Psychedelic and stimulating phenethylamines belong to the family of new psychoactive substances (NPS). The acute toxicity framework has begun to be investigated, while studies showing genotoxic potential are very limited or not available. Therefore, in order to fill this gap, the aim of the present work was to evaluate the genotoxicity by treating TK6 cells with 2C-H, 2C-I, 2C-B, 25B-NBOMe, and the popular 3,4-Methylenedioxymethylamphetamine (MDMA). On the basis of cytotoxicity and cytostasis results, we selected the concentrations (6.25-35 µM) to be used in genotoxicity analysis. We used the micronucleus (MN) as indicator of genetic damage and analyzed the MNi frequency fold increase by an automated flow cytometric protocol. All substances, except MDMA, resulted genotoxic; therefore, we evaluated reactive oxygen species (ROS) induction as a possible mechanism at the basis of the demonstrated genotoxicity. The obtained results showed a statistically significant increase in ROS levels for all genotoxic phenethylamines confirming this hypothesis. Our results highlight the importance of genotoxicity evaluation for a complete assessment of the risk associated also with NPS exposure. Indeed, the subjects who do not have hazardous behaviors or require hospitalization by using active but still "safe" doses could run into genotoxicity and in the well-known long-term effects associated.

3,4,5-Trimethoxyphenethylamine (mescaline) is a psychedelic alkaloid found in peyote cactus. Related 4-alkoxy-3,5-dimethoxy-substituted phenethylamines (scalines) and amphetamines (3C-scalines) are reported to induce similarly potent psychedelic effects and are therefore potential novel therapeutics for psychedelic-assisted therapy. Herein, several pharmacologically uninvestigated scalines and 3C-scalines were examined at key monoamine targets in vitro. Binding affinity at human serotonergic 5-HT1A, 5-HT2A, and 5-HT2C, adrenergic α1A and α2A, and dopaminergic D2 receptors, rat and mouse trace amine-associated receptor 1 (TAAR1), and human monoamine transporters were assessed using target specific transfected cells. Furthermore, activation of human 5-HT2A and 5-HT2B receptors, and TAAR1 was examined. Generally, scalines and 3C-scalines bound with weak to moderately high affinity to the 5-HT2A receptor (K i = 150-12,000 nM). 3C-scalines showed a marginal preference for the 5-HT2A vs the 5-HT2C and 5-HT1A receptors whereas no preference was observed for the scalines. Extending the 4-alkoxy substituent increased 5-HT2A and 5-HT2C receptors binding affinities, and enhanced activation potency and efficacy at the 5-HT2A but not at the 5-HT2B receptor. Introduction of fluorinated 4-alkoxy substituents generally increased 5-HT2A and 5-HT2C receptors binding affinities and increased the activation potency and efficacy at the 5-HT2A and 5-HT2B receptors. Overall, no potent affinity was observed at non-serotonergic targets. As observed for other psychedelics, scalines and 3C-scalines interacted with the 5-HT2A and 5-HT2C receptors and bound with higher affinities (up to 63-fold and 34-fold increase, respectively) when compared to mescaline.

