute infection requiring antibiotics within 4 weeks of challenge. 
 
16.  
Participating in an allergen inhalation study within 2 weeks of this 
 
challenge or use of any other investigational agent within the last 30 days. 
 
17. 
Use of tricyclic antidepressants or beta-blockers. 
 
18. 
Use of MAO inhibitors or any medications known to interfere with the 
treatment of anaphylaxis. 
 
 
19. 
Subjects with a history of immunologic disease or undergoing immune 
suppression for cancer or other diseases.  
 
 
c. Design of Study. 
 

 
 
 
 
 
February 20, 2017 version 
 
This study is designed to evaluate the effect of nasally applied D. farinae allergen extract 
on changes in nasal lavage, peripheral blood, and nasal epithelial cells in e-cigarette users 
compared to cigarette smokers and non-smokers.  The principal endpoint is 
Δeosinophils/mL of nasal lavage fluid. Nasal allergen will be administered in a graded 
fashion distributing the dose equally between both nares with 100, 500 and 1,000 AU of 
Der f allergen extract delivered via nasal meter-dosed pump.   Single-use nasal metered-
dose pumps will be purchased from Professional Compounding Center of America, 
Houston, TX.  
 
Secondary and exploratory endpoints will include nasal fluid cytokines, chemokines, 
markers of cellular activation, and mRNA from peripheral blood cells and nasal epithelial 
cells to examine gene expression profiles before and after nasal allergen challenge. 
 
d. Dosage 
 
Dermatophagoides farinae (Der f) allergen extract dilutions will be prepared in sterile 
saline by the University of North Carolina Hospitals Investigational Drug Service.  It will 
be administered from a handheld nasal metered dose sprayer. The maximum cumulative 
dose that will be administered is 1600 AU of D. farinae extract distributed equally 
between both nares.  Symptoms will be monitored and scored at regular intervals 
following allergen challenge. The challenge protocol is outlined in section “e” below. 
 
e. Observations and Measurements 
 
Observation Schedule: 
Prior to any nasal inhalation challenge, subjects will undergo a physical examination of 
the ears, nose, throat (noting the uvula) and chest and will have an assessment of vital 
signs (temperature, pulse, respiratory rate, blood pressure), Total Nasal Symptom Score 
(TNSS) assessment and undergo spirometry to rule out acute illness prior to challenge. 
All female volunteers will undergo a urine pregnancy test. After adequate lung function 
has been documented, nasal allergen challenge will take place as outlined above.  A 
physician will be immediately available during the challenge. 
 
After nasal allergen challenge is complete, the subjects will be observed in CEMALB 
until venipuncture, nasal lavage (NL), epithelial lining fluid (ELF), and nasal epithelial 
cell (NEC) sampling are complete. Subjects will not be discharged from the CEMALB 
unit following challenge until FEV1 is within 90% of baseline values or higher. A 
physician will be on call following the challenge session for telephone consultation with 
the volunteer subject at any time following the nasal challenge.   
 
Each subject will have four study visits: 
 
Visit 0: Screening allergen challenge day studies:  
1. Consent obtained 
2. Urine pregnancy test for females of child bearing potential 

 
 
 
 
