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The preclinical evaluation of moxidectin as a platform for drug development for alcohol use disorder
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Content
i
The Preclinical Evaluation of Moxidectin as a Platform for
Drug Development for Alcohol Use Disorder
by
Nhat Huynh
A dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the Requirement for the Degree
DOCTOR OF PHILOSOPHY
(CLNICAL AND EXPERIMENTAL THERAPEUTICS)
May 2018
Copyright 2018 Nhat Huynh
ii
Acknowledgements
First and foremost, I would like to thank to my family: my mother, father, grandparents,
and entire extended family who have always provided unconditional support for me and my
dreams. The work ethic and dedication they have instilled in me have allowed me to accomplish
things I would have never thought imaginable.
I would also like to thank Dr. Daryl Davies, Dr. Stan Louie and the rest of my committee
members, Dr. Liana Asatryan, Dr. Michael Jakowec, Dr. Kathleen Rodgers, and Dr. Frances
Richmond, who have provided constant encouragement, support, and mentoring during my
years at the University of Southern California.
I also want to express gratitude to all my other colleagues, in no particular order, Dr.
Sheraz Khoja, Dr. Megan Yardley, Dr. Nick Mordwinkin, Dr. Sachin Jadhav, Dr. Maira Soto, Dr.
Alick Tan, Dr. Jared Russell, Dr. Isaac Asante, Damaris Garcia, Vivek Shah, Larry Rodriguez,
Christopher Meeks, Natalie Arabian, Jamie Thuy, and Dustin Lieu. Their work hard and support
have made possible the completion of many important experiments presented in this
dissertation.
iii
Authorships
Published and in preparation works by the author incorporated into the dissertation.
Huynh N, Arabian N, Rodriguez L, Khoja S, Asante I, Louie S, Jakowec MW, Asatryan L,
Davies DL (2017). Purinergic P2X4 receptor knockout mice exhibit altered glutamatergic
signaling in the nucleus accumbens (2017). In Prepa ration. (Chapter 5)
Huynh N*, Khoja S*, Warnecke AMP, Asatryan L, Jakowec MW, Davies DL (2017).
Avermectins as a platform for drug development for alcohol use disorder. Invited Book Chapter
for Elsevier, In Preparation.
Khoja S*, Huynh N*, Warnecke AMP, Asatryan L, Jakowec MW, Davies DL ( 2017). Preclinical
evaluation of avermectins as novel therapeutic agents for alcohol use disorders. Invited Review
Article for Psychopharmacology, Under Review.
Khoja S, Huynh N, Asatryan L, Jakowec MW, Davies DL (2017). Reduced expression of
purinergic P2X4 receptors increases voluntary ethanol intake in C57BL/6J mice. Alcohol, In
Press. (Chapter 4)
Huynh N, Arabian N, Naito A, Louie S, Jakowec MW, Asatryan L, Davies DL (2017). Preclinical
development of moxidectin as a novel therapeutic for alcohol use disorder. Neuropharmacology
113(Pt A):60-70. (Chapter 2)
* Equal Contribution
Additional works by the author relevant to this dissertation but not forming part of it.
Huynh N, Arabian N, Lieu D, Asatryan L, Davies DL (2016). Utilizing an Orally Dissolving Strip
for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral
Gavage for Animals. J Vis Exp 23(109):e53770.
Yardley MM, Huynh N, Rodgers KE, Alkana RL, Davies DL (2015). Oral del ivery of ivermectin
using a fast dissolving oral film: Implications for repurposing ivermectin as a pharmacotherapy
for alcohol use disorder. Alcohol 49(6):553-559.
Yardley MM, Neely M, Huynh N, Asatryan L, Louie SG, Alkana RL, Davies DL (2014) . Multi-day
administration of ivermectin is effective in reducing alcohol intake in mice at doses shown to be
safe in humans. Neuroreport 25(13):1018-1023.
Asatryan L, Yardley MM, Khoja S, Trudell JR, Huynh N, Louie SG, Petasis NA, Alkana RL,
Davies DL (2014). Avermectins differentially affect ethanol intake and receptor function:
Implications for developing new therapeutics for alcohol use disorders. International Journal of
Neuropsychopharmacology 17(6):907-916.
Yardley MM, Wyatt L, Khoja S, Asatryan L, Ramaker MJ, Finn DA, Alkana RL, Huynh N, Louie
SG, Petasis NA, Bortolato M, Davies DL (2012). Iver mectin reduces alcohol intake and
preference in mice. Neuropharmacology 63(2):190-201 .
Research Support
NIAAA/NIH R01 AA022448 (D.L.D.) and the USC School of Pharmacy.
iv
Table of Content
Acknowledgements ii
List of Tables vi
List of Figures vii
List of Abbreviations ix
Abstract xi
Chapter 1. Introduction
Significance 13
The Drug Development Hurdle 15
Current FDA-Approved Medications for Alcohol Use Disorder 17
Off-Label Medications for Alcohol Use Disorder 19
P2X4 Receptors, an Emerging Critical Target for Ethanol 21
Avermectins, P2X4 Receptors, and Ethanol 23
Repurposing Potential of Moxidectin into a Novel Alcohol Therapeutic 26
Dissertation Hypothesis and Outline 27
Chapter 2. Preclinical Development of Moxidectin as a Novel Therapeutic
for Alcohol Use Disorder
Abstract 30
Introduction 31
Materials and Methods 33
Results
Acute administration of MOX decreased 10E intake in female mice 39
MOX decreased 10E intake in a dose-dependent manner in female
mice 40
MOX decreased 10E intake in a dose-dependent manner in male mice 41
Time course of the effect of MOX on ethanol intake in female mice 42
Multiple day dosing of MOX administration reduced ethanol intake in
female mice using a 24-h-two-bottle choice paradigm 42
Multiple day dosing of MOX administration reduced ethanol intake in
female mice using a drinking-in-the-dark paradigm 45
MOX positively modulated ATP-gated P2X4Rs and antagonized the
inhibitory effects of ethanol on P2X4R function 45
MOX positively modulated GABA
A
R activity 46
Discussion 47
Chapter 3. Moxidectin Attenuates Acute Ethanol-Induced Withdrawal Symptoms
in Mice
Abstract 53
Introduction 55
Materials and Methods 60
Results
Administration of MOX significantly reduced the severity of acute
ethanol-induced withdrawal symptoms in male and female C57BL/6J
v
mice 60
Administration of MOX significantly reduced 20E intake in
ethanol-dependent female C57BL/6J mice 61
MOX did not affect the pharmacokinetic of ethanol in male and female
C57BL/6J mice 62
Discussion 63
Chapter 4. Reduced Expression of Purinergic P2X4 Receptors Increases
Voluntary Ethanol Intake in C57BL/6J Mice
Abstract 66
Introduction 67
Materials and Methods 68
Results
P2X4R KO mice exhibited increased voluntary ethanol consumption in
the 24 hour access drinking paradigm 73
Transfection of BV-2 cells or transfusion in mouse striatum with
LV-shRNA-p2rx4 reduced P2X4R expression 74
Infusion of LV alone did not have any significant effect on ethanol intake
or preference in comparison to the naïve mice 75
The LV-shRNA-p2rx4 infused mice exhibited a higher ethanol intake as
compared to mice that only received LV infusion 76
Discussion 77
Chapter 5. Purinergic P2X4 Receptor Knockout Mice Exhibits Altered
Glutamatergic Signaling in the Nucleus Accumbens
Abstract 84
Introduction 86
Materials and Methods 88
Results
Microarray analysis of the NAc from male P2X4R KO mice after
short-term ethanol exposure 91
Bioinformatics analysis of differentially expressed genes. 92
Activation of P2X4Rs significantly inhibited the current in NMDARs 96
Pharmacological inhibition of NMDARs exhibited differential effect on 10E
intake in P2X4R KO as compared to WT mice 97
Discussion 98
Chapter 6. Overall Summary
Summary of Findings 102
Future Directions 104
Bibliography 107
vi
List of Tables
Table 1.1 Diagnostic criteria for AUD.
Table 1.2 Medications used to treat AUD.
Table 5.1 IPA-implicated altered top canonical pathways.
Table 5.2 Select differentially expressed genes.
vii
List of Figures
Figure 1.1 The drug development process.
Figure 1.2 Crystal structure of zebrafish P2X4R.
Figure 1.3 Homology model of rat P2X4R.
Figure 1.4 Avermectins/P2X4R in vitro-in vivo correlation.
Figure 2.1 Acute administration of MOX significantly reduced 10E intake in
female and male C57BL/6J mice, in a dose-dependent manner,
using a 24-h-two-bottle choice paradigm.
Figure 2.2 MOX (2.g mg/kg) significantly reduced 10 E intake approximately
4 h after administration in female mice.
Figure 2.3 Daily administration of MOX (2.5 mg/kg x 5 days) significantly
reduced 10E intake in female C57BL/6J mice using a 24-h-two
bottle choice paradigm.
Figure 2.4 Daily administration of MOX (2.5 mg/kg x 5 days) reduced 20E
intake in female C57BL/6J mice using a drinking-in-the-dark
(DID) paradigm.
Figure 2.5 MOX (0.5 and 1 µM) antagonized the inhib itory effects of ethanol
in P2X4Rs.
Figure 2.6 MOX (0.1, 0.5, and 1 µM) has a weaker mo dulatory activity in
GABA
A
Rs than ivermectin.
Figure 2.7 Structures of ivermectin and MOX.
Figure 3.1 MOX significantly attenuates ethanol-induced withdrawal symptoms
in male and female C57BL/6J mice.
Figure 3.2 MOX significantly reduced 20E intake in ethanol-dependence
female C57BL/6J mice.
Figure 3.3 MOX did not affect the pharmacokinetic profile of ethanol.
Figure 4.1 P2X4R KO mice exhibited significantly higher 10E intake compared
to WT controls and tended to have higher fluid intake without any
significant changes in 10E preference or water intake.
Figure 4.2 Microglial BV-2 cells transinfected with LV-shRNA-p2rx4 reduced
P2X4R expression by 68% and 62% as compared to non-treated (NT)
and LV alone treated cells respectively.
Figure 4.3 The LV-shRNA-p2rx4 group exhibited significantly higher 10E
viii
intake as compared to mice infused with LV alone.
Figure 5.1 The NAc of P2X4R KO and WT mice were collected after 2 weeks
of ethanol exposure using a 24-h-two-bottle choice paradigm.
Figure 5.2 IPA-derived gene network indicates interactions the differentially
expressed genes in the NAc of P2X4R KO versus WT mice after
short-term ethanol exposure.
Figure 5.3 Reduction of NMDARs’ current by P2X4Rs, tested using the
Xenopus oocyte expression system and two-electrode voltage
clamp method.
Figure 5.4 Memantine (10 mg/kg) exhibited different ial activity in P2X4R KO
versus WT mice.
ix
Abbreviations
5-HT3 receptor, 5-hydroxytryptamine receptor
10E, 10% ethanol (v/v)
20E, 20% ethanol (v/v)
AMPAR, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor
ATP, adenosine triphosphate
Arc, arcuate nucleus
AUD, alcohol use disorder
APA, American Psychological Association
BBB, blood brain barrier
BEC, blood ethanol concentration
CDC, Centers for Disease Control and Prevention
CNS, central nervous system
cRNA, complementary RNA
cDNA, complementary cDNA
DID, drinking-in-the-dark
DSM-5, Diagnostic and Statistical Manual of Mental Disorders 5
th
Edition
EC, effective concentration
FAS, fetal alcohol syndrome
FDA, Food and Drug Administration
GABA
A
R, gamma-aminobutyric acid type A receptor
GABA
B
R, gamma-aminobutyric acid type B receptor
GFP, green fluorescent protein
HIC, handling-induced convulsions
IA, intermittent access
x
IP, intraperitoneally
IVM, ivermectin
LGICs, ligand-gated ion channels
Lentiviral-shRNA, lentiviral particles containing short hairpin RNA
IND, investigational new drug application
MOX, moxidectin
NAc, nucleus accumbens
nAChR, nicotinic acetylcholine receptor
NME, new molecular entities
NIAAA, National Institute of Alcohol Abuse and Alcoholism
NMDAR, N-methyl-D-aspartate receptor (NMDAR)
P2XRs, P2X receptors
P2X4Rs, purinergic P2X4 receptors
P2X4R KO, P2X4 receptor knockout
P-gp, P-glycoprotein
PVN, paraventricular nucleus
RSA, Research Society on Alcoholism
US, United States
VTA, ventral tegmental area
WT, wildtype
xi
Abstract
Alcohol use disorder (AUD) affects over 18 million people in the United States, costing
an economic burden in excess of $220 billion and causing more than 100,000 deaths annually.
Despite considerable effort and funding that have been dedicated toward drug development
programs, currently we only have three medications approve by the Food and Drug
Administration (FDA) for the treatment of AUD. Thes e pharmacotherapies, even combined with
psychosocial support, have only produced limited results as evident by the fact that more than
90% of patients relapse at least once during treatment and that the prevalence of alcohol abuse
continues to rise. As such, the use of new approaches for the rapid development of effective
AUD therapies represents an important public health goal. Using a translational strategy, our
laboratory has been investigating the repurposing potential of compounds from the avermectin
family (i.e., ivermectin [IVM] and moxidectin [MOX] ) to be developed into novel medications to
combat alcoholism. In particular, IVM has already been safely used in humans as an anti-
parasitic agent for over two decades. With the advantage of having the lead compounds (such
as IVM and MOX) already present and the abundant availability of safety data, the drug
repurposing route is a fast and economically feasible approach for AUD drug development.
Previous investigations have established that IVM and related analogues can significantly
reduce ethanol intake in both male and female mice through multiple drinking paradigms and
the anti-alcohol effects of these avermectins, in part, comes from their activity on P2X4
receptors (P2X4Rs). Although the evidence supports the development of IVM into an anti-
alcohol medication for use in humans, there remains challenges in moving this molecule
forward. As such, we have expending our investigation regarding the use of other avermectins.
It is this quest that is the focus of my dissertation. My effort primarily focused on MOX with the
goal of demonstrating that MOX: 1) exhibits comparable or better anti-alcohol efficacy to IVM,
and 2) is more suitable for use on a chronic basis. My results, in agreement with published
xii
studies, have suggested that MOX has superior central nervous safety profile as compared to
IVM. These advantages support the further development of MOX to be used as a long-term
AUD medication. Importantly, and very timely, MOX is currently in clinical development as an
alternate anti-parasitic therapy to IVM with no clinical abnormalities reported to date. If
supported by my pre-clinical efficacy studies, MOX can be rapidly translated into human studies
for the treatment of AUD.
In the first half of my dissertation (chapters 2 & 3), I evaluated the pre-clinical efficacy of
MOX using different rodent drinking models that represent different stages of alcoholism. In
chapters 4 and 5 I began to elucidate the anti-alcohol mechanism(s) of MOX and other
avermectins. As we have previously shown that P2X4R is an important target for avermectins’
anti-alcohol effect, chapter 4 investigated the neuroanatomical specificity of P2X4Rs in relation
to ethanol behavior by utilizing a lentiviral-shRNA strategy for region-specific knockdown of
brain P2X4Rs. Chapter 5 utilized a gene-profiling approach to identify potential signaling
mechanism(s) that may explain how P2X4R regulate dr inking in mice; these observations were
further verified using electrophysiological and pharmacological methods. Overall, the positive
preclinical results, coupled with a better understanding of the drug anti-alcohol mechanism(s),
should set the stage for future human studies that will allow for the successful translation of
MOX into clinics for the treatment of AUD.
13
Chapter 1
Introduction
Significance
Alcohol use disorder (AUD) is defined as problemati c pattern of drinking, manifested by
at least two of the following symptoms (Table 1.1) that occur within a 12-month period
(American Psychological Association [APA], Diagnost ic and Statistical Manual of Mental
Disorders 5
th
Edition [DSM-5], 2013). In the United States (US), AUD has major health
implications by affecting over 18 million people, and contributing to over 100,000 deaths and an
economic burden of $200+ billion annually (Bouchery et al., 2011; Grant et al., 2004; Hardwood,
2000).
The majority of this cost comes from reduced earnings, decreased productivity, medical
expenses, accidents, violence, and premature deaths. Alcohol has been implicated as a
component of multiple diseases and can contribute to and/or exacerbate many of these
conditions such as gastrointestinal diseases (i.e., liver cirrhosis, pancreatitis), cardiovascular
Table 1.1. Diagnostic criteria for AUD. Mild: 2-3 symptoms, moderate: 4-5 symptoms, severe: 6 or more
symptoms (APA 2013, DSM-5).
1. Had times when you ended up drinking more, or longer, than you intended?
2. More than once wanted to cut down or stop drinking, or tried to, but couldn’t?
3. Spent a lot of time drinking? Or being sick or getting over other aftereffects?
4. Wanted a drink so badly you couldn’t think of anything else?
5. Found that drinking, or being sick from drinking, often interfered with taking of your home or
family? Or caused job troubles? Or school problems?
6. Continued to drink even though it was causing trouble with your family or friends?
7. Given up or cut back on activities that were important or interesting to you, or gave you
pleasure, in order to drink?
8. More than once gotten into situations while or after drinking that increased your chances of
getting hurt (such as driving, swimming, using mach inery, walking in a dangerous area, or
having unsafe sex)?
9. Continued to drink even though it was making you feel depressed or anxious or adding to
another health problem? Or after having had a memory blackout?
10. Had to drink much more than you once did to get the effect you want? Or found that your
usual number of drinks had much less effect than before?
11. Found that, when the effects of alcohol were wearing off, you had withdrawal symptoms,
such as trouble sleeping shakiness, restlessness, nausea, sweating, a racing heart, or a
seizure? Or sensed things that were not there?
14
diseases (i.e., hypertension, cardiac arrhythmia), diabetes, neuropsychiatric disorders (i.e.,
schizophrenia, anxiety, and suicidal behavior), cancer, and fetal alcohol syndrome (FAS). The
use of alcohol heavily and unrestrained has been shown to play a direct or indirect role in nearly
4% of all cancer cases (Bagnardi et al., 2015). Alc ohol is also one of the leading causes of birth
defects with 1 in 100 babies being born with fetal alcohol syndrome (FAS) as a result of the 12%
of women that drank during pregnancy (Centers for D isease Control and Prevention [CDC],
2017). In addition to harm to oneself, alcohol consumption can also lead to harm to others
through criminal acts, automobile accidents, and domestic violence (CDC, 2017; Graham and
Livingston, 2011). In 2015, there were 10,265 deaths related to alcohol-impaired driving
crashes, which account for nearly one-third of all traffic-related deaths in the US for that year
(CDC, 2017). These are some examples that illustrat e the social and economic cost associate
with the heavy use of alcohol. The rapid increase in the prevalence of alcohol abuse and misuse
was highlighted by two recent national epidemiologic surveys on alcohol and related conditions
conducted by the National Institute of Alcohol Abuse and Alcoholism (NIAAA) in 1991-1992 and
2001-2002. The surveys concluded that the prevalence of AUD has increased from 13.8 million
to 17.6 million Americans during the 10-year period (Grant et al., 2004). The incidence of
alcohol abuse was also found to increase in both males and females, and in most ethnic groups
(Grant et al., 2004).
Present treatment strategies for AUD include psychosocial therapies and
pharmacological interventions (Heilig and Elgi, 200 6; Johnson, 2010). The current drugs
approved by the Food and Drug Administration (FDA) are discussed in greater detail below. The
use of medications is intended to help reduce cravings and/or withdrawal symptoms, and to
reduce the probability of relapse during abstinence periods. Psychosocial therapies aim to
educate the patients, provide a continuing support group, and to help patients find an
appropriate rehabilitative environment away from exposure to alcohol. Psychosocial alone may
be sufficient for few patients but the majority still requires the use of pharmacological agents in
15
addition to behavioral therapies (Heilig and Elgi, 2006; Johnson, 2010). However, the
ineffectiveness of currently available medications is illustrated by the fact that more than 90% of
patients relapse at least once after undergoing treatments (NIAAA, 2000). The belief that
current available treatment options are few and only yield modest results at best are reflected by
the fact that as few as 1 in 10 patients that have AUD actually perceive the need to seek
pharmacotherapy treatment (NIAAA, 2000; Edlund et a l., 2012). With the persistence of
uncontrolled heavy drinking and the rising prevalence of alcohol abuse, the rapid development
of effective AUD therapies using more novel approaches represent an important public health
goal.
The Drug Development Hurdle
The entire process of developing a new drug and successfully launching it onto the
pharmacy shelves can take over ten years and cost more than $1 billion (Baines, 2004). This is
due to the complex nature of conventional drug development approach and the multiple tedious
steps involved (Figure 1.1). Each step is equally i mportant and is necessary to ensure the
overall safety and effectiveness of the drug of interest before it gets to patients. Conventional
drug development includes six major stages: disease discovery (i.e., target identification, target
validation), molecule discovery (i.e., identificati on of hits and leads through in silico modeling
and high-throughput screening assay), preclinical testing (toxicity and efficacy studies using
animal models), and the three phases of clinical trials that will evaluate the efficacy and safety of
the drugs in human (Baines, 2004).
16
Following promising results in preclinical testing, which include many extensive efforts
on areas such as understanding drug stability, designing appropriate manufacturing scale-up of
drug for use in large human population, and extensive pharmacological and toxicology studies
in animals, an investigational new drug application (IND) will be submitted to the Food and Drug
Administration (FDA) to request approval for testin g the drug of interest in human. The three
phases of clinical trials are even more exhaustive in time and cost and aimed at testing the
safety, effectiveness, and overall benefit/risk of the drug of the interest in humans. The high
failure rate is attributed to uncertainty in efficacy during animal and/or human studies. Only
about 1 out of 5,000-10,000 compounds screened actually becomes a successful drug (Figure
1.1) and only 22 new molecular entities (NME) were approved by the FDA in 2016 (Baines,
2004; Moore, 2003; FDA, 2017). Any availability of existing data (i.e., safety data for the same
compound tested for a different indication in other clinical trials) that can help to eliminate or
Figure 1.1. The drug development process. The entire process from initial discovery to the
availability for treating patients can take over 10 years and cost more than $1 billion (PhRMA,
2015).
17
reduce any of these steps will greatly reduce the cost and increase the efficiency of the drug
development process.
Current FDA-ApprovedMedications for Alcohol Use Disorder
In the last two decades, considerable resources have been put forth into development of
AUD pharmacotherapies. However, currently there are only three FDA-approved medications
(Table 1.2) for the treatment and/or prevention of AUD (Johnson et al., 2007). These
medications act to deter further consumption by blocking the alcohol metabolism pathway or by
targeting neurotransmitter receptor systems that are involved in the modulation of craving
and/or dependence (Gewiss et al., 1991; Johnson et al., 2007; Steensland et al., 2007).
Unfortunately, as presented above, none of the current pharmacotherapies has been particular
successful. In fact, at the present time only 1 out of 10 patients seeing a clinician for AUD is
prescribed a medication.
