UFP‐112 a Potent and Long‐Lasting Agonist Selective for the Nociceptin/Orphanin FQ Receptor

Abstract

Nociceptin/orphanin FQ (N/OFQ) controls several biological functions via selective activation of the N/OFQ peptide receptor (NOP). [(pF)Phe⁴Aib⁷Arg¹⁴Lys¹⁵]N/OFQ‐NH₂ (UFP‐112) is an NOP receptor ligand designed using a combination of several chemical modifications in the same peptide sequence that increase NOP receptor affinity/potency and/or reduce susceptibility to enzymatic degradation. In the present review article, we summarize data from the literature and present original findings on the in vitro and in vivo pharmacological features of UFP‐112. Moreover, important biological actions and possible therapeutic indications of NOP receptor agonists are discussed based on the results obtained with UFP‐112 and compared with other peptide and nonpeptide NOP receptor ligands.

Introduction

G‐protein‐coupled receptors (GPCRs) historically represent the most important target for drug discovery [1]. This is particularly true as reverse pharmacology strategies over the last 20 years have led to the deorphanization of more than 300 GPCRs [2]. Thus, novel molecules modulating these previously orphan GPCRs may open new avenues for treating human diseases in the near future. Identification of nociceptin/orphanin FQ (N/OFQ) and the N/OFQ peptide receptor (NOP) represent the first successful example of reverse pharmacology [3]. Soon after the cloning of opioid receptors that started with the delta opioid peptide (DOP) receptor [4, 5] followed by the kappa opioid peptide (KOP) [6] and mu opioid peptide (MOP) [7] receptors, different research groups started programs aimed at the identification of opioid receptor subtypes. This led, in 1994, to the simultaneous identification of a GPCR showing an overall 60% homology with classical opioid receptors which, unexpectedly, did not bind opioid ligands [8, 9, 10, 11]. This receptor, named ORL‐1 (opioid receptor like receptor 1) [12], was then used for “fishing” for its endogenous ligand, assuming for the receptor the same type of coupling (i.e., G_i‐mediated inhibition of cAMP levels) as classical opioid receptors and for its ligand the same chemical nature (i.e., a peptide) as endogenous opioids. This strategy and these assumptions were indeed correct as demonstrated, 1 year later, by the successful identification from brain extracts of a heptadecapeptide able to inhibit forskolin stimulated cAMP in cells expressing the ORL‐1 receptor but not in wild type cells [13, 14]. This peptide whose primary sequence (H‐Phe‐Gly‐Gly‐Phe‐Thr‐Gly‐Ala‐Arg‐Lys‐Ser‐Ala‐Arg‐Lys‐Leu‐Ala‐Asn‐Gln‐OH) resembles that of opioid peptides, was named nociceptin [14] or orphanin FQ [13]. Interestingly enough, nociceptin/orphanin FQ (N/OFQ) did not bind to the classical opioid receptors [13]. The N/OFQ peptide precursor (ppN/OFQ) was then cloned; it displayed organizational features that were strikingly similar to those of the genes of preproenkephalin, preprodynorphin, and preproopiomelanocortin, the precursors to endogenous opioid peptides. This suggests that the four genes have a common evolutionary origin and belong to the same family [15, 16]. Thus, the novel peptide‐receptor system is similar to classical opioid systems in terms of structure of both receptors and peptides and of receptor cellular actions (G_i/o coupling with subsequent inhibition of cAMP and calcium currents and stimulation of potassium currents [17]) while being completely different from classical opioids in terms of pharmacology; in fact, N/OFQ does not bind opioid receptors and opioid ligands (including naloxone) do not bind to the N/OFQ peptide (NOP) receptor. On this basis the NC‐IUPHAR subcommittee suggested that the N/OFQ–NOP receptor system is considered as “a nonopioid branch of the opioid family”[18].

Understanding of the biological roles played by the N/OFQ–NOP receptor system was dependent upon the generation of useful research tools, particularly transgenic animals and NOP selective ligands. Mice knockout for the N/OFQ precursor (ppN/OFQ(−/−), [19]) as well as for the NOP receptor (NOP(−/−), [20]) gene have been available since 1999 and 1997, respectively. Recently, NOP(−/−) rats were generated [21]. These transgenic animals represent very important research tools both for investigating the role played by endogenous N/OFQ in regulating various biological functions and for performing ex vivo and in vivo studies on ligand selectivity.

A wide range of NOP ligands are now available (see for recent reviews on this topic [3, 22]) and these can be divided into three groups based on their chemical nature: (1) N/OFQ‐related peptides, (2) N/OFQ unrelated short peptides, (3) small nonpeptide molecules. A series of N/OFQ‐related peptides were identified by our group using classical peptide structure–activity relationship studies and using the electrically stimulated mouse vas deferens as an N/OFQ sensitive pharmacological preparation [23, 24]. The peptide N/OFQ(1–13)‐NH₂ was soon identified as the shortest N/OFQ sequence maintaining the same affinity [25] and functional potency and efficacy as the natural ligand [24, 26]. This peptide was then used as template for performing further structure–activity relationship studies [27, 28, 29] that led to the identification of interesting pharmacological tools such as the partial agonist [Phe¹ψ(CH₂‐NH)Gly²]N/OFQ(1–13)‐NH₂ ([F/G]N/OFQ(1–13)‐NH₂, [30]), the low potency pure antagonist [Nphe¹]N/OFQ(1–13)‐NH₂[31], and the highly potent agonist [(pF)Phe⁴]N/OFQ(1–13)‐NH₂[32, 33]. It has been shown that the introduction in position 14 and 15 of N/OFQ of an extra pair of basic residues Arg, Lys generates a highly potent agonist, [Arg¹⁴Lys¹⁵]N/OFQ [34]. This chemical modification was combined with that able to eliminate efficacy (Nphe¹) to generate the highly potent and selective antagonist [Nphe¹Arg¹⁴Lys¹⁵]N/OFQ‐NH₂; UFP‐101 [35]. This peptide, whose in vitro and in vivo pharmacological properties are reviewed in [36], is among the most frequently used NOP receptor selective antagonists.

Small N/OFQ unrelated peptides were identified by screening of synthetic peptide combinatorial libraries. In this way compounds such as the nonselective NOP antagonist III‐BTD [37] and the NOP selective partial agonist hexapeptides Ac‐RYYRWK‐NH₂ and Ac‐RYYRIK‐NH₂[38] were identified. The hexapeptides were used as templates for a series of structure–activity studies [39, 40, 41, 42, 43, 44, 45], for generating useful radiolabeled probes [46], MOP/NOP bivalent ligands [47], and for the development of novel NOP ligands such as the highly potent partial agonist ZP120 [48, 49, 50, 51, 52]. This latter peptide is now under clinical development as a novel diuretic.

Small molecules acting as NOP selective ligands have been discovered by pharmaceutical industries via high throughput screening. The first molecule of this class reported in literature was the NOP selective antagonist J‐113397 (1‐[(3R,4R)‐1‐cyclooctylmethyl‐3‐hydroxymethyl‐4‐piperidyl]‐3‐ethyl‐1,3‐dihydro‐benzimidazol‐2‐one, [53]). This compound, also known as compB, represents the most frequently used nonpeptide NOP receptor ligand with more than 80 papers in the literature reporting its in vitro and in vivo activity. Structure–activity studies on J‐113397 led to the identification of an interesting achiral analog named Trap‐101 (1‐[1‐(cyclooctylmethyl)‐1,2,3,6‐tetrahydro‐5‐(hydroxymethyl)‐4‐pyridinyl]‐3‐ethyl‐1,3‐dihydro‐2H‐benzimidazol‐2‐one, [54]) whose in vivo NOP antagonist properties and antiparkinsonian actions have been recently demonstrated [55]. In 2004, GlaxoSmithKline researchers reported the identification and pharmacological characterization of a potent and selective NOP antagonist named SB‐612111 ((−)‐cis‐1‐Methyl‐7‐[[4‐(2,6‐dichlorophenyl)piperidin‐1‐yl]methyl]‐6,7,8,9‐tetrahydro‐5H‐benzocyclohepten‐5‐ol, [56]). The excellent pharmacological profile of SB‐612111 has been confirmed and extended in follow‐up studies performed by our group both in vitro[57, 58] and in vivo[59]. This molecule represents the most potent and selective nonpeptide antagonist available to date. More recently Banyu researchers identified a novel potent and selective NOP receptor antagonist, 1‐benzyl‐N‐{3‐[spiroisobenzofuran‐1(3H),4’‐piperidin‐1‐yl]propyl} pyrrolidine‐2‐carboxamide [60], Compound 24. The NOP antagonist properties of this molecule have been recently confirmed in different laboratories [61, 62, 63].

