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过表达和去极化的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。

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.