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Preclinical investigation of ivermectin as a novel therapeutic agent for treatment of alcohol use disorders

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Preclinical investigation of ivermectin as a novel therapeutic agent for treatment of alcohol use disorders

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PRECLINICAL INVESTIGATION OF IVERMECTIN AS A NOVEL THERAPEUTIC AGENT FOR
TREATMENT OF ALCOHOL USE DISORDERS

by

Sheraz Khoja

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(PHARMACEUTICAL SCIENCES)

August 2012

Copyright 2012 Sheraz Khoja

ii

ACKNOWLEDGMENTS

I would like to express my deepest gratitude to my mentors and fellow lab mates for
making my journey as a graduate student a highly memorable one. Firstly, I would like
to thank Dr. Daryl Davies for giving me the opportunity to work in his laboratory and for
allowing me to participate in one of the several significant projects that are being
executed in our laboratory. I am highly grateful to Dr. Davies for devoting his time and
effort in making me a highly competent research student. My enthusiasm and passion
for the field of research has grown solely because the amazing experience I had while
working under him.
I would also like to thank my co-mentor, Dr. Ronald Alkana for his significant
contribution to my project with respect to his ideas and comments and for serving on
my thesis committee. I am also thankful to Dr. Kathleen Rodgers for taking time out
from her busy schedule in serving on my thesis committee.
I am grateful to the collaborators for the published manuscript “Ivermectin reduc es
alcohol intake and preference” (Yardley et al., 2012) which includes Megan Yardley,
Letisha Wyatt, Dr. Liana Asatryan, Marcia J.Ramaker, Dr. Deborah Finn, Dr. Nicos
Petasis, Dr. Stan G Louie and Nhat Huynh. In addition, I am also grateful to the
collaborators for the “conditionally accepted” manuscript “Pharmacological insights into
role of P2X4 receptors in behavioral regulation: lessons from ivermectin” (Bortolato et
iii

al., 2012) which includes Dr. Marco Bortolato and Dr. Sean Godar. Some of the
experiments from both these manuscripts formed a significant core of my thesis project
and are explained in detail in various chapters of this thesis. I would also like to
acknowledge the efforts of Yara Bashawri, Dimple Shah and Michelle Lee for their
assistance in various experiments of the project.
Lastly, I would like to thank all my fellow lab mates especially Anna Naito and Karan
Muchhala for their support and for creating a suitable environment for me to carry out
my experiments.

iv

TABLE OF CONTENTS

Acknowledgments ii
List of figures vi
Abstract vii
Chapter 1: Introduction
1.1 Impact of Alcohol use disorders on society 1
1.2 Current pharmacological agents used for treatment 5
of AUDs
1.2.1 FDA approved drugs for treatment of AUDs 6
1.2.2 Drugs under current clinical trial investigations 10
1.3 Role of P2X4 in being a critical target for AUDs 14
1.4 Ivermectin: The marvelous discovery from Japan 17
1.4.1 The discovery of ivermectin 17
and its multiple uses
1.4.2 Mechanism of action of IVM’s antiparasitic 18
activity
1.4.3 IVM,P2X4Rs and ethanol 19

Chapter 2: Aim 1: Test the hypothesis by investigating the effects of IVM on
alcohol intake and preference in C57BL/6 mice using a 24 hour access two bottle choice
paradigm.
2.1 Background 22
2.2 Materials 24
2.3 Methods 26
2.4 Results 28
2.4.1 IVM significantly reduced 10E intake and preference 28
in male mice
2.4.1.1 IVM (10mg/kg) significantly reduced 10E intake 28
and preference in male mice
2.4.1.2 IVM significantly decreased 10E intake and 29
preference in a dose dependent fashion in male mice
v

2.4.1.3 Time course effect of IVM on 10E intake in male 30
mice
2.4.2 IVM significantly reduced 10E intake and preference in 34
a dose dependent manner in female mice
2.4.3 Long term dosing of IVM significantly reduced 10E intake 36
and preference in female mice
2.4.4 IVM decreased saccharin consumption and preference in 37
a dose dependent manner in female mice
2.5 Discussion 42
Chapter 3: Aim 2: Test the hypothesis that IVM does not cause behavioral effects that
would negatively impact its application to preventing or treating AUDs by conducting a
set of behavioral paradigms capturing the complementary aspects of perceptual,
emotional and cognitive regulation in mice.
3.1 Background 49
3.2 Materials 55
3.3 Methods 56
3.4 Results 59
3.4.1 IVM elicited anxiogenic-like effect in open field test for anxiety 59
3.4.2 IVM elicted anxiolytic response in marble burying 62
test for anxiety
3.4.3 IVM induced behavioral state of despair in forced swim 62
model for depression in dose dependent manner
3.4.4 IVM dose dependently reduced prepulse inhibition of acoustic 62
startle reflex
3.5 Discussion 66
Chapter 4: Conclusion 73
Bibliography 76

vi

LIST OF FIGURES

Figure 1 Effect of IVM (10mg/kg) on 10E intake and 31
preference in male C57BL/6 mice
Figure 2 Dose response study of IVM in male C57BL/6 mice 32
Figure 3 Time course effect of IVM on 10E intake 33
in male C57BL/6 mice

Figure 4 Dose response study of IVM in female C57BL/6 mice 39
Figure 5 Long term dosing of IVM in female C57BL/6 mice 40

Figure 6 Effect of IVM on saccharin (0.033%w/v) 41
intake in female C57BL/6 mice
Figure 7 Effect of IVM on anxiety-like behavior in male 61
C57BL/6 mice using open field test for anxiety

Figure 8 Effect of IVM on anxiety-like behavior in male 63
C57BL/6 mice using marble burying test for anxiety
Figure 9 Effect of IVM on stress induced depressive behavior 64
in male C57BL/6 mice using forced swim test for
depression

Figure 10 Effect of IVM on prepulse inhibition of acoustic 65
startle reflex in male C57BL/6 using prepulse
inhibition test

vii

ABSTRACT
Alcohol use disorders (AUDs) ranks third in the list of preventable causes of
morbidity and mortality in United States having a major national impact affecting
over 18 million people and causing over 100,000 deaths annually. The
prevalence of AUDs, in combination with limited clinical efficacy of currently
approved FDA drugs in the treatment of this disorder signifies the importance for
development of novel therapeutic agents. Alcohol is known to modulate a
multitude of receptors in the brain to induce its cellular and behavioral effects.
Recent electrophysiological findings from our laboratory, coupled with genomic
studies, have suggested that the ionotropic receptor, P2X4 receptor (P2X4R) is a
critical target that is modulated by ethanol. P2X4R activity has been
demonstrated to be inhibited by ethanol at intoxicating and anesthetic
concentrations. Recent evidence from our laboratory using electrophysiological
strategies has shown that the FDA approved antiparasitic agent, ivermectin
(IVM) antagonizes ethanol induced inhibition of P2X4R activity. On the basis of
our in vitro data, and suggestion from my advisors I began testing the hypothesis
that IVM represents a novel therapy that can be repurposed for treatment of
AUDs.
Aim 1 tests the hypothesis by investigating the effect of IVM on alcohol intake
and preference in male and female C57BL/6 mice. This was accomplished by
testing IVM at doses ranging from 0.65 mg/kg through 10mg/kg versus a 10%
viii

ethanol (10E) solution using a 24 hour two bottle choice paradigm. In support of
the hypothesis, we found that IVM significantly reduced alcohol intake and
preference in male and female mice. In addition, using a similar paradigm,
evaluation of IVM’s effect on s accharin intake was undertaken to determine if
IVM’s effect was specific for ethanol or if IVM was also active versus a second
substance known to have positive reinforcing effects. In agreement with our
ethanol studies, IVM significantly decreased saccharin intake and preference.
Aim 2 tests the hypothesis that IVM does not cause behavioral effects that would
negatively impact its potential therapeutic application to preventing or treating
AUDs. To this end, I tested the effects of IVM on recognized measures of anxiety
(open field, marble burying), depression (forced swim test), information
processing (prepulse inhibition test). Collaborators from my research team also
tested the elevated plus maze, tail suspension, conditioned place preference, hot
plate and object exploration. A detailed explanation of these latter studies will
be presented elsewhere but the findings will also be summarized in my thesis to
present a broad picture of the investigations. The results overall support the
hypothesis. IVM did not show rewarding effects in conditioned place preference
test, suggesting that it does not have addictive potential and had anxiolytic
effects in the marble burying test. However, it does appear to have some
negative consequences.
ix

Taken together, the findings from the alcohol drinking and behavioral studies in
mice support the hypothesis that IVM represents a novel therapy that can be
repurposed for treatment of AUDs.

1

CHAPTER 1
INTRODUCTION

1.1) Impact of Alcohol use disorders on society:

Alcohol use disorders (AUDs), which comprises of alcohol abuse and alcohol
dependence, are considered as one of the most prevalent substance
dependence disorders in today’s society (Grant et al., 2004; Johnson, 2010).
Alcoholism is a chronic relapsing disorder with serious consequences due to its
severe impact on one’s physical and mental well being (Johnson, 2010).
Alcoholism is considered a critical predisposing factor to mortality and morbidity
(Bouchery et al., 2011; Grant et al., 2004; Johnson, 2010). In 2011, the World
Health Organization published a report describing the global impact of
alcoholism on health indicating that 4% of deaths which occurred worldwide and
4.5% of the global burden of injuries (intentional and unintentional) are
attributable to alcohol use. Alcoholism succeeds childhood underweight and
unsafe sex as being a leading risk factor to disease and disability (Department of
Mental Health and Substance Abuse, 2011).
Excessive alcohol drinking can impair one’s potential to perform their tasks in
a work environment which could have a negative impact on productivity and
2

eventually result in loss of job (Bouchery et al., 2011). In addition, alcoholism
is a severe burden to government agencies at local, state and federal level
due to exorbitant economic costs associated with alcoholics (Bouchery et al.,
2011; Harwood, 2000). The economic costs; related to ambulatory care for
such individuals, productivity losses and property damage (due to vehicle
accidents, crimes) (Bouchery et al., 2011) were estimated at $148 billion in
1992 followed by a 25% increment in 1998 when the economic costs were
$184 billion (Harwood, 2000). This gradually increased to $223.5 billion in
2006 emphasizing the need for useful interventions at the behavioral and
pharmacological level for purpose of reducing the economic burden
associated with alcoholism.
Alcoholism is also one of the main contributing factors to severe disorders
including gastrointestinal diseases (pancreatitis, liver cirrhosis) (Yadav and
Whitcomb, 2010), neuropsychiatric diseases such as schizophrenia, antisocial
personality disorder, anxiety, depression, suicidal behavior (Pompili et al.,
2010; Regier et al., 1990), cardiovascular diseases (ischemic stroke, cardiac
dysrhythmia, hypertension) (Costanzo et al., 2010) , several types of cancer
including cancer of the larynx, breast, esophagus and liver (Baan et al., 2007).
Heavy alcohol drinking in pregnant women can have severe consequences on
the developing fetus which ultimately leads to irreparable defects in the
3

physical and mental development of the child (Chiriboga, 2003). Various
injuries such as road traffic accidents, drowning, poisoning, violence being
committed on others, suicidal attempts are all attributable to alcohol abuse
and dependence (Rehm et al., 2009). The high degree of association of
alcohol with several devastating disorders and injuries makes alcoholism one
of the main health risks for disease and injury in United States and abroad
(Department of Mental Health and Substance Abuse, 2011).
The two latest national surveys conducted by the National Institute of Alcohol
Abuse and Alcoholism (NIAAA) were the National Longitudinal Alcoholic
Epidemiologic Survey in 1991-1992 and National Epidemiologic Survey on
Alcohol and Related conditions in 2001-2002. These two surveys utilized the
definitions of alcohol abuse and alcohol dependence established by the
Diagnostic and Statistical Manual of Mental Disorders, (fourth addition of DSM-
IV) of the American Psychiatric Association, 1994. These two surveys concluded
that the prevalence of AUDs rose from 13.8 million Americans in 1991-1992 to
17.6 million Americans in 2001-2002. Between 1991-92 and 2001-02 a significant
increase in incidence of alcohol abuse was found in both males and females and
as far as ethnicity is concerned, the incidence of alcohol abuse has increased in
most ethnic groups (Grant et al., 2004). Another significant report of the
National Longitudinal Alcohol Epidemiological Survey, 1992 was that one out of
4

every 4 children in United States is living in a family that has 1 or more members
suffering from alcohol abuse or alcohol dependence (Grant, 2000).
The data from these two surveys conducted by NIAAA and the World Health
Organization signifies the fact that AUDs continue to be an unmet medical
challenge in United States and many other nations.
At the present time, treatment strategies for AUDs include psychosocial and
pharmacological interventions (Castro and Baltieri, 2004; Heilig and Egli, 2006;
Johnson, 2010). Psychosocial intervention primarily consists of alcoholic
undergoing two phases namely detoxification and rehabilitation. Detoxification
typically involves daily administration of a benzodiazepine until the signs and
symptoms associated with alcohol withdrawal are diminished. Alternative
methods involve administration of depressants (drugs that potentiate GABA
receptor) or anticonvulsants but benzodiazepines are more cost effective
(Hietala et al., 2006). Rehabilitative measures include interviewing with the
patients to educate them about hazards related to excessive drinking and the
deleterious effect of alcoholism on society, to suggest them with various
measures to reduce their alcohol intake, to motivate them in achieving
abstinence from excessive drinking (Johnson, 2010; Swift, 1999) Medications are
available to inhibit the craving towards alcohol and reducing the probability of
relapse during abstinence period (Heilig and Egli, 2006; Johnson, 2010; Johnson,
5

2008; Swift, 1999). For some alcohol abusing patients, psychosocial interventions
maybe sufficient for treatment of their addiction problem, but most patients
require pharmacological agents in adjunct to their behavioral therapy. However,
even with the current pharmacotherapies, the clinical outcome continues to be
quite poor with relapse rates as high as 70% (Johnson, 2010).
1.2) Current pharmacological agents used for treatment of AUDs:
At the present time, few pharmacological agents have been approved by the FDA
for treatment of AUDs. These include 3 FDA approved oral medications namely
disulfiram (1949), naltrexone (1995) and acamprosate (2004) (Castro and
Baltieri, 2004; Heilig and Egli, 2006; Johnson, 2010; Johnson, 2008). In addition,
an injectable formulation of naltrexone is now available. There are several drugs
under clinical development for treatment of this psychiatric disorder including
ondansteron, baclofen, topiramate, gabapentin but these drugs are yet to
receive approval from the FDA (Addolorato et al., 2002; Addolorato et al., 2007;
Heilig and Egli, 2006; Johnson, 2005; Johnson et al., 2007; Vengeliene et al.,
2008). Certain novel targets have been implicated in pathogenesis of alcohol
dependence. These include the cannabinoid receptors, the metobrotropic
glutaminergic system and stress related neuropeptides such as corticotrophin
releasing factor, neuropeptide Y. Validation of these targets could pave the way
for development of novel entities for treatment of alcoholism (Valdez et al.,
6

2003; Wang et al., 2003). The paucity of efficacious pharmacological agents
symbolizes the importance for development of novel agents for treatment of this
psychiatric disorder.
1.2.1) FDA approved drugs for treatment of AUDs:
a) Disulfiram:
Disulfiram was the first pharmacological agent to be approved by the FDA to
treat AUDs (Castro and Baltieri, 2004; Heilig and Egli, 2006; Swift, 1999).
Disulfiram acts upon the metabolic pathway of alcohol. Alcohol is initially
converted into acetaldehyde by alcohol dehydrogenase after which
acetaldehyde is converted into acetic acid by aldehyde dehydrogenase.
Disulfiram blocks the action of aldehyde dehydrogenase which leads to
accumulation of acetaldehyde. This results in severe consequences including
tachycardia, dyspnoea, nausea, diaphoresis and flushing. Such adverse events
compel the individual to abstain from alcohol ingestion (Swift, 1999). Disulfiram
is contraindicated in pregnant women due to high risk of fetal abnormalities,
patients with liver disease due to risk of hepatitis cirrhosis and with ischemic
heart disease (Castro and Baltieri, 2004; Swift, 1999). On the basis of results
obtained from placebo-controlled studies, disulfiram was reported to reduce the
consumption of alcohol and number of heavy drinking days but it neither
protracts the abstinence period nor reduces relapse rates (Fuller et al., 1986).
7

