Elucidating the harm potential of brorphine analogues as new synthetic opioids: Synthesis, in vitro, and in vivo characterization.

The emergence of new synthetic opioids (NSOs) has added complexity to recreational opioid markets worldwide. While NSOs with diverse chemical structures have emerged, brorphine currently remains the only NSO with a piperidine benzimidazolone scaffold. However, the emergence of new generations of NSOs, including brorphine analogues, can be anticipated. This study explored the pharmaco-toxicological, opioid-like effect profile of brorphine alongside its non-brominated analogue (orphine) and three other halogenated analogues (fluorphine, chlorphine, iodorphine). In vitro, radioligand binding assays in rat brain tissue indicated that all analogues bind to the µ-opioid receptor (MOR) with nM affinity. While analogues with smaller-sized substituents showed the highest MOR affinity, further in vitro characterization via two cell-based (HEK 293T) MOR activation (ß-arrestin 2 and mini-Gαi recruitment) assays indicated that chlorphine, brorphine, and iodorphine were generally the most active MOR agonists. None of the compounds showed significant in vitro biased agonism compared to hydromorphone. In vivo, we investigated the effects of intraperitoneal (IP) administration of the benzimidazolones (0.01-15 mg/kg) on mechanical and thermal antinociception in male CD-1 mice. Chlorphine and brorphine overall induced the highest levels of antinociception. Furthermore, the effects on respiratory changes induced by a fixed dose (15 mg/kg IP) of the compounds were investigated using non-invasive plethysmography. Fluorphine-, chlorphine-, and brorphine-induced respiratory depressant effects were the most pronounced. For some compounds, pretreatment with naloxone (6 mg/kg IP) could not reverse respiratory depression. Taken together, brorphine-like piperidine benzimidazolones are opioid agonists that have the potential to cause substantial harm to users should they emerge as NSOs. This article is part of the Special Issue on "Novel Synthetic Opioids (NSOs)".

Reinforcing effects of fentanyl analogs found in illicit drug markets.

RATIONALE: The potent synthetic opioid fentanyl, and its analogs, continue to drive opioid-related overdoses. Although the pharmacology of fentanyl is well characterized, there is little information about the reinforcing effects of clandestine fentanyl analogs (FAs). OBJECTIVES: Here, we compared the effects of fentanyl and the FAs acetylfentanyl, butyrylfentanyl, and cyclopropylfentanyl on drug self-administration in male and female rats. These FAs feature chemical modifications at the carbonyl moiety of the fentanyl scaffold. METHODS: Sprague-Dawley rats fitted with intravenous jugular catheters were placed in chambers containing two nose poke holes. Active nose poke responses resulted in drug delivery (0.2 mL) over 2 s on a fixed-ratio 1 schedule, followed by a 20 s timeout. Acquisition doses were 0.01 mg/kg/inj for fentanyl and cyclopropylfentanyl, and 0.03 mg/kg/inj for acetylfentanyl and butyrylfentanyl. After 10 days of acquisition, dose-effect testing was carried out, followed by 10 days of saline extinction. RESULTS: Self-administration of fentanyl and FAs was acquired by both male and female rats, with no sex differences in acquisition rate. Fentanyl and FAs showed partial inverted-U dose-effect functions; cyclopropylfentanyl and fentanyl had similar potency, while acetylfentanyl and butyrylfentanyl were less potent. Maximal response rates were similar across drugs, with fentanyl and cyclopropylfentanyl showing maximum responding at 0.001 mg/kg/inj, acetylfentanyl at 0.01 mg/kg/inj, and butyrylfentanyl at 0.003 mg/kg/inj. No sex differences were detected for drug potency, efficacy, or rates of extinction. CONCLUSIONS: Our work provides new evidence that FAs display significant abuse liability in male and female rats, which suggests the potential for compulsive use in humans.

Evidence for enduring cardiac and multiorgan toxicity after repeated exposure to the synthetic cannabinoid JWH-018 in male rats.

