The opioid epidemic remains a persistent public health challenge in the United States, leaving many people affected by or vulnerable to opioid use disorder (OUD). Although US Food and Drug Administration-approved medications for OUD improve the odds of recovery and reduce mortality, relapse rates remain high and access, adherence, and individual response variabilities limit their overall impact. The ongoing burden of OUD therefore underscores the need to develop additional therapeutics that target complementary biological mechanisms and support long-term recovery. One strategy of interest is to delineate the biological overlap between OUD and other motivated behaviors, particularly the neurobiology underlying appetitive and reward-driven processes. The orexigenic peptide hormone ghrelin, often referred to as the “hunger hormone,” regulates energy homeostasis, hedonic feeding, and reward sensitivity by binding to the growth hormone secretagogue receptor 1α (GHS1αR), a receptor expressed in mesolimbic circuits implicated in addiction. In the present study, we used the inverse agonist/antagonist PF5190457 to assess the impact of full GHS1αR blockade on fentanyl-mediated behaviors in preclinical models. We found that PF5190457 significantly reduced fentanyl self-administration and attenuated drug-seeking during cue tests while maintaining a favorable pharmacological and safety profile in risk assessment assays, including measures of withdrawal and respiratory depression. Collectively, these findings demonstrate a functional role for ghrelin-GHS1αR signaling in the reinforcing effects of fentanyl and positions GHS1αR inverse agonism as a promising, mechanistically novel therapeutic strategy for OUD treatment. Significance Statement The present work demonstrates that the growth hormone secretagogue receptor 1α (GHS1αR) inverse agonist/antagonist PF5190457 reduces fentanyl self-administration and drug-seeking while maintaining a favorable safety profile in male rats, supporting ghrelin-GHS1αR modulation as a promising therapeutic strategy for opioid use disorder.

Overdose fatalities involving cocaine continue to rise with over 5.3 million cocaine users reported in the United States in 2022. The abuse liability of cocaine is reliant upon inhibition of dopamine (DA) reuptake and consequent increase in DA efflux in meso-corticolimbic circuitry that controls reward and motivation. Cocaine also increases serotonin (5-HT) efflux which is integral in cocaine abuse. The 5-HT2A receptor (5-HT2AR) is a key regulator of meso-corticolimbic DA release and controls cellular mechanisms underlying cocaine effects. 5-HT2AR actions contribute importantly to psychedelic mechanisms of action, and the efficacy of these compounds in limiting cocaine intake is unknown. The present studies evaluated the efficacy of acute administration of a psychedelic to reduce cocaine intake using standard and advanced preclinical models of drug self-administration. Both a standard fixed ratio (FR) schedule and behavioral economics threshold procedure of cocaine intravenous self-administration were employed to evaluate the efficacy of the psychedelic 5-HT2AR agonist (−)-2,5-dimethoxy-4-iodoamphetamine [(−)-DOI] to decrease cocaine intake and motivation for cocaine in male rats. The 5-HT2AR-selective antagonist M100907 was utilized to explore the role of 5-HT2AR in the effects of (−)-DOI on cocaine intake. We found that (−)-DOI dose-dependently reduced intake on the FR5 schedule of cocaine IVSA and left shifted the demand curve to evoke greater sensitivity to price increases in the behavioral economics paradigm. Pretreatment with M100907 abated the efficacy of (−)-DOI on cocaine intake in both paradigms. (−)-DOI ‘devalued’ cocaine reward and motivation to take cocaine in a 5-HT2AR-dependent manner. As serotonergic psychedelics emerge as therapeutic candidates, investigations of 5-HT2AR-acting psychedelics in preclinical analyses of cocaine intake and relapse vulnerability during abstinence will be valuable as prelude to future clinical trials.