There has been a scarcity of clinical studies documenting disulfiram’s efficacy on
account of methodological issues such as the impracticality of double blinded
studies, a problem with compliance and patient treatment matching (Hughes
and Cook, 1997). A meta analysis including several clinical studies suggests that
disulfiram may be effective under special circumstances such as when given
under supervision or when given in combination with psychosocial measures or
when administered to individuals who are highly motivated to curb their
addiction (Berglund et al., 2003). On the basis of a careful review of these clinical
studies, it is now widely accepted that disulfiram does not target the
mechanisms or signaling pathways responsible for alcohol dependence but
rather has its pharmacological action through the blockade of metabolism of
alcohol.
b) Naltrexone:
Naltrexone was approved by the FDA in 1995 for treatment of alcohol
dependence (Castro and Baltieri, 2004; Heilig and Egli, 2006; Swift, 1999). The
mechanism of action of naltrexone involves antagonism at the opioid receptors
(mostly µ and δ receptors). This leads to inhibition of the release of endogenous
opioids such as encephalins and endorphins which diminishes the release of
dopamine in the nucleus accumbens (NAc) (Heilig and Egli, 2006; Herz, 1997).
Hence naltrexone most likely works by inhibiting the positive reinforcing effects
of alcohol. Initial clinical evidence reported that, naltrexone when given at
8

sufficient oral dose of 50mg on daily basis for 12 weeks was effective in reducing
the average alcohol intake per week and decreasing the rate of relapse as
compared to the placebo group (Volpicelli et al., 1992). However, despite the
strong evidence supporting its clinical efficacy, the main obstacle for its clinical
acceptance is the issue of non compliance since patients in the long run would
discontinue due to certain side effects such as nausea and hepatotoxicity
(Oncken et al., 2001). Apparently, naltrexone shows considerable efficacy in
several primary and secondary outcomes like relapse rates, number of drinking
days, time to relapse in short term studies (Volpicelli et al., 1992), but not in
case of long term studies of more than 12 months (Krystal et al., 2001). To help
improve compliance, an extended release form of naltrexone as a once in a
month injectable (380 mg) was developed. The injectable form is reported to
improve the rates of abstinence and reducing the number of drinking days as
compared to placebo group in clinical trials (O'Malley et al., 2007) but it does not
appear to be more efficacious than oral formulation (Johnson, 2008). Also it
appears that the injectable form does not greatly reduce side effects of the drug.
On a positive note, the depot formulation appears to be effective in subjects in
whom abstinence has already been established as per the clinical studies
(O'Malley et al., 2007). Due to naltrexone’s ability to antagonize opioid
receptors, it is contraindicated in patients who are heroine dependent. In
addition, naltrexone is contraindicated in patients who have hepatitis as studies
9

have reported that high doses (>300 mg) can be associated with hepatoxicity
(Swift, 1999).
c) Acamprosate:
Acamprosate is another FDA approved medication used for treatment of alcohol
dependence (Johnson, 2010; Mason and Heyser, 2010; Swift, 1999).
Acamprosate is structurally similar to GABA and taurine and it acts a partial co
agonist at the ionotropic glutamate gated channel, NMDA receptor (Littleton,
1995). Acute exposure to alcohol decreases glutaminergic activity through
inhibitory action on the glutaminergic receptors such as NMDA and AMPA
receptor (Carta et al., 2003; Lovinger et al., 1989). However, during chronic
alcohol exposure, upregulation of NMDA receptors occurs, which is one of the
neuroadaptive changes made by the body in order to restore regulation within
these systems (Vengeliene et al., 2008). Discontinuation of alcohol leads to
elevated levels of glutamate which leads to hyperexcitability of these NMDA
receptors. This leads to severe withdrawal symptoms such as disturbances in
sleep, delirium and seizures (Tsai et al., 1998) due to dysregulation within this
neurochemical system. Acamprosate normalizes the dysregulated functioning of
the glutaminergic system (Mason and Heyser, 2010). As a result of which;
acamprosate diminishes withdrawal symptoms associated with abstinence which
ultimately leads to reduction in craving for alcohol. Interestingly, the approval of
acamprosate by the FDA in 2004 was largely based on European studies where it
10

successfully maintained longer abstinence for period of 6 months than the
placebo group (Mann et al., 2004). However, in U.S based trials, acamprosate
failed to demonstrate any therapeutic benefit except in a small subpopulation of
alcoholics who were highly motivated to control their addiction (Mason et al.,
2006). Clinical data suggests that it may only be useful in prolonging the
abstinence period and thereby inhibit the craving for alcohol (Mason and Heyser,
2010). Another finding that further erodes support of acamprosate as a
beneficial anti-alcohol agent stems from clinical studies that report a failure of
any significant reduction on any drinking outcome at 12 months of treatment
(Bouza et al., 2004). Therefore, despite acamprosate’s approval for treatment of
alcohol dependence, there is minimal clinical evidence in United States
supporting the use of acamprosate for the treatment of AUDs (Johnson, 2008).
1.2.2) Drugs under current clinical trial investigations:
Ondansteron which is a 5-HT3 antagonist has shown to be effective in patients
with early onset of alcoholism based on clinical studies. Activation of 5-HT3
receptors on GABAergic interneurons by ethanol is critical for some of its
inhibitory actions through release of GABA (Lovinger, 1999). Ondansteron
inhibits 5-HT3 activation resulting in reduced levels of dopamine in the cortico-
mesolimbic system. Thus, ondansteron inhibits the positive reinforcing effects of
alcohol by acting on the dopamine mesolimbic system (Heilig and Egli, 2006). In
11

various clinical studies ranging from 6 weeks through 12 weeks, ondansteron has
shown benefit in significantly improving drinking outcomes in patients with early
onset of alcoholism (Kranzler et al., 2003; Sellers et al., 1994) suggesting its
efficacy in only certain types of alcoholics.
Additional compounds that are being evaluated in the clinic include baclofen,
topiramate and gabapentin. All these compounds have shown some
effectiveness in treatment of AUDs through various preclinical and clinical
studies (Addolorato et al., 2002; Addolorato et al., 2007; Heilig and Egli, 2006;
Johnson et al., 2007) but none have received approval from the FDA.
Baclofen which is an agonist at GABA
B
receptor has shown to be effective in
reducing alcohol consumption, inhibiting the intention to seek alcohol and
decrease relapse like binge drinking behavior in alcohol preferring rats (Colombo
et al., 2000; Colombo et al., 2003a; Colombo et al., 2003b). Activation of GABA
B

receptors is responsible for suppression of alcohol drinking through inhibition of
the cortico-mesolimbic dopamine system (Addolorato et al., 2009; Vengeliene et
al., 2008). Hence; baclofen could possibly dampen the rewarding properties
associated with heavy drinking through this mechanistic pathway. Through
activation of GABA
B
receptors in the limbic system, baclofen is effective in
reducing anxiety and possibly other withdrawal symptoms during abstinence
(Addolorato et al., 2009). This suggests baclofen’s ability in prolonging
12

abstinence in alcohol dependent subjects by suppression of symptoms
associated with alcohol withdrawal. Clinical investigations have shown baclofen
to be beneficial in reducing craving, improving drinking outcomes and anxiety
scores as compared to placebo group (Addolorato et al., 2000; Addolorato et al.,
2002).
Topiramate’s potential for treatment of alcohol dependence is explained on
basis of its ability to counteract the hyperexcitation of the glutaminergic system,
to cause potentiation of GABAergic signaling and to block the activity of L type
calcium channel (Heilig and Egli, 2006). Hyperexcitable state of GABAergic
neurons in the ventral tegmental area (VTA) due to reduced inhibition of
GABAergic neuronal activity in NAc and increased glutaminergic activity from
other limbic structures occurs during chronic alcohol exposure. This ultimately
leads to decreased levels of dopamine in the NAc on account of inhibited
dopaminergic neuronal activity in VTA (Johnson, 2004). Sudden alcohol
discontinuation would lead to precipitation of withdrawal symptoms which
ultimately leads to compulsive drug seeking behavior (Gonzales et al., 2004;
Weiss and Porrino, 2002). Topiramate may normalize the GABAergic activity in
VTA through increased GABAergic tone from NAc, inhibition of hyperexcitable
glutaminergic system and blockade of L-type calcium channel and hereby blocks
the motivation to resume alcohol drinking (Johnson, 2005, 2008). Clinical studies
have shown topiramate at doses upto 300 mg/kg to be effective in decreasing
13

craving, improving all drinking outcomes as compared to placebo in 12 weeks of
treatment (Johnson et al., 2003; Ma et al., 2006).
Small scale clinical studies have reported the effectiveness of gabapentin for
diminishing of symptoms associated with alcohol withdrawal (Bozikas et al.,
2002; Brower et al., 2008). A randomized double blind pilot study evaluating the
utility of gabapentin as an anti-alcohol agent has reported delay in relapse to
heavy drinking upto 6 weeks after treatment (Brower et al., 2008). Other small
scale pilot studies have reported the potential of gabapentin in reduction of
alcohol withdrawal symptoms including hand tremors, sweating, anxiety and
high pulse rate (Bozikas et al., 2002). Gabapentin’s ability in reducing other
drinking outcomes is yet to be investigated (Johnson, 2010; Johnson, 2005).
Similar to topiramate and baclofen, gabapentin facilitates the GABAergic activity
from NAc to VTA through increased availability of GABA. Possible mechanisms of
action of gabapentin include inhibition of GABA transaminase which is
responsible for breakdown of GABA or blockade of calcium channels or
enhanced mediation of GABA inhibitory current (Johnson, 2005).
Taken together, these aforementioned compounds that are under clinical
investigations have shown promise with limited positive results in improving
various drinking outcomes which generally includes percentage of heavy drinking
days during treatment, time to relapse, days of abstinence, total consumption
14

during treatment, number of drinks per drinking day, blood levels of markers of
heavy consumption like γ-glutamyl transferase and aspartate aminotransferase.
Notably, the ongoing studies have shown that these compounds have potential
in being therapeutic agents for treatment of AUDs but larger scale randomized
double blind placebo controlled studies for a longer duration will be required to
evaluate the true clinical efficacy of these candidates. In summary, paucity of
efficacious agents available in market alongside with alcoholism associated social
and economic burden addresses the urgency for discovery and development of
novel therapeutic agents for this psychiatric illness.
1.3) Role of P2X4R in being a critical target for AUDs:
P2X receptors (P2XRs) are non-selective fast acting cation permeable trimeric
channels that are gated by ATP (Abbracchio et al., 2009; Buell et al., 1996).
Electrophysiological findings have reported the possible role of P2XRs in
modulating ethanol induced cellular and behavioral effects since ethanol at
intoxicating and anesthetic concentrations was found to inhibit ATP activated
currents in P2XRs (Asatryan et al., 2008; Davies et al., 2005; Davies et al., 2002;
Xiong et al., 2000). Among all the P2X subtypes, the P2X4 receptor (P2X4R)
received most emphasis due to building evidence regarding the critical
association between P2X4Rs and ethanol’s induced cellular and behavioral
effects which is supported by significant findings from electrophysiological
15

studies undertaken in our laboratory and genomic studies at the behavioral level
(Asatryan et al., 2011; Kimpel et al., 2007; Ostrovskaya et al., 2011; Tabakoff et
al., 2009; Xiong et al., 2000). P2X4Rs are widely distributed in the central nervous
system including brain regions that are widely believed to be targeted by ethanol
including the hippocampus, cerebellar Purkinje cells, most of the nuclei of the
brain stem including the facial nuclei, the cortex and the amygdala (Amadio et
al., 2007; Soto et al., 1996). P2X4Rs are also expressed in the striatum which is
one of the vital regions for development of alcohol dependence (Amadio et al.,
2007).
The P2X4Rs are the most ethanol sensitive P2XRs identified to date with in vitro
studies findings that ethanol, at low concentrations of 5mM can inhibit ATP
activated currents (Davies et al., 2005; Xiong et al., 2005; Xiong et al., 2000;
Xiong et al., 1999). Various electrophysiological investigations have shown that
ethanol when co applied with low concentrations of ATP, inhibited ATP induced
currents of P2X4 in a concentration dependent manner when tested in a
mammalian cell system or Xenopus oocyte system (Davies et al., 2005;
Ostrovskaya et al., 2011; Xiong et al., 2000; Xiong et al., 1999).
Follow on studies in our laboratory has also led to the identification of two key
residues namely D331 and M336 at the ectodomain-TM 2 interface which are
responsible for controlling the deactivation rate of the receptor. Point mutations
16

at these residues with amino acids differing in physical-chemical properties were
found to reduce or eliminate the inhibitory effects of ethanol on P2X4R function
(Ostrovskaya et al., 2011; Popova et al., 2010). Aligning the sequence of rat
P2X4R with zebra sh P 2X4R through the . crystal structure led the authors to
suggest that these residues might play an important role in regulating ion flow at
entrance of channel (Ostrovskaya et al., 2011).
At the behavioral level, recent findings have linked the association of p2x4r gene
with alcohol intake and preference. First, a lower functional expression of p2x4r
gene was observed in the alcohol preferring rats than in their alcohol-non
preferring counterparts (Kimpel et al., 2007) . Second, a study to elucidate the
candidate genes responsible for variations in alcohol consumption identified the
p2x4r gene as one of the possible candidate genes linked to high alcohol intake
and preference (Tabakoff et al., 2009).
Taken together, the above findings from electrophysiological and behavioral
studies suggest an important role of P2X4R in mediating some of ethanol’s
cellular and behavioral effects. The observation of ethanol’s inhibitory effect on
ATP gated currents in P2X4Rs preludes the hypothesis that activation of P2X4Rs
in presence of ethanol could possibly interfere with or negate the development
of ethanol seeking behavior and could provide a platform for development of
novel pharmacological agents which may have potential in treatment of AUDs.
17

Unfortunately, at the present time there are no P2X4R modulators to test this
hypothesis.
1.4) Ivermectin: The marvelous discovery from Japan
1.4.1) The discovery of ivermectin and its multiple uses:
In the mid-1970, the Kitasato Research Institute in Tokyo, Japan discovered a
semi-synthetic dihydro derivative of avermectins (macrocyclic lactones) namely
ivermectin (IVM) from the soil actinomycete, Streptomyces avermitilis. Due to
the significant contribution of the parasitological team from Merck Sharp and
Dohme, New Jersey, USA who were in the quest for novel antimicrobial agents;
IVM was introduced in the markets in the 980’s for v eterinary and human
purpose (Crump and Omura, 2011; Geary, 2005; Richard-Lenoble et al., 2003).
Notably,-IVM, due to its potent microfilaricidal agent, became a popular drug in
field of veterinary and human medicine for treatment of parasites namely
Wucheria bancrofti, Brugia malayi, Onchocerca volvulus, Loa loa (Ottesen and
Campbell, 1994). Among all the deadly parasitic infections; IVM’s greatest
contribution to the world was the successful eradication of Onchocerciasis and
Lymphatic filariasis in tropical areas where these parasites were endemics
(Crump and Omura, 2011).

18

.4.2) Mechanism of action of IVM’s antiparasitic activity:
An early hypothesis of IVM’s mode of action as an anti -parasitic agent was that
the compound caused hyperpolarization of GABA
A
receptors in the somatic
neuromuscular system of the nematodes leading to muscle paralysis of parasite
(Wolstenholme and Rogers, 2005). Subsequent experimental findings indicated
that the required concentrations of IVM to inhibit GABA
A
receptors were too
high for clinical purpose suggesting a different target for IVM’s anti parasitic
activity (Martin and Pennington, 1989) . Electrophysiological investigations by
(Arena et al., 1992) in which mRNA of Caenorhabditis elegans was injected into
Xenopus oocytes led to identification of an IVM sensitive inhibitory glutamate
chloride channel. The glutamate chloride channel is expressed in the pharyngeal
muscles (responsible for pumping food into gut) (Martin, 1996) and body wall
muscles (responsible for locomotion) (Dent et al., 1997). IVM, acting as an
irreversible agonist of the glutamate chloride channel causes greater influx of
chloride ions leading to hyperpolarization of the glutamate channel. This leads to
paralysis of the pharyngeal muscles and induces starvation of the parasites
resulting in death (Wolstenholme and Rogers, 2005).
Mammalians do not have the glutamate chloride channel that is found in
parasites. However, they do have other proteins that share a similar sequence
homology and are sensitive to IVM. These include GABA
A
, glycine, and nicotinic
19

acetylcholine receptors, all of which are members of the cys-loop superfamily of
ligand gated ion channels (LGICs) (Wolstenholme and Rogers, 2005).
1.4.3) IVM, P2X4Rs and ethanol:
In mammalians, in addition to IVM’s effects on the cys -loop superfamily
members, electrophysiological studies have investigated the ability of IVM to act
as a positive allosteric modulator at P2X4R (Jelinkova et al., 2006; Priel and
Silberberg, 2004) by two distinct mechanisms. This includes an effect of IVM that
results in an increase in maximal current as a result of activation at saturating
concentrations of ATP and an effect that results in decrease in the deactivation
rate of P2X4Rs, thus increasing the affinity for ATP (Priel and Silberberg, 2004).
Interestingly, other P2X members are not sensitive to IVM (Silberberg et al.,
2007), thus allowing the use of IVM as a selective allosteric modulator of P2X4R
function and a tool that can be used to identify participation of P2X4Rs
compared to other ATP gated P2X subtypes.
Within P2X4Rs, key residues located at the TM2-ectodomain interface have been
identified to play an important role in interaction of IVM (Jelinkova et al., 2008;
Jelinkova et al., 2006; Silberberg et al., 2007). Prior studies from our laboratory
have also shown the importance of certain amino acids at the TM2-ectodomain
interface in modulating the sensitivity of P2X4R to ethanol (Ostrovskaya et al.,
2011; Popova et al., 2010). Such findings led to the proposal of IVM having some
20

modulatory effect on ethanol’s action on the P 2X4Rs by binding to the same
putative pocket to which ethanol was reported to act on or bind in.
To begin testing this hypothesis, electrophysiological studies from our laboratory
using the Xenopus oocyte system, showed that IVM at low concentrations
(0.5µM) could antagonize the inhibitory effect of ethanol at concentrations
ranging from 25-100mM (Asatryan et al., 2010). Higher concentrations of IVM
(3µM) antagonized even higher concentrations of ethanol (100-200mM).
Notably; M336 located at the ectodomain-TM2 segment was shown to play an
important role in regulating the activity of IVM as substitutions of M336 with
charged amino acids (Glu or Arg) at this position abolished the sensitivity of
channel towards IVM. M336 is also a key residue for ethanol to exert its
inhibitory effects on P2X4R (Popova et al., 2010). The observation that mutation
of M336 altered sensitivity of the channel to IVM and ethanol could possibly
explains its antagonizing action on ethanol’s inhibitory effec t on the channel
(Asatryan et al., 2010).
In summary, the critical finding from our laboratory that ethanol induces an
inhibitory effect on the P2X4R activity and that IVM has the ability to antagonize
this particular effect of ethanol provides basis for the hypothesis that IVM may
represent a novel therapy that can be repurposed for treatment of AUDs.

