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Tapentadol increases levels of noradrenaline in the rat spinal cord as measured by in vivo microdialysis
Neuroscience Letters 507 (2012) 151–155
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Tapentadol increases levels of noradrenaline in the rat spinal cord as measured by in vivo microdialysis Thomas M. Tzschentke a,∗ , Joost H.A. Folgering b , Gunnar Flik b , Jean De Vry a a b
Global Preclinical Research and Development, Grünenthal Innovation, Grünenthal GmbH, Department of Pharmacology, Zieglerstrasse 6, 52078 Aachen, Germany Brains On-line, de Mudden 16, 9741 AW Groningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 8 September 2011 Received in revised form 6 December 2011 Accepted 6 December 2011 Keywords: Descending inhibition Spinal cord Tapentadol Morphine Venlafaxine Microdialysis
a b s t r a c t Spinal noradrenaline is thought to play an important role in descending pain inhibitory pathways and the modulation of nociceptive information at the spinal level. Tapentadol is a -opioid receptor (MOR) agonist and noradrenaline reuptake inhibitor (NRI). We showed previously that tapentadol, in contrast to morphine, elevates levels of noradrenaline, but not serotonin, in the ventral hippocampus of rats. The aim of this study was to examine the effects of tapentadol, morphine and venlafaxine on spinal monoamine levels. Rats were implanted with spinal microdialysis probes. Drugs were administered intraperitoneally, and samples were collected for 3 h in isoflurane-anesthetized animals and analysed for monoamine content using HPLC–MS/MS. In terms of area-under-curve (AUC, 0–180 min), tapentadol (4.64–21.5 mg/kg) produced a dosedependent, significant increase in extracellular spinal noradrenaline levels (9275 ± 4346 min % at the highest dose versus −1047 ± 889 min % for vehicle). A maximum increase of 182 ± 32% of baseline was reached 60 min after administration of 10 mg/kg tapentadol. Venlafaxine (10 mg/kg) produced an effect of similar magnitude. In contrast, tapentadol decreased extracellular spinal serotonin levels (nonsignificantly compared to vehicle), while venlafaxine increased spinal serotonin to 267 ± 74% of baseline. In contrast to tapentadol and venlafaxine, morphine slightly decreased levels of noradrenaline and serotonin. This study demonstrates that analgesic doses of tapentadol (and venlafaxine), but not morphine, increase spinal noradrenaline levels and that tapentadol is devoid of a relevant serotonergic effect. It supports the suggestion that the NRI component of tapentadol is functionally relevant and contributes to its mechanism of action. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The descending pain modulatory system, originating in the midbrain (periaqueductal grey, PAG) and brainstem (rostroventromedial medulla, RVM) and projecting to the spinal cord dorsal horn, has a powerful influence on the modulation of nociceptive information transmitted from the periphery to the brain. The prominent role of this system in suppression of nociception has been recognized early on [6,17]. More recently, the role of descending projections in pain facilitation and the generation of hyperalgesic states has received increased attention. The descending modulatory system uses noradrenaline (NA) and serotonin (5-HT) as its main transmitters. The role of NA appears to be predominantly
∗ Corresponding author. Tel.: +49 241 5692816; fax: +49 241 5692852. E-mail address: [email protected] (T.M. Tzschentke). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.12.008
inhibitory, while the role of 5-HT appears to be bidirectional, mediating inhibitory as well as excitatory effects [20]. A number of drugs have been shown to increase the activity of the descending noradrenergic system and to increase extracellular levels of spinal NA, and this increase in noradrenergic activity has been linked to the antinociceptive/antiallodynic/antihyperalgesic effect of these treatments in acute or neuropathic pain, usually via activation of ␣2 -adrenoceptors. This was shown for morphine ([2] sheep, human; [26] sheep), fentanyl ([2] sheep), pregabalin ([22] mouse), gabapentin ([21,23] mouse; [9] human, [10] rat), nitrous oxide ([27] rat), and ketamine ([11] rat). Conversely, the pronociceptive effects of intra-PAG administration of nociceptin are associated with decreased monoamine levels in the spinal dorsal horn [14]. Tapentadol is a novel centrally acting analgesic belonging to a new pharmacological class, MOR-NRI (-opioid receptor agonist and noradrenaline reuptake inhibitor). We have shown previously that tapentadol, in contrast to morphine, elevates levels of NA, but
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not 5-HT, in the ventral hippocampus of rats [25]. The lack of significant serotonergic effects clearly differentiates tapentadol from the ‘atypical opioid’ tramadol, which produced clear increases of NA as well as 5-HT in the ventral hippocampus of rats, using identical experimental conditions [3]. The demonstration of tapentadolinduced elevation of extracellular NA levels can be taken as an in vivo proof of its NRI activity. The aim of the present study was to examine the effects of tapentadol on monoamine levels in the spinal cord dorsal horn, i.e. the site where the descending modulatory system impinges upon the ascending pain transmission pathway. Morphine, the prototypical ‘classical’ opioid, and venlafaxine, a mixed 5-HT/NA reuptake inhibitor, were used as comparators. Drug doses were chosen to fall within the analgesic dose range [18,25], and the dose of venlafaxine used was shown previously to produce significant elevations in extracellular levels of both 5-HT and NA in the ventral hippocampus of freely moving rats [3]. 