Mescaline derivative (2C phenethylamines) drugs have been modified by the introduction of a N-2-methoxybenzyl group to originate a new series of compounds with recognized and potent psychedelic effects, the NBOMe-drugs. Although they are prevalent in unregulated drug markets, their toxicity profile is still poorly understood, despite several reports highlighting cases of acute intoxication, with brain and liver toxicity. Thus, in this study, mescaline, 2C-N (insertion of a nitro in the para position of the 2C phenethylamines aromatic ring) and 2C-B (insertion of a bromide in the para position of the 2C phenethylamines aromatic ring) and their corresponding NBOMe counterparts, mescaline-NBOMe, 25N-NBOMe and 25B-NBOMe, were synthetized and the in vitro neuro- and hepatocytotoxicity evaluated in differentiated SH-SY5Y and HepG2 cell lines, respectively. Cytotoxicity, oxidative stress, metabolic and energetic studies were performed to evaluate the main pathways involved in their toxicity. Our results demonstrated that the presence of the N-2-methoxybenzyl group significantly increased the in vitro cytotoxicity of 2C phenethylamines drugs in both cell lines, with the NBOMe drugs presenting lower EC50 values when compared to their counterparts. Consistently, our data showed a correlation between the drug's lipophilicity and the EC50 values, except for 2C-B. The 2C-B presented higher cytotoxic effects in both cell lines than mescaline-NBOMe, a result that can be explained by its higher passive permeability. All the NBOMe derivatives were able to cross the blood-brain barrier. Considering metabolic studies, the cytotoxicity of these drugs was shown to be influenced by inhibition of cytochrome P450 (CYP), which suggests a potential role of this enzyme complex, especially CYP3A4 and CYP2D6 isoenzymes in SH-SY5Y cells, in their detoxification or bioactivation. Furthermore, in differentiated SH-SY5Y cells, the drugs were able to induce mitochondrial membrane depolarization, and to disrupt GSH and ATP intracellular levels, these effects being concentration dependent and more pronounced for the NBOMe derivatives. No ROS overproduction was detected for any of the drugs in the tested experimental conditions. A correlation between a drug's lipophilicity and the EC50 values in both cell lines, except for 2C-B, was also obtained. In summary, the introduction of a NBOMe moiety to the parent drugs significantly increases their lipophilicity, brain permeability and cytotoxic effects, with GSH and ATP homeostasis disruption. The inhibition of CYP3A4 and CYP2D6 emphasized that CYP-mediated metabolism impacts the toxicity of these drugs.

The popularity of new psychoactive substances (NPS) has grown in recent years, with certain NPS commonly and preferentially consumed even following the introduction of preventative legislation. With the objective to improve the knowledge on the use of NPS, a rapid and very sensitive method was developed for the determination of ten priority NPS (N-ethylcathinone, methylenedioxypyrovalerone (MDPV), methylone, butylone, methedrone, mephedrone, naphyrone, 25-C-NBOMe, 25-I-NBOMe and 25-B-NBOMe) in influent wastewater. Sample clean-up and pre-concentration was made by off-line solid phase extraction (SPE) with Oasis MCX cartridges. Isotopically labelled internal standards were used to correct for matrix effects and potential SPE losses. Following chromatographic separation on a C18 column within 6 min, the compounds were measured by tandem mass spectrometry in positive ionization mode. The method was optimised and validated for all compounds. Limits of quantification were evaluated by spiking influent wastewater samples at 1 or 5 ng/L. An investigation into the stability of these compounds in influent wastewater was also performed, showing that, following acidification at pH 2, all compounds were relatively stable for up to 7 days. The method was then applied to influent wastewater samples from eight European countries, in which mephedrone, methylone and MDPV were detected. This work reveals that although NPS use is not as extensive as for classic illicit drugs, the application of a highly sensitive analytical procedure makes their detection in wastewater possible. The developed analytical methodology forms the basis of a subsequent model-based back-calculation of abuse rate in urban areas (i.e. wastewater-based epidemiology).

Bivalent ligands--compounds incorporating two receptor-interacting moieties linked by a flexible chain--often exhibit profoundly enhanced binding affinity compared with their monovalent components, implying concurrent binding to multiple sites on the target protein. It is generally assumed that neurotransmitter sodium symporter (NSS) proteins, such as the dopamine transporter (DAT), contain a single domain responsible for recognition of substrate molecules. In this report, we show that molecules possessing two substrate-like phenylalkylamine moieties linked by a progressively longer aliphatic spacer act as progressively more potent DAT inhibitors (rather than substrates). One compound bearing two dopamine (DA)-like pharmacophoric 'heads' separated by an 8-carbon linker achieved an 82-fold gain in inhibition of [(3)H] 2beta-carbomethoxy-3beta-(4-fluorophenyl)-tropane (CFT) binding compared with DA itself; bivalent compounds with a 6-carbon linker and heterologous combinations of DA-, amphetamine- and beta-phenethylamine-like heads all resulted in considerable and comparable gains in DAT affinity. A series of short-chain bivalent-like compounds with a single N-linkage was also identified, the most potent of which displayed a 74-fold gain in binding affinity. Computational modelling of the DAT protein and docking of the two most potent bivalent (-like) ligands suggested simultaneous occupancy of two discrete substrate-binding domains. Assays with the DAT mutants W84L and D313N--previously employed by our laboratory to probe conformation-specific binding of different structural classes of DAT inhibitors--indicated a bias of the bivalent ligands for inward-facing transporters. Our results strongly indicate the existence of multiple DAT substrate-interaction sites, implying that it is possible to design novel types of DAT inhibitors based upon the 'multivalent ligand' strategy.