 
February 20, 2017 version 
 
3. Vital signs 
4. Medical history (including medications) and physical exam (including oral exam, 
noting the uvula), and a smoking use questionnaire will be obtained. 
5. Skin testing for D farinae sensitization (unless previously obtained). 
6. Baseline spirometry 
7. Lavage with normal saline to both nares to clear secretions and prepare for 
placement of allergen. 
8. Total nasal symptom score (TNSS) 
9. Peak nasal inspiratory flow (PNIF) measurement 
10. Graded nasal allergen challenge using both nares with TNSS after each dose.  
Nasal allergen challenge will be provided in a graded dose fashion 0 AU, 100 AU, 
500 AU and then 1,000 AU separated by 10 minutes.  Dilutions will be provided 
by the University of North Carolina Hospitals Investigational Drug Service. Over 
10 minutes, symptoms will be monitored and scored. Increasing concentrations of 
dust mite allergen dilution are deposited into the nose until the TNSS reaches a 
total of 8 or more (described below) and PNIF is reduced by at least 50%. An oral 
exam will occur prior to each dose. The cumulative allergen dose producing a 
TNSS of ≥ 8 and reduction in PNIF of ≥ 50% will be considered the provocative 
dose of allergen. Failure to achieve TNSS of ≥ 8 and reduction in PNIF of ≥ 50% 
will result in screen failure. 
11. Subjects will be monitored for 90 minutes from the last dose of nasal allergen. 
Oral and upper airway exam, vital signs and spirometry will be performed at 30 
and 60, and a physical exam at 90 minutes. At 90 minutes, if no significant 
findings on exam and no significant reduction in FEV1 (<10% reduction from 
baseline), the subject will be discharged. 
 
Post Nasal Allergen Challenge Telephone Follow up (24 hours after Visit 0)  
Subjects will be contacted for phone call follow-up 24 hours after allergen 
challenge (see phone script included in the study worksheets section) 
 
Visit 1:  Baseline visit (at least 7 days but within 2 months of screening)  
1. Review of medical history including present medications 
2. Obtain diaries detailing number of e-cig uses or number of cigarettes smoked per 
day (e-cig users and cigarette smokers only) 
3. Urine pregnancy test for females of child bearing potential.  
4. Nasal epithelial cell sampling of the right nare for mRNA to evaluate TLR2, 
TLR3, TLR4, TLR7, NLRP3, NLRP12, and CD14 expression) 
5. Discharge to home. 
 
Visit 2: Nasal Allergen Challenge visit (to occur at least 14 days after Visit 1) 
1. Review of medical history including present medications 
2. Obtain diaries detailing number of e-cig uses or number of cigarettes smoked per 
day (e-cig users and cigarette smokers only) 
3. Urine pregnancy test for females of child bearing potential, and urine for NNAL 
and cotinine.  

 
 
 
 
 
February 20, 2017 version 
 
4. Physical examination (PE to include vital signs, assessment of the head, neck, 
chest, and baseline oral exam including the uvula by study MD) 
5. Baseline TNSS and PNIF  
6. Spirometry  
7. Epithelial lining fluid (ELF) collection from both nares to measure baseline 
cytokines (ECP, Leukotrienes C4, D4, and E4; Prostaglandin D2, GM-CSF, Il-4, 
IL-8, IL-13, RANTES, Eotaxin, soluble CD14 (LPS-binding protein), TNF, IL1, 
IL6, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β) 
8. Nasal lavage of both nares for baseline assessment of nasal fluid cellularity 
9. Venipuncture for evaluation of baseline complete blood count and differential, 
Der f IgE levels, mRNA, cytokines, and flow cytometry for the following cell 
surface markers: CD11b, CD14, CD80/86, HLA-DR, TLR 4, FcεR1.  
10. Nasal allergen challenge with the previously demonstrated provocative dose of 
allergen administered with equal distribution between the nares. An oral exam 
will occur prior to administration of allergen. 
11. Post nasal challenge TNSS and PNIF measurements performed at 10, 30, and 60 
minutes then hourly for 4 hours. 
12. Post nasal challenge, vital signs, spirometry and oral and upper airway exam will 
be performed at 30, 60, and 90 minutes following allergen challenge.  At 4 hours 
a physical exam will be performed along with vital signs and spirometry. 
13. Nasal lavage, nasal epithelial cell sampling (from the left nare), ELF collection, 
and venipuncture will occur 4 hours after allergen challenge for measurements as 
noted above.  
14. If any reduction in FEV1≥10% from baseline is seen during the initial 90 minutes 
post-allergen challenge, spirometry will be repeated prior to discharge. If 
spirometry is not significantly reduced (reduction of <10% from baseline), the 
subject will be discharged. 
 
