Novel Use of Confocal Microscopy on Cultured Porcine Corneas for Pre-Clinical Tes

描述(由申请人提供)：重要的是需要一种筛查方法，能够检测和区分化妆品、个人护理或药物配方的早期发展阶段的相对眼睛不适或“刺痛”潜力。目前可用的唯一可靠方法是昂贵的、有时甚至是痛苦的人类临床眼睛测试，在这种测试中，测试物质被引入人的眼睛，测试对象报告感觉到的不适或刺痛。虽然这可能是眼科产品的关键最终临床测试，但处于开发和生产阶段的产品也必须在同一人体临床测试中进行筛查，因为没有其他可用的筛查选择。现有的眼部刺激测试可以表明产品或配方是“无刺激性的”，这是一个很好的开始。然而，目前的眼睛刺激测试并不能很好地预测眼睛刺痛。拉登(1973)曾报道过，刺激性低的制剂经常仍会引起刺痛。因此，一种配方的潜在刺痛性通常与刺激性无关。Van Abbe(1973)也报道了眼睛刺激对人类眼睛刺痛、疼痛和不适的不良预测作用。我们假设，这种可测量的刺激性和刺痛潜力之间的脱节是由于目前的刺激性试验缺乏敏感性。目前的测试使用不敏感的终点，例如缺乏角膜混浊(即兔子测试和BCOP)和组织范围内的大量细胞死亡(即EpiOcul.EpiOcul.Mattek Corp，Ashland，MA)是一种相对较高灵敏度的行业标准眼刺激测试。将组织暴露在测试物质中，然后通过减少代谢指示剂来测量组织的活力。从整个组织中提取并随后测量比色指示剂是整个组织刺激/细胞毒性效应的终点。一旦上眼球组织与阴性对照组织相比达到100%的活性(不可测量的组织死亡)，这些组织就不能再指示潜在的亚刺激细胞死亡，这可能是眼部刺痛的指标。MB Research开发的猪角膜共聚焦分析(PorFocal)可能具有放大的灵敏度，通过共聚焦显微镜测量每个组织体积的单个细胞死亡，可能预测人类眼睛的刺痛。PorFocal使用来自肉类行业的废弃猪角膜来分析由于共焦显微镜终点而导致的高灵敏度的个体角膜细胞死亡。在PorFocal中，测试物质直接放置在培养中的活体角膜组织上，因此测试物质的溶解度无关紧要。PorFocal培养的角膜在活体状态下保持长达7天，并每天与测试物质一起服用。这种多次曝光剂量计划允许量化极轻微的眼细胞死亡，并随着时间的推移产生相加效应。然后通过共聚焦显微镜对角膜组织中单个染色的死亡细胞进行量化来衡量这些相加效应。角膜组织以“光学组织学”的方式成像，其中一系列图像“切片”在进入角膜组织的深度不断增加时被获取。然后，这些图像可以被数字重建，以显示所成像的整个角膜组织体积(参见研究策略)。因此，极低的角膜损伤量是可以量化的，因为终点是每组织体积的实际个体死亡细胞数。在目标1中，7种测试物质将在EpiOcull和PorFocal中进行测试，以确定PorFocal分析是否比EpiOcula更敏感，后者是最敏感的体外眼刺激分析(行业标准)。这将检验一种假设，即由于缺乏敏感性，在当前的体外检测中没有检测到刺痛。如果PorFocal显示出比行业标准EpiOcle更高的敏感度，那么它可能有很大的潜力预测叮咬。在目标2中，市场上已知的无刺激性或刺激性的产品将在EpiOcul.和PorFocal中进行测试。EpiOtual“将被用于确定”刺激性“是否被行业标准视为”无刺激性“。如果PorFocal可以解决非刺激性刺激物中的刺痛，这可能允许在最终的人类临床眼睛刺痛测试之前对刺痛产品配方进行前瞻性筛选。根据PorFocal的进一步表征，这项测试可以显著减少人体试验对象的痛苦和痛苦，以及产品开发和配方期间的成本/时间支出。 公共卫生相关性：该项目将测试使用培养的猪角膜作为一种高度敏感的临床前筛查试验，以预测人类刺眼的潜力。在产品配方和开发阶段，需要进行人眼刺痛筛查试验，以便在最终的人体临床刺眼试验之前进一步筛选刺痛产品配方。这将降低总体产品开发成本和人体试验对象的痛苦和痛苦，同时促进公众健康的强大产品安全。

DESCRIPTION (provided by applicant): There is an important need for screening methods that can detect and distinguish the relative eye discomfort or "sting" potential of cosmetic, personal care, or pharmaceutical formulations early in their developmental stages. The only currently reliable method available is costly and sometimes painful human clinical eye testing, in which test substances are introduced into human eyes and test subjects report perceived discomfort or stinging. While this may be a critical final clinical test for an ocular product, products in development and formulation stages must also be screened in the same human clinical test, as there are no other available screening options. Available ocular irritation testing can indicate that a product or formulation is "non-irritating", which is a good start. However, current ocular irritation tests are not very predictive of eye sting. Laden (1973) has reported that formulations which had low irritancy often still caused stinging. Hence, the stinging potential of a formulation was often unrelated to irritancy. Van Abbe (1973) has also reported ocular irritancy to be a poor predictor of human reported eye stinging, pain, and discomfort. We hypothesize that this disconnect between measurable irritation and sting potential is due to lack of sensitivity in current irritation tests. Current tests use non-sensitive endpoints such as lack of corneal opacity (i.e. rabbit tests and BCOP) and substantial tissue-wide cell death (i.e. EpiOcular"). EpiOcular" (MatTek Corp, Ashland, MA) is an industry standard ocular irritation test with relatively high sensitivity. EpiOcular " tissues are exposed to test substances and then tissue viability is measured by reduction of