Modeling Novel Treatments for Dry Eye

描述（由申请人提供）：我们的多学科和多机构申请从我们发现的新的人类分泌丝裂原“泪泌素”的角度解决干眼症。泪素在泪腺和睑板腺以及角膜和结膜中有限制性表达，它似乎能够保护泪腺-角膜轴上皮免受炎症相关细胞死亡。干眼症患者泪液中促炎细胞因子TNFa水平升高与眼表上皮损伤有关。的确，在培养的人角膜上皮细胞中加入TNFa通过caspase -8和-3促进死亡。最近我们观察到10 nM的lacritin可以完全阻止caspase的激活和死亡。这一观察结果通过泪泌素缺失分析得到了加强，该分析揭示了泪泌素c端内的细胞保护位点。泪泌素利用一种独特的细胞靶向机制：肝素酶解除细胞表面syndecan-1 n端外域内泪泌素结合位点的阻断。结合的泪腺素随后可能被呈递给GPCR。由于肝素酶具有较低的最佳pH值，因此有可能泪泌素对假设的突然低pH值“危险信号”具有保护作用，这种信号被认为是某些个体原发性干燥综合征干眼症发病的基础。在兔临床前研究中，局部应用催泪素可以促进至少4小时的泪液流动而没有毒性-甚至超过30天的连续治疗。在细胞培养中，催泪素刺激泪腺细胞分泌眼泪，而泪腺细胞正是分泌催泪素的细胞。它还能促进角膜上皮MUC16和泪泌素的表达。由于泪泌素是一种天然泪液蛋白，这表明上游和下游的自分泌刺激机制延长了泪泌素的细胞保护和分泌作用。泪液蛋白可能具有生物活性复合物的功能。这些活性可以通过重组蛋白工程加以利用。我们的假设是，泪腺素具有天然的预防干眼炎症的作用。我们的直接目标是优化催泪素的细胞保护活性，并了解其作用机制。我们的第一个目标是设计出最小和最具细胞保护作用的泌乳素。我们的第二个目标是找出其细胞保护活性的生物学途径，以寻找治疗协同作用或反指征。我们的第三个目标是在干眼动物模型中进行局部应用或基因诱导的泪腺素临床前测试。

DESCRIPTION (provided by applicant): Our multi-disciplinary and multi-institutional application addresses dry eye syndromes from the perspective of the new human prosecretory mitogen 'lacritin' discovered by us. Lacritin is restrictively expressed in lacrimal and meibomian glands and in the cornea and conjunctiva, where it appears to be capable of protecting epithelia of the lacrimal-corneal axis against inflammation-associated cell death. Increased levels of proinflammatory cytokine TNFa in tears of dry eye patients is associated with damage to ocular surface epithelia. Indeed, adding TNFa to cultured human corneal epithelial cells promotes death via caspases-8 and -3. Recently we observed that caspase activation and death is completely prevented by inclusion of 10 nM lacritin. This observation has been reinforced by lacritin deletion analysis that reveals a cytoprotective site within lacritin's C-terminus. Lacritin's utilizes a unique cell targeting mechanism: heparanase unblocks a lacritin binding site within the N-terminal ectodomain of cell surface syndecan-1. Bound lacritin is then likely presented to a GPCR. Since heparanase has a lower pH optimum, it is possible that lacritin is protective against the hypothetical sudden low pH 'danger signal' thought to underlie the initiation of primary Sjogren's syndrome dry eye in some individuals. In rabbit preclinical studies, topical application of lacritin promotes increased tear flow for at least 4 hr without toxicity - even over 30 days of continuous treatment. In cell culture, lacritin stimulates tear secretion by lacrimal acinar cells - the same cells from which it is secreted. It also promotes corneal epithelial MUC16 and lacritin expression. Since lacritin is a natural tear protein, this suggests mechanisms of upstream and downstream autocrine stimulation that prolong lacritin's cytoprotective and prosecretory effects. Tear proteins likely function as bioactive complexes. These activities can be harnessed by recombinant protein engineering. Our working hypothesis is that lacritin is naturally protective against dry eye inflammation. Our immediate goal is to optimize lacritin's cytoprotective activity and understand its mechanism of action. Our first aim is to engineer the smallest and most cytoprotective form of lacritin. Our second aim is to work out biological pathways that underlie its cytoprotective activity in a search for treatment synergies or counterindications. Our third aim is to preclinically test topically applied or genetically induced lacritin in animal models of dry eye.