Nociceptin/Orphanin FQ Peptide Receptors: Pharmacology and Clinical Implications

Current Drug Targets, 2007, 8, 117-135 117

1389-4501/07 $50.00+.00 © 2007 Bentham Science Publishers Ltd.

Nociceptin/Orphanin FQ Peptide Receptors: Pharmacology and Clinical Implications

L.-C. Chiou1,2,*, Y.-Y. Liao

2, P.-C. Fan

2,3, P.-H. Kuo

4, C.-H. Wang

1, C. Riemer

5 and E. P. Prinssen

5

1Department and

2Institute of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan;

3Department of Pe-

diatric and 4Department of Internal Medicine, National Taiwan University Hospital, Taipei, Taiwan;

5F. Hoffmann-la Roche Ltd,

Basel, Switzerland

Abstract: The advance of functional genomics revealed the superfamily of G-protein coupled receptors (GPCRs). Hundreds of GPCRs have been cloned but many of them are orphan GPCRs with unidentified ligands. The first identified orphan GPCR is the opioid receptor

like orphan receptor, ORL1. It was cloned in 1994 during the identification of opioid receptor subtypes and was de-orphanized in 1995 by the discovery of its endogenous ligand, nociceptin or orphanin FQ (N/OFQ). This receptor was renamed as N/OFQ peptide (NOP) re-

ceptor. Several selective ligands acting at NOP receptors or other anti-N/OFQ agents have been reported. These include N/OFQ-derived peptides acting as agonists (cyclo[Cys10,Cys14]N/OFQ, [Arg14, Lys15]N/OFQ, [pX]Phe4N/OFQ(1-13)-NH2, UFP-102, [(pF)Phe4,Aib7,

Aib11,Arg14,Lys15]N/OFQ-NH2) or antagonists (Phe1 (CH2-NH)Gly2]N/OFQ(1-13)-NH2, [Nphe1]N/OFQ(1-13)-NH2, UFP-101, [Nphe1, (pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2), hexapeptides, other peptide derivatives (peptide III-BTD, ZP-120, OS-461, OS-462, OS-

500), non-peptide agonists (NNC 63-0532, Ro 64-6198, (+)-5a compound, W-212393, 3-(4-piperidinyl)indoles, 3-(4-piperidinyl) pyrrolo[2,3-b]pyridines) and antagonists (TRK-820, J-113397, JTC-801, octahydrobenzimidazol-2-ones, 2-(1,2,4-oxadiazol-5-yl)-1 H-

indole, N-benzyl-D-prolines, SB-612111), biostable RNA Spiegelmers specific against N/OFQ, and a functional antagonist, nocistatin. Buprenorphine and naloxone benzoylhydrazone are two opioid receptor ligands showing high affinity for NOP receptors. NOP receptor

agonists might be beneficial in the treatment of pain, anxiety, stress-induced anorexia, cough, neurogenic bladder, edema, drug depend-ence, and, less promising, in cerebral ischemia and epilepsy, while antagonists might be of help in the management of pain, depression,

dementia and Parkinsonism. N/OFQ is also involved in cardiovascular, gastrointestinal and immune regulation. Altered plasma levels of N/OFQ have been reported in patients with various pain states, depression and liver diseases. This review summarizes the pharmacologi-

cal characteristics of, and studies with, the available NOP receptor ligands and their possible clinical implications.

Key Words: Nociceptin/Orphanin FQ, Nocistatin, Ro 64-6198, J-113397, SB-612111, W-212393, [Nphe1]N/OFQ(1-13)-NH2, UFP-101,

UFP-102.

1. INTRODUCTION

After the human genome was revealed, hundreds of G-protein coupled receptors (GPCRs) were cloned. They are a super family of proteins with 7-transmembrane domains and believed to be the most important target for drug development [1]. The endogenous ligands for some of the cloned GPCRs have been identified to be various neurotransmitters while the nature of other cloned GPCRs is still unrevealed. These GPCRs with their binding ligands uniden-tified are called orphan GPCRs. The first identified orphan GPCR was cloned in 1994 from a search to discover subtypes of opioid receptors and was called opioid receptor like 1 (ORL1) orphan receptor because of the high homology to traditional opioid recep-tors [2]. One year later, this orphan receptor was de-orphanized by the identification of its endogenous heptadecapeptide ligand, simul-taneously by two research groups. This peptide was named, from its first and last amino acids, as orphanin FQ by Reinscheid et al. [3] and as nociceptin for its pronociceptive action by Meunier et al. [4]. The ORL1 receptor was classified as the fourth member of the opioid receptor family and renamed as N/OFQ peptide (NOP) re-ceptor by NC-IUPHAR [5].

N/OFQ is derived from a precursor protein, preproN/OFQ (ppN/OFQ) which consists of 181, 187 and 176 amino acids, re-spectively, in the rat, mouse and human [6]. The mRNA levels of ppN/OFQ are widely distributed in the brain of rats [7], humans [6] and mice [8] and their expressions correlate well with the distribu-tion of immunoreactivity of N/OFQ labeled with polyclonal antibo-dy in the rat brain [7]. The distribution of N/OFQ in the human brain as demonstrated by radioimmunoassay is also similar to that in the rat brain [9]. The distribution of NOP receptors, which was

*Address correspondence to this author at the Department of Pharmacology,

College of Medicine, National Taiwan University, No. 1, Jen-Ai Rd., Sec-

tion 1, Taipei 100, Taiwan; Tel: 886-2-2312-3456, ext. 8323; Fax: 886-2-

2341-4788; E-mail: [email protected]

revealed by in situ hybridization for the mRNA distribution [10] or immunolabeling for the protein distribution [11], or by autoradio-graphic studies using the radioligand, [

3H]N/OFQ [12] or [

125I]

[14

Tyr]N/OFQ [10], correlates mostly with that of N/OFQ in the brain of rats [10, 11], mice [12] and humans [13].

Seeing the wide distribution of N/OFQ and NOP receptors in the brain, it is not surprising that many central actions of N/OFQ have been suggested from animal studies, including supraspinal hyperalgesia, spinal analgesia, hyperphagia, depression, and inhibi-tions of anxiety, epilepsy, cough, motor activity, and learning and memory, as well as the regulation of cardiovascular, urogenital, gastrointestinal and immune systems. Many efforts have been en-deavored in the development of agonists or antagonists of this novel member of the opioid receptor family. A special issue focusing on N/OFQ and NOP receptors was published in the July issue of Pep-tides in 2000 and several comprehensive reviews have also been published [14-16]. The present review summarizes and updates the NOP receptor ligands developed so far, the physiological or patho-logical roles of N/OFQ obtained from the studies using these ligands, ppN/OFQ knockout (ppN/OFQ

-/-) mice or NOP receptor

knockout (NOP-/-

) mice, as well as the potential clinical applica-tions of NOP receptor ligands.

2. NOP RECEPTOR AGONISTS AND ANTAGONISTS

Specific NOP receptor ligands have been developed since 1998, three years after this orphan GPCR was de-orphanized. [Phe

1

(CH2-NH)Gly2]N/OFQ(1-13)-NH2, developed by Calo’s group

[17], is the first reported N/OFQ-modified ligand selective for NOP receptors. Thereafter, several peptide and non-peptide agonists or antagonists have been developed [18-21]. Currently reported puta-tive NOP receptor agonists and antagonists are summarized in Ta-bles 1 and 2, respectively. For a very comprehensive overview on recent advances in the development of new N/OFQ ligands, see also the Expert Opinion by Bignan et al. [20].

118 Current Drug Targets, 2007, Vol. 8, No. 1 Chiou et al.

2.1. NOP Receptor Agonists

2.1.1. Peptide Agonists

Since N/OFQ was identified, intensive efforts have been con-ducted to reveal the structure-activity relationship [20] of this novel opioid receptor family member and its ligands. Modified peptides have been synthesized to give a shorter aminated peptide,

N/OFQ(1-13)-NH2, which is equally potent to N/OFQ. Substituting the 14

th and 15

th residues with Arg and Lys, respectively, gave an

agonist, [Arg14

, Lys15

]N/OFQ, more potent than N/OFQ [22, 23]. Conformationally restricted peptide synthesis has also generated an agonist, cyclo[Cys

10,Cys

14]N/OFQ, with comparable affinity to

N/OFQ [24]. Calo’s group found that substituting the -F or -NO2 group at the para position of the Phe

4 residue of N/OFQ(1-13)-NH2

Table 1. Putative NOP Receptor Agonists

Ki† /EC50

‡ (nM)

Compound

NOP Other Receptors /Transporters

References

Peptide

OFQ/N 1†/1.44‡ [3]

Cyclo[Cys10,Cys14]N/OFQ 0.12†/4.37‡ [24]

[Arg14, Lys15]N/OFQ 0.32†/0.76‡ [22, 23]

[pX]Phe4N/OFQ (1-13)-NH2 0.015-71†/0.1-1047‡ [25]

[(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 (UFP-102) 0.02†/0.067-2.69‡ [26]

[(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 0.43-0.52‡ [21]

OS-461, OS-462, OS-500 0.43-0.9†/11-190‡ [27]

Non-peptide

Buprenorphine 8.4‡ 0.51‡ (MOP) [31, 203]

Ro 64-6198 0.3-0.39†/0.32‡

36-47† (MOP)*

89-214† (KOP)

1380-3787† (DOP)

[35, 183]

NNC 63-0532 7.3†/305‡

209† (D2)

133† (D3)

140† (MOP)

405† (KOP)

[39, 40]

(+)-5a compound 0.55†/65‡

537† (MOP)*

309† (KOP)

2138† (DOP)

[41]

W-212393 0.5†

76† (MOP)*

>1000† (KOP)

>1000† (DOP)

11† (SERT)

[42]

3-(4-piperidinyl)indolesa 18†/27200‡

500† (MOP)

1890† (KOP)

>5000† (DOP)

[43]

3-(4-piperidinyl)pyrrolo[2,3-b]pyridinesb 4†/4200‡

390† (MOP)

2780† (KOP)

>5000† (DOP)

[43]

†: Ki; ‡: EC50; SERT: serotonin transporter.

