Nociceptin Receptor-Related Agonists as Safe and Non-addictive Analgesics

Abstract

As clinical use of currently available opioid analgesics is often impeded by dose-limiting adverse effects, such as abuse liability and respiratory depression, new approaches have been pursued to develop safe, effective, and non-addictive pain medications. After the identification of the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor more than 25 years ago, NOP receptor-related agonists have emerged as a promising target for developing novel and effective opioids that modulate the analgesic and addictive properties of mu-opioid peptide (MOP) receptor agonists. In this review, we highlight the effects of the NOP receptor-related agonists compared with those of MOP receptor agonists in experimental rodent and more translational non-human primate (NHP) models and the development status of key NOP receptor-related agonists as potential safe and non-addictive analgesics. Several lines of evidence demonstrated that peptidic and non-peptidic NOP receptor agonists produce potent analgesic effects by intrathecal delivery in NHPs. Moreover, mixed NOP/MOP receptor partial agonists (e.g., BU08028, BU10038, and AT-121) display potent analgesic effects when administered intrathecally or systemically, without eliciting adverse effects, such as respiratory depression, itch behavior, and signs of abuse liability. More importantly, cebranopadol, a mixed NOP/opioid receptor agonist with full efficacy at NOP and MOP receptors, produces robust analgesic efficacy with reduced adverse effects, conferring promising outcomes in clinical studies. A balanced coactivation of NOP and MOP receptors is a strategy that warrants further exploration and refinement for the development of novel analgesics with a safer and effective profile.

Graphical Abstract

1. Introduction

Pain is an unpleasant sensation that alerts us of potential abnormalities in our organs, as well as to enable us to avoid tissue damage. However, as a symptom of numerous clinical disorders, intractable pain negatively affects the quality of life. It has created a huge burden to individuals and to society [1, 2]. Morphine, first isolated from opium more than 400 years ago, is among the most important naturally occurring compounds, and has impacted on a wealth of scientific knowledge and human lives [3]. Along with morphine, other drugs that also act on mu opioid peptide (MOP) receptors are the most effective and widely used analgesics in clinics to manage various pain conditions, such as postoperative pain, chronic back pain, and cancer pain [4, 5]. However, the disturbing adverse effects associated with these drugs, abuse liability and physical dependence in particular, have caused a tremendous increase in opioids abuse and overdose, leading to escalating medical and economic burdens and the opioid crisis in the global community [6–8]. Concerns for the sustained use of opioids warrant the development of safe and efficacious novel analgesics with improved side effect profiles.

Multiple scientific approaches have been used in attempts to develop safe, effective, and nonaddictive medications for pain management, yet none of the candidates developed have proven successful in human studies [9–11]. For example, studies of pain mechanisms in animal models identified serotonin-norepinephrine reuptake inhibitors and gabapentinoids as potential pain relievers [12], but they are not sufficiently efficacious for certain types of pain due to dose-limiting adverse effects or diversity of pathophysiology [1, 13]. In rodent models, transient receptor potential vanilloid 1 (TRPV1), neurokinin-1 receptor (NK1), and nerve growth factor (NGF) were identified as alternative targets to opioid receptors and with potential for pain relief [14–16]. Disappointingly, ligands targeting these receptors failed in human studies because of their lower efficacy or unexpected adverse effects [16, 17]. MOP receptor modulators and non-opioid pain targets remain as the research focus to explore strategies to combat opioid overdose and addiction and to develop alternative analgesics with opioid-sparing effects [12, 18]. Agonists targeting the other two classical opioid receptors—delta-opioid peptide (DOP) and kappa-opioid peptide (KOP) receptors—have narrow therapeutic windows and various adverse effects, such as dysphoria and convulsions [9, 19]. Over the last 20 years, a fourth opioid receptor subtype, the nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, has emerged as a promising target for developing agonists that not only are effective pain relievers, but can also modulate the pain-relieving and addictive properties of MOP agonists [20–22].

NOP receptor, initially named opioid receptor-like 1, was discovered in 1994 as a G protein-coupled receptor (GPCR) with high sequence homology to the classical opioid receptors, but lower affinity for the opioid ligands [23–27], shortly after the cloning of DOP, KOP, and MOP receptors [28–31]. An endogenous 17-amino acid peptide (FGGFTGARKSARKLANQ) selective for this newly identified receptor was soon isolated, named “nociceptin” by one group on the basis of its ability to elicit hyperalgesia following supraspinal administration in mice [32], and “orphanin FQ” by another group to recognize its first and last amino acid residues and the orphan status of the receptor [33]. The International Union of Basic and Clinical Pharmacology later named this peptide “nociceptin/orphanin FQ” (N/OFQ) and its receptor “N/OFQ peptide (NOP) receptor,” consistent with the nomenclature guidelines [34]. The identification of N/OFQ as an endogenous ligand of the NOP receptor was the first successful example of reverse pharmacology. N/OFQ is derived from a precursor peptide prepro-N/OFQ (ppN/OFQ), which displays similar structural features to precursors of classical opioid peptides, such as prepro-enkephalin, dynorphin, and opiomelanocortin [35]. Despite the high homology of amino acid sequences among the opioid peptides and among the cognate receptors, biochemical and structural studies have revealed appreciable differences in the constitution of the hydrophobic and hydrophilic parts of the binding pockets between NOP receptors and the classical opioid receptors [36–39]. These differences in chemical structure determine the marked differences in the binding affinity and selectivity of N/OFQ and classical opioid peptides toward the various opioid receptors [20, 38, 40]. The structures of human opioid receptors provide the molecular insights of the interplay between endogenous opioid peptides and their receptors to facilitate the design of safer opioid analgesics [40]. The primary structures of both N/OFQ and the NOP receptor are well conserved across different animal species [38, 41]. They are expressed widely and abundantly in the nervous system of rodents and primates, including multiple brain regions and the spinal cord, consistent with their broad biological functions and roles in integrating sensory processing, such as pain transmission and modulation [42–46].

Similar to classical opioid receptors, NOP receptors are coupled to pertussis toxin-sensitive Gi/o proteins that inhibit adenylate cyclase and voltage-gated calcium channels and activate inward potassium channels [47–49]. Upon activation of the NOP receptor by N/OFQ, synaptic transmission is reduced due to inhibition of neuronal excitability via postsynaptic NOP receptors or by a reduction in the release of neurotransmitter (e.g., glutamate, GABA, substance P, noradrenaline) via presynaptic NOP receptors [20, 50, 51]. In this manner, the N/OFQ-NOP ligand-receptor system can regulate various functions of the central nervous system, including memory, reward, mood, motor functions, and sensory processing [20, 52, 53]. Preclinical studies in animal models have provided support for the NOP receptor being singled out as a novel target for numerous therapeutic applications, with pain modulation being one of the most attractive research foci [53–56]. Interestingly, a recent study has documented that NOP receptors are present and functional in human dorsal root ganglion neurons, supporting the suggestion that NOP receptor agonists may be viable alternatives to MOP receptor agonists for pain treatment [57].

The ability of NOP receptors to inhibit excitatory glutamatergic transmission via GPCR signaling in the spinal dorsal horn and brain [58] indicates that NOP receptor activation may indeed produce antinociception. However, species differences have been noted regarding the outcomes of NOP receptor activation [19, 59]. In rodents, NOP receptor activation at the spinal or supraspinal levels produces either pronociceptive or antinociceptive effects, depending on the dose and pain modality [53]. In contrast, spinal, supraspinal, or systemic NOP receptor agonists only produce antinociception in non-human primates (NHPs) [60–62]. Furthermore, NOP receptor activation counteracted MOP receptor-mediated antinociception in mice [63], but enhanced it in NHPs [64, 65]. Considering the close genetic, physiological and behavioral similarities between NHPs and humans, these results support the interpretation that there might potentially be synergistic antinociception by the coactivation of MOP and NOP receptors in humans [21, 66]. In light of this, medicinal chemists have been studying the structure-activity relationships of NOP receptors and synthesizing novel ligands that bind to and activate both MOP and NOP receptors with different affinity and efficacy [67, 68]. Encouragingly, spinal or systemic administration of some of these newly developed NOP receptor-related ligands produce antinociceptive effects comparable to, or more potent than, those of morphine, but lack the adverse effects associated with classical opioids in NHPs [52, 66–69].

The N/OFQ-NOP receptor system has been extensively studied in the past 25 years. There are several comprehensive reviews discussing the medicinal chemistry [52, 67, 68], pharmacology [22, 70], biological functions [20, 22], and therapeutic applications [54, 56, 71] of this ligand-receptor system. There is a translational gap between rodents and primates regarding the NOP and MOP receptor functions [19, 62]. The functional profiles of clinically used opioids are similar between NHPs and humans, but some adverse effects and therapeutic windows of opioids cannot be manifested in rodents [18, 19]. The focus of this article is to discuss (1) the effects of the NOP receptor-related agonists as compared with those of MOP receptor agonists in NHP models, and (2) the research and development status of a few key NOP receptor-related agonists as safe and non-addictive analgesics.

