In vitro characterization of J-113397, a non-peptide nociceptin/orphanin FQ receptor antagonist

The lack of availability of a selective, highly potent, competitive antagonist for the nociceptin receptor (OP4) devoid of residual agonistic activity has hampered studies in this area. We report here the in vitro pharmacological properties of the novel non-peptide OP4 antagonist, J-113397, which was recently discovered by Banyu Pharmaceutical investigators. The compound was synthesized as a racemic mixture in our laboratories. J-113397 was shown to antagonize (pA2 7.52) the nociceptin-induced inhibition of cAMP formation in cells expressing the recombinant human OP4 receptor (CHOhOP4) and to displace [125I]Tyr14nociceptin from CHOhOP4 membranes with a pKi of 8.56. It also competitively antagonized the contractile actions of nociceptin in the mouse colon (pA2 8.07) and the inhibitory effect of nociceptin in electrically stimulated preparations such as the mouse vas deferens (pA2 7.85), the guinea pig ileum (7.75), and the rat vas deferens (7.77). At high concentrations (10 microM), the compound was devoid of agonist activity in the mouse vas deferens and CHOhOP4, while it contracted the mouse colon and increased the twitch response of the rat vas deferens, and produced a naloxone-sensitive inhibition of the electrically evoked twitches in the guinea pig ileum. pA2 values for the new antagonist against deltorphin I in the mouse vas deferens (OP1 receptors), or against dermorphin in the guinea pig ileum (OP3 receptors), etorphine in the rat vas deferens (OP receptors), U69593 in the rabbit vas deferens (OP2 receptors) and endomorphin 1 in the mouse colon (OP3 receptors) were lower than 6. Taken together, these data indicate that J-113397 is a high-affinity, selective and competitive antagonist of the OP4 receptor; this novel pharmacological tool will be of great value in studies directed at evaluating the physiological roles of the nociceptin/OP4 system.     

High-affinity, non-peptide agonists for the ORL1 (orphanin FQ/nociceptin) receptor

The discovery of 8-(5,8-dichloro-1,2,3,4-tetrahydro-naphthalen-2-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 1a, as a high-affinity ligand for the human ORL1 (orphanin FQ/nociceptin) receptor led to the synthesis of a series of optimized ligands. These compounds exhibit high affinity for the human ORL1 receptor, exhibit moderate to good selectivity versus opioid receptors, and behave as full agonists in biochemical assays. In this paper we present the synthesis, structure-activity relationship (SAR), and biochemical characterization of substituted 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-ones culminating in the discovery of 8-(5-methyl-1,2,3,4-tetrahydro-naphthalen-1-yl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one, 1p, and 8-acenaphten-1-yl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one 1q, two high-affinity, potent ORL1 receptor agonists with good to moderate selectivity versus the other opioid receptors.     

In vitro and in vivo pharmacological characterization of J-113397, a potent and selective non-peptidyl ORL1 receptor antagonist

1-[(3R,4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl -1, 3-dihydro-2H-benzimidazol-2-one (J-113397) was found to be the first potent nonpeptidyl ORL1 receptor antagonist (K(i): cloned human ORL1=1.8 nM) with high selectivity over other opioid receptors (K(i): 1000 nM for human mu-opioid receptor, >10,000 nM for human delta-opioid receptor, and 640 nM for human kappa-opioid receptor). In vitro, J-113397 inhibited nociceptin/orphanin FQ-stimulated [35S]guanosine 5'-O-(gamma-thio)triphosphate (GTP gamma S) binding to Chinese Hamster Ovary (CHO) cells expressing ORL1 (CHO-ORL1) with an IC(50) value of 5.3 nM but had no effect on [35S]GTP gamma S binding by itself. Schild plot analysis of the [35S]GTP gamma S binding assay and cAMP assay using CHO-ORL1 indicated competitive antagonism of J-113397 on the ORL1 receptor. In CHO cells expressing mu-, delta- or kappa-opioid receptors, J-113397 had no effects on [35S]GTP gamma S binding up to a concentration of 100 nM, indicating selective antagonism of the compound on the ORL1 receptor. In vivo, J-113397, when administered subcutaneously (s.c.), dose-dependently inhibited hyperalgesia elicited by intracerebroventricular (i.c.v.) administration of nociceptin/orphanin FQ in a tail-flick test with mice. An in vitro binding study using mouse brains indicated that J-113397 possesses high affinity for the mouse ORL1 receptor (K(i): 1.1 nM) as well as the human receptor. In summary, J-113397 is the first potent, selective ORL1 receptor antagonist that may be useful in elucidating the physiological roles of nociceptin/orphanin FQ.     

