Conditions: fMRI Alcohol Drinking Alcoholism
Keywords: Addiction Alcohol Alcohol Clamp
Allocation: RANDOMIZED | Intervention model: CROSSOVER
Enrollment: 17
Healthy volunteers: yes

Brief summary:
Background:

\- Ghrelin is a hormone in the human body that is mostly produced by the stomach. It makes people feel hungry, and also is connected with the desire to drink alcohol. Researchers want to test ghrelin to see if it can be used to control alcohol cravings and use. They will compare doses of ghrelin with a placebo in people who drink heavily.

Objectives:

\- To study the effects of ghrelin on alcohol craving and use.

Eligibility:

* Individuals between 21 and 60 years of age who are heavy drinkers but are not seeking treatment for alcohol use.
* Participants must on average have more than 20 drinks per week for men, and more than 15 drinks per week for women.

Design:

* Participants will have a screening visit, four 2-night study visits, and a follow-up visit.
* Participants will be screened with a physical exam and medical history. They will provide urine and breath samples for drug testing. They will also answer questions about mood and physical symptoms, and about alcohol and other cravings.
* At the study visits, participants will stay overnight at the National Institutes of Health clinical center. They will spend the night at the center, have tests on the next day, and go home on the following morning. At each visit, participants will receive a ghrelin or placebo infusion, and will complete a series of tasks.
* For the first and second study visits, participants will have tests of alcohol craving and use. They will be able to receive alcohol infusions through a computer program that tests response time and craving reactions. At the same time, they will have a ghrelin or a placebo infusion. Blood alcohol levels, reaction time, and craving will be studied.
* For the third and fourth study visits, participants will have a magnetic resonance imaging (MRI) study. They will have an initial MRI to provide a picture of the brain. They will then have a functional MRI during which they will respond to a computer test. The test will allow them to win points for snack food or alcohol. This test will look at the brain s response time and craving reactions.
* There will be a follow-up visit 1 week after the fourth study visit. Some of the tests from the screening visit will be repeated.

Detailed description:
Objective:

Ghrelin is a 28-amino acid peptide acting as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). Ghrelin stimulates appetite by acting on the hypothalamic arcuate nucleus (ARC), a region that controls the intake of food and other substances, including alcohol. In addition to the ARC, GHS receptors (GHS-Rs) are also highly expressed in the caudal brain stem, the ventral tegmental area (VTA), hippocampus, substantia nigra, and dorsal and medial raphe nuclei. The expression of the GHS-R in the mesolimbic dopamine (DA) pathway suggests that ghrelin could play a role in reward processing. The role of ghrelin in the DA reward processing and the role of the DA reward system in alcoholism suggest a role of ghrelin in alcoholism. Consistent with this hypothesis, preclinical studies demonstrate that both ghrelin and ethanol activate the cholinergic-dopaminergic reward link, implying neurochemical analogies between ghrelin and ethanol. This supports the hypothesis that ghrelin is involved in mediating the rewarding properties of ethanol. Additional animal experiments demonstrate that the central ghrelin action not only stimulates the reward processing but is also required for stimulation of that system by alcohol. Human studies show reduced ghrelin levels in actively drinking alcoholics; increased ghrelin levels during alcohol abstinence; and a positive correlation between ghrelin level and alcohol craving scores. More recently, a study conducted at Brown University by the PI demonstrated the safety of the administration IV of human ghrelin to non-treatment seeking alcohol-dependent heavy drinking individuals. Furthermore, a preliminary interim analysis shows that IV ghrelin administration may lead to a temporary significant increase in alcohol craving. The primary objective of this protocol is to investigate whether IV ghrelin, as compared to placebo, will increase motivation for alcohol reward, as measured by a progressive ratio (PR) schedule paradigm with IV alcohol self-infusion (primary aim). We will also assess a number of secondary aims. Specifically, we will also assess whether IV ghrelin, as compared to placebo, will also alter urges to drink and the subjective response to IV alcohol. Adverse events will also be assessed to ensure the safety of the IV coadministration of ghrelin and alcohol. During an fMRI/alcohol clamp session, fMRI will be used to see whether ghrelin affects the activation of the ventral striatum induced by acute IV alcohol administration and the incentive salience of cues associated with alcohol administration.

Study Population:

Male and female participants will be non-treatment seeking heavy drinking volunteers.

Design:

The study is designed as a within-subject, double-blind, placebo-controlled study of ghrelin. The first visit will be the initial screening visit. The second and third visits will be PR sessions with IV ethanol, during which each participant will also receive IV ghrelin (or matched placebo). The fourth and fifth visits will combine an fMRI session with an alcohol clamp session (i.e. a fixed dose of alcohol will be administered) and subjects will participate in a modified version of the monetary incentive delay (MID) task in which they will respond to cues that indicate the opportunity to press a button to gain a reward. On some trials the reward will be points that can be exchanged for snack foods while on other trials the reward will be points that will determine how much intravenous alcohol a subject will be given during the alcohol clamp procedure which will immediately follow the modified MID task. After the modified MID task is complete subjects will have a short break and then begin the alcohol IV infusion.

Outcome measures:

The primary measure of this study will be the breakpoint, which is the schedule (number of button presses) at which the individual stops to work for more alcohol. Also, the BrAC exposure measures will be determined. Alcohol craving in response to ghrelin will be measured using the Alcohol Visual Analogue Scale (A-VAS) and the Alcohol Urge Questionnaire (AUQ) during the PR sessions. Sensitivity to alcohol will be measured using the Drug Effects Questionnaire (DEQ), Biphasic Alcohol Effects Scale (BAES), and the Profile of Mood States (POMS), repeatedly during the PR sessions and the CASE Experience Questionnaire (CEQ) at the end of all sessions. fMRI BOLD signal in brain areas associated with incentive salience and areas associated with reward circuitry (including the ventral striatum) will be measured during the fMRI/alcohol clamp session. This study may facilitate the identification of a novel neuropharmacological target, thus facilitating the development of novel pharmacological treatments for alcoholism.

Arms:
arm 1: Intravenous acyl-ghrelin \[a loading dose (3 mcg/kg), followed by a continous infusion (16.9 ng/kg/min)\] was administered on the first ASA visit; Intravenous placebo \[packed in Dextrose 5% / Water USP 50mL bags and made identical to ghrelin in terms of appearance, texture, and odor\] was administered on the second ASA visit. arm 2: Intravenous placebo \[packed in Dextrose 5% / Water USP 50mL bags and made identical to ghrelin in terms of appearance, texture, and odor\] was administered on the first ASA visit; Intravenous acyl-ghrelin \[a loading dose (3 mcg/kg), followed by a continous infusion (16.9 ng/kg/min)\] was administered on the second ASA visit arm 3: Intravenous acyl-ghrelin \[a loading dose (3 mcg/kg), followed by a continous infusion (16.9 ng/kg/min)\] was administered on the first fMRI visit; Intravenous placebo \[packed in Dextrose 5% / Water USP 50mL bags and made identical to ghrelin in terms of appearance, texture, and odor\] was administered on the second fMRI visit. arm 4: Intravenous placebo \[packed in Dextrose 5% / Water USP 50mL bags and made identical to ghrelin in terms of appearance, texture, and odor\] was administered on the first fMRI visit; Intravenous acyl-ghrelin \[a loading dose (3 mcg/kg), followed by a continous infusion (16.9 ng/kg/min)\] was administered on the second fMRI visit

Interventions: intervention 1: Ghrelin intervention 2: Placebo intervention 3: fMRI intervention 4: Alcohol
Intervention descriptions: intervention 1: None intervention 2: None intervention 3: None intervention 4: None

Protocol document:
Prot_SAP_000.pdf: 
PI name: Lorenzo Leggio, MD, PhD, MSc 
                                                                               
 
 
 
Protocol number: 13-AA-0043                                                                                                                         
CNS and NIDCR IRB Protocol Template (rev.10Feb11) 
 
 
Protocol Title: Effects of Ghrelin on Alcohol Administration in non-treatment seeking heavy 
drinkers  
Abbreviated Title: Ghrelin and Alcoholism 
 
Protocol Number: 13-AA-0043 
Date of This Submission: June 24, 2016 /Version 5.0_CR 2016 
 
Principal Investigator 
Name, 
Degree 
Branch/Institute Bldg/Rm 
Phone 
E-mail 
Lorenzo 
Leggio, 
M.D., Ph.D., 
M.Sc. 
SCPN/LCTS/ 
NIAAA 
10 (CRC)/ 
Room 1-5429 
301-435-
9398 
lorenzo.leggio@nih.gov 
 
Lead AI 
Mehdi 
Farokhnia, 
M.D. 
CPN/LCTS/N
IAAA 
10/(CRC)/
1-5413 
(301) 
827-
1488
 
 
mehdi.farokhnia@nih.gov 
 
 
Associate Investigators 
Name, Degree Branch/Institu
te 
Bldg/Rm 
Phone 
E-mail 
Vijay A. 
Ramchandani, 
Ph.D. 
SHP/LCTS/ 
NIAAA 
10CRC/ 
Rm 2-
2352 
301-
402-
8527 
vijayr@mail.nih.gov 
David Ted 
George, M.D. 
CATE/LCTS/ 
NIAAA 
10CRC/ 
Rm 2-
2352 
301-
435-
9385 
tedg@mail.nih.gov 
Melanie 
Schwandt, 
Ph.D. 
LCTS/NIAAA 10-CRC/ 
1-5330 SE 
301-
451- 
6960 
Melanie.Schwandt@mail.nih.gov 
 
Reza 
Momenan, 
Ph.D. 
SBEI/LCTS/ 
NIAAA 
10-CRC/ 
1-5435 
301-
451-
6972 
rezam@nih.gov 
 
 
 
 
 
Mary R. Lee, 
M.D. 
SCPN/LCTS/
NIAAA 
10(CRC)/1
-5330 SE 
(301) 
827-
0545 
leemary@mail.nih.gov 
LaToya S. 
Sewell, 
CRNP-Family 
LCTS/NIAAA 10(CRC)/1
-5417 
(301) 
496-
1103 
Latoya.sewell@nih.gov 
 
 
 
 
 
 
 
 
 
  


2 
 
Fatemeh 
Akhlaghi, 
PhD, PharmD 
Department of 
Biomedical & 
Pharmaceutica
l Sciences, 
University of 
Rhode Island 
495A 
College of 
Pharmacy 
 
401-
874-
9205 
Fatemeh@uri.edu 
 
Research Contact 
Name, Degree 
Branch/Institute Bldg/Rm 
Phone 
E-mail 
Lorenzo 
Leggio, M.D., 
Ph.D., M.Sc. 
SCPN/LCTS/ 
NIAAA 
10CRC/ 
Room 1-5429 
301-435-
9398 
lorenzo.leggio@nih.gov 
 
 
 
Medical Advisory Investigator 
Name, 
Degree 
Branch/Institute 
Bldg/Rm 
Phone 
E-mail 
Mary R. Lee, 
M.D. 
SCPN/LCTS/NIAAA 10(CRC)/1-
5330 SE 
(301) 827-
0545 
leemary@mail.nih.gov  
 
Total requested accrual 
  (no. 00) Patients       
  (no. 62) Volunteers   
  
 
Project Uses Ionizing Radiation:    No  
 Yes (attach RSC/RDRC documentation) 
  
 
Medically-indicated only 
  
 
Research-related only 
  
 
Both 
 
IND/IDE 
 
 
 
 No   
Yes (attach FDA documentation) 
  
Drug/Device/# 117,778 
 
Durable Power of Attorney  
 
 No   
 Yes  
 
Multi-institutional Project 
 
 No   
 Yes  
 
 
 
 
 
Institution#1_________________    FWA #_________  
Date of IRB approval ______________(attach IRB documentation) 
 
 
 
Institution#2_________________    FWA #_________  
Date of IRB approval ______________(attach IRB documentation) 
 
Data and Safety Monitoring Board  
 No   
 Yes  
 
Technology Transfer Agreement  
 No   
 Yes  
Agreement type and number MTA between NIAAA and URI Expiration Date N/A 


3 
 
 
Samples are being stored  
 
 
 No   
 Yes  
 
Flesch-Kincaid reading level of consent form: 9.1 grade 
(exclude boilerplate in assessing reading level) 
 
Precis: 
Objective. Ghrelin is a 28-amino acid peptide acting as the endogenous ligand for the growth 
hormone secretagogue receptor (GHS-R). Ghrelin stimulates appetite by acting on the 
hypothalamic arcuate nucleus (ARC), a region that controls the intake of food and other 
substances, including alcohol. In addition to the ARC, GHS receptors (GHS-Rs) are also highly 
expressed in the caudal brain stem, the ventral tegmental area (VTA), hippocampus, substantia 
nigra, and dorsal and medial raphe nuclei. The expression of the GHS-R in the mesolimbic 
dopamine (DA) pathway suggests that ghrelin could play a role in reward processing. The role of 
ghrelin in the DA reward processing and the role of the DA reward system in alcoholism suggest 
a role of ghrelin in alcoholism. Consistent with this hypothesis, preclinical studies demonstrate 
that both ghrelin and ethanol activate the cholinergic-dopaminergic reward link, implying 
neurochemical analogies between ghrelin and ethanol. This supports the hypothesis that ghrelin 
is involved in mediating the rewarding properties of ethanol. Additional animal experiments 
demonstrate that the central ghrelin action not only stimulates the reward processing but is also 
required for stimulation of that system by alcohol. Human studies show reduced ghrelin levels in 
actively drinking alcoholics; increased ghrelin levels during alcohol abstinence; and a positive 
correlation between ghrelin level and alcohol craving scores. More recently, a study conducted at 
Brown University by the PI demonstrated the safety of the administration IV of human ghrelin to 
non-treatment seeking alcohol-dependent heavy drinking individuals. Furthermore, a preliminary 
interim analysis shows that IV ghrelin administration may lead to a temporary significant 
increase in alcohol craving. 
The primary objective of this protocol is to investigate whether IV ghrelin, as compared to 
placebo, will increase motivation for alcohol reward, as measured by a progressive ratio (PR) 
schedule paradigm with IV alcohol self-infusion (primary aim). We will also assess a number of 
secondary aims. Specifically, we will also assess whether IV ghrelin, as compared to placebo, 
will also alter urges to drink and the subjective response to IV alcohol. Adverse events will also 
be assessed to ensure the safety of the IV co-administration of ghrelin and alcohol. During an 
“fMRI/alcohol clamp” session, fMRI will be used to see whether ghrelin affects the activation of 
the ventral striatum induced by acute IV alcohol administration and the incentive salience of cues 
associated with alcohol administration. 
Study Population. Male and female participants will be non-treatment seeking heavy drinking 
volunteers. 
Design. The study is designed as a within-subject, double-blind, placebo-controlled study of 
ghrelin. The first visit will be the initial screening visit. The second and third visits will be PR 
sessions with IV ethanol, during which each participant will also receive IV ghrelin (or matched 
placebo). The fourth and fifth visits will combine an fMRI session with an ‘alcohol clamp’ 
session (i.e. a fixed dose of alcohol will be administered) and subjects will participate in a 
modified version of the monetary incentive delay (MID) task in which they will respond to cues 
that indicate the opportunity to press a button to gain a reward. On some trials the reward will be 
points that can be exchanged for snack foods while on other trials the reward will be points that 


4 
 
will determine how much intravenous alcohol a subject will be given during the “alcohol clamp” 
procedure which will immediately follow the modified MID task. After the modified MID task is 
complete subjects will have a short break and then begin the alcohol IV infusion. 
Outcome measures. The primary measure of this study will be the breakpoint, which is the 
schedule (number of button presses) at which the individual stops to work for more alcohol. 
Also, the BrAC exposure measures will be determined. Alcohol craving in response to ghrelin 
will be measured using the Alcohol Visual Analogue Scale (A-VAS) and the Alcohol Urge 
Questionnaire (AUQ) during the PR sessions. Sensitivity to alcohol will be measured using the 
Drug Effects Questionnaire (DEQ), Biphasic Alcohol Effects Scale (BAES), and the Profile of 
Mood States (POMS), repeatedly during the PR sessions and the CASE Experience 
Questionnaire (CEQ) at the end of all sessions. fMRI BOLD signal in brain areas associated with 
incentive salience and areas associated with reward circuitry (including the ventral striatum) will 
be measured during the “fMRI/alcohol clamp” session. This study may facilitate the 
identification of a novel neuropharmacological target, thus facilitating the development of novel 
pharmacological treatments for alcoholism. 
 
 
 
Table of Contents 
Precis 
3 
Introduction  
6 
Study objectives  
21 
Subjects 
22 
Study Design and Methods 
23 
Storage of data and samples 
32 
Additional Considerations 
32 
Risks and discomforts 
32 
Subject safety monitoring 
36 
Outcomes measures 
37 
Statistical Analysis 
37 
Human Subjects Protection 
38 
Anticipated Benefit 
40 
Classification of risk 
40 
Consent documents and process 
40 
Data and Safety monitoring 
            41 
Quality Assurance 
41 
Alternatives to participation or Alternative therapies 
41 
Confidentiality 
41 
Conflict of Interest 
42 
Technology Transfer 
42 
Compensation  
42 
References 
42 
 
 
 
 
 
List of Abbreviations 


5 
 
AE 
Adverse Events 
AD 
AEFQ 
Alcohol Dependence 
Alcohol Effects Questionnaire  
A-VAS 
Alcohol Visual Analogue Scale 
ARC 
AUDIT 
AUQ 
Arcuate Nucleus  
Alcohol Use Disorders Identification Test 
Alcohol Urge Questionnaire 
BAES 
BAS 
BIS 
BMI 
Biphasic Alcohol Effects Scale 
Behavioral Approach System 
Behavioral Inhibition System 
Body Mass Index 
BP 
Blood Pressure 
BASDA 
Brief Addictive Behavior Social Density Assessment 
BOLD 
BAC 
BrAC 
Blood Oxygen Level Dependent 
Blood Alcohol Content 
Breath Alcohol Concentration 
BrCO 
CASE 
breath carbon monoxide 
Computer-Assisted Self-infusion of Ethanol 
CBC 
CEQ 
CIWA 
CeA  
Complete Blood Count 
CASE Experience Questionnaire 
Clinical Institute Withdrawal Assessment 
central nucleus of the amygdala 
CPP 
CPRS 
Conditioned Place Preference 
Comprehensive Psychopathology Rating Scale 
CR 
CRF 
Cue-Reactivity 
Corticotropin-Releasing Factor 
C-SSRS 
Columbia Suicide Severity Rating Scale 
DA 
Dopamine 
DEQ 
FDA 
fMRI 
Drug Effects Questionnaire 
Food and Drug Administration 
functional Magnetic Resonance Imaging 
GHS-R 
HPA 
Growth Hormone Secretagogue Receptor 
hypothalamic-pituitary-adrenal 
HR 
Heart Rate 
ICV 
Intracerebral Ventricular 
IP 
IV 
Intraperitoneal 
Intravenous 
LDTg 
Laterodorsal Tegmental Area 
MCR 
Metabolic Clearance Rate 
MID 
Monetary Incentive Delay 
NAc 
Nucleus Accumbens 
nAChR 
NPY 
Nicotinic Acetylcholine Receptor 
Neuropeptide Y 
OCDS 
OFC 
P 
Obsessive-Compulsive Drinking Scale 
Orbitofrontal Cortex 
Preferring 


6 
 
PACS 
Penn Alcohol Craving Score 
PDS 
Pharmaceutical Development Service 
POMS 
Profile of Mood States 
PR 
Progressive Ratio 
QA 
Quality Assurance 
ROI’s 
Regions-of-Interest 
RYGB 
Roux-en-Y Gastric Bypass 
SAEs 
Serious Adverse Events 
SCID 
Structured Clinical Interview for DSM-IV 
TLFB 
Timeline Followback 
VTA 
UDT 
Ventral Tegmental Area 
Urine Drug Test 
 
 
 
1. Introduction 
There exist commonalities over-eating and over-consumption of alcohol.1-21 Like alcoholism, 
obesity and binge eating are complex genetic traits determined by several genes, and interacting 
with the environment. It is also notable that medications used to treat alcohol dependence (AD) 
often result in weight loss in other populations. Naltrexone, topiramate and ondansetron have all 
been utilized to treat obesity and/or eating disorders22. In recent years there has been an interest 
in the role of ghrelin in food-seeking behavior. Animal studies have shown that ghrelin is 
involved in the central dopaminergic reward processing and human studies have shown a direct 
relationship between ghrelin and alcohol craving. The overall conclusion is that the ghrelin 
system deserves human investigations in alcoholism, as detailed next. 
Ghrelin. 
Ghrelin is a 28-amino acid peptide acting as the endogenous ligand for the growth hormone 
secretagogue receptor (GHS-R), a G-protein coupled receptor that induces growth hormone (GH) 
release from the pituitary.1 The n-octanoyl bearing ghrelin is known as active ghrelin (acylated), 
although the des-acylated ghrelin is not totally inactive.23 Ghrelin was first isolated from the 
stomach in 1999.1 A hypothalamic production of ghrelin was suggested.24 Ghrelin activates 
hypothalamic orexigenic neurons and inhibits anorectic neurons to induce hunger25,26. 
Intracranial administration of ghrelin stimulates feeding in mammals27,28 and non-mammals29,30. 
Human studies have demonstrated the role of ghrelin in stimulating appetite, vasodilatation and 
gastrointestinal motility.31 Several studies have already used ghrelin intravenous (IV) both in 
healthy controls (Table 1) and in subjects with chronic diseases (Table 2). All of these studies 
have demonstrated the safety of IV ghrelin in humans as detailed next. 
 
