The NKCC1 inhibitor bumetanide as a novel therapy in TSC

NKCC1 抑制剂布美他尼作为 TSC 的新型疗法

• 批准号: 8457417
• 负责人: Frances E Jensen
• 金额: $ 24.97万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8457417
• 关键词: Address; Adjuvant; Affect; Agonist; Animal Model; Animals; Anticonvulsants; Antiepileptic Agents; Applications Grants; Area; Autistic Disorder; Benchmarking; Biopsy; Brain; Brain Pathology; Bumetanide; Cells; Child; Chloride Ion; Chlorides; Clinical; Cortical Dysplasia; Data; Disease; Diuretics; Drug Kinetics; Electroencephalography; Elements; Epilepsy; Epileptogenesis; Equilibrium; Excision; Exhibits; FDA approved; GABA Agonists; GABA Receptor; Genes; Giant Cells; Gramicidin; Henle' s loop; Hereditary Disease; Human; In Vitro; Incidence; Intellectual functioning disability; Left; Life; Measures; Mediating; Mental Retardation; Modeling; Morphology; Mus; Neonatal; Neuroglia; Neurologic; Neurons; New Agents; Operative Surgical Procedures; Outcome; Partial Epilepsies; Pathway interactions; Patients; Pharmaceutical Preparations; Phase I/II Trial; Phenobarbital; Population; Prevention; Publications; Rattus; Receptor Activation; Refractory; Relative (related person); Research; Resected; Resistance; Role; Safety; Sampling; Seizures; Sirolimus; Slice; Synapses; Techniques; Testing; Tetanus Helper Peptide; Text; Tissues; Transgenic Mice; Tuberous sclerosis protein complex; Up-Regulation; Validation; Vigabatrin; Wild Type Mouse; Work; base; clinically relevant; gamma-Aminobutyric Acid; hippocampal pyramidal neuron; human FRAP1 protein; human tissue; improved; in vivo; inhibitor/antagonist; innovation; mTOR Inhibitor; mouse model; mutant mouse model; neonate; nestin protein; new technology; novel; novel strategies; overexpression; pre-clinical; preclinical efficacy; prevent; receptor; response; sodium-potassium-chloride cotransporter 1 protein; therapeutic target; treatment strategy

DESCRIPTION (provided by applicant): This proposal aims to provide preclinical data in support of a novel therapy for epilepsy in Tuberous sclerosis complex (TSC), a disorder that includes early life epilepsy, mental retardation and autism. We propose that bumetanide, an inhibitor of the chloride cotransporter NKCC1, will be effective for the prevention of seizures in TSC alone or in combination with a canonical GABA agonist phenobarbital or the recently approved anticonvulsant vigabatrin. Our recent publication shows that NKCC1 is overexpressed in human TSC tissue, as well as in another cause of refractory epilepsy, focal cortical dysplasia Type IIb (FCD IIb). Increased NKCC1 causes enhanced intracellular chloride, and this impairs inhibitory actions of GABA receptor activation by causing it to mediate depolarization and excitation. We have recently generated a novel Tsc1cc Nestin-rtTA+ tet-OP-cre+ mutant mouse model that is epileptic and shows NKCC1 overexpression and depolarizing GABA responses. Aim 1 will use this mouse model to further text the efficacy of bumetanide alone or in combination with phenobarbital or vigabatrin in cortical slices in vitro and by treatment in vivo using EEG recordings. Aim 2 will examine the effects of bumetanide alone and in combination with phenobarbital or vigabatrin on GABA responses in brain slices acutely prepared from human TSC and FCD IIb surgical biopsy tissue. The proposed work represents a new approach in studying the mechanisms of epileptogenesis in TSC and has the potential to generate an adjuvant novel mechanism-based anticonvulsant strategy for seizure control in TSC patients. It will also provide proof-of-principle for this mechanism, as well as preclinical efficacy in a TSC mouse model with clinically relevant outcomes. Finally our proposal includes target validation as well as mechanism in human tissue. These are critical elements of a path to first-in-human trials for this compound in TSC, and may also extend to FCD IIb. PUBLIC HEALTH RELEVANCE: Epilepsy is a common disorder affecting over 1% of the population, and over 30% of patients have treatment resistant epilepsy. Some genetic conditions have even higher incidences of epilepsy: Tuberous Sclerosis Complex (TSC) is a genetic condition with and incidence of over 90% of patients with epilepsy. In addition, TSC epilepsy can be resistant to conventional anti-seizure drugs, and seizures in children with TSC can be associated with high rates of later intellectual disability. In some cases, the only treatment is surgical removal of the TSC tubers that are thought to be the foci of seizures. Here we show that a novel target, the NKCC1 protein, is expressed at higher than normal levels in human TSC brain as well as in a mouse model of TSC. The NKCC1 protein may cause seizures as well as be responsible for the resistance of TSC seizures from conventional drugs. We propose to use a blocker of NKCC1 activity, bumetanide, as add-on therapy in combination with an approved antiepileptic drugs phenobarbital and vigabatrin, to improve seizure control in a mouse model of TSC but also directly in brain slices from human TSC as well as surgical samples that are removed during surgery. We will also study tissue from patients with Focal Cortical Dysplasia Type IIb, as we have recently shown similar dysregulation of NKCC1 in these cases. As bumetanide is already FDA approved as a diuretic, if successful, the present proposal would provide important preclinical evidence and human target validation to support an early Phase I/II trial of bumetanide as an add-on therapy with conventional GABA agonist anticonvulsants like phenobarbital or other newer agents like vigabatrin for seizure control in TSC patients. Importantly, as a number of features of TSC patients are also seen in non-TSC patients with severe refractory epilepsy, such as Focal Cortical Dysplasia Type IIb, and these results may have wider application to more general cases of severe epilepsy. This grant application addresses several components of the Epilepsy Research Benchmarks (http://www.ninds.nih.gov/research/epilepsyweb/2007_benchmarks.htm) Area I. Prevent epilepsy and its progression D. Identify new ways to prevent epilepsy or stop it once it begins E. Develop new animal models to study epileptogenesis. Area II. Develop new treatment strategies and improve current approaches in order to cure epilepsy C. Improve current treatments and develop new technologies

