The Receptor Tyrosine Kinase Inhibitor, AL–39324, Completely Inhibits Preretinal NV in the Rat OIR Model

Abstract

Abstract: : Purpose: Receptor tyrosine kinases (RTKs), such as VEGFRs, FGFRs, and PDGFRs, play significant roles in tumor–induced angiogenesis and several drug candidates are being evaluated in human oncology trials. We evaluated the dose–response effect of the small molecule, receptor tyrosine kinase inhibitor, AL–39324 (KDR IC₅₀=4nM), on VEGF–induced endothelial cell proliferation/survival and preretinal NV in the rat model of oxygen–induced retinopathy (OIR). Methods: In vitro: inhibitory activity of AL–39324 on hrVEGF₁₆₅ (25ng/ml)–induced endothelial cell proliferation/survival was examined using bovine retinal microvascular ECs. AL–39324 was tested at concentrations ranging from 0.2 to 200 nM. Proliferation/survival was quantified by a Promega MTS assay. In vivo (Alcon & Vanderbilt U.): AL–39324 was tested in 2 independent rat OIR models by either a single 5 µl intravitreal injection of 0.1, 0.3, 0.6, 1 and 3%, or oral gavage at 3, 10 and 20 mg/kg/d. Neonatal pups were exposed to a 50%/10% oxygen exposure paradigm q24hrs from Day 0–14 postpartum and then placed into room air on D14, when single Ivt injections or oral dosing commenced. All animals were euthanized at Day 20 postpartum. Retinas were harvested, stained with ADPase, and viewed as whole mounts. Computerized image analysis was used to obtain a median NV clockhour score from each retina and used in statistical analyses; P < 0.05 was considered significant. Results: 20 nM–200 nM AL–39324 completely inhibited the proliferation/survival response elicited by VEGF in the BRECs, while 2 nM AL–39324 provided partial inhibition(P<0.003). In the rat OIR models, local and systemic delivery of AL–39324 significantly inhibited pathological preretinal NV in a dose–dependent manner. Moreover, eyes injected with 1–3% AL–39324 or pups treated with 20 mg/kg/d showed complete prevention of preretinal NV (P<0.002 and P<0.001, respectively). Eyes injected with 0.1% AL–39324 or pups treated with 3mg/kg/day exhibited no significant inhibition. Conclusions: AL–39324 provided potent and robust efficacy against VEGF–induced retinal endothelial cell proliferation/survival and preretinal NV in the rat OIR model. In comparison with other antiangiogenic classes tested, AL–39324 was able to provide 100% inhibition of pathologic ocular angiogenesis in the rat OIR model, which suggests that RTKi’s may be useful for antiangiogenic therapy in human posterior segment disease.

Keywords: retinal neovascularization • receptors: pharmacology/physiology