Abstracts

Rationale: Drug resistance in epilepsy remains an unsolved problem. Our previous results unveiled the presence of CYP metabolic enzymes at the human epileptic blood-brain barrier (BBB, Ghosh et al, 2009). The focus of this study is to determine whether endothelial metabolism for commonly prescribed anti-epileptic drugs (AED) takes place at the epileptic BBB. Methods: Brain resections were obtained from adult patients affected by drug resistant epilepsy. Patients received carbamazepine (CBZ) prior to surgery. Blood samples were collected at time of surgery. Each brain sample was used for: 1) in-situ drug profiling and 2) establishment of correspondent in vitro primary brain endothelial cell cultures (EPI-EC). EPI-ECs were cultured using a dynamic system (DIV) to establish the “epileptic” in vitro BBB. The DIV system features a luminal and abluminal sides to study blood-to-brain drug distribution. We evaluated by microdialysis-HPLC-Mass Spectrometry the patterns of CBZ brain penetration/metabolism in situ and in vitro. Results: As previously shown, penetration of free CBZ was negligible in the epileptic brain resections compared to historic controls (Munari et al 1980, Rambeck et al 2006). Therapeutic levels of free CBZ were measured in blood. We have recently discovered that a property of the epileptic BBB is the CYP-dependent transformation of CBZ (Ghosh et al 2009). We have now determined the resulting brain metabolic profile. These metabolites included traditional CYP CBZ products (e.g. DiOH-CBZ), previously unrecognized (e.g. cyano-derivate of iminostilbene, MW 218.51) and previously undescribed molecules. Remarkably, correspondent in vitro BBBs provided virtually identical information, that is: 1) lack of CBZ penetration across the epileptic BBB; 2) CBZ metabolic transformation. In addition the DIV provided the opportunity to measure total metabolic efficiency of brain endothelium. Conclusions: We described a novel approach to study drug pharmacokinetics in the human epileptic brain, in situ and in vitro. These preliminary results show the capacity of our experimental approach of determining the quantitative and qualitative profile of AED penetration/metabolism in the human epileptic brain. This work was supported by NIH-RO1 NS43284, NIH-RO1 NS38195, NIH-R21 HD057256 to Damir Janigro. References 1) Munari et al. Brain levels of antiepileptic drugs in man. Monogr Neural Sci. 1980;5:213-20. 2) Rambeck B et al. Comparison of brain extracellular fluid, brain tissue, cerebrospinal fluid, and serum concentrations of antiepileptic drugs measured intraoperatively in patients with intractable epilepsy. Epilepsia. 2006 Apr;47(4):681-94. 3) C. Ghosh et al. Cytochrome P450 expression at the human blood-brain barrier: CYP3A4 and its role in carbamazepine brain metabolism. (Peer Reviewed Abstract for the XXIVth International Symposium on Cerebral Blood Flow and Metabolsim, Chicago, IL, USA)