GAP JUNCTION MODULATORS AND THEIR USE FOR THE TREATMENT OF AGE-RELATED MACULAR DEGENERATION - Patent application

Abstract

Disclosed is the use of gap junction-dependent cellular modulators in the treatment or prevention of age-related macular degeneration (AMD), more specifically, their use for the treatment or prevention of dry AMD (d-AMD) and for preventing progression to wet or neovascular or advanced vascular AMD or geographic atrophy (GA).

Description

[Technical Field]

[0001] The present invention relates to gap junction-dependent cellular modulators and their use in the treatment or prevention of age-related macular degeneration (AMD). The present invention further relates to pharmaceutical compositions adapted for delivery of gap junction-dependent cellular modulators to the eye. [Background technology]

[0002] Age-related macular degeneration (AMD) is one of the most important causes of vision loss in the elderly. AMD is a progressive and degenerative disorder of the central retina that leads to vision loss and blindness in patients with a damaged macula. While age is a major risk factor for AMD, the prevalence and severity of the disease are likely to increase as human life expectancy increases, for example, in developed countries (Bandello et al., 2017).

[0003] AMD is a central retinal disorder with multifactorial etiological pathology, and can be classified as dry AMD (d-AMD; also known as non-neovascular AMD) or neovascular AMD (n-AMD; also known as wet AMD) depending on the presence of choroidal neovascularization (CNV), which is the hallmark of n-AMD and distinguishes it from non-neovascular dry AMD.Clinically, AMD generally presents in two forms: non-exudative "dry" form (i.e., d-AMD) and exudative neovascular "wet" (i.e., wet AMD) form.More than 80% of AMD patients exhibit d-AMD, also known as atrophic AMD, which is characterized by thinning of the macula, drusen deposits, and early leakage of the retina. An advanced form of dry AMD, termed geographic atrophy (GA), exhibits widespread loss of the retinal pigment epithelium (RPE) and photoreceptors; this severe stage of the disease can lead to irreversible blindness in up to 20% of patients. Currently, there are no effective treatments for d-AMD, as once the RPE or photoreceptors are substantially damaged or dead, it is extremely difficult to restore them, especially in the advanced stages of the disease (Bandello et al., 2017).

[0004] A subpopulation of d-AMD patients (10-20%) develop wet AMD later in the disease process, typically due to abnormal blood vessel growth in the retina. The primary ocular injection treatments for wet AMD are commercially available therapies (e.g., Eylea® and Lucentis®) that are the same as those used to treat patients with diabetic retinopathy (DR) accompanied by diabetic macular edema (DME). The abnormal angiogenesis that characterizes both proliferative DR and wet AMD is associated with dramatic increases in vascular leakage and edema that damage the retina and macula, resulting in debilitating vision loss. In patients with proliferative DR and DME, the primary anti-VEGF injection treatments, Eylea® and Lucentis®, have been shown to effectively reduce this pathological angiogenesis and improve vision, with optimal treatment responses observed in only approximately 40% of patients. Additionally, these treatments require several years of tedious injections with limited durability of the response. Therefore, there is a great unmet need for more effective treatments for patients with both wet and dry AMD that can effectively treat these diseases at earlier and less severe stages before disease progression and permanent, debilitating vision loss occurs.

[0005] Clinical conditions without obvious functional or visual loss, such as early and / or intermediate AMD, are characterized by the accumulation of drusen and / or retinal pigment epithelium (RPE) dysfunction or degeneration in the macular region. In later stages, the disease can progress to either geographic atrophy (GA) or neovascular AMD (n-AMD). Geographic atrophy (GA), secondary to d-AMD, occurs when the RPE begins to degenerate in the macular region, resulting in substantial loss of photoreceptors in the eye, rod and cone photoreceptor (PR) cell death, and eventual central vision loss and blindness (Naylor et al., 2020).

[0006] etiology The exact pathophysiological mechanisms behind AMD remain to be determined, but it is clear that AMD is a multifactorial pathology, with genetic and environmental risk factors playing a crucial role ( Bandello et al., 2017 ).

[0007] Several pathways have been identified that appear to play important roles in the pathogenesis of d-AMD, including oxidative stress, lipofuscin deposition, chronic inflammation, and choroidal blood flow insufficiency. These pathways represent possible targets for new therapies (Bandello et al., 2017).

[0008] For example, the blood-retinal barrier (BRB) is essential for establishing and maintaining an environment favorable for optimal retinal function (Naylor et al., 2020). The BRB consists of the inner blood-retinal barrier (iBRB) and the outer blood-retinal barrier (oBRB). The iBRB consists of retinal endothelial cells and pericytes, which form highly regulated cell-cell couplings through specialized protein complexes between endothelial cells and pericytes to maintain barrier function. The oBRB consists of retinal pigment epithelial (RPE) cells, which form highly regulated cell-cell couplings through specialized protein complexes between RPE cells to maintain barrier function. These specialized protein complexes, localized in the plasma membranes of retinal endothelium, pericytes, and epithelial cells, include gap junctions, tight junctions (TJs), and adherens junctions.

[0009] The RPE is a key homeostatic component of the oBRB, which separates the Bruch's membrane of the choroid from the photoreceptor layer of the neural retina. The epithelial cells that form the RPE monolayer are pigmented and highly polarized, with their apical surface facing the photoreceptors and their basal surface attached to Bruch's membrane. These epithelial cells are organized in a compact fashion using actin-dependent cell-cell junctions, particularly TJs. The oBRB acts to regulate and filter the movement of solutes and nutrients from the choroid into the subretinal space, and is important for maintaining retinal homeostasis. The dense, compact monolayer of RPE cells that forms the oBRB plays a key role in retinal development and maintenance, including secreting growth factors, limiting the entry of large toxic molecules from the bloodstream into the retina, regulating nutrient and fluid transport into and out of the retina, and maintaining photoreceptor biogenetic health. Loss of RPE integrity and dysfunction of the oBRB are associated with several ocular diseases, including AMD, DR, and DME. Disruption of any of these barriers can result in the accumulation of fluid, blood-borne proteins, and other potentially toxic solutes within the retina ( Naylor et al., 2020 ).

[0010] The RPE can be divided into apical and basolateral regions. The apical surface is in direct contact with photoreceptor outer segments (POS), and the basolateral region acts as a barrier in its interaction with the highly permeable and highly perfused choriocapillaris of the choroid (Naylor et al., 2020). The RPE exhibits three types of intercellular junctions: TJs, adherens junctions, and gap junctions. TJs form gates or barriers that regulate the paracellular diffusion of solutes and nutrients in the RPE. Adherens junctions provide strong mechanical attachment between adjacent RPE cells. Gap junctions enable intercellular communication and, by associating with TJs, support cell-cell coupling between RPE cells and within the RPE monolayer (Obert et al., 2017).

[0011] TJs, which connect adjacent RPE cells, block the transfer of plasma constituents and toxic molecules into the retina and allow the controlled flow of fluids and solutes across an osmotic gradient from the choroidal vasculature to the outer retina. The retina has the highest oxygen consumption per weight of any tissue in the body, and the BRBs (both outer and inner) are essential to facilitate this high metabolic rate by providing a regulated and distinct source of nutrients. TJs in both the iBRB and oBRB are complex, dynamic structures, and in the context of these barriers, the integrity of these TJs is important to ensure.

[0012] Specifically, the oBRB enables photoreceptor (PR) survival by supporting essential functions, including nutrient filtration and transport and phagocytosis of photoreceptor outer segments (POS). Other important functions of the RPE include absorption of off-focus and scattered light, retinal adhesion, and transport and processing of vitamin A, as well as the re-isomerization of all-trans-retinal to 11-cis-retinal, which is important for the visual cycle (Naylor et al., 2020). Thus, the RPE is essential for visual function, and impairment of any one of these functions can lead to retinal degeneration, loss of visual function, and ultimately blindness.

[0013] TJs, in conjunction with gap junctions and adherens junctions, allow a high degree of selectivity in paracellular barrier function in both the iBRB and oBRB. TJs are unique assemblies of transmembrane and peripheral cytoplasmic proteins. Transmembrane proteins, including claudins, occludin, the MARVEL (Mal and related proteins for vesicle trafficking and membrane ligation) family, and junctional adhesion molecules (JAMs), span the plasma membrane. Peripheral cytoplasmic proteins, such as zonula occludens-1 (ZO-1), -2 (ZO-2), and -3 (ZO-3), along with gap junctions and adherens junctions, anchor these transmembrane proteins to the cytoskeleton and are crucial for the initial formation of TJs and in different tissues (Naylor et al., 2020).

[0014] Connexin subunits of gap junctions, which are important for intercellular communication and contribute to cell-cell coupling, are tetraspanning transmembrane proteins. 21 connexin isoforms have been identified in the human genome, each with distinct spatial and temporal expression patterns (Obert et al., 2017). Six connexins assemble into a connexon (hemichannel), and two connexons from adjacent cells join to form a gap junction channel; gap junctions themselves are aggregations of such channels. Various heteromeric structures of different connexin proteins can assemble into connexon hemichannels, resulting in gap junctions with unique communication properties. For example, TJs contain at least 40 proteins, some of which are transmembrane and mediate cell-cell adhesion; others are intracellular scaffolding proteins that link junction components to the cytoskeleton. Adherens junctions have adhesion elements that are cadherin receptors that bridge the gap between adjacent cell membranes through homophilic interactions, and contain catenins as key scaffolding proteins that connect the mechanical link to the cytoskeleton ( Obert et al., 2017 ).

[0015] ZO-1 is a scaffolding protein common to all three junction types and anchors the junction macromolecular complex to cytoplasmic actin. ZO-1 belongs to the family of membrane-associated guanylate kinase-like proteins (MAGUKs) and incorporates three PDZ domains at its C-terminus: an SH3 domain, a GUK domain, and a proline-rich domain (Obert et al., 2017). Binding of ZO-1 to the PDZ2 domain of connexin 43 (Cx43) regulates the size and stability of gap junction channel aggregates. ZO-1 determines the cellular distribution of Cx43 and provides a control point for the dynamic switch between gap junction communication and non-junctional (hemichannel) communication at the perinexus, a specialized membrane domain at the periphery of gap junction channel aggregates. Disruption of ZO-1 in functional epithelial monolayers results in loss of barrier function and reorganization of apical actin and myosin (Obert et al., 2017).

[0016] Cx43 is the primary connexin involved in gap junction formation in retinal endothelial, pericyte, and epithelial cells, mediating intercellular communication by allowing the passage of small molecules for homeostatic processes such as growth, repair, and survival. Six connexin monomers form a normally closed, undocked hemichannel (HC). However, docking of two HCs from adjacent cells results in the formation of an open gap junction during physiological conditions, allowing the exchange of intracellular contents (Coutinho et al., 2020). However, during pathological conditions, the normally closed, undocked HCs are stimulated and open to the extracellular environment, ultimately leading to cell death. Sudden tissue reperfusion dramatically increases cell death and tissue damage because cells are unable to cope with the rapid ion influx during the open Cx43 HC state.

[0017] Hypoxia is often associated with the production of proinflammatory cytokines and the overexpression of proteins such as vascular endothelial growth factor (VEGF), connexin 43 (Cx43), and syndecan-4 (Coutinho et al., 2020). RPE expresses Cx43 as the most abundant connexin (Obert et al., 2017). Furthermore, VEGF is known to disrupt ZO-1 organization, leading to the disassembly of tight junctions and increased monolayer transparency. Additionally, in vitro studies using endothelial cells indicate that VEGF-mediated disruption of gap junction communication correlates with changes in Cx43 phosphorylation (Coutinho et al., 2020).

[0018] For example, in neovascular AMD (n-AMD), the uncontrolled growth of poorly formed blood vessels, known as choroidal neovascularization (CNV), leads to intraretinal hemorrhage, resulting in tissue ischemia (Coutinho et al., 2020). To compensate for disruptions in the blood / oxygen supply, VEGF is overexpressed by the retinal pigment epithelium (RPE) and contributes to the blood-retinal barrier (BRB) between the vascular choroid and neural retina (Coutinho et al., 2020). This VEGF overexpression perpetuates the formation of leaky blood vessels, induces more inflammatory factors in the environment, and causes RPE barrier dysfunction and cell death due to hypoxia, ultimately allowing blood vessel growth into the retina and resulting in vision loss.

[0019] In the aging eye, extracellular material is deposited in Bruch's membrane, resulting in increased thickness and decreased permeability (Naylor et al., 2020). Aging leads to the accumulation of oxidative damage and a concomitant decrease in protective mechanisms. Lipofuscin accumulation in the RPE has been suggested to act as a starting point (Naylor et al., 2020). Oxidative damage is thought to be an early trigger for age-related degenerative diseases such as AMD (Naylor et al., 2020). In AMD patients, the adaptive response of the RPE to stress becomes dysregulated, and an increased imbalance of protective and toxic factors contributes to macular damage and the development of retinal pathology (Naylor et al., 2020). The RPE's ability to absorb light energy is reduced and is thought to be a key factor in the cascade of events leading to AMD (Naylor et al., 2020).

[0020] The integrity of the outer blood-retinal barrier (oBRB) prevents choroidal vessels from invading the retina and transforming d-AMD into wet AMD. This loss of RPE-RPE attachment can induce VEGF overexpression (Naylor et al., 2020). Age-related RPE degeneration, tearing, drusen formation, or apoptosis can also cause loss of RPE-RPE attachment.

[0021] Diagnosis and Treatment As diagnostic tools, there are many tests available in clinics that monitor morphological changes in the retina, RPE, and choroid of d-AMD patients. For example, fundus autofluorescence and optical coherence tomography (OCT) are considered the most useful tools for diagnosing and following up on d-AMD degeneration, including monitoring the progression of atrophic areas. Another example is OCT angiography, a novel imaging tool that can add additional information in patients affected by d-AMD. OCT can also be used to diagnose and monitor macular edema, which is observed in wet AMD (Bandello et al., 2017).

[0022] Recently, intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) drugs have been introduced into the clinic, and new therapies have been developed targeting vascular maturation and remodeling to fundamentally reverse the course of the disease. These intravitreal agents that block VEGF have revolutionized the care of patients with wet AMD, reducing growth and leakage from CNV lesions and preventing moderate and severe vision loss (Naylor et al., 2020).

[0023] However, there are currently no approved therapies for geographic atrophy (GA), the later stage of d-AMD, because no treatments can reverse damage to the retinal pigment epithelium (RPE) or photoreceptors. For this reason, current treatment approaches in d-AMD can only prevent and / or slow the progression of existing atrophy (Bandello et al., 2017).

