- Email: [email protected]
Tissue uptake of ketamine and norketamine enantiomers in the rat Indirect evidence for extrahepatic metabolic inversion
Life Sciences 69 (2001) 2051–2066
Tissue uptake of ketamine and norketamine enantiomers in the rat Indirect evidence for extrahepatic metabolic inversion Stephen R. Edwards, Laurence E. Mather* Department of Anaesthesia and Pain Management, University of Sydney at Royal North Shore Hospital, St. Leonards, NSW 2065, Australia Received 16 October 2000; accepted 2 April 2001
Abstract Ketamine, used clinically as an intravenous analgetic and dissociative anaesthetic agent, is a racemate with both pharmacokinetic and pharmacodynamic enantioselectivity. S-ketamine has been found have a higher clearance and greater potency than R-ketamine as well as a greater therapeutic index. We performed a study in rats with two complementary paradigms: (i) constant rate “washin” infusion until fatal, (ii) brief infusion then “washout”. These, respectively, allowed examination of ketamine and norketamine serial plasma enantiomer concentrations and tissue distribution at maximal and minimal drug effects. Both paradigms found plasma concentrations of R-ketamine.S-ketamine; however, tissue distribution coefficients for S-ketamine.R-ketamine. For paradigm (i), plasma concentrations of R-norketamine.S-norketamine; for paradigm (ii), R-norketamine..S-norketamine initially, but S-norketamine..R-norketamine later. Comparison of distribution coefficients of ketamine and norketamine enantiomers for the two paradigms provided indirect evidence for metabolic inversion. During washin, when circulating concentrations of ketamine enantiomers were high, uptake and metabolism occurred predominantly in the kidney and to a lesser extent in liver, lung and gut, with formation of R-norketamine by a (presumed) first-order process predominating. However, following washout, when circulating concentrations of ketamine enantiomers were low, uptake and metabolism was dominated by the kidney and gut. Under these conditions inversion of R- to S-ketamine appeared to predominate with subsequent metabolism to S-norketamine by (presumed) zero-order processes. In summary, different profiles for the uptake and metabolism of ketamine enantiomers were apparent following constant rate washin, and brief infusion washout, paradigms with i.v. rac-ketamine. Uptake into most tissues, and metabolism in some tissues, was enantioselective. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Regional pharmacokinetics; Distribution coefficient; Intravenous infusion
* Corresponding author. Tel.: 161-2-99268420; fax: 161-2-99064079. E-mail address: [email protected] (L.E. Mather) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 8 7 -5
2052
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
Introduction The arylcyclohexylamine derivative, ketamine, is an N-methyl-D-aspartate (NMDA) receptor antagonist [1]. It has been used for over 30 years as an analgetic and dissociative anaesthetic where it offers the clinical advantages of administration by intravenous mode, short duration of action, lack of cardiovascular depressant effects, and rapid recovery [2–7]. However, ketamine has the clinical disadvantage of sometimes causing psychotomimetic effects [2,8,9]. Ketamine, as currently in clinical use, is a racemic drug containing equal amounts of the R- and S-ketamine enantiomers that have been found, in early animal laboratory studies, to have differences in the relative potencies and effects. As examples, the therapeutic index in rats, expressed by the ratio of the LD50 to the ED50 for hypnosis, was found to be greater for S-ketamine (510) than for either rac-ketamine (56.25) or R-ketamine (54.0); the posthypnotic psychomotor activation following administration of equihypnotic doses in rats was significantly less for S-ketamine than R-ketamine [10]; the analgesic potency of S-ketamine in mice was 3 times greater than R-ketamine, but psychomotor activation from equianalgetic doses was less for S-ketamine [11]. Similar differences in potency and potency ratios for ketamine enantiomers have been reported from human laboratory studies. As examples, R-ketamine was found to cause less slowing in the electroencephalogram (EEG) than either S- or rac-ketamine [12]; the anaesthetic potency of S-ketamine was 4 times more potent than that of R-ketamine in surgical patients, however, the psychotomimetic effects in the emergence period were more pronounced for R-ketamine, while posthypnotic analgesia was greater for S-ketamine [13]; the analgetic potency of S-ketamine was 4 times that of R-ketamine in studies of experimentally induced ischaemic pain [8]; Additionally, pharmacokinetic differences in surgical patients were found after administration of rac-ketamine, such that the plasma clearance of S-ketamine was z16% greater than that of R-ketamine [14]. Norketamine is the principal metabolite of ketamine [15]. Work with human liver microsomes indicated that the rate of N-demethylation was greater with S-ketamine than with rac- or R-ketamine [16]. An earlier clinical investigation of the pharmacokinetics and analgesic effects of ketamine and norketamine, following intramuscular and oral administration of ketamine, indicated that norketamine also had analgetic properties [17]. It was suggested, on the basis of the affinities of ketamine and norketamine enantiomers for the NMDA receptor, that S-norketamine contributes to the analgetic effect of ketamine following administration of rac-ketamine [18]. The present investigation examined further the enantioselectivity of ketamine and norketamine plasma and tissue concentrations following the administration of rac-ketamine with two complementary i.v. administration paradigms. In the first paradigm, a constant rate washin infusion until lethality was examined; in the second paradigm, a brief infusion followed by a prolonged washout was examined. Methods Animals and their preparation Adult male Wistar rats (350–400g; Gore Hill Animal Research Laboratories, Sydney, AUS) were housed in groups of four with free access to food and water on a constant 12/12
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2053
hour light dark cycle at 218C. Surgical procedures were performed under general anaesthesia (pentobarbitone, 30 mg/Kg, followed 5 min later by ketamine, 45 mg/Kg; both i.p. in 1 mL 0.9% saline). Body temperature was maintained by a heating pad and monitored with a rectal probe. Chronic indwelling cannulae were implanted into the jugular vein and carotid artery to allow simultaneous venous infusion and arterial sampling. A 1 cm incision was made just lateral to the midline, and the jugular vein and carotid artery were exposed. Silastic laboratory tubing (respectively, 0.025 in ID 3 0.047 in OD, and 0.020 in ID 3 0.037 in OD) was inserted 2.5 cm into the jugular vein, and 2.0 cm into the carotid artery. Each cannula line was tunnelled under the skin and externalised above the neck anterior to the shoulder blades and filled with a solution containing 6 g polyvinylpyrrolidone (MW 40,000, Sigma Chemical Co, St Louis, MO, USA) dissolved in 5 mL of 1000 U/mL sodium heparin (Delta West Pty Ltd, Perth, WA, AUS) in order to maintain patency. Postoperatively, buprenorphine (0.2 mg/ Kg, s.c.) was administered for pain relief, and procaine penicillin and benzathine penicillin (standard veterinary preparation, each 150 mg/mL, 0.1 mL, s.c.) was given for antimicrobial cover. Following surgery, the animals were housed individually and their post-operative body weights and fluid intake were recorded. Ketamine enantiomer assays The enantiomers of ketamine and norketamine were separated on a micro chiral-AGP column (150 mm 3 2 mm, 5 mm, Chromtech) using a Hewlett Packard series 1100 LC system, and quantitated by spectrophotometric detection at 215 nm. The method was adapted from a previously described procedure [19]. The mobile phase, containing ammonium acetate (30 mM, adjusted to pH 7.0, in 3.0% v/v isopropanol), was used at a flow rate of 0.22 mL/min. Plasma aliquots (50 ml) were extracted in Eppendorf tubes (1.5 mL) after addition of internal standard (N-benzylmethylamine, Sigma-Aldrich, 50 ml, 18.4 mM) and Na3PO4 (100 ml, 0.5M). Tissue samples were homogenised into NaH2PO4 (0.1M) to a concentration of 100 mg/mL and frozen. After thawing and resuspension tissue aliquots (200 ml) were extracted in Eppendorf tubes (2.0 mL) following the addition of internal standard (50 ml, 18.4 mM) and Na3PO4 (200 ml, 0.5M). Tubes were briefly vortex-mixed before the addition iso-amyl alcohol in cyclohexane (2% v/v, 1.0 mL). Samples were subsequently shaken for 5 min, centrifuged in a bench centrifuge (3000 rpm for 2 min), and the organic layer decanted with a Pasteur pipette into a fresh Eppendorf tube (1.5 mL). Aliquots of H3PO4 (100 ml, 0.5M) were added to each tube, the tubes were shaken and centrifuged as before, and the organic layer was discarded. Samples were then alkalinized (NaOH, 30 ml, 5M), briefly vortex-mixed, and re-extracted into iso-amyl alcohol in cyclohexane (2% v/v, 1.0 mL) by shaking as before. After centrifugation the organic layer was transferred to a fresh Eppendorf tube (1.5 mL) and dried in a rotatory vacuum bench evaporator (408C). The residue was reconstituted into ammonium acetate buffer (100 ml, 20 mM, pH 5, containing 10% v/v isopropanol), and sonicated (5 min) before transfer to polypropylene inserts (250 ml) and injection of aliquots (10 ml) onto the column. Experimental design The studies were carried out 24 hours after cannulation between 1400–1900 hours. Infusion and sampling lines (respectively, 75 and 45 cm) were attached to the venous and arterial
2054
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
lines, and the animals were allowed to settle (30 min) in a study chamber before commencement of the study. rac-Ketamine as HCl salt (Ketalar™, Parke-Davis Australia Pty Ltd, Sydney, AUS) was diluted with deionised water to 20 mg/mL (17.3 mg/mL as base) and infused at the appropriate rate. The infusions were delivered from a gas tight syringe (Hamilton, 5 mL) using a syringe driver (Harvard Apparatus Model 22). Immediately after the righting reflex was lost, following removal from the study chamber, a thermal probe was inserted rectally and body temperature was maintained with a heating lamp. Blood samples (100 ml) were collected into heparinised (50U) polyethylene microfuge tubes. At the conclusion of the study, brain, spinal cord, heart, lung, liver, kidney, gut, adductor muscle, and epididymal fat were sampled for the determination of tissue: plasma drug distribution. The brain was divided into forebrain, brainstem, and cerebellum; the spinal cord was divided into brachiothoracic and lumbo-sacral regions. In the “washin” paradigm, an infusion of rac-ketamine was administered at a constant rate of 6 mg/Kg/min until lethality ensued. Serial blood samples were taken at 2 min intervals. A consistent loss of foot withdrawal reflex upon toe pinch was used to determine the onset of anaesthesia; cessation of respiration following the agonal gasp was used as the point of lethality. In the “washout” paradigm, an infusion of rac-ketamine was administered at a constant rate of 10 mg/Kg/min for 5 min; serial blood samples were taken at 0, 1.5, 3, 5, 7, 10, 15, 20, 30, 40, 60, 80, 100, 120, 140, 160 and 180 min. Once motor function began to return the rectal probe was removed and rats were returned to the study chamber. Data analysis Data are expressed as mean (6SEM) unless described as otherwise. Area under the curve (AUC) values for each plasma concentration-time data set were determined by the linear trapezoid method. Distribution coefficients were calculated as the ratio of tissue to plasma analyte concentrations, uncorrected for residual tissue blood volume. Student’s t-test for paired data was used for the pairwise comparison of enantiomer AUC values, tissue concentrations, and distribution coefficients, while the respective R:S enantiomer ratios were compared to unity with Student’s one sample t-test. Regional CNS tissue enantiomer concentrations and distribution coefficients were compared by one-way analysis of variance (AOV) with additional two-factor AOV for comparisons between ketamine and norketamine in the washin paradigm. Individual post-hoc comparisons were performed by the method of Least Significant Difference (LSD). Log-transformed values for regional CNS distribution coefficients were compared by AOV in the two paradigms. The logtransformed values for the peripheral tissue distribution coefficients in the two paradigms were compared by Student’s two sample t-tests. Pharmacokinetic parameters for each ketamine enantiomer were determined from fitting polyexponential decay equations to the concentration-time washout curve, as previously described, from which the mean total body clearance (ClT), initial dilution volume (VC), volume of distribution at steady state equilibrium (VSS), terminal half life (T1/2) and mean residence time in the body (MRT) were calculated by conventional means [20]. Enantioselectivity in pharmacokinetics was determined from the S:R enantiomer ratio when compared to unity with Student’s one sample t-test.
