Probiotics, Immune Function, and the Brain in Alcohol Consumers
Phase: PHASE2
Conditions: Alcohol; Use, Problem
Sponsor: Brown University

COVER PAGE 
 
OFFICIAL TITLE: Probiotics, Immune Function, and the Brain in Alcohol Consumers 
 
NCT NUMBER: NCT05232682 
 
DOCUMENT DATE: October 25, 2022 

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THE BLUE TEXT IN THE FOLLOWING SECTIONS IS A GUIDE TO ENSURE ALL RELEVANT 
INFORMATION IS INCLUDED IN YOUR APPLICATION. YOU MAY DELETE THE BLUE TEXT 
BEFORE SUBMISSION 
If your study ONLY involves the use of identifiable secondary data / biospecimens, 
including coded data from which you may be able to ascertain participant identity, skip to 
PART V. Otherwise, please continue to next page. 
1. Introduction and Background. In reviewing the protocol, the IRB must consider the rationale for the
study and the importance of the knowledge that may reasonably be expected to result.
Animal models and clinical studies show preliminary support for probiotic supplementation to ameliorate
negative physiological consequences of heavy drinking, specifically inflammation. However, previous
clinical trials have focused on alcohol-related complications, usually alcoholic liver disease. Such
studies have been conducted in controlled settings with supervised abstinence from alcohol. As a
result, it is not known whether probiotics improve alcohol-related inflammation in the absence of liver
disease or in individuals who continue to drink heavily. The latter factor is important, as many
individuals who currently are not seeking to cut down on drinking may nevertheless be willing to
consume a dietary supplement to improve their health. In summary, probiotic therapy may be an
acceptable intervention to reduce alcohol-related tissue injury in individuals not seeking to change their
drinking behavior, but no clinical studies to date have addressed this population. This study will
investigate effects of probiotics on inflammation in healthy heavy drinkers who currently are not seeking
to change their alcohol use (i.e., are non-treatment-seeking).
2. Specific Aims and Study Objectives. The IRB must evaluate the objectives of the research in order to
determine whether the risks to participants are reasonable in relation to the importance of the
knowledge that may be gained.
This pilot project is a brief, open-label clinical trial of probiotics as an intervention to reduce systemic and 
neural inflammation in heavy drinkers. Heavy drinkers who do not yet show significant signs of liver disease 
also may stand to benefit from probiotics, but no clinical trials to date have addressed this population. This 
study will recruit 15 non-treatment-seeking heavy drinkers to complete an open-label within-subjects trial. Aim 
1 is to demonstrate proof-of-concept for beneficial effects of probiotic use on inflammatory processes. Aim 2 is 
to examine effects of probiotic use on brain metabolites correlated with neuroinflammation using magnetic 
resonance spectroscopy. Aim 3 is to gather preliminary data on acceptability and feasibility of the probiotic 
intervention in non-treatment-seeking heavy drinkers. 
Aim 1: Demonstrate proof-of-concept for beneficial effects of probiotic use on inflammatory processes, as 
reflected in plasma markers of microbial translocation, immune activation, systemic inflammation, and liver 
function. 
Aim 2: Examine effects of probiotic use on brain metabolites correlated with inflammation using magnetic 
resonance imaging (MRI). 
Aim 3: Gather preliminary data on acceptability and feasibility of the probiotic intervention in the specific 
population of interest, i.e., non-treatment-seeking heavy drinkers. 
PART III. RESEARCH DESIGN & METHODS 
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3. Materials, Methods and Analysis. The study design, methods and procedures must be adequately 
described in order for the IRB to understand all activities in which human subjects will participate. The 
IRB must also be able to differentiate those procedures that are performed for research purposes from 
those that are performed for routine care or evaluation. 
 