21

The ability of IVM to antagonize the inhibitory effect of ethanol on P2X4R
provided the basis for the two main aims of my thesis.
Aim 1: Test the hypothesis by investigating the effects of IVM on alcohol intake
and preference in C57BL/6 mice using a 24 hour access two bottle choice
paradigm. Results from this study would suggest the potential of IVM as a novel
therapeutic agent for AUDs. In addition to alcohol studies, a saccharin study was
also undertaken to determine if IVM’s effect was specific to ethanol or a second
substance known to have positive reinforcing effects.
Aim 2: Test the hypothesis that IVM does not cause behavioral effects that
would negatively impact its therapeutic application to preventing or treating
AUDs. This aim would be accomplished by testing a set of behavioral paradigms
capturing complementary aspects of perceptual, emotional and cognitive
regulation in mice. These studies would provide us with insights of the
behavioral effects that IVM may cause in alcohol abusing patients.

22

CHAPTER 2
AIM 1: TEST THE HYPOTHESIS BY INVESTIGATING THE EFFECTS OF IVM ON
ALCOHOL INTAKE AND PREFERENCE IN C57BL/6 MICE USING A 24 HOUR TWO
BOTTLE CHOICE PARADIGM.
Note that unless otherwise indicated, experiments presented in this chapter
represent work from our recently published manuscript entitled “Ivermectin
reduces alcohol intake and preference in mice” (Yardley et al., 2012). I was a
contributing author on this published work and significantly participated in all
the experiments that are presented in this chapter.
2.1) Background:
The observed antagonism of ethanol induced inhibition of P2X4R by IVM
provided basis for my first aim that IVM may reduce alcohol consumption in an
animal model. To test this aim, the 24 hour two bottle choice paradigm was used
in which the mice have unrestricted access to either one of the drinking tubes
containing ethanol/water solution or a second tube containing only water. This is
a commonly used model to measure the preference of mice towards different
concentrations of ethanol (Tabakoff and Hoffman, 2000) and helps in
determining the concentration at which the mice exhibit their highest
preference. To evaluate IVM’s impact on voluntary consumption and preference
23

for alcohol, this particular paradigm was chosen. One noteworthy feature of this
model is that it mimics social drinking since mice have the tendency to titrate
their intake by reducing their preference for highly concentrated alcohol
solutions (Blednov et al., 2010). In agreement with previous reports (Belknap et
al., 1993; Blednov et al., 2010), we noticed that C57BL/6 mice show a high
preference for 10% v/v solution in a 24 hour two bottle choice paradigm and
that they tend to reduce their preference for concentrations higher than 10%.
Hence, for these studies, a 0% v/v solution of ethanol was used to test IVM’s
effect on alcohol intake and preference in C57BL/6 mice. We also undertook a
saccharin study using the same paradigm on basis of prior observations that
ethanol preferring animals also display high preference for sweet solutions
(Sinclair et al., 1992; Woods et al., 2003) and that ethanol and sucrose may share
a similar pathway in inducing their rewarding effects (Lemon et al., 2004). The
saccharin study would provide us with a preliminary insight of IVM’s effect on
sensory components of alcohol perception and if IVM can block the rewarding
pathway that is linked to alcohol dependence.
In addition to the 24 hour two bottle choice method, other animal models used
in alcohol research include limited access drinking model and the operant self
administration model. The limited access drinking paradigm mimics ‘binge
drinking’ behavior seen in alcoholics since the mice are provided with high
concentrations of ethanol (usually 20% or greater) for a short period of time
24

(either 2 or 4 hours) and they attain blood ethanol concentrations that indicates
excessive alcohol drinking (Rhodes et al., 2007). In the operant self
administration model, the mice have to perform a certain task (pressing of lever)
to obtain alcohol. This model is highly critical in elucidating the motivation and
craving behavior of mice to obtain alcohol (Simms et al., 2010).
My efforts over the course of my thesis investigations, focused on the use of 24
hour two bottle choice paradigm and thus, the model is explained in detail. We
predict that using this paradigm would provide us with important insights into
the effects of IVM on alcohol consumption in mice.
2.2) Materials:
2.2.1) Drugs:
Ivermectin (IVM) (Noromectin, Norbrook Laboratories Inc, Lenexa, KS) was
supplied as a 1% sterile solution containing glycerol formula and propylene glycol
as vehicle. IVM was adjusted to various doses from 0.65mg/kg to 10mg/kg by
using 0.9% sodium chloride solution as a diluent. The final intraperitoneal
injection volume was 0.0 ml/g of the mouse’s body weight. Alcohol, 200 proof
USP (100% ethanol) (Gold Shield Chemical Company, Hayward, CA) was used for
all alcohol drinking studies. 10% ethanol solution (10E) was prepared by diluting
200 proof alcohol with tap water. Saccharin (Sigma –Aldrich, St Louis, MO) was
dissolved in tap water to a final concentration of 0.033% w/v.
25

2.2.2) Animals:
Male and female C57BL/6 ranging from 10-12 weeks in age were used for the
alcohol drinking studies. The C57BL/6 mice show high preference for 10E over
water and hence are routinely used in many alcohol intake behavioral studies.
The animals were obtained from our breeding colony at Hoffman Research
Building located at University of Southern California, Los Angeles, CA. This
breeding program is established for purpose of generating P2X4R knockout
animals using C57 as background strain. The breeding colony room follows a
12:12 light/dark cycle with lights going off at 6:00pm. The room is maintained at
a temperature of 22
o
C. The male and female breeders are purchased from
Jackson Laboratory, Bar Habour, ME generally at the age of 6 weeks. The mice
used for the experiments were allowed to acclimatize to the experimental
facilities at Mudd Memorial research building at University of Southern
California, Los Angeles, CA for a period of one week. They were housed in
polysulfone/polycarbonate cages in groups of 4-5 with ad libitum access to food
and water. After a period of 1 week; the mice were single housed in
polysulfone/polycarbonate cages having a cage top with two slots for the
graduated tubes with metal sippers. Food was distributed near both the tubes to
avoid the conflict of food associated tube preference. The experimental room is
maintained at a temperature of 22
o
C and humidity of 30-60%. The room follows
a 12h light/dark cycle with lights going off at 12:00p.m. All the procedures were
26

carried out as per the protocols established by Institutional Animal Care and Use
Committee of University of Southern California and the National Institute of
Health. The facilities at USC are accredited by Accreditation and Assessment of
Laboratory Animal Care.
2.3) Methods:
2.3.1) 24 hour access two bottle choice paradigm for alcohol drinking:
For a period of 1 week, mice were provided with water in both tubes prior to
their provision of alcohol. Food intake and body weight was recorded on a daily
basis. Once, the acclimatization period ended, one of tubes was replaced with
10% v/v solution of alcohol (10E). The positions of the bottles were alternated
every other day to avoid the issue of side preferences. The volume of 10E (or
water) was determined by measuring the lower meniscus to nearest 0.1 ml.
Drinking tubes containing 10E (or water) were replaced twice a week. Cage
changes took place twice a week except on days when the mice received IVM so
as to avoid the conflict of variation in alcohol intake due to association of
anxiety-like behavior with novel environment. Once consistent drinking levels
were established (variability of mean intake < 10% for 3 days), the mice received
saline injection via intraperitoneal route in volume of 0.01ml/g. The mice were
given i.p injections of saline until stable drinking levels have been achieved
(usually within 4-5 days from first day of injection). When the drinking levels
27

were consistent over a period of 3 days, the mice received a dose of IVM using
within subject design. In all cases of saline or IVM injections, the injections
occurred prior to the 24 hour access so that the change in alcohol intake after
IVM or saline administration could be determined. Following IVM administration,
the mice received saline injections until their drinking levels reached baseline.
This protocol of IVM administration was followed throughout the experimental
studies.
2.3.2) Saccharin studies:
The experimental protocol for determining the effect of IVM on saccharin intake
was similar to the alcohol studies with the following exception. The mice
received two tubes of metal sippers one of which contained 0.033% w/v solution
of saccharin in tap water and other contained tap water.
2.3.3) Statistical analysis:
Ethanol and saccharin consumption in terms of g/kg and mg/kg on basis of their
body weights and ethanol preference (volume of ethanol /total fluid volume)
were calculated for each IVM dose tested. Dependent variables included
ethanol, water and total fluid intake consumed (ml), ethanol dose (g/kg), ethanol
preference (%), saccharin preference (%). Total fluid refers to combined volume
of 10E and water intake. Analysis of variance (one way and two way ANOVA)
with post hoc tests (either Bonferroni or Tukey multiple comparison test as
28

indicated) were used to evaluate the IVM dose effects on the dependent
variables. IVM dose and time (pre treatment, treatment and post treatment)
were analyzed by using a repeated measure by ANOVA. Statistical significance
was set at P<0.05. Graph pad software (San Diego, CA) was used for the analysis
of IVM effects on the dependent variables.
2.4) Results:
2.4.1) IVM significantly reduced 10E intake and preference in male mice:
2.4.1.1) IVM administration (10mg/kg) significantly reduced 10E intake and
preference in male mice:
As presented in figure 1A, mean 10E intake for the 3 day period prior to
administration of 10mg/kg of IVM averaged 8.55g/kg/day (variability between
the days <10%). Administration of IVM (10mg/kg) significantly reduced (i.e. 45%)
10E intake as compared to pre IVM level [F (2,30) = 16.85), p<0.001] (Figure 1A).
10E intake reached the baseline levels on the 4th day post IVM administration.
In addition to 10E intake, IVM also significantly decreased 10E preference [F
(2,30) = 10.11, p<0.001] (Figure 1B). Moreover, IVM significantly increased water
intake by 47% [F (2,30) = 3.56, p<0.05] (Figure 2C).
We tested a separate group of animals which was used as a control group where
the mice were injected with propylene glycol (vehicle for IVM formulation).
29

Propylene glycol did not significantly affect 10E intake or preference or water
intake indicating that reduction in 10E intake and preference could not be
attributed to solvent effects (data not shown).
2.4.1.2) IVM significantly decreased 10E intake and preference in a dose
dependent fashion in male mice:
In this study, IVM was administered in doses ranging from 0.65mg/kg through
10mg/kg. As presented in figure 2A, IVM significantly decreased 10E intake in a
dose dependent fashion [F (4,100) = 5.51, p<0.001] (Figure 2A). Two way ANOVA
analysis revealed a time dependent effect of IVM 10E intake [F (2,100) = 14.37,
p<0.001]. There was a significant interaction between time and IVM dose [F
(8,100) = 5.53, p<0.001]. Bonferroni post hoc test confirms significant decrease
in 10E intake with 2.5 mg/kg (~39%, t=5.025, p<0.001) and with 10mg/kg (~45%,
t=6.161, p<0.001). The 10E intake returned to baseline by the 3rd day post IVM
administration in case of 2.5mg/kg and 4th day in case of 10 mg/kg.
In addition to 10E intake, IVM decreased 10E preference in dose dependent
fashion [F (4,100) = 3.69, p<0.05] as indicated by two way ANOVA (Figure 2B).
IVM was found to significantly decrease 10E preference in time dependent
manner [F (2,100) = 8.92, p<0.001]. There was a significant interaction between
IVM dose and time [F (8,100) = 7.40, p<0.001] with Bonferroni post hoc test
confirming reduction in 10E preference with 2.5 mg/kg (t=4.527, p<0.001) and 10
30

mg/kg (t=5.715, p<0.001). IVM did not have any significant effect on water
intake except in case of 10mg/kg of IVM where there was a significant increase
of 47% (Figure 2C). There was a significant interaction between time and IVM
dose on water intake [F (8,100) = 7.09, p<0.001] with post hoc tests confirming
that water intake increased with 10 mg/kg of IVM (t=3.430, p<0.01). A significant
reduction was seen in total fluid intake across time [F (2,100) = 13.66, p<0.001]
(Figure 2D). As indicated by two way ANOVA, IVM dose had a significant effect
on total fluid intake [F (4,100) = 3.50, p<0.05]. There was also a significant
interaction between time and IVM dose [F (8,100) = 5.40, p<0.001] with post hoc
tests showing that the fluid intake decreased with 2.5 mg/kg (t=3.526, p<0.01)
and 10 mg/kg (t=3.689, p<0.01).
2.4.1.3) Time course effect of IVM on 10E intake in male mice:
We next investigated the time course effect of IVM on 10E intake on an hourly
basis for 10 hours post IVM administration. The purpose of this study was to gain
insights regarding IVM’s time period required to exert its anti -alcohol effect. Two
way ANOVA analysis showed dose dependent effect of IVM on 10E intake [F
(1,130) = 45.44] (Figure 3). Bonferroni post hoc comparisons confirmed a
significant decrease in IVM group as compared to control group (saline group) at
7
th
hour (t=3.085, p<0.05), 8
th
hour (t=3.919, p<0.01), 9
th
hour (t=4.148, p<0.001)
and 10
th
hour (t=4.731, p<0.001).
31

Figure 1: Effect of IVM (10mg/kg) on 10E intake and preference in male C57BL/6 mice.
IVM (10mg/kg) decreased A) 10%v/v ethanol (10E) and B) preference ratio for 10E in male
C57BL/6 mice using a 24 hour access drinking paradigm. IVM was administered after attaining
stable levels for 3 consecutive days. Bars represent the levels of 10E intake from the day prior to
IVM administration (white: pre-IVM, black: IVM, grey: post IVM). Values represent the mean
SEM for 11 mice. * P<0.05, ***P<0.001 versus pre IVM, Tukey multiple comparison test.
A.
B.
Pre IVM IVM Post IVM
0
25
50
75
***
*
Preference Ratio for 10E
Pre IVM IVM Post IVM
0.0
2.5
5.0
7.5
10.0
***
*
10E Intake (g/kg/24-h)
32

Figure 2: Dose response study of IVM in male C57BL/6 mice
IVM dose response study in male C57BL/6 mice using a 24 hour access drinking paradigm. IVM
was administered after achieving stable drinking levels for 3 consecutive days. Bars represent the
10E intake from the day prior to IVM administration (white: pre IVM, black: IVM). A) IVM at
doses of 2.5 and 10mg/kg significantly reduced 10E intake and B) preference ratio for 10E. The
10mg/kg dose but not 2.5mg/kg significantly altered water intake (C) and both the doses reduced
fluid intake significantly (D). The values represent the mean SEM for 11 mice. * P<0.05,
**P<0.01, ***P<0.001 versus pre IVM, Bonferroni post hoc test.
A. B.
C. D.
0 .0 0 0 .6 5 1 .2 5 2 .5 0 1 0 .0 0
0 .0
2 .5
5 .0
7 .5
1 0 .0
Pre I V M
I V M
*** ***
IV M Dose (m g/kg)
10E In take (g /kg /24-h )
0 .0 0 0 .6 5 1 .2 5 2 .5 0 1 0 .0 0
0
10
20
30
40
50
60
70
80
*** ***
IV M Dose (m g/kg)
P r efer en ce Ratio fo r 10E
0.00 0.65 1.25 2.50 10.00
0
1
2
3
**
IV M Dose (m g/kg)
Water In take (m L )
0 .0 0 0 .6 5 1 .2 5 2 .5 0 1 0 .0 0
0
1
2
3
4
5
**
**
IV M Dose (m g/kg)
T o tal F lu id In take (m L )
33

Figure 3: Time course effect of IVM on 10E intake in male C57BL/6 mice
Time course effect of IVM on 10E intake in male C57BL/6 mice using a 24 hour access drinking
paradigm. The 10E intake was monitored every hour following administration of IVM (5mg/kg)
upto 10 hours. The maximum effect of IVM was seen at 9
th
and 10
th
hour following
administration. All values represent the mean ±SEM for 15 mice.*P<0.05, **P<0.01, ***P<0.001
versus saline group, Bonferroni post hoc test.

