Dr. Jeremy J. Day
University of Alabama at Birmingham
Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. In this presentation, I will discuss our recent efforts leveraging single-nucleus RNA-sequencing (snRNA-seq) and Assay for Transposase Accessible Chromatin (snATAC-seq) to generate a comprehensive molecular atlas of cell subtypes in the NAc, defining both sex-specific and cell type-specific responses to acute and repeated drug experience in a rat model system. Our work demonstrates that psychostimulant drugs such as cocaine recruit activity-dependent transcriptional programs in the NAc to subsequently initiate chromatin reorganization at enhancer elements near genes implicated in synaptic function. Moreover, we show that direct activation of a core dopamine-driven gene program with a multiplexed CRISPR strategy initiates a secondary synapse-centric transcriptional profile, alters striatal physiology in vitro, and enhances cocaine sensitization in vivo. Taken together, these results define the genome-wide transcriptional response to cocaine with cellular precision, and highlight the mechanisms by which drugs of abuse initiate experience-dependent chromatin remodeling.
Eric J. Nestler, M.D., Ph.D.
Nash Family Professor of Neuroscience Director
Friedman Brain Institute Dean for Academic and Scientific Affairs
Icahn School of Medicine at Mount Sinai
New York, NY USA
Nash Family Professor of Neuroscience Director
Friedman Brain Institute Dean for Academic and Scientific Affairs
Icahn School of Medicine at Mount Sinai
New York, NY USA
Drug addiction can be viewed as a stable form of drug-induced neural plasticity, whereby long-lasting changes in gene expression mediate some of the stable behavioral abnormalities that define an addicted state. Our laboratory has focused on transcriptional pathways in addiction, deduced from large RNA-sequencing datasets of RNAs that show altered expression in brain reward regions of mice as a consequence of drug self-administration, withdrawal, and relapse. Activation or induction of certain transcription factors represent homeostatic adaptations that oppose drug action and mediate aspects of drug tolerance and dependence. In contrast, induction of other transcription factors exerts the opposite effect and contributes to sensitized responses to drug exposure. Studies are underway to explore the detailed molecular mechanisms by which these various transcription factors regulate target genes and thereby contribute to the complex state of addiction. We are approaching this question by studying a range of chromatin mechanisms genome-wide, including post-translational modifications of histones, DNA methylation, nucleosome positioning, and the 3-dimensional structure of chromatin. These studies are identifying many of the molecular targets of drug self-administration in brain reward regions and the biochemical pathways most prominently affected. Among these targets are those that regulate synaptic function and plasticity as well as the morphology of drug-regulated neurons and other cell types, thus linking transcriptional and chromatin regulation to neural and behavioral plasticity. Parallel work has focused on homologous regions in the brains of addicted humans examined postmortem. This work establishes transcriptional and chromatin regulation as important mechanisms underlying the ways in which a history of drug use causes lasting changes in targeted brain reward neurons, and the circuits in which they operate, to result in addiction-related behavioral abnormalities. These advances can now be mined to develop improved diagnostic tests and treatments for addictive disorders.
Kerri D. Pryce1, Hope Kronnman1, Aarthi Ramakrishnan1, Claire Polizu, Anne Ruiz1, Sevasti Gaspari, Catherine Jensen-Pena2, Angelica Torres-Berrios1, Vassiliki Mitsi1, Matthew Jarpe3, Ralph Mazitshek3, John van Duzer3, Li Shen1, Eric Nestler1 and Venetia Zachariou1.
