Evaluation of Collagen-Derived peptide (EB-203) for Treating Wet Age-Related Macular Degeneration

Evaluation of Collagen-Derived peptide (EB-203) for Treating Wet Age-Related Macular Degeneration | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Evaluation of Collagen-Derived peptide (EB-203) for Treating Wet Age-Related Macular Degeneration Byulnim Ahn, Kyong Ah Min, Jaewook Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7970433/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 14 You are reading this latest preprint version Abstract Wet age-related macular degeneration has been one of the challenging ocular diseases due to severe ocular implications like vision loss. As a key protein factor, VEGF has been reported to be closely related with the choroid neovascularization (CNV) as a representative pathogenesis of wet AMD. Conventionally, anti-VEGF antibody therapeutics like aflibercept have been used for improving AMD with a route of intravitreal (IVT) injection. However, there have been burdensome for patients to continue the IVT therapies because of high costs and ocular inflammation issues. Up to date, different strategies using modified antibodies, genes or small molecules have been investigated along with the underlying mechanisms of wet AMD. In the present study, a novel peptide has been investigated for its anti-angiogenic activity in AMD using EA.hy926 cells treated with cobalt chloride (CoCl₂) to simulate hypoxic conditions. In result, the peptide, EB-203, showed inhibition on Hif-1α and VEGF expression in western blot. Under the hypoxia condition, tube formation of the endothelial cells was interfered with EB-203 and further, migration and invasion of endothelial cells were inhibited by EB-203 to the levels of control. Moreover, mouse AMD models intravitreally injected by EB-203 at some drug concentrations exhibited comparable improvement to aflibercept in vascular leakage and CNV area. Mouse models administered by 5% or 10% EB-203 eyedrops in twice daily dosing showed the reduction of vascular leakage and CNV area significantly. These results demonstrated that the peptide drug can contribute to improve wet AMD complications and visual acuity as an anti-VEGF inhibitor. Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Medical research Wet age-related macular degeneration Anti-angiogenic effect Hypoxia condition EB-203 Laser induced Choroidal neovascularization (CNV) mouse model Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Globally, age-related macular degeneration (AMD) has been known to cause central vision loss in elder people, decreasing the quality of life. Wet AMD is the leading cause of severe and irreversible central vision loss in the elderly. It is characterized by the growth of abnormal, leaky blood vessels beneath the macula. These new vessels readily bleed or leak fluid into the retinal layers, resulting in retinal edema, hemorrhage, and disruption of the photoreceptor architecture, which can rapidly progress to vision loss or legal blindness. Unlike dry AMD, which progresses slowly and has limited therapeutic options, wet AMD often deteriorates very quickly but can be treated pharmacologically if diagnosed early 1 – 3 . Although vascular endothelial growth factor (VEGF)-driven angiogenesis has been central to the pathogenesis of choroidal neovascularization (CNV) in neovascular (wet) AMD 4 , accumulating evidence indicates that retinal hypoxia plays a crucial role in initiating and sustaining disease progression by upregulating pro-angiogenic and inflammatory mediators. Consistent with this, recent findings highlight that AMD progression involves hypoxia, oxidative stress, inflammation, and immune dysregulation 5 – 7 . VEGF inhibition remains the cornerstone of AMD therapy. Currently, anti-VEGF treatments including aflibercept (Eylea), ranibizumab (Lucentis), brolucizumab (Beovu), and the bispecific antibody faricimab (Vabysmo) has been regarded as efficient therapies for wet AMD to improve CNV conditions 8 , 9 . Anti-VEGF treatments using antibody biologics via intravitreal (IVT) injections into eyes have been reported to resolve CNV, reduce vascular permeability, and treat the vision acuity (VA) loss 10 . The clinical outcomes depend on frequent and consistent treatments for two years. Despite of effects of those treatments, patients feel burdensome because of high costs and ocular implication issues, including intraocular inflammation, ocular (or conjunctival) hemorrhage, or increased intraocular pressure which might be related to the invasiveness of intravitreal (IVT) injections or unknown actions of macromolecule biologics. Moreover, frequent anti‑VEGF injections have been shown to cause retinal thickness fluctuations, and such anatomical variations are closely correlated with poorer long‑term visual acuity outcomes in patients with neovascular AMD 11 , 12 . To overcome the disadvantages and side effects of current intravitreal anti‑VEGF drugs, extensive efforts have been made to develop alternative therapies for neovascular AMD, including modified antibody biologics, small molecules, and gene‑based modalities and non-invasive drug delivery methods 13 – 16 . Chondrocyte-derived extracellular matrix (CDECM) has been extensively studied for its remarkable anti-angiogenic and anti-inflammatory properties. CDECM suppresses neovascularization and promotes wound healing in corneal alkali burn and glaucoma filtration surgery models in rabbits, prevents vascular invasion during mesenchymal stem cell chondrogenesis, and protects corneal epithelial cells by inhibiting NF-κB signaling pathways 17 – 21 . CDECM mainly consists of type II collagen, proteoglycans, and glycosaminoglycans, which together provide the structural and functional properties of cartilage 22 , 23 . In our previous study, we identified the anti-inflammatory and antiangiogenic effects of a type II collagen–derived peptide isolated from CDECM in animal models of dry eye disease and diabetic retinopathy 24 , 25 . Peptide therapy is an encouraging new approach to AMD with advantages of improved biocompatibility and reduced immunogenicity compared with larger protein therapy 26 . In order to develop an optimized therapeutic peptide, we conducted systematic structure–activity relationship investigations and designed a new 7-amino-acid peptide, EB-203 (INN: lorutengitide; Korean patent no. 10-2022-0053844), which was engineered for enhanced molecular stability and prolonged therapeutic duration while maintaining its anti-angiogenic potency. In this study, we evaluated the therapeutic efficacy of EB-203 in vitro and in vivo . Its anti‑angiogenic potential was assessed in hypoxic endothelial cells, and its efficacy was further examined in a laser‑induced choroidal neovascularization (CNV) mouse model in comparison with aflibercept. Results Evaluation of hypoxia-induced HIF-1α and VEGF expression EA.hy926 cells were treated with EB-203 for 24 h under hypoxia and then analyzed by western blot. As shown in Fig. 1 , EB-203 markedly suppressed hypoxia-induced HIF-1α expression following CoCl₂ treatment. Similarly, VEGF expression, which was strongly induced by CoCl₂, was reduced in the presence of EB-203. It was demonstrated that EB-203 inhibits HIF-1α and consequently down-regulates VEGF under hypoxic conditions, suggesting a potential inhibitory effect on angiogenesis Assessment of endothelial cell migration under hypoxia Wound healing assays were performed to assess the effect of EB-203 on cell migration under hypoxic conditions. EA.hy926 cells were seeded into 6-well plates and subjected to a scratch wound, followed by treatment with CoCl₂ (50 µM) and/or EB-203 (100 µM). After 24 h, CoCl₂ treatment significantly enhanced wound closure compared to the control (48% vs. 39%, p < 0.05). Co-treatment with EB-203 under hypoxic conditions reduced the wound closure percentage to 39%, which was comparable to untreated controls. Quantification of wound areas demonstrated consistent trends across three independent experiments. Representative images from each group are shown in Fig. 2 . These findings indicate that EB-203 is capable of suppressing hypoxia-induced migration of endothelial cells. Evaluation of CoCl₂-induced tube formation The anti-angiogenic effect of EB-203 was further evaluated using tube formation assays on Matrigel-coated plates. EA.hy926 cells treated with CoCl₂ (50 µM) demonstrated a significant increase in all angiogenic parameters relative to control: total tube length (130%), number of nodes (155%), total segments (165%), and number of junctions (150%) ( p < 0.01). EB-203 co-treatment markedly reduced these parameters, restoring values to near-control levels (e.g., tube length 102%). Quantitative analysis is presented in Fig. 3 , with representative micrographs shown in Fig. 2 B. Statistical analysis confirmed that the reduction in angiogenic parameters by EB-203 was significant ( p < 0.01–0.001). These data suggest that EB-203 potently inhibits CoCl₂-induced endothelial network formation. Inhibition of hypoxia-driven endothelial cell invasion Transwell invasion assays were used to assess the impact of EB-203 on EA.hy926 cell invasion under hypoxic conditions. after 24 h of CoCl₂ treatment, cell invasion increased to 170% compared to control groups ( p < 0.001). EB-203 reduced invasion to 40%. Microscopic examination supported the quantitative findings, as illustrated in Fig. 3 a. These results collectively demonstrate the robust anti-invasive activity of EB-203 against hypoxia-driven endothelial cell invasion. Evaluation of intravitreal EB-203 on vascular leakage and CNV formation To identify the therapeutic effects of EB-203 on choroidal neovascularization (CNV), compared to aflibercept (a positive control), mice received either 20 µg of aflibercept (positive control) or 20 µg or 100 µg of EB-203 via intravitreal injection one day after laser injury. Vascular leakage was evaluated by fluorescein fundus angiography (FFA) and CNV lesion area was quantified by optical coherence tomography (OCT) on Days 6 and 14 