With respect to nonpeptide NOP agonists, the first series of such compounds was reported in the literature in 1999 by Roche [64]. However, these compounds displayed only moderate NOP potency and particularly selectivity over classical opioid receptors. These pharmacological features were substantially improved with the identification of Ro 64–6198 ([(1S,3aS)‐8‐(2,3,3a,4,5,6‐hexahydro‐1H‐phenalen‐1‐yl)‐1‐phenyl‐1,3,8‐triaza‐spiro[4.5]decan‐4‐one], [65]). Ro 64–6198 behaved as a potent and selective NOP full agonist in in vitro assays and after peripheral administration mimicked the anxiolytic‐like effects of supraspinally injected N/OFQ [66, 67]. Interestingly, no signs of tolerance to Ro 64–6198 anxiolytic‐like effects were detected following 15 days of daily drug exposure in rats [68]. The pharmacokinetic and pharmacodynamic features of Ro 64–6198 as well as its biological actions have been recently reviewed [69]. Ro 64–6198 is certainly a valuable research tool that has been extensively used and characterized in several laboratories thus becoming the standard nonpeptide NOP agonist. Several nonpeptide NOP agonists have been recently reported by Schering–Plough [70, 71, 72, 73] and Pfizer [74, 75, 76] investigators. The best compounds among these series appear to be SCH 221510 (8‐[bis(2‐methylphenyl)methyl]‐3‐phenyl‐8‐azabicyclo[3.2.1]octan‐3‐ol, [72]) and MCOPPB (1‐[1‐(1‐Methylcyclooctyl)‐4‐piperidinyl]‐2‐[(3R)‐3‐piperidinyl]‐1H‐benzimidazole, [74]). Similar to N/OFQ and Ro 64–6198, both compounds are able to evoke dose dependent anxiolytic like effects in rodents. The most important research tools and selective NOP receptor ligands identified to date are summarized, in chronological order, in Table 1. Moreover, the chemical structure of the above‐mentioned nonpeptide NOP ligands is shown in Figure 1.

Table 1.

Research milestones in the N/OFQ–NOP receptor field: pharmacology and research tools

Year	Milestone	Comment	References
1994	NOP receptor	Cloning of the NOP receptor	[8, 9, 10, 11]
1995	N/OFQ	Identification of N/OFQ as the endogenous ligand of the NOP receptor	[13, 14]
1996	ppN/OFQ	Cloning of the gene coding for ppN/OFQ	[15, 16]
	mouse vas deferens	The electrically stimulated mouse vas deferens is a N/OFQ sensitive preparation	[23, 24]
	N/OFQ(1–13)‐NH2	The minimum N/OFQ sequence maintaining the same potency as the natural peptide	[24, 26]
1997	NOP(−/−) mice	Generation of mice knockout for the NOP receptor gene	[20]
	Ac‐RYYRWK‐NH2	Identification of hexapeptides acting as selective partial agonists at NOP receptors	[38]
1998	[F/G]N/OFQ(1–13)‐NH2	The first N/OFQ‐related peptide with reduced efficacy at NOP receptors	[30]
1999	ppN/OFQ(−/−) mice	Generation of mice knockout for the ppN/OFQ gene	[19]
2000	[Nphe1]N/OFQ(1–13)‐NH2	The first selective peptide antagonist	[31]
	J‐113397	The first selective nonpeptide antagonist	[53]
	Ro 64–6198	The first selective nonpeptide agonist	[67]
2002	UFP‐101	A potent and selective peptide antagonist	[35]
2004	SB‐612111	A potent and selective nonpeptide antagonist	[56]
2009	NOP(−/−) rats	Generation of rats knockout for the NOP receptor gene	[21]

Figure 1.

Chemical structure of nonpeptide NOP receptor agonists (top row) and antagonists (bottom row).

Anatomical studies have revealed high levels of expression of the ppN/OFQ and NOP receptor mRNA in various areas of the central nervous system [12, 77]. NOP mRNA and binding sites exhibit approximately the same distribution pattern, suggesting that the NOP receptor is mainly located on local neuronal circuits. The NOP receptor is also expressed in the peripheral nervous system [12]. The diffuse distribution of N/OFQ and its receptor in the brain, spinal cord and peripheral nervous system indicates that this peptidergic system may control several biological actions. This has been confirmed by a series of in vitro and in vivo studies demonstrating that N/OFQ, via selective NOP receptor activation, modulates several biological functions in the central nervous system including pain transmission, stress, anxiety and emotional states, learning and memory, locomotor activity, food intake, and the motivational properties of drugs of abuse (see for reviews on these topics [3, 22, 78, 79, 80, 81]). N/OFQ may also behave as an important regulatory signal of the functions of peripheral systems such as the cardiovascular, gastrointestinal, renal, genitourinary, respiratory, and immune systems (see for reviews [3, 22, 82, 83, 84, 85]). The most important findings in terms of biological functions regulated by the N/OFQ–NOP receptor system and possible indications for NOP selective ligands are summarized, in chronological order, in Table 2.

Table 2.

Research milestones in the N/OFQ–NOP receptor field: biological actions and putative therapeutic indications of NOP ligands

Year	Milestone	Comment	Selected references
1995	Pain	Supraspinal N/OFQ produces hyperalgesia while spinal N/OFQ naloxone insensitive analgesia. This latter effect has been confirmed in nonhuman primates. Spinal NOP receptors may be a potential target for novel analgesics.	[13, 14, 86, 148, 149]
1997	Diuresis	N/OFQ evokes aquaresis; NOP partial agonists may be indicated for patients with water‐retaining diseases.	[150, 151]
	Anxiety	N/OFQ evokes robust anxiolytic‐like effects. These results, later confirmed with Ro 64–6198, suggest NOP agonists as a novel class of anxiolytics.	[66, 67]
	Memory	N/OFQ severely impairs spatial learning after intrahippocampal administration. NOP(−/−) have greater learning ability then NOP(+/+) mice. NOP antagonists may have memory enhancing properties.	[152, 153]
1998	Micturition reflex	N/OFQ inhibits the micturition reflex in rats. Intravesical N/OFQ produces beneficial effects in patients suffering from overactive bladder. NOP agonists may represent innovative drugs for treating urinary incontinence.	[90, 154]
1999	Drug addiction	N/OFQ inhibits the rewarding properties of alcohol and morphine. NOP agonists can be developed as a novel treatment for drug addiction.	[129, 155]
2001	Cough	N/OFQ inhibits cough after both central and peripheral administration. NOP agonists as novel antitussive therapy.	[156]
2002	Depression	[Nphe1]N/OFQ(1–13)‐NH2 and J‐113397 reduced immobility time in the forced swimming test. NOP(−/−) mice display an antidepressant phenotype. NOP antagonists may act as innovative antidepressant drugs.	[157, 158]
2004	Parkinson disease	J‐113397 and UFP‐101 facilitate motor activity in normal animals and relieve akinesia in hemiparkinsonian rats. NOP(−/−) outperformed NOP(+/+) mice on the rotarod and are less sensitive to haloperidol‐induced motor depression. NOP receptor antagonists may represent a novel approach for Parkinson's disease.	[121, 122]
2008	Sepsis	UFP‐101 reduces animal mortality in a rat model of sepsis. Plasma N/OFQ levels are higher in human subjects who died as a result of sepsis. NOP antagonists as innovative drugs for the treatment of sepsis.	[159, 160]

It should, however, be emphasized that evidence for the importance of the N/OFQ–NOP receptor system in regulating these biological functions is, in most cases, restricted to rodent studies. Only for N/OFQ‐evoked spinal analgesia [86, 87] and for the beneficial effects of NOP receptor antagonists in models of Parkinson's disease [88, 89], this evidence has been confirmed in nonhuman primates. Finally, clinical studies with N/OFQ were only performed in the urological field. These studies demonstrated that both acute [90, 91] and subchronic (once a day for 10 days, [92]) intravesical instillation of N/OFQ produces beneficial effects in patients suffering from urinary incontinence due to neurogenic detrusor overactivity.

