Supplementary Material

Appendix S1. Study eligibility criteria

Inclusion Criteria:

Male or female between 21 and 65 years old Current diagnosis of alcohol dependence (AD) according to the Structured Clinical Interview for

DSM-IV-TR Axis I Disorders (SCID) A score of ≥ 40 on the trait version of Spielberger State Trait Anxiety Inventory (STAI) No or mild alcohol withdrawal symptoms, defined as Clinical Institute Withdrawal Assessment for

Alcohol-revised (CIWA-Ar) score ≤ 8 Good health as confirmed by medical history, physical examination, ECG, blood/urine lab tests Females only: postmenopausal for at least one year, surgically sterile, or practicing an effective

method of birth control before entry and throughout the study; reliable methods of birth control include: oral contraceptives or Norplant®; barrier methods such as diaphragms with contraceptive jelly, cervical caps with contraceptive jelly, condoms with contraceptive foam, or intrauterine devices; a partner with a vasectomy; abstinence from intercourse

Exclusion Criteria:

Current diagnosis of substance dependence (other than alcohol and nicotine) based on SCID Lifetime diagnosis of schizophrenia, bipolar disorder, or other psychoses; lifetime history of

attempted suicide; a diagnosis of Major Depressive Disorder (MDD) within the past 6 months History of epilepsy or alcohol-related seizures Express interest in treatment for alcohol use disorders and/or anxiety at any time during the study Positive urine test for drugs of abuse at any time during the study Females only: breast-feeding and/or positive urine pregnancy test at any time during the study Poor venous access based on physical exam and medical history History of adverse reaction or hypersensitivity to baclofen Medical contraindications for baclofen use Clinically significant ECG abnormalities, uncontrolled hypertension, creatinine > 2 mg/dL, and/or

clinically significant liver problems (i.e., liver cirrhosis, AST or ALT > 5x the upper normal limit, Hepatitis B or C)

Current use of psychotropic medications that cannot be discontinued and may have an effect on alcohol consumption or may interact with baclofen, including: naltrexone, acamprosate, alcohol dehydrogenase inhibitors, topiramate, gabapentin, ondansetron, benzodiazepines, β-blockers, H2-blockers, and α1-blockers

1

Table S1. The study drug dose schedule1

Day2 Early Morning Late Morning Evening

1-3 5 mg 5 mg 5 mg

4-73 10 mg 10 mg 10 mg

84 10 mg5 10 mg6 5 mg

9-117 5 mg 5 mg 5 mg

1 The NIH Pharmacy Department prepared 5 mg baclofen capsules by grinding commercially available baclofen tablets (Upsher-Smith Laboratories Inc., Plymouth, MN) and adding appropriate fillers. These were identical to placebo capsules in appearance, texture, taste, and odor.2 See the manuscript (Figure 1) for details3 +1-8 days to allow scheduling flexibility4 Laboratory experiment visit5 Taken at home before coming to the clinic6 Taken in the clinic, 1 hour before the laboratory experiment started7 The study medication was continued for three additional days (starting the evening after the laboratory experiment) at half dose (5 mg t.i.d) for safety reasons.

2

Appendix S2. Standardized meals served before the laboratory experiment

Breakfast (standard calorie: 400 kcal – served at 9:00 AM):

Option A : 1 cornflakes box, 1 banana, 1 apple sauce, 1 orange juice (4 fl oz), 1 non-fat milk (8 fl oz), 2 sugar packets (13% protein, 2% fat, and 85% carb).

Option B : 1 cottage cheese, 2 hardboiled eggs, 2 slices American cheese, 2 slices deli turkey, 1 bottled water (43% protein, 47% fat, and 10% carb).

Lunch (standard calorie: 400 kcal – served at 10:45 AM):

Option A : ½ sandwich (whole wheat bread, deli turkey, and American cheese), 1 potato chips bag, 1 bottled water (19% protein, 45% fat, and 36% carb).

Option B : ½ sandwich (multigrain bread, peanut butter, and strawberry jelly), 1 banana, 1 bottled water (9% protein, 26% fat, and 65% carb).

Option C : salad (green leaf lettuce, tomato, and cucumber), 1 packet of Italian salad dressing, 1 plain yogurt, 1 apple, 1 bottled water (17% protein, 29% fat, and 54% carb).

