The CYP2B6*6 allele significantly alters the N-demethylation of … · 2013. 4. 2. · bromide,...

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The CYP2B6*6 allele significantly alters the N-demethylation of ketamine

enantiomers in vitro.

Yibai Li, Janet K. Coller, Mark R. Hutchinson, Kathrin Klein, Ulrich M. Zanger,

Nathan J. Stanley, Andrew D. Abell, Andrew A. Somogyi.

Author affiliations: Discipline of Pharmacology, University of Adelaide, SA,

Australia (Y.L, J.K.C, A.A.S); Discipline of Physiology, The University of Adelaide,

SA, Australia (M.R.H); Dr. Margarete Fischer-Bosch Institute of Clinical

Pharmacology, Stuttgart, Germany and University of Tübingen, Tübingen, Germany

(K.K, U.M.Z); Department of Chemistry, University of Otago, Dunedin, New

Zealand (N.J.S); Discipline of Chemistry, The University of Adelaide, SA, Australia

(A.D.A) and Centre for Personalised Cancer Medicine, University of Adelaide, SA,

Australia (A.A.S)

DMD Fast Forward. Published on April 2, 2013 as doi:10.1124/dmd.113.051631

Copyright 2013 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on April 2, 2013 as DOI: 10.1124/dmd.113.051631

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Running title page

Running title: CYP2B6*6 and ketamine metabolism Corresponding author: Yibai Li,

• Address: Discipline of Pharmacology, School of Medical Sciences, Level 5,

Medical School North, The University of Adelaide, South Australia, Australia,

5005

• Telephone: 61 (08)83135985

• Fax: 61 (08)82240685

• Email: [email protected]

Number of text pages: 31

Number of tables: 2

Number of figures: 6

Number of references: 32

Number of words in the Abstract: 249

Number of words in the Introduction: 587

Number of words in the Discussion: 1393

Abbreviations:

CLint, intrinsic clearance; Cyt b5, cytochrome b5; HLMs, human liver microsomes;

HPLC, high-performance liquid chromatography; Km: Michaelis-Menten constant;

P450, cytochrome P450; SNP, single nucleotide polymorphism


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Abstract:

Ketamine is primarily metabolized to norketamine by hepatic cytochrome P450

(CYP) 2B6 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 Km value 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

Vmax and Km values for CYP2B6.1 were approximately 160% and 70% of those for

CYP2B6.6, respectively. 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

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Introduction: Ketamine [(RS)-2-(2-chlorophenyl)-2-(methylamino)cyclohexanone], is a non-

competitive N-methyl-D-aspartate receptor antagonist. It has been used as an

anesthetic agent and an adjuvant analgesic in sub-anesthetic doses to attenuate opioid

tolerance and opioid-induced hyperalgesia (White et al., 1982; Subramaniam et al.,

2004). Additionally, ketamine has displayed a rapid antidepressant effect and a

preventive effect on postoperative interleukin-6 inflammatory response in clinical

studies (Skolnick et al., 2009; Dale et al., 2012). However, these therapeutic

applications of ketamine are frequently restricted by considerable interindividual

variabilities in drug efficacy and undesired psychotomimetic effects (White et al.,

1982; Meyer et al., 2004).

In most countries, ketamine is marketed as a racemic mixture consisting of equal

amounts of (S)- and (R)-ketamine. (S)-Ketamine has an approximately 5-fold greater

NMDA affinity and 4-fold greater analgesic potency compared to (R)-ketamine, and

is associated with less psychotomimetic effects (White et al., 1980; Mathisen et al.,

1995; Ebert et al., 1997). Furthermore, the plasma clearance of (S)-ketamine is

approximately 22% faster in vivo (White et al., 1985). However, no definitive

evidence has been obtained from in vitro studies to support the stereoselective

difference in ketamine clearance that is seen in vivo (Yanagihara et al., 2001;

Portmann et al., 2010; Mossner et al., 2011).

Ketamine is predominantly metabolized by hepatic cytochrome P450 (CYP)-mediated

N-demethylation to norketamine, a weakly active metabolite that has approximately

one-third the anesthetic activity of its parent drug in rats (White et al., 1975). Several

studies using microsomes containing cDNA-expressed human CYP enzymes have

shown the involvement of CYP2B6, CYP2C9, CYP2C19 and CYP3A4 in ketamine


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N-demethylation. Among these CYP enzymes, CYP2B6 exhibited the highest

demethylation activity followed by CYP3A4 (Yanagihara et al., 2001; Hijazi and

Boulieu, 2002; Portmann et al., 2010). In human liver microsomes (HLMs),

Yanagihara et al. reported that CYP2B6 is the major enzyme responsible for (S)- and

(R)-ketamine N-demethylation (Yanagihara et al., 2001). However, this was not

confirmed by later studies, which identified that the primary contribution was from

CYP3A4 (Hijazi and Boulieu, 2002; Mossner et al., 2011).

CYP2B6 is responsible for the hepatic metabolism of several other clinically

important drugs including efavirenz, methadone, bupropion, propofol and

cyclophosphamide (Zanger et al., 2007; Turpeinen and Zanger, 2012). It exhibits

substantial interindividual variability with regard to both its catalytic activity and

level of expression, which can be partially explained by the genetic variability of the

highly polymorphic CYP2B6 gene. Among the currently described 30 alleles

(www.cypalleles.ki.se/cyp2b6.htm, accessed January 14th 2013), the CYP2B6*6 allele

is the most prevalent and clinically important variant. This variant is characterized as

a haplotype consisting of two linked nonsynonymous single nucleotide

polymorphisms (SNPs), c.516G>T (rs3745274) and c.785A>G (rs2279343), which

reduce the expression of functional enzyme (Hofmann et al., 2008). The CYP2B6*6

allele has previously been associated with approximately 2-fold greater plasma

concentration of efavirenz (Haas et al., 2004) and a 6-fold decrease in efavirenz 8-

hydroxylation by HLMs (Xu et al., 2012). Interestingly, the influence of the

CYP2B6*6 allele on in vitro drug metabolism appears to be substrate dependent with

bupropion and efavirenz being negatively affected while cyclophosphamide 4-

hydroxylation activity was generally found to be higher for the variant (Xie et al.,

2006; Ariyoshi et al., 2011; Xu et al., 2012). In contrast to these well investigated


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CYP2B6 substrates, nothing is known regarding the impact of the CYP2B6*6 allele

on ketamine N-demethylation.

