European Journal of Pharmacology 741 (2014) 264–271

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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Pharmacogenomic study of the role of the nociceptin/orphanin FQ receptor and opioid receptors in diabetic hyperalgesia Kris Rutten a,n, Thomas M. Tzschentke a, Thomas Koch b, Klaus Schiene a, Thomas Christoph a a b

Grünenthal GmbH, Global Preclinical Drug Development, Department of Pain Pharmacology, Zieglerstrasse 6, 52078 Aachen, Germany Grünenthal GmbH, Global Preclinical Drug Development, Department of Molecular Pharmacology, Aachen, Germany

art ic l e i nf o

a b s t r a c t

Article history: Received 13 March 2014 Received in revised form 10 July 2014 Accepted 5 August 2014 Available online 26 August 2014

Targeting functionally independent receptors may provide synergistic analgesic effects in neuropathic pain. To examine the interdependency between different opioid receptors (m-opioid peptide [MOP], δ-opioid peptide [DOP] and κ-opioid peptide [KOP]) and the nociceptin/orphanin FQ peptide (NOP) receptor in streptozotocin (STZ)-induced diabetic polyneuropathy, nocifensive activity was measured using a hot plate test in wild-type and NOP, MOP, DOP and KOP receptor knockout mice in response to the selective receptor agonists Ro65-6570, morphine, SNC-80 and U50488H, or vehicle. Nocifensive activity was similar in non-diabetic wild-type and knockout mice at baseline, before agonist or vehicle administration. STZ-induced diabetes significantly increased heat sensitivity in all mouse strains, but MOP, DOP and KOP receptor knockouts showed a smaller degree of hyperalgesia than wild-type mice and NOP receptor knockouts. For each agonist, a significant antihyperalgesic effect was observed in wildtype diabetic mice (all P o0.05 versus vehicle); the effect was markedly attenuated in diabetic mice lacking the cognate receptor compared with wild-type diabetic mice. Morphine was the only agonist that demonstrated near-full antihyperalgesic efficacy across all non-cognate receptor knockouts. Partial or near-complete reductions in efficacy were observed with Ro65-6570 in DOP and KOP receptor knockouts, with SNC-80 in NOP, MOP and KOP receptor knockouts, and with U50488H in NOP and DOP receptor knockouts. There was no evidence of NOP and MOP receptor interdependency in response to selective agonists for these receptors. These findings suggest that concurrent activation of NOP and MOP receptors, which showed functional independence, may yield an effective and favorable therapeutic analgesic profile. & 2014 Elsevier B.V. All rights reserved.

Keywords: Antihyperalgesia Knockout mice Nociceptin/orphanin FQ receptor Opioid receptors ORL1 Neuropathic pain

1. Introduction Neuropathic pain is one of the most common long-term complications of diabetes (Boulton et al., 2004; Jensen et al., 2006). Morphine and other m-opioid peptide (MOP) receptor agonists have been used to treat peripheral neuropathic pain in patients with diabetes. However, the use of MOP receptor agonists is complicated by their narrow therapeutic index due to serious side effects, such as respiratory depression, nausea, vomiting and constipation (Labianca et al., 2012; McNicol et al., 2013). The potential development of tolerance, physical dependence and addiction with longterm administration of MOP receptor agonists is also problematic (Smith, 2012; Zöllner and Stein, 2007). As a result, there is an unmet medical need for analgesic therapy for neuropathic pain that combines high efficacy with good tolerability.

n

Corresponding author. Tel.: þ 49 2415 692077; fax: þ49 2415 692852. E-mail address: [email protected] (K. Rutten).

http://dx.doi.org/10.1016/j.ejphar.2014.08.011 0014-2999/& 2014 Elsevier B.V. All rights reserved.

In addition to the MOP receptor, two other types of opioid receptor, the δ-opioid peptide (DOP) receptor and the κ-opioid peptide (KOP) receptor, as well as the functionally different nociceptin/orphanin FQ peptide (NOP) receptor (also known as opioid receptor-like 1 [ORL1] receptor) have been identified (Kieffer, 2000; Meunier et al., 1995; Pradhan et al., 2012). Although systemic administration of DOP and NOP receptor agonists is ineffective in rodent models of acute pain, data from animal studies suggest that they may be effective antihyperalgesics for inflammatory and neuropathic pain (Bie and Pan, 2007; Khroyan et al., 2011). Certain DOP receptor agonists, however, have been reported to cause convulsions in rodents (Pradhan et al., 2012). KOP receptor agonists are also being investigated as analgesics, but their therapeutic potential is limited by the occurrence of dosedependent dysphoria and diuresis (Pradhan et al., 2012). A possible future strategy in the development of opioid analgesics is to target more than one type of receptor simultaneously. Synergism between different receptor-targeted treatments could widen the therapeutic window by increasing analgesic efficacy

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and/or reducing adverse side effects compared with treatment with selective opioid receptor agonists. The availability of opioid and NOP receptor knockout mice makes it possible to study the role of individual receptors in pain generation and response to analgesic drugs. Single and combination receptor knockout mice for the three opioid receptors and the NOP receptor have been described (Kogel et al., 2011; Martin et al., 2003; Nishi et al., 1997; Pradhan et al., 2012). Although the nociceptive responses for single mutants and different mutant combinations have been characterized, the phenotypes observed were generally subtle (Bertorelli et al., 2002; Martin et al., 2003). In the present study, a model of diabetic polyneuropathy induced by streptozotocin (STZ) was used to explore further the nociceptive responses in NOP and opioid receptor knockout mice. The aims of this study were to establish the baseline heat hyperalgesic phenotypes of the three opioid and the NOP receptor knockout mouse strains after induction of diabetes. Then, for the first time, to investigate whether differential functional interdependencies exist between these different receptors by examining the impact of selective NOP, MOP, DOP and KOP receptor agonists (Ro65-6570, morphine, SNC-80 and U50488H, respectively) on diabetic heat hyperalgesia in the different strains of knockout mice.

