The nociceptin/orphanin FQ receptor: a target with broad

The nociceptin/orphanin FQ peptide (N/OFQ)1,2 is involved in a wide range of physiological responses with effects noted in the nervous system (central and peripheral), the cardiovascular system, the airways, the gastrointestinal tract, the urogenital tract and the immune system3,4 (FIG. 1). The effects in the nervous sys-tem are complex and have received much attention. It is generally accepted that spinal N/OFQ is antinociceptive with many features that are common to the classical members of the opioid family5. Nevertheless, when given supraspinally, N/OFQ reverses the effects of opioids (anti-opioid action) with a whole-animal response that manifests as hyperalgesia5 (see below for more detailed coverage of this important point). In the brain, this pep-tide is also hyperphagic and affects the response to stress, anxiety and locomotion3,4. In the cardiovascular system N/OFQ produces bradycardia and hypotension; this response is similar to that produced by classical opioids and more specifically to morphine used in the clinic6.

The N/OFQ receptor (NOP; also known as ORL1, OP4 or LC132) is a deorphanized member of the G-protein coupled receptor (GPCR) superfamily (Box 1). NOP is currently classified as a non-opioid member of the opioid receptor family by the International Union of Basic and Clinical Pharmacology (IUPHAR; see Further information and Supplementary information S1 (table)). Although NOP shares considerable structural and locali-zation features with the classical opioid receptors, NOP activity is insensitive to the opioid antagonist naloxone, an important discriminatory feature for classical opioids (there are several excellent reviews on the classical opiod

receptors m, d and k7,8). Activation with N/OFQ inhibits the formation of cyclic AMP1,2,9,10, closes voltage-gated Ca2+ channels9–11 and opens inwardly rectifying K+ channels9,10,12. The net effect of these cellular actions is to reduce neuronal excitability and neurotransmitter release. Indeed, a wide range of neurotransmitter systems are modulated by N/OFQ and these include glutamate13, catecholamines14 and tachykinins15. NOP activation also modulates mitogen-activated protein (MAP) kinase, extracellular signal-regulated kinase (ERK) and JUN activity10,16. NOP may also be involved in phospholipase C (PLC)-mediated phosphatidylinositol bisphosphate hydrolysis10,17. As the identification of N/OFQ — as the endogenous ligand for NOP — was by the process of reverse pharmacology and as subsequent characteriza-tion of the actions of NOP required a panel of N/OFQ ligands, a description of their development precedes a description of biological function.

Ligand development: peptides and relativesDevelopment of this system from an experimental point of view has been hampered, until recently, by a relative paucity of selective ligands and especially of antagonists. Several groups have active structure–activity relationship (SAR) programmes that are based on native N/OFQ, and a number of these are now generally accepted as stand-ards for characterization of this receptor by IUPHAR (see Further information). Many of the currently accepted reference molecules are from the group of G. Calo and R. Guerrini18–24. These include the trun-cated and amidated N/OFQ(1–13)-NH2 (ReF. 18); the

Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester LE1 5WW, UK.e-mail: [email protected]:10.1038/nrd2572

HyperalgesiaAn increase in pain perception above the normal response to a stimulus.

DeorphanizedThe identification of an endogenous ligand for a receptor whose structure makes it a member of a receptor family, but for which no ligand has yet been identified (that is, an orphan receptor).

The nociceptin/orphanin FQ receptor: a target with broad therapeutic potentialDavid G. Lambert

Abstract | Identification of the enigmatic nociceptin/orphanin FQ peptide (N/OFQ) in 1995 represented the first successful use of reverse pharmacology and led to deorphanization of the N/OFQ receptor (NOP). Subsequently, the N/OFQ–NOP system has been implicated in a wide range of biological functions, including pain, drug abuse, cardiovascular control and immunity. Although this could be considered a hurdle for the development of pharmaceuticals selective for a specific disease indication, NOP represents a viable drug target. This article describes potential clinical indications and highlights the current status of the very limited number of clinical trials.

R E V I E W S

694 | AUGUST 2008 | vOLUME 7 www.nature.com/reviews/drugdisc

© 2008 Macmillan Publishers Limited. All rights reserved.

http://www.nature.com/nrd/journal/v7/n8/suppinfo/nrd2572.html

mailto:[email protected]

Nature Reviews | Drug Discovery

Heart• Negative chronotrope• Negative ionotrope

Airway• Inhibits airway smooth-muscle contraction• Inhibits mechanical and capsaicin-induced cough• Antitussive

Kidney• Water diuresis• Congestive heart failure

Urogenital system• Inhibits evoked contraction of vasa deferentia• Inhibits micturition reflex• Neurogenic bladder

Immune system• Inhibits immunocyte activity

Central (brain/spinal cord)• Analgesia• Anti-opioid (hyperalgesia)• Modulation of feeding• Anxiolysis• Antidepressant• Modulation of locomoter activity• Modulation of learning and memory• Modulation of opioid tolerance• Pain• Anxiety• Depression• Anorexia/obesity• Parkinson’s disease

Gastrointestinal tract• Inhibits gastrointestinal motility

Vasculature• ‘General’ vasodilator producing hypotension• Vasodilator of the microcirculation (histamine dependent)• Heart failure• Stroke• Hypertension• Sepsis

Structure–activity relationship(SAR). Correlations that are constructed between the features of chemical structure in a set of candidate compounds and parameters of biological activity, such as potency, selectivity and toxicity.

SuperagonistA drug that can interact with a receptor and initiate a physiological or a pharmacological response that is characteristic of that receptor but with particularly high potency and/or efficacy.

partial agonist [Phe1ψ(CH2-NH)-Gly2]N/OFQ(1–13)-NH2 ([F/G]N/OFQ(1–13)NH2)

19; the superagonists [(pF)Phe4-Arg14-Lys15]N/OFQ-NH2 (UFP-102)20 and [(pF)Phe4-Aib7-Arg14-Lys15]N/OFQ-NH2 (UFP-112)21; and the antagonists [Nphe1]N/OFQ(1–13)-NH2 (ReF. 22) and [Nphe1-Arg14-Lys15]N/OFQ-NH2 (UFP-101)23,24 (Box 2). An example of a peptide SAR study performed in an industrial environment is the study carried out by C. T. Dooley, who screened a total of 52 million hexa-peptides in mixtures to isolate just five peptides25. These five peptides are also accepted as receptor standard par-tial agonists and templates for other ligands (especially ZP120)26,27 (TABLe 1).

The development of the partial agonist [F/G]N/OFQ(1–13)NH2 is worthy of further description and as a cautionary note in characterizations that are based on single endpoint screens for amplified GPCR systems. This peptide was initially described as an antagonist in the mouse vasa deferentia and in the guinea-pig ileum19. Subsequently, it was shown to be a full agonist in cells expressing recombinant human NOP28,29, as well as a full agonist for the inhibition of K+-evoked glutamate release from rat cerebrocortical slices29. At this point a couple of suggestions could be made to explain this discrepancy: species difference or differences between central and peripheral NOP. The full agonist nature of this peptide has been reported in several other systems including the mouse colon30. A simpler pharmacological explanation can be used for these data as, in general,

where full agonist behaviour was reported, either a response downstream in the signal transduction cascade (and hence amplified) was measured or there were high levels of receptor expression. Mason et al. attempted to address this, although their study was complicated by use of different species isoforms of NOP31. Using cell-based assays, in which the number of NOPs could be controlled (ecdysone-inducible expression system), it was demonstrated that the agonist/partial agonist/antagonist behaviour was dependent on the expression levels of NOP32. These data indicate (and agree with the current molecular evidence) that there is no basis for the suggestion of central and peripheral NOP subtypes, and that [F/G]N/OFQ(1–13)NH2 should be classified as a partial agonist. Moreover, novel ligands should be screened in multiple assays (including those in which receptor expression is controlled to that in the tissue of interest) before their formal classification. So far, NOP ligands can be classified pharmacologically into three categories: a pure NOP antagonist, a partial agonist or a full agonist, depending on the system studied.

Other peptide ligands of note include peptide III-BTD33 and ZP120 (ReFS 26,27). Peptide III-BTD is based on a β-turn (important in the activity of the d-opioid receptor endogenous agonist enkephalin34) and a syn-thetic peptide combinatorial library. ZP120 is based on structure-inducing probe technology. ZP120 can be described as a ‘Dooley’ peptide (that is, Ac-RYYRWK-NH2) with Lys6 added to the C terminus to improve its

Figure 1 | Pleiotropic effects of nociceptin/orphanin FQ (N/OFQ) on major organ systems. Potential clinical indications are noted in bold.

R E V I E W S

NATURE REvIEWS | drug discOvery vOLUME 7 | AUGUST 2008 | 695


α β γ


ORL1/LC132 'orphan GPCR'

AC

↓cAMP

↓cAMPMeasured asan output

No effect

Inhibits

Further separation of

No effect

Separation of pigor rat brain extract

Structure analysis of Fraction 6 yields: FGGFTGARKSARKLANQ or N/OFQ

Fraction12345678910

cAMP–––––Inhibits––––

CHO cell

Privileged structureA single molecular framework that is able to provide ligands for diverse receptors.

metabolic stability. Peptide III-BTD is a NOP antagonist with mixed agonist activity at classical opioid receptors33, and ZP120 is selective NOP partial agonist with prolonged in vivo actions26 (TABLe 1).

There is growing interest in the design and evalua-tion of peptide drugs for use in humans35. Indeed, there is increasing activity in the development of NOP peptides (see below) and the use of N/OFQ itself (for example, intravesical). Peptides offer a number of advantages including high selectivity, predictable metabolism (for example, N/OFQ is well validated in humans) and relative safety (that is, reduced side-effect profile).

Ligand development: non-peptidesEarly studies in the identification of non-peptide NOP ligands revolved around the screening of existing opioids and traditional high-throughput medicinal chemistry approaches with small-molecule libraries in N/OFQ-sensitive preparations. Of note in this area is the work showing that the existing compounds — anilidopiperidine

(lofentanil), morphinans (naloxone benzoylhydrazone and buprenorphine) and neuroleptics (spiroxatrine and pimozide) — all interacted with NOP36. However, these compounds could not be expected to display selectivity. The main chemical classes of the current non-peptides can be classified as morphinans, 4-aminoquinolines, benzimidazopiperidines, aryl-piperidines and spiropi-peridines4; these are summarized in FIG. 2 and TABLe 2. In addition, there is a recent description of a 4-aryl-tropane NOP agonist, SCH 221510 (ReF. 37).

Of particular note is that many non-peptide NOP ligands (which are both agonists and antagonists) con-tain a piperidine structural motif. This chemical motif represents a privileged structure for GPCR binding38 and the 1,4-disubstituted piperidine scaffold seems to be particularly important for NOP interaction. Chemical modulation of the substituents in position 1 and 4 of the piperidine nucleus remains an active design process by several pharmaceutical companies, and recently a novel orally active NOP agonist has been identified39.

Box 1 | Identification of N/OFQ

In 1995 Meunier et al.1 and Reinscheid et al.2 simultaneously described the nociceptin/orphanin FQ peptide (N/OFQ) as the endogenous ligand for the orphan G-protein-coupled receptor (GPCR) ORL1/LC132, now known as NOP. Their seminal work was the first example of reverse pharmacology. Traditional pharmacological identification of novel ligand–receptor families relies on screening for a target (receptor) with a pre-identified ligand. This approach is well exemplified by the description of classical opioid receptors using radiolabelled naloxone and dihydromorphine by Pert and colleagues193,194. As a result of advances in molecular cloning, a vast array of orphan receptors were identified and these can be heterologously expressed in a range of cell types with relative ease. The production of such cell lines and the coupling of this expressed protein (receptor) to a measurable effector system sets up a convenient assay with which to screen natural or fractionated tissue extracts or body fluids for activity. In the example below, Chinese hamster ovary (CHO) cells expressing the orphan ORL1/LC132 were used. Based on structural similarities with the known opioid receptors, both the chemical nature of the endogenous ligand (peptide) and the consequences of its activation (inhibition of cyclic AMP) were assumed to be similar to those of classical opioids. Consequently, cells were stimulated with forskolin to activate adenylyl cyclase (AC) and increase intracellular cAMP. As a Gi/o-coupled orphan receptor, endogenous agonists (peptides) at this receptor will inhibit the formation of cAMP. Extracts from rat brain (Meunier et al.1) or pig brain (Reinscheid et al.2) were screened using this system following a number of rounds of fractionation (the fraction numbers–separation chromatogram are illustrative and not from the original papers). Control experiments were performed with cells not expressing the recombinant receptor. A pure product with strong activity was sequenced and N/OFQ was formally identified.

R E V I E W S



H2Nf

H2N

H2N

H2Nc

d

H2Ng

H2Nh

e

H2N

H2N

H2N

b

aTruncation to amino acids 1–13 retains full activity

NH2

NH2

COOH

COOH

COOH

COOH

COOH

COOH

COOH


Y G G F T G A R K S A R K L A N Q

F G G F T G A R K S A R K

Nphe G G F T G A R K S A R K L A N Q

F G G (pF)F T G A R K S A R K L A N Q

Substitution of F with Y may produce non-selective opioid

F G G F T G A R K S A R K L A N Q

F G G F T G A R K S A R K R K N Q

Increases potency

F ψ(CH2–NH) G G F T G A R K S A R K L A N Q

Partial agonist template

Antagonist template

Nphe G G F T G A R K S A R K R K N Q

Competitive antagonist (UFP-101)

F G G (pF)F T G A R K S A R K R K N Q

Super agonist (UFP-102)

Increases affinity

Message Address

Molecular modelling studies suggest that the nitro-gen of the piperidine ring interacts with the Asp130 of NOP40; the same amino-acid residue that is involved in the interaction with the N-terminal nitrogen of the natural ligand41. These data suggest that non-peptide NOP ligands might interact with NOP amino acids that are crucial for binding the physiological peptide and add value to current peptide SAR approaches. A two-dimensional pharmacophore model for the interaction between NOP and non-peptide ligands has been proposed42. In this elegant model the three key elements (or moieties) are described as the heterocyclic A-moiety, the basic nitrogen-containing B-moiety and the lipophilic group on the basic nitrogen, the C-moiety. Moiety A is an important determinant of binding affinity and selectivity compared with classical m-opioid, d-opioid and k-opioid receptors. By contrast,

the lipophilic C moiety has a role in the intrinsic activity of the ligand at NOP. Subtle changes in the distance between the piperidine nitrogen and moiety C produce important differences in ligand efficacy. In the follow-ing section describing the role(s) of the N/OFQ–NOP system, the selective NOP agonist Ro64-6198 and antagonists J-113397 and SB-612111, together with the low-selectivity antagonist JTC-801, are described. Details of the pharmacology of these ligands can be found in TABLe 2.

