We have identified P2X4 receptors (P2X4Rs) as a target for the development of drugs to prevent and/or treat alcohol use disorder (AUD). This hypothesis is derived from compelling systems; genetic, pharmacological and behavioral evidence reporting an inverse relationship between ethanol (EtOH) intake and P2X4R activity. Supporting this hypothesis we found that p2rx4 knock-out mice (P2X4 KO) consumed significantly more EtOH than wildtype (WT) littermate controls. We and others found that ivermectin (IVM), a positive allosteric modula- tor of P2X4Rs, significantly antagonized EtOH inhibition of ATP-gated P2X4Rs and reduced EtOH intake in mice and rats. We have assembled a multidisciplinary team that together will use genetic, molecular, electrophysiological, chemical and behavioral techniques to systematically explore targets in the mesolimbic dopamine (DA) system that will be amenable for drug development. The proposed studies will translate laboratory findings into opportunities to discover and develop novel therapeutics for the prevention and treatment of AUD.
Aim 1 studies will test the hypothesis that P2X4Rs within the DA reward system regulate EtOH intake. We will use: in vivo microdialysis coupled with P2X4R modulation (Study 1) and lentiviral- mediated short hairpin RNA (shRNA) knockdown P2X4R expression in the nucleus accumbens (NAc) and/or ventral tegmental area (VTA) (Studies 2 & 3) to gain insights into specific contributions of P2X4Rs in the mesolimbic DA system to EtOH drinking. Male and female P2X4KO and/or WT mice will be tested using two drinking paradigms: (1) 24 hr access, two-bottle choice (free choice) and (2) drinking in the dark (DID; binge EtOH drinking).
Aim 2 studies will use a combination of brain slice electrophysiological and knock-out/knock- down technologies to test interrelated hypotheses where we predict that under conditions in which P2X4Rs are reduced, P2X-mediated inhibition of DA neuron firing and EtOH regulation of purinergic inhibition will be reduced or eliminated. This work will provide key information regarding the interaction of P2X4Rs and EtOH, and how it may affect both purinergic and EtOH responses of mesolimbic DA neurons.
Aim 3 studies will test the hypothesis that IVM can be used as a platform to identify and develop new compounds that positively modulate P2X4Rs and decrease EtOH-intake.
This Aim will be accomplished by designing and synthesizing a series of semisynthetic avermectin and milibemycin analogs followed by their in vitro and in vivo evaluation. Through this iterative process combining synthetic and biological evaluation, we will identify molecules that have maximal anti-alcohol effects while minimizing the IVM-like toxicities (i.e., increased therapeutic index) to yield candidates for further assessment for treating AUD. Taken together, these studies will further our long- term goal of identifying neurochemical mechanisms within key brain reward regions important for regulating EtOH intake. Moreover, this work will set the stage for future translational studies to develop novel pharmacotherapies for AUD.

Public Health Relevance

Alcohol use disorder (AUD) has a staggering socio-economic impact, yet current therapeutic strategies are largely inadequate to treat these disorders. We have identified P2X4 receptors (P2X4Rs) as a target for the development of drugs to treat alcohol use disorder (AUD). Findings from the current application will provide strong support for this hypothesis from several directions including development of lead compounds for drug development.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA022448-05
Application #
9346000
Study Section
Neurotoxicology and Alcohol Study Section (NAL)
Program Officer
Cui, Changhai
Project Start
2013-09-01
Project End
2021-06-30
Budget Start
2017-07-01
Budget End
2018-06-30
Support Year
5
Fiscal Year
2017
Total Cost
Indirect Cost
Name
University of Southern California
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90033
Khoja, Sheraz; Huynh, Nhat; Warnecke, Alicia M P et al. (2018) Preclinical evaluation of avermectins as novel therapeutic agents for alcohol use disorders. Psychopharmacology (Berl) 235:1697-1709
Khoja, Sheraz; Huynh, Nhat; Asatryan, Liana et al. (2018) Reduced expression of purinergic P2X4 receptors increases voluntary ethanol intake in C57BL/6J mice. Alcohol 68:63-70
Csóka, Balázs; Németh, Zoltán H; Szabó, Ildikó et al. (2018) Macrophage P2X4 receptors augment bacterial killing and protect against sepsis. JCI Insight 3:
Huynh, Nhat; Arabian, Natalie; Naito, Anna et al. (2017) Preclinical development of moxidectin as a novel therapeutic for alcohol use disorder. Neuropharmacology 113:60-70
Huynh, Nhat; Arabian, Natalie; Lieu, Dustin et al. (2016) Utilizing an Orally Dissolving Strip for Pharmacological and Toxicological Studies: A Simple and Humane Alternative to Oral Gavage for Animals. J Vis Exp :e53770
Xu, Ji; Bernstein, Alexander M; Wong, Angela et al. (2016) P2X4 Receptor Reporter Mice: Sparse Brain Expression and Feeding-Related Presynaptic Facilitation in the Arcuate Nucleus. J Neurosci 36:8902-20
Khoja, Sheraz; Shah, Vivek; Garcia, Damaris et al. (2016) Role of purinergic P2X4 receptors in regulating striatal dopamine homeostasis and dependent behaviors. J Neurochem 139:134-48
Guizzetti, Marina; Davies, Daryl L; Egli, Mark et al. (2016) Sex and the Lab: An Alcohol-Focused Commentary on the NIH Initiative to Balance Sex in Cell and Animal Studies. Alcohol Clin Exp Res 40:1182-91
Helms, Christa M; Bell, Richard L; Bennett, Allyson J et al. (2015) The importance of animals in advancing research on alcohol use disorders. Alcohol Clin Exp Res 39:575-8
Asatryan, Liana; Khoja, Sheraz; Rodgers, Kathleen E et al. (2015) Chronic ethanol exposure combined with high fat diet up-regulates P2X7 receptors that parallels neuroinflammation and neuronal loss in C57BL/6J mice. J Neuroimmunol 285:169-79

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