Clinical trial NCT02102100
Conditions: Nicotine Dependence
Keywords: menthol, electronic cigarette, nicotine, smoking, tobacco
Sponsor: Yale University
Start date: 2014-03-26; Completion date: 2016-07-11
Enrollment: 57 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This study will help determine if menthol administered by inhalation via electronic cigarettes (e-cigarette) changes the reinforcing effects of pure nicotine administered intravenously in cigarette smokers who smoke mentholated or non-mentholated cigarettes.

Detailed description:
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. This screening evaluation will be for the menthol / nicotine reinforcement study as described in detail in this project description. The dose-finding study was conducted at the John B. Pierce Laboratory. The dose finding study concluded with the establishment of the high and low dose of menthol that will be used in this protocol "Menthol's Effects on Nicotine Reinforcement in Smokers." The screening of these subjects used the same inclusion and exclusion criteria as described below.

Inclusion criteria: Female and male smokers, aged 18 to 30 years; 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; not seeking treatment for nicotine dependence at the time of study entry; in good health as verified by medical history, screening examination, and screening laboratory tests; for women, not pregnant as determined by pregnancy screening, nor breast feeding, and using acceptable birth control methods.

Exclusion criteria: History of major medical illnesses that the physician investigator deems as contraindicated for the patient to be in the study; regular use of psychotropic medication (antidepressants, antipsychotics, or anxiolytics); psychiatric diagnosis and / or treatment for Axis I disorders including major depression, bipolar affective disorder, schizophrenia or panic disorder in the past month and abuse of alcohol or any other recreational or prescription drugs in the past 30 days. Any allergy to propylene glycol or menthol.

This outpatient, double-blind, placebo-controlled study consisted of an adaptation session followed by three test sessions. All participants were randomized to a test session order and received control menthol (0.0% + tobacco flavor), low menthol (0.5% + tobacco flavor), and high menthol (3.2% + tobacco flavor) by standardized inhalation from an e-cigarette just prior to each nicotine infusion (a single menthol condition for each test session). Within each test session, all 3 IV nicotine conditions were tested, one hour apart, by delivering saline, nicotine at 0.25 mg nicotine/70 kg and nicotine at 0.5 mg nicotine/70 kg, in a random order, just after last inhalation. For each participant, the randomized nicotine infusion sequence was fixed across the three test sessions, each performed at least 24 hours apart.

Arms: arm 1: Each subject in this arm received a random sequence of 3 different inhaled menthol conditions across 3 test sessions (a single menthol condition for each test session). In each test session, a random order of one saline, and 2 nicotine (0.25 mg and 0.5 mg /70 kg) infusions were given one hour apart, concurrent with the randomized menthol inhalation condition for that test session. arm 2: Each subject in this arm received a random sequence of 3 different inhaled menthol conditions across 3 test sessions (a single menthol condition for each test session). In each test session, a random order of one saline, and 2 nicotine (0.25 mg and 0.5 mg /70 kg) infusions were given one hour apart, concurrent with the randomized menthol inhalation condition for that test session.
Interventions: intervention 1: nicotine intervention 2: menthol
Intervention descriptions: intervention 1: In each of 3 test sessions, subjects in each arm will receive a randomized sequence of three intravenous infusions (30 sec each), one hour apart. The infusions will consist of saline, nicotine (.25 mg / 70kg) and nicotine (0.5 mg / 70 kg). The randomized sequence will remain the same between test sessions for a given subject. intervention 2: In each of 3 test sessions, subjects will take 6 inhalations from the study e-cigarette, one inhalation every 15 seconds, of the randomized menthol condition for that test session ust prior to each intravenous infusion. The menthol conditions are: 3.2% menthol, 0.5% menthol and tobacco-flavor only (0.0% menthol)