(Patho)physiological role(s) of N/OFQ–NOPAs described in FIG. 1, N/OFQ–NOP is involved in a wide range of responses and thus has wide potential for drug development. This Review selects areas in which there is one or more of the following: good basic science evidence for involvement; clinical evidence in the form of plasma

Box 2 | Modification of N/OFQ

In common with a range of peptides nociceptin/orphanin FQ (N/OFQ) can be loosely broken into message and address domains, with the former being involved in receptor activation and the latter in receptor occupation18. There have been a number of groups engaged in peptide structure–activity relationship (SAR) studies including those of Meunier195, Civelli196 and Dooley25. Data predominantly from the group of G. Calo and R. Guerrini, using a comprehensive analysis of both message (pink) and address (blue) domains is depicted (top sequence). C-terminal truncation of the peptide results in a progressive loss of binding affinity and functional potency such that N/OFQ(1–13) is the smallest fragment (a) to retain full biological activity28,197,198. This fragment is a commonly used template for further modification. If Phe1 in N/OFQ is replaced with Tyr1 (as in classical opioids) then a previously selective NOP ligand becomes a relatively non-selective (b) opioid ligand199; although this has been questioned200,201. Some of the more important modifications of N/OFQ are depicted in the message domain (c–e) and in the address domain (f). These modifications are combined to produce at least two interesting and now widely accepted preclinical reference peptides: UFP-101 (g) and UFP-102 (h). Adding a pseudopeptide bond between Phe1 and Gly2 (Phe1ψ(CH2-NH)-Gly2) or [F/G] (c) reduces binding affinity and peptide efficacy such that at low expression in mouse vasa deferentia (mVD) the peptide has antagonist properties. However, at higher expression in Chinese hamster ovary (CHO) cells expressing the recombinant human NOP (CHOhNOP) a full agonist profile is observed. This behaviour is consistent with the partial agonist profile now ascribed to the [F/G] template (c). If the benzyl side chain on Phe1 is shifted from C to N to produce [Nphe1] (d) then a further reduction in binding affinity is observed, but more importantly agonist activity is eliminated both at low (mVD) and high (CHOhNOP) expression. This is the antagonist template. If fluorine is added to para position of the benzene ring in Phe4 to produce [(pF)Phe4] (e), an increase in binding affinity is produced. Moreover, when fluorine is introduced in combination with [F/G] and [Nphe1], higher affinity partial agonists and antagonists are produced. Addition of an Arg14-Lys15 repeat in the address domain (f) produced a marked increase in affinity and potency19,20,202,203. Arguably the most important modifications in this field are the combination of the Arg14-Lys15 repeat with [Nphe1] to produce UFP-101 (g) a highly selective, high potency competitive NOP antagonist23,24, and with [(pF)Phe4] to produce UFP-102 (h), a super high affinity/potency agonist with long-lasting actions in vivo204. There are a number of other chemical modifications along this line including use of a-aminoisobutyric acid21 and peptide cyclization205,206.

R E V I E W S



peptide measurements, studies with human tissues or for-mal clinical studies; pharmaceutical evidence with candi-date drugs suitable for development; and/or emerging new areas without an extensive literature base. Consequently, pain, mood (depression and anxiety) disorders, reward/drug abuse, cardiovascular disease, urogenital disease and immune function are selected for discussion.

Central effects of N/OFQPain. Since the identification of N/OFQ1,2 there has been intense interest in the role of this peptide in pain processing. This is based on various factors, including the similarity of distribution of receptor and peptide to classical opioids within the defined pain pathway, the structural similarity to classical opioids, and the simi-larity in post-receptor transduction to classical opioids. In addition, there is some limited association of plasma N/OFQ levels with human pain states and the paucity of current effective analgesics with activity in chronic pain states. In the original description of Meunier et al., the name nociceptin was related to the fact that intracerebroventricular administration of N/OFQ pro-duced hyperalgesia1. However, the marked differences in supraspinal and spinal effects set this peptide–receptor system apart from that of morphine and the m-opioid receptor system used in the clinic.

N/OFQ administered supraspinally reverses the effects of exogenous opioids; that is, N/OFQ is an anti-opioid peptide at this site5,43,44. In addition, acupuncture-induced antinociception is reversed45 and it has been suggested that the hyperalgesia observed in early studies1 is a result of a simple reversal of stress-induced antinociception5. Intracerebroventricular injection of N/OFQ was stressful,

resulting in the release of central endogenous opioid peptides with their effects subsequently reversed by the delivered dose of N/OFQ. In contrast to this supraspinal anti-opioid/hyperalgesic action, spinal administration produces a more classical antinociceptive effect46,47.

The neuroanatomical site underlying the anti-opioid actions of N/OFQ is the rostral ventromedial medulla where two types of cells exist: the ‘on’ and ‘off ’ cells5,44

(FIG. 3). On cells inhibit the action of off cells, and are inhibited by morphine acting at m-opioid receptors. Off cells project back to the spinal dorsal horn (known as the descending inhibitory control circuitry) to reduce ascending nociceptive information reaching third-order neurons via the ascending spinothalamic tract. When morphine inhibits the on cell this disinhibits the off cell, leading to an antinociceptive effect. N/OFQ inhibits both the on and off cell with direct inhibition of the off cell producing an increase in nociceptive traffic. Clearly, this inhibition of the off cell would reverse any actions of opioids at the on cell; producing an anti-opioid action. In vivo endogenous opioid peptides would produce a degree of antinociceptive ‘tone’, and stress (as in early studies1) would increase the local concentration of these peptides. Addition of N/OFQ under these experimental circumstances would be sufficient to produce a hyper-algesic response43,44,48. The consensus view is that spinal N/OFQ produces a classical (opioid-like) antinociceptive response by inhibiting transmitter release at primary nociceptive afferent terminals5,47,49. Indeed N/OFQ has been shown to inhibit the release of transmitters involved in this pathway including glutamate50. Interestingly, one study51 reported an increase in the release of the pro-nociceptive transmitter substance P.

Table 1 | Characteristics of non-N/OFQ-related peptides

Peptide NOP m-Opioid receptor

d-Opioid receptor

k-Opioid receptor

In vivo effects at NOP (route of administration)

‘Dooley’ peptides and derivatives*

Ac-RYYRIK-NH2‡ Partial

agonistInactive§ Inactive§ Inactive§ • Hypotension and bradycardia

(intravenous)207

Ac-RYYRWKKKKKKK-NH2 (ZP120)

Partial agonist

Inactive|| Inactive|| Inactive|| • Pro-nociceptive and decreased locomotion (intravenous)26

• Long-lasting Na+/K+-sparing aquaresis208

• Phase I/II trials in heart failure (Zealand Pharma; see Further information)

Ac-RY(3-Cl)YRWR-NH2 (Syn-1020)

Partial agonist

Inactive¶ Inactive¶ Inactive¶ • Antimorphine (intracerebroventricular)210

• Antinociception (subcutaneous)210

• Anti-allodynic (intraperitoneal)210

Other peptides

Peptide III-BTD Antagonist Partial agonist

Agonist Partial agonist#

• No data available; predict antinociceptive profile

OS-461, OS-462, OS-500

Agonist No data available

No data available

No data available

• Long-lasting hyperphagic response (intracerebroventricular)211

*See ReF. 25 for more information. ‡There are five members of this group, data for one is shown. Ac-RYYRWK-NH2 of this group is modified to produce ZP120. §Did not reverse the effects of m-opioid receptor, d-opioid receptor or k-opioid receptor-selective agonists in GTP-γ[35S] assays142. ||Implied, as effects are not reversed by naloxone26. ¶1,000-fold selectivity for NOP209.

#Very low efficacy with a of 0.1 compared with CI-977 (ReF. 33). N/OFQ, nociceptin/orphanin FQ peptide; NOP, N/OFQ receptor; OS-461, N-a-6-guanidinohexyl-l-tyrosyl-l-arginyl-l-tryptophanamide; OS-462, N-a-6-guanidinohexyl- 3,5-dimethyl-l-tyrosyl-l-tyrosyl-N-[(R)-1-(2-naphthyl)ethyl]-l-argininamide; OS-500, N-a-6-guanidinohexyl-3,5-dimethyl-l-tyrosyl-3,5-dimethyl-l-tyrosyl-N-[(R)-1-(2-naphthyl)ethyl]-l-argininamide.

R E V I E W S




N

NH2HN

OO

Et N N

O

N

OH

H

NN

HNO

N

Cl

ClHO

Me

NN

H3CO

CH3O

O

NH

ON

HOO

OH

N

HOO

N

OCH3

C Bu

OH

CH3

CH2

O

ON

N

NHO

N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamine

4-AminoquinolinesJTC-801 (Japan Tobacco)NOP antagonist

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

BenzimidazopiperidinesJ-113393 (Banyu)NOP antagonist

(–)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol

Aryl piperidinesSB-612111 (GlaxoSmithKline)NOP antagonist

[(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]

SpiropiperidinesRo64-6198 (Roche)NOP agonist

LofentanilAnilidopiperidine

Naloxone benzoylhydrazoneMorphinan

BuprenorphineMorphinan

SpiroxatrineNeuroleptic

The relative contribution of supraspinal pro-noci-ceptive and spinal antinociceptive actions of systemi-cally administered NOP agonists is worth considering. Depending on the pharmacokinetics and blood–brain barrier permeability of the ligand under study, the relative distribution will be important. On the one hand supraspinal (agonist) spread might produce a hyperalgesic response (via reversal of endogenous opioid action in the ventromedial medulla), yet if the agonist also acts at the spinal site then an inhibition of nociceptive afferent inflow would result. The net effect would therefore be difficult to predict.

Several groups have reviewed the details of intra-cerebroventricular NOP antagonism4,5,24 and there is a reasonably consistent view that peptide but not non-peptide antagonists are generally antinociceptive when administered intracerebroventricularly. Using the non-peptide data in the schematic shown in FIG. 3, it is proposed that there is a degree of N/OFQ-mediated tone in the brain and that this produces a tonic anti-(endogenous)opioid action, thus setting the pain thresh-old to a more pain phenotype. Switching this off would produce analgesia/antinociception. However, the lack of effect of intracerebroventricular non-peptide antagonists

via this route (for example, J-113397 (ReF. 52)) is at variance with this hypothesis and is hard to reconcile. In addition, the lack of antinociceptive effects of the selective non-peptide antagonists J-113397 (ReF. 53) and SB-612111 (ReF. 54) given systemically is also at variance with this general scheme. How can this complex (central compared with peripheral administration) behaviour be explained? There are a number of differences in the way the experiments are conducted. For instance, the time course of the nociceptive stimulus is variable, short-term (tail withdrawal, tail flick and hot plate) compared with long-term (formalin test), and the degree to which the experimental protocol (for example, intracerebro-ventricular injection) produces stress and hence stress-induced antinociception against which NOP antagonists can work is also variable.

The relative contribution of spinal and supraspinal actions in the overall response to systemic NOP antag-onists has been elegantly addressed in the formalin test55. Intraplantar formalin produces a biphasic pain response with an acute phase lasting approximately 10 mins and a later secondary phase starting at around 20 mins and lasting for half an hour. Intracerebroventricular administration of UFP-101 produced an antinociceptive response and

Figure 2 | Non-peptide nociceptin/orphanin FQ receptor (NOP) ligands. Examples of the existing relatively non-selective ligands (highlighted in the centre of the figure) and members of the four chemical classes (4-aminoquinolines, benzimidazopiperidines, aryl piperidines and spiropiperidines) are shown.

R E V I E W S



intrathecal administration of UFP-101 produced a pro-nociceptive response in the secondary phase. This could be predicted based on the previous discussion. Interestingly, systemic (intravenous) administration of J-113397, which would deliver this antagonist to both spinal and supraspinal sites, produced a pro-nociceptive effect. In general data obtained with NOP–/– mice and mice deficient in pre-pro-N/OFQ (ppN/OFQ–/–) agree with pharmacological findings56. The overall conclusion from this experiment is that the predominant action of systemic NOP antagonists is spinal, and that endogenous N/OFQ-mediated tone in the spinal cord is high and in the brain it is low. This contrasts with the effects seen following acute and stressful intracerebroventricular injection with an acute pain measure in which N/OFQ-mediated tone is presumed to be high and possibly an assay and stimulus-dependent phenomenon55.

It has been known for many years that peripheral terminals of nociceptive afferents express NOP. NOPs are located in skin57, bladder (see below for a more detailed consideration)58 and on lymphocytes (see below for a more detailed consideration)59. If it is assumed that peripheral inflammation upregulates NOP (in a similar manner to m-opioid receptors) coupled with lymphocyte N/OFQ release, then an additional facet to the neuroimmune axis can be proposed60. Activation of NOP at any of these peripheral sites would reduce nociceptive afferent inflow and produce peripheral analgesia/antinociception. There is some experimental evidence for this mode of action in producing antinoci-ception61–63 (FIG. 3).

Opioids are the gold standard for acute pain and are often used with some effect in chronic pain8. However, there is a trade-off between good analgesia and poor side-effect profile. Indeed, tolerance to morphine develops such that dose escalation is required, which inevitably increases the prevalence and severity of side effects8,64. Therefore, any new analgesic would have greater potential if it had a relatively low (or absent) ability to produce tolerance. There is evidence for toler-ance to the antinociceptive effects of spinal N/OFQ65. Intriguingly in animals that lack NOP66,67 or are treated with the peptide NOP antagonist [Nphe1]N/OFQ(1–13)-NH2 (ReF. 68) and the non-peptide antagonists J-113397 (ReF. 69) and SB-612111 (ReF. 54) there was a reduction in the development of tolerance to morphine. Moreover, ppN/OFQ–/– mice also display reduced liability to develop morphine tolerance69. This raises the possibility of co-administration of a m-opioid receptor agonist and a NOP antagonist. Based on the discussion above this might produce classical m-opioid receptor analgesia, reduce the anti-opioid effects of endogenous N/OFQ to potentiate morphine analgesia and produce less toler-ance, which would then enable lower doses of morphine to be used. This reduced dose might then reduce the other clinical side effects that are associated with mor-phine, such as respiratory depression and constipation. Current development for pain remains predominantly preclinical, although Phase I and II trials of the low-selectivity NOP antagonist JTC-801 (TABLe 2) are noted but there are no published data available.

Human studies of pain. There have been attempts to correlate human plasma N/OFQ levels with pain but the results of these studies have been contradictory70. An increase in N/OFQ levels in acute and chronic pain com-pared with controls was reported71. However, a decrease in N/OFQ levels was noted in fibromyalgia syndrome72 and cluster headache73. From an effect-site perspective it is difficult to reconcile plasma measurements with nociceptor-synaptic peptide concentrations. This prob-lem has been addressed by measuring cerebrospinal fluid N/OFQ in labour (requiring an epidural anaesthetic enabling sampling of the cerebrospinal fluid); no increase relative to non-labouring (elective caesarean sections) patients was reported74.

