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Branched peptides have been found to be useful in several research fields however their synthesis and purification is complicated. Here we present a novel and facile synthesis of tetra branched derivatives of nociceptin/orphanin FQ (N/OFQ). Three N/OFQ tetra branched derivatives were prepared using novel cores ( PWT1 , PWT2 and PWT3 ) containing a maleimido moiety. [Cys 18 ]N/OFQ-NH 2 was linked to the cores via thiol-Michael reaction characterized by high yield and purity of the desired final product. In the electrically stimulated mouse vas deferens PWT-N/OFQ derivatives mimicked the inhibitory action of the natural sequence showing similar maximal effects and 3 fold higher potencies. The NOP selective antagonist SB-612111 antagonized the effects of N/OFQ and PWT derivatives with similar p K B values (8.02–8.48). In vivo after supraspinal administration PWT2-N / OFQ stimulated food intake in mice mimicking the action of N/OFQ. Compared to the natural peptide PWT2-N / OFQ was 40 fold more potent and elicited larger effects. These findings suggest that the PWT chemical strategy can be successfully applied to biologically active peptides to generate, with unprecedented high purity and yield, tetra branched derivatives displaying an in vitro pharmacological profile similar to that of the natural sequence associated, in vivo, to increased potency and effectiveness.

Background and Purpose Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor antagonists have been proposed as a novel therapeutic approach to Parkinson's disease. Main limitations of previous studies were the use of structurally similar compounds and the evaluation of their acute effects only. We report here on the acute and long-term antiparkinsonian effects of the novel compound 2-[3-[4-(2-chloro-6-fluoro-phenyl)-piperidin-1-ylmethyl]-2-(morpholine-4-carbonyl)-indol-1-yl]-acetamide (NiK-21273) in comparison with the potent and selective NOP receptor antagonist SB-612111. Experimental Approach Basic pharmacological properties of NiK-21273 were studied in cell lines and isolated tissues (mouse and rat vas deferens). Antiparkinsonian effects were studied in reserpinized mice and 6-hydroxydopamine hemilesioned rats under both acute and chronic administration protocols. Key Results In vitro, NiK-21273 behaved as a potent (pA2 7.7) and selective NOP receptor antagonist. In vivo, it reduced hypokinesia in reserpinized mice at 0.1 and 1 but not 10 mg·kg−1, whereas SB-612111 (0.01–1 mg·kg−1) provided a dose-dependent antiparkinsonian effect. NiK-21273 ameliorated motor performance in 6-hydroxydopamine hemilesioned rats at 0.5 and 5 but not 15 mg·kg−1. SB-612111 replicated these effects in the 0.01–1 mg·kg−1 range without loss of efficacy. Both antagonists synergized with L-DOPA at subthreshold doses. Chronic administration of NiK-21273 provided delayed improvement in baseline activity at 0.5 and 1.5 mg·kg−1, although tolerance to the higher dose was observed. Conversely, SB-612111 (1 mg·kg−1) maintained its effects over time without modifying baseline activity. Conclusions and Implications NOP receptor antagonists provide motor benefit in parkinsonism models although the ‘therapeutic’ window and long-term effects may vary between compounds.

Background and Purpose Intrathecally (i.t.) administered nociceptin/orphanin FQ (N/OFQ) evokes antinociceptive effects in rodents. Recent studies in monkeys demonstrated that i.t. co-application of N/OFQ and morphine elicits synergistic antinociceptive actions suggesting mixed N/OFQ peptide (NOP) and μ opioid receptor agonists as innovative spinal analgesics. Thus, novel N/OFQ related peptides were synthesized in order to identify and pharmacologically characterize a mixed NOP/ μ opioid receptor agonist. Experimental Approach The following in vitro assays were used: calcium mobilization in cells expressing the human NOP or classical opioid receptors and chimeric G proteins, receptor and [35S]-GTPγS binding, [35S]-GTPγS binding in rat spinal cord membranes, guinea pig ileum bioassay. In vivo experiments were performed in monkeys using the tail withdrawal assay. Key Results From calcium mobilization studies [Dmt1]N/OFQ(1–13)-NH2 was selected as the most potent and least selective compound. The mixed NOP/opioid full agonist activity and high affinity of [Dmt1]N/OFQ(1–13)-NH2 was confirmed at human recombinant receptors in receptor binding, calcium mobilization and/or [35S]-GTPγS binding studies, at rat spinal cord receptors in [35S]-GTPγS binding experiments, and at guinea pig receptors inhibiting neurogenic contractions in the ileum. In vivo in the tail withdrawal assay in monkeys i.t. [Dmt1]N/OFQ(1–13)-NH2 was able to elicit robust and long-lasting antinociceptive effects. Conclusions and Implications Collectively, these results demonstrate that [Dmt1]N/OFQ(1–13)-NH2 behaves as NOP/opioid receptor universal agonist and substantiate the suggestion that such mixed ligands are worthy of development as innovative spinal analgesics.

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.

In this study we provided a pharmacological characterization of the recently synthesized nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) antagonist 1-[1-Cyclooctylmethyl-5-(1-hydroxy-1-methyl-ethyl)-1,2,3,6-tetrahydro-pyridin-4-yl]-3-ethyl-1,3-dihydro-benzoimidazol-2-one (GF-4) and investigated its antiparkinsonian properties. GF-4 inhibited N/OFQ binding to CHO(hNOP) cell membranes (pK(i) 7.46), and antagonized N/OFQ effects in a calcium mobilization assay and electrically stimulated isolated tissues (pK(B) 7.27-7.82), showing a approximately 5-fold selectivity over classical opioid receptors. In vivo, GF-4 dually modulated stepping activity in wild-type mice, causing facilitation in the 0.01-10mg/kg dose range and inhibition at 30mg/kg. These effects were mediated by NOP receptors since GF-4 was ineffective in NOP receptor knock-out mice. Antiparkinsonian properties of GF-4 were investigated in 6-hydroxydopamine hemilesioned rats. GF-4 ameliorated akinesia, bradykinesia and overall gait ability in the 0.1-10mg/kg dose range, but inhibited motor activity at 30mg/kg. To investigate the circuitry underlying motor facilitating and inhibitory effects of GF-4, microdialysis coupled to behavioral testing (akinesia test) was performed. An anti-akinetic dose of GF-4 (1mg/kg) reduced glutamate (GLU) and enhanced GABA release in SNr, while the pro-akinetic dose of GF-4 (30mg/kg) evoked opposite effects. Moreover, the anti-akinetic dose of GF-4 reduced GABA and increased GLU release in ventro-medial thalamus, the pro-akinetic dose decreasing GABA without affecting GLU release in this area. We conclude that GF-4 is an effective NOP receptor antagonist able to attenuate parkinsonian-like symptoms in vivo via inhibition of the nigro-thalamic pathway.