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Investigation of the anti-alcohol potential of avermectin drugs in in vitro studies of P2X4 and GABA(A) receptors

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Investigation of the anti-alcohol potential of avermectin drugs in in vitro studies of P2X4 and GABA(A) receptors

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INVESTIGATION OF THE ANTI-ALCOHOL POTENTIAL OF AVERMECTIN DRUGS IN
IN VITRO STUDIES OF P2X4 AND GABAA RECEPTORS

by

Ruowei Liu

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

May 2017

Copyright 2017 Ruowei Liu

2

DEDICATION

To my dearest parents and friends

3
ACKNOWLEDGEMENTS
I would like to express my greatest gratitude to my committee, mentors, lab mates,
friends and family for supporting and helping me all the time.

I would like to thank Dr. Daryl Davies, Dr. Liana Asatryan and Dr. Jing Liang for giving
me the opportunity to work in a new and significant field, opening my eyes to the deeper
insight of drug development and devoting time and energy on assisting me in my
experiments and dissertation.

I am grateful to my lab mates, Miriam Fine, Dr. Anna Naito, Sheraz Khoja, Dr. Nhat
Huynh, Natalie Arabian, and Larry Rodriguez, for their support and help. I would like to
especially thank Dr. Anna Naito for her guidance, patience and encouragement and
Miriam for her technical assistance.

Lastly, I want to thank Ana Cantuaria and Mengyun Wu who assisted me in collecting
data.

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TABLE OF CONTENTS
LIST OF FIGURES ............................................................................................................ 6
ABSTRACT ........................................................................................................................ 7
1. INTRODUCTION .......................................................................................................... 9
1.1 Alcohol use disorder (AUD) ......................................................................................... 9
1.1.1 Impact of AUD on individual’s health ..................................................................... 11
1.1.2 Impact of AUD on society ........................................................................................ 12
1.2 Treatments of AUD ..................................................................................................... 12
1.2.1 Approved therapies ................................................................................................... 13
1.2.2 Treatments under development ................................................................................. 14
1.3 Role of P2X4 receptors in AUD ................................................................................. 16
1.4 Role of GABA A receptors in AUD develop more ..................................................... 19
1.5 Avermectin family drugs ............................................................................................ 20
1.5.1 IVM ........................................................................................................................... 22
1.5.2 MOX ......................................................................................................................... 24
1.6 Background of the study ............................................................................................. 25
2. MATERIALS AND METHODS .................................................................................. 27
2.1 Materials ...................................................................................................................... 27
2.2 Methods ........................................................................................................................ 29
3. RESULTS ..................................................................................................................... 32
3.1 Effect of MOX and LMWI-002 on P2X4R function ................................................ 32
3.2 Effect of MOX and LMWI-002 on GABA AR function ............................................ 34
5
3.3 Effect of MOX and LMWI-002 on ethanol inhibition in P2X4Rs .......................... 35
4. DISCUSSION ............................................................................................................... 39
5. CONCLUSION ............................................................................................................. 42
References ......................................................................................................................... 43

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LIST OF FIGURES
Figure 1. Structure of IVM………………………………………………………………21
Figure 2. Structure of MOX……………………………………………………..……….25
Figure 3. Concentration-responding curves of co-application of IVM, MOX and LMWI-
002 (0.5-3 μM) with EC10 ATP.………………………………...…………….32
Figure 4. Bar graph of potencies of MOX and LMWI-002 compared with IVM in
modulating P2X4Rs.…………………………………………….……………33
Figure 5. Effect of MOX and LMWI-002 on GABAARs function.……………………...34
Figure 6a. Effects of MOX on ethanol inhibition of P2X4Rs.………...……..………….35
Figure 6b. Effect of LWMI-002 on ethanol inhibition in P2X4Rs..……………………..36
Figure 7. 0.5 μΜ MOX and LMWI-002 antagonized the inhibitory effects in P2X4Rs...37
Figure 8. 1.0 μΜ MOX and LMWI-002 antagonized the inhibitory effects.……………38

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ABSTRACT
Current therapeutic options for alcohol use disorder (AUD) are relatively inadequate,
which urges the development of novel and effective pharmacotherapies. Using a
translational approach, our laboratory has indicated that ivermectin (IVM), an FDA-
approved, human and animal anti-parasitic agent, and some other members of the
avermectin family can significantly antagonize alcohol effects in vitro and reduce ethanol
intake in mice across different drinking paradigms, showing their pharmacotherapeutic
potential to prevent and/or treat AUD. We hypothesized that structural modifications that
enhance IVM’s effects on key receptors and/or increase its brain concentration should
improve its anti-alcohol efficacy (Asatryan et al., 2014). Moxidectin (MOX) is an FDA-
approved animal anti-parasitic agent while LMWI-002 is a synthetic IVM analogue. In
this study, the in vitro anti-alcohol potential of MOX and LMWI-002 was investigated by
measuring their modulation of two important central nervous receptors, purinergic 2X4
receptors (P2X4Rs) and γ-aminobutyric acid type A receptors (GABAARs), compared
with IVM as the bench marker. Using a Xenopus oocyte expression system, MOX and
LMWI-002 were able to positively modulate P2X4R and GABAAR function and
antagonize the inhibitory effects of ethanol on ATP-activated currents in P2X4Rs.
However, the modulation of MOX on both receptors was significantly lower than IVM,
suggesting MOX had lower neurotoxicity potential, improved margin of safety, compared
to IVM. LMWI-002 showed atypical dose-response curve, indicating that it may have
other mechanisms of action. This finding represents the first report of MOX having
8
activity on P2X4Rs. Overall, the results illustrate the potential for development of MOX
as a novel pharmacotherapy for the treatment of AUD.
9
1. INTRODUCTION
1.1 Alcohol use disorder (AUD)
The history of alcohol consumption can be traced back to 10,000 B.C.(McGovern, 2009).
In ancient China, alcohol was used as the first anesthesia in surgery. Currently, global
consumption of pure alcohol is equal to 6.2 liters per year per capita aged 15 years or
older, which translates into 13.5 grams of pure alcohol per day. About 16.0% of drinkers
aged 15 years or older are in heavy episodic drinking worldwide (WHO, 2014).

Moderate consumption of alcoholic beverages may have beneficial effects on public
health (Pinder and Sandler, 2004). It has been demonstrated that wine can benefit
cardiovascular system due to its antimicrobial and antifungal properties, and may protect
against some forms of cancers (Leikert et al., 2002; Vinson et al., 2001). However, these
potential health benefits of wine may be attributed to the high content of polyphenol
antioxidants such as phenolic acids, stilbenes and flavonoids, instead of alcohol itself
(Kammerer et al., 2004). Certain compounds are also found in beer by in vitro studies
suggesting antioxidant, anti-carcinogenic, anti-inflammatory, estrogenic and/or antiviral
properties (Arranz et al., 2012). Moreover, more benefits related to moderate
consumption of alcoholic beverages are still under study (Barron et al., 2014; Mazué et
al., 2014).