0 1 2 3 4 5 6 7 8 9 10 11
0
2
4
6
8
10
12
14
Saline
IVM
*
**
***
***
Hours after injection
10E Intake(g/kg)
34

2.4.2) IVM significantly reduced 10E intake and preference in a dose dependent
manner in female mice:
A similar study was performed in female mice to investigate any sex differences
in IVM effect on 10E intake and preference. In agreement with previous reports
(Middaugh et al., 1999; Yoneyama et al., 2008), female C57BL/6J mice consumed
significantly higher levels of alcohol as compared to males in the 24 hour two
bottle choice paradigm. Differences in ethanol metabolism (Crabb, 1997; Elmer
et al., 1987) and levels of neuroactive steroids (Lancaster, 1995) that may
modulate alcohol drinking through interaction with GABA
A
have been proposed
for sex differences in alcohol drinking. This study was undertaken to determine if
the anti-alcohol action of IVM observed in males would also be present in
females.
As presented in figure 4A, IVM significantly decreased 10E intake in dose
dependent fashion [F (6,252) = 20.16, p<0.001]. Two way ANOVA analysis reveals
time dependent effect of IVM on 10E intake [F (6,252) = 79.53, p<0.001]. The
interaction between time and IVM dose was significant [F (12,252) = 7.50,
p<0.001] with post hoc tests showing that 10E intake decreased significantly
with 2.5 mg/kg (~30%, t=4.735, p<0.001), 5mg/kg (~66%, t=8.536, p<0.001), 7.5
mg/kg (~56%, t=8.318, p<0.001) and 10 mg/kg (~50%, t=8.214, p<0.001).
35

In addition, IVM significantly decreased 10E preference in dose dependent
fashion [F (6,252) = 10.45, p<0.001] as revealed by two way ANOVA analysis
(Figure 4B). When analysis was conducted across time, there was a significant
decrease in 10E preference [F (2,252) = 77.80, p<0.001]. There was significant
interaction between time and dose [F (12,252) = 10.11, p<0.001] with
Bonferroni post hoc test exhibiting decrease in 10E preference ratio for
2.5mg/kg (t=3.323, p<0.01), 5 mg/kg (t=9.650, p<0.001), 7.5 mg/kg (t=8.154,
p<0.001) and 10 mg/kg (t=9.470, p<0.001). IVM significantly increased water
intake in dose dependent manner [F (6,252) = 3.27, p<0.01] (Figure 4C). There
was significant interaction between time and IVM dose [F (12,252) = 6.55,
p<0.001] with Bonferroni post hoc test confirming increase in water intake with
5 mg/kg (t=3.752, p<0.01), 7.5 mg/kg (t=3.764, p<0.01) and 10mg/kg (t=7.196,
p<0.001). Significant dose dependent [F (6,252) = 8.13, p<0.001] and time
dependent effect [F (2,252) = 35.34, p<0.001] of IVM on fluid intake was
indicated by two way ANOVA (Figure 4D). There was significant interaction
between time and dose [F (12,252) = 4.78, p<0.001] with Bonferroni post hoc
tests showing decrease in fluid intake with 2.5mg/kg (t=3.988, p<0.001),
5mg/kg (t=5.694, p<0.001), 7.5 mg/kg (t=5.342, p<0.001).

36

2.4.3) Long term dosing of IVM significantly reduced 10E intake and preference
in female mice:
Current pharmacotherapies which have been approved by FDA for AUDs such as
disulfiram, naltrexone and acamposate are administered on daily basis for weeks
ranging from 3 -12 weeks (Castro and Baltieri, 2004; Johnson, 2010; Schuckit,
2009). The present study was undertaken to determine if long term
administration of IVM would precipitate any unwanted toxic effects due to
accumulation of drug in various tissues and to begin to elucidate the overall
safety of the drug as a therapeutic agent for treatment of alcoholism.
Upon reaching stable baseline 10E levels, we administered 1.25 mg/kg dose of
IVM (daily) for a period of 7 days in female mice. IVM significantly decreased 10E
intake over the 7 day period [F (2, 27) = 7.974, p<0.01] (Figure 5A). The
significant reduction in 10E intake ranged from 11% to 27%. The longer, daily
administration of IVM did not appear to induce any undue physical changes in
mice as measured by no significant changes in food intake or body weight or
general behavior of the mice. Decrease in 10E intake was not accompanied with
a decrease in 10E preference. The average water intake during the 7 day period
was not significantly different from pre IVM levels [F (2,27) = 3.155, p>0.05]. A
significant reduction was seen in total fluid intake during the 7 day period [F (2,
27) = 37.56, p<0.001].
37

We extended our investigations by next testing a higher dose of IVM (i.e.
5mg/kg) for a period of 7 days to determine if a higher dose would result in any
toxicity or tolerance issues. IVM significantly decreased 10E intake during the
dosing period [F (2, 27) = 16.87, p<0.001] (Figure 5B). The average 10E intake for
3 days post IVM chronic administration was significantly lower compared to pre
IVM levels [F (2,27) = 16.87, p<0.001) (Figure 5B). 10E intake reached baseline
levels on 6
th
day post IVM administration. Long term administration of IVM did
not induce any overt changes in food intake or body weight of the mice.
In contrast to our initial 1.25mg/kg/day studies, 5mg/kg/day caused a significant
reduction in 10E preference [F (2, 27) =21.97, p<0.01]. The reduction ranged
from 3% to 20% during the 7 day period. The average water consumption during
the IVM period was not significantly different from the pre IVM levels [F (2,27) =
8.369, p>0.05]. A significant reduction was observed in the total fluid intake [F
(2,27) = 42.66, p<0.001]. The reduction in fluid intake ranged from 19% to 34%.
2.4.4) IVM decreased saccharin consumption and preference in dose dependent
manner in female mice:
In this study, the effect of two doses of IVM (i.e. 2.5 and 5mg/kg) on 0.033% v/v
saccharin intake was investigated. We found a significant reduction in saccharin
intake compared to water intake at both these doses. IVM (2.5 mg/kg)
significantly reduced saccharin intake by 31% [F (2,54) = 20.82, p<0.001] (Figure
38

6A). On the other hand, the same dose did not decrease saccharin preference [F
(2,54) = 1.710, p>0.05]. A significant reduction was seen in total fluid intake by
29% [F (2,54) = 24.82, p<0.001] but no significant alteration was observed in
water intake. We next investigated the effects of 5 mg/kg on saccharin intake
and preference. This dose significantly decreased saccharin intake by 46% [F
(2,54) = 52.83, p<0.001] (Figure 6B). This decrease in saccharin intake was
accompanied by a significant reduction of saccharin preference by 7% [F (2,54) =
4.761, p<0.05]. Similar to our findings with 2.5 mg/kg study, there was significant
alteration in total fluid intake [F (2,54) = 52.70, p<0.001] and no significant
change was seen in water intake .

39

Figure 4: Dose response study of IVM in female C57BL/6 mice
IVM dose response study in female C57BL/6 mice using a 24 hour access drinking paradigm. IVM
was administered after attaining stable drinking levels for 3 consecutive days. Bars represent the
10E intake from the day prior to IVM administration (white: pre IVM, black: IVM). IVM at doses of
2.5, 5, 7.5 and 10mg/kg significantly reduced 10E intake (A) and preference ratio for 10E (B).
Doses of 5, 7.5 and 10mg/kg significantly increased water intake (C) while dose range of 2.5-
10mg/kg reduced total fluid intake significantly (D). Values represent mean ± SEM for 19 mice.
**P<0.01, ***P<0.001 versus pre IVM levels, Bonferroni post hoc test.

A.
C.
B.
D.
0 .0 0 0 .6 5 1 .2 5 2 .5 0 5 .0 0 7 .5 0 1 0 .0 0
0
1
2
3
4
5
6
***
*** ***
I VM Dose (m g/kg)
Total Fluid Intake (mL)
0 .0 0 0 .6 5 1 .2 5 2 .5 0 5 .0 0 7 .5 0 1 0 .0 0
0
10
20
30
40
50
60
70
80
**
***
***
***
I VM Dose (m g/kg)
Preference Ratio for 10E
0 .0 0 0 .6 5 1 .2 5 2 .5 0 5 .0 0 7 .5 0 1 0 .0 0
0 .0
0 .5
1 .0
1 .5
2 .0
2 .5
***
**
**
I VM Dose (m g/kg)
Water Intake (mL)
0 .6 5 1 .2 5 2 .5 0 5 .0 0 7 .5 0 1 0 .0 0
0
5
10
15
Pre IVM
IVM
*** *** *** ***
I VM Dose (m g/kg)
10E Intake (g/kg/24-h)
40

Figure 5: Long term dosing of IVM in female C57BL/6 mice
Long term dosing of IVM in female C57BL/6 mice using a 24 hour access drinking paradigm. IVM
was administered for 7 days and drinking levels were monitored throughout the period. Bars
represent the 10E intake from the day prior to IVM administration (white: pre IVM, black: mean
10E intake during IVM period, grey: post IVM. IVM at doses of 1.25mg/kg (A) and 5mg/kg (B)
significantly reduced 10E intake during the dosing period. Values represent mean ±SEM for 10
mice. **P<0.01, ***P<0.001 versus pre IVM levels, Tukey multiple comparison test.

A.
Pre IVM IVM Post IVM
0
5
10
15
**
10E Intake (g/kg/24-h)
B.
Pre IVM IVM Post IVM
0
5
10
15
***
***
10E Intake(g/kg/24-h)
41

Figure 6: Effect of IVM on saccharin (0.033%w/v) in female C57BL/6 mice
Effect of IVM on saccharin intake (0.033%w/v) in female C57BL/6 mice using a 24 hour access
drinking paradigm. IVM was administered after attaining stable drinking levels for 3 days. Bars
represent the saccharin intake from the day prior to IVM injection (white: pre IVM, black: IVM,
grey: post IVM). IVM at doses of 2.5mg/kg (A) and 5mg/kg (B) reduced saccharin intake
significantly. Values represent mean ±SEM for 19 mice. *P<0.05, ***P<0.001 versus pre IVM,
Tukey multiple comparison test.

A.
B.
Pre IVM IVM Post IVM
0
20
40
60
80
100
120
***
Saccharin Intake (mg/kg/24-h)
Pre IVM IVM Post IVM
0
20
40
60
80
100
120
***
*
Saccharin Intake (mg/kg/24-h)
42

2.5) Discussion:
The present findings suggest the potential of IVM as a novel therapeutic agent
for treatment of AUDs. Using a 24 hour access two bottle choice paradigm to
investigate the anti-alcohol effects of IVM, we found that acute and longer term
dosing of IVM significantly decreased alcohol intake and preference in male and
female mice. The significant decrease in alcohol intake and preference was not
accompanied with any significant changes in the food intake or body weight or
any untoward alteration in natural behavior of the animal. Interestingly, the
decrease in alcohol intake in female mice at 5mg/kg was greater than that
observed at 7.5 and 10mg/kg suggesting a possible saturation effect of IVM at
the receptor through which its mediates its anti-alcohol action. A significant
increase in water intake was observed at doses higher than 5mg/kg in both male
and female mice. This phenomenon indicates that IVM reduces alcohol
consumption through its pharmacological action at receptors responsible for
modulation of alcohol drinking (possibly the P2X4R as hypothesized by our
electrophysiological findings) and not through alteration of the physiological
mechanism of drinking.
A time course study of IVM effect on 10E intake in male mice showed that IVM
substantially decreased 10E intake after the 7
th
hour with maximum impact at
the 9
th
and 10
th
hour following administration. This study gives us preliminary
evidence regarding the time required for IVM to induce its anti-alcohol effects.
43

Taking into consideration that alcoholism is a chronic relapsing disorder, a longer
term IVM administration study was undertaken to gain insight into potential
toxicity or tolerance issues associated with longer term treatment of IVM. IVM
significantly reduced alcohol intake as compared to baseline levels during the
dosing period without causing any untoward changes in behavior, appearance,
food intake or body weight. As briefly stated in the result section of this study,
the 10E intake remained significantly low as compared to pre IVM levels for 6
days after the dosing period. Interestingly, during this period there was no
untoward change in the water intake. However, this does not disregard the
possibility that dehydration may occur if IVM was administered on a chronic
basis. Longer term studies of greater than 7 days would be required to
determine IVM’s effect on water intake and any other severe adverse reaction
associated with long term administration of drug.
The similar efficacy of IVM in both the male and female rules out the possibility
of sex related hormonal differences that could interfere with IVM potential to
reduce alcohol intake and preference in female mice. Although the estrous cycle
was not kept under vigilance during this study, our inference is supported by
earlier studies which report that estrous related changes in self ethanol
administration were not observed in rats that cycle freely (Ford et al., 2008;
Roberts et al., 1998) and other studies which revealed no changes in ethanol
intake for several weeks during the baseline consumption and in the control
44

group that was subjected to vehicle treatment in C57BL/6 female mice (Ford et
al., 2008). Our current findings showing that the 10E intake did not significantly
alter for several days without IVM administration and the consistency with
previous work suggest that there were no estrous cycle related factors that could
have influenced the ethanol intake following IVM administration.
A saccharin study was undertaken to observe the impact of IVM on saccharin
intake in the C57BL/6 mice. This study was carried out on basis of the
observation that alcohol and sweeteners share a common pathway for
perception of taste which could be the underlying mechanism for rewarding
effects associated with these substances (Lemon et al., 2004). This notion has
been supported by electrophysiological investigations in which alcohol
activates sweetener sensitive neurons of the nucleus solitary tract (Di Lorenzo
et al., 1986) and behavioral studies in which selective lines of high alcohol
preferring rats show high preference for sweet tastants like saccharin over
water (Sinclair et al., 1992; Woods et al., 2003). Gene mapping studies utilizing
linkage analysis have identified the gene loci that are responsible for linking
alcohol preference with saccharin preference (Bachmanov et al., 1997; Blizard
et al., 1999). Moreover, alcohol preferring mice which are null mutants for
certain genes responsible for perception of sweet taste show reduced
preference for alcohol in addition to sweet tastants (Blednov et al., 2008). Such
findings suggest the genetic association between alcohol and sweetener intake.
45

Reduction in saccharin intake at doses of 2.5 and 5 mg/kg indicates that IVM
may act on the neural circuits that are common to the gustatory processing of
sweetener and alcohol. The sweet tastant responsive pathways that play a
critical role in activation of the rewarding pathways in the CNS and induction of
compulsive drug seeking behavior have shown to be activated by ethanol as
well through an orosensory component (Lemon et al., 2004). Significant
reduction in saccharin intake by IVM in these studies suggests that IVM
probably acts on the sensory components that are critical for perception of
alcohol flavor. This could possibly lead to inhibition of positive reinforcing
effects of alcohol and diminish the craving or compulsive drug seeking behavior
which is associated with substance dependence.
The dopamine mesolimbic system is a vital site of action for many drugs of abuse
as this system makes significant contributions to development of drug seeking
behavior and association of a drug of abuse with certain environmental cues
through activation of rewarding pathways that results in release of dopamine in
extracellular space in various regions of this system including NAc, amygdala,
prefrontal cortex and hippocampus (Gonzales et al., 2004; Hyman and Malenka,
2001; Imperato and Di Chiara, 1986). The NAc is the most studied region while
elucidating the mechanism of action of drugs since this region plays a significant
role of establishment of motivational seeking behavior associated with several
drugs of abuse (Gonzales et al., 2004; Weiss and Porrino, 2002). P2XRs are
46

expressed in the neurons and glia of the mesolimbic dopamine system (Heine et
al., 2007). P2XRs interact presynaptically and postsynaptically with GABA
A
,
glycine and glutamate receptors all of which play a critical role in mediating
ethanol’s cellular and behav ioral effects (Hugel and Schlichter, 2002; Rubio and
Soto, 2001). P2XRs have shown to be affected by ethanol which modulates GABA
transmission of dopaminergic neurons in VTA (Xiao et al., 2008). Hence, these
receptors could be vital components of signaling pathways that underlie the
manifestation of alcohol’s rewarding effects that ultimately leads to compulsive
drug seeking.
IVM has been reported to act on multiple ligand gated ion channels in central
nervous system including GABA
A,
glycine and nicotinic acetylcholine receptors by
electrophysiological findings (Dawson et al., 2000; Krause et al., 1998; Shan et
al., 2001). The cys-loop superfamily members are associated with ethanol
induced cellular and behavioral effects (Davies, 2003; Vengeliene et al., 2008).
The diverse impact of IVM on multiple neurochemical targets suggests that
IVM’s anti -alcohol effect may not be solely dependent on one receptor. On basis
of findings from these studies, it is noteworthy to point out that the modulatory
effect of IVM may be dependent on the concentration since IVM was found to
potentiate the activity of these ionotropic receptors at significantly different
concentrations (Dawson et al., 2000; Krause et al., 1998; Shan et al., 2001). As far
as in vivo studies are concerned, IVM was shown to induce CNS depression in
47

rats and to potentiate thiopentone induced sleeping at doses larger than
10mg/kg, suggesting the role of GABA
A
receptors in mediating IVM induced
central depressant effects (Trailovic and Trailovic, 2010). IVM was unable to
produce these similar effects at doses below 10mg/kg (Trailovic and Trailovic,
2010).
On basis of our electrophysiological findings from (Asatryan et al., 2010), IVM
may be mediating its anti-alcohol effects in our animal model in part by
antagonizing ethanol’s inhibitory effect on P2X4Rs. Genomic studies have
reported the critical association between p2x4r gene and alcohol intake in
alcohol preferring rats (Kimpel et al., 2007; Tabakoff et al., 2009). These findings
suggest the possibility of P2X4Rs being a critical target in mediation of IVM’s
anti-alcohol effects. However, further studies will be needed to further elucidate
IVM’s mechanism of action in reducing alcohol consumption due to ethanol’s
multiple targets and the diverse pharmacological action of IVM.
The above findings suggest the potential of IVM as a novel therapeutic agent in
treatment of AUDs. IVM has shown an excellent therapeutic profile as an
antiparasitic agent (Geary, 2005) with mild and transient side effects including
nausea, headaches and dizziness at dose range of 300-1090 µg/kg which is much
higher than the actual dose administered to humans (200µg/kg) (Guzzo et al.,
2002). The limited pharmacotherapy available for treatment of AUDs along with
48

the high rate of relapse among alcoholic patients after several years of
psychosocial and pharmacological intervention mandates the need for
development of new medications or repurposing of old drugs for treatment of
alcoholism.