UFP-101, a peptide antagonist selective for the nociceptin/orphanin FQ receptor

Nociceptin/orphanin FQ modulates various biological functions at central and peripheral levels by selectively activating a G-protein coupled receptor named N/OFQ peptide (NOP) receptor. For extending our knowledge on the biological roles of the N/OFQ-NOP receptor system the identification of selective NOP ligands, especially antagonists, is mandatory. [Nphe1, Arg14, Lys15] N/OFQ-NH2 (UFP-101) is a novel NOP ligand that was designed by combining, in the same molecule, the [Nphe1] chemical modification which eliminates efficacy and the [Arg14, Lys15] substitution which increases ligand potency and duration of action in vivo. In the present article, we summarize the pharmacological features of UFP-101 as determined in a series of in vitro and in vivo assays. Moreover, some biological actions and possible therapeutic indications of NOP ligands are discussed on the basis of results obtained with UFP-101. Data obtained with this compound were compared with those generated using other NOP antagonists, especially J-113397 and [Nphe1]N/OFQ(1-13)-NH2, receptor or peptide knockout mice and other pharmacological tools useful for blocking N/OFQ - NOP receptor signaling. The analysis of the available data demonstrates that UFP-101 is a useful pharmacological tool for the investigation of the central and peripheral biological functions regulated by the N/OFQ-NOP receptor system and for defining the therapeutic potential of NOP receptor ligands.     

N- and C-terminal modifications of nociceptin/orphanin FQ generate highly potent NOP receptor ligands

Previous structure-activity studies on nociceptin/orphanin FQ (N/OFQ) identified [Phe(1)Psi(CH(2)NH)Gly(2)]N/OFQ(1-13)-NH(2) and [Nphe(1)]N/OFQ(1-13)-NH(2) as a N/OFQ peptide receptor (NOP) partial agonist and pure antagonist, respectively. The addition of fluorine to the Phe(4) or the insertion of a further pair of basic amino acids Arg(14)-Lys(15) generate potent agonists. On the basis of these findings, we combined in the N/OFQ-NH(2) template the chemical modifications Arg(14)-Lys(15) and (pF)Phe(4) that increase the agonist potency with those conferring partial agonist (Phe(1)Psi(CH(2)NH)Gly(2)) or pure antagonist (Nphe(1)) properties. Twelve peptides were synthesized and pharmacologically evaluated in Chinese hamster ovary cells expressing the human recombinant NOP and in electrically stimulated mouse vas deferens and guinea pig ileum assays. All peptides behaved as NOP ligands; the chemical modifications Arg(14)-Lys(15) and (pF)Phe(4) increased ligand affinity/potency. Peptides with the normal Phe(1)-Gly(2) peptide bond behaved as full agonists, and those with the Phe(1)Psi(CH(2)NH)Gly(2) modification behaved as partial agonists, while those with the Nphe(1) modification behaved as partial agonists or pure antagonists depending on the presence or absence of the (pF)Phe(4) modification, respectively. The full agonist [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2), the partial agonist [Phe(1)Psi(CH(2)NH)Gly(2),(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2), and the pure antagonist [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) represent the most potent peptide ligands for NOP.     

Antinociceptive effects of the ORL1 receptor agonist nociceptin/orphanin FQ in diabetic mice.

