Effects of Flavors on Nicotine Reinforcement in Smokers
Phase: EARLY_PHASE1
Conditions: Nicotine Dependence
Sponsor: Yale University

Mehmet Sofuoglu, M.D., Ph.D. 
Effects of Flavors on Nicotine Reinforcement in Smokers 
VA Healthcare System 
 
 
 
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1.  Principal Investigator: Mehmet Sofuoglu, M.D., Ph.D. 
2. Co-Investigators (including VA and WOC staff): While all research staff are trained to 
provide informed consent, those identified with an asterisk (*) will provide informed consent for 
this study.   
Co-Investigators: Joel Gelernter, M.D., *Gerald Valentine, M.D.  
 
Kevin Sevarino, M.D. 
Nursing Staff:  
*Ellen Mitchell, R.N., Angelina Genovese, R.N , Elizabeth O’Donnell R.N., 
Margaret Dion-Markovitz, R.N. 
Research Staff:  *Stacy Minnix, BSW,  *Lance Barnes, and *Chris Cryan, * Michael Stephens 
3. Title of the Project 
 
Effects of Flavors on Nicotine Reinforcement in Smokers  
 
4. Purpose, Hypothesis and Key Questions 
Specific Aim #1: To examine the acute reinforcing effects of menthol, a fruit flavor (green 
apple) or a fruit flavor plus menthol, alone or in combination with nicotine in smokers. Flavors 
will be administered by inhalation via electronic cigarettes (e-cigarettes) and nicotine will be 
administered intravenously. The reinforcing drug effects will be measured with the drug effects 
questionnaire (DEQ).  
Hypothesis: Hypothesis #1A: Combined fruit flavor (green apple) plus menthol will be more 
reinforcing than menthol alone or fruit flavor alone.  
Hypothesis #1B: Fruit flavor plus menthol will be more effective than menthol alone, or fruit 
flavor alone, in enhancing the reinforcing effects of nicotine. 
Exploratory Aims: To examine if fruit flavor plus menthol will be more effective than 
menthol or fruit flavor alone in enhancing nicotine’s effects in a) alleviating urges to smoke, b) 
suppressing withdrawal severity and c) enhancing cognitive performance. 
 
5-6. Background and Significance: 
a) Role of Flavors in Tobacco Addiction 
With the exception of menthol, all characterizing flavors have been banned from use in 
combustible tobacco cigarettes. However, flavors are not prohibited from use in other tobacco 
product categories where the majority, including e-cigarettes, cigars and cigarillos, contain a 
wide variety of flavorants. Prior studies with tobacco cigarettes have shown that the addition of 
sweet flavors increased the appeal of these products, especially among youth (King et al. 2014). 
Similar recent data for non-combustible tobacco products has informed the U.S. Food and Drug 
Administration’s (FDA) prioritization of understanding the impact of flavors on the appeal and 
use of flavored tobacco products.  
Flavors are particularly ubiquitous in e-cigarettes and refill liquids marketed for use in the 
expanding array of electronic nicotine delivery systems (ENDS). A recent study estimated that 
there are over 7,000 unique e-cigarette flavors including menthol/mint, fruit, desert/sweet, 

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tobacco, coffee/tea, nuts/spices and alcohol flavors (Zhu et al. 2014). Many survey and 
epidemiological studies found that e-cigarette users, especially youth, prefer e-liquids with 
flavors (Pesko et al. 2016; Soule et al. 2016). For example, in one survey of high school students 
who use e-cigarettes, over 70% reported having experience with flavored e-liquids and over 50% 
prefer using e-liquids with sweet flavors (Krishnan-Sarin et al. 2015). In another study with 
youth from Texas, 95 to 98% reported using flavored e-cigarette as their first e-cigarette, 
compared to 44 % of older adults in nationwide sample (Harrell et al. 2017). The most 
commonly used flavors were fruit and sweet flavors and overall, flavors were reported to be a 
common reason for use of e-cigarettes by youth (Harrell et al. 2017). These studies suggest that 
flavors increase the appeal of e-cigarettes and may therefore contribute to initiation and 
maintenance of e-cigarette use.  
b) Menthol as a Common Flavor 
Menthol is one of the most intensively studied flavors used in tobacco products. It has a well-
characterized cooling and soothing action in the airways that may enhance the appeal of menthol 
cigarettes by reducing the harshness of tobacco smoke (Wise et al. 2012). This action may be 
particularly significant for youth who are experimenting with combustible tobacco products. For 
example, in a National Survey on Drug Use and Health, 44.7% of 12 to 17 year-olds smoked 
menthol cigarettes, compared to 30.1% of adults aged 26 or older (Rock et al. 2010). In addition, 
numerous cross-sectional studies have shown that menthol cigarette smoking is a risk factor for 
the development of dependence (Collins and Moolchan 2006; Hersey et al. 2006; Muscat et al. 
2009; Wackowski and Delnevo 2007), and a prospective study of smokers aged 17 or younger 
demonstrated that smoking initiation with menthol cigarettes was associated with higher risk of 
progression to established smoking, as well as higher levels of nicotine dependence 
(Nonnemaker et al. 2012). While these studies indicate that menthol plays a role in both the 
initiation and maintenance of tobacco product use, the underlying mechanisms by which menthol 
may facilitate nicotine dependence in humans have not been fully elucidated. 
In a recent human laboratory study, we examined potential mechanisms underlying 
menthol’s effects in relation to tobacco addiction (Valentine et al. 2017). We found that menthol, 
administered by inhalation via e-cigarettes, significantly reduced urges to smoke in menthol-
preferring, but not in non-menthol-preferring smokers, suggesting that menthol may have 
negative reinforcing effects by alleviating withdrawal. In addition, menthol-preferring smokers 
reported diminished positive subjective responses to IV nicotine and less severe tobacco 
withdrawal than did non-menthol cigarette smokers. These findings are consistent with 
menthol’s effects in reducing the rate of nicotine metabolism or clearance, as indicated by a 
lower nicotine metabolite ratio, a biomarker for the rate of nicotine metabolism, in menthol-
preferring smokers (Benowitz et al. 2004; Fagan et al. 2016). These findings support previous 
data indicating that one long-term adaptation to menthol exposure may be slowing of nicotine 
metabolism (DeVito et al. 2016; Sofuoglu et al. 2012). In contrast to such findings for menthol, 
very little is known about the behavioral pharmacology of flavors or their effects on nicotine 
metabolism.  
c) Research Need for Understanding the Role of Flavors in Tobacco Addiction 
The identification of common mechanisms by which flavors may influence tobacco use and 
appeal is complicated by the availability of thousands of flavor combinations in tobacco 
products, particularly in e-liquids. To reduce the complexity of research on flavored tobacco 
products, there have been attempts to categorize e-liquid flavors. For example, one recent survey 