The study will enroll participants to complete a brief open-label clinical trial of a probiotic dietary supplement. 
Individuals who provide informed consent and are eligible for participation will attend three study visits. At Visit 
1, the research team will collect data on demographics, substance use, and medical history. Eligibility will be 
determined using the criteria listed below. Blood samples will be taken by a licensed healthcare provider. 
Eligible participants will complete a one-hour magnetic resonance imaging (MRI) scan, to occur on a separate 
day due to MRI scheduling constraints. Participants will receive a 30-day supply of the probiotic supplement 
with dosing and storage instructions. Participants will be instructed to take the standard dosage of 2 probiotic 
capsules per day. Participants will be asked to respond to a daily query from the research team confirming that 
they have taken their daily probiotic dose. Visit 2 will be scheduled 15 days (plus or minus 5 days) after the 
initiation of the probiotic supplement. The research team will collect blood samples and assess alcohol 
consumption since Visit 1, protocol adherence, and potential side effects. Participants will attend Visit 3 upon 
completion of the probiotic supplement, to occur 30 days (plus or minus 5 days) after initiation. Data will be 
collected as described for Visit 2, plus participants will complete a final one-hour MRI scan. Visit 4 will take 
place 1-10 months after completion of the study probiotic as a “washout” time point. A blood sample and 
substance use assessment will be collected at Visit 4. Appointments will take place at the Center for Alcohol 
and Addiction Studies lab space in the School of Public Health, and MRI scans will be conducted at Brown’s 
Magnetic Resonance Imaging Facility (MRF). 
 
Data to be collected at each session is described below. 
• 
Visit 1 (Baseline Screening & MRI): Breath alcohol content (BrAC) will be measured using a handheld 
digital sensor to confirm BrAC of .00 g% at the time informed consent is obtained. Age, weight, 
pregnancy status, urine drug metabolites, recent alcohol use, treatment-seeking status, MRI 
safety/compatibility, safety for blood draw, basic medical history, and medication use will be assessed 
per Eligibility Criteria listed in Section 4. The research team will check the individual’s state-issued 
photo ID to verify age and identity. Height and weight will be measured using a standard scale. Urine 
samples will be collected to test for presence of drug metabolites. Urine samples from participants who 
are able to become pregnant will be tested for pregnancy hormones. Basic demographic information, 
social history, and health-related data will be collected on questionnaires. Questionnaires on alcohol 
use and related symptoms (Alcohol Use Disorders Identification Test; AUDIT) will be administered. 
Blood samples will be taken for testing of biomarkers per the research aims. The Timeline Followback 
(TLFB) measure will be used to assess alcohol use in the past 30 days. Current or recent seeking of 
treatment for alcohol or drug use will be assessed. Participants will be interviewed for history of major 
medical conditions and current medication usage. Because it is necessary to confirm eligibility prior to 
MRI scanning and because advance notice is needed for MRI scheduling, participants will be asked to 
return on a separate day to complete a one-hour MRI scan of the brain at Brown’s MRI Research 
Facility. Participants who are able to become pregnant will be given a urine pregnancy test prior to MRI 
scanning. BrAC of .00 g% will be confirmed prior to the MRI scan. Eligible participants will be given a 
30-day supply of the probiotic supplement with dosing and storage instructions. The research team will 
collect contact information and request permission to send daily emails to confirm participants’ 
adherence to the study protocol. 
• 
Visit 2: Participants will complete questionnaires on side effects and adherence to the study protocol. 
Blood samples will be taken for testing of biomarkers. BrAC of .00 g% will be confirmed prior to study 
procedures. 
• 
Visit 3 (Day 30 of Probiotic Intervention): The TLFB will be used to assess alcohol use since Visit 1. 
Side effects, adherence data, and blood samples will be collected as before. Participants who are able 
to become pregnant will be given a urine pregnancy test prior to MRI scanning. BrAC of .00 g% will be 
confirmed prior to the MRI scan. Participants will complete a one-hour MRI scan. They will be debriefed 
about the study and given the opportunity to ask questions at study completion. 
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• 
Visit 4: A post-assessment visit will be conducted 1 to 10 months after completion of the probiotic. The 
Timeline Followback will be used to assess alcohol use in the past 30 days. A blood sample will be 
taken. 
 