Disulfiram (Antabuse) blocks the enzyme acetaldehyd e dehydrogenase and prevent the
breakdown of acetaldehyde, a metabolite of alcohol. This usually leads to an increase in the
concentration of acetaldehyde, eventually resulting in severe nausea, vomiting, and potentially
death if the patient is unable to resist further alcohol consumption (Heilig and Egli, 2006). A 52-
week multisite randomized controlled trial in alcohol dependence men found that disulfiram is
effective at preventing relapse in compliant patients, but not successful at promoting long-term
abstinence. Several other studies were also in agreement with this finding, that disulfiram will
only be effective when given under supervision or to compliant patients that are highly motivated
to curb their addiction (Berglund et al., 2003; Hei lig and Egli, 2006; Hughes and Cook, 1997).
18
Naltrexone (oral - Revia; injectable - Vivitrol) b locks opioid receptors, and consequently
results in decreased release of endogenous opioids such as endorphins and encephalins which
will diminish the release of dopamine in the nucleus accumbens (NAc) (Heilig and Egli, 2006;
Herz, 1997). As such, it is suggested that naltrexone acts by reducing the craving and the
positive reinforcing effects of alcohol. In a randomized controlled 12-week study, naltrexone
administered daily was effective at reducing average alcohol intake per week and also at
decreasing the rate of relapse as compared to placebo group (Volpicelli et al., 1992). Despite
this initial strong evidence, the main issue for long-term usage of naltrexone comes from
patients discontinuing the drug because of certain side effects such as severe nausea and
hepatotoxicity (Oncken et al., 2001). Other studies have also found that naltrexone was not
effective in long-term studies that were more than 12 months (Volpicelli et al., 1992).
Additionally, preliminary evidences have shown that it appears only a small subpopulation of
alcoholics is more responsive to naltrexone therapy, which complicates the patient selection
process and the clinical application of this drug ( Johnson, 2008).
Acamprosate acts as a partial agonist to ionotropic glutamate gated channel, N-methyl-
D-aspartate receptor (NMDAR) (Johnson, 2010; Swift, 1999). Acute exposure to alcohol causes
Drug Pharmacologic Activity
Disulfiram Alter alcohol metabolism.
Naltrexone Opioid receptor antagonist.
Acamprosate Glutamate receptor modulator.
Topiramate Glutamate and GABA
A
receptor modulator.
Gabapentin GABA transaminase inhibitor.
Baclofen GABA
B
receptor agonist.
Ondansetron Serotonin receptor modulation.
Table 1.2. Medications used to treat AUD. The first three are FDA-approved medications
while the remaining are used as off-label therapies.
19
a decrease in glutaminergic activity through inhibitions on glutamate receptors such as NMDAR
and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) (Lovinger et al.,
1998). However, prolonged alcohol exposure causes compensatory upregulation of glutamate
expression and increased activity in these receptors in order to restore homeostasis within the
brain. The discontinuation of drinking leads to elevated concentration of glutamate and
hyperexcitability in these receptors, which results in severe withdrawal symptoms such as
insomnia, nightmares, agitation, irritability, delirium, and hallucination (Vengeliene et al., 2008;
Tsai et al., 1998). Acamprosate is thought to normalize this dysregulation within glutaminergic
signaling and reduce the withdrawal symptoms during the abstinent period to prevent relapse
(Mason and Heyser, 2010). The approval of acamprosa te in the US is mainly based on
European studies where it successfully maintained abstinence for over six months as compared
to placebo group (Mann et al., 2004). However, tria ls in US have failed to demonstrate the
therapeutic efficacy of acamprosate except in a small subpopulation of alcoholics who were
highly motivated to control their addiction (Mason et al., 2006).
Off-Label Medications for Alcohol Use Disorder
Since all of the current FDA-approved therapeutic agents for the treatment of AUD only
produce limited results, clinicians have also utilized certain off-label medications (e.g.,
topiramate, gabapentin, baclofen, and ondansentron) to manage this condition. Topiramate has
two main mechanisms that may contribute to its anti-alcohol effect, by facilitating GABA
A
receptors (GABA
A
Rs) mediated inhibitory impulses and at the same time antagonizing the
AMPAR and NMDAR (Heilig and Egli, 2006). Together, the combined effect leads to
suppression of alcohol-induced NAc dopamine release and thereby reduce the reinforcing
effects associated with alcohol consumption. Clinical studies have shown that topiramate,
administered daily, can be effective at decreasing craving when compared to placebo in a 12-
week study (Johnson et al., 2003; Ma et al., 2006).
20
Gabapentin inhibits the activity of GABA transaminase and leads to an increased
availability of GABA, resulting in enhanced GABAergic-mediated inhibitory current (Johnson,
2005). Several small-scale studies have reported the effectiveness of gabapentin to attenuate
alcohol withdrawal symptoms such as tremors, sweating, anxiety, and high pulse rate, and also
a delay in relapse to heavy drinking up to six weeks after treatment was also observed (Bozikas
et al., 2002; Brower et al., 2008).
Baclofen is thought to be able to reduce alcohol intake by inhibiting mesolimbic
dopamine neurotransmission through the activation of GABA
B
receptors (GABA
B
Rs) (Colombo
et al., 2000). Several studies have shown that baclofen can prolong abstinence in alcohol-
dependent subjects by suppressing craving, anxiety, and other symptoms associated with
alcohol withdrawal (Addolorato et al., 2000).
Ondansentron is a 5-hydroxytryptamine (5-HT3) rece ptor antagonist which can reduce
level of mesolimbic dopamine (Lovinger, 1999). Thus , ondansetron can potentially reduce
consumption of alcohol by inhibiting the positive reinforcing effects of alcohol. Various clinical
studies (6 to 12 weeks) have shown significant impr oved drinking outcomes in patients with
early onset of alcoholism, suggesting that ondansetron is likely most efficacious with only
certain types of alcoholics (Sellers et al., 1994).
These aforementioned compounds have shown some positive signals in small-scale
trials and are already being used off-label to treat certain cases of AUD. Large trials with more
subjects and longer durations are still required to fully understand entire efficacy of these
candidates. The list of available medications for AUD, FDA-approved and off-label, still pales in
comparison to other disease with more established drug development programs (i.e., cancer,
diabetes). Further, these medications only show limited results, even when combined with
psychosocial support, as reflected by the rising prevalence of alcoholism (Grant et al., 2004;
NIAAA, 2000). The paucity of efficacious AUD pharmacotherapies is highlighted by the urgent
21
call by NIAAA to employ more novel approaches for the rapid development of effective
medications to treat this debilitating disease.
P2X4 Receptors, an Emerging Critical Target for Ethanol Action
Ethanol is known to act on multiple central nervous system ( ) targets, such as
transporters, neurotransmitter receptors, and ion channels to exert its physiological and
behavioral effects (Wallace and Newton, 2012). Liga nd-gated ion channels (LGICs) are widely
thought to play an important role in ethanol behavior. Research on the relationship between
LGICs and ethanol have focused on two large superfamilies: 1) nicotinic acetylcholine receptor
(nAChR) from the superfamily of cys-loop receptors that also include 5-HT
3
receptors (5-
HT
3
Rs), GABA
A
Rs, and glycine receptors (Betz, 1990; Ortells and Lunt, 1995); 2) glutamate
superfamily with members that include NMDARs and AMPARs (Monaghan et al., 1989;
Sommer and Seeburg, 1992). During the last two decades, P2X receptors (P2XRs) got
substantial attention as the third superfamily of receptors as as novel targets for AUD drug
development (Burnstock, 2008; Litten et al., 2012).
P2XRs are non-selective fast acting cation permeable channels that are gated by
adenosine triphosphate (ATP). Seven P2XRs subunits (P2X
1
-P2X
7
) have been identified to
date, and these subunits can assemble to form homomeric and heteromeric receptors (Khakh,
2001). A trimeric assembly is required for a functional P2XR (Figure 1.2; Buell et al., 1995).
22
In particular, P2X4 receptors (P2X4Rs) have receive d the most attention in relation to
ethanol intake due to strong building evidences that have implicated a critical role for this
receptor and ethanol behavior (Asatryan et al., 201 4; Wyatt et al., 2014; Yardley et al., 2013).
P2X4Rs are widely distributed throughout the brain, but more importantly, P2X4Rs are present
in regions that are considered important neural substrates for ethanol (i.e., hippocampus,
amygdala, cerebellum, NAc, and ventral tegmental area [VTA]). Within these regions, P2X4Rs
are able to modulate the activity of different neurotransmitter receptor systems (i.e., GABA,
glutamate, dopamine) that have been linked to ethanol behavior (Amadio et al., 2007; Soto et
al., 1996). To date, P2X4Rs have been identified as the most ethanol-sensitive of all the P2XRs.
In vitro studies, using slice physiology and Xenopus oocyte recombinant system, showed that
very low concentration of ethanol (5 mM) can inhibi t the ATP-activated currents in these
receptors (Davies et al., 2005; Xiong et al., 2005) . Genomic studies in different strains of rats
and mice have found an inverse relationship between the expression of p2rx4 gene and ethanol
intake/preference (Kimpel et al., 2007; Tabakoff et al., 2009). In support of this, others have also
shown that the reduction of p2rx4 genes in specific brain regions (i.e., NAc and VTA) can alter
Figure 1.2. Crystal structure of zebrafish P2X4R. Each subunit consists of
two transmembrane domains, a large extracellular domain, and intracellular
amino-(N)- and carboxy-(C)-termini (Kawate et al., 2009).
23
ethanol intake (Franklin et al., 2015; Khoja et al. , 2017). Collectively, the combined evidence
from molecular, to electrophysiological and behavioral studies support the contention that
P2X4Rs play an important role in the regulation of ethanol consumption. Therefore, P2X4Rs
can be used as targets for development of drugs to confer ethanol action.
Avermectins, P2X4 Receptors, and Ethanol
Ivermectin (IVM), a semi-synthetic dihydro derivat ive of macrocyclic lactone from the
avermectin family, was discovered in the late 1970s from soil actinomycete, Streptomyces
avermitilis. With significant work by the Merck parasitological team, who was in the quest for
novel antimicrobial agents at the time, IVM was introduced to the market for use in humans and
animals as an anti-parasitic agent in the 1980s (Cr ump and Omura, 2011; Geary, 2005). As an
anti-parasitic agent, IVM acts as an irreversible agonist on non-mammalian glutamate chloride
channel leading to greater influx of chloride ions. This results in hyperpolarization of the
glutamate channel, paralysis of the muscles, and eventually death of these parasites
(Wolstenholme and Rodgers, 2005). IVM has been safe ly used worldwide for over 25 years. It
has contributed to the successful eradication of Onchocerciasis in tropical regions where these
parasites were endemics (Crump and Omura, 2011; Gea ry, 2005). In rare instances, mild to
moderate CNS adverse events have been reported which are thought to be linked to the
alteration of P-glycoprotein (P-gp) function (Edwar ds, 2003; Geyer et al., 2009). These few
reports of adverse advents have not deterred the use of IVM as illustrated by over 69 million
individuals up till 2006 that were successful treated with this drug (Omura, 2008).
IVM can also act on several mammalian ion channels that include include nAChRs,
GABA
A
Rs, glycine receptors, and P2X4Rs (Wolstenholme and Rodgers, 2005).
Electrophysiological investigations have found that IVM can act as a positive allosteric
modulator of P2X4R function whereas other P2X members are not sensitive to IVM, thus
allowing the use IVM as a tool to differentiate the activity of P2X4Rs from other ATP-gated P2X
subtypes (Silberrberg et al., 2007).
24
IVM has been suggested to act on several sites near the transmembrane domain
interface of P2X4Rs (Figure 1.3), some of which hav e also been reported as important for
ethanol binding and activity (Met 336 and Asp 331 o n the TM2 segment) (Jelinkova et al., 2008;
Silberberg et al., 2007). This observation led us to hypothesize that IVM can potentially interfere
with ethanol’s action on P2X4Rs. Our electrophysiological studies demonstrated that when co-
applied with ethanol, IVM can significantly antagonize the ethanol inhibitory effects on P2X4R
function in a concentration-dependent manner (Asatr yan et al., 2010). We further performed
mutational studies to substitute the two identified amino acids with others that have different
properties which resulted in significant changes in sensitivity of P2X4Rs to IVM and ethanol
(Popova et al, 2013). We used these mutational resu lts and the first published crystal structure
of P2X4R in zebrafish (Figure 1.2) to construct a m olecular model of this receptor which also
revealed a putative ethanol-binding pocket that appears to be important for IVM action (Figure
1.3; Kawate et al., 2009; Asatryan et al., 2010; Popova et al., 2013). These results continue to
support the hypothesis that the binding pocket of IVM and alcohol overlap and that there is
interaction of the 2 drugs at those regions of P2X4R.
Figure 1.3. Homology model of rat P2X4R. This was constructed by threading the rat primary
sequence on the zebrafish X-ray structure in the open conformation (Asatryan et al., 2010).
25
These aforementioned findings, coupled with the widespread use of IVM in humans with
minimal reported adverse events or safety concerns set the stage for investigations to
repurpose IVM as a new pharmacological therapy for the treatment and/or prevention of AUD.
In support of this hypothesis, building evidence from the Davies group and others have
demonstrated that IVM significantly reduces ethanol intake in rodents using multiple drinking
paradigms (Asatryan et al., 2014; Franklin et al., 2015; Yardley et al., 2012; Yardley et al.,
2014). Long-term administration of IVM did not lead to any overt toxicity or histopathological
development in major organs (Yardley et al., 2015). Notably, the doses of IVM that significantly
reduce drinking also did not exert rewarding properties indicating that the psychotropic effects of
IVM are dissociated from any addiction liability that can affect therapeutic compliance (Bortolato
et al., 2013). Results from this body of evidence ( e.g. using IVM) and other avermectins (e.g.,
abamectin and selamectin) support the overall hypothesis that avermectins represent a novel
class of molecules that have the potential to be developed as novel pharmacotherapies for
AUD. Importantly, from a drug discovery standpoint, the evidence links the ability of the
avermectins to significantly reduce ethanol intake in mice to their ability to antagonize the
inhibitory effects of ethanol on P2X4R function in vitro (e.g., Figure 1.4 ; Asatryan et al., 2014,
Huynh et al., 2017). This observation, linking in vitro-in vivo findings implicates the use of
P2X4R as a screening platform for the development of novel anti-alcohol therapeutics. Notably,
this finding is a breakthrough as it would be challenging (both in time and use of animals) to test
every avermectin as a new AUD pharmacotherapy.
26
Repurposing Potential of Moxidectin into a Novel Alcohol Therapeutic
As presented above, the evidence supports the use of IVM as an effective agent for
reducing ethanol intake in mice. However, the application of IVM as a long-term AUD
pharmacotherapy presents some safety challenges (pr esented below). To this end, my work, as
presented in this body of literature began to test the hypothesis that moxidectin (MOX, an IVM
analogue) represents a lead molecule that has comparable (anti-alcohol) efficacy to IVM as well
as possessing a more advantageous safety profile. The reason I elected to focus on MOX is
due to a number of recent reports in the literature that suggest MOX exhibits lower neurotoxicity
potential as compared to IVM. This more advantageous CNS safety profile is thought to be
linked to: 1) MOX having lower potency on GABA
A
Rs as compared to IVM, and 2) the
differential transport across the blood brain barrier, with MOX being a weaker P-gp substrate
and less dependent on P-gp for removal from the brain (Janko and Geyer, 2013; Menez et al.,
2012). As such, the potential long-term use of MOX as a chronic AUD therapy is less likely to
have complications from excessive stimulation of GABA
A
Rs that can lead to CNS depression,
and also less likely to result in brain accumulation due to a deficiency in P-gp function or drug-
drug interaction. MOX is already in clinical development as an alternative therapy to IVM as an
Figure 1.4. Avermectins/P2X4R in vitro-in vivo correlation. (Left) Avermectins eliminated the ethanol
inhibitions of P2X4R function using the two-electrode voltage-clamp Xenopus oocyte system, values
represent mean ± SEM, n = 5-10 oocytes in each group, * p < 0.05 versus ethanol. (Right) Administratio n of
avermectins caused reduction in 10% ethanol (v/v) i ntake in mice using a 24-h-two-bottle choice paradigm,
mean ± SEM, n = 7-10 in each group, ** p < 0.01 versus saline-injected control. (Asatryan et al., 2014 ).
27
anti-parasitic agent for humans (Cotreau et al., 20 03; Korth-Bradley et al., 2012). To date, no
significant clinical abnormalities have been reported in any these studies. With MOX possessing
superior CNS safety profile as compared to IVM and its potential for becoming approved for
human use for a different indication, MOX may represent a lead candidate that can be rapidly
repurposed into a novel AUD pharmacotherapy.
Dissertation Hypothesis and Outline
The rapid development of more effective AUD therapies represents an important public
health goal. As already discussed, the process of conventional drug development continues to
be challenging as it is very lengthy and costly, and shadowed with a high degree of uncertainty
of whether the drug candidate will actually succeed (Baines, 2004). Using a translational
approach, which is the hallmark of drug repurposing, the Davies laboratory including my efforts
continue to investigate the potential of avermectins to be repositioned into novel AUD
medications.
The focus of my dissertation work has been on MOX due to the building evidence
suggesting superior CNS safety profile for MOX as compared to other avermectins that we have
previously investigated in our drinking studies (Ja nko and Geyer, 2013; Menez et al., 2012). As
mentioned previously, MOX is already in clinical development for a different indication with very
tolerable side effects reported to date (Cotreau et al., 2003; Korth-Bradley et al., 2012). If
supported by solid preclinical results, MOX can be rapidly translated into the clinics for the
treatment and/or prevention of alcoholism. The experiments outlined within mydissertation,
using several different rodent drinking paradigms, tested the hypothesis that MOX exhibit
efficacy in reducing ethanol intake and ethanol-related withdrawal symptoms in mice, and has
the potential to be translated into a novel AUD medication. Chapter 2 tested the efficacy of
acute and multi-day administrations of MOX in relation to ethanol intake, using the 24-h-two-
bottle choice (social drinking) and the drinking-in -the dark (DID, binge drinking) paradigm
(Belknap et al., 1993; Lowery et al., 2010; Rhodes et al., 2005; Yoneyama et al., 2008). These
28
two drinking models have been well-characterized and represent the early-staged of alcoholism
in humans. Testing the efficacy and safety of MOX in a multi-day long-term drinking study is
critical since AUD medications will likely need to be given on a chronic basis. The use of
multiple rodent drinking models is also important since no single model can represent the entire
spectrum of alcoholism. Chapter 3 tested the efficacy of MOX to attenuate acute ethanol
withdrawal symptoms that have been induced with a single injection (intraperitoneally) of
sedative concentration of ethanol (4 g/kg). The sev erity of withdrawal symptoms was scored
using the handling-induced convulsion (HIC) method. These procedures (administering a high
dose of ethanol + HIC scoring) have been routinely used to study ethanol withdrawal
mechanisms and for drug testing in mice (Martinez e t al., 2016; Finn and Crabbe, 1999).
Chapter 3 also tested the ability of MOX to reduce ethanol intake in mice that have been
rendered ethanol-dependent using an intermittent access two-bottle choice (Hwa et al., 2013).
This model has been shown to induce mice to voluntarily escalate their drinking to significantly
higher level (from established baseline level) afte r four weeks of ethanol exposure (i.e., 10
mg/kg → up to 20 mg/kg in male, 15 mg/kg → up to 30 mg/kg in female C57BL/6J mice), which
is a cardinal feature of the development of human alcohol-dependency. As the majority of
avermectins’ anti-alcohol effect comes mainly from their activity on P2X4Rs, the studies in
chapter 4 and 5 were aimed at elucidating the role of P2X4Rs in the regulation of ethanol
behavior. Chapter 4 investigated the function and the importance of the neuroanatomical
position of P2X4Rs on ethanol intake by utilizing lentiviral-shRNA mediated knockdown of
P2X4Rs in the NAc. We chose to focus on this region first as numerous literatures have
implicated its role in drug-seeking behavior (Koob and Volkow, 2010). Chapter 5 utilized
microarray technology to investigate the differentially expressed genes in P2X4R knockout mice
versus wildtype littermates under ethanol exposure to identify potential signaling mechanism(s)
in relation to ethanol intake. Our genomic finding was further validated using pharmacological
and electrophysiological methods. Chapter 6 presents a summary of my dissertation
29
investigations and discusses future investigations and new directions. Taken together, the entire
data presented in my dissertation demonstrates the efficacy and tolerable features of MOX and
also reveals a possible molecular explanation of how P2X4Rs is regulating drinking behavior.
These positive results implicate the repurposing potential of MOX to be developed into a novel
pharmacotherapy for the treatment and/or prevention of AUD. Moreover, the work continues to
support the development of novel therapeutics that target P2X4Rs.
30
Chapter 2
Preclinical Development of Moxidectin as a Novel Therapeutic for Alcohol Use Disorder
Abstract
Current pharmacotherapies for alcohol used disorder (AUD) are few and relatively
ineffective illustrating the need for the development of new, effective medications. Using a
translational approach, our laboratory reported that ivermectin, an FDA-approved, human and
animal anti-parasitic agent, can significantly reduce ethanol intake in male and female mice
across different drinking paradigms. Extending this line of investigation, the current paper
investigated the utility of moxidectin (MOX), an an alogue of ivermectin, to reduce ethanol intake.
Notably, MOX is widely held to have lower neurotoxicity potential and improved margin of safety
compared to ivermectin. Using a 24-h-two-bottle choice paradigm, MOX significantly reduced
ethanol intake in a dose dependent manner in both male and female C57BL/6J mice,
respectively (1.25 - 7.5 mg/kg) and (1.25 - 10 mg/k g). Further, multi-day administration of MOX
(2.5 mg/kg; intraperitoneal injection) for 5 consec utive days significantly reduced ethanol intake
in both the 24-h-two-bottle choice and Drinking-in-the-Dark paradigms in female mice. No overt
signs of behavioral toxicity were observed. Notably in both male and female mice, MOX
significantly reduced ethanol intake starting approximately 4 h post-injection. Using a Xenopus
oocyte expression system, we found that MOX significantly potentiated P2X4 receptors
(P2X4R) function and antagonized the inhibitory eff ects of ethanol on ATP-gated currents in
P2X4Rs. This latter finding represents the first report of MOX having activity on P2X4Rs. In
addition, MOX potentiated GABA
A
receptors, but to a lesser degree as compared to ivermectin
supporting the hypothesis that MOX would be advantageous (compared to ivermectin) with
respect to reducing contraindications. Overall, the results illustrate the potential for development
of MOX as a novel pharmacotherapy for the treatment of AUD.
31
Introduction
Alcohol use disorder (AUD) has major health implic ations in the United States, affecting
over 17 million people, causing more than 100,000 deaths and costing over $200 billion
annually (Bouchery et al., 2011; Grant et al., 2004 ; Hardwood, 2000). Despite an ongoing effort
focusing on the development of new medications for AUD, there are only three-FDA approved
pharmacotherapies available (disulfiram, naltrexone , and acamprosate), all which have yielded
limited success (Harris et al., 2010; Litten et al. , 2012). This is evident by the continual
prevalence of high rates of uncontrolled heavy drinking and high relapse rate in patients even
after long-term inpatient treatment and support (Su bstance Abuse and Mental Health Services
Administration, National Survey on Drug Use and Health, 2013). As such, the development of
effective medications to treat AUD is an important public health goal (Bouchery et al., 2011;
Heilig and Egli, 2006; Johnson et al., 2007; Johnson, 2010; Steensland et al., 2007).