a metabolic indicator. Extraction and subsequent measurement of the colorimetric indicator from the entire tissue is the endpoint for overall tissue irritation/cytotoxicity effects. Once the EpiOcular tissues reach 100% viability compared to the negative control tissues (no measurable tissue death), the tissues can no longer be indicative of potential sub-irritation cell death that may be an ocular sting indicator. The Porcine Cornea Confocal Assay, PorFocal, developed by MB Research, likely has the amplified sensitivity to potentially predict human eye sting due to measurement of individual cell death per tissue volume by confocal microscopy. The PorFocal uses waste porcine corneas from the meat industry to assay individual corneal cell death with high sensitivity due to a confocal microscopy endpoint. In PorFocal, test substances are placed directly onto living corneal tissue in culture; therefore solubility of the test substance is irrelevant. PorFocal cultured corneas are maintained in a living state for up to 7 days and are dosed daily with the test substance. This multiple-exposure dosing schedule allows for quantification of extremely mild ocular cell death with additive effects over time. These additive effects are then measured by quantification of individual stained dead cells within the corneal tissue by confocal microscopy. Corneal tissue is imaged in an "optical histological" manner where a series of image "slices" are acquired at increasing depths into the corneal tissue. The images can then be digitally reconstructed to exhibit the entire corneal tissue volume imaged (see Research Strategy). Therefore, extremely low amounts of corneal damage can be quantified because the endpoint is actual individual dead cell number per tissue volume. In Aim 1 seven test substances will be tested in both the EpiOcular " and the PorFocal to determine if the PorFocal assay is more sensitive than the EpiOcular ", the most sensitive in vitro ocular irritation assay (industry standard). This will test the hypothesis that sting is not detected in current in vitro assays due to lack of sensitivity. If PorFocal demonstrates higher sensitivity than the industry standard EpiOcular ", then it may have great potential to predict sting. In Aim 2, commercially available products that are known non-stingers or stingers will be tested in both EpiOcular " and PorFocal. The EpiOcular" will be used to establish whether "stingers" would be considered "non-irritants" by industry standards. If PorFocal can resolve stingers from non-irritants, this may allow for prospective culling of stinging product formulations before final human clinical eye sting testing. Upon further characterization of PorFocal, this test could significantly reduce human test subject pain and distress, and cost/time expenditure during product development and formulation. PUBLIC HEALTH RELEVANCE: This project will test the use of a cultured porcine cornea assay as a highly sensitive pre-clinical screen test to predict human eye-sting potential. A human eye sting screening test is needed for testing during product formulation and development phases, thus allowing for further culling of stinging product formulations before final human clinical eye sting testing. This would reduce overall product development cost and human test subject pain and distress while promoting robust product safety for public health.