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Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test
Accepted Manuscript Title: Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test Authors: A. Rizzi, C. Ruzza, S. Bianco, C. Trapella, G. Calo’ PII: DOI: Reference:
S0196-9781(17)30222-X http://dx.doi.org/doi:10.1016/j.peptides.2017.07.002 PEP 69792
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Peptides
Received date: Revised date: Accepted date:
10-5-2017 30-6-2017 3-7-2017
Please cite this article as: Rizzi A, Ruzza C, Bianco S, Trapella C, Calo’ G.Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test.Peptides http://dx.doi.org/10.1016/j.peptides.2017.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test Rizzi A1, Ruzza C1, Bianco S2, Trapella C2, and Calo’ G1 1
Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy. 2 Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, Ferrara, Italy. Corresponding author:
Girolamo Calo’, MD PhD Department of Medical Sciences, Section of Pharmacology, University of Ferrara, Via Fossato di Mortara 19, 44121 Ferrara, Italy [email protected] Highlights:
Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions, including pain.
NOP(-/-) mice displayed a pronociceptive phenotype in the orofacial formalin test (OFF)
In the OFF the NOP agonist Ro 65-6570 evoked antinociceptive effects
The effects of Ro 65-6570 and morphine were additive
The mixed NOP/opioid receptor agonist cebranopadol evoked potent antinociceptive effects
Abstract Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions, including pain transmission via selective activation of a specific receptor named NOP. The aim of this study was the investigation of the antinociceptive properties of NOP agonists and their interaction with opioids in the trigeminal territory. The orofacial formalin (OFF) test in mice was used to investigate the antinociceptive potential associated to the activation of NOP and opioid receptors. Mice subjected to OFF test displayed the typical biphasic nociceptive response and sensitivity to opioid and NSAID drugs. Mice knockout for the NOP gene displayed a robust pronociceptive phenotype. 1
The NOP selective agonist Ro 65-6570 (0.1 – 1 mg kg-1) and morphine (0.1 – 10 mg kg-1) elicited dose dependent antinociceptive effects in the OFF with the alkaloid showing larger effects; the isobologram analysis of their actions demonstrated an additive type of interaction. The mixed NOP/opioid receptor agonist cebranopadol elicited potent (0.01 – 0.1 mg kg-1) and robust antinociceptive effects. In the investigated dose range, all drugs did not modify the motor performance of the mice in the rotarod test. Collectively the results of this study demonstrated that selective NOP agonists and particularly mixed NOP/opioid agonists are worthy of development as innovative drugs to treat painful conditions of the trigeminal territory.
Key Words Orofacial formalin test; cebranopadol; mixed NOP/opioid agonists; isobolographic analysis
1. Introduction The nociceptin/orphanin FQ (N/OFQ) - NOP receptor system shares with classical opioid systems high structural and transductional similarities and, among others, the ability to modulate pain responses [1]. In rodents, N/OFQ and selective NOP receptor agonists elicit antinociceptive effects at peripheral and spinal levels but also pronociceptive effects at supraspinal sites [2-4]. Moreover, when injected systemically NOP agonists are far more effective on inflammatory or neuropathic pain than on nociceptive pain [3]. However, in nonhuman primates powerful antinociception is observed either after local [5], systemic [6], spinal [7], and intracisternal [8] administration. This suggests that systemic NOP ligands in primates may reduce pain by acting synergistically at different levels, whereas in rodents there might be opposite effects in brain and spinal cord, which result in a smaller or even absent antinociceptive effect [1, 3]. Emerging evidence suggests that mixed NOP/opioid receptor agonists may have innovative analgesic potential. In fact, a synergistic enhancement of antinociception achieved by the concurrent activation of NOP and opioid (particulary mu) receptors was demonstrated with systemically or intrathecally delivered agonists (reviewed in [1, 3]). Novel molecules acting as mixed NOP/opioid receptor agonists [9-14] are extremely useful tools for exploring the potential benefit of NOP and opioid receptor co-activation in the analgesia field. One of such ligands, cebranopadol, demonstrated high affinity and efficacy at NOP and opioid receptors and displayed robust antinociceptive properties in different rodent models of pain [13, 15-17]. Importantly the analgesic potential of cebranopadol has been confirmed in phase II clinical trials [18].
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Evidence coming from rodent [19-21] and human [22-24] studies demonstrated that NOP receptors as well as N/OFQ are expressed in trigeminal structures. Thus, the aim of the present study was the investigation of the antinociceptive action of NOP and opioid receptor selective and non selective agonists in the trigeminal territory. Hence we first set up the experimental conditions for the mouse orofacial formalin (OFF) assay by testing different doses of formalin and the effects of morphine and ibuprofen. Then we investigated in this assay the phenotype of mice knockout for the NOP receptor gene (NOP-/-) was well as the dose response curves and isobolographic analysis of morphine and Ro 65-6570 selected as standard opioid and NOP agonists, respectively. Finally we evaluated the action of cebranopadol in the mouse OFF test.
2. Methods 2.1 Animal welfare and ethical statement All procedures were performed in accordance with the European Communities Council Directive 86/6609/EEC. All animal care and experimental procedures conformed to the standards of the European Communities Council directives (2010/63/EU) and national regulations (D.L. 26/2014). Studies involving animals are reported in accordance with the ARRIVE guidelines [25]. The present study was approved by the Ethical Committee for the Use of Laboratory Animals (CEASA) of the University of Ferrara and by the Italian Ministry of Health (authorization number 316/2013-B). Male CD-1 mice (25-35 g, total number 154) were from Envigo, Natisone Udine, Italy. They were housed in plastic cages (425x266x155 mm, Techniplast, Buguggiate, Italy) with soft bedding with free access to food (standard diet, Mucedola, Settimo Milanese, Italy) and water and were maintained in climate (23 ± 1°C) and light-controlled (12/12-h dark/light cycle with light on at 7:00 am) for at least 1 week before the experiments. Each cage was provided with a mouse red house (Tecniplast, Buguggiate, Italy) and nesting materials. Test sessions took place during the light phase between 9:00 am and 3:00 pm in a quiet room maintained at 23-24°C. The test box had the dimensions of 25 × 25 × 40 cm with 3 mirrored sides. Each animal was first placed in this box for a 30-min habituation period to minimize stress. The mice did not have access to food or water during the test. Each mouse was used only once and was killed at the end of the experiment by the administration of a lethal dose of CO2. Animals were assigned randomly to treatment groups. Different treatment doses and vehicle (saline or 1% DMSO) were tested in a randomized fashion and the operators performing the behavioral tests were blinded with respect to the treatments. 2.2 Orofacial Formalin test 3
Mice received a 20 µl subcutaneous injection of diluted formalin or saline into the right upper lip, just lateral to the nose. Solutions were prepared from commercially available stock formalin further diluted in isotonic saline to 1.5%, and 2.5%. Stock formalin is an aqueous solution of 37% formaldehyde. Formalin was injected subcutaneously through a 30-gauge needle into the center of the right vibrissa pad as quickly as possible, with only minimal animal restraint. Following injection the animals were immediately placed back in the test box for a 45-min observation period. The recording time was divided into 15 blocks of 3 min each, and a nociceptive score was determined for each block by measuring the number of seconds that the animals spent grooming the injected area with the ipsilateral fore- or hindpaw. Movements of the ipsilateral forepaw were accompanied by movements of the contralateral forepaw. A videocamera was used to record the grooming response. Time (s) spent by the animal showing all these pain-related behaviours was cumulatively measured and expressed as seconds (s) of nociceptive behaviour/min. The cumulative response times during 0–9 min and during 10–45 min were regarded as the first-phase (I° phase) and secondphase (II° phase) responses, respectively. Analysis of the behavior was made by an investigator who was blinded to the animal’s group assignment. 2.3 Rotarod test To investigate potential effects on motor coordination we performed a rotarod test using a constant speed device (Ugo Basile, Varese, Italy). Mice were trained at 15 r.p.m for 120 sec 1 day before the experiment. The next day, rotarod latencies were measured 30 min after systemic administration of the compounds. Motor performance was calculated as time (sec) spent on rod measured 30 min after drug injection. A cut-off time of 120 sec was chosen.
