Clinical trial NCT04535362
Conditions: Nicotine Dependence
Sponsor: Yale University
Start date: 2021-06-28; Completion date: 2023-06-29
Enrollment: 16 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
To examine if switching from menthol to non-menthol cigarettes will change the dose-effect curves for positive subjective effects and alleviation of smoking urges as a function of nicotine delivery rate in smokers.

Detailed description:
A placebo-controlled study that will recruit male and female menthol nicotine dependent smokers. Following screening and evaluation as described above, eligible participants will be enrolled in the study which will last about 4 weeks. Eligible, participants will be randomized to menthol or non-menthol smoking condition for 2 weeks (Phase 1) and then will be switched to the alternative condition for another 2 weeks (Phase 2). The smoking condition will be open label. Participants will be provided with free cigarettes in Phases 1 and 2. For the menthol condition, participants will be provided their usual brand of menthol cigarettes and for the non-menthol condition, they will be provided a matched-brand non-menthol cigarette (e.g., Newport Non-Menthol Gold 100s for those who smoke Newport Menthol Gold 100s). In week 2 of each Phase, participants will have a test session. Each session will include 3 infusions in the same order: nicotine (1 mg per 70 kg body weight) delivered over 5 minutes, saline and nicotine (1 mg/ 70 kg) delivered over 2.5 minutes. Once the participants complete the test session, participants will be crossed-over to the alternative treatment. The period between the 2 Phases will not be longer than one week.

Arms: arm 1: Will smoke only menthol cigarettes for two weeks arm 2: Will only smoke non menthol cigarettes for two weeks
Interventions: intervention 1: Nicotine
Intervention descriptions: intervention 1: session will include 3 infusions in the same order: nicotine (1 mg per 70 kg body weight) delivered over 5 minutes, saline and nicotine (1 mg/ 70 kg) delivered over 2.5 minutes.

Protocol documents:
Prot_SAP_000.pdf: 
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ClinicalTrials.gov ID: NCT04535362    
Brief Title: Nicotine's Potential Abuse With Menthol  
Date: 6.16.2023 
Document: Protocol w SAP 
 


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1. Principal Investigator: Mehmet Sofuoglu, M.D., Ph.D. 
2. Co-Investigators While all research staff are trained to provide informed consent, those 
identified with an asterisk (*) will provide informed consent for this study. All the research staff 
will have access to the PHI 
Co- Investigator: Suprit Parida, M.D. 
Nursing Staff: *Ellen Mitchell, RN, Angelina Genovese, R.N., 
Research Staff:  *Stacy Minnix, BS, and *Chris Cryan 
 
3. Title of Project: Impact of Menthol on the Abuse Potential of Nicotine  
4.  Purpose, hypothesis and key questions: 
Specific Aim:  To examine if switching from menthol to non-menthol cigarettes will change the 
dose-effect curves for positive subjective effects and alleviation of smoking urges as a function 
of nicotine delivery rate in smokers. 
Hypothesis #1: Switching to non-menthol cigarettes, compared to smoking menthol cigarettes, 
will enhance responses to nicotine for both positive subjective effects and alleviation of smoking 
urges as reflected by a leftward shift in dose-effect curves. 
 
Exploratory Aims: 
Exploratory Aim#1: To examine if switching from menthol to non-menthol cigarettes will change 
the dose-effect curves of nicotine delivery rate for a) reinforcement (assessed with the monetary 
value of the dose from the Multiple-Choice Procedure, b) heart rate and blood pressure, and c) 
tobacco withdrawal severity (assessed with self-report symptoms and cognitive performance).  
Exploratory Aim#2: To examine the impact of plasma nicotine levels and rate of nicotine 
metabolism (measured by plasma hydroxy cotinine/cotinine ratio) on the dose-effect curves for 
the main study outcomes.  
Exploratory Aim#3: To examine sex differences for the main study outcomes. 
 
5. Background: 
Nicotine control policies to reduce the addictive potential of tobacco products:  
Benowitz and Henningfield (1) proposed that the gradual reduction of nicotine in cigarettes to an 
amount below the addiction threshold could prevent the development of nicotine addiction 
among young smokers. The Benowitz and Henningfield proposal and its subsequent appraisal 
by the American Medical Association Council on Scientific Affairs (6) have provided the 
foundation for the current nicotine control approaches being considered by the FDA (7). More 
recently, Shihadeh and Eissenberg made a similar proposal for minimizing the potential risks of 
using EC (2). After considering several EC variables including liquid composition, puff behavior 
and electrical power, they focused on nicotine flux, the rate of nicotine delivery, as the key factor 
in determining the abuse potential of a given EC. Accordingly, an EC yielding no nicotine flux 
will not maintain usage. In contrast, an EC with high nicotine flux may have high addictive 
potential and lead to negative health effects. If the nicotine flux does not produce dependence 
but is sufficient to attenuate craving for cigarettes and withdrawal symptoms among those that 
are already dependent, the product may have limited abuse potential while retaining potential 
benefits (e.g. as a smoking cessation aid). In the “nicotine flux” model, the delivery rate of 
nicotine, rather than the amount of nicotine in the products, is more likely to reflect the addictive 
potential of the product. The proposed central role of nicotine flux in determining the addictive 
potential of nicotine remains to be tested in controlled human studies.  


