The Electrophysiological Characterization Of Perampanel And The Mechanisms Of Anti-epileptic Compounds

Epilepsy is a servere neurological disorder affecting approximately 1%-2%population worldwide,which results from abnormal hyperexcitable and hypersynchronous activity of neurons.It is characterized by the loss of consciousness and periodic convulsions.Various studies in animal models and humans suggests that dysfunction of both voltage-and ligand-gated ion channels make a major contribution to the cause of many different types of epilepsy.Most of the marketly anti-epileptic drugs targert voltage-and ligand-gated ion channels,protease and synaptic vesicle protein.In the past decades,despite the increased markedly number of new anti-epilepptic drugs gaining regulatory approval,one-third of patients still have poorly controlled epilepsy and continue to experience refractory seizures.Therefore,the development of the new anti-epileptic drugs is required for most efforts.Perampanel（PRP）,approved by the FDA in 2012,was used to the treatment for the partial onset seizures in patients with epilepsy aged 12 years and older.Both fluorescence and radioligand binding assays have been used for the mechanism studies of PRP.However,there is no eletrophysiological evidence..We tested electrophysiological properties of PRP using patch-clamp technology in cultured or acute dissociated hippocampal neurons.We found,at 10μM concentration,PRP completely suppressed both the stimulated and spontaneous action potential firing.At the same concentration,PRP inhibited AMPA and kainite acid induced currents without affecting NMDA,GABA and voltage gated K⁺,Na⁺,Ca²⁺channels,indicating a high selectivity of PRP.Dose-dependent analysis revealed IC₅₀values（113.0±14.6n M and 78.3±19.1 n M）of PRP for AMPA and kainite acid.Noticeably,The EC₅₀value of AMPA was not affected by pre-applied PRP,which validates that PRP non-competitively inhibits AMPA activity.In addition,we firstly found that perampanel emerges potential neuroprotective effect against kainate-induced neuronal injury.Being a first-line antiepileptic drug,topiramate blocks both sodium channel and calcium channel.In contrast,retigabine is the first approved potassium channel agonist for the treatment for epilepsy.Both topiramate and retigabine show excellent anti-epileptic effect and pharmacokinetic properties.In our early studies,we have obtained a number of structural analogs designed by the molecular skeleton of them.Then through screening on maximal electrical stimuli（MES）and pentetrazole（PTZ）-induced animal model,three anti-epileptic candidates,ZGS03,ZGN1and ZGN2,were identified.We have demonstrated that ZGN1and ZGN2 exhited anti-epileptic effects through enhancing KCNQ chanenles.But the mechanism of ZGS03 is unclear.We examined the selectivity of ZGS03,ZGN1and ZGN2 on various channels expressed primary cultured hippocampal neurons.The results revealed that 100μM ZGS03 exerted weak inhibitory effects on calcium channel and sodium channel but did not affect other tested channels,including K⁺,GABA,AMPAR and NMDAR channels.ZGN1 and ZGN2 dose-dependent blocked calcium channel,retaining the enhancement by 10μM ZGN2 for GABA receptors.In summary,our studies demonstrated that PRP is a non-competitive AMPA receptor antagonist with high selectivity using electrophysiology technique.We also found that the neuroprotective effects of PRP may contribute to its anti-epileptic activity.In addition,the ion channel selectivity of the three anti-epileptic compounds was systematically evaluated.