49

CHAPTER 3

AIM 2: TEST THE HYPOTHESIS THAT IVM DOES NOT CAUSE BEHAVIORAL EFFECTS
THAT WOULD NEGATIVELY IMPACT ITS APPLICATION TO PREVENTING OR
TREATING AUDS BY CONDUCTING A SET OF DIFFERENT BEHAVIORAL PARADIGMS
CAPTURING COMPLEMENTARY ASPECTS OF PERCEPTUAL, EMOTIONAL AND
COGNITIVE REGULATION IN MICE.
Note that unless otherwise indicated, the experiments presented in this chapter
represent work from our “ conditionally accepted” manuscript entitled
“Pharmacological insights into the role of P2X4 receptors in behavioral
regulation: lessons from ivermectin” (Bortolato et al., 20 2). I was a contributing
author on this work and significantly participated in all the experiments that are
presented in this chapter.
3.1) Background
IVM has been reported to modulate several members of cys-loop superfamily of
ligand gated ion channels (Dawson et al., 2000; Krause et al., 1998; Shan et al.,
2001). Such members including GABA
A
, glycine, and nicotinic acetylcholine
receptors have been implicated in pathogenesis of various neurological disorders
(Albuquerque et al., 2009; Paredes and Agmo, 1992; Picciotto et al., 2008). The
50

modulatory effect of IVM on these receptors proposes the critical association of
IVM with these CNS related disorders. GABA
A
receptors have been linked to
manifestations of mood disorders including anxiety, depression, aggressive
behavior and other neurological disorders like epilepsy (Paredes and Agmo,
1992). Nicotinic acetylcholine receptors have been implicated in
neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease and
depression as well (Albuquerque et al., 2009; Picciotto et al., 2008). Examining
the outcomes produced by IVM in various behavioral paradigms would elucidate
the role of IVM in modulating aspects of behavior related to anxiety, depression
and cognition. Another interesting target of IVM which is not a cys-loop
superfamily member is P2X4R. The role of P2X4R in pathogenesis of neurological
disorders remains to be studied. Critical studies investigating P2X4R facilitated
fast synaptic transmission in CNS have shown the role of P2X4R in facilitation of
calcium influx and hormonal secretion in thyrotropin releasing hormone
responsive cells such as lactotrophs underlying their importance in hormonal
release and secretion (Zemkova et al., 2010). P2X4R may have a role in
neuropathic pain since antidepressants like paroxetine and fluvoxamine
provided relief from neuropathic pain through blockade of P2X4R (Nagata et al.,
2009). P2X4R has been demonstrated in modulation of long term potentiation
through excitatory synapses in the hippocampus which provides the platform for
learning and memory (Pankratov et al., 2002; Sim et al., 2006; Wang et al., 2004).
51

However, the precise role in this function remains controversial due to
contrasting results obtained from literature and lack of specific agonists and
antagonists. Investigating the effect of IVM on various behavioral aspects related
to emotional and cognitive regulation in our animal model may provide us with
important insights of P2X4Rs’ role in beha vioral regulation. The four behavioral
tests that will be discussed in detail and in various sections of this chapter
include open field, marble burying, forced swim and prepulse inhibition of
acoustic startle reflex.
Open field test is one of the most commonly used tests for measuring anxiety
behavior and spontaneous locomotor activity. This test involves forceful
subjection of the animal to a novel environment from which escape is
improbable (Lister, 1990; Prut and Belzung, 2003). In this test, anxiety-like
behavior is precipitated in the animal by the fact that the mouse is exposed to
surroundings to which it has no prior experience (Prut and Belzung, 2003).
Increased thigmotactic behavior (movement near the walls) or increase in
latency to move into central portion of arena is sign of anxiety like behavior
(Lister, 1990; Prut and Belzung, 2003). Notably, one should take into
consideration the predictive validity of this model since studies have shown that
open field can only be used as a model of generalized anxiety state and not
anxiety related disorders such as post traumatic disorder, social phobia or
obsessive compulsive disorder (Prut and Belzung, 2003).
52

The marble burying test exploits the defensive behavior as an index of anxiety
(Njung'e and Handley, 1991). Mice have the natural tendency to bury objects
which are perceived as a noxious stimuli and precipitation of the burying
behavior reflects the anxiety state of the mice (Broekkamp et al., 1986; Poling et
al., 1981). In the marble burying test, the marbles are perceived as objects of
aversive stimuli on basis of their novelty (Njung'e and Handley, 1991). This test
has depicted the ability of anxiolytics to reduce the number of marbles being
buried suggesting the predictive validity of this test for generalized anxiety state
or anxiety related disorders (Treit et al., 1981). However, the predictive validity
of this test for anxiety may be limited since serotonin reuptake inhibitors which
are used in the treatment of obsessive compulsive disorder also reduce the
number of marbles being buried as digging represents compulsive behavior as
well (Njung'e and Handley, 1991). Notably, the predictive validity of this test for
anxiety is greatly augmented by including the parameter of spontaneous
locomotor activity (Nicolas et al., 2006).
Prepulse inhibition of acoustic startle reflex is a measure of preattentional
function of information processing in which the actual stimulus that generates
a startle is preceded by a weak, non startling stimulus (Graham, 1975). This test
is considered a validated paradigm for identification of deficits in sensorimotor
gating which is commonly associated with individuals suffering from perceptual
disorders (Braff et al., 2001). Prepulse inhibition test is used to detect deficits in
53

the sensorimotor gating. Sensorimotor gating is an involuntary preattention
function of enabling an individual to discriminate the trivial stimulus from the
significant ones to function efficiently in a stimulus associated environment
through mediation of attention dependent cognitive processes (Braff and Light,
2004). Sensorimotor gating is an important component of preattentional
‘automatic’ sensory processing system so as to allow an individual to respond
appropriately to a sensory stimulus (Braff and Light, 2004). Deficits in the process
of information filtering are generally observed in neuropsychiatric disorders
including schizophrenia, Huntington’s disease and bipolar disorder. Such deficits
can lead to increased disorientation in a stimulus laden environment which could
possibly restrict one’s potential of performing tasks efficien tly on daily basis
(Braff et al., 2001).
Forced swim test is one of the most simple and quick paradigms for screening of
anti-depressants (Petit-Demouliere et al., 2005). In this test, a depressive
behavior is induced in the mice by subjecting them to forceful swimming in an
inescapable environment. A state of behavioral despair is attained when the
mouse ceases to struggle and remains afloat with just its head remaining above
the water surface (Porsolt et al., 1977). Antidepressants tend to reduce the
duration of immobility and increase the latency to immobility (Porsolt et al.,
1979; Porsolt et al., 1977). The face validity is somewhat controversial since
acute administration of anti depressants is required as per protocol of this test
54

but these drugs exhibit their true clinical potency when being administered on
daily basis (Dulawa et al., 2004; Petit-Demouliere et al., 2005). The construct
validity has also been criticized since state of immobility could be interpretated
as means of conserving energy for purpose of surviving in an inescapable
environment (Holmes, 2003) or lack of motivation to maintain a constant effort
of survival rather than behavioral despair (West, 1990). Subjection to a stressful
environment could lead to depression which not necessarily represents the true
etiology of depressive symptoms that is seen in humans (Willner and Mitchell,
2002). Several sensitivity and variability factors need to be taken into
consideration for precise detection of anti-depressant activity including depth
and temperature of water, cylinder diameter, time interval between drug
administration and performance of test, housing of animals, food restriction and
strain of animal being used (Petit-Demouliere et al., 2005). Despite the
controversial face and/or construct validity of forced swim test, it should not be
discouraged from being used since it allows better understanding of mechanism
of actions underlying depression through use of genetically altered mice and has
shown a high degree of sensitivity to various classes of antidepressants.
In addition to these tests, several other behavioral tests were performed such as
elevated plus maze test, tail suspension test, conditioned place preference test,
hot plate and novel object exploration test, results of which will be explained in
the discussion section to give a broad picture of the investigations.
55

3.2) Materials:
3.2.1) Drugs:
IVM (Norbrook Laboratories, Lenexa KS) was supplied as a 1% sterile solution
containing propylene glycol and glycerol formula as the vehicle. IVM was diluted
with 0.9% sodium chloride solution to obtain the required concentrations for an
injection volume of 0.01ml/g. IVM

was injected 8 hours prior to the performance
of the tests since the maximum activity and T
max
was achieved 8 hours post
administration as determined by pharmacokinetic analysis (Yardley et al., 2012).
The animals were injected with either 2.5 or 10mg/kg IVM. A control group was
also used where the animals were injected with propylene glycol.
3.2.2) Animals:
C57BL/6 male mice aged 6-10 weeks were purchased from Jackson Laboratories,
Bar Habour, ME. The animals were housed in cages made of
polycarbonate/polysulfonate in groups of 4. The animals were allowed to
acclimatize to the animal housing facility for a period of 1 week. They had
unrestricted access to food and water. The holding room followed a 12h: 12h
light/dark cycle with lights going off at 6:00p.m. The holding room was
maintained at a temperature of 22
o
C. Due to aggressive behavior displayed by
the mice when group housed, the animals were later single housed. All
procedures were carried as per the protocols established by the Institutional
56

Animal Care and Use committee of University of Southern California and
National Institute of Health.
3.3) Methods:
3.3.1) Open field test:
The mice were placed in a Plexiglass square gray arena (40 x 40 cm) which
comprised of four black walls (40 cm high). The floor was divided into two
concentric zones of equal area: a central square zone and a peripheral zone
which was adjacent to the walls. The mice were placed in the center and their
behavior was investigated for 5 minutes. The various parameters related to the
locomotor activity were being assessed by Ethovision (Noldus instruments,
Wageningen, Netherlands). Various parameters include the time spent and
distance moved, percent locomotor activity and percent of time spent in the
central and peripheral zone, the number of transitions between the zones,
number of rearing episodes and meandering.
3.3.2) Marble Burying Test:
The mice were placed in novel cages (35 x 28 cm) where they had no prior
exposure. The cages were filled with sani chips upto a depth of 3 cm followed by
2cm depth of sawdust to give final height of 5cm. The intensity of light in each
cage was adjusted to 0.5 lux. The mice were habituated to the novel
environment for a period of 30mins where they were allowed to move freely.
57

After the initial 30 minutes of habituation, the mice were transferred to separate
cages covered with polycarbonate sheets. 20 marbles were placed in the cages in
4 rows of 5 maintaining equivalent distance between each marble and sufficient
enough for the mice to move around. The marbles were arranged in such a
manner that the perimeter of the cages was covered from 3 sides so that the
mice are not given the opportunity to avoid the marbles. Each marble was
considered buried if more than 2/3 of its surface area was covered in sawdust. A
camera was positioned over the cages so as to monitor the digging and
locomotor activity of the mice. The behavior the mice was monitored for 30
minutes. The duration of digging, the number of digging bouts as well as the
number of marbles buried were analyzed.
3.3.3) Forced Swim test:
The mice were made to habituate to Plexiglass cylinders (40cm x 19cm in
diameter) that were filled upto a depth of 15cm with water for a period of 1
minute. The temperature of water was maintained at 30
o
C± 2
0
C. On the testing
day; the mice were exposed to the same conditions for a period of 5 minutes.
The behavior of the mice was carefully monitored by video camera attached over
the cylinders. The duration and frequency of immobility was analyzed. The light
was adjusted at 300lux.

58

3.3.4) Prepulse inhibition of startle acoustic reflex:
The apparatus used for identification of startle reflex (San Diego instruments,
San Diego, CA) consisted of one cage in sound attenuated chambers provided
with ventilation fan. The cage consisted of Plexiglass cylinder of 3cm in
diameter which was mounted on a piezoelectric accelerometric platform
connected to an analogue digital converter. Background noise and acoustic
bursts were discriminated by two separate speakers, each positioned in such a
manner that they produce a variation of sound within 1dB across the startle
cage. Both speakers and startle cages were connected to a main PC which
analyzed all the chamber variables with a particular software. At the beginning
of each testing session, the acoustic stimuli were calibrated by specific devices
supplied by San Diego Instruments, San Diego, CA. One day prior to the testing
session; the mice were exposed to a 5 min acclimatization period which
consisted of a 70dB white noise background that persisted throughout the
session. Each session consisted of three consecutive sequences of trials. During
the first and third session, the mice were presented with five pulse alone trials
of 115dB. The second and third session consisted of a pseudorandom sequence
of 50 trials, including 12 alone pulse trials, 30 trials preceded by 73dB, 76 dB
and 82dB (10 for each level of pre pulse loudness) and eight no stimulus trials
where only background noise was delivered. The duration of pulses and pre
pulses were 80ms and 40 ms respectively. Prepulse-pulse delays were
59

amounted to 100ms. Inter-trial intervals were randomly selected between 10s
and 15s. Percent PPI was calculated by using the formula 100- [(mean startle
amplitude for prepulse-pulse trials/mean startle amplitudes for pulse alone
trials) x 100].
3.3.5) Statistical Analyses:
Normality and homoscedasticity of data distribution was analyzed by
Kolmogorov-Smirnov and Bartlett’s test. Various b ehavioral parameters of the
marble burying and forced swim test was carefully scored using Behavior Tracker
Software,Caligari,Italy followed by one way –ANOVA using repeated measures or
independent factors when required followed by Tukey test for post hoc analysis.
Parameters for open field test were assessed by Ethovision software (Noldus
Instruments, Wageningen, Netherlands). Percent PPI was calculated by using the
above described formula. Significant threshold was set at P=0.05.
3.4) Results:
3.4.1) IVM elicited anxiogenic like effect in open field test for anxiety:
When determining the effects of IVM on the regulation of locomotor and
exploratory activity, IVM did not induce any significant changes in the locomotor
activity but one way ANOVA analysis revealed that IVM reduced the time spent
in the center [F (2, 15) =2.12, p>0.05] in a dose dependent manner (Figure 7B).
60

Post hoc tests analysis confirm significant decrease in time spent in the center at
doses of 2.5mg/kg [(F (2, 15) =8.29, p<0.05] and at 10mg/kg [F (2, 15) =8.29,
p<0.01]. IVM induced dose dependent decrease in percent locomotor activity in
the center and post hoc tests confirm decrease in percent locomotor activity at
doses of 2.5mg/kg [F (2,15) = 6.49, p<0.05] and 10mg/kg [F (2,15) = 6.49, p<0.01]
(Figure 7C). No significant changes were brought about in meandering [F (2,15) =
2.50, p>0.05] (Figure 7D), number of rears [F (2,15) = 1,8, p>0.05] (Figure 7E) and
percentage of time spent moving between the two zones [F (2,15) = 2.04,
p>0.05] (Figure 7F). These data collectively suggest a dose dependent effect of
IVM on anxiogenic-like behaviors in the mice.

61

Figure 7: Effect of IVM on anxiety-like behavior in male C57BL/6 mice using the open field test
for anxiety.
IVM at dose of 2.5 and 10mg/kg decreases the time spent in the center (B) and percent
locomotor activity in the center (C) implicating an increase in thigmotactic behavior in the mice.
At these doses, IVM did not have any significant consequence on overall locomotor activity (A)
meandering (D), number of rears (E). All values are represented as ±SEM for 30 mice. * P<0.05,
**P< 0.01 versus vehicle treated group, Tukey multiple comparison test.

VEH IVM 2.5 IVM 10
0
10
20
30
VEH IVM 2.5IVM 10
0
50
100
150
VEH IVM 2.5IVM 10
20
40
60
VEH IVM 2.5 IVM 10
0
5
10
20
25
E F
**
*
VEH IVM 2.5IVM 10
0
20
60
100
% Distance in center
*
VEH IVM 2.5IVM 10
0
20
40
60
80
100
Distance travelled (m)
Time in center (s)
0
Meandering (degrees/cm)
15
Rears
A B C
D
40
80
*
% time moving
62

3.4.2) IVM elicited anxiolytic like response in marble burying test for anxiety:
IVM at the highest dose of 10mg/kg exhibited a significant decrease in the
number of marbles buried as illustrated by post hoc tests analysis [F (2, 26)
=16.04, p<0.001] (Figure 8B). The same dose also elicited a significant decrease
in digging duration as confirmed by post hoc tests analysis [F (2,25) = 4.34,
p<0.05] (Figure 8A). However, no significant alteration was found in the number
of digging bouts as confirmed by Tukey post test [F (2,25) = 2.35, p>0.05] (Figure
8C). These results suggest induction of anxiolytic response in mice by IVM.
3.4.3) IVM induced behavioral state of despair in forced swim model of
depression in a dose dependent manner:
IVM induced a dose dependent increase in duration of immobility. One way
ANOVA analysis indicates dose dependent increase in duration of immobility
and post hoc analyses confirms a significant increase in immobility at 10mg/kg [F
(2,24) = 3.54, p<0.05] (Figure 9). Although an increase in immobility duration was
seen at 2.5 mg/kg it was not considered significant as per post hoc analyses.
3.4.4) IVM dose dependently reduced prepulse inhibition of acoustic startle
reflex
IVM did not generate any significant alterations in the magnitude of the acoustic
startle reflex [F (2, 12) =2.82, p>0.05] (Figure 10A). One way ANOVA analysis
63

reveals that IVM elicits a reduction in the prepulse inhibition of acoustic startle
reflex in dose dependent fashion and post hoc test analysis confirms a significant
alteration at 10mg/kg [F(2,12)=9.65,p<0.01] (Figure 10B).This indicates IVM
induces deficits in sensorimotor gating which is an important preattentional
function of information processing system.

