Planar culture of gastrointestinal stem cells for screening pharmaceuticals for adverse event risk

项目摘要 胃肠道副作用，如溃疡和腹泻，是最常见的不良事件来源 制药公司。GI干细胞负责修复和补充GI上皮，并提供药物 这些功能的抑制可能会导致不良事件风险。目前没有高吞吐量和 筛选对胃肠道干细胞有影响的候选疗法的经济有效的手段。动物模型令人担忧 有混淆的(例如，大鼠通常在死亡迫在眉睫之前不会表现出腹泻)和当前的体外模型 像Caco-2肿瘤细胞系一样，不包括正常干细胞群体。而3D-有机化合物培养物具有 干细胞成分，不可能接触到单层的顶端以进行化合物暴露 没有低通量的微量注射。阿尔蒂斯生物系统公司开发了一种专有的培养平台， RepliGut®Planar，使原代人类GI细胞能够形成用于药物筛选的上皮细胞。在预赛中 经过努力，我们启动了名为RepliGut®StemScreen的GI干细胞特异性平台的开发，以解决 对高通量、高成本效益的胃肠道干细胞筛查的需求尚未得到满足，包括一系列要测量的分析 可能导致不良事件的属性。这些检测包括与干细胞增殖、自身 更新和差异化能力。 在我们的第一阶段研究中，我们开发了一种为期5天、96孔的平板筛选试验(称为StemTox) 并测试了一组已知会引发胃肠道副作用的药剂。结果显示， 灵敏和剂量依赖的抑制干细胞增殖和损伤肠屏障形成。 此外，我们的化验检测到与临床密切相关的化合物的“不利”浓度。 血浆Cmax与人类临床结局相关。在此阶段2应用程序中，我们建议完成 优化StemTox筛选，生成商业验证数据集(使用多个组织捐赠者和 细胞大量横结肠)，并扩展模型，将细胞分化和长期药物暴露结合在一起。 总体而言，这项工作将导致StemTox试剂盒的商业推出，并使几个 后续实验范式，以更全面地评估非靶点药物机制，并重复- 给药试验(与当前支持IND的非临床研究要求平行)。

Project Summary GI side effects, such as ulcers and diarrhea, represent the most common source of adverse events for pharmaceuticals. GI stem cells are responsible for repairing and replenishing GI epithelium, and pharmaceutical inhibition of these functions likely contribute to adverse event risk. Currently there are no high-throughput and cost-effective means of screening candidate therapeutics for effects on GI stem cells. Animal models are fraught with confounds (e.g., rats generally do not exhibit diarrhea until death is imminent) and current in vitro models like the Caco-2 tumor cell line do not include a normal stem cell population. While 3D-organoid cultures have a stem cell component, access to the apical aspect of the monolayer for compound exposure is not possible without low-throughput microinjection. Altis Biosystems, Inc. has developed a proprietary culture platform, RepliGut® Planar, enabling primary human GI cells to form an epithelium for drug screening. In preliminary efforts, we initiated development of a GI stem cell-specific platform called RepliGut® StemScreen to address the unmet need for high-throughput, cost-effective GI stem cell screening, including a range of assays to measure properties that might lead to adverse events. These included assays related to stem cell proliferation, self- renewal, and differentiation competency. In our Phase 1 studies, we developed a 5 day, 96-well, plate-based screening assay (termed StemTox) and tested a panel of pharmaceutical agents known to trigger GI side effects. The results revealed a highly sensitive and dose-dependent inhibition of stem cell proliferation and impairment of intestinal barrier formation. Furthermore, our assay detected “adverse” concentrations for compounds that closely paralleled the clinical plasma Cmax associated with human clinical outcomes. In this Phase 2 application, we propose to complete optimization of the StemTox screen, generate a commercial validation data set (using multiple tissue donors and cell lots of transverse colon), and expand the model to integrate cell differentiation and long-term drug exposures. Collectively, this work will result in the commercial launch of the StemTox assay, as well as, enable several follow-on experimental paradigms to more comprehensively assess off-target drug mechanisms and repeat- dosing experiments (to parallel current IND-enabling nonclinical study requirements).