In vitro and in vivo pharmacological characterization of the neuropeptide s receptor antagonist [D-Cys(tBu)5]neuropeptide S.

Neuropeptide S (NPS) was identified as the endogenous ligand of an orphan receptor now referred to as the NPS receptor (NPSR). In the frame of a structure-activity study performed on NPS Gly5, the NPSR ligand [D-Cys(tBu)(5)]NPS was identified. [D-Cys(tBu)(5)]NPS up to 100 microM did not stimulate calcium mobilization in human embryonic kidney (HEK) 293 cells stably expressing the mouse NPSR; however, in a concentration-dependent manner, the peptide inhibited the stimulatory effects elicited by 10 and 100 nM NPS (pK(B), 6.62). In Schild analysis experiments [D-Cys(tBu)(5)]NPS (0.1-100 microM) produced a concentration-dependent and parallel rightward shift of the concentration-response curve to NPS, showing a pA(2) value of 6.44. Ten micromolar [D-Cys(tBu)(5)]NPS did not affect signaling at seven NPSR unrelated G-protein-coupled receptors. In the mouse righting reflex (RR) recovery test, NPS given at 0.1 nmol i.c.v. reduced the percentage of animals losing the RR in response to 15 mg/kg diazepam and their sleeping time. [d-Cys(tBu)(5)]NPS (1-10 nmol) was inactive per se but dose-dependently antagonized the arousal-promoting action of NPS. Finally, NPSR-deficient mice were similarly sensitive to the hypnotic effects of diazepam as their wild-type littermates. However, the arousal-promoting action of 1 nmol NPS could be detected in wild-type but not in mutant mice. In conclusion, [D-Cys(tBu)(5)]NPS behaves both in vitro and in vivo as a pure and selective NPSR antagonist but with moderate potency. Moreover, using this tool together with receptor knockout mice studies, we demonstrated that the arousal-promoting action of NPS is because of the selective activation of the NPSR protein.

Further studies on the pharmacological features of the nociceptin/orphanin FQ receptor ligand ZP120.

ZP120 is a nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) ligand. In previous studies, the effects of ZP120 were found to be sensitive to J-113397 in mouse tissues while resistant to UFP-101 in rat tissues. The aim of this study was to further investigate the ZP120 pharmacological profile using mouse and rat preparations, J-113397 and UFP-101, as well as NOP receptor knockout (NOP(-/-)) mice. Electrically stimulated mouse and rat vas deferens were used to characterize the pharmacology of ZP120 in vitro. For in vivo studies the tail-withdrawal assay was performed in wild type (NOP(+/+)) and NOP knockout (NOP(-/-)) mice. In the mouse and rat vas deferens ZP120 mimicked the effects of N/OFQ showing higher potency but lower maximal effects. In both preparations, J-113397 antagonized N/OFQ and ZP120 effects showing similar pK(B) values ( approximately 7.8). UFP-101 antagonized the actions of N/OFQ (pK(B) values approximately 7.3) but did not modify the effects of ZP120. The inhibitory effects of N/OFQ and ZP120 were no longer evident in vas deferens tissues taken from NOP(-/-) mice. In NOP(+/+) mice subjected to the tail-withdrawal assay, ZP120 (1 nmol) mimicked the pronociceptive action of N/OFQ (10 nmol), producing longer lasting effects. The effects of both peptides were absent in NOP(-/-) animals. The NOP receptor ligand ZP120 is a high potency NOP selective partial agonist able to evoke long-lasting effects; its diverse antagonist sensitivity in comparison with N/OFQ may derive from different modality of binding to the NOP receptor.

Altered anxiety-related behavior in nociceptin/orphanin FQ receptor gene knockout mice.

Studies showed that nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) agonists produce anxiolytic-like actions, while little is known about the effects of blockade of NOP receptor signaling in anxiety. To this aim, we investigated the behavioral phenotype of NOP receptor gene knockout mice (NOP(-/-)) in different assays. In the elevated plus-maze and light-dark box, NOP(-/-) mice displayed increased anxiety-related behavior. In the novelty-suppressed feeding behavior and elevated T-maze, NOP(-/-) mice showed anxiolytic-like phenotype, while no differences were found in the open-field, hole-board, marble-burying, and stress-induced hyperthermia. Altogether, these findings suggest that the N/OFQ-NOP receptor system modulates anxiety-related behavior in a complex manner.

In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor.

[(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC(50) 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP(-/-) mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6- and 3.5-fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1-100pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t.; in both cases, UFP-112 was approximately 100-fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP(-/-) mice. Equi-effective high doses of UFP-112 (0.1nmol) and N/OFQ (10nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16h. N/OFQ produced a clear inhibitory effect which lasted for 60min while UFP-112 elicited longer lasting effects (>6h). In conscious rats, UFP-112 (0.1 and 10nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors.

