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）：对脑药物的药理学耐药性是影响患者管理的常见临床事件；这也是神经外科干预的常见原因。特别是，在患有癫痫的患者中，耐药受试者的数量是显着的。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) 产生耐药性。对患病大脑中 AED 生物转化模式的不完全了解是新药开发的主要障碍。人们正在达成共识，即耐药表型的建模需要采用多模式实验方法，包括使用人脑组织（及其体外操作）与疾病动物模型相结合。 我们现在建议 1) 检验以下假设：在 DRE 中，AED 的大脑生物利用度受到 BBB P450 酶的影响； 2) 检验 BBB P450 产生具有神经毒性的代谢物的假设。我们还提出了一个推论假设，即协调一致的代谢转运机制决定了 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) 分离的原代内皮细胞 (EC) 中 P450 酶的转录物升高；这些包括 AED 代谢物，例如 CYP3A4、CYP2C9 等。将数据与可用的对照脑 EC（非 DRE）进行比较； b) DRE EC 中存在 PHASE II 代谢酶的转录本；这些酶负责第一代和第二代 AED 的代谢； c) CYP3A4 和 MDR1 共定位于人 DRE 大脑的 BBB（和神经元）； d) DRE EC 中 CYP3A4 的过度表达与卡马西平 (CBZ) 代谢过度相关。这种新的代谢途径产生有毒的 CBZ 代谢物喹啉酸 (QA)。使用 HPLC 加速质谱 (AMS) 体外和离体（DRE 脑标本）评估 QA 的母体 (14C CBZ) 来源。 AMS 结果通过质谱 (MS) 和两个针对 QA 检测优化的 HPLC 方案得到证实； 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大脑样本来治疗除耐药性癫痫、自视脑和由切除的 DRE 大脑中的非尖峰区域组成的“内部”对照之外的疾病，来解决人类控制脑组织的问题。为了剖析 EC 的作用，我们将使用源自切除脑标本的原代 BBB 细胞培养物。 BBB 通过基于流动的设备在体外重现。 AMS 和 MS 的组合用于确定 DRE 大脑中新代谢物的分子性质。最后，使用两种癫痫模型来研究 P450 酶的大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 DRE 中脑 P450 酶的表达和作用的首次多模式尝试。我们的最终目标是改善 DRE 的临床管理。拟议的研究将为人类 DRE 的机制提供新的见解，增进对 BBB P450 病理生理学意义的理解。 DRE BBB 建模可以作为个性化医疗和特定疾病建模（例如耐药性癫痫类型和潜在病理学）的工具，以便筛选新的 AED。