The Anticonvulsive Effects Of 4-Amino-2-Methyl-canthari-dinimide And The In-fluence On The Mdr1 MRNA And Pgp Expression In The Brain Of Pheny-toin-resistant Convulsive Rats

Objective To study the anticonvulsive effect and its toxicity and pharmacodynamics kept in body of 4-Amino-2-Methyl-canthar-idinimide(AMC).Established the penicillin(PNC)-chronic kindling phenytoin-resistant convulsive rat model, to investigate the influence of AMC on the mdr1(multidrug resistance gene 1)mRNA and the Pgp(P-glucoprotein)expression in order to explore its anti-drug-resistant mechanism on the level of gene and protein.MethodsⅠThe maximal electroshock seizure(MES) model, carbamazepine (CBZ), and topi-ramate (TPM)as the positive control, was employed to evaluate the dose and time-effect rela-tionship of AMC, and according to Bliss method to calculate the ED50 to compare their potency and efficacy after intragastrical (ig) administration drugs; Metrazol seizure test (MET) was per-formed to compare the seizure latency and the anticonvulsant rate. Meantime, the toxicity of AMC was evaluated by the rotorod test, and according to Bliss method to calculate the TD50;ⅡPharmacodynamic parameters of AMC were assayed by Pharmacological Effective Method.ⅢThe PNC-chronic kindling convulsive rat model was established by ip PNC(3×106U·kg^-1) for 13 days. phenytoin-resistant model was set up by phenytoin (PHT, 0.225g·kg^-1·d) ig for 21 days. The convulsive latency and Racine behavior classification was used to evaluate the anti-convulsive effects after ig AMC (0.175g·kg^-1, 0.0875g·kg^-1) and flunarizine (FLU, 0.0875g·kg^-1, positive control). Reverse transcription polymerase chain reaction (RT-PCR) technology was used to detect the effect of AMC and FLU on the mdr1 gene expression; Immuno histochemistry (SABC method) was assayed to detect the changes of Pgp expression in the brain of drug-resistant convulsive rat.ResultsⅠAMC, CBZ, TPM could dose-dependently antagonize MES in mice, anticonvulsive rates were respectively AMC100%, TPM 90%, CBZ 100%; The ED50 were as follows: AMC ( 0.022g·kg^-1), TPM (0.22g·kg^-1), CBZ, (0.016g·kg^-1);ⅡAMC, CBZ, TPM could prolong the convulsion latency on MET in mice, there were significantly statistical difference compared with control rats(P<0.01);ⅢAMC, CBZ, TPM all have CNS toxicity, the TD50 values were as fol-lows: AMC (0.545g·kg^-1), TPM (0.492g·kg^-1), CBZ (0.141g·kg^-1);Ⅳthe results of pharma-codynamic studies showed that the minimum effect dose from low to high as follows: AMC CBZ>TPM; the persist time from long to short is: AMC (15.6h)>TPM >CBZ; the elimina- tion rate of AMC (0.2) were smaller than the other two drugs;ⅤAfter given ip small doses of penicillin (PNC3×106U·kg^-1) for 13d, all of rats were appearance Racine IV-V-class attack;After convulsive rats given ig phenytoin 0.225g·kg^-1·d for three weeks,animals were screened by ip PNC3×106U·kg^-1; Phenytoin-resistant rats convulsion latency (20.62±9.80min) com-pared to convulsion model control group (19.27±10.55min) were no statistical signifi-cance(P>0.05), so the phenytoin-resistant convulsant rats model was accomplished. The achievement rate of the phenytoin-resistant convulsant rats model was 100%.ⅥAfter both dose of AMC and FLU ig respectively could inhibit epileptiform behavior and prolong the convulsant latency in phenytoin-resistant rats (P<0.01);ⅦThe mdr1 mRNA expression in the convulsion model group were increased and higher than the normal control group, there were significantly statistical difference (P<0.05); the content of mdr1mRNA expression in phenytoin-resistant model group were higher than the convulsion model group, there were statistically significant difference (P<0.01); Compared with phenytoin-resistant model group, both dose of AMC and FLU decreased the content of mdr1mRNA expression, there were significantly statistical differ-ence (P<0.01);ⅧThe Pgp expression levels of the convulsive model control group, compared with normal control group, were increased, there were significant statistical difference (P<0.01);The Pgp expression of phenytoin-resistant model group rats were higher than the con-vulsion model group significantly increased, there was a significant statistical difference (P <0.01); Compared with phenytoin-resistant model group, both dose of AMC and FLU decreased the Pgp levels, both have a statistically significant difference (P <0.01); but the Pgp expression of FLU group was more than the AMC group.ConclusionsⅠThe anti-MES potency of AMC is stronger than TPM, similar with CBZ; its efficacy was stronger than TPM, and similar than CBZ;ⅡAMC could antagonize MET sei-zure,the potency of AMC anti-MET attack is similar to that of CBZ and TPM;ⅢAMC, CBZ, TPM all have CNS toxicity, however, the toxicity of AMC was the lowest;ⅣCompared to CBZ and TPM, the lowest effect dose of AMC was the smallest, the t1/2 (ED) and peak time were the longest,but eliminate slow.ⅤTo use low-dose penicillin ip for 13 days could be set up convulsion model, phenytoin was gaven for 21 days, phenytoin-resistant convulsion rat model established;ⅥAdministered two doses of AMC and FLU could antagonize the pheny-toin-resistant convulsion;ⅦThe expression of mdr1 gene and Pgp in the normal rat brain only was microcontent, PNC could induce mdr1 gene and Pgp expression; phenytoin were gaven for 21 days, its expression were further significantly increased. Two doses of AMC and FLU alone could inhibit the expression of mdr1mRNA and reduce Pgp content of drug-resistant sei-zures rat. Those results shows that the anti-drug-resistance mechanism of AMC and FLU has re-lationship with the the mdr1 mRNA and Pgp expression inhibited.ⅧChange trends of AMC and FLU inhibited the Pgp expression were smaller with that of the mdr1 mRNA expression de-terminated by RT-PCR, this shows that the two drugs inhibited mdr1 gene expression are the di-rect cause of the inhibition Pgp expression. However, the anti-drug-resistant action of AMC was stronger than FLU.