Screening Inhaled Allergen Challenge for Dermatophagoides Farinae
Phase: EARLY_PHASE1
Conditions: Asthma
Sponsor: University of North Carolina, Chapel Hill

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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                                                                                   NCT03049111 
 
 
 
 
 
 
 
IDENTIFICATION OF ALLERGIC 
ASTHMATICS REACTIVE TO 
DERMATOPHAGOIDES FARINAE (HOUSE 
DUST MITE) INHALATION 
 
 
 
Regulatory Sponsor/ 
Primary Investigator:  
 
 
Michelle Hernandez, MD 
 
 
 
 
 
 
CEMALB 
 
 
 
 
 
 
104 Mason Farm Rd 
 
 
 
 
 
 
The University of North Carolina 
 
 
 
 
 
 
CB #7310 
 
 
 
 
 
 
Chapel Hill, NC 27599-7310 
 
 
 
 
 
 
(919) 962-5136 
 
 
Study Product: 
Standardized house dust mite 
(Dermatophagoides farinae) allergen extract 
at 10,000 allergen units (AU)/mL provided 
by Greer Laboratories, Lenoir, NC. 
 
 
 
 
 
 
 
 
 
 
Version:  
 
May 13, 2019 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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List of Abbreviations 
 
AE: Adverse Event 
AHR: Airway Hyperreactivity 
AU: Allergen Units 
AUC: Area Under the Curve 
CBC: Complete Blood Count 
CBER: Center for Biologics Evaluation and Research 
CEMALB: Center for Environmental Medicine, Asthma and Lung Biology 
CTRC: Clinical Translational Research Center 
DerF: Dermatophagoides farinae (house dust mite) 
ELISA: Enzyme-linked Immunosorbent Assay 
ENO: exhaled nitric oxide 
EPR: Early phase response 
FEV1: Forced Expiratory Volume in 1 Second 
FRC: Forced Residual Capacity 
FVC: Forced Vital Capacity 
HCG: Human Chorionic Gonadotropin  
HRV: Heart Rate Variability 
IgE: immunoglobulin E 
IL: Interleukin 
LAIV: Live Attenuated Influenza Virus 
LDMS: Lab Data Monitoring System 
LPR: Late phase response 
MDI: Metered Dose Inhaler 
ML: Milliliters 
MMHG: Millimeters of Mercury 
NACL: Sodium Chloride 
NO: Nitric Oxide 
NSAIDS: Non-steroidal Anti-inflammatory Drugs  
NSBR: Non-specific Bronchial Reactivity 
PD20: provocative dose of allergen resulting in ≥20% fall in FEV1 
SAE: Serious Adverse Event 
SOP: Standard Operating Procedures 
TH1/TH2: T Helper Cell Type 1/2 
TSLP: Thymic Stromal Lymphopoetin 
 
 
 
 
 
 
 
 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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1. Introduction 
This document is a protocol of a human research study. This study is to be conducted 
according to US and international standards for Good Clinical Practice (FDA Title 21 
part 312 and International Conference on Harmonization guidelines), applicable 
government regulations and Institutional research policies and procedures. 
 
1.1 Background and Rationale 
Asthma is an increasingly common chronic illness among children and adults (1), and 
allergen exposure is among the most common triggers for asthma exacerbations. 
Exacerbations of allergic asthma are characterized by an early phase response (EPR), 
mediated by release of preformed mediators like histamine from mast cells, and a late phase 
response (LPR) 3-7 hours later mediated by chemokines and cytokines that attract 
leukocytes such as neutrophils and eosinophils to the airways, increase mucus production, 
trigger airway smooth muscle contraction, and result in airway constriction and airway 
hyperreactivity (AHR). The LPR does not occur in the absence of an EPR. The LPR is 
thought to be predominantly responsible for the symptoms associated with acute 
exacerbations of allergic asthma and is often used as the measure of efficacy in trials of 
asthma therapeutics.  
O’Byrne and others have used allergen challenge to understand the pathophysiology of 
asthma and screen asthma interventions (2-6). This technique has been extensively used to 
screen a number of new asthma agents in Phase IIa studies. This list includes anti-IL5 (7), 
anti-IgE (8, 9), anti-TSLP (10), and a number of other asthma agents (4, 11-15). In a recent 
review, Gauvreau et al (16) have noted that no currently available effective asthma agents 
failed to demonstrate a physiologically important effect in Phase 2a proof of concept 
studies using an inhaled allergen/LPR model in mild allergic asthmatics. Overall, allergen-
inhalation challenge in mild asthmatic subjects has a very high negative predictive value 
and a reasonable positive predictive value for screening novel therapeutics for asthma for 
Phase III pivotal studies. 
 
Our lab has taken a particular interest in targeting an inflammatory cytokine, Interleukin-
1β, involved in both the early and late phase asthmatic responses to inhaled allergen in 
allergic asthmatics. In the lung, IL-1β is produced by numerous cell types (including 
epithelial cells, macrophages, neutrophils, eosinophils, and mast cells), where it signals 
through its receptor to induce transcription of pro-inflammatory genes (17-19). IL-1β is 
increased in bronchoalveolar lavage fluid from persons with symptomatic asthma vs. 
those with asymptomatic asthma (20); likewise, immunohistochemistry of bronchial 
biopsies of allergic asthmatics reveal increased expression of IL-1β in both bronchial 
epithelial cells and macrophages(18).  Previous studies in animal and in vitro models 
demonstrate that IL-1β can directly impact three aspects of an airway inflammatory 
response: 1). granulocyte (neutrophil/eosinophil) recruitment (21, 22); 2). non-specific 
(23, 24) and allergen-specific airway reactivity (25, 26); and 3). production and clearance 
of airway mucous (27, 28).  Supporting literature and our preliminary studies in human 
subjects further promote the study of IL-1 blockade for mitigating features of acute 
allergen-induced asthma exacerbation.   

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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The role of IL-1 in allergen challenge models has not been fully defined. In a study 
examining 12 asthmatics allergic to D. farinae at our research center, we found that 9/12 
asthmatics had a greater than 10% reduction in forced expiratory volume in 1 second 
(FEV1) after inhaled dust mite challenge (29). These individuals were considered 
responders. It was notable that when comparing post-allergen levels of cytokines between 
responders and non-responders there was a much greater concentration of IL-1β in post-
challenge sputum from responders vs. nonresponders, Furthermore, within the responders, 
post challenge IL-1β also significantly correlated with sputum eosinophil concentrations 
(r=0.83, P<0.05) and neutrophil concentrations (r=0.89, P<0.05) 24 hours after allergen 
challenge. These data suggest that IL-1 may play a role in both immediate airway 
hyperresponsiveness and the late phase recruitment of inflammatory cells (neutrophils and 
eosinophils) after inhaled allergen challenge. 
 
