Monomeric annexin A5 for use in treatment of macular edema or retinal vein occlusion

Abstract

The present invention relates to novel methods, uses and compositions of molecules consisting of the monomeric annexin A5 protein for the prevention or treatment of macular edema and / or one or more diseases, conditions or conditions associated with and / or caused by macular edema. In a preferred embodiment, the annexin A5 is administered systemically, such as intravenously.

Description

Technical Field

[0001] The present invention relates to novel methods, uses and compositions for preventing or treating macular edema and conditions associated therewith. Background Art

[0002] Macular edema is a pathological accumulation of fluid in the central retina. It is a complication of many retinal diseases, including diabetic retinopathy (DR), retinal vascular occlusion (RVO) and uveitis (UV) (Haydinger et al., 2023, Frontiers in Medicine, 10: 1128811). Macular edema causes reduced visual acuity, and when it is chronic or refractory macular edema, it can cause severe and permanent visual impairment and blindness. Combined with the underlying pathology, edema can progress to irreversible tissue damage and permanent visual impairment accompanied by retinal cell death.

[0003] In most cases, the development of macular edema is due to dysregulation of the blood-retinal barrier (BRB), a set of structures that strictly regulate the passage of proteins, salts, metabolites, and other solutes between the blood and retinal tissue under healthy conditions. Dysregulation of the BRB allows proteins and other solutes that are normally retained in the blood to infiltrate the retinal tissue (Haydinger et al., 2023, supra). Increased osmotic pressure in the tissue drives fluid accumulation.

[0004] Current treatments target blood-retinal barrier (BRB) dysfunction and include vascular endothelial growth factor (VEGF) blockers, corticosteroids, and nonsteroidal anti-inflammatory drugs (Haydinger et al., 2023, supra; Ip & Hendrick, 2018, Asia-Pacific Journal of Ophthalmology, 7 (1): 40-45). These treatments target vasoactive and inflammatory mediators that cause blood-retinal barrier disruption. Other treatments include targeted laser photocoagulation (Haydinger et al., 2023, supra; Ip & Hendrick, 2018, supra).

[0005] The first-line therapy for macular edema associated with diabetic retinopathy (DR) or retinal vascular occlusion (RVO), as well as for the treatment of RVO itself, is anti-VEGF therapy, which is sometimes augmented with intravitreal corticosteroids and / or retinal laser photocoagulation (Ehlers et al. 2022, Ophthalmology, 129:88-99; Ehlers et al., 2017, Ophthalmology, 124:1412-23; and Yeh et al., 2015, Ophthalmology, 122:769-78). The treatment of macular edema secondary to neovascular AMD also relies heavily on the use of anti-VEGF therapy (Flaxel et al., 2020, Ophthalmology, 127:P1-P65).

[0006] The established standard of care for anti-VEGF therapy for macular edema and / or RVO, and indeed in a variety of retinal diseases, is to deliver the therapeutic agent directly to the eye via intravitreal injection (Haydinger et al., 2023, supra; Ip & Hendrick, 2018, supra; Cox et al., 2021, J. Clin. Med., 10(5):981). Such drugs are injected directly into the eye because the retina is located at the back of the eye and is surrounded by several protective layers, including the cornea, iris, and lens. These layers can make it difficult for the drug to reach the retina when the drug is administered by other routes. When the drugs are injected into the eye, they are delivered directly to the retina and can act locally to target the source of the problem. For example, anti-VEGF drugs are injected into the eye to block the effects of VEGF-A, a protein that promotes abnormal blood vessel growth in the retina. By reducing the levels of VEGF-A, these drugs can help prevent the formation of new blood vessels and reduce inflammation and swelling in the eye. Eye injections are usually performed in an ophthalmologist's office or outpatient surgery center. After the injection, the patient is usually monitored for a short period of time to ensure that there are no adverse effects from the drug.

[0007] Targeting VEGF with intravitreal injections of monoclonal antibodies or high-affinity decoy receptors is an effective means of treating most diseases that complicate macular edema, including wet age-related macular degeneration (Heier et al., 2012, Ophthalmology, 119:2537-48), diabetic macular edema (Nguyen et al., 2012, Ophthalmology, 119:789-801; Korobelnik et al., 2014, Ophthalmology, 121:2247-54), and macular edema caused by central RVO (CRVO) (Brown et al., 2010, Ophthalmology, 117:1124-33), branch RVO (BRVO) (Campochiaro et al., 2010, Ophthalmology, 117:1102-12), and uveitis (Mackensen et al., 2008, Retina, 28:41-5).

[0008] However, there are ocular and systemic toxicity issues with anti-VEGF therapy, and frequent reinjections are required over a long period of time (Nguyen et al., 2010, Ophthalmology, 117:2146-51; Campochiaro et al., 2010, Ophthalmology, 117:2387-94; Saint-Geniez et al., 2008, PLoS One., 3:e3554). Potential complications associated with intravitreal injections of anti-VEGF therapy include infectious endophthalmitis, sterile intraocular inflammation, and retinal vasculitis, especially with the injection of brolucizumab (Cox et al., 2021, supra) and transient intraocular pressure (IOP) dopants (Levin et al., 2021, J Glaucoma, 30:1019-26).

[0009] Infectious endophthalmitis remains one of the most devastating complications associated with anti-VEGF therapy. Reported post-injection incidence varies from 0.008% to 0.092%, and despite their low incidence, repeated injections of large amounts of anti-VEGF mean that they account for a large proportion of infectious endophthalmitis cases (Cox et al., 2021, supra).

[0010] Sterile intraocular inflammation (SII), also known as pseudoendophthalmitis, is characterized by acute onset intraocular inflammation without infection that resolves without antibiotic treatment. The incidence of anti-VEGF-related SII reported by studies varies from 0.02% to 0.37%, with presentation typically occurring between 24 hours and seven days from the stimulating injection. Symptoms may consist of blurred vision, floaters, pain, and photophobia. Pain is present in up to 46% of patients, and its presence is significantly associated with severe vitreous or anterior chamber (AC) inflammation. Photophobia occurs in 4% to 19% of patients. In addition, visual acuity is often greatly reduced from the baseline at the time of its presentation, but it often returns to pre-injection levels after the inflammation resolves. Intraocular inflammation is present in almost every case examined. This may consist of vitrification, AC reaction, or most commonly both. Additionally, less common findings include hypopyon, fibrin, posterior corneal deposits, corneal edema, conjunctival injection, and conjunctival chemosis (Cox et al., 2021, supra).

[0011] Continuous anti-VEGF therapy via intravitreal injection is the current standard of care for the treatment of macular edema (Ip & Hendrick, 2018, supra). Indeed, it is recognized that the indications for intravitreal injections are expanding, and more and more patients may be affected by the acute and chronic effects of intravitreal injections (Levin et al., 2021, supra). However, to date no treatment for macular edema or RVO has been shown to be a reliable method for directly improving perfusion. Instead, current strategies focus on minimizing the consequences caused by RVO to minimize vision loss due to macular edema and neovascularization (Ip & Hendrick, 2018, supra).

[0012] It is in this context that there is a need in the art for improved treatments for macular edema and related disorders, including RVO. Specifically, there is a need for effective treatments that can be effectively administered to a subject without the need for direct injection (especially multiple repeated injections) into the subject's eye. Summary of the invention

[0013] Surprisingly, in this context, it has been determined that monomeric Annexin A5 protein, when systemically administered to a subject (such as by intravenous administration), is able to preferentially localize to the site of occlusion in a subject with retinal vein occlusion (RVO). It has also been determined that Annexin A5 provides biological activities that modulate factors that contribute to dysregulation of the blood-retinal barrier (BRB) in subjects with macular edema and / or RVO, as well as to other therapeutic benefits in subjects with macular edema and / or RVO.

[0014] Therefore, in a second aspect of the present invention, there is provided a molecule consisting of a monomeric Annexin A5 protein for use in preventing or treating macular edema or a condition associated therewith in a subject.

[0015] In an alternative embodiment of the first aspect, the present invention provides a method of treating or preventing macular edema or a condition associated therewith in a subject, comprising the step of administering to the subject a therapeutically effective amount of a molecule consisting of monomeric Annexin A5 protein.

[0016] In an alternative embodiment of the first aspect, the present invention provides use of a molecule consisting of monomeric Annexin A5 protein in the preparation of a medicament for preventing or treating macular edema or a disorder associated therewith in a subject.

[0017] Preferably, according to the first aspect of the present invention, the molecule consisting of the monomeric annexin A5 protein is systemically administered to the subject, preferably intravenously, subcutaneously or intramuscularly. When administered intravenously, the molecule consisting of the monomeric annexin A5 protein or the composition comprising the molecule can be used, for example, in the form of a sterile aqueous solution, which can contain other substances, such as sufficient salt or glucose to make the solution isotonic with blood. If necessary, the aqueous solution should be appropriately buffered (preferably, buffered to a pH of 3 to 9). For example, optionally, the aqueous solution is buffered at a pH selected from the following range: about pH 4 to about pH 8, about pH 5 to about pH 8, about pH 6 to about pH 8, about pH 6.5 to about pH 7.9, about pH 6.8 to about pH 7.7, about pH 7.0 to about pH 7.5, about pH 7.1 to about pH 7.4, about pH 7.1 to about pH 7.4, and optionally about pH 7.1, 7.2, 7.3 or 7.4, most preferably about pH 7.2.

[0018] Preferably, according to the first aspect of the invention, a dose of a molecule consisting of monomeric annexin A5 protein is administered to a subject for two or more consecutive days, such as three, four, five, six, seven or more consecutive days, and / or at least two, three, four, five, six or seven days in each 7-day (one week) cycle, for at least one week, two weeks, three weeks, four weeks or more.

[0019] Optionally, according to the first aspect of the invention, the daily dose of the molecule consisting of monomeric annexin A5 protein administered to the subject is as follows: a) a total daily dose of about 5 mg, about 4 mg, about 3 mg, about 2 mg or less than about 2 mg, 0.1 mg to 1.5 mg or about 1 mg; and / or b) within a range selected from the group consisting of about 50 μg-100 μg, about 40 μg-80 μg, about 30 μg-60 μg, about 20 μg-40 μg, 1 μg to 30 μg or less than about 20 μg per kg of subject body weight. The term "about" as used herein may be used herein to indicate a range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, ±4%, ±3%, ±2% or ±1% of the referenced value.

[0020] In a second aspect of the present invention, a molecule consisting of monomeric Annexin A5 protein is provided for use in preventing or treating retinal vein occlusion (RVO) or a condition associated therewith in a subject, wherein a dose of the molecule consisting of monomeric Annexin A5 protein is systemically administered to the subject and / or wherein the molecule consisting of monomeric Annexin A5 protein is administered to the subject daily for two or more consecutive days.

[0021] In an alternative embodiment of the second aspect, the present invention provides a method of treating or preventing RVO or a condition associated therewith in a subject, comprising the step of administering to the subject a therapeutically effective dose of a molecule consisting of monomeric Annexin A5 protein, and wherein the dose is administered systemically to the subject and / or wherein the dose is administered to the subject daily for two or more consecutive days.

[0022] In an alternative embodiment of the second aspect, the present invention provides use of a molecule consisting of monomeric Annexin A5 protein in the preparation of a medicament for preventing or treating RVO or a condition associated therewith in a subject, wherein a dose of the molecule consisting of monomeric Annexin A5 protein is systemically administered to the subject and / or wherein the molecule consisting of monomeric Annexin A5 protein is administered to the subject daily for two or more consecutive days.

[0023] Preferably, according to the second aspect of the present invention, the systemic administration mode of the molecule consisting of monomeric annexin A5 protein is selected from intravenous, subcutaneous or intramuscular administration. When administered intravenously, the molecule consisting of monomeric annexin A5 protein or the composition comprising the molecule can be used, for example, in the form of a sterile aqueous solution, which can contain other substances, such as sufficient salt or glucose to make the solution isotonic with blood. If necessary, the aqueous solution should be appropriately buffered (preferably, buffered to a pH of 3 to 9). For example, optionally, the aqueous solution is buffered at a pH selected from the following range: about pH 4 to about pH 8, about pH 5 to about pH 8, about pH 6 to about pH 8, about pH 6.5 to about pH 7.9, about pH 6.8 to about pH 7.7, about pH 7.0 to about pH 7.5, about pH 7.1 to about pH 7.4, about pH 7.1 to about pH 7.4, and optionally about pH 7.1, 7.2, 7.3 or 7.4, most preferably about pH 7.2.

[0024] More preferably, a dose of a molecule consisting of monomeric Annexin A5 protein is systemically administered (such as by intravenous, subcutaneous or intramuscular administration) to a subject daily for two or more consecutive days.

[0025] In one embodiment of the second aspect of the invention, a molecule consisting of monomeric Annexin A5 protein is administered to a subject daily for three, four, five, six, seven or more consecutive days, and / or at least two, three, four, five, six or seven days in each 7-day (one week) cycle, for at least one week, two weeks, three weeks, four weeks or more.

[0026] Optionally, according to the second aspect of the invention, the daily dose of the molecule consisting of monomeric annexin A5 protein administered to the subject is as follows: a) a total daily dose of about 5 mg, about 4 mg, about 3 mg, about 2 mg or less than about 2 mg, 0.1 mg to 1.5 mg or about 1 mg; and / or b) within a range selected from the group consisting of about 50 μg-100 μg, about 40 μg-80 μg, about 30 μg-60 μg, about 20 μg-40 μg, 1 μg to 30 μg or less than about 20 μg per kg of subject body weight. The term "about" as used herein may be used herein to indicate a range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, ±4%, ±3%, ±2% or ±1% of the referenced value.

[0027] The present inventors have also determined that treatment and prevention of retinal vein occlusion and / or one or more conditions associated therewith can be achieved with substantially lower doses of molecules consisting of monomeric Annexin A5 protein.

[0028] For example, a molecule consisting of monomeric annexin A5 protein can be administered to a subject at a dose of about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg, and / or wherein the molecule is administered to a subject at a dose of about 0.001 mg to about 1 mg or 1 μg to about 100 μg. As used herein, the term "about" can be used herein to represent a range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, ±4%, ±3%, ±2% or ±1% of the referenced value.

[0029] Optionally, according to the second aspect of the invention, molecules consisting of monomeric Annexin A5 protein may be used in the prevention or treatment of a condition in a subject having, suspected of having, or at risk of having retinal vein occlusion (RVO), wherein the condition is selected from the group consisting of: macular edema, retinal ischemia, iris neovascularization (optionally, iris neovascularization characterized by increased cytokines, such as VEGF-increased iris neovascularization), retinal neovascularization (optionally, retinal neovascularization characterized by increased cytokines, such as VEGF-increased retinal neovascularization), neovascular glaucoma, vitreous hemorrhage, erythrosis and retinal detachment.

[0030] Optionally, according to the first and / or second aspects of the present invention, the retinal vein occlusion may be, for example, selected from: central retinal vein occlusion (CRVO); branch retinal vein occlusion (BRVO); or hemi-retinal vein occlusion (HRVO).

[0031] Optionally, according to the first aspect and / or the second aspect of the present invention, the retinal vein occlusion may optionally be an acute retinal vein occlusion, such as: acute central retinal vein occlusion (CRVO); acute branch retinal vein occlusion (BRVO); or acute hemi-retinal vein occlusion (HRVO).

[0032] Optionally, according to an embodiment of the first aspect and / or the second aspect of the present invention, the use or method may also (or alternatively) be used to prevent or treat one or more diseases, conditions or disorders associated with and / or caused by (directly or indirectly) macular edema and / or retinal vein occlusion.

[0033] Optionally, according to the first and / or second aspects of the invention, the subject may be characterized as having about 1.0% to about 2% of Annexin A5-positive red blood cells.

[0034] Optionally, according to the first and / or second aspects of the invention, the subject may be characterised by an average number of monomeric Annexin A5 binding sites per erythrocyte of about 400 to about 1000.

[0035] Optionally, according to the first aspect and / or the second aspect of the present invention, the subject is typically a human subject, such as a male and / or female. The subject may be an adult, or less typically a teenager, a child or a newborn.

[0036] Optionally, according to a first aspect and / or a second aspect of the invention, the experimenter is an adult and / or has a body weight of about 50 kg to about 150 kg. As used herein, the term "about" can be used in this article to represent the scope of ± 50%, ± 40%, ± 30%, ± 20%, ± 10%, ± 5%, ± 4%, ± 3%, ± 2% or ± 1% of the value mentioned.

[0037] Optionally, according to the first aspect and / or the second aspect of the present invention, the subject may additionally or alternatively be characterized by one or more of the following:

[0038] a. The subject is about 50 years old or older;

[0039] b. The subject does not have or is not suspected of having cardiovascular disease (such as myocardial infarction, stroke, hypertension, hyperlipidemia, hyperhomocysteinemia, coagulopathy and / or peripheral arterial disease, and optionally excluding atherosclerosis) or diseases associated with cardiovascular disease (such as sickle cell disease, glaucoma, diabetic dyscrasia, diabetes mellitus, polycythemia vera and / or systemic inflammatory disorders (e.g. Behcet's disease ( disease, polyarteritis nodosa, sarcoidosis, Wegener's Granulomatosis, and / or Goodpasture's Syndrome), but preferably excluding polyuria and diabetes mellitus);

[0040] c. the subject has no history of cardiovascular disease (such as myocardial infarction, stroke, hypertension, hyperlipidemia, hyperhomocysteinemia, coagulopathy and / or peripheral arterial disease, and optionally excluding atherosclerosis) or cardiovascular disease-related diseases (such as sickle cell disease, glaucoma, polyuria, diabetes mellitus, polycythemia vera, and / or systemic inflammatory disorders (e.g. Behcet's disease, polyarteritis nodosa, sarcoidosis, Wegener's granulomatosis and / or Goodpasture's syndrome), but preferably excluding polyuria and diabetes mellitus);

[0041] d. the subject has no family history of cardiovascular disease (such as myocardial infarction, stroke, atherosclerosis, hypertension, hyperlipidemia, hyperhomocysteinemia, coagulopathy and / or peripheral arterial disease, and optionally excluding atherosclerosis) or cardiovascular disease-related diseases (such as sickle cell disease, glaucoma, polyuria, diabetes, polycythemia vera, and / or systemic inflammatory disorders (e.g. Behcet's disease, polyarteritis nodosa, sarcoidosis, Wegener's granulomatosis and / or Goodpasture's syndrome), but preferably excluding polyuria and diabetes); and / or

[0042] e. the subject does not have or is not suspected of having a neoplastic disorder, such as a solid tumor, a lymphoma and / or a blood cancer, for example one or more disorders selected from the group consisting of breast cancer, endometrial cancer, cervical cancer, ovarian cancer, lung cancer, head and / or neck cancer, brain cancer, thyroid cancer, esophageal cancer, stomach cancer, colon cancer and / or rectal cancer, liver cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, bone cancer, lymphoma (such as Hodgkin lymphoma and / or non-Hodgkin lymphoma) and / or blood cancer (such as acute lymphocytic leukemia, acute myeloid leukemia and / or chronic myeloid leukemia).