OS-461: N- -6-guanidinohexyl-ltyrosil-L-tyrosyl-L-arginyl-L-tryptophanamide.

OS-462: N- -6-guanidinohexyl-3,5-dimethyl-ltyrosyl-L-tyrosyl-N-[(R)-1-(2-naphthyl)ethyl]-L-argininamide.

OS-500: N- -6-guanidinohexyl-3,5-dimethyl-L-tyrosyl-3,5-dimethyl-L-tyrosyl-N-[(R)-1-(2-naphthyl)ethyl]-L-argininamide.

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.

NNC 63-0532: (8-naphthalen-1-ylmethyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl)-acetic acid methyl ester.

(+)-5a compound: (3aS,6aR)-1-(cis-4-Isopropylcyclohexyl)-5'-methyl-2'-phenylhexahydrospiro[piperidine-4,1'-pyrrolo[3,4-c]pyrrole].

W-212393: 2-{3-[1-((1R)-acenaphthen-1-yl)piperidin-4-yl]-2,3-dihydro-2-oxo-benzimidazol-1-yl}-N-methylacetamide. aThe data of the best congener, compound 11, are shown. bThe data of the best congener, compound 23, are shown.

* Nonspecific binding activity at receptors other than opioid receptors was discussed in Section 2.2.2.

Nociceptin/Orphanin FQ Peptide Receptors Current Drug Targets, 2007, Vol. 8, No. 1 119

gave a more potent and longer acting peptide agonist [25]. Thereaf-ter, they synthesized an even more potent and longer acting agonist, [(pF)Phe

4,Arg

14,Lys

15]N/OFQ-NH2 (UFP-102) [26]. It displays 200

times selectivity over other opioid receptors and has an affinity for NOP receptors 50 times higher than N/OFQ and longer in vivo ac-tivity [26]. Peng et al. [21], by substituting the 7th and 11th amino acid residues of UFP-102 with aminoisobutyric acid (Aib), devel-oped a more potent long-acting peptide agonist of NOP receptors (Table 1). Very recently, Economidou et al. verified the hyperphagic effect of 3 new peptide agonists, termed OS-500, OS-462 and OS-461 [27], which exhibit an affinity for NOP receptors in the subnanomolar, and efficacy in the low to moderate nanomolar range, but no selectivity data were provided (Table 1). They, how-ever, found that OS-500 and OS-462, but not OS-461, induced hy-perphagia [27].

2.1.2. Non-Peptide Agonists

When the NOP receptor was identified, several available ligands of other receptors have been reported to have good affinity and act as agonists at NOP receptors, including buprenorphine, lofentanil, spiroxatrin or pimozide [28, 29]. Among these, bu-prenorphine is noteworthy. It is a semisynthetic lipophilic deriva-tive of an opiate alkaloid, thebaine, and is used clinically in the pain management and for the maintenance therapy of opiate addiction. This compound has been filed for Phase II clinical trials for cocaine addiction [20]. It was also reported to reduce alcohol consumption possibly through NOP receptor activation and might be useful, in combination with naltrexone, in the treatment of severe alcoholism [30]. It was long known as a partial agonist of μ-opioid (MOP) and antagonist of -opioid (KOP) receptors but was later found to be a full agonist of NOP receptors [31]. In addition, buprenorphine also acts as an antagonist of -opioid (DOP) receptors [32]. These mul-tiple actions of buprenorphine at opioid receptors and the different pharmacological profile of its active metabolite, norbuprenorphine [32], complicate the understanding of the mechanisms that underlie its analgesic and anti-addictive effects [33, 34].

In 2000, F. Hoffmann-La Roche Ltd. presented the first syn-thetic non-peptide agonist, Ro 64-6198 (Table 1), with subnanomo-lar affinity and high selectivity for NOP receptors [35]. It has no affinity for 44 other receptors or channels and only micromolar affinity for -opioid, histamine H2, , dopamine D2 and Na

+ chan-

nel-site 2 [35]. In two assays of cAMP inhibition and GTP- -S binding at cloned human NOP receptors, Ro 64-6198 acted as a full agonist with similar potency as N/OFQ [35]. However, at native NOP receptors in the rat ventrolateral periaqueductal gray (vlPAG), this compound was shown to be a weak agonist of NOP receptors that mediate K

+ channel activation in only one third of the recorded

neurons [36]. In the same preparation, N/OFQ is effective at NOP receptors in almost all recorded neurons. In Ro 64-6198-insensitive neurons, N/OFQ is effective in activating K

+ channels through NOP

receptors [36]. These findings suggest that there are N/OFQ-sensitive but Ro-646198-insenstive NOP receptors in the rat brain. Similar functional heterogeneity of NOP receptors may be deduced from the findings that Ro 64-6198 produced some, but not all, re-sponses generated by N/OFQ in the central regulations of nocicep-tive response, motor activity, heart rate, and renal sympathetic ac-tivity as well as that in the mouse vas deferens [36, 37]. It is sug-gested that heterogeneity of NOP receptors exists in the brain and periphery and that Ro 64-6198 might act at a subset of NOP recep-tors [36]. Recently, Gehlert et al. reported that Ro-64-6198 displays similar potency and activity as N/OFQ in increasing [

35S]-GTP- -S

binding in the rat brain homogenate isolated from various brain regions [38]. However, it is interesting to note that among the vari-ous brain regions investigated, the central gray is the area in which Ro 64-6198 displayed the lowest, only one-third, activity as com-pared to N/OFQ [38].

Novo Nordisk [39], in 2000, also presented a non-peptide ago-nist, NNC 63-0532 (Table 1), with 12-fold selectivity for NOP

receptors, as compared with MOP receptors. However, Guerrini et al., [40] found that although this compound, like N/OFQ, inhibited the contraction induced by electrical stimulation in guinea pig ileum preparations, its effect was blocked by an antagonist of MOP, but

not NOP, receptors.

In 2003, F. Hoffmann-La Roche Ltd. presented another selec-tive non-peptide agonist, (+)-5a compound [41] (Table 1), which acted as a full agonist, with subnanomolar affinity, at cloned NOP receptors in two different in vitro assays and showed selectivity over 30 different receptors and channels. Only at concentrations of 10 μM or above, did this compound interact with histamine H3, muscarinic and receptors as well as Na

+ channels [41]. Interest-

ingly, we found this compound displayed similar pharmacological characteristics as Ro 64-6198 in the NOP receptors of midbrain vlPAG neurons (Liao, Prinssen, Kolczewski and Chiou, unpub-lished data). It, like Ro 64-6198, activated a subset, but not all, of NOP receptors which mediated K

+ channel activation in vlPAG

neurons.

Recently, Teshima et al. [42] revealed a brain penetrating non-peptide agonist, termed W-212393, which has subnanomolar affin-ity for NOP and similar efficacy as N/OFQ. This compound is fairly selective versus other opiate receptors, but also exhibits a Ki of 11 nM for the serotonin transporter (Table 1). When i.p. adminis-tered, this compound significantly accelerated the re-entrainment of

the body temperature rhythm to a 6h advanced light-dark cycle.

Most recently, Bignan et al. [43] presented novel piperidin-indoles and pyrrolo-pyridines as NOP receptor agonists with low nanomolar affinity and more than 25-100 times selectivity versus

other opioid receptors (Table 1).

2.2. NOP Receptor Antagonists

2.2.1. Peptide Antagonists

Through a combinational library screen, Dooley et al. [44] identified 5 acetylated hexapeptides having high affinity for NOP receptors. Among these, Ac-RYYRIK-NH2 and Ac-RYYRWK-NH2 were actively studied. Both peptides were reported to be potent NOP receptor antagonists but were later found to have intrinsic activity at NOP receptors and act as agonists or partial agonists [45-49]. Nevertheless, these hexapeptides have no in vivo activity [18]. Zealand Pharma synthesized a peptide derivative, ZP-120 (Ac-RYYRWKKKKKK-NH2) (Table 2) with the aim of an increased half life and not to penetrate the CNS. This compound acted as a partial agonist at NOP receptors with higher potency and longer duration than N/OFQ [50, 51]. In addition, a conformational re-stricted peptide, peptide III-BTD, was identified in 1999 from the combinational library to be a NOP receptor antagonist [52]. How-ever, this compound also acted as an agonist at MOP and DOP receptors [52, 53]. A retro-N/OFQ methylester with oppositely directed structure to N/OFQ was synthesized and shown to reverse N/OFQ-induced inhibition of guinea pig ileum contraction in vitro. This peptide had low affinity (submilimolar) for NOP and could not reverse N/OFQ-induced hyperalgesia in mice at the maximal solu-ble dose. When given alone, however, it produced analgesia and improved the performance in the step-through passive avoidance

test [54].

