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    Mean ± SD results for generation of the luminescence signal (ie, luciferin) in human (black bars), equine (gray bars), and canine (white bars) liver microsomes incubated with various concentrations of racemic ketamine (R-/S-ketamine; A and B) or S-ketamine alone (C) and a luminogenic substrate specific for CYP 3A4 (A and C) or CYP 2C9 (B). Ketoconazole (0.2μM) was used as an inhibitor for CYP 3A4 (ie, positive control compound), and diclofenac (20μM) was used as an inhibitor for CYP 2C9. Values for the luminescence signal represent a comparison with the value obtained with the solvent control sample (Buffer), which was assigned a value of 100%. *Values differ significantly (P < 0.05) between human and equine liver microsomes.

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    Mean ± SD results for generation of the luminescence signal for various concentrations of methadone incubated with baculovirus-insect-cell–expressed human CYP 3A4 plus P450 reductase plus cytochrome b5 (ie, human CYP 3A4 supersomes) and the luminogenic substrate specific for human CYP 3A4. Ketoconazole (0.2μM) was used as a positive control compound. See Figure 1 for remainder of key.

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    Mean ± SD results for generation of the luminescence signal for various concentrations of ibuprofen incubated with human (A) and equine (B) liver microsomes and the luminogenic substrate specific for CYP 2C9. Diclofenac (20μM) was used as a positive control compound. The luminescence signal obtained is shown, compared with the signal for the solvent control sample. See Figure 1 for remainder of key.

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    Mean ± SD concentrations of R-ketamine (circles and solid line), S-ketamine (squares and solid line), R-norketamine (circles and dashed line), and S-norketamine (squares and dashed line) when incubated with methadone and 26μM racemic ketamine in human (A), equine (B), and canine (C) liver microsomes. The incubation period was 8 minutes. Data represent results for duplicate incubations. In panels B and C, there is a significant (P < 0.05) decrease in norketamine formation with increasing concentrations of methadone.

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    Mean ± SD concentrations of R-ketamine (circles and solid line), S-ketamine (squares and solid line), R-norketamine (circles and dashed line), and S-norketamine (squares and dashed line) when incubated with xylazine and 26μM racemic ketamine in equine (A) and canine (B) liver microsomes. The incubation period was 8 minutes. Data represent results for duplicate incubations. For both species, there is a significant (P < 0.05) decrease in norketamine formation with increasing concentrations of xylazine.

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    Mean ± SD concentrations of R-ketamine (circles and solid line), S-ketamine (squares and solid line), R-norketamine (circles and dashed line), and S-norketamine (squares and dashed line) when incubated with diclofenac and 26μM racemic ketamine in equine (A) and canine (B) liver microsomes. The incubation period was 8 minutes. Data represent results for duplicate incubations. For both species, there is no significant decrease in norketamine formation with increasing concentrations of xylazine.

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    Mean ± SD concentrations of S-ketamine (squares and solid line) and S-norketamine (squares and dashed line) when 26μM racemic ketamine was incubated with methadone in equine liver microsomes and S-ketamine (circles and solid line) and S-norketamine (circles and dashed line) when 13μM S-ketamine was incubated with methadone in equine liver microsomes. The incubation period was 8 minutes. Data represent results for duplicate incubations. There is a significant (P < 0.05) decrease in S-norketamine formation with increasing concentrations of methadone. Decrease in S-norketamine formation is significant (P < 0.05) in both curves.

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In vitro evaluation of differences in phase 1 metabolism of ketamine and other analgesics among humans, horses, and dogs

Livia Capponi Dr med vet1, Andrea Schmitz Dr med vet2, Wolfgang Thormann Dr phil nat, Dr habil3, Regula Theurillat4, and Meike Mevissen Dr med vet, Dr habil5
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  • 1 Division of Veterinary Pharmacology and Toxicology, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland.
  • | 2 Division of Veterinary Pharmacology and Toxicology, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland.
  • | 3 Department of Clinical Pharmacology, Faculty of Medicine, University of Bern, 3010 Bern, Switzerland.
  • | 4 Department of Clinical Pharmacology, Faculty of Medicine, University of Bern, 3010 Bern, Switzerland.
  • | 5 Division of Veterinary Pharmacology and Toxicology, Department of Clinical Research and Veterinary Public Health, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland.

Abstract

Objective—To investigate cytochrome P450 (CYP) enzymes involved in metabolism of racemic and S-ketamine in various species and to evaluate metabolic interactions of other analgesics with ketamine.

Sample Population—Human, equine, and canine liver microsomes.

Procedures—An analgesic was concurrently incubated with luminogenic substrates specific for CYP 3A4 or CYP 2C9 and liver microsomes. The luminescence signal was detected and compared with the signal for negative control samples. Ketamine and norketamine enantiomers were determined by use of capillary electrophoresis.

Results—A concentration-dependent decrease in luminescence signal was detected for ibuprofen and diclofenac in the assay for CYP 2C9 in human and equine liver microsomes but not in the assay for CYP 3A4 and methadone or xylazine in any of the species. Coincubation of methadone or xylazine with ketamine resulted in a decrease in norketamine formation in equine and canine liver microsomes but not in human liver microsomes. In all species, norketamine formation was not affected by ibuprofen, but diclofenac reduced norketamine formation in human liver microsomes. A higher rate of metabolism was detected for S-ketamine in equine liver microsomes, compared with the rate for the S-enantiomer in the racemic mixture when incubated with any of the analgesics investigated.

