University of Southern California Dissertations and Theses

Development of ivermectin as a platform for the treatment and/or prevention of alcohol use disorders

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Development of ivermectin as a platform for the treatment and/or prevention of alcohol use disorders

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Content i

DEVELOPMENT OF IVERMECTIN AS A PLATFORM FOR THE TREATMENT
AND/OR PREVENTION OF ALCOHOL USE DISORDERS

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY & TOXICOLOGY)

July 2014

Copyright 2014 Megan M. Yardley

ii

EPIGRAPH

“The important thing in science is not so much to obtain new facts as to discover new ways of
thinking about them.”

William Bragg

iii

DEDICATION

To
Ron Alkana
An incredible man and inspiring mentor
For your endless dedication and commitment to students

iv

ACKNOWLEDGEMENTS
Personal

Deepest gratitude to…

My parents, Rita and Shawn, for the love and support you continue to show me every day

My boyfriend, Ryan, for being a constant source of laughs, love and encouragement throughout
this endeavor

My co-advisor, Ron, for your guidance and support that have been instrumental in both my
scientific career and character development

My co-advisor, Daryl, for the invaluable opportunities you have provided me and the time and
effort you put into preparing me for the next steps

My lab members and friends who have made my time at USC an unforgettable one

Statement of contributions to works contained in this dissertation

This dissertation is composed of the author’s original work and contains no material previously
published or written by any other individual except where due reference is made. All data
contained herein were collected and analyzed by M.M. Yardley. The authorship on published
manuscripts is described in greater detail on the following page. Drs. Alkana and Davies
provided discussion and revisions to the manuscript.

v

AUTHORSHIPS
Published works by the author incorporated into the dissertation
Yardley MM, Wyatt L, Khoja S, Asatryan L, Ramaker MJ, Finn DA, Alkana RL, Huynh N,
Louie SG, Petasis NA, Bortolato M, Davies DL (2012). Ivermectin reduces alcohol intake and
preference in mice. Neuropharmacology 63(2): 190-201.

Bortolato M, Yardley M, Khoja S, Godar SC, Asatryan L, Finn DA, Alkana RL, Louie SG,
Davies DL (2013). Pharmacological insights into the role of P2X4 receptors in behavioral
regulation: Lessons from ivermectin. International Journal of Neuropsychopharmacology 16(5):
1059-1070.

Asatryan L, Yardley MM, Khoja S, Trudell JR, Huynh N, Louie SG, Petasis NA, Alkana RL,
Davies DL (2014). Avermectins differentially affect ethanol intake and receptor function:
Implications for developing new therapeutics for alcohol use disorders. International Journal of
Neuropsychopharmacology 17(6): 907-16.

Yardley MM, Neely M, Huynh N, Asatryan L, Louie SG, Alkana RL, Davies DL. Multi-day
administration of ivermectin is effective in reducing alcohol intake in mice at doses shown to be
safe in humans. NeuroReport; in press PMID: 25004078.

Additional works by the author relevant to this dissertation but not forming part of it
Wyatt LR, Finn DA, Khoja S, Yardley MM, Asatryan L, Alkana RL, Davies DL (2014).
Contribution of P2X4 receptors to ethanol intake in male C57BL/6 mice. Neurochemical
Research 39(6): 1127-39.

Research Support
SC CTSI NIH/NCRR/NCATS --TL1TR000132 (M.M.Y.) and UL1TR000130 (D.L.D.),
American Foundation for Pharmaceutical Education (M.M.Y.), NIAAA/NIH K01 AA022448
(D.L.D.), NIAAA/NIH R01 AA022448 (D.L.D.) and the USC School of Pharmacy.

vi

TABLE OF CONTENTS
Epigraph ii
Dedication iii

Acknowledgements iv

List of Tables ix

List of Figures x

Abbreviations xii

Abstract xv

Chapter 1 Introduction 1
Significance 1
Drug Development Pathway Overview 3
Current Treatments for Alcohol Use Disorders 5
Pharmacokinetics of Ethanol 8
Targets of Ethanol Action 9
P2XRs and Their Role in the Effects of Ethanol 10
Support for Involvement of P2X4Rs in Ethanol Action 11
Ivermectin 12
Evidence for Antagonistic Effects of IVM in P2X4Rs in vitro 14
Conclusion: Dissertation Hypothesis and Outline 15

Chapter 2 Ivermectin reduces alcohol intake and preference in mice 18
Abstract 18
Introduction 18
Materials and Methods 21
Results 29
Single dose administration of IVM (10 mg/kg) decreased 10E 29
intake and preference
IVM decreased 10E intake and preference in a dose-dependent 30
manner in male mice
Time-course of IVM’s effects on alcohol intake in male mice 32
Pharmacokinetics of IVM in plasma and brain 33
IVM administration reduced alcohol intake and preference in 34
female mice
Multiple dosing of IVM administration reduced alcohol intake 37
and preference in female mice
IVM reduced saccharin consumption in female mice 38
IVM decrease alcohol intake by 50% in an intermittent limited 39
access paradigm in female mice
vii

Significant reduction in ethanol but not sucrose operant self- 40
administration following IVM in male mice
Discussion 43
Conclusion 49

Chapter 3 Pharmacological insights into the role of P2X4 receptors in 51
behavioral regulation: Lessons from ivermectin
Abstract 51
Introduction 52
Materials and Methods 54
Results 59
Sensorimotor functions, tactile sensitivity and thermal nociception 59
Novel open field 60
Elevated plus maze 60
Marble burying 61
Tail suspension 62
Forced swim test 63
Conditioned place preference 63
Prepulse inhibition (PPI) of the acoustic startle 63
Novel object exploration/recognition 64
Behavioral effects of IVM in P2X4 KO mice 65
Discussion 66

Chapter 4 Multi-day administration of ivermectin is effective in reducing 71
alcohol intake in mice at doses shown to be safe in humans
Abstract 71
Introduction 72
Materials and Methods 74
Results 77
Multi-day IVM administration significantly reduced alcohol 77
intake and preference in male mice
Pharmacokinetics of IVM 79
Discussion 80
Conclusion 82

Chapter 5 Avermectins differentially affect ethanol intake and receptor 83
function: Implications for developing new therapeutics for alcohol use
disorders
Abstract 83
Introduction 84
Materials and Methods 86
Results 92
IVM and ABM, but not SEL, reduced alcohol intake and 92
preference in C57BL/6J mice
Pharmacokinetics of IVM, ABM, and SEL 93
Effect of avermectin analogs on P2X4R and GABA
A
R function 94
viii

IVM and ABM, but not SEL, antagonize the effects of ethanol 96
on P2X4Rs
Discussion 98

Chapter 6 Overall Discussion and Conclusions 103
Summary of Overall Findings 103
Future Directions 105

Bibliography 108

ix

LIST OF TABLES
Table 1.1 Diagnostic criteria for AUDs. 1

Table 2.1 Three doses of IVM were evaluated to determine the IVM
disposition in both plasma and brain tissue.
34

Table 4.1 IVM disposition in the brain was analyzed at 7 time points
following the 10 days of injections: 0 (just prior to day 10
injections), 0.5, 2, 8, 32, 48 and 72.
79

Table 5.1 Pharmacokinetic parameters of avermectins. 94

x

LIST OF FIGURES
Figure 1.1 Drug development process. 4

Figure 1.2 CDER’s 10-year historic comparison on approvals of new
molecular entities (NMEs).
5

Figure 2.1 IVM (10 mg/kg) reduces A) 10% v/v ethanol (10E) intake and
B) preference ratio for 10E in male C57BL/6J mice using a 24-h
access two-bottle choice paradigm.
30

Figure 2.2 IVM dose response study in male C57BL/6J mice using a 24-h
access two-bottle choice paradigm.
32

Figure 2.3 IVM (10 mg/kg) administered to male C57BL/6J mice significantly
reduced 10E intake approximately 9 hours after IVM administration.
33

Figure 2.4 IVM AUC in plasma and brain tissue was determined following
injection of various IVM dose groups.
34

Figure 2.5 IVM dose response study in female C57BL/6J mice using a 24-h
access two-bottle choice paradigm.
36

Figure 2.6 Daily administration of IVM (1.25 mg/kg/day X 7 days) reduced
10E intake in female C57BL/6J mice using a 24-h access two-bottle
choice paradigm.
38

Figure 2.7 IVM administration reduced saccharin (0.033% w/v) intake in
female C57BL/6J mice using a 24-h access two-bottle choice
paradigm following doses of A) 2.5 mg/kg and B) 5 mg/kg.
39

Figure 2.8 10 mg/kg IVM administration reduced ethanol (10% v/v) intake in
female C57BL/6 mice using an intermittent, limited (4-h) access
paradigm.
40

Figure 2.9 Effect of IVM on operant self-administration of 10% ethanol (10E)
in male C57BL/6J mice during 60 minute sessions.
42

Figure 3.1

Effects of ivermectin (IVM) and its vehicle (VEH) on haptic
stimulation and sensorimotor coordination.

59

Figure 3.2 Ivermectin (IVM) administration increases thigmotactic behavior in
the open field.
60

Figure 3.3 Behavioral effects of ivermectin (IVM) on the elevated plus-maze. 61

Figure 3.4 Ivermectin (IVM) treatment significantly reduces marble-burying 62
xi

activity.

Figure 3.5 Ivermectin (IVM) increases depressive-related behaviors. 62

Figure 3.6 Ivermectin (IVM) treatment disrupts sensorimotor gating. 63

Figure 3.7 Ivermectin (IVM) administration had no effect on mnemonic
parameters in the object interaction and recognition test.
64

Figure 3.8 Behavioral effects of ivermectin (IVM, 10 mg/kg, i.p.)
administration in P2X4 receptor knockout mice.
65

Figure 4.1 IVM significantly reduces 10E intake in C57BL/6J mice. 78

Figure 4.2 IVM accumulates in the CNS in a time dependent manner. 80

Figure 5.1 Structures of IVM, ABM and SEL. 87

Figure 5.2

Figure 5.3

Figure 5.4
IVM, SEL, ABM (5 mg/kg) administration in male C57BL/6J
mice using a 24-h access two-bottle choice paradigm.

IVM, ABM and SEL modulation of P2X4R and GABA
A
R activity.

Effects of IVM, ABM and SEL on ethanol inhibition of P2X4R
function.
93

95

97

xii

ABBREVIATIONS
5HT
3
R, 5- hydroxytryptamine type 3 receptor

ABM, abamectin

AD, alcohol dependent

AMPAR, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors

ANOVA, analysis of variance

Asp, aspartic acid

ATP, adenosine 5’-triphosphate

AUC, area under the curve

AUD, alcohol use disorder

AUDIT, alcohol use disorder identification test

BBB, blood brain barrier

BEC, blood ethanol concentration

CNS, central nervous system

C
max
, maximum concentration

CPP, conditioned place preference test

CR, cue reactivity

DA, dopamine

DSM-V, Diagnostic and Statistical Manual of Mental Disorders, fifth edition

EC, effective concentration

FAS, fetal alcohol syndrome

FDA, Food and Drug Administration

GABA
A
R, γ-aminobutyric acid type-A receptor

xiii

GABA
B
, γ-aminobutyric acid type-B receptor

HAD, high alcohol drinking

HEK, human embryonic kidney

IND, investigational new drug

IV, intravenous

IVM, ivermectin

LAD, low alcohol drinking

LC-MS, liquid chromatography/ mass spectrometry

LD
50
, lethal dose, 50%

LGIC, ligand-gated ion channel

Met, methionine

NAc, nucleus accumbens

nAChRs, nicotinic acetylcholine receptors

NIAAA, National Institution of Alcohol Abuse and Alcoholism

NMDAR, N-methyl D-aspartate receptor

NME, new molecular entity

P2XR, P2X receptors

Pgp or P-gp, p-glycoprotein

Phe, phenylalanine

PPI, prepulse inhibition

RNA, ribonucleic acid

SAE, severe adverse event

SEL, selamectin

xiv

SGOT, serum glutamyl phosphate oxaloacetate transferase

TM, transmembrane

T
max
, time to achieve maximal concentration

Trp, tryptophan

VTA, ventral tegmental area

WT, wild type

xv

ABSTRACT
Alcohol use disorders (AUDs) affect over 18 million people in the United States alone,
cost over $235 billion, and yet only 8% of this population receives treatment and even less use a
medication approved by the U.S. Food and Drug Administration (FDA) as part of that treatment.
Despite considerable efforts focusing on new drug development to reduce ethanol abuse, high
rates of harmful drinking persist. This is, in part, due to the fact that current therapeutic strategies
are largely inadequate to treat these disorders. Thus, developing novel therapeutics for the
treatment of AUDs is of paramount importance. The working hypothesis of our laboratory is that
ivermectin (IVM) can be repurposed as a therapeutic agent for the treatment of AUDs. As IVM
is currently FDA-approved and used by millions of humans each year for other indications, the
repurposing of IVM for the treatment of AUDs represents a fast and economically feasible
approach for drug development. Initial support suggesting that IVM can be developed as a novel
drug candidate for the treatment of AUDs comes from previous work demonstrating that IVM is
able to antagonize the effect of ethanol in vitro on P2X4 receptors (PRX4Rs). Studies included in
this dissertation test the hypothesis that IVM can be repurposed as a therapeutic agent for the
treatment of AUDs using multiple preclinical mouse models of ethanol intake and behavior.
Chapter 2 describes initial efficacy studies using 3 distinct models of ethanol intake and explores
the pharmacokinetics (PK) of IVM. Chapter 3 characterizes the intrinsic properties of IVM using
a battery of behavioral paradigms to test for effects such as depression, anxiety, locomotion,
memory, and rewarding properties. Chapter 4 evaluates the sustainability and safety of multi-day
IVM administration. Finally, Chapter 5 focuses on the use of IVM as a platform for developing
novel therapeutics for AUDs by testing two related avermectins, selamectin (SEL) and
abamectin (ABM). Findings from my work support the hypothesis that IVM is able to reduce
ethanol intake using multiple murine models of ethanol intake without causing overt toxicity.
xvi

Overall, the studies presented within this dissertation set the stage for first-in-human testing of
IVM for this new indication.

1

CHAPTER 1
INTRODUCTION
Significance
Alcohol use disorders (AUDs) are defined in the American Psychiatric Association’s
Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-V) as “a problematic
pattern of alcohol use leading to clinically significant impairment or distress, as manifested by at
least two of the following, occurring within a 12-month period.” The diagnostic criteria for
AUDs are listed in Table 1.1 (Association, 2013).
Table 1.1
1. Alcohol is often taken in larger amounts or over a longer period than was intended.
2. There is a persistent desire or unsuccessful efforts to cut down or control alcohol use.
3. A great deal of time is spent in activities necessary to obtain alcohol, use alcohol, or recover from its
effects.
4. Craving, or a strong desire or urge to use alcohol.
5. Recurrent alcohol use resulting in a failure to fulfill major role obligations at work, school, or home.
6. Continued alcohol use despite having persistent or recurrent social or interpersonal problems caused or
exacerbated by the effects of alcohol.
7. Important social, occupational, or recreational activities are given up or reduced because of alcohol use.
8. Recurrent alcohol use in situations in which it is physically hazardous.
9. Alcohol use is continued despite knowledge of having a persistent or recurrent physical or psychological
problem that is likely to be have been caused or exacerbated by alcohol.
10. Tolerance, as defined by either of the following:
a. A need for markedly increase amount of alcohol to achieve intoxication or desired effects.
b. A markedly diminished effect with continued use of the same amount of alcohol.
11. Withdrawal, as manifested by either of the following:
a. The characteristic withdrawal syndrome for alcohol (refer to Criteria A and B of the criteria set
for alcohol withdrawal).
b. Alcohol (or closely related substance, such as a benzodiazepine) is taken to relieve or avoid
withdrawal symptoms.##
Table 1. Diagnostic criteria for AUDs.

AUDs have a major national impact in the United States affecting nearly 18 million
people and causing over 100,000 deaths annually (Bouchery et al., 2011; Grant et al., 2004;
Harwood, 2000). Alcoholism accounts for nearly half of the annual total cost of untreated
addiction totaling over $185 billion annually (Alcoholism, 2011). The majority of this cost is due
2

to reduced, lost, and forgone earnings. The remainder of the cost comes from medical and
treatment expenses, workforce productivity, accidents, violence, and premature death. However,
AUDs are not only a national problem; in fact, ethanol, often used interchangeably with alcohol
in popular culture, is the most commonly abused drug in the world (Wallace and Newton, 2012).
Worldwide, ethanol abuse and misuse is the third leading risk factor for premature death and
disabilities and is responsible for 4% of all deaths (2011). This is partly because ethanol is a
causal factor in 60 different diseases including cirrhosis of the liver, neuropsychiatric disorders,
gastrointestinal diseases, cancer, cardiovascular diseases, diabetes, and fetal alcohol syndrome
(FAS). Furthermore, ethanol is a component cause in over 200 additional diseases. Specifically,
heavy drinking, defined as 5 or more drinks per day at least once a week for males and 4 or more
drinks for females, is believed to almost double the incidence of high blood pressure making it
the second most common risk factor behind obesity in men (Alcoholism, 2011). Heavy drinking
also results in an increased risk for certain cancers including esophageal, mouth, and pharynx.
Overall, ethanol plays a direct or indirect role in 2-4% of all cancer cases. Ethanol is the leading
cause of birth defects with 1 in 100 babies being born with FAS as a direct result of the 12% of
women that continue to consume ethanol during pregnancy.
In addition to the harm ethanol causes the user in the form of lost productivity, lost
earnings, and disease, heavy ethanol consumption can also be harmful to others as it greatly
increases the occurrence of domestic violence, crimes, and automobile accidents. In 2008, there
was one ethanol-impaired driving fatality every 45 minutes in the United States, equaling 11,773
deaths resulting from an ethanol-related automobile accident (Alcoholism, 2011). In that same
year, 159 million people reported driving under the influence, yet only 0.88% of those drivers
were actually arrested for ethanol-impaired driving. A study conducted in the United Kingdom
3

assessed the relative harm both to self and to others of 20 drugs including ethanol, tobacco,
heroin, crack cocaine, and methamphetamine (Nutt et al., 2010). The assessment was based on
16 criteria: drug-specific mortality, drug-related mortality, drug-specific damage, drug-related
damage, dependence, drug-specific impairment of mental functioning, drug-related impairment
of mental functioning, loss of tangibles, loss of relationships, injury, crime, environmental
damage, family adversities, international damage, economic cost and community. Interestingly,
although heroin, crack cocaine and methamphetamine had a higher score for harm to users,
ethanol had a much higher score for harm to others. Taken together, ethanol was found to be the
more harmful drug with an overall harm score of 72 followed by heroin with a score of 55.
Despite the known harm caused by ethanol, even with currently available pharmacological-
psychological strategies, rates of uncontrolled heavy drinking remain high. The lack of effective
therapies is highlighted by the urgent call from the National Institute on Alcohol Abuse and
Alcoholism (NIAAA) to bring in new therapeutics to either prevent or treat ethanol-related
problems. As such, development of effective treatments for AUDs represents an important public
health goal (Bouchery et al., 2011; Heilig and Egli, 2006; Johnson, 2010; Johnson et al., 2007;
Steensland et al., 2007).

Drug Development Pathway Overview
It takes approximately 12.5 years and can cost over $1 billion to develop a drug and
successfully bring it to market (Baines, 2004). This is partly due to the fact that about 70% of
drugs do not make it past the preclinical phase. The conventional drug discovery process
includes 6 main stages: drug discovery, molecule discovery, preclinical testing, and 3 phases of
clinical trials (Fig. 1.1) (Baines, 2004). These steps are necessary to ensure that the drugs are safe
4

and effective prior to marketing as required by the Federal Food, Drug and Cosmetic Act of 1938
and the Kefauver-Harris Drug Amendments passed in 1962, respectively. (Moore, 2003)
Figure 1.1

Figure 1.1. Drug development process.

Following preclinical testing and prior to phase I clinical trials, it is necessary to
complete an investigational new drug application (IND) (1998). The purpose of the IND is to
demonstrate to the Food and Drug Administration (FDA) that the drug of interest is safe for use
in small-scale clinical studies. Specifically, the IND is comprised of 3 main sections: 1) animal
pharmacology and toxicology studies; 2) manufacturing information; and 3) clinical protocols
and investigator information. The animal pharmacology and toxicology studies normally range
from 2 weeks to 3 months and are carried out in at least one rodent species and one non-rodent
species. These studies are crucial in elucidating the pharmacological profile of the drug with
regards to absorption, distribution, metabolism, and excretion. The manufacturing section of an
IND focuses on the scale-up. Prior to initiating human studies, it is important to validate the
manufacturing process to ensure that the compound is consistent from one batch to the next.
Figure 1
Cartoon of the conventional
drug discovery process
Figure 2
Illustration of method for
adding distributions of
durations of each stage of a
process to obtain the duration
distribution for the whole
process. Shows are simplified
distributions for two stages,
and a cumulative distribution
of duration for both
10 Drug Discovery World Fall 2004
Business
Probability of success of each stage
Cost, time for each stage
Disease
discovery
• Target discovery
• genomics, proteomics
• systems biology
• Target validation
• knock-out technologies
• chemogenomics
• Disease models
• T oxicity/metabolism
models
• metabonomics
• Formal preclinical
• ‘Proof of principal in
man’
• patients,
• clinical endpoints
• X-ray SBDD
• Combichem/virtual chemistry
• HTS, high-content screening
• Validation assays
• In vitro and in silico ADME
• Volunteer studies
• PK in man
• initial efficacy
surrogate endpoints
• human MTD
estimates
• Dose validation
• Medical economics
• Formulation
• Marketing material
Molecule
discovery
Preclinical
testing
Phase I
clinical
Phase II
clinical
Phase III
clinical
If Phase I
takes 1 year
Phase I + II
time %
2 12
3 20
48
Overall
Phase I + II
time %
2 12
3 48
4 38
5 12
If Phase I
takes 2 years
Phase I + II
time %
3 18
4 30
5 12
Phase II
time %
1 30
2 50
3 20
Phase I
time %
1 40
2 60
Distribution of duration for each phase
5

Furthermore, it is necessary to demonstrate that the product is stable. The clinical protocols are
included to confirm the clinical study will not contain unnecessary risk to the participants and the
proposed design is appropriate to measure outcomes related to the safety and efficacy of the
drug. However, only 1 out of every 5,000-10,000 compounds screened is granted approval from
the FDA (Moore, 2003). For example, in 2011, 30 new molecular entities (NMEs) were
approved (Fig. 1.2) (2012). This is a direct result of the risk associated with drug development.
Figure 1.2

Figure 1.2. CDER’s 10-year historic comparison on approvals of new molecular entities (NMEs).

Current Treatments for Alcohol Use Disorders
Although treatments for AUDs have improved in past decades (Miller et al., 2001), there
is still a great need to develop more effective interventions. Pharmacotherapies for AUDs are
used less often than psychosocial interventions (Fuller and Hiller-Sturmhofel, 1999). However,
without a pharmacological adjunct to psychosocial therapy, nearly three quarters of patients
resume drinking within 1 year (Johnson, 2008). The limited use of pharmacotherapy for AUDs is
6

due, in part, to the relative lack of pharmacological options to successfully treat these disorders
(Edlund et al., 2012). As few as 1 in 10 people that have an AUD actually perceive a need for
treatment. Possible reasons for this low number include: they don’t acknowledge they have an
AUD; they believe available treatment options are ineffective; they don’t perceive a need for
treatment to recover; they don’t want to stop drinking; or they may be receiving treatment for
other co-morbid disorders and, therefore, are not receiving treatment specifically for the AUD.
The drugs currently approved for AUD management are believed to deter ethanol intake by
blocking its metabolism or by targeting the neurochemical systems in the downstream cascades
leading to craving and dependence (Colombo et al., 2007; Gewiss et al., 1991; Johnson et al.,
2007; Steensland et al., 2007). Presently there are three FDA-approved oral medications and one
FDA-approved injectable medication to treat alcohol dependence (AD): disulfiram (1949);
naltrexone (1994 oral; 2006 injectable); and acamprosate (2004) (Johnson et al., 2007).
Disulfiram (Antabuse) blocks the enzyme acetaldehyde dehydrogenase, preventing
formation of acetic acid from acetaldehyde, a metabolite of ethanol (Heilig and Egli, 2006). By
blocking this step in the normal metabolism of ethanol, there is an increase in the concentration
of acetaldehyde, leading to violent nausea and vomiting and potentially to more severe adverse
reactions, including death if the patient is unable to resist consuming ethanol. A 52-week
multisite randomized controlled trial (n=605) in AD men found that disulfiram is more effective
in preventing relapse in compliant patients but would not necessarily be successful in promoting
continued abstinence.
Naltrexone (oral- Revia; injectable- Vivitrol and Naltrel) blocks opioid receptors that
play a role in the rewarding effects of ethanol. It is thought to act by decreasing the craving for
ethanol. Support for this comes from preclinical studies that demonstrate mice lacking the mu
7

opioid receptor do not self-administer ethanol (Johnson, 2008). Similarly, mu opioid antagonists
can decrease ethanol consumption across multiple paradigms. However, clinical studies have had
mixed results, likely because naltrexone seems to be effective in only a small subtype of
alcoholics, specifically, those with a family history of AUDs. High cost and sedative properties
have also limited the usefulness of naltrexone.
Acamprosate (Campral) is believed to act by decreasing glutamate activity, ultimately
lessening the negative effects associated with ethanol withdrawal. Support for this theory comes
from a study involving transgenic mice with increased glutamate activity in the brain region (De
Witte et al., 2005). Data from this study suggest that the transgenic mice drink significantly more
compared to wild type (WT). Moreover, acamprosate was able to significantly decrease ethanol
intake in the transgenic mice but had no effect on ethanol intake in WT mice, suggesting that this
drug only acts on hyper glutamatergic and not normal glutamatergic states. There is little clinical
data to support the use of acamprosate for the treatment of AUDs. A multisite COMBINE study
in 2006 showed no difference between acamprosate and placebo on any drinking outcome
measure (Johnson, 2008). Although data from U.S. studies have been inconclusive, FDA
approval was based on results from European clinical trials (Johnson et al., 2007). Furthermore,
large and frequent dosing requirements make adherence to this therapy difficult for patients.
In addition to the approved medications for AUDs, there are drugs that are approved for
other indications currently in clinical trials to be repurposed as novel treatments for AUDs.
Ondansetron, baclofen, and topiramate are considered to be the second wave of potential
therapeutics (Heilig and Egli, 2006). Ondansetron is a 5-hydroxytryptamine type 3 (5HT
3
)
antagonist approved as an antiemetic to treat nausea and vomiting. Many of the reinforcing
effects of ethanol are mediated by 5HT
3
and dopamine (DA) interactions in the reward pathway.
8

Ondansetron is thought to act by decreasing the pleasurable effects of ethanol. Baclofen is a γ-
aminobutyric acid type-B receptor (GABA
B
R) agonist approved for the treatment of spasticity.
This drug is believed to decrease voluntary ethanol intake by altering DA metabolism and
preventing the firing of DA neurons. Topiramate is a serotonin reuptake inhibitor approved for
the treatment of epilepsy. Although the mechanism is relatively unknown, proposed targets
include sodium channels, kainate receptors and γ-aminobutyric acid type-A receptors
(GABA
A
Rs). A critical barrier to the development of new, effective medications for AUDs is the
lack of information regarding the molecular target(s) by which ethanol exerts its pharmacologic
activity.

