The Nociceptin/Orphanin FQ Receptor: a Target with Broad Therapeutic Potential

Home , Nociceptin, Nociceptin receptor, Opioid receptor

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The nociceptin/orphanin FQ receptor: a target with broad therapeutic potential

David 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.

1,2 m d k7,8 Hyperalgesia The nociceptin/orphanin FQ peptide (N/OFQ) is receptors , and ). Activation with N/OFQ inhibits 1,2,9,10 An increase in pain perception involved in a wide range of physiological responses the formation of cyclic AMP , closes voltage-gated above the normal response to with effects noted in the nervous system (central and Ca2+ channels9–11 and opens inwardly rectifying K+ a stimulus. peripheral), the cardiovascular system, the airways, channels9,10,12. The net effect of these cellular actions is the gastrointestinal tract, the urogenital tract and the to reduce neuronal excitability and neurotransmitter Deorphanized 3,4 (FIG. 1) The identification of an immune system . The effects in the nervous sys- release. Indeed, a wide range of neurotransmitter systems 13 endogenous ligand for a tem are complex and have received much attention. It is are modulated by N/OFQ and these include glutamate , receptor whose structure generally accepted that spinal N/OFQ is antinociceptive catecholamines14 and tachykinins15. NOP activation also makes it a member of with many features that are common to the classical modulates mitogen-activated protein (MAP) kinase, a receptor family, but for 5 which no ligand has yet been members of the opioid family . Nevertheless, when given extracellular signal-regulated kinase (ERK) and JUN 10,16 identified (that is, an orphan supraspinally, N/OFQ reverses the effects of opioids activity . NOP may also be involved in phospholipase C receptor). (anti-opioid action) with a whole-animal response that (PLC)-mediated phosphatidylinositol bisphosphate manifests as hyperalgesia5 (see below for more detailed hydrolysis10,17. As the identification of N/OFQ — as the coverage of this important point). In the brain, this pep- endogenous ligand for NOP — was by the process of tide is also hyperphagic and affects the response to stress, reverse pharmacology and as subsequent characteriza- anxiety and locomotion3,4. In the cardiovascular system tion of the actions of NOP required a panel of N/OFQ N/OFQ produces bradycardia and hypotension; this ligands, a description of their development precedes a response is similar to that produced by classical opioids description of biological function. and more specifically to morphine used in the clinic6. The N/OFQ receptor (NOP; also known as ORL1, Ligand development: peptides and relatives deorphanized OP4 or LC132) is a member of the G‑protein Development of this system from an experimental point Department of coupled receptor (GPCR) superfamily (Box 1). NOP of view has been hampered, until recently, by a relative Cardiovascular Sciences is currently classified as a non-opioid member of the paucity of selective ligands and especially of antagonists. (Pharmacology and structure–activity relationship Therapeutics Group), opioid receptor family by the International Union of Several groups have active Division of Anaesthesia, Basic and Clinical Pharmacology (IUPHAR; see Further (SAR) programmes that are based on native N/OFQ, and Critical Care and Pain information and Supplementary information S1 (table)). a number of these are now generally accepted as stand- Management, Although NOP shares considerable structural and locali- ards for characterization of this receptor by IUPHAR University of Leicester, zation features with the classical opioid receptors, NOP (see Further information). Many of the currently Leicester Royal Infirmary, Leicester LE1 5WW, UK. activity is insensitive to the opioid antagonist naloxone, accepted reference molecules are from the group of 18–24 e-mail: [email protected] an important discriminatory feature for classical opioids G. Calo and R. Guerrini . These include the trun- doi:10.1038/nrd2572 (Ref. 18) (there are several excellent reviews on the classical opiod cated and amidated N/OFQ(1–13)-NH2 ; the

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Airway Central (brain/spinal cord) • Inhibits airway smooth-muscle • Analgesia contraction • Anti-opioid (hyperalgesia) • Inhibits mechanical and • Modulation of feeding capsaicin-induced cough • Anxiolysis • Antitussive • Antidepressant • Modulation of locomoter activity • Modulation of learning and memory Heart • Modulation of opioid tolerance • Negative chronotrope • Pain • Negative ionotrope • Anxiety • Depression • Anorexia/obesity Kidney • Parkinson’s disease • Water diuresis • Congestive heart failure Gastrointestinal tract • Inhibits gastrointestinal motility

Urogenital system • Inhibits evoked contraction Vasculature of vasa deferentia • ‘General’ vasodilator producing • Inhibits micturition reflex hypotension • Neurogenic bladder • Vasodilator of the microcirculation (histamine dependent) Immune system • Heart failure • Inhibits immunocyte activity • Stroke • Hypertension • Sepsis

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

1 2 partial agonist [Phe ψ(CH2-NH)-Gly ]N/OFQ(1–13)- where full agonist behaviour was reported, either a 19 superagonists NH2 ([F/G]N/OFQ(1–13)NH2) ; the response downstream in the signal transduction cascade 4 14 15 20 [(pF)Phe -Arg -Lys ]N/OFQ-NH2 (UFP‑102) and (and hence amplified) was measured or there were high 4 7 14 15 21 [(pF)Phe -Aib -Arg -Lys ]N/OFQ-NH2 (UFP‑112) ; levels of receptor expression. Mason et al. attempted to 1 (Ref. 22) and the antagonists [Nphe ]N/OFQ(1–13)-NH2 address this, although their study was complicated by 1 14 15 23,24 31 and [Nphe -Arg -Lys ]N/OFQ-NH2 (UFP‑101) use of different species isoforms of NOP . Using cell- (Box 2). An example of a peptide SAR study performed based assays, in which the number of NOPs could be in an industrial environment is the study carried out by controlled (ecdysone-inducible expression system), C. T. Dooley, who screened a total of 52 million hexa- it was demonstrated that the agonist/partial agonist/ peptides in mixtures to isolate just five peptides25. These antagonist behaviour was dependent on the expression five peptides are also accepted as receptor standard par- levels of NOP32. These data indicate (and agree with the tial agonists and templates for other ligands (especially current molecular evidence) that there is no basis for ZP120)26,27 (TABLE 1). the suggestion of central and peripheral NOP subtypes,

Structure–activity The development of the partial agonist [F/G]N/ and that [F/G]N/OFQ(1–13)NH2 should be classified relationship OFQ(1–13)NH2 is worthy of further description and as as a partial agonist. Moreover, novel ligands should be (SAR). Correlations that are a cautionary note in characterizations that are based on screened in multiple assays (including those in which constructed between the single endpoint screens for amplified GPCR systems. receptor expression is controlled to that in the tissue of features of chemical structure in a set of candidate This peptide was initially described as an antagonist in interest) before their formal classification. So far, NOP 19 compounds and parameters the mouse vasa deferentia and in the guinea-pig ileum . ligands can be classified pharmacologically into three of biological activity, such Subsequently, it was shown to be a full agonist in cells categories: a pure NOP antagonist, a partial agonist or a as potency, selectivity and expressing recombinant human NOP28,29, as well as a full agonist, depending on the system studied. toxicity. full agonist for the inhibition of K+-evoked glutamate Other peptide ligands of note include peptide III- 29 33 Superagonist release from rat cerebrocortical slices . At this point BTD and ZP120 (Refs 26,27). Peptide III-BTD is based A drug that can interact a couple of suggestions could be made to explain this on a β‑turn (important in the activity of the δ-opioid with a receptor and initiate discrepancy: species difference or differences between receptor endogenous agonist enkephalin34) and a syn- a physiological or a central and peripheral NOP. The full agonist nature of thetic peptide combinatorial library. ZP120 is based pharmacological response that is characteristic of that this peptide has been reported in several other systems on structure-inducing probe technology. ZP120 can be 30 receptor but with particularly including the mouse colon . A simpler pharmacological described as a ‘Dooley’ peptide (that is, Ac-RYYRWK- high potency and/or efficacy. explanation can be used for these data as, in general, NH2) with Lys6 added to the C terminus to improve its nature reviews | drug discovery volume 7 | august 2008 | 695 © 2008 Macmillan Publishers Limited. All rights reserved.