3

Figure S1. Blood sampling, processing, and analysis

1 Lavender cap spray-coated K2EDTA 3 mL tube (BD Vacutainer®)2 This tube was pre-treated with 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (Roche Diagnostics GmbH, Germany – Pefabloc® SC), dipeptidyl peptidase IV inhibitor (EMD Millipore Corp., Billerica, MA – Cat. #DPP4-010), and a protease inhibitor cocktail (Sigma-Aldrich Inc., Saint Louis, MO – Cat. #P8340) prior to blood collection, and inverted 10 times and kept on ice after collection.3 Red cap – yellow ring Z Serum Separator Clot Activator 4 mL tubes (VACUETTE®)4 This tube was centrifuged within 30 minutes post-collection (relative centrifugal force: 1700×g, temperature: 4oC, centrifugation time: 15 minutes), and the extracted plasma sample was pipetted into 500 µL microtubes.5 This tube was centrifuged after 30 minutes post-collection (relative centrifugal force: 1000×g, temperature: 25oC, centrifugation time: 10 minutes), and the extracted serum sample was pipetted into 500 µL microtubes.6 The Millipore Human Ghrelin (Total) 96-Well Plate Enzyme-Linked Immunosorbent Assay (ELISA) kit (EMD Millipore Corp., Billerica, MA – Cat. #EZGRT-89K) was used to measure total ghrelin. The optical density of each well was determined using the GloMax®-Multi Detection System (Promega Corp., Madison, WI – Part #TM297) and a regression model was applied to calculate the concentration.

4

7 The Millipore Human Metabolic Hormone Magnetic Bead Panel 96-Well Plate MILLIPLEX® MAP kit (EMD Millipore Corp., Billerica, MA – Cat. #HMHEMAG-34K) was used to measure the following analytes: active ghrelin (here referred to as acyl-ghrelin), leptin, active amylin (here referred to as amylin), active GLP-1 (here referred to as GLP-1), and insulin. The assay was performed on fluorescence-coded magnetic beads coated with capture antibodies specific for each marker. Introduction of biotinylated detection antibody and streptavidin-phycoerythrin permitted simultaneous detection of all analytes on the MAGPIX® instrument (Luminex Corp., Austin, TX). These multiplex data were pre-processed and analyzed in the MILLIPLEX® Analyst software (Version 3.5 – EMD Millipore Corp., Billerica, MA) to calculate the concentration of each neuroendocrine marker.8 Chemiluminescence immunoassays were run on an IMMULITE® 2000 XPi (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) to measure prolactin, GH, cortisol, and ACTH concentrations at the NIH Clinical Center DLM. 9 An Electrochemiluminescence immunoassay was run on a cobas® 600 analyzer (Roche Diagnostics International Ltd., Switzerland) to measure TSH concentrations at the NIH Clinical Center DLM.

Abbreviations: ACTH: Adrenocorticotropic Hormone; CPN: Section on Clinical Psychoneuroendocrinology and Neuropsychopharmacology; DLM: Department of Laboratory Medicine; GH: Growth Hormone; GLP-1: Glucagon-Like Peptide-1; NIH: National Institutes of Health; TSH: Thyroid Stimulating Hormone.

5

Figure S2. Flow diagram of the study

Copied with permission from Farokhnia M, Schwandt ML, Lee MR, Bollinger JW, Farinelli LA, Amodio JP, Sewell L, Lionetti TA, Spero DE, Leggio L (2017). Biobehavioral effects of baclofen in anxious alcohol-dependent individuals: a randomized, double-blind, placebo-controlled, laboratory study. Transl Psychiatry, 7(4):e1108: https://www.nature.com/articles/tp201771This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License: https://creativecommons.org/licenses/by-nc-nd/4.0/

Abbreviations: BrAC: Breath Alcohol Concentration.

