Clinical trial NCT04161144
Conditions: Smoking Cessation Smoking, Cigarette Nicotine Dependence
Sponsor: Medical University of South Carolina
Start date: 2018-10-01; Completion date: 2019-12-20
Enrollment: 22 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to examine the effects of rapamycin (sirolimus) versus a placebo, an inactive substance, on responses to smoking cues in individuals with nicotine dependence. Rapamycin (sirolimus) is a FDA-approved antibiotic and immunosuppressive drug that is currently used to (a) prevent organ transplant recipients from rejecting their transplants (b) treat cardiovascular diseases, and (c) treat some forms of cancer. Rapamycin (sirolimus) is not FDA-approved for smoking cessation. The use of rapamycin (sirolimus) in this study is investigational, meaning that the study medication is not a proven treatment for nicotine dependence, however this study will examine the medication's use as a potential future treatment for nicotine dependence.

Detailed description:
The proposed study will employ 58 ND treatment-seeking smokers who will be randomly assigned to receive either 15-mg of sirolimus or placebo (group n's=29) immediately after the first of two smoking cue exposure sessions scheduled to occur on consecutive days. The first session will serve as a Retrieval session during which smoking (e.g., handling and lighting of a cigarette) cue exposure will elicit retrieval and reconsolidation of smoking-related memories; the second session will be a Test session to examine the potential modulatory role of sirolimus on the reconsolidation of memories putatively elicited during the retrieval session. Participants will be required to refrain from smoking the night before (bedtime) their first laboratory (i.e., Retrieval) session and will remain abstinent from smoking until the completion of the second laboratory (i.e., Test) session. It is posited that changes in reactivity during the test session will reflect medication effects on memory reconsolidation that occurred following the retrieval session. Measures of subjective responses (i.e., craving) and physiological reactivity (i.e., heart rate \& skin conductance) will be obtained before, during and after cue presentations in both sessions. The durability of any observed treatment effects will be assessed in a Follow-up session performed 7-days following completion of the test session. Treatment effects on self-report measures of smoking behavior during the 7-days preceding the Follow-up session will also be assessed.

Arms: arm 1: Participants will receive three 5mg pills from a nurse in the Research Nexus, some participants will receive the study medication and others will be randomized to the placebo control group. arm 2: Participants will receive three 5mg pills from a nurse in the Research Nexus, some participants will receive the study medication and others will be randomized to the placebo control group.
Interventions: intervention 1: sirolimus intervention 2: Placebo
Intervention descriptions: intervention 1: Rapamycin (sirolimus) is administered in three 5mg oral capsules. This administration happens once during the first visit. intervention 2: Placebo is administered in three 5mg oral capsules. This administration happens once during the first visit.

Protocol documents:
Prot_SAP_000.pdf: 
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 Cover Page  
 
 
 
 
Study Short Title: Enhancing Self-Regulation Among Smokers 
(Pro00076669) 
NCT04161144 
4/29/2019 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


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Enhancing self-regulation by altering memories that increase risk of relapse among smokers:  A 
translational clinical neuroscience study of a novel medication 
 
PI: Dr. Michael Saladin  
 
I. Objectives/Specific Aims 
Smoking-related nicotine dependence (ND) is the single greatest cause of addiction-related morbidity and 
mortality. Current pharmacological treatments have improved cessation rates but there is still considerable room 
for improvement. The impetus for the proposed translational HCC Team Science study (Project 2) comes 
primarily from recent neuroscience research demonstrating that the FDA-approved protein synthesis inhibitor 
rapamycin (henceforth sirolimus), administered after the reactivation (i.e., recall) of memories for learned 
associations, can result in disrupted memory reconsolidation. Importantly, the behavioral consequence of 
disrupted memory reconsolidation is diminished reactivity to stimuli/cues that previously controlled robust 
behavioral activation.  
 
The well-known ability of smoking cues (e.g., sight/smell of cigarettes) to elicit craving and other bodily 
reactions in smokers attempting to quit has long been assumed to result from learned associations that develop 
over a smoker’s extensive history of pairings between the cues and the appetitive pharmacological effects of 
nicotine. For smokers, the challenge of quitting requires that they deploy a range of self-regulatory processes 
(e.g., control the impulse to smoke) in order to sustain abstinence. Not surprisingly, encounters with smoking 
cues elicit strong cravings that represent a significant obstacle to successful self-regulation, which often elevates 
relapse risk. To address the threat to self-regulation and cessation posed by memories for these learned 
associations, the overarching goal of this project is to strategically employ sirolimus to attenuate smoking-related 
memories, thereby decreasing cue-elicited craving, physiological reactivity and smoking.  
 
The proposed study will employ 58 ND treatment-seeking smokers who will be randomly assigned to receive 
either 15-mg of sirolimus or placebo (group n’s=29) immediately after the first of two smoking cue exposure 
sessions scheduled to occur on consecutive days. The first session will serve as a Retrieval session during 
which smoking (e.g., handling and lighting of a cigarette) cue exposure will elicit retrieval and reconsolidation of 
smoking-related memories; the second session will be a Test session to examine the potential modulatory role 
of sirolimus on the reconsolidation of memories putatively elicited during the retrieval session. Participants will 
be required to refrain from smoking the night before (bedtime) their first laboratory (i.e., Retrieval) session and 
will remain abstinent from smoking until the completion of the second laboratory (i.e., Test) session. It is posited 
that changes in reactivity during the test session will reflect medication effects on memory reconsolidation that 
occurred following the retrieval session. Measures of subjective responses (i.e., craving) and physiological 
reactivity (i.e., heart rate & skin conductance) will be obtained before, during and after cue presentations in both 
sessions. The durability of any observed treatment effects will be assessed in a Follow-up session performed 
7-days following completion of the test session. Treatment effects on self-report measures of smoking behavior 
during the 7-days preceding the Follow-up session will also be assessed. 
 
