背景:在IMI2-NeuroDeRisk联盟的框架中,比较了三种体外电生理检测方法,以提高临床前对癫痫发作的预测.
方法:两种细胞模型,在两种不同的微电极阵列(MEA)平台上测试了原代大鼠皮质神经元和与hiPSC衍生的星形胶质细胞共培养的人诱导多能干细胞(hiPSC)衍生的谷氨酸能神经元,MaestroPro(AxionBiosystems)和Multiwell-MEA-System(多通道系统),在三个独立的实验室。戊四唑(PTZ)和/或微毒素(PTX)作为阳性(n=3-6孔)包括在每个板中,并且使用≤0.2%DMSO作为阴性对照(n=3-12孔)。总的来说,在0.1-30μM范围内的浓度进行了测试,锚定,如果可能,对临床相关暴露(未结合Cmax)进行了测试。药物诱导变化的活性阈值设定为20%。为了评估灵敏度,细胞模型的特异性和预测性,癫痫发作的尿源性反应定义为4个或更多终点的变化.还考虑了浓度依赖性趋势。
结果:被归类为阳性工具药物的33种化合物的神经元活性,评估了癫痫发作阳性或癫痫发作阴性化合物。将急性药物作用(<60分钟)与基线记录进行比较。<15分钟的时间点显示更强,对许多测试代理的反应变化较小。对于许多化合物,在较高测试浓度下检测到神经元活性的降低和停止。没有检测到单一的癫痫发作活动模式,即使在工具化合物中,可能是由于不同的行动机制和/或脱靶概况。提出了针对指示神经元兴奋的变化的事后分析。
结论:所有细胞模型都显示出良好的敏感性,从70%到86%不等。特异性范围为40-70%。与更常规的海马切片诱发活动测量相比,这些基于平板的模型提供了更高的通量和研究亚急性反应的潜力.然而,它们可能受到随机限制,他们的网络活动的自发性。
BACKGROUND: In the framework of the IMI2-NeuroDeRisk consortium, three in vitro electrophysiology assays were compared to improve preclinical prediction of seizure-inducing liabilities.
METHODS: Two cell models, primary rat cortical neurons and human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with hiPSC-derived astrocytes were tested on two different microelectrode array (MEA) platforms, Maestro Pro (Axion Biosystems) and Multiwell-MEA-System (Multi Channel Systems), in three separate laboratories. Pentylenetetrazole (PTZ) and/or picrotoxin (PTX) were included in each plate as positive (n = 3-6 wells) and ≤0.2% DMSO was used as negative controls (n = 3-12 wells). In general, concentrations in a range of 0.1-30 μM were tested, anchored, when possible, on clinically relevant exposures (unbound Cmax) were tested. Activity thresholds for drug-induced changes were set at 20%. To evaluate sensitivity, specificity and predictivity of the cell models, seizurogenic responses were defined as changes in 4 or more endpoints. Concentration dependence trends were also considered.
RESULTS: Neuronal activity of 33 compounds categorized as positive tool drugs, seizure-positive or seizure-negative compounds was evaluated. Acute drug effects (<60 min) were compared to baseline recordings. Time points < 15 min exhibited stronger, less variable responses to many of the test agents. For many compounds a reduction and cessation of neuronal activity was detected at higher test concentrations. There was not a single pattern of seizurogenic activity detected, even among tool compounds, likely due to different mechanisms of actions and/or off-target profiles. A post-hoc analysis focusing on changes indicative of neuronal excitation is presented.
CONCLUSIONS: All cell models showed good sensitivity, ranging from 70 to 86%. Specificity ranged from 40 to 70%. Compared to more conventional measurements of evoked activity in hippocampal slices, these plate-based models provide higher throughput and the potential to
study subacute responses. Yet, they may be limited by the random, spontaneous nature of their network activity.