Does Endotoxin Administration Increase Alcohol Consumption in Individuals With AUD?
Phase: PHASE2
Conditions: Alcohol Use Disorder
Sponsor: Yale University

Study Protocol, including Statistical Analysis Plan 
Official Title: Does endotoxin administration increase alcohol consumption in individuals 
with AUD? 
Brief Title: Does endotoxin administration increase alcohol consumption? (Terril 
Verplaetse, PhD, PI) 
ClinicalTrials.gov ID: NCT04527185 
Protocol Date: Most recent IRB approval – 4/4/2024  
 
 

Protocol and Statistical Analysis Plan for 
“Does endotoxin administration increase alcohol consumption?”  
NCT04527185 (Terril Verplaetse, PhD, PI) 
 
Scientiﬁc Background 
 
One of the principal mechanisms associated with the maintenance of and relapse to alcohol use is 
stress12-17. Neuroimmune function is a key system linked to negative reinforcement drinking and 
to the development of alcohol use disorder (AUD)18,19. Stress and chronic alcohol consumption 
have been found to increase microglial activity, a marker of neuroinflammation18-20. However, a 
provocation study to examine neuroinflammation-induced drinking has not been developed in 
humans and has direct clinical significance for the development and evaluation of 
pharmacotherapies targeting the neuroimmune system. 
 
Endotoxin, also called lipopolysaccharide (LPS), is well-known to induce neuroinflammation1 
and acute stress2, and a growing literature indicates that neuroinflammatory processes are 
involved in alcohol-motivated behavior3. In rodents, systemic administration of LPS resulted in 
prolonged increases in levels of proinflammatory cytokines, an effect potentiated by alcohol4. 
Administration of inflammatory signaling by LPS demonstrated long-lasting increases in 
voluntary alcohol intake in mice5, which may be due to the effects of acute stress induced by 
LPS – as LPS has transient effects on neuroinflammatory responses114. Proinflammatory 
cytokines that increased in rat brain following alcohol or LPS were also increased in the brain of 
humans with alcohol dependence6. In alcohol-dependent individuals, LPS stimulated 
inflammatory pathways correlated with alcohol craving and short-term alcohol withdrawal was 
associated with the recovery of LPS-dependent receptors7. Regarding stress, LPS administration 
increased plasma ACTH in alcohol-exposed rats8,9 and dose-dependently increased 
proinflammatory cytokines, cortisol, anxiety, and depressive symptoms in humans2,10; altogether, 
supporting a clear role for neuroinflammation in stress and alcohol use. To our knowledge, the 
effect of LPS, which directly induces neuroinflammation compared to other stress challenges, on 
alcohol self-administration in humans with AUD has not been studied, identifying an area of 
weakness in the scientific rigor of previous work. The first step in addressing this gap is to 
examine peripheral measures of neuroinflammation (e.g., cytokine levels) and relate 
that to drinking behavior in the human laboratory. By collecting subjective and objective 
measures of stress (e.g., mood, cortisol, ACTH), we can also determine whether LPS 
administration is related to stress-reactivity in individuals with AUD. 
 
Over the past decade, rates of AUD have continued to increase more so in women (84%) than in 
men (35%)50,51. Women also experience exacerbated health risks associated with drinking, 
including brain atrophy, liver inflammation, cardiovascular disease, and neurotoxicity when 
compared to men52-56. Identifying effective mechanisms underlying problem alcohol use in 
women vs. men remains a priority. Stress and greater neuroimmune response are associated with 
alcohol use for both women and men, but play an especially critical role in women11. Research 
identifies that alcohol administration leads to higher cortical levels of proinflammatory cytokines 
associated with oxidative stress in female compared to male mice57,58 and higher plasma 
endotoxin levels in healthy women compared to men57,59. Problematic drinking in women is also 
associated with higher plasma levels of proinflammatory cytokines compared to men60,61. 

Importantly, we plan to calculate effect sizes for the interaction between sex and treatment 
(endotoxin vs. placebo) on drinking behavior and associated mechanisms (e.g., peripheral 
cytokines, craving, cortisol, ACTH, sex hormones) during the 2-hour alcohol self-administration 
period, as these may be important mechanisms underlying drinking behavior in women vs. men. 
 
Elucidation of neuroinflammatory mechanisms underlying drinking can inform on the 
involvement of neuroinflammation in alcohol use, as well as potential sex differences in 
mechanisms underlying how neuroinflammation influences acute drinking. This data will inform 
sex-appropriate mechanisms and potential treatment targets, such as neuroimmune modulators, 
for alcohol use in individuals with AUD, as well as inform future research on disentangling the 
effect of LPS vs. psychological stress (e.g., stress vs. neutral imagery) on subsequent drinking 
behavior. 
 
