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

Sphingosine-1-phosphate (S1P) signaling as a novel therapeutic target for alcohol abuse | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Sphingosine-1-phosphate (S1P) signaling as a novel therapeutic target for alcohol abuse Irene Lorrai, Riccardo Maccioni, Chenhao Wu, Chase Shankula, Jorge Marquez-Gaytan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8653084/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 14 You are reading this latest preprint version 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 S1P 1 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. Health sciences/Diseases/Psychiatric disorders/Addiction Biological sciences/Neuroscience Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 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 2.1x50mm 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 prefrontal cortex 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. 2 A) 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. 2 B). 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] (Fig. 2 D), 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. 3 A). 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. 3 B). 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. 3 C). 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. 3 D). 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. 3 E). 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. 3 F). 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. 3 G). 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. 3 H). 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. 3 I). 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. 3 J). 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. 3 K) 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. 3 L). 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. 3 M). 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. 3 M). 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. 4 B). 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. 3 N). 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. 3 O). Similarly, in females, treatment had no significant effect on LORR duration (unpaired two-tailed t-test, t 29 = 1.05, p > 0.05) (Fig. 3 Q). 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 0.05; Interaction F (3,84) = 1.27; p > 0.05) (Fig. 3 P). For females: 2-way RM ANOVA, Time F (3,90) = 2117; p 0.05; Interaction F (3,90) = 2.17; p > 0.05) (Fig. 3 R). 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. 5 E) 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. 3 S). 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. 3 T) or females (F ( 3 , 44 ) = 0.25; p > 0.05) (Fig. 3 U). 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 0.05). 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. 4 A). 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 0.05). 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. 4 B). 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 0.05). 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. 4 C). 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 0.05). 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. 4 D). 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 0.05). 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. 4 E). 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 0.05). 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. 4 F). 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. 4 G, 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 0.05). 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. 4 G, 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. 4 H). 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 0.05) or time x treatment (F (15,100) = 0.71; p > 0.05) (Fig. 4 I). 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. 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Nat Commun. 2014;5:4859. Table Table 1 is available in the supplementary files section Additional Declarations Yes PPS is an inventor on a patent application related to this paper. The other authors declare no competing interests Supplementary Files SMLorraietal.2026.docx Supplemental Matierial Table1.tif Table 1. Effect of CYM5442 on mice exposed to CLAMS. Male mice were administered IP with CYM5442 (0-5 mg/kg) 60 min before the 3 rd h of the dark phase of the dark/light cycle. Food, water intake and respiratory exchange ratio were measured in male C57BL/6J mice (n=8 per group). Values are reported as mean ± SEM. *p<0.05, **p<0.01, ****p<0.0001 in comparison to the vehicle-treated group. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8653084","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":589290597,"identity":"bb415235-7ea6-4b3b-8d67-76f21f3cb0d5","order_by":0,"name":"Irene Lorrai","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYBACNnYwJSEHIg9AxBIIaGGGaDEmXgsDRAtDYgNCiIAWPmbmY58raizS57effXi4cEcdAz97jgEBh7ElzzxzTCJ3w5l0g8MzzxxmkOx5Q0gLjzFjAxtQiwQbw2HetgMMBjcI2gLS8k8iXX4GWEsdgz1RWhrbJBIYboC1MDMYSBDhF8bGPgnDDWfSGEB+4ZE486wArxb59ubDjA3f6uTl248xfwaGmBx/e/IGvFpQADNjAwMP8cphWkbBKBgFo2AUYAAA+lI8WHbZj3MAAAAASUVORK5CYII=","orcid":"","institution":"The Scripps Research Institute","correspondingAuthor":true,"prefix":"","firstName":"Irene","middleName":"","lastName":"Lorrai","suffix":""},{"id":589290598,"identity":"ff7fe0d6-64a8-4683-a507-339ff8ea7c8e","order_by":1,"name":"Riccardo Maccioni","email":"","orcid":"https://orcid.org/0000-0003-1349-4903","institution":"The Scripps Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Riccardo","middleName":"","lastName":"Maccioni","suffix":""},{"id":589290599,"identity":"c0dba6af-4b5a-4845-8380-b7bf25143b1c","order_by":2,"name":"Chenhao Wu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chenhao","middleName":"","lastName":"Wu","suffix":""},{"id":589290600,"identity":"9d040f26-8d9b-4887-bc6f-93d901df2349","order_by":3,"name":"Chase Shankula","email":"","orcid":"https://orcid.org/0000-0002-7922-655X","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Chase","middleName":"","lastName":"Shankula","suffix":""},{"id":589290601,"identity":"9bb3709e-94a3-44b9-bad2-47ec05b15f61","order_by":4,"name":"Jorge