The aim of this study was to a) evaluate the relative contribution of CYP2B6 and

CYP3A4 to the N-demethylation of (S)- and (R)-ketamine using HLMs and expressed

CYP2B6 and 3A4 isoforms and b) assess the impact of CYP2B6*6 allelic variant on

ketamine metabolism.

Materials and Methods

Materials

Chemicals

Boric acid, bromophenol blue, cyclopentylbromide, 2-chlorobenzonitrile, ethidium

bromide, dimethyl sulfoxide BioReagent, ethylenediaminetetraacetic acid, DL-isocitric

acid, isocitric dehydrogenase, (R,S)-ketamine hydrochloride, (S)-ketamine

hydrochloride, sodium acetate, sodium pyrophosphate, D-(-)-tartaric acid, thioTEPA

and trizma base were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia).

Bovine serum albumin (BSA), Thermolpol reaction buffer, Taq DNA polymerase,

BsrI, BsrBI, StyI and SspI restriction enzymes and corresponding reaction buffers

were purchased from New England Biolabs (distributed by Genesearch, Arundel,

QLD, Australia). Disodium hydrogen orthophosphate, dipotassium hydrogen

orthophosphate, hydrochloric acid, magnesium chloride hexahydrate, sodium

carbonate, sodium dihydrogen orthophosphate were obtained from Ajax Chemicals

(Auburn, NSW, Australia). NADP-disodium salt, potassium chloride and

triethylamine were purchased from Merck Pty. Ltd. (Kilsyth, VIC, Australia).

Oligonucleotide primers and pUC19/HpaII DNA molecular weight marker were

obtained from GeneWorks (Thebarton, SA, Australia). Acetonitrile, diethyl ether,


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hexane ethanol and sucrose were purchased from Chem-supply (Gillman, SA,

Australia). Agarose I was purchased by AMRESCO (distributed by Astral Scientific,

Gymea, NSW, Australia). Omnigel-sieve agarose was manufactured by Edwards

Instrument Co. (Narellan, NSW, Australia,). Ephedrine was obtained from Faulding

Co. Ltd. (Torrensville, SA, Australia). Butylated hydroxytoluene was from MP

Biochemicals (Irvine, CA, USA). Deoxyribonucleoside triphosphate was purchased

from Finnzymes (distributed by Genesearch). BigDye version3.0 sequencing reagents

were obtained from Applied Biosystems (Mulgrave, VIC, Australia). Maxwell® 16

tissue DNA purification kits were purchased from Promega Co. (Madison, WI, USA).

Preparation of (R)-norketamine, (S)-norketamine and (R)-ketamine

Racemic norketamine hydrochloride was prepared starting from 2-chlorobenzonitrile.

Treatment with cyclopentylmagnesium bromide in the presence of copper (I)

bromide, followed by hydrolysis, gave the cyclopentyl-(2-chlorophenyl) ketone

(Weiberth and Hall, 1987). This was brominated with N-bromosuccinimide followed

by treatment with liquid ammonia to form an imine intermediate. Thermal

rearrangement of the imine afforded (R,S)-norketamine (Figure 1). The structures of

these chemicals were confirmed by 1H and 13C NMR spectra using a Varian Gemini

(300 MHz) instrument. NMR spectra were recorded in CDCl3 solution using TMS (0

ppm) and CDCl3 (77.0 ppm) as internal standards for 1H and 13C, respectively. Optical

resolution of the enantiomers was accomplished via formation of the diastereomeric

tartrate salts (Hong and Davisson, 1982). (R)-Ketamine hydrochloride was separated

from (R,S)-ketamine hydrochloride via a modification of the published procedure for

the resolution of (S)-ketamine (Steiner et al., 2000).

Expressed proteins and monoclonal antibodies


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CYP2B6.1 and CYP2B6.6 protein variants were transiently expressed in COS-1 cells

using pCMV-derived expression vectors and microsomal fractions were prepared

according to a previously described method (Lang et al., 2004). Human CYP isoforms

(CYP3A4 and CYP2B6) coexpressed with P450 reductase and cytochrome b5 (Cyt b5)

in baculovirus-infected insect cells were commercially available from BD Gentest™

(North Ryde, NSW, Australia). Monoclonal antibodies inhibitory to human CYP3A4

and to human CYP2B6, and antibodies against human CYP3A kit (WB-MAB-3A)

and antibodies against human CYP2B6 kit (WB-2B6-PEP) for immunoblotting were

also obtained from BD Gentest™.

Liver samples

Ethical approval was obtained from the Committee on the Ethics of Human

Experimentation of the University of Adelaide and Human Ethics Committee of the

Royal Adelaide Hospital. Human liver samples were donated by 23 patients

undergoing partial hepatectomy for hepatic tumours. All liver samples were

genotyped for the major CYP2B6 alleles by assays described below and 11 liver

tissues were selected based on their CYP2B6*6 genotype. Characteristics of the 11

patients were as follows: 1) all were Caucasians; 2) ages ranged from 31-77 years; 3)

six of eleven were male; 4) four patients carrying CYP2B6*1/*1 genotype, four

carrying CYP2B6*1/*6 genotype and three carrying CYP2B6*6/*6 genotype; 5) nine

patients had normal clinical chemistry and hematology prior to surgery; one patient

had high concentrations of lactate dehydrogenase (3.4×upper limit of normal [ULN])

and transaminases (8×ULN), and one patient had high concentration of alanine

transaminase (13.6×ULN). All tissue samples were frozen in liquid nitrogen and

stored at � 80°C until used.

CYP2B6 genotyping


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Genomic DNA was isolated from liver tissues using Maxwell® 16 instrument with

Maxwell® 16 Tissue DNA purification kit according to the manufacturer’s protocol.

SNPs related to CYP2B6*5 (c.1459C>T, [rs3211371]), CYP2B6*6 (c.516G>T and

c.785A>G), CYP2B6*7 (c.516G>T, c.785A>G and c.1459C>T), CYP2B6*8

(c.415A>G, [rs12721655]) and CYP2B6*13 (c.415A>G, c.516G>T and c.785A>G)

allele were screened by previously described PCR – restriction fragment length

polymorphism (RFLP) assays (Lang et al., 2004; Nakajima et al., 2007). Genotypes of

random samples were confirmed by DNA sequencing (BigDye version 3.0).

