Development of SRPK1 inhibitors as novel anti-angiogenic therapeutics for exudative age-related macular degeneration (AMD)

Abstract

Purpose: Anti-angiogenic VEGF inhibitors are currently the standard of care for exudative AMD but must be administered monthly by intraocular injection and non-specifically inhibit all VEGF isoforms. Pathological angiogenesis occurs when the balance between pro-angiogenic VEGF-A₁₆₅a and anti-angiogenic VEGF-A₁₆₅b is switched by SRPK1-mediated differential splicing of VEGF mRNA to the proangiogenic form. We previously developed a small molecule inhibitor of SRPK1, termed SPHINX, that could inhibit SRPK1 at µM levels and were effective as eye drops in laser induced choroidal neovascularisation{Gammons, 2013 #4989}. We hypothesised that highly potent prelinical lead candidate SRPK1 inhibitors could be generated that can be given as topical eye drops and specifically inhibit pro-angiogenic VEGF-driven angiogenesis underlying wet AMD

Methods: Novel compounds were synthesized based on the structure of SPHINX. These were tested in in vitro kinase assays, thermal shift binding assays and in immunoprecipitation, immunoblotting and immunofluorescent cell based assays. Toxicity was evaluated by testing in vitro hERG activity, off-target gene splicing by RT-PCR and retinal toxicity by Ganzfeld ERG. Efficacy was tested using in vivo laser-induced CNV assay. Pharmacokinetics and bioavailability were evaluated in vivo and in an ex vivo permeability assay using mass spectrometry

Results: Novel compounds selectively bind to SRPK1 and dose-dependently inhibit SRPK1 kinase activity with IC50s <10 nM, SRSF1 phosphorylation and nuclear localization and VEGF-A₁₆₅a protein levels. We identified compounds that do not inhibit hERG activity, splicing of off target genes or retinal function in vivo, yet potently inhibit laser-induced CNV following topical eye drop administration with EC50s of <500nM (n=6-8, P<0.05, One-way ANOVA). Compounds could be detected in the sclera/choroid and retina following eye drop administration in vivo and in ex vivo permeability assays

Conclusions: We have developed novel SRPK1 inhibitors that specifically target proangiogenic VEGF and can be delivered to the retina following topical eye drop administration to inhibit choroidal neovascularization. These compounds potentially offer for more specific, efficacious and safer therapeutics for patients with exudative AMD