EGFR antagonists for treating diseases involving undesired migration, proliferation and / or metaplasia of retinal pigment epithelium (RPE) cells

Abstract

The present invention relates to a pharmaceutical composition for use in the treatment of a disease involving undesired migration, proliferation and / or metaplasia of retinal pigment epithelium (RPE) cells in a subject wherein the pharmaceutical composition comprises at least one epidermal growth factor receptor (EGFR) antagonist.

Description

Technical Field

[0001] This invention relates generally to the field of pharmaceutical compositions, and more specifically to the development of pharmaceutical compositions for treating diseases involving unwanted migration, proliferation and / or metaplasia of retinal pigment epithelial (RPE) cells in subjects. Background Technology

[0002] Age-related macular degeneration (AMD)

[0003] Age-related macular degeneration (AMD) is one of the most common causes of irreversible vision impairment and blindness worldwide (Bourne et al., 2013; Flaxman et al., 2017). AMD can differentiate into a non-exudative dry form, which features degenerative changes and ultimately leads to the loss of retinal pigment epithelial (RPE) cells, and a wet or exudative / neovascular / proliferative form (Lim et al., 2012; Mitchell et al., 2018). Changes in the RPE are a hallmark of AMD (Mitchell et al., 2018; Guymer et al., 2023). These RPE alterations include RPE cell loss associated with the development of geographic atrophy in late-stage AMD, RPE cell proliferation in the subretinal space between the retina and Bruch's membrane in neovascular or exudative forms of AMD, and RPE cell migration / proliferation to the retina in mid- and late-stage AMD (Cao et al., 2021; Fleckenstein et al., 2018; Sadda et al., 2018; Guymer et al., 2020; Spaide et al., 2018).

[0004] In the eyes of patients with non-exudative or intermediate-stage AMD, irregular pigmentation may occur in the macular region. This indicates a loss of the original function of these RPE cells in the macular region of AMD patients' eyes and the occurrence of cellular metaplasia. Cao et al. recently conducted a combined histological and clinical study, reporting the migration / proliferation of single or a few aggregated macular RPE cells into the retina in eyes without choroidal neovascularization (Cao et al., 2021). Cao et al. observed that the feather morphology of intraretinal migrating / proliferating RPE cells eventually regressed after an increase, with complete disappearance in approximately 40% of eyes. The intraretinal RPE cells lost their immunoreactivity to typical retinaldehyde markers and were immunoreactive to immunomarkers. This abnormal immunoreactivity of migrating / proliferating RPE cells extended to all aberrant phenotypes, with some RPE cells migrating towards and contacting retinal capillaries. The observations by Cao et al. are consistent with other studies describing activated RPE cells and RPE cells associated with AMD-related fibrosis (Zanzottera et al., 2015; Balaratnasingam et al., 2017; Chen et al., 2022; Curcio et al., 2017). In this manner, Jonas et al. recently observed intraretinal hyperreflective bodies in optical coherence tomography (OCT) images at locations corresponding to macular hyperpigmentation in almost all eyes with intermediate or advanced AMD and macular hyperpigmentation. These intraretinal hyperreflective bodies correspond to RPE cells located within the retina. Histological studies by Jonas et al. confirmed the findings of Cao et al., identifying intraretinal migrating / proliferating RPE cells in the eyes of patients with AMD (and other conditions) (proprietary data). Figure 1 Based on clinical studies, the presence of intraretinal migrating / proliferating retinal pemphigoid (RPE) cells in mid-stage AMD, corresponding to irregular hyperpigmentation in the macular region detectable on fundus microscopy, is a poor prognostic marker for further disease progression (Mitchell et al., 2018; Fleckenstein et al., 2018; Guymer et al., 2020; 2023; Sadda et al., 2018; Spaide et al., 2018). Intraretinal RPE cells can be considered part of the intraretinal scarring process in mid-stage AMD. Therefore, preventing unnecessary migration / proliferation of macular RPE cells into the retina is therapeutically significant.

[0005] In exudative or neovascular types or stages of AMD, RPE cell proliferation is characterized by the formation of subretinal scars in the macular region. The proliferating RPE cells typically remain in contact with or generate the periodic acid Schiff (PAS)-positive basement membrane. The finding that proliferating RPE cells can still generate their basement membrane within the subretinal scar suggests that the transformation of RPE cells is best described as fibrotic pseudometaplasia, where the RPE cells retain some of their fundamental epidermal characteristics (i.e., the generation of their basement membrane) (Jonas et al., 2021). Exudative or neovascular age-related macular degeneration (AMD) is also characterized by newly formed vessels growing from the choroid and extending across Bruch's membrane into the space beneath the retinal pigment epithelium (RPE), or, after breaching the RPE, extending into the subretinal space in the macular region of the fundus. Intravitreal injection of vascular endothelial growth factor (VEGF) antibodies for the treatment of neovascularization in exudative AMD is an important step in reducing or even completely halting neovascularization. This can slow down the rate of vision loss and even temporarily improve visual function (Rosenfeld et al., 2006; Martin et al., 2011).

[0006] Despite extensive research focusing on subretinal angiogenesis, studies to date have been limited on the second part of exudative AMD: the abnormal proliferation, migration, and / or metaplasia of retinocytes (RPEs). RPE cell proliferation, migration, and metaplasia are key features of exudative AMD and lead to subretinal scarring in the macular region. This scar separates photoreceptors from the nutrient-supplying choroidal capillaries, and scarring also results in the loss of normal RPE cells that physiologically support photoreceptor metabolism, particularly in the phagocytosis of detached photoreceptor segments and the recycling of visual pigments (Rosenfeld et al., 2006; Martin et al., 2011). Figure 2 and Figure 3 ).

[0007] To date, drugs used intravitreally, such as ranibizumab, bevacizumab, aflibercept, and fareximab, have shown high specificity in inhibiting VEGF and other growth factors (e.g., aflibercept inhibits placental growth factor, and fareximab inhibits angiopoietin 2), but none have a significant direct effect on the proliferation, migration, and metaplasia of RPE cells. RPE cells differ from vascular endothelial cells and perivascular cells in many ways, including their embryonic origin (neuroectodermal vs. mesodermal) and the expression of cell surface receptors.

[0008] Polypoid choroidal vascular lesions (PCV)

[0009] PCV is another disease occurring in the macular region characterized by subretinal choroidal neovascularization, accompanied by subretinal proliferation and scarring. It shares clinical features with exudative AMD and has similar treatments, including intravitreal injection of anti-VEGF drugs. Additionally, laser-based photodynamic therapy can be used. Similar to AMD, a major problem in PCV treatment is the abnormal proliferation of tissues, including RPE cells, below the macular RPE layer or above the subretinal RPE layer, which significantly impacts visual prognosis. As with AMD, there is currently no treatment specifically for this abnormal proliferation. This unwanted proliferation leads to scarring, increasing the distance between the choroid and the photoreceptors nourished by the choroid, and resulting in loss of RPE cell and photoreceptor function.

[0010] Myopic macular degeneration accompanied by RPE cell proliferation associated with macular neovascularization and subretinal scarring sex

[0011] In myopic macular degeneration, macular neovascularization may also occur, originating from the choroid, extending into the space beneath the retinas per the retinal plexus (RPE), and reaching the subretinal space after breaking through the RPE layer. Figure 2 and Figure 3 Similar to exudative AMD, in myopic macular neovascularization, RPE cells also proliferate, migrate, and undergo metaplasia or pseudometaplasia, leading to subretinal scarring. On optical microscopy histological examination, this subretinal scar is composed of proliferating and migrating RPE cells (Jonas et al., 2021).

[0012] Other diseases

[0013] Besides exudative AMD, myopic macular neovascularization, and PCV, there are other diseases located in the macular region characterized by neovascularization originating from the choroid and extending into the subretinal space and / or subretinal space. These conditions include traumatic choroidal rupture, to name just one example. Like exudative AMD, myopic macular neovascularization, and PCV, these diseases are also accompanied by abnormal proliferation of RPE cells, leading to unwanted scarring in the spaces beneath the RPE layer and / or the subretinal space.

[0014] Another disease involving abnormal migration, proliferation, and / or metaplasia of retinal pigment epithelium (RPE) cells is proliferative vitreoretinopathy (PVR). PVR is a disease that develops as a complication of retinal defects and rhegmatogenous retinal detachment. Approximately 5–10% of patients undergoing surgery for primary retinal detachment develop PVR, making surgical repair of rhegmatogenous retinal detachment difficult. PVR is caused by cells such as RPE cells invading the vitreous cavity through retinal defects and forming scar-like tissue within the vitreous. This process involves the formation of a membrane on the retinal surface, leading to hardening and contraction of the retina and the formation of irreversible retinal folds. PVR can be treated with vitreoretinal surgery to remove the vitreous and the membrane. By unfolding the retina, it can be repositioned. However, the visual prognosis after surgery may be limited. Many studies have explored various possible intraocular adjunctive medications for the prevention and treatment of PVR, such as methotrexate or 5-fluorouracil, but none are currently approved for intravitreal use (Schaub et al. 2022).

[0015] Intraretinal proliferation of retinal pigment epithelial (RPE) cells in the eyes of patients with retinitis pigmentosa is known and has been clinically and histologically confirmed (see Kuroda et al. 2014; Schuerch et al. 2016; Yasvoina et al. 2023).