Pharmacokinetics of Ethanol
Ethanol is a small polar molecule with the chemical formula C
2
H
5
OH (Wallace and
Newton, 2012). Ethanol is absorbed from the stomach and small intestine, therefore, the
absorption rate is different in the fed versus fasted state. In the fasted state, the maximum
concentration (C
max
) of ethanol in plasma occurs between 0.5-2 hours after consumption whereas
in the fed state, plasma C
max
occurs between 1-6 hours after consumption. The majority of
ethanol is metabolized by the enzyme alcohol dehydrogenase to acetaldehyde, which is further
metabolized by acetaldehyde dehydrogenase to acetic acid. The ethanol that is not metabolized is
excreted in the urine and lungs. Elimination of ethanol follows zero order kinetics, as the rate of
elimination is constant at approximately 10 mg dL
-1
h
-1
and not contingent on the concentration of
ethanol in the body.
Intoxication occurs because the elimination rate of ethanol is not dependent on the
plasma concentration and therefore, regardless of how much ethanol is consumed, the
9

elimination rate remains the same (Wallace and Newton, 2012). As such, accumulation of
ethanol can lead to visible signs of intoxication. For example, at a 50 mg/dL
-1
blood ethanol
concentration (BEC), non-alcoholics often experience euphoria and loss of social inhibitions
while others can display aggressive behavior. At higher BECs, ethanol begins to increasingly
affect speech, balance, posture, vision, blood pressure, and respiratory function eventually
leading to death at BECs between 300-500 mg/dL
-1
. These numbers can be significantly higher
in heavy drinkers because they have developed tolerance to ethanol.
Chronic tolerance is a reduced response to ethanol after prolonged exposure to heavy
drinking (Wallace and Newton, 2012). Intoxication in alcoholics may not even be evident at
BECs of 400 mg/dL
-1
,

which would normally be lethal in non-alcoholics. However, the opposite
can also be true. It is possible that some chronic alcoholics have a much lower tolerance than
non-alcoholics due to liver damage caused by long-term heavy drinking. Heavy drinkers
oftentimes experience withdrawal symptoms when they attempt to stop drinking. This
phenomenon occurs as a result of cellular changes that result from prolonged ethanol exposure.
Symptoms of ethanol withdrawal include tremors, convulsions, increased heart rate, increased
body temperature, circulatory collapse, and, in extreme cases, death.

Targets of Ethanol Action
Ethanol does not have a single molecular target but instead acts on a variety of different
neurotransmitter receptors, ion channels, and transporters to exert its behavioral effects (Wallace
and Newton, 2012). Ligand-gated ion channels (LGICs) are widely held to play an important role
in ethanol-induced behaviors and drinking (Cardoso et al., 1999; Davies and Alkana, 2001;
Deitrich et al., 1989; Dildy-Mayfield et al., 1996; Harris, 1999; Mihic et al., 1997; Weight et al.,
10

1992; Woodward, 2000). Research in this area has focused on investigating the effects of
ethanol on two large superfamilies of LGICs: 1) nicotinic acetylcholine receptor (nAChR)
superfamily (cys-loop) with members including nAChRs, 5-HT
3
Rs, GABA
A
Rs and glycine
receptors (Betz, 1990; Ortells and Lunt, 1995) and 2) glutamate superfamily with members
including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), kainate
receptors and N-methyl-d-aspartate receptors (NMDARs) (Monaghan et al., 1989; Sommer and
Seeburg, 1992). P2X receptors (P2XRs) constitute a third superfamily of LGICs that are
becoming a focus of investigation in neuroscience and ethanol studies (Asatryan et al., 2011;
Burnstock, 2008; Gum et al., 2012; Litten et al., 2012).

P2XRs and Their Role in the Effects of Ethanol
P2XRs are a family of cation-permeable LGICs gated by synaptically released
extracellular adenosine 5’-triphosphate (ATP). ATP directly mediates synaptic transmission by
acting on P2XRs located on postsynaptic membranes. In addition, ATP acts presynaptically on
P2XRs to modulate the action of other neurotransmitters such as GABA, glycine and glutamate
(Chizh and Illes, 2001; Deuchars et al., 2001; Hugel and Schlichter, 2002; Khakh, 2001) that are
known to play important roles in ethanol seeking behaviors. Several lines of evidence suggest
that P2XRs in the ventral tegmental area (VTA) of the brain participate in modulation of neurons
critical for controlling ethanol intake. P2XRs have been identified as potential targets for ethanol
action. Support for this comes from: 1) studies that show ATP currents recorded from native
P2XRs are inhibited by ethanol (Li et al., 1993, 1994, 1998; Weight et al., 1999; Xiao et al.,
2008); 2) investigations using heterologous expression systems such as Xenopus oocytes and
HEK293 cells that demonstrate ATP currents recorded from recombinant P2XRs are modulated
11

by ethanol (Asatryan et al., 2008; Davies et al., 2005; Davies et al., 2002; Ostrovskaya et al.,
2011; Popova et al., 2010; Xiong et al., 2000; Xiong et al., 2001); 3) evidence there is a broad
distribution of P2XRs in neurons and microglia of the mammalian brain (Franke et al., 2001;
Heine et al., 2007; Kanjhan et al., 1999; Kidd et al., 1995; Rubio and Soto, 2001). Of the seven
P2XR subtypes, P2X4 receptors (P2X4Rs) are the most abundantly expressed in the central
nervous system (CNS) and are the most ethanol sensitive P2XRs identified (Buell et al., 1996b;
Davies et al., 2005; Davies et al., 2002; Soto et al., 1996; Xiong et al., 2000).

Support for Involvement of P2X4Rs in Ethanol Action
Several lines of evidence suggest that P2X4Rs can modulate a spectrum of the effects of
ethanol. In vitro studies report that ethanol concentrations starting at approximately 5 mM
modulate ATP-activated currents in neurons (Li et al., 1993, 1994, 1998; Weight et al., 1999;
Xiao et al., 2008) and recombinant models (Asatryan et al., 2010; Asatryan et al., 2008; Davies
et al., 2005; Davies et al., 2002; Xiong et al., 2000; Xiong et al., 2001). Additionally, P2X4Rs
are located in brain regions that have been identified as neural substrates of ethanol [e.g.,
hippocampus, cerebellum, VTA and nucleus accumbens (NAc)] (Gonzales et al., 2004; McCool,
2011; Pankratov et al., 2009; Sim et al., 2006). Recent studies implicate P2X4Rs in the
regulation of multiple nervous system functions, including neuropathic pain (Tsuda et al., 2000;
Ulmann et al., 2008), neuroendocrine functions (Zemkova et al., 2010) and hippocampal
plasticity (Baxter et al., 2011; Lorca et al., 2011; Sim et al., 2006). In addition, P2X4Rs have
been recently shown to modulate the function of other major ionotropic targets, such as
GABA
A
Rs (Jo et al., 2011) and NMDARs (Baxter et al., 2011). Many of the physiological and
behavioral functions linked to P2X4Rs are affected by ethanol as well.
12

Further support for the notion that P2X4Rs are a target for ethanol action comes from
preliminary studies which found greater expression of the p2rx4 gene in the VTA of high alcohol
drinking (HAD2) vs. low alcohol drinking (LAD2) rats (Kimpel et al., 2007). Similarly, a recent
genomic/phenomic approach investigating ethanol consumption identified p2rx4 as one of the
candidate genes that predisposes to varying levels of ethanol intake across 28 recombinant inbred
strains of rats (Tabakoff et al., 2009). Combining the findings from this latter study with a
literature-based search led the authors to conclude that the interactions of the products of the
candidate genes identified in the study were involved in neurobiological pathways affecting
GABAergic neuronal activity acting in brain regions including the VTA (Tabakoff et al., 2009).
Collectively, these findings support the contention that P2X4Rs play a role in ethanol
consumption.

Ivermectin
Ivermectin (IVM), a semi-synthetic macrocyclic lactone, is a broad-spectrum
antiparasitic avermectin medicine used worldwide in humans and animals (Geary, 2005;
Molinari et al., 2010; Richard-Lenoble et al., 2003). The safety of IVM has been demonstrated
over twenty-five years where millions of humans have been treated successfully (Boxall and
Long, 2005; Burkhart, 2000; Guzzo et al., 2002; Omura, 2008). The high level of safety may be
attributed to the dosing (200 µg/kg) and the frequency of IVM treatment for parasite infections,
which is given once yearly or intermittently. However, doses more than 10X that of the
recommended dosage have been safely tested in clinical studies (Guzzo et al., 2002). In rodents,
doses less than 10 mg/kg IVM administered intravenously (IV) do not appear to cause visible
CNS depression (Trailovic and Trailovic, 2010), whereas lethality is reported to be 25 to 50
13

mg/kg (Merck et al., 1988). Although IVM has been reported to have some mild to moderate
CNS adverse events, these incidences are rare and appear to be linked to alteration of p-
glycoprotein (Pgp), which is found abundantly in the blood brain barrier (BBB) (Edwards, 2003;
Geyer et al., 2009). Overall, these rare adverse events have not deterred the use of IVM as it
appears to have a high therapeutic index and a good safety profile (Burkhart, 2000; Davis et al.,
1999) illustrated by the 69 million plus individuals that were treated with IVM in 2006 [for
review see (Omura, 2008)].
The current therapeutic potential of IVM is attributed to action on a non-mammalian,
glutamate-gated inhibitory chloride channel (Cully et al., 1994; Dent et al., 1997; Vassilatis et
al., 1997). IVM was initially used in humans to treat onchocerciasis, also known as river
blindness, caused by Onchoncerca volvulus. As an anti-parasitic agent, IVM kills the
microfilariae, possibly by paralyzing the body-wall and pharyngeal muscles and preventing it
from invading the human immune system thus disrupting the parasitic relationship (Crump and
Omura, 2011). However, since then, IVM has also been approved to treat other parasitic
infections. IVM has been shown to potentiate mammalian GABA
A
and glycine receptors in vitro
[for review see (Dawson et al., 2000; Shan et al., 2001)] and act as a weak anticonvulsant in mice
(Dawson et al., 2000). It has also recently been studied as a novel farnesoid X receptor (FXR),
often referred to as the bile acid receptor, ligand that regulates metabolism in humans (Jin et al.,
2013). Studies in humans suggest additional sites of action for IVM not related to these receptors
(Sung et al., 2009) including nAChRs (Krause et al., 1998; Sattelle et al., 2009) and P2X4Rs
(Asatryan et al., 2010). Notably, all of these receptor families have been linked to the behavioral
effects of ethanol (Asatryan et al., 2011; Davies, 2003; Perkins et al., 2010).

14

Evidence for Antagonistic Effect of IVM in P2X4 Receptors in vitro
Among P2XR family members, IVM is a selective positive modulator of P2X4Rs and has
been used to differentiate the role of P2X4Rs from other P2X family members in ATP-mediated
processes (Jelinkova et al., 2006; Khakh et al., 1999b; Silberberg et al., 2007). IVM is reported
to act on sites in or near the ectodomain-transmembrane (TM) domain interface of P2X4Rs
(Jelinkova et al., 2008; Jelinkova et al., 2006; Silberberg et al., 2007). Interestingly, some of
these sites are proximal to regions that we have previously reported as being important for
ethanol modulation, specifically, the TM2 segment of P2X4Rs (Asatryan et al., 2008; Popova et
al., 2010). Studies from our lab were conducted to test the hypothesis that IVM interferes with
and antagonizes the inhibitory effect of ethanol (Asatryan et al., 2010). Specifically, this study
demonstrates that, when applied with ethanol, IVM (0.5-10 µM) antagonizes ethanol inhibition
in a concentration-dependent manner. This effect is not due to individual actions of the two
drugs, but rather, a result of IVM and ethanol action on or close to the same site on P2X4Rs.
Previous studies reported that loss of ethanol inhibition occurs when 2 key residues, methionine
336 (Met336) and aspartic acid 331 (Asp331), near the ectodomain of the TM2 segment, were
replaced with an alanine residue, suggesting that these positions are important in ethanol action.
To test whether or not IVM antagonizes ethanol via interference at position 336, the WT
residue at this position (Met336) was mutated to other residues. Replacing Met336 with other
amino acids had considerable effects on the sensitivity of P2X4Rs to both IVM and ethanol, thus
supporting the initial hypothesis. For example, substitution with small, nonpolar amino acids
increased IVM potentiation and caused a decrease in ethanol inhibition. Substitution with large,
non-polar and polar amino acids had a similar effect on IVM potentiation but to a lesser extent.
The response to ethanol by the phenylalanine (Phe) substitution was comparable to WT whereas
15

the tryptophan (Trp) substitution and substitution with large, polar amino acids reduced
sensitivity to ethanol. Substitution with charged, positive amino acids completely extinguished
the response to both IVM and ethanol. These findings were used to construct the first molecular
model based on the recently published 3.1 Å resolution X-ray crystal structure of the zebrafish
P2X4R (Kawate et al., 2009). Interestingly, our model revealed a putative ethanol-binding
pocket that also appears to be important for IVM action on P2X4Rs (Asatryan et al., 2010;
Popova et al., 2013) and thus may represent a target for drug development for AUDs. Moreover,
with its anti-ethanol effects in vitro, these data suggest that IVM could be developed as a novel
drug candidate for the treatment of AUDs.

Conclusion: Dissertation Hypothesis and Outline
Developing effective therapeutics for the treatment of AUDs is of major importance. The
experiments contained within the chapters of my thesis will begin to address this issue by testing
the hypothesis that IVM can be repurposed as a novel treatment for AUDs. This was
accomplished by employing several preclinical models of ethanol intake and multiple behavioral
paradigms using C57BL/6 mice. First, data in Chapter 2 illustrate the effect of acute IVM
administration on three distinct models of ethanol intake: 1) 24-h access two-bottle choice model
of social drinking; 2) intermittent limited access model of binge drinking; and 3) operant self-
administration. The pharmacokinetic (PK) profile of IVM is also elucidated in this chapter using
liquid chromatography/ mass spectrometry (LC-MS) techniques. This latter work is the first to
link brain concentration of IVM to a change in behavior.
Chapter 3 experiments test the effect of IVM on mice across a wide battery of well-
validated behavioral paradigms including the sticky tape, hot plate, novel open field, elevated
16

plus maze, marble burying assay, forced swim, tail suspension, conditioned place preference,
acoustic startle, pre-pulse inhibition of the startle reflex, and novel object recognition paradigms,
for the evaluation of major domains of neuropsychiatric toxicity. These paradigms allow for a
thorough assessment of behavioral alterations consequent to IVM exposure, and highlight
whether this regimen may affect comorbid psychiatric disturbances usually observed in patients
with AUDs. These studies suggest that IVM has some anxiolytic properties but does not elicit
major changes in pain and tactile sensitivity, locomotion, or memory. Notably, IVM does not
exhibit rewarding properties, strongly suggesting that the drug is not addictive.
Chapter 4 evaluates the sustainability and safety of multi-day IVM administration in
reducing ethanol intake at a corresponding dose shown to be safe in humans. Mice were
administered IVM for 10 consecutive days and subjected to the 24-h access two-bottle choice
paradigm of social drinking. LC-MS techniques were used to determine the concentration of
IVM in the brain and plasma at multiple time points following 10-day administration of the drug.
IVM was able to successfully decrease ethanol intake throughout the study without causing overt
signs of toxicity. AUDs are a chronic disease and, therefore, a single dose of IVM would be
insufficient to successfully treat these disorders, yet only acute dosing is required for the current
approved indication of IVM. Therefore, these studies are important to determine if IVM is well
tolerated when administered over multiple days, which is critical if it is to be repurposed as a
therapeutic for AUDs.
Chapter 5 tests the hypothesis that the ability to antagonize ethanol-mediated inhibition of
P2X4R function may be a good predictor of the potential of an avermectin to reduce ethanol
intake. We tested this hypothesis by comparing the abilities of IVM and two other avermectins,
abamectin (ABM) and selamectin (SEL), to reduce ethanol intake in mice, alter modulation of
17

GABA
A
Rs and P2X
4
Rs expressed in Xenopus oocytes, and penetrate the brain. Results from this
study suggest that there is no correlation between concentration of avermectins in the brain and
anti-ethanol activity but that the ability of avermectins to antagonize P2X4Rs in vitro is a strong
predictor of anti-ethanol activity in vivo. These data support the use of avermectins as a platform
for developing novel drugs to prevent and/or treat AUDs.
Chapter 6 summarizes the conclusions from my dissertation research and discusses the
current preclinical and future clinical studies that will further test the hypothesis that IVM can be
repurposed as a therapeutic for AUDs. The ongoing preclinical study looks at the effectiveness
of alternate oral delivery methods, specifically, fast dissolving oral films. The human laboratory
study will test the safety of a single dose of IVM (30 mg) in the context of ethanol administration
and evaluate the effect of IVM on craving for ethanol. Taken together, the data presented in this
dissertation provide strong preclinical support for my hypothesis that IVM can be repurposed as
a treatment for AUDs. Specifically, these data set the stage for first-in-human testing of IVM for
this new indication.

18

CHAPTER 2
IVERMECTIN REDUCES ALCOHOL INTAKE AND PREFERENCE IN MICE

ABSTRACT
The high rate of therapeutic failure in the management of alcohol use disorders (AUDs)
underscores the urgent need for novel and effective strategies that can deter ethanol
consumption. Recent findings from our group showed that ivermectin (IVM), a broad-spectrum
anthelmintic with high tolerability and optimal safety profile in humans and animals,
antagonized ethanol-mediated inhibition of P2X4 receptors (P2X4Rs) expressed in Xenopus
oocytes. This finding prompted us to hypothesize that IVM may reduce alcohol consumption;
thus, in the present study we investigated the effects of this agent on several models of alcohol
self-administration in male and female C57BL/6 mice. Overall, IVM (1.25-10 mg/kg,
intraperitoneal) significantly reduced 24-h alcohol consumption and intermittent limited access
(4-h) binge drinking, and operant alcohol self-administration (1-h). The effects on alcohol intake
were dose-dependent with the significant reduction in intake at 9 h after administration
corresponding to peak IVM concentrations (Cmax) in the brain. IVM also produced a significant
reduction in 24-h saccharin consumption, but did not alter operant sucrose self-administration.
Taken together, the findings indicate that IVM reduces alcohol intake across several different
models of self-administration and suggest that IVM may be useful in the treatment of AUDs.

INTRODUCTION
Alcohol (ethanol) use disorders (AUDs) have a major national impact in the United
States, affecting nearly 18 million people, causing over 100,000 deaths, and costing upward of
19

$235 billion annually (Bouchery et al., 2011; Grant et al., 2004; Harwood, 2000). Alcohol abuse
and misuse also possess significant health risks worldwide where alcohol abuse and misuse is the
third leading risk factor for premature death and disabilities (World Health Organization). The
few drugs currently approved for AUD management attempt to deter alcohol intake by blocking
its metabolism or by targeting the neurochemical systems in the downstream cascades leading to
craving and dependence (Colombo et al., 2007; Gewiss et al., 1991; Johnson et al., 2007;
Steensland et al., 2007). Unfortunately, these approaches, even when applied in combination
with psychological strategies, have resulted in limited success as evidenced by the continued
high rates of AUDs. As such, development of effective treatments for AUDs represents an
important public health goal (Bouchery et al., 2011; Heilig and Egli, 2006; Johnson, 2010;
Johnson et al., 2007; Steensland et al., 2007).
In the quest to identify novel therapeutic targets for AUDs, our group and others have
found that the P2X4 receptor (P2X4R), a member of the P2X family of ATP-gated channels
abundantly expressed in the CNS (Buell et al., 1996b; Soto et al., 1996), plays a role in alcohol-
induced behaviors (Asatryan et al., 2011; Kimpel et al., 2007; Tabakoff et al., 2009). P2X4Rs are
the most abundant P2X receptor subtype expressed in the CNS (Buell et al., 1996b; Soto et al.,
1996) and are the most alcohol sensitive P2XR identified to date (Davies et al., 2005). In vitro,
P2X4Rs are inhibited by ethanol concentrations as low as 5 mM (Asatryan et al., 2010; Davies et
al., 2005; Davies et al., 2002; Popova et al., 2010; Xiong et al., 2005; Xiong et al., 2000; Xiong
et al., 1999). This concentration of ethanol is well below the 17 mM legal blood ethanol
concentration (BEC) that is considered “legal intoxication” (i.e., 0.08%) in the United States.
P2X4Rs are expressed in key brain regions implicated in the reinforcing properties of alcohol
and other drugs, such as the striatum (Amadio et al., 2007; Krügel et al., 2003). Taken together,
20

the evidence suggests that P2X4Rs may play a role in alcohol addiction through modulation of
P2X4Rs in the mesolimbic dopamine (DA) system.
Additional evidence supporting the hypothesis comes from recent studies reporting that
the p2rx4 gene may be linked to alcohol intake and/or preference (Kimpel et al., 2007; Tabakoff
et al., 2009). First, alcohol-preferring (P) rats show lower functional expression of the p2rx4
gene compared to alcohol-non-preferring (NP) rats (Kimpel et al., 2007). Second, investigations
on the genetic determinants of alcohol consumption report an inverse relationship between the
expression of the p2rx4 gene and innate alcohol preference (Tabakoff et al., 2009). Overall, these
findings suggest that alcohol intake may be modulated by ethanol acting on P2X4Rs and that
pharmacological activation of P2X4Rs may reduce alcohol consumption and preference.
While no selective agonists of P2X4Rs have been developed to date, rich evidence has
shown that ivermectin (IVM), a broad-spectrum antiparasitic dihydro avermectin derivative used
worldwide in humans and animals (Geary, 2005; Molinari et al., 2010; Richard-Lenoble et al.,
2003), acts as a potent and selective positive allosteric modulator of P2X4Rs. IVM’s selectivity
affinity (between P2XR subtypes) provides the basis for it to be routinely employed to determine
the contribution of P2X4Rs in ATP-mediated processes (Khakh et al., 1999b).
IVM is believed to act, in part, in close proximity to positions 331 and 336 in P2X4Rs
(Jelinkova et al., 2008; Jelinkova et al., 2006; Silberberg et al., 2007). Recent investigations in
our laboratory indicated that positions 331 and 336 are also important as targets for the inhibitory
actions of ethanol on P2X4Rs expressed in Xenopus oocytes (Popova et al., 2010). Further
studies demonstrated that IVM antagonized the inhibitory effects of ethanol on P2X4Rs.
Specifically, mutational studies provided evidence that IVM antagonism of ethanol involved
interference in the action of alcohol at a putative pocket at or near position 336 in these receptors
21

(Asatryan et al., 2010). This ability of IVM to antagonize the effects of ethanol on P2X4Rs,
taken in context with evidence suggesting that P2X4Rs play a role in ethanol preference
[presented above and see (Asatryan et al., 2011)], suggested that IVM may reduce ethanol intake
and preference. This notion is supported by recent findings that acute administration of IVM
reduced maintenance of alcohol self-administration in rats, but results from this work were
inconclusive (Kosten, 2011).
The present study systematically tests the hypothesis that IVM reduces ethanol intake and
preference in mice. The findings provide strong evidence that acute and short-term chronic IVM
can reduce ethanol intake. Pharmacokinetic analyses provide strong support that the actions of
IVM on ethanol intake reflect actions of IVM found in the brain. Collectively, the data indicates
that IVM holds promise as a novel therapeutic agent for the treatment of AUDs.