The pleiotropic actions evoked by N/OFQ via selective activation of the NOP receptor imply that different therapeutic indications can be addressed with NOP selective ligands. However, this also implies that a rather large number of possible side effects can be encountered by developing molecules acting at the NOP receptor. This certainly represents a challenging issue for the drug development process in this field.

Design of UFP‐112

[(pF)Phe⁴Aib⁷Arg¹⁴Lys¹⁵]N/OFQ‐NH₂ (UFP‐112, [93, 94]) is an NOP receptor ligand designed by combining several chemical modifications in the same peptide sequence (see Fig. 2) that were previously reported to increase NOP receptor affinity/potency and/or reduce susceptibility to enzymatic degradation. These chemical modifications and their effects on peptide pharmacological features are summarized below.

Figure 2.

Primary structure of UFP‐112.

As far as C‐terminal amidation of N/OFQ is concerned, this modification was reported in the first generation of structure–activity studies [25, 26]. N/OFQ‐NH₂ binds to NOP receptors expressed in the rat [25], mouse [95] and guinea pig [96] brain membranes as well as to recombinant human NOP receptors expressed in CHO cells [97] with pK_i values slightly higher than N/OFQ. These receptor binding data were confirmed in in vitro functional studies performed in different preparations measuring inhibition of cAMP accumulation in CHO_hNOP cells, glutamate release from rat cerebrocortical slices [97], and electrically induced contractions in several isolated tissues [98, 99, 100]. In all these studies N/OFQ‐NH₂ mimicked N/OFQ actions with similar maximal effects but potency values 2–8‐fold higher. Interestingly, when rat vas deferens experiments were repeated in the presence of a cocktail of peptidase inhibitors, the potency of N/OFQ was increased by 6‐fold while that of N/OFQ‐NH₂ by only 2‐fold [101] thus suggesting that amidation of the C‐terminus of N/OFQ confers higher metabolic stability to the peptide. This may explain why in some in vivo assays, that is, the mouse locomotor activity assay [102] and the rat plantar test [103], N/OFQ‐NH₂ was found to be significantly more potent than the natural peptide. Similar considerations can be drawn analyzing the pharmacological profile of N/OFQ(1–13)‐NH₂. A large series of in vitro and in vivo studies reviewed in [101] demonstrated that N/OFQ actions are mimicked by N/OFQ(1–13)‐NH₂. C‐terminal amidation of this peptide is, however, crucial for biological activity because N/OFQ(1–13)‐OH displayed strongly reduced affinity in binding studies [95, 104], very low potency in the mouse vas deferens assay [26] and inactivity in the rat in vivo[105]. Interestingly, in vivo in mice N/OFQ(1–13)‐OH is inactive under control conditions whereas it mimics the bradycardic and hypotensive action of N/OFQ and N/OFQ(1–13)‐NH₂ when injected in the presence of the peptidase inhibitor thiorphan [106]. Collectively, this evidence corroborates the findings obtained with N/OFQ‐NH₂ and demonstrates that amidation of the C‐terminus of N/OFQ counteracts the action of peptidases making the peptide more resistant to enzymatic degradation.

The (pF)Phe⁴ modification has been identified as part of a detailed study performed on the Phe⁴ residue of N/OFQ(1–13)‐NH₂ that demonstrated that the biological activity of a series of Phe⁴ substituted peptides positively correlates with the electron withdrawal properties of groups in the para position and inversely with their size [29]. The most potent compound of this series, [(pF)Phe⁴]N/OFQ(1–13)‐NH₂, was then selected for further in vitro and in vivo characterization. [(pF)Phe⁴]N/OFQ(1–13)‐NH₂ displayed higher affinity than N/OFQ(1–13)‐NH₂ in receptor binding studies performed using CHO cells expressing the recombinant human NOP (CHO_hNOP) and rat cerebrocortical membranes. In a series of functional assays (stimulation of [³⁵S]GTPγS binding in CHO_hNOP and rat cerebrocortical membranes, inhibition of cAMP accumulation in CHO_hNOP cells, bioassay in the mouse colon and in the electrically stimulated guinea pig ileum and mouse and rat vas deferens) [(pF)Phe⁴]N/OFQ(1–13)‐NH₂ behaved as full agonist showing potency values 3–10‐fold higher than N/OFQ(1–13)‐NH₂ or the natural peptide. In addition, the effects of [(pF)Phe⁴]N/OFQ(1–13)‐NH₂ were not modified by naloxone while being antagonized by [Nphe¹]N/OFQ(1–13)‐NH₂. This NOP antagonist displayed similar pA₂ values against [(pF)Phe⁴]N/OFQ(1–13)‐NH₂ and N/OFQ [32]. In vivo in the locomotor activity assay in mice, 1 nmol N/OFQ(1–13)‐NH₂ given intracerebroventricularly (i.c.v.) caused a significant decrease (about 70% inhibition) in activity for the first 15 min following injection; [(pF)Phe⁴]N/OFQ(1–13)‐NH₂ at the same dose exerted a similar inhibitory effect that lasted until the end of the observation period (30 min). This effect was prevented by 10 nmol [Nphe¹]N/OFQ(1–13)‐NH₂. In the mouse tail‐withdrawal assay, after i.c.v. administration [(pF)Phe⁴]N/OFQ(1–13)‐NH₂ mimicked the actions of N/OFQ(1–13)‐NH₂ producing pronociceptive effects when given alone and blocking morphine‐induced analgesia when administered with the alkaloid. In both experimental paradigms, the actions of [(pF)Phe⁴]N/OFQ(1–13)‐NH₂ were longer lasting (>60 min) compared to those of N/OFQ(1–13)‐NH₂ (≈30 min). In unanaesthetized normotensive mice, bolus intravenous (i.v.) injection of 100 nmol/kg of [(pF)Phe⁴]N/OFQ(1–13)‐NH₂ decreased mean blood pressure and heart rate; these effects were longer lasting than those elicited by the same dose of N/OFQ(1–13)‐NH₂. Finally, the i.c.v. administration of [(pF)Phe⁴]N/OFQ(1–13)‐NH₂ dose‐dependently stimulated feeding in rats, and the peptide was about 10‐fold more potent than N/OFQ(1–13)‐NH₂[33].

In a follow‐up study, the (pF)Phe⁴ modification was applied in the N/OFQ‐NH₂ template generating in vitro results superimposable to those obtained with [(pF)Phe⁴]N/OFQ(1–13)‐NH₂[107]. Collectively, these findings demonstrated that the (pF)Phe⁴ modification produces an increase in peptide potency and duration of action without affecting its selectivity and pharmacological activity.