Preparations of HLMs

HLMs were prepared by a previously described differential centrifugation method

(Zanger et al., 1988). Total protein content of the microsomes was quantified using a

bicinchoninic acid (BCA) colorimetric assay according to the manufacturer’s protocol.

Total P450 content of microsomes was measured using the carbon monoxide

difference spectrum assay (Omura and Sato, 1964). The CYP2B6 and CYP3A

contents were determined by quantitative Western blot analysis using WB-2B6-PEP

or WB-MAB-3A kits according to the manufacturer’s protocol. The dilutions of

primary (1°) and secondary (2°) antibodies for the detection of CYP3A and CYP2B6

in HLMs were 1° 1: 2000/2° 1:10000 and 1° 1: 1000/2° 1:1000, respectively.

Enzyme kinetics in HLMs

Norketamine formation from separate (S)- and (R)-ketamine in HLMs was carried out

as follows. All incubations were performed in duplicate at 37°C in a shaking water

bath. Incubation medium, in a final volume of 200 μl, consisted of 100 μg HLMs

protein, NADPH-regenerating system (1 mM NADP, 1 unit.ml-1 isocitrate

dehydrogenase, 5 mM DL-isocitric acid, 5 mM magnesium chloride) and 25 mM of


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disodium phosphate buffer (pH 7.4). After a 3 min preincubation, the reaction was

initiated by the addition of various concentrations of ketamine (10 concentrations,

final concentration range of 2.5 to 2000 μM of (S)- or (R)-ketamine) and terminated

by the addition of 100 μl of saturated sodium carbonate after 30 min incubation. The

mixture was then spiked with 15 μl of 50 μg/ml of ephedrine (internal standard).

Metabolites were extracted with 4 ml of 70:30 v/v hexane:ether for 20 min and back

extracted with 100 μl of 0.1 M hydrochloric acid after centrifugation at 2000×g for 10

min. The organic layer of the mixture was then aspirated and 50 μl of the residue was

injected for HPLC analysis. The NADPH-regenerating system was replaced by an

equal volume of disodium phosphate buffer in incubations with norketamine

standards. The (S)- and (R)-norketamine formations were linear with respect to

incubation time (up to 30 min), microsomal protein content (up to 200 μg) and

substrate concentration (2.5 to 2000 μM).

Enzyme kinetics in cDNA-expressed CYP enzymes and expressed CYP2B6 variants

Baculovirus-infected insect cell microsomes containing cDNA-expressed CYP2B6,

and CYP3A4 with coexpression of Cyt b5 (in a final concentration of 5 pmol) were

incubated in duplicate at five substrate concentrations for 30 min at 37°C. Each

cDNA-expressed enzyme was preincubated with NADPH-regenerating system and

disodium phosphate buffer for 3 min at 37°C before the addition of ketamine. The (S)-

and (R)-ketamine concentration ranges for the expressed CYP2B6 and CYP3A4 were

10 to 150 μM and 100 to 1500 μM, respectively. These ranges were determined

according to the average Km1 and Km2 values for the HLMs with CYP2B6*1/*1

genotype.


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N-Demethylations of (S)- and (R)-ketamine were also examined in CYP2B6.1 and

CYP2B6.6 expressing COS-1 cell microsomes. The incubation conditions were

identical to those described above.

Inhibition studies with chemical inhibitors

The effects of chemical inhibitors selective for CYP2B6 (thioTEPA, 25 μM) and

CYP3A (troleandomycin, 25 μM) on the formation of (S)- and (R)-norketamine from

both low (equivalent to respective Km1 values for each genotype group) and high

(equivalent to respective Km2 values for each genotype group) ketamine

concentrations were studied in the 11 HLMs. The effects of chemical inhibitors at

various final concentrations (5 to 100 μM) on norketamine formation were tested. The

final concentration of each inhibitor (25 μM) was selected to optimize selective

inhibition. Stock solutions of inhibitors were prepared in 0.1 M sodium phosphate

buffer (pH 7.4) with 5% methanol. All components of the incubation medium except

ketamine were preincubated with inhibitors or methanol control for 20 min at 37°C.

The reaction was then initiated by the addition of substrate and terminated after 30

min incubation. Norketamine formation was quantified by the HPLC assay as

described below.

Inhibition studies with monoclonal antibodies against CYPs

Monoclonal antibodies against CYP2B6 (MAB2B6) and CYP3A (MAB3A) were

used according to the manufacturer’s recommendation. Antibodies (50 μg/100 μg

HLMs protein) or inhibitor-free control were preincubated with an incubation medium

containing HLMs, NADPH-regenerating system, incubation buffer and 0.5 mM Tris

buffer (pH 7.5) on ice for 20 min. The reaction was initiated by the addition of (S)- or


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(R)-ketamine (identical final concentrations as assays with chemical inhibitors) and

terminated after 30 min.

HPLC conditions

Norketamine formation was quantified by a HPLC assay with UV detection that was

modified from a previously reported method (Chong et al., 2009). In brief, separation

of compounds was achieved in a 3 μm C8 reverse phase column (150×4.6 mm,

LUNA, Phenomenex, Torrance, CA, USA) using a mobile phase of 15% (v/v)

acetonitrile and 0.05% triethylamine in 20 mM dipotassium phosphate (pH 3) and the

flow rate was 0.8 ml/min. The column was protected by a guard cartridge system

packed with 4×3 mm C8 SecurityGuard cartridge (Phenomenex). Analytes were

detected at 210 nm. The peak area was calculated using Shimadzu Class-VP software

(version 6.12 SP2, Shimadzu). The retention times for ketamine, norketamine and

ephedrine were 11 min, 9 min and 4 min, respectively. Norketamine formation was

quantified with calibration curves consisting of eight standards of norketamine over

the concentration range 0.5 to 200 μM. Inter-assay and intra-assay variabilities were

determined by the analysis of duplicates of quality control norketamine samples (QC)

at three different concentrations: low (2.5 μM), medium (25 μM) and high (80 μM).

The inter-assay (n = 6) and intra-assay (n = 6) precision and inaccuracy of all QC

samples were less than 10%. The precision and inaccuracy for the limit of

quantification (n = 6) were below 10%.