[0043] In one embodiment of the first aspect and / or the second aspect of the present invention, the dose of the molecule consisting of monomeric annexin A5 protein is administered to the subject once or more per day. The dose of the molecule consisting of monomeric annexin A5 protein can be optionally administered to the subject once or more per day for one, two, three, four, five or six or more consecutive days.

[0044] In one embodiment of the first aspect and / or the second aspect of the invention, the molecule consisting of monomeric annexin A5 protein is administered to the vascular system of the subject, for example, as a systemic administration such as by intravenous administration. When administered intravenously, the molecule consisting of monomeric annexin A5 protein or a composition comprising the molecule can be used, for example, in the form of a sterile aqueous solution, which can contain other substances, such as sufficient salt or glucose to make the solution isotonic with blood. If necessary, the aqueous solution should be appropriately buffered (preferably, buffered to a pH of 3 to 9). For example, the aqueous solution is optionally buffered at a pH in a range selected from about pH 4 to about pH 8, about pH 5 to about pH 8, about pH 6 to about pH 8, about pH 6.5 to about pH 7.9, about pH 6.8 to about pH 7.7, about pH 7.0 to about pH 7.5, about pH 7.1 to about pH 7.4, about pH 7.1 to about pH 7.4, and optionally about pH 7.1, 7.2, 7.3 or 7.4, most preferably about pH 7.2.

[0045] Although disclosed as a treatment option with respect to the first and / or second aspects of the invention, it is less preferred to administer molecules consisting of monomeric annexin A5 proteins directly to the eye. For example, molecules consisting of monomeric annexin A5 proteins can be administered by intravitreal injection. In another option, molecules consisting of monomeric annexin A5 proteins can be administered directly to a subject's retinal vein (such as the central retinal vein, branch retinal veins and / or hemi-retinal veins), for example by catheterization. However, it is preferred to avoid this situation.

[0046] The monomeric Annexin A5 protein used in the first aspect and / or the second aspect of the present invention may optionally be selected from one or more of the following:

[0047] a. A protein consisting essentially of the sequence of human monomeric annexin A5 protein or consisting of the sequence of human monomeric annexin A5 protein (optionally, a protein consisting of the sequence of SEQ ID NO: 1 with or without an N-terminal methionine);

[0048] b. recombinant human monomeric annexin A5 protein;

[0049] c. the mammalian ortholog of the human monomeric annexin A5 protein;

[0050] da), b) or c) allelic variant or genetic variant;

[0051] e. A functional analog or variant of a monomeric Annexin A5 protein, which is a protein consisting of an amino acid sequence that is more than 50%, 60%, 70%, 75%, such as more than 80%, 85%, more than 90% or even more preferably more than 95% or 99% identical to a human monomeric Annexin A5 protein having a sequence defined by SEQ ID NO: 1 with or without an N-terminal methionine; and

[0052] A biologically active fragment of any one of fa) to e).

[0053] Also provided herein is a composition comprising a molecule consisting of a monomeric annexin A5 protein used according to the first aspect and / or the second aspect of the present invention. The composition may, for example, be a unit dose composition. The unit dose composition may, for example, comprise: about 5 mg, about 4 mg, about 3 mg, about 2 mg or less than about 2 mg, such as a total dose of about 0.1 mg-1.5 mg, about 1 mg or less. Optionally, the composition is a pharmaceutical composition, and further optionally, the composition also comprises one or more pharmaceutically acceptable carriers. In some embodiments, the composition consists of the following: a molecule consisting of a monomeric annexin A5 protein and one or more pharmaceutically acceptable carriers. Optionally, the composition is used as a combination therapy with another agent. Optionally, the molecule consisting of monomeric Annexin A5 protein is used as a first line treatment with or without the one or more other agents for preventing or treating macular edema and / or retinal vein occlusion (RVO) and / or one or more diseases, conditions or disorders associated with and / or caused (directly or indirectly) by macular edema and / or retinal vein occlusion.

[0054] According to the first and / or second aspects of the present invention, in some embodiments, a molecule consisting of a monomeric annexin A5 protein or a composition comprising the molecule is used and / or used as a combination therapy with another agent, wherein the molecule consisting of a monomeric annexin A5 protein is administered to a subject in the same composition, separately from one or more other agents, simultaneously with a separate formulation containing one or more other agents, or sequentially with one or more other agents. In some embodiments, the other agent may be an anti-vascular endothelial growth factor (anti-VEGF) agent. Optionally, the molecule consisting of a monomeric annexin A5 protein and the anti-VEGF agent are combined, administered simultaneously in a separate administration, or administered sequentially in a separate administration, and preferably wherein if the administration is separate and sequential, the time period between the sequential administrations is 28 days or less, such as 3 weeks or less, 2 weeks or less, 1 week or less, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day or less, such as 24 hours, 28 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour or less.

[0055] In a third aspect, the invention provides a composition (such as a unit dose composition) comprising a molecule consisting of monomeric Annexin A5 protein,

[0056] Optionally wherein the composition comprises a molecule consisting of monomeric Annexin A5 protein in an amount of about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg of the subject's body weight, and / or

[0057] Optionally wherein the composition comprises molecules consisting of monomeric Annexin A5 protein in a total amount of about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg, or about 0.001 mg to about 0.1 mg, and

[0058] Optionally wherein the composition is used in preventing or treating macular edema and / or retinal vein occlusion (RVO) in a subject and / or one or more diseases, conditions or disorders associated with and / or caused (directly or indirectly) by macular edema and / or retinal vein occlusion. The term "about" as used herein may be used herein to represent a range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, ±4%, ±3%, ±2% or ±1% of a referenced value.

[0059] In an alternative embodiment of the third aspect, the invention provides a method of treating or preventing macular edema and / or retinal vein occlusion (RVO) and / or one or more diseases, conditions or disorders associated with and / or caused (directly or indirectly) by macular edema and / or retinal vein occlusion in a subject, typically a human subject, the method comprising the step of administering a therapeutically effective amount of a composition comprising a molecule consisting of a monomeric Annexin A5 protein,

[0060] Optionally, wherein the composition comprises a molecule consisting of monomeric Annexin A5 protein in an amount of about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg of subject body weight, and / or

[0061] Optionally, wherein the composition comprises a molecule consisting of monomeric annexin A5 protein in a total amount of about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg, or about 0.001 mg to about 0.1 mg. As used herein, the term "about" may be used herein to represent a range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, ±4%, ±3%, ±2%, or ±1% of the referenced value.

[0062] In one embodiment of the third aspect of the invention, the composition is a unit dose composition and further optionally, the treatment and / or prevention may comprise or consist of administering the entire unit dose to the subject, e.g. in a single administration.

[0063] The composition used according to the third aspect of the present invention may be a pharmaceutical composition.

[0064] Optionally, the composition of the third aspect of the invention further comprises one or more pharmaceutically acceptable carriers.

[0065] For example, the composition may consist of a molecule consisting of monomeric Annexin A5 protein and one or more pharmaceutically acceptable carriers.

[0066] In a fourth aspect, the present invention provides a sterile injectable intravitreal composition (such as a unit dose of a sterile injectable intravitreal composition) comprising a molecule consisting of a monomeric Annexin A5 protein,

[0067] Typically wherein the composition is presented in a sterile syringe, and more preferably wherein the syringe contains a volume of about or less than 200 μL, preferably a volume of about or less than 100 μL of the composition, and more preferably wherein the volume is about 10 μL to about 90 μL, about 20 μL to about 80 μL, about 30 μL to about 70 μL, about 40 μL to about 60 μL or about 50 μL (wherein the term "about" in this context refers to an amount of the specified value ± 4 μL, 3 μL, 2 μL or 1 μL)

[0068] Optionally, wherein the composition comprises a molecule consisting of monomeric annexin A5 protein: (i) 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg of the subject; body weight of the subject, and / or (ii) a total amount of about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg, or about 0.001 mg to about 0.1 mg, wherein the term "about" as used in this context may be used herein to indicate a range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, ±4%, ±3%, ±2% or ±1% of the referenced value, and

[0069] Optionally wherein the composition is used in preventing or treating macular edema and / or retinal vein occlusion (RVO) and / or one or more diseases, conditions or disorders associated with and / or caused (directly or indirectly) by macular edema and / or retinal vein occlusion in a subject.

[0070] The sterile injectable intravitreal composition of the fourth aspect of the invention may be presented in the form of a kit comprising the composition and one or more additional components.

[0071] For example, the kit may comprise a composition presented in a sterile syringe and one or more sterile needles attached thereto or separately contained in the kit. Preferably, the needle or each needle may be a gauge selected from gauge 27, 28, 29 or more preferably 30 or 31. It may be preferred that the needle or each needle has a length of about 0.5 inches to 0.62 inches (12.7 mm to 15.75 mm). Longer needles may increase the risk of retinal damage if the patient accidentally moves forward during the intravitreal injection procedure.

[0072] In another example, the kit can include one or more additional compositions, each comprising one or more antibiotics, for administration to a patient during and / or after intravitreal injection.

[0073] The present application also provides a molecule consisting of monomeric Annexin A5 protein or a composition comprising the molecule, or a use or method for use substantially as described herein in conjunction with the specification.

[0074] definition

[0075] The term "molecule consisting of monomeric annexin A5 protein" as used herein is further defined below in Section B of the detailed description of the invention. It optionally also includes the meaning of a composition comprising, consisting essentially of, or consisting of a molecule consisting of monomeric annexin A5 protein. The composition may be, for example, a pharmaceutically acceptable or veterinarily acceptable composition.

[0076] A "pharmaceutically acceptable" composition may be a composition that is safe for administration to a subject, such as a human subject, by injection, such as intravenous, subcutaneous or intramuscular injection. Most preferably, the injection is intravenous.

[0077] A "veterinarily acceptable" composition may be a composition that is safe for administration to an animal subject, such as by injection (eg, by intravenous, subcutaneous or intramuscular injection). Most preferably, the injection is intravenous.

[0078] Thus, a pharmaceutical or veterinary composition according to the invention may comprise, consist essentially of or consist of a molecule consisting of an Annexin A5 protein in admixture with a pharmaceutically or veterinarily acceptable adjuvant, diluent and / or carrier, which will generally be selected according to the intended route of administration and standard pharmaceutical practice. The composition may be in the form of an immediate, sustained or controlled release application. The formulation may be a unit dose, for example a unit dose of the active ingredient containing a daily dose or unit, daily subdose or an appropriate fraction thereof.

[0079] A "sterile injectable intravitreal" composition may be a composition that is safe for administration to a subject, particularly a human subject, by direct injection into the vitreous of the subject's eye.

[0080] Thus, a "sterile injectable intravitreal composition" according to the invention may comprise, consist essentially of or consist of a molecule consisting of a monomeric annexin A5 protein in admixture with an adjuvant, diluent and / or carrier selected for compatibility and / or tolerability when introduced directly into the vitreous of the eye. The composition may be in the form of an immediate, sustained or controlled release application. The formulation may be a unit dose, e.g., a unit dose containing a daily dose or unit of active ingredient.

[0081] The phrases "pharmaceutically acceptable," "pharmacologically acceptable," and "veterinarily acceptable," where appropriate, refer to compositions that do not produce an adverse, allergic, or other untoward reaction when administered to an animal, such as, for example, a human. The same considerations apply to sterile injectable intravitreal compositions. The preparation of such compositions is known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Edition, Mack Printing Company, 1990, which is incorporated herein by reference. In addition, for animal (e.g., human) administration, it will be understood that formulations should meet sterility, pyrogenicity, general safety, and purity standards, such as those required by the FDA Office of Biological Standards.

[0082] As used herein, "pharmaceutically acceptable carriers" and "veterinarily acceptable carriers" include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, preservatives, drugs, drug stabilizers, excipients, disintegrants, such as materials and combinations thereof, as known to those of ordinary skill in the art. Except in the case where any conventional carrier is incompatible with the active ingredient, its use in treatment or pharmaceutical compositions is contemplated.

[0083] Molecules consisting of monomeric Annexin A5 protein or compositions comprising molecules consisting of monomeric Annexin A5 protein (such as mixed with one or more pharmaceutically acceptable carriers or veterinarily acceptable carriers) can be administered to a subject, for example, parenterally, e.g., intravenously, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or can be administered by infusion techniques.

[0084] According to one option, administration avoids direct administration to the eye, such as by intravitreal injection.

[0085] In another alternative, the molecule consisting of monomeric annexin A5 protein can be administered directly to the eye. For example, in an optionally preferred embodiment, the molecule consisting of monomeric annexin A5 protein can be administered to one or both eyes of a subject by intravitreal injection. According to this embodiment, the monomeric annexin A5 protein can be administered to a patient in the form of a sterile injectable intravitreal composition, such as defined according to the fourth aspect of the invention.

[0086] In another alternative, a molecule consisting of monomeric Annexin A5 protein can be administered directly to a retinal vein (such as the central retinal vein, branch retinal veins and / or hemi-retinal veins) of one or both eyes of a subject, for example, by catheterization.

[0087] Systemic administration, such as intravenous administration, is a preferred embodiment of the first, second and third aspects of the present invention. The molecule composed of monomeric annexin A5 protein or the composition comprising the molecule can be used, for example, in the form of a sterile aqueous solution, which can contain other substances, such as enough salt or glucose to make the solution isotonic with blood. If necessary, the aqueous solution should be appropriately buffered (preferably, buffered to a pH of 3 to 9). For example, optionally, the aqueous solution is buffered at a pH selected from the following range: about pH 4 to about pH 8, about pH 5 to about pH 8, about pH 6 to about pH 8, about pH 6.5 to about pH 7.9, about pH 6.8 to about pH 7.7, about pH 7.0 to about pH 7.5, about pH 7.1 to about pH 7.4, about pH 7.1 to about pH 7.4, and optionally about pH 7.1, 7.2, 7.3 or 7.4, most preferably about pH 7.2. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

[0088] Formulations suitable for parenteral administration generally include: aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that provide formulations that are isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. The formulations may be present in unit dose or multi-dose containers (e.g., sealed ampoules and vials) and may be stored under freeze-dried (lyophilized) conditions that require only the addition of a sterile liquid carrier (e.g., water for injection) just before use. Ready-to-use injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the type previously described.

[0089] In one embodiment, a pharmaceutically acceptable or veterinary acceptable composition (e.g., as can be used in any of the first, second and / or third aspects of the present invention) and / or a sterile injectable intravitreal composition (e.g., as defined according to the fourth aspect of the present invention) can be defined as safe for injection if it does not contain or is substantially free of endotoxin. Endotoxin is often used synonymously with the term lipopolysaccharide, which is the main component of the outer cell wall of Gram-negative bacteria. Lipopolysaccharide consists of a polysaccharide (sugar) chain and a lipid portion known as lipid A, which is responsible for the toxic effects observed with endotoxin. The polysaccharide chain varies greatly in different bacteria and determines the serotype of endotoxin, and the lipid component also varies greatly, so that a single endotoxin sample can contain 10 to 100 different molecular species. The size of endotoxin is about 10kDa, but large aggregates up to 1000kDa can be formed. Endotoxin is usually harmful and pyrogenic in therapeutic compositions, and regulatory agencies have imposed strict restrictions on the endotoxin levels allowed in pharmaceutical compositions. Therefore, the endotoxin level in the composition according to the present invention should be minimized, and can be less than 100 endotoxin units (EU) per dose, such as less than 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1 or less EU per dose. The concentration of endotoxin in the composition according to the present invention can be less than 200EU / m3, such as less than 150EU / m3, 100EU / m3, 90EU / m3, 80EU / m3, 70EU / m3, 60EU / m3, 50EU / m3, 40EU / m3, 30EU / m3, 20EU / m3, 10EU / m3, 5EU / m3, 4EU / m3, 3EU / m3, 2EU / m3, 1EU / m3 or less. Methods for measuring endotoxin levels such as horseshoe crab amebocyte assay (LAL) method are well known in the art.

[0090] Compositions comprising molecules consisting of monomeric annexin A5 proteins may optionally comprise molecules consisting of monomeric annexin A5 proteins as the sole active agent. In other words, in one embodiment, the composition (or pharmaceutical composition) may be composed of the following: molecules consisting of monomeric annexin A5 proteins and one or more pharmaceutically acceptable carriers or one or more or veterinarily acceptable carriers. For example, it may optionally exclude the composition of annexin A5 being formulated, conjugated to the medicament or expressing the fusion protein formed with the medicament together with one or more other proteins or non-protein agents that can provide additional therapeutic effects. The additional therapeutic effect may be present in the case of macular edema and / or retinal vein occlusion or in the case of therapeutic effects unrelated to the treatment of macular edema and / or retinal vein occlusion. For the avoidance of doubt, the molecule consisting of monomeric annexin A5 proteins is not dimeric annexin A5 (commonly referred to as "di-annexin" in the art), and is not pegylated annexin A5.

[0091] Other definitions :

[0092] The term "treatment" will be understood by those skilled in the medical field. As used herein, the term "treatment" may include any treatment of a condition (e.g., where the condition is macular edema or retinal vein occlusion) in a subject, specifically a human or other mammal, and optionally includes one or more of the following effects:

[0093] (i) inhibiting a condition (e.g., macular edema or retinal vein occlusion and / or other diseases, conditions or disorders associated with and / or caused by macular edema or RVO), i.e., slowing, reducing or arresting the progression of the condition;

[0094] (ii) alleviates a disorder (e.g., macular edema or retinal vein occlusion and / or other diseases, conditions or disorders associated with and / or caused by macular edema or RVO), i.e., causes regression of the disorder in a subject suffering from the disorder; or

[0095] (iii) curing a disorder (e.g., macular edema or retinal vein occlusion and / or other diseases, conditions or disorders associated with and / or caused by macular edema or RVO), i.e., returning a subject suffering from the disorder to a state of health in which the disorder is no longer detectable.