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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.

AC α β γ Separation of pig or rat brain extract ORL1/LC132 'orphan GPCR' ↓cAMP

Fraction cAMP CHO cell 1 – Further separation of 2 – 3 – 4 – ↓cAMP 5 – Measured as 6 Inhibits an output 7 – 8 – No effect 9 – Inhibits 10 – No effect

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

Nature Reviews | Drug Discovery metabolic stability. Peptide III-BTD is a NOP antagonist (lofentanil), morphinans (naloxone benzoylhydrazone with mixed agonist activity at classical opioid receptors33, and buprenorphine) and neuroleptics (spiroxatrine and and ZP120 is selective NOP partial agonist with prolonged pimozide) — all interacted with NOP36. However, these in vivo actions26 (TABLE 1). compounds could not be expected to display selectivity. There is growing interest in the design and evalua- The main chemical classes of the current non-peptides tion of peptide drugs for use in humans35. Indeed, there can be classified as morphinans, 4-aminoquinolines, is increasing activity in the development of NOP peptides benzimidazopiperidines, aryl-piperidines and spiropi- (see below) and the use of N/OFQ itself (for example, peridines4; these are summarized in FIG. 2 and TABLE 2. In intravesical). Peptides offer a number of advantages addition, there is a recent description of a 4‑aryl-tropane including high selectivity, predictable metabolism (for NOP agonist, SCH 221510 (Ref. 37). example, N/OFQ is well validated in humans) and relative Of particular note is that many non-peptide NOP safety (that is, reduced side-effect profile). ligands (which are both agonists and antagonists) contain a piperidine structural motif. This chemical motif Ligand development: non-peptides represents a privileged structure for GPCR binding38 and Early studies in the identification of non-peptide NOP the 1,4-disubstituted piperidine scaffold seems to be ligands revolved around the screening of existing opioids particularly important for NOP interaction. Chemical and traditional high-throughput medicinal chemistry modulation of the substituents in position 1 and 4 of the Privileged structure A single molecular framework approaches with small-molecule libraries in N/OFQ- piperidine nucleus remains an active design process by that is able to provide ligands sensitive preparations. Of note in this area is the work several pharmaceutical companies, and recently a novel for diverse receptors. showing that the existing compounds — anilidopiperidine orally active NOP agonist has been identified39.

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Box 2 | Modification of N/OFQ

In common with a range of peptides Message Address nociceptin/orphanin FQ (N/OFQ) can be loosely broken into message and address H2N FGGFTGARKSARKLANQ COOH domains, with the former being involved in receptor activation and the latter in receptor occupation18. There have been a number of a H2N FGGFTGARKSARK COOH groups engaged in peptide structure–activity Truncation to amino acids 1–13 retains full activity relationship (SAR) studies including those of Meunier195, Civelli196 and Dooley25. Data b H N YGGFTGARKSARKLANQ COOH predominantly from the group of G. Calo and 2 Substitution of F with Y may produce non-selective opioid R. Guerrini, using a comprehensive analysis of both message (pink) and address (blue) c H N F ψ(CH –NH) GGFTGARKSARKLAN Q COOH domains is depicted (top sequence). C‑terminal 2 2 Partial agonist template truncation of the peptide results in a progressive loss of binding affinity and d H N Nphe GGFTGARKSARKLANQ COOH functional potency such that N/OFQ(1–13) 2 Antagonist template is the smallest fragment (a) to retain full biological activity28,197,198. This fragment e H N FG G (pF)FTGARKSARKLANQ COOH is a commonly used template for further 2 modification. If Phe1 in N/OFQ is replaced with Increases affinity Tyr1 (as in classical opioids) then a previously f H N FGGFTGARKSARKRKNQ COOH selective NOP ligand becomes a relatively non- 2 selective (b) opioid ligand199; although this has Increases potency been questioned200,201. Some of the more g H N Nphe GGFTGARKSARKRKNQ NH important modifications of N/OFQ are 2 2 depicted in the message domain (c–e) and in Competitive antagonist (UFP-101) the address domain (f). These modifications are h H N FGG (pF)FTGARKSARKRKNQ NH combined to produce at least two interesting 2 2 and now widely accepted preclinical reference Super agonist (UFP-102) peptides: UFP‑101 (g) and UFP‑102 (h). Adding 1 2 1 2 a pseudopeptide bond between Phe and Gly (Phe ψ(CH2-NH)-Gly ) or [F/G] (c) reduces binding affinity and peptide efficacy such that at low expression in mouse vasa deferentia (mVD) the peptide has antagonistNatur eproperties. Reviews | Drug However, Discove at ry 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.

Molecular modelling studies suggest that the nitro- the lipophilic C moiety has a role in the intrinsic activity gen of the piperidine ring interacts with the Asp130 of of the ligand at NOP. Subtle changes in the distance NOP40; the same amino-acid residue that is involved between the piperidine nitrogen and moiety C produce in the interaction with the N‑terminal nitrogen of the important differences in ligand efficacy. In the follow- natural ligand41. These data suggest that non-peptide ing section describing the role(s) of the N/OFQ–NOP NOP ligands might interact with NOP amino acids system, the selective NOP agonist Ro64‑6198 and that are crucial for binding the physiological peptide antagonists J‑113397 and SB‑612111, together with and add value to current peptide SAR approaches. the low-selectivity antagonist JTC‑801, are described. A two-dimensional pharmacophore model for the Details of the pharmacology of these ligands can be interaction between NOP and non-peptide ligands has found in TABLE 2. been proposed42. In this elegant model the three key elements (or moieties) are described as the heterocyclic (Patho)physiological role(s) of N/OFQ–NOP A‑moiety, the basic nitrogen-containing B‑moiety As described in FIG. 1, N/OFQ–NOP is involved in a wide and the lipophilic group on the basic nitrogen, the range of responses and thus has wide potential for drug C‑moiety. Moiety A is an important determinant of development. This Review selects areas in which there is binding affinity and selectivity compared with classical one or more of the following: good basic science evidence µ-opioid, δ-opioid and κ-opioid receptors. By contrast, for involvement; clinical evidence in the form of plasma

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Table 1 | Characteristics of non-N/OFQ-related peptides Peptide NOP µ-Opioid δ-Opioid κ-Opioid In vivo effects at NOP receptor receptor receptor (route of administration) ‘Dooley’ peptides and derivatives*