2.4 Materials Based on the results obtained in the first series of experiments, the 2.5% formalin concentration was selected as standard noxious stimulus to evaluate the effects of systemic ibuprofen, morphine, Ro 65-6570 and cebranopadol on the rubbing response. Drugs were administered intravenously (i.v.) 100 µl/mouse into the vein caudal tail using a 30-gauge needle attached to a sterile syringe. Morphine chlorohydrate was bought from SALARAS (Como, Italy, authorization number SP/095 del 04/04/2011); ibuprofen was purchased from Sigma Chemical Co (St Louis, MO) and were dissolved in saline (0.9% NaCl solution). Ro 65-6570 and cebranopadol [26] were synthesized in house and solubilized in DMSO (10nM). 2.5 Data analysis and statistics 4
Data are presented as mean value ± sem of raw data. Sample size was calculated using GPower 3.0.10, and for obtaining an effects size equal to 0.7; α value = 0.5 and power = 0.8, 7 mice for group have been used. The ED50 defines as the dose of a drug that produces 50% of the maximal effect and the SE were calculated. Isobolografic analysis was performed according to [27]. Firstly, the potency of the individual drugs was determined. The Ro 65-6570 ED50 was plotted on the abscissa and the morphine ED50 on the ordinate. A theoretical simple additive line for a combination of the two drugs was then generated by connecting the ED50 for Ro 65-6570 with that of morphine. For the combination morphine + Ro 65-6570, the ED50 of the mixture (ED50mix) and the SE of the mixture were calculated by linear regression of the dose-response curve and resolved into its component parts according to dose ratio. The theoretical additive value (ED50add) was obtained according to the formula ED50add = ED50(morphine)/( p1 + Rp2), where R is the potency ratio of morphine to Ro-656570, p1 is the proportion of morphine in the total dose and p2 is the proportion of Ro 65-6570 in the total dose. No significant difference between ED50mix and ED50add suggests a simple additive effect of the two drugs whereas ED50mix significantly less than ED50add indicates a superadditive effect of the two agents. Differences between groups were analyzed using 1-way analysis of variance (ANOVA) followed by Dunnett t test for multiple comparisons between groups For all tests the level of significance was set at p < 0.05.
3. Results 3.1 Formalin Figure 1 (panels A and B) displays the results obtained by testing different doses of formalin in the mouse OFF test. Animal treated with vehicle did not consistently show nociceptive behaviors over the time course of the experiment. Mice injected with formalin 1.5% showed the typical biphasic effect with nociceptive behavior appearing immediately after the inject and lasting for approximately 10 min (I° phase) followed by a delayed increase evident after 15 min and lasting for approximately 15 min (II° phase) (Figure 1 panel A). Mice injected with formalin 2.5% showed a similar phase I but a longer lasting phase II compared to animals treated with lower dose (Figure 1 panel A). As shown in Figure 1 panel B both doses of formalin elicited statistically significant effects. The higher dose of formalin was selected for further studies.
3.2 Morphine and ibuprofen As shown in panels C and D of Figure 1 morphine 10 mg/kg produced a large antinociceptive effect virtually abolishing nociceptive behaviors both in I° and II° phase of the OFF test. On the 5
contrary, ibuprofen at 100 mg kg-1did not significantly modify phase I while producing a statistically significant antinociceptive action in the II° phase of the OFF test.
3.3 Knockout studies Wild type mice (NOP(+/+)) injected with formalin 2.5% displayed nociceptive behaviors very similar to CD-1 mice both in terms of time course of I° and II° phase and of amount of effect. On the contrary NOP(-/-) mice displayed a robust pronociceptive phenotype in the OFF test showing statistically significant higher nociceptive behaviors both in I° and II° phase (Figure 2).
3.4 Ro 65-6570 and morphine: dose response curves The NOP selective agonist Ro 65-6570 in the range of doses 0.1 – 1 mg kg-1 elicited a dose dependent antinociceptive action (Figure 3, panel A) causing statistically significant effects at 0.3 and 1 mg/kg doses both in I° and II° phase (Figure 3 panel B) and showing ED50 values of 0.52 and 0.44 mg kg-1for I° and II° phase, respectively. The opioid receptor agonist morphine in the range of doses 0.1 – 10 mg kg-1elicited a dose dependent antinociceptive action in the OFF test (Figure 3 panel C). In particular statistically significant effects were measured in response to the doses 1 and 10 mg/kg both in I° and II° of the OFF test (Figure 3, panel D). The ED50 value of morphine was similar for I° and II° phase approximately corresponding to 0.5 mg kg-1.
3.5 Isobolographic analysis In order to investigate the type of interaction between morphine and Ro 65-6570 in the OFF test the dose response curve to the drug combination (mix) was assessed; in particular the following doses were tested 0.1 + 0.1 mg kg-1 (mix 0.2), 0.3 + 0.3 mg/kg (mix 0.6), and 1 + 1 mg kg-1 (mix 2). As shown in Figure 4 (panels A and B) the combination of morphine and Ro 65-6570 (mix) elicited a dose dependent antinociceptive action eliciting statistically significant effect at the dose of 0.6 and of 2 mg kg-1both in I° and II° phase. As shown in the isobolographic graph the ED50 mix is superimposable to the theoretical ED50 of drug additivity for the phase I (Figure 4 panel C). As far as phase II is concerned, the ED50 mix although located in the infraadditivity area, is not statistically significant compared to the theoretical ED50 of drug additives (Figure 4 panel D).
3.6 Cebranopadol Finally as shown in Figure 5 the mixed NOP/opioid agonist cebranopadol produced in the range of doses 0.01 – 0.1 mg kg-1a dose dependent antinociceptive action in the OFF test. At all doses tested 6
cebranopadol produced statistically significant antinociceptive effect both in I° and II° phase. Of note at the higher dose tested cebranopadol virtually abolished nociceptive behaviors similar to what reported for morphine 10 mg kg-1. As far as cebranopadol potency is concerned the ED50 calculated from the present results was 0.02 mg kg-1for both phases.
3.7 Rotarod In the rotarod test, morphine (10 mg kg-1) and ibuprofen (100 mg kg-1) did modified the mouse locomotor performance. Similar results were previously obtained under identical experimental conditions testing cebranopadol (0.1 mg kg-1) and Ro-65-6570 (1 mg kg-1) [17].
4. Discussion
This study investigated the role of NOP and opioid receptors in regulating pain transmission in the trigeminal territory using the OFF test in mice. NOP knockout studies suggested an antinociceptive action of endogenous N/OFQergic signaling. In line with this finding, activation of NOP receptors in response to the NOP non peptide agonist Ro 65-6570 produced robust a dose-dependent analgesic effect similar to that obtained by activating opioid receptors with morphine. Isobolographic analysis of morphine and Ro 65-6570 actions demonstrated that simultaneous activation of opioid and NOP receptors elicits additive effects. Finally cebranopadol, a new compound acting as mixed NOP/opioid receptor agonist, elicited robust and potent antinociceptive effects in the mouse OFF test. Collectively, these results suggest that NOP agonists and particularly mixed NOP/opioids receptor agonists may represent a class of innovative medicines for the control of trigeminal pain. In the first series of experiments we set up the experimental conditions for the OFF test. As expected, control animals showed a slight nociceptive behavioral, solely due to the injection trauma. On the contrary, formalin produced a biphasic response that was longer lasting with the higher dose tested. These results are in good agreement with literature data that confirm in the trigeminal area the biphasic response to formalin in rodents [28, 29]. Another feature of the nociceptive response to formalin is the different sensitivity of the two phases to opioid and NSAID drugs: I° phase that is essentially due to transient stimulation of nociceptors resulting in nociceptive pain is exclusively sensitive to opioids while II° phase that is due to the persistent inflammatory response to formalin is sensitive to both opioid and NSAID analgesics. The results obtained in this study with morphine and ibuprofen perfectly match the above mentioned features that have been previously confirmed in the trigeminal territory both in rats [28] and mice [29]. 7
In order to investigate the role of the endogenous N/OFQ-NOP signaling in controlling pain transmission in the OFF test the phenotype of NOP(-/-) mice was investigated. Receptor knockout animals displayed a robust pronociceptive phenotype in the OFF test suggesting an antinociceptive action of endogenous N/OFQ in the trigeminal territory. This is in line with a large body of evidence obtained in the formalin test with different approach including NOP receptor knockout mice [30, 31] and rats [32], ppN/OFQ knockout mice [30] and receptor antagonist studies performed in mice [31] and rats [33]. As far as the anatomical and functional substrate of the putative antinociceptive action of endogenous N/OFQ in the trigeminal territory is concerned, the following literature findings are worthy of being mentioned. Different complimentary techniques demonstrated the expression of the NOP receptor and/or ppN/OFQ both in the trigeminal ganglion [19, 22] and spinal caudalis nucleus [20, 21, 23]. Moreover powerful inhibitory effects were reported with N/OFQ in these structures with neurochemical and electrophysiological techniques [34-37]. Collectively these findings suggest that N/OFQ may exert an important inhibitory action on pain transmission in the trigeminal territory via pre and postsynaptic mechanisms both in the trigeminal ganglion and in the spinal caudalis nucleus. The compound Ro 65-6570 was used to investigate the potential of selective NOP agonists in the control of trigeminal pain. This molecule has been identified in Roche laboratories [38] and then widely used in vitro and in vivo (see [1] for a review). Of note recent findings demonstrate that the anxiolytic action of this molecule is fully prevented by a NOP selective antagonist and no longer evident in NOP(-/-) animals [39]. In the orofacial formalin test, Ro 65-6570 produces dosedependent antinociceptive effects with an ED50 of approx. 0.5 mg kg-1. This value is perfectly in line that reported by [40] in the mouse formalin test. Of note Ro 65-6570 does not modify animal locomotor performance in the rotarod test up to 1 mg/kg while higher doses (i.e. 3 and 10 mg kg-1) elicited a robust impairment of motor performance to the rotarod [17]. The implication of these findings are twofold. On one hand these results suggest that selective NOP agonists could be developed as novel analgesics for the treatment of painful syndromes of the trigeminal area. On the other hand, it should be underlined that the ratio of doses between the therapeutic action (antinociception) and side effects (sedative action) of Ro 65-6570 is only approximately 10. This ratio is rather low thus limiting the therapeutic potential of NOP receptor agonists as innovative analgesics. In order to investigate the type of interaction between NOP and opioid induced analgesic action in the trigeminal area the isobolographic analysis of Ro 65-6570 and morphine effect was performed. As expected based on previous findings [41], morphine caused a dose-dependent robust reduction of the nociceptive behavior of mice subjected to the OFF test. In the dose range examined morphine 8