Our laboratory has been investigating the utility of different compounds from the
avermectin family of macrocyclic lactones (eg., ive rmectin, abamectin, selamectin) to be
developed into novel pharmacotherapies for AUD. This class of compounds is already
recognized for their ability to act on several CNS receptor targets (eg., GABA
A
receptors
[GABA
A
Rs], glycine receptors, and nicotinic acetylcholine receptors) in mammals, all of which
have been linked to the behavioral effects of ethanol (Dawson et al., 2000; Shan et al., 2001;
Spinosa et al., 2002). Using a combination of electrophysiology methods and rodent drinking
models, we observed that the ability of these avermectins to reduce ethanol intake in mice
appeared to be related to their ability to significantly reduce or eliminate the inhibitory effects of
ethanol on ATP-gated P2X4 receptor (P2X4R) function in vitro (Asatryan et al., 2014). This
initial in vivo-in vitro correlation implicates the use of P2X4R as a screening platform for the
development of avermectins and other related analogues into novel AUD therapeutics.
Since ivermectin is already an FDA-approved drug that has been safely used in humans
for several decades for the treatment of parasites (Guzzo et al. 2002; Omura, 2008), we have
32
been investigating the feasibility of repurposing ivermectin into a novel pharmacotherapy for
AUD. In support of this hypothesis, we demonstrated that ivermectin can significantly
antagonize the inhibitory effects of ethanol on P2X4R function in vitro (Asatryan et al., 2008,
2010, 2014) and significantly reduce ethanol intake in multiple drinking paradigms in both male
and female mice (Asatryan et al., 2014; Yardley et al., 2012, 2014, 2015; Wyatt et al., 2014).
Notably, doses of IVM as high as 10 mg/kg did not appear to cause any overt signs of organ
toxicity or exert rewarding properties indicating that the psychotropic effects of IVM are
dissociated from any addiction liability that can affect therapeutic compliance (Bortolato et al.,
2013; Yardley et al., 2015).
Although we have established that ivermectin is an effective agent for reducing ethanol
intake in mice, recently we have begun shifting our attention to moxidectin (MOX; an ivermectin
analogue) due to a number of recent reports in the literature that suggests MOX exhibits lower
neurotoxicity potential compared to ivermectin (Jan ko et al., 2012; Menez et al., 2012). This
more favorable central nervous system (CNS) safety profile is thought to be due to: 1) MOX
having lower potency on GABAARs as compared to ivermectin which should be advantageous
with respect to reducing contraindications, and 2) the differential transport across the blood
brain barrier (BBB), with MOX being a weaker P-glyc oprotein (P-gp) transporter substrate and
less dependence on P-gp for removal from the brain (Janko et al., 2012; Menez et al., 2012). As
such, the potential chronic use of MOX as a long-term AUD therapy is less likely to have
complications from excessive stimulation of GABA
A
Rs that can lead to CNS depression and
potentially coma, and also less likely to result in brain accumulation due to a deficiency in P-gp
function or drug-drug interaction with other concurrent medications that may also act as P-gp
substrates (Balayssac et al., 2005; Edwards et al., 2003; Prichard et al., 2012;). Notably, MOX
is now being developed as an alternative therapy to ivermectin as an anti-parasitic agent for
humans. To date, no significant clinical abnormalities have been reported (Cotreau at al., 2003;
33
Korth-Bradley et al., 2012). With MOX becoming approved for human use, it could represent
another avermectin candidate that could be repurposed as a pharmacotherapy for AUD.
To begin to determine if MOX has the potential to be developed into a safe and effective
pharmacotherapy for AUD, the present paper investigates the ability of MOX to reduce ethanol
intake in male and female mice using a 24-h-two-bottle choice paradigm. The lower limit of
alcohol consumed in this model suggests that it mimics social drinking (Blednov et al., 2010).
We also utilized a Drinking-in-the-Dark (DID) parad igm where the amount of alcohol consumed
is much higher in a short period of time (compared to the 24 h model) and results in blood
ethanol concentrations (BECs) similar to human bing e-like drinking (i.e., > 80 mg%) (Lowery et
al., 2010; Neasta et al., 2010; Rhodes et al., 2005). Both of these models are well-validated and
routinely used to assess changes in ethanol intake in rodents.
To begin to investigate the mechanism of MOX’s anti-alcohol effects, we used a two-
electrode voltage clamp, Xenopus oocyte expression system (Davies et al., 2002; Davies et al.,
2005) to test the ability of MOX to potentiate P2X4R function and to antagonize the inhibitory
effects of ethanol on P2X4Rs as previously demonstrated for ivermectin and abamectin
(Asatryan et al., 2014). We also compared the pote ntiating effects of MOX versus ivermectin on
GABA
A
Rs. Overall, the findings support the development of MOX as a pharmacotherapy for the
treatment of AUD.
Materials and Methods
Drugs. MOX (10 mg/kg) solution (Boehringer Ingelheim, St . Joseph, MO) was diluted in
a 0.9% sodium chloride solution (saline) to a conce ntration that would allow for an injection
volume of 0.01 mL/g of body weight. We used saline as the diluent based on previous pilot work
where we found that propylene glycol [(1,2 propaned iol), (Alfa Aesar, Ward Hill, MA)], the
solvent used by the manufacturer to dissolve MOX (w hen diluted in saline to equivalent
concentrations as in the MOX [10mg/kg] solution) showed comparable drinking level as saline
injection alone, and both the solvent and saline did not cause significant reduction in ethanol
34
intake. 190 proof USP ethanol (Koptec, King of Prus sia, PA) was diluted in water to achieve a
10% (10E) v/v solution or a 20% (20E) v/v solution (20E).
Animals. Studies were performed on drug-naïve male and female C57BL/6J mice
purchased from Jackson Laboratory (Bar Harbor, ME). All studies were conducted with mice 10-
12 weeks of age. Mice were first acclimated to the housing facility for one week and group-
housed (4 mice per cage) in polycarbonate/polysulfo ne cages at a 12 h light/dark cycle (lights
off at 12:00PM) with ad libitum access to food and water in rooms maintained at 22˚C. All
procedures in this study were performed in accordance with the NIH Guide for the Care and
Use of Laboratory Animals and all efforts were made to minimize animal suffering. The USC
Institutional Animal Care and Use Committee approved the protocols.
24-h-two-bottle choice paradigm. All experiments numbering noted in the methods
section corresponds to the figures numbering in the results section for the ease of reading (i.e.,
Experiment 2.1A corresponds to Figure 2.1A).
For our initial investigations, we used a 24-h-two-bottle choice paradigm that is widely
used to assess changes in drinking behaviors (Belkn ap et al., 1993; Yoneyama et al., 2008;
Yardley et al., 2012) with modifications as presented previously (Yardley et al., 2012). Mice
were individually housed 3 days before the start of the study and had 24 h access to two
inverted water bottles (graduated cylinders) with m etal sippers placed on the cage tops. Food
was distributed near both bottles to avoid food associated tube preferences.
Experiment 2.1A: A within-subjects design was used with one group of female mice (n =
12) that were allowed free access to two bottles, one containing 10E and the other water. 10E
position was alternated every other day during testing period to avoid for side preference. Body
weights and food intake (weight of food on day 1 su btract weight of food on day 2 = food
consumed for the 24 h period) were monitored daily during testing period. Fluid intake was
recorded (fluid level on inverted cylinder on day 2 subtract fluid level on day 1 = fluid consumed
for the 24 h period) daily during testing period. Daily observations on normal overt behaviors of
35
mice were also noted (normal: constant active movem ent, responsive to experimenter
intervention, and fur groomed; abnormal: lack of movement, hunched postured and hurdling in
corner, unresponsive to experimenter intervention, and piloerection of fur). 10E position was
also alternated every other day to avoid for side preference. Daily 10E intake was measured
until it stabilized (± 10% variability from the mea n of the last 3 days). Next mice were habituated
to saline injections (intraperitoneal; i. p.) until 10E intake again stabilized. Mice then received
one dose of MOX (5 mg/kg). Animals were injected wi th saline on the day following MOX
injection. Change in drinking over the 24 h following each injection (saline or MOX) was
measured. Pre-MOX is the day prior to MOX injection, and post-MOX is the day following MOX
injection.
Experiment 2.1B followed similar procedures as described in experiment 1a. A new
group of female mice (n = 16) were allowed free acc ess to 10E and water. After saline
habituation and obtaining stable baseline 10E intake, all mice received one injection (i. p.) of
MOX (0.65, 1.25, 2.5, 5, 7.5, or 10 mg/kg). Animals were injected with saline on subsequent
days after MOX injection until 10E drinking returned to baseline levels (which took about 1-2
days), and then mice were injected with another dose of MOX. This pattern of MOX
administration continued until all doses of a particular study were completed. The doses were
given in random fashion with no particular order.
Experiment 2.1C followed similar procedures as described in experiment 1b, except
male mice (n = 12) were used in this study and the doses of MOX tested were 1.25, 2.5, 5, and
7.5 mg/kg
Experiment 2.2: We conducted an hour by hour evaluation using the two-bottle choice
paradigm and a between-subjects design. A new group of female mice was habituated to saline
injection. After establishing baseline drinking level, mice were split into two groups based on
average 10E intake and subsequently received either one injection (i. p.) of MOX (2.5 mg/kg) or
36
saline (controls) (n = 18 MOX versus 10 saline cont rols). Following drug injection, 10E intake
was monitored for the two groups every hour up to the 9th h.
Experiment 2.3: We conducted a multi-day MOX study using the two-bottle choice
paradigm and a between-subjects design. All the female mice from experiment 2.2 received
saline injection during the washout period until 10E intake returned to baseline, then received
either daily injection (i. p.) of MOX (2.5 mg/kg) o r saline (controls) for 5 consecutive days in the
multi-day MOX study (n = 18 MOX versus 10 saline co ntrols). 10E intake was monitored for 24
h period after each injection for 5 consecutive days.
Drinking-in-the-Dark (DID). Experiment 2.4: We conducted a multi-day MOX study
using the DID paradigm and a between-subjects design. This model is widely used to assess
changes in binge-like drinking behaviors. A modified version of this procedure (Lowery et al.,
2010; Neasta et al., 2010; Rhodes et al., 2005) was utilized during which mice had daily limited
access (2 h) to one bottle containing 20E beginning at the third hour into the circadian dark
phase, which results in mice reaching high BECs in a short period of time. A new group of
female mice was used and following habituation to saline injection and establishing baseline
drinking level, mice were split into two groups based on average 20E intake and subsequently
received either one injection (i. p.) of MOX (2.5 m g/kg) or saline daily for 5 days (n = 18 MOX
versus 10 saline controls). MOX or saline was administered 4 h prior to the start of the drinking
session based on our time-course study for MOX which indicates that it took 4 h post-injection
before significant reduction in ethanol intake was observed (Figure 2). 20E intake was
monitored during the 2 h drinking session for 5 consecutive days. A single bottle of water was
continuously available between ethanol access periods.
Complementary RNA (cRNA) and complementary DNA (cDNA) injections in
Xenopus laevis oocyte. Stage V or VI Xenopus oocytes (purchased from Eco Cyte Bioscience,
Austin, TX) were injected with 20 ng cRNA of rat p2rx4 gene or 1 ng cDNA for GABAAR
(α1:β2:(2:, 1:1:10) as described previously (Asatry an et al., 2010, 2014) using the Nanoject II
37
Nanoliter injection system (Drummond Scientific, Br oomall, PA). Injected oocytes were stored at
16°C in incubation medium containing 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5
mM HEPES, and 2.5 mM pyruvic acid with 1% heat inactivated HyClone® horse serum (VWR,
San Dimas, CA) and 0.05 mg/ml gentamycin, adjusted to pH 7.5. Electrophysiological
experiments were conducted 24 – 48 h after cRNA injections.
Whole Cell Two-Electrode Voltage Clamp Recordings were performed using the OC-
725C oocyte clamp (Warner Instruments, Hamden, CT) as previously described elsewhere
(Davies et al., 2002, 2005). The oocytes were volta ge clamped at −70 mV and the currents
were recorded on a strip-chart recorder (Barnstead Instrument, Dubuqe, IA).
Experiment 2.5: P2X4R oocytes were continuously perfused at a rate of 3 - 4 mL/min at
room temperature with modified Ringers buffer containing (in mM) 110 NaCl, 2.5 KCl, 10
HEPES and 1.8 BaCl2, pH 7.5, using a peristaltic pump (Rainin Instrument, Oakland, CA). To
induce currents, submaximal concentrations (EC
10
) of adenosine 5’-triphosphate (ATP, Sigma,
St. Louis, MO) were used, which produced 10% of maximal effect induced by 100 µM ATP. We
have shown previously that the use of EC
10
can maximize the effects of ethanol and minimize
receptor desensitization (Asatryan et al., 2010; Da vies et al., 2002, 2005). Ethanol and/or drugs
were applied after stable response to ATP EC
10
was obtained. A washout period of 5 min was
allowed between each application to allow for re-sensitization of the receptor (Asatryan et al.,
2010; Davies et al., 2002, 2005; Popova et al., 2013). Effects of ethanol (25 or 50 mM) or MOX
([0.5 and 1 μM], powder dissolved in in DMSO to 10m M stock solution, then dissolved to
appropriate concentration in perfusion buffer) were tested alone and in combination during co-
application with ATP. Pilot studies determined that the effects of ethanol and MOX on P2X4R
were concentration-dependent and reversible (n = 4 - 6 oocytes).
Experiment 2.6: Effects of MOX (0.1, 0.5, and 1 µM ) or ivermectin (0.1, 0.5, and 1 µM)
on GABAARs were tested using GABA EC10 (Asatryan et al., 2014). Oocytes were perfused at
a rate of 3 - 4 mL/min at room temperature with modified Bart’s saline containing (in mM) 83
38
NaCl, 1 KCl, 10 HEPES, 0.82 MgSO4, 2.4 NaHCO3, 0.91 CaCl2, and 0.33 Ca(NO3)2, pH 7.5.
Drugs were applied after stable response to GABA EC10 was obtained. A washout period of 5
min was allowed between each GABA application. Due to irreversible and non-washable effects
on GABA-induced currents, each oocyte was tested for one concentration of MOX or ivermectin
(n = 4 - 12 oocytes per data point).
Statistical Analyses. Ethanol intake for all studies was calculated as g/kg [g of pure
ethanol per kg of body weight; 10E intake = (volume of 10E consumed in mL x 0.07893 g/mL) /
body weight in kg; 20E intake = (volume of 20E cons umed in mL x 0.15786 g/mL) / body weight
in kg]. The dependent variables include ethanol intake (g/kg), water (mL), food intake (g), and
change in mouse weight (g). Experiment 2.1a: one-wa y ANOVA was used to assess the
treatment effect of MOX with time [saline pre-treatment (pre-MOX), MOX dose, saline post-
treatment (post-MOX)] as a repeated measures factor on the dependent variable. Experiment
2.1bc: two-way ANOVA was used to assess the treatment effect of MOX with each MOX dose
(0.65 – 10 mg/kg) or time [saline pre-treatment (pr e-MOX), MOX dose, saline post-treatment
(post-MOX)] as a repeated measures factor on the de pendent variable. Experiment 2.2: two-
way ANOVA was used to assess the treatment effect of MOX between groups (MOX versus
saline-injected) or with time (for each hour post-i njection) as a repeated-measures factor on the
dependent variables. Experiment 2.3-2.4: two-way ANOVA was used to assess the treatment
effect of 5-day MOX administration between groups ( MOX versus saline-injected) or with time
(for each day of MOX administration) as a repeated- measures factor on the dependent
variables. Experiment 2.5-2.6: two-tailed, unpaired, individual Student’s t-test was used to
assess for difference between treatment groups. Significant main effects and interactions of the
ANOVAs were further investigated with post-hoc tests (i.e., Tukey’s for one way ANOVA and
Bonferroni for two-way ANOVA). For all studies significance was set at p ≤ 0.05. All graphs and
statistical analyses were generated using Prism (Gr aphPad Software Inc., San Diego, CA).
39
Results
Acute administration of MOX decreased 10E intake in female mice. Using female
C57BL/6J mice, we tested the effects of acute administration of MOX (5 mg/kg) on ethanol
intake using a 24-h-two-bottle choice paradigm (10E versus water). This dose was previously
shown to be the lowest ivermectin dose that produced maximal reduction in ethanol
consumption (Yardley et al., 2012). Baseline 10E in take was first obtained followed by
habituation to saline injections.
As illustrated in Figure 2.1A, 10E intake stabilized at 14.14 g/kg following saline
injections (pre-MOX). Acute administration of MOX ( 5 mg/kg) significantly reduced ethanol
intake in excess of 44% when analyzed across time ( pre-MOX, MOX, post-MOX) [F(2, 33) =
8.045, p = 0.0014], and 10E intake remained significantly lower than pre-MOX on the day
following MOX treatment (by more than 20%, shown as post-MOX, p < 0.05) (Figure 2.1A).
10E Intake (g/kg)
C.
10E Intake (g/kg)
A.
B.
40
MOX decreased 10E intake in a dose-dependent manner in female mice. We
extended our initial single dose MOX study to one where several doses of MOX were tested in
random fashion (saline, 5, 10, 2.5, 7.5, and 0.65 m g/kg) in order to begin to establish the dose
characteristics of MOX in relation to 10E intake. Two-way ANOVA revealed a significant effect
of MOX administration on ethanol intake when analyzed across time (pre-MOX, MOX, post-
MOX) [F(2, 312) = 84.60, p < 0.001]. The analysis o f the MOX doses (0.65 – 10mg) indicated
that MOX significantly reduced ethanol intake in a dose-dependent manner in female mice
(Figure 2.1B) [F(5, 312) = 4.299, p < 0.001]. The i nteraction between time and dose was
significant [F(10, 312), 5.057, p < 0.001]. Bonferr oni post-hoc comparisons between pre-MOX
and MOX data indicated that 2.5 mg/kg MOX was the lowest dose that caused a maximum
significant reduction in ethanol intake [~44% reduction, (t = 5.467, p < 0.001)]. For the MOX
doses that significantly reduced 10E intake, we found that 10E intake returned to comparable
pre-MOX ethanol intake levels within 1-2 days post-MOX injection (data not shown). The lowest
dose of MOX tested (0.65 mg/kg) did not significant ly reduce 10E intake compared to pre-MOX
level. In addition, following saline habituation, further injections with saline alone did not
significantly affect 10E intake (Figure 2.1B, dose 0.00 mg/kg). MOX administration did have
significant impairment on food intake when analyzed across time (pre-MOX, MOX, post-MOX)
[F(1, 242) = 84.85, p < 0.0001] and doses (0.65 - 1 0 mg/kg) [F(6, 242) = 9.710, p < 0.0001]
(data not shown). The interaction between time and dose was significant [F(6, 242) = 25.33 p <
0.0001]. Bonferroni post-hoc comparisons between pre-MOX and MOX data revealed that only
Figure 2.1. Acute administration of MOX significantly reduced 10E intake in female and male
C57BL/6J mice, in a dose-dependent manner, using a 24-h-two-bottle choice paradigm. MOX was
administered after habituation with saline injection and attaining stable drinking levels. Bars represent
levels from the day prior to MOX injection (white; pre-MOX), the day of MOX injection (black; MOX), an d
the day after MOX injection (gray; post-MOX). (A) M OX (5 mg/kg) significantly reduce 10E intake in
female mice. Values represent mean ± SEM for 12 mice, * p ˂ 0.05, # p ˂ 0.0001 versus pre-MOX,
Tukey multiple comparison post-hoc test. (B) MOX (1 .25, 2.5, 5, 7.5, and 10 mg/kg) significantly reduced
10E intake in female mice. Values represent mean ± SEM for 16 mice per dose group, * p ˂ 0.05, # p ˂
0.0001 versus respective pre-MOX condition, Bonferroni’s post-hoc test. (C) MOX (1.25, 2.5, 5, and 7.5
mg/kg) significantly reduced 10E intake in male mice. Values represent mean ± SEM for 12 mice per
dose group, ** p ˂ 0.01, # p ˂ 0.0001 versus respective pre-MOX condition, Bonferroni’s post-hoc test.
41
higher doses of MOX caused significant impairment on food intake [5 mg/kg (t = 5.918, p <
0.0001), 7.5 mg/kg (t = 8.776, p < 0.0001), 10 mg/k g (t=5.179, p < 0.0001)]. MOX (all doses
tested, pre-MOX compared to MOX) did not cause significant changes in body weight (data not
shown) or showed any signs of abnormal overt behaviors such as lack of movement with
hunched postured and hurdling in corner, unresponsive to experimenter intervention, and
piloerection of fur.
MOX decreased 10E intake in a dose-dependent manner in male mice. Extending
our line of investigation, we also tested the utility of MOX using male C57BL/6 mice. Two-way
ANOVA revealed a significant effect of MOX administration on ethanol intake when analyzed
across time (pre-MOX, MOX, post-MOX) [F(1, 11) = 68 .43, p < 0.0001]. The analysis of the
MOX doses (1.25 - 7.5 mg/kg) indicated that MOX sig nificantly reduced ethanol intake in a
dose-dependent manner in male mice (Figure 2.1C) [F (4, 44) = 4.005, p < 0.01]. The interaction
between time and dose was significant [F(4, 44), 6. 434, p = 0.0004]. Bonferroni post-hoc
comparisons between pre-MOX and MOX data indicated that 2.5 mg/kg MOX was the lowest
dose that caused a maximum significant reduction in ethanol intake [~55% reduction, (t = 6.427,
p < 0.0001)]. For the MOX doses that significantly reduced 10E intake, we found that 10E intake
returned to comparable pre-MOX ethanol intake levels within 1-2 days post-MOX injection (data
not shown). MOX administration did have significant impairment on food intake when analyzed
across time (pre-MOX, MOX, post-MOX) [F(1, 11) = 24 .75, p < 0.001] and doses (1.25 - 7.5
mg/kg) [F(4, 44) = 4.448, p < 0.01] (data not shown ). The interaction between time and dose
was significant [F(4, 44) = 4.700 p < 0.01]. Bonfer roni post-hoc comparisons between pre-MOX
and MOX data revealed that only higher doses of MOX caused significant impairment on food
intake [5 mg/kg (t = 4.912, p < 0.0001), 7.5 mg/kg (t = 4.701, p < 0.001)]. MOX (all doses tested,
pre-MOX compared to MOX) did not cause significant changes in body weight or showed any
signs of abnormal overt behaviors.
42
Time course of the effect of MOX on ethanol intake in female mice. To gain insight
to the pharmacokinetics of MOX distribution and its impact on ethanol intake, we evaluated the
time course of the effect of MOX on hourly ethanol intake using a 24-h-two-bottle choice
paradigm. Using this paradigm, we previously reported that the onset of significantly activity for
ivermectin was approximately 9 h post-administration (Figure 2.2 inset) (Yardley et al., 2012).
After stable 10E intake was obtained, either MOX (2 .5 mg/kg, previously determined as the
minimum effective dose) or saline was administered 1 h to female mice before the first reading.