Conclusions and Clinical Relevance—Enzymes of the CYP 3A4 family and orthologs of CYP 2C9 were involved in ketamine metabolism in horses, dogs, and humans. Methadone and xylazine inhibited in vitro metabolism of ketamine. Therefore, higher concentrations and diminished clearance of ketamine may cause adverse effects when administered concurrently with other analgesics.

Abstract

Objective—To investigate cytochrome P450 (CYP) enzymes involved in metabolism of racemic and S-ketamine in various species and to evaluate metabolic interactions of other analgesics with ketamine.

Sample Population—Human, equine, and canine liver microsomes.

Procedures—An analgesic was concurrently incubated with luminogenic substrates specific for CYP 3A4 or CYP 2C9 and liver microsomes. The luminescence signal was detected and compared with the signal for negative control samples. Ketamine and norketamine enantiomers were determined by use of capillary electrophoresis.

Results—A concentration-dependent decrease in luminescence signal was detected for ibuprofen and diclofenac in the assay for CYP 2C9 in human and equine liver microsomes but not in the assay for CYP 3A4 and methadone or xylazine in any of the species. Coincubation of methadone or xylazine with ketamine resulted in a decrease in norketamine formation in equine and canine liver microsomes but not in human liver microsomes. In all species, norketamine formation was not affected by ibuprofen, but diclofenac reduced norketamine formation in human liver microsomes. A higher rate of metabolism was detected for S-ketamine in equine liver microsomes, compared with the rate for the S-enantiomer in the racemic mixture when incubated with any of the analgesics investigated.

Conclusions and Clinical Relevance—Enzymes of the CYP 3A4 family and orthologs of CYP 2C9 were involved in ketamine metabolism in horses, dogs, and humans. Methadone and xylazine inhibited in vitro metabolism of ketamine. Therefore, higher concentrations and diminished clearance of ketamine may cause adverse effects when administered concurrently with other analgesics.

The CYP superfamily is involved in numerous oxidative reactions and plays a decisive role in the metabolism and kinetics of xenobiotics.1–3 Although 18 families and at least 43 subfamilies of CYPs have been identified in humans, not all are involved in the metabolism of drugs. The CYPs are found in many organs, but they are most abundantly expressed in the liver, which is a major site of drug metabolism.4 Cytochrome P450 3A4 is the predominant enzyme and provides approximately 30% of human liver microsomal enzymes; CYP 3A4 is reported to be involved in the metabolism of many drugs.5

Ketamine, a noncompetitive antagonist of the N-methyl-D-aspartate receptor, is widely used as an anesthetic in veterinary medicine.6 Ketamine is a mixture of 2 optical isomers (S- and R-ketamine). The 2 enantiomers are N-demethylated into S- and R-norketamine, respectively, by liver microsomal CYPs. It is common to routinely administer ketamine concurrently with various other and analgesics.

The effects of subanesthetic doses of ketamine on acute pain in horses have been evaluated.6 Investigators in 1 study7 detected a significant decrease in the amplitude of the nociceptive withdrawal reflex during a low-dose infusion of racemic ketamine in standing ponies. Analysis of results of another study8 suggests that perioperative administration of low doses of ketamine in dogs may augment analgesia in the postoperative period. In humans, ketamine is reported to be useful for treatment of acute postoperative pain,9 and investigators in another study10 determined that low doses of ketamine reduce acute visceral pain in humans. For > 10 years, S-ketamine has been preferentially used in human medicine because of its higher potency and the fact that it has fewer adverse effects, compared with those for the racemic mixture. A major characteristic of S-ketamine is its greater affinity for N-methyl-D-aspartate receptors, compared with the affinity of R-ketamine.11 The S-enantiomer has been successfully used in experiments involving dogs12 and other laboratory animals. Recently, S-ketamine has been introduced into veterinary practice for use in cats. Clinically, the anesthetic and analgesic potency of the S-isomer is approximately 3 to 4 times that of the R-isomer.

In horses, ketamine should not be used as the sole induction agent because its use results in extensor rigidity, dog-sitting posture, extreme muscle spasms, purposeless movements, excited facial expressions, profuse sweating and salivation, and sometimes convulsions.13 Therefore, it is recommended that ketamine be administered in combination with xylazine and diazepam. The combination of racemic ketamine with the α2-adrenergic receptor agonist xylazine has been widely used in horses to induce and maintain anesthesia.14 A more rapid recovery was evident in ponies sedated with xylazine administered in conjunction with S-ketamine, compared with recovery after administration of xylazine and racemic ketamine.15 The more rapid elimination of S-ketamine, compared with elimination of the racemic mixture, could explain the more rapid recovery detected with use of S-ketamine. In contrast, pharmacokinetic variables of racemic ketamine or the single S-enantiomer in ponies anesthetized with isoflurane did not significantly differ.16 These are indications that concurrently administered drugs are of importance for the pharmacokinetics of ketamine.