[0096] The term "prevention" will be understood by those skilled in the art of medicine. As used herein, the term "prevention" may include any prophylactic treatment of a condition (e.g., wherein the condition is retinal vein occlusion) in a subject, specifically a human or other mammal, and optionally includes one or more of the following effects:

[0097] (i) preventing a subject (e.g., a subject who may be predisposed to or at risk of developing a disorder but has not yet been diagnosed as having the disorder) from developing a disorder (e.g., macular edema or retinal vein occlusion and / or other diseases, conditions or disorders associated with and / or caused by macular edema or RVO), i.e., arresting the subject from developing the disorder;

[0098] (ii) delaying the onset of a disorder (e.g., macular edema or retinal vein occlusion and / or other diseases, conditions or disorders associated with and / or caused by macular edema or RVO) in a subject, i.e., delaying the subject from developing the disorder until later in the subject's life;

[0099] (iii) limiting the occurrence of a disorder (e.g., macular edema or retinal vein occlusion and / or other diseases, conditions or disorders associated with and / or caused by macular edema or RVO) in a subject, such as reducing the extent to which a subject is affected by the disorder; or

[0100] (iv) preventing one or more symptoms of a disorder (e.g., macular edema or retinal vein occlusion and / or other diseases, conditions or disorders associated with and / or caused by macular edema or RVO) in a subject, i.e., arresting the subject from developing one or more symptoms of the disorder.

[0101] In the case of preventing another disease, condition or disorder associated with and / or caused by macular edema or RVO, the subject may optionally have, be suspected of having, and / or be diagnosed as having macular edema or RVO.

[0102] The terms "optionally", "preferably", "such as", "for example", etc. indicate that the subsequently described feature, characteristic, event or circumstance may or may not occur, and that the description includes examples where the feature, characteristic, event or circumstance occurs and examples where the feature, characteristic, event or circumstance does not occur. For the avoidance of doubt, it should be understood that in some embodiments of the present invention, any feature, characteristic, event or circumstance described in the context of "optionally", "preferably", "such as", "for example", etc. may be excluded from the scope of protection of the claims.

[0103] The term "subject" as used herein includes the meaning of any living human or animal to which the treatment of the present invention is applied. Without limitation, exemplary subjects are further defined below in Section C of the detailed description of the present invention. Most preferably, the subject is a human subject.

[0104] As used herein, the term "elevated" includes the meaning that a substance (e.g., a cell type such as red blood cells (i.e., erythrocytes), or a marker on a cell such as an Annexin A5 binding site on red blood cells) is present in a higher amount in a subject with macular edema and / or retinal vein occlusion compared to the amount present in a subject without macular edema and / or retinal vein occlusion, or the average of a group of subjects (e.g., at least 10,000 or 1000 subjects).

[0105] As used herein, the term "distal" includes the meaning that the administration of a molecule consisting of monomeric Annexin A5 protein is not directly administered to or into an eye that has been diagnosed with macular edema and / or retinal vein occlusion.

[0106] The term "therapeutically effective amount" refers to an amount of a drug, compound, protein (e.g., a molecule consisting of monomeric Annexin A5 protein as defined herein), or pharmaceutical composition sufficient to achieve a beneficial effect or desired result, including a clinical result associated with treating or preventing retinal vein occlusion and / or other diseases, conditions or disorders associated with and / or caused by macular edema and / or retinal vein occlusion. An effective amount can be administered in one or more administrations. For the purposes of the present invention, an effective amount of a drug, compound, protein (e.g., a molecule consisting of monomeric Annexin A5 protein as defined herein), pharmaceutical composition or veterinary composition is an amount sufficient to treat, ameliorate, reduce the intensity of and / or prevent macular edema and / or retinal vein occlusion and / or other diseases, conditions or disorders associated with and / or caused by macular edema and / or retinal vein occlusion. As understood in a clinical setting, an effective amount of a drug, compound, protein (e.g., a molecule consisting of monomeric Annexin A5 protein as defined herein), or pharmaceutical composition may or may not be achieved when administered in combination (such as separately, sequentially, or simultaneously) with another drug, compound, protein, or drug, such as those described elsewhere in this application. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the desired result can be achieved or is achieved in combination with one or more other agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0107] Figure 1The results of the phosphatidylserine (PS) assay are shown. The assay uses FITC-Annexin A5 ("ANXV") and PE-anti-CD235a as positive markers for RBCs. Panel A: In the negative control, Ca was absent. 2+ , while in all other samples there was Ca 2+ To achieve ANXV-800CW binding. Panel B: shows PS expression at the following time points: screening, imaging day, follow-up.

[0108] Figure 2 Fluorescein angiography (FA) using intravenously administered fluorescein prior to systemic administration of a 1 mg steady-state dose of ANXV-800CW is shown. The red arrow marks the location where the artery extends over the vein, causing the blockage. The darker area marked by the red oval corresponds to the damaged area where bleeding is present.

[0109] Figure 3 Near infrared (NIR) fluorescence images obtained at t = 5 minutes after systemic injection of 1.0 mg ANXV-800CW are shown (Panel B). NIR image t = 229 minutes (Panel A). The red arrow indicates the occlusion site, while the yellow arrow marks the vein anterior to the occlusion site in the damaged area where bleeding is present. The light blue arrow indicates the large vein in the optic nerve. The darker area surrounded by the red oval corresponds to the damaged area where bleeding is present. The oval marks the Figure 2 The red ovals in Figure 1 are in approximately the same anatomical location. The settings used with the Heidelberg Spectralis camera system were similar for Figures A and B.

[0110] Figure 4 It is shown that application of TNFα resulted in a decrease in cell viability (column 2) compared to control cells to which TNFα had not been applied (column 1), whereas application of Annexin A5 plus TNFα inhibited cell death (column 3), resulting in cell viability comparable to levels seen in the control.

[0111] Figure 5It is shown that when treated with annexin A5 protein, cell viability increases in a dose-dependent manner. The application of IL1β leads to a decrease in cell viability. This situation is shown in the figure. For each experiment, the flow cytometry curve of the sample to which only IL1β (marked on the figure) is applied is offset to become the curve farthest from the y-axis, while the control cells to which IL1β has not yet been applied are in the center of the figure. This specific positioning of those curves shows that cell death occurs at a higher level when IL1β is applied. It is also shown that the application of only annexin A5 protein at all three doses does not cause an increase in cell death compared to the control sample, because the control curves and annexin A5 protein curves for all three doses are in the same position. Finally, the application of annexin A5 plus IL1β inhibits cell death in a dose-dependent manner, with doses of 20 μg and 5 μg showing the same level of cell viability observed in the control. This is shown as the flow cytometry curves for the 20 μg and 5 μg doses of Annexin A5 are in the center of the graph (marked on the graph), consistent with the control curve and the Annexin A5 protein only curve. The 100 ng dose also inhibited cell death, but to a lesser extent, as can be seen from the 100 ng flow cytometry curve (marked on the graph) being in the middle of the graph between the IL1-β only curve.

[0112] Figure 6 It is shown that the expression of ICAM1 is reduced when treated with Annexin A5. Experiments have been performed on cell cultures using flow cytometry. The application of TNFα increases the surface expression of ICAM1 (column 2) compared to control cells to which TNFα has not been applied (column 1). The application of Annexin A5 plus TNFα reduces the surface expression of ICAM1 (column 3).

[0113] Figure 7 It is shown that the expression of IL-6 is reduced in a dose-dependent manner when treated with Annexin A5. The application of IL-1β leads to an increase in IL-6 expression (column five) compared to the control sample to which IL-1β has not been applied (column one). The application of Annexin A5 protein alone did not increase the expression of IL-6 at all doses (columns one to four). The application of Annexin A5 plus IL-1β reduces IL-6 gene expression in a dose-dependent manner (columns five to eight). DETAILED DESCRIPTION

[0114] The established standard of care for delivering therapeutic agents (such as anti-VEGF therapeutic agents) directly to the eye for macular edema and / or RVO, and indeed in a variety of other retinal diseases, is by intravitreal injection (Haydinger et al., 2023, supra; Ip & Hendrick, 2018, supra; Cox et al., 2021, J. Clin. Med., 10(5):981). Such drugs are injected directly into the eye because the retina is located at the back of the eye and is surrounded by several protective layers, including the cornea, iris, and lens. These layers can make it difficult for the drugs to reach the retina when the drugs are administered by other routes. When the drugs are injected into the eye, they are delivered directly to the retina and can act locally to target the source of the problem. The present invention is based on the surprising determination that monomeric annexin A5 protein, when administered systemically to a subject (such as by intravenous administration), is able to preferentially localize to the site of occlusion in a subject with retinal vein occlusion (RVO).

[0115] It has also been determined that the modulation of biological activity provided by Annexin A5 has been identified as contributing to dysregulation of the blood-retinal barrier (BRB) in subjects with macular edema and / or RVO, as well as various factors that contribute to other therapeutic benefits in subjects with macular edema and / or RVO. The inventors have also determined that prevention and / or treatment of macular edema, retinal vein occlusion, and / or other diseases, conditions, or disorders associated with and / or caused by macular edema and / or retinal vein occlusion can be achieved with a range of doses of Annexin A5, including, but not limited to, low doses of monomeric Annexin A5 protein.

[0116] A. Macular edema

[0117] Macular edema is pathological fluid accumulation in the macula, which is the central area of ​​the retina essential for high-acuity vision. Macular edema is a common complication of many eye diseases, such as diabetic retinopathy, retinal vascular occlusion, uveitis, inflammation after cataract surgery (pseudophakic macular edema or Irvine-Gass syndrome), retinal dystrophy, drug reaction, intraocular tumor, serous central chorioretinopathy, radiation retinopathy and other retinal vascular abnormalities, including retinal aneurysms and retinal telangiectasia (Tranos et al., 2004, Surv Ophthalmol, 49: 470-90). Subretinal neovascular membranes observed in conditions such as age-related macular degeneration (AMD) are also associated with intraretinal and subretinal fluid accumulation (Trichonas et al., 2014, Br J Ophthalmol.98: ii24).

[0118] Symptoms of macular edema include metamorphopsia, microsomia, blurred vision, central scotoma, and decreased contrast or color sensitivity (Haydinger et al., 2023).

[0119] Optionally, according to various aspects of the invention, treatment of macular edema or a condition associated therewith begins within 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours, or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days of the onset of one or more symptoms of macular edema.

[0120] Clinical diagnosis of macular edema can be challenging in mild cases or when fundus visualization is impaired due to poor pupil dilation, cataracts, and other opacities of the ocular media. Therefore, diagnosis is usually made using fluorescein angiography and optical coherence tomography (OCT). The latter is preferred due to its non-invasiveness, accurate measurement of retinal thickness, and ability to identify other structural abnormalities (such as epiretinal membranes and vitreomacular traction), and it is the standard method for evaluating macular edema in clinical trials.

[0121] Thus, in a preferred embodiment, the subject who has been diagnosed as having macular edema is a subject in which macular edema has been diagnosed by fluorescein angiography and / or optical coherence tomography.

[0122] Optionally, according to the present invention, treatment of a subject's macular edema, or a condition associated therewith, begins within 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days of diagnosis of macular edema.

[0123] The thickness of the retina is usually measured between the inner limiting membrane and Bruch's membrane. With the current spectral domain OCT technology, the average and central (foveal) macular thickness in normal adult eyes are 334 μm and 226 μm, respectively, and men are thicker than women. Three modes of macular edema have been described using OCT: cystoid macular edema, diffuse macular edema, and subretinal detachment. Diffuse retinal thickening is the main mode in diabetic macular edema, while in retinal vascular occlusion, subretinal fluid accumulation is common. In uveitis, cystoid macular edema and diffuse macular edema are typical forms of macular edema, while subretinal detachment is seen in less than one-third of cases, usually in combination with cystoid or diffuse edema. In some embodiments of the present invention, macular edema is selected from the group consisting of one or more of the following: cystoid macular edema, diffuse macular edema, and subretinal detachment.

[0124] The deformation of the retinal structure caused by fluid accumulation in the macula leads to vision loss, and when the disease becomes chronic, it leads to potentially irreversible visual impairment due to scarring. Macular edema is the main cause of vision loss in patients with diabetes, retinal vein occlusion and uveitis. Population studies have shown that the average prevalence of diabetic macular edema is about 6% in type 1 diabetes and slightly higher in type 2 diabetes. Macular edema is extremely common in central retinal vein occlusion, while in branch retinal vein occlusion, macular edema occurs in 30% of cases. About one-third of uveitis patients develop macular edema, which is more frequent in intermediate uveitis and panuveitis and less common in anterior uveitis. The burden of macular edema is not only related to the reduced quality of life experienced by patients, but also to the long-term nature of the disease and the economic costs associated with treatment and frequent visits to medical centers. Especially in uveomacular edema, the most affected population is the working-age population, increasing the socioeconomic impact of the disease.

[0125] In some embodiments of the invention, a subject suffering from, diagnosed as suffering from, or suspected of having macular edema can be a subject suffering from, has been diagnosed as suffering from, or suspected of having one or more conditions selected from the group consisting of diabetes, diabetic retinopathy, retinal vein occlusion (e.g., CRVO, BRVO and / or HRVO) and / or uveitis (e.g., intermediate uveitis and / or panuveitis).

[0126] In some embodiments of the present invention, a subject having, diagnosed as having, or suspected of having macular edema can be a subject who has received, is receiving, or will receive a related therapy for macular edema, such as a treatment selected from the group consisting of: anti-VEGF therapy (e.g., anti-VEGF therapy delivered to the eye by intravitreal injection), corticosteroids, NSAIDs, pars plana vitrectomy, retinal laser photocoagulation, or anti-VEGF therapy enhanced with intravitreal corticosteroids and / or retinal laser photocoagulation.

[0127] Exemplary anti-VEGF therapies may include any one or more of the following:

[0128] Ranibizumab, an FDA-approved anti-VEGF drug, is a recombinant humanized antibody fragment directed against VEGF.

[0129] aflibercept, another FDA-approved anti-VEGF drug, is a recombinant protein comprising the ligand-binding domains of VEGF receptors 1 and 2 fused to the Fc portion of immunoglobulin G1,

[0130] bevacizumab, a humanized monoclonal antibody against VEGF that is widely used off-label (Haydinger et al., 2023, supra; Ip & Hendrick, 2018, supra), and / or

[0131] Brolucizumab and / or faricimab, which are newer anti-VEGF drugs.

[0132] Corticosteroids are commonly used to manage macular edema, particularly in subjects with non-infectious uveitis. Exemplary corticosteroids include difluprednate, triamcinoloneacetonide, triamcinoloneacetonide and dexamethasone, which can be used as monotherapy, or used in combination with systemic immunomodulatory therapy to achieve faster edema regression. The most common complications of topical corticosteroid treatment are increased intraocular pressure and cataract. The systemic use of corticosteroids is mainly applicable to bilateral macular edema and should be used for a limited time due to a wide range of side effects (Foster et al., 2016, Surv Ophthalmol, 61: 1-17).

[0133] Nonsteroidal anti-inflammatory drugs (NSAIDs) have an adjunctive role in treatment approaches and are primarily used for pseudophakic macular edema. Acetazolamide, a carbonic anhydrase (CA) inhibitor, is sometimes used in cases where corticosteroid therapy has failed, but is sometimes used as primary therapy for pseudophakic macular edema or macular edema associated with retinal dystrophies.

[0134] Pars plana vitrectomy is primarily reserved for treating mechanical factors that contribute to macular edema, such as epiretinal membranes and vitreomacular traction. Due to its inherent risks (including retinal breaks and detachments, vitreous hemorrhage, cataracts, glaucoma, and hypotony) and limited data on outcomes, it is a last resort for treating macular edema in the absence of mechanical factors.

[0135] Fluid can diffusely accumulate in the central retina or in cysts usually located in the inner nuclear layer or Henle's fiber layer, or accumulate in the subretinal space, causing disruption and distortion of retinal architecture and reduced visual acuity (1,2). In combination with the underlying pathology, edema can progress to irreversible tissue damage with retinal cell death and permanent visual impairment. A common feature of most diseases causing macular edema is dysfunction of the blood-retinal barrier (BRB), a set of structures that tightly regulates the passage of proteins, salts, metabolites, and other solutes between the blood and retinal tissue under healthy conditions. Angioactive growth factors and inflammatory cytokines, including vascular endothelial growth factor-A (VEGF) and tumor necrosis factor (TNF)-α, are upregulated in ocular fluid in macular edema and have been implicated in disruption of the BRB. Dysregulated entry and accumulation of solutes in the retina disrupts the balance of osmotic and hydrostatic forces and results in fluid entry when mechanisms maintaining fluid balance are overcome. Current treatments target proteins and processes involved in angiogenesis, inflammation, and blood-retinal barrier (BRB) dysfunction. This review provides an overview of the clinical aspects of macular edema and discusses the cellular and molecular mechanisms targeted by current treatments.

[0136] Conditions associated with macular edema include, but are not limited to, any one or more of the following: dysregulation of the blood-retinal barrier (BRB), such as dysregulation of the inner BRB and / or the outer BRB; dysfunction of the barrier between the vitreous and blood vessels at the ciliary body; eye diseases associated with macular edema, such as diabetic retinopathy, retinal vascular occlusion, uveitis, inflammation following cataract surgery (pseudophakic macular edema or Irvine-Gass syndrome), retinal dystrophy, drug reactions, intraocular tumors, serous central chorioretinopathy, radiation retinopathy and other retinal vascular abnormalities, including retinal macroaneurysms and retinal telangiectasias; subretinal neovascular membrane changes, metamorphopsia, microsomia, blurred vision, central scotoma, and decreased contrast or color sensitivity observed in conditions such as age-related macular degeneration (AMD).

[0137] Mechanisms of macular edema

[0138] Macular edema is the result of the failure of the mechanism that maintains fluid homeostasis in the macula (Haydinger et al., 2023, supra). The movement of fluid through capillaries throughout the body is classically described by the Starling force. The hydrostatic pressure gradient between the intravascular space and the extracellular space of the tissue tends to drive fluid into the tissue, and this is opposite to the colloid osmotic pressure (swelling pressure) gradient that tends to draw fluid from the extracellular space into the blood vessels. Cunha-Vaz's latest review discusses the formation of macular edema in the context of the classic Starling force (Ophthalmologica, 2017, 237: 1-10). The relative increase in hydrostatic pressure in the blood vessels relative to the retinal tissue and the relative increase in swell pressure in the retinal tissue relative to the blood vessels are two forces that drive edema. Among them, macular edema in most diseases is believed to be caused by an increase in tissue swell pressure due to abnormal accumulation of solutes in the extracellular space of the retina (Haydinger et al., 2023, supra). The compliant nature of the retinal tissue, which allows unimpeded expansion into the vitreous, makes it particularly susceptible to edema, as increases in tissue turgor pressure readily induce increases in tissue volume.

