Differential contribution of opioid and noradrenergic mechanisms of tapentadol in rat models of nociceptive and neuropathic pain

European Journal of Pain 14 (2010) 814–821

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Differential contribution of opioid and noradrenergic mechanisms of tapentadol in rat models of nociceptive and neuropathic pain Wolfgang Schröder *, Jean De Vry, Thomas M. Tzschentke, Ulrich Jahnel, Thomas Christoph Grünenthal GmbH, Global Preclinical Research and Development (UJ), Department of Pharmacology (TMT, TC, JDV, WS), Zieglerstrasse 6, 52078 Aachen, Germany

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Article history: Received 2 December 2009 Received in revised form 28 April 2010 Accepted 10 May 2010 Available online 11 June 2010 Keywords: Tapentadol Nociceptive pain Neuropathic pain l-Opioid receptor agonism Noradrenaline reuptake inhibition

a b s t r a c t The novel analgesic tapentadol combines l-opioid receptor agonism and noradrenaline reuptake inhibition in a single molecule and shows potent analgesia in various rodent models of pain. We analyzed the contribution of opioid and monoaminergic mechanisms to the activity of tapentadol in rat models of nociceptive and neuropathic pain. Antinociceptive efficacy was inferred from tail withdrawal latencies of experimentally naive rats using a tail flick test. Antihypersensitive efficacy was inferred from ipsilateral paw withdrawal thresholds toward an electronic von Frey filament in a spinal nerve ligation model of mononeuropathic pain. Dose–response curves of tapentadol (intravenous) were determined in combination with vehicle or a fixed dose (intraperitoneal) of the l-opioid receptor antagonist naloxone (1 mg/kg), the a2-adrenoceptor antagonist yohimbine (2.15 mg/kg), or the serotonin 5-HT2A receptor antagonist ritanserin (0.316 mg/kg). Tapentadol showed clear antinociceptive and antihypersensitive effects (>90% efficacy) with median effective dose (ED50) values of 3.3 and 1.9 mg/kg, respectively. While the antinociceptive ED50 value of tapentadol was shifted to the right 6.4-fold by naloxone (21.2 mg/kg) and only 1.7fold by yohimbine (5.6 mg/kg), the antihypersensitive ED50 value was shifted to the right 4.7-fold by yohimbine (8.9 mg/kg) and only 2.7-fold by naloxone (5.2 mg/kg). Ritanserin did not affect antinociceptive or antihypersensitive ED50 values of tapentadol. Activation of both l-opioid receptors and a2adrenoceptors contribute to the analgesic effects of tapentadol. The relative contribution is, however, dependent on the particular pain indication, as l-opioid receptor agonism predominantly mediates tapentadol’s antinociceptive effects, whereas noradrenaline reuptake inhibition predominantly mediates its antihypersensitive effects. Ó 2010 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

1. Introduction Activation of the l-opioid receptor (MOR) is a very effective way to inhibit acute nociceptive pain. However, the effectiveness and/or therapeutic window of MOR agonists may be limited in chronic neuropathic pain conditions (Mao et al., 2000; Martin and Eisenach, 2001). For instance, the potency and efficacy of intrathecally administered morphine was reduced in spinal nerve– ligated compared with naive rats (Ossipov et al., 1995). Inhibiting the reuptake of noradrenaline (NA) into axon terminals of the descending inhibitory noradrenergic pathway increases the extracellular concentration of NA in the spinal cord, producing antinociception via activation of a2-adrenoceptors. The mechanism of NA reuptake inhibition (NRI) seems to be particularly effective under conditions of chronic neuropathic pain (Carter and Sullivan, 2002). For example, the potency and efficacy of a2-adrenoceptor agonists was increased after sciatic nerve section in rats (Xu * Corresponding author. Tel.: +49 (0)241 569 2897; fax: +49 (0)241 569 2852. E-mail address: [email protected] (W. Schröder).