Ascorbic and 6-Br-ascorbic acid conjugates as a tool to increase the therapeutic effects of potentially central active drugs.

Ascorbic acid (AA) or 6-Br-ascorbate (BrAA) conjugation has been investigated as a tool to improve brain drug delivery by the Vitamin C transporter SVCT2. To this aim, the effects of AA- or BrAA-conjugation on drug affinity and uptake have been assessed in vitro, by using human retinal pigment epithelium (HRPE) cells, and compared in vivo on mice. Nipecotic, kynurenic and diclofenamic acids were chosen as model drugs. Kinetic and inhibition experiments referred to [(14)C]AA uptake into HRPE cells showed that nipecotic and kynurenic acids became able to interact with SVCT2, as competitive inhibitors, only when conjugated to AA or BrAA. Surprisingly, diclofenamic acid itself appeared able to interact with SVCT2, with an affinity that was significantly increased or decreased by AA or BrAA conjugation, respectively. HPLC analysis, performed on HRPE cells, confirmed the SVCT2 mediated transport for the BrAA-conjugate of nipecotic acid, whereas kynurenic acids conjugates although interacting with the transporter did not enter the cells. In accordance, only the nipecotic acid conjugates showed anticonvulsant activity after systemic injection in mice.

Pharmacological characterization of the novel nociceptin/orphanin FQ receptor ligand, ZP120: in vitro and in vivo studies in mice.

1 This study reports on the pharmacological characterization of ZP120, a novel ligand of the nociceptin/orphanin FQ (N/OFQ) peptide receptor, NOP. ZP120 is a structure inducing probes modified NOP ligand: Zealand Pharma proprietary SIP technology was used to increase the enzymatic stability and half-life of peptide. 2 In vitro, ZP120 mimicked the inhibitory effects of N/OFQ in the electrically stimulated mouse vas deferens, showing however higher potency (pEC(50) 8.88 vs 7.74), lower maximal effects (E(max) 69+/-5% vs 91+/-2%), and slower onset of action. Like N/OFQ, the effects of ZP120 were not modified by 1 micro M naloxone, but they were antagonized by the NOP receptor selective antagonist J-113397 (pA(2) 7.80 vs ZP120, 7.81 vs N/OFQ). 3 In vivo, ZP120 mimicked the effects of N/OFQ, producing pronociceptive effects in the tail withdrawal assay and decreased locomotor activity after i.c.v., but not after i.v. administration in mice. ZP120 elicited similar maximal effects as N/OFQ, but it was about 10 fold more potent and its effects lasted longer. 4 In conclusion, the novel NOP receptor ligand ZP120 is a highly potent and selective partial agonist of the NOP receptor with prolonged effects in vivo.

[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.

Pharmacological characterisation of [(pX)Phe4]nociceptin(1-13)NH2 analogues. 2. In vivo studies.

As part of a structure-activity study focused on the Phe(4) residue of nociceptin (NC) (1-13)NH(2), we identified two highly potent and selective agonists for the OP(4) receptor, [(pF)Phe(4)]NC(1-13)NH(2) and [(pNO(2))Phe(4)]NC(1-13)NH(2), whose in vitro pharmacological profiles have been described in the companion paper. In the present study, we investigated the actions of [(pF)Phe(4)]NC(1-13)NH(2) and compared it with those of NC(1-13)NH(2) in a battery of vivo assays. In the locomotor activity test in mice, 1 nmol NC(1-13)NH(2) given intracerebroventricularly (i.c.v.) caused a significant decrease (about 70% inhibition) in activity for the first 15 min following injection; [(pF)Phe(4)]NC(1-13)NH(2), at the same dose, exerted a similar inhibitory effect that continued until the end of the observation period (30 min). This effect was prevented by the selective OP(4) receptor antagonist [Nphe(1)]NC(1-13)NH(2) (10 nmol, i.c.v.). In the tail-withdrawal assay in mice, [(pF)Phe(4)]NC(1-13)NH(2) mimicked the effects of NC(1-13)NH(2) producing pronociceptive and antimorphine effects following i.c.v. administration. In both experimental paradigms, the actions of [(pF)Phe(4)]NC(1-13)NH(2) were longer lasting (>60 min) compared to those of NC(1-13)NH(2) (ca. 30 min). In unanaesthetised normotensive mice, bolus intravenous (i.v.) injection of 100 nmol/kg of [(pF)Phe(4)]NC(1-13)NH(2) decreased mean blood pressure and heart rate; these effects were longer lasting than those elicited by the same dose of NC(1-13)NH(2). I.c.v. administration of [(pF)Phe(4)]NC(1-13)NH(2) dose-dependently stimulated feeding in rats, and was about tenfold more potent than NC(1-13)NH(2).Collectively, the present data demonstrate that, in a variety of in vivo assays, NC(1-13)NH(2) and [(pF)Phe(4)]NC(1-13)NH(2) mimicked the actions of NC. [(pF)Phe(4)]NC(1-13)NH(2) was more potent and its in vivo effects were longer lasting than those of NC(1-13)NH(2) and NC.