[0016] CN 114 652 826 A describes anti-epidermal growth factor receptor (EGFR) antibodies, particularly cetuximab, for the treatment or prevention of diseases associated with ocular or fundus neovascularization, primarily choroidal and retinal neovascularization, including exudative AMD.

[0017] However, there is currently no way to address the non-vascular proliferative portion of AMD, myopic macular neovascularization, PCV, or any other disease located in the macular region characterized by neovascularization originating from the choroid and extending into the subretinal space and / or subretinal space. Furthermore, there is no way to address the abnormal intraretinal migration / proliferation of RPE cells in non-neovascular AMD or retinitis pigmentosa groups, nor is there an effective way to prevent vitreous scarring in patients with rhegmatogenous retinal detachment. Therefore, there is a need for a pharmaceutical composition to reduce the abnormal migration, proliferation, and / or metaplasia or pseudometaplasia of RPE cells in an attempt to halt the progression of the aforementioned ocular diseases and protect the vision of affected patients. Summary of the Invention

[0018] This invention is particularly based on the discovery that pharmaceutical compositions containing EGFR blockers or substances that stimulate EGF receptors inhibit the proliferation of retinal pigment epithelial (RPE) cells in vitro (see Example 3) and in vivo (see Examples 1 and 4), and that intravitreal application of EGFR blockers is well tolerated in patients with no specific side effects observed (see Example 2). It is also based on the discovery that intraocular injection of blockers containing substances that stimulate EGF receptors in the eyes of guinea pigs, rabbits, and monkeys resulted in well-tolerated intravitreal applications (Bikbov et al., 2022; Dong et al., 2020; Dong et al., 2022).

[0019] By targeting EGFR signaling, the pharmaceutical composition can effectively reduce or inhibit the processes in RPE cells that lead to early and intermediate AMD, late non-neovascular (non-exudative) AMD, retinitis pigmentosa group diseases, myopic macular neovascularization, PCV, or any other neovascularization located in the macular region that is characterized by originating from the choroid and extending into the subretinal space and / or subretinal space.

[0020] Specifically, it should be understood that blocking EGFR signaling affects the abnormal migration, proliferation, and metaplasia or pseudometaplasia of RPE cells. In the following text, the term "metaplasia" refers to both metaplasia and pseudometaplasia. Based on the hypothesized mechanism, it can be hypothesized that this pharmaceutical composition will also have the same effect on other diseases associated with abnormal migration, proliferation, and metaplasia of RPE cells, such as proliferative vitreoretinopathy (PVR).

[0021] Therefore, the present invention provides a pharmaceutical composition for treating diseases involving abnormal migration, proliferation, and / or metaplasia of RPE cells in subjects. The composition comprises at least one epidermal growth factor receptor (EGFR) antagonist. Attached Figure Description

[0022] Figure 1 Clinical photographs and optical coherence tomography images of an eye in intermediate AMD with macular hyperpigmentation are shown. The hyperpigmentation in the fundus photograph (yellow arrow) corresponds to a feather-like intraretinal hyperreflector (or proliferating retinal pigment epithelial cells) (yellow arrow) in the outer nuclear layer, clearly emerging from the retinal pigment epithelium.

[0023] Figure 2 Tissue photographs of highly myopic eyes with pathological myopia and myopic macular neovascularization are shown, demonstrating subretinal proliferation of retinal pigment epithelial cells in the macular region.

[0024] Figure 3Tissue photographs of highly myopic eyes with pathological myopia and myopic macular neovascularization are shown, demonstrating subretinal proliferation of retinal pigment epithelial cells in the macular region.

[0025] Figure 4 The image shows fundus photographs and optical coherence tomography (OCT) images of rabbit eyes 15 days after two applications of laser spot to the same fundus region; yellow arrows indicate laser-induced depigmentation areas; red arrows indicate hyperpigmentation areas within the depigmentation areas.

[0026] Figure 5 The graph shows the results of retinal pigment epithelial (RPE) cell culture, where the addition of epidermal growth factor (EGF) to the culture medium increases RPE cell proliferation in a dose-dependent manner, while EGF antibody reduces RPE cell proliferation in a dose-dependent manner.

[0027] Figure 6 A graph showing the results of retinal pigment epithelial (RPE) cell culture is presented. When epidermal growth factor (EGF) was added to the culture medium, the scratch length at the end of the study was significantly shorter (i.e., more covered by migrating RPE cells), while the scratch was longer when an EGF antibody was added. These results indicate that EGF increases RPE cell migration in a dose-dependent manner, while EGF inhibitors reduce RPE cell migration in a dose-dependent manner.

[0028] Figure 7 This image shows an eye in the early stages of age-related macular degeneration (AMD), with irregularities in the retinal pigment epithelium (indicated by solid white arrows) and highly reflective intraretinal objects covering the outer retinal nuclear layer ("smoke," indicated by dashed white arrows); (dashed black arrows: corresponding optical coherence tomography lines; solid black arrows: lines on the fundus photograph). Detailed Implementation

[0029] This invention relates to pharmaceutical compositions for treating diseases involving undesirable migration, proliferation, and / or metaplasia of retinal pigment epithelial (RPE) cells in a subject, the compositions comprising at least one epidermal growth factor receptor (EGFR) antagonist.

[0030] As shown in Example 1, the EGFR antagonist, namely the EGFR blocking antibody panitumab, when administered intravitreally to rabbit eyes, inhibited laser-induced proliferation of retinal pigment epithelial (RPE) cells, thereby improving a condition similar to that of mid-stage AMD. In Example 3, treatment of the human RPE cell line (ARPE-19 cells) with different doses of epidermal growth factor (EGF) and EGF antibody demonstrated that EGF increased RPE cell proliferation in a dose-dependent manner, while the EGF antibody decreased RPE cell proliferation in a dose-dependent manner.

[0031] This invention is particularly applicable to the treatment of diseases involving undesirable migration, proliferation, and / or metaplasia of retinal pigment epithelial (RPE) cells in a subject. This pathological process can lead to and / or be associated with the formation of fibrous and fibrovascular membranes, subretinal neovascularization, intraretinal RPE cell migration / proliferation, and retinal detachment, thereby causing severe damage to the retina and impairing vision. Examples of such diseases, in addition to non-neovascular (non-exudative) late-stage AMD and retinitis pigmentosa group diseases, also include early and intermediate stages of AMD, which were studied in the animal model of Example 1.

[0032] According to one embodiment, the disease is selected from non-exudative AMD, macular neovascularization in pathological myopia, polypoid choroidal vascular disease (PCV), any other disease located in the macular region characterized by neovascularization (originating from the choroid and extending into the subretinal space and / or subretinal space), proliferative vitreoretinopathy (PVR), and retinitis pigmentosa group diseases. The non-exudative AMD can be early, intermediate, or late non-exudative AMD. Preferably, it is early or intermediate AMD.

[0033] While the rabbits in Example 1 are more of a model for mid-stage AMD, their effectiveness is expected to extend to early-stage AMD as well. Recent studies have shown that intraretinal hyperreflective bodies (iHRBs) are associated with any stage of AMD, including early-stage, mid-stage, late-stage non-neovascular (non-exudative) AMD, and late-stage neovascular (exudative) AMD. Based on histological studies and optical coherence tomography (OCT), these iHRBs are substitutes for intraretinal migrating / proliferating RPE cells. In a recent analysis of the Beijing Eye Study, iHRBs were observed in 141 (34.6%) of 408 early-stage AMD eyes and 262 (79.2%) of 331 mid-stage AMD eyes (a related study has been submitted by Panda-Jonas et al.). Given the strong scientific basis and low risk of referral bias in the Beijing Eye Study, as a population-based survey, these data suggest a relatively high proportion of iHRBs (a marker of intraretinal RPE cell migration / proliferation) in early- and mid-stage AMD eyes. Therefore, OCT images show that in some eyes, the space above Bruch's membrane lacks RPE cells, while a "smoke" of iHRB formation extends from this location into the outer nuclear layer of the retina, a hallmark of RPE cell migration / proliferation. These cells have left their original location above Bruch's membrane. Interestingly, most eyes with iHRBs do not show retinal hyperpigmentation on ophthalmoscopy or fundus photography, suggesting that intraretinal RPE cell migration / proliferation occurs relatively frequently in eyes with AMD, including in the early and middle stages of AMD, particularly much more frequently than is shown in the prevalence of macular hyperpigmentation. This is another indication that iHRBs, as a marker of RPE cell migration / proliferation, are also present in the early stages of AMD.

[0034] Macular neovascularization in pathological myopia, PCV, any other disease located in the macular region characterized by neovascularization (originating from the choroid and extending into the subretinal space and / or retinitis pigmentosa), retinitis pigmentosa group diseases, and PVR: In most of these diseases, undesirable migration, proliferation, and metaplasia occur simultaneously. However, any one of migration, proliferation, and metaplasia may be less pronounced. Therefore, the present invention is not limited to diseases involving undesirable migration, proliferation, and metaplasia of RPE cells, but also to diseases involving undesirable migration, proliferation, and / or metaplasia of RPE cells. However, diseases involving undesirable migration, proliferation, and metaplasia of RPE cells are preferred.