et al., 1992). Combining both mechanisms of action (MOR agonism and NRI) in one molecule might therefore be a strategy to retain effectiveness in chronic neuropathic pain conditions. Combining MOR agonists with NA reuptake inhibitors or a2-adrenoceptor agonists additively, or even synergistically, produced analgesia after intrathecal or systemic administration in models of nociceptive and neuropathic pain (Ossipov et al., 1982, 1990; Fairbanks and Wilcox, 1999; Reimann et al., 1999). The spinal adrenergic– opioid antinociceptive synergy is mediated by concomitant activation of MOR and a2A- or a2C-adrenoceptors (Stone et al., 1997; Fairbanks et al., 2002). Likewise, the antinociceptive potency of systemic morphine was increased in NA transporter knockout mice, and this potentiation could be blocked by yohimbine (Bohn et al., 2000). The novel centrally acting analgesic tapentadol combines MOR agonism and NRI in a single molecule and exerts potent analgesia in rodent models of nociceptive and neuropathic pain (Tzschentke et al., 2006, 2007). We reported previously that the antihypersensitive effect of a high dose of intravenously administered tapentadol in spinal nerve–ligated rats was clearly inhibited by

1090-3801/$36.00 Ó 2010 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpain.2010.05.005

W. Schröder et al. / European Journal of Pain 14 (2010) 814–821

yohimbine, whereas naloxone produced only a small inhibition (Tzschentke et al., 2007). For the reasons outlined above, the relative contribution of MOR agonism and NRI to analgesia can be expected to be different in neuropathic pain compared with nociceptive pain. The aim of the present study was to determine the relative contribution of MOR agonism and NRI to the analgesic effects of tapentadol in models of nociceptive and neuropathic pain. Serotonin (5-HT) is also released from modulatory pathways descending from the brainstem to the spinal cord. Inhibition of 5-HT reuptake leads to increased activation of 5-HT receptors on spinal neurons which contributes to the modulation of spinal nociceptive processing (Bannister et al., 2009). Therefore, we also investigated whether the weak in vitro 5-HT reuptake inhibition activity of tapentadol contributes to analgesia. Dose–response curves of tapentadol in the presence of vehicle or the MOR antagonist naloxone, the a2-adrenoceptor antagonist yohimbine, and the 5-HT2A receptor antagonist ritanserin were determined and compared in the tail flick test of acute nociceptive pain and in the spinal nerve ligation (SNL) model of chronic mononeuropathic pain.

2. Methods 2.1. Animals Male Sprague–Dawley rats (Janvier, Le Genest St. Isle, France) were housed under a 12:12-h light–dark cycle (lights on at 06:00 h); room temperature 20 °C to 24 °C; relative air humidity 35–70%; 15 air changes per hour, air movement less than 0.2 m/ s. The animals had free access to standard laboratory food and tap water. There were at least 5 days between the delivery of the animals and behavioral testing or surgery. Average animals’ weights were 160–240 g for the tail flick test and 200–400 g in the SNL model (at the time of testing, 140–160 g at the time of surgery). All experiments were conducted according to the guidelines of the International Association for the Study of Pain (Zimmermann, 1983) and the German Animal Welfare Law. 2.2. Behavioral testing

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and 10, 20, 30 and 60 min after intravenous (IV) administration of the test compounds or vehicle. 2.2.3. Spinal nerve ligation The spinal nerve ligation model of neuropathic pain was adapted from Kim and Chung (1992). Under pentobarbital anesthesia (Narcoren, 60 mg/kg intraperitoneally), the left L5 and L6 spinal nerves were exposed by removing a small piece of the paravertebral muscle and a part of the left spinous process of the L5 lumbar vertebra. The L5 and L6 spinal nerves were then carefully isolated and tightly ligated with silk (NC-silk black, USP 5/0, metric 1, Braun Melsungen, Germany). After checking hemostasis, the muscle and the adjacent fascia were closed with sutures, and the skin was closed with metal clips. After surgery, animals were allowed to recover for 1 week. For the assessment of mechanical hypersensitivity, which was stable for at least 5 weeks, the rats were placed on a metal mesh covered with a plastic dome and were allowed to habituate until exploratory behavior ceased. The threshold for mechanical hypersensitivity was determined with an electronic von Frey anesthesiometer (Somedic AB, Malmö, Sweden) using the median of five consecutive measurements (inter-measurement interval 1–2 min). Animals were tested before and 0.5, 1, and 3 h after administration of the test compounds. Withdrawal thresholds of the injured paws were assessed and expressed as the percentage of MPE comparing the pre-drug threshold of spinal nerve–ligated animals (i.e., 0% MPE) and the control threshold of sham animals (i.e., 100% MPE). Drugs or vehicle were tested 2–5 weeks after surgery (1 test per week) in a counterbalanced within-group design. 2.3. Drugs Tapentadol HCl (Grünenthal GmbH, Germany), morphine HCl (Merck AG, Germany), reboxetine mesylate (Tocris), citalopram HBr (Bosche Scientific) and venlafaxine (Grünenthal GmbH, Germany) were administered intravenously into the tail vein 5 cm distal from the tail root. The antagonists naloxone (1 mg/kg), yohimbine (2.15 mg/kg), and ritanserin (0.316 mg/kg) or the respective vehicles were given intraperitoneally 5 min (SNL) or 10 min (low-intensity tail flick) prior to IV treatment. Since mechanical hypersensitivity testing in the SNL model lasted about 5 min whereas testing in the tail flick assay was performed instan-

