Conditions: Allergic Asthma
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 18
Healthy volunteers: no

Brief summary:
To study the effects of 1200mg gamma tocopherol, a form of vitamin E, given daily on the response of the airway in mild asthmatics after exposure to ozone (O3)

Detailed description:
Allergic asthma (AA) is the most commonly encountered respiratory disease in children and adults in the United States and is a leading cause of morbidity worldwide. Among the most disruptive expressions of disease in AA is acute asthma exacerbation. The Center for Disease Control (CDC) lists ambient air pollutants and environmental tobacco smoke as among the most common triggers for acute asthma exacerbation. Ozone (O3) is the most commonly encountered ambient air pollutant in the US.

Increased oxidative stress and decreased antioxidant capability have been observed in asthmatics. These pollutants are pro-inflammatory and are associated with increased oxidative stress, which would exacerbate reactive oxygen and reactive nitrogen species (ROS and RNS)-induced injury in asthmatics. O3 injures epithelial cells, releasing secondary mediators which activate inflammatory cells, in part by ligation of Toll-like receptor 4 (TLR4), the primary receptor for lipopolysaccharide (LPS). TLR4 activation of inflammatory cells activates (nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and induces oxidative stress. O3 and LPS has been associated with exacerbation of asthma, and the investigators have reported that O3 and LPS augments allergic airway inflammation in allergic asthmatics (AA). Development of interventions to mitigate these responses will greatly decrease disease morbidity. Given the role that oxidants play in the pathophysiology of asthma exacerbation, defects in antioxidant levels would increase risk for acute asthma exacerbation. Nutritional deficiencies in vitamin E, ascorbate and selenium have been linked to asthma severity, and asthmatics have decreased antioxidant levels in airway fluid. It has been shown that vitamins C and E are decreased in airway fluids of asthmatics. Additionally, genetic factors may increase risk for oxidant induced exacerbation of asthma. Many investigators have reported that persons who are homozygous for the null polymorphism of the Glutathione-S-Transferase Mu1 (GSTM1) gene and unable to produce GSTM1 protein (the GSTM1 null genotype) have increased risk of acute pollutant-induced exacerbation of asthma. This lab has shown in healthy volunteers that the GSTM1 null genotype is associated with increased inflammatory response to O3, with no impact on the nociceptive response to this pollutant.

Other researchers have shown that the GSTM1 null genotype is associated with increased response to secondhand tobacco smoke, diesel exhaust, and other particulate matter components. Romieu et al demonstrated that children with asthma in Mexico City had increased susceptibility to O3-induced exacerbations if they had the GSTM1 null genotype. This group also found that GSTM1 null AAs selectively benefited from antioxidant intervention. The GSTM1 null genotype is found in 20-40% of the population, and may be overrepresented in allergic populations. Taken together, these observations show that the sizable GSTM1 null population is at risk for pollutant-induced airway disease, and that antioxidant intervention targeting the action of ROS and RNS will benefit asthmatics and especially GSTM1 null asthmatics.

A non-exclusive list of proposed antioxidants includes radical scavengers such as ascorbate, a-tocopherol (aT), y-tocopherol (γT) or inducers of Nuclear factor erythroid-2-related factor 2 (NRF2), which activate NRF2 with subsequent broad upregulation of acute phase II and antioxidant proteins. These agents are available in naturally occurring foods and as nutritional supplements. A number of animal, cell culture and epidemiological studies support the idea that antioxidant supplementation is useful in allergic airway disease. However, despite these studies and widespread public and scientific enthusiasm regarding use of such agents in asthmatics, there are scant human data to support or refute the use of such interventions for either acute or chronic allergic airways disease.

Gamma tocopherol has both radical scavenging and anti-inflammatory actions which may play important roles in decreasing pollutant-induced and allergic injury to the airway. Like other isoforms of vitamin E, γT is a potent ROS scavenger and is also a powerful nucleophile that traps electrophiles such as peroxynitrite in lipophilic compartments. One mechanism by which γT is cytoprotective is scavenging of RNS at the un-substituted C-5 position on the hydroxy-chroman ring of γT to form 5-NO-γ-tocopherol (5-NO-γT). Overall, vitamin E provides general protection of DNA, lipids and proteins from radical stress. An example of such protection is shown in rodent studies of prostate cancer in which intake of γT is associated with decreased DNA methylation of CpG rich regions of the NRF2. NRF2 is a master regulator of numerous cytoprotective antioxidant enzymes.

Supplementation with γT also prevents protein nitration and attenuates loss of plasma vitamin C in plasma in a rodent peritonitis model of inflammation. The investigators have also shown that γT inhibits Cyclooxygenase (COX)-2 and 5-Lipoxygenase (LO) in LPS-stimulated macrophages and interleukin (IL)-1b stimulated epithelial cells. These actions are mediated primarily by the γT metabolite 2, 7, 8-trimethyl-2S-(.-carboxyethyl)-6-hydroxychroman (γ-CEHC) which requires hydroxylation of the γ-methyl group of γT by cytochrome P450 (CYP450). In carrageenan- induced inflammation in male Wistar rats, γT decreases prostaglandin (PGE2) and leukotriene (LTB4) production, suggesting that LO-mediated production of leukotrienes may also be inhibited by γT. Tocopherols, including γT, also modulate gene expression of a number of inflammatory genes. Thus, γT and other tocopherols decrease production of a number of pro-inflammatory cytokines at the transcriptional level.

In animals, it has also been shown that gamma tocopherol reduces baseline eosinophilia in the airway. In early phase I studies of gamma tocopherol-enriched mixed tocopherols (γT-mT) the investigators have shown that γT-mT inhibits monocyte induced cytokine production, decreases baseline nitrosative stress, and inhibits LPS eosinophil and neutrophil influx in healthy volunteers.

SUMMARY:

There is widespread opinion that antioxidant nutrients like gamma tocopherol ( γT, a form of Vitamin E) are an untapped and inexpensive intervention for environmentally triggered acute asthma. However, there is a crucial gap in evidence-based support of such interventions in asthma. A lack of coordinated research assessing specific antioxidant regimens from preclinical, phase I and phase II studies impedes development of phase III antioxidant trials in asthmatic populations. It is also unclear which physiological endpoints are most relevant in such studies.

Arms:
arm 1: Safflower oil, taken in 2 capsules every 12 hours for a total of 4 doses arm 2: gamma tocopherol 1400 mg, taken as 2 700 mg capsules every 12 hours for a total of 4 doses

Interventions: intervention 1: gamma tocopherol intervention 2: Placebo
Intervention descriptions: intervention 1: The effect of supplemental with gamma tocopherol on O3 response will be assessed using the difference pre and after γT dosing. intervention 2: The effect of supplemental with gamma tocopherol on O3 response will be assessed using the difference pre and after placebo dosing.

Protocol document:
Prot_000.pdf: 
 
 
Study Title 
Effect of Gamma Tocopherol Enriched Supplementation on Response to 
Inhaled O3 Exposure 
 
 
 
NCT number  NCT02911688 
Document Date    July 18, 2017 
 
 
 


 
        IND 71475, “Airway Response to Ozone Exposure”.    D. B. Peden 
 
 
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Version July 18, 2017 
 
Protocol Version Date:  July 18, 2017 
 
Summary of Changes 
 
1) 1) In Section A.3.1 under Inclusion Criteria, the definition of  of a positive methacho-
lone test (item d.) has been corrected to read "...a provocative concentration of meth-
acholine of 10mg/ml or less producing a 20% fall in FEV1...." (This was also cor-
rected in the protocol itself.) 
 
2) 2) The collection of samples of nasal epithelial cells (NEC) and epithelial lining fluid 
(ELF) is not done at the Screening Visit (V-0).  Those procedures have been removed 
from the description of that visit in Section A.4.2., and now the Application matches 
the protocol. 
 
