Conditions: Alcohol Drinking Control
Keywords: adolescent
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 57
Healthy volunteers: yes

Brief summary:
This study will examine the effect of N-Acetylcysteine (NAC), an over-the-counter antioxidant supplement, on brains of youth (ages 15-19) using magnetic resonance imaging (MRI).

Detailed description:
55 adolescents will receive, in a counterbalanced order, a 10-day course of NAC 1200 mg twice daily and a subsequent 10-day course of matched placebo twice daily, separated by 11 days. Urine and blood samples will be collected at baseline and urine samples again before and after each course of medication treatment. Participants will receive a 1- hour MRI scan at baseline and after each treatment trial.

Arms:
arm 1: Participants first received N-Acetylcysteine 600mg capsules by mouth, two pills twice daily will be taken for 10 days. After washout for 11 days, they then received placebo capsules mimicking N-Acetylcysteine (two pills, twice daily) will be taken for 10 days. arm 2: Participants first received placebo capsules mimicking N-Acetylcysteine (two pills, twice daily) will be taken for 10 days. After washout for 11 days, they then received N-Acetylcysteine 600mg capsules by mouth, two pills twice daily will be taken for 10 days.

Interventions: intervention 1: N-Acetylcysteine intervention 2: Placebo Oral Capsule
Intervention descriptions: intervention 1: N-Acetylcysteine; 600mg capsules by mouth, two pills twice daily (total 2400 mg/day). Participants will undergo MRI (including magnetic resonance imaging and functional MRI during an alcohol cue reactivity task) at baseline, after 10 days of N-Acetylcysteine and after 10 days of placebo. intervention 2: N-acetylcysteine-matched placebo tablet two pills twice daily. Participants will undergo MRI (including magnetic resonance imaging and functional MRI during an alcohol cue reactivity task) at baseline, after 10 days of N-Acetylcysteine and after 10 days of placebo.

Protocol document:
ICF_005.pdf: 
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Neuroscience-Informed Treatment Development for Adolescent Alcohol Use  
NCT03238300 
October 19, 2021 
 


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Medical University of South Carolina  
CONSENT TO BE A RESEARCH SUBJECT 
 
TITLE OF RESEARCH: 
Neuroscience-Informed Treatment Development for Adolescent Alcohol Use 
 
If participants include those under 18 years of age: 1) The subject's parent or legal guardian will 
be present when the informed consent form is provided. 2) The subject will be able to participate 
only if the parent or legal guardian provides permission and the adolescent provides his/her 
assent. 3) In statements below, the word "you" refers to your child or adolescent who is being 
asked to participate in the study. 
 
A. PURPOSE OF THE RESEARCH 
 
You are being asked to volunteer for a research study. Research studies are voluntary and 
include only people who choose to take part. Please read this consent form carefully and take 
your time making your decision. As your study doctor or study staff discusses this consent form 
with you, please ask him/her to explain any words or information that you do not clearly 
understand. 
 
The purpose of this study is to evaluate how an active medication [an over-the-counter 
supplement N-acetylcysteine (NAC)] versus an inactive medication (e.g., sugar pill) affects the 
brain functions in adolescents who may or may not use alcohol. NAC has been approved by the 
Food and Drug Administration (FDA) for use in adults and children for other uses, but little is 
known about its effect on brain function in youth. 
 
The study is sponsored by the National Institute on Alcohol Abuse and Alcoholism. The 
investigator in charge of this study is Dr. Lindsay M. Squeglia. The study is being done at the 
Medical University of South Carolina and approximately 55 people will take part. 
 
B. PROCEDURES 
 
Overview: For this study, you will first complete a screening visit to determine your eligibility 
for the study. If eligible, you will take NAC and an inactive medication given in the same form 
as the active drug for 10 days each, separated by 11 days, and you will attend a total five visits. 
You will not know when you will be receiving the NAC or the inactive medication. We will 
collect a saliva sample and a blood sample at Visit 1, Visit 2, and Visit 4. You also will be given 
breath and urine screenings at each visit. You will also receive a 1-hour MRI scan at Visit 1, 
Visit 2, and Visit 4. If you are unable to attend or complete a visit due to unexpected conflict 
(e.g., transportation issues, travel, University closings), arrangements may be made to remotely 


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complete as much of the visit procedures as possible. Finally, you will be sent text reminders 
twice a day and/or e-mail reminders while you are in the study.  
 
Specifically, if you agree to be in this study, the following will happen: 
 
Screening (~2 hours): You will come in for a screening visit to see if you qualify for the study. 
Parents/guardians of participants under 18 years old will participate in the screening, evaluation, 
and informed consent/assent procedure. The Informed Consent process may take place 
electronically and you may sign electronically. You will be given a paper or electronic copy of 
this form to keep. This visit will include a brief medical history, review of medications, and 
physical exam. Parents will not be informed about their child’s substance use and youth self-
report and lab data are confidential with the exception of any acute safety issues (e.g., 
suicidality, abuse). A urine sample will be collected for a urine drug test and pregnancy test 
(females only). The pregnancy test will be completed prior to the drug testing. Females testing 
positive or those who plan to become pregnant during the study period cannot participate in the 
study and no further study procedures will be completed. If you have recently completed the 
Youth Collaborative (YC) Intake study (Pro00094743) in the past 30 days, portions of this 
screening will have been completed. Data collected during that visit will be carried over and 
included in order to avoid repeating any data collection. Examples of such data include answers 
to completed surveys and questionnaires about your substance use. We will also check your 
pulse, blood pressure, temperature, and weight (vitals). You will be asked questions about your 
general substance use, mood, and alcohol use and craving. If you complete the full screening 
visit, it could take up to 3 hours. 
 
The research team will use several methods of contact to keep in touch with you. We will ask 
you to provide us with phone numbers, e-mail addresses, current home and work addresses, and 
contact information of family and friends who may know how best to reach you. 
 
Visit 1- Randomization 1 & Scan (2 hours): You will be asked to complete questionnaires, urine 
drug tests, provide saliva and blood samples. You will then have a 1-hour MRI scan. After, you 
will receive your first 10-day supply of study medication with instructions. 
 
You will be given both NAC and the inactive medication throughout the course of the study, but 
the study medication you receive first will be determined by chance. You will have a 50:50 
chance (like the flip of a coin) of receiving either NAC or the inactive medication during the 
first 10 days of the trial. Neither you nor your study team will know which study medication you 
are receiving; however, the researchers can find out what you are taking if there is a need to do 
so. You will take 2 capsules of study medication twice a day (2 capsules in the morning and 2 
capsules in the evening). 
 
During the scan the following will happen: 


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1. If you would like, you may first be introduced to the mock (fake) MRI scanner. This is a 
model of a scanner that is not magnetically active. It is used to introduce people (that may or 
may not have ever been in an MRI scanner) to the size, shape, and sounds associated with 
the scanning procedure. During the introduction to the mock scanner you will be asked to 
lay down on the scanner bed. This bed will then be slowly pushed back into the tube of the 
scanner and we make sure you are comfortable enough to participant in the actual scan. 
2. After we remove metal from your clothing, you will be placed in the actual MRI scanner. 
You will be lying down and your head and shoulders will be inside the opening during the 
procedure. You will be given earplugs to wear while you are in the scanner. You will be able 
to speak to and hear the research staff during the scan. While you are in the MRI scanner, we 
will take pictures of your brain. You will be asked to lay still in the MRI scanner during this 
time.  
In between each scanning session we will talk to you through the speakers in the MRI 
scanner to see how you are feeling. You will be given a pressure-sensitive ball which you 
can squeeze at any time to end the MRI scan if you feel uncomfortable. Furthermore, we 
will be able to hear you and will be talking with you over a speaker periodically to make 
sure that you are feeling well. 
 
3. Several tasks will be completed during the 1 hour MRI scan. These tasks involve looking at 
different images on a screen and occasionally providing responses with a hand pad. 
 
These procedures are completely non-invasive and painless. There is no radiation with  
MRIs. 
 
Visit 2- Scan (1.5 hours): 10 days after Visit 1, you will be asked to complete questionnaires, 
urine drug tests, provide saliva and blood samples again. You will then have your second 1-hour 
MRI scan (same procedure as the first scan). Afterwards, you will stop taking your study 
medication for 11 days. 
 
Visit 3- Randomization 2 (30 minutes): Eleven days after Visit 2, you will be asked to complete 
questionnaires and urine drug tests. After, you will receive your second 10-day supply of study 
medication with instructions. 
 
The study medication you receive will be determined by which medication you have already 
received. If you already received NAC, you will then take the inactive medication and vice versa. 
However, neither you nor your study team will know which study medication you are receiving; 
however, the researchers can find out what you are taking if there is a need to do so. You will 
again take 2 capsules of study medication twice a day (2 capsules in the morning and 2 capsules 
in the evening). 
 
Visit 4- Scan (1.5 hour): On the following Monday (10 days after Visit 3) you will be asked to 
complete questionnaires, urine drug tests, provide saliva and blood samples again. You will then 


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have your third 1-hour MRI scan (same procedure as the previous scans). Afterwards, you will 
stop taking your study medication and your participation will be complete. 
 
Daily Reminders: The day after you receive your first medication dose, you will be sent daily 
text and/or e-mails reminders to help you remember to take your medication. You may also be 
asked questions in these texts/emails. These reminders will be sent every day you are in the 
study. 
 
Medication Videos: You will be trained on how to record and upload videos of yourself taking 
your morning and evening medication doses. Study staff will look at what time you took these 
videos to confirm that you are taking your study medication within the correct time frames. 
 
Pill Tracking: In order to monitor that you are taking your study medication, we will use special 
pill bottle caps, that will send us information about when you take your medication. 
 
You may be withdrawn from this study without your consent if the researchers believe it is in 
your best interest or if you fail to follow study procedures. 
 
You may withdraw from the study at any time. If you decide to stop participating in the study, 
you are encouraged to talk to study staff first so that stopping can be done safely. Another reason 
to tell study staff that you are thinking about stopping is to discuss what follow-up care and 
testing could be most helpful to you. 
 
C. DURATION 
 
Participation in the study will include a screening visit and 4 subsequent visits over a period of 
approximately 31 days. 
 
D. RISKS AND DISCOMFORTS 
 
There are risks involved with participating in this study, including risks associated with N-
acetylcysteine (NAC), the MRI scan, study procedures, and loss of confidentiality. 
 