2. Materials and methods 2.1. General considerations All experiments were carried out at Grünenthal GmbH, Global Preclinical Drug Development, Department of Pharmacology Pain, Aachen, Germany. They were performed in accordance with the German Animal Welfare Act and the recommendations and policies of the International Association for the Study of Pain (Zimmermann, 1983), and were approved by the local government authority. 2.2. Animals 2.2.1. In vivo experiments Male C57BL/6 mice (18–20 g) were obtained from Charles River Laboratories (Sulzfeld, Germany) and were used as the wild-type control animals in all experiments. Male congenic NOP, MOP, DOP and KOP receptor knockout mice (C57BL/6J) were generated by TACONIC Europe A/S (Silkeborg, Denmark) as follows: NOP receptor knockout mice were produced by replacement of the first exon of the oprl1 gene with a β-galactosidase-neomycin resistance gene (neo) cassette (as described in Nishi et al. (1997)); MOP receptor knockout mice (Oprm1tm1Lex) were created by deletion of exons 2 and 3 of the oprm1 gene (strain information: Lexicon Pharmaceuticals, The Woodlands, TX USA); DOP receptor knockout mice (Oprd1tm1949Arte) were generated by flanking exon 2 of the oprd1 gene with loxP sites and deleting the sequence between the sites by crossing the chimaeras with C57BL/6 mice expressing Cre recombinase in the germline (strain information: Taconic, Cologne, Germany); KOP receptor knockout mice (Oprk1tm1950Arte) were generated by flanking exon 3 of the opkr1 gene with loxP sites and deleting the sequence between the sites by crossing the chimaeras with C57BL/6 mice expressing Cre recombinase in the germline (strain information: Taconic, Cologne, Germany). Mice were housed in temperature-controlled rooms (227 2 1C) with a 12-h light–dark cycle (lights on at 06:00, lights off at 18:00). Standard laboratory food (ssniff R/M-Haltung, ssniff Spezialdiäten GmbH, Soest, Germany) and water were available ad libitum in the home cage. Behavioral experiments were conducted between 07:00 and 12:00 during the light phase of the light–dark cycle.

265

Animals were allowed to acclimatize to the laboratory room for at least 1 h before the start of each experiment. 2.2.2. In vitro experiments Material for the in vitro binding affinity experiments was obtained from male Sprague-Dawley specific-pathogen-free rats (average weight: 200 g) (Charles River Laboratories, Sulzfeld, Germany). 2.3. Drugs Analgesic compounds or corresponding vehicle (10% dimethyl sulfoxide [DMSO] and 5% Cremophor EL dissolved in 5% glucose solution) were administered by intraperitoneal injection in a volume of 10 ml/kg. Doses were as follows: 0.1 mg/kg for Ro656570 (Grünenthal GmbH, Aachen, Germany), 3.16 mg/kg and 10.0 mg/kg for morphine hydrochloride (Merck, Darmstadt, Germany), 3.16 mg/kg for SNC-80 (Alexis Biochemicals, San Diego, CA, USA) and 3.16 mg/kg for U50488H (Biotrend Chemikalien GmbH, Cologne, Germany). To avoid overdosing the animals, doses were determined on the basis of an expected efficacy of 80% of the maximal possible efficacy, derived from wide-ranging previous experience with these compounds in our laboratory. Experiments were repeated at a higher dose if the expected efficacy was not reached. 2.4. STZ-induced diabetic polyneuropathy Mice were randomly assigned to intraperitoneal injection with 200 mg/kg STZ or sodium citrate buffer, pH 5. Tail vein blood glucose levels in STZ-injected mice were measured 1 week later using Hemoglukotest 20-800R glucose and a reflectance colorimeter (Roche Diagnostics, Mannheim, Germany). Animals reaching blood glucose levels above 22.2 mmol/l were considered to be diabetic. Animals injected with citrate buffer were used as nondiabetic controls. Experiments with diabetic mice were terminated at maximum 3 weeks after STZ administration because the health of the animals is known to deteriorate after that time point (demonstrated by weight loss and reduced activity). 2.5. Experimental design Diabetic heat hyperalgesia was tested as described previously (Christoph et al., 2010). An adapted version of the classical hot plate test was used to measure changes in hyperalgesic reactions in STZ-treated diabetic mice and non-diabetic control mice at different time points before and after administration of the analgesic test compounds or vehicle. Of note, in the classical hotplate test, the latency to first withdrawal response is recorded on a 48 1C hotplate (i.e. acute heat nociception). In the present study an adapted version of this test was used which allows for the measurement of hyperalgesia by means of recording the number of withdrawal reactions in 2 min on a 50 1C hotplate. This version of the hotplate test is a valid readout to compare STZ-induced behavioral phenotypes as well as measuring heat hyperalgesia and anti-hyperalgesic efficacy (Christoph et al., 2010, 2013; Kogel et al., 2011; Tzschentke et al. 2007). Mice were placed individually on a 50 1C metal plate under a transparent Plexiglas box (13  13  10 cm3) for 2-min periods at the following time points: 30 min (habituation) and 15 min (baseline) before administration of the test compound or vehicle and 15, 30, 45 and 60 min after administration of the test compound or vehicle. For each mouse, the number of nocifensive reactions (licking or shaking of the hind paws, licking of the genitals and jumping) was recorded during the 2-min period. Although the

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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 differences between the drugs. For studies conducted in MOP, DOP and KOP receptor knockout strains, each experiment included 10 animals. For studies conducted in NOP receptor knockout animals, only seven animals were available per experiment with Ro65-6570, SNC-80 and U50488H, and nine animals for the experiment with morphine because of poor breeding in this knockout strain. Animals were used for a maximum of two experiments, with a washout period of at least 7 days between tests. If a second experiment was conducted, the reversibility of effects from the first experiment was confirmed by new habituation and baseline measurements. Animals were randomly allocated to treatment groups to avoid systematic bias. 2.6. Binding affinities Binding affinities of the four test agonist compounds for the three opioid receptors and the NOP receptor were measured in order to assess the selectivity of each agonist for its cognate receptor. 2.6.1. Ro65-6570, morphine and SNC-80 The binding affinity of the analgesic compounds (Ro65-6570, morphine and SNC-80) for rat NOP, MOP and DOP receptors was investigated in receptor competition binding studies with homogenates of rat brains using the radioligands [3H]nociceptin (1 nM), [3H]DAMGO (2 nM) and [3H]deltorphin II (2 nM), respectively. Membrane suspensions were prepared from rat brain without cerebellum (for MOP receptor assays) or without pons, medulla oblongata and cerebellum (for DOP and NOP receptor assays). Test compounds were serially diluted with 25% DMSO in H2O to yield a final DMSO concentration of 0.5%, which also served as a vehicle control. The final assay volume was 250 ml per well. Assays were started by the addition of membrane suspensions. After incubation for 60 min at room temperature with Tris buffer (50 mM Tris–HCl, pH 7.4, with 0.05% sodium azide), samples were collected by rapid filtration under mild vacuum using a cell harvester (Brandel, Gaithersburg, MD, USA) and two washes with 5 ml buffer using FP-100 Whatman GF/B filter mats. All incubations were run in triplicate. The radioactivity of the samples was assessed after a stabilization and extraction period of at least 15 h using the scintillation fluid Ready Protein (Beckman Coulter, Krefeld, Germany). Complete competition curves were recorded for the tested compounds and the binding affinity (Ki) values were calculated using the Cheng–Prusoff equation (Cheng and Prusoff, 1973). 2.6.2. U50488H The binding affinity of the analgesic compound U50488H for the rat recombinant KOP receptor was assessed using a homogenous scintillation proximity assay with the radiolabelled ligand [3H] U69593 (PerkinElmer Life and Analytical Sciences, Brussels, Belgium). Membrane preparations from Chinese hamster ovary-K1 cells transfected with the rat KOP receptor (CEREP, Celle l'Evescault, France) were preloaded onto wheatgerm agglutinin-coated scintillation proximity assay beads to yield a final concentration of 5.9 mg per well. The assay volume was 250 ml per well, which included 2 nM [3H]U69593 and either test compound in dilution series or 100 mM naloxone for determination of unspecific binding. Test compounds were serially diluted with 25% DMSO in H2O to yield a final DMSO concentration of 0.5%, which also served as a vehicle control. Assays were started by the addition of beads (1 mg/well). After incubation for 90 min at room temperature with Tris buffer

