Development of tolerance upon repeated administration with the GABAB receptor positive allosteric modulator, COR659, on alcohol drinking in rodents.

Background: Recent work has demonstrated that acute administration of the novel positive allosteric modulator of the GABAB receptor, COR659, reduces several alcohol-related behaviors in rodents.Objective: To assess whether COR659 continues to lessen alcohol intake after repeated administration, a fundamental feature of drugs with therapeutic potential.Methods: Male C57BL/6J mice (n = 40) were exposed to daily 2-hour drinking sessions (20% (v/v) alcohol) under the 1-bottle "drinking in the dark" protocol and male Sardinian alcohol-preferring rats (n = 40) were exposed to daily 1-hour drinking sessions under the 2-bottle "alcohol (10%, v/v) vs water" choice regimen. COR659 (0, 10, 20, and 40 mg/kg in the mouse experiment; 0, 5, 10, and 20 mg/kg in the rat experiment) was administered intraperitoneally before 7 consecutive drinking sessions.Results: Alcohol intake in vehicle-treated mice and rats averaged 2.5-3.0 and 1.5-1.6 g/kg/session, respectively, indicative of high basal levels. In both experiments, treatment with COR659 resulted in an initial, dose-related suppression of alcohol intake (up to 70-80% compared to vehicle treatment; P < .0005 and P < .0001 in mouse and rat experiments, respectively). The magnitude of the reducing effect of COR659 on alcohol drinking diminished progressively, until vanishing over the subsequent 2-4 drinking sessions.Conclusion: COR659 effectively reduced alcohol intake in two different rodent models of excessive alcohol drinking. However, tolerance to the anti-alcohol effects of COR659 developed rapidly. If theoretically transposed to humans, these data would represent a possible limitation to the clinical use of COR659.

Marked Differences in the Submandibular Salivary Proteome between Sardinian Alcohol-Preferring and Sardinian Alcohol-Non Preferring Rats Revealed by an Integrated Top-Down-Bottom-Up Proteomic Platform.

Sardinian alcohol-preferring (sP) and Sardinian alcohol-non preferring (sNP) rats have been selectively bred for opposite alcohol preference and consumption. Aiming to verify possible differences at the proteomics level between sP and sNP rats, we investigated the salivary proteome by a a liquid chromatography-mass spectrometry top-down-bottom-up integrated approach. For this purpose, submandibular saliva was collected from alcohol-naive sP and sNP rats under isoprenaline stimulation. A total of 200 peptides and proteins were detected and quantified in the two rat lines, 149 of which were characterized in their naturally occurring structure. The data are available via ProteomeXchange with identifier PXD006997. Surprisingly, sP rats exhibited marked quantitative and qualitative differences with respect to sNP rats, namely higher levels of proteoforms originating from submandibular gland protein C, and from submandibular rat protein 2, as well as those of several unidentified peptides and proteins. sP rats expressed some proteins not detectable in sNP rats such as the glutamine and glutamic acid-rich protein (GRP)-CB. The isoform GRP-B, detectable in both rat lines, was more abundant in sNP rats. The submandibular saliva of sNP rats was also characterized by very high levels of GRP-B proteolytic peptides and rat salivary protein 1. Whether these differences could contribute to the opposite alcohol preference and consumption of sP and sNP rats is currently unknown and requires further investigation.

Liver Injury, Endotoxemia, and Their Relationship to Intestinal Microbiota Composition in Alcohol-Preferring Rats.

