Multifunctional small molecule TLR4 antagonist for treating ocular neovascularization

Abstract

Purpose: The multifactorial pathological challenge of ocular neovascularization is difficult to address so far only by anti-VEGF therapy. We have tested our hypothesis that, a novel class of natural product derived compound with toll like receptor 4 (TLR4) antagonist activity can ameliorate the hyper-inflammation produced by macrophage/macroglia over activation as well as decrease choroidal neovascularization (CNV) size in mice. Methods: Inhibition of cytokines: Mouse bone marrow derived macrophages were treated with high mobility group box1 (HMGB1, 100 ng/mL), an endogenous TLR4 ligand for 8 hours, with or without 100 mg/mL of test compounds. The mRNA levels of TNF-a, iNOS were measured by real-time RT-PCR, and normalized to the control cells. Inhibition of VEGF production: ARPE-19 cells were treated either with 100ng/mL or without HMGB1 along with the compounds (50μg/mL) for 24 h. Supernatants were collected and assayed using human VEGF ELISA kit according to manufacturer’s instructions. Experiments were repeated three times and one way ANOVA was used for statistical analysis. Inhibition of CNV: Laser CNV was induced in C57BL/6 mice (male, 10-12 weeks, n = 5). Each eye received 4 laser burns. The compounds (200 μg/mL) or BSS (vehicle) were administered by IP injection once before and once daily up to 10 days following laser injury. Fundus fluorescein angiography and optical coherence tomography was used to visualize the CNV lesions. RPE/choroid/sclera flat mounts were prepared and stained with both FITC conjugated isolectin B4 and anti-ICAM-2 antibody to quantitatively measure the size of CNV lesion. Results: Compound treatment significantly (p Conclusions: Our results are consistent with our hypothesis that this novel class of compounds will decrease ocular inflammation and neovascularization. Further structure optimization of the lead compound and TLR4 dependent and independent mechanistic investigation are underway.