HIGH-THROUGHPUT SCREEN OF TOPICALLY APPLIED THERAPEUTICS

在制药和个人护理行业中筛选分子文库往往会产生大量的先导化合物。这反过来又导致了在进行人体临床试验之前需要新的高通量方法来筛选这些化合物的潜在毒性。本提案的目标是开发一种皮肤模型和相关方法，通过监测与各种皮肤现象有关的基因信息，对局部应用的药物或其他材料进行高通量测试。该模型将由96孔格式的三维器官型人体皮肤培养物组成。空气/液体界面的培养和体内类似的屏障特性将使新药物和/或成品能够以一种局部的、现实的方式进行测试。RNA将从组织中提取，并通过工程捕获和检测探针以96格式进行具体量化。在第一阶段，将通过制备和测试P53(遗传毒性)、IL-1α(刺激)和β-肌动蛋白(对照)消息的探针来证明该方法的实用性。组织模型和方法应该适用于研究任何感兴趣的基因的信息调制，前提是可以设计出合适的探针。这些方法将在第二阶段适用于其他组织模型和基因信息。拟议的商业应用：拟议的高通量皮肤模型将应用于广泛的制药、化学和消费品公司的产品安全评估，这些公司从事高通量筛选以确定主要候选者，其产品可能用于局部使用。该模型还将适用于其他基础研究，涉及通过化学物质、紫外线辐射或其他外源治疗来调节皮肤中的基因信息。

Screening of molecular libraries in the pharmaceutical and personal care industries often results in the generation of large numbers of lead compounds. This, in turn, has resulted in a need for new high-throughput methods of screening these compounds for potential toxicity prior to conducting human clinical trials. The goal of the present proposal is to develop a skin model and associated methodology to allow high- throughput testing of topically applied pharmaceuticals or other materials by monitoring gene messages related to a broad variety of dermal phenomena. The model will be composed of three-dimensional organotypic human skin cultures in a 96-well format. Culturing at the air/liquid interface and in-vivo-like barrier properties will enable new pharmaceuticals and/or finished products to be tested in a topical, realistic manner. RNA will be extracted from the tissues and specifically quantified in a 96-format by means of engineered capture and detection probes. During Phase I, the utility of this methodology will be demonstrated by preparing and testing probes for p53 (genotoxicity), IL-1alpha (irritation), and beta-actin (control) message. The tissue model and methods should be adaptable to enable investigation of message modulation for any gene of interest provided appropriate probes can be engineered. The methods will be adapted to other tissue models and gene messages in Phase II. PROPOSED COMMERCIAL APPLICATIONS: The proposed high-throughput skin model will find application in product safety assessment for a broad variety of pharmaceutical, chemical, and consumer product companies who engage in high-throughput screening to identify lead candidates, and whose products may be intended for topical use. The model will also be adaptable for other basic studies involving modulation of gene messages in skin by chemicals, UV radiation, or other exogenous treatments.