The Aib⁷ modification was first reported by Zhang et al. [108]. These authors performed a structure–activity study in which alpha‐aminoisobutyric acid (Aib) or N‐methylalanine (MeAla) were inserted as replacement(s) for Ala⁷, Ala¹¹, or Ala¹⁵ in the N/OFQ sequence. They found that [Aib^7,11]N/OFQ‐NH₂ behaved as a highly potent NOP receptor full agonist in the [³⁵S]GTPγS binding assay. Based on the known alpha helix structure induction properties of Aib [109], the authors suggested that N/OFQ might adopt an amphipathic helix conformation in its C‐terminal sequence [108]. This suggestion was later confirmed by NMR conformational analysis studies [110]. Based on these findings, we synthesized and pharmacologically evaluated in the mouse vas deferens assay a series of N/OFQ‐NH₂ analogs substituted in position 7 and 11 with Calpha,alpha‐disubstituted cyclic, linear and branched amino acids. None of the novel N/OFQ analogs produced better results than [Aib⁷]N/OFQ‐NH₂ that displayed 3‐fold higher potency (pEC₅₀ 8.35) than the parent peptide N/OFQ‐NH₂ (pEC₅₀ 7.80) [93]. Importantly, the inhibitory effects of both [Aib⁷]N/OFQ‐NH₂ and N/OFQ‐NH₂ in the mouse vas deferens are similarly sensitive to the antagonist action of UFP‐101 while not being modified by naloxone [110].

The Arg¹⁴Lys¹⁵ modification has been reported by Okada et al. [34]. The very first structure–activity studies on N/OFQ demonstrated that the pair of dipeptides Arg‐Lys at positions 8–9 and 12–13 plays a very important role in NOP receptor binding [25, 111]. This is probably due to the formation of high energy ionic bonds between the peptide basic residues Arg‐Lys and the acidic amino acid cluster in the second extracellular loop of the NOP receptor [112]. With the aim of obtaining a N/OFQ analog that binds more strongly to the NOP receptor, Okada et al. [34] synthesized a series of N/OFQ analogs in which the Arg‐Lys dipeptide unit was placed at positions 6–7, 10–11, or 14–15 adjacent to the parent Arg‐Lys. This design strategy was indeed successful since, among these N/OFQ analogs, [Arg¹⁴Lys¹⁵]N/OFQ was found to be more potent than the natural peptide by 3‐fold in receptor binding and by 17‐fold in [³⁵S]GTPγS binding experiments [34]. These results were later confirmed and extended by us in vitro and in vivo[113]. Indeed, in isolated tissues, [Arg¹⁴Lys¹⁵]N/OFQ mimicked the effects of N/OFQ, with similar maximal effects but higher potencies (17‐fold in the mouse vas deferens, 10‐fold in the rat vas deferens, and about 5‐fold in the guinea pig ileum and mouse colon). In these preparations, the effects of [Arg¹⁴Lys¹⁵]N/OFQ were not modified by 1 μM naloxone, but were blocked by the NOP antagonists [Nphe¹]N/OFQ(1–13)‐NH₂ and J‐113397. Interestingly, in the rat vas deferens, a cocktail of peptidase inhibitors increased the potency of N/OFQ (by 4‐fold) but not that of [Arg¹⁴Lys¹⁵]N/OFQ, thus suggesting that the [Arg¹⁴Lys¹⁵] modification confers to the peptide some resistance to enzymatic degradation. This was recently corroborated by demonstrating that the half‐life of N/OFQ and [Arg¹⁴Lys¹⁵]N/OFQ in the presence of trypsins are 13 and 30 min, respectively [114]. In in vivo experiments in mice, [Arg¹⁴Lys¹⁵]N/OFQ mimicked the effects of N/OFQ administered i.c.v, producing pronociceptive effects in the tail‐withdrawal assay and inhibiting locomotor activity. In both assays, [Arg¹⁴Lys¹⁵]N/OFQ was about 30‐fold more potent than N/OFQ and produced longer lasting effects [113]. Collectively, these results demonstrate that the [Arg¹⁴Lys¹⁵] modification has a 2‐fold effect: first, it increases peptide binding to the NOP receptor and, second, it makes the peptide less susceptible to enzymatic degradation thus increasing its in vivo duration of action.

As described above and summarized in Table 3 all of these chemical modifications produced an increase in N/OFQ potency in the electrically stimulated mouse vas deferens. This increase ranged from 1.3‐fold for the C‐terminal amidation to 7‐fold for the [Arg¹⁴Lys¹⁵] substitution. When all modifications are applied in the same molecule, that is, UFP‐112, their effects on peptide potency were more than additive. In fact, the addition of the concentration ratios obtained with the single modifications yielded a number (13.3) that is far from the concentration ratio measured with UFP‐112 (62). In contrast, multiplication of the concentration ratios of the single modifications yields a number (54.6) close to the experimentally obtained concentration ratio (62) for UFP‐112. This clearly suggests that the combination of the single modifications into the same molecule produces synergistic effects on peptide potency.

Table 3.

Comparison of N/OFQ and N/OFQ‐related peptide potencies in the electrically stimulated mouse vas deferens

	N/OFQ pEC50	Peptide pEC50	CR	References
N/OFQ‐NH2	7.84	7.96	1.3	[99]
[(pF)Phe4]N/OFQ‐NH2	8.27	8.59	2	[107]
[Aib7]N/OFQ‐NH2	7.82	8.35	3	[110]
[Arg14Lys15]N/OFQ‐NH2	8.27	9.12	7	[107]
[(pF)Phe4Aib7Arg14Lys15]N/OFQ‐NH2 (UFP‐112)	7.45	9.24	62	[93]

The maximal effects evoked by N/OFQ‐related peptides were similar to those evoked by N/OFQ. CR, concentration ratio.

In vitro Pharmacological Profile of UFP‐112

In receptor binding experiments performed on CHO_hNOP cell membranes, UFP‐112 produced a concentration‐dependent inhibition of [³H]N/OFQ binding with a pK_i value (10.55) 10‐fold higher than that of the natural peptide N/OFQ (9.50). UFP‐112 displayed high (>100‐fold) selectivity over classical opioid receptors. In CHO_hNOP cell membranes, N/OFQ stimulated [³⁵S]GTPγS binding in a concentration‐dependent manner with a pEC₅₀ of 9.04 and maximal effect corresponding to approximately 8‐fold over basal values. UFP‐112 mimicked the stimulatory effect of N/OFQ producing similar maximal effects but being 30‐fold more potent [93]. Original data generated as described in [97], indicate that forskolin‐stimulated cAMP accumulation in CHO_hNOP cells is inhibited in a concentration‐dependent manner by N/OFQ with very high potency. UFP‐112 mimicked the inhibitory effects of N/OFQ displaying a superimposable concentration‐response curve (Table 4).

Table 4.

In vitro pharmacological profile of UFP‐112

Preparation	N/OFQ action	pEC50	UFP‐112 action	pEC50	References
CHOhNOP	↑[35S]GTPγS	9.04	↑[35S]GTPγS	10.55	[93]
CHOhNOP	↓ cAMP	10.29	↓ cAMP	10.34	present article
CHOhNOP‐Gαqi5	↑ intracellular calcium	9.54	↑ intracellular calcium	9.05	[58]
Mouse vas deferens (Swiss)	↓ contractions	7.45	↓ contractions	9.24	[93]
Rat vas deferens	↓ contractions	6.83	↓ contractions	8.34	[93]
Guinea pig ileum	↓ contractions	8.05	↓ contractions	9.17	[93]
Mouse vas deferens NOP(+/+)	↓ contractions	7.62	↓ contractions	9.40	[94]
Mouse vas deferens NOP(−/−)	Inactive		Inactive		[94]
Mouse lung NOP(+/+)	↓ capsaicin‐induced bronchoconstriction	≈7.5	↓ capsaicin‐induced bronchoconstriction	≈8.5	[115]
Mouse lung NOP(−/−)	Inactive		Inactive		[115]