Primary specific aim and hypothesis: 
Aim: Evaluate the effects of post-retrieval sirolimus vs. placebo on measures of craving and cue reactivity 
administered 1 day and 1 week following the medicated Retrieval session. 
Hypothesis: Compared to placebo treated smokers, sirolimus-treated smokers will evidence less craving, 
emotion/arousal and physiological reactivity to smoking cues presented during the (a) Test session and (b) 
Follow-up session. 
 
Secondary specific aim and hypothesis: 
Aim: Evaluate the effects of post-retrieval sirolimus vs. placebo on smoking behavior occurring during the 
one-week follow-up period. 
Hypothesis: Compared to placebo-treated controls, sirolimus-treated smokers will evidence changes in 
smoking behavior during follow-up as indicated on multiple measures including (i) mean cigarettes per day, 
(ii) days to first cigarette, (iii) percent days non-smoking during follow-up.  
 
Since most smoking relapse occurs in the first week of cessation, a pharmacological aid that decreases 
craving and cue reactivity may make the difference between successful self-regulation and cessation vs. failed 
self-regulation and relapse. Favorable outcomes from this study could lead to a new generation of treatment 
adjuncts that would be brief, easy to administer and cost effective. These treatments could also become a 
complementary treatment paradigm, operating synergistically with the current generation of pharmacotherapies 


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and/or cognitive behavioral treatment approaches to reduce cue-elicited craving in nicotine addicted smokers. 
Because smoking is a primary causal factor in lung and other cancers, this translational, treatment-focused 
project has the potential to significantly advance cancer prevention and control. 
 
II. Background 
A. Significance:  
Conceptual Overview: The role of basic associative learning processes in addiction has been well established 
in both theory and research1-8. In smokers, numerous pairings between smoking-related cues (e.g., pack of 
cigarettes) and the reinforcing effects of nicotine can result in cues acquiring the ability to elicit a range of 
conditioned responses, most notably craving and physiological arousal. Importantly, there is substantial evidence 
that craving is powerfully associated with smoking relapse9-15. One viable explanation for this association is that 
craving undermines the self-regulatory processes (e.g., impulse control, emotion regulation, distress 
tolerance16-18) necessary to initiate and maintain cessation. Consistent with this conceptualization are the findings 
of neuroimaging studies suggesting that failures to self-regulate may occur when connectivity between prefrontal 
control regions (e.g., lateral prefrontal cortex) and subcortical reward regions (e.g., ventral striatum) are 
diminished or otherwise impaired17. Given this conceptual model, there are at least two approaches by which 
impaired self-regulation could be treated: a) restore/strengthen prefrontal-subcortical connectivity (see Project 
1), 
and 
b) 
diminish 
the 
motivational 
properties 
of 
smoking-related cues. Project 2 
will adopt the latter of these two 
approaches by using an existing 
FDA-approved 
medication 
rapamycin (hereafter sirolimus) to 
weaken memories for the learned 
associations between smoking-related cues and nicotine reward, thereby diminishing craving and smoking 
behavior (cf., Figure 1). The supporting evidence and rationale for the Project 2 is detailed below.    
Scientific Premise: New learning is said to become stable in memory via the process of consolidation19-23. 
Reconsolidation refers to a process in which retrieval of consolidated memories begets a period of instability 
during which the memories can either be strengthened or otherwise altered and then are restabilized in long-
term storage24-30. Generally, reconsolidation begins with memory retrieval, which is initiated by the presentation 
of cues that elicit a network of target memories. There is a large body of basic neuroscience research27,31,32 
demonstrating that reconsolidation of memories for both appetitively- and aversively-motivated learning can be 
pharmacologically disrupted, leading to a decrement in, or near eradication of, behavior supported by the original 
learning. By contrast, a small body of human fear conditioning studies, using the -adrenergic antagonist 
propranolol as the disrupting agent, has yielded findings that parallel those of the animal literature33-37. 
Translational studies targeting clinical anxiety and addictive disorders are also few in number and have focused 
almost exclusively on propranolol as a disruptive agent. In the case of anxiety disorders, three published reports 
have yielded suggestive evidence of attenuated trauma-related memories in PTSD, as indicated by decreased 
emotional responsiveness and PTSD symptomatology38-40. Our research group was the first to study 
propranolol’s effects on memories for important addiction related-learning; we found that a single dose of 
propranolol following memory retrieval (via cocaine cue presentation) resulted in attenuated cocaine craving and 
physiological reactivity during a test performed the following day41. However, a recent study42 involving smokers 
failed to find any effect of propranolol on craving, emotional and physiological reactivity to smoking cues 
presented in a test performed 1-week post-treatment. These contrasting findings tentatively suggest that a single 
disrupting agent may not have uniform effects across all addictive disorders and that reconsolidation disruption 
in smokers might be more profitably pursued with a pharmacological agent other than propranolol.   
One candidate agent that has been used in animal studies to demonstrate disruption of reconsolidation (DoR) 
is the protein synthesis inhibitor, anisomycin26,43-45. While robust DoR effects have been observed with 
anisomycin, they have not been replicated in humans because of concerns about toxicity46. However, there are 
three emerging lines of evidence suggesting that the FDA-approved, protein synthesis inhibitor rapamycin 
(henceforth sirolimus) may be as effective a disrupting agent as anisomycin. First, animal studies employing fear 
conditioning procedures have reported that either systemic or intra-amygdalar injection of rapamycin can 
substantially disrupt reconsolidation of contextual and discrete cue fear memory47-50. Second, it has been shown 
that systemic sirolimus administration following re-exposure to a drug-paired environment results in lasting (14 
days) decrements in morphine-, cocaine- and alcohol-reinforced place preferences, which could not be 
reinstated with priming drug injections51. Additionally, a recent Nature Neuroscience report showed that either 
systemic or intra-amygdalar administration of sirolimus after retrieval of alcohol-related memories substantially 


