Sphingosine-1-phosphate (S1P) signaling as a novel therapeutic target for alcohol abuse

TL;DR

This study shows that S1P signaling can reduce alcohol drinking in rodents and may be a new treatment target for alcohol use disorder.

Contribution

S1P signaling is identified as a novel therapeutic target for alcohol abuse, with CYM5442 showing efficacy in multiple rodent models.

Findings

CYM5442 reduced binge drinking in mice and alcohol self-administration in dependent and non-dependent rats.

CYM5442 prevented cue-induced relapse to alcohol use and had fewer aversive effects than naltrexone.

S1P regulates a complex set of genes during the transition to alcohol dependence.

Abstract

Sphingosine-1-phosphate (S1P) is a lipid mediator signaling through broadly expressed G protein-coupled receptors. We found that S1P is regulated by alcohol and that S1P receptor agonists reduce alcohol drinking in rodent models. Specifically, we observed that two S1P receptor agonists FDA-approved for multiple sclerosis, fingolimod and ozanimod, and the more brain penetrant S1P1 receptor agonist CYM5442, reduced binge alcohol drinking in the drinking in the dark (DID) paradigm in mice. CYM5442 also reduced drinking in dependent mice in the chronic intermittent ethanol vapor paradigm of dependence-induced increased drinking paired with 2 bottle-choice (CIE-2BC) as well as in non-dependent mice. CYM5442 also reduced operant oral alcohol self-administration in both non-dependent and dependent rats made dependent by vapor exposure, and reduced motivation for alcohol in dependent rats tested in a progressive ratio schedule of reinforcement. CYM5442 significantly prevented cue-induced reinstatement of alcohol seeking behavior in alcohol-dependent rats, a model of relapse to alcohol use. CYM5442 also reduced intake of non-drug reinforcers, including sucrose, food, water and, to a lesser extent, saccharine. Notably, CYM5442 was less aversive than naltrexone, an FDA-approved medication for the treatment of alcohol use disorder that shares a similar broad reducing action on alcohol intake and non-drug reinforcers. CYM5442 had no effect on loss of righting reflex, alcohol metabolism, motor coordination or spontaneous locomotor activity in rodents. Lastly, gene expression analysis by RNA-Seq revealed that S1P regulates a complex set of genes in the transition to alcohol dependence. Overall, our results establish S1P signaling as a novel therapeutic target for alcohol use disorder.

Figures

C](#F2)). However, the reducing effect of ozanimod was not evident at the end of the 4 h drinking session [1-way ANOVA, F (2, 32) = 3.05, p > 0.05] ([Fig. 2D](#F2)), consistent with its short half-life (172).

O](#F3)). Similarly, in females, treatment had no significant effect on LORR duration (unpaired two-tailed t-test, t_29_ = 1.05, p > 0.05) ([Fig. 3Q](#F3)). Additionally, separated 2-way RM ANOVA of the BALs over time revealed a significant main effect of time in both sexes, but not significant effect of treatment or time x treatment interaction. For males: Time F (3,84) = 1401; p < 0.0001; Treatment F (1, 28) = 1.26; p > 0.05; Interaction F (3,84) = 1.27; p > 0.05) ([Fig. 3P](#F3)). For females: 2-way RM ANOVA, Time F (3,90) = 2117; p < 0.0001; Treatment F (1, 30) = 0.002; p > 0.05; Interacti

F](#F4)). On the day following the last extinction session, dependent and non-dependent rats were allocated into two groups based on their number of responses on the active lever during the last two days of extinction. ([Fig. 4G](#F4), left panel). Rats of both groups received either vehicle or CYM5442 (10 mg/kg) 60-min before the reinstatement session. Three-way ANOVA revealed a significant main effect of group (F (1,60) = 8.48; p < 0.005) and treatment (F (1,60) = 8.64; p < 0.005) but not protocol (extinction/reinstatement) (F (1,60) = 0.847; p > 0.05). No significant interactions were obser

E](#F5)) and 4 h session (1-way ANOVA F(3, 26) = 3.14; p < 0.05), although only at the highest dose of 5 mg/kg (*p < 0.05; p < 0.01, Tukey’s post hoc) (Table 1).