However, at the same time, alcohol is the most frequently abused drug in the world. The
problem of alcohol use ranges widely from mild situations to severe ones, such as
10
alcoholism, alcohol dependence and withdrawal symptoms, all of which are referred as
alcohol use disorder (AUD). According to the National Institute on Alcohol Abuse and
Alcoholism (NIAAA), precisely, AUD is defined as a chronic, relapsing disorder that
results from a variety of genetic, psychosocial, and environmental factors. It is
characterized by increased tolerance to the effects of alcohol, the presence of
characteristic withdrawal signs and symptoms, and impaired control over the quantity and
frequency of drinking. Therefore, treatment for AUD is complicated. In the 5th edition of
the Diagnostic and Statistical Manual of Mental Disorders (DSM-5), anyone who meets
at least any two out of 11 criteria is diagnosed with AUD.

Excessive alcohol consumption has been associated with deterioration of human health,
social problems, and financial burdens. According to the WHO report, AUD has been
identified as one of five major global risk factors for chronic disease and injury (Lim et
al., 2013; WHO, 2014). In the U.S. alone, AUD affects more than 18 million people and
causes around 100, 000 deaths every year, ranking third of preventable causes of
morbidity and mortality (Bouchery et al., 2011; Grant et al., 2004; Harwood, 2000;
Johnson, 2010; Litten et al., 2012; Rehm et al., 2009). Worldwide, 3.3 million people die
from harmful use of alcohol each year, contributing to 5.9% of all global death according
to Global Health Observatory (GHO) data (WHO, 2014). Excessive alcohol consumption
is also associated with many social problems, like violence, psychiatric illness, drunk
driving, drug use, unsafe sex, suicide and premature death (Hill et al., 2009; Lam and
Chim, 2010). Moreover, it is commonly accepted that the abuse of ethanol not only
damages the health of the population, but also causes social impact in economic output.
11
For example, it is estimated that 220 billion dollars loss of productivity is caused by AUD
each year in the U.S. (Holden, 1987).

Compared to the severe effect of alcohol consumption, the success rate of available drugs
is poor. Approximately 70% of patients relapse back to heavy drinking within the first
year of treatment, even combined with psychological strategies (Johnson, 2008; Litten et
al., 2012). Therefore, novel treatments for AUD are needed urgently.

1.1.1 Impact of AUD on individual’s health
Alcohol, or more specifically, ethanol, produces several effects in humans (Davies, M,
2003). Ethanol is a structurally simple compound that can significantly affect people’s
health. Ethanol is a central nervous system (CNS) depressant and can affect human brains
on memory, emotions, balance and many other cognitive functions. Chronic ethanol
consumption puts an individual at higher risk of suffering from a lot of diseases,
including but not limited to, bleeding in the digestive tract, brain cell damage, Wernicke-
Korsakoff syndrome, eye problems, cancer, changes in the menstrual cycle, delirium
tremens, dementia and memory loss, depression, erectile dysfunction, heart damage, high
blood pressure, pancreatitis, liver disease including cirrhosis, nerve damage, insomnia
and fetal alcohol syndrome. What’s more, AUD may also lead to poor nutrition, sexually
transmitted infections, congenital defects, bone damage, weakened immune system and
some safety issues. AUD is the third leading risk factor for premature death and
disabilities (WHO, 2014; Yardley et al., 2012). In 2012, 139 million disability-adjusted
12
life years (DALYs), or 5.1% of the global burden of disease and injury, were attributable
to alcohol consumption (WHO, 2014).

More importantly, ethanol is a central nervous system (CNS) depressant and shares many
of the effects of other CNS depressants, such as sedatives, hypnotics, and anesthetic
agents. So it affects memory, emotions, balance and many other cognitive functions.

1.1.2 Impact of AUD on society
Apart from the impact on individuals, ethanol also does harm to the family, community
and even the society in large. It is common that AUD damages the relationship in a
family due to the poor performance at study or work and the considerable expense on
alcohol, AUD may also worsen addicts’ financial situation. Violence and crimes
including sexual assault occur more frequently among the group with AUD. Car
accidents related to alcohol cost the nation $132 billion per year (CDC, 2016). It is
reported that in 2014, 9,967 people were killed in alcohol-impaired driving crashes,
accounting for nearly one-third (31%) of all traffic-related deaths in the U.S. (CDC,
2016).

1.2 Treatments of AUD
It is ideal to have some effective treatments for AUD to help individuals as well as to
relieve its burden on society. The current treatments for AUD focus on reducing or
stopping alcohol consumption and preventing relapse. Some of them work on withdrawal
13
symptoms as aversion therapy, and others work on alcohol action receptors of
metabolism pathways. Despite of ongoing efforts focusing on the development of new
medications, there are only a few currently approved drugs for AUD working by either
blocking its metabolism or targeting the neurochemical systems in the downstream
signaling that leads to craving and dependence (Colombo et al., 2007; Gewiss et al.,
1991; Johnson et al., 2007; Xiong et al., 1999). It is evident by the continual prevalence
of high rates of uncontrolled heavy drinking and relapsing in patients even after long-
term inpatient treatment and support (U.S. DEPARTMENT OF HEALTH AND
HUMAN SERVICES, 2014). Therefore, it is crucial for personal healthcare and public
health burden to develop more effective medications to treat AUD.
.
1.2.1 Approved therapies
Clinically available treatments for AUD include three oral medications and one injectable
agent, disulfiram (Antabuse®), naltrexone (oral Revia® and injectable Vivitrol®), and
acamprosate (Campral®) (Harris et al., 2010; Litten et al., 2012). Due to the limit of
available medications, more agents are in the pipeline to resolve the unmet market.

Alcohol is metabolized mainly by two enzymes, alcohol dehydrogenase (ADH) and
aldehyde dehydrogenase (ALDH). First, ADH metabolizes alcohol to acetaldehyde, a
highly toxic substance and known carcinogen (Edenberg, H.J, 2007). Then, acetaldehyde
is further oxidized to acetate by ALDH (Edenberg, H.J, 2007), which then is broken
down into water and carbon dioxide for easy elimination (NIAAA, 1997). Disulfiram
disrupts the normal alcohol metabolism by inhibiting ALDH activity. Apparently, it
14
causes some undesired side effects due to the toxicity of acetaldehyde. On the other hand,
unpleasant physical symptoms caused by acetaldehyde accumulation in the blood stop
further drinking (Clapp, 2012).

