Structural basis of ketamine action on human NMDA receptors.

Ketamine is a non-competitive channel blocker of N-methyl-D-aspartate (NMDA) receptors1. A single sub-anaesthetic dose of ketamine produces rapid (within hours) and long-lasting antidepressant effects in patients who are resistant to other antidepressants2,3. Ketamine is a racemic mixture of S- and R-ketamine enantiomers, with S-ketamine isomer being the more active antidepressant4. Here we describe the cryo-electron microscope structures of human GluN1-GluN2A and GluN1-GluN2B NMDA receptors in complex with S-ketamine, glycine and glutamate. Both electron density maps uncovered the binding pocket for S-ketamine in the central vestibule between the channel gate and selectivity filter. Molecular dynamics simulation showed that S-ketamine moves between two distinct locations within the binding pocket. Two amino acids-leucine 642 on GluN2A (homologous to leucine 643 on GluN2B) and asparagine 616 on GluN1-were identified as key residues that form hydrophobic and hydrogen-bond interactions with ketamine, and mutations at these residues reduced the potency of ketamine in blocking NMDA receptor channel activity. These findings show structurally how ketamine binds to and acts on human NMDA receptors, and pave the way for the future development of ketamine-based antidepressants.

An easily fabricated nanoporous Au membrane in drug detection with reusable functionality and high SERS performance.

A method for detecting methamphetamine (MET), ketamine (KET), and morphine (MOP) molecules is presented using a reusable substrate based on SERS. The SERS substrate was prepared by etching the Au/Ag alloy film to synthesize a nanoporous Au membrane (AuNPM). By optimizing the preparation conditions and using rhodamine 6G (R6G) as an analyte, the AuNPM exhibited good SERS performance with a limit of detection (LOD) of 10-9 mol L-1. A competitive immunoassay category has been applied to the detection of MET, KET, and MOP. The MET, KET, and MOP antigens were functionalized on the surface of the AuNPM to specifically bind to the related drug antibodies. The Au nanoparticles (AuNPs) modified with 4-mercaptobenzoic acid (4-MBA) and antibodies against MET, KET, and MOP were used as nanotags. The 4-MBA served as the reporting molecule and drug antibodies were used to bind to free drug molecules in the target solution. The mixture of nanotags and target solution was dropped onto the antigen-modified AuNPM (antigen/AuNPM), and the free nanotags bind to the antigen/AuNPM. By comparing the SERS intensity of 4-MBA with the presence or absence of drug molecules, the drugs were qualitatively and quantitatively identified. Through this category, the LODs for detecting MET, KET, and MOP were 0.1, 1, and 1 ng mL-1, respectively. This study proposes an effective method for constructing SERS-based detection of drug molecules with good potential for practical applications.

Design, Synthesis and Biological Evaluation of Novel Ketamine Derivatives as NMDAR Antagonists.

Depression is a chronic, severe, and often life-threatening neurological disorder. It not only causes depression in patients and affects daily life but, in severe cases, may lead to suicidal behavior and have adverse effects on families and society. In recent years, it has been found that sub-anesthetic doses of ketamine have a rapid antidepressant effect on patients with treatment-resistant depression and can significantly reduce the suicidal tendencies of patients with major depressive disorder. Current studies suggest that ketamine may exert antidepressant effects by blocking NMDAR ion channels, but its anesthetic and psychotomimetic side effects limit its application. Here, we report efforts to design and synthesize a novel series of ketamine derivatives of NMDAR antagonists, among which compounds 23 and 24 have improved activity compared with ketamine, introducing a new direction for the development of rapid-acting antidepressant drugs.

Molecular mechanisms underlying the antidepressant actions of arketamine: beyond the NMDA receptor.