In 1998, [Phe1

(CH2-NH)Gly2]N/OFQ(1-13)-NH2 was pre-

sented by Calo’s group at the University of Ferrara, Italy, as the first selective peptide antagonist of NOP receptors using bioassays with peripheral preparations [17]. However, this peptide was later found to have intrinsic activity at NOP receptors and to act either as a full agonist at cloned human NOP receptors or as a partial agonist at NOP receptors in vlPAG neurons [55]. It is now believed that this peptide can act as a full agonist, partial agonist or silent an-tagonist [14, 55, 56] depending on the level of expressed NOP re-ceptors and the downstream coupling efficiency in the assay sys-tems [57].


Calo’ et al. [58] later developed a selective peptide antagonist without intrinsic activity, [Nphe

1]N/OFQ(1-13)-NH2 (Nphe). Sev-

eral physiological or pathological roles of N/OFQ have been re-vealed by using this pure and selective antagonist (see Section 3). Thereafter, they developed another peptide antagonist, [Nphe

1,

Arg14

,Lys15

]N/OFQ-NH2 (UFP-101) with less susceptibility to the peptidase [59]. This peptide has been proven to be a more potent antagonist than [Nphe

1]N/OFQ(1-13)-NH2 and to have no intrinsic

activity at NOP receptors in several in vitro and in vivo studies [60, 61]. Recently, Peng et al. [21], by substituting the 7th and 11th amino acid residues of UFP-101 with aminoisobutyric acid (Aib) (Table 1), developed a novel peptide competitive antagonist of NOP receptors with very high potency using both in vivo and in vitro assays.

2.2.2. Non-Peptide Antagonists

Naloxone benzoylhydrazone (NalBzOH) was the only opioid receptor ligand displaying antagonistic activity at NOP receptors when this orphan receptor was identified. It had been used to reveal the functional role of N/OFQ in pain regulation [62] before more selective non-peptide antagonists were reported [55]. However, it has higher affinity at classical opioid receptors, as compared to NOP receptors [63, 64] (Table 2). A morphinan derivative, TRK-820, was reported in 1999 [65] to be an antagonist of NOP recep-tors with submicromolar binding affinity. However, this compound also acts as a KOP receptor agonist and MOP receptor partial ago-

nist [66] (Table 2). This non-specific property makes this com-pound less useful as a tool in the studies of NOP receptor-mediated functions. Nevertheless, it is interesting to note that when s.c. ad-ministered, it exhibited more potent antinociceptive activity than morphine. This compound was licensed to Daichi, Japan (Nal-furafine) and is under phase II clinical trials for the treatment of pruritus [20].

In 1999, Banyu Pharmaceutical Company, Japan, presented the first non-peptide antagonist selective for NOP receptors, 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1, 3-dihydro-2H-benzimidazol-2-one, which was coded as J-113397 [67]. Quantitative studies indicated that it is a competitive antagonist of cloned NOP receptors and native NOP receptors in brain tissues with pA2 values of 8.2 ~ 8.9 [68-70] (Table 2). Banyu Company renamed this chiral isomer as CompB, which is more potent than the racemic mixture [71]. It seems that this compound is a potent and promising antagonist as a tool to reveal the physiological or pathological roles of endogenous N/OFQ. Indeed, CompB has been used to reveal the roles of N/OFQ in the supraspinal and spinal pain regulation, morphine tolerance, and the motor activity control in the nigrostriatal dopamine system (see Section 3, Tables 5, 6). We tested J-113397 (prepared at Hoffmann-La Roche) in a broad CEREP screen (www.cerep.fr) and found that it displays affinity at receptors other than NOP receptors. At 100 nM, it had no signifi-cant binding at 71 non-opioid receptors and channels, except the

Table 2. Putative NOP Receptor Antagonists

Compound pA2†/pIC50

‡ pKi pEC50 Other Receptor Reference

Peptides

Ac-RYYRIK-NH2 / Ac-RYYRWK-NH2 8.7-9.1† 8.1-8.3 8.1-9.3 – [44, 47, 204]

ZP-120 (Ac-RYYRWKKKKKKK-NH2) 9.5† 9.6 8.88 – [50, 51]

Peptide III-BTD 6.6-6.9† – – MOP agonist; DOP agonist [53]

[Phe1 (CH2-NH)Gly2]N/OFQ(1-13)-NH2 6.7-7.6†/6.1‡ 8.9-9.6 6.9-8.5 – [14, 17, 47]

[Nphe1]N/OFQ(1-13)-NH2 6.0-8.45† – – – [58]

[Nphe1,Arg14,Lys15]N/OFQ-NH2 (UFP-101) 6.9-9.1† 10.2 – – [59, 61]

[Nphe1,(pF)Phe4,Aib7,Aib11,Arg14,Lys15]N/OFQ-NH2 8.4-8.5† – – – [21]

Non-peptides

Naloxone benzoylhydrazone 5.7-6.9† 7.3-7.6 6.1-7 MOP partial agonist; KOP agonist [55, 64, 205]

TRK-820 – 6.42 – KOP agonist; MOP antagonist [18, 65, 66]

J-113397 (CompB) 8.2-8.9†/7.6-8.3‡ 8.56 – –* [68-70]

Trap-101 (Achiral analogue of J-113397) 7.75† – – – [74]

JTC-801 5.59‡ 7.35 – –* [76]

SB-612111 8.3† 9.48 – –* [78]

Octahydrobenzimidazol-2-onesa – 8.0 – MOP antagonist [19]

2-(1,2,4-oxadiazol-5-yl)-1 H-indoleb 7.3-7.4‡ – – – [79]

N-benzyl-D-proline spiropiperidinec 9.82‡ 9.6 <6 –* [80]

†: pA2, ‡:pIC50, * Nonspecific binding activity at receptors other than NOP receptors was discussed in Section 2.2.2.

TRK-820: (-)-17-cyclopropylmethyl-3, 14b-dihydroxy-4, 5a-epoxy-6b-[N-methyl-trans-3-(3-furyl)acrylamido]morphinan hydrochloride.

J-113397: 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one.

JTC-801: N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide.

SB-612111: (-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol. aThe data of the best congener, compound 23, are shown. bThe data of the best congeners, compounds 39 and 67 , are shown. cThe data of the best congener, compound 24, are

shown.


sigma receptor at which 64 % displacement of the control binding was observed. At 10 μM, it displaced more than 50 % binding of the radioligand for 12 non-opioid receptors and channels. Among these, higher displacement bindings (>80%) were observed for dopamine D3- and D4.-, 5-HT1A- and -receptors, and Na

+ channel-

site 2 (Supplement I). Its affinities at NOP and the other opioid receptors are in agreement with the published affinities [67]. The finding of high affinity binding at -receptors is in agreement with the report of Bolser et al. [72], in which J-113397 was cited to have binding affinity at -receptors at concentrations less than 10 nM. Therefore, one has to be cautious to attribute the effect(s) of J-113397 to NOP receptors, especially in systems where -receptors may play a role. Indeed, Koizumi et al. [73] found that J-113397 stimulates mesolimbic dopamine release and is rewarding in mice through a mechanism not mediated by NOP receptors.

Later, Calo’s group synthesized a high yielding achiral ana-logue of J-113397, termed Trap-101 (Table 2), which is equipotent to the racemate of J-113397 [74]. Recently, Chen et al. at Purdue Pharma presented saturated structural congeners of J-113397 which have high (lower nanomolar range) potency at NOP receptors but only moderate (~50-fold) selectivity towards MOP receptors [19].

Japan Tobacco Inc., in 2000, presented a non-peptide antago-nist, JTC-801 [75], which displays moderate affinity/potency at NOP receptors but has limited selectivity over other opioid recep-tors, being 12, 129, and 1040 versus MOP, KOP and DOP recep-tors, respectively [76]. Nevertheless, this compound was filed for clinical trials as a novel analgesic and is orally bioavailable [77]. We also tested this compound (prepared at Hoffmann-La Roche) in a broad CEREP screen (Supplement II). Whereas, at 100 nM, no significant binding was observed at 71 non-opioid receptors and channels, at 10 μM more than 50 % binding was observed for more than 20 non-opioid receptors and channels. Among these higher bindings (>80%) were observed for dopamine D1 and D5, histamine H2, 5-HT2A and 5-HT2C, muscarinic M2, and neurokinin NK2 recep-tors, Na

+ and Ca

2+ channels, and the norepinephrine transporter

(Supplement II). Its affinities at NOP and other opioid receptors are comparable to the published affinities [75].

In 2004, a potent and selective non-peptide antagonist, SB-612111, was reported by GlaxoSmithKline Pharmaceuticals [78]. This compound displayed comparative affinity (subnanomolar) and antagonistic potency at NOP receptors as J-113397 (Table 2) with hundred-fold selectivity versus classical opioid receptors [78]. It had submicromolar binding activity at 1A-, 2A-, 2C- and 2-adrenergic and H2-histamine receptors [78].