BACKGROUND: There is strong evidence that alcoholism leads to dysbiosis in both humans and animals. However, it is unclear how changes in the intestinal microbiota (IM) relate to ethanol (EtOH)-induced disruption of gut-liver homeostasis. We investigated this issue using selectively bred Sardinian alcohol-preferring (sP) rats, a validated animal model of excessive EtOH consumption. METHODS: Independent groups of male adult sP rats were exposed to the standard, home-cage 2-bottle "EtOH (10% v/v) versus water" choice regimen with unlimited access for 24 h/d (Group Et) for 3 (T1), 6 (T2), and 12 (T3) consecutive months. Control groups (Group Ct) were composed of matched-age EtOH-naïve sP rats. We obtained samples from each rat at the end of each experimental time, and we used blood and colon tissues for intestinal barrier integrity and/or liver pathology assessments and used stool samples for IM analysis with 16S ribosomal RNA gene sequencing. RESULTS: Rats in Group Et developed hepatic steatosis and elevated serum transaminases and endotoxin/lipopolysaccharide (LPS) levels but no other liver pathological changes (i.e., necrosis/inflammation) or systemic inflammation. While we did not find any apparent alteration of the intestinal colonic mucosa, we found that rats in Group Et exhibited significant changes in IM composition compared to the rats in Group Ct. These changes were sustained throughout T1, T2, and T3. In particular, Ruminococcus, Coprococcus, and Streptococcus were the differentially abundant microbial genera at T3. The KEGG Ortholog profile revealed that IM functional modules, such as biosynthesis, transport, and export of LPS, were also enriched in Group Et rats at T3. CONCLUSIONS: We showed that chronic, voluntary EtOH consumption induced liver injury and endotoxemia together with dysbiotic changes in sP rats. This work sets the stage for improving our knowledge of the prevention and treatment of EtOH-related diseases.

Reducing effect of intragastrically administered saikosaponin A on alcohol and sucrose self-administration in rats.

Saikosaponin A (SSA) is an active ingredient of the Asian medicinal herb, Bupleurum falcatum L. When administered via the intraperitoneal (i.p.) route, SSA suppressed multiple addictive-like behaviours, including operant alcohol self-administration, in rodents. It is unknown whether these effects are retained after intragastric (i.g.) administration, a desirable prerequisite for a compound with therapeutic potential. To fill this gap, i.g. SSA (0, 50, and 100 mg/kg) was tested in Sardinian alcohol-preferring (sP) rats trained to lever-respond for oral alcohol. SSA reduced lever-responding and amount of self-administered alcohol. However, when compared to i.p. SSA, i.g. SSA resulted to be markedly less potent and effective, suggestive of reduced bioavailability after i.g. treatment. Finally, and in agreement with previous data on the suppressing effect of i.p. SSA on behaviours motivated by highly palatable foods, i.g. SSA (0, 50, and 100 mg/kg) reduced oral sucrose self-administration in a separate set of sP rats.

Reducing Effect of Cannabidiol on Alcohol Self-Administration in Sardinian Alcohol-Preferring Rats.

Introduction: Cannabidiol (CBD) is a major cannabinoid extracted from Cannabis sativa with no abuse potential. Data from recent rodent studies suggest that amelioration of alcohol-motivated behaviors may be one of the numerous pharmacological effects of CBD. This study was designed to contribute to this research, assessing the effect of CBD on operant oral alcohol self-administration in selectively bred Sardinian alcohol-preferring (sP) rats, a validated animal model of excessive alcohol consumption. In addition, this study investigated the effect of CBD on operant self-administration of a highly palatable chocolate solution in Wistar rats. Materials and Methods: Male sP rats were trained to lever respond for alcohol (15% v/v) under the fixed ratio 4 (FR4) schedule of reinforcement. Once lever responding had stabilized, rats were exposed to test sessions under the FR4 and progressive ratio (PR) schedules of reinforcement. Test sessions were preceded by acute treatment with CBD (0, 6.25, 12.5, and 25 mg/kg or 0, 25, 50, and 100 mg/kg, i.p.; each dose range was tested in an independent experiment). Male Wistar rats were trained to lever respond for a chocolate solution (5% w/v chocolate powder) under the FR10 schedule of reinforcement. Once lever responding had stabilized, rats were exposed to test sessions under the same schedule. Test sessions were preceded by acute treatment with CBD (0, 6.25, 12.5, and 25 mg/kg or 0, 25, 50, and 100 mg/kg, i.p., in two independent experiments). Results: Under the FR schedule, treatment with doses of CBD ≥12.5 mg/kg markedly reduced lever responding for alcohol and amount of self-administered alcohol. Under the PR schedule, treatment with CBD produced a slight tendency toward a decrease in lever responding and breakpoint for alcohol. Finally, no dose of CBD affected lever responding for the chocolate solution and amount of self-administered chocolate solution. Discussion: These results extend previous data on CBD ability to affect alcohol-motivated behaviors to an animal model of genetically-determined proclivity to high alcohol consumption. Because of the predictive validity of sP rats, these results may be of relevance in view of possible future studies testing CBD in patients affected by alcohol use disorder.