Probiotic Supplement Storage and Dispensing: 
• 
The probiotic product will be stored in a locked cabinet in the PI’s office until the time of dispensing to 
the participant. Participants eligible for the study will receive a 30-day supply with dosing and storage 
instructions per the manufacturer. Any unused probiotic products at the end of the study will be 
disposed of by the PI by placing the unused product in a waste bin, or taken home by study staff. This 
product is not a controlled substance or considered hazardous with regard to storage or disposal. 
 
Procedures for a Positive Breath Alcohol Reading (BrAC): 
In the event that a participant has a positive BrAC reading, the visit will be rescheduled for another date. If a 
participant has a repeated positive BrAC reading, the participant will be withdrawn from the study. If a 
participant’s BrAC exceeds the legal limit for operating a motor vehicle (>.08%) at any visit, the person will be 
asked to wait at the research site until the BrAC reading is under .08%. The person will be offered a rideshare 
for transportation home at study expense. 
 
4. Participant Population. In order to approve research, the IRB must determine that the selection of 
participants is equitable and reasonably related to the purpose and aims of the research. The IRB must 
also consider whether adequate safeguards are in place to minimize any risks that are unique to 
vulnerable populations. To make this determination, the IRB must review all methods and materials 
used to contact and recruit potential participants, including letters, flyers, emails, etc. 
 
Human participants will be healthy, non-treatment-seeking adult heavy drinkers recruited from the community. 
Participants will not be asked or encouraged to change their alcohol use as part of the study. Up to 100 
individuals will be enrolled to obtain a final sample of 15 individuals to complete the study. The sample size is 
appropriate for a pilot study intended to generate preliminary data for future full-scale work. Full eligibility 
criteria are listed below. 
Inclusion Criteria: 
1) 18-64 years of age; 
2) Able to speak and read English well enough to complete study procedures; 
3) Meets NIAAA guidelines for heavy drinking in the past 30 days, defined as at least one of the 
following: 
A) at least 4 heavy drinking episodes (≥4 drinks for women, ≥5 drinks for men on a given day); 
B) at least 2 heavy drinking weeks (>7 drinks for women, >14 drinks for men per week). 
 
Exclusion Criteria: 
4) Chronic disease requiring daily use of medication; 
5) Seeking or receiving treatment for alcohol/drug use, with exception of smoking cessation; 
6) Self-reported history of liver disease; 
7) Antibiotic or probiotic use in past 1 month, wherein probiotic use pertains only to supplements and 
not to foods that contain probiotics such as yogurt, kefir, kimchi, etc.; 
8) Positive urine test for amphetamine, cocaine, methamphetamine, opioids, or benzodiazepines 
(cannabis use will be assessed but is not an exclusion criterion); 
9) History of fainting, weakness, infection, excessive bruising, or extreme distress from blood draw; 
10) Safety contraindication for MRI (e.g., ferromagnetic implant in the body, claustrophobia); 
11) History of head trauma with loss of consciousness >10 min; 
12) For people able to become pregnant: Pregnant, breastfeeding, or not using effective birth control; 
13) Unable to complete the study visits due to time or scheduling constraints; 
14) Weight <110 lbs (due to blood draw guidelines). 
15) Conditions of immunodeficiency, such as HIV infection, primary immune deficiency, or taking 
immune-suppressant medications. 
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Vulnerable Populations: Vulnerable populations (e.g., fetuses, pregnant women, prisoners, children) will not be 
included in this study. 
 