[0139] Dysregulated solute entry into the retina occurs due to disruption of the blood-retinal barrier (BRB), and this has been the primary focus of most efforts to understand the mechanism of the disorder (Haydinger et al., 2023, supra).

[0140] The BRB is a group of structures in the eye that separates the blood from the neural retina and the retinal extracellular space. There are two components: the inner BRB and the outer BRB. The inner BRB is based on the retinal endothelial cells (REC) lining the retinal blood vessels, and the outer BRB is formed at the level of the retinal pigment epithelium (RPE) cells. Since there may be non-selective bidirectional diffusion of solutes between the retinal extracellular space and the vitreous, the barrier process between the vitreous and the blood vessels at the ciliary body may also be considered to form part of the BRB, but this area is not grouped in the inner BRB or the outer BRB.

[0141] The retinal blood vessel wall forms the inner BRB. The luminal surface of the retinal blood vessel is continuously lined with a squamous REC layer, and the membranes of adjacent RECs are kept together by a continuous network of tight junctions. RECs are not porous, and they contain very few endocytic vesicles. RECs and their connection form a barrier (Cunha-Vaz et al., 1966, Br J Ophthalmol., 50: 441-53) that promotes the regulated passage of solutes between the blood and the retina. The basement membrane completely covers the luminal surface of RECs. Evidence collected in vivo indicates that the basement membrane does not directly constitute a barrier, but some evidence indicates that it can prevent molecular tracers from diffusing in vitro. Larger blood vessels have additional layers of smooth muscle, basement membrane, and adventitia, which are structurally different according to the size and type of blood vessels.

[0142] The outer BRB is not located at the vessel wall, but is formed by the retinal pigment epithelium, which is a monolayer of RPE cells located behind the photoreceptors and directly opposite to Bruch's membrane, which separates it from the vascular bed of the choriocapillaris (Haydinger et al., 2023, supra). In contrast to the inner BRB, the vessels of the choroid have thin and highly fenestrated endothelial cells and do not form a selective barrier. Therefore, the epithelium is responsible for maintaining the regulated environment required for retinal function.

[0143] Dysfunction of the BRB is the basis for the development of macular edema (Haydinger et al., 2023, supra). The selectivity of molecular exchange between blood and retinal tissue at the level of inner or outer BRB allows proteins and other solutes to enter retinal tissue. Usually leakage is obvious before macular edema forms. When the severity of leakage overcomes the solute and fluid clearance mechanism, edema eventually occurs. The clearance rate of solutes from the subretinal space is inversely proportional to their size. Different sizes, labeled dextran tracers injected into patients with uveal macular edema show leakage rates roughly proportional to the size of the tracer. Up to 20kDa size leaks into the macula, but 150kDa does not leak (Atkinson et al., 1991, Eye, 5: 440-6). These data indicate that although the infiltration of larger solutes may pose a greater challenge to clearance, complete BRB destruction allows maximum solute non-selective entry is not necessary for edema formation.

[0144] Evidence suggests that several inflammatory cytokines play a role in disrupting BRB integrity (Haydinger et al., 2023, supra; Park et al., 2019, J Diabetes Res, 2019:813741; Noma et al., 2020, J. Clin. Med, 9(1):3457).

[0145] VEGF plays a major role in driving the progression of retinal diseases and macular edema, as irrefutably demonstrated by the broad efficacy of anti-VEGF therapy (Haydinger et al., 2023, supra; Park et al., 2019, supra; Noma et al., 2020, supra).

[0146] Intravitreal levels of VEGF and IL-6 correlate with the severity of macular edema and the size of the non-perfused area (Noma et al., 2009, Ophthalmology, 116:87-93; Noma et al., 2006, Graefes Arch Clin Exp Ophthalmol., 244:309-15). Evidence implicates IL-6 in diseases that cause macular edema (Haydinger et al., 2023, supra). It is elevated in patients with uveitis (van Kooij et al., 2006, Am J Ophthalmol. 142:192-4). It is elevated in diabetic macular edema (Funatsu et al., 2002, Am J Ophthalmol. 133:70-7) and is associated with macular thickness (Oh et al., 2010, Curr Eye Res. 35:1116-27). IL-6 is also upregulated in patients with central retinal vein occlusion, especially those with ischemia (Noma et al., 2009, Ophthalmology. 116: 87-93). Its expression is often correlated with that of VEGF (Haydinger et al., 2023, supra). In addition, it is proposed that VEGF increases vascular permeability and further increases the expression of inflammatory cytokines (including MCP-1 and ICAM-1 that destroy the BRB), leading to the onset and progression of macular edema (Noma et al., 2020, supra).

[0147] Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) have also been implicated in the mechanism of BRB dysfunction in macular edema (Haydinger et al., 2023, supra).

[0148] TNF-α is a major inflammatory cytokine that has been shown to increase the barrier permeability of REC monolayers in vitro (Haydinger et al., 2023, supra; Aveleira et al., 2010, Diabetes.59:2872-82). When injected into the vitreous of rabbits, TNF-α increases the proportion of REC tight junctions that appear to be open, and albumin is detected in these connections, indicating leakage (Luna et al., 1997, J Neurosci Res.49:268-80). In RECs in the treated eye, larger vesicles also stain positive for albumin, but not in the control. These results indicate the destruction of both transcellular and paracellular pathways. Interestingly, 24 hours after injection, the tight junction changes induced by TNF-α have been reversed, which is a promising signal for potential therapeutic targets. When injected into the vitreous of rats, it also induced barrier leakage, and the injection effect was restored after 7 days (Bamforth et al., 1996, Acta Neuropathol. 91: 624-32). Huang et al. used two diabetic mouse models to evaluate the pathological effects of TNF-α (Ophthalmol Vis Sci. (2011) 52: 1336-44); TNF-α knockout completely prevented vascular leakage in the late stage of the disease.

[0149] Along with TNF-α, another major inflammatory cytokine of increasing interest is IL-1β. Similar to TNF-α, when injected into rabbit vitreous, IL-1β increases the number of open, leaky tight junctions (140). It also increases the permeability of REC monolayers in vitro (Aveleira et al., 2010, supra). In streptozotocin-diabetic rats, retinal IL-1β expression increases between 1.5 and 4.5 months of diabetes, and IL-1β mRNA is induced in vitro by high glucose in bovine REC (Liu et al., 2012, PLoS One.7: e36949); it induces its own expression in REC, Müller cell and brain astrocytes in vitro, which is prevented by inhibiting protein kinase C.

[0150] B. Retinal vein occlusion

[0151] Types of Retinal Vein Occlusion

[0152] Aspects of the invention relate to the prevention and treatment of retinal vein occlusion and / or one or more diseases, conditions or disorders associated with and / or caused by retinal vein occlusion.

[0153] Retinal vein occlusion (sometimes called an eye stroke) is a common retinal vascular condition and is one of the most common causes of vision loss in the world. Retinal vein occlusion is generally considered to be caused by an obstruction (i.e., blockage) of the retinal venous system, such as by thrombosis. Other possible causes of retinal vein occlusion are considered to be venous obstruction caused by external pressure on the retinal veins or diseases of the retinal vein walls, such as vasculitis ("Guidelines for Retinal Vein Occlusion (RVO)", supra).

[0154] It is known that red blood cells can clot to form thrombi, and that this is sometimes mediated by exposed phosphatidylserine on the surface of red blood cells. Without being bound by theory, the applicants have characterized that the role of Annexin A5 in masking (in other words, covering or shielding) the phosphatidylserine on the surface of those red blood cells can prevent and treat thrombosis in retinal vein occlusion. By characterizing the role of exposed phosphatidylserine in thrombosis in retinal vein occlusion, the applicants have identified that patients can be treated with substantially less monomeric Annexin A5 than has previously been thought to be effective in the art.

[0155] In one embodiment, retinal vein occlusion is caused by venous obstruction. For example, retinal vein obstruction may be associated with one or more of the following: formation of one (or more) thrombi; significantly enhanced red blood cell aggregation and / or blood hyperviscosity; external pressure on the vein; and disease of the vein wall (such as vasculitis). Most preferably, retinal vein obstruction is associated with thrombus formation.

[0156] "Associated" includes that the retinal vein occlusion is caused by a specific mechanism (such as the formation of a thrombus); and / or the retinal vein occlusion is suspected to be caused by a specific mechanism; and / or the retinal vein occlusion is diagnosed to be caused by a specific mechanism. As discussed herein, those skilled in the medical field know how to identify whether the retinal vein occlusion is associated with a specific mechanism.

[0157] As known to those skilled in the medical field, thrombosis is generally considered to be a blood clot formed in a blood vessel (such as a vein) by the aggregation of platelets and / or red blood cells, which obstructs blood flow. Therefore, "thrombus" includes a blood clot that obstructs blood flow formed in a blood vessel (such as a vein).

[0158] Subjects can be classified as having significantly enhanced red blood cell aggregation and / or blood hyperviscosity by any appropriate method. The enhancement is compared with healthy subjects, or compared with the mean value of a healthy subject group (such as a group of 10, 50 or 100 healthy subjects). For example, the level of red blood cell aggregation and / or blood hyperviscosity can be measured by determining the level of adhesion of red blood cells to endothelial cells. Suitable methodology is, for example, described in Wauter et al., 2011, J. Thrombosis and Hemostasis, 9: 1049-1055, the contents of which are incorporated herein by reference for describing red blood cell adhesion methods.

[0159] More specifically, in one option, the level of adhesion of erythrocytes to endothelial cells can be determined under static conditions. For example, this can involve the use of radiometric techniques as described by Kuypers et al., 1996, Blood, 87: 1179-87 (the contents of which are incorporated herein by reference). Human microvascular endothelial cells (HMEC-1) can be cultured; washed erythrocytes are resuspended (e.g., in Hanks balanced salt solution (HBSS) plus 0.5% of an appropriate marker (e.g., with 51 The HMECs are incubated with 5% erythrocytes (e.g., chromium-labeled) human serum albumin to a hematocrit of 25%) and incubated with the HMECs (e.g., at 37° C. for 30 minutes). Non-adherent erythrocytes can be removed by washing, and the remaining adherent erythrocytes can be detected. Such detection can, for example, include lysing the remaining adherent erythrocytes (e.g., with distilled water) and measuring the radioactivity in the lysate. Using such techniques, the radioactivity can be measured, for example, per mm 2 The degree of adhesion was calculated by counting the number of adhered erythrocytes.

[0160] Under such static conditions, for example, a healthy individual may typically exhibit 3,700 ± 300 red blood cells / mm 2 , while the more common CRVO patients may demonstrate 13,500 ± 700 RBCs / mm 2 . When adhesion to endothelial cells is assessed under static conditions, e.g., when determined by the techniques described above, subjects treated in accordance with the invention can optionally be classified as having an erythrocyte count that exhibits greater than 4,000, such as about 4,500, about 5,000, about 5,500, about 6,000, about 6,500, about 7,000, about 7,500, about 8,000, about 8,500, about 9,000, about 9,500, about 10,000, about 10,500, about 11,000, about 11,500, about 12,000, about 12,500, about 13,000, about 13,500, about 14,000, about 14,500, about 15,000, about 15,500, about 16,000, about 16,500, about 17,000 or more erythrocytes / mm2 The term "about" in this context means ±500, 400, 300, 200, 100, 50 or fewer red blood cells / mm 2 The value of .

[0161] In another selection, the level of adhesion of erythrocytes to endothelial cells can be determined under flow conditions. For example, this can involve the adhesion of erythrocytes to HMEC under flow conditions, as determined according to Wautier et al., 2007, Blood, 110: 894-901, the contents of which are incorporated herein by reference. For example, HMEC can be cultured (such as in a glass capillary coated with gelatin (2%) for 24 hours), and then a microslide containing fused HMEC is fixed on the stage of a video microscope, and a red blood cell suspension (such as hematocrit 0.5%) is perfused through the microslide at a suitable flow rate (such as a rate equivalent to a wall shear stress of 0.03Pa) for a suitable time (e.g., 10 minutes), followed by elimination of non-adherent cells (e.g., 10 minutes). The wall shear stress can then be gradually increased (e.g., from 0.03Pa to 0.3Pa, increasing every 5 minutes). At each step, adherent erythrocytes can be recorded (e.g., by video imaging in 10 consecutive fields of view, where one field of view measures 0.1 mm 2 ) and counted (e.g. using a computerized image analysis system) and the results can be expressed as per mm 2 The number of red blood cells.

[0162] Under such flow conditions, for example, when adhered at 0.03 Pa and analyzed for elimination at 0.07 Pa, a healthy individual may typically exhibit 30 ± 4 erythrocytes / mm 2 , while the more common CRVO patients may show 190 ± 8 red blood cells / mm 2 When adhesion to endothelial cells is assessed under flow conditions, e.g., when determined by the techniques described above, subjects treated in accordance with the invention can optionally be classified as having an erythrocyte count greater than 35, such as about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200 or more red blood cells / mm 2 The term "about" in this context means ±5, 4, 3, 2 or 1 red blood cells / mm 2 The value of .

[0163] Under such flow conditions, for example, when adhered at 0.03 Pa and analyzed for elimination at 0.3 Pa, healthy individuals may typically demonstrate less than 1 red blood cell / mm 2 , while the more common CRVO patients may show 20 ± 8 red blood cells / mm 2 When adhesion to endothelial cells is assessed under such flow conditions, e.g., when determined by the techniques described above, subjects treated in accordance with the invention can optionally be classified as having a flow rate that exhibits greater than 1, such as about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, or more red blood cells / mm 2 The term "about" in this context means ±50%, 40%, 30%, 20%, 10% or less of the specified value of red blood cells / mm 2 The value of .

[0164] There are three veins in the retina that are commonly associated with retinal vein occlusion, these are the central retinal vein, semicentral retinal veins or branch retinal veins ("Guidelines for Retinal Vein Occlusion (RVO)", The Royal College of Ophthalmologists, July 2015).

[0165] The central, hemicentral, and branch central veins provide the names for the subtypes of retinal vein occlusion, which are central retinal vein occlusion (CRVO), hemicentral retinal vein occlusion (HRVO), and branch retinal vein occlusion (BRVO).

[0166] Central retinal vein occlusion is caused by obstruction of the central retinal vein, especially where it passes through the lamina cribosa. Hemicentral retinal vein occlusion is caused by obstruction of the retinal vein in the superior or inner retinal hemisphere. Branch retinal vein occlusion is caused by obstruction at the arteriovenous crossing, where the retinal vein and retinal artery share a common vascular sheath.

[0167] Thus, in one embodiment, the retinal vein presenting with RVO and / or targeted by the present invention for prevention or treatment is selected from one or more of the following: central vein; semi-central vein; and branch central vein. In other words, in one embodiment, the retinal vein occlusion is one or more of the following: central retinal vein occlusion, semi-central retinal vein occlusion, and branch retinal vein occlusion.

[0168] Preferably, the retinal vein occlusion is a central retinal vein occlusion.

[0169] Optionally, according to various aspects of the invention, treatment of retinal vein occlusion (RVO) or macular edema in a subject with RVO is initiated within 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days of symptom onset of the retinal vein occlusion. More preferably, according to the present invention, treatment of retinal vein occlusion or macular edema in a subject with RVO is initiated within 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, or 60 minutes, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours, or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days of diagnosis of retinal vein occlusion.

[0170] Retinal vein occlusion can also be classified as ischemic retinal vein occlusion or non-ischemic retinal vein occlusion. This classification is generally associated with areas of capillary non-perfusion, as discussed herein. Thus, in one embodiment, retinal vein occlusion is ischemic retinal vein occlusion (such as ischemic central retinal vein occlusion) or non-ischemic retinal vein occlusion (such as non-ischemic central retinal vein occlusion).

[0171] Retinal vein occlusion can also be classified as acute retinal vein occlusion. In acute retinal vein occlusion, RVO can develop due to severe grades of ischemia in the retinal veins, and the onset of symptoms is rapid. Therefore, it is necessary to provide treatment as quickly as possible after the onset and / or diagnosis of acute retinal vein occlusion. Therefore, in one embodiment, the retinal vein occlusion is acute retinal vein occlusion, such as acute central retinal vein occlusion. Preferably, according to the present invention, the treatment of acute retinal vein occlusion begins within 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours or 24 hours, or within 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days of the onset of symptoms of acute retinal vein occlusion. More preferably, according to the present invention, treatment of acute retinal vein occlusion is initiated within 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes or 60 minutes, or within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours of diagnosis of the acute retinal vein occlusion.

[0172] Retinal vein occlusion can affect one eye (or one retina) (referred to as unilateral retinal vein occlusion) or both eyes (or both retinas) (referred to as bilateral retinal vein occlusion). Thus, in one embodiment, the retinal vein occlusion is a unilateral retinal vein occlusion (such as a unilateral central retinal vein occlusion). In another embodiment, the retinal vein occlusion is a bilateral retinal vein occlusion (such as a bilateral central retinal vein occlusion).

[0173] Diagnosis and manifestations of retinal vein occlusion

[0174] It is known to those skilled in the art of medicine how to diagnose retinal vein occlusion, as well as the subtypes of central retinal vein occlusion, semi-central retinal vein occlusion, and branch retinal vein occlusion. Examples of such diagnoses are discussed in the "Guidelines for Retinal Vein Occlusion (RVO)" (supra). Typically, patients with retinal vein occlusion present to their physicians with changes in vision, such as blurred vision, loss of a portion of the visual field, floating spots in the visual field, flashes of light in the visual field, decreased or diminished vision (i.e., decreased or diminished visual acuity), and loss of vision (i.e., blindness). In some cases, the loss of vision is sudden. In addition, in some cases, the patient does not show any pain.

[0175] Some of the features of retinal vein occlusion that can be used for diagnosis are as follows: optic disc edema (i.e., swelling of the optic disc); increased dilation of the retinal veins; increased tortuosity of the retinal veins; ocular hemorrhages; macular edema; hemorrhages; "cotton-wool spots" (these are white spots on the retina that can be observed on a fundus examination of the retina); retinal edema; electrodiagnostic test (ERG) results; pupillary afferent defect; capillary nonperfusion; arteriolar stenosis; venous obstruction; and retinal pigment epithelial changes. These features can usually be observed by a physician by examining the fundus (the inner surface of the eye, opposite the lens) or the retina using ophthalmoscopy and / or fundus photography. Another diagnostic method used, especially for identifying venous obstruction, is angiography (such as angiography with a fluorescent dye (e.g., fluorescein)). In the case of fluorescent dye angiography, a dye is injected into the patient, and the behavior of the dye in the veins of the eye can be used to identify whether there is an obstruction in the veins.