2. Intrathecal Delivery

Neuraxial/spinal drug administration delivers drugs in close proximity to the spinal cord. Intrathecal delivery of opioids has become a standard procedure in perioperative analgesia—for example during cesarean section—and has been successfully used for decades under different clinical contexts in patients with acute and chronic pain [72, 73]. There are several advantages of intrathecal drug delivery. First, multiple ligands and receptors modulating pain signaling are associated with the spinal systems, i.e., dorsal root ganglion, nerve root, dorsal and/or ventral horn of the spinal cord [74, 75]. Second, delivery of the therapeutic agent directly into the spinal canal may reduce systemic drug exposure, thus permitting higher target concentrations with fewer adverse effects [76]. However, the multiple adverse side effects associated with the spinal use of MOP agonists, including itch/pruritus, warrants the development of less compromising intrathecal analgesics [77–79]. In this section, we discuss the in vivo effects of several NOP receptor-related agonists (Table 1) administered intrathecally as compared with those of the MOP receptor agonist morphine.

Table 1.

In vitro pharmacological profiles of NOP-related agonists in binding and functional assays at the opioid receptors

	Binding affinity Ki (nM) or pKi				Stimulation of [35S]GTPγS binding								References
					NOP		MOP		DOP		KOP
	NOP	MOP	DOP	KOP	EC50 (nM)	% Stim	EC50 (nM)	% Stim	EC50 (nM)	% Stim	EC50 (nM)	% Stim
Selective NOP agonist
N/OFQ	0.08	133	> 1000	247	8.1	100	–	–	–	–	–	–	[122]
UFP-112	10.6	7.1	6.4	8.4	10.6	103	–	–	–	–	–	–	[96]
PWT2-N/OFQ	10.3	6.6	< 6	6.4	9.7	3.5	–	–	–	–	–	–	[106]
Ro 64–6198	9.4	7.3	5.9	7.1	7.4		–	–	–	–	–	–	[109, 110]
SCH 221510	0.3	65	2854	131	12		693		8071		683		[140]
MCOPPB	0.09	1.1	> 667	23.1	0.4	140	34	30	4696	79	80	43	[117]
Mixed NOP/MOP agonist
[Dmt1]N/OFQ(1–13)-NH2	10.6	10.5	9.4	9.8	8.5	4.8	8.2	5.5	8.2		9.1	2.5	[120, 121]
PWT2-[Dmt1]N/OFQ(1–13)-NH2	9.1	9.3	8.4	9.1	9.6	3.2	8.5	2.8	8.2		9.2	2.4	[121]
BU08028	8.5	2.1	1.6	5.6	78.6	48	6	21.1	a	10.8	> 10,000	–	[122]
AT-121	3.7	16.5	145.6	301.3	34.7	41.1	19.6	14.2	N.D.	N.D.	N.D.	N.D.	[155]
BU10038	14.8	0.9	1.2	10.5	44	34	a	18	> 10,000	–	> 10,000	–	[69]
Mixed NOP/MOP full agonist
Cebranopadol	0.9	0.7	18	2.6	13	88.9	1.2	103.5	110	105	17	67.2	[165]

N.D. not determined

^aToo little stimulation to determine EC₅₀

Numbers of % Stim represent either the percentage of stimulation with EC₅₀ or the stimulation factor with pEC₅₀

2.1. Peptidic NOP Receptor Selective Agonists

2.1.1. N/OFQ

In rodent models of acute pain, such as the tail flick test and formalin-induced pain, intrathecal administration of N/OFQ caused biphasic effects. Ultralow femtomole doses of N/OFQ induced nociceptive responses characterized by scratching of the limbs and biting and licking of the paws and tail, and low nanomole doses produced antinociceptive effects [80–84]. Inhibition of the excitatory glutamatergic transmission via presynaptic and postsynaptic NOP receptor activation is likely to account for the spinal analgesia from N/OFQ at nanomole doses [85]. In rodent models of chronic pain, such as carrageenan- and complete Freund’s adjuvant-induced inflammatory pain and chronic constriction injury, or spinal nerve ligation-induced neuropathic pain, intrathecal N/OFQ potently produced efficacious antihyperalgesic and antiallodynic effects [86–91]. Upregulation of N/OFQ and the NOP receptors in the dorsal horn of the spinal cord may play an important role in alleviating pain in these models [59, 92]. A rodent study found a supraadditive interaction between intrathecal morphine and intrathecal N/OFQ-induced antinociception [88].

In contrast to the findings in rodent studies, intrathecal administration of N/OFQ over a wide range from femtomole to nanomole doses only produced antinociceptive, but not pronociceptive, effects in NHP models of acute pain and inflammatory pain. These effects could be reversed by a NOP receptor antagonist, indicating that the spinal NOP receptor mediates the effect [93, 94]. It is worth noting that, unlike classical opioid peptides such as β-endorphin and enkephalins, intrathecal N/OFQ exerts its antihyperalgesic effects without inducing itch scratching responses in NHPs [93, 95].

Taken together, these findings suggest that the spinal N/OFQ-NOP receptor system plays an important role in pain inhibition and set the foundation for the development of novel spinal analgesics targeting the NOP receptor-ligand system [59, 62].

2.1.2. UFP-112

In a quest to improve the potency of NOP agonists and decrease the peptidase sensitivity of N/OFQ, medicinal chemists made chemical modifications to N/OFQ and generated a derivative, UFP-112, a potent and long-lasting peptidic full agonist selective for the NOP receptor [96]. It displays a binding affinity for the NOP receptor with a pKi of 10.55, higher than that of N/OFQ (pKi 9.5) and its NOP receptor binding affinity is approximately 100-fold more selective than the classical opioid receptors. In a functional assay using cell membranes expressing the human NOP receptor, UFP-112 stimulated [³⁵S]GTPγS binding with a pEC₅₀ of 10.55, which was 32-fold more potent than that of N/OFQ (pEC₅₀ 9.04). Its maximal stimulatory effect was 103% relative to that of N/OFQ [96]. In a mouse model of acute pain, intrathecal UFP-112 exhibited antinociceptive effects with higher potency (~ 100-fold) and longer duration of action (2 h) than N/OFQ [97, 98]. In NHPs, intrathecal UFP-112 was 10 times more potent than morphine with a similar duration of action (4–5 h) in attenuating thermal nociception and capsaicin-induced thermal allodynia [65]. Models of capsaicin-induced pain have also been utilized in humans to study pain mechanisms and explore novel pharmacological pain treatments [99–101]. It was shown that capsaicin evokes pain by activating TRPV1, a receptor implicated in signal transduction of diverse pain modalities such as diabetic neuropathy [102, 103]. While the analgesic efficacy of spinal UFP-112 in the models of neuropathic pain is still uncertain, further investigation is warranted to broaden the pharmacological profile of UFP-112. Due to findings of full antiallodynic effects in primates, UFP-112 holds a promising potential for further pharmacological development as a spinal analgesic.

2.1.3. PWT2-N/OFQ

Advances in chemical research techniques allowed further development of N/OFQ derivatives. Chemists utilized peptide welding technology (PWT) to generate PWT2-N/OFQ [104, 105], a potent and selective full NOP receptor agonist [106]. This peptide displayed a high binding affinity at the NOP receptor (pKi 10.3), yet its selectivity for the NOP receptor was not inferior to that demonstrated by N/OFQ. In functional [³⁵S]GTPγS binding assay, it mimicked the stimulatory effects of N/OFQ by showing similar maximal effects but higher potency (pEC₅₀ ~ 10) [106]. In mice, PWT2-N/OFQ was 40-fold more potent and produced an extremely long-lasting antinociceptive effect compared with the natural peptide N/OFQ [107]. Translated to the NHP model, intrathecal PWT2-N/OFQ (0.3–3 nmol) was approximately 30-fold more potent than N/OFQ (10–100 nmol) and its antinociceptive effects lasted 10-fold longer (~ 24 h) than those of N/OFQ (~ 2.5 h) [94, 107]. Given the high potency, long duration of action, and lack of mu-opioid receptor-associated adverse effects [65, 107], N/OFQ derivatives such as UFP-112 and PWT2-N/OFQ are viable candidates for future spinal analgesics with improved therapeutic profiles.