Numerous IL-1 blocking agents are FDA-approved for conditions where the IL-1β 
pathway predominates disease pathophysiology such as in systemic juvenile idiopathic 
arthritis and the cryopyrin-associated periodic syndromes (30).  Anakinra is a FDA-
approved recombinant form of human IL-1 receptor antagonist (IL-1RA), a natural anti-
inflammatory cytokine that competes with agonist binding to the IL-1 receptor, 
suppressing IL-1β and IL-1 signaling.  With a fast onset of action, reaching peak 
concentrations in 3-7 hours, and a short 4-6 hour half-life, anakinra is an ideal candidate 
to test as a rescue treatment for acute asthma exacerbation.  
The purpose of this study is to identify subjects with mild allergic asthma who 
experience a LPR after inhalation of Dermatophagoides farinae (DerF). Additionally, 
in order to better understand the role of IL-1β in allergen-induced airway inflammation, 
we will obtain induced sputum to determine if higher baseline sputum IL-1β 
concentrations or larger increases in IL-1β following allergen challenge impact non-
specific airway hyperresponsiveness (via methacholine challenge), sputum granulocyte 
recruitment (neutrophil and eosinophil counts and exhaled nitric oxide (eNO), a marker 
of airway eosinophilia), sputum mucin content (MUC5AC, MUC5B), markers of 
systemic inflammation (inflammatory cytokines, absolute neutrophil or eosinophil counts 
in peripheral blood), or changes in expression of inflammatory or allergy-related genes. 
To this last point, little is known about the mechanisms contributing to response patterns 
in allergic asthmatics undergoing allergen challenge. We are particularly interested in 
gene expression changes occurring during the window of time between the EPR and 
LPR, as these expression changes may dictate whether or not a LPR occurs or to what 
extent it occurs (31, 32).  
 
Once identified, these subjects will be eligible for participation in two randomized, 
double-blinded, placebo-controlled clinical trials of Anakinra vs placebo administered 
after allergen challenge for mitigation of features of an asthma exacerbation, including 
maximum drop in FEV1 during the LPR, sputum granulocyte recruitment, mucous 
production and airway mucous clearance. The sample size needed to provide adequate 
power for the primary endpoint of each Anakinra study (maximum % fall from baseline 
in FEV1 during the LPR) is 12 subjects per study. Hence the goal of this screening 
protocol is to identify at least 25 subjects who will be eligible for enrollment in the 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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Anakinra protocols, which will be fully developed after our grant funding becomes 
available from the NHLBI. During the early phase of this project while we are 
developing the Anakinra protocol, our goal is to begin the process of screening allergic 
asthmatics for reactivity to DerF, specifically identifying those who exhibit both an EPR 
and LPR and can be later included in the Anakinra protocols.  
 
1.2 House Dust Mite allergen for challenge 
Standardized house dust mite (D. farinae) allergen extract at a potency of 10,000 AU/mL 
purchased from Greer Laboratories, Lenoir, NC.  
 
1.3 Dose Rationale and Risk/Benefits 
Dermatophagoides farinae (Der F) allergen extract dilutions will be prepared by the 
University of North Carolina Hospitals Investigational Drug Pharmacy in sterile saline by 
a licensed pharmacist and administered by a study coordinator from a DeVilbiss Ultraneb 
646 ultrasonic nebulizer.  Doses will be made up no more than 24 hours prior to 
administration. Doses of allergen will include 5 inhalations of allergen at concentrations 
of 0.25, 0.50, 1.0, 2.0, 4.0, 8.0, 16, 32, 64, 125, 250, 500, 1000, and 2000 AU/mL. Using 
an activation pressure of 20 lbs/sq. inch and duration of 0.6 sec of aerosolization, the 
nebulizer delivers 0.02 to 0.03 ml of solution.  Each volunteer will inhale 5 breaths from 
the nebulizer at each concentration starting with saline control. Increasing (doubling) 
concentrations will be inhaled until the Forced Expiratory Volume in 1 Second (FEV1) 
falls by >20% from the post saline value, the highest concentration has been inhaled, or 
the subject experiences discomfort or anxiety sufficient for the investigator or the subject 
to consider further testing unacceptable. The dose of allergen required to elicit a >20% 
drop in FEV1 from the post-saline value at the screening visit will be defined as the 
provocative dose (PD20) and marks the onset of the EPR.  The challenge will be stopped 
once the PD20 is reached. Following challenge completion, symptom scores and 
spirometry will be repeated every 10 minutes until 30 minutes, each 30 minutes until 2 
hours, each hour until 10 hours, and again the next day. The subject will be discharged 10 
hours following allergen challenge when clinically stable and when FEV1 is within 
approximately 10% of the pre-allergen baseline value. All subjects will be provided with 
rescue medications, including a short-acting bronchodilator inhaler with an aerochamber 
and oral prednisone 60 mg, at the time of discharge with detailed instructions for use in 
the event of symptoms related to bronchoconstriction and will return the following 
morning for reassessment.  
 
This dosing regimen is based on that employed in two previous studies of mild allergic 
asthmatic volunteers [4,5] defining PD20.  We have also conducted another study of 14 
subjects in which the maximum dose employed was 2,560 AU/ml to establish a PD20 
response [50].  
 
At the doses proposed in this study, the most significant predictable risk to subjects as a 
result of allergen inhalation is development of immediate airway obstruction 
(bronchospasm) during the EPR, which would cause shortness of breath, cough and/or 
wheeze. This drop in FEV1 during the EPR should be short lived (maximal 10 to 30 
minutes after allergen), and reversible with medications such as beta agonists (albuterol). 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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This effect is similar to the effect of methacholine, which is an FDA-approved agent 
employed for the diagnosis of airway reactivity and asthma.   
 
Other possible risks include development of a LPR to allergen inhalation. This occurs in 
approximately 50% of asthmatics challenged, consisting of similar symptoms described 
for the EPR.  Since a key endpoint of this study is to identify which subjects have a LPR, 
albuterol for rescue prior to the 10 hour post challenge time point will only be employed 
if the FEV1 does not show spontaneous improvement, and/or if the subject has distress 
necessitating rescue. All subjects will be given 4 puffs of albuterol at 10 hours after the 
inhaled allergen challenge.   
 
2. Study Objective 
Primary Objective: The purpose of this study is to identify subjects with mild allergic 
asthma who display a late phase response (LPR) after inhalation of Dermatophagoides 
farinae (DerF) for potential participation in two planned studies of interleukin-1 receptor 
antagonist (Anakinra) therapy against features of allergen-induced asthma exacerbation.  
 
