Studies Of Antinociceptive Site(s) And Tolerance Induced By The Bifunctional Opioid /NPFF Receptors Agonist BN-9 In Preclinical Pain Models

Bifunctional drugs had been used in the treatment of cancer,diabetes,depression and infectious diseases.In recent years,bifunctional analgesics with high efficiency and low side effects were attracted widespread attention.The novel bifucntional nonapeptide BN-9(Tyr-D.Ala-Gly-Phe-Gln-Pro-Gln-Arg-Phe-NH2)behaved as a full agonist towards ?-,?-,?-opioid,NPFF1 and NPFF2 receptors in functional assays.And BN-9 produced potent non-tolerance forming antinociception in the tail-flick test.To further validate the non-tolerance forming antinociception of BN-9,the anti-allodynic activities,site(s)of action and tolerance of BN-9 are comprehensively evaluated in the preclinical models.In the present studies,we initially chosen the classic carrageenan inflammatory pain model and investigated the analgesic sites of BN-9 by different routes of administration.The results suggest that supraspinal(i.c.v.)and spinal(i.t.)administrations of BN-9 dose-dependently decreased carrageenan-induced mechanical allodynia via opioid receptors,and the antinociceptive actions were augmented by the NPFF receptor antagonist RF9.In contrast,hindpaw(i.paw),intraperitoneal(i.p.),subcutaneous(s.c.),and intramuscular(i.m.)injection of BN-9 produced anti-allodynic effects in an opioid receptor-dependent manner,independent of the NPFF receptor.Furthermore,the anti-allodynia of systemic BN-9 was blocked by naloxone methiodide(i.p.),but not by i.c.v.injection of naloxone methiodide,indicating that anti-allodynic effect of intraperitoneal injection of BN-9 mainly mediated by peripheral opioid receptors.In addition,i.paw of naloxone can significantly block the anti-allodynic effect of BN-9(i.p.),it's also demonstrated the peripherally anti-allodynic properties of BN-9,and the peripherally anti-allodynia of BN-9 did not mediated by NPFF receptor.These results suggest that central,peripheral or systemic administrations of BN-9 exert potent analgesic activities in the inflammatory pain models.I.c.v.injection of BN-9 had the strongest analgesic effect,followed by i.t.BN-9,and the central injection BN-9 showed the opioid and NPFF receptors agonistic character.While the anti-allodynic effect induced by peripheral and systemic injection of BN-9mainly mediated by peripheral opioid receptors,and BN-9 can not cross the blood-brain barrier.In view of the above-mentioned facts that,BN-9 can not cross the blood-brain barrier and the central anti-allodynic effect analgesia is more strong.Therefore,we investigated the analgesic mechanism and tolerance in other experimental models after i.t.injection of BN-9.The results showed that BN-9 could produce dose-dependently analgesic effect in CFA inflammatory pain,incisional pain,acetic acid writhing and formalin test via different mechanisms.In the CFA inflammatory pain,opioid receptor antagonist naloxone blocked anti-allodynic effect of BN-9,and were augmented by the NPFF receptor antagonist RF9,which support the bifunctional opioid/NPFF receptors agonism character of BN-9.Both naloxone and RF9 can antagonize the anti-allodynic effect of BN-9 in the incisional pain.In acetic acid writhing test and formalin test,BN-9 produced antinociceptive effects in an opioid receptor-dependent manner,which were not modified by RF9.In addition,BN-9(1 nmol)has no effects on the mechanical latency of naive rats after consecutively 7 days treatment.However,morphine(2 nmol)induced significant allodynia after chronical administration.Repeated administration of BN-9(1 nmol)did not lead to the development of anti-allodynic tolerance in the CFA inflammatory pain and no cross-tolerance to morphine(2 nmol).In conclusion,BN-9 produced a significant analgesic effect on acute and chronic pain,which is mainly mediated by opioid receptors.And BN-9 produced potent non-tolerance forming antinociception in chronic pain models.