Comprehensive investigation on synthetic cannabinoids: Metabolic behavior and potency testing, using 5F‐APP‐PICA and AMB‐FUBINACA as model compounds

Abstract

Synthetic cannabinoids (SCs) represented 45% of new psychoactive substances seizures in Europe (data from 2016). The consumption of SCs is an issue of concern due to their still unknown toxicity and effects on human health, the great variety of compounds synthetized, and the continuous modifications being made to their chemical structure to avoid regulatory issues. These compounds are extensively metabolized in the organism and often cannot be detected as the intact molecule in human urine. The monitoring of SCs in forensic samples must be performed by the analysis of their metabolites. In this work, a workflow for the comprehensive study of SC consumption is proposed and applied to 5F‐APP‐PICA (also known as PX 1 or SRF‐30) and AMB‐FUBINACA (also known as FUB‐AMB or MMB‐FUBINACA), based not only on the elucidation of their metabolites but also including functional data using the NanoLuc approach, previously published. Both cannabinoids were completely metabolized by human hepatocytes (12 and 8 metabolites were elucidated by high resolution mass spectrometry for 5F‐APP‐PICA and AMB‐FUBINACA, respectively) and therefore suitable consumption markers are proposed. The bioassays revealed that 5F‐APP‐PICA presented lower activity than AMB‐FUBINACA at CB1 and CB2 receptors, based on the half maximal effective concentration (EC50) and the maximum response (Emax). These results are in agreement with the different intoxication cases found in the literature for AMB‐FUBINACA.

Phase I metabolism of synthetic cannabinoid receptor agonist PX‐1 (5F‐APP‐PICA) via incubation with human liver microsomes and UHPLC–HRMS

“…A recent report by Fabregat‐Safont et al () validates the primary findings of the current study; the authors determined the in vitro metabolism of PX‐1 via pooled human hepatocyte incubation over 180 min; seven Phase I metabolites were proposed. The major biotransformations reported by Fabregat‐Safont et al were consistent with those delineated in this report, including the enzymatic defluorination and monohydroxylation of the side chain (M1), amide hydrolysis (M3), and carboxypentyl (M5) as some of the most relevant metabolic products.…”

“…In addition to these metabolite types, our findings include PX‐1‐specific metabolites (M8, M9, and M10) which allow laboratories to unequivocally determine PX‐1 consumption; these are reported for the first time in this study. A fluorine‐containing metabolite was identified by (Fabregat‐Safont et al (; M3); however, this metabolite is potentially shared with 5F‐MPP‐PICA (Figure ) as outlined below. M1–M4 reported in our report (non‐specific metabolites) were also identified by Cooman and Bell (); these were the only metabolites reported.…”

Sex‐dependent pharmacological profiles of the synthetic cannabinoid MMB‐Fubinaca

“…Both cannabinoids exhibit CB 1 receptor‐dependent agonist activity profile; however, MMB‐Fubinaca exerts about two orders of magnitude more potency than WIN55,512‐2 in CB 1 ‐receptor‐mediated G‐protein activation. The higher potency of MMB‐Fubinaca is in agreement with previous findings in cells using a variety of functional assays (including [ 35 S]GTPγS) 3,6,7,32 . Thus, we might also expect higher maximal effect and potency of MMB‐Fubinaca compared with THC at this functional level in native brain tissue.…”

Detection and characterization of the new synthetic cannabinoid APP‐BINACA in forensic casework

“…The metabolism of APP‐BINACA has not been previously reported. Theoretical metabolites of APP‐BINACA were formulated based on knowledge of previously reported metabolic pathways for similar synthetic cannabinoids . The resulting theoretical metabolites were converted to formulae (and exact mass) and added to a targeted screening program.…”