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impaired relapse to alcohol self-administration for up to 14 days; these effects were similar to those observed 
when anisomycin served as the DoR agent52. Lastly, a clinical study employing Vietnam era and post-Vietnam 
era war veterans has provided the first evidence that a 15-mg dose of sirolimus vs. placebo administered 
contiguously with recall (retrieval) of war-related trauma resulted in reduced PTSD symptom score at a 1-month 
follow-up assessment, albeit only in the post-Vietnam era war veterans and not at 3-month follow-up53. 
Importantly, the authors of the study reported that no adverse medical outcomes or side-effects occurred during 
the course of the study. Collectively, these studies indicate that systemic or intracranial sirolimus (i) can 
effectively disrupt memories for both fear-based and a broad range of drug-reinforced learning and that this effect 
may be long lasting and comparable to anisomycin-induced DoR, and (ii) may be able to attenuate clinically 
important memories in the absence of side-effects.  
While sirolimus’ exact mechanism of action is unknown, it is likely that it achieves DoR by inhibiting the 
mammalian/mechanistic target of rapamycin (mTOR) kinase, which regulates phosphorylation of a large number 
of intracellular targets responsible for protein synthesis and translation54-58. Since rapamycin is a safe medication 
with minimal side effects, there are no obstacles to initiating research with human participants. Accordingly, the 
proposed research will evaluate the novel hypothesis that the strategic, post-retrieval administration of sirolimus 
can disrupt reconsolidation of memories for cue-elicited craving in smokers.  
B. Innovation:  
The hypothesis being tested is highly innovative in that it draws on an established basic neuroscience 
phenomenon, disruption of memory reconsolidation (DoR) via protein synthesis inhibition, and seeks to 
document its potential treatment utility in nicotine addicted smokers. To our knowledge, no other research group 
is evaluating this highly innovative and potentially important hypothesis. Since the study focuses on the alteration 
of memory for appetitive addictions-related memory, it could lead to the development of an entirely new 
intervention strategy that will significantly advance relapse risk reduction in smokers. Positive findings from this 
study could stimulate treatment development for a broad spectrum of substance abusing populations. 
Accordingly, the proposed research fits well with NIDA’s mission to stimulate clinical neuroscience research that 
focuses on the neurobiological mechanisms of addiction and its treatment.     
The proposed research is also novel because it is translational: it uses basic neuroscience findings as a 
springboard for the development of novel medication that targets the dampening of the motivational properties 
of memories for cue-elicited learning involved in the etiology/maintenance of smoking-related ND. The proposed 
study also challenges an existing paradigm in that it focuses on altering memories for “pathological” learning that 
subserves smoking-related ND rather than the establishment of new, extinction-based learning to oppose it. The 
overarching hypothesis is that attenuation of these memories should decrease smoking-related behavior 
including craving and cue reactivity, thereby potentially enhancing smoking outcomes.   
 
III. Inclusion and Exclusion Criteria/Study Population:  
A total of 58 treatment seeking ND men and women smokers (29 per group), aged 18 or older, will be 
randomized into the study. Participants must smoke 10+ cigarettes/day for three or more years, have a 
Fagerström Test for Nicotine Dependence score  4, be willing to make a cessation attempt, and comply with 
reinforced abstinence requirements for the three laboratory sessions described below. Participants must (i) not 
be dependent on other substances (may meet criteria for abuse), (ii) willing to use appropriate birth control 
methods during study participation including oral contraceptives, barrier methods (diaphragm or condoms with 
spermicide or both), surgical sterilization, use of an intra-uterine contraceptive device, or complete abstinence 
from sexual intercourse, (iii) remain abstinent from alcohol and all non-prescription drugs prior to medication 
administration and testing sessions, (iv) not be undergoing other smoking cessation treatment (e.g., nicotine 
replacement, Chantix), (v) not be taking medications that may interact with the study medication or alter 
responding on any study measure, (vi) live with within a 50 mile radius of our research program and have reliable 
transportation, (vii) consent to fast for a two-hour period prior to medication administration, and (viii) consent to 
random assignment to rapamycin (sirolimus) vs. placebo conditions.  Participants will be excluded if they (i) are 
undergoing other smoking cessation treatment (e.g Chantix, nicotine replacement), (ii) are pregnant, nursing, or 
of childbearing potential and not using birth control, (iii) present evidence of or a history of significant endocrine, 
cardiovascular, pulmonary, renal or neurological disease, as these conditions may affect heart rate or skin 
conductance measurement, (iv) have significant liver impairment (as indicated by alanine aminotransferase 
(ALT) and/or aspartate aminotransferase (AST) values that are three times the upper limit of normal) as 
rapamycin (sirolimus) is hepatically metabolized, (v) have an existing infection or immune system disorder as 
rapamycin (sirolimus) has known immunosuppressive properties, (vi) have a history of or current psychotic 
disorder, severe major depression as evidenced by active and profound psychomotor retardation and/or 
persistent and intense suicidal ideation, (vii) are currently taking anti-arrhythmic agents, psychostimulants or any 
other agents known to interfere with heart rate and skin conductance monitoring, (viii) have known or suspected 