Full text

Introduction

Alcohol use disorder (AUD) is a chronic, relapsing disorder characterized by the inability to stop or control alcohol use despite experiencing negative social, occupational, and health-related consequences (1). To date, only 3 medications have been approved by the Food and Drug Administration (FDA) for the treatment of AUD (disulfiram, oral and extended-release injectable naltrexone, and acamprosate) (2–4). Nalmefene has been approved in Europe and baclofen in France (2–4). Other medications such as topiramate, varenicline, ondansetron, gabapentin, aripiprazole, and prazosin/doxazosin have shown efficacy in some clinical trials (2–4). Due to AUD heterogeneity, both approved and investigational medications have limited efficacy, which reduces the confidence of clinicians in prescribing medications for AUD (4). Thus, there is pressing need to better understand the complexity of AUD and develop predicting models to guide drug selection to facilitate the utilization of existing AUD medications in clinical practice and to expand the therapeutic toolbox to better tailor AUD treatment to specific groups of patients and to identify broadly effective medications (4). The latter will require the identification and validation of new and more effective druggable targets (4).

Here we identified sphingosine-1-phosphate (S1P) signaling as a therapeutic target for AUD. S1P is a lipid mediator that affects multiple brain processes including neuroinflammation (5). S1P is derived from the phosphorylation of sphingosine by two sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, broadly expressed in the brain (6, 7). S1P can either act as a second messenger within the cells or can be released and signal through five G protein-coupled (S1P_1–5_) receptors (8) that are widely expressed in the body including in lymphocytes, neurons, astrocytes, oligodendrocytes, and microglia (9–12). S1P and its receptors are involved in physiological and pathological states including synaptic transmission, autophagy, and neuroinflammation, among others (8, 13, 14). The S1P receptors family has been identified as an important target for the treatment of chronic inflammatory states such as multiple sclerosis, ulcerative colitis, Crohn’s disease, and other conditions (15). Fingolimod, ozanimod and other S1P agonists are FDA-approved for relapsing-remitting multiple sclerosis and ozanimod also for ulcerative colitis.

Here, we investigated the role of S1P signaling in alcohol drinking and seeking. We observed that administration of alcohol modulates S1P levels in the mouse brain and that S1P agonists, including the FDA-approved medications fingolimod and ozanimod and the experimental compound CYM5442, reduce alcohol intake in rodents. These results establish S1P signaling as a therapeutic target for AUD.

Materials and Methods

Animals

Male and female C57BL/6J mice (6 weeks old, The Jackson Laboratories, USA) were either single or group housed according to the experimental design. Male Wistar rats (4 weeks old, Charles River, USA) were housed in pairs. All animals were kept in standard plastic cages under controlled temperature (21 ± 1°C) and humidity (50 ± 5%). Food and water were available ad libitum, except when specified otherwise. Behavioral experiments were conducted during the dark phase of the light/dark cycle. All procedures adhered to the National Institutes of Health guidelines for the “Care and Use of Laboratory Animals” and were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.

Drugs

Fingolimod, ozanimod, and CYM5442 (Tocris, MN, USA) were suspended in saline with 1% (w/v) Tween 80 and administered intraperitoneally (IP) 60 min prior to the behavioral experiments. The administration volumes were 10 ml/kg for mice and 2 ml/kg for rats.

Behavioral procedures

To evaluate whether pharmacological modulation of S1P signaling influences alcohol drinking, we examined the effect of S1P receptor agonists on binge-like alcohol drinking in C57BL/6J mice exposed to the drinking in the dark (DID) paradigm (16). We first assessed the effects of the two FDA-approved clinical compounds, fingolimod and ozanimod. Of note, fingolimod acts on almost all S1P receptor subtypes (S1P_1 – 5_) except S1P_2_, whereas ozanimod selectively targets S1P_1_ and S1P_5_. In contrast, CYM5442 is a highly selective agonist for the S1P_1_ receptor and has been shown to accumulate in the brain (17). Therefore, we focused subsequent studies on the pharmacological properties of CYM5442. These studies were also extended to include female mice. CYM5442 was further evaluated in i) saccharin and ii) sucrose intake in a DID-like experimental design; iii) CIE-2BC, iv) loss of righting reflex, v) conditioned place aversion, and vi) rotarod. In addition, CYM5442 was also tested in alcohol dependent and non-dependent Wistar rats exposed to fixed and progressive ratio schedules of reinforcing, cue-induced reinstatement and spontaneous locomotor activity. Details on the behavioral procedures employed in the present study are provided in the Supplementary Material.