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Policies to reduce the addictive potential of tobacco product would be strengthened if the 
thresholds for nicotine’s addictive properties were empirically defined by carefully controlled 
studies. 
Influence of menthol on nicotine sensitivity 
A large body of evidence supports the potential impact of menthol in facilitating the initiation of 
cigarette smoking (34). However, menthol’s effects on nicotine’s reinforcing and addictive 
properties are not well understood. Preclinical studies reported that menthol has inhibitory 
effects on α7, α3β4 and α4β2*nAChR subtypes (35-37), although menthol may also enhance 
nicotine’s reinforcing effect (38). To better understand the potential contribution of menthol to 
nicotine’s addictive effects, we examined if menthol administered by inhalation via an e-cigarette 
would change the subjective effects of nicotine administered intravenously in menthol and non-
menthol preferring smokers (See Preliminary Studies). We found that menthol produces minimal 
positive subjective effects, alone or in combination with nicotine. However, menthol-preferring 
smokers, compared to non-menthol preferring smokers, had diminished positive subjective 
responses to IV nicotine, and had less severe tobacco withdrawal following overnight 
abstinence. In addition, mentholated cigarette smoking was also associated with a lower 
nicotine metabolite ratio (NMR), a biomarker for the rate of nicotine metabolism (39, 40). This 
finding is consistent with previous studies demonstrating an inhibitory effect of menthol on 
nicotine metabolism (40, 41). A slower rate of nicotine metabolism, as assessed by a lower 
NMR, is associated with lower urges to smoke and less severe withdrawal symptoms following 
nicotine deprivation, and reduced positive subjective effects to IV nicotine (42, 43). Thus, the 
impact of menthol on nicotine’s addictive effects may be partly due to menthol’s inhibition of 
nicotine metabolism and this effect seems to be reversible after switching from menthol to non-
menthol cigarettes (41). In a carefully conducted study with menthol smokers, switching to non-
menthol cigarettes for one week, compared to a menthol-cigarette condition, was associated 
with  an increased rate of nicotine metabolism (41). Together, these findings support the 
modulatory effects of menthol on the abuse liability and addictive effects of nicotine. Whether 
menthol-cigarette smoking affects the sensitivity for nicotine delivery rate has not been 
determined. Similarly, it has not been examined whether switching from menthol to non-menthol 
cigarettes changes sensitivity to nicotine’s effects. This is an important knowledge gap given the 
widespread use of menthol in both tobacco cigarettes and e-cigarettes. The current study will 
address these questions by systematically examining the impact of nicotine delivery rate in both 
menthol and non-menthol smokers and by examining the impact of delivery rate after switching 
from menthol to non-menthol cigarettes. 
 
Preliminary Studies: 
 
Menthol’s effects on nicotine reinforcement in smokers [Valentine et al. (49)]. The goals of this 
TCORS project were to determine if menthol administered by inhalation via an e-cigarette would 
change the subjective effects of pure nicotine administered intravenously, and whether these 
effects would be greater in menthol-preferring smokers. A total of 57 menthol-preferring (n=32) 
and non-menthol cigarette smokers (n=25) (44 Male, 13 Female, 25 African-American and 32 
White) were enrolled in this double-blind, placebo-controlled study with 3 test sessions. 
Participants were assigned to a random sequence of three different e-cigarette conditions [0% 
(no menthol), 0.5% (low) or 3.2% (high) menthol] for the 3 test sessions (a different flavor 
condition for each session). In each test session, smokers received a random order of 1 
intravenous delivery of saline, and 2 intravenous deliveries of nicotine (0.25 mg/70 kg and 0.5 
mg/70kg), one hour apart. 


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While menthol did not change the positive subjective effects of nicotine, positive effects were 
found to be dependent on cigarette preference. Compared to menthol-preferring smokers, non-
menthol smokers reported greater positive subjective responses to IV nicotine (Figure 1), and 
had more severe tobacco withdrawal following nicotine deprivation (Figure 2) and a higher 
nicotine metabolite ratio, a biomarker for a faster rate of nicotine clearance. These findings 
suggest that mentholated cigarette smoking may influence smoking behavior through its effects 
on nicotine reward and severity of tobacco abstinence symptoms. 
 
 
6. Significance: 
The proposed study is relevant to the FDA’s overall authority to regulate the manufacture, 
marketing, and distribution of tobacco products to protect public health. Specifically, the project 
addresses the CTP’s research interest on “understanding the effect of tobacco product 
characteristics on addiction and abuse liability” and “the amounts of nicotine delivered to ENDS 
users during experimentation.” First, although enhanced nicotine delivery rate is a key feature of 
newer e-cigarettes (2-5), no human studies have systematically examined the relationship 
between nicotine delivery rates that are within the range of those achieved by tobacco and e-
cigarettes, and nicotine’s abuse potential vs. beneficial effects. We will address this question by 
using IV nicotine infusion, which closely matches the behavioral effects of inhaled nicotine and 
allows precise control over the dose and delivery rate of nicotine. Thus, by determining the 
impact of nicotine delivery rates, with the correspondent plasma nicotine levels, on the abuse 
potential vs. beneficial effects, this study will provide a benchmark for evaluating the risks from 
new nicotine delivery products. Second, the study will also examine if switching to non-menthol 
cigarettes, as a short-term model of banning menthol cigarettes, will affect the impact of delivery 
rate on nicotine’s addictive vs. beneficial effects. This is a significant goal given the widespread 
use of menthol in tobacco products and its complex interactions with nicotine. Third, this 
application will examine possible sex differences on the impact of nicotine delivery rate on the 
main study outcomes. Systematically generated data on individual differences in responses to 
nicotine delivery rate should be critical to the development of nicotine reduction policies. 
Together, this application will help the FDA in setting benchmarks for nicotine delivery rates that 
minimize the abuse potential of nicotine-delivery products and will also assess the potential 
impact of banning or limiting menthol cigarettes on these benchmarks. 
 
