Validation of an In Vitro Human Airway Model

描述（由申请人提供）：一种市售的体外人体传导气道模型（EpiAirway）已被开发并用于各种基础研究和工业药物输送应用。然而，该模型尚未正式验证毒理学应用于药物开发和风险评估。本拨款提案的目标是根据替代方法验证机构间协调委员会（iccam）和欧洲替代方法验证中心（ECVAM）指南，验证EpiAirway体外人气道模型。实验将确定20种已知的在体内传导气道毒性电位的化学物质对EpiAirway模型的体外反应。测试化学品将从职业安全与健康管理局（OSHA）根据人体和动物吸入数据制定的允许暴露限值（PELs）中选择。EpiAirway模型的体外终点将是通过MTT和ToxiLight细胞毒性试验测量的细胞毒性，以及酶联免疫吸附试验测量的炎症细胞因子/趋化因子（白细胞介素-8、白细胞介素6和粒细胞巨噬细胞刺激因子）的产生。整套实验将分别重复两次。然后对数据进行统计分析，以确定实验室内的可重复性和实验室间的可转移性。体外细胞毒性和细胞因子/趋化因子生产数据将与OSHA为测试化学品建立的体内（PELs）相关。最后，将对这些数据进行统计评估，以建立一个预测模型，将体外细胞毒性结果转化为体内PELs的预测。这些第一阶段的目标构成了预验证过程，用于建立进入完全正式验证的可行性。进入第二阶段的里程碑是可接受的可重复性、实验方案的实验室间可转移性和可接受的预测模型的发展。在第二阶段，将按照ICCVAM指南进行正式的验证研究。将扩大试验化合物和制剂的数量和类型，并在4个独立实验室进行盲法试验。研究结果将提交独立评估和监管机构接受。

DESCRIPTION (provided by applicant): A commercially available in vitro model of human conducting airways (EpiAirway) has been developed and utilized for a variety of basic research and industry drug delivery applications. However, the model has not been formally validated for toxicology applications in drug development and risk assessment. The goal of the present grant proposal is to validate the EpiAirway in vitro human airway model for these purposes following Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and European Center for the Validation of Alternative Methods (ECVAM) guidelines. Experiments will be performed to determine in vitro responses of the EpiAirway model after treatment with 20 chemicals of known in vivo conducting airway toxic potentials. Test chemicals will be chosen from those with permissible exposure limits (PELs) established by Occupational Safety and Health Administration (OSHA) based upon in vivo human and animal inhalation data. In vitro endpoints in the EpiAirway model will be cytotoxicity as measured by the MTT and ToxiLight cytotoxicity assays, and inflammatory cytokine/chemokine (interleukin-8, interleukin 6 and granulocyte macrophage stimulating factor) production as measured by enzyme linked immunosorbant assays. The entire set of experiments will be repeated two separate times. The data will then be statistically analyzed to determine intralaboratory reproducibility and interlaboratory transferability. The in vitro cytotoxicity and cytokine/chemokine production data will be correlated with in vivo (PELs) established for the test chemicals by OSHA. Finally, the data will be statistically evaluated to produce a prediction model for converting in vitro cytotoxicity results into a prediction of in vivo PELs. These Phase I goals constitute the prevalidation process for establishing the feasibility of proceeding to full formal validation. Milestones for advancement to Phase II are acceptable reproducibility, interlaboratory transferability of protocols and development of an acceptable prediction model. In Phase II, formal validation studies will be conducted following ICCVAM guidelines. The number and types of test compounds and formulations will be expanded and tested in blinded trials in 4 independent laboratories. The study results will then be submitted for independent assessment and regulatory acceptance.