Study Design and Methods 
 
We will examine whether endotoxin, known to induce acute stress and 
neuroinflammation, increases acute drinking behavior in a sex-dependent manner. We will 
recruit 32 individuals with AUD (50% women). Subjects will undergo a single laboratory session 
in which we will evaluate alcohol consumption.  
 
Subjects will be informed to not drink 24 hours prior to the laboratory session (confirmed by 
breath alcohol reading and self-report). Smokers will be instructed to smoke as they usually do 
prior to coming to the YCCI/HRU. If participants report or test positive for breath alcohol, drug 
use, alcohol withdrawal, or pregnancy they will not complete the laboratory session. If 
participants present with alcohol withdrawal (CIWA ≥ 8), the participant will be evaluated by the 
study MD for possible study removal and referral to detox. An IV cannulae will be inserted in 
the non-dominant arm to obtain blood samples throughout the session. LPS (0.4ng/kg; 
Escherichia coli) or placebo (saline) injections will be performed through the IV cannulae, 
approximately 1 hour and 45 minutes before the alcohol self-administration period. This dose has 
been safely administered to healthy humans previously (range 0.2 – 1.0ng/kg i.v. in healthy 
human subjects). 
 
Ad-libitum drinking will occur over a 2-hour period. A video camera will be present in the room 
during this session to monitor drinking behavior. Subjects will be provided a pre-determined 
amount of their preferred beverage designed to raise BALs to 0.12 g/dL. Participants will be 
provided with their preferred beverage (e.g., beer, wine, liquor, mixed drink) as a bolus at the 
start of the session. The bolus dose is provided in a plastic pitcher (e.g., beer) or a plastic carafe 
(e.g., wine, mixed drink) depending on the participant’s preferred beverage. The participant is 
also provided with a drinking vessel (e.g., plastic pint glass, plastic wine glass, plastic tumbler). 
The participant can pour as much or as little of their preferred beverage as they like from the 
pitcher or carafe into their drinking vessel, and choose to drink as much or as little as they like 
throughout the 2-hour alcohol self-administration session. 
 
Prior to discharge, serum endotoxin will be drawn. Subjects will remain in the 
YCCI/HRU overnight, at which time their breath alcohol levels will be below 0.02% (confirmed 
by two BAC readings). Participants who are deemed to be belligerent by study/HRU staff will be 

immediately discontinued at the discretion of the PI. Participants will be asked to remain in their 
room until reaching a safe BAC at which time they will be dismissed. If they continue to behave 
in a threatening manor, hospital security will be called to supervise the participant until they can 
be safely discharged from the HRU. 
 
Eligibility Criteria 
 
After obtaining written informed consent, potential subjects will be 
screened for eligibility. In other words, potential subjects will sign consent prior to beginning 
screening procedures. We will administer the Structured Clinical Interview for DSM-563 (SCID-
5) to confirm AUD and exclude other primary psychiatric and substance abuse disorders (other 
than tobacco use disorder). We will record alcohol use over the prior 90 days with the Timeline-
Follow Back Interview64 (TLFB), and quantify lifetime patterns of alcohol use with the Lifetime 
Drinking History65. The consequences of alcohol use and severity will be measured with the 
Short-Inventory of Problems for Alcohol Use66 and the Alcohol Use Disorders Test67 (AUDIT), 
respectively. The Clinical Institute Withdrawal Assessment of Alcohol Scale, Revised68 (CIWA-
R) will assess alcohol withdrawal symptoms. Patients with CIWA-R ≥ 8 will not be eligible to 
participate. Subjects will undergo medical screening, including medical history, medication use, 
allergies, a physical exam, an EKG, basic blood chemistries, urine drug toxicology screen, and a 
blood pregnancy test for women. The study physicians will review the subject’s medical status 
and medical eligibility criteria. Eligible subjects will be randomized to receive endotoxin or 
placebo. If potential participants are found to be suicidal or are experiencing any psychiatric 
symptoms or distress during the screening process, they will receive short-term support from the 
study team (including clinical psychologists and study physicians) and will be connected to a 
local emergency department and their physician or therapist for ongoing care. 
 
