Development of novel tools for dissection of central versus peripheral dopamine D2-like receptor signaling in dysglycemia.

Dopamine (DA) D2-like receptors in both the central nervous system (CNS) and the periphery are key modulators of metabolism. Moreover, disruption of D2-like receptor signaling is implicated in dysglycemia. Yet, the respective metabolic contributions of CNS versus peripheral D2-like receptors including D2 (D2R) and D3 (D3R) receptors remain poorly understood. To address this, we developed new pharmacological tools, D2-like receptor agonists with diminished and delayed blood-brain barrier capability, to selectively manipulate D2R/D3R signaling in the periphery. We designated bromocriptine methiodide (BrMeI), a quaternary methiodide analogue of D2R/D3R agonist and diabetes drug bromocriptine, as our lead compound based on preservation of D2R/D3R binding and functional efficacy. We then used BrMeI and unmodified bromocriptine to dissect relative contributions of CNS versus peripheral D2R/D3R signaling in treating dysglycemia. Systemic administration of bromocriptine, with unrestricted access to CNS and peripheral targets, significantly improved both insulin sensitivity and glucose tolerance in obese, dysglycemic mice in vivo. In contrast, metabolic improvements were attenuated when access to bromocriptine was restricted either to the CNS through intracerebroventricular administration or delayed access to the CNS via BrMeI. Our findings demonstrate that the coordinated actions of both CNS and peripheral D2-like receptors are required for correcting dysglycemia. Ultimately, the development of a first-generation of drugs designed to selectively target the periphery provides a blueprint for dissecting mechanisms of central versus peripheral DA signaling and paves the way for novel strategies to treat dysglycemia.

Development of novel tools for dissection of central versus peripheral dopamine D2-like receptor signaling in dysglycemia.

Dopamine (DA) D2-like receptors in both the central nervous system (CNS) and the periphery are key modulators of metabolism. Moreover, disruption of D2-like receptor signaling is implicated in dysglycemia. Yet, the respective metabolic contributions of CNS versus peripheral D2-like receptors including D2 (D2R) and D3 (D3R) receptors remain poorly understood. To address this, we developed new pharmacological tools, D2-like receptor agonists with diminished and delayed blood-brain barrier capability, to selectively manipulate D2R/D3R signaling in the periphery. We designated bromocriptine methiodide (BrMeI), a quaternary methiodide analogue of D2/3R agonist and diabetes drug bromocriptine, as our lead compound based on preservation of D2R/D3R binding and functional efficacy. We then used BrMeI and unmodified bromocriptine to dissect relative contributions of CNS versus peripheral D2R/D3R signaling in treating dysglycemia. Systemic administration of bromocriptine, with unrestricted access to CNS and peripheral targets, significantly improved both insulin sensitivity and glucose tolerance in obese, dysglycemic mice in vivo. In contrast, metabolic improvements were attenuated when access to bromocriptine was restricted either to the CNS through intracerebroventricular administration or delayed access to the CNS via BrMeI. Our findings demonstrate that the coordinated actions of both CNS and peripheral D2-like receptors are required for correcting dysglycemia. Ultimately, the development of a first-generation of drugs designed to selectively target the periphery provides a blueprint for dissecting mechanisms of central versus peripheral DA signaling and paves the way for novel strategies to treat dysglycemia.

Pharmacologic Characterization of Substituted Nitazenes at µ, κ, and Δ Opioid Receptors Suggests High Potential for Toxicity.

The benzimidazole opioids (substituted nitazenes) are highly potent µ opiod receptor (MOR) agonists with heroin- or fentanyl-like effects. These compounds have caused hospitalizations and fatal overdoses. We characterized the in vitro pharmacology and structure-activity relationships of 19 nitazenes with substitutions at three positions of the benzimidazole core. Affinities were assessed using agonist radioligand binding assays at human µ, κ, and Δ opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in CHO cells. Notably, for MOR binding, nine substituted nitazenes had significantly higher affinities than fentanyl including N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, and N-desethyl isotonitazene; 13 had subnanomolar affinities. Only metodesnitazene and flunitazene had significantly lower affinities than fentanyl. Affinities for the substituted nitazenes at KOR and DOR relative to MOR were 46- to 2580-fold and 180- to 1280-fold lower, respectively. Functional activities were assessed using [35S]GTPγS binding assays. Four nitazenes had subnanomolar potencies at MOR: N-pyrrolidino etonitazene, N-pyrrilidino isonitazene, N-pyrrilidino protonitazene and N-desethyl isotonitazene. Ten substituted nitazenes had significantly higher potencies than fentanyl. All tested nitazenes were full MOR agonists. Potencies at KOR and DOR relative to MOR were 7.3- to 7920-fold and 24- to 9400-fold lower, respectively. Thus, many of these compounds are high affinity/high potency MOR agonists with elevated potential to elicit toxicity and overdose at low doses. SIGNIFICANCE STATEMENT: Substituted nitazenes are a growing public health threat. Although the 19 nitazenes tested vary in their opioid receptor pharmacology, a number are very high affinity, high potency, and high efficacy compounds- higher than fentanyl. Their pharmacology suggests high potential for harm.