In humans, in vitro studies17–19 have revealed that CYP 3A4, CYP 2C9, and CYP 2B6 are responsible for N-demethylation of racemic ketamine, with CYP 3A4 being the enzyme primarily involved in biotransformation. In vitro biotransformation experiments have revealed that ketamine is demethylated in the liver and lungs of horses.20 Preliminary data from our laboratory group have confirmed that the CYP 3A4 and CYP 2C9 orthologs are the metabolizing enzymes for racemic ketamine in equine liver microsomes. Taking into account the important role of ketamine in anesthesia and analgesia in many species, it has been our intent to evaluate metabolic interactions of ketamine with routinely coadministered drugs, such as methadone, xylazine, ibuprofen, and diclofenac. Methadone is used as an analgesic in humans and many other animal species, and it can augment analgesic effects of ketamine.21 Methadone is metabolized by CYP 3A4 in humans.22 The α2-adrenergic receptor agonist xylazine is reportedly metabolized by CYP 3A in rats.23 Many NSAIDs (such as ibuprofen and diclofenac) are metabolized by CYP 2C9 in humans.24,25 Information systems on drug-drug interactions of ketamine and analgesics are available for human medicine, and information about induction or inhibition of CYP activity is also available for a number of drugs. In contrast, information on drug metabolism pathways of ketamine and possible drug-drug interactions has not been sufficiently evaluated in animal species. Therefore, it is not possible to predict interactions between drugs, and there may be adverse effects. Furthermore, an increase in metabolism as a result of interactions with concurrently administered compounds can affect treatment.

The objectives of the study reported here were to investigate by use of a luminogenic assay the metabolic activities of CYP 3A4 and CYP 2C9 and their respective orthologs toward commonly used analgesic drugs (methadone, ibuprofen, and diclofenac) and anesthetic drugs (ketamine and xylazine) in liver microsomal preparations from humans, horses, and dogs. To provide a basis on which to predict possible drug-drug interactions, investigations of the metabolism of ketamine alone and ketamine in combination with analgesic compounds were performed in vitro by use of enantioselective capillary electrophoresis for quantitation of ketamine and norketamine enantiomers in the incubation mixtures.

Materials and Methods

Sample population—Liver samples were collected from the cadavers of 3 healthy horses at a local slaughterhouse. Horses were crossbred horses or Franches-Montagnes of both sexes; horses were 13 to 30 years old. The horses had no history of drug treatment for at least 6 months preceding slaughter. Liver samples were collected within 30 minutes after the horses were stunned. Samples were immediately placed on dry ice for transportation to our laboratory. Tissues were frozen and stored at −70°C until used for microsomal preparation, as described elsewhere.26 Total CYP protein was determined by use of a method reported elsewhere.27 The final concentration of CYP protein was 461 pmol/mg of protein. Humana and canineb liver microsomes were obtained from a commercial source and stored at −70°C. Baculovirus-insect-cell–expressed human CYP 3A4 plus P450 reductase plus cytochrome b5 (referred to as the CYP 3A4 supersomes) and human CYP 1A2 plus P450 reductase plus cytochrome b5 (referred to as the CYP 1A2 supersomes) were also obtained from a commercial source.c

Luminescence signal of liver microsomes incubated with substrate specific for CYP 3A4 and CYP 2C9—A luminogenic CYP assayd couples CYP enzyme activity to the light generation of firefly luciferase.28 Luciferase uses ATP and D-luciferin to generate light in the presence of ambient oxygen and magnesium. Luminogenic CYP substrates are derivates of D-luciferin, but they are not active with luciferase.

In an initial step, human, canine, and equine liver microsomes were incubated with ketamine, methadone, xylazine, ibuprofen or diclofenac, and D-luciferin derivates for CYP 2C9 (deoxyluciferin [ie, luciferin-H]) and CYP 3A4 (ie, luciferin-BE) for 30 minutes, and the derivates were converted by the CYP enzyme to D-luciferin products when they reacted with the respective CYP. Luciferin-H is highly selective for CYP 2C9 in human microsomes, whereas luciferin-BE reacts with CYP 3A4, CYP 3A7, and CYP 4F12. However, the expression of CYP 3A7 and CYP 4F12 is minimal in the liver of humans.28 The luminescence signal was detected in a second step by the addition of luciferin detection reagent, which contained luciferase and ATP in a reaction mixture limited only for D-luciferin. Luciferin detection reagent simultaneously stopped the CYP enzymatic activity and initiated a luciferase reaction that generated an amount of light directly proportional to the luciferin product produced by the CYP. Concurrent incubation of the luminogenic substrate and the compound of interest (eg, ketamine) was expected to attenuate the luminescence signal, compared with the signal for a control substance, when the 2 substances interacted with the same CYP. Assays were conducted in accordance with the manufacturer's instructions. Positive control samples were inhibitors for human CYP 3A4 (ketoconazolee) and CYP 2C9 (diclofenacf). Potassium phosphate bufferg (100mM potassium dihydrogen phosphate mixed with 100mM dipotassium hydrogen phosphate [pH, 7.4]) was used to perform negative control reactions. Luminogenic assays were conducted in a white opaque polystyrene nontreated flat-bottom 96-well plate.h A luciferin standard curve was prepared for each assay; concentrations ranged from 0.016 to 1μM beetle luciferin.i The microsomal protein content in each well was 20 μg. Racemic ketaminej was used at final concentrations of 1, 10, 50, 100, and 200μM; S-ketaminek was used at concentrations of 0.5, 5, 25, 50, and 100μM; methadone,l xylazine hydrochloride,m and ibuprofenm were each used at final concentrations of 10, 50, 100, 200, and 300μM; and diclofenac was incubated at final concentrations of 1, 5, 10, 50, and 100μM. Ketoconazole (an inhibitor for human CYP 3A4) was used as a positive control compound at a final concentration of 0.2μM, and diclofenac (a selective inhibitor for CYP 2C9) was used at a final concentration of 20μM. Ketoconazole was dissolved in methanol to yield a final methanol concentration of 0.02% in the incubation mixture. The final concentration of the potassium phosphate buffer was 100mM. The luminescence signal was detected at 20°C by use of a luminometern and computer software.o The NADPH required for CYP activity was supplied by use of an NADPH regeneration system.p Incubations were performed in triplicate. Unspecific toxic effects of the inhibitors and ketamine against any enzymes were ruled out in preliminary experiments in which luciferin was incubated (37°C for 20 minutes) with the respective inhibitors and the commercial luciferin detection reagent containing the luciferase enzyme. In addition, incubation of racemic ketamine with human CYP 1A2 supersomes was used to test for unspecific quenching of luminogenesis by the substrate. Use of the respective substrates did not reveal a reaction.

