In Vitro Metabolic Profile Elucidation of Synthetic Cannabinoid APP-CHMINACA (PX-3)

Indazole carboxamide synthetic cannabinoids remain the most prevalent subclass of new psychoactive substances (NPS) reported internationally. However, the metabolic and pharmacological properties of many of these compounds remain unknown. Elucidating these characteristics allows members of the clinical and forensic communities to identify causative agents in patient samples, as well as render conclusions regarding their toxic effects. This work presents a detailed report on the in vitro phase I metabolism of indazole carboxamide synthetic cannabinoid APP-CHMINACA (PX-3). Incubation of APP-CHMINACA with human liver microsomes, followed by analysis of extracts via high-resolution mass spectrometry, yielded 12 metabolites, encompassing 7 different metabolite classes. Characterization of the metabolites was achieved by evaluating the product ion spectra, accurate mass and chemical formula generated for each metabolite. The predominant biotransformations observed were hydrolysis of the distal amide group and hydroxylation of the cyclohexylmethyl (CHM) substituent. Nine metabolites were amide hydrolysis products, of which five were monohydroxylated, one dihydroxylated and two were ketone products. The metabolites in greatest abundance in the study were products of amide hydrolysis with no further biotransformation (M1), followed by amide hydrolysis with monohydroxylation (M2.1). Three APP-CHMINACA-specific metabolites were generated, all of which were hydroxylated on the CHM group; one mono-, di- and tri-hydroxylated metabolite each was produced, with dihydroxylation (M6) present in the greatest abundance. The authors propose that metabolites M1, M2.1 and M6 are the most appropriate markers to determine consumption of APP-CHMINACA. The methods used in the current study have broad applicability and have been used to determine the in vitro metabolic profiles of multiple synthetic cannabinoids and other classes of NPS. This research can be used to guide analytical scientists in method development, synthesis of reference material, pharmacological testing of proposed metabolites and prediction of metabolic processes of compounds yet to be studied.