The antinociceptive potency of nociceptin/orphanin FQ, an opioid-like orphan receptor agonist, was examined using the tail-flick test and the formalin-induced nociception test in diabetic mice. Nociceptin/orphanin FQ, at doses of 0.1 to 10 nmol, intrathecal (i.t.), produced a marked and dose-dependent inhibition of the tail-flick response in both non-diabetic and diabetic mice. The antinociceptive effect of nociceptin/orphanin FQ in the tail-flick test in diabetic mice was greater than that in non-diabetic mice. The antinociceptive effect of nociceptin/orphanin FQ was not antagonized by pretreatment with either beta-funaltrexamine, a selective mu-opioid receptor antagonist, naltrindole, a selective delta-opioid receptor antagonist, or nor-binaltorphimine, a selective kappa-opioid receptor antagonist. The antinociceptive effects of nociceptin/orphanin FQ in diabetic, but not in non-diabetic mice, were abolished when mice were pretreated with capsaicin i.t. 24 h before testing. In the formalin test, nociceptin/orphanin FQ also produced a marked and dose-dependent antinociceptive effect on the first-phase response, but not the second phase-response, in both diabetic and non-diabetic mice. Furthermore, nociceptin/orphanin FQ significantly and dose-dependently reduced the flinching responses to i.t.-administered substance P in diabetic mice, but not in non-diabetic mice. The results of the present experiments clearly indicate that the antinociceptive potency of nociceptin/orphanin FQ is significantly greater in diabetic mice than in non-diabetic mice. Furthermore, the results of this study suggest that the reduction of substance P-mediated nociceptive transmission in the spinal cord may be responsible for the antinociceptive effect of nociceptin/orphanin FQ.     

Nphe1,Arg14,Lys15]nociceptin-NH2, a novel potent and selective antagonist of the nociceptin/orphanin FQ receptor

1. Nociceptin/orphanin FQ (N/OFQ) modulates several biological functions by activating a specific G-protein coupled receptor (NOP). Few molecules are available that selectively activate or block the NOP receptor. Here we describe the in vitro and in vivo pharmacological profile of a novel NOP receptor ligand, [Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-101). 2. UFP-101 binds to the human recombinant NOP receptor expressed in Chinese hamster ovary (CHO) cells with high affinity (pK(i) 10.2) and shows more than 3000 fold selectivity over classical opioid receptors. UFP-101 competitively antagonizes the effects of N/OFQ on GTPgamma(35)S binding in CHO(hNOP) cell membranes (pA(2) 9.1) and on cyclic AMP accumulation in CHO(hNOP) cells (pA(2) 7.1), being per se inactive at concentrations up to 10 microM. 3. In isolated peripheral tissues of mice, rats and guinea-pigs, and in rat cerebral cortex synaptosomes preloaded with [(3)H]-5-HT, UFP-101 competitively antagonized the effects of N/OFQ with pA(2) values in the range of 7.3 - 7.7. In the same preparations, the peptide was inactive alone and did not modify the effects of classical opioid receptor agonists. 4. UFP-101 is also active in vivo where it prevented the depressant action on locomotor activity and the pronociceptive effect induced by 1 nmol N/OFQ i.c.v. in the mouse. In the tail withdrawal assay, UFP-101 at 10 nmol produces per se a robust and long lasting antinociceptive effect. 5. UFP-101 is a novel, potent and selective NOP receptor antagonist which appears to be a useful tool for future investigations of the N/OFQ-NOP receptor system.     

Structure-activity studies of the Phe(4) residue of nociceptin(1-13)-NH(2): identification of highly potent agonists of the nociceptin/orphanin FQ receptor.