MicroRNA137-loaded lipid nanoparticles regulate synaptic proteins in the prefrontal cortex.

Genome-wide association studies indicate that allele variants in MIR137, the host gene of microRNA137 (miR137), confer an increased risk of schizophrenia (SCZ). Aberrant expression of miR137 and its targets, many of which regulate synaptic functioning, are also associated with an increased risk of SCZ. Thus, miR137 represents an attractive target aimed at correcting the molecular basis for synaptic dysfunction in individuals with high genetic risk for SCZ. Advancements in nanotechnology utilize lipid nanoparticles (LNPs) to transport and deliver therapeutic RNA. However, there remains a gap in using LNPs to regulate gene and protein expression in the brain. To study the delivery of nucleic acids by LNPs to the brain, we found that LNPs released miR137 cargo and inhibited target transcripts of interest in neuroblastoma cells. Biodistribution of LNPs loaded with firefly luciferase mRNA remained localized to the mouse prefrontal cortex (PFC) injection site without circulating to off-target organs. LNPs encapsulating Cre mRNA preferentially co-expressed in neuronal over microglial or astrocytic cells. Using quantitative proteomics, we found miR137 modulated glutamatergic synaptic protein networks that are commonly dysregulated in SCZ. These studies support engineering the next generation of brain-specific LNPs to deliver RNA therapeutics and improve symptoms of central nervous system disorders.

Monoacylglycerol Lipase Protects the Presynaptic Cannabinoid 1 Receptor from Desensitization by Endocannabinoids after Persistent Inflammation.

Cannabinoid-targeted pain therapies are increasing with the expansion of cannabis legalization, however, their efficacy may be limited by pain-induced adaptations in the cannabinoid system. Cannabinoid receptor subtype 1 (CB1R) inhibition of spontaneous, GABAergic miniature IPSCs (mIPSCs) and evoked IPSCs (eIPSCs) in the ventrolateral periaqueductal gray (vlPAG) were compared in slices from naive and inflamed male and female Sprague Dawley rats. Complete Freund's Adjuvant (CFA) injections into the hindpaw induced persistent inflammation. In naive rats, exogenous cannabinoid agonists robustly reduce both eIPSCs and mIPSCs. After 5-7 d of inflammation, the effects of exogenous cannabinoids are significantly reduced because of CB1R desensitization via GRK2/3, as function is recovered in the presence of the GRK2/3 inhibitor, Compound 101 (Cmp101). Inhibition of GABA release by presynaptic µ-opioid receptors in the vlPAG does not desensitize with persistent inflammation. Unexpectedly, while CB1R desensitization significantly reduces the inhibition produced by exogenous agonists, depolarization-induced suppression of inhibition protocols that promote 2-arachidonoylglycerol (2-AG) synthesis exhibit prolonged CB1R activation after inflammation. 2-AG tone is detected in slices from CFA-treated rats when GRK2/3 is blocked, suggesting an increase in 2-AG synthesis after persistent inflammation. Inhibiting 2-AG degradation with the monoacylglycerol lipase (MAGL) inhibitor JZL184 during inflammation results in the desensitization of CB1Rs by endocannabinoids that is reversed with Cmp101. Collectively, these data indicate that persistent inflammation primes CB1Rs for desensitization, and MAGL degradation of 2-AG protects CB1Rs from desensitization in inflamed rats. These adaptations with inflammation have important implications for the development of cannabinoid-based pain therapeutics targeting MAGL and CB1Rs.SIGNIFICANCE STATEMENT Presynaptic G-protein-coupled receptors are resistant to desensitization. Here we find that persistent inflammation increases endocannabinoid levels, priming presynaptic cannabinoid 1 receptors for desensitization on subsequent addition of exogenous agonists. Despite the reduced efficacy of exogenous agonists, endocannabinoids have prolonged efficacy after persistent inflammation. Endocannabinoids readily induce cannabinoid 1 receptor desensitization if their degradation is blocked, indicating that endocannabinoid concentrations are maintained at subdesensitizing levels and that degradation is critical for maintaining endocannabinoid regulation of presynaptic GABA release in the ventrolateral periaqueductal gray during inflammatory states. These adaptations with inflammation have important implications for the development of cannabinoid-based pain therapies.

Enantiomeric Separation, Absolute Configuration by X-ray Crystallographic Analysis, and Functional Evaluation of Enantiomers of the Dual Ligand, SYA0340 at 5-HT1A and 5-HT7A Receptors.