Post Nasal Allergen Challenge Telephone Follow up (24 hours after Visit 2)  
Subjects will be contacted for phone call follow-up 24 hours after allergen 
challenge (see phone script below) 
 
 
Discontinuation visit: (5-10 days after challenge) 
1.   Vital signs 
2.    PE (including oral exam) 
3.    Spirometry 
 
Endpoints 
The primary endpoint for this study is Δeosinophils/mL of nasal lavage fluid (post-
allergen challenge minus pre-allergen challenge values), comparing e-cigarette users to 
cigarette smokers and non-smokers. A secondary endpoint will be ΔEosinophilic cationic 
protein (ΔECP), a marker of eosinophil activation. Exploratory endpoints as well as 
safety criteria for stopping involvement of an individual in the protocol or suspending the 
overall study are outlined below.  

 
 
 
 
 
February 20, 2017 version 
 
 
Exploratory Endpoints 
Exploratory endpoints of interest from examination of epithelial lining fluid (ELF) and 
nasal lavage fluid collection include: 
1. Cytokines and chemokines: Leukotriene C4, D4, and E4, PGD2, GM-CSF, IL-4, 
IL-8 IL-13, RANTES, Eotaxin, soluble CD14, LPS-binding protein, 
Dermatophagoides Farinae (Der F) specific and total IgE , TNF, IL1, IL6, CCL2 
(MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β). 
 
Exploratory endpoints of interest from examination of nasal epithelial cell sampling 
include: 
2. mRNA to evaluate gene expression profiles: TLR2, TLR3, TLR4, TLR7, NLRP3, 
NLRP12, and CD14 expression. 
 
Exploratory endpoints of interest from examination of peripheral blood include: 
3. Complete blood count and differential 
4. mRNA from peripheral blood mononuclear cells (PMBCs) 
5. Cytokine profile examined in nasal fluid samples 
6. Flow cytometry for cell surface markers: CD11b, CD14, CD80/86, HLA-DR, 
TLR 4, FcεR1 
 
Safety measures 
Criteria for safety of a given individual following a specific challenge will include 
(failure of which would result in discontinuation of that subject from further study): 
 
1. No greater than 10% decrease in FEV1 from pre-challenge values following a 
nasal allergen challenge to D. farinae allergen.  
 
2. FEV1 must recover without treatment to within 85% of baseline within 6 hours of 
challenge without treatment, or if albuterol is employed the volunteer must 
recover to within 90% of baseline value within 20 minutes. All persons with a 
≥10% decrease in FEV1 or those with respiratory symptoms will be given 
albuterol. 
 
3. If albuterol therapy is needed more than three times within the first hour or more 
than 3 times during the observation period. 
 
4. Requirement of oral corticosteroid treatment due to asthma exacerbation or other 
serious allergic reaction associated with challenge. 
 
5. Evidence of upper airway obstruction, defined by the subject complaining of 
difficulty breathing, excessive salivation, changes in voice quality, and stridor. If 
an individual experiences evidence of upper airway obstruction, the subject will 
be treated with epinephrine via administration of an EpiPen device, as well as 
topical application of epinephrine if there is uvular involvement.  

 
 
 
 
 
February 20, 2017 version 
 
 
Criteria for safety within the entire protocol (failure of which would result in suspension 
of further study until consultation with the CBER of the FDA and the UNC IRB) will 
include the following: 
 
1. No more than 20 % of patients will fail the individual safety criteria outlined 
above. As it is anticipated that 45 subjects will be recruited into this study, then 9 
or more subjects who fail individual safety measures will result in suspension of 
further study until consultation with the CBER of the FDA. 
 
2. No occurrence of any Serious Adverse Event 
 
Subjects will be under direct supervision for at least 90 minutes post challenge and will 
have contact information for after-hours access to a study physician.  In the event of an 
unexpected event, such as wheezing, urticaria or other allergic symptoms, the physician 
will follow the usual medical standard of care to treat the subject.   
 
Confidentiality 
Risks to subject confidentiality will be minimized by storing records with personal 
identifiers in an office in CEMALB which is locked when unattended by the study 
coordinators. All samples will be stored with codes only (no personal identifiers). The 
CEMALB is located in the US Environmental Protection Agency’s Human Studies 
Facility on the UNC campus which has a security guard and limited access 24 hours/day, 
7 days/week. 
 
