Role of Cytochrome P4502B6 Polymorphisms in Ketamine Metabolism and Clearance

Lesley K. Rao, M.D., Alicia M. Flaker, A.S., Christina C. Friedel, B.S., Evan D. Kharasch, M.D., Ph.D.

ABSTRACT

Background: At therapeutic concentrations, cytochrome P4502B6 (CYP2B6) is the major P450 isoform catalyzing hepatic ketamine N-demethylation to norketamine in vitro. The CYP2B6 gene is highly polymorphic. The most common variant allele, CYP2B6*6, is associated with diminished hepatic CYP2B6 expression and catalytic activity compared with wild-type CYP2B6*1/*1. CYP2B6.6, the protein encoded by the CYP2B6*6 allele, and liver microsomes from CYP2B6*6 carriers had

diminished ketamine metabolism in vitro. This investigation tested whether humans with the CYP2B6*6 allele would have Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/125/6/1103/487909/20161200_0-00014.pdf by guest on 27 September 2021 decreased clinical ketamine metabolism and clearance. Methods: Thirty volunteers with CYP2B6*1/*1, *1/*6, or *6/*6 genotypes (n = 10 each) received a subsedating dose of oral ketamine. Plasma and urine concentrations of ketamine and the major CYP2B6-dependent metabolites were determined by mass spectrometry. Subjects’ self-assessment of ketamine effects were also recorded. The primary outcome was ketamine N-demethylation, measured as the plasma norketamine/ketamine area under the curve ratio. Secondary outcomes included plasma ketamine enantiomer and metabolite area under the plasma concentration–time curve, maximum concentrations, apparent oral clearance, and metabolite formation clearances. Results: There was no significant difference between CYP2B6 genotypes in ketamine metabolism or any of the secondary outcome measures. Subjective self-assessment did reveal some differences in energy and level of awareness among subjects. Conclusions: These results show that while the CYP2B6*6 polymorphism results in diminished ketamine metabolism in vitro, this allelic variant did not affect single, low-dose ketamine metabolism, clearance, and pharmacokinetics in vivo. While in vitro drug metabolism studies may be informative, clinical investigations in general are needed to validate in vitro observations. (Anesthesiology 2016; 125:1103-12)

etamine was originally developed in 1964 as a What We Already Know about This Topic K “dissociative” anesthetic and was Food and Drug Admin- istration approved in 1970. Since that time, it has been widely • Ketamine is used in anesthesiology, chronic pain manage- used in anesthesia, in part, because unlike most other intra- ment, psychiatry, and emergency medicine • Cytochrome P4502B6 (CYP2B6) is the major P450 isoform venous anesthetics, it does not significantly depress the respi- catalyzing ketamine metabolism to norketamine and metabo- ratory and circulatory systems. In addition, ketamine causes lism overall significant antinociceptive and antihyperalgesic (i.e., analgesic) • In vitro, CYP2B6.6 (the protein encoded by the variant effects at low doses—lower than those that cause sedation and CYP2B6*6 allele) has diminished activity toward ketamine metabolism compared with wild-type CYP2B6.1 loss of consciousness. It can be administered by intravenous, intramuscular, oral, sublingual, intranasal, and rectal routes. What This Article Tells Us That Is New It has long been thought that ketamine primarily acts • Healthy volunteers with CYP2B6*1/*1, *1/*6, or *6/*6 geno- by antagonizing N-methyl-D-aspartate receptors; how- types received a single oral ketamine dose ever, more recently, some evidence suggests that inhibi- • There was no significant difference between CYP2B6 geno- tion of hyperpolarization-activated cyclic nucleotide-gated types in ketamine or norketamine plasma concentrations or potassium channel 1 channels may also contribute to drug ketamine metabolism effects.1 Oral ketamine has been evaluated extensively for use in chronic pain management.2 The use of ketamine in Thus, there is marked interest in better understanding the pediatric sedation, analgesia, and emergency room analgesia pharmacokinetics and pharmacodynamics, as well as other is also of interest.3–6 More recently, it has been discovered applications of this drug. that ketamine may be effective in the therapy of treatment- Ketamine is administered clinically as a racemic mixture resistant depression, and with very fast response rates.7–10 of R- and S-ketamine although the S-ketamine isomer alone

This article is featured in “This Month in Anesthesiology,” page 1A. Corresponding article on page 1085. Timothy J. Brennan, Ph.D., M.D., served as Handling Editor for this article. Submitted for publication February 10, 2016. Accepted for publication August 3, 2016. From the Division of Clinical and Transla- tional Research, Department of Anesthesiology (L.K.R., A.M.F., C.C.F., E.D.K.), and Department of Biochemistry and Biophysics (E.D.K.), Washington University in St. Louis, St. Louis, Missouri; and the Center for Clinical Pharmacology, St. Louis College of Pharmacy and Wash- ington University in St. Louis School of Medicine, St. Louis, Missouri (E.D.K.). Copyright © 2016, the American Society of Anesthesiologists, Inc. Wolters Kluwer Health, Inc. All Rights Reserved. Anesthesiology 2016; 125:1103-12