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hypersensitivity to macrolide compounds (such as rapamycin/sirolimus), (ix) are taking medications that could 
adversely interact with study medication including but not limited to significant inhibitors of CYP2D6 or CYP3A4 
(voriconazole, fluconazole, itraconazole, erythromycin, clarithromycin, diltiazem, verapamil, etc.) or significant 
inducers of CYP3A4, such as anticonvulsants (carbamazepine, phenobarbital, phenytoin, etc.) and antibiotics 
(rifabutin, rifapentine, etc.),  (x) have a history of thrombocytopenia, idiopathic thrombocytopenia purpura (ITP), 
or with a current platelet count of less than 100,000 cells per mm3, (xi) have any unhealed wounds, including 
but not limited to oral ulcers, foot ulcers, or recent surgical or trauma wounds, and (x) have any planned surgeries 
within the next month, including surgical dental procedures.  
Determining Eligibility. A brief phone screening will preliminarily assess participant suitability via 
inclusion/exclusion criteria. Qualifying individuals will be scheduled to undergo an IRB-approved informed 
consent procedure. After consent, all female participants will undergo a urine pregnancy test to confirm a 
negative test result. Any female participant with a positive urine pregnancy test result will be immediately 
excluded from the study.  Participants will also provide a urine sample for a urine drug screen, a breath sample 
for a breathalyzer, and breath sample to measure carbon monoxide in the participants’ breath.  Any participant 
found to be dependent on other substances or have a CO breath sample indicating a nonsmoker (< 3ppm) will 
be excluded from the study.  (Participants can continue in the study if they test positive for cannabis/marijuana 
use, but not if they meet criteria for moderate or severe substance use disorder for cannabis/marijuana.)  The 
remainder of the in-person assessment will consist of a comprehensive assessment of substance use, nicotine 
dependence and general psychiatric functioning. It will include the following: Fagerström Test for Nicotine 
Dependence (FTND)61; Mini-International Neuropsychiatric Interview or MINI from the DSM-V62,63. The Timeline 
Follow-Back (TLFB; a calendar-based instrument with specific probes to obtain detailed information about 
substance use)64 will be used to assess smoking and other substance use (a) during the three month period 
preceding to study involvement, and (b) during the week preceding the Follow-up laboratory session. All 
participants will undergo a history and physical exam. This includes an electrocardiogram and blood chemistries, 
which will be performed by the SCTR Research Nexus. With the blood sample, a complete metabolic panel and 
a complete blood count (without differential) will be performed to ensure participants are medically fit to 
participate in the study. 
 
IV. Number of Subjects 
We expect to recruit 58 participants, equally allocated to treatment and placebo groups during the active 
treatment phase of the study. While literature is lacking on studies of the proposed treatment, we used data from 
our previous and ongoing work to derive an estimated standard deviation of 2.1 for single-item craving rating. 
Using this estimate and fixing power at 80% and =.05, we can expect to detect a minimum mean craving rating 
difference between the two treatment groups of 1.7 with 25 participants randomized to each treatment group 
(n=50). Although some attrition is expected, the proposed study uses a participant compensation strategy (see 
below) that is designed to minimize attrition. This strategy was based on one used in our previous NIDA-funded 
study of treatment-seeking smokers59. In this study, we found 5% attrition occurs between two contiguous 
laboratory sessions where abstinence was required and 15% attrition at 2-week follow-up. Extrapolating to the 
proposed study, we are confident that 29 participants randomized to each study group (n=58) will provide 
sufficient power to evaluate expected group differences in this proof of concept study.  
 
V. Setting 
 The research team will be conducting the research at the Research Nexus and the Addictions Studies suite 
of MUSC.  Both locations have individual rooms for privacy.  The Research Nexus has a specific room set up for 
collecting skin conductance and heart rate from the participants while simultaneously allowing the research 
assistant to monitor them.    
 
VI. Recruitment Methods 
Participants will be primarily recruited using local media (e.g., print, online, radio), a recruitment strategy that 
has been very successful in our previous and ongoing studies of ND smokers. We may also elect to recruit from 
the Medical University of South Carolina’s (MUSC) Center for Drug and Alcohol Programs (CDAP).  
 
VII. Consent Process 
The study PI, Co-I or other research staff will obtain informed consent. The informed consent form includes 
a detailed description of the study procedures, along with statements regarding participants’ rights to withdraw 
from the procedure at any time without consequences. The informed consent form will be explained to 
participants in easy-to-understand language, and participants will be instructed to read the form carefully prior to 


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signing it. Consent will be documented by the signature of the participant on the informed consent agreement, 
accompanied by the signature of the individual obtaining the consent.     
 