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2055
Results Ketamine enantiomer assays Plasma calibration curves (n58) were linear over an enantiomer concentration range of 0.22–43.35 mg/ml for both R- (R250.998460.0006) and S-ketamine (R250.998560.0006). The minimum limit of detectability in plasma, at a signal to noise ratio of 2:1, was 0.09 mg/ml for each ketamine enantiomer. Assay coefficients of variation were based on the slopes of the plasma calibration curves. Within-assay coefficients of variation (n56) for R- and S-ketamine were 4.3% and 4.2%, respectively; between-assay coefficients of variation (n58) were 5.5% and 5.8%, respectively. Recoveries of R- and S-ketamine from plasma were 77.061.4% and 76.961.2% at enantiomer concentrations of 2.2 mg/ml (n58); recovery was 80.761.2% and 80.461.0% respectively at enantiomer concentrations of 22 mg/ml (n57). Tissue homogenates were assayed against standards, prepared in the relevant respective drug-free tissue homogenates, to which ketamine enantiomer concentrations between 0.2 – 43.4 mg/ml had been added. Norketamine enantiomer concentrations were quantified using calibration curves derived from ketamine enantiomer standards. Washin paradigm (constant rate infusion) The anaesthetic dose to lack of response to toe pinch was 10163 mg/Kg and the lethal dose was 14767 mg/Kg. Plasma concentrations of R- and S-ketamine, and the corresponding concentrations of R- and S-norketamine, are shown in Figure 1 for the duration of the infusion. The enantiomer concentration ratios for ketamine and norketamine are shown in Figure 2. Over the duration of the infusion, the plasma concentrations and AUC values of the R-enantiomers of both ketamine and norketamine exceeded the corresponding concentrations of the S-enantiomers (P50.0011 and P50.0001), and the AUC R:S enantiomer ratios were significantly greater than unity for both ketamine (P50.0004) and norketamine (P,0.0001). The tissue concentrations and distribution coefficients for ketamine and norketamine enantiomers at the conclusion of washin infusions are shown in Table 1. In all CNS regions, the concentrations of both R-ketamine and R-norketamine were significantly greater than those of their corresponding S-enantiomers (P,0.0001). However, the corresponding distribution coefficients for S-ketamine were significantly greater than those of R-ketamine (P#0.0005), but the distribution coefficients for R-norketamine were significantly greater than those of S-norketamine (P#0.005). Whereas significant differences between regional CNS concentrations of R- and S-ketamine were apparent (respectively, P50.0008 P50.002), there were no such differences for both norketamine enantiomers. Concentrations in the pons-medulla and cerebellum significantly exceeded those in the forebrain and spinal cord for R- and S-ketamine (respectively, P50.002 and P50.005). There were also regional differences in the CNS distribution coefficients of both R- and S-ketamine (both P,0.0001). Similar differences in the regional CNS distribution coefficients for R- and Snorketamine (respectively, P50.0004 and P5 0.0012) were also apparent. Distribution coefficients were significantly greater in the pons-medulla than the forebrain and spinal cord for both Rand S-ketamine (respectively, P50.002 and P50.005), and they were significantly greater in the pons-medulla and cerebellum than the spinal cord for R- and S-norketamine (respectively, P50.005 and P50.02). Finally, the regional CNS concentrations and distribution coefficients
2056
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
Fig. 1. Plasma concentrations of ketamine and norketamine enantiomers in individual rats (n58) during infusion with rac-ketamine at a constant rate of 6 mg/Kg/min until lethality (“washin” paradigm). AUC R vs S: P50.0011 for ketamine and P50.001 for norketamine (Student’s t-test for paired data).
of both norketamine enantiomers were significantly lower than those of the corresponding ketamine enantiomers (P50.001) in all CNS regions. Concentrations of S-ketamine were significantly greater than R-ketamine in liver and fat (both P,0.0001), but significantly lower in heart (P,0.0001). However, as in CNS tissue, the distribution coefficients for S-ketamine significantly exceeded those of R-ketamine in all peripheral tissues (P#0.01). Concentrations of R-norketamine were significantly greater than S-norketamine in kidney (P,0.005), heart (P,0.0001), lung (P,0.0001), and muscle (P,0.0001). The distribution coefficient for R-norketamine was significantly greater than that of S-norketamine in kidney (P,0.01), heart (P,0.0005), and muscle (P,0.0001), but significantly less than S-norketamine in liver (P,0.001) and lung (P,0.05). Washout paradigm (brief infusion) Plasma concentrations of the ketamine and norketamine enantiomers are shown in figure 3. Again, the AUC values for R-ketamine significantly exceeded those of S-ketamine (P,0.0001). However, the AUC values for S-norketamine were significantly greater (P50.0001) than
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2057
Fig. 2. Plasma enantiomer concentrations ratios for ketamine and norketamine in individual rats (n58) during infusion with rac-ketamine at a constant rate of 6 mg/Kg/min until lethality (“washin” paradigm). AUC R vs S: P50.0004 for ketamine and P,0.0001 for norketamine (Student’s one sample t-test).
those of R-norketamine, although early concentrations of R-norketamine were significantly higher (P,0.0001) than S-norketamine (at 7 min). The AUC R:S enantiomer ratios were significantly greater than unity for ketamine (P,0.0001), but significantly less than unity for norketamine (P,0.0001). Tissue concentrations and distribution coefficients for ketamine and norketamine enantiomers, 180 min after “washout infusion”, are shown in Table 2. Due to the variance in the distribution coefficients the data were subjected to a log-transformation; the anti-logs of the mean logtransformed values for each tissue are given in Table 2 as “log-normalized” values. Whilst the concentrations of R-ketamine were significantly greater than S-ketamine in all CNS regions (P#0.01), the distribution coefficients were not significantly different (P.0.05), except for the forebrain (P,0.05). Regional differences in CNS tissue concentrations and distribution coefficients, between ketamine enantiomers, were not significant. Unfortunately, CNS and heart concentrations of norketamine were less than the limit of quantitation, thus precluding examination of enantiomer related differences. At 180 min, the plasma concentrations of R-ketamine remained significantly greater (P,0.005) than S-ketamine, but those of S-norketamine were significantly greater than those of R-norketamine (P,0.005). Concentrations of S-ketamine significantly exceeded those of R-ketamine in kidney (P,0.0001) and gut (P,0.0005), but R-ketamine remained significantly higher (P,0.0001) than S-ketamine in fat. Distribution coefficients for S-ketamine were significantly greater than those of R-ketamine in kidney (P,0.0001), heart (P,0.01), and gut (P,0.0001). Whilst the concentrations of S-norketamine were significantly greater than R-norketamine in liver (P,0.005), kidney (P,0.0001), and gut (P,0.0001), the distribution coefficients were not significantly different. Comparison of distribution coefficients from the two paradigms Log transformed values of the distribution coefficients for the CNS regions did not differ significantly between paradigms for either ketamine enantiomer; however, significant differ-
2058
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
Table 1 Mean (6SEM) of the tissue concentrations and distribution coefficients for ketamine and norketamine enantiomers in rats, following constant rate infusions of rac-ketamine at 6 mg/Kg/min until lethality (“washin” paradigm)
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2059
Fig. 3. Mean (6SEM) plasma concentrations of ketamine and norketamine enantiomers in rats (n59) following brief infusion with rac-ketamine at a constant rate of 10 mg/Kg/min over 5 min. AUC R- . S-ketamine: P,0.0001 (paired Student’s t-test) AUC R- . S-norketamine: P50.0001 (paired Student’s t-test).
