Stereoselective Ketamine Metabolism by Genetic Variants of Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase

Abstract

Editor’s Perspective What We Already Know about This Topic What This Article Tells Us That Is New Background Human ketamine N-demethylation to norketamine in vitro at therapeutic concentrations is catalyzed predominantly by the cytochrome P4502B6 isoform (CYP2B6). The CYP2B6 gene is highly polymorphic. CYP2B6.6, the protein encoded by the common variant allele CYP2B6*6, exhibits diminished ketamine metabolism in vitro compared with wild-type CYP2B6.1. The gene for cytochrome P450 oxidoreductase (POR), an obligatory P450 coenzyme, is also polymorphic. This investigation evaluated ketamine metabolism by genetic variants of human CYP2B6 and POR. Methods CYP2B6 (and variants), POR (and variants), and cytochrome b5 (wild-type) were coexpressed in a cell system. All CYP2B6 variants were expressed with wild-type POR and b5. All POR variants were expressed with wild-type CYP2B6.1 and b5. Metabolism of R- and S-ketamine enantiomers, and racemic RS-ketamine to norketamine enantiomers, was determined using stereoselective high-pressure liquid chromatography–mass spectrometry. Michaelis–Menten kinetic parameters were determined. Results For ketamine enantiomers and racemate, metabolism (intrinsic clearance) was generally wild-type CYP2B6.1 > CYP2B6.4 > CYP2B6.26, CYP2B6.19, CYP2B6.17, CYP2B6.6 > CYP2B6.5, CYP2B6.7 > CYP2B6.9. CYP2B6.16 and CYP2B6.18 were essentially inactive. Activity of several CYP2B6 variants was less than half that of CYP2B6.1. CYP2B6.9 was 15 to 35% that of CYP2B6.1. The order of metabolism was wild-type POR.1 > POR.28, P228L > POR.5. CYP2B6 variants had more influence than POR variants on ketamine metabolism. Neither CYP2B6 nor POR variants affected the stereoselectivity of ketamine metabolism (S > R). Conclusions Genetic variants of CYP2B6 and P450 oxidoreductase have diminished ketamine N-demethylation activity, without affecting the stereoselectivity of metabolism. These results suggest candidate genetic polymorphisms of CYP2B6 and P450 oxidoreductase for clinical evaluation to assess consequences for ketamine pharmacokinetics, elimination, bioactivation, and therapeutic effects.

Stereoselective Bupropion Hydroxylation by Cytochrome P450 CYP2B6 and Cytochrome P450 Oxidoreductase Genetic Variants

“…CYP2B6 is an unusual P450 isoform, in that activity of variants is clearly substrate-dependent, and generalizations about catalytic consequence or clinical implications of CYP2B6 polymorphisms are not possible. CYP2B6.4 (785G>T, K262R) had greater activity in metabolism of methadone (Gadel et al, 2015), efavirenz , artemether (Honda et al, 2011), and selegiline (Watanabe et al, 2010), and decreased activity with cyclophosphamide (Ariyoshi et al, 2011;Raccor et al, 2012), ifosfamide (Zanger and Klein, 2013;Calinski et al, 2015), ketamine (Wang et al, 2018), and nicotine (Bloom et al, 2019). Bupropion metabolism in the present study, using the CYP2B6.4 expression system used to study many of these other substrates, was slightly (10%) but not substantially greater than CYP2B6.1.…”

Nicotine oxidation by genetic variants of CYP2B6 and in human brain microsomes

“…Ketamine incubations were performed with 80 μmol L −1 ketamine, and processed and analyzed as previously described In brief, the reactions were quenched with 20% trichloroacetic acid containing norketamine‐d4. The plate was centrifuged at 900 g for 5 minutes and the supernatant was subjected to solid phase extraction .…”

“…CYP2B6 variants, reference allele P450 oxidoreductase (POR), and reference allele cytochrome b 5 vectors were previously generated, and recombinant proteins previously expressed in insect cells, as described. 17,21 Haplotype selection was based on the known clinical significance and frequency of the polymorphism, and a desire to inform the mechanistic basis for the influence of these polymorphisms on metabolism. There is growing evidence that the influence of CYP2B6 polymorphisms varies between substrates, and may vary between enantiomers of those substrates.…”

“…CYP2B6 has also been reported to be expressed in human brain tissue, unlike CYP2A6, and it has therefore been proposed that genetic variation affecting the CYP2B6 enzyme might play a significant role in nicotine‐related phenotypes . CYP2B6 is highly polymorphic, and common haplotypes, especially CYP2B6*6 and *9 , containing 516G>T (Q172H, minor allele frequency 26% in European Americans, 37% in African Americans (http://evs.gs.washington.edu/EVS/)) are associated with clinically significantly altered pharmacokinetics for different substrates, relative to the reference allele . Because the effects of CYP2B6 polymorphisms on CYP2B6 activity is substrate‐specific, activities of different CYP2B6 isoforms cannot be assumed for untested substrates, such as nicotine.…”

“…13 CYP2B6 is highly polymorphic, and common haplotypes, especially CYP2B6*6 and *9, containing 516G>T (Q172H, minor allele frequency 26% in European Americans, 37% in African Americans (http://evs.gs.washing ton.edu/EVS/)) are associated with clinically significantly altered pharmacokinetics for different substrates, relative to the reference allele. [14][15][16][17][18][19] Because the effects of CYP2B6 polymorphisms on CYP2B6 activity is substrate-specific, activities of different CYP2B6 isoforms cannot be assumed for untested substrates, such as nicotine. Therefore, we have expressed polymorphic CYP2B6 enzymes and measured their nicotine metabolism activity in vitro.…”