The use of synthetic cannabinoid receptor agonists (SCRAs) represents a public health concern. Besides abuse liability and cognitive impairments, SCRAs consumption is associated with serious medical consequences in humans, including cardiotoxicity. The precise mechanisms underlying cardiac or other toxicities induced by SCRAs are not well understood. Here, we used in silico, in vivo, and ex vivo approaches to investigate the toxicological consequences induced by exposure to the SCRA JWH-018. Along with in silico predictive toxicological screening of 36 SCRAs by MC4PC software, adult male Sprague-Dawley rats were repeatedly exposed to JWH-018 (0.25 mg/kg ip) for 14 consecutive days, with body temperature and cardiovascular parameters measured over the course of treatment. At 1 and 7 days after JWH-018 discontinuation, multiorgan tissue pathologies and heart mitochondria bioenergetics were assessed. The in silico findings predicted risk of cardiac adverse effects specifically for JWH-018 and other aminoalkylindole SCRAs (i.e., electrocardiogram abnormality and QT prolongation). The results from rats revealed that repeated, but not single, JWH-018 exposure induced hypothermia and cardiovascular stimulation (e.g., increased blood pressure and heart rate) which persisted throughout treatment. Post-mortem findings demonstrated cardiac lesions (i.e., vacuolization, waving, edema) 1 day after JWH-018 discontinuation, which may contribute to lung, kidney, and liver tissue degeneration observed 7 days later. Importantly, repeated JWH-018 exposure induced mitochondrial dysfunction in cardiomyocytes, i.e., defective lipid OXPHOS, which may represent one mechanism of JWH-018-induced toxicity. Our results demonstrate that repeated administration of even a relatively low dose of JWH-018 is sufficient to affect cardiovascular function and induce enduring toxicological consequences, pointing to risks associated with SCRA consumption.

Comparative Pharmacological Effects of Lisuride and Lysergic Acid Diethylamide Revisited.

Lisuride is a non-psychedelic serotonin (5-HT) 2A receptor (5-HT2A) agonist and analogue of the psychedelic lysergic acid diethylamide (LSD). Lisuride also acts as an agonist at the serotonin 1A receptor (5-HT1A), a property known to counter psychedelic effects. Here, we tested whether lisuride lacks psychedelic activity due to a dual mechanism: (1) partial agonism at 5-HT2A and (2) potent agonism at 5-HT1A. The in vitro effects of lisuride, LSD, and related analogues on 5-HT2A signaling were characterized by using miniGαq and ß-arrestin 2 recruitment assays. The 5-HT1A- and 5-HT2A-mediated effects of lisuride and LSD were also compared in male C57BL/6J mice. The in vitro results confirmed that LSD is an agonist at 5-HT2A, with high efficacy and potency for recruiting miniGαq and ß-arrestin 2. By contrast, lisuride displayed partial efficacy for both functional end points (6-52% of 5-HT or LSD Emax) and antagonized the effects of LSD. The mouse experiments demonstrated that LSD induces head twitch responses (HTRs)(ED50 = 0.039 mg/kg), while lisuride suppresses HTRs (ED50 = 0.006 mg/kg). Lisuride also produced potent hypothermia and hypolocomotion (ED50 = 0.008-0.023 mg/kg) that was blocked by the 5-HT1A antagonist WAY100635 (3 mg/kg). Blockade of 5-HT1A prior to lisuride restored basal HTRs, but it failed to increase HTRs above baseline levels. HTRs induced by LSD were blocked by lisuride (0.03 mg/kg) or the 5-HT1A agonist 8-OH-DPAT (1 mg/kg). Overall, our findings show that lisuride is an ultrapotent 5-HT1A agonist in C57BL/6J mice, limiting its use as a 5-HT2A ligand in mouse studies examining acute drug effects. Results also indicate that the 5-HT2A partial agonist-antagonist activity of lisuride explains its lack of psychedelic effects.

In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the µ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as µ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and ß-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and ß-arrestin2 recruitment pathways. Compared to the reference agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting ß-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and ß-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. In addition, the extremely high potency of many NSOs now infiltrating illicit drug markets further contributes to the danger posed to public health.