post-laser injury. FFA analysis revealed that both aflibercept and EB-203 significantly decreased vascular leakage compared to PBS controls. On Day 6, vascular leakage was reduced by 60–70% in the aflibercept and high-dose Laurentide groups, with sustained suppression observed at Day 14 (Figs. 5 b and 5 c). OCT images further demonstrated significant reductions in CNV area in all treatment groups versus PBS controls. On Day 6, CNV area was reduced by 40–50% in aflibercept and EB-203 groups, with consistent effects maintained through Day 14 (Figs. 5 d and 5 e). Dose-dependent effect of topical EB-203 on CNV vascular leakage and lesion area To further assess the therapeutic efficacy of EB-203 as a topical eye drop, mice received twice daily eye drop administration of EB-203 at 1%, 5%, or 10% for 13 days following laser induced CNV. Vascular leakage was evaluated by FFA and CNV lesion area was measured by OCT on Days 6 and 14 post-laser injury. FFA analysis showed only the highest concentration of EB-203 (10%) produced a statistically significant reduction in CNV vascular leakage at both Day 6 and Day 14 compared to PBS controls (Fig. 6 b and 6 c). OCT images demonstrated dose-dependent suppression of CNV area by EB-203, with significant reductions observed in the 5% and 10% groups at day 6, and in all EB-203 groups at Day 14 (Fig. 6 d and 6 e). Discussion In the present study, the peptide candidate, EB-203, was evaluated for its anti-angiogenic activity in endothelial cell cultures and potency as an ocular agent in neovascularized rodent models. Generally, HUVECs or human EA.hy926 cells (a hybrid cell line derived from HUVECs and A549 carcinoma cells) have been used to investigate anti-angiogenesis mechanisms by various agents in addition to the high throughput screening of types of angiogenic promotors or inhibitors 27 – 30 . Here, hypoxic conditions were induced using cobalt(II) chloride (CoCl₂), a chemical hypoxia mimetic that stabilizes hypoxia-inducible factor-1α (HIF-1α), resulting in elevated HIF-1α and VEGF expression in EA.hy926 cells 31 – 34 . VEGF has been known to be an important protein marker involved in the mechanisms of angiogenesis, a main cause of CNV of wet AMD 35 , 36 . For decades, antibody agents like aflibercept or ranibizumab have been used to inhibit choroid neovascularization by specifically downregulating VEGF actions 37 – 39 . In this study, Western blot analysis proved that EB-203 might be engaged in inhibiting processes of HIF-1α and VEGF induced by the hypoxia in the EA.hy926 cells. As shown in the results, CoCl₂ administration drastically increased both HIF-1α and VEGF protein expressions in comparison to the control group, while co-treatment with EB-203 significantly reduced these elevated levels. HIF-1α is a master transcriptional regulator of cellular responses to hypoxia that directly activates numerous pro-angiogenic genes, not only VEGF, but also VEGFR1/2, PDGF, angiopoietins, and matrix metalloproteinases 40 . The rationale for inhibiting HIF-1α and VEGF simultaneously has been supported by recent clinical data showing that anti-VEGF monotherapy often leads to compensatory activation of alternative angiogenic pathways 41 . Studies have demonstrated that tumors and pathologic tissues exposed to anti-VEGF agents can become resistant by upregulating alternative pro-angiogenic factors under continuous hypoxic stress 42 , 43 . By blocking HIF-1α, the upstream regulator, EB-203 may prevent such compensatory responses and have more comprehensive anti-angiogenic activity than current VEGF-targeted therapies alone. Angiogenesis includes two phases of the process, with the early angiogenesis recruiting the cell proliferation, tube formation, and cell migration, and the late angiogenesis composed of the organization of the perivascular sheath 28 , 44 – 46 . In our study, these phases were evaluated using cell-based functional assays that model each step of vascular development under hypoxic conditions. Cell migration was examined by wound healing experiments (Fig. 2 ). Under the hypoxia condition after treatment of CoCl 2 , the EA.hy926 cells showed migration into the uncoated region on plates with 1.3-fold wound closures, compared to the control cells on plates (Fig. 2 B). In Figs. 3 and 4 , in the EA.hy926 cells, EB-203 showed activities of inhibiting tube formation in culture plates, as well as cell invasion in the trans-well membrane cultures. Tube formation assays quantitatively revealed that EB-203 treatment resulted in marked reductions in total tube length, node number, segments, and junctions compared to hypoxia-exposed controls. These findings affirm that EB-203 impedes endothelial morphogenesis and network architecture, which are hallmarks of angiogenic progression (Fig. 3 ). In the invasion study (Fig. 4 ), the EA.hy926 cells were infiltrated across the matrigel of trans-well membrane and pores on the membranes (pore size: 0.4 µm). As an extracellular matrix, Matrigel is primarily composed of laminin, collagen IV, entactin, and heparan sulfate proteoglycans, providing a supportive scaffold that mimics the basement membrane environment. It has been widely applied in invasion and tube formation assays using endothelial cells such as HUVECs and EA.hy926 cells to evaluate their angiogenic or anti-angiogenic responses as described in previous reports 28 , 47 . Co-treatment of EB-203 for the cells under the hypoxia by CoCl 2 indicated the least wound closures like the control cells on plates, which means inhibiting actions of EB-203 on microvascular formation of endothelial cells. In the conventional treatment using aflibercept for patients with wet AMD, intravitreal injections of aflibercept have been done with 2 mg per eye once a month during the period of treatment. To approximate the clinical regimen in human therapy, various doses ranging from 1 µg to 50 µg per eye have been adopted in laser-induced CNV mouse 48 , 49 . In the present study, 20 µg of aflibercept was selected as a positive control dosage to ensure a robust therapeutic effect. The laser-induced CNV mouse model has been widely used as a robust and clinically relevant in vivo platform to recapitulate key pathological features of neovascular AMD, including choroidal neovascularization, rapid progression, and response to anti-angiogenic therapies 50 . In our study, the 532 nm green laser photocoagulator was used to generate four laser spots in each eye of the mice. The laser parameters (power: 200 mW, duration: 80 ms, spot size: 50 µm) were optimized according to previous reports 51 – 53 . In laser-induced CNV mouse models, successful rupture of Bruch’s membrane is critical for consistent induction of choroidal neovascularization. Only laser burns that produced an immediate vaporization bubble at the time of photocoagulation were considered successful, as the bubble formation indicates disruption of Bruch’s membrane and reliable CNV development 52 . Burns that failed to produce a bubble or caused excessive hemorrhage were excluded from analysis to maintain reproducibility and minimize variability of lesion size. In the present study, the mouse models were successfully set using the laser application for testing agents to treat wet AMD, based on FFA and OCT images. For the animal models with induced CNV, aflibercept or EB-203 in PBS solution was intravitreally injected once. As a result, the vascular leakage and CNV sizes dramatically decreased by EB-203 with similar levels to aflibercept, the anti-VEGF antibody drug (Fig. 5 ). These results suggest that at a much lower cost than the antibody drug, EB-203 could contribute to improving wet AMD in clinics. Up to the present, for IVT injections for wet AMD patients, only anti-VEGF therapeutics including aflibercept have been approved. Moreover, the results of topically administered EB-203 were promising, suggesting that EB-203 eyedrops with some concentrations could be comparable to aflibercept (Fig. 6 ). Mice treated with 10% EB-203 eyedrops with a twice-daily dosing schedule exhibited efficacy in decreasing CNV areas on Day 6 of treatment, and this therapeutic effect was maintained through Day 14. Dose-dependent effects were observed—particularly at 10% concentration—both in reductions of vascular leakage and CNV area, supporting the potential of topical EB-203 as a less invasive alternative. Taken together, these findings demonstrate that both intravitreal and topical EB-203 consistently suppressed CNV progression, with efficacy comparable to standard anti-VEGF therapy in this preclinical AMD model. Further studies will be required to confirm long-term safety, optimal dosing regimens, and clinical applicability of EB-203 either as monotherapy or in combination with standard of care therapy. In conclusion, the overall in vitro and in vivo results in the present study support that EB-203 may effectively improve vascular leakage and reduce CNV lesion area through a mode of action associated with anti‑VEGF activity. As small molecular drugs, the clinical study with squalamine lactate eyedrops, a dual inhibitor of VEGF and PDGF, failed to show substantial anatomical improvement in the choroid region or visual acuity (VA). Meanwhile, a Phase I/II study of VEGF‑A inhibitor suspension (PAN‑90806) demonstrated improvement in VA only when combined with conventional anti‑VEGF injections 54 . So far, topically administered small molecular drugs have exhibited therapeutic efficacy mainly in combination therapy with an anti‑VEGF intravitreal (IVT) agent in preclinical or clinical studies. In our study, a single administration of EB-203 eyedrops at 5% or 10% concentration with twice‑daily dosing effectively reduced vascular leakage and decreased CNV lesion area to levels comparable to those achieved by aflibercept IVT injection. EB-203 eyedrops may be applicable either as an adjunct or as a standalone therapy, depending on dosing frequency or concentration. Although most current therapies for wet AMD are administered via intravitreal injection, the development of VEGF‑inhibiting eyedrop formulations