Data analysis

Eadie-Hofstee plots were used for analysis of enzyme kinetic data. The kinetic

parameters (Km and Vmax) were estimated by one-enzyme Michaelis-Menten model

and an user-defined two-enzyme Michaelis-Menten model (ν =


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Vmax1[S]/(Km1+[S])+Vmax2[S]/(Km2+[S])) using GraphPad Prism 5 software (San

Diego, CA, USA). Goodness of fit of data was compared between the two models.

Intrinsic clearance (CLint) was calculated as Vmax/Km. Inhibition data were expressed

as a percentage of the corresponding controls. The differences in CYP protein content,

kinetic parameters and inhibitory effects on norketamine formation rate among

CYP2B6*6 genotypes were determined by Jonckheere-Terpstra test using SPSS

Statistics 19 (IBM, Armonk, NY, USA). Correlations between CYP2B6 or CYP3A4

content and maximal norketamine formation rate were assessed by Spearman’s rank

correlation. Stereoselective differences in kinetic parameters and inhibitory effects

were analyzed by 2-tailed Wilcoxon matched pairs signed rank test. Data are present

as median (range) (unless specified). The results were considered statistically

significant when P

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Non-linear Eadie-Hofstee plots indicated the participation of at least two enzymes in

the N-demethylation of (S)- and (R)-ketamine in HLMs (Figure 2). The two-enzyme

Michaelis-Menten model provided a much better fit to the data compared with the

single enzyme Michaelis-Menten model. The estimated values of kinetic parameters

for the high affinity/low capacity enzyme (Vmax1, Km1 and CLint1) and the low

affinity/high capacity enzyme (Vmax2, Km2 and CLint2) are listed in Table 1. The

variability in kinetic parameters amongst three CYP2B6 genotypes is shown in Table

1 and Figure 3. For (S)-ketamine, significant gene-dose effects in Km1 and CLint1

values among three genotype groups were identified (P = 0.03 and 0.008,

respectively). For (R)-ketamine, gene-dose effects in Km1, CLint1 and CLint2 values

were found (P = 0.008, 0.001 and 0.008, respectively).

The maximal formation rates (Vmax, pmol/min/mg protein) and intrinsic clearance

(CLint, ml/min/mg protein) of norketamine enantiomers by the high affinity/low

capacity enzyme were better correlated with the CYP2B6 content (Vmax: Spearman rs

= 0.7, P = 0.02 and rs = 0.56, P = 0.08 for (S)- and (R)-norketamine, respectively;

CLint: rs = 0.81, P = 0.004 and rs = 0.80, P = 0.005 for (S)- and (R)-norketamine,

respectively) compared with the CYP3A4 content (Vmax: rs = � 0.05, P = 0.90 and rs

= 0.33, P = 0.33 for (S)- and (R)-norketamine, respectively; CLint: rs = 0.19, P = 0.57

and rs = 0.37, P = 0.26 for (S)- and (R)-norketamine, respectively) (Figure 4 and

Figure 5). Conversely, the maximal norketamine formation rates and intrinsic

clearance by the low affinity/high capacity enzyme were better correlated with

CYP3A4 content (Vmax: rs = 0.55, P = 0.09 and rs = 0.39, P = 0.24 for (S)- and (R)-

norketamine, respectively; CLint: rs = 0.71, P = 0.02 and rs = 0.80, P = 0.005 for (S)-

and (R)-norketamine, respectively) but not with CYP2B6 content (Vmax: rs = 0.35, P =


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0.30 and rs = 0.00, P > 0.99 for (S)- and (R)-norketamine, respectively; CLint: rs = 0.20,

P = 0.56 and rs = � 0.04, P = 0.92 for (S)- and (R)-norketamine, respectively).

There were no significant differences between (S)- and (R)-ketamine N-demethylation

observed for any kinetic parameters (Wilcoxon signed ranks test, P > 0.07).

Enzyme kinetics in cDNA-expressed CYP enzymes and expressed CYP2B6 variants

Table 2 shows the kinetic parameters of metabolism of (S)- and (R)-ketamine to

norketamine by baculovirus-infected insect cell microsomes containing cDNA-

expressed CYP2B6 and CYP3A4 coexpression of Cyt b5 and by COS-1 cell expressed

CYP2B6.1 and CYP2B6.6 protein variants. The Km values for the expressed CYP2B6

were considerably lower than those for the expressed CYP3A4. The Vmax values were

not substantially different among these three isoforms.

The average Km value determined for (S)-ketamine N-demethylation by the insect cell

expressed CYP2B6 was significantly lower than that for (R)-ketamine (P = 0.03), and

a higher average CLint value for (S)-ketamine N-demethylation (P = 0.03), but not for

the COS-1 cell expressed CYP2B6.1 protein (P = 0.69). Differences in kinetic

parameters were not apparent for the two enantiomers of ketamine with other

expressed proteins (P > 0.05).

Inhibition study

Figure 6 shows the effects of CYP2B6 and CYP3A specific chemical inhibitors and

inhibitory monoclonal antibodies on the N-demethylation of ketamine at low and high

concentrations in HLMs carrying the three different CYP2B6 genotypes. At low

ketamine concentrations (equivalent to the corresponding average Km1 values for each

CYP2B6 genotype group in HLMs kinetic assay, 20 to 80 μM), the inhibitory effects

of thioTEPA (25 μM) on norketamine formation in HLMs with CYP2B6*1/*1


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genotype (67% and 62% for (S)- and (R)-norketamine, respectively) were

significantly greater than those in HLMs with CYP2B6*1/*6 (39% and 35% for (S)-

and (R)-norketamine, respectively) and CYP2B6*6/*6 genotypes (18% for both

enantiomers) (Jonckheere-Terpstra test, P = 0.007). Troleandomycin (25 μM)

significantly inhibited (S)- and (R)-norketamine formations only in HLMs with

CYP2B6*1/*6 genotype (P = 0.002 and 0.007 for (S)- and (R)-norketamine,

respectively), with median values of percentage of inhibition less than 6%. At high

ketamine concentrations (equivalent to the corresponding Km2 values, 300 to 850 μM),

troleandomycin significantly diminished (S)- and (R)-norketamine formation by 30%

to 46% (P < 0.0001), however significant gene-dose effect on the degree of inhibition

was not observed (Jonckheere-Terpstra test, P = 0.62). ThioTEPA had no effect on

norketamine formation at these substrate concentrations (P = 0.38 and 0.50 for (S)-

and (R)-norketamine, respectively).

