Formation of the Major Circulating Metabolite of Cannabidiol by Non‐P450, Cytosolic Enzymes

Cannabidiol (CBD), a natural product derived from cannabis, was recently FDA‐approved to treat epilepsy in children. CBD is extensively metabolized by hepatic cytochrome P450 (P450) enzymes to form the pharmacologically active metabolite 7‐hydroxy‐cannabidiol (7‐OH‐CBD). This metabolite is further converted to the major circulating metabolite, 7‐carboxy‐cannabidiol (7‐COOH‐CBD), prior to hepatobiliary excretion. CBD is mainly eliminated in the form of 7‐COOH‐CBD and related hydroxylated derivatives, and the plasma area under the curve of 7‐COOH‐CBD is up to 40 times higher than that of the parent compound. While prior work has shown that 7‐OH‐CBD is mainly formed by CYP2C9 and CYP2C19, the enzymes involved in 7‐COOH‐CBD formation have not been clearly established. Preliminary data from in vitro experiments with hepatocytes and hepatic subcellular fractions suggests that cytosolic enzymes may contribute to formation of 7‐COOH‐CBD. The objective of this study was to define the roles of P450 versus non‐P450 enzymes in 7‐COOH‐CBD generation. We hypothesized that 7‐OH‐CBD may be metabolized through a multi‐step reaction involving an aldehyde intermediate prior to conversion to 7‐COOH‐CBD. Reaction phenotyping experiments were performed with 7‐OH‐CBD and human liver microsomes, cytosol, and S9 fraction in the presence of selective enzyme inhibitors and cofactors to determine their effects on 7‐COOH‐CBD formation. Results show that 7‐COOH‐CBD formation is largely NAD + ‐dependent, and the cytosolic enzyme aldehyde dehydrogenase can generate 7‐COOH‐CBD. In addition, reactions performed in the presence of the trapping agent methoxylamine show decreased 7‐COOH‐CBD formation in human liver S9 fraction compared to those lacking this agent, suggesting that a reactive aldehyde intermediate may be formed from 7‐OH‐CBD. These data fill a critical research gap in our understanding of cannabinoid metabolism and help identify potential safety risks and drug‐drug interactions for patients and consumers taking CBD.