Metabolomics

Targeted LC-MS/MS quantification of S1P in the PFC of alcohol-naïve C57BL/6J mice was done at the TSRI Center for Metabolomics and Mass Spectrometry. Briefly, weighed brain tissue of about 10 mg with S1P (d17:1) as internal standard added, was extracted with cold MeOH:H2O (4:1, v/v) solvent mixture using glass beads in homogenizer and sonicated in ice bath for 10 min. The homogenized solution was then rinsed with additional 200 μL cold MeOH:H2O (4:1, v/v). To precipitate proteins, the samples were incubated at − 20°C followed by centrifugation at 13000 rpm and 4°C. The resulting supernatant was removed and evaporated to dryness in a vacuum concentrator, resuspended in 100uL MeOH, and centrifuged at 13000 rpm and 4°C to remove insoluble debris. The supernatant was transferred to autosampler vials for analysis in the Agilent 6495 triple quadrupole mass spectrometer coupled to an Agilent 1290 UPLC stack with an Agilent poroshell 2.1×50mm C18 column. Mobile phase and operating conditions were set following established protocol at TSRI Mass Spectrometry Core. Data processing was done using the Agilent Quantitative analysis software with S1P (d17:1) fixed at 500nM. Calibration curve was established using standards run from 50nM to 10uM. S1P was quantified from the standard curve and normalized to sample weight expressed as fmol/mg.

Gene expression analysis

Total RNA isolation and RNA-sequencing

Dependent rats with chronic intermittent alcohol vapor exposure, with CYM5442 or vehicle treatment, were sacrificed after the 30-minute alcohol self-administration session. Matching alcohol-naive controls (with CYM5442 or vehicle treatment) were sacrificed at the corresponding time. Brain regions were microdissected and the PFC was processed for total RNA isolation using the RNeasy mini kit (Qiagen, Redwood City, CA). Libraries were prepared with the VAHTS Universal V10 RNA-seq Library Prep Kit (stranded) for Illumina (Vazyme, San Diego, CA, USA) and subsequently sequenced on NovaSeq6000 (Illumina) at 50M reads target coverage (150 bp paired-end reads).

Gene expression measures and Gene Set Enrichment Pathway Analysis.

RNA-seq fastq raw data underwent QC and adapter trimming by fastQC and Fastp. Transcriptome mapping and annotation to the current rat genome reference GRCr8 (GCF_036323735.1) were done using Hisat2, Samtools and FeatureCounts. Differential gene expression analysis was then performed using Deseq2. GSEA prerank was used for pathway analysis with ranking being - log10(pval)*sign(log2FC) from DEA.

Results

Mice Experiments

Alcohol decreases S1P levels in the mouse prefrontalcortex

Targeted LC-MS/MS analysis was conducted to investigate the effects of alcohol on S1P levels. Male alcohol-naive C57BL/6J mice were administered with either saline or an intoxicating dose of alcohol [3.5 g/kg, 15% (w/v)]. Thirty minutes later the prefrontal cortex was collected and stored at −80°C until metabolomics analysis was performed. Statistical analysis revealed a significant decrease of S1P levels in the mouse group treated with alcohol (unpaired T-test, t_7_ = 3.12; p < 0.05) (Fig. 1).

S1P receptor agonists reduce binge-like alcohol drinking

Fingolimod. Acute administration of fingolimod effectively reduced alcohol consumption in the DID paradigm over both the first 2 h at a dose of 4 mg/kg [1-way ANOVA, F(3, 50) = 11.41, p < 0.0001] (Fig. 2A) and the entire 4 h drinking session [1-way ANOVA, F(3, 50) = 16.74, p < 0.0001; ********p < 0.0001 by Tukey’s post hoc test] in male C57BL/6J mice (Fig. 2B).

Ozanimod. Acute administration of ozanimod reduced alcohol consumption in the DID paradigm after 2 h in male C57BL/6J mice at both doses [1-way ANOVA, F (2, 32) = 5.33; p < 0.05; *****p < 0.05 by Tukey’s post hoc test] (Fig. 2C). However, the reducing effect of ozanimod was not evident at the end of the 4 h drinking session [1-way ANOVA, F (2, 32) = 3.05, p > 0.05] (Fig. 2D), consistent with its short half-life (172).