Marquez-Gaytan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jorge","middleName":"","lastName":"Marquez-Gaytan","suffix":""},{"id":589290602,"identity":"283995cb-40ff-4e9c-a785-bc50e81fb1ba","order_by":5,"name":"Itzamar Torres","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Itzamar","middleName":"","lastName":"Torres","suffix":""},{"id":589290603,"identity":"8da2e7f0-ffac-44df-8694-75a66b716ae2","order_by":6,"name":"Roberta Puliga","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Roberta","middleName":"","lastName":"Puliga","suffix":""},{"id":589290604,"identity":"98389e94-d73c-48d9-96ed-502c610c0131","order_by":7,"name":"Vez Repunte-Canonigo","email":"","orcid":"","institution":"The Scripps Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Vez","middleName":"","lastName":"Repunte-Canonigo","suffix":""},{"id":589290605,"identity":"cc304db1-b385-4d87-b45f-9e1fee135a17","order_by":8,"name":"Pietro Paolo sanna","email":"","orcid":"https://orcid.org/0000-0002-8307-2151","institution":"The Scripps Research Insititute","correspondingAuthor":false,"prefix":"","firstName":"Pietro","middleName":"Paolo","lastName":"sanna","suffix":""}],"badges":[],"createdAt":"2026-01-20 21:29:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8653084/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8653084/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102774402,"identity":"2ed9c377-cd71-4ffb-b468-2fc1cba18d95","added_by":"auto","created_at":"2026-02-16 13:31:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":53015,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAlcohol reduces S1P levels in the Prefrontal Cortex of male C57BL/6J mice. \u003c/strong\u003eMetabolomics of the prefrontal cortex (PFC) of C57BL/6J mice injected IP with alcohol (3.5 g/kg) or saline and euthanized 30 min after. Each bar represents the mean ± SEM of 4, 5 mice. (t\u003csub\u003e7\u003c/sub\u003e=3.12; p=0.017).\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-8653084/v1/8b185cc9cf7f16adf69bf5cf.png"},{"id":102962778,"identity":"0a958378-03cb-4595-9003-09bd40c30090","added_by":"auto","created_at":"2026-02-19 04:11:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":241985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eS1P receptor agonists reduce alcohol intake in the drinking-in-the-dark (DID) paradigm in male C57BL/6J mice.\u003c/strong\u003e \u003cstrong\u003eA–B:\u003c/strong\u003e Fingolimod (4 mg/kg) decreased alcohol consumption during the first 2 h (A) and across the full 4 h session \u003cstrong\u003e(B)\u003c/strong\u003e of the DID paradigm (****p \u0026lt; 0.0001). Bars represent mean ± SEM of n=13-15 mice. \u003cstrong\u003eC–D\u003c/strong\u003e: Ozanimod (0.25 and 0.5 mg/kg) reduced alcohol intake during the first 2 h of the DID session (C; *p \u0026lt; 0.05). The effect of ozanimod was not observed over the full 4 h session \u003cstrong\u003e(D)\u003c/strong\u003e. Bars represent mean ± SEM of n=11-12 mice.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-8653084/v1/b24f5435d9edc52482eae728.png"},{"id":103049269,"identity":"e8e01999-ae7e-4d92-a4ef-34488617b31c","added_by":"auto","created_at":"2026-02-20 07:39:23","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1426110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePharmacological effects of CYM5442 in male and female C57BL/6J mice.\u003c/strong\u003e \u003cstrong\u003eA–D\u003c/strong\u003e: CYM5442 reduces alcohol intake in the Drinking-in-the-Dark (DID) binge-drinking paradigm during the first 2 h \u003cstrong\u003e(A, C)\u003c/strong\u003e and across the entire 4 h session \u003cstrong\u003e(B, D) \u003c/strong\u003ein male (n=10) and female (n=8-10) mice. \u003cstrong\u003eE–H\u003c/strong\u003e: Effects on saccharin intake in the DID paradigm. CYM5442 decreases saccharin consumption in male (n=8-9) mice \u003cstrong\u003e(E, F)\u003c/strong\u003e but not in female (n=8-10) mice \u003cstrong\u003e(G, H)\u003c/strong\u003e. \u003cstrong\u003eI–L\u003c/strong\u003e: CYM5442 reduces sucrose consumption in the DID paradigm during the first 2 h \u003cstrong\u003e(I, K) \u003c/strong\u003eand across the full 4 h session \u003cstrong\u003e(J, L)\u003c/strong\u003e in both sexes (male n=8; female n=9). \u003cstrong\u003eM–N\u003c/strong\u003e: Effects of CYM5442 on alcohol drinking in alcohol-dependent (male n=10; female n=10) and non-dependent (male n=9; female n=10) mice using the chronic intermittent ethanol exposure paired with two-bottle choice paradigm (CIE-2BC). Vehicle-treated dependent males consume more alcohol than non-dependent controls \u003cstrong\u003e(M)\u003c/strong\u003e; females show a similar trend \u003cstrong\u003e(N)\u003c/strong\u003e. CYM5442 reduces alcohol intake in both groups, with larger effects in dependent mice. \u003cstrong\u003eO–R\u003c/strong\u003e: CYM5442 does not alter sensitivity to alcohol hypnotic/sedative effects or alcohol metabolism as assessed by loss of righting reflex (LORR) duration and blood alcohol levels (male n=13; female n=15). \u003cstrong\u003eS\u003c/strong\u003e: CYM5442 is less aversive than naltrexone (NTX) as assessed in the Conditioned place aversion test in male (n=9-10) mice. \u003cstrong\u003eT–U\u003c/strong\u003e: CYM5442 does not impair motor coordination on the rotarod test in male (n=12) and female (n=12) mice. Bars represent mean ± SEM. *p\u0026lt;0.05, **p\u0026lt;0.005, ***p\u0026lt;0.0005, ****p\u0026lt;0.0001 in comparison to the vehicle-treated group.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-8653084/v1/abec1d48db7570a4a2e5bc70.png"},{"id":102962448,"identity":"74734b7a-bc73-4ab8-bc45-d9081fc972ea","added_by":"auto","created_at":"2026-02-19 04:08:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":926738,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCYM5442 reduces alcohol self-administration and prevents cue-induced reinstatement in male rats without altering locomotor activity.