title: The CYP2B6*6 allele significantly alters the N-demethylation of ketamine enantiomers in vitro.
abstract: Ketamine is primarily metabolized to norketamine by hepatic CYP2B6 and CYP3A4-mediated N-demethylation. However, the relative contribution from each enzyme remains controversial. The CYP2B6*6 allele is associated with reduced enzyme expression and activity that may lead to interindividual variability in ketamine metabolism. We examined the N-demethylation of individual ketamine enantiomers using human liver microsomes (HLMs) genotyped for the CYP2B6*6 allele, insect cell-expressed recombinant CYP2B6 and CYP3A4 enzymes, and COS-1 cell-expressed recombinant CYP2B6.1 and CYP2B6.6 protein variant. Effects of CYP-selective inhibitors on norketamine formation were also determined in HLMs. The two-enzyme Michaelis-Menten model best fitted the HLM kinetic data. The Michaelis-Menten constants (K(m)) for the high-affinity enzyme and the low-affinity enzyme were similar to those for the expressed CYP2B6 and CYP3A4, respectively. The intrinsic clearance for both ketamine enantiomers by the high-affinity enzyme in HLMs with CYP2B6*1/*1 genotype were at least 2-fold and 6-fold higher, respectively, than those for CYP2B6*1/*6 genotype and CYP2B6*6/*6 genotype. The V(max) and K(m) values for CYP2B6.1 were approximately 160 and 70% of those for CYP2B6.6, respectively. N,N'N'-triethylenethiophosphoramide (thioTEPA) (CYP2B6 inhibitor, 25 μM) and the monoclonal antibody against CYP2B6 but not troleandomycin (CYP3A4 inhibitor, 25 μM) or the monoclonal antibody against CYP3A4 inhibited ketamine N-demethylation at clinically relevant concentrations. The degree of inhibition was significantly reduced in HLMs with the CYP2B6*6 allele (gene-dose P < 0.05). These results indicate a major role of CYP2B6 in ketamine N-demethylation in vitro and a significant impact of the CYP2B6*6 allele on enzyme-ketamine binding and catalytic activity.
authors.affiliation: Discipline of Pharmacology, The University of Adelaide, South Australia, Australia. yibai.li@adelaide.edu.au
authors.forename: Yibai
authors.initials: Y
authors.lastname: Li
authors.forename: Janet K
authors.initials: JK
authors.lastname: Coller
authors.forename: Mark R
authors.initials: MR
authors.lastname: Hutchinson
authors.forename: Kathrin
authors.initials: K
authors.lastname: Klein
authors.forename: Ulrich M
authors.initials: UM
authors.lastname: Zanger
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Stanley
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Abell
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Somogyi
mesh_terms: D000328:Adult; D000368:Aged; D000483:Alleles; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000378:metabolism*; D019556:COS Cells; D002522:Chlorocebus aethiops; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D005260:Female; D006801:Humans; D007313:Insecta; D007649:Ketamine / Q000737:chemistry* / Q000378:metabolism*; D008297:Male; D008875:Middle Aged; D013237:Stereoisomerism
chemical_list: D007649:Ketamine; D001189:Aryl Hydrocarbon Hydroxylases; C585599:CYP2B6 protein, human; D065702:Cytochrome P-450 CYP2B6; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human