P-glycoprotein alters blood-brain barrier penetration of antiepileptic drugs in rats with medically intractable epilepsy

Abstract

P-glycoprotein is one of the earliest known multidrug transporters and plays an important role in resistance to chemotherapeutic drugs. In this study, we detected levels of P-glycoprotein and its mRNA expression in a rat brain model of medically intractable epilepsy established by amygdala kindling and drug selection. We investigated whether inhibition of P-glycoprotein affects the concentration of antiepileptic drugs in cortical extracellular fluid. We found that levels of P-glycoprotein and its mRNA expression were upregulated in epileptic cerebral tissue compared with cerebral tissue from normal rats. The concentrations of two antiepileptic drugs, carbamazepine and phenytoin, were very low in the cortical extracellular fluid of rats with medically intractable epilepsy, and were restored after blockade of P-glycoprotein by verapamil. These results show that increased P-glycoprotein levels alter the ability of carbamazepine and phenytoin to penetrate the blood-brain barrier and reduce the concentrations of these agents in extracellular cortical fluid. High P-glycoprotein levels may be involved in resistance to antiepileptic drugs in medically intractable epilepsy.

Keywords: P-glycoprotein; amygdala kindling; antiepileptic drugs; medically intractable epilepsy; verapamil.

Figure 1

Expression of P-glycoprotein and its mRNA in cerebral tissue. MDR1 was determined using real-time reverse transcription polymerase chain reaction and P-glycoprotein using Western blotting. RNA and total protein was extracted from the hippocampus (H) and cortex (C) of control rats and kindled rats. (A) Products of PCR were run on a 1% w/w agarose gel. β-actin (lower band, 241 bp) and MDR1 (upper band, 600 bp) are shown. (B) Semiquantitative analysis using gel after densitometry. The amount of MDR1 expression is represented by the ratio of MDR1 to β-actin (mean ± standard deviation, n=8) and normalized. *P<0.05 versus control group. (C) Western blotting of P-glycoprotein, and (D) quantitative analysis. The amount of P-glycoprotein expression was normalized to β-actin (mean ± standard deviation, n=8). *P<0.05 versus control group. Abbreviation: PGP, P-glycoprotein.

Figure 2

Concentrations of carbamazepine and phenytoin in extracellular cortical fluid and serum. Notes: Serum concentrations (A,C) and the cortical extracellular fluid (B,D) of AEDs were detected at the time indicated (post-injection). The highest PHT concentration in serum and the cortical extracellular fluid was at 30 minutes and 60 minutes, respectively, after intraperitoneal injection with 50 mg/kg PHT (n=8), and the highest CBZ concentration in serum and the cortical extracellular fluid was at 15 minutes and 90 minutes, respectively, after intraperitoneal injection with 20 mg/kg CBZ (n=8). Abbreviations: CBZ, carbamazepine; PHT, phenytoin; AED, antiepileptic drug.

Figure 3

Concentrations of phenytoin (A) and carbamazepine (B) in extracellular cortical fluid as measured by high-performance liquid chromatography. Notes: Both CBZ and PHT in the cortical extracellular fluid of kindled rats were very lower than those in controls at each time point during 30–180 minutes and 30–150 minutes respectively (n=8). The administration of verapamil significantly restored PHT concentrations at each time point (A, n=8, P=0.024, 0.003, 0.015, 0.031, 0.028, 0.005, 0.008 and 0.016 versus epilepsy group, respectively), and CBZ concentrations at 45 minutes, 60 minutes and 120 minutes (B, n=8, P=0.028, 0.016 and 0.036 vs epilepsy group, respectively). Abbreviations: CBZ, carbamazepine; PHT, phenytoin.