Recently, Sugimoto et al. at Banyu Company [79] presented novel indole derivatives as N/OFQ receptor antagonists of moderate affinity. The best congeners reached a pIC50 of 7.4 (Table 2). No selectivity data or additional characterization was provided. Later, Goto et al. [80] at the same company identified structural diverse analogs of spiropiperidines for NOP receptor ligands in a 3D-pharmacophore similarity screen followed by a further optimization in a focused library approach. The best congener achieved an IC50 at NOP receptors of 0.27 nM with more than 1000-fold selectivity within the opioid receptor family and high brain penetration (Table 1). Almost simultaneously, Mustazza et al. [81] presented several spiropiperidines with a different heterocyclic core, quinazolines and triazoloquinazolines, as opposed to isobenzofuran based spi-ropiperidines from the Banyu group, as NOP receptor antagonists and mostly partial agonists. Their compounds are significantly less potent with the best congener just reaching the submicromolar range, and less selective towards other opioid receptor subtypes.

2.3. Other Anti-N/OFQ Agents

2.3.1. Functional Antagonist: Nocistatin

Nocistatin is another encoded product of ppN/OFQ, the precur-sor of N/OFQ [2]. It was initially identified in the spinal cord to be

able to counteract N/OFQ-induced antinociception without binding to NOP receptors [82]. In addition to acting as a functional antago-nist of N/OFQ in the spinal cord, nocistatin has been reported to reverse central regulatory effects of N/OFQ in pain processing, anxiety, food intake, and learning and memory, but not that in car-diovascular control (Table 3). In in vitro studies, nocistatin failed to reverse K

+ channel activation or Ca

2+ channel inhibition induced by

N/OFQ in various brain tissues, although it did produce a weak reversal of N/OFQ-induced K

+ channel activation in spinal neurons

(Table 4). We found that nocistatin was ineffective in reversing the N/OFQ-induced K

+ channel activation in most of the recorded

vlPAG neurons except a small portion of neurons (8 out 28 re-corded neurons) (Table 4). Since nocistatin does not act at NOP receptors [82], a prerequisite to have an interaction between no-cistatin and N/OFQ on the same neuron is that NOP receptors and the yet to be identified nocistatin receptors [83] have to be co-expressed on the same neuron. The reason why nocistatin reverses the effect of N/OFQ observed in our in vitro study but not in others (Table 4) is not clear. It might be that: 1) more neurons were tested in our study (n=28) than those (n=5~8) in most of other studies, and 2) the brain region studied plays a role, i.e. the PAG is an area en-riched with ppN/OFQ and NOP receptors. Therefore, the possibility of obtaining the nocistatin-sensitive neurons might be higher.

2.3.2. Biostable RNA Spiegelmers Selective for N/OFQ

It is interesting to note that two biostable RNA Spiegelmers, NOX 2137 a/b and NOX 2149, with high affinity and selectivity to N/OFQ were reported [84]. Spiegelmers are L-enantiomeric oli-gonucleotides, which are nucleotidase-resistant and can be regarded as the biostable oligonucleotide analogues of antibodies against the target proteins, and are therefore suited for diagnostic and therapeu-tic application. With the biostable characteristic and the specificity targeting to N/OFQ, the applications of these Spiegelmers could shed light on the functional roles of N/OFQ in the periphery or CNS in the future.

3. FUNCTIONAL ROLES AND CLINICAL IMPLICATIONS

OF N/OFQ

A wide range of pharmacological effects of N/OFQ and NOP receptor agonists has been reported [15, 16, 85], which are summa-rized below. The physiological or pathological roles of endogenous N/OFQ were also revealed by various approaches, includeing using NOP receptor antagonists and the antisense oligonucleotide against NOP receptors (antisense-NOP) or ppN/OFQ (antisense-ppN/OFQ), or using NOP receptor knockout (NOP

-/-) or ppN/OFQ knockout

(ppN/OFQ-/-

) mice. Tables 5 and 6 summarize the results by using these approaches, from which the roles of endogenous N/OFQ can be revealed. However, these approaches have their limitations. The limitation of using NOP receptor antagonists is their possible non-specificity or intrinsic activity at NOP receptors. On the other hand, this approach allows distinguishing the roles of N/OFQ in distinct brain regions by a direct administration in the selected region, which is impossible when using knockout mice. The conclusions made from studies using knockout mice might be confounded by possible developmental compensations. A further possible con-found using ppN/OFQ

-/- mice is the fact that nocistatin, the func-

tional antagonist of N/OFQ, has also been nullified simultaneously.

3.1. Pain Regulation

Given that NOP receptors are part of the opioid receptor family, it is not surprising that the studies in revealing the roles of endoge-nous N/OFQ have focused on pain regulation (Table 5). N/OFQ produces controversial effects in pain regulation, such as supraspi-nal hyperalgesic but spinal analgesic effects [15]. It seems that the results obtained in the studies investigating the role of N/OFQ in-pain regulation at the supraspinal level are more homogenous than that in the spinal or systemic level (Table 5). All the studies con-ducting i.c.v. blockade of NOP receptors obtained antinociceptive


responses with only one exception (Table 5). Morphine-induced supraspinal analgesia was also enhanced by Nphe and antisense-ppN/OFQ (Table 5). This may suggest that there is a basal nocicep-tive tone produced by endogenous N/OFQ at the supraspinal level. Since NOP receptor activation can reduce stress effects per se (see Section 3.3) and the hyperalgesic effect of i.c.v. N/OFQ in mice has been attributed to an inhibition of stress-induced analgesia [15], the nociceptive tone of endogenous N/OFQ might be - at least in part - an effect secondary to its anti-stress effect. The tolerance induced by low doses of morphine was reversed by all the tested NOP re-ceptor antagonists and was prevented in NOP

-/- and ppN/OFQ

-/-

mice (Table 5). However, the tolerance developed by escalating doses of morphine was not prevented in ppN/OFQ

-/- mice [86, 87].

Therefore, the supraspinal nociceptive tone of endogenous N/OFQ might contribute to the tolerance development by low doses of morphine.

At the spinal level, N/OFQ was found to have an analgesic effect in rodents [14-16, 88] and primates [89]. Ro 64-6198 (i.t.) was recently reported to have an antiallodynic effect mediated by NOP receptors in a neuropathic pain model [37]. The role of endo- geneous N/OFQ at the spinal level in pain regulation seems to be controversial (Table 5). Intrathecal Nphe increased the flexor reflex in spinalized rats [90], and N/OFQ and NOP immunoreactivities were upregulated in the dorsal root ganglia of neuropathic and in-flamed rats [91]. Intrathecal injection of UFP-101 and J-113397 in rats [92] and mice [93], respectively, enhanced the phase II noci-ceptive responses in the formalin test. However, intrathecal injec-tion of NOP receptor antagonists did not affect the nociceptive response in the acute pain models [93, 94]. Intrathecal Nphe could

block the analgesic effect induced by electroacupuncture in the inflammatory rat model [95]. These studies suggest that endoge-nous N/OFQ in the spinal cord play a protective role in the inflam-matory, but not acute, pain status. However, negative results have been reported by intrathecal injection with J-113397 in an inflam-matory pain model [95] or with Nphe [96] or JTC-801 [97] in neu-ropathic pain models. On the contrary, intrathecal injection of JTC-801 decreased the nociceptive behaviors in both phases [97] or the second phase [98] of the formalin test as well as the tactile allo-dynia in the L5/L6 nerve ligation models [98]. Intrathecal N/OFQ at lower dose, on the contrary, has a NOP receptor-mediated pronoci-ceptive effect [97, 99]. The antinociceptive effect of intrathecal JTC-801 might be mediated through blocking this pronociceptive effect of endogenous N/OFQ in the spinal cord.

The phase II nociceptive responses in the formalin test, which are generally assumed to be due to central sensitization of spinal cord nociceptive neurons [100], were also enhanced in both NOP

-/-

and ppN/OFQ-/-

mice [93, 101]. However, the nociceptive responses in acute pain models were unchanged either in NOP

-/- mice or when

NOP receptor antagonists were given systemically in most of the studies (Table 5). These studies suggest N/OFQ might have an anti-nociceptive role in the inflammatory, but not acute, pain state. On the contrary, analgesic effects have been observed after systemic administration of J-113397 [102] or SB 612-111 [78] in the in-flammatory, but not acute, pain model, while the novel peptide antagonist developed by Peng et al. had an antinociceptive effect in the mouse tail withdrawal assay [21]. Nevertheless, systemic ad-ministration of JTC-801 induced analgesic effects in all tested mod-els (Table 5). Given the above-mentioned potential non-specificity

Table 3. Interactions of Nocistatin and N/OFQ- In Vivo Studies

N/OFQ actions Route Counteracted by Nocistatin References

Spinal analgesia i.t. Yes [82]

Flexor reflex inhibition i.t. Yes [206]

Depress thermal hyperalgesia i.t. Yes [207]

Anti-opioid analgesia supraspinally i.c.v. Yes [208, 209]

Inhibition of thalamic neuron firing local Yes [210]

Hyperalgesia i.c.v. Yes* [211, 212]

Morphine tolerance i.c.v. * [213]

Potentiation of acupuncture analgesia i.c.v. * [214]

Anxiolytic effect (low dose)

Axiogenic effect (high dose)

i.c.v.

i.c.v.

Yes

Yes

[127]

[127]

Hyperphagia i.c.v. Yes [215]

Inhibition of baroreceptor reflex i.c.v. Yes [216]

Impairment of learning and memory i.c.v. Yes [151]

Improvement of scopolamine-induced amnesia (low dose) i.c.v. No [153]

Central modulation of renal sympathetic activity i.c.v. No [217]

Central cardiovascular depression i.c.v. No [217]

Transient hypotension i.v. No [218]

Inhibition of neurogenic dural vasodilation i.v. No [218]

* Nocistatin alone induced an effect opposite to that produced by N/OFQ.


