In vitro human hepatocyte incubation of 5F-MDMB-PINACA, 5F-MDMB-PICA, 5F-EMDB-PICA, 4F-MDMB-BICA, and 4F-MDMB-BINACA and cannabinoid receptor 1 activity profiling of major human metabolites

Since their first reported fatalities in 2016, fluorinated MDMB-group synthetic cannabinoid receptor agonists (SCRAs), 5F-MDMB-PINACA, 5F-MDMB-PICA, 5F-EDMB-PICA, 4F-MDMB-BICA, and 4F-MDMB-BINACA, have remained present on illegal drug markets. Detection of SCRAs in clinical and forensic casework relies on metabolite detection, yet confirmation using metabolite reference standards is often lacking. Moreover, limited knowledge is available regarding the metabolites’ activity at the cannabinoid 1 receptor (CB 1 ) and their potential contribution to cannabinoid pharmacology and toxicology. Therefore, this work aimed to confirm major metabolites of the five analogue SCRAs using in-house-synthesized reference standards and evaluate their activity at the CB 1 receptor. SCRAs (5 µM) were incubated for 3 h with human hepatocytes, and supernatants were analyzed by high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (HPLC-QToF-MS), and spectra were compared with reference standards. Parent and metabolite activity were measured using an in vitro recombinant cell AequoScreen ® assay with a concentration range of 0.0286 nM–60 µM. Sixteen of 20 assessed metabolites were confirmed, including major metabolites such as ester hydrolysis (EH), carboxylic acid formation, combinations with oxidative defluorination (OD). Ten major and rapidly formed metabolites, such as hydroxylated metabolites, OD, and EH, showed high to moderate CB 1 potency (EC 50 96.3 nM–8.07 µM), whereas metabolites with multiple biotransformations showed little to no activation. Together, this study confirms several major metabolites using reference standards and characterizes their CB 1 activity, improving confidence in SCRA detection and providing insight into metabolite contribution to cannabinoid-related activity.