5. Recruitment Methods 
Participants will be recruited from the general community using print or online advertisements (Google), 
flyers posted in public venues, listservs (Brown University listservs), postings on social media (Facebook), 
Today@Brown postings, and online bulletin boards (Craigslist). Contact information will be provided for 
follow-up. Interested individuals who contact the study will complete a brief (10-minute) telephone pre- 
screening interview administered by research staff. Individuals will be prescreened by phone to assess key 
eligibility criteria and interest in participation. Information collected from the prescreening interview will not 
be retained for research purposes. Before beginning the interview, staff will confirm that the caller is in a 
private location. Ineligible individuals will be informed of their ineligibility and thanked for their interest. 
Eligible individuals will be scheduled for Visit 1. The PI and Research Assistant will complete all applicable 
training in human subjects research protections prior to recruiting participants. The study will be advertised 
as not providing treatment or medication, such that the probiotic supplement should not be confused with 
clinical care. 
 
6. Compensation / Reimbursement 
 
Participants will be compensated for their time and effort as follows: $50 for Visit 1 screening (approximately 
2.5 hours) and $50 for Visit 1 MRI (approximately 1.75 hours); $30 for Visit 2 (approximately 1.5 hours); $80 for 
Visit 3 (approximately 3 hours); $1 per day for each day the participant responds to emails about adherence 
(maximum $30); $40 for Visit 4 (approximately 1.5 hours). Participants found not eligible to complete the study 
at Visit 1 will be given compensation prorated at $20/hour. Participants will be offered reimbursement for 
parking and/or transportation as needed. Payments will be made by electronic gift card, specifically an Amazon 
gift card. The compensation rate of approximately $20/hour is appropriate for the time, effort, and activities 
involved in participation. Participants will be paid at the completion of each visit. For the daily response 
compensation, participants will be paid at the end of the 30-day period for the total of days that they 
responded. 
 
 
7. Potential Research Risks / Discomforts to Participants. In order to approve the research, the IRB 
must consider the risks posed to participants by the research and any efforts to mitigate those risks. The 
IRB needs to determine that the risks have been both minimized and are reasonable in relation to the 
anticipated benefits to participants, as well as to the importance of the knowledge that may be gained. 
The IRB will also consider whether the informed consent process provides potential participants with an 
accurate and fair description of the risks or discomforts. 
 
1. Risk of side effects from probiotic supplementation: The study will utilize a commercially available probiotic 
supplement product (Seed DS-01 Daily Synbiotic) that has an excellent safety and tolerability profile. The 
most common side effects of probiotics are abdominal bloating and/or gas, which typically dissipate after the 
first few days of use. The Seed DS-01 Daily Synbiotic undergoes rigorous quality control testing for 
contaminants (e.g., heavy metals, pesticides) and allergens (e.g., nuts, gluten, milk) and is the subject of 
ongoing independent clinical trials for irritable bowel syndrome and post-antibiotic recovery. The product is 
vegan, preservative-free, and free of common allergens (soy, dairy, gluten). Probiotic supplements are not 
regulated by the FDA or subject to IND requirements. 
 
 
2. Risks of MRI scan: MRI has no known side effects and does not involve radiation. However, there are 
several contraindications for MRI scan. The first is metallic materials within the body, such as aneurysm clip, 
bullet or shrapnel fragment, ear implant, certain prosthetic devices, heart pacemaker, or artificial heart valve. 
Participants will be thoroughly screened, first by the researcher and again by the MRI technician, to prevent 
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admission of individuals with these contraindications to the MRI suite. The second contraindication is 
pregnancy, as risks to the fetus are unknown. Pregnancy tests will be administered to all participants able to 
become pregnant at the baseline assessment and again on the days of MRI scanning. Aside from medical 
contraindications, there is risk of subjective discomfort from being in a small space or from noise emitted by the 
scanner. Participants will be given a call button to hold during the MRI so that they can communicate distress 
to the MRI technician and research staff, and procedures can be discontinued if indicated. Participants will be 
required to wear earplugs to reduce exposure to noise from the scanner. 
 
 
3. Psychological discomfort associated with assessment: Participants will be advised during the informed 
consent process that they may choose not to answer any particular question, that their verbal and written 
responses are confidential, and that they are free to withdraw from the study at any time. All data collection 
procedures will be performed in private rooms reserved for these purposes. 
 