Protocol documents:
Prot_000.pdf: 
Mehmet Sofuoglu, M.D., Ph.D. 
 Menthol’s Effects 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 is trained to provide 
informed consent, those identified with an asterisk (*) will provide informed consent for this study.   
Co-Investigators: Aryeh Herman, Psy.D., Joel Gelernter, M.D., *Gerald Valentine, M.D.  
Elise DeVito,Ph.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, *Katherine Barrett, B.S., *Margaret Gemmell-Debiase, CATS, 
*Lance Barnes, and *Chris Cryan, *Marcedes Coffman, M.A., Elaine LaValle, M.S. 
3. Title of the project 
 
Menthol’s Effects on Nicotine Reinforcement in Smokers  
4. Purpose, hypothesis and key questions 
Purpose: To determine if menthol administered by inhalation via electronic cigarettes (e-cigarettes) 
changes the reinforcing effects of pure nicotine administered intravenously in cigarette smokers who 
smoke mentholated or non-mentholated cigarettes. The reinforcing drug effects will be measured with the 
drug effects questionnaire (DEQ).  
Hypothesis: Hypothesis #1A: Concurrent menthol and nicotine administration will lead to greater 
reinforcement than control flavor and nicotine, menthol and saline, or control flavor and saline conditions. 
Hypothesis #1B: Enhancement of nicotine reinforcement by menthol will be greater in menthol smokers 
than in non-menthol cigarette smokers.   
 
5-6. Background and Significance: 
a) Introduction 
Menthol is found as an additive in over 90 percent of cigarettes, although at widely different 
concentrations. Cigarettes that are specifically marketed as menthol cigarettes are disproportionately 
preferred by adolescent smokers and African-Americans. Evidence from epidemiological studies suggests 
that menthol is a potential contributor to the development and maintenance of tobacco addiction. Given 
that nicotine, compared to other drugs of abuse, is a weak reinforcer, it is possible that additional 
reinforcers, combined with nicotine, may facilitate the development and maintenance of addiction. The 
potential reinforcing properties of menthol have not been examined with systematic studies in controlled 
settings. Most importantly, human studies examining the reinforcing effects of menthol in combination 
with nicotine are lacking. One roadblock in determining the contribution of menthol to cigarette 
reinforcement has been the ‘brand loyalty’ of smokers: those who smoke non-mentholated cigarettes do 
not like menthol cigarettes and vice versa. This gap in our knowledge base regarding the basic behavioral 
pharmacology of menthol makes it difficult to develop science-based policies for menthol as an additive 
to tobacco products. To overcome this problem, novel approaches are needed that will minimize the 
influence of the conditioned association between nicotine and menthol formed when smoking 
mentholated cigarettes, or while using mentholated chewing tobacco. 
 