Table 4. Interactions of Nocistatin and N/OFQ- In Vitro Studies

N/OFQ actions N/OFQ

(μM)

Counteracted

by Nocistatin

Nocistatin

(μM) N number Reference

Inhibition of anococcygeus muscle contraction 0.001-1.3 No 1 5 [219]

Inhibition of glutamate release 0.1 Yes 0.1 6 [220]

Activation of K+ channels in substantia gelatinosa neurons 1 Yes 1 6 [221]

Activation of K+ channels in hippocampal CA3 neurons 1 No

1

10

100

6

4

4

[222]

Activation of K+ channels in hypothalamic neurons 1 No 10 6 [217]

Activation of K+ channels in rostral ventromedial medulla 0.3 No 1 7 [223]

Activation of K+ channels in amygdala 1 No 1

5

3

N/A [224]

Activation of K+ channels in trigeminal neurons 1 No 0.3

3

8

3 [225]

Inhibition of Ca2+ channels in locus cereulus 0.03 No 10 8 [226]

Inhibition of Ca2+ channels in rostral ventromedial medulla 0.3 No 10 8 [223]

Activation of K+ channels in PAG neurons 0.3 Partial

0.3

0.5

1

10/14*

6/8*

4/6*

*

*In a few neurons, the effect of N/OFQ was partially reversed by nocistatin. Among the tested neurons, 4, 2 and 2 neurons, respectively, were sensitive to nocistatin of 0.3, 0.5 and 1

μM, respectively (Chiou et al., unpublished data).

Table 5. Changes in Pain Regulation and Morphine Effects after N/OFQ-NOP Receptor Blockade by NOP Receptor Antagonists,

Antisense-NOP and Antisense-ppN/OFQ, or in NOP-/-

and ppN/OFQ-/-

Mice

NOP receptor antagonists

Functions

Nphe UFP-101 J-113397 JTC-801 SB 612-111 NalBzOH

NOP-/-

ppN/OFQ-/-

Antisense

NOP

Antisense

ppN/OFQ

Pain Regulation

Supraspinal

pain [58]a

[59]a

[93]g

[102]h # [92]e

[227]e

[137] b

Spinal pain

[96]f

[90]i

[94]d

# [93]g [102]h, e

# [92]e

[97]f

[97]g

# [98]e, f

General

pain

[68, 102]c

[92]e

[102]h, e, f

[103]j

[93]g

[75]b

[228]f

[76]f, b, d

[78]c

[78]h [62]a

[62, 138,

229, 230]a, c

# [101]g

[93]g

[231]a

# [101]g

[86]

Morphine effect

Analgesia [232]g [163]b

Tolerance [232]

[114]

[86]

[78] [114]

[86]

[87]*

[86]*

: increase; : no change; : decrease; a: mouse tail flick test; b: rat tail flick test; c: mouse hot plate test; d: rat hot plate test; e: rat formalin test; f: never injury in rats; g: mouse forma-lin test; h: rat carrageenin test; i: rat flexor reflex; j: thermal nociception in monkey; # phase II only; *: High doses of morphine.


Table 6. Functional Changes after N/OFQ-NOP Receptor Blockade by NOP Receptor Antagonists, Antisense-NOP and Antisense-

ppN/OFQ, or in NOP-/-

and ppN/OFQ-/-

Mice

NOP receptor antagonist

Functions

Nphe UFP-101 J-113397

NOP-/-

ppN/OFQ-/-

Antisense

NOP

Antisense

ppN/OFQ

Drug dependence

Morphine [114] [114] [87]

[86]*

Alcohol [124]

Dopamine level (Mesolimbic) [233] [233]

Stress

Anxiety-like [138] [87, 135, 136] [137]

Corticosterone level [140] [135] [137]

Feeding

Normal [147] [27]

Under Stress [147]

Striatal activity

Motor activity [159] [159] [159] [161]

[162] [137] [163]

Dopamine level (Striatal) [159] [159] [159]

Others

Depression-like [234] [142, 144] [142]

Learning and Memory [150, 152, 156] [154]

[155]

Body temperature [140] [137]

Water intake [137]

Seizures [166] [167] [167, 168]

: increase; : no change; : decrease *: high doses of morphine.

of these tools, the role of NOP receptor blockade in these effects needs to be further characterized.

It has been reported that antinociceptive responses can be in-duced by activating peripheral NOP receptors with local admini-stration of N/OFQ in capsaicin-induced nociceptive responses in monkeys [103] and mice [104], or with Ro 64-6198 in the rat neu-ropathic pain model [37]. However, N/OFQ was found to induce hyperaemia by topical administration to the inflamed joints of rats [105]. This effect was found to be mediated through activating the NOP receptors on synovial mast cells and leukocytes leading to the release of proinflammatory mediators which subsequently stimulate sensory neuropeptides release which results in vasodilatation. En-dogenous N/OFQ does not seem to play a role in the periphery since J-113397, even though it antagonized the effect of locally administered N/OFQ, had no effect per se in the allodynic response in the monkey [103] (Table 5).

Human plasma and CSF levels of N/OFQ have been measured by radioimmunoassay by several groups in different pain states [106] (Table 7). No significant change was found in the CSF of the subjects with labor pain, low back pain or fibromyalgia. However,

the plasma level of N/OFQ was significantly lower in fibromyalgia patients and in patients during cluster headache attacks and mi-graine attacks (Table 7). Interestingly, the basal plasma level in migraine patients in headache-free periods was lower than that of normal subjects [107]. However, the serum levels of N/OFQ were elevated in patients either in acute or chronic pain states and the latter was higher than the former (Table 7). Given that N/OFQ would be subjected to peptidase hydrolysis, the levels measured might be far lower than that at the site of generation, especially when samples were taken from the plasma.

The role of N/OFQ in pain regulation is still elusive after 10 years of active studies. A novel mechanism, inducing N-type Ca

2+

channel internalization, has been proposed to contribute to the antinociceptive action of N/OFQ [108]. Nevertheless, many non-peptide and peptide-modified molecules have been explored in the pharmaceutical industries and patents have been filed for the man-agement of migraine, neuropathic pain, pain associated with diabe-tes or Herpes zoster, or even for pruritus [20]. Among these, JTC-801 and TRK-820 have been filed for Phase II clinical trials [20].


3.2. Drug Dependence

N/OFQ plays an important modulatory role on the rewarding system [15]. Although Malin et al. [112] found N/OFQ (i.c.v.) pro-duced morphine-like abstinence signs, N/OFQ (i.c.v.) alone did not induce conditioned place preference (CPP) [109, 110], nor did Ro 64-6198. However, many studies indicated that i.c.v. injection of N/OFQ has an inhibitory effect on rewarding drugs. It reduced morphine withdrawal [113, 114] and the CPP induced by morphine [115], cocaine [115, 116], amphetamine [117] and alcohol [118], but not heroin [119]. Ro 64-6198 (i.p.), as effective as naltrexone, inhibited alcohol-induced acute reinforcing and relapse-like behav-iors in rats [120]. These inhibitory effects of N/OFQ might be attri-buted to its inhibition of mesolimbic dopamine release [121]. Alter-natively, it could be explained by the finding that N/OFQ inhibits GABAergic transmission and blocks ethanol-induced increase of GABA release in the central amygdala [122]. These findings sug-gest a potential for NOP receptor agonists in the treatment of drug

dependence (Table 8). Interestingly, Ciccocioppo et al. [123] found that buprenorphine, a partial agonist at MOP and NOP receptors, increased alcohol intake at lower doses through MOP receptors while decreased it at higher doses through NOP receptors. It is sug-gested that the therapeutic potential of buprenorphine in drug addic-tion might be attributed to its NOP receptor activation, but not to its activation of classical opioid receptors (see Section 2.1.2.). How-ever, the antinociceptive effect of systemic buprenorphine was found to be mainly mediated through MOP receptors [34].

The role of endogenous N/OFQ in the rewarding system is less clear. Although Kest et al. [87] reported that morphine withdrawal symptoms were enhanced in ppN/OFQ

-/- mice after continuous

morphine administration, Chung et al. [86] found less jumps in ppN/OFQ

-/- mice treated with escalating doses of morphine, as

compared with the wild type. Similarly, less withdrawal symptoms were observed in NOP

-/- and J-113397-treated mice (Table 6).