MATERIALS AND METHODS
Drugs. IVM [10 mg/ml] (Norbrook Inc., Lenexa, KS) was diluted in a 0.9% sodium
chloride solution (saline) to a concentration that would allow for an injection volume of 0.01
ml/g of body weight. Propylene glycol (1,2 Propanediol – solvent used by the manufacturer to
dissolve the IVM), purchased from Alfa Aesar (Ward Hill, MA) was diluted in saline at a
concentration equivalent to the amount used in the 10 mg/kg dose of IVM. Ethanol was diluted
in tap water using either Everclear, a 150 proof solution (Luxco, St. Louis, MO) or Gold Shield
Alcohol, a 200 proof USP solution (Gold Shield Chemical Company, Hayward, CA) to achieve a
10% v/v solution (10E). Sucrose (Sigma-Aldrich, St. Louis, MO) was prepared as a 2% w/v (2S)
solution in tap water, whereas saccharin (Sigma-Aldrich, St. Louis, MO) was prepared at a
0.033% w/v solution in tap water.
22

Animals. Studies were performed, as described in specific experiments, on drug-naïve,
C57BL/6J male mice purchased at 6 weeks of age (Jackson Laboratory, Bar Harbor, ME, USA
for studies conducted in Portland, OR) or on male and female mice from our internal breeding
colony at the University of Southern California (USC). For the studies conducted at USC,
C57BL/6J breeders were obtained from the Jackson Laboratory, and new breeders were replaced
every 3 generations. Mice were acclimated to the housing facility for one week and group-
housed (2-4 mice per cage) in polycarbonate/polysulfone cages at a 12 h light/dark cycle with
lights off at 12:00PM (USC) or 0:600PM (Portland) with ad libitum access to food and water.
The holding room was maintained at approximately 22°C. All procedures in this study were
performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and
all efforts were made to minimize animal suffering. The USC Institutional Animal Care and Use
Committee approved the protocols. The operant self-administration study was conducted in
Portland, Oregon, and the Institutional Animal Care and Use Committee at the Portland VA
Medical Center approved this work.
IVM effects on 24-h access ethanol and tastant preference drinking in mice. The 24-
h access model [e.g., (Belknap et al., 1993; McClearn, 1959; Middaugh et al., 1999; Rodgers,
1966; Yoneyama et al., 2008) is widely used to assess changes in drinking behaviors. We used a
modification of the procedure employed by Yoneyama et al. (2008). Briefly, mice (individually
housed 3 days before the start of the study) had 24-h access to two inverted bottles with metal
sippers placed on the cage tops. Food was distributed near both bottles to avoid food associated
tube preferences.
Alcohol studies. One tube contained tap water and the other a 10E solution in tap water.
Mice were given free access to 10E with bottle positions alternated every other day. Fresh fluids
23

were provided twice a week when cages were changed. Body weights were recorded daily.
Every morning daily fluid intake (to the nearest 0.1 ml) was recorded from both bottles by
measuring the level of the meniscus on the graduated drinking tube. Daily 10E intake was
measured until it stabilized (+/- 10% variability from the mean dose of the last 3 days). After
establishing stable alcohol drinking levels (usually within one week), mice received daily saline
injections (intraperitoneal; i.p.) until 10E intake stabilized (which averaged between 4-5 days of
saline injections). In all cases, injections were administered immediately prior to the period of 24
h access to 10E versus tap water, so that the change in drinking over 24 h after IVM
administration was measured. Mice then received one injection (i.p.) with either IVM (0.65-10
mg/kg) or saline (control for injection effect, per se) daily for the remainder of the study using a
within subjects design. Animals were injected with saline on subsequent days until 10E drinking
stabilized at baseline levels, and then mice were injected with another dose of IVM. This pattern
of IVM administration continued until all doses of a particular study were complete.
Consumption of 10E returned to baseline levels prior to the administration of each subsequent
dose of IVM tested. A pilot study determined that the effects of IVM on 10E intake following
IVM doses less than 10 mg/kg were not significantly altered within 3 days post IVM
administration, suggesting a return to baseline 10E intake.
Saccharin studies. Upon conclusion of one IVM/alcohol study, a tastant study was
conducted similar to that for ethanol, with the exception that the 10E solution was replaced with
a bottle containing a saccharin solution (0.033% w/v). Daily saccharin intake was measured until
it stabilized (+/- 10% variability from the mean dose of the last 3 days). After establishing stable
drinking levels, mice received daily saline injections (i.p.) until intake stabilized. Mice then
24

received one injection (i.p.) with either IVM (2.5, 5.0 mg/kg) or saline (control for injection
effect, per se) daily for the remainder of the study using a within subjects design.
IVM effects on intermittent limited access drinking in mice. A modified version of
drinking in the dark (DID) procedure (Rhodes et al., 2005) was used, following intermittent
limited access procedures similar to recent studies reported by others (Neasta et al., 2010).
Briefly, mice had 4-h access to one bottle containing 10E every other day. Alcohol drinking
sessions began 3 h into the circadian dark on Monday, Wednesday, and Friday with a 48-h
(weekend) alcohol deprivation period. A single bottle of water was continuously available
between drug access periods. The volume from the alcohol-containing bottle was measured
immediately prior to the start and immediately following the 4-h drinking period. Following the
establishment of stable baseline alcohol drinking levels and habituation to saline injections, a
single 10 mg/kg dose of IVM was administered at 6 h prior to the start of the drinking session.
Based on our pharmacokinetic analysis of IVM in plasma and brain, initiation of the limited
access procedures 6 hours after IVM administration allowed for the evaluation of IVM on 10E
intake as IVM approached peak brain concentrations, at peak, and after peak IVM
concentrations.
IVM effects on operant ethanol and sucrose self-administration in mice. Daily
sessions were carried out Monday to Friday in 8 modular chambers (21.6 x 17.8 x 12.7 cm) with
stainless steel grid floors (Med Associates, Inc., St. Albans, VT), according to published
procedures (Ford et al., 2007a; Ford et al., 2009; Ford et al., 2007b; Tanchuck et al., 2011). Each
chamber contained a house light, two ultra-sensitive retractable levers, and a retractable sipper
apparatus that held a 10 ml graduated pipette with a double ball bearing sipper tube that allowed
volume measurements to the nearest 0.05 ml. A lickometer circuit was connected to each metal
25

sipper to monitor lick patters via MED-PC software (Med Associates, Inc.). Each chamber was
housed within a wooden cabinet (61 x 38 x 33 cm; Fisher Custom Woodworking, Portland, OR)
that contained a fan to minimize outside noise and to facilitate airflow.
The present study utilized the “sipper” model of operant self-administration, which we
have described in detail elsewhere (Ford et al., 2009; Ford et al., 2007b). Advantages to the use
of the “sipper” model are that the appetitive and consummatory phases of self-administration can
be procedurally separated [e.g., (Samson et al., 2000; Samson et al., 1998)] and that C57BL/6
mice exhibit a heightened appetitive drive to acquire ethanol access and more than a two-fold
increase in ethanol consumption with the “sipper” procedure than when responding on a fixed
ratio schedule (Ford et al., 2007a). Briefly, separate groups of male C57BL/6J mice were trained
to respond for access to either a 10E or 2S solution via sucrose fading. Upon completion of
training, a single response requirement of 16 presses on the active lever (RR16) resulted in 60
min of continual access to the fluid reinforcer. A 20 min time limit to complete the RR16
schedule was imposed. The first study was conducted in male C57BL/6J mice that had a 6
month history of ethanol or sucrose self-administration and prior drug exposure, and the second
study was conducted in C57BL/6J mice that were experimentally naïve.
In the first study, C57BL/6J male mice were 8 weeks old upon purchase and had
approximately 6 months of experience with either ethanol (n =7) or sucrose (n = 10) self-
administration before testing in the present study. Mice had previously been tested for effects of
the synthetic neurosteroid ganaxolone on ethanol and sucrose self-administration in sessions
where animals had 30 min of continual access to the reinforcer, and each mouse had the same
history of treatment exposure (Ramaker, Ford and Finn, in preparation). Then, mice were
maintained on an operant self-administration procedure without any drug injections before the
26

initiation of this study. Mice were then habituated to saline injections (6.5 – 8.5 h pretreatment
time was selected to be similar to the 6 h drug pre-treatment in the DID alcohol study and to be
compatible with the time to achieve maximal concentration, which was found to be at 8-10 h
following IVM administration) and exhibited stable baselines of 10E or 2S with less than 10%
variability between sessions. An initial pilot study determined that there were minimal effects of
IVM (0.65, 2.5, or 10 mg/kg versus saline, within-subjects design) when animals had 30 min of
continual access to 10E or 2S (data not shown). Thus, session length was increased to 60 min of
continual reinforcer access (10E or 2S) over a period of approximately 2 weeks, and animals did
not receive any injections during this time. Because mice already were habituated to saline
injections, they received a single injection of saline and then IVM (10 mg/kg) a few days later
during the final week of testing.
In the second study, C57BL/6J male mice were 8 weeks old upon purchase. Mice were
trained to respond for access to a 10E solution as described above. Upon completion of training,
a single response requirement of 16 presses on the active lever (RR16) resulted in 60 min of
continual access to the 10E solution. Once the animals exhibited stable baselines of 10E with
less than 10% variability between sessions, mice were then habituated to saline injections (6.25 –
8.5 h pretreatment time) during one week of testing. The following week, mice received a single
injection of saline and then IVM (10 mg/kg) a few days later.
Quantification of IVM using liquid chromatography with tandem mass
spectrometry (LC-MS/MS). Brain tissue and plasma samples were collected from separate
groups of animals at 1, 6 and 8 h after being administered with IVM (0, 0.25, 2.5, or 10 mg/kg,
i.p.). Samples were extracted, using either 100 mg of brain sample or 50 µL of plasma. The
whole brain sample was excised and placed into liquid nitrogen for 1 min to flash freeze. The
27

sample was then pulverized, where 50 µL of 500 ng/mL abamectin (ABM) was added as an
internal standard. The entire mixture was extracted using 1 mL ethyl acetate, and the samples
vigorously mixed and centrifuged at 5000 rpm for 5 min. This process was performed 4 times,
and the supernatants were collected and evaporated to dryness using a steady stream of nitrogen
gas.
To 50 µL plasma, 50 µL of 500 ng/mL ABM was added, and the samples were extracted
using 4 mL of ethyl acetate and centrifuged at 13,000 rpm for 10 min. The organic layer was
then collected and evaporated to dryness using a steady stream of nitrogen. The evaporated
residues were all reconstituted in 100 µL of 0.1% formic acid in acetonitrile. A 30 µL aliquot
was injected into Agilent 1100 HPLC system linked into an AB Sciex API 3000 turboionspray
mass spectrometer. The analytes were separated using an ACE C18 column, with a gradient
mobile phase system. The mobile phase consisted of component A, which was 0.1% formic
acid, and component B, which was 0.1% formic acid in acetonitrile. The gradient program was
set at 50% component B for 1 min, and then a gradient from 50% to 95% component B for the
next 5 minutes. The amount of IVM and ABM were quantified using the mass spectrometer set
in the positive mode and monitoring the following multiple reaction monitoring (MRM) using
parent to transition ions of 892 →307.2 and 867.6→453.3, respectively. The lowest level of
detection was 2 ng/mL.
Pharmacokinetic Analysis. The concentration of IVM in brain and plasma were
analyzed using a non-compartment model. Serial blood and tissue IVM quantification was used
to calculate the pharmacokinetic parameters such as the maximum IVM concentration (Cmax),
time to achieve maximal IVM (Tmax), half-life, elimination constant, and area under the curve
(AUC).
28

Statistical analyses. For each study, consumption of each solution corrected for body
weight (ethanol = g/kg, saccharin = mg/kg, sucrose = g/kg), and ethanol preference ratio (mls
ethanol ÷ total mls) were calculated for each IVM/saline dose tested. ANOVA assessed IVM
dose effects with IVM dose or time [saline pre-treatment (pre IVM), IVM dose, saline post-
treatment (post IVM)] as a repeated measures factor on the dependent variables (ethanol and
water intake in mls, ethanol intake in g/kg, and ethanol preference ratio). Separate analyses were
conducted for each sex, since males and females were tested in separate studies. For the operant
self-administration data, cumulative records of lever and sipper (lick) responding were recorded
by MED-PC IV software (MED Associates, Inc.). Measures of appetitive (response rate, latency
to first press, latency to first bout) and consummatory (bout frequency, size, duration and lick
rate) processes were calculated from these cumulative records via a custom data analysis
program (http://www.r-project.org). Based on our previous work, a bout was defined as ≥ 20
licks with < a 60 sec pause between successive licks (Finn et al., 2008; Ford et al., 2007a; Ford et
al., 2009; Ford et al., 2007b). ANOVA with repeated measures was used to examine IVM
effects on active and inactive lever responses, intakes of 10E and 2S (volume and dose),
latencies to reinforcer access, and the bout parameters mentioned above. Since we were
predicting a differential effect of IVM on intake of 10E and 2S, planned comparisons were
conducted in the absence of a significant interaction. Significant main effects and interactions of
the ANOVAs were further investigated with post-hoc tests (i.e., Tukey’s test, t-tests with
Bonferroni correction, paired t-tests). For all studies, significance set at p ≤ 0.05.

29

RESULTS
Single dose administration of IVM (10 mg/kg) decreased 10E intake and preference.
We initiated our investigation by examining the impact of acute administration of IVM (10
mg/kg) on alcohol intake using a 24-h access two-bottle choice paradigm (10E versus tap water).
The lower limit of alcohol consumed by mice using this model suggests that this model mimics
social drinking (Blednov et al., 2010). We started with the 10 mg/kg dose in order to maximize
the chance of observing an effect of treatment. The 10 mg/kg dose was the highest dose that
could be given without introducing possible CNS toxicity (Lerchner et al., 2007; Merck et al.,
1988).
Prior to the initiation of saline injections, baseline 10E intake during the 24-h period was
13.47 ± 0.61 g/kg (n = 11). As illustrated in Fig 2.1A, 10E intake stabilized at 8.67 g/kg
following saline injections. Acute administration of 10 mg/kg IVM was analyzed (pre IVM,
IVM, post IVM) and significantly reduced alcohol intake in excess of 45%, compared with pre
IVM injections (Fig. 2.1A) [F(2,30) = 16.85, p<0.001], and 10E intake remained significantly
lower pre IVM levels on the day following IVM treatment (by more than 25%, shown as post
IVM injections in Fig. 2.1A). The reduction in 10E intake induced by IVM was paralleled by a
35% decrease in ethanol preference [F(2,30) = 10.11, p<0.001] (Fig. 2.1B). This dose of IVM
also significantly increased water consumption by about 47%, when compared to pre-IVM
injections [F(2,30) = 3.577, p<0.05] but there was no change in total fluid intake (data not
shown). Using a separate group of animals, we also tested the effects of the vehicle alone (i.e.,
propylene-glycol) on 10E intake to ensure that the changes observed in this first study could not
be attributed to an action of the vehicle, per se. The vehicle did not significantly alter 10E
intake, water intake, fluid intake or preference ratio (data not shown) giving us confidence that
30

the changes in alcohol drinking were attributed to the drug (IVM) and not the vehicle
(propylene-glycol). Thus, subsequent studies used saline as the vehicle treatment.

Figure 2.1

Figure 2.1. IVM (10 mg/kg) reduces A) 10% v/v ethanol (10E) intake and B) preference ratio for 10E in male
C57BL/6J mice using a 24-h access two-bottle choice paradigm. After attaining stable drinking levels for 3
consecutive days, IVM was administered. Bars represent levels from the day prior to IVM injection (white; Pre
IVM), the day of the IVM injection (black; IVM) and the day after the IVM injection (gray; Post IVM). Values
represent the mean ±SEM for 11 mice.*P<0.05, ***P<0.001 versus Pre IVM, Tukey multiple comparison post-hoc
test.

IVM decreased 10E intake and preference in a dose-dependent manner in male
mice. We extended the initial single dose IVM experiment to one where several doses of IVM
(0.65 – 10 mg/kg) were tested for their effects on 10E intake. When the analysis was conducted
across time (pre-IVM, IVM, post-IVM), there was a significant effect of IVM on ethanol intake
[F(2,100) = 14.37, p<0.001]. The analysis of IVM dose indicated that IVM significantly reduced
alcohol intake in a dose dependent manner (Fig. 2.2A) [F(4,100) = 5.51, p<0.001]. The
interaction between time and dose was significant [F(8,100) = 5.53, p<0.001]. Bonferroni post-
hoc comparisons versus pre-IVM indicated that 2.5 mg/kg IVM was the lowest dose of IVM that
significantly reduced alcohol intake in this study [i.e., 2.5 mg/kg ~39% (t=5.025, p<0.001)]. The
31

two lowest doses of IVM tested (i.e., 0.65 mg/kg and 1.25 mg/kg) did not significantly alter 10E
intake. In addition, saline injections alone did not significantly alter 10E intake. For the doses of
IVM that significantly reduced 10E intake, we found that 10E intake returned to comparable pre-
IVM intake levels by the third day post-IVM injection. The exception was 10 mg/kg IVM. For
this dose, we found that 10E intake did not fully return to pre-IVM levels until 4-5 days post-
IVM injection (data not shown).
The investigation also found that the reductions in ethanol intake induced by IVM were
accompanied by decrease in ethanol preference, which was revealed when the analysis was
conducted across time [F(2,100) = 8.92, p<0.001]. Moreover, the analysis of dose indicated that
IVM significantly reduced alcohol intake in a dose dependent manner [F(4,100) = 3.69, p<0.05].
The interaction between time and IVM dose was significant [F(8,100) = 7.40, p<0.001], with
Bonferroni post-hoc tests confirming a significant decrease in ethanol preference ratio following
doses of 2.5 mg/kg (t=4.527, p<0.001) and 10 mg/kg (t=5.715, p<0.001) IVM (Fig. 2.2B).
While there was no main effect of IVM to significantly alter water intake when the analyses were
conducted across time or dose, there was a significant interaction between time and IVM dose
[F(8,100) = 7.09, p<0.001]. Post-hoc comparisons confirmed that water intake was increased
significantly with the IVM dose of 10 mg/kg (t=3.430 p<0.01; Fig. 2.2C). Investigating the
effects of IVM on total fluid intake (Fig. 2.2D) across time we found a significant reduction
[F(2,100) = 13.66, p<0.001]. The analysis of IVM dose indicated that dose also had a significant
effect on fluid intake [F(4,100) = 3.50, p<0.05]. Furthermore, there was a significant interaction
between time and dose [F(8,100) = 5.40, p<0.001], with post-hoc comparisons confirming that
fluid intake decreased after IVM doses of 2.5 mg/kg (t=3.526, p<0.01) and 10 mg/kg (t=3.689,
p<0.01)
32

Figure 2.2

Figure 2.2. IVM dose response study in male C57BL/6J mice using a 24-h access two-bottle choice paradigm. Each
dose of IVM was administered after achieving stabilized drinking for 3 consecutive days. Bars represent levels from
the day prior to IVM injection (white; Pre IVM) and the day of the IVM injection (black; IVM). A) IVM (2.5 and
10 mg/kg) significantly reduced 10E intake. B) IVM (2.5 and 10 mg/kg) significantly reduced preference ratio for
10E. The effects of IVM on water and total fluid intake are presented in panels C and D, respectively. Values
represent the mean ±SEM for 11 mice per dose group. **P<0.01, ***P<0.001 versus respective pre IVM condition,
Bonferroni’s post-hoc test.

Time-course of IVM’s effect on alcohol intake in male mice. IVM has been previously
reported to have slow absorption, with a wide distribution. The clearance of IVM is slow, which
may be attributed to a low level of metabolism (Merck et al., 1988). The normal anti-parasitic
dosage is 200 µg/kg. However, for this study we evaluated the time course of IVM’s effect on
hourly ethanol intake (10E versus water) when used at a dose of 10 mg/kg. IVM or saline was
administered 1h before the first reading. As expected, 10E intake increased significantly across
time [F(9,190) = 80.89, p<0.001] and was significantly reduced following IVM pre-treatment
33

[F(1,190) = 37.33, p<0.001]. The interaction between time and IVM dose was not significant.
We conducted planned comparisons, which confirmed that ethanol consumption was
significantly decreased at 9 and 10 h after the administration of IVM (Fig. 2.3). This correlated
well with the time to achieve maximal concentration or Tmax, which was found to be at 8-10 h
following IVM administration.
Figure 2.3
0 1 2 3 4 5 6 7 8 9 10 11
0
1
2
3
4
5
6
7
Saline
IVM
**
**
Time After Injection (h)
10E Intake (g/kg/24-h)

Figure 2.3. IVM (10 mg/kg) administered to male C57BL/6J mice significantly reduced 10E intake approximately 9
hours after IVM administration. The intake was measured on an hourly basis, up to 10 hours following IVM (closed)
or saline (open) administration. Values represent the mean ±SEM cumulative intake for 10-11 male mice per
treatment group. **P<0.01 versus saline-treated group, Bonferroni’s post-hoc test.

Pharmacokinetics of IVM in plasma and brain. To determine a pharmacokinetic-
pharmacodynamics (PK/PD) relationship, we conducted an optimal dose finding study, where
six mice were assigned to each dosage group (0, 0.25, 2.5 and 10 mg/kg). The AUC for plasma
and brain IVM was dosage proportional (Fig 2.4). Plasma and brain IVM AUC also correlated
with the percentage reduction of ethanol consumption (Table 2.1). Specifically, the lowest IVM
dosage leading to detectable IVM in the brain was found at 2.5 mg/kg, which corresponded to
lowest IVM dosage to reduce ethanol consumption. At this dosage, the IVM AUC in plasma and
brain was 155 ng*hr/mL and 0.28 ng*hr/mg of tissue, respectively.
34

Figure 2.4

Figure 2.4. IVM AUC in plasma and brain tissue was determined following injection of various IVM dose groups.
A dose-dependent IVM AUC was found in the plasma (open bars) and brain tissue (black bars).

Table 2.1
IVM Dose
(mg/kg)
% Reduction of
Ethanol
Consumption
IVM Plasma
AUC
0-8

(ng*hr/mL)
IVM Brain
AUC
(ng*hr/mg)
0.25 No Effect 48.91 9.6X10
-05

2.5 32 % 155.56 2.8X10
-01

10 > 50% 2928.31 2.20X10
00

Table 2.1. Three doses of IVM were evaluated to determine the IVM disposition in both plasma and brain tissue.
IVM produced a dose-dependent increase in detectable IVM levels in plasma and brain tissue.

IVM administration reduced alcohol intake and preference in female mice. The
effects of IVM on alcohol intake and preference were also examined in female mice, as they
typically drink higher levels of alcohol than that of males (Finn et al., 2004; Yoneyama et al.,
2008). In agreement with previous investigations (Finn et al., 2004; Yoneyama et al., 2008), the
degree of 10E intake in female mice was greater than what we observed in male mice (females
~15 g/kg; males ~10 g/kg). As presented in Fig. 2.5A, IVM significantly reduced ethanol intake
in a dose dependent manner [F(6,252) = 20.16, p<0.001]. When the analysis was conducted
0.00 0.25 2.50 10.00
0.001
0.01
0.1
1
10
100
1000
10000
IVM Plasma AUC
0-8
(ng/mL*hr)
IVM Brain AUC
0-8
(ng/mg*hr)
IVM Dose (mg/kg)
AUC
IVM
(ng*hr/mL)
35

across time (pre-IVM, IVM, post-IVM), there was a significant effect of IVM on ethanol intake
[F(2,252) = 79.53, p<0.001]. The interaction between time and dose was significant [F(12,252)
= 7.50, p<0.001]. Bonferroni post-hoc comparisons versus pre-IVM indicated that the highest
dose of IVM tested (10 mg/kg) significantly reduced ethanol intake by approximately 50%
(t=8.214, p<0.001). Lower doses of IVM also significantly reduced ethanol intake (i.e., 2.5
mg/kg ~30% (t=4.735, p<0.001), 5.0 mg/kg ~66% (t=8.536, p<0.001), 7.5 mg/kg ~56%
(t=8.318, p<0.001). Intake of 10E remained significantly lower than pre IVM levels on the day
after IVM injection of the 5.0, 7.5 and 10 mg/kg doses (data not shown). However, over the
following 3-day period (post IVM), 10E intake in all groups of animals was similar to their pre
IVM values (pre IVM exposure), and was significantly higher than 10E intake in presence of
IVM (data not shown).
In addition, we found that IVM decreased 10E preference. Analysis of the data across
time revealed a significant decrease in preference for ethanol [F(2,252) = 77.80, p<0.001].
Moreover, the analysis of IVM dose indicated that IVM significantly reduced alcohol preference
in a dose dependent manner [F(6,252) = 10.45, p<0.001]. There was a significant interaction
between time and IVM dose [F(12,252) = 10.11, p<0.001], with Bonferroni post-hoc tests
confirming a significant decrease in ethanol preference ratio following IVM doses of 2.5 mg/kg
(t=3.323, p<0.01), 5.0 (t=9.650, p<0.001), 7.5 (t=8.154, p<0.001) and 10 mg/kg (t=9.470,
p<0.001) (Fig. 2.5B). With regard to the effects of IVM on water intake, conducting the
analyses across IVM dose, [F(6,252) = 3.27, p<0.01] and time [F(2,252) = 17.80, p<0.001]
revealed significant effects of IVM on water intake. The interaction between time and IVM dose
also was significant [F(12,252) = 6.55, p<0.001]. Post hoc tests revealed a significant difference
in water intake following IVM doses of 5.0 mg/kg (t=3.752, p<0.01), 7.5 mg/kg (t=3.764,
36

p<0.01) and 10 mg/kg (t=7.196, p<0.001) (Fig. 2.5C). Finally, when investigating the effects of
IVM on total fluid intake across time, we found a significant reduction [F(2,252) = 35.34,
p<0.001]. The analysis of IVM dose also confirmed a significant effect of IVM on fluid intake
[F(6,252) = 8.13, p<0.001]. Furthermore, there was a significant interaction between time and
dose [F(12,252) = 4.78, p<0.001], with post-hoc comparisons confirming that fluid intake
decreased after IVM doses of 2.5 mg/kg (t=3.988, p<0.001), 5.0 mg/kg (t=5.694, p<0.001), and
7.5 mg/kg (t=5.342, p<0.001) (Fig. 2.5D).
Figure 2.5

37

Figure 2.5. IVM dose response study in female C57BL/6J mice using a 24-h access two-bottle choice paradigm.
Each dose of IVM was administered after achieving stabilized drinking for 3 consecutive days. Bars represent levels
from the day prior to IVM injection (white; Pre IVM) and the day of the IVM injection (black; IVM). A) IVM (2.5-
10 mg/kg) significantly reduced 10E intake and B) preference ratio for 10E. The effects of IVM on water and total
fluid intake are presented in panels C and D, respectively. Values represented the mean ±SEM for 19 mice per dose
group. **P<0.01, ***P<0.001 pre IVM condition, Bonferroni’s post-hoc test.