1Nash Family Department of Neuroscience and Friedman Brian Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029
2Princeton Neuroscience Institute, Princeton, New Jersey
3Regenancy Pharmaceutics. Boston MA, USA
1Nash Family Department of Neuroscience and Friedman Brian Institute, Icahn School of Medicine at Mount Sinai, New York, NY, 10029
2Princeton Neuroscience Institute, Princeton, New Jersey
3Regenancy Pharmaceutics. Boston MA, USA
The development of physical dependence and addiction disorders due to misuse of opioid analgesics is a major concern with current pain therapeutics. In this study, we developed a mouse model of oxycodone misuse, in order to gain insight on genes and pathways in reward-related brain regions that are affected by physical dependence to oxycodone in the presence and in the absence of chronic neuropathic pain. Our RNA-Sequencing (RNA-Seq) and bioinformatic analysis reveal that spontaneous oxycodone withdrawal triggers robust gene expression adaptations in the medial prefrontal cortex (mPFC), in the nucleus accumbens (NAc) and in the ventral tegmental area (VTA), while several genes and pathways are selectively affected under peripheral nerve injury states. Our pathway analysis revealed that Histone deacetylase 1 (HDAC1), an epigenetic modifier with a prominent role in striatal plasticity is a top upstream regulator with opioid withdrawal in both the NAc and the mPFC. Indeed, treatment with the novel HDAC1/2 inhibitor RCY1305 attenuates sensory and emotional manifestations of oxycodone withdrawal. Overall, our studies highlight novel intracellular pathways affected by oxycodone abstinence in reward circuitry, providing novel treatment possibilities for pain patients transitioning to non-opioid analgesics, as well as for non-pain subjects with oxycodone use disorders.
Dr. Jennifer Tuscher
University of Alabama at Birmingham
The ventral tegmental area (VTA) is a complex brain region that is essential for reward function but is also implicated in neuropsychiatric diseases including substance abuse. While decades of research on VTA function have focused on the role of dopaminergic neurons, recent evidence has identified critical roles for VTA GABAergic and glutamatergic neurons in reward processes as well. Interestingly, molecular characterization has revealed that subsets of these neurons express genes involved in the transport, synthesis, and vesicular packaging of multiple neurotransmitters, providing evidence for the presence of co-release neurons. However, these studies have largely relied on low-throughput methods, and the molecular architecture of the VTA has not been comprehensively examined. Here, we performed single nucleus RNA-sequencing (snRNA-seq) on 21,600 nuclei from male and female Sprague-Dawley rats to generate a transcriptional atlas of the rat VTA. We identified 16 transcriptionally distinct cell types, including 7 dissociable neuronal populations. Further subclustering revealed several VTA populations harboring markers for more than one neurotransmitter system, including a cluster exhibiting high expression levels of genes involved in the synthesis and transport of GABA, glutamate, and dopamine. Between-cluster statistical comparisons were leveraged to identify novel molecular markers for dopamine neurons, which may provide improved selectivity relative to canonical markers observed across multiple cell populations. Finally, we used SNP-level gene set analysis to examine cell-type specific enrichment of GWAS risk-associated genes within the VTA, highlighting the contribution of distinct cell populations to polygenic traits across several clinical disorders. Together, these results highlight the heterogeneity of cellular populations in the VTA and identify novel markers and disease-linked genes enriched in distinct neuronal subtypes.
Dr. Linda Dwoskin
University of Kentucky
Methamphetamine use disorder is a primary diagnosis, associated secondarily with other neuropsychiatric conditions. Diagnosis is restricted to long-term misuse leading to impaired social and/or occupational function. Deaths from methamphetamine overdose continue to escalate both with and without opioid involvement, yet no medication has been approved by the Food and Drug Administration for the treatment of methamphetamine use disorder. We have focused our iterative drug discovery program on a novel target, the vesicular monoamine transporter-2 (VMAT2), with the goal of discovering a pharmacotherapy to treat methamphetamine use disorder. A number of lead compounds have been identified during our quest, including lobeline, mesotransdiene, lobelane, and GZ-793A; however, each of these lead compounds was associated with an insurmountable hurdle (i.e., development of tolerance or hERG channel activity) precluding advancement to human clinical testing. More recently, GZ-11608 was identified as a promising lead, exhibiting high affinity and high selectivity for VMAT2 over off targets, i.e., hERG channels, dopamine transporters, serotonin transporters, and nicotinic acetylcholine receptors. GZ-11608 specifically decreases methamphetamine-sensitized locomotor activity, methamphetamine self-administration and reinstatement of drug seeking induced by either methamphetamine or methamphetamine-associated cue. GZ-11608 does not exacerbate methamphetamine-induced striatal dopamine depletion and tolerance does not develop to GZ-11608 efficacy. Further, GZ-11608 does not substitute for methamphetamine in self-administering rats and is not self-administered in drug naïve rats, suggesting low abuse liability. Unfortunately, GZ-11608 exhibits relatively high clearance, a short half-life and low (3%) oral bioavailability, limiting its utility as a therapeutic. Moving forward, we continue our search through iteration on the GZ-11608 structural scaffold and we have identified UKy-21, with potent affinity at VMAT2, good selectivity over hERG, and importantly good oral bioavailability (62%). UKy-21 is currently being evaluated in preclinical behavioral studies. Supported by NIH U01 DA043908 and U01HL152392.