However, the mesolimbic dopamine release was not enhanced by

Table 7. Human N/OFQ Levels Measured at Various Disease States by Radioimmunoassay

N/OFQ (pg/ml)

Status

Normal Disease

Sample References#

Pain

Fibromyalgia 8.65 + 4.74 (7) 4.37 + 1.23 (8)* Plasma #

Fibromyalgia 5.65 (2.65-12.04a) (6) 4.27 (3.22-5.66a) (14) CSF [235]

Low back pain 4.52 (3.12-6.55a) (10) CSF [235]

Labor painb 52.49 + 34.25 (10) 63.39 + 33.26 (10) CSF #

7.59 + 21.58 (10) 13.73 + 23.79 (10) Plasma #

Pain-Acute 10.64 + 6.25 ( 10) 16.65 + 8.01 (30)* Serum #

-Subacute 10.39 + 5.28 ( 10) 20.66 + 8.98 (20)*

-Chronic 24.44 + 13.60 (20)*

Cluster headachec 9.58 + 2.57 (14) 4.91 + 1.96 (14)* Plasma #

After headache 8.60 + 1.47 (14)

Migraine Plasma [107]

Normal 9.74 + 2.57 (24) 5.79 + 1.82 (18)*, d

Ictal attack 1.04 + 0.17 (18)*

Hepatic diseases

HCC 9.2 + 1.8 (29) 105.9 + 14.4 (18)* Plasma #

Wilson disease 14.0 + 2.7 (26)*

Liver cirrhosis 12.8 + 4.0 (15)*

Biliary cirrhosis 12.1 + 3.2 (21)*

Chronic hepatitis 10.2 + 3.6 (18)

HCCe 0.01 pg/mg (1) 0.16 pg/mg (1)* Liver #

Postpartum depression 10.4 + 3.7 (25) 28.5 + 5.8 (21)* Plasma #

HCC: hepatic hepatocellular carcinoma. Data are mean + S.D. (n) from normal and patient groups, unless indicated. a: 95 % confidence interval. b: Elective Caesarean patients were

taken as the normal group, compared with the established labor group. c: Patients at normal and disease attack states were compared. d: Migraine patients at interictal state. e: Autop-

sic samples of normal and carcinoma liver tissues taken from a HCC subject were compared. #: For the original reports, unless indicated, refer to the editorial article [106].

*Significant difference v.s. the normal group.


UFP-101 and unchanged in NOP-/-

mice (Table 6). The alcohol preference intake was not changed by i.c.v. Nphe [124].

3.3. Anxiety and Stress

N/OFQ (i.c.v.) produced anxiolytic-like effects in several dif-ferent anxiety paradigms in mice, such as light-dark preference, pharmacological anxiogenesis, operant conflict [125] and mouse defense test battery [126], although in the latter paradigm, effects were observed only after very high stress. Kamei et al. [127] also reported that a low dose of N/OFQ has an anxiolytic effect in the hole-board test, possibly via the activation of serotoninergic func-tion in the hippocampus, while a high dose of N/OFQ has an anxio-genic effect, possibly via the inhibition of serotoninergic function in the amygdala. Similarly, in rats, N/OFQ produced anxiolytic-like effects in a conditioned lick paradigm [128] and elevated plus-maze [125] when given by i.c.v. injection, and in a social interaction paradigm after infusion into the basolateral amygdala [129]. Sur-prisingly, Fernandez et al. [130] observed anxiogenic-like effects of N/OFQ given by i.c.v. injection in several anxiety-related proce-dures (i.e., open field, elevated plus maze, and dark-light prefer-ence) in rats. Explanations given by the authors are a difference in baseline stress level between studies, and/or strain differences. Even though a control experiment suggested that the anxiogenic-like effects were -at least in part- independent of effects on locomo-tion [130], a recent study by Vitale et al. [131] may suggest the opposite. They replicated the anxiogenic-like effects of N/OFQ in the rat elevated plus-maze test, but observed anxiolytic-like effects after 2 subsequent administrations of N/OFQ [131]. This change from anxiogenic- to anxiolytic-like effect of N/OFQ was accompa-nied by a tolerance to the hypolocomotor effects of N/OFQ, sug-gesting that the anxiolytic-like effects of acute N/OFQ were masked by hypolocomotor effects.

In rats, the non-peptide NOP receptor agonist, Ro 64-6198 (i.p.) produced anxiolytic-like effects in a variety of paradigms such as the elevated plus maze [35], social interaction [129], pup isola-tion-induced ultrasonic vocalization [132], fear-potentiated startle [35], Geller-Seifter conflict [35], conditioned lick suppression [132], and Vogel conflict drinking (Prinssen, unpublished data) tests. The anxiolytic-like effects in the elevated plus maze of Ro 64-6198 did not show tolerance during a 2 week study [133]. In mice, the anxiolytic-like effects of Ro 64-6198 were more difficult to demonstrate because of increased disruptive effects [35]. However, recent data showed selective anxiolytic-like effects in a combined marble burying/locomotor activity test [134], and in Geller-Seifter conflict [132] and stress-induced hyperthermia (Spooren and Prins-sen, unpublished data) tests.

Several lines of evidence also suggest that endogenous N/OFQ has an important role in anxiety and stress regulation. Enhanced anxiety was shown in ppN/OFQ

-/- mice [87, 135, 136] and an-

tisense-NOP treated rats [137], but not in NOP-/-

mice [138] (Table 6). Interestingly, the impact of ppN/OFQ deletion on the anxiety-like behaviors is more significant in group-housed mice, as com-pared with individual-housed ones, and male mice were more sus-ceptible than females [136]. In mice, differences in anxiety state are associated with differences in G protein coupling efficiency in the nucleus accumbens (but not in 12 other brain regions) [139]. A likely explanation of this finding is that the observed increase in coupling in non-anxious mice leads to increased N/OFQ-mediated transmission and thus protects from anxiety [139].

Interestingly, endogenous N/OFQ may play a role in the modu-lation of the hypothalamus-pituitary-adrenal cortex (HPA) axis, but this role is rather complex and possibly species-dependent. For example, although the corticosterone level in NOP

-/- mice was not

changed [140], it was elevated in ppN/OFQ-/-

mice [135]. Con-versely, N/OFQ (i.c.v.) decreased the elevated plasma corticoster-one induced by manual restraint [141]. In rats, plasma corticoster-one was found to be decreased in antisense-NOP treated rats [137]

whereas i.c.v. injection of N/OFQ elevated circulating corticoster-one [130, 131]. It is interesting to note that, in the study by Vitale et al. [131], the effect of N/OFQ on corticosterone showed tolerance already after 2 injections. Taken together, this suggests that N/OFQ modulates the HPA axis, apparently in a manner opposite in mice and rats, although it is also possible that methodological differences play an important role in these differences. Comparing these find-ings with those in anxiety models, it seems that HPA axis activity and anxiety are regulated in an independent manner.

Although not directly related to anxiety, it is interesting to note that N/OFQ (i.c.v.) decreases core body temperature [15]. This hypothermic tone of endogenous N/OFQ can be revealed from NOP

-/- mice and antisense-N/OFQ-treated rats (Table 6). The hy-

perthermia observed in NOP-/-

mice is independent of cortisol regu-lation [140].

3.4. Depression

NOP receptor antagonists, including Nphe, racemic J-113397 and UFP-101 reduced the immobility times in both the forced swim and tail suspension tests (Table 6). N/OFQ (i.c.v.) alone did not affect the immobility time. Whereas the immobility time in NOP

-/-

mice is less than that in the wild type, the antidepressant-like effects of NOP receptor antagonists were not observed in NOP

-/- mice

[142], suggesting that endogenous N/OFQ plays a role in those depression-like behaviors. It is interesting to note that the plasma N/OFQ level was elevated in postpartum depressive women [143] (Table 7). Thus, NOP receptor antagonists may have the potential to be novel antidepressants (Table 8). The antidepressant-like effect of UFP-101 in the forced swimming test in mice might be me-diated by activating serotoninergic but not noradrenergic systems [144].

Table 8. Possible Clinical Indications for NOP Receptor

Ligands

Functions NOP Agonists NOP Antagonists

Sensation Neuropathic pain

Migraine General analgesic

Pruritus

Mood Anxiety Depression

Feeding Anorexia

Drug dependence

Alcohol addiction

Morphine addiction

Cocaine addiction

Amphetamine addiction

Urination Neurogenic bladder

Respiration Cough

Asthma

Circulation

Edema (Heart failure)

Hypertension

Impotency

Learning Memory Dementia

Motor activity Parkinsonism

Cerebrocirculation Stroke

Neuron excitability Seizures


3.5. Feeding

N/OFQ increased food consumption in satiated rats and food-deprived rats. The orexigenic action of N/OFQ is suggested to be attributed to both the inhibition of anorexigenic systems and the activation of orexigenic systems [145] although the former is be-lieved to be prominent. N/OFQ has been found to inhibit pathways that promote termination of food intake in the hypothalamic satiety centers, such as oxytocinergic neurons in

the paraventricular nu-

cleus and -MSH neurons in the arcuate nucleus [145]. Besides, Ciccocioppo et al. [146] found that N/OFQ, at the doses without hyperphagic effect, inhibited stress-induced anorexia and that this anti-anorexic effect is due to the fact that N/OFQ acts as a func-tional antagonist of corticotrophin-releasing factor at the bed nu-cleus of the stria terminalis [146]. Nphe did not affect food con-sumption per se in satiated rats, but reduced that in food-deprived rats [147]. UFP-101 also did not affect free feeding in the rat [27] (Table 6). It is suggested that endogenous N/OFQ plays an orexi-genic tone in response to food deprivation but not in normal feed-ing. Olszewske et al. [148] found that nocistatin also reduced dep-rivation-induced food consumption and depressed the orexigenic effect of N/OFQ.

3.6. Learning and Memory

N/OFQ (i.c.v.) impairs learning and memory performance in mice and rats in water maze [149], fear conditioning [150], Y-maze [151, 152] and passive avoidance [151, 152] tests. Biphasic effects of N/OFQ, enhancement at low doses but impairment at high doses, were observed in the rat water maze test when it was given by intra-hippocampal injection [149]. Hirmatsu and Inoue [153] also found that, in the Y-Maze and passive avoidance tests, N/OFQ impaired the task performances at nmol doses in normal mice but improved them at lower (doses down to fmol) in scopolamine-impaired mice.