The discovery of robust antidepressant actions exerted by the N-methyl-D-aspartate receptor (NMDAR) antagonist (R,S)-ketamine has been a crucial breakthrough in mood disorder research. (R,S)-ketamine is a racemic mixture of equal amounts of (R)-ketamine (arketamine) and (S)-ketamine (esketamine). In 2019, an esketamine nasal spray from Johnson & Johnson was approved in the United States of America and Europe for treatment-resistant depression. However, an increasing number of preclinical studies show that arketamine has greater potency and longer-lasting antidepressant-like effects than esketamine in rodents, despite the lower binding affinity of arketamine for the NMDAR. In clinical trials, non-ketamine NMDAR-related compounds did not exhibit ketamine-like robust antidepressant actions in patients with depression, despite these compounds showing antidepressant-like effects in rodents. Thus, the rodent data do not necessarily translate to humans due to the complexity of human psychiatric disorders. Collectively, the available studies indicate that it is unlikely that NMDAR plays a major role in the antidepressant action of (R,S)-ketamine and its enantiomers, although the precise molecular mechanisms underlying antidepressant actions of (R,S)-ketamine and its enantiomers remain unclear. In this paper, we review recent findings on the molecular mechanisms underlying the antidepressant actions of (R,S)-ketamine and its potent enantiomer arketamine. Furthermore, we discuss the possible role of the brain-gut-microbiota axis and brain-spleen axis in stress-related psychiatric disorders and in the antidepressant-like action of arketamine. Finally, we discuss the potential of arketamine as a treatment for cognitive impairment in psychiatric disorders, Parkinson's disease, osteoporosis, inflammatory bowel diseases, and stroke.

Study on the Mass Spectrometry Fragmentation Patterns for Rapid Screening and Structure Identification of Ketamine Analogues in Illicit Powders.

Ketamine analogues have been emerging in recent years and are causing severe health and social problems worldwide. Ketamine analogues use 2-phenyl-2-aminocyclohexanone as the basic structure and achieve physiological reactions similar to or even more robust than the prototype of ketamine by changing the substituents on the benzene ring (R1 and R2) and amine group (RN1). Therefore, the mass spectrometry (MS) fragmentation pathways and fragments of ketamine analogues have certain regularity. Eight ketamine analogues are systematically investigated by GC-QTOF/MS and LC-Q-Orbitrap MS/MS with the positive mode of electrospray ionization. The MS fragmentation patterns of ketamine analogues are summarized according to high-resolution MS data. The α-cleavage of carbon bond C1-C2 in the cyclohexanone moiety and further losses of CO, methyl radical, ethyl radical and propyl radical are the characteristic fragmentation pathways of ketamine analogues in EI-MS mode. The loss of H2O or the sequential loss of RN1NH2, CO and C4H6 are the distinctive fragmentation pathways of ketamine analogues in ESI-MS/MS mode. Moreover, these MS fragmentation patterns are first introduced for the rapid screening of ketamine analogues in suspicious powder. Furthermore, the structure of the ketamine analogue in suspicious powder is 2-(Methylamino)-2-(o-tolyl)cyclohexan-1-one, which is further confirmed by NMR. This study contributes to the identification of the chemical structure of ketamine analogues, which can be used for the rapid screening of ketamine analogues in seized chemicals.

Antidepressant-relevant concentrations of the ketamine metabolite (2R,6R)-hydroxynorketamine do not block NMDA receptor function.

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine's antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.

(2R,6R)-hydroxynorketamine exerts mGlu2 receptor-dependent antidepressant actions.