Multiple dosing of IVM administration reduced alcohol intake and preference in
female mice. The aforementioned studies focused on acute administration of IVM (i.e., a single
administration of individual IVM doses). However, alcoholism is a chronic disorder, and as
such, treatment strategies most likely would need to be long lasting. To begin to investigate the
utility of IVM for use as an anti-alcohol agent, we tested the effects of IVM (1.25 mg/kg/day)
administered daily for 7 days (i.p.) on 10E intake and preference. As presented in Fig. 2.6, 7
daily administrations of IVM significantly reduced alcohol intake, when average 10E intake
across the 7 days was analyzed (pre IVM, IVM, post IVM) [F(2,27) = 7.974, p<0.01]. The
degree of reduced ethanol drinking over the 7 day period of IVM administration was somewhat
varied, ranging from approximately 11-27%, but did not significantly differ between days and no
overt changes in behavior, food intake or water intake were observed (data not shown). IVM
(1.25 mg/kg), however, did not significantly reduce 10E preference (data not shown). The
reduction in alcohol intake was accompanied by increases in average water consumption that
ranged from no change to 26%, but these changes were not significantly different from pre IVM
levels (data not shown). There was a significant change in average total fluid intake [F(2,27) =
8.862, p<0.001], with a degree of day to day variability ranging from a 20% decrease to a 5%
increase over the 7 day testing period (data not shown).

38

Figure 2.6
Pre IVM IVM Post IVM
0
5
10
15
**
10E Intake (g/kg/24-h)

Figure 2.6. Daily administration of IVM (1.25 mg/kg/day X 7 days) reduced 10E intake in female C57BL/6J mice
using a 24-h access two-bottle choice paradigm. After achieving stable drinking levels for 3 consecutive days, IVM
was administered for 7 consecutive days. Bars represent levels from the day prior to IVM injections (white; Pre
IVM), the 7 day average of the IVM injection (black; IVM) and the day after the IVM injections (gray; Post IVM).
Values represented the mean ±SEM for 11 mice. **P<0.01 versus Pre-IVM, Tukey multiple comparison post-hoc
test.

IVM reduced saccharin consumption in female mice. To begin to determine whether
the effects of IVM were selective for alcohol or could be generalized to other potential
reinforcers, we investigated the ability of IVM to reduce saccharin (0.033%) intake and
preference. As presented in Fig. 2.7A, we found that 2.5 mg/kg IVM resulted in a significant
reduction in saccharin consumption by approximately 31% [F(2,54) = 20.82, p<0.001]. In
contrast, 2.5 mg/kg IVM did not significantly decrease saccharin preference (data not shown).
This dose of IVM did not significantly affect water intake (data not shown), but did significantly
reduce total fluid intake by approximately 29% [F(2,54) = 24.82, p<0.001]. A second study
extended our investigation by testing the effects of 5 mg/kg of IVM on saccharin intake. We
found that 5 mg/kg IVM significantly reduced saccharin consumption by more than 45%
[F(2,54) = 52.83, p<0.001] (Fig. 2.7B). In addition, preference for the saccharin solution was
significantly decreased by about 7% [F(2,54) = 4.761, p<0.05] (data not shown). In agreement
with our 2.5 mg/kg saccharin intake study, 5.0 mg/kg IVM did not significantly affect water
intake (data not shown), but did significantly reduce total fluid intake by approximately 42%
39

[F(2,54) = 52.70, p<0.001)]. IVM did not significantly alter animal weight or food intake as
compared to baseline (data not shown).
Figure 2.7

Figure 2.7. IVM administration reduced saccharin (0.033% w/v) intake in female C57BL/6J mice using a 24-h
access two-bottle choice paradigm following doses of A) 2.5 mg/kg and B) 5 mg/kg. After achieving stable
drinking levels for 3 consecutive days, IVM was administered (separate studies for each dose). Bars represent levels
from the day prior to IVM injection (white; Pre IVM), the day of the IVM injection (black; IVM) and the day after
the IVM injection (gray; Post IVM). Values represent the mean ±SEM for 19 mice per dose group. ***P<0.001
versus Pre IVM, Tukey multiple comparison post-hoc test.

IVM decreased alcohol intake by 50% in an intermittent limited access paradigm in
female mice. We extended our investigation to a second model of alcohol intake with the use of
an intermittent limited access paradigm. Using this model, the animals have access to ethanol for
4 h during the circadian dark phase (Lowery et al., 2010; Neasta et al., 2010; Rhodes et al.,
2005), which results in the mice reaching high, pharmacologically active blood ethanol
concentrations (BECs) in a short period of time as found in binge-like drinking behavior (i.e.,
>80 mg%). We utilized the 10 mg/kg dose, since it was the most effective dose in the 24 h two-
bottle choice studies, and it was the highest dose that could be given without introducing possible
CNS toxicity (Lerchner et al., 2007; Merck et al., 1988).
Prior to the initiation of saline injections, baseline 10E intake during the 4h session was
5.12 ± 0.22 g/kg in the female mice (n = 8). Control, saline injections did not significantly alter
40

baseline alcohol intake in these mice (Fig. 8). Acute administration of 10 mg/kg IVM
(administered 6-h prior to the start of the alcohol drinking session) was analyzed (pre IVM, IVM,
post IVM) and found to significantly reduced alcohol intake in excess of 45%, when compared
with pre IVM injections [F(2,23) = 17.64, p<0.001] (Fig. 8). We found that the mice resumed
drinking 10E at levels comparable with their pre IVM drinking levels within 1 drinking session
post IVM treatment. This contrasts with our findings from the above 24 h access studies, where
the drinking levels did not return to pre IVM levels until 4-5 days post IVM treatment, and
suggests that there may be differences in the persistence of IVM’s suppression in ethanol intake
with continuous versus intermittent and limited access procedures.
Figure 2.8
Pre IVM IVM Post IVM
0.0
1.0
2.0
3.0
4.0
5.0
6.0
***
Treatment
10E Intake (g/kg/4hr)

Figure 2.8. 10 mg/kg IVM administration reduced ethanol (10% v/v) intake in female C57BL/6 mice using an
intermittent, limited (4-h) access paradigm. After attaining stable drinking levels, IVM was administered. Bars
represent levels from the day prior to IVM injection (white; Pre IVM), the day of the IVM injection (black; IVM)
and the day after the IVM injection (gray; Post IVM). Values represent the mean + SEM for 8 mice. ***P<0.001
versus Pre IVM, Tukey multiple comparison post-hoc test.

Significant reduction in ethanol but not sucrose operant self-administration
following IVM in male mice. Prior to the initiation of saline injections, baseline intake during
the 30 min session was 1.00 ± 0.14 g/kg for the 10E reinforced mice (n=7) and was 0.49 ± 0.06
g/kg for the 2S reinforced mice (n=10). Pretreatment with IVM did not significantly alter
responding on the active lever, as all animals were able to complete the RR16 and gain access to
the reinforcer. However, IVM did not alter intake of the 2S or 10E solutions (data not shown)
41

during the 30 min sessions. Analysis of first bout dynamics revealed no effect of IVM on any
bout parameters examined (not shown).
One advantage of the 30 min operant sessions (following access to the reinforcer) is the
ability to capture changes in ethanol intake when using drugs with a short half-life. Because this
was not a concern with IVM and because we thought that that the low intake and short time
frame inherent in a 30 min session might decrease the ability to detect a change in self-
administration following IVM, we tested the effects of the 10 mg/kg IVM dose (most effective
dose in other models) in a 60 min operant self-administration session in the same animals.
Mice rapidly adjusted to the 60 min operant sessions, and baseline intake was 1.16 ± 0.18
g/kg for the ethanol-reinforced mice (n=7) and was 0.73 ± 0.07 g/kg for the sucrose-reinforced
mice (n=8). Pretreatment with IVM did not significantly alter responding on the active lever, as
all animals were able to complete the RR16 and gain access to the reinforcer. Planned
comparisons revealed a trend for IVM to decrease intake of 10E by 13% (p=0.06, Fig. 2.9A),
while having no effect on sucrose intake (data not shown). Analysis of first bout dynamics
revealed that IVM differentially affected latency to reinforcer in the 10E- versus the 2S-
reinforced mice (a significant interaction between group and IVM [F(1,12) = 5.97, p<0.05]; not
shown). Subsequent analyses revealed that IVM produced a significant 30% decrease in latency
to reinforcer only in the 10E reinforced mice (p<0.05; not shown). Similar results were seen
with analysis of latency to first bout (not shown). However, first bout size was not significantly
altered by IVM, nor was there a significant alteration in the average bout size, bout frequency, or
lick rate (not shown). Finally, bin analysis was conducted to examine how IVM was affecting
the pattern of reinforcer consumption across 10 min bins (not shown). Importantly, the
correlation between licks and intake (g/kg; across the two days when animals received saline or
42

10 mg/kg IVM) was highly correlated for the 10E- (r=0.93, p=0.003, n=7) and 2S-reinforced
(r=0.85, p=0.007, n=8). The high correlations confirmed that the analysis of lick behavior was
highly predictive of reinforcer consumption. Bin analysis revealed that IVM altered 10E licks in
a consistent (no bin by dose interaction) and modest manner (trend for effect of IVM dose =
F(1,6) = 3.75, p=0.10) across the 10 min bins. There was no main effect of IVM or interaction
between IVM and bin on 2S licks (not shown).
Figure 2.9

Figure 2.9. Effect of IVM on operant self-administration of 10% ethanol (10E) in male C57BL/6J mice during 60
minute sessions. IVM tended to decrease 10E intake in animals with a long history of ethanol self-administration
(panel A) or in animals that were experimentally naïve (panel B). Importantly, the data from the two cohorts did not
differ, so the combined data indicated that IVM significantly decreased 10E intake (panel C). Panel D depicts the
effect of IVM injection on the temporal distribution of 10E licks in 10 min intervals across the 60 min session in the
combined data from the two cohorts (n=16). Values represent the mean ± SEM for the numbers of animals in
parentheses for panels A-C.
+
p ≤ 0.10, *p < 0.05 versus saline (0 mg/kg), paired t-test.

Due to concern that the prolonged ethanol history in the above cohort may have blunted
the effects of IVM, a second study examined the effect of the 10 mg/kg IVM dose (or saline) on
43

self-administration of 10E during a 60 min session in experimentally naïve mice. Baseline
intake was 1.06 ± 0.22 g/kg (n = 9), and pretreatment with IVM did not significantly alter
responding on the active lever. All mice were able to complete the RR16 and gain access to the
10E solution. IVM reduced 10E intake by 31% [F(1,8) = 3.30, p=0.10] (Fig. 2.9B). No bout
parameters reached statistical significance with the exception of a trend for a decrease in first
bout size (p<0.10). Bin analysis (not shown) revealed a bin by dose interaction [F(5,40) = 9.14,
p=0.004], where 10 mg/kg IVM decreased intake primarily during bin 1 (p=0.025) and bin 2
(p=0.005).
Importantly, the difference in ethanol history between the 2 cohorts did not appear to
significantly alter the effect of IVM on 10E intake. This conclusion is supported by analyses that
were conducted across studies (referred to as “pass”) to determine whether the data could be
collapsed. There was no dose by pass interaction [F(1,14) = 0.666, p=0.43] or a main effect of
pass (p = 0.46) on g/kg intake. Similarly, bin analysis of the effect of pass revealed no dose by
bin by pass interaction [F(5,70) = 0.80, p=0.554] or a main effect of pass (p=0.91). Analysis on
the collapsed data revealed that IVM significantly decreased 10E intake by 23% [F(1,15) = 5.71,
p=0.03] (Fig. 2.9C). Additionally, there was a significant dose by bin interaction (Fig. 2.9D),
with post hoc tests indicating that 10 mg/kg IVM significantly decreased licks during bin1
(p=0.003) and bin 2 (p=0.05).

DISCUSSION
The present study tested the hypothesis that IVM reduces ethanol intake and preference in mice
and used several animal models of ethanol self-administration. In support of the hypothesis, we
found that IVM significantly reduced alcohol intake following acute IVM administration using
44

24-h access and intermittent limited access procedures as well as in an operant self-
administration procedure. Interestingly, administration of a sub-threshold IVM dose daily for 7
days also significantly decreased 24 h ethanol intake. Pharmacokinetic studies demonstrated that
IVM was detected in brain tissues. Brain IVM was correlated to the anti-alcohol intake effects
of IVM at a dosage of 2.5 mg/kg and the effects of IVM on ethanol intake correlated with the
presence of drug. Taken together, the findings indicate that IVM reduces alcohol intake across
several different models of self-administration, albeit with differences in efficacy, and support
the notion that IVM may be useful in the treatment of AUDs.
Acute administration of IVM decreased 24-h ethanol intake and preference in male and
female mice in a dose-dependent manner. A separate study confirmed that the reduction in
alcohol intake reached statistical significance approximately 9 h after IVM administration, which
corresponded with the Tmax (8-10 h) in both the plasma and brain compartments. Moreover,
IVM plasma half-life of 8-12 h was compatible with the duration of the decrease in ethanol
consumption, because ethanol intake did not return to baseline levels until approximately 72 h
post-IVM dosage. At this time point, IVM is expected to be over 99% eliminated (Merck et al.,
1988).
Acute administration of IVM also significantly reduced higher levels of alcohol drinking
when tested using an intermittent limited-access model. This second model results in BECs that
mimic excessive or high levels of alcohol drinking. For instance, 4 h ethanol intake of 7 ± 2 g/kg
in male C57BL/6J mice was associated with a BEC of 97 ± 9 mg% (Neasta et al., 2010) in one
recent study. Similar results were reported by a different group in C57BL/6J male mice. In this
study 4 h ethanol intake resulted in approximately 5-6 g/kg and was associated with BECs of
approximately 100-125 mg% (Lowery et al., 2010). In female C57BL/6J mice, 4 h ethanol
45

intake of 7.3 ± 0.6 g/kg was associated with a BEC of 92 ± 16 mg% (Rhodes et al., 2007). Since
we did not measure BECs in the present study, we do not know if the IVM-induced reduction in
high alcohol intake corresponded to a decrease in BEC from binge to non-binge levels. Future
studies will be necessary to examine the therapeutic potential of IVM to reduce binge drinking,
based on a reduction in BEC to non-binge levels.
IVM significantly reduced alcohol-drinking behavior with similar efficacy in male and
female mice. This lack of difference suggests that sex-related hormonal differences do not
influence IVM’s reduction in alcohol drinking. We did not monitor the stage of the estrous cycle
in the female mice during testing. We based this decision on previous studies in which we did
not observe systematic changes in ethanol intake across weeks of baseline consumption or
following repeated vehicle injections in female C57BL/6 mice [e.g., (Ford et al., 2008)] and on
earlier reports that estrous cycle-related changes in operant ethanol self-administration were not
observed in female rats that were allowed to cycle freely (Roberts et al., 1998). Thus, it is
unlikely that an estrous cycle-related change in ethanol intake in the control conditions
confounded interpretations of the data following IVM pretreatment. More important, the
findings suggest that estrous cycle may not influence the clinical application of IVM for
treatment of AUDs.
IVM significantly reduced ethanol intake in the operant self-administration paradigm,
when the data were collapsed across studies. Importantly, the difference in ethanol history
between the 2 cohorts did not appear to significantly alter the effect of IVM on 10E intake.
However, the reduction in ethanol self-administration was not of the same magnitude as was
seen with other measures of ethanol intake where fluid availability was not behaviorally
contingent. We believe that these differences reflect the short duration of the operant sessions,
46

rather than whether (or not) instrumental responding was required by the animal to gain fluid
access. That is, the operant self-administration procedure resulted in 1-h of fluid access versus
the home cage drinking with 24-h or intermittent (4-h) access procedures. This is supported by
the bin analysis, which showed a decrease in ethanol self-administration that was still suppressed
at 60 min. Further, this modest effect of a 23% decrease in ethanol intake was consistent with
recent work in rats where the 10 mg/kg IVM dose produced a 17.8% decrease in the number of
sweetened ethanol reinforcers (10E+2S; but intake not reported) earned on a progressive ratio
schedule when animals were responding in 3 h sessions (Kosten, 2011). In the present 1-h
sessions, all animals were able to complete the response requirement to gain access to the
reinforcer following the 10 mg/kg IVM dose, suggesting that IVM was not exerting a non-
specific effect on motor activity with the 6 – 8 h pretreatment time. Taken in conjunction with
the lengths of the single published operant self-administration study [3-h; (Kosten, 2011)] and
our current findings with two drinking models (4-h and 24-h), it is possible that IVM would have
exerted a greater reduction in operant ethanol self-administration during a longer session.
Another consideration with regard to oral consumption of ethanol in animals is that
sensory modalities such as taste, olfaction, and chemosensory irritation play an important role in
the acceptance or rejection of the alcohol solution [e.g.,(Bachmanov et al., 2003)]. Introducing
ethanol into a sweet solution facilitated the initiation of reliable operant ethanol self-
administration in rodents in the absence of factors such as food or fluid restriction or post-
prandial sessions [e.g., (Middaugh and Kelley, 1999; Samson, 1986) and the present study] and
increased voluntary ethanol consumption in alcohol-avoiding mouse strains [e.g., (Belknap et al.,
1993; Yoneyama et al., 2008)]. Additionally, mutant mice with a deletion in a gene critical in
taste transduction significantly decreased preference for alcohol and saccharin solutions without
47

altering consumption of sodium chloride (Blednov et al., 2008). These findings are consistent
with studies demonstrating a strong association between consumption of ethanol and sweetened
fluids [e.g., (Belknap et al., 1993; Kampov-Polevoy et al., 1999)]. Related to this point, we
found that the regimens of IVM that attenuated alcohol consumption also significantly decreased
saccharin intake. These data suggest that the ability of IVM to reduce alcohol intake may
partially reflect IVM’s ability to negatively modulate the reinforcing and/or chemosensory
properties of several rewarding stimuli. This possibility is strengthened by the finding that water
intake was significantly increased by IVM, which rules out that the observed phenomena may
stem from a generalized hypodipsia. Nonetheless, a future study that examines the manner in
which the 10 mg/kg IVM dose alters the dose-response function for ethanol and saccharin by
testing across different concentrations of these fluids would provide insight regarding the
interaction between IVM and taste sensitivity.
The mesolimbic DA reward system plays a critical role in directing reward seeking and
motivational behavior through the regulated release of DA (Di Chiara and Imperato, 1988;
Gonzales et al., 2004; Koob, 2009; Weiss et al., 1993). And, recent preclinical studies clinical
trials have begun focusing on pharmacotherapies that exert their effects by cortico-mesolimbic
DA system modulation [for review see (Johnson, 2010)]. P2X4Rs are expressed in the cortico-
mesolimbic DA system (Amadio et al., 2007; Krügel et al., 2003) and are sensitive to
physiologically relevant concentrations of ethanol (Ostrovskaya et al., 2011; Xiao et al., 2008).
As such, it is plausible to propose that P2X4Rs play a role in the signaling cascades involved in
alcohol consumption and addiction. In support of this contention, we recently reported that
P2XRs can modulate ethanol’s effect on GABAergic synaptic transmission of DA neurons in the
VTA (Xiao et al., 2008). Additional evidence supporting the importance of P2X4Rs comes from
48

recent studies reporting that low functional expression of p2rx4 gene is associated with high
alcohol preference (Kimpel et al., 2007; Tabakoff et al., 2009). This concept appears to be
supported by our preliminary findings on male p2rx4 knockout (KO) mice, which exhibit
significant increases in alcohol intake (unpublished data). Taken together, the findings suggest
that the efficacy of IVM in reducing alcohol intake involves, in part, its ability to block the
action of ethanol on P2X4Rs.
In mammals, IVM is also purported to act on GABA
A
and glycine receptors [e.g., see
(Dawson et al., 2000; Shan et al., 2001; Spinosa et al., 2002)] and nicotinic acetylcholine
receptors (nAChRs) (Krause et al., 1998; Sattelle et al., 2009). All of these receptor families
have been linked to the behavioral effects of ethanol (Asatryan et al., 2011; Davies, 2003;
Kimpel et al., 2007; Perkins et al., 2010; Vengeliene et al., 2008). Although the current findings
do not shed light on the role that other receptors may play in causing the IVM-mediated anti-
alcohol effects, it is possible that the reduction in alcohol intake may reflect a combined effect of
IVM on several receptors, including GABA
A
Rs, nAChRs and P2X4Rs. Additional studies are
necessary before definitive conclusions can be drawn.
Collectively, the present findings indicate that IVM might be useful in the treatment of
AUDs. The potential translation of the present findings and repurposing of IVM for use in this
manner is aided by the twenty plus year history of IVM’s use to treat parasitic diseases in
millions of humans (Burkhart, 2000; Guzzo et al., 2002; Omura, 2008). IVM has an excellent
safety profile when used as an anthelmintic (Burkhart, 2000; Davis et al., 1999; Omura, 2008).
Accordingly, the literature on IVM attests to the safety and relative lack of toxicity of IVM over
this period during which billions of doses of IVM-containing products have been used
worldwide (Boxall and Long, 2005). Mild to moderate CNS adverse events have been
49

associated with IVM. However, the incidences of these adverse events are rare and appear to be
linked to alteration of p-glycoprotein (Pgp) expression, which is found abundantly in the blood
brain barrier (BBB) (Edwards, 2003; Geyer et al., 2009; Lespine et al., 2009; Lespine et al.,
2007; Sun et al., 2010).
The lack of adverse events with IVM in its current use as an anti-parasitic may be
attributed to the manner in which it is dosed—200 mg once yearly or intermittently. However,
doses up to 10X the recommended dosage (i.e., 2.0 mg/kg/day) have been safely tested in
clinical trials (Guzzo et al., 2002). In rodents, doses less than 10 mg/kg IVM IV did not cause
visible CNS depression (Trailovic and Trailovic, 2010), whereas lethality (i.e., LD
50
) is reported
to be between 25-50 mg/kg depending on the sex and species tested (Merck et al., 1988).
The current studies tested doses more like the non-toxic rodent studies described in the
previous paragraph. Importantly, we did not observe behavioral changes or other overt signs of
toxicity in our studies in which multiple day (7 d) dosing of 1.25 mg/kg IVM elicited a continued
reduction in drinking levels. This initial finding, combined with the safety record and other
studies of IVM described above suggests that the longer term IVM dosing as would be expected
for treating AUDs, would not produce significant adverse effects. However, this issue requires
further investigation.

CONCLUSION
The present findings indicate that IVM reduces alcohol intake across several different models of
self-administration in both male and female mice. This suggests that IVM may be useful in the
treatment of AUDs. The ongoing widespread commercial use of IVM treatments for parasitic
infections has led recent efforts to expand the approved uses of IVM in humans as noted by
50

searching the Clinicaltrials.gov Website. However, it is important to point out that these efforts
are focusing on using IVM for new indications and new dosing strategies (including multiple day
dosing strategies) linked to various antiparasitic actions and not for affecting alcohol
consumption.
In view of the current widespread use of IVM as an anthelmintic drug and recent efforts
to expand the use of IVM for other indications, the development of IVM as an anti-alcohol agent
may represent a fast and economically advantageous approach. In addition, our results highlight
IVM as a novel lead structure for the development of novel anti-alcohol agents. Future
preclinical and clinical research will be necessary to elucidate the significance of IVM-mediated
effects and the potential clinical use of this drug in alcohol use.

