In vitro and in vivo metabolism of psilocybin's active metabolite psilocin.

In vivo, psilocybin is rapidly dephosphorylated to psilocin which induces psychedelic effects by interacting with the 5-HT2A receptor. Psilocin primarily undergoes glucuronidation or conversion to 4-hydroxyindole-3-acetic acid (4-HIAA). Herein, we investigated psilocybin's metabolic pathways in vitro and in vivo, conducting a thorough analysis of the enzymes involved. Metabolism studies were performed using human liver microsomes (HLM), cytochrome P450 (CYP) enzymes, monoamine oxidase (MAO), and UDP-glucuronosyltransferase (UGT). In vivo, metabolism was examined using male C57BL/6J mice and human plasma samples. Approximately 29% of psilocin was metabolized by HLM, while recombinant CYP2D6 and CYP3A4 enzymes metabolized nearly 100% and 40% of psilocin, respectively. Notably, 4-HIAA and 4-hydroxytryptophol (4-HTP) were detected with HLM but not with recombinant CYPs. MAO-A transformed psilocin into minimal amounts of 4-HIAA and 4-HTP. 4-HTP was only present in vitro. Neither 4-HIAA nor 4-HTP showed relevant interactions at assessed 5-HT receptors. In contrast to in vivo data, UGT1A10 did not extensively metabolize psilocin in vitro. Furthermore, two putative metabolites were observed. N-methyl-4-hydroxytryptamine (norpsilocin) was identified in vitro (CYP2D6) and in mice, while an oxidized metabolite was detected in vitro (CYP2D6) and in humans. However, the CYP2D6 genotype did not influence psilocin plasma concentrations in the investigated study population. In conclusion, MAO-A, CYP2D6, and CYP3A4 are involved in psilocin's metabolism. The discovery of putative norpsilocin in mice and oxidized psilocin in humans further unravels psilocin's metabolism. Despite limitations in replicating phase II metabolism in vitro, these findings hold significance for studying drug-drug interactions and advancing research on psilocybin as a therapeutic agent.

Pharmacokinetics and Pharmacodynamics of Oral Psilocybin Administration in Healthy Participants.

Psilocybin is being investigated as a potential treatment for psychiatric and neurological disorders. Only a few studies have evaluated the pharmacokinetics (PKs) of psilocybin and have used body weight-adjusted dosing. Data on PKs and the PK-pharmacodynamic (PD) relationship of fixed doses that are commonly used are unavailable. The present study characterized the PKs and PK-PD relationship of 15, 25, and 30 mg of orally administered psilocybin in 28, 23, and 28 healthy subjects, respectively. Plasma levels of unconjugated psilocin (the psychoactive metabolite of psilocybin) and corresponding subjective effects were repeatedly assessed up to 24 hours. PK parameters were determined using compartmental modeling. Concentration-subjective effect relationships were described using PK-PD modeling. Mean (95% confidence interval) maximal psilocin concentrations were 11 ng/mL (10-13), 17 ng/mL (16-19), and 21 ng/mL (19-24) after the administration of 15, 25, and 30 mg psilocybin, respectively. Maximal concentrations were reached after an average of 2 hours. Elimination half-lives were 1.8 hours (1.7-2.0), 1.4 hours (1.2-1.7), and 1.8 hours (1.6-1.9) for 15, 25, and 30 mg psilocybin, respectively. Mean (± SD) durations of subjective effects were 5.6 ± 2.2 hours, 5.5 ± 1.6 hours, and 6.4 ± 2.2 hours, and maximal effects ("any drug" effects) were 58% ± 25%, 73% ± 27%, and 80% ± 18% after 15, 25, and 30 mg psilocybin, respectively. Psilocin exhibited dose-proportional PKs. The duration and intensity of subjective effects were dose-dependent. Body weight did not influence pharmacokinetics or the response to psilocybin. These data may serve as a reference for future clinical trials.

Liquid chromatography-tandem mass spectrometry method for the bioanalysis of N,N-dimethyltryptamine (DMT) and its metabolites DMT-N-oxide and indole-3-acetic acid in human plasma.