VIII. Study Design/Methods 
A. Screening and Diagnostic/Abstinence Assessment: 
Screening.  All participants will be screened in two parts.  First through a phone screen followed by an in-
person screening.  The screenings are both detailed above in the “determining eligibility” section.  
Smoking and Other Drug Abstinence Assessment. Participants will be required to remain abstinent from 
smoking beginning at bedtime on the night before the (first) Retrieval cue exposure session and remain abstinent 
through the completion of the (second) Test session. Participants failing to meet the CO criterion at the Retrieval 
session will be allowed to reschedule their participation one time; however, if a participant fails the abstinence 
assessment on the second day, they will be dropped from the study because abstinence violators would be 
different from abstainers (a) with respect to the reconditioning of the putative learning/memory processes under 
study, and (b) amount of time elapsing between the Retrieval and Test sessions. Lastly, there will be no 
abstinence requirement for the 7-day period between the Test and Follow-up sessions; however, participants 
will be required to abstain from smoking starting at bedtime the night before the Follow-up session.  
Briefly, participants will be informed that their participation (and associated compensation) in any given 
laboratory session will necessitate that they provide a breath sample that conforms with a CO criterion of  10 
ppm. The full schedule of compensation is described in section F below. 
Additionally, abstinence from alcohol and other drug use prior to all laboratory sessions will be confirmed via 
expired breath alcohol (BAC) and urine drug screen (UDS), respectively. A positive test will result in 
rescheduling/termination as described above for smoking; however, participants may test positive for 
cannabis/marijuana and continue to participate.  
B.  Cue Reactivity Session Measures: (This team has extensive experience using all listed measures). 
Self-Report Measures: The key measure that will be used to quickly and unobtrusively assess smoking craving 
is: single-item verbal report of subjective craving to smoke. Prior to any stimulus presentation, participants will 
be trained to provide a verbal report of a numeric value between 0 (none) and 20 (extreme) that best represents 
their current level of craving (“My urge to smoke right now is”). In addition to single item self-reports, brief multi-
item assessments of craving to smoke and emotion will be performed: The Questionnaire of Smoking Urges- 
Brief (QSU)65 is a 10-item self-rating questionnaire for assessment of reward-related and withdrawal-related 
craving. The Mood Form66 is a 9-item immediate assessment of current negative and positive emotional states.  
In the interest of harmonizing measures across projects, we will obtain several additional measures including 
the Cognitive Emotion Regulation Questionnaire (CERQ), Profile of Mood States (POMS), Sensitivity to 
Punishment and Sensitivity to Reward Questionnaire (SPSRQ), Barratt Impulsiveness Scale (BIS) and the 
Toronto Alexithymia Scale (TAS-20). 
Physiological Measures: Heart rate and skin conductance will be measured as indices of physiological 
arousal during each cue exposure session. Heart rate (HR) will be collected via two electrodes along the bottom 
of the participant’s ribcage, rather than on the participant’s forearm, to minimize movement artifacts. Skin 
conductance (SC) will be recorded using Ag/AgCl electrodes attached to the second phalanx of the first and third 
fingers of the non-dominant hand. HR and SC signals will be amplified using ECG 100c and GSR 100c Biopac 
Modules and interfaced with the Biopac MP100 data acquisition system. 
C.  Smoking Cue Reactivity Stimuli: 
Because we have been successful eliciting smoking craving in previous work with ND smokers67, we will 
adopt a similar strategy here. In particular, smoking cues will consist of the handling and lighting of the 
participant’s preferred brand of cigarettes. Participants will be instructed to (a) look at the cigarette pack/lighter, 
(b) take one cigarette from the pack and hold it in hand as they normally do, (c) handle/smell the cigarette, (d) 
pick up the lighter and flick the lighter without actually lighting the cigarette, and (e) light the cigarette without 
smoking and then extinguish it. This exposure sequence will take approximately 4 minutes.  
D.  Randomization, Rapamycin Dosing, Preparation and Administration: 
Randomization. Stratified block randomization will be used to assign participants to groups while balancing 
treatment assignment based on gender and Fagerström Test for Nicotine Dependence (FTND) score.  Both are 
known to influence smoking cue reactivity and/or behavior.  Dependence will be balanced on FTND score ≤5 
or >5. This method, when used with smaller block sizes, is appropriate for small study sample sizes68 and has 
been used in a number of previous and ongoing studies by our research group.  
Sirolimus Dosage Rationale and Potential Side Effects. The decision to employ a 15-mg dose of rapamycin 
(sirolimus) in the proposed study was based on the following rationale. First, as noted above, Suris et al, 2013 
employed 15 mg of rapamycin to disrupt trauma-related memories in combat veterans. The study results 
suggested, in a subsample of non-Vietnam era veterans, that 15 mg of rapamycin vs. placebo administered in 


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conjunction with trauma memory reactivation was associated with decreased PTSD symptomatology at a 1-
month follow-up assessment. Importantly, they also documented that this dose was not associated with any 
adverse medical consequences or side effects.  
There is strong animal and clinical pharmacokinetic (PK) and pharmacodynamic (PD) data to demonstrate the 
proposed 15-mg dosing regimen will demonstrate clinically relevant outcomes. Results from a number of clinical 
PK studies demonstrate that sirolimus is rapidly absorbed after oral ingestion, with peak whole blood 
concentrations occurring 1.0 to 1.3 hours after administration69-71. Additionally, sirolimus adequately crosses the 
blood brain barrier, with an estimated blood to brain ratio of 1:372. In a clinical PK study in health males, an 8 
mg/m2 one-time oral dose produced a whole blood Cmax of 115.2 ng/mL at 1.0 hours after ingestion. This 
corresponds to an approximate peak brain concentration of 38.4 ng/mL60. Additionally, the organ transplant 
animal and clinical data strongly support the proposed dosing strategy. Studies conducted in rats using sirolimus 
to prevent organ rejection utilized dosing ranges between 0.5 to 50 mg/kg per day73. However, maximum 
pharmacodynamic activity with sirolimus monotherapy in rat kidney and heart transplant models was 
demonstrated with a dose of 8 mg/kg intravenously. This dose correlated with a mean AUC0-24h of 27012 
g/L*h74. By using the trapezoidal rule, the AUC0- in rats for an 8 mg/kg dose would approximate 350 g/L*h. 
This dose of rapamycin (sirolimus) also demonstrated maximal lymphocyte protein kinase inhibition at 2 hours 
post-dose (i.e., maximal protein synthesis inhibition), with return of full function occurring at 12 to 24 hours post-
dose. Therefore, based on the above noted literature, a one-time 15-mg dose should produce substantial protein 
kinase inhibition in the brain within 1-1.3 hours of administration, while also being well-tolerated, with mild-
transient side effects. The current clinical use of sirolimus in organ transplantation also supports using the 15-
mg oral dose in the proposed study. The pivotal trials used to gain approval for sirolimus in transplantation had 
an arm that received a loading dose of 15 mg orally during the perioperative period75-77.  
Lastly, we recently received (January 2018) both Investigational New Drug (IND) approval from the FDA and 
MUSC IRB approval for a similar study involving a single 15-mg administration of sirolimus in individuals who 
misuse alcohol.   
The recently-updated labeling for sirolimus (dated 1/2018) includes warnings regarding health consequences 
associated with sirolimus use. These warnings include increased susceptibility to infection and possible 
development of lymphoma due to the medication’s immunosuppressive properties. The warnings that are 
potentially applicable to this protocol also include hypersensitivity reactions, angioedema, impaired or delayed 
wound healing, increased risk of opportunistic infections, and embryo-fetal toxicity. 
However, many of these health consequences were observed with regular sirolimus use rather than a one-
time dose, as will be the case in this study. Participants who are not willing to use an effective form of birth control 
during the course of the study and for twelve weeks after will be excluded from participating given the risk of 
embryo-fetal toxicity. Female participants will be pregnancy tested twice (including once on the same day as 
medication administration) and immediately excluded if the result is positive in order to avoid the risk of embryo-
fetal toxicity. Participants will receive a maximum of 15mg of sirolimus under medically supervised conditions 
Medication Preparation & Administration. Sirolimus (15-mg) and placebo will be compounded, packaged and 
dispensed by the MUSC Investigational Drug Service (IDS). The IDS and study Statistician (Baker) will oversee 
the randomization procedures. Immediately after the cue exposure in the Retrieval session, Sirolimus or a 
matching placebo will be administered.  
E.  Laboratory Session Procedures: 
Session Preparation. Abstinence will be assessed (see above A, Abstinence Assessment) at each laboratory 
session visit. If abstinence is confirmed, participants will be escorted to MUSC’s CTRC waiting room where they 
will remain until the laboratory is ready for cue exposure administration (approximately noon). Next, they will be 
escorted to the laboratory where they will sit quietly/read in order to acclimate to the environment until the cue 
exposure procedures begin. Following the acclimation period, HR and SC sensors will be placed, sound-
attenuating headphones will be fitted, and baseline measures of subjective and physiological measures will be 
collected (see GENERAL PROCEDURES TABLE below).    


