Enhanced Phototoxicity Assay in Reconstituted Skin

描述（由申请人提供）：我们已经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试验（EPARS）”。化学品、化妆品、膳食补充剂和药品的潜在光毒性是消费品行业日益关注的主要问题。光毒性动物模型是昂贵的，缓慢的，主观的，并且不能修改高通量。缺乏快速、经济、可靠的光毒性筛选试验抑制了许多新产品的开发和商业化。目前在美国，没有经过验证的监管机构接受的替代品或体外光毒性测试。3T3小鼠成纤维细胞中性红色光毒性试验（3T3 NRU）在欧洲验证机构ECVAM预验证后，目前正在icvam考虑作为一种替代的光毒性方法。然而，这种测试有几个缺点。EPARS测试克服了3T3 NRU测试的许多局限性，其中：1)EPARS采用与人体皮肤形态非常相似的多层组织，而不是成纤维细胞单层；2)可以测试非水溶性配方，与3T3 NRU试验相反，在3T3 NRU试验中，测试物质需要通过培养基给药；3)与小鼠肿瘤细胞系相比，人原代角化细胞为基础的组织是一个更相关的模型。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活力，并比较辐照和未辐照的组织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，如PGE2释放、MHC表达、细胞增殖和炎症细胞因子产生（IL-1a、lL1-ra、TNF-a和IFN-g），以提高检测的敏感性和特异性。

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.