N/OFQ has been administered in humans in two studies: intramuscular injection in volunteers75 and intra-vesical instillation in urology patients (see below). The two volunteer studies involved increasing doses of intra-muscular N/OFQ into the left and right temporal muscle in random and as an open label or as a balanced placebo-controlled intramuscular injection into the non-dominant trapezius. There was no pain noted in either study but in the latter study there was an increase in local tenderness.

Anxiety and depression. The current market has a wide range of drugs for the treatment of anxiety and depression, but these are often characterized by poor and/or variable efficacy, long run in to peak behavioural effect and, from a patients perspective, a wide range of side effects leading to tolerability and compliance problems76,77.

There is now good evidence from animal work for a role for the N/OFQ–NOP system in emotional dis-orders78 including anxiety79,80 and depression81–83. NOP and N/OFQ are located in areas that are crucial to mood control including but not limited to amygdala, hippo-campus, thalamus and cortical processing areas78. At relatively low doses, N/OFQ79 and several non-peptide agonists from Roche (Ro65-6570 and Ro64-6198) are generally reported as an anxiolytic84–86, although others87 have questioned this. It has been reported that repeated administration of Ro64-6198 for 15 days failed to induce tolerance to the anxiolytic actions of this molecule88, perhaps setting Ro64-6198 aside from more traditional anxiolytics. In a recent study from Schering–Plough the non-peptide agonist SCH 221510 was shown to be anxiolytic but with a reduced side-effect profile when compared with benzodiazepines37. Moreover, ppN/OFQ–/–

mice display an anxiogenic phenotype89. Interestingly, there was some modulation of pain in this knockout model, underscoring a close link between stress and pain89,90. Use of NOP antagonists does not modify the level of anxiety and implies a lack of N/OFQ-mediated tone in the control of this behaviour. There is no consensus on the mechanism(s) underlying this behavioural response, although modulation of 5-hydroxytryptamine (5-HT)91, functional coriticotropin-releasing factor (CRF) antago-nism79 and recently a dependence on GABAA (γ-amino-butyric acid A) receptors92 has been suggested. In contrast to this lack of antagonist effect per se in this model of anxiety, NOP antagonists display an antidepressant pro-file81–83 and indicate central N/OFQ-mediated tone in the

R E V I E W S



control of depression. Again, there is no consensus on the mechanism(s) underlying this behavioural response. However, with the wealth of information on the role of catecholamines in depression93, the well-known observa-tion that N/OFQ inhibits catecholamine release14, the fact that NOP antagonists will switch off N/OFQ-mediated signalling combined with observations that N/OFQ acting at postsynaptic NOP increases K+ conductance and reduces the firing of locus coeruleus (noradrener-gic)94 and dorsal raphe (serotonergic)12 nuclei, a simple catecholamine hypothesis can be advanced (FIG. 4).

Taken at face value these data indicate that NOP agonists are anxiolytic and that antagonists are anti-depressant. However, there are several problems and areas in which detailed information is lacking: most animal studies involve a short-term exposure to the compound, whereas longer-term treatments in humans are more usual (>4 weeks for selective serotonin reuptake inhibitor effect); the precise neuroanatomical sites(s) of the anxiolytic/antidepressant behaviours are largely unknown; and there is an almost complete lack of clinical information.

Table 2 | Selected characteristics of some common and novel non-peptide NOP ligands

class and compound

Pharmacological profile In vivo effects

Morphinans (poor selectivity)

TRK820 (Toray/Acologix)

NOP antagonist, m-opioid receptor partial agonist, k-opioid receptor agonist212

• Antinociceptive in mice213 and monkeys214

• Antipruritic in monkeys215

• In Phase III as antipruritic in patients with uraemia‡

• No specific NOP-related clinical development

Buprenorphine Non-selective m-opioid receptor and NOP partial agonist216

• In widespread clinical use for moderate to severe pain • Analgesia via m-opioid receptor217

• Also under evaluation by Samyang as a patch§

Naloxone benzoylhydrazone

Non-selective m-opioid receptor antagonist, k-opioid receptor agonist219 and NOP partial agonist (very low efficacy)32

• Antinociceptive activity in mice, which is lost in NOP–/– mice218

4-Aminoquinolines

JTC-801 (Japan Tobacco)

NOP antagonist, low selectivity over m-opioid receptor220

• Systemic220 and spinal antinociceptive in mice221

• Phase I (oral) and Phase II pain (injectable) trials have been done (no published details)

• Some actions may be mediated by m-opioid receptor

Benzimidazopiperidines*

J-113397 (Banyu) Highly selective NOP antagonist53,222,223

• Complex behaviour; antinociceptive and pro-nociceptive5

• Trap-101 is an achiral analogue of J-113397 (ReF. 224); not evaluated in vivo

Aryl-piperidines

SB-612111 (GlaxoSmithKline)

Highly selective NOP antagonist54,182

• Reduces N/OFQ-induced pro-nociceptive and antinociceptive and orexigenic responses

• Has antidepressant profile225

• Phase I trials in parkinsonism expected in 2008||

Series of compounds (Schering)

Highly selective NOP agonist226,227 • Not extensively evaluated; displays antitussive activity in capsaicin-induced cough in guinea-pig227

Spiropiperidines*

Ro64-6198 (Roche) Selective NOP agonist96 • Anxiolytic and hyperphagic effects in rats (improved version of Ro65-6570)96

NNC-63-0532 (Novo) Low selectivity NOP agonist228 • Not evaluated in vivo

NNC-63-0780 (Novo) Selective NOP antagonist229 • Not evaluated in vivo

Series of compounds (Schering)

Highly selective NOP agonist230 • Not extensively evaluated

*In addition to those mentioned here, Pfizer has patents for benzimidazopiperidines231 and spiropiperidines232–234. ‡According to Acologix (see Further information). §According to Samyang (see Further information). ||According to Brane Discovery (see Further information). 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; 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; NNC-63-0780: (3R,4S)-3-((2-tert-butylphenoxy)methyl)-1-methyl-4-phenylpiperidine; Ro64-6198, (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; SB-612111, (–)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (also known as BND-10001; NiKem-Brane code for SB-612111); TRK820, (–)-17-cyclopropylmethyl-3,14b-dihydroxy-4,5a-epoxy-6b-[N-methyl-trans-3-(3-furyl)acrylamido]morphinan hydrochloride. In a very recent paper a 4-aryl-tropane is described, with the nociceptin/orphanin FQ peptide (N/OFQ) receptor (NOP) agonist SCH 221510 as an example37.

R E V I E W S



Conditioned place preference (CPP) testAn experimental animal is presented with a positive stimulus (in this case a drug of abuse) in conjunction with a specific cue. The animal develops an association between the place and preference for the positive stimulus. The amount of time spent in the place previously associated with the stimulus can be used as an index of the rewarding properties of the original stimulus.

Marchigian Sardinian alcohol-preferring (mSP) ratA genetically selected strain of rat with a natural preference for ethanol. This is used as a model of human alcoholism.

GTPγ [35S] bindingAn in vitro assay used to monitor G-protein coupled receptor-mediated guanine nucleotide exchange at G proteins.

Human study of depression. There is just one human study95 in which plasma N/OFQ and 5-HT levels were measured: 21 patients with post-partum depression were compared with 25 healthy controls. In controls, plasma N/OFQ levels were 10.4 ± 3.7 pg ml–1 (remarkably con-sistent with previously measured values70) and this was elevated to 28.5 ± 5.8 pg ml–1 in the post-partum depres-sion group. By contrast, 5-HT levels in the post-partum depression group were lower (1.0 ± 0.3 mmol l–1 com-pared with 1.4 ± 0.4 mmol l–1). These limited small study data agree with the notion that post-partum depression results from reduced 5-HT levels and that this is accom-panied by elevated N/OFQ with the increase in N/OFQ possibly causing the fall in 5-HT. There is currently no major clinical development in this area, but based on this small study larger studies are required to confirm this interesting and important finding95. Studies of clinically useful antagonists (such as SB-612111, awaiting trials in parkinsonism, TABLe 2) are eagerly awaited. In terms of development of anxiolytic agonists Ro64-6198 seems an ideal candidate (TABLe 2), but the future of this molecule according to Shoblock96 “is uncertain” owing to potential side-effect problems.

Modulation of the rewarding properties of drugs of abuse. Drug abuse (including alcohol) has a major socioeconomic impact and is relatively difficult to treat. According to estimations from the UK government (Neighbourhood.gov.uk; see Further information), the misuse of drugs costs the UK economy between UK£10–18 billion per year. The major pharmacological treatment strategies are based on reducing the impact of the symptoms of dependence, allowing abstinence and prevention of relapse. There are a limited number of such treatments available, with varying efficacy, including methadone and buprenorphine (for opiate abuse)97 and naltrexone and the antiglutamatergic agent acompros-tate (for alcohol abuse)98. The value of addressing the psychological component of dependence has also been described99,100.

In animal models aimed at elucidating the rewarding properties of drugs of abuse the conditioned place preference (CPP) test is commonly used. In this assay N/OFQ has been shown to reduce CPP to alcohol101–103, ampheta-mines104, cocaine105,106 and morphine106, indicating that this peptide was reducing reward to these stimuli. N/OFQ alone was inactive. A role for the endogenous N/OFQ–NOP system was recently examined in a complex study107 that assessed hedonic state (which they define as “a bal-anced affective, emotional and motivational state”) in methamphetamine and ethanol-conditioned wild-type and NOP–/– mice. As N/OFQ appeared to suppress an enhancement of hedonic state the authors suggest that the endogenous N/OFQ–NOP system might facilitate the development of addiction107.

The neuroanatomical substrates for these responses include the ventral tegmental area, nucleus accumbens, amygdala and medial prefrontal cortex; all of these areas express NOP. In anaesthetized rats intracerebroventricular administration108 and intrategmental administration109 of N/OFQ suppressed dopamine release from the nucleus

accumbens. In addition, intracerebroventricular adminis-tration of N/OFQ reduced morphine-induced110,111 and cocaine-induced112 dopamine release from this brain structure. Using electrophysiological techniques, gluta-mate and GABA release in the amygdala of the rat are inhibited by N/OFQ113. In a more recent study, N/OFQ was shown to decrease GABAA receptor-mediated inhibi-tory postsynaptic currents and importantly to prevent ethanol-induced augmentation of inhibitory postsynaptic currents in the central amygdala of the rat114. In the prefrontal cortex N/OFQ inhibits the release of noradrena-line115; data for the major reward neurotransmitters is lacking.

A substantial amount of current information on addiction and reward comes from studies using alcohol. In this respect the Marchigian Sardinian alcohol-preferring (msP) rat has provided some interesting insights101,102,116–118. In msP rats intracerebroventricular administration of the peptides OS-462, UFP-102 and UFP-112 reduced alcohol consumption. But interestingly and in contrast to the later work in normal Wistar rats of Kuzmin119 the non-peptide Ro64-6198 given intraperitoneally increased alcohol consumption, an effect the authors ascribe to m-opioid receptor residual agonism120. In this model low doses of the mixed m-opioid receptor/NOP agonist buprenorphine increased alcohol consumption (in a naltrexone-sensitive manner) and reduced consumption at higher doses (in a UFP-101-sensitive manner)121. As buprenorphine is currently available for use in humans it may form an interesting alternative or adjunct to current therapy for treating alcoholism.

In an elegant study comparing msP rats and normal Wistar rats it was shown that the N/OFQ-mediated reduction in alcohol drinking behaviour of the msP (but not normal Wistar) rat corresponded with an increase in NOP and ppN/OFQ mRNA and NOP pro-tein in reward areas of the brain122. In addition, there was an increases in receptor function (as measured by GTPγ[35S] binding) in these areas of msP rat brains with the notable exception of central amygdaloid nucleus where there was a decrease. Direct injection of N/OFQ into this region markedly reduced ethanol consump-tion. In the basolateral amygdala and the bed nucleus of the stria terminalis (where GTPγ[35S] binding was increased) direct injection of N/OFQ did not reduce alcohol consumption122. Based on the following evi-dence — one there is a upregulation of CRF receptor activity in the central amygdaloid nucleus in the msP model, and two there is a compensatory increase in NOP signalling in other brain areas except the central amygdaloid nucleus — the authors speculate that an imbalance between CRF and N/OFQ–NOP signalling underlie the increased alcohol drinking in msP rats and that restoration of this imbalance with CRF antagonists and/or NOP agonists might represent a mechanism-driven treatment strategy.

Human studies of drug dependence. There are two studies examining an association between single nucleotide polymorphisms (SNPs) in the NOP gene (ORPL1) and ppN/OFQ gene (PNOC) and alcohol/

R E V I E W S



On Off


–

–

–

–

–

–

–

+

Aδ/C

Morphine

Morphine

N/OFQ

N/OFQ

–N/OFQ

Skin

Peripheral organs (bladder)

PBMC

Spinal

Supraspinal

Inflamedterminal

–N/OFQ

–– N/OFQ

Peripheral input

Descending

Ascending

RVM

N/OFQrelease

SympatholyticAn agent that decreases the activity of the sympathetic nervous system, for example, guanethidine.

illicit drug dependence123,124. One study examined 10 SNPs covering OPRL1 and the adjacent regulator of G-protein signalling RGS19 and 15 SNPs covering PNOC in European American subjects. The authors attempted to correlate genotype with alcohol and illicit drug (marijuana, cocaine, stimulant, sedative or opioid) dependence. The authors reported no convincing asso-ciation between alcohol dependence and either OPRL1 or PNOC polymorphisms. However, they did report a marginal association between two (alcohol depend-ence) and one (illicit drug dependence) of 15 non-coding PNOC SNPs123. A second recent study examined 18 OPRL1 SNPs in Scandinavians with alcohol depend-ence. One SNP (rs6010718) was associated with alcohol dependence, more so in females124. These studies offer a tantalising early insight into a genetic role for

NOP–N/OFQ in drug dependence. However, several questions remain: how widespread is the association? Is it dependent on race and sex? What is the role of an association with SNPs in non-coding regions? Are there any differences in functional receptor or peptide production? There is currently no indication of new pharmaceutical development beyond preclinical phases for this indication.