[0035] Specifically, it should be understood that blocking EGFR signaling affects the abnormal migration, proliferation, and metaplasia or pseudometaplasia of RPE cells. Based on the hypothesized mechanism, it can be assumed that the drug composition will have the same effect on other diseases associated with undesirable migration, proliferation, and metaplasia of RPE cells, such as proliferative vitreoretinopathy (PVR) and retinitis pigmentosa.

[0036] In the context of this invention, wet or exudative / neovascular / proliferative AMD is referred to as "exudative AMD".

[0037] CN 114 652 826 A has proposed the use of the epidermal growth factor receptor (EGFR) antibody cetuximab for the treatment of exudative AMD. Therefore, according to one embodiment, diseases involving undesirable migration, proliferation, and / or metaplasia of RPE cells are not exudative AMD.

[0038] The experimental study described in CN 114 652 826 A used a model of choroidal neovascularization in the macular region. A defect was created on the Bruch's membrane using a relatively high-intensity laser, causing choroidal vessels to grow into the subretinal and / or subretinal space, resulting in proliferative fibrous scarring below the retina. Using this method, CN 114 652826 A established an exudative AMD model in rabbits.

[0039] In contrast, the laser intensity used in the experimental design of Example 1 was significantly lower than that of CN 114 652 826 A. The reduced laser power in Example 1 resulted in heat-related damage and activation of RPE cells, leading to localized RPE cell proliferation and the formation of locally hyperpigmented tissue in the subretinal space. Therefore, Example 1 is a dry (non-exudative) AMD model rabbit without any neovascularization.

[0040] Therefore, although CN 114 652 826 A generally mentions AMD as one of the diseases that cetuximab can treat, it is clear from its embodiments that CN 114 652 826 A only addresses the exudative form of AMD. From the research findings and mechanisms presented in this document, the applicant of CN 114 652 826 A concludes that other diseases, such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, retinal phlebitis, high myopia macular hemorrhage, and central exudative chorioretinitis, are not related to the proliferation of RPE cells.

[0041] While the non-exudative or "dry" form or stage of age-related macular degeneration (AMD) can be a precursor to the exudative or "wet" stage or form of AMD, the two forms differ significantly in morphology, clinical presentation, response to treatment, further progression, and prognosis. In the exudative form of AMD, newly formed vessels grow from the choroidal space through a often imperceptible defect in Bruch's membrane into the space beneath the retinal pigment epithelium (RPE) or between the RPE and the outer retina. This results in fluid accumulation (edema) in the space beneath the RPE and / or subretinal, and relatively commonly, fluid accumulation also occurs within the retina. RPE cell proliferation often follows, forming subretinal fibrosis or scarring between the photoreceptor layer of the retina and Bruch's membrane. This form of AMD, particularly in its neovascularization aspect, is sensitive to intravitreal application of anti-vascular endothelial growth factor (VEGF) antibodies.

[0042] In contrast, non-exudative AMD presents with an intact Bruch's membrane, no neovascularization, and no fluid or edema in the subretinal space, subretinal space, or intraretinal space. It is characterized by the formation and deposition of so-called macular drusen beneath the RPE basement membrane above Bruch's membrane, and the formation and accumulation of so-called pseudodrusen in the space between the RPE and the photoreceptor layer. RPE cells migrate and / or proliferate from their location on Bruch's membrane into the deep and intermediate retinal layers, ultimately leading to RPE cell loss and resulting in so-called geographic atrophy, a hallmark of late-stage non-neovascular or non-exudative AMD. The non-exudative form of late-stage AMD is unresponsive to anti-VEGF drugs, but intravitreal application of complement factor inhibitors has only a slight effect.

[0043] Therefore, exudative AMD differs significantly from non-exudative AMD in morphology, treatability, and final morphological outcome. Similar to the major difference in treatment availability between the two types of AMD (exudative AMD uses anti-VEGF drugs, while non-exudative AMD uses complement factor antibodies), this may suggest that findings from studies on exudative AMD cannot be applied to ocular conditions of non-exudative AMD. Therefore, the results presented in CN 114 652 826 A do not necessarily imply the subject matter of this invention.

[0044] "EGFR antagonists" are any proteins or other molecules that can block EGFR or EGFR activators (such as members of the EGF family) or eliminate EGFR expression, thereby reducing the activity of downstream EGFR signaling.

[0045] Compared to normal physiological levels, EGFR antagonists can reduce EGFR signaling by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.25%, at least 99.5%, or at least 99.75%.

[0046] Various methods are known in the art for quantifying the reduction of EGFR signaling, such as Western blotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemistry, and cell-based assays.

[0047] Western blotting can be used to detect the expression levels of EGFR and its downstream signaling proteins in cells or tissues. By comparing the levels of these proteins in control and treated samples, those skilled in the art can assess the extent of EGFR signaling reduction. ELISA is a quantitative technique used to measure the content of EGFR ligands or other signaling proteins in a sample. By comparing the levels of these proteins in control and treated samples, those skilled in the art can determine the degree of reduction in epidermal growth factor receptor (EGFR) signaling. Immunohistochemistry can be used to visualize the location and distribution of EGFR and its downstream signaling proteins in cells or tissues. By comparing the staining intensity or pattern of these proteins in control and treated samples, those skilled in the art can assess the extent of EGFR signaling reduction. Cell-based detection methods can be used to measure the activity of EGFR and its downstream signaling pathways in response to ligand stimulation or other stimuli. By comparing the cellular responses in control and treated samples, those skilled in the art can determine the degree of EGFR signaling reduction.

[0048] It is worth noting that EGFR belongs to the ErbB receptor (ErbBs) family, which consists of four transmembrane receptors belonging to the receptor tyrosine kinase (RTK) superfamily, including EGFR (ErbB1 / HER1), ErbB2 / Neu / HER2, ErbB3 / HER3, and ErbB4 / HER4 (see Bublil et al., 2007). All four ErbB receptors share a common structure, including a large extracellular ligand-binding region, a single transmembrane region, a homologous cytoplasmic protein tyrosine kinase domain, and a C-terminal tail with multiple phosphorylation sites.

[0049] The extracellular regions of EGFR family members contain two homologous ligand-binding domains (domains I and III) and two cysteine-rich domains (domains II and IV) (Ferguson 2008).

[0050] The encoded mRNA sequence was determined by the National Center for Biotechnology Information (NCBI) reference sequence: NM_001346897.2 (SEQ ID NO: 1). The amino acid sequence of EGFR was determined by UniProtKB database P00533, sequence version 2, entry 285 (SEQ ID NO: 2).

[0051] For example, when epidermal growth factor (EGF) or transforming growth factor-α (TGFα) binds to the first three domains of EGFR to form a dimer complex, the growth factor binding site is far from the dimer interface and does not directly participate in the interactions within the dimer. All interactions at the dimer interface are mediated by the receptor's domain II—making these dimers "receptor-mediated" rather than "ligand-mediated" (Schlessinger 2002). The β-hairpin structure in domain II, known as the "dimerizing arm," interacts extensively with the domain II of its binding partner.

[0052] EGFR activation is a key step in many cellular signaling pathways, including the following steps: a) ligand binding; b) receptor dimerization; c) activation of intracellular kinase domains; d) recruitment of signaling molecules; and e) signal transduction and cellular responses.

[0053] Specifically, EGFR activation begins with the binding of EGF family ligands (such as epidermal growth factor (EGF), transforming growth factor-α (TGF-α), or bimodalin) to the extracellular domain of the receptor. This interaction is highly specific due to the complementary structure and charge distribution between the ligand and receptor binding pockets.

[0054] Upon ligand binding, EGFR undergoes a conformational change, promoting the formation of receptor dimers. These dimers can be homodimers (two identical EGFR molecules) or heterodimers (EGFR paired with another member of the ErbB receptor family, such as ErbB2, ErbB3, or ErbB4).

[0055] Dimerization brings the intracellular kinase domains of the two receptor molecules close together. This spatial arrangement allows specific tyrosine residues at the cytoplasmic tail of each receptor molecule to undergo trans-autophosphorylation, thereby activating the receptor's intrinsic kinase activity.

[0056] Phosphorylated tyrosine residues in the cytoplasmic tail of the receptor serve as docking sites for various signaling molecules, such as proteins containing Src homology 2 (SH2) and phosphotyrosine-binding (PTB) domains. This recruitment initiates downstream signaling cascades, including the mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3K) / Akt pathway, and the phospholipase C-γ (PLCγ) pathway.

[0057] Activation of these downstream signaling pathways ultimately leads to changes in gene expression, protein synthesis, and post-translational modifications, which in turn regulate various cellular processes such as proliferation, differentiation, migration, and survival.

[0058] EGFR antagonists may include, but are not limited to: antibody-based molecules that can bind to EGFR and reduce EGFR signaling; antibody-based molecules that can bind to EGF family members and reduce EGFR signaling; small molecule EGFR tyrosine kinase inhibitors; peptide inhibitors that can bind to EGFR and reduce EGFR signaling; antibody-based molecules that can bind to EGF family members and reduce EGFR signaling; small molecule interfering RNA (siRNA) reagents that can reduce EGFR expression; CRISPR / Cas9 constructs that can knock out the EGFR gene and act directly on EGFR; and mRNA molecules that encode antibody-based molecules that can bind to EGFR and reduce EGFR signaling or encode peptide inhibitors that can bind to EGFR and reduce EGFR signaling.

