Identifying cytochrome P450s involved in oxidative metabolism of synthetic cannabinoid N‐(adamantan‐1‐yl)‐1‐(5‐fluoropentyl)‐1H‐indole‐3‐carboxamide (STS‐135)

Synthetic cannabinoids (SCBs), designer drugs marketed as legal alternatives to marijuana, act as ligands to cannabinoid receptors; however, they have increased binding affinity and potency, resulting in toxicity symptoms such as cardiovascular incidents, seizures, and potentially death. N‐(adamantan‐1‐yl)‐1‐(5‐fluoropentyl)‐1H‐indole‐3‐carboxamide (STS‐135) is a third generation SCB. When incubated with hepatocytes, it undergoes oxidation, hydrolysis, and glucuronidation, resulting in 29 metabolites, with monohydroxy STS‐135 (M25) and dihydroxy STS‐135 (M21) being the predominant metabolites. The enzymes responsible for this oxidative metabolism were unknown. Thus, the aim of this study was to identify the cytochrome P450 (P450s or CYPs) enzymes involved in the oxidative metabolism of STS‐135. In this study, STS‐135 was incubated with liver, intestinal, and brain microsomes and recombinant P450s to determine the enzymes involved in its metabolism. Metabolite quantification was carried out using ultra‐performance liquid chromatography. STS‐135 was extensively metabolized in HLMs and HIMs. Screening assays indicated CYP3A4 and CYP3A5 could be responsible for STS‐135’s oxidation. Through incubations with genotyped HLMs, CYP3A4 was identified as the primary oxidative enzyme. Interestingly, CYP2J2, a P450 isoform expressed in cardiovascular tissues, showed high activity towards the formation of M25 with a K (m) value of 11.4 μmol/L. Thus, it was concluded that STS‐135 was primarily metabolized by CYP3A4 but may have extrahepatic metabolic pathways as well. Upon exposure to STS‐135, individuals with low CYP3A4 activity could retain elevated blood concentration, resulting in toxicity. Additionally, CYP2J2 may aid in protecting against STS‐135‐induced cardiovascular toxicity.