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Synergistic interaction between the agonism of cebranopadol at nociceptin/orphanin FQ and classical opioid receptors in the rat spinal nerve ligation model

Abstract Cebranopadol (trans‐6′‐fluoro‐4′,9′‐dihydro‐N,N‐dimethyl‐4‐phenyl‐spiro[cyclohexane‐1,1′(3′H)‐pyrano[3,4‐b]indol]‐4‐amine) is a novel analgesic nociceptin/orphanin FQ opioid peptide (NOP) and classical opioid receptor (MOP, DOP, and KOP) agonist with highly efficacious and potent activity in a broad range of rodent models of nociceptive, inflammatory, and neuropathic pain as well as limited opioid‐type side effects such as respiratory depression. This study was designed to explore contribution and interaction of NOP and classical opioid receptor agonist components to cebranopadol analgesia in the rat spinal nerve ligation (SNL) model. Assessing antihypersensitive activity in SNL rats intraperitoneal (IP) administration of cebranopadol resulted in ED 50 values of 3.3 and 3.58 μg/kg in two independent experiments. Pretreatment (IP) with J‐113397 (4.64 mg/kg) a selective antagonist for the NOP receptor or naloxone (1 mg/kg), naltrindole (10 mg/kg), or nor‐BNI (10 mg/kg), selective antagonists for MOP, DOP, and KOP receptors, yielded ED 50 values of 14.1, 16.9, 17.3, and 15 μg/kg, respectively. This 4‐5 fold rightward shift of the dose‐response curves suggested agonistic contribution of all four receptors to the analgesic activity of cebranopadol. Combined pretreatment with a mixture of the antagonists for the three classical opioid receptors resulted in an 18‐fold potency shift with an ED 50 of 65.5 μg/kg. The concept of dose equivalence was used to calculate the expected additive effects of the parent compound for NOP and opioid receptor contribution and to compare them with the observed effects, respectively. This analysis revealed a statistically significant difference between the expected additive and the observed effects suggesting intrinsic synergistic analgesic interaction of the NOP and the classical opioid receptor components of cebranopadol. Together with the observation of limited respiratory depression in rats and humans the synergistic interaction of NOP and classical opioid receptor components in analgesia described in the current study may contribute to the favorable therapeutic index of cebranopadol observed in clinical trials.

the synergistic interaction of NOP and classical opioid receptor components in analgesia described in the current study may contribute to the favorable therapeutic index of cebranopadol observed in clinical trials.

K E Y W O R D S
cebranopadol, nociceptin/orphanin FQ, rat, spinal nerve ligation, synergism

| INTRODUCTION
Cebranopadol is a first-in-class analgesic with agonistic activity at the nociceptin/orphanin FQ opioid peptide (NOP) receptor and the classical μ-opioid peptide (MOP), κ-opioid peptide (KOP), and δopioid peptide (DOP) receptors. 1,2 It has subnanomolar affinity for the human and rat NOP and MOP receptors and low nanomolar affinity for the KOP and DOP receptors. 2 After systemic administration, cebranopadol exerted highly efficacious analgesic effects in rodent models of nociceptive, inflammatory, bone cancer, and chronic mono-and polyneuropathic pain that were 2-3 orders of magnitude more potent than those of morphine. Recently, we demonstrated that equianalgesic doses of cebranopadol produced less respiratory depression than fentanyl because the NOP receptor agonistic component of cebranopadol exerted a protective role by intrinsically counteracting MOP receptor-mediated respiratory depression in rats. 3 This finding suggests a subadditive interaction of the NOP and opioid receptor components of action of cebranopadol when it comes to this prototypic MOP receptor related side effect.
On the other hand, activation of both NOP and MOP receptors contributed to antihypersensitive activity of cebranopadol in rat models of spinal nerve ligation (SNL)-induced mono-neuropathic pain 2 and complete Freund's adjuvant (CFA)-induced knee joint arthritis. 4 Interestingly, and unlike morphine, cebranopadol was about 10-fold more potent in rodent models of chronic neuropathic 2,5 or persistent pain 6 as compared to other more acute pain conditions. This increase in potency in neuropathic pain models might be a result of functional NOP receptor upregulation at peripheral, 7-9 spinal 8 , and supraspinal 10 levels combined with synergistic interaction of activation of NOP and the classical opioid receptors, although recent data also discuss alternative contribution of the endogenous NOP system and a potential role of spinal interneurons. 11 While agonistic activity at all four opioid receptors contribute to the in vitro profile of cebranopadol and NOP and MOP receptor-mediated analgesic efficacies have been proven in neuropathic 2 and inflammatory pain models 4 in rodents, neither DOP nor KOP contributions have been assessed in vivo. Concomitant activation of NOP and MOP receptors produced additive antinociception in acute pain models in rodents 12,13 and interacted synergistically to produce antihypersensitive and antinociceptive effects in rodent models of neuropathic pain 14 and non-human primate models of acute pain, 15 respectively (for review see 16 ).
The concept of dose equivalence was successfully used over the last years to analyze and describe the nature of pharmacological interaction both for combinations of independent drugs and drugs featuring inherent combination of two mechanisms of action. 17 This approach enables differentiation between subadditive, additive, and supra-additive interaction comparing experimental potency and efficacy with the theoretically additive interaction of two independent drugs or mechanisms. Numerous examples are published supporting the value of this concept in preclinical models of experimental pain. [18][19][20][21][22] The application of this concept to a compound like cebranopadol targeting all four opioid receptors crucially depends on carefully controlled experimental conditions. Recently, in the rat SNL model with mechanical readout, we analyzed the interaction of opioid receptor agonists and antagonists for efficacy and selectivity. 23  The aim of the present study was to further characterize the mode of action of cebranopadol in SNL rats by exploring the role of DOP and KOP receptors and to elucidate the way activation of NOP and classical opioid receptors interact to produce antihypersensitivity.