 
4. Breach of confidentiality of personal identifiable information (PII): Several steps will be taken to protect 
against loss of confidentiality. All data will be collected and stored using technology that employs the highest 
industry standards for security. Data will be stored in de-identified, password-protected files on Brown 
University’s secure servers. Participant data and samples will be identified using a unique study ID code not 
related to their PII. Participants’ personal identifying information will be kept in a separate location from their 
response data. At the conclusion of the study, the file that contains links of personal identifiers with unique 
study IDs will be destroyed by the study PI. Biological samples will be identified with the participant’s study ID 
and will not be associated with PII. With these precautions, loss of confidentiality is considered very unlikely; 
however, participants will be informed of the potential risk at the time they provide consent. 
 
 
5. Risks associated with blood draw procedures. During the informed consent process, participants will be 
advised of the necessity of obtaining blood samples throughout the trial. The study will exclude individuals who 
report physical or psychological discomfort caused by blood draw or the sight of blood, or who report history of 
excessive bruising, fainting, or weakness following standard blood draw procedures. Blood draw procedures 
will follow World Health Organization guidelines for safe and well-tolerated procedures to minimize risk of 
infection or other complications. A licensed research nurse will collect the blood samples. Risk reduction 
strategies are to use sterile, single-use implements; surface disinfectant; proper hand hygiene; application of 
70% isopropyl alcohol to the blood draw site; minimal vein probing; application of pressure after drawing blood; 
and non-latex, vinyl gloves. 
 
 
6. Risk of coercion from compensation received for participation: The risk of coercion from compensation for 
participation is low, as the total amount of compensation is modest. The hourly compensation rate for the study 
sessions translates to approximately $20/hour, which is consistent with rates offered by similar studies in the 
Brown University area and is intended to offset the effort required of participants outside of study sessions (i.e., 
daily adherence to the probiotic protocol). 
 
 
7. Risk of adverse reaction to the probiotic supplement: In the unlikely event that a participant reports adverse 
reaction to the probiotic beyond the mild side effects noted in #1 above, the participant will be instructed to 
discontinue the probiotic and will be withdrawn from the study. 
 
 
Standard of Care: There is no established “standard of care” for healthy heavy drinkers who are not seeking 
treatment. The probiotic supplement does not replace or otherwise alter any clinical care that participants 
would receive. At the conclusion of the study, participants will be given an NIAAA pamphlet on the risks of 
heavy drinking. 
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PART IV. INFORMED CONSENT 
Informed consent is a process, not just a form. The IRB must ensure the informed consent process clearly 
discloses and facilitates the understanding of all information needed to make an informed decision to 
participate while promoting the voluntariness of participation. 
Please use the Brown consent / assent templates and related guidance on the HRPP Forms & Templates page 
to develop your consent forms. 
DSMP/DSMB: The DSMP is attached and contains information on the DSMB. 
 
8. Potential Benefits of the Research. NOTE: Compensation for participation is not a benefit and 
should not be included in this section. In order to approve this research, the IRB must determine that 
the potential benefits to research participants are reasonable in relation to the potential risks. Very 
often, research at Brown does not include potential direct benefits to participants, but may only benefit 
society as a whole by helping researchers. 
 
Some individuals experience a noticeable benefit in health when using probiotics. Many subjects in this study 
are unlikely to benefit directly from participation. Benefits to society are expected to accrue through generation 
of scientific knowledge about effects of probiotics on inflammation in heavy drinkers. 
 
 
 
 
1. Describe the informed consent process, including: 
 
Informed consent will be obtained at Visit 1. BrAC of .00 g% will be confirmed at the time that consent is 
obtained. The nature of the study will be described verbally and in writing. Potential participants will be 
informed that study participation is voluntary and that they have the right to terminate study procedures at any 
time. Participants will be informed that the alternative is to not participate. Written informed consent will be 
obtained by the PI or trained Research Assistant. The Research Assistant will receive one-on-one training from 
the PI in consent procedures. The names and contact information of the PI (Dr. Monnig) and study staff will be 
provided on the consent document in case questions arise. Participants will receive a copy of the signed consent 
document, and the original form will be kept in a locked filing cabinet. 
 
