Inhibition of cytochrome P450 enzymes involved in ketamine metabolism by use of liver microsomes and specific cytochrome P450 enzymes from horses, dogs, and humans

To identify and characterize cytochrome P450 enzymes (CYPs) responsible for the metabolism of racemic ketamine in 3 mammalian species in vitro by use of chemical inhibitors and antibodies.

Enantioselective capillary electrophoresis for the assessment of CYP3A4-mediated ketamine demethylation and inhibition in vitro

Enantioselective CE with sulfated cyclodextrins as chiral selectors was used to determine the CYP3A4-catalyzed N-demethylation kinetics of ketamine to norketamine and its inhibition in the presence of ketoconazole in vitro. Ketamine, a chiral phencyclidine derivative, was incubated with recombinant human CYP3A4 from a baculovirus expression system as racemic mixture and as single enantiomer. Alkaline liquid/liquid extracts of the samples were analyzed with a pH 2.5 buffer comprising 50 mM Tris and phosphoric acid together with either multiple isomer sulfated β-cyclodextrin (10 mg/mL) or highly sulfated γ-cyclodextrin (2%, w/v). Data obtained in the absence of ketoconazole revealed that the N-demethylation occurred stereoselectively with Michaelis-Menten (incubation of racemic ketamine) and Hill (separate incubation of single enantiomers) kinetics. Data generated in the presence of ketoconazole as the inhibitor could best be fitted to a one-site competitive model and inhibition constants were calculated using the equation of Cheng and Prusoff. No stereoselective difference was observed, but inhibition constants for the incubation of racemic ketamine were found to be larger compared with those obtained with the incubation of single ketamine enantiomers.

Comparison of the effects of racemic ketamine and S-ketamine for anesthesia in Rheem gazelles (Gazella subgutturosa marica) and Subgutturosa gazelles (Gazella subgutturosa subgutturosa)

OBJECTIVE: To evaluate effects of racemic ketamine and S-ketamine in gazelles. ANIMALS: 21 male gazelles (10 Rheem gazelles [Gazella subgutturosa marica] and 11 Subgutturosa gazelles [Gazella subgutturosa subgutturosa]), 6 to 67 months old and weighing (mean+/-SD) 19 +/- 3 kg. PROCEDURES: In a randomized, blinded crossover study, a combination of medetomidine (80 mug/kg) with racemic ketamine (5 mg/kg) or S-ketamine (3 mg/kg) was administered i.m.. Heart rate, blood pressure, respiratory rate, rectal temperature, and oxygen saturation (determined by means of pulse oximetry) were measured. An evaluator timed and scored induction of, maintenance of, and recovery from anesthesia. Medetomidine was reversed with atipamezole. The alternate combination was used after a 4-day interval. Comparisons between groups were performed with Wilcoxon signed rank and paired t tests. RESULTS: Anesthesia induction was poor in 2 gazelles receiving S-ketamine, but other phases of anesthesia were uneventful. A dominant male required an additional dose of S-ketamine (0.75 mg/kg, i.m.). After administration of atipamezole, gazelles were uncoordinated for a significantly shorter period with S-ketamine than with racemic ketamine. Recovery quality was poor in 3 gazelles with racemic ketamine. No significant differences between treatments were found for any other variables. Time from drug administration to antagonism was similar between racemic ketamine (44.5 to 53.0 minutes) and S-ketamine (44.0 to 50.0 minutes). CONCLUSIONS AND CLINICAL RELEVANCE: Administration of S-ketamine at a dose 60% that of racemic ketamine resulted in poorer induction of anesthesia, an analogous degree of sedation, and better recovery from anesthesia in gazelles with unremarkable alterations in physiologic variables, compared with racemic ketamine.