Objectives/Goals: One in 14 individuals have a substance use disorder (SUD). We suggest that a trait of poor impulse control, or high impulsivity, may predict relapse risk. We explore how changes in brain structure linked to decision-making and reward might drive high impulsivity, helping create a “biosignature” to identify those most at risk and guide treatment choices. Methods/Study Population: Male rats were phenotyped as high impulsive (HI) or low impulsive (LI) based on premature responses on the one-choice serial reaction time (1-CSRT) task. Rats then received an intracranial infusion of a retrograde virus (AAVr2) in the nucleus accumbens (NAc) to trace corticoaccumbens neurons back to the medial prefrontal cortex (mPFC). After impulsivity phenotyping (ITI8), another cohort of animals performed cocaine self-administration followed by 30 days of abstinence. Cue reactivity, a measure of relapse-like behaviors, was performed on abstinence day 30. Analyses of microtubule-associated protein 2 (MAP2), a cytoskeletal marker of dendrites, spines, and somas was performed with western blotting and fluorescent images of brain slices after phenotyping and cocaine abstinence. Results/Anticipated Results: HI rats made greater premature responses, a marker of impulsive action vs. LI rats at baseline (p Discussion/Significance of Impact: Poor inherent impulse control and drug cues heighten relapse risk. We found high impulsivity linked to brain structure differences and lower protein markers of synaptic (units supporting signaling) strengthening. Future investigations into brain-behavior links with impulsivity may further identify a SUD relapse vulnerability biosignature.

Abstract ID 98540Poster Board 087AIMS: The 5-HT2A receptor (5-HT2AR) is a promising therapeutic target for neuropsychiatric disorders. There is renewed therapeutic interest in psychedelic 5-HT2AR agonists; however, their abuse liability and ancillary pharmacological actions for off-target toxicities are of concern. Targeting 5-HT2AR allosteric sites spatially distinct from the conserved orthosteric site that binds 5-HT creates new opportunities to precisely enrich 5-HT2AR signaling via selective 5-HT2AR positive allosteric modulators (PAMs). We have identified novel 5-HT2AR PAMs, unlocking an entirely new and innovative line of investigation. These studies are focused to assess in vitro and in vivo profiles of putative 5-HT2AR PAMs.METHODS: Newly synthesized molecules (CTW0404, CTW0419) were screened for efficacy to enhance 5-HT-induced intracellular calcium release in stable cell lines expressing the human (h) 5-HT2AR, h5-HT2BR or h5-HT2CR and evaluated in in vitro drug metabolism and pharmacokinetics (DMPK) assays and secondary competition binding to CNS targets prior to scale-up for in vivo DMPK and blood-brain barrier penetration analyses. CTW0404 and CTW0419 were further evaluated in male Sprague-Dawley rats trained to discriminate the 5-HT2AR agonist 2,5-dimethoxy-4-iodoamphetamine [(-)-DOI] from saline. Rats were also monitored for head twitches following treatment with 5-HT2AR PAMs to gauge hallucinogenic potential. In silico molecular docking with receptor X-ray crystal structures was used to identify potential PAM binding sites.RESULTS: CTW0404 and CTW0419 exhibited 5-HT2AR PAM efficacy without intrinsic agonist activity, were devoid of actions at homologous 5-HT2R subtypes and possessed minimal binding to off-target CNS receptors. In vitro DMPK demonstrated CTW0404 and CTW0419 possess various drug-like qualities; less favorable measures leave room for continued optimization. In vivo DMPK studies detected both compounds in rat brain and a greater brain to plasma ratio for CTW0404 vs. CTW0419. CTW0404 and CTW0419 did not substitute for the interoceptive cue of (−)-DOI and did not evoke head twitches. Intriguingly, CTW0404 and CTW0419 significantly blunted the (−)-DOI-evoked head twitch response. In silico studies revealed that the 5-HT2AR PAMs occupied a putative 5-HT2AR allosteric pocket at the top of the seven transmembrane (TM) bundle comprising extracellular loop 2 (ECL2) and the TM helices I-III and V-VII.CONCLUSIONS: We designed, synthesized and evaluated a novel series of 5-HT2AR PAMs with promising profiles. The disparate, complex behavioral findings of CTW0404 and CTW0419 necessitate further in vivo and in vitro mechanism of action studies. In the future, we will optimize these 5-HT2AR PAMs with improved drug-like properties and analyze select molecules in proof-of-concept in vivo assays as new pharmacological tools to develop novel candidate medications with reduced hallucinogenic properties.Supported by R01DA038446, T32DA007287 and the UTMB Center for Addiction Sciences and Therapeutics.