2.2.1. Experimental procedures Animals were assigned randomly to treatment groups. Different doses and vehicle were tested in a randomized fashion. Although the operators performing the behavioral tests were not formally ‘‘blinded” with respect to the treatment, they were not aware of the study hypothesis or the nature of the differences between drugs. 2.2.2. Low-intensity tail flick test The tail flick test was carried out in rats using a modification of the method described by D’Amour and Smith (1941). The tail flick latency, defined by the time (in seconds) to withdraw the tail from a radiant heat source, was measured using a semi-automated device (Rhema Labortechnik, Germany). The rat was placed in a plexiglas restrainer, and a low-intensity radiant heat beam (48 °C) was focused onto the dorsal surface of the tail root. The stimulus intensity was adjusted to result in a mean pre-drug control latency of 7 s, thus also allowing a supraspinal modulation of the spinally mediated acute nociceptive reflex. A cutoff time of 30 s was applied to avoid tissue damage. The increase in tail flick latency was defined as anti-nociception and calculated as the percentage of maximum possible effect (MPE) according to the following formula: MPE [%] = (tl tc)/(tcutoff tc)  100% (tl, withdrawal latency; tc, control latency; and tcutoff, cutoff time). Animals were tested before

Fig. 1. Dose- and time-dependent antinociceptive effect of tapentadol in the lowintensity tail flick test in rats. Data are presented as% MPE (mean ± SEM). P < 0.05 vs. corresponding vehicle. % MPE, percentage of maximum possible effect; SEM, standard error of the mean.

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taneously, the administration time point of the antagonists relative to the agonists and reuptake inhibitors was adapted accordingly in the SNL model. The doses of the reference agonists (morphine, reboxetine, citalopram/venlafaxine) were chosen as maximally effective. Antagonist drug doses were in turn carefully chosen to reach full antagonism of these maximally active doses of the reference agonists without confounding analgesic effect (Figs. 4 and 8, Table 2). Saline (NaCl 0.9%) was used as a vehicle except for ritanserin, in which 5% DMSO, 10% Cremophor EL in 5% glucose solution was used. The volume of administration was 5 mL/kg. Drugs were purchased from Sigma (Deisenhofen, Germany) unless otherwise stated. All doses refer to the respective salt form as indicated above. 2.4. Data analysis Data were analyzed by means of a 1- or 2-factor analysis of variance with or without repeated measures, depending on the experimental design, with a post hoc Bonferroni test. Significance of treatment, time, or treatment  time interaction effects was ana-

Fig. 4. Naloxone, yohimbine and ritanserin antagonized the antinociceptive effect of morphine, reboxetine and citalopram, respectively, in the low-intensity tail flick test in rats. Data are presented as% MPE (mean + SEM) 30 min after administration of the agonists. P < 0.05 vs. corresponding agonist + vehicle group. % MPE, percentage of maximum possible effect; SEM, standard error of the mean; IP, intraperitoneal; IV, intravenous.

lyzed by means of Wilks K (lambda) statistics. In case of a significant treatment effect, pair-wise comparison was performed at the time of maximal effect by the Fisher least significant difference test. Results were considered statistically significant if P < 0.05. Median effective dose (ED50) values and 95% confidence intervals (95% CIs) were calculated by linear regression using the percentage of MPE values obtained at 30 min after IV compound administration. Median effective dose values with non-overlapping 95% CIs were considered to be significantly different. Each group included 10 rats. 3. Results 3.1. Low-intensity tail flick

Fig. 2. Naloxone produced a clear, while yohimbine produced a moderate, rightward shift of the antinociceptive dose–response curve of tapentadol in the low-intensity tail flick test in rats. Data are presented as% MPE (mean ± SEM) 30 min after administration of tapentadol. P < 0.05 vs. corresponding vehicle. % MPE, percentage of maximum possible effect; SEM, standard error of the mean; IP, intraperitoneal; IV, intravenous.