[0024] Cx43 hemichannel (HC) blockers have been shown to prevent vascular leakage, support the repair of leaky vessels, and promote tissue repair in many animal models (Coutinho et al., 2020). In chronic hypoxic or inflammatory conditions, Cx43 HCs are also referred to as "pathological pores" because they are involved in the activation of the inflammatory cascade via the Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome complex, which leads to the production of inflammatory cytokines and thus perpetuates the inflammatory environment (Coutinho et al., 2020). Blocking open Cx43 HCs during injury using Cx43 mimetic peptides, such as Gap27 and Pepide5, has been shown to promote cell survival and tissue repair in cardiac, spinal cord injury, and ocular models. However, one issue with these peptides is their action at the extracellular motif of Cx43, potentially affecting gap junction function, which is necessary for cell survival, when used at high concentrations and / or for long exposure periods (Coutinho et al., 2020).

[0025] Gap19 is a HC blocker derived from the second cytoplasmic loop of Cx43 and does not interfere with gap junction function. However, it must enter cells to bind to the corresponding sequence in the cytoplasmic tail of Cx43 (Coutinho et al., 2020). Due to its poor cell penetration, high concentrations have previously been used, but with limited efficacy. Therefore, cell-penetrating peptides (CPPs) are being explored to improve the transport of cargo molecules across cell membranes. For example, Xentry, a CPP derived from the X-protein of hepatitis B virus, has been shown to improve the transport of a range of molecules into cells via an endocytic mechanism by binding to syndecan-4, which is expressed on the cell surface (Coutinho et al., 2020). Because syndecan-4 is not expressed on circulating monocytes and erythrocytes, sequestration through the circulation is prevented when delivered systemically, and uptake into cells overexpressing syndecan-4 is increased. Coutinho et al. found that conjugation of Xentry to Gap19 (XG19) could increase the cellular uptake of Gap19 and block Cx43 HC-mediated injury in hypoxic cells at low peptide concentrations ( Coutinho et al., 2020 ).

[0026] Obert et al. (2017) hypothesized that targeting ZO-1 signaling maintains BRB integrity and alleviates RPE pathophysiology by stabilizing gap junctions and / or TJs. They developed a Connexin 43-based peptide mimetic, alpha connexin carboxyl terminal 1 (αCT1), to competitively block ZO-1 interaction at the PDZ2 domain, thereby inhibiting ligands that selectively bind to this domain. αCT1 differs from several other Cx43 mimetics, which mimic the Cx43 COOH-terminal PDZ-binding domain and are thought to cause attenuation of Cx43 channel activity, or target the Cx43 microtubule-binding domain and attenuate hemichannel activity. αCT1 was thought to maintain BRB integrity and alleviate RPE pathophysiology by stabilizing gap junctions and / or TJs. In experiments, RPE cell barrier dysfunction was induced in mice using laser photocoagulation, which causes choroidal neovascularization (CNV), or bright light exposure, which results in morphological damage. αCT1 treatment reduced CNV development and fluid leakage as determined by optical coherence tomography, and the damage correlated with disruption of the cellular integrity of surrounding RPE cells. Pretreatment with αCT1 via eye drops prevented light damage but typically significantly disrupted RPE cell morphology. In vitro experiments using RPE and Madin-Darby canine kidney (MDCK) monolayers showed that αCT1 stabilized tight junctions, regardless of its effect on Cx43. Taken together, αCT1-mediated stabilization of intercellular junctions was effective in improving RPE dysfunction in a model of AMD-like pathology. Obert et al. concluded that αCT1-mediated stabilization of tight junctions could serve as a novel treatment for both wet and d-AMD.

[0027] King et al. (2021) also provide an overview of different Cx43 targeting molecules, describing danegaptide. However, other peptides disclosed herein fail to effectively block hemichannels and simultaneously stabilize cell-cell coupling via gap junctions under stress conditions in AMD. Summary of the Invention [Problem to be solved by the invention]

[0028] Thus, there is a need in the art for additional treatments for age-related macular degeneration (AMD), particularly dry AMD (d-AMD), especially before it progresses to advanced geographic atrophy (GA) or wet AMD or neovascular AMD (n-AMD). [Means for solving the problem]

[0029] Generally, the present invention relates to compounds that are gap junction-dependent cell modulators, such as gap junction intercellular communication (GJIC) modulators and cell-cell coupling modulators, for use in methods for treating or preventing age-related macular degeneration (AMD), typically in human patients.AMD can be dry AMD (d-AMD), advanced GA, wet AMD, or neovascular AMD (n-AMD).In some preferred aspects, the present invention is particularly relevant to the treatment or prevention of dry AMD (d-AMD), especially just before the condition worsens to the point where the patient develops wet AMD or advanced GA.

[0030] While the role of gap junction intercellular communication (GJIC) has been widely studied in various tissues, the present invention relates to its involvement in the structural integrity of the external BRB, including the barrier integrity of the RPE, and functional impairment in AMD patients. Gap junction channels allow the passage of ions, nutrients, and other signaling molecules (up to 1 kDa) between adjacent cells. Importantly, connexin 43 (Cx43)-mediated GJIC plays a key role in regulating cell growth, vascular tone, barrier integrity, and cell death in the retina, and is therefore an essential factor in maintaining retinal metabolism and vascular homeostasis. Cx43 is abundantly expressed in the retina, suggesting substantial gap junction-dependent coupling, particularly in the structures of both the iBRB and oBRB. However, under conditions of oxidative stress, ischemic stress, hypoxia, high glucose, and diabetes, Cx43-dependent cell-cell coupling is downregulated and functionally impaired, resulting in impaired gap junction activity in RPE and retinal vascular cells. Furthermore, oxidative stress, ischemic stress, hypoxia, high glucose, and diabetes-induced Cx43 downregulation, as well as decreased GJIC activity, play a key role in the increased RPE and vascular cell death in the retinas of diabetic mice, rats, and humans, which exhibit a significantly increased number of acellular capillaries and pericyte loss.

[0031] Furthermore, it is known that blood vessels, including those in the eye, are composed of two interacting cell types (Song et al., 2005). Thus, endothelial cells form the inner layer of the blood vessel wall, while pericytes surround the surface of blood vessels, supporting and maintaining vascular structure and barrier integrity and helping to prevent leakage from blood vessels. Therefore, pericytes are functionally significant because, if blood vessels lose pericytes, they bleed and become highly dilated, causing fluid leakage to exceed the fluid absorption capacity of the RPE, resulting in conditions such as vascular leakage and edema, which can lead to loss of vision and eventual blindness. In addition to the effects of gap junction modulators on retinal endothelial cells and RPE, this application demonstrates that RPE barrier integrity can be protected from oxidative and high-glucose stress, as well as swelling of the outer retina, in rats, and that a diabetic retinopathy model can be significantly improved by administering gap junction modulators.

[0032] Furthermore, the combination of maintaining structural cell-cell coupling and hemichannel closure provides unique and improved advantages of the compounds described herein, such as danegaptide, over the previously disclosed therapeutic benefits of molecules that primarily target hemichannel closure (e.g., peptide 5 and tonabersat).

[0033] Without wishing to be bound by theory, the inventors have observed in the BRB model of AMD and DME that the MoA (mode of action) for the effects of the compounds described herein, such as danegaptide, is primarily due to the compound's therapeutic / protective effect on gap junction (linked to TJ)-dependent cell-cell coupling in the presence of high glucose, reactive oxygen species (RoS), and metabolic / bioenergenic (i.e., oxidative phosphorylation (OxPos), tricarboxylic acid cycle (TCA), and glycolysis) and other cellular stressors. The compounds described herein can also prevent the pathological opening of gap junction hemichannels in AMD, which can cause cell loss and cell-cell uncoupling. This MoA may be due to the increased expression of small ions (e.g., Ca) in certain tissues where cell-cell coordination is crucial (e.g., cardiac cardiomyocytes). 2+Beyond its role in classical gap junction intercellular communication (GJIC) signaling involving VEGF-1 and other small molecules, it is believed to have an additional important structural role in maintaining BRB integrity. This MoA is upstream of major anti-VEGF therapies and acts at the core pathology of DR, DME, and AMD. Therefore, compounds described herein, such as danegaptide, may be useful in preventing the onset of such diseases and in treating such diseases once they have developed.

[0034] The compounds described herein are gap junction modulators. Danegaptide described herein is the compound (2S,4R)-1-(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid (Butera et al., 2009).

[0035] Thus, in a first aspect, the present invention provides a compound of formula (I):

[0036] [ka]

[0037] or a pharmaceutically acceptable salt or hydrate thereof, wherein the method comprises administering to a subject a therapeutically effective amount of the compound; or a pharmaceutically acceptable salt thereof.

[0038] Preferably, the compound is (2S,4R)-1-(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid; or a pharmaceutically acceptable salt or hydrate thereof.

[0039] In some embodiments, the compound or a pharmaceutically acceptable salt or hydrate thereof is administered locally to the eye. In some embodiments, the compound or a pharmaceutically acceptable salt or hydrate thereof is administered locally in the eye via intravitreal injection.

[0040] In some embodiments, the compound or its pharmaceutically acceptable salt or hydrate is administered systemically.In some embodiments, the compound or its pharmaceutically acceptable salt or hydrate is administered systemically via oral, subcutaneous, transdermal or intravenous administration.Preferably, the compound or its pharmaceutically acceptable salt or hydrate is administered systemically via oral administration.

[0041] In some embodiments, the method is for the prevention of AMD in a human subject. In some embodiments, the method is for the prevention of the progression of AMD in a human subject. Preferably, the compound is for use in a method for preventing the progression of dry AMD to wet AMD in a human subject. Preferably, the method is for preventing the progression of moderate dry AMD to an advanced form of dry AMD in a human subject. Preferably, AMD is characterized as early dry AMD. Preferably, AMD is characterized as intermediate dry AMD. Preferably, AMD is characterized as advanced dry AMD. Preferably, advanced dry AMD is geographic atrophy (GA). These embodiments are advantageous because there is no known treatment for dry AMD.

[0042] In some embodiments, the AMD is characterized as wet, neovascular, or advanced vascular AMD. In some embodiments, the method is for preventing the development of choroidal neovascularization in a human subject.

[0043] In some embodiments, the patient has a comorbid condition.For example, in some embodiments, the patient has chronically high blood glucose levels, including patients who are in glycemic control.In some embodiments, the patient also has type 1 diabetes or type 2 diabetes.

[0044] In some embodiments, the patient also has hypertension or chronically high blood pressure, including patients who achieve normal blood pressure control. In some embodiments, the patient achieves normal blood pressure control through hypertension treatment, glycemic control, or cholesterol reduction. In some embodiments, the patient has high cholesterol.

[0045] In some embodiments, the patient also has diabetic retinopathy or diabetic macular edema. In some embodiments, the patient has retinal vein occlusion (RVO) eye disease. In some embodiments, the patient has glaucoma with or without pathological intraocular pressure. In some embodiments, the patient has uveitis or other forms of inflammatory eye disease.

[0046] In some aspects, the patient has or has had drusen deposits or protein exudates in the eye. In some embodiments, the compounds inhibit dysfunction of the RPE (retinal pigment epithelium) or outer blood-retinal barrier (oBRB), including loss of barrier integrity between RPE cells and loss of RPE cells. In some embodiments, the compounds inhibit stress-induced dysfunction of the retina or choroid layer in the eye. In some embodiments, the compounds inhibit loss of photoreceptors in the eye, such as the retina. In some embodiments, the compounds inhibit retinal vascular leakage, including vascular leakage that leads to macular edema. In some embodiments, the compounds improve fluid extraction from retinal tissue through the retinal pigment epithelium. In some embodiments, the compounds inhibit death or loss of retinal endothelial cells, choroidal endothelial cells, pericytes, or epithelial cells.

[0047] In some embodiments, the compound inhibits pathological hemichannel opening and / or ATP release, e.g., connexin 43-mediated opening and / or ATP release. The pathological effects of hemichannel opening and ATP release (or other such small, sub-1 kDa signaling molecules) can be both autocrine and paracrine.

[0048] In some embodiments, a concentration of about 50 nM to about 150 nM, e.g., about 100 nM, of the administered compound, or a pharmaceutically acceptable salt, hydrate, or formulation thereof, e.g., oral, systemic, and sustained-release compound formulations, is achieved in the ocular microenvironment.

[0049] In some embodiments, the compound is administered to the patient once or twice daily, hi some embodiments, the compound is administered to the patient up to four times daily. In a further aspect, the present invention provides pharmaceutical compositions adapted for delivery of compounds to the eye.

[0050] The compounds described herein can enable cells to protect themselves during age-related macular degeneration (AMD). Without wishing to be bound by any particular theory, the inventors speculate that the compounds may have a stabilizing effect on cells, reduce the tendency of mitochondria to become leaky and / or reduce the tendency of cells to develop leaky outer cell membranes, and / or improve the intercellular and / or structural coupling of cells. The compounds can provide better intercellular coupling, allowing cells to share available energy (ATP) and enable intercellular signaling, including calcium signaling. The compounds can also reduce pathological stress-induced ATP leakage into the extracellular compartment, which leads to inflammation. The compounds can also functionally improve the integrity of the barrier between iBRB and oBRB, and / or improve the fluid absorption capacity of RPE.

[0051] In a further aspect, the present invention provides a compound of formula (I):

[0052] [ka]

[0053] or a pharmaceutically acceptable salt or hydrate thereof, wherein the method comprises administering to a subject a therapeutically effective amount of the compound, or a pharmaceutically acceptable salt or hydrate thereof.

[0054] In a further aspect, the invention provides a method of treating or preventing age-related macular degeneration (AMD) in a human subject, comprising administering to the human subject a therapeutically effective amount of a compound, or a pharmaceutically acceptable salt or hydrate thereof; The compound has the formula (I):

[0055] [ka]

[0056] or a pharmaceutically acceptable salt or hydrate thereof. In a further aspect, the present invention provides a pharmaceutical composition comprising a compound described herein for use in a method described herein, the pharmaceutical composition comprising the compound and a pharmaceutically acceptable excipient. Preferably, the compound is for systemic administration, oral administration, subcutaneous administration, transdermal administration, nasal spray, eye drops, or via contact lenses in an eye drop formulation, via contact lenses, via nasal spray, or via injection, for example, intravitreal injection.

[0057] Aspects of the present invention will now be described by way of example, and not by way of limitation, with reference to the accompanying drawings. However, various further aspects and embodiments of the present invention will be apparent to those skilled in the art in light of the present disclosure.

[0058] "And / or," as used herein, shall be a specific disclosure of each of two specific features or components with or without the other. For example, "A and / or B" shall be a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually set forth herein.

