Tramadol, (1RS, 2RS)-2-dimethylaminomethyl-1-(3-methoxyphenyl)cyclohexanol, is a centrally acting analgesic used clinically for the treatment of postoperative and cancer pain. Tramadol binds μ-opioid receptors with low affinity and inhibits reuptake of monoamines such as norepinephrine and serotonin in the central nervous system (CNS), resulting in the activation of the descending inhibitory system (1,2). These actions are believed to primarily contribute to tramadol's antinociceptive effect. Its major active metabolite, O-desmethyltramadol (M1 metabolite), also has analgesic potency. M1 metabolite is a demethylated compound of tramadol (Fig. 1). Similar to the parent molecule, M1 metabolite has an agonistic effect at the μ-opioid receptor, but with a higher affinity than tramadol, and inhibits monoamine reuptake (3). Although the administration of opioids is accompanied by several adverse effects, including respiratory suppression, hypnosis, dependence, and abuse potential, these are uncommon with the administration of tramadol at equipotent doses (4). Additionally, tramadol is effective in treating acute pain alone, whereas tricyclic antidepressants, which are classic monoamine reuptake inhibitors, are generally ineffective (5). Furthermore, although opioids have little effect on touch-evoked pain (allodynia), tramadol has been proven effective against allodynia (6). Therefore, it is conceivable that there might be other action site(s) of tramadol, but little is known about other antinociceptive mechanisms or pharmacological actions of tramadol or M1 metabolite.
Neurotransmitter-gated ion channels, including glycine, γ-aminobutyric acidA (GABAA), and N-methyl-d-aspartate (NMDA) glutamate receptors, are thought to be sensitive to most anesthetics (7). Although investigators have studied the effects of most anesthetics on these channels, the effects of analgesics are not fully understood. Glycine receptors are the major inhibitory neurons in the spinal cord and GABAA receptors are distributed mainly in the brain. Both receptors are involved in pain modulation in the CNS (8). However, NMDA receptors are distributed in the CNS; they mediate pain perception and are thought to transmit allodynia in the spinal cord (9). In the present study, we sought other plausible targets for tramadol's actions and tested the effects of tramadol and M1 metabolite on human recombinant neurotransmitter-gated ion channels, including α1 glycine, α1β2γ2S GABAA, and NR1/NR2A NMDA receptors expressed in Xenopus oocytes. The subunit compositions of the recombinant receptors were chosen on the basis of the predominant subunit distributions in the CNS.
This study was approved by the Ethics Committee of Animal Care and Experimentation, University of Occupational and Environmental Health, Japan.
Xenopus laevis female frogs were purchased from Seac Yoshitomi (Fukuoka, Japan). GABA, glycine, and l-glutamate were obtained from Sigma (St. Louis, MO). Racemic compounds of tramadol hydrochloride and M1 metabolite were generous gifts from Nippon Sinyaku (Kyoto, Japan).
The cDNA encoding the human α1 glycine receptor subunit in pBK-CMV vector, the cDNAs of human α1, β2, and γ2S GABAA receptor subunits in pBK-CMV, pCDM8, and pCIS2 vectors, respectively, and the cDNAs of human NR1 and NR2A NMDA receptor subunits in pcDNA Amp vector were used for nuclear injections. Surgical procedures were performed on frogs after being anesthetized in water with 3-aminobenzoic acid ethyl ester (240 mg/200 mL water). The isolation of X. laevis oocytes was conducted as described previously (10). The isolated oocytes were placed in modified Barth's saline (MBS) containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.91 mM CaCl2, 0.33 mM Ca(NO3)2, and 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) adjusted to pH 7.5. The cDNAs for the α1 glycine receptor subunit (1 ng/30 nL), the α1, β2, and γ2S GABAA receptor subunits (2 ng/30 nL in a 1:1:2 molar ratio), or the NR1 and NR2A NMDA receptor subunits (1.5 ng/30 nL in a 1:1 molar ratio), were injected into the animal poles of oocytes by a blinded method (11). The injected oocytes were singly placed in Corning cell wells (Corning Glass Works, Corning, NY) containing incubation medium (sterile MBS supplemented with 10 mg/L streptomycin, 100,000 U/L penicillin, 50 mg/L gentamycin, 90 mg/L theophylline, and 220 mg/L pyruvate) and incubated at 15°–19°C. At 2–5 days after injection, oocytes were used for electrophysiological recordings (12).