Currently approved antidepressant drugs often take months to take full effect, and ∼30% of depressed patients remain treatment resistant. In contrast, ketamine, when administered as a single subanesthetic dose, exerts rapid and sustained antidepressant actions. Preclinical studies indicate that the ketamine metabolite (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a rapid-acting antidepressant drug candidate with limited dissociation properties and abuse potential. We assessed the role of group II metabotropic glutamate receptor subtypes 2 (mGlu2) and 3 (mGlu3) in the antidepressant-relevant actions of (2R,6R)-HNK using behavioral, genetic, and pharmacological approaches as well as cortical quantitative EEG (qEEG) measurements in mice. Both ketamine and (2R,6R)-HNK prevented mGlu2/3 receptor agonist (LY379268)-induced body temperature increases in mice lacking the Grm3, but not Grm2, gene. This action was not replicated by NMDA receptor antagonists or a chemical variant of ketamine that limits metabolism to (2R,6R)-HNK. The antidepressant-relevant behavioral effects and 30- to 80-Hz qEEG oscillation (gamma-range) increases resultant from (2R,6R)-HNK administration were prevented by pretreatment with an mGlu2/3 receptor agonist and absent in mice lacking the Grm2, but not Grm3-/-, gene. Combined subeffective doses of the mGlu2/3 receptor antagonist LY341495 and (2R,6R)-HNK exerted synergistic increases on gamma oscillations and antidepressant-relevant behavioral actions. These findings highlight that (2R,6R)-HNK exerts antidepressant-relevant actions via a mechanism converging with mGlu2 receptor signaling and suggest enhanced cortical gamma oscillations as a marker of target engagement relevant to antidepressant efficacy. Moreover, these results support the use of (2R,6R)-HNK and inhibitors of mGlu2 receptor function in clinical trials for treatment-resistant depression either alone or in combination.

Antidepressant Actions of Ketamine: Potential Role of L-Type Calcium Channels.

Recently, the United States Food and Drug Administration approved esketamine, the S-enantiomer of ketamine, as a fast-acting therapeutic drug for treatment-resistant depression. Although ketamine is known as an N-methyl-d-aspartate (NMDA) receptor antagonist, the underlying mechanisms of how it elicits an antidepressant effect, specifically at subanesthetic doses, are not clear and remain an advancing field of research interest. On the other hand, high-dose (more than the anesthetic dose) ketamine-induced neurotoxicity in animal models has been reported. There has been progress in understanding the potential pathways involved in ketamine-induced antidepressant effects, some of which include NMDA-receptor antagonism, modulation of voltage-gated calcium channels, and brain-derived neurotrophic factor (BDNF) signaling. Often these pathways have been shown to be linked. Voltage-gated L-type calcium channels have been shown to mediate the rapid-acting antidepressant effects of ketamine, especially involving induction of BDNF synthesis downstream, while BDNF deficiency decreases the expression of L-type calcium channels. This review focuses on the reported studies linking ketamine's rapid-acting antidepressant actions to L-type calcium channels with an objective to present a perspective on the importance of the modulation of intracellular calcium in mediating the effects of subanesthetic (antidepressant) versus high-dose ketamine (anesthetic and potential neurotoxicant), the latter having the ability to reduce intracellular calcium by blocking the calcium-permeable NMDA receptors, which is implicated in potential neurotoxicity.

Stereoselective ketamine effect on cardiac output: a population pharmacokinetic/pharmacodynamic modelling study in healthy volunteers.

BACKGROUND: Ketamine has cardiac excitatory side-effects. Currently, data on the effects of ketamine and metabolite concentrations on cardiac output are scarce. We therefore developed a pharmacodynamic model derived from data from a randomised clinical trial. The current study is part of a larger clinical study evaluating the potential mitigating effect of sodium nitroprusside on the psychedelic effects of ketamine. METHODS: Twenty healthy male subjects received escalating esketamine and racemic ketamine doses in combination with either placebo or sodium nitroprusside on four visits: (i) esketamine and placebo, (ii) esketamine and sodium nitroprusside, (iii) racemic ketamine and placebo, and (iv) racemic ketamine and sodium nitroprusside. During each visit, arterial blood samples were obtained and cardiac output was measured. Nonlinear mixed-effect modelling was used to analyse the cardiac output time-series data. Ketamine metabolites were added to the model in a sequential manner to evaluate the effects of metabolites. RESULTS: A model including an S-ketamine and S-norketamine effect best described the data. Ketamine increased cardiac output, whereas modelling revealed that S-norketamine decreased cardiac output. No significant effects were detected for R-ketamine, metabolites other than S-norketamine, or sodium nitroprusside on cardiac output. CONCLUSIONS: S-Ketamine, but not R-ketamine, increased cardiac output in a dose-dependent manner. In contrast to S-ketamine, its metabolite S-norketamine reduced cardiac excitation in a dose-dependent manner. CLINICAL TRIAL REGISTRATION: Dutch Cochrane Center 5359.