has recently been accelerated to improve patient compliance worldwide. MG‑O‑1002 and KHK4951 eyedrops, both targeting VEGF signaling, are currently in Phase II clinical trials for neovascular (wet) AMD and diabetic macular edema (DME) 55 , 56 . Ultimately, it can be concluded that EB-203, as a small peptide drug, represents a promising and cost‑effective therapeutic candidate for wet AMD, offering a safer and less invasive route of administration. Materials & Methods Drug Preparation EB-203 was provided by EYEBIOKOREA Inc. (Busan, Republic of Korea). For in vitro experiments, the compound was dissolved in sterile phosphate‑buffered saline (PBS, pH 7.4) to prepare a stock concentration of 100 mM, which was stored at − 20°C until use. Working solutions were freshly diluted to 100 µM, a concentration confirmed to be non‑cytotoxic to EA.hy926 cells (data not shown). For in vivo studies, EB-203 was freshly dissolved in PBS immediately and administered either by intravitreal injection at doses of 20 µg or 100 µg per eye, or as topical eye drops formulated at 1%, 5%, and 10% (w/v). Aflibercept (Eylea®, Bayer AG) was purchased commercially and diluted in PBS to deliver 20 µg per eye as a positive control. Cell culture The American Type Culture Collection (ATCC) EA.hy926 endothelial cells (CRL-2922; ATCC, Manassas, VA, USA) were used in the in vitro evaluation experiments. The EA.hy926 cells were cultured in Dulbecco Modified Eagle Medium (DMEM, GibcoTM; Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific). It was cultured in humidified air with 5% CO 2 at 37 ℃. Western blot Western blot analysis was performed to determine whether EB-203 regulates angiogenic factors in EA.hy926 cells under hypoxic conditions. The cells were seeded at a density of 2 × 10⁵ cells per well in 6‑well plates (SPL life sciences, Pocheon, Republic of Korea). After 24 h, the cells were treated with 100 µM EB-203 for 24 h in the presence of 50 µM cobalt(II) chloride (CoCl₂; Sigma‑Aldrich, St. Louis, MO, USA) to chemically induce hypoxia. Cells were lysed using RIPA buffer on ice, and the lysates were centrifuged at 13,500 rpm for 15 min at 4°C. Equal amounts of protein were separated by 10% SDS‑PAGE and transferred onto a polyvinylidene difluoride (PVDF; Sigma‑Aldrich) membrane. The membrane was blocked with 5% non‑fat dry milk in TBS-T (TBS with 0.1% Tween‑20) and incubated overnight at 4°C with primary antibodies against HIF-1α (ab179483; Abcam, Cambridge, UK), VEGFA (ab46154; Abcam) (1:1000 dilution) and GAPDH (sc-47724; Santa Cruze, Dallas, TX, USA) (1:5000 dilution) After washing, membranes were incubated with horseradish peroxidase‑conjugated secondary antibodies and visualized using Western Lighting™ ECL (enhanced chemiluminescence) solution (Advansta, San Jose, CA, USA) Migration assay To examine whether EB-203 inhibits cell wound close in hypoxia, a migration assay was performed by inducing hypoxic conditions with CoCl 2 at 50 µM. EA.hy926 cells were seeded in a 12-well plate (SPL life sciences) at 3 × 10 5 cells per well. After 24 hours, the monolayer was scraped cross-center of the wells using a 200 µL pipette tip. After scratching, cells were treated with 100 µM of EB-203 and/or 50 µM of CoCl 2 in DMEM containing 1% FBS. After treatment for 24 h, it was photographed under a microscope. The area where the cells migrated was measured using ImageJ software (v1.53), and the percentage of wound closure was calculated relative to the initial area. Tube formation assay Tube formation assay was performed by inducing hypoxic conditions with CoCl 2 to confirm whether EB-203 inhibits angiogenesis under hypoxia. Growth factor-reduced Matrigel (Corning Life Sciences, Tewksbury, MA, USA) was added to µ-Slide Angiogenesis (Ibidi GmBH, Gräfelfing, Germany) at 10 µL per well, and incubated at 37°C for 30 min. EA.hy926 cells were seeded at 2 × 10⁴ cells per well, treated were treated with 100 µM EB-203 and/or 50 µM CoCl₂, and incubated at 37°C for 6 h. After incubation, the formation of capillary‑like junctions was observed and photographed under a microscope. Quantitative analysis was performed using the AngioTool plugin in ImageJ software (v1.53), measuring total tube length, number of nodes, total segments, and number of junctions. All data were normalized to the control group (without CoCl₂ treatment) and expressed as a percentage. Invasion assay An invasion assay was performed to confirm the change in the invasiveness of cells by EB-203 under hypoxia. The coating buffer was prepared by filtering the mixed solution of 0.01 M Tris (pH 8.0) and 0.7% NaCl, followed by filtration through a 0.2 µm filter. Matrigel was diluted 10 times with the coating buffer, and 200 µL of the diluted Matrigel was added to each transwell inserts (12-well plate, 10.5mm of diameter, 0.4µm of pore sizes of polycarbonate membrane, SPL life sciences). The coated inserts were incubated at 37℃ for 2 h to polymerize. EA.hy926 cells (4 × 10 4 cells/insert) in serum-free medium were seeded into the upper chamber of each trans-well. The lower chamber was filled with 500 µL of complete medium containing 10% FBS as a chemoattractant. Cells were treated with 100 µM EB-203 and/or 50 µM CoCl₂ immediately after seeding and incubated at 37°C for 24 h. After incubation, non-invaded cells on the upper membrane surface were removed with a cotton swab. The inserts were washed with PBS, fixed with 10% formaldehyde for 15 minutes, and stained with 0.1% Crystal Violet (Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes. Invaded cells on the lower membrane surface were photographed under a bright-field microscope (10× magnification). For quantification, ImageJ software (v1.53) was used to count cells in insert. Data were normalized to the untreated control group and expressed as invasion percentage (%). Laser induced Choroidal neovascularization (CNV) in mouse model Seven-week-old female C57BL/6 mice were purchased from Koatech Inc. (Koatech, Inc., Pyeongtaek, South Korea). The care and use of the animals were approved by the Institutional Animal Care and Use Committee of Inje Busan Paik Hospital (approval ID: IJUBPH-2023-003-02). All mice were provided ad libitum access to food and water and reared in a specific-pathogen-free facility, maintaining standardized environmental conditions with 12-h light and 12-h dark cycles. After 1 week of acclimatization, CNV was induced. The pupils of mice were dilated with tropherine ophthalmic solution (Hanmi Pharmaceutical Co., Ltd, Seoul, South Korea) and anesthetized using an intraperitoneal injection of ketamine hydrochloride (25 mg/kg body weight, Huons, Jacheon, South Korea) and xylazine hydrochloride (11.66 mg/kg body weight, Bayer Korea Ltd, South Korea). After mice were placed on a custom stage, both eyes were lubricated with hypromellose ophthalmic solution. The 532 nm Green Laser Photocoagulator (Phoenix Research Labs, CA, USA) was used to generate 4 laser spots in each eye (power: 200 mW, duration: 80 ms, a spot size: 50 µm). Only laser burns that resulted in the formation of a bubble at the moment of photocoagulation, indicating the Bruch's membrane rupture, were considered for inclusion in this study. Any spots exhibiting hemorrhage at the laser site were excluded from the analysis. A total of 6–7 mice were assigned to each experimental group. Intravitreal injection Two days after laser induction (Day3), the mice received a single intravitreal injection of aflibercept (20 µg) or EB-203 (20, 100 µg). After an eye drop of 0.5% paracaine (Hanmi Pharmaceutical Co., Ltd), 1 µL of aflibercept or EB-203 were delivered slowly into the vitreous. The volume of injectate was controlled by a micro controller (Micro 4. World Precision Instruments, sarasota, FL, USA) under magnification of a zoom stereo microscope (Leica, Wetzlar, Germany). Topical administration of EB-203 EB-203 was prepared by dissolving it in PBS. The eye drops containing EB-203 at doses of 1%, 5%, and 10% were administered twice daily from Day 3 to Day 14 after laser injury. To apply the test substance, 5 µL of the drug solution at each concentration was directly instilled onto the superior corneal surface of each eye using a 10 µL pipette, without the use of anesthesia. Subsequently, the mice were held still for 20 sec to facilitate the penetration of the eye drops. Fundus Fluorescein Angiography (FFA) and Optical coherence tomography (OCT) The mice's pupils were dilated and anesthesia was induced by a mixture of ketamine and xylazine hydrochloride. Subsequently, the mice were intraperitoneal injected with 2% Fluorescein (Alcon Laboratories, Inc.,Texas, USA) and their eyes were lubricated with hypromellose ophthalmic solution. After a waiting period of 2 to 3 min, the laser-induced neovasculature was captured using a Micron IV imaging system (Phoenix Research Labs). The assessment of vascular leakage involved measuring the size of the choroidal neovascularization (CNV) area and analyzing fluorescence intensity using the ImageJ software program. OCT imaging was performed in the laser-induced eye immediately after FFA. The OCT beam was directed horizontally and imaged at the highest point of the CNV area. FFA and OCT were performed simultaneously on days 2 (baseline), 6 and 14 after CNV induction. Statistical Analysis All data are presented as the mean ± standard error of the mean (S.E.M). Data normality was assessed using the Kolmogorov–Smirnov test. For statistical comparisons, one-way ANOVA or the Kruskal–Wallis test was applied according to data normality, followed by Tukey’s or Dunn’s post hoc test to evaluate differences relative to the control group. Statistical analyses were performed using GraphPad Prism software (Version 10), and a p -value less than 0.05 was considered statistically significant. Declarations Funding This work was supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: RS-2021-KH113823), and by the Bio Industry Technology Development Project (grant number: 20008870) through the Korea Evaluation Institute of Industrial Technology (KEIT), funded by the Ministry of Trade, Industry & Energy, Republic of Korea.