51

CHAPTER 3
PHARMACOLOGICAL INSIGHTS INTO THE ROLE OF P2X4 RECEPTORS IN
BEHAVIORAL REGULATION: LESSONS FROM IVERMECTIN

ABSTRACT
Purinergic ionotropic P2X receptors are a family of cation-permeable channels that bind
extracellular adenosine 5’-triphosphate (ATP). In particular, convergent lines of evidence have
recently highlighted P2X4 receptors as a potentially critical target in the regulation of multiple
nervous and behavioral functions, including pain, neuroendocrine regulation and hippocampal
plasticity. Nevertheless, the role of the P2X4 receptor in behavioral organization remains
poorly investigated. To study the effects of P2X4 activation, we tested the acute effects of its
potent positive allosteric modulator ivermectin (IVM, 2.5-10 mg/kg, i.p.) on a broad set of
paradigms capturing complementary aspects of perceptual, emotional and cognitive regulation
in mice. In a novel open field, IVM did not induce significant changes in locomotor activity,
but increased the time spent in the peripheral zone. In contrast, IVM produced anxiolytic- like
effects in the elevated plus maze and marble burying tasks, as well as depression- like behaviors
in the tail-suspension and forced swim tests. The agent induced no significant behavioral
changes in the conditioned place preference test and in the novel object recognition task.
Finally, the drug induced a dose-dependent decrease in sensorimotor gating, as assessed by
prepulse inhibition (PPI) of the acoustic startle reflex. In P2X4 knockout mice, the effects of
IVM in the open field and elevated plus maze were similar to those observed in wild type mice;
conversely, the drug significantly increased startle amplitude and failed to reduce PPI. Taken
52

together, these results suggest that P2X4 receptors may play a role in the regulation of
sensorimotor gating.

INTRODUCTION
Purinergic ionotropic P2X receptors are a family of hetero- and homotrimeric cation-
permeable channels that bind extracellular adenosine 5’-triphosphate (ATP) (North, 2002). The
biophysical and pharmacological properties of P2X receptors are defined by their subunit
composition (Khakh and North, 2006). In particular, of the seven P2X subunits characterized to
date (termed P2X1 through P2X7), recent evidence has highlighted P2X4 subunits as a
potentially interesting target in the regulation of nervous functions. P2X4 subunits form
homotrimeric receptors with distinct functional properties (Bo et al., 1995; Soto et al., 1996), as
well as heteromers with other P2X subunits (Guo et al., 2007; Nicke et al., 2005; Ormond et al.,
2006) (for contrasting results, see (Antonio et al., 2011)). P2X4 receptors are abundant in the
central nervous system (Rubio and Soto, 2001; Tsuda et al., 2003) and involved in the regulation
of neuropathic pain (Tsuda et al., 2003; Ulmann et al., 2008; Zhang et al., 2006), neuroendocrine
regulation (Zemkova et al., 2010) and hippocampal plasticity (Baxter et al., 2011; Lorca et al.,
2011; Sim et al., 2006). Furthermore, P2X4 receptors have been recently shown to mediate
inhibition of γ-amino-butyric acid (GABA)
A
(Jo et al., 2011) and modulation of N-methyl-D-
aspartate glutamate (Baxter et al., 2011) receptors. Recent studies have documented that some
antidepressants may inhibit P2X4 receptors (Nagata et al., 2009), although this evidence remains
controversial in view of contrasting findings (Sim and North, 2010; Toulme et al., 2010).
Furthermore, converging lines of in vitro, and in vivo evidence suggest that P2X4 receptors may
53

be an important therapeutic target for alcohol use disorders (Asatryan et al., 2011; Asatryan et
al., 2010; Kimpel et al., 2007; Popova et al., 2010; Tabakoff et al., 2009).
In contrast with this background, the pharmacological armamentarium to probe P2X4
receptors is very limited. P2X receptors are poorly sensitive to P2X receptor antagonists, such as
suramin and pyridoxal phosphate-6-azo-(benzene-2,4-disulfonic acid) (PPADS) (Buell et al.,
1996a). Conversely, research has shown that the antiparasitic ivermectin (IVM) acts as a potent
positive allosteric regulator of P2X4 in either its homomeric or heteromeric configurations, but
does not interact with other P2X receptors (Khakh et al., 1999b; Priel and Silberberg, 2004).
IVM is a mixture of semisynthetic macrocyclic lactone disaccharides derived by hydroxylation
of avermectins B
1a
and B
1b
, natural fermentation products of the actinomycete Streptomyces
avermitilis. IVM is widely adopted in human and veterinarian medicine as a broad-spectrum
anthelmintic and insecticide agent (Geary, 2005; Richard-Lenoble et al., 2003), with high
therapeutic index and limited side effects. The antiparasitic mechanism of IVM is based on the
activation of glutamate-gated chloride channels specific to invertebrates (Cully et al., 1994;
Vassilatis et al., 1997).
In mammalians, in addition to its action on P2X4 receptors, IVM has been shown to
activate GABA-A receptors and exert weak effects on the positive modulation of other
ionotropic channels, including glycine, and acetylcholine nicotinic receptors (Collins and Millar,
2010; Dawson et al., 2000; Krause et al., 1998; Sattelle et al., 2009; Shan et al., 2001; Sung et
al., 2009).
Previous studies have revealed that long-term administration of IVM has subtle
behavioral effects on locomotor and sensory reactivity of mice (Davis et al., 1999); furthermore,
this substance has been shown to exert anxiolytic-like properties in rats (Spinosa et al., 2002).
Capitalizing on our discovery that IVM antagonizes ethanol-mediated inhibition of P2X4
54

receptors (Asatryan et al., 2010 ), we recently found that this compound reduces alcohol
consumption (Yardley et al., 2012). Nevertheless, to the best of our knowledge, the acute
behavioral effects of IVM have not been the object of systematic investigations. Thus, in
consideration of the potential role of P2X4 receptors in the regulation of brain functions, we
addressed the present study to determine the perceptual, emotional and cognitive impact of IVM
in C57/BL6 mice, using a broad spectrum of well-validated paradigms targeting complementary
aspects of behavioral regulation.

MATERIALS AND METHODS

Animals. Studies were performed on drug-naïve, C57BL/6J (Jackson Laboratory, Bar
Harbor, ME) wild type (WT) and P2X4 knockout (KO) male mice, aged 6-10 weeks at the time
of testing, were used. P2X4 KO mice were generated and genotyped according to previous
protocols (Sim et al., 2006). Animals were acclimated to the housing facility for a minimum of
one week and group-housed (4 mice per cage) in polycarbonate cages, with ad libitum access to
food and water. The holding room was maintained at approximately 22°C with a 12:12h
light:dark cycle with lights on at 6:00 AM. All procedures were in compliance with the National
Institute of Health guidelines, and the protocols were approved by the University of Southern
California Institutional Animal Care and Use Committee.
Generation and identification of knockout P2X4
-/-
mice. The current P2X4 KO colony
in our laboratory was started by rederivation (USC transgenic core, Los Angeles, CA) of frozen
heterozygous P2X4
+/-
embryos (via surrogate female C57BL/6 mice (Jackson Labs, Bar Harbor,
ME). Presently, we are maintaining the P2X4R KO colony using 6
th
generation heterozygous
P2X4
+/-
backcrossed onto C57BL/6J background. Upon weaning, offspring are genotyped,
separated by sex, and maintained in groups of 4 in individually ventilated cages with free access
to food and water under a 12:12 hr light/dark cycle at 26 ± 1°C.
55

Drugs. IVM (Norbrook Inc.,Lenexa, KS) and its vehicle propylene glycol (Alfa Aesar,
Ward Hill, MA) were diluted in a 0.9% saline solution, to a concentration that would allow for
an injection volume of 0.01 ml/g of body weight. Throughout all experiments, IVM was injected
8 h before behavioral testing, as our previous studies documented that the maximal efficacy and
the Tmax of the drug in brain and plasma was reached at this time (Yardley et al., 2012).
Preliminary physical assessment. The impact of IVM in mice was tested on posture,
gait, heart rate, breathing frequency and neurological reflexes (righting reflex, postural reflex,
eye-blink reflex and whisker-orienting reflex) (Davis et al., 1999).
Sticky tape test. Mice were briefly restrained and a circular piece of tape was placed on
the bottom of each forepaw. Mice were then released, and the latency to remove the first piece
of tape was recorded.
Hot plate. Mice were individually exposed to a hot plate (IITC Life Science, Woodland
Hills, CA) at 47.5°C and 50 °C, and the latency to lick their paws was measured. We used a cut-
off time of 40 s to eliminate any potential tissue damage.
Novel open field. The open field was a Plexiglas square grey arena (40 x 40 cm)
surrounded by 4 black walls (40 cm high). On the floor, two concentric zones of equivalent areas
were defined: a central square quadrant and a peripheral frame directly adjacent to the walls.
Mice were placed in the center and their behavior was monitored for 5 min. Analysis of
locomotor activity was performed using Ethovision (Noldus Instruments, Wageningen, The
Netherlands). Behavioral measures included the distance travelled, duration in the central zone,
percent activity in the center (defined as the distance travelled in the center divided by the total
distance travelled), meandering (defined as the ratio of the turn-angle degrees over total distance)
(Kalueff et al., 2007), number of rearing episodes and percent time moving (calculated as the
time spent moving over the total time).
Elevated plus-maze. Behavior in the elevated-plus maze was monitored as described
elsewhere (Bortolato et al., 2009). The apparatus was black Plexiglas with a light grey floor and
56

consisted of two open (25 × 5 cm) and two closed arms (25 × 5 × 5 cm), which extended from a
central platform (5 × 5 cm) at 60 cm from the ground. Mice were individually placed on the
central platform facing an open arm, and their behavior was observed for 5 min by an
experimenter unaware of the genotype. An arm entry was counted when all four paws were
inside the arm. Behavioral measures included: time spent and entries into each partition of the
elevated plus-maze; number of head dips, stretch-attend postures both defined as in (Rodgers et
al., 1992) and rears.
Marble burying. Marble burying was tested as previously described (Bortolato et al.,
2009). Briefly, mice were acclimated to novel cages (35 x 28 cm), filled with sawdust for 10
min. At the end of this phase, they were briefly removed and 20 glass marbles were placed on the
surface of the cage at even distances. Animals were then reintroduced into the cages and their
behavior was monitored for the following 10 min. The number marbles buried, as well as the
frequency and overall duration of digging was scored. A marble was considered buried if at least
two-thirds of its surface area was covered in sawdust.
Forced swim test. The forced swim test was performed as previously described (Gobbi
et al., 2005). Briefly, mice were habituated to clear Plexiglas cylinders (40 cm x 19 cm in
diameter) filled to 15 cm with water for 1 min. The water temperature was maintained at 30°C.
On the following day, mice were re-exposed to the cylinder using the above mentioned
conditions for 5 min. Environmental light was kept at 300 lux. Animals were video recorded,
and the duration of immobility (s) and the latency to immobility (s) were measured.
Tail suspension test. The tail suspension test was performed as described elsewhere
(Scott et al., 2008). Mice were individually suspended by the tail using medical tape affixed to a
hook, at 30 cm from the floor. Environmental light was kept at 300 lux. Animals were
videorecorded for 6 min, and the duration of immobility (s), the latency to immobility (s) and
number of fecal boli were measured.
57

Conditioned place preference (CPP). CPP was evaluated as previously described
(Bortolato et al., 2006; Maldonado et al., 1997). The apparatus consisted of two different
compartments (15 × 15 × 15 cm) separated by a central neutral area (start compartment), through
two guillotine doors. The two compartments featured different visual cues (striped or triangular
patterns) on the walls and tactile cues (square or checkered grid) on the floor. The combinations
of visual and tactile cues were present in the compartments in a counterbalanced order. The
experiment was conducted with a biased design and consisted of three consecutive phases. In
phase I (pre-conditioning), the animals were habituated to the apparatus for 2 days, and their
initial side preferences were recorded in a 15-min trial. Phase II (training) lasted 10 days. On odd
days, mice received IVM or vehicle and were placed in the non-preferred (NP) compartment
(with the guillotine door closed) for 15 min; conversely, on even days, mice received vehicle and
were placed in the preferred (P) compartment for 15 min. On the test day (post-conditioning,
phase III), the animals were given no treatment and were placed in the start compartment, with
free access to both sides for 15 minutes. IVM preference was measured as the difference between
the time spent in the drug-paired compartment (NP) during the post-conditioning and pre-
conditioning phases.
Acoustic startle and prepulse inhibition (PPI) of the startle reflex. Acoustic startle
reflex and PPI were tested as previously described (Bortolato et al., 2005). The apparatus used
for detection of startle reflex (San Diego Instruments, San Diego, CA) consisted of one standard
cage placed in sound-attenuated chambers with fan ventilation. Each cage consisted of a
Plexiglas cylinder of 3 cm diameter, mounted on a piezoelectric accelerometric platform
connected to an analog-digital converter. Background noise and acoustic bursts were conveyed
by two separate speakers, each one properly placed so as to produce a variation of sound within 1
dB across the startle cage. Both speakers and startle cages were connected to a main PC, which
detected and analyzed all chamber variables with specific software. Before each testing session,
acoustic stimuli were calibrated via specific devices supplied by San Diego Instruments (San
Diego, CA). Mice were placed in a cage for a 5-min acclimatization period with a 70 dB white
58

noise background, which continued for the remainder of the session. Each session consisted of
three consecutive sequences of trials (periods). Unlike the first and the third period - during
which mice were presented with only five pulse-alone trials of 115 dB - the second period
consisted of a pseudorandom sequence of 40 trials, including 12 pulse-alone trials, 30 trials of
pulse preceded by 73, 76 and 82 dB pre-pulses (respectively defined as PP3, PP6 and PP12; 10
for each level of pre-pulse loudness), and eight no stimulus trials, where only the background
noise was delivered. The duration of pulses and prepulses was 80 and 40 ms, respectively.
Prepulse–pulse delay amounted to 100 ms. Inter-trial intervals were selected randomly between
10 and 15 s. Percent PPI was calculated using the following formula: 100-[(mean startle
amplitude for pre-pulse pulse trials/mean startle amplitude for pulse alone trials) x 100].
Novel object exploration and recognition. We used a modified version of the protocol
described in Bortolato et al (2010). Mice were individually acclimatized to Makrolon cages for
15 min each. The day after, animals were exposed to two novel black plastic cylinders (8 cm tall
x 3.5 cm in diameter), affixed to the floor and symmetrically placed at 6 cm from the two nearest
walls. Mice were placed in a corner, facing the center and at equal distance from the two
objects. Their start position was rotated and counterbalanced for each treatment throughout the
test. Twenty-four h later, mice were placed in the same cage for long-term memory testing. One
of the cylinders was replaced by a novel plastic rectangular block (6 cm tall x 3 x 3cm), which
was placed in a counterbalanced fashion to avoid experimental bias. Behaviors for both sessions
were videotaped for 15 min. Analysis included the number and total duration of exploratory
approaches between novel and familiar objects. Exploration was defined as sniffing or touching
either of the two objects with the snout; sitting on the object was not considered exploration. In
the second exploration trial, an object exploration index was calculated as the ratio of the
duration of the exploratory approaches targeting the novel objects over the time of exploration of
both objects. The locomotor activity was defined as the number of crossings on a grid
superimposed onto the image of each cage in a video monitor.
59

Statistical analyses. Normality and homoscedasticity of data distribution were verified
using the Kolmogorov-Smirnov and Bartlett’s test. Parametric analyses were performed with
one-way ANOVAs (for repeated measures or independent factors, as appropriate), followed by
Tukey’s test with Spjøtvoll-Stoline correction for post-hoc comparisons. Nonparametric
comparisons were carried out by Kruskal-Wallis test, followed by Nemenyi’s test for post-hoc
comparisons. Significance threshold was set at P = 0.05.

RESULTS
Sensorimotor functions, tactile sensitivity and thermal nociception. In an initial set of
experiments, we ascertained that IVM did not exert any significant effect on reflex and home-
cage activity (data not shown). We then studied the ability of IVM to modify haptic perception
and somatosensory coordination in the sticky tape test. The P2X4 positive allosteric modulator
did not significantly affect the latency to remove the tape from the forepaws of the mice (Fig.
3.1A) [F(2, 15) = 0.02; NS]. Furthermore, in consideration of the role of P2X in pain
modulation (Toulme et al., 2010), we tested its effects on acute thermal nociception in the hot-
plate test. However, IVM did not modify the latency to the first paw-licking episode in mice, at
either 47.5°C (Fig. 3.1B) [F(2, 17) = 1.51; NS] or 50°C (Fig. 3.1C) [F(2, 17) = 1.69; NS].
Figure 3.1

Figure 3.1. Effects of ivermectin (IVM) and its vehicle (VEH) on haptic stimulation and sensorimotor coordination.
IVM did not alter the latencies to remove a sticky tape (A) or to lick the paw in a hot plate at 47.5 ºC (B) and 50 ºC
(C). IVM doses are expressed in mg/kg (i.p.). Values represented as mean ± S.E.M. n=6-8 per treatment group for
sticky tape and hot plate tests.
60

Novel open field. We evaluated the effects of IVM in the regulation of the locomotor and
exploratory responses within a novel open field. While IVM did not induce significant variations
of locomotor activity [F(2,15) = 2.12, NS] (Fig. 3.2A), it dose-dependently reduced the time
spent in the center (Fig. 3.2B) [F(2, 15) = 8.29; P<0.01], as well as the percent locomotor activity
in the center (Fig. 3.2C) [F(2, 15) = 6.46; P<0.01]. In contrast, no differences were detected
between treatment groups in meandering (Fig. 3.2D) [F(2, 15) = 2.50; NS], number of rears (Fig.
3.2E) [F(2, 15) = 1.80; NS], or the percentage of time spent moving (Fig. 3.2F) [F(2, 15) = 2.04;
NS]. Collectively, these data indicate that IVM enhanced the thigmotactic responses evoked in
mice by a novel open arena in mice, without modifying their exploratory behavior.
Figure 3.2

Figure 3.2. Ivermectin (IVM) administration increases thigmotactic behavior in the open field. While IVM did not
affect the overall locomotor activity (A), the time spent (B) and percent activity (C) in the central zone were
significantly reduced following IVM treatment. Conversely, drug administration did not produce any changes in
meandering (D), number of rears (E) or percent time moving (F) between groups. IVM doses are expressed in
mg/kg (i.p.). Values represented as mean ± S.E.M. for n=10 per treatment group. VEH, vehicle; *P<0.05 and
**P<0.01 compared to VEH-treated mice.

Elevated plus maze. To further qualify the role of IVM in the regulation of anxiety-like
responses, we tested a separate group of mice in the elevated plus-maze paradigm. The highest
IVM dose significantly increased the time spent in open arms (Fig. 3.3A) [F(2,20) = 5.73,
61

p<0.05]. This effect was accompanied by a significant decrease in the closed arm duration (Fig.
3.3B) [F(2,20) = 8.00, p<0.01] and an increase in the time spent on the central platform (Fig.
3.3C) [F(2,20) = 10.17, p<0.01]. Administration of the highest dosage of IVM also significantly
elevated the number of total entries (Fig. 3.3D) [F(2,20) = 10.84, p<0.001]. Additionally,
treatment with the 10 mg/kg dose of IVM resulted in increments of head dips (Fig. 3.3E)
[F(2,20) = 5.54, p<0.05] and rears (Fig. 3.3F) [F(2,20) = 6.10, p<0.01]. Conversely, the 2.5
mg/kg dose of IVM did not elicit any significant effect in any of the aforementioned parameters.
No significant differences were found in stretch-attend postures (data not shown). Taken
together, these data showed that the 10 mg/kg dose of IVM increased the exploratory activity and
reduced anxiety-like reactions in the elevated plus maze, without exerting significant effects on
risk assessment.
Figure 3.3

Figure 3.3. Behavioral effects of ivermectin (IVM) on the elevated plus-maze. (A-C) IVM increased the time spent
in open arms and on central platform, but reduced in the time spent in the closed arms. (D-F) IVM treatment at high
doses also enhanced the total number of entries, as well as exploratory head dips and rearing behavior. IVM doses
are expressed in mg/kg (i.p.). Values are indicated as mean ± S.E.M. for n=7-8 per treatment group. VEH, vehicle;
*P<0.05 and **P<0.01 in comparison with VEH-treated animals;
#
P<0.05 and
##
P<0.01 in comparison with IVM at
2.5 mg/kg.

Marble burying. In line with the effects of IVM in the elevated plus-maze, the highest
dose of this agent (10 mg/kg, i.p.) elicited a significant decrease in marble-burying activity (Fig.
3.4A) [F(2, 26) = 16.04, p<0.001]. The 10 mg/kg dose also induced a decrease in digging
62

duration (Fig. 3.4B) [F(2, 25) = 4.34; p<0.05], but not number of digging bouts (Fig. 3.4C) [F(2,
25) = 2.35; NS].
Figure 3.4

Figure 3.4. Ivermectin (IVM) treatment significantly reduces marble-burying activity. (A-C) IVM significantly
decreased marble-burying behavior and the duration of digging time, but not digging frequency. IVM doses are
expressed in mg/kg (i.p.). Values represented as mean ± S.E.M. for n=10 per treatment group. VEH, vehicle;
**P<0.01 and ***P<0.001 compared to VEH-treated mice;
###
P<0.001 compared to IVM at 2.5 mg/kg.

Tail suspension. We proceeded with the assessment of IVM’s effects on depression-
related responses in mice. In the tail-suspension paradigm, the 2.5 mg/kg dose of the drug
significantly increased immobility time in comparison with both the vehicle and the 10 mg/kg
dose (Fig. 3.5A) [F(2,18)=11.17]. No significant difference, however, was found between
animals treated with the 10 mg/kg dose and vehicle. IVM did not induce significant changes in
latency to immobility (data not shown) [H(2, 21) = 2.14; NS].
Figure 3.5

Figure 3.5. Ivermectin (IVM) increases depressive-related behaviors. (A) In the tail-suspension test, IVM
significantly increased immobility time at 2.5 mg/kg (i.p.), but not at 10 mg/kg (i.p.). (B) Conversely, high, but not
low dose administration of IVM increased immobility in the forced swim test. (C) IVM treatment did not produce
any alterations in conditioned place preference compared to vehicle treatment. IVM doses are expressed in mg/kg
63

(i.p.). Values represented as mean ± S.E.M. n=7-10 per treatment group for tail suspension, forced swim, and
conditioned place preference. VEH, vehicle; *P<0.05 compared to VEH-treated mice;
#
P<0.05 compared to IVM at
2.5 mg/kg.

Forced swim test. In the forced swim paradigm, IVM treatment induced a dose-
dependent increase in the duration of immobility (Fig. 3.5B) [F(2, 24) = 3.54; P<0.05]. Post-hoc
analyses revealed that this effect was due to the difference between the 10 mg/kg dose and the
vehicle (P<0.05). A statistical trend was also detected between the 2.5 mg/kg dose and the
vehicle (P<0.10). No significant difference was found between the groups treated with the two
IVM doses. Similar to the results in the tail-suspension tests, IVM failed to affect the latency to
immobility [F(2, 25) = 0.10; NS].
Conditioned place preference. In the CPP test, IVM did not elicit any overt rewarding
or aversive effect, as indicated by the absence of significant changes in preference for the drug-
paired compartments (Fig. 3.5C) [F(2, 19) = 0.24; NS]. No significant differences were found in
either locomotor activity during the pre- and post-conditioning phases or in the differences of
time spent in the other compartment of the apparatus (data not shown).
Prepulse inhibition (PPI) of the acoustic startle. Treatment with IVM did not induce
significant alterations of acoustic startle magnitude [F(2, 12) = 2.82; NS] (Fig. 3.6A). The drug,
however, elicited a significant PPI reduction (Fig. 3.6B) [treatment: F(2, 12) = 9.65; p<0.01].
Post-hoc analysis revealed that mice treated with 10 mg/kg IVM showed a significantly lower
PPI than both the groups treated with vehicle (p<0.01) and 2.5 mg/kg IVM (p<0.05).
Figure 3.6

Figure 3.6. Ivermectin (IVM) treatment disrupts sensorimotor gating. IVM did not produce any alterations in the
acoustic startle response (A), but impaired prepulse inhibition (PPI) compared to the vehicle (VEH) in a dose-
64

dependent fashion (B). Prepulses (PP) are indicated by the intensity corresponding to decibels above background
noise. IVM doses are expressed in mg/kg (i.p.). Values represented as mean ± S.E.M. for n=9-10 per treatment
group. **P<0.01 compared to VEH-treated mice.
#
P<0.05 compared to IVM at 2.5 mg/kg.