Novel Benzofuran Derivatives Induce Monoamine Release and Substitute for the Discriminative Stimulus Effects of 3,4-Methylenedioxymethamphetamine.

3,4-Methylenedioxymethamphetamine (MDMA) has shown efficacy as a medication adjunct for treating post-traumatic stress disorder (PTSD). However, MDMA is also used in nonmedical contexts that pose risk for cardiovascular and neurologic complications. It is well established that MDMA exerts its effects by stimulating transporter-mediated release of the monoamines 5-hydroxytryptamine (5-HT), norepinephrine, and dopamine. Current research efforts are aimed at developing MDMA-like monoamine releasers with better efficacy and safety profiles. To this end, we investigated neurochemical and behavioral effects of novel analogs of the designer drug 5-(2-methylaminopropyl)benzofuran (5-MAPB). We used in vitro transporter assays in rat brain synaptosomes to examine transmitter uptake inhibition and releasing properties for enantiomers of 5-(2-methylaminobutyl)benzofuran (5-MABB) and 6-(2-methylaminobutyl)benzofuran (6-MABB) compared with MDMA. We then tested these same compounds in male Sprague-Dawley rats trained to discriminate MDMA (1.5 mg/kg) from saline. In vitro results revealed that S isomers of 5- and 6-MABB are efficacious releasing agents at transporters for 5-HT (SERT), norepinephrine (NET), and dopamine (DAT). By contrast, R isomers are efficacious releasers at SERT and partial releasers at NET but lack releasing activity at DAT. In vivo results showed that all compounds produce dose-dependent increases in MDMA-lever responding and full substitution at the highest dose tested. The diminished NET and DAT releasing activities for R isomers of 5- and 6-MABB are associated with reduced potency for inducing behavioral effects. Collectively, these findings indicate that the aminoalkyl benzofuran scaffold may be a viable template for developing compounds with MDMA-like properties. SIGNIFICANCE STATEMENT: Despite the clinical utility of 3,4-methylenedioxymethamphetamine (MDMA), the drug is associated with certain cardiovascular risks and metabolic side effects. Developing a therapeutic alternative with MDMA-like monoamine releasing activity is of interest. Our in vitro and in vivo findings indicate that the aminoalkyl benzofuran scaffold may be useful for developing compounds with MDMA-like properties.

Psychedelic-like Activity of Norpsilocin Analogues.

Primary metabolites of mushroom tryptamines, psilocybin and baeocystin (i.e., psilocin and norpsilocin), exhibit potent agonist activity at the serotonin 2A receptor (5-HT2A) in vitro but differ in their 5-HT2A-mediated effects in vivo. In particular, psilocin produces centrally mediated psychedelic effects in vivo, whereas norpsilocin, differing only by the loss of an N-methyl group, is devoid of psychedelic-like effects. These observations suggest that the secondary methylamine group in norpsilocin impacts its central nervous system (CNS) bioavailability but not its receptor pharmacodynamics. To test this hypothesis, eight norpsilocin derivatives were synthesized with varied secondary alkyl-, allyl-, and benzylamine groups, primarily aiming to increase their lipophilicity and brain permeability. Structure-activity relationships for the norpsilocin analogues were evaluated using the mouse head-twitch response (HTR) as a proxy for CNS-mediated psychedelic-like effects. HTR studies revealed that extending the N-methyl group of norpsilocin by a single methyl group, to give the corresponding secondary N-ethyl analogue (4-HO-NET), was sufficient to produce psilocin-like activity (median effective dose or ED50 = 1.4 mg/kg). Notably, N-allyl, N-propyl, N-isopropyl, and N-benzyl derivatives also induced psilocin-like HTR activity (ED50 = 1.1-3.2 mg/kg), with variable maximum effects (26-77 total HTR events). By contrast, adding bulkier tert-butyl or cyclohexyl groups in the same position did not elicit psilocin-like HTRs. Pharmacological assessments of the tryptamine series in vitro demonstrated interactions with multiple serotonin receptor subtypes, including 5-HT2A, and other CNS signaling proteins (e.g., sigma receptors). Overall, our data highlight key structural requirements for CNS-mediated psychedelic-like effects of norpsilocin analogues.