(50 mM Tris–HCl, pH 7.4, with 0.05% sodium azide), samples were centrifuged for 20 min at 500 rpm. Radioligand concentrations were measured by liquid scintillation count. A dissociation constant (Kd) of 2.00 nM was predetermined for the membrane preparations (lot no. 190209BP) and was used to calculate the Ki values based on the Cheng–Prusoff equation (Cheng and Prusoff, 1973). 2.7. Statistical analysis Statistical analysis was carried out at Grünenthal GmbH using SYSTAT 13 (Systat Software GmbH, Erkrath, Germany). Data were analyzed using a repeated measures analysis of variance (ANOVA) and post-hoc Bonferroni test. P valueso0.05 were considered significant. The main outcome variable was the number of nocifensive reactions observed over a 2-min period. Baseline values for the diabetic and non-diabetic controls were defined as 0% and 100% of the maximum possible antihyperalgesic effect (MPE), respectively. The %MPE for STZ and citrate animals was calculated as follows: % MPE STZ ¼

ðSTZBL – STZTestÞ  100%; ðSTZBL – CitrateBLÞ

where STZBL¼ individual baseline nocifensive reaction measurements in diabetic mice; STZTest¼individual post-baseline measurements in diabetic mice and CitrateBL ¼mean baseline nocifensive reaction measurements in citrate mice experiments. % MPE Citrate ¼

ðCitrateBL –CitrateTestÞ  100%; ðCitrateBLÞ

where CitrateBL ¼individual baseline nocifensive reaction measurements in citrate mice and CitrateTest¼ individual postbaseline measurements in citrate mice. Values above 100% were considered as 100%. Mean %MPE and standard error of the mean (SEM) were calculated for each of the four agonist compounds at five time points (baseline, and 15, 30, 45 and 60 min after administration of the agonist compound or vehicle) in the wild-type and receptor knockout mouse strains. Area under the data (AUD) was calculated for %MPE values from 0 to 60 min after treatment with agonist as follows: ðð%MPE15 minÞ  15Þ ðð%MPE15 min þ %MPE30 minÞ  15Þ þ 2 2 ðð%MPE30 min þ%MPE45 minÞ  15Þ þ 2 ðð%MPE45 min þ%MPE60 minÞ  15Þ þ 2 Maximal area under the dose (AUDmax) was determined by ∑AUDt2max  t1max, thus (((0þ100)  15)/2)þ(((100þ100)  15)/2)þ (((100þ100)  15)/2)þ(((100þ 100)  15)/2)¼750þ 1500 þ1500 þ1500¼5250. Thus %AUD¼AUD/AUDmax  100%¼ AUD/5250  100%. Mean %AUD and SEM were calculated for each of the four agonist compounds in the wild-type and receptor knockout mouse strains.

3. Results 3.1. Baseline heat sensitivity Fig. 1 shows the mean number of nocifensive reactions/2 min assessment period at baseline in non-diabetic and diabetic wildtype and knockout mice, as measured using a hot plate test. In nondiabetic animals, sensitivity to heat was similar in wild-type and knockout mice, with mean number of nocifensive reactions/2 min ranging from 13.8 to 15.4 in the different strains. STZ-induced diabetic polyneuropathy caused significant increases in heat sensitivity in all mouse strains. Compared with non-diabetic controls, the mean number of nocifensive reactions/2 min following induction of

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Mean number of nocifensive reactions/2 min

40

267

Non-diabetic mice Diabetic mice

35 30 25 20 15 10 5 0

Wild-type

NOP receptor knockouts

MOP receptor knockouts

DOP receptor knockouts

KOP receptor knockouts

Fig. 1. Mean number of nocifensive reactions per 2-min assessment period at baseline in non-diabetic and diabetic wild-type and knockout mice. Error bars show standard error of the mean. nPo 0.05. DOP, δ-opioid peptide; KOP; κ-opioid peptide; MOP, m-opioid peptide; NOP, nociceptin/orphanin FQ peptide.

diabetes was increased 2.4 times in wild-type mice, 2.7 times in NOP receptor knockout mice, 2.0 times in MOP and KOP receptor knockout mice, and 1.8 times in DOP receptor knockout mice. Nocifensive reactions differed significantly by genotype in diabetic mice (ANOVA: F(4,404)¼128.2; Po0.05). Bonferroni-corrected pairwise analysis showed that the mean number of nocifensive reactions was significantly lower in DOP than KOP receptor knockout mice and in KOP than MOP receptor knockout mice (Po0.05), but did not differ significantly between MOP and NOP receptor knockout mice. 3.2. Effects of agonist compounds on heat sensitivity Fig. 2 shows the %MPE values at 15, 30, 45 and 60 min after treatment with agonist compounds or vehicle in diabetic and with vehicle in non-diabetic wild-type and knockout animals. %AUD were calculated for integrated values and are depicted in Fig. 3. In wild-type diabetic mice, each of the selective receptor ligands produced statistically significant (Po0.05) antihyperalgesic effects compared with vehicle at all post-baseline time points (Fig. 2, top row; Fig. 3). Furthermore, with the exception of 3.16 mg/kg morphine, all of the agonists reached the target efficacy of 80% MPE at one or more time points. The peak %MPE (and overall antihyperalgesic response, %AUD) for each agonist was 84.173.3% (75.573.8%) with Ro65-6570, 78.675.2% (66.173.9%) with 3.16 mg/kg morphine, 96.872.2% (94.072.2%) with SNC-80 and 87.873.9% (82.772.8%) with U50488H. When repeated at a dose of 10 mg/kg, morphine reached a peak %MPE (%AUD) of 95.372.4% (93.772.2%). Treatment with vehicle did not have a marked effect on nocifensive behavior in diabetic and non-diabetic wild-type and knockout mice (Fig. 2). Overall, the %AUD differed significantly by genotype in diabetic knockout mice following administration of agonists (ANOVA, Ro656570: F[4,42]¼21.7 [Po0.05]; morphine 3.16 mg/kg: F[4,44]¼ 27.3 [Po0.05], morphine 10.0 mg/kg: F[3,37]¼78.0 [Po0.05]; SNC-80: F[4,42]¼287.4 [Po0.05]; U50488H: F[4,42]¼ 25.5 [Po0.05]) (Fig. 3). For each of the agonists, the antihyperalgesic effect was markedly attenuated compared with wild-type when assessed in diabetic mice that lacked the cognate receptor (Fig. 2, shaded graphs) and %AUD was decreased significantly versus wild-type (all Po0.05; Fig. 3). The peak %MPE (%AUD) was 44.5712.1% (21.077.9%) with Ro656570 in NOP receptor knockout mice, 27.379.8% (16.076.4%) with 3.16 mg/kg morphine and 8.374.4% (4.073.4%) with 10.0 mg/kg