CYM5442. We observed that acute administration of CYM5442 resulted in a statistically significant reduction of alcohol intake (1-way ANOVA: F(3, 36) = 13.66; p < 0.0001) in the mouse groups treated with 2.5 and 5 mg/kg in comparison to the mouse vehicle-treated group (**p < 0.005; ****p < 0.0001 by Tukey’s post hoc test) during the first 2 h of the drinking session (Fig. 3A). Furthermore, the alcohol-reducing effect of CYM5442 persisted through the entire 4 h session at the highest administered dose [1-way ANOVA: F (3, 36) = 17.46; p < 0.0001; ****p < 0.0001 by Tukey’s post hoc test] (Fig. 3B). A similar pattern was observed in female mice with a stronger reduction of alcohol intake during the first 2 h session following administration of 2.5 and 5 mg/kg CYM5442 [1-way ANOVA: F (3, 32) = 15.51; p < 0.0001; ****p < 0.0001 by Tukey’s post hoc test] (Fig. 3C). At the end of the 4 h session, alcohol consumption remained significantly reduced at the highest dose [1-way ANOVA: F (3, 32) = 6.30; p < 0.0005; **p < 0.005, by Tukey’s post hoc test] (Fig. 3D).

CYM5442 reduces non-drug reinforcers intake in the drinking in the dark paradigm in male and female C57BL/6J mice

To delineate the specificity of CYM5442 effects, we tested it on two palatable non-alcohol liquid reinforcers with different caloric value, saccharine and sucrose, in male and female mice in a DID-like experimental design. We found that in male mice, saccharine intake (ml/30g) was slightly reduced following administration of CYM5442 during the first 2 h session (1-way ANOVA: F (3, 31) = 4.51; p < 0.05). Tukey’s post hoc analysis indicated that the 2.5 and 5 mg/kg doses produced statistically significant reductions (**p < 0.005 and *p < 0.05, respectively). (Fig. 3E). By the end of the 4 h session, a significant decrease in saccharine intake remained evident at the 5 mg/kg dose [1-way ANOVA F (3, 31) = 4.88; p < 0.01; ****p < 0.01 by Tukey’s post hoc test] (Fig. 3F). In female mice, CYM5442, at the highest dose of 5 mg/kg, significantly reduced saccharine intake during the first 2 h session (1-way ANOVA F (3, 40) = 5.83; p < 0.005; *p < 0.05 by Tukey’s post hoc test) (Fig. 3G). At the end of the entire 4h a slight reduction in saccharin intake was still observed (1-way ANOVA F (3, 40) = 3.23; p < 0.05), however, post hoc analysis did not reveal any statistically significant differences between groups (Fig. 3H).

A similar pattern was observed with sucrose intake. Specifically, in male mice, administration of CYM5442 reduced sucrose intake (ml/30g) during the first 2 h session (1-way ANOVA: F (3, 28) = 8.62; p < 0.0005), particularly at the 2.5 and 5 mg/kg doses, as indicated by Tukey’s post hoc test (**p < 0.01 and ***p < 0.001, respectively) (Fig. 3I). At the end of the entire 4 h session, only the highest dose (5 mg/kg) produced a significant reduction in sucrose intake (1-way ANOVA: F (3, 28) = 6.85; p < 0.005; **p < 0.005 by Tukey’s post hoc test) (Fig. 3J). In female mice, only the highest dose of CYM5442 (5 mg/kg) significantly reduced mice sucrose intake during both the first 2 hours (1-way ANOVA F (3, 32) = 6.16; p < 0.005; ***p < 0.001 by Tukey’s post hoc test) (Fig. 3K) and the entire 4 h session (1-way ANOVA F (3, 32) = 7.41; p < 0.005; ***p < 0.001 by Tukey’s post hoc test) (Fig. 3L).

CYM5442 reduces alcohol drinking in 2-bottle choice (2BC) after chronic intermittent ethanol (CIE) in male and female C57BL/6J mice