Classic psychedelics are substances of paramount cultural and neuroscientific importance. A distinctive feature of psychedelic drugs is the wide range of potential subjective effects they can elicit, known to be deeply influenced by the internal state of the user ("set") and the surroundings ("setting"). The observation of cross-tolerance and a series of empirical studies in humans and animal models support agonism at the serotonin (5-HT)2A receptor as a common mechanism for the action of psychedelics. The diversity of subjective effects elicited by different compounds has been attributed to the variables of "set" and "setting," to the binding affinities for other 5-HT receptor subtypes, and to the heterogeneity of transduction pathways initiated by conformational receptor states as they interact with different ligands ("functional selectivity"). Here we investigate the complementary (i.e., not mutually exclusive) possibility that such variety is also related to the binding affinity for a range of neurotransmitters and monoamine transporters including (but not limited to) 5-HT receptors. Building on two independent binding affinity datasets (compared to "in silico" estimates) in combination with natural language processing tools applied to a large repository of reports of psychedelic experiences (Erowid's Experience Vaults), we obtained preliminary evidence supporting that the similarity between the binding affinity profiles of psychoactive substituted phenethylamines and tryptamines is correlated with the semantic similarity of the associated reports. We also showed that the highest correlation was achieved by considering the combined binding affinity for the 5-HT, dopamine (DA), glutamate, muscarinic and opioid receptors and for the Ca+ channel. Applying dimensionality reduction techniques to the reports, we linked the compounds, receptors, transporters and the Ca+ channel to distinct fingerprints of the reported subjective effects. To the extent that the existing binding affinity data is based on a low number of displacement curves that requires further replication, our analysis produced preliminary evidence consistent with the involvement of different binding sites in the reported subjective effects elicited by psychedelics. Beyond the study of this particular class of drugs, we provide a methodological framework to explore the relationship between the binding affinity profiles and the reported subjective effects of other psychoactive compounds.

Natural phenethylamines are trace amine neurotransmitters associated with dopamine transmission and related illnesses such Parkinson's disease, and addiction. Synthetic phenethylamines can have psychoactive and hallucinogenic effects due to their high affinity with the 5-HT2A receptor. Evidence indicates phenethylamines can directly alter the microtubule cytoskeleton being structurally similar to the microtubule destabilizing agent colchicine, however little work has been done on this interaction. As microtubules provide neuron structure, intracellular transport, and influence synaptic plasticity the interaction of phenethylamines with microtubules is important for understanding the potential harms, or potential pharmaceutical use of phenethylamines. We investigated 110 phenethylamines and their interaction with microtubules. Here we performed molecular docking of these compounds at the colchicine binding site and ranked them via binding energy. The top 10% of phenethylamines were further screened based on pharmacokinetic and physicochemical properties derived from SwissADME and LightBBB. Based on these properties 25B-NBF, 25C-NBF, and DMBMPP were tested in in vitro microtubule polymerization assays showing that they alter microtubule polymerization dynamics in a dose dependent manner. As these compounds can rapidly cross the blood brain barrier and directly affect cytoskeletal dynamics, they have the potential to modulate cytoskeletal based neural plasticity. Further investigations into these mechanisms are warranted.