描述（由申请人提供）：本提案旨在提供临床前数据，以支持脑硬化症（TSC）癫痫的新疗法，TSC是一种包括早期癫痫、精神发育迟滞和自闭症的疾病。我们建议，布美他尼，氯化物协同转运蛋白NKCC 1的抑制剂，将有效地预防癫痫发作的TSC单独或与经典的GABA激动剂苯巴比妥或最近批准的抗惊厥药氨己烯酸。我们最近的出版物表明，NKCC 1在人类TSC组织中过表达，以及在难治性癫痫的另一个原因中，局灶性皮质发育不良IIb型（FCD IIb）。增加NKCC 1导致增强的细胞内氯化物，并且这通过使其介导去极化和兴奋而损害GABA受体活化的抑制作用。我们最近产生了一种新的Tsc 1cc Nestin-rtTA+ tet-OP-cre+突变小鼠模型，该模型具有癫痫性，并显示NKCC 1过表达和去极化GABA反应。目的1将使用该小鼠模型进一步测试布美他尼单独或与苯巴比妥或氨己烯酸组合在体外皮质切片中以及通过使用EEG记录的体内治疗的功效。目的2将检查布美他尼单独和与苯巴比妥或氨己烯酸组合对从人TSC和FCD IIb手术活检组织急性制备的脑切片中GABA反应的影响。 拟议的工作代表了一种新的方法，在研究癫痫发作的机制，在TSC患者，并有可能产生一个辅助的新的机制为基础的抗惊厥策略癫痫发作控制。它还将为该机制提供原理证明，以及在具有临床相关结果的TSC小鼠模型中的临床前疗效。最后，我们的建议包括目标验证以及在人体组织中的机制。这些是该化合物在TSC中首次人体试验的关键要素，也可能扩展到FCD IIb。 公共卫生关系：癫痫是一种常见的疾病，影响超过1%的人口，超过30%的患者患有难治性癫痫。一些遗传性疾病甚至具有更高的癫痫发病率：多发性硬化症（TSC）是一种遗传性疾病，其发病率超过90%的癫痫患者。此外，TSC癫痫对传统的抗癫痫药物具有耐药性，TSC儿童的癫痫发作可能与后期智力残疾的高发生率有关。在某些情况下，唯一的治疗方法是手术切除被认为是癫痫发作病灶的TSC结节。在这里，我们表明，一种新的目标，NKCC 1蛋白，表达高于正常水平，在人类TSC大脑以及在小鼠模型的TSC。NKCC 1蛋白可能引起癫痫发作，也可能是TSC癫痫发作对常规药物产生耐药性的原因。我们建议使用NKCC 1活性的阻断剂布美他尼作为添加治疗，与批准的抗癫痫药物苯巴比妥和氨己烯酸组合，以改善TSC小鼠模型中的癫痫发作控制，但也直接在人TSC的脑切片以及手术期间取出的手术样本中。我们还将研究IIb型局灶性皮质发育不良患者的组织，因为我们最近在这些病例中显示了类似的NKCC 1失调。由于布美他尼已经被FDA批准为利尿剂，如果成功，本提案将提供重要的临床前证据和人体靶点验证，以支持布美他尼作为传统GABA激动剂抗惊厥药物（如苯巴比妥）或其他新药物（如氨己烯酸）的添加治疗的早期I/II期试验，用于TSC患者的癫痫发作控制。重要的是，由于TSC患者的许多特征也见于患有严重难治性癫痫的非TSC患者，例如局灶性皮质发育不良IIb型，这些结果可能更广泛地应用于更一般的严重癫痫病例。本资助申请涉及癫痫研究基准（http：//www.ninds.nih.gov/research/epilepsyweb/2007_benchmarks.htm）区域I的几个组成部分。预防癫痫及其进展D.确定新的方法来预防癫痫或停止它一旦开始E。开发新的动物模型研究癫痫发生。区域二。发展新的治疗策略和改善现有的方法，以治愈癫痫C。改进现有治疗方法并开发新技术