 
 


 
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Version July 18, 2017 
 
 
Protocol Version Date:  February 17th, 2017 
 
Summary of Changes 
 
1)  Based on published data1, the pulmonary function criteria have been relaxed, including: 
a.  
the minimum allowable volume in liters of forced expiratory volume in the 
first second (FEV1) has been decreased from 80% to 75% of predicted; and 
b. 
the FEV1/FVC (forced vital capacity) ratio will no longer be used for determi-
nation of eligibility because the value can be artificially low when a person 
has an exceptionally large FVC. 
Our original restrictions on spirometry measures for study inclusion were very conservative.  
Since the approval of the original ozone IND, we have found that the mean drop (from that 
day’s baseline values) in FEV1 is 13.6%, and mean drop in FVC is 11%.  These changes are 
transient, evident immediately after subjects exit the chamber, and resolve to within 90% of 
that day’s baseline by 4 hours after the end of the ozone exposure.  For a subject to be re-
leased from the CEMALB after ozone exposure, their spirometry must show these measures 
to be within 90% of that day’s baseline.  
Inhaled allergen challenge requires a drop in FEV1 of 20% to be achieved, and presents a 
much longer period of bronchoconstriction, particularly in those with a late phase response 
(onset evident 3-7 hours after the initial drop in FEV1).  Inclusion criteria for inhaled aller-
gen challenge requires an FEV1 of at least 70% of predicted with no restrictions on 
FEV1/FVC ratios.  Given that the ozone effects on spirometry are milder than those seen 
with inhaled allergen challenge, we feel that it is safe to slightly relax our spirometry criteria.  
However, our entrance criteria for FEV1 are still more conservative than those used by others 
with inhaled allergen challenge. 
1 Diamant, et al. Inhaled allergen bronchoprovocation tests. J Allergy Clin Immunol 2013; 132:1045-
55 
2)  The Ozone Symptom Questionnaire score is removed from the list of inclusion criteria. 
3)  The consent form and the application have been revised to indicate the following changes: 
a.  
Now that several subjects have gone through the various visits, we are revis-
ing the estimated duration of some of the visits – some longer and some 
shorter. The estimated total duration has not changed 
b.  
The description of Study Visit 7 (Final Study Visit) now includes, "This visit 
will occur 5 to 10 days after your final ozone exposure, and ...." 
c.  
Section A.6.10 of the application has been revised to include new information, 
and in the consent form, Risks associated with Vitamin E administration now 
reads: "In a previous study, nausea, vomiting, bloating and/or diarrhea were 


 
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Version July 18, 2017 
commonly reported.  These symptoms generally occurred in the first day or 
two of dosing. You are encouraged to take the study treatment with food con-
taining fat, such as cheese or peanut butter, to minimize these effects." 
 
 


 
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Protocol Version Date:  January 13, 2017 
 
Description of Change 
 
Assessment of the impact of gamma tocopherol on inflammatory gene expression in 
peripheral blood mononuclear cells (PBMCs) is added to the protocol. 
 
 
 


 
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Protocol Version Date:  October 28, 2016 
 
Summary of Protocol Changes  
1. Spirometry prior to exercise, vital sign measurement after exercise, and a pregnancy test 
(as applicable) have been added to the Training/ Baseline Visit (Visit 0a) to optimize sub-
ject safety.  
2. “Use of investigational drugs within 6 weeks of screening” has been added to the list of 
exclusion criteria.  
3. The Asthma Symptom Score has been removed from the list of inclusion criteria and 
from the list of assessments performed at Visits 0, 1 and  4; it has been renamed Ozone 
Symptom questionnaire at Visits 2, 3, 5, 6 and 7.  (The questionnaire to be used has not 
changed, and is attached to the application.)  
4. The target minute ventilation has been decreased from 25L/ min/ BSA(m2) to 
20L/min/BSA. It was decided that the lower rate is sufficient to assess the effect of ozone 
on the airway. 
5. The duration of participation has been changed to “6 weeks to 5 months;” practically 
speaking, 6 weeks is about as little time as it would take for the participant to complete 
all visits.  
6. A blood draw for clinical lab tests (CBC, PT/INR, aPTT and C-reactive protein [CRP]) 
and biomarkers has been added to the 24 Hr Post-Exposure Visits (Visits 3 and 6), to op-
timize subject safety and to add an extra timepoint for  evaluation of the effects of γ-to-
copherol on inflammation.  (A sample for CRP has also been added to the Screening and 
pre-exposure lab assessments on Exposure Visits 2 and 5 to establish baseline values.)   
7. The final follow-up/End of Study Visit (Visit 7) will include a blood draw for CBC, PT, 
and/or aPTT, “if [there were] abnormal values at Visit 6.”   
 
 
 
 


 
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Protocol Version Date August 17, 2016 
 
Summary of protocol changes: 
1.  Sputum induction - this procedure has been added to the treatment initiation visits of both 
treatment periods (initial and cross-over, Visits 1 and 4). The rationale for this addition is 
two-fold.  the time between sputum inductions and the mucocilliary (MCC) scans will be ap-
proximately the same (48 hours) for each subject, and the extra specimens will provide more 
baseline data on the primary endpoint. 
2.  Blood pressure will be monitored during the 3-hour O3 exposure, with measurements 
taken at least once every hour. 
 
 


 
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Protocol 4:   Effect of gamma tocopherol enriched supplementation on response to inhaled 
O3 exposure 
 
a. Objectives and Purpose. 
To test the hypothesis that gamma tocopherol supplementation inhibits ozone induced 
airways inflammation in allergic asthmatics 
 
b. Investigators, Facilities, and Institutional Review Board 
 
 
Role in project 
Name and Address 
Title 
Principal 
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 
 
 
 
 
Neil Alexis, Ph.D. 
CEMLB, 104 Mason Farm Road 
The University of North Carolina 
CB#7310 
Chapel Hill, NC 27599-7310 
Professor of Pediatrics 
University of North Carolina 
Co-Investigator 
 
Michelle Hernandez, MD 
CEMALB, 104 Mason Farm Road 
The University of North Carolina 
CB#7310 
Chapel Hill, NC 27599-7310 
Associate Professor of Pediatrics 
University of North Carolina 
Co-Investigator 
William Bennett, Ph.D 
CEMALB, 104 Mason Farm Road 
The University of North Carolina 
CB#7310 
Chapel Hill, NC 27599-7310 
Professor of Medicine & 
University of North Carolina 
 
Curriculum Vitae for Drs., Peden, Alexis, Hernandez and Bennett are appended. 
 
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 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. Boulevard, Chapel Hill, NC 27599-7097 prior to enrolling 
the first subject. 


 
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c. Patient selection criteria, exclusion criteria and numbers. 
Up to 32 mild allergic asthmatic volunteers ages 18-45 will be recruited, to determine the 
comparative effect of 0.25 parts per million ozone (O3) in persons receiving 2 capsules of 
700mg each gamma tocohpherol (yT) supplement for 48 hours.  Subjects will undergo 
baseline measurements including venipuncture, induced sputum and mucociliary clearance. 
After 48 hours of treatment subjects will undergo O3 exposure, followed a few hours later by 
repeating the venipuncture, induced sputum and mucociliary clearance scan. Subjects will 
have a minimum 3 week washout and repeat the procedures, in a double blind, crossover 
fashion, with safflower oil as the placebo control. 
There will no gender or ethnic restrictions.  Spirometry will be performed to determine the 
current level of lung function. Women of childbearing potential will not undergo the 
methacholine challenge or mucocilliary clearance scans unless a urine pregnancy test 
confirms the absence of pregnancy. 
Subjects for this study will be otherwise healthy, allergic asthmatic 18-45 year old males and 
females.  Our recruitment will include up to 32 volunteers, and at least 50% female subjects 
with at least a 30% minority representation. It is anticipated that the demographics of our 
subject population will reflect those of the local university community.  Subjects are recruited 
through a separate screening protocol, IRB # 98-0799, which is performed to identify 
subjects for potential participation in experimental challenge protocols such as this one. 
 
Specific Inclusion Criteria: 
a) Age 18-45 of both genders 
b) Negative pregnancy test for females who are not s/p hysterectomy with oopherectomy 
c) History of episodic wheezing, chest tightness, or shortness of breath consistent with 
asthma, or physician diagnosed asthma. 
d) Positive methacholine test. A positive test is defined as a provocative concentration of 
methacholine of 10 mg/ml or less producing a 20% fall in FEV1 (PC20 methacholine) by the 
method used in a separate screening protocol; OR physician diagnosed asthma with a history 
consistent with mild, intermittent asthma after age 6; OR a positive bronchodilator challenge. 
Subjects with regular use of inhaled corticosteroids may be included, these volunteers must 
be able to come off of the ICS for 2 weeks without increased symptoms or increased need for 
beta agonist rescue medication. 
e) FEV1 of at least 75% of predicted without use of bronchodilating medications for 12 hours 
or long acting beta agonists for 24 hours 
f) Allergic sensitization to at least one of the following allergen preparations: (House Dust 
Mite f, House dust mite p, Cockroach, Tree mix, Grass Mix, Weed Mix, Mold Mix 1, Mold 
Mix 2, Rat, Mouse, Guinea Pig, Rabbit, Cat or Dog) confirmed by positive immediate skin 
test response; OR a history of seasonal or perennial allergy symptoms such as sneezing, or 
itchy and watery eyes. 
h) subjects must be willing to avoid caffeine for 12 hours prior to all visits. 
i) subjects must be willing to avoid non-steroidal anti-inflammatory medications for 4 days 
prior to all visits 
 
