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

Therapeutic potential of VEGF and PlGF inhibition in scar formation after glaucoma filtration surgery

Glaucoma is a neurodegenerative disease and the second most important cause of irreversible blindness. This disease is characterized by a raised intraocular pressure (IOP) and by progressive retinal ganglion cell degeneration, resulting in irreversible visual field loss. Filtration surgery (trabeculectomy) remains the most effective therapy to reduce IOP in glaucoma patients. However, in 30% of the cases this surgery fails due to subconjunctival wound healing. Pharmacological enhancement of trabeculectomy using different anti-scarring agents was found to significantly improve rates of surgical success. However, the nonspecific mechanism of action of these agents may result in severe side-effects. Therefore, there is still a need for alternative strategies to prevent filtration failure. Vascular endothelial growth factor (VEGF) plays an important role in both physiological and pathological angiogenesis. VEGF-R2 mediates most biologically relevant VEGF responses, including vascular permeability, cell migration and proliferation. Alternative splicing of a single VEGF-gene results in multiple isoforms, such as VEGF121, VEGF165 and VEGF189. Different anti-VEGF treatments are used in the ophthalmological practice, some of which have more selective properties. Pegaptanib (Macugen , Pfizer) is an aptamer that selectively prevents the binding of VEGF165 to its receptor, whereas bevacizumab (AvastinTM; Genentech) is a humanized, monoclonal full-length antibody against all isoforms. Placental growth factor (PlGF) is a VEGF-homologue which only binds to VEGF-R1. Therefore, this growth factor only acts on pathological angiogenesis and inflammation and is not involved in physiological angiogenic processes. A monoclonal anti-PlGF antibody against mouse PlGF-2 was developed by ThromboGenics NV. In this thesis work, we explored the hypothesis that VEGF and PlGF are playing an important role in scar formation after glaucoma filtration surgery and that they may be targets for improvement of filtration surgery outcome. The exact role of the different VEGF isoforms in the process of wound healing was also studied. The effect of non-selective and selective VEGF inhibitors and inhibition of PlGF on ocular wound healing was investigated in vivo using different animal models of glaucoma filtration surgery. In the first part of this thesis, we demonstrated that VEGF and PlGF are important players in scar formation in vitro. Aqeuous humor of glaucoma patients was collected and aqueous levels of VEGF and PlGF were significantly upregulated compared to control (cataract). Tenon fibroblasts (TF) and endothelial cells, both regarded as key players in wound healing after glaucoma surgery, were cultured and the expression of VEGF, PlGF and their receptors was determined. Quantitative real-time RT-PCR showed that both growth factors and their receptors (VEGF-R1 and VEGF-R2) were indeed expressed on these cells. Moreover, VEGF and PlGF were able to significantly stimulate proliferation of Tenon fibroblasts.  In a second chapter, we showed that the different VEGF isoforms were differentially involved in ocular wound healing. We demonstrated that VEGF121 and VEGF165 significantly stimulated proliferation of endothelial cells, by activating the ERK pathway. PD98059, an inhibitor of the ERK pathway, significantly inhibited VEGF121 and VEGF165-induced endothelial cell proliferation. We also reported the effect of the different isoforms on TF growth. We showed that TF proliferation was mainly induced by VEGF121 and VEGF189, associated with an activation of the ERK pathway. These proliferative effects were significantly reduced by the ERK pathway inhibitor PD98059. Thus, our results indicated that VEGF isoforms play a differential role not only in ocular angiogenesis but also in wound healing. VEGF165 was found to be the most important isoform in inducing endothelial cell proliferation, while VEGF189 was the most prominent in inducing TF proliferation. VEGF121 clearly affected both cell types. In a third section, the therapeutic potential of selective versus non-selective VEGF inhibition (pegaptanib versus bevacizumab) was investigated in vitro and in vivo by analyzing the area and survival of the filtration bleb area and process of wound healing of rabbit eyes following surgery. Proliferation assays on endothelial cells and TF suggested that non-selective VEGF inhibition might be anti-angiogenic and anti-fibrotic, while selective VEGF165 blockage might mainly inhibit angiogenesis. Indeed, these results were confirmed in our rabbit model for glaucoma surgery. A single administration of bevacizumab at the time of trabeculectomy could improve the surgical outcome by reducing postoperative angiogenesis during the initial phase, and collagen deposition at later stages. Pegaptanib injection(s) improved surgery outcome less efficiently by reducing angiogenesis only. Thus, our in vitro and in vivo data indicated that selective VEGF165 inhibition by pegaptanib was less effective than non-selective VEGF inhibition by bevacizumab in reducing scarring after glaucoma surgery, presumably due to retained action of VEGF121 and VEGF189 isoforms, which had a more pronounced effect on the Tenon fibroblasts as compared to VEGF165. Finally, in the last part, the therapeutic potential of PlGF inhibition was investigated. We showed that anti-VEGF therapy prevented post-surgical scarring by inhibiting angiogenesis and collagen deposition, but did not influence inflammation. Therefore, in collaboration with ThromboGenics NV, the known anti-angiogenic and anti-inflammatory, and possibly anti-fibrotic properties of anti-PlGF-antibody were studied in vitro and in vivo in a mouse model of glaucoma surgery. Administration of anti-PlGF antibody could reduce proliferation of TF in vitro and could improve the surgical outcome in vivo. Postoperative inflammation and angiogenesis was reduced during the initial phase and collagen deposition at later time points. Direct comparison showed that inhibition of PlGF possibly seemed to be even more effectively than inhibition of VEGF-R2, which only had an effect on angiogenesis and fibrosis. In conclusion, we showed that VEGF and PLGF are playing an important role in the process of wound healing after glaucoma surgery. Our study also sheds new light on the differential function of the VEGF isoforms in the mechanisms of angiogenesis and wound healing. VEGF165 and VEGF121 predominantly affect blood vessel growth, whereas VEGF189 seems to be more important in fibrosis. These insights may have important therapeutic implications for glaucoma patients. VEGF treatment - particularly non-selective - could improve the surgical outcome in glaucoma patients by reducing angiogenesis and fibrosis. On the other hand, inhibition of PlGF seems effective in improving surgical outcome by reducing inflammation, angiogenesis and collagen deposition. The insights generated within this thesis may open new perspectives not only for glaucoma surgery, but also for modulation of postoperative scarring in general.status: publishe