MAB2B6 inhibited norketamine formation at low ketamine concentrations (P <

0.0001 for both enantiomers), with a higher median percentage inhibition in HLMs

with CYP2B6*1/*1 (57% and 46% for (S)- and (R)-norketamine, respectively)

compared to HLMs with CYP2B6*1/*6 (27% and 37% for (S)- and (R)-norketamine,

respectively) and CYP2B6*6/*6 genotypes (19% and 30% for (S)- and (R)-

norketamine, respectively). MAB2B6 did not cause inhibition at high ketamine

concentrations (P = 0.77 and 0.41 for (S)- and (R)-norketamine, respectively).

MAB3A4 inhibited norketamine formations by 30-50% at high substrate

concentrations (P < 0.005) but it had no effect at low substrate concentrations. A

significant gene-dose effect was not observed with MAB3A4 inhibition (P = 0.65 and

0.39 for (S)- and (R)-norketamine, respectively).


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None of the four inhibitors exhibited stereoselective preference on norketamine

formation at either substrate concentration.

Discussion

The present study provides the first evidence that the CYP2B6*6 genetic variant has a

major impact on the N-demethylation of ketamine to norketamine in vitro, which is

likely due to impairment in both enzyme-substrate binding and catalytic activity. The

estimated Km values for (S)- and (R)-norketamine formation by the high affinity/low

capacity enzyme and the low affinity/high capacity enzyme in HLMs with

CYP2B6*1/*1 genotype were similar to those values for the insect cell expressed

CYP2B6 and CYP3A4, respectively. Hence the high affinity/low capacity enzyme

very likely corresponds to CYP2B6 and the low affinity/high capacity to CYP3A4.

This finding was also supported by the results of Spearman correlation analysis

between the values of Vmax or CLint and CYP protein expression. The Km values for

the high affinity/low capacity enzyme in HLMs with CYP2B6*1/*1 genotype were at

least 3-fold and 2.7-fold lower, respectively, than those for HLMs with CYP2B6*1/*6

and CYP2B6*6/*6 genotypes. Similarly, significantly lower Km values for the COS-1

cell expressed CYP2B6.1 protein compared with the CYP2B6.6 variant were

observed. These results suggest an influence of the CYP2B6*6 allele on enzyme-

ketamine binding. In addition to the increase in Km values, Vmax values for (S)- and

(R)-norketamine formation in CYP2B6.6 variant significantly decreased by 41% and

35%, respectively, compared to CYP2B6.1, despite the minor difference in CYP2B6

expression levels between the two proteins. Therefore, the genetic impact of the

CYP2B6*6 allele on Vmax values is likely attributed to an impairment in catalytic

activity rather than in enzyme expression level. In human liver microsomal assays,


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however, no gene-dose effect in Vmax values was observed. This may be due to the

low level of CYP2B6 as a percentage of total hepatic CYP content, and as these Vmax

values were normalized to total microsomal CYP content, they might not accurately

reflect the influence of the mutation in norketamine formation rate. As a consequence

of the reduction in both enzyme-substrate binding and catalytic activity, intrinsic

clearance rates of ketamine enantiomers by the high affinity/low capacity enzyme

were decreased by at least 62% in HLMs with CYP2B6*1/*6 genotype and 84% in

HLMs with CYP2B6*6/*6 genotype. This is consistent with the results for the COS-1

cell expressed CYP2B6 protein variants. Surprisingly, a significant gene-dose effect

of the CYP2B6*6 allele on the intrinsic clearance rates of (R)-ketamine by the low

affinity/high capacity enzyme was observed, which is possibly due to a small sample

size and large variability in CYP3A4 content between each HLMs. The impact of the

CYP2B6*6 allele on ketamine metabolism was further confirmed by inhibition assays,

where the inhibitory effect of thioTEPA and MAB2B6 on norketamine formation

from low ketamine concentrations substantially reduced in HLMs carrying

CYP2B6*1/*6 (by 40 to 50%) and CYP2B6*6/*6 genotypes (by 60 to 70%) compared

with HLMs with CYP2B6*1/*1 genotype.

The CYP2B6*6 allele has been previously associated with an increase in Km values

and decrease in Vmax values for efavirenz 8-hydroxylation and bupropion 4-

hydroxylation (Xu et al., 2012). In contrast, it has also been associated with decreased

Km values for cyclophosphamide 4-hydroxylation (Ariyoshi et al., 2011). The

substrate-dependent effects of the allele on enzyme-substrate binding and catalytic

activity suggest that this influence of the CYP2B6*6 allele is more complicated than a

decrease in enzyme expression. A previous report has shown that the c.516G>T

polymorphism, one of the two nonsynonymous SNPs of the allele, induced high levels


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of an alternative splicing variant that may be responsible for altered enzyme activity

(Hofmann et al., 2008). However, whether the distinct substrate-dependent genetic

impact is caused by the c.516G>T polymorphism as well as its molecular mechanism

needs to be further investigated.

Another major finding of the current study is that the human liver microsomal N-

demethylation of ketamine, at clinically relevant concentrations, is predominantly

mediated by CYP2B6. By comparison, CYP3A4 appears to be a dominant contributor

at much higher substrate concentrations. At low ketamine concentrations (20 to 80

μM) that are similar to the extrapolated peak hepatic concentration (approximately 50

μM) after an intravenous dose of 2 mg/kg of racemic ketamine for anesthesia (Hijazi

and Boulieu, 2002), substantial inhibition of (S)- and (R)-norketamine formation by

the CYP2B6 inhibitor thioTEPA and MAB2B6 but minor inhibition by

troleandomycin and MAB3A4 were found, suggesting a major participation of

CYP2B6 but not CYP3A4 in ketamine N-demethylation. The effects of CYP2B6

inhibitors on norketamine formation in HLMs with CYP2B6*1/*1 genotype were in

good agreement with previous investigations using pooled HLMs (Yanagihara et al.,

2001; Hijazi and Boulieu, 2002; Mossner et al., 2011). At high ketamine

concentrations (300 to 850 μM), significant inhibition by troleandomycin and

MAB3A4 but not by thioTEPA or MAB2B6, indicates that the predominant CYP

isoform responsible for ketamine N-demethylation at high concentrations is more

likely to be CYP3A4.