Efficacy Measure 
The primary endpoint for efficacy of the nasal allergen challenge will be 
Δeosinophils/mL of nasal lavage fluid, comparing e-cigarette users to cigarette smokers 
and non-smokers. 
 
Analysis Plan 
For analysis of nasal eosinophil responses, we will use (i) descriptive analysis and (ii) 
two-sample comparison tests. Specifically, we will use the tabular and graphic methods 
to describe/explore the basic relationship among the groups. We will then follow up by 
comparing the Δ (post-allergen)-(baseline) responses of the e-cigarette and tobacco 
smoker groups using two-sample tests, either parametric (t test) or non-parametric (Mann 
Whitney U), depending on whether the normality assumption is met. We will transform 
the data to achieve normality if needed. These analyses will be repeated for comparisons 
of e-cigarette users to non-smokers and for tobacco smokers to non-smokers. In all 
analyses, criterion for significance will be p ≤ 0.05. Dr. Haibo Zhou, the biostatistician 
for the CEMALB, will oversee all statistical analysis.  To support the primary endpoint, 
we will compare the mean change in NELF eosinophilic cationic protein (ECP) at 4 hours 
post-allergen challenge from baseline between the e-cigarette users versus tobacco 
smokers, between the e-cigarette users versus non-smokers, and between tobacco 

 
 
 
 
 
February 20, 2017 version 
 
smokers versus non-smokers using ANCOVA to adjust for baseline NELF ECP.  The 
NELF ECP AUC will be calculated using the trapezoidal rule. 
 
 
Procedures to be Performed 
Nasal allergen challenge procedure: Allergen dilutions will be prepared from a 30,000 
AU/mL extract solution purchased from Greer® Labs and loaded into nasal actuator 
devices by a licensed pharmacist in the UNC Investigational Drug Service. Specific lot 
numbers will be recorded, and CBER will be informed of any lot extract changes. The 
subject will be instructed to inhale to total lung capacity (TLC).  When at TLC, a single-
use nasal metered dose pump will then be placed at the base of the subject’s naris and 
activated delivering 100 uL of volume per spray, without an inhalation from the subject.  
After the correct dose is given, a timer will be set for 10 seconds.  At the end of the time 
the subject will be asked to gently expel excess fluid from the nose into a cup which will 
be discarded.  The technician will spray the solution sequentially distributing between 
both nares with 0 AU (glycerinated vehicle control), 100 AU (2 sprays or 200 uL of a 
solution of Der F extract of 500 AU/ml), 500 AU (2 sprays or 200 uL of a solution of 
Der F extract of 2500 AU/mL) and 1,000 AU of Der F allergen extract (2 sprays or 200 
uL of a solution of Der f extract of 5000 AU/ml). Once the solution is delivered, the 
subject will be asked to exhale.  The subject will NOT “sniff” the solution.  TNSS will be 
assessed 10 minutes after each dose of the graded nasal dose challenge. Challenge will be 
stopped when a TNSS of 8 or greater and reduction in PNIF of at least 50% are achieved, 
or the maximal dose (1,000 AU) is administered.  
 
Subjects will be attended for a minimum of 90 minutes following allergen challenge.  An 
oral exam will be performed prior to each successive dose of allergen and at 30, 60 and 
90 minutes post-allergen challenge. 
 
 
Total Nasal Symptom Score (TNSS): 
Nasal Allergen Symptom score (scale 0-none, 1-mild, 2-moderate, 3-severe): 
Sneezing____, Congestion____, Nasal Itching_____, Rhinorrhea (Runny nose) ____,  
 
The highest possible score will be 12 and the lowest is 0.  A mild score for every criteria 
is 4, moderate is 8 and severe is 12. 
 
Symptom score describer definitions: 
 
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”) 

 
 
 
 
 
February 20, 2017 version 
 
 
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). 
 