The indole alkaloid N,N-dimethyltryptamine (DMT) induces psychedelic effects in humans. In addition to ceremonial and recreational use, DMT is subject to clinical investigations. Sensitive bioanalytical methods are required to assess the pharmacokinetics of DMT and its metabolites in human plasma. Here, a high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of DMT and its major metabolites indole-3-acetic acid (IAA) and DMT-N-oxide (DMT-NO) was developed and validated. As IAA is an endogenous component of human plasma, 13C6-IAA was used to determine IAA concentrations. After simple protein precipitation with methanol, analytes were separated on a pentafluorophenyl column. A gradient consisting of 0.1% (v/v) formic acid in a methanol-water mixture was applied for analyte separation. The analytes were detected by positive electrospray ionization followed by multiple reaction monitoring. The calibration range of the assay was 0.25-250 ng/mL for DMT, 0.1-100 ng/mL for DMT-NO, and 25-25,000 ng/mL for 13C6-IAA. The intra- and inter-assay accuracy was 93-113% for all analytes at all quality control levels, with coefficient of variation ≤ 11%. All analytes were stable under storage conditions relevant for the analysis of large batches of study samples. The validated method was capable of assessing pharmacokinetic (PK) parameters of DMT and its metabolites in study participants intravenously perfused with 1 mg/min DMT for 90 min. Overall, the developed method is easy-to-use, has a short run time, and qualifies for PK and metabolism studies of DMT in clinical settings.

Pharmacological characterization of 3,4-methylenedioxyamphetamine (MDA) analogs and two amphetamine-based compounds: N,α-DEPEA and DPIA.

3,4-methylenedioxyamphetamine (MDA) is a psychoactive compound chemically related to the entactogen MDMA. MDA shares some of the entactogenic effects of MDMA but also exerts stimulant effects and psychedelic properties at higher doses. Here, we examined the pharmacological properties of MDA analogs and related amphetamine-based compounds detected in street drug samples or in sport supplements. We examined the key pharmacological mechanisms including monoamine uptake inhibition and release using human embryonic kidney 293 cells stably transfected with the respective human transporters. Additionally, we assessed monoamine transporter and receptor binding and activation properties. MDA, its fluorinated analogs, as well as the α-ethyl containing BDB and the dimeric amphetamine DPIA inhibited NET with the greatest potency and preferentially inhibited 5-HT vs. dopamine uptake. The ß­methoxy MDA analog 3C-BOH and the amphetamine-based N,α-DEPEA inhibited NET and preferentially inhibited dopamine vs. 5-HT uptake. The test drugs mediated efflux of at least one monoamine with the exception of DPIA. Most compounds bound to 5-HT2A and 5-HT2C receptors (Ki ≤ 10 µM) and several substances activated the 5-HT2A and 5-HT2B receptor as partial or full agonists. Furthermore, several compounds interacted with adrenergic receptors and the trace amine-associated receptor 1 (TAAR1) in the micromolar range. The pharmacological profiles of some fluorinated and nonfluorinated MDA analogs resemble the profile of MDMA. In contrast, 3C-BOH and N,α-DEPEA displayed more pronounced dopaminergic activity similar to amphetamine. Pharmacokinetics and pharmacodynamics studies are necessary to better establish the risks and therapeutic potential of the tested drugs.

Direct comparison of the acute effects of lysergic acid diethylamide and psilocybin in a double-blind placebo-controlled study in healthy subjects.

