Halogen Substitution Influences Ketamine Metabolism by Cytochrome P450 2B6: In Vitro and Computational Approaches

Ketamine is analgesic at anesthetic and subanesthetic doses, and it has been used recently to treat depression. Biotransformation mediates ketamine effects, influencing both systemic elimination and bioactivation. CYP2B6 is the major catalyst of hepatic ketamine N-demethylation and metabolism at clinically relevant concentrations. Numerous CYP2B6 substrates contain halogens. CYP2B6 readily forms halogen-protein (particularly Cl-π) bonds, which influence substrate selectivity and active site orientation. Ketamine is chlorinated, but little is known about the metabolism of halogenated analogs. This investigation evaluated halogen substitution effects on CYP2B6-catalyzed ketamine analogs N-demethylation in vitro and modeled interactions with CYP2B6 using various computational approaches. Ortho phenyl ring halogen substituent changes caused substantial (18-fold) differences in K_m, on the order of Br (bromoketamine, 10 μM) < Cl < F < H (deschloroketamine, 184 μM). In contrast, V_max varied minimally (83-103 pmol/min/pmol CYP). Thus, apparent substrate binding affinity was the major consequence of halogen substitution and the major determinant of N-demethylation. Docking poses of ketamine and analogs were similar, sharing a π-stack with F297. Libdock scores were deschloroketamine < bromoketamine < ketamine < fluoroketamine. A Bayesian log K_m model generated with Assay Central had a ROC of 0.86. The probability of activity at 15 μM for ketamine and analogs was predicted with this model. Deschloroketamine scores corresponded to the experimental K_m, but the model was unable to predict activity with fluoroketamine. The binding pocket of CYP2B6 also suggested a hydrophobic component to substrate docking, on the basis of a strong linear correlation ( R² = 0.92) between lipophilicity ( Alog P) and metabolism (log K_m) of ketamine and analogs. This property may be the simplest design criteria to use when considering similar compounds and CYP2B6 affinity.