Inclusion Criteria: 
1) Age 21-65; 
2) Able to read and write English; 
3) Meets DSM-5 criteria for current (past 6 months) moderate to severe alcohol use disorders; 
4) Drinking criteria: Males - Drinks > 14 drinks per week and exceeds 4 drinks per day at least 
twice per week; Females -Drinks > 7 drinks per week and exceeds 3 drinks per day at least twice 
per week. 
5) Must meet drinking criteria during a consecutive 30-day period within the 90 days prior to 
baseline; 
6) Laboratory sessions will be scheduled such that subjects will not have major responsibilities 
on the following day which might limit drinking during the self-administration session (e.g., job 
interview, exam); 
7) Negative urine pregnancy test for women. 
 
Exclusion Criteria: 
1) Participants with any significant current medical conditions (neurological, cardiovascular 
[including hypertension or hypotension: sitting BP >160/100 or <90/60mmHg at baseline 
screening], endocrine, thyroid, renal, liver), seizures, delirium or hallucinations, or other unstable 
medical conditions including HIV; 
2) Current DSM-5 substance use disorders, other than alcohol or nicotine; 

3) A positive test result at intake appointment on urine drug screens conducted for illicit drugs, 
excluding cannabis; 
4) Past 30 day use of psychoactive drugs including anxiolytics and antidepressants; 
5) Women who are pregnant or nursing, or fail to use one of the following methods of birth 
control unless she or partner is surgically sterile or she is postmenopausal (hormone 
contraceptives [oral, 
implant, injection, patch, or ring], contraceptive sponge, double barrier [diaphragm or condom 
plus spermicide], or IUD); 
6) Suicidal, homicidal or evidence of current (past 6-month) severe mental illness such as 
schizophrenia, bipolar disorder or major depression, or anxiety disorders; 
7) Subjects treatment-seeking or who are currently in treatment for alcohol use; 
8) Subjects with medical conditions contraindicating alcohol use (e.g., liver enzymes ≥3× 
normal); 
9) Subjects likely to exhibit clinically significant alcohol withdrawal during the study. We will 
exclude subjects who a) have a history of perceptual distortions, seizures, delirium, or 
hallucinations upon withdrawal, or b) have a score of > 8 on the Clinical Institute Withdrawal 
Assessment scale at intake appointments; 
10) Participation within the past 8 weeks in other studies that involve additive blood sampling 
and/or interventional measures that would be considered excessive in combination with the 
current application. 
11) Subjects >38 on the Alcohol Use Disorders Identification Test (AUDIT) 
12) Subjects with resting pulse >100 at challenge 
13) Subjects with recent (past 2 weeks) acute illness or vaccination 
14) Subjects with >Grade 2 laboratory abnormalities on screening 
 
Statistical Considerations 
 
To determine whether endotoxin vs. placebo will increase milliliters of alcohol consumed in 
women and men. Linear mixed models with intervention condition (0.4ng/kg i.v. endotoxin or 
placebo) and sex as between subject factors will be used to evaluate milliliters consumed during 
ad-libitum drinking collected in the laboratory. All interactions will be considered. The best 
fitting variance-covariance structure will be assessed using information criteria. The mixed 
effects approach is advantageous in that it is unaffected by randomly missing data and allows 
greater flexibility in modeling the correlation structure of repeated measures data. This data will 
generate effect sizes for the interaction between sex and intervention (endotoxin vs. placebo) on 
alcohol use.  
 
We will evaluate the safety and tolerability of endotoxin in combination with alcohol in 
individuals with AUD. Participants will be assessed for altered alcohol intoxication and 
risks associated with endotoxin (e.g., flu-like symptoms [chills, nausea]). We will take extra 
safety precautions when assessing adverse effects of LPS and alcohol co-administration, 
including drawing serum endotoxin during alcohol self-administration and discharge. 
 