We have previously identified 5-chloro-2-methyl-2-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-2,3-dihydro-1H-inden-1-one (SYA0340) as a dual 5-HT1A and 5-HT7 receptor ligand, and we posited such ligands might find utility in the treatment of various CNS related illnesses including cognitive and anxiolytic impairments. However, SYA0340 has a chiral center and its enantiomers may confound the readouts for their functional characteristics. Thus, in this study, we resynthesized SYA0340, separated the enantiomers, identified the absolute configurations, and evaluated their binding affinities and functional characteristics at both the 5-HT1A and 5-HT7A receptors. The results of this study show that the (+)-SYA0340-P1 [specific rotation [α] = +18.4 (deg.mL)/(g.dm)] has a binding affinity constant, Ki = 1.73 ± 0.55 nM at 5-HT1AR and Ki = 2.20 ± 0.33 nM at 5-HT7AR and (-)-SYA0340-P2 [specific rotation [α] = -18.2 (deg.mL)/(g.dm)] has Ki = 1.06 ± 0.32 nM (5-HT1AR) and 4.7 ± 1.1 nM (5-HT7AR). Using X-ray crystallographic techniques, the absolute configuration of the P2 isomer was identified as the S-enantiomer and, therefore, the P1 isomer as the R-enantiomer. Functionally, both SYA0340-P1 (EC50 = 1.12 ± 0.41 nM; Emax = 94.6 ± 3.1%) and SYA0340-P2 (EC50 = 2.21 ± 0.59 nM; Emax = 96.8 ± 5.1%) display similar agonist properties at the 5-HT1AR while both enantiomers display antagonist properties at the 5-HT7AR with P1 (IC50 = 32.1 ± 9.2 nM) displaying over 8 times greater potency as P2 (IC50 = 277 ± 46 nM). Thus, based on the functional evaluation results, SYA0340-P1 is considered as the eutomer of the pair of enantiomers of SYA0340. It is expected that these enantiomers will serve as new pharmacological probes for the 5-HT1A and 5-HT7A receptors.

Pharmacologic Activity of Substituted Tryptamines at 5-Hydroxytryptamine (5-HT)2A Receptor (5-HT2AR), 5-HT2CR, 5-HT1AR, and Serotonin Transporter.

Novel psychoactive substances, including synthetic substituted tryptamines, represent a potential public health threat. Additionally, some substituted tryptamines are being studied under medical guidance as potential treatments of psychiatric disorders. Characterizing the basic pharmacology of substituted tryptamines will aid in understanding differences in potential for harm or therapeutic use. Using human embryonic kidney cells stably expressing 5-hydroxytryptamine (5-HT)1A, 5-HT2A, and 5-HT2C receptors (5-HT1AR, 5-HT2AR, and 5HT2CR, respectively) or the serotonin transporter (SERT), we measured affinities, potencies and efficacies of 21 substituted tryptamines. With the exception of two 4-acetoxy compounds, substituted tryptamines exhibited affinities and potencies less than one micromolar at the 5-HT2AR, the primary target for psychedelic effects. In comparison, half or more exhibited low affinities/potencies at 5-HT2CR, 5-HT1AR, and SERT. Sorting by the ratio of 5-HT2A to 5-HT2C, 5-HT1A, or SERT affinity revealed chemical determinants of selectivity. We found that although 4-substituted compounds exhibited affinities that ranged across a factor of 100, they largely exhibited high selectivity for 5-HT2ARs versus 5-HT1ARs and 5-HT2CRs. 5-substituted compounds exhibited high affinities for 5-HT1ARs, low affinities for 5-HT2CRs, and a range of affinities for 5-HT2ARs, resulting in selectivity for 5-HT2ARs versus 5-HT2CRs but not versus 5-HT1ARs. Additionally, a number of psychedelics bound to SERT, with non-ring-substituted tryptamines most consistently exhibiting binding. Interestingly, substituted tryptamines and known psychedelic standards exhibited a broad range of efficacies, which were lower as a class at 5-HT2ARs compared with 5-HT2CRs and 5-HT1ARs. Conversely, coupling efficiency/amplification ratio was highest at 5-HT2ARs in comparison with 5-HT2CRs and 5-HT1ARs. SIGNIFICANCE STATEMENT: Synthetic substituted tryptamines represent both potential public health threats and potential treatments of psychiatric disorders. The substituted tryptamines tested differed in affinities, potencies, and efficacies at 5-hydroxytryptamine (5-HT)2A, 5-HT2C, and 5HT1A receptors and the serotonin transporter (SERT). Several compounds were highly selective for and coupled very efficiently downstream of 5-HT2A versus 5-HT1A and 5-HT2C receptors, and some bound SERT. This basic pharmacology of substituted tryptamines helps us understand the pharmacologic basis of their potential for harm and as therapeutic agents.