Anesthesiology, V 125 • No 6 1103 December 2016

Copyright © 2016, the American Society of Anesthesiologists, Inc. Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Role of CYP2B6 Polymorphisms in Ketamine Metabolism

is used in some countries outside the United States. First- several substrates, compared with wild-type CYP2B6*1/*1 pass metabolism of oral ketamine is considerable; thus, it is carriers. A recent in vitro investigation demonstrated only about 15% bioavailable.11 Ketamine undergoes exten- that CYP2B6.6 (the protein encoded by the CYP2B6*6 sive metabolism, primarily via N-demethylation to norket- allele) has diminished catalytic activity toward ketamine 12–15 14–18 amine and to several other metabolites (fig. 1 ). The N-demethylation compared with wild-type CYP2B6.1, primary metabolites, norketamine and hydroxyketamine, and liver microsomes from humans heterozygous or homo- are rapidly further metabolized to hydroxynorketamine and zygous for the CYP2B6*6 allele also had diminished cata- dehydronorketamine. There are minor stereoselective differ- lytic activity toward ketamine N-demethylation, compared ences in ketamine enantiomer metabolism and disposition. with CYP2B6*1/*1 genotypes.20 It has been suggested It has recently been established that cytochrome P4502B6 that CYP2B6*6 carriers have diminished clinical ketamine (CYP2B6) is the major isoform catalyzing both ketamine Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/125/6/1103/487909/20161200_0-00014.pdf by guest on 27 September 2021 N-demethylation.20 N-demethylation and ketamine metabolism overall in vitro Nonetheless, there is no formal evaluation of ketamine at therapeutic concentrations14,16,17,19,20 and clinically.21 A recent clinical investigation showed the predominant role pharmacokinetics, metabolism, or clearance in CYP2B6*6 of CYP2B6 and lack of significant CYP3A involvement in carriers. This investigation tested the hypothesis that ketamine pharmacokinetics and metabolism.21 CYP2B6 variants (CYP2B6*6 hetero- or homozygotes) in CYP2B6 is a highly polymorphic enzyme.22 The most vivo will have decreased ketamine metabolism and clearance common variant allele, CYP2B6*6, found mostly in Afri- and potentially greater clinical effects. Better understanding cans, African-Americans, and some Asian populations, is of interpatient variability in drug metabolism and clearance associated with both diminished hepatic CYP2B6 enzyme would potentially allow for more accurate dosing to achieve expression and diminished CYP2B6 catalytic activity toward clinical effectiveness and avoid side effects.

Fig. 1. Hepatic biotransformation of ketamine and responsible enzymes in humans at therapeutic concentrations. R- and S- ketamine enantiomers are N-demethylated to the major primary metabolite enantiomers R- and S-norketamine. A minor initial route of metabolism is 6-hydroxylation, yielding two pairs of diasteromers (2S,6S-, 2S,6R-, 2R,6R-, and 2R,6S-hydroxyketamine). The primary metabolite(s) R- and S-norketamine undergo further metabolism to the secondary metabolite enantiomers R- and S-dehydronorketamine. The primary metabolites may also undergo further N-demethylation or (4, 5, or 6)-hydroxylation to six pairs of diasteromeric hydroxynorketamine metabolites. In human liver microsomes, the major hydroxynorketamine formed from ketamine is 4-hydroxynorketamine, the major hydroxynorketamine from norketamine is 5-hydroxynorketamine, and the major hydroxynorketamine from hydroxyketamine is 6-hydroxynorketamine. After intravenous infusion of 0.5 mg/kg ketamine in humans, the major circulating metabolites were R- and S-norketamine, 2S,6S;2R,6R-hydroxynorketamine, 2S,5R;2R,5S- hydroxynorketamine, and R- and S-dehydronorketamine, with negligible concentrations of hydroxyketamine. Overall, cytochrome P450 (CYP)2B6 is the major isoform responsible for the metabolism of both R- and S-ketamine at therapeutic concentrations. Rates of S-ketamine metabolism are moderately greater than R-ketamine, but relative amounts of metabolites formed and responsible CYPs are not substantially different between enantiomers except where shown. Based on previous reports.14–18

Anesthesiology 2016; 125:1103-12 1104 Rao et al.

Materials and Methods with blood sampling, subjective self-assessment of drug effect was performed using a visual analog scale for levels Clinical Protocol of alertness/sedation (almost asleep to wide awake), energy This was a single-center, single-session, open-label study of level (no energy to full of energy), confusion (confused to oral ketamine pharmacokinetics. The Institutional Review clear headed), clumsiness (extremely clumsy to well coor- Board of Washington University in St. Louis (Missouri) dinated), anxiety (calm/relaxed to extremely nervous), approved the protocol, and the investigation was regis- and nausea (no nausea to worst nausea). Subjects used an tered (ClinicalTrials.gov identifier: NCT01988922). All unruled 100-mm slider to indicate their response. After subjects provided written informed consent. Subjects were the response was given, a numerical score was assigned recruited from the greater St. Louis community and had according to the location of the cursor on the ruler. Clini-