ences were apparent for peripheral tissues. Log transformed distribution coefficients for the kidney were significantly greater following the washout paradigm (P,0.0001) for both ketamine enantiomers, whereas they were significantly less in the gut for R-ketamine (P,0.01) but significantly greater for S-ketamine (P,0.05). Log transformed distribution coefficients were significantly greater after washout for both R- and S-norketamine in the liver (respectively, P,0.0001 and P,0.05), gut (P,0.0001 and P,0.0002), and fat (P,0.001 and P,0.0001). The R:S ratios for the tissue concentrations and distribution coefficients of ketamine and norketamine enantiomers are summarized for the two paradigms in Table 3. By the conclusion of washin, the R:S ratios for CNS tissue concentrations of ketamine and norketamine enantiomers were significantly greater (P,0.0001) than unity. In peripheral tissues the R:S ratio for tissue concentrations of ketamine enantiomers was significantly greater than unity in plasma and heart (both P,0.0001), but significantly less than unity in liver and fat (P,0.0001). The R:S ratio for concentrations of norketamine enantiomers, however, was significantly greater than unity in all tissues (P#0.005), except gut. The R:S ratio of the distribution coefficients for ketamine enantiomers was significantly less (P#0.001) than unity in all tissues examined, while for norketamine, it was significantly less than unity in liver (P,0.0005), lung (P,0.05), gut (P,0.005) and fat (P,0.0001), but significantly greater than unity in kidney (P,0.01) and heart (P,0.001) and muscle (P,0.05). After washout, the R:S ratio for ketamine enantiomer concentrations was significantly greater than unity in all CNS regions (P,0.005). However, the R:S ratio of the log-transformed values for the distribution coefficients did not differ significantly from unity. In peripheral tis-
2060
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
Table 2 Mean (6SEM) of the tissue concentrations and log-normalised distribution coefficients, at 180 min, for ketamine and norketamine enantiomers in rats, following brief infusions of ketamine racemate at 10 mg/Kg/min for 5 min (“washout” paradigm)
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2061
Table 3 Mean (6SEM) of the R:S enantiomer ratios for the tissue concentrations and tissue distribution coefficients in rats following infusions with rac-ketamine at either a constant rate of 6 mg/Kg/min until lethality (“washin” paradigm), or at 180 min after a brief infusion of 10 mg/Kg/min for 5 min (“washout” paradigm)
2062
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
sues R:S enantiomer concentration ratios were significantly greater in plasma (P,0.05), and fat (P,0.0001), but significantly less in kidney (P,0.0001) and gut (P,0.0001), while for norketamine, the R:S enantiomer ratio of tissue concentrations was significantly less than unity in plasma, liver, kidney, and gut (P,0.0005). The R:S ratio of the distribution coefficients for ketamine enantiomers was significantly less than unity in kidney (P,0.0001), heart (P,0.005), lung (P,0.05), and gut (P,0.0001). Ketamine pharmacokinetics during washout Pharmacokinetic parameters for the two ketamine enantiomers, derived from the plasma concentration-time washout data, are shown in table 4. The S:R enantiomer ratio was significantly greater than unity for ClT (P,0.0002), Vc (P,0.002), and Vss (P,0.02). Discussion Two dose paradigms were used in this study in an attempt to study distribution under two complementary conditions. The washin paradigm provided the maximal effects of the drug; the washout paradigm provided the minimal effects of the drug. This allowed influences on drug distribution by way of acute drug effect on the circulatory system to be elucidated. Similarly, the dual effects on norketamine by way of distribution and regional formation also should have been elucidated. Regrettably, the assay sensitivity limited the achievement of the latter objective. Nevertheless, new insights about ketamine biodisposition have been revealed. Ketamine produces sympathomimetic effects mediated by direct effects within the CNS but has direct myocardial depressant properties [2]. Lethality following constant rate infusions of rac-ketamine resulted from respiratory failure, presumably preceded by myocardial depression. The therapeutic index for S-ketamine, reported in earlier studies [10], suggests
Table 4 Pharmacokinetic parameters (mean6SEM) for ketamine enantiomers following bolus infusions of ketamine racemate at 10 mg/Kg/min over 5 min (“washout” paradigm)
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2063
that S-ketamine produces less myocardial depression than R-ketamine. The distribution coefficient into myocardial tissue, following washout, was significantly greater for S-ketamine, suggesting a longer residence time for this enantiomer in the myocardium. However, in view of the previously reported much higher therapeutic index for S-ketamine [10], the longer residence time and higher tissue uptake for S-ketamine do not appear to contribute disproportionately to the depressant effects of rac-ketamine on the myocardium. Significantly higher plasma concentrations of R- than of S-ketamine, found over the duration of the washin infusions, are consistent with the significantly greater tissue:plasma distribution coefficients found for S-ketamine, along with the greater values of Vc (14%),.ClT (15%) and VSS (10%) found during washout and are analogous to values found in humans [14]. Although the difference in plasma ketamine enantiomer concentrations was small under the conditions of this investigation, during procedures such as target controlled infusions this disparity in enantiomer concentrations could be expected to become more substantial as shown previously in an analogous study with thiopentone [25]. Plasma concentrations of R-norketamine, found over the duration of the washin infusions, were significantly higher than S-norketamine and, by the conclusion of the infusions, were almost twice those of S-norketamine. Moreover, in contrast to R-ketamine, the distribution coefficients of R-norketamine were significantly greater than S-norketamine indicating its greater uptake into CNS and myocardial tissue. Regional differences were also apparent, between brain and spinal cord, in both tissue concentrations and distribution coefficients. At the conclusion of the washin infusions, the distribution of ketamine and norketamine was highest into the kidney. Although the concentrations of ketamine enantiomers in renal tissue did not differ significantly, the concentration of R-norketamine was significantly higher (3-fold) than S-norketamine, suggesting that there was metabolism of both ketamine enantiomers to R-norketamine, and this is supported by the significantly greater (2-fold) distribution coefficient for R-norketamine. Similarly, there appeared to be preferential metabolism of both ketamine enantiomers to R-norketamine in the lung, although the distribution coefficients indicate that tissue uptake of S-norketamine exceeded the formation of R-norketamine. Enantiomer concentrations for ketamine and norketamine in liver tissue suggest a more rapid metabolism of R-ketamine to R-norketamine, but the distribution coefficients for norketamine enantiomers again suggest that tissue uptake of S-norketamine exceeded formation of R-norketamine. Tissue concentrations and distribution coefficients in the gut further suggest there was enantioselective tissue uptake of S-ketamine, but the tissue concentrations and distribution coefficients for norketamine enantiomers suggest that there was preferential formation of S-norketamine. Ketamine is a lipophilic drug, with predominantly blood flow-limited redistribution kinetics, [4,21], and has the capacity to alter its own regional disposition through its haemodynamic changes [22]. Thus, the use of the two infusion paradigms was useful to determine the concentrations and distribution coefficients for ketamine and norketamine enantiomers under maximal and minimal changes. Following the washin infusions, concentrations were lowest in the more poorly perfused tissues of muscle and fat. This is consistent with delayed redistribution due to reduced cardiac output associated with myocardial depression. For ketamine, uptake into muscle and fat was significantly higher for the S-enantiomer; for norketamine uptake into muscle was significantly higher for the R-enantiomer, while in fat uptake was
2064
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
