The Selection Of Opioid Receptor Subtypes In The Sedative Of FENT-001 And Mechanisms Of Its Actions

Opioidsrepresented as analgesic drugs are an irreplaceable cure for pain therapy and anesthesia.Fentanyl and its derivatives achieve the ideal analgesic effect,but also have the advantage with low active metabolites and side effect.FENT-001,as an analog of the synthetic fentanyl,has a high potency and short effective time.Like fentanyl,FENT-001 can activate the G protein(mainly Gi protein)by acting on the opioid receptors(μ,δ,κ)subtypes of the membrane and affect the downstream signal molecules to produce physiological and pharmacological function.At present,many studies have focused on the analgesic effect of fentanyl,while the sedative effect of fentanyl is rarely known.It is unclear whether the analgesic effect of FENT-001 and sedation are activatingonthe same receptor subtype,or a common signaling pathway,or independent of each other.In this study,we attempt to explain the relationship between the sedation of FENT-001 and the subtypes of opioid receptors,and to explore the mechanism of sedation in order to support the data of sedation of opioids.Objective:The main purpose of the study isto explore the binding affinity andagonistic activity of FENT-001 with opioid receptor subtypes,andto clarify the selectivity and contribution of opioid receptor subtypes of FENT-001-induced sedation.Methods:(1)To evaluate the sedative effect of FENT-001 through the locomotor activity,the rotarod test,the loss of righting reflex(LORR)and the respirationparameter test.(2)Pretreatment with different opioid receptor subtypes antagonists were performed to observe the effect of FENT-001-induced sedation through the above mentioned animal models.And weusedμ-opioid receptor knockout mice to confirm the sedationrelated opioid receptor subtypes of the FENT-001.(3)The radioactivity ligand binding assay and PKA redistribution experiment were used to investigate thebinding affinity and efficacy of FENT-001 on CHO-PKAcat-EGFP-m,CHO-PKAcat-EGFP-d and CHO-PKAcat-EGFP-k stable transfected cell.Results:(1)In the locomotor activity model,the locomotor activity of mice was reduced to 60% compared with that of saline group after FENT-001(80 μg/kg,s.c.)treatment.In the rotarod test,FENT-001(20 μg/kg,s.c.)treatment reduced the time on the rotarod from 180 s to 50 safter 15 min of administration.In LORR test,the EC50 value in mice was 119.0 μg/kg.And FENT-001(3.2-10.00μg/kg,i.v.)dose-dependently reduced induction time and prolonged immobilization time in rats.In pulmonaryrespiratoryparameters test,FENT-001(5-40 μg/kg,i.v.)dose-dependently induce respiratory depressionand decreased to 40% compared with that of baseline in 10 min.(2)Preatment with CTOP(160 ng/mouse,i.c.v.)or NTI(3 mg/kg,s.c.)antagonizedthe effect of FENT-001-induced(5-80 μg/kg,s.c.)locomotor activity,whereas nor-BNI(0.5 mg/kg,s.c.)had no effect.Preatment withCTOP(160ng/mouse,i.c.v.)or NTI(3 mg/kg,s.c.)significantly reversed the time on the rotarod induced byFENT-001(20 μg/kg,s.c.),while nor-BNI(0.5 mg/kg,s.c.)had no effect.Preatment withCTOP(2 μg/rat,i.c.v.)increased induction time and shortened immobilization time ofLORRinduced by FENT-001(8 μg/kg,i.v.),but immobilization time was not statistically significant.While preatment with NTI(5 mg/kg,s.c.)or nor-BNI(5 mg/kg,s.c.)had no effect on induction time and immobilization timeinduced by FENT-001(8 μg/kg,i.v.).Preatment with CTOP(2 μg/rat,i.c.v.)improvedrespiratory depression induced by FENT-001(5μg/kg,i.v.).Preatment with NTI(5 mg/kg,s.c.)or nor-BNI(5 mg/kg,s.c.)did not show antagonism to FENT-001-induced respiratory depression.Treatment with FENT-001 not influenced locomotor activity and the time on the rotarod in the rotarod test with m opioid receptor knockout mice.Treatment with FENT-001 notaffected LORR on m opioid receptor knockout mice.(3)The results of radioligand receptor saturation test showed that[3H]diprenorphine(0.0625-2 nM)had high saturation binding with μ,δ and κopioid receptors,with theKd values as1.09 ± 0.60 nM,1.76 ± 1.51 nM and0.61 ±0.84 nM;Bmax values as1.76 ± 1.46 pmol/mg protein,9.70 ± 8.24pmol/mgprotein and1.28 ± 1.72 pmol/mg protein,respectively.The binding of radioactive ligand receptors showed that FENT-001 had high affinity forμopioid receptor,with Ki value as 0.03 ± 0.11 nM.For δ and κ opioid receptors,the Ki values were 20 ± 3.3 nM and 71 ± 17 n M,respectively.PKA redistribution experiments showed that FENT-001 had high efficacy with μ,δ,κ opioid receptors,the EC50 values were 0.9 ± 0.5 nM,8.7 ± 1.3 nM and44.2 ± 3.6 nM,respectively.Conclusion:(1)μ opioid receptor plays a key role in the sedation of FENT-001,and δopioid receptor maybe involved in the sedation ofFENT-001,andκ opioid receptor not participate in the sedation of FENT-001.(2)FENT-001 has high affinity and efficacy with mopioid receptor.