2. Materials and methods 2.1. Animals Male adult Wistar rats (280–350 g; Harlan, Horst, the Netherlands) were used for the experiments and were kept under standard, constant conditions (lights on 07.00–19.00, temperature 22 ± 2 ◦ C, humidity 55 ± 10%). Experiments were conducted in accordance with the Declaration of Helsinki and were approved by the Institutional Animal Care and Use Committee of the University of Groningen. 2.2. Surgery Rats were anesthetized using isoflurane (2%; 800 ml/min O2 ). Bupivacaine and adrenaline were used for local anesthesia. Animals were placed in a stereotaxic frame (Kopf Instruments, USA). The dorsal skin was incised, and the spinal cord vertebrae were exposed. A small hole was drilled in the area between T12 and T13 with an angle of around 45◦ towards the spinal cord inside T13, and I-shaped probes (Hospal AN69 membrane; Brainlink, the Netherlands; 3 mm exposed surface) slowly inserted. 2.3. Drugs Tapentadol (Grünenthal GmbH, Aachen, Germany; batch GRTA19586-1-1), morphine (BUFA, Ijsselstein, the Netherlands; batch 99H17FO) and venlafaxine (Teva, Tikva, Israel; batch 330770503) were dissolved in saline. Solutions were injected intraperitoneally (i.p.) at 2 mL/kg (tapentadol, morphine) or 1 mL/kg (venlafaxine). Doses of all drugs refer to and were administered as their free base. 2.4. Experiment
Samples were mixed with 4 L internal standards mix (noradrenaline-d6, 5 × 10−8 M; serotonin-d4, 5 × 10−5 M), and were derivatized with SymDAQ in the autosampler (SIL-10ADvp, Shimadzu, Japan), by addition of 40 L of reagent to the sample vial. After 2 min, 50 L of the mixture was injected into the LC system by an automated sample injector (SIL-10ADvp, Shimadzu, Japan). Chromatographic separation was performed on a reversed phase Phenomenex Synergi MAX-RP 100 mm × 3.0 mm (2.5 m particle size) held at 35 ◦ C. Components were separated using a gradient of UP/ACN (98/2) 0.1% FA (mobile phase A) and UP/ACN 0.1% FA (30/70) (mobile phase B) (flow rate 0.3 mL/min) according to the following scheme: time (min)/% mobile phase B: 0/0; 4/40; 5.5/100; 7/2; 9/end. A post-column make-up flow of 0.15 mL/min was added to the flow of the LC, which was diverted to the waste for 2.85 min, after which it was switched to the MS for detection of the neurotransmitters. MS analyses were performed using an API4000 MS/MS system consisting of an API4000 MS/MS detector and a Turbo Ion Spray interface (both from Applied Biosystems, the Netherlands). Acquisitions were performed in positive ionization mode, with ionization spray voltage set at 4 kV and a probe temperature of 200 ◦ C. The instrument was operated in multiple-reaction-monitoring (MRM) mode. The collision gas (nitrogen) pressure was held at 2 psig. Data were calibrated and quantified using the AnalystTM data system (Applied Biosystem, version 1.4.2). 2.6. Statistical evaluation Three consecutive pre-treatment microdialysis samples with less than 50% variation were taken as baseline and their mean was set at 100%. Drug effects were expressed as percentages of basal level (mean ± SE) within the same subject. Statistical analysis was performed using Sigmastat for Windows (SPSS Corporation). Treatment and dose effects were compared, using two-way (time × dose) ANOVA for repeated measurements followed by Student Newmann Keuls post hoc test (except for venlafaxine where a one-way ANOVA for repeated measures over time was performed). Overall drug effects were expressed as area under curve (AUC) within the same subject, as calculated using the linear trapezoidal method. Treatment effects (AUC) were compared to vehicle using one-way ANOVA followed by Student–Newmann–Keuls post hoc test. The level of statistical significance was set at P < 0.05. 3. Results 3.1. Basal output Basal output values (mean ± SEM) obtained in dialysis samples from the spinal cord were 5.81 ± 0.60 fmol/sample for NA and 41.0 ± 11.1 fmol/sample for 5-HT (based on N = 38 rats each). 3.2. Tapentadol
Microdialysis probes were connected with flexible PEEK tubing to a microperfusion pump (Syringe pump UV8301501, TSE, Univentor, Malta) and perfused with artificial CSF, containing (in mM): NaCl 147, KCl 3, CaCl2 1.2 and MgCl2 1.2, at a flow rate of 1.5 L/min (dialysis flow). Samples were collected by an automated refrigerated fraction collector (CMA142, Sweden) for 15 min periods into mini-vials already containing 7.5 L 20 mM formic acid/0.04% ascorbic acid, and stored at −80 ◦ C until analysis. 2.5. Analysis of neurotransmitters Concentrations of NA and 5-HT were determined by HPLC with tandem mass spectrometry (MS/MS) detection.
3.2.1. Noradrenaline Fig. 1A shows the time course effects of 0, 4.64, 10 or 21.5 mg/kg tapentadol (i.p.) on the levels of NA in the spinal cord. There was a significant effect of time [F(12,227) = 8.225; P < 0.001], and dose × time interaction [F(36,227) = 3.145; P < 0.001]. Post hoc analysis revealed that extracellular NA levels were not significantly increased compared to basal levels after 0 or 4.64 mg/kg tapentadol. However, 10 mg/kg tapentadol significantly increased NA levels from t = 30 to t = 105 min compared to basal levels. After administration of 21.5 mg/kg tapentadol, NA levels were significantly increased from t = 30 to 180 min compared to basal levels.