Specific Exclusion Criteria:     
1. Clinical contraindications: 


 
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a) Any chronic medical condition considered by the PI as a contraindication to the exposure 
study including significant cardiovascular disease, diabetes, chronic renal disease, chronic 
thyroid disease, history of chronic infections/immunodeficiency, history of tuberculosis 
b) Physician directed emergency treatment for an asthma exacerbation within the preceding 3 
months 
c) Moderate or Severe asthma 
d) Exacerbation of asthma more than 2x/week 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. 
e) 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). 
f) Viral upper respiratory tract infection within 2 weeks of challenge. 
g) Any acute infection requiring antibiotics within 4 weeks of exposure or fever of unknown 
origin within 4 weeks of challenge. 
i) 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. 
j) Medications which may impact the results of the O3 exposure, interfere with any other 
medications potentially used in the study (to include systemic steroids, beta antagonists, non-
steroidal anti-inflammatory agents) 
k) Any history of smoking in the year prior to study enrollment; lifetime smoking history > 
10 pack years 
l) 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 
m) Allergy/sensitivity to study drugs, or their formulations. 
n) Known hypersensitivity to methacholine or to other parasympathomimetic agents 
o) History of intubation for asthma 
p) Unwillingness to use reliable contraception if sexually active (IUD, birth control 
pills/patch, condoms). 
q) Abnormal PT or aPTT values at screening or during the treatment period. Normal values 
will be those published by the clinical lab (Labcorp, INC). 
r) Any bleeding disorder 
s) Orthopedic conditions which would prevent the volunteer from performing moderate 
exercise on a treadmill. 
t) Radiation exposure history will be collected. Subjects whose exposure history within the 
past twelve months would cause them to exceed their annual limits will be excluded. 
u) Recent nasal surgery 
 
2. Pregnant women: Pregnant women will be excluded due to the radiation exposure 
associated with the mucociliary clearance scans.  Pregnant women and children (<18 years as 
this is the age of majority in NC) will also be excluded since the risks associated with O3 
exposure to the fetus or child, respectively, are unknown and cannot be justified for this non-


 
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therapeutic protocol.   Individuals over 45 years of age will not be included due to the 
increased possibility of co-morbidities and need for prohibited medications. 
 
3. Use of the following medications: 
a) Use of systemic steroid therapy within the preceding 3 months for an asthma exacerbation. 
All use of systemic steroids in the last year will be reviewed by a study physician. 
b) Use of inhaled steroids, cromolyn or leukotriene inhibitors (Montelukast or Zafirkulast) 
except for use of cromolyn exclusively prior to exercise.  Subjects who are prescribed ICS 
must demonstrate the ability to withhold these medications for 2 weeks prior to any visit 
without increased symptoms or increased requirement for beta agonist rescue. 
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 immunomodulatory therapy within the preceding 12 months 
f) Use of beta blocking medications 
g) Non-steroidal anti-inflammatory drugs in the 4 days prior to the baseline visit as well as 4 
days prior to the pre-exposure days. Subjects will not be allowed to use NSAIDs during the 
treatment periods 
h) use of any investigational agent within 6 weeks of Screening. 
 
 
4. Allergy/sensitivity to study drugs or their formulations: Known IgE mediated 
hypersensitivity to tocopherols, albuterol, diphenhydramine or corticosteroids. 
 
5. Physical/laboratory indications (may be temporary exclusions): 
a) Abnormalities on lung auscultation 
b) Temperature > 37.8 
c) Systolic BP >150 mm hg or < 90 mm Hg or diastolic BP>90 mm Hg or < 50 
d) Oxygen saturation of < 94% 
e) Abnormal PT or PTT values at screening or during the treatment period. Normal values 
will be those published by the clinical lab (Labcorp, INC). 
 
6. Inability or unwillingness of a participant to give written informed consent. 
 
d. Design of Study. 
This will be an intention-to-treat study. The effect of gamma tocopherol (yT) on O3 response 
of continuous variables (e.g., PMNs in sputum, cytokines, mucins and MCC) will be assessed 
using the difference (delta) from baseline (i.e. post –O3 minus baseline) values, comparing 
yT versus placebo treatment periods,  
Potential subjects will be seen for an initial visit at which time informed consent will be 
obtained.  Once the subject is deemed eligible by clinical lab and sputum values, they will 
return to the research lab for treadmill training and baseline muco-ciliary clearance (MCC) 
lung scan including a lung transmission scan.  Treadmill training is required to determine the 
treadmill settings for the exposure day, and will be done prior to MCC to control for exercise 


 
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effects on the baseline MCC measurements.  Baseline nasal endpoints will also be collected 
to determine pre and post O3 nasal epithelial cell gene expression levels and cytokine 
production.  On the following day, subjects will return to the imaging lab for a 30-minute 
follow-up MCC scan. 
Within 30 days of the screening visit, subjects will come in for randomization to placebo or 
gamma tochopherol (yT) supplement. At this visit, vital signs will be measured and 
spirometry will be performed, then subjects will undergo sputum induction.  They will be 
instructed to begin the study drug the evening of the visit, then take it every 12 hours for a 
total of four doses (the last dose being taken the morning of O3 exposure). They will arrive 
on the exposure day and undergo baseline measurements including venipuncture, spirometry, 
and NO.  Subjects will then undergo O3 exposure, followed by mucociliary clearance, 
venipuncture, and induced sputum.  Subjects will return to the research lab the next morning 
for a 24 hour follow up.  Subjects will have a 3 week-3 month washout and repeat the 
procedures during the second treatment period, with placebo or yT, depending on 
randomization. 
Subjects will be queried at all visits for any unexpected or adverse events, general health 
status, medication and supplement use, and for any recent injections. 
After completion of 48 hours of treatment, subjects will undergo baseline (pre O3) measures, 
including vital signs with symptom scoring, urine pregnancy test for women of childbearing 
potential, spirometry, venipuncture for CBC, PT, aPTT, and C-reactive protein (CRP), as well 
as study endpoints, up to about 50cc of blood.  Subjects will then undergo exposure to 
0.25ppm of O3 in a chamber, with intermittent exercise for three hours.  Spirometry and 
symptom scoring will be performed immediately after exposure, and 30 to 45 minutes after 
exposure.  Subjects will undergo (MCC) lung scan 1 hour after completion of exposure, 
followed by venipuncture for approximately 50cc blood, and by sputum induction (with 
albuterol pretreatment) and nasal samples at the completion of MCC. 
The following day the subject will return for vital signs; symptom scoring; spirometry; 
venipuncture for CBC, PT, aPTT, (CRP), and study endpoints; and a 30 minute follow-up 
MCC scan. 
Subjects will be sent home with a diary to complete each day for 4 days.  A follow up phone 
call will be made to the subject within 7 days of completing the first O3 exposure to review 
the diaries, following a phone script (attached).  If there are any health concerns from either 
the subject or the study team member, subjects will be asked to come to the research lab for 
evaluation.   
Subjects will be given a minimum of 3 week, maximum of 3 month washout and then repeat 
the series with crossover study medication. Samples for analyses will be collected and the 
MCC repeated at the same time points as those used in the initial sequence 
Subjects will return the next week after completing the second session for a check out visit 
which will include vital signs, symptom scoring, spirometry and if any values are out of the 
normal range, or if the subject has health symptoms or concerns, a physical examination and 
evaluation by a study physician. 
Randomization will be determined by Dr. Haibo Zhou, and the randomization will be given 
to IDS. Randomization blocks will be n=4. 