morphine in MOP receptor knockout mice, 9.474.0% (7.573.0%) with SNC-80 in DOP receptor knockout mice and 27.4714.1% (20.679.1%) with U50488H in KOP receptor knockout mice. When receptor ligands were assessed in diabetic mice that lacked a receptor other than the cognate receptor of the agonist tested (Fig. 2, rows 2–5, unshaded graphs; Fig. 3), only morphine was able to demonstrate near-full antihyperalgesic activity across all noncognate receptor knockout mice at all post-baseline time points. In NOP receptor knockout mice, the peak %MPE was 86.572.9% (%AUD: 82.172.8%) with 3.16 mg/kg morphine (the 10 mg morphine dose was not tested in this strain because of the small number of NOP receptor knockout mice available). In DOP and KOP receptor knockout mice, morphine at the 10 mg/kg dose achieved a peak %MPE (%AUD) of 83.874.0% (80.476.9%) and 89.073.6% (78.974.7%), respectively. At the 3.16 mg/kg dose, morphine achieved a peak %MPE (%AUD) of 54.378.0% (37.674.4%) in DOP receptor knockout mice and of 49.377.0% (43.975.69%) in KOP receptor knockout mice. Ro65-6570 demonstrated near-full antihyperalgesic efficacy in MOP receptor knockout mice (peak %MPE: 90.0 75.45 [%AUD: 69.2 76.4%]), a partial reduction in efficacy in DOP receptor knockout mice (peak %MPE: 45.9 712.0% [%AUD: 30.6 77.3%]) and a near-complete reduction of efficacy in KOP receptor knockout mice (peak %MPE: 24.0 7 6.6% [%AUD: 18.2 7 3.9%]). A near-complete reduction of antihyperalgesic efficacy was observed with SNC-80 in all three non-DOP receptor knockout mice (peak %MPE [%AUD] – NOP receptor knockout mice: 6.6 7 6.8% [2.8 72.5%]; MOP receptor knockout mice: 15.9 75.0% [11.372.7%]; KOP receptor knockout mice: 8.3 74.4% [4.6 71.4%]). U50488H demonstrated near-full efficacy in MOP receptor knockout mice (peak %MPE: 85.077.4% [%AUD: 63.474.5%]) and a partial reduction in NOP receptor knockout mice (peak %MPE: 51.8710.8% [%AUD: 31.076.4%]). In addition, peak %MPE was reached at a later time point in these knockout mice than in the wild-type mice. In DOP receptor knockout mice, U50488H showed near-complete reduction of efficacy (peak %MPE: 20.177.6% [%AUD: 15.474.8%]). 3.3. Binding affinity to rat brain homogenates In vitro data demonstrated high binding affinities of the four receptor agonists for their cognate receptors (Table 1). Ligands showed high specificity for their respective target receptor: for

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NOP receptor agonist Ro65-6570 0.1 mg/kg

%MPE

*

*

*

*

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100

3.16

*

*

*

*

*

*

%MPE

*

*

50

50

*

50 3.16 *

100

100

DOP receptor knockouts %MPE

0

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*

*

*

*

*

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10.0 *

*

*

*

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*

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50

*

* 50 *

0

0

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100

%MPE

*

50 *

10.0 0

*

*

*

*

*

* 3.16

*

50

100 MOP receptor knockouts

*

*

*

KOP receptor agonist U50488H 3.16 mg/kg

10.0 50

0

KOP receptor knockouts

100

50

NOP receptor knockouts %MPE

Wild-type

100

DOP receptor agonist SNC-80 3.16 mg/kg

MOP receptor agonist morphine 3.16 and 10.0 mg/kg

50

*

*

3.16

* 10.0 * 3.16

50 *

0

0

0 15

30

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Time (minutes)

60

15

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0 15

Time (minutes)

Agonist-treated diabetic mice

30

45

Time (minutes)

Vehicle-treated diabetic mice

60

15

30

Time (minutes)

Vehicle-treated non-diabetic mice

Fig. 2. Percentage maximum possible antihyperalgesic effect (%MPE) of the receptor agonists Ro65-6570, morphine, SNC-80 and U50488H in the different receptor knockout strains at 15, 30, 45 and 60 min after agonist treatment. Error bars show standard error of the mean. Shaded panels depict %MPE data of receptor agonists in the cognate receptor knockout mice. DOP, δ-opioid peptide; KO, knockout; KOP; κ-opioid peptide; MOP, m-opioid peptide; NOP, nociceptin/orphanin FQ peptide. nPo 0.05 versus vehicle.

each agonist compound, the binding affinity for its cognate receptor was at least two orders of magnitude higher than for the three other receptors, with the exception of Ro65-6198, which had an only 10-fold selectivity over MOP receptor binding. 4. Discussion To our knowledge, this is the first study to use a combination of systemically administered selective receptor agonists, strains of knockout mice bearing deletions of the different opioid receptors and the NOP receptor, and STZ-induced diabetic heat hyperalgesia in order to investigate functional receptor interdependencies. The interdependencies between receptors appeared to be most prominent for DOP–KOP, NOP–KOP and NOP–DOP receptor combinations. In contrast, there was only limited functional interdependency for the MOP–KOP receptor combination, and no functional interaction was observed for the MOP–NOP receptor combination. In wild-type

diabetic mice, all four agonists were found to be highly effective, with each of them inducing strong antihyperalgesic effects. Knocking out any of the opioid receptors or the NOP receptor had limited effects on baseline nocifensive behavior, and all knockout animals were viable and had normal and comparable weights. Other studies have used quantitative autoradiography to assess receptor expression in knockout mice. MOP receptor knockout mice showed no major differences in radioligand binding at brain KOP or NOP receptor, although DOP receptor binding was downregulated in several regions (Kitchen et al., 1997; Slowe et al., 1999). KOP receptor knockout mice displayed no changes in MOP receptor binding, but DOP receptor binding was upregulated and NOP receptor binding was downregulated (Slowe et al., 2001, 1999). In DOP receptor knockout mice, MOP receptor binding was significantly downregulated, KOP receptor binding was upregulated or downregulated (depending on the ligand used) and NOP receptor binding was upregulated (Goody et al., 2002). In contrast, no

