Study On The Pharmacokinetics And Anti-drug Resistance Mechanism Of Compound Danshen Dripping Pills Combined With Carbamazepine In The Treatment Of Refractory Epileptic Rats

ObjectiveThis study was designed to assess the effects of compound danshen dripping pills（CDDP）combined with carbamazepine（CBZ）on the brain distribution and pharmacokinetic of CBZ in normal and refractory epilepsy rats and to investigate the effect of CDDP combined with CBZ on the expression of P-gp and Mrp1 protein and related m RNA in the hippocampus of epileptic rats to study its possible anti-drug resistance mechanism in a rat model of kainic acid（KA）-induced refractory epilepsy.Methods1.Wistar male rats were divided into normal rats and model rats.Normal rats were divided into control group and CDDP group.Model rats were divided into control group,CDDP group and verapamil group.The animal model of refractory epilepsy was induced by intrahippocampal injection of KA.After the success of modeling,CDDP group was given CDDP（85mg/kg）,verapamil group was given verapamil（20mg/kg）for 10d,then each group was given CBZ（100mg/kg）respectively.Brain tissue samples were collected at different time.After the samples were pretreated,the concentration of CBZ in the brain tissue was detected by HPLC-MS/MS.2.The rats were grouped and given medicine according to the above-mentioned methods.The blood samples were collected at different time.After the samples were pretreated,the concentration of CBZ in the plasma was detected by HPLC-MS/MS and the pharmacokinetic parameters were calculated using DAS3.0.software program.3.Wistar rats were selected to create a refractory epilepsy model by intrahippocampal injection of KA and then were randomly divided into five groups of equal size:control（sham operated）,model,CDDP,CBZ and CDDP+CBZ.Subsequently,each group was divided into 45d and 90d subgroups according to postoperation time.Rats were gavaged with same volume of physiologic saline in the control and model groups and were gavaged with corresponding medicines in other groups.After 45 d and 90 d,three rats were randomly selected from each group and their brains were quickly removed after deeply anaesthetised and then their dextral hippocampus were freshly isolated.The expression of MDR1 and Mrp1 m RNA of each group were detected by RT-PCR.4.The dextral hippocampus of rats in each group were isolated according to the above-mentioned methods.Then the expression of P-gp and Mrp1 proteins was detected by western blotting.Results1.When using carbamazepine alone,the concentration of CBZ in normal rats was obviously higher than epileptic rats.Compared with using carbamazepine alone,CDDP could significantly increase the concentration of CBZ in normal and epileptic rats.For the epileptic rats,the enhancing effect of CDDP to the cerebral distribution of CBZ was similar to that of P-glycoprotein inhibitor verapamil.CDDP could enhance the permeability of blood-brain barrier to CBZ and increase the cerebral distribution of CBZ.2.In the normal physiological status,CDDP could increase the greatest concentration of CBZ but could not change the pharmacokinetic process of CBZ in the plasma of normal rats.In the epileptic status,CDDP could increase the area under the curve（AUC）,extend the half-life(t_1/2)and reduce the clearance rate.The positive control group（verapamil group）could extend the t_maxax of CBZ and also could reduce clearance rate,These results indicate that both CDDP and verapamil can affect the pharmacokinetic process of CBZ.CDDP combined with CBZ can improve the bioavailability,promote the absorption and slow the clearance rate of CBZ.3.MDR1 and Mrp1 mRNA expression in the hippocampal area was evaluated by RT-PCR method.We found that in the control group,there were only a little expression of MDR1 and Mrp1,however in the model and CBZ groups there were a large body of these genes expression.The expression of MDR1 and Mrp1 mRNA in the CDDP and CDDP+CBZ groups showed an obvious decrease compared to the model group.With the extention of treatment time,CDDP alone and in combination with CBZ could obviously weaken the increasing trend of MDR1 and Mrp1 m RNA expression.4.The expression of P-gp and Mrp1 proteins in the hippocampal area was evaluated by western blotting method.We found that in the control group,there were only a little expression of P-gp and Mrp1 proteins,however in the model and CBZ groups there were a large body of these proteins expression compared with the control group.The P-gp and Mrp1 proteins expression in the CDDP and CDDP+CBZ groups showed a clear decrease over the model and CBZ groups.With the extention of treatment time,CDDP alone and in combination with CBZ could obviously weaken the increasing trend of P-gp and Mrp1 proteins expression.These findings are consistent with the results of the RT-PCR experiment.ConclusionIn conclusion,combined administration of CDDP with CBZ could enhance the cerebral distribution of CBZ in normal and epileptic rats and could change the pharmacokinetic process of CBZ in the plasma of epileptic rats but had no obvious impact on the plasma of normal rats.CDDP showed a significant effect on regulate the expression of the multidrug resistant transporter P-gp and Mrp1 proteins.CDDP combined with CBZ could led to an obvious decrease in P-gp and Mrp1 proteins expression.This inhibitory effect of CDDP combined with CBZ on the overexpression of P-gp and Mrp1 proteins might be one of the possible mechanisms for the treatment of refractory epilepsy.