Engineered Intestinal Microenvironments as Preclinical Drug Screening Platforms

产品说明：工程化肠道微环境作为临床前药物筛选平台将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识别具有高市场转化概率的候选药物。治疗从心脏病和糖尿病到慢性疼痛和感染的各种疾病的口服药物必须首先被身体吸收才能在生理上有效，而不管其作用的解剖位置如何。目前，最广泛使用的体外吸收模型是Caco-2单层试验。该测定的一个关键限制是与健康小肠组织相比，通过Caco-2细胞之间紧密连接的细胞旁转运水平可忽略不计。这种不准确性导致错误地放弃有希望的药物分子，这是由于错误地预测不良的药代动力学参数。我们建议开发一种工程化细胞外基质（eECM），以取代Caco-2测定中通常使用的I型胶原基质。我们假设基质生物化学（目标1）和生物力学（目标2）的工程将可重复地控制粘着斑形成和细胞骨架组织，导致形成更具有生理相关性的紧密连接，并能够模拟细胞旁转运。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工业实验室环境。一直 然而，利用生物材料工程策略引导Caco-2细胞行为沿着沿着更生理相关的途径是一个焦点。使用重组技术，我们合成模块化的eECM材料含有弹性蛋白样结构域和细胞结合位点来自天然ECM蛋白。这种策略使解耦控制和基质生物化学和生物力学的调查。在目的1中，系统地改变细胞结合位点的特性和浓度以影响Caco-2单层的成熟和渗透性，如通过整合素接合研究、细胞增殖速率、粘着斑的数量和大小、细胞密度、紧密连接蛋白和上皮标志物的表达和组织以及模型药物的细胞旁转运测量所定量的。在目标2中，基质生物力学的改变独立于基质生物化学，以调节细胞-基质牵引力（如通过牵引力显微镜测量的），从而调节粘着斑和紧密连接形成以及Caco-2单层渗透性（如目标1中定量测量的）。在这两个目标中，将细胞密度、上皮标志物的表达、紧密连接蛋白的表达和组织以及细胞旁转运率与人小肠组织的值进行比较。这项工作将导致体外临床前吸收模型的开发，该模型在协议格式内具有改进的生理准确性，该协议格式可以通过用新型eECM材料简单地替换胶原蛋白而被工业实验室容易地采用。

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.