The effects of CYM5442 were also evaluated in the chronic intermittent ethanol vapor paradigm of dependence-induced increased drinking paired with 2 bottle-choice (CIE-2BC) (18, 19) in male and female mice. In male mice, two-way RM ANOVA of alcohol intake (g/kg) during the limited 2BC session revealed a significant main effect of alcohol vapor exposure (F (1, 17) = 7.74; p < 0.05) and treatment (F (1, 17) = 13.97; p < 0.005) as well as significant interaction (F (1, 17) = 8.30; p < 0.05). As expected, post hoc analysis indicated a significant difference in alcohol intake between the two control groups, with the dependent mice consuming an average of 4.30 g/kg and the non-dependent mice consuming an average of 1.50 g/kg (**p < 0.005 by Šidák post hoc test) (Fig. 3M). Administration of CYM5442 (5 mg/kg), drastically reduced alcohol intake in both groups (0.42 g/kg vs 1.15 g/kg, respectively). However, statistical significance was reached only in the dependent group (***p < 0.0005, Šidák post hoc test) (Fig. 3M). In female mice, two-way RM ANOVA of alcohol intake (g/kg) during the limited 2BC session revealed a significant main effect of alcohol vapor exposure (F (1, 18) = 4.74; p < 0.05) and treatment (F (1, 18) = 36.23; p < 0.0001) with a trend toward a significant interaction (F (1, 18) = 3.75; p = 0.06) (Fig. 4B). Post hoc analysis showed that control dependent mice consumed more alcohol than non-dependent mice (6.11 g/kg vs. 4.11 g/kg, respectively). Administration of CYM5442 significantly reduced alcohol consumption in both non-dependent (*p < 0.05) and dependent (****p < 0.0001) groups compared to their vehicle-treated control groups (Šidák post hoc test) (Fig. 3N).

CYM5442 does not affect loss of righting reflex (LORR) or alcohol metabolism in male and female mice

To determine whether CYM5442 affects mouse sensitivity to the sedative and hypnotic effects of alcohol, male and female alcohol-naive mice were tested in the LORR paradigm. Statistical analysis indicated that duration of LORR was comparable between male mice treated with either 0 or 2.5 mg/kg CYM5442 and no significant difference was observed (unpaired two-tailed t-test, t_26_ = 1.38, p > 0.05) (Fig. 3O). Similarly, in females, treatment had no significant effect on LORR duration (unpaired two-tailed t-test, t_29_ = 1.05, p > 0.05) (Fig. 3Q). Additionally, separated 2-way RM ANOVA of the BALs over time revealed a significant main effect of time in both sexes, but not significant effect of treatment or time x treatment interaction. For males: Time F (3,84) = 1401; p < 0.0001; Treatment F (1, 28) = 1.26; p > 0.05; Interaction F (3,84) = 1.27; p > 0.05) (Fig. 3P). For females: 2-way RM ANOVA, Time F (3,90) = 2117; p < 0.0001; Treatment F (1, 30) = 0.002; p > 0.05; Interaction F (3,90) = 2.17; p > 0.05) (Fig. 3R).

CYM5442 reduces food, water intake, and energy metabolism in male C57BL/6J mice

CYM5442 effect was also tested in feeding, water intake and energy metabolism in male alcohol-naive mice. Statistical analysis revealed that food and water intake were reduced by administration of CYM5442 over both the first 2 [food: 1-way ANOVA (F(3, 26) = 7.87; p = 0.0007), water (F(3, 26) = 8.155; p = 0.0005) (Table 1) and the entire 4 h session (food: 1-way ANOVA F(3, 26) = 3.014; p < 0.05), water (F(3, 26) = 3.147; p < 0.05)), (*p < 0.05; **p < 0.01; ***p < 0.0005; Tukey’s post hoc) (Table 1). In addition, CYM5442 significantly decreased mouse respiratory exchange ratio (RER) over both the first 2 h [1-way ANOVA F(3, 26) = 4.22; p < 0.05) (Fig. 5E) and 4 h session (1-way ANOVA F(3, 26) = 3.14; p < 0.05), although only at the highest dose of 5 mg/kg (*p < 0.05; **p < 0.01, Tukey’s post hoc) (Table 1).

CYM5442 is less aversive than naltrexone in the conditioned place aversion paradigm

Alcohol-naive male mice were tested in the conditioned place aversion paradigm to evaluate potential aversive effects of CYM5442. A 2-way RM ANOVA revealed a significant main effect of time (F (1, 26) = 16.03, p < 0.001), but not treatment, although a trend toward significance was observed (F (2, 26) = 2.96, p = 0.07). In addition, a significant treatment x time interaction (F (2, 26) = 6.71, p < 0.005) was also found. Post hoc analysis showed a highly significant difference between pre and post conditioning in the naltrexone-treated group (***p = 0.0001, by Šidák post hoc test) and a moderately significant difference was observed in the CYM5442-treated group (p = 0.045, by Šidák post hoc test) (Fig. 3S). Overall, these data suggests that naltrexone produces a stronger aversive effect compared to CYM5442.

CYM5442 does not alter mouse motor coordination

Potential unspecific effects of CYM5442 were assessed using the rotarod apparatus in both male and female alcohol-naive C57BL/6J mice. Administration of CYM5442 had no effect on motor performance in either sex. Accordingly, 1-way ANOVA revealed no significant differences in latency to fall among treatment groups for males [F (3, 44) = 0.11; p > 0.05] (Fig. 3T) or females (F (3, 44) = 0.25; p > 0.05) (Fig. 3U).