 
 


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7. Subjects: 
Smokers will be recruited from the New Haven area through newspaper, and radio 
advertisements and fliers. The study will be described over the telephone. Subjects will be given 
a brief tobacco use history and medical screening questionnaire. Eligible subjects will be invited 
to a screening evaluation. The screening evaluation will include the following: a) obtaining an 
informed consent; b) tobacco use history, the Fagerstrom Test of Nicotine Dependence (FTND) 
and DSM-5 Tobacco Use Disorder (TUD) criteria; c) urine cotinine levels determined with a 
NicAlert test strip; d) complete physical and psychiatric examination including the structured 
clinical interview (SCID) for DSM-5 criteria; e) laboratory examination including CBC, TSH, ALT, 
AST, GGT, alkaline phosphatase, glucose, BUN and creatinine; f) urine analysis for drug 
screening, and for women, urine pregnancy test. 
 
To minimize the risk of COVID-19 transmission, every attempt will be made to conduct the 
screening procedures that do not require in-person interaction, like physical and laboratory 
examination, remotely by video or phone. Consent form and other self-report forms will be 
mailed to the participants before the sessions and they will be asked to return these documents 
when they come to the medical center for screening.    
Inclusion criteria: 1) Female and male smokers, aged 21 to 59 years, who have been smoking 
tobacco cigarettes for at least a year; 2) smoke ≥ 5 and less than 30 cigarettes per day; 3) urine 
cotinine levels > 100 ng/mL consistent with nicotine intake of an active smoker (23); 4) not 
seeking treatment at the time of the study for nicotine dependence; 5) in good health as verified 
by medical history, screening examination, and screening laboratory tests; and 6) 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 or psychiatric disorders that the physician 
investigator deems as contraindicated for the subject to be in the study; 2) regular use of 
psychotropic medication (antidepressants, antipsychotics, or anxiolytics); 3) current alcohol or 
substance dependence for any other recreational or prescription drugs other than nicotine; 5) 
urine drug screening indicating recent illicit drugs use (with the exception of marijuana). 
8: Recruitment:  
Approximately, 50 subjects will be recruited, to have 38 completers. 
9. Research Plan: 
 
A) Measures 
Screening Measures: PATH (Population Assessment of Tobacco Health Study) – we will 
use a subset of Wave 1 items from the PATH to gather demographic data and assess 
tobacco product use history (67). SCID (Structured Clinical Interview for DSM-5) - semi-
structured diagnostic interview for Axis I psychiatric disorders (68). CES-D (Center for 
Epidemiologic Studies Depression Scale) – a 20-item self-report scale of depressive 
symptoms to control for baseline differences in mood state (69). 
Measures of Tobacco Product Use, Urges and Dependence: TLFB (Time Line Follow-
Back) - will be used for monitoring tobacco, alcohol and drug use during the study 
participation (70). FTND (Fagerstrom Test of Nicotine Dependence) - self-report measure 
that assesses severity of nicotine dependence (71). MNWS (Minnesota Nicotine Withdrawal 
Scale) – 8-item scale that assesses DSM-IV symptoms of nicotine withdrawal (72). BQSU 
(Brief Questionnaire on Smoking Urges) –a 10-item scale that reliably reflects levels of 
nicotine deprivation (73).     
 


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Biomarkers: Plasma and urine cotinine, 3-hyroxycotinine (3HC), and nicotine levels: 
Plasma and urine cotinine levels of these biomarkers will be obtained at screening and 
before each test session to quantify and confirm the prior level of nicotine. During each test 
session, samples for determining plasma nicotine levels will be collected just before, during 
and after nicotine infusion. Consistent with previous studies, both peak nicotine 
concentration and the area under the plasma nicotine concentration time curve (AUC) 
values will be included in the analysis.  
The peak plasma concentrations of nicotine are expected to increase with increasing 
delivery rates of nicotine, as demonstrated in previous studies (28). In contrast, the AUC, 
which will include nicotine levels at time points before, during, and after nicotine delivery, 
reflects the total dose of nicotine independent of the delivery rate (74, 75). 3-hyroxycotinine 
is the main metabolite of cotinine and the ratio of 3HC/cotinine [nicotine metabolite ratio 
(NMR)], reflects the activity of cytochrome P450 (CYP) 2A6 and can therefore be used as a 
correlate of the rate of nicotine clearance (76). The NMR has been shown to be stable in 
smokers during ad lib and reduced smoking (77). Plasma samples for 3HC will be obtained 
at baseline and the NMR will be included as a covariate in our analyses. Urine menthol 
glucuronide (MG): In phase 1 and 2, urine samples will be obtained in the screening visit 
and before each test session to quantify MG levels at baseline and during study 
participation. In study 2, urine samples for MG will also be collected weekly to monitor 
compliance with switching to non-menthol cigarettes. Alveolar carbon monoxide (CO): The 
CO measurement taken before the sessions will help to verify compliance with smoking 
abstinence. Abstinence will be verified with CO or plasma nicotine as recommended by the 
SRNT Subcommittee on Biochemical Verification (56). The CO levels will be subsequently 
confirmed by the baseline nicotine levels, which will also account for all tobacco and nicotine 
product use. Serum estradiol and progesterone analysis (females only): Serum estradiol 
and progesterone levels will be collected before each test session for use as covariates in 
our analysis, since female sex hormones may contribute to sex differences in nicotine 
responses (54). 
 