໿ Author Contribution B.A conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. K.M. contributed to data interpretation and critically revised the manuscript. J.Y. supervised the study and served as the corresponding author. All authors read and approved the final manuscript. 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JoVE e58591. 10.3791/58591 (2019). Aranda, E. & Owen, G. I. A semi-quantitative assay to screen for angiogenic compounds and compounds with angiogenic potential using the EA.hy926 endothelial cell line. Biol. Res. 42 , 377–389 (2009). Nardinocchi, L., Pantisano, V., Puca, R., Porru, M. & Aiello, A. Zinc Downregulates HIF-1a and Inhibits Its Activity in Tumor Cells In Vitro and In. (2010). Thomas, P., Bansal, A., Singh, M., Shukla, D. & Saxena, S. Preconditioning effect of cobalt chloride supplementation on hypoxia induced oxidative stress in male albino rats. Biomed. Prev. Nutr. 1 , 84–90. https://doi.org/10.1016/j.bionut.2010.10.003 (2011). Yang, C. et al. N-Acetylcysteine protects against cobalt chloride-induced endothelial dysfunction by enhancing glucose-6-phosphate dehydrogenase activity. FEBS Open. Bio . 12 , 1475–1488. 10.1002/2211-5463.13449 (2022). Li, Q., Ma, R. & Zhang, M. CoCl(2) increases the expression of hypoxic markers HIF-1α, VEGF and CXCR4 in breast cancer MCF-7 cells. Oncol. Lett. 15 , 1119–1124. 10.3892/ol.2017.7369 (2018). Spilsbury, K., Garrett, K. L., Shen, W. Y., Constable, I. J. & Rakoczy, P. E. Overexpression of vascular endothelial growth factor (VEGF) in the retinal pigment epithelium leads to the development of choroidal neovascularization. Am. J. Pathol. 157 , 135–144. 10.1016/s0002-9440(10)64525-7 (2000). Witmer, A. N., Vrensen, G. F. J. M., Van Noorden, C. J. F. & Schlingemann, R. O. Vascular endothelial growth factors and angiogenesis in eye disease. Prog. Retin. Eye Res. 22 , 1–29. https://doi.org/10.1016/S1350-9462(02)00043-5 (2003). Balaratnasingam, C., Dhrami-Gavazi, E., McCann, J. T., Ghadiali, Q. & Freund, K. B. Aflibercept: a review of its use in the treatment of choroidal neovascularization due to age-related macular degeneration. Clin. Ophthalmol. 9 , 2355–2371. 10.2147/OPTH.S80040 (2015). Stewart, M. W. Clinical and differential utility of VEGF inhibitors in wet age-related macular degeneration: focus on aflibercept. Clin. Ophthalmol. 6 , 1175–1186. 10.2147/OPTH.S33372 (2012). Cheng, S. et al. Treatment of neovascular age-related macular degeneration with anti-vascular endothelial growth factor drugs: progress from mechanisms to clinical applications. Front. Med. (Lausanne) . 11 , 1411278. 10.3389/fmed.2024.1411278 (2024). Zimna, A., Kurpisz, M. & Hypoxia-Inducible Factor-1 in Physiological and Pathophysiological Angiogenesis: Applications and Therapies. Biomed Res Int 549412, (2015). 10.1155/2015/549412 (2015). Montemagno, C. & Pagès, G. Resistance to Anti-angiogenic Therapies: A Mechanism Depending on the Time of Exposure to the Drugs. Front. Cell. Dev. Biol. 8 , 584. 10.3389/fcell.2020.00584 (2020). Sharma, D. et al. VEGF inhibition increases expression of HIF-regulated angiogenic genes by the RPE limiting the response of wet AMD eyes to aflibercept. Proc. Natl. Acad. Sci. U S A . 121 , e2322759121. 10.1073/pnas.2322759121 (2024). Gacche, R. N. Compensatory angiogenesis and tumor refractoriness. Oncogenesis 4 , e153–e153. 10.1038/oncsis.2015.14 (2015). Kubis, N. & Levy, B. I. Vasculogenesis and Angiogenesis: Molecular and Cellular Controls. Part 2: Interactions between Cell and Extracellular Environment. Interv Neuroradiol. 9 , 239–248. 10.1177/159101990300900302 (2003). Barrasa-Ramos, S., Dessalles, C. A., Hautefeuille, M. & Barakat, A. I. Mechanical regulation of the early stages of angiogenesis. J. R Soc. Interface . 19 , 20220360. 10.1098/rsif.2022.0360 (2022). Peng, Z. et al. CCL2 promotes proliferation, migration and angiogenesis through the MAPK/ERK1/2/MMP9, PI3K/AKT, Wnt/β–catenin signaling pathways in HUVECs. Exp. Ther. Med. 25 , 77. 10.3892/etm.2022.11776 (2023). Hall, D. M. & Brooks, S. A. In vitro invasion assay using matrigel™: a reconstituted basement membrane preparation. Methods Mol. Biol. 1070 , 1–11. 10.1007/978-1-4614-8244-4_1 (2014). Nirmal, J. et al. Potential of subconjunctival aflibercept in treating choroidal neovascularization. Exp. Eye Res. 199 , 108187. 10.1016/j.exer.2020.108187 (2020). Zhang, Z., Shen, M. M. & Fu, Y. Combination of AIBP, apoA-I, and Aflibercept Overcomes Anti-VEGF Resistance in Neovascular AMD by Inhibiting Arteriolar Choroidal Neovascularization. Invest. Ophthalmol. Vis. Sci. 63 , 2. 10.1167/iovs.63.12.2 (2022). Takata, S. et al. The effect of triamcinolone acetonide on laser-induced choroidal neovascularization in mice using a hypoxia visualization bio-imaging probe. Sci. Rep. 5 , 9898. 10.1038/srep09898 (2015). Lambert, V. et al. Laser-induced choroidal neovascularization model to study age-related macular degeneration in mice. Nat. Protoc. 8 , 2197–2211. 10.1038/nprot.2013.135 (2013). Gong, Y. et al. Optimization of an Image-Guided Laser-Induced Choroidal Neovascularization Model in Mice. PLoS One . 10 , e0132643. 10.1371/journal.pone.0132643 (2015). Shah, R. S., Soetikno, B. T., Lajko, M. & Fawzi, A. A. A Mouse Model for Laser-induced Choroidal Neovascularization. J. Vis. Exp. e53502 10.3791/53502 (2015). Hussain, R. M., Shaukat, B. A., Ciulla, L. M., Berrocal, A. M. & Sridhar, J. Vascular Endothelial Growth Factor Antagonists: Promising Players in the Treatment of Neovascular Age-Related Macular Degeneration. Drug Des. Devel Ther. 15 , 2653–2665. 10.2147/dddt.S295223 (2021). Okada, H. et al. in American Conference of Pharmacometrics. (International Society of Pharmacometrics). Lin, J. B. & Apte, R. S. The landscape of vascular endothelial growth factor inhibition in retinal diseases. Investig. Ophthalmol. Vis. Sci. 66 , 47–47 (2025). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7970433","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":603267923,"identity":"48439684-594c-4ab1-8742-6f597b401b27","order_by":0,"name":"Byulnim Ahn","email":"","orcid":"","institution":"Inje University Busan Paik Hospital","correspondingAuthor":false,"prefix":"","firstName":"Byulnim","middleName":"","lastName":"Ahn","suffix":""},{"id":603267924,"identity":"610b89ac-9c15-4cde-af84-53fc3edba6ee","order_by":1,"name":"Kyong Ah Min","email":"","orcid":"","institution":"Inje University","correspondingAuthor":false,"prefix":"","firstName":"Kyong","middleName":"Ah","lastName":"Min","suffix":""},{"id":603267925,"identity":"e749dbcb-2c79-42db-a85b-3814b001c6b3","order_by":2,"name":"Jaewook Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYHACxgMMDBZgxgMgwcNHjB6gFgkGBjYGZgOQFjZStLBJgHgEtfCznzE48HGHhLz8/B6zyq85djJA2x4+uoFHi2RPjsHBmWckDDcc4zG7LbstGegwNmPjHDxaDG7wGBzmbZNg3MAG1CK5jRmohYdNmhgt9vPbeMyKJbfVE68lsQHoMMaP2w4T1iLZk1YA8kvyhmNpxdKM247zsDET8As/++GNDz7usLGd33x448ef26rt+dmbHz7GpwUMGBtAJIcBMw+IZiakHKGF/QHjD2JUj4JRMApGwYgDAOOPRGG5I2/BAAAAAElFTkSuQmCC","orcid":"","institution":"Inje University Busan Paik Hospital","correspondingAuthor":true,"prefix":"","firstName":"Jaewook","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2025-10-28 16:50:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7970433/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7970433/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104396464,"identity":"097c248b-2581-415b-ba30-a11e8875b7d6","added_by":"auto","created_at":"2026-03-11 11:12:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":340283,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEB-203 suppresses hypoxia-induced expression of HIF-1α and VEGF in EA.hy926 cells. \u003c/strong\u003eEA.hy926 cells were incubated under normoxic conditions or treated with 50 µM CoCl₂ for 24 h to induce hypoxia, with or without 100 µM EB-203. Whole cell lysates were analyzed by western blotting for HIF-1α, VEGF, and GAPDH (loading control). Original blots are presented in Supplementary Figure 1 and 2.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7970433/v1/e227d005964e6ab61f2a559b.png"},{"id":104396466,"identity":"958b3c74-d135-4d8b-ba0f-421580ba472f","added_by":"auto","created_at":"2026-03-11 11:12:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3810976,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEB-203 inhibits hypoxia-induced migration of EA.hy926 cells. \u003c/strong\u003e(a) Representative wound healing images at 0 h and 24 h after scratch under indicated treatments (normoxia, CoCl₂ 50 µM, EB-203 100 µM). (b) Quantification of wound closure (%) after 24 h (one-way ANOVA followed by Tukey’s post hoc test). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 vs CoCl\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7970433/v1/de706e9fdb31c78cc90cb146.png"},{"id":104396496,"identity":"fe7ae44c-3d9d-438d-9afa-0bbb99667580","added_by":"auto","created_at":"2026-03-11 11:12:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3493584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEB-203 inhibits CoCl₂-induced tube formation in EA.hy926 cells. \u003c/strong\u003e(a) Representative images of tube formation assays performed under normoxic and hypoxic (50 µM CoCl₂) conditions, with or without 100 µM EB-203. (b) Quantitative analysis of total tube length and number of nodes (one-way ANOVA followed by Tukey’s post hoc test), total segments and number of junctions (Kruskal–Wallis test followed by Dunn’s multiple comparisons test). Data shown as percent of normoxic group (mean ± SEM). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs CoCl\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7970433/v1/7e0993425dc9c4564a0508a1.png"},{"id":104396438,"identity":"1c30d2a3-af26-4d1f-98a2-72a2abc41647","added_by":"auto","created_at":"2026-03-11 11:12:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4894904,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEB-203 inhibits hypoxia-induced invasion of EA.hy926 cells. \u003c/strong\u003e(a) Representative bright-field images showing Matrigel invasion by EA.hy926 cells under normoxic, hypoxic (50 µM CoCl₂), and hypoxic conditions with EB-203 (100 µM). (b) Quantification of invaded cell numbers normalized to the normoxic group (one-way ANOVA followed by Tukey’s post hoc test). *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 vs CoCl₂.