\u003c/strong\u003e \u003cstrong\u003eA–B\u003c/strong\u003e: Administration of CYM5442 60 min prior to a 30-min fixed-ratio (FR) alcohol self-administration session decreased the number of responses on the alcohol lever \u003cstrong\u003e(A)\u003c/strong\u003e and the amount of self-administered alcohol \u003cstrong\u003e(B)\u003c/strong\u003e in both non-dependent and dependent rats (**p\u0026lt;0.005, ***p\u0026lt;0.001, ****p\u0026lt;0.0001; n = 14–15). \u003cstrong\u003eC–E\u003c/strong\u003e: In a progressive-ratio (PR) session, CYM5442 reduced breakpoint value \u003cstrong\u003e(C)\u003c/strong\u003e, lever responses on the alcohol lever \u003cstrong\u003e(D)\u003c/strong\u003e, and number of alcohol rewards earned \u003cstrong\u003e(E)\u003c/strong\u003e (*p\u0026lt;0.05, **p\u0026lt;0.005, ***p\u0026lt;0.0005; n=12). \u003cstrong\u003eF–G\u003c/strong\u003e: During an extinction training, dependent and non-dependent rats progressively reduced lever responding over 5 consecutive 30-min sessions (\u003cstrong\u003eF\u003c/strong\u003e; *p\u0026lt;0.05, ***p\u0026lt;0.0005, ****p\u0026lt;0.0001; non-dependent n=28, dependent n=34). Re-exposure to alcohol-paired cues reinstated alcohol-seeking behavior in dependent rats and it was prevented by administration of CYM5442 (10 mg/kg) 60 min before testing (\u003cstrong\u003eG\u003c/strong\u003e; #p\u0026lt;0.05, $p\u0026lt;0.005, ****p\u0026lt;0.0001; non-dependent n=14, dependent n=17). \u003cstrong\u003eH–I\u003c/strong\u003e: CYM5442 does not alter spontaneous locomotor activity in alcohol-naive male rats. Total distance \u003cstrong\u003e(H)\u003c/strong\u003e and distance across six 10-min intervals \u003cstrong\u003e(I)\u003c/strong\u003e were unaffected by CYM5442 (n=6 per group). Bars represent mean ± SEM.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-8653084/v1/416bb5f8513e40851c085672.png"},{"id":103056299,"identity":"17981cff-a076-4a1a-9994-cbde654b8e85","added_by":"auto","created_at":"2026-02-20 09:05:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2315628,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePathway analysis of CYM5442-treated rats exposed to CIE. A)\u003c/strong\u003e The GSEA algorithm was used for pathway analysis of gene expression changes induced by CYM5442 in rats exposed to CIE vs. vehicle-treated rats exposed to CIE. On the X axis is the pathway analysis of CIE-exposed (dependent) rats vs alcohol naive control rats (D-VEH vs. N-VEH); on the Y axis is the pathway analysis of CIE-exposed CYM5442-treated rats vs. alcohol naive control rats (D-CYM vs N-VEH). Concordantly regulated pathways in vehicle-treated CIE-exposed rats vs. controls and CYM5442-treated CIE-exposed rats vs. controls are shown on the top right (red: induced) and bottom left (blue: decreased) quadrants. On the top left (turquoise) and bottom right (yellow) quadrants are the pathways specifically regulated by CYM5442. These pathways are discordantly regulated by vapor in CYM5442 treated vs. vehicle treated rats. The normalized enrichment scores (NES) are shown for pathways that are significantly differential regulated (p\u0026lt;0.05). \u003cstrong\u003eB) \u003c/strong\u003ePathways specifically regulated by CYM5442 in CIE exposed rats, represented on the top left and bottom right quadrants in A include pathways involved of signal transduction, neuronal function, synaptic and structural neuronal plasticity, and regulation of gene expression.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-8653084/v1/667a4ee23e5f806e5a6aede9.png"},{"id":103056694,"identity":"ffe0d868-e0a9-433e-bf12-fb1f907d252b","added_by":"auto","created_at":"2026-02-20 09:24:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6288036,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8653084/v1/130931d4-3ce4-4e6c-85fa-c728b8c46650.pdf"},{"id":102962476,"identity":"b5367bac-f79a-4212-9e4e-ffde217f3ef0","added_by":"auto","created_at":"2026-02-19 04:09:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":249956,"visible":true,"origin":"","legend":"Supplemental Matierial","description":"","filename":"SMLorraietal.2026.docx","url":"https://assets-eu.researchsquare.com/files/rs-8653084/v1/9449ccc699d1809f19265b11.docx"},{"id":102962416,"identity":"648935f5-f4a1-41e0-9994-7f7caf851bc7","added_by":"auto","created_at":"2026-02-19 04:08:18","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":254352,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1. Effect of CYM5442 on mice exposed to CLAMS.\u003c/strong\u003e Male mice were administered IP with CYM5442 (0-5 mg/kg) 60 min before the 3\u003csup\u003erd\u003c/sup\u003e h of the dark phase of the dark/light cycle. \u003cstrong\u003eFood, water intake and respiratory exchange ratio were measured in male C57BL/6J mice (n=8 per group).\u003c/strong\u003e Values are reported as mean ± SEM. *p\u0026lt;0.05, **p\u0026lt;0.01, ****p\u0026lt;0.0001 in comparison to the vehicle-treated group.\u003c/p\u003e","description":"","filename":"Table1.tif","url":"https://assets-eu.researchsquare.com/files/rs-8653084/v1/96310eb59689b39f6445eba7.tif"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e\nPPS is an inventor on a patent application related to this paper. The other authors declare no competing interests","formattedTitle":"\u003cp\u003eSphingosine-1-phosphate (S1P) signaling as a novel therapeutic target for alcohol abuse\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlcohol 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 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). 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) (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Nalmefene has been approved in Europe and baclofen in France (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Other medications such as topiramate, varenicline, ondansetron, gabapentin, aripiprazole, and prazosin/doxazosin have shown efficacy in some clinical trials (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Due to AUD heterogeneity, both approved and investigational medications have limited efficacy, which reduces the confidence of clinicians in prescribing medications for AUD (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). 