 
 
 
 
Table 1. Studies in healthy controls 
# 
N 
IV Ghrelin dose 
AEs 
32 
6 
10 mcg/Kg (B) 
decreased BP 
33 
18 
1 or 5 pmol/kg/min (I) 
somnolence,  mild glucosuria, fever, diarrhoea 
34 
17 
5 pmol/Kg/min (I) 
None 
35 
8 
1 or 3 mcg/kg (B) 
None 


7 
 
36 
4 
1 mcg/kg (B) 
None 
37 
9 
5 pmol/kg/min (I) 
None 
38 
7 
1 mcg/kg (B) 
None 
39 
11 
1 mcg/kg (B) 
None 
40 
7 
50 mcg X 4 (B) 
None 
41 
7 
1 mcg/kg (B) 
None 
42 
34 
1 mcg/kg (B) 
None 
43 
7 
1 mcg/kg (B) 
None 
44 
7 
1 mcg/kg (B) 
Flushing 
45 
6 
1 mcg/kg (B) 
None 
46 
12 
1 or 5 pmol/kg/min (I) 
None 
47 
9 
100 mcg (B) 
fatigue; elevated mood; vertigo; emesis; flush 
48 
9 
1 mcg/kg (B) 
None 
49 
8 
10 pmol/kg/min(I) 
None 
50 
9 
40 mcg (I) 
None 
51
52 
10 
5 pmol/Kg/min (I) 
None 
53 
21 
0.3 mcg/kg (B) 
facial warmth 
54 
21 
0.3 mcg/kg (B) 
facial and/or neck flushing 
55 
15 
5 pmol/Kg/min (I) 
None 
56 
12 
50 mcg X 4 (B) 
sweating  
57 
10 
50 mcg X 4 (B) 
None 
58 
8 
5 pmol/Kg/min (I) 
None 
59 
7 
1 mcg/kg (B) 
None 
60 
12 
1 mcg/kg (B) 
None 
61 
9 
1 mcg/kg (I) 
None 
62 
20 
0.5 – 2.4 μg/kg (I) 
flushing, facial warmth 
63 
8 
0.5 mug/kg/h (I) 
None 
64 
6 
1 mcg/kg (B) 
facial flushing 
65 
9 
5 pmol/Kg/min (I) 
None 
66 
20 
50 mcg X 4 (B) 
None 
67 
10 
1 mcg/kg (B) 
None 
68 
10 
40 mcg (I) 
None 
69 
5 
2.5, 5 & 10 pmol/kg/min (I) 
Warmth 
Legend for Table 1: #: reference number; N: number of subjects; AEs: adverse events; 
B: bolus; I: Infusion; BP: blood pressure; PCOS: polycystic ovary syndrome 
 
 
 
 
 
Pharmacokinetic Profile. Because ghrelin is degraded in the digestive tract, ghrelin cannot be 
administered orally. Studies in healthy controls32-35 have used bolus IV ghrelin doses range from 
1 to 10 μg/kg, and continuous ghrelin infusion rates from 3.4 to 16.9 ng/kg/min. 
The concentration-dependence of ghrelin kinetics may contribute to some disparities in results, 
as suggested by Paulo et al35. Mean terminal half-life estimates for total ghrelin vary from as low 
as 10 min to as high as 47 min after bolus injection and 146 min after constant infusion. After 
constant infusion, the peak plasma concentrations (Cmax) are 1058.7 (882.6 - 1234.8) fmol/ml 


8 
 
when 1 mcg/kg ghrelin is infused and 6597.9 (5919 - 7276.6) fmol/ml when 5 mcg/kg total 
ghrelin is infused33. After bolus injection, Cmax is of 4438 ± 407pg/mL after 1 mcg/kg ghrelin is 
injected, and of 8176 ± 273 after 3 mcg/kg ghrelin in injected35. Bolus and steady-state infusions 
of ghrelin yield concentration-dependent (median) estimates of the metabolic clearance rate 
(MCR) of blood ghrelin35. 
 
 
IV Ghrelin in healthy subjects (Table 1 above).32-69 
Studies testing IV ghrelin in healthy subjects demonstrate that IV ghrelin reproduces 
physiological effects similar to that of endogenous ghrelin, e.g.: increased appetite, 
gastrointestinal motility. All the studies clearly demonstrate the safety of IV ghrelin either 
infused over time or given as a bolus. Described adverse events (AEs, e.g.:  
hypotension, diarrhoea, flushing, somnolence) were sporadic and transitory with a rapid and 
spontaneous resolution. There were no serious adverse events (SAEs) after IV ghrelin, nor AEs 
requiring medical intervention or the cessation of IV ghrelin infusion. 
 
IV Ghrelin in non-healthy subjects (Table 2 below)44-46;48,49,51,52,67,69,70-89. 
Based on the physiological actions of ghrelin and its safety in healthy controls, a growing 
number of studies have tested IV ghrelin in subjects with severe chronic diseases, including 
patients afflicted with cancer cachexia. For example, IV ghrelin significantly decreased blood 
pressure (BP) without significant changes in heart rate (HR) in patients with chronic heart 
failure70. IV Ghrelin also reverses endothelium dysfunction in patients with metabolic 
syndrome77 and increases gastric emptying in patients with diabetic gastroparesis78. Studies on 
the role of IV ghrelin in appetite are described below. 
 
 
 
 
 
 
 
 
 
 
 
 
 


9 
 
 
Rationale for studying the ghrelin system in alcohol dependence. 
Ghrelin stimulates appetite by acting on the hypothalamic arcuate nucleus (ARC), a region that 
controls the intake of food and other substances, including alcohol. Opioidergic neurons, which 
play a role in the reinforcing effects of alcohol, also are located in the ARC90. Plasma levels of 
ghrelin are higher under fasting conditions and prior to meals, indicating that ghrelin transmits a 
hunger signal from the periphery to the brain and participates in meal initiation91. Plasma ghrelin 
levels then fall to minimum levels within 1 hour after eating92. In addition to the ARC, GHS 
receptors (GHS-Rs) are also highly expressed in the caudal brain stem, the ventral tegmental area 
(VTA), hippocampus, substantia nigra, and dorsal and medial raphe nuclei93-95. The expression 
of the GHS-R in the mesolimbic dopamine (DA) pathway suggests that ghrelin could play a role 
in the reward processing.3,4  
Feeding behaviour, DA release and locomotor stimulation are triggered following intra-VTA 
microinfusion of ghrelin into either the VTA or nucleus accumbens (NAc)96 and intracerebral 
ventricular (ICV) administration97. Kawahara and colleagues98 demonstrated that ghrelin: i) 
administered to rats into the VTA induced DA release in the NAc and food consumption; and ii) 
administered to rats (without food deprivation) peripherally induced a robust increase of DA 
levels in the NAc and this stimulatory effect of ghrelin primarily required activation of GHS-Rs 
in the VTA. Furthermore, subcutaneous administration of ghrelin in rats produced a significant 
increase in extracellular levels of DA in the NAc shell, but not in the NAc core99. These results 
suggest that the mesolimbic DA processing is implicated in the effects of ghrelin on food-
seeking behaviour2. The role of the DA processing in the neurobiology of alcohol and other drug 
dependence is well known. In particular, cortico-mesolimbic DA pathways may mediate 
Table 2. Studies in non-healthy subjects 
# 
N 
chronic disease(s) 
Ghrelin dose i.v. 
AEs 
44 
9 
Obesity 
1 mcg/kg (B) 
flushing 
45 
6 
Obesity 
1 mcg/kg (B) 
none 
46 
12 
Obesity 
1 or 5 pmol/kg/min (I) 
none 
48 
7 
Bulimia nervosa 
1 mcg/kg (B) 
none 
49 
6 
GH-deficiency 
10 pmol/kg/min (I) 
none 
51,52 
10 
Anorexia nervosa 
5 pmol/Kg/min (I) 
none 
67 
8 
Gastrectomy 
5 pmol/Kg/min (I) 
none 
67 
9 
Obesity 
5 pmol/Kg/min (I) 
none 
69 
5 
Geriatric frailty 
2.5, 5 & 10 pmol/kg/min (I) 
warmth 
70 
6 
Chronic heart failure 
0.1 mcg/kg (I) 
warmth; sleepness 
71 
6I 
Idiopathic GH-deficiency 
1 mcg/kg (B) 
none 
72 
6 
GH-deficiency 
1 mcg/kg (B) 
none 
73 
8 
GH-deficiency 
1 mcg/kg (B) 
none 
74 
9 
Anorexia nervosa 
1 mcg/kg (B) 
none 
75 
7 
Cancer 
5 pmol/kg/min (I) 
none 
76 
6 
Idiopathic gastroparesis 
40 mcg (I) 
none 
77 
18 
Metabolic syndrome 
200 mcg/min (I) 
none 
78 
10 
Diabetic gastroparesis 
5 pmol/kg/min (I) 
none 
79 
9 
Malnourished peritoneal dialysis patients 
3.6 nmol/kg (SC) 
decreased BP 
80 
10 
Gastrectomy 
5 pmol/Kg/min (I) 
none 
81 
25 
Obesity 
0.33 mcg/kg (B) 
none 
82 
15 
Obesity & PCOS 
1 mcg/Kg (B) 
none 
83 
6 
Dyspepsia 
3 mcg/Kg (I) 
abdominal pain; flushing; 
somnolence; hyperhidrosis 
84 
35 
Obesity & PCOS 
1 mcg/Kg (B) 
none 
85 
15 
Primary hyperparathyroidism 
1 mcg/kg (B) 
none 
86 
7 
Obesity & PCOS 
1 mcg/kg (B) 
none 
87 
12 
Malnourished dialysis patients 
12 mcg/kg (SC) 
abdominal discomfort, decreased, 
BP, lethargy, sleepiness 
88 
8 
Obesity 
100 and 200 mcg (B) 
flushing, dizziness 
89 
5 
Anorexia nervosa 
3 mcg/kg b.i.d. for 14 days 
loose stools, warmth, sweating 
Legend for Table 2: #: reference number; N: number of subjects; AEs: adverse events; B: bolus; I: Infusion; SC: 
subcutaneous, BP: blood pressure; PCOS: polycystic ovary syndrome. 


10 
 
alcohol’s rewarding effects (including craving) associated with its abuse liability20,100-102. In vivo 
microdialysis studies have shown that ethanol administration in non-dependent rats is associated 
with increased NAc DA concentrations, and these DA rises are greater in ethanol-preferring 
compared with wild-strain animals103-105. Increased extracellular DA levels in the NAc shell have 
been typically associated with the acute reinforcing effects of alcohol and addictive drugs102. 
Indeed, baseline extracellular DA concentrations and the rate of rise following ethanol self-
administration may predict alcohol preference in animals106. Clinical studies further support the 
preclinical evidence reported above. Increased appetite is the most common effect in studies 
testing IV ghrelin. For example, Schmid et al.47 demonstrated that a single bolus injection of 
ghrelin acutely increases appetite, as well as ideas about food in healthy subjects. IV Ghrelin also 
increases appetite in patients with chronic diseases, such as cancer-related anorexia75, dyspepsia-
related anorexia83, obesity46 and geriatric frailty69. 
In summary: i) in rats, ghrelin increases food consumption and in humans, IV ghrelin increases 
appetite; ii) in rats, ghrelin acts centrally modulating the DA reward processing; and iii) the DA 
reward processing regulates appetite.107 Therefore, in humans ghrelin could increase appetite via 
the DA reward processing. Moreover: i) alcohol and food-seeking behaviour share the same 
neurobiological mechanisms;108 and ii) both alcohol and food exert their reinforcing effects in 
part by increasing DA in limbic regions. Thus, the role of ghrelin in the DA reward processing 
and the role of the DA reward processing in alcoholism suggest a role of ghrelin in alcoholism. It 
is important to keep in mind that the mechanisms regulating alcohol-seeking behaviours are 
multiple and complex. In fact, alcohol and drugs of abuse activate the mesolimbic DA 
processing, but much evidence suggests that dopamine-independent reinforcement occurs at the 
level of the nucleus accumbens, suggesting multiple inputs to the activation of critical 
reinforcement circuitry in these brain regions.109 Brain arousal/stress systems in the extended 
amygdala may be key components of the negative emotional states that drive dependence on 
alcohol and drugs of abuse. Furthermore, the central nucleus of the amygdala (CeA) also plays a 
key role in the acute reinforcing actions of drugs of abuse; in fact, compulsive alcohol and drug 
use is mediated by not only loss of function of reward systems but also recruitment of brain 
stress systems such as corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) in areas 
such as the amygdala. 110,111 CRF is associated with an increased stress response and negative 
affect, and NPY with anxiolytic properties. As widely described by Koob’s lab,109,112 
dysregulation of the CRF and NPY systems plays an important role in the motivational basis of 
continued alcohol-seeking behavior. In particular, NPY regulates the anxiolytic effects of 
ethanol, thus the role of NPY is crucial in the motivational basis to alcohol intake. Interestingly, 
Skibicka and colleagues113 performed a series of experiments with rats trained in a progressive 
ratio sucrose-induced operant schedule to measure food reward/motivation behavior and reported 
that the ventricular ghrelin-induced increase in sucrose-motivated behavior and chow intake 
were suppressed by ICV pretreatment with either an NPY-Y1R antagonist or naltrexone. 
Furthermore, ghrelin infusion was associated with elevated VTA-opioid receptor expression. As 
such, this study provided evidence of the role of central NPY and opioid signaling as key 
mediators of food intake and reward effects of ghrelin. On the other and, Naleid and colleagues 
114 demonstrated that ghrelin had a significant, short-term effect on reward-based food intake 
when injected into the VTA and that this effect on feeding behavior was independent from 
opioid signaling in the Acb. Additional evidence was provided by Spencer and colleagues115 who 
used ghrelin knockout mice to investigate anxious behavior and hypothalamic-pituitary-adrenal 
axis (HPA) responses to acute stress. In this study, the authors found that ghrelin reduces anxiety 


11 
 
after acute stress by stimulating the HPA axis at the level of the anterior pituitary.116 This 
additional evidence is of interest given that HPA dysregulation may contribute to alcohol 
consumption. In summary, the neurobiological mechanisms of alcohol dependence are complex 
and while there is robust evidence to support the role of the DA system in the mechanisms of 
ghrelin modulation of alcohol reward, on the other hand alternative hypotheses cannot rule out 
and deserve further investigations. 
 
Preclinical Studies. 
Ghrelin activates the cholinergic-dopaminergic reward link. 
Ghrelin administration into the VTA increases extracellular concentrations of accumbal DA in 
mice4. The non-selective nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine 
attenuates the stimulatory and DA-enhancing effects of ghrelin infused into the third ventricle97, 
indicating that nAChRs mediates these neurochemical properties of ghrelin; nAChRs also 
mediate the locomotor stimulatory and DA-enhancing properties of ghrelin administered into the 
VTA117. Taken together, these studies suggest that ghrelin activates the cholinergic-
dopaminergic reward link and that ventral tegmental nAChRs have a central role for the DA-
enhancing properties of ghrelin. Only some specific nAChR subtypes (alpha3-beta2, beta3, 
alpha6) are implicated in the central rewarding actions of ghrelin117. Interestingly, the same 
nAchRs (alpha3-beta2, beta3) mediate an increase in ventral tegmental ACh and accumbal DA 
levels after voluntary ethanol consumption118-119. Ghrelin administered into the lateral 
hypothalamus or paraventricular nucleus has no effects on ethanol consumption120, confirming 
that ghrelin works in specific brain reward nodes (i.e., VTA). Peripherally injected ghrelin also 
activates brain reward parameters, such as locomotor activity, accumbal-DA release and 
conditioned place preference (CPP)121. 
In summary, these preclinical studies demonstrate that both ghrelin and ethanol activate the 
cholinergic-dopaminergic reward link, supporting the hypothesis that ghrelin is involved in 
mediating the rewarding properties of ethanol.  
 
Requirement of central ghrelin signaling for alcohol reward. 
Another set of experiments122 demonstrated that: i) alcohol-induced brain reward parameters, 
such as enhanced extracellular accumbal DA overflow (a measure reflecting synaptic DA 
release), locomotor stimulation and CPP were consistently abolished or attenuated by two GHS-
R1A antagonists in wild-type mice and were abolished in GHS-R1A knockout mice; ii) ICV 
administration of ghrelin to mice significantly increased alcohol consumption compared to 
vehicle treatment in a 2-bottle (alcohol/water) free choice limited access paradigm. Bilateral 
administration of ghrelin into either the VTA or the laterodorsal tegmental area (LDTg) also 
increased alcohol consumption in comparison to vehicle. The percentage increase in alcohol 
consumption was significantly greater following administration to the VTA or the LDTg 
compared to the ICV route. Food intake (normal chow) was increased by ICV ghrelin 
administration in comparison to vehicle but was not affected by bilateral ghrelin administration 
into either the VTA or the LDTg. This observation is particularly relevant as it may shed light on 
the specific role of ghrelin in alcohol-seeking behavior, as opposed to a non-specific role of 
ghrelin in appetitive behaviors. In fact, ICV administration of ghrelin significantly increased 
alcohol consumption as well as food intake. On the other hand, when ghrelin was administered 
directly into either the VTA or the LDTg, then only alcohol consumption was increased, while 
food intake was not affected. Interestingly, the percentage increase in alcohol consumption was 


12 
 
significantly greater following administration to the VTA or the LDTg compared to the ICV 
route, thus suggesting that ghrelin’s actions via reward areas (VTA, LDTg) are somewhat 
specifically related to alcohol reward and not simply secondary to its effect on appetite. 
Alcohol intake in the 2-bottle (alcohol/water) free choice limited access paradigm was 
suppressed in mice by both the GHS-R1A antagonists [delivered either ICV or intraperitoneal 
(IP)]. The effects of ICV ghrelin on alcohol intake were absent in GHS-R1A knockout mice. 
In summary, using genetic and pharmacological models of suppressed ghrelin signaling, these 
studies demonstrate that the central ghrelin action not only stimulates the reward system but is 
also required for stimulation of that system by alcohol.  
 
Human Studies on Ghrelin in alcoholic individuals: 
Ghrelin, alcohol intake and craving. 
Alcohol, compared to water, significantly reduces ghrelin levels in healthy subjects123-124. 
Zimmermann et al.125 showed a significant reduction of blood ghrelin levels after alcohol 
ingestion in healthy subjects but unchanged ghrelin levels after a laboratory stressor exposure. 
Preliminary studies reported that actively drinking alcohol-dependent patients (with an alcohol 
intake within 24 hrs) show lower plasma ghrelin levels compared to controls5,126. On the other 
hand, other studies demonstrated a significant increase of ghrelin in abstinent alcoholics127-128. 
All together, these studies suggest that ghrelin is suppressed by acute alcohol intake and 
increases during abstinence. One of these studies also reported a significant positive correlation 
between plasma ghrelin levels and the Obsessive-Compulsive Drinking Scale (OCDS) craving 
scores in actively drinking alcoholics5. Interestingly, ghrelin was positively correlated with the 
Compulsive but not with the Obsessive subscore of the OCDS5. This last finding is consistent 
with the role of ghrelin in modulating food intake and with the observation that compulsion 
reflects the behavioral reward mechanisms of addictive disorders like eating disorders129. 
Successive human studies have confirmed, at least partially, the relationship between plasma 
ghrelin levels and alcohol craving. In particular, Hillemacher et al.130 reported a significant 
relationship between ghrelin and OCDS score in an alcoholic subtype (Lesch type 1) 
characterized by a positive family history of alcoholism. Wurst et al.131 found a relationship 
between ghrelin levels and the OCDS compulsive subscore in female alcoholics. Recently, the PI 
completed a study with alcohol-dependent individuals132, where plasma ghrelin levels were 
determined several times – the first longitudinal study testing repeated blood ghrelin 
determinations in a sample of alcohol-dependent individuals. Baseline ghrelin (T0) was taken 
after 72 hrs of alcohol abstinence, in order to avoid an acute effect of alcohol on ghrelin levels as 
a possible confounding factor. Then, ghrelin was re-tested after 2 weeks (T1), 6 weeks (T2) and 
12 weeks (T3). Results showed a relationship between ghrelin, alcohol intake and craving.132 In 
fact, a comparison of non-abstinent vs. abstinent (during the 12 weeks) subjects showed that: 
i) non-abstinent subjects had higher ghrelin levels, which went down during the 12-week period 
(consistent with the inhibitory effect of alcohol on ghrelin5,123-126); ii) in abstinent subjects 
ghrelin levels increased during the 12-week period, as previously shown127-128; iii) there was a 
significant difference in the changes of ghrelin between the two groups (Fbetween 
groups=4.913;p=0.012; Figure 1 below) and the difference in baseline ghrelin levels was 
statistically significant (p=0.035).  
 
Figure 1 


13 
 
 
Legend: a comparison of non-abstinent alcohol-dependent subjects (n = 13) and abstinent alcohol-dependent 
subjects (n = 19) indicated a significant difference in the changes of ghrelin between the two groups (p=0.012) and 
a significant difference in baseline ghrelin levels (p=0.035).  
 
These findings suggest that baseline ghrelin levels play an important role in alcoholic 
individuals; those patients with higher ghrelin levels at T0, in fact, were those who relapsed 
during the 12-week period. We also found a statistically significant positive correlation between 
ghrelin at baseline (T0) and craving during the 12-week period (T1-3), measured by the OCDS 
and the Penn Alcohol Craving Score (PACS). 
 
Additional translational studies. 
Consistent with the preclinical and clinical data presented above and with our overall hypothesis 
that the ghrelin plays a key role in alcohol reward and consumption, Davis and colleagues133 at 
the University of Cincinnati analyzed self-report of ethanol intake in 6,165 bariatric patients 
before and following a Roux-en-Y gastric bypass (RYGB) surgery. Patients that reported 
frequent consumption of ethanol before RYGB reported decreased consumption following 
RYGB surgery. The same group also examined the hypothesis that RYGB surgery attenuates 
ethanol intake and reward in the context of frequent ethanol consumption utilizing a rodent 
model of RYGB and examined ethanol consumption and ethanol reward in male ethanol-
preferring (P) rats. Interestingly, the RYGB procedure decreased ethanol intake and the 
reinforcing properties of ethanol in P rats. Of special interest for this protocol, pharmacologic 
replacement of the gut hormone ghrelin restored drinking behavior in P rats following RYGB. 
 
Genetic polymorphisms of the ghrelin gene. 
In another study132, our group explored two polymorphisms of the ghrelin gene (i.e. G152A 
(Arg51Gln) and C214A (Leu72Met); these two polymorphisms were investigated previously in 
patients with binge eating disorders) in alcoholics and controls. Statistical comparisons of BMI 
values by means of two-way ANOVA revealed a main effect of 
genotype (F = 7.013, P = 0.009) with no main effect of diagnosis (F = 0.705, P = 0.402) and a 
trend toward a significant diagnosis–genotype interaction (F = 0.096, P = 
0.0757). Furthermore, Landgren and colleagues134 reported that SNP rs2232165 of the GHS-R1A 
gene was associated with heavy alcohol consumption and SNP rs2948694 of the same gene as 
well as haplotypes of both the pro-ghrelin and the GHS-R1A genes were associated with body 


14 
 
mass in heavy alcohol consuming individuals. In summary, very preliminary data suggest a 
possible role of the genes encoding ghrelin and/or its receptor in alcohol use, but these findings 
are not conclusive and suggest the need to further explore this aspect. 
 
Summary. 
In summary, human studies show: i) reduced ghrelin levels in actively drinking alcoholics; ii) 
increased ghrelin levels during alcohol abstinence; iii) a positive correlation between ghrelin 
level and alcohol craving scores. In keeping with the pre-clinical studies, the human studies 
suggest that ghrelin represents a potential new target for treatment of alcoholism. 
 