2.2. Non-Peptidic NOP Receptor Selective Agonists

2.2.1. Ro 64–6198

Although N/OFQ derivatives exhibit improved potency and long-lasting antinociceptive effects, the inability of these peptides to cross the blood–brain barrier may limit their systemic administration for therapeutic purposes. In the 1990s, scientists discovered several non-peptidic NOP receptor ligands with better metabolic stability by using high-throughput screening from compound libraries. Subsequently, these lead non-morphinan compounds were used for the synthesis of new NOP receptor ligands [108], resulting in the development of the first non-peptidic high affinity NOP agonist, Ro 64–6198 [109, 110]. It binds to the NOP receptor with a higher affinity (pKi 9.4) than the MOP, KOP, and DOP receptors and shows full NOP agonist activity in the functional [³⁵S]GTPγS binding assay (pEC₅₀ 7.4) [109, 110]. Ro 64–6198 has been a pivotal pharmacological tool for exploring potential therapeutic applications of NOP receptor agonists when administered systemically. Only one study could be found that examined the analgesic effect of intrathecal Ro 64–6198 in a rat model of neuropathic pain. Ro 64–6198 did not change the threshold to elicit pain-related behaviors in naïve animals, but exhibited antiallodynic activity in neuropathic rats with lower potency than N/OFQ [111].

2.2.2. MCOPPB

Studies conducted soon after the discovery of N/OFQ and the NOP receptor demonstrated the anxiolytic activities of N/OFQ and Ro 64–6198 in rodent models after intracerebroventricular or intraperitoneal administration [109, 112–115]. However, the development of these ligands into anxiolytics was hindered due to their poor bioavailability and side effects that included motor impairment [116]. In search of an orally potent anxiolytic with better metabolic stability and an improved side effect profile, chemists designed and synthesized a series of 1,2-disubstituted benzimidazole derivatives that meet the criteria as NOP receptor agonists. This resulted in the identification of a highly potent non-peptidic NOP receptor selective full agonist, 1-[1-(1-methylcyclooctyl)-4-piperidinyl]-2-[(3R)-3-piperidinyl]-1H-benzimidazole 1 (MCOPPB) [117, 118].

In vitro assays revealed a high affinity of MCOPPB for the human NOP receptor (Ki 0.086 nM) and weaker affinities for the MOP (Ki 1.05 nM), KOP (Ki 23.1 nM), and DOP (Ki > 667 nM) receptors, approximately 12-, 270-, and >1000-fold more selective for the NOP receptor over the MOP, KOP, and DOP receptors, respectively. In the [³⁵S] GTPγS binding assay, MCOPPB displayed full agonist efficacy at the human NOP receptor (140% stimulation) and partial efficacy at the human MOP (30% stimulation), KOP (43% stimulation), and DOP (79% stimulation) receptors as compared with their respective control ligand. MCOPPB exhibits the highest potency at the NOP receptor, followed by the MOP, KOP, and DOP receptors with EC₅₀ of 0.39, 34, 80, and 4696 nM, respectively [117]. An ex vivo binding study revealed oral MCOPPB has adequate blood–brain barrier penetration in mice [118]. Accordingly, in vivo, MCOPPB produced anxiolytic-like effects in the Vogel conflict test (comparable with those of diazepam) in mice when administered orally. Of note, MCOPPB did not significantly impair motor and memory function, suggesting its potential as a new drug therapy for anxiety [118].

Considering the need for a novel spinal analgesic and the role of the spinal NOP receptor in pain modulation, Centrexion Therapeutics in the USA conducted a rodent study to assess the analgesic efficacy of CNTX-3001 (MCOPPB). Intrathecal administration of CNTX-3001 at 1 and 10 μg, as compared with morphine 15 μg, into the lumbar region of the spinal cord improved the lameness and gait movement in a rodent model of osteoarthritis. Given its potency and receptor binding selectivity for the NOP receptors, this molecule is currently under development as a spinal analgesic delivered by the intrathecal pump for patients suffering from intractable pain [119].

2.3. Mixed NOP/MOP Receptor Agonists

NOP receptor selective agonists, such as Ro 64–6198, produced synergistic antinociception with buprenorphine without inducing undesirable side effects in NHPs [64]. The synergism of NOP and MOP receptor coactivation occurs at both systemic and spinal levels in NHPs, but not systemically in rodents [65, 88, 93]. More importantly, ample evidence indicates that activation of central NOP receptors does not counteract MOP receptor-mediated antinociceptive effects in NHPs and humans [61, 62]. Ligands with mixed NOP and MOP receptor agonist actions may be developed as safer analgesics with a wider therapeutic window and a slower development of tolerance to analgesic efficacy [21, 66]. To investigate the pharmacological profile of a single molecule with mixed NOP/MOP receptor agonist activity, several mixed NOP/MOP receptor agonists have been identified.

2.3.1. [Dmt1]N/OFQ(1–13)-NH2

Aided by the knowledge that the residues at the N-terminal of N/OFQ determine its receptor selectivity, researchers generated [Dmt1]N/OFQ(1–13)-NH2 by introducing a 2,6-dimethyltyrosine (Dmt) residue at the N-terminal of N/OFQ. In vitro, this peptide ligand displays a high binding affinity at the NOP and MOP receptors with a pKi of 10.59 and 10.48, respectively. In the [³⁵S]GTPγS binding assay, it mimics the stimulating effects of the natural peptides N/OFQ or opioid peptide dermorphin at the NOP or MOP receptor, respectively, by showing similar maximal effects at similar or even higher potency [120]. The in vivo effects of [Dmt1] N/OFQ(1–13)-NH2 on pain transmission were evaluated in NHPs after spinal administration. At 1–10 nmol, intrathecal [Dmt1]N/OFQ(1–13)-NH2 produced dose-dependent antinociceptive effects without causing significant itch/scratching responses. The minimal dose to elicit maximal antinociceptive effects was 10 nmol, approximately 30-fold more potent than N/OFQ. At supramaximal doses of 30 nmol and 100 nmol, this peptide still produced full antinociceptive effects that were, however, associated with robust scratching responses [120].

2.3.2. PWT2-[Dmt1]N/OFQ(1–13)-NH2

Using the same PWT technology that produced PWT2-N/OFQ, PWT2-[Dmt1]N/OFQ(1–13)-NH2 was synthesized. It is a tetrameric ligand with mixed NOP/MOP agonist actions. In vitro, PWT2-[Dmt1]N/OFQ(1–13)-NH2 displays a significant decrease in affinity at the NOP receptor (pKi 9.13) when compared with the parent compound [Dmt1]N/OFQ(1–13)-NH2. It behaved similarly as [Dmt1] N/OFQ(1–13)-NH2 in terms of potency and maximal effects in the stimulation of [³⁵S]GTPγS binding at both the NOP and MOP receptors. In vivo, intrathecal administration of PWT2-[Dmt1]N/OFQ(1–13)-NH2 in NHPs produced antinociception against an acute nociceptive thermal stimulus in a dose- and time-dependent manner. The minimal dose to achieve full antinociception was 3 nmol, with antinociceptive effects lasting 24 h without eliciting significant scratching responses or causing any overt side effects, such as sedation or motor impairment. The effects of PWT2-[Dmt1]N/OFQ(1–13)-NH2 were more potent and longer lasting than the parent compound [Dmt1]N/OFQ(1–13)-NH2. Such effects were blocked by the NOP receptor antagonist J-113397, but not by the MOP receptor antagonist naltrexone. Despite its limited in vitro selectivity for NOP over opioid receptors, the spinal analgesic effects of PWT2-[Dmt1] N/OFQ(1–13)-NH2 in NHPs were found to be exclusively due to NOP receptor activation in the respective doses studied [121]. Thus, PWT2-[Dmt1]N/OFQ(1–13)-NH2 obtained with the PWT technology maintains the in vitro pharmacological profile of the parent peptide; however, it displays higher potency and longer lasting actions in vivo. As noted, the above-mentioned NOP receptor-related peptides have been patented and are under development by the company, UFPeptides s.r.l., in Italy.

2.3.3. BU08028

BU08028 is a recently developed buprenorphine analog. It has binding affinity (Ki 1–10 nM) for the NOP and classical opioid receptors, but only has detectable efficacy on the NOP and MOP receptors [122]. The antinociceptive effects of spinal BU08028 were studied in mouse models of pain [123]. Intrathecal BU08028 (0.001–1 μg) reduced thermal hyperalgesia in mice with carrageenan-induced paw inflammation, a model of inflammatory pain [123]. At higher doses of 0.03–1 μg, intrathecal BU08028 attenuated tactile allodynia in mice in the chronic constriction injury model for neuropathic pain model. The antiallodynic effects were mediated by both NOP and MOP receptors in the spinal cord, as revealed by the complete blockade of allodynia after coadministration of NOP- and MOP-selective antagonists. Both antiallodynic and antihyperalgesic effects lasted up to 24 h at the minimal doses to produce the full effects. The potency of BU08028 to produce antihypersensitive effects is higher than that of selective NOP or MOP receptor agonists [123].