K. Rutten et al. / European Journal of Pharmacology 741 (2014) 264–271

269

Wild-type NOP receptor knockouts MOP receptor knockouts DOP receptor knockouts KOP receptor knockouts

100 90 80 70

% AUD

60 50 40 30 20 10 0

Morphine 3.16 mg/kg

Ro65-6570 0.1 mg/kg

na Morphine 10.0 mg/kg

SNC-80 3.16 mg/kg

U50488H 3.16 mg/kg

Fig. 3. Percentage area under the data (%AUD) of the receptor agonists Ro65-6570, morphine, SNC-80 and U50488H in the cognate receptor knockout mice strains. %AUD was calculated for data from 0 to 60 min after treatment with agonist. Error bars show standard error of the mean. n P o0.05 versus wild-type (Dunnet test). # 0.05 o P o0.1 versus wild-type (Dunnet test). DOP, δ-opioid peptide; KOP; κ-opioid peptide; MOP, m-opioid peptide; na, not assessed; NOP, nociceptin/orphanin FQ peptide.

Table 1 In vitro binding affinity data in recombinant rat receptors. Receptor type

rNOP rMOP rDOP rKOP

Ki (lM) Ro65-6570

Morphine

SNC-80

U50488H

0.0008 0.011 0.81 0.032

9.0 0.0021 0.20 0.20

2.8 1.5 0.0004 2.5

11 0.41 6.6 0.0017

Shaded areas depict Ki of receptor agonists in the cognate rat receptor. Ki, binding constant (concentration required to produce half-maximum inhibition). rDOP, rat δ-opioid peptide; rKOP; recombinant rat κ-opioid peptide; rMOP, rat m-opioid peptide; rNOP, rat nociceptin/orphanin FQ peptide.

differences in MOP, DOP or KOP receptor binding were observed in NOP receptor knockout mice (Clarke et al., 2001). These expression data provide support for functional interactions between MOP, DOP and KOP receptors, and suggest that the NOP receptor is independent. The data from the present study provide evidence for functional receptor interdependencies in a disease model for diabetic polyneuropathy. Presumably as a result of the development of diabetic polyneuropathy, administration of STZ produced clear, measurable hyperalgesia in wild-type and all knockout mouse strains, although the degree of STZ-induced hyperalgesia was slightly less in MOP, DOP or KOP receptor knockout mice than in wild-type and NOP receptor knockout mice. Irrespective of these differences, rates of baseline nocifensive reactions were significantly increased for each of the different strains. The effects on increased thermal sensitivity of knocking out opioid receptors have been described in the literature (Gendron et al., 2007; Nadal et al., 2006). Unlike the present study, which assessed the extent of the nocifensive response (i.e. hyperalgesia), these previous studies measured the latency to the first pain (paw withdrawal) response (i.e. hypersensitivity). Sciatic nerve injury-induced neuropathic pain led to decreases in time to respond

to a thermal stimulus in both wild-type and DOP receptor knockout mice, and DOP receptor knockout mice showed a significantly greater decrease in paw withdrawal latency than wild-type mice (Nadal et al., 2006). In a model of inflammatory pain induced by Complete Freund's Adjuvant, the latency to paw withdrawal did not differ among wildtype, MOP and KOP receptor knockout mice (Gendron et al., 2007). However, the antihypersensitive efficacy of the DOP receptor agonist deltorphin II was significantly reduced in MOP receptor knockout mice, but not in KOP receptor knockout mice, compared with wildtype mice (Gendron et al., 2007), perhaps due to the requirement of MOP receptors for DOP receptor trafficking to the cell surface (Vanderah, 2010). In non-diabetic control animals no changes in baseline hyperalgesic responses were observed between wild-type and knockout strains. Interestingly, we observed reduced hyperalgesic responses in diabetic opioid receptor knockout animals compared to diabetic wild-type or NOP receptor knockout animals (i.e. diabetic MOP, DOP and KOP knockout mice had a lower hyperalgesic response than NOP KO and wild-type mice). The reason for the reduced diabetic hyperalgesia in these opioid knockout strains is thus far unknown. However, it is becoming generally accepted that G protein-coupled receptors (GPCRs) including opioid receptors interact with each other to form heteromers with new pharmacological properties and differences in signaling and trafficking compared to homomers (Pasternak and Pan, 2011). Furthermore, MOP displays optimal coupling and signaling, leading to the “classical” (G protein-mediated) MOP response. MOP, when complexed with DOP, displays impaired coupling and signaling, leading to a “nonclassical” (beta-arrestinmediated) MOP response. Interestingly, this heterodimer/betaarrestin-mediated “nonclassical” signaling can be reversed to G protein-mediated “classical” signaling by a DOP-selective antagonist (Rozenfeld et al., 2007). Thus, the lack of the formation of distinct signaling complexes via GPCR-heterodimerization might be a possible explanation for the observed lower hyperalgesic effect in MOP, DOP, and KOP knockout mice. The present findings suggest important functional interactions between the different opioid receptors and the NOP receptor

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NOP receptor signaling (agonist: Ro65-6570) KOP receptor signaling (agonist: U50488H)

MOP receptor signaling (agonist: morphine) DOP receptor signaling (agonist: SNC-80)

Efficacy of agonist in knockout strain Near-full efficacy Partial reduction Near-complete reduction Fig. 4. Proposed interactions between opioid and NOP receptors. Based on the antihyperalgesic effect of selective ligands for receptor types in streptozotocininduced diabetic hyperalgesia in mice with deletions of the opioid and NOP receptors. Antihyperalgesic efficacy of an agonist in each knockout strain was classified as follows: near-full efficacy, %AUD or highest %MPE 480%; partial reduction in efficacy, %AUD and highest %MPE 20–80%; near-complete reduction in efficacy, %AUD or highest %MPE o 20%. AUD, area under the data; DOP, δ-opioid peptide; KOP; κ-opioid peptide; MOP, m-opioid peptide; MPE, maximum possible antihyperalgesic effect; NOP, nociceptin/ orphanin FQ peptide.