Additional clinical data: preliminary findings with IV Ghrelin in alcohol-dependent individuals 
While at Brown University, Dr. Leggio received funding from NIAAA (AA019709) to conduct 
the first study ever administering IV human ghrelin to non-treatment seeking heavy drinking 
alcohol-dependent individuals. The study – clinicaltrials.gov NCT01190085 – was also reviewed 
by the FDA (IND# 109242; sponsor and holder: Dr. Leggio). The experimental design is a 
between-subject 3-arm randomized double-blind placebo-controlled laboratory. Human ghrelin 
or saline solution (placebo) represents the between subjects factor. Each participant is randomly 
assigned to receive IV ghrelin 1 microg/kg or ghrelin 3 microg/kg or saline solution and then 
participates in a cue-reactivity (CR) experiment. Cue reactivity has demonstrated utility in 
eliciting urge to drink in alcoholics135-137. Exposure to alcohol cues (e.g.: the sight and smell of 
an individual’s preferred beverage), reliably elicits increased urge to drink alcohol, increased 
salivation, and increased attention to the cues135-137 when compared with control cues. 
The two main endpoints of the trial performed at Brown University were: (1) whether IV ghrelin, 
as compared to IV saline, dose-dependently results in increased CR responses to alcohol cues in 
terms of urge to drink; and (2) whether IV ghrelin, as compared to IV saline, does not 
significantly increase frequency and intensity of Adverse Events (AEs). 
The main findings of this trial were: 
- 
Forty-five (n = 45) non-treatment seeking alcohol-dependent heavy drinking individuals were 
randomized and received administration of either IV ghrelin 1 microg/kg, IV ghrelin 3 
microg/kg or placebo (IV saline). There were no serious adverse events (SAEs), unexpected 
AEs or severe AEs. There were only a few AEs, all were in the mild-moderate range and 
they were all expected. AEs were only transitory, and were all resolved without sequelae. It 
should be noted that the most common AE, i.e. increase in appetite, is in actuality a highly 
expected effect of ghrelin, given that ghrelin physiologically increases appetite (indeed, 
current studies are investigating the therapeutic role of ghrelin to increase appetite, for 
example, in patients with cancer). The increase in appetite was only transitory and resolved 
quickly and without sequelae. Notably, although this study enrolled heavy drinkers, none of 
them reported flushing after the infusion IV of ghrelin. 
- 
 We found no significant differences (p > 0.5) in the frequency AEs among the 3 arms of the 
study. In a repeated measures ANCOVA with the baseline SAFTEE score entered as 
covariate, dose did not significantly predict adverse events for the 3 post-injection 
assessments [F(2,40) = 0.97, p = .39].  Due to the short half-life of ghrelin, we also ran a 
parallel ANCOVA on just the first two post-injection assessments and this was also non-
signficant [F(2,40) = 1.37, p = .27].  
- 
Repeated measures ANCOVAs were conducted for the increase in alcohol urge during the 
CR procedure relative to the pre-medication urge level. Covariates included age, weight, 


15 
 
gender, and ethnicity/race. Pairwise comparisons revealed that alcohol urge was significantly 
greater for high dose ghrelin (3 mcg/kg) than for the placebo [p = .04, Least Significant 
Difference procedure (low vs. High dose: p = .19, placebo vs. Low dose: p = .52)] (Figure 2). 
The effect size (d) for the increase in alcohol urge for the high dose ghrelin versus placebo 
was 0.77. Notably, while CR studies usually use water as a neutral cue, here we used juice as 
a non-alcoholic appetitive control cue, and we didn’t find a significant effect of ghrelin in 
increasing urge to juice, thus suggesting the specificity of ghrelin’s effects on urge to alcohol.  
 
 
 
 
Figure 2 
A-VAS score for 
alcohol craving 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Legend: significant increase in A-VAS score for craving in subjects receiving IV ghrelin 3 mcg/kg (n = 14)  vs. 
those receiving IV ghrelin 1 mcg/kg (n = 13) or placebo (n = 18). 
 
Summary of studies of ghrelin in alcoholism and questions that need to be answered. 
Both animal and human studies provide strong evidence that the ghrelin system influences not 
only hunger but also has a clear role in the search for rewarding substances such as alcohol. 
Taken together, the preclinical studies provide strong evidence that ghrelin works not only on 
homeostatic energy-regulatory centers (e.g., hypothalamus and hindbrain), thus increasing the 
research of food, but also on reward centers (i.e., mesolimbic DA pathways), thus playing a role 
on the reward value of substances, such as food and alcohol. As reported above, animal studies 
suggest that ghrelin activates the cholinergic-dopaminergic reward link and that ventral 
tegmental nAChRs have a central role for the DA-enhancing properties of ghrelin. 
Similarly, human studies have provided important information, showing that alcoholic 
individuals with higher plasma ghrelin levels have higher alcohol craving. These experiments 
suggest a model wherein elevated ghrelin levels might contribute to craving for substances such 
as alcohol. While the previous human studies have only tested plasma ghrelin levels, the study 
NCT01190085 conducted at Brown University (PI: Leggio) represents a translational research 
investigating the direct effect of exogenous ghrelin administration IV on alcohol-seeking 
behaviors. In addition to showing the safety of ghrelin administered IV to alcohol-dependent 
heavy drinkers, study NCT01190085 provides preliminary evidence that, as demonstrated in 
animal models, ghrelin may represent a novel pharmacological target to treat alcoholism.  
While ghrelin antagonists are not available (but their development for other medical diseases is 
in progress), there is a crucial need to provide additional human data using IV ghrelin 
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
CR trial 1 (alcohol)
CR trial 2 (alcohol)
placebo
Ghrelin 1 mcg/Kg
Ghrelin 3 mcg/Kg


16 
 
administration that can corroborate the animal studies performed to date and can provide 
evidence that ghrelin plays an important role in alcohol-seeking behavior. While study 
NCT01190085 provides safety data and a preliminary evidence that ghrelin may increase acutely 
alcohol craving, a logical next step of this research program is to test the effects of IV ghrelin 
administration on alcohol administration and on the behavioral effects of alcohol. Furthermore, it 
is important to investigate the CNS areas involved with alcohol reward via imaging tools. In the 
proposed study, we wish to provide direct, controlled clinical evidence that ghrelin is involved in 
the behavioral and central effects of alcohol administration. We propose to assess this using 1) a 
progressive ratio (PR) schedule paradigm with IV alcohol infusion, a paradigm that this research 
team has already used and validated (as detailed next), for example in protocol 08-AA-0178; and 
then 2) an “fMRI/alcohol clamp” paradigm that this research group has already validated and 
currently using, for example, in a protocol assessing varenicline (08-AA-0137). 
 
PR IV Alcohol Procedure 
The goal of the first paradigm is to investigate whether ghrelin alters alcohol’s pharmacologic 
effects, including motivation for alcohol reward, as well as subjective measures of craving, and 
the biphasic effects of alcohol. The progressive ratio (PR) schedule paradigm requires 
participants to press the button an increasing number of times for each subsequent alcohol 
exposure (for e.g., 10, 20, 40, 80, etc., button presses for the 1st, 2nd, 3rd, 4th, etc. infusions). 
Outcome measures of this paradigm are the “breakpoint”, i.e., the point at which the individual 
stops to work for more alcohol, as well as the average and peak BrACs achieved during the 
study. The PR schedule paradigm is an operant paradigm that is expected to assess motivation 
for reward (in this case, alcohol), and are based on the principle that people will work harder for 
greater rewards. There is much animal work to suggest the utility of operant paradigms in 
characterizing motivation for reward.18 These operant paradigms have been used extensively in 
animal models and in a human studies to evaluate self-administration behavior for other drugs of 
abuse such as heroin and cocaine. Our research team at LCTS has experience using the PR 
paradigm, e.g. 08-AA-0178. The PR schedule paradigm represents an interesting and novel 
extension of the current development of IV alcohol self-administration methods extensively used 
by our research team (e.g.138,139).  Notably, since the alcohol administration is not oral, it avoids 
the cues associated with oral alcohol administration that can confound the interpretation of 
laboratory studies employing drinking paradigms. Finally, during the PR sessions, the safety of 
ghrelin and alcohol, when co-administered IV will be evaluated, therefore the PR sessions will 
assure the safety of alcohol and ghrelin co-administered before performing the second paradigm 
(“fMRI/alcohol clamp”) with the same volunteers (in fact, we will employ a within-subject 
design, as detailed after).Our team has recently demonstrated (PI: Vijay Ramchandani, Ph.D.; 
unpublished) the validity and utility of this procedure in a study whose primary objective was to 
examine alcohol intake behavior and the motivation for reward using subjective measures of 
alcohol effects and personality measures in a PR schedule of IV alcohol self-administration in 28 
social drinkers. In this study, we estimated the correlations between sessions for number of 
button presses (Fig. 3A), total rewards earned (Fig. 3B), peak BrAC (Fig. 3C), and average 
BrAC (Fig. 3D); the correlation coefficients were high, demonstrating that this method has good 
test-retest reliability. 
 
 
 


17 
 
 
Figure 3. 
  3A 
    3C 
  3B 
   3D 
Legend: between-sessions correlations for number of button presses (3A), total rewards earned (3B), peak BrAC 
(3C), and average BrAC (3D). 
 
Furthermore, total ethanol self-administered during the PR schedule was significantly associated 
with drinking days measured with the TimeLine Follow-Back (TLFB) (Figure 4), thus indicating 
the clinical relevance of this procedure given its ability to reproduce in the human lab setting, the 
alcohol use in the naturalistic milieu. 
 
 
     Figure 4 
 
 
Legend: significant association between total ethanol self-administered during the PR schedule and drinking days. 
Additionally, in this study we also found that total ethanol self-administered during the whole PR 
schedule was significantly associated with the Drug Effects Questionnaire (DEQ) score 
(considering its peak value) for measures of “wanting” (Fig. 5A), as well as with the DEQ score 
for “wanting” following the priming (Fig. 5B). 
 
 
 
 
 
 
 


18 
 
Figure 5 
 
 
5A 
 
 
 
 
5B 
 
Legend: significant association between total ethanol self-administered and both DEQ “Want” score during the 
whole PR procedure (A) and following the priming (B). 
 
Additional interesting results of this study were that total ethanol self-administered during the PR 
schedule was significantly associated with DEQ scores for measures of “feeling” and “liking” 
drug effects, as well as “high” and “intoxication” were positively associated with number of 
button presses.  Alcohol Urge Questionnaire (AUQ) maximum measures were significantly 
associated with total ethanol and number of button presses. Biphasic Alcohol Effects Scale 
sedation measure after priming was strongly associated with peak BrAc, average BrAC, and total 
rewards earned. 
In summary, these results demonstrate that the PR IV alcohol infusion procedure has good test-
retest reliability and demonstrate that this method is sensitive to the rewarding and motivational 
properties of alcohol. 
 
fMRI/alcohol clamp 
The goal of the second paradigm is to investigate possible mechanisms of action of ghrelin 
though the use of two slightly different but related fMRI procedures. Specifically: 
a) We will determine if ghrelin increases the incentive salience of alcohol predicting cues 
by using a modified version of the monetary incentive delay (MID) task.  In this task 
subjects will button press to earn credits associated with subsequent alcohol 
administration, instead of monetary, reward.  The MID task has been used extensively in 
neuroeconomic imaging research and the difference in BOLD signal in the ventral 
striatum (VS) between anticipation of responding for $0 and anticipation of responding 
for $5 is thought to measure the incentive salience of cues associated with monetary 
reward.140,141 We propose that the difference in striatal BOLD signal between anticipation 
of responding for a cue predicting no alcohol delivery and cues predicting delivery of 
alcohol will provide an objective measure of the incentive salience of cues associated 
with alcohol reward.  Our hypothesis is that ghrelin increases the motivational value of 
alcohol cues. In addition to the VS, other fMRI regions-of-interest (ROI’s) for this study 
are the amygdala, insula and orbitofrontal cortex (OFC), as IV ghrelin has already shown 
its ability to activate these areas,142 and all these areas play an important role in the neural 
systems model of alcoholism. 20,101,109,112,143 
b) In addition to using fMRI to examine ghrelin’s effect on the incentive salience of alcohol 
cues, we will also use fMRI procedures we have developed144 to examine the effect of 
ghrelin on the brain’s response to IV alcohol.  Our previous work has shown that IV 


19 
 
alcohol administration using the “alcohol clamp” procedure (NIAAA protocol 04-AA-
0060) results in a robust increase in BOLD signal in the human ventral striatum.  Thus, 
by administering alcohol during functional imaging we should be able to determine if 
ghrelin enhances the direct pharmacological effect of alcohol on the brain’s reward 
circuitry. 
Our team has validated the procedures we propose here for the fMRI/clamp sessions. Dr. 
Hommer of the Section of Brain Electrophysiology and Imaging has used and validated different 
versions of the MID task in the last decade and during the last years these procedures have been 
widely used in the functional neuroimaging imaging community to study reward processing. In 
the basic version of the task subjects see a cue indicating that on the current trial they are 
working for money (e.g., $5.00, $1.00, or $.20) or for no money; next they wait for a variable 
delay, and then press a button in response to presentation of a target. If they respond before the 
target disappears, they win the money indicated at the start of the trial.  If they are too slow they 
win nothing. The interval between appearance of the initial cue and the target cue signaling the 
need to make a response is considered to manifest the brain state underlying the motivation for 
the specific action and involves both elements of motor preparation as well as anticipation of 
gaining reward. Striatal activation during this interval can also be considered a measure of 
incentive salience.  Finally the subjects get visual feedback indicating if they successfully 
responded, how much they won or lost, and their total earnings. One of the major goals has been 
to develop ways of using a version of MID task to directly measure motivation to experience 
alcohol’s effects in humans and to use this model to study pharmacotherapies. In fact, Dr. 
Ramchandani of the Section on Human Psychopharmacology is currently using this new 
approach in an experimental medicine study to evaluate the efficacy of varenicline for the 
reduction of excessive alcohol use in non-treatment seeking heavy drinkers. In this study, 
subjects are initially trained to button press to obtain intravenously administered ethanol. Then 
they are scanned while they  
perform a MID task during which they can button press to 
obtain points that determine how much intravenous alcohol or 
snack food they will be given at the conclusion of the scan. 
Thus, instead of measuring brain activation while anticipating 
working for money, we can measure brain activation while 
anticipating working for alcohol or food. Our group has 
recently conducted preliminary analysis (PI: Vijay 
Ramchandani, PhD; unpublished) of the data obtained from 
subjects on placebo (n=7); this analysis indicated significantly 
higher BOLD activation in striatal regions during anticipation 
of working for alcohol reward under placebo conditions. 
Preliminary analysis of data from subjects receiving varenicline 
(n=8) compared to placebo (n=7) show lower  
 
Figure 6: decreased BOLD 
activation in striatum (blue region) 
seen in patients on varenicline 
compared to placebo during 
anticipation for alcohol reward in 
Alcohol-MID task. 
BOLD activation in striatum during anticipation of working for alcohol (p<0.01, uncorrected, 
k>10) (Figure 6). These results suggest that the Alcohol-MID task can be used to measure 
motivation or desire for alcohol as well as the effect of a drug on this measure. 
 
Innovation of the study 
This protocol is not only a logical ‘next step’ of Dr. Leggio’s NIAAA-funded study with ghrelin 
performed at Brown University, but also there are several new and innovative aspects, i.e. 1) the 


20 
 
study at Brown University only assessed alcohol craving (i.e. alcohol was not administered), 
while this study will test for the first time the role of IV ghrelin on alcohol administration. Using 
a model already validated by our team (the PR paradigm with IV alcohol administration; 08-AA-
0178), this protocol represents a direct translational study of the preclinical studies conducted by 
Jerlhag and colleagues.3,4,117,121,122 In fact, the use of the PR administration will provide novel 
information, that is if IV ghrelin alters motivation for alcohol reward; 2) this study will provide, 
for the first time, information on the effect of ghrelin on the brain’s response to alcohol; 3) the 
study at Brown University only assessed the effects of a bolus on alcohol craving, while this 
protocol will assess the effects of ghrelin for a longer period of time. In fact, after a bolus of 
ghrelin, then an IV infusion of ghrelin will take place during the entire duration of the 
experiment; 4) there are only a few and preliminary data on the role of genetic polymorphisms of 
the ghrelin and ghrelin receptor genes on alcohol consumption; also, none of the previous studies 
was conducted in subjects receiving an IV administration of ghrelin, thus this protocol will test 
genetic polymorphisms related to the ghrelin system in heavy drinkers receiving IV ghrelin; and 
5) although the well-known role of ghrelin in appetite and food intake, and while several data 
suggest that sweet preference may represent an important phenotype in alcoholic individuals, this 
protocol will explore if and how sweet-liking status (a sweet preference test will be performed, 
as detailed below) may affect ghrelin levels and behavioral responses in heavy drinkers receiving 
IV ghrelin. 
In summary, this project proposes to use validated approaches (i.e. IV alcohol infusion and 
fMRI) to provide a direct and objective evidence of the central role of ghrelin in alcohol reward 
and administration. The overall goal is to provide evidence of a novel neuropharmacological 
target to treat alcoholism, i.e. the ghrelin system. If our hypothesis will be demonstrated, then 
this study will serve as the platform to test ghrelin antagonists/inverse agonists as new 
pharmacotherapies for alcoholism. Additionally, this study may provide evidence of the use of 
IV ghrelin as a ‘challenge/provocative test’ for future behavioral and pharmacological studies. 
 
2. Study objectives  
The primary objective of this protocol is to investigate whether ghrelin alters alcohol’s 
pharmacologic effects, including motivation for alcohol reward, as well as subjective measures 
of craving, and the subjective responses to alcohol in heavy drinkers receiving IV 
administrations of ghrelin and alcohol. 
 
 
a. Primary objectives 
The primary measure of this study will be the breakpoint, which is the schedule (number of 
button presses) at which the individual stops to work for more alcohol. 
 
b. Secondary objectives 
Secondarily, the BrAC exposure measures will also be determined, that is the highest (max) 
BrAC, average BrAC and the area under the BrAC-time curve determined for each subject. Urge 
to drink and alcohol sensitivity will be measured during the PR session. Adverse events will also 
be assessed in order to assess the safety of the IV co-administration of ghrelin and alcohol. Also, 
during the “fMRI/alcohol clamp” session, fMRI will be used to see whether ghrelin affects the 
activation of the ventral striatum (VS) in anticipation to work for alcohol reward and the 
incentive salience of cues in subjects who will subsequently receive IV alcohol administration. In 


21 
 
addition to the VS, other fMRI ROI’s are the amygdala, insula and OFC. Adverse events will 
also be assessed during the fMRI/alcohol clamp sessions. 
Furthermore, an additional secondary aim will be an exploratory analysis of the role of genetic 
polymorphisms of both ghrelin and ghrelin receptor genes – detailed in page 14 – as possible 
moderators of ghrelin’s effects on the primary aim (‘breakpoint’, i.e. number of button presses in 
the IV alcohol PR procedure). 
 
3. Subjects: 
 a.  Description of study populations 
Male and female adults will be eligible to participate in this study. Participants will be non-
treatment seeking heavy drinking volunteers. Our goal is to screen 124 subjects and recruit 62 
individuals. Assuming a 20% drop-out rate, we will reach 50 completers. 
 
b. Inclusion criteria 
1. Male and female participants between 21-60 years of age. 
2. Good health as determined by medical history, physical exam, ECG and lab tests. 
3. Creatinine ≤ 2 mg/dl. 
4. Female must have a negative urine pregnancy (hCG) test at the start of each study 
session. Females of childbearing potential who are sexually active and have not been 
surgically sterilized must agree to use an adequate method of birth control during the 
study.  Adequate methods of contraception for sexually active women are having a male 
sexual partner(s) who is surgically sterilized prior to inclusion; having a sexual partner(s) 
who is/are exclusively female; using oral contraceptives (either combined or 
progestrogen only) with a single-barrier method of contraception consisting of spermicide 
and condom or diaphragm; using double-barrier contraception, specifically, a condom 
plus spermicide and a female diaphragm or cervical cap plus spermicide; or using an 
approved intrauterine device (IUD) with established efficacy. 
5. Participants must drink alcohol regularly at a heavy level, on average greater than 20 
drinks per week for men, and greater than 15 drinks per week for women, and not be 
seeking help for alcohol-related problems. 
6. Participant must be willing to receive two IV lines. 
 
c. Exclusion criteria 
1) Current or prior history of any clinically significant disease, including CNS, 
cardiovascular, respiratory, gastrointestinal, hepatic, renal, endocrine, or reproductive 
disorders.  
2) Specific exclusion criteria related to the administration of ghrelin, are chronic 
inflammatory diseases (e.g., Crohn’s disease, ulcerative colitis, celiac disease), diabetes, 
obesity (BMI ≥ 30 kg/m2), weight ≥ 120 Kg, high triglycerides level (> 350 mg/dL), 
history of clinically significant hypotension (e.g.: history of fainting and/or syncopal 
attacks) and/or resting systolic BP < 100 mmHg. 
3) Positive hepatitis or HIV test at screening. 
4) Current clinically significant major depression or anxiety; or prior clinically significant 
psychiatric problems, including eating disorders, schizophrenia, bipolar disorder, 
obsessive compulsive disorder.      
5) Current diagnosis of substance dependence (other than alcohol or nicotine). 


22 
 
6) Currently seeking treatment for alcohol use disorder. 
7) History of significant withdrawal symptoms or presence of clinically significant 
withdrawal symptoms (Clinical Institute Withdrawal Assessment (CIWA) score > 8) at 
screening. 
8) Non-drinkers (alcohol-naïve individuals or current abstainers) or no experience drinking 
5 or more drinks on one occasion. 
9) Unable to provide a negative urine drug screen.  
10) Pregnancy or intention to become pregnant for women. Female participants will undergo 
a urine beta-hCG test to ensure they are not pregnant. 
11) Use of prescription or OTC medications known to interact with alcohol within 2 weeks of 
the study. These include, but may not be limited to: isosorbide, nitroglycerine, 
benzodiazepines, warfarin, anti-depressants such as amitriptyline, clomipramine and 
nefazodone, anti-diabetes medications such as glyburide, metformin and tolbutamide, 
H2-antagonists for heartburn such as cimetidine and ranitidine, muscle relaxants, anti-
epileptics including phenytoin and phenobarbital codeine, and narcotics including 
darvocet, percocet and hydrocodone. Drugs known to inhibit or induce enzymes that 
metabolize alcohol should not be used for 4 weeks prior to the study. These include 
chlorzoxazone, isoniazid, metronidazole and disulfiram. Cough-and-cold preparations, 
which contain anti-histamines, pain medicines and anti-inflammatories such as aspirin, 
ibuprofen, acetaminophen, celecoxib and naproxen, should be withheld for at least 72 
hours prior to each study session. 
12) Current or prior history of alcohol-induced flushing reactions. 
13) Contraindications for MRI scanning, including metal in body that are contraindicated for 
MRI (such as implants, pacemaker, prostheses, shrapnel, irremovable piercings), left-
handedness, and claustrophobia. 
 
Please also see eligibility checklist (Attachment). 
 
Clearance for eligibility to this protocol will be consistent with the recent “NIAAA/LCTS Patient 
Eligibility Checklist Process” policy implemented by the NIAAA Clinical Director. As such, 
eligibility criteria will be reviewed by a team of three individuals: research nurse coordinator (or 
designee), a nurse practitioner and PI or AI designated by the PI. All three signatures will be 
required in order to establish participants’ eligibility to this protocol. 
 