Structure-activity relationships for locomotor stimulant effects and monoamine transporter interactions of substituted amphetamines and cathinones.

Substitutions to the phenethylamine structure give rise to numerous amphetamines and cathinones, contributing to an ever-growing number of abused novel psychoactive substances. Understanding how various substitutions affect the pharmacology of phenethylamines may help lawmakers and scientists predict the effects of newly emerging drugs. Here, we established structure-activity relationships for locomotor stimulant and monoamine transporter effects of 12 phenethylamines with combinations of para-chloro, ß-keto, N-methyl, or N-ethyl additions. Automated photobeam analysis was used to evaluate effects of drugs on ambulatory activity in mice, whereas in vitro assays were used to determine activities at transporters for dopamine (DAT), norepinephrine (NET), and 5-HT (SERT) in rat brain synaptosomes. In mouse studies, all compounds stimulated locomotion, except for 4-chloro-N-ethylcathinone. Amphetamines were more potent stimulants than their ß-keto counterparts, while para-chloro amphetamines tended to be more efficacious than unsubstituted amphetamines. Para-chloro compounds also produced lethality at doses on the ascending limbs of their locomotor dose-effect functions. The in vitro assays showed that all compounds inhibited uptake at DAT, NET, and SERT, with most compounds also acting as substrates (i.e., releasers) at these sites. Unsubstituted compounds displayed better potency at DAT and NET relative to SERT. Para-chloro substitution or increased N-alkyl chain length augmented relative potency at SERT, while combined para-chloro and N-ethyl substitutions reduced releasing effects at NET and DAT. These results demonstrate orderly SAR for locomotor stimulant effects, monoamine transporter activities, and lethality induced by phenethylamines. Importantly, 4-chloro compounds produce toxicity in mice that suggests serious risk to humans using these drugs in recreational contexts.

Unique pharmacodynamic properties and low abuse liability of the µ-opioid receptor ligand (S)-methadone.

(R,S)-methadone ((R,S)-MTD) is a µ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers. (S)-MTD is being developed as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. We compared the pharmacology of (R)-MTD and (S)-MTD and found they bind to MORs, but not NMDARs, and induce full analgesia. Unlike (R)-MTD, (S)-MTD was a weak reinforcer that failed to affect extracellular dopamine or induce locomotor stimulation. Furthermore, (S)-MTD antagonized motor and dopamine releasing effects of (R)-MTD. (S)-MTD acted as a partial agonist at MOR, with complete loss of efficacy at the MOR-galanin Gal1 receptor (Gal1R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use. One-sentence summary: (S)-MTD, like (R)-MTD, binds to and activates MORs in vitro, but (S)-MTD antagonizes the MOR-Gal1R heteromer, decreasing its abuse liability.

The in vitro functional profiles of fentanyl and nitazene analogs at the µ-opioid receptor - high efficacy is dangerous regardless of signaling bias.

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as the µ-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G protein biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at MOR using adenylate cyclase inhibition and ß-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and ß-arrestin2 recruitment pathways. Compared to the reference agonist DAMGO, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting ß-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and ß-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. Instead, our results show that, regardless of bias, opioids with sufficiently high intrinsic efficacy can be lethal, especially given the extremely high potency of many of these compounds that are now pervading the illicit drug market.

Subtle Structural Modification of a Synthetic Cannabinoid Receptor Agonist Drastically Increases its Efficacy at the CB1 Receptor.