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suggested that the majority of e-liquid flavors used by consumers can be categorized as either 
tobacco (23.7%), fruit (20.3%), dessert/sweets (20.7%) or menthol/mint (14.8%) varieties 
(Yingst et al. 2017). However, as compared to menthol, there is very limited information about 
the behavioral effects of these other flavor categories. It is important to note that most e-liquid 
flavors were developed as food additives (e.g., watermelon, apple, cherry, cotton candy or 
bubble gum), but have not been examined for inhalation use (Tierney et al. 2016). For example, 
it is unknown if menthol is uniquely different than other flavors that have been used in tobacco 
products or if flavors have similar effects as menthol. Although many commercially available e-
liquid products include a combination of different flavors (e.g., menthol plus fruit or sweet 
flavors), it is unknown if different flavors have additive or synergistic effects as reinforcers.  
Proposed Study 
In this study, we will compare the effects of flavored e-liquids that contain menthol alone, a 
fruit flavor (green apple), and a fruit flavor plus menthol, either alone or in combination with 
intravenously administered nicotine. Study outcomes will include aversive and positive 
subjective effects, withdrawal severity, urges to smoke, cognitive performance and 
cardiovascular measures. We will enroll only menthol-preferring smokers because in our recent 
study on the acute effects of inhaled menthol, effects were more prominent in menthol-preferring 
smokers compared to the non-menthol-preferring sample. We chose to use green apple because it 
represents one of the most commonly consumed e-liquid flavors. The use of menthol, green 
apple and green apple plus menthol flavors will allow the effects of menthol and fruit flavor to 
be compared in a systematic way.   
 
7. Subjects: 
Young adult male and female smokers will be recruited from the New Haven area through 
newspaper advertisements, radio advertisements, and fliers. Interested subjects will have the 
study described over the telephone, and they will be asked to answer a brief tobacco use history 
and medical screening questionnaire. If subjects pass the telephone screening, they will be 
invited to come to the West Haven VA clinic for a screening evaluation.  
Inclusion criteria: 1) Female and male mentholated cigarette smokers, aged 18 to 30 years; 2) 
history of smoking for the past 12 months, at least one cigarette per day; smoking status is 
verified with urinary cotinine levels above 10 ng/ml; 3) not seeking treatment for nicotine 
dependence at the time of study entry; 4) in good health as verified by medical history, screening 
examination, and screening laboratory tests; 5) for women, not pregnant as determined by 
pregnancy screening, nor breast feeding, and using acceptable birth control methods.    
Exclusion criteria: 1) History of major medical illnesses that the physician investigator deems as 
contraindicated for the patient to be in the study; 2) regular use of psychotropic medication 
(antidepressants, antipsychotics, or anxiolytics); 3) psychiatric diagnosis and / or treatment for 
Axis I disorders including major depression, bipolar affective disorder, schizophrenia or panic 
disorder in the past month; 4) abuse of alcohol or any other recreational or prescription drugs in 
the past 30 days; 5) any allergy to propylene glycol or menthol; and 6) aversion to green apple 
flavor.  
 
8. Privacy: Participation in research may involve a loss of privacy. To prevent this, research 
records will be kept as confidential as possible. Only a code number will identify the individual 

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research records.  The code number will not be based on any information that could be used to 
identify the subject (for example, social security number, initials, birth date, etc.)  The master list 
linking names to code numbers will be kept separately from the research data. All research 
information will be kept in locked files at all times.  Subject identity will not be revealed in any 
reports or publications resulting from this study. Only authorized research staff will have access 
to the information gathered in this study.   
While filling out study forms, or during the psychiatric examination, some participants may feel 
uncomfortable disclosing personal thoughts and feelings. Careful efforts aimed at maintaining 
confidentiality have been effective in previous research, and only participants’ code numbers 
will be recorded on the forms themselves to protect confidentiality. 
 
9. Selection: Interested subjects will have the study briefly described over the telephone, and if 
interested, they will be asked to answer a brief tobacco use history and medical screening 
questionnaire. If subjects are eligible for the study based on the telephone screening, they will be 
invited to come to the clinic for a screening evaluation. The screening evaluation will include the 
following: a) obtaining informed consent; b) smoking history and assessment of nicotine 
dependence using the Fagerstrom Test of Nicotine Dependence (FTND) c) complete physical 
and psychiatric examination including the structured clinical interview (SCID) for DSM-IV; d) 
laboratory examination including complete blood count, liver and kidney function tests, and 
glucose; e) urine analysis, drug screen, and for women, urine pregnancy test and f) urine cotinine 
and urine menthol glucuronide levels.  
 
10. Recruitment: A total of 50 smokers will be recruited from the New Haven area by 
newspaper advertisements and fliers. 
 