As expected, 10E intake increased significantly across time (hourly) [F(9,190) = 78.52, p <
0.001] and was significantly reduced following MOX treatment [F(1,190) = 36.33, p < 0.001]
(Figure 2.2). The interaction between time and MOX dose was not significant. We conducted
planned comparison which showed that ethanol intake was significantly decreased starting at 4
h after MOX administration (Figure 2.2).
Multiple day dosing of MOX administration reduced ethanol intake in female mice
using a 24-h-two-bottle choice paradigm. Alcoholism is a chronic disorder. As such, an
effective pharmacotherapy for AUD will mostly need to be taken chronically. To begin to
investigate the utility of MOX for chronic use, we tested the effects of MOX (2.5 mg/kg)
Figure 2.2. MOX (2.5 mg/kg) significantly reduced 10E intake approximately 4 h after
administration in female mice. The intake was measured on an hourly basis, up to the 9th h
following MOX (square) or saline (circle) administr ation. The inset was reproduced from Figure 3
in Yardley et al., 2012, which shows ivermectin began to significantly reduced 10E intake
approximately 9 h after administration. Values represent mean ± SEM cumulative intake for MOX
(18 mice) and saline (10 mice), IVM (11 mice) and s aline (10 mice), * p ˂ 0.05, ** p < 0.01, # p ˂
0.0001 versus saline-treated group, Bonferroni’s post-hoc test. group, ** p ˂ 0.01, # p ˂ 0.0001
versus respective pre-MOX condition, Bonferroni’s post-hoc test.
43
administered for 5 consecutive days in female mice using a 24-h-two-bottle choice paradigm.
On the day prior to drug/vehicle treatment, there was no significant difference in average 10E
intake for the MOX (12.26 g/kg) or control (12.68 g /kg) groups. We tested the effect of MOX
versus saline, administered daily, on 10E intake over a 5-day period (Figure 2.3A). When
analyzed across the 5-day treatment period, we found that MOX significantly reduced ethanol
intake compared to the saline-injected control group [F(1,24) = 26.35, p < 0.001]. There was
also a significant effect on 10E intake when analyzed across time (day) [F(4, 96) = 8.730, p <
0.001]. The interaction between treatment and time was not significant.
MOX treatment did not significantly change either water or food intake between the two
groups when analyzed across 5-day treatment period and time (day) (Figure 2.3B - 2.3C). There
was a significant effect of MOX treatment [F(1, 24) = 24.4, p < 0.0001] and time (day) [F(4, 96)
= 5.951, p < 0.001] on change in body weight with no significant interaction between treatment
and time (Figure 2.3d).
A.
B.
C.
D.
44
Multiple day dosing of MOX administration reduced ethanol intake in female mice
using a drinking-in-the-dark paradigm. We extended our multiple dosing of MOX
administration using a modified version of the DID paradigm in female mice (Lowery et al.,
2010; Neasta et al., 2010; Rhodes et al., 2005). One day prior to treatment, there was no
significant difference in average 20E intake for the MOX (2.91 g/kg) and control (3.02 g/kg)
groups. We tested the effect of MOX (2.5 mg/kg) ver sus saline, administered daily, 4 h prior to
the start of the drinking sessions on 20E intake over a 5-day period (Figure 2.4a). MOX
administration consistently reduced 20E intake across the testing period, and the reduction was
significant when analyzed across time (day) [F(3,27 ) = 8.862, p < 0.001] but not across the 5-
day treatment period. There was no interaction between treatment and time.
MOX treatment did not cause any significant changes in food intake between the two
groups (Figure 2.4b) when analyzed across the 5-day treatment period and time (day). We did
observe a significant effect of MOX treatment on body weight [F(1, 24) = 12.01, p < 0.001) but
no significant effect of time (day) on change in bo dy weight (Figure 2.4c), and there was no
interaction between treatment and time.
Figure 2.3. Daily administration of MOX (2.5 mg/kg x 5 days) significantly
reduced 10E intake in female C57BL/6J mice using a 24-h-two-bottle choice
paradigm. Following habituation with saline injection and attaining stable drinking
levels, MOX was administered for 5 consecutive days. Squares represent MOX
and circles represent saline. (A) MOX (2.5 mg/kg) s ignificantly reduced 10E
intake across the 5 treatment days. The effects of water intake, food intake, and
weight are presented in panels, (B), (C), and (D), respectively. Values represent
mean ± SEM cumulative intake for MOX (18 mice) and saline (10 mice), ** p ˂
0.01, *** p ˂ 0.001, # p < 0.0001 versus saline-treated group, Bonferroni’s post-
hoc test.
45
MOX positively modulated ATP-gated P2X4Rs and antagonized the inhibitory
effects of ethanol on P2X4R function. We investigated the effects of 0.5 μM and 1 μM MOX
alone, and in combination with behaviorally relevant concentrations of ethanol (25 and 50 mM)
on ATP-induced currents in P2X4Rs in vitro. In agreement with previous studies (Asatryan et
al., 2010, 2014; Davies et al., 2005, 2010; Popova et al., 2010), ethanol (25 and 50 mM)
significantly inhibited ATP-gated P2X4R currents (F igure 2.5A – 2.5B). In the presence of ATP,
MOX (0.5 and 1 μM) produced a comparable degree of potentiation of P2X4R activity (Figure 5a
- 5b). When tested in the presence of ethanol, 0.5 μM MOX significantly reduced the inhibitory
effect caused by 25 mM (p = 0.003) but not 50 mM et hanol on ATP-gated P2X4R activated
currents (Figure 5a). In contrast, 1 μM MOX elimina ted the inhibitory effect of ethanol at both
concentrations, 25 (p = 0.0028) and 50 mM (p = 0.02 9) (Figure 4b).
Figure 2.4. Daily administration of MOX (2.5 mg/kg x 5 days) reduced 20E intake
in female C57BL/6J mice using a drinking-in-the-dark (DID) paradigm. Following
habituation with saline injection and attaining stable drinking levels, MOX was
administered for 5 consecutive days. Squares represent MOX and circles represent
saline. (A) MOX (2.5 mg/kg) consistently reduced 20 E intake across the 5 treatment
days. The effects of food intake and weight are presented in panels, (B) and (C),
respectively. Values represent mean ± SEM cumulative intake for MOX (18 mice) and
saline (10 mice).
A.
B.
C.
46
MOX positively modulated GABA
A
R activity. We also tested the effects of MOX on
GABA
A
Rs since we, and others have previously reported that ivermectin and other related
avermectins have significant GABAergic activity (As atryan et al., 2014; Janko et al., 2013;
Menez et al., 2012). We used α1β2γ2 GABA
A
Rs due to their predominant expression in the
CNS in mammals (Sigel and Steinmann, 2012). As illu strated in Figure 2.6, we found that MOX
and ivermectin each significantly increased GABAAR function. There was no significant
difference between the extent of potentiation of GABAAR function between MOX and ivermectin
at 0.1 µM. However, the degree of MOX potentiation was significantly less (compared to
ivermectin) when tested at higher concentrations [0.5 µM (p = 0.0072 and 1 µM (p = 0.00092)].
Figure 2.5. MOX (0.5 and 1 µM) antagonized the inhibitory effects of ethanol in P2X4Rs.
Exposure to (A) 0.5 µM MOX and (B) 1 µM MOX potenti ates P2X4Rs and significantly
eliminated the inhibitory effect of 25 and 50 mM ethanol on EC5 ATP-gated currents in
P2X4Rs. Values represent mean ± SEM for 4 to 6 oocytes per data point, * p ˂ 0.05, ** p <
0.01 versus respective control, two-tailed unpaired Student’s t-test.
A.
B.
ATP-Induced Currents,
% Modulation
ATP-Induced Currents,
% Modulation
47
Discussion
The present study was the first investigation to test the utility of MOX as a
pharmacotherapy for AUD. Several key findings came from this work. Testing the hypothesis
that administration of MOX can reduce ethanol intake in male and female mice, we found that
acute and repeated administration of MOX significantly reduced ethanol intake using two well-
established drinking paradigms. Using a Xenopus oocyte expression system, we found that
MOX significantly reduced the inhibitory effects of ethanol on P2X4Rs suggesting that at least a
portion of MOX’s anti-ethanol effects may be linked to activity on P2X4Rs. Using a 5-day MOX
administration treatment regimen with a 24-h-two-bottle choice paradigm, we found that the
reduction in ethanol intake was significant and remained consistent over the 5-day period (with
an average reduction of approximately 40% compared to saline-injected controls). Importantly,
no significant decrease in food consumption or weight loss, or other abnormalities in overt
behavior (i.e., lack of movement and constant hurdl ing in corner, unresponsive to experimenter
intervention, and piloerection of fur) were noted. These findings are well aligned with our
previous work demonstrating that chronic administration of ivermectin was efficacious in
reducing ethanol intake and well tolerated (Yardley et al., 2012, 2014, 2015). Finally, MOX also
consistently reduced ethanol intake when tested using a 5-day DID paradigm (average
reduction approximately 30%).
Figure 2.6. MOX (0.1, 0.5, and 1 µM) has a weaker modulatory activity in GABA ARs than
ivermectin. At low 0.1 µM concentration, MOX potentiated GABAARs to similar extent as
ivermectin. At 0.5 µM and above, the effect of MOX was significantly smaller compared to that
of ivermectin. Values represent mean ± SEM for 4 to 12 oocytes per data point, ** p ˂ 0.01, ***
p < 0.001 versus respective control, two-tailed unpaired Student’s t-test.
48
Several reports have suggested that different rodent drinking models involve overlapping
and distinct neurobiological mechanisms (Crabbe et al., 2011; McBride and Li, 1998). MOX has
been purported to act several different neurotransmitter targets that can regulate ethanol
behavior through multiple mechanisms (Menez et al., 2012, Wolstenholme and Rogers, 2005;
Yamaguchi et al., 2012). Thus, it is possible that a higher dose of MOX may exert a different
degree of activity on a single or combination of these targets leading to more pronounced
reduction in ethanol intake using the DID paradigm. Collectively, these results support the
development of MOX as a novel pharmacotherapy for the treatment of AUD and illustrate the
potential for P2X4Rs as a novel target for AUD drug development.
The fact that the administration of MOX as illustrated in our multi-day studies did not
result in any consistent, significant effect on water or food intake suggests the drug is well
tolerated. As shown, we did find that MOX did cause some reduction in weight and the change
remained stable over the 5-day period. We do not know the reason for this change, but it could
be due to a loss of caloric intake linked to the decreased ethanol consumption that was caused
by MOX. Notably, all animals appeared healthy and remained alive at the end of the study,
suggesting that MOX does not have any unexpected interactions with ethanol that could lead to
lethality or negative changes in behavior.
In the current investigation, we found that the reduction in ethanol intake reached
significance approximately 4 h after MOX administration. This represents a significant
improvement in onset of effect as compared to the 9 h window necessary for initial onset of
ivermectin activity (Yardley et al., 2012). This fi nding suggests that MOX can reach effective
therapeutic (anti-alcohol effect) brain concentrati ons more rapidly versus ivermectin and is
consistent with previous findings suggesting that MOX has improved BBB penetration compared
to ivermectin (Prichard et al., 2012, Kiki-Mvouaka et al., 2010).
The faster onset to the initial reduction of ethanol intake may be explained by the
differences in structural features and physiochemical properties of ivermectin and MOX.
49
Ivermectin and MOX share a common macrocyclic lactone ring and are distinguishable by
specific substituents at the C13, C23, and C25 position (Figure 2.7). These substituents play a
role in influencing the lipophilicity and affinity for P-gp transporters. The higher lipophilicity of
MOX compared to ivermectin (log PMOX = 6, Pivermect in = 4.8) is consistent with its higher
entrance into the brain, greater accumulation in adipose tissue, and longer retention time in the
organism (Baoliang et al., 2006; Lanusse et al., 19 97; Prichard et al., 2012). In addition, MOX
has been shown to have a weaker affinity for P-gp compared to ivermectin and other
avermectins based on structural and biochemical studies. The disaccharide sugar moiety found
on ivermectin is absent on MOX. This moiety is thought to govern the affinity for P-gp (Lespine
et al., 2007).
Interestingly, despite reaching higher brain levels, MOX is predicted to have a better
neurotoxicity profile when compared to ivermectin, which is thought to be due to 1) the
differential transport across the blood brain barrier (BBB), with MOX being less dependent on P-
gp for removal from the brain and less likely to accumulate due to a deficiency in P-gp function
or drug-drug interaction arising from concurrent medications that may also act as P-gp
C13
C23
C25
C13
C23
C25
Figure 2.7. Structures of (Left) ivermectin and (Right) MOX. Major structural differences are noted. C13:
ivermectin contains a disaccharide while MOX is protonated; C23: MOX has a methoxime; C25: ivermectin
is a mixture of C25-ethyl (~10%) or C25-methyl (~90 %) groups while MOX has a dimethyl-butyl substituent.
50
substrates (Kiki-Mvouaka et al., 2010; Menez et al. , 2012), and 2) the differential interaction with
GABAARs, with MOX exhibiting lower activity at these receptors (Janko et al., 2012; Menez et
al., 2012). In agreement with the latter, our results also indicated that the degree of MOX
potentiation of GABA
A
Rs was significantly less as compared to ivermectin. At higher
concentrations, we found that ivermectin continued to increase the degree of GABAARs activity
whereas the effects of MOX quickly reached a plateau. This result, coupled with the
aforementioned benefits of MOX (weak P-gp target; b etter BBB), suggests that chronic use of
MOX as a long-term treatment for AUD should be more favorable as compared to ivermectin
because there should be less complications arising from potential brain accumulation and/or
over-stimulation of GABAARs that can lead to CNS depression and potentially coma. (Prichard
et al., 2012; Balayssac et al., 2005).
Importantly, MOX is currently undergoing clinical development as an alternative to
ivermectin for treating the parasite Onchocerca volvulus, and to date, no significant clinical
abnormalities or serious adverse events have been reported over for these investigations
(Cotreau et al., 2003; Korth-Bradley et al., 2012). The faster onset time, robust efficacy and
favorable safety profile continue to support the development of MOX as a novel
pharmacotherapy for the treatment of AUD.
In addition to our in vivo results, as reported above, we found that MOX positively
modulated ATP-gated currents in P2X4Rs and antagonized the inhibitory effects of ethanol on
P2X4R function. This is the first evidence demonstrating that a compound from the milbermycin
subfamily of macrocyclic lactones can act on P2X4Rs in vitro. This finding is in good agreement
with our earlier investigations where we demonstrated that both ivermectin and abamectin
significantly antagonized the inhibitory effects of ethanol on P2X4R function (Asatryan et al.,
2014).
In addition to activity on P2X4Rs, we (in this stu dy) and others report that MOX can also
act on other brain targets including GABA
A
Rs, glycine, and nAChRs (Menez et al., 2012,
51
Wolstenholme et al., 2005; Yamaguchi et al., 2012). Notably, all of these receptors have been
linked to the modulation of mesolimbic dopamine activity and regulation of ethanol behavior
(Davies, 2003; Xiao et al., 2008). Thus, it is like ly that the reduction of ethanol intake, by MOX,
as presented in the current investigation reflects a cumulative effect of MOX activity on several
different classes of receptors including P2X4Rs. Additional studies are necessary before definite
conclusions can be drawn.
In that this was the first investigation focusing on MOX, several limitations should be
noted. First, the majority of the results presented here were conducted using female mice and
we did not monitor for the effects of estrous cycle, which could potentially confound the
interpretation of the results. However, in our dose-response study we used both male and
female mice (Figures 1b - 1c). As presented, there was a significant reduction of ethanol intake
by MOX in both male and female mice and the degree of reduction was similar for both groups.
In addition, we also utilized a saline-treated control group in both of the 5-day investigations
using female mice (24-h-bottle-bottle-choice and DI D studies), it is unlikely that the differences
in 10E and 20E intake between the MOX and saline group could be attributed to the estrous
cycle. This conclusion is similar to previous work where no systematic changes in ethanol intake
across weeks of baseline consumption in female C57BL/6 mice were observed (Ford et al.,
2008). Collectively, these results suggests that the estrous cycle is unlikely to have an impact
on the anti-alcohol effects of MOX and further supports the utility of MOX as a pharmacotherapy
for AUD in both male and female. In future investigations, we plan to continue evaluating the
effects of MOX administration on ethanol intake in both male and female mice using long-term
drinking paradigms.
Second, we did not test other tastants such as sucrose or quinine in this study.
Previously, we reported that ivermectin produced a significant reduction in 24 h saccharin
consumption, but did not significantly alter operant sucrose self-administration (Yardley et al.,
2012). Based on this finding, it would not be unreasonable to predict that MOX may also act on
52
other tastants, but this should not reduce the utility of MOX for its anti-alcohol effects. Third, in
the DID investigation, we did not measure BECs. As such, we cannot definitively conclude that
the BEC levels achieved in our DID study reached binge-like drinking levels. However, the
levels of ethanol intake achieved in our study were similar to levels of ethanol intake reported by
others where BECs were recorded (Wilcox et al., 201 4; Rhodes et al., 2005).
The present findings support the development of MOX as a novel pharmacotherapy for
the treatment of AUD. We presented solid evidence showing that both acute and repeated
administration of MOX can reduce ethanol intake across different models of self-administered
drinking paradigms. Importantly, we observed no signs of overt toxicity and all animals remained
alive at the end of the study. Of note, the safety and initial efficacy of co-administration of a
single 30-mg dose of ivermectin and intravenous alcohol infusion in alcoholic patients was
recently tested. In this study, ivermectin (30 mg) was found to be safe and well tolerated where
the number and severity of reported adverse effects were low and did not differ from the
placebo session (Roche et al., 2016). Although iver mectin did not differ from placebo in regards
to reducing alcohol cue-induced craving or basal alcohol craving, the study represented an
important first step in developing this class of molecules as pharmacotherapies for AUD. In that
MOX is currently being developed for use in humans as an alternative for ivermectin as an anti-
parasitic coupled with the better pharmacokinetics and margin of safety (as compared to
ivermectin) once fully approved for human use MOX should have the potential to be repurposed
and rapidly advanced to the clinic for the treatment and/or prevention of AUD.
53
Chapter 3
Moxidectin Attenuates Acute Ethanol-Induced Withdrawal Symptoms in Mice
Abstract
Alcohol use disorder (AUD) is a spectrum disorder t hat is characterized by the excessive
craving and inability to stop drinking. Individuals with more severe AUD can also experience
withdrawal symptoms (i.e., tremor, delirium, seizur e) upon cessation of drinking, which further
reinforce the drinking behavior. Although current medications used to treat ethanol withdrawal
symptoms are numerous, they also carry significant liabilities that include high abuse potential
and major toxicities (i.e., respiratory depression, coma) when used concurrently with ethanol or
other substance of abuse. To address this issue of rapidly discovering more efficacious and
safe medications that can reduce drinking and manage ethanol withdrawal, I have been
investigating the potential of moxidectin (MOX) to be developed into a novel therapeutic for
AUD. Our previous works with ivermectin (IVM, an an alog of MOX) and related compounds
have identified purinergic P2X4 receptors (P2X4Rs) as an emerging novel target for AUD drug
development and that certain avermectins (e.g., IVM , MOX) can significantly reduce ethanol
intake in male and female mice through the modulation of P2X4R function. In that MOX is
already in clinical trial for another indication with excellent central nervous safety profile and no
significant clinical abnormalities reported to date, strong efficacy results from my preclinical
studies can set the stage for the rapid translation MOX into clinic to combat alcoholism. In
support of this goal, I have initially shown that MOX can significantly reduce ethanol intake in
both male and female mice using a 24-h-two-bottle choice (social drinking) and drinking-in-the-
dark (binge drinking) paradigms. These two drinking models are well-validated and routinely use
in early-staged AUD drug testing, and represent the early stages of human alcoholism.
Medications that can effectively reduce ethanol consumption in this population of patients can
also prevent the progression to full-blown AUD. The experiments outlined in this chapter will
54
employ another well-characterized mouse model of ethanol withdrawal (single injection of
sedative-hypnotic dose of ethanol [4 g/kg]) to further test if MOX can reduce the severity of
acute ethanol withdrawal symptoms. To this end, my results demonstrated that the
administration (intraperitoneally) of 5 mg/kg MOX s ignificantly reduced the severity of ethanol
withdrawal in both male and female mice as compared to saline-injected control. This positive
result, in addition to our previous findings, illustrate the potential of MOX to be developed into a
novel AUD pharmacotherapy that can be applied to a broad population of patients suffering from
this debilitating disease.
55
Introduction
Alcohol use disorder (AUD) continues to have a majo r impact in the United States,
affecting over 18 million people and imposing an economic burden of more than $220 billion
annually (Bouchery et al., 2011; Grant et al., 2004 ; Hardwood and Lewin, 2000). Further, each
year approximately 500,000 AUD patients experience clinically relevant episode(s) of
withdrawal, which have a detrimental effect on the function and well-being of these patients, and
continue to reinforce further drinking behavior (NI AAA, 2010). In the last two decades, despite
significant improvement in treatments, ethanol withdrawal remains a significant clinical problem
(NIAAA, 2010). This is partially due to the fact th at many current available medications also
possess major side effects (e.g., high abuse potent ial, respiratory depression, coma) that can
affect compliance and treatment outcome. This highlights the urgent need to employ novel
strategies for the rapid development of safe and effective medications to reduce drinking and
manage ethanol withdrawal symptoms. Using a translational approach, the Davies’ laboratory
has been investigating the potential of compounds from the avermectin family (e.g., ivermectin,
moxidectin [MOX]) to be developed into novel AUD therapies. To date, we have shown that: 1)
certain avermectins can significantly reduce ethanol intake in male and female mice by mainly
acting on purinergic P2X4 receptors (P2X4Rs)
(Asatryan et al., 2010, 2014; Davies et al., 2005;
Wyatt et al., 2014; Yardley et al., 2012, 2014, 2015), 2) the safety and tolerability of chronic
administration of avermectins in our long-term drinking studies where no overt toxicity or
histopathological development in major organs was observed
(Yardley et al., 2015), and 3) the
dosages of avermectins that significantly reduce ethanol intake do not exhibit addictive
properties
(Bortolato et al., 2013).
The current focal point of our investigation has been to understand the full utility of MOX
using multiple rodent drinking paradigms that represent the different stages of alcoholism. The
use of different drinking paradigms are critical for understanding the entire efficacy and
appropriate clinical application of MOX since no single rodent drinking model can represent the
56
entire spectrum of alcoholism. We chose to focus on MOX since several emerging literatures
have suggested that it has superior central nervous system (CNS) safety profile compared to
the other avermectins that we have tested (Janko an d Geyer, 2013), which will make MOX
highly suitable as a long-term pharmacotherapy for AUD. Further, MOX is already in clinical
development for a different indication with excellent CNS safety signals and no significant
clinical abnormalities reported to date
(Cotreau et al., 2003; Korth-Bradley et al., 2012), if
supported by my preclinical data, MOX can be rapidly translated into clinic settings to combat
alcoholism. In support of this hypothesis, I have recently reported that MOX can significantly
reduce ethanol intake in male and female mice using two well-characterized paradigms that
mimics human social drinking and binge-like behavior (Huynh et al., 2017). It is well known that
the development of alcoholism usually begins with the occasional drinks at social gatherings
that can spiral into more frequent episodes of binging, and eventually leading to
dependency/addiction. Thus far, my preclinical results indicate that MOX has the potential to
treat early-staged at risk patients by reducing binge behavior and preventing the progression to
full-blown AUD.
