Purpose. To test the efficacy of a synthetic antisense oligonucleotide inhibitor of an intracellular signal transduction protein, C-raf-1 kinase, as an inhibitor of ocular neovascularization.Methods. A 2'methoxyethyl, 2'deoxy chimeric 20-nucleotide sequence containing a uniform phosphorothioate backbone was synthesized which targets the 3'untranslated region of porcine C-raf-1 mRNA (ISIS 107189). Efficacy of mRNA inhibition was tested in vitro in porcine vascular endothelial cells and treated pigs by Northern blotting. In a pig model of intraocular neovascularization induced by branch retinal vein occlusion, intravitreal injection of ISIS 107189 compound (8 muM calculated intraocular concentration) at baseline and on days 14, 42, and 70, was tested against vehicle as control for inhibition of neovascularization. After enucleation on day 84, ocular tissues were analyzed for ISIS 107189 content by solid-phase extraction and capillary gel electrophoresis. Cryostat sections were immunostained for C-raf-1 kinase protein.Results. The antisense oligonucleotide demonstrated high potency for inhibition of C-raf-1 kinase in the porcine cells lines. Levels of C-raf-1 kinase were also decreased in the retina of pigs following a single 180 mug dose. Pig eyes injected with the multiple doses of 180 mug oligonucleotide demonstrated a marked decrease in neovascularization due to branch retinal vein occlusion 12 weeks after treatment (p = 0.05, Mann-Whitney U-test). Posterior subcapsular cataracts were noted in the treated eyes. Concentrations of oligonucleotide in retina ranged from 2-12 muM in the treated eyes. Qualitative assessment of the expression of C-raf-1 kinase via immunohistostaining of frozen sections demonstrated inhibition of expression in the treated eyes compared to controls.Conclusions. ISIS 107189 successfully inhibited neovascularization in this model, which was correlated with an inhibition of expression of C-raf-1 kinase. While not proven in these studies, these results suggest that C-raf kinase may be important in angiogenesis. Antisense therapy has potential applicability in the therapy of ocular neovascular diseases.

Aim: To validate the first non-invasive measurements of volumetric ophthalmic artery blood flow in humans.Methods: The ophthalmic arteries of healthy normal adults were examined by Advanced Technology Laboratories (ATL, a subsidiary of Phillips Medical Systems Inc) high definition imaging (HDI) 5000 colour Doppler imaging ultrasound with a 5-12 MHZ probe. A group of 14 subjects for experiment 1 and a group of 10 subjects for experiments 2 and 3 were selected, with the examined eye chosen randomly. Peak systolic velocities (PSV) and end diastolic velocities (EDV) of the ophthalmic artery and central retinal artery were measured and recorded. Cineloops (cinegraphic videos) of the ophthalmic arteries were then recorded with the ATL HDI 5000 and values for ophthalmic artery blood flow were produced offline using experimental analysis software. Multiple regression analysis was used to compare blood flow measurements with PSV and EDV measurements in the ophthalmic artery. In two follow up experiments, intraobserver variation in obtaining cineloops and the interanalyser variability in cineloop analysis were studied.Results: Volumetric flow correlated with ophthalmic artery PSV and EDV (p = 0.02, r(2) = 0.5). There was no correlation with the cental retinal artery. The intraobserver coefficient of variation in obtaining cineloops was 29.89% for blood flow, 19.07% for diameter, and 22.27% for velocity. The coefficients of variation of the measurements of the two cineloop analysers were 40.21% for blood flow, 22.71% for diameter, and 26.34% for velocity.Conclusion: Cineloop analysis produces ophthalmic artery flow measurements which correlate with PSV and EDV, suggesting validity. The intraobserver variation and cineloop analyser variation were found to be in the acceptable range.