(summarized in Fig. 4). For example, the antihyperalgesic effect of the KOP receptor agonist U50488H was largely absent in DOP receptor knockout mice, and the effect of the DOP receptor agonist SNC-80 was absent in KOP receptor knockout mice. In contrast, there was no evidence of interdependency between NOP and MOP receptors in response to their selective agonists: the NOP receptor agonist Ro65-6570 showed near-full efficacy in MOP receptor knockout mice and morphine approached full efficacy in NOP receptor knockout mice. In line with our findings, morphine and U50488H have been shown to have similar efficacies in wild-type and NOP receptor knockout mice when assessed in tail flick and acetic acid-induced writhing tests (Noda et al., 1998). Only limited functional interdependency was observed between MOP and KOP receptors in response to their selective agonists: at the original dose (3.16 mg/kg), morphine had a markedly more pronounced antihyperalgesic effect in NOP than KOP receptor knockout mice, suggesting that the MOP–KOP receptor combination, but not the MOP–NOP receptor combination, requires a higher morphine dose to overcome functional dependence. Morphine was the only analgesic tested that showed near-full efficacy across all noncognate (i.e. DOP, KOP and NOP) receptor knockout mice in the present study, suggesting that its efficacy as an antihyperalgesic is independent of the other opioid receptors and the NOP receptor. From the literature it is known that antihyperalgesic effects of agonists in their respective cognate receptor knockout mice are largely absent. Thus, the antinociceptive effect of morphine, as well as its rewarding effects and its potential to induce physical dependence, were abolished in MOP receptor knockout mice (Matthes et al., 1996). Likewise, SNC-80 was efficacious in mouse models of persistent inflammatory and neuropathic pain, but these effects were absent in mice with the DOP receptor knocked out in primary afferent NaV1.8 neurons (Gaveriaux-Ruff et al., 2011). Furthermore, the antinociceptive effect of U50488H, as well as its hypolocomotor and aversive effects, were abolished in KOP receptor knockout mice (Simonin et al., 1998). Finally, the bidirectional changes in pain sensitivity observed in wild-type mice after treatment with the NOP receptor agonist Ro64-6198 were

abolished in NOP receptor knockout mice (Reiss et al., 2008). Our findings in diabetic polyneuropathic mice fully corroborate these earlier findings. Binding experiments indicated that each ligand was at least 100-fold selective for its cognate receptor versus other receptors, with the exception of Ro65-6570, which had an only 10-fold selectivity over MOP receptor binding. Therefore, some remaining weak activities (e.g. of Ro65-6570 in diabetic MOP receptor knockout mice) might be attributable to low, offtarget affinity for other receptors. However, the minimal antihyperalgesic activity of the compounds in the cognate receptor knockout mice suggests that such off-target activity is limited. Although binding experiments were performed in rat tissue, opioid pharmacology is well-conserved across species (Kalvass et al., 2007; Schattauer et al., 2012; Wallisch et al., 2007). Similar to opioid receptors, NOP receptors have been shown to couple to pertussis toxin-sensitive Gi/o proteins to inhibit adenylate cyclase, activate inwardly rectifying potassium channels and inhibit calcium channels (Ma et al., 1997; Margas et al., 2008). However, unlike opioid receptors, NOP receptors can also couple to pertussis toxin-insensitive G proteins, including Gz, G16 and Gs (Chan et al., 1998; Klukovits et al., 2010). Furthermore, although NOP receptors use many of the same G-protein-mediated signaling pathways as opioid receptors, their endogenous peptide agonists appear to be located in separate neuronal circuits (Meunier, 1997; Neal et al., 1999). These differences in network effects and anatomical distribution may explain, in part, the differences in functional interdependency seen between receptors in the present study, in particular the observed lack of interaction between MOP and NOP receptors. Targeting multiple receptors may provide synergistic analgesic effects and a more favorable side effect profile (e.g. Schröder et al., 2011). In the present study, no functional interaction was observed for MOP and NOP receptors, suggesting that these independent receptors may be suitable targets for concurrent activation. Indeed, co-activation of NOP and MOP receptors may yield an effective and favorable therapeutic analgesic profile (for review see: Schröder et al. (2014)). Although only limited interdependence was seen for MOP and KOP receptors, combined targeting of these receptors may be less attractive owing to known side effects of KOP receptor activation (Pradhan et al., 2012). Combined activation of spinal NOP and MOP receptors has been reported to lead to dose-dependent, synergistic antihypersensitive effects in the rat chronic constriction injury model (Courteix et al., 2004). Along similar lines, concurrent activation of spinal NOP and MOP receptors resulted in improved antiallodynic efficacy and potency and, promisingly, to a reduced development of tolerance in a mouse chronic constriction injury model (Sukhtankar et al., 2013). Furthermore, combined systemic administration of the MOP receptor agonist buprenorphine and the NOP receptor agonist Ro64-6198 resulted in synergistic antinociceptive effects in non-human primates, without the side effects (respiratory depression, itching/scratching) that were observed when buprenorphine was administered alone (Cremeans et al., 2012). In rats, Ro65-6570 attenuated the rewarding side-effects of opioids and psychostimulants, and knockout of the NOP receptor potentiated the rewarding effects of morphine, suggesting that NOP receptor agonists may counteract MOP receptor-related side effects (Rutten et al., 2010, 2011). In conclusion, findings from this study are suggestive of functional interactions between MOP, KOP and DOP receptors. Although there was only limited functional interdependency between MOP and KOP receptors, the unfavorable side effects produced by KOP receptor activation make this receptor combination an unattractive target for the development of novel therapies. As the only pair of receptors that appear to be independent of each other, concurrent activation of MOP and NOP receptors may be a useful strategy in the development of novel therapies for neuropathic pain.