[0059] Unless the context indicates otherwise, the feature descriptions and definitions outlined above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described. [Brief explanation of the drawings]

[0060] [Figure 1]Danegaptide protects against DR and AMD-mimicking injury mediated by decreased permeability in stressed human retinal pigment epithelial cell monolayers. Human RPE (retinal pigment epithelial) cell line, ARPE-19, grown to confluent cell monolayers showed increased permeability at 48 hours following sublethal oxidative injury with tBHP (tert-butyl hydroperoxide) in high glucose conditions, which was prevented by danegaptide treatment using two different permeability markers (6-CF and RhoB). Data are presented as mean ± SEM with each data point representing a separate experimental condition and analyzed using one-way ANOVA with Holm-Sidak multiple comparisons test; Papp (apparent permeability coefficient), 6-CF (6-carboxyfluorescein; top graph A), RhoB (rhodamine B; bottom graph B), *p<0.05, **p<0.01, ***p<0.001, n=6–8. [Figure 2] Danegaptide protects against pseudo-DR and AMD damage mediated through improved tight junction organization ratio (TiJOR) and improved cell-cell coupling between human retinal pigment epithelial cells in stressed monolayers. Following oxidative injury with sublethal tBHP (tert-butyl hydroperoxide) in high glucose, 48 hours after treatment, the human RPE (retinal pigment epithelial) cell line ARPE-19, grown in confluent monolayers, exhibited elevated cell-cell uncoupling and tight junction (TJ) disorganization measured by immunocytochemistry using anti-zonula occludens-1 (ZO-1) immunostaining (lower image B; left = control; middle = high glucose and 200 μM tBHP; right = high glucose, 200 μM tBHP, and 100 nM danegaptide), and tight junction organization ratio (TiJOR; upper bar graph A). This decline was prevented by danegaptide treatment. Data are presented as mean ± SEM with data points representing separate TiJOR analyses and analyzed using one-way ANOVA with Holm-Sidak multiple comparisons test; TiJOR (tight connective tissue index); HG (hyperglycemia); tBHP (tert-butyl hydroperoxide); DGP (danegaptide), ***p<0.001, n=13–16. [Figure 3] A. Blood glucose levels (mmol / l) and B. Blood glycated hemoglobin (HbA1c) levels (mmol / mol) during in vivo studies. Data are presented as mean ± SD from 15–18 rats per group. Data were analyzed by two-way ANOVA (blood glucose, p<0.001; and blood HbA1c, p<0.001) followed by Tukey's post-hoc test versus the untreated group. ***p<0.001. [Figure 4] Cataract scores during in vivo studies. Cataracts were scored from 0 (normal lens) to 3 (severe opacity involving more than 75% of the lens). Data are presented as mean ± SEM from 15-18 rats per group. Data were analyzed by two-way ANOVA (p<0.001) followed by Tukey's post-hoc test against the untreated group. ***p<0.001. [Figure 5A] Danegaptide protects against outer retinal thickening in rats with DR after streptozotocin (STZ) induction. Danegaptide prevented thickening in the outer blood-retinal barrier, and spectral-domain optical coherence tomography (SD-OCT) imaging was used to measure the outer plexiform and nuclear layers, inner and outer segments, and RPE / choroid layers. SD-OCT retinal scans were segmented, and the thickness of the outer retinal layers was analyzed using a convolutional neural network algorithm. A. STZ induction in Brown Norway rats resulted in a statistically significant increase in outer retinal thickness in STZ + vehicle (n = 21) retinas 9 weeks after induction when compared with untreated (n = 16; t test p < 0.01). B. Danegaptide treatment with localized 100 nM targeted ocular injections weekly for 3 weeks prevented this increase in outer retinal thickness (p<0.05), whereas anti-VEGF and anti-VEGF + danegaptide IVT injection treatments had no effect. Data are expressed as mean ± SEM. *p<0.05; **p<0.01. C. Representative SD-OCT scans of outer retinal thickness from left and right eyes 9 weeks after STZ induction. [Figure 5B]Danegaptide protects against outer retinal thickening in rats with DR after streptozotocin (STZ) induction. Danegaptide prevented thickening in the outer blood-retinal barrier, and spectral-domain optical coherence tomography (SD-OCT) imaging was used to measure the outer plexiform and nuclear layers, inner and outer segments, and RPE / choroid layers. SD-OCT retinal scans were segmented, and the thickness of the outer retinal layers was analyzed using a convolutional neural network algorithm. A. STZ induction in Brown Norway rats resulted in a statistically significant increase in outer retinal thickness in STZ + vehicle (n = 21) retinas 9 weeks after induction when compared with untreated (n = 16; t test p < 0.01). B. Danegaptide treatment with localized 100 nM targeted ocular injections weekly for 3 weeks prevented this increase in outer retinal thickness (p<0.05), whereas anti-VEGF and anti-VEGF + danegaptide IVT injection treatments had no effect. Data are expressed as mean ± SEM. *p<0.05; **p<0.01. C. Representative SD-OCT scans of outer retinal thickness from left and right eyes 9 weeks after STZ induction. [Figure 5C]Danegaptide protects against outer retinal thickening in rats with DR after streptozotocin (STZ) induction. Danegaptide prevented thickening in the outer blood-retinal barrier, and spectral-domain optical coherence tomography (SD-OCT) imaging was used to measure the outer plexiform and nuclear layers, inner and outer segments, and RPE / choroid layers. SD-OCT retinal scans were segmented, and the thickness of the outer retinal layers was analyzed using a convolutional neural network algorithm. A. STZ induction in Brown Norway rats resulted in a statistically significant increase in outer retinal thickness in STZ + vehicle (n = 21) retinas 9 weeks after induction when compared with untreated (n = 16; t test p < 0.01). B. Danegaptide treatment with localized 100 nM targeted ocular injections weekly for 3 weeks prevented this increase in outer retinal thickness (p<0.05), whereas anti-VEGF and anti-VEGF + danegaptide IVT injection treatments had no effect. Data are expressed as mean ± SEM. *p<0.05; **p<0.01. C. Representative SD-OCT scans of outer retinal thickness from left and right eyes 9 weeks after STZ induction. [Figure 6] Schematic diagram showing the regions used for regional analysis of retinal thickness. ST: superior temporal; SN, superior nasal; IT, inferior temporal; IN, inferior nasal. [Figure 7A] Danegaptide protects against outer retinal thickening throughout the retina. A-D. Outer retinal thickness was statistically significantly increased in STZ + vehicle-treated rats compared to untreated rats in all regions: superior temporal (A), superonasal (B), inferotemporal (C), and inferonasal (D). Data were analyzed by unpaired t-test. E-H. Danegaptide prevented outer retinal thickening in all regions: superior temporal (E), superonasal (F), inferotemporal (G), and inferonasal (H), as determined by one-way ANOVA or Kruskal-Wallis (p<0.01) followed by Tukey or Dunn's multiple comparisons test. Data are presented as mean ± SEM from n = 12–18 rats per group. *p<0.05; **p<0.01; ***p<0.001. [Figure 7B]Danegaptide protects against outer retinal thickening throughout the retina. A-D. Outer retinal thickness was statistically significantly increased in STZ + vehicle-treated rats compared to untreated rats in all regions: superior temporal (A), superonasal (B), inferotemporal (C), and inferonasal (D). Data were analyzed by unpaired t-test. E-H. Danegaptide prevented outer retinal thickening in all regions: superior temporal (E), superonasal (F), inferotemporal (G), and inferonasal (H), as determined by one-way ANOVA or Kruskal-Wallis (p<0.01) followed by Tukey or Dunn's multiple comparisons test. Data are presented as mean ± SEM from n = 12–18 rats per group. *p<0.05; **p<0.01; ***p<0.001. [Figure 7C] Danegaptide protects against outer retinal thickening throughout the retina. A-D. Outer retinal thickness was statistically significantly increased in STZ + vehicle-treated rats compared to untreated rats in all regions: superior temporal (A), superonasal (B), inferotemporal (C), and inferonasal (D). Data were analyzed by unpaired t-test. E-H. Danegaptide prevented outer retinal thickening in all regions: superior temporal (E), superonasal (F), inferotemporal (G), and inferonasal (H), as determined by one-way ANOVA or Kruskal-Wallis (p<0.01) followed by Tukey or Dunn's multiple comparisons test. Data are presented as mean ± SEM from n = 12–18 rats per group. *p<0.05; **p<0.01; ***p<0.001. [Figure 7D]Danegaptide protects against outer retinal thickening throughout the retina. A-D. Outer retinal thickness was statistically significantly increased in STZ + vehicle-treated rats compared to untreated rats in all regions: superior temporal (A), superonasal (B), inferotemporal (C), and inferonasal (D). Data were analyzed by unpaired t-test. E-H. Danegaptide prevented outer retinal thickening in all regions: superior temporal (E), superonasal (F), inferotemporal (G), and inferonasal (H), as determined by one-way ANOVA or Kruskal-Wallis (p<0.01) followed by Tukey or Dunn's multiple comparisons test. Data are presented as mean ± SEM from n = 12–18 rats per group. *p<0.05; **p<0.01; ***p<0.001. [Figure 7E] Danegaptide protects against outer retinal thickening throughout the retina. A-D. Outer retinal thickness was statistically significantly increased in STZ + vehicle-treated rats compared to untreated rats in all regions: superior temporal (A), superonasal (B), inferotemporal (C), and inferonasal (D). Data were analyzed by unpaired t-test. E-H. Danegaptide prevented outer retinal thickening in all regions: superior temporal (E), superonasal (F), inferotemporal (G), and inferonasal (H), as determined by one-way ANOVA or Kruskal-Wallis (p<0.01) followed by Tukey or Dunn's multiple comparisons test. Data are presented as mean ± SEM from n = 12–18 rats per group. *p<0.05; **p<0.01; ***p<0.001. [Figure 7F]Danegaptide protects against outer retinal thickening throughout the retina. A-D. Outer retinal thickness was statistically significantly increased in STZ + vehicle-treated rats compared to untreated rats in all regions: superior temporal (A), superonasal (B), inferotemporal (C), and inferonasal (D). Data were analyzed by unpaired t-test. E-H. Danegaptide prevented outer retinal thickening in all regions: superior temporal (E), superonasal (F), inferotemporal (G), and inferonasal (H), as determined by one-way ANOVA or Kruskal-Wallis (p<0.01) followed by Tukey or Dunn's multiple comparisons test. Data are presented as mean ± SEM from n = 12–18 rats per group. *p<0.05; **p<0.01; ***p<0.001. [Figure 7G] Danegaptide protects against outer retinal thickening throughout the retina. A-D. Outer retinal thickness was statistically significantly increased in STZ + vehicle-treated rats compared to untreated rats in all regions: superior temporal (A), superonasal (B), inferotemporal (C), and inferonasal (D). Data were analyzed by unpaired t-test. E-H. Danegaptide prevented outer retinal thickening in all regions: superior temporal (E), superonasal (F), inferotemporal (G), and inferonasal (H), as determined by one-way ANOVA or Kruskal-Wallis (p<0.01) followed by Tukey or Dunn's multiple comparisons test. Data are presented as mean ± SEM from n = 12–18 rats per group. *p<0.05; **p<0.01; ***p<0.001. [Figure 7H]Danegaptide protects against outer retinal thickening throughout the retina. A-D. Outer retinal thickness was statistically significantly increased in STZ + vehicle-treated rats compared to untreated rats in all regions: superior temporal (A), superonasal (B), inferotemporal (C), and inferonasal (D). Data were analyzed by unpaired t-test. E-H. Danegaptide prevented outer retinal thickening in all regions: superior temporal (E), superonasal (F), inferotemporal (G), and inferonasal (H), as determined by one-way ANOVA or Kruskal-Wallis (p<0.01) followed by Tukey or Dunn's multiple comparisons test. Data are presented as mean ± SEM from n = 12–18 rats per group. *p<0.05; **p<0.01; ***p<0.001. [Figure 8A] Regional analysis of inner retinal thickness. STZ-induced animals exhibited thinner retinas compared with untreated animals (A–D), but no statistically significant differences were identified between treatment groups (E–H). Superotemporal (A and E), superonasal (B and F), inferotemporal (C and G), and inferonasal (D and H). Data are presented as mean ± SD from 12–18 rats per group. Untreated and STZ + vehicle data were analyzed by unpaired t-test. *p<0.05; **p<0.01; ***p<0.001. STZ-treated group data were analyzed by one-way ANOVA or Kruskal-Wallis ANOVA; p values ​​were greater than 0.05 in all regions. [Figure 8B] Regional analysis of inner retinal thickness. STZ-induced animals exhibited thinner retinas compared with untreated animals (A–D), but no statistically significant differences were identified between treatment groups (E–H). Superotemporal (A and E), superonasal (B and F), inferotemporal (C and G), and inferonasal (D and H). Data are presented as mean ± SD from 12–18 rats per group. Untreated and STZ + vehicle data were analyzed by unpaired t-test. *p<0.05; **p<0.01; ***p<0.001. STZ-treated group data were analyzed by one-way ANOVA or Kruskal-Wallis ANOVA; p values ​​were greater than 0.05 in all regions. [Figure 8C]Regional analysis of inner retinal thickness. STZ-induced animals exhibited thinner retinas compared with untreated animals (A–D), but no statistically significant differences were identified between treatment groups (E–H). Superotemporal (A and E), superonasal (B and F), inferotemporal (C and G), and inferonasal (D and H). Data are presented as mean ± SD from 12–18 rats per group. Untreated and STZ + vehicle data were analyzed by unpaired t-test. *p<0.05; **p<0.01; ***p<0.001. STZ-treated group data were analyzed by one-way ANOVA or Kruskal-Wallis ANOVA; p values ​​were greater than 0.05 in all regions. [Figure 8D] Regional analysis of inner retinal thickness. STZ-induced animals exhibited thinner retinas compared with untreated animals (A–D), but no statistically significant differences were identified between treatment groups (E–H). Superotemporal (A and E), superonasal (B and F), inferotemporal (C and G), and inferonasal (D and H). Data are presented as mean ± SD from 12–18 rats per group. Untreated and STZ + vehicle data were analyzed by unpaired t-test. *p<0.05; **p<0.01; ***p<0.001. STZ-treated group data were analyzed by one-way ANOVA or Kruskal-Wallis ANOVA; p values ​​were greater than 0.05 in all regions. [Figure 8E] Regional analysis of inner retinal thickness. STZ-induced animals exhibited thinner retinas compared with untreated animals (A–D), but no statistically significant differences were identified between treatment groups (E–H). Superotemporal (A and E), superonasal (B and F), inferotemporal (C and G), and inferonasal (D and H). Data are presented as mean ± SD from 12–18 rats per group. Untreated and STZ + vehicle data were analyzed by unpaired t-test. *p<0.05; **p<0.01; ***p<0.001. STZ-treated group data were analyzed by one-way ANOVA or Kruskal-Wallis ANOVA; p values ​​were greater than 0.05 in all regions. [Figure 8F]Regional analysis of inner retinal thickness. STZ-induced animals exhibited thinner retinas compared with untreated animals (A–D), but no statistically significant differences were identified between treatment groups (E–H). Superotemporal (A and E), superonasal (B and F), inferotemporal (C and G), and inferonasal (D and H). Data are presented as mean ± SD from 12–18 rats per group. Untreated and STZ + vehicle data were analyzed by unpaired t-test. *p<0.05; **p<0.01; ***p<0.001. STZ-treated group data were analyzed by one-way ANOVA or Kruskal-Wallis ANOVA; p values ​​were greater than 0.05 in all regions. [Figure 8G] Regional analysis of inner retinal thickness. STZ-induced animals exhibited thinner retinas compared with untreated animals (A–D), but no statistically significant differences were identified between treatment groups (E–H). Superotemporal (A and E), superonasal (B and F), inferotemporal (C and G), and inferonasal (D and H). Data are presented as mean ± SD from 12–18 rats per group. Untreated and STZ + vehicle data were analyzed by unpaired t-test. *p<0.05; **p<0.01; ***p<0.001. STZ-treated group data were analyzed by one-way ANOVA or Kruskal-Wallis ANOVA; p values ​​were greater than 0.05 in all regions. [Figure 8H] Regional analysis of inner retinal thickness. STZ-induced animals exhibited thinner retinas compared with untreated animals (A–D), but no statistically significant differences were identified between treatment groups (E–H). Superotemporal (A and E), superonasal (B and F), inferotemporal (C and G), and inferonasal (D and H). Data are presented as mean ± SD from 12–18 rats per group. Untreated and STZ + vehicle data were analyzed by unpaired t-test. *p<0.05; **p<0.01; ***p<0.001. STZ-treated group data were analyzed by one-way ANOVA or Kruskal-Wallis ANOVA; p values ​​were greater than 0.05 in all regions. [Figure 9]Danegaptide and anti-VEGF treatment attenuate Evans blue (EB) extravasation in the diabetic retina. A. Induction of hyperglycemia with STZ resulted in a significant 6.28-fold increase in EB extravasation 9 weeks after induction (100 ± 13.5% for untreated vs. 628 ± 212.5% ​​for STZ + vehicle, n = 10-11, t-test p < 0.01). B. Anti-VEGF (p < 0.01), danegaptide (p < 0.05), and combined treatment (p < 0.05) resulted in a statistically significant reduction in EB extravasation of 82.3%, 68.9%, and 65.6%, respectively. Data from the STZ-induced group passed normality using the D'Agostino and Pearson tests and were analyzed by one-way ANOVA (p < 0.01) followed by Holm-Sidak multiple comparisons test. Data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. [Figure 10] Danegaptide prevents pericyte loss 9 weeks after STZ induction. A. Induction of DR with STZ resulted in a statistically significant increase in pericyte ghosts after 9 weeks (t-test, p<0.05, n=45-67). B. The number of pericyte ghosts in the STZ group was compared using one-way ANOVA (p<0.001) followed by Dunnett's multiple comparison test. Both AF564 (p<0.01, n=45) and danegaptide (p<0.001, n=58) resulted in a statistically significant decrease in pericyte ghosts. Combined treatment with AF564 and danegaptide had no effect on the presence of pericyte ghosts (p=0.11; n=61). Data are expressed as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. [Figure 11-1] Representative images from trypsin digestion showing protection from pericyte loss by danegaptide. PAS staining was used to assess vascular abnormalities, including acellular capillaries (arrows) and pericyte ghosts (arrowheads). Pericytes are not shown. Scale bar = 100 μm. [Figure 11-2]Representative images from trypsin digestion showing protection from pericyte loss by danegaptide. PAS staining was used to assess vascular abnormalities, including acellular capillaries (arrows) and pericyte ghosts (arrowheads). Pericytes are not shown. Scale bar = 100 μm. [Figure 11-3] Representative images from trypsin digestion showing protection from pericyte loss by danegaptide. PAS staining was used to assess vascular abnormalities, including acellular capillaries (arrows) and pericyte ghosts (arrowheads). Pericytes are not shown. Scale bar = 100 μm. DETAILED DESCRIPTION OF THE INVENTION