A new therapeutic approach for treating methamphetamine intoxication
Xinhua Li, Mitchell Zakin, and John Marota
Clear Scientific, Inc., 737 Concord Ave, Cambridge, MA 02138
Methamphetamine is the fastest growing drug of abuse in the U.S., causing an estimated 280,000 hospitalizations and 20,000 fatal overdoses each year. Methamphetamine use disorder costs the healthcare system more than $3 billion annually. There are no FDA-approved therapies available for treating methamphetamine intoxication. Current standard of care is to treat symptoms only. Our therapeutic platform uses sequestrants with a proven mechanism of action to bind, inactivate, and rapidly eliminate harmful compounds from the body via the kidneys. Our lead program, funded by NIH/NIDA, is to develop a first-in-kind therapeutic, CS-1103, to treat methamphetamine overdose, an urgent Public Health crisis. In non-clinical studies, CS-1103 significantly accelerated clearance of methamphetamine and rapidly reversed acute toxic effects. We will present study results for CS-1103 and its potential impact to address the methamphetamine crisis.
Shoptaw, S; Clark JL; Goodman
Objective: There are no rationally derived novel therapeutics for methamphetamine use disorder. This presentation reviews targets and corresponding outcomes that can guide rational development of a novel methamphetamine medication for use in overdose. Methods: The presentation provides a brief review of trial designs, primary and secondary outcomes, and results for medication approaches with statistically significant findings for methamphetamine use disorder. Together with meta-analyses outcomes for behavioral therapies (contingency management, cognitive behavioral therapy), evidence also is provided to support using medications as treatment for methamphetamine use disorder. Results: There are no FDA-approved medications for methamphetamine use disorder. Two medication approaches, both using a repurposing strategy of currently approved medications (mirtazapine (30mg/d) and combination extended release naltrexone and high dose bupropion), show ~15-20% reduction of methamphetamine use over 12 weeks when evaluated in outpatient trials. No medications produce sustained methamphetamine abstinence. Medications are currently used for overdose to treat behavioral agitation and cardiovascular symptoms. No medications for reversal or for removing methamphetamine from the body exist for the setting of overdose. Conclusions: Scientific evidence supports use of two medication approaches for reducing methamphetamine use, with potential to increase impact by combining these with behavioral therapies. A gap exists with no medication currently that removes methamphetamine from the body, which would have great utility in treating overdose. Clear Scientific-1103 represents a first rationally derived small molecule that has capacity for removing methamphetamine from the body.
Dr. Sarah Withey
Behavioral Biology Program, McLean Hospital, Belmont, Massachusetts (F.B.d.M., S.L.W., J.B.) and Department of Psychiatry, Harvard Medical School, Boston, Massachusetts (F.B.d.M., S.L.W., J.B.).
Opioid addiction is a chronic relapsing disorder and yet little research has focused on understanding the ability of pharmacotherapies to protect against relapse to drug-seeking. This study was conducted to directly assess the effects fentanyl on relapse-related behavior and reward circuitry in non human primates. Preliminary experiments investigate the effects of naltrexone on fentanyl-induced relapse-related behavior and patters of neural activation. At baseline, fentanyl reinstated drug-seeking behavior and produced a functional inhibition of activity in multiple brain regions known to be involved in the reward-related effects of opioid agonists, including the cingulate cortex, nucleus accumbens, and thalamus. Results suggest that this type of combined approach using both behavioral and MRI techniques, can be utilized to investigate changes in neural circuitry following opioid administration and, perhaps, to investigate medication-related changes in opioid-induced behavior and neural activity.