As a functional N-methyl-D-aspartate receptor (NMDA) modulator, acamprosate works
to antagonize NMDA glutamate receptors, which restores the balance between excitatory
and inhibitory central nervous system (Bouza et al., 2004; Clapp, 2012; Davies et al.,
2013).

Naltrexone, an opioid antagonist, blocks the release of alcohol-induced dopamine,
thereby reducing the stimulus and reinforcing effects of ethanol (Bouza et al., 2004). It
also avoids the craving for alcohol and loss of control.

However, these treatments are not satisfying. They are only effective to certain patient
subgroups because of the heterogeneity and complex causes of alcoholism (Addolorato et
al., 2013; Franck and Jayaram-Lindstrom, 2013).

1.2.2 Treatments under development
Due to the limited treatments for AUD, some other compounds are undergoing
investigations to get approved by the FDA. They either target certain receptors, like 5-
HT3 receptors and GABAA receptors, or try to balance the inhibitory and excitatory
nervous system. However, they all focus on particular types of alcoholics.
15

Ondansetron is a 5-HT3 antagonist that shows efficacy for patients with early onset of
alcoholism (Kranzler et al., 2003; Sellers et al., 1994). The inhibition of 5-HT3 activation
results in reduced dopamine in the cortico-mesolimbic system, therefore, inhibits the
positive reinforcing effects of alcohol by acting on the dopamine mesolimbic system
(Heilig and Egli, 2006). It can be a promising drug for early-stage patients to avoid
deeper addiction.

Baclofen is an agonist of γ-aminobutyric acid type B (GABAB) receptor, showing
efficacy in reducing alcohol consumption, inhibiting the intention to seek alcohol and
decreasing relapse like binge drinking behavior in alcohol-preferring rats (Colombo et al.,
2000; Colombo et al., 2003a; Colombo et al., 2003b). Baclofen may reduce the rewarding
properties related to heavy drinking by activating GABAB receptors, which is responsible
for suppressing alcohol drinking through inhibition of the cortico-mesolimbic dopamine
system (Addolorato et al., 2009; Vengeliene et al., 2008). Besides, by activating GABAB
receptors in the limbic system, baclofen can reduce anxiety and other withdrawal
symptoms (Addolorato et al., 2009). Therefore, baclofen may be effective in relieving
alcohol dependence and withdrawal symptoms.

Topiramate counteracts the hyperexcitation of the glutaminergic system, causing
potentiation of GABAergic signaling and blocking the activity of calcium channel (Heilig
and Egli, 2006). Therefore, it blocks the motivation to consume alcohol. Clinical studies
have shown that topiramate at doses up to 300 mg/kg is effective in decreasing craving
16
and improves all drinking outcomes after 12 weeks of treatment compared to placebo
(Johnson et al., 2003; Ma et al., 2006).

There are more anti-alcohol agents being investigated. But all these agents have their own
drawbacks. Either they only work for certain types of symptoms, or they have side effects
issues. Furthermore, the mechanisms of some agents are still not clear. Therefore, further
understanding of the mechanism of alcohol action, and developing more effective agents
are urgently needed.
1.3 Role of P2X4 receptors in AUD
Adenosine 5’-triphosphate (ATP), a purine nucleotide, was recognized as a
neurotransmitter in 1972 (Asatryan et al., 2011). Adenosine signaling has been implicated
in the pathophysiology of many CNS disorders including sleep disorders, anxiety, and
alcoholism (Burnstock, 2008; Dunwiddie and Masino, 2001; Fredholm, 2010; Fredholm
et al., 2005). Also, the interaction between adenosine receptors and other G-protein
coupled receptors (GPCRs) in the striatum regulates motor and motivational behaviors
(Ferre et al., 2008; Ferre et al., 1997; Ferre et al., 2002; Ferre et al., 1996).

ATP-gated P2X receptors (P2XRs) are a superfamily of ligand-gated ion channels
(LGICs) (Khakh et al., 2001; North, 2002), and are being a focus of research in alcohol
studies. There are seven subunits of the P2XRs (P2X1-7) that can form homo- or
heteromeric channels, e.g., P2X1/4, P2X1/5, P2X2/3, P2X4/6, when expressed in
17
Xenopus oocytes or mammalian cell lines (Lalo et al., 2008; Le et al., 1998; Lewis et al.,
1995; Nicke et al., 2005).

Structurally, each P2XR subunit consists of 2 α-helical transmembrane (TM) segments, a
large extracellular domain (ectodomain), and an intracellular location of amino and
carboxy terminals (North, 2002).

P2XRs are widely distributed in the CNS on neurons (Surprenant and North, 2009) and
on glial cells (Heine et al., 2007; Inoue, 2008; Trang et al., 2006) in the mesolimbic
dopamine (DA) system, while the DA system plays a major role in ethanol consumption,
ethanol addiction, and reinforcement (Gonzales et al., 2004; Spanagel and Weiss, 1999).
The functions of P2XRs include learning and memory (Labrousse et al., 2009; Sim et al.,
2006; Wang et al., 2004), depression and anxiety (Basso et al., 2009), pain perception
(Honore et al., 2006; Jarvis et al., 2002; Tsuda et al., 2009; Ulmann et al., 2008), and
vascular tone (Yamamoto et al., 2006), demonstrated by multiple technologies, for
example, brain slice preparations, dissociated neuronal cultures, and P2XR knockout
(KO) mouse models (Surprenant and North, 2009). Furthermore, P2XRs are involved in
hormonal control of temperature regulation, food and water intake, sexual behavior, and
emotional responses (Stojilkovic, 2009), which are also implicated in AUD (Asatryan et
al., 2011). In addition, P2XRs modulate ethanol’s effect on GABAergic synaptic
transmission in the ventral tegmental area (VTA) (Xiao et al., 2008) and the activity of
dopaminergic neurons that are necessary for controlling ethanol intake (Asatryan et al.,
2011).
18

Among all the subunits of P2XRs, P2X4Rs are the most abundant subtype expressed in
the CNS (Buell et al., 1996; Soto et al., 1996) and associated with alcohol consumption
(Asatryan et al., 2011). The previous studies from Davies’ laboratory and colleagues have
shown that P2X4Rs expressed in Xenopus oocytes are sensitive to ethanol at intoxicating
and anesthetic concentrations (Davies et al., 2005; Davies et al., 2002; Xiong et al.,
2000). In P2X4Rs, residues contained within the ectodomain-TM interfaces (W46, D331,
M336) are crucial for causing or modulating the effects of ethanol (Jelinkova et al., 2008;
Popova et al., 2010).