[0061] definition Unless otherwise specified, the following definitions are provided for specific terms. Standard three-letter and one-letter abbreviations for natural amino acids are used in the present specification and claims. As used herein, the term "peptide" refers to a chain of two or more amino acid moieties (amino acid residues) linked by peptide bonds. In general, a peptide may contain one or more naturally occurring amino acids and / or one or more non-naturally occurring amino acids.

[0062] In the present context, the term "naturally occurring amino acid" refers to one of the following 20 amino acids: Ala (A), Cys (C), Ser (S), Thr (T), Asp (D), Glu (E), Asn (N), Gln (Q), His (H), Arg (R), Lys (K), Ile (I), Leu (L), Met (M), Val (V), Phe (F), Tyr (Y), Trp (W), Gly (G), and Pro (P). In naturally occurring peptide molecules, these amino acids (except Gly, which lacks a chiral center) generally occur in the form of L-amino acid residues, although suitable compounds for use in the present invention include peptides comprising D-amino acid residues.

[0063] The three letter abbreviations for amino acids are as used in the art: Hyp refers to 4-hydroxyproline. Compounds for use in the present invention may contain two or more asymmetric atoms (also called chiral centers), giving rise to the possibility of the occurrence of diastereomers. Compounds suitable for use in the present invention include such diastereomers.

[0064] As used herein, the term "vascular cells" includes endothelial cells and pericytes, including retinal endothelial cells and retinochoroidal endothelial cells. Age-related macular degeneration (AMD) AMD is a degenerative disorder of the central retina (central vision is processed in an area known as the macula). The macula is important for straight ahead vision, which is used for reading, recognizing faces, and driving. AMD can be classified as dry AMD (d-AMD; also known as non-neovascular AMD or non-exudative AMD) or neovascular AMD (n-AMD; also known as wet AMD or exudative AMD).

[0065] Typically, wet AMD usually begins as the dry form of AMD. The typical stages of AMD are as follows: 1. Early dry AMD, which does not contain pigmentary changes or abnormalities but is characterized by medium drusen (63-125 microns or μm) (Ferris et al., 2013). This stage is also referred to as early AMD.

[0066] 2. Intermediate dry AMD, characterized by either large drusen (greater than 125 microns or μm) containing at least medium drusen or pigmentary changes / abnormalities. This stage is also referred to as moderate AMD.

[0067] 3. Advanced dry AMD, characterized by lesions associated with the development of tissue atrophy and / or progression of atrophic areas in or near the macula. This stage of d-AMD includes geographic atrophy (GA), characterized by atrophy of the outer retinal tissue, retinal pigment epithelium, photoreceptors, and / or choriocapillaris. This stage is also referred to as late AMD. Geographic atrophy (GA) is a chronic, progressive degeneration of the macula as part of late-stage age-related macular degeneration (AMD). The disease is characterized by localized, well-defined atrophy of the outer retinal tissue, retinal pigment epithelium, and choriocapillaris.

[0068] 4. Wet or neovascular or advanced vascular AMD. Characterized by choroidal neovascularization (CNV). Neovascularization is the growth or angiogenesis of new blood vessels. These new blood vessels are immature, fragile, and easily leak fluid and blood. They can also create scar tissue, resulting in decreased vision or retinal detachment. Wet AMD refers to the component of leaky blood vessels, but excessive vascular leakage can lead to the formation of retinal edema. New blood vessel formation can occur with or without substantial edema, and edema can occur without substantial neovascularization or angiogenesis. These stages are also called exudative AMD and late AMD.

[0069] Because the present invention targets the role of gap junctions, in preferred aspects, the present invention relates to therapies that can treat patients with AMD, i.e., stages 1-4 above, more preferably stages 1-3 above (i.e., dry AMD), or prevent further progression of AMD to more advanced or later stages. The present invention also relates to preventative therapies for patients with other vascular stressors, such as diabetes, who are at risk of developing AMD.

[0070] Diabetic patients at risk of developing AMD include those with all types of diabetes (type 1, type 2, and gestational diabetes). The risk of developing AMD increases the long-term risk that people have diabetes. Therefore, the present invention can be used to treat all of these patient types. Although the pathophysiological connection between diabetes and AMD is not fully understood, the high glucose stress observed in stressed diabetes and more leaky blood vessels may lead to increased drusen deposition, ROS, and hypoxia, and VEGF is an important pathological growth factor common to both diseases.

[0071] In addition, the medical use and method of the present invention can be utilized for preventive use in patients who are trying to achieve glycemic control.In this treatment, when patients with chronically high blood glucose levels are trying to achieve glycemic control, ocular tissues can be used to high glucose utilization rate, and stress can be applied to achieve normal glucose levels.These patients are at risk of experiencing the worsening of AMD, and can be protected by controlling blood glucose.Therefore, the present invention can be used as a protective treatment for patients who are trying to achieve glycemic control.

[0072] Diabetic retinopathy (DR) involves changes to retinal blood vessels that can cause bleeding or fluid leakage, distorting vision. Diabetic retinopathy is the most common cause of vision loss among people with diabetes and is a leading cause of blindness in working-age adults. Diabetic macular edema is a result of diabetic retinopathy causing swelling in the macular region of the retina.

[0073] Diabetic macular edema (DME) is the accumulation of fluid (edema) in the macula. DME is the most common cause of vision loss in people with diabetic retinopathy. Approximately half of all people with diabetic retinopathy will develop DME at some point in their lifetime. DME can occur at any stage of the disease, although it becomes more likely to occur as diabetic retinopathy worsens.

[0074] Patients with diabetic retinopathy and / or diabetic macular edema are also at risk of developing AMD.As mentioned above, diabetic patients have a high risk of developing AMD, and this risk is even higher when they also develop diabetic retinopathy or diabetic macular edema.In both conditions, the internal and external blood-retinal barriers are attacked by pathological stress conditions and become leaky.Therefore, the present invention can be used to treat or prevent patients with diabetic retinopathy or diabetic macular edema.

[0075] Retinal vein occlusion (RVO) is a blockage in the arteries or veins (called blockage or stroke) that control blood flow to and from the retina. This typically occurs when a blood clot blocks the retinal vein. Patients with retinal vein occlusion (RVO) are also at risk for developing AMD. The ischemic condition resulting from RVO is known to drive pathologically high VEGF levels, and high VEGF levels in the eye can stimulate or worsen the development of AMD, including neovascular AMD and wet AMD. The extremely high VEGF levels observed in the eyes of RVO patients are known to cause significant vascular leakage and edema formation. Attenuating vascular leakage and strengthening both the iBRB and oBRB using gap junction modulating agents as disclosed herein is beneficial. Therefore, the present invention can be used as a treatment or preventative measure in patients with retinal vein occlusion (RVO) eye disease.

[0076] Glaucoma is a common eye condition in which the optic nerve connecting the eye to the brain is damaged. It is usually caused by fluid accumulation in the anterior part of the eye, increasing intraocular pressure. However, glaucoma can occur without increased intraocular pressure. If undiagnosed and untreated early, glaucoma can result in vision loss. Patients with glaucoma, both with and without pathological intraocular pressure, are at risk for developing AMD. The retinal pigment epithelium (RPE) plays a key role in removing fluid from the retina and the eye in general. When fluid drainage from the anterior part of the eye is reduced, as is often observed in glaucoma, removal of fluid from the posterior chamber via the RPE becomes increasingly important to avoid elevated intraocular pressure. Therefore, therapeutic interventions involving fluid removal from the eye, such as the compounds described herein, including danegaptide, that can stabilize RPE cell connections and improve their function, may be beneficial for glaucoma patients and those at risk of developing AMD. In addition, it is observed that nerve tissue and optic nerve are stressed in patients with glaucoma and patients at risk of developing nerve tissue atrophy over time.In addition, therapeutic interventions that can protect RPE and external blood-retinal barrier (oBRB), such as the compounds described herein, including danegaptide, help RPE deliver functional and metabolic support to nerve tissue, including photoreceptors, rods and cones.Therefore, the present invention can be used as treatment or prevention in patients with glaucoma, with or without pathological intraocular pressure.

[0077] Inflammatory eye diseases, such as uveitis, occur in response to inflammation, allergies, autoimmune diseases, irritation, injury, or trauma to the eye, eyelid, or surrounding tissues. Uveitis is inflammation that occurs in the middle layer of tissue in the wall of the eye (uvea). Patients with uveitis or other forms of inflammatory eye disease are at risk for developing AMD. The inflammatory state observed in uveitis is known to stimulate pathologically elevated levels of several cytokines and growth factors, including VEGF. These signaling molecules can further stress the RPE, reducing barrier integrity and the function of the RPE and therefore the outer retinal layers. In addition, pathological stress on the RPE can lead to Cx43-based hemichannel opening and excessive ATP leakage into the extracellular matrix. ATP is known to be involved in the stimulation of purinergic receptors, again playing a key role in inflammation and angiogenesis (Clapp et al., 2019). Therefore, therapeutic interventions that can stabilize RPE cell intercellular coupling and prevent the pathological opening of Cx43-based hemichannels, such as the compounds described herein, including danegaptide, are beneficial for patients with uveitis and those at risk of developing AMD. Thus, the present invention can be used to treat or prevent patients with uveitis or other forms of inflammatory eye disease.

[0078] Drusen are a hallmark of macular degeneration. Drusen are tiny yellow or white accumulations of extracellular material that accumulate between the eye's Bruch's membrane and the retinal pigment epithelium (RPE). Drusen contain proteins and lipids (naturally occurring molecules, including fats). Patients with drusen deposits or other protein exudates in the eye are at risk for developing AMD. Drusen accumulation reduces the efficiency of oxygen, nutrient, and waste exchange between the RPE and the retina-choroidal plexus. Given that the retina and photoreceptors have very high energy expenditures and require effective oxygenation, any decline in the RPE's ability to support photoreceptors and neural tissue can result in stress on the retinal tissue and increase the risk of developing AMD. Therefore, therapeutic interventions that can stabilize RPE and improve metabolic function and the exchange of oxygen, nutrients and waste products between RPE and the retina-choroidal plexus, such as the compounds described herein, including danegaptide, are beneficial for patients with drusen or at risk of developing drusen accumulation, such as the drusen accumulation observed in AMD.Therefore, the present invention can be used to treat or prevent patients who also have drusen deposits or protein exudates in the eye or at risk of developing drusen accumulation.