3. Study Design 
 
3.1 General Design 
This will be a non-blinded study to identify mild allergic asthmatics with measurable 
LPR to inhaled Dermatophagoides farinae allergen extract. The principal endpoint will 
be the presence of a LPR, defined as a decline in FEV1 of ≥15% from baseline values 3-
10 hours after the onset of the EPR (which typically occurs within 30 minutes of allergen 
challenge).  
 
Prior to enrollment, subjects will undergo a screening visit to establish eligibility (part of 
a separate screening protocol IRB#98-0799). Study visits will consist of a baseline visit 
to determine baseline values for study endpoints (Visit 1), a pre-allergen challenge visit 
to determine pre-challenge values for study endpoints (Visit 2), an allergen challenge 
visit (Visit 3), a visit 24 hours post-challenge to evaluate symptoms (Visit 4), and a final 
study discontinuation visit 5-10 days following allergen challenge. See Table in section 
5.1.7. 
 
3.2. Primary Study Endpoints 
The principal endpoint of this screening inhaled allergen challenge protocol will be to 
identify those subjects who experience a greater than or equal to 15% drop in FEV1 from 
baseline within 3-10 hours after the early phase response. 
 
3.3 Secondary Study Endpoints 
In addition to identifying subjects for participation in the planned studies of Anakinra vs 
placebo for mitigating features of asthma exacerbation, the additional purpose of this 
study is to determine if IL-1β concentrations in the sputum at baseline before challenge 
are predictive of key asthma outcomes following inhaled allergen challenge, specifically: 
 
a) maximum drop in %FEV1 during LPR 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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b). Change in methacholine reactivity from baseline pre-allergen measurement to 
 
24 hours after allergen challenge. 
 
c). exhaled nitric oxide (eNO) level 
 
d). induced sputum: 
i. 
Granulocyte (neutrophils/eosinophils) numbers and percentages 
ii. 
Mucins (secretion of MUC5AC and MUC5B determined by Western blot 
and ELISA) 
 
Additionally, as exploratory endpoints, we will assess the effects of allergen challenge on 
markers of systemic inflammation and changes in inflammatory gene expression as 
described below: 
a). venipuncture for assessment of systemic inflammatory  markers (CBC with 
differential for absolute neutrophil count and absolute eosinophil count), pro-
inflammatory cytokine levels such as IL-1β, IL-6, IL-8)  
 
b). gene array analysis from blood and induced sputum at the baseline visit, 
again on the day of the challenge and, finally, at 24 hours post challenge.  To 
identify signaling pathways potentially impacted by allergen challenge, we will 
use Nanostring® technology to measure differential expression of a panel of 594 
immunology-related genes. This will be accomplished by collecting and isolating 
peripheral blood mononuclear cells from venous blood samples collected before 
allergen challenge, during allergen challenge, and 24 hours after allergen 
challenge.  
c). Heart rate variability.  We will place an ambulatory heart rate variabilty 
monitor on subjects before the challenge and monitor the heart rate for the 
duration of the post challenge monitoring, to investigate any acute cardiac 
changes. 
 
3.4 Primary Safety Endpoints 
 
3.4.1. Criteria to be met prior to initiation of allergen challenge  
(Subjects not meeting these criteria on the morning of the challenge will not proceed with 
the allergen challenge). 
 
1.   FEV1 of at least 80% of predicted and a ratio of FEV1 to Forced Vital Capacity 
(FVC) of at least .70 (without use of bronchodilating medications for 8 hours, 
anticholinergic therapy (ipratropium ) within 2 hours, or antihistamines within 5 
days prior to challenge). 
 
2. Baseline oxygen saturation of at least 95%. 
 
3. No history of asthma exacerbation requiring systemic corticosteroid treatment, 
visit to an emergency room or hospitalization within 12 months of inhalation 
challenge. 
 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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4. No current history of requiring short-acting beta agonists more frequently than 
every 8 hours for asthma symptoms.  Any use of albuterol in the week prior to 
allergen challenge will be reviewed by the study physician. 
 
5. No history of viral respiratory tract symptoms within 4 weeks of challenge. 
 
3.4.2. Criteria for safety within the entire protocol  
(failure of which would result in subject withdrawal or suspension of further study) will 
include the following: 
 
1. No occurrence of any Serious Adverse Event 
 
2. No more than 20% of subjects will fail the individual safety criteria outlined below. 
As it is anticipated that 100 subjects will be recruited for this screening protocol, then 
20 or more subjects who fail individual safety measures will result in suspension of 
further study until consultation with the CBER of the FDA. Additionally, in order to 
further monitor safety, we will assess this after completion of 20 subjects, and 
annually. 
 
a. 
Less than or equal to 40% decrease in FEV1 or FVC from pre-challenge 
values following EPR or the LPR to Dermatophagoides farinae allergen 
(the target endpoint is a >20% decrease in FEV1 from baseline during the 
EPR and a >15% decrease in FEV1 from baseline during the LPR).  
 
b. 
If albuterol is employed as rescue, FEV1 must recover to within 90% of 
baseline value within 20 minutes. 
 
c. 
Oxygen saturation should remain >93% throughout the challenge period and 
must be no lower than 2% of baseline measure. 
 
d. 
Albuterol for rescue therapy should be required no more than three times 
within the first hour after challenge  
 
e. 
Albuterol for rescue therapy should be required no more than three times 
within 12 hours of discharge.  
 
f. 
FEV1 should return to within 90% of baseline value at the study 
discontinuation visit which occurs within 10 days after allergen challenge. 
 
g. 
No more than 1 dose of oral corticosteroid (prednisone 60mg) should be 
required to treat an asthma exacerbation associated with inhalation challenge  
 
4. Subject Selection  
 
One hundred mild extrinsic (allergic) non-smoking asthmatics ages 18-45 will be 
recruited. There will be no gender or ethnic restrictions. Prior to enrollment in this 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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protocol, subjects will have undergone allergy skin testing as part of our IRB-approved 
general screening protocol. Only extrinsic asthmatics sensitive to house dust mite (D. 
farinae) will be selected for participation.  Spirometry will be performed to determine the 
current level of lung function. Those with an FEV1 of at least 80% predicted and an FEV1 
to FVC ratio of at least .70 off bronchodilators for at least 8 hours will have previously 
completed a standard graded dose methacholine bronchoprovocation challenge test to 
determine NSBR during the IRB-approved general screening protocol.  
 