Rat experiments

CYM5442 reduces alcohol self-administration on a fixed ratio 1 (FR1) and a progressive ratio (PR) schedule of reinforcement in non-dependent and dependent Wistar rats

We then investigated whether the ability of CYM5442 to reduce binge-like alcohol drinking in mice, extended to a well-established operant paradigm of alcohol self-administration on FR1 in non-dependent and dependent male Wistar rats. Statistical analysis revealed that alcohol dependent rats exhibited significantly higher responding on the alcohol lever compared to non-dependent rats (63.6 vs 37.0 lever presses over 30-min session; main effect of group: F (1,103) = 20.66; p < 0.0001). Two-way ANOVA revealed that CYM5442 administration significantly reduced alcohol self-administration in both non-dependent and dependent rat groups (main effect of treatment: F (3, 103) = 18.78; p < 0.0001). No significant interaction between treatment and group was observed (F (3,103) = 1.467; p > 0.05). Post-hoc analysis indicated that only the 10 mg/kg dose of CYM5442 significantly reduced alcohol lever-responding in both non-dependent and dependent rat groups (****p < 0.0001; **p < 0.005, Tukey’s post hoc test) (Fig. 4A). Consistently, alcohol dependent rats self-administered greater amounts of alcohol than non-dependent rats (1.0 vs 0.6 g/kg/30-min session; main effect of group: F (1,103) = 21.85; p < 0.0001). Two-way ANOVA revealed a significant main effect of CYM5442 treatment on alcohol intake (F (3,103) = 18.73; p < 0.0001) with no significant interaction between group and treatment (F (3,103) = 1.553; p > 0.05). Post-hoc comparisons revealed that only 10 mg/kg dose of CYM5442 significantly decreased alcohol self-administration in both non-dependent and dependent rat groups (****p < 0.0001; **p < 0.005, by Tukey’s post hoc test) (Fig. 4B).

To determine whether CYM5442 also affected the motivational properties of alcohol, non-dependent and dependent rats were exposed to a progressive ratio schedule of reinforcement. We observed that alcohol dependent rats exhibited a significantly higher motivation to obtain alcohol compared to non-dependent rats, as indicated by higher break point (BP) value (19.3 vs 11.7). Two-way ANOVA revealed significant main effects of both group (F (1,66) = 22.13; p < 0.0001)) and treatment (F (2,66) = 8.26; p < 0.001) with no significant interaction between the two factors (F (2,66) = 0.32; p > 0.05). Post-hoc analysis showed that only the 10 mg/kg dose of CYM5442 significantly reduced the motivation for alcohol in both non-dependent and dependent rat groups (**p < 0.005; *p = 0.05 by Tukey’s post hoc test) (Fig. 4C).

Consistently, the number of responses on the active lever was significantly higher in the alcohol dependent rat group than the non-dependent rat group (76.3 vs 33.1 responses over 60-min session). Two-way ANOVA revealed significant main effects of group (F (1,66) = 24.04; p < 0.0001) and treatment (F (2,66) = 5.98; p < 0.005), but no significant interaction (F (2,66) = 1.06; p > 0.05). Post-hoc analysis revealed that both 5 and 10 mg/kg doses of CYM5442 significantly reduced lever-responding for alcohol in the dependent rat group only (**p < 0.005; *p < 0.05 by Tukey’s post hoc test) (Fig. 4D). Similar results were observed on number of alcohol rewards earned. Two-way ANOVA revealed significant main effects of group (F (1,66) = 21.33; p < 0.0001) and treatment (F (2,66) = 8.11; p < 0.005), but no significant interaction (F (2,66) = 0.28; p > 0.05). Post-hoc analysis revealed that 10 mg/kg dose of CYM5442 significantly reduced lever-responding for alcohol in both the non-dependent and dependent rat groups (**p < 0.005; *p < 0.05 by Tukey’s post hoc test) (Fig. 4E).