does not affect the behavior of the animals on the rotarod thus excluding confounding effects. This result together with large preclinical and clinical evidence suggests that classical opioids are clearly able to control pain transmission in the trigeminal territory; however a wider use of such drugs in trigeminal painful syndromes is limited by the major side effects that are typically associated to opioid drugs. The isobolographic analysis of the Ro 65-6570/morphine interaction displayed no significant differences between experimental and theoretical ED50 thus suggesting that the interaction between the two drugs in controlling orofacial pain is simple additive. This is in line with results reported by [42] in the mouse hot plate test, where an additive interaction was demonstrated between morphine and the NOP agonist Ro 64-6198. However other findings suggest a supraadditive interaction between NOP and opioid receptors. In fact, Courteix and colleagues (2004) demonstrated that the co-administration of intrathecal morphine and N/OFQ evokes a synergistic analgesic effect in two models of neuropathic pain in rats [43]. In addition to rodent studies, the interaction of NOP and opioid receptors was also investigated in non-human primates. The systemic administration of buprenorphine and NOP selective agonists promoted a synergistic antinociceptive effects with no opioid side effects (respiratory depression, pruritus) [44]. Finally, Hu and colleagues (2010) showed that the spinal coadministration of inactive doses of UFP-112 (a peptide NOP selective agonist) and morphine produced a robust antinociceptive effect [45]. Altogether the present results along with those from literature suggest that the analgesic response to the simultaneous activation of NOP and opioid receptors may be additive or supraadditive depending on the route of administration, the pain model, and the animal species. Based on the above mentioned evidence, it can be reasonably assumed that mixed NOP/opioid agonists may evoke robust analgesic effects associate to a lower number and/or magnitude of side effects. Some examples of such compounds have been described in literature yielding encouraging results (see [1] for a recent review). In particular, cebranopadol is characterized by similar high affinity and agonist activity at NOP and opioid receptors and favorable pharmacokinetic characteristics [13, 17]. In the rat, this new compound evokes powerful antinociceptive effects in several models of acute and chronic pain being more potent than morphine particularly in models of chronic neuropathic pain [15]. Importantly compared to morphine cebranopadol displayed longer lasting action, a better safety profile, and reduced tolerance liability [15]. In addition a recent study demonstrated that cebranopadol is also effective after local administration in nociceptive and chronic neuropathic pain models [16]. In the range of doses tested, cebranopadol did not modify the animal performance in the rotarod test [17], but was able to elicit a robust and dose dependent antinociceptive effect in the mouse OFF test. The value of potency of cebranopadol obtained in the present study (ED50 0.02 mg kg-1) is in line with those previously obtained in other in vivo tests 9
both in mice [17] and rats [15]. Thus powerful and potent antinociceptive action of cebranopadol is clearly evident also in the trigeminal territory. The cebranopadol in vivo action derives from simultaneous activation of NOP and opioid receptors as demonstrated by selective antagonist studies both in rats [15] and mice [17]. Thus the analgesic action evoked by cebranopadol in the OFF test may result from the simultaneous activation of NOP and opioid receptors along different levels of trigeminal nociceptive pathways including peripheral nociceptors, the neurons of the Gasser ganglion, and those of the trigeminal caudalis nucleus. Altogether, the data obtained in this study for the trigeminal territory and the literature evidence show that cebranopadol is a very effective and promising compound in animal models of nociceptive, inflammatory, and neuropathic pain. In addition, the ratio of doses between therapeutic and unwanted cebranopadol effects is larger than for classical opioids. These findings candidates cebranopodal as a prototype of a new class of analgesic drugs. Phase II clinical studies confirm the efficacy and tolerability of cebranopadol in patients with osteoarthritis pain, low back pain, and diabetic neuropathy [18]. Moreover the results of the present work indicate that cebranopadol is worthy of being evaluated in clinical trials enrolling patients suffering of trigeminal painful syndromes.
Conflict of interest The authors declare no conflict of interest.
Acknowledgements This work was financially supported by funds from the University of Ferrara (FAR grant to G.C.) and the Italian Ministry of Research (PRIN 2015WX8Y5B grant to G.C.). C.R holds a MSD bourse of the Italian Society of Pharmacology.
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References
[1] Toll L, Bruchas MR, Calo G, Cox BM, Zaveri NT. Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems. Pharmacological reviews. 2016;68:419-57. [2] Lee H. Effects of co-administration of intrathecal nociceptin/orphanin FQ and opioid antagonists on formalin-induced pain in rats. Yonsei medical journal. 2013;54:763-71. [3] Schroder W, Lambert DG, Ko MC, Koch T. Functional plasticity of the N/OFQ-NOP receptor system determines analgesic properties of NOP receptor agonists. British journal of pharmacology. 2014;171:3777-800. [4] Zeilhofer HU, Calo G. Nociceptin/orphanin FQ and its receptor--potential targets for pain therapy? J Pharmacol Exp Ther. 2003;306:423-9. [5] Ko MC, Naughton NN, Traynor JR, Song MS, Woods JH, Rice KC, et al. Orphanin FQ inhibits capsaicin-induced thermal nociception in monkeys by activation of peripheral ORL1 receptors. British journal of pharmacology. 2002;135:943-50. [6] Ko MC, Woods JH, Fantegrossi WE, Galuska CM, Wichmann J, Prinssen EP. Behavioral effects of a synthetic agonist selective for nociceptin/orphanin FQ peptide receptors in monkeys. Neuropsychopharmacology. 2009;34:2088-96. [7] Ko MC, Wei H, Woods JH, Kennedy RT. Effects of Intrathecally Administered Nociceptin/Orphanin FQ in Monkeys: Behavioral and Mass Spectrometric Studies. J Pharmacol Exp Ther. 2006;318:1257-64. [8] Ding H, Hayashida K, Suto T, Sukhtankar DD, Kimura M, Mendenhall V, et al. Supraspinal actions of nociceptin/orphanin FQ, morphine and substance P in regulating pain and itch in non-human primates. British journal of pharmacology. 2015;172:3302-12. [9] Sobczak M, Cami-Kobeci G, Salaga M, Husbands SM, Fichna J. Novel mixed NOP/MOP agonist BU08070 alleviates pain and inhibits gastrointestinal motility in mouse models mimicking diarrhea-predominant irritable bowel syndrome symptoms. European journal of pharmacology. 2014;736:63-9. [10] Khroyan TV, Zaveri NT, Polgar WE, Orduna J, Olsen C, Jiang F, et al. SR 16435 [1-(1(bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a novel mixed nociceptin/orphanin FQ/mu-opioid receptor partial agonist: analgesic and rewarding properties in mice. J Pharmacol Exp Ther. 2007;320:934-43.
11
[11] Molinari S, Camarda V, Rizzi A, Marzola G, Salvadori S, Marzola E, et al. [Dmt1]N/OFQ(113)-NH2: a potent nociceptin/orphanin FQ and opioid receptor universal agonist. British journal of pharmacology. 2013;168:151-62. [12] Zaveri NT, Jiang F, Olsen C, Polgar WE, Toll L. Designing bifunctional NOP receptor-mu opioid receptor ligands from NOP receptor-selective scaffolds. Part I. Bioorganic & medicinal chemistry letters. 2013;23:3308-13. [13] Schunk S, Linz K, Hinze C, Frormann S, Oberbörsch S, Sundermann B, et al. Discovery of a Potent Analgesic NOP and Opioid Receptor Agonist: Cebranopadol. ACS Med Chem Lett. 2014;in press. [14] Bird MF, Cerlesi MC, Brown M, Malfacini D, Vezzi V, Molinari P, et al. Characterisation of the Novel Mixed Mu-NOP Peptide Ligand Dermorphin-N/OFQ (DeNo). PloS one. 2016;11:e0156897. [15] Linz K, Christoph T, Tzschentke TM, Koch T, Schiene K, Gautrois M, et al. Cebranopadol: a novel potent analgesic nociceptin/orphanin FQ peptide and opioid receptor agonist. J Pharmacol Exp Ther. 2014;349:535-48. [16] Tzschentke TM, Linz K, Frosch S, Christoph T. Antihyperalgesic, Antiallodynic, and Antinociceptive Effects of Cebranopadol, a Novel Potent Nociceptin/Orphanin FQ and Opioid Receptor Agonist, after Peripheral and Central Administration in Rodent Models of Neuropathic Pain. Pain practice : the official journal of World Institute of Pain. 2017. [17] Rizzi A, Cerlesi MC, Ruzza C, Malfacini D, Ferrari F, Bianco S, et al. Pharmacological characterization of cebranopadol a novel analgesic acting as mixed nociceptin/orphanin FQ and opioid receptor agonist. Pharmacology research & perspectives. 2016;4:e00247. [18] Lambert DG, Bird MF, Rowbotham DJ. Cebranopadol: a first in-class example of a nociceptin/orphanin FQ receptor and opioid receptor agonist. British journal of anaesthesia. 2015;114:364-6. [19] Xie GX, Meuser T, Pietruck C, Sharma M, Palmer PP. Presence of opioid receptor-like (ORL1) receptor mRNA splice variants in peripheral sensory and sympathetic neuronal ganglia. Life Sci. 1999;64:2029-37. [20] Neal CR, Jr., Mansour A, Reinscheid R, Nothacker HP, Civelli O, Akil H, et al. Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding. J Comp Neurol. 1999;412:563-605.
