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In vitro and in vivo pharmacological characterization of a neuropeptide S tetrabranched derivative.

Ruzza C, Rizzi A, Malfacini D, Pulga A, Pacifico S, Salvadori S, Trapella C, Reinscheid RK, Calo G, Guerrini R - Pharmacol Res Perspect (2015)

Bottom Line: This PWT1-NPS stimulant effect was no longer evident in mice lacking the NPSR receptor.The PWT1 technology can be successfully applied to the NPS sequence.In vivo PWT1-NPS mimicked NPS effects showing higher potency and long-lasting action.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara 44121, Ferrara, Italy.

ABSTRACT
The peptide welding technology (PWT) is a novel chemical strategy that allows the synthesis of multibranched peptides with high yield, purity, and reproducibility. With this approach, a tetrabranched derivative of neuropeptide S (NPS) has been synthesized and pharmacologically characterized. The in vitro activity of PWT1-NPS has been studied in a calcium mobilization assay. In vivo, PWT1-NPS has been investigated in the locomotor activity (LA) and recovery of the righting reflex (RR) tests. In calcium mobilization studies, PWT1-NPS behaved as full agonist at the mouse NPS receptor (NPSR) being threefold more potent than NPS. The selective NPSR antagonists [ (t) Bu-D-Gly(5)]NPS and SHA 68 displayed similar potency values against NPS and PWT1-NPS. In vivo, both NPS (1-100 pmol, i.c.v.) and PWT1-NPS (0.1-100 pmol, i.c.v.) stimulated mouse LA, with PWT1-NPS showing higher potency than NPS. In the RR assay, NPS (100 pmol, i.c.v.) was able to reduce the percentage of mice losing the RR after diazepam administration and their sleep time 5 min after the i.c.v. injection, but it was totally inactive 2 h after the injection. On the contrary, PWT1-NPS (30 pmol, i.c.v.), injected 2 h before diazepam, displayed wake-promoting effects. This PWT1-NPS stimulant effect was no longer evident in mice lacking the NPSR receptor. The PWT1 technology can be successfully applied to the NPS sequence. PWT1-NPS displayed in vitro a pharmacological profile similar to NPS. In vivo PWT1-NPS mimicked NPS effects showing higher potency and long-lasting action.

No MeSH data available.


Recovery of righting reflex test performed in CD-1 mice (A and B) and in NPSR(-/-) and NPSR(−/−) mice (C and D). The percent of animals losing the RR after diazepam (15 mg/kg, i.p.) administration is shown in A and C, while B and D display mice sleep time. In CD-1, mice sleep time, two-way ANOVA followed by the Bonferroni’s post hoc test, revealed aneffect of NPS (100 pmol, i.c.v) and PWT1-NPS (30 pmol, i.c.v., F(2,44) = 13.89), an effect of time (F(2,44) = 5.60) and a significant interaction treatment × time (F(2,44) = 7.25; B). Data are mean ± SEM of 8–9 mice per group, *P < 0.05 vs. saline. In NPSR(-/-) and NPSR(−/−) mice, two-way ANOVA followed by the Bonferroni’s post hoc test, revealed an effect of PWT1-NPS on mice sleep time (F(1,21) = 9.47) and a significant interaction PWT1-NPS × genotype (F(1,21) = 15.31, D). Data are mean ± SEM of 8–9 mice per group, *P < 0.05 vs. saline, #P < 0.05 vs. NPSR(-/-).
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fig06: Recovery of righting reflex test performed in CD-1 mice (A and B) and in NPSR(-/-) and NPSR(−/−) mice (C and D). The percent of animals losing the RR after diazepam (15 mg/kg, i.p.) administration is shown in A and C, while B and D display mice sleep time. In CD-1, mice sleep time, two-way ANOVA followed by the Bonferroni’s post hoc test, revealed aneffect of NPS (100 pmol, i.c.v) and PWT1-NPS (30 pmol, i.c.v., F(2,44) = 13.89), an effect of time (F(2,44) = 5.60) and a significant interaction treatment × time (F(2,44) = 7.25; B). Data are mean ± SEM of 8–9 mice per group, *P < 0.05 vs. saline. In NPSR(-/-) and NPSR(−/−) mice, two-way ANOVA followed by the Bonferroni’s post hoc test, revealed an effect of PWT1-NPS on mice sleep time (F(1,21) = 9.47) and a significant interaction PWT1-NPS × genotype (F(1,21) = 15.31, D). Data are mean ± SEM of 8–9 mice per group, *P < 0.05 vs. saline, #P < 0.05 vs. NPSR(-/-).