Drug reinforcement and subjective effects: The MCP (Multiple-Choice Procedure) was 
developed and validated by Roland Griffiths and colleagues as an efficient tool to assess 
drug reinforcement in humans, including nicotine reinforcement (6, 50, 78). For each test 
session (6 total), participants will have choices between forfeiting or receiving escalating 
sums of money on a scale of values between -$20.00 and $20.00, or re-receiving that trial’s 
infusion. The monetary scale will include values at $0.25 increments between $2.00 and -
$2.00 and values at $0.50 increments from $2.00 to $20.00 and from -$2.00 to -$20.00. A 
crossover point, the value at which the participant chooses money rather than infusion, will 
be determined for each session. No additional money or drug infusions will be provided 
based on these choices. DEQ (Drug Effects Questionnaire). Participants will rate on a 100 
mm scale, from "not at all" to "extremely, 10 items that are related to nicotine’s subjective 
effects. The items are 1) feel the “drug strength,” 2) feel “good” drug effects, 3) feel “bad” 
drug effects, 4) like the drug effects, 5) feel high, 6) feel stimulated, 7) feel anxious, 8) feel 
down, 9) want more. This instrument is for a rapid detection of nicotine’s effects and is 
adapted from a VAS (51, 79). Good drug effects and drug liking are items that are 
recommended for the assessment of abuse potential of drugs (10, 80). 
 
Cognitive Performance: CPT (Continuous Performance Test): Cognitive performance will 
be assessed with the continuous performance test (CPT) from the ANAM battery (University 
of Oklahoma): This test was chosen because of its sensitivity to tobacco withdrawal (52, 53). 
CPT assesses sustained attention, concentration, and working memory. For CPT, the main 
outcome measures will be percent correct responses and reaction time (52). 
 
Physiological: Heart rate and blood pressure readings will be taken at intake for 
screening purposes, and during the test sessions to monitor nicotine’s effects.  


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Adverse Events: SAFTEE (Systematic Assessment for Treatment Emergent Events): To 
monitor adverse events from nicotine, the SAFTEE will be administered before and after 
each IV nicotine session. The SAFTEE has been used in a number of pharmacotherapy 
trials (81).  
 
B) Drugs 
Nicotine: administration: Nicotine bitartrate will be obtained from Interchem Corporation, 
Paramus, NJ, and solutions for IV injection will be prepared by U.S. Specialty Formulations, 
Bethlehem, PA. On the morning of each test session, weight-adjusted nicotine and saline 
solutions will be prepared by the West Haven VA research pharmacy. Nicotine and saline will 
be administered using an infusion pump connected to an IV catheter located in a forearm vein. 
We have followed these procedures in our previous studies, which were completed without 
any serious adverse effects attributed to nicotine or other safety concerns. Dr. Sofuoglu holds 
an ITP (Investigational Tobacco Product) for IV nicotine administration. A new ITP application 
has been submitted to the FDA for this project. 
 
In each test session, there will be a total of 3 infusions. Each infusion duration will be 10 minutes 
long, which will be achieved by infusing saline for the reminder of the time following the 2.5- and 
5-minute nicotine infusions. A research nurse, will program 2 infusion pumps to administer 
nicotine or saline solutions. For the 2.5 and 5 min infusion conditions, participants will first receive 
nicotine for 2.5 and 5 minutes, followed by saline infusions. For the saline condition, participants 
will receive saline for 10 minutes. The participants will be blind to the randomization. The order of 
infusions will be: 1) 5-min or 2.5-min nicotine infusion, 2) saline infusion and 5-min or 2.5-min 
infusion. This infusion order was chosen to minimize carry over effects between nicotine 
infusions. Participants will be blind to the order of nicotine infusions. 
Justification for the nicotine doses and infusion rates: The total nicotine dose for each test 
session will be 2 mg/70 kg body weight, a dose within the range of nicotine delivered by 
smoking 1 or 2 tobacco cigarettes or e-cigarettes (21). For safety reasons, the maximum dose 
of nicotine for each infusion will be 1 mg.  The infusion rates will be 0.096 and 0.048 
µg/kg/second over 2.5 and 5 minutes, respectively.  
Menthol and Non-Menthol Cigarettes:  Participants will be provided with free cigarettes in 
Phases 1 and 2 of the study. For the menthol condition, participants will be provided their usual 
brand of menthol cigarettes and for the non-menthol condition, they will be provided a matched-
brand non-menthol cigarette (e.g., Newport Non-Menthol Gold 100s for those who smoke 
Newport Menthol Gold 100s). Participants will be given a two- week supply of their assigned 
cigarettes based on their reported cigarettes per day. If they run out of cigarettes earlier than 
expected, they will be given additional cigarettes not to exceed 20 % higher than the reported 
daily cigarette use. 
Justification for age criteria: The study will enroll young adults between the ages of 21 to 59 
because this age group captures the majority of smokers who smoke menthol cigarettes . 
Limiting enrollment to this age group will also minimize variation in the number of years of 
smoking that can influence measures of dependence, including withdrawal severity. The lower 
age limit is 21 because purchasing cigarettes below this age is illegal in Connecticut. 
 