In addition to the cravings that most AUD individuals experience, those with more severe
AUD will also experience withdrawal symptoms when they attempt to reduce or stop consuming
ethanol. These symptoms include, among others, delirium, anxiety, insomnia, hallucinations,
tremors, seizures (Kosten and O’Connor, 2003). The exact pathophysiology of ethanol
withdrawal is still not completely understood; however, one widely accepted explanation is the
increased excitatory glutamatergic and reduced inhibitory GABAergic activity in the CNS that
develop over prolonged period of drinking (Mckeon e t al., 2008). The onset of acute symptoms
have been reported to vary between 2 and 24 hours following cessation of drinking, even when
significant amount of blood ethanol concentration ( BEC) was still present, making ethanol
withdrawal a common reason for emergency department or inpatient clinic visits (Cooper and
Vernon, 2013). Current clinical standard of care use long-acting benzodiazepines for outpatient
57
treatment of mild symptoms (Blondell 2005), or shor t-acting benzodiazepines in combination
with anti-seizure medications (e.g., carbamazepine, valproic acid, phenobarbital) and close
monitoring for inpatient treatment of more severe symptoms (Perry, 2014). Although current
medications are numerous, the most effective ones also possess high abuse potential and
significant risk of toxicities, especially when used concurrently with ethanol. Thus, more effective
and safer medications are needed to address this issue. We have established that the ability of
MOX (and other averrmectins) to significantly reduc e ethanol intake comes from their activity on
P2X4Rs (Yardley et al., 2012, 2014, 2015; Wyatt et al., 2014), where these receptors are
potentially acting to reduce the excitatory glutamatergic tone (detailed mechanism will be
discussed in chapter 5 below). We have also shown that the anti-alcohol doses of these
avermectins do not elicit rewarding properties (Bor tolato et al., 2013). This led us to hypothesize
that in addition to being an effective medication at curbing the urge and cravings to drink, MOX
may also be an effective treatment for the management ethanol withdrawal.
The similarity of ethanol withdrawal signs between humans and rodents have been
demonstrated previously, which led to the use of inbred mouse strains for the development of
animal models of ethanol withdrawal (i.e., injectio n of sedative dose(s) of ethanol, continuous
exposure to high concentration of ethanol vapor) and the severity of symptoms can be reliably
scored using the handling-induced convulsions (HIC) system. These procedures are routinely
used to study the mechanism of ethanol withdrawal and to test novel lead compounds (Buck et
al., 1997; Crabbe 2014; Crabbe et al., 1980; Goldstein, 1973; Goldstein and Pal, 1971; Kozell et
al., 2005). The experiments outlined here will employ these methods to test the hypothesis that
MOX can attenuate ethanol withdrawal symptoms caused by a bolus injection (intraperitoneally,
i.p.) of sedative-hypnotic concentration of ethanol. Given that sex is an important determination
in many ethanol actions, we used both male and female mice in the withdrawal study.
Materials & Methods
58
Drugs. MOX (10 mg/kg) solution (Boehringer Ingelheim, St . Joseph, MO) was diluted in
a 0.9% sodium chloride solution (saline) to a conce ntration that would allow for an injection
volume of 0.01 mL/g of body weight. We used saline as the diluent based on previous pilot work
where we found that propylene glycol [(1,2 propaned iol), (Alfa Aesar, Ward Hill, MA)], the
solvent used by the manufacturer to dissolve MOX (w hen diluted in saline to equivalent
concentrations as in the MOX [10mg/kg] solution) showed comparable drinking level as saline
injection alone, and both the solvent and saline did not cause significant reduction in ethanol
intake. 190 proof USP ethanol (Koptec, King of Prus sia, PA) was diluted in water to achieve a
3% (3E) v/v solution, 6% (6E) v/v solution, 10% (10 E) v/v solution, or a 20% (20E) v/v solution
(20E).
Animals. Studies were performed on drug-naïve male and female C57BL/6J mice
purchased from Jackson Laboratory (Bar Harbor, ME). All studies were conducted with mice 10-
12 weeks of age. Mice were first acclimated to the housing facility for one week and group-
housed (4 mice per cage) in polycarbonate/polysulfo ne cages at a 12 h light/dark cycle (lights
off at 12:00PM) with ad libitum access to food and water in rooms maintained at 22˚C. All
procedures in this study were performed in accordance with the NIH Guide for the Care and
Use of Laboratory Animals and all efforts were made to minimize animal suffering. The USC
Institutional Animal Care and Use Committee approved the protocols.
Assessment of ethanol withdrawal symptoms by the handling-induced
convulsions (HIC) method. HIC scores were measured prior to concurrent administration (i.p.)
of 4 g/kg ethanol (20E) and 5 mg/kg MOX, and every 2 hours thereafter for 14 hours total. Both
male (n = 10 MOX, n = 10 saline control) and female (n = 10 MOX, n = 10 saline control) mice
were tested. The scoring system was similar to that previously described (Finn and Crabbe,
1999). Briefly, mice were picked up from their home cage by the tail, observed for ~1 second,
and then gently spun 180° bidirectionally, or in other words, a full 360°. Mouse's withdrawal
symptoms were scored using the following scale: 0 - no withdrawal signs, 1 - tonic convulsion
59
after 360° spin, 2 - tonic-clonic convulsion after 360° spin, 3 – tonic convulsion after tail lift, 4 -
tonic-clonic convulsion after tail lift. The scores were tabulated and then averaged for each time
point. AUC was calculated by adding the scores across the 14-hour time course.
Intermittent-access (IA) two-bottle choice paradigm. Adapted to Hwa et al., 2011.
On week 1 of the experiment, on Monday, Wednesday, and Friday, female mice (n = 10-11)
were given 3E, 6E, and 10E in one bottle and water in a second bottle. Each drinking session
lasted 24 hours. Week 2-5, female mice received one bottle of 20E solution and one bottle of
water every Monday, Wednesday, and Friday for 24 hours. During week 5, mice also were
administered (i.p.) 5 mg/kg MOX at the start of eac h drinking session. Amount of consumption
for each drinking session (over the 24 hour) was me asured.
Blood ethanol concentration (BEC) analysis. Male (n = 2) and female (n = 2) mice
were concurrently injected (i.p.) with a single dos e of 5 mg/kg MOX and 4 g/kg ethanol (20E).
Submandibular blood samples (50 µL) were taken at t ime point (30, 60, 120, and 180 minutes)
after injection. BECs (mg/dL) were determined by t he Analox Alcohol Analyzer (Analox
Instruments, Luneburg, MA).
Statistical Analysis. HIC scores for each sex were analyzed with two-way ANOVA to
determine the effect of treatment between groups (M OX versus saline-injected control) with time
(every 2 hour post injection of MOX + ethanol) as a repeated measure on the dependent
variables. Ethanol intake was calculated in g/kg, average intakes for week 4 (non-treated) and
week 5 (MOX-treated) were compared with two-tailed, unpaired, t-test to determine the effect of
MOX treatment on drinking. BECs for each sex were analyzed by two-way ANOVA to determine
the effect of MOX treatment (MOX versus saline-inje cted control) on the pharmacokinetic of
ethanol with time (at 30, 60, 120, 180 minutes). Significant main effects and interactions of
ANOVAs were further investigated with post-hoc tests. For all studies, significance was set at p
≤ 0.05. All graphs and statistical analyses were generated using Prism (GraphPad Software
Inc., San Diego, CA).
60
HIC Score
HIC AUC
0
1
2
3
4
5
MOX + Ethanol
Saline + Ethanol
Male
*
Results
Administration of MOX significantly reduced the severity of acute ethanol-induced
withdrawal symptoms in male and female C57BL/6J mice. Using a mouse model of ethanol
withdrawal (induced by a single injection (i.p.) of sedative concentration of ethanol [4 g/kg]), we
tested the ability of acute administration (i.p.) o f 5 mg/kg MOX to reduce withdrawal symptoms
in both male and female C57BL/6J mice. We chose to use the dose of 5 mg/kg because this
dose is within the minimum effective dose range (2. 5 - 5 mg/kg) in our previously tested drinking
studies (Huynh et al, 2017). MOX administered at th e same time with ethanol reduced peak
magnitude (Figure 3.1A) and significantly reduced t he overall severity (shown as total area
under the curve [AUC]) of withdrawal related convulsions in male mice (Figure 3.1B, t = 2.541, p
= 0.0235). HIC scores also changed significantly across time (F [7, 126] = 21.88, p < 0.000)
with no significant interaction between treatment and time. Female mice treated with 5 mg/kg
MOX showed similar results, with reduced peak magnitude (Figure 3.1C) and significantly
reduced overall severity of withdrawal symptoms (Fi gure 3.1D, t = 2.770, p = 0.0150). Female
HIC scores also changed significantly across time ( F [7, 126] = 18.65, p < 0.0001) with no
significant interaction between treatment and time. The onset of withdrawal symptoms, defined
as an increase above baseline, was similar in both MOX-treated and saline-injected control for
each sex; however male mice did exhibit earlier onset than female mice (Figure 3.1AC, 6
th
hour
versus 8
th
hour).
A.
B.
Male
61
Week of Ethanol Access
Ethanol Intake (g/kg/24h)
0
10
20
30
40
1 2 3 4
3% 6%10% 20%
***
5
5 mg/k MOX
Administration of MOX significantly reduced 20E intake in ethanol-dependent
female C57BL/6J mice. In addition to ethanol-related withdrawal symptoms, individuals with
severe AUD may also experience excessive cravings which further contribute to the inability to
stop drinking. We also tested the ability of MOX to reduce drinking in mice that have been
C. D.
Figure 3.1. MOX significantly attenuates ethanol-induced withdrawal symptoms in male and
female C57BL/6J mice. (A-B) 5 mg/kg MOX (i.p.) reduced peak magnitude an d overall severity of
withdrawal in male mice. (C-D) Similar results were observed in female mice. Values represent mean
± SEM for MOX (n = 10) and saline (n =10 mice) for each sex, * p ˂ 0.05 versus saline-treated group.
Figure 3.2. MOX significantly reduced 20E intake in ethanol-dependence female C57BL/6J mice
Female mice were rendered ethanol-dependent using the intermittent access two-bottle-choice model,
which induced the animals to gradually escalate their drinking level to a daily stable high of 26.9 mg/kg
20E intake (compared to ~25 mg/kg established daily value). Administration (i.p.) of 5 mg/kg MOX
during week 5 significantly reduced ethanol intake across the 3-day testing period.
Female
62
rendered ethanol-dependent and highly addictive, using the intermittent access (IA) two-bottle
choice model. This drinking paradigm has been shown to be able to induce mice to voluntarily
escalate their drinking to a significantly higher daily intake level as compared to established
baseline (Hwa et al., 2011; 10 mg/kg → up to 20 mg/ kg for male and 15 mg/kg → 30 mg/kg for
female C57BL/6J mice with exposure to 10E-20E ethanol solution). This voluntarily escalation in
drinking is a cardinal feature in human ethanol dependency and mice undergoing this paradigm
has been shown to exhibit similar neuroadaptations and neurochemistry changes that closely
matched the development of human alcoholism (Griffi n, 2014; Jesse et al., 2016). As shown in
Figure 3.2, female mice gradually elevated their daily drinking level to a stable high of 26.6
mg/kg 20E by week 3 of the experiment. The administration (i.p.) of 5 mg/kg MOX was able to
significantly reduced ethanol intake in these ethanol-dependence female mice consistently over
the three testing days in week 5 (Figure 3.2, avera ge week 4 versus week 5, t = 4.470, p =
0.0002).
MOX did not affect the pharmacokinetic of ethanol in male and female C57BL/6J
mice. We have previously shown that the anti-alcohol effect of MOX is by mainly by modulating
P2X4R function in the central nervous system (Wyatt et al., 2014; Khoja et al., 2017). Here we
further showed that MOX anti-alcohol activity is not related to any effects on the
pharmacokinetic profile and elimination of ethanol. Analysis of blood ethanol concentrations
(BECs) in male and female C57BL/6J mice following s ingle-dose injection (i.p.) of 4 g/kg ethanol
concurrently with 5 mg/kg MOX revealed no effect of MOX on BEC levels (Figure 3.3A-B). Two-
way ANOVA revealed no significant effect of treatment, and no interaction between treatment
and time.
63
Discussion
One of the main reason people relapse following treatment is the combination of
excessive cravings and the severe side effects from ethanol-related withdrawal symptoms.
Although treatments for ethanol withdrawal are numerous, they mostly carry high risks of liability
that include toxicity when used concurrently with ethanol and the high potential of abuse.
Developing safer and more efficacious AUD medication remains an important public health goal.
The experiments described in this chapter address this issue by testing the hypothesis that
MOX can reduce drinking and withdrawal symptoms in severe cases of AUD. We have
previously shown that MOX can significantly reduce ethanol intake using a 24-h-two-bottle
choice and DID paradigms, without exhibiting addictive properties, which makes MOX highly
suited as a long-term pharmacotherapy for AUD.
Our result here further demonstrated that the administration (i.p.) of 5 mg/kg MOX
significantly reduced acute ethanol-induced withdrawal symptoms across the 14-hour testing
Figure 3.3. MOX did not affect the pharmacokinetic profile of ethanol. (A) Concurrent administration
(i.p.) of 5 mg/kg with 4 g/kg ethanol (20E) in male C57BL/6J mice did not have any effect on the elimination
of ethanol. (B) Female C57BL/6J mice showed simila r result. Values represent mean ± SEM for MOX (n =
2) and saline (n =2) at each time point for both se xes.
Male Female A. B.
64
period, and overall severity (total AUC) was also s ignificantly lower in MOX-treated group as
compared saline-injected control (Figure 3.1). The ability of MOX to attenuate ethanol-induced
withdrawal was similar in both male and female mice. While the exact mechanism of how MOX
can attenuate ethanol withdrawal symptoms is still not clearly understood, our ongoing
investigations indicated that the potentiation of P2X4Rs by MOX may also indirectly lead to
inhibition of NMDAR function (will be discussed in chapter 5 below). This is in line with current
evidences implicating the role of NMDARs in treating ethanol withdrawal symptoms. Ethanol is a
CNS depressant that inhibits NMDARs and potentiates GABA
A
Rs. Prolonged drinking will lead
to the development of tolerance and physical dependence, which will result in compensatory
function changes by the downregulation of GABA
A
Rs and increased expression of NMDARs
with the production of more glutamate to maintain CNS homeostasis. Abrupt cessation of
chronic alcohol consumption unmasks these changes leading to a glutamatergic dominant
environment resulting in CNS overactivity and neuropsychiatric complications such as delirium
and seizures that exhibit behavioral phenotype of tonic/tonic-clonic seizures. Pharmacological
agents that can restore homeostasis in the glutamatergic system, either by restoring synaptic
glutamate concentration or by regulating receptor expression/activity can also potentially reduce
the severity of ethanol withdrawal symptoms and/or urges to continue drinking (Griffin, 2014;
Jesse et al., 2016).
We also use the IA two-bottle access model that alternates access and deprivation in
female mice to render them ethanol-dependent. Female mice were induced to voluntarily
escalate their drinking to a significantly higher daily consumption level (as compared to their
established baseline value). This voluntarily significant increase in drinking is a hallmark feature
of human alcoholism. Several studies have shown that increased in drinking in ethanol-
dependence mice models, such as the IA paradigm employed here, is likely driven by
neuroadaptations in the glutamatergic system. The dysregulation of these systems parallels
findings in human alcoholics, where magnetic resonance spectroscopy studies have shown
65
elevated glutamate activity in the NAc of human alcoholics (Griffin et al., 2014). Other reports
from animal studies have confirmed that there is increased glutamatergic transmission in the
nucleus accumbens (NAc) of ethanol-dependence mice and pharmacologically increasing the
glutamate concentration further increased consumption (Griffin et al., 2014). Our results
indicated that MOX has the potential to effectively reduce drinking in individuals with severe
AUD.
Identification of new treatments for ethanol withdrawal is an important public health goal.
The current treatments are numerous but are ridden with major limitations. Long-acting
benzodiazepines are effective against withdrawal seizures, but the concurrent use with alcohol
can be dangerous and even deadly (Cooper and Vernon , 2013). As a consequence, the
treatment with benzodiazepines should be limited to in-patient facilities only. MOX has the
potential to be a novel therapeutic for the treatment of alcoholism including the ability to
attenuate withdrawal symptoms and reduce drinking in individuals with severe AUD. More
importantly, MOX can suppress drinking and reduce withdrawal symptoms without exhibiting
abuse potential or toxicity like benzodiazepines.
66
Chapter 4
Reduced Expression of Purinergic P2X4 Receptors
Increases Voluntary Ethanol Intake in C57BL/6J Mice
Abstract
Purinergic P2X4 receptors (P2X4Rs) belong to the P2 X superfamily of ionotropic
receptors that are gated by adenosine-5’-triphosphate (ATP). Accumulating evidence indicates
that P2X4Rs play an important role in regulation of ethanol intake. At the molecular level,
ethanol’s inhibitory effects on P2X4Rs are antagonized by ivermectin (IVM), in part; via action
on P2X4Rs. Behaviorally, male mice deficient in p2rx4 gene [P2X4R knockout (KO)] have been
shown to exhibit a transient increase in ethanol intake over a period of 4 days as demonstrated
by social and binge drinking paradigms. Furthermore, IVM reduced ethanol consumption in
male and female rodents, whereas, male P2X4R KO mice were less sensitive to anti-alcohol
effects of IVM compared to wildtype (WT), further s upporting a role for P2X4Rs as targets of
IVM’s action. The current investigation extends testing the hypothesis that P2X4Rs play a role in
regulation of ethanol intake. First, we tested the response of P2X4R KO mice to ethanol for a
period of 5 weeks. Second, to gain insights into the changes in ethanol intake, we employed a
lentivirus-shRNA (LV-shRNA) methodology to selectiv ely knockdown P2X4R expression in the
nucleus accumbens (NAc) core in male C57BL/6J mice. In agreement with our previous study,
male P2X4R KO mice exhibited higher ethanol intake than WT mice. Additionally, reduced
expression of P2X4Rs in NAc core significantly increased ethanol intake and preference.
Collectively, the findings support the hypothesis that P2X4Rs play a role in regulation of ethanol
intake and that P2X4Rs represent a novel drug target for treatment of alcohol use disorder.
67
Introduction
P2X receptors (P2XRs) are becoming a focus of inves tigation in neuroscience and
ethanol studies (Litten et al., 2012; Burnstock 200 8; Asatryan et al., 2011; Gum et al., 2012;
Franklin et al., 2014; Xu et al., 2016). P2XRs are fast acting cation-permeable ion channels that
are gated by synaptically released extracellular adenosine 5’-triphosphate (ATP) (Chizh & Illes
2001; Khakh 2001; North 2002). In the CNS, ATP directly mediates fast excitatory synaptic
transmission by acting on P2XRs located on postsynaptic membranes. In addition, ATP can
produce neuromodulator responses by promoting neurotransmitter release of other major
ionotropic targets (e.g., GABA and glutamate), know n to play important roles in ethanol drinking
and other behaviors by acting on P2XRs located on pre- and postsynaptic membranes (Khakh
2001; Jo & Schlichter 1999; Hugel & Schlichter 2002; Baxter et al., 2011; Xu et al., 2016).
P2X4Rs are the most abundantly expressed P2XR subtype in the CNS ranging from
neurons to microglia (Buell et al., 1996; Soto et a l., 1996) and are the most sensitive P2XR
subtype to ethanol. In vitro studies report that ethanol concentrations starting at approximately 5
mM modulate ATP-activated currents in neurons (Li e t al., 1994; Li et al., 1998; Li et al., 1993;
Weight et al., 1999; Xiao et al., 2008) and recombinant models (Xiong et al., 2000; Xiong et al.,
2001; Davies et al., 2002; Davies et al., 2005; Asatryan et al., 2008; Asatryan et al., 2010). This
concentration of ethanol is well below the 17 mM (i .e., 0.08%) blood ethanol concentration
(BEC) that is considered “under the influence” in t he U.S. In addition, P2X4Rs are located in
brain regions identified as neural substrates of ethanol [e.g., hippocampus, cerebellum, ventral
tegmental area (VTA), nucleus accumbens (NAc), hypo thalamic nuclei including paraventricular
nucleus (PVN) and arcuate nucleus (Arc)] (McCool 20 11; Pankratov et al., 2009; Sim et al.,
2006; Gonzales et al., 2004; Xu et al., 2016).
Recent studies implicate P2X4Rs in the regulation of multiple CNS functions including
neuropathic pain (Tsuda et al., 2000; Ulmann et al. , 2008), neuroendocrine functions (Zemkova
et al., 2010) and hippocampal plasticity (Baxter et al., 2011; Lorca et al., 2011; Sim et al., 2006).
68
In addition, P2X4Rs have been recently shown to modulate the function of other major
ionotropic targets, such as γ-amino butyric acid receptors (GABA
A
Rs) (Jo et al., 2011) and
glutamate N-Methyl-D-aspartate receptors (NMDA) (Ba xter et al., 2011) receptors. Many of
these physiological and behavioral functions linked to P2XRs are known to be affected by
ethanol.
Building evidence links P2X4Rs to ethanol consumption including investigations using
microarray techniques, which found an inverse relationship between p2rx4 gene expression and
innate rodent intake and preference for ethanol (Ki mpel et al., 2007; Tabakoff et al., 2009). In
agreement with this hypothesis, we recently demonstrated that male P2X4 knockout (KO) mice
(i.e., p2rx4 deleted) consumed significantly more e thanol than wildtype (WT) controls (Wyatt et
al., 2014). The present paper extends the investigation of the role of P2X4Rs in ethanol intake
and addresses two unresolved questions from the recent Wyatt et al paper. First, we
significantly increased the length of time of the ethanol investigation to gain insights regarding
the transient nature of the increased drinking reported by Wyatt and colleagues (Wyatt et al.,
2014). This was accomplished by testing male P2X4KO mice and WT littermates for changes in
ethanol intake and preference for 5 weeks using a 24 hr access two-bottle choice paradigm.
Second, in that the increase in ethanol intake previously measured in male P2X4KO mice could
partially reflect compensatory developmental changes, we also utilized a lentiviral-mediated
shRNA knockdown strategy (LV-shRNA) to knockdown P2 X4R expression in the NAc core and
measured changes in ethanol intake and preference.
Materials and Methods
Animals. We used experimentally naïve 2-3 month old male WT and P2X4R KO mice
from our breeding colony at the University of Southern California. The generation of P2X4R KO
mice and the breeding scheme has been described previously (Sim et al. 2006, Wyatt et al.
2013). For the LV-shRNA experiments, 2-3 month old male C57BL/6 mice were obtained from
Jackson laboratories (Bar Harbor, ME). Mice were gr oup housed (i.e. 5 per cage) in the
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vivarium maintained at 22 0C and a 12 hr/12 hr light: dark cycle with free access to food and
water. All procedures are carried out in compliance with the guidelines of National Institute of
Health and approved by the Institutional Animal Care and Use Committee of University of
Southern California.