[0059] Antibody-based molecules, in particular, can reduce EGFR signaling by blocking the binding of EGF family members to EGFR. Antibody-based molecules can directly bind to EGFR or a member of the EGF family. For example, blocking the binding of EGF family members can be achieved by binding to the ligand-binding domains (I and III) of the extracellular region of EGFR. Alternatively, it can be achieved by binding to the EGFR-binding motif of an EGF family member.

[0060] Antibody-based molecules can reduce EGFR signaling by inhibiting EGFR dimerization or inhibiting EGFR tyrosine kinase activity. For example, inhibiting EGFR dimerization can be achieved by binding to the dimerization arm of extracellular domain II. Similarly, inhibiting tyrosine kinase activity can be achieved by binding to the intracellular tyrosine kinase domain.

[0061] According to one implementation scheme, the antibody-based molecule is selected from antibody-based molecules that block the binding of EGF family members to EGFR, antibody-based molecules that inhibit EGFR dimerization, or antibody-based molecules that inhibit EGFR tyrosine kinase activity.

[0062] Besides EGF itself, other members of this family include: heparin-bound EGF-like growth factor (HB-EGF), transforming growth factor-α (TGF-α), amphotericin (AR), epithelial regulatory protein (EPR), epithelial cells, β-cytokinin (BTC), neuroregulatory protein-1 (NRG1), neuroregulatory protein-2 (NRG2), neuroregulatory protein-3 (NRG3), and neuroregulatory protein-4 (NRG4). Members of the EGF family share highly similar structural and functional characteristics. The ability of EGF family members to bind EGFR is based on one or more shared "EGFR-binding motif" repeat sequences, whose conserved amino acid sequences are:

[0063] CX7CX 4-5 CX 10-13 CXCX5GXRC (SEQ ID NO: 3).

[0064] Where X represents any amino acid.

[0065] The sequence contains six cysteine ​​residues that form three intramolecular disulfide bonds. The formation of these disulfide bonds creates three structural loops, which are essential for the high-affinity binding of EGF family members to their cell surface receptors.

[0066] According to one implementation, an EGFR antagonist is an antibody-based molecule capable of binding to EGF family members selected from EGF, HB-EGF, TGF-α, AR, EPR, epigenes, BTC, NRG1, NRG2, NRG3, and NRG4 and reducing EGFR signaling.

[0067] According to one implementation scheme, the antibody-based molecules are selected from antibodies, antibody fragments, and antibody mimics.

[0068] According to one implementation scheme, the antibody is preferably selected from panitumumab, cetuximab, nimotuzumab, materutuzumab, pertuzumab, and trastuzumab. Cetuximab (Erbitux) ® Panitumumab is a chimeric (mouse / human) monoclonal antibody that targets the extracellular domain of EGFR. By binding to the receptor, it prevents the binding of endogenous ligands and subsequent receptor activation, thereby inhibiting downstream signaling pathways. Cetuximab is used to treat metastatic colorectal cancer (mCRC) with the wild-type KRAS gene and in combination with radiotherapy or chemotherapy for the treatment of locally or regionally advanced head and neck squamous cell carcinoma (HNSCC). It is also used to treat recurrent or metastatic HNSCC that has progressed after platinum-based therapy (see, for example, Cunningham et al., 2004). Panitumumab (Vectib) ®Panitumumab is a fully humanized monoclonal antibody that also targets the extracellular domain of EGFR, blocking ligand binding and receptor activation. It is used as monotherapy for metastatic colorectal cancer (mCRC) with the wild-type KRAS gene when the disease progresses after chemotherapy. Panitumumab has also been investigated in combination with other therapies for the treatment of various solid tumors (see, for example, Hecht et al., 2007). Nimotuzumab (Tageva) ® BIOMAb EGFR ® Matozumab (EMD 72000) is a humanized monoclonal antibody that, similar to cetuximab and panitumumab, targets the extracellular domain of EGFR. It has been approved in several countries for the treatment of head and neck cancer, glioma, and other cancers (see, for example, Ramakrishnan et al., 2009). ® Pertuzumab is another humanized monoclonal antibody targeting EGFR. It has been used in clinical trials to treat various solid tumors, including non-small cell lung cancer (NSCLC), colorectal cancer, and ovarian cancer. However, it has not shown significant clinical benefit in these trials, and its development has been terminated (see, for example, Vanhoefer et al., 2004). ® (This is a humanized monoclonal antibody that targets the extracellular dimerization domain of human epidermal growth factor receptor 2 (HER2), and is another member of the ErbB family. It is similar to trastuzumab (Herceptin).) ® Trastuzumab is used in combination with chemotherapy to treat HER2-positive breast cancer (see, for example, Swain et al., 2015). Trastuzumab is a humanized monoclonal antibody that targets HER2. It is widely used to treat HER2-positive breast cancer and HER2-positive metastatic gastric or gastroesophageal junction adenocarcinoma (see Piccart-Gebhart et al., 2005).

[0069] According to one embodiment, the EGFR antagonist is an antibody fragment. According to one embodiment, the antibody fragment is selected from Fab fragments, F(ab')2 fragments, and Fab' fragments. The Fab fragment is an antibody fragment consisting of variable regions of the antibody heavy and light chains, and the first constant region of the heavy chain. The Fab fragment can be prepared by enzymatic digestion of a full-length antibody with papain. The F(ab')2 fragment is an antibody fragment consisting of two Fab fragments linked together by disulfide bonds. The F(ab')2 fragment can be prepared by enzymatic digestion of a full-length antibody with pepsin. The Fab' fragment is an antibody fragment consisting of variable regions of the antibody heavy and light chains, and a portion of the constant region of the heavy chain. The Fab' fragment can be prepared by first enzymatically digesting the full-length antibody with papain, and then reducing the disulfide bonds linking the heavy chain. These fragments are commonly used in research and diagnostic applications, and the methods for preparing the antibody fragments are not particularly limited and are known in the art.

[0070] According to one embodiment, the EGFR antagonist is an antibody mimic. The antibody mimic according to the invention can be selected from the group consisting of: single-chain variable fragments (scFv), single-domain antibodies, avidins, avidins, avidin proteins, avidin proteins, anti-carrier proteins, design-anchor repeat proteins (DARPins), single-domain antibodies, and peptide aptamers.

[0071] Single-chain variable fragments (scFvs) are antibody fragments composed of variable domains of the antibody heavy and light chains linked together by short peptide linkers. They can be produced in bacterial, yeast, or mammalian cells. Single-domain antibodies, also known as nanobodies, are antibody fragments composed of a single variable domain of the antibody heavy or light chain. They are smaller and more stable than traditional antibodies. Affinities are engineered small protein scaffolds capable of binding to specific targets with high affinity and specificity. They are based on the B domain of protein A, which is the natural ligand for the antibody's Fc region. Avidin is an engineered small protein scaffold capable of binding to specific targets with high affinity and specificity. It is based on the cystatin protein family, which are natural protease inhibitors. Affinity proteins are engineered protein scaffolds capable of binding to specific targets with high affinity and specificity. They are based on proteins called staphylococcal nucleases. Affinity proteins are engineered protein scaffolds capable of binding to specific targets with high affinity and specificity. Based on a protein called ExbB, which is involved in iron transport in bacteria, these proteins can be used for research, diagnostic, and therapeutic applications. Anticarrier proteins are engineered protein scaffolds capable of binding to specific targets with high affinity and specificity. They are based on proteins called lipid carrier proteins, which are involved in the transport of small hydrophobic molecules. Designed ankyrin repeat proteins (DARPins) are engineered protein scaffolds capable of binding to specific targets with high affinity and specificity. Based on the ankyrin repeat protein family, they can be used for research, diagnostic, and therapeutic applications. Single-domain antibodies are antibody mimics composed of engineered single protein domains capable of binding to specific targets with high affinity and specificity. Based on the fibronectin type III domain, they can be used for research, diagnostic, and therapeutic applications. Peptide aptamers are protein scaffolds composed of engineered short peptide sequences capable of binding to specific targets with high affinity and specificity. They are typically generated using phage display or other screening methods and can be used for research, diagnostic, and therapeutic applications. There are no particular limitations on the corresponding antibody mimics and their preparation methods, and they are known in the art.

[0072] According to one implementation scheme, the epidermal growth factor receptor (EGFR) antagonist is a small molecule EGFR tyrosine kinase inhibitor (TKI). Small molecule TKIs are a class of targeted cancer therapies specifically designed to interfere with EGFR enzyme activity.

[0073] Small molecule TKIs are small molecules that selectively bind to the intracellular EGFR kinase domain (typically at the ATP-binding site) and block its tyrosine kinase activity. By inhibiting EGFR kinase activity, these TKIs can disrupt downstream signaling responsible for tumor growth and survival. Because small molecule TKIs successfully reduce EGFR signaling, they are also suitable for treating diseases involving unwanted migration, proliferation, and / or metaplasia of the retinal pigment epithelium (RPE).

