Mechanisms of hepatocellular toxicity associated with new psychoactive synthetic cathinones

Synthetic cathinones are a new class of psychostimulant substances. Rarely, they can cause liver injury but associated mechanisms are not completely elucidated. In order to increase our knowledge about mechanisms of hepatotoxicity, we investigated the effect of five frequently used cathinones on two human cell lines. Bupropion was included as structurally related drug used therapeutically. In HepG2 cells, bupropion, MDPV, mephedrone and naphyrone depleted the cellular ATP content at lower concentrations (0.2-1mM) than cytotoxicity occurred (0.5-2mM), suggesting mitochondrial toxicity. In comparison, methedrone and methylone depleted the cellular ATP pool and induced cytotoxicity at similar concentrations (≥2mM). In HepaRG cells, cytotoxicity and ATP depletion could also be demonstrated, but cytochrome P450 induction did not increase the toxicity of the compounds investigated. The mitochondrial membrane potential was decreased in HepG2 cells by bupropion, MDPV and naphyrone, confirming mitochondrial toxicity. Bupropion, but not the other compounds, uncoupled oxidative phosphorylation. Bupropion, MDPV, mephedrone and naphyrone inhibited complex I and II of the electron transport chain, naphyrone also complex III. All four mitochondrial toxicants were associated with increased mitochondrial ROS and increased lactate production, which was accompanied by a decrease in the cellular total GSH pool for naphyrone and MDPV. In conclusion, bupropion, MDPV, mephedrone and naphyrone are mitochondrial toxicants impairing the function of the electron transport chain and depleting cellular ATP stores. Since liver injury is rare in users of these drugs, affected persons must have susceptibility factors rendering them more sensitive for these drugs

Monoamine Transporter and Receptor Interaction Profiles in Vitro Predict Reported Human Doses of Novel Psychoactive Stimulants and Psychedelics

Pharmacological profiles of new psychoactive substances can be established rapidly in vitro and provide information on potential psychoactive effects in humans. The present study investigated whether specific in vitro monoamine transporter and receptor interactions can predict effective psychoactive doses in humans.; We correlated previously assessed in vitro data of stimulants and psychedelics with human doses that are reported on the Internet and in books.; For stimulants, dopamine and norepinephrine transporter inhibition potency was positively correlated with human doses, whereas serotonin transporter inhibition potency was inversely correlated with human doses. Serotonin 5-hydroxytryptamine-2A (5-HT2A) and 5-HT2C receptor affinity was significantly correlated with psychedelic doses, but 5-HT1A receptor affinity and 5-HT2A and 5-HT2B receptor activation potency were not.; The rapid assessment of in vitro pharmacological profiles of new psychoactive substances can help to predict psychoactive doses and effects in humans and facilitate the appropriate scheduling of new psychoactive substances

Monoamine receptor interaction profiles of 4-thio-substituted phenethylamines (2C-T drugs)

4-Thio-substituted phenethylamines (2C-T drugs) are potent psychedelics with poorly defined pharmacological properties. Because of their psychedelic effects, 2C-T drugs are sometimes sold as new psychoactive substances (NPSs). The aim of the present study was to characterize the monoamine receptor and transporter interaction profiles of a series of 2C-T drugs.; We determined the binding affinities of 2C-T drugs at monoamine receptors and transporters in human cells that were transfected with the respective receptors or transporters. We also investigated the functional activation of serotonergic 5-hydroxytryptamine 2A (5-HT2A) and 5-HT2B receptors, activation of human trace amine-associated receptor 1 (TAAR1), and inhibition of monoamine uptake transporters.; 2C-T drugs had high affinity for 5-HT2A and 5-HT2C receptors (1-54 nM and 40-350 nM, respectively). With activation potencies of 1-53 nM and 44-370 nM, the drugs were potent 5-HT2A receptor and 5-HT2B receptor, respectively, partial agonists. An exception to this were the benzylthiophenethylamines, which did not potently activate the 5-HT2B receptor (EC50 > 3000 nM). Furthermore, the compounds bound to serotonergic 5-HT1A and adrenergic receptors. The compounds had high affinity for the rat TAAR1 (5-68 nM) and interacted with the mouse but not human TAAR1. The 2C-T drugs did not potently interact with monoamine transporters (Ki > 4000 nM).; The receptor binding profile of 2C-T drugs predicts psychedelic effects that are mediated by potent 5-HT2 receptor interactions