Oocytes expressing the GABAA or glycine receptors were placed in a rectangular chamber (approximately 100-μL volume) and perfused (2 mL/min) with MBS. Oocytes expressing the NMDA receptors were perfused with Ba2+ Ringer's solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM BaCl2, and 10 mM HEPES adjusted to pH7.4) to minimize the effects of secondarily activated Ca2+-dependent Cl− currents. The animal poles of oocytes were impaled with 2 glass electrodes (0.5–10 MΩ) filled with 3 M KCl, and the oocytes were voltage-clamped at −70 mV using a Warner Instruments model OC-752B (Hamden, CT) oocyte clamp. Glycine (for the glycine receptors) or GABA (for the GABAA receptors) dissolved in MBS or l-glutamate with 10 μM glycine (for the NMDA receptors) dissolved in Ba2+ Ringer's solution was applied for 20 s. To test the effects of different concentrations of tramadol or M1 metabolite, experiments were performed at the EC5 of GABA or glycine, i.e., the agonist concentration producing 5% of the maximal current produced by 1 mM GABA or glycine. For the NMDA receptors, experiments were performed at the EC50 of glutamate, i.e., the concentration producing 50% of the maximal current produced by glutamate. To obtain a control response, the agonists were repeatedly applied until a consistent response was observed. Tramadol or M1 metabolite was dissolved in MBS or Ba2+ Ringer's solution and preapplied for 1 min before being coapplied with agonists. For the NMDA receptors, 10 and 30 μM tramadol or 30 μM M1 metabolite were applied to study the effects on the agonist (glutamate) concentration-response relationship. A 10-min washout period was provided between drug applications.
The effects of the drugs were expressed as the fraction of control response. Data were obtained from five to eight oocytes taken from at least three different frogs. The values of the EC50, the Hill coefficient, and the half-maximal inhibition concentration (IC50) for tramadol and M1 metabolite were calculated by nonlinear regression using GraphPad Prism software version 3.0 (GraphPad Inc., San Diego, CA). Data are represented as means ± sem. Statistical analysis was performed by one-way analysis of variance for multiple comparisons and unpaired t-test for comparisons between two groups. Differences were considered statistically significant at P < 0.05. All experiments were performed at room temperature (23°C).
In a number of investigations with the recombinant α1 glycine and α1β2γ2S GABAA receptors, inward chloride currents were observed in response to the application of agonists (Fig. 2A). Oocytes expressing the NR1/NR2A NMDA receptors yielded inward cation currents (Fig. 2B). Control currents in the glycine and GABAA receptors in response to the EC5 of agonists were 760 ± 90 and 620 ± 80 nA, respectively. The control current in the NMDA receptors in response to the EC50 of the agonist was 2730 ± 250 nA.
Neither tramadol nor M1 metabolite at concentrations of up to 100 μM had any effect on the glycine receptors (Fig. 3). Neither compound influenced the GABAA receptors at small concentrations, but at a concentration of 100 μM, both significantly inhibited the current responses (Fig. 4). Both tramadol and M1 metabolite, at concentrations from 0.1 to 100 μM, suppressed the NMDA receptors in a concentration-dependent manner (Fig. 5); nonlinear regression analysis yielded IC50 values of 16.4 and 16.5 μM, respectively. At a concentration of 10 μM, tramadol and M1 metabolite significantly inhibited the NMDA receptor currents by 13% ± 2% and 19% ± 9%, respectively. Different concentrations of glutamate (1 nM–100 μM) with 10 μM glycine were applied to obtain the agonist concentration-response relationship of the NMDA receptors (Fig. 6). Tramadol at 10 μM inhibited the agonist response without changing the EC50 value or the Hill coefficient (control: EC50, 0.27 μM; Hill coefficient, 1.1 ± 0.1 and tramadol: EC50, 0.29 μM; Hill coefficient, 1.0 ± 0.1). Tramadol at 30 μM also inhibited the agonist response without changing the EC50 value or the Hill coefficient (control: EC50, 0.25 μM; Hill coefficient, 1.0 ± 0.1 and tramadol: EC50, 0.27 μM; Hill coefficient, 0.9 ± 0.1). Similarly, M1 metabolite inhibited the agonist response but did not alter the EC50 value or the Hill coefficient (control: EC50, 0.26 μM; Hill coefficient, 0.9 ± 0.1 and M1 metabolite: EC50, 0.28 μM; Hill coefficient, 1.0 ± 0.1), indicating a noncompetitive inhibition of the NMDA receptors by tramadol and M1 metabolite. Neither tramadol nor M1 metabolite influenced the basal currents of any receptors tested in this study.