Central and peripheral actions of N/OFQThe control of the cardiovascular and renal system. Based on statistics collated by The British Heart Foundation (see Further information) 49% of all deaths in Europe were as a consequence of cardiovascular disease. With the financial impact of cardiovascular disease in Europe estimated at 192 billion euros there is a substantial market for novel, high efficacy, low side-effect drugs to treat heart (failure) and vessel disease. In anaesthetized and conscious laboratory animals N/OFQ produces hypotension and bradycardia6,125. This occurs following intravenous126 and intracerebroventricular127 administra-tion and is absent in NOP–/– mice128. The effects occur at both central and peripheral sites. The most compelling evidence for peripheral effects is the hypotension and bradycardia produced by intravenous administration of N/OFQ, a peptide that does not cross the blood–brain barrier. A role for reflex loops cannot, however, be completely excluded. It has been suggested that as the sympatholytic guanethidine reduced the hypotensive effects of N/OFQ, then this peptide acts to inhibit sympa-thetic control of the cardiovascular system129. In addition, this study showed that the bradycardic effects of N/OFQ were reduced by vagotomy, indicating that N/OFQ increased parasympathetic activity. Direct injection of N/OFQ into the rostral ventrolateral medulla produced bradycardia and hypotension130. Moreover, intravenous infusion of N/OFQ produces a diuresis and antinatriuresis, and decreases renal sympathetic nerve activity125,126,131.

Intravenous N/OFQ produces vasodilation132–134. Nitric oxide is not involved in this dilator response134. In a series of studies of the cerebral circulation, N/OFQ was shown to have little effect under normal physiological conditions, but in brain injury (experimental ischaemia or trauma) cerebral blood flow is reduced135. In a rat mesenteric microcirculation model, intravenous admin-istration of N/OFQ dilated arterioles and venules136. Dilation of these non-innervated vessels was blocked by histamine antagonists and mast-cell stabilizers, suggesting that the N/OFQ-mediated dilation of the microcirculation is secondary to mast-cell release of histamine. N/OFQ has been shown to release histamine in the brain following intracerebroventricular injection137 and directly from rat peritoneal mast cell preparations138.

Sepsis and its progression to septic shock is a condition in which there is marked hypotension and an associated high mortality rate139. Using the caecal ligation-puncture model of sepsis in the rat it has been recently reported that the mortality rate of 70% was markedly reduced to 40% following treatment with the peptide NOP antagonist UFP-101 (Gavioli et al., personal communication). The implication of this study is that N/OFQ concentrations

Figure 3 | schematic to describe the interrelationship between the anatomical site(s) underlying the actions of N/OFQ on pain. Nociceptin/orphanin FQ peptide (N/OFQ) produces supraspinal hyperalgesia/anti-opioid actions by inhibiting both on and off cells in the rostral ventromedial medulla (RVM). On cells inhibit off cells, and are inhibited by morphine acting at m-opioid receptors. Off cells project back to the spinal dorsal horn and reduce ascending nociceptive information. When classical opioids like morphine inhibit the on cell this disinhibits the off cell, leading to an antinociceptive or analgesic effect. By contrast, N/OFQ (exogenously added or endogenously produced) inhibits both the on and off cell with direct inhibition of the off cell producing an increase in nociceptive traffic. Clearly, inhibition of the off cell would reverse any actions of opioids at the on cell; producing an anti-opioid (exogenously added or endogenously produced) action. At the spinal level, N/OFQ produces classical opioid analgesia by inhibiting nociceptive afferent inflow. Inhibition of nociceptive afferent inflow can occur in the periphery in tissues such as skin and bladder, and possibly via an interaction with the neuroimmune axis. PBMC, peripheral blood mononuclear cells. Exogenous N/OFQ is in the blue box.

R E V I E W S



Chronotropic effectIn the context of the cardiovascular system, this refers to heart rate. opioids slow heart rate and thus have a negative chronotropic effect.

Hyponatraemia Low plasma sodium concentration.

Capsaicin-sensitive primary afferentsThese are primary nociceptive afferent fibres of the Ad and C-fibre type. They express TRPV1 receptors and hence are capsaicin sensitive. As TRPV1 receptors are also activated by heat and low pH, these channels convey a polymodal activation profile to Ad and C-fibres.

Detrusor hyperreflexiaIncreased contractility of the detrusor muscle of the bladder (idiopathic), often as a result of neurological disease (neurogenic), leading to urinary incontinence.

Afferent fibre switchingAfferent signals from the bladder are conveyed by Ad and C-fibres, with the latter being ‘silent’. In spinal-cord injury, C-fibre activity is unmasked, leading to fibre switching.

might be elevated in sepsis. In support of this notion, N/OFQ concentrations in 21 patients in intensive care with a diagnosis of sepsis were measured. In this small study four patients died and this was associated with elevated N/OFQ levels when compared with those who survived140. Based on these complementary pieces of evidence it is tempting to suggest that NOP antagonists might be a useful adjunct in the management of sepsis, and larger studies in humans are warranted.

Direct effects of N/OFQ on the heart are controversial. In the adult there is a single study reporting evidence of NOP receptors in heart tissue141, albeit with differing ligand affinity and binding capacities in comparison with other ex vivo studies. The authors did report some limited PCR for NOP transcripts and noted that in spon-taneously hypertensive rats there was an increase in the number of high-affinity N/OFQ binding sites. These receptors may be on cardiac neural tissue. In 1–2 day old cardiomyocytes isolated from Wistar rats N/OFQ displayed a positive chronotropic effect with a maximum 65% of that produced by isoprenaline142. Again in neo-natal cardiomyocytes N/OFQ increased atrial natriuretic peptide secretion and inhibited cAMP formation143. Pharmacological analysis using antagonists for classi-cal opioid and NOP receptors are strongly suggestive of expression in this tissue.

Although the general hypotensive, bradycardic and vasodilator actions of N/OFQ hold, there are several apparently contradictory pieces of data. First, in con-scious sheep N/OFQ increases blood pressure and heart rate144. Second, injection of N/OFQ into the nucleus of the solitary tract (the termination point for cardiac sensory afferents) increases heart rate and blood pressure145. Last, in cultured neonatal rat myocytes exogenous N/OFQ increased contraction rate142.

Human studies of the cardiovascular and renal system. Of particular note in this area is the work with the peptide ZP120. Based on animal work, ZP120 is best described as an aquaretic as it promotes excretion of water while sparing electrolyte loss26,27,146. Coupled with vasodilatory actions147 this peptide displays a clinical profile that is compatible with use in congestive heart failure (reduction in oedema and correction of hyponatraemia). Phase I and II trials have been completed; these data are in abstract form and can be found at the Zealand Pharma web site (see Further information) and TABLe 1. ZP120 is described as “safe and well tolerated in patients with chronic heart failure” with a nonlinear pharmacokinetic profile.

Peripheral effects of N/OFQThe urogenital system. Overactive bladder affects approximately 16% of the European population >40 years of age and Ad and C-fibre afferents are variably implicated148. Traditional use of anticholinergics and more recently targeting transient receptor potential cation channel, subfamily v, member 1 (TRPv1) receptors is of variable efficacy148. One of the earliest ex vivo bioassays for NOP was in the mouse vas deferens in which N/OFQ inhibits electrically evoked contraction149,150. In the rat, NOP receptors located on capsaicin-sensitive primary afferent

fibres of the bladder inhibits the volume-evoked mictu-rition reflex58,151–153. At the time of writing there are no studies detailing NOP expression in bladder tissue of human origin. However, based on animal data a series of elegant clinical studies have been performed using intravesical N/OFQ by Lazzeri and colleagues, which are described next.

Human studies of the urogenital system. In a pilot study of 14 patients, five normal controls and nine with detrusor hyperreflexia, intravesical instillation of 1 mM N/OFQ increased bladder capacity and the volume required for detrusor hyperreflexia. This effect lasted 24–48 hours and was not present in the control group. Interestingly, one patient had previously received intravesical capsaicin and the effects of N/OFQ were minimal, indicating a capsaicin-sensitive target154. In a later follow-up study, N/OFQ was compared with the structurally similar but inactive [desPhe1]N/OFQ (a major metabolite of N/OFQ, which does not bind to NOP; that is, a placebo-controlled study) in 14 patients with detrusor overactivity due to spinal-cord damage. Intravesical administration of 1 mM N/OFQ but not [desPhe1]N/OFQ increased bladder capacity and volume required for detrusor hyperreflexia155. In a further study of 18 patients with neurogenic detrusor overactivity incontinence who were capable of self catheterization, half of the group instilled 1 mg of N/OFQ and the remainder instilled saline at the first catheterization of the morning for a period of 10 days. In agreement with previous studies bladder capacity increased following N/OFQ treatment. From a patient perspective the number of daily urine leakage episodes were reduced in the N/OFQ (but not saline) group. There did not appear to be any major practical problems related to use, and the effects of a single instill-ation in the morning appeared to last the whole day156.

Collectively, these studies broadly agree with studies in rats that NOP is probably expressed on capsaicin-sensitive primary afferents and their activation inhibits the micturition reflex. However, basic work has only been done in normal rats, and N/OFQ is only effective in spinally injured patients where there may be afferent fibre switching157,158. N/OFQ and N/OFQ mimetics have a potential role in the treatment of overactive bladder and their use is not limited to specialist centres capable of intravesical administration. However, it is most likely that any potential development (of which there is cur-rently none) will be for patients resistant to the more conventional use of anticholinergics148.

The immune response. It is well documented that opioids depress the immune system159,160. As early as 1950 Hussey and Katz described a series of 102 opioid addicts with infection as a result of their addiction161. Despite this clear and well-known observation there is much controversy as to the precise site(s) of this interaction. Opioids can depress the immune response via the hypothalamic–pituitary–adrenal axis via increased glucocorticoid production162. However with regard to the classical opioid receptors (m, d, k) there is much controversy as to a direct interaction with cells of the immune system.

R E V I E W S




• NOP–/– • ppN/OFQ–/–

• Antidepressant phenotype• Not done

• Some anxious features• Anxiogenic phenotype

Depression Anxiety

Depression Anxiety

Knockout mice

• Agonist• Antagonist

• No direct effect• Antidepressant

• Anxiolytic• No direct effect

NOPligands

• Supporting evidence• Preferred NOP ligand

• N/OFQ elevated (28.5 pg ml–1) in post- partum depression (10.4 pg ml–1 in control)• Antagonist

• None

• Agonist

Clinical

Tonic N/OFQ reducescatecholamine levels

Possible modulation of 5-HT and CRF levels, and GABAA receptors

NOP antagonist reverses tonicinhibition to elevatecatecholamines andincrease mood control

N/OFQ signallingNOP receptor

Neuron

NOPantagonist

Consensus is that under resting conditions immuno-cytes do not express classical opioid receptors163. However, one limited PCR study showed m-opioid receptor upregulation following pretreatment with interleukin 4 (IL4), which suggests that in sepsis m-opioid receptors may be upregulated164. That circulating lymphocytes contain and release opioid peptides is also clear60. Of importance are studies that propose a neuroimmune axis in which peripheral inflammation upregulates m-opioid receptors on neurons at the site of inflammation, and infiltration of white cells releases opioids to produce a degree of peripheral analgesia60. NOP and N/OFQ are expressed on crude cultures of peripheral blood mono-nuclear cells163, monocytes165 and neutrophils166. It is tempting to add an N/OFQ–NOP component to the neuroimmune axis, where, in addition to inhibition of peripheral neuronal activity, released N/OFQ may feed-back control lymphocyte function. Indeed, increased monocyte chemotaxis165 and T-cell proliferation have been reported in response to N/OFQ treatment167.

It remains to be determined whether peripheral inflammation upregulates neuronal NOP in a similar manner to m-opioid receptors. However, recent studies have shown upregulation of N/OFQ in the rat in response to peripheral inflammation with bacterial lipopolysac-charide168, and a modulation of immune function in response to staphylococcal enterotoxin A administration in mice169. It is possible that the elevated N/OFQ levels

reported in non-surviving septic patients140 originates from increased immunocyte release, and it is also possible that there may be some interplay with the vasculature via a neuro–vascular–immune axis. In a mouse model of inflammatory bowel disease (dextran sulphate sodium administration) in which there is an infiltration of inflam-matory cells and an upregulation of N/OFQ expression, NOP knockout prevents the development of colitis, thus indicating a further link between N/OFQ and the immune system170.

Airway. The cough reflex results from an afferent impulse (originating in the airway) via the nodose ganglion to the nucleus of the solitary tract in the medulla oblon-gata171. N/OFQ inhibits cough in guinea-pigs172 and in cats173, and inhibits ex vivo airway contractility in various species including humans174. As N/OFQ does not cross the blood–brain barrier, the in vivo effects are peripheral. NOP is located on capsaicin-sensitive affer-ents and causes an inhibition of tachykinin release174. The antitussive effects can be mimicked by the non-peptide agonist Ro64-6198 in a J-113397-sensitive man-ner175. Codeine is the current gold-standard antitussive agent but has a poor side-effect profile that is typical of m-opioid receptor agonists (such as nausea, constipa-tion, tolerance and dependence). So, orally active NOP agonists represent a viable alternative for the treatment of cough.

Figure 4 | involvement of N/OFQ–NOP in depression and anxiety: NOP antagonists are antidepressant and agonists are anxiolytic. The mechanism of the antidepressant actions of nociceptin/orphanin FQ (N/OFQ) receptor (NOP) antagonists are best understood. If we accept that in depression synaptic (and plasma) catecholamine (especially 5-hydroxytryptamine; 5-HT) concentrations are reduced then a mechanism involving a reversal of this fall is logical. As N/OFQ is known to depress catecholamine release from a variety of preparations, and this peptide is elevated in (at least) post-partum depression, then a NOP antagonist would reverse the inhibitory actions of N/OFQ to raise catecholamine levels (5-HT levels were elevated in post-partum depression). Therefore, the elevated mood produced by NOP antagonists may result from a reduction of central N/OFQ-mediated signalling. At present there is no concrete information as to whether these actions are presynaptic or postsynaptic (for simplicity the picture shows a presynaptic action). The precise mechanism(s) of the anxiolytic effects of NOP agonists are presently unknown, but may involve modulation of 5-HT and corticotrophin-releasing factor (CRF) levels and GABAA (γ-aminobutyric acid A) receptors. ppN/OFQ, pre-pro-N/OFQ.

R E V I E W S



Gastrointestinal tract. N/OFQ inhibits contractility of the gastrointestinal tract in a wide range of species and at most sites along the gastrointestinal tract176. In addition to the well-characterized inhibition of gastric motility, N/OFQ prevents gastric damage that is induced by intragastric ethanol177 and cold-restraint stress178, and a role for NOP in inflammatory bowel disease has been described above170. There is evidence for central and peripheral components to the regulation of gastrointes-tinal function179,180. Indeed, an elegant study addressed this using the peptides UFP-112 and N/OFQ adminis-tered intracerebroventrically and intraperitoneally on gastrointestinal function in the rat. When administered intracerebroventrically both peptides (UFP-112 is more potent) inhibited gastric emptying, inhibited gastric acid secretion and reduced ethanol-induced intragastric lesions. However, when the peptides were administered intraperitoneally, reduced gastric emptying was absent and the inhibitory action on acid secretion was con-verted to enhanced secretion179. The effects of N/OFQ in simple contractility bioassays can be mimicked by the non-peptide agonist Ro64-6198 (ReF. 181) and can be reversed by the non-peptide antagonist SB-612111 (ReF. 182).