Crocins, the Bioactive Components of Crocus sativus L., Counteract the Disrupting Effects of Anesthetic Ketamine on Memory in Rats.

Consistent experimental evidence suggests that anesthetic doses of the non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist ketamine cause severe memory impairments in rodents. Crocins are among the various bioactive ingredients of the plant Crocus sativus L., and their implication in memory is well-documented. It has not yet been elucidated if crocins are able to attenuate the memory deficits produced by anesthetic ketamine. The present study was undertaken aiming to clarify this issue in the rat. For this aim, the object recognition, the object location and the habituation tests, reflecting non-spatial recognition memory, spatial recognition memory and associative memory, respectively, were utilized. A post-training challenge with crocins (15-30 mg/kg, intraperitoneally (i.p.), acutely) counteracted anesthetic ketamine (100 mg/kg, i.p.)-induced performance impairments in all the above-mentioned behavioral memory paradigms. The current findings suggest that crocins modulate anesthetic ketamine's amnestic effects.

Pharmacokinetics of ketamine and its major metabolites norketamine, hydroxynorketamine, and dehydronorketamine: a model-based analysis.

BACKGROUND: Recent studies show activity of ketamine metabolites, such as hydroxynorketamine, in producing rapid relief of depression-related symptoms and analgesia. To improve our understanding of the pharmacokinetics of ketamine and metabolites norketamine, dehydronorketamine, and hydroxynorketamine, we developed a population pharmacokinetic model of ketamine and metabolites after i.v. administration of racemic ketamine and the S-isomer (esketamine). Pharmacokinetic data were derived from an RCT on the efficacy of sodium nitroprusside (SNP) in reducing the psychotomimetic side-effects of ketamine in human volunteers. METHODS: Three increasing i.v. doses of esketamine and racemic ketamine were administered to 20 healthy volunteers, and arterial plasma samples were obtained for measurement of ketamine and metabolites. Subjects were randomised to receive esketamine/SNP, esketamine/placebo, racemic ketamine/SNP, and racemic ketamine/placebo on four separate occasions. The time-plasma concentration data of ketamine and metabolites were analysed using a population compartmental model approach. RESULTS: The pharmacokinetics of ketamine and metabolites were adequately described by a seven-compartment model with two ketamine, norketamine, and hydroxynorketamine compartments and one dehydronorketamine compartment with metabolic compartments in-between ketamine and norketamine, and norketamine and dehydronorketamine main compartments. Significant differences were found between S- and R-ketamine enantiomer pharmacokinetics, with up to 50% lower clearances for the R-enantiomers, irrespective of formulation. Whilst SNP had a significant effect on ketamine clearances, simulations showed only minor effects of SNP on total ketamine pharmacokinetics. CONCLUSIONS: The model is of adequate quality for use in future pharmacokinetic and pharmacodynamic studies into the efficacy and side-effects of ketamine and metabolites. CLINICAL TRIAL REGISTRATION: Dutch Cochrane Center 5359.

A Fast and Validated High Throughput Bar Adsorptive Microextraction (HT-BAµE) Method for the Determination of Ketamine and Norketamine in Urine Samples.

