Systematic Toxicological and Pharmacological Assessment of Synthetic Cannabinoid Receptor Agonists and their Metabolites using In Vitro Techniques

Synthetic cannabinoid receptor agonists (SCRAs) are potent new psychoactive substances (NPS) that are a major public health problem, particularly amongst vulnerable prison and rough-sleeping populations. Although designed as mimics of Δ9-tetrahydrocannabinol (Δ9-THC), a partial agonist of the cannabinoid receptors, SCRAs are full agonists and activation of cannabinoid receptor type 1 (CB1) is associated with a number of adverse outcomes, including intoxications and death. There is a lack of toxicological and pharmacological data regarding these compounds, especially as new SCRAs continually emerge. This study investigated the physicochemical properties and pharmacokinetics (lipophilicity, plasma protein binding and metabolic stability) of numerous SCRAs, involving the development and validation of a reversed-phase high-performance liquid chromatography (RP-HPLC) method to determine lipophilicity. The metabolic profile of the newly emerged SCRA ADB-HEXINACA was elucidated using human hepatocyte (HHep) incubations and liquid chromatography coupled to high-resolution mass spectrometry (LC–HRMS). Structure-metabolism relationships were elucidated for 11 established or prophetic SCRAs incorporating 4-pentenyl tails and a range of head groups, using HHep incubations and LC–HRMS. This enabled biomarkers of their consumption to be predicted. The potency and efficacy of ADB-HEXINACA, several structural analogues and some of the known or expected key metabolites were determined using a CB1 activity assay. This study, using in vitro techniques, provides a better understanding of the likely in vivo behaviour of SCRAs, knowledge that can be applied to analytical methods and results interpretation in forensic toxicology casework.