11. Research Plan 
A. Overview 
We propose a double-blind, crossover study that enrolls young adult smokers who prefer 
menthol cigarettes. The study will consist of an adaptation session and three test sessions. In the 
adaptation session, participants will practice using the e-cigarette by sampling the flavors to be 
used in the test sessions. The test sessions will be performed following overnight abstinence from 
tobacco. Across the three test sessions, participants will be assigned to a random sequence of the 
three different e-cigarette conditions: menthol, green apple or menthol plus green apple, a 
different flavor condition for each test session. In each test session, just after the assigned flavor 
is delivered via the e-cigarette, participants will receive a random order of one intravenous 
delivery of saline, and two intravenous deliveries of nicotine (3.6 mcg/kg and 7 mcg/kg or 0.25 
mg/70 kg and 0.5 mg/70kg), one hour apart. The test sessions will be performed at least 24 hours 
apart to minimize carryover nicotine effects. The main outcome measure will be subjective drug 
effects as measured with the Drug Effects Questionnaire (DEQ). Other outcomes include 
cardiovascular measures, cognitive performance, and self-report measures of nicotine withdrawal 
and craving. Cardiovascular measures include heart rate, systolic and diastolic blood pressure. 
Cognitive performance will be assessed with the Stroop test, mathematical processing test 
(MPT), and continuous performance test (CPT). Nicotine withdrawal measures will be measured 
with the Minnesota Nicotine Withdrawal Symptom Checklist (M-NWSC) and the Brief 

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Questionnaire on Smoking Urges (BQSU). 
B. Medical Monitoring and Safety 
Subjects will be given a thorough physical examination prior to study entry and a physician 
will be present at all test sessions. Subjects will be attached to blood pressure and heart rate 
monitoring devices throughout each test session and an IV catheter will remain in place until the 
end of each test session. Subjects will be administered nicotine only if the systolic blood pressure 
is <150 mmHg and heart rate <100 beats/minute. The subject’s participation will be terminated if 
their blood pressure at any time is >170/110 mm Hg, the heart rate is >120 beats/min, or if the 
subjects develop signs and symptoms compatible with nicotine toxicity. Subjects will be 
monitored for two hours after the last nicotine administration.   
C. Measures 
a) Physiological: 
• Heart rate and blood pressure: Heart rate and blood pressure readings will be taken at 
screening, and throughout each test session to measure nicotine’s effect on these 
parameters. 
b) Biochemical: 
• Serum estradiol and progesterone analysis (females only): Serum estradiol and 
progesterone levels will be measured before each test session, and will be used as 
covariates in our analysis because female sex hormones may contribute to sex 
differences in nicotine responses (Lynch et al, 2010). 
• Plasma cotinine and 3-hyroxycotinine (3HC): Plasma cotinine and 3-hydroxycotinine 
levels will be measured from blood samples obtained during the three test sessions to 
determine the nicotine metabolite ratio (NMR). The NMR is the ratio of 3HC (the 
primary metabolite of cotinine) to cotinine, and reflects the activity of cytochrome P450 
(CYP) 2A6 and therefore, the rate of nicotine clearance (Dempsey et al, 2004). NMR 
has been shown to be stable in smokers during ad-lib and reduced smoking (Mooney et 
al, 2008). Plasma samples for 3HC will be obtained at baseline, and the NMR will be 
included as a covariate in our analyses. 
• Plasma nicotine: Plasma nicotine levels will be measured from blood that was obtained 
prior to the first experimental vaping session on each test day to characterize the 
subject’s smoking status.  
• Urinary cotinine levels: A semi-quantitative rapid urine level of cotinine will be 
obtained at screening to confirm smoking status at study entry (NicAlert).  
• Plasma menthol glucuronide levels: Blood samples collected during each test session will 
also be used to determine the amount of menthol delivered by the e-cigarette. Samples will 
be obtained just prior to each of the three experimental vaping sessions (baseline), at 2 time 
points after each nicotine or saline infusion, and just prior to removing the IV at the end of 
the study. The plasma levels of menthol glucuronide measured at different time points will 
be used to generate concentration-time curves for each menthol delivery to characterize the 
pharmacokinetics of menthol, and to account for differing menthol exposure between 
subjects.  

Mehmet Sofuoglu, M.D., Ph.D. 
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• Alveolar carbon monoxide: CO readings ≤ 10ppm will be used to verify overnight 
abstinence from smoking as recommended by the SRNT Subcommittee on Biochemical 
Verification (Benowitz et al, 2002).  
c) Subjective: 
Measures of Dependence:  
• DSM-5 tobacco use disorder items.  
• Structured Clinical Interview for DSM-5 (SCID) for Axis I disorders: SCID is a semi-
structured interview based on DSM-IV (American Psychiatric Association, 1994) and 
will be performed to diagnose Axis I psychiatric disorders. 
•  Fagerstrom Test of Nicotine Dependence (FTND): This self-report measure assesses the 
degree of nicotine dependence and has been used widely in smoking studies (Heatherton 
et al, 1991).  
Measures of Affective State:  
• Center for Epidemiologic Studies Depression (CES-D) scale: The CES-D is a 20-item 
self-report measure of depressive symptoms (Radloff, 1977). This scale will be used at 
intake to control for baseline differences in depressive symptoms. 
• Positive and Negative Affect Schedule (PANAS): The PANAS is a 20-item scale that 
assesses both negative and positive affective states (Watson et al, 1988). This scale is 
sensitive to the affective symptoms of tobacco withdrawal and predicts relapse to 
smoking (Kenford et al, 2002).  
Measures of Acute Drug Effects: 
Subjective Drug Effects:  
• Drug Effects Questionnaire (DEQ), adapted from Soria (1996), will be used during 
each test session to measure acute drug responses including: liking, cooling, irritation, 
strength, feeling, stimulation, good effects, bad effects, head rush, and want more. 
Each item is rated on a 100 mm scale, from ‘not at all’ to ‘extremely’. 
Measures of withdrawal and negative affect:  
• Minnesota Nicotine Withdrawal Symptom Checklist (M-NWSC): Smokers will be 
asked to rate several nicotine withdrawal symptoms on a 100 mm scale, from ‘not at 
all’ to ‘extremely’. The items are derived from the M-NWSC (Hughes and Hatsukami, 
1986) and have been used in previous human laboratory studies (Eissenberg et al, 
1996). The items include cigarette craving, irritability/anger, anxiety/tension, difficulty 
concentrating, restlessness, increased appetite, depressed mood, and insomnia. 
• Brief Questionnaire on Smoking Urges (BQSU): This 10-item scale, originally 
developed by Tiffany and Drobes (Tiffany and Drobes, 1991), has been found to be 
highly reliable and reflects levels of nicotine deprivation (Bell et al, 1999; Morgan et 
al, 1999). This scale will be used to measure changes in cigarette craving. 
d) Measures of Cognitive Performance:  

