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

癫痫是最常见的神经系统疾病之一。尽管在过去的20多年里，许多新的抗癫痫药物被开发出来，但大约30%的患者并没有完全摆脱癫痫发作，即对药物耐药。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疗效。因此，开发更有效的抗癫痫药物的新策略是医学上的迫切需要。此外，临床上迫切需要开发防止脑损伤后癫痫发展的药物，即发挥预防(抗癫痫)作用。利尿药布美他尼是一种选择性的钠钾氯转运体(NKCCs)抑制剂，多年来一直被认为是一种非常有趣的抗癫痫和抗癫痫化合物，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的发生和作为新生儿和成人癫痫耐药的机制中发挥着重要作用。布美他尼的实验研究似乎支持这一观点；然而，在患有新生儿癫痫的儿童中进行的第一次临床试验是否定的。这是布美他尼在血浆中的高电离率造成的明显后果，导致大脑渗透不良，这是L团队在2010年首次报道的。因此，该小组开发了布美他尼的亲脂性前体药物，这种药物可以渗透到大脑并释放布美他尼。然而，这一策略不能解决布美他尼的一些显著缺点，例如缺乏对肾脏NKCC2和神经元NKCC1的选择性。本申请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性布美他尼衍生物。这一目标的起点是布美塔尼衍生物，它是我们的合作伙伴Peter Feit在布美塔尼开发过程中合成的，使用的衍生物在结构和利尿剂性质上都与布美塔尼有显著不同。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试验来确定。在这些实验中，我们还将研究NKCC1剪接变体NKCC1b，它主要在大脑中表达。最有趣的衍生物将在小鼠和大鼠的活体模型中进行测试，以测试(A)脑渗透，(B)利尿剂效力，以及(C)抗癫痫和抗癫痫作用。从这些综合研究中，我们期望不仅能优化布美他尼的结构，用于治疗脑部疾病，而且能更好地了解NKCC1在癫痫发生和细胞发生中的作用。

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.