AMA0076, a Novel, Locally Acting Rho Kinase Inhibitor, Potently Lowers Intraocular Pressure in New Zealand White Rabbits with Minimal Hyperemia

PURPOSE. To determine whether ROCK inhibition for the treatment of glaucoma can be improved by using novel, locally acting Rho kinase (ROCK) inhibitors, such as AMA0076, that lower IOP without inducing hyperemia. METHODS. On-target potency of AMA0076 was compared with other ROCK inhibitors (Y-27632 and Y-39983) and conversion of AMA0076 into its functionally inactive metabolite was evaluated in rabbit eye tissues. Human trabecular meshwork (HTM) cell morphology, actin filaments, and focal adhesion were studied in vitro after exposure to AMA0076. The effect of AMA0076 on IOP was investigated in normotensive rabbits and a new, acute hypertensive rabbit model. Intraocular pressure lowering efficacy of AMA0076 was compared with pharmacologic treatments. Hyperemia after single topical dosing of AMA0076 and Y-39983 was scored. RESULTS. AMA0076 and Y-39983 showed similar on-target potency. AMA0076 was most stable in aqueous humor and converted into its metabolite in other eye tissues. Exposure of HTM cells to AMA0076 led to significant and reversible changes in cell shape and a decrease in actin stress fibers and focal adhesions. Both AMA0076 and Y-39983 provided an equivalent IOP control. Compared with latanoprost and bimatoprost, AMA0076 was more potent in preventing the IOP elevation in the acute hypertensive rabbit model. The degree of hyperemia was significantly lower in rabbits treated with AMA0076 then with Y-39983. CONCLUSIONS. AMA0076 is a locally acting ROCK inhibitor that is able to induce altered cellular behavior of HTM cells. Administration of AMA0076 effectively reduces IOP in ocular normotensive and acute hypertensive rabbits without causing distinct hyperemia

Rho kinase inhibition, a versatile therapy for modulating wound healing in corneal pathologies

Nowadays, over 10 million patients worldwide suffer from corneal blindness. Ocular trauma or surgery is often followed by the dynamic process of excessive wound healing, which results in corneal edema, neovascularization (NV) and fibrosis and eventually leads to vision loss. Current pharmacological therapy options include steroids (e.g. dexamethasone), anti-VEGF agents (e.g. bevacizumab) and cytostatics (e.g. mitomycin-C). However, these pharmacological treatment options remain limited or insufficient and therefore corneal transplantation (keratoplasty) is frequently required. New therapeutic approaches may allow modulation of wound healing, resulting in safer and more efficient outcomes. Several molecular biology studies have suggested an association of corneal scarring and neovascularization with cytoskeletal regulators. Because of the complexity of the corneal wound healing process, it is important to identify possible alternative treatments, which reach the efficacy level that is necessary for corneal function recovery. Finding drugs, with pleiotropic working mechanisms, (which affect both inflammatory, angiogenic and fibrotic component of wound healing after trauma) would be a major benefit, since this might offer improved therapeutic opportunities. Rho proteins are a subfamily within the Ras superfamily of small monomeric GTPase binding proteins, which exert pleiotropic cellular functions. Three main GTPases can be identified: RhoA, RhoB and RhoC, which share 85% amino acid sequence identity. The best-characterized downstream effectors of RhoA are the rho-associated kinase proteins (ROCK1/2 isoforms), which are serine/threonine protein kinases. Rho/ROCK signaling responds to cell surface receptors for various cytokines, adhesion molecules and multiple growth factors. Through substrate phosphorylation, the serine/threonine protein kinase, ROCK has pleiotropic functions and controls a diverse array of cellular processes including stress fibre assembly and focal adhesions formation, cell polarity, cell adhesion, motility, cytokinesis and apoptosis. In recent studies, inhibition of ROCK strongly disrupts VEGF-mediated angiogenesis and has shown to induce an additional anti-inflammatory and anti-fibrotic reaction. In this thesis work, we explored the hypothesis that ROCK plays an important role in corneal scar formation and neovascularization and that it may be a target for controlling and improving corneal healing after trauma. Rho-kinase (ROCK) inhibitors, as possible candidate drugs, distinguish themselves from other agents by their efficacy and pleiotropic working mechanisms. Therefore, in collaboration with Amakem Therapeutics, the effect of a selective ROCK inhibitor, AMA0526 on corneal wound healing was investigated in vitro with multiple cell culture assays and in vivo using different animal models for corneal trauma. Due to its local mode of action, AMA0526 might have an additional therapeutic benefit compared to current therapy (such as anti-VEGF therapy and steroids), minimizing systemic side effects (safe profile). The first specific object of this research was to explore the potential anti-angiogenic effects of ROCK-inhibition in vitro on vascular endothelial cells (HUVEC & HBMEC) and in vivo in the micropocket NV murine model. We demonstrated that ROCK inhibition significantly reduced proliferation and migration of vascular endothelial cells in a dose-dependent manner (using a WST-1 assay, Boyden chamber and scratch migration assays). Importantly, a caspase 3/7 apoptosis assay showed no toxic effects of the compound. Formation of new blood vessels was induced in vivo by incorporating fibroblast growth factor (bFGF) in the central stroma. Topical treatment (TID) of both AMA0526 and bevacizumab (anti-VEGF therapy as a positive control) showed a large decrease in corneal neovascularization compared to vehicle treated eyes. Additionally, AMA0526 also diminished the inflammatory reaction whereas bevacizumab had no anti-inflammatory effects. Thus, these first experiments suggested a strong anti-angiogenic with also an added anti-inflammatory effect of the ROCK inhibitor. In the next section within this PhD project, in vitro and in vivo experiments revealed a strong anti-fibrotic effect of AMA0526. In vitro data were generated by using primary (rabbit) stromal fibroblasts (RCF, regarded as key players during corneal scarring) to assess the effects of ROCK inhibition. Multiple fibroblast functions (apoptosis, proliferation, migration and morphology changes) were significantly diminished after AMA0526 exposure. Immunostaining for actin and vinculin revealed a decrease in actin bundles and focal adhesions and thus altered cell morphology when fibroblasts were treated with the ROCK inhibitor. Also, fibroblast-to-myofibroblast transdifferentiation of the stromal cells (stimulated by TGFβ1) was reversed after ROCK inhibitor exposure. A second murine model was employed to elucidate the effects of ROCK inhibition on the general processes of wound healing in vivo. The therapeutic potential of the selective ROCK inhibitor, AMA0526 was investigated by analyzing the corneal opacity, neovascularization (score), NV area and the processes of wound healing (inflammation, angiogenesis en collagen III deposition, histologically) of mouse eyes following an alkali induced corneal burn injury. ROCK activity measurements revealed an upregulation of ROCK activity in corneal tissue after chemical burn, suggesting a significant role of the Rho-associated kinase protein during wound healing. Indeed, the positive in vitro results were confirmed in this mouse model for acute chemical trauma. Topical treatment (once daily) of AMA0526 could strongly reduce corneal opacity and NV and improve the outcome by reducing post-traumatic inflammation, neovascularization and collagen III deposition in comparison to vehicle. Comparison to dexamethasone revealed AMA0526 to be as effective as topical steroids, the current golden standard therapy in reducing inflammation and angiogenesis and more efficient in diminishing fibrosis. Controlling wound healing is an important step during corneal healing, however even if excessive scarring is avoided, it is crucial to preserve the functionality of the specialised corneal cell layers, more specifically the corneal endothelial monolayer. The endothelium is of utmost importance for the function of the cornea by maintaining a tight barrier and its pump capacities in order to keep the excessive fluid (aqueous humor) out of the cornea and thus ensuring its transparency. In the last part of this thesis, the therapeutic potential of ROCK inhibition to recover the corneal endothelium was explored in a rabbit model using a transcorneal freeze (TCF) injury. Preliminary results showed an accelerated reduction of corneal thickness (at early time points) and higher corneal transparency after topical AMA0526 treatment. Histologically, endothelial cell density showed to be higher at early time points compared to vehicle, suggesting a possible beneficial effect of the ROCK inhibitor on endothelial recovery. In conclusion, we showed that ROCK signaling plays an important role in the process of wound healing after corneal trauma. Our study sheds new light on the differential effect of ROCK inhibition in various cell types important for the mechanisms of angiogenesis and corneal wound healing. These insights may have significant therapeutic implications for patients with corneal trauma, infection, refractive surgery and/or other keratopathies and whose vision is at risk. The insights generated within this thesis may open new perspectives not only for corneal wound healing but also for modulation of postoperative and posttraumatic scarring in general.status: publishe