Microsomal incubations with ketamine and other compounds—The incubation mixture (250 μL) comprised human, equine, or canine liver microsomes (0.5 mg/mL of microsomal protein), racemic ketamine at a concentration of 26μM or S-ketamine at a concentration of 13μM, potassium phosphate buffer (100mM [pH, 7.4]), and the NADPH regenerating systemp (1.3mM NADP+, 3.3mM glucose-6-phosphate, glucose-6-phosphate dehydrogenase [0.4 U/mL], 3.3mM μgCl2, and 5μM sodium citrate). The mean value for the Michaelis constant (ie, Km) for racemic ketamine was calculated to be approximately 65μM. Racemic ketamine or S-ketamine was incubated concurrently with xylazine or ibuprofen at concentrations ranging from 0 to 300μM, with methadone at concentrations ranging from 0 to 200μM, or with diclofenac at concentrations ranging from 0 to 100μM. The incubation mixture was vortexed and preincubated for 3 minutes at 37°C. Microsomal protein was added to achieve a final concentration of 0.5 mg/mL in a final volume of 250 μL, and the enzymatic reaction was started at 37°C. Samples were incubated for 8 minutes without shaking. An incubation period of 8 minutes was chosen on the basis of preliminary experiments conducted with equine and canine microsomes. Incubations were stopped by the addition of 500μL of 0.2M NaOH,g and vials were placed on ice. Incubations were performed in duplicate.

Analytic procedure and assay specifications—Enantioselective analysis of ketamine and norketamine was performed by use of capillary electrophoresis29 with modifications described in another report30 and conditions used in the in vitro experiments of other investigators.20 The assay was based on liquid-liquid extraction at alkaline pH followed by capillary electrophoresis analysis of the reconstituted extract by use of a Tris-phosphate buffer (50mM [pH, 2.5]) containing 10 mg of sulfated β-cyclodextrin/mL as a chiral selector. The β-cyclodextrin was a mixture (35 mg from 1 lotq and 15 mg from a second lot,r which was dissolved in 5 mL of Tris-phosphate buffer). To avoid an overlap of peaks between R-ketamine and xylazine, only β-cyclodextrin from 1 lotq was used (50 mg, which was dissolved in 5 mL of Tris-phosphate buffer that consisted of 0.303 g of Trisg and 144 μL of orthophosphoric acid [85%]s in 50 mL of distilled water with xylazine) as the chiral selector for measuring the incubations. Briefly, samples (200 μL) were mixed with 0.5 mL of 0.2M sodium hydroxide containing 30 μL of the internal standard (+)-pseudoephedrine hydrochloride.s Samples were evaporated, and remaining residues were dissolved in 30 μL of Tris-phosphate buffer (5mM), A capillary electrophoresis instrumentt with an on-column variable wavelength detector set to 195 nm and a 50-μm (internal diameter) fused-silica capillaryu with a total length of 45 cm (effective length, approx 34 cm) was used. Applied voltage was −20 kV, and temperature of the circulating cooling fluid in the capillary cartridge and around the sample trays was set at 20°C. Samples were injected with a vacuum of 6.895 kPa for 5 to 7 seconds; run time was 15 to 20 minutes. Quantitation was based on multilevel internal calibration by use of corrected peak areas.20 All chemicals were analytic grade.

Data analysis—All values were reported as mean and SD. Data were statistically analyzed by use of an ANOVA for repeated measures followed by the Bonferroni multiple comparison test; statistical analyses were conducted by use of commercially available software.v Values of P < 0.05 were considered significant.

Results

Identification of CYP orthologs involved in metabolism of ketamine and analgesics in human, equine, and canine species—In the first part of our study, our intent was to identify CYP orthologs involved in the metabolism of ketamine and analgesic compounds in human, canine, and equine liver microsomes. Luminogenic CYP assays were used to accomplish this objective.

Concurrent incubation of racemic ketamine, the substrate for CYP 3A4, and liver microsomes from all 3 species resulted in a significant (P = 0.01) concentration-dependent decrease in the luminescence signal in human and equine microsomes (Figure 1). No significant concentration-dependent results were obtained in canine liver microsomes. No marked differences were detected among species. Ketoconazole, an inhibitor of CYP 3A4, caused an inhibition of the luminescence signal of approximately 30% in all 3 species. Inhibition of the luminescence signal of the CYP 2C9 substrate was significantly more pronounced for equine liver microsomes, compared with that for human liver microsomes, for all concentrations of ketamine, except 10μM, which was not significantly (P = 0.08) different. Diclofenac, an inhibitor of CYP 2C9,4 caused an inhibition of the luminescence signal of approximately 35% in the human and equine liver microsomes. Incubations with canine liver microsomes were not performed because the canine orthologs of CYP 2C9 (ie, CYP 2C21 and CYP 2C41) do not metabolize the substrate for human CYP 2C9.