[0079] Various routes of administration can be utilized in connection with the method of the present invention, including but not limited to intraocular injection, systemic administration, oral administration, subcutaneous administration, transdermal administration, nasal spray, eye drops, or contact lenses.In some embodiments, the compound or its pharmaceutically acceptable salt or hydrate is a drug formulation for its therapeutic use that is administered locally to the eye (for example, intravitreal injection) or systemically (for example, oral, subcutaneous, transdermal or intravenous administration).Preferably, the compound or its pharmaceutically acceptable salt or hydrate is administered systemically via oral administration.

[0080] In this regard, it may be desirable to achieve a concentration of the compound (or a pharmaceutically acceptable salt or hydrate thereof) administered to a subject's plasma in the range of 50 nM to 5 μM. The in vitro studies provided herein demonstrated that optimal effects on RPE cells were observed when a target concentration of 100 nM of the gap junction modulator compound was achieved in the microenvironment of RPE cell cultures. Additionally, in vivo studies performed in diabetic rats support the finding that intravenous (IVT) injection of danegaptide at a dose selected to achieve 100 nM in retinal tissue also confers protective effects on the outer retinal layers, including the RPE. For example, these therapeutic and protective effects may be achieved when a plasma concentration of 50 to 150 nM is targeted by an osmotic pump or by local injection into the eye that targets a higher concentration in the vitreous, allowing for a concentration gradient across retinal blood vessels. These data suggest effective doses to be administered to achieve 50 to 150 nM in the microenvironment of retinal tissue, including the RPE.

[0081] Compounds suitable for use according to the present invention An example of a compound that is particularly suitable for use according to the invention is 1-(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid, such as its (2S,4R) diastereomer [i.e., (2S,4R)-1-(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid], or a pharmaceutically acceptable salt or hydrate thereof. An example of an alternative name for this compound is (2S,4R)-1-(2-aminoacetyl)-4-benzamidopyrrolidine-2-carboxylic acid.

[0082] Other diastereomers of the latter compound (ie, the 2S4S, 2R4R, 2S4R or 2R4S diastereomers) may also be of value for use in the context of the present invention. This particular form of the compound may also be called danegaptide.

[0083] Pharmaceutically acceptable salts of danegaptide include danegaptide hydrochloride. The compounds described herein are gap junction-dependent cell modulators. In some embodiments, the compounds can inhibit RPE barrier dysfunction, including the loss of barrier integrity between RPE (retinal pigment epithelium) cells and the loss of RPE cells. In some embodiments, the compounds can inhibit retinal or choroidal layer dysfunction in the eye. In some embodiments, the compounds can inhibit the loss of photoreceptors in the retina. In some embodiments, the compounds can inhibit retinal vascular leakage, including vascular leakage that leads to macular edema. In some embodiments, the compounds can improve barrier integrity and RPE function, thereby improving fluid reabsorption from retinal tissue, thereby helping to treat and alleviate macular edema. In some embodiments, the compounds can inhibit the death or loss of retinal endothelial cells, choroidal endothelial cells, pericytes, or epithelial cells, including RPE cells. In some embodiments, the compounds can inhibit pathological hemichannel opening and / or ATP release, for example, connexin 43-mediated hemichannel opening and / or ATP release. The pathological effects of hemichannel opening and / or ATP release (or other such small signaling molecules) can be both autocrine and paracrine.

[0084] For example, Squires et al. (2021) demonstrated that danegaptide can reduce ATP leakage from renal epithelial cells. Mugisho et al. (2019) demonstrated that peptide 5, a connexin 43 hemichannel blocker, reduced inflammatory cytokine and ATP release, which may be useful for treating inflammatory diseases such as AMD and DR. Gonzalez-Casanova et al. (2021) suggested that connexin hemichannel blockers or drugs that improve gap junction intercellular communication (GJIC) may be useful for treating DR. Subauste (2019) studied the CD40-ATP-P2X7 receptor pathway and linked it to inflammatory responses and endothelial cell death. Clapp et al. (2019) suggested that the protective role of P2X7 receptor blockade in DR and / or AMD may be due to inhibition of inflammatory cytokine release and VEGF release.

[0085] A review by King et al. (2021) disclosed different Cx43-targeting molecules, but these molecules did not block hemichannels and simultaneously did not stabilize gap junctions under stress conditions in AMD. In contrast, the compounds disclosed herein, including danegaptide, have the competitive advantage of not only blocking hemichannels but also effectively stabilizing gap junction-dependent intercellular coupling under stress conditions in AMD.

[0086] In addition to the compounds described above, further compounds that may be suitable for use in the context of the present invention include certain other gap junction modulating compounds, such as the antiarrhythmic peptides AAP (Aonuma et al., 1980), AAP10 (Dhein et al., 1994; Muller et al., 1997), HP5 (disclosed in U.S. Pat. No. 4,775,743) and other antiarrhythmic peptides, as disclosed in WO02 / 077017, WO2007 / 078990, or WO2018 / 202865.

[0087] It will be understood that the compounds described herein can be used in combination, for example, two or more gap junction modulator compounds can be administered in the methods described herein either simultaneously or sequentially.

[0088] pharmaceutically acceptable salts Pharmaceutically acceptable salts of compounds suitable for use according to the present invention that contain an acidic moiety can be formed using organic or inorganic bases. Suitable salts formed with bases include metal salts, such as alkali metal or alkaline earth metal salts, such as sodium, potassium, or magnesium salts; ammonia salts; and organic amine salts, such as those formed with morpholine, thiomorpholine, piperidine, pyrrolidine, mono-, di-, or tri-lower alkylamines (e.g., ethyl-tert-butyl-, diethyl, diisopropyl, triethyl, tributyl, or dimethylpropylamine), or mono-, di-, or trihydroxy-lower alkylamines (e.g., mono-, di-, or triethanolamine). Internal salts can also be formed. When a compound suitable for use according to the present invention contains a basic moiety (e.g., as in the case of 1-(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid and its described diastereomers), salts can be formed using organic or inorganic acids. For example, salts can be formed from the following acids: acetic acid, propionic acid, lactic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, malonic acid, mandelic acid, malic acid, phthalic acid, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, methanesulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, or camphorsulfonic acid. Other known pharmaceutically acceptable acids can also be used. As previously mentioned (see above), the preferred salt form of the (2S,4R) diastereomer of 1-(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid is the hydrochloride monohydrate.

[0089] The present teachings can also be extended to the use of prodrugs of the compounds disclosed herein that are suitable for use according to the present invention. As used herein, "prodrug" refers to a moiety that, when administered to a mammalian subject, particularly a human subject, produces, generates, or releases a compound of the disclosed type. Prodrugs can be prepared by modifying functional groups present in the compound in such a way that the modification is cleaved from the parent compound either by routine manipulation or in vivo. Examples of prodrugs include compounds disclosed herein that contain one or more molecular moieties attached (bound) to a hydroxy, amino, sulfhydryl, or carboxy group of the compound, which, when administered to a treated subject, are cleaved in vivo to form the free hydroxy, amino, sulfhydryl, or carboxy group, respectively. Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol and amine functional groups in the compounds disclosed herein for use according to the present invention. Examples of preferred prodrugs include oxazolidinone or imidazolidinone prodrugs. Ester prodrugs are esters of lower alcohols, e.g., C 1~6 The preparation and use of prodrugs are discussed in T. Higuchi and V. Stella, "Prodrugs as Novel Delivery Systems," ACS Symposium Series, Vol. 14, and Bioreversible Carriers in Drug Design, edited by Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0090] Pharmaceutical Composition The compounds, or pharmaceutically acceptable salts or hydrates thereof, utilized by the present invention can be administered in the form of suitable pharmaceutical compositions, and can be administered alone or in combination by any acceptable method known in the art. Pharmaceutical compositions relevant in this context can include the compounds disclosed herein for use according to the present invention in combination with one or more pharmaceutically acceptable carriers, diluents, vehicles, or excipients. Generally, the pharmaceutical compositions used by the present invention can be adapted for administration of the compounds via eye drops, contact lenses, nasal sprays, intravitreal or systemic administration.

[0091] Useful formulations may include formulations that provide sustained release of the compounds of the present teachings. These may be particularly useful for subsequent administrations (after the first administration). The compositions are preferably in the form of liquid formulations, and methods for their preparation are generally described in "Remington's Pharmaceutical Sciences," 17th ed., Alfonso R. Gennaro (ed.), Mark Publishing Company, Easton, PA, USA, 1985. Such compositions generally contain an effective amount of one or more active compounds of the present teachings together with a suitable carrier to provide a dosage in a form compatible with the selected administration route. Preferably, the carrier is in the form of a vehicle, diluent, buffer, tonicity adjuster, preservative, inhibitor, and / or stabilizer. The excipients that make up the carrier should be compatible with the active pharmaceutical ingredient and preferably be capable of stabilizing the compound without being harmful to the subject to be treated.

[0092] Repository or sustained-release formulations can be used to deliver a therapeutically effective amount of the preparation to the bloodstream for extended periods of time or even days following administration of the compound or composition, for example, by transdermal injection or deposition. Formulations suitable for sustained release can include biodegradable polymers, such as L-lactic acid, D-lactic acid, DL-lactic acid, glycolide, glycolic acid, and isomers thereof. Similarly, the carrier or diluent can include any sustained-release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

[0093] Other sustained release formulations may include, but are not limited to, liposomes, microspheres, emulsions or micelles and formulations comprising at least one of the compounds disclosed herein in combination with a liquid stabilizer.

[0094] The administration of the compound according to the present invention (or its pharmaceutical salt or hydrate) can be carried out in a single unit dosage form (for example, in the form of a bolus), or as a continuous therapy in the form of multiple doses over time. Alternatively, a continuous infusion system or a slow-release depot preparation can be utilized. Two or more compounds (or pharmaceutical compositions thereof) for use according to the present invention can be co-administered simultaneously or sequentially in any order. In addition, the compounds and compositions can be administered in a similar manner for the purpose of prevention, for example, when a diabetic patient or a patient with drusen deposits is considered to be at risk of developing AMD or the worsening of AMD or macular edema associated with AMD. Ultimately, the best dosing regimen is determined by the attending physician for each patient individually.

[0095] therapeutic use Conditions that can be treated or prevented according to the present invention using the compounds specified herein include, inter alia, age-related macular degeneration (AMD) in human subjects.

[0096] According to the present invention, one or more of the compounds, or a pharmaceutically acceptable salt or hydrate thereof (e.g., in the form of a suitable pharmaceutical composition), may be administered in a therapeutically effective amount to an individual in need thereof.

[0097] As used herein, "therapeutically effective amount" refers to an amount that can alleviate the symptoms of a given neurovascular condition or pathology in the eye, and preferably can partially or completely normalize the physiological response in a subject with the condition or pathology.The alleviation of symptoms or normalization of physiological response can be determined using methods known in the art, and may vary depending on the given condition or pathology.The effective amount is determined by those skilled in the art, taking into account factors such as the efficacy of the drug, the age and constitution of the patient, body weight, and the pharmacokinetic profile of the drug, and generally, the drug is prescribed to each patient or group of patients.

[0098] The effective amount of the compound can be at least about 10 μg / kg body weight / day, for example, at least about 100 μg / kg body weight / day, at least about 300 μg / kg body weight / day, and at least about 1000 μg / kg body weight / day.On the other hand, the effective amount of the compound or dimer can be at most about 100 mg / kg body weight / day, for example, at most about 50 mg / kg body weight / day and at most about 10 mg / kg body weight / day.The effective amount of the compound is expected to be about 100 μg / kg body weight / day, about 300 μg / kg body weight / day, or about 1000 μg / kg body weight.

[0099] Dose findings provided by experiments performed in the in vitro system described herein (cell-coupled assay / SLDT) indicated that concentrations of 50-150 nM were optimal in the microenvironment, and preliminary in vivo studies showed that 1000 nM injections were better than 200 nM injections when targeting 100 nM in the ocular microenvironment.

[0100] In some embodiments, the compound is administered to a patient once or twice daily, sometimes referred to as QD (quaque die) or BID (bis in die), respectively. In some embodiments where the compound is administered to a patient twice daily (BID), the dosage is about 75 mg / kg per administration. In some embodiments, the compound is administered to a patient four times daily, sometimes referred to as QID (quater in die). In some embodiments where the compound is administered to a patient four times daily (QID), the dosage is about 75 mg / kg to about 125 mg / kg per administration.

[0101] Experimental Example Gap junction and hemichannel modulator compounds The compounds (peptides) for use according to the invention may be suitably synthesized by solid-phase or solution-phase synthesis. In this context, reference may be made, for example, to Fields et al., "Principles and practice of solid-phase peptide synthesis", Synthetic peptides (2002, 2nd edition).

[0102] With respect to the preparation of 1-(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid, for example its (2S,4R) diastereomer, suitable methods for its synthesis and purification are described in WO2007 / 078990, where the (2S,4R) isomer is designated "Compound 2" (WO2007 / 078990 is incorporated by reference in its entirety).

[0103] An example of a useful salt form of the (2S,4R) diastereomer is the hydrochloride monohydrate, the preparation of which is described in WO2008 / 079266 and is also referred to herein as Compound X (WO2008 / 079266 is incorporated by reference in its entirety).

[0104] In vitro studies - Figures 1 and 2 Cell lines and cell culture Human retinal pigment epithelial cells (ARPE-19, CRL-2302™, American Tissue Type Collection, ATTC, Manassas, VA) were used. Cells were passaged and maintained according to the supplier's instructions. Briefly, cells were cultured in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium containing 1.2 g / L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES, and 0.5 mM sodium pyruvate, and fetal bovine serum at a final concentration of 10%, at 37°C in a 5% CO2 / 95% humidified atmosphere. Penicillin / streptomycin was added to the cell culture medium at a final concentration of 100 U / ml.

[0105] Cells were maintained in T25 and T75 tissue culture flasks (TPP Techno Plastic Products AG, Trasadingen, Switzerland). For experiments, cells were seeded onto Transwell inserts (150,000–260,000 cells / well; 12 mm Transwell® with 0.4 μm pore size polyester membrane inserts, Corning, Corning, NY) or multichamber slides (100,000 cells per well; Nunc® Lab-Tek IT 8-well chamber slides, Thermo Fisher Scientific, Waltham, MA).

[0106] Permeability Test For permeability experiments, ARPE-19 cells were grown to confluency in Transwell inserts for 48–72 hours. Cells were then pretreated with danegaptide (DGP; 100 nM; dissolved in saline) or vehicle control for 24 hours. Cells were then challenged with a combination of sublethal oxidative stress (250 μM tert-butylhydroperoxide; Millipore Sigma, St. Louis, MO) and hyperglycemia (30 mM glucose; Millipore Sigma, St. Louis, MO) in the continued presence of 100 nM DGP or vehicle for 48 hours.