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Retrieval session 1 procedure. The primary objective of this session is to elicit the retrieval of smoking-
related 
memories. 
To 
achieve 
this 
objective, 
participants 
will 
be 
exposed to the smoking 
cues as described above 
(view, handle, light and 
extinguish the cigarette). A 
craving 
rating 
will 
be 
obtained midway through 
the smoking cue exposure 
(i.e., the inquiry will be 
made by research staff via headphones). Immediately following smoking cue presentation, participants will 
provide the craving rating and complete the other study measures.  Participants will receive medication 
immediately after the measures have been obtained. Collection of all study measures will occur every 10 min 
after medication administration and on one final occasion at 50-min post-medication (see GENERAL PROCEDURES 
TABLE above). Although HR and SC will be collected continuously during the smoking cue exposure, only single 
time point measures will be collected thereafter. Possible medication side effects will be assessed using the 
Monitoring of Side Effects Scale (MOSES) and will be managed by a study physician (Gray) if necessary. 
Approximately one hour after medication administration, participants will have blood drawn for the purpose of 
determining sirolimus levels; this will permit subsequent assessment of the association between blood levels of 
sirolimus and primary/secondary outcomes. Immediately after the blood draw, participants will be asked to 
complete a brief questionnaire that asks whether or not they thought they received study medication or placebo. 
Lastly, the participant will be reminded of the smoking abstinence requirement and instructed not to smoke or 
use other substances (abstinence serves to mitigate the confounding reconditioning effects of smoking on the 
reconsolidation processes initiated in this session). 
Test session 2 procedure. This session provides an opportunity to test the hypothesis that rapamycin will 
acutely attenuate craving and physiological reactivity to smoking cues. Test session 2 will be identical to session 
1 with the following exceptions. First, no medication will be administered. Second, at the end of the session, 
participants will receive a smoking diary in which to record the occurrence of any smoking behavior over the 
week follow-up period (abstinence is not required during the week preceding Follow-up session). Finally, the 
experimenter will provide compensation and schedule the Follow-up cue reactivity session. 
Follow-up session procedure. This session affords the opportunity to assess the maintenance of any effects 
observed in Test session 2 and the effects of treatment on smoking behavior. In the Follow-up session,  
participants will experience the same session preparation and Retrieval session 1 procedures as described 
above with the following exceptions. First, no medication will be administered. Second, smoking diary data will 
be collected (research staff will perform a TLFB assessment of smoking behavior if participants do not present 
with their smoking dairy). Finally, debriefing will take place to address questions or concerns that participants 
may be experiencing (blinding will be maintained). Referral for additional cessation treatment will be provided.  
F. Timeline: 
Since we have ongoing studies with a variety of substance using populations, an active recruitment network 
and extensive experience using cue reactivity paradigms, we anticipate start up to occur in approximately 4-
months. During this period, research staff will be hired and trained, the protocol will be IRB approved at MUSC, 
an IND application will be approved by the FDA, and laboratory procedures and database(s) will be established. 
We will actively recruit participants for 18 months and plan to allow two months for final data cleaning/reduction, 
analysis and manuscript preparation (albeit manuscript preparation will begin sooner). At a recruitment rate of 
approximately 2-3 participants per month, we should have no difficulty in completing the study in the two-year 
timeframe. With regard to recruitment milestones, we anticipate being able to recruit approximately 26 ND 
smokers in the 8-month recruitment period of the first year and the remaining 32 participants in the 10-month 
recruitment period of the 2nd year. 
G. Participant Compensation:   
The compensation strategy for this study is designed to (i) provide remuneration for participant time and 
effort, (ii) maintain adherence to laboratory session schedule, and (iii) minimize abstinence violation and 
participant attrition. We have used this compensation strategy in a previous study (noted above) involving 
treatment-seeking smokers and have had very high levels of compliance with smoking and other substance 
abstinence.  


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Participants will be compensated as follows: Screening and assessment = $50.00; Lab sessions 1 & 2 
(Retrieval and Test) = $75.00 each; 1-week Follow-up session = $100.00. Maximum compensation for 
participation is $300.00.  
 