been previously genotyped for CYP2B6. Eligible subjects cally detectable ketamine effects were not expected given Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/125/6/1103/487909/20161200_0-00014.pdf by guest on 27 September 2021 were 18- to 50-year-old volunteers with CYP2B6*1/*1, the ketamine dose, and were recorded so that any effects CYP2B6*1/*6, or CYP2B6*6/*6 genotypes who were in detected in certain genetic variants could be followed up on good health without any remarkable medical conditions with subsequent studies. Two hours after dosing, subjects 2 and with a body mass index less than 33 kg/m (table 1). were free to move around and were given a standard meal. 23 Genotyping was done as described previously. Exclusion They had free access to food and water during the study criteria included known history of liver or kidney disease; session. Urine was continuously collected for 24 h after ket- use of prescription or nonprescription medications, herb- amine administration. als, foods, or chemicals known to be metabolized by or affecting CYP2B6; females who were pregnant or nurs- Analytical Methods ing; known history of alcohol or drug addiction; or having Ketamine, norketamine, and dehydronorketamine concentra- direct physical access to and routine handling of addicting tions in plasma and urine were determined by enantioselective drugs in the regular course of duty. Each subject filled out high-performance liquid chromatography (HPLC)–tandem a health self-assessment, including a structured interview mass spectrometry, using solid-phase extraction, based on a to screen for history of substance abuse or addiction. The modification of a published method.25 Ketamine (undeuter- investigation was a pilot study, and 30 subjects, 10 with ated, d0 and deuterated, d4), norketamine (undeuterated, each with of the genotypes CYP2B6*1/*1, CYP2B6*1/*6, d0 and deuterated, d4), and dehydronorketamine were from and CYP2B6*6/*6, were studied. Cerilliant (Round Rock, USA). Strata-X 33u (30 mg) solid- Subjects were required to abstain from (1) alcohol for phase extraction plates were from Phenomenex (USA). Other 48 h before and during study days; (2) caffeine-contain- reagents were from Sigma-Aldrich (USA). ing beverages the day of study drug administration; (3) To thawed subject plasma, calibration, or quality con- grapefruit, oranges, apples, or their juices for 5 days before trol samples (250 μl), an internal standard (1.25 ng RS- the study day; (4) food or liquids after midnight the day ketamine-d4 and 1.25 ng RS-norketamine-d4) and 0.75 ml before drug administration (to eliminate food influence 10 mM ammonium acetate in water were added (pH 9.5). on oral drug absorption); and (5) nonstudy medications Solid-phase extraction plates were conditioned with 1 ml (including over the counter and/or herbal medications) for methanol, water, and then 10 mM ammonium acetate in 3 days before the study visit, without previous principal water (pH 9.5). Plasma samples were loaded under soft investigator approval. (5 mmHg) vacuum at 0.5 ml/min and the plate was washed Subjects had a peripheral intravenous catheter inserted with water and then completely dried under high vacuum for blood sampling. They were orally administered 0.4 mg/ (10 to 15 mmHg for 2 to 5 min). Samples were eluted with kg of a parenteral formulation of racemic ketamine 0.5 ml methanol by gravity for 15 min and then under with 200 ml water (average dose, 31 ± 6 mg). This dose vacuum (10 to 15 mmHg) and then evaporated to dryness was intended to have a small detectable pharmacologic under nitrogen at 35°C. For analysis, samples were resus- effect, but be subsedating, and was based on other stud- pended in 200-μl mobile phase (10 mM ammonium acetate, ies in healthy volunteers.21,24 Venous blood samples were pH 7.6). Calibration samples contained 0, 0.1, 0.25, 0.5, 1, drawn before and 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, 2.5, 5, 10, 25, 50, 100, and 250 ng/ml racemic ketamine, 10, and 12 h after ketamine administration. Coincident norketamine, and dehydronorketamine (base). Quality

Table 1. Study Subject Demographics

CYP2B6 Genotype Sex (M:F) Age (yr) Weight (kg) Caucasian African-American Asian Other/Unknown

*1/*1 7:3 25 ± 6 77 ± 30 7 0 3 0 *1/*6 6:4 27 ± 11 75 ± 23 8 1 1 0 *6/*6 6:4 37 ± 13 78 ± 20 6 4 0 0

F = female; M = male.

Anesthesiology 2016; 125:1103-12 1105 Rao et al. Copyright © 2016, the American Society of Anesthesiologists, Inc. Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Role of CYP2B6 Polymorphisms in Ketamine Metabolism

control samples contained 0.5, 5, and 25 ng/ml racemic ket- curtain gas 20, gas 1 70, gas 2 10, collision gas medium. amine, norketamine, and dehydronorketamine (base). Calibration curves of peak area ratio versus analyte con- To thawed subject urine, calibration, or quality concentration were fit using linear least-squares analysis and trol samples (25 μl), an internal standard (1.25 ng RS- 1/x2 weighting. Analyte concentrations in patient samples ketamine-d4 and 1.25 ng RS-norketamine-d4) and 1,000 were quantified using calibration curves for ketamine and units β-glucuronidase in 100 μl 100 mM ammonium ace- norketamine (for norketamine, hydroxynorketamine, and tate (pH 5.0) were added; samples were incubated at 37°C dehydronorketamine). overnight and then diluted with 1 ml 10 mM ammonium acetate in water (pH 9.5). Solid-phase extraction was per- Data and Statistical Analysis formed as described above. Calibration samples contained Sample sizes for this pilot study were determined after 0, 20, 50, 100, 200, 500, 1,000, 1,500, and 2,000 ng/ a priori power calculations. Pharmacokinetic data were Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/125/6/1103/487909/20161200_0-00014.pdf by guest on 27 September 2021 ml racemic ketamine, norketamine, and dehydronorket- analyzed using noncompartmental methods (Phoenix; amine (base). Quality control samples contained 200 and Pharsight Corp, USA), assuming complete absorption, 1,000 ng/ml racemic ketamine, norketamine, and dehy- as described previously.26 Apparent oral clearance (CL/F) dronorketamine (base). was dose divided by concentration–time area under the