Fig. 3. Ritanserin did not affect the antinociceptive dose–response curve of tapentadol in the low-intensity tail flick test in rats. Data are presented as% MPE (mean ± SEM) 30 min after administration of tapentadol. P < 0.05 vs. corresponding vehicle. % MPE, percentage of maximum possible effect; SEM, standard error of the mean; IP, intraperitoneal; IV, intravenous.

Tapentadol induced potent (ED50, 3.3 [2.9–3.6] mg/kg IV) doseand time-dependent anti-nociception in the tail flick model of acute thermal nociception (treatment: F(4,45) = 139.0, P < 0.001; time: F(1,135) = 180.5, P < 0.001; interaction: F(12,135) = 33.4, P < 0.001). Full efficacy, 30 min after administration, was reached at 10 mg/kg IV (Fig. 1). The MOR antagonist naloxone significantly shifted the dose–response curve of tapentadol to the right by about a factor of 6.4 (ED50, 3.3 vs. 21.2 [17.4–27.6] mg/kg, [treatment: F(4,45) = 38.1, P < 0.001; time: F(3,135) = 2.95, P < 0.035; interaction: F(12,135) = 0.54, P = 0.888]; statistical evaluation relates to the within-group effect of tapentadol, and differences between groups were assessed based on CI overlap, see Section 2). The a2-adrenoceptor antagonist yohimbine slightly, but significantly, shifted the dose–response curve of tapentadol to the right by about a factor of 1.7 (ED50, 3.3 vs. 5.6 [4.8–6.5] mg/kg, [treatment: F(3,36) = 65.1, P < 0.001; time: F(3,108) = 94.7, P < 0.001; interaction: F(9,108) = 21.0, P < 0.001], Fig. 2). In contrast, the dose–response curve of tapentadol remained unchanged when tapentadol was combined with vehicle or the 5-HT2A receptor antagonist ritanserin (ED50, 3.8 [3.4–4.2] mg/kg [treatment: F(3,35) = 323.6, P < 0.001; time: F(3,105) = 105.5, P < 0.001; interaction: F(9,105) = 37.1, P < 0.001] vs. 4.2 [3.4–5.3] mg/kg, [treatment: F(3,36) = 136.1, P < 0.001; time: F(3,108) = 104.3, P < 0.001; interaction: F(9,108) = 39.8, P < 0.001], respectively; Fig. 3). ED50 values were calculated 30 min after tapentadol administration. The antinociceptive effects of the MOR agonist morphine, the NA reuptake inhibitor reboxetine, and the selective 5-HT reuptake inhibitor citalopram were completely antagonized by naloxone, yohimbine, and ritanserin, respectively, thus confirming the appro-

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priateness of the antagonist doses used (Fig. 4). The target affinities of all drugs used are presented in Table 1. It should be noted that only about half-maximal efficacy was reached in the tail flick test with reboxetine and citalopram because of the occurrence of dose-limiting side effects, such as accelerated respiration and hypersensitivity to touch. Administration of vehicle or antagonists alone did not produce antinociceptive effects (Table 2). 3.2. Spinal nerve ligation Tapentadol showed potent (ED50, 1.9 [1.7–2.1] mg/kg IV) doseand time-dependent inhibition of mechanical hypersensitivity in the SNL model of mononeuropathic pain (treatment: F(3,36) = 33.7, P < 0.0001; time: F(2,72) = 223.0, P < 0.0001; interaction: F(6,72) = 35.0, P < 0.0001). Full efficacy, 30 min after administration, was reached at 4.64 mg/kg IV (Fig. 5). The time course of the antihypersensitive effect of tapentadol in the SNL model was longer than that of the antinociceptive effect in the tail flick test. Therefore, testing in the SNL model was extended to 3 h. Prior administration of naloxone led to a significant, but moderate,

Table 1 MOR, a2-adrenoceptor and 5-HT2A receptor binding affinities and inhibition of synaptosomal monoamine uptake by tapentadol and reference compounds. Rat MOR Binding Assay (Ki Values ± SEM [lM])

Rat Synaptosomal Monoamine Uptake Assays (Ki Values ± SEM [lM])