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7970433/v1/0f928e096f130b93e57f419b.png"},{"id":104396507,"identity":"b4d324c5-c6b3-4709-9d31-d100bac67ed9","added_by":"auto","created_at":"2026-03-11 11:12:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8979069,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntravitreal EB-203 reduces vascular leakage and CNV area in a mouse model as effectively as aflibercept. \u003c/strong\u003e(a) Experimental timeline for laser-induced CNV and drug treatment. (b,c) Representative FFA images and quantification of relative CNV leakage on Days 2, 6, 14 after PBS, aflibercept, or EB-203 injection (d,e). Representative OCT images and quantification of CNV area (normalized by Day 2) at Days 6 and 14. Statistical analysis: Kruskal–Wallis test and Dunn’s multiple comparisons test. ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 vs PBS group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7970433/v1/e88b5e1d33456ef7b1dacc98.png"},{"id":104396493,"identity":"73ed0728-d284-40d8-8b15-c0c5983f41ff","added_by":"auto","created_at":"2026-03-11 11:12:10","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":13787014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTopical EB-203 eyedrops dose-dependently suppress vascular leakage and CNV area in a mouse CNV model\u003c/strong\u003e. (a) Experimental timeline for laser CNV induction and eyedrop dosing. (b,c) Representative FFA images and quantification of relative CNV leakage at Days 2, 6, 14 for PBS and EB-203 groups (1%, 5%, 10%) (d,e). Representative OCT images and quantification of CNV area (normalized by Day 2) at Days 6 and 14. Statistical analysis: Kruskal–Wallis test and Dunn’s multiple comparisons test. *p \u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 vs PBS group.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7970433/v1/17a70902bccd65669c1db557.png"},{"id":104396614,"identity":"7123d474-1d5b-4f77-bb05-aeee770a4a32","added_by":"auto","created_at":"2026-03-11 11:12:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":44751579,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7970433/v1/651a38ce-92f5-471d-8523-0a3e12c1e008.pdf"},{"id":104396465,"identity":"5459e338-b2e6-4207-93c8-f191be214564","added_by":"auto","created_at":"2026-03-11 11:12:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":414652,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryfigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7970433/v1/3acb00bb79927672afaff9a5.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Evaluation of Collagen-Derived peptide (EB-203) for Treating Wet Age-Related Macular Degeneration","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGlobally, age-related macular degeneration (AMD) has been known to cause central vision loss in elder people, decreasing the quality of life. Wet AMD is the leading cause of severe and irreversible central vision loss in the elderly. It is characterized by the growth of abnormal, leaky blood vessels beneath the macula. These new vessels readily bleed or leak fluid into the retinal layers, resulting in retinal edema, hemorrhage, and disruption of the photoreceptor architecture, which can rapidly progress to vision loss or legal blindness. Unlike dry AMD, which progresses slowly and has limited therapeutic options, wet AMD often deteriorates very quickly but can be treated pharmacologically if diagnosed early\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAlthough vascular endothelial growth factor (VEGF)-driven angiogenesis has been central to the pathogenesis of choroidal neovascularization (CNV) in neovascular (wet) AMD\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, accumulating evidence indicates that retinal hypoxia plays a crucial role in initiating and sustaining disease progression by upregulating pro-angiogenic and inflammatory mediators. Consistent with this, recent findings highlight that AMD progression involves hypoxia, oxidative stress, inflammation, and immune dysregulation\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. VEGF inhibition remains the cornerstone of AMD therapy. Currently, anti-VEGF treatments including aflibercept (Eylea), ranibizumab (Lucentis), brolucizumab (Beovu), and the bispecific antibody faricimab (Vabysmo) has been regarded as efficient therapies for wet AMD to improve CNV conditions\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Anti-VEGF treatments using antibody biologics via intravitreal (IVT) injections into eyes have been reported to resolve CNV, reduce vascular permeability, and treat the vision acuity (VA) loss\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. The clinical outcomes depend on frequent and consistent treatments for two years. Despite of effects of those treatments, patients feel burdensome because of high costs and ocular implication issues, including intraocular inflammation, ocular (or conjunctival) hemorrhage, or increased intraocular pressure which might be related to the invasiveness of intravitreal (IVT) injections or unknown actions of macromolecule biologics. Moreover, frequent anti‑VEGF injections have been shown to cause retinal thickness fluctuations, and such anatomical variations are closely correlated with poorer long‑term visual acuity outcomes in patients with neovascular AMD\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTo overcome the disadvantages and side effects of current intravitreal anti‑VEGF drugs, extensive efforts have been made to develop alternative therapies for neovascular AMD, including modified antibody biologics, small molecules, and gene‑based modalities and non-invasive drug delivery methods\u003csup\u003e\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eChondrocyte-derived extracellular matrix (CDECM) has been extensively studied for its remarkable anti-angiogenic and anti-inflammatory properties. CDECM suppresses neovascularization and promotes wound healing in corneal alkali burn and glaucoma filtration surgery models in rabbits, prevents vascular invasion during mesenchymal stem cell chondrogenesis, and protects corneal epithelial cells by inhibiting NF-κB signaling pathways\u003csup\u003e\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eCDECM mainly consists of type II collagen, proteoglycans, and glycosaminoglycans, which together provide the structural and functional properties of cartilage \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In our previous study, we identified the anti-inflammatory and antiangiogenic effects of a type II collagen\u0026ndash;derived peptide isolated from CDECM in animal models of dry eye disease and diabetic retinopathy\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePeptide therapy is an encouraging new approach to AMD with advantages of improved biocompatibility and reduced immunogenicity compared with larger protein therapy\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. In order to develop an optimized therapeutic peptide, we conducted systematic structure\u0026ndash;activity relationship investigations and designed a new 7-amino-acid peptide, EB-203 (INN: lorutengitide; Korean patent no. 10-2022-0053844), which was engineered for enhanced molecular stability and prolonged therapeutic duration while maintaining its anti-angiogenic potency.\u003c/p\u003e \u003cp\u003eIn this study, we evaluated the therapeutic efficacy of EB-203 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Its anti‑angiogenic potential was assessed in hypoxic endothelial cells, and its efficacy was further examined in a laser‑induced choroidal neovascularization (CNV) mouse model in comparison with aflibercept.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of hypoxia-induced HIF-1α and VEGF expression\u003c/h2\u003e \u003cp\u003eEA.hy926 cells were treated with EB-203 for 24 h under hypoxia and then analyzed by western blot. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, EB-203 markedly suppressed hypoxia-induced HIF-1α expression following CoCl₂ treatment. Similarly, VEGF expression, which was strongly induced by CoCl₂, was reduced in the presence of EB-203. It was demonstrated that EB-203 inhibits HIF-1α and consequently down-regulates VEGF under hypoxic conditions, suggesting a potential inhibitory effect on angiogenesis\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAssessment of endothelial cell migration under hypoxia\u003c/h3\u003e\n\u003cp\u003eWound healing assays were performed to assess the effect of EB-203 on cell migration under hypoxic conditions. EA.hy926 cells were seeded into 6-well plates and subjected to a scratch wound, followed by treatment with CoCl₂ (50 \u0026micro;M) and/or EB-203 (100 \u0026micro;M). After 24 h, CoCl₂ treatment significantly enhanced wound closure compared to the control (48% vs. 39%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Co-treatment with EB-203 under hypoxic conditions reduced the wound closure percentage to 39%, which was comparable to untreated controls. Quantification of wound areas demonstrated consistent trends across three independent experiments. Representative images from each group are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. These findings indicate that EB-203 is capable of suppressing hypoxia-induced migration of endothelial cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eEvaluation of CoCl₂-induced tube formation\u003c/h3\u003e\n\u003cp\u003eThe anti-angiogenic effect of EB-203 was further evaluated using tube formation assays on Matrigel-coated plates. EA.hy926 cells treated with CoCl₂ (50 \u0026micro;M) demonstrated a significant increase in all angiogenic parameters relative to control: total tube length (130%), number of nodes (155%), total segments (165%), and number of junctions (150%) ( \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). EB-203 co-treatment markedly reduced these parameters, restoring values to near-control levels (e.g., tube length 102%). Quantitative analysis is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, with representative micrographs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB. Statistical analysis confirmed that the reduction in angiogenic parameters by EB-203 was significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u0026ndash;0.001). These data suggest that EB-203 potently inhibits CoCl₂-induced endothelial network formation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eInhibition of hypoxia-driven endothelial cell invasion\u003c/h3\u003e\n\u003cp\u003eTranswell invasion assays were used to assess the impact of EB-203 on EA.hy926 cell invasion under hypoxic conditions. after 24 h of CoCl₂ treatment, cell invasion increased to 170% compared to control groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). EB-203 reduced invasion to 40%. Microscopic examination supported the quantitative findings, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea. These results collectively demonstrate the robust anti-invasive activity of EB-203 against hypoxia-driven endothelial cell invasion.