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[21] Neal CR, Jr., Mansour A, Reinscheid R, Nothacker HP, Civelli O, Watson SJ, Jr. Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat. J Comp Neurol. 1999;406:503-47. [22] Hou M, Uddman R, Tajti J, Edvinsson L. Nociceptin immunoreactivity and receptor mRNA in the human trigeminal ganglion. Brain Res. 2003;964:179-86. [23] Witta J, Palkovits M, Rosenberger J, Cox BM. Distribution of nociceptin/orphanin FQ in adult human brain. Brain Res. 2004;997:24-9. [24] Mork H, Hommel K, Uddman R, Edvinsson L, Jensen R. Does nociceptin play a role in pain disorders in man? Peptides. 2002;23:1581-7. [25] Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG. Animal research: reporting in vivo experiments: the ARRIVE guidelines. British journal of pharmacology. 2010;160:1577-9. [26] Fantinati A, Bianco S, Guerrini R, Salvadori S, Pacifico S, Cerlesi MC, et al. A diastereoselective synthesis of Cebranopadol, a novel analgesic showing NOP/mu mixed agonism. Scientific reports. 2017;7:2416. [27] Tallarida RJ. Statistical analysis of drug combinations for synergism. Pain. 1992;49:93-7. [28] Raboisson P, Dallel R. The orofacial formalin test. Neurosci Biobehav Rev. 2004;28:219-26. [29] Luccarini P, Childeric A, Gaydier AM, Voisin D, Dallel R. The orofacial formalin test in the mouse: a behavioral model for studying physiology and modulation of trigeminal nociception. J Pain. 2006;7:908-14. [30] Depner UB, Reinscheid RK, Takeshima H, Brune K, Zeilhofer HU. Normal sensitivity to acute pain, but increased inflammatory hyperalgesia in mice lacking the nociceptin precursor polypeptide or the nociceptin receptor. Eur J Neurosci. 2003;17:2381-7. [31] Rizzi A, Nazzaro C, Marzola GG, Zucchini S, Trapella C, Guerrini R, et al. Endogenous nociceptin/orphanin FQ signalling produces opposite spinal antinociceptive and supraspinal pronociceptive effects in the mouse formalin test: pharmacological and genetic evidences. Pain. 2006;124:100-8. [32] Rizzi A, Molinari S, Marti M, Marzola G, Calo G. Nociceptin/orphanin FQ receptor knockout rats: in vitro and in vivo studies. Neuropharmacology. 2011;60:572-9. [33] Yamamoto T, Sakashita Y, Nozaki-Taguchi N. Antagonism of ORLI receptor produces an algesic effect in the rat formalin test. Neuroreport. 2001;12:1323-7. [34] Capuano A, Curro D, Dello Russo C, Tringali G, Pozzoli G, Di Trapani G, et al. Nociceptin (113)NH2 inhibits stimulated calcitonin-gene-related-peptide release from primary cultures of rat trigeminal ganglia neurones. Cephalalgia. 2007;27:868-76.
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[35] Borgland SL, Connor M, Christie MJ. Nociceptin inhibits calcium channel currents in a subpopulation of small nociceptive trigeminal ganglion neurons in mouse. J Physiol. 2001;536:35-47. [36] Jennings EA. Postsynaptic K+ current induced by nociceptin in medullary dorsal horn neurons. Neuroreport. 2001;12:645-8. [37] Wang XM, Zhang KM, Long LO, Mokha SS. Orphanin FQ (nociceptin) modulates responses of trigeminal neurons evoked by excitatory amino acids and somatosensory stimuli, and blocks the substance P-induced facilitation of N-methyl-D-aspartate-evoked responses. Neuroscience. 1999;93:703-12. [38] Wichmann J, Adam G, Rover S, Cesura AM, Dautzenberg FM, Jenck F. 8-acenaphthen-1-yl-1phenyl-1,3,8-triaza-spiro[4.5]decan-4-one derivatives as orphanin FQ receptor agonists. Bioorganic & medicinal chemistry letters. 1999;9:2343-8. [39] Asth L, Ruzza C, Malfacini D, Medeiros I, Guerrini R, Zaveri NT, et al. Beta-arrestin 2 rather than G protein efficacy determines the anxiolytic-versus antidepressant-like effects of nociceptin/orphanin FQ receptor ligands. Neuropharmacology. 2016;105:434-42. [40] Byford AJ, Anderson A, Jones PS, Palin R, Houghton AK. The hypnotic, electroencephalographic, and antinociceptive properties of nonpeptide ORL1 receptor agonists after intravenous injection in rodents. Anesth Analg. 2007;104:174-9. [41] Bornhof M, Ihmsen H, Schwilden H, Yeomans DC, Tzabazis A. The orofacial formalin test in mice revisited--effects of formalin concentration, age, morphine and analysis method. J Pain. 2011;12:633-9. [42] Reiss D, Wichmann J, Tekeshima H, Kieffer BL, Ouagazzal AM. Effects of nociceptin/orphanin FQ receptor (NOP) agonist, Ro64-6198, on reactivity to acute pain in mice: comparison to morphine. European journal of pharmacology. 2008;579:141-8. [43] Courteix C, Coudore-Civiale MA, Privat AM, Pelissier T, Eschalier A, Fialip J. 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. 2004;110:236-45. [44] Cremeans CM, Gruley E, Kyle DJ, Ko MC. Roles of mu-opioid receptors and nociceptin/orphanin FQ peptide receptors in buprenorphine-induced physiological responses in primates. J Pharmacol Exp Ther. 2012;343:72-81. [45] Hu E, Calo G, Guerrini R, Ko M. Long lasting antinociceptive spinal effects in primates of the novel nociceptin/orphanin FQ receptor agonist UFP-112. Pain. 2010;148:107-13.
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S0196-9781(17)30222-X http://dx.doi.org/doi:10.1016/j.peptides.2017.07.002 PEP 69792
To appear in:
Peptides
Received date: Revised date: Accepted date:
10-5-2017 30-6-2017 3-7-2017
Please cite this article as: Rizzi A, Ruzza C, Bianco S, Trapella C, Calo’ G.Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test.Peptides http://dx.doi.org/10.1016/j.peptides.2017.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antinociceptive action of NOP and opioid receptor agonists in the mouse orofacial formalin test Rizzi A1, Ruzza C1, Bianco S2, Trapella C2, and Calo’ G1 1
Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara, Ferrara, Italy. 2 Department of Chemical and Pharmaceutical Sciences and LTTA, University of Ferrara, Ferrara, Italy. Corresponding author:
Girolamo Calo’, MD PhD Department of Medical Sciences, Section of Pharmacology, University of Ferrara, Via Fossato di Mortara 19, 44121 Ferrara, Italy [email protected] Highlights:
Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions, including pain.
NOP(-/-) mice displayed a pronociceptive phenotype in the orofacial formalin test (OFF)
In the OFF the NOP agonist Ro 65-6570 evoked antinociceptive effects
The effects of Ro 65-6570 and morphine were additive
The mixed NOP/opioid receptor agonist cebranopadol evoked potent antinociceptive effects
Abstract Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions, including pain transmission via selective activation of a specific receptor named NOP. The aim of this study was the investigation of the antinociceptive properties of NOP agonists and their interaction with opioids in the trigeminal territory. The orofacial formalin (OFF) test in mice was used to investigate the antinociceptive potential associated to the activation of NOP and opioid receptors. Mice subjected to OFF test displayed the typical biphasic nociceptive response and sensitivity to opioid and NSAID drugs. Mice knockout for the NOP gene displayed a robust pronociceptive phenotype. 1
The NOP selective agonist Ro 65-6570 (0.1 – 1 mg kg-1) and morphine (0.1 – 10 mg kg-1) elicited dose dependent antinociceptive effects in the OFF with the alkaloid showing larger effects; the isobologram analysis of their actions demonstrated an additive type of interaction. The mixed NOP/opioid receptor agonist cebranopadol elicited potent (0.01 – 0.1 mg kg-1) and robust antinociceptive effects. In the investigated dose range, all drugs did not modify the motor performance of the mice in the rotarod test. Collectively the results of this study demonstrated that selective NOP agonists and particularly mixed NOP/opioid agonists are worthy of development as innovative drugs to treat painful conditions of the trigeminal territory.
