The NKCC1 inhibitor bumetanide as a novel therapy in TSC

描述（由申请人提供）：本申请旨在为结节性硬化症（TSC）癫痫的新疗法提供临床前数据支持，TSC是一种包括早期癫痫、智力迟钝和自闭症的疾病。我们建议，氯共转运体NKCC1的抑制剂布美他尼单独或与典型的GABA激动剂苯巴比妥或最近批准的抗惊厥药维加巴林联合使用，可有效预防TSC癫痫发作。我们最近的研究表明，NKCC1在人类TSC组织以及另一种难治性癫痫的病因局灶性皮质发育不良IIb型（FCD IIb）中过表达。NKCC1的增加引起细胞内氯离子的增强，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 nesting - rtta + tet-OP-cre+突变小鼠模型，该模型是癫痫，显示NKCC1过表达和去极化GABA反应。目的1将使用该小鼠模型进一步验证布美他尼单独使用或与苯巴比妥或维加巴林联合使用在体外皮质切片中的疗效，并通过脑电图记录在体内治疗。目的2将研究布美他尼单独使用以及与苯巴比妥或维加巴林联合使用对从人TSC和FCD IIb手术活检组织中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癫痫发生机制的新途径，并有可能为TSC患者的癫痫控制产生一种基于辅助机制的新型抗惊厥药物策略。它还将为这一机制提供原理证明，并在具有临床相关结果的TSC小鼠模型中提供临床前疗效。最后，我们的建议包括靶点验证以及在人体组织中的机制。这些是该化合物在TSC中进行首次人体试验的关键因素，也可能扩展到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. 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