Figure 1—
Figure 1—

Mean ± SD results for generation of the luminescence signal (ie, luciferin) in human (black bars), equine (gray bars), and canine (white bars) liver microsomes incubated with various concentrations of racemic ketamine (R-/S-ketamine; A and B) or S-ketamine alone (C) and a luminogenic substrate specific for CYP 3A4 (A and C) or CYP 2C9 (B). Ketoconazole (0.2μM) was used as an inhibitor for CYP 3A4 (ie, positive control compound), and diclofenac (20μM) was used as an inhibitor for CYP 2C9. Values for the luminescence signal represent a comparison with the value obtained with the solvent control sample (Buffer), which was assigned a value of 100%. *Values differ significantly (P < 0.05) between human and equine liver microsomes.

Citation: American Journal of Veterinary Research 70, 6; 10.2460/ajvr.70.6.777

A significant (P = 0.01) concentration-dependent reduction in the luminescence signal was obtained when the luminogenic substrate for CYP 3A4 was concurrently incubated with S-ketamine (Figure 1). This effect was evident with human, canine, and equine liver microsomes, and no marked difference was evident among the 3 species. At the highest S-ketamine concentration (100μM), inhibition of the luminescence signal was approximately 25% in all species investigated. At the lowest concentration investigated (0.5μM), activity of the signal was approximately 85% for human and canine liver microsomes, whereas no effect was evident for equine liver microsomes. No difference was detected for the 3 species for data obtained with incubations consisting of 50μM S-ketamine and 100μM racemic ketamine.

Human, equine, and canine liver microsomes were incubated with the substrate for CYP 3A4 and various concentrations of methadone or xylazine. No effect on luminescence was obtained for methadone in human, equine, or canine liver microsomes at any concentration investigated (data not shown). With respect to the limited specificity of the CYP 3A4 substrate provided by the supplier, methadone was incubated with human CYP 3A4 supersomes to provide evidence that this enzyme is involved in methadone biotransformation. A significant (P < 0.001) concentration-dependent inhibition of the luminescence signal was detected (Figure 2). Inhibition of the signal was approximately 25% at the lowest concentration used (10μM). The decrease in activity was 45% at a concentration of 50μM, and no further decrease in the luminescence signal was detected at the higher concentrations (100 to 300μM).

Figure 2—
Figure 2—

Mean ± SD results for generation of the luminescence signal for various concentrations of methadone incubated with baculovirus-insect-cell–expressed human CYP 3A4 plus P450 reductase plus cytochrome b5 (ie, human CYP 3A4 supersomes) and the luminogenic substrate specific for human CYP 3A4. Ketoconazole (0.2μM) was used as a positive control compound. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 70, 6; 10.2460/ajvr.70.6.777

Minor inhibition of the luminescence signal was only detected at the highest concentration (300μM) when xylazine was incubated with human and equine liver microsomes and the substrate for CYP 3A4. With canine liver microsomes, no decrease in the luminescence signal was detected. A decrease of 23% and 13% in the luminescence signal was detected at the highest concentration investigated for human and equine liver microsomes, respectively (Table 1). Inhibition of the luminescence signal of the positive control compound (0.2μM ketoconazole) was 51%, 35%, and 26% for human, equine, and canine liver microsomal preparations, respectively.

Table 1—

Results for incubation of human, equine, and canine liver microsomes with xylazine, diclofenac, and CYP inhibitors.*

CYPCompoundConcentration (μM)Luminescence signal (%)
   HumanEquineCanine
3A4Ketoconazole0.249 ± 16.065 ± 11.474 ± 8.2
Xylazine0100 ± 6.1100 ± 2.8100 ± 6.0
1094 ± 5.1100 ± 3.585 ± 14.6
5094 ± 5.197 ± 3.499 ± 6.3
100102 ± 4.594 ± 5.197 ± 5.6
20094 ± 8.991 ± 6.485 ± 6.7
30077 ± 24.287 ± 4.097 ± 8.7
2C9Ibuprofen20086 ± 9.670 ± 9.6ND
Diclofenac0100 ± 6.7100 ± 10.1ND
181 ± 8.3106 ± 9.6ND
5101 ± 5.098 ± 7.5ND
1098 ± 4.592 ± 12.1ND
5059 ± 4.953 ± 3.3ND
10039 ± 7.934 ± 5.2ND

Values reported are mean ± SD of triplicate incubations.

Ketoconazole is a standard inhibitor for CYP 3A4, and ibuprofen is a standard inhibitor for CYP 2C9.

ND = Not determined.

Incubation of human and equine liver microsomes with various concentrations of the NSAID ibuprofen and the substrate for CYP 2C9 resulted in a significant (P < 0.001) concentration-dependent inhibition of the luminescence signal for both species (Figure 3). In equine liver microsomes, inhibition of the signal was more pronounced, compared with that for human liver microsomes; inhibition differed significantly between the 2 species at 3 concentrations of ibuprofen. Incubation of the NSAID diclofenac with the substrate specific for human CYP 2C9 resulted in a significant (P < 0.001) concentration-dependent inhibition of the luminescence signal in human and equine liver microsomes (Table 1). The decrease in activity was approximately 65% at the highest concentration of diclofenac in human and equine liver microsomes, compared with results for the solvent control sample (which was assigned a value of 100%). Ibuprofen (200μM) was used as a positive control compound, and inhibition of approximately 20% and 30% was evident in human and equine liver microsomes, respectively, compared with results for the solvent control sample.