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 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The latter will require the identification and validation of new and more effective druggable targets (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHere 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 (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). S1P is derived from the phosphorylation of sphingosine by two sphingosine kinase (SphK) isoenzymes, SphK1 and SphK2, broadly expressed in the brain (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). S1P can either act as a second messenger within the cells or can be released and signal through five G protein-coupled (S1P\u003csub\u003e1-5\u003c/sub\u003e) receptors (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e) that are widely expressed in the body including in lymphocytes, neurons, astrocytes, oligodendrocytes, and microglia (\u003cspan additionalcitationids=\"CR10 CR11\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). S1P and its receptors are involved in physiological and pathological states including synaptic transmission, autophagy, and neuroinflammation, among others (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). 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\u0026rsquo;s disease, and other conditions (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Fingolimod, ozanimod and other S1P agonists are FDA-approved for relapsing-remitting multiple sclerosis and ozanimod also for ulcerative colitis.\u003c/p\u003e \u003cp\u003eHere, 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.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale 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\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;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 \u0026ldquo;Care and Use of Laboratory Animals\u0026rdquo; and were approved by the Institutional Animal Care and Use Committee of The Scripps Research Institute.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDrugs\u003c/h3\u003e\n\u003cp\u003eFingolimod, 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.\u003c/p\u003e\n\u003ch3\u003eBehavioral procedures\u003c/h3\u003e\n\u003cp\u003eTo 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 (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). 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\u003csub\u003e1\u0026thinsp;\u0026minus;\u0026thinsp;5\u003c/sub\u003e) except S1P\u003csub\u003e2\u003c/sub\u003e, whereas ozanimod selectively targets S1P\u003csub\u003e1\u003c/sub\u003e and S1P\u003csub\u003e5\u003c/sub\u003e. In contrast, CYM5442 is a highly selective agonist for the S1P\u003csub\u003e1\u003c/sub\u003e receptor and has been shown to accumulate in the brain (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). 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.\u003c/p\u003e\n\u003ch3\u003eMetabolomics\u003c/h3\u003e\n\u003cp\u003eTargeted LC-MS/MS quantification of S1P in the PFC of alcohol-na\u0026iuml;ve 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 \u0026micro;L cold MeOH:H2O (4:1, v/v). To precipitate proteins, the samples were incubated at \u0026minus;\u0026thinsp;20\u0026deg;C followed by centrifugation at 13000 rpm and 4\u0026deg;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\u0026deg;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 2.1x50mm 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.\u003c/p\u003e\n\u003ch3\u003eGene expression analysis\u003c/h3\u003e\n\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eTotal RNA isolation and RNA-sequencing\u003c/h2\u003e \u003cp\u003eDependent 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).\u003c/p\u003e \u003cp\u003e \u003cem\u003eGene expression measures and Gene Set Enrichment Pathway Analysis.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eRNA-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.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eMice Experiments\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003eAlcohol decreases S1P levels in the mouse prefrontal cortex\u003c/h2\u003e \u003cp\u003eTargeted 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\u0026deg;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\u003csub\u003e7\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.12; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eS1P receptor agonists reduce binge-like alcohol drinking\u003c/h2\u003e \u003cp\u003eFingolimod. 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(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;11.41, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and the entire 4 h drinking session [1-way ANOVA, F(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;16.74, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; \u003cb\u003e****\u003c/b\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 by Tukey\u0026rsquo;s post hoc test] in male C57BL/6J mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOzanimod. 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 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;5.33; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; \u003cb\u003e*\u003c/b\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by Tukey\u0026rsquo;s post hoc test] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, the reducing effect of ozanimod was not evident at the end of the 4 h drinking session [1-way ANOVA, F (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.05, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), consistent with its short half-life (172).