Giuliano Marzola

Characterization of [Nphe1]nociceptin(1‐13)NH2, a new selective nociceptin receptor antagonist

Nociceptin (orphanin FQ) is a novel neuropeptide capable of inducing a variety of biological actions via activation of a specific G‐protein coupled receptor. However, the lack of a selective nociceptin receptor antagonist has hampered our understanding of nociceptin actions and the role of this peptide in pathophysiological states. As part of a broader programme of research, geared to the identification and characterization of nociceptin receptor ligands, we report that the novel peptide [Nphe1]nociceptin(1‐13)NH2 acts as the first truly selective and competitive nociceptin receptor antagonist and is devoid of any residual agonist activity. [Nphe1]nociceptin(1‐13)NH2 binds selectively to recombinant nociceptin receptors expressed in Chinese hamster ovary (CHO) cells (pKi 8.4) and competitively antagonizes the inhibitory effects of nociceptin (i) on cyclic AMP accumulation in CHO cells (pA2 6.0) and (ii) on electrically evoked contractions in isolated tissues of the mouse, rat and guinea‐pig with pA2 values ranging from 6.0 to 6.4. [Nphe1]nociceptin(1‐13)NH2 is also active in vivo, where it prevents the pronociceptive and antimorphine actions of intracerebroventricularly applied nociceptin, measured in the mouse tail withdrawal assay. Moreover, [Nphe1]nociceptin(1‐13)NH2 produces per se a dose dependent, naloxone resistant antinociceptive action and, at relatively low doses, potentiates morphine‐induced analgesia. Collectively our data indicate that [Nphe1]nociceptin(1‐13)NH2, acting as a nociceptin receptor antagonist, may be the prototype of a new class of analgesics.

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

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, [Nphe1,Arg14,Lys15]N/OFQ‐NH2 (UFP‐101). UFP‐101 binds to the human recombinant NOP receptor expressed in Chinese hamster ovary (CHO) cells with high affinity (pKi 10.2) and shows more than 3000 fold selectivity over classical opioid receptors. UFP‐101 competitively antagonizes the effects of N/OFQ on GTPγ35S binding in CHOhNOP cell membranes (pA2 9.1) and on cyclic AMP accumulation in CHOhNOP cells (pA2 7.1), being per se inactive at concentrations up to 10 μM. In isolated peripheral tissues of mice, rats and guinea‐pigs, and in rat cerebral cortex synaptosomes preloaded with [3H]‐5‐HT, UFP‐101 competitively antagonized the effects of N/OFQ with pA2 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. 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. 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.

Neuropeptide S is a stimulatory anxiolytic agent: a behavioural study in mice

Neuropeptide S (NPS) was recently identified as the endogenous ligand of an orphan receptor, now referred to as the NPS receptor. In vivo, NPS produces a unique behavioural profile by increasing wakefulness and exerting anxiolytic‐like effects. In the present study, we further evaluated the effects of in vivo supraspinal NPS in mice.

Pharmacological characterization of the nociceptin receptor mediating hyperalgesia in the mouse tail withdrawal assay

1 The newly discovered neuropeptide nociceptin (NC) has recently been reported to be the endogenous ligand of the opioid‐like orphan receptor. Despite its structural similarity to opioids, when injected intracerebroventricularly (i.c.v.) in the mouse, NC exerts a direct hyperalgesic effect and reverses opioid‐induced analgesia. In the present investigation, these two effects of NC were evaluated under the same experimental conditions; in addition, a pharmacological characterization of the receptor mediating these central effects of NC was attempted. 2 NC caused a dose dependent (0.1–10 nmol/mouse), naloxone‐insensitive reduction of tail withdrawal latency with a maximal effect of about 50% of the reaction time observed in saline injected mice. In the same range of doses, NC inhibited morphine (1 nmol/mouse) induced analgesia. 3 The effects of the natural peptide were mimicked by NCNH2 and NC(1–13)NH2 (all tested at 1 nmol/mouse) while 1 nmol NC(1–9)NH2 was found to be inactive either in reducing tail withdrawal latency or in preventing morphine analgesia. 4 [Phe1ψ(CH2‐NH)Gly2]NC(1–13)NH2 ([F/G]NC(1–13)NH2), which has been shown to antagonize NC effects in the mouse vas deferens, acted as an agonist, mimicking NC effects in both the experimental paradigms. In addition, when NC and [F/G]NC(1–13)NH2 were given together, their effects were additive. 5 These results demonstrate that both the direct hyperalgesic action and the anti‐morphine effect of NC can be studied under the same experimental conditions in the mouse tail withdrawal assay. Moreover, the pharmacological characterization of the NC functional site responsible for these actions compared with the peripherally active site, indicates the existence of important differences between peripheral and central NC receptors.