In vitro, P2X4Rs are inhibited by ethanol concentrations as low as 5 mM (Asatryan et al.,
2010; Davies et al., 2005; Davies et al., 2002; Popova et al., 2010; Xiong et al., 2005;
Xiong et al., 1999), which concentration is below the 17.4 mM legal blood ethanol
concentration (BEC), as known as “legal intoxication” (i.e., 0.08%) in the United States
(Yardley et al., 2012). In vivo, lower functional expression of p2rx4 gene was observed in
alcohol-preferring rats compared to non-preferring group (Kimpel et al., 2007). p2rx4
gene may be linked to alcohol intake and/or preference, and there is an inverse
relationship between alcohol preference and the expression of the p2rx4 gene (Kimpel et
al., 2007; Tabakoff et al., 2009). Taken together, alcohol intake may be modulated by
ethanol acting on P2X4Rs and that pharmacological activation of P2X4Rs may reduce
alcohol consumption and preference (Yardley et al., 2012).

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To sum up, the findings in cellular and behavioral levels suggest that P2X4R plays an
important role in alcohol consumption. The inhibitory effect of ethanol on ATP-gated
P2X4R provides us a possible perspective that P2X4R can be a target and platform for
developing novel pharmacological agents that may have potential in the treatment for
AUD.
1.4 Role of GABAA receptors in AUD develop more
GABAARs are one of the primary inhibitory ligand-gated ion channels (LGICs) in the
CNS that have been demonstrated in affecting many acute and chronic behavioral effects
of ethanol, including tolerance, dependence, reward, anxiolysis, motor ataxia, impaired
cognition, sedation, and aggression (Deitrich et al., 1989; Dutertre et al., 2012; Follesa et
al., 2006; Grobin et al., 1998; Kumar et al., 2009; Lobo and Harris, 2008; Olsen et al.,
2007). They are pharmacologically activated by GABA and its selective agonist,
muscimol, blocked by bicuculline and picrotoxin, and modulated by benzodiazepines,
barbiturates, and certain other CNS depressants (Macdonald and Olsen, 1994; Sieghart et
al., 1999).

The extracellular loop 2 of GABAARs has been identified as a crucial site of ethanol
action and been found that structural modifications in this region influence receptor
sensitivity to ethanol (Crawford et al., 2007; Davies et al., 2004; Davies et al., 2003;
Mascia et al., 1996; Perkins et al., 2008). It was found that the ethanol sensitivity can be
significantly increased by replacing loop 2 of γ2 GABAAR subunits with loop 2 of a
more ethanol-sensitive δ GABAAR (Perkins et al., 2009). And it was identified that there
20
are important physical-chemical properties of loop 2 that alter receptor sensitivity to
ethanol and agonist (Crawford et al., 2008).

Currently, 19 homologous subunits of GABAAR are found. These subunits form
numerous, mostly hetero-oligomeric, pentamers (Olsen and Sieghart, 2008). The most
common native GABAARs are made up of two α, two β, and one γ or δ subunits
(McKernan and Whiting, 1996). The major GABAA in the CNS, approximately 75 to
80%, contain γ2 subunit, and this is the most abundant subunit in rat brain (Laurie et al.,
1992; Persohn et al., 1991, 1992; Wisden et al., 1992). Among all subunit combinations,
α1β2γ2 GABAARs are the most prevalent isoform of GABAARs.
1.5 Avermectin family drugs
Avermectins are a series of 16-membered macrocyclic lactone derivatives with potent
anthelmintic and insecticidal properties (Ōmura and Shiomi, 2007; Pitterna et al., 2009).
They are extracted from Streptomyces avermitilis, a soil actinomycete. So far, eight
different avermectins were isolated in four pairs of homologous compounds, with a major
(a-component) and minor (b-component) in a ratio of 80:20-90:10 (Pitterna et al., 2009)
(Figure 1).
21

Figure 1. Structure of IVM

Half of the 2015 Nobel Prize in Physiology or Medicine was awarded to the discoverers
of avermectin, William C. Campbell, and Satoshi Ōmura, because “avermectin, in
chemically modified form, ivermectin, proved effective against elephantiasis and river
blindness. Drugs based on these discoveries have led to the survival and improved health
of millions of people” (Foundation, 2015).

The primary mechanism of action of avermectins as antiparasitic agents is that they block
the transmission of electrical activity in invertebrate nerve and muscle cells by positively
modulating glutamate-gated chloride channel, which doesn’t exist in vertebrates
(Bloomquist, 1996, 2003; Cully et al., 1994). This mechanism leads to an influx of
chloride ions into the cells, causing hyperpolarization and subsequent paralysis of
22
invertebrate neuromuscular systems. However, some avermectins also modulate GABA
receptors because GABA receptors are the homologues in vertebrates of glutamate-gated
receptors in invertebrates.
1.5.1 IVM
IVM is a semisynthetic macrocyclic lactone, a dihydro avermectin derivative, used as a
broad spectrum anthelmintic both on humans and animals (Geary, 2005; Omura, 2008;
Richard-Lenoble et al., 2003). The mechanism of action of IVM as an antiparasitic agent
is attributed to action on a nonmammalian, glutamate-gated inhibitory chloride channel as
well (Cully et al., 1994; Dent et al., 1997; Le et al., 1998). On the other hand, IVM can
also positively modulate mammalian GABAA and glycine receptors in vitro (Dawson et
al., 2000; Shan et al., 2001). IVM exerts anticonvulsant and anxiolytic effects in rodents
which are linked to its action on GABAARs (Dawson et al., 2000; de Souza Spinosa et
al., 2002).

Some studies in humans suggest that IVM also affects other LGICs including nicotinic
acetylcholine receptor (Krause et al., 1998; Sattelle et al., 2009) and P2X4Rs (Khakh et
al., 1999). In fact, among all 7 P2XRs, IVM specifically modulates P2X4Rs, whch is the
basis for the utility of IVM as a tool to differentiate involvement of other P2X family
members in ATP-mediated processes (Khakh et al., 1999).

Based on the previous studies, IVM antagonizes ethanol in a dose-dependent manner
(Asatryan et al., 2010; Popova et al., 2010). It is illustrated that there are some
23
overlapping sites of action for ethanol and IVM (i.e., D331, M336) on P2X4Rs (Asatryan
et al., 2010; Jelinkova et al., 2008; Jelínková et al., 2006; Silberberg et al., 2007),
suggesting that the ectodomain-TM interface of P2X4Rs is a site of action and/or
modulation for both ethanol and IVM (Asatryan L, et al., 2011). More specifically, IVM
antagonized ethanol at a putative pocket at or near position 336 of P2X4Rs (Asatryan et
al., 2010). Therefore, P2X4Rs can be a potential target for the development of medication
for prevention/treatment of AUD (Asatryan et al., 2011).

