Saline IVM Saline 0123 *** Treatment W a t e r I n t a k e ( m L )Saline IVM Saline 0123456 *** Treatment F l u i d I n t a k e ( m L ) Saline IVM Saline

M Saline 0 10 20 30 40 50 60 70 *** Treatment P r e f e r e n c e f o r E t O H ( % )Saline IVM Saline 0.0 2.5 5.0 7.5 10.0 12.5 *** Treatment E t O H I n t a k e ( g / k g ) Saline IVM Saline 05 10 15 *** Treatment E t O H I n t a k e ( g / k g )Saline IVM Saline 0.0 2.5 5.0 7.5 10.0 *** Treatment E t O H I n t a k e ( g / k g )
ABSTRACT
This study is the first to show that IVM can reduce
ethanol intake over a prolonged period and provides
additional support for translation from rodent to human
for the use of IVM in the prevention and/or treatment of
AUDs.

We have shown that orally delivered IVM reduces 10E
intake and 10E preference (****p < 0.0001; Figure 1 A-B)
when administered orally for 14 consecutive days at a
concentration (3.1 mg/kg) that correlates with an already
established safe dosage in humans (30 mg PO).

Longer-term, orally delivered IVM administered at a higher
dosage (5.0 mg/kg/day) significantly reduced 10E intake
(****p < 0.0001; Figure 2 A-B), and 10E preference (***p <
0.001; Figure 2 C) without causing any signs of toxicity.
The effects of IVM were still significant on day 39 (****p
< 0.0001), suggesting a lack of tolerance to IVM.
Overall, there was a 46% (4.88 g/kg/24-h) reduction of
alcohol intake over the 39 day treatment period.
Importantly, no overt changes in behavior, water intake,
weight, or food intake were observed over the course of
the study (Figure 3).

IVMs ability to reduce alcohol intake and preference in
mice may be linked to its actions on multiple receptor
families including purinergic and GABA-ergic systems.

The use of IVM, an FDA approved compound used by
millions of humans as an anthelmintic agent, for the
treatment of AUDs is an innovative approach.
Repurposing IVM as a potential new therapeutic for
AUDs could be implemented in a shorter time frame
and at lower cost than required for the development of
entirely new chemical entities.

IVM may serve as a platform for further development of
new agents with potentially greater efficacy and safety
than IVM in preventing and treating AUDs.



1. NIH. Alcohol Facts and Statistics [Internet]. Bethesda, MD: National Institute on Drug Abuse. 2. Davies DL, Bortolato M,
Finn DA, Ramaker MJ, Barak S, Ron D, Liang J, Olsen RW (2012) Recent Advances in the Discovery and Preclinical Testing of
Novel Compounds for the Prevention and/or Treatment of Alcohol Use Disorders. Alcohol Clin Exp Res doi: 10.1111/j.1530-
0277.2012.01846.x [Epub ahead of print]. 3. Asatryan L, Popova M, Perkins DI, Trudell JR, Alkana RL, Davies DL (2010)
Ivermectin antagonizes ethanol inhibition in P2X4 receptors. Pharmacol Exp Ther 334:720728. 4. Bortolato M, Yardley M,
Khoja S, Godar SC, Asatryan L, Finn DA, Alkana RL, Louie SG, Davies DL: Pharmacological insights into the role of P2X4
receptors in behavioral regulation: lessons from Ivermectin. In.; conditionally accepted, International Journal of
Neuropsychopharmacology. 5. Yardley MM, Wyatt L, Khoja S, Asatryan L, Ramaker MJ, Finn DA, Alkana RL, Huynh N, Louie
SG, Petasis NA, Bortolato M, Davies DL (2012) Ivermectin reduces alcohol intake and preference in mice. Neuropharmacology
63(2): 190-201.
24-h two-bottle choice paradigm
This paradigm was performed as previously described (Yardley et al 2012). Mice WT
C57BL/6 mice (n=11 female, n=22 male) were individually housed in a Lucite box with
sawdust and cotton bedding. The box was covered by a metal grid cage top containing
food and two graduated bottles of solution, 10% v/v ethanol solution (10E) in tap water,
and tap water. Fluid levels were recorded daily by measuring the meniscus (0.1
mL). Additionally, the orientation of the bottles was switched every other day in order to
account for orientation-based fluid intake, and a control cage was present to measure
the amount of solution leakage and evaporation that could be present. Access to food
and both bottles of solution was available ad libitum and food and weight
measurements were recorded daily to observe any possible toxicity related problems.
Subjects were given one week to get acclimated to their environment during which
daily 10E intake was measured until it stabilized (10% variability from the mean dose of
the last 3 days). After establishing stable alcohol drinking levels, mice received the
vehicle via daily intragastric delivery (gavage) until 10E intake stabilized. Mice were
then gavaged with IVM (5.0 mg/kg/day female, 3.1 mg/kg/day male) for the duration of
the study (39 days females 14 days male). All intragastric delivery procedures were
performed with a 20 gauge stainless steel needle and occurred immediately prior to the
period of 24 h access to 10E versus tap water.