significantly higher for the S-enantiomer. The distribution coefficients for ketamine and norketamine enantiomers demonstrate lower tissue uptake into fat than muscle for the less lipophilic metabolite, norketamine, but higher tissue uptake into fat than muscle for the more lipophilic parent drug, ketamine. In contrast to the washin infusions, the plasma concentrations of R-norketamine in the washout paradigm significantly exceeded those of S-norketamine for only a brief period. Concentrations of S-norketamine peaked at 60 min after the infusions and, by 180 min, remained significantly higher (4-fold) than R-norketamine. This could indicate that differing routes of metabolism were available to ketamine under the two paradigms, such that both concentration dependent N-demethylation to R-norketamine, as well as concentration independent metabolic inversion of R-norketamine to S-norketamine, were occurring. At the conclusion of washout period, ketamine and norketamine enantiomer concentrations were again highest in the kidney. However, unlike the constant rate infusions, concentrations of the S-enantiomers of ketamine and norketamine significantly exceeded (4-fold) those of the R-enantiomers, suggesting that inversion of R-ketamine to S-ketamine preceded metabolism to S-norketamine. The distribution coefficients demonstrate tissue uptake of ketamine enantiomers was highest in renal tissue, as well as further indicating that metabolic inversion of R-ketamine to S-ketamine occurred. The lack of significant difference in the distribution coefficients for norketamine enantiomers, in renal tissue, is consistent with metabolic formation rather than tissue uptake, but also indicates renal tissue was contributing to the circulating concentrations of norketamine enantiomers. Although ketamine enantiomer concentrations were substantially lower in gut than in kidney following washout, the tissue concentrations of S-ketamine were significantly higher (4-fold) than R- ketamine indicating metabolic inversion occurred in the gut as well. The norketamine enantiomer concentrations suggest that substantial uptake of ketamine enantiomers occurred in the gut, but the substantially lower ketamine enantiomer concentrations suggest that metabolism was more rapid here. As in renal tissue, significantly higher (4-fold) concentrations of S-norketamine were present in the gut, and the significantly higher (4-fold) distribution coefficient for S-ketamine in the gut suggests further evidence of metabolic inversion to S-ketamine. The measured distribution coefficients for norketamine enantiomers include a function of any local metabolism, and their lack of significant difference suggests that the gut also contributed to circulating concentrations of norketamine enantiomers. The liver appeared to be of a lesser importance in the uptake and metabolism of ketamine enantiomers following the brief infusion, and the higher tissue concentrations of S-norketamine probably only reflect the higher circulating concentrations of S-norketamine. The measured distribution coefficients suggest that uptake into the liver was not enantioselective for either ketamine or norketamine. Although tissue uptake of ketamine enantiomers in the lung was relatively high it was not enantioselective, and there appeared to be minimal metabolism to norketamine. In this investigation total ketamine concentrations in fat were only z1/10 of those present in kidney, unlike the earlier work where tissue concentrations of ketamine in fat were equivalent to those in kidney by 180 min [21]. However, in this study adipose tissue was sampled from the epididymal fat pad, whereas perinephric fat was sampled [21], and the difference in sampling sites may account for these differences in findings. Lastly, concentrations in the CNS are considered: this is the accepted site of ketamine (and nor-
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2065
ketamine) action. Concentrations of R-ketamine remained significantly higher than S-ketamine in CNS tissue at the conclusion of the washout period, and the significantly higher distribution coefficient for R-ketamine in the forebrain suggests a longer residence time for R-ketamine in this CNS region. Mean plasma concentrations for the S-enantiomers of ketamine and norketamine at 60 min were, respectively, 0.8 mg/mL (3.4 mM) and 1.4 mg/mL (6.3 mM), whilst those for the R-enantiomers were, respectively, 0.9 mg/mL (3.9 mM) and 0.4 mg/mL (1.8 mM). The Ki values for the S- and R-enantiomers of ketamine and norketamine at the NMDA receptor have been reported to be 0.3, 1.4, 1.7, and 13 mM, respectively [18]. Other investigators found Ki values, for S- and R-ketamine of 0.6 mM and 2.0 mM, at the NMDA receptor, but 145 mM and 35 mM, at the sigma receptor, and the enantiomer potency ratio for inhibition at the NMDA receptor corresponded to the enantiomer potency ratio for analgesia in exercise-induced ischaemic pain [23]. In the present investigation, posthypnotic behavioural arousal, characterised by increased motor activity with amphetamine-like stereotypic head and forepaw movements, was observed to be maximal 60–80 min after the brief infusion of rac-ketamine. It would appear, therefore, that the psychostimulant effect of rac-ketamine during washout was predominantly caused by the action of S-ketamine at the NMDA receptor with S-norketamine contributing to a lesser degree. It would seem that ketamine enantiomer concentrations by 60 min were too low for activity at the sigma receptor. In summary, different profiles for the uptake and metabolism of ketamine enantiomers were apparent following constant rate washin, and brief infusion washout, paradigms of i.v. rac-ketamine. Enantioselectivity was found in regional distribution and clearance, including indirect evidence for extrahepatic metabolic inversion. Others, using different paradigms and species, had previously not found evidence for enantioselective distribution [24] or inversion [14]. However, regional pharmacokinetics had not previously been examined for enantioselectivity. During washin, when circulating concentrations of ketamine enantiomers were high, uptake and metabolism occurred predominantly in the kidney and to a lesser extent in liver, lung and gut, with formation of R-norketamine by a (presumed) first-order process predominating. However, following washout, when circulating concentrations of ketamine enantiomers were much lower, uptake and metabolism was dominated by the kidney and gut. Under these conditions inversion of R- to S-ketamine appeared to predominate with subsequent metabolism to S-norketamine by (presumed) zero-order processes. Acknowledgments The authors acknowledge with pleasure the grant support of the Australian New Zealand College of Anaesthetists and the expert technical advice of Ms XQ Gu. References 1. Church J and Lodge D. N-methyl-D-aspartate antagonism is central to the actions of ketamine and other phencyclidine receptor ligands. In: Domino EF, editor. Status of Ketamine in Anesthesiology. Ann Arbor:NPP Books, 1990. pp.501–19. 2. White PF, Way WL, and Trevor AJ. Ketamine - its pharmacology and therapeutic uses. Anesthesiology 1982;56:119–36.
2066
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
3. Backonja M, Arndt G, Gombar KA, Check B, and Zimmermann M. Response of chronic neuropathic pain syndromes to ketamine: a preliminary study Pain 1994;56:51–7. 4. Clements JA and Nimmo WS. Pharmacokinetics and analgesic effect of ketamine in man. Br. J. Anaesth. 1981;53:27–30. 5. Eide PK, Stubhaug A, Oye I, and Breivik H. Continuous subcutaneous administration of the NMDA receptor antagonist ketamine in the treatment of post-herpetic neuralgia. Pain 1995; 61:221–8. 6. Maurset A, Skoglund LA, Hustveit O, and Oye I. Comparison of ketamine and pethidine in experimental and postoperative pain. Pain 1989;36:37–41. 7. Slogoff S, Allen GW, Wessels J, and Cheney DH. Clinical experience with subanesthetic ketamine. Anesth. & Analg. 1974;53:354–8. 8. Klepstad P, Maurset A, Moberg ER, and Oye I. Evidence of a role for NMDA receptors in pain perception. Eur. J. Pharmacol. 1990;187:513–8. 9. Oye I, Paulsen O, and Maurset A. Effects of ketamine on sensory perception: evidence for a role of NMDA receptors. J. Pharmacol. Exp. Ther. 1992;260:1209–13. 10. Marrietta MP, Way WL, Castagnoli N, and Trevor AJ. On the pharmacology of the ketamine enantiomorphs in the rat. J. Pharmacol. Exp. Ther. 1977;202:157–65. 11. Ryder S, Way WL, and Trevor AJ. Comparative pharmacology of the optical isomers of ketamine in mice. Eur. J. Pharmacol. 1978;49:15–23. 12. White PF, Schuttler J, Shafer A, Stanski DR, Horai Y, and Trevor AJ. Comparative pharmacology of the ketamine isomers. Br. J. Anaesth. 1985;57:197–203. 13. White PF, Ham J, Way WL, and Trevor AJ. Pharmacology of ketamine isomers in surgical patients. Anesthesiology 1980;52:231–39. 14. Geisslinger G, Hering W, Thomann P, Knoll R, Kamp HD, and Brune K. Pharmacokinetics and pharmacodynamics of ketamine enantiomers in surgical patients using a stereoselective analytical method. Br. J. Anaesth. 1993;70:666–71. 15. Woolf TF and Adams JD. Biotransformation of ketamine, (Z)-6-hydroxyketamine, and (E)-6-hydroxyketamine by rat, rabbit, and human liver microsomal preparations. Xenobiotica 1987;17:839–47. 16. Karasch ED and Labroo R. Metabolism of ketamine stereoisomers by human liver microsomes. Anesthesiology 1992;77:1201–07. 17. Grant IS, Nimmo WS, and Clements JA. Pharmacokinetics and analgesic effects of i.m. and oral ketamine. Br. J. Anaesth. 1981;53:805–10. 18. Ebert B, Mikkelsen S, Thorkildsen C, and Borgbjerg FM. Norketamine, the main metabolite of ketamine, is a non-competitive NMDA receptor antagonist in the rat cortex and spinal cord. Eur. J. Pharmacol. 1997;156:177–80. 19. Geisslinger G, Menzel-Soglowek S, Kamp HD, and Brune K. Stereoselective high-performance liquid chromatographic determination of the enantiomers of ketamine and norketamine in plasma. J. Chromatography B 1991;568:165–76. 20. Mather LE, Upton RN, Huang JL, Ludbrook GL, Gray E, and Grant C. The systemic and cerebral kinetics of thiopental in sheep: enantiomeric analysis. J. Pharmacol. Exp. Ther. 1996;279:291–7. 21. White PF, Marietta MP, Pudwill CR, Way WL, and Trevor AJ. Effects of halothane anesthesia on the biodisposition of ketamine in rats. J. Pharmacol. Exp. Ther. 1976; 196:545–5. 22. Bjorkman S, Akeson J, Nilsson F, Messeter K, and Roth B. Ketamine and midazolam decrease cerebral blood flow and consequently their own rate of transport to the brain: an application of mass balance pharmacokinetics with a changing regional blood flow. J. Pharmacokin. Biopharm. 1992;20:637–52. 23. Maurset A, Moberg R, and Oye I. The analgesic effect of ketamine: evidence for a non-opioid, PCP receptor mediated mechanism. In: Domino EF, editor. Status of Ketamine in Anesthesiology. Ann Arbor:NPP Books, 1990. pp. 239–46. 24. Henthorn TK, Krejcie TC, Niemann CU, Renders-Klein C, Shanks CA, and Avram MJ. Ketamine distribution described by a recirculatory pharmacokinetic model is not stereoselective. Anesthesiology 1999;91:1733–43. 25. Mather LE, Edwards SR, Duke CC, and Cousins MJ. Enantioselectivity of thiopental distribution into the central neural tissue of rats: an interaction with halothane. Anesth. Analg. 1999;89:230–5.