[0074] Several small molecule TKIs have been developed to target dysregulated EGFR in various cancers. Examples include gefitinib, erlotinib, lapatinib, icotinib, afatinib, neratinib, dacomitinib, ametinib, osimertinib, brigatinib, vandetanib, and pyrotinib. Lapatinib (Talisar) ® Or Taiweida ® Icotinib (Cemexinib) is a dual EGFR and HER2 TKI used to treat HER2-positive breast cancer, often in combination with other treatments such as capecitabine. ® Neratinib (Helian) is a small molecule EGFR TKI primarily used to treat non-small cell lung cancer (NSCLC) with activating EGFR mutations. ® Dacomitinib (Dazortinib) is a pan-HER inhibitor that targets EGFR, HER2, and HER4, and is used for extended adjuvant therapy in early-stage HER2-positive breast cancer. ® Ametinib is an irreversible pan-HER inhibitor that targets EGFR, HER2, and HER4, and is used as a first-line treatment for metastatic non-small cell lung cancer (NSCLC) with EGFR exon 19 deletion or exon 21 L858R substitution mutations. ® Osimertinib (BI 1482694 or HM61713) is a third-generation small-molecule EGFR TKI used to treat advanced non-small cell lung cancer (NSCLC) with EGFR T790M mutations that has progressed during or after EGFR TKI treatment. Brigatinib (Brigatinib) is also a third-generation small-molecule EGFR TKI under development for the treatment of NSCLC with EGFR T790M mutations. ® Vandetanib (caprixacin) is a TKI that targets anaplastic lymphoma kinase (ALK) and EGFR for the treatment of metastatic non-small cell lung cancer (NSCLC) with ALK rearrangements that has progressed or is intolerant to crizotinib. ® Pyrotinib (Ayrinib) is a multi-target TKI that inhibits EGFR, vascular endothelial growth factor receptor (VEGFR), and RET kinase, and is used to treat patients with symptomatic or progressive, unresectable, locally advanced, or metastatic medullary thyroid carcinoma.® Small molecule EGFR TKIs are irreversible pan-HER inhibitors that target EGFR, HER2, and HER4 for the treatment of HER2-positive breast cancer, often used in combination with other treatments such as capecitabine. There are no particular restrictions on the preparation methods of these small molecule EGFR TKIs, and they are known in the art.

[0075] According to one embodiment, the EGFR antagonist is a peptide inhibitor. According to one embodiment, the peptide inhibitor is selected from the group consisting of peptides that inhibit the binding of epidermal growth factor (EGF) family members to EGFR, peptides that inhibit EGFR dimerization, or peptides that inhibit EGFR tyrosine kinase activity. For example, a peptide that inhibits the binding of EGF family members to EGFR may bind to ligand-binding domains (I and III) of the extracellular region of EGFR. Alternatively, a peptide that inhibits the binding of EGF family members to EGFR may bind to the EGFR-binding motif of an EGF family member. According to one embodiment, the peptide inhibitor has a length of up to 80 amino acids. The peptide inhibitor can be 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, or 80 amino acids in length. According to one embodiment, the peptide inhibitor has a length of up to 60 amino acids. According to one embodiment, the peptide inhibitor has a length of up to 40 amino acids. According to one embodiment, the peptide inhibitor has a length of up to 20 amino acids. Peptide inhibitors are described, for example, in Ahsan et al., 2014. Methods for generating peptide inhibitors are not particularly limited and are known in the art.

[0076] According to one implementation, EGFR antagonists are small interfering RNA (siRNA) agents capable of reducing EGFR expression. siRNA is a double-stranded RNA molecule, typically approximately 21-23 nucleotides in length, involved in the RNA interference (RNAi) pathway. RNAi is a natural mechanism by which cells silence gene expression by degrading messenger RNA (mRNA) molecules that carry genetic information from DNA to ribosomes (the site of protein synthesis). siRNA molecules are designed to be complementary to specific mRNA sequences, leading to their degradation and subsequently inhibiting protein synthesis. In siRNA, the antisense strand is the RNA strand complementary to the target mRNA molecule; it is this strand responsible for base pairing with the mRNA to induce its degradation. The antisense strand of siRNA is typically 21-23 nucleotides in length and is usually designed to be perfectly complementary to the target mRNA sequence, except for a two-nucleotide overhang at the 3' end. This overhang is important for determining the specificity of the siRNA and preventing off-target effects.

[0077] According to one embodiment, the siRNA agent comprises an antisense strand of 21, 22, or 23 nucleotides in length. Furthermore, the antisense strand is designed to be perfectly complementary to an EGFR mRNA sequence (SEQ ID NO: 1). The antisense strand also includes a 3' end with a two-nucleotide overhang that does not match a segment of SEQ ID NO: 1.

[0078] According to one embodiment, the EGFR antagonist is an mRNA molecule. According to one embodiment, the mRNA molecule encodes an antibody. According to one embodiment, the antibody encoded by the mRNA is selected from panitumumab, cetuximab, nimotuzumab, materutuzumab, pertuzumab, and trastuzumab. According to one embodiment, the mRNA molecule encodes a peptide inhibitor as described above. The process involved in administering EGFR-targeting antibodies using an mRNA-based approach differs slightly from the traditional mRNA vaccines developed by Moderna and BioNTech. Instead of encoding an antigen to stimulate an immune response, the mRNA encodes the antibody itself.

[0079] The process is briefly outlined below. A) Design and Production: The mRNA sequence encoding the desired antibody that specifically targets EGFR, as described above, is synthesized. This sequence typically consists of the heavy and light chains of the antibody, optimized for improved stability, efficient translation, and reduced immunogenicity. B) Delivery System: Similar to mRNA vaccines, the mRNA encoding the antibody can be encapsulated in lipid nanoparticles (LNPs) to protect it from degradation and facilitate its entry into host cells. C) Administration: The mRNA-antibody construct is injected into the recipient, typically via intramuscular or subcutaneous routes. In this case, one of the administration routes listed below is used, such as intraocular administration. The mRNA is taken up by cells at the injection site. D) Translation and Secretion: Once inside the host cell, the mRNA is released from the LNP and translated by cellular mechanisms to produce the antibody. The heavy and light chains of the antibody assemble into a functional antibody, which is then secreted from the cell into the extracellular space and subsequently enters the intraocular compartment and the bloodstream. E) Binding and Neutralization: The synthesized antibody specifically binds to EGFR, which may lead to a reduction in receptor function.

[0080] According to one embodiment, the use is for therapeutic or prophylactic treatment. The pharmaceutical composition containing an EGFR agonist can be administered intravitreally, through the corneal epithelium, across the cornea, across the sclera, across the conjunctiva, subconjunctivally, intraocularly, or by injection into the Tenon capsule.

[0081] Another administration option is the PLGA microparticle Densomere administration method for the sustained release of biotherapeutic antibodies, as described by Peterson et al., 2023. To prepare Densomere, an antibody solution is mixed with a PLGA solution and processed using proprietary technology. Densomere microparticle carriers (DMCs) can be suspended in a range of sizes for injection or inhalation, or shaped into implantable solid forms for local delivery to various tissues. In this study conducted by Peterson and colleagues, a DMC suspension was administered as a single subconjunctival injection in a rabbit corneal model to inhibit neovascularization.

[0082] Needles used for administering the pharmaceutical composition are known in the art. Possible needle sizes, according to the Birmingham gauge system, are 25, 26, 27, 28, 29, 30, 31, 32, 33, and 34. According to one embodiment, the needle size is selected from 28, 29, 30, 31, and 32. According to one embodiment, the needle size is 30. Syringes used for administering the pharmaceutical composition are known in the art. Suitable syringe sizes are 0.5 ml, 1.0 ml, 2.5 ml, or 5 ml. Specifically, a tuberculin syringe (1 ml volume) is particularly suitable.

[0083] Preferably, the pharmaceutical composition is administered intravitreal. For intravitreal application, anesthesia is recommended. Specifically, the cornea and conjunctiva are anesthetized by applying local anesthetic eye drops (such as oxybuprocaine). The outer surface of the eye, the eyelid margin, and the eyelid are disinfected, and an eyelid retractor is inserted.

[0084] The pharmaceutical composition can be injected at different locations in the eye. According to one embodiment, the pharmaceutical composition is injected into the inferotemporal quadrant. According to one embodiment, the pharmaceutical composition is injected behind the limbus. According to one embodiment, the pharmaceutical composition is injected into the vitreous cavity through the conjunctiva, sclera, and pars plana of the ciliary body.

[0085] According to one embodiment, the pharmaceutical composition is injected 1 mm to 6 mm posterior to the limbus. This distance can be, for example, 1.0 mm, 1.2 mm, 1.4 mm, 1.6 mm, 1.8 mm, 2.0 mm, 2.2 mm, 2.4 mm, 2.6 mm, 2.8 mm, 3.0 mm, 3.2 mm, 3.4 mm, 3.6 mm, 3.8 mm, 4.0 mm, 4.2 mm, 4.4 mm, 4.6 mm, 4.8 mm, 5.0 mm, 5.2 mm, 5.4 mm, 5.6 mm, 5.8 mm, or 6.0 mm. According to one embodiment, the pharmaceutical composition is injected 2 mm to 5 mm posterior to the limbus. According to one embodiment, the pharmaceutical composition is injected 3 mm to 4 mm posterior to the limbus.

[0086] After the needle is removed, an ointment containing antibiotics and anti-inflammatory agents can be applied.

