Enhanced Phototoxicity Assay in Reconstituted Skin

描述（由申请方提供）：我们设想了一种高通量体外光毒性筛选试验，命名为“重建皮肤中的增强光毒性试验（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。光毒性的动物模型是昂贵的、缓慢的、主观的，并且不适于高通量。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认可的替代品或体外光毒性试验。3 T3小鼠成纤维细胞中性红光毒性试验（3 T3 NRU）在经过欧洲验证机构ECVAM的预验证后，目前正在被ICCVAM考虑作为替代光毒性方法。然而，这个测试有几个缺点。EPARS试验克服了3 T3 NRU试验的许多局限性，因为：1）EPARS采用紧密平行于人皮肤形态的多层组织，而不是成纤维细胞单层; 2）与3 T3 NRU试验相反，可以测试非水溶性制剂，在3 T3 NRU试验中，测试物质需要通过培养基给药; 3）基于人原代角质形成细胞的组织是比小鼠肿瘤细胞系更相关的模型。在EPARS中，将供试品局部应用于重建的人类皮肤模型，有和没有紫外线照射。使用MU活力测定法测定组织活力，并比较辐照和未辐照组织活力以测定光毒性效应。此外，可以测量与光毒性相关的其他分子和机制终点，例如PGE 2释放、MHC表达、细胞增殖和炎性细胞因子产生（IL-1 a、IL 1-ra、TNF-α和IFN-γ），以增加测试的灵敏度和特异性。 拟定商业应用：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服务。 对我们客户的调查表明，对快速和具有成本效益的光毒性测试的需求日益增加，并且需要更多基于机械的测试，以提供定性和定量信息。我们认为流式细胞术和DNA微阵列在毒性方面没有得到充分利用，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试验。

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.