The effects of N/OFQ and UFP‐112 were also compared measuring calcium mobilization in CHO_hNOP cells stably expressing the Gα_qi5 chimeric protein that forces the NOP receptor to couple to calcium signaling [58]. In this kind of experiment, UFP‐112 mimicked N/OFQ effects both in terms of shape and kinetics of the calcium transients and of maximal effects. However, UFP‐112 potency was unexpectedly lower than that of the natural peptide although only by 3‐fold. UFP‐112 effects were also evaluated in isolated tissues where N/OFQ inhibits electrically induced contractions. In the mouse and rat vas deferens and guinea pig ileum N/OFQ and UFP‐112, concentration‐dependently inhibited electrically induced twitches eliciting similar maximal effects. However, UFP‐112 was found to be 60‐, 30‐, and 10‐fold more potent than the natural peptide [93]. It is worthy of mention that the kinetics of the inhibitory effects evoked by N/OFQ and UFP‐112 were very different in these tissues. The inhibitory effects induced by N/OFQ occurred rapidly after adding the peptide (≈3 min) to the bath and were immediately reversible after washing. In contrast, UFP‐112 displayed a slower onset of action and its inhibitory effects reached a plateau only after ≈10 min; moreover, tissues treated with UFP‐112 did not fully recover to the control twitch even after washing the tissue for more than 2 h (data not shown). As an example of these different kinetics, an original tracing obtained in the electrically stimulated mouse vas deferens with equieffective concentrations of N/OFQ and UFP‐112 is reported in Figure 3.

Figure 3.

Typical tracing showing the kinetics of the inhibitory effects of equieffective concentrations of N/OFQ and UFP‐112 in the electrically stimulated mouse vas deferens.

In order to establish the receptor mechanism involved in their action, the effects of N/OFQ and UFP‐112 in the electrically stimulated mouse vas deferens were challenged with receptor antagonists and reassessed in tissues taken from NOP(+/+) and NOP(−/−) mice. The nonselective opioid receptor antagonist naloxone at 1 μM did not affect the concentration‐response curve to either N/OFQ or UFP‐112. In contrast UFP‐101 displaced to the right the concentration‐response curve to N/OFQ and UFP‐112 to a similar extent without modifying their maximal effects and with superimposable pK_B values (6.81 and 6.91, respectively). The effects of the two peptides in tissues taken from NOP(+/+) mice were very similar to those previously described in tissues from Swiss mice that is, no difference in maximal effects and UFP‐112 more potent than N/OFQ by approximately 70‐fold. In contrast, in parallel experiments performed on tissues from NOP(−/−) mice both N/OFQ and UFP‐112 were found inactive up to 1 μM. The selective DOP agonist [D‐Pen²,D‐Pen⁵]‐enkephalin (DPDPE) produced identical inhibitory effects in tissues taken from both NOP(+/+) and NOP(−/−) mice [94]. Finally, in the isolated and perfused mouse lung, capsaicin‐induced bronchoconstriction was reversed by N/OFQ in a concentration‐dependent manner. UFP‐112 mimicked the effects of the natural peptide being, however, at least 10‐fold more potent. The inhibitory effects of both N/OFQ and UFP‐112 were not present in lungs taken from NOP(−/−) mice [115]. The in vitro actions of UFP‐112 in comparison with those evoked by the natural NOP agonist N/OFQ are summarized in Table 4.

Thus, these in vitro findings clearly demonstrated that UFP‐112 behaves (1) as NOP receptor full agonist, since its maximal effects were always superimposable with those of N/OFQ, (2) as a highly selective NOP ligand, as demonstrated by receptor binding studies and receptor antagonist and knockout studies in the mouse vas deferens and lung, (3) as an NOP ligand characterized by slow onset and long duration of action, (4) as a highly potent NOP ligand with 10‐fold higher affinity in receptor binding and 10–70‐(depending on the preparation) fold higher potency in functional studies. The only exceptions to the latter statement are data obtained in the cAMP and calcium assays and these findings deserve a comment. Both assays are characterized by a high efficiency of the stimulus/response coupling due to the presence of amplification phenomena. Indeed values for N/OFQ potency in these particular assays are higher than in the other assays (Table 4). Thus ceiling effects may prevent identification of ligands with potency higher than N/OFQ in these particular tests. However, this consideration does not explain why in the Gα_qi5 NOP receptor calcium assay UFP‐112 displayed slightly lower potency than that of N/OFQ. In this assay all the NOP receptor antagonists tested displayed potency values perfectly in line with the literature [58]. This also applies to most full and partial agonists tested. Only a subset of NOP ligands including the full agonist Ro 64–6198 and the partial agonist ZP120 displayed in this assay, similar to UFP‐112, values of potency significantly lower than expected [58]. The chemical nature of the small molecule Ro 64–6198, that of the N/OFQ analog UFP‐112, and that of the Dooley hexapeptide derivative ZP120 are very different suggesting that chemical features are not relevant. It is worthy of mention that isolated tissue experiments demonstrated an important characteristic common to the three ligands. As depicted in Figure 3 for UFP‐112 and described in detail in previous publications for Ro 64–6198 [116] and ZP120 [48, 52], the kinetics of the inhibitory effect elicited by N/OFQ on the electrically induced twitch response is rapid and immediately and completely reversible after washing while that of UFP‐112, Ro 64–6198, and ZP120 is characterized by slow onset, and slow and partial reversibility after washing. The slow kinetics of action of these ligands may be relevant for the estimation of their potency in the Gα_qi5 NOP receptor calcium assay. Indeed, the long time required to obtain activation of NOP receptors with these agonists may be incompatible with the rapid kinetics that characterizes the calcium transient response. This might be the reason for the underestimation of the UFP‐112, Ro 64–6198, and ZP120 potencies in the Gα_qi5 NOP receptor calcium assay. For details and discussion of this issue see [58].

The conclusion that UFP‐112 behaves in vitro as a potent and selective full agonist for the NOP receptor is corroborated by the findings by the group of Wang [117] who synthesized a series of N/OFQ analogs, including the peptide [(pF)Phe⁴Aib⁷Aib¹¹Arg¹⁴Lys¹⁵]N/OFQ‐NH₂, which is very similar to UFP‐112. [(pF)Phe⁴Aib⁷Aib¹¹Arg¹⁴Lys¹⁵]N/OFQ‐NH₂ evaluated in receptor binding (rat brain membranes) and bioassay (mouse vas deferens) experiments displayed an affinity of 10.78 and an agonist potency of 9.37. These values are virtually superimposable to those obtained in our laboratories with UFP‐112.

Finally, the degradation half‐life (T_1/2) of N/OFQ and UFP‐112 in mouse plasma and brain homogenates was evaluated in HPLC studies by least‐square linear regression analysis of peptide peak area versus time [94]. Results of this analysis indicate that N/OFQ showed a relatively long half‐life in plasma (about 1 h) compared to that obtained in brain homogenate (approximately 3 min). UFP‐112 exhibited significantly longer half‐lives compared to the natural peptide. In particular, the plasma T_1/2 of UFP‐112 is about 3‐fold longer than that of N/OFQ and this difference was even more pronounced in the mouse brain homogenate [94]. Thus, UFP‐112 seems to associate to full agonist activity, slow onset and long duration of action, high NOP potency and selectivity another desirable characteristic: low susceptibility to enzymatic degradation. This latter feature might be relevant for the interpretation of the in vivo pharmacological actions of UFP‐112 discussed below.

