Modeling Novel Treatments for Dry Eye

描述(由申请人提供)：我们的多学科和多机构的应用从我们发现的新的人类前分泌有丝分裂原‘lacritin’的角度来解决干眼症。Lacritin在泪腺和眉板腺以及角膜和结膜中有限制性表达，它似乎能够保护泪液-角膜轴上皮细胞免受炎症相关细胞死亡的影响。干眼症患者泪液中促炎症细胞因子TNFa水平升高与眼表上皮损伤有关。事实上，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caspase的激活和死亡完全可以通过包含10 nM的lacritin来阻止。这一观察结果得到了撕裂蛋白缺失分析的加强，该分析揭示了撕裂蛋白的C末端有一个细胞保护部位。Lacritin‘s利用一种独特的细胞靶向机制：乙酰肝素酶解锁细胞表面Syndecan-1的N-末端胞外区内的一个催乳素结合位点。然后，结合的催乳素很可能被呈递给GPCR。由于乙酰肝素酶的最适pH值较低，因此，拉西丁有可能对假想的突然低pH“危险信号”具有保护作用，这种信号被认为是某些人发生原发性干燥综合征干眼的基础。在兔的临床前研究中，局部应用lacritin可促进泪液流量增加至少4小时，而不会产生毒性--即使在连续治疗30天后也是如此。在细胞培养中，催泪素刺激泪腺泡细胞分泌泪液，而泪腺泡细胞正是分泌泪液的细胞。它还促进角膜上皮细胞MUC16和Lacritin的表达。由于Lacritin是一种天然的泪液蛋白，这暗示了上游和下游自分泌刺激的机制，延长了Lacritin的细胞保护和前分泌作用。泪液蛋白可能作为生物活性复合体发挥作用。重组蛋白工程可以利用这些活性。我们的工作假设是，催乳素对干眼炎有天然的保护作用。我们的近期目标是优化Lacritin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催乳素。我们的第二个目标是找出支持其细胞保护活性的生物学途径，以寻找治疗协同效应或反适应症。我们的第三个目标是在干眼的动物模型中进行临床前的局部应用或基因诱导的催乳素的测试。

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.