The emergence of synthetic cannabinoid receptor agonists (SCRAs) as illicit psychoactive substances has posed considerable public health risks, including fatalities. Many SCRAs exhibit much higher efficacy and potency compared with the phytocannabinoid Δ9-tetrahydrocannabinol (THC) at the cannabinoid receptor 1 (CB1R), leading to dramatic differences in signaling levels that can be toxic. In this study, we investigated the structure-activity relationships of aminoalkylindole SCRAs at CB1Rs, focusing on 5F-pentylindoles containing an amide linker attached to different head moieties. Using in vitro bioluminescence resonance energy transfer assays, we identified a few SCRAs exhibiting significantly higher efficacy in engaging the Gi protein and recruiting ß-arrestin than the reference CB1R full agonist CP55940. Importantly, the extra methyl group on the head moiety of 5F-MDMB-PICA, as compared to that of 5F-MMB-PICA, led to a large increase in efficacy and potency at the CB1R. This pharmacological observation was supported by the functional effects of these SCRAs on glutamate field potentials recorded in hippocampal slices. Molecular modeling and simulations of the CB1R models bound with both of the SCRAs revealed critical structural determinants contributing to the higher efficacy of 5F-MDMB-PICA and how these subtle differences propagated to the receptor-G protein interface. Thus, we find that apparently minor structural changes in the head moiety of SCRAs can cause major changes in efficacy. Our results highlight the need for close monitoring of the structural modifications of newly emerging SCRAs and their potential for toxic drug responses in humans.

Alkoxy chain length governs the potency of 2-benzylbenzimidazole 'nitazene' opioids associated with human overdose.

RATIONALE: Novel synthetic opioids (NSOs) are emerging in recreational drug markets worldwide. In particular, 2-benzylbenzimidazole 'nitazene' compounds are problematic NSOs associated with serious clinical consequences, including fatal respiratory depression. Evidence from in vitro studies shows that alkoxy chain length can influence the potency of nitazenes at the mu-opioid receptor (MOR). However, structure-activity relationships (SARs) of nitazenes for inducing opioid-like effects in animal models are not well understood compared to relevant opioids contributing to the ongoing opioid crisis (e.g., fentanyl). OBJECTIVES: Here, we examined the in vitro and in vivo effects of nitazene analogues with varying alkoxy chain lengths (i.e., metonitazene, etonitazene, isotonitazene, protonitazene, and butonitazene) as compared to reference opioids (i.e., morphine and fentanyl). METHODS AND RESULTS: Nitazene analogues displayed nanomolar affinities for MOR in rat brain membranes and picomolar potencies to activate MOR in transfected cells. All compounds induced opioid-like effects on locomotor activity, hot plate latency, and body temperature in male mice, and alkoxy chain length markedly influenced potency. Etonitazene, with an ethoxy chain, was the most potent analogue in MOR functional assays (EC50 = 30 pM, Emax = 103%) and across all in vivo endpoints (ED50 = 3-12 µg/kg). In vivo SARs revealed that ethoxy, isopropoxy, and propoxy chains engendered higher potencies than fentanyl, whereas methoxy and butoxy analogues were less potent. MOR functional potencies, but not MOR affinities, were positively correlated with in vivo potencies to induce opioid effects. CONCLUSIONS: Overall, our data show that certain nitazene NSOs are more potent than fentanyl as MOR agonists in mice, highlighting concerns regarding the high potential for overdose in humans who are exposed to these compounds.

In silico characterization of ligand-receptor interactions for U-47700, N,N-didesmethyl-U-47700, U-50488 at mu- and kappa-opioid receptors.

The increasing misuse of novel synthetic opioids (NSOs) represents a serious public health concern. In this regard, U-47700 (trans-3,4-dichloro-N-[2-(dimethylamino)cyclohexyl]-N-methylbenzamide) and related "U-compounds" emerged on recreational drug markets as synthetic substitutes for illicit heroin and constituents of counterfeit pain medications. While the pharmacology of U-compounds has been investigated using in vitro and in vivo methods, there is still a lack of understanding about the details of ligand-receptor interactions at the molecular level. To this end, we have developed a molecular modeling protocol based on docking and molecular dynamics simulations to assess the nature of ligand-receptor interactions for U-47700, N,N-didesmethyl U-47700, and U-50488 at the mu-opioid receptor (MOR) and kappa-opioid receptor (KOR). The evaluation of ligand-receptor and ligand-receptor-membrane interaction energies enabled the identification of subtle conformational shifts in the receptors induced by ligand binding. Interestingly, the removal of two key methyl groups from U-47700, to form N,N-didesmethyl U-47700, caused a loss of hydrogen bond contact with tryptophan (Trp)229, which may underlie the lower interaction energy and reduced MOR affinity for the compound. Taken together, our results are consistent with the reported biological findings for U-compounds and provide a molecular basis for the MOR selectivity of U-47700 and KOR selectivity of U-50488.