The anti-alcohol effect of IVM has been demonstrated. First, it antagonizes ethanol-
mediated inhibition of P2X4Rs expressed in Xenopus oocytes. Second, it reduces ethanol
consumption in several mouse models (Yardley et al., 2012). In vitro, IVM can
significantly antagonize the inhibitory effects of ethanol on P2X4R function (Asatryan et
al., 2010; Asatryan et al., 2008; Asatryan et al., 2014). In vivo, IVM significantly reduces
ethanol intake and preference in mice across several validated alcohol drinking
paradigms (Asatryan et al., 2014; Wyatt et al., 2014; Yardley et al., 2012). More
importantly, IVM significantly reduce alcohol intake using a 24h access model (Yardley
et al., 2012) that mimics “social” or non-intoxicating levels of alcohol drinking (Blednov
et al., 2010) and using the intermittent limited-access model, which mimics binge-like
drinking (Yardley et al., 2012). IVM is a positive modulator of P2X4Rs but can be an
agonist of P2X4Rs at higher doses, e.g. 10 mM and higher. Notably, IVM has good
safety profile, not exhibiting any addiction potential, significant changes in pain
sensitivity, motor competency or memory, nor any visible signs of organ toxicity at as
high as 10 mg/kg (Bortolato et al., 2013; Yardley et al., 2012), but it slightly increased
24
the incidence of microcellular necrosis or perivascular inflammation (Yardley et al.,
2015).

However, due to the lipophilic nature of IVM, it can pass the blood-brain -arrier (BBB),
but does not readily reach high brain concentration because it is easily exported by P-
glycoprotein (P-gp). Therefore, structural modifications are needed to improve its brain
concentration and safety profile (Lespine et al., 2007; Menez et al., 2012).
1.5.2 MOX
MOX, an ivermectin analogue, is also a member of avermectins. (Figure 2) Some reports
in the literature suggest that MOX exhibits lower neurotoxicity potential compared to
IVM (Janko and Geyer, 2013; Menez et al., 2012), because 1) MOX has lower potency
on GABAARs as compared to IVM, which should be advantageous with respect to
reducing contraindications (Huynh et al., 2016), and 2) MOX is a weaker P-gp
transporter substrate and less dependent on P-gp for removal from the brain (Janko and
Geyer, 2013; Menez et al., 2012). Currently, MOX is being developed as an alternative
therapy to IVM as an anti-parasitic agent for humans with no significant clinical adverse
events reported (Cotreau et al., 2003; Korth-Bradley 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 GABAARs 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
25
P-gp substrates (Balayssac et al., 2005; Edwards, 2003; Prichard et al., 2012). Therefore,
MOX can be a potential candidate for AUD treatment.

Figure 2. Structure of MOX

1.6 Background of the study
AUD is a serious issue in the U.S. and in the world. Current therapies are undesirable
regarding efficacy and relapse rate. Considering both situations, it is necessary to develop
more effective agents to prevent/treat AUD.

Avermectins are a family of drugs that have been applied widely to animals or humans
with confirmed safety profile. Our laboratory has investigated the anti-alcohol effect of
26
IVM and some other avermectin compounds, i.e. Abamectin (ABM) and Selamectin
(SEL). IVM showed strong anti-alcohol effect, with potent modulation on P2X4Rs and
GABAARs, which may lead to side effects in CNS and complicated contraindications.
ABM significantly reduced ethanol intake and antagonized the inhibitory effects of
ethanol on P2X4Rs function with poor pharmacokinetics (PK) parameter in the brain
(Asatryan L, et al., 2014). SEL had great concentration and half-life, but the efficacy
wasn’t ideal. In a word, they have either potentially undesired side effects, limited
concentration in the brain, or insufficient efficacy. Therefore, we are exploring other
compounds, MOX and LMWI-002, trying to find a better candidate for AUD using
P2X4R as an in vitro platform and IVM as a benchmark.

Like IVM, MOX is also an FDA-approved agent with reported lower neurotoxicity
potential (Janko and Geyer, 2013; Menez et al., 2012). Therefore, to determine if MOX
has the potential to be developed into a safe and effective pharmacotherapy for AUD, this
study investigates the ability of MOX to modulate P2X4Rs and GABAARs and to
antagonize the inhibitory effects of alcohol.

On the other hand, based on the structure-efficacy relationships that we learnt from the
previous study of ABM and SEL, we collaborated with Dr. Kevin Gaffney in Dr.
Kathleen Roger’s laboratory (USC School of Pharmacy) to synthesize a novel compound,
named LMWI-002. This study also tested the anti-alcohol potential of LMWI-002.

27
2. MATERIALS AND METHODS
2.1 Materials
Buffers: Five-time concentrated Modified Barth’s solution (MBS) contains (in millimolar
(mM) concentration) 88 NaCl, 1.8 KCl, 5 HEPES, 0.82 Mg2SO4•7H2O, 2.4 NaHCO3,
0.41 CaCl2•2H2O, and 0.33 Ca(NO3)2•4H2O, adjusted to PH 7.5 with 5M NaOH. One-
time concentrated MBS was diluted from five-time MBS with purified water immediately
prior to using. Ten-times P2X buffer contains (in mM concentration) 110 NaCl, 2.5 KCl,
10 HEPES, 1.8 BaCl2, adjusted to PH 7.5 with 5M NaOH. One-time concentrated P2X
buffer was diluted from five-time MBS with purified water immediately prior to using.
Incubation medium contains (in mM concentration) 96 NaCl, 2 KCl, MgCl2, 1 CaCl2, 5
HEPES, and 2.5 pyruvic acid with 1% heat inactivated HyClone
®
horse serum (VMR,
San Dimas, CA) and 0.05 mg/ml gentamycin, adjusted to pH 7.4, which can be used
within two weeks.