Reducing effect of the novel positive allosteric modulator of the GABAB receptor, COR659, on binge-like alcohol drinking in male mice and rats.

RATIONALE: Binge drinking (BD) is a widespread drinkingpattern that may contribute to promote the development of alcohol use disorder (AUD). The comprehension of its neurobiological basis and the identification of molecules that may prevent BD are critical. Preclinical studies demonstrated that positive allosteric modulators (PAMs) of the GABAB receptor effectively reduced, and occasionally suppressed, the reinforcing and motivational properties of alcohol in rodents, suggesting their potential use as pharmacotherapy for AUD, including BD. Recently, we demonstrated that COR659, a novel GABAB PAM, effectively reduced (i) alcohol drinking under the 2-bottle choice regimen, (ii) alcohol self-administration under both fixed and progressive ratio schedules of reinforcement, and (iii) cue-induced reinstatement of alcohol-seeking behavior in Sardinian alcohol-preferring (sP) rats. OBJECTIVES: The present study investigated whether the "anti-alcohol" properties of COR659 extend to binge-like drinking in rodents. METHODS: COR659 was tested on the "drinking in the dark" (DID) paradigm in C57BL/6J mice and the 4-bottle "alcohol [10%, 20%, 30% (v/v)] versus water" choice regimen with limited and unpredictable access to alcohol in sP rats. RESULTS: Acute administration of non-sedative doses of COR659 (10, 20, and 40 mg/kg; i.p.) effectively and selectively suppressed the intake of intoxicating amounts of alcohol (> 2 g/kg) consumed by C57BL/6J mice and sP rats exposed to these binge-like drinking experimental procedures. CONCLUSIONS: The present data demonstrate the ability of COR659 to suppress binge-like drinking in rodents and strengthen the hypothesis that GABAB PAMs may represent a potentially effective pharmacotherapy for alcohol misuse.

The GABAB receptor positive allosteric modulator COR659: In vitro metabolism, in vivo pharmacokinetics in rats, synthesis and pharmacological characterization of metabolically protected derivatives.

We report an in vitro phase I metabolism study on COR659 (1), a 2-acylaminothiophene derivative able to suppress alcohol and chocolate self-administration in rats, likely via positive allosteric modulation of the GABAB receptor and antagonism/inverse agonism at the cannabinoid CB1 receptor. Given the identification of the methyl ester group at C-3 of the thiophene ring as a metabolic soft spot, we also report the chemical optimization project aimed to balance metabolic stability with in vitro and in vivo potency on a set of 3-substituted COR659 analogues. High performance liquid chromatography coupled to tandem and high resolution mass spectrometry was employed for the characterization of in vitro metabolism and in vivo pharmacokinetics of COR659 in rats. In vitro [35S]GTPγS binding assays on stimulated GABAB and CB1 receptors, in combination with alcohol and chocolate self-administration experiments in rats, were employed to assess the pharmacological profile of this novel set of analogues, using COR659 as reference compound. Eight metabolites of COR659 were discovered in liver microsomal incubates; two of them (M1, M2) were identified by comparison with synthetic reference standards. M2, oxidation product of methyl group at C-5 of the thiophene ring, was a major metabolite in vitro, but showed a low systemic exposure in vivo. M1, cleavage product of the methyl ester group at C-3, revealed in vitro an unusual mechanism of metabolism by a NADPH-dependent route and, in vivo, it maintained high and persistent levels in plasma, which could represent a potential pharmacokinetic and toxicological issue. In the novel set of COR659 analogues, those bearing branched alkyl substituents on the ester group, showed an improved in vitro metabolic stability (2-4), had an in vitro GABAB PAM (2-4) and/or CB1 partial agonist/antagonist profile (2-3) and maintained the ability to reduce alcohol (2-4) and/or chocolate (4) self-administration in rats. Both PK and PD data ruled out any involvement of metabolite M1 in the in vivo potency of COR659 and 4. The present results, therefore, highlight the importance to design and synthesize novel compounds endowed with the dual activity profile and devoid of metabolic liabilities.