Figure 3—
Figure 3—

Mean ± SD results for generation of the luminescence signal for various concentrations of ibuprofen incubated with human (A) and equine (B) liver microsomes and the luminogenic substrate specific for CYP 2C9. Diclofenac (20μM) was used as a positive control compound. The luminescence signal obtained is shown, compared with the signal for the solvent control sample. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 70, 6; 10.2460/ajvr.70.6.777

Drug-drug interactions of analgesics with ketamine in human, equine, and canine liver microsomes—Increasing methadone concentrations resulted in a decrease in metabolite formation (S- and R-norketamine) in equine and canine microsomal preparations; consequently, significantly (P < 0.001) higher amounts of ketamine were detected in the reaction mixture (figure 4). In contrast, increasing concentrations of methadone decreased norketamine formation in human liver microsomes only at the 2 highest concentrations investigated, and results did not differ significantly. In human, equine, and canine liver microsomes, R-ketamine concentrations exceeded S-ketamine concentrations when ketamine was incubated alone or concurrently with methadone. Concentrations of S-norketamine were larger than concentrations of R-norketamine in all cases.

Figure 4—
Figure 4—

Mean ± SD concentrations of R-ketamine (circles and solid line), S-ketamine (squares and solid line), R-norketamine (circles and dashed line), and S-norketamine (squares and dashed line) when incubated with methadone and 26μM racemic ketamine in human (A), equine (B), and canine (C) liver microsomes. The incubation period was 8 minutes. Data represent results for duplicate incubations. In panels B and C, there is a significant (P < 0.05) decrease in norketamine formation with increasing concentrations of methadone.

Citation: American Journal of Veterinary Research 70, 6; 10.2460/ajvr.70.6.777

Coincubation of xylazine and ketamine resulted in a decrease in norketamine formation in equine liver microsomes; this was a significant (P = 0.01) concentration-dependent effect. Consequently, higher amounts of ketamine were detected with increasing xylazine concentrations (Figure 5). No change was detected for human liver microsomes (data not shown), and the effect on norketamine formation was small but significant for canine liver microsomes. Concentrations of R-ketamine > 10μM in combination with high xylazine concentrations could not be accurately determined, which explained missing R-ketamine data. The amount of S-norketamine exceeded the amount of R-norketamine at all xylazine concentrations investigated in equine and canine liver microsomes, whereas this effect was not evident in human liver microsomes (data not shown).

Figure 5—
Figure 5—

Mean ± SD concentrations of R-ketamine (circles and solid line), S-ketamine (squares and solid line), R-norketamine (circles and dashed line), and S-norketamine (squares and dashed line) when incubated with xylazine and 26μM racemic ketamine in equine (A) and canine (B) liver microsomes. The incubation period was 8 minutes. Data represent results for duplicate incubations. For both species, there is a significant (P < 0.05) decrease in norketamine formation with increasing concentrations of xylazine.

Citation: American Journal of Veterinary Research 70, 6; 10.2460/ajvr.70.6.777

Norketamine formation was not affected by the concentration of ibuprofen in all 3 species investigated (data not shown). Diclofenac had no significant effect on norketamine formation at any concentration investigated when equine and canine liver microsomes were incubated with racemic ketamine. A small but significant decrease in norketamine formation with increasing diclofenac concentrations was detected for human liver microsomes. Higher R-ketamine concentrations were obtained, compared with S-ketamine concentrations, for equine and canine liver microsomes (Figure 6). Biotransformation rates were higher for S-ketamine than for R-ketamine at all concentrations of diclofenac in equine and canine liver microsomes but not in human liver microsomes (data not shown).

Figure 6—
Figure 6—

Mean ± SD concentrations of R-ketamine (circles and solid line), S-ketamine (squares and solid line), R-norketamine (circles and dashed line), and S-norketamine (squares and dashed line) when incubated with diclofenac and 26μM racemic ketamine in equine (A) and canine (B) liver microsomes. The incubation period was 8 minutes. Data represent results for duplicate incubations. For both species, there is no significant decrease in norketamine formation with increasing concentrations of xylazine.

Citation: American Journal of Veterinary Research 70, 6; 10.2460/ajvr.70.6.777

Concentrations of the norketamine enantiomers after incubation with human liver microsomes for 8 minutes were approximately 70% to 95% lower than the norketamine enantiomer concentrations of the equine and canine liver microsomes when incubated with ketamine. This difference was also detected when the incubation included ketamine with methadone, xylazine, and ibuprofen.

A higher rate of metabolism was found for S-ketamine in equine liver microsomes, compared with the metabolic rate for the S-enantiomer, in the racemic mixture when concurrently incubated at various concentrations of methadone, xylazine, ibuprofen, and diclofenac. However, there was a decrease in the biotransformation rate of S-ketamine, compared with the rate for racemic ketamine, in equine liver microsomes incubated with increasing concentrations of the various compounds (Figure 7).

Figure 7—
Figure 7—

Mean ± SD concentrations of S-ketamine (squares and solid line) and S-norketamine (squares and dashed line) when 26μM racemic ketamine was incubated with methadone in equine liver microsomes and S-ketamine (circles and solid line) and S-norketamine (circles and dashed line) when 13μM S-ketamine was incubated with methadone in equine liver microsomes. The incubation period was 8 minutes. Data represent results for duplicate incubations. There is a significant (P < 0.05) decrease in S-norketamine formation with increasing concentrations of methadone. Decrease in S-norketamine formation is significant (P < 0.05) in both curves.