References 
 
1. Sandiego CM, Gallezot J-D, Pittman B, et al. Imaging robust microglial activation after 

lipopolysaccharide administration in humans with PET. Proceedings of the National Academy of 
Sciences. 2015;112(40):12468-12473. 
2. Grigoleit J-S, Kullmann JS, Wolf OT, et al. Dose-dependent effects of endotoxin on 
neurobehavioral functions in humans. PLoS One. 2011;6(12):e28330. 
3. Hillmer A, Sandiego C, Hannestad J, et al. In vivo imaging of translocator protein, a marker of 
activated microglia, in alcohol dependence. Molecular psychiatry. 2017;22(12):1759. 
4. Qin L, He J, Hanes RN, Pluzarev O, Hong J-S, Crews FT. Increased systemic and brain 
cytokine production and neuroinflammation by endotoxin following ethanol treatment. Journal 
of neuroinflammation. 2008;5(1):10. 
5. Blednov Y, Benavidez JM, Geil C, Perra S, Morikawa H, Harris R. Activation of 
inflammatory signaling by lipopolysaccharide produces a prolonged increase of voluntary 
alcohol intake in mice. Brain, behavior, and immunity. 2011;25:S92-S105. 
6. He J, Crews FT. Increased MCP-1 and microglia in various regions of the human alcoholic 
brain. Experimental neurology. 2008;210(2):349-358. 
7. Leclercq S, De Saeger C, Delzenne N, de Timary P, Stärkel P. Role of inflammatory 
pathways, blood mononuclear cells, and gut-derived bacterial products in alcohol dependence. 
Biological psychiatry. 2014;76(9):725-733. 
8. Lee S, Rivier C. Gender differences in the effect of prenatal alcohol exposure on the 
hypothalamic-pituitary-adrenal axis response to immune signals. Psychoneuroendocrinology. 
1996;21(2):145-155. 
9. Lee S, Schmidt D, Tilders F, Rivier C. Increased activity of the hypothalamic-pituitary-adrenal 
axis of rats exposed to alcohol in utero: role of altered pituitary and hypothalamic function. 
Molecular and Cellular Neuroscience. 2000;16(4):515-528. 
10. Hannestad J, Subramanyam K, DellaGioia N, et al. Glucose metabolism in the insula and 
cingulate is affected by systemic inflammation in humans. Journal of nuclear medicine: official 
publication, Society of Nuclear Medicine. 2012;53(4):601. 
11. Koob GF, White A. Alcohol and the Female Brain. Paper presented at: 2017 National 
Conference on Alcohol and Opioid Use in Women & Girls: Advances in prevention, Treatment, 
and Recovery Research2017; Washington D.C. 
12. Brady KT, Sonne SC. The role of stress in alcohol use, alcoholism treatment, and relapse. 
Alcohol Research & Health. 1999;23(4):263-. 
13. Pohorecky LA. Stress and alcohol interaction: an update of human research. Alcoholism: 
Clinical and Experimental Research. 1991;15(3):438-59. 
14. Sayette MA. An appraisal-disruption model of alcohol's effects on stress responses in social 
drinkers. Psychological bulletin. 1993;114(3):459. 
15. Sher KJ, Levenson RW. Risk for alcoholism and individual differences in the stress-response 
dampening effect of alcohol. Journal of Abnormal Psychology. 1982;91(5):350. 
16. Zywiak WH, Connors GJ, Maisto SA, Westerberg VS. Relapse research and the Reasons for 
Drinking Questionnaire: A factor analysis of Marlatt's relapse taxonomy. Addiction. 
1996;91(12s1):121-30. 
17. Marlatt GA, Donovan DM. Relapse prevention: Maintenance strategies in the treatment of 
addictive behaviors: Guilford Press; 2005. 
18. Hillmer AT, Sandiego CM, Hannestad J, Angarita GA, Kumar A, McGovern EM, Huang Y, 
O'Connor KC, Carson RE, O'Malley SS, Cosgrove KP. In vivo imaging of translocator protein, a 
marker of activated microglia, in alcohol dependence. Mol Psychiatry. 2017;22(12):1759-1766. 
19. Kalk NJ, Guo Q, Owen D, Cherian R, Erritzoe D, Gilmour A, Ribeiro AS, McGonigle J, 

