Abstracts

Rationale: Drug delivery to the drug resistant epileptic brain may be hampered by the overexpression of multiple drug transporter proteins at the blood-brain barrier (BBB). We hypothesize that the BBB may also act as metabolic barrier. Thus, while cytochrome P450 (CYPs) enzymes play a major role in the metabolism of drugs in the liver, it is not clear whether these enzymes are also functionally expressed by human epileptic BBB. Methods: Primary endothelial cultures were obtained from brain specimens of drug resistant epileptic subjects (EPI-EC). Human-derived brain endothelial cells (HBMEC) and non-brain endothelial cells (HUVEC) were used as control. CYPs and multi-drug transporter transcript levels were assessed by cDNA microarrays. Results were confirmed and quantified by western blot on endothelial cells. The effect of laminar flow on gene transcription was evaluated using a dynamic in vitro model (DIV) capable of generating shear stress. Human derived hepatocytes were used as positive control for CYP expression. Results: mRNA levels of CYP1A1,1B1, 2A6, 2B6, 2C, 2C9, 2E1, 2J2, 3A4, 4A11 and 11b were significantly elevated in EPI-EC. We also confirmed the endothelial increase in the transcript levels of drug transporters (MDR1, MRP1 and RLIP76). Exposure to laminar flow significantly increased the transcript levels of the CYPs and drug transporters above their correspondent thresholds, suggesting of a positive effect of flow on gene transcription. The comparison between mRNA levels in EPI-EC and HBMEC unveiled cell specific differences in flow response. Specifically, CYP3A4 protein, involved in the metabolism of anti-epileptic drugs, was overexpressed in EPI-EC (290 ± 30%) compared to control and expressed at levels comparable to those observed in human hepatocytes. Exposure to laminar flow did not affect CYP expression in hepatocytes. Conclusions: Our results show the expression of P450 enzymes at the human epileptic BBB and the regulatory effect of flow. Previous evidences demonstrated that exposure to laminar flow affects the regulation of transcription and protein synthesis by BBB endothelial cells. Hyper/hypo-perfusion is associated with ictal/interictal events in the epileptic brain and it is plausible to hypothesize that hemodynamic changes could cyclically modulate protein expression and function at the epileptic BBB. Key words: blood-brain barrier - epilepsy - drug metabolism - laminar flow Acknowledgements This work was supported by NIH-RO1 NS43284, NIH-RO1 NS38195, NIH-R21 HD057256 to Damir Janigro.