The effect of bevacizumab on human Tenon fibroblasts in ocular wound healing

No abstract available.status: publishe

What is the Potential of Rho Kinase Inhibitors in the Treatment of Glaucoma

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Complementary effects of bevacizumab and MMC in the improvement of surgical outcome after glaucoma filtration surgery

PURPOSE: To determine the optimum administration route of bevacizumab after glaucoma filtering surgery (GFS) and to investigate whether a reduced dose of mitomycin-C (MMC) in combination with bevacizumab could improve surgical outcome with a reduced incidence of side-effects. METHODS: Plasma levels of bevacizumab were determined via ELISA after intracameral (IC), subconjunctival (SC) and intravitreal (IV) injections in mice, subjected to a mouse model of GFS. Application of MMC was compared to bevacizumab (SC, 25 μg) and to the combined use of both adjuvants. Surgical sponges soaked in MMC 0.02% or 0.01% were exposed to the sclera for 1 or 2 min. Treatment outcome was studied by bleb investigation. RESULTS: The three administration routes of bevacizumab equally improved surgical outcome. The VEGF antibody was detected at relatively high levels in plasma shortly after IV injection, whereas it was minimally absorbed after IC and SC injections. Both bevacizumab (SC) and MMC 0.02% (2 min) similarly increased bleb area. As compared to MMC, the combined injection with bevacizumab induced an additional effect on surgical outcome. Exposure of MMC 0.02% for 1 or 2 min together with bevacizumab equally improved surgical outcome, but 2 min application induced corneal toxicity. The combined use of bevacizumab and 1-min MMC 0.01% also improved surgical outcome compared to monotherapy, although to a lesser extent than the combination with 1-min MMC 0.02%. CONCLUSIONS: Adjunctive bevacizumab not only enhances the beneficial effect of MMC on surgical outcome, but also allows reducing the administration time of MMC 0.02%, thereby eliminating its toxic effects on the cornea.status: publishe

Modulation of wound healing after glaucoma surgery

Abstract not provided.status: publishe

Improving patient outcomes following glaucoma surgery: state of the art and future perspectives

Of all the treatments currently used to lower intraocular pressure in glaucoma patients, filtration surgery is known to be the most effective. However, in a significant percentage of cases, the constructed channel closes due to excessive scar formation, resulting in surgical failure. The process of postoperative wound healing is characterized by the coagulative and inflammatory phase, followed by the proliferative and repair phase, and finally the remodeling phase. Perioperative antimitotic agents, such as mitomycin C and 5-fluorouracil, are known to modulate the process of wound healing and to improve surgical outcome, but they carry a risk of vision-threatening complications. New alternative strategies to prevent filtration failure, such as inhibition of transforming growth factor-β, vascular endothelial growth factor, and placental growth factor, have shown promising results in the improvement of surgical success. However, it remains necessary to broaden the therapeutic approach by focusing on combined therapies and on extended drug delivery.status: publishe

The Combination of PlGF Inhibition and MMC as a Novel Anti-Scarring Strategy for Glaucoma Filtration Surgery

The complementary effects of mitomycin-C (MMC) and anti-placental growth factor (PlGF) therapy were explored and compared to the combined administration of MMC and aflibercept. Additionally, the effect of PlGF (inhibition) on IOP was investigated, since aqueous PlGF is known to be upregulated in glaucoma patients.status: publishe

ROCK inhibition improves glaucoma surgery outcome in a rabbit model

status: publishe

Rho kinase inhibitor AMA0526 improves surgical outcome in a rabbit model of glaucoma filtration surgery

First, to elucidate the effect of Rho kinase inhibitor, AMA0526, on Human Tenon Fibroblast (HTF) proliferation and transdifferentiation to myofibroblasts. Second, the effects of ROCK inhibition on the wound healing process and surgical outcome were investigated in a rabbit model of glaucoma filtration surgery.status: publishe