Concluding thoughtsIt is clear that N/OFQ exhibits a vast range of biological activities and as such has potential for clinical develop-ment. The challenge will be to be selective for a particular disease although not necessarily selective for NOP. The effects in some disease indications can be described as central, peripheral or mixed. For example, a centrally active NOP antagonist might be antidepressant with analgesic properties (some antidepressants are already widely used for treating chronic pain183). The downside of administering an antagonist that is able to access the CNS in humans is that it would also produce elevated circulating NOP antagonist levels, which might be problematic in patients with heart failure, hyperreactive airways or urinary incontinence. Of course this would only be a concern if N/OFQ levels are elevated and, at present, there is little evidence for this.

Preventing passage across the blood–brain barrier, which would lead to peripheral targeting, may be easier to achieve and this has been done with molecules such as methyl naltrexone for the treatment of opioid-induced constipation184. A peripheral NOP agonist might prove useful in heart failure or for the treatment of urinary incontinence. Opioids have been delivered to the spinal cord for pain relief for decades and are often combined with local anaesthetics for use in labour185. As intrathecal

N/OFQ is antinocieptive47 it is tempting to suggest that this route be utilized in humans.

There has always been a desire to create highly selec-tive drugs. Indeed, in the opioid field there is the com-monly held clinical view that there is a need for selective morphine-like molecules that have reduced side-effect profiles, often achieved by modification of the pharma-cokinetic behaviour. A good example of this approach is the highly successful remifentanil, which, like morphine, targets m-opioid receptors, but is metabolized by plasma esterases to inactive metabolites. The effects of this are a rapid off-set of action186. However, there is now grow-ing interest in the design and evaluation of non-selective molecules. One of the most compelling pieces of infor-mation from the opioid field in favour of this approach is the observation that in mice lacking d-opioid recep-tors or wild-type mice treated with d-opioid receptors antagonists, tolerance to morphine does not develop187,188. Clearly a mixed m-opioid receptor agonist/d-opioid recep-tor antagonist would have tremendous clinical potential. Such molecules already exist for experimental use, for example, H-Dmt-Tic-Gly-NH-CH2-Ph (UFP-505)189 and [Dmt1,D-1-Nal4]endomorphin-1 (ReF.190). From the description above there would be some advantage in ligands with mixed activity at NOP and classical opioid receptors. If we consider the observation that NOP–/– animals display reduced morphine tolerance66,67 then a mixed NOP antagonist and m-opioid receptor agonist has potential in terms of reduced tolerance and lower dosage (as NOP antagonists per se might be antinociceptive). Do such molecules exist? In a recent study the interaction of a range of classical opioids and five non-peptides of the sequence from SRI International was characterized191. One particular molecule, SR-14148, behaves as a NOP antagonist and as a m-opioid receptor partial agonist. Although the authors extensively discuss the relative merits of different assays this work is promising for the future design of higher-affinity NOP antagonists with improved m-opioid receptor efficacy. As an interesting addition to the general concept of mixed molecules the same group described SR-16435, which behaved as a mixed m-opioid receptor/NOP partial agonist. This molecule produced m-opioid receptor-mediated antino-ciception with reduced development of tolerance192.

It has now been 13 years since the truly seminal work of Meunier and colleagues1 and Reinscheid colleagues2. The pharmacological and physiological groundwork has been done with a wide range of characterized mol-ecules and pharmaceutical leads available. It is now time to begin the handover to our clinical colleagues to see these evaluated and put to good use.

1. Meunier, J. C. et al. Isolation and structure of the endogenous agonist of opioid receptor-like ORL1 receptor. Nature 377, 532–535 (1995).

2. Reinscheid, R. K. et al. Orphanin FQ: a neuropeptide that activates an opioidlike G protein-coupled receptor. Science 270, 792–794 (1995).These two references represent the beginning of the N/OFQ–NOP field. In addition, they are of interest to the general pharmacologist as they are the first example of successful reverse pharmacology (deorphanization).

3. Mogil, J. S. & Pasternak, G. W. The molecular and behavioral pharmacology of the orphanin FQ/nociceptin peptide and receptor family. Pharmacol. Rev. 53, 381–415 (2001).

4. Chiou, L. C. et al. Nociceptin/orphanin FQ peptide receptors: pharmacology and clinical implications. Curr. Drug Targets. 8, 117–135 (2007).

5. Zeilhofer, H. U. & Calo, G. Nociceptin/orphanin FQ and its receptor — potential targets for pain therapy? J. Pharmacol. Exp. Ther. 306, 423–429 (2003).

6. Malinowska, B., Godlewski, G. & Schlicker, E. Function of nociceptin and opioid OP4 receptors in the regulation of the cardiovascular system. J. Physiol. Pharmacol. 53, 301–324 (2002).

7. Dhawan, B. N. et al. International Union of Pharmacology. XII. Classification of opioid receptors. Pharmacol. Rev. 48, 567–592 (1996).

8. Zollner, C. & Stein, C. Opioids. Handb. Exp. Pharmacol. 177, 31–63 (2007).

9. Hawes, B. E., Graziano, M. P. & Lambert, D. G. Cellular actions of nociceptin: transduction mechanisms. Peptides 21, 961–967 (2000).

R E V I E W S



10. New, D. C. & Wong, Y. H. The ORL1 receptor: molecular pharmacology and signalling mechanisms. Neurosignals 11, 197–212 (2002).

11. Knoflach, F., Reinscheid, R. K., Civelli, O. & Kemp, J. A. Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons. J. Neurosci. 16, 6657–6664 (1996).

12. Vaughan, C. W. & Christie, M. J. Increase by the ORL1 receptor (opioid receptor-like1) ligand, nociceptin, of inwardly rectifying K conductance in dorsal raphe nucleus neurones. Br. J. Pharmacol. 117, 1609–1611 (1996).

13. Nicol, B., Lambert, D. G., Rowbotham, D. J., Smart, D. & McKnight, A. T. Nociceptin induced inhibition of K+ evoked glutamate release from rat cerebrocortical slices. Br. J. Pharmacol. 119, 1 081–1083 (1996).

14. Marti, M. et al. Pharmacological profiles of presynaptic nociceptin/orphanin FQ receptors modulating 5-hydroxytryptamine and noradrenaline release in the rat neocortex. Br. J. Pharmacol. 138, 91–98 (2003).

15. Giuliani, S. & Maggi, C. A. Inhibition of tachykinin release from peripheral endings of sensory nerves by nociceptin, a novel opioid peptide. Br. J. Pharmacol. 118, 1567–1569 (1996).

16. Armstead, W. M. Differential activation of ERK, p38, and JNK MAPK by nociceptin/orphanin FQ in the potentiation of prostaglandin cerebrovasoconstriction after brain injury. Eur. J. Pharmacol. 529, 129–135 (2006).

17. Chan, J. S. et al. Pertussis toxin-insensitive signaling of the ORL1 receptor: coupling to Gz and G16 proteins. J. Neurochem. 71, 2203–2210 (1998).

18. Guerrini, R. et al. Address and message sequences for the nociceptin receptor: a structure–activity study of nociceptin-(1–13)-peptide amide. J. Med. Chem. 40, 1789–1793 (1997).

19. Guerrini, R. et al. A new selective antagonist of the nociceptin receptor. Br. J. Pharmacol. 123, 163–165 (1998).

20. Guerrini, R. et al. N- and C-terminal modifications of nociceptin/orphanin FQ generate highly potent NOP receptor ligands. J. Med. Chem. 48, 1421–1427 (2005).

21. Arduin, M. et al. Synthesis and biological activity of nociceptin/orphanin FQ analogues substituted in position 7 or 11 with Ca, a-dialkylated amino acids. Bioorg. Med. Chem. 15, 4434–4443 (2007).

22. Calo, G. et al. Characterization of [Nphe1]nociceptin(1–13)NH2, a new selective nociceptin receptor antagonist. Br. J. Pharmacol. 129, 1183–1193 (2000).

23. Calo, G. et al. [Nphe1, Arg14, Lys15]nociceptin-NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor. Br. J. Pharmacol. 136, 303–311 (2002).

24. Calo, G. et al. UFP-101, a peptide antagonist selective for the nociceptin/orphanin FQ receptor. CNS Drug Rev. 11, 97–112 (2005).

25. Dooley, C. T. et al. Binding and in vitro activities of peptides with high affinity for the nociceptin/orphanin FQ receptor, ORL1. J. Pharmacol. Exp. Ther. 283, 735–741 (1997).

26. Rizzi, A. et al. Pharmacological characterization of the novel nociceptin/orphanin FQ receptor ligand, ZP120: in vitro and in vivo studies in mice. Br. J. Pharmacol. 137, 369–374 (2002).

27. Larsen, B. D., Petersen, J. S., Harlow, K. & Kapusta, D. R. Novel peptide conjugates. WO 01/98324 (2001).

28. Butour, J. L., Moisand, C., Mazarguil, H., Mollereau, C. & Meunier, J. C. Recognition and activation of the opioid receptor-like ORL 1 receptor by nociceptin, nociceptin analogs and opioids. Eur. J. Pharmacol. 321, 97–103 (1997).

29. Okawa, H. et al. Comparison of the effects of [Phe1psi(CH2-NH)Gly2]nociceptin(1–13)NH2 in rat brain, rat vas deferens and CHO cells expressing recombinant human nociceptin receptors. Br. J. Pharmacol. 127, 123–130 (1999).

30. Rizzi, A. et al. [Nphe1]nociceptin-(1–13)-NH2 antagonizes nociceptin effects in the mouse colon. Eur. J. Pharmacol. 385, R3–R5 (1999).

31. Mason, S. L., Ho, M., Nicholson, J. & McKnight, A. T. In vitro characterization of Ac-RYYRWK-NH2, Ac-RYYRIK-NH2 and [Phe1CPsi(CH2-NHGly2] nociceptin(1–13)NH2 at rat native and recombinant ORL(1) receptors. Neuropeptides 35, 244–256 (2001).

32. McDonald, J. et al. Partial agonist behaviour depends upon the level of nociceptin/orphanin FQ receptor expression: studies using the ecdysone-inducible mammalian expression system. Br. J. Pharmacol. 140, 61–70 (2003).Much of the early ligand classification was based on different end points in systems with different levels of expression. This study addresses this problem using an inducible expression system for NOP. Of note is that the variably classified ligand [F/G]N/OFQ(1–13)NH2 could be made to behave as full agonist, partial agonist and antagonist as a function of receptor density. This ligand is now classified as a partial agonist.

33. Becker, J. A. et al. Ligands for kappa-opioid and ORL1 receptors identified from a conformationally constrained peptide combinatorial library. J. Biol. Chem. 274, 27513–27522 (1999).

34. Picone, D., D’Ursi, A., Motta, A., Tancredi, T. & Temussi, P. A. Conformational preferences of [Leu5]enkephalin in biomimetic media. Investigation by 1H NMR. Eur. J. Biochem. 192, 433–439 (1990).

35. Hruby, V. J. Designing peptide receptor agonists and antagonists. Nature Rev. Drug Discov. 1, 847–858 (2002).

36. Zaveri, N. Peptide and nonpeptide ligands for the nociceptin/orphanin FQ receptor ORL1: research tools and potential therapeutic agents. Life Sci. 73, 663–678 (2003).

37. Varty, G. B. et al. The anxiolytic-like effects of the novel, orally active nociceptin NOP receptor agonist, 8-[bis(2-methylphenyl)methyl]-3-phenyl-8- azabicyclo[3.2.1]octan-3-ol (SCH 221510). J. Pharmacol. Exp. Ther. 20 May 2008 (doi:10.1124/jpet.108.136937).

38. Blakeney, J. S., Reid, R. C., Le, G. T. & Fairlie, D. P. Nonpeptidic ligands for peptide-activated G protein-coupled receptors. Chem. Rev. 107, 2960–3041 (2007).

39. Hirao, A. et al. Pharmacological properties of a novel nociceptin/orphanin FQ receptor agonist, 2-(3,5-dimethylpiperazin-1-yl)-1-[1-(1-methylcyclooctyl)piperidin-4-yl]-1H-benzim idazole, with anxiolytic potential. Eur. J. Pharmacol. 579, 189–195 (2008).

40. Broer, B. M., Gurrath, M. & Holtje, H. D. Molecular modelling studies on the ORL1-receptor and ORL1-agonists. J. Comput. Aided Mol. Des. 17, 739–754 (2003).

41. Topham, C. M., Mouledous, L., Poda, G., Maigret, B. & Meunier, J. C. Molecular modelling of the ORL1 receptor and its complex with nociceptin. Protein Eng. 11, 1163–1179 (1998).

42. Zaveri, N., Jiang, F., Olsen, C., Polgar, W. & Toll, L. Small-molecule agonists and antagonists of the opioid receptor-like receptor (ORL1, NOP): ligand-based analysis of structural factors influencing intrinsic activity at NOP. AAPS J. 7, e345–e352 (2005).

43. Heinricher, M. M., McGaraughty, S. & Grandy, D. K. Circuitry underlying antiopioid actions of orphanin FQ in the rostral ventromedial medulla. J. Neurophysiol. 78, 3351–3358 (1997).

44. Pan, Z., Hirakawa, N. & Fields, H. L. A cellular mechanism for the bidirectional pain-modulating actions of orphanin FQ/nociceptin. Neuron 26, 515–522 (2000).Central NOP activation produces pro- and antinociceptive effects when administered supraspinally and spinally, respectively. This important paper describes a mechanism (based in the medulla) to explain the pro-nociceptive or anti-opioid actions of supraspinal N/OFQ.

45. Tian, J. H. et al. Endogenous orphanin FQ: evidence for a role in the modulation of electroacupuncture analgesia and the development of tolerance to analgesia produced by morphine and electroacupuncture. Br. J. Pharmacol. 124, 21–26 (1998).

46. King, M. A., Rossi, G. C., Chang, A. H., Williams, L. & Pasternak, G. W. Spinal analgesic activity of orphanin FQ/nociceptin and its fragments. Neurosci. Lett. 223, 113–116 (1997).

47. Ko, M. C., Wei, H., Woods, J. H. & Kennedy, R. T. Effects of intrathecally administered nociceptin/orphanin FQ in monkeys: behavioral and mass spectrometric studies. J. Pharmacol. Exp. Ther. 318, 1257–1264 (2006).

48. Mogil, J. S. et al. Orphanin FQ is a functional anti-opioid peptide. Neuroscience 75, 333–337 (1996).

49. Inoue, M. et al. In vivo pain-inhibitory role of nociceptin/orphanin FQ in spinal cord. J. Pharmacol. Exp. Ther. 305, 495–501 (2003).