A highly D3R-selective and efficacious partial agonist (S)-ABS01-113 compared to its D3R-selective antagonist enantiomer (R)-ABS01-113 as potential treatments for opioid use disorder.

The non-medical use of opioids has become a national crisis in the USA. Developing non-opioid pharmacotherapies for controlling this opioid epidemic is urgent. Dopamine D3 receptor (D3R) antagonists and low efficacy partial agonists have shown promising profiles in animal models of opioid use disorders (OUD). However, to date, advancement to human studies has been limited. Here we report the effects of (S)- and (R)-enantiomers of (±)-ABS01-113, structural analogs of the D3R partial agonist, (±)-VK4-40, in which the 3-OH in the linking chain is replaced by 3-F group. (S)- and (R)-ABS01-113 are identical in chemical structure but with opposite chirality. In vitro receptor binding and functional assays indicate that (S)-ABS01-113 is an efficacious (55%) and potent (EC50 = 7.6 ± 3.9 nM) D3R partial agonist, while the (R)-enantiomer is a potent D3R antagonist (IC50 = 11.4 nM). Both (S)- and (R)-ABS01-113 bind with high affinity to D3R (Ki = 0.84 ± 0.16 and 0.37 ± 0.06 nM, respectively); however, the (S)-enantiomer is more D3/D2-selective (>1000-fold). Pharmacokinetic analyses indicate that both enantiomers display excellent oral bioavailability and high brain penetration. Systemic administration of (S)- or (R)-ABS01-113 alone failed to alter open-field locomotion in male rats and mice. Interestingly, pretreatment with (S)- or (R)-ABS01-113 attenuated heroin-enhanced hyperactivity, heroin self-administration, and (heroin + cue)-induced reinstatement of drug-seeking behavior. Together, these findings reveal that both enantiomers, particularly the highly selective and efficacious D3R partial agonist (S)-ABS01-113, demonstrate promising translational potential for the treatment of OUD.

Development of a Translational Model to Assess the Impact of Opioid Overdose and Naloxone Dosing on Respiratory Depression and Cardiac Arrest.

In response to a surge of deaths from synthetic opioid overdoses, there have been increased efforts to distribute naloxone products in community settings. Prior research has assessed the effectiveness of naloxone in the hospital setting; however, it is challenging to assess naloxone dosing regimens in the community/first-responder setting, including reversal of respiratory depression effects of fentanyl and its derivatives (fentanyls). Here, we describe the development and validation of a mechanistic model that combines opioid mu receptor binding kinetics, opioid agonist and antagonist pharmacokinetics, and human respiratory and circulatory physiology, to evaluate naloxone dosing to reverse respiratory depression. Validation supports our model, which can quantitatively predict displacement of opioids by naloxone from opioid mu receptors in vitro, hypoxia-induced cardiac arrest in vivo, and opioid-induced respiratory depression in humans from different fentanyls. After validation, overdose simulations were performed with fentanyl and carfentanil followed by administration of different intramuscular naloxone products. Carfentanil induced more cardiac arrest events and was more difficult to reverse than fentanyl. Opioid receptor binding data indicated that carfentanil has substantially slower dissociation kinetics from the opioid receptor compared with nine other fentanyls tested, which likely contributes to the difficulty in reversing carfentanil. Administration of the same dose of naloxone intramuscularly from two different naloxone products with different formulations resulted in differences in the number of virtual patients experiencing cardiac arrest. This work provides a robust framework to evaluate dosing regimens of opioid receptor antagonists to reverse opioid-induced respiratory depression, including those caused by newly emerging synthetic opioids.

Fentanyl causes naloxone-resistant vocal cord closure: A platform for testing opioid overdose treatments.

BACKGROUND: High doses of the synthetic opioid fentanyl cause rapid and sustained vocal cord closure (VCC) leading to airway obstruction that prevents overdose victims from breathing. This airway effect is not caused by morphine-derived opiates (e.g. heroin), is distinct from respiratory depression, resistant to naloxone, and can be lethal. However, VCC has not been previously included in animal models of opioid overdose. METHODS: Video laryngoscopy was used to monitor vocal cord movement in anesthetized Sprague-Dawley rats. Rats were administered saline, fentanyl (5, 25, or 50 µg/kg) or morphine (5 mg/kg) in an intravenous (IV) bolus delivered over a 10 s period. The mu opioid receptor (MOR) antagonist naloxone was administered as a pre-treatment (1 mg/kg, IV) 5 min prior to fentanyl (25 µg/kg) or a post-treatment (1 and 2 mg/kg) 1 min after fentanyl (25 µg/kg). RESULTS: Fentanyl (25 and 50 µg/kg) caused sustained and lethal VCC within 10 s. Morphine (5 mg/kg) and fentanyl (5 µg/kg) caused only brief laryngospasm with full recovery. Pre-treatment with naloxone (1 mg/kg) prevented fentanyl-induced VCC, but naloxone (1 and 2 mg/kg) was unable to reverse VCC when administered after fentanyl. CONCLUSIONS: These results indicate sustained VCC is a lethal physiological reaction, specific to fentanyl and resistant to naloxone treatment. While pre-treatment with naloxone prevented fentanyl-induced VCC, naloxone was unable to reverse the effect, suggesting a non-opioid receptor-mediated mechanism. These findings demonstrate the necessity of VCC inclusion in animal models of synthetic opioid overdose and the urgent need for more effective treatments for fentanyl-related overdoses.

