Engineered Intestinal Microenvironments as Preclinical Drug Screening Platforms

将一种药物推向市场是一个密集的过程，耗资8亿美元，耗时12年。因此，临床前筛选在很大程度上依赖于确定具有高市场翻译概率的候选药物。口服药物用于治疗心脏病、糖尿病、慢性疼痛和感染等多种疾病，无论其作用的解剖位置如何，必须首先被身体吸收，才能在生理上有效。目前，应用最广泛的体外吸收模型是Caco-2单层法。该试验的一个关键限制是，与健康小肠组织相比，Caco-2细胞之间紧密连接的细胞旁运输水平可以忽略不计。这种不准确性导致由于对不良药代动力学参数的错误预测而错误地放弃有前途的药物分子。我们建议开发一种工程化的细胞外基质（eECM）来取代Caco-2试验中通常使用的I型胶原基质。我们假设，基质生物化学工程（目标1）和生物力学工程（目标2）将可重复地控制病灶黏附形成和细胞骨架组织，导致紧密连接的形成，这些紧密连接更具有生理学相关性，能够模拟细胞旁运输。虽然其他人试图解决Caco-2测定的局限性，但他们通常依赖于化学试剂、细胞共培养系统或原代细胞的使用。虽然在科学上很有趣，但不幸的是，这些策略在技术上很麻烦，因此不容易转化为高通量的工业实验室环境。有

DESCRIPTION: Engineered Intestinal Microenvironments as Preclinical Drug Screening Platforms Getting a drug to market is an intensive process costing $800 million and taking 12 years. Therefore, preclinical screening is heavily relied upon to identify drug candidates with a high probability of market translation. Orally administered drugs, which treat myriad conditions ranging from heart disease and diabetes to chronic pain and infection, must first be absorbed by the body to be physiologically effective, regardless of their anatomical location of action. Currently, the most widely used in vitro absorption model is the Caco-2 monolayer assay. A key limitation of this assay is a negligible level of paracellular transport through tight junctions between Caco-2 cells compared to healthy small intestinal tissue. This inaccuracy results in erroneous abandonment of promising drug molecules due to the false prediction of poor pharmacokinetic parameters. We propose development of an engineered extracellular matrix (eECM) to replace the collagen type I matrix typically used in the Caco-2 assay. We hypothesize that engineering of the matrix biochemistry (Aim 1) and biomechanics (Aim 2) will reproducibly control focal adhesion formation and cytoskeletal organization, leading to the formation of tight junctions that are more physiologically relevant and capable of modeling paracellular transport. While others have tried to address the limitations of the Caco-2 assay, they have typically relied on use of chemical agents, cellular co-culture systems, or primary cells. While scientifically interesting, unfortunately these strategies are technically cumbersome and therefore not readily translatable to high-throughput industrial laboratory settings. There has yet to be a focus on utilizing biomaterials engineering strategies to guide Caco-2 cellular behavior along a more physiologically relevant pathway. Using recombinant techniques, we synthesize modular eECM materials containing elastin-like structural domains and cell-binding sites derived from native ECM proteins. This strategy enables decoupled control and investigation of matrix biochemistry and biomechanics. In Aim 1, cell-binding site identity and concentration are systematically altered to affect Caco-2 monolayer maturation and permeability, as quantified via integrin engagement studies, cell proliferation rate, number and size of focal adhesions, cellular density, expression and organization of tight junction proteins and epithelial markers, and paracellular transport measurements of model drugs. In Aim 2, matrix biomechanics is altered independently of matrix biochemistry to regulate cell-matrix traction forces (as measured by traction force microscopy) and hence focal adhesion and tight junction formation and Caco-2 monolayer permeability (quantitatively measured as in Aim 1). In both aims, cell density, expression of epithelial markers, expression and organization of tight junction proteins, and paracellular transport rates will be compared to values for human small intestinal tissue. This work will result in the development of an in vitro preclinical absorption model with improved physiological accuracy within a protocol format that can be easily adopted by industrial laboratories through the simple replacement of collagen with a novel eECM material.