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Probenecid Reduces Alcohol Drinking in Rodents. Is Pannexin1 a Novel Therapeutic

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Probenecid Reduces Alcohol Drinking in Rodents. Is Pannexin1 a Novel Therapeutic Alcohol and Alcoholism, 2019, 54(5) 497–502 doi: 10.1093/alcalc/agz054 Article Article Probenecid Reduces Alcohol Drinking in Rodents. Is Pannexin1 a Novel Therapeutic Target for Alcohol Use Disorder? Downloaded from https://academic.oup.com/alcalc/article/54/5/497/5571632 by guest on 23 September 2021 Brendan J. Tunstall1,†, Irene Lorrai2,3,†, Sam A. McConnell1, Katrina L. Gazo1, Lia J. Zallar1,4, Giordano de Guglielmo5, Ivy Hoang2, Carolina L. Haass-Koffler6, Vez Repunte-Canonigo2, George F. Koob1, Leandro F. Vendruscolo1,‡, and Pietro Paolo Sanna2,‡* 1National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, MD, USA, 2Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA, 3Department of Biomedical Sciences, University of Cagliari, Monserrato, Cagliari, Italy, 4National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA, 5Department of Psychiatry, University of California San Diego, La Jolla, CA, USA, and 6Center for Alcohol and Addiction Studies; Department of Psychiatry and Human Behavior; Department of Behavioral and Social Sciences, Brown University, Providence, RI, USA *Corresponding author: Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA. E-mail: [email protected] †Contributed equally. ‡Co-Senior authors. Received 10 May 2019; Revised 3 June 2019; Editorial Decision 4 June 2019; Accepted 2 July 2019 Abstract Aims: The development of novel and more effective medications for alcohol use disorder (AUD) is an important unmet medical need. Drug repositioning or repurposing is an appealing strategy to bring new therapies to the clinic because it greatly reduces the overall costs of drug development and expedites the availability of treatments to those who need them. Probenecid, p-(di-n-propyl- sulfamyl)-benzoic acid, is a drug used clinically to treat hyperuricemia and gout due to its activity as an inhibitor of the kidneys’ organic anion transporter that reclaims uric acid from urine. Probenecid also inhibits pannexin1 channels that are involved in purinergic neurotransmission and inflammation, which have been implicated in alcohol’s effects and motivation for alcohol. Therefore, we tested the effects of probenecid on alcohol intake in rodents. Methods: We tested the effects of probenecid on operant oral alcohol self-administration in alcohol-dependent rats during acute withdrawal as well as in nondependent rats and in the drinking-in-the-dark (DID) paradigm of binge-like drinking in mice. Results: Probenecid reduced alcohol intake in both dependent and nondependent rats and in the DID paradigm in mice without affecting water or saccharin intake, indicating that probenecid’s effect was selective for alcohol and not the result of a general reduction in reward. Conclusions: These results raise the possibility that pannexin1 is a novel therapeutic target for the treatment of AUD. The clinical use of probenecid has been found to be generally safe, suggesting that it can be a candidate for drug repositioning for the treatment of AUD. © The Author(s) 2019. Medical Council on Alcohol and Oxford University Press. All rights reserved. 497 498 Alcohol and Alcoholism, 2019, Vol. 54, No. 5 INTRODUCTION testing. Male C57BL/6 J mice, 7 weeks old upon arrival (The Jackson Laboratory, Bar Harbor, ME, USA), were individually Alcohol is the most prevalent substance misused in the United States housed in standard plastic cages and kept under a reverse 12 h/12 h (SAMHSA, 2013). Approved medications for alcohol use disorder light/dark cycle with ad libitum access to food and water except dur- (AUD) have efficacy but are prescribed for fewer than 10% of US ing behavioral testing. The room was controlled for temperature patients with AUD (Jonas et al., 2014). Thus, the development of and humidity. novel and more efficacious medications for AUD is a pressing med- All procedures adhered to the National Institutes of Health ical need (Litten et al., 2012). Guide for the Care and Use of Laboratory Animals (8th edition) and Drug repositioning or repurposing is an appealing strategy to were approved by the Institutional Animal Care and Use Committee bring new therapies to the clinic because it greatly reduces the over- of The Scripps Research Institute and the National Institute on Drug all costs of drug development and expedites the availability of treat- Abuse Intramural Research Program. ments to those who need them (Nosengo, 2016). We recently observed that the glycyrrhetinic acid derivative carbenoxolone β Drugs (CBX; 3 -hydroxy-11-oxoolean-12-en-30-oic acid 3-hemisuccinate), Downloaded from https://academic.oup.com/alcalc/article/54/5/497/5571632 