Abstracts

Rationale: Nuclear receptors (NRs) and cytochrome P450 (CYP) regulate liver metabolism of many drugs. We reported overexpression of NRs, pregnane X (PXR) and glucocorticoid receptor (GR) at the neurovascular unit in human epileptic brain. In the present study, we asked whether silencing GR in human epileptic endothelial cells impacts drug bioavailability by affecting P450 expression or function. Methods: Surgical brain specimens were obtained from drug-resistant epileptic patients who underwent temporal lobectomies to relieve drug-resistant seizures. Primary cultures of epileptic brain microvascular endothelial cells isolated from brain resections (EPI-EC, n = 12) and commercially available control brain microvascular endothelial cells (HBMECs, n = 12) were used. Expression of PXR/GR proteins was analyzed by western blot and immunohistochemistry. PXR and GR silencing was performed in EPI-ECs, and CYP expression was determined. GR silenced, non-silenced EPI-ECs or HBMECs were seeded in a flow-based dynamic system (DIV-BBB) to model the BBB in vitro. ECs were co-cultured with human astrocytes. We explored the metabolism of oxcarbazepine (OXC, a second-generation anti-epileptic drug) at the BBB. OXC and metabolite levels in the blood and brain were compared across experiments (n = 4 each; with or without GR silencing of EPI-ECs) and measured by using high performance liquid chromatography. Results: CYP-GR co-localization was found in ECs and neurons in the human epileptic brain. GR was highly expressed in regions showing reactive gliosis. GR silencing in EPI-ECs led to decreased CYP3A4, CYP2C9, and PXR expression. PXR silencing in EPI-ECs resulted in specific downregulation of CYP3A4 expression and negligible alteration of GR expression. OXC bioavailability evaluated across the DIV-BBB showed a progressive decrease of intravascular OXC levels (0-24 hr) in EPI-non-siRNA > EPI-GR siRNA > HBMECs. Increased levels of brain-side OXC (*p < 0.05) were observed at 24 hr in EPI-GR siRNA and HBMECs. When compared to EPI-GR siRNA or HBMEC BBB, negligible OXC penetration was measured across the EPI-non-siRNA BBB, accompanied by increased levels of the OXC metabolite licarbazepine measured in the brain-side. Conclusions: Our results suggest that modulation of GR/PXR expression at the epileptic BBB impacts local drug brain metabolism, as a consequence of abnormal levels of P450 enzymes. Knowledge of the GR-P450 regulation of antiepileptic drugs at the BBB may be exploited for future drug design. Funding: This work is supported by NIH grants R01NS078307, R01NS095825, and UH4TR000491; Brain & Behavior Foundation (NARSAD); American Heart Association National Center Scientist Development Grant (13SDG13950015); and the Alternatives Research & Development Foundation.