Vascular Normalization by ROCK Inhibitor: Therapeutic Potential of Ripasudil (K-115) Eye Drop in Retinal Angiogenesis and Hypoxia

Abstract

PURPOSE. In this study, we investigated the therapeutic potential of a Rho-associated coiledcoil-containing protein kinase (ROCK) inhibitor ripasudil (K-115) eye drop on retinal neovascularization and hypoxia.METHODS. In vitro, human retinal microvascular endothelial cells (HRMECs) were pretreated with ripasudil and then stimulated with VEGF. ROCK activity was evaluated by phosphorylation of myosin phosphatase target protein (MYPT)-1. Endothelial migration and cell viability were assessed by cell migration and MTT assay, respectively. The concentration of ripasudil in the retina was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In vivo, normal saline, 0.4%, or 0.8% ripasudil were administered three times a day to mice with oxygen-induced retinopathy (OIR). The areas of neovascularization and avascular retina were also quantified with retinal flat-mounts at postnatal day (P) 15, P17, or P21. The retinal hypoxic area was evaluated using hypoxia-sensitive drug pimonidazole by immunohistochemistry at P17. The vascular normalization was also evaluated by immunohistochemistry at P17. RESULTS.Ripasudil but not fasudil significantly reduced VEGF-induced MYPT-1 phosphorylation in HRMECs at 30 lmol/L. Ripasudil significantly inhibited VEGF-induced HRMECs migration and proliferation. The concentration of ripasudil in the retina was 3.8 to 10.4 lmol/ L and 6.8 to 14.8 lmol/L after 0.4% and 0.8% ripasudil treatment, respectively. In the 0.4% and 0.8% ripasudil treated OIR mice, the areas of neovascularization as well as avascular area in the retina was significantly reduced compared with those of saline-treated mice at P17 and P21. Pimonidazole staining revealed that treatment with 0.4% and 0.8% ripasudil significantly inhibited the increase in the hypoxic area compared with saline. 0.8% ripasudil could cause intraretinal vascular sprouting and increase retinal vascular perfusion.CONCLUSIONS. Novel ROCK inhibitor ripasudil eye drop has therapeutic potential in the treatment of retinal hypoxic neovascular diseases via antiangiogenic effects as well as vascular normalization.

ROCK inhibitors in ocular disease

“…Moreover, the benefit of ROCK inhibition may be increased by the fact that the inhibitors often target pathological change specifically and have no effect on normal processes. For instance, ROCK inhibition has no effects on control cornea [37], normal blood flow [84] and normal angiogenesis [10]. Constitutive ROCK activity may be low in many ocular tissues.…”

“…For instance, topically instilled 0.4 % and 0.8 % ripasudil were shown to reach the posterior segment in sufficient concentration to affect neovascularization, whereas 0.4 % fasudil did not. Preparation of 0.8 % fasudil eye drops was not feasible because of poor solubility at this concentration [10]. Further chemical modification of drugs such as liposomal encapsulation [101,102], or other types of conjugations [103], may be able to enhance in vivo efficacy in the future.…”

“…In contrast, 0.4 % fasudil eye drops did not show any effect. Additional experiments which tested normal vascularization found that ROCK inhibitors did not affect physiological angiogenesis, although they did modify pathological vascular growth in a retinopathy model [10]. …”

“…In 2008 was the first report, from in vivo mouse cornea, that fasudil had inhibitory potential in ocular angiogenesis [38]. Additional evidence from human foreskin [78] and retinal microvascular endothelial cell cultures [10] has demonstrated that RhoA or Rho kinases are involved in VEGF-induced cytoskeletal changes and cell migration. Increased RhoA activity was accompanied by elevated phosphorylation and activity of the VEGF receptor-2, which might be the connection between RhoA pathways and VEGF effects [78].…”

“…Both fasudil and Y27632 were effective at reducing IOP in animal models [6], however the stability of the solutions was poor [9,10]. The Y27632-derivative Y39983 was stabile in solution and shown to have 30 times more ROCK inhibition and to be 10 fold more effective at lowering IOP than Y27632 in animal models [9].…”