C) Study procedures 
 
General procedures: Before the test sessions, smokers will be required to abstain from 
smoking for 10 h, which will be verified by expired air CO levels ≤8 ppm (56). Participants will be 
asked to refrain from consuming alcoholic beverages and drugs during study participation, 
which will be verified by urine drug screening and breathalyzer measurements before the 


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sessions. In our prior studies, the adherence rate with overnight abstinence has been over 95 
percent. If results indicate non-adherence with these study procedures, the session will be 
rescheduled. Repeatedly non-adherent participants will be discharged from the study. 
Participants will be instructed to drink their typical number of caffeinated beverages in the 
morning to minimize caffeine withdrawal that could confound interpretation of study outcomes. 
Participants will be instructed not to eat for 4 hours before the sessions to minimize heartburn or 
nausea that can be associated with IV nicotine administration.  
Medical monitoring: Participants will be given a thorough physical examination prior to study 
entry. For nicotine administration sessions, participants will be attached to a cardiac monitor as 
well as a blood pressure and heart rate monitoring device. An IV catheter for nicotine delivery 
will be in place throughout each test session. Participants will be administered nicotine only if 
the systolic blood pressure is <150 mmHg and heart rate is <90 beats/minute. Participants will 
be terminated from the study if the blood pressure at any time is >170/110 mm Hg, if the heart 
rate is >130 beats/min, or if they develop signs and symptoms consistent with nicotine toxicity. 
We have not encountered any cases of nicotine toxicity in our nicotine infusion studies. These 
study procedures were developed as part of our Investigational Tobacco Product (ITP) 
application to the FDA for IV nicotine. 
Overview of the study: We propose a placebo-controlled study that will recruit male and female 
menthol nicotine dependent smokers. Following screening and evaluation as described above, 
eligible participants will be enrolled in the study which will last about 4 weeks. Eligible, 
participants will be randomized to menthol or non-menthol smoking condition for 2 weeks 
(Phase 1) and then will be switched to the alternative condition for another 2 weeks (Phase 2). 
The smoking condition will be open label. Participants will be provided with free cigarettes in 
Phases 1 and 2. For the menthol condition, participants will be provided their usual brand of 
menthol cigarettes and for the non-menthol condition, they will be provided a matched-brand 
non-menthol cigarette (e.g., Newport Non-Menthol Gold 100s for those who smoke Newport 
Menthol Gold 100s). In week 2 of each Phase, participants will have a test session.  Each 
session will include 3 infusions in the following order: nicotine (1 mg per 70 kg body weight to a 
maximum dose of 1 mg) delivered over 2.5 or 5 minutes, saline delivered over 10 minutes, and 
nicotine (1 mg/ 70kg to a maximum dose of 1 mg) delivered over 2.5 or 5 minutes). For safety 
reasons, the maximum dose of nicotine for each infusion will be 1 mg. The infusion rates will be 
0.096 and 0.048 μg/kg/second over 2.5 and 5 minutes, respectively. Once the participants 
complete the test session, participants will be crossed over to the alternative treatment. The 
period between the 2 Phases will not be longer than one week.  
  
Table 1: Study Overview  
 
Study Phases 
Procedures 
Phase 1 (Days 0 to 14) 
Phase 2 (Day 0 to 
14) 
Smoking assignment 
Menthol vs. Non-
Menthol* 
Menthol vs. Non-
Menthol 
Phone Check-ins 
Daily (Days 1 to 14) 
Daily (Days 1 to 14) 
Outpatient Visits 
Day 1  
Day 1 
Test Sessions (IV 
nicotine infusion) 
1 session (between Days 
8 to 14) 
1 session (between 
Days 8 to 14) 
*Menthol cigarettes will be their preferred menthol cigarettes. Non-Menthol will be a brand 
matched non-menthol cigarettes. 
 
Daily Phone Check-in Measures: Participants will receive daily phone calls in Phase 1 and 
Phase 2 to assess: 1) the number of cigarettes smoked of the cigarettes provided to the 


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participants and the number of other cigarettes smoked each day, and 2) other tobacco product 
use including e-cigarettes and other product types.  
Outpatient Visits: Participants will have an outpatient visit on the first day of Phases 1 and 2. 
Participants will be provided the assigned cigarettes and the following measures will be 
collected: CO, urine cotinine, urine menthol glucuronide, BQSU, MNWS, heart rate/blood 
pressure and SAFTEE.  
 
Adherence: Adherence to use of the assigned cigarettes during the 2 Phases of the study 
will be closely monitored using multiple measures. First, participants will report the total number 
of cigarettes consumed each day. Second, urine samples will be obtained for menthol 
glucuronide levels, to quantify menthol intake during each phase of the study. To reduce the 
impact of other mentholated products on the total menthol intake, participants will be provided 
non-menthol toothpaste and will be instructed to avoid using typical menthol-containing 
products. Lastly, participants will be paid for completing the daily assessments in a timely 
manner.   
Test Session: The schedule of events during the test sessions is shown in Table 2. The 
sessions will begin around 8 AM, following overnight nicotine abstinence. Before the session 
begins, participants will have two indwelling catheters placed in antecubital veins, one for 
administering infusions and one for collecting blood samples. A blood sample will be drawn prior 
to the first infusion to measure baseline levels of plasma nicotine and menthol glucuronide and 
for women, additional samples for the measurement of estradiol and progesterone levels will be 
collected. Baseline measurements will then be collected including heart rate, blood pressure, 
self-report assessments and cognitive testing. Participants will then receive the assigned infusion 
over a period of ten minutes. Psychometric assessments and blood sampling will then continue 
for the next 120 minutes. Participants will be discharged after evaluation by the study physician. 
Participants will also receive their assigned cigarettes at the end of the session. 
Table 2. Schedule of Events: Test Session* 
Time point 
(min) 
Measures and Events 
Baseline 
CO, urine and blood samples, HR/BP, M-NWSC, BQSU, SAFTEE, CPT 
0  
IV Infusion 1 (nicotine over 2.5 min or 5min) starts 
1  
HR, DEQ, plasma nicotine 
3  
HR, DEQ, plasma nicotine  
5  
HR, DEQ  
6  
plasma nicotine 
8  
HR, DEQ 
10  
IV Infusion 1 ends, HR, DEQ, BQSU, 
12  
plasma nicotine  
13  
HR, DEQ,  
15 
HR, DEQ, plasma nicotine  
18 
HR, DEQ  
20 
HR, DEQ, plasma nicotine 
25 
HR, DEQ, 
30  
HR/BP, DEQ, BQSU, CPT, M-NWSC, MCP 
45 
HR, DEQ, BQSU, M-NWSC CPT, plasma nicotine 
60   
IV Infusion 2 (Saline) starts 
61  
HR, DEQ, plasma nicotine 
63  
HR, DEQ, plasma nicotine  