Tissue uptake of ketamine and norketamine enantiomers in the rat Indirect evidence for extrahepatic metabolic inversion Stephen R. Edwards, Laurence E. Mather* Department of Anaesthesia and Pain Management, University of Sydney at Royal North Shore Hospital, St. Leonards, NSW 2065, Australia Received 16 October 2000; accepted 2 April 2001
Abstract Ketamine, used clinically as an intravenous analgetic and dissociative anaesthetic agent, is a racemate with both pharmacokinetic and pharmacodynamic enantioselectivity. S-ketamine has been found have a higher clearance and greater potency than R-ketamine as well as a greater therapeutic index. We performed a study in rats with two complementary paradigms: (i) constant rate “washin” infusion until fatal, (ii) brief infusion then “washout”. These, respectively, allowed examination of ketamine and norketamine serial plasma enantiomer concentrations and tissue distribution at maximal and minimal drug effects. Both paradigms found plasma concentrations of R-ketamine.S-ketamine; however, tissue distribution coefficients for S-ketamine.R-ketamine. For paradigm (i), plasma concentrations of R-norketamine.S-norketamine; for paradigm (ii), R-norketamine..S-norketamine initially, but S-norketamine..R-norketamine later. Comparison of distribution coefficients of ketamine and norketamine enantiomers for the two paradigms provided indirect evidence for metabolic inversion. During washin, when circulating concentrations of ketamine enantiomers were high, uptake and metabolism occurred predominantly in the kidney and to a lesser extent in liver, lung and gut, with formation of R-norketamine by a (presumed) first-order process predominating. However, following washout, when circulating concentrations of ketamine enantiomers were low, uptake and metabolism was dominated by the kidney and gut. Under these conditions inversion of R- to S-ketamine appeared to predominate with subsequent metabolism to S-norketamine by (presumed) zero-order processes. In summary, different profiles for the uptake and metabolism of ketamine enantiomers were apparent following constant rate washin, and brief infusion washout, paradigms with i.v. rac-ketamine. Uptake into most tissues, and metabolism in some tissues, was enantioselective. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Regional pharmacokinetics; Distribution coefficient; Intravenous infusion
* Corresponding author. Tel.: 161-2-99268420; fax: 161-2-99064079. E-mail address: [email protected] (L.E. Mather) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 2 8 7 -5
2052
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
Introduction The arylcyclohexylamine derivative, ketamine, is an N-methyl-D-aspartate (NMDA) receptor antagonist [1]. It has been used for over 30 years as an analgetic and dissociative anaesthetic where it offers the clinical advantages of administration by intravenous mode, short duration of action, lack of cardiovascular depressant effects, and rapid recovery [2–7]. However, ketamine has the clinical disadvantage of sometimes causing psychotomimetic effects [2,8,9]. Ketamine, as currently in clinical use, is a racemic drug containing equal amounts of the R- and S-ketamine enantiomers that have been found, in early animal laboratory studies, to have differences in the relative potencies and effects. As examples, the therapeutic index in rats, expressed by the ratio of the LD50 to the ED50 for hypnosis, was found to be greater for S-ketamine (510) than for either rac-ketamine (56.25) or R-ketamine (54.0); the posthypnotic psychomotor activation following administration of equihypnotic doses in rats was significantly less for S-ketamine than R-ketamine [10]; the analgesic potency of S-ketamine in mice was 3 times greater than R-ketamine, but psychomotor activation from equianalgetic doses was less for S-ketamine [11]. Similar differences in potency and potency ratios for ketamine enantiomers have been reported from human laboratory studies. As examples, R-ketamine was found to cause less slowing in the electroencephalogram (EEG) than either S- or rac-ketamine [12]; the anaesthetic potency of S-ketamine was 4 times more potent than that of R-ketamine in surgical patients, however, the psychotomimetic effects in the emergence period were more pronounced for R-ketamine, while posthypnotic analgesia was greater for S-ketamine [13]; the analgetic potency of S-ketamine was 4 times that of R-ketamine in studies of experimentally induced ischaemic pain [8]; Additionally, pharmacokinetic differences in surgical patients were found after administration of rac-ketamine, such that the plasma clearance of S-ketamine was z16% greater than that of R-ketamine [14]. Norketamine is the principal metabolite of ketamine [15]. Work with human liver microsomes indicated that the rate of N-demethylation was greater with S-ketamine than with rac- or R-ketamine [16]. An earlier clinical investigation of the pharmacokinetics and analgesic effects of ketamine and norketamine, following intramuscular and oral administration of ketamine, indicated that norketamine also had analgetic properties [17]. It was suggested, on the basis of the affinities of ketamine and norketamine enantiomers for the NMDA receptor, that S-norketamine contributes to the analgetic effect of ketamine following administration of rac-ketamine [18]. The present investigation examined further the enantioselectivity of ketamine and norketamine plasma and tissue concentrations following the administration of rac-ketamine with two complementary i.v. administration paradigms. In the first paradigm, a constant rate washin infusion until lethality was examined; in the second paradigm, a brief infusion followed by a prolonged washout was examined. Methods Animals and their preparation Adult male Wistar rats (350–400g; Gore Hill Animal Research Laboratories, Sydney, AUS) were housed in groups of four with free access to food and water on a constant 12/12
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2053
hour light dark cycle at 218C. Surgical procedures were performed under general anaesthesia (pentobarbitone, 30 mg/Kg, followed 5 min later by ketamine, 45 mg/Kg; both i.p. in 1 mL 0.9% saline). Body temperature was maintained by a heating pad and monitored with a rectal probe. Chronic indwelling cannulae were implanted into the jugular vein and carotid artery to allow simultaneous venous infusion and arterial sampling. A 1 cm incision was made just lateral to the midline, and the jugular vein and carotid artery were exposed. Silastic laboratory tubing (respectively, 0.025 in ID 3 0.047 in OD, and 0.020 in ID 3 0.037 in OD) was inserted 2.5 cm into the jugular vein, and 2.0 cm into the carotid artery. Each cannula line was tunnelled under the skin and externalised above the neck anterior to the shoulder blades and filled with a solution containing 6 g polyvinylpyrrolidone (MW 40,000, Sigma Chemical Co, St Louis, MO, USA) dissolved in 5 mL of 1000 U/mL sodium heparin (Delta West Pty Ltd, Perth, WA, AUS) in order to maintain patency. Postoperatively, buprenorphine (0.2 mg/ Kg, s.c.) was administered for pain relief, and procaine penicillin and benzathine penicillin (standard veterinary preparation, each 150 mg/mL, 0.1 mL, s.c.) was given for antimicrobial cover. Following surgery, the animals were housed individually and their post-operative body weights and fluid intake were recorded. Ketamine enantiomer assays The enantiomers of ketamine and norketamine were separated on a micro chiral-AGP column (150 mm 3 2 mm, 5 mm, Chromtech) using a Hewlett Packard series 1100 LC system, and quantitated by spectrophotometric detection at 215 nm. The method was adapted from a previously described procedure [19]. The mobile phase, containing ammonium acetate (30 mM, adjusted to pH 7.0, in 3.0% v/v isopropanol), was used at a flow rate of 0.22 mL/min. Plasma aliquots (50 ml) were extracted in Eppendorf tubes (1.5 mL) after addition of internal standard (N-benzylmethylamine, Sigma-Aldrich, 50 ml, 18.4 mM) and Na3PO4 (100 ml, 0.5M). Tissue samples were homogenised into NaH2PO4 (0.1M) to a concentration of 100 mg/mL and frozen. After thawing and resuspension tissue aliquots (200 ml) were extracted in Eppendorf tubes (2.0 mL) following the addition of internal standard (50 ml, 18.4 mM) and Na3PO4 (200 ml, 0.5M). Tubes were briefly vortex-mixed before the addition iso-amyl alcohol in cyclohexane (2% v/v, 1.0 mL). Samples were subsequently shaken for 5 min, centrifuged in a bench centrifuge (3000 rpm for 2 min), and the organic layer decanted with a Pasteur pipette into a fresh Eppendorf tube (1.5 mL). Aliquots of H3PO4 (100 ml, 0.5M) were added to each tube, the tubes were shaken and centrifuged as before, and the organic layer was discarded. Samples were then alkalinized (NaOH, 30 ml, 5M), briefly vortex-mixed, and re-extracted into iso-amyl alcohol in cyclohexane (2% v/v, 1.0 mL) by shaking as before. After centrifugation the organic layer was transferred to a fresh Eppendorf tube (1.5 mL) and dried in a rotatory vacuum bench evaporator (408C). The residue was reconstituted into ammonium acetate buffer (100 ml, 20 mM, pH 5, containing 10% v/v isopropanol), and sonicated (5 min) before transfer to polypropylene inserts (250 ml) and injection of aliquots (10 ml) onto the column. Experimental design The studies were carried out 24 hours after cannulation between 1400–1900 hours. Infusion and sampling lines (respectively, 75 and 45 cm) were attached to the venous and arterial
2054
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
lines, and the animals were allowed to settle (30 min) in a study chamber before commencement of the study. rac-Ketamine as HCl salt (Ketalar™, Parke-Davis Australia Pty Ltd, Sydney, AUS) was diluted with deionised water to 20 mg/mL (17.3 mg/mL as base) and infused at the appropriate rate. The infusions were delivered from a gas tight syringe (Hamilton, 5 mL) using a syringe driver (Harvard Apparatus Model 22). Immediately after the righting reflex was lost, following removal from the study chamber, a thermal probe was inserted rectally and body temperature was maintained with a heating lamp. Blood samples (100 ml) were collected into heparinised (50U) polyethylene microfuge tubes. At the conclusion of the study, brain, spinal cord, heart, lung, liver, kidney, gut, adductor muscle, and epididymal fat were sampled for the determination of tissue: plasma drug distribution. The brain was divided into forebrain, brainstem, and cerebellum; the spinal cord was divided into brachiothoracic and lumbo-sacral regions. In the “washin” paradigm, an infusion of rac-ketamine was administered at a constant rate of 6 mg/Kg/min until lethality ensued. Serial blood samples were taken at 2 min intervals. A consistent loss of foot withdrawal reflex upon toe pinch was used to determine the onset of anaesthesia; cessation of respiration following the agonal gasp was used as the point of lethality. In the “washout” paradigm, an infusion of rac-ketamine was administered at a constant rate of 10 mg/Kg/min for 5 min; serial blood samples were taken at 0, 1.5, 3, 5, 7, 10, 15, 20, 30, 40, 60, 80, 100, 120, 140, 160 and 180 min. Once motor function began to return the rectal probe was removed and rats were returned to the study chamber. Data analysis Data are expressed as mean (6SEM) unless described as otherwise. Area under the curve (AUC) values for each plasma concentration-time data set were determined by the linear trapezoid method. Distribution coefficients were calculated as the ratio of tissue to plasma analyte concentrations, uncorrected for residual tissue blood volume. Student’s t-test for paired data was used for the pairwise comparison of enantiomer AUC values, tissue concentrations, and distribution coefficients, while the respective R:S enantiomer ratios were compared to unity with Student’s one sample t-test. Regional CNS tissue enantiomer concentrations and distribution coefficients were compared by one-way analysis of variance (AOV) with additional two-factor AOV for comparisons between ketamine and norketamine in the washin paradigm. Individual post-hoc comparisons were performed by the method of Least Significant Difference (LSD). Log-transformed values for regional CNS distribution coefficients were compared by AOV in the two paradigms. The logtransformed values for the peripheral tissue distribution coefficients in the two paradigms were compared by Student’s two sample t-tests. Pharmacokinetic parameters for each ketamine enantiomer were determined from fitting polyexponential decay equations to the concentration-time washout curve, as previously described, from which the mean total body clearance (ClT), initial dilution volume (VC), volume of distribution at steady state equilibrium (VSS), terminal half life (T1/2) and mean residence time in the body (MRT) were calculated by conventional means [20]. Enantioselectivity in pharmacokinetics was determined from the S:R enantiomer ratio when compared to unity with Student’s one sample t-test.