IX. Data Management 
Primary Hypothesis. Mixed effects models will be used to test the hypothesized group differences at the Test 
(acute effect) and 1-week Follow-up (maintenance effect) laboratory sessions. Models will be developed to 
additionally assess the effects of potential covariates (e.g., age) on the single item craving outcomes and 
possible effect modification (through interaction terms).  Other self-report measures (e.g., QSU, affect ratings, 
etc.) and HR/SC measures will be analyzed in a similar manner. Additional measures of craving, such as Area 
Under the Curve (AUC) and/or maximum within-session decrement in craving may be considered as outcomes. 
It may also be of interest to fit a covariance pattern model, analyzing craving over time, while accounting for 
treatment and session (Retrieval, Test & Follow-up Sessions). Secondary Hypothesis.  Treatment group 
differences on various indices of smoking behavior (time to first cigarette, % days abstinent, CPD) will be 
assessed using general linear regression models while controlling for potential covariates. Continuous outcomes 
(% days abstinent, CPD) will be analyzed using normal linear models while time to first cigarette will be assessed 
using time to event models for efficacy analysis (Log-rank, Cox PH Models).  Binary outcomes will be assessed 
using Logistic regression models. While only large group differences are likely to be statistically detectable, 
analysis of smoking behavior is consistent with the interventional focus of this research and will provide efficacy 
estimates to inform sample size estimation for a larger study. 
 
X. Provisions to Monitor the Data to Ensure the Safety of Subjects 
Data and Safety Monitoring Board.  A Data and Safety Monitoring Board (DSMB) will be created to ensure 
the safety of participants and the validity and integrity of data collected during the project.  The board will consist 
of Drs. Hugh Myrick, Bryan Tolliver and Karen Hartwell, all board-certified addiction psychiatrists, and Royce 
Sampson, R.N., MSN, the Director of the Regulatory Core for the CTRC. The board will meet as needed to 
review the adverse events related to the study, and recommendations for appropriate action to maintain a 
favorable safety profile will be made. The board will also ensure that all serious adverse events have been 
followed to resolution, and that the appropriate agencies (including the IRB and internal funding agency) have 
been informed of the event. 
 
Adverse Event Monitoring 
Adverse events will be monitored throughout the study and all events will be followed to resolution or 
stabilization. All serious adverse events will be collected and reported immediately to the IRB and the internal 
funding agency (Hollings). Serious adverse events related to rapamycin (sirolimus) will be reported to the FDA.  
A serious adverse event is one that meets any of the following criteria: 
1. Fatal or life threatening 
2. Requires or prolongs inpatient hospitalization 
3. Results in persistent or significant disability/incapacity 
4. Congenital anomaly 
5. Medical event that may jeopardize the participant or require intervention to mitigate serious outcome 
6. Cancer 
7. Overdose 
8. Results in the development of drug dependency or abuse. 
 
XI. Risks to Subjects 
Rapamycin (Sirolimus; Rapamune®) Dosage and Potential Side Effects. The decision to employ a 15 mg 
dose of rapamycin (sirolimus) in the proposed study was based on the following rationale. First, as noted in the 
main application, there was a dosing precedent established in the clinical literature. Specifically, the previously 
noted study by Suris et al, 2013 employed 15 mg of rapamycin to disrupt trauma-related memories in combat 
veterans. The study results suggested, in a subsample of non-Vietnam era veterans, that 15 mg of rapamycin 
vs. placebo administered in conjunction with trauma memory reactivation was associated with decreased PTSD 
symptomatology at a 1-month follow-up assessment. Importantly, they also documented that this dose was not 
associated with any adverse medical consequences or side effects. 
In addition to the precedent set by the Suris et al study, there is strong animal and clinical pharmacokinetic 
(PK) and pharmacodynamic (PD) data to demonstrate the proposed dosing regimen will demonstrate clinically 
relevant outcomes. Results from a number of clinical PK studies demonstrate that sirolimus is rapidly absorbed 
after oral ingestion, with peak whole blood concentrations occurring 1.0 to 1.3 hours after administration1-3  