HPLC–mass spectrometry analysis was performed on curve (AUC)0–∞. Metabolite formation clearance was an ultrafast liquid chromatography system (Shimadzu Sci- the percentage of the ketamine dose excreted in urine entific Instruments, USA) with a CMB-20A system con- as metabolite multiplied by ketamine CL/F. Results are troller, two LC-20ADXR pumps, DGU-20A3 degasser, reported as the arithmetic mean ± SD. The primary out- SIL-20AC autosampler, FCV-11AL solvent selection come measure was ketamine N-demethylation, measured module, and CTO-20A column oven (30°C) containing a as the plasma norketamine/ketamine concentration–time ChiralPak AGP analytical column (100 × 2.0 mm; 5 μm) AUC0–∞ ratio (and also as the sum of N-demethylated and AGP guard column (10 × 2.0 mm; Chiral Technolo- ketamine metabolites/ketamine). Secondary outcomes gies, USA), coupled to an API 4000 QTrap LC-MS/MS were plasma ketamine enantiomer and metabolite linear ion trap triple-quadrupole tandem mass spectrom- AUC, maximum concentrations, apparent ketamine eter (Applied Biosystems/MDS Sciex, USA). For both oral clearance, and metabolite formation clearances. plasma and urine, the mobile phase (0.22 ml/min) was Differences between CYP2B6*1/*1, CYP2B6*1/*6, and 10 mM ammonium acetate in water (pH 7.60; A) and CYP2B6*6/*6 genotypes for pharmacokinetic parameters isopropanol (B). The column was equilibrated with 4% were analyzed using one-way ANOVA followed by the B, maintained after injection for 0.1 min, then a lin- Student–Newman–Keuls test for multiple comparisons ear gradient to 16.8% B was applied for 12 min, then (Sigmaplot 12.5; Systat Software, Inc, USA). Nonnormal reverted back to 4% B for 0.5 min and reequilibrated data were log transformed for analysis but reported as for 3 min. Total run time was 16 min. Under these con- the nontransformed results. Statistical significance was ditions, approximate retention time for each compound assigned at P < 0.05. was 7.0 and 8.1 min for S- and R-ketamine, 4.9 and 6.9 min for S- and R-norketamine, 4.1 and 7.3 min for Results S- and R-dehydronorketamine, and 2.4 min for both Plasma ketamine and metabolite concentrations are shown hydroxynorketamine isomers (fig. 2). The mass spec- in figure 3 for the three genotype groups (CYP2B6*1/*1, trometer electrospray ion source was operated in posi- CYP2B6*1/*6, and CYP2B6*6/*6). Enantiomeric data are tive-ion multiple reaction monitoring mode. The [M + shown for ketamine, norketamine, and dehydronorket- H]+ transitions were optimized for each analyte as fol- amine, which were chromatographically resolvable, while lows: m/z, 238.0 → 125.1 and 242.1 → 129.2 for d0- hydroxynorketamine diastereomers were nonresolvable and and d4-ketamine, 224.0 → 125.0 and 228.2 → 129.1 are quantified together (fig. 2). Pharmacokinetic parameters for d0- and d4-norketamine, and 222.1 → 142.1 for are provided in table 2. There was no significant difference dehydronorketamine. Because a standard for hydroxynor- in ketamine N-demethylation, for either R- or S-ketamine ketamine was not available, the transition m/z 240.0 → measured by plasma norketamine/ketamine AUC ratios 25 125.0 was based on previous reports, and the retention (or norketamine/ketamine Cmax ratios, not shown), in time was confirmed by incubating ketamine with human CYP2B6*6 carriers (CYP2B6*6 hetero- or homozygotes) liver microsomes. No stereochemistry was assigned to the compared to the wild-type CYP2B6*1/*1 genotype (fig. 3). hydroxynorketamine peak. Mass spectrometer settings for There was also no significant difference between CYP2B6 the declustering potential (36 to 70 V), collision energy genotypes in ketamine N-demethylation, measured by nor- (30 to 39 V), entrance potential (10 V), and collision cell ketamine formation clearance (fig. 4) or by other measures of exit potential (12 to 22 V) were optimized for each transi- CYP2B6-dependent metabolism that incorporate secondary tion. Optimized global parameters were: source tempera- metabolism (dehydronorketamine/ketamine, norketamine ture, 350°C; ionspray voltage, 5,500 V; nitrogen (psig) plus dehydronorketamine/ketamine, hydroxynorketamine/

Anesthesiology 2016; 125:1103-12 1106 Rao et al.

also no significant difference in the maximum plasma concentration, AUC, elimination half-life, or formation clearance of the primary metabolite norketamine or the secondary metabolites, dehydronorketamine and hydroxynorketamine. Ketamine clinical effects were measured by subjects’ subjective self-assessment (fig. 5). The dose was deliberately chosen for safety reasons to have a small (and nonsedating) pharmacologic effect but sufficient to reveal clinical effects if there were genotype-dependent increases in plasma concentration. There were essentially no significant effects, compared with predrug baseline, in alertness, energy, confusion, clumsiness, Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/125/6/1103/487909/20161200_0-00014.pdf by guest on 27 September 2021 anxiety, or nausea, in any genotype group, and no significant differences between groups in any assessment scale.

