Evaluation of Acute and Subacute Effects of Nicotine Free Electronic Cigarette(NCFE) Vapors

The recent introduction of electronic cigarettes (ECs) to provide habitual smokers a source
of nicotine without the need to inhale tobacco smoke is thought to reduce toxicity to airway
epithelial cells. The use of ECs decreases use of traditional cigarettes diminishes nicotine
withdrawal symptoms leads to opposite effects on exhaled NO levels compared to tobacco and
may diminish stimulation of reward centers in the brain leading to less dependence due to the
lack of other tobacco-derived chemicals. The manufacturers of ECs tout comparative safety as
a reason to switch from traditional tobacco cigarettes to their products. However the dearth
of data supporting this claim led the FDA to issue warning letters to these companies. For
now, the World Health Organization recommends against EC use until ECs have been properly
evaluated. On the other hand, the American Association of Public Health Physicians support
the use of ECs as a means of "Harms Reduction" since ECs provide nicotine without the myriad
of other toxic chemicals produced from burning tobacco. These recommendations are mostly due
to theoretical considerations and expert opinion. Due to the novelty of the ECs, many
concerns of their safety on health and long-term nicotine addiction remain unanswered due to
a lack of studies that comprehensively evaluate their toxicity in the airways.

Moreover, ECs produce vapor mainly from propylene glycol (PG) or vegetable glycerin (VG) that
are blended at different concentrations, making comprehensive testing more difficult.

The lack of consistent results demands a full evaluation of EC toxicity in the airway even
when nicotine is not inhaled.

In our laboratory using cultures of primary normal human bronchial epithelial(NHBE) cells
(either differentiating or fully differentiated) exposed to EC vapor or an equivalent volume
of filtered air we have demonstrated that EC vapor can be delivered to fully differentiated
NHBE cells and that high, but realistic puff numbers decrease ciliary beat frequency (CBF) as
well as Large, Ca2+-activated K+ channels (BK channel) (the pore forming α subunit KCNMA1)
and Forkhead box protein (FOXJ)-1 expression. We also have preliminary data of smoke exposure
in vitro and in vivo, showing a negative effect of cigarette smoke and nicotine on parameters
of mucociliary clearance (MCC) and nasal potential difference (NPD), a way to assess ion
transport in vivo. These changes occurred in a TGF-β-dependent manner.

This study is designed to evaluate the airway toxicity of NFECs (Aim 2.1 of the project
"Adverse effects of inhaled nicotine from tobacco and e-cigarettes").

PRIMARY ENDPOINT As the primary endpoint, we will assess the consequences of vaping EC liquid
with no nicotine (NFEC) on cystic fibrosis transmembrane (CFTR)- and calcium-activated
chloride channel (CaCC)-mediated chloride conductance in lifetime non-smokers using nasal
potential difference (NPD) measurements.

We will use NPD as a primary endpoint because changes in most clinical parameters will only
be seen after months or even years of exposure. For that particular reason, there is a recent
surge to find alternative biomarkers that may be used as surrogate endpoints in shorter
clinical trials. Since NPD directly measures the changes in ion transport expected to
influence MCC and therefore overall outcome and since changes of NPD measurements are
indirectly linked to lung function changes and MCC in trials with cystic fibrosis (CF)
patients, we believe that NPD lends itself as a reasonable surrogate for MCC for this study.

SECONDARY ENDPOINTS Nasal nitric oxide and TGF-ß levels in nasal lavage and cells will be
also collected to correlate with the changes on NPD results after exposure to NFEC.

STUDY DESIGN This aim will examine whether acute and subacute exposures to NFEC vapors have
adverse effects on lifetime non-smokers by measuring changes in nasal ion transport and TGF-β
levels. Nasal ion transport will be assessed by nasal potential difference (NPD), which
measures the voltage potential resulting from epithelial ion fluxes (both Na+ absorption and
Cl- and K+ secretion at the mucosal surface in vivo. In normal airway epithelia, Na+
absorption is the primary ion transport activity so that the resulting airway surface
potential difference is negative with reference to the interstitium. Ion transport across
nasal epithelia is representative for findings in distal airways. We also believe that NPD is
a reasonably sensitive biomarker for this study as discussed below. Therefore, we will
evaluate the acute effects of NFEC vapor generated from the nicotine-carrier system
("e-liquid" without nicotine) and the subacute effect after a 7-days exposure to the NFEC.

Since the major difference between the ECs is the amount of vapor produced per puff, we will
focus on a commonly used mini and mid-sized EC: Halo (The Halo company, USA) and eGo-T®
(Joyetech Co., Ltd., ShenZhen China), and examine different vapor fluids or "e-liquids" (100%
PG, 50% PG/50% VG and 100% VG) without nicotine.

Inclusion Criteria:

INCLUSION CRITERIA:

- Subjects will be over 18 years of age.

- Healthy life-time non-smokers

Exclusion Criteria:

- Presence of airflow obstruction (COPD, asthma, bronchiectasis)

- Other concomitant inflammatory pulmonary disorders

- Subjects with known pulmonary malignancies within 5 years

- Subjects with prior thoracic surgery

- Subjects with respiratory infection that required use of oral corticosteroids and/or
antibiotics within the prior 3 months

- Subjects with allergies to study medications

- Subjects incapable of providing informed consent.