In vivo Actions of UFP‐112

Pain transmission—N/OFQ modulates pain transmission in a complex manner. Although some conflicting results are reported in the literature most of the available evidence indicates that N/OFQ produces inhibitory effects on nociception (antinociceptive action) at peripheral and spinal levels while facilitates pain transmission (pronociceptive action) in the brain [3, 78]. In line with this view, experiments performed in our laboratories with the mouse tail‐withdrawal assay demonstrated pronociceptive effects of i.c.v N/OFQ [118] and antinociceptive effects when the peptide is given intrathecally (i.t.) [119]. Both of these actions are resistant to naloxone, antagonized by UFP‐101 and SB‐612111 and not observed in NOP(−/−) mice [35, 52, 59, 118, 119]. Under the same experimental conditions, the effects of i.c.v. and i.t. UFP‐112 were assessed in the mouse tail‐withdrawal assay [94]. UFP‐112 (1–100 pmol) produced dose‐dependent pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t. over the same dose range. Thus, UFP‐112 mimicked the actions of N/OFQ. However, UFP‐112 was approximately 100‐fold more potent than the natural peptide, and clearly produced longer lasting effects. In fact, the effects induced by UFP‐112 in the tail‐withdrawal assay were still statistically significant 120 min after i.c.v. or i.t. injection of the peptide, while those elicited by N/OFQ lasted for only 15–30 min [118, 119]. Interestingly, the antinociceptive properties of spinally administered N/OFQ were confirmed in nonhuman primates [86, 87]. These studies demonstrated that i.t. N/OFQ produces naltrexone resistant and J‐113397 sensitive antinociceptive effects that lasted for about 120 min. Moreover, N/OFQ is able to potentiate morphine antinociceptive action. Studies on the effects of UFP‐112 on pain transmission in monkeys are currently underway in the laboratories of Dr Ko at the Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan, USA. Preliminary results (Ko MC, personal communication) demonstrated that i.t. UFP‐112 is able to produce dose‐dependent antinociceptive effects that are similar to those evoked by N/OFQ and blocked by J‐113397. However, in this assay UFP‐112 is about 10‐fold more potent than N/OFQ and, more importantly, produces longer lasting effects (statistically significant effects are recorded 4.5 h after i.t. injection). Collectively, these findings demonstrate that the spinal antinociceptive actions of N/OFQ and UFP‐112 are similar in rodents and nonhuman primates. Moreover, these results suggest that spinal NOP receptors represent a promising target for innovative analgesic drugs and indicate that UFP‐112 is worthy of further development for this particular indication.

Locomotor activity—one of the first biological actions described in response to the i.c.v. administration of N/OFQ in mice was a reduction of locomotor activity [13]. This effect was later confirmed in rats [120] and the involvement of the NOP receptor in this N/OFQ action was demonstrated by receptor antagonist [35, 102] and knockout [20] studies. UFP‐112 was compared with N/OFQ for its ability to inhibit locomotor activity in mice. A 10 nmol N/OFQ given i.c.v. produced a clear inhibitory effect on spontaneous locomotor activity that lasted for only 60 min. In contrast, UFP‐112 at 100‐fold lower doses (i.e., 0.1 nmol) elicited long lasting effects, inhibiting locomotor activity for approximately 6 h [94].

Endogenous N/OFQ—NOP receptor signaling also seems to play an important inhibitory role on motor behavior in pathological conditions. Indeed, NOP receptor antagonists (UFP‐101, J‐113397, and Trap‐101) produce beneficial effects in rodent models of Parkinson's disease [55, 88, 121, 122, 123, 124]. These results were recently confirmed in nonhuman primates [88, 89]. Moreover, recent studies [125] implicate the N/OFQ–NOP receptor system in L‐DOPA‐induced dyskinesia, a long‐term side effect of the therapy of Parkinson's disease. In fact, in dyskinetic rats i.c.v. treatment with N/OFQ, as well as systemic treatment with the NOP agonist Ro 65–6570, significantly attenuated abnormal involuntary movements and produced a full recovery of the animals motor performance on the rotarod. These same effects were observed in response to i.c.v. UFP‐112, which was at least 10‐fold more potent than the natural peptide. These antidyskinetic properties of NOP agonists were no longer observed in rats treated with the antagonist J‐113397 demonstrating the exclusive involvement of the NOP receptor in their actions [125]. Collectively, these findings demonstrated that NOP receptor antagonists are worth evaluating as novel treatments for Parkinson's disease while NOP receptor agonists may represent an innovative strategy for controlling L‐DOPA‐induced dyskinesia.

Food intake—several studies demonstrated that i.c.v. N/OFQ is able to stimulate food intake in rats (for recent reviews see [80, 126]). A similar hyperphagic effect could be measured after i.c.v. administration of N/OFQ in mice [59, 94]. N/OFQ was inactive at 0.1 nmol, while at 1 nmol produced a statistically significant orexigenic effect. Increasing the dose of peptide to 10 nmol resulted in a loss of effect, thus making the dose‐response curve to N/OFQ bell shaped. The involvement of NOP receptors in this action of N/OFQ was demonstrated by its sensitivity to the antagonist action of SB‐612111 [59]. UFP‐112 mimicked the hyperphagic effect of N/OFQ producing a bell‐shaped dose–response curve with the maximum reached at 10 pmol. Thus UFP‐112 was found to be approximately 100‐fold more potent than N/OFQ in this assay [94]. In addition, it is worthy of mention that the amount of the orexigenic effect elicited by UFP‐112 was approximately double that evoked by N/OFQ. Since food intake was measured cumulatively over the 60 min time course of the experiment, the larger orexigenic effect of UFP‐112 can be interpreted assuming a more prolonged stimulation of the NOP receptor by the synthetic peptide than by N/OFQ. The involvement of the NOP receptor in the orexigenic action of N/OFQ and UFP‐112 was demonstrated in knockout studies. Indeed, 1 nmol N/OFQ and 10 pmol UFP‐112 elicited a robust hyperphagic effect in NOP(+/+) mice while the two peptides were found completely inactive in NOP(−/−) animals [94].

Recently, experiments were carried out to investigate the effects of i.c.v. injections of UFP‐112 on food intake in rats. These studies were performed using the protocols and experimental conditions described in details in [127].

The results obtained indicate that the peptide exerts a potent and very pronounced hyperphagic effect following i.c.v. administration in rats (Fig. 4). A statistically significant hyperphagic effect was even observed at the dose of 0.03 nmol, while 2.1 nmol of N/OFQ were required. Therefore, the present findings are in keeping with previous reports in mice showing that UFP‐112 is about 100 times more potent than N/OFQ [94]. Both the effect of UFP‐112 and that of N/OFQ appears to be behaviorally selective since they are not accompanied by evident modifications of the gross behavior of the animals. Moreover, other aspects of the ingestive behavior such as water drinking are not significantly modified following single i.c.v. injection.

Figure 4.

Cumulative food intake following i.c.v. injection of different doses of N/OFQ (top panel) and UFP‐112 (bottom panel). Values are means ± sem of four separate experiments. *P< 0.05 versus control, according to ANOVA followed by Dunnett test for multiple comparisons. *indicates that all the data points of the relative treatment are statistically different from control.

In addition, the effect of UFP‐112 appears to be much longer lasting than that of N/OFQ. In fact, the hyperphagic effect was still detected when food was presented even 6 h after the i.c.v. injection of UFP‐112 (Fig. 5), while the hyperphagic effect of N/OFQ was completely over 1 h after administration (data not shown). These data perfectly matched those obtained in similar experiments comparing the time course of N/OFQ and UFP‐112 effects on locomotor activity in mice [94], and may likely explain why the intensity of the hyperphagic effect of UFP‐112 was markedly more pronounced than that of N/OFQ.

Figure 5.

Thirty‐minute food intake in rats treated with 0.05 nmol UFP‐112 i.c.v. at different times (1–8 h) before access to food. Values are means ± sem of four separate experiments. *P < 0.05 versus control, according to the Student's t‐test for unpaired data.

The experiment in which the NOP receptor antagonist UFP‐101 was given before UFP‐112 administration provides evidence that the hyperphagic effect of this molecule is completely dependent upon activation of NOP receptors. In fact, when UFP‐101 was given in two injections of 20 nmol the 15 and 30 min food intake of UFP‐112‐treated rats was not statistically different from that of controls (Fig. 6). On the other hand, even in rats treated with UFP‐101 (2 × 20 nmol) UFP‐112 was able to produce a delayed stimulation of food intake. This finding can be interpreted considering the lower susceptibility to enzymatic degradation of the agonist than the antagonist.

Figure 6.