 
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Efficacy Measures: 
We expect that treatment with yT will increase airway sputum PMN and eosinophil response 
to inhaled O3. We also expect similar effects of yT on all exploratory measures, especially 
mucin secretion and MCC consistent with our preclinical data. We will use regression 
modeling to assess the impact of the GSTM1 null genotype on response to O3 and efficacy of 
yT supplementation. Our published data on the effect of this GSTM1 null genotype on 
response to LPS indicates that n=32 volunteers will yield 15 GSTM1 null participants. We 
expect enhanced response to O3 in this subgroup and selective efficacy of antioxidant 
interventions in this group. 
Pharmacology measures: 
We will also examine baseline and post treatment levels of alpha and gamma tocopherol and 
alpha and gamma CEHC (an active metabolite of tocopherol) 
Sample Size Estimates:  
Sample size for induced sputum granulocyte response: Our primary endpoint will focus on 
the effect of γT on reducing ozone-induced neutrophilic inflammation, defined by 
neutrophils/mg of sputum (PMNs/mg).  We are comparing the post-O3/pre-O3 ratio of log 
PMNS/mg as the data are not normally distributed.  Using data from our study of the effect 
of 2 mg of Fluticasone Propionate (FP) on O3 response, we observed in the placebo group 
that the Post/Pre ratio in log PMNs/mg has a mean 1.205, and a standard deviation 0.225. In 
the treatment group, the post/pre ratio has a mean 1.005, and a standard deviation 0.206 If 
γT-mT is 50% as effective as 2mg FP, the correlation between active treatment and placebo 
responses is  0.5,  and ß= 0.80 and α=0.05, then the estimated sample size is n=29.  We have 
inflated the sample size to 32 for potential drop-outs seen with crossover studies.   
Sample size for MCC: In a study of healthy subjects (n=16) we found a standard deviation of 
the delta between 2 repeat MCC measures of 8.7 % (SD) in average clearance through 90 
minutes (Ave90Clr) (i.e. the average of %clearance ((1-retention) X100) over all 10 min data 
points from 10-90 min). In a study of 18 healthy subjects challenged with LPS in a repeated, 
crossover design, we observed a decrease from 13% to 8% Ave90Clr compared to no LPS. If 
yT treatment completely blocks O3 induced decrease in MCC, using an estimated SD of 
8.7 % for repeat measures, a decrement of 5% in Ave90Clr (or a 40% decrement in MCC) 
should be seen with n=24 volunteers (ß= 0.80 and a=0.05).  The overall sample size of 32 for 
the primary endpoint should provide sufficient power to detect changes in this secondary 
endpoint. 
Safety measures: 
Criteria for safety within the entire protocol (failure of which would result in suspension 
of further study) will include the following: 
1. No more than 20 % (n=6) of patients will fail the individual safety criteria outlined 
below. Safety data endpoints will be assessed after every 10 subjects, and all adverse 
events will be reported to CBER, the UNC IRB and NIEHS. 
2. No occurrence of any Serious Adverse Event 
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 40% decrease in FEV1 or FVC from pre- O3 challenge values, which do 
not improve within the first 2 hours of challenge.  


 
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2. FEV1 must recover without treatment to within 90% of baseline within 3 hours of 
challenge without treatment. (Ibuprofen 800mg will be employed as rescue medication if 
the FEV1 does not return to 90% of baseline, or if symptoms persist.) 
3. Specifically no symptom score greater than “moderate” (3 on a scale of 0-4) for shortness 
of breath or cough throughout the protocol and no assessment of severe (4 on a scale of 
0-4) for any other individual item in the symptom score criteria. 
4. Oxygen saturation of >93% throughout the exposure period and must be at least 2% of 
baseline measure. 
5. If albuterol therapy is needed more than once during an observation period. 
6. Any increase in total symptom score over baseline within 96 hours after challenge which 
are greater than 6 out of a possible 24. 
7. Any significant abnormalities in PT and PTT during the treatment periods will be 
assessed and reported to CBER, IRB and NIEHS. 
 
 
Protocol Outline: 
I.  SCREENING (Visit 0): 
 
1. Vitals signs will be collected temperature, pulse, systolic and diastolic BP, respiratory 
rate, and SpO2 (oxygen saturation),  
2. A brief physical exam by a study physician. 
3. Urine pregnancy tests, using a commercially available testing device, will be performed 
on all women of childbearing potential. Pregnancy tests are considered valid for 7 days. 
4. FeNO will be collected. 
5. Spirometry will be performed following ATS/ERS standard procedures to obtain FVC 
and FEV1. 
6. A standard 12 lead ECG will be obtained. 
7. Venipuncture will be performed. CBC, PT/INR and aPTT, and CRP will be collected and 
sent to Labcorp, Inc. Additional blood will be collected for study endpoints, a total of 
about 50cc will be collected. 
8. Subjects will undergo sputum induction.  Subjects will be pretreated with 2-4 puffs of 
albuterol prior to sputum induction, and post albuterol spirometry will be collected. 
Subjects will inhale 3% saline for 7 minutes from an ultrasonic nebulizer. After the 
inhalation is complete, subjects will be asked to "cleanse" the upper airway by rinsing the 
mouth with water, gargling, clearing the throat, then blowing the nose. This is followed 
by a voluntary cough. Sputum will be placed into a sample cup. Spirometry will again be 
performed to ensure that the FEV1 does not fall more than 20% from the post albuterol 
value. The subject will then repeat the procedure with 4% and then with 5% saline. FEV1 
must be within 5 % of baseline before a subject will be discharged from the research lab. 
If the sputum sample is adequate for required analysis, the subject is potentially eliglible 
for continued study participation. 
 
II. TRAINING (V0a/b): 
All subjects who successfully complete the screening procedures will return to the research 
lab for a training visit, prior to V1.  The purpose of this visit is to establish the treadmill 
settings required for the volunteer to breathe at 20 L/min/BSA in m2 (DuBois). After baseline 


 
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vital signs the subject will be shown the treadmill and instructed in proper and safe use.  The 
settings will be increased until the desired minute ventilation is reached.  A urine pregnancy 
test will be repeated on those for whom it is applicable.  Vitals signs, including temperature 
HR, RR, BP, and SpO2, will be collected before and after treadmill exercise.  Spirometry will 
be performed before exercise begins.  Subjects will exercise for 15 minutes, during which 
time the treadmill will be adjusted to the subjects comfort and to reach the desired minute 
ventilation. They will then rest for 15 minutes, followed by another 15 minute exercise 
period to confirm the degree of exercise and the treadmill settings.  Subjects will be 
monitored with cardiac telemetry during the treadmill period, and vital signs will be 
measured after exercise is complete. 
 
A baseline MCC scan, including a transmission scan, will be completed following the 
treadmill training. (Full description in section f.) 
 
Nasal epithelial lining fluid (ELF) samples will be obtained from both nares.  Nasal epithelial 
cells (NEC) will be obtained from the right nare.   
 
Subjects will be required to return to the research lab 24 hours after the baseline MCC for a 
30 minute follow up scan. (V0b). 
 
III. DOSING: 
(Visit 1 and 4) 
At Visit 1, subjects will be randomized to receive either active gamma tocopherol or 
safflower oil placebo.  Visit 1 must occur within one month of the baseline visit.  Visit 1 may 
be combined with Visit 0a. 
1. Vitals signs will be measured. 
2. Urine pregnancy test will be performed on all women of childbearing potential. 
3. Spirometry will be performed. 
4. Exhaled NO will be measured. 
5. Sputum induction will be performed. 
6. Subjects will be given study drug prepared by the Investigational Drug Service and 
instructed to take the first dose of study drug the evening of the visit, 2 days prior to the 
planned exposure.  The second dose will be taken 12 hours later, in the morning on the 
day prior to the exposure.  Dose three will be taken 24 hours after the first dose.  The 
final dose will be taken on the morning of the exposure, 12 hours after dose 3. 
 
IV.   OZONE CHALLENGE (after 4 doses of study drug) 
(Visit 2 and 5): 
Pre-Exposure 
1. Urine pregnancy testing if applicable 
2. Vital signs 
3. Ozone Symptom questionnaire 
4. Exhaled NO measurement 
5. Spirometry   
6. Subject will undergo brief physical examination prior to exposure 


 
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7. Blood samples obtained for phagocytic function, γΤ,γΤ metabolite, αT, cytokines, 
proteins, immune and inflammatory markers, eicosanoids, glutahione levels, PT, aPTT, 
CBC with differential, CRP, plasma lipids, and other antioxidants, as well as flow 
cytometry studies; RT-PCR will also be performed for gene expression studies. 
8. ELF samples will be collected. 
9. Standard telemetry monitoring will commence 
 
Ozone exposure (0.25 ppm)  
1. The subject enters the chamber, and proceeds to the treadmill.  This is time point 0 for data 
collection.  Exercise starts at the settings determined on the training day.  
2. Minute ventilation is measured at 8 minutes into the exercise period, and again at 12 
minutes, for 2 minutes each time.  For subsequent exercise sessions, minute ventilation will 
only be measured at the 12 minute mark. 
3. The subject will exercise for 15 minutes, followed by 15 minutes of rest.  This cycle is 
repeated for a total of 3 hours, for 6 exercise and 6 rest periods. 
4. Blood pressure will be measured at least once an hour while they are in the chamber. 
5. Symptom scoring will be done immediately prior to exiting the chamber. 
Post-Exposure 
1. Spirometry and vital signs will be measured immediately post exposure. 
2. Elf samples will be collected. 
3. Spirometry will be collected again at approximately 30-45 minutes post completion of the 
exposure. 
4. One hour after O3 exposure is completed, the subject begins the MCC scan procedures.  
Spirometry will only be collected during MCC scanning if the subject has distress. 
5. At completion of MCC, vital signs and spirometry will be obtained.  This should 
approximately 6 hours after the beginning of the exposure time point. 
6. Post-exposure blood samples will be collected. 
7. Sputum induction, with albuterol pre-treatment. 
8. Elf samples will be collected. 
9. NEC will be collected from the left nare. 
10. Subjects will return to the gamma camera for a final 10-minute scan. 
11. Discharge home with contact information for a study physician. 
 
