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

共聚焦显微镜在培养猪角膜上的临床前测试的新用途

• 批准号: 8252737
• 负责人: George L. DeGeorge
• 金额: $ 10.86万
• 依托单位: MB RESEARCH LABORATORIES, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-16 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8252737
• 关键词: Benzalkonium Chloride; Biological Assay; Cell Count; Cell Death; Cells; Cessation of life; Clinical; Confocal Microscopy; Cornea; Corneal Opacity; Cosmetics; Data; Data Analyses; Development; Distress; Dose; Drug Formulations; Exhibits; Expenditure; Eye; Family suidae; Grant; Human; Image; Image Analysis; In Vitro; Individual; Industry; Laboratories; Life; Materials Testing; Measurable; Measurement; Measures; Meat; Metabolic; Methods; Optics; Oryctolagus cuniculus; Pain; Pharmacologic Substance; Phase; Protocols documentation; Public Health; Relative (related person); Reporting; Research; Safety; Schedule; Screening procedure; Self Care; Series; Slice; Sodium Dodecyl Sulfate; Solubility; Staging; Staining method; Stains; Sting Injury; Sunscreening Agents; Testing; Time; TimeLine; Tissue Viability; Tissues; Writing; base; cost; cytotoxicity; in vitro Assay; irritation; novel; pre-clinical; product development; prospective; research clinical testing; titanium dioxide; wasting

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.

描述（由申请人提供）：非常需要能够在化妆品、个人护理或药物制剂开发阶段早期检测和区分相对眼睛不适或“刺痛”可能性的筛选方法。目前唯一可靠的方法是昂贵且有时痛苦的人体临床眼部测试，其中将测试物质引入人眼，测试对象报告感觉到不适或刺痛。虽然这可能是眼科产品的关键最终临床测试，但处于开发和配制阶段的产品也必须在相同的人体临床测试中进行筛选，因为没有其他可用的筛选选项。可用的眼部刺激测试可以表明产品或配方“无刺激性”，这是一个好的开始。然而，目前的眼部刺激测试并不能很好地预测眼睛刺痛。 Laden (1973) 报道说，低刺激性的制剂通常仍然会引起刺痛。因此，制剂的刺痛潜力通常与刺激性无关。 Van Abbe (1973) 还报告说，眼部刺激性不能很好地预测人类报告的眼睛刺痛、疼痛和不适。我们假设可测量的刺激和刺痛潜力之间的这种脱节是由于当前刺激测试缺乏敏感性。目前的测试使用非敏感终点，例如缺乏角膜混浊（即兔子测试和 BCOP）和大量组织范围的细胞死亡（即 EpiOulous”）。EpiOulous”（MatTek Corp，Ashland，MA）是行业标准眼部刺激测试具有相对较高的灵敏度。 EpiOcular“组织暴露于测试物质，然后通过代谢指示剂的还原来测量组织活力。从整个组织中提取并随后测量比色指示剂是整体组织刺激/细胞毒性效应的终点。一旦 EpiOcular 组织达到 100与阴性对照组织（无可测量的组织死亡）相比，该组织不再指示可能是眼部刺痛指标的潜在亚刺激细胞死亡。 MB Research 开发的共聚焦检测 PorFocal 可能具有更高的灵敏度，可以通过共聚焦显微镜测量单位体积的单个细胞死亡来预测人眼刺痛。PorFocal 使用来自肉类工业的废弃猪角膜来分析单个角膜细胞。由于共聚焦显微镜终点，测试物质被直接置于培养的活角膜组织上，因此具有高灵敏度的死亡。因此测试物质的溶解度是无关紧要的。 PorFocal 培养角膜可维持长达 7 天的活状态，并每天服用测试物质。这种多次暴露的给药方案可以量化极轻微的眼细胞死亡，并随着时间的推移产生附加效应。然后通过共聚焦显微镜对角膜组织内单个染色的死细胞进行定量来测量这些附加效应。角膜组织以“光学组织学”方式成像，其中在角膜组织中不断增加的深度处采集一系列图像“切片”。然后可以对图像进行数字重建，以显示成像的整个角膜组织体积（参见研究策略）。因此，可以量化极少量的角膜损伤，因为终点是每组织体积的实际个体死细胞数。在目标 1 中，将在 EpiOulous 和 PorFocal 中对七种测试物质进行测试，以确定 PorFocal 测定是否比最灵敏的体外眼刺激测定（行业标准）EpiOulous 更敏感。这将检验以下假设：由于缺乏敏感性，目前的体外测定中未检测到刺痛。如果 PorFocal 表现出比行业标准 EpiOcular 更高的灵敏度，那么它可能具有预测刺痛的巨大潜力。在目标 2 中，将在 EpiOcular 和 PorFocal 中测试已知的非刺痛或刺痛的市售产品。 EpiOcular”将用于确定行业标准中的“毒刺”是否被视为“无刺激物”。如果 PorFocal 能够将毒刺与非刺激物区分开来，这可能允许在最终人类临床眼睛刺痛之前前瞻性地剔除刺痛产品配方根据 PorFocal 的进一步表征，该测试可以显着减少人类测试对象的疼痛和痛苦，以及产品开发和配制过程中的成本/时间支出。 公共健康相关性：该项目将测试使用培养猪角膜测定作为高度敏感的临床前筛选测试，以预测人类眼睛刺痛的可能性。在产品配方和开发阶段需要进行人眼刺痛筛选测试，从而可以在最终的人体临床眼刺痛测试之前进一步剔除刺痛产品配方。这将降低总体产品开发成本和人体测试对象的痛苦和困扰，同时促进公共卫生的强大产品安全。