Follow-up Symptom Score (administered by telephone for post-challenge observation): 
1. On a scale of 0 to 3, with 0=no symptoms, 1=mild symptoms, 2=moderate symptoms, 
and 3=severe symptoms, please rate the severity of each of the following symptoms:  
 
Sneezing (0-3)____ 
Congestion (0-3) ____ 
Nasal Itching(0-3) _____ 
Rhinorrhea(0-3)______ 
 
2. Do you have any health concerns since we saw you on the day of the challenge? 
 
3. Have you needed to see a doctor for any reason since your last visit? 
 
4. Have you needed to use any over the counter medications? 
 
5. Have you had any specific problems with nasal discomfort, cough, wheezing or have 
you needed any extra allergy or asthma medicines? 
 
If the symptom score is  6 (out of 12), if any single symptom is scored at 3, or if the 
answers to questions 2, 3, 4, or 5 are “yes”, then subjects will be offered the opportunity 
to come back to the laboratory for follow-up assessment. 
 
Nasal lavage procedure:  
Both nares will be lavaged. A standard amount of saline will be sprayed into each nare 
using a nasal meter dosed sprayer which delivers 100ul/actuation. Each lavage is 
comprised of eight sets of five sprays.  Lavage fluid will be recovered by forceful 
expulsion of lavage fluid into a specimen cup immediately after each set of 5 actuations. 
Samples will then be transported on ice to the laboratory where slides for differential cell 
counts of uncentrifuged NLF will be made.  The remainder of each specimen will be 
centrifuged and stored at -70o C until further analysis.   
 
Nasal epithelial lining fluid (ELF) collection procedure: 
ELF is obtained by spraying the nostril with 0.9% sterile, normal saline irrigation 
solution (about 100 μl per nostril) one time per nostril.  One 10x55mm strip of filter 
paper, cut from Leukosorb paper (Pall Scientific, Port Washington, NY) on a laser cutter, 
is inserted into the anterior part of the inferior nasal turbinate of each nostril.  The nostrils 
are clamped shut using a padded nose clip for two minutes.  Strips are then removed from 
the nostril and collected in 1.5ml tubes.  They are then stored in strips in a -20°C freezer 
until elution.  

 
 
 
 
 
February 20, 2017 version 
 
 
Venipuncture:  
Venous blood will be obtained by venipuncture for various exploratory endpoints and for 
cotinine and NNAL levels to detect recent tobacco or e-cigarette use. 
 
Spirometry and Peak Expiratory Flow (PEF) Measurement: Standard methodology 
conforming to the American Thoracic Society guidelines for measurement of spirometry 
and peak exploratory flow will be used.   
 
Allergy Skin Testing: 
Allergy skin testing is performed epicutaneously, examining wheal and flare reactions to 
histamine, a glycerol control, and various allergens. A Greer® Pick® device that has 
been immersed in a solution of allergen extract purchased from Greer® Laboratories is 
used to apply allergen epicutaneously by placing the device at a 45 degree angle to the 
skin and gently lifting up to create a skin prick. Reagents are obtained from Greer 
Laboratories and tests are performed as outlined for FDA approved procedures for 
evaluation of allergy. All testing instruments are single patient use. Each patient will 
undergo skin prick testing with the following allergen preparations: House Dust Mite (D. 
farinae), House dust mite p (D. pteronyssinus), Cockroach, Tree mix, Grass Mix, Weed 
Mix, Mold Mix 1, Mold Mix 2, Rat, Mouse, Guinea Pig, Rabbit, Cat and Dog.  The 
allergens will be placed on the forearm.  After 15 minutes, a research coordinator will 
compare the wheal size (a raised bump surrounded by red itchy skin) for each allergen to 
that of the negative and positive control (histamine). A positive skin test is defined by a 3 
mm wheal greater than the saline control. For a volunteer to be considered non-allergic, 
the histamine control must be reactive and all others must be negative. 
 