4.1 Inclusion Criteria 
1. Age range 18-45 years, inclusive 
2. FEV1 of at least 80% of predicted and FEV1/FVC ratio of at least 0.7 (without use 
of bronchodilator medications for 8 hours or long acting beta agonists for 24 
hours), consistent with lung function of persons with no more than mild 
intermittent or mild persistent asthma.  
3. Physician diagnosis of asthma 
4. Allergic sensitization to house dust mite (D. farinae) as confirmed by positive 
immediate skin prick test response 
5. Negative pregnancy test for females who are not s/p hysterectomy with 
oophorectomy or who have been amenorrheic for 12 months or more. 
6. Oxygen saturation of >94% and blood pressure within the following limits: 
Systolic between 150-90 mmHg and Diastolic between 90-60 mmHg. 
 
4.2 Exclusion Criteria 
1. Clinical contraindications: 
a. Any chronic medical condition considered by the PI as a 
contraindication to participation in the study including significant 
cardiovascular disease, diabetes, chronic renal disease, chronic thyroid 
disease, history of chronic infections or immunodeficiency.  
b. Physician directed emergency treatment for an asthma exacerbation 
within the preceding 12 months. 
c. Exacerbation of asthma more than 2x/week which could be 
characteristic of a person of moderate or severe persistent asthma as 
outlined in the current NHLBI guidelines for diagnosis and 
management of asthma. 
d. Daily requirements 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). 
e. Viral upper respiratory tract infection within 4 weeks of challenge. 
f. Any acute infection requiring antibiotics within 6 weeks of exposure 
or fever of unknown origin within 6 weeks of challenge. 
g. Severe asthma 
h. 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. 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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i. Cigarette smoking >1 pack per month 
j. 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. 
k. Allergy/sensitivity to study drugs or their formulations 
l. Known hypersensitivity to methacholine or to other 
parasympathomimetic agents 
m. History of intubation for asthma 
n. Unwillingness to limit coffee, tea, cola drinks, chocolate, or other 
foods containing caffeine after midnight on the days that methacholine 
challenge testing is to be performed. 
o. Unwillingness in females to use reliable contraception if sexually 
active (IUD, birth control pills/patch, condoms). 
 
2. Pregnancy or nursing a baby. Female volunteers will be asked to use effective 
birth control (stable regimen of hormonal contraceptive use for at least 3 
months, intrauterine device placement, tubal ligation or endometrial ablation 
for at least 3 months through at least one week after study completion) and 
will provide a urine sample to test for pregnancy on study days. If the test is 
positive or the subject has reason to believe she may be pregnant, she will be 
dismissed from the study. Women who have been amenorrheic for 12 months 
may participate.  
 
3. Usage of the following medications: 
a. Use of systemic steroid therapy within the preceding 12 months for an 
asthma exacerbation. All use of systemic steroids in the last year will 
be reviewed by a study physician. 
b. Subjects who are prescribed daily inhaled corticosteroids, cromolyn, or 
leukotriene inhibitors (Montelukast or Zafirlukast) will be required to 
discontinue these medications at least 2 weeks prior to their screening 
visit. 
c. Use of daily theophylline within the past month. 
d. 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). 
e. Use of any immunosuppressant therapy within the preceding 12 
months will be reviewed by the study physician.  
f. Receipt of LAIV (Live Attenuated Influenza Vaccine), also known as 
FluMist®, or any other live viral vaccine within the prior 30 days, or 
any vaccine for at least 5 days 
g. Use of beta blocking medications 
h. Antihistamines in the 5 days prior to allergen challenge 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
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i. Routine use of NSAIDs, including aspirin. 
 
4. Physical/laboratory indications: 
a. Abnormalities on lung auscultation 
b. Temperature >37.8 C 
c. Oxygen saturation of <94% 
d. Systolic BP>150 mmHg or <90 mmHg or diastolic BP>90 mmHg or 
<60 mmHg 
 
5. Inability or unwillingness of a participant to give written informed consent. 
 
5. Study Procedures 
 
5.1. Schedule of Events 
 
5.1.1. Visit 1: Baseline visit (within 12 months of completing screening protocol 
IRB#98-0799) 
1. Consent will be obtained 
2. Review of subject’s medical history and current medications 
3. Vital sign measurements (temperature, pulse, respiratory rate, blood pressure), 
oxygen saturation, and symptom scoring 
4. Urine pregnancy test for women of child bearing potential 
5. eNO 
6. Spirometry 
7. Venipuncture for CBC with Diff, Total and der f IgE, cytokines, and gene 
expression  
8. Physical exam of the ears, nose, throat and chest  
9. Sputum induction 
 
5.1.2. Visit 2: 24-48 hours prior to challenge visit (at least 2 days but less than 2 weeks 
after the baseline visit) 
1. Review any change in medical status since last visit 
2. Vital signs, oxygen saturation, and symptom score 
3. Urine pregnancy test 
4. eNO 
5. Spirometry 
6. Physical exam of the ears, nose, throat and chest 
7. Methacholine challenge 
 
 
5.1.3. Visit 3: Allergen challenge day 
1. Review any change in medical status since last visit 
2. Vital signs, oxygen saturation, and symptom score 
3. Spirometry 
4. eNO 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
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5. Placement of HRV electrodes 
6. If above measures are acceptable, allergen challenge will be performed as 
described.  
a. If subject is asymptomatic and FEV1 drops <10% at the completion of 
allergen challenge, subjects will be monitored for 2 hours post challenge 
and will be discharged following post-challenge venipuncture with rescue 
medications, including albuterol with aerochamber and oral prednisone 60 
mg, instructions for use of these medications in the event of symptoms 
related to bronchoconstriction, and contact information for the on-call 
physician. Subject will resume protocol beginning with Study visit 4 
detailed below.  
b. Any subject whose FEV1 drops >10% will stay for the 10 hour post-
allergen monitoring period as outlined below.  
7. Post-challenge monitoring of vital signs every 10 minutes for the 1st hour, then 
every 30 minutes until 2 hours post-challenge, then hourly until 10 hours post-
challenge 
8. Spirometry when vital signs are obtained 
9. eNO hourly between 2 and 10 hours post-challenge 
10. Venipuncture for CBC with diff, cytokines, and gene expression 10 hours post-
challenge 
11. All subjects will receive 4 puffs of albuterol 10 hours post-challenge. Spirometry 
will be performed 15 minutes after albuterol if post-challenge FEV1 at 10 hours 
was not approximately within 10% of baseline.  
a. Albuterol will be repeated every 20 minutes up to three doses if repeat 
FEV1 is not within approximately 10% of baseline value or if subject 
remains in distress.  
b. If needed, after three inhaled albuterol treatments, additional medications, 
including nebulized albuterol and atrovent and oral prednisone 60mg, will 
be given per physician discretion.  
12. Subjects will be discharged 10 hours following allergen challenge when clinically 
stable and when FEV1 is within approximately 10% of baseline FEV1. HRV 
monitoring will be discontinued prior to discharge.  At discharge, all subjects will 
be provided with an albuterol inhaler and aerochamber, a single dose of oral 
prednisone 60 mg, clear instructions for use of rescue medications in the event of 
symptoms related to bronchoconstriction, and contact information for the on-call 
physician. Subjects will reside overnight at their home if their residence is within 
10 miles of UNC or a local hotel within 10 miles of UNC.  
 