[0087] If the epidermal growth factor receptor (EGFR) antagonist is an antibody-based molecule, the total dose of the EGFR antagonist per eye can range from 0.1 mg to 6 mg. The total dose can be 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2.0 mg, 2.2 mg, 2.4 mg, 2.6 mg, 2.8 mg, 3.0 mg, 3.2 mg, 3.4 mg, 3.6 mg, 3.8 mg, 4.0 mg, 4.2 mg, 4.4 mg, 4.6 mg, 4.8 mg, 5.0 mg, 5.2 mg, 5.4 mg, 5.6 mg, 5.8 mg, or 6.0 mg. No significant therapeutic or preventative effect was detected at doses below 0.1 mg. Doses higher than 6.0 mg do not further improve drug efficacy and may instead increase the risk of side effects. According to one embodiment, the dosage of the antibody-based molecule in the pharmaceutical composition ranges from 0.30 µg to 3 mg. According to another embodiment, the dosage of the antibody-based molecule in the pharmaceutical composition ranges from 0.5 mg to 2 mg.

[0088] The pharmaceutical composition may be applied at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, or at least 40 times.

[0089] The side effects of EGFR antagonist administration are expected to be minimal. As demonstrated in Examples 3 and 4, no injection-related effects were observed in either rabbits or guinea pigs, such as retinal cell loss, changes in retinal thickness measurements based on intravitreal optical coherence tomography (OCT), increased number of apoptotic retinal cells, ciliary body atrophy or swelling, astrocyte proliferation induction, or changes in intraocular pressure. Similarly, no signs of intraocular inflammation were detected during either biopsy or ocular histology. These findings support the view that repeated intravitreal administration of EGFR antagonists does not lead to intraocular inflammation or toxicity.

[0090] The pharmaceutical composition can be any dosage form suitable for administration to a patient, such as crystals, solutions, or lyophilized formulations. According to one embodiment, the pharmaceutical composition is a solution or lyophilized formulation.

[0091] In a preferred embodiment, an EGFR antagonist or a pharmaceutically acceptable salt thereof is formulated in a pharmaceutical composition together with one or more pharmaceutically acceptable excipients and / or carriers.

[0092] Lyophilized proteins can be reconstituted in sterile buffer. Suitable buffer components include citrate or sodium phosphate. Sodium phosphate buffer consists of sodium dihydrogen phosphate (NaH2PO4) and disodium hydrogen phosphate (Na2HPO4).

[0093] To improve the stability of EGFR antagonists in solution and extend shelf life, stabilizers can be added to the buffer solution. Suitable stabilizers include sucrose, dextran, and carrier proteins such as heat-inactivated fetal bovine serum (FCS) or tissue culture-grade bovine serum albumin (BSA). According to one embodiment, the lyophilized EGFR antagonist is reconstituted in water for injection. Therefore, in the pharmaceutical composition, the EGFR antagonist can be dissolved in water for injection.

[0094] According to another implementation, the EGFR antagonist is formulated with sucrose, dextran, and sodium phosphate buffer.

[0095] The injection volume can range from 30 µL to 300 µL. For example, the volume can be 30 µL, 40 µL, 50 µL, 60 µL, 70 µL, 80 µL, 90 µL, 100 µL, 120 µL, 140 µL, 160 µL, 180 µL, or 200 µL. According to one embodiment, the volume is in the range of 50 µL to 100 µL. For volumes exceeding 100 µL, anterior chamber paralysis should be performed prior to injection to release fluid from the anterior chamber and reduce intraocular volume. Anterior chamber paralysis is a method known to those skilled in the art.

[0096] The treatment can be a single application. Preferably, the pharmaceutical composition is administered multiple times. The time interval between applications can range from 2 days to 12 months. For example, the time intervals between applications are 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 3 months, 4 months, 5 months, 6 months, 9 months, and 12 months. According to one embodiment, the time interval between applications ranges from 2 weeks to 7 months. According to one embodiment, the time interval between applications ranges from 4 weeks to 6 months.

[0097] Example

[0098] Example 1 – Laser-induced proliferation of rabbit retinal pigment epithelial (RPE) cells as a treatment for a mid-stage model of age-related macular degeneration (AMD), wherein the Bruch membrane was intact and no neovascularization was observed.

[0099] method

[0100] This experimental study included rabbits (Soviet Grey Chinchilla Rabbits; age: 3-4 months; weight: 2.5-3 kg). All animal handling complied with the Association for Research in Vision and Ophthalmology (ARVO) Statement on the Use of Animals in Ophthalmic and Visual Research. The study was approved by the Biomedical Ethics Committee of the Ufa Institute of Ophthalmology and was reported in accordance with the Guidelines for Animal Research: In Vivo Experiment Reporting (ARRIVE). Animals purchased from a commercial supplier (Russian Federal State-Owned Single Enterprise "Research and Production Association of Microbial Immunotherapy Agents" under the Ministry of Health of the Russian Federation, Ufa, Bashkirstan, Russia) were housed in a constant temperature (22±1℃) and light-controlled environment (lighting time from 7:00 AM to 7:00 PM) with free access to food and water.

[0101] In rabbits, posterior pole laser-induced coagulation was performed using a 500 mW argon laser coagulation point with a spot diameter of 100 μm and a duration of 0.5 seconds. This was repeated twice, with a 2-minute interval, at the same location on the posterior retina to induce laser-induced proliferation of retinal pigment epithelial (RPE) cells, serving as a rabbit mid-stage AMD model in which the Bruch's membrane remained intact and neovascularization was absent (“dry” AMD). Immediately after injection, the rabbits received an intravitreal injection of 0.10 mL containing 1 mg panitumab. The left eye received an intravitreal injection of 0.10 mL of Ringer's solution (Gematek OOO Company, Moscow, Russia). Intraocular pressure (IOP) was measured immediately after each injection. Injections and retinal laser coagulation were performed under the same setup and under general anesthesia, administered via intramuscular (biceps femoris) injections of Zoletil (15 mg / kg) (a mixture of teretamine and zolazepam; Valdepharm Co., France) and Xyla (20 mg / kg) (Interchemie Werken, De Adelaar BV, Netherlands). Topical anesthetic eye drops (0.4% oxabcaine, Inocaine) were also applied. ® ; Promed Exports Ltd., New Delhi, India (Promed Exports Ltd.). The injection is performed in the superior temporal quadrant, 3 to 4 mm from the limbus. This technique has been described in detail recently (Bikbov et al., 2020; Bikbov et al., 2022).

[0102] Panitumumab and phosphate-buffered saline (PBS) injections were repeated twice every three days. Animals were re-examined at baseline, at the time of re-injection, and three days after the final (third) injection at the end of the study by external eye examination, intraocular pressure measurement (Auto-2Ref / Keratometer HRK-7000A from Huvitz Co., Ltd., Gyeonggi Province, South Korea), fundus photography (VISUCAM 500 from Carl Zeiss Meditec AG, Jena, Germany), and optical coherence tomography (OCT) of the laser spot. Using morphometry from fundus photographs and OCT images, we measured the size of the depigmented areas and the size of the hyperpigmented areas within the depigmented areas. The final examination was performed 15 days after laser irradiation at the end of the study. Figure 4 ).

[0103] Using statistical software (SPSS for Windows, version 27.0, IBM-SPSS, Chicago, Illinois, USA), we determined the mean ± standard deviation of the primary outcome parameters: the size of the depigmented area and the size of the hyperpigmented area within the laser spot irradiation region. We applied the Wilcoxon-Mann-Whitney test for unpaired samples to compare the parameters between the study and control groups. A two-sided p-value less than 0.05 was considered statistically significant.

[0104] result

[0105] Seven rabbits were included in this study and randomly divided into a study group (n=3) and a control group (n=4). There was no significant difference in body weight between the two groups (P<0.05). There was also no significant difference in the area of ​​laser-induced depigmented patches between the two groups (1.74±1.65 mm). 2 Comparison: 1.56 ± 0.42 mm 2 P=0.29). The pigmentation area, a surrogate indicator of retinal pigment epithelium (RPE) proliferation, was significantly smaller in the study group than in the control group (0.35±0.19 mm). 2 Comparison: 0.42 ± 0.49 mm 2 (P=0.03). The ratio of pigmented area to depigmented area was significantly smaller in the study group than in the control group (0.05±0.04 vs. 0.23±0.09; P=0.03). There were no significant differences between the two groups in anterior chamber flare, cells, vitreous opacity, vitreous hemorrhage, ciliary injection, and intraocular pressure as examined by slit-lamp biometry.

[0106] Example 2 – Safety of Intravitreal Administration of EGFR Antibody for the Treatment of Patients with High Myopia and Myopic Macular Degeneration

[0107] method

[0108] This is a single-center, phase 1, open-label, multiple-dose study designed to evaluate the safety and tolerability of intravitreal panitumumab in adult patients with high myopia and myopic macular degeneration. The study was approved by the Ethics Committee of the Academic Committee of the Ufa Eye Institute (November 2, 2021) and confirmed that it adheres to the principles of the Declaration of Helsinki (Registration Trial No.: DRKS00027302). All study participants signed informed consent forms. Recruitment began in November 2021.

[0109] Inclusion criteria were age ≥50 years, axial length ≥26.5 mm, stage 4 myopic macular degeneration with patchy atrophy of the fovea as defined by the meta-analysis of the Pathological Myopia Study Group, best corrected visual acuity (BCVA) ≥1.0 logMAR (minimum resolving angle logarithm) (equivalent to 20 / 200 on the Snellen chart), and clear refractive media to allow imaging of the macula and optic disc (Ohno-Matsui et al., 2015).