V.  FOLLOW-UP: 
(Visit 3 and 6) 
Subjects will arrive in the research lab approximately 24 hours after the start of the exposure. 
Subjects will undergo:  
1. 30 minute follow up MCC scan.  
2. Vital sign collection and symptom scoring 
3. Blood sample collection 
4. Exhaled NO 
5. ELF 
6. Spirometry  
Subjects will be discharged with Ozone Symptom questionnaires to be filled out on each of 
the 4 days following the Follow-Up Visit, and with contact information for a study physician. 


 
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A follow up phone call will be made to the subject approximately 7 days after discharge. 
 
VI.  WASHOUT PERIOD 
Subject will not take any study treatment for a minimum of 3 weeks and a maximum of 3 
months. 
 
VII.  CROSSOVER   
Subject will repeat steps II and III with the SO (placebo) if they received yT before 
crossover, or the subject will receive yT if they received SO before crossover.   
 
VIII.  END OF STUDY VISIT:  
(Visit 7) 
Within 5-10 days of the final challenge dose, each subject will be asked to return for a study 
discontinuation visit. At that time, the following assessments will be performed:  vital sign 
measurement including O2 saturation; lung function (FVC and FEV1); CBC, PT and aPTT (if 
abnormal values at Visit 6) and Ozone Symptom questionnaire. If any of these suggest 
lingering effects of ozone exposure, medical evaluation as directed by the study physician 
will be undertaken. 
 
The table below summarizes the procedures at each visit: 
 
Assessment/ 
Procedure 
V0 
V0a 
V0b 
V1 
V2 
 
V3 
 
V4 
V5 
V6 
V7 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
 
 
 
Review history/AE’s 
 
X 
 
X 
X 
X 
X 
X 
X 
X 
Record Concomitant meds 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
Urine pregnancy test 
X 
X 
 
X 
X 
 
X 
X 
 
 
Vital signs 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
12 lead ECG 
X 
 
 
 
 
 
 
 
 
 
Spirometry 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
Physical exam 
X 
 
 
 
X 
 
 
X 
 
X*** 
Blood draw 
X 
 
 
 
X* 
X 
 
X* 
X 
X*** 
Ozone Symptom 
Questionnaire 
 
 
 
 
X** 
X 
 
X** 
X 
X 
Sputum Induction 
X 
 
 
X 
X 
 
X 
X 
 
 
Exhaled NO 
X 
X 
 
X 
X 
X 
X 
X 
X 
 
ELF 
 
X 
 
 
 X** 
X 
 
X** 
X 
 
NEC 
 
X 
 
 
X 
 
 
X 
 
 
Telemetry 
 
X 
 
 
X 
 
 
X 
 
 
Treadmill training 
 
X 
 
 
 
 
 
 
 
 
Study drug dispensed 
 
 
 
X 
 
 
X 
 
 
 
Medication Diary disp. 
 
 
 
X 
 
 
X 
 
 
 


 
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Lung transmission scan 
 
X 
 
 
X 
 
 
X 
 
 
MCC 
 
X 
 
 
X 
 
 
X 
 
 
Follow-up scan  
 
 
X 
 
 
X 
 
 
X 
 
Ozone challenge  
 
 
 
 
X 
 
 
X 
 
 
*These assessments are performed twice at this visit – before ozone exposure and after ozone exposure 
**These are performed three times at this visit – pre-exposure, immediately post- and 6 hours post- exposure 
*** These are done as needed, as determined by the study doctor. 
 
e. Dosage: 
1.  Gamma Tocopherol: 
γT geltabs (and identical matching safflower oil placebo geltabs) will be provided by Andreas 
M. Papas PhD, President of Callion Health, Inc. and Adjunct Professor, East Tennessee State 
University, Johnson City, TN..  Subjects will take 2 geltabs (1200 mg/dose) every 12 hours 
for a total of 4 doses..  Each geltab will be composed of 612 mg γ-tocopherol, 7 mg d-α-
tocopherol, 28 mg d-β-tocopherol, and 8 mg d-δ-tocopherol. This γT preparation is currently 
in use by our group under a separate IND.  The geltabs will be taken with meals to maximize 
bile secretion in order to enhance absorption of the lipid material including tocopherols 
The 48 hour supply of study drug will be dispensed from the Investigational Drug Pharmacy 
to the CEMALB study staff who will provide the drug to the volunteers,   Subjects will be 
instructed to take the first dose in the evening at approximately 8pm, 2 days prior to the 
planned exposure.  They will be instructed to take dose 2 at 8am the next day, at 8pm that 
same day and at 8am the following day.  The 4th dose will be taken at the CEMALB.  
Subjects will be randomized to either orally administered gamma tocopherol or safflower oil 
during the initial study period, and will receive the alternative drug during the crossover 
period.  
Subjects will be allocated to the first treatment group using permuted block randomization 
with a block of size 4.  Thus after every 5th subject is entered, there will be two subjects in 
the placebo group for every 2 subjects in the gamma tocopherol group.  The randomization 
list will be prepared by a biostatistician and provided to the pharmacy.  Only the pharmacist 
and the statistician will have access to the randomization schedule.  Once a patient is found 
to be eligible for randomization, the clinician will contact the pharmacist and ask for 
medication.  The pharmacist will provide the medication as the next allocation on the 
randomization list.   If it is necessary to break the blind, the pharmacy will be contacted and 
will provide the treatment allocation.  During the crossover period, the pharmacist will 
provide the alternative study medication to a subject (i.e. if the subject was randomized to 
receive gamma tocopherol during the initial treatment period, they will be provided placebo 
during the crossover period. 
This section outlines CMC information on the ozone used as a challenge agent in a dose-
ranging clinical study to assess the effect of pre-treatment with a single dose of inhaled 
fluticasone propionate on lung inflammation following challenge with inhaled ozone and 
intermittent exercise in healthy volunteers, relative to placebo.  This amendment includes 
details of the description, composition, and manufacturer, method of manufacture, 
specifications and analytical methods. 
 


 
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2. Ozone (Challenge Agent) 
Description 
As described in the IND, Ozone is a highly reactive gas that is commonly found as a major 
component of photochemical smog and acute exposure to ozone is known to cause airway 
inflammation.  For the purpose of this challenge study, exposures to ozone via inhalation are 
conducted in a 4 x 6 x 3.2 m stainless-steel chamber with continuous reconditioning of the 
chamber air through high-efficiency particle filters.  Ozone is generated by exposure of 
oxygen to a silent electric arc in an ozone generator.  The concentration of the ozone is 
continuously monitored to ensure that the appropriate concentration is maintained. 
Composition 
The chamber atmosphere will be maintained by continuous conditioning of the chamber air 
to ensure a composition of ozone of 0.25 ppm (± 0.0125 ppm) over 3 hours at 22°C ± 1.1°C 
and 40% RH ± 5% RH. 
Manufacturer(s) 
The ozone is made in-situ for use in the environmental chamber that is located as follows: 
EPA Human Studies Facility 
104 Mason Farm Road 
Chapel Hill 
NC 27599-7315 
Method of Manufacture 
Ozone is generated by a Fischer, model 502 ozone generator (Meckenheim/Bonn, Germany).  
The generator uses oxygen which passes through a silent electric discharge to generate 
ozone.  The ozone concentration and output is controlled by varying the flow of oxygen 
through the generator and by varying the power of the silent electric arc.  The ozone is mixed 
with the air to obtain a desired concentration.  The resulting ozone enriched air is then 
introduced into the environmental chamber to allow exposure via inhalation. 
The concentration of ozone is continuously1 monitored using UV photometric analyzers.  The 
analyzers are connected to the Analyzer Support System comprising of: 
(1) Analyzer Function Support System.  This provides vacuum and gas shut-off 
valves, and potentiometers for the mass-flow controllers;. 
(2) Analyzer Calibration System.  This consists of equipment to provide accurate cal-
ibration of the gas analyzer;  
(3) Analyzer Alarm System.  Comprises computer software which controls alarms 
(both sound and visual (light) warnings) and gas shut-off valves. 
Description of the Manufacturing Process 
The ozone delivery system comprises the following components: 
Gas supply bottles and manifolds in the bottle house with oxygen cylinders; 
 
Gas delivery panels; 
 
Gas injection system; 
 
Ozone generator; 
 
Deionized water system; 
 
Computers and computer interfaces. 