Receptor Interaction Profiles of 4-Alkoxy-3,5-Dimethoxy-Phenethylamines (Mescaline Derivatives) and Related Amphetamines

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-HT; 1A; , 5-HT; 2A; , and 5-HT; 2C; , adrenergic α; 1A; and α; 2A; , and dopaminergic D; 2; 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-HT; 2A; and 5-HT; 2B; receptors, and TAAR1 was examined. Generally, scalines and 3C-scalines bound with weak to moderately high affinity to the 5-HT; 2A; receptor (; K; i; = 150-12,000 nM). 3C-scalines showed a marginal preference for the 5-HT; 2A; vs the 5-HT; 2C; and 5-HT; 1A; receptors whereas no preference was observed for the scalines. Extending the 4-alkoxy substituent increased 5-HT; 2A; and 5-HT; 2C; receptors binding affinities, and enhanced activation potency and efficacy at the 5-HT; 2A; but not at the 5-HT; 2B; receptor. Introduction of fluorinated 4-alkoxy substituents generally increased 5-HT; 2A; and 5-HT; 2C; receptors binding affinities and increased the activation potency and efficacy at the 5-HT; 2A; and 5-HT; 2B; receptors. Overall, no potent affinity was observed at non-serotonergic targets. As observed for other psychedelics, scalines and 3C-scalines interacted with the 5-HT; 2A; and 5-HT; 2C; receptors and bound with higher affinities (up to 63-fold and 34-fold increase, respectively) when compared to mescaline

Receptor interaction profiles of novel N-2-methoxybenzyl (NBOMe) derivatives of 2,5-dimethoxy-substituted phenethylamines (2C drugs)

N-2-methoxybenzyl-phenethylamines (NBOMe drugs) are newly used psychoactive substances with poorly defined pharmacological properties. The aim of the present study was to characterize the receptor binding profiles of a series of NBOMe drugs compared with their 2,5-dimethoxy-phenethylamine analogs (2C drugs) and lysergic acid diethylamide (LSD) in vitro.; We investigated the binding affinities of 2C drugs (2C-B, 2C-C, 2C-D, 2C-E, 2C-H, 2C-I, 2C-N, 2C-P, 2C-T-2, 2C-T-4, 2C-T-7, and mescaline), their NBOMe analogs, and LSD at monoamine receptors and determined functional 5-hydroxytryptamine-2A (5-HT2A) and 5-HT2B receptor activation. Binding at and the inhibition of monoamine uptake transporters were also determined. Human cells that were transfected with the respective human receptors or transporters were used (with the exception of trace amine-associated receptor-1 [TAAR1], in which rat/mouse receptors were used).; All of the compounds potently interacted with serotonergic 5-HT2A, 5-HT2B, 5-HT2C receptors and rat TAAR1 (most Ki and EC50: <1 μM). The N-2-methoxybenzyl substitution of 2C drugs increased the binding affinity at serotonergic 5-HT2A, 5-HT2C, adrenergic α1, dopaminergic D1-3, and histaminergic H1 receptors and monoamine transporters but reduced binding to 5-HT1A receptors and TAAR1. As a result, NBOMe drugs were very potent 5-HT2A receptor agonists (EC50: 0.04-0.5 μM) with high 5-HT2A/5-HT1A selectivity and affinity for adrenergic α1 receptors (Ki: 0.3-0.9 μM) and TAAR1 (Ki: 0.06-2.2 μM), similar to LSD, but not dopaminergic D1-3 receptors (most Ki:>1 μM), unlike LSD.; The binding profile of NBOMe drugs predicts strong hallucinogenic effects, similar to LSD, but possibly more stimulant properties because of α1 receptor interactions