Figure 8: Effect of IVM on anxiety-like behavior in male C57BL/6 mice using the marble burying
test for anxiety.
IVM at dose of 10mg/kg significantly reduces the number of marbles buried (A) and the digging
duration (B) but not the frequency of digging bouts. All values represent ±SEM for 28 mice.
*P<0.05, ***P<0.001 versus vehicle treated group
###
P<0.001 versus 2.5 mg/kg IVM, Tukey
multiple comparison test.

VEH IVM 2.5 IVM 10
0
2
4
6
8
10
Buried marbles
VEH IVM 2.5 IVM 10
0
50
100
150
Digging duration (s)
VEH IVM 2.5 IVM 10
0
10
20
30
40
Digging bouts
A B C
***
*
###
64

Figure 9: Effect of IVM on stress induced depressive behavior in male C57BL/6 mice using
forced swim test for depression.

IVM at highest dose of 10mg/kg increased the duration of immobility implicating signs of
depressive behavior in the mice. All values represent ±SEM for 28 mice. *P<0.05 versus vehicle
treated group, Tukey multiple comparison test.

VEH IVM 2.5 IVM 10
0
100
200
300
400
500
Immobility duration (s)
*
65

Figure 10: Effect of IVM on prepulse inhibition of acoustic startle reflex in male C57BL/6 mice
using the prepulse inhibition test.
IVM did not have any impact on acoustic startle magnitude (A) but the highest dose of 10mg/kg
significantly reduced the prepulse inhibition suggesting aberrations in sensorimotor gating (B). All
values represent ±SEM for 28 mice. **P<0.01 versus vehicle treated group
#
P<0.05 versus 2.5
mg/kg IVM, Tukey multiple comparison test.

0
200
400
600
800
Acoustic startle
VEH IVM 2.5 IVM 10
A
B
VEH IVM 2.5 IVM 10
0
20
40
60
80
100
PP3
PP6
PP12
% PPI
**
#
66

3.5) Discussion:
The above mentioned behavioral paradigms were performed with the aim to
better understand the role of IVM on certain aspects of behavior related to
emotionality and cognition. These behavioral paradigms focused on different
aspects of behavior related to anxiety, depression and preattentional function of
information processing. The inferences drawn from these studies suggest the
diverse and unique role of IVM in modulating these aspects of behavior. IVM
elicited anxiogenic –like response in mice in the open field test as depicted by
increased thigmotactic behavior in the mice. In contrast, IVM induced anxiolytic
response in the marble burying test. IVM increased duration of immobility which
is a sign of depression in the forced swim test. IVM reduced prepulse inhibition
of acoustic startle which suggests that IVM may have a deleterious effect on the
function of sensorimotor gating.
IVM exhibited contrasting effects in the open field and marble burying tests,
both of which are models for studying anxiety-like behavior. The anxiogenic
response induced by IVM in the open field is in striking contradiction to the
results reported by (Spinosa et al., 2002) where IVM elicited anxiolytic response
in the same test. However, the variation in the dosing regimen of IVM, strain of
animal and the analysis of various behavioral parameters should be taken into
consideration for explanation of differential results. The open field and marble
67

burying differ in terms of the environmental cues that elicit an anxiogenic
stimulus and the parameters (Belzung and Le Pape, 1994) that are used to
measure the behavior of the animal in response to the stimulus (Lister, 1990;
Njung'e and Handley, 1991; Prut and Belzung, 2003). Avoidance of novel
environment in open field and burying of marbles in marble burying are highly
correlated parameters since they both represent the phenomenon of neophobia.
However, these two tests assess different forms of anxiety-related behavior
which could possibly explain the differential effects of IVM. In open field test, the
animal is exposed to novel environment which basically represents state anxiety
whereas in marble burying, the mouse is confronted with a novel situation which
represents trait anxiety (Belzung and Le Pape, 1994; Lister, 1990). On account
our limited understanding of the complex neurobiological mechanisms
underlying the different forms of anxiety; it is highly important to attempt to
develop a single model that reproduces the true features of anxiety (Belzung and
Le Pape, 1994; Lister, 1990). Anxiolytic compounds such as the classical
benzodiazepines which are used for generalized anxiety disorder, exhibit positive
effects in the open field test whereas anti-anxiety drugs such as the
triazolobenzodiazepines (alprazolam and adinozolam) which are used for panic
attacks failed to elicit an anxiolytic response in the open field test (Prut and
Belzung, 2003). This indicates the limitation of open field test for assessing
different forms of anxiety. Marble burying test responds positively not only to
68

anxiolytics such as the classical benzodiazepines but also to selective serotonin
reuptake inhibitors that are used for treatment of obsessive compulsive
disorder. Thus, the predictive validity of this paradigm applies to different forms
of anxiety (Njung'e and Handley, 1991). The findings from these two models of
anxiety suggest that IVM may have a critical role in treatment of pathological
anxiety such as obsessive compulsive disorder and probably not generalized
anxiety but due to limited predictive validity of both the tests, further studies
will be required before concrete conclusions can be drawn in this regard.
IVM induced depression-like reactions in the forced swim test as indicated by
increase in immobility duration. The main finding is the dose dependent increase
in immobility duration. Antidepressants like fluvoxamine and paroxetine were
shown to inhibit the P2X4R through measurement of calcium levels by real time
calcium imaging systems in human cell lines (Nagata et al., 2009). This finding in
conjunction with IVM’s outcome in for ced swim test suggests that IVM may
induce depression atleast in part through potentiation at P2X4R. Due to IVM’s
multiple pharmacological targets; it could precipitate this state of behavior
through modulation of other receptors. IVM exerts central depressant effects
similar to those of general anesthetics on the CNS at doses higher than 10mg/kg.
In these studies, the central depressant effects of IVM were attributable to its
interaction with the GABA
A
channel since IVM potentiated thiopentone induced
sleeping in rats and the benzodiazepine antagonist, flumazenil inhibited IVM’s
69

central depressant actions (Trailovic and Trailovic, 2010). However, it is
controversial to provide the same explanation for IVM’s induced depressive state
in the forced swim test since doses lower than 10mg/kg were tested.
Nevertheless, one cannot disregard the possible diverse and complex interaction
of IVM with several receptors for manifestation of depressive behavior in this
paradigm.
An interesting finding of the behavioral studies is reduction in prepulse tone by
IVM. Decrease in prepulse inhibition of the acoustic startle reflects IVM’s
potential in inducing deficits in the preattentional functioning of sensorimotor
gating. (Davis et al., 1999). This could eventually lead to obliteration of sensory
information processing system which may lead to improper allocation of
attention dependent cognitive resources that are needed for appropriate
response to a sensory stimulus. (Braff and Light, 2004). Thus, IVM may have the
propensity to induce cognitive deficits which is one of consequences in various
neuropsychiatric disorders including schizophrenia, Huntington’s disease , bipolar
disorder (Braff et al., 2001). Notably, these findings are in concordance with
(Davis et al., 1999) where IVM had the same effect on the acoustic startle
magnitude and prepulse inhibition of the startle reflex. Surprisingly; this study
also suggested IVM did not have any effect on learning and memory process as
indicated by the Morris maze test (Davis et al., 1999). Such observations suggest
the need for further studies to better understand the effects of IVM on the
70

process of cognition and learning. The mechanism of action by which IVM
decreases prepulse inhibition remains to be investigated. It was previously
described that the function of prepulse inhibition is controlled by elements of
cortico-striato-pallido-thalamic circuitry of CNS (Penney and Young, 1983). Thus;
IVM could possibly have some modulatory impact on one or some of the
components in this system.
There were other behavioral studies that were performed by my collaborators to
understand the multifaceted role of IVM on regulation of diverse behavioral
aspects. In addition to the two models of anxiety that I used, the elevated plus
maze test was also performed wherein at a dose of 10mg/kg, the mice spent
more amount of time in the open arms and in the central platform which is a
sign of enhanced exploratory activity and anxiolytic behavior. These findings are
consistent with studies conducted by (Spinosa et al., 2002). Thus, the similar
outcome produced by IVM in the marble burying and elevated plus maze test
suggests the notion that possible anxiolytic effects of IVM may be attributable to
its potentiating activity at GABA
A
receptors which is actively investigated
(Dawson et al., 2000). In the tail suspension paradigm for depression, IVM (2.5
mg/kg) significantly increased the duration of immobility but a higher dose of
10mg/kg failed to reproduce a similar effect. The results from forced swim and
tail suspension both of which have high predictive validity (Petit-Demouliere et
al., 2005; Steru et al., 1987) for depression indicate the possibility of IVM
71

inducing CNS depressant effects in reaction to stressful environmental cues.
Findings from conditioned place preference test suggest that IVM lacks positive
reinforcing effects. Findings from the hot plate test indicate that IVM does not
affect thermal nociception. In the novel object exploration test, IVM (10mg/kg)
decreased the exploration time of novel objects but no alteration in the novel
exploration index citing that IVM does not affect the mnemonic encoding of
novel objects.
Induced anxiolytic effects of IVM in the elevated plus maze and marble burying
test represents behavioral outcomes of GABA
A
activation (Davis et al., 1999)
suggesting that IVM may induce such responses through GABA
A
receptors. On
the other hand; anxiogenic effects in the open field test, attenuation of prepulse
inhibition, induced depressive like behavior in forced swim and tail suspension
test and lack of rewarding effect in conditioned place preference are
contradictory to what is observed in GABA
A
agonists (Borsini and Meli, 1988;
Steru et al., 1987) suggesting the notion that these behavioral effects may be
manifested partially through modulation of P2X4R since IVM is an allosteric
modulator at this receptor. The contrasting effects of IVM across these
behavioral paradigms could probably be explained by the negative interaction
between P2X4R and GABA
A
receptors in the ventromedial nucleus of
hypothalamus which could influence the synaptic strength at the hypothalamic
synapse. P2X4R may play a critical role in ATP facilitated GABAergic transmission
72

by a presynaptic mechanism in the neurons of spinal cord dorsal horn due to
expression of this receptor in the spinal cord dorsal horn in addition to other P2X
subtypes (Hugel and Schlichter, 2002; Jo et al., 2011; Jo and Schlichter, 1999).
The behavioral effects of IVM across these behavioral paradigms provides us
with some critical insights into understanding its diverse role in modulating
emotional and cognition related aspects of behavior. The observed anxiolytic
effects in marble burying and elevated plus maze tests suggest the potential role
of IVM in treatment of anxiety related disorders which are comorbid with
alcoholism. However, due to the anxiogenic effects observed in open field test,
further investigations will be required in this regard. The findings from the
paradigms for measuring depressive behavior and prepulse inhibition reveal the
possible adverse effects associated with IVM. Further studies will be
necessitated to understand the mechanisms by which IVM induces such adverse
events. The unique role of IVM on modulating various neurochemical targets
such as GABA (Dawson et al., 2000), nicotinic acetylcholine receptors (Krause et
al., 1998) and P2X4R (Asatryan et al., 2010) which are associated with regulation
of certain behavioral aspects explains the complex yet intriguing outcomes
produced by IVM in these paradigms. The data obtained from these studies
suggests IVM’s possible additional effects if administered to alcohol dependent
patients for treatment of their addiction problem.
73

CHAPTER 4
CONCLUSION
Alcoholism is considered to be a major risk factor to more than 60 diseases and
is reported to cause approximately 2.5 million deaths per year by the World
Health Organization in 2011. Despite the major economic and social burden
caused by AUDs, this problem has received very little attention in health policy
(Department of Mental Health and Substance Abuse, 2011). At present, there
are few compounds that have been approved by FDA for AUDs and there are
some compounds that are currently in the pipeline and awaiting approval (Heilig
and Egli, 2006; Johnson, 2010; Johnson, 2008; Swift, 1999). Several novel targets
linked to alcohol addiction and compulsive use have been identified and
validated by preclinical models (Heilig and Egli, 2006), which will lay the
foundation for discovery and development of novel therapeutic agents for AUDs
in the near future.
We demonstrated that IVM significantly reduced alcohol consumption and
preference in male and female mice using a 24 hour access two bottle choice
model (Chapter 2). IVM also significantly reduced saccharin intake which
suggests that IVM may reduce reward by acting on the neural circuits that are
linked to various drugs of abuse. However, further studies will be needed before
74

conclusions could be drawn with this regard. Further studies will be required to
determine if IVM has any effect on the compulsive drug seeking behavior
associated with alcohol withdrawal. Such parameters are highly critical since
relapse is a major concern that limits the pharmacotherapeutic efficacy of any
anti-alcohol agent.
The results from Chapter 3 suggest that IVM induced anxiogenic effects in the
open field test but in contrast, it induced anxiolytic effects in the marble burying
test. IVM also induced depression in the forced swim model for depression.
Notably, IVM reduced the prepulse inhibition of acoustic startle reflex which
suggests that IVM may induce deficits in sensorimotor gating. The observed
anxiolytic effect in marble burying paradigm suggests that IVM may have some
potential to treat anxiety related disorders which are comorbid with alcoholism
but due to contrasting results from the open field test, further examination in
this context will be required. The ability of IVM to treat comorbid disorders that
are seen in alcoholics will further boost the therapeutic value of this drug for
treatment of AUDs.
The lack of efficacious drugs for treatment of AUDs alongwith the formidable
burden of AUDs on human health and economic status in many countries
emphasizes the urgency for development and approval of new drugs for this
chronic relapsing disorder. IVM’s ability to reduce alcohol intake in combination
75

with its excellent safety profile in humans (Burkhart, 2000; Geary, 2005) suggests
that IVM may be repurposed as an effective new agent for treating and/or
preventing AUDs and could serve as the platform for development of novel
compounds for this purpose.

76

BIBLIOGRAPHY
Abbracchio, M. P., Burnstock, G., Verkhratsky, A., Zimmermann, H., 2009.
Purinergic signalling in the nervous system: an overview. Trends in neurosciences
32, 19-29.

Addolorato, G., Caputo, F., Capristo, E., Colombo, G., Gessa, G. L., Gasbarrini, G.,
2000. Ability of baclofen in reducing alcohol craving and intake: II--Preliminary
clinical evidence. Alcoholism, clinical and experimental research 24, 67-71.

Addolorato, G., Caputo, F., Capristo, E., Domenicali, M., Bernardi, M., Janiri, L.,
Agabio, R., Colombo, G., Gessa, G. L., Gasbarrini, G., 2002. Baclofen efficacy in
reducing alcohol craving and intake: a preliminary double-blind randomized
controlled study. Alcohol Alcohol 37, 504-508.

Addolorato, G., Leggio, L., Cardone, S., Ferrulli, A., Gasbarrini, G., 2009. Role of
the GABA(B) receptor system in alcoholism and stress: focus on clinical studies
and treatment perspectives. Alcohol 43, 559-563.

Addolorato, G., Leggio, L., Ferrulli, A., Cardone, S., Vonghia, L., Mirijello, A.,
Abenavoli, L., D'Angelo, C., Caputo, F., Zambon, A., Haber, P. S., Gasbarrini, G.,
2007. Effectiveness and safety of baclofen for maintenance of alcohol abstinence
in alcohol-dependent patients with liver cirrhosis: randomised, double-blind
controlled study. Lancet 370, 1915-1922.

Albuquerque, E. X., Pereira, E. F., Alkondon, M., Rogers, S. W., 2009. Mammalian
nicotinic acetylcholine receptors: from structure to function. Physiol Rev 89, 73-
120.

Amadio, S., Montilli, C., Picconi, B., Calabrei, P., C., V., 2007. Mapping P2X and
P2Y receptor proteins in striatum and substantia nigra: An immunohistological
study. Purinergic Signal 3 389-398.

Arena, J. P., Liu, K. K., Paress, P. S., Schaeffer, J. M., Cully, D. F., 1992. Expression
of a glutamate-activated chloride current in Xenopus oocytes injected with
Caenorhabditis elegans RNA: evidence for modulation by avermectin. Brain Res
Mol Brain Res 15, 339-348.

77

Asatryan, L., Nam, H. W., Lee, M. R., Thakkar, M. M., Dar, M. S., Davies, D. L.,
Choi, D. S., 2011. Implication of the purinergic system in alcohol use disorders.
Alcoholism, clinical and experimental research 35, 584-594.

Asatryan, L., Popova, M., Perkins, D. I., Trudell, J. R., Alkana, R. L., Davies, D. L.,
2010. Ivermectin antagonizes ethanol inhibition in P2X4 receptors. J. Pharmacol.
Exp. Ther. 334, 720-728.

Asatryan, L., Popova, M., Woodward, J. J., King, B. F., Alkana, R. L., Davies, D. L.,
2008. Roles of ectodomain and transmembrane regions in ethanol and agonist
action in purinergic P2X2 and P2X3 receptors. Neuropharmacology 55, 835-843.