\u003c/p\u003e\n\u003ch3\u003eEvaluation of intravitreal EB-203 on vascular leakage and CNV formation\u003c/h3\u003e\n\u003cp\u003eTo identify the therapeutic effects of EB-203 on choroidal neovascularization (CNV), compared to aflibercept (a positive control), mice received either 20 \u0026micro;g of aflibercept (positive control) or 20 \u0026micro;g or 100 \u0026micro;g of EB-203 via intravitreal injection one day after laser injury. Vascular leakage was evaluated by fluorescein fundus angiography (FFA) and CNV lesion area was quantified by optical coherence tomography (OCT) on Days 6 and 14 post-laser injury. FFA analysis revealed that both aflibercept and EB-203 significantly decreased vascular leakage compared to PBS controls. On Day 6, vascular leakage was reduced by 60\u0026ndash;70% in the aflibercept and high-dose Laurentide groups, with sustained suppression observed at Day 14 (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003eb and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). OCT images further demonstrated significant reductions in CNV area in all treatment groups versus PBS controls. On Day 6, CNV area was reduced by 40\u0026ndash;50% in aflibercept and EB-203 groups, with consistent effects maintained through Day 14 (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ed and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDose-dependent effect of topical EB-203 on CNV vascular leakage and lesion area\u003c/h2\u003e \u003cp\u003eTo further assess the therapeutic efficacy of EB-203 as a topical eye drop, mice received twice daily eye drop administration of EB-203 at 1%, 5%, or 10% for 13 days following laser induced CNV. Vascular leakage was evaluated by FFA and CNV lesion area was measured by OCT on Days 6 and 14 post-laser injury. FFA analysis showed only the highest concentration of EB-203 (10%) produced a statistically significant reduction in CNV vascular leakage at both Day 6 and Day 14 compared to PBS controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003eb and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). OCT images demonstrated dose-dependent suppression of CNV area by EB-203, with significant reductions observed in the 5% and 10% groups at day 6, and in all EB-203 groups at Day 14 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ed and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003ee).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, the peptide candidate, EB-203, was evaluated for its anti-angiogenic activity in endothelial cell cultures and potency as an ocular agent in neovascularized rodent models. Generally, HUVECs or human EA.hy926 cells (a hybrid cell line derived from HUVECs and A549 carcinoma cells) have been used to investigate anti-angiogenesis mechanisms by various agents in addition to the high throughput screening of types of angiogenic promotors or inhibitors\u003csup\u003e\u003cspan additionalcitationids=\"CR28 CR29\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Here, hypoxic conditions were induced using cobalt(II) chloride (CoCl₂), a chemical hypoxia mimetic that stabilizes hypoxia-inducible factor-1α (HIF-1α), resulting in elevated HIF-1α and VEGF expression in EA.hy926 cells\u003csup\u003e\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eVEGF has been known to be an important protein marker involved in the mechanisms of angiogenesis, a main cause of CNV of wet AMD\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. For decades, antibody agents like aflibercept or ranibizumab have been used to inhibit choroid neovascularization by specifically downregulating VEGF actions\u003csup\u003e\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, Western blot analysis proved that EB-203 might be engaged in inhibiting processes of HIF-1α and VEGF induced by the hypoxia in the EA.hy926 cells. As shown in the results, CoCl₂ administration drastically increased both HIF-1α and VEGF protein expressions in comparison to the control group, while co-treatment with EB-203 significantly reduced these elevated levels. HIF-1α is a master transcriptional regulator of cellular responses to hypoxia that directly activates numerous pro-angiogenic genes, not only VEGF, but also VEGFR1/2, PDGF, angiopoietins, and matrix metalloproteinases\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The rationale for inhibiting HIF-1α and VEGF simultaneously has been supported by recent clinical data showing that anti-VEGF monotherapy often leads to compensatory activation of alternative angiogenic pathways\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Studies have demonstrated that tumors and pathologic tissues exposed to anti-VEGF agents can become resistant by upregulating alternative pro-angiogenic factors under continuous hypoxic stress\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. By blocking HIF-1α, the upstream regulator, EB-203 may prevent such compensatory responses and have more comprehensive anti-angiogenic activity than current VEGF-targeted therapies alone.\u003c/p\u003e \u003cp\u003eAngiogenesis includes two phases of the process, with the early angiogenesis recruiting the cell proliferation, tube formation, and cell migration, and the late angiogenesis composed of the organization of the perivascular sheath\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. In our study, these phases were evaluated using cell-based functional assays that model each step of vascular development under hypoxic conditions. Cell migration was examined by wound healing experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Under the hypoxia condition after treatment of CoCl\u003csub\u003e2\u003c/sub\u003e, the EA.hy926 cells showed migration into the uncoated region on plates with 1.3-fold wound closures, compared to the control cells on plates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e, in the EA.hy926 cells, EB-203 showed activities of inhibiting tube formation in culture plates, as well as cell invasion in the trans-well membrane cultures. Tube formation assays quantitatively revealed that EB-203 treatment resulted in marked reductions in total tube length, node number, segments, and junctions compared to hypoxia-exposed controls. These findings affirm that EB-203 impedes endothelial morphogenesis and network architecture, which are hallmarks of angiogenic progression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the invasion study (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003e), the EA.hy926 cells were infiltrated across the matrigel of trans-well membrane and pores on the membranes (pore size: 0.4 \u0026micro;m). As an extracellular matrix, Matrigel is primarily composed of laminin, collagen IV, entactin, and heparan sulfate proteoglycans, providing a supportive scaffold that mimics the basement membrane environment. It has been widely applied in invasion and tube formation assays using endothelial cells such as HUVECs and EA.hy926 cells to evaluate their angiogenic or anti-angiogenic responses as described in previous reports\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Co-treatment of EB-203 for the cells under the hypoxia by CoCl\u003csub\u003e2\u003c/sub\u003e indicated the least wound closures like the control cells on plates, which means inhibiting actions of EB-203 on microvascular formation of endothelial cells.