4. Study Design and Methods: 
a. Study overview  
The study is designed as a within-subject, double-blind, placebo-controlled randomized clinical 
trial of ghrelin with non-treatment seeking heavy drinking volunteers. Including screening and 
follow-up evaluations, the study lasts about 8 weeks and will require five visits to the NIH CRC. 
The five visits will be as follow and performed in a counter-balanced order across participants: 
Visit #1 - Initial screening visit (under the NIAAA 98-AA-0009 or 14-AA-0181 
protocol); 
Visit #2 - Alcohol PR + ghrelin/placebo; 
Visit #3 - Alcohol PR + placebo/ghrelin; 
Visit #4 - fMRI/alcohol clamp + ghrelin/placebo; 
Visit #5 - fMRI/alcohol clamp + placebo/ghrelin; 


23 
 
Visit #6 - Post-study follow-up. 
 
b. Recruitment 
Participants will be recruited through referrals from the NIH Volunteer Office, and from the 
Patient Recruitment and Public Liaison (PRPL) Office as well as through ResearchMatch.org 
(facilitated through PRPL). ResearchMatch is a voluntary service that matches people interested 
in being research participants with researchers conducting a wide range of studies. 
ResearchMatch is currently used for other protocols of our Clinical Program at NIAAA, as well 
as for many other NIH protocols. ResearchMatch is a Clinical and Translational Science Awards 
(CTSA) initiative funded by the National Center for Advancing Translational Sciences, part of 
the National Institutes of Health. 
Furthermore, participants will also be recruited by word of mouth and through local 
advertisements. Advertisement language will be used as flyers with tear off tabs, posted on the 
NIH campus, campuses of universities and colleges in the greater Washington DC area, as well 
as in billboards of public places and public transportation services in the greater Washington DC 
area. Advertisements will also be posted in electronic and printed local media, including 
newsletters, websites and local newspapers in the greater Washington DC area (for example, the 
‘Express’). Listserv ad will be used on craigslist.org, under the volunteers’ section, as well as in 
NIH and other local email distribution lists, that are moderated, and following approval from the 
managers of these lists. ‘ClinicalTrials.gov’ may also represent a source of recruitment. 
Finally, participants will also be recruited via the NIAAA Screening Protocol (98-AA-0009, PI: 
Reza Momenan, Ph.D.), or 14-AA-0181 as the 98-AA-0009 and 14-AA-0181 protocols serve as 
the screening platform not only for this study but also for any other NIAAA protocol.. 
 
 
c. Screening  
Candidates will first be evaluated over the phone. Qualified candidates will come to the NIH 
CRC for a screening visit (Visit #1) during which they will be enrolled in the NIAAA screening 
protocol (98-AA-0009 or 14-AA-0181). If a clinical procedure is repeated (e.g. blood/urine lab 
work, EKG) under the screening protocol, exclusion/inclusion criteria will be based on the most 
recent clinical values. The screening procedures covered under the NIAAA screening protocol 
include (details of the procedures are in the 98-AA-0009 protocol or 14-AA-0181 protocol) (a) 
medical history, (b) drinking history, using the timeline followback method (TLFB), (c) family 
history of alcoholism, using the Family Tree Questionnaire, (d) Structured Clinical Interview for 
DSM-IV (SCID) psychiatric diagnoses including alcohol or drug dependence, (e) physical 
examination, including ECG, (f) blood tests for routine blood chemistry, complete blood count 
(CBC), liver enzymes, hepatitis and HIV screen, (g) urine tests to screen for illicit drugs, (h) the 
NEO-PI personality inventory, and (i) an Alcohol Flushing Questionnaire145-146 to identify 
individuals who might have a flushing response (including facial flushing, palpitations, 
drowsiness and other unpleasant symptoms) when drinking alcoholic beverages. The visit may 
last up to 4 hours.  
Normally, the results of this evaluation, including possible diagnoses of alcohol use disorders, 
will be discussed with the subject after the study is completed. However, if an alcohol-related 
problem is found that requires immediate clinical attention, or if the subject requests it, the 
diagnostic information will be given immediately and the subject will have an additional 
opportunity to consider whether they would like to receive treatment. 


24 
 
Given that the 98-AA-0009 or 14-AA-0181 protocol serves as the screening platform for this 
study, identified data obtained under this protocol may be shared with the data collected under 
the AA-98-0009 or 14-AA-0181 protocol and may be combined for analysis. The 98-AA-0009 
and 14-AA-0181 protocol are also protected by Certificate of Confidentiality. 
Eligible participants will be randomized to 1 of 2 counterbalanced arms to receive first either 
ghrelin or placebo during the first PR session. Participant then will receive the alternate 
condition during the second PR session. Similarly, participants will receive ghrelin first and then 
placebo (or vice versa) during the third and fourth sessions, namely the two “fMRI/alcohol 
clamp” sessions. Eligible participants will be randomized to receive either ghrelin or placebo 
during the study according to gender, based on a stratified block design. The Pharmaceutical 
Development Service (PDS) of the NIH CRC will develop the randomization schedule and 
maintain the blind; they will also be responsible for determining subject allocation. The 
allocation code will be kept in a safe location at the pharmacy, with access only for the 
responsible pharmacist. In emergency medical situations, which may require premature breaking 
of the code, the PI will be able to obtain access to the code table through the pharmacist on duty; 
this will then be documented, and reported to the IRB.    
 
d. Study Procedures 
Visits and procedures 
 
IV Ghrelin infusion: dose justification 
Overview. 
After a 10 minute bolus of IV ghrelin or matched placebo (saline solution), IV ghrelin or placebo 
infusion will last 120 min during visits #2 and #3 (120 minutes is the duration of the alcohol PR 
session) and 65 min during visits #4 and #5 (the total duration of the fMRI/alcohol clamp is 95 
min with 35min for the alcohol clamp – details below and in appendix flow sheets). The 
Pharmaceutical Development Service (PDS) of the NIH CRC will prepare the ghrelin bags ready 
for IV infusion. Vials of ghrelin and saline will be indistinguishable visually.  
 
Dose. 
A bolus of IV ghrelin followed by an infusion of IV ghrelin will be employed, as detailed next. 
- 
An IV administration of 3 mcg/kg bolus of ghrelin. The injection of the bolus will start 10 
minutes before the start of the other lab procedures (PR for Visits #2 and #3; “fMRI/alcohol 
clamp” for Visits #4 and #5) and will last 10 minutes; 
- 
An infusion of 5 pmol/Kg/min (that is 16.9 ng/kg/min) of ghrelin for 120 minutes (Visits #2 
and #3) or 65 minutes (Visits #4 and #5). In fact, the infusion will last for the duration of the 
PR (120 minutes at Visits #2 and #3) and for a portion of the 95 min “fMRI/alcohol clamp” 
(65 minutes at at Visits #4 and #5). 
 
 
Dose justification. 
1. 
Ghrelin dose is adjusted per weight. Although some studies used a fixed dose47, the 
majority of the studies used a dose of ghrelin adjusted per weight (see Tables 1-2). 
Consistent with the majority of the literature, we will use a dose of ghrelin based on body 
weight. To now, there are no published data showing that ghrelin dose should also be based 
on other parameters, such as body water and/or gender. However, it is worth noting that 


25 
 
two studies showed that IV ghrelin effects are independent of gender42,47. We will exclude 
subjects affected by obesity (BMI ≥ 30 Kg/m2) or subjects with weight ≥ 120 Kg, for at 
least three reasons: 1) to keep doses of ghrelin administered at a low-medium level, given 
we will adjust the dose per weight; 2) to avoid enrolling subjects with a possible ‘ghrelin 
resistance’ which may mask the effects of IV ghrelin administration; and 3) to avoid 
enrolling subjects with other psychiatric disorders (e.g. binge eating), as we want to focus 
specifically on the effects of ghrelin on alcohol reward. 
2. 
Ghrelin is administered as a bolus followed by an infusion. In the previous study at Brown 
University, only a bolus of 1 mcg/kg or 3 mcg/kg was used because a longer exposure to 
ghrelin was not needed given the short duration of the human lab session (e.g. the cue-
reactivity experiment last ~30 minutes). By contrast, in this protocol, it is important that 
ghrelin’s effects last longer in order to fully investigate the possible effects of ghrelin on 
alcohol reward, alcohol craving, alcohol sensitivity and fMRI outcomes during the entire 
duration of the human lab sessions performed at Visits #2-5. We will still employ an initial 
bolus, as the preliminary data from Brown University (see Figure 2) suggest that a bolus 
might acutely and temporarily increase alcohol craving. Specifically, we will use a bolus of 
3 mcg/kg because the preliminary data (see Figure 1 above) might suggest a possible 
(albeit not significant - interim analysis) dose-response effect on alcohol craving, thus the 
higher dose should be used to maximize the probability of finding a drug effect. After the 
10-minute ghrelin bolus, an infusion of 5 pmol/Kg/min of ghrelin for 120 minutes (Visits 
#2 and #3) or 65 minutes (Visits #4 and #5) will take place. The dose of 5 pmol/Kg/min (= 
16.9 ng/kg/min) is that most frequently used in previous studies with healthy controls or 
subjects with other chronic conditions, as detailed in the Tables 1 and 2. For example, 
Stephen Bloom’s lab at the Imperial College of London has demonstrated that 5 
pmol/Kg/min (as compared not only to placebo, but also to 1 pmol/Kg/min) is able to 
reproduce the physiological effects of ghrelin. Although our dose schedule includes a bolus 
of 3 microg/Kg before the infusion of 5 pmol/Kg/min, it is important to note that other 
studies have used even higher doses of ghrelin (e.g. higher total daily exposure) and still 
with a very safe profile. For example, Lavin et al.49 used an IV ghrelin dose of 10 
pmol/Kg/min for 180 minutes, and participants did not report any side-effect. Anna 
Cappola’s lab at UPenn has recently performed a 3-hr IV ghrelin study69 where ghrelin was 
infused at a rate of 2.5pmol/kg/min for the first 60 minutes, 5 pmol/Kg/min for the second 
60 minutes, and 10 pmol/kg/min for the last 60 minutes. The only transitory side-effect was 
a feeling of warmth. Notably, Cappola’s study was conducted in individuals who were 
generally more prone to develop side-effects, e.g. individuals > 70 years old with geriatric 
frailty (while here we will exclude anyone > 60-yr-old). In this study, we propose a dose 
whose total daily exposure is significantly lower compared to the studies conducted by 
Lavin’s49 and Cappola’s lab69. 
 
PR sessions (visits #2 & #3) 
Breath ethanol levels will be measured, a urine drug screen and, in women, a urine pregnancy 
test will be performed at the start of these sessions. Information regarding alcohol drinking, 
cigarette smoking, mood and anxiety states and symptoms will be collected. Food craving and 
urge to drink alcohol will also be collected. 
Prior to the start of the session, two indwelling IV catheters are inserted using sterile technique. 
One of the IV catheters is used for the ethanol IV infusion, the other for the ghrelin IV infusion. 


26 
 
One of the IV catheters includes a bidirectional valve that allows for both infusion and blood 
sampling during the experiment. The subject is seated in a comfortable chair, out of sight of the 
infusion pumps, and instructed in the procedures. The experimenter will be available to answer 
any questions raised by the participants and will occasionally inquire about the well-being of the 
participant. 
 
Progressive Ratio Schedule Self-administration (Visits #2 and #3). 
The PR schedule self-administration will be conducted using the Computer-Assisted Self-
infusion of Ethanol (CASE) system (currently being used in two NIH protocols 08-AA-0137 and 
08-AA-0178). Just prior to the start of the PR schedule session, the subject will be reminded of 
the procedure for obtaining alcohol during the session. Following a 15-min priming interval (that 
will not applied here, as our ‘priming’ is the IV bolus of ghrelin), the PR period lasting 120 
minutes begins. Each standardized exposure will follow a slopelet pattern (linear increase of 7.5 
mg% over 2.5 min followed by a linear decrease of 0.5 mg%/min until the next exposure). The 
PR schedule will escalate geometrically from using a progressive ratio that will be empirically 
established. An initial PR schedule will be established based on simulations using the model 
published by Richardson and Roberts.147 For practical considerations, we will select a schedule 
that requires 2 button presses for the first exposure and escalates exponentially to requiring 
~15,000 button presses for the 20th exposure. This initial schedule will be tested in the first 6 
subjects, and the range of break-points obtained will be evaluated. If there is insufficient 
variability in the break-points across the first 6 subjects, the schedule may be adjusted before 
proceeding with the enrollment of other subjects. Within 0.5 seconds of pushing the drink button, 
the CASE system software will begin infusing the alcohol necessary to produce the desired 
increase in BrAC exposure from the BrAC level at the time the button was pushed. Also, if the 
CASE system predicts that an additional drink selected by the participant would result in a peak 
BrAC 0.12 (which is the upper limit for BrACs achieved in this study), then the push button will 
be inactivated, with the subject’s knowledge, until the time when the predicted peak BrAC is 
below the upper limit.  
 
In an attempt to control the ambient environment during the self-administration session, 
participants will be offered one of five choices of activity: 1) listening to music; 2) watching 
videos of television comedy shows; 3) watching videos of nature documentaries; 4) watching 
sports videos; or 5) browsing the internet on a laptop or tablet. The participant’s choice will be 
documented and any effect of the participant’s activity on self-administration measures will be 
explored. The experimenter will be available to answer any questions raised by the participants 
and will occasionally enquire about the well-being of the participant. At 15-minute intervals, the 
experimenter will obtain a BrAC reading. Participants will be asked to complete a Drug Effects 
Questionnaire (DEQ) which assesses feelings of “high” and “intoxication” and how much they 
“like” the feelings they perceive using visual analog scales,148 and the Biphasic Alcohol Effects 
Scale (BAES)149 to assess the stimulant and sedative effect of alcohol. Participants will also 
complete the Alcohol Visual Analogue Scale (A-VAS), the Alcohol Urge Questionnaire (AUQ) 
to assess the urge to consume alcohol,150 a mood questionnaire (Profile of Mood States, 
POMS151), and the CASE Experience Questionnaire (CEQ) to determine craving and if 
participant’s can gauge the amount of ethanol infused. At the end of the session, the infusion 
pump will be disconnected and the IV catheters will be removed from the participant’s arm. 
Lunch will be provided and serial breathalyzer tests will be done to track the BrAC level until it 


27 
 
reaches 0.00%. Then, participant will be asked to stay in the Clinical Center overnight and the 
morning after participant will have breakfast, receive debriefing and then released. 
 
Infusion profile computation using the CASE system: The infusion rate profile for each 
participant is calculated using CASE system software, which exercises a modification of the 
physiologically-based pharmacokinetic model of ethanol distribution and elimination being used 
in completed and ongoing LCTS alcohol infusion studies (03-AA-0283, 04-AA-0060, 07-AA-
0026, 08-AA-0125, 08-AA-0137, 08-AA-0178, 11-AA-0180). Currently, physiologically-based 
pharmacokinetic model parameters are computed for each individual participant by 
transformation of the subject’s age, height weight and gender into the physiologic parameters of 
the model.  Using a high gain on the simulated error between model and desired BrAC as the 
infused alcohol, the model is forced to follow the desired time course of BrAC, thereby 
computing the prescribed infusion rate profile as a continuous variable. A pump driver software 
provides the interface between the buffer and the infusion pumps, using the computer’s real-time 
system clock to administer the profile during the experimental session.  Since the modeling 
solutions are calculated at a thousand-times real-time, the delay between the subject electing 
another drink and the consequent adjustment to the infusion profile is negligible. The software 
will manage adherence to the limits on availability of another drink by computing the predicted 
peak BrAC for an additional drink and making the push button unavailable until the predicted 
peak BrAC is below the pre-set upper limit for BrAC exposure. 
 
Additional baseline assessments performed at Visit# 2 for exploratory analyses. 
Sweet Preference Test. In this study, we will explore if responses to ghrelin administration differ 
between sweet-liking and sweet-disliking individuals. A growing body of evidence in fact 
indicates that the hedonic response to sweet taste is associated with genetic risk of alcohol 
dependence (AD) in humans. Given that we are investigating a peptide that physiologically is 
involved with food reward and appetitive behaviors, it will be scientifically interesting to explore 
possible differences between sweet-liking and sweet-disliking individuals in this study. 
Sweet preference testing will be conducted as previously described (e.g.152.153). Each subject will 
be instructed to sip, swish around his/her mouth and then spit out five different concentrations of 
a sweet solution (0.05, 0.10, 0.21, 0.42 and 0.83 M) each of which will be presented five times in 
a pseudorandom order for a total of 25 tastings. After each tasting, participants will rinse their 
mouths with distilled water before proceeding to the next solution. The participants then will rate 
the intensity and pleasurableness of each tasting using a 200 mm analog scale. Subjects will be 
assigned to one of two categories— sweet-liking or sweet-disliking —based upon their hedonic 
response to the various sucrose concentrations. To be categorized as a sweet liker, a subject must 
rate the highest concentration of sucrose (0.83 M) as the most pleasurable. Sweet-dislikers could 
prefer any of the other four concentrations. Such categorization has been shown to be stable over 
time. 
 
Brief Addictive Behavior Social Density Assessment (BASDA). We will administer the alcohol 
component of the abbreviated version of the BASDA, as recently developed at the University of 
Georgia.154 The BASDA measures the perception of alcohol use for each participant’s 1st, 2nd, 
3rd, & 4th closest non-biological associates, mother, father, and two closest siblings in age, as 
self-reported by the study participant. The BASDA alcohol use subscale consists of the first three 
questions of the Alcohol Use Disorders Identification Test (AUDIT). The goal of this assessment 


28 
 
is to explore the social milieu that surrounds the participant and if this information might help to 
predict different responses to drinking behavior in our human laboratory setting. The BASDA is 
a self-reported assessment where participants self-report on their closest associates’ drinking 
behaviors. Participants are not asked to provide the names of their participants’ closest biological 
and non-biological associates. 
 
Alcohol effects questionnaire (AEFQ) version s. The alcohol effects questionnaire is a revision of 
the alcohol expectancy questionnaire and is used to assess the positive and negative effects 
people expect to experience from alcohol. The brief questionnaire consists of 40 true/false items 
with 8 subscales that aims to assess personal beliefs regarding the both the reinforcing and 
undesirable effects of alcohol. The 8 subscales are: global positive, social and physical pleasure, 
sexual enhancement, power and aggression, social expressiveness, relaxation and tension 
reduction, cognitive and physical impairment, and careless unconcern. In research studies, AEFQ 
expectancy scores have correlated with drinking problems, alcohol cue reactivity, and number of 
lever presses during IV alcohol self-administration (unpublished data from Dr. Ramchandani’s 
lab).   
 
Behavioral approach system (BAS)/behavioral inhibition system (BIS) scale. The BIS/BAS scale 
was developed to measure individual differences in the sensitivity of the BIS and BAS.155 The 
scale is divided into 4 subscales: BAS Drive, BAS Fun Seeking, BAS Reward Responsiveness, 
and BIS. Much research suggests these systems play an important role in vulnerability to 
addiction. In fact, high BAS Fun Seeking scores have been shown to be associated with lifetime 
drug abuse/dependence,156 and high BAS Drive scores have been shown to predict alcohol 
craving during exposure to alcohol cues.157 The individual differences in reward sensitivity 
measured by these scales are especially relevant to addiction when examining the role of the DA 
reward system. The goal of this assessment in the current study is to further explore how BAS 
and BIS personality traits relate to alcohol craving and the activation of reward circuitry. 
 
Finally, although this protocol will not assess formally smoking-related outcomes, we are also 
aware of the high comorbidity between alcoholism and smoking. Therefore, at Visit 2 (as well as 
at Visits 3, 4 and 5), we will assess breath carbon monoxide (BrCO) for possible between-group 
differences and possible post-hoc analyses. 
 
fMRI/alcohol clamp sessions (visits #4 & #5) 
Breath ethanol levels will be measured, a urine drug screen and, in women, a urine pregnancy 
test will be performed at the start of these sessions. The two “fMRI/alcohol clamp” sessions will 
be identical (but the double-blind drug condition will be ghrelin or placebo) and they will be 
scheduled at approximately the same time of the day with at least 3 days washout window 
between them. A washout period of at least 3 days will also be applied between visits #3 and #4. 
fMRI scanning in this protocol is an investigational procedure. The administration of alcohol is 
performed following the standardized alcohol clamp paradigm for which our research team at 
LCTS has considerable experience (e.g. 03-AA-0283, 04-AA-0060, 07-AA-0026, 08-AA-0125 
and 11-AA-0180). The fMRI procedures will be similar to those used in protocols 98-AA-0056 
and 04-AA-0060. Also, the combined “fMRI/alcohol clamp” procedures will be identical to 
those used in the current varenicline protocol (08-AA-0137), therefore the research team has 
already experience in this combined paradigm. BrAC testing will be done immediately prior to 


29 
 
the scan and subjects whose BrAC exceeds 0.000% will not be scanned. An IV catheter will be 
inserted in each forearm; one of the IV catheters is used for the ethanol IV infusion, the other for 
the ghrelin IV infusion. The IV catheter includes a bidirectional valve that allows for both 
infusion (e.g. ethanol) and blood sampling during the experiment.  
Imaging will take place in the NIH NMR center using one of the center’s 3T scanners. An initial 
structural scan will be collected on each subject for later co-registration of functional images. 
The structural scan will last less than 10 minutes. Functional scans will be acquired using a T2*-
EPIRT sequence that measure changes in blood oxygen level dependent (BOLD) contrast. Visual 
stimuli will be projected onto an opaque screen at the foot of the imaging machine via a liquid 
crystal diode projector. Subjects will be able to view the images via a mirror placed above their 
head in the scanner. Subjects will have an opportunity for a break between structural and 
functional imaging, and will remain in contact with the experimenters at all times via an 
intercom system built into the scanner. The  functional scans, (see appendix flow sheet for scan 
times), will be acquired while the subjects are at rest and while they participate in a modified 
version of the MID task in which they will respond to cues that indicate the opportunity to press 
a button to gain a reward. On some trials the reward will be points that can be exchanged for 
snack foods (subjects can choose items from a hamper containing candy bars, bags of chips and 
crackers provided by the Nutrition Department of the NIH Clinical Center) while on other trials 
the reward will be points that will determine how much intravenous alcohol a subject will be 
given during the “alcohol clamp” procedure which will immediately follow the modified MID 
task. In the previous IV ghrelin alcohol study at Brown, we included a palatable nonalcoholic 
beverage to investigate whether the effects of IV ghrelin occur across caloric appetitive beverage 
cues. For the same reasons, and consistent with the role of ghrelin in controlling food intake, in 
this protocol, participants will be exposed to both alcoholic and food-related cues in order to 
tease a part possible non-specific effects of ghrelin on appetitive behaviors vs. specific effects on 
alcohol-related behaviors. 
After completion of the baseline modified MID task, subjects will receive the ghrelin/placebo 
bolus and followed by a continuous infusion as described above.  During ghrelin/placebo 
infusion subjects will repeat the MID task and resting scans.  Next, alcohol infusion begins, 
which will be given as described in protocol 08-AA-0137. Ethanol will be infused as a 6% v/v 
solution in saline. The infusion rates will be based on a physiologically-based pharmacokinetic 
model for ethanol – the same model used to compute the infusion rates for the alcohol self-
administration sessions described above. Blood samples will be obtained during the clamp. The 
alcohol clamp method has been used successfully in several studies of the pharmacokinetics and 
pharmacological effects of ethanol in humans,158-165 and is has been used, for example, in 
NIAAA protocols listed above.  
During the alcohol clamp, subjects will have resting scans and repeat the MID task.  (See fMRI 
appendix flow sheet for details). At the end of the infusion, the catheters will be removed, lunch 
will be served and repeated BrACs will be performed until BrAC returns to 0.00 g%. 
 