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2055
Results Ketamine enantiomer assays Plasma calibration curves (n58) were linear over an enantiomer concentration range of 0.22–43.35 mg/ml for both R- (R250.998460.0006) and S-ketamine (R250.998560.0006). The minimum limit of detectability in plasma, at a signal to noise ratio of 2:1, was 0.09 mg/ml for each ketamine enantiomer. Assay coefficients of variation were based on the slopes of the plasma calibration curves. Within-assay coefficients of variation (n56) for R- and S-ketamine were 4.3% and 4.2%, respectively; between-assay coefficients of variation (n58) were 5.5% and 5.8%, respectively. Recoveries of R- and S-ketamine from plasma were 77.061.4% and 76.961.2% at enantiomer concentrations of 2.2 mg/ml (n58); recovery was 80.761.2% and 80.461.0% respectively at enantiomer concentrations of 22 mg/ml (n57). Tissue homogenates were assayed against standards, prepared in the relevant respective drug-free tissue homogenates, to which ketamine enantiomer concentrations between 0.2 – 43.4 mg/ml had been added. Norketamine enantiomer concentrations were quantified using calibration curves derived from ketamine enantiomer standards. Washin paradigm (constant rate infusion) The anaesthetic dose to lack of response to toe pinch was 10163 mg/Kg and the lethal dose was 14767 mg/Kg. Plasma concentrations of R- and S-ketamine, and the corresponding concentrations of R- and S-norketamine, are shown in Figure 1 for the duration of the infusion. The enantiomer concentration ratios for ketamine and norketamine are shown in Figure 2. Over the duration of the infusion, the plasma concentrations and AUC values of the R-enantiomers of both ketamine and norketamine exceeded the corresponding concentrations of the S-enantiomers (P50.0011 and P50.0001), and the AUC R:S enantiomer ratios were significantly greater than unity for both ketamine (P50.0004) and norketamine (P,0.0001). The tissue concentrations and distribution coefficients for ketamine and norketamine enantiomers at the conclusion of washin infusions are shown in Table 1. In all CNS regions, the concentrations of both R-ketamine and R-norketamine were significantly greater than those of their corresponding S-enantiomers (P,0.0001). However, the corresponding distribution coefficients for S-ketamine were significantly greater than those of R-ketamine (P#0.0005), but the distribution coefficients for R-norketamine were significantly greater than those of S-norketamine (P#0.005). Whereas significant differences between regional CNS concentrations of R- and S-ketamine were apparent (respectively, P50.0008 P50.002), there were no such differences for both norketamine enantiomers. Concentrations in the pons-medulla and cerebellum significantly exceeded those in the forebrain and spinal cord for R- and S-ketamine (respectively, P50.002 and P50.005). There were also regional differences in the CNS distribution coefficients of both R- and S-ketamine (both P,0.0001). Similar differences in the regional CNS distribution coefficients for R- and Snorketamine (respectively, P50.0004 and P5 0.0012) were also apparent. Distribution coefficients were significantly greater in the pons-medulla than the forebrain and spinal cord for both Rand S-ketamine (respectively, P50.002 and P50.005), and they were significantly greater in the pons-medulla and cerebellum than the spinal cord for R- and S-norketamine (respectively, P50.005 and P50.02). Finally, the regional CNS concentrations and distribution coefficients
2056
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
Fig. 1. Plasma concentrations of ketamine and norketamine enantiomers in individual rats (n58) during infusion with rac-ketamine at a constant rate of 6 mg/Kg/min until lethality (“washin” paradigm). AUC R vs S: P50.0011 for ketamine and P50.001 for norketamine (Student’s t-test for paired data).
of both norketamine enantiomers were significantly lower than those of the corresponding ketamine enantiomers (P50.001) in all CNS regions. Concentrations of S-ketamine were significantly greater than R-ketamine in liver and fat (both P,0.0001), but significantly lower in heart (P,0.0001). However, as in CNS tissue, the distribution coefficients for S-ketamine significantly exceeded those of R-ketamine in all peripheral tissues (P#0.01). Concentrations of R-norketamine were significantly greater than S-norketamine in kidney (P,0.005), heart (P,0.0001), lung (P,0.0001), and muscle (P,0.0001). The distribution coefficient for R-norketamine was significantly greater than that of S-norketamine in kidney (P,0.01), heart (P,0.0005), and muscle (P,0.0001), but significantly less than S-norketamine in liver (P,0.001) and lung (P,0.05). Washout paradigm (brief infusion) Plasma concentrations of the ketamine and norketamine enantiomers are shown in figure 3. Again, the AUC values for R-ketamine significantly exceeded those of S-ketamine (P,0.0001). However, the AUC values for S-norketamine were significantly greater (P50.0001) than
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2057
Fig. 2. Plasma enantiomer concentrations ratios for ketamine and norketamine in individual rats (n58) during infusion with rac-ketamine at a constant rate of 6 mg/Kg/min until lethality (“washin” paradigm). AUC R vs S: P50.0004 for ketamine and P,0.0001 for norketamine (Student’s one sample t-test).
those of R-norketamine, although early concentrations of R-norketamine were significantly higher (P,0.0001) than S-norketamine (at 7 min). The AUC R:S enantiomer ratios were significantly greater than unity for ketamine (P,0.0001), but significantly less than unity for norketamine (P,0.0001). Tissue concentrations and distribution coefficients for ketamine and norketamine enantiomers, 180 min after “washout infusion”, are shown in Table 2. Due to the variance in the distribution coefficients the data were subjected to a log-transformation; the anti-logs of the mean logtransformed values for each tissue are given in Table 2 as “log-normalized” values. Whilst the concentrations of R-ketamine were significantly greater than S-ketamine in all CNS regions (P#0.01), the distribution coefficients were not significantly different (P.0.05), except for the forebrain (P,0.05). Regional differences in CNS tissue concentrations and distribution coefficients, between ketamine enantiomers, were not significant. Unfortunately, CNS and heart concentrations of norketamine were less than the limit of quantitation, thus precluding examination of enantiomer related differences. At 180 min, the plasma concentrations of R-ketamine remained significantly greater (P,0.005) than S-ketamine, but those of S-norketamine were significantly greater than those of R-norketamine (P,0.005). Concentrations of S-ketamine significantly exceeded those of R-ketamine in kidney (P,0.0001) and gut (P,0.0005), but R-ketamine remained significantly higher (P,0.0001) than S-ketamine in fat. Distribution coefficients for S-ketamine were significantly greater than those of R-ketamine in kidney (P,0.0001), heart (P,0.01), and gut (P,0.0001). Whilst the concentrations of S-norketamine were significantly greater than R-norketamine in liver (P,0.005), kidney (P,0.0001), and gut (P,0.0001), the distribution coefficients were not significantly different. Comparison of distribution coefficients from the two paradigms Log transformed values of the distribution coefficients for the CNS regions did not differ significantly between paradigms for either ketamine enantiomer; however, significant differ-
2058
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
Table 1 Mean (6SEM) of the tissue concentrations and distribution coefficients for ketamine and norketamine enantiomers in rats, following constant rate infusions of rac-ketamine at 6 mg/Kg/min until lethality (“washin” paradigm)
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2059
Fig. 3. Mean (6SEM) plasma concentrations of ketamine and norketamine enantiomers in rats (n59) following brief infusion with rac-ketamine at a constant rate of 10 mg/Kg/min over 5 min. AUC R- . S-ketamine: P,0.0001 (paired Student’s t-test) AUC R- . S-norketamine: P50.0001 (paired Student’s t-test).