A subtle structural modification of a synthetic cannabinoid receptor agonist drastically increases its efficacy at the CB1 receptor.

The emergence of synthetic cannabinoid receptor agonists (SCRAs) as illicit psychoactive substances has posed considerable public health risks that include fatalities. Many SCRAs exhibit much higher efficacy and potency, compared with the phytocannabinoid Δ9-tetrahydrocannabinol (THC), at the cannabinoid receptor 1 (CB1R), a G protein-coupled receptor involved in modulating neurotransmitter release. In this study, we investigated structure activity relationships (SAR) of aminoalkylindole SCRAs at CB1Rs, focusing on 5F-pentylindoles containing an amide linker attached to different head moieties. Using in vitro bioluminescence resonance energy transfer (BRET) assays, we identified a few of SCRAs exhibiting significantly higher efficacy in engaging the Gi protein and recruiting ß-arrestin than the reference CB1R full agonist CP55940. Importantly, adding a methyl group at the head moiety of 5F-MMB-PICA yielded 5F-MDMB-PICA, an agonist exhibiting a large increase in efficacy and potency at the CB1R. This pharmacological observation was supported by a functional assay of the effects of these SCRAs on glutamate field potentials recorded in hippocampal slices. Molecular modeling and simulations of the CB1R bound with either of the SCRAs revealed critical structural determinants contributing to the higher efficacy of 5F-MDMB-PICA, and how these subtle differences propagated to the receptor-G protein interface. Thus, we find that apparently minor structural changes in the head moiety of SCRAs can cause major changes in efficacy. Our results highlight the need for close monitoring of structural modifications of newly emerging SCRAs and their potential for toxic drug responses in humans.

Comparative neuropharmacology of structurally distinct non-fentanyl opioids that are appearing on recreational drug markets worldwide.

BACKGROUND: The emergence of novel synthetic opioids (NSOs) is contributing to the opioid overdose crisis. While fentanyl analogs have historically dominated the NSO market, a shift towards non-fentanyl compounds is now occurring. METHODS: Here, we examined the neuropharmacology of structurally distinct non-fentanyl NSOs, including U-47700, isotonitazene, brorphine, and N-desethyl isotonitazene, as compared to morphine and fentanyl. Compounds were tested in vitro using opioid receptor binding assays in rat brain tissue and by monitoring forskolin-stimulated cAMP accumulation in cells expressing the human mu-opioid receptor (MOR). Compounds were administered subcutaneously to male Sprague-Dawley rats, and hot plate antinociception, catalepsy score, and body temperature changes were measured. RESULTS: Receptor binding results revealed high MOR selectivity for all compounds, with MOR affinities comparable to those of morphine and fentanyl (i.e., nM). All drugs acted as full-efficacy MOR agonists in the cyclic AMP assay, but nitazene analogs had greater functional potencies (i.e., pM) compared to the other drugs (i.e., nM). When administered to rats, all compounds induced opioid-like antinociception, catalepsy, and body temperature changes, but nitazenes were the most potent. Similar to fentanyl, the nitazenes had faster onset and decline of in vivo effects when compared to morphine. In vivo potencies to induce antinociception and catalepsy (i.e., ED50s) correlated with in vitro functional potencies (i.e., EC50s) but not binding affinities (i.e., Kis) at MOR. CONCLUSIONS: Collectively, our findings indicate that non-fentanyl NSOs pose grave danger to those individuals who use opioids. Continued vigilance is needed to identify and characterize synthetic opioids as they emerge in clandestine drug markets.

Receptor Binding Profiles for Tryptamine Psychedelics and Effects of 4-Propionoxy-N,N-dimethyltryptamine in Mice.