 
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Stainless steel gas plumbing is used to deliver oxygen to the pollutant delivery system on 
demand.  Mass-flow controller precisely control the amount of oxygen that is supplied to the 
zone generator and subsequently mixed with facility makeup air.  To ensure that the flow of 
oxygen is not interrupted, each gas manifold (equipped with a pressure regulator) will 
automatically change from one bank of cylinders to another.  The line pressure, of 
approximately 50 psig, is monitored by a computer.  Deionized water is used to humidify the 
air. 
f.  Observations and Measurements 
 
1. Primary Endpoints:  The primary efficacy endpoint (airway neutrophils/mg in the 
sputum) will be assessed by post-O3/pre-O3 ratio of log sputum PMNS/mg, comparing the 
ratios during the placebo treatment to the active treatment arms.  
2. Safety measurements: Symptom scores, lung function tests, PT and aPTT and platelet 
counts will be assessed as safety measures. Tocopherols in high doses may rarely have an 
effect on coagulation. 
3. Exploratory Endpoints:  
Cytokines and other proteins at baseline and in response to ozone: We will examine sputum 
and plasma samples for levels of inflammatory cytokines, including (but not limited to) GM-
CSF, IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, RANTES, eotaxin and TNFα. Also to be 
examined are metalloproteinases, mast cell tryptase, eosinophil cationic protein and 
eosinophil derived neurotoxin. CRP will also be measured at baseline and 24hr post-
exposure. 
Airway and blood phagocyte function: We have observed that inhalation of inflammatory 
stimuli (including ozone and ozone) is associated with changes in cell surface markers of 
monocytes, macrophages, and neutrophils, especially CD11b (CR3 receptor, associated with 
response to complement and egress of cells from the circulation to tissue),  CD16 (FcRγI 
receptor for IgG), CD64 (FcRγIII receptor for IgG), CD45 (pan leukocyte marker), CD80 and 
CD86, MHC class II molecules, and CD14 (an endotoxin receptor).  We have also observed 
that the ability of monocytes and macrophages to phagocytize opsonized zymosan is blunted 
following challenge with inhaled inflammatory stimuli (1). Similar observations have been 
made in asthmatics with increased eosinophilia (2). We will examine the effect of γΤ 
supplementation on these important parameters of host defense. 
Microarray analysis: We will analyze mRNA isolated from sputum macrophages and 
granulocytes using DNA microarray technology to assess genes involved in innate immune 
and oxidant stress responses before and after ozone challenge.  We will also analyze protein 
mediator profiles of these cells using the sputum supernatants.  These techniques will allow us 
to examine if γΤ supplementation modifies innate immune and oxidant stress responses. 
Immune Markers: We will also examine cell surface markers of lymphocyte subsets (CD3, 
CD4, CD8, CD45), monocytes (CD14), NK cells (CD16/56), B cells (CD19/20) and markers 
relevant to allergic responses (CD23, FcΕRI)    


 
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Eicosanoids: We will examine sputum and plasma samples for levels of cyclooxygenase 
pathway products (PGE2, PGF2α) and lipoxygenase pathway products (LTB4, LTC4, LTD4, 
LTE4) as γΤ has observed actions on eicasanoid production. 
Plasma lipids:   Panel will include cholesterol, LDL, VLDL and HDL and triglyercides.  
Markers of inflammatory cell activity: To assess neutrophil activity in the airway, we will 
assay sputum for meyloperoxidase (MPO) and 5-chlorouracil (a product of the action of HOCL 
derived from neutrophil MPO and nucleobases of injured cells, and a marker of PMN oxidative 
burst). To assess for eosinophil activity, we will assay sputum for eosinophil cationic protein 
(ECP) and 5-Bromouracil (a product of the action of HOBr derived from eosinophil peroxidase 
and nucleobases of injured cells, and a marker of eosinophil oxidative burst). Reactive nitrogen 
oxide species (RNOS) such as peroxynitrite are generated by inflammatory cells following 
stimulation with inflammatory irritants and  sputum and plasma samples will be assayed for 5-
Nitro-γ-tocopherol, a product of the action of RNOS on γΤ. 
Nasal epithelial lining fluid:  We will examine epithelial lining fluid for levels of 
inflammatory cytokines, including (but not limited to) GM-CSF, IL-1β, IL-4, IL-6, IL-8, IL-
13, IL-17, RANTES, eotaxin and TNFα. We will also examine levels of metalloproteinases 
and eosinophil cationic protein. 
Nasal epithelial cells:  We will analyze mRNA isolated from nasal epithelial cells using a 
customized Nanostring immune panel to assess expression levels of genes involved in innate 
immune and oxidant stress responses before and after ozone challenge. Genes examined will 
include those implicated in various aspects of epithelial-driven inflammatory responses, such 
as those associated with epithelial barrier integrity (apical junction complex proteins), 
epithelial-derived danger signals, pro-inflammatory chemokines/cytokines, PRRs (pattern 
recognition receptors), downstream targets of PRR signaling, and genes associated with the 
antioxidant response element.   
Other Antioxidants in plasma and sputum samples:  These measures will be provided by 
the Biochemistry Core of the UNC Clinical Nutrition Research Unit and may include 
reduced and oxidized glutathione, catalase, glutathione peroxidase, glutathione reductase, 
and superoxide dismutase. 
4. Hypertonic Saline Induced Sputum procedure: 
FEV1 and force vital capacity (FVC) are measured before ozone challenge to determine the 
post bronchodilator baseline FEV1, and FVC values. The FEV1 values that match a 10% and 
20% fall from post bronchodilator baseline are determined. An ultrasonic nebulizer is filled 
with 15cc of 3% hypertonic saline (inhalation grade for respiratory use only, 3% NaCl) to 
begin the test. The nebulizer is set to the maximum output setting and turned on. The subject 
is instructed to latch his/her mouth onto the nebulizer mouthpiece and breath normally (i.e., 
tidal breaths) for 7 minutes. The saline is nebulized through the mouthpiece in a jet stream 
and inhaled. The nose is not occluded for this procedure. The subject is 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 are 
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 is expectorated into a sterile specimen jar 
and capped. Following the measurement of FEV1 after the first 7 minute inhalation period, 


 
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the concentration of saline is increased from 3% to 4%, provided the FEV1 falls by < 10% 
from the post-bronchodilator value. A volume of 15cc fills the nebulizer well on each 
occasion. If the FEV1 falls between 10-20% of the post bronchodilator value, the test 
proceeds but the concentration of saline remains the same If the FEV1 falls by > 20% or if 
troublesome symptoms occur, the nebulization is discontinued and albuterol is available if 
necessary to relieve symptoms. Troublesome symptoms included cough, chest tightness, and 
general discomfort. The same procedure is followed for the final 7 minute inhalation period 
using 5% hypertonic saline provided the FEV l safety parameters described above have been 
met. The nebulization is stopped after 21 minutes or earlier if a sputum sample of good 
quality is obtained (i.e. visible sputum plugs). 
5. Spirometry:  
Standard methodology conforming to the American Thoracic Society guidelines for 
measurement of spirometry will be used.  
6. Fraction of Exhaled Nitric Oxide 
Airway inflammation will be evaluated using the NIOX MINO device, following ATS, 2005 
recommendations and the manufacturer’s instructions. Since spirometry can potentially 
impact the nitric oxide (NO) measurement, the FeNO test needs be completed prior to 
spirometry. In addition, subjects should not eat or drink 1 hour prior to having the FeNO, as 
this may affect the results. 
7. Nasal Epithelial Lining Fluid (ELF):  Nasal ELF will be sampled with a small strip of 
Leukosorb paper inserted gently by means of direct vision into the nasal cavity laterally against 
the anterior portion of the inferior turbinate. Sterile saline solution is transferred into a mist 
bottle (the same one used for NL) and subjects are asked to moisten their nasal passages with 
a single spay into each nostril.  A nasal clip will be applied, and the Leukosorb paper will be 
left for absorption for 2 minutes. Subjects will be required to breathe though the mouth during 
this time.  Thereafter, the Leukosorb strip will be removed and immediately stored at 80°C. 
8. Nasal Epithelial Cells (NEC):  a non-invasive biopsy procedure will be 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.  This maneuver will be 
repeated on the contralateral nasal cavity using a fresh curette.  The nasal biopsy may only be 
obtained from 1 nostril during a visit.   
9. Regional Deposition and Mucociliary Clearance (MCC) procedures: 
Prior to the each MCC study a transmission Co57 scan will be performed to define the lung 
boundaries, to assign regions of interest, and to normalize these regions for lung volume 
differences. A rectangular phantom containing the radioisotope Co57 (< 25 mCi) will be placed 
in front (5cm) of the subject sitting with his/her back to the gamma camera for 30 seconds. The 
transmission scan has been used by us and others to provide a delineation of lung boundaries 
for assessing regional deposition/clearance of the inhaled radioaerosol.  Prior to the 
transmission scan on each study day we will place 2 spot markers of Americium241 (0.9 