This study shows the effects of tramadol and its M1 metabolite on the neurotransmitter-gated ion channels. Grond et al. (13) reported that the therapeutic (minimal effective) serum concentration of tramadol is 2.0 ± 1.4 μM, which yields an M1 concentration of 0.29 ± 0.12 μM, being 7 times smaller than tramadol. Accordingly, we regard the concentrations of tramadol up to 10 μM for tramadol and 1 μM for M1 metabolite as clinically relevant.
Glycine receptors are distributed mainly in the spinal cord and have been considered a primary target for anesthetics (7). Glycinergic neurons are the major inhibitory system in the spinal cord and brainstem, and these regions are thought to mediate immobility by anesthetics in response to painful stimulation (14,15). Additionally, our recent study (16) implied the possibility that glycine receptors are a partial mediator of immobility produced by inhaled anesthetics. Furthermore, the intrathecal administration of strychnine, an antagonist of the glycine receptor, induces allodynia or hyperalgesia (17). In the present study, tramadol and M1 metabolite at concentrations of up to 100 μM presented no effect on the glycine receptors expressed in oocytes, indicating that the glycine receptors do not mediate any pharmacological effect of tramadol or M1 metabolite.
GABAA receptors were not affected by tramadol or M1 metabolite at clinically relevant concentrations, but were significantly inhibited only at a large concentration, 100 μM. Thus, it seems that the GABAA receptors do not mediate the antinociceptive action of tramadol. The lack of action of the GABAA receptors is consistent with the clinical finding that tramadol does not induce unconsciousness. These effects on the GABAA receptors are quite different from those of most anesthetics that enhance GABAA receptor activity (7).
NMDA receptors are involved in nociceptive neurotransmission. In particular, the NMDA receptors have a crucial role in the hypersensitization of the nociceptive neurons in the spinal cord, resulting in allodynia or hyperalgesia (9). Tramadol and M1 metabolite concentration-dependently inhibited the NMDA receptors in this study. Significant inhibition was observed at 10 μM, indicating that the NMDA receptors mediate, at least in part, the antinociceptive effect of tramadol. The present result may explain the clinical finding that tramadol relieves allodynia (6). Ketamine is a noncompetitive inhibitor of the NMDA receptors; it reduces the function of mouse NMDA receptors expressed in Xenopus oocytes by >80% at 10 μM, the anesthetic EC50 (18). Liu et al. (19) reported that ketamine suppresses human NMDA receptors by approximately 40% at 10 μM. Because analgesic effects of ketamine are seen at much smaller concentrations than those required for the anesthetic effect (immobility), even a mild inhibition (<20%) of the NMDA receptors by tramadol may contribute to its analgesic effect. To address the inhibitory mechanism of tramadol and M1 metabolite for the NMDA receptors, we studied the effects on the agonist concentration-response relationship. This study demonstrated that tramadol and M1 metabolite noncompetitively inhibit the NMDA receptors.
However, a previous study (2) reported that Ki values of tramadol for μ-opioid receptor binding and norepinephrine uptake were 2.1 and 0.79 μM, respectively, both of which are less than the IC50 of tramadol for the NMDA receptor (16 μM) in the current study. Accordingly, the inhibition of the NMDA receptors may be responsible for the antinociceptive effects at relatively large concentrations. In previous studies, we found that tramadol suppressed the function of muscarinic acetylcholine (ACh) receptors and nicotinic ACh receptors at clinically relevant concentrations, with IC50 values of 3.4 and 1.2 μM, respectively (20,21). Although the physiological roles of the inhibitory effects on these ACh receptors are not clear, the hypnosis and dizziness observed with tramadol might be associated with this inhibition. Because the clinical concentration of M1 metabolite is much less than that of the parent molecule (13), the effect of M1 metabolite on the NMDA receptors is unlikely to be involved in the clinical actions of tramadol administration.