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Fig. 2. Effects of tapentadol (0, 4.64, 10 or 21.5 mg/kg; i.p. at t = 0) on 5-HT levels in spinal cord, (A) in terms of time course data, (B) in terms of AUC0–180 data. N = 8, 5, 5 and 5 animals for 0, 4.64, 10 and 21.5 mg/kg, respectively. Fig. 1. Effects of tapentadol (0, 4.64, 10 or 21.5 mg/kg; i.p. at t = 0) on NA levels in spinal cord, (A) in terms of time course data, (B) in terms of AUC0–180 data. N = 8, 5, 5 and 5 animals for 0, 4.64, 10 and 21.5 mg/kg, respectively.
Fig. 1B shows the NA response in terms of AUC(0–180 min) . There was a significant difference between the AUCs of the different doses of tapentadol [F(3,19) = 3.134; P = 0.050]. However, post hoc analysis between the individual dose groups did not reveal a specific dose to be different from any of the other doses. 3.2.2. Serotonin Fig. 2A shows the time course effects of 0, 4.64, 10 or 21.5 mg/kg tapentadol (i.p.) on the levels of 5-HT in the spinal cord. There was a significant effect of time on 5-HT levels after the administration of tapentadol [F(12,225) = 8.699; P < 0.001]. Post hoc analysis revealed a decrease in 5-HT levels at t = 90 and 180 min after administration. However, this decrease could not be attributed to a specific dose. Fig. 2B shows the 5-HT response in terms of AUC(0–180 min) . There was no significant difference between the AUCs of the different doses of tapentadol [F(3,19) = 1.899; P = 0.166]. 3.3. Morphine 3.3.1. Noradrenaline Fig. 3A shows the time course effects of 0, 1, 3, 10 mg/kg morphine (i.p.) on levels of NA in the spinal cord. There was a significant effect of dose [F(3,190) = 8.380; P = 0.001], time [F(12,190) = 5.323; P < 0.001] and dose × time interaction [F(36,190) = 1.760; P < 0.01]. Post hoc analysis revealed a decrease in NA levels compared to baseline after 3 mg/kg morphine at t = 60 min and 105 min. None of the other doses (including vehicle) showed a significant change compared to baseline.
Fig. 3B shows the response of NA levels in terms of AUC(0–180 min) . There was a significant difference between the AUCs of the different doses of morphine [F(3,18) = 12.265; P < 0.001]. Post hoc analysis showed that AUC values for all doses were significantly different from the vehicle AUC.
3.3.2. Serotonin Fig. 4A shows the effects of 0, 1, 3, 10 mg/kg morphine (i.p.) on the extracellular levels of 5-HT. There was a significant effect of time on 5-HT levels after the administration of morphine [F(12,213) = 5.858; P < 0.001]. Post hoc analysis revealed a decrease in 5-HT levels from t = 45 min up to and including 180 min. However, this decrease could not be attributed to a specific dose. Fig. 4B shows the 5-HT response in terms of AUC(0–180 min) . There was no significant difference between the AUCs of the different doses of morphine [F(3,19) = 0.310; P = 0.818]. 3.4. Venlafaxine After administration of venlafaxine (10 mg/kg; i.p.) (data not shown), there was a significant effect over time on NA levels [F(11,43) = 6.821; P < 0.001; N = 5]. Post hoc analysis revealed a significant increase of NA levels compared to baseline from t = 30 min up to and including t = 165 min. There was also a significant effect over time on 5-HT levels [F(11,51) = 6.850; P < 0.001; N = 6]. Post hoc analysis revealed a significant increase of 5-HT levels compared to baseline from t = 30 min up to and including t = 105 min and at t = 135 min. As these experiments were done as a pilot study to demonstrate method sensitivity for measuring changes in spinal monoamine levels, no separate vehicle group was run.
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Fig. 3. Effects of morphine (0, 1, 3, 10 mg/kg; i.p. at t = 0) on NA levels in spinal cord, (A) in terms of time course data, (B) in terms of AUC0–180 data. N = 8, 4, 5 and 4 animals for 0, 4.64, 10 and 21.5 mg/kg, respectively.
Fig. 4. Effects of morphine (0, 1, 3, 10 mg/kg; i.p. at t = 0) on 5-HT levels in spinal cord, (A) in terms of time course data, (B) in terms of AUC0–180 data. N = 8, 4, 5 and 4 animals for 0, 4.64, 10 and 21.5 mg/kg, respectively.