Novel object exploration/recognition. As shown in Fig. 3.7A, IVM decreased the
duration of novel object exploration [F(2, 18) = 3.49, p=0.05]. This effect was due to a
significant difference between the IVM doses of 2.5 and 10 mg/kg (p<0.05). While the drug did
not have any effect on the number of exploratory approaches directed towards the objects (Fig.
3.7B) [F(2, 18) = 0.58; NS], it did affect the mean duration of each sniffing episode [F(2, 19) =
3.75, p<0.05] (data not shown). Further scrutiny of this latter effect depended on a significant
difference between the two doses of IVM (p<0.05), as well as by a statistical trend (p<0.10)
between vehicle and IVM 2.5 mg/kg (Tukey). No difference in preference between the two
objects was detected in any of the treatment groups.
The P2X4 receptor activator failed to affect the mnemonic encoding of novel objects, as
revealed by the equivalence of the novel exploration index at 24 h after the first exposure (Fig.
3.7C) [F(2, 18) = 0.82; NS]. In line with the results from the open field, no significant
differences were observed in locomotor activity between treatment groups (data not shown).
Figure 3.7

Figure 3.7. Ivermectin (IVM) administration had no effect on mnemonic parameters in the object interaction and
recognition test. (A) IVM treatment significantly decreased exploration duration as compared to vehicle (VEH)-
treated mice. (B-C) Conversely, IVM did not affect the total number of exploratory approaches or the object
recognition index. IVM doses are expressed in mg/kg (i.p.). Values represented as mean ± S.E.M. for n=7-8 per
treatment group.
#
P<0.05 compared to IVM at 2.5 mg/kg.
65

Behavioral effects of IVM in P2X4 KO mice. To verify whether the
observed behavioral effects of IVM were mediated by P2X4 receptors, we tested the high dose
(10 mg/kg, i.p.) of this compound in P2X4 KO mice across the main paradigms significantly
affected by it. In the open field, IVM evoked a spectrum of responses similar to those observed
in WT mice, including the lack of changes in overall distance [F(1, 12) = 0.04; NS] (Fig. 3.8A),
as well as significant reductions in time [F(1, 13) = 22.61; P<0.001] (Fig. 3.8B) and percent
distance travelled in the center quadrant [F(1, 12) = 54.76; P<0.001] (Fig, 3.8C).
Figure 3.8

Figure 3.8. Behavioral effects of ivermectin (IVM, 10 mg/kg, i.p.) administration in P2X4 receptor knockout mice.
In the open field (A-C), IVM did not affect the overall locomotor activity (A), but reduced the time spent (B) and
percent activity in the central quadrant (C). In the elevated plus-maze (D-G) IVM significantly increased the time
spent in open arms (D), reduced the time spent in the closed arms (E), increased the total numbers of entries (F) and
head dips (G). IVM significantly increased startle amplitude (H), but did not significantly affect the prepulse
inhibition (PPI) of the startle (I). Prepulses (PP) are indicated by the intensity corresponding to decibels above
66

background noise. The IVM dose is expressed in mg/kg. Values represented as mean ± S.E.M. for n=8 per
treatment group. VEH, vehicle; *P<0.05, **P<0.01, ***P<0.001 compared to VEH-treated mice.

In the elevated plus maze, IVM significantly increased the duration of time spent in the
open arms (Fig. 3.8D) [F(1, 13) =6.04; P<0.05], and reduced the time spent in the closed arms by
P2X4 KO mice (Fig. 3.8E) [F(1, 13) = 14.49; P<0.01]. In addition, IVM-treated P2X4 KO mice
exhibited significant enhancements in the total number of arm entries (Fig. 3.8F) [F(1, 13) =
11.43; P<0.01] and head dips (Fig. 3.8G) [F(1, 13) = 11.75; P<0.01]. In contrast with the effects
of IVM in WT mice, this drug significantly increased startle amplitude (Fig. 3.8E) [F(1, 11) =
6.25; P<0.05], but did not reduce %PPI (Fig. 3.8F) [F(3, 9) = 1.30; NS].

DISCUSSION
The results of the present study document that acute administration of IVM resulted in a
complex set of effects on the regulation of emotional, cognitive and perceptual functions. In
particular, the drug exhibited a multifaceted, complex profile with respect to complementary
aspects of anxiety-like responsiveness. IVM induced a highly heterogeneous array of behavioral
responses across different anxiety-related tasks. In a novel open field, mice treated with this drug
displayed a dose-dependent increase in thigmotaxis, which was not accompanied by any other
significant variation in locomotor and exploratory parameters. While these results are generally
associated with anxiogenic-like effects (Prut and Belzung, 2003), this interpretation is in
stark contrast with the finding that the 10 mg/kg (but not 2.5 mg/kg) dose of IVM exerted a
marked reduction of anxiety-like behaviors in the elevated plus- maze and marble-burying
assays.
The discrepancies among IVM-induced effects on anxiety-like responses are likely to
reflect the differential impact of this drug on diverse aspects of anxiety-related and exploratory
responses. Although the ethological construct underlying most paradigms for anxiety testing in
rodents is based on the conflict between exploration and neophobia in response to different
67

environmental cues, several studies have documented that each test captures different dimensions
of anxiety-related responsiveness {Belzung, 1994 #6553;(Ramos et al., 1997);Carola, 2002 #25}.
Additionally, IVM increased depression-associated indices in both the tail suspension and
forced swim tests, and reduced sensorimotor gating in the PPI assay. These results suggest that
IVM induced depression-like reactions (albeit at different doses) in response to acute,
inescapable stressors. Both assays carry high predictive validity for depression, and treatments
that increase immobility duration are generally interpreted to worsen mood, particularly if, like
IVM, elicit no intrinsic hypolocomotion or sedation. Although previous reports indicated that
very high doses of IVM induced signs of depression in animals (Basudde, 1989), the increased
immobility in these studies is unlikely to reflect the same phenomena, since they were elicited by
the low IVM dose in the tail suspension, and not associated by any sign of acute toxicity in this
or other studies (Yardley et al., 2012). Notably, we found divergent dose-response relations of
IVM-induced depressive-like effects in the tail-suspension test (in which immobility was
observed in response to the 2.5 mg/kg, but not 10 mg/kg, dose) and in the forced swim test
(which resulted in immobility only at the dose of 10 mg/kg). The differences between these two
results are currently unknown, but they may reflect different sensitivities of these two paradigms
to the various neurotransmitter systems that regulate stress reactivity. Indeed, although both
paradigms are based on the same ethological construct, based on the immobility caused by
inescapable situations (Castagné et al., 2010), they have been shown to respond differently to
several pharmacological agents (Chatterjee et al., 2012).
Another interesting finding of our studies was that, in WT mice, IVM induced a dose-
dependent reduction in PPI, without affecting acoustic startle magnitude, signifying that this drug
has an acute impact on sensorimotor gating and information processing. This finding is in line
with previous reports (Davis et al., 1999), which documented PPI deficits following long-term
administration of IVM in drinking water to mice. These impairments are connected to a broad set
of neuropsychiatric deficits, ranging from psychotic disorders to obsessive-compulsive disorder
and attention-deficit hyperactivity disorder. In addition, pharmacological disruptors of PPI in
68

rodents have been shown to elicit psychotomimetic effects in humans. Irrespective of these
considerations, it is interesting to notice that IVM-induced gating impairments did not overtly
affect declarative mnemonic encoding, as revealed by the novel object recognition test. This
observation tempers the possibility that the observed changes in PPI may have a repercussion on
cognition and learning.
It is relevant to note that IVM did not elicit overt rewarding and/or aversive actions in
conditioned place preference test. As the rewarding effects of a drug are regarded as a necessary
premise for abuse and dependence, these data strongly suggest that IVM does not significantly
modify incentive behavior. Given the biased design of our protocol (which was used to enhance
the sensitivity of the test to the potential rewarding effects of IVM), the apparent lack of aversive
effects remains inconclusive, as it may be masked by potential floor effects. Irrespective of this
issue, the lack of inherent addictive potential of IVM is in line with the absence of clinical notes
on its recreational consumption in humans, and is particularly important in view of the recent
finding that this drug reduces alcohol intake in mice, as measured in several models (Yardley et
al., 2012).
To our knowledge, few studies have been published on the behavioral effects of IVM in
rodents. In line with our findings, previous work showed the lack of effects of this drug on
general parameters of health and motor coordination in mice and rats (Davis et al., 1999; Nafstad
et al., 1991). Davis and colleagues (1999) found that prolonged administration of IVM in tap
water elicited significant psychotropic effects in several murine strains, including an
enhancement in motor and acoustic reactivity as well as an attenuation of gating, but that it did
not alter spatial learning. Furthermore, intraperitoneal injections of low IVM doses were found to
exert rapid anxiolytic-like properties in the elevated plus-maze and Vogel test in rats (Spinosa et
al., 2002). Although these findings cannot be directly compared to our results, in view of
differences in regimens of administration and species (or strains) tested, our study confirmed that
IVM exerts psychotropic properties and induces a complex array of effects on emotional
regulation.
69

The multifaceted behavioral profile of IVM across the tests in this study is likely to
reflect the involvement of multiple, divergent neurochemical targets of this drug, also in relation
to the different sensitivity of each paradigm to different systems. In mammals, high-affinity
binding sites for IVM have been characterized on GABA
A
(Huang and Casida, 1997) and P2X4
receptors (Priel and Silberberg, 2004).
Given the lack of available effective P2X4 receptor antagonists, we tested the effects of
P2X4 KO mice to identify the specific contributions of this target to IVM-induced behavioral
effects. The effects of IVM remained substantially unaltered in the open field and elevated plus
maze, suggesting that P2X4 receptors do not play a critical role in the modulation of anxiety-
related responses in paradigms based on the conflict between approach and avoidance with
respect to unprotected environments. Thus, it is likely that the apparently antithetical effects of
IVM in the open field and elevated plus-maze may reflect the impingement of other receptors.
For example, the behavioral changes in the elevated plus-maze are in line with the anxiolytic-like
properties of GABA
A
receptor agonists (Davis et al., 1999).
In contrast, IVM elicited significantly divergent effects in WT and P2X4 KO mice with
respect to startle and PPI. In particular, IVM enhanced the startle amplitude of P2X4 KO mice,
suggesting a role of these receptors in the regulation of acoustic startle response. Further research
will be needed to fully understand the specific neurobiological underpinnings of the role of these
targets in the modulation of startle. In addition, we found that IVM did not induce any
impairments of PPI in P2X4 KO mice, suggesting that the deficits in sensorimotor gating caused
by this drug were in fact mediated by P2X4 receptor activation.
Although the tail-suspension effects of IVM were not tested in P2X4 KO mice, the
enhancement of tail-suspension immobility by IVM is also unlikely to strictly reflect the
outcomes of GABA
A
activation, as this effect is dose-dependent and reflects a reduction in
overall locomotor activity (Steru et al., 1987). Given that tail-suspension and forced swim
behaviors are poorly sensitive to the outcomes of GABA
A
receptor activation (Borsini and Meli,
1988; Lalonde and Strazielle, 2010; Steru et al., 1987), it is possible that IVM-induced responses
70

may reflect a more prominent influence by P2X4 receptors. These results indicate that positive
allosteric modulation of P2X4 receptors may directly modulate emotional reactivity in response
to acute stressors, possibly inducing anxiety- and depression-like responses. Complementary to
this idea, previous studies suggest that the behavioral effects of some antidepressant agents may
in fact be mediated by the blockade of P2X4 receptors, (Sim and North, 2010; Toulme et al.,
2010) (for contrasting results see Nagata et al., 2009).
Recent evidence suggests that P2X4 receptors may negatively interact with the effects of
GABA
A
receptor activation (Jo et al., 2011), providing a possible explanation for the contrasting
outcomes observed across different tests. In particular, it is possible that P2X4 receptor
activation may induce aversive effects, which may contrast the rewarding properties of GABA
A

receptor activation. Thus, the simultaneous stimulation of both targets may reduce the addictive
potential associated with GABA
A
receptor agonists.
The identifications of novel pharmacological targets for behavioral regulation is a task of
paramount importance, in consideration of the largely inadequate therapeutic armamentarium for
neuropsychiatric conditions. The translational implications of our data afford a broader
perspective on the behavioral effects of IVM. Further studies are necessary to qualify the nature
of the potentially untoward effects documented by this study, such as the increase in depression-
like responses and the sensorimotor gating deficits. Our results point to a role of P2X4 receptors
in the modulation of startle responsiveness, as well as sensorimotor gating. Future studies with
selective antagonists or small interfering RNAs for P2X4 receptors will be critical to ascertain
the implication of the activation of these targets. Irrespective of these aspects, the lack of
rewarding properties of IVM in the conditioned place preference paradigm, its anxiolytic-like
properties in two complementary paradigms for anxiety-related behaviors and its ability to
attenuate alcohol intake (Yardley et al., 2012) strongly suggest that IVM and its derivatives may
be of great interest for the development of novel therapeutic avenues in neuropsychiatric therapy.

71

CHAPTER 4
MULTI-DAY ADMINISTRATION OF IVERMECTIN IS EFFECTIVE IN REDUCING
ALCOHOL INTAKE IN MICE AT DOSES SHOWN TO BE SAFE IN HUMANS

ABSTRACT
Ivermectin (IVM), an FDA approved anthelmintic agent, can significantly reduce ethanol
intake in mice following acute administration. The current study evaluates the sustainability and
safety of multi-day IVM administration in reducing 10E intake in mice at a dose shown to be
safe in humans. We tested the effect of 10-day administration of IVM (3.0 mg/kg/day; i.p.) on
reducing 10% v/v alcohol (10E) intake in C57BL/6J mice using a 24-h, two-bottle choice
paradigm. On the 10
th
day of IVM administration, mice were sacrificed at 0, 0.5, 2, 8, 32, 48 and
72 hours post-injection. Brain tissue and plasma samples were collected and analyzed using
liquid chromatography with tandem mass spectrometry (LC-MS/MS). Analysis of variance
(ANOVA) was used to assess the effect of 10-day IVM administration on 10E intake, 10E
preference, water intake and total fluid intake with Dunnett’s Multiple Comparison post-hoc test.
Individual student’s t-tests were also used to further quantify changes in these dependent
variables. IVM significantly decreased 10E intake over a 9-day period (p<0.01). Pre IVM 10E
intake was 9.1 + 3.2 g/kg/day. Following the 9
th
day of IVM injections, intake dropped by almost
30% (p<0.05). IVM had no effect on total water intake or mouse weight throughout the study;
however, there was a significant decrease in both preference for 10E (p<0.01) and total fluid
intake (p<0.05). Multi-day administration of IVM significantly reduces 10E intake and
preference in animals without causing any apparent adverse effects at a dose shown to be safe in
humans.
72

INTRODUCTION
Alcohol use disorders (AUDs) rank third on the list of preventable causes of morbidity
and mortality in the United States, affecting over 18 million people and causing over 100,000
deaths annually (Bouchery et al., 2011; Grant et al., 2004; Johnson, 2010). The economic
burden to society for AUDs is in excess of $200 billion/year (Bouchery et al., 2011), which
exceeds the costs of other leading preventable causes of death such as cigarette smoking and
physical inactivity (Naimi, 2011). According to the Global Status Report on Alcohol and Health
2011, nearly 4% of all deaths worldwide were alcohol-related (2011). Current FDA approved
clinical treatment options include three FDA-approved oral medications and one FDA-approved
injectable agent: disulfiram, naltrexone (both oral and injectable), and acamprosate (Harris et al.,
2010; Litten et al., 2012). Data collected from The National Survey on Drug Use and Health
(2006) and the National Epidemiologic on Alcohol and Related Conditions (2001-2002) found
only 8.2% of subjects with 12-month AUDs had received treatment for AUDs (Edlund et al.,
2012). These statistics reflect, in part, the fact that current therapeutic strategies are largely
inadequate for the management of AUDs. Despite considerable efforts focusing on drug
development to reduce ethanol abuse, high rates of uncontrolled heavy ethanol drinking persist.
We propose that ivermectin (IVM), a drug used by millions of humans for treatment of
parasites (Geary, 2005; Molinari et al., 2010) can be repositioned as a novel pharmacotherapy to
treat and/or prevent excess alcohol consumption and abuse. In P2X4 receptors (P2X4Rs), IVM
is used to selectively identify the participation of P2X4Rs from other P2X family members in
adenosine 5’-triphosphate (ATP)-mediated processes (Jelinkova et al., 2006; Khakh et al., 1999a;
Silberberg et al., 2007). Interestingly, sites of action of IVM are proximal to regions that we
have previously reported as being important for ethanol modulation (Asatryan et al., 2008;
73

Popova et al., 2010). Studies from our lab demonstrated that IVM is able to antagonize the
inhibitory effect of ethanol in vitro (Asatryan et al., 2010).
Further support for the repositioning of IVM is drawn from a number of studies showing
that IVM significantly reduces ethanol intake and preference in mice as determined across
several validated ethanol drinking paradigms (Asatryan et al., 2014; Wyatt et al., 2014; Yardley
et al., 2012). This work found that IVM doses ranging from 1.25 to 10.0 mg/kg can be safely
administered and can significantly reduce alcohol intake using a 24-h access two-bottle choice
model (2011; Yardley et al., 2012) that mimics “social” or non-intoxicating levels of alcohol
drinking (Blednov et al., 2010).
We also found that acute administration of IVM can significantly reduce higher levels of
ethanol intake using the intermittent limited-access model, which mimics binge-like drinking
(Yardley et al., 2012). Importantly, in humans, young adults who participate in binge or heavy
drinking are more likely to progress to alcohol abuse or dependence than age-matched
counterparts (Johnson, 2010). Further, individuals participating in binge-like drinking behavior
and/or drinking to intoxication is associated with significant increases in vehicle accidents,
injuries, date rape and other types of violence, pregnancy, and blackouts (for review see
(Johnson, 2010)). Our findings that IVM significantly reduces binge-like drinking in mice
(Yardley et al., 2012) further supports the development of IVM as a new pharmacotherapeutic
agent for treatment and/or prevention of AUDs.
The current approved dosing and administration regimen for IVM is based on acute use
of the drug in human subjects. However, chronic administration would be anticipated in patients
for treatment of AUDs. Several pieces of information support the safety of the chronic
administration of IVM. First, doses up to 10 times that of the recommended dosage (i.e., 2.0
74

mg/kg/day) have been safely tested in human clinical trials (Guzzo et al., 2002). Second, in
rodents, doses less than 10 mg/kg IVM do not cause detectable CNS depression (Trailovic and
Trailovic, 2010), and is more than 2.5 fold lower than the LD
50
(25- 50 mg/kg) (Merck et al.,
1988). Third, allometric scaling identified a dose of 3.1 mg/kg/day IVM in mice that corresponds
to an oral dose (30 mg or approximately 0.5 mg/kg) already shown to be safe in humans (Guzzo
et al., 2002). Fourth, a case-control study reported that there were no significant increases in
severe adverse events (SAEs) for patients that had self-reported consuming alcoholic beverages
at the time of IVM administration (Takougang et al., 2008). Collectively, these findings point to
IVM as an attractive agent for the treatment of AUDs, which appears to have a good margin of
safety and tolerability. The present study tests the hypothesis that multi day dosing of IVM is
well tolerated and has sustainable effects in reducing ethanol intake in mice.

MATERIALS AND METHODS
Animals. Studies were performed on drug naïve C57BL/6J male mice that were 8 weeks
old upon purchase (Jackson Laboratory, Bar Harbor, ME, USA). Mice were singly housed in
polycarbonate/polysulfone cages at a 12 h light/dark cycle with lights off at 12:30PM. The
holding room was maintained at approximately 22°C. All procedures in this study were
performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and
all efforts were made to minimize animal suffering. The USC Institutional Animal Care and Use
Committee approved the protocols.
Drugs. IVM was administered via daily intraperitoneal (IP) injections. Noromectin (10
mg/ml in 60% propylene glycol) (Norbrook Inc, Lenexa, KS) was used for IVM injections. The
noromectin was diluted using a 0.9% sodium chloride solution (saline) to a concentration that
75

would allow for an injection volume of 0.01 ml/g of body weight. Gold Shield Alcohol (200
proof USP solution, Gold Shield Chemical Company, Hayward, CA) was diluted in water to
achieve a 10% v/v ethanol solution (10E).
Alcohol Intake Study. The 24-h access model [e.g., (Belknap et al., 1993; McClearn,
1959; Middaugh et al., 1999; Rodgers, 1966; Yoneyama et al., 2008) is widely used to assess
changes in drinking behaviors. We used a modification of the procedure employed by
Yoneyama et al. (Yoneyama et al., 2008). Briefly, using a within subjects design, 24 male mice
had 24-h access to two inverted bottles of water with metal sippers placed on the cage tops for
one day. One bottle of water was replaced with a bottle containing 10E. Food was distributed
near both bottles to avoid food associated tube preferences. Alcohol determinations followed
previously described procedures (Yardley et al., 2012). Briefly, daily 10E intake was measured
until it stabilized (+ 10% variability from the mean dose over 3 consecutive days). After
establishing stable alcohol drinking level (after 7 days) mice received daily saline injections
(intraperitoneal; i.p.) until 10E intake stabilized again (after 3 days). Mice then received one
injection (i.p.) of IVM (3.0 mg/kg) daily for 10 consecutive days. The data reflects 9 days
because mice were sacrificed post day 10 injections therefore, no drinking data was obtained for
the mice sacrificed 0-8 hours after day 10 injections.
Analysis of brain concentrations of IVM using liquid chromatography with tandem
mass spectrometry (LC-MS/MS). Brain samples were collected just prior to day 10 injections
(t=0), and 0.5, 2, 8, 32, 48 and 72 hours (n=3-4/time point) after day 10 IVM injections. Brain
samples were analyzed as previously described (Asatryan et al., 2014; Yardley et al., 2012). To
150 mg of brain, 2 µg of abamectin was added as internal standard. To each brain sample, 1
scoop of 1mm zirconium beads was added with 1 mL of acetonitrile, and sample was vigorously
76

homogenized using the bullet blender for 5 minutes. Samples were then centrifuged at 10,000
rpm for 5 minutes and supernatant was collected. This was repeated with another 1 mL of fresh
acetonitrile, and the combined 2 mL supernatant were evaporated to dryness using a steady
stream of dried air. Evaporated residues of brain samples were reconstituted in 100uL of
acetonitrile : water (90:10 v/v) . A 40 µL aliquot was injected into the Agilent 1100 HPLC
System linked to an AB Sciex API 3000 Turboion spray mass spectrometer (Agilent
Technologies, Santa Clara, CA USA). The analytes were separated using an ACE C18 column
(Advanced Chromatography Technologies, Aberdeen Scotland) with an isocratic mobile phase
consisting of 0.1% formic acid in acetonitrile : 0.1% of acid in water (90:10 v/v). IVM and
abamectin (ABM) was quantified in positive mode with multiple reaction monitoring using
parent to transition ions of 888.8 → 551.5 and 890.1 → 449.6 respectively.

Data Analyses
Alcohol Intake Study. Ethanol dose (g/kg) and ethanol preference (mls ethanol/total mls
x 100) were calculated daily. The dependent variables included 10E intake (g/kg), 10E
preference (%), water (ml) and total fluid intake (ml). One-way analysis of variance (ANOVA)
was used to assess the effect of 10-day IVM administration on the 4 dependent variables with
Dunnett’s Multiple Comparison post-hoc test. Outliers were defined as data points + 2 standard
deviations from the daily mean of each dependent variable and were removed from the analysis.
Based on previous investigations, we predicted, a priori, that IVM would significantly reduce
10E intake and preference (Asatryan et al., 2014; Yardley et al., 2012) therefore; one-tailed,
unpaired, individual Student’s t-test was used to analyze 10E intake and preference for 10E.
Changes in total fluid intake on the other hand, have been somewhat variable from study to
77

study, therefore, two-tailed, unpaired, individual Student t-test was used to analyze total fluid
intake. The significance level was set at p ≤ 0.05.
Pharmacokinetic Analysis. The pharmacokinetics (PK) of IVM were analyzed using
non-compartmental PK modeling. Serial blood and tissue IVM quantification was used to
calculate PK parameters such as maximum drug concentration (Cmax), time to achieve maximal
drug exposure (Tmax), half-life, elimination constant and area under the curve (AUC).

RESULTS
Multi-day IVM administration significantly reduced alcohol intake and preference
in male mice. The effect of 3.0 mg/kg/day IVM administered for 10 consecutive days using a
24-h access two-bottle choice alcohol paradigm (10E versus tap water) in C57BL/6J mice was
evaluated. This study was designed based on our previous investigation that tested the effects of
IVM on ethanol intake and preference (Yardley et al., 2012). Additionally, our PK modeling
using allometric scaling identified 3.1 mg/kg as the dosage that corresponds to a dose of IVM
used safely in humans (Guzzo et al., 2002).
Prior to the initiation of saline injections, the 3-day average 10E intake was 9.0 + 3.7
g/kg/24-h in the absence of vehicle or drug injections. The 3-day average 10E intake with saline
injections was 9.1 + 3.2 g/kg/24-h. A one-way ANOVA revealed that IVM (3.0 mg/kg/day x 10
days) significantly decreased 10E intake over a 9-day period (p<0.01; Fig. 4.1a). Dunnett's
Multiple Comparison Test revealed a significant reduction in ethanol intake on days 4 (p<0.05;
reduction of 27% in 10E intake), 6 (p<0.05; reduction of 28%) and 9 (p<0.05; reduction of 30%)
compared to the day immediately prior to the start of IVM injections (pre IVM; day 0). One-
tailed, unpaired, individual student’s t-tests, identified 3 additional days where ethanol intake
78

was significantly reduced. These included days 1 (t=2.488, p<0.05; reduction of 26%), 3
(t=1.847, p<0.05; reduction of 19%) and 7 (t=2.190, p<0.05; reduction of 25%), with a trend for
decreased 10E intake on day 8 (p=0.06; reduction of 17%) compared to pre IVM levels. 48-h
after the final administration of IVM, 10E intake increased to 8.4 + 3.3 g/kg/24-h (n=6). 72-h
after the final administration of IVM, 10E intake returned to baseline levels with an average of
10E intake of 9.7 + 3.4 g/kg/24-h 72-h (n=3).
Figure 4.1

Fig 4.1. IVM significantly reduces 10E intake in C57BL/6J mice. The graph illustrates the effects of IVM (3.0
mg/kg) administration in male mice using 24-h access two-bottle choice paradigm. Day 0 represents the saline
control the day prior to the initiation of IVM administration for (a) 10E intake, (b) preference for 10E, (c) water
intake, and (d) total fluid intake. Values represent the mean + SEM for 24 mice. Values represent the mean + SEM
for 24 mice. *p<0.05 vs. respective day 0 value (represented by the dotted line) as determined by Dunnett’s Multiple
Comparison Test and
+
p<0.05 as determined by individual student’s t-tests.