Discussion Previous investigations established that CYP2B6 is the major enzyme catalyzing hepatic ketamine N-demethylation and ketamine metabolism at clinically relevant concentrations. Human liver microsomal ketamine N-demethylation is catalyzed by a high-affinity (low Michaelis-Menten constant, Km) CYP and a low-affinity (high Km) CYP.17,20,27 Although both expressed CYP2B6 and CYP3A4 can N-demethylate ketamine at high substrate concentrations, CYP2B6 pre- dominates at low, clinically relevant concentrations.17–20 Sev- eral investigations using expressed enzymes and human liver microsomes showed that CYP2B6 and CYP3A4 were the low-Km (clinically relevant) and high-Km enzymes, respectively, that the activity (intrinsic clearance) of CYP2B6 is 6 to 8 times greater than that of CYP3A4, and that inhibitors (chemical and antibody) of CYP2B6 but not CYP3A4 diminished ketamine N-demethylation at clinically relevant concentrations.14,17–20 Available clinical studies corroborate these findings, as the CYP2B6 inhibitor ticlopidine, but not the CYP3A4 inhibitor itraconazole, diminished oral ketamine N-demethylation and increased plasma ketamine concentrations,21 although some results are inconsistent.28 CYP3A4 in the intestine may participate in intestinal presystemic Fig. 2. Chromatogram of a plasma sample obtained after ket- metabolism of oral ketamine, at high local concentrations,29 amine administration. Ketamine (m/z, 238.0 → 125.1), norket- as intestinal CYP2B6 expression is low or nonexistent.30 amine (m/z, 224.0 → 125.0), dehydronorketamine (m/z, 222.1 → 142.1), and hydroxynorketamine (m/z, 240.0 → 125.0). cps The CYP2B6 gene is highly polymorphic, with some 22 = counts per second; DHNK = dehydronorketamine. alleles causing altered activity. The most common variant allele, CYP2B6*6, found mostly in Africans, African- Americans, and some Asian populations, is associated ketamine, or norketamine plus dehydronorketamine plus with both diminished hepatic CYP2B6 enzyme expression hydroxynorketamine/ketamine AUC ratios; table 2). These and diminished CYP2B6 catalytic activity toward several ratios are less reliable, however, because S-dehydronorket- substrates, compared with wild-type CYP2B6*1/*1 carri- amine and hydroxynorketamine are elimination-rate limited ers. Additionally, the rate of ketamine N-demethylation rather than formation-rate limited (half-life is longer than by recombinant-expressed CYP2B6.6 (the enzyme coded that of the parent drug). There was no significant influence of for by CYP2B6*6) is considerably less than by wild-type CYP2B6*6 hetero- or homozygote genotypes on maximum CYP2B6.1, and ketamine N-demethylation in liver micro- ketamine enantiomer plasma concentration, time to maxi- somes from individuals hetero- or homozygous for the mum plasma concentration, ketamine AUC, CL/F, apparent CYP2B6*6 allele was significantly decreased compared volume of distribution, elimination half-life, or renal clear- with wild-type CYP2B6*1/*1.19,20 Interindividual variabil- ance. Excepting the AUC for hydroxynorketamine, there was ity in ketamine metabolism and plasma concentrations has

Anesthesiology 2016; 125:1103-12 1107 Rao et al. Copyright © 2016, the American Society of Anesthesiologists, Inc. Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Role of CYP2B6 Polymorphisms in Ketamine Metabolism Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/125/6/1103/487909/20161200_0-00014.pdf by guest on 27 September 2021

Fig. 3. Plasma concentrations of ketamine and metabolite enantiomers. Results are presented as mean ± SD (n = 10). Some SDs are omitted for clarity. CYP2B6 = cytochrome P4502B6.

been thought likely to CYP2B6 genetic polymorphisms.31 either as the plasma norketamine/ketamine AUC ratio or A preliminary finding of a reduced plasma norketamine norketamine formation clearance. Because norketamine also to ketamine concentration ratio in CYP2B6*6 carriers was undergoes further biotransformation, CYP2B6-dedependent reported.20 The purpose of this investigation was to deter- ketamine metabolism was also assessed using both primary mine if the CYP2B6*6 polymorphism influenced clinical and secondary metabolites. Although potentially influenced ketamine plasma concentrations and metabolism. by the slower elimination of the secondary metabolites, the The major finding of this investigation was that there was metabolite/parent plasma AUC area ratios reflecting all no significant difference between CYP2B6*1/*1 (wild-type), CYP2B6-dependent metabolites, including dehydronorket- CYP2B6*1/*6, and CYP2B6*6/*6 subjects in the plasma amine/ketamine, norketamine+dehydronorketamine/ket- concentrations of ketamine, the primary metabolite norketamine, hydroxynorketamine/ketamine, and norketamine+ amine, or the secondary metabolite dehydronorketamine, dehydronorketamine+hydroxynorketamine/ketamine, were for either ketamine enantiomer. There was also no genetic also not different from wild-type in CYP2B6*6 carriers. The difference in the N-demethylation of ketamine, assessed major conclusion is that the CYP2B6*6 polymorphism did

Anesthesiology 2016; 125:1103-12 1108 Rao et al.