 
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microcurie (uCi) each, gamma 66 KeV) on the upper and lower back of each subject during 
scanning (both Tc99m-SC deposition/retention and Co57 transmission). With dual isotope 
imaging, these spot markers will allow alignment of images for more accurate determination 
of regional deposition/retention. These very low radiation sources have been obtained from 
commercially available home smoke alarms. The placement of these markers will be 
determined to be outside the lung field during the transmission scan. Their location will be 
marked in semi-permanent ink for later placement during Tc99m deposition/retention scans. 
The shielded side of this source will be placed/taped onto the subject’s skin. 
Radiolabeled Tc99m-sulfur colloid will be delivered using a modified Pari-LL nebulizer 
(MMAD 9.5 um).  This is a closed delivery system that produces 80 ml/sec air flow, and 
therefore limits the inspiratory flow rate to this value.  While seated in front of a gamma camera 
subjects will perform single inhalations lasting ~10 seconds each from the delivery system, 
and will exhale at 500 ml/sec (using feedback from a flow meter in the breathing circuit).  
Approximately 5-10 of these inhalation maneuvers will be required to deposit an adequate 
radioisotope dose to the lung.  Subjects will be allowed to breathe normally (off the nebulizer) 
in between each inspiratory maneuver. Each volunteer will practice these maneuvers prior to 
the actual radioaerosol inhalation to guarantee his/her proficiency.   The activity of Tc99m-SC 
loaded in the nebulizer will be adjusted to provide an estimated 40 uCi deposited in the lung 
for each MCC scan. A single crystal detector will be placed at the subject’s back during 
inhalation to monitor dose to the lung. Total inhalation time should be less than 5 minutes in 
all cases. Immediately following radioisotope inhalation, the subject will gargle and drink 
water to clear activity that deposited in the mouth into the stomach. The subject will then 
(within a minute of final inhalation maneuver) be seated in front of a large-field-of-view 
gamma camera to begin acquiring particle retention images.  Serial, 2-minute gamma images 
will be captured continuously for the first 34 minutes following isotope delivery.  Thereafter, 
2 consecutive 2-minute images will be obtained at the start of every 10 minute period until 2 
hours post isotope inhalation. The subject will also return the following day to obtain a 30 
minute scan of 24 hour lung activity. 
Whole lung clearance is strongly dependent on the site of particle deposition within the lung. 
To characterize regional deposition in the lung a transmission scan of the subject’s lungs will 
be obtained on each MCC study day to outline the area of the whole lung. Central (C) vs. 
peripheral (P) deposition will be determined by region of interest (ROI) analysis and 
normalized to C/P for the transmission scan. A whole lung ROI bordering the right lung will 
be used to determine, by computer analysis, the whole lung retention, as a fraction of the initial 
counts in the right lung, over the two hour clearance period and at 24 hours. The computer 
program decay-corrects the Tc99m counts throughout the clearance period. Both C and P lung 
retention will also be determined to allow comparisons of MCC from a region with a 
preponderance of large, bronchial airways to a region lacking such airways.  
10. Symptom Scores: 
Subjects will be asked to rate a variety of possible symptoms during the dosing period, 
including the following: headache, irritation of the nose stuffy nose/sinus congestion, runny 
nose, dry/sore throat, pain on deep inspiration, unusual fatigue or tiredness, eye irritation, 
sneezing, shortness of breath, cough, wheezing/whistling in chest, chest tightness, sweating 
and other. 
The following symptom score assessment will be made: 


 
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0 = NONE 
 
 
 
(not present) 
1 = TRACE/NOTICED 
 
(barely detectable) 
2 = MILD/LIGHT 
 
 
(present, but not annoying) 
3 = MODERATE 
 
 
(present, but somewhat annoying) 
4 = SEVERE/HEAVY 
 
(present and very annoying and painful) 
The highest possible score will be 60 and the lowest is 0.  A mild score for every criteria is 
15, moderate is 30 and severe is 60. 
11. Venipuncture:  Blood samples totaling approximately 50ml will be taken each week for 
a total of 150ml.  An additional volume of 6ml will be taken during each O3 exposure. 
 
Observation Schedule: Prior to any inhalation challenge, subjects will undergo a physical 
examination of the ears, nose, throat and chest and will have an assessment of vital signs 
(temperature, pulse, respiratory rate, blood pressure), oxygen saturation, symptom score 
assessment and undergo spirometry to rule out acute illness prior to challenge.  All female 
volunteers will undergo a urine pregnancy test.  The timing for collecting study endpoints in 
relation to the exposure is that time 0 is when the subject enters the exposure chamber.  
Induced sputum will be obtained on the screening day and again at six hours after each 
exposure. Sputum will be analyzed for PMN content, CD14 expression on airway 
macrophages and monocytes, soluble CD14 levels, cytokine levels in sputum and products of 
inflammatory cells (eosinophil cationic protein, myeloperoxidase). Blood will be collected 
for a CBC and differential and other exploratory endpoints when the pre- challenge induced 
sputum is obtained and 6 hours after challenge. 
Any subject who is asked to withhold ICS for the purpose of this study will have baseline 
asthma symptom scoring done.  These subjects will complete scoring each day that they 
withhold medication.  They will be given contact information for a study physician who will 
be available 24 hours a day.  They will have rescue albuterol available.  Subjects will be 
discontinued from the study and returned to prior dosing regimen if they have sustained 
increase in asthma symptoms.  Subjects will be treated with prednisone if needed for an 
exacerbation.  
 
Post-Challenge Observations Reporting: 
All volunteers will be discharged home after each challenge day with contact information for 
a study physician.      
Each volunteer will be given a symptom scoring sheet for each day up to 96 hours (4 days) 
after challenge. The following instructions will be written on the sheet: 
Please record your symptom score today at noon based on the scale above. Please call the 
study coordinator or physician for the following reasons: 
1. If your total score is >6 plus your baseline score.  
2. If any of your individual symptoms equals 4.  
3. If you are concerned about symptoms regardless of the score you assign them. 
Each sheet will include the name and phone number of the study MD.  Sample home 
symptom scoring sheets and instructions are included with this protocol. 


 
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Study discontinuation visit (Visit 7): 
Within 5-10 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 SpO2 (oxygen saturation), CBC, PT and aPTT and symptoms 
scores will be assessed and, if abnormal, medical evaluation as directed by the study 
physician will be undertaken. 
Definition of an unanticipated problem, or UP, (previously referred to adverse event 
AE) and Serious Adverse Event (SAE): 
An unanticipated problem for a given volunteer will be defined as failure of any of the safety 
criteria outlined in section “d.” 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 “d” will be considered an adverse event.  Failure of a total symptom score to return 
to no greater than 6 above baseline within 96 hours will be considered an adverse event. Any 
symptoms that induce a volunteer to seek medical attention from any provider within 96 
hours of challenge will be considered an adverse event. 
A significant 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).   
 
g. Monitoring and risk minimization 
Risks to subjects: At the doses proposed in this study, the most significant predictable risk to 
subjects as a result of ozone inhalation is development of airway inflammation which would 
cause shortness of breath, cough or wheeze. Ozone is known to cause a temporary inhibition 
in the ability to take a deep breath – this is transient and typically declines within 2 hours of 
completion of exposure, though it may take up to 72 hours for complete resolution.  At high 
doses, tocopherols can increase coagulability, though we have not observed this in other 
studies using gamma tocopherol.  Nausea and upset stomach have been reported, subjects are 
asked to take the supplement with food.  Sputum induction may induce bronchospasm.  
Venipuncture carries a risk for hematoma.   There is a slight risk of sneezing with ELF 
collection, subjects who may not be able to tolerate the paper will not be asked to continue 
with this measurement.  Nasal epithelial biopsy is a transiently irritating procedure, and it 
presents a rare risk of nose bleed. 
Measures to minimize risk: Only volunteers who have normal lung function 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 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 ozone inhalation challenge, will be a basis for 
exclusion. As outlined above in section “d”, all subjects will need to fulfill objective lung 
function and symptom criteria prior to initiating a specific challenge study.  Subjects will be 
questioned about their previous radiation exposure history. Subjects whose exposure history 


 
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within the past twelve months would cause them to exceed their annual limits will be excluded. 
Upon being accepted into the study each subject will be advised to avoid radiation doses of a 
comparable magnitude within the next 12 months unless there is a diagnostic or therapeutic 
necessity. Female volunteers will be asked to use effective birth control 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.  Nasal samples will not 
be collected in subjects who have had recent nasal surgery. 
 