Drugs. The ethanol solution was prepared as a 10% v/v ethanol solution in tap water
from 190 proof USP grade ethanol solution (Koptec, King of Prussia, PA).
Short hairpin RNA (shRNA) constructs and LV production. cDNA encoding two
shRNA sequences targeting different regions of P2X4 mRNA (S1 and S2) were subcloned into
the Clontech biscistronic pLVX-shRNA2 vector where the shRNA expression was driven by
human U6 promoter, located just upstream of the MCS (Mountain View, CA). The vector also
expressed ZsGreen1 reporter, a human codon optimized variant of the coral reef green
fluorescent protein (GFP), under CMV promoter contr ol. The shRNA sequences were 5’-
CCACAAATACTCAGGGTTG-3’ and 5’-CTCAGATGGGCTTCAGATA-3’. We have observed
that the simultaneous use of both the sequences resulted in higher extent inhibition of P2X4R
expression. LV was produced by mixing both shRNA constructs with psPAX2 and pMD2.G
packaging vectors obtained from Addgene (Cambridge, MA) and transfected HEK 293T cells.
Virus-containing supernatant was collected, concentrated and resultant viral titers were
determined via the ELISA method. Concentrator and titration kits were obtained from Clontech
Laboratories (Mountain View, CA).
Stereotaxic surgery and microinjection procedure. Mice were anesthetized with a
ketamine/xylazine cocktail and placed in a mouse stereotaxic frame (David Kopf Instruments,
Tujunga, CA). A small incision was made to the skin exposing the skull. Bregma and lambda
were measured to ensure an even plane and a small area of dura removed in the area for
microinjection. A ten-microliter syringe (Hamilton, Reno, NV) was used to deliver 1μL of LV (4.1
x 107 to 5.3 x 109 IU/mL) to each NAc (bregma coord inates: anteriorposterior 1.2 mm;
mediolateral 1.0 mm; dorsoventral 4.5 mm) at a rate of 0.1 μL/min. After infusion, the syringe
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was left in place for a further 5 min. Mice were allowed to recover from surgery in cages placed
on heating pads for 2 days, after which they were transported to the vivarium. Mice were
subsequently single housed in the vivarium with ad libitum access to food and water during their
resting period for 1 week prior to start of the ethanol drinking experiments.
Anatomical verification of LV microinfusion into the NAc core by fluorescence
microscopy. Following the stereotaxic surgery and microinjection of LV, transcardial perfusion
was performed on mice using 0.9% NaCl followed by 4% phosphate buffered
paraformaldehyde. Brains were post fixed in 4% phosphate buffered paraformaldehyde
overnight followed by storage in 20% sucrose for 48 hr and frozen in 4-Methylbutane on dry ice.
Striatal sections were cut coronally at 25 μm thickness in a cryostat and later stored in a
cryoprotective solution containing 30% sucrose in PBS at 4 0C until further use. The striatal
sections were then examined for GFP immunofluorescence under a fluorescent microscope
(Olympus BX61 microscope, Shinjuku, Tokyo, Japan).
Verification of LV-shRNA mediated knockdown of P2X4Rs in vitro and in vivo
using Western immunoblotting. BV-2 transduction: Knockdown of P2X4Rs by LV-shRNA
strategy in vitro was verified through transduction of mouse microglial BV-2 cells, which have a
high endogenous P2X4R expression. BV-2 cells were cultured in 6-well plates in DMEM/F12
medium supplemented with penicillin/streptomycin and fetal bovine serum until they reached
approximately 80% confluence. Cells were transduced with 106 infectious units/ml of shRNA-
based LV. Confirmation of virus expression was visualized by GFP fluorescence after 48 hr.
Microinjection into NAc core. P2X4R knockdown in mouse nucleus accumbens core
by LV-shRNA methodology was verified at 14 days post infusion of LV-shRNA. 2 and 3 mice
were stereotactically injected with the LV alone and LV-shRNA-p2rx4 respectively. A separate
cohort of mice that have never undergone surgery (w ill be described as naïve mice) was used
as a positive control for this study. After surgery, mice were allowed to rest for period of 1 week.
At the end of the recovery period, the mice remained in their cages for period of 14 and 42 days
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during which they had ad libitum access to food and water. We chose 14 day time-point since
the mice are exposed to ethanol 2 weeks post surgery and 42 day time-point since the final
week during which they receive ethanol is 6 weeks post surgery. At their respective time points,
mice were euthanized using CO2 asphyxiation and striatum was dissected out as per landmarks
described in the mouse brain atlas (Franklin & Paxi nos 2007).
BV-cell lysate or striatal tissue homogenate preparation. Cells or striatal tissues
were treated with lysis buffer containing 50mM tris-HCl pH (7.4), 150 mM NaCl, 0.5% sodium
deoxycholate, 1% Triton-X-100, 0.1% SDS, 1% proteinase inhibitor cocktail (Millipore,
Temecula,CA). BV-2 cell lysates were spun at 13,000 rpm for 10 min at 4 0C and the protein-
containing supernatant was collected. Protein content for BV-2 cells and ventral striatum was
measured by using BCA protein assay (Thermo Scienti fic, Rockford, IL).
Immunoblotting procedure. Striatal homogenates or cell lysates (respectivel y of 50 μg
and 10 μg per lane) ran on 10% SDS PAGE gels and transferred onto polyvinylidine fluoride
membranes using a semi-dry transfer method (Trans t urbo blot, BioRad, Hercules, CA). Non-
specific binding was blocked using 5% non-fat dry milk (BioRad, Hercules, CA) followed by
incubation with rabbit anti-P2X4 receptor antibody (Alomone Labs, Jerusalem, Israel) overnight
at 4.0° C. Membranes were then incubated with goat anti-rabbit secondary antibody for 1 hr at
room temperature. In between incubation steps for antibodies and blocking, membranes were
washed 3 times, 5 min each time, with TBS containing 0.05% Tween-20. After secondary
antibody incubation, membranes were incubated with ECL substrate, (BioRad, Hercules, CA)
and bands were visualized using chemilumenescent method (ChemiDoc system, BioRad,
Hercules, CA).
24 hr access two-bottle choice paradigm. The 24 hr access two bottle choice
paradigm is a model that mimics social drinking and is used to investigate differences in ethanol
intake in genetically modified animals or upon pharmacological treatment (Yoneyama et al.,
2008; Middaugh & Kelley 1999; Belknap et al., 1993). We used the procedure previously
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described (Wyatt et al., 2014; Yardley et al., 2012 ; Asatryan et al., 2014). Briefly, singly housed
WT/P2X4R KO or naïve/LV alone/LV-shRNA-p2rx4 infused mice had 24 hr access to 2 inverted
graduated tubes (25 mL) with metal sippers position ed on stainless steel cage tops. Food was
evenly distributed on the cage tops to avoid association with either of the tubes. The mice had
access to tubes containing water only for the first week post acclimation. In the second week,
one of the tubes contained water and the other with 10% ethanol solution (10E). 10E and water
intake was recorded by measuring the lower meniscus. It was ensured that positions of the
tubes were switched every alternate day to avoid side preferences. Mice were given fresh
solution of 10E and water once a week. Body weights were measured and used to calculate the
g/kg/24hr ethanol intake. Percent ethanol preference was determined by multiplying the ratio of
volume of 10E intake (mL) over total fluid intake ( 10E + water) by 100.
For the LV-shRNA-p2rx4 drinking experiment, mice underwent a baseline drinking
session post acclimation during which their voluntary consumption of ethanol, water, total fluid
intake and ethanol preference was measured. Upon stable baseline drinking, mice were
randomly assigned to one of the three treatment options: 1) LV-shRNA-p2rx4, LV alone or naïve
mice (i.e. mice that have never undergone the surge ry). ANOVA was used to ensure that the
ethanol intake, preference, water or total fluid intake did not significantly differ between the three
treatment groups.
Statistical Analyses. Repeated measures two way ANOVA (genotype x week) w as
used to investigate differences in 10E intake, 10E preference, water and total fluid intake
between WT and P2X4R KO mice, followed by Bonferroni post hoc test for multiple
comparisons. For the LV-shRNA drinking studies, separate two-way ANOVAs followed by
Bonferroni post hoc comparisons were conducted between LV alone and LV-shRNA-p2rx4
groups to analyze the effect of LV-shRNA on drinking behavior as well as between naïve mice
and LV alone group to determine whether injection of LV alone has any impact on drinking
behavior. One-way ANOVA with Tukey’s post hoc test was also used to compare the efficiency
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of LV-shRNA-p2rx4 transfusion and transfection on P2X4R knockdown in mouse striatum and
BV-2 cells. Significance was set at p < 0.05. All data was analyzed using Graph Pad software
(Prism, San Diego, CA).
Results
P2X4R KO mice exhibited increased voluntary ethanol consumption in the 24 hour
access drinking paradigm. We tested the effects of global knockout of p2rx4 gene on ethanol
intake using a 24 hr two bottle choice paradigm (10 E versus water). As illustrated in Figure
4.1A, there was a significant effect of genotype [F(1,16) = 4.88, p < 0.05], but not week or
genotype x week interaction for 10E intake. There was no significant effect of genotype, week or
genotype x week interaction for 10E preference or water intake between WT and P2X4R KO
mice (Figure 4.1B & 4.1C). There was a non-signific ant trend towards effect of genotype
[F(1,16) = 3.28, p = 0.0891] but not week or genoty pe x week interaction for total fluid intake.
(Figure 4.1D). Considering that there were changes in 10E intake, but not preference, we
evaluated the effect of genotype and week on body weight. There was a significant effect of
genotype [F(1,16) = 4.68, p < 0.05] since the P2X4R KO mice weighed significantly more than
their WT counterparts. There was also significant effect of week [F(4,64) = 59.08, p < 0.001] on
body weight. There was no significant interaction between the two factors on body weight
between WT and P2X4R KO mice.
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Transfection of BV-2 cells or transfusion in mouse striatum with LV-shRNA-p2rx4
reduced P2X4R expression. We first investigated the efficiency of knockdown of P2X4Rs
using a LV-shRNA strategy in BV-2 cells. As depicted in Figure 4.2A, LV-shRNA treatment
significantly reduced P2X4R expression [F(2,4) = 27 .88, p < 0.01] in BV-2 cells. Tukey’s post
hoc test confirmed that LV-shRNA treatment significantly reduced P2X4R expression as
compared to untreated cells (q = 9.875, p < 0.01) a nd cells treated with LV alone (q = 7.377, p <
0.05). We next tested the efficiency of LV-shRNA-p2rx4 infusion on P2X4R knockdown in the
mouse striatum at 14 and 42 days post infusion. As illustrated in Figure 4.2C, LV-shRNA
significantly reduced P2X4R expression in the striatum at 14 days post infusion [F(2,12) =
9.266, p < 0.01].Tukey’s post hoc test confirmed significant reduction in P2X4R expression in
Figure 4.1. P2X4R KO mice exhibited significantly higher 10E intake compared to WT controls (A)
and tended to have higher total fluid intake (D) without any significant changes in 10E preference
(B) or water intake (C). For each week, the 10E intake, preference and water intake was measured as
an average of 5 days. Values represent mean ± SEM for a duration of 5 days each week for 8 WT and 10
P2X4R KO mice. * p <0.05 versus WT mice, two-way ANOVA.
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mice that received LV-shRNA-p2rx4 treatment as compared to both naive mice (q = 5.565, p <
0.01) and mice that only received the LV infusion after a period of 14 days (q = 4.889, p < 0.05).
In Figure 4.2D, LV-shRNA treatment significantly reduced P2X4R expression at 42 days post
infusion [F(2,14) = 10.37, p < 0.05] with Tukey’s p ost hoc text confirming significant reduction in
P2X4R expression in mice injected with LV-shRNA-p2rx4 in comparison to naïve controls (q =
4.300, p < 0.05) and mice that received LV infusion alone (q = 6.435,p < 0.01).
Infusion of LV alone did not have any significant effect on ethanol intake or
preference in comparison to the naïve mice. There was non-significant trend towards effect
of week [F(4,76) = 2.35, p = 0.0618] without any si gnificant effect of treatment or week x
treatment interaction for 10E intake in mice infused with LV alone relative to naïve mice (Figure
4.3A). Similarly, in the context of 10E preference, there was a non-significant trend towards
effect of week [F(4,76) = 2.03, p = 0.0982] without any significant effect of treatment or week x
treatment interaction (Figure 4.3B). There was a si gnificant effect of week [F(4,76) = 2.59, p <
0.05] but not treatment and the week x treatment interaction trended towards significance
[F(4,76) = 2.19, p = 0.0782] for water intake betwe en the two groups (Figure 4.3C). There was a
non-significant trend towards effect of week [F(4,7 6) = 2.21, p = 0.0753] and treatment [F(1,19)
= 3.86, p = 0.0642] on total fluid intake between the two groups of mice. However, there was a
significant week x treatment interaction [F(4,76) = 2.75, p < 0.05] with Bonferroni post hoc test
indicating reduced total fluid intake in mice receiving LV infusion relative to naïve mice at week
5 (t = 3.329, p < 0.01) (Figure 4.3D). Finally, the re was a non-significant trend towards effect of
week on body weight [F(4,76) = 2.49, p = 0.0503] wi thout any significant effect of treatment or
week x treatment interaction between LV alone infused mice and naïve mice.
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The LV-shRNA-p2rx4 infused mice exhibited a higher ethanol intake as compared
to mice that only received LV infusion. There was a significant effect of week [F(4,80) = 3.87,
p < 0.01] and treatment [F(1,20) = 6.08, p < 0.05] on 10E intake in the LV-shRNA-p2rx4 group
as compared to the group that received LV infusion, without any significant week x treatment
interaction. (Figure 4.3A). There was a significant effect of week [F(4,80) = 3.96, p < 0.01] but
not treatment or week x treatment interaction for 10E preference between the two groups
(Figure 4.3B). Similarly, there was a significant e ffect of week [F(4,80) = 5.57, p < 0.001] but not
treatment or week x treatment interaction for water intake (Figure 4.3C). There was no
significant effect of week, treatment or week x treatment interaction for total fluid intake (Figure
Figure 4.2. Microglial BV-2 cells transinfected with LV-shRNA-p2rx4 reduced P2X4R expression by
68% and 62% as compared to non-treated cells (NT) and LV alone treated cells respectively (A).
Microinfusion of LV into the NAc core was verified by detecting ZsGreen1 immunofluorescence (B).
Stereotaxic injection of LV-shRNA-p2rx4 in NAc core significantly reduced P2X4R expression as compared
to naïve mice and LV alone infused mice respectively after 14 (C) and 42 days (D). Values represent me an
± SEM for 3-8 mice per treatment group for (C) and (D), ** p < 0.01 versus non-treated cells, # p < 0. 05
versus LV alone treated cells for (A), * p < 0.05, ** p < 0.01 versus naïve controls, # p < 0.05, ## p < 0.01
versus LV alone group for (C) and (D), Tukey’s post hoc test.
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4.3D). There was a significant effect of week [F(4, 80) = 16.60, p < 0.001],but not treatment on
body weight between two groups. However, there was a significant week x treatment interaction
[F(4,80) = 3.39, p < 0.05] for body weight since LV-shRNA-p2rx4 infused mice gained more
weight across the 5 weeks period.
Discussion
The current study investigated the role of P2X4Rs in regulation of ethanol drinking
behavior. Overall, the findings support the hypothesis that P2X4Rs play an important role in the
regulation of ethanol intake by demonstrating that reduced P2X4R expression results in
changes in ethanol drinking behavior. Using a global knockout strategy, we demonstrated that
Figure 4.3. The LV-shRNA-p2rx4 group exhibited significantly higher 10E intake as compared to mice
infused with LV alone (A). No significant changes in 10E preference (B), wat er intake (C) or total fluid intake
(D) between the groups. Values represent mean ± SEM for a duration of 5 days for each week for 10 mice
infused with LV alone and 14 mice infused with LV-shRNA-p2rx4. * p < 0.05, versus LV alone infused mice,
two-way ANOVA.
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P2X4R KO mice exhibited significantly increased ethanol intake. The increased body weights of
P2X4R KO mice may partially contribute to their increased ethanol intake as suggested by lack
of significant change in ethanol preference. On the other hand, there was no significant change
in water intake suggesting that p2rx4 deficiency affects mechanism of ethanol drinking, without
perturbing the physiolgy of drinking per se. These results are in good agreement with our
previous study wherein we showed that male P2X4R KO mice exhibited higher ethanol intake
over a period of 4 days without changes in ethanol preference or water intake as compared to
their WT littermates (Wyatt et al., 2014).
Taking into consideration the complex compensatory changes that occur in a knockout
mouse model, the constitutive deficiency of P2X4Rs does not necessarily represent the full
pharmacological blockade of the receptor. At present, we do not have any selective P2X4R
antagonists that can be used to provide a direct link between P2X4R antagonism and increased
ethanol consumption. As a complementary strategy, we employed a LV-shRNA methodology to
address this issue. In the present investigation, we targeted the NAc core as the site for the LV-
shRNA injection since this is a critical site of the dopamine (DA) mesolimbic circuitry for various
drugs of abuse including ethanol to induce their reinforcing and rewarding effects (Bassareo et
al., 2017; Cador et al., 1991; Corbit et al., 2016; Di Chiara 2002). Moreover, P2X4Rs are
expressed in the striatum (Amadio et al., 2007) and endogenous ATP (possibly via activation of
P2XRs) has been implicated in modulation of DA neurotransmission in various regions of the
mesolimbic circuitry including the VTA and NAc (Xia o et al., 2008; Krugel et al., 2001). In
agreement with our previous and current findings from the male P2X4R KO study, we found that
mice with reduced P2X4R expression (via LV-sh-RNA-p 2rx4 infusion) exhibited greater ethanol
consumption relative to naïve mice and mice infused with LV alone. There were no significant
changes in ethanol intake upon infusion of LV alone in relation to naïve mice indicating that the
increased ethanol intake in mice with reduced P2X4R expression is due to shRNA mediated
knockdown of P2X4Rs and not infusion of the virus alone. The coherence in findings from both
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the P2X4R KO and LV-shRNA-p2rx4 studies indicates that functional deletion of p2rx4 gene
increases ethanol intake. Nevertheless, considering there have been compensatory changes in
receptors such as NMDARs, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors
(AMPARs) as well as DA receptors in P2X4R KO mice ( Wyatt et al., 2013; Khoja et al., 2016), it
is possible that alterations in these receptor systems may occur upon infusion of LV-shRNA in
the NAc core.Hence, future studies involving measuring expression levels of glutamatergic and
dopaminergic receptors in LV-shRNA-p2rx4 infused mice would be warranted to delieanate the
interaction of P2X4Rs with other neurotransmitter systems in regulation of ethanol intake.
Additionally, shRNA mediated knockdown of P2X4Rs significantly increased ethanol
preference relative to naïve mice but not mice infused with LV alone. The increased ethanol
preference in LV-shRNA-p2rx4 infused mice may account for the increased ethanol intake
relative to naïve mice since there were no significant differences in body weights between these
group. Unlike in the LV-shRNA methodology where there is knockdown of a particular gene at
the adult stage, the P2X4R KO mice may exhibit neurodevelopmental adaptations to
compensate for constitutive deficiency of p2rx4 gene, and such adaptations could nullify the
effect of p2rx4 knockout on ethanol preference. Moreover, the p2rx4 knockdown is in the NAc
core which is a key brain region for expression of ethanol reinforcement and has an important
role in acquisition and maintenance of ethanol seeking behavior (Gonzales et al., 2004). Thus,
potential neurodevelopmental changes in this brain region of P2X4R KO mice could significantly
interfere with motivational behavior towards seeking ethanol. The somewhat differing data with
respect to ethanol preference from P2X4R KO and LV-shRNA-p2rx4 infused mice suggests the
need for additional investigations using operant chamber technique to monitor self-
administration or conditioned place preference in P2X4R KO mice or LV-shRNA-p2rx4 infused
mice to better understand the role of P2X4Rs in ethanol seeking behavior.
The findings from LV-shRNA-p2rx4 and P2X4R KO experiments are in agreement with
mutiple studies that have reported an inverse correlation between P2X4R expression and
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ethanol consumption. For example, Kimpel and colleagues compared gene expression in brain
areas associated with reward in inbred alcohol preferring (iP) v/s non-preferring (iNP) rat lines
and found that functional p2rx4 expression was significantly reduced in iP rats (Kimpel et al.,
2007). Similarly, Tabakoff and colleagues found lower levels (i.e., inverse relationship) of whole
brain expression of p2rx4 mRNA in inbred rats that display a high ethanol-drinking phenotype
compared to those with a lower ethanol-drinking phenotye (Tabakoff et al., 2009). On the other
hand, McBride and colleagues reported that p2rx4 gene expression was significantly increased
in high alcohol drinking female (HAD2) rats, relati ve to their low-alcohol drinking (LAD2)
counterparts (McBride et al., 2012). In addition, i ncreased expression of P2X4Rs was detected
in the periaqueductal gray (PAG), a region associat ed with fear and anxiety (Behbehani 1995),
in adolescent male P rats (McClintick et al., 2016) . To further validate the correlation of
increased P2X4R expression with increased ethanol intake, LV-shRNA mediated knockdown of
P2X4R expression in the posterior VTA was recently shown to significantly decrease ethanol
intake in female HAD2 rats (Franklin et al., 2015). Although there are differences regarding the
direction of change in drinking behavior in these reports, a common theme emerging from these
investigations is that manipulation of p2rx4 expression is associated with significant changes in
ethanol consumption.
Multiple investigations from our laboratory as well as others have supported the
hypothesis that inhibition of P2X4R activity increases ethanol drinking behavior and potentiation
of P2X4Rs reduces ethanol intake as well as the propensity to seek ethanol Based on recent
work, we suggested that ethanol acts as an open channel blocker of P2X4Rs (Ostrovskaya et
al., 2011, Popova et al., 2010) and that positive modulation of P2X4Rs by ivermectin (IVM)
antagonized ethanol induced inhibition of P2X4Rs (A satryan et al., 2010). Positive modulation of
P2X4Rs by IVM can reduce ethanol drinking behavior as illustrated in previous reports showing
that IVM administration in C57BL/6J mice reduced ethanol intake and preference using variety
of paradigms that mimic social drinking and motivation to seek ethanol (Yardley et al., 2012;
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Asatryan et al., 2014; Wyatt et al., 2014).The anti-alcohol effects of IVM can be, in part, linked to
P2X4R activity in that the degree of reduction of ethanol intake was decreased in male P2X4R
KO mice (Wyatt et al., 2014). Similar findings were obtained from Franklin and colleagues,
where IVM significantly reduced ethanol intake in male and female HAD-1 and HAD-2 rats.