by guest on 23 September 2021 a medication used to treat gastritis and peptic ulcer (Bertaccini and Probenecid (Sigma Aldrich, St. Louis, MO) was administered in a Coruzzi, 1985), reduced both dependent and nondependent alcohol Latin-square design at doses previously shown to be effective in intake in rodents (Sanna et al., 2016). CBX inhibits 11β-hydroxy- studies targeting the central nervous system in mice (Dossi et al., steroid dehydrogenase (11β-HSD) isozymes, shaping cellular 2018) and rats (Mousseau et al., 2018). responses to glucocorticoids (Chapman et al., 2013), which are functionally involved in alcohol dependence in rats and AUD in Operant alcohol self-administration and alcohol vapor humans (Vendruscolo et al., 2012, 2015; Repunte-Canonigo et al., exposure 2015). In particular, 11β-HSD1 is broadly expressed in brain Oral alcohol self-administration experiments were conducted as regions relevant to alcohol’s reinforcing properties, such as the previously described using standard operant chambers (Med amygdala (Pelletier et al., 2007). CBX also inhibits pannexin1 chan- Associates, St. Albans, VT) fittedwith2retractableleversanda nels that contribute to adenosine triphosphate (ATP) release into the dual-cup liquid receptacle (Vendruscolo and Roberts, 2014). After extracellular space (Dahl, 2015). Because probenecid was also initial training to acquire operant responding for alcohol shown to inhibit pannexin1 channels (Silverman et al., 2008), we reinforcement, rats were split into 2 groups matched for alcohol tested its effect on alcohol intake, with the hypothesis that pannex- consumption. In the next phase (vapor exposure) and for the in1 channel blockade may contribute to reducing alcohol drinking. remainder of these experiments, one group of rats (nondependent In the extracellular space, ATP is hydrolyzed to adenosine group) was exposed to air without alcohol, whereas the other diphosphate (ADP) and adenosine, which contribute to the activation group of rats (dependent group) was exposed to alcohol vapor in of purinergic receptors and neuroinflammation (Silverman et al., daily cycles designed to cause intoxication (14 h vapor ‘on’; 2009; Velasquez and Eugenin, 2014). Alcohol has been shown to 200 mg/dl target blood alcohol levels [BALs]) and withdrawal increase adenosine in the extracellular space, and adenosine neuro- (10 h vapor ‘off’) to induce alcohol dependence (Vendruscolo and transmission contributes to alcohol intoxication and reinforcement Roberts, 2014). During initial training and during the vapor (Ruby et al., 2010). ATP that is released into the extracellular space exposure phase, rats in both groups were allowed 30-min operant in turn inhibits pannexin1 channel opening (Dahl, 2015). Despite sessions to lever press for alcohol (10%, w/v; 0.1 ml) and water their controlled opening, pannexin1 channels have been shown to (0.1 ml) on separate levers according to a concurrent fixed-ratio 1 promote neuronal excitability after N-methyl-D-aspartate (NMDA) (FR1) schedule of reinforcement (i.e. each lever press on each lever glutamate receptor activation (Thompson et al., 2008) and seizure resulted in fluid delivery). Operant alcohol self-administration ses- activity (Dossi et al., 2018). sions were conducted 2–3 sessions per week during the 10 h ‘off’ Probenecid is a medication used in the clinic primarily to period, 6–8 h into withdrawal. After each training session, the liquid increase uric acid excretion in the urine in hyperuricemic conditions, receptacle and surrounding area were inspected to confirm the con- such as gout, through its activity as a competitive substrate for sumption of earned reinforcers. Probenecid was injected acutely organic anion transporters (OATs) in the kidney (Pascale et al., intraperitoneally 60 min prior to behavioral testing. 1952). We observed that, similarly to CBX, probenecid reduced alcohol intake both in dependent and nondependent rats and in the drinking-in-the-dark (DID) paradigm of binge-like drinking in mice Non-drug reinforcement: saccharin self-administration (Rhodes et al., 2005). These results suggest that probenecid, a gener- in rats ally safe medication that has long been used in humans, is a candi- Rats were trained to self-administer saccharin (0.1% w/v) in 30 min date drug for repositioning for AUD and indicate that pannexin1 sessions (FR1) until all rats responded over 100 times per session on may be a potential therapeutic target for the treatment of AUD. two occasions. Next, all rats were given a baseline session of sac- charin self-administration, followed by testing a range of probenecid doses administered in a Latin-square design on consecutive days. METHODS Animals Mouse drinking-in-the-dark paradigm Male Wistar rats (Charles River, Kingston, NY), weighing 225
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