A total of 32 compounds was prepared to investigate the functional role of Phe(4) in NC(1-13)-NH(2), the minimal sequence maintaining the same activity as the natural peptide nociceptin. These compounds could be divided into three series in which Phe(4) was replaced with residues that would (i) alter aromaticity or side chain length, (ii) introduce steric constraint, and (iii) modify the phenyl ring. Compounds were tested for biological activity as (a) inhibitors of the electrically stimulated contraction of the mouse vas deferens; (b) competitors of the binding of [(3)H]-NC-NH(2) to mouse brain membranes; and (c) inhibitors of forskolin-stimulated cAMP accumulation in CHO cells expressing the recombinant human OP(4) receptor. Results indicate that all compounds of the first and second series were inactive or very weak with the exception of [N(CH(3))Phe(4)]NC(1-13)-NH(2), which was only 3-fold less potent than NC(1-13)-NH(2). Compounds of the third series showed higher, equal, or lower potencies than NC(1-13)-NH(2). In particular, [(pF)Phe(4)]NC(1-13)-NH(2) (pF) and [(pNO(2))Phe(4)]NC(1-13)-NH(2) (pNO(2)) were more active than NC(1-13)-NH(2) by a factor of 5. In the mVD, these compounds showed the following order of potency: (pF) = (pNO(2)) > or = (pCN) > (pCl) > (pBr) > (pI) = (pCF(3)) = (pOCH(3)) > (pCH(3)) > (pNH(2)) = (pOH). (oF) and especially (mF) maintained high potencies but were less active than (pF). Similar orders of potency were observed in binding competition and cAMP accumulation studies. There was a strong (r(2) > or = 0.66) correlation between data observed in these assays. Biological activity data of compounds of the third series were plotted against some Hansch parameters that are currently used to quantify physicochemical features of the substituents. In the three biological assays agonist potency/affinity positively correlates with the electron withdrawal properties of the groups in the p-position of Phe(4) and inversely with their size.     

The role of the ORL1 receptor in the modulation of spinal neurotransmission by nociceptin/orphanin FQ and nocistatin

Nociceptin/orphanin FQ and nocistatin are two neuropeptides with opposing effects on spinal neurotransmission and nociception. Nociceptin/orphanin FQ selectively suppresses excitatory glutamatergic neurotransmission, while nocistatin selectively interferes with glycinergic and gamma-aminobutyric acid (GABA)-ergic transmission. Here, we performed whole-cell patch-clamp recordings from superficial rat spinal cord dorsal horn neurons to investigate the role of the opioid receptor-like (ORL)1 receptor for modulatory actions of these peptides. The partial ORL1 receptor antagonist [phe1psi(CH(2)-NH)Gly(2)]nociceptin-(1-13)NH(2) competitively reversed the effects of nociceptin/orphanin FQ on excitatory neurotransmission (estimated pA(2) 6.43), but left the suppression of inhibitory synaptic transmission by nocistatin unaffected. These results indicate that the inhibitory action of nociceptin/orphanin FQ on glutamatergic transmission is mediated via ORL1 receptors, while nocistatin acts via a different so far unidentified receptor.     

1,4-Dioxolane-triazaspirodecanone derivatives as nociceptin/orphanin FQ receptor ligands

A series of N-substituted analogs based upon the spiropiperidine core of the lead compound Spiroxatrine was synthesized. In particular, the new compounds were obtained by replacing the benzodioxane moiety of the Spiroxatrine with several 2-substituted 1,3-dioxolanes. Thus the designed derivatives were synthesized and evaluated as possible NOP receptor ligands. As a conclusion of these studies, the new triazaspirodecanone derivatives showed unique and significant SAR as NOP receptor agonists. In particular, the present study demonstrated that 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one portion together with appropriate 1,3-dioxolane substituents could lead to a new promising class of NOP receptor ligands.     