5.1.4. Visit 4: 24 hours post challenge 
1. Vital signs, oxygen saturation, and symptom score 
2. eNO 
3. Spirometry 
4. Venipuncture for CBC with Diff, gene expression, cytokines 
5. Methacholine challenge 
6. Sputum induction 
 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
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5.1.5. Post Challenge Observations/Reporting 
1. Subjects will be contacted for phone call follow-up 24 hours after post-challenge 
sputum induction (see phone script included in the study worksheets section) 
2. Subjects who require treatment with prednisone will be contacted by phone within 
24 hours to assess asthma symptoms.   
3. Any subject who requires a dose of steroids will return to the lab 48-72 hours 
after the steroid dose for evaluation of symptoms, spirometry and a physical exam 
to assess the need for further steroid treatment. 
4. Each volunteer will be given a symptom scoring sheet for each day up to 96 hours 
(4 days) after challenge (see symptom scoring sheet in section 5.2.7.).  
 
5.1.6. Study discontinuation visit within 10 days of the final challenge dose: 
1. Vital signs, oxygen saturation, and symptom score 
2. eNO 
3. Spirometry 
4. If any findings are abnormal, medical evaluation as directed by the study 
physician will be undertaken. 
 
5.1.7. Table of Study Procedures 
 Procedures to be performed at various time points are listed in the following table.  
 
baseline 
24-48 hrs  
pre-
challenge 
allergen  
challenge 
day 
24 hrs 
post- 
challenge 
discontinuation visit 
consent 
X 
 
 
 
 
review history/AE’s 
X 
X 
X 
X 
X 
Concomitant meds 
X 
X 
X 
X 
X 
urine HCG  
X 
X 
X 
 
 
vital signs 
X 
X 
X 
X 
X 
spirometry 
X 
X 
X 
X 
X 
physical exam 
X 
X 
 
 
 
Methacholine 
challenge 
 
X 
 
X 
 
sputum induction 
X 
 
 
X 
 
symptom score 
X 
X 
X 
X 
X 
allergen challenge 
 
 
X 
 
 
overnight in CTRC 
 
 
X 
 
 
Venipuncture 
X 
 
X 
X 
 
Exhaled nitric oxide 
X 
X 
X 
X 
X 
HRV 
 
 
X 
 
 
 
 
5.2. Protocols for Measurements and Observations 
 
5.2.1. Allergen challenge procedure: 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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Immediately prior to allergen challenge, determination of adequate lung function without 
use of bronchodilating medications for 8 hours will be made via spirometry with FEV1 of 
at least 80% of predicted and FEV1/FVC ratio of at least .70, consistent with lung 
function of persons with mild intermittent or mild persistent asthma being considered 
minimal acceptable lung function.    
 
Doses will be administered from a DeVilbiss 646 nebulizer as previously described.  
Each volunteer will inhale 5 breaths from the nebulizer at each concentration starting 
with saline control. After administration of the control diluent, concentrations of allergen 
will be administered starting with solutions of zero (saline), 0.25, 0.50, 1.0, 2.0, 4.0, 8.0, 
16, 32, 64, 125, 250, 500, 1000, and 2000 AU/ml via nebulizer.  During the inhalation 
challenge the subject will be under direct observation of a physician experienced in 
treating asthma.  Epinephrine and diphenhydramine hydrochloride for intramuscular 
administration and albuterol for inhalation will be immediately available.  The challenges 
will be performed in the Clinical and Translational Research Center (CTRC) of the 
University of North Carolina at Chapel Hill.  Full emergency equipment and nursing 
personnel are immediately available at this location. 
 
The FEV1 will be measured prior to and 10 minutes after each aerosol inhalation.  
Starting with the control (saline diluent) solution, increasing concentrations of allergen 
will be inhaled at 10-minute intervals starting with 0.25 AU/ml.  The FEV1 measurement 
obtained after inhalation of the control solution (saline) will be considered the baseline 
value. If the FEV1 has declined by less than 10% of baseline after a given concentration 
of allergen is inhaled, the next higher concentration of allergen will be given. If after 
inhalation of an allergen dose, the FEV1 declines by ≥10% but <20% from the baseline 
FEV1 measurement, spirometry will be repeated every 5 minutes for 15 minutes or until 
a clear nadir in the decline has been reached. If the nadir after 15 minutes is a decline in 
FEV1 of less than 10% of baseline, the next higher concentration of allergen will be 
given.If, however, the nadir after 15 minutes is a decline in FEV1 ≥ 10% but < 20% from 
the baseline measurement, the previous allergen dose will be repeated. . To prioritize the 
safety of subjects and to prevent severe or precipitous decline in lung function, decisions 
to repeat doses of allergen (rather than escalate dosing) or perform additional spirometry 
measurements will be made in real time at the discretion of the supervising physician, 
who will be physically present throughout the allergen challenge procedure. Once a 
decline in FEV of ≥ 20% is reached, the challenge will be stopped. These activities are 
summarized in Figure 1. Once the challenge is stopped, FEV1 will be monitored every 10 
minutes for the first hour, then every 30 minutes until 2 hours post-challenge, and then 
hourly until 10 hours post-challenge.    

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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Oxygen saturation by pulse oximeter will be measured during the challenge and with 
each spirometric evaluation.  The physician responsible for the study will determine if 
any medication is necessary based on the subject's lung function and symptoms. 
Vital sign measurements including pulse oximetry evaluation will continue to be 
performed at hourly intervals during and for 10 hours after the exposure. Oxygen 
saturation by pulse oximeter will be measured during the challenge and with each 
spirometric evaluation.  All subjects will receive 4 puffs of albuterol 10 hours after 
allergen challenge. If clinically stable and if FEV1 is approximately within 10% of 
baseline prechallenge FEV1, subject will be discharged with an albuterol inhaler with 
aerochamber and a single oral dose of prednisone 60 mg, clear instructions on use of 
medications in the event of symptoms related to bronchoconstriction, and contact 
information for the on-call physician. The subject will reside in either a local hotel within 
10 miles of UNC or their home if their residence is within a 10 mile radius of UNC.  
 