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65  
HR, DEQ  
66  
plasma nicotine 
68  
HR, DEQ 
70  
IV Infusion 2 ends, HR, DEQ, BQSU, 
72  
plasma nicotine  
73  
HR, DEQ,  
75  
HR, DEQ, plasma nicotine  
78  
HR, DEQ  
80  
HR, DEQ, plasma nicotine 
85  
HR, DEQ, 
90  
HR/BP, DEQ, BQSU, CPT, M-NWSC 
105  
HR, DEQ, BQSU, M-NWSC CPT, plasma nicotine 
120  
IV Infusion 3 (nicotine over 2.5 min or 5min) starts 
121  
HR, DEQ, plasma nicotine 
123  
HR, DEQ, plasma nicotine  
125  
HR, DEQ  
126  
plasma nicotine 
128   
HR, DEQ 
130  
IV Infusion 3 ends, HR, DEQ, BQSU, 
132  
plasma nicotine  
133  
HR, DEQ,  
135  
HR, DEQ, plasma nicotine  
138  
HR, DEQ  
140  
HR, DEQ, plasma nicotine 
145  
HR, DEQ, 
150  
HR/BP, DEQ, BQSU, CPT, M-NWSC 
165  
HR, DEQ, BQSU, M-NWSC CPT, plasma nicotine 
180  
End of session, HR/BP, DEQ, plasma nicotine, M-NWSC, BQSU, SAFTEE, MCP 
190   
Discharge  
*Saline infusions will follow each nicotine infusion to maintain a 10-min total infusion time for 
each test session. Abbreviations: CO: Alveolar carbon monoxide; HR/BP: Heart rate/Blood 
pressure; M-NWSC: Minnesota Nicotine Withdrawal Symptom Checklist; BQSU: Brief 
Questionnaire of Smoking Urges; CPT: Continuous Performance Test; DEQ: Drug Effects 
Questionnaire; SAFTEE: Systematic Assessment for Treatment Emergent Events. MCP: Money 
choice procedure. 
 
 
7) Statistical analysis and sample size estimates 
Specific Aim #1: To examine if switching from menthol to non-menthol cigarettes will change 
the dose-effect curves for positive subjective effects and alleviation of smoking urges as a 
function of nicotine delivery rate.   
Positive subjective effects of nicotine will be assessed with the “Like the Drug Effects” and 
“Good Drug Effects” items from the DEQ, consistent with the FDA Guidance on abuse potential 
assessment (10). These items have been shown to correlate with the abuse potential of drugs 
of abuse. Effects will be tested using linear mixed effects models with peak change on each 
DEQ item as a response variable, cigarette-assignment (menthol vs. non-menthol), phase (1 
vs.2) and nicotine delivery condition as within-subject factors and their interaction. Random 
effects for subject and condition within subject will be used to account for correlations between 


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repeated measures. Because of the potential skewed distributions of the subjective effects data, 
transformations will be applied as necessary. We will evaluate the main and interactive effects 
of cigarette assignment and delivery rate. We anticipate statistically significant main effects of 
cigarette assignment, nicotine delivery rate and a significant interaction between cigarette 
assignment and nicotine delivery rate. To better understand the dose effects of nicotine delivery 
rate and whether they are moderated by cigarette assignment, we will construct specific 
contrasts among the four doses to test whether there is an indication of significant linear, 
quadratic, linear on log-scale or sigmoidal dose-response relationships across, or within 
menthol-preference groups. The possible dose-response curves identified by the specific mean 
contrasts will be compared by fitting linear and non-linear (as necessary) dose-response models 
with continuous dose and selecting the best-fitting model by the Akaike’s Information Criterion 
(AIC). To test for an effect of delivery rate on smoking urges, assessed by the BQSU (Factor 1 
and 2) scores, we will use similar modeling approach described for subjective effects, but we 
will also include time as a within-subject factor with outcomes for minute 0, 10, 30, 60 and 120. 
We anticipate a significant main effect of menthol-preference, nicotine delivery rate and 
potentially a significant interaction between cigarette assignment and nicotine delivery rate.  
Exploratory Aim#1: To explore the dose-effect curves of nicotine delivery rate for a) 
reinforcement (assessed with the Multiple-Choice Questionnaire), b) heart rate and blood 
pressure, and c) tobacco withdrawal severity, assessed with self-report symptoms and cognitive 
performance.  
The primary outcome for the analysis of reinforcement will be the crossover point 
determined by the monetary choice procedure for the four infusion conditions. The crossover 
point is the monetary value at which a subject chooses money over receiving the infusion for 
that condition. It is a continuous measure (dollar amount) from -20 to +20 with values > $0 
indicating reinforcement. Tobacco withdrawal severity is measured by the MNWS and CPT 
taken at 0, 30, 60 and 120 min after the beginning of infusion. The models will be similar to the 
one described for Specific Aim #1 with time as an additional within-subject factor for heart rate, 
blood pressure and tobacco withdrawal severity. 
Exploratory Aim#2: To examine the impact of plasma nicotine levels and rate of nicotine 
metabolism (measured by plasma hydroxy cotinine/cotinine ratio) on the dose-effect curves for 
the main study outcomes.  
To address this aim, we will use a similar mixed model approach to the one described for 
specific aim #1. For plasma nicotine levels, we will include peak plasma and area under the 
curve (AUC) nicotine plasma concentration as a continuous predictor instead of nicotine delivery 
condition in separate mixed models. The AUC will be calculated using the nine nicotine 
concentrations collected across the 120-minute sampling period.  
For the rate of nicotine metabolism, the plasma hydroxy cotinine/cotinine ratio will be 
included as a continuous predictor in the model. 
Exploratory Aim#3: To examine sex differences for the main study outcomes. 
To evaluate whether sex moderates’ menthol and nicotine delivery rate effects we will add 
sex as an additional factor in all models above.  
Rationale for sample size: With 30 individuals, the study will have over 80% power to detect 
large effect sizes (f=0.4) for the comparisons of the menthol and non-menthol conditions (f=0.6 
or higher) on alleviation of the subjective effects and similarly large effect sizes for the 
interaction between condition and nicotine delivery rate (f=0.65 or higher) at Bonferroni-adjusted 
level of 0.025. To account for 20% dropout, we will recruit approximately 38 subjects. 
  