6

Table S2. Demographic characteristics, baseline blood tests1, and comparison of these variables between the two study groups (baclofen vs. placebo)

Variable1 Placebo Group Baclofen Group StatisticsAge, years, M (SEM) 42.06 (2.69) 46.44 (2.09) t = -1.29, p = 0.20

Gender, males, n (%) 13 (81.25) 14 (77.77) X2 = 0.06, p = 0.80

BMI, kg/m2, M (SEM) 27.99 (1.01) 31.11 (1.99) t = -1.34, p = 0.18

Race, African Americans, n (%) 12 (75.00) 11 (61.11) X2 = 0.93, p = 0.63

Cigarette smokers, n (%) 7 (43.75) 12 (66.66) X2 = 1.80, p = 0.18

Average drinks per drinking days2, M (SEM) 9.11 (1.62) 8.61 (1.12) t = -0.26, p = 0.80

Trait STAI score, M (SEM) 48.81 (1.71) 46.00 (1.39) t = -1.28, p = 0.21

Glucose, mg/dL, M (SEM) 95.06 (4.26) 94.47 (3.43) t = 0.10, p = 0.91

AST, U/L, M (SEM) 19.94 (1.77) 21.65 (1.80) t = -0.67, p = 0.50

ALT, U/L, M (SEM) 26.44 (11.84) 28.41 (15.59) t = -0.40, p = 0.68

Alkaline phosphatase, U/L, M (SEM) 67.81 (19.60) 72.94 (17.45) t = -0.79, p = 0.43

Total bilirubin, mg/dL, M (SEM) 0.41 (0.04) 0.56 (0.09) t = -1.42, p = 0.16

Direct bilirubin, mg/dL, M (SEM) 0.04 (0.01) 0.17 (0.01) t = -0.08, p = 0.93

BUN, mg/dL, M (SEM) 12.81 (1.05) 13.00 (0.83) t = -0.14, p = 0.88

Creatinine, mg/dL, M (SEM) 0.90 (0.05) 0.91 (0.4) t = -0.24, p = 0.80

Total Ghrelin, pg/ml, M (SEM) 388.57 (78.57) 281.21 (28.71) t = 1.34, p = 0.18

Acyl-Ghrelin, pg/ml, M (SEM) 86.77 (13.99) 67.31 (13.55) t = 0.99, p = 0.32

Leptin, pg/ml, M (SEM) 4398.18 (1538.15) 6048.19 (1195.72) t = -0.85, p = 0.40

Amylin, pg/ml, M (SEM) 6.59 (1.24) 4.13 (1.03) t = 1.53, p = 0.13

GLP-1, pg/ml, M (SEM) 5.84 (1.40) 6.00 (2.24) t = -0.05, p = 0.95

Insulin, pg/ml, M (SEM) 838.47 (225.48) 549.48 (116.82) t = 1.15, p = 0.25

Prolactin, ng/ml, M (SEM) 5.98 (0.84) 4.07 (0.41) t = 2.03, p = 0.05

TSH, mcIU/ml, M (SEM) 1.22 (0.24) 1.79 (0.24) t = -1.66, p = 0.10

GH (Log10), ng/ml, M (SEM) -0.84 (0.16) -0.94 (0.12) t = 0.49, p = 0.62

Cortisol, mcg/dl, M (SEM) 9.86 (0.79) 9.74 (1.11) t = 0.08, p = 0.93

ACTH, pg/ml, M (SEM) 19.95 (2.09) 19.48 (2.31) t = 0.14, p = 0.881Baseline blood tests were performed on Day 1; 2Based on alcohol Timeline Followback 90 days prior to the screening visit

Abbreviations: ACTH: Adrenocorticotropic Hormone; ALT: Alanine Transaminase; AST: Aspartate Transferase; BMI: Blood Mass Index; BUN: Blood Urea Nitrogen; GH: Growth Hormone; GLP-1: Glucagon-Like Peptide-1; STAI: Spielberger State Trait Anxiety Inventory; TSH: Thyroid Stimulating Hormone.

7

Figure S3. Blood concentrations of neuroendocrine markers during the outpatient phase. Statistics for each neuroendocrine outcome are presented in the main manuscript (Table 1).

8

Figure S3 – Continued

9

Figure S4. Blood concentrations of neuroendocrine markers during the human laboratory experiment. Statistics for each neuroendocrine outcome are presented in the main manuscript (Table 2).

10

Figure S4 – Continued

11

Table S3. Participants’ preferred alcohol type used during the human laboratory experiment

Alcohol Type1 Placebo Group (n = 16)

Baclofen Group (n = 18) Statistics

Beer, n (%) 5 (31.2) 3 (16.7)

X2 = 2.47, p = 0.47Malt liquor, n (%) 1 (6.2) 1 (5.6)

Wine, n (%) 1 (6.2) 0

Spirits, n (%) 9 (56.2) 14 (77.8)

1Information on participants’ preferred alcohol was collected on Day 1.

12