Probenecid Reduces Alcohol Drinking in Rodents. Is Pannexin1 a Novel Therapeutic Target for Alcohol Use Disorder?

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-propylsulfamyl)-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.

Figures

Fig. 1.

Probenecid reduces oral alcohol self-administration in rats. (A) Dependent rats self-administered greater amounts of alcohol than nondependent rats across testing (two-way RM ANOVA; Group: F1,22 = 14.88, P < 0.001). Probenecid dose-dependently decreased alcohol drinking in both groups (Dose: F3,66 = 13.54, P < 0.0001), with a significant effect at the 100 mg/kg dose compared to vehicle (P < 0.0001). (B) Dependent and nondependent rats did not differ in their water drinking during testing (two-way RM ANOVA; Group: F1,22 = 0.0036, P = 0.9528), nor did probenecid significantly alter water drinking (Dose: F3,66 = 0.9853, P = 0.4052; Group x Dose Interaction: F3,66 = 0.0128, P = 0.9980). (C) In rats trained to self-administer saccharin, probenecid treatment did not alter saccharin consumption (one-way RM ANOVA; Dose: F3,42 = 0.2238, P = 0.8793). Nondep = Nondependent, Dep = Dependent. ****P < 0.0001 compared to vehicle, ###P < 0.001 compared to Nondependent group.

Fig. 2.

Probenecid reduces alcohol drinking in the drinking-in-the-dark paradigm in mice. (A) Effects of probenecid on alcohol intake in the first 2 h of the 4-h drinking session (one-way RM ANOVA, F3,33 = 19.27; P < 0.0001, followed by Bonferroni post hoc). (B) Probenecid effect on alcohol intake in the 4-h drinking session (one-way RM ANOVA, F3,33 = 10.07, P < 0.0001, followed by Bonferroni post hoc). (C) Probenecid effect on blood alcohol levels after the 4-h drinking session (one-way RM ANOVA, F3,33 = 3.17, P = 0.05, followed by Bonferroni post hoc). ****P < 0.0001; *P < 0.05.

Fig. 3.

Probenecid does not reduce saccharin intake in a 2-day drinking-in-the-dark paradigm in mice. (A) Probenecid effect on saccharin intake in the first 2 h of the 4-h drinking session (two-tailed paired t-test, t = 1.510, df = 11; P = 0.1593). (B) Probenecid effect on saccharin intake in the 4-h drinking session (two-tailed paired t-test, t11 = 1.357, P = 0.2019).

Fig. 4.

Schematic representation of some of the mechanisms implicated in the release of ATP and purinergic neurotransmission in the brain. (A) Pannexin1 channels release ATP into the extracellular space, which in turn inhibits their opening (Dahl, 2015). (B) ATP can also be released from connexin hemichannels. Connexin hemichannel opening has been associated with cerebral insults and brain injury. (C) ATP is also released into the extracellular space by vesicular release along neurotransmitters, including acetylcholine, dopamine, serotonin, and norepinephrine. (D) The equilibrative nucleoside transporter type 1 (ENT1) allows adenosine to move in either direction, driven by the gradient. Alcohol is believed to inhibit ENT1 in vivo, resulting in increased adenosine in the extracellular space (Ruby et al., 2010). (E) Ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase1)/CD39 hydrolyzes ATP to ADP and ADP to AMP. Ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) can also hydrolyze ATP to AMP. Ecto-5’-nucleotidase/CD73 hydrolyzes adenosine monophosphate (AMP) to adenosine (Yegutkin, 2008) (F). Purinergic receptors include the ionotropic P2X (G) and metabotropic P2Y (H) receptors for ATP/ADP and (I) adenosine receptors (ARs) for adenosine.