The pharmacological properties and potencies of tramadol and M1 metabolite on the neurotransmitter-gated ion channels tested were very similar in this study. As shown in Figure 1, chemical structures of the compounds are slightly different, and the methyl group of tramadol is not involved in the specific interactions of the compounds with the NMDA and the GABAA receptors. The influence of tramadol or M1 metabolite on the other NR2 subunits was not determined in this study. Recently, NR2B subunits were reported to be associated mainly with antinociception (22). Some investigations using the oocyte expression system have shown that the effects of anesthetics (19,23) and psychotropic drugs (24) on the NR1/NR2A receptors are very similar to effects on the NR1/NR2B receptors. Further experiments are needed to determine precisely how tramadol and M1 metabolite modulate glutamatergic neurotransmission in the CNS in vivo.
Some other clinical aspects of tramadol administration should be discussed here. Tramadol has been proven to induce seizures, especially at large concentrations (25). The inhibition of GABAergic neurons and the activation of glutamatergic neurons lead to seizures. This study demonstrated that tramadol and M1 metabolite inhibit the GABAA receptors at large concentrations and inhibit the NMDA receptors at clinically relevant concentrations, which is compatible with the anticonvulsive and proconvulsive properties of tramadol (26). The clinical significance of the slight inhibition of the GABAA receptors at large concentrations is still unclear; however, the inhibition might decrease a threshold of the seizure even if other systems are simultaneously involved in the seizure.
The racemic compounds of tramadol and M1 were used in this study. It is reported that their enantiomers have different pharmacokinetic-pharmacodynamic properties as to an affinity for μ-opioid receptor, an ability to inhibit norepinephrine or serotonin uptake, and antinociceptive effects (27–29). Further experiments using the enantiomers are required for our understanding of how the chirality influences the neurotransmitter-gated ion channels, especially the NMDA receptors.
In conclusion, this study demonstrated that tramadol inhibits the NMDA receptors at clinically relevant concentrations, and the GABAA receptors at large concentration. These findings may explain some of the clinical properties of tramadol, including its antinociceptive effect and side effects.
We thank Dr. Paul J. Whiting for kindly providing GABAA and NMDA receptor subunit cDNAs, and Dr. Heinrich Betz for glycine receptor subunit cDNA. We also thank Dr. R. Adron Harris for careful attention to this study.
1. Raffa RB, Friderichs E, Reimann W, et al. Opioid and nonopioid components independently contribute to the mechanism of action of tramadol, an ‘atypical' opioid analgesic. J Pharmacol Exp Ther 1992;260:275–85.
2. Driessen B, Reimann W, Giertz H. Effects of the central analgesic tramadol on the uptake and release of noradrenaline and dopamine in vitro. Br J Pharmacol 1993;108:806–11.
3. Goeringer KE, Logan BK, Christian GD. Identification of tramadol and its metabolites in blood from drug-related deaths and drug-impaired drivers. J Anal Toxicol 1997;21:529–37.
4. Lehmann KA. Tramadol in acute pain [in French]. Drugs 1997;53(Suppl 2):25–33.
5. Monks R, Merskey H. Psychotropic drugs. In: Wall PD, Melzack R, eds. Textbook of pain. London: Churchill Livingstone, 1999:1155–86.
6. Sindrup SH, Andersen G, Madsen C, et al. Tramadol relieves pain and allodynia in polyneuropathy: a randomised, double-blind, controlled trial. Pain 1999;83:85–90.
7. Yamakura T, Bertaccini E, Trudell JR, Harris RA. Anesthetics and ion channels: molecular models and sites of action. Annu Rev Pharmacol Toxicol 2001;41:23–51.
8. Cousins M, Power I. Acute and postoperative pain. In: Wall PD, Melzack R, eds. Textbook of pain. London: Churchill Livingstone, 1999:447–91.
9. Doubell TP, Mannion RJ, Woolf CJ. The dorsal horn: state-dependent sensory processing, plasticity and the generation of pain. In: Wall PD, Melzack R, eds. Textbook of pain. London: Churchill Livingstone, 1999:165–81.