Pharmacological profile of mephedrone analogs and related new psychoactive substances

Mephedrone is a synthetic cathinone and one of the most popular recreationally used new psychoactive substances. The aim of the present study was to characterize the in vitro pharmacology of novel analogs of mephedrone and related newly emerged designer stimulants.; We determined norepinephrine, dopamine, and serotonin transporter inhibition potencies and monoamine release in transporter-transfected human embryonic kidney 293 cells. We also assessed monoamine receptor and transporter binding affinities.; Mephedrone analogs potently inhibited the norepinephrine transporter and, with the exception of 3-methylmethcathinone (3-MMC), inhibited the serotonin transporter more potently than the dopamine transporter. Similar to classic amphetamines, mephedrone analogs were substrate-type monoamine releasers. 5-(2-Aminopropyl)indole (5-IT) was a highly potent monoamine transporter inhibitor and a releaser of dopamine and serotonin. 4-Methylamphetamine (4-MA) mediated efflux of all three monoamines and inhibited the serotonin transporter more potently than the dopamine transporter, unlike amphetamine. N-methyl-2-aminoindane (N-methyl-2-AI) was a selective norepinephrine transporter inhibitor and norepinephrine releaser, whereas 5-methoxy-6-methyl-2-aminoindane (MMAI) was a selective serotonin transporter inhibitor and serotonin releaser. All of the drugs interacted with monoamine receptors.; The predominant actions on serotonin vs. dopamine transporters suggest that dimethylmethcathinones, 4-MA, and MMAI cause entactogenic effects similar to 3,4-methylenedioxymethamphetamine, whereas 3-MMC, 5-IT, and N-methyl-2-AI have more stimulant-type properties like amphetamine. Because of pharmacological and structural similarity to mephedrone, similar health risks can be expected for these analogs

Pharmacological profile of methylphenidate-based designer drugs

Methylphenidate-based designer drugs are new psychoactive substances (NPS) that are used outside medical settings and their pharmacology is largely unexplored. The aim of the present study was to characterize the pharmacology of methylphenidate-based substances in vitro.; We determined the potencies of the methylphenidate-based NPS N-benzylethylphenidate, 3,4-dichloroethylphenidate, 3,4-dichloromethylphenidate, ethylnaphthidate, ethylphenidate, 4-fluoromethylphenidate, isopropylphenidate, 4-methylmethylphenidate, methylmorphenate, and propylphenidate and the potencies of the related compounds cocaine and modafinil with respect to norepinephrine, dopamine, and serotonin transporter inhibition in transporter-transfected human embryonic kidney 293 cells. We also investigated monoamine efflux and monoamine receptor and transporter binding affinities. Furthermore, we assessed the cell integrity under assay conditions.; All methylphenidate-based substances inhibited the norepinephrine and dopamine transporters 4 to >1000-fold more potently than the serotonin transporter. Similar to methylphenidate and cocaine, methylphenidate-based NPS did not elicit transporter-mediated efflux of monoamines. Besides binding to monoamine transporters, several test drugs had affinity for adrenergic, serotonergic, and rat trace amine-associated receptors but not for dopaminergic or mouse trace amine-associated receptors. No cytotoxicity was observed after drug treatment at assay concentrations.; Methylphenidate-based substances had pharmacological profiles similar to methylphenidate and cocaine. The predominant actions on dopamine transporters vs. serotonin transporters may be relevant when considering abuse liability

Stereochemistry of phase-1 metabolites of mephedrone determines their effectiveness as releasers at the serotonin transporter