[0176] In one embodiment, the subject has been diagnosed with retinal vein occlusion; such as central retinal vein occlusion, semi-central retinal vein occlusion, and branch retinal vein occlusion; preferably, central retinal vein occlusion. In an alternative embodiment, the subject is suspected of having retinal vein occlusion; such as central retinal vein occlusion, semi-central retinal vein occlusion, and branch retinal vein occlusion; preferably, central retinal vein occlusion.

[0177] In one embodiment, the subject suffers from one or more symptoms of retinal vein occlusion; such as central retinal vein occlusion, semicentral retinal vein occlusion, and branch retinal vein occlusion; preferably, central retinal vein occlusion.

[0178] In one embodiment, retinal vein occlusion is characterized by changes in the subject's vision, such as one or more of the following: blurred vision, loss of a portion of the visual field, floating spots in the visual field, flashing lights in the visual field, decreased or reduced vision, and loss of vision. Typically, retinal vein occlusion is characterized by decreased or loss of vision, most often sudden decreased or loss of vision.

[0179] In another embodiment, retinal vein occlusion is characterized by a significant change in the subject's vision that occurs within seven days or less, such as: six days or less; five days or less; four days or less; three days or less; two days or less; or one day or less. This may optionally be a feature of acute RVO.

[0180] Otherwise, typically, the loss of vision or reduced or diminished vision occurs over about seven days or more, such as: about eight days or more; about nine days or more; about ten days or more; about 11 days or more; about 12 days or more; about 13 days or more; about 14 days or more; about 15 days or more; about 16 days or more; about 17 days or more; about 18 days or more; about 19 days or more; about 20 days or more; about 21 days or more; about 22 days or more; about 23 days or more; about 24 days or more; about 25 days or more; about 26 days or more; about 27 days or more; about 28 days or more; about 29 days or more; about 30 days or more; about 31 days or more; about one month or more; about two months or more; about three months or more; about four months or more; about five months or more; about six months or more; about seven months or more; about eight months or more; about nine months or more; about ten months or more; about 11 months or more; about 12 months or more; about 13 months or more; about 14 months or more; about 15 months or more; about 16 months or more; about 17 months or more; about 18 months or more; about 19 months or more; about 20 months or more; about 21 months or more; about 22 months or more; about 23 months or more; about 24 months or more; about 36 months or more; about three years or more; about four years or more; about five years or more; about six years or more; about seven years or more; about eight years or more; about nine years or more; or about ten years or more.

[0181] During the period just mentioned, vision may be reduced or diminished until vision is lost (i.e., blindness) or until vision is at a certain percentage of normal vision, at which point that percentage of vision is maintained. Typically, the percentage of reduced or diminished vision is about 95% or less of normal vision, such as: about 90% or less of normal vision; about 85% or less; about 80% or less; about 75% or less; about 70% or less; about 69% or less; about 68% or less; about 67% or less; about 66% or less; about 65% or less; about 64% or less; about 63% or less; about 62% or less; about 61% or less; about 60% or less; about 59% or less; about 58% or less; about 57% or less; about 56% or less; about 55% or less; about 54% or less; about 53% or less; about 52% or less; about 53% or less; about 54% or less; about 55% or less; about 56% or less; about 57 ... We include normal vision as the subject's vision before the onset of retinal vein occlusion or normal vision for the general population. If the retinal vein occlusion is unilateral, normal vision may be the vision in the eye not affected by the retinal vein occlusion.

[0182] In one embodiment, the subject does not exhibit any pain in the eye, and / or does not exhibit any pain in the retina.

[0183] In one embodiment, retinal vein occlusion is characterized by one or more of the following: optic disc edema; increased retinal vein dilation; increased retinal venous tortuosity; hemorrhage; "cotton-wool spots"; retinal edema; electrodiagnostic testing (ERG); pupillary afferent defect; capillary nonperfusion; arteriolar stenosis; and retinal pigment epithelial changes.

[0184] In one embodiment, retinal vein occlusion (such as central retinal vein occlusion, preferably ischemic central retinal vein occlusion) is characterized by one or more of the following:

[0185] Poor visual acuity (e.g., about 95% or less of normal vision, such as: about 90% or less of normal vision; about 85% or less; about 80% or less; about 75% or less; about 70% or less; about 69% or less; about 68% or less; about 67% or less; about 66% or less; about 65% or less; about 64% or less; about 63% or less; about 62% or less; about 61% or less; about 60% or less; about 59% or less; about 58% or less; about 57% or less; about 56% or less; about 55% or less; about 54% or less; about 53% or less; about 52% or less; about 51% or less; about about 50% or less; about 49% or less; about 48% or less; about 47% or less; about 46% or less; about 45% or less; about 44% or less; about 43% or less; about 42% or less; about 41% or less; about 40% or less; about 39% or less; about 38% or less; about 37% or less; about 36% or less; about 35% or less; about 34% or less; about 33% or less; about 32% or less; about 31% or less; about 30% or less; about 25% or less; about 20% or less; about 15% or less; about 10% or less; or about 5% or less - preferably, about 44% or less of normal vision);

[0186] Afferent pupillary defect

[0187] The presence of one or more retinal hemorrhages, such as one or more intraretinal hemorrhages (e.g., two or more; three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; 11 or more; 12 or more; 13 or more; 14 or more; 15 or more; 16 or more; 17 or more; 18 or more; 19 or more; 20 or more retinal hemorrhages);

[0188] The presence of one or more "cotton-like spots" (e.g., two or more; three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; 11 or more; 12 or more; 13 or more; 14 or more; 15 or more; 16 or more; 17 or more; 18 or more; 19 or more; 20 or more "cotton-like spots");

[0189] Dilated retinal veins

[0190] Retinal venous tortuosity;

[0191] Angiography showing one or more areas of retinal capillary non-perfusion (e.g., two or more; three or more; four or more; five or more; six or more; seven or more; eight or more; nine or more; ten or more; 11 or more; 12 or more; 13 or more; 14 or more; 15 or more; 16 or more; 17 or more; 18 or more; 19 or more; 20 or more areas of retinal capillary non-perfusion - preferably 10 or more areas of retinal capillary non-perfusion)

[0192] • Electrodiagnostic testing (ERG) presenting with one or more of the following: decreased b-wave amplitude, decreased b-wave:a-wave ratio; and prolonged b-wave implicit time; or decreased b-wave:a-wave ratio and prolonged b-wave implicit time.

[0193] Subgroups of patients with retinal vein occlusion

[0194] In addition to the aforementioned patients with different subtypes of retinal vein occlusion (eg, central retinal vein occlusion), retinal vein occlusion patients may also be identified by different characteristics.

[0195] It is known that in patients with retinal vein occlusion, about 1% to about 2% of the red blood cells are Annexin A5 positive (i.e., have monomeric Annexin A5 binding sites, such as phosphatidylserine on the outside of the red blood cell membrane), while typically in "normal" patients (such as patients without retinal vein occlusion) about 0.7% of the red blood cells are Annexin A5 positive. By comparison, in patients with sickle cell disease, typically about 2.84% of the red blood cells are Annexin A5 positive. Similarly, it is known that in patients with retinal vein occlusion, there are typically an average of about 400 to about 1000 monomeric Annexin A5 binding sites on each Annexin A5 positive red blood cell, while in "normal" patients, there are typically an average of about 280 monomeric Annexin A5 binding sites on each Annexin A5 positive red blood cell. By comparison, in patients with sickle cell disease, there are typically an average of about 12,340 monomeric Annexin A5 binding sites on each Annexin A5 positive red blood cell.

[0196] There may also be significant variability between subjects in the average number of monomeric Annexin A5 binding sites exposed on the surface of vascular endothelial cells.

[0197] Microparticles are submicron vesicles that are shed from various cell types (including, for example, erythrocytes and / or vascular endothelial cells) and released into the extracellular environment (Hargett and Bauer, 2013, Pulm. Circ., 3(2):329-340). They are typically detectable in the circulating plasma of a subject. The average number of monomeric annexin A5 binding sites exposed on the surface of the microparticles may also vary between subjects, for example, based on the level of binding sites in the subject's erythrocytes and / or vascular endothelial cells responsible for microparticle release.

[0198] Thus, in one embodiment, a subject is characterized as having about 1% (or 1.0%) to about 2% of cells (e.g., erythrocytes (i.e., red blood cells) and / or vascular endothelial cells) and / or microparticles that are positive for Annexin A5 (e.g., about 1.1% to about 2%, about 1.2% to about 2%, about 1.3% to about 2%, about 1.4% to about 2%, about 1.5% to about 2%, about 1.6% to about 2%, about 1.7% to about 2%, about 1.8% to about 2%, or about 1.9% to about 2% of cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles that are positive for Annexin A5. Preferably, a subject is characterized as having about 1.7% to about 1.9% of cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles that are positive for Annexin A5.

[0199] Thus, the subject may be characterized in that about 1%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2% of the cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles are positive for Annexin A5. Preferably, about 1.8% of the cells (erythrocytes and / or vascular endothelial cells) and / or microparticles are positive for Annexin A5.

[0200] Additionally or alternatively, a subject may be characterized as having an average number of about 400 to about 1000 monomeric Annexin A5 binding sites per cell (eg, erythrocyte and / or vascular endothelial cell) and / or microparticle.

[0201] For example, a subject can be characterized as having an average number of monomeric annexin A5 binding sites per cell (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles of about 410 to about 1000, about 420 to about 1000, about 430 to about 1000, about 440 to about 1000, about 450 to about 1000, about 460 to about 1000, about 470 to about 1000, about 480 to about 1000, about 490 to about 1000, about 500 to about 1000, about 510 to about 1000, about 520 to about 1000, about 530 to about 1000, about 540 to about 1000, about 550 to about 1000, about 560 to about 1000, about 570 to about 1000, about 580 to about 1000, about 590 to about 1000, about 600 to about 1000, about 610 to about 1000, about 620 to about 1000, about 630 to about 1000, about 640 to about 1000 about 1000, about 520 to about 1000, about 530 to about 1000, about 540 to about 1000, about 550 to about 1000, about 560 to about 1000, about 570 to about 1000, about 580 to about 1000, about 590 to about 1000, about 600 to about 1000, about 610 to about 1000, about 620 to about 1000, about 630 to about 1000, about 640 to about 1000, about 650 to about 1000, about 660 to about 1000, about 670 to about 1000, and about 680 to about 1000. 0 to about 1000, about 680 to about 1000, about 690 to about 1000, about 700 to about 1000, about 710 to about 1000, about 720 to about 1000, about 730 to about 1000, about 740 to about 1000, about 750 to about 1000, about 760 to about 1000, about 770 to about 1000, about 780 to about 1000, about 790 to about 1000, about 800 to about 1000, about 810 to about 1000, about 820 to about 1000, about 8 The invention relates to a novel nanostructured carbon foam and a nanostructured carbon foam. The nanostructured carbon foam and the nanostructured carbon foam are preferably shaped like a nanostructured carbon foam. The nanostructured carbon foam and the nanostructured carbon foam are preferably shaped like a nanostructured carbon foam.

[0202] Preferably, the subject can be characterized as having an average number of about 700 to about 800, or about 700 to about 740, and most preferably about 710 to about 730 monomeric Annexin A5 binding sites per cell (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles.

[0203] Stated another way, a subject can be characterized as having an average number of monomeric Annexin A5 binding sites per cell (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles of about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 510, about 520, about 530, about 540, about 550, about 560, about 570, about 580, about 590, about 600, about 610, about 620, about 630, about 640, about 650, about 660, about 670, about 680 about 900, about 910, about 920, about 930, about 940, about 950, about 960, about 970, about 980, about 990 - preferably about 720 monomeric Annexin A5 binding sites per cell (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles.

[0204] Including the binding site on the cell (e.g., erythrocyte and / or vascular endothelial cell) and / or microparticle is on the outside (i.e., adventitia) of the surface membrane of the cell or microparticle. Preferably, in the embodiments outlined immediately above, each monomeric Annexin A5 binding site of the cell (e.g., erythrocyte and / or vascular endothelial cell) and / or microparticle is on a cell and / or microparticle that is Annexin A5 positive.

[0205] It is known to those skilled in the art of molecular biology how to identify the number of annexin A5-positive cells and / or microparticles in a patient, for example by determining the number of annexin A5-positive red blood cells and / or microparticles in the patient's blood. Various methods are available that allow for counting cells and / or microparticles based on markers on the surface of cells and / or microparticles (such as flow cytometry). See, for example, Hargett and Bauer, 2013, Pulm.Circ., 3(2):329-340, the contents of which are incorporated herein by reference. There are also many variants of annexin A5 that are conjugated with fluorescent markers that allow such analysis. As known to the skilled person, a simple example of a possible method is to obtain a sample of the subject's blood, label it with fluorescent annexin A5, and use a flow cytometer to count the number of annexin A5-positive cells and / or microparticles. Therefore, "annexin A5-positive" includes cells (such as red blood cells) that can be bound by annexin A5 (especially monomeric annexin A5).

[0206] In one embodiment, a cell (e.g., an erythrocyte and / or a vascular endothelial cell) and / or microparticle is considered positive for Annexin A5 if it has 290 or more monomeric Annexin A5 binding sites, such as: 300 or more; 310 or more; 320 or more; 330 or more; 340 or more; 350 or more; 360 or more; 370 or more; 380 or more; 390 or more; or 400 or more Annexin A5 binding sites.

[0207] However, for the same sample, the average number of monomeric annexin A5 binding sites determined per cell and / or microparticle may vary depending on the specific determination method used. Thus, in another embodiment, cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles are considered to be positive for annexin A5 if they have more than 100% of the average number of annexin A5 binding sites of corresponding cells (such as erythrocytes and / or vascular endothelial cells) and / or microparticles in healthy subjects. For example, the average number of monomeric annexin A5 binding sites per erythrocyte that is specific to RVO may be in the region of about 1.4 times to about 4 times (such as about 2 times to about 3 times) the average number of monomeric annexin A5 binding sites per erythrocyte in healthy people.

[0208] One of the molecules that can be bound by annexin A5 is phosphatidylserine. A monomeric annexin A5 binding site is about 30 to about 60 phosphatidylserine molecules, and a monomeric annexin A5 binding site is about 3,100 angstroms on the surface membrane of cells and / or microparticles. to about In addition, the monomeric annexin A5 binding site is a region of about 40 to about 60 phospholipid heads on the surface membrane of cells and / or microparticles.

[0209] In one embodiment, the term "Annexin A5 binding site" refers to an area of ​​about 30 to about 60 phosphatidylserine molecules on the surface membrane of a cell (e.g., an erythrocyte and / or a vascular endothelial cell) and / or a microparticle, such as: about 31 to about 59; about 32 to about 58; about 33 to about 57; about 34 to about 56; about 35 to about 55; about 36 to about 54; about 37 to about 53; about 38 to about 52; about 39 to about 51; about 40 to about 41; about 42 to about 43; about 44 to about 46; about 47 to about 48; about 49 to about 50; about 51 to about 52; about 53 to about 54; about 55 to about 55; about 56 to about 54; about 57 to about 56; about 57 to about 57; about 57 to about 51; about 55 to about 55; about 56 to about 54; about 57 to about 53; about 57 to about 52; about 53 to about 53; about 53 to about 52; about 53 to about 52; about 53 to about 51; about 53 to about 52 ... about 41 to about 49; about 42 to about 48; about 43 to about 47; or about 44 to about 46 phosphatidylserine molecules on the surface membrane of cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles, preferably about 35 to about 45 phosphatidylserine molecules on the surface membrane of cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles, more preferably about 42 to about 57 phosphatidylserine molecules on the surface membrane of cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles, and most preferably about 40 phosphatidylserine molecules on the surface membrane of cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles.

[0210] In an additional or alternative embodiment, the term "Annexin A5 binding site" refers to a region on the surface membrane of a cell (e.g., an erythrocyte and / or a vascular endothelial cell) and / or a microparticle approximately to about such as: approximately on the surface membrane of cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles to about about to about about to about about to about about to about about to about about to about about to about about to about preferably about 100 μm on the surface membrane of cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles. to about More preferably, about 100 μm in the surface membrane of cells (e.g., erythrocytes and / or vascular endothelial cells) and / or microparticles. area.

[0211] In another embodiment, the term "Annexin A5 binding site" refers to an area of ​​about 40 to about 60 phospholipid heads on an area on the surface membrane of a cell (e.g., an erythrocyte and / or a vascular endothelial cell) and / or a microparticle, such as: about 41 to about 59; about 42 to about 58; about 43 to about 57; about 44 to about 56; about 45 to about 55; about 46 to about 54; about 47 to about 53; about 48 to about 52; about 49 to about 51 phospholipid heads on an area on the surface membrane of a cell (e.g., an erythrocyte and / or a vascular endothelial cell) and / or a microparticle, preferably about 45 to 55 phospholipid heads on an area on the surface membrane of a cell (e.g., an erythrocyte and / or a vascular endothelial cell) and / or a microparticle, more preferably about 50 phospholipid heads on an area on the surface membrane of a cell (e.g., an erythrocyte and / or a vascular endothelial cell) and / or a microparticle.