\u003c/p\u003e \u003cp\u003eIn the conventional treatment using aflibercept for patients with wet AMD, intravitreal injections of aflibercept have been done with 2 mg per eye once a month during the period of treatment. To approximate the clinical regimen in human therapy, various doses ranging from 1 \u0026micro;g to 50 \u0026micro;g per eye have been adopted in laser-induced CNV mouse \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. In the present study, 20 \u0026micro;g of aflibercept was selected as a positive control dosage to ensure a robust therapeutic effect. The laser-induced CNV mouse model has been widely used as a robust and clinically relevant \u003cem\u003ein vivo\u003c/em\u003e platform to recapitulate key pathological features of neovascular AMD, including choroidal neovascularization, rapid progression, and response to anti-angiogenic therapies\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our study, the 532 nm green laser photocoagulator was used to generate four laser spots in each eye of the mice. The laser parameters (power: 200 mW, duration: 80 ms, spot size: 50 \u0026micro;m) were optimized according to previous reports\u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. In laser-induced CNV mouse models, successful rupture of Bruch\u0026rsquo;s membrane is critical for consistent induction of choroidal neovascularization. Only laser burns that produced an immediate vaporization bubble at the time of photocoagulation were considered successful, as the bubble formation indicates disruption of Bruch\u0026rsquo;s membrane and reliable CNV development\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Burns that failed to produce a bubble or caused excessive hemorrhage were excluded from analysis to maintain reproducibility and minimize variability of lesion size. In the present study, the mouse models were successfully set using the laser application for testing agents to treat wet AMD, based on FFA and OCT images. For the animal models with induced CNV, aflibercept or EB-203 in PBS solution was intravitreally injected once. As a result, the vascular leakage and CNV sizes dramatically decreased by EB-203 with similar levels to aflibercept, the anti-VEGF antibody drug (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e). These results suggest that at a much lower cost than the antibody drug, EB-203 could contribute to improving wet AMD in clinics. Up to the present, for IVT injections for wet AMD patients, only anti-VEGF therapeutics including aflibercept have been approved. Moreover, the results of topically administered EB-203 were promising, suggesting that EB-203 eyedrops with some concentrations could be comparable to aflibercept (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Mice treated with 10% EB-203 eyedrops with a twice-daily dosing schedule exhibited efficacy in decreasing CNV areas on Day 6 of treatment, and this therapeutic effect was maintained through Day 14. Dose-dependent effects were observed\u0026mdash;particularly at 10% concentration\u0026mdash;both in reductions of vascular leakage and CNV area, supporting the potential of topical EB-203 as a less invasive alternative. Taken together, these findings demonstrate that both intravitreal and topical EB-203 consistently suppressed CNV progression, with efficacy comparable to standard anti-VEGF therapy in this preclinical AMD model. Further studies will be required to confirm long-term safety, optimal dosing regimens, and clinical applicability of EB-203 either as monotherapy or in combination with standard of care therapy.\u003c/p\u003e \u003cp\u003eIn conclusion, the overall in vitro and in vivo results in the present study support that EB-203 may effectively improve vascular leakage and reduce CNV lesion area through a mode of action associated with anti‑VEGF activity. As small molecular drugs, the clinical study with squalamine lactate eyedrops, a dual inhibitor of VEGF and PDGF, failed to show substantial anatomical improvement in the choroid region or visual acuity (VA). Meanwhile, a Phase I/II study of VEGF‑A inhibitor suspension (PAN‑90806) demonstrated improvement in VA only when combined with conventional anti‑VEGF injections\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. So far, topically administered small molecular drugs have exhibited therapeutic efficacy mainly in combination therapy with an anti‑VEGF intravitreal (IVT) agent in preclinical or clinical studies.\u003c/p\u003e \u003cp\u003eIn our study, a single administration of EB-203 eyedrops at 5% or 10% concentration with twice‑daily dosing effectively reduced vascular leakage and decreased CNV lesion area to levels comparable to those achieved by aflibercept IVT injection. EB-203 eyedrops may be applicable either as an adjunct or as a standalone therapy, depending on dosing frequency or concentration. Although most current therapies for wet AMD are administered via intravitreal injection, the development of VEGF‑inhibiting eyedrop formulations has recently been accelerated to improve patient compliance worldwide. MG‑O‑1002 and KHK4951 eyedrops, both targeting VEGF signaling, are currently in Phase II clinical trials for neovascular (wet) AMD and diabetic macular edema (DME)\u003csup\u003e\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUltimately, it can be concluded that EB-203, as a small peptide drug, represents a promising and cost‑effective therapeutic candidate for wet AMD, offering a safer and less invasive route of administration.\u003c/p\u003e"},{"header":"Materials \u0026 Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDrug Preparation\u003c/h2\u003e \u003cp\u003eEB-203 was provided by EYEBIOKOREA Inc. (Busan, Republic of Korea). For \u003cem\u003ein vitro\u003c/em\u003e experiments, the compound was dissolved in sterile phosphate‑buffered saline (PBS, pH 7.4) to prepare a stock concentration of 100 mM, which was stored at \u0026minus;\u0026thinsp;20\u0026deg;C until use. Working solutions were freshly diluted to 100 \u0026micro;M, a concentration confirmed to be non‑cytotoxic to EA.hy926 cells (data not shown). For \u003cem\u003ein vivo\u003c/em\u003e studies, EB-203 was freshly dissolved in PBS immediately and administered either by intravitreal injection at doses of 20 \u0026micro;g or 100 \u0026micro;g per eye, or as topical eye drops formulated at 1%, 5%, and 10% (w/v). Aflibercept (Eylea\u0026reg;, Bayer AG) was purchased commercially and diluted in PBS to deliver 20 \u0026micro;g per eye as a positive control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eThe American Type Culture Collection (ATCC) EA.hy926 endothelial cells (CRL-2922; ATCC, Manassas, VA, USA) were used in the \u003cem\u003ein vitro\u003c/em\u003e evaluation experiments. The EA.hy926 cells were cultured in Dulbecco Modified Eagle Medium (DMEM, GibcoTM; Thermo Fisher Scientific, Waltham, MA, USA) medium supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific). It was cultured in humidified air with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37 ℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eWestern blot analysis was performed to determine whether EB-203 regulates angiogenic factors in EA.hy926 cells under hypoxic conditions. The cells were seeded at a density of 2 \u0026times; 10⁵ cells per well in 6‑well plates (SPL life sciences, Pocheon, Republic of Korea). After 24 h, the cells were treated with 100 \u0026micro;M EB-203 for 24 h in the presence of 50 \u0026micro;M cobalt(II) chloride (CoCl₂; Sigma‑Aldrich, St. Louis, MO, USA) to chemically induce hypoxia. Cells were lysed using RIPA buffer on ice, and the lysates were centrifuged at 13,500 rpm for 15 min at 4\u0026deg;C. Equal amounts of protein were separated by 10% SDS‑PAGE and transferred onto a polyvinylidene difluoride (PVDF; Sigma‑Aldrich) membrane. The membrane was blocked with 5% non‑fat dry milk in TBS-T (TBS with 0.1% Tween‑20) and incubated overnight at 4\u0026deg;C with primary antibodies against HIF-1α (ab179483; Abcam, Cambridge, UK), VEGFA (ab46154; Abcam) (1:1000 dilution) and GAPDH (sc-47724; Santa Cruze, Dallas, TX, USA) (1:5000 dilution) After washing, membranes were incubated with horseradish peroxidase‑conjugated secondary antibodies and visualized using Western Lighting\u0026trade; ECL (enhanced chemiluminescence) solution (Advansta, San Jose, CA, USA)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMigration assay\u003c/h2\u003e \u003cp\u003eTo examine whether EB-203 inhibits cell wound close in hypoxia, a migration assay was performed by inducing hypoxic conditions with CoCl\u003csub\u003e2\u003c/sub\u003e at 50 \u0026micro;M. EA.hy926 cells were seeded in a 12-well plate (SPL life sciences) at 3 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well. After 24 hours, the monolayer was scraped cross-center of the wells using a 200 \u0026micro;L pipette tip. After scratching, cells were treated with 100 \u0026micro;M of EB-203 and/or 50 \u0026micro;M of CoCl\u003csub\u003e2\u003c/sub\u003e in DMEM containing 1% FBS. After treatment for 24 h, it was photographed under a microscope. The area where the cells migrated was measured using ImageJ software (v1.53), and the percentage of wound closure was calculated relative to the initial area.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTube formation assay\u003c/h2\u003e \u003cp\u003eTube formation assay was performed by inducing hypoxic conditions with CoCl\u003csub\u003e2\u003c/sub\u003e to confirm whether EB-203 inhibits angiogenesis under hypoxia. Growth factor-reduced Matrigel (Corning Life Sciences, Tewksbury, MA, USA) was added to \u0026micro;-Slide Angiogenesis (Ibidi GmBH, Gr\u0026auml;felfing, Germany) at 10 \u0026micro;L per well, and incubated at 37\u0026deg;C for 30 min. EA.hy926 cells were seeded at 2 \u0026times; 10⁴ cells per well, treated were treated with 100 \u0026micro;M EB-203 and/or 50 \u0026micro;M CoCl₂, and incubated at 37\u0026deg;C for 6 h. After incubation, the formation of capillary‑like junctions was observed and photographed under a microscope. Quantitative analysis was performed using the AngioTool plugin in ImageJ software (v1.53), measuring total tube length, number of nodes, total segments, and number of junctions. All data were normalized to the control group (without CoCl₂ treatment) and expressed as a percentage.