Baan, R., Straif, K., Grosse, Y., Secretan, B., El Ghissassi, F., Bouvard, V., Altieri, A.,
Cogliano, V., Group, W. H. O. I. A. f. R. o. C. M. W., 2007. Carcinogenicity of
alcoholic beverages. Lancet Oncol 8, 292-293.

Bachmanov, A. A., Reed, D. R., Ninomiya, Y., Inoue, M., Tordoff, M. G., Price, R.
A., Beauchamp, G. K., 1997. Sucrose consumption in mice: major influence of
two genetic loci affecting peripheral sensory responses. Mamm Genome 8, 545-
548.

Belknap, J. K., Crabbe, J. C., Young, E. R., 1993. Voluntary consumption of ethanol
in 15 inbred mouse strains. Psychopharmacology (Berl). 112 503-510.

Belzung, C., Le Pape, G., 1994. Comparison of different behavioral test situations
used in psychopharmacology for measurement of anxiety. Physiol Behav 56, 623-
628.

Berglund, M., Thelander, S., Salaspuro, M., Franck, J., Andreasson, S., Ojehagen,
A., 2003. Treatment of alcohol abuse: an evidence-based review. Alcoholism,
clinical and experimental research 27, 1645-1656.

Blednov, Y. A., Ozburn, A. R., Walker, D., Ahmed, S., Belknap, J. K., Harris, R. A.,
2010. Hybrid mice as genetic models of high alchol consumption. Behav Genet
40 93-110.

Blednov, Y. A., Walker, D., Martinez, M., Levine, M., Damak, S., Margolskee, R. F.,
2008. Perception of sweet taste is important for voluntary alcohol consumption
in mice. Genes Brain Behav. 7 1-13.

78

Blizard, D. A., Kotlus, B., Frank, M. E., 1999. Quantitative trait loci associated with
short-term intake of sucrose, saccharin and quinine solutions in laboratory mice.
Chem Senses 24, 373-385.

Borsini, F., Meli, A., 1988. Is the forced swimming test a suitable model for
revealing antidepressant activity? Psychopharmacology 94, 147-160.

Bouchery, E. E., Harwood, H. J., Sacks, J. J., Simon, C. J., Brewer, R. D., 2011.
Economic costs of excessive alcohol consumption in the U.S., 2006. Amer. J.
Prevent. Med. 41, 516-524.

Bouza, C., Angeles, M., Munoz, A., Amate, J. M., 2004. Efficacy and safety of
naltrexone and acamprosate in the treatment of alcohol dependence: a
systematic review. Addiction 99, 811-828.

Bozikas, V., Petrikis, P., Gamvrula, K., Savvidou, I., Karavatos, A., 2002. Treatment
of alcohol withdrawal with gabapentin. Prog Neuropsychopharmacol Biol
Psychiatry 26, 197-199.

Braff, D. L., Geyer, M. A., Swerdlow, N. R., 2001. Human studies of prepulse
inhibition of startle: normal subjects, patient groups, and pharmacological
studies. Psychopharmacology 156, 234-258.

Braff, D. L., Light, G. A., 2004. Preattentional and attentional cognitive deficits as
targets for treating schizophrenia. Psychopharmacology 174, 75-85.

Broekkamp, C. L., Rijk, H. W., Joly-Gelouin, D., Lloyd, K. L., 1986. Major
tranquillizers can be distinguished from minor tranquillizers on the basis of
effects on marble burying and swim-induced grooming in mice. European journal
of pharmacology 126, 223-229.

Brower, K. J., Myra Kim, H., Strobbe, S., Karam-Hage, M. A., Consens, F., Zucker,
R. A., 2008. A randomized double-blind pilot trial of gabapentin versus placebo
to treat alcohol dependence and comorbid insomnia. Alcoholism, clinical and
experimental research 32, 1429-1438.

Buell, G., Collo, G., Rassendren, F., 1996. P2X receptors: an emerging channel
family. The European journal of neuroscience 8, 2221-2228.

Burkhart, C. N., 2000. Ivermectin: an assessment of its pharmacology,
microbiology and safety. Vet.Hum.Toxicol. 42, 30-35.
79

Carta, M., Ariwodola, O. J., Weiner, J. L., Valenzuela, C. F., 2003. Alcohol potently
inhibits the kainate receptor-dependent excitatory drive of hippocampal
interneurons. Proceedings of the National Academy of Sciences of the United
States of America 100, 6813-6818.

Castro, L. A., Baltieri, D. A., 2004. [The pharmacologic treatment of the alcohol
dependence]. Rev Bras Psiquiatr 26 Suppl 1, S43-46.

Chiriboga, C. A., 2003. Fetal alcohol and drug effects. Neurologist 9, 267-279.

Colombo, G., Agabio, R., Carai, M. A., Lobina, C., Pani, M., Reali, R., Addolorato,
G., Gessa, G. L., 2000. Ability of baclofen in reducing alcohol intake and
withdrawal severity: I--Preclinical evidence. Alcoholism, clinical and experimental
research 24, 58-66.

Colombo, G., Serra, S., Brunetti, G., Vacca, G., Carai, M. A., Gessa, G. L., 2003a.
Suppression by baclofen of alcohol deprivation effect in Sardinian alcohol-
preferring (sP) rats. Drug and alcohol dependence 70, 105-108.

Colombo, G., Vacca, G., Serra, S., Brunetti, G., Carai, M. A., Gessa, G. L., 2003b.
Baclofen suppresses motivation to consume alcohol in rats. Psychopharmacology
167, 221-224.

Costanzo, S., Di Castelnuovo, A., Donati, M. B., Iacoviello, L., de Gaetano, G.,
2010. Alcohol consumption and mortality in patients with cardiovascular disease:
a meta-analysis. J Am Coll Cardiol 55, 1339-1347.

Crabb, D. W., 1997. First pass metabolism of ethanol: gastric or hepatic,
mountain or molehill? Hepatology 25, 1292-1294.

Crump, A., Omura, S., 2011. Ivermectin, 'wonder drug' from Japan: the human
use perspective. Proc Jpn Acad Ser B Phys Biol Sci 87, 13-28.

Davies, D. L., Kochegarov, A. A., Kuo, S. T., Kulkarni, A. A., Woodward, J. J., King,
B. F., Alkana, R. L., 2005. Ethanol differentially affects ATP-gated P2X(3) and
P2X(4) receptor subtypes expressed in Xenopus oocytes. Neuropharmacology
49, 243-253.

80

Davies, D. L., Machu, T. K., Guo, Y., Alkana, R. L., 2002. Ethanol sensitivity in ATP-
gated P2X receptors is subunit dependent. Alcoholism, clinical and experimental
research 26, 773-778.

Davies, M., 2003. The role of GABAA receptors in mediating the effects of alcohol
in the central nervous system. Journal of psychiatry & neuroscience : JPN 28,
263-274.

Davis, J. A., Paylor, R., McDonald, M. P., Libbey, M., Ligler, A., Bryant, K., Crawley,
J. N., 1999. Behavioral effects of ivermectin in mice. Lab Anim Sci. 49 288-296.

Dawson, G. R., Wafford, K. A., Smith, A., Marshall, G. R., Bayley, P. J., Schaeffer, J.
M., Meinke, P. T., McKernan, R. M., 2000. Anticonvulsant and adverse effects of
avermectin analogs in mice are mediated through the gamma-aminobutyric acid
A receptor. Journal of Pharmacology and Experimental Therapeutics 295, 1051-
1060.

Dent, J. A., Davis, M. W., Avery, L., 1997. avr-15 encodes a chloride channel
subunit that mediates inhibitory glutamatergic neurotransmission and
ivermectin sensitivity in Caenorhabditis elegans. EMBO J 16, 5867-5879.

Department of Mental Health and Substance Abuse, W., 2011. Global status
report on alcohol and health. World Health Organization.

Di Lorenzo, P. M., Kiefer, S. W., Rice, A. G., Garcia, J., 1986. Neural and behavioral
responsivity to ethyl alcohol as a tastant. Alcohol 3, 55-61.

Dulawa, S. C., Holick, K. A., Gundersen, B., Hen, R., 2004. Effects of chronic
fluoxetine in animal models of anxiety and depression.
Neuropsychopharmacology 29, 1321-1330.
Elmer, G. I., Meisch, R. A., George, F. R., 1987. Differential concentration-
response curves for oral ethanol self-administration in C57BL/6J and BALB/cJ
mice. Alcohol 4, 63-68.

Ford, M. M., Beckley, E. H., Nickel, J. D., Eddy, S., Finn, D. A., 2008. Ethanol intake
patterns in female mice: influence of allopregnanolone and the inhibition of its
synthesis. Drug Alcohol Depend. 97, 73-85.

Fuller, R. K., Branchey, L., Brightwell, D. R., Derman, R. M., Emrick, C. D., Iber, F.
L., James, K. E., Lacoursiere, R. B., Lee, K. K., Lowenstam, I., et al., 1986.
81

Disulfiram treatment of alcoholism. A Veterans Administration cooperative
study. JAMA : the journal of the American Medical Association 256, 1449-1455.

Geary, T. G., 2005. Ivermectin 20 years on: maturation of a wonder drug. Trends
in Parasitology 21, 530-532.

Gonzales, R. A., Job, M. O., Doyon, W. M., 2004. The role of mesolimbic
dopamine in the development and maintenance of ethanol reinforcement.
Pharmacology & Therapeutics 103, 121-146.

Graham, F. K., 1975. Presidential Address, 1974. The more or less startling effects
of weak prestimulation. Psychophysiology 12, 238-248.

Grant, A., 2000. Tenth Special Report to the U.S. Congress on Alcohol and Health.

Grant, B. F., Dawson, D. A., Stinson, F. S., Chou, P., Dufour, M. C., Pickering, R. P.,
2004. The 12-month prevalence and trends in DSM-IV alcohol abuse and
dependence: United States, 1991-1992 and 2001-2002. Drug and alcohol
dependence 74 223-234.

Guzzo, C. A., Furtek, C. I., Porras, A. G., Chen, C., Tipping, R., Clineschmidt, C. M.,
Sciberras, D. G., Hsieh, J. Y., Lasseter, K. C., 2002. Safety, tolerability, and
pharmacokinetics of escalating high doses of ivermectin in healthy adult
subjects. The Journal of Clinical Pharmacology 42, 1122-1133.

Harwood, H. J., 2000. Updating estimates of the economic costs of alcohol abuse
in the United States: Estimates, update methods, and data. . In: The Lewin, G.,
(Ed). National Institute on Alcohol Abuse and Alcoholism.

Heilig, M., Egli, M., 2006. Pharmacological treatment of alcohol dependence:
target symptoms and target mechanisms. Pharmacol Ther. 111, 855-876.

Heine, C., Wegner, A., Grosche, J., Allgaier, C., Illes, P., Franke, H., 2007. P2
receptor expression in the dopaminergic system of the rat brain during
development. Neuroscience 149, 165-181.
Herz, A., 1997. Endogenous opioid systems and alcohol addiction.
Psychopharmacology 129, 99-111.

Hietala, J., Koivisto, H., Anttila, P., Niemela, O., 2006. Comparison of the
combined marker GGT-CDT and the conventional laboratory markers of alcohol
82

abuse in heavy drinkers, moderate drinkers and abstainers. Alcohol Alcohol 41,
528-533.

Holmes, P. V., 2003. Rodent models of depression: reexamining validity without
anthropomorphic inference. Crit Rev Neurobiol 15, 143-174.

Hugel, S., Schlichter, R., 2002. Presynaptic P2X receptors facilitate inhibitory
GABAergic transmission between cultured rat spinal cord dorsal horn neurons. J
Neuroscience 20, 2121-2130.

Hughes, J. C., Cook, C. C., 1997. The efficacy of disulfiram: a review of outcome
studies. Addiction 92, 381-395.

Hyman, S. E., Malenka, R. C., 2001. Addiction and the brain: the neurobiology of
compulsion and its persistence. Nature reviews. Neuroscience 2, 695-703.

Imperato, A., Di Chiara, G., 1986. Preferential stimulation of dopamine release in
the nucleus accumbens of freely moving rats by ethanol. Journal of
Pharmacology and Experimental Therapeutics 239, 219-228.

Jelinkova, I., Vavra, V., Jindrichova, M., Obsil, T., Zemkova, H. W., Zemkova, H.,
Stojilkovic, S. S., 2008. Identification of P2X(4) receptor transmembrane residues
contributing to channel gating and interaction with ivermectin. Pflugers Arch.
456, 939-950.

Jelinkova, I., Yan, Z., Liang, Z., Moonat, S., Teisinger, J., Stojilkovic, S. S., Zemkova,
H., 2006. Identification of P2X4 receptor-specific residues contributing to the
ivermectin effects on channel deactivation. Biochem Biophys Res Commun. 349
619-625.

Jo, Y. H., Donier, E., Martinez, A., Garret, M., Toulme, E., Boue-Grabot, E., 2011.
Cross-talk between P2X4 and gamma-aminobutyric acid, type A receptors
determines synaptic efficacy at a central synapse. The Journal of biological
chemistry 286, 19993-20004.

Jo, Y. H., Schlichter, R., 1999. Synaptic corelease of ATP and GABA in cultured
spinal neurons. Nature Neurosci. 2, 241-245.

Johnson, B., 2010. Medication treatment of different types of alcoholism. Am J
Psychiatry 167, 630-639.

83

Johnson, B. A., 2004. Progress in the development of topiramate for treating
alcohol dependence: from a hypothesis to a proof-of-concept study. Alcoholism,
clinical and experimental research 28, 1137-1144.

Johnson, B. A., 2005. Recent advances in the development of treatments for
alcohol and cocaine dependence: focus on topiramate and other modulators of
GABA or glutamate function. CNS Drugs 19, 873-896.

Johnson, B. A., 2008. Update on neuropharmacological treatments for
alcoholism: Scientific basis and clinical findings. Biochemical Pharmacology 75,
34-56.

Johnson, B. A., Ait-Daoud, N., Bowden, C. L., DiClemente, C. C., Roache, J. D.,
Lawson, K., Javors, M. A., Ma, J. Z., 2003. Oral topiramate for treatment of
alcohol dependence: a randomised controlled trial. Lancet 361, 1677-1685.

Johnson, B. A., Rosenthal, N., Capece, J. A., Wiegand, F., Mao, L., Beyers, K.,
McKay, A., Ait-Daoud, N., Anton, R. F., Ciraulo, D. A., Kranzler, H. R., Mann, K.,
O'Malley, S. S., Swift, R., 2007. Topiramate for treating alcohol dependence: A
randomized controlled trial. JAMA : the journal of the American Medical
Association 298 1641-1651.

Kimpel, M. W., Strother, W. N., McClintick, J. N., Carr, L. G., Liang, T., Edenberg,
H. J., McBride, W. J., 2007. Functional gene expression differences between
inbred alcohol-preferring and -non-preferring rats in five brain regions. Alcohol
41, 95-132.

Kranzler, H. R., Pierucci-Lagha, A., Feinn, R., Hernandez-Avila, C., 2003. Effects of
ondansetron in early- versus late-onset alcoholics: a prospective, open-label
study. Alcoholism, clinical and experimental research 27, 1150-1155.

Krause, R. M., Buisson, B., Bertrand, S., Corringer, P. J., Galzi, J. L., Changeux, J.
P., Bertrand, D., 1998. Ivermectin: A positive allosteric effector of the alpha 7
meuronal nicotinic acetylcholine receptor. Molecular pharmacology 53, 283-294.

Krystal, J. H., Cramer, J. A., Krol, W. F., Kirk, G. F., Rosenheck, R. A., 2001.
Naltrexone in the treatment of alcohol dependence. N Engl J Med 345, 1734-
1739.

Lancaster, F. E., 1995. Gender differences in animal studies. Implications for the
study of human alcoholism. Recent Dev Alcohol 12, 209-215.
84

Lemon, C. H., Brasser, S. M., Smith, D. V., 2004. Alcohol activates a sucrose-
responsive gustatory neural pathway. J Neurophysiol 92, 536-544.

Lister, R. G., 1990. Ethologically-based animal models of anxiety disorders.
Pharmacol Ther 46, 321-340.

Littleton, J., 1995. Acamprosate in alcohol dependence: how does it work?
Addiction 90, 1179-1188.

Lovinger, D. M., 1999. 5-HT3 receptors and the neural actions of alcohols: an
increasingly exciting topic. Neurochem Int 35, 125-130.

Lovinger, D. M., White, G., Weight, F. F., 1989. Ethanol inhibits NMDA-activated
ion current in hippocampal neurons. Science 243, 1721-1724.

Ma, J. Z., Ait-Daoud, N., Johnson, B. A., 2006. Topiramate reduces the harm of
excessive drinking: implications for public health and primary care. Addiction
101, 1561-1568.

Mann, K., Lehert, P., Morgan, M. Y., 2004. The efficacy of acamprosate in the
maintenance of abstinence in alcohol-dependent individuals: results of a meta-
analysis. Alcoholism, clinical and experimental research 28, 51-63.

Martin, R. J., 1996. An electrophysiological preparation of Ascaris suum
pharyngeal muscle reveals a glutamate-gated chloride channel sensitive to the
avermectin analogue, milbemycin D. Parasitology 112 ( Pt 2), 247-252.

Martin, R. J., Pennington, A. J., 1989. A patch-clamp study of effects of
dihydroavermectin on Ascaris muscle. British journal of pharmacology 98, 747-
756.