Table 2. Oral Ketamine Pharmacokinetic Parameters

CYP2B6*1/*1 CYP2B6*1/*6 CYP2B6*6/*6 CYP2B6*1/*1 CYP2B6*1/*6 CYP2B6*6/*6

R-ketamine S-ketamine

Cmax (ng/ml) 14 ± 4 16 ± 8 21 ± 13 11 ± 3 12 ± 7 15 ± 8 tmax (h) 0.7 ± 0.3 0.5 ± 0.2 0.6 ± 0.3 0.7 ± 0.3 0.6 ± 0.2 0.5 ± 0.1 −1 29 ± 10 26 ± 11 35 ± 21 21 ± 7 19 ± 10 23 ± 13 AUC0–∞ (ng · h · ml ) CL/F (ml · kg−1 · min−1) 131 ± 57 147 ± 66 132 ± 79 182 ± 79 211 ± 103 194 ± 122 Vz/F (l/kg) 33 ± 17 35 ± 12 37 ± 21 31 ± 13 40 ± 11 46 ± 22

Elimination t1/2 (h) 2.9 ± 0.8 2.9 ± 0.6 3.3 ± 0.8 2.1 ± 0.9 2.5 ± 0.9 3.0 ± 1.1

Renal clearance (ml · kg−1 · min−1) 3.2 ± 1.3 4.0 ± 3.0 3.7 ± 2.2 3.6 ± 1.6 4.6 ± 3.6 4.3 ± 2.7 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/125/6/1103/487909/20161200_0-00014.pdf by guest on 27 September 2021

R-norketamine S-norketamine

Cmax (ng/ml) 90 ± 25 91 ± 45 99 ± 27 82 ± 18 78 ± 39 86 ± 24 −1 328 ± 61 307 ± 115 297 ± 75 315 ± 58 280 ± 82 269 ± 59 AUC∞ (ng · h · ml )

Elimination t1/2 (h) 3.5 ± .0.6 4.3 ± 1.3 3.7 ± 0.7 3.8 ± 0.7 4.7 ± 0.9 4.0 ± 0.6 13 ± 5 12 ± 4 11 ± 5 15 ± 5 14 ± 4 13 ± 7 AUC∞ (norketamine/ketamine) Formation clearance (ml · kg−1 · min−1) 18 ± 7 24 ± 17 18 ± 10 32 ± 10 44 ± 30 36 ± 18

R-dehydronorketamine S-dehydronorketamine

Cmax (ng/ml) 34 ± 13 38 ± 20 50 ± 20 15 ± 8 20 ± 11 26 ± 12 −1 166 ± 48 187 ± 67 205 ± 67 84 ± 26 117 ± 48 127 ± 45 AUC∞ (ng · h · ml )

Elimination t1/2 (h) 3.5 ± 0.7 4.4 ± 1.5 3.6 ± 0.5 4.3 ± 0.7 5.8 ± 3.0 4.4 ± 1.0 6.9 ± 4.2 7.7 ± 3.2 9.0 ± 8.1 4.8 ± 2.9 6.7 ± 2.5 7.9 ± 6.8 AUC∞ (dehydronorketamine/ketamine) 20 ± 9 20 ± 6 20 ± 13 21 ± 8 23 ± 7 22 ± 14 AUC∞ (norketamine plus dehydronorketamine/ketamine) Formation clearance (ml · kg−1 · min−1) 10 ± 9 12 ± 5 12 ± 8 12 ± 11 17 ± 8 17 ± 12

Hydroxynorketamine

Cmax (ng/ml) 23 ± 7 17 ± 6 19 ± 5 −1 224 ± 48 182 ± 39* 173 ± 36* AUC∞ (ng · h · ml )

Elimination t1/2 (h) 7.7 ± 2.7 8.2 ± 2.3 7.6 ± 2.2 5.0 ± 2.0 4.5 ± 1.7 3.8 ± 1.9 AUC∞ (hydroxynorketamine/RS-ketamine) 25 ± 10 26 ± 7 25 ± 15 AUC∞ (RS-norketamine plus RS-dehydronorketamine plus hydroxynorketamine/RS-ketamine) Formation clearance (ml · kg−1 · min−1) 36 ± 22 43 ± 20 36 ± 20

Subjects received 0.4 mg/kg oral ketamine HCl. Results are presented as arithmetic mean ± SD (n = 10; except renal clearances and formation clearances, n = 9). *Significantly different compared with CYP2B6*1/*1 subjects (P < 0.05).