All the studies investigating the role of endogenous N/OFQ in the regulation of learning and memory were conducted in knockout mice; none was performed with NOP receptor antagonists (Table 6). In ppN/OFQ

-/- mice, although the water maze performances

were similar to that in the wild type mice [154], the passive avoid-ance performances were enhanced [155]. In NOP

-/- mice, the per-

formances were better than that in the wild types in the water maze [156], passive avoidance [152], fear conditioning [150] tests, but not in the Y-maze test [152]. The theta rhythm and ACh release in the hippocampus, both involved in learning memory function, have been found to be increased in NOP

-/- mice [157]. These findings

suggest that NOP receptor antagonists might be of use in the treat-ment of cognitive impairment [20].

3.7. Motor Activity

N/OFQ (i.c.v.) increased motor activity at low doses but de-creased it at high doses [158]. When microinjected into substantia nigra, N/OFQ reduced dopamine release in the striatum and loco-motor activity [159], which was associated with a reduction of glu-tamate release in the substantia nigra. Conversely, the NOP receptor antagonists, J-113397 and UFP-101, injected in the substantia nigra, enhanced striatal dopamine release and facilitated motor perform-ance [159]. Microinjection of UFP-101 into the substantia nigra also reversed the akinesia in haloperidol-treated [160] or 6-hyd- roxydopamine-hemilesioned rats [161]. Enhancement of N/OFQ expression and release was observed in the latter parkinsonism model [161]. Haloperidol-induced motor impairment and the do-paminergic neuronal toxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, but not methamphetamine, were par-tially abolished in ppN/OFQ

-/- mice [161, 162]. Increased locomo-

tor activity was observed in NOP-/-

mice [159] and in rats treated with antisense-NOP [137] or antisense-ppN/OFQ [163]. These studies suggest that endogenous N/OFQ might have a negative regulation in the striatal dopamine level and motor activity (Table 6). Therefore, NOP receptor antagonists might be beneficial in alle-

viating extrapyramidal side effects of classical antipsychotics as well as in the treatment of Parkinsonism (Table 8), which is one of the claimed indications in patent applications for NOP receptor antagonists [20]. This negative regulatory role of endogenous N/OFQ on dopamine levels in the striatal system does not seem to exist in the mesolimbic system since UFP-101 alone did not in-crease mesolimbic dopamine release [121] (Table 6).

3.8. Epilepsy/Seizures

N/OFQ is suggested to have antiepileptic activity since it inhib-its glutamate release and blocks T-type Ca

2+ channels, which are

important in regulating neuronal excitability [14, 164]. It did raise the convulsive threshold in seizures induced by pentylenetetrazole, N-methyl D-aspartic acid, bicucculine [165] and penicillin [166], but not by electrical shock [165]. Paradoxically, susceptibility to kainate seizures was decreased in J-113397-treated [167] and ppN/OFQ

-/- mice [167, 168] (Table 6). It is unclear if these observa-

tions could suggest that endogenous N/OFQ has a facilitatory role in the expression of limbic seizures since J-113397 might have affinity at receptors other than NOP receptors (see Section 2.2.2.) and nocistatin might also be deleted in ppN/OFQ

-/- mice. Nphe

(i.c.v.) per se did not change the seizure activity induced by penicil-lin [166] (Table 6). Interestingly, elevated N/OFQ release and ppN/OFQ mRNA expression in the hippocampus and thalamus have been found in animals with kainate-induced seizures [167, 169]. This elevation could be either a cause or an effect of seizures.

3.9. Cardiovascular System

3.9.1. Cardiovascular Depression

N/OFQ induces bradycardia and hypotension via both central and peripheral actions [170]. The rostral ventrolateral medulla might be involved in the central cardiodepressive effect of N/OFQ [171], although microinjection of N/OFQ into the rostral nucleus tractus solitarius and nucleus ambiguus was found to produce car-diostimulation [172]. Peripherally, N/OFQ can inhibit norepineph-rine release from sympathetic nerve terminals in the heart [173] and blood vessels [174]. NOP receptors are functionally expressed in endothelial cells of human aortic artery, rat aorta [175], and post-ganglionic sympathetic nerve terminals which innervate cardiac muscle. Activation of postsynaptic NOP receptors by N/OFQ in rat stellate ganglionic neurons inhibits cardiac sympathetic activity via an inhibition of Ca

2+ currents [176]. The vasodilatatory effect of

N/OFQ makes NOP receptor agonists being claimed to have a clinical potential in the management of hypertension and impotency [20]. The latter indication was implicated from the finding that N/OFQ induced a pronounced and long-lasting erectile response when injected into the cavernosal cavity of the cat [177].

3.9.2.Water Diuresis

N/OFQ increased water excretion (water diuresis) but decreased sodium excretion (antinatriuresis) through both central and periph-eral actions [45, 50]. Activation of NOP receptors in paraventricular nucleus (PVN) of the hypothalamus by N/OFQ produces bradycar-dia, renal sympathoinhibition, and water diuresis. Recently, the group of Kapusta [178] proved that endogenous N/OFQ produces a tonic inhibition on the PVN activity since UFP-101, when injected into the PVN, increased heart rate and renal sympathetic nerve ac-tivity and decreased urine flow rate. Interestingly, ZP-120, a pe-ripherally acting partial agonist of NOP receptors, when given in-travenously, induced significant water diuresis without bradycardia and hypotension, which are induced by i.v. injection of N/OFQ [50]. This suggests that the water diuretic effect is partly of periph-eral origin. ZP-120 was, hence, filed for phase II clinical trials for the treatment of acute decompensated heart failure [20].

3.9.3. Cerebral Circulation

Animal studies indicate that N/OFQ seems to contribute to the reduction of cerebral blood

flow observed following hypoxia/


Supplement I. Radioligand displacement studies were conducted by a broad to CEREP screen to determine the binding affinity of

J-113397 at various receptors, channels and transporters.

% Control % Control

Target

100nM 10 M

Target

100nM 10 M

A1 (h) 119 108 M4 (h) 100 36

A2A (h) 100 91 M5 (h) 99 84

A3 (h) 98 71 NK1 (h) 103 68

1 (non-sel) 100 48 NK2 (h) 96 26

2 (non-sel) 98 68 NK3 (h) 88 101

1 (h) 104 93 Y1 (h) 98 86

2 (h) 96 71 Y2 (h) 101 106

AT1 (h) 97 98 NT1 (h) (NTS1 ) 86 98

AT2 (h) 100 99 2 (h) (DOP) 103 85

GABAA-BZD 93 96 (KOP) 84 7

BZD (peripheral) 106 95 μ (h) (MOP) 76 2

BB (non-sel) 98 89 NOP (h) (ORL1 ) 18 0.4

B2 (h) 119 109 PACAP (PAC1) 97 108

CGRP (h) 105 108 PCP 101 111

CB1 (h) 111 58 TXA2 /PGH2 (h) 102 98

CCKA (h) 85 95 PGI2 (h) 87 87

CCKB 85 105 P2X 97 99

D1 (h) 117 110 P2Y 94 104

D2S (h) 100 23 5-HT1A (h) 86 6

D3 (h) 97 9 5-HT1B 116 66

D4.4 (h) 87 8 5-HT2A (h) 100 67

D5 (h) 96 89 5-HT2C (h) 85 63

ETA (h) 105 99 5-HT3 (h) 96 83

ETB (h) 93 99 5-HT5A (h) 99 30

GABA (non-sel) 102 95 5-HT6 (h) 89 87

GAL1 (h) 85 94 5-HT7 (h) 101 54

GAL2 (h) 93 110 (non-sel) 36 -2

PDGF 100 100 sst (non-sel) 103 79

IL-8B (h)

(CXCR2) 109 93

VIP1 (h)

(VPAC1) 104 105

TNF (h) 102 104 V1A (h) 105 80

CCR1 (h) 99 79 Ca2+ channel (L, verapamil site) 100 54

H1 (central) 92 40 K+ V channel 102 104

H2 73 31 SK+ Ca channel 97 95

MC4 (h) 103 90 Na+ channel (site 2) 73 -2


(Suppl. I) contd….

% Control % Control

Target

100nM 10 M

Target

100nM 10 M

ML1 96 94 Cl- channel 119 101

M1 (h) 118 56 NE transporter (h) 108 72

M2 (h) 109 30 DA transporter 90 107

M3 (h) 87 71

Data are % of control radioligand activity : the lower the number the more radioligand was displaced. The number lower than 50 % is considered meaningful and is shown in bold.

Two concentrations, 100nM and 10 μM, of J-113397 were tested in 75 receptors, channels and transporters. (h) : human ; (non-sel) = non-selective. For more information on the

binding sites, see www.cerep.fr.

ischemia or traumatic brain injury, especially in neonates [179].

However, N/OFQ has little effect on normal physiological cerebral

hemodynamics. NOP receptors were found to be functionally ex-pressed in endothelial cells of rat brain microvessels [175] and N/OFQ was found to inhibit ischemia-induced glutamate efflux from rat cerebrocortical slices [164]. This suggests that NOP recep-tor agonists might be beneficial in stroke patients [20].