Mephedrone (4-methyl-N-methylcathinone) is a psychostimulant that promotes release of monoamines via the high affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). Metabolic breakdown of mephedrone results in bioactive metabolites that act as substrate-type releasers at monoamine transporters and stereospecific metabolism of mephedrone has been reported. This study compared the effects of the enantiomers of the phase-1 metabolites nor-mephedrone, 4-hydroxytolyl-mephedrone (4-OH-mephedrone) and dihydro-mephedrone on (i) DAT, NET and SERT mediated substrate fluxes, (ii) determined their binding affinities towards a battery of monoamine receptors and (iii) examined the relative abundance of the enantiomers in human urine. Each of the enantiomers tested inhibited uptake mediated by DAT, NET and SERT. No marked differences were detected at DAT and NET. However, at SERT, the S-enantiomers of nor-mephedrone and 4-OH-mephedrone were several times more potent than the corresponding R-enantiomers. Moreover, the R-enantiomers were markedly less effective as releasers at SERT. S-nor-mephedrone displayed moderate affinities towards human alpha; 1A; , human 5-HT; 2A; and rat and mouse trace amine-associated receptor 1. These results demonstrate that stereochemistry dictates the pharmacodynamics of the phase-1 metabolites of mephedrone at SERT, but not at DAT and NET, which manifests in marked differences in their relative potencies, i.e. DAT/SERT ratios. Chiral analysis of urine samples demonstrated that nor-mephedrone predominantly exists as the S-enantiomer. Given the asymmetric abundance of the enantiomers in biological samples, these findings may add to our understanding of the subjective effects of administered mephedrone, which indicate pronounced effects on the serotonergic system

The psychostimulant (±)-cis-4,4'-dimethylaminorex (4,4'-DMAR) interacts with human plasmalemmal and vesicular monoamine transporters

(±)-cis-4,4'-Dimethylaminorex (4,4'-DMAR) is a new psychoactive substance (NPS) that has been associated with 31 fatalities and other adverse events in Europe between June 2013 and February 2014. We used in vitro uptake inhibition and transporter release assays to determine the effects of 4,4'-DMAR on human high-affinity transporters for dopamine (DAT), norepinephrine (NET) and serotonin (SERT). In addition, we assessed its binding affinities to monoamine receptors and transporters. Furthermore, we investigated the interaction of 4,4'-DMAR with the vesicular monoamine transporter 2 (VMAT2) in rat phaeochromocytoma (PC12) cells and synaptic vesicles prepared from human striatum. 4,4'-DMAR inhibited uptake mediated by human DAT, NET or SERT, respectively in the low micromolar range (IC; 50; values < 2 μM). Release assays identified 4,4'-DMAR as a substrate type releaser, capable of inducing transporter-mediated reverse transport via DAT, NET and SERT. Furthermore, 4,4'-DMAR inhibited both the rat and human isoforms of VMAT2 at a potency similar to 3,4-methylenedioxymethylamphetamine (MDMA). This study identified 4,4'-DMAR as a potent non-selective monoamine releasing agent. In contrast to the known effects of aminorex and 4-methylaminorex, 4,4'-DMAR exerts profound effects on human SERT. The latter finding is consistent with the idea that fatalities associated with its abuse may be linked to monoaminergic toxicity including serotonin syndrome. The activity at VMAT2 suggests that chronic abuse of 4,4'-DMAR may result in long-term neurotoxicity

Monoamine Transporter and Receptor Interaction Profiles in Vitro Predict Reported Human Doses of Novel Psychoactive Stimulants and Psychedelics

Abstract Background Pharmacological profiles of new psychoactive substances can be established rapidly in vitro and provide information on potential psychoactive effects in humans. The present study investigated whether specific in vitro monoamine transporter and receptor interactions can predict effective psychoactive doses in humans. Methods We correlated previously assessed in vitro data of stimulants and psychedelics with human doses that are reported on the Internet and in books. Results For stimulants, dopamine and norepinephrine transporter inhibition potency was positively correlated with human doses, whereas serotonin transporter inhibition potency was inversely correlated with human doses. Serotonin 5-hydroxytryptamine-2A (5-HT2A) and 5-HT2C receptor affinity was significantly correlated with psychedelic doses, but 5-HT1A receptor affinity and 5-HT2A and 5-HT2B receptor activation potency were not. Conclusions The rapid assessment of in vitro pharmacological profiles of new psychoactive substances can help to predict psychoactive doses and effects in humans and facilitate the appropriate scheduling of new psychoactive substances