[0212] In one embodiment of any one of the first, second, third or fourth aspects of the invention, the subject is characterized by one or more of the following:

[0213] a. the subject is about 50 years of age or older, such as at least 55, 60, 65, 70, 75 or 80 years of age (optionally 90 years of age or younger, such as about 89 years of age or younger; about 88 years of age or younger; about 87 years of age or younger; about 86 years of age or younger; about 85 years of age or younger; about 84 years of age or younger; about 83 years of age or younger; about 82 years of age or younger; about 81 years of age or younger; about 80 years of age or younger; about 79 years of age or younger; about 78 years of age or younger; about 77 years of age or younger; about 76 years of age or younger; about 75 years of age or younger; about 74 years of age or younger; about 73 years or younger; about 72 years or younger; about 71 years or younger; about 70 years or younger; about 69 years or younger; about 68 years or younger; about 67 years or younger; about 66 years or younger; about 65 years or younger; about 64 years or younger; about 63 years or younger; about 62 years or younger; about 61 years or younger; about 60 years or younger; about 59 years or younger; about 58 years or younger; about 57 years or younger; about 56 years or younger; about 55 years or younger; about 54 years or younger; about 53 years or younger; about 52 years or younger; about 51 years or younger);

[0214] b. the subject does not have or is not suspected of having cardiovascular disease (such as myocardial infarction, stroke, hypertension, hyperlipidemia, hyperhomocysteinemia, coagulopathy and / or peripheral arterial disease, and optionally excluding atherosclerosis) or diseases associated with cardiovascular disease (such as sickle cell disease, glaucoma, polyuria, diabetes mellitus, polycythemia vera and / or systemic inflammatory disorders (e.g. Behcet's disease, polyarteritis nodosa, sarcoidosis, Wegener's granulomatosis and / or Goodpasture's syndrome), but preferably excluding polyuria and diabetes mellitus);

[0215] c. the subject has no history of cardiovascular disease (such as myocardial infarction, stroke, hypertension, hyperlipidemia, hyperhomocysteinemia, coagulopathy and / or peripheral arterial disease, and optionally excluding atherosclerosis) or cardiovascular disease-related diseases (such as sickle cell disease, glaucoma, polyuria, diabetes mellitus, polycythemia vera, and / or systemic inflammatory disorders (e.g. Behcet's disease, polyarteritis nodosa, sarcoidosis, Wegener's granulomatosis and / or Goodpasture's syndrome), but preferably excluding polyuria and diabetes mellitus);

[0216] d. the subject has no family history of cardiovascular disease (such as myocardial infarction, stroke, hypertension, hyperlipidemia, hyperhomocysteinemia, coagulopathy and / or peripheral arterial disease, and optionally excluding atherosclerosis) or cardiovascular disease-related diseases (such as sickle cell disease, glaucoma, polyuria, diabetes, polycythemia vera, and / or systemic inflammatory disorders (e.g. Behcet's disease, polyarteritis nodosa, sarcoidosis, Wegener's granulomatosis and / or Goodpasture's syndrome), but preferably excluding polyuria and diabetes); and / or

[0217] e. the subject does not have or is not suspected of having a neoplastic disorder, such as a solid tumor, a lymphoma and / or a blood cancer, for example one or more disorders selected from the group consisting of breast cancer, endometrial cancer, cervical cancer, ovarian cancer, lung cancer, head and / or neck cancer, brain cancer, thyroid cancer, esophageal cancer, stomach cancer, colon cancer and / or rectal cancer, liver cancer, pancreatic cancer, skin cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, bone cancer, lymphoma (such as Hodgkin lymphoma and / or non-Hodgkin lymphoma) and / or blood cancer (such as acute lymphocytic leukemia, acute myeloid leukemia and / or chronic myeloid leukemia).

[0218] A subject's "family history" can be readily determined by a person skilled in the art of medicine. In one embodiment, family history includes the history of one or more of the following relatives: a parent (such as a mother or father); a grandparent (such as a grandmother or grandfather); a sibling (such as a brother or sister); a child (such as a son or daughter); and / or a grandchild (such as a grandson or granddaughter).

[0219] In one embodiment, if the subject does not exhibit one or more symptoms of cardiovascular disease or a disease associated with cardiovascular disease, then the subject does not have or is not suspected of having these diseases. The symptoms of these diseases are known to those skilled in the medical field.

[0220] In one embodiment, the coagulation disorder is not retinal vein occlusion, such as central retinal vein occlusion, branch retinal vein occlusion, or hemi-retinal vein occlusion.

[0221] Preferably, the subject is characterized as:

[0222] a. About 50 years old or above;

[0223] b. the subject does not suffer from or is not suspected of suffering from one or more conditions selected from hypertension, polyuria and / or sickle cell disease;

[0224] c. the subject has no history of one or more conditions selected from hypertension, polyuria and / or sickle cell disease; and

[0225] d. The subject has no family history of one or more conditions selected from hypertension, polyuria and / or sickle cell disease.

[0226] In an alternative embodiment, the subject may be (i) 50 years of age or older and / or (ii) suspected of having, having a medical history of, and / or having a family history of, one or more conditions selected from hypertension, polyuria, and / or sickle cell disease.

[0227] In one embodiment, the subject does not have or is not suspected of having a condition characterized by increased extracellular Annexin A5. Preferably, the increased extracellular Annexin A5 is increased when compared to a patient not having a condition characterized by increased extracellular Annexin A5. In one embodiment, the condition characterized by increased extracellular Annexin A5 is not retinal vein occlusion and / or a retinal vein occlusion-related disease.

[0228] It has been demonstrated that a subgroup of patients with retinal vein occlusion may be genetically predisposed to developing retinal vein occlusion (i.e., carry a genetic risk factor for developing retinal vein occlusion). For example, genetically predisposed patients may produce red blood cells that preferentially form blood clots in the veins of the eye, particularly in the retina. The development of retinal vein occlusion in these genetically predisposed patients is less likely to be associated with other cardiovascular disease.

[0229] One skilled in the medical arts would know how to identify a patient as being genetically predisposed to developing a particular disease, such as retinal vein occlusion.

[0230] Thus, in one embodiment, the subject is genetically predisposed to retinal vein occlusion, or is suspected of being genetically predisposed to retinal vein occlusion. Preferably, the subject is genetically predisposed to form a thrombus (or multiple thrombi) in the ocular veins (especially the retina). Most preferably, the subject is genetically predisposed to produce red blood cells that preferentially form a thrombus (or multiple thrombi) in the ocular veins (interestingly, the retina). For example, it is known from studies of in vitro bone marrow cultures of CRVO patients that approximately 27% of CRVO patients exhibit spontaneous growth of erythroid precursors in the absence of any detectable myeloproliferative conditions (Heron et al., 2007, Ophthalmology, 114: 2155-61). Thus, in one embodiment, the subject may be a subject that exhibits spontaneous in vitro growth of erythroid precursors from the bone marrow in the absence of any detectable myeloproliferative conditions.

[0231] Typically, the subject is a human subject.

[0232] In one embodiment, the subject (especially a human subject) has a body weight of about 10 kg to about 110 kg, such as: about 20 kg to about 100 kg; about 30 kg to about 90 kg; about 40 kg to about 80 kg; or about 50 kg to about 70 kg. In a preferred embodiment, the subject has a body weight of about 40 kg to about 80 kg. In another preferred embodiment, the subject has a body weight of about 50 kg to about 70 kg. In a more preferred embodiment, the subject has a body weight of about 60 kg. As used in this context, the term "about" may be used herein to represent a range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, ±4%, ±3%, ±2% or ±1% of the value mentioned.

[0233] Diseases, conditions or disorders associated with and / or caused by retinal vein occlusion

[0234] Various diseases, conditions or disorders are associated with and / or caused by RVO and may also be treated by the present invention.

[0235] Thus, in one embodiment, the invention provides for the prevention or treatment of one or more diseases, conditions or disorders associated with and / or caused by RVO.

[0236] For example, when a retinal vein is blocked during retinal vein occlusion, blood cannot drain properly and it can leak into the retina. If it leaks into the macula, this can produce macular edema (ME). The severity of the blockage, how many veins are involved, and the pressure inside the veins make the leakage worse.

[0237] Macular edema (ME) is the accumulation of fluid in the macula (the area in the center of the retina). The retina is the light-sensitive tissue at the back of the eye, and the macula is the part of the retina responsible for clear, straight-ahead vision. Fluid accumulation causes the macula to swell and thicken, which distorts vision. It usually occurs when there is leakage of fluid and blood into the retina due to increased venous and / or capillary pressure causing vascular permeability. Reduction, impairment or loss of vision in retinal vein occlusion is often associated with macular edema.

[0238] Thus, in one embodiment, the present invention provides a molecule consisting of monomeric Annexin A5 protein for use in preventing or treating macular edema in a subject, and preferably a subject characterized as having RVO, or suspected of having RVO, or at risk of having RVO.

[0239] Other diseases, conditions or disorders associated with and / or caused by RVO include retinal ischemia, iris neovascularization, retinal neovascularization, neovascular glaucoma, vitreous hemorrhage, erythropoiesis, and retinal detachment.

[0240] That is, in a separate implementation:

[0241] The present invention provides a molecule consisting of a monomeric Annexin A5 protein for use in preventing or treating retinal ischemia in a subject, and preferably a subject characterized as having RVO, or suspected of having RVO, or at risk of having RVO,

[0242] The present invention provides a molecule consisting of monomeric Annexin A5 protein for use in preventing or treating diabetic iris neovascularization in a subject, and preferably a subject characterized as having RVO, or suspected of having RVO, or at risk of having RVO,

[0243] The present invention provides a molecule consisting of a monomeric annexin A5 protein for use in preventing or treating retinal neovascularization in a subject, and preferably a subject characterized as having RVO, or suspected of having RVO, or at risk of having RVO,

[0244] The present invention provides a molecule consisting of a monomeric annexin A5 protein for use in preventing or treating neovascular glaucoma in a subject, and preferably a subject characterized as having RVO, or suspected of having RVO, or at risk of having RVO,

[0245] The present invention provides a molecule consisting of a monomeric Annexin A5 protein for use in preventing or treating vitreous hemorrhage in a subject, and preferably a subject characterized as having RVO, or suspected of having RVO, or at risk of having RVO,

[0246] The present invention provides a molecule consisting of a monomeric annexin A5 protein for use in preventing or treating erythropoiesis in a subject, and preferably a subject characterized as having RVO, or suspected of having RVO, or at risk of having RVO, and

[0247] The present invention provides a molecule consisting of monomeric Annexin A5 protein for use in preventing or treating retinal detachment in a subject, and preferably a subject characterized as having RVO, or suspected of having RVO, or at risk of having RVO.

[0248] Retinal ischemia is local ischemia caused by nonperfusion of retinal veins and capillaries due to retinal vein occlusion.

[0249] Iris neovascularization and retinal neovascularization are the growth of new blood vessels in the eye. These neovascularizations cause an increase in cytokines (such as VEGF), which act in a positive feedback loop to further promote new growth of blood vessels.

[0250] One or more of retinal ischemia, iris neovascularization, and retinal neovascularization can lead to neovascular glaucoma (i.e., optic nerve damage), vitreous hemorrhage (i.e., leakage of blood into the vitreous humor of the eye), rubeosis (i.e., formation of new blood vessels in the iris), and / or retinal detachment (i.e., detachment of the retina from its blood supply) (Guidelines for Retinal Vein Occlusion (RVO) supra).

[0251] Thus, the present invention also provides for the prevention or treatment of one or more diseases, conditions or disorders selected from the group consisting of retinal ischemia, iris neovascularization, retinal neovascularization, neovascular glaucoma, vitreous hemorrhage, erythrosis and retinal detachment, preferably in a subject characterized as having RVO or suspected of having RVO or at risk of having RVO.

[0252] In one embodiment, iris neovascularization and / or retinal neovascularization is characterized by an increase in cytokines, such as an increase in VEGF. In one embodiment, retinal vein occlusion is central retinal vein occlusion, and retinal vein occlusion-related diseases are iris neovascularization. In some embodiments, retinal vein occlusion is branch retinal vein occlusion, and retinal vein occlusion-related diseases are retinal neovascularization. In one embodiment, retinal vein occlusion is hemicentral vein occlusion, and retinal vein occlusion-related diseases are erythropoiesis. Preferably, retinal vein occlusion-related diseases are macular edema.

[0253] In a fifth aspect, the present invention provides a molecule consisting of a monomeric Annexin A5 protein for use in preventing or treating a disease, condition or disorder associated with and / or caused by RVO in a subject,

[0254] Optionally wherein the composition comprises a molecule consisting of monomeric Annexin A5 protein in an amount of about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg of the subject's body weight, and / or

[0255] Optionally wherein the composition comprises molecules consisting of monomeric Annexin A5 protein in a total amount of about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg or about 0.001 mg to about 0.1 mg.

[0256] In an alternative embodiment of the fifth aspect, the present invention provides a method of treating or preventing a disease, condition or disorder associated with and / or caused by RVO in a subject, the method comprising the step of administering to the subject a therapeutically effective amount of a molecule consisting of a monomeric Annexin A5 protein,

[0257] Optionally wherein the composition comprises a molecule consisting of monomeric Annexin A5 protein in an amount of about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg of the subject's body weight, and / or

[0258] Optionally wherein the composition comprises molecules consisting of monomeric Annexin A5 protein in a total amount of about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg or about 0.001 mg to about 0.1 mg.

[0259] In an alternative embodiment of the fourth aspect, the present invention provides a use of a molecule consisting of a monomeric Annexin A5 protein in the preparation of a medicament for preventing or treating a disease, condition or disorder associated with and / or caused by RVO in a subject,

[0260] Optionally wherein the composition comprises a molecule consisting of monomeric Annexin A5 protein in an amount of about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg of the subject's body weight, and / or

[0261] Optionally wherein the composition comprises molecules consisting of monomeric Annexin A5 protein in a total amount of about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg or about 0.001 mg to about 0.1 mg.

[0262] Unless otherwise stated, embodiments of the invention discussed herein in relation to the first, second, third and fourth aspects of the invention may apply to the fifth aspect of the invention.

[0263] In a sixth aspect, the present invention provides a composition comprising a molecule consisting of monomeric Annexin A5 protein,

[0264] Optionally wherein the composition comprises a molecule consisting of monomeric Annexin A5 protein in an amount of about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg of the subject's body weight, and / or

[0265] Optionally wherein the composition comprises molecules consisting of monomeric Annexin A5 protein in a total amount of about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg, or about 0.001 mg to about 0.1 mg, and

[0266] Optionally, the composition is used in preventing or treating one or more diseases, conditions or disorders associated with and / or caused by RVO in a subject.

[0267] In an alternative embodiment of the fifth aspect, the present invention provides a method of treating or preventing one or more diseases, conditions or disorders associated with and / or caused by RVO in a subject, the method comprising the step of administering a therapeutically effective amount of a composition comprising a molecule consisting of a monomeric Annexin A5 protein,

[0268] Optionally wherein the composition comprises a molecule consisting of monomeric Annexin A5 protein in an amount of about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg of the subject's body weight, and / or

[0269] Optionally wherein the composition comprises molecules consisting of monomeric Annexin A5 protein in a total amount of about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg or about 0.001 mg to about 0.1 mg.

[0270] Unless otherwise stated, embodiments of the first, second, third, fourth and fifth aspects of the invention discussed herein are applicable to the sixth aspect of the invention.

[0271] C. Annexin A5

[0272] The molecule consisting of monomeric Annexin A5 protein used in all aspects of the invention may consist essentially of or consist of a protein consisting of the sequence of Annexin A5, such as the sequence of human Annexin A5.

[0273] For the avoidance of doubt, a molecule consisting of monomeric Annexin A5 protein is not dimeric Annexin A5 (commonly referred to in the art as "di-Annexin"), and is not PEGylated Annexin A5.

[0274] Preferably, the molecule consisting of human monomeric Annexin A5 protein is a protein consisting of or essentially consisting of a polypeptide molecule consisting of the amino acid sequence of SEQ ID NO: 1 as shown below, with or without an N-terminal methionine. Preferably, the molecule consisting of or essentially consisting of a monomeric Annexin A5 protein is a recombinant human monomeric Annexin A5 protein, for example, a protein produced by recombinantly expressing a gene encoding a protein having the sequence of SEQ ID NO: 1 in a recombinant host cell organism such as a recombinant E. coli host, preferably followed by recovery and purification of the monomeric Annexin A5 protein therefrom.

[0275] The sequence of the protein encoded by the human Annexin A5 gene is defined by the following SEQ ID NO: 1:

[0276]

[0277] In another embodiment, the monomeric Annexin A5 protein may include, consist essentially of, or consist of a variant or mutant of the sequence of Annexin A5, such as human Annexin A5 (optionally, a protein consisting of the sequence of SEQ ID NO: 1 with or without an N-terminal methionine). For example, the sequence of the variant or mutant may differ from the sequence of SEQ ID NO: 1 (with or without the N-terminal methionine) at any one or more positions, such as at or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160 or more positions.

[0278] In another embodiment, the monomeric Annexin A5 protein may include, consist essentially of, or consist of a biologically active fragment or variant or mutant of mature Annexin A5 such as human Annexin A5 (optionally, a protein consisting of the sequence of SEQ ID NO: 1 with or without an N-terminal methionine).

[0279] Biologically active fragments of Annexin A5 share functional properties or binding properties with full-length Annexin A5. Epitope fragments of Annexin A5 bind to monoclonal antibodies that bind to full-length Annexin A5. An "activity" of Annexin A5 refers to any binding function performed by the protein, additional examples of which are further defined below.

[0280] Biologically active fragments of monomeric annexin A5 proteins that can be used in the present invention can include at least about 50 consecutive amino acids, generally at least about 100 consecutive amino acids, at least about 150 consecutive amino acids, at least about 200 consecutive amino acids, at least about 250 consecutive amino acids, at least about 260 consecutive amino acids, at least about 270 consecutive amino acids, at least about 280 consecutive amino acids, at least about 290 consecutive amino acids, at least about 300 consecutive amino acids, at least about 310 consecutive amino acids, and can include up to about 320 consecutive amino acids of annexin A5 protein or modifications thereof, including but not limited to human annexin A5 protein (optionally, a protein consisting of the sequence of SEQ ID NO: 1 with or without N-terminal methionine), and in addition to the provided sequence, can further include fusion polypeptides known in the art. The annexin A5 sequence can be from any mammalian or avian species, such as primate species, particularly humans; rodents, including mice, rats and hamsters; rabbits; horses, cattle, dogs, cats; and the like. Of particular interest is the human Annexin A5 protein (optionally, a protein consisting of the sequence of SEQ ID NO: 1 with or without an N-terminal methionine).

[0281] In one embodiment, the biologically active fragment of the monomeric Annexin A5 protein is effective for treating retinal vein occlusion or retinal vein occlusion-related diseases; preferably, the efficiency is 10% or more, such as: the efficiency is 20% or more; 30% or more; 40% or more; 50% or more; 60% or more; 70% or more; 80% or more; 90% or more, compared to the human monomeric Annexin A5 protein, i.e., a polypeptide molecule consisting of the amino acid sequence of SEQ ID NO: 1 with or without N-terminal methionine.