2.3.4. BU10038

BU10038 is a naltrexone-derived analog with mixed NOP/MOP receptors partial agonist activity. Intrathecal administration of BU10038 potently produced thermal antinociception and attenuated capsaicin-induced thermal allodynia without significant scratching responses in NHPs [69]. In particular, intrathecal BU10038 (0.3–3 μg) potently produced antinociceptive effects against an acute noxious stimulus. The duration of antinociceptive action produced by the minimum effective dose (3 μg) of BU10038 was 30 h and subsided by 48 h, much longer than that of morphine 30 μg. Intrathecal BU10038 also displayed a similar duration of action against capsaicin-induced thermal allodynia. Although intrathecal BU10038 produced potent antinociception and antihypersensitivity, it did not significantly increase scratching responses compared with morphine [69, 124, 125]. Such findings support the hypothesis that coactivation of NOP and MOP receptors synergistically exerts analgesia with fewer side effects [21, 66]. Taking these results together, BU10038 displays a promising spinal analgesic profile in primates.

Collectively, these findings in NHPs provide a promising profile of NOP receptor-related agonists (both selective and mixed action) as spinal analgesics without the itch side effect (Table 2). The spinal dorsal horn is the major locus for the integration of peripheral sensory input, the modulation of descending supraspinal signaling, and the regulation of peripherally and centrally elicited nociceptive information [126, 127]. Given that intrathecal drug delivery can provide effective pain intervention as an alternative delivery route, mixed NOP/MOP receptor agonists or selective NOP receptor agonists could represent a significant medical advance as spinal analgesics.

Table 2.

Effects of NOP-related agonists in NHPs in comparison with MOP agonists

Intrathecal delivery	Analgesia	Itch	References
MOP receptor agonist
Morphine	✓	✓	[124, 125]
Selective NOP receptor agonist
N/OFQ	✓	−	[94, 95]
UFP-112	✓	−	[65]
PWT2-N/OFQ	✓	−	[107]
Mixed NOP/MOP agonist
[Dmt1]N/OFQ(1–13)-NH2	✓	✓	[120]
PWT2-[Dmt1]N/OFQ(1–13)-NH2	✓	−	[121]
BU10038	✓	−	[69]
Mixed NOP/MOP full agonist
Cebranopadol	✓	−	[170]
Systemic delivery	Analgesia	Abuse potential	Respiration depression	Physical dependence	Itch	Sedation	References
MOP receptor agonist
Oxycodone	✓	+ + +	N.D.	✓	✓	−	[124, 151, 152]
Fentanyl	✓	+ + +	✓	N.D.	✓	−	[145, 170, 174]
Heroin	✓	+ + +	✓	✓	N.D.	−	[8, 155, 173]
Selective NOP receptor agonist
Ro 64–6198	✓/−	−	−	N.D.	−	✓	[60, 139, 172]
SCH 221510	✓	N.D.	−	−	−	N.D.	[64, 143, 145]
Mixed NOP/MOP partial agonist
BU08028	✓	−	−	−	−	−	[145]
AT-121	✓	−	−	−	−	−	[155]
BU10038	✓	−	−	−	−	−	[69]
Mixed NOP/MOP full agonist
Cebranopadol	✓	+	−	N.D.	−	−	[170]

(✓) detected, (−) not detected, (+++/+) degree of abuse potential, N.D. not determined

3. Systemic Delivery

3.1. Non-Peptidic NOP Receptor-Selective Agonists

3.1.1. Ro 64–6198

Since its discovery, N/OFQ has been the leading NOP receptor agonist studied for the functional consequences of NOP receptor activation. N/OFQ and other peptidic N/OFQ derivatives played important roles in paving the road to studying the N/OFQ-NOP receptor system. However, the peptidic ligands are in part limited therapeutically by metabolism, due to peptidases and poor bioavailability following systemic delivery in treating diseases of the central nervous system. In attempting to overcome challenges posed by peptidic ligands, Hoffmann-La Roche patented nonpeptide 8-substituted-1,3,8-triazaspiro[4.5]decan-4-one derivatives as non-peptidic ligands at the NOP receptor for therapeutic treatments related to anxiety, stress, pain, addiction, and several other areas [128]. After a series of structure–activity relationship studies [129], Ro 64–6198 was developed as the first non-peptidic agonist with the highest affinity for the NOP receptor, and 100-fold better selectivity for the NOP receptor over the classical opioid receptors [110, 116]. Ro 64–6198 became the most extensively studied non-peptidic NOP receptor selective agonist and a valuable pharmacological tool in exploring the NOP receptor for potential therapeutics.

Although intrathecal Ro 64–6198 showed antiallodynic efficacy in a rat model of neuropathic pain [111], early studies of systemic Ro 64–6198 in rodent pain models generated disappointing results. Systemic Ro 64–6198 did not elicit responses in various assays, such as tail flick, tail immersion, tactile stimulation, and foot shock [109, 111, 112, 115, 130]. However, it did produce antinociceptive effects in the mouse hot plate test [131]. The overall null effect of systemic Ro 64–6198 could be due to (1) the integration of spinal antinociceptive and supraspinal pronociceptive actions of the N/OFQ-NOP receptor signaling [53], and (2) being a partial agonist in the periaqueductal gray, which is a major site for pathways acting via the NOP receptor to modulate pain [132–134]. In addition to being ineffective in modulating basal levels of nociception, Ro 64–6198 was also reported to block morphine-induced antinociception in a tail-withdrawal assay in mice [130], similar to the effect of N/OFQ [135]. Yet coadministration of subthreshold doses of Ro 64–6198 and morphine acted in an additive, rather than a synergistic, manner to reduce pain sensitivity in the mouse hot-plate test [131]. These studies suggest that systemic Ro 64–6198 may have pain modality-dependent antinociceptive effects in rodents.

In NHPs, systemic Ro 64–6198 produced antinociceptive effects against noxious thermal stimulus [60], potently inhibited capsaicin-induced thermal allodynia [60], and attenuated carrageenan-induced hyperalgesia in an inflammatory pain model [136]. Since capsaicin-induced transductions of pain signals are also involved in diverse nociceptive conditions [99, 137], the ability of Ro 64–6198 to attenuate capsaicin-induced allodynia indicates that NOP agonists may be effective for treating pain of different nociceptive origins. The antinociceptive effects of Ro 64–6198 were antagonized by the NOP antagonist, J-113397, but not by the MOP antagonist, naltrexone. Conversely, the antinociceptive effects of alfentanil, a MOP agonist, were antagonized by naltrexone, but not by J-113397 [60]. Such cross examinations using receptor selective antagonists demonstrate that equal effectiveness of antinociception can be achieved by activation of these two independent receptors in NHPs.

Besides the antinociceptive effects of Ro 64–6198 acting alone, Ro 64–6198 in combination with buprenorphine synergistically enhanced antinociception in NHPs [64], contrary to its antimorphine effect observed in rodents. It is encouraging that the enhancement of buprenorphine-induced antinociception by Ro 64–6198 in NHPs occurred without concurrent respiratory depression and itch/scratching associated with typical MOP receptor agonists [64]. More importantly, Ro 64–6198 lacks the reinforcing effects (abuse liability) observed with clinically used MOP receptor agonists. This is supported by both rodent and NHP studies, indicating that NOP agonists hold great potential to be developed as novel analgesics for humans without abuse liability, one of the biggest concerns with the use of opioid analgesics.

The main side effects of Ro 64–6198 observed in rodents include motor impairment and deficits in learning and memory [115, 116, 138]. The potency at which Ro 64–6198 produces these side effects varies between rat and mouse [115, 116, 138]. In NHPs, although Ro 64–6198 produced antinociception without the MOP receptor agonist-associated side effects, it did cause sedation at a dose that was approximately 30-fold higher than its antinociceptive dose [139]. This indicates a wider therapeutic window of a NOP receptor agonist relative to MOP receptor agonists, but the undesirable sedative effect must be considered during drug development. How these therapeutic and sedative effects compare with those of other non-opioid pain medications (i.e., gabapentin or pregabalin) remains to be explored.

3.1.2. SCH 221510

SCH 221510 is another non-peptidic NOP receptor agonist with a chemical structure that differs from Ro 64–6198. It binds to the NOP receptor with an affinity of 0.3 nM, exhibiting 217-, 437-, and > 9500-fold binding selectivity versus MOP, KOP, and DOP receptors, respectively. In the [³⁵S]GTPγS binding assay, SCH 221510 stimulates the NOP receptor with an EC₅₀ of 12 nM, exhibiting 58-, 57-, and > 673-fold functional selectivity versus MOP, KOP, and DOP receptors, respectively [140]. It produced antiinflammatory and antihyperalgesic effects in the rodent model of trinitrobenzene sulfonie acid-induced inflammatory pain [141, 142]. Such findings support the notion that selective NOP receptor agonists may provide clinical utility as analgesics for the treatment of chronic inflammatory pain [53].