‡ § § § Ac-RYYRIK-NH2 Partial Inactive Inactive Inactive • Hypotension and bradycardia agonist (intravenous)207 Ac- Partial Inactive|| Inactive|| Inactive|| • Pro-nociceptive and decreased RYYRWKKKKKKK- agonist locomotion (intravenous)26 + + 208 NH2 (ZP120) • Long-lasting Na /K -sparing aquaresis • Phase I/II trials in heart failure (Zealand Pharma; see Further information) Ac-RY(3-Cl)YRWR- Partial Inactive¶ Inactive¶ Inactive¶ • Antimorphine 210 NH2 (Syn-1020) agonist (intracerebroventricular) • Antinociception (subcutaneous)210 • Anti-allodynic (intraperitoneal)210 Other peptides Peptide III-BTD Antagonist Partial Agonist Partial • No data available; predict agonist agonist# antinociceptive profile OS-461, OS-462, Agonist No data No data No data • Long-lasting hyperphagic response OS-500 available available available (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 µ-opioid receptor, δ-opioid receptor or κ-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 α of 0.1 compared with CI-977 (Ref. 33). N/OFQ, nociceptin/orphanin FQ peptide; NOP, N/OFQ receptor; OS-461, N-α-6-guanidinohexyl-l-tyrosyl-l-arginyl-l-tryptophanamide; OS-462, N-α-6-guanidinohexyl- 3,5-dimethyl-l-tyrosyl-l-tyrosyl-N-[(R)-1-(2-naphthyl)ethyl]-l-argininamide; OS-500, N-α-6-guanidinohexyl-3,5-dimethyl-l- tyrosyl-3,5-dimethyl-l-tyrosyl-N-[(R)-1-(2-naphthyl)ethyl]-l-argininamide.

peptide measurements, studies with human tissues or for- resulting in the release of central endogenous opioid mal clinical studies; pharmaceutical evidence with candi- peptides with their effects subsequently reversed by the date drugs suitable for development; and/or emerging new delivered dose of N/OFQ. In contrast to this supraspinal areas without an extensive literature base. Consequently, anti-opioid/hyperalgesic action, spinal administration pain, mood (depression and anxiety) disorders, reward/ produces a more classical antinociceptive effect46,47. drug abuse, cardiovascular disease, urogenital disease and The neuroanatomical site underlying the anti-opioid immune function are selected for discussion. actions of N/OFQ is the rostral ventromedial medulla where two types of cells exist: the ‘on’ and ‘off’ cells5,44 Central effects of N/OFQ (FIG. 3). On cells inhibit the action of off cells, and are Pain. Since the identification of N/OFQ1,2 there has inhibited by morphine acting at µ-opioid receptors. Off been intense interest in the role of this peptide in pain cells project back to the spinal dorsal horn (known as processing. This is based on various factors, including the descending inhibitory control circuitry) to reduce the similarity of distribution of receptor and peptide to ascending nociceptive information reaching third-order classical opioids within the defined pain pathway, the neurons via the ascending spinothalamic tract. When structural similarity to classical opioids, and the simi- morphine inhibits the on cell this disinhibits the off cell, larity in post-receptor transduction to classical opioids. leading to an antinociceptive effect. N/OFQ inhibits In addition, there is some limited association of plasma both the on and off cell with direct inhibition of the off N/OFQ levels with human pain states and the paucity cell producing an increase in nociceptive traffic. Clearly, of current effective analgesics with activity in chronic this inhibition of the off cell would reverse any actions of pain states. In the original description of Meunier opioids at the on cell; producing an anti-opioid action. et al., the name nociceptin was related to the fact that In vivo endogenous opioid peptides would produce a intracerebroventricular administration of N/OFQ pro- degree of antinociceptive ‘tone’, and stress (as in early duced hyperalgesia1. However, the marked differences in studies1) would increase the local concentration of these supraspinal and spinal effects set this peptide–receptor peptides. Addition of N/OFQ under these experimental system apart from that of morphine and the µ-opioid circumstances would be sufficient to produce a hyper- receptor system used in the clinic. algesic response43,44,48. The consensus view is that spinal N/OFQ administered supraspinally reverses the effects N/OFQ produces a classical (opioid-like) antinociceptive of exogenous opioids; that is, N/OFQ is an anti-opioid response by inhibiting transmitter release at primary peptide at this site5,43,44. In addition, acupuncture-induced nociceptive afferent terminals5,47,49. Indeed N/OFQ has antinociception is reversed45 and it has been suggested been shown to inhibit the release of transmitters involved that the hyperalgesia observed in early studies1 is a result in this pathway including glutamate50. Interestingly, one of a simple reversal of stress-induced antinociception5. study51 reported an increase in the release of the pro- Intracerebroventricular injection of N/OFQ was stressful, nociceptive transmitter substance P.

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4-Aminoquinolines Benzimidazopiperidines JTC-801 (Japan Tobacco) J-113393 (Banyu) NOP antagonist NOP antagonist

NH2 H N Et NN N

O N O O OH N-(4-amino-2-methylquinolin-6-yl)-2- 1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4- (4-ethylphenoxymethyl)benzamine piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one

N O CH3 OH H3CO N HO N O O O N N O N CH2 H NH Lofentanil Naloxone benzoylhydrazone O Anilidopiperidine Morphinan N HO CH3 N O CBu O Buprenorphine OH Spiroxatrine Morphinan OCH3 Neuroleptic Aryl piperidines Spiropiperidines SB-612111 (GlaxoSmithKline) Ro64-6198 (Roche) H NOP antagonist Cl NOP agonist O N

Cl N HO N N H

Me (–)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1- [(1S,3aS)-8-(2,3,3a,4,5,6-hexahydro-1H-phenalen-1 yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol -yl)-1-phenyl-1,3,8-triaza-spiro[4,5]decan-4-one] 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. Nature Reviews | Drug Discovery

The relative contribution of supraspinal pro-noci- via this route (for example, J‑113397 (Ref. 52)) is at ceptive and spinal antinociceptive actions of systemi- variance with this hypothesis and is hard to reconcile. cally administered NOP agonists is worth considering. In addition, the lack of antinociceptive effects of the Depending on the pharmacokinetics and blood–brain selective non-peptide antagonists J‑113397 (Ref. 53) and barrier permeability of the ligand under study, the SB-612111 (Ref. 54) given systemically is also at variance relative distribution will be important. On the one hand with this general scheme. How can this complex (central supraspinal (agonist) spread might produce a hyperalgesic compared with peripheral administration) behaviour be response (via reversal of endogenous opioid action in explained? There are a number of differences in the way the ventromedial medulla), yet if the agonist also acts at the experiments are conducted. For instance, the time the spinal site then an inhibition of nociceptive afferent course of the nociceptive stimulus is variable, short- inflow would result. The net effect would therefore be term (tail withdrawal, tail flick and hot plate) compared difficult to predict. with long-term (formalin test), and the degree to which Several groups have reviewed the details of intra the experimental protocol (for example, intracerebro cerebroventricular NOP antagonism4,5,24 and there is a ventricular injection) produces stress and hence stress- reasonably consistent view that peptide but not non- induced antinociception against which NOP antagonists peptide antagonists are generally antinociceptive when can work is also variable. administered intracerebroventricularly. Using the The relative contribution of spinal and supraspinal non-peptide data in the schematic shown in FIG. 3, it actions in the overall response to systemic NOP antag is proposed that there is a degree of N/OFQ-mediated onists has been elegantly addressed in the formalin test55. tone in the brain and that this produces a tonic anti- Intraplantar formalin produces a biphasic pain response (endogenous)opioid action, thus setting the pain thresh- with an acute phase lasting approximately 10 mins and a old to a more pain phenotype. Switching this off would later secondary phase starting at around 20 mins and lasting produce analgesia/antinociception. However, the lack of for half an hour. Intracerebroventricular administration effect of intracerebroventricular non-peptide antagonists of UFP‑101 produced an antinociceptive response and