3.10. Urinary Incontinence

N/OFQ inhibits the micturition reflex [180] and has been re-ported to be in clinical trials (Phase II) for the treatment of urinary incontinence [20]. In a clinical trial in patients with neurogenic bladder due to spinal cord injury, intravesical infusion of N/OFQ has been found to increase bladder capacitance and decease bladder pressure [180].

3.11. Cough

N/OFQ or Ro 64-6198 inhibited the cough responses provoked by capsaicin in guinea pigs or by mechanical stimulation of intra-thoracic airways in cats [181-183]. These antitussive actions might be mediated by both central and peripheral actions. In the airways, N/OFQ inhibited

the contractions of isolated airway preparations

through a pre-junctional inhibition of ACh and/or sensory peptide release in guinea pigs, rats [184] or humans [185]. N/OFQ and Ro 64-6198 decreased capsaicin-induced Ca

2+ influx in nodose ganglia

[183], the sensory ganglia involved in cough reflex [186], and the airway contraction in a manner blocked by tertiapin, an inwardly rectifying K

+ channel blocker [187]. In the brain, there are dense

NOP receptors in the medullar nucleus tractus solitarius [11], where pulmonary and extrapulmonary afferent fibers terminate and pro-vide polysynaptic inputs to second-order neurons which modulate the respiratory neuron activities [186]. Recently, N/OFQ was found to inhibit airway microvascular leakage induced by intra-esophageal acid instillation [188]. The inhibitory effect of N/OFQ on acid-induced cough in guinea pigs may result from a decrease of periph-eral C-fiber activity through an inhibition of the vallinoid receptor on airway-specific capsaicin-sensitive jugular ganglia [189]. There-fore, NOP receptor agonists, in addition to their antitussive poten-tial, might be of help in the clinical management of cough related to gastro-esophageal reflux disorders. NOP receptor agonists, devoid of side effects of classical opioids, might be superior to codeine as potential antitussive agents [20].

3.12. Immunoregulation

NOP receptors and N/OFQ are widely distributed throughout the immune system. NOP receptor mRNA and protein have been found in a variety of immune cells including mouse lymphocytes [190], human peripheral blood mononuclear cells (PBMC’s) [191] and human circulating granulocytes, lymphocytes and monocytes

[192-194]. Neutrophils are thought to be a source of N/OFQ in inflammatory tissues [193]. N/OFQ can function as an immunosup-pressant. It suppressed the antibody production in mouse lympho-cytes [190], decreased the proliferation of phytohemagglutinin-stimulated PBMC’s [195] and inhibited mast cell function [196]. In human monocytes, however, N/OFQ failed to affect LPS-induced cytokine production [194]. On the other hand, a pro-inflammatory role of N/OFQ has also been suggested in the experiments using mast cells [197] and neutrophils [198]. N/OFQ enhanced TNF- and IFN- transcripts in the spleen when injected prior to staphylo-coccal enterotoxin A (SEA) challenge. After SEA challenge, the TNF- and IFN- mRNA induction in the spleen was diminished in ppN/OFQ

-/- mice [199]. Recently, Waits et al. [200] demonstrated

that N/OFQ modulated T cell activation bidirectionally. It enhanced T cell proliferation and increased TNF- secretion by up-regulating marker CD28 activation. However, it inhibited the proliferation of re-stimulated T cells by up-regulating CTLA-4 expression. The exact role of N/OFQ in immunoregulation needs to be further elu-cidated.

It is interesting to note that the plasma levels of N/OFQ in pa-tients with hepatic hepatocellular carcinoma (HCC) were markedly elevated. Much higher N/OFQ in the carcinoma tissue, compared with the normal hepatic tissue from the same subject, was observed in the postmortem liver (Table 7). Higher N/OFQ plasma levels were also found in patients with Wilson disease and hepatic cirrho-sis, but not in those with chronic hepatitis. However, no correlation was found between the plasma levels of N/OFQ and clinical and laboratory features in patients with hepatocellular carcinoma (HCC) in an association study with HCC (n=28) and liver cirrhosis (n=30) patients, and normal subjects (n=25) [201].

In addition, N/OFQ also affects circadian rhythms, the gastroin-testinal and vestibular systems, and might be involved in hearing control [15]. Indeed, several indications were, therefore, claimed by patents on NOP receptor ligands [202], including tinnitus, GI disor-der or hearing disorder.

4. CONCLUSIONS

Ten years after being de-orphanized, the NOP receptor has been found to be involved in a variety of biological functions, and many agonists and antagonists of NOP receptors have been developed. Using these ligands and antisense oligonucleotides as tools, as well as the use of NOP

-/- or N/OFQ

-/- mice, several physiological or

pathological roles of N/OFQ and the NOP receptor system have been suggested. Several clinical indications (Table 8) have been proposed, mostly based on animal studies. Whereas there is strong interest in silent antagonists and full agonists, possibly, in clinical applications, a partial agonist might be beneficial if the proposed therapeutic effects could be maintained and side effects could be reduced. Some of the NOP receptor ligands have been filed for


Supplement II. Radioligand displacement studies were conducted by a broad to CEREP screen to determine the binding affinity of

JTC-801 at various receptors, channels and transporters.

% Control* % Control*

Target

100nM 10 M

Target

100nM 10 M

A1 (h) 91 29 M4 (h) 102 101

A2A (h) 91 20 M5 (h) 94 63

A3 (h) 116 94 NK1 (h) 90 24

1 (non-sel) 109 47 NK2 (h) 76 15

2 (non-sel) 112 68 NK3 (h) 101 101

1 (h) 102 62 Y1 (h) 103 20

2 (h) 80 76 Y2 (h) 105 68

AT1 (h) 116 63 NT1 (h) (NTS1 ) 103 40

AT2 (h) 100 81 2 (h) (DOP) 98 75

GABAA-BZD 92 74 (KOP) 93 37

BZD (peripheral) 96 83 μ (h) (MOP) 87 4

BB (non-sel) 104 110 NOP (h) (ORL1 ) 47 2

B2 (h) 96 114 PACAP (PAC1) 100 113

CGRP (h) 106 107 PCP 118 101

CB1 (h) 97 89 TXA2 /PGH2 (h) 104 87

CCKA (h) 123 44 PGI2 (h) 101 139

CCKB 97 96 P2X 124 158

D1 (h) 87 2 P2Y 106 104

D2S (h) 90 23 5-HT1A (h) 98 59

D3 (h) 97 70 5-HT1B 107 125

D4.4 (h) 93 69 5-HT2A (h) 78 2

D5 (h) 96 12 5-HT2C (h) 79 0.7

ETA (h) 91 103 5-HT3 (h) 102 85

ETB (h) 96 86 5-HT5A (h) 100 30

GABA (non-sel) 108 118 5-HT6 (h) 96 31

GAL1 (h) 81 77 5-HT7 (h) 78 27

GAL2 (h) 110 81 (non-sel) 107 43

PDGF 109 118 sst (non-sel) 103 67

IL-8B (h)

(CXCR2)

112 73 VIP1 (h)

(VPAC1)

109 125

TNF (h) 104 91 V1A (h) 106 77

CCR1 (h) 102 94 Ca2+ channel (L, verapamil site) 92 4

H1 (central) 99 68 K+ V channel 116 101

H2 83 2 SK+ Ca channel 100 103


(Suppl. II) contd….

% Control* % Control*

Target

100nM 10 M

Target

100nM 10 M

MC4 (h) 107 40 Na+ channel (site 2) 69 -3

ML1 102 43 Cl- channel 109 94

M1 (h) 92 39 NE transporter (h) 66 8

M2 (h) 99 16 DA transporter 93 32

M3 (h) 100 98

Data are % of control radioligand activity : the lower the number the more radioligand was displaced. The number lower than 50 % is considered meaningful and is shown in bold.

Two concentrations, 100nM and 10 μM, of JTC-801 were tested in 75 receptors, channels and transporters. (h) : human ; (non-sel) = non-selective. For more information on the

binding sites, see www.cerep.fr.

clinical trials. Nevertheless, the further development of promising small molecule ligands with high selectivity at NOP receptors is expected to further enhance our understanding of the functional roles of N/OFQ and NOP receptors as well as their possible clinical applications in the future.

ACKNOWLEDGEMENTS

This work was supported by the grants from National Health Research Institutes, Taiwan (NHRI-EX90-9005NC, NHRI-EX91-9005NC, NHRI-EX92-9005NC, NHRI-EX93-9005NC, NHRI-EX94-9005NC and NHRI-EX95-9506NI), National Science Coun-cil, Taiwan (NSC89-2320-B002-273, NSC92-2320-B002-088, NSC93-2320-B002-117 and NSC 94-2320-B002-034) and National Bureau of Controlled Drug, Department of Health, Taiwan (DOH 94-NNB-1019 and DOH 95-NNB-1025). We would like to thank Mrs. Kuang-Chieh Chuang, Shu-Huai Fan, and Ms. Chia-Ju Kuo for their help in preparing the manuscript as well as Drs Grund-schober and Wichmann for helpful comments and critical reading.

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Received: October 19, 2005 Accepted: March 26, 2006 Updated: October 6, 2006

Nociceptin/Orphanin FQ Peptide Receptors: Pharmacology and Clinical Implications

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Transcript of Nociceptin/Orphanin FQ Peptide Receptors: Pharmacology and Clinical Implications