Among 14 subtypes of serotonin receptors (5-HTRs), 5-HT2AR plays important roles in drug addiction and various psychiatric disorders. Agonists for 5-HT2AR have been classified into three structural groups: phenethylamines, tryptamines, and ergolines. In this study, the structure-activity relationship (SAR) of phenethylamine and tryptamine derivatives for binding 5-HT2AR was determined. In addition, functional and regulatory evaluation of selected compounds was conducted for extracellular signal-regulated kinases (ERKs) and receptor endocytosis. SAR studies showed that phenethylamines possessed higher affinity to 5-HT2AR than tryptamines. In phenethylamines, two phenyl groups were attached to the carbon and nitrogen (R3) atoms of ethylamine, the backbone of phenethylamines. Alkyl or halogen groups on the phenyl ring attached to the β carbon exerted positive effects on the binding affinity when they were at para positions. Oxygen-containing groups attached to R3 exerted mixed influences depending on the position of their attachment. In tryptamine derivatives, tryptamine group was attached to the β carbon of ethylamine, and ally groups were attached to the nitrogen atom. Oxygen-containing substituents on large ring and alkyl substituents on the small ring of tryptamine groups exerted positive and negative influence on the affinity for 5-HT2AR, respectively. Ally groups attached to the nitrogen atom of ethylamine exerted negative influences. Functional and regulatory activities of the tested compounds correlated with their affinity for 5-HT2AR, suggesting their agonistic nature. In conclusion, this study provides information for designing novel ligands for 5-HT2AR, which can be used to control psychiatric disorders and drug abuse.

Psychedelics alter the perception of reality through agonist or partial agonist interaction with the 2A serotoninergic receptor. They are classified as phenethylamines, tryptamines and lysergamides. These classes, according to the United Nations Office on Drugs and Crime (UNODC) and European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), account for an important percentage of the new psychoactive substances (NPS) current scenario.The paper aimed at: a) identifying and categorising psychedelic molecules from a list of psychonaut websites and NPS online resources; and b) comparing the NPSfinderⓇ results with those from the European and United Nations databases. A crawling software (i.e. 'NPSfinderⓇ') was created to automatically scan, 24/7, a list of URLs and to extract a range of information (chemical/street names, chemical formulae, etc.) to facilitate NPS identification. Data collected were manually analysed and compared with the EMCDDA and UNODC databases.The overall number of psychedelic NPS detected by NPSfinderⓇ (November 2017-February 2020) was 1344, almost ten-times higher than that reported by the UNODC and EMCDDA combined. Of these, 994 previously unknown molecules were identified as (potential) novel psychedelics, suggesting a strong discrepancy between online and real-world NPS scenarios. The results show the interest of psychonauts, and maybe of the much larger community of 'recreational' drug users, towards psychedelics. Moreover, examining online scenario may help in assessing the availability in the real world of psychedelic NPS; understanding drug trends; and in possibly predicting future drug scenarios.

Preworkout supplements ("boosters") are used to enhance physical and mental performance during workouts. These products may contain various chemical substances with undefined pharmacological activity. We investigated whether substances that are contained in commercially available athletic multiple-ingredient preworkout supplements exert amphetamine-type activity at norepinephrine, dopamine, and serotonin transporters (NET, DAT, and SERT, respectively). We assessed the in vitro monoamine transporter inhibition potencies of the substances using human embryonic kidney 293 cells that expressed the human NET, DAT, and SERT. The phenethylamines β-phenethylamine, N-methylphenethylamine, β-methylphenethylamine, N-benzylphenethylamine, N-methyl-β-methylphenethylamine, and methylsynephrine inhibited the NET and less potently the DAT similarly to D-amphetamine. β-phenethylamine was the most potent, with IC50 values of 0.05 and 1.8 μM at the NET and DAT, respectively. These IC50 values were comparable to D-amphetamine (IC50 = 0.09 and 1.3 μM, respectively). The alkylamines 1,3-dimethylbutylamine and 1,3-dimethylamylamine blocked the NET but not the DAT. Most of the phenethylamines interacted with trace amine-associated receptor 1, serotonin 5-hydroxytryptamine-1A receptor, and adrenergic α1A and α2A receptors at submicromolar concentrations. None of the compounds blocked the SERT. In conclusion, products that are used by athletes may contain substances with mainly noradrenergic amphetamine-type properties.

