Enhanced Phototoxicity Assay in Reconstituted Skin

重建皮肤中增强的光毒性测定

• 批准号: 6550218
• 负责人: George L. DeGeorge
• 金额: $ 18.87万
• 依托单位: MB RESEARCH LABORATORIES, INC.
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6550218
• 关键词: artificial skin; biotechnology; cytotoxicity; flow cytometry; high throughput technology; microarray technology; skin irritation /irritant; technology /technique development; ultraviolet radiation

DESCRIPTION (provided by applicant): We have conceived a high-throughput in vitro screening test for Phototoxicity designated the "Enhanced Phototoxicity Assay in Reconstituted Skin (EPARS). The phototoxic potential of chemicals, cosmetics, dietary supplements and pharmaceuticals are a growing major concern in the consumer products industry. Animal models of phototoxicity are expensive, slow, subjective, and not amendable to high throughput. The lack of rapid, economical, reliable phototoxicity screening tests inhibits the development and commercialization of many new products. Currently in the US, there are no validated regulatory agency-accepted alternatives or in vitro phototoxicity tests. The 3T3 Mouse Fibroblast Neutral Red Phototoxicity Test (3T3 NRU) is now under consideration by ICCVAM as an alternative phototoxicity method after having been pre-validated by Europe's validation agency, ECVAM. This test, however, has several weaknesses. The EPARS test overcomes many of the limitations of the 3T3 NRU test in that: 1) EPARS employs multi-layer tissues that closely parallel human skin morphology, instead of a fibroblast monolayer; 2) non-aqueous soluble formulations can be tested, in contrast to the 3T3 NRU Assay, in which test substances need to dosed via the culture media; 3) the human primary keratinocyte-based tissues are a more relevant model than a mouse tumor cell line. In the EPARS, the test substance is applied topically to the reconstructed human skin models, with and without UV irradiation. The viability of the tissues is the determined using the MU viability assay, and the irradiated and non-irradiated tissue viability is compared to determine phototoxic effects. In addition, other molecular and mechanistic endpoints relevant to phototoxicity such as PGE2 release, MHC expression, cellular proliferation and inflammatory cytokine production (IL-1a, lL1-ra, TNF-a and IFN-g) can be measured to increase the sensitivity and specificity of the test. PROPOSED COMMERCIAL APPLICATION: MB Research will offer the service of an alternative phototoxicity assay to members of the pharmaceutical, biotech, cosmetic and chemical industry. A survey of our clients indicates that there is an increasing demand for rapid and cost-effective phototoxicity tests, and a need for more mechanistic-based tests which provide both qualitative and quantitative information, In addition, we believe that flow cytometry and DNA microarrays are underutililized in the toxicity-screening field and that this technology can be used to develop other commercially viable in vivo and in vitro toxicity tests at our company.

描述（由申请人提供）：我们构思了一种高通量体外光毒性筛选试验，称为“重建皮肤增强光毒性测定（EPARS）”。化学品、化妆品、膳食补充剂和药品的光毒性潜力日益受到消费品行业的关注。光毒性动物模型昂贵、缓慢、主观，且不适合高通量。 缺乏快速、经济、可靠的光毒性筛选测试阻碍了许多新产品的开发和商业化。目前在美国，还没有经过验证的监管机构认可的替代品或体外光毒性测试。 ICCVAM 目前正在考虑将 3T3 小鼠成纤维细胞中性红光毒性试验 (3T3 NRU) 作为一种替代光毒性方法。 由欧洲验证机构 ECVAM 预先验证。然而，该测试有几个缺点。 EPARS 测试克服了 3T3 NRU 测试的许多局限性，因为： 1) EPARS 采用与人类皮肤形态非常相似的多层组织，而不是单层成纤维细胞； 2) 与3T3相比，可以测试非水溶制剂 NRU 测定，其中测试物质需要通过培养基添加； 3) 人类原代角质形成细胞组织是比小鼠肿瘤细胞系更相关的模型。在 EPARS 中，测试物质局部应用于重建的人体皮肤模型，有或没有紫外线照射。使用 MU 活力测定法测定组织的活力，并且照射和未照射的组织 比较活力以确定光毒性效应。此外，还可以测量与光毒性相关的其他分子和机制终点，例如 PGE2 释放、MHC 表达、细胞增殖和炎症细胞因子产生（IL-1a、IL1-ra、TNF-a 和 IFN-g），以提高测试的灵敏度和特异性。 拟议的商业应用：MB Research 将为制药、生物技术、化妆品和化学行业的成员提供替代光毒性测定的服务。 对我们客户的一项调查表明，对快速且具有成本效益的光毒性测试的需求不断增加，并且需要更多基于机械的测试来提供定性和定量信息。此外，我们相信流式细胞术和DNA微阵列在毒性筛选领域中尚未得到充分利用，并且该技术可用于在我们公司开发其他商业上可行的体内和体外毒性测试。