Functional evaluation of metabolites of synthetic cannabinoid receptor agonists

Abstract

Synthetic cannabinoid receptor agonists (SCRAs) are commonly abused new psychoactive substances (NPS). The human body extensive metabolizes these SCRAs, to facilitate clearance and excretion. In certain cases, bio-activation of these compounds and/or generation of active metabolites has been reported, which might contribute to toxicological effects. Moreover, these metabolites have also already been detected as main ingredients in some “legal highs”, but their toxicological profile remains poorly characterized. The activity of seven common hydrolysis metabolites of fifteen SCRAs, featuring scaffolds based on valine or tert-leucine, was systematically investigated in direct comparison to their parent compound. An activity-based cannabinoid receptor 1 (CB1) bio-assay was used for activity profiling of SCRAs and their metabolites in a stable HEK293 T cell system. The recruitment of β-arrestin 2 to the activated CB1 (each fused to one part of a split Nanoluciferase) was provoked by adding the (putative) SCRAs. Luminescence of the functionally complemented luciferase was monitored by a 96-well plate-reader. The major hydrolysis metabolites of 5F-AB-PINACA, ADB-CHMICA, ADB-CHMINACA, ADB-FUBICA, and their methyl- and ethylester derivatives showed no detectable CB1 activation at concentrations up to 1 μM. On the other hand, metabolites of 5F-ADB-PINACA, AB-CHMINACA, ADB-FUBINACA did retain activity, although significantly reduced as compared to the parent compounds (EC 50 values > 150 nM). The present study provides critical and missing data related to the potential toxicological characteristics of common hydrolysis metabolites of 15 SCRAs featuring scaffolds based on L-valine and L- tert -leucine. Results indicate overall severely impaired activity of these metabolites at CB 1 . These data highlight that the hydrolysis metabolites of closely related SCRAs can possess markedly distinct pharmacological characteristics, ranging from no activity detected to EC 50 values around 150 nM. The bioassay can serve as a tool to determine the activity of SCRAs and their metabolites at CB1. The assay may allow a better insight into the complex interplay between SCRAs and their metabolites in intoxications involving SCRAs.