Diet (visits #2-5) 
The night before each session, participants are admitted to the Clinical Center and will receive a 
standardized dinner. After overnight stay, participants will receive a fixed breakfast in the 
morning. These two courses will be standardized in agreement with the NIH CC Nutrition 
Service. Then, before starting the session, a fixed light snack is served (≈167 Kcal; 
approximately 62% carbohydrate, 13% protein, 25% fat) to standardize food intake before IV 


30 
 
ghrelin. The use of a standardized caloric food intake before the experiment: i) is a procedure 
used in human studies testing ghrelin i.v.35,37,75,; ii) is consistent with studies testing the role of 
ghrelin on alcohol reward in animals without food deprivation effect98,122; iii) avoids the 
presence of elevated baseline ghrelin levels (present in food deprivation conditions34,74,81,91), 
which may be a confounding factor for our controlled experiment (exogenous ghrelin vs. saline 
solution); iv) does not represent a confounding factor for the ethanol administration, as it is 
infused IV and not orally; and v) it is consistent with our similar procedure used in the IV ghrelin 
alcohol study at Brown University. Lunch will be provided after the end of each session. Water 
will be permitted ad libitum, while other beverages (e.g. soda) will not be permitted. 
 
In order to standardize the appetitive/metabolic conditions of the study participants, we also plan 
the following: 
- 
Nutrition Department staff at the NIH Clinical Center will meet with the participants to 
perform a 24-hour recall of everything the participant consumed prior to admission. 
Participants will also be instructed to eat as similarly as possible during the 24 hours prior to 
each of their visits #2-5; capturing the 24-hr recall will allow for checking compliance with 
this. (This is not meant to say that all participants have to eat the same foods, just that 
participant ‘001’ should eat the same things each time and participant ‘002’ should eat the 
same things each time, even though ‘001’ and ‘002’ will be eating different things.). 
Information on food intake and related calorie intake during the entire stay in our Unit at the 
Clinical Center will be also recorded. 
- 
Participants will be given a choice of a few lunch and breakfast options, but all options will 
be the same in kcal and macronutrient content.  Tentatively, each participant will receive the 
same meals for all the visits (e.g., if participant ‘001’ picks lunch A for the first visit, s/he 
will get lunch A for all her/his visits.), although participants’ requests to change their choice 
among the given options may be satisfied.   
 
Blood Sampling: 
Furthermore, at admission at each visit (Visit #2-5), we will also collect blood samples in order 
to control appetitive/metabolic indicators prior to the initiation of the dietary regimen. 
Specifically, during the PR sessions and the fMRI/alcohol clamp sessions, at baseline we will 
collect: 
*Blood Test Panel I: blood glucose, cholesterol, triglycerides, GH, insulin, IGF-1, cortisol, 
ACTH, PRL, FSH, LH, testosterone total and free, T3-free, T4-free, TSH; 
**Blood Test Panel II: GH, insulin, cortisol, PRL and other feeding-related (or stress-related 
hormones).  During ghrelin/placebo/ETOH infusions, Blood Test Panel II will be repeated every 
30 minutes during the PR sessions and every 15 minutes during the fMRI/alcohol clamp 
sessions. In addition, BAC will be measured twice while participants are in the scanner. 
 
The timepoints for blood collection in the flowsheets of this protocol represent the maximum 
number of blood sampling for this protocol. While our ideal goal is to collect all of them, we 
appreciate that occasionally blood collection may be missed due to expected technical problems 
during the study procedures. Furthermore, if there are blood draw issues with both IV lines, we 
might also attempt to draw blood using regular or butterfly needles.  
  


31 
 
Samples will be stored for subsequent analyses of ghrelin levels and other appetitive hormones 
(and stress hormones), including (but not limited to) insulin, leptin and GLP-1. Some of these 
stored samples will be shipped in a fully de-identified form to AI Fatemeh Akhlaghi’s laboratory 
to be processed for a metabolic panel that includes appetitive and stress peptides like ghrelin, 
insulin, leptin, GLP-1 and others. No samples will be shipped until MTA is fully executed 
between NIAAA and URI. Furthermore, additional metabolic indicators (e.g. glucose, 
cholesterol, triglycerides, thyroid hormones) will also be assessed prior to the initiation of the 
dietary regimen. 
 
Overnight stays (visits #2-5) 
This study will enroll non-treatment seeking heavy drinking outpatient individuals. Nonetheless, 
two overnight stays will take place in the Clinical Center (e.g. Metabolic Unit or NIAAA-LCTS 
inpatient unit, pending on availability) per each session: 
- 
The night before each session, i.e. Visits #2-5. This allows for controlling for two important 
factors in this study: 
i) Alcohol consumption before each session, which includes the IV administration of 
alcohol and ghrelin. In fact, while the baseline BrAC allows for controlling a recent 
consumption of alcohol, the overnight staying allows for a much longer and thus 
more rigorous control of alcohol consumption, thus standardizing alcohol 
consumption across all participants. Additionally, the overnight stay before Visits #2-
5 increases the feasibility of the study as it does assure that baseline BrAC will be 
0.00 before each session. In fact, if participants came to the clinic on the same day of 
the session, there is a risk that their BrAC could be positive, which, in turn, would 
require delaying the session or indeed withdraw the subject if BrAC was > 0.08; 
ii) The night before each session, each participant will receive a standard pre-fixed 
dinner. Given the key role of ghrelin in food intake and metabolism, the overnight 
stay allows for controlling carefully caloric intake during the night before the 
experiment, thus standardizing food intake across all participants.  
- 
The night following each session. This overnight will permit additional monitoring for safety 
reasons. Specifically, after the session is complete and subjects have lunch, vital signs and 
general well-being will be assessed approximately every 30 minutes until BrAC will zero 
out. A non-standardized dinner will served in the evening. Finally, participants will be 
debriefed on the following morning before being sent home. 
 
e. End of Participation 
Follow-up (visit #6) (lasting 30 minutes). One week after the last fMRI/alcohol clamp session 
(Visit #5), subjects will return to the NIH CRC for clinical evaluation, including assessment for 
compliance with the previous recommendations to reduce alcohol consumption. 
At this visit they will be informed about the results of their clinical evaluation for alcohol use 
disorders. We will follow the clinical guidelines for treatment of alcohol use disorders as 
described in the NIAAA publication "Helping patients who drink too much: a clinician's guide". 
These guidelines prescribe a "Brief Intervention" during which the patient is informed of the 
diagnosis and a clear recommendation to stop drinking alcohol is made. The possible use of 
medication to reduce alcohol use as well as possible participation in a self-help group will be 
discussed, and a follow-up appointment in the NIAAA outpatient clinic will be scheduled if 
desired. After the follow-up visit, subjects will be considered to have completed the protocol. 


32 
 
Only information that a subject specifically requests in writing will be shared with other 
healthcare providers or third parties. 
 
f. Future Contact  
While all steps are always taken to ensure quality data from all participants, we may ask 
participants who generate unusable data (or if there is an equipment failure) on one of their 
experimental sessions to repeat that session.  This will have the effect of maximizing the 
risk/benefit ratio as it will prevent having to discard usable data from one session and will 
minimize the number of participants recruited overall to obtain sufficient data to meet power 
requirements.  We may ask the participant to return at a later date or to stay an additional night 
on the clinic unit.  Subjects will receive additional compensation if experimental sessions are 
repeated; compensation for this as needed research procedures may vary based on the 
inconvenience units and will be based on the NIDA/NIAAA recommendations.  
 
5. Storage of data and samples 
Data will be stored on an NIH data server, under the management of the ORIT, NIAAA. The 
existence and types of information contained in the data management system have been publicly 
reported as required by the FOIA. 
 
It should be mentioned that the timepoints for the data collection outlined in the flowsheets of 
this protocol represent the maximum number that will be collected. While our ideal goal is to 
collect all of them, we appreciate that occasionally questionnaires and other data collection may 
be missed (missing data) as in any clinical study, due to time constraints resulting from possible 
technical problems during the study procedures (e.g., a problem with the IV line or with the 
computer used to administer questionnaires, etc.). 
 
Plasma and serum for blood levels of ghrelin, GH and other feeding-related and stress-related 
hormones will be collected from each subject on repeated occasions, as detailed in the study flow 
sheets. Blood not processed via the Department of Laboratory Medicine at the Clinical Center or 
will be in -80 freezers. All biological specimens obtained under this protocol will be stored in 
coded form (protocol plus subject number) in freezers located in the access-controlled laboratory 
area of LCTS. 
 
Additional blood samples will be collected for DNA extraction and future genetic analysis under 
the NIAAA Screening Protocol (PI: Reza Momenan, Ph.D.Genetic analyses include loci 
encoding pro-ghrelin (GHRL) and its receptor (GHSR). 
 
6. Additional Considerations 
The present protocol will be submitted for review to the FDA. Dr. Kenna holds an IND for the 
use of IV ghrelin in alcohol-dependent heavy drinkers for the trial performed at Brown 
University. The FDA submission for this protocol will cross-reference the previous IND 
(notably, we will use ghrelin from the same lot#). Furthermore, Dr. Ramchandani holds an IND 
for the use of IV ethanol, which will be cross-referenced as well. 
 


33 
 
There is a possibility that subjects might identify if they received IV ghrelin or placebo. 
Therefore, at the end of each session, we will ask subjects if they believe they received ghrelin or 
placebo and this information will be recorded. 
 
7. Risks and discomforts 
 
IV Ghrelin administration 
There is a potential risk of side-effects from infusion of ghrelin, although side-effects have not 
been frequent, and when side-effects were reported, they were all in the mild range (see Tables 1 
and 2 above). No clinically significant, serious or unexpected side-effects have ever been reported 
in the published literature to data. It should also be noted that the previous study conducted at 
Brown by Dr. Leggio shows a very safe profile of IV ghrelin when administered to non-treatment 
seeking heavy drinking alcohol-dependent volunteers. 
Risks will be minimized through careful and detailed screening of participants to minimize 
recruitment of those who might be at higher risk for adverse events, as well as careful and detailed 
monitoring of adverse events and participant well-being during the laboratory sessions. 
1. Consistent with the well-known effect of ghrelin on food intake, hunger and craving for 
food may occur after IV ghrelin administration. 
2. IV Ghrelin has been associated with flushing, facial warmth and sweating. This side-effect, 
however, was never reported in the IV ghrelin study with alcoholic individuals at Brown 
University, and in that study history of flushing (e.g. alcohol-induced flushing reactions) 
was not an exclusion criterion. Nonetheless, in this study involving the IV administrations 
of ghrelin and alcohol, potential individuals with current or prior history of alcohol-induced 
flushing reactions will be excluded. 
3. IV Ghrelin has been associated with decreased arterial pressure and secondary increased 
heart rate can also occur. In this study, we will exclude individuals with history of 
hypotension clinically significant (e.g.: history of fainting and/or syncopal attacks) as well 
as those with resting systolic BP < 100mg. 
4. Rarely, elevated mood, abdominal discomfort, somnolence, fever, diarrhoea, mild 
glucosuria, vertigo and emesis have been reported in individuals receiving IV ghrelin. 
Patients with chronic intestinal diseases and/or medical history of diarrhoea and metabolic 
disorders, including diabetes and obesity will be excluded. Furthermore, in order to 
maximize safety, blood panels will include assessments of several metabolic hormones in 
order to monitor ghrelin’s effects on their levels. 
5. There are no enough data on the exposure of elder individuals to IV ghrelin. Specifically, 
among all of the studies testing IV ghrelin in humans (Tables 1-2), only one study69 
included individuals > 70 years. Therefore, consistent with the majority of the published 
literature, participants > 70 years old will be excluded in the present study (i.e. we will not 
enroll individuals who are > 60). 
6. Beyond the risks already described when ghrelin was either infused or injected, we are 
aware that consistent with the main hypothesis of this study, IV ghrelin may, at least 
temporarily, increase alcohol craving (a symptom provocation study). Therefore, this study 
includes a post-session debriefing after each of the four sessions. The Investigators have 
all experience in the management of this population, including non-treatment seekers 
taking part in alcohol laboratory studies. In particular, all NIAAA LCTS Investigators of 
this protocol have experience with challenge experiments (for example, alcohol, meta-


34 
 
chlorophenylpiperazine, yohimbine, lipopolysaccharide) that have been used previously or 
currently in patients with addictive disorders within the NIAAA intramural program, and 
no serious adverse events have been encountered in those studies. Notably, unlike other 
compounds used for challenge studies, ghrelin is a naturally occurring hormone. 
Importantly, since the brief half-life of IV ghrelin, we do not expect that the effects of the 
peptide on craving will be too long once the IV administration is over. Safety is maximized 
by the fact that participants will be kept overnight at the NIH Clinical Center after each 
session. This will allow for continued monitoring as needed, and the morning after the 
session, subjects will be debriefed on their drinking and receive counseling, using 
motivational enhancement techniques, aimed at enhancing their readiness for behavioral 
change and seeking treatment if needed, as recommended by the NIAAA guidelines.166 
Prior to discharge, subjects will be asked about the severity of their urges to drink, as well 
as any untoward symptoms they might be experiencing. Recommendations to reduce or 
stop drinking again will be given to them prior to discharge. We also note that subjects will 
receive a meal right after each session, and it is demonstrated that food suppresses plasma 
ghrelin levels.  
It should be also noted that we are applying a conservative approach, e.g. we will keep participants 
overnight at the NIH Clinical Center after each alcohol/ghrelin infusion. Dr. Leggio’s study at 
Brown was an outpatient study and, there was no need to hospitalize any of the participants 
(although included in the protocol as a possible additional safety action). No serious adverse events 
(SAEs) occurred during the ghrelin study at Brown University. All debriefing sessions were 
conducted successfully, e.g. all participants were released after A-VAS craving score was equal or 
lower to the baseline (pre-infusion IV ghrelin) A-VAS craving. There were no cases of persistent 
high craving at the end of the session. 
 
It is unknown if administration of ghrelin to pregnant women might cause harm to the fetus. No 
fetus will be exposed to exogenous ghrelin. A urine pregnancy test will be administered during 
each study visit block to all women. A positive test for beta-hCG would exclude participation in 
the study. Should a positive test occur, the investigators will be informed, and the PI or Medical 
Investigator will be involved in gently informing the participant of the fact, and will offer referral 
to an obstetrician. 
 
IV alcohol administration 
    There is a potential risk of side-effects from infusion of alcohol. Ethanol is primarily a 
depressant of the Central Nervous System. Its effects, in general, are proportional to the BrAC 
reached after alcohol administration. Alcohol may produce motor in-coordination, deficits in 
concentration, memory impairment and a feeling of expansiveness or euphoria, possibly 
followed by sleepiness. Nausea and vomiting could rarely occur.  
1. The maximum BrACs that may be achieved during the IV alcohol PR sessions will be 
0.12, while during the fMRI/alcohol clamp sessions the max will be 0.08. This research 
team has reached these levels in previous/ongoing studies, and no subject has reported 
nausea, experienced vomiting or other clinically important side-effects. 
2. The alcohol infusion procedure is considered to be relatively safe: the concentration of 
alcohol in the infusate is never greater than 6% v/v and the limit on the amount of alcohol 
that can be infused is determined by hanging only two 1000 ml capacity bags of the 
infusate (6% v/v alcohol ~ 4 oz. ethanol at most). 


35 
 
3. During all alcohol exposure sessions, participants will be monitored by the nursing and 
research staff for any untoward effect and the session will be terminated if any major or 
unexpected adverse events occur. The physician will be on-call to respond immediately 
to any problems. 
4. Participants will be kept overnight at the hospital after each session. The Investigators 
have several years of experience in studies administering intravenous alcohol to human 
participants without any major adverse events, and the nursing staff is well experienced 
in managing intoxicated participants.  
5. The procedures are consistent with the "Recommended Guidelines on Ethyl Alcohol 
Administration in Human Experimentation" prepared by the National Advisory Council 
on Alcohol Abuse and Alcoholism, Dept. of Health and Human Services.  
 
Administration of alcohol to pregnant women might cause harm to the fetus. No fetus will be 
exposed to ethanol. A urine pregnancy test will be administered during each study visit block to 
all women.  A positive test for beta-hCG would exclude participation in the study. Should a 
positive test occur, the investigators will be informed, and the PI or Medical Investigator will be 
involved in gently informing the participant of the fact, and will offer referral to an obstetrician. 
 
IV Ghrelin and IV alcohol co-administration 
There is a potential risk for additive effects of ghrelin and alcohol co-administered, such as: 
- 
Flushing and facial warmth; thus, we will exclude individuals with current or prior history of 
alcohol-induced flushing reactions. 
- 
Sedation; thus, we will exclude subjects who take medications that may amplify alcohol’s 
sedative effects and we will only enroll individuals who are not older than 60. 
- 
Decreased arterial pressure; thus, we will exclude individuals with history of hypotension 
clinically significant, and blood pressure and continuous heart rate will be monitored during 
the lab and fMRIsessions. Furthermore, subjects whose screening will show resting systolic 
BP < 100 mmHg are not enrolled in this study. 
As shown in Tables 1 and 2 above, the IV administration of ghrelin has a very safe profile, and 
we anticipate that its administration together with alcohol administration will be safe and well-
tolerated. Also, subjects will be continuously monitored during the lab sessions and alcohol 
and ghrelin administrations will be stopped (and the IRB notified) if a subject should develop 
any significant symptoms requiring to do so. Not only the sessions are performed in a well-
controlled environment in the NIH Clinical Center, but subjects will be kept overnight at the 
hospital and only released the morning after in order to maximize their safety and monitoring. 
 
Risk of fMRI 
1. Anxiety about enclosed spaces- Some individuals experience claustrophobia when placed 
inside the magnet bore of the scanner. Participants will be warned about this in advance and 
advised not to participate if they are prone to experiencing anxiety in enclosed places.   
2.  Nerve stimulation- The use of a 3T field strength magnet sometimes induces “peripheral 
nerve stimulation,” in which peripheral muscles twitch in association with scanning. Subjects 
will be notified of this possibility in the consent form. The twitching does not occur in 
everyone, is not painful, and usually lasts for a short time. Some individuals may find this 
mildly annoying and we will terminate the scanning if they feel uncomfortable. Finally, 


36 
 
fMRI is a noisy process, so we require participants to wear foam earplugs while inside the 
scanner. 
 
Insertion of IV lines 
The total amount of blood to be drawn for this protocol will be approximately 428cc (=  87 US 
teaspoons). There is a slight risk of bruising or, rarely, infection from insertion of indwelling 
peripheral venous catheters for infusion and blood sampling. Risks from IV catheterization are 
minimized by experienced medical personnel who will perform catheter placement using sterile 
technique and following universal precautions. Participants will be seated or supine when 
catheters are placed. 
 
8. Subject Safety monitoring 
Subjects will be monitored regularly and frequently during their participation in the study. They 
will undergo standard assessment battery (described below) at each study visit. 
 
Parameters to be monitored (see Study flow sheet for times of monitoring individual items): 
1. Weight 
2. Vital signs 
3. Continuous heart rate monitoring will be recorded during IV Ghrelin and IV alcohol co-
administration.  
4. BrAC, breath carbon monoxide (BrCO); Blood Alcohol Content (BAC) when 
participants are in the fMRI scanner. 
5. Drug use: Urine Drug Test (UDT) 
6. Blood Test Panel I: blood glucose, cholesterol, triglycerides, GH, insulin, IGF-1, cortisol, 
ACTH, PRL, FSH, LH, testosterone total and free, T3-free, T4-free, TSH. 
Blood Test Panel II: GH, insulin, cortisol, PRL (and other feeding and stress related 
hormones). 
7. Urinalysis 
8. Pregnancy: urine pregnancy testing 
9. Mood and anxiety level and suicidality (Comprehensive Psychopathology Rating Scale – 
CPRS), Columbia Suicide Severity Rating Scale (C-SSRS). 
10. Alcohol consumption- TLFB and alcohol craving (A-VAS and AUQ) 
11. NMR Safety Questionnaire- questionnaire developed by NMR Center to verify absence 
of any exclusions to MRI scanning (to be completed at screening and visits 4 and 5).  
 
Toxicity tables/criteria to be used 
NCI Common Terminology Criteria for Adverse Events v. 3.0 will be used for grading and 
reporting adverse events.167 
 
Criteria for individual subject withdrawal 
- 
A Serious Adverse Event (SAE) that is judged by the Investigators as being related to the 
study drug; 
- 
Pregnancy; 
- 
At the discretion of the Principal Investigator (PI) based on adverse event (AE) severity; 
- 
Non-compliance with protocol procedures or investigator request(s); 
- 
Patient request. 


37 
 
 
Enrolled subjects who show up with positive UDT or BrAC ≥ 0.08% at one of the four 
experimental sessions (excluding the night before visits 2-5) will not perform the session. In this 
case, the experimental session may be re-scheduled at the PI discretion. Additionally, a session 
may be rescheduled for other medical reasons for which the PI and/or MAI think it is in the best 
interest of the participant to re-schedule the session. However, if the subject will repeatedly show 
up with positive UDT or BrAC  ≥ 0.08% at repeated visits, the PI may withdraw him/her from 
the study. 
 
9. Outcome measures 
a. Primary outcome measures 
The primary measure of this study will be the breakpoint, which is the schedule (number of 
button presses) at which the individual stops to work for more alcohol during the PR sessions. 
 
b. Secondary outcome measures 
Alcohol exposure will be measured via BrAC exposure measures, i.e. highest BrAC, average 
BrAC and the area under the BrAC-time curve and by BAC when subjects are in the fMRI 
scanner. Alcohol craving will be measured using the Alcohol Visual Analogue Scale (A-VAS) 
and the Alcohol Urge Questionnaire (AUQ). Sensitivity to alcohol will be measured using the 
Drug Effects Questionnaire (DEQ), Biphasic Alcohol Effects Scale (BAES), and the Profile of 
Mood States (POMS), repeatedly during the PR session and the CASE Experience Questionnaire 
(CEQ)                                                                                                                                                                          
at the end of all sessions. fMRI BOLD signal in brain areas associated with incentive salience 
and areas associated with reward circuitry (including the ventral striatum) will be measured 
during the “fMRI/alcohol clamp” session. 
 
10. Statistical Analysis 
1. Analysis of data/ study outcomes 
Analysis of data/study outcomes 
    Outcome data will be examined for homogeneity of variance, and if necessary transformed to 
meet this criterion. Most of the analyses will consist of repeated measures ANCOVAs to 
accommodate dependent measures assessed at multiple time points. When the dependent 
measure is just assessed once then a regular ANCOVA will be used. We will examine 
medication main effects across several different dependent measures. 
 