We developed, optimized and validated a fast analytical cycle using high throughput bar adsorptive microextraction and microliquid desorption (HT-BAµE-µLD) for the extraction and desorption of ketamine and norketamine in up to 100 urine samples simultaneously, resulting in an assay time of only 0.45 min/sample. The identification and quantification were carried out using large volume injection-gas chromatography-mass spectrometry operating in the selected ion monitoring mode (LVI-GC-MS(SIM)). Several parameters that could influencing HT-BAµE were assayed and optimized in order to maximize the recovery yields of ketamine and norketamine from aqueous media. These included sorbent selectivity, desorption solvent and time, as well as shaking rate, microextraction time, matrix pH, ionic strength and polarity. Under optimized experimental conditions, suitable sensitivity (1.0 µg L-1), accuracy (85.5-112.1%), precision (≤15%) and recovery yields (84.9-105.0%) were achieved. Compared to existing methods, the herein described analytical cycle is much faster, environmentally friendly and cost-effective for the quantification of ketamine and norketamine in urine samples. To our knowledge, this is the first work that employs a high throughput based microextraction approach for the simultaneous extraction and subsequent desorption of ketamine and norketamine in up to 100 urine samples simultaneously.

Pharmacokinetics of S-ketamine and R-ketamine and their active metabolites after racemic ketamine or S-ketamine intravenous administration in dogs sedated with medetomidine.

OBJECTIVE: To assess the differences in the pharmacokinetic profiles of S-ketamine, R-ketamine and their metabolites, S-norketamine and R-norketamine, and to measure relevant physiologic variables after intravenous administration of racemic (RS) ketamine or S-ketamine alone in Beagle dogs sedated with medetomidine. STUDY DESIGN: Experimental, blinded and randomized crossover study. ANIMALS: A total of six (three female and three male) adult Beagle dogs. METHODS: Medetomidine (450 µg m-2) was administered intramuscularly, followed by either S-ketamine (2 mg kg-1) or RS-ketamine (4 mg kg-1) 20 minutes later, both administered intravenously. Blood samples were collected before medetomidine administration and at multiple time points 1-900 minutes following the ketamine administration. Plasma samples were analysed using liquid chromatography-tandem mass spectrometry. Heart rate, respiratory rate, noninvasive blood pressure, haemoglobin saturation with oxygen and body temperature were measured at baseline, before ketamine administration, and 1, 2, 5, 10, 15, 20 and 30 minutes after ketamine administration. All cardiovascular variables, blood glucose, haemoglobin and lactate concentrations were analysed using different linear mixed effects models; the significance was set at p < 0.05. RESULTS: S-ketamine showed a two-compartment kinetic profile; no statistically significant differences were observed between its concentrations or in the calculated pharmacokinetic parameters following S- or RS-ketamine. When the racemic mixture was administered, no differences were detected between R- and S-ketamine concentrations, but the area under the curve (AUC) for R-norketamine was significantly lower than that for S-norketamine. Clinically relevant physiologic variables did not show statistically significant differences following the administration of the racemic mixture or of S-ketamine alone. CONCLUSIONS AND CLINICAL RELEVANCE: This study performed in dogs showed that RS-ketamine and S-ketamine combined with medetomidine showed enantioselective pharmacokinetics as S- and R-norketamine AUCs were different, but S-ketamine levels were identical.

Structure-Activity Relationships for the Anaesthetic and Analgaesic Properties of Aromatic Ring-Substituted Ketamine Esters.

A series of benzene ring substituted ketamine N-alkyl esters were prepared from the corresponding substituted norketamines. Few of the latter have been reported since they have not been generally accessible via known routes. We report a new general route to many of these norketamines via the Neber (oxime to α-aminoketone) rearrangement of readily available substituted 2-phenycyclohexanones. We explored the use of the substituents Cl, Me, OMe, CF3, and OCF3, with a wide range of lipophilic and electronic properties, at all available benzene ring positions. The 2- and 3-substituted compounds were generally more active than 4-substituted compounds. The most generally acceptable substituent was Cl, while the powerful electron-withdrawing substituents CF3 and OCF3 provided fewer effective analogues.

