The Antiepileptic Effect Of KCNQ Potassium Channel Opener

KCNQ type potassium channels belong to voltage dependent potassiumchannel family(Kv7) group, and have been found five subtypes(KCNQ1~5)up to now. KCNQ2and KCNQ3subtypes have constitute M channel andexpresses in the nerve system, which is bound up with convulsive andepileptic symptom in case of gene mutation or M channel’s dysfunction.KCNQ potassium channel have became a key target for screening antiepilepticdrug, such as the first M-channel opener Retigabine (RTG) explored byGlaxoSmithKline and Valeant company. We have found a new kind ofchemical compound, pyrazolo [1,5-a] pyrimidin-7(4H)-one, and it has a goodpotential to open the KCNQ2/3channel(ED500.21μM). This study focuses onmultiple epileptic animal model and establishing in vivo methods forscreening antiepileptic drug. On the other hand, EEG was recorded by abio-signal telemetry system following with the different model operation, andcompared with each other, in order to set up efficient and safe animal testmethods to evaluate the KCNQ potassium channel opener.1The effect of KCNQ channel opener on electrical seizures animal modelsObjective: To observe the effect of KCNQ channel opener on electricalseizures animal models and compare with each compound’s function(Retigabine and QO-58lysine).Methods:(1) Maximal electroshock seizure test (MES)：Forty KM mice, weight22-26g, were divided into four groups randomly (n=10) and administrated byintraperitoneal injection (ip) of the following drugs. Two group wereQO-58lysine with the dosage12.5、25mg/kg respectively, and one group wasRTG with a dosage25mg/kg, and another was the solvent (15%PEG). Theanticonvulsant activity of KCNQ opener was evaluated in the maximalelectroshock seizure model (MES) by calculating the negative-respond mice number or the protection rate, using S48physiological and pharmacologicalmulti-instrument (frequency50Hz, stimulus duration0.2s, alternating current25mA, bandwidth0.1s). Each mouse used only once and all tests wereperformed between9a.m. and14p.m. after administration QO-58lysine30min and RTG15min we determined. The second part animals were orallyadministrated by QO-58lysinate compounds with the dosage25and50mg/kg,and RTG25mg/kg. After administration QO-58lysin50-60min and RTG30min all animals were used the same stimulation parameters to measure theantiepileptic ratio (or called protection rate).(2) Absence epileptic stimulation test (6Hz stimulation). Fifty KM mice,weight22-26g, were divided into5groups randomly and each group had thesame number10animals. The plan of administration was similar to theprevious test (i.p) and levetiracetam (LEV)60mg/kg (i.g) was added as apositive control group compounds. After administration QO-58lysinate(i.p)，RTG(i.p)30min and LEV (i.g)60min,6Hz seizure stimulation parameters setup as the following content: frequency6Hz, stimulus duration:3s, alternatingcurrent32mA, bandwidth0.2ms, to measure the antiepileptic ratio.Results:(1) Protection rate of mice in MES test, with the i.p administrationmethod, QO-58lysine had no significant differences with the control group(P>0.05) and the RTG had different significantly with the control group(P<0.01); In i.g administration test, QO-58lysine had no significant differencewith the control data (P>0.05) and RTG had an obvious difference comparingwith the control data (P<0.01).(2) In6Hz seizure test, there was no different changes between theQO-58lysinate and RTG groups, had no significant differences comparingwith the solvent group (P>0.05)，while LEV group was very obviouslydifferent comparing with the solvent group.Conclusions: Consistent with previous reports, RTG had significanteffect on the convulsions in MES but the QO-58lysinate had no effect. In6Hztest, the two anticonvulsant drug had no effect on the epileptic animal model. 2The effect of KCNQ channel opener on metrazol maximal seizure model（MMS）Objective: To observe the effect of KCNQ channel opener on chemicalcompound seizures animal models test.Methods:(1) Sixty KM mice, weight18-22g, were divided into five groupsrandomly, three of them were administrated with QO-58lysine6.25,12.5,25mg/kg, one was RTG12.5mg/kg, control group was solvent. After injectingintraperitoneally QO-58lysine and RTG30minutes, the metrazol was appliedto observe the threshold of systemic clonic seizures, and record thenegative-respond number in30minutes by injecting PTZ s.c.85mg/kg on theback or neck in mice. The second part of animals was divided five groups andadministrated by oral with25,50,100mg/kg QO-58lysine，12.5mg/kg RTGand solvent PEG（15%）. After60minutes, metrazol was applied to make anepileptic animal model, and measured the antiepileptic ratio.(2) Eight KM mice, weight22-26g, was divided into4groups randomly(n=20), all the animals was respectively administrated once by intraperitonealinjection of12.5,25mg/kg QO-58lysine and12.5mg/kg Retigabine, andsolvent (NS). After administration30min,1%PTZ solution was infused at aconstant rate of0.3ml/min using infusion pump, then observed the epilepticsymptom and gave a score as an evaluation on the convulsive extent, tocalculate the protecting duration.Results:(1) MMS test, Intraperitoneal injection test results：compared withsolvent group, there were obvious different QO-58lysine and RTG (p<0.05，0.01). Three dosage of QO-58lysine and RTG groups had the following resultabout protection ratio8.3%,25%,42%,50%, and middle dosage ofQO-58lysine and RTG had significant differences (P<0.05,0.01). Orallyadministration test results: comparing with solvent group, the middle dosageof QO-58lysine and RTG groups had obviously prolonged the threshold ofbursting epileptic symptoms (P<0.05), while the small and large dosage of QO-58lysine had no any ovbious changes. The three dosage of QO58lysineand RTG had the following result about protection ratio:25%,58%,25%,50%, and the middle dosage QO58lysine and RTG had significant differencecomparing with solvent group (P<0.01) respectively.(2) PTZ administrating by tail vein infusion test, RTG could prolong thelatent of epileptic symptoms significantly (P<0.01), while QO-58lysine had noobvious effect.Conclusions: RTG had a significant effect on the metrazol maximalseizure model, QO-58lysine50mg/kg (i.g) and12.5mg/kg(i.p) also appearedobviously effect on MMS animal.3The effect of KCNQ opener on PTZ kindling seizures animal model andthe change of TrkA and KCNQ2expressing level.Objective: To observe the effect of KCNQ openers on PTZ kindlingseizures animal model and the changes of TrkA and KCNQ2protein’sexpression level.Methods: Sprague-Dawley (SD) rats, weight150-200g, five rats selectedas control group, others injected PTZ30mg/kg intraperitoneally every day,and record the score evaluating the grade of epileptic symptoms, afteradministration30minutes. Choosing the similar28rats were grouped fourparts as the following drugs administrated intraperitoneally: RTG5mg/kg,QO-58lysine12.5mg/kg,25mg/kg and solvent (N.S). To observe what was thegrade and how long the epileptic duration after kindling30minutes using PTZwhich injected7days time. At the end of experiment all animals wereanesthetized to separate the brain for immunohistochemical detecting theexpression of KCNQ2and TrkA protein.Results: Eighty percent of animals were smoothly kindled out typicalepileptic symptoms, after injection30mg/kg PTZ twenty days. Comparingwith control group, RTG could decrease the evaluating score on the4thday（P<0.05）with the efficiency100%. QO-58lysine (12.5mg/kg) began to havean efficient action on the5thday and the evaluating score reduce from the6thday with the efficiency75%, while the higher dosage QO-58lysine (25mg/kg) appeared lower efficiency57.1%. The immunohistochemical result showedthat RTG could broadly decrease the TrkA expression in PTZ inducedepileptic test, while the QO58lysine had little effect on model animals. BothRTG and QO-58lysine had no effect on the KCNQ2protein expression.Conclusions: RTG and QO-58lysine had a clear effect on the PTZkindling model, one side was both drugs could control the epileptic symptomsefficient, the other side was RTG could decrease the expression of TrkAprotein significantly more than QO-58lysine.4The effect of KCNQ channel opener on epileptic animal’selectroencephalogram (EEG)Objective: To observe characters of epileptic animal models’ EEGrecorded by a telemetry system and to find some valuable parameters forscreening some potential epileptic drugs.Methods: SD rats, weight180-220g, planted two screw electrodes torecord EEG of conscious rats after operation one week. In this way, we wereable to record EEG activity and evaluate seizure stage at the same time. Weobserved the changes of EEG pre and after injection PTZ, KA or pilocarpinewhich could lead to seizure reactions. After administration of RTG，QO-58lysine and other antiepileptic drugs three days, recorded EEG and toanalysis the relevance with generalized behaviors.Results:The EEG of rats had a significant changes pre and after injection PTZ.The typical spike waves showed in epileptic seizure, the Phenobarbital sodium(PBT), Carbamazepine (CBZ) and RTG can reduce generalized seizuredurations and spikes numbers. However, the QO-58lysine is instability.In the KA test, the RTG and QO-58lysine can relieve seizure spikes, butnot obvious, so we need further study. In PILO model, the seizure was soserious that could not cure.The animal EEG unlike humans, the α wave was the main band inhuman EEG, but the animal was the low-frequency wave band δandθ. Thechange of EEG induced by PTZ, KA and PILO was different, but all the increased in the proportion of high-frequency waves.Conclusions: EEG prompted, the PTZ, KA and PILO models induceddifferently change in power bands. The EEG can be as an important means ofevaluation of drug efficacy.5Determine the concentration of QO-58lysine in brain and bloodObjective: To determine the concentration of QO-58lysine in brain andblood with HPLC, to preliminary determinate the influence of dfficacy.Methods:22SD rats, weight180-220g, were divided into2groupsrandomly, and each group had11animals. Each group12.5and25.0mg/kg ofthe QO-58lysine administrated continuously3days (two times a day), afterthree days of i.p. administration, detected the concentration of QO-58lysine inbrain and blood.Results: In the HPLC test, the concentration of QO-58lysine in brain andblood were different in rats.Conclusions: HPLC showed that QO-58lysine so unstable effect shouldhave a great relationship with the absorption.