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intrathecal administration of UFP‑101 produced a Human studies of pain. There have been attempts to pro-nociceptive response in the secondary phase. This correlate human plasma N/OFQ levels with pain but the could be predicted based on the previous discussion. results of these studies have been contradictory70. An Interestingly, systemic (intravenous) administration of increase in N/OFQ levels in acute and chronic pain com- J‑113397, which would deliver this antagonist to both pared with controls was reported71. However, a decrease spinal and supraspinal sites, produced a pro-nociceptive in N/OFQ levels was noted in fibromyalgia syndrome72 effect. In general data obtained with NOP–/– mice and and cluster headache73. From an effect-site perspective mice deficient in pre-pro-N/OFQ (ppN/OFQ–/–) agree it is difficult to reconcile plasma measurements with with pharmacological findings56. The overall conclusion nociceptor-synaptic peptide concentrations. This prob- from this experiment is that the predominant action of lem has been addressed by measuring cerebrospinal fluid systemic NOP antagonists is spinal, and that endogenous N/OFQ in labour (requiring an epidural anaesthetic N/OFQ-mediated tone in the spinal cord is high and in enabling sampling of the cerebrospinal fluid); no increase the brain it is low. This contrasts with the effects seen relative to non-labouring (elective caesarean sections) following acute and stressful intracerebroventricular patients was reported74. injection with an acute pain measure in which N/OFQ- N/OFQ has been administered in humans in two mediated tone is presumed to be high and possibly an studies: intramuscular injection in volunteers75 and intra- assay and stimulus-dependent phenomenon55. vesical instillation in urology patients (see below). The It has been known for many years that peripheral two volunteer studies involved increasing doses of intra terminals of nociceptive afferents express NOP. NOPs muscular N/OFQ into the left and right temporal muscle are located in skin57, bladder (see below for a more in random and as an open label or as a balanced placebo- detailed consideration)58 and on lymphocytes (see below controlled intramuscular injection into the non-dominant for a more detailed consideration)59. If it is assumed trapezius. There was no pain noted in either study but in that peripheral inflammation upregulates NOP (in a the latter study there was an increase in local tenderness. similar manner to µ-opioid receptors) coupled with lymphocyte N/OFQ release, then an additional facet to Anxiety and depression. The current market has a wide the neuroimmune axis can be proposed60. Activation range of drugs for the treatment of anxiety and depression, of NOP at any of these peripheral sites would reduce but these are often characterized by poor and/or variable nociceptive afferent inflow and produce peripheral efficacy, long run in to peak behavioural effect and, from analgesia/antinociception. There is some experimental a patients perspective, a wide range of side effects leading evidence for this mode of action in producing antinoci to tolerability and compliance problems76,77. ception61–63 (FIG. 3). There is now good evidence from animal work for Opioids are the gold standard for acute pain and are a role for the N/OFQ–NOP system in emotional dis- often used with some effect in chronic pain8. However, orders78 including anxiety79,80 and depression81–83. NOP there is a trade-off between good analgesia and poor and N/OFQ are located in areas that are crucial to mood side-effect profile. Indeed, tolerance to morphine control including but not limited to amygdala, hippo develops such that dose escalation is required, which campus, thalamus and cortical processing areas78. At inevitably increases the prevalence and severity of side relatively low doses, N/OFQ79 and several non-peptide effects8,64. Therefore, any new analgesic would have agonists from Roche (Ro65‑6570 and Ro64‑6198) are greater potential if it had a relatively low (or absent) generally reported as an anxiolytic84–86, although others87 ability to produce tolerance. There is evidence for toler- have questioned this. It has been reported that repeated ance to the antinociceptive effects of spinal N/OFQ65. administration of Ro64‑6198 for 15 days failed to induce Intriguingly in animals that lack NOP66,67 or are treated tolerance to the anxiolytic actions of this molecule88, with the peptide NOP antagonist [Nphe1]N/OFQ(1–13)- perhaps setting Ro64‑6198 aside from more traditional (Ref. 68) NH2 and the non-peptide antagonists J‑113397 anxiolytics. In a recent study from Schering–Plough (Ref. 69) and SB‑612111 (Ref. 54) there was a reduction in the non-peptide agonist SCH 221510 was shown to be the development of tolerance to morphine. Moreover, anxiolytic but with a reduced side-effect profile when ppN/OFQ–/– mice also display reduced liability to compared with benzodiazepines37. Moreover, ppN/OFQ–/– develop morphine tolerance69. This raises the possibility mice display an anxiogenic phenotype89. Interestingly, of co-administration of a µ-opioid receptor agonist and there was some modulation of pain in this knockout a NOP antagonist. Based on the discussion above this model, underscoring a close link between stress and might produce classical µ-opioid receptor analgesia, pain89,90. Use of NOP antagonists does not modify the level reduce the anti-opioid effects of endogenous N/OFQ to of anxiety and implies a lack of N/OFQ-mediated tone in potentiate morphine analgesia and produce less toler- the control of this behaviour. There is no consensus on ance, which would then enable lower doses of morphine the mechanism(s) underlying this behavioural response, to be used. This reduced dose might then reduce the although modulation of 5-hydroxytryptamine (5‑HT)91, other clinical side effects that are associated with mor- functional coriticotropin-releasing factor (CRF) antago- 79 phine, such as respiratory depression and constipation. nism and recently a dependence on GABAA (γ-amino Current development for pain remains predominantly butyric acid A) receptors92 has been suggested. In contrast preclinical, although Phase I and II trials of the low- to this lack of antagonist effect per se in this model of selectivity NOP antagonist JTC‑801 (TABLE 2) are noted anxiety, NOP antagonists display an antidepressant pro- but there are no published data available. file81–83 and indicate central N/OFQ-mediated tone in the

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Table 2 | Selected characteristics of some common and novel non-peptide NOP ligands Class and Pharmacological profile In vivo effects compound Morphinans (poor selectivity) TRK820 (Toray/ NOP antagonist, µ-opioid • Antinociceptive in mice213 and monkeys214 Acologix) receptor partial agonist, κ-opioid • Antipruritic in monkeys215 receptor agonist212 • In Phase III as antipruritic in patients with uraemia‡ • No specific NOP-related clinical development Buprenorphine Non-selective µ-opioid receptor • In widespread clinical use for moderate to severe pain and NOP partial agonist216 • Analgesia via µ-opioid receptor217 • Also under evaluation by Samyang as a patch§ Naloxone Non-selective µ-opioid receptor • Antinociceptive activity in mice, which is lost in benzoylhydrazone antagonist, κ-opioid receptor NOP–/– mice218 agonist219 and NOP partial agonist (very low efficacy)32 4-Aminoquinolines JTC-801 (Japan NOP antagonist, low selectivity • Systemic220 and spinal antinociceptive in mice221 Tobacco) over µ-opioid receptor220 • Phase I (oral) and Phase II pain (injectable) trials have been done (no published details) • Some actions may be mediated by µ-opioid receptor Benzimidazopiperidines* J-113397 (Banyu) Highly selective NOP • Complex behaviour; antinociceptive and antagonist53,222,223 pro-nociceptive5 • Trap-101 is an achiral analogue of J-113397 (Ref. 224); not evaluated in vivo Aryl-piperidines SB-612111 Highly selective NOP • Reduces N/OFQ-induced pro-nociceptive and (GlaxoSmithKline) antagonist54,182 antinociceptive and orexigenic responses • Has antidepressant profile225 • Phase I trials in parkinsonism expected in 2008|| Series of compounds Highly selective NOP agonist226,227 • Not extensively evaluated; displays antitussive activity (Schering) 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 Highly selective NOP agonist230 • Not extensively evaluated (Schering) *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.