CYM5442 prevents cues-induced reinstatement of alcohol seeking behavior in alcohol dependent rats

After the PR test, rats completed 10 regular 30-min sessions of alcohol self-administration and then underwent an extinction responding training. As expected, alcohol dependent and non-dependent rats progressively extinguished their alcohol seeking behavior over five consecutive days. Two-way ANOVA revealed significant main effects of group (F (1,62) = 25.86; p < 0.0001) and day [F (4,247) = 22.07; p < 0.0001) but no significant interaction between these factors (F (4,247) = 1.53; p > 0.05). Post hoc analysis indicated that lever-responding was higher in dependent rats compared to non-dependent rats during extinction days 1–3 (****p < 0.0001, ***p < 0.0005, *p < 0.05 by Tukey’s post hoc test) (Fig. 4F). On the day following the last extinction session, dependent and non-dependent rats were allocated into two groups based on their number of responses on the active lever during the last two days of extinction. (Fig. 4G, left panel). Rats of both groups received either vehicle or CYM5442 (10 mg/kg) 60-min before the reinstatement session. Three-way ANOVA revealed a significant main effect of group (F (1,60) = 8.48; p < 0.005) and treatment (F (1,60) = 8.64; p < 0.005) but not protocol (extinction/reinstatement) (F (1,60) = 0.847; p > 0.05). No significant interactions were observed for protocol x treatment (F (1,60) = 0.04; p > 0.05), protocol x group (F (1,60) = 21.57; p > 0.05), treatment x group (F (1,60) = 0.949; p > 0.05), or protocol x group x treatment (F (1,60) = 2.38; p > 0.05). Post hoc analysis revealed that CYM5442 significantly prevented cue-induced reinstatement in the dependent rat group ($p < 0.005; #p < 0.05; ***p < 0.0001, by Bonferroni post hoc). (Fig. 4G, right panel).

CYM5442 does not alter rat spontaneous locomotor activity

As for mice, potential secondary effects of CYM5442 were assessed on spontaneous locomotor activity of alcohol-naive rats. One-way ANOVA of the total ambulatory distance traveled by rats during a 60-minute session showed that administration of CYM5442 did not significantly affect locomotor activity (F (3, 20) = 0.59; p > 0.05) (Fig. 4H). Furthermore, two-way RM ANOVA of ambulatory distance across six 10-min intervals, revealed a significant effect of time (F(5,100) = 156.7; p < 0.0001), but no significant effects of treatment (F (3, 20) = 0.59; p > 0.05) or time x treatment (F (15,100) = 0.71; p > 0.05) (Fig. 4I).

S1P regulates a complex set of genes in the transition to alcohol dependence.

We carried out RNA-Seq of the PFC of CYM5442 and vehicle treated rats with histories of CIE and alcohol-naive controls. Pathway analysis by gene set enrichment analysis (GSEA) revealed that treatment with CYM5442 affected several key pathways related to signal transduction, neuronal function, synaptic and structural neuronal plasticity, and regulation of gene expression. These results suggest that S1P1-regulated pathways are complex gene expression programs with the potential to substantially affect neuronal states consistent with the role of S1P1 in the transition to escalated (dependent) alcohol intake (Fig. 5).

Discussion

Here we show that S1P levels are decreased after administration of an intoxicating dose of alcohol in the mouse prefrontal cortex (PFC), a key brain region in the shift from moderate to compulsive alcohol drinking and taking (20–29). Also relevantly, ceramide, the precursor of sphingosine, was reduced in the forebrain of male selectively bred alcohol-preferring rats with a history of chronic intermittent drinking of 20% alcohol (30).

High levels of S1P_1_ receptor have been identified in the prefrontal cortex and striatum, two brain regions involved in alcohol use and abuse (31). In human alcoholics, prefrontal cortex deficits are believed to contribute to excessive drinking and increased vulnerability to relapse (22–29, 32). Rodents with a history of alcohol dependence exhibit cognitive impairment that reflects prefrontal cortex dysfunction (26). Fronto-striatal circuits are implicated in the loss of control and enhanced motivation to drink that characterize AUD (33–36).

Acute administration of CYM5442 effectively prevented the consumption of intoxicating amounts of alcohol in male and female C57BL/6J mice exposed to the DID paradigm. Alcohol intake was reduced in a dose-dependent manner, with a significant effect observed at both 2.5 and 5 mg/kg CYM5442. Notably, the effect of 5 mg/kg CYM5442 persisted throughout the 4 h drinking session in both sexes despite its short half-life due to rapid clearance from circulation (17). Furthermore, the highest dose of CYM5442 (5 mg/kg) also reduced alcohol intake in both non-dependent and dependent male and female C57BL/6J mice in the 2-bottle choice paradigm after chronic intermittent exposure to alcohol vapor. CYM5442 did not alter the hypnotic/sedative effects of alcohol as assessed by the loss of righting reflex or interfere with its metabolism in male and female C57BL/6J mice. Notably, in the conditioned place aversion test, CYM5442 was less aversive than naltrexone, an FDA-approved medication for AUD in humans.