Systemic administration of SCH 221510 produced antinociceptive and antihyperalgesic effects in the NHP models of acute pain, capsaicin-induced allodynia, and carrageenan-induced inflammatory pain [64, 143]. It also synergistically enhanced buprenorphine-induced antinociception without eliciting other side effects such as respiratory depression [64]. Besides its effectiveness in reflex-based assays [136], SCH 221510 produced morphine-like antinociceptive effects in a NHP “operant” nociceptive assay with behavioral selectivity [144]. Such findings further support the analgesic potential of NOP receptor selective agonists and point out the importance of including different outcome measures when assessing the analgesic efficacy in different pain modalities. In the assessment of opioid-induced constipation, SCH 221510 at the analgesic dose did not change bowel motility, whereas the equianalgesic dose of morphine significantly prolonged the gastrointestinal transit function in NHPs [64, 143]. Furthermore, repeated systemic administration of SCH 221510 at its analgesic dose for a short-term exposure of 3 days did not lead to the development of physical dependence as measured by the antagonist-precipitated withdrawal signs [145].

There are several other selective NOP receptor agonists reported in the literature, such as Ro 65–6570, MCOPPB, and AT-403 [108, 118, 146]. These compounds have not yet been studied in NHP models. Nonetheless, it is worth noting that differing abilities of these compounds at inducing G protein versus β-arrestin 2 recruitment did not distinguish their antinociceptive efficacy versus motor impairment [147]. It has been known that a G protein signaling-biased MOP receptor agonist without β-arrestin recruitment may exert enhanced analgesia with fewer side effects in rodents [148]. However, such findings cannot be translated to large animals such as NHPs, as the newly discovered G-protein signaling-biased MOP receptor agonist PZM21 still produced oxycodone-like reinforcing effects (abuse potential) in NHPs [124]. It is reasonable to note that the low intrinsic efficacy of G-protein biased agonists has contributed to their improved side effect profiles [149, 150].

3.2. Non-Peptidic Mixed NOP/MOP Receptor Partial Agonists

3.2.1. BU08028

BU08028 was developed as a novel orvinol analog along with the hypothesis that NOP receptor agonism may enhance MOP receptor-medicated analgesia while ameliorating MOP receptor-mediated adverse effects [21]. BU08028 has a binding affinity of less than 10 nM at each of the four opioid receptor subtypes, making it the first universally high-affinity opioid ligand [122]. Despite its structural similarity to buprenorphine, the binding affinity of BU08028 for NOP receptor (8 nM) is almost 10-fold higher than that of buprenorphine (77 nM). In vitro functional assays showed that BU08028 has no efficacy at DOP and KOP receptors and that it is a partial agonist for MOP and NOP receptors (Table 1). Its efficacy at MOP receptors is very similar to that of buprenorphine with 21% stimulation of [³⁵S]GTPγS binding relative to DAMGO. At NOP receptors, BU08028 showed 48% stimulation relative to N/OFQ, significantly higher than buprenorphine (15% stimulation), indicating a higher efficacy. Based on these results, the pharmacological actions and behavioral outcomes produced by BU08028 are expected to have more NOP receptor agonist activity when compared with buprenorphine.

In NHP models, BU08028 was approximately 10-fold more potent than buprenorphine (ED₅₀ of 0.003 mg/kg versus 0.03 mg/kg) in producing antinociception against a noxious thermal stimulus. The duration of its antinociceptive action was also longer than that of buprenorphine (> 24 h versus 1–6 h), which could be attributed to the high logP value of BU08028. Such antinociceptive effect was equally antagonized by NOP and MOP receptor antagonists [145], in contrast to rodent studies showing that BU08028-induced antinociception was potentiated by a NOP receptor antagonist [122]. These findings indicate that MOP and NOP receptors contribute equally to the antinociceptive effects of BU08028 in primates.

The therapeutic window of BU08028 is much wider compared with opioids currently used in the clinic. At its antinociceptive dose, fentanyl, a MOP receptor agonist, causes respiratory depression in NHPs. Unlike fentanyl, BU08028 at doses up to 10-fold higher than the antinociceptive dose, did not cause significant decreases in respiratory functions or changes in cardiovascular activities, which lends support to BU08028 being a safer analgesic than standard MOP receptor agonists [145]. On the basis of intravenous drug self-administration assays, buprenorphine produced a mild-to-moderate reinforcing strength in NHPs under a progressive ratio schedule of reinforcement. However, in the same group of NHPs, BU08028 produced no reinforcing effects, comparable with the results of saline administration trials [145]. The value of a progressive ratio schedule is that it can distinguish between abused drugs that function as reinforcers. For instance, oxycodone and buprenorphine exert strong and mild-to-moderate reinforcing strengths, respectively, in NHPs [124, 145, 151, 152], which is concordant with their abuse liabilities in humans [153, 154]. BU08028’s lack of reinforcing effects may indicate this compound is devoid of abuse liability in humans. Furthermore, administration of both MOP and NOP receptor antagonists did not precipitate withdrawal signs in NHPs that were given repeated doses of BU08028 for a short-term period, indicating lower liability to develop physical dependence on BU08028 [145]. These recent findings strongly support the therapeutic potential of ligands with mixed NOP and MOP receptor agonist activities as safe and non-addictive analgesics in humans.

3.2.2. AT-121

Using receptor structure-guided drug design and by exploring rational structure-activity relationships, chemists developed a bifunctional NOP/MOP partial agonist, AT-121. It binds with significantly higher affinity at the NOP (Ki 3.7 nM) and MOP (Ki 16.5 nM) receptors than at the DOP or KOP receptors (Ki > 100 nM). [³⁵S]GTPγS binding assays demonstrated its high potency and partial agonist efficacy at both NOP and MOP receptors (EC₅₀ 35 nM and 41% stimulation relative to N/OFQ at the NOP receptor, EC₅₀ 20 nM and 14% stimulation relative to DAMGO at the MOP receptor) [155]. The suitable pharmacokinetics and brain permeability of AT-121 in rats and its stability in monkey plasma suggested that further in vivo investigations were warranted. AT-121 has been studied as a prototype to explore the hypothesis that bifunctional NOP/MOP receptor agonists with an appropriate balance of action on NOP and MOP receptors could yield better analgesics with reduced side effects [155].

The pharmacological profiles of AT-121 were characterized in preclinical NHP models [155]. AT-121 (ED₅₀ 0.01 mg/kg) produced much more potent antinociceptive effects than morphine (ED₅₀ 1 mg/kg). It also exerted a dose-dependent inhibitory effect on capsaicin-induced thermal allodynia. An antagonist study revealed that both NOP and MOP receptors contributed to the antinociceptive effects of AT-121 in the absence of itch scratching responses as compared with morphine. Higher doses of AT-121 did not significantly alter respiratory and cardiovascular activities (respiration rate, minute volume, heart rate, and blood pressure), whereas heroin, an illicit MOP receptor agonist, rapidly caused respiratory depression that required reversal with naltrexone. Besides having a safer and wider therapeutic window than currently approved opioids on the market, AT-121 after a short-term exposure (1–3 days) did not cause opioid-induced hyperalgesia and physical dependence. After a long-term exposure (4 weeks), NHPs did not develop tolerance to the antinociceptive effects of AT-121 as they did when they were treated with morphine for the same period. This observation supports the notion that coactivation of NOP and MOP receptors may not deplete the reservoir of functional receptors to the same degree as activation of a single receptor type, and repeated administration of a bifunctional NOP/MOP agonist may cause a smaller degree of receptor desensitization [21, 66, 156].

In addition to their functional efficacy in modulating pain sensitivity, NOP/MOP receptor partial agonists present wider opportunities for treatment applications. AT-121 attenuated the reinforcing effects of oxycodone in intravenous drug self-administration assays, comparable to the effectiveness of buprenorphine and naltrexone. This attenuation was selective against oxycodone, as the food-maintained operant behavior was not disrupted [155], making AT-121 the first reported bifunctional NOP/MOP receptor agonist to show functional efficacy in blocking the reinforcing effect of a prescription opioid. Recently, BU08028 was found to selectively decrease alcohol drinking without altering the reinforcing effects of food pellets in NHPs [157]. As AT-121 and BU08028 alone did not produce reinforcing effects [145, 155], these studies demonstrated the potential of bifunctional NOP/MOP receptor partial agonists in treating substance use disorders. Partial agonism at both NOP and MOP receptors may underlie the favorable pharmacological profile of such agonists with mixed actions. Indeed, opioid and non-opioid “partial” agonists generally have proven therapeutic efficacy with favorable safety and tolerability [158–160]. Taken together, current literature supports bifunctional NOP/MOP receptor agonists, not only as safer, non-addictive analgesics with a wider therapeutic window [21, 62], but also as promising drug candidates with dual therapeutic actions for treatment of pain and opioid/substance addiction [22, 66].