50. Faber, E. S., Chambers, J. P., Evans, R. H. & Henderson, G. Depression of glutamatergic transmission by nociceptin in the neonatal rat hemisected spinal cord preparation in vitro. Br. J. Pharmacol. 119, 189–190 (1996).

51. Inoue, M. et al. Dose-related opposite modulation by nociceptin/orphanin FQ of substance P nociception in the nociceptors and spinal cord. J. Pharmacol. Exp. Ther. 291, 308–313 (1999).

52. Yamamoto, T., Sakashita, Y. & Nozaki-Taguchi, N. Antagonism of ORLI receptor produces an algesic effect in the rat formalin test. Neuroreport 12, 1323–1327 (2001).

53. Ozaki, S. et al. In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist. Eur. J. Pharmacol. 402, 45–53 (2000).

54. Zaratin, P. F. et al. Modification of nociception and morphine tolerance by the selective opiate receptor-like orphan receptor antagonist (–)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol (SB-612111). J. Pharmacol. Exp. Ther. 308, 454–461 (2004).

55. Rizzi, A. et al. Endogenous nociceptin/orphanin FQ signalling produces opposite spinal antinociceptive and supraspinal pronociceptive effects in the mouse formalin test: pharmacological and genetic evidences. Pain 124, 100–108 (2006).

56. Depner, U. B., Reinscheid, R. K., Takeshima, H., Brune, K. & Zeilhofer, H. U. Normal sensitivity to acute pain, but increased inflammatory hyperalgesia in mice lacking the nociceptin precursor polypeptide or the nociceptin receptor. Eur. J. Neurosci. 17, 2381–2387 (2003).

57. Andoh, T., Yageta, Y., Takeshima, H. & Kuraishi, Y. Intradermal nociceptin elicits itch-associated responses through leukotriene B4 in mice. J. Invest. Dermatol. 123, 196–201 (2004).

58. Giuliani, S., Lecci, A., Tramontana, M. & Maggi, C. A. The inhibitory effect of nociceptin on the micturition reflex in anaesthetized rats. Br. J. Pharmacol. 124, 1566–1572 (1998).

59. Peluso, J. et al. Distribution of nociceptin/orphanin FQ receptor transcript in human central nervous system and immune cells. J. Neuroimmunol. 81, 184–192 (1998).

60. Stein, C., Schafer, M. & Machelska, H. Attacking pain at its source: new perspectives on opioids. Nature Med. 9, 1003–1008 (2003).

61. Ko, M. C. et al. Orphanin FQ inhibits capsaicin-induced thermal nociception in monkeys by activation of peripheral ORL1 receptors. Br. J. Pharmacol. 135, 943–950 (2002).

62. Obara, I., Przewlocki, R. & Przewlocka, B. Spinal and local peripheral antiallodynic activity of Ro64-6198 in neuropathic pain in the rat. Pain 116, 17–25 (2005).

63. Kolesnikov, Y. A. & Pasternak, G. W. Peripheral orphanin FQ/nociceptin analgesia in the mouse. Life Sci. 64, 2021–2028 (1999).

64. Collett, B. J. Opioid tolerance: the clinical perspective. Br. J. Anaesth. 81, 58–68 (1998).

65. Hao, J. X., Wiesenfeld-Hallin, Z. & Xu, X. J. Lack of cross-tolerance between the antinociceptive effect of intrathecal orphanin FQ and morphine in the rat. Neurosci. Lett. 223, 49–52 (1997).

66. Ueda, H. et al. Partial loss of tolerance liability to morphine analgesia in mice lacking the nociceptin receptor gene. Neurosci. Lett. 237, 136–138 (1997).

67. Ueda, H., Inoue, M., Takeshima, H. & Iwasawa, Y. Enhanced spinal nociceptin receptor expression develops morphine tolerance and dependence. J. Neurosci. 20, 7640–7647 (2000).

68. Rizzi, A. et al. The nociceptin/orphanin FQ receptor antagonist, [Nphe1]NC(1–13)NH2, potentiates morphine analgesia. Neuroreport 11, 2369–2372 (2000).

69. Chung, S., Pohl, S., Zeng, J., Civelli, O. & Reinscheid, R. K. Endogenous orphanin FQ/nociceptin is involved in the development of morphine tolerance. J. Pharmacol. Exp. Ther. 318, 262–267 (2006).References 66 and 69 illustrate the effects of NOP and ppN/OFQ knockout on morphine tolerance. Addressing the system from both ‘ends’ indicates that blockade of NOP–N/OFQ signalling via knockout reduces morphine tolerance, a response also seen with NOP antagonists. This gives some credibility to the development of m-opioid receptor agonist/NOP antagonist chimeric molecules.

70. Barnes, T. A. & Lambert, D. G. Editorial III: Nociceptin/orphanin FQ peptide-receptor system: are we any nearer the clinic? Br. J. Anaesth. 93, 626–628 (2004).

R E V I E W S



71. Ko, M. H., Kim, Y. H., Woo, R. S. & Kim, K. W. Quantitative analysis of nociceptin in blood of patients with acute and chronic pain. Neuroreport 13, 1631–1633 (2002).

72. Anderberg, U. M., Liu, Z., Berglund, L. & Nyberg, F. Plasma levels on nociceptin in female fibromyalgia syndrome patients. Z. Rheumatol. 57 (Suppl. 2), 77–80 (1998).

73. Ertsey, C., Hantos, M., Bozsik, G. & Tekes, K. Circulating nociceptin levels during the cluster headache period. Cephalalgia 24, 280–283 (2004).

74. Brooks, H. et al. Identification of nociceptin in human cerebrospinal fluid: comparison of levels in pain and non-pain states. Pain 78, 71–73 (1998).

75. Mork, H., Hommel, K., Uddman, R., Edvinsson, L. & Jensen, R. Does nociceptin play a role in pain disorders in man? Peptides 23, 1581–1587 (2002).

76. Wong, M. L. & Licinio, J. From monoamines to genomic targets: a paradigm shift for drug discovery in depression. Nature Rev. Drug Discov. 3, 136–151 (2004).

77. Christmas, D. M. & Hood, S. D. Recent developments in anxiety disorders. Recent Patents CNS Drug Discov. 1, 289–298 (2006).

78. Gavioli, E. C. & Calo, G. Antidepressant- and anxiolytic-like effects of nociceptin/orphanin FQ receptor ligands. Naunyn Schmiedebergs Arch. Pharmacol. 372, 319–330 (2006).

79. Jenck, F. et al. Orphanin FQ acts as an anxiolytic to attenuate behavioral responses to stress. Proc. Natl Acad. Sci. USA 94, 14854–14858 (1997).

80. Gavioli, E. C., Rae, G. A., Calo, G., Guerrini, R. & De Lima, T. C. Central injections of nocistatin or its C-terminal hexapeptide exert anxiogenic-like effect on behaviour of mice in the plus-maze test. Br. J. Pharmacol. 136, 764–772 (2002).

81. Redrobe, J. P., Calo, G., Regoli, D. & Quirion, R. Nociceptin receptor antagonists display antidepressant-like properties in the mouse forced swimming test. Naunyn Schmiedebergs Arch. Pharmacol. 365, 164–167 (2002).

82. Gavioli, E. C. et al. Blockade of nociceptin/orphanin FQ-NOP receptor signalling produces antidepressant-like effects: pharmacological and genetic evidences from the mouse forced swimming test. Eur. J. Neurosci. 17, 1987–1990 (2003).

83. Gavioli, E. C. et al. Antidepressant-like effects of the nociceptin/orphanin FQ receptor antagonist UFP-101: new evidence from rats and mice. Naunyn Schmiede-bergs Arch. Pharmacol. 369, 547–553 (2004).

84. Wichmann, J. et al. 8-acenaphthen-1-yl-1-phenyl- 1,3,8-triaza-spiro[4.5]decan-4-one derivatives as orphanin FQ receptor agonists. Bioorg. Med. Chem. Lett. 9, 2343–2348 (1999).

85. Jenck, F. et al. A synthetic agonist at the orphanin FQ/nociceptin receptor ORL1: anxiolytic profile in the rat. Proc. Natl Acad. Sci. USA 97, 4938–4943 (2000).

86. Varty, G. B. et al. Characterization of the nociceptin receptor (ORL-1) agonist, Ro64-6198, in tests of anxiety across multiple species. Psychopharmacology (Berl.) 182, 132–143 (2005).

87. Fernandez, F., Misilmeri, M. A., Felger, J. C. & Devine, D. P. Nociceptin/orphanin FQ increases anxiety-related behavior and circulating levels of corticosterone during neophobic tests of anxiety. Neuropsychopharmacology 29, 59–71 (2004).

88. Dautzenberg, F. M. et al. Pharmacological characterization of the novel nonpeptide orphanin FQ/nociceptin receptor agonist Ro 64-6198: rapid and reversible desensitization of the ORL1 receptor in vitro and lack of tolerance in vivo. J. Pharmacol. Exp. Ther. 298, 812–819 (2001).Ro64-6198 is arguably one of the most important non-peptide agonists in the NOP field. In this early paper the Roche group describe anxiolysis without tolerance.

89. Ouagazzal, A. M., Moreau, J. L., Pauly-Evers, M. & Jenck, F. Impact of environmental housing conditions on the emotional responses of mice deficient for nociceptin/orphanin FQ peptide precursor gene. Behav. Brain Res. 144, 111–117 (2003).

90. Koster, A. et al. Targeted disruption of the orphanin FQ/nociceptin gene increases stress susceptibility and impairs stress adaptation in mice. Proc. Natl Acad. Sci. USA 96, 10444–10449 (1999).

91. Le Maitre, E., Vilpoux, C., Costentin, J. & Leroux-Nicollet, I. Opioid receptor-like 1 (NOP) receptors in the rat dorsal raphe nucleus: evidence for localization on serotoninergic neurons and functional adaptation after 5,7-dihydroxytryptamine lesion. J. Neurosci. Res. 81, 488–496 (2005).

92. Gavioli, E. C. et al. GABAA signalling is involved in N/OFQ anxiolytic-like effects but not in nocistatin anxiogenic-like action as evaluated in the mouse elevated plus maze. Peptides 20 May 2008 (doi:10.1016/j.peptides.2008.04.004).

93. Belmaker, R. H. & Agam, G. Major depressive disorder. N. Engl. J. Med. 358, 55–68 (2008).

94. Connor, M., Vaughan, C. W., Chieng, B. & Christie, M. J. Nociceptin receptor coupling to a potassium conductance in rat locus coeruleus neurones in vitro. Br. J. Pharmacol. 119, 1614–1618 (1996).

95. Gu, H. et al. Changes and significance of orphanin and serotonin in patients with postpartum depression. Zhonghua Fu Chan Ke Za Zhi 38, 727–728 (2003) (in Chinese).

96. Shoblock, J. R. The pharmacology of Ro 64-6198, a systemically active, nonpeptide NOP receptor (opiate receptor-like 1, ORL-1) agonist with diverse preclinical therapeutic activity. CNS Drug Rev. 13, 107–136 (2007).

97. Gonzalez, G., Oliveto, A. & Kosten, T. R. Combating opiate dependence: a comparison among the available pharmacological options. Expert Opin. Pharmacother. 5, 713–725 (2004).

98. Spanagel, R. & Kiefer, F. Drugs for relapse prevention of alcoholism: ten years of progress. Trends Pharmacol. Sci. 29, 109–115 (2008).

99. Amato, L. et al. Psychosocial and pharmacological treatments versus pharmacological treatments for opioid detoxification. Cochrane Database Syst. Rev. CD005031 (2004).

100. Assanangkornchai, S. & Srisurapanont, M. The treatment of alcohol dependence. Curr. Opin. Psychiatry 20, 222–227 (2007).

101. Ciccocioppo, R., Panocka, I., Polidori, C., Regoli, D. & Massi, M. Effect of nociceptin on alcohol intake in alcohol-preferring rats. Psychopharmacology (Berl.) 141, 220–224 (1999).

102. Ciccocioppo, R. et al. Pharmacological characterization of the nociceptin receptor which mediates reduction of alcohol drinking in rats. Peptides 23, 117–125 (2002).

103. Kuzmin, A., Sandin, J., Terenius, L. & Ogren, S. O. Acquisition, expression, and reinstatement of ethanol-induced conditioned place preference in mice: effects of opioid receptor-like 1 receptor agonists and naloxone. J. Pharmacol. Exp. Ther. 304, 310–318 (2003).

104. Kotlinska, J. et al. Nociceptin inhibits acquisition of amphetamine-induced place preference and sensitization to stereotypy in rats. Eur. J. Pharmacol. 474, 233–239 (2003).

105. Kotlinska, J., Wichmann, J., Legowska, A., Rolka, K. & Silberring, J. Orphanin FQ/nociceptin but not Ro 65-6570 inhibits the expression of cocaine-induced conditioned place preference. Behav. Pharmacol. 13, 229–235 (2002).

106. Sakoori, K. & Murphy, N. P. Central administration of nociceptin/orphanin FQ blocks the acquisition of conditioned place preference to morphine and cocaine, but not conditioned place aversion to naloxone in mice. Psychopharmacology (Berl.) 172, 129–136 (2004).

107. Sakoori, K. & Murphy, N. P. Endogenous nociceptin (orphanin FQ) suppresses basal hedonic state and acute reward responses to methamphetamine and ethanol, but facilitates chronic responses. Neuropsychopharmacology 33, 877–891 (2008).

108. Murphy, N. P., Ly, H. T. & Maidment, N. T. Intracerebroventricular orphanin FQ/nociceptin suppresses dopamine release in the nucleus accumbens of anaesthetized rats. Neuroscience 75, 1–4 (1996).

109. Murphy, N. P. & Maidment, N. T. Orphanin FQ/nociceptin modulation of mesolimbic dopamine transmission determined by microdialysis. J. Neurochem. 73, 179–186 (1999).

110. Di Giannuario, A. & Pieretti, S. Nociceptin differentially affects morphine-induced dopamine release from the nucleus accumbens and nucleus caudate in rats. Peptides 21, 1125–1130 (2000).

111. Di Giannuario, A., Pieretti, S., Catalani, A. & Loizzo, A. Orphanin FQ reduces morphine-induced dopamine release in the nucleus accumbens: a microdialysis study in rats. Neurosci. Lett. 272, 183–186 (1999).

112. Lutfy, K., Do, T. & Maidment, N. T. Orphanin FQ/nociceptin attenuates motor stimulation and changes in nucleus accumbens extracellular dopamine induced by cocaine in rats. Psychopharmacology (Berl.) 154, 1–7 (2001).

