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， 大药厂胃肠道干细胞负责修复和补充胃肠道上皮， 这些功能的抑制可能导致不良事件风险。目前没有高通量和 筛选候选治疗剂对GI干细胞的影响的成本效益的手段。动物模型充满了 具有混淆（例如，大鼠通常不会出现腹泻，直到死亡迫在眉睫）和当前的体外模型 如Caco-2肿瘤细胞系不包括正常干细胞群。虽然3D类器官培养物具有 干细胞组分，无法进入单层的顶面进行化合物暴露 没有低通量显微注射。Altis生物系统公司开发了一个专有的文化平台， DiGut ® Planar，使原代人GI细胞能够形成用于药物筛选的上皮。初步 为了解决这一问题，我们开始开发一种名为<$Gut ® StemScreen的GI干细胞特异性平台， 对高通量、具有成本效益的GI干细胞筛选的未满足需求，包括一系列测定 可能导致不良事件的性质。这些包括与干细胞增殖、自身免疫和细胞周期相关的分析。 更新和差异化能力。 在我们的1期研究中，我们开发了一种5天，96孔，基于平板的筛选试验（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， 敏感的和剂量依赖性的干细胞增殖抑制和肠屏障形成的损害。 此外，我们的检测方法检测到了化合物的“不利”浓度， 血浆Cmax与人类临床结局相关。在第二阶段的申请中，我们建议完成 优化StemTox筛选，生成商业验证数据集（使用多个组织供体和 横结肠的细胞批次），并扩展模型以整合细胞分化和长期药物暴露。 总的来说，这项工作将导致StemTox检测试剂盒的商业化推出，并使几个 后续实验范式，以更全面地评估脱靶药物机制和重复- 给药实验（平行于当前IND使能非临床研究要求）。

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).