Drugs: 10 mM stock solutions of IVM (powder from Sigma, USA), MOX (powder from
Sigma, USA), LMWI-002 (powder synthesized by Dr. Kevin Gaffney in Dr. Kathleen
Roger’s laboratory) were dissolved in Dimethyl sulfoxide (DMSO) and kept at -20ºC
until use (See Fig. 1 for chemical structures. The structure of LMWI-002 was
confidential). The highest DMSO concentration in the final solution was 0.1%, which did
not show significant effect with or without agonist on α1β2γ2 GABAARs nor P2X4Rs
currents in wild-type (WT) receptors from preliminary experiments. Compounds
solutions were diluted with MBS or P2X buffer, depending on receptors, immediately
28
prior to testing. 10 mM stock solution of GABA (powder from Sigma, USA) was
dissolved in MBS and kept fresh at 4ºC for two weeks. 100 mM ATP (powder from
Sigma, USA) solution was dissolved in P2X buffer and kept at -20ºC, then 10 mM stock
solution of ATP was diluted from 100 mM ATP solution with P2X buffer and kept at 4ºC
for 2 weeks. Ethanol (190 proof USP, Sigma, USA) was diluted to different
concentrations with MBS or P2X Buffer as needed.

Xenopus oocytes were purchased from Ecocyte Bioscience LLC, USA.
Electrophysiological experiments were conducted 24 hours after complementary RNA
(cRNA) injection and 48 hours after complementary DNA (cDNA) injection.

cRNA and cDNA synthesis: I use α1β2γ2 GABAAR as the wildtype (WT) model of
GABAARs because it’s the most prevalent isoform of GABAARs. The cDNAs of rat
P2X4R (GenBank accession No. X87763) and that of rat α1, β2 and γ2 subunits of
GABAAR were subcloned into the pcDNA3 vector (Invitrogen, USA). The DNA
containing p2rx4 gene was then linearized and transcribed using the mMESSAGE
mMACHINE kit (Ambion, USA) to result in cRNA, which was stored at -70ºC until
injection.

cRNA and cDNA injections: The oocytes were injected with 10 ng cRNA or 1 ng of
DNA mixture of α1: β2: γ2 GABAAR subunits at 1:1:10 ratio using Nanoject II Nanoliter
injection system (Drummond Scientific, USA). The oocytes were incubated with
29
incubation medium at 17ºC and used in electrophysiological recordings for 3-7 days after
injections.

Whole cell voltage clamp recordings: Two-electrode voltage clamp (TEVC) recordings
were performed using the Warner instrument model OC-725C oocyte clamp (USA). The
oocytes were voltage clamped at -70mV, and the currents were recorded on a chart
recorder (Barnstead/Thermolyne, USA).
2.2 Methods
P2X4Rs: Oocytes were continuously perfused at a rate of 3-4 ml/min with P2X buffer
using a peristaltic pump (Rainin Instrument, USA or Harvard Apparatus, USA). Ca
2+
in
the solution was replaced with Ba
2+
to prevent the activation of Ca
2+
-dependent Cl
-

channels (Khakh et al., 1999). All experiments were performed at room temperature (20-
23ºC).

To induce currents, submaximal concentrations (i.e. EC10) of ATP were used. Normally,
P2X4Rs EC10 is at 1 μM ATP. Using EC10 has been previously shown to maximize the
effects of ethanol while causing minimal receptor desensitization (Davies et al., 2005;
Davies et al., 2002)Ethanol and drugs were applied after stable responses to EC10 ATP
were obtained, which means at least three currents that the changes were no more that
10% compared to the last response. There was a washout period of 5 min between each
application to allow for resensitization of the receptor (Asatryan et al., 2010; Davies et
al., 2005; Davies et al., 2002; Popova et al., 2013).
30

Effects of ethanol (25, 50, 100 or 200 mM) or IVM, MOX and LMWI-002 (0.1-3 μM)
were tested alone and in combination with ATP for 25 seconds. ATP currents were
measured before and after each drug application in order to confirm the existence of a
stable baseline response. Pilot studies determined that the drugs did not have an effect on
the membrane potential of uninjected cells, nor did the drugs produce currents when
applied in the absence of agonist.

GABAARs: Oocytes were perfused at a rate of 3-4 ml/min with MBS using a peristaltic
pump (Rainin Instrument, USA). Similar to the approach for P2X4Rs, EC10 concentration
of GABA (i.e. 5 μΜ) was used to test the effect of the drugs. A washout period of 5 min
was allowed between each GABA application.

IVM, MOX and LMWI-002 were applied with GABA EC10 after a stable response to
GABA EC10 was obtained, which means at least three currents that the changes were no
more that 10% compared to the last response. Each oocyte was tested for one
concentration of drugs as avermectins caused irreversible effects that were not washable.
The second consecutive response to the application of the drug was always larger;
therefore, only the average currents of three stable responses prior the drug application
were counted into data analysis, and each oocyte was tested for one concentration of
drugs.

31
Two-electrodes whole-cell voltage clamp recording: Oocytes were put in a small
depression in an oocyte-recording chamber (total volume 100 μl) and were impaled with
two glass electrodes, which were pulled from 1.2 mm thick-walled glass capillaries (WPI,
Sarasota, FL). The electrodes had a resistance of 0.5-2 MΩ when filled with 3 M KCl and
voltage-clamped at -70 mV (Warner Instruments model OC-725C oocyte clamp,
Hamden, CT). Oocytes were perfused continuously at the rate of 3-4 ml/min by
extracellular bathing solution via roller pump (Rainin Instrument, USA or Harvard
Apparatus, USA) through 18-gauge polyethylene tubing (Becton Dickinson, Sparks,
MD). A chart recorder (Barnstead/Thermolyne, Dubuque, IA) continuously recorded the
clamping currents. The peaks were measured and used in data analysis.

Data analyses: Data were obtained from at least two batches of oocytes and were
expressed as mean+/-S.E.M. for an indicated number of test. The results were presented
as the percentage change in agonist EC10 alone. To access concentration-response
relationships, data were fitted to a concentration-response curve by using the following
logistic equation: I=Imax * [drug]/([drug]+(EC50)drug), where I is the percentage of the
maximum obtainable response (Imax), EC50 is the drug concentration producing a half-
maximal response. Bar graphs were used to compare the effects of ethanol with and
without compounds on P2X4Rs. GraphPad Prism Software (USA) was used for data
analysis and curve fitting. Statistical analysis was performed using unpaired t-tests with
significance set and p≤0.05.
32
3. RESULTS
3.1 Effect of MOX and LMWI-002 on P2X4R function
To determine the effect of MOX and LMWI-002 on P2X4 receptor function, I conducted
experiments testing MOX, LMWI-002 versus IVM. Prior findings demonstrated that
IVM (0.5, 1, 3, 10 μM) potentiated ATP-gated currents in P2X4Rs expressed in Xenopus
oocytes (Asatryan et al., 2010; Khakh et al., 1999; Priel and Silberberg, 2004). Therefore,
to test the effects of MOX and LMWI-002 on P2X4Rs, I chose to use a similar series of
concentrations (0.5, 1.0, 3.0, 10.0 μM) that was used in the previous IVM study
(Asatryan et al., 2010). I compared the ability of MOX, LMWI-002 versus IVM to
modulate ATP-gated currents in P2X4Rs. (Figure 3, 4)
Avermectin Concentration ( M)
ATP-induced Currents,
% Modulation
0 1 2 3
0
20
40
60
MOX
IVM
LMWI-002

Figure 3. Concentration-responding curves of co-application of IVM, MOX and LMWI-
002 (0.5-3 μM) with EC10 ATP.
33
Avermectin Concentration ( M)
ATP-induced Currents,
% Modulation
0
100
200
300
MOX
IVM
LMWI-002
0.5 1.0 3.0 10.0
***
*
***
***
****

Figure 4. Bar graph of potencies of MOX and LMWI-002 compared with IVM in
modulating P2X4Rs.