Mehmet Sofuoglu, M.D., Ph.D. 
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b) Why Study Menthol?   
Menthol is a flavoring agent that is used in a wide range of products. It has also commonly been used 
as an additive in cigarettes since the 1920s. Menthol has a well-described cooling and soothing action in 
the airways that may reduce the harshness of tobacco smoke. These effects make menthol cigarettes 
particularly appealing among youth who are experimenting with cigarettes. In fact, among 12 to 17 year-
olds, 44.7% smoke menthol cigarettes, compared to 30.1% of adults aged 26 or older (Rock et al, 2010). 
Among young smokers, menthol cigarette use is especially common in African-Americans (71.9%), 
compared to Hispanic (47%) or Non-Hispanic Caucasians (41%) (Rock et al, 2010). In several cross-
sectional studies, menthol cigarette smoking has been found to be a risk factor for development of 
dependence (Collins and Moolchan, 2006; Hersey et al, 2006; Muscat et al, 2009; Wackowski and 
Delnevo, 2007). More recently in a prospective study in smokers 17 or younger, initiation of smoking 
with menthol cigarettes was associated with higher risk of progression to established smoking and higher 
levels of nicotine dependence (Nonnemaker et al, 2012). A recent report by the Tobacco Product 
Scientific Advisory Committee (TPSAC) concluded that there was evidence to support the relationship 
between menthol cigarettes and smoking uptake, especially in African-Americans (Benowitz and Samet, 
2011). Collectively, these studies demonstrate an association of menthol cigarettes with smoking 
initiation, higher levels of dependence, and difficulty in quitting smoking. Thus, there is a clear need for 
further research on the role of menthol in multiple aspects of nicotine dependence in order to provide 
guidance for developing policies regarding menthol.   
c) Menthol’s Biological Effects - Published Studies  
Although menthol use is ubiquitous, beyond its local sensory actions, surprisingly little is known 
about menthol’s biological and behavioral effects. The well-known cooling effect of menthol (Green, 
1992;1985; Eccles, 2003) is likely mediated by a subtype of the transient receptor potential channels 
(TRPM8) that are found in the airways (Bautista et al, 2007). The TRP channels act like microscopic 
thermometers that sense hot and cold, as well as different tastes and pressure. Menthol, however, also acts 
on the other TRP channels, as well as other neurotransmitter receptors including opioid, GABAA, and 
nicotinic acetylcholine receptors (nAChR) (Corvalan et al, 2009; Zhang et al, 2008). Behaviorally, 
menthol has locomotor activating effects that are likely mediated by brain dopamine activation (Umezu, 
2009). Most relevant to nicotine addiction is whether menthol interacts with nicotine. Only one published 
study in humans has demonstrated a significant desensitizing effect of menthol on sensations evoked by 
nicotine on the tongue (Dessirier et al., 2001). A few studies point to an interaction between menthol and 
nicotine, both at the nAChR (Hans et al, 2012) and the TRP channels (Hans et al, 2012). In a recent study 
using patch clamp technique, menthol blocked the activation of alpha4beta2 nAChR, suggesting a direct 
interaction of menthol and nicotine at the nAChR level. At the behavioral level, nicotine antagonized 
menthol-induced increases in body temperature in rats (Ruskin et al, 2007, 2008). However, virtually no 
published studies have examined the reinforcing effects of menthol, alone or in combination, with 
nicotine.   
d) Menthol’s Effects in Humans - Tobacco Company Documents  
Given the lack of published literature on the reinforcing effects of menthol, an intriguing question is 
what led the tobacco companies to add menthol in varying concentrations to more than 90% of the 
cigarettes sold in the U.S. In fact, tobacco companies have conducted key studies examining the 


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reinforcing effects of menthol as an additive in cigarettes (Yerger, 2011). These studies are mentioned in 
brief memos that are included in tobacco company documents. For example, a study conducted by Philip 
Morris systematically examined the ‘impact’ of cigarettes containing different amounts of nicotine (0.08, 
0.41, and 0.91 mg/cig) and menthol (0.0, 0.41, 0.85, and 1.95 mg/cig) in a factorial design. The study 
concluded that menthol increased the ‘impact’ or liking of cigarettes that had low nicotine. In all 
cigarettes, medium amounts of menthol produced the highest liking scores (Carchman and Southwick, 
1990). Studies conducted by Philip Morris also supported the effectiveness of menthol in enhancing the 
impact of low-nicotine or low-tar cigarettes, suggesting that menthol has reinforcing effects independent 
of nicotine. Together, these studies clearly demonstrated that the addition of menthol enhances the appeal 
and likability of cigarettes. 
e) Research Need for Understanding the Effects of Menthol in Smokers 
One key question that remains to be answered is whether menthol increases the reinforcing effects of 
nicotine. Pure nicotine, similar to other drugs of abuse, is reinforcing. It is self-administered by cigarette 
smokers as well as by rodents and primates (Corrigall, 1999; Donny et al, 1995; Henningfield et al, 1983; 
Le Foll et al, 2007; Sofuoglu et al, 2008). However, the primary reinforcing effects of nicotine are less 
robust compared to other drugs of abuse like cocaine, amphetamines, or opioids. Thus, in addition to 
nicotine, other stimuli associated with smoking, including the sight, taste, and smell of cigarettes, may 
also serve as conditioned reinforcers (Sorge et al, 2009). Nicotine provides dual-reinforcement. In 
addition to its primary reinforcing effects, nicotine also enhances the motivational effects of these 
conditioned reinforcers (Caggiula et al, 2009). Thus, if environmental stimuli are intrinsically reinforcing, 
nicotine self-administration may reflect both the primary reinforcing, and the reinforcement-enhancing, 
effects of nicotine (Paterson, 2009). Accordingly, if menthol has intrinsic reinforcing properties, then its 
combination with nicotine may enhance reinforcement. Alternatively, if menthol has no intrinsic 
reinforcing properties, its combination with nicotine should not enhance reinforcement. To test the 
validity of these alternatives, carefully controlled studies delivering nicotine and menthol are needed.   
Although there are established human laboratory methods to determine drug reinforcement, the study 
of the relative reinforcing effects of menthol and nicotine present some unique challenges. First, direct 
comparison of the reinforcing effects of menthol and non-menthol cigarettes in smokers is confounded by 
the strong conditioning of smokers for either menthol or non-menthol cigarettes. This ‘brand loyalty’ 
makes blinding unfeasible in a direct comparison study with smokers. Second, while the local sensory 
effects of menthol are well recognized, little is known about the behavioral effects of menthol in humans, 
and how these effects change with menthol dose. Third, it is unclear if menthol is uniquely different than 
other flavors that have been used in tobacco products. This gap in our knowledge allows tobacco 
companies to persistently claim that menthol is just a flavor without any behavioral effects of its own 
(Heck, 2010).  
7. Subjects: 
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. This screening evaluation will be for the menthol / nicotine 
reinforcement study as described in detail in this project description.  The dose-finding study was 
conducted at the John B. Pierce Laboratory. The dose finding study concluded with the establishment of 
the high and low dose of menthol that will be used in this protocol “Menthol’s Effects on Nicotine 
Reinforcement in Smokers.” The screening of these subjects used the same inclusion and exclusion 


