Structure-based Optimization And Pharmacological Characterization Of Novel Multi-target Opioid And Neuropeptide FF Agonists

Pain is an unpleasant sensory and emotional experience and serves a protective role to warn us of potential injuries or diseases.However,chronic pain can significantly influence the individual’s quality of life,and millions of people are suffering from various pains worldwide.The treatment of pain,especially chronic pain,is a major challenge in the clinic.Although opioid analgesics play important roles in the treatment of severe acute pain and chronic pain,currently available opioid analgesics have a number of unwanted side-effects that hamper their clinical use,particularly in chronic pain management.Therefore,the need to develop novel analgesics with enhanced efficacy and limited side effects is becoming more and more urgent.Recently,the developments of multi-targets opioid drugs have emerged as an attractive therapeutic strategy to reduce or eliminate opioid-related side effects.In the previous studies,mounting evidences indicated neuropeptide FF(NPFF)has important modulatory effects on opioid activities,including antinociception,antinociceptive tolerance,and physical dependence et al.Our recent study had successfully developed an opioid and neuropeptide FF pharmacophore-containing chimeric peptide BN-9,which was shown to produce potent non-tolerance forming analgesia.In the present study,eleven analogues of BN-9 were designed and synthesized,and the in vitro and in vivo pharmacology activities were also characterized.An in vitro cAMP assay demonstrated that these analogues behaved as multifunctional agonists at both opioid and NPFF receptors.In mouse tail-flick test,most of the analogues produced potent non-tolerance forming analgesic effect,except analogues 2 and 3.Notably,analogue 11(DN-9)was 33-fold more potent than BN-9.The supraspinal analgesic effect of DN-9 could be completely blocked by pretreatments with μ-and κ-opioid receptors selective antagonist β-FNA and nor-BNI,respectively.However,the NPFF receptors antagonist RF9 could significantly argumented the supraspinal analgesic effect of DN-9.These results indicated that the supraspinal analgesic effect was mediated by μ-and κ-opioid receptors and NPFF receptors had anti-opioid activities at DN-9 supraspinal analgesic effect.In addition,i.c.v.administration of DN-9 also produced powerful analgesic effects in the formalin pain and CFA-induced chronic inflammatory pain models.Strikingly,following its repeated administration for 6 days,DN-9 did not produce antinociceptive tolerance in the tail-flick test and CFA-induced pain model.At the spinal level,DN-9 could also produce powerful analgesia effects in various pain models.In mouse tail-flick test,the antinociceptive effects of DN-9 was 263-fold more potent than BN-9,which also did not produce antinociceptive tolerance.We got the similar results with i.c.v.administration using antagonists,the antinociceptive effects of intrathecal injection of DN-9 were also mediated by opioid receptors,and NPFF receptors also participanted in the spinal antinociception of DN-9.Intrathecally injection of DN-9 showed potent analgesia in a series of preclinical pain models,including post-operative pain,acetic acid writhing test,formalin test,carrageenan induced inflammatory pain and neuropathic pain model.The antinociceptive ED50 values in these pain models were all at pmol level.The antinociceptive effects of DN-9 after systemic administration were also investigated in the present studies.Intraperitoneally injection of DN-9 could also produce significant antinociceptive effects in the mouse tail-flick test,formalin test,acetic acid writhing test,and post-operative pain model.Experiments results using opioid and NPFF receptors antagonists indicated the antinociceptive effects of DN-9 after i.p.administration were mainly mediated by opioid receptors.However,the NPFF receptors antagonist RF9 could not modulate the systemic antinociception of DN-9.Moreover,DN-9 showed limited side effects in gastrointestinal transit inhibition,conditioning place preference,cardiovascular,body temperature regulation and,locomotor function and coordination.Taken together,in the present study,we synthesized and pharmacologically characterized a series of multifunctional opioid/NPFF receptors agonists,especially DN-9,which produced robust,nontolerance-forming analgesia with limited side effects.DN-9 might be a promising compound for developing novel multifunctional opioid analgesics which devoided adverse effects.The present work also indicates that it is reasonable to design multifunctional peptide ligands for opioid and NPFF receptors in a single molecule to producing effective non-tolerance forming antinociception.