 
2. Facilitate Understanding 
 
Participants will be encouraged to ask questions to clarify the nature of the study and to ensure understanding. 
Participants who decide to withdraw from the study at any point prior to completion will be paid for their 
participation up to that point. 
 
 
3. Documentation 
 
Participants will indicate informed consent by signing the consent form. 
 
4. Additional Considerations 
Brown University IRB Original Approval: 10/21/2021 
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STATISTICAL ANALYSIS PLAN 
 
The goal of this analysis was to observe the effect of the probiotic on plasma concentrations of sCD14, 
sCD163, LBP, IL-6, IL-8, IL-10, MCP-1, and TNF-a. Of the 16 eligible participants who initiated the probiotic, 
14 participants completed the probiotic course, and two were lost to follow-up prior to completion. Ten 
participants returned after the washout period for Visit 4. Descriptive statistics were obtained on demographic 
characteristics and drinking history. All collected datapoints were considered for analysis regardless of probiotic 
completion or loss to follow-up. Biomarker concentrations greater than three standard deviations from the mean 
were excluded to limit the impact of statistical outliers on the analysis. Regarding undetectable biomarker 
values, sCD14 had 3.7% missing data (n=2), IL-6 had 35% missing data (n=19), IL-10 had 5.6% missing data 
(n=3), LBP had 1.8% missing data (n=1), and MCP-1 had 5.6% missing data (n=3). There were no undetectable 
biomarker values for IL-8, sCD163, or TNF-a. Regarding outlier exclusions, IL-6 had one outlier value, IL-8 
had two outlier values, LBP had one outlier value, sCD163 had one outlier value, and TNF-a had one outlier 
value. Distributions for sCD14, IL-10, and MCP had no outlier values.  
Linear mixed models were used to assess changes in biomarkers over the study period. Models included 
the effect of alcohol use variables (assessed at Visit 1, Visit 3, and Visit 4) as time-varying covariates, 
specifically, number of heavy drinking days, total number of drinking days in, total number of standard drinks, 
and days since last drink. Self-reported alcohol use variables were treated as continuous. Self-reported drinking 
variables were assessed for collinearity and included in the final model if they met an acceptable variance 
inflation factor (VIF) <10. Total number of standard drinks was dropped from the final model due to high VIF.  
Despite prior evidence for sex effects on alcohol consumption and its impacts, there were too few females to 
test for this effect, and sex was not included in models. Models were specified to identify participant as the 
subject variable and visit number (1, 3, and 4) as the repeated measure, and with fixed slope and intercept. An 
autoregressive variance-covariance structure was selected for optimal model fit based on the Akaike 
Information Criterion (AIC). In the primary model (Model 1), the trajectory of biomarker concentrations before, 
during, and after probiotic use were examined, with alcohol variables (number of heavy drinking days, number 
of drinking days, and days since last drinks) included as time-varying covariates. In Model 1, biomarker 

concentrations below the threshold of detectability were treated as missing data in analyses. Of note, maximum 
likelihood estimation as applied in the linear mixed model approach is able to handle missing data without 
excluding cases listwise or imputing data, thus making full use of the available data and minimizing bias in 
parameter estimates (Baraldi & Enders, 2010). To assess for non-linear changes in biomarker concentrations, a 
secondary model (Model 2) was tested to include a quadratic effect of time for the biomarker outcomes. Finally, 
a sensitivity analysis model (Model 3) was tested to assess for the effect of data missing at random. In Model 3, 
data below the detectability threshold of the assay (i.e., missing) were imputed at half the value of the threshold 
per the manufacturer. All analyses in this paper were conducted using SAS software, version 9.4 for Windows 
(SAS Institute, Cary NC.)