Abstracts

Increased MDR1 expression of in endothelial cells (EC) of epileptic human blood-brain barrier (BBB) supports the hypothesis that lack of appropriate pharmacodistribution leads to suboptimal CNS AED levels. MDR1 expression in astrocytes from epileptic brain is associated with loss of p53 signaling, suggesting that MDR1confers resistance to apoptosis. We investigated the functional significance of MDR1 expression in parenchymal cells from drug resistant epileptic human brain. We tested the hypothesis that while MDR1 expression at the BBB correlates with poor AED penetration, neuronal and glial MDR1 expression relates to abnormal sensitivity to apoptosis.
Brain samples were taken during surgical resections in patients (n=14) administered with carbamazepine (CBZ). Astrocyte cultures were established from cortical tissue of epileptic (n= 8) or non-epileptic (n=2) patients. Control and epileptic glial cells were exposed to therapeutic concentration of phenytoin or doxorubicin, in the presence or absence of MDR inhibitors.
The brain-to-plasma ratio for CBZ was 1.3+- 0.07 (n=14) compared to distribution across the rodent BBB of 2.4+-0.4) suggesting limited CNS drug access in epileptic patients. MDR1 mRNA expression in resected tissue revealed higher levels in samples with the largest accumulation of AED (p=0.08). Levels of P-gp in epileptic vs. normal glia revealed a 5-fold increase in epileptic tissue. P-gp immunocytochemistry in brain sections from 8 drug-refractory patients showed staining of EC and parenchymal cells. 64% of neurons (n= 264) weere P-gp+ as compared to 91% astrocytes (n= 107). Cultured epilpetic astrocytes exhibited a significant increase in AED extrusion as compared to astrocytes isolated from non-epileptic brain (n=3) demonstrating a decreased phenytoin uptake by epileptic astrocytes compared with non-epileptic glia (15.03+-0.38 vs. 17.43+-0.39 fmol/cell). MDR inhibitors abolished these differences, indicating that active drug extrusion by epileptic glia was due to MDR. Cryopreserved sections from human epileptic tissue were immunostained for P-gp and nuclear integrity was assessed by DAPI. Regardless of cell type, MDR1 expression was associated with DNA integrity: 85% of glia and 66% of neurons expressing MDR1 were devoid of DNA damage.
These results suggest that AED extrusion by epileptic glia may ultimately enhance interstitial drug levels. Thus, the diminished AED penetration into the brain may be determined by P-gp overexpression by epileptic BBB EC while P-gp in glia and/or neurons may subserve a novel role independent of drug resistance related to neuron/glia protection from apoptotic cell death.
[Supported by: HL51614, NS43284, NS38195]