[0107] To assess permeability, the apical-to-basolateral movement of low-permeability and high-permeability dyes, 6-carboxyfluorescein (6-CF; Thermo Fisher Scientific, Waltham, MA) and rhodamine B (RhoB; Thermo Fisher Scientific, Waltham, MA), respectively, were quantified. To this end, dyes diluted in Hank's Balanced Salt Solution (HBSS, Corning, Corning, NY) were added to the apical compartment of the cells (upper insert, donor chamber; 6-CF: 100 μM, RhoB: 50 μM). At regular intervals (10, 20, 30, 45, 60, 90, and 120 min), samples (100 μl) from the basolateral compartment (lower well, receiver chamber) were collected. The sampled volume was replaced with fresh buffer.

[0108] Cumulative concentrations of 6-CF and RhoB were calculated using standard curves (0–10 μM) established using a microplate reader (Cytation 5, Agilent, Santa Clara, CA), taking into account the volume removed and replaced with medium in the final calculation. 6-CF was quantified using excitation 490 nm / emission 520 nm, and RhoB was quantified using excitation 533 nm / emission 627 nm. See Figure 1.

[0109] Quantification of tight junction tissue For immunocytochemistry, ARPE-19 cells were grown to confluency in chamber slides for 72 hours. Cells were then pretreated with danegaptide (DGP; 100 nM) or vehicle control for 24 hours. Cells were then challenged with a combination of sublethal oxidative stress (250 μM tert-butylhydroperoxide; Millipore Sigma, St. Louis, MO) and hyperglycemia (30 mM glucose; Millipore Sigma, St. Louis, MO) in the continued presence of 100 nM DGP or vehicle for 48 hours.

[0110] Cells were fixed with 4% paraformaldehyde for 15 minutes, then blocked and permeabilized with blocking solution (10% normal goat serum, 1% bovine serum albumin, and 0.5% Triton X-100 in 0.1 M phosphate buffer, pH 7.4). Cells were labeled with anti-ZO-1 antibody (mouse anti-human 70-1, 1 pg / ml, catalog number 339100, Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) in antibody solution (3% normal goat serum, 1% bovine serum albumin, 0.5% Triton X-100, and 0.05% sodium azide in 0.1 M phosphate buffer, pH 7.4) for 2 hours at room temperature. After washing three times with phosphate-buffered saline, cells were labeled with a fluorescently conjugated goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody (AlexaFluor® 488, 1:2,000 dilution) at room temperature for 1 hour. Nuclei were co-stained with 0.1 pg / ml of 4',6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma-Aldrich) and washed with phosphate-buffered saline. Chamber slides were coverslipped using Aqua / Poly-Mount (Polysciences, Inc., Warrington, PA). Images were obtained using a Leica SPE confocal microscope (Leica Microsystems, Buffalo Grove, IL). See Figure 2.

[0111] Data analysis Apparent permeability coefficient (P app , cm / s) was calculated by the following equation (Ziniauskaite et al., 2019):

[0112]

number

[0113] (In the formula: ΔQr / Δt = the slope of the linear region of the cumulative amount of test compound in the receiver chamber versus time plot C d = initial donor concentration of test compound A=surface area).

[0114] See Figure 1. The tight junction tissue ratio (TiJOR) was calculated from confocal images as previously described ( Terryn et al., 2013 ), see Figure 2 .

[0115] Data were plotted and analyzed using Prism 9 (GraphPad, Inc., La Jolla, CA). Data are presented as mean ± SEM, with each data point representing a separate experimental condition. Data were analyzed using one-way ANOVA, group analysis, along with the Holm-Sidak multiple comparison test.

[0116] In vivo test - Figures 3 to 11 animal All animals were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, EC Directive 2010 / 63 / EU of the European Parliament and of the Government for the Protection of Animals Used for Scientific Purposes and using protocols approved and monitored by the Finnish Board of Animal Experiments (animal license number ESAVI-9520-2020).

[0117] For these animal studies, Brown Norway rats (strain: BN / Crl rats; Charles River Laboratories, Germany), 9–10 weeks old at the time of challenge, were housed in individually ventilated cages with nutrient enrichment, poplar bedding, nesting material (Populus tremula, Tapvei®, Estonia OU), and red polycarbonate tubes (Datesand group), in a dimly lit, controlled environment (lights on from 7 AM to 7 PM) at constant temperature (22 ± 1 °C), relative humidity (50 ± 10%), and with free access to food (Rat / Mouse Maintenance V1534-000, ssniff Spezialdiaten GmbH) and tap water. Experiments began after one week of isolation and acclimation in the animal facility.

[0118] The body weight of all animals was monitored at baseline and twice weekly throughout the study period. Animal welfare checks were performed daily, and if an animal was identified with a welfare problem, medical care was provided under the guidance of the attending veterinarian in collaboration with the study director.

[0119] To prevent excessive weight loss after induction, rats were fed a wet diet (regular diet hydrated with tap water) and a supportive diet (Solid Drink-Diet Bio Cup, Triple A Trading) daily. Animals with weight loss greater than 15 percent received additional hydration with daily subcutaneous (sc) injections (Ringer-Lactat Animalcare, Ecuphar NV). Paraphimosis and urinary retention were treated with lidocaine hydrochloride monohydrate (2%, Xylocain, AstraZeneca). Minor skin infections occurred and were treated with fusidic acid (1%, Isathal®, Dechr).

[0120] No other medical treatments were administered to the animals during the study. A total of 111 rats were used in the study. From these, 28 rats were sacrificed prior to the endpoint due to various welfare issues, primarily severe weight loss (>25% from baseline), as commonly observed in STZ-treated diabetic Brown Norway rats. Animals were randomized into treatment groups based on glucose levels and ocular health. At the end of the study (week 9), a total of 83 rats were included in the study, distributed across five treatment groups: Group 1: untreated for tissue sampling (n = 18); Group 2: STZ + vehicle (n = 15); Group 3: STZ + AF564 (or anti-VEGF) (n = 17); Group 4: STZ + danegaptide (n = 17); Group 5: STZ + AF564 (or anti-VEGF) + danegaptide (n=16).

[0121] Anesthesia and reversal For all procedures, rats were anesthetized with a subcutaneous injection of a mixture containing ketamine (30 mg / kg; Ketaminol vet 50 mg / ml, Intervet) and medetomidine hydrochloride (0.2 mg / kg; Cepetor vet 1 mg / ml; CP-Pharma Handelsgesellschaft mbH). Anesthesia was reversed with an α2-antagonist for medetomidine, atipamezole (1.0 mg / kg; Revertor™ 5 mg / mL; CP-Pharma Handelsgesellschaft mbH). All anesthetic reagents were diluted to working stocks in a dosable saline solution. Lactated Ringer's solution was administered during anesthesia to prevent dehydration.

[0122] Diabetic retinopathy induction and blood glucose measurement Before induction, animals were weighed and blood glucose was measured using a rapid meter (AlphaTRAK 2; Zoetis). Rats were fasted for 4 hours before induction.

[0123] Diabetes was induced in Brown Norway rats (9–10 weeks old) with a single dose of streptozotocin (STZ; 65 mg / kg, Sigma) in 10 mM sodium citrate buffer, pH 4.5. After STZ injection, rats were kept in their cages containing ad libitum food and a 10% (w / v) sucrose solution in tap water for 48 hours. The following morning, 5% glucose (1 ml) was injected intraperitoneally or subcutaneously into all induced rats. Four days after the first STZ injection, the success of the induction was monitored by measuring blood glucose levels from the saphenous vein. Diabetes induction was considered successful if blood glucose levels measured by a rapidometer were 16 mmol / L (288 mg / dL) or higher. Animals that were not diabetic based on this criterion received a second STZ injection 96 hours after the first STZ injection.

[0124] Selected rats were monitored for blood glucose levels and animal health for 6 weeks and then treated with intravitreal (IVT) injections of vehicle or test compound once a week for 3 weeks for a total of 3×IVT ocular injections per eye per animal.

[0125] Blood glucose levels were monitored weekly throughout the follow-up period. If blood glucose measurements fell within the range of the rapidometer (>41.7 mmol / L), plasma samples were collected and analyzed using a colorimetric assay (Rat Glucose Assay Kit, Crystal Chem). See Figure 3A.

[0126] Plasma samples for glycated hemoglobin (HbA1c) measurement were collected 5 and 9 weeks after induction of DR. HbA1c was measured using a commercially available turbidimetric immunoassay (Konelab Prime 60i; Thermo Scientific). See Figure 3B.

[0127] Preparation of test solution Vehicle Solution A vehicle solution containing D-mannitol and trisodium citrate dihydrate was prepared as follows:

[0128] [Table 1]

[0129] The sterile solution was aliquoted into 15 ml Falcon tubes and two further samples of 0.5 ml were stored in standard amber tubes as holdout samples. All aliquots were stored protected from light at +4°C.

[0130] Danegaptide solution A 0.3 mg / ml stock solution of danegaptide was prepared weekly from the first day of dosing as follows:

[0131] [Table 2]

[0132] Two 0.5 ml aliquots were stored in standard amber tubes as holdout samples. The remainder of the solution was used to prepare dosing solutions that week (to be administered within 2 days). The stock solution and aliquots were stored at +4°C protected from light.

[0133] The 0.3 mg / ml danegaptide stock solution is diluted to 9 μg / ml as follows:

[0134] [Table 3]

[0135] Two aliquots of 0.5 ml were stored in standard amber tubes as retention samples. The aliquots were stored protected from light at +4° C. The 9 μg / ml danegaptide solution was further used to prepare a 0.3 μg / ml working solution as follows, and the remainder was discarded.

[0136] [Table 4]

[0137] Two aliquots of 0.5 ml were stored in standard amber tubes as retention samples. The aliquots were stored protected from light at +4° C. The remainder from the working solution was discarded daily. The 9 μg / ml danegaptide solution was further used to prepare a 0.6 μg / ml solution that was used to prepare treatments for the groups "STZ+AF564+danegaptide" (see, e.g., Figures 3B, 4, 5C, 9B, and 10B), "STZ+anti-VEGF+danegaptide" (see, e.g., Figure 5B), or "STZ+AF564+Dgp" (see, e.g., Figures 7E-H, and 8E-H).

[0138] [Table 5]

[0139] Two aliquots of 0.5 ml were stored in standard amber tubes as retention samples. The aliquots were stored protected from light at +4° C. The remainder from the working solution was discarded daily. Rat VEGF antibody (AF564) solution For administration of anti-VEGF treatment ("STZ + AF564" in Figures 3B, 4, 5C, 7E-H, 8E-H, 9B, and 10B, or "STZ + anti-VEGF" in Figure 5B), rat anti-VEGF antibody (AF564; R&D Systems) was dissolved in vehicle solution at a concentration of 0.675 mg / ml. Dosing solutions were prepared by dissolving one vial of 100 μg of antibody in 150 μl of vehicle solution.

[0140] For co-administration of danegaptide and anti-VEGF antibody ("STZ + AF564 + danegaptide" in Figures 3B, 4, 5C, 9B, and 10B; "STZ + anti-VEGF + danegaptide" in Figure 5B; or "STZ + AF564 + Dgp" in Figures 7E-H and 8E-H), one vial of antibody (100 μg) was dissolved in 75 μl of vehicle to create a 2× AF564 stock solution, which was then combined with an equal volume of 2× working stock of danegaptide (0.6 μg / ml) to achieve final concentrations of 0.675 mg / ml AF564 antibody and 0.3 μg / ml danegaptide in the dosing solution.

[0141] Five microliters of the dosing solution was delivered to rat eyes via intravitreal injection. Rat eyes contain approximately 20-25 μl of vitreous humor. Therefore, delivery of 5 μl of test article results in a dilution to a final concentration of anti-VEGF antibody in the eye of 0.135-0.1125 mg / ml, similar to the human clinical dose of 0.125 mg / ml, according to Filek et al. (2019).

[0142] Intravitreal (IVT) treatment For IVT treatment administration, animals were anesthetized and placed under a stereoscope (Leica Microsystems). A drop of iodine (Minims povidone-iodine 5%, Bausch & Lomb) was applied to the cornea and allowed to spread evenly. A small incision in the choroid exposing the vitreous chamber was made using a 30G needle near the limbus. Compounds were injected into the intravitreal space using a microinjector with an attached 33G needle (Hamilton Bonaduz AG, Bonaduz). The test solution was injected into the intravitreal space for 10 seconds and left in place for another 30 seconds before removing the needle to prevent backflow of the compound. Chloramphenicol ointment was applied after injection (Oftan Chlora, Santen Oy). Different treatments were administered three times, at weeks 6, 7, and 8 after STZ induction, in a volume of 5 μl for both intravitreal injections.

[0143] In vivo imaging Following the development of diabetic retinopathy and associated vascular leakage, spectral-domain optical coherence tomography (SD-OCT) was performed 9 weeks after DR induction. In vivo imaging analysis was performed on a subset of study animals (9–14 animals per treatment group). In addition to in vivo imaging, diabetic cataract development in all animals was assessed at 6, 7, 8, and 9 weeks after induction.

[0144] Spectral Domain Optical Coherence Tomography (SD-OCT) Anesthetized rats were imaged with SD-OCT (Envisu R2200. Bioptigen Inc. / Leica Microsystems) retinal scans at 9 weeks, shown in Figure 5C. The scanned area was a 2.4 × 2.4 mm section of the retina centered around the optic nerve. 2 Each scan consisted of 100 B-scans, each of which consisted of 1000 A-scans. SD-OCT was performed on both eyes at 9 weeks after DR induction, before sacrifice and sampling.

[0145] Retinal thickness at 9 weeks was analyzed using a convolutional neural network based on U-Net construction using a transfer learning approach. The resulting segmentation mask was used to measure layer thickness in each pixel-wide column, i.e., a-scan, resulting in 1,000 measurement points per layer per b-scan. Only measurement points that passed a set of automated quality control criteria were used for calculations. Thickness results were expressed as SD-OCT measurements of the inner retina, including the ganglion cell layer, inner plexiform and inner nuclear layers; and the outer retina, including the outer plexiform layer, outer nuclear layer, inner and outer segments, and RPE / choroid. Retinal thickness measurements, such as changes in retinal thickness measurements, were obtained by calculating the average of all measurements per scan or by detailed analysis of the superior temporal, superior nasal, inferior temporal, and inferior nasal regions (see Figure 6 for a schematic diagram showing these regions of the eye). See Figures 5, 7, and 8 for SD-OCT results.