[3H] Naloxone

[3H] NA

[3H] 5-HT

0.48 ± 0.11 >100 0.0085b 4 ± 1c 0.45 ± 0.03a 0.64 (0.50–0.84)e

2.37 ± 0.54 >100 6.9b 0.0013 ± 0.0002c 0.062 ± 0.013a 0.21 (0.15–0.28)e

Agonists and reuptake inhibitors Tapentadola 0.096 ± 0.009 Morphinea 0.0022 ± 0.0001 Reboxetine N.D. Citalopram N.D. Venlafaxine N.D.a >1d

Fig. 5. Dose- and time-dependent antihypersensitive effect of tapentadol in spinal nerve–ligated rats. Data are presented as% MPE (mean ± SEM). P < 0.05 vs. corresponding vehicle. % MPE, percentage of maximum possible effect; SEM, standard error of the mean.

rightward shift (factor of 2.5; ED50, 1.9 vs. 5.2 [4.4–6.1] mg/kg, [treatment: F(4,45) = 19.5, P < 0.0001; time: F(2,90) = 104.2, P < 0.0001; interaction: F(8,90) = 26.6, P < 0.0001]) of the dose–response curve of tapentadol. Yohimbine significantly shifted the dose–response curve of tapentadol farther to the right than naloxone, by about a factor of 4.5 (ED50, 1.9 vs. 8.9 [8.2–9.6] mg/kg, [treatment: F(4,45) = 32.7, P < 0.0001; time: F(2,90) = 265.1, P < 0.0001; interaction: F(8,90) = 57.9, P < 0.0001]; Fig. 6). As seen in the low-intensity tail flick test, the dose–response curve of tapentadol remained unchanged when tapentadol was combined

Rat Receptor Binding Assay (Ki Values ± SEM [lM]) MOR a2-adrenoceptors 5-HT2A receptor Antagonists Naloxone Yohimbine

0.0025 ± 0.0001f N.D.

Ritanserin

1.74j

n.e.g a2A: 0.05; a2C: 0.0028h 0.086 ± 0.002k

N.D. 1i 0.00025l

N.D., not determined; n.e., no effect. a Data from Tzschentke et al. (2007). b IC50 values, Miller et al. (2002). c Richelson and Pfenning (1984). d Bymaster et al. (2001). e IC50 (95% CI) values, Muth et al. (1986). f IC50 value, Villiger et al. (1983). g Mouse receptor, Hocker et al. (2009). h Uhlen et al. (1998). i Johnson et al. (1996). j Leysen et al. (1993). k IC50 value, Doble et al. (1992). l Albinsson et al. (1994).

Fig. 6. Naloxone produced a moderate, while yohimbine produced a clear, rightward shift of the antihypersensitive dose–response curve of tapentadol in spinal nerve–ligated rats. Data are presented as% MPE (mean ± SEM) 30 min after administration of tapentadol. P < 0.05 vs. corresponding vehicle. % MPE, percentage of maximum possible effect; SEM, standard error of the mean; IP, intraperitoneal; IV, intravenous.

Table 2 Administration of vehicle and antagonists alone did not elicit antinociceptive or antihypersensitive effects in the tail flick test and the spinal nerve ligation model, respectively. Data are presented as% MPE (mean ± SEM) 30 min after administration of saline. Saline IP + saline IV Tail flick SNL

1.4 ± 3.1 0.3 ± 4.6

Naloxone IP 1 mg/kg + saline IV 0.8 ± 2.3 2.0 ± 3.0

Yohimbine IP 2.15 mg/kg + saline IV 2.0 ± 2.1 1.1 ± 4.3

DMSO + Cremophor IP + saline IV

Ritanserin IP 0.316 mg/kg + saline IV

2.7 ± 2.4 7.4 ± 3.1

MPE, percentage of maximum possible effect; SEM, standard error of the mean; IP, intraperitoneal; IV, intravenous; SNL, spinal nerve ligation.

1.6 ± 4.3 5.2 ± 5.6

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Fig. 7. Ritanserin did not affect the antihypersensitive dose–response curve of tapentadol in spinal nerve–ligated rats. Data are presented as% MPE (mean ± SEM) 30 min after administration of tapentadol. P < 0.05 vs. corresponding vehicle. % MPE, percentage of maximum possible effect; SEM, standard error of the mean; IP, intraperitoneal; IV, intravenous.