Nasal Epithelial Biopsy:   
This is a non-invasive biopsy procedure performed to retrieve a small cluster of cells 
from each of the nasal cavities.   For the nasal biopsy procedure, the subject will be 
seated comfortably in a straight-backed chair or reclining on an examination table with 
the head tilted as far back as possible while remaining comfortable.  Subjects will be 
given no medication, sedation, or anesthesia for this procedure.  A short, sterile plastic 
sampling device called a curette will be inserted into one of the nasal cavities and the 
surface of the nasal cavity will be stroked several times for approximately 5 seconds in 
order to obtain a small cluster of cells. Due to the potential for the biopsy itself to create a 
local inflammatory response, we will ensure that visit 2 occurs no sooner than 14 days 
after visit 1 to allow for dissipation of this inflammatory response before collection of 
nasal fluid samples. We will sample nasal epithelium from the right nare during visit 1 
and from the left nare during visit 2 to ensure we obtain adequate epithelial samples on 
each occasion. 
 
Table of Study Procedures 
 Procedures to be performed at various time points are listed in the following table and in 
the accompanying study worksheets. 
 

 
 
 
 
 
February 20, 2017 version 
 
 
Screening 
day 
24 hrs 
post 
challenge 
(phone) 
Baseline 
Visit 
Allergen 
Challenge 
day 
24 hrs 
post 
challenge 
(phone) 
Discontinuation 
visit 
Consent 
X
Collect smoking diaries (if 
applicable)
 
 
X 
X 
 
 
Review history/AE’s 
X 
X 
 
X 
X 
X 
Follow up symptom score 
 
X 
 
 
X 
X 
Urine HCG (prior to 
challenge)
X 
 
X 
X 
 
 
Vital signs 
X 
 
X 
X 
 
X 
Spirometry/PEF 
X 
 
 
X 
 
X 
Physical exam 
X
X
X
Total nasal symptom score 
X
X
Peak nasal inspiratory flow 
rate
X 
 
 
X 
 
 
Allergen challenge 
X 
 
 
X 
 
 
Nasal lavage 
 
 
 
X 
 
 
Nasal epithelial cell 
collection
 
 
X 
X 
 
 
Epithelial lining fluid 
collection
 
 
 
X 
 
 
Venipuncture 
X
X
 
Post Challenge Observations: Reporting 
Within 24 hours after allergen challenge, each volunteer will be evaluated using the 
Follow up Symptom Score described above. If the symptom score is 6 (out of 12), if any 
single symptom is scored at 3, or if the answers to questions 2, 3, 4, or 5 are “yes”, then 
subjects will be offered the opportunity to come back to the laboratory for follow-up 
assessment. 
 
Study discontinuation visit:  
Within 10 days of the final challenge dose, each subject will be asked to return for a 
study discontinuation visit. At that time temperature, pulse, systolic and diastolic BP, 
respiratory rate, FVC and FEV1, and symptoms scores will be assessed and, if abnormal, 
medical evaluation as directed by the study physician will be undertaken.  
 
Definition of an adverse event (AE) and Serious Adverse Event (SAE): 
An adverse event for a given volunteer will be defined as failure of any of the safety 
criteria outlined in section “e”. Additionally, minor upper respiratory tract infections 
occurring within 96 hours of the exposure will be considered adverse events.  Other, non-
specified clinical illnesses, which occur within 96 hours of each challenge, will also be 
reported as an AE.  Any decrease in lung function or increase in symptom score, as 
outlined in section “e” will be considered an adverse event. Any symptoms that induce a 

 
 
 
 
 
February 20, 2017 version 
 
volunteer to seek medical attention from any provider within 96 hours of challenge will 
be considered an adverse event. 
 
Adverse events will be graded using Common Terminology Criteria for Adverse Events 
v3.0, (CTCAE website at http://ctep.cancer.gov/reporting/ctcnew.html and 
http://safetyprofiler-ctep.nci.nih.gov/CTC/CTC.aspx). Symptom scores will be graded as 
outlined elsewhere in this application. 
  