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Cognitive performance will be assessed with 3 tests from the ANAM battery (University 
of Oklahoma): the Stroop test, the mathematical processing test (MPT), and the 
continuous performance test (CPT). These 3 tests were chosen because of their sensitivity 
to tobacco withdrawal and nicotine administration (Heishman et al, 2010; Mancuso et al, 
1999; Myers et al, 2008). 
• Stroop Test: This test assesses processing speed, selective attention, and interference 
(Spreen, 1991). It is a computerized version of the classical Stroop Test (Reeves, 2002). 
The Stroop design consists of one three-minute block of congruent stimuli, and one 
three-minute block of incongruent stimuli.  
• Mathematical Processing Test (MPT): This test assesses basic computational skills, 
concentration, and working memory.  An arithmetic problem involving three single-
digit numbers and two operators is displayed (e.g., "5 - 2 + 3 ="). The subject presses 
buttons to indicate whether the answer to the problem is less than five or greater than 
five.  
• Continuous Performance Test (CPT): This test assesses sustained attention, 
concentration, and working memory. A target character is displayed for memorization.  
Then, as individual characters are displayed in sequence, the user presses a designated 
button only when the displayed character is the target letter.  
e) Other Measures: 
• E-cigarette Questionnaire: This 10-item self-report questionnaire was developed by our 
research group to characterize the study populations’ experience with, and attitude 
about, e-cigarettes. It will also include a response item that will identify aversion to 
green apple flavor. This questionnaire will be administered during the adaptation 
session.  
• Drug Use History: Self-reports of cigarette smoking and use of other tobacco products, 
alcohol and drugs (cocaine, alcohol, opioids, marijuana, and other illicit drugs) will be 
assessed with Time Line Follow-Back, which has been shown to be a reliable and valid 
method for monitoring substance use and other outcomes in longitudinal studies (Miller 
and DelBoca, 1994; Sobell and Sobell, 1992). The Time Line Follow-Back will be 
administered at intake. 
• Adverse events: SAFTEE: In order to monitor adverse events from the study 
medications, the SAFTEE will be administered before and after each session. The 
SAFTEE has been used in a number of pharmacotherapy trials (Levine and Schooler, 
1986).  
f) Genetic Markers:  
We recognize the value of banking biological samples for future genetic, epigenetic and 
biomarker analyses given the care taken to establish clinical phenotypes. For example, 
results from genetic studies may confirm the importance of certain TRP-ion channels, or 
sweet taste receptors, in the effects of menthol and sweeteners on initiation and 
maintenance of nicotine addiction. These findings would provide a rationale for examining 
established and novel genetic variants of TRP-channels, and sweet taste receptors, as 
determinants of responses to flavors in the laboratory.  Whenever specific hypotheses are 
generated in the future that require targeted genetic analysis, this protocol will be amended 

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to describe the purpose and use of such analyses.  
D. Drugs 
a) Nicotine: Nicotine bitartrate will be obtained from Interchem Corporation, Paramus, NJ 
and nicotine solutions for intravenous injection will be prepared by U.S. Specialty 
Formulations, Bethlehem, PA. On the morning of each test session, syringes containing 
5 ml of each infusate will be prepared by the West Haven VA research pharmacy. 
Common side effects of nicotine include nausea, vomiting, heartburn, elevated heart 
rate and increased blood pressure.   
• IV Nicotine Administration: Nicotine will be administered over 30 seconds using a 
syringe that is attached to an IV catheter located in a forearm vein. We have 
followed these procedures in our previous studies (Sofuoglu et al. 2005, 2006b; 
2008a, 2009a,b,c) which were completed without any serious and unexpected 
adverse effects attributed to nicotine, or with other safety concerns. After the last 
dose, the subjects will stay in the lab for at least two hours, which has been shown 
to be sufficient for the nicotine-induced changes in heart rate, blood pressure, and 
subjective effects to return to baseline (Sofuoglu et al, 2005; Sofuoglu et al, 2006).  
• Justification for the nicotine doses: For this proposal, we chose two dose sequences 
of nicotine (3.6 mcg/kg and 7 mcg/kg or 0.25mg/70 kg and 0.5mg/70kg) with a 
maximum dose of 0.3 mg and 0.6 mg, respectively, regardless of body weight. In 
our previous studies, these nicotine doses produced robust subjective and 
physiological responses (Sofuoglu et al, 2009a, Valentine et al, 2017). However, 
the doses used in this study are much lower than doses used in previous studies with 
addicted smokers (up to 3 mg per injection). Therefore, we believe that the doses 
used in this study represent an appropriate balance between being sufficiently high 
to answer the research questions, but low enough to reduce risks to the study 
participants.  
E. Electronic Cigarette 
a)  Hardware: A Joyetech eGo, tank-type e-cigarette with a single coil atomizer (2.2 ohm) 
operating at 3.7V (6.2W) will be used as in our previous studies. The e-cigarette will be 
fitted with a 2 ml plastic disposable tank that allows for loading of the assigned e-liquids 
into the e-cigarette just prior to each test session.  
b) Flavors: Three different flavored e-liquids: 1) menthol, 2) green apple, and 3) green 
apple chill will be obtained from the AmericaneLiquidStoreTM, a large internet-based 
vendor that prepares its products in Wauwatosa, Wisconsin. They claim to be the first e-
liquid manufacturer in the United States to obtain International Organization for 
Standardization (ISO) 9001:2008 and Current Good Manufacturing practices (cGMP) 
certification. As is common for many large vendors, consumers are able to specify the 
nicotine level, base liquid mixture and bottle size for each e-liquid product. For this 
experiment, we will specify e-liquids that are nicotine-free (0.0% nicotine) in a 50%: 
50% base mixture of propylene glycol (PG) and vegetable glycerin (VG). The West 
Haven VA research pharmacy will prepare sealed, 2 ml aliquots of each e-liquid from 
the stock 132 ml bottles, and store them in a cool dark location until the morning of each 
test session. These procedures have been successfully deployed in 3 prior e-cigarette 
studies.  