Many ring-substituted phenethylamines exert psychedelic effects that are thought to be primarily mediated by interactions with serotonergic 5-hydroxytryptamine 2 (5-HT2A) receptors. The 2,5-dimethoxyphenethylamine (2C derivative) core structure with small lipophilic substituents at the 4-position seems to be particularly favorable for psychedelic effects. In contrast, 2C derivatives with bulky lipophilic substituents at the 4-position of the phenyl ring tend to display antagonist behavior at serotonin 5-HT2 receptor sites. To gain a better understanding of agonist and antagonist behavior of substituted phenethylamines, binding affinities and functional activation and inhibition of a series of 4'-aryl substituted 2,5-dimethoxyphenethylamine (2C-BI derivatives) at various monoamine receptors were determined. In addition, the interactions of the compounds with monoamine transporters were assessed. Various 2C-BI derivatives potently bound to human serotonergic and adrenergic receptors and to rat and mouse trace amine-associated receptor 1. Additionally, 2C-BI-8 and 2C-BI-12 activated serotonin 5-HT2A and 5-HT2B receptors at submicromolar concentrations. 2C-BI-1 and 2C-BI-7 were the only 2C-BI derivatives to activate human trace amine-associated receptor 1. 2C-BI-3 and 2C-BI-4 interacted with monoamine transporters but with low overall potency. In conclusion, the tested 2C-BI derivatives displayed diverse pharmacological profiles. The relatively high affinities of various 2C-BI derivatives at the serotonin 5-HT2A receptor indicate a high steric tolerance of the binding pocket. Potent partial activation of the serotonin 5-HT2A receptor by 2C-BI-8 and 2C-BI-12 suggests that these substances may potentially exert psychedelic effects similar to other compounds of the 2C family.

Background: 2,4,5-Trimethoxyamphetamine (TMA-2) is a potent psychedelic compound. Structurally related 4-alkyloxy-substituted 2,5-dimethoxyamphetamines and phenethylamine congeners (2C-O derivatives) have been described but their pharmacology is mostly undefined. Therefore, we examined receptor binding and activation profiles of these derivatives at monoamine receptors and transporters. Methods: Receptor binding affinities were determined at the serotonergic 5-HT1A, 5-HT2A, and 5-HT2C receptors, trace amine-associated receptor 1 (TAAR1), adrenergic α1 and α2 receptors, dopaminergic D2 receptor, and at monoamine transporters, using target-transfected cells. Additionally, activation of 5-HT2A and 5-HT2B receptors and TAAR1 was determined. Furthermore, we assessed monoamine transporter inhibition. Results: Both the phenethylamine and amphetamine derivatives (K i = 8-1700 nM and 61-4400 nM, respectively) bound with moderate to high affinities to the 5-HT2A receptor with preference over the 5-HT1A and 5-HT2C receptors (5-HT2A/5-HT1A = 1.4-333 and 5-HT2A/5-HT2C = 2.1-14, respectively). Extending the 4-alkoxy-group generally increased binding affinities at 5-HT2A and 5-HT2C receptors but showed mixed effects in terms of activation potency and efficacy at these receptors. Introduction of a terminal fluorine atom into the 4-ethoxy substituent by trend decreased, and with progressive fluorination increased affinities at the 5-HT2A and 5-HT2C receptors. Little or no effect was observed at the 5-HT1A receptor for any of the substances tested (K i ≥ 2700 nM). Phenethylamines bound more strongly to the TAAR1 (K i = 21-3300 nM) compared with their amphetamine analogs (K i = 630-3100 nM). Conclusion: As seen with earlier series investigated, the 4-alkyloxy-substituted 2,5-dimethoxyamphetamines and phenethylamines share some trends with the many other phenethylamine pharmacophore containing compounds, such as when increasing the size of the 4-substituent and increasing the lipophilicity, the affinities at the 5-HT2A/C subtype also increase, and only weak 5-HT2A/C subtype selectivities were achieved. At least from the binding data available (i.e., high affinity binding at the 5-HT2A receptor) one may predict mainly psychedelic-like effects in humans, at least for some of the compound investigated herein.