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Acknowledgments We are grateful for the excellent technical assistance of Elke Schumacher and for valuable comments for the discussion of the manuscript by Dr Wolfgang Schröder. Writing support was provided by Dr Anja Becher of Oxford PharmaGenesis™ Ltd, Oxford, UK, and was funded by Grünenthal GmbH, Aachen, Germany. This study was funded by Grünenthal GmbH. References Bertorelli, R., Bastia, E., Citterio, F., Corradini, L., Forlani, A., Ongini, E., 2002. Lack of the nociceptin receptor does not affect acute or chronic nociception in mice. Peptides 23, 1589–1596. Bie, B., Pan, Z.Z., 2007. Trafficking of central opioid receptors and descending pain inhibition. Mol. Pain 3, 37. Boulton, A.J., Malik, R.A., Arezzo, J.C., Sosenko, J.M., 2004. Diabetic somatic neuropathies. Diabetes Care 27, 1458–1486. Chan, J.S., Yung, L.Y., Lee, J.W., Wu, Y.L., Pei, G., Wong, Y.H., 1998. Pertussis toxininsensitive signaling of the ORL1 receptor: coupling to Gz and G16 proteins. J. Neurochem. 71, 2203–2210. Cheng, Y., Prusoff, W.H., 1973. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108. Christoph, T., De Vry, J., Tzschentke, T.M., 2010. Tapentadol, but not morphine, selectively inhibits disease-related thermal hyperalgesia in a mouse model of diabetic neuropathic pain. Neurosci. Lett. 470, 91–94. Christoph, T., Schröder, W., Tallarida, R.J., De Vry, J., Tzschentke, T.M., 2013. Spinalsupraspinal and intrinsic μ-opioid receptor agonist-norepinephrine reuptake inhibitor (MOR-NRI) synergy of tapentadol in diabetic heat hyperalgesia in mice. J. Pharmacol. Exp. Ther. 347, 794–801. Clarke, S., Chen, Z., Hsu, M.S., Pintar, J., Hill, R., Kitchen, I., 2001. Quantitative autoradiographic mapping of the ORL1, mu-, delta- and kappa-receptors in the brains of knockout mice lacking the ORL1 receptor gene. Brain Res. 906, 13–24. Courteix, C., Coudore-Civiale, M.A., Privat, A.M., Pelissier, T., Eschalier, A., Fialip, J., 2004. Evidence for an exclusive antinociceptive effect of nociceptin/orphanin FQ, an endogenous ligand for the ORL1 receptor, in two animal models of neuropathic pain. Pain 110, 236–245. Cremeans, C.M., Gruley, E., Kyle, D.J., Ko, M.C., 2012. Roles of mu-opioid receptors and nociceptin/orphanin FQ peptide receptors in buprenorphine-induced physiological responses in primates. J. Pharmacol. Exp. Ther. 343, 72–81. Gaveriaux-Ruff, C., Nozaki, C., Nadal, X., Hever, X.C., Weibel, R., Matifas, A., Reiss, D., Filliol, D., Nassar, M.A., Wood, J.N., Maldonado, R., Kieffer, B.L., 2011. Genetic ablation of delta opioid receptors in nociceptive sensory neurons increases chronic pain and abolishes opioid analgesia. Pain 152, 1238–1248. Gendron, L., Pintar, J.E., Chavkin, C., 2007. Essential role of mu opioid receptor in the regulation of delta opioid receptor-mediated antihyperalgesia. Neuroscience 150, 807–817. Goody, R.J., Oakley, S.M., Filliol, D., Kieffer, B.L., Kitchen, I., 2002. Quantitative autoradiographic mapping of opioid receptors in the brain of delta-opioid receptor gene knockout mice. Brain Res. 945, 9–19. Jensen, T.S., Backonja, M.M., Hernandez Jimenez, S., Tesfaye, S., Valensi, P., Ziegler, D., 2006. New perspectives on the management of diabetic peripheral neuropathic pain. Diab. Vasc. Dis. Res. 3, 108–119. Kalvass, J.C., Olson, E.R., Cassidy, M.P., Selley, D.E., Pollack, G.M., 2007. Pharmacokinetics and pharmacodynamics of seven opioids in P-glycoprotein-competent mice: assessment of unbound brain EC50,u and correlation of in vitro, preclinical, and clinical data. J. Pharmacol. Exp. Ther. 323, 346–355. Khroyan, T.V., Polgar, W.E., Orduna, J., Montenegro, J., Jiang, F., Zaveri, N.T., Toll, L., 2011. Differential effects of nociceptin/orphanin FQ (NOP) receptor agonists in acute versus chronic pain: studies with bifunctional NOP/mu receptor agonists in the sciatic nerve ligation chronic pain model in mice. J. Pharmacol. Exp. Ther. 339, 687–693. Kieffer, B.L., 2000. Opioid receptors: from genes to mice. J. Pain 1, 45–50. Kitchen, I., Slowe, S.J., Matthes, H.W., Kieffer, B., 1997. Quantitative autoradiographic mapping of mu-, delta- and kappa-opioid receptors in knockout mice lacking the mu-opioid receptor gene. Brain Res. 778, 73–88. Klukovits, A., Tekes, K., Gunduz Cinar, O., Benyhe, S., Borsodi, A., Deak, B.H., Hajagos-Toth, J., Verli, J., Falkay, G., Gaspar, R., 2010. Nociceptin inhibits uterine contractions in term-pregnant rats by signaling through multiple pathways. Biol. Reprod. 83, 36–41. Kogel, B., De Vry, J., Tzschentke, T.M., Christoph, T., 2011. The antinociceptive and antihyperalgesic effect of tapentadol is partially retained in OPRM1 (mu-opioid receptor) knockout mice. Neurosci. Lett. 491, 104–107. Labianca, R., Sarzi-Puttini, P., Zuccaro, S.M., Cherubino, P., Vellucci, R., Fornasari, D., 2012. Adverse effects associated with non-opioid and opioid treatment in patients with chronic pain. Clin. Drug Investig. 32 (Suppl. 1), S53–S63. Ma, L., Cheng, Z.J., Fan, G.H., Cai, Y.C., Jiang, L.Z., Pei, G., 1997. Functional expression, activation and desensitization of opioid receptor-like receptor ORL1 in neuroblastoma x glioma NG108-15 hybrid cells. FEBS Lett. 403, 91–94. Margas, W., Sedeek, K., Ruiz-Velasco, V., 2008. Coupling specificity of NOP opioid receptors to pertussis-toxin-sensitive Galpha proteins in adult rat stellate

