Chemical Construction And Biological Activity Evaluation Of VF-13,A Chimeric Peptide Of Cannabinoid Peptide （m）VD-Hpα And Neuropeptide VF

Opioids are often used clinically to treat moderate and severe pain,but the side effects of which greatly limit its clinical application,such as tolerance,addiction,and respiratory depression.It is urgent to develop a novel analgesic with high efficacy and low side effects.Cannabinoid receptor are popular targets for the development of new analgesic drugs.Clinically,cannabinoid drugs can not only treat cancer pain and neuropathic pain,but also for nausea and vomiting caused by chemotherapy,epilepsy,colitis,liver fibrosis and other symptoms.Because the analgesic effects of most cannabinoid drugs depend on the activation of central cannabinoid receptors,they are often accompanied by central side effects such as addiction,hypothermia,and hypoactivity.The cannabinoid receptor peptide agonists（m）VD-Hpαisolated and identified from the enzymatically hydrolyzed fragments of mouse hemoglobin can mediate potent analgesia.In addition,compared with traditional cannabinoid receptor agonists,（m）VD-Hpαat an effective analgesic dose has reduced central side effects such as hypothermia,hypoactivity and constipation,but still produces analgesic tolerance.In the previous research of our laboratory,it was found that there is a functional interaction between the NPFF system and the cannabis system.NPFF-related peptides NPVF enhanced the analgesic effects and attenuate the constipation and analgesic tolerance induced by（m）VD-Hpα.Multi-target drugs can activate multiple targets that are related to pain and are interconnected functionally,so as to exert a potent analgesic effect while reducing its side effects.Based on the above research,this study chose the N-terminal of cannabinoid peptide（m）VD-Hpαand the C-terminal of NPVF as templates,designed and synthesized the chimeric peptide VF-13 through molecular chimerization.Cannabinoid peptide（m）VD-Hpα-NH₂ was used as a reference compound.Systematic biological activity evaluation and pharmacological mechanism research had been carried out on it.In vitro biological activities of（m）VD-Hpα-NH₂ and VF-13 were evaluated by Western blotting analysis,c AMP function assay and neurite growth experiments,and its agonistic activity for each receptor subtype were determined through selective antagonists of cannabinoid receptors and NPFF receptors.In vivo behavioral experiments,the analgesic effect and analgesic tolerance of intracerebroventricular（m）VD-Hpα-NH₂ and VF-13 were evaluated by acute and inflammatory pain model,and the effects of intracerebroventricular（m）VD-Hpα-NH₂and VF-13 on motor function,body temperature,gastrointestinal motility,food intake and sedation were evaluated in mice.The results of in vitro experiments showed that VF-13 significantly increased the phosphorylation of extracellular signal-regulated kinase 1/2（ERK1/2）by activating the CB₁receptor,and VF-13 concentration-dependently inhibited forskolin-induced c AMP accumulation via the NPFF₁ and NPFF₂ receptor.In addition,VF-13 could promote neurite outgrowth of mouse neuroblastoma N2A cells by activating CB₁ and NPFF receptors.These results indicated that the new chimeric peptide VF-13 behaves as a multifunctional agonist for CB₁,NPFF₁ and NPFF₂ receptors in in vitro experiments,while the control compound（m）VD-Hpα-NH₂ was shown as an agonist for CB₁ receptors.Neither of these two peptide compounds activated CB₂ receptors.In vivo experiments showed that intracerebroventricular injection of VF-13through TRPV1 produced dose-dependent analgesia in both acute and inflammatory pain,independent of cannabinoid receptors;and intracerebroventricular injection of the control compound（m）VD-Hpα-NH₂ produced CB₁ receptor-dependent analgesia.Further side-effect evaluation showed that intracerebroventricular injection of VF-13has no analgesic tolerance,while（m）VD-Hpα-NH₂ could produce analgesic tolerance.The intracerebroventricular injection of VF-13 produced a significant hypothermia effect,but did not cause catalepsy and motor inhibition.In addition,supraspinal administration of VF-13 had no significant effect on gastrointestinal motility,pentobarbital-induced sedation,food intake and motor coordination.In summary,the new chimeric peptide VF-13 based on cannabinoid peptide（m）VD-Hpαand neuropeptide NPVF had been shown to be a multifunctional agonist of CB₁,NPFF₁ and NPFF₂ receptors in in vitro functional experiments.Furthermore,VF-13 could produce potent and non-tolerance forming analgesia in acute and inflammatory pain models,and its side effects were significantly reduced.The results of this research suggest that the chimeric peptide VF-13 has potential application prospects in the development of new cannabinoid analgesics with high efficiency and low side effects.