OBJECTIVES/GOALS: Neuropsychiatric disorders classified as synaptopathies are marked by a glutamate-associated hypofrontality which impacts decision making and impulsivity. We hypothesized that behavioral efficacy of the psychoplastogen ketamine is mediated in part through lasting promotion of markers of synaptic strength in corticoaccumbens circuit. METHODS/STUDY POPULATION: Male, Sprague-Dawley rats received an intraperitoneal (i.p.) injection of saline, single ketamine (10 mg/kg; 1x/day), or repeated ketamine (10 mg/kg; 1x/day for three days). Twenty-four hrs following the dosing regimen, animals were euthanized, and brains dissected to harvest corticoaccumbens structures including the medial prefrontal cortex (mPFC) and the nucleus accumbens (NAc). mRNA was extracted and converted to cDNA. Levels of brain derived neurotrophic factor (BDNF) exon II mRNA were quantified using reverse transcriptase polymerase chain reaction (RT-PCR); cyclophilin A (PPIA) was used as a loading control. Gene expression differences in ketamine-treated rats were identified versus saline-treated rats. BDNF protein levels were quantified using capillary-electrophoresis immunoblotting. RESULTS/ANTICIPATED RESULTS: Repeated, but not single, ketamine administration decreased mPFC, but increased NAc, BDNF exon II mRNA levels versus saline (p<0.05). Single and repeated ketamine administration increased NAC BDNF protein (p<0.05), while both dosing paradigms induced a trend towards an increase in mPFC BDNF levels. DISCUSSION/SIGNIFICANCE: We discovered a dosing regimen-dependent and sustained effects of ketamine administration on BDNF levels in the rodent brain. Taken together, ketamine-mediated BDNF levels may sustain synaptic strengthening mechanisms supporting future investigation into the utility of ketamine for diseases characterized by synaptopathies.

The drug overdose crisis has spawned serious health consequences, including the increased incidence of substance use disorders (SUDs), conditions manifested by escalating medical and psychological impairments. While medication management is a key adjunct in SUD treatment, this crisis has crystallized the need to develop additional therapeutics to facilitate extended recovery from SUDs. The “hunger hormone” ghrelin acts by binding to the growth hormone secretagogue receptor 1α (GHS1αR) to control homeostatic and hedonic aspects of food intake and has been implicated in the mechanisms underlying SUDs. Preclinical studies indicate that GHS1αR antagonists and inverse agonists suppress reward-related signaling associated with cocaine and opioids. In the present study, we found that the GHS1αR antagonist JMV2959 was efficacious to suppress both cue-reinforced cocaine and oxycodone drug-seeking, but not cocaine or oxycodone self-administration in male Sprague-Dawley rats. These data suggest a role of the ghrelin-GHS1αR axis in mediating overlapping reward-related aspects of cocaine and oxycodone and premises the possibility that a GHS1αR antagonist may be a valuable therapeutic strategy for relapse vulnerability in SUDs.

Opioid misuse and opioid-involved overdose deaths are a massive public health problem involving the intertwined misuse of prescription opioids for pain management with the emergence of extremely potent fentanyl derivatives, sold as standalone products or adulterants in counterfeit prescription opioids or heroin. The incidence of repeated opioid overdose events indicates a problematic use pattern consistent with the development of the medical condition of opioid use disorder (OUD). Prescription and illicit opioids reduce pain perception by activating µ-opioid receptors (MOR) localized to the central nervous system (CNS). Dysregulation of meso-corticolimbic circuitry that subserves reward and adaptive behaviors is fundamentally involved in the progressive behavioral changes that promote and are consequent to OUD. Although opioid-induced analgesia and the rewarding effects of abused opioids are primarily mediated through MOR activation, serotonin (5-HT) is an important contributor to the pharmacology of opioid abused drugs (including heroin and prescription opioids) and OUD. There is a recent resurgence of interest into psychedelic compounds that act primarily through the 5-HT2A receptor (5-HT2AR) as a new frontier in combatting such diseases (e.g., depression, anxiety, and substance use disorders). Emerging data suggest that the MOR and 5-HT2AR crosstalk at the cellular level and within key nodes of OUD circuitry, highlighting a major opportunity for novel pharmacological intervention for OUD. There is an important gap in the preclinical profiling of psychedelic 5-HT2AR agonists in OUD models. Further, as these molecules carry risks, additional analyses of the profiles of non-hallucinogenic 5-HT2AR agonists and/or 5-HT2AR positive allosteric modulators may provide a new pathway for 5-HT2AR therapeutics. In this review, we discuss the opportunities and challenges associated with utilizing 5-HT2AR agonists as therapeutics for OUD.