4. Discussion The current set of experiments was performed to evaluate the effects of tapentadol on changes in spinal cord levels of NA and
5-HT in rats. Administration of tapentadol resulted in increases in NA levels after 10 and 21.5 mg/kg of tapentadol compared to baseline. 5-HT levels were decreased by tapentadol and morphine in a non-dose-dependent, statistically non-significant manner. A comparable downward ‘drift’ of 5-HT was also observed after vehicle treatment, and such an effect has been previously observed and described [16]; thus, this effect may be non-specific, and the reason for and relevance of this observation remains unclear. Nevertheless, this study clearly extends the functional in vivo proof of tapentadol’s NRI activity from the ventral hippocampus to the spinal cord dorsal horn, a structure of central importance for the modulation of pain signals by the descending modulatory system. It also confirms the lack of a relevant SRI activity of tapentadol. There is good evidence that NA reuptake inhibition plays a greater role than 5-HT reuptake inhibition in antinociception [1,8,15]. Thus, tapentadol’s monoaminergic activity is selective for the more relevant antinociceptive system (NA), and has no impact on the less relevant antinociceptive system (5-HT). That our current experimental protocol is able to demonstrate SRI activity was shown with venlafaxine. In contrast to tapentadol and venlafaxine, morphine slightly decreased levels of noradrenaline. The finding that tapentadol elevates spinal levels of NA also corroborates our interpretation of the intrinsic synergy between its two mechanisms of action demonstrated previously [19]. Through its opioid activity tapentadol would activate the descending noradrenergic projections at the supraspinal level, and its NRI activity would then amplify the effect of the opioid-mediated increase in NA release at the spinal level, by blocking the reuptake of NA released due to supraspinal activation of the descending noradrenergic projections. An increase in spinal noradrenergic transmission may be particularly relevant in the context of chronic pain, since there is evidence that under conditions of chronic pain, the balance of excitatory and inhibitory supraspinal input to the spinal cord is disrupted, such that excitatory, pain-facilitatory mechanisms prevail – either due to an enhancement of facilitatory serotonergic processes, or due to a diminution of inhibitory noradrenergic processes – and contribute to the development and/or maintenance of central sensitization (see [1,5,20,24]). Under such circumstances, restoring the balance by selectively enhancing NA-mediated inhibition (and not increasing serotonergic activity) may be a particularly valuable approach. In fact, tapentadol shows a relatively higher potency compared to pure opioids in animal models of chronic neuropathic pain. While in models of acute nociceptive pain tapentadol is 2–3 times less potent than morphine, in models of chronic neuropathic pain tapentadol is at least as potent as morphine, if not more potent [4,12,18,25]. That tapentadol is so potent despite the fact that its MOR affinity is 50 times less than that of morphine, further underlines the importance of its NRI activity. The magnitude of drug effects on transmitter levels in the spinal cord of anesthetized rats observed in the present study is smaller than the magnitude observed in our previous studies in the ventral hippocampus of freely moving rats at comparable drug doses [3,25]. The two obvious differences between the two sets of studies are the different sampled CNS regions and the absence or presence of anesthesia. Upon inspection of the limited literature on spinal monoamine microdialysis in rats, it appears that the drug effect size in the spinal cord does not exceed 250–300% of baseline, in both anesthetized (present study (tapentadol, venlafaxine); [10] gabapentin) and freely moving animals ([13] desipramine); [27] nitrous oxide; [11] ketamine), whereas supraspinally, e.g. in the ventral hippocampus, increases of 400–800% of baseline are frequently observed. Thus, it appears likely that the relatively small effect sizes seen in the present study are mainly related to the spinal location of the microdialysis probe. Although it cannot be excluded that anesthesia had an influence on basal and/or
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drug-induced monoamine release in the spinal cord, anesthesia abolished the potentially confounding factor of locomotor activity. As Gerin and Privat [7] have shown, locomotor activity can influence monoamine levels in the (ventral horn of the) spinal cord. The present study did not find a morphine-induced increase in spinal levels of NA. In fact, a small decrease in NA (and 5-HT) was observed. While this finding is consistent with the lack of NRI (and SRI) activity of morphine, it appears to be at odds with the concept of supraspinal activation of descending inhibitory projections and with the previously reported increase in spinal NA levels by morphine and fentanyl [2,26]. However, these studies were conducted in sheep. Our study is the first to use microdialysis to examine the effects of morphine on spinal NA in the rat, and presently, species differences in the response to morphine cannot be ruled out. In conclusion, this present study showed that tapentadol increases spinal NA levels while being devoid of a relevant serotonergic effect. It supports the suggestion that the NRI component of tapentadol is functionally relevant and contributes to its potent analgesia and broad efficacy range. Acknowledgement This study was funded by Grünenthal GmbH. References [1] K. Bannister, L.A. Bee, A.H. Dickenson, Preclinical and early clinical investigations related to monoaminergic pain modulation, Neurotherapeutics 6 (2009) 703–712. [2] H. Bouaziz, C. Tong, Y. Yoon, D.D. Hood, J.C. Eisenach, Intravenous opioids stimulate norepinephrine and acetylcholine release in spinal cord dorsal horn. Systematic studies in sheep and an observation in a human, Anesthesiology 84 (1996) 143–154. [3] P. Bloms-Funke, E. Dremencov, T.I. Cremers, T.M. Tzschentke, Tramadol increases extracellular levels of serotonin and noradrenaline as measured by in vivo microdialysis in the ventral hippocampus of freely-moving rats, Neurosci. Lett. 490 (2011) 191–195. [4] T. Christoph, J. De Vry, T.M. Tzschentke, Tapentadol, but not morphine, selectively inhibits disease-related thermal hyperalgesia in a mouse model of diabetic neuropathic pain, Neurosci. Lett. 470 (2010) 91–94. [5] R. D’Mello, A.H. Dickenson, Spinal cord mechanisms of pain, Br. J. Anaesth. 101 (2008) 8–16. [6] H.L. Fields, A.I. Basbaum, Brainstem control of spinal pain-transmission neurons, Annu. Rev. Physiol. 40 (1978) 217–248. [7] C. Gerin, A. Privat, Direct evidence for the link between monoaminergic descending pathways and motor activity. II. A study with microdialysis probes implanted in the ventral horn of the spinal cord, Brain Res. 794 (1998) 169–173. [8] F.S. Hall, J.M. Schwarzbaum, M.T.G. Perona, J.S. Templin, M.G. Caron, K.P. Lesch, D.L. Murphy, G.R. Uhl, A greater role for the norepinephrine transporter than the serotonin transporter in murine nociception, Neuroscience 175 (2011) 315–327.