If the subject has wheezing on physical examination the morning after the inhaled 
allergen and/or has an FEV1 that is >15% from baseline measures (indicating that they 
are still experiencing a late phase response), they will be a single dose of prednisone 60 
mg and continue following instructions from the provided asthma action plan for 
albuterol use. These subjects will be contacted by phone within 24 hours of receiving the 
dose to assess asthma symptoms and will be evaluated in person within 48-72 hours of 
the dose to monitor FEV1 and to assess the need for continued therapy.   
 
5.2.2. Hypertonic Saline Induced Sputum procedure:  
•
Administer allergen dose
•
10 min later, perform 
spirometry
Proceed to next doubling-
dose of allergen
Repeat spirometry 
q5 min x 3, or 
until FEV1 nadir 
reached
Challenge 
Terminated
Repeat previous 
dose of allergen*
Challenge 
Terminated
Proceed to the 
next doubling-
dose of allergen
in FEV1 <10%
in FEV1 ≥10%
but <20%
in FEV1 ≥20%
in FEV1 <10%
in FEV1 ≥10% 
but <20%
in FEV1 ≥20%
*At discretion of supervising physician
Figure 1. Inhaled allergen challenge schematic.

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
16 
 
Prior to sputum induction, subjects will receive 4 inhalations of albuterol from an MDI 
attached to a spacer device. FEV1 and FVC will be measured after this step to determine 
the post-bronchodilator baseline 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 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). 
 
5.2.3. Spirometry:  
 Standard methodology conforming to the American Thoracic Society guidelines for     
 measurement of spirometry will be used.      
 
5.2.4. Methacholine Challenge: 
This test measures the responsiveness of the airways to a standard cholinergic 
bronchoconstriction agent.  Methacholine challenge testing is a standard clinical 
procedure to determine airway reactivity in patients with known or suspected airway 
disease. Subjects will be asked to limit caffeine on the day of testing.  Subjects will inhale 
5 breaths from the nebulizer at each concentration starting with saline control.  Each 
breath is started from resting end-expiratory lung volume (Forced Residual Capacity) and 
continued until a maximum inhalation is reached.  Increasing (doubling) concentrations 
are inhaled until the FEV1 falls by at least 20% from the post saline value, the highest 
concentration has been inhaled, or the subject experiences discomfort or anxiety 
sufficient for the investigator or subject to consider further testing unacceptable.  
Methacholine concentrations will be 0.075, 0.156, 0.312, 0.625, 1.25, 2.5, 5.0, and 10.0 
mg/ml.  If subjects experience symptoms that do not rapidly and spontaneously remit at 
the end of the procedure, he/she will receive 2 puffs of an inhaled bronchodilator 
(albuterol) MDI.  If needed an additional 2 puffs or a nebulizer treatment with standard 
dose albuterol (0.083mg) will be given.  The subject will not be discharged until 
spirometry levels return to baseline.   

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
17 
 
 
5.2.5. Venipuncture: 
Blood will be drawn to evaluate a complete blood count with differential and also to 
determine what genes may play a role in a person’s response to allergen challenge.  At 
the time of each venipuncture, up to 50 cc will be drawn, and the total for the study will 
be less than 150 cc.  
 
5.2.6. Exhaled nitric oxide measurement: 
We will measure the amount of nitric oxide (NO) present in orally expired air. An 
increased concentration of NO in exhaled air may be found in normal persons with acute 
inflammation.  Thus, measurement of the concentration of NO in expired air may be 
useful as an indirect assessment of airway inflammation. Lung production of NO will be 
measured by asking subjects to exhale briefly into a mouthpiece. 
 
5.2.7. 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: 
 
 
       
Waking up at night due to cough (0-3): ______ 
 
 
Shortness of Breath (0-3): ________ 
 
 
Cough at rest (0-3): _________ 
 
 
Cough with Exercise (0-3): _________ 
 
 
Wheeze at Rest (0-3): _________ 
 
 
Wheeze with Exercise (0-3): _________ 
 
 
 
 
 
Total________ 
 
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 cough, wheezing, or needed any extra 
allergy or asthma medications? 
 
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 the subject will be offered the 
opportunity to come back to the laboratory for follow up assessment by a study 
physician. 
 
6. Statistical Plan 
 
6.1. Sample Size Determination 
 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
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Sample size was calculated based on the sample required for our upcoming randomized 
placebo-controlled crossover studies of anakinra for mitigating features of allergen-
induced asthma exacerbation. We hypothesize that anakinra treatment reduces the LPR, 
as assessed by the change in maximum percentage fall in %FEV1 during hours 3-10 
following allergen challenge.  We will be developing two anakinra treatment clinical 
trials, each of which will incorporate a crossover study design: one will provide anakinra 
treatment (v. placebo) immediately after the EPR, and one will provide anakinra 
treatment (v. placebo) at the onset of the LPR (i.e. 3 hours after the EPR).   
 
We calculated the sample size for the anakinra treatment protocols using a model of 
inhaled allergen challenge similar to what we propose in this project (69) based on a 
paired t test. The maximal drop in %FEV1 during the LPR was 25.6% + 9.8%(SD). To 
detect a 50% attenuation of the drop in %FEV1 during the LPR (12.8%) by anakinra v. 
placebo treatment, assuming that group-wise standard deviations are similar (10.28% for 
anakinra; 9.8% for placebo), with β=0.9 & α=0.05, a sample size of 6.4 would be 
required for each anakinra protocol.  We estimate that 25% of subjects will require rescue 
short-acting beta agonist treatment during the EPR or the LPR, and will additionally 
account for a 25% dropout rate for a crossover study, inflating our required sample size to 
at least N=12 for each anakinra treatment protocol.   
 
Previous studies of inhaled allergen challenge models in allergic asthmatics have found 
that 50-70% of subjects who undergo challenge will experience an EPR (33, 34). 
Gauvreau et al (16) reported that >75% of D. farinae challenged volunteers who display 
an EPR will also have a LPR. Using a more conservative estimate of 50% of our screened 
volunteers with a EPR and 50% of those with a LPR, we will screen up to 100 subjects to 
identify at least 25 LPR subjects eligible to enroll into the anakinra treatment studies. 
Screening will stop following identification of 25 LPR subjects. 
 
6.2. Statistical Methods 
Our analytical plan was developed with Dr. Zhou, the biostatistician for the CEMALB who 
will oversee all statistical analysis. In all analyses, criterion for significance will be p ≤0.05.  
 
Our primary endpoint is identification of subjects who achieve a ≥15% fall in FEV1 during 
hours 3-10 following allergen challenge. We will subtract the pre-allergen challenge 
%FEV1 value from the post-allergen %FEV1 value to make this determination.  
 