Parallel studies in male Wistar rats confirmed the ability of CYM5442 to interfere with the reinforcing and motivational properties of alcohol as evidenced by reduced self-administration under both fixed and progressive ratio schedule of reinforcement in non-dependent and dependent rats. Additionally, in dependent rats, CYM5442 prevented cues-induced reinstatement of alcohol seeking behavior, a validated model of loss of control over alcohol and relapse into heavy alcohol drinking (37).

To evaluate CYM5442 specificity, we tested CYM5442 effects on saccharin and sucrose intake, two palatable reinforcers with different caloric value, using an experimental design paralleling the DID paradigm, with independent groups of male and female mice. CYM5442 significantly reduced saccharin intake in both sexes, but only at a higher dose than that effective in reducing alcohol intake. In contrast, sucrose intake was reduced in males at 2.5 and 5 mg/kg whereas in females only at 5 mg/kg, with no effect detected at the end of the 4 h drinking session. Additionally, CLAMS data indicate that acute CYM5442 also affects food, water intake, and respiratory exchange ratio. Importantly, CYM5442 at doses between 1.25–5 mg/kg did not alter mouse motor coordination in the rotarod apparatus, ruling out nonspecific effects such as sedation or malaise as explanations for reduced drinking and feeding.

Overall, these results indicating CYM5442 effects on mouse drinking (drug, non-drug reinforcers, and water) and feeding, suggest that the S1P_1_ receptor could be also involved in general consummatory behavior. That is akin to other drugs that reduce alcohol intake. Importantly, naltrexone, an FDA-approved medication for the treatment of AUD, reduces alcohol, sucrose, and saccharin intake of male C57BL/6J mice exposed to the DID paradigm (38). However, in contrast with the observed CYM5442 properties, naltrexone also induces conditioned place aversion (39). A similar ability to affect broad reduction of rodent consummatory behavior has been shown for cannabinoids 1 (CB_1_) receptors (CB_1_-R) antagonists such as rimonabant, AM6527, and AM4113 (40, 41). Interestingly, it has been reported that S1P and its analog, the non-selective S1P agonist fingolimod, interact with the CB_1_-Rs, suggesting that molecules belonging to the same pharmacological class could potentially be provided with CB_1_-R activity (42). However, we found that CYM5442 does not (see Supplementary material), supporting that the effect of S1P agonists on alcohol drinking is independent of CB_1_-R. Other drugs that cause broad inhibition of consummatory behaviors include the CRF1-selective antagonist NBI-27914 and the immune-targeting compound tacrolimus that reduced both alcohol and saccharin intake in the DID paradigm (43). The glucagon-like peptide-1 (GLP-1) analogue semaglutide has been shown to reduce alcohol, water, saccharin, maltodextrin and corn oil intake (44). Overall, the present findings are consistent with the overlapping neurobiological and chemosensory mechanisms that regulate food, drug reward, and consummatory behavior (45). Consistently, preclinical and clinical data indicate a strong correlation between excessive alcohol and sweet food consumption in rodents and humans (46–48), and drug abuse and binge eating disorders have been shown to share imbalances in brain systems that regulate motivation, reward saliency, decision-making, and self-control (45, 49, 50).

In the present study, we observed that acute administration of CYM5442 effectively reduced water, food intake and RER in mice, supporting a role for S1P_1_ also in energy balance and metabolism. This effect could be explained by the activation of S1P_1_ receptors localized at the level of the hypothalamus, the regulatory center of feeding and drinking. In this regard, recent studies have shown high levels of S1P_1_ protein in all hypothalamic nuclei with a predominance in the arcuate, dorsomedial, and ventromedial nuclei. In addition, the S1P_1_ receptor was found predominantly in anorexigenic but not in orexigenic neurons in the arcuate nucleus (51, 52).

In conclusion, we show that the selective S1P receptor agonists reduced alcohol intake and consummatory behavior, particularly on caloric reinforcers with low aversion potential. These results establish S1P signaling as a therapeutic target for AUD.

Supplementary Material

This is a list of supplementary files associated with this preprint. Click to download.

SMLorraietal.2026.docx

Table1.tif

Supplementary information is available at MP’s website.