Mehmet Sofuoglu, M.D., Ph.D. 
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VA Healthcare System 
 
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criteria as described below.  
Inclusion criteria: 1) Female and male 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 and 4) abuse of alcohol or any other recreational or prescription drugs in the past 30 days. 5) 
Any allergy to propylene glycol or menthol.  
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 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) urinary cotinine and urine menthol glucuronide levels.  
10. Recruitment: A total of 210 smokers will be recruited from the New Haven area by newspaper 
advertisements and fliers. 
11. Research Plan 
A. Overview 
We are proposing a double-blind, placebo-controlled study that enrolls young adult smokers stratified for 
menthol preference. Menthol and control flavor will be delivered via an e-cigarette just prior to saline or 
IV nicotine infusion.  This menthol / nicotine reinforcement study was preceded by a separate menthol 
dose-finding study that determined the low and high doses of menthol to be administered by the e-


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cigarette. For this dose-finding study, subjects were screened at the West Haven site where the IV 
nicotine study will be performed, but the dose-finding study was conducted at the John Pierce Labs 
located on the Yale Campus located about 2 miles from the West Haven site. The screening procedure for 
the dose-finding study was described in a separate screening consent form. The dose-finding study did not 
administer any nicotine. 
 
For this menthol / nicotine reinforcement study, the Test Sessions will be conducted after an Adaptation 
Session is completed where subjects will learn to use the e-cigarette for delivering the menthol or control 
(tobacco) flavor. A short e-cigarette questionnaire will also be administered during the Adaptation 
Session. Then, the nicotine Test Sessions will be performed following overnight abstinence from tobacco. 
Smokers will be assigned a random sequence of three different e-cigarette conditions (low menthol (0.5% 
or 5 mg/mL) plus tobacco flavor, high dose menthol (3.5% or 35 mg/mL) plus tobacco flavor  or tobacco  
flavor alone for each of the three Test Sessions (a different menthol condition for each session). The 
concentration of tobacco flavor will be identical across all conditions. The amount of menthol in high 
dose condition is the same as those found in commercially available mentholated e-cigarette solutions 
(AmericaneLiquidStoreTM). The low dose menthol solution contains menthol that is minimally perceptible 
by smokers and is in the range of e-cigarette solutions targeting smokers with a preference for low 
menthol impact in the e-cigarette vapor. In each Test Session, smokers 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), just after the menthol or control flavor is delivered via the e-cigarette. 
The intravenous dose deliveries will be one hour apart to provide sufficient time for the nicotine and 
menthol effects to wear off.  The nicotine infusion sessions will be 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. 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 Nicotine Withdrawal Symptom Checklist (NWSC) and the Brief 
Questionnaire on Smoking Urges (BQSU). 
 
