Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Portmann, Simone; Kwan, Hiu Ying; Theurillat, Regula; Schmitz, Andrea; Mevissen, Meike; Thormann, Wolfgang

doi:10.1016/j.chroma.2010.06.028

Cited by 91 publications

(152 citation statements)

References 30 publications

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“…Accordingly, our results provide little evidence supporting the stereoselectivity in ketamine clearance that has been reported clinically. The K m value for (R)-ketamine metabolism was 1.3-fold higher than that for (S)-ketamine, leading to an approximately 27% decrease in the CL int 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). Of interest, the COS-1 cell-expressed CYP2B6.1 protein did not exhibit stereoselectivity in ketamine Ndemethylation.…”

Section: Discussionsupporting

confidence: 79%

“…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).…”

Section: Introductionmentioning

confidence: 97%

“…Several studies using microsomes containing cDNA-expressed human P450 enzymes have shown the involvement of CYP2B6, CYP2C9, CYP2C19, and CYP3A4 in ketamine 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).…”

Section: Introductionmentioning

confidence: 99%

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TheCYP2B6*6Allele Significantly Alters theN-Demethylation of Ketamine Enantiomers In Vitro

Coller

Hutchinson

et al. 2013

Drug Metab Dispos

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Ketamine is primarily metabolized to norketamine by hepatic CYP2B6 and CYP3A4-mediated N-demethylation. However, the relative contribution from each enzyme remains controversial. The CYP2B6*6 allele is associated with reduced enzyme expression and activity that may lead to interindividual variability in ketamine metabolism. We examined the N-demethylation of individual ketamine enantiomers using human liver microsomes (HLMs) genotyped for the CYP2B6*6 allele, insect cell-expressed recombinant CYP2B6 and CYP3A4 enzymes, and COS-1 cell-expressed recombinant CYP2B6.1 and CYP2B6.6 protein variant. Effects of CYP-selective inhibitors on norketamine formation were also determined in HLMs. The two-enzyme Michaelis-Menten model best fitted the HLM kinetic data. The Michaelis-Menten constants (K m ) for the highaffinity enzyme and the low-affinity enzyme were similar to those for the expressed CYP2B6 and CYP3A4, respectively. The intrinsic clearance for both ketamine enantiomers by the high-affinity enzyme in HLMs with CYP2B6*1/*1 genotype were at least 2-fold and 6-fold higher, respectively, than those for CYP2B6*1/*6 genotype and CYP2B6*6/*6 genotype. The V max and K m values for CYP2B6.1 were approximately 160 and 70% of those for CYP2B6.6, respectively. N,N9N9-triethylenethiophosphoramide (thioTEPA) (CYP2B6 inhibitor, 25 mM) and the monoclonal antibody against CYP2B6 but not troleandomycin (CYP3A4 inhibitor, 25 mM) 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 (genedose P < 0.05). These results indicate a major role of CYP2B6 in ketamine N-demethylation in vitro and a significant impact of the CYP2B6*6 allele on enzyme-ketamine binding and catalytic activity.

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Section: Discussionsupporting

confidence: 79%

Section: Introductionmentioning

confidence: 97%

Section: Introductionmentioning

confidence: 99%

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TheCYP2B6*6Allele Significantly Alters theN-Demethylation of Ketamine Enantiomers In Vitro

Coller

Hutchinson

et al. 2013

Drug Metab Dispos

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“…4-Hydroxychalcone was identified as inhibitor with an IC 50 value in the submillimolar range. The stereospecific N-demethylation of the drug ketamine to norketamine by cytochrome P450 (CYP) isoenzymes was studied by Portmann and colleagues [39]. Separation of the analyte enantiomers was performed in a 45/34 cm, 50 μm id, fused-silica capillary employing either 35 or 50 mM Tris/phosphoric acid buffer, pH 2.5, containing 10 mg mL −1 sulfated β-CD as chiral selector for the determination of the metabolite pattern and the kinetic data, respectively.…”