control of depression. Again, there is no consensus on Taken at face value these data indicate that NOP the mechanism(s) underlying this behavioural response. agonists are anxiolytic and that antagonists are anti However, with the wealth of information on the role of depressant. However, there are several problems and catecholamines in depression93, the well-known observa- areas in which detailed information is lacking: most tion that N/OFQ inhibits catecholamine release14, the fact animal studies involve a short-term exposure to the that NOP antagonists will switch off N/OFQ-mediated compound, whereas longer-term treatments in humans signalling combined with observations that N/OFQ are more usual (>4 weeks for selective serotonin acting at postsynaptic NOP increases K+ conductance reuptake inhibitor effect); the precise neuroanatomical and reduces the firing of locus coeruleus (noradrener- sites(s) of the anxiolytic/antidepressant behaviours are gic)94 and dorsal raphe (serotonergic)12 nuclei, a simple largely unknown; and there is an almost complete lack catecholamine hypothesis can be advanced (FIG. 4). of clinical information.

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Human study of depression. There is just one human accumbens. In addition, intracerebroventricular adminis study95 in which plasma N/OFQ and 5‑HT levels were tration of N/OFQ reduced morphine-induced110,111 and measured: 21 patients with post-partum depression were cocaine-induced112 dopamine release from this brain compared with 25 healthy controls. In controls, plasma structure. Using electrophysiological techniques, gluta- N/OFQ levels were 10.4 ± 3.7 pg ml–1 (remarkably con- mate and GABA release in the amygdala of the rat are sistent with previously measured values70) and this was inhibited by N/OFQ113. In a more recent study, N/OFQ –1 elevated to 28.5 ± 5.8 pg ml in the post-partum depres- was shown to decrease GABAA receptor-mediated inhibi- sion group. By contrast, 5‑HT levels in the post-partum tory postsynaptic currents and importantly to prevent depression group were lower (1.0 ± 0.3 µmol l–1 com- ethanol-induced augmentation of inhibitory postsynaptic pared with 1.4 ± 0.4 µmol l–1). These limited small study currents in the central amygdala of the rat114. In the data agree with the notion that post-partum depression prefrontal cortex N/OFQ inhibits the release of noradrena results from reduced 5‑HT levels and that this is accom- line115; data for the major reward neurotransmitters is panied by elevated N/OFQ with the increase in N/OFQ lacking. possibly causing the fall in 5-HT. There is currently no A substantial amount of current information on major clinical development in this area, but based on this addiction and reward comes from studies using alcohol. small study larger studies are required to confirm this In this respect the Marchigian Sardinian alcohol-preferring interesting and important finding95. Studies of clinically (msP) rat has provided some interesting insights101,102,116–118. useful antagonists (such as SB‑612111, awaiting trials in In msP rats intracerebroventricular administration of the parkinsonism, TABLE 2) are eagerly awaited. In terms of peptides OS‑462, UFP‑102 and UFP‑112 reduced alcohol development of anxiolytic agonists Ro64‑6198 seems an consumption. But interestingly and in contrast to the later ideal candidate (TABLE 2), but the future of this molecule work in normal Wistar rats of Kuzmin119 the non-peptide according to Shoblock96 “is uncertain” owing to potential Ro64‑6198 given intraperitoneally increased alcohol side-effect problems. consumption, an effect the authors ascribe to µ-opioid receptor residual agonism120. In this model low doses of Modulation of the rewarding properties of drugs of the mixed µ-opioid receptor/NOP agonist buprenorphine abuse. Drug abuse (including alcohol) has a major increased alcohol consumption (in a naltrexone-sensitive socioeconomic impact and is relatively difficult to treat. manner) and reduced consumption at higher doses (in According to estimations from the UK government a UFP‑101-sensitive manner)121. As buprenorphine is (Neighbourhood.gov.uk; see Further information), currently available for use in humans it may form an the misuse of drugs costs the UK economy between interesting alternative or adjunct to current therapy for UK£10–18 billion per year. The major pharmacological treating alcoholism. treatment strategies are based on reducing the impact of In an elegant study comparing msP rats and normal the symptoms of dependence, allowing abstinence and Wistar rats it was shown that the N/OFQ-mediated prevention of relapse. There are a limited number of reduction in alcohol drinking behaviour of the msP such treatments available, with varying efficacy, including (but not normal Wistar) rat corresponded with an methadone and buprenorphine (for opiate abuse)97 and increase in NOP and ppN/OFQ mRNA and NOP pro- 122 Conditioned place naltrexone and the antiglutamatergic agent acompros- tein in reward areas of the brain . In addition, there preference (CPP) test tate (for alcohol abuse)98. The value of addressing the was an increases in receptor function (as measured by An experimental animal is psychological component of dependence has also been GTPγ[35S] binding) in these areas of msP rat brains with presented with a positive described99,100. the notable exception of central amygdaloid nucleus stimulus (in this case a drug of abuse) in conjunction with In animal models aimed at elucidating the rewarding where there was a decrease. Direct injection of N/OFQ a specific cue. The animal properties of drugs of abuse the conditioned place preference into this region markedly reduced ethanol consump- develops an association (CPP) test is commonly used. In this assay N/OFQ has tion. In the basolateral amygdala and the bed nucleus between the place and been shown to reduce CPP to alcohol101–103, ampheta- of the stria terminalis (where GTPγ[35S] binding was preference for the positive 104 105,106 106 stimulus. The amount of time mines , cocaine and morphine , indicating that increased) direct injection of N/OFQ did not reduce 122 spent in the place previously this peptide was reducing reward to these stimuli. N/OFQ alcohol consumption . Based on the following evi- associated with the stimulus alone was inactive. A role for the endogenous N/OFQ– dence — one there is a upregulation of CRF receptor can be used as an index of NOP system was recently examined in a complex study107 activity in the central amygdaloid nucleus in the msP the rewarding properties that assessed hedonic state (which they define as “a bal- model, and two there is a compensatory increase in of the original stimulus. anced affective, emotional and motivational state”) in NOP signalling in other brain areas except the central Marchigian Sardinian methamphetamine and ethanol-conditioned wild-type amygdaloid nucleus — the authors speculate that an alcohol-preferring (mSP) rat and NOP–/– mice. As N/OFQ appeared to suppress an imbalance between CRF and N/OFQ–NOP signalling A genetically selected strain of enhancement of hedonic state the authors suggest that underlie the increased alcohol drinking in msP rats and rat with a natural preference for ethanol. This is used as a the endogenous N/OFQ–NOP system might facilitate that restoration of this imbalance with CRF antagonists 107 model of human alcoholism. the development of addiction . and/or NOP agonists might represent a mechanism- The neuroanatomical substrates for these responses driven treatment strategy. GTPγ [35S] binding include the ventral tegmental area, nucleus accumbens, An in vitro assay used to amygdala and medial prefrontal cortex; all of these areas Human studies of drug dependence. There are two monitor G-protein coupled receptor-mediated guanine express NOP. In anaesthetized rats intracerebroventricular studies examining an association between single 108 109 nucleotide exchange at G administration and intrategmental administration of nucleotide polymorphisms (SNPs) in the NOP gene proteins. N/OFQ suppressed dopamine release from the nucleus (ORPL1) and ppN/OFQ gene (PNOC) and alcohol/

