A Unidirectional 96-Well Fluidic Culture Platform for Upstream Cell Dosing with Subsequent Downstream Nonlinear and Ascending Exposure Gradients for Real-Time and Cell-Based Toxicity Screening Environments.

Introduction: Because of the importance to create in vitro screening tools that better mimic in vivo models, for exposure responses to drugs or toxicants, reproducible and adaptable culture platforms must evolve as approaches to replicate functions that are native to human organ systems. The Stairstep Waterfall (SsWaterfall) Fluidic Culture System is a unidirectional, multiwell, gravity-driven, cell culture system with micro-channels connecting 12 wells in each row (8-row replicates). Materials and Methods: The construct allows for the one-way flow of medium, parent and metabolite compounds, and the cellular signaling between connected culture wells while simultaneously operating as a cascading flow and discretized nonlinear dosing device. Initial cell seeding in SsWaterfall mimics traditional static plate protocols but thereafter functions with controlled flow and ramping concentration versus time exposure environments. Results: To investigate the utility of a microfluidic system for predicting drug efficacy and toxicity, we first delineate device design, fabrication, and characterization of a disposable dosing and gradient-exposure platform. We start with detailed characterizations by demarcating various features of the device, including low nonspecific binding, wettability, biocompatibility with multiple cell types, intra-well and inter-well flow, and efficient auto-mixing properties of dose compounds added into the platform. Discussion: We demonstrate the device utility using an example in sequential testing-screening drug toxicity and efficacy across wide-ranging inducible exposures, 0 → IC100, featuring real-time assessments. Conclusion: The integrated auto-gradient technology, gravity flow with stairstep pathways, offers end-users an easy and quick alternative to evaluate broad-ranging toxicity of new compound entities (e.g., pharmaceutical, environmental, agricultural, cosmetic) as opposed to traditional/arduous manual drug dilutions and/or expensive robotic technology.

The discovery of potent blockers of the canonical transient receptor channels, TRPC3 and TRPC6, based on an anilino-thiazole pharmacophore.

Lead optimization of piperidine amide HTS hits, based on an anilino-thiazole core, led to the identification of analogs which displayed low nanomolar blocking activity at the canonical transient receptor channels 3 and 6 (TRPC3 & 6) based on FLIPR (carbachol stimulated) and electrophysiology (OAG stimulated) assays. In addition, the anilino-thiazole amides displayed good selectivity over other TRP channels (TRPA1, TRPV1, and TRPV4), as well as against cardiac ion channels (CaV1.2, hERG, and NaV1.5). The high oxidation potential of the aliphatic piperidine and aniline groups, as well as the lability of the thiazole amide group contributed to the high clearance observed for this class of compounds. Conversion of an isoquinoline amide to a naphthyridine amide markedly reduced clearance for the bicyclic piperidines, and improved oral bioavailability for this compound series, however TRPC3 and TRPC6 blocking activity was reduced substantially. Although the most potent anilino-thiazole amides ultimately lacked oral exposure in rodents and were not suitable for chronic dosing, analogs such as 14-19, 22, and 23 are potentially valuable in vitro tool compounds for investigating the role of TRPC3 and TRPC6 in cardiovascular disease.