Effect of Gamma Tocopherol Enriched Supplementation on Response to Inhaled O3 Exposure

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 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 TLR4, the primary receptor for LPS.
TLR4 activation of inflammatory cells activates NF-kB and induces oxidative stress. O3 and
LPS has been associated with exacerbation of asthma, and we 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 were had
increases 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 (yT) or inducers of 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, yT is a potent ROS scavenger and is also a powerful nucleophile that
traps electrophiles such as peroxynitite in lipophilic compartments. One mechanism by which
y-T is cytoprotective is scavenging of RNS at the un-substituted C-5 position on the
hydroxy-chroman ring of y-T to form 5-NO-y-tocopherol (5-NO-yT). 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 yT 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 yT also prevents protein nitration and attenuates loss of plasma vitamin
C in plasma in a rodent peritonitis model of inflammation. Our group has also shown that yT
inhibits COX-2 and 5-LO in LPS-stimulated macrophages and IL-1b stimulated epithelial cells.
These actions are mediated primarily by the yT metabolite 2, 7,
8-trimethyl-2S-(.-carboxyethyl)-6-hydroxychroman (y-CEHC) which requires hydroxylation of the
y-methyl group of yT by cytochrome P450 (CYP450). In carrageenan- induced inflammation in
male Wistar rats, yT decreases prostaglandin (PGE2) and leukotriene (LTB4) production,
suggesting that LO-mediated production of leukotrienes may also be inhibited by yT.
Tocopherols, including yT, also modulate gene expression of a number of inflammatory genes.
Thus, yT 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 our early phase I studies of gamma tocopherol-enriched mixed tocopherols (gT-mT)
we have shown that gT-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 ( yT, 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.

Inclusion Criteria:

1. Age 18-45 of both genders

2. Negative pregnancy test for females who are not s/p hysterectomy with oopherectomy

3. History of episodic wheezing, chest tightness, or shortness of breath consistent with
asthma, or physician diagnosed asthma.

4. Positive methacholine test. A positive test is defined as a provocative concentration
of methacholine of 10 mg/ml or less producing a 15% fall in FEV1 (PC20 methacholine)
by the method used in a separate screening protocol or a PRE and POST bronchodilator
challenge used to determine reversible lung function. Reversibility is confirmed with
a 10-12% increase in FEV1 15 minutes after inhaling 4 puffs of albuterol with a
spacer. Reversibility is used in the same separate screening protocol; or a clinical
history of asthma after the age of 6.

5. FEV1 of at least 80% of predicted and FEV1/FVC ratio of at least .70 (without use of
bronchodilating medications for 12 hours or long acting beta agonists for 24 hours),
consistent with lung function of persons with no more than mild episodic or mild
persistent asthma.

6. 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 clinical history consistent with seasonal or
perennial allergy symptoms.

7. Symptom Score (this will be submitted as an attachment) no greater than 20 (out of a
possible 60) for total symptom score with a value no greater than 3 for any one score.
No more than one score may be greater or equal than 3.

8. subjects must be willing to avoid caffeine for 12 hours prior to all visits.
Methacholine challenge and allergy skin testing are performed as part of IRB 98-0799,
which a subject must complete in order to be considered for this protocol.

9. for subjects who are prescribed inhaled corticosteroids. 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 prior to screening and throughout the course of the
study.

Exclusion Criteria:

1. 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

2. Physician directed emergency treatment for an asthma exacerbation within the preceding
3 months

3. Moderate or Severe asthma

4. 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.

5. 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).

6. Viral upper respiratory tract infection within 4 weeks of challenge.

7. Any acute infection requiring antibiotics within 4 weeks of exposure or fever of
unknown origin within 4 weeks of challenge.

8. Severe asthma

9. 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.

10. 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)

11. Any history of smoking in the year prior to study enrollment; lifetime smoking history
> 10 pack years

12. 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

13. Allergy/sensitivity to study drugs, or their formulations.

14. Known hypersensitivity to methacholine or to other parasympathomimetic agents

15. History of intubation for asthma

16. Unwillingness to use reliable contraception if sexually active (IUD, birth control
pills/patch, condoms).

17. Abnormal PT or aPTT values at screening or during the treatment period. Normal values
will be those published by the clinical lab (Labcorp, INC).

18. Any bleeding disorder

19. Orthopedic conditions which would prevent the volunteer from performing moderate
exercise on a treadmill.

20. 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.