ences were apparent for peripheral tissues. Log transformed distribution coefficients for the kidney were significantly greater following the washout paradigm (P,0.0001) for both ketamine enantiomers, whereas they were significantly less in the gut for R-ketamine (P,0.01) but significantly greater for S-ketamine (P,0.05). Log transformed distribution coefficients were significantly greater after washout for both R- and S-norketamine in the liver (respectively, P,0.0001 and P,0.05), gut (P,0.0001 and P,0.0002), and fat (P,0.001 and P,0.0001). The R:S ratios for the tissue concentrations and distribution coefficients of ketamine and norketamine enantiomers are summarized for the two paradigms in Table 3. By the conclusion of washin, the R:S ratios for CNS tissue concentrations of ketamine and norketamine enantiomers were significantly greater (P,0.0001) than unity. In peripheral tissues the R:S ratio for tissue concentrations of ketamine enantiomers was significantly greater than unity in plasma and heart (both P,0.0001), but significantly less than unity in liver and fat (P,0.0001). The R:S ratio for concentrations of norketamine enantiomers, however, was significantly greater than unity in all tissues (P#0.005), except gut. The R:S ratio of the distribution coefficients for ketamine enantiomers was significantly less (P#0.001) than unity in all tissues examined, while for norketamine, it was significantly less than unity in liver (P,0.0005), lung (P,0.05), gut (P,0.005) and fat (P,0.0001), but significantly greater than unity in kidney (P,0.01) and heart (P,0.001) and muscle (P,0.05). After washout, the R:S ratio for ketamine enantiomer concentrations was significantly greater than unity in all CNS regions (P,0.005). However, the R:S ratio of the log-transformed values for the distribution coefficients did not differ significantly from unity. In peripheral tis-
2060
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
Table 2 Mean (6SEM) of the tissue concentrations and log-normalised distribution coefficients, at 180 min, for ketamine and norketamine enantiomers in rats, following brief infusions of ketamine racemate at 10 mg/Kg/min for 5 min (“washout” paradigm)
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2061
Table 3 Mean (6SEM) of the R:S enantiomer ratios for the tissue concentrations and tissue distribution coefficients in rats following infusions with rac-ketamine at either a constant rate of 6 mg/Kg/min until lethality (“washin” paradigm), or at 180 min after a brief infusion of 10 mg/Kg/min for 5 min (“washout” paradigm)
2062
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
sues R:S enantiomer concentration ratios were significantly greater in plasma (P,0.05), and fat (P,0.0001), but significantly less in kidney (P,0.0001) and gut (P,0.0001), while for norketamine, the R:S enantiomer ratio of tissue concentrations was significantly less than unity in plasma, liver, kidney, and gut (P,0.0005). The R:S ratio of the distribution coefficients for ketamine enantiomers was significantly less than unity in kidney (P,0.0001), heart (P,0.005), lung (P,0.05), and gut (P,0.0001). Ketamine pharmacokinetics during washout Pharmacokinetic parameters for the two ketamine enantiomers, derived from the plasma concentration-time washout data, are shown in table 4. The S:R enantiomer ratio was significantly greater than unity for ClT (P,0.0002), Vc (P,0.002), and Vss (P,0.02). Discussion Two dose paradigms were used in this study in an attempt to study distribution under two complementary conditions. The washin paradigm provided the maximal effects of the drug; the washout paradigm provided the minimal effects of the drug. This allowed influences on drug distribution by way of acute drug effect on the circulatory system to be elucidated. Similarly, the dual effects on norketamine by way of distribution and regional formation also should have been elucidated. Regrettably, the assay sensitivity limited the achievement of the latter objective. Nevertheless, new insights about ketamine biodisposition have been revealed. Ketamine produces sympathomimetic effects mediated by direct effects within the CNS but has direct myocardial depressant properties [2]. Lethality following constant rate infusions of rac-ketamine resulted from respiratory failure, presumably preceded by myocardial depression. The therapeutic index for S-ketamine, reported in earlier studies [10], suggests
Table 4 Pharmacokinetic parameters (mean6SEM) for ketamine enantiomers following bolus infusions of ketamine racemate at 10 mg/Kg/min over 5 min (“washout” paradigm)
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2063
that S-ketamine produces less myocardial depression than R-ketamine. The distribution coefficient into myocardial tissue, following washout, was significantly greater for S-ketamine, suggesting a longer residence time for this enantiomer in the myocardium. However, in view of the previously reported much higher therapeutic index for S-ketamine [10], the longer residence time and higher tissue uptake for S-ketamine do not appear to contribute disproportionately to the depressant effects of rac-ketamine on the myocardium. Significantly higher plasma concentrations of R- than of S-ketamine, found over the duration of the washin infusions, are consistent with the significantly greater tissue:plasma distribution coefficients found for S-ketamine, along with the greater values of Vc (14%),.ClT (15%) and VSS (10%) found during washout and are analogous to values found in humans [14]. Although the difference in plasma ketamine enantiomer concentrations was small under the conditions of this investigation, during procedures such as target controlled infusions this disparity in enantiomer concentrations could be expected to become more substantial as shown previously in an analogous study with thiopentone [25]. Plasma concentrations of R-norketamine, found over the duration of the washin infusions, were significantly higher than S-norketamine and, by the conclusion of the infusions, were almost twice those of S-norketamine. Moreover, in contrast to R-ketamine, the distribution coefficients of R-norketamine were significantly greater than S-norketamine indicating its greater uptake into CNS and myocardial tissue. Regional differences were also apparent, between brain and spinal cord, in both tissue concentrations and distribution coefficients. At the conclusion of the washin infusions, the distribution of ketamine and norketamine was highest into the kidney. Although the concentrations of ketamine enantiomers in renal tissue did not differ significantly, the concentration of R-norketamine was significantly higher (3-fold) than S-norketamine, suggesting that there was metabolism of both ketamine enantiomers to R-norketamine, and this is supported by the significantly greater (2-fold) distribution coefficient for R-norketamine. Similarly, there appeared to be preferential metabolism of both ketamine enantiomers to R-norketamine in the lung, although the distribution coefficients indicate that tissue uptake of S-norketamine exceeded the formation of R-norketamine. Enantiomer concentrations for ketamine and norketamine in liver tissue suggest a more rapid metabolism of R-ketamine to R-norketamine, but the distribution coefficients for norketamine enantiomers again suggest that tissue uptake of S-norketamine exceeded formation of R-norketamine. Tissue concentrations and distribution coefficients in the gut further suggest there was enantioselective tissue uptake of S-ketamine, but the tissue concentrations and distribution coefficients for norketamine enantiomers suggest that there was preferential formation of S-norketamine. Ketamine is a lipophilic drug, with predominantly blood flow-limited redistribution kinetics, [4,21], and has the capacity to alter its own regional disposition through its haemodynamic changes [22]. Thus, the use of the two infusion paradigms was useful to determine the concentrations and distribution coefficients for ketamine and norketamine enantiomers under maximal and minimal changes. Following the washin infusions, concentrations were lowest in the more poorly perfused tissues of muscle and fat. This is consistent with delayed redistribution due to reduced cardiac output associated with myocardial depression. For ketamine, uptake into muscle and fat was significantly higher for the S-enantiomer; for norketamine uptake into muscle was significantly higher for the R-enantiomer, while in fat uptake was
2064
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
