Effects Of Central And Peripheral Administration Of The Bifunctional Opioid/npff Receptors Agonist DN-9 On Gastrointestinal Transit In Mice

Opioids（morphine,fentanyl,etc.）are widely used to treat moderate and severe pain,but its clinical application is limited by opioid-like side effects.Almost all opioid analgesics have side effect on constipation,which is a serious trouble for doctors and patients.In recent years,study of bifunctional opioid ligands has provided novel ideas for development of the new analgesics with reduced side effects,such as tolerance and addiction.Our group synthesized several bifunctional opioid/NPFF receptors agonists in previous studies,and discoverd the bifunctional agonist DN-9that simultaneously activated mu recetor,delta recetor,kappa recetor,NPFF₁ and NPFF₂ receptors.In addition,DN-9 produces powerful,nontolerance-forming antinociception with limited side effects.To further evaluate the effects of the bifunctional agonist DN-9 on gastrointestinal motility,in the present study,the effect of DN-9 on upper gastrointestinal transit and colonic bead expulsion were systematically explored in mice.In addition,to evaluate the role of NPFF receptor in the regulation of gastrointestinal motility induced by DN-9,we used its analogue[Phg⁹]-DN-9 as the reference compound,which can activate MOR,DOR and KOR,but not NPFF receptors.The data obtained from the upper gastrointestinal transit experiment in mice showed that:（1）intracerebroventricular administration of DN-9 and morphine significantly inhibited gastrointestinal motility via opioid receptors in the brain,and the gastrointestinal motility inhibition induced by DN-9 was mainly mediated by MOR and KOR.Furthermore,the important role of MOR in the gastrointestinal motility inhibition of central DN-9 was also confirmed in MOR^-/-mice.（2）At the supraspinal level,the gastrointestinal motility inhibition induced by DN-9 was significantly enhanced by the NPFF receptors antagonist RF9.At the same dose,[Phg⁹]-DN-9 produed a more potent gastrointestinal motility inhibition compared to DN-9.However,central administration the NPFF agonists including NPFF,NPVF and dNPA,alone have no significant effect on gastrointestinal motility in mice.（3）Intraperitoneal injection of DN-9 and morphine significantly inhibited the gastrointestinal motility in mice,which was blocked by intraperitoneal administration of the opioid receptor antagonists,naloxone and naloxone methiodide,but not by intracerebroventricular injection of the peripherally acting opioid antagonist naloxone methiodide.Furthermore,the important role of MOR in the gastrointestinal motility inhibition of peripheral DN-9 was also confirmed in MOR^-/-mice.（4）At the peripheral level,the gastrointestinal motility inhibition induced by DN-9 was significantly enhanced by the NPFF receptors antagonist RF9.At the same dose,[Phg⁹]-DN-9 produed a more potent gastrointestinal motility inhibition compared to DN-9.However,peripheral administration the NPFF agonists including NPFF,NPVF and dNPA,alone have no significant effect on gastrointestinal motility in mice.The data obtained from the colonic bead expulsion experiment in mice showed that:（1）intracerebroventricular administration of DN-9 and morphine significantly inhibited colonic motility via opioid receptors in the brain,and the colonic motility inhibition induced by DN-9 was mainly mediated by MOR and KOR.（2）At the supraspinal level,the colonic motility inhibition induced by DN-9 was significantly enhanced by the NPFF receptors antagonist RF9.At the same dose,[Phg⁹]-DN-9produed a more potent colonic motility inhibition compared to DN-9.However,central administration the NPFF agonists including NPFF and dNPA with low and high dose have no significant effect on colonic bead expulsion in mice,but NPVF has significant effect with high dose.（3）Intraperitoneal injection of DN-9 and morphine significantly inhibited the colonic motility in mice,which was blocked by intraperitoneal administration of the opioid receptor antagonists,naloxone and naloxone methiodide,but only morphine can be blockde by intracerebroventricular injection of the peripherally acting opioid antagonist naloxone methiodide.（4）At the peripheral level,the colonic motility inhibition induced by DN-9 was significantly enhanced by the NPFF receptors antagonist RF9.At the same dose,[Phg⁹]-DN-9produed a more potent colonic motility inhibition compared to DN-9.However,peripheral administration the NPFF agonists including NPFF,NPVF and dNPA,alone have no significant effect on colonic motility in mice.In summary,the research of this paper show that DN-9 can inhibite movement of small intestine and colon through the receptors of central and peripheral respectively on mice,it suggests that can’t pass blood-brain barrier（BBB）.NPFF has little effection on small intestine and colon itself,but it can bate the opioid-reduced gastrointestinal motility inhibition.So,the activation of NPFF on DN-9 can effectively reduce the constipation.