Cytochrome P450-mediated drug interactions at the human blood-brain barrier

摘要 脑内药物相互作用和活性代谢物在脑内分布的机制 药物治疗在很大程度上是未知的。细胞色素P450(CYP)药物代谢酶已知的功能 活跃在血脑屏障(BBB)。血脑屏障上的多药转运蛋白(如mdr1)曾经被认为是 抗癫痫药物(AED)治疗难治性癫痫(DRE)失败的主要机制。 然而，我们报道了BBB通过CYP介导的机制参与这一过程 将治疗剂转化为神经毒性分子。发现CYP-MDR1共同表达增加 癫痫脑内皮细胞和神经元。我们还发现神经毒性相互作用 卡马西平(CBZ，一种AED)和舍曲林(抗抑郁药)-CYP介导的产物 反应性CBZ代谢物和氧化应激。联合处方药物的范围、相关性和地形图 癫痫脑内的新陈代谢和潜在的神经毒性仍未被研究。其中一些药物是诱导性的 和/或被CYP抑制；我们必须设计方法来防止随后的神经元变化。 我们的多学科团队将测试中心假设，即两种联合处方的AEDs将具有竞争性 与人血脑屏障内皮细胞相互作用调节神经功能和CYP-MDR机制影响药物- DRE大脑中的代谢物水平。我们将研究一个同质的患者群体(由于皮质原因，局限性DRE 发育不良，FCD)共同处方的CYP和/或非CYP调节的AED的组合。具体目标将是 通过使用这些多方面的方法来实现：(1)评估CYP对神经毒性和代谢的贡献 AEDs在人源化的体外神经血管单位(NVU)中的作用；(2)确定调控因素及其程度 CYP通过使用(A)DRE合并FCD脑切除的EPI-ECs来代谢AEDs，形成NVU和 (B)从患者个人(体外)切除的癫痫(发育不良)和非癫痫(非发育不良)组织；和 (3)研究CYP/MDR1的原位表达与AED组织生物利用度、反应性代谢产物的形成、 以及癫痫患者与非癫痫患者切除的DRE脑区的细胞损伤程度。人体组织将会是 通过侵入性SEEG或硬膜下栅格记录表现出组织学和电生理学特征。 将使用一种新的人源化NVU，具有初级对照或患者来源的DRE脑细胞。药效 血脑屏障的相互作用和实时神经元效应将被评估为 疾病模型。进一步的测量包括药物/代谢物的高效液相-质谱法(药代动力学)；神经毒性；无 自由基化验；线粒体(药效学)和基因表达变化(药物基因组学)。分子 对脑细胞的操纵(通过转基因/抑制剂)将在正常、 DRE脑内耐药血脑屏障和CYP-MDR1调节(癫痫/非癫痫)。合在一起，将会有足够的 最大限度减少CYP依赖的神经毒性和预防综合疗法副作用的信息。

ABSTRACT The mechanisms involved in brain drug interactions and reactive metabolites distribution in the brain during pharmacotherapy is largely unknown. Cytochrome P450 (CYP) drug-metabolizing enzymes are known to be functionally active at the blood-brain barrier (BBB). Multidrug transporter proteins (e.g., MDR1) at the BBB were once thought to be the main mechanism of failed antiepileptic drug (AED) therapy in patient suffering from drug-resistant epilepsy (DRE). However, we reported that the BBB participates in this process via CYP-mediated mechanism through the conversion of a therapeutic agent into a neurotoxic molecule. Increased CYP-MDR1 co-expression was found in epileptic brain endothelial cells (EPI-ECs) and neurons. We also found neurotoxic interactions between carbamazepine, (CBZ, an AED) and sertraline (antidepressant) – a consequence of CYP-mediated production of reactive CBZ metabolites and oxidative stress. The extent, relevance and topography of co-prescribed drug metabolism and potential neurotoxicity in the epileptic brain remain unexplored. Some of these drugs are induced and/or inhibited by CYP; we must devise ways to prevent subsequent neuronal changes. Our multidisciplinary team will test the central hypothesis that two co-prescribed AEDs will competitively interact in human BBB endothelial cells to regulate neuronal function and a CYP-MDR mechanism affects drug- metabolite levels in DRE brain. We will study a homogeneous patient population (with focal DRE due to cortical dysplasia, FCD) co-prescribed combination of CYP- and/or non-CYP-regulated AEDs. The Specific Aims will be achieved by using these multifaceted approaches to: (1) assess CYP’s contribution to neurotoxicity and metabolism of AEDs in a humanized in vitro neurovascular unit (NVU); (2) determine the regulatory factors and the extent to which CYP metabolize AEDs by using (a) EPI-ECs derived from brain resections of DRE with FCD to form a NVU and (b) epileptic (dysplastic) and nonepileptic (nondysplastic) tissue resected from the individual patient (ex vivo); and (3) study in situ CYP/MDR1 expression correlated to AED tissue bioavailability, formation of reactive metabolites, and extent of cellular damage in epileptic vs. nonepileptic resected DRE brain regions. The human tissue will be characterized histologically and electrophysiologically through invasive SEEG or subdural grid recording. A novel humanized NVU with primary control or patient-derived DRE brain cells will be used. Drug interactions at the BBB and real-time neuronal effects will be evaluated as a “personalized medicine” tool for disease modeling. Further measurement includes drug/metabolites by HPLC-MS (pharmacokinetics); neurotoxicity; free radical assay; and mitochondrial (pharmacodynamics) and gene expression changes (pharmacogenomics). Molecular manipulations of brain cells (by transfection/inhibitors) will validate CYP-mediated AED interactions in normal, drug-resistant BBB and CYP-MDR1 regulation in DRE brain (epileptic/nonepileptic). Together, there will be ample information to minimize CYP-dependent neurotoxicity and prevent side-effects of a polytherapy.