Dr. Ikhlas Khan
University of Mississippi
Finding a cure in the nature is nothing new, it has been proven time and time again. The natural world offers to us a host of potent compounds, some of which we know very well, but many others remain to be recognized and eventually understood. The derivatives of plants and fungi that humans have explored for millennia in order to alter consciousness can generally be categorized by their pharmacological or psychotropiceffects. Some, such as opium, the source of the pain-killer morphine, or plants like kava kava and kratom induce a combination of effects or variable effects. Each plant has its own story, such as the hallucinogenic rainforest shrub Tabernanthe iboga that was first recognized for ritual use and is now promoted as a treatment for drug dependence. Examples of plants and fungi categorized by their perceived effects will be presented.
Mahmoud A. ElSohly, Ph.D, BCFM, BCFE
University of Mississippi
This presentation will give an overview of the different cannabis and cannabinoid activities relevant to the NIDA Drug Supply program and the products manufactured under the NIDA contract to provide Researchers across the USA and beyond. These activities include production of the cannabis biomass, cannabis extract and purified cannabinoids. A quick review of the botanical and chemical aspects of the plant will be presented as well as analytical procedures used to standardize the biomass and extracts and to monitor the potency of confiscated cannabis products. Furthermore, a summary of the development of a new THC-derivative and its formulation in suppositories, transmucosal delivery system and in ophthalmic preparation, will be presented.
Dr. Ziva Cooper
UCLA Cannabis Research Initiative, Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California
Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California
Department of Anesthesiology and Perioperative Medicine, David Geffen School of Medicine, University of California
Novel pharmacotherapeutic strategies are urgently needed to curb reliance on opioids for pain relief. This presentation will explore several lines of evidence from preclinical investigations to population-based studies suggesting that cannabinoids and other cannabis constituents may have potential analgesic properties and may serve as adjunct or substitutes to opioids for pain management. This talk will highlight current controlled human laboratory studies probing both the analgesic and adverse effects (specifically abuse liability) of cannabis constituents including delta-9-tetrahydrocannabinol, cannabidiol, and specific terpenes.
Dr. David E Olson
UC Davis
Neural plasticity—broadly defined as the ability of the brain to change and adapt—is of fundamental importance to a properly functioning nervous system. It is the basis for learning and memory and enables our brains to recover from the pathological changes that underlie neuropsychiatric diseases such as depression, post-traumatic stress disorder, and substance use disorder. Recently, our group discovered that psychedelic natural products and related compounds, such as LSD, DMT, and ibogaine, rapidly promote structural and functional neural plasticity in rodents. These compounds are capable of re-wiring neural circuitry to produce long-lasting antidepressant, anxiolytic, and anti-addictive behavioral responses. Psychedelics have inspired our total synthesis and medicinal chemistry efforts to develop safer and more effective neurotherapeutics, and they serve as key chemical tools in our studies to understand the fundamental biochemical mechanisms that give rise to neural plasticity.
Eleana Solakidou1,3, Monica H. Dawes2, Chandrashekhar Honrao1, Xiaoyu Ma1, Alexandros Makriyannis1, Elena H. Chartoff2, Rajeev I. Desai1,2
1Northeastern University, Center for Drug Discovery, Boston, MA
2Harvard Medical School-McLean Hospital, Belmont, MA
3University of Crete, Medical School, Heraklion, Greece
Oxycodone is considered to be the most commonly abused prescription opioid over the last decade, with women being disproportionally affected by opioid use disorder (OUD) compared to men. To further investigate sex-differences in oxycodone’s effects, we used in vivo microdialysis coupled with LC-MS/MS analysis to measure oxycodone and dopamine levels in key reward-related brain regions of female and male rats. This talk will provide insights on sex-differences in brain levels of oxycodone and its impact on dopamine responsiveness.