AUC = area under the curve; CL/F = apparent oral clearance; Cmax = maximum plasma concentration; elimination t1/2 = elimination half-life; tmax = time of maximal plasma concentration; Vz/F = apparent volume of distribution.

not affect oral ketamine metabolism, clearance, and pharma- confirmed.31 The expectation is that given the lack of effect of cokinetics in healthy human volunteers in vivo. the CYP2B6*6 polymorphism on oral ketamine disposition, Oral ketamine undergoes extensive first-pass metabolism this polymorphism would not affect metabolism, clearance, and has a high CL/F. The CL/F of R- and S-ketamine observed and pharmacokinetics of intravenous ketamine in vivo. was 131 ± 57 and 182 ± 79 ml · kg−1 · min−1, respectively. Because CYP2B6 is the major isoform involved in Assuming hepatic blood flow of approximately 20 ml · kg−1 clinical ketamine metabolism and only CYP2B6-catalyzed · min−1 in healthy subjects, representing maximum clearance, metabolic pathways were of interest, only CYP2B6-depen- the bioavailability of R- and S-ketamine was only approxi- dent metabolites (norketamine, deydronorketamine, and mately 15% and 10%, respectively, which is in accordance hydroxynorketamine) were studied. There is also some with previously reported racemic bioavailability of approxi- CYP3A-mediated metabolism of ketamine to hydroxyket- mately 16 to 20%11,20 and an extraction ratio of 0.85. For amine. However, CYP3A involvement is not thought to be drugs with high extraction and extensive hepatic metabolism, significantly involved in ketamine pharmacokinetics and changes in metabolism, clearance, and plasma concentra- metabolism.12–15 Moreover, knowledge of hydroxyketamine tions will be greater for oral than intravenous administra- concentrations would not inform or alter the conclusion of tion.32 Specifically, for ketamine, this has been proposed20 and this investigation.

Anesthesiology 2016; 125:1103-12 1109 Rao et al. Copyright © 2016, the American Society of Anesthesiologists, Inc. Wolters Kluwer Health, Inc. Unauthorized reproduction of this article is prohibited. Role of CYP2B6 Polymorphisms in Ketamine Metabolism ) -1 120 •min -1

g R-ketamine 100 R-norketamine R-dehydronorketamine 80 S-ketamine S-norketamine 60 S-dehydronorketamine rac-ketamine 40 rac-norketamine rac-dehydronorketamine 20 rac-hydroxynorketamine

0 Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/125/6/1103/487909/20161200_0-00014.pdf by guest on 27 September 2021 CYP2B6*1/*1CYP2B6*1/*6 CYP2B6*6/*6 Renal or formation clearance (ml•k Fig. 4. Formation clearance for ketamine and metabolite enantiomers. CYP2B6 = cytochrome P4502B6.

Assay of ketamine and metabolite enantiomers and This investigation was a pilot study, and a small number diasteromers is challenging, and until recently, most phar- of subjects were studied. However, based on conventional, macokinetic studies evaluated only ketamine and norket- a priori, sample size calculations, a clinically significant dif- amine, without chiral discrimination. The analytical method ference could be expected with this number of subjects, if developed and implemented by Wainer and colleagues14,15 such a difference in metabolism did exist. It is unlikely that enabling quantification of several ketamine and metabolite evaluation of more subjects would change the results or enantiomers and diastereomers, was a major advance. That conclusions. assay used achiral analysis for total metabolite quantifica- The results of this investigation in healthy volunteers

tion, followed by chiral analysis using an α1-acid glycopro- differ from a recent report on ketamine disposition and tein HPLC column to determine the relative enantiomeric CYP2B6 polymorphisms in chronic pain patients34 who concentrations of R- and S-ketamine, R- and S-norket- received a 24-h subcutaneous ketamine infusion. A single amine, and R- and S-dehydronorketamine. Because the cur- steady-state plasma ketamine and norketamine achiral rent investigation did not aim to quantify the minor and concentration was determined and used to calculate clear- non-CYP2B6–dependent metabolite hydroxyketamine, nor ance. In CYP2B6*6 carriers, compared with CYP2B6*1/*1 did it aim to individually quantify the various hydroxynor- patients, ketamine clearance was lower, with a gene dose ketamine regioisomers (4, 5, and 6-hydroxylation) and dia- effect, and the plasma norketamine/ketamine ratio was lower stereomers, all analyses and quantification were performed in CYP2B6*6/*6 patients compared with CYP2B6*1/*6 and