Fig. 8. Naloxone, yohimbine, and ritanserin antagonized the antihypersensitive effect of morphine, reboxetine, and venlafaxine, respectively, in spinal nerve– ligated rats. Data are presented as% MPE (mean + SEM) 30 min after administration of the agonists. P < 0.05 vs. corresponding agonist + vehicle group. % MPE, percentage of maximum possible effect; SEM, standard error of the mean; IP, intraperitoneal; IV, intravenous.

with vehicle or ritanserin (ED50, 1.8 [1.7–2.0] mg/kg, [treatment: F(3,36) = 66.5, P < 0.0001; time: F(2,72) = 311.8, P < 0.0001; interaction: F(6,72) = 48.3, P < 0.0001] vs. 1.7 [1.5–1.8] mg/kg, [treatment: F(3,36) = 62.3, P < 0.0001; time: F(2,72) = 234.0, P < 0.0001; interaction: F(6,72) = 36.8, P < 0.0001], respectively; Fig. 7). ED50 values were calculated 30 min after tapentadol administration. The antihypersensitive effects of morphine, reboxetine, and the mixed NA/5-HT reuptake inhibitor venlafaxine were completely antagonized by naloxone, yohimbine, and ritanserin, respectively (Fig. 8). Notably, the antihypersensitive efficacy of reboxetine in the SNL model was much greater than its antinociceptive effect in the tail flick test (Figs. 4 and 8). Citalopram had no effect in the SNL model (data not shown). Administration of vehicle or antagonists alone did not produce antihypersensitive effects (Table 2). 4. Discussion and conclusions Tapentadol inhibited acute thermal nociception as well as nerve injury–induced tactile hypersensitivity in a dose- and time-depen-

dent manner after intravenous administration, as demonstrated in the low-intensity tail flick test of nociceptive pain and in the SNL model of neuropathic pain, respectively. Tapentadol was slightly, though significantly, more potent in the SNL model and reached full efficacy in both models without producing side effects. In a previous study, tapentadol was found to be effective in a broad range of acute nociceptive and chronic neuropathic pain models after intraperitoneal and intravenous administration, and both MOR agonism and NRI were shown to contribute to the antihypersensitive efficacy of a high IV dose of tapentadol in the SNL model (Tzschentke et al., 2007). However, the relative contribution of MOR agonism and NRI to tapentadol-mediated anti-nociception was not investigated so far. In the present study, dose–response curves of tapentadol after intravenous administration generated in the presence of the MOR antagonist naloxone or the a2-adrenoceptor antagonist yohimbine revealed a different relative contribution of MOR and a2-adrenoceptor activation to the analgesic effect of tapentadol in the tail flick test and the SNL model. This differential contribution of the two mechanisms of action might also contribute to the different time course of the analgesic effect of tapentadol in both models. Significant time dependency was demonstrated together with dose dependency by means of the statistical analysis. Dose response relationships generated at 30 min after administration were used (corresponding to the time point of maximal efficacy in the SNL model) in order to optimally visualize the primary issue of interest of the present study, i.e. the relative contribution of MOR agonism and NRI to the effect of tapentadol in both models. The acute antinociceptive effect of tapentadol in the tail flick test was predominantly mediated by MOR activation, whereas a2-adrenoceptor activation secondary to NRI only had a minor contribution, as inferred from the large and the small rightward shift of the dose–response curve produced by naloxone and yohimbine, respectively. On the other hand, the antihypersensitive effect of tapentadol after spinal nerve ligation depended primarily on activation of a2-adrenoceptors, whereas MOR activation appeared to be less important, as yohimbine induced a greater rightward shift of the dose–response curve than naloxone. Nevertheless, although the relative contribution of MOR activation and a2-adrenoceptor activation was different under conditions of nociceptive and neuropathic pain, tapentadol inhibited both pain states with similar efficacy. These findings are in agreement with the concept that the functional down-regulation of MOR observed under conditions of neuropathic pain is compensated for by a functional upregulation of a2-adrenoceptors (Xu et al., 1992; Ossipov et al., 1995), suggesting that tapentadol’s equivalent effectiveness in both nociceptive and neuropathic pain states relies on its unique combination of MOR agonism with NRI. Mechanical hypersensitivity in the SNL model was chosen since this stimulus is considered to be of greater clinical relevance for neuropathic pain conditions as compared to heat hypersensitivity. In contrast, acute heat nociception is a well accepted surrogate for acute nociceptive pain. Indeed, this results in the dilemma that we describe two different modalities, which might complicate the discussion of the results. However, published data on tapentadol (ED50 0.32 mg/kg i.v.) and morphine (ED50 0.65 mg/kg i.v.) regarding heat hypersensitivity in diabetic mice (Christoph et al., 2010) and acute heat nociception in naïve mice (ED50 tapentadol 4.2 mg/kg i.v., ED50 morphine 1.4 mg/kg i.v., Tzschentke et al., 2006) reveal a higher potency of tapentadol as compared to morphine in this neuropathic pain state and hence, suggest a differential activity of tapentadol in nociceptive and neuropathic pain similar to the data presented here, irrespective of the chosen stimulus. Both MOR agonists (Stein and Lang, 2009) and the NA reuptake inhibitor desipramine (Dina et al., 2008) as well as the a2-adreno-