3.2.3. BU10038

BU10038 is a naltrexone derivative with binding affinity for all four opioid receptor subtypes (Ki 1–15 nM) but has better binding affinity for the NOP receptor (14.8 nM) than naltrexone (> 10,000 nM). Although BU10038 has binding affinity for the DOP and KOP receptors, it does not have detectable efficacy at these two receptors. As noted, chronic pain models could be used to explore if the KOP receptor antagonist property of BU10038 can modulate the affective dimension of pain. In the [³⁵S]GTPγS binding assay, BU10038 has ~ 18% stimulation relative to DAMGO at the MOP receptor and ~ 34% stimulation relative to N/OFQ at the NOP receptor [69]. It is being developed as a novel NOP/MOP receptor partial agonist that may be a good candidate for pain modulation [69, 161] and cocaine use disorder [162]. Focusing on its potential in providing pain relief identified in this review, systemic administration of BU10038 in NHPs produced potent and long-lasting (up to 30 h) antinociceptive and antiallodynic effects [69]. Even at a dose ten times higher than the minimum dose to produce full antinociception, BU10038 did not compromise respiratory and cardiovascular function, demonstrating a wider safety window. It did not produce reinforcing effects in NHPs that otherwise self-administered the opioid analgesic oxycodone. The analgesic effects of BU10038 without abuse potential have been confirmed by another laboratory working with NHPs [161]. In addition, after receiving BU10038 daily for 3 days, NHPs did not develop dependence on BU10038, as no significant changes in physiological parameters (i.e., physical withdrawal signs) were detected after being challenged with a combination of MOP and NOP receptor antagonists (naltrexone and J-113397). After receiving systemic or intrathecal BU10038 for 4 weeks, NHPs did not develop tolerance to the antinociceptive effects of BU10038 [69]. These findings indicate the advantages of mixed NOP/MOP partial agonists such as BU10038 over morphine in short-term or chronic dosing regimens. BU10038 (PPL-138) is currently licensed and developed by Phoenix PharmaLabs in the USA.

Collectively, current studies support the notion that the mixed NOP/MOP receptor partial agonists demonstrate higher potency, a wider therapeutic window, and fewer side effects (Table 2), and thus, such ligands may be innovative, safer, non-addictive opioid analgesics in humans. As noted, the ratio of intrinsic efficacies at the NOP versus the MOP receptor is a major factor determining the functional profiles of newly developed NOP receptor-related ligands. The degree by which these ligands access central versus peripheral sites of action may also determine the functional efficacy and therapeutic window in the treatment of pain and substance abuse. Future studies are warranted to determine the brain/plasma ratios of such compounds in NHPs, along with other behavioral and physiological measurements.

4. Cebranopadol

NOP and MOP receptors may modulate pain via independent mechanisms in partly distinct neuronal networks [60, 163]. In previous sections, we discuss the enhanced antinociceptive action and reduced adverse effects by mixed NOP/MOP receptor partial agonists. In this section, we highlight the pharmacological and therapeutic profile of a NOP receptor-related agonist, cebranopadol, which is currently under development as an innovative analgesic.

4.1. In Vitro Binding Affinity and Functional Efficacy of Cebranopadol

In 2014, Grünenthal GmbH, a pharmaceutical company in Germany, reported a potent NOP and opioid receptor agonist, cebranopadol, which was identified from a novel chemical series of spiro[cyclohexane-dihydropyrano[3,4-b]indol]-amines [164]. In vitro radioligand binding assays determined that it binds with high affinity to the human NOP (Ki 0.9 nM) and MOP (Ki 0.7 nM) receptors, which are approximately 3–4- or 20–26-fold stronger than its affinity at KOP (Ki 2.6 nM) or DOP (Ki 18 nM) receptors, respectively [165]. In the [³⁵S]GTPγS binding assay, cebranopadol showed full agonist efficacy at the human MOP receptor (104% stimulation relative to DAMGO) and DOP receptor (105% stimulation relative to the DOP receptor-selective agonist SNC80), almost full efficacy at the human NOP receptor (89% stimulation relative to N/OFQ), and partial efficacy at the human KOP receptor (67% stimulation relative to the KOP receptor-selective agonist U69,593). Its agonistic potency is the highest at the MOP receptor, followed by NOP, KOP, and DOP receptor with an E C₅₀ of 1.2, 13, 17, and 110 nM, respectively [165]. The off-target profile of cebranopadol is deemed favorable as its binding affinities to other neuronal receptors, ion channels, and enzymes are much lower and it does not show functional agonist efficacy at much higher concentrations [165]. These in vitro data demonstrate that in comparison with NOP/MOP receptor partial agonists such as BU08028, AT-121, and BU10038, cebranopadol is a unique mixed NOP/opioid receptor agonist with potent and full, or nearly full, efficacy at MOP and NOP receptors.

4.2. Preclinical Rodent Studies with Cebranopadol

The potential of cebranopadol as an efficacious analgesic was first investigated in rodent studies. In rats, intravenous cebranopadol was rapidly absorbed and extensively distributed with a half-life of 4.5 h. Its oral bioavailability in rats was 13–23% [165]. Cebranopadol showed consistent analgesic efficacy across a broad range of rat and mouse models of acute and chronic pain, including acute nociception, neuropathic allodynia and hyperalgesia, visceral allodynia, inflammatory hypersensitivity, and bone cancer-induced allodynia [164–167]. It is both highly potent and efficacious. For instance, in a rat tail-flick test for acute nociception, the antinociceptive potency of intravenous cebranopadol (ED₅₀ 5.6 μg/kg) is approximately 200-fold higher than that of morphine (ED₅₀ 1.1 mg/kg) and in the same range as fentanyl [165]. What is even more remarkable is the increased potency of cebranopadol in the models of neuropathic pain. In the rat spinal nerve ligation model, cebranopadol abolished mechanical hypersensitivity with an E D₅₀ of 0.8 μg/kg via an intravenous route [165], which is close to 5000-fold more potent than morphine (ED₅₀ 3.7 mg/kg) [168]. Therefore, in comparison with selective MOP receptor agonists, cebranopadol was more potent in models of neuropathic pain than acute pain, suggesting its potential advantage as an effective treatment for moderate to severe chronic pain. At the cellular level, antagonism studies in the spinal nerve ligation model revealed the involvement of both NOP and MOP receptors in the antihypersensitivity effects of cebranopadol [165]. In clear contrast to morphine, cebranopadol did not disrupt motor coordination and respiratory function at doses within and exceeding the analgesic dose range. In the chronic constriction injury model, development of tolerance to the cebranopadol-induced antiallodynic effect was delayed compared with tolerance to an equianalgesic dose of morphine [165]. Collectively, cebranopadol is a highly potent and efficacious analgesic in various rodent pain models with a favorable side effect profile [165, 167, 169].

4.3. Preclinical NHP Studies with Cebranopadol

To further explore the receptor components contributing to cebranopadol-induced pain relief and the abuse liability of cebranopadol, researchers have conducted experiments in NHP models with more translational relevance [170]. In NHPs, intrathecal cebranopadol (1 μg) exerted full antinociception more potently, with minimal itch scratching responses, in contrast to intrathecal morphine (30 μg). This finding supports the notion that ligands targeting more than one receptor may have enhanced analgesic potency and improved side-effect profiles [66, 171]. Subcutaneous cebranopadol produced more potent antinociceptive and antihypersensitive effects (ED₅₀ 2.9 μg/kg) than fentanyl (ED₅₀ 15.8 μg/kg). The full efficacy and high potency of cebranopadol in the NHP model of acute pain were consistent with its analgesic efficacy in rodents, whereas the duration of action (3 h) was shorter than that in the rat/mouse tail-flick assay (7 h) [165, 170]. The antagonist studies revealed a larger contribution from the MOP receptor than the NOP receptor, and no involvement of DOP and KOP receptors in cebranopadol-induced antinociception in NHPs. This is different from the equal NOP and MOP receptor antagonist effects toward BU08028-, AT-121-, and BU10038-induced antinociception [69, 145, 155]. The analgesic action of cebranopadol may imply that MOP receptor full agonists are more efficacious than agonists selective for other opioid receptor subtypes in suppressing this nociceptive response in primates [62, 172]. It is worth noting that DOP receptor agonists are weak analgesics and KOP receptor agonist-induced antinociception is associated with sedation and dysphoria in primates [19, 136]. Although the MOP receptor component may be largely responsible for the antinociceptive effects of cebranopadol, it did not lead to significant itch scratching responses after intrathecal or systemic administration of cebranopadol [170]. This improved side effect profile of cebranopadol supports the early NHP studies that a combined administration of MOP and NOP receptor agonists produced synergistic antinociception without eliciting scratching responses [21, 64].

