Drug brain biotransformation in human refractory epilepsy

人类难治性癫痫的药物脑生物转化

• 批准号: 8890897
• 负责人: Chaitali Ghosh
• 金额: $ 34.34万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8890897
• 关键词: ABCB1 gene; Acids; Affect; Agonist; Amygdaloid structure; Animal Disease Models; Animal Model; Antiepileptic Agents; Biological Availability; Blood - brain barrier anatomy; Brain; Brain Diseases; CYP2C9 gene; CYP3A4 gene; Carbamazepine; Carrier Proteins; Cell Culture Techniques; Clinical; Clinical Management; Comorbidity; Consensus; Cytochrome P450; Data; Detection; Development; Devices; Disease; Disease model; Drug resistance; Electric Stimulation; Electroencephalography; Endothelial Cells; Enzymes; Epilepsy; Event; Experimental Models; Failure; Generations; Goals; Hepatocyte; High Pressure Liquid Chromatography; Human; In Vitro; Injection of therapeutic agent; International; Intervention; Investigation; Knowledge; Levetiracetam; Mass Spectrum Analysis; Medical; Metabolic; Metabolic Biotransformation; Metabolic Pathway; Metabolism; Methods; Modeling; Molecular; Monitor; N-Methylaspartate; Nature; Neurons; Operative Surgical Procedures; Parents; Pathology; Patients; Pattern; Penetration; Peritoneal; Pharmaceutical Preparations; Phase; Phenotype; Pilocarpine; Property; Protocols documentation; Publishing; Rattus; Refractory; Research Personnel; Resected; Resistance; Role; Sampling; Seizures; Specimen; Testing; Therapeutic; Transcript; base; brain surgery; brain tissue; cerebrovascular; drug distribution; improved; in vitro Model; insight; internal control; lamotrigine; mind control; multi drug transporter; multidisciplinary; neurotoxic; non-drug; overexpression; personalized medicine; screening; stem; tool

DESCRIPTION (provided by applicant): Pharmacological resistance to brain drugs is a common clinical event affecting patient management; it is also a common cause for neuro-surgical interventions. In particular, the number of drug resistant subjects is significant among those suffering from epilepsy. The International League against Epilepsy estimates that 20-25% of epileptic subjects are resistant to available anti-epileptic drugs (AED). Incomplete understanding of the pattern of brain AED biotransformation in the diseased brain represents a major hindrance to the development of new drugs. Consensus is gathering on the fact that modeling of the drug resistant phenotype requires a multi-modal experimental approach, including the use of human brain tissue (and of their in vitro manipulation) paired with animal models of disease. We now propose to 1) Test the hypothesis that, in DRE, the brain bioavailability of AEDs is affected by BBB P450 enzymes; 2) Test the hypothesis that BBB P450 produce metabolites with neurotoxic properties. We also propose the corollary hypothesis that a concerted metabolic-transport mechanism determines AED bioavailability in the DRE brain. Our recent published data and preliminary results show that: a) Transcripts of P450 enzymes are elevated in primary endothelial cells (EC) isolated from drug resistant epileptic patients (DRE); these include AED- metabolizers such as CYP3A4, CYP2C9, etc. Data were compared to available, control brain EC (non-DRE); b) Transcripts for PHASE II metabolic enzymes are present in DRE EC; these enzymes are responsible for the metabolism of 1st and 2nd generation AEDs; c) CYP3A4 and MDR1 co-localize at the BBB (and neurons) in human DRE brain; d) Overexpression of CYP3A4 in DRE EC is associated with exaggerated carbamazepine (CBZ) metabolism. This new metabolic pathway produces the toxic CBZ metabolite quinolic acid (QA). The parent (14C CBZ) origin of QA was evaluated using HPLC-Accelerated Mass Spectrometry (AMS) in vitro and ex vivo (DRE brain specimens). AMS results were corroborated by mass spectroscopy (MS) and by two HPLC protocols optimized for QA detection; e) DRE endothelial cells metabolize lamotrigine (LMT) and levetiracetam (LEV). In our proposal we will approach the issue of human control brain tissues by using brain samples resected to treat diseases other than drug resistant seizures, autoptic brain, and "internal" controls consisting of non-spiking regions in resected DRE brains. To dissect the role of EC, we will use primary BBB cell cultures derived from resected brain specimens. The BBB is recapitulated in vitro by a flow-based device. A combination of AMS and MS is used to determine the molecular nature of new metabolites in the DRE brain. Finally, two models of epilepsy are used to study the temporal and topographic pattern of brain expression and function of P450 enzymes. To our knowledge, these studies represent the first multimodal attempt to elucidate the expression and the role of brain P450 enzymes in DRE. Our ultimate goal is improved clinical management of DRE. The proposed studies will provide new insight into the mechanisms contributing to human DRE, improving the understanding of the pathophysiological significance of BBB P450. Modeling of the DRE BBB may serve as a tool for personalized medicine and specific disease modeling (e.g., type of drug resistant epilepsy and underlying pathology) allowing for the screening of new AED.