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c) Ingredients:  
Propylene glycol (USP). PG is the original base liquid for which most e-cigarettes were 
designed and remains the most common ingredient in base liquids. PG is also credited 
with producing a throat sensation (‘throat hit’) that mimics the feel of smoking a 
cigarette.  
Vegetable glycerin (USP). VG is commonly included in e-liquid bases to enhance the 
volume of vapor production, giving a greater sensory illusion of smoking.  
Menthol: Widely used in a range of healthcare products and classified by the FDA as 
compound that is Generally Recognized as Safe (GRAS), menthol has mild anesthetic, 
antiseptic and counter irritant properties and is safe for oral ingestion, topical 
administration and inhalation. 
Justification for menthol doses: The menthol present in the two menthol-containing e-
liquids is marketed to consumers who prefer light menthol impact. The concentration 
of menthol in these e-liquids will not exceed 2.0% (20 mg/ml), levels well below 
those associated with human toxicity. The concentrations of menthol in the stock 
solutions procured from the AmericaneLiquidStoreTM will be verified using an assay 
developed by Dr. Peter Jatlow.   
Green apple flavorant: This proprietary fruit flavor is composed of simple esters and 
aldehydes dissolved in the base liquid for a final flavor concentration of approximately 2% 
(net wt. %). Green apple flavorants are commonly used chemicals used in a wide array of 
food and cosmetic products. Although rare, allergic reactions to green apple flavorants can 
occur, and any subject reporting intolerance to products with green apple flavor or aroma 
will be excluded from study participation. Green apple flavor constituents are classified by 
the FDA as GRAS.  
Justification for green apple flavor: Although thousands of different flavor 
combinations are currently available to e-cigarette consumers, current evidence 
indicates that second to tobacco flavors, fruit flavors are the most commonly used 
flavor category in e-liquids upon initiation of e-cigarette use (Farsalinos et al. 2013).  
Among fruit flavors, green apple flavor is identified by the AmericaneLiquidStoreTM 
as one of their best selling products (also confirmed by personal communication from 
Dr. Tony Pace, PEC, Wisconsin). 
F. Genotyping 
DNA Extraction and Genotyping: 
DNA will be extracted from peripheral blood using a commercial kit (Paxgene™; 
PreAnalytiX Qiagen/BD, Germany). We will genotype variations that may modulate nicotine 
responses using a 384-well plate format. Briefly, DNA samples will be genotyped in 2 µl-
reactions. Each reaction will be prepared with 1 ng genomic DNA, 0.05 µl of 20× (or 0.025 
µl of 40×) minor groove binder (MGB) probes and primers (hCV8950074 and 
hCV26000428) (supplied as pre-validated SNP Assays on Demand, Applied Biosystems) 1 
µl of 2× TaqMan Universal PCR Master Mix, and 0.004 µl of 100× bovine serum albumin 
(New England Biolabs). Reactions will be genotyped for an initial denaturation at 95°C for 
10 min followed by 60 cycles of 92°C for 15 s and 60°C for 1 min. Discrimination will be 
performed using ABI PRISM 7900 Sequence Detection System (Applied Biosystems). All 

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samples will be genotyped in duplicate. We will also examine messenger RNA (mRNA) 
levels extracted from the peripheral blood. We are especially interested to examine the 
changes in mRNA levels related to menthol’s effects (TRP-ion channels) and to the effects of 
nicotine. About 2.5 ml of blood will be drawn into the PAXgene tubes at baseline, prior to 
each IV infusion, and at the end of the session. Extracted mRNA samples will be stored at -
80°C in the Genetics Laboratory. 
 
G. Study Procedures 
For 24 hours prior to each test session, subjects will be asked to avoid using products 
containing menthol such as mint teas, cough lozenges, and mint-flavored gum. A trial-size 
tube of toothpaste that does not contain menthol will be provided for use the morning of each 
test session. Subjects will be required to abstain from smoking for 10 hours. Abstinence will 
be verified by expired air CO levels of < 10 ppm (Benowitz et al. 2002) measured on the 
morning of testing. Subjects will also be asked to refrain from consuming alcoholic 
beverages and drugs during study participation. Abstinence will be verified by urine drug 
screening and Breathalyzer measurements before the test sessions. If results indicate non-
compliance with these study procedures, the session will be rescheduled. Repeatedly non-
compliant subjects will be discharged from the study. Subjects will be instructed to drink 
their typical amount of caffeinated beverages in the morning to minimize caffeine 
withdrawal, which could confound interpretation of study measures.  Subjects will be given a 
light breakfast before each test session and a lunch will be provided at the end of each test 
session.  
a) Adaptation Session:  The goal of this session is to familiarize participants with the study 
procedures including the use of e-cigarettes, and to gather information regarding 
participants’ use and knowledge about e-cigarettes. This session will also allow for a 
determination of the participants tolerance of, or aversion to, any of the flavors prior to 
participation in a test session. Participants will be given an e-cigarette and instructed to 
inhale more softly, but for a longer duration (3-4 seconds), than is customary for a 
standard cigarette. Additionally, a two-phase inhalation is suggested where the vapor from 
this elongated inhalation is initially gathered in the mouth (as one might do with pipe 
tobacco) but then further inhaled into the lungs. The e-cigarettes used in this adaptation 
session will be the same as those used in the test sessions. Although the familiarization 
with this technique will occur during the adaptation session, participants will be reminded 
of this inhalation technique during each test session just prior to e-cigarette use. 
Participants will be informed that the schedule of flavor delivery prior to each IV nicotine 
infusion will consist of 6 inhalations at a rate of one inhalation every 15 seconds. 
b) Test Sessions: Experimental sessions will start around 8:30 AM. As summarized in 
Table 2, blood samples for baseline biochemical measures, including plasma cotinine, 
nicotine, and for women, estradiol and progesterone levels, will be obtained just prior to 
the collection of baseline subjective ratings and cognitive testing. Subjects will then start 
inhaling from the e-cigarette with the assigned flavor condition (menthol, green apple or 
menthol plus green apple), one inhalation every 15 seconds for 90 second as practiced in 
the adaptation session (6 inhalations total (Vansickel et al, 2012). The e-liquids used in 
the e-cigarette will be prepared just prior to each test session by the VA Research 