For analysis of secondary endpoints, we will first use linear regression to determine if there 
is a linear relationship between baseline sputum IL-1β concentration (pg/mL) and asthma 
outcomes (maximum fall in %FEV1 during LPR, methacholine reactivity defined by the 
dose required to produce a ≥20% drop in FEV1, sputum granulocyte content both in terms 
of % of total cell count and cells/mg of sputum, exhaled nitric oxide concentration) 
following allergen challenge, adjusting for potential confounders. The point estimate and 
confidence interval will be reported for the effect of baseline sputum IL-1β on asthma 
outcomes. If the normality assumption is violated or there exists a non-linear relationship, 
we will calculate the Spearman correlation coefficient between baseline sputum IL-1β 
concentration and asthma outcomes.   

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
19 
 
 
For analysis of exploratory endpoints, we will use two sample comparison tests to compare 
systemic inflammatory markers and gene expression levels before and after allergen 
challenge. We will use two-sample t tests or Mann Whitney U tests depending on whether 
the normality assumption is met. The point estimate and confidence interval will be 
reported for mean changes. We will transform the data to achieve normality if needed.  
 
As we have considered additional individuals in our sample size calculation, we do not 
anticipate missing data will be a major issue affecting our study power. 
 
6.3. Data Management 
Study coordinators responsible for efforts and computations for database management 
will include Carole Robinette, MS; Martha Almond, RRT; and Katherine Mills, BA. Data 
will be initially collected by study coordinators on paper documents. Symptom 
questionnaires will be completed by the subject and collected by the coordinator. Clinical 
data such as health history and smoking history will be collected by study staff as 
interview or directly measured, such as vital signs, spirometry, etc. All paper forms 
(source documentation) is maintained by a study coordinator or PI in a binder, 1 binder 
per subject. All data will be entered into REDCap by a study coordinator, within 2 weeks 
of collection and will be verified by a 2nd person within 4 weeks to ensure accuracy and 
completeness. REDCap is a secure web application developed at Vanderbilt University 
and can be used to collect virtually any type of data (including 21 CFR Part 11, FISMA, 
and HIPAA-compliant environments). It is specifically geared to support online or offline 
data capture for research studies and operations. Missing data will be noted and a 
comment made in the database to explain the missing data. Time stamps will be used 
when appropriate. At minimum, the visit number will be indicated.  
 
Biological samples collected by coordinators will be delivered to the CEMALB lab by 
the coordinators in properly labeled containers. On receipt in the lab, samples are initially 
entered into a software program called LDMS (Lab Data Management System) which 
tracks samples. Processing of these samples is done per the SOP maintained in the 
CEMALB quality assurance plan, as some samples are processed immediately and others 
are processed in batches at later time points. This data is entered into REDCap at 
appropriate intervals, based on sample analysis. 
 
We will use the data dictionary or REDCap codebook as our course for data codes. All 
data will be collected by qualified staff using CEMALB SOPs and will be entered into 
REDCap. Once the data is entered and confirmed by a 2nd person, it will be considered 
“locked” and will not be changed without documentation with the reason for the change. 
REDCap limits and monitors who can change data, a feature developed to ensure data 
integrity. 
 
7. Monitoring and Risk Minimization 
 
7.1. Definition of Adverse Event (AE) and Serious Adverse Event (SAE): 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
20 
 
An adverse event for a given volunteer will be defined as failure of any of the safety 
criteria outlined in section 3.4.2. Additionally, minor upper respiratory tract infections 
occurring within 10 days of the exposure will be considered adverse events.  Other, non-
specified clinical illnesses, which occur within 10 days of each challenge will also be 
reported as an AE.  Specific potential adverse events which may occur with allergen 
challenge include the following which are defined in the Common Terminology Criteria 
for Adverse Events developed by the NCI: Allergic Reaction, Cough, Dyspnea, Urticaria, 
Syncope, and Voice Changes 
 
Also, any decrease in lung function or increase in symptom score, as outlined in section 
3.4.2. will be considered an adverse event.  Failure of a total symptom score to return to 
no greater than 6 above baseline within 10 days will be considered an adverse event. Any 
symptoms that induce a volunteer to seek medical attention from any provider within 10 
days 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 (CTCAE Grade 4), or death (CTCAE Grade 5), if these events occur within 10 
days of challenge (or if the clinical scenario leading up to hospitalization, illness, injury 
or death begins within 10 days of a challenge).   
 
7.2. Risk to Subjects 
 
At the doses proposed in this study, the most significant predictable risk to subjects as a 
result of allergen inhalation is development of immediate airway obstruction 
(bronchospasm) during the EPR, which would cause shortness of breath, cough and/or 
wheeze. This drop in FEV1 should be short lived (maximal 10 to 30 minutes after 
allergen) and reversible with medications such as beta agonists (albuterol).  This effect is 
similar to the effect of methacholine, which is an FDA-approved agent employed for the 
diagnosis of airway reactivity and asthma.   
 
Other possible risks include development of a LPR to allergen inhalation. This occurs in 
approximately 50% of asthmatics challenged, consisting of similar symptoms described 
for the EPR.  Since an important secondary endpoint of this study is to identify which 
subjects have a late asthmatic response, albuterol for rescue prior to the 10 hour post 
challenge time point will only be employed if the FEV1 does not show spontaneous 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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improvement, and/or if the subject has distress and requests rescue. All subjects will be 
given 4 puffs of albuterol 10 hours post-challenge with additional doses given based on 
FEV1 and subject distress.   
 
Sputum induction may induce cough, chest tightness or bronchospasm, and albuterol will 
be immediately available.   
 
Venipuncture carries a risk of hematoma, and this procedure will be performed only by 
trained staff members.    
 
There is no risk to measurement of eNO since subjects are simply asked to exhale into a 
mouthpiece. 
 
7.3. Measures to Minimize Risk 
 
Only clinically healthy volunteers will be recruited for this project. 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 4 weeks after the last dose of 
antibiotics), 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 3.4.1., all subjects will need to fulfill objective lung function 
and symptom criteria prior to initiating the challenge study.  Methacholine challenge is 
employed prior to the allergen challenge to ensure that the subject has a repeatable, 
within one-doubling dose response to the methacholine (compared to the previous 
methacholine challenge performed during the separate general screening protocol 
IRB#98-0799) to help rule out an exacerbation prior to the allergen challenge.   
 
Female subjects of child-bearing potential will undergo urine HCG testing for pregnancy 
within 48 hours prior to administration of any challenge.  
 