Therapeutic Mechanisms of Ketamine.

Major depressive disorder is the greatest burden of developed countries in the context of morbidity caused by mental disorders. Until recent, ketamine has been mostly used for anesthesia, analgesia, sedation and treatment of chronic pain syndromes. However, unique pharmacodynamic properties of ketamine have increased interests in it's use for treatment of depression. It is assumed that ketamine reverses synaptic chronic stress pathology within one day of administration by postsynaptic glutamate activation, providing synaptic connectivity restoration that last for days or weeks. Potential glutamatergic agents, in context of treatment of major depressive disorder are not entirely novel phenomenon. Considering the aforementioned, current neurobiological view of depression as a solely monoaminergic phenomenon should be reassessed in order to prompt discovery of putative antidepressant drugs of novel generation. Acute side effects, such as increased salivation, increase in heart rate, systemic arterial pressure and intracranial pressure necessitate careful monitoring during intravenous administration of ketamine, even in subanesthetic doses. However, major burden of ketamine administration lies in it's ability to produce psychotomimetic side effects and emergence delirium. Esketamine nasal spray has now been widely approved and is considered safe in terms of acute side effects, tolerability and consistent therapeutic benefit.

Enantioselective capillary electrophoresis for pharmacokinetic analysis of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equines anesthetized with ketamine and isoflurane.

An enantioselective assay for the determination of methadone and its main metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in equine plasma based on capillary electrophoresis with highly sulfated γ-cyclodextrin as chiral selector and electrokinetic analyte injection is described. The assay is based on liquid/liquid extraction of the analytes at alkaline pH from 0.1 mL plasma followed by electrokinetic sample injection of the analytes from the extract across a buffer plug without chiral selector. Separation occurs cationically at normal polarity in a pH 3 phosphate buffer containing 0.16% (w/v) of highly sulfated γ-cyclodextrin. The developed assay is precise (intra- and interday RSD < 4% and < 7%, respectively), is capable to determine enantiomer levels of methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in plasma down to 2.5 ng/mL, and was successfully applied to monitor enantiomer drug and metabolite levels in plasma of a pony that was anesthetized with racemic ketamine and isoflurane and received a bolus of racemic methadone and a bolus followed by constant rate infusion of racemic methadone. The data suggest that the assay is well suited for pharmacokinetic purposes.

Cognitive Impairment That Is Induced by (R)-Ketamine Is Abolished in NMDA GluN2D Receptor Subunit Knockout Mice.

Although the N-methyl-D-aspartate receptor antagonist ketamine has attracted attention because of its rapid and sustained antidepressant effects in depressed patients, its side effects have raised some concerns. Ketamine is a racemic mixture of equal amounts of the enantiomers (R)-ketamine and (S)-ketamine. The neural mechanisms that underlie the differential effects of these enantiomers remain unclear. We investigated cognitive impairment that was induced by ketamine and its enantiomers in N-methyl-D-aspartate GluN2D receptor subunit knockout (GluN2D-KO) mice. In the novel object recognition test, (RS)-ketamine and (S)-ketamine caused cognitive impairment in both wild-type and GluN2D-KO mice, whereas (R)-ketamine induced such cognitive impairment only in wild-type mice. The present results suggest that the GluN2D subunit plays an important role in cognitive impairment that is induced by (R)-ketamine, whereas this subunit does not appear to be involved in cognitive impairment that is induced by (RS)-ketamine or (S)-ketamine.

Halogen Substitution Influences Ketamine Metabolism by Cytochrome P450 2B6: In Vitro and Computational Approaches.