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ceptor agonist clonidine (Lavand’homme et al., 2002) have been shown to produce analgesia also at peripheral sites. Since tapentadol has been shown to easily pass the blood brain barrier and to be effective after intrathecal and intracerebroventricular administration (unpublished results, and see also Tzschentke et al. (2006, 2007)) its analgesic effects are likely to be mediated by both peripheral and central activity at spinal and supraspinal sites. Spinal MORs are expressed presynaptically on the central terminals of glutamatergic primary afferent C-fibers (Besse et al., 1990; Abbadie et al., 2002) and postsynaptically on both glutamatergic interneurons (Arvidsson et al., 1995; Trafton et al., 2000) and on wide dynamic range neurons (Herrero et al., 2000; Guan et al., 2006). Spinal nerve ligation has been shown to reduce the expression of MOR protein in the spinal dorsal horn and in dorsal root ganglion neurons ipsilateral to the ligation (Porreca et al., 1998; Kohno et al., 2005). This is accompanied by a reduction in DAMGO-induced inhibition of the amplitude and frequency of excitatory postsynaptic currents recorded from lamina II spinal cord neurons (Kohno et al., 2005). Furthermore, the antinociceptive potency and efficacy of intrathecally administered morphine is reduced after SNL, reflecting reduced MOR function in a neuropathic pain model also at the behavioral level (Ossipov et al., 1995). Besides the development of tolerance (Ballantyne and Mao, 2003), these functional changes may also contribute to the limited responsiveness of chronic neuropathic pain states to selective MOR agonists in the clinic that in turn requires dose escalation to ensure adequate analgesia. However, dose escalation is often limited by intolerable side effects (Portenoy et al., 1990; Portenoy, 1996; Benedetti et al., 1998; Kalso et al., 2004). Indeed, in the present study, intravenous morphine reached full efficacy in the tail flick as well as in the SNL model, with a higher dose being required in the model of neuropathic pain. Of the three subtypes of a2-adrenoceptors, the a2A and a2C subtypes have been implicated to contribute to analgesia in the adult rat spinal cord (Pertovaara, 2006). The a2A-adrenoceptor subtype is located presynaptically on the central terminals of primary afferent C-fibers (Stone et al., 1998) and on Ad fibers where they inhibit glutamatergic nociceptive transmission (Kawasaki et al., 2003). The a2C subtype is expressed on axon terminals of excitatory interneurons that innervate nociceptive projection neurons in the dorsal spinal cord (Olave and Maxwell, 2003). Both a2A- and a2C-adrenoceptors have been shown to mediate spinal adrenergic–opioid synergy in the substance P assay of acute nociceptive pain (Stone et al., 1997; Fairbanks et al., 2002). Synergistic antinociceptive interactions between clonidine and morphine also occur in rats with nerve ligation injury (Ossipov et al., 1997). The spinal adrenoceptor system is subject to morphological and functional changes after SNL. First, SNL surgery increased a2-adrenoceptor G-protein coupling in the spinal cord of rats (Bantel et al., 2005). This was hypothesized to underlie the increased potency and efficacy of clonidine after nerve injury in rats (Xu et al., 1992). Second, while reduced a2A immunoreactivity was seen, increased a2C immunoreactivity was observed in the rat spinal cord after SNL (Stone et al., 1999). Third, a2A- and a2non-A-adrenoceptors mediated the mechanical anti-nociception of intrathecally administered clonidine in experimentally naive rats. However, the clonidine-mediated inhibition of tactile hypersensitivity in the SNL model has been suggested to completely depend on a2adrenoceptors other than a2A, most likely of the a2C subtype as demonstrated by the use of a2 subtype–preferring antagonists (Duflo et al., 2002). Considering this and the fact that yohimbine binding is about 18-fold more potent at recombinant rat adrenoceptors of the a2C than of the a2A subtype (Uhlen et al., 1998), this may well explain why yohimbine induced a greater rightward shift of the tapentadol dose–response curve in the SNL model than in the tail flick test. The higher efficacy of systemic reboxetine in