Mentions: As demonstrated in previous studies, in the RR, 0.1 nmol NPS, injected i.c.v. 5 min before diazepam, was able to reduce the percentage of mice losing the RR and to reduce the sleep time of those mice responding to diazepam. This effect of NPS was no longer evident when NPS was injected 2 h before diazepam administration. On the contrary, PWT1-NPS (0.03 pmol, i.c.v.), given 5 min before diazepam, did not change the percentage of mice losing the RR but was able to significantly reduce the sleep time induced by diazepam. When PWT1-NPS was injected 2 h before diazepam administration it reduced both the percentage of mice losing the RR and the sleep time of those mice responding to diazepam (Fig.6A and B). PWT1-NPS was not able to produce any statistically significant effects 4 h after the injection (data not shown). Under the present experimental conditions, no differences were measured between NPSR(-/-) and NPSR(−/−) mice in terms of sensitivity to diazepam. PWT1-NPS (0.03 pmol, i.c.v., 2 h of pretreatment) elicited a robust wake-promoting effect in NPSR(-/-) mice; this action of PWT1-NPS was no longer evident in mice lacking the NPSR receptor (Fig.6C and D).


In vitro and in vivo pharmacological characterization of a neuropeptide S tetrabranched derivative.

Ruzza C, Rizzi A, Malfacini D, Pulga A, Pacifico S, Salvadori S, Trapella C, Reinscheid RK, Calo G, Guerrini R - Pharmacol Res Perspect (2015)