New dual 5-HT1A and 5-HT7 receptor ligands derived from SYA16263.

We have previously reported that dual 5-HT1A and 5-HT7 receptor ligands might find utility as treatment options for various CNS related conditions including cognitive and anxiolytic impairments. We have also more recently reported that SYA16263 has antipsychotic-like properties with an absence of catalepsy in animal models ascribed to its ability to recruit ß-arrestin to the D2 receptor. However, SYA16263 also binds with very high affinity to 5-HT1AR (Ki = 1.1 nM) and a moderate affinity at 5-HT7R (Ki = 90 nM). Thus, it was of interest to exploit its pharmacophore elements in designing new dual receptor ligands. Using SYA16263 as the lead molecule, we have conducted a limited structure-affinity relationship (SAFIR) study by modifying various structural elements in the arylalkyl moiety, resulting in the identification of a new dual 5-HT1AR and 5-HT7R ligand, 6-chloro-2-methyl-2-(3-(4-(pyridin-2-yl)piperazin-1-yl)propyl)-2,3-dihydro-1H-inden-1-one (21), which unlike SYA16263, has a sub-nanomolar (5-HT1AR, Ki = 0.74 nM) and a low nanomolar (5-HT7R, Ki = 8.4 nM) affinity for these receptors. Interestingly, 21 is a full agonist at 5-HT1AR and antagonist at the 5-HT7R, functional characteristics which point to its potential as an antidepressant agent.

Affinity, potency, efficacy, selectivity, and molecular modeling of substituted fentanyls at opioid receptors.

Substituted fentanyls are abused and cause rapid fatal overdose. As their pharmacology is not well characterized, we examined in vitro pharmacology and structure-activity relationships of 22 substituted fentanyls with modifications of the fentanyl propyl group, and conducted in silico receptor/ligand modeling. Affinities for mu, kappa, and delta opioid receptors (MOR, KOR, and DOR, respectively) heterologously expressed in mammalian cells were assessed in agonist radioligand binding assays. At MOR, furanyl fentanyl had higher affinity than fentanyl, while acryl, isobutyryl and cyclopropyl fentanyls had similar affinities. Comparing affinities, thiophene and methoxyacetyl fentanyls had highest selectivity for MOR (2520- and 2730-fold compared to KOR and DOR, respectively). Functional activities were assessed using [35S]GTPγS binding assays. At MOR, furanyl fentanyl had higher potency and 11 substituted fentanyls had similar high potencies compared to fentanyl. Eight compounds were full agonists of MOR and twelve compounds were partial agonists, with efficacies from 8.8% (phenyl fentanyl) to 60.2% (butyryl fentanyl). All efficacious compounds had selective functional potency for MOR. The predicted binding poses of flexible fentanyl and rigid morphine against MOR show partially overlapping binding pockets, with fentanyl maintaining additional interaction with the transmembrane (TM) 2 helix. Subsequent molecular dynamics simulations revealed a predominant fentanyl binding pose involving various TM interactions. The piperidine nitrogen of substituted fentanyls establishes a salt-bridge with the conserved D-1473.32 residue and the propanamide carbonyl group establishes a hydrogen bond with the indole side-chain (-NH) of W-3187.35. The simulation suggests theN-linked phenethyl group may regulate the rotameric switch of W-2936.48. The predicted binding pose, in conjunction with in vitro binding affinity, clarified the molecular basis of the binding/selectivity profile of furanyl fentanyl and other derivatives at the sequence level. In summary, substituted fentanyls with high MOR potencies, selectivities, and efficacies are likely to have abuse and overdose potential. The work presented here is a prototype to investigate fentanyl derivatives and their abuse potential.

Fentanyl but not Morphine Interacts with Nonopioid Recombinant Human Neurotransmitter Receptors and Transporters.