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Additionally, sirolimus adequately crosses the blood brain barrier, with an estimated blood to brain ratio of 1:34. 
In a clinical PK study in health males, an 8-mg/m2 one-time oral dose produced a whole blood Cmax of 115.2 
ng/mL at 1.0 hour after ingestion. This corresponds to an approximate peak brain concentration of 38.4 ng/mL60. 
Additionally, the organ transplant animal and clinical data strongly support the proposed dosing strategy. Studies 
conducted in rats using sirolimus to prevent organ rejection utilized dosing ranges between 0.5 to 50 mg/kg per 
day5. However, maximum pharmacodynamic activity with sirolimus monotherapy in rat kidney and heart 
transplant models was demonstrated with a dose of 8 mg/kg intravenously. This dose correlated with a mean 
AUC0-24h of 27012 
g/L*h6. By using the trapezoidal rule, the AUC0- in rats for an 8 mg/kg dose would 
approximate 350 g/L*h. This dose of sirolimus also demonstrated maximal lymphocyte protein kinase inhibition 
at 2 hours post-dose (i.e., maximal protein synthesis inhibition), with return of full function occurring at 12 to 24 
hours post-dose. Therefore, based on the above noted literature, a one-time 15 mg dose should produce 
substantial protein kinase inhibition in the brain within 1-1.3 hours of administration, while also being well 
tolerated, with mild-transient side effects. The current clinical use of sirolimus in organ transplantation also 
supports using the 15 mg oral dose in this proposed study, as it is commonly given as a loading dose of 15 mg 
orally during the perioperative period6.    
Other Considerations. Participants may experience some craving and arousal with exposure to smoking 
cues.  However, the length of each cue exposure is brief, and no participants among the several hundreds tested 
with similar procedures in our previous research in this area reported feelings of prolonged distress. Therefore, 
participant risk is considered minimal, and there are no relevant alternative procedures that would be more 
advantageous for participants. If a participant is intolerant of the interview or cue reactivity procedures, the 
participant will be withdrawn from the study and appropriate therapeutic measures (brief intervention performed 
by senior investigator, appropriate clinical referral, etc) will be undertaken. In the event that a participant 
experiences elevated craving at the end of a cue reactivity session (i.e., report a post-session craving score  
20% above baseline), they will be asked to remain in the SCTR Research Nexus until their craving subsides. A 
member of the research staff will be available to discuss management of craving/urges. Confidentiality will be 
maintained at all times. Participants who would like research staff to discuss their study participation with their 
personal therapist will be required to sign a release of information form.   
Protection Against Risk.  Participants will be informed of the potential side effects of rapamycin/sirolimus 
and will be closely followed by either a study physician, Pharm.D. or other member of the research team. 
Rapamycin (sirolimus) administration will occur in a fully staffed clinical environment (MUSC’s CTRC). 
Drs. Saladin, Gray or Brady will monitor all study participants for psychiatric stability, and Drs. Gray, Taber and 
Brady will monitor for medical stability. Adverse Events (AEs) will be assessed by the medical clinician at all 
visits using the Monitoring of Side Effects Scale (MOSES). The medical clinician will be available 24/7 by 
phone/pager, and study staff will have phone contact with participants at least once during the follow-up period. 
Adverse events will be coded at all visits using Medical Dictionary for Regulatory Activities (MedDRA) rules, and 
entered into a database. For each study meeting, study staff will prepare a summary of all AEs, including their 
severity and presumed relation to study medication. The PI will review this at the weekly study meeting (or before 
if more urgent). 
Guidelines have been developed for managing and reporting of AEs, including serious adverse events (SAE; 
defined as any untoward medical occurrence that results in death, is life-threatening, requires or prolongs 
hospitalization, causes persistent or significant disability/incapacity, results in congenital anomalies/birth defects, 
or in the opinion of the investigators represents other significant hazards or potentially serious harm to research 
subjects or others).  If an AE is non-serious (self-limited with no intervention needed), no further action will be 
necessary.  However, in the case of a serious, unresolved event, an AE follow-up log will be completed. The first 
member of the research team to become aware of a SAE will notify the study physician and PI immediately. Study 
staff will record the information on SAE Notification Form. The clinician will forward hard copies of the complete 
report (SAE Notification Form, Concomitant Medication Log, and AE Log) to Dr. Saladin, who will, in turn notify 
the IRB and DSMB about the SAE. If the event is “Serious, Unexpected and Associated” (an SAE is 
considered unexpected if it is not described in the Package Insert), Dr. Saladin will complete Food and Drug 
Administration (FDA) Form 3500A and will forward it to the FDA. Dr. Saladin also will inform the IRB about the 
SAE. In all of these reviews and reports, strict patient confidentiality will be maintained. 
The instrumentation used for physiological recordings meets all safety standards for non-invasive recordings, 
and participants are located out of reach of any AC-powered devices in the laboratory.  


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All sessions will be conducted under the supervision of trained personnel. In the unlikely event that crisis 
intervention is necessary, senior staff will be available to evaluate the participant and provide an intervention or 
referral.  
All participants will be fully informed that they may withdraw from the experiment at any time without penalty. 
All participant records will be kept in a locked filing cabinet, and confidentiality of all materials will be maintained.  
Offices also will be locked when not in use. 
To ensure confidentiality, participant data will be number coded, and only the investigators will have access 
to the master lists of codes. All participant records will be kept in an office that will be locked at times when not 
in use. The research staff understands the importance of maintaining confidentiality. This method of maintaining 
confidentiality has been used for several years by our research group and has been effective.  
 
XII. Potential Benefits to Subjects or Others 
Possible risks to study participants include distress, craving and arousal as a result of exposure to smoking 
cues. Additionally, adverse reactions to rapamycin (sirolimus) are possible (though this medication is known to 
be very well tolerated at the proposed single dose, as noted above). Benefits include detailed assessments of 
smoking and other substance use; participants will also receive appropriate professional referral as dictated by 
the outcome of their clinical/diagnostic assessment. Moreover, participants may benefit from their experience 
during the cue reactivity procedure and these benefits may include a potential decrease/elimination of craving to 
smoking cues and/or smoking. The minimal risks are reasonable in relation to the benefits to be gained from the 
investigation. The clinical assessment, cue reactivity procedures and/or medication interventions may provide 
important information that can guide treatment for future patients with nicotine dependence. This information can 
be provided to participants' health care providers (with signed consent) if individual participants make this 
request. In addition, understanding the mechanisms underlying memories for, and responses to, smoking cues 
is important for the development of more effective treatment for nicotine dependence. Thus, the benefits of this 
study outweigh the risks. 
Importance of the Knowledge to be Gained.  This study may provide important information that can improve 
treatment for individuals with nicotine dependence. More specifically, this research could lead to the development 
a new class of therapeutic agents to be used in the treatment of nicotine dependence. It is also possible that 
rapamycin (sirolimus) may be shown in the future to be similarly beneficial in the treatment of persons with other 
substance use disorders (i.e., cocaine, methamphetamine, marijuana, etc.).  
 
XIII. Future Directions  
If sirolimus exerts the expected disruptive effect on memory reconsolidation, then attenuated craving and 
other cue-elicited reactions should be observed during subsequent presentations of the smoking-related cues. 
Additionally, it is possible that reduced craving and cue reactivity will translate into changes in smoking behavior. 
Favorable outcomes from this study would set the stage for a larger, phase II RCT within the context of a NIH 
program project grant and could lead to a new generation of brief and strategic neuropharmacological 
interventions that substantially improve cessation rates by reducing craving-related obstacles to self-regulation. 
This type of intervention could be especially important early during a quit attempt when craving-related risk of 
relapse is greatest. It could also become a complementary treatment paradigm that would operate synergistically 
with the current generation of pharmacological and behavioral interventions to reduce the cancer burden posed 
by this most prevalent form of addiction. More broadly, this project fits within our larger Team Science application, 
which proposes a translational framework to identify/develop (Projects 1 & 2) or apply (Project 3) novel, evidence-
based treatments that either (1) directly target processes subserving self-regulation (Project 1) or target 
obstacles to self-regulation (Projects 2 & 3) in the interest of advancing smoking cessation. We believe this 
collection of studies will put us in strong position for future development of program project funding. 


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