[0110] Exclusion criteria included a history of vitreoretinal or retinal surgery (excluding peripheral retinal laser coagulation), intravitreal therapy with vascular endothelial growth factor (VEGF) inhibitors within three months prior to enrollment, active choroidal neovascularization, subretinal or intraretinal edema in the macular region, pregnancy, being premenopausal without reliable contraception, and known allergy to panitumumab (Vectibix). ® Intolerance or allergy to its components, participation in another clinical trial (concurrent participation or being in the washout period of the previous trial), or inability to understand and sign a written informed consent form.

[0111] The study population was divided into three dose groups with progressively increasing panitumumab doses (0.6 mg, 1.2 mg, or 1.8 mg). Between each dose phase, a comprehensive assessment of injection safety and tolerability was conducted, taking into account adverse events and the results of a detailed post-injection ophthalmological examination, including the sclera and cornea, conjunctiva at the injection site, lens, and fundus. Since no formal statistical comparisons were planned, and it was considered sufficient that at least three participants in each cohort could proceed to higher dose levels without finding dose-limiting outcomes, the inclusion of 11 patients in the study was deemed adequate.

[0112] At baseline, the patient underwent a detailed ophthalmological examination, including automated and subjective refraction (performed three times at different time points, possibly on the same day), BCVA measurement (using the standard ETDRS (Early Treatment of Diabetic Retinopathy Study) testing protocol), visual field testing (PTS-1000, Optopol Technology, Zawielce, Poland), axial length measurement using laser interferometry (AL-Scan; Nidek Co., Ltd., Japan), slit-lamp examination of the anterior and posterior segments, with particular attention to signs of intraocular inflammation, intraocular pressure measurement (Tonoref III, Nidek Co., Ltd., Japan), fundus photography, optical coherence tomography (OCT) of the macular and optic disc, evaluation of fundus autofluorescence (SS-OCT (DRI-OCT, Triton; Topcon Inc., Tokyo, Japan)), and electroretinography (“Neiro- ERG (Neirosoft, Russia). This series of examinations is repeated at each re-injection and at the end of the observation period. This complete set of examinations is repeated on day 1 and day 7, 1 month and 2 months after intravitreal injection, and at each re-injection and at the end of the observation period.

[0113] Intravitreal injection of panitumumab is performed under sterile operating room conditions and topical anesthesia, similar to the intravitreal injection of traditional anti-VEGF (vascular endothelial growth factor) drugs. The first step involves anterior chamber paracentesis to release approximately 0.1 to 0.2 mL of aqueous humor to reduce intraocular volume, preparing for the subsequent intravitreal injection of panitumumab (Vectibix). ® Create space. Collected aqueous humor samples are cryopreserved for subsequent biochemical analysis. Intravitreal injection is performed via the conjunctiva at a distance of 3.0 to 3.5 mm from the limbus in the inferotemporal quadrant. Note that the conjunctiva should be slightly moved before injection to ensure the conjunctival perforation site is not aligned with the scleral perforation site. Inject 60 µL (0.6 mg panitumumab), 120 µL (1.2 mg panitumumab), and 180 µL (1.8 mg panitumumab), respectively, towards the center of the vitreous cavity. Depending on the intraocular pressure and the location of the scleral perforation at the end of the injection, some vitreous fluid may reflux into the subconjunctival region. A manual test is performed after intravitreal injection. Apply an eye ointment containing topical antibiotics and topical steroids (levofloxacin 0.5%, dexamethasone 0.1% from Lekko, Russia; Belmedpreparaty, Belarus).

[0114] Dosage considerations:

[0115] In animal studies, 20 μg of EGFR antibody (molecular weight: 175 kDa; #2232, Cell Signaling Technology, Danvers, Massachusetts, USA) was administered to a guinea pig eye with a diameter of approximately 8 mm, equivalent to an intraocular volume of approximately 268 mm². 3 (Jiang et al., 2017; Dong et al., 2019; Dong et al., 2020; Dong et al., 2022). The corresponding intraocular concentration was 0.07 μg EGFR antibody / mm². 3 Assuming the eyeball is spherical, the axial length is 27 mm or the intraocular volume is approximately 10,306 mm. 3 For highly myopic adult eyes, this is equivalent to a 0.72 mg dose of EGFR antibody. Considering the molecular weight of panitumumab (144.3 kDa) compared to the molecular weight of the antibody used in guinea pig studies (175 kDa), an equimolar dose of panitumumab is 0.59 mg. According to the product information sheet (SmPC), the systemic dose of panitumumab (Vectibix®) for cancer treatment, administered intravenously every two weeks (6 mg panitumumab / kg body weight, approximately 420 mg panitumumab for a 70 kg patient), is approximately 1 / 700th of that dose (Douillard et al., 2013). The pharmaceutical company provides panitumumab (Vectibix®) at a concentration of 100 mg / 5 mL (i.e., 20 mg / mL), which needs to be diluted to 10 mg / mL. Therefore, when panitumumab is injected intravitreally at doses of 0.6 mg, 1.2 mg, and 1.8 mg, the injection volumes are 60 µL, 120 µL, and 180 µL, respectively.

[0116] Based on global experience with intravitreal bevacizumab (Avastin®) for the treatment of neovascular macular degeneration and other retinal diseases, intravitreal injection of 1.25 mg bevacizumab (molecular weight: 149 kDa), equivalent to an 8 nmol molecular dose, is well-tolerated both intraocularly and systemically (Martin et al., 2011). Similarly, intravitreal injection of ranibizumab (Lucentis®) (also used for the treatment of exudative macular degeneration) at a dose of 0.50 mg (molecular weight: 48 kDa) (equivalent to a 10 nmol molecular dose) is well-tolerated both intraocularly and systemically (Martin et al., 2011). 0.6 mg panitumumab (molecular weight: 144.3 kDa), equivalent to 4 nmol of panitumumab, is approximately half the molar dose of intravitreal ranibizumab. In oncology, Avastin is administered intravenously every three weeks.® (Bevacizumab), at a dose of 15 mg / kg body weight (1.2 g bevacizumab for a patient weighing 80 kg) (Hurwitz et al., 2004). Therefore, the routine intravitreal injection of 1.25 mg bevacizumab is approximately 1 / 1,000 of the intravenous dose used in oncology.

[0117] result

[0118] A total of 11 patients (2 males and 9 females) were included, with a mean age of 66.8 ± 6.3 years (median: 65.7 years; range: 57.6 to 80.2 years). The mean best corrected visual acuity was 1.62 ± 0.47 logMAR (median: 1.52 logMAR; range: 1.00 to 2.30).

[0119] All patients participating in the study self-identified as Russian (n=6), Tatar (n=3), or other ethnicities (n=2). The mean height was 1.67 ± 0.10 m (median: 1.70 m; range: 1.46–1.80 m), the mean weight was 66.6 ± 8.2 kg (median: 65.0 kg; range: 53–80 kg), and the mean body mass index was 24.0 ± 2.4 kg / m². 2 (Median: 24.7 kg / m³) 2 Range: 20.1-27.6 kg / m³ 2 The follow-up period ranged from one week to 11.9 months (mean: 5.6 ± 3.4 months; median: 6.6 months).

[0120] All patients had received at least one intravitreal injection of panitumumab. Patients received 0.6 mg (4 eyes; 1 eye, 3 eyes, 2 doses), 1.2 mg (4 eyes; 1 eye, 2 eyes, 3 doses), and 1.8 mg (3 eyes; 1 eye, 2 eyes, 2 doses) of panitumumab (Table 1). The mean interval between the first and second injections was 3.6 ± 1.8 months (median: 2.6 months; range: 2.1 to 6.3 months), and the interval between the second and third injections was 4.7 months.

[0121] No systemic adverse events occurred during treatment in any participant, although subconjunctival hemorrhage is a common side effect of intravitreal injection procedures. All subconjunctival hemorrhages resolved spontaneously. One patient reported floaters and subjective visual impairment one month after the second injection, but visual impairment was not significant. Subjective symptoms resolved within four weeks. No intraocular inflammatory reactions, such as vitreous inflammation or retinal vasculitis, occurred in any eye postoperatively. No cases of endophthalmitis or choroidal neovascularization occurred. Specifically, apart from the aforementioned subconjunctival hemorrhage, no abnormalities were detected on the ocular surface, including the cornea and conjunctiva. Intraocular pressure was below 25 mmHg when measured approximately 15 minutes after injection.

[0122] During the study period, the BCVA remained constant (P=0.08), with a mean BCVA of 1.62±0.47 logMAR at baseline and 1.28±0.59 logMAR at the end of the study (median: 1.10; range: 0.10 to 2.30). Similarly, there was no significant difference in intraocular pressure (IOP) between baseline and the end of the study (13.8±2.4 mmHg vs 14.3±2.6 mmHg; P=0.20) (Table 1). During the study period, IOP in all eyes did not exceed 21 mmHg.