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Tapentadol increases levels of noradrenaline in the rat spinal cord as measured by in vivo microdialysis Thomas M. Tzschentke a,∗ , Joost H.A. Folgering b , Gunnar Flik b , Jean De Vry a a b
Global Preclinical Research and Development, Grünenthal Innovation, Grünenthal GmbH, Department of Pharmacology, Zieglerstrasse 6, 52078 Aachen, Germany Brains On-line, de Mudden 16, 9741 AW Groningen, The Netherlands
a r t i c l e
i n f o
Article history: Received 8 September 2011 Received in revised form 6 December 2011 Accepted 6 December 2011 Keywords: Descending inhibition Spinal cord Tapentadol Morphine Venlafaxine Microdialysis
a b s t r a c t Spinal noradrenaline is thought to play an important role in descending pain inhibitory pathways and the modulation of nociceptive information at the spinal level. Tapentadol is a -opioid receptor (MOR) agonist and noradrenaline reuptake inhibitor (NRI). We showed previously that tapentadol, in contrast to morphine, elevates levels of noradrenaline, but not serotonin, in the ventral hippocampus of rats. The aim of this study was to examine the effects of tapentadol, morphine and venlafaxine on spinal monoamine levels. Rats were implanted with spinal microdialysis probes. Drugs were administered intraperitoneally, and samples were collected for 3 h in isoflurane-anesthetized animals and analysed for monoamine content using HPLC–MS/MS. In terms of area-under-curve (AUC, 0–180 min), tapentadol (4.64–21.5 mg/kg) produced a dosedependent, significant increase in extracellular spinal noradrenaline levels (9275 ± 4346 min % at the highest dose versus −1047 ± 889 min % for vehicle). A maximum increase of 182 ± 32% of baseline was reached 60 min after administration of 10 mg/kg tapentadol. Venlafaxine (10 mg/kg) produced an effect of similar magnitude. In contrast, tapentadol decreased extracellular spinal serotonin levels (nonsignificantly compared to vehicle), while venlafaxine increased spinal serotonin to 267 ± 74% of baseline. In contrast to tapentadol and venlafaxine, morphine slightly decreased levels of noradrenaline and serotonin. This study demonstrates that analgesic doses of tapentadol (and venlafaxine), but not morphine, increase spinal noradrenaline levels and that tapentadol is devoid of a relevant serotonergic effect. It supports the suggestion that the NRI component of tapentadol is functionally relevant and contributes to its mechanism of action. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction The descending pain modulatory system, originating in the midbrain (periaqueductal grey, PAG) and brainstem (rostroventromedial medulla, RVM) and projecting to the spinal cord dorsal horn, has a powerful influence on the modulation of nociceptive information transmitted from the periphery to the brain. The prominent role of this system in suppression of nociception has been recognized early on [6,17]. More recently, the role of descending projections in pain facilitation and the generation of hyperalgesic states has received increased attention. The descending modulatory system uses noradrenaline (NA) and serotonin (5-HT) as its main transmitters. The role of NA appears to be predominantly
∗ Corresponding author. Tel.: +49 241 5692816; fax: +49 241 5692852. E-mail address: [email protected] (T.M. Tzschentke). 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.12.008
inhibitory, while the role of 5-HT appears to be bidirectional, mediating inhibitory as well as excitatory effects [20]. A number of drugs have been shown to increase the activity of the descending noradrenergic system and to increase extracellular levels of spinal NA, and this increase in noradrenergic activity has been linked to the antinociceptive/antiallodynic/antihyperalgesic effect of these treatments in acute or neuropathic pain, usually via activation of ␣2 -adrenoceptors. This was shown for morphine ([2] sheep, human; [26] sheep), fentanyl ([2] sheep), pregabalin ([22] mouse), gabapentin ([21,23] mouse; [9] human, [10] rat), nitrous oxide ([27] rat), and ketamine ([11] rat). Conversely, the pronociceptive effects of intra-PAG administration of nociceptin are associated with decreased monoamine levels in the spinal dorsal horn [14]. Tapentadol is a novel centrally acting analgesic belonging to a new pharmacological class, MOR-NRI (-opioid receptor agonist and noradrenaline reuptake inhibitor). We have shown previously that tapentadol, in contrast to morphine, elevates levels of NA, but
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not 5-HT, in the ventral hippocampus of rats [25]. The lack of significant serotonergic effects clearly differentiates tapentadol from the ‘atypical opioid’ tramadol, which produced clear increases of NA as well as 5-HT in the ventral hippocampus of rats, using identical experimental conditions [3]. The demonstration of tapentadolinduced elevation of extracellular NA levels can be taken as an in vivo proof of its NRI activity. The aim of the present study was to examine the effects of tapentadol on monoamine levels in the spinal cord dorsal horn, i.e. the site where the descending modulatory system impinges upon the ascending pain transmission pathway. Morphine, the prototypical ‘classical’ opioid, and venlafaxine, a mixed 5-HT/NA reuptake inhibitor, were used as comparators. Drug doses were chosen to fall within the analgesic dose range [18,25], and the dose of venlafaxine used was shown previously to produce significant elevations in extracellular levels of both 5-HT and NA in the ventral hippocampus of freely moving rats [3]. 