113. Meis, S. & Pape, H. C. Control of glutamate and GABA release by nociceptin/orphanin FQ in the rat lateral amygdala. J. Physiol. 532, 701–712 (2001).

114. Roberto, M. & Siggins, G. R. Nociceptin/orphanin FQ presynaptically decreases GABAergic transmission and blocks the ethanol-induced increase of GABA release in central amygdala. Proc. Natl Acad. Sci. USA 103, 9715–9720 (2006).

115. Okawa, H. et al. Effects of nociceptinNH2 and [Nphe1]nociceptin(1–13)NH2 on rat brain noradrenaline release in vivo and in vitro. Neurosci. Lett. 303, 173–176 (2001).

116. Ciccocioppo, R., Angeletti, S., Panocka, I. & Massi, M. Nociceptin/orphanin FQ and drugs of abuse. Peptides 21, 1071–1080 (2000).

117. Ciccocioppo, R., Economidou, D., Fedeli, A. & Massi, M. The nociceptin/orphanin FQ/NOP receptor system as a target for treatment of alcohol abuse: a review of recent work in alcohol-preferring rats. Physiol. Behav. 79, 121–128 (2003).

118. Ciccocioppo, R. et al. Attenuation of ethanol self-administration and of conditioned reinstatement of alcohol-seeking behaviour by the antiopioid peptide nociceptin/orphanin FQ in alcohol-preferring rats. Psychopharmacology (Berl.) 172, 170–178 (2004).

119. Kuzmin, A., Kreek, M. J., Bakalkin, G. & Liljequist, S. The nociceptin/orphanin FQ receptor agonist Ro 64-6198 reduces alcohol self-administration and prevents relapse-like alcohol drinking. Neuropsychopharmacology 32, 902–910 (2007).

120. Economidou, D. et al. Effect of novel nociceptin/orphanin FQ-NOP receptor ligands on ethanol drinking in alcohol-preferring msP rats. Peptides 27, 3299–3306 (2006).

121. Ciccocioppo, R. et al. Buprenorphine reduces alcohol drinking through activation of the nociceptin/orphanin FQ-NOP receptor system. Biol. Psychiatry 61, 4–12 (2007).

122. Economidou, D. et al. Dysregulation of nociceptin/orphanin FQ activity in the amygdala is linked to excessive alcohol drinking in the rat. Biol. Psychiatry 24 Mar 2008 (doi:10.1016/j.biopsych.2008.02.004).

123. Xuei, X. et al. Association analysis of genes encoding the nociceptin receptor (OPRL1) and its endogenous ligand (PNOC) with alcohol or illicit drug dependence. Addict. Biol. 13, 80–87 (2008).

124. Huang, J., Young, B., Pletcher, M. T., Heilig, M. & Wahlestedt, C. Association between the nociceptin receptor gene (OPRL1) single nucleotide polymorphisms and alcohol dependence. Addict. Biol. 13, 88–94 (2008).

125. Kapusta, D. R. Neurohumoral effects of orphanin FQ/nociceptin: relevance to cardiovascular and renal function. Peptides 21, 1081–1099 (2000).

126. Bigoni, R. et al. Characterization of nociceptin receptors in the periphery: in vitro and in vivo studies. Naunyn Schmiedebergs Arch. Pharmacol. 359, 160–167 (1999).

127. Kapusta, D. R., Chang, J. K. & Kenigs, V. A. Central administration of [Phe1psi(CH2-NH)Gly2]nociceptin(1–13)-NH2 and orphanin FQ/nociceptin (OFQ/N) produce similar cardiovascular and renal responses in conscious rats. J. Pharmacol. Exp. Ther. 289, 173–180 (1999).

128. Burmeister, M. A., Ansonoff, M. A., Pintar, J. E. & Kapusta, D. R. Nociceptin/orphanin FQ (N/OFQ)-evoked bradycardia, hypotension and diuresis are absent in N/OFQ peptide (NOP) receptor knockout (NOP–/–) mice. J. Pharmacol. Exp. Ther. 6 Jun 2008 (doi:10.1124/jpet.107.135905v1).

129. Giuliani, S., Tramontana, M., Lecci, A. & Maggi, C. A. Effect of nociceptin on heart rate and blood pressure in anaesthetized rats. Eur. J. Pharmacol. 333, 177–179 (1997).

130. Chu, X., Xu, N., Li, P., Mao, L. & Wang, J. Q. Inhibition of cardiovascular activity following microinjection of novel opioid-like neuropeptide nociceptin (orphanin FQ) into the rat rostral ventrolateral medulla. Brain Res. 829, 134–142 (1999).

131. Kapusta, D. R. et al. Functional selectivity of nociceptin/orphanin FQ peptide receptor partial agonists on cardiovascular and renal function. J. Pharmacol. Exp. Ther. 314, 643–651 (2005).

132. Champion, H. C. et al. Nitric oxide release mediates vasodilator responses to endomorphin 1 but not nociceptin/OFQ in the hindquarters vascular bed of the rat. Peptides 19, 1595–1602 (1998).

133. Champion, H. C., Pierce, R. L. & Kadowitz, P. J. Nociceptin, a novel endogenous ligand for the ORL1 receptor, dilates isolated resistance arteries from the rat. Regul. Pept. 78, 69–74 (1998).

R E V I E W S



134. Champion, H. C. et al. Role of nitric oxide in mediating vasodilator responses to opioid peptides in the rat. Clin. Exp. Pharmacol. Physiol. 29, 229–232 (2002).

135. Armstead, W. M. Role of Nociceptin/orphanin FQ in the physiologic and pathologic control of the cerebral circulation. Exp. Biol. Med. (Maywood) 227, 957–968 (2002).

136. Brookes, Z. L. et al. Proinflammatory and vasodilator effects of nociceptin/orphanin FQ in the rat mesenteric microcirculation are mediated by histamine. Am. J. Physiol. Heart Circ. Physiol. 293, H2977–H2985 (2007).

137. Tekes, K. et al. Stimulating effect of nociceptin on histamine release in the rat brain? Inflamm. Res. 54 (Suppl. 1), S38–S39 (2005).

138. Kimura, T. et al. Intradermal application of nociceptin increases vascular permeability in rats: the possible involvement of histamine release from mast cells. Eur. J. Pharmacol. 407, 327–332 (2000).

139. O’Brien, J. M., Jr, Ali, N. A., Aberegg, S. K. & Abraham, E. Sepsis. Am. J. Med. 120, 1012–1022 (2007).

140. Williams, J. P. et al. Nociceptin and urotensin-II concentrations in critically ill patients with sepsis. Br. J. Anaesth. 100, 810–814 (2008).

141. Dumont, M. & Lemaire, S. Characterization of the high affinity [3H]nociceptin binding site in membrane preparations of rat heart: correlations with the non-opioid dynorphin binding site. J. Mol. Cell Cardiol. 30, 2751–2760 (1998).

142. Berger, H., Albrecht, E., Wallukat, G. & Bienert, M. Antagonism by acetyl-RYYRIK-NH2 of G protein activation in rat brain preparations and of chronotropic effect on rat cardiomyocytes evoked by nociceptin/orphanin FQ. Br. J. Pharmacol. 126, 555–558 (1999).

143. Kim, K. W. et al. Nociceptin/orphanin FQ increases ANP secretion in neonatal cardiac myocytes. Life Sci. 70, 1065–1074 (2002).

144. Arndt, M. L., Wu, D., Soong, Y. & Szeto, H. H. Nociceptin/orphanin FQ increases blood pressure and heart rate via sympathetic activation in sheep. Peptides 20, 465–470 (1999).

145. Mao, L. & Wang, J. Q. Microinjection of nociceptin (orphanin FQ) into nucleus tractus solitarii elevates blood pressure and heart rate in both anesthetized and conscious rats. J. Pharmacol. Exp. Ther. 294, 255–262 (2000).

146. Hadrup, N. et al. Differential down-regulation of aquaporin-2 in rat kidney zones by peripheral nociceptin/orphanin FQ receptor agonism and vasopressin type-2 receptor antagonism. J. Pharmacol. Exp. Ther. 323, 516–524 (2007).

147. Simonsen, U., Laursen, B. E. & Petersen, J. S. ZP120 causes relaxation by pre-junctional inhibition of noradrenergic neurotransmission in rat mesenteric resistance arteries. Br. J. Pharmacol. 153, 1185–1194 (2008).

148. Lazzeri, M. & Spinelli, M. The challenge of overactive bladder therapy: alternative to antimuscarinic agents. Int. Braz. J. Urol. 32, 620–630 (2006).

149. Calo, G. et al. The mouse vas deferens: a pharmacological preparation sensitive to nociceptin. Eur. J. Pharmacol. 311, R3–R5 (1996).

150. Berzetei-Gurske, I. P., Schwartz, R. W. & Toll, L. Determination of activity for nociceptin in the mouse vas deferens. Eur. J. Pharmacol. 302, R1–R2 (1996).

151. Giuliani, S., Lecci, A., Tramontana, M. & Maggi, C. A. Nociceptin protects capsaicin-sensitive afferent fibers in the rat urinary bladder from desensitization. Naunyn Schmiedebergs Arch. Pharmacol. 360, 202–208 (1999).

152. Lecci, A. et al. Tachykinin-mediated effect of nociceptin in the rat urinary bladder in vivo. Eur. J. Pharmacol. 389, 99–102 (2000).

153. Lecci, A., Giuliani, S., Meini, S. & Maggi, C. A. Nociceptin and the micturition reflex. Peptides 21, 1007–1021 (2000).

154. Lazzeri, M. et al. Urodynamic and clinical evidence of acute inhibitory effects of intravesical nociceptin/orphanin FQ on detrusor overactivity in humans: a pilot study. J. Urol. 166, 2237–2240 (2001).

155. Lazzeri, M. et al. Urodynamic effects of intravesical nociceptin/orphanin FQ in neurogenic detrusor overactivity: a randomized, placebo-controlled, double-blind study. Urology 61, 946–950 (2003).

156. Lazzeri, M. et al. Daily intravesical instillation of 1 mg nociceptin/orphanin FQ for the control of neurogenic detrusor overactivity: a multicenter, placebo controlled, randomized exploratory study. J. Urol. 176, 2098–2102 (2006).

Third in a series of clinical papers examining the effects of intravesical administration of N/OFQ on bladder function in neurogenic detrusor overactivity incontinence. This study is important as it describes a technique that can be used by patients at home with a single daily administration increasing bladder capacity and decreasing the number of urine leakages lasting a full day.

157. Fowler, C. J., Griffiths. D. & de Groat, W. C. The neural control of micturition. Nature Rev. Neurosci. 9, 453–466 (2008).

158. de Groat, W. C. et al. Mechanisms underlying the recovery of urinary bladder function following spinal cord injury. J. Auton. Nerv. Syst. 30 (Suppl.), S71–S77 (1990).

159. Welters, I. D. Is immunomodulation by opioid drugs of clinical relevance? Curr. Opin. Anaesthesiol. 16, 509–513 (2003).

160. Budd, K. Pain management: is opioid immunosuppression a clinical problem? Biomed. Pharmacother. 60, 310–317 (2006).

161. Hussey, H. H. & Katz, S. Infections resulting from narcotic addiction; report of 102 cases. Am. J. Med. 9, 186–193 (1950).

162. Wei, G., Moss, J. & Yuan, C.-S. Opioid-induced immunosuppression: is it centrally mediated or peripherally mediated? Biochem. Pharmacol. 65, 1761–1766 (2003).

163. Williams, J. P. et al. Human peripheral blood mononuclear cells express nociceptin/orphanin FQ, but not m, d, or k opioid receptors. Anesth. Analg. 105, 998–1005 (2007).

164. Kraus, J. et al. Regulation of m-opioid receptor gene transcription by interleukin-4 and influence of an allelic variation within a STAT6 transcription factor binding site. J. Biol. Chem. 276, 43901–43908 (2001).

165. Trombella, S. et al. Nociceptin/orphanin FQ stimulates human monocyte chemotaxis via NOP receptor activation. Peptides 26, 1497–1502 (2005).

166. Fiset, M. E., Gilbert, C., Poubelle, P. E. & Pouliot, M. Human neutrophils as a source of nociceptin: a novel link between pain and inflammation. Biochemistry 42, 10498–10505 (2003).

167. Waits, P. S., Purcell, W. M., Fulford, A. J. & McLeod, J. D. Nociceptin/orphanin FQ modulates human T cell function in vitro. J. Neuroimmunol. 149, 110–120 (2004).

168. Acosta, C. & Davies, A. Bacterial lipopolysaccharide regulates nociceptin expression in sensory neurons. J. Neurosci. Res. 86, 1077–1086 (2008).This is an interesting paper for a number of reasons. It describes an upregulation of N/OFQ in sensory neurons in response to lipopolysaccharide and it suggests a link (involving N/OFQ) between the nervous and immune systems. This paper has wider implications for a neuroimmune axis.

169. Goldfarb, Y., Reinscheid, R. K. & Kusnecov, A. W. Orphanin FQ/nociceptin interactions with the immune system in vivo: gene expression changes in lymphoid organs and regulation of the cytokine response to staphylococcal enterotoxin A. J. Neuroimmunol. 176, 76–85 (2006).

170. Kato, S. et al. Role of nociceptin/orphanin FQ (Noc/oFQ) in murine experimental colitis. J. Neuroimmunol. 161, 21–28 (2005).

171. McLeod, R. L. et al. Antitussive effect of nociceptin/orphanin FQ in experimental cough models. Pulm. Pharmacol. Ther. 15, 213–216 (2002).

172. Lee, M. G., Undem, B. J., Brown, C. & Carr, M. J. Effect of nociceptin in acid-evoked cough and airway sensory nerve activation in guinea pigs. Am. J. Respir. Crit. Care Med. 173, 271–275 (2006).

173. Bolser, D. C., McLeod, R. L., Tulshian, D. B. & Hey, J. A. Antitussive action of nociceptin in the cat. Eur. J. Pharmacol. 430, 107–111 (2001).

174. Faisy, C. et al. Nociceptin inhibits vanilloid TRPV-1-mediated neurosensitization induced by fenoterol in human isolated bronchi. Naunyn Schmiedebergs Arch. Pharmacol. 370, 167–175 (2004).

175. McLeod, R. L. et al. Antitussive profile of the NOP agonist Ro-64-6198 in the guinea pig. Pharmacology 71, 143–149 (2004).

176. Osinski, M. A. & Brown, D. R. Orphanin FQ/nociceptin: a novel neuromodulator of gastrointestinal function? Peptides 21, 999–1005 (2000).

177. Morini, G., De Caro, G., Guerrini, R., Massi, M. & Polidori, C. Nociceptin/orphanin FQ prevents ethanol-induced gastric lesions in the rat. Regul. Pept. 124, 203–207 (2005).