[0146] Animal sacrifice and tissue collection No samples were collected from animals found dead or sacrificed prior to the study endpoint.

[0147] Samples for retinal vasculature analysis At the endpoint, six rats per treatment group were sacrificed by overdose anesthesia and perfused transcardially with 0.9% NaCl solution (10 ml / min for 3 min, 120S / DV Manual Control Variable Speed ​​Pump, Watson-Marlow Pumps). Eyes (n=12) were enucleated, marked for orientation, and placed on 4% PFS before retinal flat mounting.

[0148] Evans blue retinal leakage measurement Prior to sacrifice, five rats per treatment group were intravenously injected via the tail vein with 4% Evans Blue solution (100 μl / 100 g). Two hours later, plasma samples were collected, and the animals were transcardially perfused with saline solution at a flow rate of 10 ml / min for 30 minutes (120 S / DV Manual Control Variable Speed ​​Pump). Eyes (n=10) were enucleated, and retinas were dissected, rapidly frozen, and stored at -80°C until analysis of Evans Blue extravasation. See Figure 9 for results.

[0149] Tissue processing PAS histological staining and acellular capillary and pericyte analysis Retinal flat mounts were trypsinized to isolate the retinal vasculature and stained using periodic acid Schiff (PAS). Briefly, retinal flat mounts were placed in distilled water overnight and then incubated with 4% trypsin in 0.1 M Tris-HCl buffer (pH 7.8) at 37°C for 1.5 hours. The samples were carefully washed with distilled water to detach the retinal cell layer from the vasculature. The vascular flat mounts were dried onto microscope slides and processed for PAS staining. The samples were oxidized, rinsed with dH2O, placed in Schiff's reagent, washed, counterstained with hematoxylin, differentiated in acid alcohol, dehydrated, and mounted in Depex.

[0150] Four to six individual images from the central and peripheral regions of the retinal vasculature were acquired using a Leica Thunder 3D histoscope (Leica Microsystems). The numbers of acellular capillaries, pericytes, and pericyte ghosts were counted manually by investigators blinded to the experimental group assignment.

[0151] The number of data points per group was as follows: untreated (n=67), STZ+vehicle (n=59), STZ+AF564 (n=45), STZ+danegaptide (n=58), STZ+AF564+danegaptide (n=61).

[0152] See Figure 11 for representative PAS images and Figure 10 for results. Evans blue retinal extravasation measurement Retinas were homogenized in formamide at a 1:10 ratio (w / v) and incubated overnight at +70°C. The homogenate was centrifuged at 20,800 x g for 45 minutes, and the supernatant was transferred to a normal tube. 30 μl of the supernatant was pipetted into a 384-well plate in triplicate. The absorbance (λ = 620 nm) of the samples was measured using a plate reader (Cytation3, BioTek Instruments Inc., Winooski, VT), and the concentration of Evans Blue in each sample was calculated against an Evans Blue standard curve and normalized to tissue weight. See Figure 9 for results.

[0153] Data analysis Quantitative data were graphed, analyzed, and expressed as mean ± standard deviation (SD) or standard error of the mean (SEM), as indicated. Normality was assessed, and data were log-transformed to achieve a Gaussian distribution, if necessary. Outliers were identified using the ROUT method with a Q factor of 1%. Differences were considered statistically significant at the p<0.05 level. Data were analyzed using GraphPad Prism software (v9.1.2. GraphPad Inc., La Jolla, CA) using appropriate statistical tests, as indicated.

[0154] result The results of the experimental tests are shown in Figures 1-11. Figure 1: Danegaptide protects against pseudo-DR and AMD injury mediated as decreased permeability in stressed human retinal pigment epithelial cell monolayers.

[0155] Confluent monolayers of human retinal epithelial cells (ARPE-19) in Transwell plates were treated with either vehicle (complete medium) or danegaptide (DGP, 100 nM) for 24 hours. The medium was then replaced with either (i) vehicle (complete medium; Figures 1A and 1B, left column, "-,-"); ​​(ii) medium supplemented with 30 mM high glucose (hyperglycemic stress) and a sublethal concentration of 200 μM tert-butyl hydroperoxide (tBHP) (oxidative stress) (Figures 1A and 1B, middle column, "+,-"); ​​or (iii) medium supplemented with 30 mM glucose, a sublethal concentration of tert-butyl hydroperoxide (tBHP, 200 μM), and 100 nM danegaptide (Figures 1A and 1B, right column, "+,+") and incubated for 48 hours.

[0156] The combination of 30 mM high glucose (hyperglycemic stress) and oxidative stress caused RPE barrier dysfunction and increased permeability across the RPE monolayer, as demonstrated using two fluorescent permeability markers, 6-carboxyfluorescein (6-CF; Figure 1A) or rhodamine B (RhoB; Figure 1B). The increase in RPE monolayer permeability due to hyperglycemic and oxidative stress conditions, and the protection afforded by danegaptide, were measured by measuring the amount of permeability marker efflux into the lower chamber of the Transwell plate and the basolateral side of the RPE monolayer, after 10, 20, 30, 45, 60, 90, and 120 minutes following addition of the permeability marker to the apical side of the Transwell chamber insert and RPE monolayer.

[0157] The cumulative concentrations of 6-CF and RhoB present in the lower chamber were calculated by measuring the amount of fluorescent signal based on a standard curve established using a microplate reader (Cytation5, Agilent, Santa Clara, CA). The amount of paracellular permeability was calculated as the apparent permeability coefficient (P app) and calculated to determine steady-state flux. Hyperglycemia and oxidative stress insults in pseudo-DR and AMD significantly increased permeability of both 6-CF and RhoB (n = 6-8) across the RPE monolayer (Figures 1A and 1B, middle column, "+,-"; compared to left column, "-,-"). Treatment with 100 mM danegaptide completely protected against this increase in permeability (n = 6-8) caused by these RPE barrier stressors (Figures 1A and 1B, right column, "+,+").

[0158] Figure 2: Danegaptide protects against pseudo-DR and AMD injury mediated through improved tight junction organization (TiJOR) and improved cell-cell coupling between human retinal pigment epithelial cells in stressed monolayers.

[0159] Human retinal epithelial cells (ARPE-19) were seeded and cultured on microscope chamber slides and grown to confluency to form a tightly adherent monolayer visualized by RPE cell-cell conjugates in either vehicle (complete medium) or danegaptide (DGP, 100 nM) treatment for 24 hours. The medium was then replaced with (i) vehicle (complete medium; Figure 2A, left column "-,-"; Figure 2B, left image); (ii) medium supplemented with 30 mM glucose (hyperglycemic stress) and a sublethal concentration of tert-butyl hydroperoxide (tBHP, 200 µM) (oxidative stress) (Figure 2A, middle column "+,-"; Figure 2B, middle image); or (iii) medium supplemented with 30 mM glucose, a sublethal concentration of tert-butyl hydroperoxide (tBHP, 200 µM), and 100 nM danegaptide (Figure 2A, right column "+,+"; Figure 2B, right image) and incubated for 48 hours. The results are shown in the upper graph of Figure 2 (Figure 2A).

[0160] Tight junction organization was assessed by immunocytochemistry using anti-zonula occludens 1 (ZO-1) immunostaining (Figure 2, bottom image; Figure 2B), a major component of tight junctions that binds to the cytoplasmic domains of both the gap junction transmembrane protein, Cx43, and the TJ transmembrane proteins, occludin and claudin. The tight junction organization index (TiJOR) was calculated according to Terryn et al. (2013) and measures the degree of TJ-dependent cell-cell coupling. Hyperglycemia and oxidative stress in the pseudo-DR and AMD insults significantly reduced TiJOR (n = 13–16). Treatment with 100 nM danegaptide completely prevented this loss of tight junction organization (n = 13–16) and RPE cell-cell uncoupling, correlating with danegaptide's ability to protect the RPE barrier from dysfunction and pathological RPE paracellular permeability caused by the combination of these hyperglycemic and oxidative cellular stressors.

[0161] High glucose and oxidative stress (i.e., cellular stress) lead to the breakdown of tight junctions (i.e., a decrease in TiJOR) and cell-cell coupling. These results indicate that danegaptide can protect cells from this uncoupling under stress conditions. That is, danegaptide can improve TiJOR and maintain cell-cell coupling between RPE cells exposed to stress.

[0162] Figure 3: Blood glucose levels (mmol / l) and blood glycated hemoglobin (HbA1C) levels (mmol / mol) during the in vivo study. Blood glucose levels were measured by Rapidometer every two weeks after DR induction with STZ in all animals. If the Rapidometer readings were out of range, additional plasma samples were collected and measured using fluorometry. Blood glucose levels were significantly increased on day 4 after induction compared with the untreated group (two-way ANOVA, p<0.001) (Figure 3A).

[0163] Glycated hemoglobin (HbA1c) levels increased significantly on day 35 of the study and remained at similar levels at the end of the study (day 60) (two-way ANOVA, p<0.001) (Figure 3B).

[0164] Figure 4: Cataract scores during the in vivo study. The appearance of hyperglycemia-induced cataracts is a common phenotype in the STZ model and was scored on the day of test article administration and at the end of the study period. Cataract appearance was scored from 0, representing a normal lens, to 3, representing opacification covering more than 75% of the lens. Untreated animals did not have any cataracts or opacities at any time during the study. The various STZ-induced groups showed similar cataract scores throughout the study period after IVT administration, with mild cataracts appearing on day 42 becoming more severe, reaching moderate severity on day 60 (Figure 4). However, there were no significant differences in cataracts between STZ-treated groups.

[0165] Figure 5: Danegaptide protects outer retinal thickening in rats with DR after STZ induction. Increased outer retinal thickness is typically observed during early STZ-induced DR in Brown Norway rats. Despite small absolute increases, typically in the 3-5% range, state-of-the-art in vivo imaging using SD-OCT can reveal these biologically relevant differences, which are indicative of edema, or cell swelling, indicative of an impaired outer blood-retinal barrier.

[0166] SD-OCT scans were performed 9 weeks before sampling. Retinal scans were segmented and thickness analyzed using a convolutional neural network algorithm. Outer retinal thickness was quantified from SD-OCT images from 24 locations to generate a mean outer retinal thickness. Outliers were removed using the GROUT method (Q = 1%), resulting in a total of 5 outliers removed (untreated: 0, STZ + vehicle: 1; STZ + AF564: 1; STZ + danegaptide: 1; STZ + AF564 + danegaptide: 2). When tested for normality, the SD-OCT data showed a normal distribution.

[0167] Streptozotocin (STZ) induction in Brown Norway rats resulted in a statistically significant increase in outer retinal thickness 9 weeks after induction in retinas with hyperglycemia, DR, and STZ + vehicle (123.8 ± 0.9 μm; n = 21; t-test p < 0.01) compared with untreated animals that did not undergo STZ induction and did not have DR (119.8 ± 0.5 μm; n = 16; t-test p < 0.01), as measured by SD-OCT imaging and quantification of changes in the outer plexus, outer nuclear layer, inner and outer segments, and RPE / choroid layers with a neural network algorithm (Figure 5A).

[0168] Danegaptide treatment by localized intravitreal (IVT) injection of 100 nM danegaptide in the eye weekly for 3 weeks prevented this increase in outer retinal thickness as measured by SD-OCT imaging (119.7 ± 0.8 μm; n = 27; p < 0.05), whereas treatment with an anti-VEGF compound (AF564; 122.7 ± 1.2 μm; n = 25; p = 0.60) and anti-VEGF plus danegaptide injection treatment (122.3 ± 1.1 μm; n = 17; p = 0.60) had no protective effect (Figure 5B). Pathological thickening of the oBRB is caused by RPE barrier dysfunction, a hallmark of DME and AMD. The ability of danegaptide to prevent this thickening of the outer retinal layers in these diabetic rats with DR indicates that danegaptide may be an effective therapy for AMD as well as DR and DME (as shown above).

[0169] Representative SD-OCT scans of outer retinal thickness from left and right eyes 9 weeks after STZ induction are shown in Figure 5C. Without wishing to be bound by theory, the inventors believe that the increased thickness of the outer retina caused by STZ induction represents cell swelling as a result of cellular stress. This is thought to be due to the opening of Cx43 hemichannels, allowing water to penetrate the cells. Danegaptide may close Cx43 hemichannels, thereby protecting subjects from cell swelling and resulting thickening of the outer retinal layer.

[0170] Figure 7: Danegaptide protects against outer retinal thickening throughout the retina. Outer retinal thickness was analyzed in different regions of the retina, specifically the superonasal (SN), superior temporal (ST), inferior nasal (IN), and inferior temporal (IT), with eight locations contributing to each measurement (Fig. 7 and Table 1) (see Fig. 6 for locations).

[0171] [Table 6]

[0172] Outer retinal thickness was statistically significantly increased in STZ + vehicle-treated rats compared to untreated rats in all regions (Figure 7A-D; see also Table 1, "STZ + vehicle" compared to "untreated"). Danegaptide prevented outer retinal thickening in all regions (Figure 7E-H; see also Table 1, "STZ + danegaptide" compared to "STZ + vehicle").

[0173] Figure 8. Regional analysis of inner retinal thickness (and total retinal thickness). SD-OCT scans were performed 9 weeks prior to sampling. Retinal scans were segmented, and thickness was analyzed using a convolutional neural network algorithm. Inner retinal thickness was analyzed by region (Figure 8). The analyzed regions were superonasal (SN), superior temporal (ST), inferior nasal (IN), and inferior temporal (IT) (see Figure 6 for locations).

[0174] The retina was significantly thinner in STZ-induced eyes compared to untreated animals (Figure 8A-D), but no statistically significant differences were identified between treatment groups (Figure 8E-H). Analysis of total retinal thickness in STZ animals is confounded by a reduction in inner retinal thickness and a concomitant increase in outer retinal thickness and therefore typically does not provide sufficient granularity to analyze the pharmacological effects of treatment. Total retinal thickness measurements are outlined in Table 2.

[0175] [Table 7]

[0176] Figure 9: Danegaptide and anti-VEGF treatment attenuates Evans Blue (EB) extravasation in the diabetic retina. Evans blue extravasation is a highly recognized measurement that quantifies vascular leakage in multiple organ systems. In the case of the retina, Evans blue extravasation is detectable in tissue lysates when either the inner or outer retinal blood barrier (or both) is impaired.

[0177] To determine the effects of danegaptide alone or in combination with an anti-VEGF antibody, Evans blue was injected intravenously and allowed to perfuse for 2 hours. After this period, blood samples were collected to determine the amount of Evans blue in the plasma, which served as a normalization factor for quantifying Evans blue extravasation in the retina. The values ​​presented are the normalized amount of Evans blue, derived as the concentration of Evans blue per milligram of tissue weight (ng / ml) divided by the concentration of Evans blue in plasma.

