Anticonvulsant screening using chronic epilepsy models

摘要 NINDS抗惊厥药物筛选计划(ASP)已经确定了大多数临床使用的抗惊厥药物 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 关于使正常动物处于急性惊厥状态而导致的癫痫。我们已经开发出一种 用于慢性癫痫患者自发性发作的新的体内外检测系统的补充 准备工作。这种两阶段筛查系统为反复发作的自发性癫痫提供了独特的重点。 慢性癫痫模型。第一阶段是由器官型海马片组成的体外实验。 培养，在第一次培养时产生电惊厥活性和相应的生化生物标志物 体外培养一周。第二阶段是由红藻氨酸癫痫模型组成的体内试验。 使用连续遥测监测自发性癫痫发作，并进行监督、盲目、计算机化 癫痫检测。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定制复合体集合。我们发现了一种先导化合物，塞来昔布，然后证实了这一点 采用体内随机双盲交叉试验进行第二阶段试验。塞来昔布不起作用 对正常脑组织急性应用惊厥药所致癫痫发作的研究 抗惊厥特性是慢性癫痫所特有的，这增加了其作用范围的可能性 将与ASP协议发现的抗惊厥药物不同。 下一步的开发是药物化学，以优化塞来昔布的抗惊厥疗效。这是 最可行的是通过UH2/UH3蓝图神经治疗网络完成的。因为我们的讨论 随着BPN计划官员的明确，为了有效地利用BPN药物化学计划，我们必须进一步 开发体外和体内测试，并获得有关我们的先导化合物的更多数据。UH2/3 该机制被NINDS项目官员认为是最合适的筹资机制。在 R21期，我们将延长塞来昔布和2，5 二甲基塞来昔布，一种衍生物，不抑制COX2，但在体外具有同等的抗惊厥效果。我们 然后将表征该分析的重复性和Z因子。我们还将建立药物的剂量反应关系 体内检测塞来昔布，并将体外和体内部位的数量分别增加到两个，以便 提高稳健性和吞吐量，并聘请优秀的年轻研究人员参与这一努力。在 R33阶段的建议，我们将通过确定COX2是否为先导化合物进一步表征 在体外和体内试验中，抑制是抗惊厥活性所必需的。

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.