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）：有一个重要的筛选方法，可以检测和区分化妆品，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”的潜力。目前唯一可靠的方法是昂贵的，有时是痛苦的人类临床眼科测试，其中将测试物质引入人的眼睛，测试对象报告感到不适或刺痛。虽然这可能是眼科产品的关键最终临床试验，但由于没有其他可用的筛选选择，处于开发和配方阶段的产品也必须在相同的人体临床试验中进行筛选。现有的眼部刺激测试可以表明产品或配方是“无刺激性的”，这是一个良好的开端。然而，目前的眼部刺激试验并不能很好地预测眼睛刺痛。拉登（1973）报告说，低刺激性的配方仍然经常引起刺痛。因此，一种配方的刺痛电位通常与刺激性无关。Van Abbe（1973）也报道了眼刺激是人类报告的眼睛刺痛、疼痛和不适的一个很差的预测指标。我们假设这种可测量刺激和刺痛电位之间的脱节是由于当前刺激试验缺乏敏感性。目前的测试使用非敏感的终点，如角膜不透明（如兔试验和BCOP）和组织范围内的大量细胞死亡（如EpiOcular）。EpiOcular （MatTek Corp, Ashland， MA）是一种具有较高灵敏度的行业标准眼部刺激测试。眼表组织暴露于测试物质中，然后通过代谢指标的降低来测量组织活力。从整个组织中提取和随后测量比色指示剂是整个组织刺激/细胞毒性作用的终点。与阴性对照组织（无可测量的组织死亡）相比，一旦眼表组织达到100%的活力，这些组织就不能再表明潜在的亚刺激细胞死亡，而亚刺激细胞死亡可能是眼部刺痛的一个指标。猪角膜共聚焦测定法（PorFocal）是由MB Research开发的，由于共聚焦显微镜可以测量每组织体积的单个细胞死亡，因此可能具有更高的灵敏度，可以潜在地预测人眼蜇伤。PorFocal使用来自肉类行业的废弃猪角膜来检测个体角膜细胞死亡，由于共聚焦显微镜的终点，它具有高灵敏度。在PorFocal中，测试物质直接放置在培养的活体角膜组织上；因此，测试物质的溶解度是无关的。将原位培养的角膜维持在活体状态长达7天，并每天服用试验物质。这种多重暴露剂量表允许对极轻微的眼细胞死亡进行量化，并随时间推移产生累加效应。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量化来测量这些加性效应。以“光学组织学”方式对角膜组织成像，其中在角膜组织中增加深度处获得一系列图像“切片”。然后，这些图像可以进行数字重建，以显示成像的整个角膜组织体积（见研究策略）。因此，极少量的角膜损伤可以量化，因为终点是每组织体积的实际个体死亡细胞数。在Aim 1中，七种测试物质将在EpiOcular和PorFocal中进行测试，以确定PorFocal法是否比EpiOcular法更敏感，EpiOcular法是最敏感的体外眼刺激试验（行业标准）。这将验证由于缺乏敏感性而在目前的体外测定中未检测到刺痛的假设。如果PorFocal的灵敏度高于行业标准EpiOcular，那么它可能具有很大的预测刺痛的潜力。在目标2中，将在EpiOcular和PorFocal中测试已知的无刺针或刺针的市售产品。EpiOcular将用于确定“毒刺”是否被工业标准视为“非刺激物”。如果PorFocal可以从非刺激物中解决刺痛，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。在对PorFocal进行进一步表征后，该测试可以显著减少人类测试对象的痛苦和痛苦，以及产品开发和配方过程中的成本/时间支出。