 B. Dose Finding Study 
Psychophysical testing of menthol delivered by an e-cigarette established the menthol concentrations 
for the e-cigarette delivery system (see below). After recruitment and initial screening for this dose-
finding study at VACHS (see VACHS consent form), young-adult smokers  were referred to the testing 
site at The John B. Pierce Laboratory (immediately adjacent to the Yale Medical School campus) for two, 
90 min sessions performed on separate days. Subjects signed a separate Yale consent form for all 
procedures conducted at the John B. Pierce Laboratory. Smokers were required to abstain from smoking 
on the day of testing and were given a breath CO test (MicroDirect CO Monitor) to insure compliance.  
The first dose-finding session began with a brief training period: (1) to teach participants how to inhale e-
cigarettes in the way they will be sampled in the Test Sessions with IV nicotine (see Experimental 
Sessions below); and (2) to familiarize them with the sensory (gLMS) and hedonic (LHS) rating tasks. 
The same type of e-cigarette was used during psychophysical testing as will be used in the Test Sessions, 
because this type of device allows for the customization of flavor and menthol concentrations. During the 
practice session, a tobacco flavored commercial product without menthol or nicotine were used to avoid 
interfering with subsequent measurements of menthol sensitivity. Once subjects mastered the delivery 
system, they received standard instructions in, and practice with, the rating scales (Lim et al., 2009; Green 


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et al., 1993). Testing with menthol began using an ascending method of limits (AMOL) that  included a 
presentation of 5 concentrations of menthol plus a no-menthol control, which was sampled first. The 
menthol used in this study was prepared and dispensed according to John B. Pierce Laboratory protocol. 
The menthol concentrations tested were chosen from pilot tests so as to encompass the level of menthol 
experienced in popular menthol-flavored e-cigarettes. The AMOL, in which the stimuli are presented in 
increasing intensity across trials, were used to reduce possible effects of sensitization and desensitization 
of menthol’s sensory irritation (Cliff & Green, 1991). After a concentration was sampled, subjects made 4 
separate subjective ratings: overall intensity of the menthol sensation, using the general version of the 
Labeled Magnitude Scale [gLMS; (Green et al., 1996;1993;Bartoshuk et al., 2002)]; degree of 
liking/disliking of the menthol sensation, using the Labeled Hedonic Scale [LHS;(Lim et al., 2009)]; and 
separate ratings of the perceived intensities of cooling and irritation (burning, stinging), also using the 
gLMS. There was a 5-min pause between trials to allow the menthol sensation to dissipate. After 
sampling the 5 concentrations and the no menthol control, there was a 15-min rest period before the 
subject sampled a popular commercial e-cigarette and rated it in the same manner as the test samples. 
Subjects returned for a second session in which the measurements were repeated to provide replicate 
ratings of the test stimuli and the commercial product.   
 
 
 
 
 
 
 
 
 
Table 1. Schedule of Events: Dose-Finding Session    
Time  
Measures and Events 
 0 
CO measurement 
  5 min 
E-cigarette training and practice (reminder in session 2) 
15 min 
Psychophysical scale instructions and practice (reminder in session 2) 
45 min 
Ascending method of limits trials (6) with sensation intensity and hedonic ratings 
60 min 
15 min break 
75 min 
Commercial e-cigarette sensation intensity and hedonic ratings 
85 min 
Discharge 
Determination of the menthol doses: Once a subject completed the testing, a grand log-means of the 
intensity ratings were used to determine the menthol test concentration that best matched the overall 
intensity of the popular commercial product. This was determined to be the high dose of menthol. The 
low dose was determined as the menthol concentration at which subjects could discriminate effects vs. the 
no menthol control condition, but at which clear differences in ratings were observed as compared to a 


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clustering of responses (response plateau) at higher menthol concentrations. In addition to these studies, 
Dr. Peter Jatlow’s laboratory analyzed the concentrations of menthol in the commercially available 
mentholated e-cigarette solution (about 35 mg/cc). Based on these findings, we chose two menthol doses 
that will be used in our study. While the high dose will have the same concentrations of menthol in 
mentholated e-cigarette solution (AmericaneLiquidStoreTM), the low dose will be minimally recognizable 
by participants as menthol flavor (about 5 mg/cc). 
 