11. Risks and benefits: 


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Potential risks 
There are potential risks, discomforts and inconveniences associated with the participation in 
this study. These may be due to nicotine administration, blood drawing, and other study 
procedures. 
1) The administration of nicotine may cause cardiovascular, autonomic, and gastrointestinal 
complications. Large doses of nicotine may cause nausea, vomiting, abdominal pain, 
hypersalivation, diarrhea, dizziness, confusion, hearing and vision problems, syncope, seizures, 
hypotension, irregular pulse, and death. However, these toxic effects occur at doses 10-20 
times that which will be used in our study.  Other potential risks from this study include 
administering a drug that has addictive potential. However, since only subjects with a history of 
cigarette use are to be included, we will not be exposing subjects to the risks of nicotine for the 
first time. Additionally, we are not enrolling subjects who are seeking treatment to quit smoking. 
Over the last 5 years, we have administered nicotine intravenously to more than 100 smokers 
and have not encountered any adverse events from nicotine. 
2) Blood Drawing: Subjects will have less than 300 ml of blood drawn as a result of their 
participation in the study. Blood drawing can cause some pain and may result in bruising.  
3) Study procedures: On the test days, subjects will not be able to smoke for 10 hours. During 
this cigarette abstinence period, subjects may experience symptoms of nicotine withdrawal such as 
craving cigarettes, mild anxiety, restlessness, irritability, difficulty concentrating, loss of energy, and 
excessive hunger.     
4) Protection of Subjects: 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 
conducting the test sessions 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.   
 
 
12. Safety 
Prior to initiating any research activity, each subject must give informed consent. Before the 
study, the subjects will be informed about all potential risks of the study. Our inclusion and 
exclusion criteria will be applied by experienced professionals who will be carefully trained and 
monitored to accept only appropriate subjects into the study. Thus, effective screening will 
exclude subjects who would be placed at a greater risk. Eligibility is determined by the medical 
and psychiatric history, drug use history, the physical examination, and the laboratory studies 
done prior to beginning this research protocol. 
For nicotine administration sessions, a physician or a nurse will be present. Subjects will be 
attached to a cardiac monitor as well as a blood pressure and heart rate monitoring device. Two 
IV catheters will be in place in each arm throughout the session. Subjects will be administered 
nicotine only if the systolic blood pressure is <150 mmHg and heart rate is < 90 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 > 130 beats/min, or if they develop signs and symptoms compatible with 
nicotine toxicity. Subjects will remain at the test session site (Biostudies unit, 9th floor - Bldg.1, 
VA Medical Center, West Haven) for at least an hour after the last nicotine administration. 
These procedures have been developed as part of our Investigational Tobacco Product (ITP) for 
IV nicotine.  


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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.    
12.1 Potential benefits of the proposed research to the subjects and others.  There will be 
no direct benefit to subjects participating in this study. However, subjects will receive complete 
medical and psychological evaluation. Cigarette smokers will be given anti-smoking literature 
and treatment resources will be provided for smoking cessation.  
12.2 Importance of the knowledge to be gained. This proposed study may help to develop 
new and more effective tobacco control policies. We believe that the risk/benefit ratio for this 
study is acceptable, and that the benefits of the proposed studies outweigh the potential risks to 
subjects.  
12.3 Data safety and monitoring plan: 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 and Yale IRB, 
and NIDA. We will directly report to the FDA, whenever their magnitude or frequency exceeds 
expectations.   
The risk associated with participating in this study is moderate, because nicotine administered 
may be associated with mild side effects. Serious adverse effects associated with nicotine 
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This project will be monitored by a 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 statistics (Dr. Hanga Galfalvy, Associate Professor of Biostatistics, 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. 
In order for the DSMB to fulfill its mission of assuring the safety of human subjects and the 
scientific integrity of the studies conducted, the Board will have access to accumulating study 
outcome data in a manner that will protect its confidentiality and preserve its statistical integrity. 
The Board will examine accumulating data to assure that the risks and benefits of participation 
remain acceptable and that the results of the study will be considered scientifically reliable. The 
conditions under which the Board will examine this data are described below. 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 study will be monitored for safety in an ongoing way as well as three times each year 
formally by the DSMB.  The P.I. will attend an initial part of this meeting to present the study’s 
adverse events and ongoing subject accrual, as well as any potential study design changes 
under consideration.  The remainder of the meeting will not include any direct study personnel 
until the end of the meeting, when the DSMB will convey directly to the P.I. any safety or study 
conduct concerns, as well as requests for potential interim analyses.    
Following each DSMB meeting written minutes will be prepared and distributed summarizing 
any recommendations. These written reports will insure timely communication with the study P.I. 
with preparation of any protocol amendments necessary. After each DSMB meeting, this written 
report will describe all recommendations including additional safety steps.  
The FDA adverse drug experience reporting timelines will be utilized as timelines to disseminate 
feedback from the DSMB to the principal investigator and sub investigators. That is, three days 
for acute circumstances and ten days for non-acute circumstances.  
 