In this study, we present the synthesis of five novel compounds by combining flurbiprofen with various substituted 2-phenethylamines. The synthesized derivatives underwent comprehensive characterization using techniques such as 1H- and 13C-NMR spectroscopy, UV-Vis spectroscopy, and high-resolution mass spectrometry (HRMS). Detailed HRMS analysis was performed for each of these newly created molecules. The biological activities of these compounds were assessed through in vitro experiments to evaluate their potential as anti-inflammatory and antioxidant agents. Furthermore, the lipophilicity of these derivatives was determined, both theoretically using the cLogP method and experimentally through partition coefficient (RM) measurements. To gain insights into their binding affinity, we conducted an in silico analysis of the compounds' interactions with human serum albumin (HSA) using molecular docking studies. Our findings reveal that all of the newly synthesized compounds exhibit significant anti-inflammatory and antioxidant activities, with results statistically comparable to the reference compounds. Molecular docking studies further explain the observed in vitro results, shedding light on the molecular mechanisms behind their biological activities. Using in silico method, toxicity was calculated, resulting in LD50 values. Depending on the administration route, the novel flurbiprofen derivatives show lower toxicity compared to the standard flurbiprofen.

1. The structural determinants of catechol hydroxyl interactions with adrenergic receptors were examined using 12 alpha2-adrenergic agonists and a panel of mutated human alpha2A-adrenoceptors. The alpha2ASer201 mutant had a Cys --> Ser201 (position 5.43) amino-acid substitution, and alpha2ASer201Cys200 and alpha2ASer201Cys204 had Ser --> Cys200 (5.42) and Ser --> Cys204 (5.46) substitutions, respectively, in addition to the Cys --> Ser201 substitution. 2. Automated docking methods were used to predict the receptor interactions of the ligands. Radioligand-binding assays and functional [35S]GTPgammaS-binding assays were performed using transfected Chinese hamster ovary cells to experimentally corroborate the predicted binding modes. 3. The hydroxyl groups of phenethylamines were found to have different effects on ligand affinity towards the activated and resting forms of the wild-type alpha2A-adrenoceptor. Substitution of Ser200 or Ser204 with cysteine caused a deterioration in the capability of catecholamines to activate the alpha2A-adrenoceptor. The findings indicate that (i) Cys201 plays a significant role in the binding of catecholamine ligands and UK14,304 (for the latter, by a hydrophobic interaction), but Cys201 is not essential for receptor activation; (ii) Ser200 interacts with the meta-hydroxyl group of phenethylamine ligands, affecting both catecholamine binding and receptor activation; while (iii) substituting Ser204 with a cysteine interferes both with the binding of catecholamine ligands and with receptor activation, due to an interaction between Ser204 and the para-hydroxyl group of the catecholic ring.

5-Methoxy-N,N-Dimethyltryptamine (5-MeO-DMT) is a tryptamine with ultra-rapid onset and short duration of psychedelic effects. Prospective studies for other tryptamines have suggested beneficial effects on mental health outcomes. In preparation for a study in patients with depression, the present study GH001-HV-101 aimed to assess the impact of four different dose levels of a novel vaporized 5-MeO-DMT formulation (GH001) administered via inhalation as single doses of 2 (N = 4), 6 (N = 6), 12 (N = 4) and 18 mg (N = 4), and in an individualized dose escalation regimen (N = 4) on the safety, tolerability, and the dose-related psychoactive effects in healthy volunteers (N = 22). The psychedelic experience was assessed with a novel Peak Experience Scale (PES), the Mystical Experience Questionnaire (MEQ), the Ego Dissolution Inventory (EDI), the Challenging Experience Questionnaire (CEQ), and the 5-Dimensional Altered States of Consciousness Questionnaire (5D-ASC). Further aims were to assess the impact of 5-MeO-DMT on cognitive functioning, mood, and well-being. Higher doses of 5-MeO-DMT produced significant increments in the intensity of the psychedelic experience ratings as compared to the lowest 2 mg dose on all questionnaires, except the CEQ. Prominent effects were observed following single doses of 6, 12, and 18 mg on PES and MEQ ratings, while maximal effects on PES, MEQ, EDI, and 5D-ASC ratings were observed following individualized dose escalation of 5-MeO-DMT. Measures of cognition, mood, and well-being were not affected by 5-MeO-DMT. Vital signs at 1 and 3 h after administration were not affected and adverse events were generally mild and resolved spontaneously. Individualized dose escalation of 5-MeO-DMT may be preferable over single dose administration for clinical applications that aim to maximize the experience to elicit a strong therapeutic response.