Key Words Orofacial formalin test; cebranopadol; mixed NOP/opioid agonists; isobolographic analysis
1. Introduction The nociceptin/orphanin FQ (N/OFQ) - NOP receptor system shares with classical opioid systems high structural and transductional similarities and, among others, the ability to modulate pain responses [1]. In rodents, N/OFQ and selective NOP receptor agonists elicit antinociceptive effects at peripheral and spinal levels but also pronociceptive effects at supraspinal sites [2-4]. Moreover, when injected systemically NOP agonists are far more effective on inflammatory or neuropathic pain than on nociceptive pain [3]. However, in nonhuman primates powerful antinociception is observed either after local [5], systemic [6], spinal [7], and intracisternal [8] administration. This suggests that systemic NOP ligands in primates may reduce pain by acting synergistically at different levels, whereas in rodents there might be opposite effects in brain and spinal cord, which result in a smaller or even absent antinociceptive effect [1, 3]. Emerging evidence suggests that mixed NOP/opioid receptor agonists may have innovative analgesic potential. In fact, a synergistic enhancement of antinociception achieved by the concurrent activation of NOP and opioid (particulary mu) receptors was demonstrated with systemically or intrathecally delivered agonists (reviewed in [1, 3]). Novel molecules acting as mixed NOP/opioid receptor agonists [9-14] are extremely useful tools for exploring the potential benefit of NOP and opioid receptor co-activation in the analgesia field. One of such ligands, cebranopadol, demonstrated high affinity and efficacy at NOP and opioid receptors and displayed robust antinociceptive properties in different rodent models of pain [13, 15-17]. Importantly the analgesic potential of cebranopadol has been confirmed in phase II clinical trials [18].
2
Evidence coming from rodent [19-21] and human [22-24] studies demonstrated that NOP receptors as well as N/OFQ are expressed in trigeminal structures. Thus, the aim of the present study was the investigation of the antinociceptive action of NOP and opioid receptor selective and non selective agonists in the trigeminal territory. Hence we first set up the experimental conditions for the mouse orofacial formalin (OFF) assay by testing different doses of formalin and the effects of morphine and ibuprofen. Then we investigated in this assay the phenotype of mice knockout for the NOP receptor gene (NOP-/-) was well as the dose response curves and isobolographic analysis of morphine and Ro 65-6570 selected as standard opioid and NOP agonists, respectively. Finally we evaluated the action of cebranopadol in the mouse OFF test.
2. Methods 2.1 Animal welfare and ethical statement All procedures were performed in accordance with the European Communities Council Directive 86/6609/EEC. All animal care and experimental procedures conformed to the standards of the European Communities Council directives (2010/63/EU) and national regulations (D.L. 26/2014). Studies involving animals are reported in accordance with the ARRIVE guidelines [25]. The present study was approved by the Ethical Committee for the Use of Laboratory Animals (CEASA) of the University of Ferrara and by the Italian Ministry of Health (authorization number 316/2013-B). Male CD-1 mice (25-35 g, total number 154) were from Envigo, Natisone Udine, Italy. They were housed in plastic cages (425x266x155 mm, Techniplast, Buguggiate, Italy) with soft bedding with free access to food (standard diet, Mucedola, Settimo Milanese, Italy) and water and were maintained in climate (23 ± 1°C) and light-controlled (12/12-h dark/light cycle with light on at 7:00 am) for at least 1 week before the experiments. Each cage was provided with a mouse red house (Tecniplast, Buguggiate, Italy) and nesting materials. Test sessions took place during the light phase between 9:00 am and 3:00 pm in a quiet room maintained at 23-24°C. The test box had the dimensions of 25 × 25 × 40 cm with 3 mirrored sides. Each animal was first placed in this box for a 30-min habituation period to minimize stress. The mice did not have access to food or water during the test. Each mouse was used only once and was killed at the end of the experiment by the administration of a lethal dose of CO2. Animals were assigned randomly to treatment groups. Different treatment doses and vehicle (saline or 1% DMSO) were tested in a randomized fashion and the operators performing the behavioral tests were blinded with respect to the treatments. 2.2 Orofacial Formalin test 3
Mice received a 20 µl subcutaneous injection of diluted formalin or saline into the right upper lip, just lateral to the nose. Solutions were prepared from commercially available stock formalin further diluted in isotonic saline to 1.5%, and 2.5%. Stock formalin is an aqueous solution of 37% formaldehyde. Formalin was injected subcutaneously through a 30-gauge needle into the center of the right vibrissa pad as quickly as possible, with only minimal animal restraint. Following injection the animals were immediately placed back in the test box for a 45-min observation period. The recording time was divided into 15 blocks of 3 min each, and a nociceptive score was determined for each block by measuring the number of seconds that the animals spent grooming the injected area with the ipsilateral fore- or hindpaw. Movements of the ipsilateral forepaw were accompanied by movements of the contralateral forepaw. A videocamera was used to record the grooming response. Time (s) spent by the animal showing all these pain-related behaviours was cumulatively measured and expressed as seconds (s) of nociceptive behaviour/min. The cumulative response times during 0–9 min and during 10–45 min were regarded as the first-phase (I° phase) and secondphase (II° phase) responses, respectively. Analysis of the behavior was made by an investigator who was blinded to the animal’s group assignment. 2.3 Rotarod test To investigate potential effects on motor coordination we performed a rotarod test using a constant speed device (Ugo Basile, Varese, Italy). Mice were trained at 15 r.p.m for 120 sec 1 day before the experiment. The next day, rotarod latencies were measured 30 min after systemic administration of the compounds. Motor performance was calculated as time (sec) spent on rod measured 30 min after drug injection. A cut-off time of 120 sec was chosen.
2.4 Materials Based on the results obtained in the first series of experiments, the 2.5% formalin concentration was selected as standard noxious stimulus to evaluate the effects of systemic ibuprofen, morphine, Ro 65-6570 and cebranopadol on the rubbing response. Drugs were administered intravenously (i.v.) 100 µl/mouse into the vein caudal tail using a 30-gauge needle attached to a sterile syringe. Morphine chlorohydrate was bought from SALARAS (Como, Italy, authorization number SP/095 del 04/04/2011); ibuprofen was purchased from Sigma Chemical Co (St Louis, MO) and were dissolved in saline (0.9% NaCl solution). Ro 65-6570 and cebranopadol [26] were synthesized in house and solubilized in DMSO (10nM). 2.5 Data analysis and statistics 4
Data are presented as mean value ± sem of raw data. Sample size was calculated using GPower 3.0.10, and for obtaining an effects size equal to 0.7; α value = 0.5 and power = 0.8, 7 mice for group have been used. The ED50 defines as the dose of a drug that produces 50% of the maximal effect and the SE were calculated. Isobolografic analysis was performed according to [27]. Firstly, the potency of the individual drugs was determined. The Ro 65-6570 ED50 was plotted on the abscissa and the morphine ED50 on the ordinate. A theoretical simple additive line for a combination of the two drugs was then generated by connecting the ED50 for Ro 65-6570 with that of morphine. For the combination morphine + Ro 65-6570, the ED50 of the mixture (ED50mix) and the SE of the mixture were calculated by linear regression of the dose-response curve and resolved into its component parts according to dose ratio. The theoretical additive value (ED50add) was obtained according to the formula ED50add = ED50(morphine)/( p1 + Rp2), where R is the potency ratio of morphine to Ro-656570, p1 is the proportion of morphine in the total dose and p2 is the proportion of Ro 65-6570 in the total dose. No significant difference between ED50mix and ED50add suggests a simple additive effect of the two drugs whereas ED50mix significantly less than ED50add indicates a superadditive effect of the two agents. Differences between groups were analyzed using 1-way analysis of variance (ANOVA) followed by Dunnett t test for multiple comparisons between groups For all tests the level of significance was set at p < 0.05.
3. Results 3.1 Formalin Figure 1 (panels A and B) displays the results obtained by testing different doses of formalin in the mouse OFF test. Animal treated with vehicle did not consistently show nociceptive behaviors over the time course of the experiment. Mice injected with formalin 1.5% showed the typical biphasic effect with nociceptive behavior appearing immediately after the inject and lasting for approximately 10 min (I° phase) followed by a delayed increase evident after 15 min and lasting for approximately 15 min (II° phase) (Figure 1 panel A). Mice injected with formalin 2.5% showed a similar phase I but a longer lasting phase II compared to animals treated with lower dose (Figure 1 panel A). As shown in Figure 1 panel B both doses of formalin elicited statistically significant effects. The higher dose of formalin was selected for further studies.