Synthetic opioids, including fentanyl and its analogs, have therapeutic efficacy in analgesia and anesthesia. However, their illicit use in the United States has increased and contributed to the number one cause of death for adults 18-50 years old. Fentanyl and the heroin metabolite morphine induce respiratory depression that can be treated with the µ opioid receptor (MOR) antagonist naloxone. With higher or more rapid dosing, fentanyl, more than morphine, causes chest wall rigidity and can also induce rapid onset laryngospasm. Because non-MORs could mediate differing clinical manifestations, we examined the interactions of fentanyl and morphine at recombinant human neurotransmitter transporters, G protein-coupled receptors, and the N-methyl-D-aspartate glutamate receptor. Both drugs were agonists at MOR, κ, and δ opioid receptors. Morphine had little or no affinity at other human receptors and transporters (K i or IC50 value >100 µM). However, fentanyl had K i values of 1407 and 1100 nM at α 1A and α 1B adrenoceptor subtypes, respectively, and K i values of 1049 and 1670 nM at dopamine D4.4 and D1 receptor subtypes, respectively; it also blocked [3H]neurotransmitter uptake by the vesicular monoamine transporter 2 (IC50 = 911 nM). Pharmacokinetic models indicate that these Ki and IC50 values are pharmacologically relevant. Fentanyl had little affinity for other receptors or transporters. Thus, noradrenergic disposition at specific receptor subtypes in relevant organs may play a role in respiratory and cardiothoracic effects of fentanyl. Data suggest that less selective fentanyl receptor pharmacology could play a role in the different clinical effects of morphine compared with fentanyl, including fentanyl-induced deaths after illicit use. SIGNIFICANCE STATEMENT: The synthetic opioid fentanyl induces different clinical effects, including rapid onset muscular rigidity, vocal cord closure, and rapid death, than the heroin metabolite morphine. Our data indicate for the first time that the two drugs have very different effects at recombinant human neurotransmitter receptors and transporters that might explain those clinical differences.

Methamphetamine use alters human plasma extracellular vesicles and their microRNA cargo: An exploratory study.

Methamphetamine (MA) is the largest drug threat across the globe, with health effects including neurotoxicity and cardiovascular disease. Recent studies have begun to link microRNAs (miRNAs) to the processes related to MA use and addiction. Our studies are the first to analyse plasma EVs and their miRNA cargo in humans actively using MA (MA-ACT) and control participants (CTL). In this cohort we also assessed the effects of tobacco use on plasma EVs. We used vesicle flow cytometry to show that the MA-ACT group had an increased abundance of EV tetraspanin markers (CD9, CD63, CD81), but not pro-coagulant, platelet-, and red blood cell-derived EVs. We also found that of the 169 plasma EV miRNAs, eight were of interest in MA-ACT based on multiple statistical criteria. In smokers, we identified 15 miRNAs of interest, two that overlapped with the eight MA-ACT miRNAs. Three of the MA-ACT miRNAs significantly correlated with clinical features of MA use and target prediction with these miRNAs identified pathways implicated in MA use, including cardiovascular disease and neuroinflammation. Together our findings indicate that MA use regulates EVs and their miRNA cargo, and support that further studies are warranted to investigate their mechanistic role in addiction, recovery, and recidivism.

Trace amine-associated receptor gene polymorphism increases drug craving in individuals with methamphetamine dependence.

BACKGROUND: Methamphetamine (MA) is a potent agonist at the trace amine-associated receptor 1 (TAAR1). This study evaluated a common variant (CV) in the human TAAR1 gene, synonymous single nucleotide polymorphism (SNP) V288V, to determine the involvement of TAAR1 in MA dependence. METHODS: Participants (n = 106) with active MA dependence (MA-ACT), in remission from MA dependence (MA-REM), with active polysubstance dependence, in remission from polysubstance dependence, and with no history of substance dependence completed neuropsychiatric symptom questionnaires and provided blood samples. In vitro expression and function of CV and wild type TAAR1 receptors were also measured. RESULTS: The V288V polymorphism demonstrated a 40% increase in TAAR1 protein expression in cell culture, but message sequence and protein function were unchanged, suggesting an increase in translation efficiency. Principal components analysis resolved neuropsychiatric symptoms into four components, PC1 (depression, anxiety, memory, and fatigue), PC2 (pain), PC3 (drug and alcohol craving), and PC4 (sleep disturbances). Analyses of study group and TAAR1 genotype revealed a significant interaction for PC3 (craving response) (p = 0.003). The control group showed no difference in PC3 associated with TAAR1, while adjusted mean craving for the MA-ACT and MA-REM groups, among those with at least one copy of V288V, was estimated to be, respectively, 1.55 (p = 0.036) and 1.77 (p = 0.071) times the adjusted mean craving for those without the TAAR1 SNP. CONCLUSIONS: Neuroadaptation to chronic MA use may be altered by TAAR1 genotype and result in increased dopamine signaling and craving in individuals with the V288V genotype.

Noradrenergic Mechanisms in Fentanyl-Mediated Rapid Death Explain Failure of Naloxone in the Opioid Crisis.

