Abstract
Ocular neovascular diseases like wet age-related macular degeneration are a major cause of blindness. Novel therapies are greatly needed for these diseases. One appealing antiangiogenic target is reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease 1 (Ref-1/APE1). This protein can act as a redox-sensitive transcriptional activator for NF-κB and other pro-angiogenic transcription factors. An existing inhibitor of Ref-1’s function, APX3330, previously showed antiangiogenic effects. Here, we developed improved APX3330 derivatives and assessed their antiangiogenic activity. We synthesized APX2009 and APX2014 and demonstrated enhanced inhibition of Ref-1 function in a DNA-binding assay compared to APX3330. Both compounds were antiproliferative against human retinal microvascular endothelial cells (HRECs; GI50 APX2009: 1.1 µM, APX2014: 110 nM) and macaque choroidal endothelial cells (Rf/6a; GI50 APX2009: 26 µM, APX2014: 5.0 µM). Both compounds significantly reduced the ability of HRECs and Rf/6a cells to form tubes at mid nanomolar concentrations compared to control, and both significantly inhibited HREC and Rf/6a cell migration in a scratch wound assay. Ex vivo, both APX2009 and APX2014 inhibited choroidal sprouting at low micromolar and high nanomolar concentrations respectively. In the laser-induced choroidal neovascularization mouse model, intraperitoneal APX2009 treatment significantly decreased lesion volume by 4-fold compared to vehicle (p < 0.0001, ANOVA with Dunnett’s post hoc tests), without obvious intraocular or systemic toxicity. Thus, Ref-1 inhibition with APX2009 and APX2014 blocks ocular angiogenesis in vitro and ex vivo, and APX2009 is an effective systemic therapy for CNV in vivo, establishing Ref-1 inhibition as a promising therapeutic approach for ocular neovascularization.
- AMD
- age-related macular degeneration
- ANOVA
- analysis of variance
- AP-1
- activator protein 1
- APE1
- apurinic/apyrimidinic endonuclease 1
- APX2009
- (E)-N,N-diethyl-2-((3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylene)pentanamide
- APX2014
- (E)-N-methoxy-2-((3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylene)pentanamide
- APX3330
- (2E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)methylene]-undecanoic acid
- ATCC
- American Type Culture Collection
- BSA
- bovine serum albumin
- CNV
- choroidal neovascularization
- DMEM
- Dulbecco’s modified Eagle’s medium
- DMSO
- dimethyl sulfoxide
- EBM-2
- endothelial basal medium 2
- EGM-2
- endothelial growth medium 2
- EMSA
- electrophoretic mobility shift assay
- FDA
- Food and Drug Administration
- FA
- fluorescein angiography
- GCL
- ganglion cell layer
- GI50
- median growth inhibitory concentration
- GS-IB4
- isolectin B4 from Griffonia simplicifolia
- HIF-1α
- hypoxia inducible factor 1α
- HRECs
- human retinal microvascular endothelial cells
- INL
- inner nuclear layer
- L-CNV
- laser-induced choroidal neovascularization
- NF-κB
- nuclear factor κ light-chain-enhancer of activated B cells
- OCT
- optical coherence tomography
- ONL
- outer nuclear layer
- PBS
- phosphate buffered saline
- PDR
- proliferative diabetic retinopathy
- PKT
- propylene glycol, Kolliphor HS15, Tween 80
- Ref-1
- reduction-oxidation factor 1
- ROP
- retinopathy of prematurity
- RP2D
- recommended Phase II dose
- RPE
- retinal pigment epithelium
- STAT3
- signal transducer and activator of transcription 3
- VEGF
- vascular endothelial growth factor
- VLDLR
- very low density lipoprotein receptor