In vitro pharmacological characterization of novel psychoactive substances and in vivo analysis of psilocin for clinical applications

In Chapter 2.1, the metabolites of three popular amphetamine analogues containing a 3,4-methylenedioxy ring (MDMA, methylone, and MDPV) were examined at the human monoamine uptake transporters and various human monoamine receptors, using transfected cell lines. The aim of the study was to determine the pharmacological interactions of the different metabolites compared to their parent compounds with the human monoaminergic system.
The N-demethylated metabolites of MDA and MDC exhibited similar inhibition profiles at the transporters as their parents with a potent inhibition at the NET (IC50 = 0.38 − 2.3 μM), and selectivity for the SERT (DAT/SERT ratio < 1). The overall inhibition profile of MDC exhibited a slight decrease in potency when compared to methylone, whereas for MDA it remained unchanged when compared to MDMA. Similar to MDMA, MDA also exhibit relevant binding affinity to the 5-HT2A receptor (Ki = 3.2 μM). The O-demethylenated metabolites of the three amphetamine analogues (e.g., HHMA and HHA) displayed a reduction in SERT inhibition potency, while maintaining their NET and DAT inhibition potency. The O-methylated metabolites (e.g., HMMA and HMA) however exhibited a significant attenuation of inhibition potency at the NET (IC50 = 1.7 − 30 μM). Taking all the findings together, several metabolites exhibited relevant interactions at the human monoamine transporters and receptors. The N-demethylated metabolites of methylone and MDMA were the most pharmacologically relevant metabolites as they are not inactivated in humans by conjugation with glucuronic acid. In conclusion, these metabolites may potentially contribute to the activity associated with their parent compounds in users.
In Chapter 2.2, numerous 4-alkyloxy-substituted 2,5-dimethoxyamphetamine and phenethylamine derivatives of TMA-2 were examined for their receptor binding and activation properties at the human monoamine receptors and transporters. The aim of the investigation was to determine the pharmacological profile of these derivatives specifically at the serotonergic 5-HT2A receptor to reveal their structure-activity relationship as well as to predict their potential psychedelic activity.
All derivatives exhibited moderate to high affinity to the 5-HT2A receptor (Ki = 8− 1700 nM), with a selectivity for the 5-HT1A vs. 5-HT2C receptors. Extension of the 4-alkoxy group enhanced the 5-HT2A and 5-HT2c receptor binding affinities, with mixed effects on 5-HT2A receptor activation potency (amount of drug need to elicit response) and efficacy (maximal drug effect). Presence of fluoro substituent at the 4-alkoxy group attenuated the 5-HT2A and 5-HT2c receptor binding affinities, however additional fluorination had the opposite effect at the receptors. The 4-allyl and 4-methallyl derivatives (e.g., 2C-O-16, MALM, 2C-O-3, and MMALM) exhibited the most promising profiles at the 5-HT2A receptor (highest affinities, activation potencies, and efficacies) and were predicted to produce psychedelic-like effects in humans.
In Chapter 4.1, a bioanalytical method to quantify psilocybin’s main metabolites, psilocin, 4-HIAA, and psilocin glucuronide in human plasma was developed and validated according to regulatory guidelines. The purpose of the project was to develop a method that was user-friendly, sensitive, and overall reliable. Moreover, the method was developed to use only small amounts of sample and to minimize the run time. The developed method was used in order to quantify samples from two ongoing clinical studies investigating the pharmacokinetics of psilocybin in healthy volunteers.
Psilocin and 4-HIAA were detected by multiple reaction monitoring in positive and negative ionisation modes, respectively. The method showed a linear relationship between concentration and signal intensity in the range observed in clinical study samples. The method was accurate and precise with an inter-assay accuracy (100 − 109%) and precision (≤8.9%) measured in three separate validation runs. A simple methanol protein precipitation extraction method was employed to process the plasma samples, with almost complete analyte recovery (≥94.7%). The matrix effect was consistent across several plasma batches and importantly, did not interfere with the analysis of the analytes. Both analytes showed little degradation (≤10%) after three thaw-freeze cycles, room temperature for 8 h and 1 month storage at -20 oC. Moreover, the conjugation of the analytes was examined using Escherichia coli β-glucuronidase. The clinical application of the method was examined by analysing samples from three healthy volunteers treated with an oral dose of 25 mg psilocybin. The maximal plasma concentration (Cmax) of psilocin and 4-HIAA was on average 19.2 ng/ml and 137 ng/ml, respectively. Time to achieve maximal plasma concentration (Tmax) was observed between 120 − 140 min post treatment. Psilocin underwent glucuronidation reaching maximal plasma concentrations of 78.3 ng/ml after 220 min, whereas 4-HIAA was not conjugated.
In conclusion, my PhD thesis furthered the pharmacological characterization of several popular NPS metabolites and potentially psychedelic novel derivatives of TMA-2 at the monoamine uptake transporters and receptors. The in vitro pharmacological knowledge that was gained can be used to understand the pharmacological and potential toxicological properties that may be associated with some of these compounds when ingested by recreational users. Moreover, the monitoring bodies can better control compounds which are associated with a higher risk of abuse (e.g., more dopaminergic). Furthermore, my PhD thesis also furthered the quantification of psilocybin’s metabolites in plasma by the development and validation of an optimized bioanalytical method. The method was used to investigate the dose-response relationship of psilocybin and was also used to investigate drug-drug interactions of escitalopram and psilocybin in healthy subjects. As a result, the method has furthered the knowledge of psilocybin’s pharmacokinetics in vivo.