Antidepressant- and anxiolytic-like effects of nociceptin/orphanin FQ receptor ligands

Many studies point toward the nociceptin/orphanin FQ (N/OFQ) and the N/OFQ peptide receptor (NOP) as targets for the development of innovative drugs for treating affective disorders. It has been reported that the activation of NOP receptors produces anxiolytic-like effects in rodents in a large series of behavioral assays, i.e., elevated plus maze, light-dark aversion, operant conflict, fear-potentiated startle, pup ultrasonic vocalizations, and hole board tests. In contrast, the blockade of N/OFQ signaling obtained with NOP-selective antagonists promotes antidepressant-like effects in the forced swimming and tail suspension tests. In these assays, N/OFQ is inactive per se, but reverses the antidepressant-like effects of NOP antagonists. NOP receptor knockout mice show an antidepressant-like phenotype, and NOP antagonists are inactive in these animals. Thus, the activation of the NOP receptor seems to evoke anxiolytic-like effects while its blockade antidepressant-like effects. This appears to be a rather unique behavioral profile since the activation or the blockade of a given neuropeptide receptor produces, in most of the cases, both antidepressant- and anxiolytic-like effects. This particular behavioral profile, the possible mechanisms of action, and the therapeutic potential of NOP receptor ligands for the treatment of depression and anxiety disorders are discussed in this review article.     

Enhanced nicotine sensitivity in nociceptin/orphanin FQ receptor knockout mice

The opioid peptide nociceptin (orphanin FQ) has been implicated in reward, reinforcement and addiction. The current study sought evidence of a role of endogenous nociceptin in nicotine responses by studying nociceptin receptor (NOP) knockout mice. The results were: (1) NOP receptor knockout mice showed enhanced anxiety-like behavior on an elevated plus maze. Whereas nicotine (0.05-0.5 mg/kg) tended to be anxiogenic in wild-type mice, NOP receptor KO mice were resistant to this effect, though interpretation was confounded by their stronger anxiety-like behavior. (2) When presented increasing nicotine concentrations (3-50 μg/ml) in a bottle choice drinking paradigm, there were no genotype-dependent differences in nicotine preference. However, NOP receptor knockout mice consumed more 3 μg/ml nicotine solution when considered in absolute terms. (3) NOP receptor knockout mice showed stronger hypothermic responses to nicotine (1 or 2 mg/kg) administration. (4) There was modest evidence that NOP receptor KO mice showed attenuated behavioral sensitization to a low dose of nicotine (0.05 mg/kg) during repeated daily treatment. (5) NOP receptor knockout mice more rapidly tolerated the sedative effect of nicotine (1 mg/kg), due partially to slightly lower locomotion on first treatment. (6) NOP receptor knockout mice, unlike wild-type mice, showed a significant mecamylamine (2.5 mg/kg) induced conditioned place aversion to nicotine (24 mg/kg/day) withdrawal. These results show that mice lacking the influence of endogenous N/OFQ mice are hypersensitive to nicotine in most measures, showing a role of endogenous nociceptin in modulating or mediating the acute effects of nicotine, and possibly nicotine addiction.     

Novel hexahydrospiro[piperidine-4,1'-pyrrolo[3,4-c]pyrroles]: highly selective small-molecule nociceptin/orphanin FQ receptor agonists

Novel hexahydrospiro[piperidine-4,1'-pyrrolo[3,4-c]pyrroles that act as potent and selective orphanin FQ/nociceptin (N/OFQ) receptor (NOP) agonists were identified. The best compound, (+)-5a, potently inhibited 3H-N/OFQ binding to the NOP receptor (K(i) = 0.49 nM) but was >1000-fold less potent in binding to MOP, KOP, and DOP opiate receptors. Further, (+)-5a potently stimulated GTP gamma S binding to NOP membranes (EC50 = 65 nM) and inhibited forskolin-mediated cAMP accumulation in NOP-expressing cells (EC50 = 9.1 nM) with a potency comparable to that of the natural peptide agonist N/OFQ. These results indicate that (+)-5a is a highly selective and potent small-molecule full agonist of the NOP receptor.     