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Pharmacy. Each e-cigarette tank will contain 2 mL of the e-liquid and the study 
personnel will load the tank into the e-cigarette. Just after the last of the 6 flavor 
inhalations, participants will then receive the first delivery of the assigned nicotine 
infusion (saline, 0.25 mg/70 kg or 0.5 mg/70 kg nicotine). Each infusion will be 60 
minutes apart to provide sufficient time for the physiological and subjective nicotine 
effects to return to baseline. Following each infusion, physiological, subjective, and 
cognitive outcome measures will be obtained.  
c) Medical Monitoring: For all nicotine test sessions, subjects will be attached to a blood 
pressure and heart rate/rhythm device (3-lead EKG) that will be monitored by the study 
physician. An IV catheter will be in place throughout the session. Subjects will be 
administered nicotine only if the systolic blood pressure is <150 mmHg and heart rate is 
<100 beats/minute. Subjects will be terminated from the study if the blood pressure at any 
time is >170/110 mm Hg, the heart rate is >120 beats/min, or if they develop signs and 
symptoms compatible with nicotine toxicity. Subjects will remain in the laboratory for two 
hours after the last nicotine administration. These procedures have been developed as part 
of our IND application for IV nicotine.  
 
 

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Table 2. Schedule of Events: Experimental Session*    
Time  
Measures and Events 
8:00 AM 
(Building 36) CO level, urine sample, BAL, and light standard breakfast 
9:00 AM 
(Biostudies Unit) blood sample, HR/BP, EKG, M-NWSC, BQSU, SAFTEE, Cognitive 
Testing 
10:00 AM  
Flavor (menthol, green apple or green apple + menthol)  
DEQ + IV Saline or Nicotine (0.25 or 0.5mg/70 kg) blood draw 
10:01 
HR/BP, DEQ 
10:02  
HR/BP 
10:03  
HR/BP, DEQ,  
10:05  
HR/BP, DEQ 
10:08 
HR/BP, DEQ blood draw 
10:10  
HR/BP, DEQ 
10:15  
HR/BP, DEQ, Cognitive Testing 
10:30 
HR/BP, DEQ blood draw 
10:45  
HR/BP, DEQ 
10:55  
HR/BP, DEQ, BQSU 
11:00  
Flavor (menthol, green apple or green apple + menthol) DEQ + IV Saline or Nicotine 
(0.25 or 0.5 mg/70 kg), blood draw 
12:00 
Flavor (menthol, green apple or green apple + menthol) DEQ + IV Saline or Nicotine 
(0.25 or 0.5 mg/70 kg), blood draw 
13:00  
14:00 
HR/BP, EKG, M-NWSC, BQSU, SAFTEE, blood draw 
Discharge 
*The same measures will be obtained following saline and each nicotine administration. For 
brevity, only the measures after saline are shown. Abbreviations: CO: Alveolar carbon monoxide; 
HR/BP: Heart rate/Blood pressure; EKG: Electrocardiogram; M-NWSC: Minnesota Nicotine 
Withdrawal Symptom Checklist; BQSU: Brief Questionnaire of Smoking Urges; DEQ: Drug 
Effects Questionnaire; SAFTEE: Systematic Assessment for Treatment Emergent Events. 
 
12. Data Analysis Methods 
Data analyses: The primary analyses will be intent-to-treat and will include all available data 
on subjects who complete at least one test day. Mixed-effects models will be used to test the 
study hypotheses. These models allow for different numbers of observations per subject, use all 
available data on each subject, and are unaffected by randomly missing data. They also provide 
flexibility in modeling the correlation structure of the data. All data will be checked for 

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normality and transformations will be applied as necessary. Significance level of 0.05 will be 
used for the main hypotheses and Bonferroni correction will be applied for post-hoc tests.  
Specific Aim #1: To examine the acute reinforcing effects of menthol, a fruit flavor (green 
apple) or a fruit flavor plus menthol, alone or in combination with nicotine in smokers. Menthol 
and fruit flavor will be administered by inhalation via electronic cigarettes (e-cigarettes) and 
nicotine will be administered intravenously. The reinforcing drug effects will be measured with 
the drug effects questionnaire (DEQ).  
Hypothesis:  
Hypothesis #1A: Green apple plus menthol will be more reinforcing than menthol alone or 
green apple alone. Hypothesis #1B: Green apple plus menthol will be more effective than 
menthol alone or green apple alone in enhancing the reinforcing effects of nicotine. 
The main outcome measures will be the “good effects” and “drug liking” items of the DEQ. 
Mixed effects models will include the within-subject factors nicotine dose (0.25, 0.5 mg nicotine 
or placebo), flavor (menthol, green apple or menthol + green apple) and time of measurement 
(+1, +3,+5,+10,+15,+30,+45,+55 minutes) and possible interactions among these factors. We 
will also consider order effects for nicotine dose (1, 2 or 3) and session day (1, 2 or 3) and the 
effects of potential covariates (race, sex, baseline plasma NMR, FTND scores). These effects 
will be included in the final model if significant. Estradiol and progesterone levels will also be 
considered as potential covariates in a secondary analysis of women only. We will consider 
several different correlation structures for the repeated measures within individuals, and will 
select the best fitting one based on Schwartz Bayesian information criterion. SAS PROC MIXED 
will be used for the mixed-model analyses.  
Rationale for sample size: The study will have 80% power to detect large effect sizes (f=0.4) 
for the interactions of interest in repeated measures analysis for 30 subjects at 0.05 significance 
level. Our preliminary data for both outcome measures show larger magnitude effects for the 
nicotine effects (all f>0.5) and flavor effects are expected to be similarly strong (Sofuoglu et al. 
2009a). Applying Bonferroni correction and using an alpha level of 0.01 we have 80% power to 
detect post-hoc comparisons of medium to large effect size d’=0.7 for flavor and nicotine effects. 
Our previous study shows larger effects of nicotine d’>1.0. To account for up to 20% dropout we 
will recruit 36 subjects for a target of 30 completers.    
 