| Animals
Two hundred and twenty-eight male Sprague-Dawley rats were used (body weight 140-160 g; Janvier Labs, Le Genest Saint Isle, France).
Animals were housed under standard conditions (room temperature 20°C-24°C, 12 hour light−dark cycle, relative air humidity 35%-70%, 10-15 air changes per hour, air movement <0.2 m/sec) with food and water available ad libitum in the home cage. Animals were assigned randomly to treatment groups. Ten rats were used per group. Different doses and vehicles were tested in a randomized fashion. Animals were tested repeatedly with a washout period of at least 1 week between tests. Although the operators performing the behavioral tests were not formally "blinded" with respect to the treatment, they were not aware of the study hypothesis or the nature of differences between drugs.
Animal testing was performed in accordance with the recommendations and policies of the International Association for the Study of Pain 24 and the German Animal Welfare Law. All study protocols were approved by the local government authority for animal research, which are advised by an independent Ethics Committee.

| Experimental preparation
Under pentobarbital anesthesia (Narcoren ® 60 m/kg IP; Merial GmbH, Hallbergmoos, Germany), the L5/L6 spinal nerves were tightly ligated according to the method by Kim and Chung. 25 The left L5 and L6 spinal nerves were exposed by removing a small piece of the paravertebral muscle and a part of the left spinous process of the L5 lumbar vertebra. The L5 and L6 spinal nerves were then carefully isolated and tightly ligated with silk (NC-silk black, USP 5/0, metric 1, Braun Melsungen AG, Melsungen, Germany). After checking hemostasis, the muscle and the adjacent fascia were closed with sutures and the skin was closed with sutures. After surgery, animals were allowed to recover for 1 week.

| Antihypersensitive testing
Animals developed tactile hypersensitivity which was stable for at least 5 weeks. For the assessment of tactile hypersensitivity, rats were placed on a metal mesh covered with a plastic dome and were allowed to habituate until the exploratory behavior diminished.

| Data analysis
Data were analyzed by means of two-factor analysis of variance (ANOVA), with repeated measures. Significance of treatment, time, or treatment by time interaction effects was analyzed by means of Wilks' Lambda. In case of a significant treatment effect, pairwise comparisons were performed by post hoc analysis using the Bonferroni test. Results were considered statistically significant if P < 0.05.
ED 50 values and 95% confidence intervals (CIs) were determined at the time of the peak effect by linear regression analysis based on % MPE data.

| Analysis of interaction between NOP and opioid receptor agonistic components of cebranopadol
The concept of dose equivalence 17   For each dose of cebranopadol, the calculated expected (additive) effect was compared with the observed effect ( Table 2). The resulting data were analyzed by Student's t test for paired data (E additive vs E observed ).

| Drugs and chemicals
The following drugs were used: cebranopadol hemi-citrate

| Antagonism of antihypersensitivity by NOP and opioid receptor antagonists
The antagonist doses used in this study were previously demon-

| Synergistic interaction between NOP and opioid receptor agonistic components of cebranopadol
The two components of action (ie, NOP receptor agonism and classical opioid receptor agonism) are a feature of the parent compound.
In experimental settings, these two components can be viewed the same way as that one would deal with two different drugs. Thus, the concept of dose equivalence, which is also the basis of isobolographic analysis, could be used to analyze the nature of interaction (ie, additive, synergistic, subadditive) between the NOP receptor agonistic and the opioid receptor agonistic component of cebranopadol to produce antiallodynic efficacy. We based our analysis on a comparison of observed and expected (additive) effect scales of cebranopadol according to [17]. To this end, the dose-effect relations of the two individual components had to be obtained by   A comparison of the observed (experimental) and calculated (additive) effects is given in Table 2. Statistical testing was performed by means of a two-sided Student's t test for paired data according to the procedure described in [17].