A physician familiar with the protocol will be available for all challenge procedures. 
Emergency treatment with albuterol (via MDI or nebulizer), oxygen, oral corticosteroids 
as well as epinephrine (subcutaneously or intramuscularly) and diphenhydramine (for 
intramuscular injection or intravenous administration) will be available to those subjects 
who require such therapy.  An emergency “crash cart” with standard emergency 
medications, IV fluids and a defibrillator are is also readily available in the UNC CTRC 
as well as at CEMALB in the unlikely event of a medical emergency during any 
challenge or study visit. 
 
If the covering physician feels that the subject’s respiratory status is such that providing 
an induced sputum sample would place them at increased risk for significant 
bronchospasm, we will prioritize spirometry assessments (as part of routine clinical care) 
and not obtain induced sputum samples for that subject. 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
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All subjects will be monitored in the CTRC for 10 hours following allergen challenge as 
outlined in previous sections. Upon discharge to either the subject’s home or local hotel, 
subjects will be provided with an albuterol inhaler with aerochamber and a single dose of 
oral prednisone 60 mg, clear instructions for use of these medications in the event of 
bronchoconstriction, and the contact information for the on-call physician. In the event of 
post-challenge bronchoconstriction that does not improve with albuterol, subject will be 
instructed to present to the UNC emergency department for further evaluation and 
management. Subjects will also be assessed the morning after the allergen challenge in 
the CEMALB. If the subject has wheezing on physical examination the morning after the 
inhaled allergen and/or has an FEV1 that is >15% from baseline measures (indicating that 
they are still experiencing a late phase response), they will be given a single dose of 
prednisone 60 mg and continue following instructions from the provided asthma action 
plan for albuterol use. These subjects will be contacted by phone within 24 hours of 
receiving the dose to assess asthma symptoms and will be evaluated in person within 48-
72 hours of the dose to monitor FEV1 and to assess the need for continued therapy.   
 
In addition, a follow-up call will be made 48 hours after challenge (24 hours after the 
follow-up sputum induction) 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.  
 
If the subject requires albuterol for >48 hours after the inhaled allergen challenge, they 
will be a single dose of prednisone 60 mg and continue following instructions from the 
provided asthma action plan for albuterol use. These subjects will be contacted by phone 
within 24 hours of receiving the dose to assess asthma symptoms and will be evaluated in 
person within 48-72 hours of the dose to monitor FEV1 and to assess the need for 
continued therapy.   
 
Subjects who are withdrawn for medical concerns related to the study, such as but not 
limited to prolonged decreased FEV1 or persistent wheezing, will be followed by study 
physicians until resolution of these events.  As this is a screening protocol, these subjects 
will not be replaced. 
 
A Data Safety Monitoring Board (DSMB) will be established through the NC TraCS 
Institute to independently review all research activities outlined for the study protocol.  
 
To ensure correct reporting, with each Subject Binder (in the Case Report Form) we have 
added a discharge worksheet to capture the above information to ensure availability in 
real time and to guarantee that we do not overlook a violation of safety criteria. 
 
We will use the NC TraCS DSMB to assess for possible changes to the overall risk of the 
study using inhaled allergen challenge.  The DSMB will have full access to research 
records and source documents.  Data regarding unanticipated problems will be provided 
to them as it is reported to the IRB and to the FDA. They will review all adverse events, 
enrollment, and protocol deviations after the first 6 subjects have completed the inhaled 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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allergen protocol, and then every 6 months thereafter. Any serious adverse events or 
major protocol deviations will be reported immediately to the DSMB via email, which 
may lead to an earlier review. They will communicate with the PI and the appropriate 
NIH Medical Officer regarding any safety issues. 
Withdrawal or Termination 
Participants will be terminated early from the study for the following reasons: 
1. The participant elects to withdraw consent from all future study activities 
including follow up. 
2. The participant is “lost to follow up” (i.e., no further follow up is possible because 
attempts to reestablish contact with the participant have failed). 
3. The participant dies. 
4. The participant develops a medical condition or is started on a new medication(s) 
not previously mentioned in the list of prohibited medications that, in the opinion 
of the investigator, may pose additional risks from participation in the study, may 
interfere with the participant’s ability to comply with study requirements or that 
may impact the quality of the data obtained from the study. 
5. The participant no longer meets inclusion or exclusion criteria as defined in 
Section 4.1 and 4.2 or if the participant does not meet the previously defined 
safety criteria (see Section 3.4.2). 
6. The study is terminated for any reason. 
 
7.4. Reporting of AEs and SAEs: 
UNC-Chapel Hill Human Research Protection Program Standard Operating Procedures 
will be followed for reporting adverse events/unanticipated problems to the Biomedical 
IRB and NC TraCS DSMB.  All SAEs will be reported to the CBER of the FDA as well 
as to the UNC OHRE, DSMB, and the NIH sponsor within 24 hours of recognition of the 
event, including the filing of an IND safety report. 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, the UNC IRB, and the NC 
TraCS DSMB.  
 
7.5. 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 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
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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. 
Withdrawal will also not impact the subject’s status as a UNC student or employee.   
Subjects may be withdrawn if the study physician feels it is in the best interest of the 
subject. 
 
7.6 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.  Records will be kept for 2 years after completion of data collection.  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.   
 
7.7. 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 
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 
Assistant Professor of Pediatrics 
UNC School of Medicine 
Department of Pediatrics 
Division of Allergy, 
Immunology, and Rheumatology 
Co-Investigator 
Haibo Zhou, PhD 
3104C McGavran-Greenberg Hall 
The University of North Carolina 
CB #7420 
Chapel Hill, NC 27599 
Professor of Biostatistics 
Director of the Biostatistics 
Core, CEMALB 

Identification of allergic asthmatics reactive to Dermatophagoides farinae (house dust mite) inhalation  
Michelle Hernandez, MD 
 
 
 
25 
 
Co-Investigator 
Terry Noah, MD 
450 MacNider – The UNC School 
of Medicine 
CB 7217  
Chapel Hill, NC 27599-7217 
Professor and Senior Vice Chair, 
UNC Department of Pediatrics 
Division Chief, UNC Pediatric 
Pulmonology Investigator 
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 undergo challenge procedures at the Clinical 
and Translational Research Center (CTRC) at the University of North Carolina in Chapel 
Hill, North Carolina, 27599-7310.  Additional subject visits and test procedures will be 
performed at the University of North Carolina Center for Environmental Medicine, 
Asthma and Lung Biology (CEMALB). All necessary clinical research equipment, 
medical equipment, and laboratory equipment is located at the CTRC and within the 
CEMALB. 
 
Institutional Review Board. This study has been submitted to the following IRB:  Office 
of Human Research Ethics 720 Martin Luther King, Jr. Blvd. 
Bldg# 385, Second Floor Chapel Hill, NC 27599-7097. This study has received IRB 
approval, which will be maintained throughout the life of the study.