Antinociceptive effects, metabolism and disposition of ketamine in ponies under target-controlled drug infusion

Ketamine is widely used as an anesthetic in a variety of drug combinations in human and veterinary medicine. Recently, it gained new interest for use in long-term pain therapy administered in sub-anesthetic doses in humans and animals. The purpose of this study was to develop a physiologically based pharmacokinetic (PBPk) model for ketamine in ponies and to investigate the effect of low-dose ketamine infusion on the amplitude and the duration of the nociceptive withdrawal reflex (NWR). A target-controlled infusion (TCI) of ketamine with a target plasma level of 1 microg/ml S-ketamine over 120 min under isoflurane anesthesia was performed in Shetland ponies. A quantitative electromyographic assessment of the NWR was done before, during and after the TCI. Plasma levels of R-/S-ketamine and R-/S-norketamine were determined by enantioselective capillary electrophoresis. These data and two additional data sets from bolus studies were used to build a PBPk model for ketamine in ponies. The peak-to-peak amplitude and the duration of the NWR decreased significantly during TCI and returned slowly toward baseline values after the end of TCI. The PBPk model provides reliable prediction of plasma and tissue levels of R- and S-ketamine and R- and S-norketamine. Furthermore, biotransformation of ketamine takes place in the liver and in the lung via first-pass metabolism. Plasma concentrations of S-norketamine were higher compared to R-norketamine during TCI at all time points. Analysis of the data suggested identical biotransformation rates from the parent compounds to the principle metabolites (R- and S-norketamine) but different downstream metabolism to further metabolites. The PBPk model can provide predictions of R- and S-ketamine and norketamine concentrations in other clinical settings (e.g. horses).

Characterization of the stereoselective biotransformation of ketamine to norketamine via determination of their enantiomers in equine plasma by capillary electrophoresis

A robust CE method for the simultaneous determination of the enantiomers of ketamine and norketamine in equine plasma is described. It is based upon liquid-liquid extraction of ketamine and norketamine at alkaline pH from 1 mL plasma followed by analysis of the reconstituted extract by CE in the presence of a pH 2.5 Tris-phosphate buffer containing 10 mg/mL highly sulfated beta-CD as chiral selector. Enantiomer plasma levels between 0.04 and 2.5 microg/mL are shown to provide linear calibration graphs. Intraday and interday precisions evaluated from peak area ratios (n = 5) at the lowest calibrator concentration are < 8 and < 14%, respectively. The LOD for all enantiomers is 0.01 microg/mL. After i.v. bolus administration of 2.2 mg/kg racemic ketamine, the assay is demonstrated to provide reliable data for plasma samples of ponies under isoflurane anesthesia, of ponies premedicated with xylazine, and of one horse that received romifidine, L-methadone, guaifenisine, and isoflurane. In animals not premedicated with xylazine, the ketamine N-demethylation is demonstrated to be enantioselective. The concentrations of the two ketamine enantiomers in plasma are equal whereas S-norketamine is found in a larger amount than R-norketamine. In the group receiving xylazine, data obtained do not reveal this stereoselectivity.

Enantioselective analysis of ketamine and its metabolites in equine plasma and urine by CE with multiple isomer sulfated beta-CD

CE with multiple isomer sulfated beta-CD as the chiral selector was assessed for the simultaneous analysis of the enantiomers of ketamine and metabolites in extracts of equine plasma and urine. Different lots of the commercial chiral selector provided significant changes in enantiomeric ketamine separability, a fact that can be related to the manufacturing variability. A mixture of two lots was found to provide high-resolution separations and interference-free detection of the enantiomers of ketamine, norketamine, dehydronorketamine, and an incompletely identified hydroxylated metabolite of norketamine in liquid/liquid extracts of the two body fluids. Ketamine, norketamine, and dehydronorketamine could be unambiguously identified via HPLC fractionation of urinary extracts and using LC-MS and LC-MS/MS with 1 mmu mass discrimination. The CE assay was used to characterize the stereoselectivity of the compounds' enantiomers in the samples of five ponies anesthetized with isoflurane in oxygen and treated with intravenous continuous infusion of racemic ketamine. The concentrations of the ketamine enantiomers in plasma are equal, whereas the urinary amount of R-ketamine is larger than that of S-ketamine. Plasma and urine contain higher S- than R-norketamine levels and the mean S-/R-enantiomer ratios of dehydronorketamine in plasma and urine are lower than unity and similar.