Risks from MCC measures 
The radiation doses for up to 120 µCi of inhaled Tc99m-SC (3exposures of 40 µCi over the 
three month period) to various organs (other than the lungs) are listed below: 
ORGAN DOSE (mRem)  
Kidneys 12  
Bladder Wall 9 
Stomach Wall 30 
Thyroid(unblocked) 18 
Testes 1.2  
Ovaries 5.4 
Red Bone Marrow 2.4 
Upper Large Intestine 15 
Lower Large Intestine 1  
Whole Body 1.8 
These doses were computed assuming an instantaneous uptake of free TcO4(99m) from the 
systemic circulation. This simulates the situation where all of the label breaks free from sulfur 
colloid and is transported instantaneously to the systemic circulation. These doses represent 
extreme cases that rarely, if ever, occur in practice. Previous studies show no Tc99m activity 
in the bloodstream of dogs over a 3 hour period after depositing Tc99m-sulfur colloid on the 
airway surfaces of dogs. TcO4(99m) is secreted by the gastrointestinal tract and there is 
minimal reabsorption. Thus, if all the label were to break free from the sulfur colloid, the 
stomach, large intestine, thyroid and kidney which see a large blood flow would receive the 
highest exposure from TcO4(99m). Since the sulfur colloid particles are not water soluble they 
are not cleared by dissolution. 
For our inhalation conditions 30-60% of the inhaled particles will likely clear by mucociliary 
clearance. Particles that clear by this mechanism will be swallowed and eliminated by the GI 
tract. Most of the remaining deposited particles will be in alveoli or small airways and clear by 
much slower processes with a half-time of approximately 60 days. Considering the short 
physical half-life of Tc99m one need not consider the radiation doses to organs, other than the 
lungs and GI tract, from Tc99m bound to sulfur colloid. There is potential for enhanced 
radiation doses to large airway epithelia. The local radiation dose depends on the pattern of 
particle deposition and on the rate of mucociliary clearance. Using published data and 
assuming a mucus transport rate in the trachea of 1.0 mm/min (representing the lowest limit 
measured in normal humans), the highest radiation dose delivered to the trachea will be 0.8 
Rems for a single 40uCi exposure. The radiation dose will decline towards the more peripheral 
portions of the lung reaching 0.016 Rems in the parenchyma or alveolar regions. The 
unweighted average dose to all tissue compartments will be 0.32 Rems (for a single 40uCi 


 
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exposure). These computed doses represent the upper limits since normal tracheal mucus 
velocity is approximately 5 mm/minute.  
Transmission scans will also be obtained to identify non-ventilated lung regions. 
These scans will be used to outline/define the whole lung regions for region-of-interest analysis 
of the Tc99m-SC radioaerosol scans on each study day. For the lung transmission scan 
procedure, a planar, solid sheet containing the radioisotope Co57 (< 25 mCi) will be placed in 
front (5cm from body) of the patient sitting with his/her back to the gamma camera while a 30 
sec gamma camera scan is acquired. In this case the dose from the Co57 exposure is from an 
external source (i.e. not inhaled) and is similar over all portions of the lung tissue (i.e. similar 
to a chest X-ray). The dose to the surface of the chest and the lungs (assuming no chest wall 
attenuation) under these conditions is 7.5 mRad for a single scan, a relatively small dose 
compared to the inhaled radioaerosol. As the Co57 source decays over time (270-day half-life) 
we will increase the time for the transmission scan accordingly to achieve comparable scans 
of the lung while not exceeding the 7.5 mRad dose per scan.  
The transmission scan has been used by us and others to provide a delineation of lung 
boundaries for assessing regional deposition/clearance of the inhaled radioaerosol. The 
absorbed radiation doses from the low activity (<1uCi) /energy (66Kev) Americium241 
sources, placed on the subjects backs for alignment of images, is insignificant relative to the 
Tc99m and Cobalt57 doses associated with these experiments. 
The maximum doses that we have computed for the lungs (combined  three Tc99m and  three 
Co57 transmission), 2.4 Rem local dose and 0.98 Rem average for whole lung, are well under 
the maximum single dose of 5 Rems for a normal adult volunteer as set forth in Federal 
regulations, and well under the 15 Rem maximum for the annual dose commitment. The doses 
to all other organs and the whole body are all far less than these maximum doses. The total 
effective dose equivalent (based on the unweighted average to the whole lung of  983 
mRem) is 133 mRem. In considering these dose estimates and comparing it with other nuclear 
medicine procedures, it should be emphasized that we have gone to considerable lengths in 
calculating local tissue doses. Most often average doses to organs are used which would be 
considerably less than any local doses. 
While the radiation exposure associated with our measures of MCC represents a potential risk 
to the human volunteers in this study, the risk is very low. The effective dose for the  
threemeasures of MCC (including Co57 transmission scans) is approximately 133 mRem. This 
is less than the natural environmental radiation that adults receive every year, which in Chapel 
Hill is about 300 mRems. The risk from the radiation dose received from this procedure is too 
small to be detected.  In addition, the dose of Tc99m- sulfur colloid to be deposited in the lung 
for each measure of MCC is 10% of that recommended as the adult oral dose for diagnosing 
pulmonary aspiration by gamma scintigraphy (CIS-US,Inc). 
A physician familiar with the protocol will be available for all challenge procedures. 
Emergency treatment with albuterol, oxygen, 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 or exposure study. 


 
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All subjects will be will be monitored as outlined in previous sections and will return for 
assessment 24 hours after the challenge. All subjects will be provided with a contact 
telephone number for access to a study physician who is on call 24 hours/day. 
Reporting of AEs and SAEs 
All SAEs will be reported to the CBER of the FDA as well as to the UNC Biomedical 
Institutional Review Board (IRB) within 24 hours of recognition of the event. Adverse events 
will be reported to both the FDA and UNC IRB on an annual 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 unexpected 
problem related to the study will be evaluated by a study physician, and if needed the subject 
will be examined by the study physician. All assessments will include the same lung function 
and temperature 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) event will be 
reported to both the FDA and the UNC IRB. 
 
 
 
 
 
 
 
 
 
 


 
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SAP_001.pdf: 
 
 
 
 
 
 
Study Title 
Effect of Gamma Tocopherol Enriched Supplementation on Response to Inhaled O3 Exposure 
 
 
 
NCT number 
 NCT02911688 
 
Document Date December 17th, 2019 
 
 
 
 
 
 
 
 
 
 
 


 
Statistical Analysis Plan 
For 
Effect of Gamma Tocopherol Enriched Supplementation on Response to Inhaled O3 Exposure 
 
Statistical Analysis:  
The effect of gamma tocopherol (γT) on ozone (O3) response of continuous variables (e.g., 
PMNs in sputum, cytokines, mucins) will be assessed using the difference pre O3 values after γT 
and placebo dosing for a given variable. To analyze cross-over clinical trial data, graphical 
presentations will be used to highlight potential order and treatment effects and use rigorous 
testing to confirm these effects. For these graphical presentations, the two plots that depict period 
1 responses against period 2 responses for both arm sequence groups (Placebo-Active and 
Active-Placebo) will be examined to confirm the presence of absence of period effect. The 
response centroids of each sequence group will be calculated to determine if a direct treatment 
effect of γT exists. These observations will be tested with a paired t-test or Wilcoxon signed rank 
test, depending on normality of the data, to evaluate the significance level of the treatment, the 
period and the carryover effects.  
For analysis of mucociliary clearance outcomes, linear regression models will be fitted that 
account for the initial regional lung deposition of radioaerosol (the central-to-peripheral, or C/P, 
deposition ratio) as a covariate to determine if γT treatment impacts O3-induced changes in 
MCC compared to placebo. Dr. Zhou, the biostatistician for the CEMALB will lead the 
statistical team overseeing data analysis and guide the statistical analysis. 
As an alternative analysis approach, regression modeling techniques will be employed to assess 
genotype effects (including the GSTM1 null genotype) on response to pollutants. For this study, 
a linear mixed model approach will be used that considers individual tests in a global, unified 
way where all data are used at the same time. This method should be more powerful than the two 
sample t-tests, especially when there is a significant carryover effect. This approach 
will also allow us to account for the influence of gender, race, BMI, and analysis of the effect of 
the GSTM1 null genotype on response to O3 and effect of γT on this response. 
To address potential missing data, we will employ multiple imputation methods to address this 
issue when appropriate.