13. Risks and benefits 
Potential risks: 
There are potential risks, discomforts and inconveniences associated with participation in this 
study. These may be due to nicotine or menthol administration, blood drawing, other study 
procedures and potential loss of confidentiality. 
1) Common side effects of nicotine include nausea, vomiting, heartburn, and elevated heart 
rate and blood pressure. Toxic doses of nicotine may cause abdominal pain, hyper 
salivation, diarrhea, dizziness, confusion, hearing and vision problems, syncope, seizures, 
hypotension, irregular pulse, and death. However, these toxic effects occur at doses 40 to 
50 times that which will be used in our study. Other potential risks from this study include 
administering a drug that has addictive potential.  

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2) Menthol administration produces typical sensory effects in the mouth and throat. The 
doses of menthol that are found in cough drops, ranging 1 to 10 mg are regarded to be safe 
(Sweetman, 2011). In spite of ubiquitous use of menthol in a wide range of products, only 
few cases of menthol poisoning has been described in the literature following very high 
doses of menthol ingestion, 200 mg or more.  Menthol poisoning reported to cause ataxia, 
confusion, coma, nausea, and vomiting. However, these toxic effects occur at doses 20 to 
30 times that which will be used in our study.  
3) Inhalation of two of the e-liquid flavors will contain a fruit flavor. All flavorants and 
sweeteners contained in the fruit flavor are classified by the FDA as Generally 
Recognized As Safe (GRAS). Fruit flavors are commonly used chemicals used in a wide 
array of food and cosmetic products. Although it is rare, allergic reactions to fruit 
flavors can occur so if a subject has a bad reaction or are allergic to products with the 
relevant fruit flavor or aroma, they will be excluded from study participation for their 
safety.  However, the administrations will be closely monitored for any signs that this 
unique combination of IV nicotine and inhaled flavorants results in unexpected findings. 
  
4) Propylene glycol and vegetable glycerin are categorized by the FDA as compounds that 
are Generally Recognized As Safe (GRAS). They are both commonly used ingredients in 
food products, pharmaceuticals and in personal care products. They are also now used as 
the base liquid ingredient for e-cigarettes. Although e-cigarettes are reported to be helpful 
for smoking cessation, the FDA has not approved e-cigarettes for this use, and the long-
term consequences of e-cigarette use have not been assessed. 
5) Blood Drawing: Subjects will have approximately 260 mL of blood drawn as a result of 
their participation in the study. Blood drawing can cause some pain and may result in 
bruising.  
6) Study procedures: On the test days, subjects will not be able to smoke for more than 10 
hours. During this cigarette abstinence period, subjects may experience symptoms of nicotine 
withdrawal such as craving for cigarettes, anxiety, restlessness, irritability, difficulty 
concentrating, loss of energy, and excessive hunger. In addition, smokers will be exposed to 
e-cigarettes and e-cigarette mixtures.  
7) Loss of confidentiality: It is possible that participation in the study may make others, 
including friends or family members, be aware of the participant’s tobacco use status. The 
potential participants will be told that if they do not feel comfortable with this then they 
should not participate in the project. Participants will also be told that if they report any 
information to us about abuse or homicidal/suicidal behavior we will be required to report 
this information to the appropriate authorities. There is also a potential risk of loss of 
confidentiality due to data sharing. 
14. Protection of Subjects:    
Before initiating any research activity, each subject must give informed consent that will 
detail the risks of study participation. They will also be advised of the data sharing plans for 
the study. Eligibility will be determined by the medical and psychiatric history, drug use 
history, the physical examination, and the laboratory studies done prior to beginning this 
research protocol. This study will enroll young adults between the ages of 18 to 30 because 
of the high prevalence of use of mentholated tobacco products in this age group (Rock et al, 

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2010). To minimize the risks associated with administering a drug that has addictive 
potential, we will only enroll those individuals with a consistent history of cigarette smoking 
as verified by a careful history, questionnaires, and urinary cotinine levels. Additionally, we 
will not enroll treatment-seeking smokers. 
As mentioned previously, the most common expected adverse events related to IV nicotine 
administration are nausea, vomiting, heartburn, and elevated heart rate and blood pressure. 
To prevent nausea, subjects will be asked not to eat before the sessions. In addition, subjects 
will sit in a comfortable reclining chair during the test sessions that minimizes heartburn. 
Subjects will also be carefully screened for the presence of high blood pressure or other 
cardiovascular disorders to minimize cardiovascular adverse events. 
During the test sessions, a physician will closely monitor all subjects. Subjects will be 
attached to a cardiac monitor as well as a blood pressure and heart rate monitoring device. 
An IV catheter will be in place throughout the session. Stopping rules are in place as 
described above (Study Procedures - Test Session).  
Over the last 6 years, we have administered nicotine intravenously to more than 400 smokers 
and have not encountered any adverse events attributed to nicotine administration. The most 
common adverse events, or reasons for discharges, in these studies include difficulty finding 
venous access, high blood pressure before nicotine administration, the use of drugs of abuse 
or alcohol during study participation, and adverse events attributed to other study 
medications. Similar to our experience, in a genetic study, Gary Swan et al. administered IV 
nicotine to 278 smokers and non-smokers via slow infusion (Cancer Epidemiol Biomarkers 
Prev 2007;16(6):1057–64). This procedure was also well tolerated in both smokers and non-
smokers, further supporting the safety and tolerability of IV nicotine administration. 
The close monitoring of subjects during the sessions will also help to detect any adverse 
events from menthol or e-cigarette administration. 
Confidentiality will be protected by having records identified by code number only with the 
master list including names kept in a sealed envelope in a locked file in the Principal 
Investigator's office and by the pharmacy. Subjects will be given telephone numbers to call in 
case of emergency, 24 hours a day.  The potential risk of loss of confidentiality due to data 
sharing will be minimized by creating de-identified data sets that exclude direct and indirect 
identifiers. In addition, researchers requesting data will be required to enter into a data 
sharing agreement.   
In order to participate in a study, each subject must give informed consent. All potential risks 
will be described in detail to the subjects in the consent form.  The personnel in the 
laboratory have been certified in either Advance Cardiac Life Support (ACLS) or Basic Life 
Support. If a problem arises, the subject will be treated immediately.        
Confidentiality in this study is of the utmost importance to us. All information obtained will 
be stored in coded form. The names of the subjects will be used in hospital records.   
The risk associated with participating in this study is moderate, because the nicotine 
administered may be associated with mild side effects. Serious side effects associated with 
this treatment are not expected. This project will be monitored by the Center’s Data and 
Safety Monitoring Board (DSMB) because the study involves double-blind treatment of 
smokers with nicotine. This board is composed of persons not otherwise affiliated with the 