3.2 Morphine and ibuprofen As shown in panels C and D of Figure 1 morphine 10 mg/kg produced a large antinociceptive effect virtually abolishing nociceptive behaviors both in I° and II° phase of the OFF test. On the 5
contrary, ibuprofen at 100 mg kg-1did not significantly modify phase I while producing a statistically significant antinociceptive action in the II° phase of the OFF test.
3.3 Knockout studies Wild type mice (NOP(+/+)) injected with formalin 2.5% displayed nociceptive behaviors very similar to CD-1 mice both in terms of time course of I° and II° phase and of amount of effect. On the contrary NOP(-/-) mice displayed a robust pronociceptive phenotype in the OFF test showing statistically significant higher nociceptive behaviors both in I° and II° phase (Figure 2).
3.4 Ro 65-6570 and morphine: dose response curves The NOP selective agonist Ro 65-6570 in the range of doses 0.1 – 1 mg kg-1 elicited a dose dependent antinociceptive action (Figure 3, panel A) causing statistically significant effects at 0.3 and 1 mg/kg doses both in I° and II° phase (Figure 3 panel B) and showing ED50 values of 0.52 and 0.44 mg kg-1for I° and II° phase, respectively. The opioid receptor agonist morphine in the range of doses 0.1 – 10 mg kg-1elicited a dose dependent antinociceptive action in the OFF test (Figure 3 panel C). In particular statistically significant effects were measured in response to the doses 1 and 10 mg/kg both in I° and II° of the OFF test (Figure 3, panel D). The ED50 value of morphine was similar for I° and II° phase approximately corresponding to 0.5 mg kg-1.
3.5 Isobolographic analysis In order to investigate the type of interaction between morphine and Ro 65-6570 in the OFF test the dose response curve to the drug combination (mix) was assessed; in particular the following doses were tested 0.1 + 0.1 mg kg-1 (mix 0.2), 0.3 + 0.3 mg/kg (mix 0.6), and 1 + 1 mg kg-1 (mix 2). As shown in Figure 4 (panels A and B) the combination of morphine and Ro 65-6570 (mix) elicited a dose dependent antinociceptive action eliciting statistically significant effect at the dose of 0.6 and of 2 mg kg-1both in I° and II° phase. As shown in the isobolographic graph the ED50 mix is superimposable to the theoretical ED50 of drug additivity for the phase I (Figure 4 panel C). As far as phase II is concerned, the ED50 mix although located in the infraadditivity area, is not statistically significant compared to the theoretical ED50 of drug additives (Figure 4 panel D).
3.6 Cebranopadol Finally as shown in Figure 5 the mixed NOP/opioid agonist cebranopadol produced in the range of doses 0.01 – 0.1 mg kg-1a dose dependent antinociceptive action in the OFF test. At all doses tested 6
cebranopadol produced statistically significant antinociceptive effect both in I° and II° phase. Of note at the higher dose tested cebranopadol virtually abolished nociceptive behaviors similar to what reported for morphine 10 mg kg-1. As far as cebranopadol potency is concerned the ED50 calculated from the present results was 0.02 mg kg-1for both phases.
3.7 Rotarod In the rotarod test, morphine (10 mg kg-1) and ibuprofen (100 mg kg-1) did modified the mouse locomotor performance. Similar results were previously obtained under identical experimental conditions testing cebranopadol (0.1 mg kg-1) and Ro-65-6570 (1 mg kg-1) [17].
4. Discussion
This study investigated the role of NOP and opioid receptors in regulating pain transmission in the trigeminal territory using the OFF test in mice. NOP knockout studies suggested an antinociceptive action of endogenous N/OFQergic signaling. In line with this finding, activation of NOP receptors in response to the NOP non peptide agonist Ro 65-6570 produced robust a dose-dependent analgesic effect similar to that obtained by activating opioid receptors with morphine. Isobolographic analysis of morphine and Ro 65-6570 actions demonstrated that simultaneous activation of opioid and NOP receptors elicits additive effects. Finally cebranopadol, a new compound acting as mixed NOP/opioid receptor agonist, elicited robust and potent antinociceptive effects in the mouse OFF test. Collectively, these results suggest that NOP agonists and particularly mixed NOP/opioids receptor agonists may represent a class of innovative medicines for the control of trigeminal pain. In the first series of experiments we set up the experimental conditions for the OFF test. As expected, control animals showed a slight nociceptive behavioral, solely due to the injection trauma. On the contrary, formalin produced a biphasic response that was longer lasting with the higher dose tested. These results are in good agreement with literature data that confirm in the trigeminal area the biphasic response to formalin in rodents [28, 29]. Another feature of the nociceptive response to formalin is the different sensitivity of the two phases to opioid and NSAID drugs: I° phase that is essentially due to transient stimulation of nociceptors resulting in nociceptive pain is exclusively sensitive to opioids while II° phase that is due to the persistent inflammatory response to formalin is sensitive to both opioid and NSAID analgesics. The results obtained in this study with morphine and ibuprofen perfectly match the above mentioned features that have been previously confirmed in the trigeminal territory both in rats [28] and mice [29]. 7
In order to investigate the role of the endogenous N/OFQ-NOP signaling in controlling pain transmission in the OFF test the phenotype of NOP(-/-) mice was investigated. Receptor knockout animals displayed a robust pronociceptive phenotype in the OFF test suggesting an antinociceptive action of endogenous N/OFQ in the trigeminal territory. This is in line with a large body of evidence obtained in the formalin test with different approach including NOP receptor knockout mice [30, 31] and rats [32], ppN/OFQ knockout mice [30] and receptor antagonist studies performed in mice [31] and rats [33]. As far as the anatomical and functional substrate of the putative antinociceptive action of endogenous N/OFQ in the trigeminal territory is concerned, the following literature findings are worthy of being mentioned. Different complimentary techniques demonstrated the expression of the NOP receptor and/or ppN/OFQ both in the trigeminal ganglion [19, 22] and spinal caudalis nucleus [20, 21, 23]. Moreover powerful inhibitory effects were reported with N/OFQ in these structures with neurochemical and electrophysiological techniques [34-37]. Collectively these findings suggest that N/OFQ may exert an important inhibitory action on pain transmission in the trigeminal territory via pre and postsynaptic mechanisms both in the trigeminal ganglion and in the spinal caudalis nucleus. The compound Ro 65-6570 was used to investigate the potential of selective NOP agonists in the control of trigeminal pain. This molecule has been identified in Roche laboratories [38] and then widely used in vitro and in vivo (see [1] for a review). Of note recent findings demonstrate that the anxiolytic action of this molecule is fully prevented by a NOP selective antagonist and no longer evident in NOP(-/-) animals [39]. In the orofacial formalin test, Ro 65-6570 produces dosedependent antinociceptive effects with an ED50 of approx. 0.5 mg kg-1. This value is perfectly in line that reported by [40] in the mouse formalin test. Of note Ro 65-6570 does not modify animal locomotor performance in the rotarod test up to 1 mg/kg while higher doses (i.e. 3 and 10 mg kg-1) elicited a robust impairment of motor performance to the rotarod [17]. The implication of these findings are twofold. On one hand these results suggest that selective NOP agonists could be developed as novel analgesics for the treatment of painful syndromes of the trigeminal area. On the other hand, it should be underlined that the ratio of doses between the therapeutic action (antinociception) and side effects (sedative action) of Ro 65-6570 is only approximately 10. This ratio is rather low thus limiting the therapeutic potential of NOP receptor agonists as innovative analgesics. In order to investigate the type of interaction between NOP and opioid induced analgesic action in the trigeminal area the isobolographic analysis of Ro 65-6570 and morphine effect was performed. As expected based on previous findings [41], morphine caused a dose-dependent robust reduction of the nociceptive behavior of mice subjected to the OFF test. In the dose range examined morphine 8