Previous studies using inhibition assays were inconsistent and inconclusive regarding

the relative contribution of CYP3A4 to ketamine N-demethylation at clinically

relevant concentrations in HLMs. This discrepancy may be due to the selectivity of

chemical inhibitors. In the two studies that described a major role of CYP3A4 in


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ketamine metabolism, the CYP3A4 inhibitor ketoconazole at 10 μM exhibited the

greatest inhibitory effects on norketamine formation from 25 μM or 50 μM of

ketamine (Hijazi and Boulieu, 2002; Mossner et al., 2011). In contrast, another

CYP3A4 inhibitor cyclosporin A and monoclonal antibodies against CYP3A4 failed

to produce any inhibition on norketamine formation (Yanagihara et al., 2001;

Mossner et al., 2011). This discrepancy may be a result of ketoconazole also being a

potent inhibitor of other CYP isoforms including CYP2B6, CYP2C9, CYP2C19 and

CYP2D6, with the half maximal inhibitory concentration and the inhibition constant

for CYP2B6 being 2.3 μM and 1.4 μM, respectively (Perloff et al., 2009). Thus its

effects on ketamine metabolism might be not only attributed to the inhibition of

CYP3A4. In the current study, thioTEPA and troleandomycin at 25 μM were used as

selective inhibitors of CYP2B6 and CYP3A4, respectively. ThioTEPA is a CYP2B6

chemical inhibitor with the highest selectivity, while troleandomycin has not been

reported to inhibit other CYP isoforms (Turpeinen and Zanger, 2012).

Differences in kinetic parameters for two enantiomers of ketamine were only

observed in the insect cell expressed CYP2B6 isoform with coexpression of Cyt b5

but not in HLMs or COS-1 cell expressed CYP2B6 enzyme. Accordingly, our results

provide little evidence supporting the stereoselectivity in ketamine clearance that has

been reported clinically. The Km value for (R)-ketamine metabolism was 1.3-fold

higher than that for (S)-ketamine leading to an approximately 27% decrease in the

CLint value for (R)-ketamine metabolism, which is similar to the data previously

reported by Portmann et al., who examined ketamine metabolism using the same

recombinant CYP2B6 system (Portmann et al., 2010). Interestingly, the COS-1 cell

expressed CYP2B6.1 protein did not exhibit stereoselectivity in ketamine N-

demethylation. This conflicting result is possibly the consequence of the Cyt b5-


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induced conformational changes on CYP2B6 protein. Although this effect on

CYP2B6 has not been reported, Cyt b5 has been associated with conformational

changes in CYP3A4 and CYP2C9 protein that can increase the collision between the

substrate and the active-oxygen species at the active site of enzyme (Perret and

Pompon, 1998; Locuson et al., 2007).

The present results are consistent with the finding of our preliminary clinical data, that

is, a significant reduction in the plasma norketamine/ketamine concentration ratio in

the CYP2B6*6 carriers (unpublished data). Although further follow-up work is

required to examine the influence of this variant of on ketamine metabolism in vivo,

our in vitro findings imply that genotyping of CYP2B6*6 allele may be useful in

estimating ketamine clearance rate and for predicting drug interactions that might be

attributed to CYP2B6 and CYP3A4 and hence may help to guide dosing decision and

establish a safer dosage regimen. As ketamine is a high hepatically cleared drug with

low (~25%) oral bioavailability, this proposal would be more relevant when ketamine

is given orally.

In conclusion, here we demonstrate that the most common allelic variant of CYP2B6

gene, CYP2B6*6, is associated with the decrease in both enzyme ketamine binding

and N-demethylation activity. To the best of our knowledge, this is the first report

showing the impact of a CYP2B6 genetic polymorphism on in vitro ketamine N-

demethylation. Our data also show that CYP2B6 but not CYP3A4 is the major

isoform responsible for the human liver microsomal ketamine N-demethylation at

clinically relevant concentrations. Nevertheless, the role CYP3A4 increases as

ketamine concentration rises. In addition, the ketamine metabolism to norketamine

mediated by CYP2B6 and CYP3A4 was not stereoselective.


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Acknowledgements:

The authors thank Mr. Joel Colvill (University of Adelaide) for assistance with HPLC assay, Dr. Daniel Barratt (University of Adelaide) for help in genotyping assays and Dr. Benjamin Lewis (Flinders University) for help in carbon monoxide difference spectrum assay.


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Authorship Contributions:

Participated in research design: Li, Coller, Hutchinson, Klein, Zanger and Somogyi Conducted experiments: Li Contributed new reagents or analytic tools: Coller, Klein, Zanger, Stanley, Abell and Somogyi Performed data analysis: Li, Coller and Somogyi Wrote or contributed to the writing of the manuscript: Li, Coller, Hutchinson, Klein, Zanger, Stanley, Abell and Somogyi


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Hijazi Y and Boulieu R (2002) Contribution of CYP3A4, CYP2B6, and CYP2C9 isoforms to N-demethylation of ketamine in human liver microsomes. Drug Metab Dispos 30:853-858.

Hofmann MH, Blievernicht JK, Klein K, Saussele T, Schaeffeler E, Schwab M, and Zanger UM (2008) Aberrant splicing caused by single nucleotide polymorphism c.516G>T [Q172H], a marker of CYP2B6*6, is responsible for decreased expression and activity of CYP2B6 in liver. J Pharmacol Exp Ther 325:284-292.

Hong SC and Davisson JN (1982) Stereochemical studies of demethylated ketamine enantiomers. J Pharm Sci 71:912-914.

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Locuson CW, Wienkers LC, Jones JP, and Tracy TS (2007) CYP2C9 Protein Interactions with Cytochrome b5: Effects on the Coupling of Catalysis. Drug Metabolism and Disposition 35:1174-1181.

Mathisen LC, Skjelbred P, Skoglund LA, and Oye I (1995) Effect of ketamine, an NMDA receptor inhibitor, in acute and chronic orofacial pain. Pain 61:215-220.

Meyer S, Aliani S, Graf N, and Gottschling S (2004) Inter- and intraindividual variability in ketamine dosage in repetitive invasive procedures in children with malignancies. Pediatr Hematol Oncol 21:161-166.

Mossner LD, Schmitz A, Theurillat R, Thormann W, and Mevissen M (2011) Inhibition of cytochrome P450 enzymes involved in ketamine metabolism by use of liver microsomes and specific cytochrome P450 enzymes from horses, dogs, and humans. Am J Vet Res 72:1505-1513.