Blockade of nociceptin/orphanin FQ–NOP receptor signalling produces antidepressant-like effects: pharmacological and genetic evidences from the mouse forced swimming test

Nociceptin/orphanin FQ (N/OFQ), the endogenous ligand of the NOP receptor, regulates several central functions such as pain transmission, learning and memory, fear and anxiety and feeding and locomotor activity. It has been recently reported that NOP receptor antagonists induce antidepressant‐like effects in the mouse forced swimming test (FST), i.e. reduce immobility time. This assay was used in the present study for further investigating the involvement of the NOP receptor in depression states. In male Swiss mice, intracerebroventricular injection (i.c.v) of the novel NOP receptor antagonist, UFP‐101 (1–10 nmol) dose‐dependently reduced the immobility time (control 192 ± 14 s, UFP‐101 91 ± 15 s). The effect of 3 or 10 nmol UFP‐101 was fully or partially reversed, respectively, by the coadministration of 1 nmol N/OFQ, which was inactive per se. NOP receptor knockout mice showed a reduced immobility time compared with their wild‐type littermates (wild‐type 215 ± 10 s, knockout 143 ± 12 s). Moreover, i.c.v. injected UFP‐101 (10 nmol) significantly reduced immobility time in wild‐type mice but not in NOP receptor knockout animals. In conclusion, these results, obtained using a combined pharmacological and genetic approach, indicate that blockade of the N/OFQ‐NOP receptor signalling in the brain produces antidepressant‐like effects in the mouse FST. These findings support the NOP receptor as a candidate target for the development of innovative antidepressant drugs.

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.

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 μM 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 (pKB, 6.62). In Schild analysis experiments [d-Cys(tBu)5]NPS (0.1–100 μM) produced a concentration-dependent and parallel rightward shift of the concentration-response curve to NPS, showing a pA2 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.

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.

Endogenous nociceptin/orphanin FQ signalling produces opposite spinal antinociceptive and supraspinal pronociceptive effects in the mouse formalin test: Pharmacological and genetic evidences

Abstract Nociceptin/orphanin FQ (N/OFQ) has been demonstrated to modulate nociceptive transmission via selective activation of N/OFQ peptide (NOP) receptors. Despite huge research efforts, the role(s) of the endogenous N/OFQ–NOP receptor system in pain processing remains incompletely understood. In the present study, we investigated the role of endogenous N/OFQ in the processing of tonic nociceptive input. To address this issue the effects of NOP‐selective antagonists [Nphe1,Arg14,Lys15]N/OFQ‐NH2 (UFP‐101) and J‐113397 on nociceptive behaviour, and the nociceptive phenotype of NOP receptor‐deficient mice were tested in the mouse formalin test. Twenty microliters of 1.5% formalin solution was injected subcutaneously into the right hind paw causing a characteristic pattern of nociceptive behaviours (licking, biting and lifting of the injected paw). In control mice, the injection of formalin resulted in a classical biphasic nociceptive response with the first phase lasting from 0 to 10 min and the second phase from 15 to 45 min. UFP‐101 at 10 nmol/mouse (but not at 1 nmol/mouse) produced antinociceptive action when injected intracerebroventricularly and a pronociceptive action when given intrathecally. Systemic administration of J‐113397 (10 mg/kg, intravenously) and the genetic ablation of the NOP receptor gene both produced a significant increase of mouse nociceptive behaviour. Collectively, these results demonstrate that endogenous N/OFQ–NOP receptor signalling is activated during the mouse formalin test producing spinal antinociceptive and supraspinal pronociceptive effects. The overall effect of blocking NOP receptor signalling, by either systemic pharmacological antagonism or genetic ablation, indicates that the spinal antinociceptive action prevails over supraspinal pronociceptive effects.

GABA induced changes in acetylcholine release from slices of guinea-pig brain

SummaryThe effect of GABA on acetylcholine (ACh) release was investigated on superfused slices of guinea-pig cerebral cortex (CC), caudate nucleus (CN), tuberculum olfactorium and brain stem.GABA (1–6×10−3 mol/l) increased the spontaneous and KCl-evoked ACh overflow in CC and CN, reduced the electrically-evoked release in all areas tested (most evidently in CC and CN) and lowered the threshold of electric stimulation-induced ACh release in CC. These effects were also caused by 3-amino-1-propane sulphonic acid (1×10−3mol/l) and ethanolamine-O-sulphate (2×10−4mol/l), were reduced by bicuculline (1×10−4mol/l) and fully antagonized by picrotoxin (8×10−5mol/l), but they were not influenced by phentolamine, methysergide, spiroperidol or strychnine.Tetrodotoxin (TTX) (5×10−7mol/l) blocked the facilitation of spontaneous ACh release by GABA only when the slices were perfused with normal Krebs solution, but not when perfused with a KCl-enriched medium. These results suggest that GABA affects the cholinergic transmitter release through bicuculline- and picrotoxin-sensitive receptors, showing low affinity toward the agonist. Moreover GABA modulation of resting ACh release requires action potentials only in normal [K+]0, but not in high [K+]0, suggesting that GABA-receptive sites are located at cholinergic terminals.