2. Materials and methods 2.1. Animals Male adult Wistar rats (280–350 g; Harlan, Horst, the Netherlands) were used for the experiments and were kept under standard, constant conditions (lights on 07.00–19.00, temperature 22 ± 2 ◦ C, humidity 55 ± 10%). Experiments were conducted in accordance with the Declaration of Helsinki and were approved by the Institutional Animal Care and Use Committee of the University of Groningen. 2.2. Surgery Rats were anesthetized using isoflurane (2%; 800 ml/min O2 ). Bupivacaine and adrenaline were used for local anesthesia. Animals were placed in a stereotaxic frame (Kopf Instruments, USA). The dorsal skin was incised, and the spinal cord vertebrae were exposed. A small hole was drilled in the area between T12 and T13 with an angle of around 45◦ towards the spinal cord inside T13, and I-shaped probes (Hospal AN69 membrane; Brainlink, the Netherlands; 3 mm exposed surface) slowly inserted. 2.3. Drugs Tapentadol (Grünenthal GmbH, Aachen, Germany; batch GRTA19586-1-1), morphine (BUFA, Ijsselstein, the Netherlands; batch 99H17FO) and venlafaxine (Teva, Tikva, Israel; batch 330770503) were dissolved in saline. Solutions were injected intraperitoneally (i.p.) at 2 mL/kg (tapentadol, morphine) or 1 mL/kg (venlafaxine). Doses of all drugs refer to and were administered as their free base. 2.4. Experiment
Samples were mixed with 4 L internal standards mix (noradrenaline-d6, 5 × 10−8 M; serotonin-d4, 5 × 10−5 M), and were derivatized with SymDAQ in the autosampler (SIL-10ADvp, Shimadzu, Japan), by addition of 40 L of reagent to the sample vial. After 2 min, 50 L of the mixture was injected into the LC system by an automated sample injector (SIL-10ADvp, Shimadzu, Japan). Chromatographic separation was performed on a reversed phase Phenomenex Synergi MAX-RP 100 mm × 3.0 mm (2.5 m particle size) held at 35 ◦ C. Components were separated using a gradient of UP/ACN (98/2) 0.1% FA (mobile phase A) and UP/ACN 0.1% FA (30/70) (mobile phase B) (flow rate 0.3 mL/min) according to the following scheme: time (min)/% mobile phase B: 0/0; 4/40; 5.5/100; 7/2; 9/end. A post-column make-up flow of 0.15 mL/min was added to the flow of the LC, which was diverted to the waste for 2.85 min, after which it was switched to the MS for detection of the neurotransmitters. MS analyses were performed using an API4000 MS/MS system consisting of an API4000 MS/MS detector and a Turbo Ion Spray interface (both from Applied Biosystems, the Netherlands). Acquisitions were performed in positive ionization mode, with ionization spray voltage set at 4 kV and a probe temperature of 200 ◦ C. The instrument was operated in multiple-reaction-monitoring (MRM) mode. The collision gas (nitrogen) pressure was held at 2 psig. Data were calibrated and quantified using the AnalystTM data system (Applied Biosystem, version 1.4.2). 2.6. Statistical evaluation Three consecutive pre-treatment microdialysis samples with less than 50% variation were taken as baseline and their mean was set at 100%. Drug effects were expressed as percentages of basal level (mean ± SE) within the same subject. Statistical analysis was performed using Sigmastat for Windows (SPSS Corporation). Treatment and dose effects were compared, using two-way (time × dose) ANOVA for repeated measurements followed by Student Newmann Keuls post hoc test (except for venlafaxine where a one-way ANOVA for repeated measures over time was performed). Overall drug effects were expressed as area under curve (AUC) within the same subject, as calculated using the linear trapezoidal method. Treatment effects (AUC) were compared to vehicle using one-way ANOVA followed by Student–Newmann–Keuls post hoc test. The level of statistical significance was set at P < 0.05. 3. Results 3.1. Basal output Basal output values (mean ± SEM) obtained in dialysis samples from the spinal cord were 5.81 ± 0.60 fmol/sample for NA and 41.0 ± 11.1 fmol/sample for 5-HT (based on N = 38 rats each). 3.2. Tapentadol
Microdialysis probes were connected with flexible PEEK tubing to a microperfusion pump (Syringe pump UV8301501, TSE, Univentor, Malta) and perfused with artificial CSF, containing (in mM): NaCl 147, KCl 3, CaCl2 1.2 and MgCl2 1.2, at a flow rate of 1.5 L/min (dialysis flow). Samples were collected by an automated refrigerated fraction collector (CMA142, Sweden) for 15 min periods into mini-vials already containing 7.5 L 20 mM formic acid/0.04% ascorbic acid, and stored at −80 ◦ C until analysis. 2.5. Analysis of neurotransmitters Concentrations of NA and 5-HT were determined by HPLC with tandem mass spectrometry (MS/MS) detection.
3.2.1. Noradrenaline Fig. 1A shows the time course effects of 0, 4.64, 10 or 21.5 mg/kg tapentadol (i.p.) on the levels of NA in the spinal cord. There was a significant effect of time [F(12,227) = 8.225; P < 0.001], and dose × time interaction [F(36,227) = 3.145; P < 0.001]. Post hoc analysis revealed that extracellular NA levels were not significantly increased compared to basal levels after 0 or 4.64 mg/kg tapentadol. However, 10 mg/kg tapentadol significantly increased NA levels from t = 30 to t = 105 min compared to basal levels. After administration of 21.5 mg/kg tapentadol, NA levels were significantly increased from t = 30 to 180 min compared to basal levels.