Criteria for significance 
    A probability level of 0.05 or lower will be considered significant. 
 
2. Power analysis  
Power analysis 
A generic calculation of power for a two-means comparison to detect an effect size of Cohen’s f 
≥ 0.4 (large effect size) with a sample size of 50 yields a power estimate ≥ 0.80 at alpha=0.05. 
The relative effect size chosen (“large” effect size) for the power calculation is based on the 
observation that laboratory studies typically yield larger effect sizes than clinical trials, 
including, for example, our recent (unpublished; PI: Ramchandani) human lab studies using the 


38 
 
IV Alcohol PR procedure, described above and representing the study our primary aim (number 
of button presses) is based upon.  
Furthermore, the sample proposed here provides sufficient power (power estimate ≥ 0.80 at 
alpha=0.05) to detect ghrelin’s effects on fMRI BOLD activation in the VS, consistent with the 
unpublished data (PI: Ramchandani; data already mentioned above) with varenicline (n = 8) vs. 
placebo (n = 7) indicating significantly higher BOLD activation in the varenicline group. 
Accrual number request, taking into account screening/ dropouts 
In the Brown University study with IV ghrelin, inclusionary/exclusionary criteria were similar 
and about 50% of the subjects screened were eligible and randomized. Consistent with other 
human laboratory studies with a similar study length, we also expect that a number up to 20% 
will not complete the study. Therefore, we anticipate a number-needed-to-screen of 124 subjects, 
with 62 being randomized and 50 completers. 
 
11. Human Subjects Protection 
a. Subject selection 
Adults who fulfill the qualification criteria will be included, regardless of race, ethnicity, sex, 
or religious affiliation. 
 
b. Justification for inclusion of children 
Children will not be included because the study proposed involves the administration of alcohol. 
It is illegal to give alcohol to individuals below 21 years of age. 
 
c. Justification for inclusion or exclusion of other vulnerable subjects  
Vulnerable participants will not be enrolled. Females who show a positive urine pregnancy test 
will be withdrawn from the study. Women of child-bearing potential (i.e. who are pregnant or 
who are trying to become pregnant) will not be included, because it is inappropriate and 
unethical to give alcohol to women who are pregnant because of the risk of fetal alcohol 
syndrome. Females of childbearing potential who are sexually active and have not been 
surgically sterilized must agree to use a reliable method of birth control during the study (details 
above in section 3b, Inclusion criteria, #4). Furthermore, a urine pregnancy test will be 
performed at each session. 
 
d. Justification of sensitive procedures 
This study employs the use of placebo to evaluate the effects of ghrelin. This is the required 
standard set by the FDA for evaluation of drugs. The previous outpatient human laboratory study 
conducted at Brown University was a placebo-controlled study and FDA reviewed its protocol. 
 
The hypothesis of this protocol is that IV ghrelin will temporarily increase the likehood that 
participants will self-administer alcohol during the PR session and will increase craving. The 
possible temporary increase in alcohol administration and craving is a necessary challenge to 
evaluate the ghrelin system as a novel neuropharmacological target for alcoholism. It should be 
noted that at this stage ghrelin administration cannot be considered a standard provocative test. 
Although very preliminary data from the Brown study suggest so, this is a very early stage 
research. The possibility that IV ghrelin increases temporarily alcohol administration and craving 
is in fact the main objective measure of this project. The study at Brown was an outpatient study 


39 
 
and its safety profile was excellent. This protocol will enroll outpatients who, however, will stay 
overnight at the NIH Clinical Center before and after each session (two nights per session). 
 
e. Safeguards for vulnerable populations 
Vulnerable populations will not be studied. Urine pregnancy tests will ensure that alcohol is not 
administered to women who may be pregnant.  
 
f. Qualifications of investigators 
The NIAAA medications development program at the Laboratory of Clinical and Translational 
Studies is among the leading sites worldwide for this type of translational medicine studies. The 
program has expertise in addiction medicine, general psychiatry and internal medicine, 
pharmacology and is supported by a nursing staff with combined expertise in medicine and 
behavioral health. The investigators have also several years of experience in conducting alcohol 
infusion studies and neuro-imaging studies. 
 
Lorenzo Leggio, M.D., Ph.D., M.Sc., is the Chief of the Section of Psychoneuroendocrinology 
and Neuropsychopharmacology, LCTS.  He has several years of clinical and academic 
experience in the field of addiction medicine. He has extensive experience in direct patient care, 
and direction and execution of clinical trials. Before joining NIH, he was Core Faculty of the 
Brown University Center for Alcohol and Addiction Studies and Assistant Professor in the 
Brown University Medical School. In this capacity, he served as PI for several clinical trials 
funded by NIAAA or Foundations. Dr. Leggio has considerable experience with 
pharmacotherapy human laboratory studies, including a NIAAA-funded study testing IV ghrelin 
in alcoholic individuals and conducted at Brown University. Dr. Leggio is the PI of this study 
and will be responsible for the conduct, analysis and publication of the results of the study. He 
will obtain consent for this study. 
 
Vijay A. Ramchandani, Ph.D., is the Chief of the Section of Human Psychopharmacology, 
Laboratory of Clinical and Translational Studies.  He is a clinical pharmacologist with several 
years of experience conducting alcohol administration studies, particularly using the alcohol 
infusion method, which he co-developed while at Indiana University, prior to joining NIH. He 
will obtain consent for this study.  
 
 
Mary R. Lee M.D. is a Staff Clinician in the Section on Clinical Psychoneuroendocrinology and 
Neuropsychopharmacology (SCPN), LCTS, NIAAA. She completed medical school and internal 
medicine residency at Columbia Presbyterian Medical Center in New York and psychiatry 
residency at George Washington University Medical Center.   She is board-certified in internal 
medicine and addiction medicine.  She has over 15 years experience in psychiatric and addiction 
research. Dr. Lee will assist the PI in conducting experimental procedures and publishing findings 
from the study. She will also serve as Medical Advisory Investigator (MAI) and will obtain 
consent. 
 
David T. George, M.D. is a Board Certified Psychiatrist and Chief of the Section of Clinical 
Assessment and Treatment Evaluation, Laboratory of Clinical and Translational Studies. He has 
more than 20 years experience in alcoholism research and treatment. Dr. George will support the 


40 
 
MAI in the conduct of the study and will be involved in aspects of clinical care. Dr. George will 
obtain consent for this study. 
 
Melanie Schwandt, Ph.D., is a Staff Scientist in the Laboratory of Clinical and Translational 
Studies. She has more than 10 years experience constructing, managing and analyzing large 
datasets that combine behavioral and physiological outcomes, including over 5 years experience 
in pre-clinical research involving alcohol-related phenotypes and more than 2 years experience 
analyzing data from clinical experimental medicine studies. She will assist the PI in the analysis 
of the data. She will not obtain consent for the study. 
 
Reza Momenan, Ph.D., is a Staff Scientist in the Section of Brain Electrophysiology and 
Imaging, Laboratory of Clinical and Translational Studies. He has extensive experience in 
human brain imaging including PET, MRI and fMRI and will guide the image processing and 
image analysis methods. He will obtain consent for this study.  
 
LaToya S. Sewell, MSN, CRNP is a Family Nurse Practitioner in the Laboratory of Clinical and 
Translational Studies. She completed her graduate nursing education at the University of 
Maryland, Baltimore and has extensive training at the NIH in the areas of research and 
Neuroscience. Her background includes clinical management of patients with autonomic 
function disorders, as well as expertise in neuroimaging analysis. Within this lab Nurse 
Practitioner Sewell serves on the research team as a clinical provider in support of many NIAAA 
studies.  Ms. Sewell will obtain informed consent. 
 
 
Mehdi Farokhnia, M.D is a Post-Doctoral fellow in the Section on Clinical 
Psychoneuroendocrinology and Neuropsychopharmacology (CPN), LCTS, NIAAA. He 
completed medical school at Tehran University of Medical Sciences and has over 5 years 
experience in psychiatric research. During medical school and after that, he has been involved in 
conducting a variety of research projects while working as a research associate at the Psychiatric 
Research Center, Roozbeh Psychiatric Hospital. Dr. Farokhnia will assist the PI serving in the 
capacity of Lead Associate Investigator in conducting experimental procedures, analyzing the 
data, and publishing findings from the study. He will also obtain informed consent. 
 
Fatemeh Akhlaghi, Pharm. D., Ph.D., is a tenured full professor in the College of Pharmacy, University of 
Rhode Island (URI) and adjunct professor of Medicine at Brown University Medical School. Her area of 
specialty is clinical pharmacology in diabetes and transplantation; she also conducts clinical 
pharmacokinetics research at Rhode Island Hospital, Providence, RI. Dr. Akhlaghi is also a member of 
the IRB at URI. Dr. Akhlaghi is the Co-PI, together with Dr. Leggio, of the multiple-PI UH2/UH3 grant 
funded by NCATS. Her laboratory at URI will receive samples that are de-identified from this study. She 
or her lab personnel will not have any means of linking the study codes to subjects’ personal information. 
She will not obtain consent for this study. 
 
 
 
12. Anticipated Benefit 
This study does not offer direct benefit to participants but is likely to yield generalizable 
knowledge about the possible role of ghrelin in alcohol-related behaviors (e.g. drinking, craving, 


41 
 
sensitivity). This will markedly facilitate the identification of a novel neuropharmacological 
target, thus facilitating the development of novel alcoholism treatments. 
 
13. Classification of risk 
Overall risk and discomfort in this study is “more than minimal”. The increase over minimal 
risk, discomfort is minor, and every precaution and protection to minimize any risks will be 
taken as described in a previous section. This level is justified, because it will markedly facilitate 
medication development for alcoholism, and thus contribute to addressing major unmet medical 
needs. As such, the benefit:risk ratio of the proposed study is favorable. 
 
14. Consent documents and process 
a. Designation of those obtaining consent 
Study investigators designated as able to obtain consent in section ‘11f’ above, will obtain 
informed consent for this protocol. 
 
b. Consent procedures  
At Visit 1 (Screening), subjects are screened throughout the NIAAA Screening protocol 98-AA-
0009 (PI: Reza Momenan, Ph.D.) or 14-AA-0181 after consenting participants with an already 
IRB-approved consent form, specific for the 98-AA-0009 or 14-AA-0181 protocol. 
Eligible subjects are scheduled for Visit 2; the study-specific consent form for ghrelin is obtained 
at Visit 2 before starting the other procedures outlined for Visit 2. Nonetheless, at Visit 1, if all 
information necessary to determine eligibility is obtained and the participant is still in our Clinic, 
then the ghrelin-specific consent form can be obtained at the end of Visit 1 (although we predict 
this will be not a frequent scenario). In summary, the ghrelin-specific consent form will be 
typically obtained at Visit 2, and occasionally at the end of the Visit 1. Participants who might be 
eligible at the end of Visit 1 (but we will make clear that their eligibility will have to be 
confirmed) will be allowed to take the consent document home before Visit 2 is scheduled. As 
such, potential participants have the option to read the consent home, call us with questions, and 
consult (if they want) family members and/or their private health care professional. The ghrelin-
specific consent form will be obtained before starting the procedures scheduled for Visit 2, and 
all procedures of Visit 2 will always take place on a different subsequent day after Visit 1.  
All participants will receive a verbal explanation in terms suited to their comprehension of the 
purposes, procedures and potential risks of the study and of their rights as research participants. 
Participants will have the opportunity to carefully review the written consent form and ask 
questions regarding this study prior to signing.  Participants will be asked to sign and date the 
informed consent. A copy of the signed and dated consent will be provided to each participant. 
In regards to consenting participants who might be impaired, our standard procedure is for the 
nurses to do a breathalyzer test upon arrival (covered generally under the standard hospital 
consent that all subjects admitted to the NIH Clinical Center sign when they check in at 
admissions). If the results is >0.03, then the subject is informed that we cannot proceed, and they 
are given the option of waiting until their level falls below 0.02 and/or sending them back in a 
cab. If the reading is >0.08, we strongly encourage them to stay at least until they drop below the 
legal limit (and if participants came by car and don’t want to wait until < 0.08, then the NIH 
police is notified, as already specified in the consent). If the reading is ≤0.03, we let the subject 
know that we will need to wait 1-2 hours to ensure their readings are back to zero before they 
can sign the consent form and proceed. 


42 
 
After negative breathalyzer is obtained, the consent process will take place and then study 
procedures will start. The NIH CC nursing staff in charge of the participant will be required to 
first complete and sign a 2-item checklist [i.e.: a) ask to the participant whether or not they have 
been consented; and b) verify consent was signed] before study procedures take place at the time 
of the admission (Visit 1).As part of the consent procedure, participants’ ability to understand the 
consent will be evaluated using the ‘NIDA/NIAAA Evaluation of Potential Research 
Participants’ Ability to Consent’. 
 
 
c. Consent documents 
The consent form attached in the Appendix contains all required elements. The original consent 
is filed with their NIH medical record and a digital copy is uploaded into the CRIS system. A 
copy of the signed consent is also made for the subject's CRF binder for regulatory purposes. 
 
Data and Safety monitoring 
d. Data and Safety monitor 
Data and safety monitoring commensurate with the degree of risk involved in participation in a 
small, single-site trial like the present one and will be performed by an independent safety 
monitor (ISM). 
 
e. Data and safety monitoring plan 
The ISM is a physician with relevant expertise, whose primary responsibility is to provide 
independent safety monitoring in a timely fashion. To ensure expertise in clinical addiction 
research as well as independence, the ISM for the current study will be a physician from a 
different Section of NIAAA. The ISM responsibilities will be assumed by Nancy Diazgranados, 
M.D., NIAAA or Falk Lohoff, M.D. NIAAA. 
The ISM evaluation includes the progress of the research study as documented in the “ISM 
Monitoring Report and Log”, data quality from review of adverse events, lab data and relevant 
clinical data collected immediately after they occur or are reported, with follow-up through 
resolution, and participant accrual in the Screening and Enrollment Log. The ISM reviews on an 
ongoing basis individual and cumulative participant data, and makes recommendations regarding 
the safe continuation of the study, or its termination. The ISM may break the study blind if 
needed. Weekly safety data monitoring reports are generated by NIAAA Staff Scientist and sent 
via encrypted email to the MAI, ISM and PI for review and consideration. Appropriate action is 
then conveyed to the research team. 
The ISM will submit their review findings on the “Independent Safety Monitoring Report and 
Log” (stored in regulatory binder) for the PI and MAI to review.  The PI will submit all findings 
from this review at the time of continuing review and/or in the case of a PR report the timing of 
which is in compliance with OHSRP guidelines.  
Adverse Event reporting and/or Problem Reporting will be performed consistent with current 
OHSRP and NIH SOP Guidelines including; report submission in PTMS for OCD and IRB 
review, email notification to the study sponsor if this is a separate individual from the PI, hard 
copy notification in triplicate via Fed Ex/U.S. Mail to the FDA.  
 
 


43 
 
 
 
Criteria for stopping the study or suspending enrollment or procedures 
The study will be terminated if more than five individuals experience serious adverse events 
(SAEs) related to the study drug [> 5 SAEs].  
There is no plan at this stage to conduct an interim analysis. 
f. Criteria for suspending study procedures on an individual basis:  Administration of 
study medication or alcohol will be suspended on a case- by-case basis as per the PI and 
MAI request in accordance with the following criteria  
 
Checklist: 
o Clinically significant findings including changes in mood or behavior such as 
aggression, agitation, depression or suicidal ideation or suicidal behavior. 
o Changes in concomitant medications that may interact with study medication or 
alcohol. 
o At the discretion of the Principal Investigator (PI) and Medical Investigator, based 
on adverse event (AE) severity 
o Non-compliance with protocol procedures or investigator request(s) 
o Patient request 
o Equipment failure 
 
Please see also “Criteria for individual subject withdrawal”, Section 8. Subject 
Safety monitoring 
 
 
 
15. 
  Clinical Monitoring/Quality Assurance 
Quality assurance will be monitored by the PI and research team and the 
NIMH/NIAAA/NIDA Combined Monitoring Plan, Intramural Research Program 
Auditing Committee (IRPAC) (Please see Appendix (E) – Standard Operating 
Procedures for the Intramural NIMH, NIAAA and NIDA Combined Monitoring Plan) 
coordinated by the Offices of the Clinical Director at NIMH,NIAAA and NIDA. 
The NIMH/NIAAA/NIDA Combined Monitoring Plan, Intramural Research Program 
Auditing Committee (IRPAC) monitors intramural research studies to ensure 
compliance with GCP, organizational policies and applicable federal, state and local 
laws and the reliability of study data.    
 
a. Quality assurance monitor 
 Quality assurance (QA) will be performed by the PI, as well as by an 
independent QA monitor, which according to established practice is 
considered sufficient for small, single-site trials, like the present project. 
The QA monitor for this study will have considerable expertise in clinical 
trials in alcohol dependence, and will have completed training in Clinical 
Research Monitoring and GCP Compliance. 
 
 


44 
 
b. Quality assurance plan 
 
All participants’ data and binders will be reviewed on a monthly basis. 
 
16. Adverse event and unanticipated problem reporting 
 
Adverse events, protocol deviations, unanticipated problems (UP), Unanticipated Adverse 
Device Effects (UADEs), serious adverse events are defined as described in NIH HRPP SOP 16 
(“Reporting Requirements for Unanticipated Problems, Adverse Events and Protocol 
Deviations.”).   The Principal Investigator is responsible for detecting, documenting, and 
reporting unanticipated problems, adverse events (AEs), including serious adverse events 
(SAEs), and deviations in accordance with NIH policy, IRB requirements, and federal 
regulations. Relatedness to the research of all serious adverse events will be determined by the PI 
in consultation with the CD. 
 Serious unanticipated problems and serious protocol deviations will be reported to the IRB and 
CD as soon as possible and in writing on the Problem Report Form not more than 7 days after 
the PI first learns of the event.  Not serious unanticipated problems and not serious deviations 
will be reported to the IRB and CD as soon as possible and in writing on the Problem Report 
Form not more than 14 days after the PI first learns of the event.  Deaths will be reported to the 
Clinical Director within 7 days after the PI first learns of the event.  All adverse events, 
deviations, and unanticipated problems will be summarized and reported at the time of 
Continuing Review. 
 
 
17. Alternatives to participation or Alternative therapies 
To be eligible for this study, subjects must not be seeking help for drinking problems. Those who 
are seeking help for a drinking problem will be referred to standard therapy for heavy drinking 
and will not be allowed to participate in this study. 
 
18.  Confidentiality 
This protocol is covered by a Certificate of Confidentiality (CoC) issued by the US Department 
of Health and Human Services.  Strict subject confidentiality will be maintained throughout the 
study. Confidentiality and information technology standards are in place at the NIAAA/LCTS 
intramural program to protect electronic repositories of patient data as well as other clinical 
patient related material. It is reasonably expected that these safeguards will protect participants' 
medical and personal health information, ensuring their privacy.   
Information obtained in the course of being screened for or participating in this protocol will 
become part of the patient's NIH medical record. This includes potentially sensitive information, 
such as urine tests positive for illicit drugs. Access to this information may be requested by third 
parties. Such access will not be granted without the explicit, written consent of the subjects.  
Samples and data will be stored using codes that we assign.  Data will be kept in password 
protected computers.  Samples will be kept in locked storage.  Only study personnel will have 
access to the samples and data. 
 
20. Conflict of Interest 
a.   Distribution of NIH Guidelines 


45 
 
NIH guidelines on conflict of interest have been distributed to all investigators. 
 
b.  Conflict of interest  
There are no conflicts-of-interest to report. 
 
c.  Role of a commercial company or sponsor  
There is no drug company involved. 
 
21. Technology Transfer 
No technology will be subject to transfer to agencies outside the US Government.  
 
22. Compensation  
Volunteers will be compensated for time and research-related inconveniences, to the extent to 
which they complete them, as follows: 
 
Visit # 
Compensation 
Visit 2 (alcohol PR) 
$250 
Visit 3 (alcohol PR) 
$250 
Visit 4 (fMRI/alcohol clamp) 
$250 
Visit 5 (fMRI/alcohol clamp) 
$250 
Visit 6 (Follow-up: $25 + bonus for completing all parts of 
the study: $100) 
$125 
TOTAL 
$1,125 
    Compensation will be prorated for parts completed if subjects do not complete the 
    study. If needed, subjects will be provided with a taxi paid for by NIH. 
 
Visit 1 (Screening) is done under a separate NIAAA protocol, i.e. the NIAAA Screening 
Protocol 98-AA-0009, (PI: Reza Momenan, Ph.D.) or 14-AA-0181. The 98-AA-0009 protocol 
has been routinely used in the NIAAA Clinical Program for several years to screen non-
treatment seeking individuals who are then enrolled - pending on their eligibility - in more 
specific studies, such as this proposed study with ghrelin. Therefore, all human lab studies 
with non-treatment populations performed in the NIAAA Clinical Program enroll participants 
who were first screened via the 98-AA-0009 protocol or the 14-AA-0181. 
 