Predisposition to Alcohol Drinking and Alcohol Consumption Alter Expression of Calcitonin Gene-Related Peptide, Neuropeptide Y, and Microglia in Bed Nucleus of Stria Terminalis in a Subnucleus-Specific Manner.

Excessive alcohol consumption is often linked to anxiety states and has a major relay center in the anterior part of bed nucleus of stria terminalis (BNST). We analyzed the impact of (i) genetic predisposition to high alcohol preference and consumption, and (ii) alcohol intake on anterior BNST, namely anterolateral (AL), anteromedial (AM), and anteroventral (lateral + medial subdivisions: AVl, AVm) subnuclei. We used two rat lines selectively bred for low- and high-alcohol preference and consumption, named Sardinian alcohol-non preferring (sNP) and -preferring (sP), respectively, the latter showing also inherent anxiety-related behaviors. We analyzed the modulation of calcitonin gene-related peptide (CGRP; exerting anxiogenic effects in BNST), neuropeptide Y (NPY; exerting mainly anxiolytic effects), and microglia activation (neuroinflammation marker, thought to increase anxiety). Calcitonin gene-related peptide-immunofluorescent fibers/terminals did not differ between alcohol-naive sP and sNP rats. Fiber/terminal NPY-immunofluorescent intensity was lower in BNST-AM and BNST-AVm of alcohol-naive sP rats. Activation of microglia (revealed by morphological analysis) was decreased in BNST-AM and increased in BNST-AVm of alcohol-naive sP rats. Prolonged (30 consecutive days), voluntary alcohol intake under the homecage 2-bottle "alcohol vs. water" regimen strongly increased CGRP intensity in BNST of sP rats in a subnucleus-specific manner: in BNST-AL, BNST-AVm, and BNST-AM. CGRP area sum, however, decreased in BNST-AM, without changes in other subnuclei. Alcohol consumption increased NPY expression, in a subnucleus-specific manner, in BNST-AL, BNST-AVl, and BNST-AVm. Alcohol consumption increased many size/shapes parameters in microglial cells, indicative of microglia de-activation. Finally, microglia density was increased in ventral anterior BNST (BNST-AVl, BNST-AVm) by alcohol consumption. In conclusion, genetic predisposition of sP rats to high alcohol intake could be in part mediated by anterior BNST subnuclei showing lower NPY expression and differential microglia activation. Alcohol intake in sP rats produced complex subnucleus-specific changes in BNST, affecting CGRP/NPY expression and microglia and leading to hypothesize that these changes might contribute to the anxiolytic effects of voluntarily consumed alcohol repeatedly observed in sP rats.

The long pursued Holy Grail of the true "alcoholic" rat.

An anthology of microdialysis and electrophysiological studies on ethanol effect on mesolimbic dopaminergic neurons is presented. The usefulness of rats with innate preference for ethanol, such as the Sardinian alcohol-preferring (sP), in studying ethanol rewarding and reinforcing effects is signaled. The generation of the long sought "alcoholics rat" from sP rats is announced. Rats of the sP line avoid the shortcomings of using rats non selected for ethanol preference.

R(+)-Baclofen, but Not S(-)-Baclofen, Alters Alcohol Self-Administration in Alcohol-Preferring Rats.