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Fig. 2. Effects of tapentadol (0, 4.64, 10 or 21.5 mg/kg; i.p. at t = 0) on 5-HT levels in spinal cord, (A) in terms of time course data, (B) in terms of AUC0–180 data. N = 8, 5, 5 and 5 animals for 0, 4.64, 10 and 21.5 mg/kg, respectively. Fig. 1. Effects of tapentadol (0, 4.64, 10 or 21.5 mg/kg; i.p. at t = 0) on NA levels in spinal cord, (A) in terms of time course data, (B) in terms of AUC0–180 data. N = 8, 5, 5 and 5 animals for 0, 4.64, 10 and 21.5 mg/kg, respectively.
Fig. 1B shows the NA response in terms of AUC(0–180 min) . There was a significant difference between the AUCs of the different doses of tapentadol [F(3,19) = 3.134; P = 0.050]. However, post hoc analysis between the individual dose groups did not reveal a specific dose to be different from any of the other doses. 3.2.2. Serotonin Fig. 2A shows the time course effects of 0, 4.64, 10 or 21.5 mg/kg tapentadol (i.p.) on the levels of 5-HT in the spinal cord. There was a significant effect of time on 5-HT levels after the administration of tapentadol [F(12,225) = 8.699; P < 0.001]. Post hoc analysis revealed a decrease in 5-HT levels at t = 90 and 180 min after administration. However, this decrease could not be attributed to a specific dose. Fig. 2B shows the 5-HT response in terms of AUC(0–180 min) . There was no significant difference between the AUCs of the different doses of tapentadol [F(3,19) = 1.899; P = 0.166]. 3.3. Morphine 3.3.1. Noradrenaline Fig. 3A shows the time course effects of 0, 1, 3, 10 mg/kg morphine (i.p.) on levels of NA in the spinal cord. There was a significant effect of dose [F(3,190) = 8.380; P = 0.001], time [F(12,190) = 5.323; P < 0.001] and dose × time interaction [F(36,190) = 1.760; P < 0.01]. Post hoc analysis revealed a decrease in NA levels compared to baseline after 3 mg/kg morphine at t = 60 min and 105 min. None of the other doses (including vehicle) showed a significant change compared to baseline.
Fig. 3B shows the response of NA levels in terms of AUC(0–180 min) . There was a significant difference between the AUCs of the different doses of morphine [F(3,18) = 12.265; P < 0.001]. Post hoc analysis showed that AUC values for all doses were significantly different from the vehicle AUC.
3.3.2. Serotonin Fig. 4A shows the effects of 0, 1, 3, 10 mg/kg morphine (i.p.) on the extracellular levels of 5-HT. There was a significant effect of time on 5-HT levels after the administration of morphine [F(12,213) = 5.858; P < 0.001]. Post hoc analysis revealed a decrease in 5-HT levels from t = 45 min up to and including 180 min. However, this decrease could not be attributed to a specific dose. Fig. 4B shows the 5-HT response in terms of AUC(0–180 min) . There was no significant difference between the AUCs of the different doses of morphine [F(3,19) = 0.310; P = 0.818]. 3.4. Venlafaxine After administration of venlafaxine (10 mg/kg; i.p.) (data not shown), there was a significant effect over time on NA levels [F(11,43) = 6.821; P < 0.001; N = 5]. Post hoc analysis revealed a significant increase of NA levels compared to baseline from t = 30 min up to and including t = 165 min. There was also a significant effect over time on 5-HT levels [F(11,51) = 6.850; P < 0.001; N = 6]. Post hoc analysis revealed a significant increase of 5-HT levels compared to baseline from t = 30 min up to and including t = 105 min and at t = 135 min. As these experiments were done as a pilot study to demonstrate method sensitivity for measuring changes in spinal monoamine levels, no separate vehicle group was run.
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Fig. 3. Effects of morphine (0, 1, 3, 10 mg/kg; i.p. at t = 0) on NA levels in spinal cord, (A) in terms of time course data, (B) in terms of AUC0–180 data. N = 8, 4, 5 and 4 animals for 0, 4.64, 10 and 21.5 mg/kg, respectively.
Fig. 4. Effects of morphine (0, 1, 3, 10 mg/kg; i.p. at t = 0) on 5-HT levels in spinal cord, (A) in terms of time course data, (B) in terms of AUC0–180 data. N = 8, 4, 5 and 4 animals for 0, 4.64, 10 and 21.5 mg/kg, respectively.
