Department of Life Sciences, Glasgow Caledonian University, Glasgow, United Kingdom.
Topivert Pharma Limited, London, United Kingdom.
Invest Ophthalmol Vis Sci. 2018 Mar 1;59(3):1443-1453. doi: 10.1167/iovs.17-23479.
The purpose of this study is to determine the potential of narrow spectrum kinase inhibitors (NSKIs) to treat inflammatory eye disorders.
Human conjunctival epithelial (HCE) cells were retrieved from subjects via impression cytology. Real-time quantitative PCR (qPCR) was performed on HCE cells to determine gene expression of NSKI kinase targets and proinflammatory cytokines in dry eye disease (DED) patients versus healthy controls. qPCR also assessed p38α expression in hyperosmolar-treated Chang conjunctival epithelial cells. Interaction of NSKI TOP1362 with the kinases was evaluated in ATP-dependent Z-LYTE and competition binding assays. Anti-inflammatory activity was assessed in human peripheral blood mononuclear cells and primary macrophages. In an endotoxin-induced uveitis (EIU) study, lipopolysaccharide (LPS) was administered intravitreally to Lewis rats. TOP1362, dexamethasone, or vehicle was administered topically, and inflammatory cytokine levels were measured 6 hours after LPS injection.
HCE cells from DED patients showed significantly increased expression of p38α, spleen tyrosine kinase (Syk), Src, lymphocyte-specific protein tyrosine kinase (Lck), interleukin one beta (IL-1β), interleukin eight (IL-8), monocyte chemotactic protein-1 (MCP-1), and matrix metalloproteinase-9 (MMP-9). TOP1362 strongly inhibited the kinase targets p38α, Syk, Src, and Lck, blocked the rise in p38α expression in hyperosmolar Chang cells, and potently reduced inflammatory cytokine release in cellular models of innate and adaptive immunities. In the EIU model, TOP1362 dose-dependently attenuated the LPS-induced rise in inflammatory cell infiltration and ocular cytokine levels with efficacy comparable to that of dexamethasone.
TOP1362 is a potent inhibitor of kinases upregulated in DED and markedly attenuates proinflammatory cytokine release in vitro and in vivo, highlighting the therapeutic potential of NSKIs for treating ocular inflammation, such as that observed in DED.
本研究旨在确定窄谱激酶抑制剂（NSKIs）治疗炎症性眼病的潜力。
通过印迹细胞学从受试者中获取人结膜上皮（HCE）细胞。对 HCE 细胞进行实时定量 PCR（qPCR），以确定干眼症（DED）患者与健康对照相比，NSKI 激酶靶标和促炎细胞因子的基因表达。qPCR 还评估了高渗处理的 Chang 结膜上皮细胞中 p38α 的表达。在 ATP 依赖性 Z-LYTE 和竞争结合测定中评估 NSKI TOP1362 与激酶的相互作用。在人外周血单核细胞和原代巨噬细胞中评估抗炎活性。在脂多糖（LPS）诱导的葡萄膜炎（EIU）研究中，将 LPS 眼内注射到 Lewis 大鼠中。局部给予 TOP1362、地塞米松或载体，在 LPS 注射后 6 小时测量炎症细胞因子水平。
DED 患者的 HCE 细胞显示 p38α、脾酪氨酸激酶（Syk）、Src、淋巴细胞特异性蛋白酪氨酸激酶（Lck）、白细胞介素 1β（IL-1β）、白细胞介素 8（IL-8）、单核细胞趋化蛋白 1（MCP-1）和基质金属蛋白酶-9（MMP-9）的表达明显增加。TOP1362 强烈抑制激酶靶标 p38α、Syk、Src 和 Lck，阻断高渗 Chang 细胞中 p38α 表达的升高，并在固有和适应性免疫细胞模型中强力抑制炎症细胞因子的释放。在 EIU 模型中，TOP1362 剂量依赖性地减弱了 LPS 诱导的炎症细胞浸润和眼内细胞因子水平的升高，其疗效与地塞米松相当。
TOP1362 是 DED 中上调的激酶的有效抑制剂，可显著减少体外和体内促炎细胞因子的释放，突出了 NSKI 治疗眼部炎症（如 DED 中观察到的炎症）的治疗潜力。