Racemic baclofen [(±)-baclofen] has repeatedly been reported to suppress several -alcohol-motivated behaviors, including alcohol drinking and alcohol -self-administration, in rats and mice. Recent data suggested that baclofen may have bidirectional, stereospecific effects, with the more active enantiomer, R(+)-baclofen, suppressing alcohol intake and the less active enantiomer, S(-)-baclofen, stimulating alcohol intake in mice. The present study was designed to investigate whether this enantioselectivity of baclofen effects may also extend to the reinforcing properties of alcohol in rats. To this end, selectively bred Sardinian alcohol-preferring (sP) rats were initially trained to lever respond on a fixed ratio 4 (FR4) schedule of reinforcement for alcohol (15%, v/v) in daily 30-min sessions. Once responding had stabilized, rats were tested with vehicle, (±)-baclofen (3 mg/kg), R(+)-baclofen (0.75, 1.5, and 3 mg/kg), and S(-)-baclofen (6, 12, and 24 mg/kg) under the FR4 schedule of reinforcement. Treatment with 3 mg/kg (±)-baclofen reduced the number of lever responses for alcohol and estimated amount of self-administered alcohol by approximately 60% in comparison to vehicle treatment. R(+)-baclofen was approximately twice as active as (±)-baclofen: treatment with 1.5 mg/kg R(+)-baclofen decreased both variables to an extent similar to that of the decreasing effect of 3 mg/kg (±)-baclofen. Conversely, treatment with all doses of S(-)-baclofen failed to affect alcohol self administration. These results (a) confirm that non-sedative doses of (±)-baclofen effectively suppressed the reinforcing properties of alcohol in sP rats and (b) apparently do not extend to operant alcohol self-administration in sP rats the capability of S(-)-baclofen to stimulate alcohol drinking in mice.

Suppression by γ-Hydroxybutyric Acid of "Alcohol Deprivation Effect" in Rats: Preclinical Evidence of its anti-Relapse Properties.

γ-Hydroxybutyric acid (GHB) reduces (a) alcohol intake and alcohol motivational properties in alcohol-preferring rats and (b) alcohol drinking and craving for alcohol in human alcoholics. The present study was designed to extend to relapse-like drinking the capacity of GHB to suppress different alcohol-related behaviors in alcohol-preferring rats. The "alcohol deprivation effect," defined as the temporary increase in alcohol intake occurring in laboratory animals after a period of alcohol deprivation, was used as model of alcohol relapse. Acute administration of non-sedative doses of GHB (0, 100, 200, and 300 mg/kg, i.p.) resulted in the complete suppression of the extra-amount of alcohol consumed by Sardinian alcohol-preferring rats during the first hour of re-access to alcohol after a 14-day period of deprivation. These data demonstrate that GHB suppressed relapse-like drinking in a rat model of excessive alcohol consumption.

Anti-Alcohol and Anxiolytic Properties of a New Chemical Entity, GET73.

N-[(4-trifluoromethyl)benzyl]4-methoxybutyramide (GET73) is a newly synthesized compound structurally related to the clinically used, alcohol-substituting agent, gamma-hydroxybutyric acid (GHB). The present study was designed to assess whether GET73 may share with GHB the capacity to reduce alcohol intake in rats. Additionally, the effect of treatment with GET73 on anxiety-related behaviors and cognitive tasks in rats was investigated. A series of in vitro binding assays investigated the capacity of GET73 to bind to the GHB binding site and multiple other receptors. GET73 (10(-9)-10(-3) M) failed to inhibit [(3)H]GHB binding at both high- and low-affinity GHB recognition sites in rat cortical membranes. GET73 displayed minimal, if any, binding at dopamine, serotonin, GABA, and glutamate receptors in membranes from different rat brain areas. Acute treatment with low-to-moderate, non-sedative doses of GET73 (5-50 mg/kg, i.g. or i.p.) (a) reduced alcohol intake and suppressed "alcohol deprivation effect" (a model of alcohol relapse) in selectively bred, Sardinian alcohol-preferring (sP) rats, (b) exerted anxiolytic effects in Sprague-Dawley (SD) and sP rats exposed to the Elevated Plus Maze test, and (c) tended to induce promnestic effects in SD rats exposed to a modified water version of the Hebb-Williams maze test. Although the mechanism of GET73 action is currently unknown, the results of the present study suggest that GET73 has a multifaceted pharmacological profile, including the capacity to reduce alcohol drinking and anxiety-related behaviors in rats.