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）：该提案旨在提供临床前数据，支持结节性硬化症（TSC）癫痫的新疗法，TSC 是一种包括早期癫痫、智力低下和自闭症的疾病。我们建议布美他尼（氯协同转运蛋白 NKCC1 的抑制剂）单独使用或与经典 GABA 激动剂苯巴比妥或最近批准的抗惊厥药氨己烯酸联合使用可有效预防 TSC 癫痫发作。我们最近发表的文章表明，NKCC1 在人类 TSC 组织以及难治性癫痫的另一种原因——局灶性皮质发育不良 IIb 型 (FCD IIb) 中过度表达。 NKCC1 增加会导致细胞内氯离子增加，从而通过介导去极化和兴奋来损害 GABA 受体激活的抑制作用。我们最近生成了一种新型 Tsc1cc Nestin-rtTA+ tet-OP-cre+ 突变小鼠模型，该模型患有癫痫，并显示 NKCC1 过度表达和去极化 GABA 反应。目标 1 将使用该小鼠模型进一步证明布美他尼单独使用或与苯巴比妥或氨己烯酸联合使用在体外皮质切片中的功效以及通过脑电图记录进行体内治疗。目标 2 将检查布美他尼单独使用以及与苯巴比妥或氨己烯酸联合使用对由人类 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。 这项工作代表了研究 TSC 癫痫发生机制的新方法，并有可能为 TSC 患者的癫痫发作控制产生一种基于辅助新机制的抗惊厥策略。它还将为该机制提供原理验证，以及 TSC 小鼠模型的临床前疗效和临床相关结果。最后，我们的建议包括目标验证以及人体组织中的机制。这些是该化合物在 TSC 中进行首次人体试验的关键要素，也可能扩展到 FCD IIb。 公共卫生相关性：癫痫是一种常见疾病，影响超过 1% 的人口，超过 30% 的患者患有难治性癫痫。有些遗传性疾病的癫痫发病率甚至更高：结节性硬化症 (TSC) 是一种遗传性疾病，超过 90% 的癫痫患者发病率较高。此外，TSC 癫痫可能对传统的抗癫痫药物产生耐药性，并且 TSC 儿童的癫痫发作可能与后期智力障碍的高发生率相关。在某些情况下，唯一的治疗方法是手术切除被认为是癫痫病灶的 TSC 结节。在这里，我们展示了一种新的靶点，即 NKCC1 蛋白，在人类 TSC 大脑以及 TSC 小鼠模型中的表达水平高于正常水平。 NKCC1 蛋白可能引起癫痫发作，并导致 TSC 癫痫发作对传统药物产生抵抗。我们建议使用 NKCC1 活性阻断剂布美他尼作为附加疗法，与已批准的抗癫痫药物苯巴比妥和氨己烯酸联合使用，以改善 TSC 小鼠模型中的癫痫发作控制，也可以直接在人类 TSC 和 TSC 的脑切片中改善癫痫发作控制。手术期间取出的手术样本。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发现了类似的 NKCC1 失调。由于布美他尼已被 FDA 批准为利尿剂，如果成功，目前的提案将提供重要的临床前证据和人体靶标验证，以支持布美他尼作为传统 GABA 激动剂抗惊厥药（如苯巴比妥或苯巴比妥）的附加疗法的早期 I/II 期试验。其他较新的药物，如氨己烯酸，用于控制 TSC 患者的癫痫发作。重要的是，由于 TSC 患者的许多特征也可见于患有严重难治性癫痫的非 TSC 患者，例如 IIb 型局灶性皮质发育不良，因此这些结果可能更广泛地应用于更一般的严重癫痫病例。该拨款申请涉及癫痫研究基准 (http://www.ninds.nih.gov/research/epilepsyweb/2007_benchmarks.htm) 的几个组成部分 I. 预防癫痫及其进展 D. 确定预防或停止癫痫的新方法E. 开发新的动物模型来研究癫痫发生。二区。开发新的治疗策略并改进当前的方法以治愈癫痫 C. 改进当前的治疗并开发新技术