The NKCC1 inhibitor bumetanide as a novel therapy in TSC

描述(由申请人提供)：本提案旨在提供临床前数据，以支持结节性硬化症(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。我们认为，氯离子转运体NKCC1的抑制剂布美他尼单独或与经典的GABA激动剂苯巴比妥或最近批准的抗惊厥药物Vigabatrin联合使用将有效地预防TSC的癫痫发作。我们最近发表的文章显示，NKCC1在人类TSC组织中过表达，以及在另一种难治性癫痫的病因-局灶性皮质发育不良IIb型(FCD IIb)中过度表达。NKCC1的增加导致细胞内氯离子的增加，这通过使GABA受体介导去极化和兴奋而削弱了GABA受体激活的抑制作用。我们最近建立了一种新的Tsc1cc Nestin-RTTA+tet-op-cre+突变小鼠模型，该模型是癫痫，并显示NKCC1过表达和去极化GABA反应。目的1利用这一小鼠模型进一步验证布美他尼单独或与苯巴比妥或威巴比林合用的疗效，并通过脑电记录在体内进行治疗。目的2将研究布美他尼单独以及与苯巴比妥或Vigabatrin合用对人TSC和FCD IIb手术活检组织急性脑片中GABA反应的影响。这项工作代表了研究TSC癫痫发生机制的新途径，并有可能为TSC患者的癫痫控制提供一种新的基于机制的辅助抗惊厥策略。它还将为这一机制提供原理证明，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，我们的建议包括靶点验证以及在人体组织中的机制。这些都是这种化合物在TSC进行首次人体试验的关键要素，也可能延伸到FCD IIb。 公共卫生相关性：癫痫是一种常见的疾病，影响着超过1%的人口，超过30%的患者患有难治性癫痫。某些遗传性疾病的癫痫发病率更高：结节性硬化症(TSC)是一种遗传性疾病，占癫痫患者的90%以上。此外，TSC癫痫可能对传统的抗癫痫药物产生耐药性，TSC儿童的癫痫发作可能与后期智力残疾的高比率有关。在某些情况下，唯一的治疗方法是手术切除被认为是癫痫灶的TSC结节。在这里，我们显示了一个新的靶点，NKCC1蛋白，在人类TSC脑中的表达水平高于正常水平，在TSC的小鼠模型中也是如此。NKCC1蛋白可能导致癫痫发作，也可能与常规药物对TSC癫痫发作的抵抗有关。我们建议将NKCC1活性阻滞剂布美他尼与已获批准的抗癫痫药物苯巴比妥和Vigabatrin结合使用作为附加治疗，以改善TSC小鼠模型的癫痫控制，并直接在TSC的脑片和手术中取出的手术样本中使用。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NKCC1调节异常。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类靶点验证，以支持布美他尼作为常规GABA激动剂抗惊厥药物(如苯巴比妥)或其他较新药物(如Vigabatrin)用于TSC患者癫痫控制的早期I/II期试验。重要的是，由于TSC患者的许多特征也可见于有严重难治性癫痫的非TSC患者，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。这项赠款申请涉及癫痫研究基准(http://www.ninds.nih.gov/research/epilepsyweb/2007_benchmarks.htm)区的几个组成部分。预防癫痫及其进展。确定预防癫痫的新方法或一旦癫痫开始就停止它。发展新的动物模型来研究癫痫的发生。领域二.制定新的治疗策略并改进现有方法，以治愈癫痫C.改进现有治疗方法并开发新技术

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