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Nakajima M, Komagata S, Fujiki Y, Kanada Y, Ebi H, Itoh K, Mukai H, Yokoi T, and Minami H (2007) Genetic polymorphisms of CYP2B6 affect the pharmacokinetics/pharmacodynamics of cyclophosphamide in Japanese cancer patients. Pharmacogenet Genomics 17:431-445.

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Portmann S, Kwan HY, Theurillat R, Schmitz A, Mevissen M, and Thormann W (2010) Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro. J Chromatogr A 1217:7942-7948.

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Zanger UM, Klein K, Saussele T, Blievernicht J, Hofmann MH, and Schwab M (2007) Polymorphic CYP2B6: molecular mechanisms and emerging clinical significance. Pharmacogenomics 8:743-759.

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Footnotes

a) Financial support: This work was supported by University of Adelaide, School of

Medical Sciences funding, Adelaide Graduate Fee Scholarship; the Australian

Research Council Australian Research Fellowship [DP110100297]; FTT Fricker

Research Fellowship [Medical Endowment Funds, University of Adelaide]; the

Robert Bosch Foundation, Stuttgart, Germany.

b) Parts of this work were previously presented at the 19th International Symposium

on Microsomes and Drug Oxidations and 12th European ISSX Meeting (June 17–21,

2012, Noordwijk aan Zee, the Netherlands) and the Joint ASCEPT-APSA 2012

Conference (December 2–5, 2012, Sydney, Australia)

c) Corresponding author for reprint request: Yibai Li,

Address: Discipline of Pharmacology, School of Medical Sciences, Level 5, Medical

School North, The University of Adelaide, South Australia, Australia, 5005

Email: [email protected]


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Figure legends

Figure 1. Scheme of (R,S)-norketamine synthesis. Intermediates: 1) cyclopentyl-(2-

chlorophenyl) ketone; 2) α-Bromo-(2-chlorophenyl)cyclopentyl ketone; 3) 1-[(2-

chlorophenyl)iminomethyl]cyclopentanol; 4) (R,S)-norketamine hydrochloride.

Reaction conditions: a) Magnesium, iodine crystals, anhydrous ether, 1 h; b) 2-

chlorobenzonitrile, copper (I) bromide, anhydrous ether, 16 h; c) N-

bromosuccinimide, p-toluenesulfonic acid, dichloromethane, 6 h; d) liquid ammonia, -

78 °C, 18 h; e) isopropanol, reflux, 5 days.

Figure 2. Example of kinetics of (R)- and (S)-norketamine formation by HLM

(HLM#44, CYP2B6*1/*1). The data were best fitted using a modified two-enzyme

Michaelis-Menten model. Each value represents the mean ± S.D.

Figure 3. Influence of CYP2B6*6 allelic variant on kinetic parameters of (R)- and (S)-

ketamine N-demethylation in HLMs by the high affinity/low capacity enzyme

(A,B,C) and by the low affinity/high capacity enzyme (D,E,F). Line represents

median. Gene-dose effects between CYP2B6 genotype and values of kinetic

parameters were analyzed by Jonckheere-Terpstra test (P-values are provided under

figures)

Figure 4. Spearman’s rank correlation between the maximal formation rate of

norketamine enantiomers and CYP2B6 or CYP3A4 content (n = 11). A & B,

correlation between Vmax1 (maximal formation rate of (S)- and (R)-norketamine by the

high affinity/low capacity enzyme) and CYP content. C&D, correlation between


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Vmax2 (maximal formation rate of (S)- and (R)-norketamine by the low affinity/high

capacity enzyme) and CYP content.

Figure 5. Spearman’s rank correlation between intrinsic clearance values of

norketamine enantiomers and CYP2B6 or CYP3A4 content (n = 11). A & B,

correlation between CLint1 (intrinsic clearance values of (S)- and (R)-norketamine by

the high affinity/low capacity enzyme) and CYP content. C&D, correlation between

CLint2 (intrinsic clearance values of (S)- and (R)-norketamine by the low affinity/high

capacity enzyme) and CYP content.

Figure 6. Effect of CYP2B6 and CYP3A4 inhibitors on norketamine formation from

both low and high ketamine concentrations in HLMs with CYP2B6*1/*1 (n = 4),

CYP2B6*1/*6 (n = 4) and CYP2B6*6/*6 (n = 3) genotypes. Low substrate

concentrations were equivalent to the relative Km1 values, high substrate

concentrations were equivalent to the relative Km2 values. TAO, troleandomycin,

MAB-3A, monoclonal antibody against CYP3A, MAB-2B6, monoclonal antibody

against CYP2B6. Results represent the mean ± S.D. # significant inhibition P < 0.05,

** significant gene-dose effect on inhibitory effects among three genotype groups P <

0.01.


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Tables:

Table 1. Two-enzyme Michaelis-Menten kinetic parameters for norketamine formation from (S)- and (R)-ketamine by the high affinity/low

capacity enzyme (Vmax1, Km1 and CLint1) and the low affinity/high capacity enzyme (Vmax2, Km2 and CLint2) in 11 HLMs with CYP2B6*1/*1 (n =

4), *1/*6 (n = 4) and *6/*6 genotypes (n = 3). CLint = Vmax/Km, data are medians (range).

Vmax1 Km1 CLint1 Vmax2 Km2 CLint2

(pmol/min/pmol CYP) (μM) (ml/min/pmol CYP) (pmol/min/pmol CYP) (μM) (ml/min/pmol CYP)

(S)-ketamine

*1/*1 3.9 (2.1-12) 28 (12-43) 226 (68-281) 31 (11-43) 471 (375-1673) 52 (22-79)

*1/*6 7.4 (5.3-9.4) 86 (63-139) 84 (51-121) 16 (5.7-20) 374 (213-532) 36 (27-51)

*6/*6 2.6 (1.9-4.7) 78# (71-85) 37# (24-56) 15 (12-26) 934 (638-969) 19 (16-27)

(R)-ketamine

*1/*1 3.3 (2.3-8.1) 20 (6.9-42) 228 (108-333) 21 (13-71) 339 (230-1475) 64 (32-82)

*1/*6 6.3 (4.6-9.5) 69 (52-98) 98 (71-105) 19 (15-23) 612 (400-825) 36 (18-46)

*6/*6 2.0 (1.9-4.1) 74 (66-91) 26 (22-61) 5.7 (4.9-23) 840 (621-1046) 8# (5.5-27)

# significant gene-dose effects among the three genotype groups, Jonckheere-Terpstra test, p

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Table 2. Single-enzyme kinetic parameters for the N-demethylation of (S)- and (R)-ketamine in insect cell expressed CYP2B6 and CYP3A4

enzymes and COS-1 cell expressed CYP2B6 variants. CLint = Vmax/Km, data are medians (range).