Besides pruritus, other side effects commonly associated with MOP receptor agonists, such as respiratory depression and abuse liability, have also been evaluated with cebranopadol in the NHP models [151, 170, 173, 174]. At a dose approximately 2-fold of its antinociceptive dose, fentanyl rapidly decreased respiration rate after systemic administration. In contrast to fentanyl, cebranopadol did not significantly change the respiratory rate and minute volume when given at the antinociceptive dose 5.6 μg/kg or doses approximately 2–3-fold of its antinociceptive dose (10 and 18 μg/kg) [170]. Therefore, cebranopadol may function as a safer analgesic than fentanyl, and this finding is consistent with its safety profile in humans [175]. However, comparable to fentanyl [174], cebranopadol produced reinforcing effects under the fixed-ratio schedule of reinforcement in the intravenous drug self-administration assay, but the reinforcing strength of cebranopadol was lower than that of fentanyl under the progressive-ratio schedule in NHPs [170]. These findings differ from those obtained in rodent studies, which showed ambiguous rewarding effects of cebranopadol in the conditioned place preference paradigm [176, 177]. Nonetheless, such findings are similar to those from a recent human study, in which it was reported that cebranopadol produced some drug-liking effects, but has lower abuse potential than a MOP agonist, hydromorphone [178]. Under the same NHP drug self-administration procedure, the mixed NOP/MOP partial agonists AT-121, BU08028, and BU10038 did not exert reinforcing effects. Such a contrast between agonists with partial or full efficacy at MOP receptors indicates that NOP receptor activation might not be sufficient to completely block full MOP receptor agonist-associated abuse potential. Clearly, the ratio of intrinsic efficacy at NOP and MOP receptors is an important factor contributing to the different pharmacologic profiles of mixed NOP/opioid receptor agonists, abuse liability in particular. Future studies using more centrally penetrating agonists with different intrinsic efficacies at NOP versus MOP receptors will advance our understanding of the functional role of NOP receptors in modulating the reinforcing effects of MOP agonists. Although cebranopadol, a mixed NOP/MOP full agonist, has been developed as a safe analgesic [167, 169], its mild reinforcing strength could be smaller than or similar to the mild-to-moderate abuse potential of the MOP receptor partial agonist buprenorphine in humans [145, 153] and warrants caution for its clinical use in a broader therapeutic context [170].

4.4. Clinical Trials with Cebranopadol

Cebranopadol is currently under the development for the treatment of pain. According to a search on the website clinicaltrials.gov in January 2023, 14 clinical studies on cebranopadol have been conducted in patients with different pain modalities and in healthy subjects to evaluate its analgesic efficacy, safety, and side effects. Table 3 summarizes the key information of these trials. Here we discuss the published information related to these trials.

Table 3.

Clinical trials of cebranopadol

Identifier	Condition or disease	Study phase	Study start date	Status	Location(s)	Original sponsor	References
NCT00872885	Post operative pain	II	March 2009	Completed	USA	Grünenthal GmbH	[182]
	Brief title: bunionectomy trial with GRT6005
	Brief summary: the purpose of the trial is to determine whether the new centrally acting analgesic is effective in comparison with placebo and an active comparator (morphine)
NCT00878293	Diabetic polyneuropathy	II	April 2009	Completed	Germany, UK	Grünenthal GmbH
	Brief title: painful diabetic polyneuropathy trial with a new centrally acting analgesic
	Brief summary: the purpose of this trial is to determine whether the new centrally active analgesic and MS Continus are effective in the treatment of painful diabetic polyneuropathy
NCT01347671	Pain, diabetic polyneuropathies	II	May 2011	Completed	Bulgaria, Germany, Romania	Grünenthal GmbH
	Brief title: assessment of GRT6005 in painful osteoarthritis of the knee
	Brief summary: the purpose of this trial is to investigate the efficacy and safety of GRT6005 in patients with painful diabetic neuropathy
NCT01357837	Osteoarthritis of the knee	II	May 2011	Completed	Austria, Poland, Spain	Grünenthal GmbH
	Brief title: assessment of GRT6005 in painful osteoarthritis of the knee
	Brief summary: the purpose of the trial is to determine whether GRT6005 is effective in patients with pain due to osteoarthritis of the knee
NCT01709214	Moderate to severe chronic pain due to osteoarthritis of the knee	II	December 2012	Completed	USA	Forest Laboratories
	Brief title: safety and efficacy study of GRT6005 in patients with osteoarthritis (OA) knee pain
	Brief summary: the purpose of this study is to evaluate the safety and efficacy of GRT6005 compared with placebo in patients with moderate to severe chronic pain due to osteoarthritis (OA) of the knee. This study includes a maximum 21-day screening period followed by a 15-week double-blind treatment period and a 4–7-day safety follow-up period. Patients who are eligible for the double-blind treatment period will be randomized to one of following treatment groups: GRT6005 high-dose range (400, 600, or 800 μg), GRT6005 low-dose range (200, 300, or 400 μg), oxycodone controlled release (CR) dose range (10, 20, 30, 40, or 50 mg), or placebo
NCT01725087	Low back pain	II	November 2012	Completed	Europe, multinational	Grünenthal GmbH	[180]
	Brief title: efficacy and safety of GRT6005 in patients with chronic low back pain
	Brief summary: the purpose of this trial is to evaluate the safety and efficacy of once daily orally administered GRT6005 in a total of three fixed doses compared with placebo in subjects with moderate to severe chronic low back pain (LBP). The study includes a maximum of 21-day screening period, followed by a 2-week titration period, 12-week maintenance double-blind treatment period, and a 10–14 day safety follow up period. Patients who are eligible for the double-blind treatment period will be randomized to one of the following treatment groups: GRT6005 low-dose, GRT6005 medium dose, GRT6005 high-dose, tapentadol, or placebo
NCT01939366	Chronic pain, diabetic neuropathies, diabetes mellitus	II	September 2013	Completed	Europe, USA	Grünenthal GmbH	Posted study results
	Brief title: cebranopadol efficacy and safety in diabetic patients suffering from chronic pain caused by damage to the nerves
	Brief summary: the purpose of this trial is to evaluate if cebranopadol is safe and can decrease pain in patients when compared with placebo (a tablet that does not contain active product) and when compared with a marketed product containing pregabalin (Lyrica). Furthermore, this trial will be undertaken to find out if the patient’s general health and well-being improves under trial treatment. The concentrations of cebranopadol in the blood will be investigated to get a better understanding of how it is absorbed from the gut, distributed and broken down in the body, and eliminated from the body
NCT01964378	Pain, neoplasms, chronic pain	III	October 2013	Completed	Multinational	Grünenthal GmbH	[181]
	Brief title: CORAL—cebranopadol versus morphine prolonged-release in patients with chronic moderate to severe pain related to cancer (CORAL)
	Brief summary: the purpose of this trial was to find out how well cebranopadol is tolerated, and how often, and which adverse reactions occur when it is taken every day for a longer period of time. In addition, information was collected on how cebranopadol affects pain and well-being in patients suffering from cancer-related pain
NCT02031432	Pain, neoplasms, chronic pain	III	December 2013	Completed	Multinational	Grünenthal GmbH	[183]
	Brief Title: CORAL XT—open-label extension trial of the CORAL trial (CORAL XT)
	Brief summary: the purpose of this trial was to find out how well cebranopadol is tolerated, and how often, and which adverse reactions occur when it is taken every day for a longer period of time. In addition, information was collected how cebranopadol affects pain and well-being in patients suffering from cancer-related pain
NCT03757559	Abuse, drug	I	April 2013	Completed	Canada	Grünenthal GmbH
	Brief title: a trial to evaluate the abuse potential of three doses of GRT6005 in adult non-dependent recreational opioid users
	Brief summary: the primary objective of this study was to evaluate the abuse potential of single doses of cebranopadol (GRT6005) relative to hydromorphone [immediate-release formulation (IR)] and placebo in 48 non-dependent recreational opioid users. Secondary objectives were to evaluate the abuse potential of hydromorphone IR compared with placebo (trial validation), to evaluate the safety and tolerability of single doses of cebranopadol (200, 400, and 800 μg), and to evaluate pharmacokinetics (PK) of cebranopadol, and optionally, some of its metabolites
NCT03882762	Pharmacokinetic	I	June 2013	Completed	USA	Grünenthal GmbH
	Brief title: a clinical study to evaluate the effect of renal impairment on the pharmacokinetics of cebranopadol
	Brief summary: the objective of this study was to evaluate the pharmacokinetics (PK), safety, and tolerability profile of cebranopadol (GRT6005) in patients with varying degrees of renal impairment and participants with normal renal function after an single dose, oral administration. This study was a phase I, multicenter, non-randomized, open-label, parallel group, single-dose study in up to 24 male and female patients with varying degrees of renal impairment and participants with normal renal function
NCT03958123	Prolonged QTc interval, pharmacokinetic	I	July 2013	Completed	USA	Grünenthal GmbH
	Brief title: evaluation of the effects of multiple doses of cebranopadol on the electrical activity of the heart in healthy subjects
	Brief summary: the objective of this study was to evaluate the effects of cebranopadol (GRT6005) on the electrical activity of the heart in healthy participants. The study consisted of a screening period within 21 days before the first dose of investigational medicinal product (IMP) (between day –25 and day –4), during which informed consent was obtained and the general suitability of the participants for the trial was assessed according to the inclusion/exclusion criteria
NCT05256108	Human abuse potential	I	March 2022	Completed	USA	Tris Pharma, Inc.
	Brief title: assessment of abuse potential of cebranopadol in humans
	Brief summary: this study will be conducted to evaluate the abuse potential of single doses of cebranopadol as compared with oxycodone, tramadol, and matching placebo in recreational drug users
NCT05491785	Pain	I	July 2022	Recruiting	Netherlands	Tris Pharma, Inc.
	Brief title: cebranopadol effects on ventilatory drive, central nervous system (CNS), and pain
	Brief summary: opioids are potent painkillers, but come with serious adverse effects ranging from addiction to potentially lethal respiratory depression via activation of mu-opioid receptors (MOP) at specific sites in the central nervous system. Cebranopadol is a first-in-class investigational drug to treat patients with acute and chronic pain. The molecule dually activates the Nociceptin/Orphanin FQ peptide (NOP) receptor and the classical MOP receptor. This is a unique mechanism of action and has demonstrated efficacy in multiple phase II and phase III clinical studies, across several nociceptive and neuropathic indications, as well as having demonstrated a superior safety profile, low potential for abuse, and minimal risk of physical dependence. In animal studies, cebranopadol produced considerably less respiratory depression at comparably analgesic doses of oxycodone and fentanyl, and appeared to have a ceiling to its respiratory effects. Preliminary clinical trials have suggested that these results will be similar in humans. The present study is designed to investigate if: (1) cebranopadol produces less respiratory depression than oxycodone, (2) cebranopadol respiratory effects have a ceiling at very high doses, and (3) cebranopadol does not produce significant respiratory depression, as measured in this study design with 30 subjects, at any dose in the VRH model