does not affect the behavior of the animals on the rotarod thus excluding confounding effects. This result together with large preclinical and clinical evidence suggests that classical opioids are clearly able to control pain transmission in the trigeminal territory; however a wider use of such drugs in trigeminal painful syndromes is limited by the major side effects that are typically associated to opioid drugs. The isobolographic analysis of the Ro 65-6570/morphine interaction displayed no significant differences between experimental and theoretical ED50 thus suggesting that the interaction between the two drugs in controlling orofacial pain is simple additive. This is in line with results reported by [42] in the mouse hot plate test, where an additive interaction was demonstrated between morphine and the NOP agonist Ro 64-6198. However other findings suggest a supraadditive interaction between NOP and opioid receptors. In fact, Courteix and colleagues (2004) demonstrated that the co-administration of intrathecal morphine and N/OFQ evokes a synergistic analgesic effect in two models of neuropathic pain in rats [43]. In addition to rodent studies, the interaction of NOP and opioid receptors was also investigated in non-human primates. The systemic administration of buprenorphine and NOP selective agonists promoted a synergistic antinociceptive effects with no opioid side effects (respiratory depression, pruritus) [44]. Finally, Hu and colleagues (2010) showed that the spinal coadministration of inactive doses of UFP-112 (a peptide NOP selective agonist) and morphine produced a robust antinociceptive effect [45]. Altogether the present results along with those from literature suggest that the analgesic response to the simultaneous activation of NOP and opioid receptors may be additive or supraadditive depending on the route of administration, the pain model, and the animal species. Based on the above mentioned evidence, it can be reasonably assumed that mixed NOP/opioid agonists may evoke robust analgesic effects associate to a lower number and/or magnitude of side effects. Some examples of such compounds have been described in literature yielding encouraging results (see [1] for a recent review). In particular, cebranopadol is characterized by similar high affinity and agonist activity at NOP and opioid receptors and favorable pharmacokinetic characteristics [13, 17]. In the rat, this new compound evokes powerful antinociceptive effects in several models of acute and chronic pain being more potent than morphine particularly in models of chronic neuropathic pain [15]. Importantly compared to morphine cebranopadol displayed longer lasting action, a better safety profile, and reduced tolerance liability [15]. In addition a recent study demonstrated that cebranopadol is also effective after local administration in nociceptive and chronic neuropathic pain models [16]. In the range of doses tested, cebranopadol did not modify the animal performance in the rotarod test [17], but was able to elicit a robust and dose dependent antinociceptive effect in the mouse OFF test. The value of potency of cebranopadol obtained in the present study (ED50 0.02 mg kg-1) is in line with those previously obtained in other in vivo tests 9
both in mice [17] and rats [15]. Thus powerful and potent antinociceptive action of cebranopadol is clearly evident also in the trigeminal territory. The cebranopadol in vivo action derives from simultaneous activation of NOP and opioid receptors as demonstrated by selective antagonist studies both in rats [15] and mice [17]. Thus the analgesic action evoked by cebranopadol in the OFF test may result from the simultaneous activation of NOP and opioid receptors along different levels of trigeminal nociceptive pathways including peripheral nociceptors, the neurons of the Gasser ganglion, and those of the trigeminal caudalis nucleus. Altogether, the data obtained in this study for the trigeminal territory and the literature evidence show that cebranopadol is a very effective and promising compound in animal models of nociceptive, inflammatory, and neuropathic pain. In addition, the ratio of doses between therapeutic and unwanted cebranopadol effects is larger than for classical opioids. These findings candidates cebranopodal as a prototype of a new class of analgesic drugs. Phase II clinical studies confirm the efficacy and tolerability of cebranopadol in patients with osteoarthritis pain, low back pain, and diabetic neuropathy [18]. Moreover the results of the present work indicate that cebranopadol is worthy of being evaluated in clinical trials enrolling patients suffering of trigeminal painful syndromes.
Conflict of interest The authors declare no conflict of interest.
Acknowledgements This work was financially supported by funds from the University of Ferrara (FAR grant to G.C.) and the Italian Ministry of Research (PRIN 2015WX8Y5B grant to G.C.). C.R holds a MSD bourse of the Italian Society of Pharmacology.
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References
[1] Toll L, Bruchas MR, Calo G, Cox BM, Zaveri NT. Nociceptin/Orphanin FQ Receptor Structure, Signaling, Ligands, Functions, and Interactions with Opioid Systems. Pharmacological reviews. 2016;68:419-57. [2] Lee H. Effects of co-administration of intrathecal nociceptin/orphanin FQ and opioid antagonists on formalin-induced pain in rats. Yonsei medical journal. 2013;54:763-71. [3] Schroder W, Lambert DG, Ko MC, Koch T. Functional plasticity of the N/OFQ-NOP receptor system determines analgesic properties of NOP receptor agonists. British journal of pharmacology. 2014;171:3777-800. [4] Zeilhofer HU, Calo G. Nociceptin/orphanin FQ and its receptor--potential targets for pain therapy? J Pharmacol Exp Ther. 2003;306:423-9. [5] Ko MC, Naughton NN, Traynor JR, Song MS, Woods JH, Rice KC, et al. Orphanin FQ inhibits capsaicin-induced thermal nociception in monkeys by activation of peripheral ORL1 receptors. British journal of pharmacology. 2002;135:943-50. [6] Ko MC, Woods JH, Fantegrossi WE, Galuska CM, Wichmann J, Prinssen EP. Behavioral effects of a synthetic agonist selective for nociceptin/orphanin FQ peptide receptors in monkeys. Neuropsychopharmacology. 2009;34:2088-96. [7] Ko MC, Wei H, Woods JH, Kennedy RT. Effects of Intrathecally Administered Nociceptin/Orphanin FQ in Monkeys: Behavioral and Mass Spectrometric Studies. J Pharmacol Exp Ther. 2006;318:1257-64. [8] Ding H, Hayashida K, Suto T, Sukhtankar DD, Kimura M, Mendenhall V, et al. Supraspinal actions of nociceptin/orphanin FQ, morphine and substance P in regulating pain and itch in non-human primates. British journal of pharmacology. 2015;172:3302-12. [9] Sobczak M, Cami-Kobeci G, Salaga M, Husbands SM, Fichna J. Novel mixed NOP/MOP agonist BU08070 alleviates pain and inhibits gastrointestinal motility in mouse models mimicking diarrhea-predominant irritable bowel syndrome symptoms. European journal of pharmacology. 2014;736:63-9. [10] Khroyan TV, Zaveri NT, Polgar WE, Orduna J, Olsen C, Jiang F, et al. SR 16435 [1-(1(bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a novel mixed nociceptin/orphanin FQ/mu-opioid receptor partial agonist: analgesic and rewarding properties in mice. J Pharmacol Exp Ther. 2007;320:934-43.
11
[11] Molinari S, Camarda V, Rizzi A, Marzola G, Salvadori S, Marzola E, et al. [Dmt1]N/OFQ(113)-NH2: a potent nociceptin/orphanin FQ and opioid receptor universal agonist. British journal of pharmacology. 2013;168:151-62. [12] Zaveri NT, Jiang F, Olsen C, Polgar WE, Toll L. Designing bifunctional NOP receptor-mu opioid receptor ligands from NOP receptor-selective scaffolds. Part I. Bioorganic & medicinal chemistry letters. 2013;23:3308-13. [13] Schunk S, Linz K, Hinze C, Frormann S, Oberbörsch S, Sundermann B, et al. Discovery of a Potent Analgesic NOP and Opioid Receptor Agonist: Cebranopadol. ACS Med Chem Lett. 2014;in press. [14] Bird MF, Cerlesi MC, Brown M, Malfacini D, Vezzi V, Molinari P, et al. Characterisation of the Novel Mixed Mu-NOP Peptide Ligand Dermorphin-N/OFQ (DeNo). PloS one. 2016;11:e0156897. [15] Linz K, Christoph T, Tzschentke TM, Koch T, Schiene K, Gautrois M, et al. Cebranopadol: a novel potent analgesic nociceptin/orphanin FQ peptide and opioid receptor agonist. J Pharmacol Exp Ther. 2014;349:535-48. [16] Tzschentke TM, Linz K, Frosch S, Christoph T. Antihyperalgesic, Antiallodynic, and Antinociceptive Effects of Cebranopadol, a Novel Potent Nociceptin/Orphanin FQ and Opioid Receptor Agonist, after Peripheral and Central Administration in Rodent Models of Neuropathic Pain. Pain practice : the official journal of World Institute of Pain. 2017. [17] Rizzi A, Cerlesi MC, Ruzza C, Malfacini D, Ferrari F, Bianco S, et al. Pharmacological characterization of cebranopadol a novel analgesic acting as mixed nociceptin/orphanin FQ and opioid receptor agonist. Pharmacology research & perspectives. 2016;4:e00247. [18] Lambert DG, Bird MF, Rowbotham DJ. Cebranopadol: a first in-class example of a nociceptin/orphanin FQ receptor and opioid receptor agonist. British journal of anaesthesia. 2015;114:364-6. [19] Xie GX, Meuser T, Pietruck C, Sharma M, Palmer PP. Presence of opioid receptor-like (ORL1) receptor mRNA splice variants in peripheral sensory and sympathetic neuronal ganglia. Life Sci. 1999;64:2029-37. [20] Neal CR, Jr., Mansour A, Reinscheid R, Nothacker HP, Civelli O, Akil H, et al. Opioid receptor-like (ORL1) receptor distribution in the rat central nervous system: comparison of ORL1 receptor mRNA expression with (125)I-[(14)Tyr]-orphanin FQ binding. J Comp Neurol. 1999;412:563-605.
