Study Of PGP And MRP1 On The Drug-resistance Mechanisms Of Medically Intractable Epilepsy

Background:Although a series of new antiepileptic drugs (AEDs) have been launched in the last two decades, drug-refractoriness remains a major problem. About 30% of patients are refractory to treatment using more than one AEDs, which is called medically intractable epilepsy. It has poor prognosis with increased morbidity and mortality. Therefore it is far-reaching important to investigate the drug resistance mechanism of medically intractable epilepsy, and develop new strategies for treatment. Researchers found that many patients with tumor would yield resistance to drugs of chemotherapy. An important characteristic of pharmacoresistance is that most patients are resistant to one drug in the beginning, and it will be found that the other drugs will also be noneffective, even though these drugs act by different mechanisms. Which is called multidrug resistance(MDR). The following research indicate that membrane of cells of tumor express multidrug transporters, they act as drug efflux pump and restrict penetration of drug into cells, which produce chemotherapy-resistance in tumor. In the same way, there are the same pathophysiological mechanisms of drug resistance in medically intractable epilepsy. A series of researches concerning the relationship between multidrug transporters and drug resistance in medically intractable epilepsy have been done. The results suggest that several kinds of multidrug transporters are expressed in capillary endothelial cells and astrocytes of normal blood-brain barrier. There is accumulating evidence demonstrating that multidrug transporters are over-expressed in capillary endothelial cells and astrocytes in epileptogenic brain tissue surgically resected from patients with medically intractable epilepsy. Multidrug transporters in the BBB are thought to act as an active defense mechanism, restricting the penetration of lipophilic substances into the brain. Over-expression of such transporters in epileptogenic tissue is thus likely to reduce the amount of drug that reaches the epileptic neurons, which would be a likely explanation for pharmacoresistance. P-glycoprotein (PGP) and multidrug resistance-associated protein 1(MRP1) are candidates to cause AEDs resistance. They are ATP-binding cassette superfamily, and can accept a wide range of ADEs with different mechanisms as their potential substrates, and actively efflux drugs out of the brain. PGP and MRP1can be blocked by specific inhibitors which raises the option to use such inhibitors as adjunctive treatment for medically refractory epilepsy. In this study, we investigated P-gp and MRPl expression, localization in brain of patients and rats with medically intractable epilepsy. Then the functional activity of PGP and MRPl was research by specific inhibitors in vitro and vivo. In view of its form and function , we intended to study the role of PGP and MRPl on the drug resistance mechanisms of medically intractable epilepsy.Materials and methods:We divided this research into four sections. In the first section, the model of medically intractable epilepsy was established by kindling of amygdale. Brain tissue surgically resected from 20 patients with medically intractable epilepsy and 20 controls were collected. The expression of mRNA and protein of PGP and MRPl was investigated in cortex and hippocampus of rats and patients. In the second section, microdialysis and high-performance liquid chromatography (HPLC) was used to observe the distribution of phenytoin(PHT) and carbamazepine(CBZ) in extracellular fluid of cortex in normal and epileptic rats after AEDs i.p. Next, after PGP inhibitor-verapamil and MRPl inhibitor-probenecid was applied to the cortex through microdialysis probe, the concentration change of PHT and CBZ in extracellular fluid of cortex was observed in epileptic rats. In the third section, the multidrug resistance(overexpression of PGP)cell line K562/Dox was established. The intracellular AEDs accumulation in MDR cell line and non-MDR cell line was observed. After PGP inhibitor-verapamil was applied to the two cell lines, the concentration change of PHT and CBZ in MDR cell was observed. In the four section, PGP inhibitor-verapamil was applied to PHT-CBZ resistant rats, and after discharge threshold(ADT), after discharge duration(ADD) and seizure activity was observed in amygdaloid kindled rats.Results:The results of first section: 1. Compared with that of control groups, PGP expression in cortex and hippocampus of rats and patients with intractable epilepsy showed an obviously statistical increasing at both the mRNA and the protein levels (P<0.05). 2. Compared with that of control groups, MRPl expression in cortex and hippocampus of rats with intractable epilepsy showed an obviously statistical increasing (P<0.05). MRPl expression in cortex of patients was higher than that in control (P<0.05). There was no MRPl immmunoreactive cells in hippocampus ofnormal human , and just a few astrocytes expressed MRP1 in hippocampus of patients with intractable epilepsy.The results of second section: 1. The concentration of PHT in extracellular fluid of cortex in epileptic rats was lower than that in normal rats during 30-180min after i.p. (P<0.05); The concentration of CBZ in extracellular fluid of cortex in epileptic rats was lower than that in normal rats during 30-150min after i.p. injection (P<0.05). 2. Compared with control group, verapamil significantly enhanced the concentration of PHT in extracellular fluid in cortex of epileptic rats during 15-180 min after i.p. (P<0.05), CBZ was also enhanced at 45min, 60min and 120min after i.p. (P<0.05). 3. Compared with control group, probenecid significantly enhanced the concentration of PHT in extracellular fluid in cortex of epileptic rats during 15-180min after i.p. injection (P<0.05), CBZ was also enhanced during 45 -120min after i.p. (P<0.05).The results of third section: 1. Compared with non-MDR cell line K562, IC50 was significantly increased in MDR cell line K562/Dox after PHT and CBZ was applied; verapamil could decease significantly the level of IC50 in MDR cell line K562/Dox, and the reversal index were 2.5 and 1.5. 2. The concentration of PHT and CBZ in MDR cell line K562/Dox was lower than that in non-MDR cell line K562, and verapamil significantly enhanced the concentration of PHT and CBZ in MDR cell line K562/Dox(P0.05).The results of last section: 1. Compared with the control group, the ADT was higher in PHT-CBZ resistant rats after verapamil was applied by i.p. (P<0.05). 2. Compared with the control group, verapamil could shorten the ADD, reduce the Racine's degree (P<0.05).Conclusions:1. The expression of PGP and MRP1 are significantly increased in cortex and hippocampus of rats and patients with medically intractable epilepsy.2. PGP and MRP1 limit the penetration of PHT and CBZ through blood-brain barrier, and reduce the concentration of AEDs in extracellular fluid of focal cortex. They are concerned with drug resistance of medically intractable epilepsy.3. Specific inhibitors of PGP and MRP1 could enhance the concentration of PHT and CBZ in extracellular fluid in cortex of epileptic rats and MDR cell line K562/Dox, and improve the electrobiological activity and seizure behavior in PHT-CBZ resistant rats. So inhibition or down-regulation of PGP and MRP1 willbe benefit to treat medically intractable epilepsy.