Anaesthesia for castration of piglets: comparison between intranasal and intramuscular application of ketamine, climazolam and azaperone

The aim of this study was to compare the effect of an anaesthetic combination given either intramuscularly (IM) or intranasally (IN) for castration of piglets. Forty piglets aged 4 to 7 days were randomly assigned to receive a mixture of ketamine 15 mg kg-1, climazolam 1.5 mg kg-1 and azaperone 1.0 mg kg-1, IN or IM, 10 minutes prior to castration. Physiological parameters were measured. Castration was videotaped for evaluation by 3 independent observers using a scoring system. Reaction and vocalization to the skin incision and cutting of spermatic cord was evaluated and scored (0 = no reaction, 16 = strong reaction). The IN group had a significantly higher (P < 0.01) castration score, compared to the IM group. There was an association between castration score and room temperature in the IN group (with temperatures below 18 "C associated with a higher castration scores (P < 0.001). Heart rate was significantly higher 10 minutes after castration in the IN group (P < 0.05). Respiratory rate was significantly higher in the IM group at time points -5, -1, 10, 20 and 30 (P < 0.05).The IN group was walking significantly (P < 0.0001) faster than the IM group. In conclusion, this combination provides effective anaesthesia for routine castration of newborn piglets when administered IM. IN administration provided shorter recovery times but had significantly higher castration scores.

Stereoselective pharmacokinetics of ketamine and norketamine after racemic ketamine or S-ketamine administration in Shetland ponies sedated with xylazine

The pharmacokinetics of ketamine and norketamine enantiomers after administration of intravenous (IV) racemic ketamine (R-/S-ketamine; 2.2mg/kg) or S-ketamine (1.1mg/kg) to five ponies sedated with IV xylazine (1.1mg/kg) were compared. The time intervals to assume sternal and standing positions were recorded. Arterial blood samples were collected before and 1, 2, 4, 6, 8 and 13min after ketamine administration. Arterial blood gases were evaluated 5min after ketamine injection. Plasma concentrations of ketamine and norketamine enantiomers were determined by capillary electrophoresis and were evaluated by non-linear least square regression analysis applying a monocompartmental model. The first-order elimination rate constant was significantly higher and elimination half-life and mean residence time were lower for S-ketamine after S-ketamine compared to R-/S-ketamine administration. The maximum concentration of S-norketamine was higher after S-ketamine administration. Time to standing position was significantly diminished after S-ketamine compared to R-/S-ketamine. Blood gases showed low-degree hypoxaemia and hypercarbia.

Stereoselective pharmacokinetics of ketamine and norketamine after racemic ketamine or S-ketamine administration during isoflurane anaesthesia in Shetland ponies

BACKGROUND: The arterial pharmacokinetics of ketamine and norketamine enantiomers after racemic ketamine or S-ketamine i.v. administration were evaluated in seven gelding ponies in a crossover study (2-month interval). METHODS: Anaesthesia was induced with isoflurane in oxygen via a face-mask and then maintained at each pony's individual MAC. Racemic ketamine (2.2 mg kg(-1)) or S-ketamine (1.1 mg kg(-1)) was injected in the right jugular vein. Blood samples were collected from the right carotid artery before and at 1, 2, 4, 8, 16, 32, 64, and 128 min after ketamine administration. Ketamine and norketamine enantiomer plasma concentrations were determined by capillary electrophoresis. Individual R-ketamine and S-ketamine concentration vs time curves were analysed by non-linear least square regression two-compartment model analysis using PCNonlin. Plasma disposition curves for R-norketamine and S-norketamine were described by estimating AUC, C(max), and T(max). Pulse rate (PR), respiratory rate (R(f)), tidal volume (V(T)), minute volume ventilation (V(E)), end-tidal partial pressure of carbon dioxide (PE'(CO(2))), and mean arterial blood pressure (MAP) were also evaluated. RESULTS: The pharmacokinetic parameters of S- and R-ketamine administered in the racemic mixture or S-ketamine administered separately did not differ significantly. Statistically significant higher AUC and C(max) were found for S-norketamine compared with R-norketamine in the racemic group. Overall, R(f), V(E), PE'(CO(2)), and MAP were significantly higher in the racemic group, whereas PR was higher in the S-ketamine group. CONCLUSIONS: Norketamine enantiomers showed different pharmacokinetic profiles after single i.v. administration of racemic ketamine in ponies anaesthetised with isoflurane in oxygen (1 MAC). Cardiopulmonary variables require further investigation.