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Peripheral input N/OFQ Supraspinal 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 Skin – – an association with SNPs in non-coding regions? Are there any differences in functional receptor or peptide – – Morphine On Off production? There is currently no indication of new – RVM N/OFQ pharmaceutical development beyond preclinical phases for this indication. Descending Central and peripheral actions of N/OFQ Peripheral organs (bladder) The control of the cardiovascular and renal system. Based on statistics collated by The British Heart Foundation Spinal (see Further information) 49% of all deaths in Europe Morphine – were as a consequence of cardiovascular disease. With the financial impact of cardiovascular disease in Europe Aδ/C Ascending 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 N/OFQ + and conscious laboratory animals N/OFQ produces hypotension and bradycardia6,125. This occurs following intravenous126 and intracerebroventricular127 administra- N/OFQ tion and is absent in NOP–/– mice128. The effects occur at PBMC release both central and peripheral sites. The most compelling evidence for peripheral effects is the hypotension and Inflamed terminal bradycardia produced by intravenous administration of – – N/OFQ, a peptide that does not cross the blood–brain – N/OFQ N/OFQ barrier. A role for reflex loops cannot, however, be completely excluded. It has been suggested that as the Figure 3 | Schematic to describe the interrelationship between the anatomical sympatholytic guanethidine reduced the hypotensive site(s) underlying the actions of N/OFQ on pain. Nociceptin/orphaninNature Reviews | Drug FQ peptide Discovery (N/OFQ) produces supraspinal hyperalgesia/anti-opioid actions by inhibiting both on effects of N/OFQ, then this peptide acts to inhibit sympa- 129 and off cells in the rostral ventromedial medulla (RVM). On cells inhibit off cells, and thetic control of the cardiovascular system . In addition, are inhibited by morphine acting at µ-opioid receptors. Off cells project back to the this study showed that the bradycardic effects of N/OFQ spinal dorsal horn and reduce ascending nociceptive information. When classical were reduced by vagotomy, indicating that N/OFQ opioids like morphine inhibit the on cell this disinhibits the off cell, leading to an increased parasympathetic activity. Direct injection of antinociceptive or analgesic effect. By contrast, N/OFQ (exogenously added or N/OFQ into the rostral ventrolateral medulla produced endogenously produced) inhibits both the on and off cell with direct inhibition of the bradycardia and hypotension130. Moreover, intravenous off cell producing an increase in nociceptive traffic. Clearly, inhibition of the off cell infusion of N/OFQ produces a diuresis and antinatriuresis, would reverse any actions of opioids at the on cell; producing an anti-opioid and decreases renal sympathetic nerve activity125,126,131. (exogenously added or endogenously produced) action. At the spinal level, N/OFQ Intravenous N/OFQ produces vasodilation132–134. produces classical opioid analgesia by inhibiting nociceptive afferent inflow. 134 Inhibition of nociceptive afferent inflow can occur in the periphery in tissues such Nitric oxide is not involved in this dilator response . In as skin and bladder, and possibly via an interaction with the neuroimmune axis. a series of studies of the cerebral circulation, N/OFQ was PBMC, peripheral blood mononuclear cells. Exogenous N/OFQ is in the blue box. 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- illicit drug dependence123,124. One study examined istration of N/OFQ dilated arterioles and venules136. 10 SNPs covering OPRL1 and the adjacent regulator Dilation of these non-innervated vessels was blocked by of G‑protein signalling RGS19 and 15 SNPs covering histamine antagonists and mast-cell stabilizers, suggesting PNOC in European American subjects. The authors that the N/OFQ-mediated dilation of the microcirculation attempted to correlate genotype with alcohol and illicit is secondary to mast-cell release of histamine. N/OFQ has drug (marijuana, cocaine, stimulant, sedative or opioid) been shown to release histamine in the brain following dependence. The authors reported no convincing asso- intracerebroventricular injection137 and directly from rat ciation between alcohol dependence and either OPRL1 peritoneal mast cell preparations138. or PNOC polymorphisms. However, they did report a Sepsis and its progression to septic shock is a condition marginal association between two (alcohol depend- in which there is marked hypotension and an associated ence) and one (illicit drug dependence) of 15 non-coding high mortality rate139. Using the caecal ligation-puncture PNOC SNPs123. A second recent study examined 18 model of sepsis in the rat it has been recently reported Sympatholytic OPRL1 SNPs in Scandinavians with alcohol depend- that the mortality rate of 70% was markedly reduced to An agent that decreases the activity of the sympathetic ence. One SNP (rs6010718) was associated with alcohol 40% following treatment with the peptide NOP antagonist 124 nervous system, for example, dependence, more so in females . These studies UFP‑101 (Gavioli et al., personal communication). The guanethidine. offer a tantalising early insight into a genetic role for implication of this study is that N/OFQ concentrations