Cumulative food intake following i.c.v. injection of 0.05 nmol UFP‐112 alone or with 2×20 nmol UFP‐101 (the first injection 15 min before and the second just before injection of UFP‐112). Values are means ± sem of four separate experiments. *P < 0.05 versus control, according to ANOVA followed by Dunnett test for multiple comparisons.

Interestingly, the same pretreatment with UFP‐101 did not significantly modify the hyperphagic effect of i.c.v. injection of NPY, 6 μg/rat (data not shown), thus providing further evidence on the high selectivity of action of this NOP receptor antagonist [36].

Alcohol intake—in contrast to opioids N/OFQ does not produce conditioned place preference per se[128] while it is able to counteract the rewarding properties of drugs of abuse including alcohol [3]. In fact N/OFQ reduced ethanol intake in genetically selected alcohol‐preferring rats [129] and this effect was prevented by the NOP antagonist [Nphe¹]N/OFQ(1–13)NH₂[130]. Moreover, N/OFQ abolished conditioned place preference induced by ethanol [129] and inhibited reinstatement of alcohol‐seeking behavior induced by electric footshock stress, as well as that induced by ethanol‐paired cues [131]. Converging evidence indicates that the central amygdala may likely represent the key area for the action of N/OFQ on alcohol dependence [132, 133]. Interestingly, the inhibitory effect of N/OFQ on ethanol intake is mimicked by different NOP receptor agonists including N/OFQ‐NH₂, N/OFQ(1–13)‐NH₂, OS‐426 and UFP‐102 [130, 134] and, but only at high doses, by the nonselective agonist of buprenorphine [135]. The involvement of the NOP receptor in the effect of high doses buprenorphine was demonstrated with the use of the NOP selective antagonist UFP‐101 [135]. Finally, conflicting results were reported on this topic on the effects of the nonpeptide NOP agonist Ro 64–6198 [134, 136, 137].

UFP‐112 was reported to mimic the reduction in ethanol intake of genetically selected alcohol‐preferring rats evoked by N/OFQ [134]. In fact subchronic i.c.v. treatment with UFP‐112 produced statistically significant inhibitory effects on ethanol intake already at the low dose of 10 ng/rat after 4 days while higher doses (i.e., 50 ng/rat) evoked significant effects after 2 days of treatment [134]. Neither food nor water intake were modified by UFP‐112 in this range of doses. Thus UFP‐112 behaved as a potent and selective agonist at NOP receptors controlling ethanol intake mimicking the inhibitory effects elicited by N/OFQ at 500 ng/rat [129] at doses 50‐fold lower (i.e., 10 ng/rat). These data obtained with UFP‐112 substantiate the proposal that NOP receptor selective agonists are worthy of development for the treatment of alcohol abuse.

Cardiovascular and renal function—N/OFQ controls cardiovascular and renal function both acting in the brain and in the periphery [85]. The cardiovascular and renal effects of N/OFQ and UFP‐112 were compared after i.v. bolus administration [94]. At 10 nmol/kg N/OFQ produced a slight but significant reduction in arterial pressure and a slight non significant reduction in heart rate. Similarly, immediately following drug injection, a 100‐fold lower dose of UFP‐112 (0.1 nmol/kg, i.v.) produced a comparably small but significant hypotension and bradycardia. However, when administered at the same dose as N/OFQ (i.e., 10 nmol/kg), UFP‐112 profoundly reduced arterial pressure and heart rate. In addition to increased potency, UFP‐112 also produced markedly longer cardiovascular responses than N/OFQ. In the same animals i.v. bolus injection of UFP‐112 (0.1 and 10 nmol/kg) also produced a concurrent diuretic response. At the higher dose tested, the UFP‐112‐induced increase in urine flow rate was delayed in onset (approximately, 30 min), of relatively long duration (50–60 min), and associated with a reduction in urinary sodium excretion (not statistically significant). Of note, similar diuretic responses could be obtained with N/OFQ only after i.v. infusion but not bolus injection [85]. Thus UFP‐112 given i.v. bolus not only mimicked the effects of N/OFQ on cardiovascular function with higher potency and longer lasting effects but it was also able to evoke renal responses (i.e., diuresis associated with antinatriuresis) which can be obtained only after i.v. infusion of N/OFQ. This different action may likely derive from the ability of UFP‐112, but not N/OFQ, to reach peripheral sites (e.g., the kidney [50]) at pharmacologically relevant concentrations when administered as an i.v. bolus. This can likely be attributed to the lower susceptibility to enzymatic degradation of UFP‐112 compared to N/OFQ.

No data are yet available on the receptor mechanism mediating the cardiovascular and renal actions of UFP‐112. Future studies performed with NOP knockout animals and selective NOP antagonists will demonstrate if these effects of UFP‐112 could be solely attributed to NOP receptor activation.

Gastrointestinal function—both ppN/OFQ and the NOP receptor are widely expressed in the intestinal tract and N/OFQ has been reported to modulate stomach, small intestine and colon contractility in several species [138]. In vitro studies demonstrated that N/OFQ‐inhibited neurogenic contractions of the stomach and small intestine while in the colon the peptide evokes direct contraction probably via inhibition of tonic NO release from enteric neurons [138, 139]. These effects of N/OFQ are very robust and some intestinal preparations such as the mouse colon and particularly the electrically stimulated guinea pig ileum are widely used N/OFQ sensitive pharmacological assays. Data obtained in electrically stimulated guinea pig ileum are available for all the NOP ligands listed in Table 1. In addition, the effects of UFP‐112 in this preparation are displayed in Table 4. As far as the in vivo actions of N/OFQ are concerned, several studies suggest an important role of this peptide in the central and peripheral control of different gastrointestinal functions including gastric empting, secretion, and damage (induced by ethanol or stress), gastrointestinal transit and colonic propulsor motility. In some recent studies, the gastrointestinal actions of N/OFQ were compared to those evoked by UFP‐112. Following i.c.v. injection, N/OFQ dose‐dependently delayed gastric emptying of a phenol red meal, decreased gastric secretion in water‐loaded rats and reduced ethanol‐induced gastric lesions. All these actions were mimicked by UFP‐112 that was 20‐ to 50‐fold more potent than the natural peptide and produced longer lasting effects. The action of N/OFQ at 2.5 nmol on gastric emptying was short lasting (10 min) while the same effect elicited by UFP‐112 at 0.1 nmol was still statistically significant 2 h after injection. In addition, the effects of UFP‐112 were sensitive to the NOP receptor antagonist, UFP‐101 [140]. When the peptides were given intraperitoneally (i.p.) they evoked a different pattern of action: no effect on gastric emptying, a gastric hypersecretory response and antiulcer effects. This suggests that central and peripheral NOP receptors have distinct roles in controlling gastric function [140]. The protective role of peripheral NOP receptor activation was confirmed on gastric damage induced by cold‐restraint stress. Again this effect of N/OFQ was sensitive to UFP‐101 and mimicked by lower doses of UFP‐112 [141]. As far as colonic functions are concerned, these appear to be regulated by NOP receptor signaling both under normal and pathological conditions [142]. Both i.c.v. or i.p., N/OFQ and UFP‐112 increased bead expulsion time in a statistically significant and dose‐related manner and reduced the percentage of rats with castor oil‐induced diarrhea. UFP‐112 showed greater efficacy, higher potency and longer‐lasting inhibitory effects than N/OFQ. These effects of UFP‐112 were sensitive to the antagonist action of UFP‐101. When injected i.c.v., N/OFQ and UFP‐112 inhibited corticotrophin releasing factor‐ and restrain stress‐stimulated fecal pellet excretion in a dose‐related manner. Conversely, when injected peripherally both peptides inhibited colonic propulsive motility only partially and this was not dose‐related [142].