Antidepressant-like effects of the nociceptin/orphanin FQ receptor antagonist UFP-101: new evidence from rats and mice

Receptor antagonist and knockout studies have demonstrated that blockade of signalling via nociceptin/orphanin FQ (N/OFQ) and its receptor (NOP) has antidepressant-like effects in mice submitted to the forced swimming test (FST). The aim of the present study was to explore further the antidepressant-like properties of the NOP antagonist UFP-101 in different species (mouse and rat) and using different assays [FST and tail suspension test (TST)], and to investigate the mechanism(s) involved in its actions.UFP-101 (10 nmol i.c.v.) reduced immobility time of Swiss mice in the TST (mean±SEM) from 179±11 to 111±10 s. N/OFQ (1 nmol i.c.v.) was without effect per se, but fully prevented the effect of UFP-101. The spontaneous immobility time of NOP^–/– CD1-C57BL/6J-129 mice in the TST was much lower than that of wild-type (NOP^+/+) littermates (75±11 vs. 144±17 s) or of Swiss mice. UFP-101 (10 nmol i.c.v.) decreased immobility time (–65%) and increased climbing time (71%) in rats submitted to the FST. In rat brain slices, N/OFQ (100 nM) triggered robust K⁺-dependent hyperpolarizing currents in locus coeruleus and dorsal raphe neurons. UFP-101 (3 µM) fully prevented N/OFQ-induced currents, but was inactive per se. Fluoxetine, desipramine (both 30 mg/kg i.p.) and UFP-101 (10 nmol i.c.v.) reduced immobility time of mice in the FST. The serotonin synthesis inhibitor p-chlorophenylalanine methylester (PCPA, 4×100 mg/kg per day i.p.) prevented the antidepressant-like effects of fluoxetine and UFP-101 (but not desipramine), whereas N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, neurotoxic for noradrenergic neurons; 50 mg/kg i.p., 7 days beforehand), suppressed only the effect of desipramine. Neither pretreatment affected spontaneous immobility time per se.Thus, UFP-101 exhibits pronounced antidepressant-like effects in different species and animal models, possibly by preventing the inhibitory effects of endogenous N/OFQ on brain monoaminergic (in particular serotonergic) neurotransmission. Participation of the N/OFQ-NOP receptor system in mood modulation sets new potential targets for antidepressant drug development.     

Effects of chronic nociceptin/orphanin FQ exposure on cAMP accumulation and receptor density in Chinese hamster ovary cells expressing human nociceptin/orphanin FQ receptors

Since the therapeutic efficacy of Li+ in the treatment of mood disorder is observed only after chronic administration, we examined whether long-term Li+ treatment with a therapeutic concentration affected the elevation of intracellular-free Ca2+ concentration ([Ca2+]i) induced by carbachol, a muscarinic receptor agonist, in 1321N1 human astrocytoma cells. Carbachol caused [Ca2+]i elevation through phosphoinositide hydrolysis in a concentration-dependent manner. Treatment of the cells with phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, for 2 min resulted in a reduction of the carbachol-induced [Ca2+]i elevation. However, PMA did not reduce the carbachol-induced [Ca2+]i elevation in cells treated with PMA for 48 h, reflecting the down-regulation of protein kinase C. Although Li+ at a therapeutic concentration (1 mM) did not affect the carbachol-induced [Ca2+]i elevation in normal cells, it potently inhibited the [Ca2+]i elevation in protein kinase C down-regulated cells. This inhibitory action of Li+ was observed in a concentration- and time-dependent manner. When protein kinase C activity was directly determined, Li+ treatment did not restore protein kinase C activity in protein kinase C down-regulated cells. [3H]Quinuclidinyl benzylate, a muscarinic receptor ligand, bound to membranes derived from normal and protein kinase C down-regulated cells with a similar Kd and Bmax, and Li+ did not affect these parameters of [3H]quinuclidinyl benzylate binding. These results indicated that Li+ at a therapeutic concentration reduced the muscarinic receptor-mediated increased in [Ca2+]i under the protein kinase C-deficient condition without affecting muscarinic receptor or protein kinase C activity.     

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

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.     