178. Grandi, D. et al. Nociceptin/orphanin FQ prevents gastric damage induced by cold-restraint stress in the rat by acting in the periphery. Peptides 28, 1572–1579 (2007).

179. Broccardo, M., Guerrini, R., Petrella, C. & Improta, G. Gastrointestinal effects of intracerebroventricularly injected nociceptin/orphaninFQ in rats. Peptides 25, 1013–1020 (2004).

180. Ishihara, S. et al. Gastric acid secretion stimulated by centrally injected nociceptin in urethane-anesthetized rats. Eur. J. Pharmacol. 441, 105–114 (2002).

181. Rizzi, D. et al. Effects of Ro 64-6198 in nociceptin/orphanin FQ-sensitive isolated tissues. Naunyn Schmiedebergs Arch. Pharmacol. 363, 551–555 (2001).

182. Spagnolo, B. et al. Pharmacological characterization of the nociceptin/orphanin FQ receptor antagonist SB-612111 [(–)-cis-1-Methyl-7-[[4-(2,6-dichlorophenyl) piperidin-1-yl]methyl]-6,7,8,9 -tetrahydro-5H- benzocyclohepten-5-ol]: in vitro studies. J. Pharmacol. Exp. Ther. 321, 961–967 (2007).

183. Jann, M. W. & Slade, J. H. Antidepressant agents for the treatment of chronic pain and depression. Pharmacotherapy 27, 1571–1587 (2007).

184. Yuan, C. S. & Foss, J. F. Oral methylnaltrexone for opioid-induced constipation. JAMA 284, 1383–1384 (2000).

185. DeBalli, P. & Breen, T. W. Intrathecal opioids for combined spinal-epidural analgesia during labour. CNS Drugs 17, 889–904 (2003).

186. Servin, F. S. & Billard, V. Remifentanil and other opioids. Handb. Exp. Pharmacol. 182, 283–311 (2008).

187. Abdelhamid, E. E., Sultana, M., Portoghese, P. S. & Takemori, A. E. Selective blockage of d opioid receptors prevents the development of morphine tolerance and dependence in mice. J. Pharmacol. Exp. Ther. 258, 299–303 (1991).

188. Zhu, Y. et al. Retention of supraspinal d-like analgesia and loss of morphine tolerance in d opioid receptor knockout mice. Neuron 24, 243–252 (1999).

189. Balboni, G. et al. Evaluation of the Dmt-Tic pharmacophore: conversion of a potent d-opioid receptor antagonist into a potent d agonist and ligands with mixed properties. J. Med. Chem. 45, 713–720 (2002).

190. Fichna, J. et al. Synthesis and characterization of potent and selective m-opioid receptor antagonists, [Dmt1, D-2-Nal4]endomorphin-1 (Antanal-1) and [Dmt1, D-2-Nal4]endomorphin-2 (Antanal-2). J. Med. Chem. 50, 512–520 (2007).

191. Spagnolo, B. et al. Activities of mixed NOP and m-opioid receptor ligands. Br. J. Pharmacol. 153, 609–619 (2008).

192. Khroyan, T. V. et al. SR 16435 [1-(1-(bicyclo[3.3.1]nonan-9-yl)piperidin-4-yl)indolin-2-one], a novel mixed nociceptin/orphanin FQ/m-opioid receptor partial agonist: analgesic and rewarding properties in mice. J. Pharmacol. Exp. Ther. 320, 934–943 (2007).

193. Pert, C. B. & Snyder, S. H. Opiate receptor: demonstration in nervous tissue. Science 179, 1011–1014 (1973).

194. Pert, C. B., Pasternak, G. & Snyder, S. H. Opiate agonists and antagonists discriminated by receptor binding in brain. Science 182, 1359–1361 (1973).

195. Lapalu, S. et al. Comparison of the structure–activity relationships of nociceptin and dynorphin A using chimeric peptides. FEBS Lett. 417, 333–336 (1997).

196. Reinscheid, R. K., Ardati, A., Monsma, F. J. Jr. & Civelli, O. Structure–activity relationship studies on the novel neuropeptide orphanin FQ. J. Biol. Chem. 271, 14163–14168 (1996).

197. Dooley, C. T. & Houghten, R. A. Orphanin FQ: receptor binding and analog structure activity relationships in rat brain. Life Sci. 59, PL23–PL29 (1996).

198. Calo, G. et al. Pharmacological characterization of nociceptin receptor: an in vitro study. Can. J. Physiol. Pharmacol. 75, 713–718 (1997).

199. Varani, K. et al. Pharmacology of [Tyr1]nociceptin analogs: receptor binding and bioassay studies. Naunyn Schmiedebergs Arch. Pharmacol. 360, 270–277 (1999).

200. Mollereau, C. et al. Distinct mechanisms for activation of the opioid receptor-like 1 and k-opioid receptors by nociceptin and dynorphin A. Mol. Pharmacol. 55, 324–331 (1999).

201. Reinscheid, R. K., Higelin, J., Henningsen, R. A., Monsma, F. J., Jr & Civelli, O. Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities. J. Biol. Chem. 273, 1490–1495 (1998).

R E V I E W S



202. Guerrini, R. et al. Further studies on nociceptin-related peptides: discovery of a new chemical template with antagonist activity on the nociceptin receptor. J. Med. Chem. 43, 2805–2813 (2000).

203. Guerrini, R. et al. Structure–activity studies of the Phe4 residue of nociceptin(1–13)-NH2: identification of highly potent agonists of the nociceptin/orphanin FQ receptor. J. Med. Chem. 44, 3956–3964 (2001).

204. Carra, G. et al. [(pF)Phe4, Arg14, Lys15]N/OFQ-NH2 (UFP-102), a highly potent and selective agonist of the nociceptin/orphanin FQ receptor. J. Pharmacol. Exp. Ther. 312, 1114–1123 (2005).

205. Kitayama, M. et al. Pharmacological profile of the cyclic nociceptin/orphanin FQ analogues c[Cys10,14]N/OFQ(1–14)NH2 and c[Nphe1, Cys10,14]N/OFQ(1–14)NH2. Naunyn Schmiedebergs Arch. Pharmacol. 368, 528–537 (2003).

206. Kitayama, M. et al. In vitro pharmacological characterisation of a novel cyclic nociceptin/orphanin FQ analogue c[Cys7,10]N/OFQ(1–13)NH2. Naunyn Schmiedebergs Arch. Pharmacol. 375, 369–376 (2007).

207. Malinowska, B., Kozlowska, H., Berger, H. & Schlicker, E. Acetyl-RYYRIK-NH2 is a highly efficacious OP4 receptor agonist in the cardiovascular system of anesthetized rats. Peptides 21, 1875–1880 (2000).

208. Kapusta, D. R. et al. Pharmacodynamic characterization of ZP120 (Ac-RYYRWKKKKKKK-NH2), a novel, functionally selective nociceptin/orphanin FQ peptide receptor partial agonist with sodium-potassium-sparing aquaretic activity. J. Pharmacol. Exp. Ther. 314, 652–660 (2005).Describes an interesting approach to peptide protection and a new aquaretic molecule that is currently in clinical evaluation for heart failure.

209. Judd, A. K. et al. Structure–activity studies on high affinity NOP-active hexapeptides. J. Pept. Res. 64, 87–94 (2004).

210. Khroyan, T. V. et al. Anti-nociceptive and anti-allodynic effects of a high affinity NOP hexapeptide [Ac-RY(3-Cl)YRWR-NH2] (Syn 1020) in rodents. Eur. J. Pharmacol. 560, 29–35 (2007).

211. Economidou, D. et al. Effect of novel NOP receptor ligands on food intake in rats. Peptides 27, 775–783 (2006).

212. Seki, T. et al. Pharmacological properties of TRK-820 on cloned m-, d- and k-opioid receptors and nociceptin receptor. Eur. J. Pharmacol. 376, 159–167 (1999).

213. Mizoguchi, H. et al. Blockade of m-opioid receptor-mediated G-protein activation and antinociception by TRK-820 in mice. Eur. J. Pharmacol. 461, 35–39 (2003).

214. Endoh, T. et al. TRK-820, a selective k-opioid agonist, produces potent antinociception in cynomolgus monkeys. Jpn. J. Pharmacol. 85, 282–290 (2001).

215. Wakasa, Y. et al. Inhibitory effects of TRK-820 on systemic skin scratching induced by morphine in rhesus monkeys. Life Sci. 75, 2947–2957 (2004).

216. Bloms-Funke, P., Gillen, C., Schuettler, A. J. & Wnendt, S. Agonistic effects of the opioid buprenorphine on the nociceptin/OFQ receptor. Peptides 21, 1141–1146 (2000).

217. Yamamoto, T., Shono, K. & Tanabe, S. Buprenorphine activates m and opioid receptor like-1 receptors simultaneously, but the analgesic effect is mainly mediated by m receptor activation in the rat formalin test. J. Pharmacol. Exp. Ther. 318, 206–213 (2006).

218. Noda, Y. et al. Loss of antinociception induced by naloxone benzoylhydrazone in nociceptin receptor-knockout mice. J. Biol. Chem. 273, 18047–18051 (1998).

219. Berzetei-Gurske, I. P. et al. The in vitro pharmacological characterization of naloxone benzoylhydrazone. Eur. J. Pharmacol. 277, 257–263 (1995).

220. Yamada, H., Nakamoto, H., Suzuki, Y., Ito, T. & Aisaka, K. Pharmacological profiles of a novel opioid receptor-like1 (ORL1) receptor antagonist, JTC-801. Br. J. Pharmacol. 135, 323–332 (2002).

221. Muratani, T. et al. Characterization of nociceptin/orphanin FQ-induced pain responses by the novel receptor antagonist N-(4-amino-2-methylquinolin- 6-yl)-2-(4-ethylphenoxymethyl) benzamide monohydrochloride. J. Pharmacol. Exp. Ther. 303, 424–430 (2002).

222. Bigoni, R. et al. In vitro characterization of J-113397, a non-peptide nociceptin/orphanin FQ receptor antagonist. Naunyn Schmiedebergs Arch. Pharmacol. 361, 565–568 (2000).

223. Hashiba, E. et al. Characterisation and comparison of novel ligands for the nociceptin/orphanin FQ receptor. Naunyn Schmiedebergs Arch. Pharmacol. 363, 28–33 (2001).

224. Trapella, C. et al. Identification of an achiral analogue of J-113397 as potent nociceptin/orphanin FQ receptor antagonist. Bioorg. Med. Chem. 14, 692–704 (2006).

225. Rizzi, A. et al. Pharmacological characterization of the nociceptin/orphanin FQ receptor antagonist SB-612111 [(–)-cis-1-methyl-7-[[4-(2,6- dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol]: in vivo studies. J. Pharmacol. Exp. Ther. 321, 968–974 (2007).

226. Ho, G. D. et al. Synthesis and structure–activity relationships of 4-hydroxy-4-phenylpiperidines as nociceptin receptor ligands: part 1. Bioorg. Med. Chem. Lett. 17, 3023–3027 (2007).

227. Ho, G. D. et al. Synthesis and structure–activity relationships of 4-hydroxy-4-phenylpiperidines as nociceptin receptor ligands: part 2. Bioorg. Med. Chem. Lett. 17, 3028–3033 (2007).

228. Thomsen, C. & Hohlweg, R. (8-Naphthalen-1- ylmethyl-4-oxo-1-phenyl-1,3,8-triaza-spiro[4.5]dec-3-yl)-acetic acid methyl ester (NNC 63-0532) is a novel potent nociceptin receptor agonist. Br. J. Pharmacol. 131, 903–908 (2000).

229. Bignan, G. C., Connolly, P. J. & Middleton, S. A. Recent advances towards the discovery of ORL-1 receptor agonists and antagonists. Expert Opin. Ther. Pat. 15, 357–388 (2005).

230. Caldwell, J. P., Matasi, J. J., Zhang, H., Fawzi, A. & Tulshian, D. B. Synthesis and structure–activity relationships of N-substituted spiropiperidines as nociceptin receptor ligands. Bioorg. Med. Chem. Lett. 17, 2281–2284 (2007).

231. Ito, F. 2-Benzimidazolylamine ORL1 receptor agonists. EP01069124 (A1) (2001).

232. Ito, F., Koike, H., Masaki, S., Yamagishi, T. & Ando, K.

Spiropiperidine derivatives as ligands for ORL-1 receptors. WO03000677 (2003).

233. Ito, F., Koike, H. & Morita, A. N-substituted spiropiperidine compounds as ligands for ORL-1 receptor. WO03064425 (2003).

234. Hirota, M. et al. Alpha aryl or heteroaryl methyl beta piperidino propanamide compounds as ORL1-receptor antagonist. WO2005092858 (2005).

AcknowledgementsI am indebted to my collaborators G. Calo and R. Guerrini (University of Ferrara, Italy) for advice in the construction of this Review and for critically reading several versions. I would also like to thank R. Ciccocioppo, Department of Pharmaco-logical Sciences and Experimental Medicine, University of Camerino, Italy, for advice on the reward/abuse section and J. McDonald for help in manuscript preparation. Over the years work on N/OFQ in my laboratory has been funded by the British Journal of Anaesthesia and the Royal College of Anaesthetists, The Wellcome Trust and Pfizer (Sandwich, UK). A PubMed search for “nociceptin” or “orphanin” yields ~1,300 hits, so I apologize to all those authors whose work I could not cite due to limitations of space.

FURTHER INFORMATIONAcologix: www.acologix.com/pipeline_trk-820.htmlBrane Discovery: http://www.branediscovery.com/pipeline.htmIUPHAR Receptor Database: www.iuphar-db.org/GPCR/ChapterMenuForward?chapterID=1295Neighbourhood.gov.uk: www.neighbourhood.gov.uk/page.asp?id=684 Samyang: www.samyangpharm.com/rnd/bupre.aspThe British Heart Foundation: www.heartstats.org/homepage.aspZealand Pharma: www.zp.dk/Product-Pipeline/ZP120

SUPPLEMENTARY INFORMATIONSee online article: S1 (table)

All liNks Are Active iN the ONliNe PdF

R E V I E W S



http://www.acologix.com/pipeline_trk-820.html

http://www.branediscovery.com/pipeline.htm

http://www.iuphar-db.org/GPCR/ChapterMenuForward?chapterID=1295

http://www.iuphar-db.org/GPCR/ChapterMenuForward?chapterID=1295

http://www.neighbourhood.gov.uk/page.asp?id=684

http://www.samyangpharm.com/rnd/bupre.asp

http://www.heartstats.org/homepage.asp

http://www.zp.dk/Product-Pipeline/ZP120

http://www.nature.com/nrd/journal/v7/n8/suppinfo/nrd2572.html

Documents

The nociceptin/orphanin FQ receptor: a target with broad