Abstract
Purpose: :
To determine the effect of CLT-005, a novel small molecule inhibitor of Stat3, on reduction of retinal vascular leakage following intravitreal administration.
Methods: :
Human vascular endothelial cells (HUVEC) were stimulated with Leptin to induce Stat3 activation and treated with 2, 4, or 8 µM of CLT-005. Immunofluorescence was performed with antibodies specific to Stat3 and phosphorylated Stat3 in order to gauge nuclear translocation of Stat3. The effect of CLT-005 on reduction of retinal vascular permeability and pro-angiogenic factors in a streptozotocin-diabetic (STZ) rats was also examined. CLT-005 was administered at doses of 1, 5, 10, or 25 µg to STZ-rats by means of an intravitreal injection. For all experiments, the contralateral eye served as a control and received an equal volume of the vehicle. At 7 days post-injection, retinal vascular permeability was measured by the Evans blue extravasation assay. At 14 days post-injection, immunoblot analyses were performed to determine the expression levels of vascular endothelial growth factor (VEGF), monocyte chemoattractant protein 1 (MCP-1), and other pro-angiogenic and pro-inflammatory proteins.
Results: :
At all doses tested, CLT-005 demonstrated robust inhibition of Stat3 nuclear translocation in HUVEC cells. Intravitreal administration of CLT-005 was well tolerated and all doses tested conferred a large decrease in retinal vascular permeability. At 14-days post-injection, the expression level of VEGF was nearly undetectable in eyes receiving CLT-005, compared to fellow eyes where significant levels of expression were observed. Similarly, the expression of MCP-1 was greatly reduced in eyes receiving intravitreal administration of CLT-005.
Conclusions: :
These studies demonstrate that CLT-005 is potent inhibitor of Stat3 and inhibition of this pathway in the diabetic eye causes a significant reduction in retinal vascular leakage. In addition, the in-vivo administration of CLT-005 was sufficient to reduce the expression of pro-angiogenic and pro-inflammatory genes that are regulated by Stat3. These results demonstrate the preclinical therapeutic efficacy of CLT-005 as an effective treatment for Diabetic Macular Edema, and possibly other ocular indications where neovascularization and inflammation are pathogenic features.
Keywords: diabetes • transcription factors • retina