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might be elevated in sepsis. In support of this notion, fibres of the bladder inhibits the volume-evoked mictu- N/OFQ concentrations in 21 patients in intensive care rition reflex58,151–153. At the time of writing there are no with a diagnosis of sepsis were measured. In this small studies detailing NOP expression in bladder tissue of study four patients died and this was associated with human origin. However, based on animal data a series elevated N/OFQ levels when compared with those who of elegant clinical studies have been performed using survived140. Based on these complementary pieces of intravesical N/OFQ by Lazzeri and colleagues, which evidence it is tempting to suggest that NOP antagonists are described next. might be a useful adjunct in the management of sepsis, and larger studies in humans are warranted. Human studies of the urogenital system. In a pilot study Direct effects of N/OFQ on the heart are controversial. of 14 patients, five normal controls and nine with detrusor In the adult there is a single study reporting evidence of hyperreflexia, intravesical instillation of 1 µM N/OFQ NOP receptors in heart tissue141, albeit with differing increased bladder capacity and the volume required for ligand affinity and binding capacities in comparison detrusor hyperreflexia. This effect lasted 24–48 hours with other ex vivo studies. The authors did report some and was not present in the control group. Interestingly, limited PCR for NOP transcripts and noted that in spon- one patient had previously received intravesical capsaicin taneously hypertensive rats there was an increase in the and the effects of N/OFQ were minimal, indicating a number of high-affinity N/OFQ binding sites. These capsaicin-sensitive target154. In a later follow-up study, receptors may be on cardiac neural tissue. In 1–2 day N/OFQ was compared with the structurally similar but old cardiomyocytes isolated from Wistar rats N/OFQ inactive [desPhe1]N/OFQ (a major metabolite of N/OFQ, displayed a positive chronotropic effect with a maximum which does not bind to NOP; that is, a placebo-controlled 65% of that produced by isoprenaline142. Again in neo- study) in 14 patients with detrusor overactivity due natal cardiomyocytes N/OFQ increased atrial natriuretic to spinal-cord damage. Intravesical administration peptide secretion and inhibited cAMP formation143. of 1 µM N/OFQ but not [desPhe1]N/OFQ increased Pharmacological analysis using antagonists for classi- bladder capacity and volume required for detrusor cal opioid and NOP receptors are strongly suggestive of hyperreflexia155. In a further study of 18 patients with expression in this tissue. neurogenic detrusor overactivity incontinence who were Although the general hypotensive, bradycardic and capable of self catheterization, half of the group instilled vasodilator actions of N/OFQ hold, there are several 1 mg of N/OFQ and the remainder instilled saline at Chronotropic effect apparently contradictory pieces of data. First, in con- the first catheterization of the morning for a period of In the context of the scious sheep N/OFQ increases blood pressure and heart 10 days. In agreement with previous studies bladder cardiovascular system, this rate144. Second, injection of N/OFQ into the nucleus of the capacity increased following N/OFQ treatment. From refers to heart rate. Opioids slow heart rate and thus have solitary tract (the termination point for cardiac sensory a patient perspective the number of daily urine leakage 145 a negative chronotropic effect. afferents) increases heart rate and blood pressure . Last, episodes were reduced in the N/OFQ (but not saline) in cultured neonatal rat myocytes exogenous N/OFQ group. There did not appear to be any major practical Hyponatraemia increased contraction rate142. problems related to use, and the effects of a single instill Low plasma sodium 156 concentration. ation in the morning appeared to last the whole day . Human studies of the cardiovascular and renal system. Collectively, these studies broadly agree with studies Capsaicin-sensitive primary Of particular note in this area is the work with the peptide in rats that NOP is probably expressed on capsaicin- afferents ZP120. Based on animal work, ZP120 is best described sensitive primary afferents and their activation inhibits These are primary nociceptive as an aquaretic as it promotes excretion of water while the micturition reflex. However, basic work has only afferent fibres of the Aδ and 26,27,146 C-fibre type. They express sparing electrolyte loss . Coupled with vasodilatory been done in normal rats, and N/OFQ is only effective 147 TRPV1 receptors and hence actions this peptide displays a clinical profile that is in spinally injured patients where there may be afferent are capsaicin sensitive. compatible with use in congestive heart failure (reduction fibre switching157,158. N/OFQ and N/OFQ mimetics have As TRPV1 receptors are also in oedema and correction of hyponatraemia). Phase I and a potential role in the treatment of overactive bladder activated by heat and low pH, these channels convey a II trials have been completed; these data are in abstract and their use is not limited to specialist centres capable polymodal activation profile form and can be found at the Zealand Pharma web site of intravesical administration. However, it is most likely to Aδ and C-fibres. (see Further information) and TABLE 1. ZP120 is described that any potential development (of which there is cur- as “safe and well tolerated in patients with chronic heart rently none) will be for patients resistant to the more Detrusor hyperreflexia failure” with a nonlinear pharmacokinetic profile. conventional use of anticholinergics148. Increased contractility of the detrusor muscle of the bladder (idiopathic), often as a result Peripheral effects of N/OFQ The immune response. It is well documented that opioids of neurological disease The urogenital system. Overactive bladder affects depress the immune system159,160. As early as 1950 Hussey (neurogenic), leading to approximately 16% of the European population >40 and Katz described a series of 102 opioid addicts with urinary incontinence. years of age and Aδ and C‑fibre afferents are variably infection as a result of their addiction161. Despite this clear 148 Afferent fibre switching implicated . Traditional use of anticholinergics and and well-known observation there is much controversy Afferent signals from the more recently targeting transient receptor potential cation as to the precise site(s) of this interaction. Opioids can bladder are conveyed by Aδ channel, subfamily V, member 1 (TRPV1) receptors is of depress the immune response via the hypothalamic– and C-fibres, with the latter variable efficacy148. One of the earliest ex vivo bioassays pituitary–adrenal axis via increased glucocorticoid being ‘silent’. In spinal-cord 162 injury, C-fibre activity is for NOP was in the mouse vas deferens in which N/OFQ production . However with regard to the classical opioid 149,150 unmasked, leading to fibre inhibits electrically evoked contraction . In the rat, receptors (µ, δ, κ) there is much controversy as to a direct switching. NOP receptors located on capsaicin-sensitive primary afferent interaction with cells of the immune system.

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Depression Anxiety Knockout • NOP–/– • Antidepressant phenotype • Some anxious features mice • ppN/OFQ–/– • Not done • Anxiogenic phenotype

NOP • Agonist • No direct effect • Anxiolytic ligands • Antagonist • Antidepressant • No direct effect

Clinical • Supporting • N/OFQ elevated (28.5 pg ml–1) in post- • None evidence partum depression (10.4 pg ml–1 in control) • Preferred • Antagonist • Agonist NOP ligand

Neuron

Tonic N/OFQ reduces Possible catecholamine levels Depression Anxiety modulation of 5-HT and CRF NOP antagonist NOP receptor levels, and reverses tonic N/OFQ signalling GABAA receptors inhibition to elevate catecholamines and increase mood control NOP antagonist

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 (andNatur plasma)e Reviews catecholamine | Drug Discove ry (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.

Consensus is that under resting conditions immuno reported in non-surviving septic patients140 originates cytes do not express classical opioid receptors163. However, from increased immunocyte release, and it is also possible one limited PCR study showed µ-opioid receptor that there may be some interplay with the vasculature upregulation following pretreatment with interleukin 4 via a neuro–vascular–immune axis. In a mouse model (IL4), which suggests that in sepsis µ-opioid receptors of inflammatory bowel disease (dextran sulphate sodium may be upregulated164. That circulating lymphocytes administration) in which there is an infiltration of inflam- contain and release opioid peptides is also clear60. Of matory cells and an upregulation of N/OFQ expression, importance are studies that propose a neuroimmune axis NOP knockout prevents the development of colitis, thus in which peripheral inflammation upregulates µ-opioid indicating a further link between N/OFQ and the immune receptors on neurons at the site of inflammation, and system170. infiltration of white cells releases opioids to produce a degree of peripheral analgesia60. NOP and N/OFQ are Airway. The cough reflex results from an afferent impulse expressed on crude cultures of peripheral blood mono- (originating in the airway) via the nodose ganglion to nuclear cells163, monocytes165 and neutrophils166. It is the nucleus of the solitary tract in the medulla oblon- tempting to add an N/OFQ–NOP component to the gata171. N/OFQ inhibits cough in guinea-pigs172 and neuroimmune axis, where, in addition to inhibition of in cats173, and inhibits ex vivo airway contractility in peripheral neuronal activity, released N/OFQ may feed- various species including humans174. As N/OFQ does back control lymphocyte function. Indeed, increased not cross the blood–brain barrier, the in vivo effects are monocyte chemotaxis165 and T‑cell proliferation have peripheral. NOP is located on capsaicin-sensitive affer- been reported in response to N/OFQ treatment167. ents and causes an inhibition of tachykinin release174. It remains to be determined whether peripheral The antitussive effects can be mimicked by the non- inflammation upregulates neuronal NOP in a similar peptide agonist Ro64‑6198 in a J‑113397-sensitive man- manner to µ-opioid receptors. However, recent studies ner175. Codeine is the current gold-standard antitussive have shown upregulation of N/OFQ in the rat in response agent but has a poor side-effect profile that is typical of to peripheral inflammation with bacterial lipopolysac- µ-opioid receptor agonists (such as nausea, constipa- charide168, and a modulation of immune function in tion, tolerance and dependence). So, orally active NOP response to staphylococcal enterotoxin A administration agonists represent a viable alternative for the treatment in mice169. It is possible that the elevated N/OFQ levels of cough.