22. References 
 
1. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-
hormone-releasing acylated peptide from stomach. Nature 402: 656-60, 1999. PMID: 
10604470. 
2. Cummings DE, Naleid AM, Figlewicz Lattemann DP. Ghrelin: a link between energy 
homeostasis and drug abuse? Addict Biol 12: 1-5, 2007. PMID: 17407491. 
3. Jerlhag E, Egecioglu E, Dickson SL, Andersson M, Svensson L, Engel JA. Ghrelin Stimulates 
Locomotor Activity and Accumbal Dopamine-Overflow via Central Cholinergic Systems in 
Mice: Implications for its Involvement in Brain Reward. Addict Biol 11: 45-54, 2006. PMID: 
16759336 


46 
 
4. Jerlhag E, Egecioglu E, Dickson SL, Douhan A, Svensson L, Engel JA. Ghrelin 
administration into tegmental areas stimulates locomotor activity and increases extracellular 
concentration of dopamine in the nucleus accumbens. Addict Biol 12: 6-16, 2007. PMID: 
17407492. 
5. Addolorato G, Capristo E, Leggio L, Ferrulli A, Abenavoli L, Malandrino N, Farnetti S, 
Domenicali M, D'Angelo C, Vonghia L, Mirijello A, Cardone S, Gasbarrini G. Relationship 
between ghrelin levels, alcohol craving, and nutritional status in current alcoholic patients. 
Alcohol Clin Exp Res 30: 1933-7, 2006. PMID: 17067359. 
6. Hillemacher T, Kraus T, Rauh J, Weiss J, Schanze A, Frieling H, Wilhelm J, Heberlein A, 
Gröschl M, Sperling W, Kornhuber J, Bleich S. Role of appetite-regulating peptides in 
alcohol craving: an analysis in respect to subtypes and different consumption patterns in 
alcoholism. Alcohol Clin Exp Res 31: 950-4, 2007. PMID: 17433008.  
7. Wurst FM, Graf I, Ehrenthal HD, Klein S, Backhaus J, Blank S, Graf M, Pridzun L, Wiesbeck 
GA, Junghanns K. Gender differences for ghrelin levels in alcohol-dependent patients and 
differences between alcoholics and healthy controls. Alcohol Clin Exp Res 31:2006-11, 2007. 
PMID: 17949465. 
8. Anton RF. What is craving? Models and implications for treatment. Alcohol Res Health 
23:165-73, 1999. PMID: 10890811. 
9. Monti PM, Rohsenow DJ, Hutchison KE. Toward bridging the gap between biological, 
psychobiological and psychosocial models of alcohol craving. Addiction 95(Suppl 2):229S-
236S, 2000. PMID: 11002917. 
10. Hutchison KE, Wooden AL, Swift RM Smolen A, McGeary JE, Adler L, Paris L. Olanzapine 
reduces craving for alcohol: a DRD4 VNTR polymorphism by pharmacotherapy interaction. 
Neuropsychopharmacology 28: 1882-8, 2003. PMID: 12888781. 
11. National Institute on Alcohol Abuse and Alcoholism (2000). Tenth Special Report to the U.S. 
Congress on Alcohol and Health, Washington, DC: U.S. Department of Health and Human 
Services. 
12. Swift RM. Drug therapy for alcohol dependence. New Engl J Med 340:1482-90, 1999. PMID: 
10320388. 
13. Addolorato G, Abenavoli L, Leggio L, Gasbarrini G. How many cravings? Pharmacological 
aspects of craving treatment in alcohol addiction: a review. Neuropsychobiology 51: 59-66, 
2005. PMID: 15741745. 
14. Miller WR, Walters ST, Bennett ME. How effective is alcoholism treatment in the United 
States? J Stud Alcohol 62: 211-20, 2001. PMID: 11327187. 
15. Garbutt JC, West SL, Carey TS, Lohr KN, Crews FT. Pharmacological treatment of alcohol 
dependence: A Review of the Evidence, JAMA 281:1318-25, 1999. PMID: 10208148. 
16. Kranzler H, Van Kirk J. Efficacy of naltrexone and Acamprosate for alcoholism treatment: A 
meta analysis. Alcohol Clin Exp Res 25:1335-41, 2001. PMID: 11584154. 
17. Litten RZ, Allen JP. Reducing the desire to drink. Pharmacology and neurobiology. Recent 
Dev Alcohol 11:325-44, 1993. PMID: 8234930. 
18. Heilig M, Egli M. Pharmacological treatment of alcohol dependence: target symptoms and 
target mechanisms. Pharmacol Ther 111:855-76, 2006. PMID: 16545872. 
19. Arora S, Anubhuti. Role of neuropeptides in appetite regulation and obesity--a review. 
Neuropeptides 40:375-401, 2006. PMID: 16935329. 
20. Addolorato G, Leggio L, Abenavoli L, Gasbarrini G. Neurobiochemical and clinical aspects 
of craving in alcohol addiction: a review. Addict Behav 30:1209–24, 2005. PMID: 15925129. 


47 
 
21. Blum K, Braverman ER, Holder JM, Lubar JF, Monastra VJ, Miller D, Lubar JO, Chen TJ, 
Comings DE. Reward deficiency syndrome: a biogenetic model for the diagnosis and 
treatment of impulsive, addictive, and compulsive behaviors. J Psychoactive Drugs 32(Suppl. 
i-iv):1–112, 2000. PMID: 11280926. 
22. Ramoz N, Versini A, Gorwood P. Eating disorders: an overview of treatment responses and 
the potential impact of vulnerability genes and endophenotypes. Expert Opin Pharmacother; 
8: 2029-44,  2007. PMID: 17714058. 
23. Aydin S. Discovery of ghrelin hormone, research and clinical applications. Turkish J Biochem 
32:76-89, 2007. 
24. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role 
for ghrelin in the central regulation of feeding. Nature 409:194–8, 2001. PMID: 11196643. 
25. Toshinai K, Date Y, Murakami N, Shimada M, Mondal MS, Shimbara T, Guan JL, Wang QP, 
Funahashi H, Sakurai T, Shioda S, Matsukura S, Kangawa K, Nakazato M. Ghrelin-induced 
food intake is mediated via the orexin pathway. Endocrinology 144:1506–12, 2003. PMID: 
12639935. 
26. Solomon A, De Fanti BA, Martinez JA. Peripheral ghrelin participates in glucostatic feeding 
mechanisms and in the anorexigenic signalling mediated by CART and CRF neurons. Nutr 
Neurosci 8:287–95, 2005. PMID: 16669599. 
27. Cowley MA, Smith RG, Diano S, Tschop M, Pronchuk N, Grove KL, Strasburger CJ, 
Bidlingmaier M, Esterman M, Heiman ML, Garcia-Segura LM, Nillni EA, Mendez P, Low 
MJ, Sotonyi P, Friedman JM, Liu H, Pinto S, Colmers WF, Cone RD, Horvath TL. The 
distribution and mechanism of action of ghrelin in the CNS demonstrates a novel 
hypothalamic circuit regulating energy homeostasis. Neuron 37:649–61, 2003. PMID: 
12597862. 
28. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 407:908–
13, 2000. PMID: 11057670. 
29. Furuse M, Tachibana T, Ohgushi A, Ando R, Yoshimatsu T, Denbow DM. 
Intracerebroventricular injection of ghrelin and growth hormone releasing factor inhibits food 
intake in neonatal chicks. Neurosci Lett 301:123–6, 2001. PMID: 11248438. 
30. Unniappan S, Lin X, Cervini L, Rivier J, Kaiya H, Kangawa K, Peter RE. Goldfish ghrelin: 
molecular characterization of the complementary deoxyribonucleic acid, partial gene structure 
and evidence for its stimulatory role in food intake. Endocrinology 143:4143–6, 2002. PMID: 
12239128. 
31. Tritos NA, Kokkotou EG. The physiology and potential clinical applications of ghrelin, a 
novel peptide hormone. Mayo Clin Proc 81(5):653-60, 2006. PMID: 16706263. 
32. Nagaya N, Kojima M, Uematsu M, Yamagishi M, Hosoda H, Oya H, Hayashi Y, Kangawa K. 
Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. Am J Physiol 
Regul Integr Comp Physiol 280: R1483–R1487, 2001. PMID: 11294772. 
33. Akamizu T, Takaya K, Irako T, Hosoda H, Teramukai S, Matsuyama A, Tada H, Miura K, 
Shimizu A, Fukushima M, Yokode M, Tanaka K, Kangawa K. Pharmacokinetics, safety, and 
endocrine and appetite effects of ghrelin administration in young healthy subjects. Eur J 
Endocrinol 150: 447–55, 2004. PMID: 15080773. 
34. Vestergaard ET, Hansen TK, Gormsen LC, Jakobsen P, Moller N, Christiansen JS,  Jorgensen 
JO.  Constant intravenous ghrelin infusion in healthy young men: clinical pharmacokinetics 
and metabolic effects. Am J Physiol Endocrinol Metab 292:E1829-36, 2007. PMID: 
17311892. 


48 
 
35. Paulo RC, Brundage R, Cosma M, Mielke KL, Bowers CY, Veldhuis JD. Estrogen Elevates 
the Peak Overnight Production Rate of Acylated Ghrelin. J Clin Endocrinol Metab 93: 4440-
7, 2008. PMID: 18697865. 
36. Arvat E, Di Vito L, Broglio F, Papotti M, Muccioli G, Dieguez C, Casanueva FF, Deghenghi 
R, Camanni F, Ghigo E. Preliminary evidence that Ghrelin, the natural GH secretagogue 
(GHS)-receptor ligand, strongly stimulates GH secretion in humans. J Endocrinol Invest 23: 
493-5, 2000. PMID: 11021763. 
37. Wren AM, Seal LJ, Cohen MA, Brynes AE, Frost GS, Murphy KG, Dhillo WS, Ghatei MA, 
Bloom SR. Ghrelin enhances appetite and increases food intake in humans. J Clin Endocrinol 
Metab 86: 5992, 2001. PMID: 11739476. 
38. Arvat E, Maccario M, Di Vito L, Broglio F, Benso A, Gottero C, Papotti M, Muccioli G, 
Dieguez C, Casanueva FF, Deghenghi R, Camanni F, Ghigo E. Endocrine activities of 
ghrelin, a natural growth hormone secretagogue (GHS), in humans: comparison and 
interactions with hexarelin, a nonnatural peptidyl GHS, and GH-releasing hormone. J Clin 
Endocrinol Metab 86: 1169-74, 2001. PMID: 11238504. 
39. Broglio F, Arvat E, Benso A, Gottero C, Muccioli G, Papotti M, van der Lely AJ, Deghenghi 
R, Ghigo E.  Ghrelin, a natural GH secretagogue produced by the stomach, induces 
hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 86:5083-6, 
2001. PMID: 11600590. 
40. Weikel JC, Wichniak A, Ising M, Brunner H, Friess E, Held K, Mathias S, Schmid DA, Uhr 
M, Steiger A. Ghrelin promotes slow-wave sleep in humans. Am J Physiol Endocrinol Metab 
284:E407-15, 2003. PMID: 12388174. 
41. Broglio F, Gottero C, Benso A, Prodam F, Casanueva FF, Dieguez C, van der Lely AJ, 
Deghenghi R, Arvat E, Ghigo E. Acetylcholine does not play a major role in mediating the 
endocrine responses to ghrelin, a natural ligand of the GH secretagogue receptor, in humans. 
Clin Endocrinol (Oxf) 58: 92-8, 2003. PMID: 12519418. 
42. Broglio F, Benso A, Castiglioni C, Gottero C, Prodam F, Destefanis S, Gauna C, van der Lely 
AJ, Deghenghi R, Bo M, Arvat E, Ghigo E. The endocrine response to ghrelin as a function of 
gender in humans in young and elderly subjects. J Clin Endocrinol Metab 88: 1537-42, 2003. 
PMID: 12679436. 
43. Broglio F, Gottero C, Benso A, Prodam F, Destefanis S, Gauna C, Maccario M, Deghenghi R, 
van der Lely AJ, Ghigo E. Effects of ghrelin on the insulin and glycemic responses to glucose, 
arginine, or free fatty acids load in humans. J Clin Endocrinol Metab 88: 4268-72, 2003. 
PMID: 12970297. 
44. Tassone F, Broglio F, Destefanis S, Rovere S, Benso A, Gottero C, Prodam F, Rossetto R, 
Gauna C, van der Lely AJ, Ghigo E, Maccario M. Neuroendocrine and metabolic effects of 
acute ghrelin administration in human obesity. J Clin Endocrinol Metab 88: 5478-83, 2003. 
PMID: 14602793. 
45. Alvarez-Castro P, Isidro ML, Garcia-Buela J, Leal-Cerro A, Broglio F, Tassone F, Ghigo E, 
Dieguez C, Casanueva FF, Cordido F. Marked GH secretion after ghrelin alone or combined 
with GH-releasing hormone GHRH) in obese patients. Clin Endocrinol (Oxf) 61: 250-5, 2004. 
PMID: 15272922. 
46. Druce MR, Wren AM, Park AJ, Milton JE, Patterson M, Frost G, Ghatei MA, Small C, 
Bloom SR. Ghrelin increases food intake in obese as well as lean subjects. Int J Obes (Lond) 
29(9):1130-6, 2005. PMID: 15917842. 


49 
 
47. Schmid DA, Held K, Ising M, Uhr M, Weikel JC, Steiger A. Ghrelin stimulates appetite, 
imagination of food, GH, ACTH, and cortisol, but does not affect leptin in normal controls. 
Neuropsychopharmacology 30: 1187-92, 2005. PMID: 15688086. 
48. Fassino S, Daga GA, Mondelli V, Pierò A, Broglio F, Picu A, Giordano R, Baldi M, Arvat E, 
Ghigo E, Gianotti L. Hormonal and metabolic responses to acute ghrelin administration in 
patients with bulimia nervosa. Psychoneuroendocrinology 30: 534-40, 2005. PMID: 
15808922. 
49. Levin F, Edholm T, Schmidt PT, Grybäck P, Jacobsson H, Degerblad M, Höybye C, Holst JJ, 
Rehfeld JF, Hellström PM, Näslund E. Ghrelin stimulates gastric emptying and hunger in 
normal-weight humans. J Clin Endocrinol Metab 91: 3296-302, 2006. PMID: 16772353. 
50. Tack J, Depoortere I, Bisschops R, Delporte C, Coulie B, Meulemans A, Janssens J, Peeters 
T. Influence of ghrelin on interdigestive gastrointestinal motility in humans. Gut 55: 327-33, 
2006. PMID: 16216827. 
51. Miljic D, Djurovic M, Pekic S, Doknic M, Stojanovic M, Milic N, Casanueva FF, Ghatei M, 
Popovic V. Glucose metabolism during ghrelin infusion in patients with anorexia nervosa. J 
Endocrinol Invest 30:771-5, 2007. PMID: 17993770. 
52. Miljic D, Pekic S, Djurovic M, Doknic M, Milic N, Casanueva FF, Ghatei M, Popovic V. 
Ghrelin has partial or no effect on appetite, growth hormone, prolactin, and cortisol release in 
patients with anorexia nervosa. J Clin Endocrinol Metab 91: 1491-5, 2006. PMID: 16449333. 
53. Veldhuis JD, Keenan DM, Iranmanesh A, Mielke K, Miles JM, Bowers CY. Estradiol 
potentiates ghrelin-stimulated pulsatile growth hormone secretion in postmenopausal women. 
J Clin Endocrinol Metab 91: 3559-65, 2006. PMID: 16804038. 
54. Veldhuis JD, Iranmanesh A, Mielke K, Miles JM, Carpenter PC, Bowers CY. Ghrelin 
potentiates growth hormone secretion driven by putative somatostatin withdrawal and resists 
inhibition by human corticotropin-releasing hormone. J Clin Endocrinol Metab 91: 2441-6, 
2006. PMID: 16537682. 
55. Vestergaard ET, Andersen NH, Hansen TK, Rasmussen LM, Moller N, Sorensen KE, Sloth 
E, Jorgensen JO.  Cardiovascular effects of intravenous ghrelin infusion in healthy young 
men. Am J Physiol Heart Circ Physiol 293: H3020-6, 2007. PMID: 17873022. 
56. Kluge M, Schüssler P, Zuber V, Kleyer S, Yassouridis A, Dresler M, Uhr M, Steiger A. 
Ghrelin enhances the nocturnal secretion of cortisol and growth hormone in young females 
without influencing sleep. Psychoneuroendocrinology 32: 1079-85, 2007. PMID: 17945435. 
57. Kluge M, Schüssler P, Bleninger P, Kleyer S, Uhr M, Weikel JC, Yassouridis A, Zuber V, 
Steiger A. Ghrelin alone or co-administered with GHRH or CRH increases non-REM sleep 
and decreases REM sleep in young males. Psychoneuroendocrinology 33: 497-506, 2008. 
PMID: 18329818. 
58. Vestergaard ET, Djurhuus CB, Gjedsted J, Nielsen S, Møller N, Holst JJ, Jørgensen JO, 
Schmitz O.  Acute effects of ghrelin administration on glucose and lipid metabolism. J Clin 
Endocrinol Metab 93: 438-44, 2008. PMID: 18042651. 
59. Lanfranco F, Bonelli L, Baldi M, Me E, Broglio F, Ghigo E. Acylated ghrelin inhibits 
spontaneous LH pulsatility and responsiveness to naloxone but not that to GnRH in young 
men: evidence for a central inhibitory action of ghrelin on the gonadal axis J Clin Endocrinol 
Metab 93: 3633-9, 2008 PMID: 18559917. 
60. Coiro V, Saccani-Jotti G, Rubino P, Manfredi G, Vacca P, Volta E, Chiodera P. Oxytocin 
inhibits the stimulatory effect of ghrelin on circulating neuropeptide Y levels in humans. J 
Neural Transm 115:1265-7, 2008. PMID: 18461271. 


50 
 
61. Veldhuis JD, Reynolds GA, Iranmanesh A, Bowers CY. Twenty-Four Hour Continuous 
Ghrelin Infusion Augments Physiologically Pulsatile, Nycthemeral and Entropic (Feedback-
Regulated) Modes of GH Secretion. J Clin Endocrinol Metab 93: 3597–603, 2008. PMID: 
18593763. 
62. Kok P, Paulo RC, Cosma M, Mielke KL, Miles JM, Bowers CY, Veldhuis JD. Estrogen 
Supplementation Selectively Enhances Hypothalamo-Pituitary Sensitivity to Ghrelin in 
Postmenopausal Women. J Clin Endocrinol Metab 93:4020-6, 2008. PMID: 18611978. 
63. Broglio F, Prodam F, Riganti F, Gottero C, Destefanis S, Granata R, Muccioli G, Abribat T, 
van der Lely AJ, Ghigo E. The continuous infusion of acylated ghrelin enhances growth 
hormone secretion and worsens glucose metabolism in humans. J Endocrinol Invest 31: 788-
94 2008. PMID: 18997491. 
64. Benso A, Gramaglia E, Prodam F, Riganti F, Gigliardi VR, Lucatello B, Olivetti I, St Pierre 
D, Ghigo E, Broglio F. Beta-adrenergic agonism does not impair the GH response to acylated 
ghrelin in humans. Clin Endocrinol (Oxf) 71: 234-6, 2009. PMID: 19067721. 
65. Huda MS, Dovey T, Wong SP, English PJ, Halford J, McCulloch P, Cleator J, Martin B, 
Cashen J, Hayden K, Wilding JP, Pinkney J. Ghrelin restores 'lean-type' hunger and energy 
expenditure profiles in morbidly obese subjects but has no effect on postgastrectomy subjects. 
Int J Obes 33: 317-25. PMID: 19188925. 
66. Kluge M, Gazea M, Schüssler P, Genzel L, Dresler M, Kleyer S, Uhr M, Yassouridis A, 
Steiger A. Ghrelin increases slow wave sleep and stage 2 sleep and decreases stage 1 sleep 
and REM sleep in elderly men but does not affect sleep in elderly women. 
Psychoneuroendocrinology Jul 31, 2009. PMID: 19647945. 
67. Messini CI, Dafopoulos K, Chalvatzas N, Georgoulias P, Messinis IE. Effect of ghrelin on 
gonadotrophin secretion in women during the menstrual cycle. Hum Reprod 24: 976-81, 
2009. PMID: 19095668. 
68. Ang D, Nicolai H, Vos R, Mimidis K, Akyuz F, Kindt S, Vanden Berghe P, Sifrim D, 
Depoortere I, Peeters T, Tack J. Influence of ghrelin on the gastric accommodation reflex and 
on meal-induced satiety in man. Neurogastroenterol Motil 21: 528-33, e8-9. 2009 PMID: 
19210631. 
69. Burns CM, Ratcliffe SJ, Gooneratne N, Scattergood T, Waring AC, Cappola AR. Effects of 
ghrelin administration in frail and healthy older women. The Endocrine Society’s Annual 
Meeting, Washington, D.C., Abstract#P3-495; 12 June 2009. 
70. Nagaya N, Miyatake K, Uematsu M, Oya H, Shimizu W, Hosoda H, Kojima M, Nakanishi N, 
Mori H, Kangawa K. Hemodynamic, renal, and hormonal effects of ghrelin infusion in 
patients with chronic heart failure. J Clin Endocrinol Metab 86: 5854-9, 2001. PMID: 
11739451. 
71. Aimaretti G, Baffoni C, Broglio F, Janssen JA, Corneli G, Deghenghi R, van der Lely AJ, 
Ghigo E, Arvat E. Endocrine responses to ghrelin in adult patients with isolated childhood-
onset growth hormone deficiency. Clin Endocrinol (Oxf) 56: 765-71, 2002. PMID: 12072046. 
72. Janssen JA, Poldermans D, Hofland LJ, Vourvouri EC, Muller AF, Bax JJ, Deghenghi R, 
Broglio F, Ghigo E, van der Lely AJ. There are no acute cardiac effects of a single IV dose of 
human ghrelin in severe growth hormone deficient patients. J Endocrinol Invest 27: 659-64, 
2004. PMID: 15505990. 
73. Gauna C, Meyler FM, Janssen JA, Delhanty PJ, Abribat T, van Koetsveld P, Hofland LJ, 
Broglio F, Ghigo E, van der Lely AJ. Administration of acylated ghrelin reduces insulin 


51 
 
sensitivity, whereas the combination of acylated plus unacylated ghrelin strongly improves 
insulin sensitivity. J Clin Endocrinol Metab 89: 5035-42, 2004. PMID: 15472202. 
74. Broglio F, Gianotti L, Destefanis S, Fassino S, Abbate Daga G, Mondelli V, Lanfranco F, 
Gottero C, Gauna C, Hofland L, Van der Lely AJ, Ghigo E. The endocrine response to acute 
ghrelin administration is blunted in patients with anorexia nervosa, a ghrelin hypersecretory 
state. Clin Endocrinol (Oxf) 60: 592-9, 2004. PMID: 15104562. 
75. Neary NM, Small CJ, Wren AM, Lee JL, Druce MR, Palmieri C, Frost GS, Ghatei MA, 
Coombes RC, Bloom SR. Ghrelin increases energy intake in cancer patients with impaired 
appetite: acute, randomized, placebo-controlled trial. J Clin Endocrinol Metab 89: 2832-6, 
2004. PMID: 15181065. 
76. Tack J, Depoortere I, Bisschops R, Verbeke K, Janssens J, Peeters T. Influence of ghrelin on 
gastric emptying and meal-related symptoms in idiopathic gastroparesis. Aliment Pharmacol 
Ther 22:847-53, 2005. PMID: 16225494. 
77. Tesauro M, Schinzari F, Iantorno M, Rizza S, Melina D, Lauro D, Cardillo C. Ghrelin 
improves endothelial function in patients with metabolic syndrome. Circulation 112: 2986-92, 
2005. PMID: 16260640. 
78. Murray CD, Martin NM, Patterson M, Taylor SA, Ghatei MA, Kamm MA, Johnston C, 
Bloom SR, Emmanuel AV. Ghrelin enhances gastric emptying in diabetic gastroparesis: a 
double blind, placebo controlled, crossover study. Gut 54: 1693-8, 2005. PMID: 16085693. 
79.  Wynne K, Giannitsopoulou K, Small CJ, Patterson M, Frost G, Ghatei MA, Brown EA, 
Bloom SR, Choi P. Subcutaneous ghrelin enhances acute food intake in malnourished patients 
who receive maintenance peritoneal dialysis: a randomized,  placebo-controlled trial. J Am 
Soc Nephrol 16:2111-8, 2005. PMID: 15888560. 
80. Damjanovic SS, Lalic NM, Pesko PM, Petakov MS, Jotic A, Miljic D, Lalic KS, Lukic L, 
Djurovic M, Djukic VB. Acute effects of ghrelin on insulin secretion and glucose disposal 
rate in gastrectomized patients. J Clin Endocrinol Metab 91:2574-81, 2006. PMID: 16621911. 
81. Cremonini F, Camilleri M, Vazquez Roque M, McKinzie S, Burton D, Baxter K, Zinsmeister 
AR. Obesity does not increase effects of synthetic ghrelin on human gastric motor functions. 
Gastroenterology 131: 1431-9, 2006. PMID: 17101319. 
82. Fusco A, Bianchi A, Mancini A, Milardi D, Giampietro A, Cimino V, Porcelli T,  Romualdi 
D, Guido M, Lanzone A, Pontecorvi A, De Marinis L.  Effects of ghrelin administration on 
endocrine and metabolic parameters in obese women with polycystic ovary syndrome. J 
Endocrinol Invest 30: 948-56, 2007. PMID: 18250617. 
83. Akamizu T, Iwakura H, Ariyasu H, Hosoda H, Murayama T, Yokode M, Teramukai S, Seno 
H, Chiba T, Noma S, Nakai Y, Fukunaga M, Nakai Y, Kangawa K, FD Clinical Study Team. 
Repeated administration of ghrelin to patients with functional dyspepsia: its effects on food 
intake and appetite. Eur J Endocrinol 158: 491-8, 2008. PMID: 18362296. 
84. Guido M, Romualdi D, De Marinis L, Porcelli T, Giuliani M, Costantini B, Lanzone A. 
Administration of exogenous ghrelin in obese patients with polycystic ovary syndrome: 
effects on plasma levels of growth hormone, glucose, and insulin. Fertil Steril 88: 125-30, 
2007. PMID: 17276431. 
85. Cecconi E, Bogazzi F, Morselli LL, Gasperi M, Procopio M, Gramaglia E, Broglio F, 
Giovannetti C, Ghigo E, Martino E. Primary hyperparathyroidism is associated with marked 
impairment of GH response to acylated ghrelin. Clin Endocrinol (Oxf). 69: 197-201, 2008. 
PMID: 18221398. 