In December 2018, the Centers for Disease Control declared fentanyl the deadliest drug in America. Opioid overdose is the single greatest cause of death in the United States adult population (ages 18-50), and fentanyl and its analogs [fentanyl/fentanyl analogs (F/FAs)] are currently involved in >50% of these deaths. Anesthesiologists in the United States were introduced to fentanyl in the early 1970s when it revolutionized surgical anesthesia by combining profound analgesia with hemodynamic stability. However, they quickly had to master its unique side effect. F/FAs can produce profound rigidity in the diaphragm, chest wall and upper airway within an extremely narrow dosing range. This clinical effect was called wooden chest syndrome (WCS) by anesthesiologists and is not commonly known outside of anesthesiology or to clinicians or researchers in addiction research/medicine. WCS is almost routinely fatal without expert airway management. This review provides relevant clinical human pharmacology and animal data demonstrating that the significant increase in the number of F/FA-induced deaths may involve α-adrenergic and cholinergic receptor-mediated mechanical failure of the respiratory and cardiovascular systems with rapid development of rigidity and airway closure. Although morphine and its prodrug, heroin, can cause mild rigidity in abdominal muscles at high doses, neither presents with the distinct and rapid respiratory failure seen with F/FA-induced WCS, separating F/FA overdose from the slower onset of respiratory depression caused by morphine-derived alkaloids. This distinction has significant consequences for the design and implementation of new pharmacologic strategies to effectively prevent F/FA-induced death. SIGNIFICANCE STATEMENT: Deaths from fentanyl and F/FAs are increasing in spite of availability and awareness of the opioid reversal drug naloxone. This article reviews literature suggesting that naloxone may be ineffective against centrally mediated noradrenergic and cholinergic effects of F/FAs, which clinically manifest as severe muscle rigidity and airway compromise (e.g., wooden chest syndrome) that is rapid and distinct from respiratory depression seen with morphine-derived alkaloids. A physiologic model is proposed and implications for new drug development and treatment are discussed.

Activation of Trace Amine-Associated Receptor 1 Stimulates an Antiapoptotic Signal Cascade via Extracellular Signal-Regulated Kinase 1/2.

Methamphetamine (MA) is highly addictive and neurotoxic, causing cell death in humans and in rodent models. MA, along with many of its analogs, is an agonist at the G protein-coupled trace amine-associated receptor 1 (TAAR1). TAAR1 activation protects against MA-induced degeneration of dopaminergic neurons, suggesting that TAAR1 plays a role in regulating MA-induced neurotoxicity. However, the mechanisms involved in TAAR1's role in neurotoxicity and cell death have not been described in detail. In this study, we investigated the apoptosis pathway in Taar1 wild-type (WT) and knockout (KO) mice and in cells expressing the recombinant receptor. Bcl-2, an antiapoptotic protein, was upregulated ∼3-fold in the midbrain area (substantial nigra and ventral tegmental area) in Taar1 KO compared with WT mice, and MA significantly increased Bcl-2 expression in WT mice but decreased Bcl-2 expression in KO mice. The proapoptotic protein Bax did not differ across genotype or in response to MA. Bcl-2 expression was significantly upregulated by the TAAR1 agonist RO5166017 ((S)-4-[(ethyl-phenyl-amino)-methyl]-4,5-dihydro-oxazol-2-ylamine) in cells expressing the recombinant mouse TAAR1. Additionally, activation of TAAR1 by RO5166017 increased phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, and protein kinase B (AKT), but only inhibition of ERK1/2 phosphorylation prevented TAAR1-induced increases in Bcl-2 levels, indicating that TAAR1 activation increases Bcl-2 through an ERK1/2-dependent pathway. All changes to ERK1/2 pathway intermediates were blocked by the TAAR1 antagonist, N-(3-ethoxyphenyl)-4-(1-pyrrolidinyl)-3-(trifluoromethyl) benzamide. These findings suggest that TAAR1 activation protects against MA-induced cell apoptosis and TAAR1 may play a role in cell death in neurodegenerative diseases. SIGNIFICANCE STATEMENT: Methamphetamine stimulates TAAR1, a G protein-coupled receptor. The role and mechanisms for TAAR1 in methamphetamine-induced neurotoxicity are not known. Here, we report that, in genetic mouse models and cells expressing the recombinant receptor, TAAR1 activates the ERK1/2 pathway but not the AKT pathway to upregulate the antiapoptotic protein Bcl-2, which protects cells from drug-induced toxicity.

The Role of Biogenic Amine Transporters in Trace Amine-Associated Receptor 1 Regulation of Methamphetamine-Induced Neurotoxicity.