Section: Pre-capillary Enzyme Assaysmentioning

confidence: 99%

Advances in Capillary Electrophoresis-Based Enzyme Assays

Scriba

Belal

2015

Chromatographia

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IntroductionEnzymes catalyze metabolic reactions in cells regulating homeostasis, growth and reproduction of living organisms. Enzymes are also often involved in human disease. The analysis of the human genome has revealed that within the so-called druggable genome a significant portion (in some analyses more than half) of the potential drug targets are enzymes [1][2][3]. Consequently, enzymes are important targets in the development of new drugs. Moreover, enzymes play a role in the clinical diagnosis of diseases. Therefore, effective methods for characterization of enzymes as well as for the determination of their activity are important, for example, for the understanding of enzyme-mediated metabolic reactions or the identification of substrates and inhibitors for the treatment of human diseases.Capillary electrophoresis (CE) has been applied to enzyme analysis for more than 20 years since the first report by Banke et al. on an assay of alkaline protease [4]. Since then, all aspects of enzyme-related analysis have been studied by CE methods including the evaluation of enzymatic activity, enzyme kinetics, identification and characterization of enzyme inhibitors and activators as well as metabolic pathways as, for example, summarized in [5][6][7][8][9][10][11][12].Generally, CE-based enzyme assays can be divided in three groups, pre-capillary (offline) assays, in-capillary (online) assays and post-capillary assays. In the pre-capillary assays, all required components including enzyme, substrate, co-factors, etc., are mixed in a vial and incubated for an intended period of time. Subsequently, the reaction is quenched and an aliquot is subjected to CE analysis for the determination of substrate(s) and/or co-substrate(s) and/ or product(s). Multiple sampling or repeated sampling in combination with sequential runs for the determination of Abstract Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis-Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CEbased enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic a...

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“…It is formed via metabolism from norketamine or hydroxyketamine [19,20]. Its pharmacological properties have been only scarcely studied because initial experiments showed that (R,S)-ketamine and (R,S)-norketamine produced anaesthesia and increased spontaneous locomotor activity during recovery from anaesthesia, whereas 6-hydroxynorketamine had no effects.…”

mentioning

confidence: 99%

Interactions of (2S,6S;2R,6R)‐Hydroxynorketamine, a Secondary Metabolite of (R,S)‐Ketamine, with Morphine

Lilius

Viisanen

Jokinen

et al. 2017

Basic Clin Pharma Tox

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Ketamine and its primary metabolite norketamine attenuate morphine tolerance by antagonising N-methyl-D-aspartate (NMDA) receptors. Ketamine is extensively metabolized to several other metabolites. The major secondary metabolite (2S,6S;2R,6R)-hydroxynorketamine (6-hydroxynorketamine) is not an NMDA antagonist. However, it may modulate nociception through negative allosteric modulation of a7 nicotinic acetylcholine receptors. We studied whether 6-hydroxynorketamine could affect nociception or the effects of morphine in acute or chronic administration settings. Male Sprague Dawley rats received subcutaneous 6-hydroxynorketamine or ketamine alone or in combination with morphine, as a cotreatment during induction of morphine tolerance, and after the development of tolerance induced by subcutaneous minipumps administering 9.6 mg morphine daily. Tail flick, hot plate, paw pressure and rotarod tests were used. Brain and serum drug concentrations were quantified with high-performance liquid chromatography-tandem mass spectrometry. Ketamine (10 mg/kg), but not 6-hydroxynorketamine (10 and 30 mg/kg), enhanced antinociception and decreased rotarod performance following acute administration either alone or combined with morphine. Ketamine efficiently attenuated morphine tolerance. Acutely administered 6-hydroxynorketamine increased the brain concentration of morphine (by 60%), and brain and serum concentrations of 6-hydroxynorketamine were doubled by morphine pre-treatment. This pharmacokinetic interaction did not, however, lead to altered morphine tolerance. Co-administration of 6-hydroxynorketamine 20 mg/kg twice daily did not influence development of morphine tolerance. Even though morphine and 6-hydroxynorketamine brain concentrations were increased after co-administration, the pharmacokinetic interaction had no effect on acute morphine nociception or tolerance. These results indicate that 6-hydroxynorketamine does not have antinociceptive properties or attenuate opioid tolerance in a similar way as ketamine.Opioids are widely used to manage severe acute and cancer pain. However, prolonged opioid use in chronic non-cancer pain may have severe drawbacks, including the development of tolerance and dependence. In the United States, eighty per cent of new heroin users have previously misused prescription opioids, and the opioid epidemic has been declared a national emergency [1]. Long-term opioid use may even lead to opioid-induced hyperalgesia that can compromise opioid analgesia and lead to further increases in opioid doses [2]. New approaches are needed to provide safer opioid analgesia with lower doses.Several N-methyl-D-aspartate (NMDA) receptor antagonists have been shown to attenuate opioid tolerance [3-5] mainly via pharmacodynamic interactions. Ketamine, a clinically used NMDA antagonist, produces anaesthesia and also analgesia at lower doses. Clinical evidence supports the perioperative use of low-dose ketamine with morphine to decrease the need for morphine [6], whereas there is lack of evidence to supp...

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Enantioselective capillary electrophoresis for identification and characterization of human cytochrome P450 enzymes which metabolize ketamine and norketamine in vitro

Cited by 91 publications

References 30 publications

TheCYP2B6*6Allele Significantly Alters theN-Demethylation of Ketamine Enantiomers In Vitro

TheCYP2B6*6Allele Significantly Alters theN-Demethylation of Ketamine Enantiomers In Vitro

Advances in Capillary Electrophoresis-Based Enzyme Assays

Interactions of (2S,6S;2R,6R)‐Hydroxynorketamine, a Secondary Metabolite of (R,S)‐Ketamine, with Morphine

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