Citation: American Journal of Veterinary Research 70, 6; 10.2460/ajvr.70.6.777

Discussion

In the study reported here, racemic and S-ketamine competed for the substrate specific for human CYP 3A4 in human, equine, and canine liver microsomes, which supported the hypothesis that CYP 3A4 is involved in biotransformation in all 3 species. Other studies17–19 revealed that CYP 3A4, CYP 2C9, and CYP 2B6 were the enzymes responsible for N-demethylation of racemic ketamine, with CYP 3A4 being the major pathway. No effect on the luminescence signal was detected when methadone or xylazine was concurrently incubated with the substrate for CYP 3A4 in the 3 species investigated, even though methadone is metabolized via CYP 3A4 in humans22 and xylazine is metabolized via CYP 3A in rats.23 Methadone is metabolized by CYP 3A4, CYP 2B6, CYP 2C8, CYP 2D6, CYP 2C9, and CYP 2C19 in humans,22,31–33 whereas the major pathways are linked to CYP 3A4 and CYP 2B6.31,33,34 It can be concluded that the substrate provided by the manufacturer is not selective for CYP 3A4, and therefore, biotransformation by CYPs other than CYP 3A4 can be assumed. Even though there is competition between the substrate for CYP 3A4 and methadone, the amount of luminescence may not be inhibited because the substrate can be metabolized by other CYPs to form luciferin. We must emphasize that it can only be suggested that orthologs of human CYP 3A4 metabolize the same compounds in other species. Although CYP enzymes are classified into distinct subfamilies on the basis of amino acid sequence,35 a high degree of sequence identity does not necessarily indicate similar catalytic specificity.36 Substitution of a single amino acid can cause a change in substrate specificity.37 Therefore, investigations that use single CYPs are needed to identify a specific CYP involved in biotransformation. For this reason, we incubated human single CYP 3A4 supersomes with methadone and the substrate for CYP 3A4, and a concentration-dependent decrease in the resulting luminescence signal was obtained. These results support the hypothesis that CYP 3A4 is involved in metabolism of methadone, but CYP 3A4 was not detected in the luminogenic assay because the luminogenic substrate was not specific for CYP 3A4.

Xylazine is reported to be metabolized by CYP 3A in rats,23 but evidence for involvement of CYP 3A4 in xylazine metabolism is still lacking. In the study reported here, no effect on generation of the luminescence signal was obtained when the substrate for CYP 3A4 was incubated concurrently with xylazine in human, equine, and canine liver microsomes. Again, a lack of specificity of the substrate may have been the reason for the lack of effect.

Equine liver microsomes are able to metabolize substrates that are markers for human CYP 3A4 and CYP 2C9 activity.38 Analysis of results for the study reported here revealed an inhibition in luminescence signal when racemic ketamine was concurrently incubated with human and equine liver microsomes and a luminogenic substrate for human CYP 2C9. This result was evidence that CYP 2C9 is involved in biotransformation in these 2 species. Investigations on canine liver microsomes with the substrate for CYP 2C9 could not be performed because canine microsomes do not metabolize the substrate specific for human CYP 2C9.39 Additional investigations would be needed with canine liver microsomes and a substrate specific for canine CYP 2C21, which is the equivalent for human CYP 2C9 in dogs.

It has been reported24,25 that CYP 2C9 is involved in biotransformation of ibuprofen and diclofenac in humans, and results of our study are in agreement with these findings. Ibuprofen caused a concentration-dependent decrease in luminescence signal in human and equine liver microsomes. A concentration-dependent decrease in the resulting luminescence signal was obtained for diclofenac in human and equine liver microsomes, which indicated that CYP 2C9 is involved in diclofenac biotransformation in the liver of these 2 species.

Our intent in the second part of the study was to identify and characterize possible drug-drug interactions between ketamine and other analgesic drugs. Incubations of equine and canine liver microsomes with racemic ketamine and methadone or xylazine followed by stereoselective analysis of the enantiomers of ketamine and its most important metabolite norketamine revealed that increasing concentrations of methadone and xylazine resulted in a decrease in formation of R- and S-norketamine. Increasing methadone concentrations resulted in a decrease in metabolite formation (S- and R-norketamine) in equine and canine liver microsomes, whereas methadone had only a minor effect on norketamine formation in human liver microsomes at the concentrations investigated. A possible reason may have been a difference in affinity of the CYPs for these compounds. Furthermore, methadone reportedly is metabolized by CYP pathways that are not used for ketamine biotransformation. The norketamine concentrations were approximately 80% less when ketamine was concurrently incubated with methadone and human liver microsomes, compared with concentrations when incubated with equine and canine liver microsomes. It can be assumed that a longer period is necessary for human liver microsomes to metabolize ketamine into norketamine, compared with the interval for metabolism by equine and canine liver microsomes. The CYP content of equine and canine liver microsomes is approximately 460 and 330 pmol/mg of protein,b respectively, whereas human liver microsomes have a metabolic rate of 240 to 250 pmol/mg of protein.a This may explain the differences in the intensity of metabolism among the 3 species.

