Zhao_2025_Forensic.Toxicol__

PURPOSE: In recent years, analogues of delta(9)-tetrahydrocannabinol (delta(9)-THC) have been widely distributed in Japan via the internet. Hexahydrocannabinol (HHC), synthesized by reducing THC, was controlled as a designated substance under the Pharmaceutical and Medical Device Act in Japan in 2022. However, other semi-synthetic cannabinoids, such as acetyl derivatives of THC and HHC, appeared soon. Herein, we examined whether the enzymatic hydrolysis of acetylated forms of delta(9)-THC, delta(8)-THC 11-alpha-HHC, and 11-beta-HHC by human liver microsomes (HLM) occurs. METHODS: The hydrolysis reaction was accomplished with HLM. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to determine products. Recombinant enzymes carboxylesterase 1C (CES1c), carboxylesterase 2 (CES2), and carboxylesterase inhibitor bis-(4-nitrophenyl) phosphate (BNPP) were used to clarify the principal hydrolysis enzymes for acetylated cannabinoids. RESULTS: The acetylated form underwent hydrolysis with HLM time-dependently, with almost no acetylated product remaining after 60 min. Furthermore, results from LC-MS showed that only the deacetylated form was present after hydrolysis. Although hydrolysis did not occur when HLM was pre-incubated with the carboxylesterase inhibitor BNPP, it was observed when CES1c or CES2 was used for in vitro experiments. CONCLUSION: This is the first time that it is elucidated that delta(9)-THC-O, delta(8)-THC-O, 11-alpha-HHC-O, and 11-beta-HHC-O are enzymatically hydrolyzed with HLM to produce delta(9)-THC, delta(8)-THC, 11-alpha-HHC, and 11-beta-HHC, respectively. Our results also support that CES1c and CES2 were the main enzymes involved in the hydrolysis of the acetylated cannabinoids. This study provides scientific support for the metabolism of newly regulated acetylated cannabinoids to cause the parent compound in vivo.