Waldman A, Matthews P, Cavanagh J, McInnes I, Dar K, Gunn R, Rabiner EA, Lingford- 
Hughes AR. Decreased hippocampal translocator protein (18 kDa) expression in alcohol 
dependence: a [(11)C]PBR28 PET study. Transl Psychiatry. 2017;7(1):e996. 
20. Wohleb ES, Terwilliger R, Duman CH, Duman RS. Stress-Induced Neuronal Colony 
Stimulating Factor 1 Provokes Microglia-Mediated Neuronal Remodeling and Depressive-like 
Behavior. Biol Psychiatry. 2018;83(1):38-49. 
47. Higley AE, Crane NA, Spadoni AD, Quello SB, Goodell V, Mason BJ. Craving in response 
to stress induction in a human laboratory paradigm predicts treatment outcome in alcohol 
dependent individuals. Psychopharmacology. 2011;218(1):121-9. 
50. Grant B.F., Chou S.P., Saha T.D. Prevalence of 12-month alcohol use, high-risk drinking, 
and DSM-IV alcohol use disorder in the United States, 2001-2002 to 2012-2013: results from the 
National Epidemiologic Survey on Alcohol and Related Conditions. JAMA psychiatry. 
2017;74(9):911–923. 
51. Dawson DA, Goldstein RB, Saha TD, Grant BF. Changes in alcohol consumption: United 
States, 2001-2002 to 2012-2013. Drug and Alcohol Dependence. 2015; 148: 56-61. 
52. National Institute of Alcohol Abuse and Alcoholism . 2017. Women and Alcohol. 
53. Agabio R., Pisanu C., Luigi Gessa G., Franconi F. Sex differences in alcohol use disorder. 
Curr. Med. Chem. 2017;24(24):2661–2670. 
54. Szabo G. Women and alcoholic liver disease — warning of a silent danger. Nature Reviews 
Gastroenterology & Hepatology. 2018;15:253. 
55. Agabio R., Campesi I., Pisanu C., Gessa G.L., Franconi F. Sex differences in substance use 
disorders: focus on side effects. Addict. Biol. 2016;21(5):1030–1042. 
56. National Cancer Institute Alcohol and cancer risk. 2013. 
https://www.cancer.gov/aboutcancer/causes-prevention/risk/alcohol/alcohol-fact-sheet 
57. Pascual M, Montesinos J, Marcos M, Torres JL, Costa‐Alba P, García‐García F et al. Gender 
differences in the inflammatory cytokine and chemokine profiles induced by binge ethanol 
drinking in adolescence. Addiction biology 2016. 
58. Alfonso-Loeches S, Pascual M, Guerri C. Gender differences in alcohol-induced 
neurotoxicity and brain damage. Toxicology 2013; 311(1-2): 27-34. 
59. Bala S, Marcos M, Gattu A, Catalano D, Szabo G. Acute Binge Drinking Increases Serum 
Endotoxin and Bacterial DNA Levels in Healthy Individuals. PLOS ONE 2014; 9(5): e96864. 
60. Kirpich IA, McClain CJ, Vatsalya V, Schwandt M, Phillips M, Falkner KC et al. Liver Injury 
and Endotoxemia in Male and Female Alcohol-Dependent Individuals Admitted to an Alcohol 
Treatment Program. Alcoholism: Clinical and Experimental Research 2017; 41(4): 747-757. 
61. Wilhelm CJ, Fuller BE, Huckans M, Loftis JM. Peripheral immune factors are elevated in 
women with current or recent alcohol dependence and associated with altered mood and 
memory. Drug and alcohol dependence 2017; 176: 71-78. 
62. Anton RF, O'Malley SS, Ciraulo DA, Cisler RA, Couper D, Donovan DM, Gastfriend DR, 
Hosking JD, Johnson BA, LoCastro JS, Longabaugh R, Mason BJ, Mattson ME, Miller WR, 
Pettinati HM, Randall CL, Swift R, Weiss RD, Williams LD, Zweben A; COMBINE Study 
Research Group. Combined pharmacotherapies and behavioral interventions for alcohol 
dependence: the COMBINE study: a randomized controlled trial. JAMA. 2006 May 
3;295(17):2003-17. 
63. First MB. (2015). Structured Clinical Interview for the DSM (SCID). In The Encyclopedia of 
Clinical Psychology (eds R. L. Cautin and S. O. Lilienfeld). 
doi:10.1002/9781118625392.wbecp351 

64. Sobell LC, Sobell MB. Timeline follow-back. Measuring alcohol consumption: Springer; 
1992. p. 41-72. 
65. Skinner HA, Sheu W-J. Reliability of alcohol use indices. The Lifetime Drinking History and 
the MAST. Journal of studies on alcohol. 1982;43(11):1157-70. 
66. Miller WR, Tonigan JS, Longabaugh R. The Drinker Inventory of Consequences (DrInC): 
An instrument for assessing adverse consequences of alcohol abuse: Test manual: US 
Department of Health and Human Services, Public Health Service, National Institutes of Health, 
National Institute on Alcohol Abuse and Alcoholism; 1995. 
67. Saunders JB, Aasland OG, Babor TF, De la Fuente JR, Grant M. Development of the alcohol 
use disorders identification test (AUDIT): WHO collaborative project on early detection of 
persons with harmful alcohol consumption‐II. Addiction. 1993;88(6):791-804. 
68. Sullivan JT, Sykora K, Schneiderman J, Naranjo CA, Sellers EM. Assessment of alcohol 
withdrawal: the revised clinical institute withdrawal assessment for alcohol scale (CIWA‐Ar). 
British journal of addiction. 1989;84(11):1353-7.