Chemical Construction And Pharmacological Characterization Of OFP022,a Cyclic DN-9 Analogue

Opioids such as fentanyl and morphine are commonly used in clinical analgesics,mainly for the treatment of patients with moderate and severe pain.However,long-term use of such drugs is often accompanied by side effects such as constipation,respiratory depression,addiction and tolerance,which severely limits the clinical use of such drugs.Therefore,the development of new analgesic drugs with low or no side effects is of great scientific significance and potential clinical application value.In recent years,multi-target analgesic compounds based on opioid pharmacophore and NPFF pharmacophore have made important progress in the development of novel analgesic molecules with high efficiency and low side effects.DN-9 is a multi-target compound constructed by our research group using the key pharmacophore of opioid and neuropeptide FF.Pharmacological results showed that peripheral injection of DN-9 could not pass the blood-brain barrier（BBB）,and it was easily degraded by enzymes in vivo.The analgesic effect lasted for 90 min,and the analgesic activity of oral injection was weak.In order to further improve the druggability of DN-9,a series of multi-target analogues were obtained by using amide bond cyclization.It was found that cyclic analogues can act on both opioid and NPFF receptors,and the analgesic activity of peripheral injection is significantly improved,resulting in more long-lasting analgesic effect.It is noteworthy that oral analogizes c[D-Lys²,Asp⁵]DN-9 have oral analgesic activity.Further study on c[D-Lys²,Asp⁵]DN-9 found that the analgesic activity of analogens gradually decreased with the size of amide ring decreasing from 17 to 14,indicating that the size of amide ring formation had a significant impact on the analgesic activity of analogens.When the size of amide bond ring formation was 17,c[D-Lys²,Asp⁵]DN-9showed the best analgesic activity.Therefore,in this study,the amide bond modified c[D-Lys²,Asp⁵]DN-9（amide ring formation is 17）is used as the chemical template molecule.Further,by introducing the unnatural amino acid D-type ornithine（D-Orn）,Its analogue OFP022（Tyr-c[D-Orn-Gly-N-Me-Phe-Glu]-Pro-Gln-Arg-Phe-NH₂）was constructed by ring modification of 2-position D-Orn and 5-position glutamic acid（Glu）amide bond,and was chemically synthesized.And in vitro and in vivo pharmacological activity identification.Firstly,the pharmacological properties of OFP022 were evaluated by c AMP in vitro function assay.The experimental results showed that compound OFP022 could simultaneously activate Mu-（MOR）,Delta-（DOR）,Kappa-（KOR）opioid receptors,as well as NPFF₁and NPFF₂receptors.Compared with the parent DN-9,the excitatory activity of KOR and NPFF₁receptor increased 13.6 times and 22.7 times,respectively,while the excitatory activity of MOR,DOR and NPFF₂receptor decreased slightly,that is,2.1 times,2.7 times and 1.8 times,respectively.Thus,like its parent,OFP022 is a multitarget agonist for opioids and NPFF receptors.In vivo experiments in mice showed that subcutaneous injection and oral administration of compound OFP022 produced dose-dependent analgesia in radiant heat tail flick test.The analgesic ED₅₀values of OFP022 were 0.017 nmol/kg and 0.14nmol/kg,respectively.Compared with c[D-Lys²,Asp⁵]DN-9,the analgesic ED₅₀of OFP022 by subcutaneous injection and oral administration is 16.5 times and 10 times,respectively,and the analgesic time can reach 240 min.In addition,further pharmacological results showed that the oral compound OFP022 produced dose-dependent analgesia in both inflammatory and neuralgia models,with analgesia duration exceeding 180 min.Finally,the effects of OFP022 on analgesic tolerance,constipation and addiction in mice were systematically evaluated.In radiant heat tail flick test,no analgesic tolerance was observed in OFP022 with the highest oral analgesic dose for 8consecutive days.In vivo gastrointestinal motility experiments,oral compound OFP022 inhibited upper gastrointestinal motility dose-dependent,but had no significant effect on gastrointestinal motility in mice at effective analgesic doses.The psychological and physiological addictiveness of oral OFP022 was evaluated by CPP test,open field test and naloxone-induced opioid withdrawal test.In the open field test,oral OFP022 did not significantly enhance motor activity in mice.In the CPP experiment,the compound OFP022 did not show conditional place preference.In naloxone-induced opioid withdrawal trials,oral compound OFP022 did not show withdrawal symptoms.Therefore,at effective analgesic doses,oral OFP022 showed no significant opioid side effects such as analgesic tolerance,constipation and addiction.In summary,OFP022 is a novel structure of amide-bond cyclization DN-9analogue with multi-target excitatory activity of opioid and NPFF receptors.Compared with maternal c[D-Lys²,Asp⁵]DN-9,OFP022 produces stronger analgesic activity by subcutaneous injection and oral administration.OFP022 also retains the long-acting oral analgesic effects of maternal c[D-Lys²,Asp⁵]DN-9,without significant opioid side effects such as analgesic tolerance,constipation,and addiction.Therefore,this study further confirmed that the amide bond cyclization strategy can effectively improve the druggability of opioid/NPFF receptor multi-target peptide molecules,which is expected to provide a new idea for the development of new analgesic peptide drugs with long effect and low side effects.