79

Preference for 10E was significantly affected by IVM treatment (p<0.01; Fig.4.1b).
Dunnett's Multiple Comparison Test showed a significant decrease in 10E preference on day 6
only (p<0.05). One-tailed, unpaired, individual student’s t-tests identified additional days when a
significant decrease in 10E preference was observed including days 1 (t=2.801, p<0.01), 4
(t=2.346, p<0.05), 7 (t=1.883, p<0.05) and 8 (t=1.696, p<0.05) with a trend for decreased 10E
preference on day 3 (p=0.07).
There was no significant effect of treatment on water intake (Fig. 4.1c). There was a
significant effect of treatment on fluid intake (p<0.05) with Dunnett's Multiple Comparison Test
revealing a significant decrease in fluid intake on day 9 of IVM injections compared to pre IVM
fluid intake (p<0.05; Fig.4.1d). Additionally, two-tailed, unpaired, individual student’s t-tests
revealed a trend for decreased fluid intake on days 3 (p=0.09) and 7 (p=0.07).
Pharmacokinetics of IVM. Brain samples were collected at designated time points of 0,
0.5, 2, 8 32, 48, and 72 h where time zero was defined as just prior to administration of the last
dose. The PK parameters from this multi-day dosing study are summarized in Table 4.1.
Table 4.1
Brain Pharmacokinetic
Parameters Mean SD
Cmax (ng/mg of tissue) 4.02 0.35
Tmax (h) 8 n/a
Half-life (h) 9.75 1.57
AUC
0-t
(ng x h/mg of tissue) 87.7 9.51

Table 4.1. IVM disposition in the brain was analyzed at 7 time points following the 10 days of injections: 0 (just
prior to day 10 injections), 0.5, 2, 8, 32, 48 and 72.

The results indicate there is accumulation of IVM in the brain with chronic dosing, however,
most of the drug is cleared within 2-3 days after the last dose, as IVM was undetected at 48 and
72 h (Fig. 4.2).
80

Figure 4.2
0 0.5 2 8 32 48 72
0
1
2
3
4
5
Time After Final IVM Injection (h)
Brain IVM
Concentration (ng/mg)

Figure 4.2. IVM accumulates in the CNS in a time dependent manner. The graph illustrates the IVM Cmax
levels in the brain tissue at the indicated time points following 10 consecutive days of IVM administration correlated
with time to effect. Time 0 represents the Cmax levels 24-h post day 9 IVM administration. Values represent the
mean + SEM for 3-4 mice/ time point. **p<0.01 vs. time 0 value as determined by Dunnett’s Multiple Comparison
Test. The Cmax correlated to onset of IVM activity. Additionally IVM half-life in the brain is 9.7 hours, where
correlates with the reduction in brain concentration after 32 hours.

DISCUSSION
The present study tested the hypothesis that multi day dosing of IVM is safe and
sustainable in reducing alcohol intake in mice. The dose of IVM that we tested in mice
corresponds to a dose already shown to be safe in humans. In support of this hypothesis, we
found that administration of IVM for 9 days, reduced 10E intake by approximately 25%
compared to baseline measures (Fig. 4.1a). There were no overt changes in behavior or
significant changes in body weight over the 9-day period suggesting that longer term IVM
administration is well tolerated in mice. In support of this latter notion, we found that IVM
administration over a period of 9 days did not significantly affect water intake. This finding
further supports the contention that the effect of IVM is not a result of a generalized hypodipsia,
which is consistent with previous studies testing the effect of acute IVM administration
(Bortolato et al., 2013; Yardley et al., 2012). The significant decrease in total fluid intake
measured on day 9 of the study may have been due, in part, to the significant decrease in alcohol
intake alone. This finding is consistent with previous results (Yardley et al., 2012). PK studies
81

revealed the average brain Cmax of IVM 32-h post day 10 injections was 0.73 ng/mg almost 3
times greater than the brain Cmax of IVM after a single dose of 2.5 mg/kg (0.261 ng/mg), the
minimum effective single dose necessary to significantly decrease 10E intake in a 24-h two-
bottle choice paradigm (Yardley et al., 2012).
IVM is a semi-synthetic macrocyclic lactone that is currently used worldwide as a broad-
spectrum antiparasitic agent. The safety of IVM has been demonstrated for over 30 years where
millions of humans have been treated successfully with good tolerability and few severe adverse
events (for review see (Crump and Omura, 2011; Omura, 2008)). IVM is also widely used for
veterinary purposes in animals that are raised for food thus requiring significant studies
reviewing the long-term safety of oral exposure to IVM as a food additive. This latter effort,
based on available toxicological data by the World Health Organization (WHO), identified an
acceptable daily intake (ADI) of 1-10 µg/kg/day based upon a No Observed Effect Level
(NOEL) for maternal toxicity in reproductive toxicology studies in the most sensitive species
(CD1 mouse) as well as a safety margin of 50-500 fold for the absence of neurological effects in
dogs.
Importantly, single doses of up to 120 mg, a dose 4 times greater than the dose tested in
the current study, have been safely administered to human subjects for other indications with no
reported adverse experience (Guzzo et al., 2002). In this same study, a 30 mg dose in humans
(equivalent to a 3.1 mg/kg dose in mice), was administered three times in the first week (days 1,
4, and 7) followed by a washout period of 1 week and then a single dose of IVM (30 mg). Of
these patients receiving IVM, 33.3% (n=5) had one or more adverse experience compared to
35.3% of the placebo group (n=6). There was no apparent evidence of CNS toxicity associated
with IVM administration in this study at either dose measured, assessed using quantitative
82

pupillometry. This suggests that IVM appears to have a good therapeutic index as a treatment for
AUDs since the minimum effective dose necessary to decrease alcohol intake is far lower than
the lethal dose 50 (LD50) of IVM which is approximately 25-50 mg/kg in mice (Merck et al.,
1988).
The potential for repositioning IVM as an anti-alcohol therapy is further supported by
previous investigations that reported that IVM, at doses needed to produce the anti-alcohol
effects in C57BL/6 mice, did not induce overt signs of toxicity across a wide range of well-
validated behavioral paradigms for the assessment of sensory, motor and cognitive competence
(Bortolato et al., 2013). IVM has been reported to elicit anxiolytic-like effects when tested using
the elevated plus maze and marble burying assays. However unlike other anxiolytic therapies
such as benzodiazepines, the same IVM regimen did not exert rewarding properties in the
conditioned place preference test. The latter strongly suggested that the psychotropic effects of
IVM are dissociated from any addiction liability or potential issues of therapeutic compliance
(Bortolato et al., 2013).

CONCLUSION
Findings from the present investigation demonstrate that IVM, administered for 10 days
using a dose of IVM that consistently reduces ethanol intake and preference in mice, is well
tolerated. Importantly, this dose of IVM corresponds to a dose already shown to be safe in
humans. Collectively, current and previous findings support the contention that IVM
administered at doses required to produce the anti-alcohol effects in mice should be safe and
effective in humans for the treatment of AUDs. Future studies will focus on the safety and
efficacy of chronic, oral delivery of IVM as a therapy to prevent and/or treat AUDs.
83

CHAPTER 5
AVERMECTINS DIFFERENTIALLY AFFECT ETHANOL INTAKE AND RECEPTOR
FUNCTION: IMPLICATIONS FOR DEVELOPING NEW THERAPEUTICS FOR
ALCOHOL USE DISORDERS

ABSTRACT
Our laboratory is investigating ivermectin (IVM) and other members of the avermectin
family as new pharmaco-therapeutics to prevent and/or treat alcohol use disorders (AUDs). Prior
work found that IVM significantly reduced ethanol intake in mice and that this effect likely
reflects IVM’s ability to modulate ligand-gated ion channels. We hypothesized that structural
modifications that enhance IVM’s effects on key receptors and/or increase its brain concentration
should improve its anti-alcohol efficacy. We tested this hypothesis by comparing the abilities of
IVM and two other avermectins, abamectin (ABM) and selamectin (SEL), to reduce ethanol
intake in mice, to alter modulation of GABA
A
Rs

and P2X4Rs expressed in Xenopus oocytes and
to increase their ability to penetrate the brain. IVM and ABM significantly reduced ethanol
intake and antagonized the inhibitory effects of ethanol on P2X4R function. In contrast, SEL did
not affect either measure, despite achieving higher brain concentrations than IVM and ABM. All
three potentiated GABA
A
receptor function. These findings suggest that chemical structure and
effects on receptor function play key roles in the ability of avermectins to reduce ethanol intake
and that these factors are more important than brain penetration alone. The direct relationship
between the effect of these avermectins on P2X4R function and ethanol intake suggest that the
ability to antagonize ethanol-mediated inhibition of P2X4R function may be a good predictor of
84

the potential of an avermectin to reduce ethanol intake and support the use of avermectins as a
platform for developing novel drugs to prevent and/or treat AUDs.

INTRODUCTION
Alcohol use disorders (AUDs) rank third on the list of preventable causes of morbidity and
mortality in the United States, affecting over 18 million people, causing over 100,000 deaths
annually (Bouchery et al., 2011; Grant et al., 2004; Johnson, 2010) and costing in excess of $220
billion (Bouchery et al., 2011). This exceeds the costs of other leading preventable causes of
death such as cigarette smoking and physical inactivity (Naimi, 2011). Presently, the only
pharmacotherapeutic agents approved by the United States Food and Drug Administration
(FDA) for the treatment of AUDs are disulfiram (Antabuse
®
), naltrexone (Revia® and
Vivitrol®), and acamprosate (Campral®), with Vivitrol
®
being an extended-release injectable
formulation of naltrexone (Harris et al., 2010; Litten et al., 2012). These drugs attempt to deter
alcohol intake by blocking its metabolism or by targeting the neurochemical and neuropeptide
systems in the downstream cascades leading to craving and dependence (Colombo et al., 2007;
Gewiss et al., 1991; Johnson, 2010; Litten et al., 2012; Steensland et al., 2007). However, their
success rate even when combined with psychotherapy, has been limited with approximately 70%
of patients relapsing back into heavy drinking within one year (Johnson, 2008; Litten et al.,
2012). Thus, the development of new drugs that will more effectively treat AUDs is of
paramount importance.
Our team is investigating ivermectin (IVM) as a potential platform for developing novel
agents for preventing or treating AUDs. IVM is a FDA approved drug that is currently used
worldwide as a broad-spectrum antiparasitic agent (Crump and Omura, 2011; Omura, 2008). We
recently demonstrated that IVM significantly reduces ethanol intake in both male and female
85

mice across several models of self-administration (Yardley et al., 2012). Doses of IVM that
significantly reduced ethanol intake also produced significant, dose-dependent anxiolytic
responses in these animals without exhibiting any addiction potential (Bortolato et al., 2013).
Moreover, IVM did not cause significant changes in pain sensitivity, motor competency or
memory (Bortolato et al., 2013) and did not cause any obvious signs of toxicity (Bortolato et al.,
2013; Yardley et al., 2012). Overall, these findings indicate that IVM reduces ethanol intake and
has an excellent safety profile with good tolerability; thus pointing to this agent and potentially
other related avermectins as novel therapeutic agents for the prevention and/or treatment of
AUDs.
The mechanism(s) by which IVM reduces ethanol intake is/are not known. The current
therapeutic application of IVM as an antihelmentic is attributed to action on a non-mammalian,
glutamate-gated inhibitory chloride channel (Crump and Omura, 2011; Cully et al., 1994; Dent et
al., 1997; Vassilatis et al., 1997). Thus, action on this channel cannot contribute to its anti-
alcohol effect. On the other hand, IVM does potentiate mammalian ligand-gated ion channels,
including gamma-aminobutyric acid A (GABA
A
)

and glycine receptors (Dawson et al., 2000;
Krusek and Zemkova, 1994; Shan et al., 2001), and has been shown in rodents to have
anticonvulsant and anxiolytic properties linked to its action on GABA
A
receptors (GABA
A
Rs)
(Dawson et al., 2000; Spinosa et al., 2002). More recent studies indicate that IVM acts on several
other ligand-gated ion channel proteins in the mammalian central nervous system (CNS) (Sung
et al., 2009) including nicotinic acetylcholine (Krause et al., 1998; Sattelle et al., 2009) and
P2X4 receptors (P2X4Rs) (Khakh et al., 1999b). We recently reported that IVM antagonizes
ethanol inhibition of P2X4Rs expressed in Xenopus oocytes (Asatryan et al., 2010; Popova et al.,
2013), suggesting that P2X4Rs may play an important role in the anti-alcohol intake effects of
86

IVM. In support of the hypothesis that P2X4Rs are important in the anti-alcohol intake effects of
IVM, preliminary investigations in our laboratory found that IVM did not significantly reduce
ethanol intake in P2X4 knockout (KO) mice, but did reduce alcohol intake in wild type (WT)
controls.
In summary, available evidence indicates that IVM can reduce alcohol intake. Given that
IVM is already approved for use in humans, IVM has the potential for rapid repurposing as a
novel treatment for AUDs. The anti-alcohol actions of IVM likely reflect its ability to modulate
one or more ligand-gated ion channels in the brain, but this hypothesis has yet to be tested. IVM,
due to its lipophilic nature, should pass the blood brain barrier (BBB), but does not readily
achieve high brain concentration, ostensibly due to its high efflux by P-glycoprotein (P-gp).
Therefore, structural modifications that reduce its P-gp substrate recognition should increase
brain concentration (Lespine et al., 2007; Menez et al., 2012; Yardley et al., 2012) and should
positively impact its ability to reduce ethanol intake (Yardley et al., 2012). Likewise, structural
changes that alter its interaction with targeted brain receptors should also impact its efficacy in
this regard. The present study investigates these possibilities by comparing the effects of IVM
with two IVM-related macrocyclic lactones, abamectin (ABM) and selamectin (SEL), for their
abilities to reduce alcohol intake in mice and to alter modulation of GABA
A
Rs and P2X4Rs
expressed in Xenopus oocytes.

MATERIALS AND METHODS
Drugs. In vitro 10 mM stock solutions of IVM (powder from Sigma, St. Louis, MO), ABM
(powder from Sigma (Supelco), St. Louis, MO) were dissolved in DMSO and kept at -20ºC until
use (See Fig. 5.1 for chemical structures). The highest DMSO concentration in the final solution
87

was 0.1 %. SEL was kindly provided by Pfizer Pharmaceuticals (Groton, CT) as a 1% solution
suspended in propylene glycol. In vivo: drugs were administered via daily intraperitoneal (IP)
injections. Noromectin (10 mg/ml in 60% propylene glycol (Norbrook Inc, Lenexa, KS) was
used for IVM injections. Drugs were diluted using a 0.9% sodium chloride solution (saline) to a
concentration that would allow for an injection volume of 0.01 ml/g of body weight. Ethanol
(190 proof USP, Sigma, St. Lois, MO) was used in in vitro studies. For drinking studies, Gold
Shield Alcohol (200 proof USP solution, Gold Shield Chemical Company, Hayward, CA) was
diluted in water to achieve a 10% v/v solution (10E).
Figure 5.1

Fig 5.1. Structures of IVM, ABM and SEL. Both IVM and ABM feature a spiroketal moiety with a mixture of
isopropyl/isobutyl substituents. (A) ABM differs from IVM with the presence of an unsaturated double bond at the
C22-23 position. SEL has three structural differences from IVM and ABM: (B) it has a cyclohexyl ring in place of
the isopropyl/isobutyl substituent on IVM and ABM; (C) it has a ketoxime substituent in the place of the hydroxyl
group of the tetrahydro-benzofuran unit on IVM and ABM; and (D) it lacks the second carbohydrate unit that is
present in both IVM and ABM.

Animals. Studies were performed on C57BL/6J male mice that were 8 weeks old upon
purchase (Jackson Laboratory, Bar Harbor, ME, USA). Mice were singly housed in
polycarbonate/polysulfone cages at a 12 h light/dark cycle with lights off at 12:30PM. The
holding room was maintained at approximately 22°C. Mice had been tested for the effects of
88

IVM on ethanol intake using the drinking in the dark paradigm for approximately 2 months
before testing in the present study. Prior to the onset of the present experiment, mice were
acclimated to 24-h two-bottle choice ethanol paradigm as described below for five days. All
procedures in this study were performed in accordance with the NIH Guide for the Care and Use
of Laboratory Animals and all efforts were made to minimize animal suffering. The USC
Institutional Animal Care and Use Committee approved the protocols.
24-h access two-bottle choice paradigm. Individually housed mice had 24-h access to two
inverted bottles with metal sippers placed on the cage tops with one tube containing tap water
and the other a 10% v/v solution in tap water (10E). Alcohol determinations followed previously
described procedures (Yardley et al., 2012).
Ethanol intake studies with avermectins. Following 5 days of acclimation to the two-bottle
choice paradigm, mice were injected daily with saline until ethanol intake on the 2-bottle choice
stabilized (+/- 10% variability from the mean dose of the last 3 days). Then, mice were assigned
to drug treatment groups and were injected with 5 mg/kg of IVM, ABM or SEL according to a
within subjects design. Mice were injected with saline on subsequent days until drinking once
again stabilized.
Analysis of brain concentrations of IVM, ABM and SEL using liquid chromatography
with tandem mass spectrometry (LC-MS/MS). Plasma and brain samples were collected from
separate groups of animals at 0.25, 1, 4, 8, 10 and 32 hours (n=1-2/time point) after
administration of 5 mg/kg (i.p.) of IVM, ABM or SEL. To each 100 µL plasma or 150 mg of
brain, 2 µg IVM was added as internal standard for samples containing either ABM or SEL. For
samples containing IVM, 2 µg ABM was added as internal standards. Plasma samples were
mixed with 2 mL of acetonitrile, centrifuged at 10,000 rpm for 5 min with the organic layer
89

collected and air dried. To brain samples, 1 scoop of 1 mm zirconium bead and 1 mL of
acetonitrile were added, and vigorously homogenized using the bullet blender for 5 min. Samples
were then centrifuged at 10,000 rpm for 5 min, where the supernatant was collected. The above
steps were repeated with another 1mL of fresh acetonitrile, and the combined 2 mL supernatant
were dried by using a steady stream of filtered and dried air. Both the evaporated residues of
plasma and brain samples were reconstituted in 100 µL of acetonitrile : water (90:10 v/v).
The LC-MS method was validated using a calibration curve of known standard solutions
with the lower detection limit found to be 5 ng/mL. To each 100 µL plasma or 150 mg of brain,
2 µg of IVM was added as internal standard. The samples were extracted as described above. A
40 !L aliquot was injected into the Agilent 1100 HPLC System linked to an AB Sciex API 3000
turboion spray mass spectrometer. The analytes were separated using an ACE C18 column with
an isocratic mobile phase consisting of acetonitrile/0.1% formic acid : water/0.1% formic acid
(90 v : 10 v). The amount of IVM, ABM and SEL was quantified using the mass spectrometer
set in positive mode with multiple reaction monitoring using the parent to transition ions of 888.8
→ 551.5, 890.1 → 449.6 and 764.0→338.0 respectively.
Preparation of Xenopus oocytes. Xenopus oocytes isolation and maintenance followed
procedures described previously (Asatryan et al., 2010; Davies et al., 2005).
cRNA synthesis, cRNA and cDNA injections. The cDNAs of rat P2X4R (GenBank
accession No. X87763) and of rat α1, β2 and γ2 subunits of GABA
A
R were sub-cloned into
pcDNA3 vector (Invitrogen, Carlsbad, CA). The DNA containing P2X4R gene was then
linearized and transcribed using the mMESSAGE mMACHINE kit (Ambion, Austin, TX) to
result in cRNA, which was stored at -70ºC until injection. Twenty four hours post isolation the
oocytes were injected with 10 or 20 ng cRNA or 1 ng of DNA mixture of GABA
A
R subunits at
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1:1:1 ratio using Nanoject II Nanoliter injection system (Drummond Scientific, Broomall, PA).
The oocytes were incubated at 17ºC and used in electrophysiological recordings for 3-7 days
after injections.
Whole cell voltage clamp recordings. Two electrode voltage clamp recordings were
performed using the Warner instrument model OC-725C oocyte clamp (Hamden, CT) following
previously described procedures (Davies et al., 2005; Davies et al., 2002). The oocytes were
voltage clamped at -70 mV and the currents were recorded on a strip-chart recorder
(Barnstead/Thermolyne, IA).
P2X4Rs. Oocytes were continuously perfused at a rate of 3-4 ml/min with extracellular
modified Ringers buffer containing (in mM) 110 NaCl, 2.5 KCl, 10 HEPES and 1.8 BaCl
2
, pH
7.5, using a peristaltic pump (Rainin Instrument, Oakland, CA). Ca
2+
in the solution was
replaced with Ba
2+
to prevent the activation of Ca
2+
-dependent Cl
−
channels (Khakh et al.,
1999b). All experiments were performed at room temperature (20-23ºC).
To induce currents, submaximal concentrations (i.e. EC
10
) of adenosine 5’-triphosphate
(ATP, Sigma, St. Lois, MO) were used. Normally, P2X4Rs EC
10
is at 1 µM ATP. Using EC
10

has been previously shown to maximize the effects of ethanol while causing minimal receptor
desensitization (Davies et al., 2005; Davies et al., 2002). Ethanol and drugs were applied after
stable responses to EC
10
ATP were obtained. A washout period of 5 min was allowed between
each application to allow for re-sensitization of the receptor (Asatryan et al., 2010; Davies et al.,
2005; Davies et al., 2002; Popova et al., 2013).
Effects of ethanol (50, 100 or 200 mM) or IVM, ABM and SEL (0.5 - 30 µM) were tested
alone and in combination during co-application with ATP for 20 seconds. ATP currents were
measured before and after each drug application in order to confirm the existence of a stable
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baseline response. Pilot studies determined that the drugs did not have an effect on the membrane
potential of uninjected cells, nor did the drugs produce currents when applied in the absence of
agonist.
Experiments on GABA
A
Rs. Oocytes were perfused at a rate of 3-4 ml/min at room
temperature with modified Bart’s saline containing in mM (83 NaCl, 1 KCl, 10 HEPES, 0.82
MgSO
4
, 2.4 NaHCO
3
, 0.91 CaCl
2
, and 0.33 Ca(NO
3
)
2
, pH 7.5). Similar to the approach for
P2X4Rs, EC
10
concentration of GABA was used to test the effects of the drugs, i.e. IVM, ABM
and SEL. A washout period of 5 min was allowed between each GABA application.
IVM, ABM and SEL were applied with GABA EC
10
after a stable response to GABA EC
10

was obtained. Each oocyte was tested for one concentration of avermectins since these drugs
caused irreversible effects that were not washable. The second consecutive response to the
application of the drug was always larger; therefore, both responses in the presence of the drug
were averaged for data analysis.
Data Analyses In vivo studies. Ethanol dose (g/kg) and ethanol preference ratio (mls
ethanol/total mls) were calculated for each drug. The dependent variables included 10E intake
(g/kg), 10E preference (%), water (ml) and total fluid intake (ml). Two-tailed t-tests were used to
assess the effects of drug treatment groups (IVM, SEL, ABM) versus the respective saline
injected pre-drug control groups for each dependent variable. The significance level was set at p
≤ 0.05.
Data Analyses In vitro studies. Data were obtained from batches of oocytes from at least 3
different frogs and are expressed as mean ± SEM for an indicated number of tests. The results
are presented as the percentage change in agonist EC
10
activated currents after normalizing these
with the response of the EC
10
alone. To assess concentration response relationships, data were
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fitted to a concentration-response curve by using the following logistic equation: I = I
max
*
[drug]/([drug] +(EC
50
)
drug
), where I is the percentage of the maximum obtainable response (I
max
),
EC
50
is the drug concentration producing a half-maximal response. Bar graphs were used to
compare the effects of ethanol with and without IVM, ABM or SEL on P2X4Rs. GraphPAD
Prism software (San Diego, CA) was used for data analysis and curve fitting. Statistical analysis
was performed using unpaired t-tests with significance set at p ≤ 0.05.
Pharmacokinetic Analysis. The pharmacokinetics (PK) of IVM, SEL and ABM in plasma
and brain were analyzed using non-compartmental PK modeling. Serial blood and tissue IVM,
SEL and ABM quantification was used to calculate PK parameters such as maximum drug
concentration (Cmax), time to achieve maximal drug exposure (Tmax), half-life, elimination
constant and area under the curve (AUC).