The severity grades of AE’s are defined as follows: 
Grade 1: Mild AE  
Grade 2: Moderate AE 
Grade 3: Severe AE  
 
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 challenge (or if the clinical 
scenario leading up to hospitalization, illness, injury or death begins within 96 hours of a 
challenge).   
 
f. Monitoring and risk minimization 
 
Risks to subjects: In previous studies performed in our laboratory (three published 
studies), which includes over 70 nasal allergen challenges in volunteers with mild allergic 
asthma, approximately half of these attained the 1000 AU dose (with a total cumulative 
dose of 1100 AU). The most commonly encountered (and anticipated) effect was 
increased nasal allergic symptoms. These were typically short term symptoms (resolved 
within 24 to 48 hours) which rarely required a short course of antihistamines.  While 
these studies did not involve regular measures of lung function after nasal challenge, 
there were no complaints of increased bronchial symptoms and no subjects required 
inhaled bronchodilators or inhaled or oral corticosteroids. There has been no episode of 
systemic anaphylaxis or acute bronchospasm associated with nasal allergen challenge.  
 
One subject had an episode of allergic uvulitis following nasal allergen challenge 
(discussed further in section 9, Previous Human Experience).  We suspect that the 
sniffing that occurred with the challenge may have contributed to that risk, in addition to 
the subject having laid recumbent immediately after the nasal allergen challenge was 
finished.  Therefore, in this study the subject will not sniff, rather the allergen will be 
delivered to the nose in such a fashion as to avoid deposition of the allergen in the 
pharynx.  Additionally, subjects will be advised not to lie recumbent for 4 hours after the 
completion of the last challenge dose. 
 
The most likely and significant risk to subjects will be increase in allergic rhinitis 
symptoms, including congestion, rhinorrhea, nasal itching and sneezing. Other 
possible, although unlikely, effects include increased cough, and increased 
occurrence and severity of bronchospasm.  Hypothetical risks, which we have not 

 
 
 
 
 
February 20, 2017 version 
 
observed in our previous studies, include generalized allergic symptoms, including 
urticaria, flushing, hypotension and anaphylaxis.  
 
Measures to minimize risk: Only people with allergic rhinitis and/or mild asthma, as 
characterized the NHLBI guidelines for the diagnosis and management of asthma, will be 
recruited for this project. Please note that our exclusion criteria for subjects include 
symptom or medication use criteria that would result in classification of an asthmatic as 
moderate or severe. Further, subjects will be deferred for challenge until 4 weeks after 
complete resolution of each of the following acute illnesses:  viral respiratory tract 
infection, pneumonia or bronchitis requiring antibiotic therapy (must be off antibiotics 
and well for 2 weeks after the last dose of antibiotics, 4 weeks in the case of azithromycin 
due to its prolonged half-life), or acute illness resulting in fever.  Also, unspecified 
illnesses, which in the judgment of the investigator increase the risks associated with 
allergen inhalation challenge, will be a basis for exclusion. As outlined above in section 
e, all subjects will need to fulfill objective lung function and symptom criteria prior to 
initiating a specific challenge study.  
 
A physician familiar with the protocol will be available for all challenge procedures. 
Emergency treatment with topical and intramuscular epinephrine, albuterol, oxygen, 
antihistamines and oral corticosteroids will be available to those patients who require 
such therapy.  The human exposure laboratory is also equipped with an emergency “crash 
cart” with standard emergency medications, IV fluids and a defibrillator in the unlikely 
event of a medical emergency for any challenge study.  
 
All subjects will be will be monitored as outlined in previous sections. A follow-up call 
will be made 24 hours after challenge to ensure that subjects are well, and all subjects 
will be provided with a contact telephone number for access to a study physician who is 
on call 24 hours/day.   Subject may be withdrawn from the study at any point if the 
physician determines it is in the best interest of the subject. 
 
Reporting of AEs and SAEs 
  
All SAEs will be reported to the CBER of the FDA as well as to the UNC Biomedical 
IRB within 24 hours of recognition of the event. Adverse events will be reported to both 
the FDA and UNC IRB on no less than a quarterly basis, or when the protocol is 
completed.  If criteria for suspension of the protocol are met, then the FDA and UNC IRB 
will be notified within 24 hours.  
 