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Gastrointestinal tract. N/OFQ inhibits contractility of the N/OFQ is antinocieptive47 it is tempting to suggest that gastrointestinal tract in a wide range of species and at this route be utilized in humans. most sites along the gastrointestinal tract176. In addition There has always been a desire to create highly selec- to the well-characterized inhibition of gastric motility, tive drugs. Indeed, in the opioid field there is the com- N/OFQ prevents gastric damage that is induced by monly held clinical view that there is a need for selective intragastric ethanol177 and cold-restraint stress178, and morphine-like molecules that have reduced side-effect a role for NOP in inflammatory bowel disease has been profiles, often achieved by modification of the pharma- described above170. There is evidence for central and cokinetic behaviour. A good example of this approach is peripheral components to the regulation of gastrointes- the highly successful remifentanil, which, like morphine, tinal function179,180. Indeed, an elegant study addressed targets µ-opioid receptors, but is metabolized by plasma this using the peptides UFP‑112 and N/OFQ adminis- esterases to inactive metabolites. The effects of this are tered intracerebroventrically and intraperitoneally on a rapid off-set of action186. However, there is now grow- gastrointestinal function in the rat. When administered ing interest in the design and evaluation of non-selective intracerebroventrically both peptides (UFP‑112 is more molecules. One of the most compelling pieces of infor- potent) inhibited gastric emptying, inhibited gastric mation from the opioid field in favour of this approach acid secretion and reduced ethanol-induced intragastric is the observation that in mice lacking δ-opioid recep- lesions. However, when the peptides were administered tors or wild-type mice treated with δ-opioid receptors intraperitoneally, reduced gastric emptying was absent antagonists, tolerance to morphine does not develop187,188. and the inhibitory action on acid secretion was con- Clearly a mixed µ-opioid receptor agonist/δ-opioid recep- verted to enhanced secretion179. The effects of N/OFQ tor antagonist would have tremendous clinical potential. in simple contractility bioassays can be mimicked by Such molecules already exist for experimental use, for (Ref. 181) 189 the non-peptide agonist Ro64‑6198 and can example, H‑Dmt-Tic-Gly-NH-CH2-Ph (UFP‑505) be reversed by the non-peptide antagonist SB‑612111 and [Dmt1,D‑1-Nal4]endomorphin‑1 (Ref.190). From (Ref. 182). the description above there would be some advantage in ligands with mixed activity at NOP and classical opioid Concluding thoughts receptors. If we consider the observation that NOP–/– It is clear that N/OFQ exhibits a vast range of biological animals display reduced morphine tolerance66,67 then a activities and as such has potential for clinical develop- mixed NOP antagonist and µ-opioid receptor agonist has ment. The challenge will be to be selective for a particular potential in terms of reduced tolerance and lower dosage disease although not necessarily selective for NOP. The (as NOP antagonists per se might be antinociceptive). Do effects in some disease indications can be described as such molecules exist? In a recent study the interaction of central, peripheral or mixed. For example, a centrally a range of classical opioids and five non-peptides of the active NOP antagonist might be antidepressant with sequence from SRI International was characterized191. analgesic properties (some antidepressants are already One particular molecule, SR‑14148, behaves as a NOP widely used for treating chronic pain183). The downside antagonist and as a µ-opioid receptor partial agonist. of administering an antagonist that is able to access the Although the authors extensively discuss the relative CNS in humans is that it would also produce elevated merits of different assays this work is promising for the circulating NOP antagonist levels, which might be future design of higher-affinity NOP antagonists with problematic in patients with heart failure, hyperreactive improved µ-opioid receptor efficacy. As an interesting airways or urinary incontinence. Of course this would addition to the general concept of mixed molecules only be a concern if N/OFQ levels are elevated and, at the same group described SR-16435, which behaved as present, there is little evidence for this. a mixed µ-opioid receptor/NOP partial agonist. This Preventing passage across the blood–brain barrier, molecule produced µ-opioid receptor-mediated antino- which would lead to peripheral targeting, may be easier ciception with reduced development of tolerance192. to achieve and this has been done with molecules such as It has now been 13 years since the truly seminal work methyl naltrexone for the treatment of opioid-induced of Meunier and colleagues1 and Reinscheid colleagues2. constipation184. A peripheral NOP agonist might prove The pharmacological and physiological groundwork useful in heart failure or for the treatment of urinary has been done with a wide range of characterized mol- incontinence. Opioids have been delivered to the spinal ecules and pharmaceutical leads available. It is now time cord for pain relief for decades and are often combined to begin the handover to our clinical colleagues to see with local anaesthetics for use in labour185. As intrathecal these evaluated and put to good use.

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Cl)YRWR-NH2] (Syn 1020) in rodents. Eur. J. 692–704 (2006). Acologix: www.acologix.com/pipeline_trk-820.html Pharmacol. 560, 29–35 (2007). 225. Rizzi, A. et al. Pharmacological characterization Brane Discovery: 211. Economidou, D. et al. Effect of novel NOP receptor of the nociceptin/orphanin FQ receptor antagonist http://www.branediscovery.com/pipeline.htm ligands on food intake in rats. Peptides 27, 775–783 SB‑612111 [(–)‑cis‑1‑methyl‑7-[[4‑(2,6- IUPHAR Receptor Database: www.iuphar-db.org/GPCR/Ch (2006). dichlorophenyl)piperidin‑1‑yl]methyl]‑6,7,8,9- apterMenuForward?chapterID=1295 212. Seki, T. et al. Pharmacological properties of TRK‑820 tetrahydro‑5H‑benzocyclohepten‑5‑ol]: in vivo Neighbourhood.gov.uk: on cloned µ‑, δ- and κ-opioid receptors and nociceptin studies. J. Pharmacol. Exp. Ther. 321, 968–974 www.neighbourhood.gov.uk/page.asp?id=684 receptor. Eur. J. Pharmacol. 376, 159–167 (1999). (2007). Samyang: www.samyangpharm.com/rnd/bupre.asp 213. Mizoguchi, H. et al. Blockade of µ-opioid receptor- 226. Ho, G. D. et al. Synthesis and structure–activity The British Heart Foundation: mediated G‑protein activation and antinociception by relationships of 4‑hydroxy‑4-phenylpiperidines as www.heartstats.org/homepage.asp TRK‑820 in mice. Eur. J. Pharmacol. 461, 35–39 nociceptin receptor ligands: part 1. Bioorg. Med. Zealand Pharma: www.zp.dk/Product-Pipeline/ZP120 (2003). Chem. Lett. 17, 3023–3027 (2007). 214. Endoh, T. et al. TRK‑820, a selective κ-opioid 227. Ho, G. D. et al. Synthesis and structure–activity SUPPLEMENTARY INFORMATION agonist, produces potent antinociception in relationships of 4‑hydroxy‑4-phenylpiperidines as See online article: S1 (table) cynomolgus monkeys. Jpn. J. Pharmacol. 85, nociceptin receptor ligands: part 2. Bioorg. Med. All links are active in the online pdf 282–290 (2001). Chem. Lett. 17, 3028–3033 (2007).