[0282] In one embodiment, the monomeric Annexin A5 protein consists of about 320 amino acids or less, such as: about 319 amino acids or less; about 318 amino acids or less; about 317 amino acids or less; about 316 amino acids or less; about 315 amino acids or less; about 314 amino acids or less; about 313 amino acids or less; about 312 amino acids or less; about 311 amino acids or less; about 310 amino acids or less; about 305 amino acids or less; about 300 amino acids or less; about 295 amino acids or less; about 290 amino acids or less; about 285 amino acids or less; about 280 amino acids or less; about 270 amino acids or less; about 260 amino acids or less; or about 250 amino acids or less.

[0283] In one embodiment, the recombinant monomeric Annexin A5 protein consists of 319 amino acids, such as: about 318 amino acids or less; about 317 amino acids or less; about 316 amino acids or less; about 315 amino acids or less; about 314 amino acids or less; about 313 amino acids or less; about 312 amino acids or less; about 311 amino acids or less; about 310 amino acids or less; about 305 amino acids or less; about 300 amino acids or less; about 295 amino acids or less; about 290 amino acids or less; about 285 amino acids or less; about 280 amino acids or less; about 270 amino acids or less; about 260 amino acids or less; or about 250 amino acids or less.

[0284] The monomeric Annexin A5 protein may include one or more of the following:

[0285] a. A protein consisting essentially of the sequence of human monomeric annexin A5 protein or consisting of the sequence of human monomeric annexin A5 protein (optionally, a protein consisting of the sequence of SEQ ID NO: 1 with or without an N-terminal methionine);

[0286] bb recombinant human monomeric annexin A5 protein;

[0287] c. the mammalian ortholog of the human monomeric annexin A5 protein;

[0288] da), b) or c) allelic variant or genetic variant;

[0289] e. A functional analog or variant of a monomeric Annexin A5 protein, which is a protein consisting of an amino acid sequence that is more than 50%, 60%, 70%, 75%, such as more than 80%, 85%, more than 90% or even more preferably more than 95% or 99% identical to a human monomeric Annexin A5 protein having a sequence defined by SEQ ID NO: 1 with or without an N-terminal methionine; and

[0290] A biologically active fragment of any one of fa) to e).

[0291] A molecule consisting of a monomeric Annexin A5 protein may also be referred to herein as an "agent consisting of a monomeric Annexin A5 protein" or a "therapeutic agent consisting of a monomeric Annexin A5 protein", and may be referred to as "monomeric Annexin A5". The term "molecule consisting of a monomeric Annexin A5 protein" is intended to exclude molecules that are not monomeric Annexin A5, such as PEGylated Annexin A5, dimeric monomeric Annexin A5 proteins (also referred to as di-Annexin) and Annexin A5 fusion proteins, especially Annexin A5 fusion proteins having a higher weight average molecular weight and / or plasma half-life than human monomeric Annexin A5 protein, which is a polypeptide molecule consisting of the amino acid sequence of SEQ ID NO: 1 with or without an N-terminal methionine.

[0292] Thus, a molecule consisting of monomeric Annexin A5 protein is not a PEGylated Annexin A5, a dimeric Annexin A5 protein, or an Annexin A5 fusion protein. For the avoidance of doubt, in one embodiment, a molecule consisting of monomeric Annexin A5 protein is not a "modified Annexin" as defined in US 9,463,217, the contents of which are incorporated herein by reference in their entirety.

[0293] In a particular embodiment, the functional analogue, mutant or variant of Annexin A5 according to the present invention has an identity of more than 50%, 60%, 70%, 75% with human Annexin A5 SEQ ID NO: 1 with or without N-terminal methionine, such as more than 80%, 85%, more than 90% or even more preferably more than 95% or 99%.

[0294] The percent identity between two amino acid sequences is determined as follows. First, the amino acid sequence is compared to, for example, SEQ ID NO: 1, using the BLAST 2 sequence (Bl2seq) program from an independent version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. The independent version of BLASTZ can be obtained from the website of the National Center for Biotechnology Information of the U.S. government, ncbi.nlm.nih.gov. Instructions on how to use the Bl2seq program can be found in the readme file attached to BLASTZ. Bl2seq uses the BLASTP algorithm to compare two amino acid sequences. In order to compare two amino acid sequences, the Bl2seq options are set as follows: - set i to the file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt); - set j to the file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt); - set p to blastp; - set o to any desired file name (e.g., C:\output.txt); and keep all other options at their default settings. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If the two compared sequences share homology, the specified output file will present those homologous regions as aligned sequences. If the two compared sequences do not share homology, the specified output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where the same nucleotide or amino acid residue is present in the two sequences.

[0295] The percent identity is determined by dividing the number of matches by the length of the sequence shown in the identified sequence and then multiplying the resulting value by 100. For example, if a sequence is compared to the sequence shown in SEQ ID NO: 1 (the length of the sequence shown in SEQ ID NO: 1 is 320) and the number of matches is 288, then the percent identity of the sequence to the sequence shown in SEQ ID NO: 1 is 90 (i.e., 288 ÷ 320 * 100 = 90).

[0296] Thus, a functional analog, mutant or variant of Annexin A5 may be a protein having conservative or non-conservative amino acid insertions, deletions or substitutions at one or more positions, provided that such changes result in a protein whose basic properties that function in the same manner as Annexin A5 are not significantly altered. "Significantly" in this context means that a person skilled in the art would consider that the properties of the variant may still be different from those of the original protein, but not unnoticeably.

[0297] "Conservative substitutions" refer to combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.

[0298] Such variants and mutants can be prepared using protein engineering and site-directed mutagenesis methods well known in the art.

[0299] Functional analogs, mutants, variants or biologically active fragments of Annexin A5 may consist of the sequence of human Annexin A5 (such as SEQ ID NO: 1), optionally with or without an N-terminal methionine, having no more than 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 continuous or non-continuous additional amino acids; and / or no more than 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 continuous or non-continuous amino acid deletions; and / or no more than 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 continuous or non-continuous amino acid substitutions.

[0300] D. Subjects of treatment

[0301] The subject treated according to the invention may be a human subject or a non-human subject.Preferably, the subject is characterized as having or suspected of having, or being at risk of having, macular edema and / or RVO.

[0302] In a preferred embodiment, the subject is a human subject.

[0303] In another embodiment, the subject is a non-human subject, and may be selected from the group consisting of: a non-human mammalian subject; a bird; an amphibian; and a reptile.

[0304] The non-human mammalian subject may be selected from the list consisting of: equine (such as: horse or donkey); cattle (such as: cow; buffalo; bison; or yak); camel; pig; llama; alpaca; bird (such as: ostrich; chicken; goose; duck; turkey; quail; or pigeon); sheep; goat; canine; feline; reptile (such as: snake; lizard; crocodile; alligator; turtle; or turtle); rabbit; amphibian (such as: frog; or salamander) or rodent (such as: mouse; rat; chinchilla; guinea pig; or squirrel).

[0305] E. Administration regimen

[0306] Dosage and schedule :

[0307] According to all aspects of the invention, a molecule consisting of monomeric Annexin A5 protein may be administered to a subject:

[0308] Optionally, the dosage is determined based on the subject's body weight, and the dosage of the molecule is about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg of the subject's body weight, and / or

[0309] Optionally, independent of the subject's weight, the total dose is about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg, or about 0.001 mg to about 0.1 mg, as defined above.

[0310] (a) Dose per unit body weight

[0311] For the avoidance of doubt, it is to be understood that the term "mg / kg body weight" refers to mg of molecules consisting of monomeric Annexin A5 protein per kg body weight of the subject.

[0312] Preferred dosages may be selected from one or more of about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg, or about 0.01 μg / kg to about 1 μg / kg, such as about 0.01 μg / kg, about 0.02 μg / kg, about 0.03 μg / kg, about 0.04 μg / kg, about 0.05 μg / kg, about 0.06 μg / kg, about 0.07 μg / kg, about 0.08 μg / kg, about 0.09 μg / kg and / or about 0.1 μg / kg.

[0313] In some embodiments, the dose can be selected from one or more of about 0.1 μg / kg, about 0.2 μg / kg, about 0.3 μg / kg, about 0.4 μg / kg, about 0.5 μg / kg, about 0.6 μg / kg, about 0.7 μg / kg, about 0.8 μg / kg, about 0.9 μg / kg and / or about 1 μg / kg.

[0314] In other embodiments, the dose can be selected from one or more of about 1 μg / kg, about 2 μg / kg, about 3 μg / kg, about 4 μg / kg, about 5 μg / kg, about 6 μg / kg, about 7 μg / kg, about 8 μg / kg, about 9 μg / kg and / or about 10 μg / kg.

[0315] A pharmaceutically or veterinarily acceptable composition comprising a molecule consisting of monomeric Annexin A5 protein may also be administered as defined above, wherein the composition is administered such that the amount of the molecule consisting of monomeric Annexin A5 protein in the composition is about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg, or about 0.001 mg to about 0.1 mg of the molecule.

[0316] In one embodiment, a molecule consisting of monomeric Annexin A5 protein is administered to a subject in an amount from about 0.00001 mg / kg body weight to about "X" mg / kg body weight, wherein "X" is about 1 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, 0.09 mg / kg, 0.08 mg / kg, 0.07 mg / kg, 0.06 mg / kg, 0.05 mg / kg, 0.04 0.004 mg / kg or less; about 0.003 mg / kg or less; about 0.002 mg / kg or less; preferably about 0.003 mg / kg or less; about 0.002 mg / kg or less; about 0.008 mg / kg or less; about 0.007 mg / kg or less; about 0.006 mg / kg or less; about 0.005 mg / kg or less; about 0.004 mg / kg or less; about 0.003 mg / kg or less; about 0.002 mg / kg or less; preferably ... Preferably, about 0.001 mg / kg body weight or less; 0.0009 mg / kg body weight or less, such as: about 0.0008 mg / kg body weight or less; about 0.0007 mg / kg body weight or less; about 0.0006 mg / kg body weight or less; about 0.0005 mg / kg body weight or less; about 0.0004 mg / kg body weight or less; about 0.0003 mg / kg body weight or less; about 0.0002 mg / kg body weight or less; about 0.0001 mg / kg g body weight or less; about 0.00009 mg / kg body weight or less; about 0.00008 mg / kg body weight or less; about 0.00007 mg / kg body weight or less; about 0.00006 mg / kg body weight or less; about 0.00005 mg / kg body weight or less; about 0.00004 mg / kg body weight or less; about 0.00003 mg / kg body weight or less; about 0.00002 mg / kg body weight or less of a molecule composed of monomeric membrane-attached protein A5.

[0317] In an additional or alternative embodiment, the dosage form is about "Y" mg / kg body weight to about 0.01 mg / kg body weight, preferably about "Y" mg / kg body weight to about 1 mg / kg, 0.5 mg / kg, 0.4 mg / kg, 0.3 mg / kg, 0.2 mg / kg, 0.1 mg / kg, 0.09 mg / kg, 0.08 mg / kg, 0.07 mg / kg, 0.06 mg / kg, 0.05 mg / kg, 0.04 mg / kg, 0.03 mg / kg, 0. In some embodiments, a molecule consisting of monomeric annexin A5 protein is administered to a subject in an amount of about 0.02 mg / kg or 0.001 mg / kg body weight, wherein "Y" is about 0.00002 mg / kg body weight or more, such as: about 0.00003 mg / kg body weight or more; about 0.00004 mg / kg body weight or more; about 0.00005 mg / kg body weight or more; about 0.00006 mg / kg body weight or more; about 0.00007 mg / kg body weight or more; about 0.00008 mg / kg body weight or more; kg body weight or more; about 0.00009 mg / kg body weight or more; about 0.0001 mg / kg body weight or more; about 0.0002 mg / kg body weight or more; about 0.0003 mg / kg body weight or more; about 0.0004 mg / kg body weight or more; about 0.0005 mg / kg body weight or more; about 0.0006 mg / kg body weight or more; about 0.0007 mg / kg body weight or more; about 0.0008 mg / kg body weight or more; or about 0.0009 mg / kg body weight or more; g / kg body weight or more; about 0.001mg / kg body weight or more; about 0.002mg / kg body weight or more; about 0.003mg / kg body weight or more; about 0.004mg / kg body weight or more; about 0.005mg / kg body weight or more; about 0.006mg / kg body weight or more; about 0.007mg / kg body weight or more; about 0.008mg / kg body weight or more; about 0.009mg / kg body weight or more of a molecule composed of monomeric membrane-attached protein A5.

[0318] In an additional or alternative embodiment, a molecule consisting of monomeric Annexin A5 protein is administered to a subject in an amount of about "X" mg / kg body weight to about "Y" mg / kg body weight, wherein "X" and "Y" are each values ​​as defined above.

[0319] In one embodiment, the molecule is administered to a subject in an amount selected from about 0.0004 mg / kg body weight to about 0.0002 mg / kg body weight, about 0.0005 mg / kg body weight to about 0.0001 mg / kg body weight, about 0.0006 mg / kg body weight to about 0.00009 mg / kg body weight, about 0.0007 mg / kg body weight to about 0.00008 mg / kg body weight, or about 0.0008 mg / kg body weight to about 0.00007 mg / kg body weight, about 0.0009 mg / kg body weight to about 0.00006 mg / kg body weight, or about 0.001 mg / kg body weight to about 0.00005 mg / kg body weight.

[0320] In another embodiment, the molecule is administered to a subject in an amount of about 0.0003 mg / kg body weight to about 0.0002 mg / kg body weight, about 0.0004 mg / kg body weight to about 0.0001 mg / kg body weight, about 0.0005 mg / kg body weight to about 0.00009 mg / kg body weight, about 0.0006 mg / kg body weight to about 0.00008 mg / kg body weight, about 0.0007 mg / kg body weight to about 0.00007 mg / kg body weight, about 0.0008 mg / kg body weight to about 0.00006 mg / kg body weight, about 0.0009 mg / kg body weight to about 0.00005 mg / kg body weight, or about 0.001 mg / kg body weight to about 0.00004 mg / kg body weight.

[0321] In another embodiment, the molecule is administered to a subject in an amount of about 0.000257 mg / kg body weight to about 0.000255 mg / kg body weight, about 0.000258 mg / kg body weight to about 0.000254 mg / kg body weight, about 0.000259 mg / kg body weight to about 0.000253 mg / kg body weight, about 0.00026 mg / kg body weight to about 0.000252 mg / kg body weight, about 0.000261 mg / kg body weight to about 0.000251 mg / kg body weight, about 0.000262 mg / kg body weight to about 0.000255 mg / kg body weight, about 0.000263 mg / kg body weight to about 0.000264 mg / kg body weight 0.000249 mg / kg body weight, about 0.000264 mg / kg body weight to about 0.000248 mg / kg body weight, about 0.000265 mg / kg body weight to about 0.000247 mg / kg body weight, about 0.000266 mg / kg body weight to about 0.000246 mg / kg body weight, about 0.000267 mg / kg body weight to about 0.000245 mg / kg body weight, about 0.000268 mg / kg body weight to about 0.000244 mg / kg body weight, about 0.000269 mg / kg body weight to about 0.000243 mg / kg body weight or about 0.00027 mg / kg body weight to about 0.000242 mg / kg.

[0322] In a preferred embodiment, the molecule is administered to a subject in an amount of about 0.0004 mg / kg body weight to about 0.0002 mg / kg body weight. In another preferred embodiment, the molecule is administered to a subject in an amount of about 0.0003 mg / kg body weight to about 0.0002 mg / kg body weight. In another preferred embodiment, the molecule is administered to a subject in an amount of about 0.000257 mg / kg body weight to about 0.000255 mg / kg body weight. In an even more preferred embodiment, the molecule is administered to a subject in an amount of about 0.0003 mg / kg body weight. In another even more preferred embodiment, the molecule is administered to a subject in an amount of about 0.000256 mg / kg body weight.

[0323] The aforementioned dosage per unit body weight may be particularly suitable for human subjects.

[0324] (b) Total dose :

[0325] Additionally or alternatively, the dosage may be determined independently of the subject's body weight.

[0326] Preferably the dose may be a total dose selected from one or more of the following: about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg, or about 0.001 mg to about 0.1 mg. Optionally, the dose may be:

[0327] about 1 μg, about 2 μg, about 3 μg, about 4 μg, about 5 μg, about 6 μg, about 7 μg, about 8 μg, about 9 μg and / or about 10 μg;

[0328] about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg and / or about 100 μg; or

[0329] About 100 μg, about 200 μg, about 300 μg, about 400 μg, about 500 μg, about 600 μg, about 700 μg, about 800 μg, about 900 μg and / or about 1 mg.

[0330] The foregoing total doses may be particularly suitable for human subjects, especially adult subjects and / or human subjects weighing from about 30 kg to about 200 kg, such as from about 40 kg to about 150 kg, or from 50 kg to about 120 kg, for example from about 60 kg to about 100 kg.

[0331] In an additional or alternative embodiment, a molecule consisting of monomeric Annexin A5 protein is administered to a subject in an amount of about 0.001 mg to about "A" mg, wherein "A" is about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.9 mg or less, such as: about 0.8 mg or less; about 0.7 mg or less; about 0.6 mg or less; about 0.5 mg or less; about 0.4 mg or less; about 0.3 mg or less; about 0.2 mg or less; and more preferably about 0.1 mg or less; about 0.09 mg or less , such as: about 0.08 mg or less; about 0.07 mg or less; about 0.06 mg or less; about 0.05 mg or less; about 0.04 mg or less; about 0.03 mg or less; about 0.02 mg or less; about 0.01 mg or less; about 0.009 mg or less; about 0.008 mg or less; about 0.007 mg or less; about 0.006 mg or less; about 0.005 mg or less; about 0.004 mg or less; about 0.003 mg or less; or about 0.002 mg or less of a molecule consisting of monomeric Annexin A5 protein.

[0332] In an additional or alternative embodiment, a molecule consisting of monomeric Annexin A5 protein is administered to a subject in an amount of about "B" mg to about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg or about 0.1 mg, wherein "B" is about 0.002 mg or more, such as: about 0.003 mg or more; about 0.004 mg or more; about 0.005 mg or more; about 0.006 mg or more; about 0.007 mg or more; about 0.008 mg or more; about 0.009 mg or more; about 0.01 mg or more; about 0.02 mg or more; about 0.03 mg or more; about 0.04 mg or more; about 0.05 mg or more; about 0.06 mg or more; about 0.07 mg or more; about 0.08 mg or more or about 0.09 mg or more of a molecule consisting of monomeric Annexin A5 protein.