The pharmacokinetic properties of cebranopadol were assessed in phase I and phase II clinical trials. Cebranopadol reaches maximum plasma concentration 4–6 h after oral administration of the immediate-release formulation and has a half-life of 14–15 h [169, 179]. Multiple once-daily dosing in patients determined its operational half-life of 24 h. Therefore, the pharmacokinetic study demonstrated that cebranopadol is a viable once-daily medication option for the treatment of chronic pain [179].

Three phase II clinical trials have been conducted and published to evaluate the analgesic efficacy, safety, and tolerability of cebranopadol in treating postoperative pain, chronic lower back pain, and cancer pain [180–182]. In patients suffering moderate to severe acute pain following bunionectomy, cebranopadol at single oral doses of 400 or 600 μg induced more effective analgesia, while being safe, compared with morphine on the primary efficacy endpoint. Both cebranopadol doses and morphine ensured adequate 24-h pain relief. However, cebranopadol was better tolerated and received a better overall rating by the patients [182]. In patients with moderate-to-severe chronic lower back pain with and without a neuropathic pain component, cebranopadol 200, 400, and 600 μg was administered once daily for 14 weeks. It displayed analgesic efficacy at the primary efficacy endpoints assessing the changes of pain from baseline to the weekly average 24-h pain during the entire 12 weeks and during week 12 of the maintenance phase. Cebranopadol also positively modulated sleep and functionality [180]. Overall, cebranopadol treatment was safe with an acceptable tolerability profile. Only at higher doses were there treatment discontinuations because of adverse events, which occurred mostly during titration [180]. In patients with moderate-to-severe cancer-related pain, non-inferiority of cebranopadol with superiority over morphine prolonged release on the primary endpoint were demonstrated in a non-inferiority trial for its efficacy, in which oral cebranopadol was taken in the dose range of 200–1000 μg once daily for up to 7 weeks [181]. In an extension trial, prolonged treatment with cebranopadol for up to 26 weeks was found to be safe and well tolerated in patients who completed the proceeding trial [183]. The most frequently reported treatment-emergent adverse events in these clinical trials included nausea, vomiting, dizziness, headache, somnolence, and constipation at a low incidence rate. Although there were some limitations of these clinical studies and further titration to the optimal dose for individual patients is necessary, cebranopadol is a promising drug candidate with a novel mechanistic approach for potential treatment of various moderate-to-severe postoperative and chronic pain [167, 180–183].

In addition to evaluating the analgesic efficacy of cebranopadol in patients suffering from pain, clinical trials have also assessed its respiratory effects and abuse liability in healthy volunteers and opioid users [175, 178]. In a phase I pharmacokinetic–pharmacodynamic study to quantify respiratory effects, oral cebranopadol at a single dose of 600 μg in healthy adults produced a ceiling effect of respiratory depression [175]. This is a major advantage over classic opioids, including fentanyl and morphine, which produce apnea at high concentrations. Although this finding is encouraging, respiratory effects of cebranopadol at higher doses and in relevant patient populations need to be investigated before a definitive conclusion can be drawn. In a 2017 trial reported by Christoph et al., physical dependence on cebranopadol was assessed using the Clinical Opiate Withdrawal Scale. In line with the weak withdrawal effects observed in preclinical rodent studies, only 4.6–6.5% of patients reported mild withdrawal symptoms following abrupt cessation at the end of the treatment period [180]. Furthermore, a phase I clinical trial was conducted in non-dependent recreational opioid users to evaluate the abuse potential of cebranopadol. Low (200 μg) and medium (400 μg) doses of oral cebranopadol did not produce drug-liking effects different from placebo. A higher dose (800 μg) showed significantly greater effects compared with placebo. However, the drug-liking effects of cebranopadol was smaller and delayed when compared with the MOP receptor agonist hydromorphone, indicating a lower abuse potential of cebranopadol [178]. This is consistent with a preclinical NHP study that reported a lower reinforcing strength of cebranopadol relative to fentanyl [170].

Collectively, cebranopadol is an effective analgesic with an improved side-effect profile. It is anticipated to advance to phase III trials in the future [167, 169]. Findings from preclinical and clinical studies both support the coactivation of NOP and MOP receptors as a viable and innovative approach for enhanced pain relief with reduced side effects.

5. Conclusion

NOP receptor activation at the spinal cord exerts a significant analgesic effect in primates, as demonstrated in assorted experimental animal models studying various types of NOP receptor selective agonists. Several peptidic and non-peptidic NOP receptor agonists with different binding affinities and efficacies produce potent analgesic effects by intrathecal delivery in preclinical studies. These findings suggest that there are numerous candidate molecules as novel analgesics targeting spinal NOP receptors. Further, considering pharmacokinetic parameters, non-peptidic NOP ligands are suitable for both intrathecal and systemic delivery. As a result, several different types of non-peptidic NOP ligands are under development and have been subject to functional scrutiny using in vitro and in vivo pharmacological assays. In particular, mixed NOP/MOP receptor partial agonists (e.g., BU08028, BU10038, AT-121) have been shown to display morphine-comparable analgesic effects through the activation of both NOP and MOP receptors. Further, at therapeutic doses, these compounds did not elicit dose-limiting adverse effects, such as respiratory depression, abuse liability, and itch sensation, which are seen with opioid use in preclinical rodent and NHP studies. Given the clinical utility of buprenorphine for the treatment of pain and opioid addiction, these mixed NOP/MOP receptor partial agonists may hold potential for clinical application as analgesics and/or treatment of opioid use disorder. Notably, cebranopadol displays full agonist activity for NOP and MOP receptors, and has shown potent and efficacious analgesic effects in preclinical rodent and NHP studies, along with a favorable side effect profile. Even though cebranopadol displays reinforcing effects, not only in preclinical but also in ongoing clinical studies, these were less intense when compared with the currently available MOP receptor agonists, indicating a lower abuse potential.

Given the similarities of the anatomical and neurochemical features between humans and NHPs, determining the functional profiles of NOP/MOP receptor-related agonists in NHPs provides robust translational evidence for their suitability as safer and efficacious analgesics. Furthermore, the use of NHPs for mechanistic studies aimed at elucidating the balance of activation of the NOP–MOP receptor system seems critical for developing novel analgesics with a wider therapeutic window.

In summary, coactivation of NOP and MOP receptors by mixed agonists with appropriate binding affinities and intrinsic efficacies for these targets seems to produce potent analgesia with fewer classic opioid-related adverse effects. We believe that the use of more translational systems, namely NHPs, would enhance the scientific rigor of future functional studies of these promising novel compounds and will facilitate the development of safer, non-addictive analgesics.

Key points.

Non-human primate studies provide robust translational evidence that NOP receptor-related agonists, especially mixed NOP/MOP receptor partial agonists, are safe and non-addictive analgesics.

Findings from preclinical and clinical studies both support the coactivation of NOP and MOP receptors as a viable and innovative approach for enhanced pain relief with reduced side effects.

Cebranopadol is currently developed as a safe and effective analgesic with an improved side-effect profile.

Data availability statement

Not applicable.