Furthermore, intracerebroventricular (ICV) administ ration of IVM significantly reduced ethanol
intake in female HAD2 rats (Franklin et al., 2015). Although previous findings from our
laboratory have suggested IVM as a positive modulator of P2X4Rs, IVM has been reported to
be a positive modulator of ligand gated ion channels (LGICs) belonging to Cys-loop superfamily
such as GABAARs (Dawson et al., 2000), glycine rece ptors (GlyRs) (Shan et al., 2001) and
nicotininc acetylcholine receptors (nAchRs) (Krause et al., 1998). Thus, the role of other
ionotropic receptors in the behavioral effects of IVM cannot be disregarded. The physiological
significance of ethanol induced inhibition of P2X4Rs within the mesolimbic circuitry is unknown
and currently under investigation in our laboratory. However, based on previous reports, it is
thought that ethanol induced inhibition of P2XRs on the GABA releasing terminals in the VTA is
linked to disinhibition of VTA DA neurons as suggested previously (Xiao et al., 2008). Therefore,
the study by Xiao and colleagues provides indirect evidence for P2X4Rs’s role in modulation of
firing of DA neurons in VTA. One possible mechanism for P2X4Rs in modulation of firing of DA
neurons is via its localization in the Arc region of the hypothalamus. P2X4Rs have been
reported to be involved in regulating presynaptic GABA release onto proopiomelanocortin
(POMC) neurons in the Arc region (Xu et al., 2016) and GABAergic activity in this region has
been associated with an inhibitory influence on the VTA DA neurons (Tabakoff et al., 2009).
However, additional electrophysiological studies would be needed to test this hypothesis before
definitive conclusions can be drawn. Overall, we do not have sufficient data to conclusively state
that there is a direct link between P2X4R potentiation/inhibition and firing of DA neurons in the
VTA. This is an area of work that is ongoing. Moreover, elucidating the GABAergic tone or firing
of VTA DA neurons in P2X4R KO mice or mice that received LV-shRNA-p2rx4 infusion would
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also be important to help provide novel insights into the mechanism leading to increased
ethanol intake in these mice.
While the aforementioned studies highlight the importance of neuronal P2X4Rs in
regulation of ethanol intake, the role of microglial P2X4Rs in mediating ethanol induced
responses cannot be excluded. P2X4Rs have been implicated in regulation of mciroglial
function and have been associated with pathophysiology of several neurodegenerative and
neuroimmune disorders (Burnstock 2008; Potucek et a l., 2006; Tsuda et al., 2013). Ethanol has
been shown to upregulate P2X4R mRNA and protein expression in microglial cells, suggesting
involvement of P2X4Rs in microglial responses (Gofm an et al., 2014). For instance, P2X4Rs
have been reported to play a role in mediating ethanol-induced macrophage and microglial
phagocytosis as well as microglial migration (Gofma n et al., 2014). In addition, P2X4Rs have
also been reported to regulate alcohol induced effects on signaling molecules such as
phosphotidylinositol-3-kinase (PI3K-Akt), extracell ular regulated kinase 1/2 (ERK 1/2) and
transcription factors such as cyclic-AMP regulated phosphoprotein (CREB) (Gofman et al.,
2016), all of which are linked to multiple physiological functions such as proliferation,
differentiation, neurodevelopment and inflammation (Kim & Choi 2010). Interestingly, we
observed significant increase in CREB phosphorylation in ventral striatum of P2X4R KO mice
(Khoja et al. 2016), indicating a role for CREB act ivation in P2X4Rs’ effects on ethanol drinking
behavior. The in vitro findings reported above suggest that alcohol can regulate mitogen
activated protein kinase (MAPK) signaling pathways in microglial cells in brain sites linked to
reward circuitry. Thus, infusion of LV-shRNA-p2rx4 could possibly lead to perturbation of
ethanol-dependent MAPK signaling pathways in microgilal cells in the NAc core, as a result of
which the LV-shRNA-p2rx4 infused mice consume significantly more ethanol than those injected
with LV alone. Hence, the contribution of P2X4Rs to ethanol intake in each cell type needs to be
thoroughly examined in future investigations.
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In conclusion, the results from our previous work using P2X4R KO mouse model
coupled with current LV-shRNA studies which corroborate findings from others, supports r the
hypothesis that P2X4Rs play an important role in regulation of ethanol intake. Moreover, the
results support the discovery and development of P2X4R allosteric modulators as novel
therapeutic agents for treatment of AUD.
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Chapter 5
Purinergic P2X4 Receptor Knockout Mice Exhibits Altered
Glutamatergic Signaling in the Nucleus Accumbens
Abstract
P2X receptors are a family of ligand-gated ion channels activated by synaptically
released adenosine 5’-triphosphate. The P2X4 receptors (P2X4Rs) subtype is most abundantly
expressed in the central nervous system and is also present in regions that are considered
important neural substrates for ethanol. Converging evidences have suggested that P2X4Rs
play a role in the regulation of ethanol behavior, where an inverse relationship was found
between the expression of p2rx4 and ethanol intake in different strains of mice and rats. In
agreement with these results, our investigations also found that the ablation of p2rx4 gene (via
P2X4R knockout [KO] mice) or reducing P2X4R expression (via lentiviral-shRNA microinjected
into mouse’s nucleus accumbens [NAc]) also resulted in significantly increased ethanol intake.
To date, several studies have shown that P2X4Rs can modulate the function of other
neurotransmitter receptor systems such as GABA, glutamate, and dopamine, all of which are
known to play a role in in ethanol behavior. However, the exact mechanism of how P2X4Rs can
regulate drinking is still not clearly understood. The current study was undertaken to begin to
elucidate the potential signaling mechanism(s) of P 2X4Rs in relation to ethanol intake.
Microarray analysis of differentially expressed genes in the NAc of P2X4R KO versus wildtype
mice (WT) after short-term ethanol exposure identif ied GRIN1 as one of the top upregulated
genes that encode for a receptor/ion channel (encod e for GluN1 subunit of NMDAR).
Electrophysiological study revealed that the P2X4Rs can significantly inhibit the current in
NMDARs. Pharmacological manipulation using the NMDAR antagonist memantine showed
differential activity in relation to drinking in KO versus WT mice. Taken together, these results
suggested that the lack of P2X4Rs can induce a change in expression and/or activity of
85
NMDARs, which may partially explained why P2X4R KO consume significantly greater amount
of ethanol.
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Introduction
Alcohol use disorder (AUD) constitutes a major nati onal health problem that affects over
18 million people and imposes an economic burden in excess of $200 billion annually (Bouchery
et al., 2011; Grant et al., 2004; Hardwood and Lewin, 2000). Despite considerable funding and
focus on identifying the different neural sites that are involved in ethanol actions, current
treatment options are limited to three FDA-approved medications (disulfiram, naltrexone, and
acamprosate) and these pharmacotherapies only show modest success even when combined
with psychosocial support (Harris et al., 2010; Lit ten et al., 2012). This highlights the importance
of identifying additional targets that can translate into effective medications for the treatment
and/or prevention of AUD.
Building investigations over the past decade have focused on the role of purinergic P2X
receptors (P2XRs) in the regulation of the action o f ethanol. These fast-acting cation channels
are gated by synaptically released extracellular adenosine 5’-triphosphate (ATP) and have been
shown to interact with several neurotransmitter systems (e.g., GABA, glycine, and glutamate)
that are known to modulate ethanol consumption (Chi zh and Illes, 2001; Jo and Schlichter,
1999; Khakh, 2001). In particular, results from our laboratory and by others have implicated
P2X4 receptors (P2X4Rs) as a promising target for t he preclinical development of novel AUD
agents. Of the seven P2X subtypes, P2X4Rs are most abundantly expressed in the central
nervous system (CNS) and are most ethanol-sensitive P2X subtypes (Buell et al., 1996; Soto et
al., 1996; Xiao et al., 2008). In vitro, ethanol at concentrations which represent doses well below
the legal-intoxicating limit negatively modulate ATP-activated currents thus inhibit P2X4R
function (Davies et al., 2002; Davies et al., 2005). In vivo, male P2X4R knock out (KO) mice
consume a significantly greater amount of ethanol compared to wildtype (WT) controls (Wyatt et
al., 2014). In addition, knockdown of P2X4Rs in the nucleus accumbens (NAc) via lentiviral
delivery of shRNA in male C57Bl/6J mice also resulted in a significant increase in ethanol intake
(Khoja et al., 2017). Collectively, these results a re in agreement with microarray genetic studies
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that have found an inverse relationship between the expression of p2rx4 gene and ethanol
consumption in different strains of mice and rats ( Kimpel et al., 2007; Tabakoff et al., 2009).
That is, the higher expression and/or function of P2X4Rs results in reduced ethanol
consumption. In support of this hypothesis, ivermectin and related avermectins can potentiate
P2X4R function and significantly antagonize the ethanol inhibition on P2X4R function in vitro
and also reduce ethanol intake in mice (Asatryan et al., 2010, 2014; Popova et al., 2013;
Yardley et al., 2012, 2014, 2015). Collectively, these findings support ongoing investigations
focusing on P2X4Rs as a potential novel target for AUD drug development.
Overall, P2X4Rs are implicated to play a role in a multitude of neural circuitries that are
involved in regulating different CNS functions such as ethanol behavior, neuropathic pain,
neuroendocrine functions, and inflammatory response in the spinal cord (Lorca et al., 2011;
Tsuda et al., 2000; Ulmann et al., 2008; Zemkova et al., 2010). Several studies have suggested
that P2X4Rs can interact with these circuitries by modulating pre-synaptic release of certain
neurotransmitters and/or by regulating post-synaptic currents on specific receptors (Andries et
al., 2007; Baxter et al., 2011; Hugel and Schlichter, 2002; Jo et al., 2011). However, these
evidences are still inadequate to explain the exact mechanism(s) of how P2X4Rs mediate
ethanol intake. As P2X4Rs are emerging as a promising target for AUD drug development, a
better understanding of these signaling mechanism(s ) by would greatly improve the drug
development process that focus on P2X4Rs since preclinical mechanistic knowledge is always
invaluable and beneficial before a lead compound enters into the uncertainty world of human
studies.
The current study was undertaken to investigate the potential signaling pathway(s)
associate with P2X4R mediation of ethanol behavior. We have performed microarray analysis of
the differentially expressed genes in the NAc of male P2X4R KO versus WT mice after two
weeks of voluntary ethanol exposure. We then validated our finding using electrophysiological
and pharmacological manipulation methods. Overall, the results from this work have provided a
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plausible mechanistic insight into why the lack of P2X4Rs can induce a natural propensity in
mice to voluntarily consume more ethanol, and continue to support the hypothesis that P2X4Rs
represents a novel target for the development of effective AUD therapies.
Materials and Methods
Animals. Ethanol-naïve male P2X4R KO and WT mice were obtained from our breeding
colony at the University of Southern California (US C). The breeding colony was established
from a previous P2X4R KO colony that was maintained on a C57BL/6 background (Wyatt et al.,
2014). The breeding scheme for generation of P2X4R KO mice and genotyping has been
described previously (Wyatt et al., 2014). Mice wer e housed in groups of 5 per cage in rooms
maintained at 22°C with 12/12 h light/dark cycle and ad libitum access to food and water. All
experiments were undertaken in compliance to guidelines established by National Institute of
Health (NIH) and approved by the Institutional Anim al Care and Use Committee of University of
Southern California.
Materials. 190 proof USP ethanol (Koptec, King of Prussia, PA) was diluted in water to
achieve a 10% (10E) v/v solution. Memantine (Sigma- Aldrich, St. Louis, MO) were diluted in
0.9% saline solution to appropriate concentration, and injected at a volume of 0.01 mL/g of body
weight.
Drinking study and statistical analysis. Brain collection for microarray samples:
Prior to testing, male P2X4R KO (n = 9) and WT (n = i) mice, aged 10-week old, were
individually housed for one week with ad libitum access to food and two bottles of water. The
24-h-two-bottle choice paradigm was performed as previously described (Yardley et al., 2012).
At the start of the experiment, one bottle of water was replaced with 10% ethanol solution (v/v,
10E). Both KO mice had continuous access with one bottle of 10E and one bottle of water with
ad libitum access to food throughout the two weeks testing period. Bottle volumes were
measured daily each morning with the positions switched every other day to avoid side
preference. Body weights were measured daily to calculate the g/kg/24-h intake of ethanol. At
89
the end of the study, the NAc were collected as per the neuroanatomical landmarks described in
the mouse brain atlas (Franklin and Paxinos, 2007). Repeated-measures two-way ANOVA was
used to analyze the effect of genotype and week on 10E intake. The significance level was set
at p ≤ 0.05.
Ethanol/drug (memantine) treatment studies: Using the two-bottle-choice, once
stable drinking level (10E intake ± 10% variability from the mean of the last 3 days) had been
achieved for the KO (n = 6) and WT (n = 6), animals were habituated to saline injections
(intraperitoneal – i.p.) until 10E intake again sta bilized. Both groups then received one dose of
memantine. Change in ethanol intake over the 24-h period following drug administration was
calculated as g/kg. Two-tailed, unpaired, individual Student's t-test was used to assess for the
effect of drug treatment (saline pre-treatment [pre -drug] vs. drug dose) on 10E intake. The
significance level was set at p ≤ 0.05.
RNA Isolation and Microarray Hybridization. At the end of 2-week drinking study
described above, mice were sacrificed by CO
2
administration and cervical dislocation, and the
nucleus accumbens were dissected out. Total RNA was extracted using Purelink RNA Kit
(ThermoFisher, Waltham, Massachusetts) using manufa cturer’s protocol. RNA samples were
then treated using DNA-free Kit (Ambion, Carlsbad, California). RNA quality was assessed
using the Agilent 2100 Bioanalyzer (Santa Clara, CA ) and all samples used for microarray had
RNA integrity number above 7. RNA concentration was measured using the ND-1000
spectrophotometer (Nanodrop Technologies, Wilmingto n, DE). Total RNA was transcribed into
double-stranded cDNA, biotin-labeled, cRNA was synthesized from these cDNA, purified and
fragmented using the reagents from the WT Pico Kit (Affymetrix, Santa Clara, CA). Labeled
cRNA from individual mice (n = 3-4 for each group, WT and KO) was hybridized to a single
Affymetrix Clariom D array. Array hybridization and scanning were performed according to
manufacturer’s protocol.
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Microarray Data Analysis. Microarray data were processed using GeneChip Operating
Software v4.1 (GCOS, Affymetrix). The raw microarray data were initially background corrected,
normalized using quantile normalization, and further log2 transformed. Significantly up- or
downregulated genes were identified using GCOS, and a score to each gene on the basis of a
change in gene expression relative to the standard deviation of repeated measurements. Cutoff
was set at p ≤ 0.05 and foldchange > 1.5 and < -1.5. Bioinformatics analysis of gene lists was
performed with Ingenuity Pathway Analysis (Ingenuit y® Systems, www.ingenuity.com), which
utilizes biomedical literature associations to annotate genes with biological functions and cellular
components. IPA also generates networks of interrelated genes based on their curated
knowledge base.
Complementary RNA (cRNA) and complementary DNA (cDNA) injections in
Xenopus laevis oocyte. Stage V or VI Xenopus oocytes (purchased from Eco Cyte Bioscience,
Austin, TX) were injected with 20 ng cRNA of rat p2rx4 gene or 1 ng cDNA for NMDAR
(ϵ1/ζ1;1:1) using the Nanoject II Nanoliter injecti on system (Drummond Scientific, Broomall, PA).
Injected oocytes were stored at 16°C in incubation medium containing 96 mM NaCl, 2 mM KCl,
1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, and 2.5 mM pyruvic acid with 1% heat inactivated
HyClone® horse serum (VWR, San Dimas, CA) and 0.05 mg/ml gentamycin, adjusted to pH
7.5. Electrophysiological experiments were conducted 24 – 48 h after cRNA injections.
Whole Cell Two-Electrode Voltage Clamp Recordings were performed using the OC-
725C oocyte clamp (Warner Instruments, Hamden, CT) as previously described elsewhere
(Davies et al., 2002, 2005). The oocytes were volta ge clamped at −70 mV and the currents
were recorded on a strip-chart recorder (Barnstead Instrument, Dubuqe, IA). Oocytes were
continuously perfused at a rate of 3 - 4 mL/min at room temperature with modified Ringers
buffer containing (in mM) 110 NaCl, 2.5 KCl, 10 HEP ES and 1.8 BaCl2, pH 7.5, using a
peristaltic pump (Rainin Instrument, Oakland, CA). To induce currents in P2X4R, submaximal
concentrations (EC
10
) of adenosine 5’-triphosphate (ATP) (Sigma, St. Lo uis, MO) were used,
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which produced 10% of maximal effect induced by 100 µM ATP. We have shown previously that
the use of EC
10
can maximize the effects of ethanol and minimize receptor desensitization
(Asatryan et al., 2010; Davies et al., 2002, 2005). To induce a current in NMDAR, 100 mM
glutamate (EC
10
) was used. A washout period of 5 min was allowed between each application to
allow for re-sensitization of the receptor (Asatrya n et al., 2010; Davies et al., 2002, 2005;
Popova et al., 2013).
Results
Microarray analysis of the NAc from male P2X4R KO mice after short-term ethanol
exposure. To begin to elucidate the mechanism(s) involving P2X4Rs mediation of drinking, we
initiated the microarray investigation on differentially expressed genes in male P2X4R KO
versus WT mice after short-term ethanol exposure using a 24-h two bottle choice paradigm
(Yardley et al., 2012; Huynh et al., 2017). This pa radigm allows the mice to voluntarily choose
between 10E and water for the duration of the two-week testing period. Our focus was to
understand the initial changes in gene expression during the first ethanol exposure, and using
this gene data set enrichment analysis to identify potential focus gene(s) and signaling
mechanism(s) that can explain why the KO mice rapid ly and voluntarily develop a preference of
ethanol over water. In agreement with our previous findings, the KO mice consistently and
significantly consumed more ethanol over the entire two weeks (Figure 5.1A). Two-way ANOVA
indicated a significant effect of genotype (F(1,8)= 32.87, p<0.0004), but not week or genotype x
week interaction on 10E intake. At the end of the drinking study, the NAc were dissected, based
on neuroanatomical landmarks, for microarray analysis (Figure 5.1B). This revealed 2908
differentially expressed genes with 1743 upregulated and 1165 downregulated. These results
were input into IPA for further bioinformatics analysis of the biological significances relating to
these differentially expressed genes.
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Bioinformatics analysis of differentially expressed genes. In our data set, we
identified the top 10 upregulated and 10 downregulated genes of which approximately 1/3 are
representd by various microRNA (Table 5.1). The alt ered expression of these microRNA genes
have been suggested to contribute to the development of different forms of cancer such as
ovarian and colorectal cancer (Duan et al., 2013; L i et al., 2017). It appears that the lack of
P2X4R under ethanol exposure has reinforced the dysregulated in expression of these genes.
P2X4R KO under ethanol exposure also exhibits a trend toward the pathological development of
other diseases, indicated by the altered expression of genes such as TLX3 (leukemia) and
CD2BP2 (dysplasia formation). In particular, we wer e interested in identifying GRIN1 on this list.
GRIN1 encodes for GluN1 subunit of NMDAR and has been widely implicated to play a role in
the regulation of ethanol behavior and also other substance of abuse (Xiang et al., 2015).
We further used IPA proprietary knowledge base to identify potential changes in
signaling pathway(s) in the KO versus WT mice. We i dentified the top 5 affected canonical
pathways, ranked by p-value (Table 5.2). Cdk5 (cycl in-dependent kinase 5) signaling pathway
was significantly impacted (p = 0.00635), this path way is important for neurodevelopment and
Figure 5.1. The NAc of P2X4R KO and WT mice were collected after 2 weeks of ethanol exposure using
the 24-h-two-bottle choice paradigm. (A) P2X4R KO mice consumed significantly more 10E during the 2-
week period. Values represent mean ± SEM for 8-9 mice per group, *** p < 0.001 versus WT, two-way ANOVA.
(B) The NAc from these mice were dissected based on neuroanatomical landmark as described in the mouse
brain atlas (Franklin and Paxinos, 2007).
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deregulation of this pathway has been linked to many neurodegenerative diseases such as
Parkinson’s and Alzheimer’s Diseases (Shu et al, 20 16). Production of reactive oxygen species
in macrophages was also significantly impacted (p=0 .0108), suggesting that the KO mice may
be experiencing more brain oxidative stress as compared to WT controls. Of interest and in
relation to ethanol behavior, dopamine-DARPP32 (dop amine- and cAMP-regulated
phosphoprotein) feedback signaling was significantly altered (p=0.00993). This pathway is
widely accepted to be important in modulating drug-seeking behavior. Surprisingly, when
analyzing the list of altered genes associated with these pathways, we again saw GRIN1 as the
only gene encoding a receptor/ion channel that was associated with this altered canonical
pathway. We did not find any significant changes in dopaminergic receptor genes.
We also use IPA to predict interacting gene networks to further evaluate the broader
potential effects of impact on the function of CNS due to the lack of P2X4R. Analysis of the one
of the top gene networks generated revealed several hub molecules, one of which is again
GRIN1, which serve as one central molecule to connect several potentially interacting gene
molecules (Figure 5.2). Based on the biological fun ction associated with these altered genes,
this gene network reveals that the lack of P2X4R KO can potentially lead to the development of
other neurological conditions that include, seizure, cognitive impairment, Parkinson’s, and
schizophrenia. Overall, these results suggested that the lack of P2X4R can impact certain
genes and signaling pathways that are central to CNS and overall health, and that GRIN1
appears to be an important gene that was altered in P2X4R KO under short-term ethanol
exposure. With our vast experience in studying ion channels using electrophysiological and
pharmacological manipulations, we decided to probe this focus molecule as a potential signaling
pathway through which P2X4R can regulation ethanol behavior.
94
Direction
of Change
Accession
Number
Gene
Symbol
Gene Name Fold
Change
Up NR_029766 mir-148a microRNA 148a 2.040
NM_001285905 CD2BP2 CD2 cytoplasmic tail binding protein 2 1.850
NM_130863 GRK2 G protein-coupled receptor kinase 2 1.650
NR_029901 mir-133 microRNA 133b 2.900
NR_029564 mir-154 microRNA 154 3.370
NM_001033633 SLC2A1 solute carrier family 2 member 1 1.800
NM_011596 ATP6V0A2 ATPase H+ transporting V0 subunit a2 1.610
NM_019916 TLX3 T-cell leukemia homebox 3 1.520
NM_016913 PORCN porcupine homolog 1.770
NM_001177656 GRIN1 glutamate ionotropic receptor NMDA type 1.720
Down NR_029562 mir-15 microRNA 15a 2.730
NM_001003685 GHRH growth hormone releasine hormone 1.960
NM_030704 HSPB8 heat shock protein family B (smal l) member 8 1.610
NM_008237 HES3 hes family bHLH transcription factor 3 1.530
NR_030571 mir-467 microRNA 467 1.574
NR_105850 mir-455 microRNA 455 4.320
NM_018853 Rplp1 ribosomal protein, large P1 2.920
NM_008250 HLX H2.0 like homebox 1.650
NM_001287386 GCK glucokinase 1.530
NM_00910 CXCR3 C-X-C motif chemokine receptor 3 1.990
Table 5.1. Select differentially expressed genes. Top 10 upregulated and downregulated genes in the NAc of
male P2X4R KO versus WT mice under short-term ethanol exposure. The majority of these genes have been
implicated to play be involved in development of different pathological conditions.