All medications may cause side effects and there are several known risks and discomforts 
associated with NAC. Your condition will be monitored closely by the medical clinician 
associated with the study. If the medical clinician or you decide to stop your participation in the 
study due to unwanted experiences or side effects, you will receive appropriate follow-up care 
as determined by the medical clinician. 
 
1) Adverse Effects of N-acetylcysteine (NAC) 
In this study, you will be taking NAC orally (by mouth). The most common side effects 
seen when NAC is taken by mouth include diarrhea, nausea, vomiting, and headache. 


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These side effects are usually mild and go away even with continued use of NAC. There 
is also a risk of a skin reaction, such as flushing, itching, or rash. The dose of NAC 
(2400 mg daily) used in this study is much less than the dose patients receive for 
acetaminophen overdose. In addition, NAC has been used up to 3500 mg in adult 
subjects orally in the treatment of Alzheimer’s disease. Although we have observed 
mostly mild side effects of 2400 mg NAC in our studies involving adolescents and 
adults, and no significant differences in side effects between NAC and the inactive 
medication, it does not rule out the possibility that more serious side effects may occur. 
Some patients who have taken intravenous NAC (liquid NAC in a vein) for the treatment 
of acetaminophen overdose have had more serious reactions. Allergic reactions have 
occurred in about 5% of patients taking intravenous NAC. These reactions may be mild, 
consisting of flushing, rash, and itching. Less common side effects include trouble 
breathing, low or high blood pressure, fever, and hives. If untreated, such a reaction 
could lead to death. Even more rare serious side effects of intravenous NAC are 
irritability, confusion, and seizures. These reactions (severe allergic reaction or seizures) 
have never been reported when NAC is taken orally (by mouth), as it will be in this 
study. 
 
It is highly unlikely (probably less than one chance in several thousand) that you will 
have a severe allergic reaction to NAC. In the event that you begin to experience mild 
itching or localized rash, we will advise you to take an antihistamine medicine, like 
Benadryl (diphenhydramine). If you have trouble breathing, the emergency medical 
system (EMS – call 911) should be called, and you should be transported to the nearest 
emergency room for immediate treatment. The likelihood of a seizure is probably even 
less than the likelihood of a serious allergic reaction. 
 
Because NAC has not been evaluated in combination with many other medications, it 
will be important for you to report to the research staff any medications you may be 
taking before, during, or after the treatment phase. Medications include prescription 
medications from a doctor, over-the-counter medications that you may buy in a drug 
store, herbal medications that you may buy in a health food store, or “street” drugs. 
 
It is unknown how the drugs in this research study will affect an unborn baby. If you are 
a female able to have children, you should not become pregnant or nurse a baby while in 
this research study. If you become pregnant during the study, you must notify the 
research staff immediately. The research staff will wish to follow the outcome of any 
pregnancy and condition of any newborn and report this to the study sponsor. In 
addition, if you are a female able to have children, you must agree to use at least one of 
the following methods of birth control: 
• oral contraceptives (birth control) 
• contraceptive patch 
• barrier (diaphragm or condom) 


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• intrauterine contraceptive system 
• levonorgestrel implant 
• medroxyprogesterone acetate contraceptive injection 
• complete abstinence from sexual intercourse 
• hormonal vaginal contraceptive ring 
2) Risks associated with MRI 
The risks associated with MRI are minimal for individuals who do not have metal and are 
not claustrophobic. Some discomfort may result from lying in the scanner for up to 60 
minutes. You will be welcome to take short breaks between the various tasks. You also 
may skip questions and/or stop the questions or tasks at any time if you feel 
uncomfortable. During the MRI scan, there are potential risk that are detailed below. 
 
Potential Risks of MRI: 
• Although the risks from magnetic resonance imaging (MRI) are low, it is critical 
that participants do not have metal in the skull, metal implants, a cardiac or brain 
pacemaker, or old metal fragments in the eye or retina. If participants have a 
question about metal their body, they should inform the researchers and they will 
determine whether it is safe in an MRI scan.  
• Some discomfort may occur from having to remain still while in the MRI scanner. 
During the scanning, an emergency ball will be given to squeeze if there are any 
feelings of discomfort or desire to be pulled out of the scanner. 
• The MRI scanner is noisy and there is a risk of hearing damage if participants do 
not wear earplugs. To eliminate this risk, earplugs will be given to wear during 
each scan. 
• Although the MRI scanner is open on both ends, some people become anxious or 
claustrophobic (fear of small spaces) when entering the MRI scanner due to the 
feeling of being enclosed. If participants have a history of experiencing this, they 
should inform the study personnel. 
• If pregnant, participants will not be eligible to continue in the study. 
• Unknown Risks: The experimental procedures may have unknown side effects. 
The researchers will let you know if they learn any new information regarding 
side effects. 
3) Interviews/Rating Scales (Questionnaires) 
The questions that will be asked may be sensitive in nature and make you feel 
uncomfortable. You may be asked personal questions that you find distressing. You may 
refuse to answer any question(s) that you do not wish to answer. 
 
4) Phlebotomy 
Risks associated with drawing blood from your arm include momentary discomfort 
and/or bruising. Infection, excess bleeding, clotting, or fainting is possible, although 
unlikely. 
 
5) Risk of Loss of Confidentiality 


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All study records will be placed in a locked, secure, limited access location. Your 
participation in the study and the information you provide will be treated as confidential. 
The information we collect will contain a code and not your name to protect your 
confidentiality. Codes linking numbers and names will be kept in a locked secure 
location and will not be accessible to anyone outside the research team. 
 
The information you share with us is confidential. That means we do not share 
information you tell us with anyone outside our research team. Likewise, we do not 
share with you any information they provide us. This helps us maintain privacy so we 
are able to get honest answers. However, you should also know that if you threaten to 
harm yourself or others or give information about child or elder abuse or neglect, this 
information will be reported to appropriate clinical staff and other persons outside the 
research program as necessary to protect yourself and others as mandated by law. 
 
6) Unknown Risks 
The experimental treatments may have unknown side effects. The researchers will let 
you know if they learn anything during the course of the study that might make you 
change your mind about participating in the study. 
 
E. BENEFITS 
 
You may benefit from participation in this study. However, given the investigational nature of 
this study, benefit cannot be guaranteed or promised. Information gained from this study may 
help other investigators have a better understand how NAC affects the brain. 
 
While the MRI scan is a research scan and these images do not have the same quality or 
specifications as a clinical scan, you may benefit from knowing that one of the medical staff on 
this study will briefly review the structural MRI scan to determine if there are any obvious 
structural abnormalities of your brain. You will be contacted by the PI and/or the 
neuroradiologist if there are obvious abnormalities. If by chance something abnormal is 
observed, one of the Investigators on this protocol will contact you and counsel you as to what 
clinical referral would be appropriate. If the possibility of such a situation is too stressful for 
you, your child should not participate in our study. It is important to understand that this 
research MRI scan is not the same as a clinical MRI scan, and should not be thought of as one. It 
is not a full clinical study and will not be read by a radiologist. 
 
F. COSTS 
 
There will be no cost to you as a result of participation in this study. You will be receiving text 
reminders twice a day, which could result in charges on your phone service plan. If problems 
arise with this, we can send the reminder via e-mail instead. 
 


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G. PAYMENT TO PARTICIPANTS 
 
In return for your time and effort, you will receive $460 for completing all 5 visits. Also, you 
can earn up to a $40 bonus ($2 per day that you are receiving medication) if you take each dose 
of medication, or provide staff with an explanation for why the medication was not taken, within 
a 4 hour requested time window. If you do not take the medication due to adverse side effects, 
you will need to discuss these side effects with research staff. Participants experiencing adverse 
side effects interfering with their ability to participate may be taken off the medication. If you 
are unable to finish the study, you will be prorated for number of completed visits. You are also 
eligible for a $50 referral bonus for each individual you refer who successfully randomizes into 
this study. Mileage reimbursement may be available. 
 
If the total amount of payment you receive from MUSC reaches or exceeds $600.00 in a 
calendar year, you will be issued a tax form (Form 1099). 
 
H. ALTERNATIVES 
 
Participants who wish to receive additional information on substance use treatments will be 
referred clinically. Participation in this study is voluntary, and you may refuse to participate or 
discontinue participation at any time. If you choose not to participate, it will not affect your 
relationship with any current treatment provider or your right to health care or other services to 
which you are otherwise entitled. 
 
I. CERTIFICATE OF CONFIDENTIALITY 
 
To help us protect your privacy, we have obtained a Certificate of Confidentiality from the 
National Institutes of Health. The researchers can use this Certificate to legally refuse to disclose 
information that may identify you in any federal, state, or local civil, criminal, administrative, 
legislative, or other proceedings, for example, if there is a court subpoena. The researchers will 
use the Certificate to resist any demands for information that would identify you, except as 
explained below. 
 
The Certificate cannot be used to resist a demand for information from personnel of the United 
States Government that is used for auditing or evaluation of federally funded projects or for 
information that must be disclosed in order to meet the requirements of the study sponsor and 
related agents participating in the study. 
 
A Certificate of Confidentiality does not prevent you or a member of your family from 
voluntarily releasing information about yourself or your involvement in this research. If an 


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insurer, employer, or other person obtains your written consent to receive research information, 
then the researchers may not use the Certificate to withhold that information. The Certificate of 
Confidentiality will not be used to prevent disclosure to state or local authorities of child abuse 
and neglect, or harm to self or others. 
 
J. SIGNIFICANT NEW FINDINGS 
 
If there are significant new findings during the course of the study, you will be notified. 
 
K. STUDENT PARTICIPATION 
 
Your participation or discontinuance will not constitute an element of your academic 
performance, nor will it be a part of your academic record at this Institution. 
 
 
L. EMPLOYEE PARTICIPATION 
 
Your participation or discontinuance will not constitute an element of your job performance or 
evaluation, nor will it be a part of your personnel record at this Institution. 
 
M. CLINICAL TRIALS.GOV 
 
A description of this clinical trial will be available on http://www.ClinicalTrials.gov, as required 
by U.S. Law. This Web site will not include information that can identify you. At most, the Web 
site will include a summary of the results. You can search this Web site at any time. 
 