Growing interest has been seen in using lysergic acid diethylamide (LSD) and psilocybin in psychiatric research and therapy. However, no modern studies have evaluated differences in subjective and autonomic effects of LSD and psilocybin or their similarities and dose equivalence. We used a double-blind, randomized, placebo-controlled, crossover design in 28 healthy subjects (14 women, 14 men) who underwent five 25 h sessions and received placebo, LSD (100 and 200 µg), and psilocybin (15 and 30 mg). Test days were separated by at least 10 days. Outcome measures included self-rating scales for subjective effects, autonomic effects, adverse effects, effect durations, plasma levels of brain-derived neurotrophic factor (BDNF), prolactin, cortisol, and oxytocin, and pharmacokinetics. The doses of 100 and 200 µg LSD and 30 mg psilocybin produced comparable subjective effects. The 15 mg psilocybin dose produced clearly weaker subjective effects compared with both doses of LSD and 30 mg psilocybin. The 200 µg dose of LSD induced higher ratings of ego-dissolution, impairments in control and cognition, and anxiety than the 100 µg dose. The 200 µg dose of LSD increased only ratings of ineffability significantly more than 30 mg psilocybin. LSD at both doses had clearly longer effect durations than psilocybin. Psilocybin increased blood pressure more than LSD, whereas LSD increased heart rate more than psilocybin. However, both LSD and psilocybin showed comparable cardiostimulant properties, assessed by the rate-pressure product. Both LSD and psilocybin had dose-proportional pharmacokinetics and first-order elimination. Both doses of LSD and the high dose of psilocybin produced qualitatively and quantitatively very similar subjective effects, indicating that alterations of mind that are induced by LSD and psilocybin do not differ beyond the effect duration. Any differences between LSD and psilocybin are dose-dependent rather than substance-dependent. However, LSD and psilocybin differentially increased heart rate and blood pressure. These results may assist with dose finding for future psychedelic research.Trial registration: ClinicalTrials.gov identifier: NCT03604744.

Acute Effects of Psilocybin After Escitalopram or Placebo Pretreatment in a Randomized, Double-Blind, Placebo-Controlled, Crossover Study in Healthy Subjects.

The psychedelic psilocybin is being investigated for the treatment of depression and anxiety. Unclear is whether antidepressant treatments interact with psilocybin. The present study used a double-blind, placebo-controlled, crossover design with two experimental test sessions to investigate the response to psilocybin (25 mg) in healthy subjects after pretreatment with escitalopram or placebo. The treatment order was random and counterbalanced. Pretreatment consisted of 10 mg escitalopram daily for 7 days, followed by 20 mg daily for 7 days, including the day of psilocybin administration, or 14 days of placebo pretreatment before psilocybin administration. Psilocybin treatments were separated by at least 16 days. The outcome measures included self-rating scales that evaluated subjective effects, autonomic effects, adverse effects, plasma brain-derived neurotrophic factor (BDNF) levels, electrocardiogram QTc time, whole-blood HTR2A and SCL6A4 gene expression, and pharmacokinetics. Escitalopram pretreatment had no relevant effect on positive mood effects of psilocybin but significantly reduced bad drug effects, anxiety, adverse cardiovascular effects, and other adverse effects of psilocybin compared with placebo pretreatment. Escitalopram did not alter the pharmacokinetics of psilocin. The half-life of psychoactive free (unconjugated) psilocin was 1.8 hours (range 1.1-2.2 hours), consistent with the short duration of action of psilocybin. Escitalopram did not alter HTR2A or SCL6A4 gene expression before psilocybin administration, QTc intervals, or circulating BDNF levels before or after psilocybin administration. Further studies are needed with a longer antidepressant pretreatment time and patients with psychiatric disorders to further define interactions between antidepressants and psilocybin.

The Pharmacological Profile of Second Generation Pyrovalerone Cathinones and Related Cathinone Derivative.

Pyrovalerone cathinones are potent psychoactive substances that possess a pyrrolidine moiety. Pyrovalerone-type novel psychoactive substances (NPS) are continuously detected but their pharmacology and toxicology are largely unknown. We assessed several pyrovalerone and related cathinone derivatives at the human norepinephrine (NET), dopamine (DAT), and serotonin (SERT) uptake transporters using HEK293 cells overexpressing each respective transporter. We examined the transporter-mediated monoamine efflux in preloaded cells. The receptor binding and activation potency was also assessed at the 5-HT1A, 5-HT2A, 5-HT2B, and 5-HT2C receptors. All pyrovalerone cathinones were potent DAT (IC50 = 0.02-8.7 µM) and NET inhibitors (IC50 = 0.03-4.6 µM), and exhibited no SERT activity at concentrations < 10 µM. None of the compounds induced monoamine efflux. NEH was a potent DAT/NET inhibitor (IC50 = 0.17-0.18 µM). 4F-PBP and NEH exhibited a high selectivity for the DAT (DAT/SERT ratio = 264-356). Extension of the alkyl chain enhanced NET and DAT inhibition potency, while presence of a 3,4-methylenedioxy moiety increased SERT inhibition potency. Most compounds did not exhibit any relevant activity at other monoamine receptors. In conclusion, 4F-PBP and NEH were selective DAT/NET inhibitors indicating that these substances likely produce strong psychostimulant effects and have a high abuse liability.