4. Discussion The current set of experiments was performed to evaluate the effects of tapentadol on changes in spinal cord levels of NA and
5-HT in rats. Administration of tapentadol resulted in increases in NA levels after 10 and 21.5 mg/kg of tapentadol compared to baseline. 5-HT levels were decreased by tapentadol and morphine in a non-dose-dependent, statistically non-significant manner. A comparable downward ‘drift’ of 5-HT was also observed after vehicle treatment, and such an effect has been previously observed and described [16]; thus, this effect may be non-specific, and the reason for and relevance of this observation remains unclear. Nevertheless, this study clearly extends the functional in vivo proof of tapentadol’s NRI activity from the ventral hippocampus to the spinal cord dorsal horn, a structure of central importance for the modulation of pain signals by the descending modulatory system. It also confirms the lack of a relevant SRI activity of tapentadol. There is good evidence that NA reuptake inhibition plays a greater role than 5-HT reuptake inhibition in antinociception [1,8,15]. Thus, tapentadol’s monoaminergic activity is selective for the more relevant antinociceptive system (NA), and has no impact on the less relevant antinociceptive system (5-HT). That our current experimental protocol is able to demonstrate SRI activity was shown with venlafaxine. In contrast to tapentadol and venlafaxine, morphine slightly decreased levels of noradrenaline. The finding that tapentadol elevates spinal levels of NA also corroborates our interpretation of the intrinsic synergy between its two mechanisms of action demonstrated previously [19]. Through its opioid activity tapentadol would activate the descending noradrenergic projections at the supraspinal level, and its NRI activity would then amplify the effect of the opioid-mediated increase in NA release at the spinal level, by blocking the reuptake of NA released due to supraspinal activation of the descending noradrenergic projections. An increase in spinal noradrenergic transmission may be particularly relevant in the context of chronic pain, since there is evidence that under conditions of chronic pain, the balance of excitatory and inhibitory supraspinal input to the spinal cord is disrupted, such that excitatory, pain-facilitatory mechanisms prevail – either due to an enhancement of facilitatory serotonergic processes, or due to a diminution of inhibitory noradrenergic processes – and contribute to the development and/or maintenance of central sensitization (see [1,5,20,24]). Under such circumstances, restoring the balance by selectively enhancing NA-mediated inhibition (and not increasing serotonergic activity) may be a particularly valuable approach. In fact, tapentadol shows a relatively higher potency compared to pure opioids in animal models of chronic neuropathic pain. While in models of acute nociceptive pain tapentadol is 2–3 times less potent than morphine, in models of chronic neuropathic pain tapentadol is at least as potent as morphine, if not more potent [4,12,18,25]. That tapentadol is so potent despite the fact that its MOR affinity is 50 times less than that of morphine, further underlines the importance of its NRI activity. The magnitude of drug effects on transmitter levels in the spinal cord of anesthetized rats observed in the present study is smaller than the magnitude observed in our previous studies in the ventral hippocampus of freely moving rats at comparable drug doses [3,25]. The two obvious differences between the two sets of studies are the different sampled CNS regions and the absence or presence of anesthesia. Upon inspection of the limited literature on spinal monoamine microdialysis in rats, it appears that the drug effect size in the spinal cord does not exceed 250–300% of baseline, in both anesthetized (present study (tapentadol, venlafaxine); [10] gabapentin) and freely moving animals ([13] desipramine); [27] nitrous oxide; [11] ketamine), whereas supraspinally, e.g. in the ventral hippocampus, increases of 400–800% of baseline are frequently observed. Thus, it appears likely that the relatively small effect sizes seen in the present study are mainly related to the spinal location of the microdialysis probe. Although it cannot be excluded that anesthesia had an influence on basal and/or
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drug-induced monoamine release in the spinal cord, anesthesia abolished the potentially confounding factor of locomotor activity. As Gerin and Privat [7] have shown, locomotor activity can influence monoamine levels in the (ventral horn of the) spinal cord. The present study did not find a morphine-induced increase in spinal levels of NA. In fact, a small decrease in NA (and 5-HT) was observed. While this finding is consistent with the lack of NRI (and SRI) activity of morphine, it appears to be at odds with the concept of supraspinal activation of descending inhibitory projections and with the previously reported increase in spinal NA levels by morphine and fentanyl [2,26]. However, these studies were conducted in sheep. Our study is the first to use microdialysis to examine the effects of morphine on spinal NA in the rat, and presently, species differences in the response to morphine cannot be ruled out. In conclusion, this present study showed that tapentadol increases spinal NA levels while being devoid of a relevant serotonergic effect. It supports the suggestion that the NRI component of tapentadol is functionally relevant and contributes to its potent analgesia and broad efficacy range. Acknowledgement This study was funded by Grünenthal GmbH. References [1] K. Bannister, L.A. Bee, A.H. Dickenson, Preclinical and early clinical investigations related to monoaminergic pain modulation, Neurotherapeutics 6 (2009) 703–712. [2] H. Bouaziz, C. Tong, Y. Yoon, D.D. Hood, J.C. Eisenach, Intravenous opioids stimulate norepinephrine and acetylcholine release in spinal cord dorsal horn. Systematic studies in sheep and an observation in a human, Anesthesiology 84 (1996) 143–154. [3] P. Bloms-Funke, E. Dremencov, T.I. Cremers, T.M. Tzschentke, Tramadol increases extracellular levels of serotonin and noradrenaline as measured by in vivo microdialysis in the ventral hippocampus of freely-moving rats, Neurosci. Lett. 490 (2011) 191–195. [4] T. Christoph, J. De Vry, T.M. Tzschentke, Tapentadol, but not morphine, selectively inhibits disease-related thermal hyperalgesia in a mouse model of diabetic neuropathic pain, Neurosci. Lett. 470 (2010) 91–94. [5] R. D’Mello, A.H. Dickenson, Spinal cord mechanisms of pain, Br. J. Anaesth. 101 (2008) 8–16. [6] H.L. Fields, A.I. Basbaum, Brainstem control of spinal pain-transmission neurons, Annu. Rev. Physiol. 40 (1978) 217–248. [7] C. Gerin, A. Privat, Direct evidence for the link between monoaminergic descending pathways and motor activity. II. A study with microdialysis probes implanted in the ventral horn of the spinal cord, Brain Res. 794 (1998) 169–173. [8] F.S. Hall, J.M. Schwarzbaum, M.T.G. Perona, J.S. Templin, M.G. Caron, K.P. Lesch, D.L. Murphy, G.R. Uhl, A greater role for the norepinephrine transporter than the serotonin transporter in murine nociception, Neuroscience 175 (2011) 315–327.
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