ID 27882 Poster Board 3 Aims: Drug overdose deaths within the U.S. reached an unprecedented high, surpassing 100,000 in 2021. Cocaine overdoses account for ∼15,000 deaths each year and cocaine use disorder (CUD) remains a challenge to treat, with the current standard of care reliant primarily on psychosocial interventions, such as cognitive behavioral therapy. Efficacious pharmacotherapeutic support for CUD is an important goal in our field. In the last decade, there has been a resurgence of interest in serotonergic psychedelics as potential therapeutics for substance use and mental disorders, including CUD. Agonist actions at the serotonin (5-HT) 5-HT2A receptor (5-HT2AR) contribute importantly to psychedelic mechanisms of action, and the efficacy of these compounds in limiting cocaine intake is unknown. In the present studies, we tested the hypothesis that the psychedelic 5-HT2AR agonist R-(-)2,5-dimethoxy-4-iodoamphetamine (DOI) will decrease cocaine demand in a rodent behavioral economics model. Methods: Male, Sprague-Dawley rats (n = 28) were trained to self-administer cocaine (0.75 mg/kg) for daily 180-min sessions until stability on an FR3 schedule of reinforcement. Upon stability, daily sessions on the threshold procedure were initiated. During each 110-min session, progressively smaller doses of cocaine were available. The duration of infusion decreased by 0.25 log per block across 11 blocks lasting 10-min each, decreasing the dose of drug earned per infusion from 0.75 mg/kg/inf to 0.003 mg/kg/inf. Pharmacological testing with DOI treatment (0.03, 0.1, 0.3 mg/kg; s.c., 20 min prior to session) began upon establishment of stable demand elasticity (α) and demand intensity (Q0). Treatment with the selective 5-HT2AR antagonist M100907 (0.01 mg/kg; i.p., 30 min prior) was employed to assesses the role of the 5-HT2AR in the behavioral effects of DOI. Demand curves for each subject were plotted with number of reinforcers earned[KC1] (consumption, mg) as a function of FR requirement (price) and the data were fitted using an exponential model. The effect of DOI on measurements of cocaine demand was analyzed with an experimentwise error rate of α=0.05. Results: Analysis indicated that DOI treatment dose-dependently decreased cocaine demand elasticity, price point at maximum expenditure, maximum expenditure, essential value, breakpoint, and the area under the demand curve (p < 0.05) without altering measurements of task disruption. DOI did not significantly alter cocaine demand intensity. The selective 5-HT2AR antagonist M100907 prevented DOI-induced suppression of most measures in the cocaine demand paradigm (p < 0.05[KC2] ). Conclusions: The current studies reveal that the psychedelic DOI decreases the motivational effects of cocaine, which were reversed by the 5-HT2AR antagonist M100907. These data suggest that while cocaine may still be reinforcing[ANC3] at minimal prices, the rats are less willing to work for cocaine after DOI pretreatment, and provide support for expanded interrogation of the efficacy of 5-HT2AR agonist psychedelics in counteracting the behavioral effects of cocaine. This work was supported by Center for Addiction Research at the University of Texas Medical Branch.