Nociceptin/orphanin FQ receptor knockout rats: in vitro and in vivo studies.

Nociceptin/orphanin FQ (N/OFQ) regulates several biological functions via selective activation of the N/OFQ peptide (NOP) receptor. Recently knockout rats for the NOP receptor gene (NOP(-/-)) have been generated; these animals were used in the present study to investigate their emotional (open field, elevated plus maze, and forced swimming test), locomotor (drag and rotarod test), and nociceptive (plantar and formalin test) phenotypes in comparison with their NOP(+/+) littermates. In addition, N/OFQ sensitivity has been assessed in electrically stimulated vas deferens tissues taken from NOP(+/+) and NOP(-/-) rats. In the elevated plus maze and forced swimming tests NOP(-/-) rats showed anxiety- and anti-depressant-like phenotype, respectively. No differences were found in the open field test. NOP(-/-) rats outperformed their NOP(+/+) littermates in two motor behaviour assays. Genetic ablation of the NOP receptor gene produced a statistically significant increase in nociceptive behaviour of the mutant rats in the formalin test. Finally, in the electrically stimulated rat vas deferens taken from NOP(+/+) tissues, N/OFQ inhibited in a concentration-dependent manner the electrically induced twitches while the peptide was inactive in tissues taken from NOP(-/-) animals. These results, in line with previous findings obtained with selective NOP receptor antagonists in mice and rats and with mouse knockout studies, clearly indicate that endogenous N/OFQ-NOP receptor signalling plays an important role in controlling anxiety- and mood-related behaviours, exercise-driven locomotor activity and nociception. These observations are relevant for defining the therapeutic indications (and contraindications) of NOP receptor antagonists.

Anti-inflammatory and analgesic effects displayed by peptides derived from PKI55 protein, an endogenous protein kinase C inhibitor.

We recently characterized the PKI55 protein as an endogenous protein kinase C (PKC) inhibitor and investigated, in vitro, the potential anti-inflammatory actions of its N-terminal peptides 1-16 (peptide 5), 1-8 (peptide 8) and 1-5 (peptide 9). We showed their ability to inhibit chemotaxis in human polymorphonuclear leukocytes activated by the N-formyl tripeptide for-Met-Leu-Phe-OMe. In this work, we evaluated the anti-inflammatory and the analgesic effects of the selected peptides by in vivo experiments carried out in the mouse. The peptides 5, 8 and 9 (0.1 and 10 nmol i.c.v.) were effective in both the parameters chosen to test the anti-inflammatory activity, i.e., the xylene-induced ear edema and the acetic acid-induced infiltration of neutrophils in the peritoneal cavity. In addition, they displayed analgesic effect, evaluated by the acetic acid-induced writhing test. All the peptides' effects were shared by the reference compounds, dexamethasone and indomethacin (10 mg kg(-1) i.p.), but not by the 9-scramble peptide (10 nmol i.c.v.). The peptide 9, which represents the shortest active sequence of the PKI55 protein, was tested in the ear edema model even following intraperitoneal (i.p.) administration and proved to be effective in the range doses 3-30 mg kg(-1). Moreover, an increase in plasma corticosterone levels was detected in mice treated with the peptide 9, but not with the 9-scramble peptide (both at 10 nmol i.c.v.). The anti-inflammatory and analgesic effects of the PKI55-derived synthetic peptides, possibly related both to PKC inhibition and hypothalamic-pituitary-adrenal axis activation, deserve further investigation in view of potential therapeutic exploitation.

Pharmacological characterization of the nociceptin/orphanin FQ receptor antagonist SB-612111 [(-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol]: in vivo studies.

The excellent pharmacological profile displayed by the selective nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor antagonist SB-612111 [(-)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol] in vitro prompted us to investigate the actions of this compound in vivo. In the mouse tail withdrawal assay, SB-612111 given i.p. up to 3 mg/kg did not modify per se tail withdrawal latencies but was able to prevent the pronociceptive and the antinociceptive action of 1 nmol of N/OFQ given i.c.v. and i.t., respectively. In food intake studies performed in sated mice, SB-612111 (1 mg/kg i.p.) had no effect on food consumption but fully prevented the orexigenic effect of 1 nmol of N/OFQ i.c.v. In 17-h food-deprived mice, the opioid receptor antagonist naltrexone (1 mg/kg s.c.), but not SB-612111 (1 and 10 mg/kg i.p.), produced a statistically significant reduction of food intake. In the mouse forced swimming and tail suspension tests, SB-612111 (1-10 mg/kg) reduced immobility time. The antidepressant-like effect elicited by SB-612111 in the forced swimming test was reversed by the i.c.v. injection of 1 nmol of N/OFQ and no longer evident in mice knockout for the NOP receptor gene. In conclusion, the present findings demonstrate that SB-612111 behaves in vivo as a potent and selective NOP antagonist and suggest that the N/OFQ-NOP receptor endogenous system plays an important role in regulating mood-related behaviors. The use of SB-612111 in future pathophysiological studies will certainly contribute to define the therapeutic potential of selective NOP receptor antagonists in different disease areas.