12
[21] Neal CR, Jr., Mansour A, Reinscheid R, Nothacker HP, Civelli O, Watson SJ, Jr. Localization of orphanin FQ (nociceptin) peptide and messenger RNA in the central nervous system of the rat. J Comp Neurol. 1999;406:503-47. [22] Hou M, Uddman R, Tajti J, Edvinsson L. Nociceptin immunoreactivity and receptor mRNA in the human trigeminal ganglion. Brain Res. 2003;964:179-86. [23] Witta J, Palkovits M, Rosenberger J, Cox BM. Distribution of nociceptin/orphanin FQ in adult human brain. Brain Res. 2004;997:24-9. [24] Mork H, Hommel K, Uddman R, Edvinsson L, Jensen R. Does nociceptin play a role in pain disorders in man? Peptides. 2002;23:1581-7. [25] Kilkenny C, Browne W, Cuthill IC, Emerson M, Altman DG. Animal research: reporting in vivo experiments: the ARRIVE guidelines. British journal of pharmacology. 2010;160:1577-9. [26] Fantinati A, Bianco S, Guerrini R, Salvadori S, Pacifico S, Cerlesi MC, et al. A diastereoselective synthesis of Cebranopadol, a novel analgesic showing NOP/mu mixed agonism. Scientific reports. 2017;7:2416. [27] Tallarida RJ. Statistical analysis of drug combinations for synergism. Pain. 1992;49:93-7. [28] Raboisson P, Dallel R. The orofacial formalin test. Neurosci Biobehav Rev. 2004;28:219-26. [29] Luccarini P, Childeric A, Gaydier AM, Voisin D, Dallel R. The orofacial formalin test in the mouse: a behavioral model for studying physiology and modulation of trigeminal nociception. J Pain. 2006;7:908-14. [30] Depner UB, Reinscheid RK, Takeshima H, Brune K, Zeilhofer HU. Normal sensitivity to acute pain, but increased inflammatory hyperalgesia in mice lacking the nociceptin precursor polypeptide or the nociceptin receptor. Eur J Neurosci. 2003;17:2381-7. [31] Rizzi A, Nazzaro C, Marzola GG, Zucchini S, Trapella C, Guerrini R, et al. Endogenous nociceptin/orphanin FQ signalling produces opposite spinal antinociceptive and supraspinal pronociceptive effects in the mouse formalin test: pharmacological and genetic evidences. Pain. 2006;124:100-8. [32] Rizzi A, Molinari S, Marti M, Marzola G, Calo G. Nociceptin/orphanin FQ receptor knockout rats: in vitro and in vivo studies. Neuropharmacology. 2011;60:572-9. [33] Yamamoto T, Sakashita Y, Nozaki-Taguchi N. Antagonism of ORLI receptor produces an algesic effect in the rat formalin test. Neuroreport. 2001;12:1323-7. [34] Capuano A, Curro D, Dello Russo C, Tringali G, Pozzoli G, Di Trapani G, et al. Nociceptin (113)NH2 inhibits stimulated calcitonin-gene-related-peptide release from primary cultures of rat trigeminal ganglia neurones. Cephalalgia. 2007;27:868-76.
13
[35] Borgland SL, Connor M, Christie MJ. Nociceptin inhibits calcium channel currents in a subpopulation of small nociceptive trigeminal ganglion neurons in mouse. J Physiol. 2001;536:35-47. [36] Jennings EA. Postsynaptic K+ current induced by nociceptin in medullary dorsal horn neurons. Neuroreport. 2001;12:645-8. [37] Wang XM, Zhang KM, Long LO, Mokha SS. Orphanin FQ (nociceptin) modulates responses of trigeminal neurons evoked by excitatory amino acids and somatosensory stimuli, and blocks the substance P-induced facilitation of N-methyl-D-aspartate-evoked responses. Neuroscience. 1999;93:703-12. [38] Wichmann J, Adam G, Rover S, Cesura AM, Dautzenberg FM, Jenck F. 8-acenaphthen-1-yl-1phenyl-1,3,8-triaza-spiro[4.5]decan-4-one derivatives as orphanin FQ receptor agonists. Bioorganic & medicinal chemistry letters. 1999;9:2343-8. [39] Asth L, Ruzza C, Malfacini D, Medeiros I, Guerrini R, Zaveri NT, et al. Beta-arrestin 2 rather than G protein efficacy determines the anxiolytic-versus antidepressant-like effects of nociceptin/orphanin FQ receptor ligands. Neuropharmacology. 2016;105:434-42. [40] Byford AJ, Anderson A, Jones PS, Palin R, Houghton AK. The hypnotic, electroencephalographic, and antinociceptive properties of nonpeptide ORL1 receptor agonists after intravenous injection in rodents. Anesth Analg. 2007;104:174-9. [41] Bornhof M, Ihmsen H, Schwilden H, Yeomans DC, Tzabazis A. The orofacial formalin test in mice revisited--effects of formalin concentration, age, morphine and analysis method. J Pain. 2011;12:633-9. [42] Reiss D, Wichmann J, Tekeshima H, Kieffer BL, Ouagazzal AM. Effects of nociceptin/orphanin FQ receptor (NOP) agonist, Ro64-6198, on reactivity to acute pain in mice: comparison to morphine. European journal of pharmacology. 2008;579:141-8. [43] Courteix C, Coudore-Civiale MA, Privat AM, Pelissier T, Eschalier A, Fialip J. 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. 2004;110:236-45. [44] Cremeans CM, Gruley E, Kyle DJ, Ko MC. Roles of mu-opioid receptors and nociceptin/orphanin FQ peptide receptors in buprenorphine-induced physiological responses in primates. J Pharmacol Exp Ther. 2012;343:72-81. [45] Hu E, Calo G, Guerrini R, Ko M. Long lasting antinociceptive spinal effects in primates of the novel nociceptin/orphanin FQ receptor agonist UFP-112. Pain. 2010;148:107-13.
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Figure 1. Panel A: Time course of the face-rubbing activity observed after subcutaneous injection of saline (control) or different concentrations of formalin into the upper lip. Panel B: formalin-induced pain behaviour during the I° (F(2,18) = 25.75) and II° phases (F(2,18) = 56.85). Panel C: Time course of the face-rubbing activity observed after subcutaneous injection of saline (control) or morphine 10 mg kg-1 or ibuprofen 100 mg kg-1. Panel D: formalin-induced pain behaviour during the I° (F(2,18) = 11.84) and II° phases (F(2,18) = 290.6). Each points represents the mean (7 animals per group) and the vertical bars indicate the sem. * p < 0.05 vs control according to ANOVA followed by the Dunnett’s test.
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Figure 2. Panel A: Time course of the face-rubbing activity observed after subcutaneous injection of formalin 2.5% in NOP(+/+) and NOP(-/-) mice into the upper lip. Panel B: formalin-induced pain behaviour during the I° (T = 3.97 df = 12) and II° phases (T = 3.90 df = 12). Each points represents the mean (7 animals per group) and the vertical bars indicate the sem. * p < 0.05 vs NOP(+/+) mice according to Student t test for unpaired data.
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Figure 3. Panel A: Time course of formalin-induced face-rubbing activity in mice treated vehicle (control) or with Ro 65-6570 (0.1 - 1 mg kg-1, panel A) or morphine (0.01 – 10 mg kg-1, panel C). Panel B: formalin-induced pain behaviour during the I° (F(3,24) = 14.28) and II° phases (F(3,24) = 7.02). Panel D: formalin-induced pain behaviour during the I° (F(4,30) = 12.47) and II° phases (F(4,30) = 15.82). Each points represents the mean (7 animals per group) and the vertical bars indicate the sem. * p < 0.05 vs vehicle according to ANOVA followed by the Dunnett’s test.
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Figure 4. Panel A: Time course of formalin-induced face-rubbing activity in mice treated with vehicle (control ) or a mix of morphine and Ro 65-6570 (0.1 + 0.1 mg kg-1 0.3 + 0.3 mg kg-1 1 + 1 mg kg-1). Panel B: formalin-induced pain behaviour during the I° (F(3,24) = 9.05) and II° phases (F(3,24) = 6.13). Each points represents the mean (7 animals per group) and the vertical bars indicate the sem. * p < 0.05 vs vehicle according to ANOVA followed by the Dunnett’s test. Isobologram for the effect of the combination of morphine and Ro 65-6570 on formalin-induced face-rubbing activity in mice during the I° phase (panel C) and II° phase (panel D) . The dashed line represents the theoretical additive interaction. The interception of the dashed lines on the ordinate and abscissa is the observed ED50 value for morphine and Ro 65-6570, respectively. The open symbol represents the ED50mix for the combination of morphine and Ro 65-6570, the ED50 add is represented by solid symbol. The standard errors for morphine and Ro 65-6570 are resolved into morphine (ordinate scale) and Ro 65-6570 (abscissa scale) components and shown by vertical and horizontal bars, respectively.
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II° phase
Figure 5. Panel A: Time course of formalin-induced face-rubbing activity in mice treated vehicle (control) or with cebranopadol (0.01 - 0.1 mg kg-1) Panel B: formalin-induced pain behaviour during the I° (F(3,24) = 25.878) and II° phases (F(3,24) = 19.59). Each points represents the mean (7 animals per group) and the vertical bars indicate the sem. * p < 0.05 vs vehicle according to ANOVA followed by the Dunnett’s test.
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