The nociceptin/orphanin FQ receptor (NOP) as a target for drug abuse medications

Several studies show that the nociceptin receptor NOP plays a role in the regulation of reward and motivation pathways related to substance abuse. Administration of the NOP's natural peptide ligand, Nociceptin/Orphanin FQ (N/OFQ) or synthetic agonist Ro 64-6198 has been shown to block rewarding effects of cocaine, morphine, amphetamines and alcohol, in various behavioral models of drug reward and reinforcement, such as conditioned place preference and drug self-administration. Administration of N/OFQ has been shown to reduce drug-stimulated levels of dopamine in mesolimbic pathways. The NOP-N/OFQ system has been particularly well examined in the development of alcohol abuse in animal models. Furthermore, the efficacy of the mixed-action opioid buprenorphine, in attenuating alcohol consumption in human addicts and in alcohol-preferring animal models, at higher doses, has been attributed to its partial agonist activity at the NOP receptor. These studies suggest that NOP receptor agonists may have potential as drug abuse medications. However, the pathophysiology of addiction is complex and drug addiction pharmacotherapy needs to address the various phases of substance addiction (craving, withdrawal, relapse). Further studies are needed to clearly establish how NOP agonists may attenuate the drug addiction process and provide therapeutic benefit. Addiction to multiple abused drugs (polydrug addiction) is now commonplace and presents a treatment challenge, given the limited pharmacotherapies currently approved. Polydrug addiction may not be adequately treated by a single agent with a single mechanism of action. As with the case of buprenorphine, a mixed-action profile of NOP/opioid activity may provide a more effective drug to treat addiction to various abused substances and/or polydrug addiction.     

High affinity conformationally constrained nociceptin/orphanin FQ(1-13) amide analogues

A series of cyclic analogues with a lactam linkage were prepared by solid phase peptide synthesis to explore possible biologically active conformation(s) of nociceptin/orphanin FQ (N/OFQ). cyclo[D-Asp(7),Lys(10)]- and cyclo[Asp (6),Lys(10)]N/OFQ(1-13)NH2 exhibit high affinity (Ki = 0.27 and 0.34 nM, respectively) and high potency in the GTPgammaS assay (EC 50 = 1.6 and 4.1 nM, respectively) at human nociceptin/orphanin FQ peptide (NOP) receptors. These analogues exhibit 2- to 3-fold higher affinity and 2- to 5-fold higher potency than the parent peptide.     

Modulation of synaptic transmission by nociceptin/orphanin FQ and nocistatin in the spinal cord dorsal horn of mutant mice lacking the nociceptin/orphanin FQ receptor

Nociceptin/orphanin FQ (N/OFQ) and nocistatin (NST) are two neuropeptides derived from the same precursor protein that exhibit opposing effects on spinal neurotransmission and nociception. Here, we have used whole-cell, patch-clamp recordings from visually identified neurons in spinal cord dorsal horn slices of genetically modified mice to investigate the role of the N/OFQ receptor (N/OFQ-R) in the modulatory action of both peptides on excitatory glutamatergic and inhibitory glycinergic and gamma-aminobutyric acid (GABA)-ergic synaptic transmission. In wild-type mice, N/OFQ selectively suppressed excitatory transmission in a concentration-dependent manner but left inhibitory synaptic transmission unaffected. In contrast, NST reduced only inhibitory but not alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor-mediated excitatory synaptic transmission. N/OFQ-mediated inhibition of excitatory transmission was completely absent in N/OFQ-R receptor-deficient (N/OFQ-R(-/-)) mice and significantly reduced in heterozygous (N/OFQ-R(+/-)) mice, whereas the action of NST on inhibitory neurotransmission was completely retained. To test for the relevance of these results for spinal nociception, we investigated the effects of intrathecally injected N/OFQ in the mouse formalin test, an animal model of tonic pain. N/OFQ (3 nmol/mouse) induced significant antinociception in wild-type mice, but had no antinociceptive effects in N/OFQ-R(-/-) mice. These results indicate that the inhibitory action of N/OFQ on excitatory glutamatergic synaptic transmission and its spinal antinociceptive action are mediated via the N/OFQ receptor, whereas the action of NST is independent of this receptor.