描述（由申请方提供）：脑药物的药理学耐药性是影响患者管理的常见临床事件;也是神经外科干预的常见原因。特别是，耐药性受试者的数量在患有癫痫的受试者中是显著的。据国际抗癫痫联盟估计，20-25%的癫痫患者对现有的抗癫痫药物（AED）具有耐药性。不完全理解脑AED在患病脑中的生物转化模式是新药开发的主要障碍。耐药表型的建模需要多模式实验方法，包括使用人脑组织（及其体外操作）与疾病的动物模型配对，这一事实正在形成共识。 我们现在建议：1）检验在DRE中，AED的脑生物利用度受BBB P450酶影响的假设; 2）检验BBB P450产生具有神经毒性特性的代谢物的假设。我们还提出了一个必然的假设，即一个协调的代谢运输机制决定AED的生物利用度在DRE的大脑。我们最近发表的数据和初步结果表明：a）P450酶的转录在从耐药癫痫患者（DRE）分离的原代内皮细胞（EC）中升高;这些包括AED代谢者，如CYP 3A 4、CYP 2C 9等。这些酶负责第一代和第二代AED的代谢; c）CYP 3A 4和MDR 1共定位于人DRE脑中的BBB（和神经元）; d）DRE EC中CYP 3A 4的过表达与过度的卡马西平（CBZ）代谢有关。这种新的代谢途径产生毒性CBZ代谢物喹啉酸（QA）。使用HPLC-加速质谱法（AMS）在体外和离体（DRE脑标本）中评价QA的母体（14 C CBZ）来源。AMS结果通过质谱法（MS）和两种针对QA检测优化的HPLC方案证实; e）DRE内皮细胞代谢拉莫三嗪（LMT）和左乙拉西坦（LEV）。 在我们的提案中，我们将通过使用切除的脑样本来治疗耐药性癫痫发作、尸检脑和由切除的DRE脑中的非尖峰区域组成的“内部”对照以外的疾病来处理人类对照脑组织的问题。为了剖析EC的作用，我们将使用来自切除的脑标本的原代BBB细胞培养物。通过基于流动的装置在体外重现BBB。AMS和MS的组合用于确定DRE脑中新代谢物的分子性质。最后，两种癫痫模型被用来研究大脑的P450酶的表达和功能的时间和地形图模式。据我们所知，这些研究代表了第一个多模式的尝试，以阐明表达和脑P450酶在DRE的作用。我们的最终目标是改善DRE的临床管理。拟议的研究将为人类DRE的机制提供新的见解，提高对BBB P450病理生理意义的理解。DRE BBB的建模可以用作个性化医学和特定疾病建模（例如，耐药性癫痫的类型和潜在的病理学），允许筛查新的AED。