Nitric Oxide, LPS and the Pathogenesis of Asthma Phase II

(IRB# Pro00005035)

The purpose of the study is to determine the role of nitric oxide (NO) in asthma and to
characterize the symptoms associated with inhaled endotoxin in normal subjects. The effect
of inhaled endotoxin on exhaled NO is determined in healthy African Americans, with and
without NOS2 promoter polymorphisms. The protocol involves the use of NIH Clinical Center
Reference Endotoxin which has been approved by the FDA under IND BB-IND-10035.

Background & significance: Asthma is a significant cause of morbidity and mortality for
African Americans and asthma is increasing in prevalence. Both environmental and genetic
factors contribute to the pathogenesis of asthma. In addition to allergens, environmental
lipopolysaccharide (LPS) or endotoxin plays an important role in the chronic inflammation
associated with asthma. There is convincing evidence that endotoxin exacerbates airflow
obstruction and airway inflammation in allergic asthmatics. Endotoxin increases inducible
nitric oxide synthase (NOS2) expression and nitric oxide (NO) production in vitro and in
vivo in humans.

In asthma, NOS2 expression is upregulated in several cell types including bronchial
epithelium, macrophages and other inflammatory cells. The increase in NOS2 expression is
associated with increases in exhaled NO in individuals with asthma as compared to healthy
individuals.

This prompted our search for NOS2 single nucleotide polymorphisms (SNPs) and the subsequent
identification of three NOS2 promoter SNPs. Two of the SNPs are associated with increased
systemic NO levels. Another SNP creates a predicted binding site for the human homolog of
delta EF1, which is a transcriptional repressor and we predicted that this SNP would be
associated with low NO levels. Importantly, the three NOS2 promoter SNPs are not linked, do
not segregate together and are present primarily in individuals of African ancestry.

Design & procedures: The study of exhaled NO levels in individuals with asthma is
confounded by the presence of cell types besides bronchial epithelium that produce NO, by
differences in the severity of asthma and by the use of medications such as corticosteroids
which alter exhaled NO levels. Therefore, in this protocol exhaled NO levels are measured
in asymptomatic healthy African Americans.

Thirty individuals (10 individuals with each polymorphism) who are heterozygous for a NOS2
promoter SNPs and 10 individuals without these NOS2 promoter SNPs will be studied. The
decision to study 10 individuals in each group was based on a power analysis (80% power,
0.05 alpha) using available data on NO levels in normal individuals following endotoxin
challenge. Because basal NO levels in patients with asthma are approximately twice that of
normal individuals, we decided that a two-fold difference in exhaled NO levels following
endotoxin challenge represents a meaningful difference.

The general design of this study is to identify healthy nonasthmatic, nonatopic, never
cigarette smokers with no history of airway reactivity, and subject them to a specific
incremental challenge with inhaled LPS to determine the airway reactivity to inhaled
endotoxin. Upon recruitment, each study subject will be scheduled for seven separate
visits, with the option of 3 additional visits after each challenge. During the initial
visit, each study participant will undergo a thorough clinical evaluation including 2
questionnaires, a physical examination, skin tests to assess the presence of atopy, full
pulmonary function testing, a chest x-ray, an EKG, and a methacholine challenge test to
assess airway hyperreactivity, exhaled nitric oxide and exhaled breath condensate samples to
measure inflammatory mediators. During the challenge visits, each subject will undergo a
specific incremental challenge with inhaled endotoxin or the vehicle (saline). The initial
evaluation and the endotoxin inhalation challenge will be separated by a minimum of two
weeks. A brief follow up visits will be scheduled to collect blood samples, exhaled gases
24, 48, 72hours and 7 days after endotoxin challenge and to obtain full pulmonary function
testing with airway resistance performed at baseline. To avoid problems with diurnal
variation in pulmonary function, all testing sessions will begin at 8:00 AM. All testing
will be performed in the Rankin Duke Clinical Research Unit at Duke University. Serum
pregnancy testing will be performed during the initial visit and the days of each endotoxin
and saline inhalation challenge to exclude pregnant women from this study. Administration
of endotoxin or saline will not occur until results of the pregnancy test have returned
negative.

In addition to serum pregnancy testing, blood samples will be obtained to determine IgE
levels (to determine whether subject has allergies; determined at visit 1 only), cotinine
levels (a metabolite of nicotine and used to determine whether subject smokes; determined at
visits 1, 2, 4 and 6), CRP and cytokine levels (markers of inflammation used to determine
whether systemic responses to inhaled endotoxin occur; determined at visits 2, 3, 4, 5, 6
and 7), and RNA levels (to be analyzed using gene arrays and used to determine whether
systemic responses to inhaled endotoxin occur; determined at visits 2, 3, 4, 5, 6 and 7). On
visits 2, 4 and 6, CRP, cytokine and RNA levels will be determined three times, initially to
establish baseline levels and 2 and 6 hours after endotoxin or saline challenge. On visits
3, 5 and 7, CRP, cytokine and RNA levels will be determined once. The URI questionnaire
will be administered at the beginning of all visits to assess whether subjects have symptoms
consistent with a URI or cold.