Ketamine is analgesic at anesthetic and subanesthetic doses, and it has been used recently to treat depression. Biotransformation mediates ketamine effects, influencing both systemic elimination and bioactivation. CYP2B6 is the major catalyst of hepatic ketamine N-demethylation and metabolism at clinically relevant concentrations. Numerous CYP2B6 substrates contain halogens. CYP2B6 readily forms halogen-protein (particularly Cl-π) bonds, which influence substrate selectivity and active site orientation. Ketamine is chlorinated, but little is known about the metabolism of halogenated analogs. This investigation evaluated halogen substitution effects on CYP2B6-catalyzed ketamine analogs N-demethylation in vitro and modeled interactions with CYP2B6 using various computational approaches. Ortho phenyl ring halogen substituent changes caused substantial (18-fold) differences in Km, on the order of Br (bromoketamine, 10 µM) < Cl < F < H (deschloroketamine, 184 µM). In contrast, Vmax varied minimally (83-103 pmol/min/pmol CYP). Thus, apparent substrate binding affinity was the major consequence of halogen substitution and the major determinant of N-demethylation. Docking poses of ketamine and analogs were similar, sharing a π-stack with F297. Libdock scores were deschloroketamine < bromoketamine < ketamine < fluoroketamine. A Bayesian log Km model generated with Assay Central had a ROC of 0.86. The probability of activity at 15 µM for ketamine and analogs was predicted with this model. Deschloroketamine scores corresponded to the experimental Km, but the model was unable to predict activity with fluoroketamine. The binding pocket of CYP2B6 also suggested a hydrophobic component to substrate docking, on the basis of a strong linear correlation ( R2 = 0.92) between lipophilicity ( Alog P) and metabolism (log Km) of ketamine and analogs. This property may be the simplest design criteria to use when considering similar compounds and CYP2B6 affinity.

Predosing Chemical Stability of Admixtures of Propofol, Ketamine, Fentanyl, and Remifentanil.

Admixtures of propofol-ketamine, propofol-ketamine-fentanyl, and propofol-ketamine-remifentanil were subjected to various clinically relevant conditions to study their chemical stability. A novel high-performance liquid chromatography-mass spectrometry method revealed no degradation of any compound by incubation at 37°C, constant mixing, or table-top storage for 6- and 24-hour time periods, except variable recovery of both propofol and fentanyl in the admixtures of propofol-ketamine-fentanyl suggesting possible degradation.

Quality control and stability of ketamine, remifentanil, and sufentanil syringes in a pediatric operating theater.

BACKGROUND: Transforming a drug from its commercial form into a ready-to-use drug is common practice, especially in pediatrics. However, the risk of compounding error is real and data on drug stability in practice are not always available. AIMS: The aim of this study was to assess, in real conditions, both the error rate and stability of three drugs: ketamine, remifentanil, and sufentanil. METHODS: A new rapid and easy-to-use high-performance liquid chromatography method with a diode array detector has been developed and validated to quantify these drugs and detect their degradation products. Over a 1-month period, 151 syringes were collected in the postanesthesia care unit. Seventy-three were stock solution syringes containing a 10-fold dilution of commercial drugs and 78 were serial dilution syringes made from successive dilutions of stock solutions. A comparison between real and expected concentrations as well as the detection of possible degradation products was carried out on these samples. RESULTS: All stock solution syringes had good chemical stability throughout the working day. A 4-µg/mL remifentanil serial dilution syringe, however, had to be discarded as a degradation peak was detected. Overall, 15.3% (95% CI, 9.5-21.1%) of syringes had a drug concentration outside the ±10% acceptability range, that is, 11.0% (95% CI, 3.7-18.2%) and 19.5% (95% CI, 10.6%-28.4%) of stock and diluted syringes respectively, with drug amounts ranging from -25.3% to 22.0%. The highest error rates were observed with sufentanil syringes: 20% and 28% for stock solution and serial dilution, respectively. CONCLUSION: The study shows that stock solution syringes prepared in advance are chemically stable throughout the day, unlike certain serial dilution syringes, indicating that the latter should be prepared just before administration to ensure chemical stability. Our results show that the error rate for serial dilution syringes is twice that of stock solution. Different safety measures are under discussion and have to be further studied.