Cardiovascular Disease Biomarkers in Smokers and Moist Snuff Consumers

This single site, observational study will provide an increased understanding of how
consumption of different tobacco products (i.e., cigarettes and moist snuff compared to no
tobacco use) affects 1) CVD-related physiological assessments and 2) CVD-related biomarkers
of tobacco effect (i.e., proteins, lipids, and cellular components). A recent policy
statement from the American Heart Association provides a review and analysis of the impact
of smokeless (ST) use on cardiovascular disease (CVD) (Piano et al. 2010). The authors
acknowledge that the evidence is consistent with the suggestion that the cardiovascular
risks from ST products are markedly lower than those from cigarette smoking. Despite the
potential risk reduction in transitioning from cigarettes to ST consumption, few studies
have directly compared biomarkers of tobacco effect (BioEff) among smokers, moist snuff
consumers (MSC) and non-tobacco consumers (NTC).

Furthermore, this study will measure biomarkers of tobacco exposure to assess their ability
to differentiate the three tobacco consumer groups (smokers, moist snuff consumers,
non-tobacco consumers) based on product use. Estimating exposures to combustion-related
compounds found in tobacco smoke is best accomplished using biomarkers. A key advantage of
human exposure biomarkers is that they are considered reliable metrics of the levels of
exposure that consumers actually experience when using tobacco products (Hecht et al.,
2010). Because combustion does not occur during ST use, ST products lack most of the
combustion-related compounds found in tobacco smoke. Biomarker differences found between
different tobacco use groups to harmful or potentially harmful constituents may indicate
differences in subsequent health risks (Rodu and Godshall, 2006; Hatsukami et al., 2006).

Epidemiological data demonstrate that the health risks associated with cigarettes are
significantly greater than those associated with the use of non-combustible tobacco and
nicotine products (Surgeon General, 2010). On a relative risk continuum, cigarette smoking
presents a significantly greater risk to tobacco users than use of non-combustible smokeless
products. ST products, which are consumed orally, do no generate chemicals associated with
the burning of tobacco, and thus, present a reduced toxicant profile compared to smoking.

To address the purpose and objectives of this study, the study was conducted as follows:

- Subjects were consented for the study prior to any procedures being performed and
screened on study-specific inclusion/exclusion criteria to determine subject
eligibility (within 28 days of study check-in).

- Eligible subjects were admitted to the clinical research unit between 12:00 noon and
5:00 p.m. on Day 1 and confined for one overnight stay (approximately 18-23 hours).

- After all study procedures were completed on Day 1 and Day 2, appropriate basic safety
assessments were made and subjects were discharged approximately at 12:00 noon on Day
2.

A brief description of the study procedures performed is listed below.

- After check-in on Day 1, eligible subjects observed a 45-minute tobacco abstention
period, followed by use of a single unit of their usual brand (UB) tobacco product,
referred to as a "Challenge." For smokers, the "challenge" was smoking one UB cigarette
in their usual manner; for moist snuff consumers, a 30-minute use of one typical pinch
of their UB moist snuff.

- Fifteen minutes after the end of UB use, the following procedures were performed
sequentially: ECO; blood samples for biomarkers of exposure (serum nicotine and
cotinine, percent carboxyhemoglobin in whole blood); and ABI.

- At 30 minutes post-UB use, FMD was measured followed by administration of
health-related questionnaires. The non-tobacco consumers had no product "Challenge".
The completion of study questionnaires served as the reference point for collection of
ECO, ABI and FMD.

- Blood biomarkers of tobacco exposure were collected on Day 1 following product
"Challenge" and on Day 2 following an overnight tobacco abstention and fast.

- Urine biomarkers of tobacco exposure and effect were collected on both Day 1
post-"Challenge" and on Day 2 fasting.

- Blood biomarkers of tobacco effect were only collected on Day 2 fasting, followed by
the re-assessment of the physiological measures (ECO, ABI, FMD) and assessment of CIMT.

Inclusion Criteria:

- Smokers: exclusive FF (full flavor; > 13.0 mg FTC "tar" ) or FFLT (full flavor, low
"tar" [6.0 to 13.0 mg FTC "tar"]) smokers who reported smoking at least 15 cigarettes
daily for at least three years prior to Day 1 and whose ECO was 10 to 100 ppm (ranges
of 2 to 9 ppm and 101 to 125 ppm were allowed upon joint review by the Sponsor and
Investigator).

- Moist Snuff Consumers: exclusive oral smokeless tobacco users of any brand
(Copenhagen, Skoal, Grizzly, Kodiak, Timber Wolf, Longhorn, Red Man, Levi Garrett,
Beech-Nut, Chattanooga Chew, Kayak, etc.), any style (snuff cut, long cut, fine cut,
pouch, loose, or plug) and any flavor (natural, straight, mint, wintergreen, etc.)
who reported using at least two cans or packages per week for at least three years
prior to Day 1 and whose ECO was 0 to 5 ppm (a range of 6 to 10 ppm was allowed upon
joint review by the Sponsor and Investigator).

- Non-tobacco Consumers: never-smokers/never-ST users whose ECO was 0 to 5 ppm (a range
of 6 to 10 ppm was allowed upon joint review by the Sponsor and Investigator).

- Male, between 26 and 49 years of age, inclusive (on Day 1 check-in).

- Free of clinically significant health problems in the opinion of the Investigator.

- Forced expiratory volume exhaled in one second (FEV1) ≥70% of predicted at Screening.

- Willing to undergo all study procedures during confinement.

- Not taking medication on a daily basis for chronic medical disorders deemed
clinically significant by the Investigator.

- Willing to suspend usage of daily aspirin or over-the-counter (OTC) medication seven
days prior to Day 1.

- Not taking any creatine supplements.

- Negative tests for selected drugs of abuse and alcohol at Screening and at Day 1
check-in.

- Able to read and comprehend questionnaires in English.

- Able to comprehend and willing to sign an Informed Consent Form (ICF).

Exclusion Criteria:

- At Screening, a BP that exceeds 140/90.

- <70% predicted FEV1 from three acceptable maneuvers.

- Unwilling to have the FMD procedure performed two or more times during confinement.

- Unwilling to have the ABI procedure performed two times during confinement.

- For the FMD determination, poor brachial artery visualization due to extremely deep
position or severe artifacts (noise) due to overlying muscle that, in the
sonographer(s)' opinion, would result in an inferior, unreadable or unobtainable
brachial artery image.

- A donation of blood from 30 days prior to Screening through Day 1, inclusive, or of
plasma from two weeks prior to Screening through Day 1, inclusive.

- Receipt of blood products within two months prior to Day 1 check-in.

- Evidence of visible oral cancer, as found in an oral health examination at Screening
or based on oral health questions at Day 1 check-in.

- Subject who is an employee of the clinical site.

- Subject who has participated in any other investigational study drug or product trial
in which receipt of an investigational study drug or product occurred within 30 days
prior to Day 1 check-in, inclusive.