Concurrent incubation of xylazine and ketamine resulted in a decrease in norketamine formation in equine and canine liver microsomes. The major enzyme for metabolism of xylazine in rats is CYP 3A.23 In the study reported here, we determined that a higher xylazine concentration resulted in formation of a lower amount of norketamine; consequently, more parent compound remained in the incubation mixture. Xylazine inhibited the CYP-mediated metabolism of ketamine, which could lead to higher ketamine concentrations in the body and may cause adverse effects in vivo. Ketamine and xylazine are both metabolized by CYP 3A4 in humans and CYP 3A in rats. Because the major pathway for both compounds is probably the same, it can be assumed that the CYP 3A family is also involved in biotransformation of xylazine in horses and dogs. We confirmed that biotransformation of ketamine into norketamine was inhibited by xylazine for the equine and canine liver microsomes, and we presume that both are metabolized by the same CYP.

In our study, increasing concentrations of ibuprofen or diclofenac had no or only a small effect on norketamine formation in human, equine, and canine liver microsomes. It can be concluded that diclofenac and ibuprofen concentrations investigated in this study do not affect ketamine biotransformation under the experimental conditions used. Based on the assumption that CYP 2C9 is involved in the biotransformation of these compounds, ketamine could be transformed into norketamine by other pathways, including that of CYP 3A4.

Analysis of our data revealed that N-demethylation of ketamine is stereoselective. Stereoselective N-demethylation of ketamine has also been hypothesized in an in vivo study40 in horses and an in vitro study19 in human liver microsomes. A higher clearance of the S-enantiomer in the absence of the R-enantiomer was reported for an in vivo study41 with human volunteers and surgical patients, compared with clearance for the S-enantiomer in the racemic mixture. Another study20 conducted by our laboratory group revealed stereoselective biotransformation of ketamine to norketamine in equine liver and lung microsomes, with slower elimination of S-ketamine in the presence of R-ketamine. In the study reported here, a higher rate of metabolism of S-ketamine in the absence of R-ketamine, compared with the metabolism rate for S-enantiomer in the racemic mixture, was detected at all concentrations of methadone, xylazine, ibuprofen, and diclofenac in equine liver microsomes. Ponies sedated with xylazine have a lower elimination half-life and mean residence time for S-ketamine administered as S-ketamine alone, compared with results for S-ketamine administered in a racemic mixture.15 This is in agreement with the findings of the study reported here. We hypothesize that this phenomenon is attributable to mutual inhibition of S- and R-ketamine via the same enzymatic pathways. In addition, the CYP enzymes involved may have a higher affinity for the S-enantiomer.

An enzyme-substrate competition between R- and S-ketamine can delay the N-demethylation of S-ketamine to S-norketamine, which would lead to slower elimination of this enantiomer when administered as part of a racemic mixture.16,19 Conversely, ponies anesthetized with isoflurane did not have significant differences for the pharmacokinetic variables estimated for the S-enantiomer of ketamine and norketamine after administration of a combination of R- and S-ketamine or S-ketamine alone.16

It has been reported that S-ketamine has a more prolonged analgesic effect (lasting 4 times as long as racemic ketamine42) and is approximately twice as potent as the racemic mixture for inhibiting a central summation of pain.43 This indicates advantages for administration of the S-enantiomer (instead of a racemic mixture) to horses. Ponies anesthetized with xylazine and S-ketamine achieve a standing position during recovery significantly faster than ponies anesthetized with xylazine and racemic ketamine.15 The authors of that study15 concluded that the more rapid elimination of S-ketamine and its active metabolite S-norketamine could explain the faster recovery detected for S-ketamine. Investigations on the biotransformation rate of S-ketamine in other species are needed.

ABBREVIATIONS

CYP

Cytochrome P450

NADPH

Reduced form of nicotinamide adenine dinucleotide phosphate

a.

No. 70196, Gentest, Basel, Switzerland.

b.

No. 73746, Gentest, Basel, Switzerland.

c.

Gentest, Woburn, Mass.

d.

P450-Glo assay, Promega, Madison, Wis.

e.

Janssen Research Foundation, Beerse, Belgium.

f.

Sigma-Aldrich Chemie, Schnelldorf, Germany.

g.

Merck Inc, Darmstadt, Germany.

h.

Catalogue No. 675095, Greiner Bio-one, Frickenhausen, Germany.

i.

Luciferin potassium salt, Promega, Madison, Wis.

j.

Racemic ketamine hydrochloride, Pharmacy of the Inselspital, Bern, Switzerland.

k.

Provided by Dr. E. Graeub AG, Bern, Switzerland.

l.

Racemic methadone hydrochloride, Pharmacy of the Inselspital, Bern, Switzerland.

m.

Sigma, Buchs, Switzerland.

n.

Synergy TM HAT, Bio-Tek Instruments, Winooski, Vt.

o.

KC4 software, Bio-Tek Instruments, Winooski, Vt.

p.

BD Biosciences, Woburn, Mass.

q.

Sulfated β-cyclodextrin sodium salt, lot No. 13112JD, Sigma-Aldrich Chemie, Schnelldorf, Germany.

r.

Sulfated β-cyclodextrin sodium salt, lot No. 13307MA, Sigma-Aldrich Chemie, Schnelldorf, Germany.

s.

Fluka, Buchs, Switzerland.

t.

Proteome Lab PA 800, Beckman Coulter, Fullerton, Calif.

u.

Polymicro Technologies, Phoenix, Ariz.

v.

NCSS statistical software, version 2001, NCSS, Kaysville, Utah.

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Contributor Notes

Supported by a grant from the Vetsuisse Faculty, University of Bern, and by the Swiss National Science Foundation.

Address correspondence to Dr. Mevissen.