13. Informed consent: Subjects will be recruited from the New Haven area by newspaper 
advertisements and fliers. Interested subjects will be informed about the study over the 
telephone and asked for current use of drugs and medical problems. If subjects are interested, 
they will then come into the clinic for a full screening evaluation. Upon arrival, a research 
assistant will review 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.   
14. Information Security/Confidentiality: Confidentiality in this study is of the utmost 
importance to us. All information obtained will be stored in coded form. 
  


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Personally identifiable information (PII) and protected health information (PHI) that is obtained 
from subjects or about them will be protected by the research team who will follow all guidance 
provided in the following VHA HANDBOOKS and DIRECTIVE: 
 
• 
VHA HANDBOOK 1605.1, PRIVACY AND RELEASE OF INFORMATION Dated May 
17, 2006 
 
• 
VHA HANDBOOK 1907.01, HEALTH INFORMATION MANAGEMENT AND HEALTH 
RECORDS Dated August 25, 2006 
 
• 
VHA HANDBOOK 6500, INFORMATION SECURITY PROGRAM Dated September 18, 
2007 
 
• 
MEMORANDUM FOR UNDER SECRETARIES, ASSISTANT SECRECTARIES AND 
OTHER KEY OFFICIALS, SUBJECT: Protecting Information Security and Privacy Dated 
February 27, 2009 
 
• 
VHA HANDBOOK 1200.12, USE OF DATA AND DATA REPOSITORIES IN VHA 
RESEARCH Dated March 9, 2009 
 
All data collected will be the property of the Department of Veterans Affairs whether in paper or 
electronic form and will be secured utilizing the following methods: 
 
Paper:  
• 
All paper documents will be stored on VA property unless authorized by the Director, 
Information Security Officer, and Privacy Officer in writing.  
• 
All paper documents will be locked in an approved file cabinet with only members of the 
research team having access.  
 
Electronic: 
• 
All information in electronic form will be stored on VA servers behind the VA firewall. 
• 
All portable media will meet or exceed FIPS 140-2 compliance and encryption. And will 
be approved in writing by the Information Security Officer.  
 
The location(s) where information will be stored is (are) Building 35, rooms 19 and 39 
The people or agencies that will have access to the information are the study investigators. 
 
No information related to this research will be released to any third party or disclosed outside of 
the VHA – except as required or permitted by law. 
 
 
Research records will have identifiers removed and will be stored with a code number linked to 
subjects.  The code will not be derived from any personal identifiers.  The key to the code will be 
kept in a locked file cabinet, located in Bldg. 35, room 19. Subjects’ identity will not be revealed 
in any reports or publications resulting from this study. 
15. Location of Study: This study will be conducted in Ward G9W (the Biostudies Unit) located 
in Building 1 and in Bldg. 36 room 116 at the West Haven VA Medical Center. 
 


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16. Payment: Subjects will be paid cash; $30 for participating in the screening, $5.00 per phone 
call for approximately 28 phone calls, $40 for each of the 2 outpatient visits and $200 for each of 
the first test sessions and $250 for the second  test session. In addition, to help offset 
transportation costs, subjects will be paid $40 for each of the 2 test sessions. If the subject is 
asked to return in addition to these visits for any reason, they will be paid $20 for travel. If the 
subjects choose to terminate a session prematurely, or a session is terminated early for medical 
reasons, they will receive full payment for that day. If they become ineligible to continue in the 
study due to non-compliance with study procedures, they will only be paid for the portions of the 
study in which they have participated. Subjects may be paid up to $700.00 if all parts of the 
study are completed. Payment may exceed $700.00 if the subject is asked to return in addition 
for any reason. The payment will increase by $20 for each additional visit. 
Visits 
Amount paid 
Total  
Screening Visit 
$30.00 
$30.00 
Phone checks  
$5.00 per phone call or 
message 
28 phone calls= $140.00 
Outpatient visits  
$40.00 each visit  
2 outpatient visits: $80.00 
Test sessions  
$200.00 for the first test 
session 
$250.00 for the next test 
sessions 
$200.00 for first Test session  
$250.00 for next Test session 
= $450 
Travel to test sessions 
40.00 for each test session 
Travel to 2= $80.00 
 
 
Max Total: $780.00 
If subject is asked to return 
for additional testing or visit  
$20 for travel  
$20.00 
 
 
17. Funding Source: An R03 DA043004-01 grant from NIDA (pending). 
18. Duration: The entire study will take approximately 2 years to complete. 
 
 
 
 
 
 
 
 
 
 
    


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