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clinical study who are experienced in various aspects of the conduct of clinical trials for the 
treatment of addictive disorders. The Yale TCORS Independent Data Safety Monitoring Board 
includes experts in the field of tobacco use behaviors and challenge studies (Chair: Dr. Tony 
George, FRCPC, Professor and Co-Director, Division of Brain and Therapeutics, Dept. of 
Psychiatry, U of Toronto; Dr. Thomas Brandon, Professor and Chair, Department of Health 
Outcomes & Behavior, H. Lee Moffitt Cancer Center & Research Institute) and in statistics 
(Dr. Hanga Galfalvy, Assistant Professor of Neurobiology, Columbia University). The 
members of the DSMB, and all study Investigators, will complete Conflict of Interest forms 
created by Yale’s IRB in accordance with NIH guidelines.  
We will report recruitment, follow-up, and adverse events to this panel on a bi-annual basis 
with serious adverse events reported within 48 hours. Prior to study initiation, critical 
parameters for collection of side effects and for study discontinuation will be recommended 
to the DSMB who may use these or other measures to monitor safety of the ongoing trial. 
The DSMB will be available to convene outside of scheduled meetings, if necessary, due to 
concerns regarding a particular subject or due to any troublesome developments in subjects' 
experiences during the study. The DSMB will make appropriate recommendations for 
changes in the study protocol, if needed.   
This monitoring will be consistent with NIH policy regarding the protection of human 
subjects in research, and FDA guidance on statistical practices for clinical trials (ICH E9) 
and good clinical practices (ICH E6). In general, the data to be reviewed will include 
screening data, baseline data, efficacy data, and safety data.   
The Principal Investigator will conduct a review of all adverse events and determine the 
attribution and grade of severity of the adverse event by using the following scales: 
Attribution of Risk Categories: 
Definite:  Adverse event(s) will clearly be related to investigational agent(s) or other 
intervention 
Probable: Adverse event(s) will likely be related to investigational agent(s) 
Possible:   Adverse event(s) may be related to investigational agent(s) 
Unlikely:  Adverse event(s) will doubtfully be related to investigational agent(s) 
Unrelated: Adverse event(s) will clearly not be related to the investigational agents(s) 
Grades of Risk: 
0: No adverse event or within normal limits 
1:  Mild adverse event 
2:  Moderate adverse event 
3:  Severe adverse event resulting in hospitalization or prolongation of existing 
hospitalization, a persistent or significant disability/incapacity, or a congenital 
anomaly/birth defect  
4:  Life-threatening or disabling adverse event 
5:  Fatal adverse event 
Serious adverse events (SAEs) include any untoward medical occurrence that at any dose 
results in death or the immediate risk of death, hospitalization or the prolonging of an 

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existing hospitalization, persistent or significant disability/incapacity or a congenital 
anomaly/birth defect. Subjects will be terminated from participation if the investigator feels 
that subjects' health or well-being may be threatened by continuation in the study. Serious 
unanticipated and anticipated adverse events will be reported within 48 hours to the VA 
Hospital, Yale IRB, the DSMB, and NIH. We will directly report to the FDA, whenever their 
magnitude or frequency exceeds expectations.   
 
15. Informed Consent: Recruitment will be by word-of-mouth, referrals from area programs 
and by advertisement. A research staff member will interview individuals that are interested in 
participating in the study over the phone. If subjects pass the initial screening for the study, they 
will then come into the clinic for a full screening evaluation. Upon arrival, research assistant will 
read the detailed consent form and will ask questions to make sure that the subjects understand 
the procedure, and their rights and informed consent will be obtained. 
 
16. Confidentiality: Only authorized research personnel will have access to the information 
gathered in this study. Patient names or other identifiers such as social security number, initials, 
and birthdate will not be used to identify paper or electronic records. Only a code number will 
identify the individual research records.  The code number will not be based on any information 
that could be used to identify the subjects (for example, social security number, initials, 
birthdate, etc.).  The master list linking names to code numbers will be kept separately from the 
research data and will be stored in an electronic file behind the VA firewall. All research 
information obtained will be kept in locked cabinets located on VA property or in electronic files 
behind the VA firewall. The names of the subjects will be used in VA electronic records, CPRS.  
 
17. Location of Study: This study will be conducted in Ward G9W (the Biostudies Unit) located 
in Building 1 at the West Haven VA Medical Center.   
 
18. Payment: Subjects will be paid $30.00 for attending the screening visit and $30 for the 
Adaptation Session.  Subjects will be paid $150 for each of the three Test Sessions. In addition, 
subjects will be paid $20 to cover transportation costs for the Adaptation Session and for each 
Test Session. Completing all components of the study will result in a maximum total payment of 
$590.  In addition, subjects may also earn $20 for referring people they know who also smoke 
cigarettes and are eligible for study participation.  A contingency payment of $20 will be given 
for transportation if a subject needs to be re-contacted. We will not share any information with 
the subject who makes the referral about the ultimate enrollment status or study outcome of the 
referred individuals.  
 
 
 
 
 
 
 

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Payment Schedule  
Visits 
Amount Paid 
Total  
Screening Visit 
$30.00 
$30.00 
Adaptation Visit 
$30.00 
$30.00 
Travel for Adaptation, and 
Test days 1,2, and 3 
$20.00 each visit 
$80.00 
Test days 1,2 and 3 
$150.00 each test day 
$450.00 
 
Max Total Payment  
$590.00 
Referral  
$20.00 
$20.00 
 
 
19. Funding Source: Provided by an NIH Center Grant. 
 
20. Duration: The entire study will take approximately one and a half years to complete.   
 
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