The Chemical Building And Characterization Of BN-9,a Bifunctional Opioid/NPFF Receptors Agonist With Nontolerance-forming Antinociception

The opioid analgesics are the first-line drugs for the clinical treatment of moderate to severe pain.However,their long-term therapeutic uses are associated with various adverse effects,including constipation,respiratory depression,the development of tolerance and physical dependence,thus restricting the scopes of their clinical treatment.Recently,the research of bifunctional opioid drugs has provided an attractive therapeutic strategy for the development of novel potent analgesic drugs with limited side effects.Neuropeptide FF(NPFF)system belongs to a neuropeptide family which includes two precursors(pro-NPFFA and pro-NPFFB)and two G-protein coupled receptors(NPFF1 and NPFF2),and was characterized as an endogenous opioid-modulating peptide.Previous study in our lab has shown that a novel chimeric peptide EN-9,a mixed endomorphin-2 and NPFF agonist,produced non-tolerence antinociception.On basis of the above facts,in the present study,we designed and chemically built a novel bifunctional peptide BN-9 based on opioid peptide biphalin and NPFF.Furthermore,BN-9 was pharmacologically characterized by in vitro cAMP assays and in vivo experiments of antinociception and antinociceptive tolerance.Moreover,its opioid-like side effects,including constipation,addiction and movement coordination,were further evaluated.Our present results showed that BN-9 acted as a bifunctional opioid/NPFF receptors agonist at μ-,δ-,κ-,NPFF1 and NPFF2 receptors in in vitro cAMP functional assays.In the tail-flick test,BN-9 produced dose-related antinociception which was approximately equipotent to morphine.Moreover,this antinociception was blocked by selective μ-and κ-opioid receptor antagonists and significantly augmented by the selective NPFF receptors antagonist RF9,supporting the bifunctional agonistic properties of BN-9.In addition,when injected intraperitoneally,the antinociceptive effects of systemic BN-9 were fully blocked by systemic pretreatment with the peripheral opioid receptor antagonist naloxone methiodide,but not by supraspinal pretreatment with naloxone methiodide,implying that BN-9 was unable to cross the blood-brain barrier and thus systemic BN-9-induced antinociception was mainly mediated by the peripheral opioid receptors.Furthermore,BN-9 also induced dose-dependent analgesia in the mouse models of formalin pain,acetic acid-induced visceral pain and post-operative pain.Systemic antinociception of BN-9 in these pain models was mediated by the peripheral opioid receptors,independently of NPFF receptors.Notably,repeated administration of BN-9 did not result in antinociceptive tolerance over 8 days at the supraspinal,spinal or systemic levels.However,BN-9 produced a progressive development of tolerance when intracerebroventricularly co-injected with the NPFF receptor antagonist RF9,implying the involvement of NPFF receptors in the development of central tolerance to BN-9-induced analgesia.Compared with morphine,BN-9 exerted weaker inhibition of gastrointestinal transit.In addition,BN-9 had little influence on motor function,and intracerebroventricular injection of BN-9 could induce conditioned place preference.In conclusion,the present work successfully built a bifunctional opioid/NPFF receptors agonist BN-9,which produced a robust,nontolerance-forming analgesia with limited inhibition of gastrointestinal transit.Moreover,intraperitoneal administration of BN-9 induced potent antinociception in a range of preclinical pathological pain models.In a word,this work provides a novel lead compound and a new strategy for the development of novel potent opioid analgesics with minimal side effects.