Potential additional learning from sensory testing: In addition to establishing menthol concentrations for 
clinical testing, the psychophysical data will also enable preliminary evaluation of potential sex and/or 
race differences in sensory and hedonic responses to menthol. The discovery of such differences would 
yield novel data on how inhaled menthol is perceived differently between these groups, and how these 
differences might contribute to variations in the enhancing effects of menthol on nicotine reinforcement. 
C. Medical Monitoring and Safety 
Subjects will be given a thorough physical examination prior to entry in the study. 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. 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 <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.   
D. Measures 
a) Physiological: 
• 
Heart rate and blood pressure: Heart rate and blood pressure readings will be taken at 
screening, and during 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, since 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 obtained during the three Test Sessions to to determine the 
nicotine metabolite ratio (NMR). The NMR is the ratio of 3HC, the main metabolite of 
cotinine, to cotinine and reflects the activity of cytochrome P450 (CYP) 2A6 and thus, 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.  
 
 


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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.  
 
• 
Urine menthol glucuronide levels: Urine samples will be obtained at intake to confirm the 
menthol preference of smokers (Benowitz et al, 2010). Additional urine samples will be 
collected throughout each test session to measure the amount of menthol exposure (via 
urinary menthol glucuronide levels) that was provided by the e-cigarette.  
  
• 
Plasma menthol glucuronide levels: Blood samples will be obtained throughout each test 
session to determine the amount of menthol delivered by the e-cigarette. Blood samples will be 
obtained prior to each of the three experimental vaping sessions, and at 2 time points after each 
nicotine or saline infusion. An additional blood sample will be obtained just prior to removing 
the IV. Plasma levels of menthol glucuronide will be measured at different time points to 
generate concentration-time curves for each menthol delivery. This will help characterize the 
pharmacokinetics of menthol and account for differing menthol exposure between subjects.  
 
• 
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:  
• 
1) DSM-IV Nicotine Dependence Questionnaire.  
• 
2) Structured Clinical Interview for DSM-IV (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. 
• 
 3) 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 withdrawal and negative affect:  
• 
1) 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. 
• 
 2) 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).  
• 
3) 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 


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reflects levels of nicotine deprivation (Bell et al, 1999; Morgan et al, 1999). This scale will 
be used to monitor cigarette craving. 
• 
 4) 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. 
• 
5) 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. This questionnaire will be administered during the Adaptation Session.  
 
• 
Subjective Drug Effects: 1) Drug Effects Questionnaire (DEQ), adapted from Soria (1996), 
will be used during the 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’. 
 
• 
Drug Use: 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).  
• 
Cognitive Performance: 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.  
 
 
d)  
Other measures: 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 


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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 to describe the purpose and use of such 
analyses.  
E. Drugs 
a) Nicotine: Nicotine bitartrate will be obtained from Interchem Corporation, Paramus, NJ. 
Nicotine samples will be prepared by the research pharmacy at the West Haven VA. 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 three 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 a previous 
study, these nicotine doses produced robust subjective and physiological responses 
(Sofuoglu et al, 2009a). 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.  
b) Menthol: USP grade natural menthol flavor (Flavor SavorTM) will be dissolved in the 
AmericaneLiquidStoreTM  menthol e-cigarette solutions.. Menthol is widely used in a range of 
healthcare products and is 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. 
  