ID 24537 Poster Board 547 Transmission via 5-HT2A receptors (5-HT2AR) in medial prefrontal cortex (mPFC) is implicated in the regulation of impulsive action which can be assessed in models which evaluate inhibitory control over prepotent motor responses. Systemic administration of a selective 5-HT2AR antagonist consistently attenuates impulsive action in choice serial reaction time (CSRT) tasks of motor impulsivity. Intra-mPFC 5-HT2AR blockade induces subtle enhancements in inhibitory control. Intriguingly, high-impulsive rats exhibit higher levels of 5-HT2AR expression and function. Here, we tested the hypothesis that stable knockdown of the 5-HT2AR locally in the mPFC regulates inhibitory control involving cues in the sucrose-reinforced 1-choice CSRT task. Methods: A short hairpin RNA (5-HT2AR-shRNA) that efficiently knocks down over 90% of 5-HT2AR mRNA in vitro was designed, validated, and packaged into an adeno-associated viral (AAV) vector; a non-silencing control (NSC) hairpin was designed and assessed to have no effect on 5-HT2AR mRNA expression. Male Sprague-Dawley rats were trained to stability in a sucrose-reinforced 1-CSRT task with a cued inter-trial interval (ITI) of 5-sec (ITI-5s) and challenged in sessions with ITI 8-sec (ITI-8s) prior to surgical delivery of viral vectors. Rats received bilateral stereotaxic infusions of 5-HT2AR-shRNA-eGFP AAV (n=8) or NSC-eGFP-AAV (n=8) into the mPFC (AP: +3.0 mm; ML: +1.4 mm; DV: -5.1, -4.1, -3.1 mm; relative to Bregma ). After recovery, rats were restabilized in the 1-CSRT task with ITI-5s with two post-surgery probes in ITI-8s. Results: Statistical analysis of performance on the ITI-5s and ITI-8s parameters did not yield significant effects of the knockdown of 5-HT2AR in mPFC. Further analysis centered on the effects of increased ITI challenges revealed that, normalized to the ITI-5s baseline, intra-mPFC 5-HT2AR knockdown significantly increased ITI-8s premature responding as compared to pre-surgery baselines. Thus, intra-mPFC 5-HT2AR knockdown revealed an increased impact of longer cued ITIs (ITI-8s) relative to baseline (ITI-5s) on premature responding for sucrose in 1CSRT task. Conclusions: These results suggest that 5-HT2AR transmission in the mPFC modulates impulsive action related to cues associated with a task and/or reward. Therefore, identification of 5-HT2AR distribution along neural circuits governing inhibitory control of motivated behavior could lead to a new mechanistic understanding of behavioral impulsivity. We are currently conducting tract-tracing studies to determine the anatomical distribution of 5-HT2AR-containing mPFC afferents to uncover the involvement of downstream connectivity that regulates impulsive behavior. Financial Support: T32DA07287, P50DA033935

The serotonin 5-HT2A receptor (5-HT2AR) and 5-HT2CR localize to the brain and share overlapping signal transduction facets that contribute to their roles in cognition, mood, learning, and memory. Achieving selective targeting of these receptors is challenged by the similarity in their 5-HT orthosteric binding pockets. A fragment-based discovery approach was employed to design and synthesize novel oleamide analogues as selective 5-HT2CR or dual 5-HT2CR/5-HT2AR positive allosteric modulators (PAMs). Compound 13 (JPC0323) exhibited on-target properties, acceptable plasma exposure and brain penetration, as well as negligible displacement to orthosteric sites of ∼50 GPCRs and transporters. Furthermore, compound 13 suppressed novelty-induced locomotor activity in a 5-HT2CR-dependent manner, suggesting 5-HT2CR PAM, but not 5-HT2AR, activity at the level of the whole organism at the employed doses of 13. We discovered new selective 5-HT2CR PAMs and first-in-class 5-HT2CR/5-HT2AR dual PAMs that broaden the pharmacological toolbox to explore the biology of these vital receptors.

ADVERTISEMENT RETURN TO BOOKPREVChapterNEXTTHE SEROTONIN 5-HT2A RECEPTOR AS AN EVOLVING NEUROTHERAPEUTIC TARGETAndrew A. BolingerAndrew A. BolingerCenter for Addiction Sciences and Therapeutics, Department of Pharmacology and Toxicology, University of Texas Medical Branch (UTMB), Galveston, Texas, U.S.A.More by Andrew A. Bolinger, Noelle C. AnastasioNoelle C. AnastasioCenter for Addiction Sciences and Therapeutics, Department of Pharmacology and Toxicology, University of Texas Medical Branch (UTMB), Galveston, Texas, U.S.A.More by Noelle C. Anastasio, Kathryn A. CunninghamKathryn A. CunninghamCenter for Addiction Sciences and Therapeutics, Department of Pharmacology and Toxicology, University of Texas Medical Branch (UTMB), Galveston, Texas, U.S.A.More by Kathryn A. Cunningham, and Jia ZhouJia ZhouCenter for Addiction Sciences and Therapeutics, Department of Pharmacology and Toxicology, University of Texas Medical Branch (UTMB), Galveston, Texas, U.S.A.More by Jia ZhouDOI: 10.1021/mc-2023-vol58.ch03Publication Date (Web):November 16, 2023Publication History Published online16 November 2023Request reuse permissions Copyright © 2023 MEDI, Inc. Published by American Chemical Society.2023 Medicinal Chemistry ReviewsChapter 3pp 53-81Medicinal Chemistry ReviewsVol. 58ISBN13: 9781734427462eISBN: 9781734427462Article Views98Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (1 MB) Get e-Alerts