The in vivo actions of UFP‐112 in comparison to those evoked by the natural NOP agonist N/OFQ are summarized in Table 5. Analysis of this series of data suggests the following. UFP‐112 always mimicked N/OFQ actions showing, with few exceptions, 10–100‐fold higher potencies than the natural peptide. UFP‐112 consistently evoked longer lasting effects and, in some assays (indicated in Table 5 by an asterisk), it elicited larger effects than N/OFQ. The 10‐fold higher affinity reported for UFP‐112 compared to N/OFQ in receptor binding studies [93] could, at least in part, explain its high potency but not the high duration of action and size of effect. Thus, other factors should be considered. One of these is likely represented by the lower susceptibility of UFP‐112 to enzymatic degradation as demonstrated in experiments using mouse plasma and brain homogenate where UFP‐112 displayed 3–4 fold longer half‐lives than N/OFQ [94]. Another factor possibly relevant to this issue is the different kinetics of interaction with the NOP receptor suggested by isolated tissue experiments (see Fig. 3). Therefore, the combination of higher NOP affinity, lower susceptibility to protease action, and a slow onset and long‐lasting kinetics of interaction with the NOP receptor may likely be relevant to explain the in vivo features of UFP‐112. These features that is, high potency associated with the ability to induce long‐lasting effects, appear to be very consistent among species (mouse, rat, monkeys), route of administration (i.c.v., i.t., i.v., i.p.), and target organ (brain, spinal cord, airways, gut, kidney, cardiovascular system). Data obtained in vivo with the peptide [(pF)Phe⁴Aib⁷Aib¹¹Arg¹⁴Lys¹⁵]N/OFQ‐NH₂ (which is very similar to UFP‐112) measuring its ability to evoke pronociceptive effects in mice after i.c.v. administration and hypotensive effects in rats after i.v. injection [143] are virtually superimposable to those measured in response to UFP‐112: very high potency and longer lasting effects.

Table 5.

In vivo pharmacological profile of UFP‐112

Test/Assay	N/OFQ action	Effective dose	UFP‐112 action	Effective dose	References
Tail‐withdrawal (m, icv)	↓ TW latencies	1 nmol	↓ TW latencies	0.01 nmol	[94]
Tail‐withdrawal (m, it)	↑ TW latencies	1 nmol	↑ TW latencies	0.01 nmol	[94]
Locomotion (m, icv)	↓ spontaneous LA	10 nmol	↓ spontaneous LA	0.1 nmol	[94]
Rotarod (dyskinetic r, icv)	↑ motor performance	0.1 nmol	↑ motor performance	0.01 nmol	[125]
AIM (dyskinetic r, icv)	attenuation of AIM	0.1 nmol	attenuation of AIM	0.01 nmol	[125]
Food intake (Swiss m, icv)	↑ food intake	1 nmol	↑ food intake*	0.01 nmol	[94]
Food intake (NOP(+/+)m, icv)	↑ food intake	1 nmol	↑ food intake*	0.01 nmol	[94]
Food intake (NOP(−/−)m, icv)	inactive	1 nmol	Inactive	0.01 nmol	[94]
Food intake (r, icv)	↑ food intake	2.1 nmol	↑ food intake*	0.03 nmol	present article
Ethanol intake (msP r; icv)	↓ ethanol consumption	500 ng	↓ ethanol consumption	10 ng	[129, 134]
HR and BP (r, iv bolus)	↓ HR and BP	10 nmol/kg	↓ HR and BP*	0.1 nmol/kg	[94]
Diuresis (r, iv bolus)	inactive	10 nmol/kg	↑ dieresis	0.1 nmol/kg	[94]
Gastric emptying (r, icv)	↓ gastric emptying	100 pmol	↓ gastric emptying	2 pmol	[140]
Gastric secretion (r, icv)	↓ acid secretion	500 pmol	↓ acid secretion	30 pmol	[140]
Gastric secretion (r, ip)	↑ acid secretion	1000 pmol	↑ acid secretion	30 pmol	[140]
Gastric damage (r, icv)	↓ alcohol‐ induced lesions	1000 pmol	↓ alcohol‐induced lesions	100 pmol	[140]
Gastric damage (r, ip)	↓ alcohol‐ induced lesions	2000 pmol	↓ alcohol‐induced lesions	20 pmol	[140]
Gastric damage (r, ip)	↓ stress‐induced lesions	1 μg/kg/h	↓ stress‐induced lesions	0.3 μg/kg/h	[141]
Colon propulsion (r, icv)	↑ mean expulsion bead time	10 pmol	↑ mean expulsion bead time*	10 pmol	[142]
Colon propulsion (r, ip)	↑ mean expulsion bead time	10 nmol	↑ mean expulsion bead time*	1 pmol	[142]
Castor oil‐induced diarrhea (r, icv)	↓% rats with diarrhea	3000 pmol	↓% rats with diarrhea	300 pmol	[142]
Castor oil‐induced diarrhea (r, ip)	↓% rats with diarrhea	100 pmol	↓% rats with diarrhea	10 pmol	[142]
CRF‐induced fecal output (r, icv)	↓ n fecal pellet	500 pmol	↓ n fecal pellet	50 pmol	[142]
CRF‐induced fecal output (r, ip)	↓ n fecal pellet	500 pmol	↓ n fecal pellet	250 pmol	[142]
RS‐induced fecal output (r, icv)	↓ n fecal pellet	100 pmol	↓ n fecal pellet	2 pmol	[142]

m, mouse; r, rat; gp, guinea‐pig; msP r, Marchigian Sardinian alcohol‐preferring rats; icv, intracerebrentricular; it, intrathecal; iv, intravenous; TW, tail‐withdrawal; LA, locomotor activity; AIMs, abnormal involuntary movements in hemiparkinsonian rats that become dyskinetic after chronic treatment with L‐DOPA; HR, heart rate; BP, blood pressure; CRF, corticotropin releasing factor; RS, restrein stress; *indicates that the maximal effects elicited by UFP‐112 in these assays are significantly higher than those produced by N/OFQ.

Finally, it should be mentioned that the available data on the pharmacological features of UFP‐112 were mainly obtained after acute administration. The only exception is represented by alcohol intake studies where UFP‐112 has been given once a day for 6 days [134]. UFP‐112 behaves as a full agonist at NOP receptors and tolerance to long‐term exposure to agonists is a rather general phenomenon in the GPCR field; thus chronic studies with UFP‐112 are mandatory in order to establish the therapeutic potential of NOP agonists in those conditions in which a chronic treatment is required.

Conclusions

In conclusion, the present review suggests that UFP‐112 is a highly potent and selective full agonist for the NOP receptor, partially resistant to enzymatic degradation and able to produce long lasting effects in vivo. UFP‐112 can represent a useful research tool to be used together with Ro 64–6198 [69] and the recently identified nonpeptide NOP agonists SCH 221510 [72] and MCOPPB [74] in future studies aimed at identification of the potential for NOP agonists as an innovative drug class. These studies should particularly investigate those conditions and states in which a selective and prolonged stimulation of the NOP receptor is beneficial, including anxiety [79], drug addiction [81, 144], stress‐induced anorexia [145], cough and possibly other respiratory diseases [84], and visceral hypersensitivity triggered by inflammation or stress [146]. Last but not least, UFP‐112 may represent more than a simple research tool for some selected therapeutic indications. In fact, the peptide nature of this molecule does not limit its usefulness and possible drug development for those conditions in which systemic administration of drugs is not required; these include urinary incontinence due to overactive bladder where UFP‐112 can be administered intravesically [92] or chronic pain states in patients intolerant or refractory to systemic opioids where the peptide can be administered via implantable intrathecal pumps. This latter strategy was recently demonstrated to be clinically successful with the omega conotoxin analog peptide Ziconotide [147].

Disclosures

The following authors Girolamo Calo’, Domenico Regoli, Severo Salvadori, and Remo Guerrini are inventors of the patent application (WO2006087340) that includes UFP‐112 and are founders of the University of Ferrara spin off company UFPeptides s.r.l. the assignee of such patent application.

Conflict of Interest

The authors have no conflict of interest.