Development and validation of an LC-MS/MS method for the bioanalysis of psilocybin's main metabolites, psilocin and 4-hydroxyindole-3-acetic acid, in human plasma.

Psilocin is the active metabolite of psilocybin, a serotonergic psychedelic substance. It is used recreationally and investigated in substance-assisted psychotherapy. The pharmacokinetic properties of psilocin are only partially characterized. Therefore, we developed and validated a rapid LC-MS/MS method to quantify psilocin and its metabolite 4-hydroxyindole-3-acetic acid (4-HIAA) in human plasma. Plasma samples were processed by protein precipitation using methanol. The injected sample was mixed with water in front of a C18 analytical column to increase retention of the analytes. Psilocin and 4-HIAA were detected by multiple reaction monitoring (MRM) in positive and negative electrospray ionisation mode, respectively. An inter-assay accuracy of 100-109% and precision of ≤8.7% was recorded over three validation runs. The recovery was near to complete (≥94.7%) and importantly, consistent over different concentration levels and plasma batches (CV%: ≤4.1%). The plasma matrix caused negligible ion suppression and endogenous interferences could be separated from the analytes. Psilocin and 4-HIAA plasma samples could be thawed and re-frozen for three cycles, kept at room temperature for 8 h or 1 month at -20 °C without showing degradation (≤10%). The linear range (R ≥ 0.998) of the method covered plasma concentrations observed in humans following a common therapeutic oral dose of 25 mg psilocybin and was therefore able to assess the pharmacokinetics of psilocin and 4-HIAA. The LC-MS/MS method was convenient and reliable for measuring psilocin and 4-HIAA in plasma and will facilitate the clinical development of psilocybin.

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-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.

Receptor Interaction Profiles of 4-Alkoxy-Substituted 2,5-Dimethoxyphenethylamines and Related Amphetamines.

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.

Metabolites of the ring-substituted stimulants MDMA, methylone and MDPV differentially affect human monoaminergic systems.

BACKGROUND: Amphetamine analogs with a 3,4-methylenedioxy ring-substitution are among the most popular illicit drugs of abuse, exerting stimulant and entactogenic effects. Enzymatic N-demethylation or opening of the 3,4-methylenedioxy ring via O-demethylenation gives rise to metabolites that may be pharmacologically active. Indeed, previous studies in rats show that specific metabolites of 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxymethcathinone (methylone) and 3,4-methylenedioxypyrovalerone (MDPV) can interact with monoaminergic systems. AIM: Interactions of metabolites of MDMA, methylone and MDPV with human monoaminergic systems were assessed. METHODS: The ability of parent drugs and their metabolites to inhibit uptake of tritiated norepinephrine, dopamine and serotonin (5-HT) was assessed in human embryonic kidney 293 cells transfected with human monoamine transporters. Binding affinities and functional activity at monoamine transporters and various receptor subtypes were also determined. RESULTS: MDMA and methylone displayed greater potency to inhibit norepinephrine uptake as compared to their effects on dopamine and 5-HT uptake. N-demethylation of MDMA failed to alter uptake inhibition profiles, whereas N-demethylation of methylone decreased overall transporter inhibition potencies. O-demethylenation of MDMA, methylone and MDPV resulted in catechol metabolites that maintained norepinephrine and dopamine uptake inhibition potencies, but markedly reduced activity at 5-HT uptake. O-methylation of the catechol metabolites significantly decreased norepinephrine uptake inhibition, resulting in metabolites lacking significant stimulant properties. CONCLUSIONS: Several metabolites of MDMA, methylone and MDPV interact with human transporters and receptors at pharmacologically relevant concentrations. In particular, N-demethylated metabolites of MDMA and methylone circulate in unconjugated form and could contribute to the in vivo activity of the parent compounds in human users.

Pharmacological profile of mephedrone analogs and related new psychoactive substances.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'