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）：筛查方法的重要需求可以检测和区分化妆品，个人护理或药品制剂的相对不适或“刺痛”潜力。当前唯一可用的可用方法是昂贵的，有时是痛苦的人类临床眼测试，其中将测试物质引入人眼中，测试受试者报告说感到不适或刺痛感。尽管这可能是对眼部产品的关键最终临床测试，但由于没有其他可用的筛查选项，还必须在同一人类的临床测试中筛选开发和配方阶段的产品。可用的眼部刺激测试可以表明产品或配方是“不侵蚀”的，这是一个很好的开始。但是，当前的眼部刺激测试不是很好地预测眼刺。 Laden（1973）报告说，刺激性低的配方通常仍会引起刺痛。因此，公式的刺痛潜力通常与刺激无关。范·阿贝（Van Abbe，1973）还报道了眼部刺激是人类报道的眼睛刺痛，疼痛和不适的不良预测指标。我们假设可测量的刺激和刺伤潜力之间的这种断开是由于当前刺激测试缺乏灵敏度。当前的测试使用非敏感终点，例如缺乏角膜不透明（即兔子测试和BCOP）和大量组织范围内的细胞死亡（即表皮”）。上皮“（马特克公司，阿什兰，马萨诸塞州）是一种行业标准的ORITARITION ICORITTION测试，具有相对较高的敏感性。外周的组织暴露于测试物质中，然后通过减少代谢指标来测量组织生存力。从整个组织中提取和随后测量比色指示剂是整体组织刺激/细胞毒性效应的整体组织刺激性/细胞毒性效应的终点。一旦在不可能的情况下与100％的无限制组织相比，无效的均可受到量度的影响。亚灌溉细胞死亡可能是眼部刺痛指标。端点。因此，测试物质的溶解度无关紧要。孢子型培养的角膜保持在生命状态长达7天，并每天用测试物质剂量。这种多种暴露给药时间表可以随着时间的推移定量具有添加剂效应的极度轻度细胞死亡。然后，通过共聚焦显微镜定量角膜组织中的单个染色死细胞来测量这些添加剂。角膜组织以“光学组织学”方式成像，其中一系列图像“切片”被增加到角膜组织的深度增加。然后可以将图像进行数字重建以展示成像的整个角膜组织体积（请参阅研究策略）。因此，可以量化极低的角膜损伤，因为终点是每个组织体积的实际单个死细胞数。在AIM 1中，将在上周围的“和孔雀斑”中测试七个测试物质，以确定高焦点测定是否比上皮更敏感”，这是最敏感的体外眼部刺激测定法（行业标准）。这将检验以下假设：由于缺乏敏感性，在当前的体外测定中未检测到刺激。如果孔雀斑显示出比行业标准表周围更高的灵敏度”，那么它可能具有预测刺激的巨大潜力。在AIM 2中，将在上周围的商业上测试已知的非刺或刺痛者的市售产品”和Porfocal。上皮“将用于确定“刺痛者”是否按行业标准视为“非辐射药”。如果孔雀焦点可以解决非灌溉剂的刺痛，这可能允许前瞻性地剔除刺ing子制剂，然后在最终的人类临床眼睛刺痛测试之前进行最终的临床眼睛刺激测试，以实现人类的进一步表征。在测试中进行验证和成本较大和成本，并成本和成本。在验证和成本中，并降低了成本和成本和成本。 公共卫生相关性：该项目将测试使用培养的猪角膜测定作为高度敏感的临床前筛查测试，以预测人类的眼睛刺激潜力。在产品配方和开发阶段进行测试需要进行人眼刺激测试，从而在最终的人类临床眼刺测试之前进一步挖掘刺激产品制剂。这将降低整体产品开发成本和人类测试主题痛苦和困扰，同时促进公共卫生的强大产品安全。