The Antinociception Of The Nociception Receptor Agonist NOP01 On Orofacial Pain And Its Interaction With The Mu Opioid Receptor Agonist

Orofacial pain is a general term involving many inflammatory and neuropathic pain in hard tissue and soft tissue of the head,face and neck.The published data show that about a quarter of adults in the United States suffered from orofacial pain.Medications used to treat orofacial pain include non-steroidal anti-inflammatory drugs（NSAIDs）,antidepressants,anticonvulsants,opioids and so on.However,the clinical applications of these medications are severely limited by their side effects.Therefore,it is important to develop novel potent analgesics with low side effects for the treatment of orofacial pain.The nociceptin/orphanin FQ peptide receptor（NOR）is the fourth member of the opioid receptor family,and distributes in both the central and peripheral nervous system.The previous reports show that NOR agonists could induce effective antinociception on chronic pain models,such as inflammatory pain and neuropathic pain.Moreover,the researches on nonhuman primates（NHPs）show that NOR agonists fail to produce side effects,including addiction,respiratory depression,and itching.Orofacial pain is often caused by orofacial inflammation and nerve damage,so NOR may be a potential target for orofacial pain treatment.Based on the structure-activity relationship studies for NOR ligands,Chang et al.reported a potent NOR peptide agonist NOP01([（p F）Phe~4,Aib~7,Aib¹¹,Arg¹⁴,Lys¹⁵]N/OFQ-NH₂),which shows a more potent agonistic activity than parent N/OFQ in both in vitro and in vivo experiments.In this article,NOP01 was used as a pharmacological tool to further evaluate the mechanism of the NOR system in orofacial pain.The antinociceptive effect of NOP01 in orofacial formalin pain model（a trigeminal nerve persistent pain model）were evaluated systematically at different levels.Its antinociceptive site（s）and related pharmacological mechanism was also explored in antagonism experiments and immunofluorescence experiment.Our results showed that central（intrathecal）,peripheral（local subcutaneous）and systemic（intraperitoneal）injections of NOP01produced dose-dependent antinociceptive effects in the mouse orofacial formalin pain model.Furthermore,antagonism experiments showed that central and peripheral NOP01-induced antinociceptive effect were mediated by NOR.Moreover,NOP01 didn’t cross the blood-brain barrier,and the antinociceptive effect of systemic NOP01 was mediated by peripheral NOR.In addition,immunofluorescence experiments show that local subcutaneous injection of NOP01could significantly reduce the up-regulation of c-Fos protein expression induced by formalin in ipsilateral TG neurons,implying that NOP01 can inhibit the hyperexcitability of TG neurons induced by formalin,which is consistent with the behavioral experiment results in orofacial formalin pain models.Recent studies show that drug combination can effectively reduce the dosage of a single drug,and therefore exhibit improved efficacy and reduced side effects.To evaluate whether NOP01interacted with mu opioid receptor（MOR）agonist in the orofacial formalin pain model,a peptide agonist DAMGO-NH₂ was designed and synthesized in this study.The results showed that DAMGO-NH₂ behaved as a potent agonist for MOR receptor(EC₅₀=1.39 n M,E_max=102.1%)in in vitro functional experiments（calcium mobilization assay）.Moreover,local subcutaneous injection of DAMGO-NH₂ produced dose-dependent antinociceptive effect through peripheral MOR in orofacial formalin pain model.More importantly,isobolographic analysis showed that NOP01 and DAMGO-NH₂ induced synergistic antinociception in the present orofacial formalin pain model.In conclusion,NOP01,a potent NOR peptide agonist produces antinociceptive effect on orofacial pain at both central and peripheral levels,which are mediated by NOR.NOP01 doesn’t cross the blood-brain barrier.Moreover,the combination between NOP01 and the MOR agonist DAMGO-NH₂ could produce synergistic antinociception at peripheral level.Therefore,this work will provide the theoretical basis and experimental support for the studies of pathological mechanism of orofacial pain and the development of analgesic drugs on orofacial pain.