ROCK inhibition improves glaucoma surgery outcome in a rabbit model

status: publishe

Bevacizumab revisited: its use in different mouse models of ocular pathologies

Previous reports have yielded conflicting data on the activity of bevacizumab (Avastin), a recombinant humanized monoclonal antibody against VEGF-A, in the mouse. The current study was designed to further explore the use of this VEGF inhibitor in various murine models of ocular diseases and compare it to the widely used murine anti-VEGF-R2 neutralizing antibody (DC101).peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=icey20status: publishe

Inhibition of Rho-Associated Kinase Prevents Pathological Wound Healing and Neovascularization After Corneal Trauma

To investigate the effect of AMA0526, a specific inhibitor of rho-associated protein kinase (ROCK), on corneal neovascularization (NV) and scarring in different in vitro and in vivo experimental models.status: publishe

AMA0076, a novel, locally acting rho kinase inhibitor, potently lowers intraocular pressure in New Zealand white rabbits with minimal hyperemia

PURPOSE: To determine whether ROCK inhibition for the treatment of glaucoma can be improved by using novel, locally acting Rho kinase (ROCK) inhibitors, such as AMA0076, that lower IOP without inducing hyperemia. METHODS: On-target potency of AMA0076 was compared with other ROCK inhibitors (Y-27632 and Y-39983) and conversion of AMA0076 into its functionally inactive metabolite was evaluated in rabbit eye tissues. Human trabecular meshwork (HTM) cell morphology, actin filaments, and focal adhesion were studied in vitro after exposure to AMA0076. The effect of AMA0076 on IOP was investigated in normotensive rabbits and a new, acute hypertensive rabbit model. Intraocular pressure lowering efficacy of AMA0076 was compared with pharmacologic treatments. Hyperemia after single topical dosing of AMA0076 and Y-39983 was scored. RESULTS: AMA0076 and Y-39983 showed similar on-target potency. AMA0076 was most stable in aqueous humor and converted into its metabolite in other eye tissues. Exposure of HTM cells to AMA0076 led to significant and reversible changes in cell shape and a decrease in actin stress fibers and focal adhesions. Both AMA0076 and Y-39983 provided an equivalent IOP control. Compared with latanoprost and bimatoprost, AMA0076 was more potent in preventing the IOP elevation in the acute hypertensive rabbit model. The degree of hyperemia was significantly lower in rabbits treated with AMA0076 then with Y-39983. CONCLUSIONS: AMA0076 is a locally acting ROCK inhibitor that is able to induce altered cellular behavior of HTM cells. Administration of AMA0076 effectively reduces IOP in ocular normotensive and acute hypertensive rabbits without causing distinct hyperemia.status: publishe

Inhibition of placental growth factor improves surgical outcome of glaucoma surgery

status: publishe