52 
 
86. Romualdi D, De Marinis L, Campagna G, Proto C, Lanzone A, Guido M. Alteration of 
ghrelin-neuropeptide Y network in obese patients with polycystic ovary syndrome: role of 
hyperinsulinism. Clin Endocrinol (Oxf) 69: 562-7. 2008. PMID: 18248643. 
87. Ashby DR, Ford HE, Wynne KJ, Wren AM, Murphy KG, Busbridge M, Brown EA, Taube 
DH, Ghatei MA, Tam FW, Bloom SR, Choi P. Sustained appetite improvement in 
malnourished dialysis patients by daily ghrelin treatment. Kidney Int 76:199-206, 2009. 
PMID: 19387475. 
88. Kiewiet RM, van Aken MO, van der Weerd K, Uitterlinden P, Themmen AP, Hofland  LJ, de 
Rijke YB, Delhanty PJ, Ghigo E, Abribat T, van der Lely AJ. Effects of acute administration 
of acylated and unacylated ghrelin on glucose and insulin concentrations in morbidly obese 
subjects without overt diabetes. Eur J Endocrinol 161: 567-73, 2009. PMID: 19628651. 
89. Hotta M, Ohwada R, Akamizu T, Shibasaki T, Takano K, Kangawa K. Ghrelin Increases 
Hunger and Food Intake in Patients with Restricting-type Anorexia Nervosa: A Pilot Study. 
Endocr J Sep 16, 2009. PMID:19755753. 
90. Loose MD, Ronnekleiv OK, Kelly MJ. Neurons in the rat arcuate nucleus are hyperpolarized 
by GABAB and mu-opioid receptor agonists: evidence for convergence at a ligand-gated 
potassium conductance. Neuroendocrinology 54: 537-44, 1991. PMID: 1664497. 
91. Cummings DE, Purnell JQ, Frayo RS, Schmidova K, Wisse BE, Weigle DS. A preprandial 
rise in plasma ghrelin levels suggests a role in meal initiation in humans. Diabetes 50, 1714-9, 
2001. PMID: 11473029. 
92. Tschop M, Wawarta R, Riepl RL, Friedrich S, Bidlingmaier M, Landgraf R, Folwaczny C. 
Post-prandial decrease of circulating human ghrelin levels. J Endocrinol Invest 24: RC19–
RC21, 2001. PMID: 11434675. 
93. Guan XM, Yu H, Palyha OC, McKee KK, Feighner SD, Sirinathsinghji DJ, Smith RG, Van 
der Ploeg LH, Howard AD. Distribution of mRNA encoding the growth hormone 
secretagogue receptor in brain and peripheral tissues. Brain Res Mol Brain Res 48: 23-9, 
1997. PMID: 9379845. 
94. Katayama M, Nogami H, Nishiyama J, Kawase T, Kawamura K. Developmentally and 
regionally regulated expression of growth hormone secretagogue receptor mRNA in rat brain 
and pituitary gland. Neuroendocrinology 72:333–40, 2000. PMID: 11146416. 
95. Zigman JM, Jones JE, Lee CE, Saper CB, Elmquist JK. Expression of ghrelin receptor mRNA 
in the rat and the mouse brain. J Comp Neurol 494:528–48, 2006. PMID: 16320257. 
96. Naleid AM, Grace MK, Cummings DE, Levine AS. Ghrelin induces feeding in the 
mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens. 
Peptides 26:2274–9, 2005. PMID: 16137788. 
97. Jerlhag E, Grøtli M, Luthman K, Svensson L, Engel JA. Role of the subunit composition of 
central nicotinic acetylcholine receptors for the stimulatory and dopamine-enhancing effects 
of ethanol. Alcohol Alcohol 41, 486-93, 2006. PMID: 16799162. 
98. Kawahara Y, Kawahara H, Kaneko F, Yamada M, Nishi Y, Tanaka E, Nishi A. Peripherally 
administered ghrelin induces bimodal effects on the mesolimbic dopamine system depending 
on food-consumptive states. Neuroscience. 161:855-64. 2009. PMID: 19362120. 
99. Quarta D, Di Francesco C, Melotto S, Mangiarini L, Heidbreder C, Hedou G. Systemic 
administration of ghrelin increases extracellular dopamine in the shell but not the core 
subdivision of the nucleus accumbens. Neurochem Int. 54:89-94. 2009. PMID: 19118592. 
100. Tupala E, Tiihonen J. Dopamine and alcoholism: neurobiological basis of ethanol abuse. Prog 
Neuropsychopharmacol Biol Psychiatry 28: 1221-47, 2004. PMID: 15588749. 


53 
 
101.  Koob GF. Neural mechanisms of drug reinforcement. Ann N Y Acad Sci 654:171-91, 1992. 
PMID: 1632582. 
102.  Wise RA. Neurobiology of addiction. Curr Opin Neurobiol 6: 243-51, 1996. PMID: 
8725967. 
103. Melendez RI, Rodd-Henricks ZA, Engleman EA, Li TK, McBride WJ, Murphy JM. 
Microdialysis of dopamine in the nucleus accumbens of alcohol-preferring (P) rats during 
anticipation and operant self-administration of ethanol. Alcohol Clin Exp Res 26: 318-25, 
2002. PMID: 11923583. 
104. Yan QS. Extracellular dopamine and serotonin after ethanol monitored with 5-minute 
microdialysis. Alcohol 19:1-7, 1999. PMID: 10487381. 
105. Nurmi M, Ashizawa T, Sinclair JD, Kiianmaa K. Effect of prior ethanol experience on 
dopamine overflow in  accumbens of AA and ANA rats. Eur J Pharmacol 315:277-83, 1996. 
PMID: 8982665. 
106. Hemby SE. Neurobiology of alcoholism. In: Johnson BA, Ruiz P, Galanter M, eds. Handbook 
of clinical alcoholism treatment. Baltimore, MD: Lippincott Williams & Wilkins; 10-8, 2003. 
107. Devlin MJ. Is there a place for obesity in DSM-V. Int J Eat Disord 40 Suppl: S83-8, 2007. 
PMID: 17683083 
108. Volkow ND, Wang GJ, Fowler JS, Telang F. Overlapping neuronal circuits in addiction and 
obesity: evidence of systems pathology. Philos Trans R Soc Lond B Biol Sci 363:3191-200, 
2008. PMID: 18640912. 
109. Koob GF. Drugs of abuse: anatomy, pharmacology and function of reward pathways. Trends 
Pharmacol Sci. 1992;13:177-84. 
110. Heilig M, Koob GF. A key role for corticotropin-releasing factor in alcohol dependence. 
Trends Neurosci. 2007;30:399-406. 
111. Pandey SC, Carr LG, Heilig M, Ilveskoski E, Thiele TE. Neuropeptide y and alcoholism: 
genetic, molecular, and pharmacological evidence. Alcohol Clin Exp Res. 2003;27:149-54. 
112. Koob GF. Brain stress systems in the amygdala and addiction. Brain Res. 2009; 13;1293:61-
75. 
113. Skibicka KP, Shirazi RH, Hansson C, Dickson SL. Ghrelin Interacts with Neuropeptide Y Y1 
and Opioid Receptors to Increase Food Reward. Endocrinology 2012; 153: 1194-1205. 
114. Naleid AM, Grace MK, Cummings DE, Levine AS. Ghrelin induces feeding in the 
mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens. 
Peptides 2005;26:2274-9. 
115. Spencer SJ, Xu L, Clarke MA, Lemus M, Reichenbach A, Geenen B, Kozicz T, Andrews ZB. 
Ghrelin Regulates the Hypothalamic-Pituitary-Adrenal Axis and Restricts Anxiety After 
Acute Stress. Biol Psychiatry 2012 Apr 20. [Epub ahead of print] PMID: 22521145. 
116. Clarke TK, Treutlein J, Zimmermann US, Kiefer F, Skowronek MH, Rietschel M, Mann K, 
Schumann G. HPA-axis activity in alcoholism: examples for a gene-environment interaction. 
Addict Biol. 2008 Mar;13(1):1-14 
117. Jerlhag E, Egecioglu E, Dickson SL, Svensson L, Engel JA. Alpha-conotoxin MII-sensitive 
nicotinic acetylcholine receptors are involved in mediating the ghrelin-induced locomotor 
stimulation and dopamine overflow in nucleus accumbens. Eur Neuropsychopharmacol 18: 
508-18, 2008. PMID: 18343642. 
118. Larsson A, Edström L, Svensson L, Söderpalm B, Engel JA. Voluntary ethanol intake 
increases extracellular acetylcholine levels in the ventral tegmental area in the rat. Alcohol 
Alcohol 40, 349-58, 2005. PMID: 16043436. 


54 
 
119. Larsson A, Jerlhag E, Svensson L, Söderpalm B, Engel JA. Is an alpha-conotoxin MII 
sensitive mechanism involved in the neurochemical, stimulatory and rewarding effects of 
ethanol. Alcohol 34, 239-46, 2004. PMID: 15902919. 
120. Schneider ER, Rada P, Darby RD, Leibowitz SF, Hoebel BG. Orexigenic peptides and 
alcohol intake: differential effects of orexin, galanin, and ghrelin. Alcohol Clin Exp Res 31, 
1858-65, 2007. PMID: 17850217. 
121. Jerlhag E. Systemic administration of ghrelin induces conditioned place preference and 
stimulates accumbal dopamine. Addict Biol 13: 358-63, 2008. PMID: 18358991. 
122. Jerlhag E, Egecioglu E, Landgren S, Salomé N, Heilig M, Moechars D, Datta R, Perrissoud 
D, Dickson SL, Engel JA. Requirement of central ghrelin signaling for alcohol reward. Proc 
Natl Acad Sci U S A. 106:11318-23. 2009. PMID: 19564604. 
123. Calissendorff J, Danielsson O, Brismar K, Röjdmark S. Inhibitory effect of alcohol on ghrelin 
secretion in normal man. Eur J Endocrinol 152: 743-7, 2005. PMID: 15879360. 
124. Calissendorff J, Danielsson O, Brismar K, Röjdmark S. Alcohol ingestion does not affect 
serum levels of peptide YY but decreases both total and octanoylated ghrelin levels in healthy 
subjects. Metabolism 55: 1625-9, 2006. PMID: 17142135. 
125. Zimmermann US, Buchmann A, Steffin B, Dieterle C, Uhr M. Alcohol administration acutely 
inhibits ghrelin secretion in an experiment involving psychosocial stress. Addict Biol 12: 17-
21, 2007. PMID: 17407493. 
126. Badaoui A, De Saeger C, Duchemin J, Gihousse D, de Timary P, Stärkel P. Alcohol 
dependence is associated with reduced plasma and fundic ghrelin levels. Eur J Clin Invest 38: 
397-403, 2008. PMID: 18422979. 
127. Kim DJ, Yoon SJ, Choi B, Kim TS, Woo YS, Kim W, Myrick H, Peterson BS, Choi YB, Kim 
YK, Jeong J. Increased fasting plasma ghrelin levels during alcohol abstinence. Alcohol 
Alcohol 40: 76-9, 2005. PMID: 15520048. 
128. Kraus T, Schanze A, Gröschl M, Bayerlein K, Hillemacher T, Reulbach U, Kornhuber J, 
Bleich S. Ghrelin levels are increased in alcoholism. Alcohol Clin Exp Res 29: 2154-7, 2005. 
PMID: 16385185. 
129. Cassin SE, von Ranson KM. Personality and eating disorders: a decade in review. Clin 
Psychol Rev 25:895-916, 2005. PMID: 16099563. 
130. Hillemacher T, Kraus T, Rauh J, Weiss J, Schanze A, Frieling H, Wilhelm J, Heberlein A, 
Gröschl M, Sperling W, Kornhuber J, Bleich S. Role of appetite-regulating peptides in 
alcohol craving: an analysis in respect to subtypes and different consumption patterns in 
alcoholism. Alcohol Clin Exp Res 31:950-4. 2007. PMID: 17433008. 
131. Wurst FM, Graf I, Ehrenthal HD, Klein S, Backhaus J, Blank S, Graf M, Pridzun L, Wiesbeck 
GA, Junghanns K. Gender differences for ghrelin levels in alcohol-dependent patients and 
differences between alcoholics and healthy controls. Alcohol Clin Exp Res 31:2006-11. 2007. 
PMID: 17949465. 
132. Leggio L, Ferrulli A, Cardone S, Nesci A, Miceli A, Malandrino N, Capristo E,  
Canestrelli B, Monteleone P, Kenna GA, Swift RM, Addolorato G. Ghrelin system in  
alcohol-dependent subjects: role of plasma ghrelin levels in alcohol drinking and 
craving. Addict Biol. 2012 Mar;17(2):452-64. 
133. Davis JF, Schurdak JD, Magrisso IJ, Mul JD, Grayson BE, Pfluger PT, Tschoep MH, Seeley 
RJ, Benoit SC. Gastric Bypass Surgery Attenuates Ethanol Consumption in Ethanol-
Preferring Rats. Biol Psychiatry. 2012 Mar 21. PMID: 22444202. 
134. Landgren S, Jerlhag E, Zetterberg H, Gonzalez-Quintela A, Campos J, Olofsson 


55 
 
U, Nilsson S, Blennow K, Engel JA. Association of pro-ghrelin and GHS-R1A gene 
polymorphisms and haplotypes with heavy alcohol use and body mass. Alcohol Clin 
Exp Res. 2008 Dec;32(12):2054-61. PMID: 18828808. 
135. Rohsenow DJ, Monti PM, Rubonis AV, Sirota AD, Niaura RS, Colby SM, Wunschel 
SM, Abrams DB. Cue reactivity as a predictor of drinking among male alcoholics. J 
Consult Clin Psychol. 1994 Jun;62(3):620-6. PMID: 8063989 
136. Monti PM, Rohsenow DJ, Hutchison KE, Swift RM, Mueller TI, Colby SM, Brown RA, 
Gulliver SB, Gordon A, Abrams DB. Naltrexone's effect on cue-elicited craving 
among alcoholics in treatment. Alcohol Clin Exp Res. 1999 Aug;23(8):1386-94. 
PMID: 10470982. 
137. Monti PM, Rohsenow DJ, Hutchison KE. Toward bridging the gap between biological, 
psychobiological and psychosocial models of alcohol craving. Addiction. 2000 Aug;95 Suppl 
2:S229-36. Review. PubMed PMID: 11002917. 
138. Ramchandani VA, Bolane J, Li TK, O'Connor S. A physiologically-based pharmacokinetic 
(PBPK) model for alcohol facilitates rapid BrAC clamping. Alcohol Clin Exp Res. 
1999;23:617-23. 
139. Plawecki MH, DeCarlo RA, Ramchandani VA, O'Connor S. Estimation of ethanol infusion 
profile to produce specified BrAC time course using physiologically-based pharmacokinetic 
(PBPK) models. Conf Proc IEEE Eng Med Biol Soc. 2004;1:778-81. 
140. Knutson B, Adams CM, Fong GW, Hommer D. Anticipation of increasing monetary reward 
selectively recruits nucleus accumbens. J Neurosci. 2001;21. 
141. Bjork JM, Knutson B, Fong GW, Caggiano DM, Bennett SM, Hommer DW. Incentive-
elicited brain activation in adolescents: similarities and differences from young adults. J 
Neurosci. 2004;24. 
142. Malik S, McGlone F, Bedrossian D, Dagher A. Ghrelin modulates brain activity in areas that 
control appetitive behavior. Cell Metab. 2008;7:400-9. 
143. Hommer DW. Functional imaging of craving. Alcohol Res Health 1999;23:187-96. 
144. Gilman JM, Ramchandani VA, Davis MB, Bjork JM, Hommer DW. Why we like to drink: a 
functional magnetic resonance imaging study of the rewarding and anxiolytic effects of 
alcohol. J Neurosci. 2008;28:4583-91. 
145. Yokoyama, A., Muramatsu, T., Ohmori, T., Kumagi, Y., Higuchi, S., and Ishii, H. Reliability 
of a flushing questionnaire and the ethanol patch text in screening for inactive aldehyde 
dehydrogenase-2 and alcohol-related cancer risk. Cancer Epidemiol Biomarkers Prev 6(12), 
1105-7. 1997. Ref Type: Generic 
146. Johnson RC, Nagoshi CT, Schwitters SY, Bowman KS, Ahern FM, Wilson JR. Further 
investigation of racial/ethnic differences and of familial resemblances in flushing in response 
to alcohol. Behav Genet. 1984;14:171-78. 
147. Richardson NR, Roberts DC. Progressive ratio schedules in drug self-administration studies in 
rats: a method to evaluate reinforcing efficacy. J Neurosci Methods 1996;66:1-11. 
148. de Wit H, McCracken SG. Ethanol self-administration in males with and without an alcoholic 
first-degree relative. Alcohol Clin Exp Res. 1990;14:63-70. 
149. Bohn MJ, Krahn DD, Staehler BA. Development and initial validation of a measure of 
drinking urges in abstinent alcoholics. Alcohol Clin Exp Res. 1995;19:600-606. 
150. Martin CS, Earleywine M, Musty RE, Perrine MW, Swift RM. Development and validation 
of the Biphasic Alcohol Effects Scale. Alcohol Clin Exp Res. 1993;17:140-146. 


56 
 
151. McNair, D., Lorr, M., Dropplemen, L. Edits manual: Profile of mood states. San Diego: 
Educational and Industrial Testing Services, 1971. 
152. Kampov-Polevoy AB, Garbutt JC, Janowsky DS. Association between preference for sweets 
and excessive alcohol intake: a review of animal and human studies. Alcohol Alcohol 34: 
386-95, 1999. PMID: 10414615. 
153. Garbutt JC, Osborne M, Gallop R, Barkenbus J, Grace K, Cody M, Flannery B, Kampov-
Polevoy AB. Sweet liking phenotype, alcohol craving and response to naltrexone treatment in 
alcohol dependence. Alcohol Alcohol. 2009;44:293-300 
154. Bollinger, J., Acker, J., & MacKillop, J. (2012). An Initial Validation of the Brief Behavior 
Social Density Assessment (BASDA) in an Undergraduate Sample. Poster presented at the 
35th Annual Convention of the Behavioral Sciences, Athens, GA, March 2012 
155. Carver, CS, & White, TL. Behavioral inhibition, behavioral activation, and affective 
responses to impending reward and punishment: The BIS/BAS scales. Journal of Personality 
and Social Psychology. 1994;67:319-333. 
156. Johnson, SL, Turner RJ, & Iwata, N. BIS/BAS Levels and Psychiatric Disorder: An 
Epidemiological Study. Journal of Psychopathology and Behavioral Assessment. 2003;25:25-
26 
157. Franken, IHA. Behavioral approach system (BAS) sensitivity predicts alcohol craving. 
Personality and Individual Differences. 2002; 32:349-355. 
158. Blekher T, Ramchandani VA, Flury L, Foroud T, Kareken D, Yee RD et al. Saccadic eye 
movements are associated with a family history of alcoholism at baseline and after exposure 
to alcohol. Alcohol Clin Exp Res. 2002;26:1568-73. 
159. Gilman JM, Ramchandani VA, Davis MB, Bjork JM, Hommer DW. Why we like to drink: a 
functional magnetic resonance imaging study of the rewarding and anxiolytic effects of 
alcohol. J Neurosci. 2008;28:4583-91. 
160. Kwo PY, Ramchandani VA, O'Connor S, Amann D, Carr LG, Sandrasegaran K et al. Gender 
differences in alcohol metabolism: relationship to liver volume and effect of adjusting for 
body mass. Gastroenterology. 1998;115:1552-57. 
161. Morzorati SL, Ramchandani VA, Flury L, Li TK, O'Connor S. Self-reported subjective 
perception of intoxication reflects family history of alcoholism when breath alcohol levels are 
constant. Alcohol Clin Exp Res. 2002;26:1299-306. 
162. Ramchandani VA, O'Connor S, Blekher T, Kareken D, Morzorati S, Nurnberger J, Jr. et al. A 
preliminary study of acute responses to clamped alcohol concentration and family history of 
alcoholism. Alcohol Clin Exp Res. 1999;23:1320-1330. 
163. Ramchandani VA, Kwo PY, Li TK. Effect of food and food composition on alcohol 
elimination rates in healthy men and women. J Clin Pharmacol. 2001;41:1345-50. 
164. Ramchandani VA, Flury L, Morzorati SL, Kareken D, Blekher T, Foroud T et al. Recent 
drinking history: association with family history of alcoholism and the acute response to 
alcohol during a 60 mg% clamp. J Stud Alcohol. 2002;63:734-44. 
165. Ramchandani VA, O'Connor S, Neumark Y, Zimmermann US, Morzorati SL, de Wit H. The 
alcohol clamp: applications, challenges, and new directions--an RSA 2004 symposium 
summary. Alcohol Clin Exp Res. 2006;30:155-64. 
166. NIAAA Guidelines: http://www.niaaa.nih.gov/research/guidelines-and-
resources/administering-alcohol-human-studies 
167. Cancer Therapy Evaluation Program. Common Terminology Criteria for Adverse Events v3.0 
(CTCAE).  8-9-2007. DCTD, NCI, NIH, DHHS.