• 
Menthol Administration: Participants will be instructed to take 6 inhalations, one 
inhalation every 15 seconds, for 90 seconds just prior to each nicotine / saline infusion 
(Vansickel et al, 2012). As described more fully below in the section Adaptation 
Session, subjects will be asked to use light, long inhalations of 3-4 seconds with vapor 
being pulled into the mouth initially then into the lungs at the end of the inhalation.  
The menthol / e-liquid base mixture used in the e-cigarette will be prepared just prior to 
each Test Session by the VA Research Pharmacy. Each e-cigarette cartridge will contain 
2mL of the e-liquid. The cartridge will then be loaded into the e-cigarette device by the 
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a. Justification for menthol doses: For this proposal, the concentrations of the high and 
low dose menthol solutions were determined in the dose-finding study. These 
concentrations were found to be different in overall sensory impact from the control 
flavor and from each other. The target concentrations for this study are 3.5% (35 
mg/mL) and 0.5% (5 mg/mL). Human toxicity only occurs at concentrations well 
above those to be used in this study.  The concentrations of menthol in the stock 
solutions procured from the AmericaneLiquidStoreTM    will be verified using an assay 
developed by Dr. Peter Jatlow.   
 
 
F. Electronic Cigarette 
 
• 
Hardware: The Joyetech eGo-CTM  e-cigarette will be used since it appears to be among 
the most widely used e-cigarette type and allows for flexibility in the type of e-liquid 
administered.  
• 
Ingredients: E-liquid will be obtained from AmericaneLiquidStoreTM, which prepares 
all 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.   
The components of the e-liquid - propylene glycol (PG) USP, vegetable glycerin (VG) 
USP, and tobacco flavor – are premixed as commercially available solutions for e-
cigarettes. The e-liquid will consist of the concentrated flavor liquid (menthol and 
tobacco flavor) added to a base liquid. The e-liquid base will itself consist of a mix of 
70% PG and 30% VG. While e-liquids on the market range in their relative proportions 
of PG and VG, this 70/30 ratio is widely used. PG is the original base liquid for which 
most e-cigarettes were designed and remains the most common ingredient in e-liquid 
bases. PG is also credited with producing a throat sensation (‘throat hit’) that mimics 
the feel of smoking a cigarette. VG is commonly included in e-liquid bases to enhance 
the volume of vapor production, giving a greater sensory illusion of smoking.  
The menthol-flavor liquid was added at different concentrations to produce the low and 
high dose menthol conditions. As the control flavor, we chose ‘Burley’ tobacco flavor 
from the AmericaneLiquidStoreTM, rather than other flavors (e.g., cherry or chocolate), 
because the Burley tobacco flavor mimics the taste and smell of tobacco products to 
which smokers are accustomed. The concentration of tobacco flavor and the ratio of 
PG/VG will be the same across all three e-liquid conditions while the concentration of 
menthol will be different for the low and high dose menthol condition. The non-
menthol condition will consist only of tobacco flavor concentrate plus PG/VG base 
liquid.  
 
 
 
 
 
 


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G. 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 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 opioid 
and related genes. About 2.5 ml of blood will be drawn into the PAXgene tubes at baseline, prior to each 
IV infusion, and end session. Extracted mRNA samples will be stored at -80°C in the Genetics 
Laboratory. 
 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. If needed, 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 ≤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.  
 
 
 
 
 
 


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a) 
Adaptation Session:  This session will take place at the West Haven VA prior to the nicotine 
Test Sessions. The goal of the Adaptation 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.  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-cigarette used in this Adaptation Session will not 
contain nicotine, or menthol, only the control flavor (tobacco) mixed with the base liquid. 
Although the familiarization with this technique will occur during the Adaptation Session, 
participants will be reminded of this technique during each test session prior to the e-cigarette 
administration. In total, the e-cigarette vaping procedure consists 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, 
baseline biochemical measures will include urine menthol glucuronide and cotinine, plasma 
nicotine, and for women, estradiol and progesterone levels. These samples will be obtained 
followed by cognitive testing. Subjects will then start inhaling from the e-cigarette with the 
assigned menthol condition (control flavor, low or high dose of menthol), one inhalation every 
15 seconds as practiced in the adaptation session. It will take approximately 90 seconds to 
complete 6 inhalations.  Subjects will then receive the first delivery of the assigned injection 
(saline, 0.25 mg/70 kg or 0.5 mg/70 kg nicotine).  Each study injection will be 60 minutes apart 
to provide sufficient time for the physiological and subjective nicotine effects to return to 
baseline. Following each injection, physiological, subjective, and cognitive outcome measures 
will be obtained.  
c) 
Medical monitoring: For nicotine test sessions, a physician will be present and subjects will be 
attached to a blood pressure and heart rate monitoring device. 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 