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eInvasion assay\u003c/h2\u003e \u003cp\u003eAn invasion assay was performed to confirm the change in the invasiveness of cells by EB-203 under hypoxia. The coating buffer was prepared by filtering the mixed solution of 0.01 M Tris (pH 8.0) and 0.7% NaCl, followed by filtration through a 0.2 \u0026micro;m filter. Matrigel was diluted 10 times with the coating buffer, and 200 \u0026micro;L of the diluted Matrigel was added to each transwell inserts (12-well plate, 10.5mm of diameter, 0.4\u0026micro;m of pore sizes of polycarbonate membrane, SPL life sciences). The coated inserts were incubated at 37℃ for 2 h to polymerize. EA.hy926 cells (4 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/insert) in serum-free medium were seeded into the upper chamber of each trans-well. The lower chamber was filled with 500 \u0026micro;L of complete medium containing 10% FBS as a chemoattractant. Cells were treated with 100 \u0026micro;M EB-203 and/or 50 \u0026micro;M CoCl₂ immediately after seeding and incubated at 37\u0026deg;C for 24 h. After incubation, non-invaded cells on the upper membrane surface were removed with a cotton swab. The inserts were washed with PBS, fixed with 10% formaldehyde for 15 minutes, and stained with 0.1% Crystal Violet (Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes. Invaded cells on the lower membrane surface were photographed under a bright-field microscope (10\u0026times; magnification). For quantification, ImageJ software (v1.53) was used to count cells in insert. Data were normalized to the untreated control group and expressed as invasion percentage (%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eLaser induced Choroidal neovascularization (CNV) in mouse model\u003c/h2\u003e \u003cp\u003eSeven-week-old female C57BL/6 mice were purchased from Koatech Inc. (Koatech, Inc., Pyeongtaek, South Korea). The care and use of the animals were approved by the Institutional Animal Care and Use Committee of Inje Busan Paik Hospital (approval ID: IJUBPH-2023-003-02). All mice were provided ad libitum access to food and water and reared in a specific-pathogen-free facility, maintaining standardized environmental conditions with 12-h light and 12-h dark cycles. After 1 week of acclimatization, CNV was induced. The pupils of mice were dilated with tropherine ophthalmic solution (Hanmi Pharmaceutical Co., Ltd, Seoul, South Korea) and anesthetized using an intraperitoneal injection of ketamine hydrochloride (25 mg/kg body weight, Huons, Jacheon, South Korea) and xylazine hydrochloride (11.66 mg/kg body weight, Bayer Korea Ltd, South Korea). After mice were placed on a custom stage, both eyes were lubricated with hypromellose ophthalmic solution. The 532 nm Green Laser Photocoagulator (Phoenix Research Labs, CA, USA) was used to generate 4 laser spots in each eye (power: 200 mW, duration: 80 ms, a spot size: 50 \u0026micro;m). Only laser burns that resulted in the formation of a bubble at the moment of photocoagulation, indicating the Bruch's membrane rupture, were considered for inclusion in this study. Any spots exhibiting hemorrhage at the laser site were excluded from the analysis. A total of 6\u0026ndash;7 mice were assigned to each experimental group.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIntravitreal injection\u003c/h2\u003e \u003cp\u003eTwo days after laser induction (Day3), the mice received a single intravitreal injection of aflibercept (20 \u0026micro;g) or EB-203 (20, 100 \u0026micro;g). After an eye drop of 0.5% paracaine (Hanmi Pharmaceutical Co., Ltd), 1 \u0026micro;L of aflibercept or EB-203 were delivered slowly into the vitreous. The volume of injectate was controlled by a micro controller (Micro 4. World Precision Instruments, sarasota, FL, USA) under magnification of a zoom stereo microscope (Leica, Wetzlar, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eTopical administration of EB-203\u003c/h2\u003e \u003cp\u003eEB-203 was prepared by dissolving it in PBS. The eye drops containing EB-203 at doses of 1%, 5%, and 10% were administered twice daily from Day 3 to Day 14 after laser injury. To apply the test substance, 5 \u0026micro;L of the drug solution at each concentration was directly instilled onto the superior corneal surface of each eye using a 10 \u0026micro;L pipette, without the use of anesthesia. Subsequently, the mice were held still for 20 sec to facilitate the penetration of the eye drops.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eFundus Fluorescein Angiography (FFA) and Optical coherence tomography (OCT)\u003c/h2\u003e \u003cp\u003eThe mice's pupils were dilated and anesthesia was induced by a mixture of ketamine and xylazine hydrochloride. Subsequently, the mice were intraperitoneal injected with 2% Fluorescein (Alcon Laboratories, Inc.,Texas, USA) and their eyes were lubricated with hypromellose ophthalmic solution. After a waiting period of 2 to 3 min, the laser-induced neovasculature was captured using a Micron IV imaging system (Phoenix Research Labs). The assessment of vascular leakage involved measuring the size of the choroidal neovascularization (CNV) area and analyzing fluorescence intensity using the ImageJ software program. OCT imaging was performed in the laser-induced eye immediately after FFA. The OCT beam was directed horizontally and imaged at the highest point of the CNV area. FFA and OCT were performed simultaneously on days 2 (baseline), 6 and 14 after CNV induction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (S.E.M). Data normality was assessed using the Kolmogorov\u0026ndash;Smirnov test. For statistical comparisons, one-way ANOVA or the Kruskal\u0026ndash;Wallis test was applied according to data normality, followed by Tukey\u0026rsquo;s or Dunn\u0026rsquo;s post hoc test to evaluate differences relative to the control group. Statistical analyses were performed using GraphPad Prism software (Version 10), and a \u003cem\u003ep\u003c/em\u003e-value less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by a grant from the Korea Health Technology R\u0026amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health \u0026amp; Welfare, Republic of Korea (grant number: RS-2021-KH113823), and by the Bio Industry Technology Development Project (grant number: 20008870) through the Korea Evaluation Institute of Industrial Technology (KEIT), funded by the Ministry of Trade, Industry \u0026amp; Energy, Republic of Korea.໿\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eB.A conceived and designed the study, performed the experiments, analyzed the data, and wrote the manuscript. K.M. contributed to data interpretation and critically revised the manuscript. J.Y. supervised the study and served as the corresponding author. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHobbs, S. D., Tripathy, K. \u0026amp; Pierce, K. in \u003cem\u003eStatPearls\u003c/em\u003e (StatPearls Publishing Copyright \u0026copy; 2025, StatPearls Publishing LLC., (2025).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmbati, J. \u0026amp; Fowler, B. J. 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Sci.\u003c/em\u003e \u003cb\u003e66\u003c/b\u003e, 47\u0026ndash;47 (2025).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Wet age-related macular degeneration, Anti-angiogenic effect, Hypoxia condition, EB-203, Laser induced Choroidal neovascularization (CNV) mouse model","lastPublishedDoi":"10.21203/rs.3.rs-7970433/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7970433/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWet age-related macular degeneration has been one of the challenging ocular diseases due to severe ocular implications like vision loss. As a key protein factor, VEGF has been reported to be closely related with the choroid neovascularization (CNV) as a representative pathogenesis of wet AMD. Conventionally, anti-VEGF antibody therapeutics like aflibercept have been used for improving AMD with a route of intravitreal (IVT) injection. However, there have been burdensome for patients to continue the IVT therapies because of high costs and ocular inflammation issues. Up to date, different strategies using modified antibodies, genes or small molecules have been investigated along with the underlying mechanisms of wet AMD.\u003c/p\u003e \u003cp\u003eIn the present study, a novel peptide has been investigated for its anti-angiogenic activity in AMD using EA.hy926 cells treated with cobalt chloride (CoCl₂) to simulate hypoxic conditions. In result, the peptide, EB-203, showed inhibition on Hif-1α and VEGF expression in western blot. Under the hypoxia condition, tube formation of the endothelial cells was interfered with EB-203 and further, migration and invasion of endothelial cells were inhibited by EB-203 to the levels of control. Moreover, mouse AMD models intravitreally injected by EB-203 at some drug concentrations exhibited comparable improvement to aflibercept in vascular leakage and CNV area. Mouse models administered by 5% or 10% EB-203 eyedrops in twice daily dosing showed the reduction of vascular leakage and CNV area significantly. These results demonstrated that the peptide drug can contribute to improve wet AMD complications and visual acuity as an anti-VEGF inhibitor.\u003c/p\u003e","manuscriptTitle":"Evaluation of Collagen-Derived peptide (EB-203) for Treating Wet Age-Related Macular Degeneration","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-11 11:09:41","doi":"10.21203/rs.3.rs-7970433/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-23T06:31:24+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-23T02:21:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-20T05:53:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-15T07:13:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"317102386657815815206147757135019835906","date":"2026-03-11T04:47:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325556141539592797669406311325645359306","date":"2026-03-09T15:54:51+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-09T11:50:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175338995318164106631209579790611205707","date":"2026-03-09T10:37:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"71494387532592309731508533366694562044","date":"2026-03-06T14:42:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-06T07:45:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-24T09:11:52+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-07T18:27:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-06T05:22:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-11-06T05:18:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a56353d7-f3b9-4eb6-900a-2e17a176be73","owner":[],"postedDate":"March 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":64193041,"name":"Health sciences/Diseases"},{"id":64193042,"name":"Biological sciences/Drug discovery"},{"id":64193043,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-04-23T06:40:26+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-11 11:09:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7970433","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7970433","identity":"rs-7970433","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}