MOX and LMWI-002 both potentiated ATP-induced currents in P2X4Rs in a
concentration-dependent manner (Figure 3, 4). The degree of MOX potentiation was
significantly less than that of IVM and LMWI-002 (p<0.05), especially at concentrations
higher than 1 μΜ (Figure 4). LMWI-002 showed significantly less potentiation at 0.5 μΜ
only (p<0.05). At concentrations 1 μΜ and above, the extent of LMWI-002 potentiation
was similar to that of IVM (Figure 4). It demonstrated that IVM, MOX and LMWI-002
were positive modulators. The modulation of MOX was the weakest, while that of
LMWI-002 was modest but still weaker than IVM.

34
3.2 Effect of MOX and LMWI-002 on GABAAR function
I also tested the modulation of MOX and LMWI-002 on GABAARs at 0.1, 0.5, 1.0, 3.0
μΜ, because some studies demonstrate that IVM and other related avermectins have
significant GABAergic activity (Harris et al., 2010; Janko and Geyer, 2013; Menez et al.,
2012). I used α1β2γ2 GABAAR because of its predominant expression in the mammalian
CNS
Avermectin Concentration ( M)
GABA-Induced Current,
% Potentiation
0
100
200
300
400
500
IVM
MOX
LMWI-002
0.1 0.5 1.0 3.0
**

Figure 5. Effect of MOX and LMWI-002 on GABAARs function.

As illustrated in Figure 5, at 1.0 μΜ, MOX showed significantly less positive modulation
than IVM. Otherwise, there was no statistical difference between IVM and MOX at other
concentrations. There was no significant difference between the degree of modulation of
GABAAR function between IVM and LMWI-002. At lower concentrations (0.1, 0.5 and
1.0 μΜ), IVM showed stronger modulation. To sum up, both MOX and LMWI-002
significantly enhanced GABAAR function.
35

3.3 Effect of MOX and LMWI-002 on ethanol inhibition in P2X4Rs
I extended this investigation including concentration-response studies for MOX and
LMWI-002 antagonism of ethanol. To accomplish this, a similar approach used in our
prior IVM studies was used (Asatryan et al., 2010; Popova et al., 2013). It was completed
by testing a concentration range (0.5, 1, 3, 10 μΜ) of MOX and LMWI-002 on the effects
of ethanol (25, 50, 100, 200 mM) in P2X4Rs (Figure 6a). With the increase of the
concentration of MOX, the curve showed a parallel right-shift, which meant the anti-
alcohol effect of MOX was dose-dependent.

EtOH Concentration (mM)
ATP-induced currents, % modulation
0 25 50 75 100 125 150 175 200
-60
-40
-20
0
20
40
0 M MOX
0.5 M MOX
1.0 M MOX
3.0 M MOX
10.0 M MOX

Figure 6a. Effects of MOX on ethanol inhibition of P2X4Rs.

36
Compared to MOX, the modulation curves of LMWI-002 were not very stable and
typical (Figure 6b). But it still basically showed a parallel right-shift. However, with the
increase of the concentration of LMWI-002, the positive modulation was remarkable,
suggesting potential side effects. In addition, at lower concentrations (0.5 and 1.0 μΜ), it
showed limited modulation, especially at 1.0 μΜ. Because it wasn’t quite dose-
dependent, there was a possibility of unknown mechanisms of action.

EtOH Concentration (mM) ATP-induced Currents, % Modulation
0 25 50 75 100 125 150 175 200
-50
0
50
100
150
200
0 M LWMI-002
0.5 M LWMI-002
1.0 M LWMI-002
3.0 M LWMI-002
10.0 M LWMI-002

Figure 6b. Effect of LWMI-002 on ethanol inhibition in P2X4Rs.

Further, I compared the anti-alcohol effect of MOX and LMWI-002 at lower
concentrations (0.5 and 1.0 μΜ) on P2X4Rs by behaviorally relevant ethanol
concentrations (25 and 50 mM) (Figure 7 and 8). MOX at 0.5 μΜ completely
37
antagonized inhibition of P2X4Rs function by behaviorally relevant ethanol
concentration, 25 mM (Figure 7). It also significantly reduced inhibition of P2X4R
function caused by higher 50 mM ethanol concentration (Figure 7). MOX was able to
completely antagonize the 50 mM ethanol-induced inhibition at higher 1.0 μΜ
concentration (Figure 8).
EtOH Concentration (mM)
ATP-induced Currents, % Modulation
-15
0
15
30
0.5 M MOX
0.5 M LMWI-002
EtOH
MOX+EtOH
LMWI-002+EtOH
25 50
****
***
*
****

Figure 7. 0.5 μΜ MOX and LMWI-002 antagonized the inhibitory effects in P2X4Rs.

38
EtOH Concentration (mM)
-30
-15
0
15
30
1.0 M MOX
1.0 M LWMI-002
EtOH
MOX+EtOH
LWMI-002+EtOH
*
***
**
**
25 50

Figure 8. 1.0 μΜ MOX and LMWI-002 antagonized the inhibitory effects.

For behaviorally relevant ethanol concentrations, LMWI-002 showed antagonism to
ethanol inhibitory effect. At 0.5 μΜ, LMWI-002 could completely reverse the ethanol
caused inhibition. However, it could antagonize more for 50 mM ethanol than 25 mM
(Figure 7). Also, interestingly, at higher concentration, 1.0 μΜ, LMWI-002 could not
completely reverse the ethanol inhibitory effect, but significantly antagonized it (Figure
8).