Recovery of righting reflex test performed in CD-1 mice (A and B) and in NPSR(-/-) and NPSR(−/−) mice (C and D). The percent of animals losing the RR after diazepam (15 mg/kg, i.p.) administration is shown in A and C, while B and D display mice sleep time. In CD-1, mice sleep time, two-way ANOVA followed by the Bonferroni’s post hoc test, revealed aneffect of NPS (100 pmol, i.c.v) and PWT1-NPS (30 pmol, i.c.v., F(2,44) = 13.89), an effect of time (F(2,44) = 5.60) and a significant interaction treatment × time (F(2,44) = 7.25; B). Data are mean ± SEM of 8–9 mice per group, *P < 0.05 vs. saline. In NPSR(-/-) and NPSR(−/−) mice, two-way ANOVA followed by the Bonferroni’s post hoc test, revealed an effect of PWT1-NPS on mice sleep time (F(1,21) = 9.47) and a significant interaction PWT1-NPS × genotype (F(1,21) = 15.31, D). Data are mean ± SEM of 8–9 mice per group, *P < 0.05 vs. saline, #P < 0.05 vs. NPSR(-/-).
© Copyright Policy - open-access
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fig06: Recovery of righting reflex test performed in CD-1 mice (A and B) and in NPSR(-/-) and NPSR(−/−) mice (C and D). The percent of animals losing the RR after diazepam (15 mg/kg, i.p.) administration is shown in A and C, while B and D display mice sleep time. In CD-1, mice sleep time, two-way ANOVA followed by the Bonferroni’s post hoc test, revealed aneffect of NPS (100 pmol, i.c.v) and PWT1-NPS (30 pmol, i.c.v., F(2,44) = 13.89), an effect of time (F(2,44) = 5.60) and a significant interaction treatment × time (F(2,44) = 7.25; B). Data are mean ± SEM of 8–9 mice per group, *P < 0.05 vs. saline. In NPSR(-/-) and NPSR(−/−) mice, two-way ANOVA followed by the Bonferroni’s post hoc test, revealed an effect of PWT1-NPS on mice sleep time (F(1,21) = 9.47) and a significant interaction PWT1-NPS × genotype (F(1,21) = 15.31, D). Data are mean ± SEM of 8–9 mice per group, *P < 0.05 vs. saline, #P < 0.05 vs. NPSR(-/-).
Mentions: As demonstrated in previous studies, in the RR, 0.1 nmol NPS, injected i.c.v. 5 min before diazepam, was able to reduce the percentage of mice losing the RR and to reduce the sleep time of those mice responding to diazepam. This effect of NPS was no longer evident when NPS was injected 2 h before diazepam administration. On the contrary, PWT1-NPS (0.03 pmol, i.c.v.), given 5 min before diazepam, did not change the percentage of mice losing the RR but was able to significantly reduce the sleep time induced by diazepam. When PWT1-NPS was injected 2 h before diazepam administration it reduced both the percentage of mice losing the RR and the sleep time of those mice responding to diazepam (Fig.6A and B). PWT1-NPS was not able to produce any statistically significant effects 4 h after the injection (data not shown). Under the present experimental conditions, no differences were measured between NPSR(-/-) and NPSR(−/−) mice in terms of sensitivity to diazepam. PWT1-NPS (0.03 pmol, i.c.v., 2 h of pretreatment) elicited a robust wake-promoting effect in NPSR(-/-) mice; this action of PWT1-NPS was no longer evident in mice lacking the NPSR receptor (Fig.6C and D).

Bottom Line: This PWT1-NPS stimulant effect was no longer evident in mice lacking the NPSR receptor.The PWT1 technology can be successfully applied to the NPS sequence.In vivo PWT1-NPS mimicked NPS effects showing higher potency and long-lasting action.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Sciences, Section of Pharmacology and National Institute of Neuroscience, University of Ferrara 44121, Ferrara, Italy.

ABSTRACT
The peptide welding technology (PWT) is a novel chemical strategy that allows the synthesis of multibranched peptides with high yield, purity, and reproducibility. With this approach, a tetrabranched derivative of neuropeptide S (NPS) has been synthesized and pharmacologically characterized. The in vitro activity of PWT1-NPS has been studied in a calcium mobilization assay. In vivo, PWT1-NPS has been investigated in the locomotor activity (LA) and recovery of the righting reflex (RR) tests. In calcium mobilization studies, PWT1-NPS behaved as full agonist at the mouse NPS receptor (NPSR) being threefold more potent than NPS. The selective NPSR antagonists [ (t) Bu-D-Gly(5)]NPS and SHA 68 displayed similar potency values against NPS and PWT1-NPS. In vivo, both NPS (1-100 pmol, i.c.v.) and PWT1-NPS (0.1-100 pmol, i.c.v.) stimulated mouse LA, with PWT1-NPS showing higher potency than NPS. In the RR assay, NPS (100 pmol, i.c.v.) was able to reduce the percentage of mice losing the RR after diazepam administration and their sleep time 5 min after the i.c.v. injection, but it was totally inactive 2 h after the injection. On the contrary, PWT1-NPS (30 pmol, i.c.v.), injected 2 h before diazepam, displayed wake-promoting effects. This PWT1-NPS stimulant effect was no longer evident in mice lacking the NPSR receptor. The PWT1 technology can be successfully applied to the NPS sequence. PWT1-NPS displayed in vitro a pharmacological profile similar to NPS. In vivo PWT1-NPS mimicked NPS effects showing higher potency and long-lasting action.

No MeSH data available.