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.

疼痛会导致人类的痛苦和整个社会的生产力丧失。阿片类药物，广泛规定 治疗疼痛是添加剂的，导致阿片类药物过量导致药物滥用和死亡的全国激增。 该阶段I SBIR提案，响应RFA-NS-23-006（HEL HEAS INTIATIVE： 开发针对增强疼痛管理的疗法和技术）将启动发展 “盘子测定中的疼痛”（PIDA）可以筛选化合物以识别潜在的镇痛药以服务 作为杀菌剂的替代品。该项目将是Vala Sciences Inc.之间的合作，专门从事 自动化数字显微镜和基于细胞的药物发现的测定法，以及解剖结构化，该测定法 源自人类诱导多能干细胞（HIPSC）的制造商神经元。专家托尼·雅克什（Tony Yaksh）博士 关于临床前疼痛研究，将成为顾问。解剖学已经开发了功能性HIPSC-NOCPTORS（称为配音） Realdrgs），代表专门对有害刺激的外周神经元的细胞体 居住在背根神经节中。与主要DRG相似，Realdrgs对有害的刺激作出反应（例如辣椒素， 辣椒中的活性成分）在细胞内钙中大大增加。解剖学也发起了 代表椎板I和II的背角神经元的神经元的发展（称为HIPSC-DLNS） 神经元疼痛传播的下一步（伤害感受器连接到DLN）。 Vala的动力学图像细胞仪（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 在96或384孔菜肴中培养的神经元。对于PIDA，Realdrgs将带有钙指标，并且 潜在的疼痛调节剂对KIC的反应对有害刺激的反应的影响。此外， 将开发PIDA的高级版本，其中realdrgs和Hipsc-dlns在 具有通过微型隧道连接的两个井的设备，这将使Realdrgs建立突触 与HIPSC-DLNS连接。这将使这些细胞之间的神经元传播可视化 响应于对雷德格的有害刺激而发起的类型。 PIDA会发现疼痛 减少伤害感受器对有害刺激的反应的调节器，或者减少信号的传播 伤害感受器和DLN，这种机制对于疼痛管理而言相对出乎意料 没有体外模型。 PIDA将具有人类神经元，这可能会更具临床性的预测 效果与动物模型，从而导致更有效的临床前药物发现和减少动物的使用。 PIDA，REALDRGS，HIPSC-DLNS和KIC将销售给制药公司，并用于合同 开发新型非阿片类镇痛药的研究。