Baculovirus-insect cell expressed CYP isoforms with coexpression of Cyt b5

(S)-ketamine (R)-ketamine

Vmax Km CLint Vmax Km CLint

(pmol/min/pmol CYP) (μM) (ml/min/pmol CYP) (pmol/min/pmol CYP) (μM) (ml/min/pmol CYP)

CYP2B6 88 (83-90) 51 (50-54) 1752 (1567-1797) 92 (68-101) 70† (51-78) 1278† (1248-1415)

CYP3A4 100 (99-132) 453 (324-949) 221 (139-305) 107 (105-111) 536 (447-772) 199 (144-234)

COS-1 cell expressed CYP2B6 variants

CYP2B6.1a 75 (68-86) 50 (38-63) 1466 (1374-1862) 81 (76-83) 53 (48-59) 1519 (1380-1592)

CYP2B6.6b 47∆ (45-49) 71∆ (64-79) 663∆ (626-711) 49∆ (46-50) 69∆ (66-76) 699∆ (647-751)

∆ significant difference compared to corresponding values for CYP2B6.1 group Mann-Whitney U test, p


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0 500 1000 1500 2000 25000

100

200

300

Ketamine conc. (µM)

Nor

keta

min

e fo

rmat

ion

rate

(pm

ol/m

in/p

mol

CY

P)(S)-ketamine (R)-ketamine

0.00 0.03 0.06 0.090

5

10

15

V/S

V

Eadie-Hofstee plot

Figure 2


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0

5

10

15V

max

1 (p

mol

/min

/pm

ol C

YP)

*1/*1 *1/*6 *6/*6(S)-ketamine, P = 0.51(R)-ketamine, P = 0.41

A

Figure 3

0

50

100

150

Km

1

(µM

)


B

0

100

200

300

400

CLin

t1(m

l/min

/pm

ol C

YP)


C

0

20

40

60

80

Vm

ax2

(pm

ol/m

in/p

mol

CY

P)


D

0

500

1000

1500

2000

Km

2

(µM

)*1/*1 *1/*6 *6/*6

(S)-ketamine, P = 0.41(R)-ketamine, P = 0.10

E

0

20

40

60

80

100

CLin

t2(m

l/min

/pm

ol C

YP)


F


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0.0 0.5 1.0 1.5 2.0 2.5 3.00

50

100

150

200

Vmax1 of (R)-norketamine formation (nmol/min/mg protein)

CYP

cont

ent

(pm

ol C

YP/

mg

prot

ein)

CYP2B6CYP3A4

r = 0.56, P = 0.06r = 0.33, P = 0.33

B

Figure 4

0 2 4 6 80

50

100

150

200

Vmax2 of (S)-norketamine formation (nmol/min/mg protein)

CYP

cont

ent

(pm

ol C

YP/

mg

prot

ein)

CYP2B6CYP3A4

r = 0.35, P = 0.30r = 0.55, P = 0.09

C

0 2 4 6 80

50

100

150

200

Vmax2 of (R)-norketamine formation (nmol/min/mg protein)

CYP

cont

ent

(pm

ol C

YP/

mg

prot

ein)

CYP2B6CYP3A4

r = 0.00, p > 0.99r = 0.39, p = 0.24

D

0.0 0.5 1.0 1.5 2.0 2.50

30

60

90

120

150

Vmax1 of (S)-norketamine formation (nmol/min/mg protein)

CYP

cont

ent

(pm

ol C

YP/

mg

prot

ein)

r = 0.70, P = 0.02r = -0.05, P = 0.90

ACYP3A4CYP2B6


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CLint1 of (S)-norketamine formation(ml/min/mg protein)

CYP

cont

ent

(pm

ol C

YP/

mg

prot

ein)

0 15 30 45 600

50

100

150

200

250CYP2B6CYP3A4

r = 0.81, P = 0.004r = 0.19, P = 0.57

A

Figure 5

CLint1 of (R)-norketamine formation(ml/min/mg protein)

CYP

cont

ent

(pm

ol C

YP/

mg

prot

ein)

0 50 100 150 2000

50

100

150

200

250CYP2B6CYP3A4

r = 0.80, P = 0.005r = 0.37, P = 0.26

B

CLint2 of (S)-norketamine formation(ml/min/mg protein)

CYP

cont

ent

(pm

ol C

YP/

mg

prot

ein)

0 5 10 15 200

50

100

150

200

250CYP2B6CYP3A4

r = 0.20, P = 0.56r = 0.71, P = 0.02

C

CLint2 of (R)-norketamine formation(ml/min/mg protein)

CYP

cont

ent

(pm

ol C

YP/

mg

prot

ein)

0.0 0.5 1.0 1.5 2.0 2.50

50

100

150

200

250CYP2B6CYP3A4

r = -0.04, P = 0.92r = 0.80, P = 0.005

D


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-20 0 20 40 60 80 100

% Inhibition


*1/*1

*1/*6

*6/*6

*1/*1

*1/*6

*6/*6

*1/*1

*1/*6

*6/*6

*1/*1

*1/*6

*6/*6

Low substrate concentrations

Thio-TEPA# (25 µM)

MAB2B6# (0.5 µg/1 µg

HLM)

Troleandomycin (25 µM)

MAB3A4 (0.5 µg/1 µg

HLM)

**

**

A

Figure 6-20 0 20 40 60 80 10

0

% Inhibition


High substrate concentrations*1/*1*1/*6

*6/*6

*1/*1

*1/*6

*6/*6

*1/*1

*1/*6

*6/*6

*1/*1

*1/*6

*6/*6

Thio-TEPA (25 µM)

MAB2B6 (0.5 µg/1 µg

HLM)

Troleandomycin# (25 µM)

MAB3A4# (0.5 µg/1 µg

HLM)

B


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The CYP2B6*6 allele significantly alters the N-demethylation of … · 2013. 4. 2. · bromide,...

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