using an α1-acid glycoprotein HPLC column. It has been CYP2B6*1/*1 genotypes. An explanation for the very differ- reported previously that the major circulating hydroxynor- ent results of the current investigation is not clearly apparent. ketamine diastereomers are 2S,6S;2R,6R>2S,5R;2R,5S.15 The difference cannot be attributed to the use of enantiose- Knowledge of individual hydroxynorketamine diastereomer lective versus achiral analysis since CYP2B6*6 affected nei- concentrations would not inform or alter the conclusion of ther R- nor S-ketamine in the current investigation. The this investigation. route of ketamine administration did differ between stud- Although the CYP2B6*6 polymorphism results in dimin- ies. Conceptually, pharmacogenetic influences on hepatic ished ketamine metabolism in vitro,20 this allelic variant did metabolism should be even greater with oral than parenteral not affect ketamine metabolism, clearance, and pharma- dosing, given extensive ketamine first-pass metabolism as cokinetics in vivo in the current investigation. A potential was observed with methadone, another CYP2B6 substrate.23 explanation why in vitro genetic differences did not translate Possibly, CYP2B6 genetic influences on ketamine hepatic to in vivo differences is that ketamine is a high-extraction first-pass metabolism could be masked by non-CYP2B6 drug (extraction ratio, 0.85), rendering it less sensitive to (i.e., CYP3A4)–dependent intestinal metabolism affecting variations in hepatic enzyme activity than intermediate- or bioavailability, although systemic (hepatic) ketamine elimi- low-extraction drugs.32 Nevertheless, inhibition of hepatic nation rates were not different between CYP2B6 genotypes. CYP2B6 activity (by ticlopidine) did diminish clinical Perhaps the apparent influence of CYP2B6*6 polymorphisms S-ketamine N-demethylation and increase plasma ketamine differs between pharmacokinetic methods (metabolite and concentrations after oral administration.21 Possibly, this parent drug parameter determinations using single-point ticlopidine effect was mediated partially through transport- concentrations vs. multipoint AUCs). It is conceivable that ers,33 as well as by CYP2B6 inhibition. Nevertheless, as a the apparent influence of CYP2B6*6 polymorphisms differs “positive control,” it strengthens the current conclusion that between (1) single-dose and steady-state dosing; (2) study reduced hepatic CYP2B6 activity due to the CYP2B6*6 populations of healthy volunteers versus chronic and can- polymorphism did not affect low-dose oral ketamine metab- cer pain patients, as inflammation and cancer are associated olism, clearance, and pharmacokinetics in humans in vivo. with down-regulation of hepatic and extrahepatic P450s35;

Anesthesiology 2016; 125:1103-12 1110 Rao et al.

Li et al.34 found an age-dependent decline in ketamine clearance, and the very small number of CYP2B6*6 subjects in their study were also the oldest; hence, there may be con- founding between an age-dependent effect and an apparent genetic effect on ketamine metabolism and clearance. The oral ketamine dose used in this investigation was intended to have a small pharmacologic effect, based on other studies in healthy volunteers.21,24 It was conservatively chosen, anticipating potentially higher plasma concentrations and clinical effects in CYP2B6*6 carriers. Clinically measureable effects of ketamine were not observed, however, in any genotype Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/125/6/1103/487909/20161200_0-00014.pdf by guest on 27 September 2021 group. This may be due to the deliberately small dose chosen or the different effect measures used herein and previously.21 In summary, in carriers of the CYP2B6*6 polymorphism, which results in diminished ketamine metabolism in vitro, compared with CYP2B6*1/*1 wild-type subjects, there was no significant difference in plasma ketamine or metabolite concentrations or in ketamine N-demethylation, measured as the plasma norketamine/ketamine AUC ratio or norketamine formation clearance, after single, low-dose oral ketamine administration. These results do not support the hypothesis that CYP2B6*6 pharmacogenetics affect single, low-dose oral ketamine metabolism, clearance, and pharmacokinetics in vivo. Similarly, CYP2B6*6 allele status would appear not to be a fac- tor in single, low-dose oral ketamine dose or patient selection.

Research Support Supported in part by the Department of Anesthesiology, Washington University in St. Louis School of Medicine (St. Louis, Missouri) and grant R01-DA14211 from the Na- tional Institutes of Health (Bethesda, Maryland).

Competing Interests The authors declare no competing interests.

Reproducible Science Full protocol available from Dr. Rao: [email protected]. Fig. 5. Subjects’ self-assessment of ketamine effects. Ef- Raw data available from Dr. Rao: [email protected]. fect scales were alertness/sedation (almost asleep to wide awake), energy level (no energy to full of energy), clumsiness (extremely clumsy to well coordinated), confusion (confused Correspondence to clear headed), anxiety (calm/relaxed to extremely nervous), Address correspondence to Dr. Rao: Department of An- or nausea (no nausea to worst nausea). Results are presented esthesiology, Washington University in St. Louis School of as mean ± SD (n = 10). Some SD data are omitted for clarity. Medicine, 660 South Euclid Avenue, Campus Box 8054, St. *P < 0.05 versus predrug baseline. There were no significant Louis, MO 63110. [email protected]. This article may be differences between CYP2B6*6 homozygotes or heterozy- accessed for personal use at no charge through the Journal gotes compared to CYP2B6*1/*1 wild-types. CYP2B6 = cyto Web site, www.anesthesiology.org. chrome P4502B6. References 1. Chen X, Shu S, Bayliss DA: HCN1 channel subunits are a (3) route of administration (thus determining intravenous molecular substrate for hypnotic actions of ketamine. clearance vs. CL/F); (4) ketamine doses and resulting plasma J Neurosci 2009; 29:600–9 concentrations, which were substantially greater in the study 2. Blonk MI, Koder BG, van den Bemt PM, Huygen FJ: Use of by Li et al.,34 and (5) norketamine/ketamine concentration oral ketamine in chronic pain management: A review. Eur J Pain 2010; 14:466–72 ratios, which were much higher in the current investigation 34 3. Anand KJ: Pharmacological approaches to the management (7 at Cmax) compared with the study by Li et al. (approxi- of pain in the neonatal intensive care unit. J Perinatol 2007; mately 1 at steady state). It is also potentially pertinent that 27(Suppl 1):S4–S11

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Anesthesiology 2016; 125:1103-12 1112 Rao et al.