The Influence Of Rational Combination Of AMC And FLU On P-gp Expression In The Brain Of VAP-resistant Convulsive Rats

Objective To explore the best combination of 4-Amino-2-Methyl-Cantharidinimide(AMC) and Flunarizine (FLU) in solo-or co-application in different portion.To observe the anticonvulsive effect of solo-or co-application groups on penicillin (PNC)-chronic kindling Valproate-resistant epilepsy rats. And expression of P-gp in the brain of VPA-resistance rats. To explore the pharmaco-resistance mechanism in the protein level.Methods (1) Used the maximal electroshock seizure (MES) model,analysis the anticonvulsant rate of the intragastrical (ig) mice with solo-or co-application of the two compounds in diffe-rent combinations (comb I, AMC:FLU=1:1; combⅡ,AMC:FLU=1:4,combⅢ,AMC:FLU=4:1), ED50 of anti-MES was calculated according to the Bliss's method.(2) The roll club method was adopted to analysis the toxic effect of intragastrical (ig) in solo-or co-application of the two co-mpounds in different combinations. Then we calculated TD50 according to the Bliss's method. (3)Used isobolographic analysis to determine the best combination of synergistic effect and ant-agonistic toxicity. (4) The PNC-chronic kindling convulsive rat model was established by ip PNC (3×1 06U·kg-1·d) for 13 days. Valproate-resistant model was set up by Valproate (VPA, 250mg·kg-l·d) ig for 21 days.The convulsion seizure latency and Racine behavior study graduation were used to evaluate drug efficacy. The expression of P-gp in brain of epileptic rats were determinated by immunohistochemistry technology. Assay the influence of AMC, FLU monotherapy and different prescriptions on the expression of P-gp in Pharmoco-resistance convulsive rats.Results (1) Each combination of AMC and FLU could antagonize MES in mice dose-dependly, the ED50 values were as follows:FLU (29 mg·kg-1), Combll (26 mg·kg-1), AMC(25 mg·kg-1), CombⅢ(25 mg·kg-1), CombⅠ(18 mg·kg-1); (2) The groups of AMC, FLU, CombⅠ, CombⅡand CombⅢhave CNS toxicity, the TD50 values decreasing order:CombⅢ(779 mg·kg-1),AMC (545 mg·kg-1), FLU (479 mg·kg-1), CombⅠ(470 mg·kg-1), CombⅡ(452 mg·kg-1); (3) In CombⅠgroup, the equivalent points of ED50, TD50 were beneath and on the equivalent line; In CombⅡgroup, the equivalent points of ED50. TD50 were respectively beneath and on the equivalent line respectively; In CombⅢgroup, the ED50 equivalent points were on the equivalent line, while the TD50 equivalent points were far above from the equivalent line; (4) Preparation of PNC-kindling and VPA-resistance rat models in 48 rats, except 6 rats as normal control, the remained 42 rats were administrated(i.p.) with small doses of PNC (3×106 U·kg-1), the rats ranged RacineⅣ-Ⅴ. and seizured 6-8 times or more were considerated to be successful seizure model.In 42 convulsive rats, except 6 rats as seizure models control, the other 36 convulsive rats were administrated with VPA at the dose of 25 mg·kg-1·d, one time a da weeks.24 h after the last administration.The PNC at the dose of 3×106 U·kg-1 were applied to select the VPA-resistant convulsive rats. All the 36 convulsive rats were resistant to VPA. (5) The anti-drug-resistant function of the groups of AMC, FLU and the different combinations:The convulsive latency period could be significantly extended by intragastric administration with AMC, FLU.and the 3 combinations of FLU and AMC. There were statically differences between before and after administration (P<0.05) in each group. (6) the expression brain P-gp, although the P-gp was numerically higher in convulsive model control group than the normal control group, there were no significant difference between them (P> 0.05); it was significantly increased in VPA-resistant convulsive group than that in seizure model groups (P<0.01); It was statistically decreased in AMC, FLU, and the 3 combinations of FLU and AMC compared to VPA-resistant convulsive model (P<0.01).The decrease of P-gp level was the most significantly in combⅢgroup,which was statistical difference compared to that of combⅠand comb II groups (P＜0.01)Conclusions (1) The different combination of AMC and FLU displayed the dose-depend in the anti-MES attack. valency was the maximum in combⅠ.TDso was the maximum in combⅢ, which indicated the minimum toxicity,TD50 was the minimum in combⅡ, indicating it was the most toxic in the treatment groups; Both the effect and toxicity were collaborated in combⅠand combⅡgroups, the effect was collaborated and the toxicity was antagonised in combⅢgroup, which was the best compatibility program; (2) After intraperitoneal injection of small doses of penicillin for 13 days, the constant partial attack-with secondary generalized seizures were presented on rats (Ⅳ～Ⅴgrade). Intragastric administration with sodium valproate for 21 days produced VPA-resistant seizures rats, the achievement ratio of VPA-resistant seizure was 100%; (3) Intragastric administration respectively with AMC, FLU, and three combinations of AMC and FLU were able to anti-convulsion, the mechanism of anti-parmaco-resisitant seizures was related to decrease of expression of P-gp in rat brain, the combⅢgroup (combination of 4:1) displayed the strongest anti-effect on the P-gp expression.In the 3 combinations, combⅢgroup(4:1)showed the greatest inhibition on the expression of P-gp.