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the SNL than in the tail flick model found in the present study lends further support to the important role the noradrenergic analgesic system plays for the attenuation of chronic neuropathic but not acute nociceptive pain (Jasmin et al., 2003; Nielsen et al., 2005; Schreiber et al., 2009). Besides the functional up-regulation of spinal a2-adrenoceptors, increased descending inhibitory noradrenergic innervation of the spinal dorsal horn after peripheral nerve injury is well documented (Ma and Eisenach, 2003). This may be a further mechanism underlying the observation that the antihypersensitive effect of tapentadol in the SNL model was primarily mediated by its noradrenergic component. To our knowledge, no data exist on the regulation of the NA transporter itself under conditions of neuropathic pain. The present study clearly shows that the weak in vitro 5-HT reuptake inhibition component of tapentadol did not contribute to its antinociceptive or antihypersensitive effects because the potent and selective 5-HT2A receptor antagonist ritanserin (Albinsson et al., 1994) did not produce a shift in the dose–response curve of tapentadol in either model. We showed that ritanserin was able to completely inhibit both the antinociceptive effect of the potent and selective 5-HT reuptake inhibitor citalopram (Richelson and Pfenning, 1984) in the tail flick test, as well as the antihypersensitive effect of the mixed NA/5-HT reuptake inhibitor venlafaxine (Muth et al., 1986) in the SNL model, thus confirming the appropriateness and effectiveness of the dose used. The fact that citalopram was ineffective whereas venlafaxine was able to produce a clear antihypersensitive effect in the SNL model suggests that modulating serotoninergic transmission alone is not sufficient to inhibit nerve injury–induced tactile hypersensitivity. Likewise, the selective 5HT reuptake inhibitors paroxetine and fluvoxamine did not inhibit tactile allodynia whereas the mixed NA/5-HT reuptake inhibitor milnacipran dose-dependently inhibited tactile allodynia in the chronic constriction injury model of neuropathic pain (Ikeda et al., 2009). This is consistent with clinical observations in neuropathic pain conditions showing a relative lack of efficacy of selective 5-HT reuptake inhibitors and a moderate efficacy of mixed NA/ 5-HT reuptake inhibitors, such as venlafaxine (Max et al., 1992; Briley, 2004; Otto et al., 2008). The opioid and the noradrenergic analgesic systems do not only interact at the level of the spinal cord as pointed out above, but they were also shown to interact at supraspinal sites. For example, MOR agonists activate the spinally projecting noradrenergic inhibitory pathway by disinhibiting GABAergic interneurons in the rat periaqueductal grey (Fields et al., 1991; Osborne et al., 1996; Vaughan and Christie, 1997). The antinociceptive synergy observed with concurrent intracerebroventricular and intrathecal administration of morphine in the rat tail flick and hot plate test (Yeung and Rudy, 1980) could be related to the interaction of spinally released NA acting on a2-adrenoceptors and spinal MOR activation (Wigdor and Wilcox, 1987). This complex supraspinal–spinal synergy between the opioid and the noradrenergic system most likely also contributed to the desipramine-induced augmentation in morphine analgesia obtained after systemic and central administration (Ossipov et al., 1982). Whether the MOR agonism and the NRI component of tapentadol contribute synergistically to its analgesic effects will be a topic for future research. In conclusion, tapentadol reached full efficacy in models of nociceptive and neuropathic pain, with a slightly higher potency in conditions of neuropathic compared to nociceptive pain. This analgesic profile is the result of the bifunctional mode of action of tapentadol, combining MOR agonism with NRI. Whereas MOR agonism predominates in inhibiting nociceptive pain, NRI predominates in attenuating neuropathic pain. This analgesic profile is supposed to be superior to that of classical MOR agonists, as their clinical effectiveness may be limited in conditions of neuropathic

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