Managing chronic whiplash associated pain with a combination of low-dose opioid (remifentanil) and NMDA-antagonist (ketamine)

The aim was to investigate the efficacy of a combination of low-dose remifentanil (REMI) and ketamine (KET) compared to the single drugs and placebo (P) on whiplash associated pain (WAD) in a double-blind, randomized, placebo-controlled, cross-over study. Twenty patients with chronic (>1 year) WAD were included. Four different drug combinations were tested in four sessions: placebo/placebo (P/P), placebo/remifentanil (P/REMI), ketamine/placebo (KET/P) and ketamine/remifentanil (KET/REMI). Target concentrations were 1 and 2ng/ml (stepwise) for remifentanil and 100ng/ml for ketamine. Habitual pain intensity was assessed on a visual analogue scale (VAS). Experimental pain was assessed with electrical stimulation (single and repeated) of tibialis anterior (TA) muscle, pressure pain algometry applied over infraspinatus (IS) and TA muscles and VAS scores after intramuscular hypertonic saline infusion in TA. KET/REMI significantly reduced habitual pain. KET/REMI infused at low REMI target concentration (1ng/ml) significantly elevated electrical intramuscular pain thresholds (single and repeated). Pain thresholds to electrical stimulation were similarly increased by both P/REMI and KET/REMI at 2ng/ml target concentration. Pressure pain thresholds were increased by both KET/REMI and P/REMI. VAS-scores after intramuscular saline were also similarly decreased by both REMI combinations. Seven out of 20 subjects were non-responders (<50% pain relief). No correlation was found between effects on spontaneous pain and experimental pain. KET/REMI showed an analgesic effect on habitual pain. Experimental pain was attenuated by both combinations containing the opioid, however, KET seemed to enhance the effect of REMI on electrical pain thresholds when a low REMI target concentration was used.

Chronic phantom limb pain: the effects of calcitonin, ketamine, and their combination on pain and sensory thresholds

BACKGROUND: Calcitonin was effective in a study of acute phantom limb pain, but it was not studied in the chronic phase. The overall literature on N-methyl-D-aspartate antagonists is equivocal. We tested the hypothesis that calcitonin, ketamine, and their combination are effective in treating chronic phantom limb pain. Our secondary aim was to improve our understanding of the mechanisms of action of the investigated drugs using quantitative sensory testing. METHODS: Twenty patients received, in a randomized, double-blind, crossover manner, 4 i.v. infusions of: 200 IE calcitonin; ketamine 0.4 mg/kg (only 10 patients); 200 IE of calcitonin combined with ketamine 0.4 mg/kg; placebo, 0.9% saline. Intensity of phantom pain (visual analog scale) was recorded before, during, at the end, and the 48 h after each infusion. Pain thresholds after electrical, thermal, and pressure stimulation were recorded before and during each infusion. RESULTS: Ketamine, but not calcitonin, reduced phantom limb pain. The combination was not superior to ketamine alone. There was no difference in basal pain thresholds between the amputated and contralateral side except for pressure pain. Pain thresholds were unaffected by calcitonin. The analgesic effect of the combination of calcitonin and ketamine was associated with a significant increase in electrical thresholds, but with no change in pressure and heat thresholds. CONCLUSIONS: Our results question the usefulness of calcitonin in chronic phantom limb pain and stress the potential interest of N-methyl-D-aspartate antagonists. Sensory assessments indicated that peripheral mechanisms are unlikely important determinants of phantom limb pain. Ketamine, but not calcitonin, affects central sensitization processes that are probably involved in the pathophysiology of phantom limb pain.

Is the combination of morphine with ketamine better than morphine alone for postoperative intravenous patient-controlled analgesia?