[0123] Nine of the eleven patients were followed up for at least three months (mean: 6.7 ± 2.7 months; median: 6.6 months; range: 3.1–11.9 months). These nine patients received panitumumab at doses of 0.6 mg (3 eyes; 3 doses of 2 injections), 1.2 mg (4 eyes; 1 dose of 1 injection, 2 doses of 2 injections, 1 dose of 3 injections), and 1.8 mg (2 eyes; 2 doses of 2 injections) (Table 1).

[0124] Only nine patients with a follow-up period of at least 3 months were included in the analysis. During the study period, best-corrected visual acuity (BCVA) remained unchanged (P=0.15), with a mean BCVA of 1.65±0.51 logMAR at baseline (median: 1.52; range: 1.00 to 2.30) and 1.31±0.65 at the end of the study (median: 1.10; range: 0.10 to 2.30). Similarly, there was no significant difference in intraocular pressure between baseline and the end of the study (13.9±2.6 mmHg vs 14.3±2.8 mmHg; P=0.37) (Table 1). Likewise, there was no significant difference in the amplitude of the b-wave on electroretinogram between baseline and the end of the study.

[0125] In the nine eyes followed up for more than 3 months, there was no significant difference in axial length measured at baseline compared to that at the end of the study (30.73±1.03 mm vs 30.77±1.19 mm; P=0.56). The mean axial length during the study period was 0.04±0.21 mm (median: 0.07 mm; range: -0.33 to +0.30 mm) (Table 1). Univariate analysis showed that changes in axial length increased with increasing baseline axial length (standardized regression coefficient β: 0.70; P=0.04) and decreased age (β: -0.68; P=0.04), but were not statistically associated with follow-up time (β: 0.34; P=0.37). In multivariate analysis, changes in axial length were not associated with baseline axial length (P=0.20), age (P=0.12), or follow-up time (P=0.25).

[0126] There was no significant difference in the amplitude of the b wave on electroretinogram between baseline and end-of-study measurements (P=0.59).

[0127] When the systemic dose of 420 mg panitumumab is compared with the intravitreal dose of 0.6 mg panitumumab, a roughly similar ratio is obtained.

[0128] Example 3 – Effects of Epidermal Growth Factor (EGF) and Epidermal Growth Factor Receptor (EGFR) Blockers on the Proliferation of Retinal Pigment Epithelial (RPE) Cells in Vitro

[0129] like Figure 5 and Figure 6 As shown, human RPE cell line (ARPE-19 cells) was treated with different doses of EGF and EGF antibody, respectively. As described by Dong et al., 2020, cell proliferation was detected by Cell Counting Kit-8 (CCK-8), and cell migration was examined by scratch assay.

[0130] EGF promoted RPE cell proliferation in a dose-dependent manner, while EGF antibody inhibited RPE cell proliferation in a dose-dependent manner (both P < 0.001). Figure 5 Correspondingly, EGF promoted RPE cell migration in a dose-dependent manner, while EGF antibody inhibited RPE cell migration (both P < 0.001). Figure 6 ).

[0131] Example 4 – Intravitreal injection of EGFR blocker for the treatment of adult patients with stage 4 myopic macular degeneration

[0132] In a clinical, single-center, open-label, multi-dose phase 1 study, adult patients with stage 4 myopic macular degeneration received intravitreal injections of panitumumab at intervals ranging from 2.1 to 6.3 months. Specifically, the study included 11 patients (age: 66.8 ± 6.3 years) who received panitumumab at doses of 0.6 mg (4 eyes; one-dose, three-dose), 1.2 mg (4 eyes; one-dose, two-dose, one-dose), and 1.8 mg (3 eyes; one-dose, two-dose). During follow-up of these patients, a reduction appeared in the degree of intraretinal hyperpigmentation (as a surrogate indicator of migrating / proliferating RPE cells) and / or the number of intraretinal hyperreflective spheres (iHRBs) on optical coherence tomography (OCT) images.

[0133] This indicates that intravitreal EGF receptor blockers have a beneficial effect on the migration / proliferation of RPE cells in the patient's retina.

[0134] Many modifications and other embodiments of the invention set forth herein will be apparent to those skilled in the art to which this invention pertains, upon which the foregoing description and the accompanying drawings will be appreciated. Therefore, it should be understood that the invention is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terminology is used herein, it is used in a general and descriptive sense only and not for limiting purposes.

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Claims

1. A pharmaceutical composition for treating a disease in a subject involving unwanted migration, proliferation, and / or metaplasia of retinal pigment epithelial (RPE) cells, the composition comprising at least one epidermal growth factor receptor (EGFR) antagonist.

2. The pharmaceutical composition for use according to claim 1, wherein the disease is selected from non-exudative early age-related macular degeneration (AMD), non-exudative mid-stage AMD, macular neovascularization in pathological myopia, polypoid choroidal vascular disease (PCV), any other disease located in the macular region and characterized by neovascularization, originating from the choroid and extending to the subretinal space and / or to the subretinal space, proliferative vitreoretinopathy (PVR), and diseases other than those in the retinitis pigmentosa group.

3. The pharmaceutical composition for use according to claim 1 or 2, wherein the disease is not exudative AMD.

4. The pharmaceutical composition for use according to any one of claims 1 to 3, wherein the EGFR antagonist reduces EGFR signaling by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92.5%, at least 95%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.25%, at least 99.5%, or at least 99.75% compared to normal physiological levels.

5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the EGFR antagonist is selected from the group consisting of: (a) An antibody-based molecule that can bind to EGFR and reduce EGFR signaling; (b) An antibody-based molecule that can bind to EGF family members and reduce EGFR signaling; (c) Small molecule EGFR tyrosine kinase inhibitors; (d) A peptide inhibitor that can bind to EGFR and reduce EGFR signaling; (e) Small interfering RNA (siRNA) agents, said siRNA agents being able to reduce EGFR expression; (F) A CRISPR / Cas9 construct capable of knocking out the EGFR gene, which will directly act on the EGFR; and (g) mRNA molecules, said mRNA molecules encoding antibody-based molecules capable of binding to EGFR and reducing EGFR signaling, or encoding peptide inhibitors capable of binding to EGFR and reducing EGFR signaling.

6. The pharmaceutical composition for use according to any one of the preceding claims, wherein the antibody-based molecule is selected from antibody-based molecules that block the binding of the EGF family members to EGFR, antibody-based molecules that can bind to EGF family members and reduce EGFR signaling, antibody-based molecules that inhibit EGFR dimerization, or antibody-based molecules that inhibit EGFR tyrosine kinase activity.

7. The pharmaceutical composition for use according to any one of the preceding claims, wherein the antibody-based molecule is selected from antibodies, antibody fragments, and antibody mimics.

8. The pharmaceutical composition for use according to claim 7, wherein the antibody is preferably selected from panitumumab, cetuximab, nimotuzumab, mateuzumab, pertuzumab, and trastuzumab.

9. The pharmaceutical composition for use according to claim 7, wherein the antibody fragment is selected from the group consisting of: Fab fragment, F(ab')2 fragment and Fab' fragment, and / or the antibody mimic is selected from the group consisting of: single-chain variable fragment (scFv), single-domain antibody, avidin, avidin, affinity molecule, affinity protein, anticarrier protein, DARPin, monomeric antibody and peptide aptamer.

10. The pharmaceutical composition for use according to any one of claims 1 to 5, wherein the small molecule EGFR tyrosine kinase inhibitor is selected from gefitinib, erlotinib, lapatinib, icotinib, afatinib, neratinib, dacomitinib, ametinib, ometinib, osimertinib, brigatinib, vandetanib, and pyrotinib.

11. The pharmaceutical composition for use according to any one of claims 1 to 5, wherein the peptide inhibitor is selected from peptides that inhibit the binding of the EGF family members to EGFR, peptides that inhibit EGFR dimerization, or peptides that inhibit EGFR tyrosine kinase activity, and wherein the length of the peptide inhibitor is at most 80 amino acids, preferably at most 60 amino acids, more preferably 40 amino acids, and most preferably 20 amino acids.

12. The pharmaceutical composition for use according to any one of claims 1 to 5, wherein the antisense strand of such siRNA agent, i.e. the strand targeting EGFR mRNA, comprises a segment of at least 50 consecutive nucleic acids having one of the nucleic acid sequences of SEQ ID NO:

1.

13. The pharmaceutical composition for use according to any one of claims 1 to 5, wherein the mRNA molecule encodes an antibody selected from panitumumab, cetuximab, nimotuzumab, mateuzumab, pertuzumab, and trastuzumab, or a peptide inhibitor according to claim 7.

14. The pharmaceutical composition for use according to any one of the preceding claims, wherein the use comprises intravitreal administration of the pharmaceutical composition, preferably in the infratemporal quadrant of the eye, more preferably through the conjunctiva, sclera and pars plana into the vitreous cavity.

15. The pharmaceutical composition for use according to any one of the preceding claims, wherein the use comprises applying the pharmaceutical composition at a distance of 1 mm to 6 mm from the limbus, more preferably at a distance of 2 mm to 5 mm from the limbus, and most preferably at a distance of 3 mm to 4 mm behind the limbus.

16. The pharmaceutical composition for use according to any one of claims 6 to 9, wherein the use comprises administering the pharmaceutical composition having the antibody-based molecule at a total dose of 0.1 mg to 6 mg per eye, preferably in the range of 0.30 µg to 3 mg, more preferably in the range of 0.5 mg to 2 mg.

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