The Pain in a Dish Assay (PIDA): a high throughput system featuring human stem cell-derived nociceptors and dorsal horn neurons to test compounds for analgesic activity

疼痛在整个社会造成广泛的人类痛苦和生产力损失。阿片类药物，广泛用于 治疗疼痛，是上瘾的，导致全国药物滥用和阿片类药物过量死亡的激增。 本第一阶段SBIR提案，响应征集RFA-NS-23-006(REAL倡议： 针对加强疼痛管理的疗法和技术的开发)，将启动开发 的“餐盘疼痛测试”(PIDA)，以筛选化合物以确定潜在的镇痛剂。 作为阿片类药物的替代品。该项目将由Vala Sciences Inc.合作完成，该公司专门从事 用于药物发现的自动数字显微镜和基于细胞的分析，以及解剖公司，该公司 制造来自人类诱导多能干细胞(HiPSCs)的神经元。托尼·雅克什博士，专家 在临床前疼痛研究方面，将担任顾问。解剖学公司已经开发出功能性的HiPSC-伤害性感受器(称为 RealDRGs)，代表专门对伤害性刺激做出反应的外周神经元，其胞体 存在于背根神经节。与初级DRG类似，RealDRGs对有害刺激(例如，辣椒素， 辣椒中的有效成分)细胞内钙大量增加。解剖学也启动了 代表板层I和II背角神经元的神经元的发育(称为HiPSC-DLN)，它们是 神经元疼痛传递的下一步(伤害性感受器连接到DLN)。瓦拉(氏)动态图像细胞仪(KIC) 是一种自动显微镜，旨在量化化合物对钙和电压瞬变的影响 神经元在96孔或384孔培养皿中培养。对于PIDA，RealDRGs将加载一个钙指示器，以及 潜在的痛觉调节剂对伤害性刺激反应的影响将用KIC进行量化。此外， 将开发PIDA的高级版本，其中RealDRGs和HiPSC-DLN在 一种设备，其特点是通过微隧道连接两个井，使RealDRGs能够建立突触 与HiPSC-DLN的连接。这将使可视化这些细胞之间的神经元传输成为可能 应用于RealDRGs的响应伤害性刺激的类型。PIDA将使人们能够发现疼痛 减少伤害感受器对伤害性刺激的反应的调制器，或减少信号在 伤害性感受器和DLN，一种相对未被探索的疼痛管理机制，对于 目前还没有体外模型。PIDA将以人类神经元为特色，这可能会更好地预测临床 效果与动物模型相比，导致更有效的临床前药物发现，并减少动物的使用。这个 PIDA、RealDRGs、HiPSC-DLN和KIC将向制药公司销售并用于合同 研究开发新型的非阿片类止痛药。

Pain causes widespread human suffering and loss of productivity across society. Opioids, widely prescribed to treat pain, are addictive, leading to a national surge in drug abuse and deaths resulting from opioid overdose. This Phase I SBIR proposal, submitted in response to solicitation RFA-NS-23-006 (HEAL INITIATIVE: Development of Therapies and Technologies Directed at Enhanced Pain Management), will initiate development of the “Pain in a Dish Assay” (PIDA) to enable screening of compounds to identify potential analgesics to serve as alternatives to opioids. The project will be a collaboration between Vala Sciences Inc., which specializes in automated digital microscopes and cell-based assays for drug discovery, and Anatomic Incorporated, which manufactures neurons derived from human induced pluripotent stem cell (hiPSCs). Dr. Tony Yaksh, an expert on preclinical pain research, will be a consultant. Anatomic has developed functional hiPSC-nociceptors (dubbed RealDRGs), which represent peripheral neurons specialized to respond to noxious stimuli, whose cell bodies reside in the dorsal root ganglia. Similar to primary DRGs, RealDRGs respond to noxious stimuli (e.g., capsaicin, the active ingredient in chili peppers) with large increases in intracellular calcium. Anatomic has also initiated development of neurons that represent dorsal horn neurons of lamina I and II (dubbed hiPSC-DLNs), which are the next step in neuronal pain transmission (nociceptors connect to DLNs). Vala’s Kinetic Image Cytometer (KIC) is an automated microscope designed to quantify effects of compounds on calcium and voltage transients for neurons cultured in 96- or 384-well dishes. For the PIDA, RealDRGs will be loaded with a calcium indicator, and effects of potential pain modulators on responses to noxious stimuli will be quantified with the KIC. Furthermore, an advanced version of the PIDA will be developed in which RealDRGs and hiPSC-DLNs are cocultured in a device that features two wells connected via micro tunnels which will enable the RealDRGs to establish synaptic connections with the hiPSC-DLNs. This will enable visualization of neuronal transmission between these cell types initiated in response to noxious stimuli applied to the RealDRGs. The PIDA will enable discovery of pain modulators that reduce responses of nociceptors to noxious stimuli, or reduce transmission of signals between nociceptors and DLNs, a mechanism that has been relatively unexplored for pain management and for which there are no in vitro models. The PIDA will feature human neurons, which will likely be more predictive of clinical effects vs. animal models, leading to more efficient preclinical drug discovery, and reduced use of animals. The PIDA, RealDRGs, hiPSC-DLNs, and KIC will be marketed to pharmaceutical companies and used for contract research to develop novel, non-opioid, analgesics.