BACKGROUND: The addition of ketamine to morphine for patient-controlled analgesia (PCA) is supported by previous basic and clinical research, but has been challenged by subsequent negative studies. Important limitations of previous studies are the low number of patients analyzed, the use of morphine-ketamine combinations that may not the optimal, and that not all the relevant outcomes have been analyzed. In this study, we compared the combination of morphine and ketamine with morphine alone for postoperative PCA in large patient groups. We used a morphine-ketamine combination identified by an optimization procedure in our previous study. METHODS: After major elective orthopedic surgery, 352 patients received either PCA with morphine bolus 1.5 mg (Group M, n = 176) or a bolus of morphine plus ketamine 1.5 mg each (Group MK, n = 176) in a randomized, double-blind fashion. Unsatisfactory treatment was defined as the occurrence of either inadequate analgesia or unacceptable side effects. In addition, total consumption of PCA drugs, duration of PCA use, direct medical costs, and number of patients with chronic postoperative pain 3 and 6 mo after operation were recorded. RESULTS: The incidence of unsatisfactory treatment was 33.0% in Group M and 36.9% in Group MK (P = 0.50). No significant differences were found between the groups with respect to secondary end points. CONCLUSIONS: Small-dose ketamine combined with morphine for PCA provides no benefit to patients undergoing major orthopedic surgery and cannot be recommended for routine use.

Stereoselective biotransformation of ketamine in equine liver and lung microsomes

Stereoselectivity has to be considered for pharmacodynamic and pharmacokinetic features of ketamine. Stereoselective biotransformation of ketamine was investigated in equine microsomes in vitro. Concentration curves were constructed over time, and enzyme activity was determined for different substrate concentrations using equine liver and lung microsomes. The concentrations of R/S-ketamine and R/S-norketamine were determined by enantioselective capillary electrophoresis. A two-phase model based on Hill kinetics was used to analyze the biotransformation of R/S-ketamine into R/S-norketamine and, in a second step, into R/S-downstream metabolites. In liver and lung microsomes, levels of R-ketamine exceeded those of S-ketamine at all time points and S-norketamine exceeded R-norketamine at time points below the maximum concentration. In liver and lung microsomes, significant differences in the enzyme velocity (V(max)) were observed between S- and R-norketamine formation and between V(max) of S-norketamine formation when S-ketamine was compared to S-ketamine of the racemate. Our investigations in microsomal reactions in vitro suggest that stereoselective ketamine biotransformation in horses occurs in the liver and the lung with a slower elimination of S-ketamine in the presence of R-ketamine. Scaling of the in vitro parameters to liver and lung organ clearances provided an excellent fit with previously published in vivo data and confirmed a lung first-pass effect.

Effects of a low dose infusion of racemic and S-ketamine on the nociceptive withdrawal reflex in standing ponies

OBJECTIVE: To investigate the effect of plasma concentrations obtained by a low dose constant rate infusion (CRI) of racemic ketamine or S-ketamine on the nociceptive withdrawal reflex (NWR) in standing ponies. STUDY DESIGN: Prospective, blinded, cross-over study. ANIMALS: Six healthy 5-year-old Shetland ponies. METHODS: Ponies received either 0.6 mg kg(-1) racemic ketamine (group RS) or 0.3 mg kg(-1) S-ketamine (group S) intravenously (IV), followed by a CRI of 20 microg kg(-1)minute(-1) racemic ketamine (group RS) or 10 microg kg(-1)minute(-1) S-ketamine (group S) for 59 minutes. The NWR was evoked by transcutaneous electrical stimulation of a peripheral nerve before drug administration, 15 and 45 minutes after the start of the bolus injection and 15 minutes after the end of the CRI. Electromyographic responses were recorded and analysed. Arterial blood was collected before stimulation and plasma concentrations of ketamine and norketamine were measured enantioselectively using capillary electrophoresis. Ponies were video recorded and monitored to assess drug effects on behaviour, heart rate (HR), mean arterial blood pressure (MAP) and respiratory rate. RESULTS: The NWR was significantly depressed in group RS at plasma concentrations between 20 and 25 ng mL(-1) of each enantiomer. In group S, no significant NWR depression could be observed; plasma concentrations of S-ketamine (9-15 ng mL(-1)) were lower, compared to S-ketamine concentrations in group RS, although this difference was not statistically significant. Minor changes in behaviour, HR and MAP only occurred within the first 5-10 minutes after bolus drug administration in both groups. CONCLUSION: Antinociceptive activity in standing ponies, demonstrated as a depression of the NWR, could only be detected after treatment with racemic ketamine. S-ketamine may have lacked this effect as a result of lower plasma concentrations, a more rapid metabolism or a lower potency of S-ketamine in Equidae so further investigation is necessary.