The Duke Investigational Pharmacy will prepare the inhaled solution of endotoxin according
to a standard protocol which has been used successfully by other investigators, as well as
in our own laboratory. Each subject will receive an initial dose of Saline Solution as a
baseline. During the second visit, inhalations will contain increasing concentrations of
endotoxin (Clinical Center Reference Endotoxin (CCRE), a lot of endotoxin prepared from the
bacterial strain E. Coli O:113 and maintained by the Pharmaceutical Development Service,
Warren Grant Magnuson Clinical Center, National Institutes of Health) according to the
following schedule: 5,000 EU (endotoxin units); 10,000 EU; and 20,000 EU. During the higher
dose challenge visit, inhalations will contain 40,000 EU and 80,000 EU of endotoxin.
Following inhalation of each dose of endotoxin or each saline challenge, we will obtain
several spirometric measures of airflow. Airflow will be assessed 1, 10, 20, and 30 minutes
following each dose of inhaled LPS or saline. If the study subject's FEV1 is more than 80%
of the baseline measurement, the inhalation challenge will continue and the next higher dose
of endotoxin will be administered. During the control visit, saline control will be
administered on three occasions. After each dose of inhaled endotoxin or saline,
non-pulmonary adverse events will be assessed and graded. The protocol will conclude if any
of the following criteria have been met: 1) subject does not wish to continue for any
reason; 2) subject's FEV1 has decreased 20% from baseline saline inhalation; 3) total dose
of 35,000 units (low dose challenge visit) or 120,000 units (high dose challenge visit) has
been achieved; or 4) the subject experiences any adverse event rated as severe or any other
adverse event deemed significant in the opinion of the investigator. Following completion
of the inhaled endotoxin or saline challenge, pulmonary function studies, vital signs and
symptom assessments will be performed to assess the safety of the inhaled endotoxin
procedure (see attached IND protocol for details). We will also collect exhaled breath
condensate samples to measure inflammatory mediators including leukotrienes and to control
for the confounding effects of airway inflammation on exhaled NO levels. Exhaled breath
condensate will be collected from subjects by having them breathe normally for approximately
10 minutes into an apparatus that traps condensation by cooling the exhaled breath. We will
measure airway resistance at 1, 2, 6 and 24 hours after endotoxin or saline challenge.
Measurement of spirometry, lung volumes, diffusing capacity will be repeated at 6 hours and
24 hours post challenge.

We will measure exhaled NO levels using an online chemiluminescent detector (Sievers 280i)
prior to and 1, 2 and 6 hours after endotoxin or saline challenge. We will also measure
exhaled NO levels 24 hours after exposure to endotoxin or saline.

Subjects will be requested to come for additional optional physiologic measurements at 48,
72 and 168 hours post challenge to gather data further in the time course. For the optional
visits after each challenge, subjects will have vital signs measured, CRP and serum for
other inflammatory markers drawn. Subjects will perform spirometry, lung volumes, diffusing
capacity, exhaled NO levels, airway resistance, and complete a short questionnaire.

NOTE:

In a separate related IRB protocol prior to the above study:

In Phase 1, (IRB # Pro00005046) we will determine the effect of promoter polymorphisms in
the gene for the NO producing enzyme, nitric oxide synthase (NOS2), on exhaled NO in healthy
African Americans. Individuals consented during Phase 1 will be asked if they are willing
to be contacted about future studies including those described in Phase 2 of this study. 30
individuals (10 individuals with each polymorphism) identified in Phase 1 who are
heterozygous for a NOS2 promoter SNPs and 10 individuals without these NOS2 promoter SNPs
will be studied.

Because SNPs in the TLR4 gene result in a lack of airway obstruction in response to
endotoxin, we will genotype the 1000 DNA samples for the Asp299Gly and Thr399Ile TLR4
polymorphisms in Phase 1 and exclude these individuals from the studies proposed in Phase 2.
Likewise, CD14 serves as a coreceptor for endotoxin and polymorphisms in the CD14 gene and
promoter have been associated with asthma and responsiveness to endotoxin. Therefore, to
control for this potential confounder in our analysis, we will also test whether
polymorphisms in the CD14 gene and its promoter are associated with responsiveness to
inhaled endotoxin.

Inclusion Criteria:

- Willing/able to give informed consent & adhere to visit/protocol schedules

- non-atopic, non-asthmatic by PFT and allergy skin testing

- never cigarette smoker,

- no significant occupational exposure to respiratory irritants or toxins,

- no chronic illness

- no chronic use of medications (excluding contraceptive medication),

- no systemic corticosteroid use in the previous month,

- no historical or physical examination evidence of unstable cardiac or severe lung
disease,

- Women of childbearing potential must have a negative serum pregnancy test

- baseline FEV1 > 80% of the predicted value,

- no clinically significant abnormalities on the chest x-ray or EKG

Exclusion Criteria:

- occupational exposure to hay or grain

- smoked 20 or more packs of cigarettes in a lifetime.

- prior allergen immunotherapy

- Allergy to potential study medications acetaminophen and albuterol

- Subjects who abuse alcohol or illicit substances will be excluded

- Viral respiratory infection within the previous 14 days

- Students or employees under direct supervision by protocol investigators are
ineligible

- Nursing mothers

- Other investigational medication within the last 30 days

- Other medical or psychological conditions which, in the opinion of the investigator,
might create undue risk to the subject or interfere with the subject's ability to
comply with the protocol requirements