Statistical Analysis
Values for all parameters were summarized using means SEM across groups. For
each parameter analyzed, values obtained during the drug treatment period were
averaged (multiple comparison tests were used to exclude significantly different values
that were obtained during the drug treatment period). This average was then compared
to the values obtained during the last day of the vehicle administration period (pre
IVM). A paired t-test was used to calculate the significance. GraphPAD Prism software
(San Diego, CA) was used for data analysis. For all studies, the confidence interval
was set to 95%.
Implications of our preclinical findings suggest that
Ivermectin (IVM), an FDA approved broad spectrum
anthelmintic agent and purigenic P2X4 receptor (P2X4R)
modulator, may be an effective compound for the treatment
of AUDs
2
. Phase 1 testing aimed at repositioning IVM is
currently underway based on our thorough preclinical
assessment which yielded the following results:
In vitro, IVM antagonizes the inhibitory effects of ethanol on
P2X4 receptors expressed throughout the mesolimbic
dopamine pathway
3
.
In vivo, acute administration of IVM (1.25 to 10.0 mg/kg i.p.)
significantly: 1) Reduces ethanol intake without exerting
rewarding properties
4,5
; 2) Reduces anxiety
4
; 3) Does not
cause any overt signs associated with IVM toxicity when
assessed across a wide range of well-validated behavioral
assessments of sensory, motor, and cognitive competence
4
(see table 1).

Collectively, our initial murine data point to IVM as a safe,
tolerable, and effective agent for the treatment of AUDs.
However, AUDs are chronic in nature. Hence, effective
treatments would require longer term dosing strategies and
would be most convenient if effective in an oral dose
formulation. The current study tested the hypothesis that
chronic IVM administration could reduce alcohol intake in mice
without causing significant changes in animal behavior or
toxicity. This was accomplished by administering IVM
(5mg/kg), orally for 39 days.
Table 1.

IVM (5.0 mg/kg/day) Delivered Long-Term (39 days) via Oral Gavage
Significantly Reduces Ethanol Intake
Behavioral Assessment Result
Startle Reactivity Startle
Visual Cliff (visual acuity) No Change
Sticky Tape (tactile sensitivity) No Change
Hot Plate (analgesia) No Change
Open Field (locomotion) No Change
Elevated Plus Maze Anxiety
Marble Burying Anxiety
Tail Suspension No Change
Novel Object Exploration No Change
Novel Object Recognition No Change
Conditioned Place Preference No Effect

Alcohol use disorders (AUD) are a health crisis of unmet need. We
propose that ivermectin (IVM), an FDA approved drug, can be
repurposed to address this problem. Our laboratory recently
reported that acute administration of IVM (1.25 - 10.0 mg/kg i.p.)
significantly reduces murine ethanol intake without causing any overt
signs of toxicity, suggesting that IVM represents a safe, tolerable,
and effective agent for the treatment of AUDs. However, efficacious
treatment regimens would require longer term dosing strategies that
would be most convenient if effective in an oral dose formulation.
The current study tested the hypothesis that long term oral
administration of IVM reduces alcohol intake. We examined the
effects of orally delivered IVM (3.1mg/kg/day) in male alcohol
preferring C57BL/6 mice over a 14 day treatment period. This dose
was selected based on allometric scaling where we identified 3.1
mg/kg as one that correlates to an already established safe dosage
tested in humans (30 mg). Alcohol consumption was measured
using a 24 h, two-bottle choice (one bottle contained tap water, one
bottle contained a 10% v/v ethanol solution [10E]) paradigm. Overall
found that IVM significantly reduced alcohol intake and preference
without any overt signs of toxicity. Taken together, the findings
provide support for the development of IVM for the prevention and/or
treatment of AUDs.
ABSTRACT
A) Bars represents ethanol intake levels from the period prior to IVM administration (black; vehicle), and each day of the IVM treatment
period (grey; IVM days 1-39). B-C) Red points represent pre-IVM values; black points represent the average values from the first five weeks
of the IVM treatment period (1-5). Values represent the mean SEM for 11 mice.
W
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Values are the mean SEM for n=11 female mice. After achieving stable drinking levels for 3 consecutive days, IVM was administered for
39 consecutive days. Bars represent levels from the day prior to IVM injections (white; Pre IVM), and the 39 day average of each mouse
during the IVM administration period (black; IVM). Values analyzed via paired T-Test. ***P<0.001.
IVM (5.0 mg/kg/day) Delivered Long-Term (39 days) via Oral Gavage Did Not
Cause Any Overt Signs of Toxicity
Figure 1.
Figure 3.
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Over 18 million Americans are afflicted with an alcohol
use disorder (AUD)
1
.
Due to the limited efficacy of current pharmacological
treatment options, a staggering 85% of sufferers fail to
ever seek treatment
1
.
A)
B)
C)
Figure 2.
Orally Delivered IVM (3.1 mg/kg/day) Significantly Reduces Ethanol Intake and
Preference in Male Mice at Concentrations that Correspond to Doses Already
Established to be Safe in Humans
No Gavage Corn Oil IVM 3.1
0
2
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Copy of 10E Intake IVM 3.1
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****
Bars represent the mean SEM n=22 male mice across the 14 day treatment period. ****P<0.0001

Treatment Period
10E Preference
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IVM 3.1
No Gavage
****
ORAL IVERMECTIN (IVM) TREATMENT SIGNIFICANTLY REDUCES
ALCOHOL INTAKE IN MALE C57BL/6 MICE

Natalie M. Arabian
1
, Megan M. Yardley
1
, Liana Asatryan
1
, Nhat Huynh
1
, Stan G. Louie
1
, Mike N. Neely
2
, Ronald L. Alkana
1
, Daryl L. Davies
1


1
University of Southern California, School of Pharmacy, Los Angeles, CA,
2
University of Southern California, Keck School of Medicine, Los Angeles, CA

P r e I V M 1 2 3 4 5
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*** ** * ** *** **
RESULTS
INTRODUCTION
DISCUSSION
Treatment Period
****P<0.0001 versus Pre IVM
REFERENCES
Evaluation of
Neurological Safety
Pharmacology and
Systemic Toxicity
After Acute IVM
(10 mg/kg i.p.)
Administration
METHODS