N. FUTURE CONTACT 
 
The researcher in charge of this study might like to contact you in the future about other 
research opportunities. Please initial by your choice below or if consenting electronically, scroll 
down to the bottom of the screen and select your choice: 
 
_____Yes, I agree to be contacted 
 
_____No, I do not agree to be contacted 
 
Results of this research will be used for the purposes described in this study. This information 
may be published, but you will not be identified. Information that is obtained concerning this 
research that can be identified with you will remain confidential to the extent possible within 
State and Federal law. The sponsor and the Food and Drug Administration (FDA) will receive 
copies of the research records. The investigators associated with this study, the FDA, and the 
MUSC Institutional Review Board for Human Research will 
have access 


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to identifying information. All records in South Carolina are subject to subpoena by a court of 
law. 
 
In the event that you are injured as a result of participation in this study, you should immediately 
go to the emergency room of the Medical University Hospital, or in case of an emergency go to 
the nearest hospital, and tell the physician on call that you are in a research study. They will call 
your study doctor who will make arrangements for your treatment. The Medical University 
Hospital and the physicians who render treatment to you will bill your insurance company. If 
your insurance company denies coverage or insurance is not available, you will be responsible 
for payment for all services rendered to you. 
 
Your participation in this study is voluntary. You may refuse to take part in or stop taking part in 
this study at any time. You should call the investigator in charge of this study if you decide to do 
this. Your decision not to take part in the study will not affect your current or future medical 
care or any benefits to which you are entitled. 
 
The investigators and/or the sponsor may stop your participation in this study at any time if they 
decide it is in your best interest. They may also do this if you do not follow the investigator’s 
instructions.


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Volunteer’s Statement 
 
 
I have been given a chance to ask questions about this research study. These questions have 
been answered to my satisfaction. If I have any more questions about my participation in this 
study or study related injury, I may contact Dr. Lindsay M. Squeglia at (843) 792-5451. I may 
contact the Medical University of SC Hospital Medical Director (843) 792-9537 concerning 
medical treatment. 
 
If I have any questions, problems, or concerns, desire further information, or wish to offer input, 
I may contact the Medical University of SC Institutional Review Board for Human Research 
IRB Manager or the Office of Research Integrity Director at (843) 792-4148. This includes any 
questions about my rights as a research subject in this study. 
 
I agree to participate in this study. I have been given a copy of this form for my own records. 
 
If you wish to participate, you should sign below or if consenting electronically scroll to the 
bottom of the screen and sign. 
 
 
_________________________  ___________    _________________________  __________ 
Signature of Parent/Legal Guardian      Date 
        Signature of Person Obtaining              Date 
(if under age 18 only) 
 
 
 
        Consent 
 
___________________________    
       _____________________________   
Printed Name of Parent/Legal Guardian 
 
        Printed Name of Person Obtaining Consent 
 
 
 
___________________________  ___________   
Signature of Adult Participant or             Date 
Printed Name of Minor Participant  
 
 
________________________________ 
Printed Name of Adult Participant  
*15-17 years of age: 
“My participation has been explained to me, and all of my questions have been answered. I am willing to 
participate.” 
 
Signature: ___________________________   
 
Age: __________ Date of Birth:______________   
 

Prot_SAP_003.pdf: 
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Neuroscience-Informed Treatment Development for Adolescent Alcohol Use  
NCT03238300 
October 19, 2021 
 


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PROTOCOL TITLE: Neuroscience-Informed Treatment Development for Adolescent Alcohol Use 
 
PRINCIPAL INVESTIGATOR: Lindsay M. Squeglia, Ph.D.  
 
Objectives / Specific Aims 
Adolescence marks a key period in the development of problematic alcohol use, with approximately 15% 
of 18 year olds meeting criteria for an alcohol use disorder. Decreasing drinking at an early age could 
offer substantial advantages for neurocognitive and psychosocial outcomes and could protect against the 
development of habitual drinking. Efforts to prevent or diminish adolescent heavy drinking have been 
only modestly effective, with up to 86% of youth returning to substance use within 12 months following 
treatment. Inadequate treatment for adolescents is an important public health concern given that alcohol 
use during this time is highly predictive of long-term problematic drinking. While several medications 
have been efficacious in treating adult alcohol dependence, pharmacotherapy research focused on 
adolescent alcohol use has been sparse. Evaluation of alternative and more efficacious treatments for 
adolescent alcohol users is warranted. 
N-acetylcysteine (NAC) is an over-the-counter antioxidant supplement with glutamatergic properties that 
has shown promise in treating marijuana dependence in adolescents. In a double-blind randomized 
placebo-controlled study, youth in the NAC group had more than double the odds of negative urine 
cannabinoid tests during treatment compared to the placebo group. Preclinical findings suggest NAC 
works through restoring glutamate homeostasis disrupted by addiction, a finding replicated across 
multiple substances of abuse. No published clinical trials to date have examined the efficacy of NAC on 
alcohol use, despite glutamate’s established role in alcohol use disorders. This is surprising, given 
previous promising NAC findings in marijuana-dependent adolescents and the safety, tolerability, and 
affordability of this over-the-counter supplement. 
This K23 application proposes to examine the effects of NAC on adolescent glutamatergic systems and 
drinking behaviors in a randomized, double-blind, placebo-controlled within-subjects crossover study. In 
counterbalanced order, 55 participants will receive a 10-day course of NAC 1200 mg twice daily and a 
subsequent 10-day course of matched placebo twice daily, separated by a washout period. Human 
laboratory and imaging procedures will be conducted after each course of medication treatment. 
Functional magnetic resonance imaging (fMRI) will examine alcohol reactivity and magnetic resonance 
spectroscopy (MRS) will examine glutamate levels in the anterior cingulate after NAC versus placebo.  
Aim 1: Quantify the effect of NAC versus placebo on alcohol cue reactivity in adolescent heavy 
drinkers. Using fMRI, we will determine the effect of NAC on alcohol cue reactivity. 
Hypothesis: Heavy drinking youth with show decreasing alcohol cue reactivity from baseline in key 
reward regions while on NAC compared to placebo. 
Aim 2: Quantify the effect of NAC versus placebo on glutamate levels in adolescent heavy 
drinkers and non-drinkers. Using MRS, we will determine the effect of NAC versus placebo on 
modulating anterior cingulate glutamate levels in heavy alcohol-using adolescents and non-alcohol using 
adolescents.  
Hypothesis: Heavy drinking youth will show increasing levels of glutamate from baseline in the anterior 
cingulate while on NAC compared to placebo. 
Exploratory Aim: Examine the effect of NAC on adolescent drinking. Given the short dosing periods 
(~10 days), we do not expect dramatic changes in drinking behavior in heavy alcohol-using adolescents. 
However, drinking days and drinks per drinking day will be assessed during the NAC, placebo, and 
washout periods. Given the findings with marijuana dependent adolescents, we anticipate a potential 
decrease in alcohol use while participants are on NAC versus placebo. 
Background 
A. SIGNIFICANCE 
Alcohol use during adolescence is problematic and has long-term consequences. Adolescence is 
a key period in the development of alcohol use disorders, with nearly 15% of youth meeting diagnostic 


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criteria for an alcohol use disorder by age 18 (Swendsen et al. 2012). Heavy adolescent alcohol use is 
related to serious psychosocial problems, including comorbid psychopathology (Deas and Thomas 2002; 
Rowe et al. 2004), poorer academic success (Kristjansson et al. 2013), and detrimental neurocognitive 
consequences (Squeglia et al. 2014). Furthermore, binge drinking patterns initiated during adolescence 
hold into adulthood (Degenhardt et al. 2013) and significantly increase risk for subsequent adult alcohol 
use disorders and related problems (Hingson et al. 2006a; b).  
Existing adolescent substance use treatments are inadequate. Decreasing substance use at this 
early stage could have significant long-term implications; however, efforts to prevent or decrease heavy 
alcohol use during adolescence have only been modestly effective (Jensen et al. 2011; Tripodi et al. 
2010), with up to 86% of youth returning to some alcohol or drug use at least once within 12 months 
following treatment (Chung and Maisto 2006; Tanner-Smith et al. 2013; Winters et al. 2009). While 
several medications have been efficacious in treating adult alcohol dependence, pharmacotherapy 
research focused on adolescent alcohol use has been sparse (Miranda et al. 2014). This interferes with 
treatment, as safety and efficacy of medications for adolescents cannot be inferred from adult studies 
(Bridge et al. 2007). Evaluation of alternative and more efficacious treatments for adolescent alcohol 
users is warranted.  
N-acetylcysteine has potential for reducing adolescent alcohol use. Based on preclinical findings, 
glutamate has emerged as a potential pharmacotherapeutic target in the treatment of addictions (Kalivas 
2009; Kalivas and Volkow 2011). N-acetylcysteine (NAC) is an over-the-counter antioxidant supplement 
with glutamatergic properties that has shown promise in treating addictive disorders (Deepmala et al. 
2015) by restoring glutamate homeostasis disrupted by addiction, a finding replicated across multiple 
substances of abuse (McClure et al. 2014; Olive et al. 2012). In a double-blind randomized placebo-
controlled study of marijuana-dependent youth, those in the NAC group had greater than double the 
odds of negative urine cannabinoid tests during treatment compared to the placebo group (Gray et al. 
2012). No published clinical trials to date have examined the efficacy of NAC on alcohol use in any age 
group.  
NAC has potential for reducing neuroinflammation associated with alcohol use. Recent evidence 
from human and animal research has converged to support the role of alcohol in promoting deleterious 
adaptations in inflammatory signaling cascades. Specifically, alcohol appears to induce a number of pro-
inflammatory effects throughout the CNS and periphery, including upregulating pro-inflammatory 
cytokines such as IL-1B, IL-6 and TNFα, and increasing high-mobility group protein B1 (HMGB1), which 
is a chromatin protein that regulates gene transcription, and is produced by immune cells in response to 
an immune challenge (i.e., alcohol exposure) and endogenous pro-inflammatory cytokines (Yu et al., 
2006). One recent human post-mortem study found that earlier age of drinking onset is associated with 
increased expression of HMGB1 in the orbitofrontal cortex (Vetreno et al., 2013). Binge-like ethanol 
administration in adolescent rats increased the inflammatory cytokines TNFα and IL-1β (Pascual et al., 
2014), and another recent rodent study found that intermittent ethanol treatment in adolescent mice was 
associated with learning deficits and increased perseverative behavior in adulthood, as well as increased 
frontal cortical HMGB1 (Vetreno and Crews, 2012). Further, alcohol-induced HMGB1 signaling is 
associated with induction of the inflammatory cytokine IL-1β in the brain (Crews et al., 2013). 
Uncontrolled activation of such inflammatory mediators (as may be the case in heavy or chronic alcohol 
exposure) is associated with increased neuroimflammation, reduced neuroprotection and neuronal 
repair, and increased neurodegeneration (Guerri and Pascual, 2013). One hypothesized consequence of 
such disrupted immune signaling is neuronal cell death in frontal control regions of the brain, consistent 
with the deficits in inhibitory control observed over the course of AUDs (Li et al., 2009). Thus, 
understanding the role of inflammatory signaling in adolescent alcohol users may have significant 
implications for understanding the behavioral sequelae of alcohol use and potentially improving 
treatments. Further, given that NAC is an anti-oxidant and has therapeutic potential in terms of reducing 
inflammation (Zafarullah et al., 2003), measuring levels of inflammatory markers before and after NAC 
treatment could provide added insight in terms of informing the development of future treatment options 
for adolescent drinkers. 
 


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NAC has potential for improving gut dysbiosis. Accumulating evidence suggests that NAC may have 
the potential to reshape microbiome structure and benefit gut health. Several studies have shown that 
NAC exhibits an antibiofilm effect towards harmful bacteria and promotes the growth of beneficial 
bacteria suggesting a curative effect on microbial imbalances (Zheng et al. 2019, Xu et al. 2014, 
Eroshekno et al. 2017). In addition to this, NAC reduces oxidative stress and inflammation, two markers 
that have been reported as elevated in patients with AUD (Hou et al. 2015, Kamal et al. 2020, Ozaras et 
al. 2003). In fact, one study showed that pretreatment with NAC blocked oxidative stress marker proteins 
and prevented binge alcohol-induced gut leakiness in mice (Abdelmegeed et al. 2013). Collection of 
salivary microbiome may be a favorable alternative to classic stool collection as some studies have 
reported that the oral microbiome closely represents the diversity and composition of the upper 
gastrointestinal tract (Kodukula et al. 2017), displays changes with alcohol consumption (Fan et al. 
2018), and displays some of the same genera with stool samples of AUD participants (Ames et al. 2020). 
The collection of salivary microbiota will allow for the investigation into the relationship between 
microbiota and adolescent alcohol use as well as provide feasibility data for future studies. 
 
NAC is safe and tolerable for adolescents. Part of the appeal of NAC is its long-established safety 
record in children and adults, with FDA approval since 1963. NAC has been used safely for several 
decades in children and adults, often at doses greatly exceeding those proposed for our study 
(Mucomyst Package Insert, 2004). A meta-analysis of NAC studies found that this supplement was well 
tolerated, with generally mild side-effects, most commonly gastrointestinal adverse effects that did not 
require treatment interruption (Grandjean et al. 2000). Systemic allergic reactions to NAC have been 
observed, but only with intravenous administration (Bailey and McGuigan 1998). Reflecting its safety 
profile, NAC is available over-the-counter as a supplement, which further increases its potential 
acceptability and accessibility. 
Summary. Given the safety profile of NAC and preclinical evidence of its actions on the glutamatergic 
system and reversing addiction pathology, it is a strong candidate medication targeting adolescent heavy 
alcohol use. Thus, the primary aim of this study is to examine the effects of NAC on alcohol cue reactivity 
and glutamatergic functioning in heavy alcohol-using adolescents, and obtain preliminary data for 
estimating sample sizes needed to adequately power subsequent clinical trials. Understanding the 
biological mechanisms of behavioral change is fundamental in making substantive advances in the field 
of adolescent addiction treatment. 
B. INNOVATION 
1. NAC’s effect on adolescent drinking has not been examined. Despite the success of NAC 
increasing abstinence rates two-fold in marijuana-dependent adolescents (Gray et al. 2012), there are no 
studies examining the effect of NAC on adolescent alcohol use, which is the most commonly used 
substance during adolescence. Effective interventions during adolescence could have substantial long-
term implications by reducing acute and enduring negative social, academic, and cognitive 
consequences related to heavy teen drinking (Squeglia et al. 2014). 
2. Magnetic resonance spectroscopy is an innovative neuroimaging technique that will help 
elucidate NAC’s mechanism of action. Further, we will be using innovative neuroimaging techniques 
including magnetic resonance spectroscopy (MRS) to understand the neurological underpinnings of 
NAC’s mechanism of action. With this knowledge, we will be well positioned to develop refined circuit-
specific treatment strategies for adolescents with substance use disorders. 
Given NAC’s accessibility as an over-the-counter supplement, safety and tolerability with youth, and low 
cost, this medication holds great promise as a potential treatment option for substance using 
adolescents.  
C. PRELIMINARY STUDIES 


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NAC may reduce co-occurring alcohol use in marijuana-
dependent adolescents. To date, no published clinical trials have 
examined the effect of NAC on alcohol use. Therefore, secondary 
analyses were performed on an adolescent marijuana dependence 
NAC clinical trial dataset (Gray et al. 2012) to examine the effect of 
NAC or placebo on co-occurring alcohol use over the 8-week 
treatment course. Participants (ages 15-21; N=116) interested in 
marijuana dependence treatment were randomized to 1200 mg of 
NAC twice daily (same dosing proposed in this study) or matched 
placebo for 8 weeks. Of the 116 participants randomized in the study, 
89 (77%) returned for at least one study treatment visit and had 
recorded alcohol use data (even if abstinent from alcohol use) and 
were included in the longitudinal analysis. Of those 89, 45 were 
randomized to receive NAC and 44 to placebo. Out of the 89 
participants with study data available, 77 (87%) reported at least one 
drink during the 30 days prior to study entry and 69 (77%) noted at least one drink within a week of study 
entry; 36 (80%) in the NAC group and 33 (75%) in the placebo group (p=0.572). There were no 
differences between study groups with respect to age, race, gender, use characteristics, or psychiatric 
comorbidities. Less marijuana use [based on urine cannabinoid tests (UCT) and creatinine adjusted 
cannabinoid levels (CC)] was associated with less alcohol use in the NAC treated group but not in the 
placebo treated group [UCT: interaction t86=2.44, p=0.016]. In other words, participants who reduced 
marijuana use while using NAC also had a significant reduction in alcohol use; this was not the case in 
the placebo condition. This suggests that NAC may be effective in reducing adolescent alcohol use. 
Heavy-drinking youth display robust activation in reward regions during the Alcohol Cue 
Reactivity Task. In a pilot study using the same fMRI alcohol cue reactivity task proposed for this 
application (Schacht et al. 2011), 11 heavy drinking youth displayed robust activation in several brain 
regions including the anterior cingulate, insula, striatum, and amygdala during alcohol vs. non-alcohol 
cue trials (see Figure 1), which is highly consistent with activation patterns seen in adult studies (Schacht 
et al. 2013a). Understanding the role of NAC on reducing cue reactivity as well as glutamatergic levels 
(via MRS) will be the primary aims of this application.  
 
Intervention to be studied  
PHARMACOLOGICAL INTERVENTION 
This double-blind crossover trial will compare NAC (1,200mg twice daily) and placebo. If assessment 
procedures reveal that a participant meets inclusion criteria and none of the exclusion criteria, the 
participant will be randomized to each condition for 10 days in a counterbalanced order. There will be a 
minimum 11-day washout period between conditions to allow for the clearance of NAC (Holdiness 1991). 
Medication response and tolerability/adverse events will be assessed at each clinic visit. Medication videos 
will be uploaded by participants to track medication compliance. Reminders to take medication will be sent 
via email or text message to increase compliance. Primary mentor Dr. Gray has a current IND for using 
NAC with adolescent substance users (#78,927).  
Study personnel will review medication logs and data from the Medication Event Monitoring System 
(MEMSCapTM) that track when the pill bottles are opened, as well as perform pill counts throughout 
treatment to monitor medication adherence. Medication tolerability and effects will be systematically 
assessed. Participants will be encouraged to contact study personnel between visits to address any 
immediate concerns regarding adverse effects. If a participant experiences intolerable medication-related 
adverse effects at any point during the study, a dose reduction to 600 mg twice daily may be undertaken. 
The dosage may be increased back to 1200 mg twice daily at the discretion of the medical clinician. 
However, if a participant is unable to tolerate the reduced dose, medication will be discontinued, and the 
participant will continue to come in for study visits. 
 
Inclusion and Exclusion Criteria/ Study Population 
Individuals responding to recruitment materials or otherwise referred to the study will be pre-screened on 
Figure 1. Robust activation was 
elicited from alcohol, compared to 
neutral, stimuli in heavy drinking 
youth in the striatum (shown here) as 


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the phone or in person to ascertain preliminary eligibility status. They may have completed the YC Intake 
Protocol (PRO# 94743) prior to the formal study screening. A series of questions will determine 
preliminary eligibility, and formal screening appointments will be scheduled for those who meet these 
eligibility criteria. The comprehensive assessment will include a medical history and physical 
examination, urine drug testing, semi-structured psychiatric diagnostic interview, and detailed 
assessment of alcohol and other substance use history. No information obtained during the pre-
screening will be used as research data. All study related procedures will take place on the MUSC 
campus within the designated office areas of the Addiction Sciences Division and the Center for 
Biomedical Imaging. 
Inclusion criteria: Participants 
(N=105; 50% female) will be 
between the ages of 15-19 
and may or may not use 
alcohol. The age of interest 
corresponds to a time when 
many adolescents initiate and 
escalate heavy binge drinking 
(Johnston et al. 2015), and is 
therefore the optimal time to 
intervene. All participants in the alcohol-using group must meet criteria for heavy drinking, based on 
quantity and frequency of drinking (Squeglia et al. 2011; Squeglia et al. 2012) (see Figure 2). Binge 
drinking criteria will be used to classify heavy drinking youth, as opposed to alcohol dependence 
diagnosis, as diagnostic criteria may not be optimal in capturing problematic drinking in youth (Winters et 
al. 2011). This criteria will be used for the recruitment for all alcohol using participants.  Inclusion criteria 
for non-alcohol-using group: has used alcohol <10 times in their life and has never had a binge drinking 
episode (4+ females, 5+ males), <5 lifetime experiences with marijuana and none in the past three 
months; <5 lifetime cigarette use; and no history of other intoxicant use. 
Exclusionary criteria: To maximize our ability to detect effects of interest and minimize potential 
confounds, we will use rigorous screening criteria. Exclusion criteria for the alcohol-using group will 
include: not having a parent to consent (for those under age 18); history of alcohol treatment or 
treatment-seeking; current DSM-5 diagnosis of moderate or severe substance use disorder other than 
alcohol or cannabis (American Psychiatric Association 2013); positive urine toxicology screen for 
narcotics, amphetamines, sedatives, hypnotics, or opiates (not prescribed by a doctor); alcohol 
withdrawal (> 10 on the Clinical Institute Withdrawal Assessment for Alcohol (Sullivan et al. 1989); 
medical conditions or medications that contraindicate taking NAC; current use of N-acetylcysteine or any 
supplement containing N-acetylcysteine (must agree not to take any such supplement throughout study 
participation);medical history of severe asthma (uncontrolled with medication); history of a serious 
medical, psychiatric, or neurological problem that could affect neural response, brain development, or 
study participation, including diabetes, seizure disorder, and severe head injury with loss of 
consciousness; history of learning disability, pervasive developmental disorder, or other condition 
requiring special education; current use of psychoactive medications that affect cerebral blood flow; non-
correctable visual or hearing problems; non-fluent in English; MRI contraindications (e.g., braces, 
claustrophobia, irremovable metal implants or piercings); (for females) pregnancy or refusal to use 
reliable methods of birth control; refusal of blood draw, abstinence from alcohol for >14 days before 
participation, and use of alcohol <12 hours before scanning (confirmed with breathalyzer). While 
cigarette and marijuana use will not be exclusionary, we will exclude any participants who are daily users 
of cannabis or tobacco. Exclusionary criteria for the control group will include all of the above 
exclusionary criteria except for abstinence from alcohol for >14 days before participation. 
 
Rationale for vulnerable populations (children and alcohol users). 45 participants in the alcohol-
using group will be between the ages of 15 and 19 and heavy alcohol users. We will include one control 
group of 10 non-alcohol using youth between the ages of 15-19 to test the effect of NAC on glutamate 
levels in non-alcohol users. We will also include a second control group of 30 non-alcohol using youth 
Figure 2. Drinking classification. Only heavy drinkers will be recruited for this proposal. 


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and a second alcohol using group of 20 youth between the ages of 15-19 who will only complete 
baseline assessments to investigate potential neural and microbiome differences within binge drinking 
adolescents as compared to non-drinking controls. Creation of alcohol-focused interventions during 
adolescence could have substantial long-term implications by reducing acute and long-term negative 
social, academic, and cognitive consequences related to heavy teen drinking and by reducing the rate of 
youth transitioning from heavy drinking to more problematic alcohol dependence. While participants in 
this study are not treatment seeking, all participants will be given treatment referrals at the conclusion of 
the study. 
 
Diverse Population: In this cohort, approximately 50% of the participants will be female, and we aim to 
recruit participants to approximate the racial and ethnic composition of Charleston County.  
 
Number of Subjects 
We will recruit 105 15-19 year-olds (50% female).  
 
Setting 
Visits will take place at MUSC in the Institute of Psychiatry and the Biomedical Imaging MRI research 
facility at 30 Bee St (for those visits requiring an MRI).  If a participant is unable to attend or complete a 
visit due to unexpected conflict (e.g., transportation issues, travel, University closings), arrangements 
may be made to remotely complete as much of the visit procedures as possible to maintain data 
collection and study engagement. 
 
Recruitment Methods 
Recruitment and process for obtaining informed consent. Adolescents will be recruited from 
the Charleston area. We have received approval from the Charleston County School District to 
recruit through local high schools. Study staff will make initial contact with school leadership to 
arrange classroom presentations for high school students. We will also canvas flyers in the 
Charleston area, although not on the school grounds or college campuses. We will also recruit 
through paid online advertisement sources such as Facebook, Instagram, Craigslist, etc. The 
fliers and advertisements include IRB-approved materials that describe the research opportunity, 
basic inclusion criteria, monetary compensation, and study contact information. Interested youth 
or parents call our office and verbal consent/assent is obtained from the parents and teens 
respectively (or simply consents for 18 year olds). The project is described in more detail. The 
teen is screened with basic questions approved for telephone administration by our IRB. If the 
teen remains eligible and interested, HIPAA and informed consent (parents and youth over age 
18) or assent (youth under age 18) forms are reviewed. 
Consent Process 
Circumstances under which consent will be sought and obtained, who will seek it, information 
provided to prospective subjects, and method of documenting consent: At study entry, any teen 
who responds to the recruitment fliers or advertisements, appears interested and eligible, and provides 
verbal consent/assent, are scheduled for an initial appointment. At the screening visit, which will take 
place in a private office of a research staff within the Addiction Sciences Division, the complete 
procedures of the study, as described in the consent/assent forms, are provided to prospective subjects 
and their parents. Parents/guardians of participants under 18 years old will participate with the 
adolescent in the screening, evaluation, and informed consent/assent procedure. Participants 18 years 
and older will be able to provide their own informed consent. If still interested, they sign the HIPAA and 
consent and assent documents and keep the extra copy provided. Trained research staff members 
complete online and lab-based training in HIPAA policy, human subjects’ research, and management of 
other research issues. During the consent phase, participants and parents are informed that all 
information provided is confidential within ethical and legal limits to facilitate disclosure. During the 
consenting process, parents are told that they will not be informed about their child’s substance use and 
that youth self-report and lab data are confidential, with the exception of any acute safety issues (e.g., 
suicidality, abuse). The forms are reviewed with the interested parties and filed in a locked file cabinet in 


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a locked office. Trained research staff members complete online and lab-based training in HIPAA policy, 
human subjects’ research, and management of other research issues.  
 
We may also use several different methods to complete electronic informed consent, if applicable and 
necessary, that include the following: 1) REDCap electronic consent (e-consent) combined with a video 
discussion or 2) via MUSC’s doxy.me system (teleconsent). As a last resort, we will also email the 
consent document to the participant and conduct the informed consent via video chat.  Participants can 
then email or mail the signed consent back to the research team if needed. Video chat functionality will 
only be used if all parties have the capability.   
 
E-consent via REDCap will be saved in a separate informed consent database.  All doxy.me signed 
consent forms will be saved as PDF files within our study records. Using these systems, signatures on 
the consent form may be obtained electronically via REDCap/doxy.me. In the case that participants mail 
back hard copies of the consent (in rare instances), those will be stored in locked file cabinets in the 
offices of research staff. 
 
Study Design / Methods 
Overview: Our primary goal is to assess the effects of NAC versus placebo on alcohol cue reactivity and 
glutamate levels in adolescent heavy drinkers. A randomized, double-blind, placebo-controlled within-
subjects crossover design will be utilized. In counterbalanced order, 55 adolescents will receive a 10-day 
course of NAC 1200 mg twice daily and a subsequent 10-day course of matched placebo twice daily, 
separated by 11 days. To allow subjects for flexibility in case of scheduling difficulties, the pill bottle will 
also include extra doses of medication for each 10-day course. Medication will be prepared and 
packaged by Pitt St. Pharmacy. Medication will be provided in pharmacy grade bottles. Bottles of 
medication will use the MEMSCapTM, which are child resistant and record a timestamp when the bottle is 
opened. The MEMSCap system will be used to monitor medication adherence during the trial. Biomarker 
data will be conducted at baseline and before and after each course of medication treatment and 
neuroimaging will be collected at baseline and after each treatment trial. Functional magnetic resonance 
imaging (fMRI) will examine alcohol reactivity, and magnetic resonance spectroscopy will examine 
glutamate levels in the anterior cingulate, post- NAC and placebo. A secondary goal is to assess the 
microbiome and neural differences between binge drinking adolescents and non-using controls; 
therefore, an age- and gender-matched control group (n=30) will be recruited to complete Visit 1, only. 
We will also include a second alcohol using group of 20 youth between the ages of 15-19 who will only 
complete baseline assessments to investigate potential neural and microbiome differences within binge 
drinking adolescents as compared to non-drinking controls. Below describes the detailed procedures for 
interviews, self-report, neuroimaging, biomarker, and medication administration. 
 
Consent and interview (30 minutes): Following the phone (or in person) screen, eligible adolescents 
will be invited to participate in a screening visit. Youth will be asked questions regarding demographic 
and background information, medication use, physical health, neurological history, family history of 
substance use, and personal substance use history. The youth Structured Clinical Interview (SCI; Brown 
et al. 1994) will ascertain information on psychosocial functioning (e.g., academic/social functioning) and 
health and medication history. The Mini International Neuropsychiatric Interview (MINI), a structured 
interview designed to ascertain a current or lifetime history of the major psychiatric disorders in DSM-5 
and ICD-10, will be used to assess psychopathology (Sheehan et al. 1998; Sheehan et al. 2010).  
Self-report measures (30 minutes): Substance use assessment: Youth typically provide valid self-


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reports of alcohol and other drug use (Winters et al. 1990-91), but to maximize accuracy, we will provide 
a comfortable context for the interview and assure confidentiality of substance use information. Biological 
samples will be collected to confirm self-reported alcohol use (see below). At baseline (pre-intervention), 
substance use histories are assessed using a modified 90-day Timeline Follow-back (Sobell and Sobell 
1992) (TLFB) to obtain information on typical use of alcohol, nicotine, marijuana and other illicit drug 
types, including binge drinking quantity and frequency. For follow-up substance use assessments the 
TLFB will be administered to obtain daily drinking and other substance use since baseline.  
Table 1. Assessment Instruments and Timeline 
Instrument Name 
Purpose/Domain 
Informed Consent 
Obtain informed consent 
Youth Interview 
Characterize sample 
MacArthur Socioeconomic Scale 
Assess socioeconomic status 
The MINI International Neuropsychiatric Interview 
Assess DSM-5 psychiatric disorders 
Pregnancy Test for Females 
Study and neuroimaging eligibility 
Clinical Institute Withdrawal Assessment of Alcohol-Revised: CIWA 
Study eligibility, safety
Urine Drug Screen tests: UDS 
Study eligibility, assess drug use 
Vital signs 
Medical safety 
Adverse Events 
Monitor AEs and safety 
Concurrent Medications Form 
Monitor medications 
Medication Adherence Log 
Assess medication compliance 
*Time Line Follow-Back: TLFB 
Primary Outcome; alcohol use (amount and 
frequency) 
Adolescent-Obsessive Compulsive Drinking Scale 
Assess alcohol craving 
Breathalyzer 
Assess alcohol use (biological) 
Alcohol Use Disorders Identification Test: AUDIT 
Assess drinking behavior & problems 
Alcohol Dependence Scale: ADS 
Assess alcohol use severity 
Rutgers Alcohol Problem Index: RAPI 
Assess alcohol problems 
Ethyl glucuronide (EtG) 
Biomarker of alcohol use 
Phosphatidylethanol (PEth) 
Biomarker of alcohol use 
IL-6, IL-10, IL-1B, HMGB1 
Biomarkers of neuroinflammation 
Cannabis Use Disorder Identification Test-Revised: CUDIT-R 
Assess marijuana use & problems 
Marijuana Craving Questionnaire: MCQ 
Assess marijuana craving 
Questionnaire of Smoking Urges: QSU 
Assess nicotine craving 
Fagerstrom Test 
Assess nicotine dependence 
Alcohol Craving Questionnaire 
Assess alcohol craving 
Beck Depression Inventory-II: BDI-lI 
Measure depression 
Columbia Suicide Severity Rating Scale 
Assess for suicidality 
State Trait Anxiety Inventory: STAI 
Measure anxiety 
Barratt Impulsiveness Scale 
Measure impulsivity 
Pittsburgh Sleep Quality Index 
Assess sleep functioning 
Risk Behavior Survey: RBS (selected questions) 
Assess risky behaviors 
Pubertal Development Scale: PDS 
Assess pubertal development stage 
Treatment Services Review 
Monitor services utilization 
Penetration of the blind 
Assess blinding procedures 
Diagnostic Interview Schedule for Children: DISC-IV (selected questions)  
Assess disruptive disorders 
D-KEFS Color-Word Interference Test 
Measure executive functioning 
WRAT-3 Reading Subtest 
Measure cognitive ability 


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Biological markers of alcohol use (10 minutes). Biological samples will be collected to verify self-
reported alcohol use. Specifically, blood phosphatidylethanol (B-PEth) (which will detect persistent 
moderate to heavy drinking in the weeks before testing), urinary Ethylglucuronide (EtG) (which will detect 
any drinking within 2 to 3 days before testing), and blood neuroinflammation markers: IL-6, IL-10, IL-1B, 
and HMGB1,which will detect neuroinflammation both before and after NAC treatment will be assayed by 
the Clinical Neurobiology Lab (CNL) which is a CAP/CLIA certified clinical laboratory directed by Dr. 
Anton (co-mentor) and the laboratory of Dr.Wei Jiang. Urine EtG is measured by an immunoassay 
(Microgenics Corp. – Fremont CA) that has excellent correlation with LC-MS methods (Böttcher et al. 
2008) and can be positive (> 100 ng/ml) up to 72 hours after alcohol consumption (Helander et al. 2009).  
Saliva Collection (10 minutes). Saliva will be collected for oral microbiome analysis. All-in-one collection 
kits will be used for the collection and stabilization of microbial DNA from saliva. DNA extraction and 
Microbiome 16S rRNA Sequencing will be performed. 
Neuroimaging protocol (1 hour). Participants will undergo 3 neuroimaging scans (1 scan at baseline 
and 1 scan after both placebo and NAC conditions). 
Mock scanner: On Visit 1, participants will be introduced to the mock scanner to help them become 
acclimated to the MR environment and give them the opportunity to practice remaining still while in the 
magnet, which will minimize motion-related confounds in the imaging data. 
Scan session: Breath and urine toxicology samples will be collected before every scan. Pre-scan state 
and craving measures are given. Imaging sessions (~60 minutes total including set-up) consist of: 
1. T1-weighted structural: A high-resolution anatomical scan will be acquired, to allow subsequent 
registration to functional images and region-of-interest (ROI) definition. 
2. The Alcohol Cue Reactivity Task: (Schacht et al. 2011; 
Schacht et al. 2013b). During the alcohol cue reactivity 
task, participants are shown pseudorandomly interspersed 
images of alcoholic (i.e., beer, wine, and hard liquor) and 
non-alcoholic (e.g., soft drink, juice) beverages, visual 
control images (i.e., blurred images), and a fixation cross 
(see Figure 3). A magnetic fieldmap will also be acquired 
to allow geometric unwarping and cost-function masking of 
EPI images induced by magnetic field inhomogeneities.  
3. Magnetic Resonance Spectroscopy: The magnetic resonance spectroscopy protocol proposed will be 
utilizing previously published methods used by Drs. Truman Brown and James Prisciandaro.  
NAC or placebo administration. This double blind crossover trial will compare NAC (1,200mg twice 
daily) and placebo. Participants will be randomized to each condition for 10 days in a counterbalanced 
order, with a minimum 11-day washout period between conditions to allow for the clearance of NAC 
(Holdiness 1991). Medication response and tolerability/adverse events will be assessed at each clinic 
visit. Reminders to take medication will be sent via email and/or text message to increase compliance. 
Participants will also be required to upload videos of themselves taking their morning and evening 
medication doses. Participants will be trained on how to record and upload these videos and study staff 
will extract timestamps from these videos to monitor that medication is being taken within the correct time 
frame. 
Participants will be compensated $460 for completing all 5 visits. Also, they can earn up to a $40 bonus 
($2 per day while receiving medication) if they take each dose of medication, or provide staff with an 
Cash Choice Task 
Measure motivation, inhibition, impulsivity 
Alcohol Purchase Task (17-item) 
Measure alcohol purchasing behavior 
Beliefs about Medicine Questionnaire 
Measure medication attitudes  
Med Adherence Video Feedback Questionnaire 
Measure medication adherence attitudes 
Microbiome Feasibility Questionnaire 
Assess feasibility of stool collection for future studies 
Figure 3. Alcohol cue reactivity paradigm. BOLD 
response during alcohol (e.g., beer) vs. neutral 
(e.g., juice) cues will be the main contrast of 
interest for Aim 1. 


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explanation for why the medication was not taken, within a 4 hour requested time window. If they do not 
take the medication due to adverse side effects, they will need to discuss these side effects with 
research staff. Participants experiencing adverse side effects interfering with their ability to participate 
may be taken off the medication. If they are unable to finish the study, they will be prorated for number of 
completed visits. Participants will also be eligible for a $50 referral bonus for each individual they refer 
who successfully randomizes into this study. Mileage reimbursement may also be available to 
participants.  
Data Management  
Sample Size Determination. Crossover designs are highly cost-efficient and powerful designs (Wellek 
and Blettner 2012). Previous research has shown NAC has an effect size of d =1.13 versus placebo on 
glutamatergic levels in the ACC in cocaine dependent adults in a similar randomized, double-blind, 
placebo-controlled within-subjects crossover study (Schmaal et al. 2012). With 32 participants, the 
proposed study will have 85% power to detect such effects (α = 0.05, two-tailed) (Erdfelder et al. 1996). 
Having 50 heavy drinking participants will allow for 20% participant drop out, which is consistent with the 
drop out rate at the 4 week mark in Dr. Gray’s NAC adolescent marijuana trial, as well as exclusion of 
participants due to motion in scanner (Gray et al., 2012). 
 
Analysis and approach. (1) BOLD response to alcohol vs. non-alcohol cues in the regions of 
interest (ACC, insula, striatum, amygdala) and (2) glutamate levels in the ACC will be the primary 
contrasts of interest to test Aims 1 and 2. A generalized linear mixed effect model will examine the effect 
of NAC vs placebo on alcohol cue reactivity and glutamate. The primary model will contain the main 
effect of treatment (NAC vs. placebo), as well as day (scan 1 vs. scan 2) and order (NAC vs. placebo) to 
ensure the crossover design and washout period were successful. During model development, random 
intercepts will be included to account for variations in baseline response levels for individual participants 
and random slopes to account for varying responses to treatment. Model fit will be tested using likelihood 
ratio tests and Akaike Information Criterion values. Baseline levels of clinical and demographic 
characteristics (sex, age, alcohol use characteristics) will be tested for univariate associations with study 
outcomes; when associated, these variables will be included in the adjusted model development 
strategy. Parameter estimates and associated 95% confidence intervals for treatment effects on cue 
reactivity and glutamate will be reported. Model based parameter estimates across groups (means and 
standard deviations) will be used to estimate treatment effect sizes, in order to inform power analysis for 
a subsequent full-scale randomized controlled trial. Power analyses and predetermined statistical 
analyses will ensure findings are robust and unbiased. 
 
We will also analyze the effects of adolescent binge drinking and sex as a biological variable (SABV), as 
compared to a non-drinking control group (1:1 sex ratio), to assess the alcohol-related effects within (1) 
glutamate, GABA, and NAA levels in the ACC, (2) BOLD response to alcohol v. non-alcohol cues in the 
regions of interest (ACC, insula, striatum, amygdala), and (3) microbiome composition. Two-way analysis 
of variance (ANOVA) will be used to test the main effects of group and SABV, as well as their interaction 
(group × SABV). Resulting significant interaction terms will be further tested to assess the effect of group 
within sex (e.g., difference in glutamate levels between drinking and non-drinking females) and the effect 
of sex within group (e.g., difference in glutamate levels between male and female heavy drinkers). 
Clinical and demographic characteristics (age, alcohol use characteristics, GM:BM for MRS data) will be 
tested for univariate associations with study outcomes; when associated, these variables will be included 
in an analysis of covariance (ANCOVA). Power analyses and predetermined statistical analyses will 
ensure findings are robust and unbiased. Summary statistics across groups (means and standard 
deviations) will be used to estimate SABV and alcohol effect sizes, in order to inform power analysis for a 
subsequent full-scale studies. SAS will be used for data analysis with a significance level of α = 0.05, 
corrected for multiple comparisons. 
 
Biological markers. Changes in blood phosphatidylethanol (PEth), urinary Ethylglucuronide (EtG), and 
blood inflammatory marker (IL-6, IL-10, IL-1B, HMGB1) levels from pre- to post-treatment will be 
explored in relation to participants’ change in drinking behaviors. This data can be used to inform power 
analysis for a subsequent full-scale randomized controlled trial. 


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Interpretation of results and future studies. We believe the proposed study is important and will 
inform future treatment options for adolescent substance use. Given the promising findings from 
marijuana-using adolescents (Gray et al. 2012), it is important to examine the effect of NAC on alcohol 
cue reactivity and glutamatergic systems, as modifying these neural systems via pharmacotherapy could 
decrease alcohol use during adolescence. However, some limitations exist. We may find that NAC does 
not affect alcohol cue reactivity or glutamate levels compared to placebo, which in itself would be 
important information and clarify the underlying mechanism of NAC. Regardless of NAC effects, 
understanding how varying levels of drinking affect glutamate will be important in informing future 
targeted therapies. The majority of the preclinical NAC literature was performed on male rodents; sex 
could play a factor in the response to the treatment and will be explored in analyses, as we will be 
collecting data on an equal number of male and female participants. NAC has the potential to 
significantly advance treatment options for adolescent heavy drinkers and could provide a safe, tolerable, 
accessible, and affordable option for adolescents seeking alcohol treatment. 
The long-term goals of this work are to determine if NAC is a promising treatment for adolescent heavy 
alcohol use, which is consistent with the trans-NIH initiative to identify neurally-informed novel substance 
use treatments for adolescents.  
 
Provisions to Monitor the Data to Ensure the Safety of Subjects  
(a) Trial Management: All aspects of the study will be run through the MUSC Department of 
Psychiatry and Behavioral Sciences, where the PI holds her faculty appointment. 
(b) Data Management and Analysis: Data will be collected using standardized paper or 
electronic forms and will only be identified with the study’s ID of the participant. The codes 
linking the name of the participant to the study ID will be kept confidential in a secured cabinet 
by the PI. Collected forms will be securely transported to the PI’s data entry center. Research 
assistants will enter data in REDCap (Research Electronic Data Capture), a secure, web-
based application designed exclusively to support data capture for research studies. REDCap 
provides: 1) an intuitive interface for data entry (with data validation); 2) audit trails for tracking 
data manipulation and export procedures; 3) automated export procedures for seamless data 
downloads to common statistical packages (SPSS, SAS, Stata, R); 4) procedures for 
importing data from external sources; and 5) advanced features, such as branching logic and 
calculated fields. These procedures are effective in minimizing data entry errors (e.g., missing 
or errant data). When possible, participant direct entry into REDCap (rather than paper forms) 
will be utilized.  
(c) Regulatory Issues: The protocol will be registered on the clinical trials registry 
(clinicaltrials.gov). All serious adverse events will be reported to the MUSC Institutional 
Review Board (IRB) within 24 hrs. Follow-up of all serious adverse events will be reported as 
well. All adverse events are reviewed weekly by the PI and yearly by both the DSMB and the 
IRB. Any significant actions taken by the local IRB, including significant protocol changes, will 
be relayed to NIAAA. We anticipate the serious adverse event rate to be extremely low. If 
monitoring indicates otherwise, we will convene a special meeting of the DSMB. 
(d) Trial Safety: The potential risks and benefits and methods to minimize these risks are 
outlined above. Guidelines have been developed for managing and reporting of adverse 
events (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). Drs. Squeglia and Gray will serve as 
the Program Managers for AEs. 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 within 24 hours of the start of the SAE. Relevant 
information will be recorded on SAE Notification Form. The complete report (SAE Notification 
Form, Concomitant Medication Log, and AE Log) will be reviewed with Dr. Gray, and Dr. 
Squeglia will notify the IRB, Data and Safety Monitoring Board (DSMB), and NIH about the 


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SAE. Additionally, Dr. Squeglia will communicate summary reports of DSMB discussion of the 
SAE, or any deliberations of IRB regarding the review of the SAE or the trial itself, to NIH. If 
the event is “Serious, Unexpected and Associated” (an SAE is considered unexpected if it is 
not described in the Package Insert), Dr. Squeglia will complete Food and Drug 
Administration (FDA) Form 3500A and will forward it to the FDA with Dr. Gray’s assistance. 
Dr. Squeglia also will inform the IRB and the study participants (and parents/guardians, as 
appropriate) about the SAE. In all of these reviews and reports, strict patient confidentiality 
will be maintained. 
 
AEs will be coded on a weekly basis using Medical Dictionary for Regulatory Activities 
(MedDRA) rules and entered into a database. For each weekly study meeting, Dr. Squeglia or 
research staff will prepare a summary of all AEs, including their severity and presumed 
relation to study medication. Drs. Squeglia and Gray will review this at their weekly meetings 
(or before if more urgent). 
 
Study procedures will follow as much as possible the FDA’s Good Clinical Practice 
Guidelines. We will encourage participants (and parents/guardians as appropriate) to notify 
their physicians that a) they are in a research study evaluating N-acetylcysteine, and b) the 
physician should contact the PI directly if the physician has any questions. 
 
The research assistants will be instructed not to reveal whether a person is a participant in the 
study and will report to the PI any outside requests for information about a participant or any 
breaches in confidentiality. All requests by participants’ physicians and other medical 
providers will be referred directly to the PI.  
 
(f) Trial Efficacy: The Data and Safety Monitoring Board may request a blinded interim 
efficacy report (blinded to the PI and research team) for review while the trial is ongoing. Final 
(fully unblinded) analyses will occur after all participants have completed all visits. 
 
(g) Data and Safety Monitoring Plan Administration: The PI will be responsible for 
monitoring the trial and will examine the database regularly for missing data, unexpected 
distributions or responses, and outliers. The PI will prepare the weekly adverse event 
database immediately prior to the weekly meetings with Dr. Gray to a) see if any particular 
MedDRA categories are being endorsed more frequently than anticipated, and b) determine if 
any adverse event symptom checklist scores are higher than expected. A DSM report will be 
filed with the IRB and NIAAA on a yearly basis, unless greater than expected problems occur. 
The report will include participant characteristics, retention and disposition of study 
participants, quality assurance issues and reports of adverse events, significant/unexpected 
adverse events and serious adverse events.  
 
Data and Safety Monitoring Board: We will create a DSMB, comprised of multidisciplinary 
faculty with expertise in pharmacotherapy and behavioral treatment trials (youth and/or 
addiction-related). The DSMB will meet annually (more frequently as needed for emergent 
situations) to review any AEs related to the study, as well as review any data management 
related errors. The board may be called at any point if needed for serious adverse events, etc. 
Modification will be made in the procedures and/or the protocol if necessary based on the 
findings of the board. 
 
Risks to Subjects 
Questionnaires and interviews are all non-invasive and, as such, involve minimal physical risk to 
participants.  
In order to address any concerns regarding coercion, adolescents and parents will be informed that they 
are free to choose not to participate and may withdraw at any time (this is included in the consent and 


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assent forms). Since this study involves minors, particular caution will be exercised in obtaining informed 
adolescent assent separately and independently from parental consent. To this end, an initial step in 
subject recruitment involves obtaining parental/legal guardian permission for participation by the 
adolescent. Once parental consent is secured, subjects will be asked separately and independently for 
informed consent (i.e., parental consent will not be used to persuade teens to participate). This approach 
is considered very effective in minimizing coercion to participate. 
 Potential risks incurred by participants include: 
1. Adverse events related to study medication 
2. Loss of confidentiality 
3. Adverse events related to MRI 
Adverse events related to study medication. N-acetylcysteine has a generally benign adverse effect 
profile. A meta-analysis of studies evaluating long-term oral treatment with N-acetylcysteine for prevention 
of chronic bronchitis found that N-acetylcysteine was well tolerated, with generally mild, most commonly 
gastrointestinal adverse effects that did not require treatment interruption (Grandjean et al. 2000). Our 
experience to date with N-acetylcysteine at the same dose proposed in this trial in youth with cannabis use 
disorder suggests a benign adverse event profile (Gray et al. 2012). 
Some patients who have taken intravenous N-acetylcysteine for the treatment of acetaminophen overdose 
have had more serious reactions. Allergic reactions have occurred in about 5% of patients taking 
intravenous N-acetylcysteine (Bailey & McGuigan 1998). These reactions may be mild, consisting of 
flushing, rash, and itching. Less common side effects include trouble breathing, low or high blood pressure, 
fever, and hives. If untreated, such a reaction could lead to death. Even more rare serious side effects of 
intravenous N-acetylcysteine are irritability, confusion, and seizures. These reactions (severe allergic 
reaction or seizures) have never been reported when N-acetylcysteine is taken orally, as it will be in this 
study. As a precaution, we will exclude individuals with a recent history of asthma, as they are believed to 
possess a higher risk of allergic reaction to N-acetylcysteine. We will also exclude individuals with a history 
of seizure disorder. 
Loss of confidentiality. Any communication of personal information carries the potential risk of breach of 
confidentiality. 
Adverse events related to MRI. Risks associated with MRI (including fMRI) are minimal for individuals 
who do not have metal and are not claustrophobic. Some discomfort may result from lying in the scanner 
for up to 60 minutes.  
Procedures for protecting against potential risks  
Adverse events related to study medication. The informed consent process will be used to thoroughly 
educate participants and parents/guardians about potential medication-related risks, including adverse 
events. This discussion will include thorough review of adverse events associated with oral NAC 
treatment. Rigorous screening procedures and strict exclusion criteria are designed to exclude potential 
participants at elevated risk for adverse events. Drs. Squeglia and Gray will conduct serial adverse 
events monitoring as part of medication management. Participants and parents/guardians will have 
access to the study medical clinician 24 hours, 7 days a week for emergencies. Participants experiencing 
intolerable adverse events will have the opportunity to reduce dose or discontinue medication altogether, 
while remaining in the study for ongoing monitoring. Primary mentor Dr. Gray has full hospital admitting 
privileges in the event of an adverse event requiring hospitalization. Urine pregnancy tests will be 
conducted for female participants. 
 
Adverse events related to confidentiality. Participants will be assured that all records will be kept 
confidential in research files located in a locked office and entered into a password-protected computer 
located behind a secure and maintained firewall. Breach of confidentiality is highly unlikely because all 
personally identifying information will be kept separate from data collected and will be linked only by a 
master subject identification list which is maintained by the PI.  
 


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Adverse events related to MRI. Participants will be told about the potential discomforts associated with 
lying in the scanner and their heads and necks will be supported for maximum comfort. Earplugs and 
headphones will be provided to reduce the noise of the scanner to a safe decibel range. Pregnancy 
testing with immediate results will ensure that no pregnant adolescent receives an MRI. Any positive 
pregnancy test results would be discussed with the adolescent, and counseling will be provided should 
the participant indicate distress concerning the result. 
 
Ensuring professional intervention in the event of adverse effects: If a participant should become 
uncomfortable with any procedures, he/she can withdraw from the study at any time. If any medical 
problems arise, 911 will be phoned immediately. If psychological distress, suicidal ideation, or another 
problem should occur, the clinically-licensed PI or a designated backup licensed professional will be 
contacted immediately. Paperwork detailing the appropriate steps to handling suicidal/homicidal ideation 
or abuse/neglect cases is posted above every research assistant’s phone, along with contact numbers of 
the PI and Co-Is. 
 
Policy on neuroimaging data: Participants and their family (for those under age 18) are informed that 
this is a research study and therefore does not include clinical imaging to confirm clinical findings. 
Therefore, we cannot provide them with medical reports. If the research staff note a structural irregularity 
on a scan, a neuroradiologist will review it (fully de-identified) and note if it is of clinical significance or 
not. If the finding has the potential of clinical significance, the volunteer or parents (depending on age) 
will be notified to follow up with his or her doctor. There is a minimal risk of undue stress or concern if the 
finding is determined to be benign or not clinically significant. Furthermore, there may be costs 
associated with the recommended evaluations which are not covered by our research protocol. We 
include the following in the consent: “If the possibility of such a situation is too stressful for you, your child 
should not participate in our study.” 
 
Potential Benefits to Subjects or Others 
Potential benefits of the research to subjects and others: Participation in this study involves minimal 
risk for participants. Benefits of participation include providing substantial long-term benefits toward 
understanding the extent to which NAC can affect underlying neural circuitry during adolescence and 
alcohol use.  
 
Why risks to subjects are reasonable in relation to anticipated benefits: Given the minimal risks to 
participants involved, we believe the risk/benefit ratio is acceptable. Youth in the study may decrease 
their alcohol use which could positively affect other aspects of their lives. 
 
 
 


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SAP_004.pdf: 
 
 
 
1 
Neuroscience-Informed Treatment Development for Adolescent Alcohol Use  
NCT03238300 
November 30, 2022 
 
 


 
 
 
2 
Aim 1:  
 
Hypothesis/aim 
Primary:  
1a. Quantify the effect of NAC versus placebo on glutamate levels in adolescent heavy drinkers. 
Using MRS, we will determine the effect of NAC versus placebo on modulating anterior cingulate 
glutamate levels in heavy alcohol-using adolescents.  
 
Hypothesis: Heavy drinking youth will show decreasing levels of glutamate from baseline in the anterior 
cingulate while on NAC compared to placebo. 
 
Sample size for aim 
N= 31 – all with 4 sessions (randomization visit 1, MRI visit 2 & 4, washout visit 3) 
 
Independent variable(s) 
NAC v. Placebo: both NAC and placebo were administered twice daily (2400 mg of NAC/day, 
administered as 1200 mg twice/day) over 10 days 
Dependent variable(s) 
dACC glutamate levels  
Handling of missing data 
Only participants with complete data will be included (N=31) 
 
Complete data = screener, visit 1-4 
How outliers and excluded 
observations will be defined 
Participants with all 4 sessions (visit 1-4) will be included.  
 
MRS data will be examined for quality using:  
1. Linewidth (exclusion: >3 SD from mean) 
2. Signal-to-noise ratio (SNR) (exclusion: >3 SD from mean) 
 
No other outliers will be removed.  
Software and specific 
software modules or 
packages 
Software: R 
Packages: Tidyverse, lmer4 OR glmer4 & lmerTest 
Analytic procedure(s) 
Generalized linear mixed effect models will examine the effect of NAC vs placebo on 
neurometabolites (Primary: glutamate) within the adolescent heavy drinking group. 
 
The primary model will contain the main effect of treatment (NAC vs. placebo), as well as day 
(period: scan 1 vs. scan 2) and order (sequence: NAC/placebo vs. placebo/NAC) to ensure the 
crossover design and washout period were successful.  
 
During model development, random intercepts will be included to account for variations in 
baseline response levels for individual participants (1|id) 
 


 
 
 
3 
Covariance structure will be determined based on best fit after trying standard options (e.g., 
unstructured, Toeplitz, autoregressive, etc.) 
 
Model fit will be tested using likelihood ratio tests, Akaike Information Criterion values, and 
interclass correlation.   
 
Parameter estimates and associated 95% confidence intervals for treatment effects on DVs.  
 
Steps to confirm data 
assumptions are met 
Generalized linear effect mixed models 
-Check normality of data using Shapiro-Wilks test &/or QQ plot 
-Divide generalized chi-square by its degrees of freedom to check for dispersion 
-Compare averages of outcomes to predicted values to check appropriateness of link function 
-Transform data (if needed)   
-Check for multicollinearity among predictor variables 
-Check for carry over effect in crossover models 
 
Generalized linear models 
-Plot deviance residuals against fitted values to evaluate linearity for GLM 
-Look for outliers using leverage & Cook’s distance &/or a QQ plot for GLM 
-Transform data if needed but only on predictor variables 
A priori covariates and/or 
means for identifying 
important covariates  
Baseline levels of clinical and demographic characteristics will be tested for univariate 
associations with study outcomes; when associated, these variables will be included in the 
adjusted model development strategy.  
 
Primary:  
• 
Age 
• 
Sex 
• 
Alcohol use variables  
 
Exploratory: 
• 
Cannabis use variables  
A priori interactions to be 
examined and rules for 
retaining interaction terms 
None. 
Planned subgroup or 
sensitivity analyses 
N/A 
Level of significance and 
whether/what corrections for 
multiple testing will be used 
P<0.05, no correction for multiple comparisons  


 
 
 
4 
(if null hypothesis 
significance testing is used) 
Required data 
cleaning/formatting steps 
RedCap:  
1. Download baseline variables 
a. Age 
b. Sex 
c. Group (drinker v. control) 
d. Mental health measures (anxiety, depression) 
2. Download drinking variables from TLFB 
a. Total drinking days (visit 1-4) 
b. Total drinks/drinking day (visit 1-4) 
c. Total binge occasions (visit 1-4) 
3. Download other substance use variables from TLFB 
a. Total cannabis use days (visit 1-4) 
b. Total cannabis use/use day (visit 1-4) 
c. Total vaping days (visit 1-4) 
d. Total cigarette days (visit 1-4) 
 
MRS: 
4. Combine excel spreadsheets for all visits and metabolites  
5. Remove unnecessary variables 
a. Final variables: 
i. Metabolite levels (I.U.) 
ii. Linewidth 
iii. SNR 
 
Combine RedCap + MRS Data: 
6. Merge RedCap and MRS data sheets 
7. Remove participants without full data (final N=41) 
8. Remove observations that don’t meet criteria for inclusion 
 
 
 
 
 
 
 
 
 
 
 


 
 
 
5 
Aim 2:  
 
Hypothesis/aim 
Primary Aim #1: 
Aim: Quantify the effect of NAC versus placebo on alcohol cue reactivity in adolescent heavy drinkers 
Hypothesis 1: Heavy drinking youth with show decreasing alcohol cue reactivity from baseline in key reward 
regions (insula, striatum, and amygdala) while on NAC compared to placebo 
 
Sample size for aim 
Initial sample size: n = 31 (heavy drinkers; 17 female & 14 male) 
Sample size for complete data: n = 27 (14 female & 13 male) 
Independent variable(s) 
Medication: NAC vs. Placebo  
Dependent variable(s) 
Primary Aim #1:  
Primary contrast: BOLD response (alcohol v. non-alcohol cues) in 10 regions of interest (left and right): 
amygdala, insula, caudate, nucleus accumbens, and putamen  
Handling of missing data 
Multiple imputation will be used for missing data (‘mice’ package) on independent variables only.  
How outliers and excluded 
observations will be defined 
Participants with excess head motion (defined as mean relative motion >0.2 mm) and missing cue-reactivity 
data at baseline will be removed (n = 4).  
Outliers will be checked with Q-Q plots and examination of residuals.  
Software and specific software 
modules or packages 
Software: R 
Packages: Tidyverse, lrtest, LME, Performance  
Analytic procedure(s) 
Generalized linear mixed effect model will examine the effect of NAC vs placebo on alcohol cue reactivity 
- 
The primary model will contain the main effect of treatment (NAC vs. placebo), as well as day (scan 
1 (visit 2) vs. scan 2 (visit 4)) and order (NAC/placebo vs. placebo/NAC) to ensure the crossover 
design and washout period were successful. 
- 
During model development, random intercepts will be included to account for variations in baseline 
response levels for individual participants 
- 
Model fit will be tested using likelihood ratio tests, Akaike Information Criterion values, and 
interclass correlation.  
- 
Model based parameter estimates across groups (means and standard deviations) will be used to 
estimate treatment effect sizes 
  
Steps to confirm data 
assumptions are met 
Check for normality of data with Q-Q plot 
Check for appropriate link function by comparing averages of outcomes versus predicted values 
Check for overdispersion by dividing generalized chi-square by degrees of freedom.  
Check for multicollinearity among predictors with VIF (cut-off of 2) 
A priori covariates and/or 
means for identifying important 
covariates  
Day and Order will be included in all models. 
 
Aim #1 and exploratory covariates: Baseline BOLD response (alcohol v. non-alcohol cues) in relevant 
region of interest (i.e. in models with ACC as the outcome, BOLD response at baseline in ACC will be the 
covariate), alcohol use (average standard drinks per drinking day, total quantity of standard drinks, number 
of drinking days, and number of binge episodes), AUD status (Yes/No defined by meeting DSM-5 criteria 
at screening) or AUD severity (none, mild, moderate, or severe defined by DSM-5 criteria at screening), 
cannabis use days, age, sex, and race. 


 
 
 
6 
 
‘Performance’ package will be used to determine best fitting model with select covariates to reduce issues 
related to multicollinearity – only 1 alcohol covariate will be used, and will be specific to each model 
A priori interactions to be 
examined and rules for 
retaining interaction terms 
N/A 
Planned subgroup or sensitivity 
analyses 
Sensitivity analysis will compare results from complete data only model (n = 27) and imputed model (n = 
31) to confirm the imputed data does not significantly change the results.   
 
 
Level of significance and 
whether/what corrections for 
multiple testing will be used (if 
null hypothesis significance 
testing is used) 
p < 0.05, no correction for multiple comparisons 
Required data 
cleaning/formatting steps 
1. Complete second-level cue-reactivity data processing in Freesurfer and extract relevant data for 
models (average percent change in BOLD signal between alcohol vs. non-alcohol cues from the  
ROIs) 
2. Download data for covariates from Redcap and look at descriptive statistics to assess accuracy 
and distributions 
3. Download raw TLFB data from Box 
4. Calculate TLFB summary variables: average standard drinks per drinking day, total quantity of 
standard drinks, number of drinking days, and number of binge episodes during 10-days on 
treatment 
5. Combine data from Redcap and output from fMRI preprocess pipeline and Freesurfer 
6. Complete multiple imputation for missing data 
7. Run models on imputed dataset and completers only dataset to check for any differences