\u003c/p\u003e \u003cp\u003eCYM5442. We observed that acute administration of CYM5442 resulted in a statistically significant reduction of alcohol intake (1-way ANOVA: F(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;13.66; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the mouse groups treated with 2.5 and 5 mg/kg in comparison to the mouse vehicle-treated group (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.005; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 by Tukey\u0026rsquo;s post hoc test) during the first 2 h of the drinking session (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, the alcohol-reducing effect of CYM5442 persisted through the entire 4 h session at the highest administered dose [1-way ANOVA: F (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;17.46; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 by Tukey\u0026rsquo;s post hoc test] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;15.51; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 by Tukey\u0026rsquo;s post hoc test] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). At the end of the 4 h session, alcohol consumption remained significantly reduced at the highest dose [1-way ANOVA: F (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;6.30; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.005, by Tukey\u0026rsquo;s post hoc test] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eCYM5442 reduces non-drug reinforcers intake in the drinking in the dark paradigm in male and female C57BL/6J mice\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.51; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Tukey\u0026rsquo;s post hoc analysis indicated that the 2.5 and 5 mg/kg doses produced statistically significant reductions (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.005 and *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively). (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.88; p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 by Tukey\u0026rsquo;s post hoc test] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;5.83; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by Tukey\u0026rsquo;s post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). At the end of the entire 4h a slight reduction in saccharin intake was still observed (1-way ANOVA F (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.23; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), however, post hoc analysis did not reveal any statistically significant differences between groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH).\u003c/p\u003e \u003cp\u003eA 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;8.62; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005), particularly at the 2.5 and 5 mg/kg doses, as indicated by Tukey\u0026rsquo;s post hoc test (**p\u0026thinsp;\u0026lt;\u0026thinsp;0.01 and ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eI). 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;6.85; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.005 by Tukey\u0026rsquo;s post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eJ). 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;6.16; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 by Tukey\u0026rsquo;s post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK) and the entire 4 h session (1-way ANOVA F (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;7.41; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 by Tukey\u0026rsquo;s post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eL).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCYM5442 reduces alcohol drinking in 2-bottle choice (2BC) after chronic intermittent ethanol (CIE) in male and female C57BL/6J mice\u003c/em\u003e \u003c/p\u003e \u003cp\u003eThe 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) (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e) 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 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;7.74; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and treatment (F (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;13.97; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) as well as significant interaction (F (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;8.30; p\u0026thinsp;\u0026lt;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;0.005 by Šid\u0026aacute;k post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). 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\u0026thinsp;\u0026lt;\u0026thinsp;0.0005, Šid\u0026aacute;k post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eM). 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 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.74; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and treatment (F (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;36.23; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with a trend toward a significant interaction (F (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.75; p\u0026thinsp;=\u0026thinsp;0.06) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). 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\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and dependent (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) groups compared to their vehicle-treated control groups (Šid\u0026aacute;k post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eN).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eCYM5442 does not affect loss of righting reflex (LORR) or alcohol metabolism in male and female mice\u003c/em\u003e \u003c/p\u003e \u003cp\u003eTo 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\u003csub\u003e26\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.38, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eO). Similarly, in females, treatment had no significant effect on LORR duration (unpaired two-tailed t-test, t\u003csub\u003e29\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.05, p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eQ). 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)\u0026thinsp;=\u0026thinsp;1401; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Treatment F (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;1.26; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Interaction F (3,84)\u0026thinsp;=\u0026thinsp;1.27; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eP). For females: 2-way RM ANOVA, Time F (3,90)\u0026thinsp;=\u0026thinsp;2117; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Treatment F (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.002; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Interaction F (3,90)\u0026thinsp;=\u0026thinsp;2.17; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eR).