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（由申请人提供）：对脑药物的药理抵抗力是影响患者管理的常见临床事件；这也是神经手术干预措施的常见原因。特别是，患有癫痫患者的耐药受试者的数量很大。国际反对癫痫联盟估计，20-25％的癫痫受试者对可用的抗癫痫药（AED）有抵抗力。对患病大脑中大脑AED生物转化模式的不完全理解代表了对新药开发的主要障碍。共识是关于耐药表型的建模需要多模式实验方法的事实，包括使用人脑组织（及其体外操纵）与疾病动物模型配对。 现在，我们建议1）检验以下假设：在DRE中，AEDS的脑生物利用度受BBB P450酶的影响； 2）检验BBB P450产生具有神经毒性特性的代谢物的假设。我们还提出了必然的假设，即协同的代谢转移机制决定了DRE大脑中的AED生物利用度。我们最近发表的数据和初步结果表明：a）从耐药性癫痫患者（DRE）中分离出的原代内皮细胞（EC）中P450酶的转录物升高；这些包括AED代谢物，例如CYP3A4，CYP2C9等。将数据与可用的Control Brain EC（非DRE）进行了比较； b）DRE EC中存在II期代谢酶的转录本；这些酶是第一和第二代AED的新陈代谢的原因。 c）在人类DRE脑中的BBB（和神经元）共定位于CYP3A4和MDR1； d）DRE EC中CYP3A4的过表达与夸张的卡马西平（CBZ）代谢有关。这种新的代谢途径会产生有毒的CBZ代谢产物喹酸（QA）。使用HPLC加速质谱法（AMS）在体外和Ex Vivo（DRE脑样本）中评估了质量检查的母体（14C CBZ）。 AMS结果通过质谱（MS）和两种用于QA检测的HPLC方案证实。 e）DRE内皮细胞代谢Lamotrigine（LMT）和Levetiracetam（LEV）。 在我们的提案中，我们将通过使用切除的脑样本来处理人类控制脑组织的问题，以治疗耐药性癫痫发作，自动脑和“内部”控制，由切除的DRE大脑中的非尖刺区域组成。为了剖析EC的作用，我们将使用源自切除的大脑标本的原代BBB细胞培养物。 BBB通过基于流的设备在体外概括。 AMS和MS的组合用于确定DRE脑中新代谢产物的分子性质。最后，使用两个模型来研究P450酶的脑表达和功能的时间和地形模式。据我们所知，这些研究代表了阐明脑p450酶在DRE中的表达和作用的第一次多模式尝试。我们的最终目标是改善DRE的临床管理。拟议的研究将为有助于人类DRE的机制提供新的见解，从而提高对BBB P450病理生理意义的理解。 DRE BBB的建模可以作为个性化医学和特定疾病建模的工具（例如，耐药性癫痫的类型和潜在的病理学），允许筛查新AED。