The Mutli-Target Opioid/NPFF Receptors Agonist DN-9:Peripheral Antinociception And Opioid-Like Side Effects

Pain is an unpleasant physical feeling and psychological experience related to tissue injury or potential injury.Opioids are considered as the first-line therapeutic drugs of analgesia for moderate and severe pain are generally used in clinic,but the abuse and addiction of opioids limit their clinical application,we developed a novel multi-target opioid/neuropeptide FF agonist named DN-9 that can activate the opioid and NPFF receptors.DN-9 behaved as a multifunctional agonist atμ-,δ-andκ-opioid,NPFF₁ and NPFF₂ receptors in cAMP assay.Supraspinal DN-9 administration exhibited potent nontolerance-forming antinociception in the tail-flick test.Our preliminary results demonstrated that DN-9 did not cross blood-brain barrier（BBB）.Here we evaluated antinociceptive effects of peripheral DN-9 and systematically studied its side effects in mice to expand our knowledge of peripherally acting opioids,especially opioid ligands targeting multiple receptors.Subcutaneous DN-9administration can produce dose-dependent analgesic effect in tail-flick test.To further determine whether opioid and NPFF receptors are involved in DN-9-induced peripheral antinociception,mice were pretreated with the non-selective opioid receptor antagonist naloxone or the peripherally acting opioid antagonist naloxone methiodide,the result suggest that the peripheral antinociception of DN-9 is mainly mediated by the peripheral opioid receptors,especiallyμandκreceptors in the peripheral nervous system,and independent of NPFF receptors.Furthermore,DN-9elicits potent analgesia in inflammatory and neuropathic pain models after subcutaneous administration.And it is in line with the results obtained from MOR^-/-mice in which DN-9-induced anti-allodynic effects were significantly attenuated compared with the wild type mice.Our results also demonstrate that DN-9-induced peripheral antinociception has no sex difference in mouse models for acute,inflammatory,and neuropathic pain.and subcutaneous DN-9 produces antinociceptive responses that are equal or greater to those produced by morphine.Treatment with DN-9 did not affect the endurance time on the rotating rod even 40 min after injection,and there were no significant differences when compared to saline-treated mice.The development of tolerance to the antinociceptive efficacy of peripheral DN-9was examined in models for acute,inflammatory,and neuropathic pain.our results showe that subcutaneous injection of DN-9 produces a non-tolerance-forming analgesic effect,but the antinociceptive potency of morphine gradually declined during chronic exposure.In addition,we are highly encouraged by the observation that the antinociceptive effects of DN-9 in inflammatory or neuropathic pain models were also observed in morphine-tolerant mice on Day 8,which suggests that mice do not experience cross-tolerance to DN-9 following exposure to morphine.Unlike morphine,chronic treatment with DN-9 did not significantly activate microglial cells in the spinal cord.Furthermore,we use the classical responses of opioids to acute hyperlocomotion,conditioned place preference（CPP）,and naloxone-precipitated withdrawal.to evaluate the abuse liability of peripheral DN-9.The results showed that DN-9 had no apparent effect on locomotion activity and was far less potent than morphine in assays designed to measure CPP and withdrawal response.In addition,DN-9 produced a significant,dose-dependent delay in upper gastrointestinal transit.But morphine exhibited a more potent effect on GIT than DN-9 when equianalgesic doses were compared.Taken together,the present work provides significant evidence that DN-9 did not cross BBB,and peripheral administration of DN-9 produces antinociceptive effects that are comparable to or greater than those induced by morphine in several preclinical pain models.And analgesic effects of DN-9 are mainly mediated by peripheralμandκopioid receptors.Subcutaneous administration of DN-9 produces a non-tolerance-forming analgesic effect and does not produce cross tolerance with morphine.Compared with morphine,peripheral administration of DN-9 also significantly reduces opioid side effects such as addiction and constipation.Therefore,the multi-target peptide DN-9 as a potential candidate with high efficiency and low side effects have potential application in the future.