New psychoactive substances that have been modified and developed to mimic the effects of already prohibited drugs are an increasingly global problem. Among them, 2-(2,5-dimethoxy-4-nitrophenyl)-N-(2-methoxybenzyl)ethanamine (25 N-NBOMe) belonging to the N-methoxybenzyl-phenethylamines (NBOMes) class has recently emerged as a new psychoactive substance. However, the rewarding effects of 25 N-NBOMe have not yet been studied. Here, we investigated the addictive potential of 25 N-NBOMe using conditioned place preference and self-administration in rodents. We also evaluated the effects of 25 N-NBOMe on the dopaminergic system using Western blot analysis. We found that 25 N-NBOMe at 3 mg/kg significantly increased conditioned place preference in mice and 25 N-NBOMe at 0.01 mg/kg/infusion significantly enhanced self-administration in rats. In addition, repeated administration of 25 N-NBOMe did not affect the expression of the dopamine 1 receptor but significantly reduced the expression of the dopamine 2 receptor in both the nucleus accumbens (NAc) and the dorsal striatum (DSt). We also found that 25 N-NBOMe significantly decreased the expression of the dopamine transporter only in the NAc, while increasing the expression of the phosphorylated dopamine transporter in both the NAc and the DSt. Furthermore, 25 N-NBOMe significantly reduced the expression of tyrosine hydroxylase in the NAc but not in the DSt. Taken together, these findings suggest that 25 N-NBOMe has abuse potential via dopaminergic system.

N-Benzyl-substituted 2C class phenethylamines (NBOMes) are psychoactive designer drugs, with strong hallucinogenic and stimulant effects, even at low doses. The designer drug, 2-(4-bromo-2, 5-dimethoxyphenyl)-N-(2-methoxybenzyl) ethanamine (25B-NBOMe) is considered to be one of the most potent agonists of the serotonin-2A (5-HT2A) receptor. Recently, we reported the first lethal case of 25B-NBOMe intoxication with severe rhabdomyolysis, concluded by clinical, pathological and toxicological analyses. There are currently no good animal models that closely recapitulate serotonin receptor-dependent rhabdomyolysis. In the present study, we created animal models of rhabdomyolysis using zebrafish larvae to study the pathomechanism of rhabdomyolysis, and demonstrated that 25B-NBOMe can simulate lethal rhabdomyolysis in this animal. Treatment of the larvae with 25B-NBOMe decreased their survival rate, locomotion, altered birefringence of the skeletal muscle and immunostainings for dystroglycan (a myoseptal protein) and myosin heavy chain (a myofibril protein), which were consistent with rhabdomyolysis. This 25B-NBOMe-induced rhabdomyolysis was inhibited by the 5-HT2A receptor antagonists ritanserin and aripirazole, but not by the 5-HT1A + 5-HT1B receptor antagonist propranolol and the 5-HT3 receptor antagonist granisetron, indicating 5-HT2A-dependent rhabdomyolysis. The 25B-NBOMe-treated zebrafish is, therefore, a highly useful model of rhabdomyolysis for studying the pathomechanism of rhabdomyolysis as well as for therapeutic drug screening.

