In Vitro Model of the Human Alveolar Epithelium

描述(由申请人提供)： 远端气管具有多种特征，这使得它们成为系统性治疗的一个有吸引力的场所。这些包括非常薄的上皮，较大的表面积，避免肝脏首过代谢和广泛的血管形成。因此，肺部给药被越来越多地考虑作为候选药物的替代方案，这些候选药物要么不稳定，要么在胃肠道(如蛋白质或多肽)吸收不良。据估计，目前已有数百种生物工程蛋白和多肽上市或正在进行临床研究。此外，即使是小分子药物也可以从几乎瞬间通过肺泡上皮实现的给药中受益。因此，一个强大的、商业上可用的肺泡转运和毒性的体外模型可能会对新治疗产品的开发做出重大贡献。然而，目前还不存在这样的模式。目前拨款提案的目标是通过开发和生产商业的人肺泡上皮体外模型来满足这一未得到满足的需求。在更昂贵、更耗时的活体动物研究之前，该模型将是一个有用的筛选/研究工具，减少了进行的动物研究的数量和范围。除了在临床前药物开发期间的运输和毒理学应用外，该模型还将用于与环境毒理学相关的应用(例如，石棉、柴油废气、香烟烟雾、臭氧等)。或与哮喘等人类肺部疾病相关的基础研究应用。正常的人小气道上皮细胞(SAEC)将被培养在微孔膜插入物上，以产生类似于体内的人肺泡上皮模型。在第一阶段，通过光镜、透射电子显微镜、跨皮细胞电阻、钙调蛋白-1(AT1标记物)、P-糖蛋白(AT1标记物)、表面活性蛋白-C(AT2标记物)和多肽转运体PEPT2(AT2标记物)表达、凝集素结合(AT1和AT2特异性)、不同分子量亲水性药物的通透性、多肽和P-糖蛋白底物的表达等方法来表征模型的细胞结构和功能。将在第二阶段研究中根据ICCVAM指南对模型进行长期的批次到批次的重复性和多实验室验证。

DESCRIPTION (provided by applicant): The distal airways possess a variety of features, which make them an attractive site for systemic delivery of therapeutics. These include a very thin epithelium, large surface area, avoidance of hepatic first pass metabolism and extensive vascularization. Pulmonary delivery is therefore being increasingly considered as an alternative for drug candidates, which are either unstable or poorly absorbed in the gastrointestinal tract (e.g. protein or peptides). It is estimated that hundreds of bioengineered proteins and peptides are currently either already on the market or are undergoing clinical investigation. Furthermore, even small molecule drugs can benefit from the nearly instantaneous delivery potentially achievable through the alveolar epithelium. Thus, a robust, commercially available in vitro model of alveolar transport and toxicity could significantly contribute to the development of new therapeutic products. However, such a model does not currently exist. The goal of the present grant proposal is to fulfill this unmet need by developing and producing a commercial in vitro model of the human alveolar epithelium. This model will be a useful screening/research tool prior to more expensive, time-consuming in vivo animal studies, reducing the number and scope of animals studies conducted. In addition to transport and toxicology applications during preclinical drug development, the model will find utility for applications related to environmental toxicology (e.g. asbestos, diesel exhaust, cigarette smoke, ozone, etc.) or basic research applications related to human lung diseases such as asthma. Normal human small airway epithelial cells (SAEC) will be cultured on microporous membrane inserts to produce an in vivo-like model of the human alveolar epithelium. During Phase I, the model will be characterized in terms of appropriate cell structure and function by methods including light microscopy, transmission electron microscopy, transepithelial electrical resistance, calveolin-1 (AT1 marker), P-glycoprotein (AT 1 marker), surfactant protein-C (AT2 marker) and peptide transporter PEPT2 (AT2 marker) expression, lectin binding (AT1 and AT2 specific), permeability to model hydrophilic drugs of various molecular weights, peptides and P-glycoprotein substrates. Longer-term lot-to-lot reproducibility and multi-laboratory validation of the model according to ICCVAM guidelines will be conducted in Phase II studies.