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will remain in the laboratory for three hours after the last nicotine administration. These 
procedures have been developed as part of our IND application for IV nicotine.  
 
 
 
 
 
 
 
 
 
 
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 
11:00 AM  
Menthol (control, high or low Menthol) DEQ + IV Saline or Nicotine (0.25 or 0.5mg/70 kg) 
blood draw 
11:01 
HR/BP, DEQ 
11:02  
HR/BP 
11:03  
HR/BP, DEQ,  
11:05  
HR/BP, DEQ 
11:08 
HR/BP, DEQ blood draw 
11:10  
HR/BP, DEQ 
11:15  
HR/BP, DEQ, Cognitive Testing 
11:30 
HR/BP, DEQ blood draw 
11:45  
HR/BP, DEQ 
11:55  
HR/BP, DEQ, BQSU 
12:00  
Menthol (control, high or low Menthol) DEQ + IV Saline or Nicotine (0.25 or 0.5 mg/70 kg), 
blood draw 
13:00 
Menthol (control, high or low Menthol) DEQ + IV Saline or Nicotine (0.25 or 0.5 mg/70 kg), 
blood draw 
14:00 noon 
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15:00 PM 
End of session, collection of urine sample, 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 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 determine if menthol administered via e-cigarettes will change the reinforcing 
effects of pure nicotine administered intravenously in cigarette smokers who smoke mentholated or non-
mentholated cigarettes. The reinforcing drug effects will be measured with the DEQ.  
Hypothesis #1A: Concurrent menthol and nicotine administration will lead to greater reinforcement 
than nicotine and control flavor, saline and menthol, or saline and control flavor conditions. 
Hypothesis #1B: Enhancement of nicotine reinforcement by menthol will be greater in menthol 
smokers than in non-menthol cigarette smokers.   
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), 
menthol dose (low dose of menthol, high dose of menthol, or control flavor) and time of measurement 
(+1,+3,+5,+10,+15,+30,+45,+55 minutes); the between-subject factor types of cigarette preference 
(menthol vs. non-menthol) and all 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.  
A significant interaction between nicotine dose and menthol dose, with significantly higher response 
to menthol + nicotine compared to control flavor + nicotine or control flavor + saline will be considered 
supportive of hypothesis 1A. A significant interaction between nicotine dose, menthol dose, and group 
(menthol vs. non-menthol smokers), with significantly stronger effects in menthol smokers than in non-
menthol smokers, will be considered supportive of hypothesis 1B. We expect significant effects for both 
doses and will also test for dose-dependent effects.  


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Rationale for sample size: Both studies have 80% power to detect large effect sizes (f=0.4) for the 
interactions of interest in repeated measures analysis with 30 subjects per group for a total of 60 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 menthol 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 the menthol and nicotine effects. Our 
previous study shows larger effects of nicotine d’>1.0. To account for up to 70% dropout we will recruit 
160 subjects for a target of 60 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.  
 
 
 
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) The combination of intravenous nicotine and inhaled menthol has not been previously examined to 
the best of our knowledge. We have no reason to believe there is an increased risk from the combination 
used in this study as compared to the widespread use of mentholated cigarettes, menthol e-cigarettes or   
smoking while using a menthol-containing lozenge. However, the administrations will be closely 
monitored for any signs that this unique combination of IV nicotine and inhaled menthol 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. 


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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. 
 
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, 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, 


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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 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 


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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 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.     


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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 $200 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 $740.  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.  
19. Funding Source: Provided by an NIH Center Grant. 
20. Duration: The entire study will take approximately two and a half years to complete.   
  
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