Anticonvulsant screening using chronic epilepsy models

摘要 NINDS抗惊厥药筛选计划（ASP）已经确定了临床使用的大多数抗惊厥药 今天然而，三分之一的癫痫患者对这些药物没有反应。ASP协议基于 对正常动物在急性惊厥条件下诱发的癫痫发作的影响。我们已经开发出一种 慢性癫痫患者自发性发作的新的体外和体内测定的互补系统 准备工作这两个阶段的筛选系统提供了一个独特的重点反复自发性癫痫发作， 慢性癫痫模型。第一阶段是体外试验，包括器官型海马切片 培养，其在第一阶段产生电图癫痫发作活动和相应的生化生物标志物。 体外一周。第二阶段是由癫痫的红藻氨酸盐模型组成的体内测定，其中 使用连续遥测和监督、盲法、计算机化监测自发性癫痫发作 癫痫发作检测我们使用快速体外试验筛选了400多种化合物浓度组合 来自NINDS自定义化合物收藏。我们发现了一种先导化合物塞来昔布， 由随机双盲体内交叉试验的第二阶段测试领导。塞来昔布没有效果 对正常脑组织急性应用惊厥药引起的癫痫发作的影响，表明其 抗惊厥特性是慢性癫痫所独有的，并提高了其作用谱的可能性。 将与ASP方案发现的抗惊厥药不同。 开发的下一步是药物化学，以优化塞来昔布的抗惊厥功效。这是 最可行的是通过UH 2/UH 3蓝图神经治疗网络实现。我们的讨论 随着BPN项目官员的澄清，为了有效地利用BPN药物化学项目，我们必须进一步 开发体外和体内试验，并获得有关我们的先导化合物的更多数据。UH2/3 NINDS项目官员认为，该机制是最合适的供资机制。在 在本提案的R21阶段，我们将扩展塞来昔布和2，5 二甲基塞来昔布，一种衍生物，不抑制COX 2，但在体外具有相同的抗惊厥功效。我们 然后将表征测定的重现性和Z因子。我们还将建立剂量反应的 塞来昔布的体内试验，并将体外和体内部位的数量增加到各两个， 提高稳健性和吞吐量，并让杰出的年轻研究人员参与这项工作。在 R33阶段的建议，我们将进一步表征的铅化合物，通过确定是否COX 2 在体外和体内试验中，抑制对于抗惊厥活性是必需的。

Abstract The NINDS Anticonvulsant Screening Program (ASP) has identified most of the anticonvulsants in clinical use today. However, one third of epileptic patients do not respond to these drugs. The ASP protocols are based on seizures induced by subjecting normal animals to acute convulsant conditions. We have developed a complimentary system of novel in vitro and in vivo assays of spontaneous seizures in chronically epileptic preparations. This two-stage screening system provides a unique focus on recurrent spontaneous seizures in chronic epilepsy models. The first stage is an in vitro assay comprised of the organotypic hippocampal slice culture, which develops electrographic seizure activity and corresponding biochemical biomarkers over the first week in vitro. The second stage is an in vivo assay comprised of the kainate model of epilepsy in which spontaneous seizures are monitored using continuous telemetry and supervised, blinded, computerized seizure detection. We used the rapid in vitro assay to screen over 400 compound-concentration combinations from the NINDS Custom Compound Collection. We found a lead compound, celecoxib, and then verified this lead by the second-stage testing in a randomized double blind in vivo crossover trial. Celecoxib had no effect on seizures induced by acute application of convulsants to normal brain tissue, suggesting that its anticonvulsant properties are unique to chronic epilepsy, and raising the possibility that its spectrum of action will be distinct from anticonvulsants discovered by the ASP protocols. The next step in development is medicinal chemistry to optimize celecoxib’s anticonvulsant efficacy. This is most feasibly accomplished through the UH2 / UH3 Blueprint Neurotherapeutics Network. As our discussions with BPN program officers clarified, to efficiently utilize the BPN medicinal chemistry program we must further develop the in vitro and in vivo assays and acquire additional data on our lead compound. The UH2/3 mechanism was considered the most appropriate funding mechanism by the NINDS program officer. In the R21 phase of this proposal, we will extend the in vitro assay’s concentration-response for celecoxib and 2,5 dimethyl celecoxib, a derivative that does not inhibit COX2 but has equal anticonvulsant efficacy in vitro. We will then characterize the assay’s reproducibility and Z factor. We will also establish the dose-response of the in vivo assays for celecoxib, and increase the number of in vitro and in vivo sites to two each in order to improve robustness and throughput, as well as engage outstanding younger investigators in this effort. In the R33 phase of the proposal, we will further characterize the lead compound by determining whether COX2 inhibition is necessary for anticonvulsant activity in the in vitro and in vivo assays.