| DISCUSSION
The novel centrally acting analgesic cebranopadol is a first-in-class potent NOP and opioid receptor agonist that displayed broad analgesic activity in preclinical models of acute, inflammatory, and chronic neuropathic pain and is currently under clinical development for the treatment of severe chronic nociceptive and neuropathic pain. 1,2 Previously, we showed that intravenous administration of cebranopadol exerted potent and fully efficacious antiallodynic activity that was dose-dependently inhibited by the NOP receptor antagonist F I G U R E 2 Dose-and time-dependent antiallodynic effect of intraperitoneal cebranopadol after IP pretreatment with a triple combination of opioid receptor antagonists (naloxone 1 mg/kg, naltrindole 10 mg/kg, and nor-BNI 10 mg/kg, (A)) and the corresponding vehicle control (B). Dose-response curves of cebranopadol after pretreatment with vehicle or antagonists 20 minutes after agonist administration (C). *P < 0.05 vs vehicle antagonists delineated the spinal cord as one site of (synergistic) interaction, whereas the pharmacogenomics study based on global NOP and opioid receptor knockout did not allow drawing any conclusion on the anatomical substrate(s) where the complex NOPopioid receptor interaction occurred. Likewise, we cannot ascribe the precise site(s) of synergistic interaction between cebranopadol's NOP and classical opioid receptor agonistic mechanisms of action as both cebranopadol and antagonists were administered systemically in the present study. Although cebranopadol was demonstrated to produce antihypersensitive efficacy after peripheral, spinal, and supraspinal administration in rodent models of chronic neuropathic pain, the site-specific relative contribution and way of interaction between NOP and classical opioid receptor agonistic MoA still remains elusive as no antagonism experiments were conducted in the context of that study. 28 In addition, also site-site interactions might contribute to produce NOP and opioid receptor synergism of cebranopadol as has been described for the MOR-NRI mediated intrinsic synergism of tapentadol. 19 Intrinsic synergism of a compound such as cebranopadol when it would be based on interaction of NOP and opioid agonistic efficacy at multiple sites relevant to pain processing requires equal distribution throughout the different compartments within the body. In fact, the pharmacokinetic profile of cebranopadol in rats suggests rapid absorption and extensive distribution 2 enabling equal NOP and opioid receptor activation at potential sites of synergism such as the spinal cord.
The complexity of local and site-site activation of NOP and classical opioid receptors might well lay the ground to the analgesic synergism detected in the current study. Furthermore, the nature of molecular receptor activation might contribute to the beneficial therapeutic index of cebranopadol. In fact, functional studies revealed a G protein biased signaling of cebranopadol at the NOP and at a reduced degree at the MOP receptor. 6 Reduced ß-arrestin recruitment and preferred G protein activation are discussed as contributor for reduction of opioid-type side effects such as respiratory depression and gastrointestinal dysfunction. 29 Respiratory depression is a clinical issue of pure MOP receptor agonists like morphine and fentanyl. 30 Thus, an obvious question based on the present finding is whether synergistic interaction between NOP and classical opioid receptor agonists is also reflected in an increase in opioid-type side effects. Notably, cebranopadol was largely devoid of a respiratory depressant effect in the clinic. 31 In a preclinical model in rats, the NOP receptor agonistic component of cebranopadol was demonstrated to counteract MOP receptormediated respiratory depression. 3 Impairment of motor coordination is another opioid-type side effect targeting the central nervous system in rodents. Similar to the situation in the respiratory system, cebranopadol does not show efficacy in the rotarod test at doses which exceed antinociceptive or antihypersensitive doses in rats 2 or mice. 6 This finding suggests lack of confounding motor effects in behavioral assays increasing the confidence in the current data set. More importantly, this data corroborates the finding on respiration, that is lack of synergism in opioidtype side effects as compared to synergistic interaction in analgesia.
The scope of the current study was to elucidate the interaction of NOP and classical opioid receptor agonism for cebranopadol. The in vitro binding profile shows predominant binding to NOP and MOP and weaker affinity to DOP and KOP, 2 which is also reflected in functional efficacies. 2,6 Hence, we first analyzed the functional contribution of all four receptors in vivo before we assessed the interaction of NOP and classical opioid receptors by the concept of dose equivalence. Interestingly, when using isolated antagonists the shift of the dose-response curves was similar in magnitude for all four receptors despite differential affinities and potencies in vitro.
The data suggest a complex interaction between the different opioid receptors which also is reflected in the outcome of genetic models in mice 27 and antagonism studies in rats. 23 Further dissection of this complex opioid receptor interaction might be possible in a similar experimental setup in vivo but was out of the scope of the current study and would require considerably higher numbers of animals contradicting the 3Rs principles of animal welfare.
Thus, NOP receptor agonism of cebranopadol both afforded intrinsic limitation of MOP receptor-mediated respiratory depression and motor impairment and contributed synergistically to opioid receptor-mediated antiallodynic efficacy. This two pronged beneficial effect of the NOP receptor agonistic component is therefore believed to contribute to the favorable therapeutic index of cebranopadol in the clinic. 32,33