39
4. DISCUSSION
The present study is the first in vitro investigation to test the utility of MOX as a
pharmacotherapy for AUD, providing insights into the mechanisms and important
structural features of MOX responsible for its potential to reduce ethanol effect that can
inform future development. We also evaluated the anti-alcohol potential of another newly
synthesized avermectin LMWI-002. The findings indicate that the structure and ability to
modulate P2X4Rs and antagonize ethanol effects in P2X4Rs are important determinants
for predicting the potential of avermectins to reduce ethanol effects. Using a Xenopus
oocyte expression system combined with two-electrode voltage clamp electrophysiology,
we found that MOX and LMWI-002 significantly potentiated P2X4R and GABAAR
function. In addition, low concentrations of MOX (0.5 and 1 μΜ) eliminated the
inhibitory effects of ethanol inP2X4Rs at behaviorally relevant concentrations (25 and 50
mM). These findings support more beneficial effects of MOX versus IVM combined with
the enhanced ability to penetrate the BBB. Based on the overall weaker action on P2X4R
and not significantly stronger action on GABAAR , the effects of MOX will be limited
regarding secondary effects on other receptors as well neurotoxicity which are typical for
IVM.

The differences in the modulating effects on P2X4R and GABAAR function and in the
antagonistic potential on P2X4R of MOX and IVM may be explained by the differences
in structural features and physical-chemical properties of IVM and MOX. IVM and MOX
share a common macrocyclic lactone ring and are different by specific substituents at the
C13, C23, and C25 positions (Figure 1 and Figure 2). These substituents play a role in
40
influencing the lipophilicity and affinity for P-gp transporters. The higher lipophilicity of
MOX compared to IVM (log PMOX= 6, log P IVM= 4.8) is consistent with its higher
entrance into the brain, greater accumulation in adipose tissue, and longer retention time
in the organism (Chen et al., 2005; Lanusse et al., 1997; Menez et al., 2012). In addition,
MOX has been indicated to have a lower affinity for P-gp compared with IVM and other
avermectins based on structural and biochemical studies. The disaccharide sugar moiety
found on IVM is absent on MOX. This moiety is thought to govern the affinity for P-gp
(Lespine et al., 2007). Thus, it appears that the pharmacokinetics of MOX is influenced
by higher lipophilicity and lower affinity for P-gp compared to IVM’s lower
hydrophobicity and higher affinity for P-gp (Chen et al., 2005; Lanusse et al., 1997;
Lespine et al., 2007; Menez et al., 2012).

Though to date the exact moieties of avermectins including IVM and MOX responsible
for the interaction with P2X4Rs and GABAARs are not defined, it is plausible to assume
that the absence of the disaccharide group may render MOX’s weaker interaction with
the brain receptors. On the other hand, smaller molecular weight and higher lipophilicity
may result in longer retention of MOX in the action pockets of these receptors. My
findings suggest that at least a portion of MOX’s and IVM’s anti-alcohol effects are
linked to their antagonistic action in P2X4Rs. As ethanol exerts its action through filling
and modifying cavities in these receptors, MOX can effectively displace ethanol from
these “action” pockets and thus be efficient in antagonizing effects of ethanol at
behaviorally relevant concentrations. Another potential mechanism of MOX and IVM’s
41
anti-alcohol effect might be that they active receptors without actually displacing EtOH,
which has been observed at high dose, e.g. 10 mM.

Compared to MOX, LMWI-002 showed potent modulation in P2X4Rs but with weaker
anti-alcohol effect, which suggests that LMWI-002 may be not a good candidate. Also,
the stock solution of LMWI-002 had some physical change after several months, with the
possible chemical change because it did not show the previous effect. It was noticed
because the stock solution was stored in -20-degree freezer and was supposed to be solid,
but it became liquid suggesting some change might have happened. This phenomenon
suggests that it should be made relatively fresh. Furthermore, further stability test for
DMSO solutions should be conducted for new synthetic compounds.

At present, the findings are an important first step and support the development of the
treatment for AUD as a novel pharmacotherapy. Currently, MOX is being developed for
use in humans as an alternative for IVM, once fully approved for human use, it should
have the potential to be repurposed and quickly advanced to the clinic for the treatment
and/or prevention of AUD.
42
5. CONCLUSION
The results demonstrate that MOX and LMWI-002 both shows positive modulation on
P2X4Rs and GABAARs and anti-alcohol effects. Overall, MOX shows better potential to
be a treatment for AUD because of moderate modulation, active anti-alcohol effect, and
stable physical-chemical properties.
43
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Abstract (if available)

Abstract Current therapeutic options for alcohol use disorder (AUD) are relatively inadequate, which urges the development of novel and effective pharmacotherapies. Using a translational approach, our laboratory has indicated that ivermectin (IVM), an FDA-approved, human and animal anti-parasitic agent, and some other members of the avermectin family can significantly antagonize alcohol effects in vitro and reduce ethanol intake in mice across different drinking paradigms, showing their pharmacotherapeutic potential to prevent and/or treat AUD. We hypothesized that structural modifications that enhance IVM’s effects on key receptors and/or increase its brain concentration should improve its anti-alcohol efficacy (Asatryan et al., 2014). Moxidectin (MOX) is an FDA-approved animal anti-parasitic agent while LMWI-002 is a synthetic IVM analogue. In this study, the in vitro anti-alcohol potential of MOX and LMWI-002 was investigated by measuring their modulation of two important central nervous receptors, purinergic 2X4 receptors (P2X4Rs) and γ-aminobutyric acid type A receptors (GABAARs), compared with IVM as the bench marker. Using a Xenopus oocyte expression system, MOX and LMWI-002 were able to positively modulate P2X4R and GABAAR function and antagonize the inhibitory effects of ethanol on ATP-activated currents in P2X4Rs. However, the modulation of MOX on both receptors was significantly lower than IVM, suggesting MOX had lower neurotoxicity potential, improved margin of safety, compared to IVM. LMWI-002 showed atypical dose-response curve, indicating that it may have other mechanisms of action. This finding represents the first report of MOX having activity on P2X4Rs. Overall, the results illustrate the potential for development of MOX as a novel pharmacotherapy for the treatment of AUD.

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Asset Metadata

Creator Liu, Ruowei (author)

Core Title Investigation of the anti-alcohol potential of avermectin drugs in in vitro studies of P2X4 and GABA(A) receptors

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 02/15/2017

Defense Date 10/24/2016

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

Tag anti-alcohol,avermectin,GABA(A) receptor,in vitro,OAI-PMH Harvest,P2X4 recptor

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Stallcup, Michael (committee chair)

Creator Email ruowei.liu@med.usc.edu,ruoweili@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-339027

Unique identifier UC11258184

Identifier etd-LiuRuowei-5073.pdf (filename),usctheses-c40-339027 (legacy record id)

Legacy Identifier etd-LiuRuowei-5073.pdf

Dmrecord 339027

Document Type Thesis

Rights Liu, Ruowei

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA