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Animal Model Of Epilepsy And Evaluation Of Antiepileptic Drugs

Posted on:2024-01-23Degree:MasterType:Thesis
Country:ChinaCandidate:S XieFull Text:PDF
GTID:2544307157455004Subject:Pharmacology
Abstract/Summary:PDF Full Text Request
Epilepsy is a temporary malfunction caused by abnormally excessive or synchronized firing of neurons in the brain.At present,the main means of clinical treatment of epilepsy is drug therapy,but there are still 30%of patients in the world are difficult to cure,so it is of great significance to find effective antiepileptic drugs.With the exception of some early treatments,including bromide and phenobarbital,the antiepileptic activity of all clinically used agents has been largely determined by acute seizure models in rodents.Clinically,commonly used antiepileptic drugs include phenytoin sodium,carbamazepine,topiramate,sodium valproate,etc.Studying the effects of these classical antiepileptic drugs on different animal models of acute epilepsy expands the treatment options,provides a lot of help for those who need to change the medical regimen,and is conducive to the targeted drug use and new drug research and development.The novel compound QO-83 is a novel drug with novel structure designed and synthesized by our laboratory.It has complete independent intellectual property rights and is a candidate compound for anti-epilepsy innovative drug.In this study,the antiepileptic effect of compound QO-83 on the whole animal level will be investigated using different animal models of epilepsy.Part one:The preparation of animal model of epilepsy and the influence of classical antiepileptic drugs on animal model of epilepsyOne:Preparation of animal model of epilepsy and the influence of classical antiepileptic drugs on animal model of epilepsyObjective:To establish a standard operation method for the preparation of various acute animal models of epilepsy,observe the effects of different classical antiepileptic drugs on different animal models of epilepsy,and screen out positive drugs that have effects on different models.Methods:1.Explore the best dosage of antiepileptic drugs pentetetrazole,penicillin,hymenic acid and pilocarpine,and then study the efficacy of antiepileptic drugs phenytoin sodium,sodium valproate,carbamazepine and topiramate in different doses to find the best dosage of antiepileptic drugs.2.An animal model of Pentylenetetrazol(PTZ)acute epilepsy:male C57mice were randomly divided into 5 groups:Saline group(NS),Phenytoin sodium group(PHT),Valproate sodium group(VPA),Carbamazepine group(CBZ),Topiramate group(TPM).The normal saline group was intraperitoneally injected with pentatetrazole to establish an animal model of acute epilepsy,and the normal saline was injected 30 min before pentatetrazole.Phenytoin sodium group was intraperitoneally injected at the dose of 50 mg/kg.Sodium valproate group was intraperitoneally injected at the dose of 300 mg/kg.Carbamazepine group was given 80 mg/kg intragastric administration;Topiramate group was intraperitoneally injected at a dose of 50mg/kg.The solvent was normal saline,and 80 mg/kg pentatetrazole was injected intraperitoneally 30 min after administration.After 60 min of video observation,epileptic behavior was scored according to Racine scale.3.An animal model of Penicillin(PNC)acute epilepsy:male C57 mice were intraperitoneally injected penicillin in the normal saline group to develop penicillin acute epilepsy animal models,which were divided into the normal saline group,phenytoin sodium group,sodium valproate group,carbamazepine group,topiramate group,and the administration method was the same as the pentatetrazole model.After 60 min of video observation,epileptic behavior was scored according to Racine scale.4.An animal model of Pilocarpine(PILO)acute epilepsy:male C57 mice were divided into normal saline group,phenytoin sodium group,valproate sodium group,carbamazepine group and topiramate group.In the normal saline group,1 mg/kg racemic anisodamine hydrochloride was intrabitoneal injected to antagonize peripheral cholinergic reaction.15 min later,350 mg/kg racemic anisodamine hydrochloride was intrabitoneal injected to establish pilocarpine epilepsy model.15 min before racemic anisodamine hydrochloride was injected,normal saline solvent was injected.Administration method were the same as the pentatetrazol model.After 60 min of video observation,epileptic behavior was scored according to Racine scale.5.An animal model of Kainic acid(KA)acute epilepsy:male C57 mice were intraperitoneally injected with KA to establish the animal model of acute epilepsy.The grouping method and administration method were the same as the pentatetrazol model.Epileptic behavior was scored on Racine scale.6.Maximal electroshock seizure model(MES):male C57 mouse,grouped in the same way as pentatetrazole model.The positive standard was hind limb stiffness after electrical stimulation.The number of mice without tetanic convulsion and the number of deaths were recorded,and the maximum electroconvulsive protection rate and death rate were calculated.Results:1.The optimal dosage for epileptogenic agents was 80mg/kg pentatetrazole,800×104U/kg penicillin,20mg/kg hyacinic acid and 350mg/kg pilocarpine.The optimal dosage of antiepileptic drugs is 50mg/kg phenytoin,300mg/kg valproate,80mg/kg carbamazepine and 25mg/kg topiramate.2.Animal model of acute epilepsy with pentatetrazole:compared with normal saline group,the tertiary incubation period was significantly prolonged in sodium valproate group,but there was no statistical difference,there was no significant change in phenytoin sodium group,carbamazepine group and topiramate group.Stage IV incubation period and stage V incubation period in sodium valproate group were significantly longer,with statistical significance(***P<0.001);Stage IV incubation period in phenytoin sodium group,carbamazepine group and topiramate group had no significant change compared with saline group;Stage IV incubation period in carbamazepine group and topiramate group was longer than that in saline group,but there was no statistical difference.The attack grade and attack rate of sodium valproate group were significantly lower than that of saline group,with statistical significance(***P<0.001),but there was no statistical difference in the attack grade and attack rate of phenytoin sodium group,carbamazepine group and topiramate group.The number of attacks decreased in both the valproate and phenytoin groups compared with normal saline(***P<0.001,**P<0.01).3.Animal model of penicillin acute epilepsy:compared with normal saline group,except for phenytoin sodium group,valproate sodium group,carbamazepine group and topiramate group showed a trend of prolongation of stage III,IV and V latency,but there was no statistical difference,the attack frequency and attack rate of phenytoin sodium group were significantly increased(*P<0.05)(**P<0.01);the number of attacks,duration and mortality in the sodium valproate group showed a decreasing trend,but there was no statistical difference,the attack frequency,duration,attack grade and mortality in carbamazepine group all showed a downward trend,but there was no statistical difference,and the attack rate in carbamazepine group was significantly decreased(*P<0.05).4.Animal model of acute epilepsy with pilocarpine:Compared with the normal saline group,the tertiary incubation period of sodium valproate group had a prolonged trend,but there was no statistical difference,the third-order latency of phenytoin sodium group,carbamazepine group and topiramate group all showed a trend of shortening,but there was no statistical difference,the fourth stage incubation period in sodium valproate group was significantly prolonged,with statistical difference(**P<0.01),the latent period of stage V in sodium valproate group and carbamazepine group had a longer trend,but there was no statistical difference.There was no significant change in attack grade and attack rate.5.Animal model of acute epilepsy with Hyman acid:Compared with the normal saline group,the tertiary incubation period of phenytoin sodium group and carbamazepine group had a longer trend,but there was no statistical difference.There were no significant changes in stage IV latency,stage V latency,attack grade and attack time in phenytoin sodium group,carbamazepine group and topiramate group,the attack frequency of sodium valproate group was significantly increased,with statistical difference(**P<0.01),but there was no significant change in phenytoin sodium group,sodium valproate group and carbamazepine group.The attack rate of the topiramate group was lower than that of the saline group,but there was no statistical difference.6.Maximum electroshock model:Compared with normal saline group,the maximum protection rate of electroshock was increased in all administration groups,and the difference was statistically significant in sodium valproate group(*P<0.05),there was a trend of decrease in mortality but no statistical difference.Conclusions:The results of epileptic ethology show that not every classical antiepileptic drug has anticonvulsive and antiepileptic effects on all acute epileptic models.Effective drugs for pentatetrazole model were sodium valproate,effective drugs for penicillin model were sodium valproate and carbamazepine,effective drugs for pilocarpine model were sodium valproate,effective drugs for hymenic acid model were sodium valproate and topiramate,effective drugs for maximum electroshock model were sodium valproate.Two:The animal model of epilepsy and positive antiepileptic drugs were verifiedObjective:To further validate the effect of positive antiepileptic drugs,and to identify positive antiepileptic drugs that are effective to each animal model of epilepsy.Methods:1.Animal model of acute epilepsy with pentatetrazole:Male C57 mice were divided into NS group and VPA group.NS group was intraperitoneally injected with pentetrazole to establish an animal model of acute epilepsy,and normal saline was injected 30 min before pentetrazole injection.The VPA group was intraperitoneally injected at 300 mg/kg.After 60 min video observation,epileptic behavior was scored according to Racine grading criteria.2.Animal model of penicillin acute epilepsy:male C57 mice were divided into NS group,VPA group and CBZ group.NS group was intraperitoneally injected with PNC,and normal saline was injected 30 min before PNC injection.VPA was intraperitoneally administered at a dose of 300mg/kg,and CBZ was intragastric administered at a dose of 80 mg/kg.After 60min video observation,epileptic behavior was scored according to Racine grading criteria.3.Animal model of acute epilepsy of pilocarpine:male C57 mice were divided into NS group and VPA group.NS group was treated with 1 mg/kg racanisodamine hydrochloride intrabitoneal injection to antagonize peripheral cholinergic reaction.15 min later,350 mg/kg racanisodamine hydrochloride intrabitoneal injection was used to establish pilocarpine epilepsy model,and15 min before racanisodamine hydrochloride injection,normal saline was injected.The VPA group was intraperitoneally injected at 300 mg/kg.After 60min video observation,epileptic behavior was scored according to Racine grading criteria.4.Animal model of acute epilepsy with Hyman acid:male C57 mice were divided into NS group,VPA group and TPM group.NS group was intraperitoneally injected with KA;Intraperitoneal injection of VPA and TPM.After 60 min video observation,epileptic behavior was scored according to Racine grading criteria.5.Maximum electroshock model:male C57 mice were divided into NS group and VPA group.NS group was intraperitoneally injected with normal saline.The VPA group was intraperitoneally injected at 300 mg/kg.The number of mice without tetanic convulsion and the number of deaths were recorded,and the maximum electroconvulsive protection rate and death rate were calculated.Results:1.Animal model of acute epilepsy with pentatetrazole:compared with saline group,VPA significantly prolonged the incubation period of grade 4 and5(*P<0.05),and reduced the seizure grade(***P<0.001).2.Penicillin acute epilepsy animal model:compared with saline group,VPA and carbamazepine prolonged stage IV latency(*P<0.05)(***P<0.001)and reduced mortality(**P<0.01).3.Animal model of acute epilepsy with pilocarpine:compared with normal saline group,VPA prolonged stage 4 incubation period(**P<0.01)and stage 5 incubation period(*P<0.05),and reduced the seizure grade(**P<0.01)and the number of seizures(**P<0.01).4.Animal model of acute epilepsy with hymenic acid:compared with normal saline group,VPA and TPM did not show better antiepileptic effects.5.Maximum electroconvulsive model:Compared with normal saline group,VPA could increase maximum electroconvulsive protection rate(***P<0.001)and reduce mortality rate(**P<0.01).Conclusions:Different animal models of epilepsy were successfully verified.Positive antiepileptic drugs were found for different animal models of epilepsy,and their antiepileptic effects were further verified.Part two:The antiepileptic effect evaluation of the novel KCNQ channel opener QO-83One:Evaluation of antiepileptic effect of QO-83Objective:To observe the antiepileptic effects of the novel compound QO-83 on different epilepsy models at the animal level.Methods:The antiepileptic effect of the new compound QO-83 on different epileptic animal models was observed by using the stable animal model of epilepsy prepared in the first part.1.Animal model of acute epilepsy with pentatetrazole:Male C57 mice were divided intoβ-cyclodextrin group(β-CD)and compound QO-83 group(1.5 mg/kg)(QO-83 was prepared withβ-cyclodextrin).Compound QO-83group andβ-cyclodextrin group were injected intraperitoneum 30 min before PTZ administration,and epileptic behavior was observed 60 min by video recording.Score on Racine scale.2.Animal model of penicillin acute epilepsy:Male C57 mice were divided intoβ-cyclodextrin group(β-CD)and compound QO-83 group(1.5mg/kg).PNC modeling dose was 8.5 million U/kg.QO-83 group andβ-cyclodextrin group were intraperitoneal injected 30 min before PNC administration.Epileptic behavior was observed by video for 60 min and scored according to Racine grading criteria.3.Animal model of acute epilepsy with Pilocarpine:Male C57 mice were divided intoβ-cyclodextrin group(β-CD)and compound QO-83 group(1.5mg/kg).The mice were intraperitoneally injected with 1 mg/kg racanisodamine hydrochloride to antagonize the peripheral cholinergic reaction.15 minutes later,the mice were intraperitoneally administered with350 mg/kg pilocarpine.QO-83 group was intrabitoneally injected with compound QO-83(1.5 mg/kg)15 min before the injection of racanisoamine hydrochloride,and the epileptic behavior was observed for 60 min by video recording,and scored according to Racine grading criteria.4.Animal model of acute epilepsy with hymenic acid:Male C57 mice were divided intoβ-cyclodextrin group(β-CD)and compound QO-83 group(1.5 mg/kg).The mice were intraperitoneically injected with 50 mg/kg hydrochloric acid,and were injected withβ-cyclodextrin and compound QO-83(1.5 mg/kg)30min before hydrochloric acid injection.Epileptic behavior was observed by video for 60 min and scored according to Racine grading criteria.5.Maximum electroshock model:Male C57 mice were divided intoβ-cyclodextrin group(β-CD)and compound QO-83 group(1.5 mg/kg),and intraperitoneally injectedβ-cyclodextrin and compound QO-83(1.5 mg/kg)30min before electrical stimulation.The number of mice without tetanic convulsion and the number of deaths were recorded,and the maximum electroconvulsive protection rate and death rate were calculated.Results:1.Pentatetrazole acute epilepsy animal model:compared withβ-cyclodextrin group,compound QO-83 group significantly extended the third,fourth and fifth order incubation periods(*P<0.05)(**P<0.01)(***P<0.001),shortened the duration and frequency of seizures,and reduced the mortality.2.Penicillin acute epilepsy animal model:compared withβ-cyclodextrin group,compound QO-83 group significantly prolonged the third and fourth incubation periods(**P<0.01)(***P<0.001),and the fifth incubation period had little change.3.Animal model of acute epilepsy of pilocarpine:compared withβ-cyclodextrin group,compound QO-83 group showed a trend of prolongation of tertiary and tertiary incubation periods,but there was no statistical difference,the quaternary latency was significantly prolonged(*P<0.05).There was a downward trend in mortality,but no statistical difference.4.Animal model of acute epilepsy with hydmanic acid:compared withβ-cyclodextrin group,the tertiary incubation period of compound QO-83group was significantly prolonged(*P<0.05),which was statistically significant;The seizure grade was significantly lower(*P<0.05).5.Maximum electroshock model:Compared withβ-cyclodextrin group,compound QO-83 showed no significant difference in maximum electroshock protection rate and death rate.Conclusions:The novel compound QO-83 was effective in acute epileptic model induced by pentatetrazole,penicillin,pilocarpine and hymenic acid,and had obvious antiepileptic effect,but had no obvious effect on maximal electroconvulsive model.Two:Evaluation of antiepileptic effect of novel compound QO-83 in different dosage formsObjective:To evaluate the antiepileptic effects of different oral preparations of the novel compound QO-83 by using the maximum electroshock model in mice and the pentatetrazole model in rats,and to provide evidence for the development of innovative drug formulations.Methods:1.Maximum electroconvulsive model(MES)of mice:Kunming(KM)mice were used as experimental animals,divided into control group and drug administration group,and the maximum electroconvulsive protection rate of MES model of mice with different doses and different time was investigated.2.Rat models of acute epilepsy with pentatetrazole were divided into control group(CMC)and administration group.CMC group was intragastric with solvent,and the administration group was intragastric with coprovidone for 100 mesh hot melt extrusion(preparation 1),the dose was 15 mg/kg,and90min later,60 mg/kg pentatetrazole was intraperitoneally injected.Results:1.The maximum electroshock model of mice was as follows:(1)The protection rate of 10 mg/kg during hot melt extrusion of 100 mesh(preparation 1)at 60 min and 90 min was 0 and 70%,respectively.Compared with CMC group,the protection rate of 10 mg/kg for 90 min was increased(***P<0.001).The protection rates of 20 mg/kg copolymerovidone were100%and 90%at 90 min and 120 min,respectively,which were increased compared with CMC group(***P<0.001).At 90 min,the protection rates of10,20,30 and 50 mg/kg were 70%,100%,100%and 100%,respectively,which were increased compared with CMC group(***P<0.001).The protection rate of compound QO-83 was 30%as the bulk drug(30 mg/kg).Compared with the bulk drug,the protection rate of 20,30 and 50 mg/kg(100%)was increased at 90 min after hot melt extrusion of 100 mesh(preparation 1)(##P<0.01).(2)When the dosage of copovidone hot melt extrusion micropowder(preparation 2)was 1,2.5,5,10,20,50 mg/kg,the protection rate was 0,20%,55.6%,75%,100%,100%,ED50was 4.49 mg/kg,compared with CMC group,there was significantly increased(**P<0.01,***P<0.001).(3)The protection rates of 10 mg/kg hot melt extrusion 1:3(preparation 3)for 45 min and 90 min were 33.3%and 37.5%,respectively.There was no significant difference compared with CMC group.(4)The protection rates of Soluplus hot melt extrusion 1:4(preparation 4)10 mg/kg at 45 min and 90 min were 12.5%and 25%,respectively.Compared with CMC group,there was not statistically different.(5)The protection rate of 15 mg/kg QO-83 dihydrochloride(preparation 5)was 57.1%,and there was no statistical difference between CMC group and CMC group.(6)The protection rate of 1:2.5(preparation 6)hot melt extrusion at 10mg/kg for 90 min was 30%,and that of 1:3(preparation 7)hot melt extrusion at 5 mg/kg for 90 min was 40%.The protection rate of 1:3(preparation 8)90min at 10 mg/kg was 40%,the protection rate of QO-83(0.5%CMCNA:0.5%TW80)7.63mg/ml(D90=344n M)(preparation 9)90 min at 10 mg/kg was 40%.Compared with CMC group,the protection rate of preparations 7,8 and 9 was significantly increased(*P<0.05).2.Acute epilepsy model of rats with pentatetrazole:compared with CMC group,the incubation period of stage III,IV and V was prolonged by hot melt extrusion of coprovidone at 100 mesh(preparation 1)15 mg/kg,but there was no statistical difference.The number,duration and grade of attacks all decreased,but there was no statistical difference.Conclusions:Copovidone hot melt extrusion preparation has the best effect,the dose dependence is obvious,the onset concentration is the lowest,the onset time is late.Compared with copolavidone hot melt extrusion of 100mesh particles,the hot melt extruded micropowder of copovidone has certain advantages.
Keywords/Search Tags:acute epilepsy model, Classical antiepileptic drugs, QO-83, Maximum electroshock model
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