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

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).