All subjects with a non-fatal SAE will be evaluated medically by a study physician, in 
concert with their own physician as appropriate. Likewise all subjects with an adverse 
event will be examined and evaluated by a study physician. All assessments will include 
the same lung function and vital sign assessments outlined for challenge observation. 
Other assessments will be undertaken as needed. Any unspecified event, which in the 
judgment of the PI of the study, constitutes an unusual, unexpected or prolonged event 
(greater than 96 hours) will be reported to both the FDA and the UNC IRB.  

 
 
 
 
 
February 20, 2017 version 
 
 
Curriculum Vitae for investigators are appended to this application. 
 
6.2. Informed Consent 
Investigators and study staff will explain all study procedures and the benefits and risks 
of the study to potential participants as part of obtaining informed consent. Subjects may 
withdraw their consent at any time during the study.  If they withdraw from the study it 
will not impact the care they receive at UNC or its affiliated hospitals and clinics.  
 
 
 
 
 
 
6.3. Investigators, Facilities, and Institutional Review Board 
 
Role in project 
Name and Address 
Title 
Principal 
Investigator 
Michelle L. Hernandez, MD 
CEMALB, 104 Mason Farm Road 
The University of North Carolina 
CB#7310  
Chapel Hill, NC 27599-7310  
Associate Professor of Pediatrics 
Chief Medical Officer, Center 
for Environmental Medicine, 
Asthma and Lung Biology 
Co-investigator 
Robert Tarran, PhD 
7102 Marsico Hall 
The University of North Carolina 
CB#7248 
Chapel Hill, NC 27599-7248 
Associate Professor of Cell 
Biology and Physiology 
Director, UNC Center for 
Tobacco Regulatory Science and 
Lung Health 
Co-Investigator 
David B. Peden, MD, MS 
CEMALB, 104 Mason Farm Road 
The University of North Carolina 
CB#7310  
Chapel Hill, NC 27599-7310 
Professor of Pediatrics & 
Director, Center for 
Environmental Medicine, 
Asthma and Lung Biology 
Co-Investigator 
Allison J. Burbank, MD 
CEMALB, 104 Mason Farm Road 
The University of North Carolina 
CB#7310 
Chapel Hill, NC 27599-7310 
Post-doctoral fellow 
UNC School of Medicine 
Department of Pediatrics 
Division of Allergy, 
Immunology, and Rheumatology 

 
 
 
 
 
February 20, 2017 version 
 
Co-Investigator 
Charity G. Duran, PhD 
CEMALB, 104 Mason Farm Road 
The University of North Carolina 
CB#7310 
Chapel Hill, NC 27599-7310 
Post-doctoral fellow 
UNC School of Medicine 
Department of Pediatrics 
Division of Allergy, 
Immunology, and Rheumatology 
Co-Investigator 
Chanchaldeep (Amika) K Sood, 
M.D. 
CEMALB, 104 Mason Farm Road 
The University of North Carolina 
CB#7310 
Chapel Hill, NC 27599-7310 
Post-doctoral fellow 
UNC School of Medicine 
Department of Pediatrics 
Division of Allergy, 
Immunology, and Rheumatology 
Facilities. Volunteers for these studies will be recruited, screened and undergo challenge 
procedures at the Center for Environmental Medicine, Asthma and Lung Biology, 
CB#7310, 104 Mason Farm Road, University of North Carolina at Chapel Hill, Chapel 
Hill, North Carolina 27599-7310. All necessary clinical research equipment, medical 
equipment, and laboratory equipment is located within the Center. 
Institutional Review Board. This study and all changes will be reviewed and approved by 
the Biomedical IRB for the UNC School of Medicine, The University of North Carolina 
at Chapel Hill CB# 7097 720 Martin Luther King, Jr. Blvd. Bldg# 385, Second Floor 
Chapel Hill, NC 27599-7097