The inclusion of new psychoactive substances (NPS) in the wastewater-based epidemiology approach presents challenges, such as the reduced number of users that translates into low concentrations of residues and the limited pharmacokinetics information available, which renders the choice of target biomarker difficult. The sampling during special social settings, the analysis with improved analytical techniques, and data processing with specific workflow to narrow the search, are required approaches for a successful monitoring. This work presents the application of a qualitative screening technique to wastewater samples collected during a city festival, where likely users of recreational substances gather and consequently higher residual concentrations of used NPS are expected. The analysis was performed using liquid chromatography coupled to high-resolution mass spectrometry. Data were processed using an algorithm that involves the extraction of accurate masses (calculated based on molecular formula) of expected m/z from an in-house database containing about 2,000 entries, including NPS and transformation products. We positively identified eight NPS belonging to the classes of synthetic cathinones, phenethylamines and opioids. In addition, the presence of benzodiazepine analogues, classical drugs and other licit substances with potential for abuse was confirmed. The screening workflow based on a database search was useful in the identification of NPS biomarkers in wastewater. The findings highlight the specific classical drugs and low NPS use in the Netherlands. Additionally, meta-chlorophenylpiperazine (mCPP), 2,5-dimethoxy-4-bromophenethylamine (2C-B), and 4-fluoroamphetamine (FA) were identified in wastewater for the first time.

Two new psychoactive substances (NPSs) classified as phenethylamines, namely 2-((2-(4-Iodo-2,5-dimethoxyphenyl)ethylamino)methyl)phenol (25I-NBOH) and 2-(((2-(4-chloro-2,5-dimethoxyphenyl)ethyl)amino)methyl)phenol (25C-NBOH), are being abused by people seeking recreational hallucinogens. These NPSs may cause serious health problems as their adverse effects are not known in most cases. Therefore, in the present study, we evaluated the cardiotoxicity of 25I-NBOH and 25C-NBOH using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, rat electrocardiography (ECG), Langendorff test, and human ether-a-go-go-related gene (hERG) assay. Furthermore, we analyzed the expression levels of p21 CDC42/RAC1-activated kinase 1 (PAK1), which is known to play various roles in the cardiovascular system. In the MTT assay, treatment with 25I-NBOH or 25C-NBOH dramatically decreased viability of H9c2 cardiomyocytes. Meanwhile, these two compounds significantly increased QT intervals and RR intervals in the rat ECG measurement. 25I-NBOH down-regulated the PAK1 protein expression in rat primary cardiomyocytes as well as H9c2 cells. However, 25C-NBOH had no effect on the PAK1 expression in H9c2 cells. In an in-depth study, 25I-NBOH inhibited potassium channels in the hERG assay, but in ex vivo test, the substance did not affect the left ventricular developed pressure (LVDP) and heart rate of the isolated rat hearts. Taken together, these results suggest that both 25I-NBOH and 25C-NBOH may have adverse cardiovascular effect. Further investigation would be needed to determine which factors mainly influence the relationship between PAK1 expression and cardiotoxicity.

Serotonergic psychedelics are described to have activation of the serotonin 2A receptor (5-HT2A) as their main pharmacological action. Despite their relevance, the molecular mechanisms underlying the psychedelic effects induced by certain 5-HT2A agonists remain elusive. One of the proposed hypotheses is the occurrence of biased agonism, defined as the preferential activation of certain signaling pathways over others. This study comparatively monitored the efficiency of a diverse panel of 4-position-substituted (and N-benzyl-derived) phenylalkylamines to induce recruitment of β-arrestin2 (βarr2) or miniGαq to the 5-HT2A, allowing us to assess structure-activity relationships and biased agonism. All test compounds exhibited agonist properties with a relatively large range of both EC50 and Emax values. Interestingly, the lipophilicity of the 2C-X phenethylamines was correlated with their efficacy in both assays but yielded a stronger correlation in the miniGαq- than in the βarr2-assay. Molecular docking suggested that accommodation of the 4-substituent of the 2C-X analogues in a hydrophobic pocket between transmembrane helices 4 and 5 of 5-HT2A may contribute to this differential effect. Aside from previously used standard conditions (lysergic acid diethylamide (LSD) as a reference agonist and a 2 h activation profile to assess a compound's activity), serotonin was included as a second reference agonist, and the compounds' activities were also assessed using the first 30 min of the activation profile. Under all assessed circumstances, the qualitative structure-activity relationships remained unchanged. Furthermore, the use of two reference agonists allowed for the estimation of both "benchmark bias" (relative to LSD) and "physiology bias" (relative to serotonin).