Mason, B. J., Goodman, A. M., Chabac, S., Lehert, P., 2006. Effect of oral
acamprosate on abstinence in patients with alcohol dependence in a double-
blind, placebo-controlled trial: the role of patient motivation. J Psychiatr Res 40,
383-393.

Mason, B. J., Heyser, C. J., 2010. Acamprosate: a prototypic neuromodulator in
the treatment of alcohol dependence. CNS & neurological disorders drug targets
9, 23-32.

85

Middaugh, L. D., Kelley, B. M., Bandy, A. L., McGroarty, K. K., 1999. Ethanol
consumption by C57BL/6 mice: influence of gender and procedural variables.
Alcohol 17, 175-183.

Nagata, K., Imai, T., Yamashita, T., Tsuda, M., Tozaki-Saitoh, H., Inoue, K., 2009.
Antidepressants inhibit P2X4 receptor function: a possible involvement in
neuropathic pain relief. Mol Pain 5, 20.

Nicolas, L. B., Kolb, Y., Prinssen, E. P., 2006. A combined marble burying-
locomotor activity test in mice: a practical screening test with sensitivity to
different classes of anxiolytics and antidepressants. European journal of
pharmacology 547, 106-115.

Njung'e, K., Handley, S. L., 1991. Evaluation of marble-burying behavior as a
model of anxiety. Pharmacology, biochemistry, and behavior 38, 63-67.

O'Malley, S. S., Garbutt, J. C., Gastfriend, D. R., Dong, Q., Kranzler, H. R., 2007.
Efficacy of extended-release naltrexone in alcohol-dependent patients who are
abstinent before treatment. J Clin Psychopharmacol 27, 507-512.

Oncken, C., Van Kirk, J., Kranzler, H. R., 2001. Adverse effects of oral naltrexone:
analysis of data from two clinical trials. Psychopharmacology 154, 397-402.

Ostrovskaya, O., Asatryan, L., Wyatt, L., Popova, M., Li, K., Peoples, R. W., Alkana,
R. L., Davies, D. L., 2011. Ethanol is a fast channel inhibitor of purinergic P2X4
receptors. J. Pharm. Exp. Ther. 337, 171-179.

Ottesen, E. A., Campbell, W. C., 1994. Ivermectin in human medicine. J
Antimicrob Chemother 34, 195-203.

Pankratov, Y. V., Lalo, U. V., Krishtal, O. A., 2002. Role for P2X receptors in long-
term potentiation. J Neuroscience 22, 8363-8369.

Paredes, R. G., Agmo, A., 1992. GABA and behavior: the role of receptor
subtypes. Neuroscience and biobehavioral reviews 16, 145-170.

Penney, J. B., Jr., Young, A. B., 1983. Speculations on the functional anatomy of
basal ganglia disorders. Annu Rev Neurosci 6, 73-94.

Petit-Demouliere, B., Chenu, F., Bourin, M., 2005. Forced swimming test in mice:
a review of antidepressant activity. Psychopharmacology 177, 245-255.
86

Picciotto, M. R., Addy, N. A., Mineur, Y. S., Brunzell, D. H., 2008. It is not
"either/or": activation and desensitization of nicotinic acetylcholine receptors
both contribute to behaviors related to nicotine addiction and mood. Prog
Neurobiol 84, 329-342.

Poling, A., Cleary, J., Monaghan, M., 1981. Burying by rats in response to aversive
and nonaversive stimuli. J Exp Anal Behav 35, 31-44.

Pompili, M., Serafini, G., Innamorati, M., Dominici, G., Ferracuti, S., Kotzalidis, G.
D., Serra, G., Girardi, P., Janiri, L., Tatarelli, R., Sher, L., Lester, D., 2010. Suicidal
behavior and alcohol abuse. Int J Environ Res Public Health 7, 1392-1431.

Popova, M., Asatryan, L., Ostrovskaya, O., Wyatt, R. L., Li, K., Alkana, R. L., Davies,
D. L., 2010. A point mutation in the ectodomain-transmembrane 2 interface
eliminates the inhibitory effects of ethanol in P2X4 receptors. J.Neurochem. 112,
307-317.

Porsolt, R. D., Bertin, A., Blavet, N., Deniel, M., Jalfre, M., 1979. Immobility
induced by forced swimming in rats: effects of agents which modify central
catecholamine and serotonin activity. European journal of pharmacology 57,
201-210.

Porsolt, R. D., Bertin, A., Jalfre, M., 1977. Behavioral despair in mice: a primary
screening test for antidepressants. Arch Int Pharmacodyn Ther 229, 327-336.

Priel, A., Silberberg, S. D., 2004. Mechanism of Ivermectin Facilitation of Human
P2X4 Receptor Channels. The Journal of General Physiology 123, 281-293.

Prut, L., Belzung, C., 2003. The open field as a paradigm to measure the effects of
drugs on anxiety-like behaviors: a review. European journal of pharmacology
463, 3-33.

Regier, D. A., Farmer, M. E., Rae, D. S., Locke, B. Z., Keith, S. J., Judd, L. L.,
Goodwin, F. K., 1990. Comorbidity of mental disorders with alcohol and other
drug abuse. Results from the Epidemiologic Catchment Area (ECA) Study. JAMA :
the journal of the American Medical Association 264, 2511-2518.

Rehm, J., Mathers, C., Popova, S., Thavorncharoensap, M., Teerawattananon, Y.,
Patra, J., 2009. Global burden of disease and injury and economic cost
attributable to alcohol use and alcohol-use disorders. Lancet 373, 2223-2233.
87

Rhodes, J. S., Ford, M. M., Yu, C. H., Brown, L. L., Finn, D. A., Garland, T., Jr.,
Crabbe, J. C., 2007. Mouse inbred strain differences in ethanol drinking to
intoxication. Genes Brain Behav. 6 1-18.

Richard-Lenoble, D., Chandenier, J., Gaxotte, P., 2003. Ivermectin and filariasis.
Fundam Clin Pharmacol 17, 199-203.

Roberts, A. J., Smith, A. D., Weiss, F., Rivier, C., Koob, G. F., 1998. Estrous cycle
effects on operant responding for ethanol in female rats. Alcoholism, clinical and
experimental research 22, 1564-1569.

Rubio, M. E., Soto, F., 2001. Distinct localization of P2X receptors at excitatory
postsynaptic specializations. Journal of Neuroscience 21, 641-653.

Schuckit, M. A., 2009. Alcohol-use disorders. Lancet 373, 492-501.

Sellers, E. M., Toneatto, T., Romach, M. K., Somer, G. R., Sobell, L. C., Sobell, M.
B., 1994. Clinical efficacy of the 5-HT3 antagonist ondansetron in alcohol abuse
and dependence. Alcoholism, clinical and experimental research 18, 879-885.

Shan, Q., Haddrill, J. L., Lynch, J. W., 2001. Ivermectin, an unconventional agonist
of the glycine receptor chloride channel. J. Biol. Chem. 276, 12556-12564.

Silberberg, S. D., Li, M., Swartz, K. J., 2007. Ivermectin interaction with
transmembrane helices reveals widespread rearrangements during opening of
P2X receptor channels. Neuron 54, 263-274.

Sim, J. A., Chaumont, S., Jo, J., Ulmann, L., Young, M. T., Cho, K., Buell, G., North,
R. A., Rassendren, F., 2006. Altered hippocampal synaptic potentiation in P2X4
knock-out mice. Journal of Neuroscience 26, 9006-9009.

Simms, J. A., Bito-Onon, J. J., Chatterjee, S., Bartlett, S. E., 2010. Long-Evans rats
acquire operant self-administration of 20% ethanol without sucrose fading.
Neuropsychopharmacology 35, 1453-1463.

Sinclair, J. D., Kampov-Polevoy, A., Stewart, R., Li, T. K., 1992. Taste preferences
in rat lines selected for low and high alcohol consumption. Alcohol 9, 155-160.

88

Soto, F., Garcia-Guzman, M., Karschin, C., Stnhmer, W., 1996. Cloning and tissue
distribution of a novel P2X receptor from rat brain. Biochemical and Biophysical
Research Communications 223, 456-460.

Spinosa, H. S., Stilck, S. R. A. N., Bernardi, M. M., 2002. Possible anxiolytic effects
of ivermectin in rats. Vet Res Commun 26 309-321.

Steru, L., Chermat, R., Thierry, B., Mico, J. A., Lenegre, A., Steru, M., Simon, P.,
Porsolt, R. D., 1987. The automated Tail Suspension Test: a computerized device
which differentiates psychotropic drugs. Prog Neuropsychopharmacol Biol
Psychiatry 11, 659-671.

Swift, R. M., 1999. Drug therapy for alcohol dependence. N Engl J Med 340,
1482-1490.
Tabakoff, B., Hoffman, P. L., 2000. Animal models in alcohol research. Alcohol
Res Health 24, 77-84.

Tabakoff, B., Saba, L., Printz, M., Flodman, P., Hodgkinson, C., Goldman, D., Koob,
G., Richardson, H., Kechris, K., Bell, R. L., H�bner, N., Heinig, M., Pravenec, M.,
Mangion, M., Legault, L., Dongier, M., Conigrave, K. M., Whitfield, J. B., Saunders,
J. B., Grant, B., Hoffman, P. L., 2009. Genetical genomic determinants of alcohol
consumption in rats and humans BMC Biology 7, 70

Trailovic, A. M., Trailovic, N., 2010. Central and Peripheral Neurotoxic effects of
ivermectin in rats. J Met Med Sci 73, 591-599.

Treit, D., Pinel, J. P., Fibiger, H. C., 1981. Conditioned defensive burying: a new
paradigm for the study of anxiolytic agents. Pharmacology, biochemistry, and
behavior 15, 619-626.

Tsai, G. E., Ragan, P., Chang, R., Chen, S., Linnoila, V. M., Coyle, J. T., 1998.
Increased glutamatergic neurotransmission and oxidative stress after alcohol
withdrawal. Am J Psychiatry 155, 726-732.
Valdez, G. R., Zorrilla, E. P., Roberts, A. J., Koob, G. F., 2003. Antagonism of
corticotropin-releasing factor attenuates the enhanced responsiveness to stress
observed during protracted ethanol abstinence. Alcohol 29, 55-60.

Vengeliene, V., Bilbao, A., Molander, A., Spanagel, R., 2008. Neuropharmacology
of alcohol addiction. British journal of pharmacology 154, 299-315.

89

Volpicelli, J. R., Alterman, A. I., Hayashida, M., O'Brien, C. P., 1992. Naltrexone in
the treatment of alcohol dependence. Arch Gen Psychiatry 49, 876-880.

Wang, L., Liu, J., Harvey-White, J., Zimmer, A., Kunos, G., 2003. Endocannabinoid
signaling via cannabinoid receptor 1 is involved in ethanol preference and its
age-dependent decline in mice. Proceedings of the National Academy of Sciences
of the United States of America 100, 1393-1398.

Wang, Y., Haughey, N. J., Mattson, M. P., Furukawa, K., 2004. Dual effects of ATP
on rat hippocampal synaptic plasticity. NeuroReport 15.

Weiss, F., Porrino, L. J., 2002. Behavioral neurobiology of alcohol addiction:
recent advances and challenges. The Journal of neuroscience : the official journal
of the Society for Neuroscience 22, 3332-3337.

West, A. P., 1990. Neurobehavioral studies of forced swimming: the role of
learning and memory in the forced swim test. Prog Neuropsychopharmacol Biol
Psychiatry 14, 863-877.

Willner, P., Mitchell, P. J., 2002. The validity of animal models of predisposition
to depression. Behav Pharmacol 13, 169-188.

Wolstenholme, A. J., Rogers, A. T., 2005. Glutamate-gated chloride channels and
the mode of action of the avermectin/milbemycin anthelmintics. Parasitology
131 Suppl, S85-95.

Woods, J. E., 2nd, McKay, P. F., Masters, J., Seyoum, R., Chen, A., La Duff, L.,
Lewis, M. J., June, H. L., 2003. Differential responding for brain stimulation
reward and sucrose in high-alcohol-drinking (HAD) and low-alcohol-drinking
(LAD) rats. Alcoholism, clinical and experimental research 27, 926-936.

Xiao, C., Zhou, C., Li, K., Davies, D. L., Ye, J. H., 2008. Purinergic type 2 receptors
at GABAergic synapses on ventral tegmental area dopamine neurons are targets
for ethanol action. Journal of Pharmacology and Experimental Therapeutics 327,
196-205.

Xiong, K., Hu, X. Q., Stewart, R. R., Weight, F. F., Li, C., 2005. The mechanism by
which ethanol inhibits rat P2X4 receptors is altered by mutation of histidine 241.
Br.J.Pharmacol. 145, 576-586.

90

Xiong, K., Li, C., Weight, F. F., 2000. Inhibition by ethanol of rat P2X4 receptors
expressed in Xenopus oocytes. Br. J. Pharmacol. 130, 1394-1398.

Xiong, K. M., Li, C., Weight, F. F., 1999. Differential ethanol sensitivity of P2X 4
and P2X 4 /P2X 6 receptors expressed in Xenopus oocytes. Alcoholism, clinical
and experimental research 23, 8A.

Yadav, D., Whitcomb, D. C., 2010. The role of alcohol and smoking in
pancreatitis. Nat Rev Gastroenterol Hepatol 7, 131-145.

Yardley, M. M., Wyatt, L., Khoja, S., Asatryan, L., Ramaker, M. J., Finn, D. A.,
Alkana, R. L., Huynh, N., Louie, S. G., Petasis, N. A., Bortolato, M., Davies, D. L.,
2012. Ivermectin reduces alcohol intake and preference in mice.
Neuropharmacology 63, 190-201.

Yoneyama, N., Crabbe, J. C., Ford, M. M., Murillo, A., Finn, D. A., 2008. Voluntary
ethanol consumption in 22 inbred mouse strains. Alcohol 42 149-160.

Zemkova, H., Kucka, M., Li, S., Gonzalez-Iglesias, A. E., Tomic, M., Stojilkovic, S. S.,
2010. Characterization of purinergic P2X4 receptor channels expressed in
anterior pituitary cells. American journal of physiology. Endocrinology and
metabolism 298, E644-651.

Abstract (if available)

Abstract Alcohol use disorders (AUDs) ranks third in the list of preventable causes of morbidity and mortality in United States having a major national impact affecting over 18 million people and causing over 100,000 deaths annually. The prevalence of AUDs, in combination with limited clinical efficacy of currently approved FDA drugs in the treatment of this disorder signifies the importance for development of novel therapeutic agents. Alcohol is known to modulate a multitude of receptors in the brain to induce its cellular and behavioral effects. Recent electrophysiological findings from our laboratory, coupled with genomic studies, have suggested that the ionotropic receptor, P2X4 receptor (P2X4R) is a critical target that is modulated by ethanol. P2X4R activity has been demonstrated to be inhibited by ethanol at intoxicating and anesthetic concentrations. Recent evidence from our laboratory using electrophysiological strategies has shown that the FDA approved antiparasitic agent, ivermectin (IVM) antagonizes ethanol induced inhibition of P2X4R activity. On the basis of our in vitro data, and suggestion from my advisors I began testing the hypothesis that IVM represents a novel therapy that can be repurposed for treatment of AUDs. ❧ Aim 1 tests the hypothesis by investigating the effect of IVM on alcohol intake and preference in male and female C57BL/6 mice. This was accomplished by testing IVM at doses ranging from 0.65 mg/kg through 10mg/kg versus a 10% ethanol (10E) solution using a 24 hour two bottle choice paradigm. In support of the hypothesis, we found that IVM significantly reduced alcohol intake and preference in male and female mice. In addition, using a similar paradigm, evaluation of IVM’s effect on saccharin intake was undertaken to determine if IVM’s effect was specific for ethanol or if IVM was also active versus a second substance known to have positive reinforcing effects. In agreement with our ethanol studies, IVM significantly decreased saccharin intake and preference. ❧ Aim 2 tests the hypothesis that IVM does not cause behavioral effects that would negatively impact its potential therapeutic application to preventing or treating AUDs. To this end, I tested the effects of IVM on recognized measures of anxiety (open field, marble burying), depression (forced swim test), information processing (prepulse inhibition test). Collaborators from my research team also tested the elevated plus maze, tail suspension, conditioned place preference, hot plate and object exploration. A detailed explanation of these latter studies will be presented elsewhere but the findings will also be summarized in my thesis to present a broad picture of the investigations. The results overall support the hypothesis. IVM did not show rewarding effects in conditioned place preference test, suggesting that it does not have addictive potential and had anxiolytic effects in the marble burying test. However, it does appear to have some negative consequences. ❧ Taken together, the findings from the alcohol drinking and behavioral studies in mice support the hypothesis that IVM represents a novel therapy that can be repurposed for treatment of AUDs.

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Creator Khoja, Sheraz (author)

Core Title Preclinical investigation of ivermectin as a novel therapeutic agent for treatment of alcohol use disorders

School School of Pharmacy

Degree Master of Science

Degree Program Pharmaceutical Sciences

Publication Date 08/01/2012

Defense Date 06/22/2012

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

Tag alcohol use disorders,behavioral pharmacology,drug development for alcohol use disorders,ethanol,OAI-PMH Harvest

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Alkana, Ronald L. (committee chair), Davies, Daryl L. (committee member), Rodgers, Kathleen E. (committee member)

Creator Email sheraz.khoja@gmail.com,skhoja@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-80601

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Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

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