10. Hara K, Harris RA. The anesthetic mechanism of urethane: the effects on neurotransmitter-gated ion channels. Anesth Analg 2002;94:313–8.
11. Colman A. Expression of exogenous DNA in Xenopus
oocytes. In: Hames BD, Higgins SJ, eds. Transcription and translation: a practical approach. Washington DC: Oxford Press, 1984:49–59.
12. Hara K, Eger EI II, Laster MJ, Harris RA. Nonhalogenated alkanes cyclopropane and butane affect neurotransmitter-gated ion channel and G-protein-coupled receptors: differential actions on GABAA
and glycine receptors. Anesthesiology 2002;97:1512–20.
13. Grond S, Meuser T, Uragg H, et al. Serum concentrations of tramadol enantiomers during patient-controlled analgesia. Br J Clin Pharmacol 1999;48:254–7.
14. Antognini JF, Schwartz K. Exaggerated anesthetic requirements in the preferentially anesthetized brain. Anesthesiology 1993;79:1244–9.
15. Rampil IJ. Anesthetic potency is not altered after hypothermic spinal cord transection in rats. Anesthesiology 1994;80:606–10.
16. Zhang Y, Laster MJ, Hara K, et al. Glycine receptors mediate part of the immobility produced by inhaled anesthetics. Anesth Analg 2003;96:97–101.
17. Sorkin LS, Puig S. Neuronal model of tactile allodynia produced by spinal strychnine: effects of excitatory amino acid receptor antagonists and a mu-opiate receptor agonist. Pain 1996;68:283–92.
18. Yamakura T, Mori H, Masaki H, et al. Different sensitivities of NMDA receptor channel subtypes to non-competitive antagonists. Neuroreport 1993;4:687–90.
19. Liu HT, Hollmann MW, Liu WH, et al. Modulation of NMDA receptor function by ketamine and magnesium. Part I. Anesth Analg 2001;92:1173–81.
20. Shiraishi M, Minami K, Uezono Y, et al. Inhibition by tramadol of muscarinic receptor-induced responses in cultured adrenal medullary cells and in Xenopus laevis
oocytes expressing cloned M1 receptors. J Pharmacol Exp Ther 2001;299:255–60.
21. Shiraishi M, Minami K, Uezono Y, et al. Inhibitory effects of tramadol on nicotinic acetylcholine receptors in adrenal chromaffin cells and in Xenopus oocytes expressing alpha 7 receptors. Br J Pharmacol 2002;136:207–16.
22. Petrenko AB, Yamakura T, Baba H, Shimoji K. The role of N-methyl-D-aspartate (NMDA) receptors in pain: a review. Anesth Analg 2003;97:1108–16.
23. Hollmann MW, Liu HT, Hoenemann CW, et al. Modulation of NMDA receptor function by ketamine and magnesium. Part II. Interactions with volatile anesthetics. Anesth Analg 2001;92:1182–91.
24. Levine JB, Martin G, Wilson A, Treistman SN. Clozapine inhibits isolated N-methyl-D-aspartate receptors expressed in Xenopus oocytes in a subunit specific manner. Neurosci Lett 2003;346:125–8.
25. Tobias JD. Seizure after overdose of tramadol. South Med J 1997;90:826–7.
26. Potschka H, Friderichs E, Loscher W. Anticonvulsant and proconvulsant effects of tramadol, its enantiomers and its M1 metabolite in the rat kindling model of epilepsy. Br J Pharmacol 2000;131:203–12.
27. Valle M, Garrido MJ, Pavon JM, et al. Pharmacokinetic-pharmacodynamic modeling of the antinociceptive effects of main active metabolites of tramadol, (+)-O-desmethyltramadol and (−)-O-desmethyltramadol, in rats. J Pharmacol Exp Ther 2000;293:646–53.
28. Frink MC, Hennies HH, Englberger W, et al. Influence of tramadol on neurotransmitter systems of the rat brain. Arzneimittelforschung 1996;46:1029–36.
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29. Bamigbade TA, Davidson C, Langford RM, Stamford JA. Actions of tramadol, its enantiomers and principal metabolite, O-desmethyltramadol, on serotonin (5-HT) efflux and uptake in the rat dorsal raphe nucleus. Br J Anaesth 1997;79:352–6.