Drug brain biotransformation in human refractory epilepsy

描述(由申请人提供)：脑部药物的药理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，在癫痫患者中，耐药受试者的数量是相当大的。国际抗癫痫联盟估计，20-25%的癫痫受试者对现有的抗癫痫药物(AED)具有耐药性。对脑AED在病脑中的生物转化模式的不完全理解是新药开发的主要障碍。人们正在就这样一个事实达成共识，即对耐药表型的建模需要一种多模式的实验方法，包括使用人脑组织(及其体外操作)和疾病的动物模型。我们现在建议1)测试假设，在DRE中，AEDs的大脑生物利用度受到BBB P450酶的影响；2)测试BBB P450产生具有神经毒性特性的代谢物的假设。我们还提出了一个推论假设，即协调的代谢-运输机制决定了AED在DRE脑中的生物利用度。我们最近公布的数据和初步结果显示：a)从耐药癫痫患者(DRE)分离的原代内皮细胞(EC)中P450酶的转录本增加；这些转录本包括AED代谢物，如CYP3A4、CYP2C9等。将数据与现有的对照脑内皮细胞(非DRE)进行比较；b)在DRE EC中存在II相代谢酶的转录本；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脑中的血脑屏障(BBB)(和神经元)；d)CyP3A4在DRE EC中的过度表达与卡马西平(CBZ)代谢的夸大有关。这一新的代谢途径产生了毒性的CBZ代谢物奎酚酸(QA)。采用高效液相色谱加速质谱仪(AMS)对QA的亲本(14C-CBZ)来源进行了体外和体外研究。质谱学(MS)和两种用于QA检测的高效液相色谱方法证实了AMS的结果：(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处理人类控制脑组织的问题，这些样本用于治疗抗药性癫痫、尸检大脑和由切除的DRE大脑中的非尖峰区域组成的“内部”对照。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。血脑屏障在体外由一种基于流动的装置重现。AMS和MS的结合被用来确定DRE脑中新代谢物的分子性质。最后，利用两种癫痫模型研究了P450酶在脑内表达和功能的时间和地形图模式。据我们所知，这些研究是首次多模式尝试阐明脑P450酶在DRE中的表达和作用。我们的最终目标是改善DRE的临床管理。所提出的研究将为人类DRE的发病机制提供新的见解，提高对BBB P450的病理生理意义的理解。DRE BBB的建模可以作为个性化药物和特定疾病建模的工具(例如，耐药癫痫的类型和潜在的病理)，从而允许筛选新的AED。 公共卫生相关性：产生新的脑疗法的失败源于对在病理条件下调节药物脑分布的机制缺乏了解。因此，相当大比例的大脑疾病患者在不同程度上对现有的药物治疗产生了抗药性。例如，20-25%的癫痫患者对两种或两种以上单独或联合使用的抗癫痫药物(AED)没有反应。在这些情况下，脑部手术仍然是唯一的医疗干预措施。我们希望验证这样的假设，即在耐药形式的癫痫中，脑血管P450酶代谢AED降低了它们的药理效果。我们将验证脑血管P450酶产生可能具有神经毒性作用的代谢物的假设。我们还提出，P450酶与药物转运蛋白协同作用，影响AED脑可利用性的模式。我们的建议有可能阐明脑耐药所隐含的脑生物转化机制，并建立模拟人类耐药的方法，允许筛选新的AED以改善难治性癫痫的临床治疗。

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. PUBLIC HEALTH RELEVANCE: Failure of generating new brain therapeutics stems from an insufficient knowledge of the mechanisms regulating drug brain distribution under pathological conditions. As a result, a significant percentage of subjects affected by brain disorders are, to various extents, resistant to available drug treatment. For example, 20-25% of subjects with epilepsy do not respond to two or more anti-epileptic drugs (AED), administered alone or in combination. In these cases, brain surgery remains the only medical intervention. We wish to test the hypothesis that, in drug resistant forms of epilepsy, cerebrovascular P450 enzymes metabolize AED reducing their pharmacological efficacy. We will test the hypothesis that cerebrovascular P450 enzymes produce metabolites which may have neurotoxic effect. We also propose that P450 enzymes act in concert with drug transporter proteins, affecting the pattern of AED brain availability. Our proposal has the potential to elucidate mechanisms of brain biotransformation implied in brain drug resistance and to establish methods modeling human drug resistance, allowing for the screening of new AED for the improved clinical management of refractory forms of epilepsy.