95
Altered Canonical Pathways p-value Focus Molecules
Cancer Drug Resistance by Drug
Efflux
0.00457 ABCC2, mir-130, mir-133, mir-154, mir-181, mir-379,
PIK3R2
CDK5 Signaling 0.00635 ADCY1, ADCY9, EGR1, MAPK4, MAPK11, PPP1CA,
PPP1R3D, PPP2R2C, PPP2R3A, PPP2R5A
Xenobiotic Metabolism Signaling 0.00851 ABCC2, CAMK1D, HDAC4, HSP90AA1, HSP90B1,
MAP3K5, MAP3K10, MAP3K13, MAPK11, MGST1,
MGST3, NDST4, PIK3R2, PPP2R2C, PPP2R3A,
PPP2R5A, PRKCH, SULT1E1
Dopamine-DARPP32 Feedback
in cAMP Signaling
0.0099 ADCY1, ADCY9, CSNK1G2, GRIN1, GUCY1B3, KCNJ2,
PLCL2, PPP1CA, PPP1R3D, PPP2R2C, PPP2R3A,
PPP2R5A, PRKCH
Production of Nitric Oxide and
Reactive Oxygen Species in
Macrophages
0.0108 MAP3K5, MAP3K10, MAP3K13, MAP11, PIK3R2,
PPP1CA, PPP1R3D, PPP2R2C, PPP2R3A, PPP2R5A,
PRKCH, RHOT2, RHOU, SERPINA1
Triacylglycerol Biosynthesis 0.0198 DGAT2, LPCAT4, PLPP2, PLPPR2, PORCN
GDP-mannose Biosynthesis 0.0245 GMPPA, MPI
GPCR-Mediated Nutrient
Sensing in Enteroendocrine Cells
0.0386 ADCY1, ADCY9, CCK, FFAR1, PLCL2, PRKCH,
RAPGEF4
Mitotic Roles of Polo-Like Kinase 0.0397 HSP90AA1, HSP90B1, PLK2, PPP2R2C, PPP2R3A,
PPP2R5A
Spliceosomal Cycle 0.0428 U2AF2
Table 5.2. IPA-implicated top altered canonical pathways. Top 10 potentially altered canonical pathways (rank ed
by p-value) that are associated with the differentially expressed genes in the NAc of male P2X4R KO versus WT
mice under short-term ethanol exposure.
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Activation of P2X4Rs significantly inhibited the current in NMDARs. Our genomic
data indicated that the lack of P2X4R perturbs the expression and/or activity of NMDARs
(GRIN1). To investigate the potential crosstalk int eraction between these receptors, we co-
expressed them using the Xenopus oocyte system. Stable current in both receptors were
achievable with the application of 100 mM glutamate for NMDARs and 5 µM ATP for P2X4Rs
(Figure 5.3). However, the activation of P2X4Rs sig nificantly reduced the current in NMDARs
Figure 5.2. IPA-derived gene network indicates interactions between the differentially expressed genes
in the NAc of P2X4R KO versus WT mice after short-term ethanol exposure. Focus molecules (colored)
are genes from the data set, red indicates significantly upregulated gene (p < 0.05), green indicates
significantly downregulated genes (p < 0.05), and w hite indicates molecules added from IPA knowledge base.
Solid-line arrow indicates direct interaction, and dashed-line arrow indicates indirect relationship. Analysis of
one of the top networks generated reveals that the lack of P2X4Rs may also lead to other CNS disorders that
include Parkinson’s, schizophrenia, seizure disorder, cognitive impairment, and degeneration of neurons.
97
with subsequent application of 100 mM glutamate. This inhibition is reversible following a 15
minutes washout period.
Pharmacological inhibition of NMDARs exhibited differential effects on 10E Intake
in P2X4R KO as compared to WT mice. Based on our findings discussed above that
demonstrated a potential interaction between P2X4Rs and NMDARs, and that P2X4R KO mice
had an increased in GRIN1 expression (encodes for G luN1 of NMDAR), we hypothesize that
these KO mice may also have altered NMDAR activity/function. We tested this by blocking
NMDARs using the non-selective antagonist memantine, and found that the 10 mg/kg
memantine exhibited differential activity in KO versus WT mice (Figure 5.4). Although
memantine was able to significantly reduce ethanol intake in both KO (t = 3.266, p = 0.0085)
and WT (t = 5.273, p = 0.0004) mice, the reduction in KO mice was significantly less when
compared to WT mice (59% WT versus 38% KO, t = 2.52 6, p = 0.0301). This implicates there
may be a differential effect on NMDARs activity/signaling between KO and WT mice in relation
to ethanol intake
Figure 5.3. Cross inhibition of NMDAR function by P2X4R, tested using the Xenopus oocyte expression
system and the two-electrode voltage clamp method. Traces showing the activation of P2X4Rs significantly
inhibited the function of NMDARs, with gray bar indicating application of 100 mM glutamate and black bar indicating
application of 5 µM ATP.
NMDAR
P2X4R NMDAR
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Discussion
The results in this chapter provide new insights regarding how P2X4R can regulate
ethanol behavior. We used a combination of different methods to confirm and validate our
findings. Our gene-profiling experiment identified a list of differentially expressed genes in the
NAc of P2X4R KO versus WT mice under short-term ethanol exposure. Analyzing this dataset
using several different approaches (i.e., top alter ed canonical pathways and associated focus
molecules, gene networks indicating interaction between focus molecules, top
upregulated/downregulated gene) have repeatedly identified GRIN1, which encodes for the
GluN1 subunit of the NMDAR, as an important molecule. Since this receptor/ion channel has
already been widely accepted as an important mediator of drug seeking behavior, we chose to
start probing for potential interaction between NMDAR and P2X4R to elucidate the signaling
mechanism(s) of how P2X4Rs mediate drinking.
Figure 5.4. Memantine (10 mg/kg) exhibited differential activity in P2X4R KO versus WT mice. 10
mg/kg memantine significantly reduced 10E intake in both KO and WT mice, however, the percentage of
reduction was significantly greater in WT mice (59% WT versus 38% KO). Values represent mean ± SEM for
6 mice in each group, ** p < 0.01, *** p < 0.001 versus respective pre-drug, # p < 0.05 versus memantine
treated KO mice.
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Our microarray results suggest that the lack of P2X4R under ethanol exposure leads to
an increased in expression and possibly activity of GRIN1 (NMDAR). In support of this
hypothesis, using a Xenopus oocyte expression system to probe the interaction between
P2X4R and NMDAR, we found that the activation of P2X4R significantly inhibited the current of
NMDAR (which was reversible following a long 15 min utes washout period). This finding is in
line with previous literature that indicates P2Xs can inhibit the function of NMDAR, using
hippocampal slices (Pankratov et al., 2002). Furthe r support of our hypothesis can be gleaned
from our genomic and electrophysiological findings, as well as our behavioral studies. We tested
the effect of memantine (NMDAR antagonist) in relat ion to ethanol intake in KO and WT mice.
We found that memantine was able to significantly reduce drinking in both groups. However, the
reduction was significantly LESS in P2X4R KO as compared to WT controls. Collectively, these
results suggest that the lack of P2X4Rs under ethanol exposure may induce a change in
expression/activity of NMDAR, or mainly increasing the glutamatergic tone in the KO mice.
The glutamatergic receptor system is known to have a significant role in mediating
tolerance and dependence to ethanol. Adaptive upregulation of glutamate activity after
prolonged ethanol exposure is well-document in several animal studies (Gass and Olive, 2008).
Microdialysis in rodents has revealed significantly elevated extracellular levels of glutamate
following chronic ethanol treatment in several brain regions that include the dorsal striatum,
NAc, and hippocampus. This elevated level persisted long after the acute ethanol withdrawal
period (Gass and Olive, 2008; Griffin et al., 2014) , which may explained why these animals
consume even greater amount of ethanol when re-expose to the substance. In agreement with
these findings, other investigation that microinjected selective glutamate reuptake antagonist
into the NAc of non-dependence ethanol-naïve mice also resulted in these animals drinking
excessively when first expose to ethanol as compared to saline-injected controls (Griffin et al.,
2014). Taken together, these findings implicate that glutamate activity in the NAc can play an
important role in the motivation and/or maintenance of ethanol consumption. These are in line
100
with our own finding that indicated the ethanol-naïve P2X4R KO already has an increased
glutamatergic state in the NAc, which may partially explained why these mice voluntarily
consumed greater ethanol as compared to WT littermates.
We also saw several top altered canonical pathways as implicated by IPA, one of which
was dopamine-DARPP32. Associated with this pathways again was GRIN1 and several other
gene molecules involved in the signaling cascade for this pathway (Cepeda et al., 2009;
Fernandez et al., 2006). DARPP-32 was initially discovered as the main target for dopaminergic
signaling, however subsequent studies have shown that DARPP-32 is also involved with
numerous other pathways including glutamate signaling (Greengard et al., 1998). When
phosphorylated at Thr-34 by protein kinase A (PKA), DARPP-32 becomes a potent inhibitor of
protein phosphatase 1 (PP1), which amplifies the ab ility of PKA to phosphorylate other
substrates such as NMDARs and GABA
A
Rs. DARPP-32 is also phosphorylated at other sites by
several different kinases that include casein kinase 1 (CK1), casein kinase 2 (CK2), and cyclin-
dependent kinase 5 (Cdk5). Phosphorylation at Thr- 75 by Cdk5 causes inhibition of PKA, while
phosphorylation at Ser-97 by CK2 increases the efficacy of DARPP-32 Thr-34 phosphorylation
by PKA, and phosphorylation of Ser-130 by CK1 decreases the rate of Thr-34
dephosphorylation by calcineurin (Nishi et al., 201 6). Earlier studies in striatal slices found that
the Ca
2+
-dependent actions of glutamate activates calcineurin (protein phosphatase 2, PP2) to
dephosphorylates phospho-Thr-34-DARPP32 and to counteract dopaminergic signaling that
leads to phosphorylation of Thr-34 by PKA (Greengar d et al., 1999). However, subsequent
studies found that glutamate and dopamine can also act synergistically, and glutamate activity
can induce a rapid increase in Thr-34 phosphorylation via activation of NMDARs (Nishi et al.,
2005). Future studies will be needed to fully elucidate the mechanism responsible for the
complex interactions with P2X4R KO mice under ethanol exposure. This should include further
biochemical and electrophysiological characterization to dissect what is happening in the
dopamine-DARPP32 pathway. Nevertheless, the present results suggest that P2X4R KO mice
101
under ethanol exposure exhibit significant changes in glutamate receptor activity which may
impacts (recruit/inhibit) other pathways such as do pamine-DARPP32 to regulate ethanol
behavior.
Further analyzing our differentially expressed gene dataset revealed that the P2X4R KO
mice has a potential to develop other CNS disorders (i.e., learning impairment, schizophrenia,
Parkinson’s), and that GRIN1 may be a central molecule for these pathological
neurodevelopments. In agreement this finding, a recent clinical retrospective study used
molecular and clinical data from several diagnostic and research cohorts to investigate the
functional consequences of different form GRIN1 mutation. The study found that these
individual all had distinct phenotype of profound developmental delay, severe intellectual
disability with absent of speech, muscular hypotonia, hyperkinetic movement disorder, cortical
blindness, cerebral atrophy, and epilepsy (Lemke et al., 2016). Thus, it is clear that a disruption
to GRIN1 normal function will likely lead to severe neurodevelopmental disorders.
Overall, the present findings provide new and unrecognized insights that begin to explain
how P2X4Rs regulate ethanol intake. Moreover, this work further supports evidence that P2X4R
play a role in the development of other CNS disorders. With further research P2X4R can be
positioned as a novel target for drug development for alcoholism and several other important
CNS conditions.
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Chapter 6
Overall Summary
Summary of Findings
The findings presented in my dissertation provide the preclinical support for the efficacy
of MOX in reducing ethanol intake and attenuating ethanol-induced withdrawal symptoms, using
several different rodent models of alcoholism. Moreover, this work highlights the importance of
P2X4Rs and the role these receptors play in AUD and possibly other CNS disorders. To briefly
summarize, Chapter 2 demonstrated that administration of MOX significantly reduced ethanol
intake in both male and female C57BL/6J mice in a dose-dependent manner, using the 24-two-
bottle choice and DID paradigms. We identified 2.5 mg/kg MOX as the minimum effective dose,
and when this was administered in a multi-days drinking study, MOX also consistently and
significantly reduced ethanol intake without signs of tolerance or overt toxicity. In agreement
with earlier work on other related analogs (Asatrya n et al., 2014), I also showed that MOX was
able to significantly antagonize ethanol inhibition on P2X4R function in vitro.
Chapter 3 investigated the efficacy of MOX using other rodent alcohol models that
represent more severe case of human alcohol-dependency. First, I showed that MOX was able
to significantly attenuate the ethanol-related symptoms induced by a single injection of a
sedative-hypnotic dose of ethanol (4 g/kg). Concurr ent injections of MOX with ethanol reduce
both the peak magnitude and overall severity of withdrawal. As ethanol action has been shown
to vary between sexes, I tested both male and female and found similar results. Second, we
showed that MOX was also able to significantly reduce ethanol intake in a mouse model of
ethanol-dependent. I used female mice that were rendered ethanol dependent by the IA two-
bottle choice. This model induced mice to voluntarily elevate their drinking to a significantly
higher level as compared to established baseline and this process is a cardinal feature of the
development of human alcoholism. Animal undergoing this paradigm also showed similar
103
changes in neurochemistry and neuroadaptations as in humans that are spiraling toward
addiction (Griffin et al., 2014). Administration ( i.p.) of 5 mg/kg MOX significantly and
consistently reduced ethanol intake in these mice during the entire testing period. Finally, I also
demonstrated that the anti-alcohol action of MOX in relation to ethanol intake and ethanol
withdrawal symptoms focus on the modulation of CNS targets rather than by affecting the
pharmacokinetic of ethanol. Analysis of BEC levels after concurrent administration (i.p.) of 5
mg/kg MOX and 4 g/kg ethanol showed similar changes in both male and female mice.
Chapter 4 focused on understanding the neuroanatomical specificity of P2X4R and its
relation to the regulation of ethanol intake. Using a lentiviral-shRNA methodology that reduced
P2X4R expression in the NAc core, I found a significant increase in ethanol intake. Of note,
stereotaxic injection of the viral construct did not affect body weight and food intake. These
results are in agreement with published genomic studies where an inverse correlation was
found between ethanol intake and the expression of p2rx4 gene, using different strains of mice
and rats (Tabakoff et al., 2009). Conversely, this result also reinforces our previous studies that
demonstrated the potentiation of P2X4R function by avermectins (e.g., IVM, MOX) can
significantly reduce ethanol intake in mice (Asatry an et al., 2014).
The exact mechanism underlying P2X4R’s anti-alcohol action is not fully understood.
However, building evidence suggests that P2X4Rs interacts with multiple receptor
neurotransmitter systems (e.g., GABA, glutamate, do pamine). These targets are well
established as playing a role in ethanol behavior. Chapter 5 employed several strategies that
began to elucidate the signaling mechanism(s) of P2 X4R in relation to ethanol intake. First, I
used a gene profiling approach to identify differentially expressed genes in the NAc of male
P2X4R KO mice versus WT under ethanol exposure (two -bottle choice for 2 weeks). I identified
significant upregulation of GRIN1 (encode GluN1 sub unit of NMDAR) in the list of top ten
upregulated genes. This is an interesting findings in that the role of NMDARs in alcoholism has
104
been suggested in several preclinical and human studies. In fact, compounds targeting the
glutamate systems are being used to treat alcoholism (Mason & Heyser, 2010).
Collectively, first, I found that P2X4R play a role in drinking behavior in mice by
regulating the expression and/or activity of NMDARs. Using in-vitro electrophysiology I
investigated the interaction between P2X4Rs and NMDARs. This initial work found that the
activation of P2X4Rs significantly inhibits NMDAR function (in a reversible manner), suggesting
that P2X4R KO mice may have increased activity in their NMDARs. Next, I used the NMDAR
antagonist in P2X4R KO versus WT mice, I found a significant difference in the degree of
ethanol intake. Finally using IPA proprietary knowledge base, I found linkage between altered
genes in P2X4R KO mice versus WT controls suggesting that P2X4Rs may be implicated in
several other CNS disorders including Parkinson’s, schizophrenia, learning ability and cognition,
which is in line with findings by our own and by others. Overall, these findings suggested that
P2X4R regulate ethanol intake by acting on NMDARs indirectly. In addition to emerging as a
novel target for AUD drug development, P2X4Rs may also serve as a novel target for several
other CNS conditions.
Future Directions
We have shown that MOX administration has consistent efficacy in reducing ethanol
intake across multiple acute and multi-day drinking paradigms with no signs of overt toxicity.
Clinical studies testing the acute administration of MOX (36 mg) in humans for a different
indication has also been shown to be very safe and well tolerated (Cotreau et al., 2003).
If MOX does become approved for the treatment of AUD, it will likely be used on a
chronic basis. As such, it is still necessary to evaluate the toxicity and efficacy in chronic
MOX/ethanol studies. To achieve the most accurate results, the experiment should be
performed using the same route that is also intended for clinical administration. I will utilize the
fast-dissolving film method to orally delivery MOX in these chronic efficacy and toxicology
studies (Yardley et al., 2015). This method was dev eloped by me and a former graduate
105
student, where we have shown that it can produce the same effect on ethanol intake that is
comparable to our previous studies where drugs were administered intrapertioneally, with
results that are reliable and reproducible (e.g., p lacebo oral-film-treated mice had similar
drinking level versus non-treated mice). With this approach, a specific dose of MOX will be
embedded on the fast-dissolving films, and when dip in a sucrose solution and present to
animals on a gavage needle tip, will result in the animals swallowing and instantaneously self-
administering the films (Yardley et al., 2015). Som e common methods that are routinely used to
orally administer drugs in rodents include oral gavage, peanut butter mixture, or adding the drug
to drinking water. In all of these methods, it is difficult to obtain reliable and reproducible results
(Gastrell & Crawley, 2013; Zhang, 2011). Several st udies have shown that it was difficult to
force the animals to consume the entire food/drug mixture during each drug administration
session. Repeat dosing using the gavage method have also been shown to increase mortality in
animals and/or caused irritation that can jeopardize the integrity of the results. As such, using
our oral-film delivery method will not only allow for ease of administration but will also produce
highly accurate results.
Multiple doses of MOX (1.25, 2.5, 5 mg/kg) will be administered daily for 3 months with
the Drinking-in-the-Dark paradigm in two groups (dr ug versus placebo). We chose this model
because it replicates human binge-liked behavior and will allow for the efficacy/toxicity of MOX
to be tested with a high-dose of ethanol. In addition to measuring the ethanol intake level daily,
we will also monitor for any sign of overt toxicity (e.g., abnormal home cage behavior, significant
reduction in weight/food intake), tolerance (e.g., the need to increase dosage to maintain
consistent reduction in drinking), and or dependence (e.g., physiological signs of
withdrawal/dependence upon cessation of drug treatment). At the end of each week, blood
samples will be collected for hematological and biochemical evaluation for sign of abnormality.
At the end of the study, tissues and major organs will be harvested for gross pathological and
histological examination (Parasuraman, 2011). These results will provide convincing evidences
106
that the long-term administration of MOX for the treatment and/or prevention of AUD is
efficacious and safe.
In parallel to the chronic studies described above, a multi-doses pharmacokinetic (PK)
study of MOX will also be conducted. We will be using several doses (acute and repeated
administration) revolving around the minimum effective dose (MED) for MOX anti-alcohol
activity that was previously identified (chapter 2) and also doses that are 2x-3x the MED. Animal
specimens (plasma and brain) will be collected at s equential time points and MOX level will be
quantified using a validated liquid chromatography-mass spectrometry (LC-MS) method. We will
use these PK results with all of our drinking data to develop a
pharmacokinetic/pharmacodynamics model. Any observed toxicity (from the higher dose range)
will also be built into the model. These results will help to further our understanding on the
dosing properties in relation to ethanol intake and will allow for the identification of appropriate
dosing regimen for future human studies. Overall, the data presented in this dissertation and
results from future proposed experiments will give a detailed understanding on the efficacy and
toxicity of MOX on treating the different stages of alcoholism, and will set the stage for the
clinical translation of MOX into clinic for the treatment and/or prevention of AUD.
107
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Abstract (if available)
Abstract
Alcohol use disorder (AUD) affects over 18 million people in the United States, costing an economic burden in excess of $220 billion and causing more than 100,000 deaths annually. Despite considerable effort and funding that have been dedicated toward drug development programs, currently we only have three medications approve by the Food and Drug Administration (FDA) for the treatment of AUD. These pharmacotherapies, even combined with psychosocial support, have only produced limited results as evident by the fact that more than 90% of patients relapse at least once during treatment and that the prevalence of alcohol abuse continues to rise. As such, the use of new approaches for the rapid development of effective AUD therapies represents an important public health goal. Using a translational strategy, our laboratory has been investigating the repurposing potential of compounds from the avermectin family (i.e., ivermectin [IVM] and moxidectin [MOX]) to be developed into novel medications to combat alcoholism. In particular, IVM has already been safely used in humans as an anti-parasitic agent for over two decades. With the advantage of having the lead compounds (such as IVM and MOX) already present and the abundant availability of safety data, the drug repurposing route is a fast and economically feasible approach for AUD drug development. Previous investigations have established that IVM and related analogues can significantly reduce ethanol intake in both male and female mice through multiple drinking paradigms and the anti-alcohol effects of these avermectins, in part, comes from their activity on P2X4 receptors (P2X4Rs). Although the evidence supports the development of IVM into an anti-alcohol medication for use in humans, there remains challenges in moving this molecule forward. As such, we have expending our investigation regarding the use of other avermectins. It is this quest that is the focus of my dissertation. My effort primarily focused on MOX with the goal of demonstrating that MOX: 1) exhibits comparable or better anti-alcohol efficacy to IVM, and 2) is more suitable for use on a chronic basis. My results, in agreement with published studies, have suggested that MOX has superior central nervous safety profile as compared to IVM. These advantages support the further development of MOX to be used as a long-term AUD medication. Importantly, and very timely, MOX is currently in clinical development as an alternate anti-parasitic therapy to IVM with no clinical abnormalities reported to date. If supported by my pre-clinical efficacy studies, MOX can be rapidly translated into human studies for the treatment of AUD. ❧ In the first half of my dissertation (chapters 2 & 3), I evaluated the pre-clinical efficacy of MOX using different rodent drinking models that represent different stages of alcoholism. In chapters 4 and 5 I began to elucidate the anti-alcohol mechanism(s) of MOX and other avermectins. As we have previously shown that P2X4R is an important target for avermectins’ anti-alcohol effect, chapter 4 investigated the neuroanatomical specificity of P2X4Rs in relation to ethanol behavior by utilizing a lentiviral-shRNA strategy for region-specific knockdown of brain P2X4Rs. Chapter 5 utilized a gene-profiling approach to identify potential signaling mechanism(s) that may explain how P2X4R regulate drinking in mice
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Huynh, Nhat Huu
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The preclinical evaluation of moxidectin as a platform for drug development for alcohol use disorder
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Clinical and Experimental Therapeutics
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05/10/2018
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