[(pF)Phe4,Arg14,Lys15]N/OFQ-NH2 (UFP-102), a highly potent and selective agonist of the nociceptin/orphanin FQ receptor.

A novel ligand for the nociceptin/orphanin FQ (N/OFQ) receptor (NOP), [(pF)Phe(4),Arg(14),Lys(15)]N/OFQ-NH(2) (UFP-102), has been generated by combining in the N/OFQ-NH(2) sequence two chemical modifications, [Arg(14),Lys(15)] and [(pF)Phe(4)], that have been previously demonstrated to increase potency. In vitro, UFP-102 bound with high affinity to the human NOP receptor, showed at least 200-fold selectivity over classical opioid receptors, and mimicked N/OFQ effects in CHO(hNOP) cells, isolated tissues from various species, and mouse cortical synaptosomes releasing 5-hydroxytryptamine. UFP-102 showed similar maximal effects but higher potency (2- to 48-fold) relative to N/OFQ. The effects of UFP-102 were sensitive to NOP-selective antagonists J-113397 [(+/-)-trans-1-[1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one] (pA(2) = 7.75-8.12) and UFP-101 ([Nphe(1),Arg(14),Lys(15)]N/OFQ-NH(2))(pA(2) = 6.91-7.33) but not to naloxone, and no longer observed in tissues taken from NOP receptor knockout mice (NOP(-/-)). In vivo, UFP-102 (0.01-0.3 nmol i.c.v.) mimicked the pronociceptive action of N/OFQ (0.1-10 nmol i.c.v.) in the mouse tail withdrawal assay, displaying higher potency and longer lasting effects. The action of UFP-102 was not apparent in NOP(-/-) mice. Similar results were obtained measuring locomotor activity in mice. In conscious rats, UFP-102 (0.05 nmol i.c.v.) produced a marked and sustained decrease in heart rate, mean arterial pressure, and urinary sodium excretion and a profound increase in urine flow rate. These effects were comparable with those evoked by N/OFQ at 5 nmol. Collectively, these findings demonstrate that UFP-102 behaves as a highly potent and selective NOP receptor agonist that produces long-lasting effects in vivo.

[Arg(14),Lys(15)]nociceptin, a highly potent agonist of the nociceptin/orphanin FQ receptor: in vitro and in vivo studies.

The nociceptin (NC)/orphanin FQ analog, [Arg(14),Lys(15)]NC, has been recently demonstrated to behave as a potent agonist at the human recombinant NC receptors (OP(4)). In this study, we evaluated the pharmacological profile of [Arg(14),Lys(15)]NC in vitro on the native OP(4) receptors expressed in isolated tissues and in vivo in the locomotor activity and the tail-withdrawal assays in mice. On isolated tissues, [Arg(14),Lys(15)]NC mimicked the effects of NC, showing similar maximal effects but higher potencies (17-fold in the mouse vas deferens, 10-fold in the rat vas deferens, and about 5-fold in the guinea pig ileum and mouse colon). In these preparations, the effects of [Arg(14),Lys(15)]NC were not modified by 1 microM naloxone, although antagonized by the OP(4) receptor antagonists [Nphe(1)]NC(1-13)NH(2) (pA(2) congruent with 6) and (+/-)trans-1-[1-cyclooctylmethyl-3hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (J-113397) (pA(2) congruent with 8). In the rat vas deferens, a cocktail of peptidase inhibitors increased the maximal effects of NC, its analog, and the pEC(50) of NC (by 4-fold); the potency of [Arg(14),Lys(15)]NC was not significantly modified by peptidase inhibitors. In in vivo experiments, [Arg(14),Lys(15)]NC mimicked the effects of NC, producing, after intracerebroventricular administration, pronociceptive effects in the tail-withdrawal assay and inhibiting the locomotor activity of the mice. In both assays, [Arg(14),Lys(15)]NC was about 30-fold more potent than NC and produced longer lasting effects. Taken together, the present data demonstrate that [Arg(14),Lys(15)]NC behaves as a highly potent agonist of the OP(4) receptor and is able to produce long-lasting effects in vivo, compared with the natural ligand NC.