Methamphetamine (MA) impairs vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) function and expression, increasing intracellular DA levels that lead to neurotoxicity. The trace amine-associated receptor 1 (TAAR1) is activated by MA, but when the receptor is not activated, MA-induced neurotoxicity is increased. To investigate interactions among TAAR1, VMAT2, and DAT, transporter function and expression were measured in transgenic Taar1 knockout (KO) and wild-type (WT) mice 24 hours following a binge-like regimen (four intraperitoneal injections, 2 hours apart) of MA (5 mg/kg) or the same schedule of saline treatment. Striatal synaptosomes were separated by fractionation to examine the function and expression of VMAT2 localized to cytosolic and membrane-associated vesicles. DAT was measured in whole synaptosomes. VMAT2-mediated [3H]DA uptake inhibition was increased in Taar1 KO mice in synaptosomal and vesicular fractions, but not the membrane-associated fraction, compared with Taar1 WT mice. There was no difference in [3H]dihydrotetrabenazine binding to the VMAT2 or [125I]RTI-55 binding to the DAT between genotypes, indicating activation of TAAR1 does not affect VMAT2 or DAT expression. There was also no difference between Taar1 WT and KO mice in DAT-mediated [3H]DA uptake inhibition following in vitro treatment with MA. These findings provide the first evidence of a TAAR1-VMAT2 interaction, as activation of TAAR1 mitigated MA-induced impairment of VMAT2 function, independently of change in VMAT2 expression. Additionally, the interaction is localized to intracellular VMAT2 on cytosolic vesicles and did not affect expression or function of DAT in synaptosomes or VMAT2 at the plasmalemmal surface, i.e., on membrane-associated vesicles. SIGNIFICANCE STATEMENT: Methamphetamine stimulates the G protein-coupled receptor TAAR1 to affect dopaminergic function and neurotoxicity. Here we demonstrate that a functional TAAR1 protects a specific subcellular fraction of VMAT2, but not the dopamine transporter, from methamphetamine-induced effects, suggesting new directions in pharmacotherapeutic development for neurodegenerative disorders.

Taar1 gene variants have a causal role in methamphetamine intake and response and interact with Oprm1.

We identified a locus on mouse chromosome 10 that accounts for 60% of the genetic variance in methamphetamine intake in mice selectively bred for high versus low methamphetamine consumption. We nominated the trace amine-associated receptor 1 gene, Taar1, as the strongest candidate and identified regulation of the mu-opioid receptor 1 gene, Oprm1, as another contributor. This study exploited CRISPR-Cas9 to test the causal role of Taar1 in methamphetamine intake and a genetically-associated thermal response to methamphetamine. The methamphetamine-related traits were rescued, converting them to levels found in methamphetamine-avoiding animals. We used a family of recombinant inbred mouse strains for interval mapping and to examine independent and epistatic effects of Taar1 and Oprm1. Both methamphetamine intake and the thermal response mapped to Taar1 and the independent effect of Taar1 was dependent on genotype at Oprm1. Our findings encourage investigation of the contribution of Taar1 and Oprm1 variants to human methamphetamine addiction.

Structure-activity relationships of bath salt components: substituted cathinones and benzofurans at biogenic amine transporters.

RATIONALE: New psychoactive substances (NPSs), including substituted cathinones and other stimulants, are synthesized, sold on the Internet, and ingested without knowledge of their pharmacological activity and/or toxicity. In vitro pharmacology plays a role in therapeutic drug development, drug-protein in silico interaction modeling, and drug scheduling. OBJECTIVES: The goal of this research was to determine mechanisms of action that may indicate NPS abuse liability. METHODS: Affinities to displace the radioligand [125I]RTI-55 and potencies to inhibit [3H]neurotransmitter uptake for 22 cathinones, 6 benzofurans and another stimulant were characterized using human embryonic kidney cells stably expressing recombinant human transporters for dopamine, norepinephrine, or serotonin (hDAT, hNET, or hSERT, respectively). Selected compounds were tested for potencies and efficacies at inducing [3H]neurotransmitter release via the transporters. Computational modeling was conducted to explain plausible molecular interactions established by NPS and transporters. RESULTS: Most α-pyrrolidinophenones had high hDAT potencies and selectivities in uptake assays, with hDAT/hSERT uptake selectivity ratios of 83-360. Other substituted cathinones varied in their potencies and selectivities, with N-ethyl-hexedrone and N-ethyl-pentylone having highest hDAT potencies and N-propyl-pentedrone having highest hDAT selectivity. 4-Cl-ethcathinone and 3,4-methylenedioxy-N-propylcathinone had higher hSERT selectivity. Benzofurans generally had low hDAT selectivity, especially 1-(2,3-dihydrobenzofuran-5-yl)-N-methylpropan-2-amine, with 25-fold higher hSERT potency. Consistent with this selectivity, the benzofurans were releasers at hSERT. Modeling indicated key amino acids in the transporters' binding pockets that influence drug affinities. CONCLUSIONS: The α-pyrrolidinophenones, with high hDAT selectivity, have high abuse potential. Lower hDAT selectivity among benzofurans suggests similarity to methylenedioxymethamphetamine, entactogens with lower stimulant activity.