Multi-targeted Cyclic Peptides Of Opioid And Neuropeptide FF Receptors:Design,Synthesis And Pharmacological Characterization

Classical opioid analgesics,including morphine,fentanyl,and oxycodone are widely used in the treatment of clinical pain,and effective to treat moderate-to-severe pain.In general,most opioids used in clinical therapy are Mu-opioid receptor agonists.Although they have potent analgesic effects,they also cause troublesome side effects,such as respiratory depression,tolerance,constipation,and abuse.Especially,in recent years,the phenomenon of overuse and abuse of opioids in the United States,known as the"opioid crisis",has developed into the"most serious public health crisis"and caused an increasing number of deaths per year.Therefore,the developments of novel analgesics with limited side effects have important scientific significance and potential application prospects.Recently,multi-targeted opioid agonists have been proved to effectively reduce opioid-like adverse effects,such as tolerance and addiction.Neuropeptide FF(NPFF)is an endogenous opioid-modulating peptide and plays complex modulating roles in opioid analgesia and side effects.In our previous studies,two multi-targeted peptide agonists of opioid/NPFF receptor,named BN-9 and DN-9,were characterized to simultaneously activate opioid and NPFF receptors,and produce dose-dependent analgesia with diminished side effects in different pain models after central and peripheral injections.Pharmacological results indicated that BN-9 and DN-9 could not penetrate blood-brain barrier and produced analgesia lasting only for 90 min after peripheral injection.Therefore,to improve the druggability of BN-9 and DN-9,the cyclization modification strategies were used in this paper.The disulfide-bridged cyclic BN-9 analogs,disulfide-bridged cyclic DN-9 molecule(OFP011)and lactam-bridged cyclic DN-9 analogs were designed and synthesized to improve their metabolic stability and prolong their analgesic duration.In this study,the peptide molecules were synthesized by using the solid synthesis method,and their activity screening and pharmacological identification were conducted by in vitro c AMP function assay and in vivo radiant heat tail-flick test.Further,the analgesic pharmacodynamics in different pathological pain models,preliminary pharmacokinetics and opioid-like side effects of the optimized compounds were evaluated systematically.Our previous studies have shown that the analgesic potencies of subcutaneous BN-9 and DN-9 were much weaker than that of centrally administered these two peptides in various pain models.These results imply that analogs of BN-9 or DN-9with increased BBB penetrability may ameliorate its antinociceptive potency.The four disulfide-bridged cyclic analogs and four their linear counterparts were designed via the incorporation of Cys or D-Cys at position 2 and 5 of template molecule BN-9.In vitro c AMP assays demonstrated that these analogs behaved as multi-targeted agonists at both opioid and NPFF receptors.All analogs produced more potent analgesic effects than the parent peptide BN-9 without tolerance development in the tail-flick test.Notably,the optimized analog 6 exhibited 7-fold more potent Mu-opioid receptor agonistic activity in comparison to BN-9 in vitro.In addition,cyclic analog 6possessed an improved stability in the brain and an increased blood-brain barrier permeability compared to the parent peptide BN-9,and produced more potent analgesia with improved duration of action of 240 min after intracerebroventricular or subcutaneous injection.Furthermore,analgesic tolerance of cyclic analog 6 was reduced compared to the classical opioid analgesic after subcutaneous injection fentanyl,as was the rewarding effect,withdrawal reaction,and gastrointestinal inhibition.Furthermore,the strategy of cyclic disulfide modification in BN-9 to obtain the optimized compound was applied in the multi-targeted peptide DN-9,and the cyclic disulfide analog of DN-9 named OFP011 was obtained and systematically pharmacological characterization.The cyclic disulfide peptide OFP011 exhibited multi-targeted agonistic efficacies at opioid and NPFF receptors in in vitro c AMP assays.The pharmacokinetic,animal imaging and pharmacological experiments demonstrated the BBB permeability of OFP011 after systemic injection.In addition,subcutaneous OFP011 produced potent and long-lasting analgesia via the central Mu-and Kappa-opioid receptors in various nociceptive models.Furthermore,at the highest analgesic doses,subcutaneous OFP011 induced reduced adverse effects including tolerance,gastrointestinal transit,motor function,addiction,reward,and respiration depression.Notably,OFP011 achieved oral analgesic activity in different pain models.Based on the above studies,the lactam-bridged cyclization modification strategy was further applied to the multi-targeted peptide DN-9,and eight lactam-bridged cyclic analogs were designed and synthesized by changing the L/D-configuration of the amino acid residues at positions 2 and 5 and the size of ring.In vitro c AMP assays revealed that these analogs were multi-targeted ligands at opioid and NPFF receptors.In comparison to DN-9,the Kappa-opioid and NPFF₂receptors agonism of analog 1was improved approximately 38-fold and 6-fold,respectively.In addition,all analogs exhibited potent,long-lasting peripherally restricted analgesia in the tail-flick test without tolerance development after subcutaneous injection.Interestingly,all these lactam-bridged cyclic analogs produced oral analgesic activities.Furthermore,oral injection of the optimized compound analog 1 exhibited powerful,peripherally restricted analgesic effects in mouse models of acute,inflammatory,and neuropathic pain.Furthermore,orally administered analog 1 had no significant side effects,such as tolerance,dependence,constipation or respiratory depression,at effective analgesic doses.Collectively,a series of multi-targeted agonists of opioid and NPFF were obtained by disulfide-and lactam-bridged cyclic modifications.Interestingly,disulfide-bridged cyclic modification enhances the BBB permeability of multi-targeted molecules,whereas lactam-bridged cyclization modification peptides are unable to cross BBB similar to its parent peptide.All the optimized compounds can produce efficient,long-lasting analgesia with limited adverse effects.It is notable that disulfide-and lactam-bridged cyclic analogs of DN-9 produced oral analgesic activity.Therefore,cyclization modification of multi-targeted peptides of opioid and NPFF receptors can effectively improve their druggability and analgesic activity while retaining the pharmacological properties of multi-targeted agonists,and this work is helpful to provide a lead compound for the development of novel analgesic drugs with high efficiency and low side effects.