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eCYM5442 reduces food, water intake, and energy metabolism in male C57BL/6J mice\u003c/h2\u003e \u003cp\u003eCYM5442 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(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;7.87; p\u0026thinsp;=\u0026thinsp;0.0007), water (F(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;8.155; p\u0026thinsp;=\u0026thinsp;0.0005) (Table\u0026nbsp;1) and the entire 4 h session (food: 1-way ANOVA F(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.014; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), water (F(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.147; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)), (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005; Tukey\u0026rsquo;s post hoc) (Table\u0026nbsp;1). In addition, CYM5442 significantly decreased mouse respiratory exchange ratio (RER) over both the first 2 h [1-way ANOVA F(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;4.22; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE) and 4 h session (1-way ANOVA F(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;3.14; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), although only at the highest dose of 5 mg/kg (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Tukey\u0026rsquo;s post hoc) (Table\u0026nbsp;1).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCYM5442 is less aversive than naltrexone in the conditioned place aversion paradigm\u003c/h2\u003e \u003cp\u003eAlcohol-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 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;16.03, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but not treatment, although a trend toward significance was observed (F (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;2.96, p\u0026thinsp;=\u0026thinsp;0.07). In addition, a significant treatment x time interaction (F (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;6.71, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) was also found. Post hoc analysis showed a highly significant difference between pre and post conditioning in the naltrexone-treated group (***p\u0026thinsp;=\u0026thinsp;0.0001, by Šid\u0026aacute;k post hoc test) and a moderately significant difference was observed in the CYM5442-treated group (p\u0026thinsp;=\u0026thinsp;0.045, by Šid\u0026aacute;k post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eS). Overall, these data suggests that naltrexone produces a stronger aversive effect compared to CYM5442.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCYM5442 does not alter mouse motor coordination\u003c/h2\u003e \u003cp\u003ePotential 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.11; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05] (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eT) or females (F (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.25; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eU).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRat experiments\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCYM5442 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\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe 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)\u0026thinsp;=\u0026thinsp;20.66; p\u0026thinsp;\u0026lt;\u0026thinsp;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)\u0026thinsp;=\u0026thinsp;18.78; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). No significant interaction between treatment and group was observed (F (3,103)\u0026thinsp;=\u0026thinsp;1.467; p\u0026thinsp;\u0026gt;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.005, Tukey\u0026rsquo;s post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Consistently, alcohol dependent rats self-administered greater amounts of alcohol than non-dependent rats (1.0 \u003cem\u003evs\u003c/em\u003e 0.6 g/kg/30-min session; main effect of group: F (1,103)\u0026thinsp;=\u0026thinsp;21.85; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Two-way ANOVA revealed a significant main effect of CYM5442 treatment on alcohol intake (F (3,103)\u0026thinsp;=\u0026thinsp;18.73; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) with no significant interaction between group and treatment (F (3,103)\u0026thinsp;=\u0026thinsp;1.553; p\u0026thinsp;\u0026gt;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.005, by Tukey\u0026rsquo;s post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo 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)\u0026thinsp;=\u0026thinsp;22.13; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001)) and treatment (F (2,66)\u0026thinsp;=\u0026thinsp;8.26; p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) with no significant interaction between the two factors (F (2,66)\u0026thinsp;=\u0026thinsp;0.32; p\u0026thinsp;\u0026gt;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;0.005; *p\u0026thinsp;=\u0026thinsp;0.05 by Tukey\u0026rsquo;s post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). 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)\u0026thinsp;=\u0026thinsp;24.04; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and treatment (F (2,66)\u0026thinsp;=\u0026thinsp;5.98; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), but no significant interaction (F (2,66)\u0026thinsp;=\u0026thinsp;1.06; p\u0026thinsp;\u0026gt;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;0.005; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by Tukey\u0026rsquo;s post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Similar results were observed on number of alcohol rewards earned. Two-way ANOVA revealed significant main effects of group (F (1,66)\u0026thinsp;=\u0026thinsp;21.33; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and treatment (F (2,66)\u0026thinsp;=\u0026thinsp;8.11; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005), but no significant interaction (F (2,66)\u0026thinsp;=\u0026thinsp;0.28; p\u0026thinsp;\u0026gt;\u0026thinsp;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\u0026thinsp;\u0026lt;\u0026thinsp;0.005; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by Tukey\u0026rsquo;s post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCYM5442 prevents cues-induced reinstatement of alcohol seeking behavior in alcohol dependent rats\u003c/h2\u003e \u003cp\u003eAfter 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)\u0026thinsp;=\u0026thinsp;25.86; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and day [F (4,247)\u0026thinsp;=\u0026thinsp;22.07; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) but no significant interaction between these factors (F (4,247)\u0026thinsp;=\u0026thinsp;1.53; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Post hoc analysis indicated that lever-responding was higher in dependent rats compared to non-dependent rats during extinction days 1\u0026ndash;3 (****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0005, *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 by Tukey\u0026rsquo;s post hoc test) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, 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)\u0026thinsp;=\u0026thinsp;8.48; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) and treatment (F (1,60)\u0026thinsp;=\u0026thinsp;8.64; p\u0026thinsp;\u0026lt;\u0026thinsp;0.005) but not protocol (extinction/reinstatement) (F (1,60)\u0026thinsp;=\u0026thinsp;0.847; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). No significant interactions were observed for protocol x treatment (F (1,60)\u0026thinsp;=\u0026thinsp;0.04; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), protocol x group (F (1,60)\u0026thinsp;=\u0026thinsp;21.57; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), treatment x group (F (1,60)\u0026thinsp;=\u0026thinsp;0.949; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05), or protocol x group x treatment (F (1,60)\u0026thinsp;=\u0026thinsp;2.38; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Post hoc analysis revealed that CYM5442 significantly prevented cue-induced reinstatement in the dependent rat group (\u003cspan\u003e$\u003c/span\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.005; #p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, by Bonferroni post hoc). (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, right panel).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eCYM5442 does not alter rat spontaneous locomotor activity\u003c/h2\u003e \u003cp\u003eAs 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 (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.59; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Furthermore, two-way RM ANOVA of ambulatory distance across six 10-min intervals, revealed a significant effect of time (F(5,100)\u0026thinsp;=\u0026thinsp;156.7; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but no significant effects of treatment (F (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e)\u0026thinsp;=\u0026thinsp;0.59; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) or time x treatment (F (15,100)\u0026thinsp;=\u0026thinsp;0.71; p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003cem\u003eS1P regulates a complex set of genes in the transition to alcohol dependence.\u003c/em\u003e \u003c/p\u003e \u003cp\u003eWe 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.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere 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 (\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). 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 (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHigh levels of S1P\u003csub\u003e1\u003c/sub\u003e receptor have been identified in the prefrontal cortex and striatum, two brain regions involved in alcohol use and abuse (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). In human alcoholics, prefrontal cortex deficits are believed to contribute to excessive drinking and increased vulnerability to relapse (\u003cspan additionalcitationids=\"CR23 CR24 CR25 CR26 CR27 CR28\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). Rodents with a history of alcohol dependence exhibit cognitive impairment that reflects prefrontal cortex dysfunction (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Fronto-striatal circuits are implicated in the loss of control and enhanced motivation to drink that characterize AUD (\u003cspan additionalcitationids=\"CR34 CR35\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAcute 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 (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003eParallel 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 (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo 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\u0026ndash;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.\u003c/p\u003e \u003cp\u003eOverall, these results indicating CYM5442 effects on mouse drinking (drug, non-drug reinforcers, and water) and feeding, suggest that the S1P\u003csub\u003e1\u003c/sub\u003e 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 (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). However, in contrast with the observed CYM5442 properties, naltrexone also induces conditioned place aversion (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). A similar ability to affect broad reduction of rodent consummatory behavior has been shown for cannabinoids 1 (CB\u003csub\u003e1\u003c/sub\u003e) receptors (CB\u003csub\u003e1\u003c/sub\u003e-R) antagonists such as rimonabant, AM6527, and AM4113 (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Interestingly, it has been reported that S1P and its analog, the non-selective S1P agonist fingolimod, interact with the CB\u003csub\u003e1\u003c/sub\u003e-Rs, suggesting that molecules belonging to the same pharmacological class could potentially be provided with CB\u003csub\u003e1\u003c/sub\u003e-R activity (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). However, we found that CYM5442 does not (see Supplementary material), supporting that the effect of S1P agonists on alcohol drinking is independent of