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

皿中疼痛测定 (PIDA)：一种高通量系统，具有人类干细胞来源的伤害感受器和背角神经元，用于测试化合物的镇痛活性

• 批准号: 10759735
• 负责人: PATRICK M MCDONOUGH
• 金额: $ 35.01万
• 依托单位: VALA SCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10759735
• 关键词: Acute Pain; Adult; Analgesics; Anatomy; Animal Model; Animals; Axon; Binding; Biological Assay; Brain; Calcium; Capsaicin; Cells; Cervical spine; Cessation of life; Chili Pepper; Coculture Techniques; Collaborations; Communication; Communities; Complex; Dendrites; Development; Devices; Doctor of Philosophy; Drug abuse; Embryo; Engineering; Funding; Future; Goals; Helping to End Addiction Long-term; Human; Image; Induced pluripotent stem cell derived neurons; Inflammation; Interneurons; Ion Channel; Kinetics; Marketing; Methods; Microglia; Microscope; Modeling; Molecular Target; Monitor; Neurons; Nociception; Nociceptors; Opioid; Pain; Pain Research; Pain management; Pathway interactions; Peripheral; Persons; Pharmacologic Substance; Phase; Phenotype; Posterior Horn Cells; Production; Protocols documentation; Quality Control; Research; Research Contracts; Science; Side; Signal Transduction; Small Business Innovation Research Grant; Societies; Specificity; Spinal; Spinal Ganglia; Stimulant; Stimulus; Synapses; System; Technology; Testing; Toxicity Tests; Veratridine; Vertebral column; Visualization; antagonist; calcium indicator; candidate identification; cell preparation; cell type; channel blockers; chemical addition; chronic pain; clinical effect; clinical predictors; design; digital; donor stem cell; dorsal horn; drug candidate; drug discovery; efficacy testing; human stem cells; improved; in vitro Model; induced pluripotent stem cell; innovation; instrument; manufacture; movie; neural circuit; neuron development; neurotransmission; non-opioid analgesic; novel; opioid overdose; overdose death; pain reduction; pre-clinical; pre-clinical research; productivity loss; response; screening; single-cell RNA sequencing; small molecule libraries; stem cells; therapy development; transcriptome; transmission process; voltage

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.

疼痛导致广泛的人类痛苦和整个社会生产力的丧失，阿片类药物被广泛用于治疗。 治疗疼痛，容易上瘾，导致全国药物滥用和阿片类药物过量导致的死亡激增。 此第一阶段 SBIR 提案是为了响应 RFA-NS-23-006 征集而提交的（HEAL INITIATIVE： 开发针对增强疼痛管理的疗法和技术），将启动开发 “盘中疼痛测定”（PIDA），可筛选化合物以识别潜在的镇痛药 作为阿片类药物的替代品，该项目将是 Vala Sciences Inc. 之间的合作，该公司专门从事阿片类药物的研究。 用于药物发现的自动数字显微镜和基于细胞的分析，以及 Anatomic Incorporated，该公司 制造源自人类诱导多能干细胞 (hiPSC) 的神经元，专家 Tony Yaksh 博士。 Anatomic 开发了功能性 hiPSC 伤害感受器（称为疼痛感受器）。 RealDRGs），代表专门对有害刺激做出反应的周围神经元，其细胞体 与初级 DRG 类似，RealDRG 驻留在背根神经节中，对有害刺激（例如辣椒素、 辣椒中的活性成分）随着细胞内钙的大量增加也开始发生。 代表 I 和 II 层背角神经元（称为 hiPSC-DLN）的神经元的发育，它们是 神经元疼痛传递的下一步（伤害感受器连接到 Vala 的动态图像细胞仪 (KIC)）。 是一款自动显微镜，旨在量化化合物对钙和电压瞬变的影响 在 96 或 384 孔培养皿中培养的神经元 对于 PIDA，RealDRG 将装载钙指示剂，并且 潜在的疼痛调节剂对有害刺激反应的影响将通过 KIC 进行量化。 将开发 PIDA 的高级版本，其中 RealDRG 和 hiPSC-DLN 在 该设备具有通过微隧道连接的两个井，这将使 RealDRG 能够建立突触 与 hiPSC-DLN 的连接这将使这些细胞之间的神经传递可视化。 针对应用于 RealDRG 的有害刺激而启动的类型 PIDA 将能够发现疼痛。 减少伤害感受器对有害刺激的反应，或减少伤害感受器之间的信号传输的调制器 伤害感受器和 DLN，这是一种在疼痛管理方面尚未被探索的机制，其 PIDA 没有体外模型，以人类神经元为特征，这可能更能预测临床。 与动物模型相比的效果，导致更有效的临床前药物发现，并减少动物的使用。 PIDA、RealDRG、hiPSC-DLN 和 KIC 将向制药公司销售并用于合同 研究开发新颖的非阿片类镇痛药。