| Pathological pain as one of the major clinical problems threatens for human health in today’s society,and it is an unmet medical need.The fundamental mechanism underlying the pain is characterized by neuronal hyperexcitability and repetitive firings.Ion channels serve as a molecular basis of neuronal excitability and pain signaling.Voltage-gated sodium channel Nav1.7 is robustly expressed in peripheral nociceptive neurons.Studies have demonstrated that the Nav1.7 mutation produces a distinct human pain syndrome and is currently considered as a therapeutic target for pathological pain,and targeting Nav1.7 by selective inhibition may be beneficial for therapy of pathological pain.Here we reported the anti-nociceptive activity of our novel identified small molecule QLS-81 that was modified based on drug candidate ralfinamide currently in phase II clinical trial for chronic pain.QLS-81 dose-dependently inhibited the current of Nav1.7 channel,also displayed efficacious anti-nociceptive activity in a dose-dependent manner in mice models of spared nerve injury(SNI)for neuropathic pain and formalin-induced inflammatory pain.These results indicate that QLS-81 has potential to be developed to treat the diseases related to the neuronal pain,in order to provide experimental basis for therapeutic targets of neuropathic pain.Objective:The aim of this study is to study the characteristics of QLS-81 on Nav1.7 channels and to evaluate the analgesic effect of QLS-81 in mice models of inflammatory pain and neuropathic pain.Methods:The recording of QLS-81 on Nav1.7 stably transfected HEK293 was applied using whole-cell patch clamp technique.The analgesic effect of QLS-81 was tested in neuropathic pain and inflammatory pain animal models.Results:1.The results showed that 10 μM QLS-81 can significantly inhibited the peak current of Nav1.7 channel in the state of inactivation.2.QLS-81 inhibited the Nav1.7 current in a dose-dependent manner with an IC50 of 3.52±1.52 μM,as compared with ralfinamide at IC50of 32.17±7.24 μM.3.The half activatin voltage(V1/2)values for Nav1.7 were not significantly shifted in the presence of QLS-81 at 10 μM V1/2=-26.77±0.76 mV,compared with control of V1/2=-28.01±0.41 mV.4.Perfusion of 10 μM QLS-81 caused a significant leftward-shift of Nav1.7 channel fast and slow inactivation to more hyperpolarizing membrane potential at-107.00±1.17 mV and-71.49±3.81 mV from-99.50±2.04 mV and-46.34±4.78 mV,respectively.5.QLS-81 at 10 μM significantly prolonged the channel recovery time constant from fast inactivation and slow inactivation at 14.59±0.77 s and 60.07±6.41 s,as compared with that of the control at 8.37±0.37 s and 46.37±5.52 s.6.In the absence of QLS-81,Nav1.7 channel was not significantly inhibited under 3 Hz,5 Hz,10 Hz stimulation,and the inhibition was only 18.46±2.52%under 30 Hz stimulation.In the presence of QLS-81,the currents were inhibited by 4.44%±2.11%,13.61±4.45%,29.44%±5.63%,and 79.23±6.03%,when stimulated at 3 Hz,5 Hz,10 Hz and 30 Hz,respectively.It suggests that the inhibition effect of QLS-81 on Nav1.7 channel is frequence depentdent.7.In the chronic neuropathic pain spared nerve injury pain model,compared with vehicle group mechanical threshold of 0.03±0.01 g,and intraperitoneal injection of 2,5,and 10 mg·kg-1 QLS-81-81 and 10 mg-kg-1 Ralfinamide increased the average mechanical threshold to 0.25±0.01,0.45±0.03,0.98±0.03 g and 0.52±0.02 g,respectively.It suggested that the anti-nociceptive activity of QLS-81 is about 10 folds efficious than ralfinamide.8.In the formalin-induced inflammatory pain model,QLS-81 has no obvious analgesic effect on phase I.The total claw licking time of injection of 2,5,10 mg·kg-1 QLS-81 and 10 mg-kg-1 Ralfinamide in phase Ⅰ reaction was 27.3±6.02 s,13.6±4.86 s,16.3±5.57 s and 18.20±3.96 s,compared with the total paw licking time of the control group with 26.2±5.71 s.The phase Ⅱ reaction total licking time was 291.00±46.19 s,201.2±30.29 s,164.80±26.04 s and 294.90±51.94 s,compared with the control group with 392.75±42.26 s.QLS-81 could dose-dependently inhibit the Ⅱ phase inflammatory pain caused by formalin-induced inflammatory pain,while ralfinamide has no obvious effect.9.The selectivity of the compounds to other ion channels was evaluated;QLS-81 inhibited the peak current of Nav1.4 with an IC50 of 37.28±7.33 μM.QLS-81 inhibited the peak current of Nav1.5 with an IC50 of 15.39±1.64 μM.QLS-81 was not significantly affected for TRPV1 channel in the presence of QLS-81 at 30 μM.10.The results showed that 100 μM QLS-81 inhibited about 21.49±2.61%of the hERG channel current,suggested that QLS-81 may not impose a significant risk on the heart.Conclusions:No significant inhibition effects were observed on the activation of Nav1.7 channel by QLS-81,and it shifted the fast and slow inactivation curves of Nav1.7 to hyperpolarization direction and also caused the delay of fast and slow inactivation recovery.In addition,QLS-81 inhibited Nav1.7 in both dose-dependent and use-dependent manners.Intraperitoneal administration of QLS-81 displayed efficacious anti-nociceptive activity in a dose-dependent manner in mice models of spared nerve injury for neuropathic pain and formalin-induced inflammatory pain,as 10 folds more efficacious than ralfinamide.Our results demonstrate that the small molecule QLS-81 may hold promise for therapeutic potential of chronic pain. |
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