Metabolism of MMB022 and identification of dihydrodiol formation in vitro using synthesised standards.

MMB022 (methyl 3-methyl-2-[1-(pent-4-en-1-yl)-1H-indole-3-carboxamido]butanoate) is a new synthetic cannabinoid with an alkene at the pentenyl side chain, a rare functional group for synthetic cannabinoids. Metabolite identification is an important step for detection of synthetic cannabinoids in humans, since they are generally extensively metabolised. The aims of the study were to tentatively identify in vitro phase I metabolites, to confirm major metabolites using synthesised metabolites, to examine metabolic pathways thoroughly, to study metabolic stability and to suggest metabolites appropriate for urine screening. MMB022 and its synthesised metabolites were incubated with human liver microsomes (HLM) and the supernatants were analysed by liquid chromatography-quadrupole time-of-flight mass spectrometry. Sixteen metabolites were identified, which were generated via dehydrogenation, dihydrodiol formation, ester hydrolysis, hydroxylation, and combinations thereof. A major biotransformation of the alkene at the pentenyl side chain was confirmed to be dihydrodiol formation. The major metabolites were ester hydrolysis (M15) and dihydrodiol (M8) metabolites, whereas the metabolite derived from the combination of ester hydrolysis and dihydrodiol (M5) was the 4th most abundant metabolite. The metabolic pathways were investigated using synthesised metabolites and revealed that M5 is an end product of the pathways, indicating that it might become a more abundant metabolite in vivo depending on the rate of metabolism in humans. The major pathway of MMB022 to M5 was determined to be via M8 formation. Intrinsic clearance of MMB022 was determined to be 296 mL/min/kg and t1/2 was 2.1 min indicating a low metabolic stability. M15, M8 and potentially M5 are suggested as suitable urinary targets.