271

ganglion neurons using small interference RNA. J. Neurophysiol. 100, 1420–1432. Martin, M., Matifas, A., Maldonado, R., Kieffer, B.L., 2003. Acute antinociceptive responses in single and combinatorial opioid receptor knockout mice: distinct mu, delta and kappa tones. Eur. J. Neurosci. 17, 701–708. Matthes, H.W., Maldonado, R., Simonin, F., Valverde, O., Slowe, S., Kitchen, I., Befort, K., Dierich, A., Le Meur, M., Dolle, P., Tzavara, E., Hanoune, J., Roques, B.P., Kieffer, B.L., 1996. Loss of morphine-induced analgesia, reward effect and withdrawal symptoms in mice lacking the mu-opioid-receptor gene. Nature 383, 819–823. McNicol, E.D., Midbari, A., Eisenberg, E., 2013. Opioids for neuropathic pain. Cochrane Database Syst. Rev., 8. Meunier, J.C., 1997. Nociceptin/orphanin FQ and the opioid receptor-like ORL1 receptor. Eur. J. Pharmacol. 340, 1–15. Meunier, J.C., Mollereau, C., Toll, L., Suaudeau, C., Moisand, C., Alvinerie, P., Butour, J.L., Guillemot, J.C., Ferrara, P., Monsarrat, B., et al., 1995. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377, 532–535. Nadal, X., Banos, J.E., Kieffer, B.L., Maldonado, R., 2006. Neuropathic pain is enhanced in delta-opioid receptor knockout mice. Eur. J. Neurosci. 23, 830–834. Neal Jr., C.R., Mansour, A., Reinscheid, R., Nothacker, H.P., Civelli, O., Watson Jr., S.J., 1999. Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat. J. Comp. Neurol. 406, 503–547. Nishi, M., Houtani, T., Noda, Y., Mamiya, T., Sato, K., Doi, T., Kuno, J., Takeshima, H., Nukada, T., Nabeshima, T., Yamashita, T., Noda, T., Sugimoto, T., 1997. Unrestrained nociceptive response and disregulation of hearing ability in mice lacking the nociceptin/orphaninFQ receptor. EMBO J. 16, 1858–1864. Noda, Y., Mamiya, T., Nabeshima, T., Nishi, M., Higashioka, M., Takeshima, H., 1998. Loss of antinociception induced by naloxone benzoylhydrazone in nociceptin receptor-knockout mice. J. Biol. Chem. 273, 18047–18051. Pasternak, P., Pan, Y., 2011. Mix and match: heterodimers and opioid tolerance. Neuron 69, 6–8. Pradhan, A.A., Smith, M.L., Kieffer, B.L., Evans, C.J., 2012. Ligand-directed signalling within the opioid receptor family. Br. J. Pharmacol. 167, 960–969. Reiss, D., Wichmann, J., Tekeshima, H., Kieffer, B.L., Ouagazzal, A.M., 2008. Effects of nociceptin/orphanin FQ receptor (NOP) agonist, Ro64-6198, on reactivity to acute pain in mice: comparison to morphine. Eur. J. Pharmacol. 579, 141–148. Rozenfeld, R., Abul-Husn, N.S., Gomes, I., Devi, L.A., 2007. An emerging role for the delta opioid receptor in the regulation of mu opioid receptor function. Sci. World J. 7 (S2), S64–S73. Rutten, K., De Vry, J., Bruckmann, W., Tzschentke, T.M., 2010. Effects of the NOP receptor agonist Ro65-6570 on the acquisition of opiate- and psychostimulantinduced conditioned place preference in rats. Eur. J. Pharmacol. 645, 119–126. Rutten, K., De Vry, J., Bruckmann, W., Tzschentke, T.M., 2011. Pharmacological blockade or genetic knockout of the NOP receptor potentiates the rewarding effect of morphine in rats. Drug Alcohol Depend. 114, 253–256. Schattauer, S.S., Miyatake, M., Shankar, H., Zietz, C., Levin, J.R., Liu-Chen, L.Y., Gurevich, V.V., Rieder, M.J., Chavkin, C., 2012. Ligand directed signaling differences between rodent and human kappa-opioid receptors. J. Biol. Chem. 287, 41595–41607. Schröder, W., Tzschentke, T.M., Terlinden, R., De Vry, J, Jahnel, U., Christoph, T., Tallarida, R.J., 2011. Synergistic interaction between the two mechanisms of action of tapentadol in analgesia. J. Pharmacol. Exp. Ther. 337, 312–320. Schröder, W., Lambert, D.G., Ko, M.C., Koch, T., 2014. Functional plasticity of the N/ OFQ-NOP receptor system determines analgesic properties of NOP receptor agonists. Br. J. Pharmacol 171, 3777–3800. Simonin, F., Valverde, O., Smadja, C., Slowe, S., Kitchen, I., Dierich, A., Le Meur, M., Roques, B.P., Maldonado, R., Kieffer, B.L., 1998. Disruption of the kappa-opioid receptor gene in mice enhances sensitivity to chemical visceral pain, impairs pharmacological actions of the selective kappa-agonist U-50,488H and attenuates morphine withdrawal. EMBO J. 17, 886–897. Slowe, S.J., Clarke, S., Lena, I., Goody, R.J., Lattanzi, R., Negri, L., Simonin, F., Matthes, H. W., Filliol, D., Kieffer, B.L., Kitchen, I., 2001. Autoradiographic mapping of the opioid receptor-like 1 (ORL1) receptor in the brains of mu-, delta- or kappa-opioid receptor knockout mice. Neuroscience 106, 469–480. Slowe, S.J., Simonin, F., Kieffer, B., Kitchen, I., 1999. Quantitative autoradiography of mu-,delta- and kappa1 opioid receptors in kappa-opioid receptor knockout mice. Brain Res. 818, 335–345. Smith, H.S., 2012. Opioids and neuropathic pain. Pain Physician 15, ES93–110. Sukhtankar, D.D., Zaveri, N.T., Husbands, S.M., Ko, M.C., 2013. Effects of spinally administered bifunctional nociceptin/orphanin FQ peptide receptor/mu-opioid receptor ligands in mouse models of neuropathic and inflammatory pain. J. Pharmacol. Exp. Ther. 346, 11–22. Tzschentke, T.M., Christoph, T., Kögel, B., Schiene, K., Hennies, H.H., Englberger, W., Haurand, M., Jahnel, U., Cremers, T.I., Friderichs, E., De Vry, J., 2007. (–)-(1R,2R)3-(3-dimethylamino-1-ethyl-2-methyl-propyl)-phenol hydrochloride (tapentadol HCl): a novel mu-opioid receptor agonist/norepinephrine reuptake inhibitor with broad-spectrum analgesic properties. J. Pharmacol. Exp. Ther. 323, 265–276. Vanderah, T.W., 2010. Delta and kappa opioid receptors as suitable drug targets for pain. Clin. J. Pain 26 (Suppl. 10), S10–S15. Wallisch, M., Nelson, C.S., Mulvaney, J.M., Hernandez, H.S., Smith, S.A., Olsen, G.D., 2007. Effects of chronic opioid exposure on guinea pig mu opioid receptor in Chinese hamster ovary cells: comparison with human and rat receptor. Biochem. Pharmacol. 73, 1818–1828. Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain in conscious animals. Pain 16, 109–110. Zöllner, C., Stein, C., 2007. Opioids. Handb. Exp. Pharmacol., 31–63.