Dr. Kenzie L. Preston
NIDA
Opioid and cocaine use disorders are complex, have multiple etiologies, and require multifaceted treatments. The best prospect for developing effective treatments comes from translating research across multiple disciplines. Research using broad range of clinical methodologies and collaboration with preclinical scientists to investigate and improve addiction treatment will be presented. In addition to human laboratory studies and randomized clinical trials, data from intensive longitudinal monitoring with mobile devices are being used to answer clinically relevant question, better understand the factors underlying drug use and relapse and identify pathways to improving treatment.
Investigation of the Endocannabinoid-regulating Catabolic Enzyme Monoacylglycerol Lipase in Treating Chronic Pain in a Humanized Mouse Model of Sickle Cell Disease
Kennedy Goldsborough1, Hamid Akbarali1, Karan Muchhala1, Donovan Argueta2, Kalpna Gupta2, and Aron H. Lichtman1,3
1Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA
2Hematology/Oncology, Department of Medicine, University of California, Irvine and Southern California Institute for Research and Education, VA Medical Center, Long Beach, California
3Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA
1Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA
2Hematology/Oncology, Department of Medicine, University of California, Irvine and Southern California Institute for Research and Education, VA Medical Center, Long Beach, California
3Department of Medicinal Chemistry, Virginia Commonwealth University, Richmond, VA
Sickle Cell Disease (SCD) is caused by a recessive mutation resulting in abnormal hemoglobin in which red blood cells undergo sickling. This sickling leads to recurrent, acute vaso-occlusive crises and results in a complex pathobiology that includes inflammation, vascular dysfunction, ischemia/reperfusion injury, neuropathy, and pain, which diminish quality of life. While opioids are the primary analgesic prescribed for treating severe pain associated with SCD, serious side effects (e.g., respiratory depression, tolerance, abuse liability, and constipation) necessitate the development of effective non-opioid analgesics to treat this condition. Berkeley (HbSS-BERK) mice, which express >99% human sickle hemoglobin and display increased nociceptive behavior, represent a useful model to investigate new potential nonaddictive analgesics for SCD. Here we tested whether MJN110 a selective inhibitor of monoacylglycerol lipase (MAGL), the primary degradative enzyme of the endogenous cannabinoid 2-arachidonoylglycerol would produce antinociception in HbSS-BERK mice compared with mice expressing normal human hemoglobin (HbAA-BERK). HbSS-BERK mice displayed a profound hypersensitivity to mechanical stimulation (von Frey filaments) and heat (hot plate test), as well as functional deficits in grip strength, inverted screen, and nesting assays, and hyperexcitable sensory neurons harvest from dorsal root ganglia. MJN110 ameliorated this pronociceptive phenotype in HbSS-BERK mice. Because chronic pain in SCD patients requires prolonged treatment of analgesics, we also evaluated whether the antinociceptive effects of MJN110 would undergo tolerance following daily administration for week. Importantly, its antinociceptive effects were retained following repeated administration. Ongoing studies are examining the mechanisms underlying the antinociceptive effects of this MAGL inhibitor, which include CB1 and CB2 receptor activation as well as reductions in the primary 2-AG metabolite arachidonic acid and its proinflammatory metabolites. Ultimately, this work will ascertain whether clinic trials should be pursued to investigate whether MAGL inhibitors offers a viable alternative to opioids in treating chronic pain in SCD in patients.