Analogues of 4-phosphoryloxy-N,N-dimethyltryptamine (psilocybin) are being sold on recreational drug markets and developed as potential medications for psychedelic-assisted therapies. Many of these tryptamine-based psilocybin analogues produce psychedelic-like effects in rodents and humans primarily by agonist activity at serotonin 2A receptors (5-HT2A). However, the comprehensive pharmacological target profiles for these compounds compared to psilocybin and its active metabolite 4-hydroxy-N,N-dimethyltryptamine (psilocin) are unknown. The present study determined the receptor binding profiles of various tryptamine-based psychedelics structurally related to psilocybin across a broad range of potential targets. Specifically, we examined tryptamine psychedelics with different 4-position (hydroxy, acetoxy, propionoxy) and N,N-dialkyl (dimethyl, methyl-ethyl, diethyl, methyl-propyl, ethyl-propyl, diisopropyl, methyl-allyl, diallyl) substitutions. Further, the psilocybin analogue 4-propionoxy-N,N-dimethyltryptamine (4-PrO-DMT) was administered to mice in experiments measuring head twitch response (HTR), locomotor activity, and body temperature. Overall, the present pharmacological profile screening data show that the tryptamine psychedelics target multiple serotonin receptors, including serotonin 1A receptors (5-HT1A). 4-Acetoxy and 4-propionoxy analogues of 4-hydroxy compounds displayed somewhat weaker binding affinities but similar target profiles across 5-HT receptors and other identified targets. Additionally, differential binding screen profiles were observed with N,N-dialkyl position variations across several non-5-HT receptor targets (i.e., alpha receptors, dopamine receptors, histamine receptors, and serotonin transporters), which could impact in vivo pharmacological effects of the compounds. In mouse experiments, 4-PrO-DMT displayed dose-related psilocybin-like effects to produce 5-HT2A-mediated HTR (0.3-3 mg/kg s.c.) as well as 5-HT1A-mediated hypothermia and hypolocomotion (3-30 mg/kg s.c.). Lastly, our data support a growing body of evidence that the 5-HT2A-mediated HTR induced by tryptamine psychedelics is attenuated by 5-HT1A receptor agonist activity at high doses in mice.

Are psychedelic medicines the reset for chronic pain? Preliminary findings and research needs.

Chronic pain is a leading cause of disability, reduced productivity, healthcare seeking behavior, and a contributor to opioid overdose in the United States. For many people, pain can be satisfactorily managed by existing medicines and comprehensive psychosocial treatments. For others, available treatments are either ineffective or not acceptable, due to side effects and concerns about risks. Preliminary evidence suggests that some psychedelics may be effective for certain types of pain and/or improved quality of life with increased functionality and reduced disability and distress in people whose pain may never be completely relieved. Efficacy in these quality-of-life related outcomes would be consistent with the 'reset in thinking' about chronic pain management being increasingly called for as a more realistic goal for some people as compared to complete elimination of pain. This commentary summarizes the rationale for conducting more basic research and clinical trials to further explore the potential for psychedelics in chronic pain management. Additionally, if shown to be effective, to then determine whether the effects of psychedelics are primarily due to direct antinociceptive or anti-inflammatory mechanisms, or via increased tolerability, acceptance, and sense of spirituality, that appear to at least partially mediate the therapeutic effects of psychedelics observed in psychiatric disorders such as major depression. This commentary represents a collaboration of clinical and more basic scientists examining these issues and developing recommendations for research ranging from neuropharmacology to the biopsychosocial treatment factors that appear to be as important in pain management as in depression and other disorders in which psychedelic medicines are under development. This article is part of the Special Issue on "National Institutes of Health Psilocybin Research Speaker Series".

Binding preference at the µ-opioid receptor underlies distinct pharmacology of cyclopropyl versus valeryl analogs of fentanyl.

