Development and characterization of bumetanide derivatives with selective effect on the neuronal cation-chloride cotransporter NKCC1 for studies on the role of NKCC1 in ictogenesis and epileptogenesis

癫痫病是最常见的神经系统疾病之一。尽管在过去20年中开发了许多新的抗癫痫药物，但约30%的患者并没有完全消除癫痫发作，即产生耐药性。对于某些类型的癫痫发作，例如新生儿癫痫发作，情况甚至更糟，因为现有的药物都没有发挥足够的功效。因此，开发更有效的抗癫痫药物的新策略是迫切的医疗需求。此外，临床迫切需要开发药物，以防止脑损伤后癫痫的发展，即发挥预防（抗癫痫）作用。作为一种na - k - cl共转运体（NKCCs）的选择性抑制剂，利尿剂布美他尼作为一种非常有趣的抗癫痫和抗癫痫化合物已被讨论多年，因为神经元NKCC1表达的增加和由此导致的GABA功能的改变似乎在癫痫发生中起重要作用，并作为新生儿和成人癫痫耐药的机制。布美他尼的实验研究似乎支持这一观点；然而，对新生儿癫痫患儿的首次临床试验结果为阴性。这是布美他尼在血浆中电离率高的明显后果，导致对大脑的渗透能力差，这是Löscher小组在2010年首次报道的。结果，这组人产生了亲脂性的布美他尼前药，这种药能进入大脑并释放布美他尼。然而，这一策略并不能解决布美他尼的一些显著缺点，例如，对肾脏NKCC2和神经元NKCC1缺乏选择性。本应用程序中描述的计划研究的目的是开发和表征对神经元NKCC1具有高度选择性作用的新型脑穿透布美他胺衍生物。我们的出发点是布美他尼衍生物，我们的合作伙伴Peter Feit在开发布美他尼的过程中合成了布美他尼衍生物，这些衍生物在结构和利尿性能上都与布美他尼有很大的不同。根据这些衍生物的数据，我们的合作伙伴Thomas Erker将合成更多的化合物。这些化合物对NKCC1和NKCC2的抑制效力将通过卵母细胞表达测定来确定。在这些实验中，我们还将研究主要在大脑中表达的nkcc1剪接变体NKCC1b。最有趣的衍生物将在小鼠和大鼠体内模型中进行测试(a)脑穿透，(b)利尿效力，(c)抗癫痫和抗癫痫作用。通过这些综合研究，我们期望不仅能优化布美他尼治疗脑部疾病的结构，还能更好地了解NKCC1在癫痫发生和ictogenesis中的作用。

Epilepsies are one of the most frequent neurological diseases. Despite the development of numerous new antiepileptic drugs in the last some 20 years, about 30% of the patients do not become seizure-free, i.e., are pharmacoresistant. For some types of epileptic seizures, e.g., neonatal seizures, the situation is even worse, because none of the available drugs exert any sufficient efficacy. Thus, novel strategies for development of more efficacious antiepileptic drugs are an urgent medical need. Furthermore, there is an urgent clinical need to develop drugs that prevent development of epilepsies after brain insults, i.e., exert a prophylactic (antiepileptogenic) effect. The diuretic drug bumetanide, a selective inhibitor of Na-K-Cl-cotransporters (NKCCs), has been discussed for years as a highly interesting antiepileptic and antiepileptogenic compound, because increased expression of neuronal NKCC1 and the resulting shift in GABA functionality seems to play an important role in epileptogenesis and as a mechanism of pharmacoresistance in neonatal and adult epilepsies. Experimental studies with bumetanide seemed to support this idea; however, first clinical trials in children with neonatal seizures were negative. This is an obvious consequence of the high ionization rate of bumetanide in plasma, resulting in poor penetration into the brain, which was first reported by the Löscher group in 2010. As a consequence, this group developed lipophilic prodrugs of bumetanide, which penetrate into the brain and release bumetanide. However, this strategy could not resolve some significant disadvantages of bumetanide, e.g., the lack of selectivity for renal NKCC2 vs. neuronal NKCC1. Aim of the planned studies described in this application is the development and characterization of novel brain-penetrating bumetanide derivatives with highly selective effect on neuronal NKCC1. Starting point for this aim are bumetanide derivatives, which have been synthesized by our cooperation partner Peter Feit during development of bumetanide, using derivatives that are significantly different from bumetanide in both structural and diuretic properties. Based on data with these derivatives, additional compounds will by synthesized by our cooperation partner Thomas Erker. The inhibitory potency of these compounds on NKCC1 vs. NKCC2 will be determined by an oocyte expression assay. In these experiments, we will also study the NKCC1-splice variant NKCC1b, which is predominantly expressed in the brain. The most interesting derivatives will be tested in in vivo models in mice and rats for (a) brain penetration, (b) diuretic potency, and (c) antiepileptic and antiepileptogenic effects. From these comprehensive studies, we expect not only an optimization of the bumetanide structure for treatment of brain diseases but also a better understanding of the role of NKCC1 in epileptogenesis and ictogenesis.