CB\u003csub\u003e1\u003c/sub\u003e-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 (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). The glucagon-like peptide-1 (GLP-1) analogue semaglutide has been shown to reduce alcohol, water, saccharin, maltodextrin and corn oil intake (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Overall, the present findings are consistent with the overlapping neurobiological and chemosensory mechanisms that regulate food, drug reward, and consummatory behavior (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Consistently, preclinical and clinical data indicate a strong correlation between excessive alcohol and sweet food consumption in rodents and humans (\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), 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 (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we observed that acute administration of CYM5442 effectively reduced water, food intake and RER in mice, supporting a role for S1P\u003csub\u003e1\u003c/sub\u003e also in energy balance and metabolism. This effect could be explained by the activation of S1P\u003csub\u003e1\u003c/sub\u003e 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\u003csub\u003e1\u003c/sub\u003e protein in all hypothalamic nuclei with a predominance in the arcuate, dorsomedial, and ventromedial nuclei. In addition, the S1P\u003csub\u003e1\u003c/sub\u003e receptor was found predominantly in anorexigenic but not in orexigenic neurons in the arcuate nucleus (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn 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.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e PPS is an inventor on a patent application related to this paper. The other authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e. This work was supported by NIH Grant AA021667; IL was partially supported by training grant T32 AA007456.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003eSupplementary information is available at MP’s website.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eKoob GF, Vendruscolo L. Theoretical Frameworks and Mechanistic Aspects of Alcohol Addiction: Alcohol Addiction as a Reward Deficit/Stress Surfeit Disorder. 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JCI Insight. 2023;8(12).\u003c/li\u003e\n \u003cli\u003eVolkow ND, Wang GJ, Tomasi D, Baler RD. Obesity and addiction: neurobiological overlaps. Obes Rev. 2013;14(1):2-18.\u003c/li\u003e\n \u003cli\u003eKampov-Polevoy A, Garbutt JC, Janowsky D. Evidence of preference for a high-concentration sucrose solution in alcoholic men. Am J Psychiatry. 1997;154(2):269-70.\u003c/li\u003e\n \u003cli\u003eKampov-Polevoy AB, Garbutt JC, Janowsky DS. Association between preference for sweets and excessive alcohol intake: a review of animal and human studies. Alcohol Alcohol. 1999;34(3):386-95.\u003c/li\u003e\n \u003cli\u003eLeggio L, Addolorato G, Cippitelli A, Jerlhag E, Kampov-Polevoy AB, Swift RM. Role of feeding-related pathways in alcohol dependence: A focus on sweet preference, NPY, and ghrelin. Alcohol Clin Exp Res. 2011;35(2):194-202.\u003c/li\u003e\n \u003cli\u003eVolkow ND, Wise RA, Baler R. The dopamine motive system: implications for drug and food addiction. Nat Rev Neurosci. 2017;18(12):741-52.\u003c/li\u003e\n \u003cli\u003eWiss DA, Avena N, Rada P. Sugar Addiction: From Evolution to Revolution. Front Psychiatry. 2018;9:545.\u003c/li\u003e\n \u003cli\u003eSilva VR, Katashima CK, Bueno Silva CG, Lenhare L, Micheletti TO, Camargo RL, et al. Hypothalamic S1P/S1PR1 axis controls energy homeostasis in Middle-Aged Rodents: the reversal effects of physical exercise. Aging (Albany NY). 2016;9(1):142-55.\u003c/li\u003e\n \u003cli\u003eSilva VR, Micheletti TO, Pimentel GD, Katashima CK, Lenhare L, Morari J, et al. Hypothalamic S1P/S1PR1 axis controls energy homeostasis. Nat Commun. 2014;5:4859.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the supplementary files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8653084/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8653084/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSphingosine-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 S1P\u003csub\u003e1\u003c/sub\u003e 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.\u003c/p\u003e","manuscriptTitle":"Sphingosine-1-phosphate (S1P) signaling as a novel therapeutic target for alcohol abuse","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-16 13:31:13","doi":"10.21203/rs.3.rs-8653084/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-03-09T16:24:50+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-03-02T00:31:15+00:00","index":3,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-24T04:29:17+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-23T17:17:05+00:00","index":4,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-17T23:40:38+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-12T01:36:08+00:00","index":4,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-11T07:05:09+00:00","index":3,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-11T04:00:46+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-02-11T02:18:43+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-02-11T02:01:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-22T16:48:50+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-22T16:14:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Psychiatry","date":"2026-01-21T17:06:53+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2026-01-21T11:17:45+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b457863d-c0e3-458a-a197-42a3a9cc2f57","owner":[],"postedDate":"February 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":63011769,"name":"Health sciences/Diseases/Psychiatric disorders/Addiction"},{"id":63011770,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-03-09T16:42:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-16 13:31:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8653084","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8653084","identity":"rs-8653084","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}