significantly higher for the S-enantiomer. The distribution coefficients for ketamine and norketamine enantiomers demonstrate lower tissue uptake into fat than muscle for the less lipophilic metabolite, norketamine, but higher tissue uptake into fat than muscle for the more lipophilic parent drug, ketamine. In contrast to the washin infusions, the plasma concentrations of R-norketamine in the washout paradigm significantly exceeded those of S-norketamine for only a brief period. Concentrations of S-norketamine peaked at 60 min after the infusions and, by 180 min, remained significantly higher (4-fold) than R-norketamine. This could indicate that differing routes of metabolism were available to ketamine under the two paradigms, such that both concentration dependent N-demethylation to R-norketamine, as well as concentration independent metabolic inversion of R-norketamine to S-norketamine, were occurring. At the conclusion of washout period, ketamine and norketamine enantiomer concentrations were again highest in the kidney. However, unlike the constant rate infusions, concentrations of the S-enantiomers of ketamine and norketamine significantly exceeded (4-fold) those of the R-enantiomers, suggesting that inversion of R-ketamine to S-ketamine preceded metabolism to S-norketamine. The distribution coefficients demonstrate tissue uptake of ketamine enantiomers was highest in renal tissue, as well as further indicating that metabolic inversion of R-ketamine to S-ketamine occurred. The lack of significant difference in the distribution coefficients for norketamine enantiomers, in renal tissue, is consistent with metabolic formation rather than tissue uptake, but also indicates renal tissue was contributing to the circulating concentrations of norketamine enantiomers. Although ketamine enantiomer concentrations were substantially lower in gut than in kidney following washout, the tissue concentrations of S-ketamine were significantly higher (4-fold) than R- ketamine indicating metabolic inversion occurred in the gut as well. The norketamine enantiomer concentrations suggest that substantial uptake of ketamine enantiomers occurred in the gut, but the substantially lower ketamine enantiomer concentrations suggest that metabolism was more rapid here. As in renal tissue, significantly higher (4-fold) concentrations of S-norketamine were present in the gut, and the significantly higher (4-fold) distribution coefficient for S-ketamine in the gut suggests further evidence of metabolic inversion to S-ketamine. The measured distribution coefficients for norketamine enantiomers include a function of any local metabolism, and their lack of significant difference suggests that the gut also contributed to circulating concentrations of norketamine enantiomers. The liver appeared to be of a lesser importance in the uptake and metabolism of ketamine enantiomers following the brief infusion, and the higher tissue concentrations of S-norketamine probably only reflect the higher circulating concentrations of S-norketamine. The measured distribution coefficients suggest that uptake into the liver was not enantioselective for either ketamine or norketamine. Although tissue uptake of ketamine enantiomers in the lung was relatively high it was not enantioselective, and there appeared to be minimal metabolism to norketamine. In this investigation total ketamine concentrations in fat were only z1/10 of those present in kidney, unlike the earlier work where tissue concentrations of ketamine in fat were equivalent to those in kidney by 180 min [21]. However, in this study adipose tissue was sampled from the epididymal fat pad, whereas perinephric fat was sampled [21], and the difference in sampling sites may account for these differences in findings. Lastly, concentrations in the CNS are considered: this is the accepted site of ketamine (and nor-
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
2065
ketamine) action. Concentrations of R-ketamine remained significantly higher than S-ketamine in CNS tissue at the conclusion of the washout period, and the significantly higher distribution coefficient for R-ketamine in the forebrain suggests a longer residence time for R-ketamine in this CNS region. Mean plasma concentrations for the S-enantiomers of ketamine and norketamine at 60 min were, respectively, 0.8 mg/mL (3.4 mM) and 1.4 mg/mL (6.3 mM), whilst those for the R-enantiomers were, respectively, 0.9 mg/mL (3.9 mM) and 0.4 mg/mL (1.8 mM). The Ki values for the S- and R-enantiomers of ketamine and norketamine at the NMDA receptor have been reported to be 0.3, 1.4, 1.7, and 13 mM, respectively [18]. Other investigators found Ki values, for S- and R-ketamine of 0.6 mM and 2.0 mM, at the NMDA receptor, but 145 mM and 35 mM, at the sigma receptor, and the enantiomer potency ratio for inhibition at the NMDA receptor corresponded to the enantiomer potency ratio for analgesia in exercise-induced ischaemic pain [23]. In the present investigation, posthypnotic behavioural arousal, characterised by increased motor activity with amphetamine-like stereotypic head and forepaw movements, was observed to be maximal 60–80 min after the brief infusion of rac-ketamine. It would appear, therefore, that the psychostimulant effect of rac-ketamine during washout was predominantly caused by the action of S-ketamine at the NMDA receptor with S-norketamine contributing to a lesser degree. It would seem that ketamine enantiomer concentrations by 60 min were too low for activity at the sigma receptor. In summary, different profiles for the uptake and metabolism of ketamine enantiomers were apparent following constant rate washin, and brief infusion washout, paradigms of i.v. rac-ketamine. Enantioselectivity was found in regional distribution and clearance, including indirect evidence for extrahepatic metabolic inversion. Others, using different paradigms and species, had previously not found evidence for enantioselective distribution [24] or inversion [14]. However, regional pharmacokinetics had not previously been examined for enantioselectivity. During washin, when circulating concentrations of ketamine enantiomers were high, uptake and metabolism occurred predominantly in the kidney and to a lesser extent in liver, lung and gut, with formation of R-norketamine by a (presumed) first-order process predominating. However, following washout, when circulating concentrations of ketamine enantiomers were much lower, uptake and metabolism was dominated by the kidney and gut. Under these conditions inversion of R- to S-ketamine appeared to predominate with subsequent metabolism to S-norketamine by (presumed) zero-order processes. Acknowledgments The authors acknowledge with pleasure the grant support of the Australian New Zealand College of Anaesthetists and the expert technical advice of Ms XQ Gu. References 1. Church J and Lodge D. N-methyl-D-aspartate antagonism is central to the actions of ketamine and other phencyclidine receptor ligands. In: Domino EF, editor. Status of Ketamine in Anesthesiology. Ann Arbor:NPP Books, 1990. pp.501–19. 2. White PF, Way WL, and Trevor AJ. Ketamine - its pharmacology and therapeutic uses. Anesthesiology 1982;56:119–36.
2066
S.R. Edwards, L.E. Mather / Life Sciences 69 (2001) 2051–2066
3. Backonja M, Arndt G, Gombar KA, Check B, and Zimmermann M. Response of chronic neuropathic pain syndromes to ketamine: a preliminary study Pain 1994;56:51–7. 4. Clements JA and Nimmo WS. Pharmacokinetics and analgesic effect of ketamine in man. Br. J. Anaesth. 1981;53:27–30. 5. Eide PK, Stubhaug A, Oye I, and Breivik H. Continuous subcutaneous administration of the NMDA receptor antagonist ketamine in the treatment of post-herpetic neuralgia. Pain 1995; 61:221–8. 6. Maurset A, Skoglund LA, Hustveit O, and Oye I. Comparison of ketamine and pethidine in experimental and postoperative pain. Pain 1989;36:37–41. 7. Slogoff S, Allen GW, Wessels J, and Cheney DH. Clinical experience with subanesthetic ketamine. Anesth. & Analg. 1974;53:354–8. 8. Klepstad P, Maurset A, Moberg ER, and Oye I. Evidence of a role for NMDA receptors in pain perception. Eur. J. Pharmacol. 1990;187:513–8. 9. Oye I, Paulsen O, and Maurset A. Effects of ketamine on sensory perception: evidence for a role of NMDA receptors. J. Pharmacol. Exp. Ther. 1992;260:1209–13. 10. Marrietta MP, Way WL, Castagnoli N, and Trevor AJ. On the pharmacology of the ketamine enantiomorphs in the rat. J. Pharmacol. Exp. Ther. 1977;202:157–65. 11. Ryder S, Way WL, and Trevor AJ. Comparative pharmacology of the optical isomers of ketamine in mice. Eur. J. Pharmacol. 1978;49:15–23. 12. White PF, Schuttler J, Shafer A, Stanski DR, Horai Y, and Trevor AJ. Comparative pharmacology of the ketamine isomers. Br. J. Anaesth. 1985;57:197–203. 13. White PF, Ham J, Way WL, and Trevor AJ. Pharmacology of ketamine isomers in surgical patients. Anesthesiology 1980;52:231–39. 14. Geisslinger G, Hering W, Thomann P, Knoll R, Kamp HD, and Brune K. Pharmacokinetics and pharmacodynamics of ketamine enantiomers in surgical patients using a stereoselective analytical method. Br. J. Anaesth. 1993;70:666–71. 15. Woolf TF and Adams JD. Biotransformation of ketamine, (Z)-6-hydroxyketamine, and (E)-6-hydroxyketamine by rat, rabbit, and human liver microsomal preparations. Xenobiotica 1987;17:839–47. 16. Karasch ED and Labroo R. Metabolism of ketamine stereoisomers by human liver microsomes. Anesthesiology 1992;77:1201–07. 17. Grant IS, Nimmo WS, and Clements JA. Pharmacokinetics and analgesic effects of i.m. and oral ketamine. Br. J. Anaesth. 1981;53:805–10. 18. Ebert B, Mikkelsen S, Thorkildsen C, and Borgbjerg FM. Norketamine, the main metabolite of ketamine, is a non-competitive NMDA receptor antagonist in the rat cortex and spinal cord. Eur. J. Pharmacol. 1997;156:177–80. 19. Geisslinger G, Menzel-Soglowek S, Kamp HD, and Brune K. Stereoselective high-performance liquid chromatographic determination of the enantiomers of ketamine and norketamine in plasma. J. Chromatography B 1991;568:165–76. 20. Mather LE, Upton RN, Huang JL, Ludbrook GL, Gray E, and Grant C. The systemic and cerebral kinetics of thiopental in sheep: enantiomeric analysis. J. Pharmacol. Exp. Ther. 1996;279:291–7. 21. White PF, Marietta MP, Pudwill CR, Way WL, and Trevor AJ. Effects of halothane anesthesia on the biodisposition of ketamine in rats. J. Pharmacol. Exp. Ther. 1976; 196:545–5. 22. Bjorkman S, Akeson J, Nilsson F, Messeter K, and Roth B. Ketamine and midazolam decrease cerebral blood flow and consequently their own rate of transport to the brain: an application of mass balance pharmacokinetics with a changing regional blood flow. J. Pharmacokin. Biopharm. 1992;20:637–52. 23. Maurset A, Moberg R, and Oye I. The analgesic effect of ketamine: evidence for a non-opioid, PCP receptor mediated mechanism. In: Domino EF, editor. Status of Ketamine in Anesthesiology. Ann Arbor:NPP Books, 1990. pp. 239–46. 24. Henthorn TK, Krejcie TC, Niemann CU, Renders-Klein C, Shanks CA, and Avram MJ. Ketamine distribution described by a recirculatory pharmacokinetic model is not stereoselective. Anesthesiology 1999;91:1733–43. 25. Mather LE, Edwards SR, Duke CC, and Cousins MJ. Enantioselectivity of thiopental distribution into the central neural tissue of rats: an interaction with halothane. Anesth. Analg. 1999;89:230–5.