[0178] One data point (in the "STZ + danegaptide" group) from the entire dataset was eliminated based on outlier analysis using the GROUT method with a Q value of 1%. The single outlier was considered a technical artifact, as this single value was biased by more than 30-fold from measurements of two additional technical replicates of the same sample. The data were then analyzed for normality using the Kolmogorov-Smirnov test. Because the majority of the experimental groups did not follow a normal distribution (Table 3), the data were log-transformed.

[0179] [Table 8]

[0180] Given the variability in EB extravasation, typical of the STZ model in Brown Norway rats, we analyzed the effect size of the various treatment groups. First, data were normalized to the untreated group to determine the fold increase in EB extravasation following STZ (Figure 9A). STZ induction resulted in a more than 5-fold increase in EB extravasation 9 weeks after induction (100 ± 13.5% in untreated vs. 628 ± 212.5% ​​in STZ + vehicle, n = 10-11, t-test p < 0.01).

[0181] The data were then normalized to the STZ + vehicle group to determine the relative effective magnitude of the IVT treatment intervention. Data were assessed for normality using the D'Agostino and Pearson test on the relative dataset, and all STZ groups passed normality.

[0182] Anti-VEGF treatment with AF564 resulted in a statistically significant reduction in EB extravasation (17.8±4.3% of STZ+vehicle, n=10, one-way ANOVA, p<0.01, Holm-Sidak multiple comparison test, p<0.01), corresponding to an 82.3% reduction (Figure 9B, "STZ+AF564" compared to "STZ+vehicle").

[0183] Similarly, danegaptide showed a statistically significant reduction in EB extravasation (31.1±5.3% of STZ+vehicle, n=9, one-way ANOVA, p<0.01, Holm-Sidak multiple comparison test, p<0.05), corresponding to a 68.9% reduction (Figure 9B, "STZ+danegaptide" compared to "STZ+vehicle").

[0184] The effect of combined treatment with anti-VEGF and danegaptide was similar to that of danegaptide alone (34.4±10.3%, n=10, p<0.05), equivalent to a 65.6% reduction (Figure 9B, "STZ+AF564+danegaptide" compared to "STZ+vehicle").

[0185] Figures 10 and 11: Danegaptide prevents pericyte loss 9 weeks after STZ induction. The effects of vascular abnormalities and STZ-induced DR were also assessed by quantification of acellular capillaries, pericytes, and pericyte ghosts in trypsin-digested retinal whole mounts. For quantification, 4–15 individual images were quantified for each animal, and data from each image were included in the analysis.

[0186] Based on our previous studies conducted in the STZ-induced DR model in Brown Norway rats, we found that the most reliable readout for determining vascular abnormalities is quantification of pericyte ghosts. Pericyte ghosts appear early in the disease and correspond to the loss of retinal pericytes, whereas acellular capillaries are typically observed at later time points. Specifically, our preliminary studies determined that induction of DR with STZ resulted in a statistically significant increase in pericyte ghosts after 12 weeks. Therefore, we expected to detect the presence of pericyte ghosts in normal pericyte counts at 9 weeks.

[0187] These observations were confirmed in the present study, as no differences in the total number of pericytes (the sum of viable pericytes and pericyte ghosts) or acellular capillaries were observed in STZ-induced animals (Table 4; "STZ + vehicle" compared to "untreated"). Furthermore, no significant differences were identified between the various treatment groups (Table 4).

[0188] [Table 9]

[0189] There was no statistically significant difference in the total number of pericytes, quantified as the sum of the number of pericytes and pericyte ghosts (one-way ANOVA, p=0.51).Similarly, there was no statistically significant difference in the number of acellular capillaries (one-way ANOVA, p<0.001 followed by Holm-Sidak multiple comparison test), except for a statistical trend toward fewer acellular capillaries in the anti-VEGF-treated group (STZ+AF564; p=0.06) compared to vehicle-treated (STZ+Vehicle) eyes.

[0190] The number of pericyte ghosts was then quantified. Outliers were removed from the dataset using the GROUT method (Q = 1%), resulting in the removal of 19 out of a total of 324 data points collected.

[0191] To validate the model based on the presence of pericyte ghosts, we compared the number of pericyte ghosts in the retinas of untreated animals with the number in the retinas of STZ-induced animals that were initially treated with vehicle. STZ induction resulted in a statistically significant increase in the number of pericyte ghosts in the retinas compared to untreated rats (Figure 10A).

[0192] The STZ groups were then compared by one-way ANOVA (p<0.05) followed by Dunnett's multiple comparison test (Figure 10B). Treatment with both anti-VEGF antibody (AF564; p<0.01) and danegaptide (p<0.001) resulted in a statistically significant reduction in pericyte ghosts, whereas combined treatment had no statistical effect (p=0.11).

[0193] A representative example of a trypsin-digested, PAS-stained retinal whole mount is shown in FIG. 11, showing acellular capillaries (arrows), pericytes (not indicated), and pericyte ghosts (arrowheads). conclusion Exposure of ARPE-19 cells (48 h) to sublethal oxidative stress and hyperglycemia resulted in P app Pretreatment with danegaptide (100 nM) completely protected against this increase in permeability (Figures 1A and 1B, right column, "+,+"; compared with control left column, "-,-"). Danegaptide also partially protected against tight junction disruption and cell-cell coupling disruption, as assessed by quantification of TiJOR (Figure 2A, right column, "+,+"; compared with control left column, "-,-" and middle column, "+,-").

[0194] STZ produced a reliable induction of hyperglycemia, as quantified by persistently elevated glucose (Figure 3A) and hemoglobin A1c levels (Figure 3B) throughout the study. STZ-induced cataracts were common in this model and moderately developed by the end of the study (Figure 4). All test articles were well tolerated, and no signs of ocular toxicity were identified by gross ophthalmic examination. The test articles did not exert differential effects on cataract development (Figure 4).

[0195] Induction of DR by STZ resulted in a statistically significant increase in outer retinal thickness (Figure 5B; "STZ + Vehicle" compared to "Untreated") that was completely reversed by danegaptide (p<0.05) (Figure 5B; "STZ + Danegaptide"). In contrast, AF564 (p=0.60) (Figure 5B; "STZ + AF564") and combined treatment (p=0.60) (Figure 5B; "STZ + AF564 + Danegaptide") had no effect. This reduction in outer retinal thickness by danegaptide was demonstrated throughout the retina (Figure 7).

[0196] The positive control, anti-VEGF antibody AF564, resulted in a statistically significant 82.3% reduction in EB extravasation (p<0.01) (Figure 9B; "STZ+AF564" compared to "STZ+Vehicle"). Similarly, danegaptide resulted in a 68.9% reduction in EB extravasation (p<0.05) (Figure 9B; "STZ+Danegaptide" compared to "STZ+Vehicle"). Combined treatment with AF564 and danegaptide showed a similar effect on EB extravasation as danegaptide alone (65.6% reduction, p<0.05) (Figure 9B; "STZ+AF564+Danegaptide" compared to "STZ+Vehicle" and "STZ+Danegaptide").

[0197] The number of pericyte ghosts was significantly increased in STZ rats (p<0.05) (Figure 10A; "STZ+vehicle" compared to "untreated"). Both AF564 (p<0.01) (Figure 10B; "STZ+AF564") and danegaptide (p<0.001) (Figure 10B; "STZ+danegaptide") induced a statistically significant reduction in pericyte ghosts, whereas combined treatment had no effect (p=0.11) (Figure 10B; "STZ+AF564+danegaptide").

[0198] No biologically relevant effects of danegaptide were identified on inner retinal thickness. These combined findings that danegaptide can protect against RPE barrier dysfunction as demonstrated by its ability to prevent hyperglycemic and oxidative stress-induced increases in RPE monolayer permeability in a TJ-related manner and its ability to prevent thickening of the outer retinal layers in rats with DR (Figures 1-11) indicate that danegaptide may be an effective treatment for patients with d-AMD, wet AMD, or neovascular AMD.

[0199] Danegaptide protects against loss of blood-retinal barrier (BRB) integrity, in part through connexin 43 (Cx43) at gap junctions by: · Protection from pericyte and endothelial cell loss and vascular leakage, not only observed in the internal BRB:DR but also recently recognized in patients with AMD (see, e.g., Hudson et al., 2020 and Hadziahmetovic et al., 2021); and · External BRB: Protection from loss of retinal pigment epithelial cell conjugate barrier integrity resulting in a decline in the barrier integrity and associated functions of the RPE cell layer, including active water movement and vascular leakage from the subretinal space causing wet AMD.

[0200] Thus, danegaptide exerts potent protection against experimental conditions mimicking the known pathologies of diabetic retinopathy (DR) and age-related macular degeneration (AMD), which are associated with the disruption of tight junctions and cell-cell coupling. Additionally, by protecting against pathological Cx43-based hemichannel opening, danegaptide can protect against cell swelling, thickening of the outer retinal cell layer, and pathological ATP leakage into the extracellular compartment, which is known to cause inflammation. Danegaptide was well tolerated in rats and provided significant protection against stress- and DR-associated pericyte loss, retinal vascular leakage, and blood-retinal barrier breakdown (both inner and outer BRB). The level of protection induced by danegaptide in vascular leakage and pericyte loss was similar to that observed with anti-VEGF treatment. Additionally, danegaptide, but not anti-VEGF treatment, protected against stress- and DR-induced increases in outer retinal thickness.

[0201] References All publications, patents and patent applications mentioned herein or filed in this application are incorporated by reference in their entirety, including any references submitted as part of the Information Disclosure Statement.

[0202] [Table 10]

Claims

1. 1. A compound of formula (I): 【Chemistry 1】 or a pharmaceutically acceptable salt or hydrate thereof, wherein said method comprises administering to said patient a therapeutically effective amount of said compound, or a pharmaceutically acceptable salt or hydrate thereof.

2. 2. The pharmaceutical composition for use in the method of treatment or prevention according to claim 1, wherein the compound is (2S,4R)-1-(2-aminoacetyl)-4-benzoylamino-pyrrolidine-2-carboxylic acid, or a pharmaceutically acceptable salt or hydrate thereof.

3. The compound or a pharmaceutically acceptable salt or hydrate thereof is administered topically to the eye; Optionally, the compound or a pharmaceutically acceptable salt or hydrate thereof is administered locally in the eye via intravitreal injection.

3. A pharmaceutical composition for use in the method of treatment or prevention according to claim 1 or 2.

4. The compound or a pharmaceutically acceptable salt or hydrate thereof is administered systemically; Optionally, the compound or a pharmaceutically acceptable salt or hydrate thereof is administered systemically via oral, subcutaneous, transdermal, or intravenous administration; Optionally, the compound or a pharmaceutically acceptable salt or hydrate thereof is administered systemically via oral administration.

3. A pharmaceutical composition for use in the method of treatment or prevention according to claim 1 or 2.

5. The method comprises: (i) for the prevention of AMD in human patients; (ii) for the prevention of the progression of AMD in human patients; (iii) for the prevention of progression from dry to wet AMD in human patients; (iv) for the prevention of progression from moderate dry AMD to advanced forms of dry AMD in human patients; and / or (v) for the prevention of the development of choroidal neovascularization in human patients; 3. A pharmaceutical composition for use in the method of treatment or prevention according to claim 1 or 2.

6. The AMD is (i) characterized as early dry AMD; (ii) characterized as intermediate-stage dry AMD; (iii) characterized as advanced dry AMD, optionally wherein the advanced dry AMD is geographic atrophy; or (iv) characterized as wet, neovascular, or advanced vascular AMD; 3. A pharmaceutical composition for use in the method of treatment or prevention according to claim 1 or 2.

7. The patient: (i) have chronically elevated blood glucose levels, including patients with glycemic control; (ii) have type 1 or type 2 diabetes; (iii) have hypertension or chronically high blood pressure, including patients with normal blood pressure control; (iv) leading to normal blood pressure control through antihypertensive treatment, glycemic control, or cholesterol reduction; (v) having high cholesterol; (vi) have diabetic retinopathy or diabetic macular edema; (vii) have retinal vein occlusion (RVO) eye disease; (viii) having glaucoma disease with or without pathological intraocular pressure; (ix) have uveitis or other forms of inflammatory eye disease; and / or (x) has or has had drusen deposits or protein exudates in the eye; 3. A pharmaceutical composition for use in the method of treatment or prevention according to claim 1 or 2.

8. The compound is (i) inhibiting RPE (retinal pigment epithelium) or outer blood-retinal barrier (oBRB) dysfunction; (ii) inhibiting the loss of barrier integrity between RPE cells and the loss of RPE cells; (iii) inhibiting stress-induced dysfunction of the retina or choroid layer in the eye; (iv) inhibiting the loss of photoreceptors in the eye; optionally, inhibiting the loss of photoreceptors in the retina; (v) inhibiting retinal vascular leakage; (vi) inhibiting vascular leakage that leads to macular edema; (vii) improving fluid extraction from retinal tissue through the retinal pigment epithelium; (viii) inhibiting the death or loss of retinal endothelial cells, choroidal endothelial cells, pericytes, or epithelial cells; (ix) inhibiting pathological hemichannel opening; and / or (x) inhibiting pathological hemichannel ATP release; optionally, said pathological hemichannel opening and / or ATP release is connexin 43-mediated opening and / or ATP release; 3. A pharmaceutical composition for use in the method of treatment or prevention according to claim 1 or 2.

9. A pharmaceutical composition for use in the method of treatment or prevention of claim 1 or 2, wherein the pharmaceutical composition is an oral, systemic, and sustained release compound formulation.

10. the concentration of the administered compound, or a pharmaceutically acceptable salt or hydrate thereof, reaches about 50 nM to about 150 nM in the ocular microenvironment; Optionally, the concentration of the administered compound, or a pharmaceutically acceptable salt or hydrate thereof, achieves about 100 nM in the ocular microenvironment.

3. A pharmaceutical composition for use in the method of treatment or prevention according to claim 1 or 2.

11. The compound is administered to the patient once or twice daily; or the compound is administered to the patient four times daily; 3. A pharmaceutical composition for use in the method of treatment or prevention according to claim 1 or 2.

12. For the preparation of a medicament for the treatment or prevention of age-related macular degeneration (AMD) in a human patient, a compound of formula (I): 【Chemistry 2】 or a pharmaceutically acceptable salt or hydrate thereof, wherein said medicament comprises a therapeutically effective amount of said compound, or a pharmaceutically acceptable salt or hydrate thereof, for administration to said patient.

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