Illicitly manufactured fentanyl is driving the current opioid crisis, and various fentanyl analogs are appearing in recreational drug markets worldwide. To assess the potential health risks posed by fentanyl analogs, it is necessary to understand structure-activity relationships for these compounds. Here we compared the pharmacology of two structurally related fentanyl analogs implicated in opioid overdose: cyclopropylfentanyl and valerylfentanyl. Cyclopropylfentanyl has a three-carbon ring attached to the carbonyl group on the fentanyl scaffold, whereas valerylfentanyl has a four-carbon chain at the same position. In vitro assays examining µ-opioid receptor (MOR) coupling to G proteins in CHO cells showed that cyclopropylfentanyl is a full agonist (EC50 = 8.6 nM, %Emax = 113%), with potency and efficacy similar to fentanyl (EC50 = 10.3 nM, %Emax = 113%). By contrast, valerylfentanyl is a partial agonist at MOR (EC50 = 179.8 nM, %Emax = 60%). Similar results were found in assays assessing MOR-mediated ß-arrestin recruitment in HEK cells. In vivo studies in male CD-1 mice demonstrated that both fentanyl analogs induce naloxone-reversible antinociception and respiratory suppression, but cyclopropylfentanyl is 100-times more potent as an antinociceptive agent (ED50 = 0.04 mg/kg, s. c.) than valerylfentanyl (ED50 = 4.0 mg/kg, s. c.). Molecular simulation results revealed that the alkyl chain of valerylfentanyl cannot be well accommodated by the active state of MOR and may transition the receptor toward an inactive state, converting the fentanyl scaffold to a partial agonist. Taken together, our results suggest that cyclopropylfentanyl presents much greater risk of adverse effects when compared to valerylfentanyl. Moreover, the summed findings may provide clues to the design of therapeutic opioids with reduced adverse side effects.

Persistent binding at dopamine transporters determines sustained psychostimulant effects.

Psychostimulants interacting with the dopamine transporter (DAT) can be used illicitly or for the treatment of specific neuropsychiatric disorders. However, they can also produce severe and persistent adverse events. Often, their pharmacological properties in vitro do not fully correlate to their pharmacological profile in vivo. Here, we investigated the pharmacological effects of enantiomers of pyrovalerone, α-pyrrolidinovalerophenone, and 3,4-methylenedioxypyrovalerone as compared to the traditional psychostimulants cocaine and methylphenidate, using a variety of in vitro, computational, and in vivo approaches. We found that in vitro drug-binding kinetics at DAT correlate with the time-course of in vivo psychostimulant action in mice. In particular, a slow dissociation (i.e., slow koff) of S-enantiomers of pyrovalerone analogs from DAT predicts their more persistent in vivo effects when compared to cocaine and methylphenidate. Overall, our findings highlight the critical importance of drug-binding kinetics at DAT for determining the in vivo profile of effects produced by psychostimulant drugs.

Characterization of the Native Disulfide Isomers of the Novel χ-Conotoxin PnID: Implications for Further Increasing Conotoxin Diversity.

χ-Conotoxins are known for their ability to selectively inhibit norepinephrine transporters, an ability that makes them potential leads for treating various neurological disorders, including neuropathic pain. PnID, a peptide isolated from the venom of Conus pennaceus, shares high sequence homology with previously characterized χ-conotoxins. Whereas previously reported χ-conotoxins seem to only have a single native disulfide bonding pattern, PnID has three native isomers due to the formation of different disulfide bond patterns during its maturation in the venom duct. In this study, the disulfide connectivity and three-dimensional structure of these disulfide isomers were explored using regioselective synthesis, chromatographic coelution, and solution-state nuclear magnetic resonance spectroscopy. Of the native isomers, only the isomer with a ribbon disulfide configuration showed pharmacological activity similar to other χ-conotoxins. This isomer inhibited the rat norepinephrine transporter (IC50 = 10 ± 2 µM) and has the most structural similarity to previously characterized χ-conotoxins. In contrast, the globular isoform of PnID showed more than ten times less activity against this transporter and the beaded isoform did not display any measurable biological activity. This study is the first report of the pharmacological and structural characterization of an χ-conotoxin from a species other than Conus marmoreus and is the first report of the existence of natively-formed conotoxin isomers.