RESULTS
IVM and ABM, but not SEL, reduced alcohol intake and preference in C57BL/6J mice. We
tested the effects of acute administration of IVM, SEL, and ABM using a 24-h access two-bottle
choice alcohol paradigm (10E versus tap water) in C57BL/6J mice. This model was selected
based on our initial investigation that tested the effects of IVM on ethanol intake and preference
(Yardley et al., 2012). We used 5 mg/kg of IVM and analogs based on our prior study where
doses ranging from 2.5 to 10.0 mg/kg provided significant reductions in drinking levels without
any significant behavioral toxicity (Yardley et al., 2012).
Ethanol intake for the saline controls the day before injection of IVM, SEL and ABM were
comparable (Fig 5.2A). IVM and ABM significantly reduced 10E intake versus their respective
controls. In contrast, SEL did not have a significant effect on ethanol intake. For all drugs, 10E
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intake returned to comparable pre drug intake levels one-day post drug injection (data not
shown).
Similarly, IVM and ABM reduced preference for 10E versus saline injection (Fig. 5.2B).
SEL had no effect on this parameter. In addition, ABM significantly increased water intake (Fig.
5.2C). There was a trend to increase water intake for IVM (p<0.06), whereas SEL had no effect
on this measure (Fig. 5.2C). Lastly, only IVM decreased total fluid intake (Fig. 5.2D).
Figure 5.2

Fig. 5.2. IVM, SEL, ABM (5 mg/kg) administration in male C57BL/6J mice using a 24-h access two-bottle choice
paradigm. Bars represent levels from the saline control the day prior to drug injection (Pre Drug) and the day of the
drug injection (Drug) for A) 10E intake, B) preference for 10E, C) water intake, and D) total fluid intake. Values
represent the mean +/-SEM for 7-8 mice per dose group. *P<0.05, **P < 0.01 versus respective pre drug condition.

Pharmacokinetics of IVM, ABM and SEL. To determine whether relative differences in
avermectin penetration across the BBB influenced the brain concentration of IVM, ABM and
SEL and might explain the differences in the respective effects of these agents on ethanol intake
found in the in vivo studies described above, we measured both plasma and brain concentrations
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at the designed time points of 0.25, 1, 4, 8, 10 and 32 hours after i.p administration of 5 mg/kg of
each. The findings are shown in Table 5.1. Brain AUCs for IVM, ABM and SEL were 1.81
ng*hr/mL, 40.1 ng*hr/mL and 69.3 ng*hr/mL respectively, where SEL and ABM had the
highest brain concentrations. Interestingly, ABM and SEL AUCs were 22- and 38-times higher
than those for IVM.
Table 5.1

Table 5.1. Pharmacokinetic parameters of avermectins. The plasma exposure of the three avermectins was similar as
determined by mean AUC
0−t
. Despite similar plasma exposures, there were marked differences in brain disposition
in this study, where brain AUC
0−t
for abamectin (ABM) and selamectin (SEL) was 22- and 38-fold higher than
ivermectin (IVM).

Effect of avermectin analogs on P2X4R and GABA
A
R function. In order to determine the
effect of avermectin analogs on receptor function, we performed several experiments testing
IVM, ABM and SEL on two ion channels that are linked to IVM’s behavioral effects, P2X4Rs
and GABA
A
Rs (Bortolato et al., 2013; Yardley et al., 2012). Previous findings demonstrated that
IVM (0.5 – 10 µM) potentiated ATP-gated currents in P2X4Rs expressed in oocytes (Asatryan et
al., 2010; Khakh et al., 1999b; Priel and Silberberg, 2004). Therefore, to test the effects of ABM
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and SEL on P2X4Rs, we elected to use a similar concentration range (0.5 – 10 µM) that was
used in our prior IVM study (Asatryan et al., 2010). We compared the ability of ABM and SEL
versus IVM to modulate ATP-gated currents in P2X4Rs.
ABM (0.5 – 10 µM) significantly potentiated ATP-induced currents in P2X4Rs in a
concentration-dependent manner (Fig. 5.3A). The degree of ABM potentiation was significantly
greater than that of IVM at concentrations ≥ 3 µM; i.e. reaching 100-fold greater activity at 10
µM (Fig. 5.3A). However, at lower concentrations, i.e. 0.5 and 1 µM, the degree of ABM and
IVM potentiation did not significantly differ (Fig. 5.3B). ABM, at higher concentrations (3 and
10 µM), directly induced P2X4R currents in the absence of ATP and this effect was
concentration-dependent (Fig. 5.3C). In addition, there was a residual effect on post ATP
currents (Fig. 5.3C, final tracings in each row) suggesting that ABM was slow to washout.
Figure 5.3

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Fig. 5.3. IVM, ABM and SEL modulation of P2X4R and GABA
A
R activity. (A) Concentration-response curves of
co-application of avermectins (0. 5 -10 µM) with EC
10
ATP demonstrate that IVM and ABM are potent positive
modulators whereas SEL is a weak modulator of P2X4R activity. (B) ABM potentiation was significantly greater
than that of IVM at concentrations ≥ 3 µM but not at lower 0.5 and 1 µM concentrations. (C) ABM directly induces
currents in P2X4Rs at higher concentrations (3 and 10 µM) in a concentration-dependent manner. (D) All
avermectins significantly potentiated GABA
A
R function. (E) At low 0.5 µM concentration, the effect of SEL was
smaller compared to that of IVM but did not reach statistical significance. Data are presented as mean ± SEM of 4-
12 oocytes per data point.

In contrast to IVM and ABM, SEL poorly potentiated ATP-gated currents in P2X4Rs (Fig.
5.3A) and had markedly lower responses than either IVM or ABM across all concentrations
tested (0.5 - 10 µM) with the response of SEL at 10 µM being similar to IVM and ABM at 0.5
µM. Therefore, we tested a higher concentration of SEL to see if we could elicit responses more
comparable to IVM and ABM. We found that 30 µM SEL induced a significantly higher degree
of potentiation compared to 10 µM SEL (78.8 ± 24.2 vs 42.4 ± 12.7 % of control, P < 0.05).
However, the SEL response at 30 µM was still 5-fold less in magnitude compared to the degree
of potentiation induced by 10 µM IVM (IVM 405 ± 36 % vs SEL 78.8 ± 24.2 %).
We also tested the effects of IVM, ABM and SEL on GABA
A
Rs. For this study we used
α1β2γ2 GABA
A
Rs due to their predominant expression in the CNS. As illustrated (Fig. 5.3D),
IVM and ABM significantly potentiated GABA
A
R function. The effects of ABM and IVM were
similar in the concentration range of 0.5 - 3 µM. At 10 µM IVM, the extent of IVM potentiation
was significantly greater compared to the effect of ABM. The greater degree of potentiation by
10 µM IVM may be due to IVM acting as a partial agonist for GABA
A
Rs. The degree of
potentiation of GABA-induced currents by SEL on GABA
A
R function tested at 0.5 µM did not
significantly differ from IVM and ABM. However, the effects of SEL were significantly less
than IVM and ABM at concentrations ≥ 1.0 µM (Fig. 5.3E).
IVM and ABM, but not SEL, antagonize the effects of ethanol on P2X4Rs. Our previous
work found that 0.5 µM IVM significantly antagonized the effects of ethanol caused by
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intoxicating (25 mM or 0.1 %) and higher (up to 100 mM) ethanol concentrations (Asatryan et
al., 2010). The current study extended this work by testing concentrations of ABM and SEL that
induced a similar degree of potentiation of P2X4R function as that of 0.5 µM IVM (i.e. 0.5 µM
for ABM and 10 µM for SEL). We selected 100 mM ethanol since it caused a robust inhibition
of ATP-induced currents.
ABM (0.5 µM) significantly reduced 100 mM ethanol inhibition and the degree of
antagonism by ABM was similar to that of IVM (Fig. 5.4A). On the other hand, we found that a
20X higher concentration of SEL was required in order for SEL to reach a similar degree of
potentiation of P2X4R function as that of IVM and ABM. Notably, even at this higher
concentration, SEL did not significantly antagonize the effects of ethanol on P2X4R function
(Fig. 5.4A).
Figure 5.4

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Fig. 5.4. Effects of IVM, ABM and SEL on ethanol inhibition of P2X4R function. (A) IVM and ABM at 0.5 µM
antagonize the 100 mM ethanol inhibition. SEL at 10 µM is not able to antagonize the effect of 100 mM ethanol.
There was a right-shift in the ethanol concentration-response curves for ABM (B) but not for SEL (C). Data are
presented as mean ± SEM of 5-17 oocytes per experimental point. * P < 0.05 compared to the ATP + 100 mM
ethanol.

We extended this investigation to include concentration-response studies for ABM and SEL
antagonism of ethanol using methods similar to our recent IVM studies (Asatryan et al., 2010;
Popova et al., 2013). This was accomplished by testing a concentration range (0.5 – 3 µM) of
ABM and SEL on the effects of ethanol (50, 100 and 200 mM) on P2X4Rs. ABM (Fig. 5.4B),
but not SEL (Fig. 5.4C), antagonized ethanol inhibition of P2X4R function as indicated by a
parallel right-shift in the ethanol concentration-response curves.

DISCUSSION
The present study provides insights into the mechanisms and important structural features of
IVM responsible for its ability to reduce ethanol intake that can inform future development of
more effective agents. Using three related, but structurally distinct avermectins (IVM, ABM, and
SEL) the findings indicate that 1) the structure and 2) ability to modulate P2X4Rs and
antagonize ethanol effects in P2X4Rs are important determinants for predicting the ability of
avermectins to reduce ethanol intake.
Using an in vivo continuous access two-bottle choice drinking paradigm, the present study
found that IVM significantly reduced ethanol consumption. IVM also reduced total fluid intake,
however this probably reflected the decrease in ethanol intake since IVM did not affect water
intake. These findings are in good agreement with our previous work (Yardley et al., 2012) and
extend this investigation, showing that ABM also significantly reduced ethanol intake, but to a
lesser extent than did IVM. In contrast, SEL did not significantly reduce ethanol consumption.
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The disposition of the avermectins in plasma and brain were evaluated by LC-MS/MS to
determine whether brain disposition might play a role in determining the efficacy of IVM, ABM,
and SEL. The results indicate that there was minimal difference between IVM, ABM, and SEL
in their plasma concentrations, as defined by the area under the curve (AUC). In contrast, the
brain disposition differed considerably between the three avermectins. SEL had the highest brain
concentration which was followed by ABM and then IVM. Thus, the degree of BBB penetration
and resultant brain concentration of the respective drugs alone cannot explain the differences in
the ability of these avermectins to reduce alcohol intake. Rather, these differences reflect their
ability to modulate or offset underlying neurochemical effects of ethanol.
In agreement with this contention, we identified substantial differences between the abilities
of avermectins to modulate P2X4R function and to antagonize the inhibitory effects of ethanol
on P2X4Rs in vitro. Specifically, IVM and ABM significantly potentiated P2X4R function and
antagonized the inhibitory effects of ethanol at therapeutically relevant concentrations. In
contrast, SEL, even at much higher concentrations, showed only minimal activity to potentiate
P2X4R function and to antagonize the effects of ethanol, in vitro. The right-shift in the ethanol
concentration-response with ABM supports a competitive mechanism for the ethanol
antagonism. Notably, the findings with ABM are similar to our previous findings with IVM
(Asatryan et al., 2010; Popova et al., 2013). On the other hand, the absence of significant
reductions in ethanol response or a right shift for the concentration-response curves for SEL
further supports the notion that SEL lacks the ability to antagonize the inhibitory effects of
ethanol on P2X4Rs.
SEL appeared to be equally potent to IVM and ABM in its ability to positively modulate
GABA
A
Rs when tested at concentrations up to 1 µM. On the other hand, at concentrations ≥ 1.0
100

µM, SEL was less effective in potentiating GABA
A
R function as compared to IVM and ABM.
Taken together, the findings suggest that the significant differences in structure between IVM
and ABM, compared to SEL, play an important role in the differences in the compounds ability
to positively modulate P2X4R and to a lesser extent, GABA
A
R function.
IVM belongs to a class of lipophilic, water insoluble compounds, the avermectins that are
produced by the soil microorganisms Streptomyces avermitilis (for review see Omura, 2008;
(Crump and Omura, 2011; Omura, 2008). The structural basis of IVM as an anti-parasitic agent
has been linked to the hexahydro-benzofuran site, but not the spiroketal group of the molecule
(Michael et al., 2001). In addition, the size of the carbohydrate side chain is suggested to be
important in the interaction with the P-gp and should influence the ability of P-gp to remove
avermectins that penetrate the BBB (Lespine et al., 2007). ABM and IVM are structurally
similar; differing only by a double bond at C22-23 (Fig. 5.1). ABM is a mixture of the
fermentation natural products avermectin B1a and B1b containing a double bond at C22-23 of
the spiroketal unit, while IVM is the product of selective chemical hydrogenation of ABM at
C22-23. SEL is a synthetic analog that is structurally different from both ABM and IVM. SEL,
in addition to having a saturated spiroketal moiety that is similar to IVM, also has a cyclohexyl
ring in place of the isopropyl/isobutyl substituent on IVM and ABM. The absence of the C22-
C23 double bond in IVM alters the conformation of the spiroketal moiety and modifies its
metabolism in comparison with ABM (Halley et al., 1992). However, given the comparable
ability of both IVM and ABM to antagonize the inhibitory effects of ethanol in P2X4Rs more
effectively than SEL, it seems that their spiroketal differences are not the primary determinant
for this effect.
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There are two other major structural differences between IVM and ABM, as compared to
SEL. First, SEL features an unsaturated ketoxime substituent in the place of the simple allylic
hydroxyl group that is found on IVM and ABM. Second, SEL contains only one carbohydrate
moiety rather than two as in IVM and ABM (Fig. 5.1). The latter feature is likely responsible for
reduced interaction of SEL with P-gp (Lespine et al., 2007) and as a result the higher brain
concentrations seen with SEL versus IVM and ABM. On the other hand, despite higher brain
concentrations, SEL did not reduce ethanol intake. Together, these findings suggest three
possibilities: 1) the presence of the two carbohydrates is required for the anti-alcohol effects of
IVM and ABM; 2) the presence of the ketoxime in the structure of SEL is sufficient to eliminate
this effect or 3) both of these structural differences are important in determining the anti-alcohol
efficacy of avermectins.
The identified structural modifications in SEL (Fig. 5.1) may be important pharmacophore
sites required for the greater anti-alcohol potency of IVM and ABM. In support of this notion,
recent studies identified an overlapping putative binding pocket for IVM and ethanol, an alcohol-
IVM pocket, in P2X4Rs (Asatryan et al., 2010; Popova et al., 2013). In this context, we propose
that the second sugar moiety that is present in both IVM and ABM, but lacking in SEL is
important for the affinity of IVM and ABM for this alcohol/IVM pocket that is key for the
efficacy of avermectins in blocking the actions of ethanol on P2X4Rs and reducing alcohol
intake. The presence/absence of this structural element may also play an important role for the
interaction of IVM and ABM with other receptors systems such as GABA
A
Rs. Future studies
will begin to address these issues by investigating structure-function interactions including
molecular modeling and extensive docking of avermectins at the identified putative binding site.
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Taken together, the findings suggest that chemical structure and effects on receptor function
play key roles in the ability of avermectins to reduce ethanol intake and that these factors are
more important than brain penetration alone. The direct relationship between the effect of these
avermectins on P2X4R function and ethanol intake suggest that the ability to antagonize ethanol-
mediated inhibition of P2X4R function may be a good predictor of the potential of an avermectin
to reduce ethanol intake and support the use of avermectins as a platform for developing novel
drugs to prevent and/or treat AUDs.

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CHAPTER 6
OVERALL DISCUSSION AND CONCLUSIONS

Summary of Overall Findings
The findings presented in my dissertation provide preclinical support for the efficacy of
IVM in reducing ethanol consumption as measured across multiple mouse models of ethanol
intake using different dosing regimens. To briefly summarize, Chapter 2 demonstrates that acute
administration of IVM (2.5-10 mg/kg) is able to reduce ethanol intake in both male and female
mice using a within subjects design. The highest dose of IVM tested (10 mg/kg) reduces ethanol
intake by almost 50% compared to pre IVM intake levels in both sexes. A similar reduction is
also seen in preference for ethanol. Importantly, IVM is able to decrease ethanol intake by the
same percentage using an intermittent limited access model of binge drinking. Furthermore, the
plasma and brain IVM AUC correlates with percent reduction of ethanol consumption and the
lowest dose of IVM leading to detectable IVM levels in the brain is the same as the lowest dose
required to significantly reduce ethanol consumption.
Chapter 3 describes studies looking at the intrinsic properties of IVM and represents the
first comprehensive investigation on the potential of IVM to alter CNS driven behaviors. Results
from these studies suggest that acute administration of IVM (10 mg/kg) results in anxiolytic-like
responses in C57BL/6J mice, as evaluated by the elevated plus maze and the marble burying
assays. As approximately 29-37% of people with an AUD also suffer from anxiety, the
anxiolytic properties of IVM could prove to be beneficial to this population of alcoholics
(Petrakis et al., 2002). Notably, unlike other currently available anxiolytic agents such as
diazepam, IVM does not appear to exhibit rewarding properties in the conditioned place
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preference paradigm, strongly suggesting that the therapeutic potential of this agent is not
associated with addiction liability. IVM does not cause significant changes in pain sensitivity, as
tested in the hot-plate assay, or in motor competency, as signified by the lack of variations in
locomotor activity and absence of catalepsy. Furthermore, acute IVM administration does not
impact short- or long-term mnemonic integrity in the novel object recognition test, an assay with
high predictive validity for the assessment of declarative memory. Taken together, the findings
from Chapter 3 support the contention that IVM appears to have an excellent safety profile and
good tolerability thus pointing to this agent as an attractive therapy for AUDs and comorbid
psychiatric diagnoses, such as anxiety.
AUDs are chronic disorders and, as such, a single dose of IVM would be insufficient for
this new indication. In Chapter 4, the effect of IVM administered over multiple days is tested.
Consecutive day dosing of IVM (3.0 mg/kg x 10 days) using a 24-h two-bottle choice paradigm
significantly decreases ethanol intake over a 9-day period without causing overt signs of toxicity,
including no change in body weight or water consumption. These results indicate that the ability
of IVM to decrease ethanol intake is sustainable over a prolonged period of time.
Chapter 5 details the possibility of using IVM as a platform for developing novel
therapeutics. To this end, these studies focus on testing the hypothesis that structural
modifications that enhance the effect of IVM on key receptors and/or increase the concentration
of drug in the brain improve the anti-ethanol effect. Although there is no correlation between
concentration of avermectins in the brain and anti-ethanol activity, these findings did suggest that
the ability of avermectins to antagonize P2X4Rs in vitro is a strong predictor of anti-ethanol
activity in vivo. Overall, these studies support the use of avermectins as a platform for drug
development of novel therapeutics to treat AUDs.
105

Collectively, the findings, as presented in my dissertation, provide strong preclinical
support for the repurposing of IVM as a therapeutic agent for the treatment of AUDs and lay the
groundwork for transitioning from preclinical to clinical studies. First, acute and multi-day IVM
administration reduces ethanol intake in both female and male mice. Second, behavioral studies
support the contention that IVM appears to have an excellent safety profile and good tolerability,
thus pointing to this agent as an attractive therapy for AUDs and comorbid psychiatric diagnoses.
Lastly, doses that have been shown to be effective in rodents correspond to doses reported to be
safe in humans. Overall, the studies presented above help set the stage for first-in-human testing
of IVM as a treatment for AUDs.

Future Directions
Chronic preclinical studies and first-in-human clinical studies are currently underway.
The preclinical study tests the hypothesis that chronic (30 days) oral administration (via fast
dissolving oral film) of IVM (0.21 mg) reduces ethanol intake in mice using a model of social
drinking and that this dosing regimen of IVM does not lead to overt signs of toxicity. The
clinical study tests the safety of IVM (30 mg) in the context of moderate doses of ethanol (0.02-
0.08 g/dL) in AD individuals (n=10). This study will also test the hypothesis that IVM reduces
the reinforcing effects of ethanol and ethanol craving.
Since AUDs are chronic and oral delivery is the ideal route of administration, the effects
of IVM administered via fast dissolving oral films over multiple days was tested. IVM was given
to C57BL6/J mice for 30 days, 5 times per week for 6 weeks. There were 3 groups in this study:
1) administered key lime flavored oral film with 0.21 mg of IVM (IVM group), 2) administered
key lime flavored oral film with no drug (placebo group) and 3) given no oral film at all (control
106

group). The IVM group drank significantly less ethanol over the 30-day period compared to the
control and placebo group. Preference for ethanol was also significantly less in the IVM group.
Perhaps to account for the decrease in ethanol intake, the IVM group drank significantly more
water but, overall, the fluid intake among the 3 groups was comparable. Change in body weight
was similar across the 3 groups; however, the IVM group consumed significantly more food,
possibly in an attempt to counteract the caloric loss resulting from the decrease in ethanol
consumption. In half of the animals from each group, brain, liver, left and right kidney, spleen,
heart and the left tibia were collected. The organ weights were normalized to the length of the
left tibia for each animal. For each of the 6 organs, there were no differences in organ weight to
tibia length ratio between the 3 groups. Although histological analyses are ongoing, these data
strongly suggest that chronic oral administration of IVM is both effective in reducing ethanol
intake and is well tolerated.
Recruitment for the clinical portion is currently underway, although, it is not expected
that exposure to ethanol will increase the risk of severe adverse events (SAEs) associated with
IVM treatment (Takougang et al., 2008). A study conducted by Takougang et al included people
in savannah and forest-savannah zones of Africa receiving IVM treatment to test the hypothesis
that SAEs experienced during IVM treatment were independent of ethanol intake. Participants
who experienced SAEs (n=36) were the cases and participants who did not experience SAEs
(n=43) were the controls. Both groups completed the AUD identification test (AUDIT), an
ethanol consumption history, and gave blood samples to determine serum glutamyl phosphate
oxaloacetate transferase (SGOT) levels, used to provide an indication of chronic alcoholism.
They found that the major risk factor in developing SAEs was the microfilariae count, not
ethanol consumption. In fact, there was no correlation between ethanol consumption and SAEs at
107

all. Homeida et al confirmed these findings when they tested the hypothesis that ethanol-induced
increases in bioavailability of IVM is responsible for an increased risk of SAEs in 10 Sudanese
men (Homeida et al., 2013). Blood was collected at 0, 1, 2, 3, 4, 5, 6, 8, 12, 28, and 30 hours
after IVM administration. There were 3 groups: 1) given 150 µg/kg IVM in a fasting state; 2)
given 150 µg/kg IVM with 600 g of a local Sudanese meal; and 3) given150 µg/kg IVM with
200 mL of a local brew. They found no significant difference between the groups except at hour
2 when the IVM plasma concentration was significantly lower for group 3, the group given the
local brew, compared to the other 2 groups. However, it is important to note that other studies
found the opposite to be true; co-administration of IVM with ethanol does increase the plasma
concentration of IVM (Canga et al., 2008; Shu et al., 2000). Despite conflicting views on plasma
concentration levels, the former studies suggest that IVM, in the context of ethanol, is not a risk
factor for increased SAEs.
Future clinical studies will expand on the results from the pilot study and test the
hypothesis that IVM (30 mg) decreases ethanol self-administration in non-treatment seeking
individuals with AD that are on a normal diet (n=32). The proposed placebo-controlled
randomized pilot safety trial has 2 aims: 1) test the hypothesis that IVM will reduce ethanol self-
administration, compared to placebo, in non-treatment seeking individuals with AD and 2) test
the hypothesis that IVM will dampen ethanol craving, compared to placebo, using the cue
reactivity (CR) paradigm. Results from these studies will then be used to inform a large-scale
clinical trial of IVM for the treatment of AUDs.

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Abstract (if available)

Abstract Alcohol use disorders (AUDs) affect over 18 million people in the United States alone, cost over $235 billion, and yet only 8% of this population receives treatment and even less use a medication approved by the U.S. Food and Drug Administration (FDA) as part of that treatment. Despite considerable efforts focusing on new drug development to reduce ethanol abuse, high rates of harmful drinking persist. This is, in part, due to the fact that current therapeutic strategies are largely inadequate to treat these disorders. Thus, developing novel therapeutics for the treatment of AUDs is of paramount importance. The working hypothesis of our laboratory is that ivermectin (IVM) can be repurposed as a therapeutic agent for the treatment of AUDs. As IVM is currently FDA‐approved and used by millions of humans each year for other indications, the repurposing of IVM for the treatment of AUDs represents a fast and economically feasible approach for drug development. Initial support suggesting that IVM can be developed as a novel drug candidate for the treatment of AUDs comes from previous work demonstrating that IVM is able to antagonize the effect of ethanol in vitro on P2X4 receptors (PRX4Rs). Studies included in this dissertation test the hypothesis that IVM can be repurposed as a therapeutic agent for the treatment of AUDs using multiple preclinical mouse models of ethanol intake and behavior. Chapter 2 describes initial efficacy studies using 3 distinct models of ethanol intake and explores the pharmacokinetics (PK) of IVM. Chapter 3 characterizes the intrinsic properties of IVM using a battery of behavioral paradigms to test for effects such as depression, anxiety, locomotion, memory, and rewarding properties. Chapter 4 evaluates the sustainability and safety of multi‐day IVM administration. Finally, Chapter 5 focuses on the use of IVM as a platform for developing novel therapeutics for AUDs by testing two related avermectins, selamectin (SEL) and abamectin (ABM). Findings from my work support the hypothesis that IVM is able to reduce ethanol intake using multiple murine models of ethanol intake without causing overt toxicity. Overall, the studies presented within this dissertation set the stage for first-in-human testing of IVM for this new indication.

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Asset Metadata

Creator Yardley, Megan M. (author)

Core Title Development of ivermectin as a platform for the treatment and/or prevention of alcohol use disorders

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology and Toxicology

Publication Date 01/18/2016

Defense Date 05/19/2014

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag alcoholism therapy,drug repositioning,medications development,OAI-PMH Harvest

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Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Cadenas, Enrique (committee chair), Davies, Daryl L. (committee chair), Neely, Michael J. (committee member), Richmond, Frances J. (committee member)

Creator Email mmyardley@gmail.com,yardley@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-444768

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