Brenda Winn1, Chungsik Kim2, Meng Cui1, Roman Manetsch1,2, Diomedes Logothetis1
1Department of Pharmaceutical Sciences, Bouvé College of Health Sciences, Northeastern University, 360 Huntington Ave, Boston 02115
2Department of Chemistry and Chemical Biology, College of Sciences, Northeastern University, 360 Huntington Ave, Boston 02115
Class A G protein-coupled receptors (GPCRs) have been reported by multiple groups to form receptor heteromers and serve as allosteric regulators of one another. Yet their mere existence has become a highly contested topic. We have employed a minimally invasive approach of introducing unnatural amino acids (UAAs), using amber codon suppression, at strategic positions predicted to form the heteromeric interface of the dopamine 2 (D2) and serotonin 2A (2A) receptor (R) heteromer that has been implicated in schizophrenia. Photoaffinity labeling (PAL) is a technique to investigate binding interactions by forming a covalent bond between two entities via irradiation of a photoactivatable group. UAAs can function as photoaffinity probes. By irradiation of the strategically incorporated UAA, the singlet carbene that is produced is crosslinked with the corresponding receptor to form a covalently bonded dimer. We show that when an azido-phenylalanine UAA replaces D2R(Y199) in TM5, trans-signaling to 2AR is potentiated upon UV irradiation, as does its interacting residue 2AR(F244) that is also in the TM5. Either the D2R(Y199L) or the 2AR(F244L) mutant expressed with the corresponding azido-phenylalanine UAA incorporated in the partner subunit of the heteromer did not show trans-signaling potentiation upon UV irradiation. These results suggest that the TM5-TM5 interface stabilizes the heteromer in the active state. We are in the process of expanding this overall approach to test whether crosslinking the TM5-TM6 interface of the two receptors stabilizes an interface that prevents trans-signaling, stabilizing the heteromer in the inactive state.
Mohammed Mustafa, Joel Schlosburg, Jayden Elmer, and Aron H. Lichtman
Department of Pharmacology & Toxicology, Virginia Commonwealth University, Richmond, VA
The approval by the Food and Drug Administration of epidiolex (cannabidiol; CBD) for the treatment of severe seizure disorders has put this phytocannabinoid in the forefront of biomedical research. Other potential therapeutic applications of CBD supported by preclinical research, human studies, and anecdotal reports include pain, inflammation, and anxiety. CBD affects a diversity of pharmacological targets (e.g., 5-HT1a, GPR55, PPARỿ) as well as within the endocannabinoid system (FAAH inhibition, CB1 allosteric modulation). Although traditionally referred to as a non-psychoactive compound, vaporized CBD elicits “drug liking” and “pleasant” subjective effects in humans (Spindle et al. (2020) Drug Alcohol Depend, 211:107937, PMCID: PMC7414803). Here we used the drug discrimination paradigm to test: 1) whether mice could learn to discriminate CBD from vehicle; and 2) whether CBD behaves as a negative allosteric modulator of CB1 by eliciting a rightward shift of the generalization dose-response curve of the subjective effects of the high efficacy CB1 orthosteric agonist CP55,940. An initial experiment in food-restricted C57BL6/J male mice trained to nose-poke on a fixed-ratio of 10 (FR10) for food reinforcement found that CBD (30-100 mg/kg) did not produce rate suppression. Subsequently, a training dose of 100 mg/kg CBD was selected in which male C57BL/6J mice failed to reach training criteria (correct first food reinforcement (FFR), ≥ 80% treatment-associated responses) to discriminate from vehicle. Although these experiments showed that mice select the injection appropriate apparatus with an average of 80% or greater correct responding, the first food reinforcement (FFR) accuracy of each mouse was ~50%, suggesting that the drug’s subjective effects are not driving the operant responses. Therefore, on day 75 of training, we increased the dose of CBD to 200 mg/kg, with the aim to increase the FFR accuracy. Like the initial dose, CBD 200 mg/kg did not produce a reliable discriminative stimulus, determined by passing training criteria on 9 of 10 consecutive sessions. Current studies include testing whether CP55,940 produces a discriminative stimulus in these same subjects, and whether CBD (200 mg/kg) modulates the dose-response relationship of CP55,940 in a separate group of animals. In summary, mice did not learn to discriminate CBD from vehicle. Instead, they may have learned a win-stay or lose-shift strategy to receive food reinforcement that resulted in high performance during training sessions. However, altering training parameters (e.g., dose, route of administration, components of the operant task) may reveal a CBD discriminative stimulus. An outcome in which subjects learn to discriminate CBD from vehicle would provide a powerful in vivo approach to study both the pharmacology and underlying mechanisms of action of this drug.
This work supported by R01DA039942 and P30DA033934.