[0333] In an additional or alternative embodiment, a molecule consisting of monomeric Annexin A5 protein is administered to a subject in an amount of about "B" mg to about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, wherein "B" is about 0.002 mg or more, such as: about 0.003 mg or more; about 0.004 mg or more; about 0.005 mg or more; about 0.006 mg or more; about 0.007 mg or more; about 0.008 mg or more; about 0.009 mg or more; about 0.01 mg or more ; about 0.02 mg or more; about 0.03 mg or more; about 0.04 mg or more; about 0.05 mg or more; about 0.06 mg or more; about 0.07 mg or more; about 0.08 mg or more; about 0.09 mg or more; about 0.1 mg or more; about 0.2 mg or more; about 0.3 mg or more; about 0.4 mg or more; about 0.5 mg or more; about 0.6 mg or more; about 0.7 mg or more; about 0.8 mg or more; about 0.9 mg or more of a molecule consisting of monomeric Annexin A5 protein.

[0334] In an additional or alternative embodiment, a molecule consisting of monomeric Annexin A5 protein is administered to a subject in an amount of about "A" mg to about "B" mg, wherein "A" and "B" are each values ​​as defined above.

[0335] In an alternative embodiment, about 0.03 mg to about 0.02 mg of the molecule, or about 0.04 mg to about 0.01 mg of the molecule, or about 0.05 mg to about 0.009 mg of the molecule, or about 0.06 mg to about 0.008 mg of the molecule, or about 0.07 mg to about 0.007 mg of the molecule, or about 0.08 mg to about 0.006 mg of the molecule, or about 0.09 mg to about 0.005 mg of the molecule, or about 0.1 mg to about 0.004 mg of the molecule is administered to the subject.

[0336] In another embodiment, about 0.03 mg to about 0.01 mg of the molecule, or about 0.04 mg to about 0.009 mg of the molecule, or about 0.05 mg to about 0.008 mg of the molecule, or about 0.06 mg to about 0.007 mg of the molecule, or about 0.07 mg to about 0.006 mg of the molecule, or about 0.08 mg to about 0.005 mg of the molecule, or about 0.09 mg to about 0.004 mg of the molecule, or about 0.1 mg to about 0.003 mg of the molecule is administered to the subject.

[0337] In another embodiment, about 0.016 mg to about 0.014 mg of the molecule, or about 0.017 mg to about 0.013 mg of the molecule, or about 0.018 mg to about 0.012 mg of the molecule, or about 0.019 mg to about 0.011 mg of the molecule, or about 0.02 mg to about 0.01 mg of the molecule, or about 0.021 mg to about 0.0099 mg of the molecule, or about 0.022 mg to about 0.0098 mg of the molecule, or about 0.02 0.0097 mg of the molecule, or about 0.024 mg to about 0.0096 mg of the molecule, or about 0.025 mg to about 0.0095 mg of the molecule, or about 0.026 mg to about 0.0094 mg of the molecule, or about 0.027 mg to about 0.0093 mg of the molecule, or about 0.028 mg to about 0.0092 mg of the molecule, or about 0.029 mg to about 0.0091 mg of the molecule, or about 0.03 mg to about 0.009 mg of the molecule.

[0338] In a preferred embodiment, about 0.03 mg to about 0.02 mg of the molecule is administered to a subject. In another preferred embodiment, about 0.03 mg to about 0.01 mg of the molecule is administered to a subject. In another preferred embodiment, about 0.016 mg to about 0.014 mg of the molecule is administered to a subject. In another preferred embodiment, about 0.01 mg to about 0.001 mg of the molecule is administered to a subject. In an even more preferred embodiment, about 0.015 mg of the molecule is administered to a subject. In another even more preferred embodiment, about 0.02 mg of the molecule is administered to a subject. In another even more preferred embodiment, about 0.01 mg of the molecule is administered to a subject.

[0339] Unless otherwise stated, embodiments of the invention discussed herein in relation to all other aspects of the invention may apply to the sixth aspect of the invention.

[0340] Route of administration :

[0341] In a preferred embodiment, the molecule consisting of monomeric annexin A5 protein or a composition thereof is administered distally to the eye. Distal administration has the advantage of avoiding administration of a molecule consisting of monomeric annexin A5 protein to the subject's eye, which may be unpleasant to the subject. Specifically, there are many potential complications associated with intravitreal injections (e.g., using established anti-VEGF therapies to treat macular edema and / or RVO), including infectious endophthalmitis, sterile intraocular inflammation, and retinal vasculitis, particularly with injections of bucizumab (Cox et al., 2021, supra) and transient intraocular pressure (IOP) dopants (Levin et al., 2021, J Glaucoma, 30: 1019-26).

[0342] According to a preferred option, the molecule consisting of monomeric annexin A5 protein or a composition thereof is administered systemically. Preferably, the molecule consisting of monomeric annexin A5 protein or a composition thereof is administered to the vascular system of the subject. Most preferably, the molecule consisting of monomeric annexin A5 protein or a composition thereof is administered to the blood of the subject.

[0343] In one embodiment, the molecule consisting of monomeric Annexin A5 protein or a composition thereof is administered by injection, infusion or perfusion; such as intravenous or intraarterial administration (particularly intravenous injection or intraarterial injection).

[0344] In a preferred embodiment, the molecule consisting of monomeric Annexin A5 protein or composition thereof is administered by infusion.

[0345] In another preferred embodiment, the molecule consisting of monomeric Annexin A5 protein or composition thereof is administered by intravenous injection.

[0346] In a more preferred embodiment, a molecule consisting of monomeric Annexin A5 protein or a composition thereof is administered into the blood of a subject by infusion (eg, by intravenous injection).

[0347] In another embodiment, a molecule consisting of monomeric Annexin A5 protein or a composition thereof is administered parenterally, intrathecally, intraperitoneally, intraarticularly, intramuscularly, subcutaneously or topically (although as described above, in one embodiment, direct administration to the eye may optionally be avoided).

[0348] In another alternative, although less preferred, a molecule consisting of a monomeric annexin A5 protein may be administered directly to the eye. For example, in an optionally preferred embodiment, a molecule consisting of a monomeric annexin A5 protein may be administered to one or both eyes of a subject by intravitreal injection. According to this embodiment, the monomeric annexin A5 protein may be administered to a patient in the form of a sterile injectable intravitreal composition, such as defined according to the fourth aspect of the invention.

[0349] The composition for intravitreal injection can be presented, for example, in a form ready for injection (e.g., without the need for reconstitution with a carrier or diluent) in a sterile syringe. Preferably, the syringe contains a volume of about or less than 200 μL, preferably a volume of about or less than 100 μL of the composition, and more preferably, a volume of about 10 μL to about 90 μL, about 20 μL to about 80 μL, about 30 μL to about 70 μL, about 40 μL to about 60 μL, or about 50 μL (wherein the term "about" in this context refers to an amount of ±4 μL, 3 μL, 2 μL, or 1 μL of the specified value).

[0350] Compositions for intravitreal injection may be presented, for example, in the form of a kit comprising the composition and one or more additional components.

[0351] For example, the kit may include a composition presented in a sterile syringe and one or more sterile needles attached thereto or separately included in the kit. Likewise, the uses and methods of various aspects of the invention may include intravitreally injecting a composition comprising an effective amount of a molecule consisting of a monomeric Annexin A5 protein into one or both eyes of a subject via a sterile needle.

[0352] Preferably, the or each needle may be of a gauge selected from gauge 27, 28, 29 or more preferably 30 or 31. It may be preferred that the or each needle is about 0.5 inch to 0.62 inch (12.7 mm to 15.75 mm) in length. Longer needles may increase the risk of retinal damage if the patient accidentally moves forward during the intravitreal injection procedure.

[0353] In less preferred embodiments where the composition is administered to the subject by intravitreal injection, the procedure may include one or more pre-injection risk management steps selected from the following: 1) application of a local anesthetic; 2) application of an agent suitable for reducing the risk of bacterial colonization (povidone-iodine drops and / or periocular povidone-iodine eyelid preparations may be particularly suitable); 3) insertion of a sterile speculum to separate the eyelids; and 4) optionally, re-application of an agent suitable for reducing the risk of bacterial colonization (e.g., povidone-iodine) to the injection site immediately prior to injection.

[0354] In embodiments where the intravitreal injection is performed under local anesthesia, local anesthetic drops may be conveniently employed.

[0355] Any agent suitable for reducing the risk of bacterial colonization can be applied to the eye as part of a pre-injection risk management regimen. Povidone-iodine is a preferred choice. Povidone-iodine has been shown to reduce the risk of bacterial colonization and endophthalmitis. It is recommended to apply povidone-iodine to the conjunctival surface, eyelids, and eyelashes before intravitreal injection, and preferably also before the introduction of a sterile speculum. (The speculum prevents the needle tip from contacting the eyelids or eyelashes prior to needle insertion.) Povidone-iodine can optionally be used in the form of a 5% to 10% solution. Studies have found that a 5% povidone-iodine solution is as effective as a 10% and is less irritating to the eye. Optionally, an agent suitable for reducing the risk of bacterial colonization (e.g., povidone-iodine) is reapplied to the injection site immediately before injection.

[0356] Antibiotics may be used before, during, and / or after intravitreal injection. Any suitable antibiotic may be used. An exemplary embodiment is a fourth generation fluoroquinolone.

[0357] In another example, the kit can include one or more additional compositions, each comprising one or more antibiotics, for administration to a patient during and / or after intravitreal injection.

[0358] Those skilled in the art will be able to select an appropriate intravitreal injection site. For example, in one exemplary embodiment, the patient is instructed to direct their gaze away from the needle entry site and the injection is performed at about 3 mm to about 3.5 mm posterior to the limbus (e.g., for aphakic or pseudophakic eyes), or about 3.5 mm to about 4 mm posterior to the limbus (e.g., for phakic eyes). Injection in the inferior temporal quadrant is common, but any quadrant (e.g., superior temporal quadrant) may be used.

[0359] In another alternative, a molecule consisting of monomeric Annexin A5 protein can be administered directly to a retinal vein (such as the central retinal vein, branch retinal veins and / or hemi-retinal veins) of one or both eyes of a subject, for example, by catheterization.

[0360] Application plan :

[0361] In light of the present disclosure, and applying their common general knowledge, the skilled artisan can readily determine an appropriate dosage to achieve the desired therapeutic effect, optionally within the range of about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg, or about 0.01 μg / kg to about 1 μg / kg of subject body weight, and / or about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg, or about 0.001 mg to about 0.1 mg of the molecule. For example, a suitable dose will generally provide and maintain an amount of molecules consisting of monomeric Annexin A5 protein sufficient to treat or prevent macular edema and / or retinal vein occlusion or related conditions. A suitable dose may be intended to achieve and / or maintain a level of Annexin A5 protein in the subject's plasma that is above the naturally occurring physiological level of Annexin A5 in plasma.

[0362] In other embodiments, the administration of a dose is carried out over an extended period of time, for example, wherein the daily dose is delivered over a period of 30 minutes or more, such as: one hour or more; two hours or more; three hours or more; four hours or more; five hours or more; six hours or more; seven hours or more; eight hours or more; nine hours or more; 10 hours or more; 11 hours or more; 12 hours or more; 13 hours or more; 14 hours or more; 15 hours or more; 16 hours or more; 17 hours or more; 18 hours or more; or up to 24 hours. In a preferred embodiment, the daily dose is delivered by infusion over a period of time.

[0363] The treatment regimen may, for example, involve continuous infusion of Annexin A5 protein to the subject, or may involve one or more administrations of a dose consisting of an amount of a molecule consisting of monomeric Annexin A5 protein as defined with respect to the preceding embodiments, e.g., once, twice, three times, four times or more per day.

[0364] In some cases, the therapeutically effective dose can be optionally administered to a subject with two or more gradually increasing concentrations of dose (i.e., gradually increasing dose), wherein (i) all doses are therapeutic doses, or wherein (ii) a subtherapeutic dose (or two or more subtherapeutic doses) is initially given and the therapeutic dose is achieved by gradually increasing. As a non-limiting example for illustrating the gradually increasing concentration (i.e., gradually increasing dose), a therapeutically effective dose can be administered daily from a subtherapeutic dose, and each subsequent dose can increase a specific increment or a variable increment until the therapeutic dose is reached, at which point administration can stop or can continue (e.g., a continuous therapeutic dose). In some embodiments, administration of a therapeutically effective dose can be continuous infusion, and the dose can change over time (e.g., increase). In some embodiments, combination therapy is also administered.

[0365] In any case, the doctor will determine the actual dosage that is most suitable for any individual patient, and this actual dosage will vary with the age, weight and response of the particular patient. The above dosage is an example of a general situation. Of course, there may also be individual cases in which higher or lower dosage ranges are justified, and this dosage range is within the scope of the present invention.

[0366] For veterinary use, the Annexin A5 protein is administered as a suitably acceptable formulation in accordance with normal veterinary practice, and the veterinarian will determine the dosing regimen and route of administration most suitable for the particular animal.

[0367] Treatment cycle :

[0368] The treatment regimen may continue for the period of time for which the treatment is beneficial.

[0369] In one embodiment, treatment may consist of a single administration of the Annexin A5 composition.

[0370] In another embodiment, multiple administrations may be performed to a subject. For example, a dose consisting of a certain amount of molecules consisting of monomeric annexin A5 protein as defined in relation to the preceding embodiments may be administered once or more per day, such as: twice or more per day; three or more times; four or more times; five or more times; six or more times; seven or more times; eight or more times; nine or more times; or ten or more times, preferably once per day.

[0371] In one embodiment, the dosage of a molecule consisting of monomeric Annexin A5 protein is administered one or more times every two days, one or more times every three days, one or more times every four days, one or more times every five days, one or more times every six days, one or more times every seven days, one or more times every week, one or more times every two weeks, one or more times every three weeks, one or more times every four weeks, one or more times every month, one or more times every two months, one or more times every three months, one or more times every four months, one or more times every five months, one or more times every six months, one or more times every seven months, one or more times every eight months, one or more times every nine months, one or more times every 10 months, one or more times every 11 months, one or more times every 12 months, one or more times every two years, one or more times every three years, one or more times every four years, one or more times every five years, or one or more times every 10 years.

[0372] In one embodiment, the dose of a molecule consisting of monomeric Annexin A5 protein is administered daily for one day or more, such as: two consecutive days or more; three consecutive days or more; four consecutive days or more; five consecutive days or more; six consecutive days or more; seven consecutive days or more; eight consecutive days or more; nine consecutive days or more; ten consecutive days or more; 11 consecutive days or more; 12 consecutive days or more; 13 consecutive days or more; two consecutive days or more; three consecutive days or more; four consecutive days or more; two consecutive days or more; three consecutive days or more; four consecutive days or more; five consecutive days or more; six consecutive days or more; seven consecutive days or more; eight consecutive days or more; nine consecutive days or more; ten consecutive days or more; 11 consecutive days or more; 12 consecutive days or more; 15 consecutive days or more 18 months for multiple consecutive days or more; 21 months for multiple consecutive days or more; 24 months for multiple consecutive days or more; 30 months for multiple consecutive days or more; 3 years for multiple consecutive days or more; 42 months for multiple consecutive days or more; 4 years for multiple consecutive days or more; 5 years for multiple consecutive days or more; 6 years for multiple consecutive days or more; 7 years for multiple consecutive days or more; 8 years for multiple consecutive days or more; 9 years for multiple consecutive days or more; 10 years for multiple consecutive days or more; 11 years for multiple consecutive days or more; 2 years for consecutive days or more; 13 years for consecutive days or more; 14 years for consecutive days or more; 15 years for consecutive days or more; 16 years for consecutive days or more; 17 years for consecutive days or more; 18 years for consecutive days or more; 19 years for consecutive days or more; 20 years for consecutive days or more; 25 years for consecutive days or more; 30 years for consecutive days or more; 35 years for consecutive days or more; 40 years for consecutive days or more; or 50 years for consecutive days or more. In a preferred embodiment, the molecule consisting of monomeric annexin A5 protein is administered daily for six or more consecutive days. In an alternative preferred embodiment, the molecule consisting of monomeric annexin A5 protein is administered daily for seven or more consecutive days. In a more preferred embodiment, the annexin A5 protein is administered daily for six consecutive days or seven consecutive days. In a most preferred embodiment, the annexin A5 protein is administered daily for six consecutive days. In an alternative and most preferred embodiment, the annexin A5 protein is administered daily for seven consecutive days.

[0373] In one embodiment, a molecule consisting of a monomeric Annexin A5 protein is administered two or more times daily for one week or more, e.g., two weeks or more; three weeks or more; four weeks or more; two months or more; three months or more; four months or more; five months or more; six months or more; seven months or more; eight months or more; nine months or more; ten months or more; 11 months or more; 12 months or more; 15 months or more; 18 months or more; 21 months or more; 24 months or more; 30 months or more; three years or more; 42 months or more; four years or more; five years or more; six years or more; seven years or more; eight years or more; nine years or more; ten years or more; 11 years or more; 12 years or more; 13 years or more; 14 years or more; 15 years or more; 16 years or more; 17 years or more; 18 years or more; 19 years or more; 20 years or more; 25 years or more; 30 years or more; 35 years or more; 40 years or more; or 50 years or more.

[0374] In one embodiment, a molecule consisting of a monomeric Annexin A5 protein is administered once a week for one week or more, e.g., two weeks or more; three weeks or more; four weeks or more; two months or more; three months or more; four months or more; five months or more; six months or more; seven months or more; eight months or more; nine months or more; ten months or more; 11 months or more; 12 months or more; 15 months or more; 18 months or more; 21 months or more; 24 months or more; 30 months or more; three years or more; 42 months or more; four years or more; five years or more; six years or more; seven years or more; eight years or more; nine years or more; ten years or more; 11 years or more; 12 years or more; 13 years or more; 14 years or more; 15 years or more; 16 years or more; 17 years or more; 18 years or more; 19 years or more; 20 years or more; 25 years or more; 30 years or more; 35 years or more; 40 years or more; or 50 years or more.

[0375] In one embodiment, a molecule consisting of monomeric Annexin A5 protein is administered two or more times per week for one week or more, e.g., two weeks or more; three weeks or more; four weeks or more; two months or more; three months or more; four months or more; five months or more; six months or more; seven months or more; eight months or more; nine months or more; ten months or more; 11 months or more; 12 months or more; 15 months or more; 18 months or more; 21 months or more; 24 months or more; 30 months or more; three years or more; 42 months or more; four years or more; five years or more; six years or more; seven years or more; eight years or more; nine years or more; ten years or more; 11 years or more; 12 years or more; 13 years or more; 14 years or more; 15 years or more; 16 years or more; 17 years or more; 18 years or more; 19 years or more; 20 years or more; 25 years or more; 30 years or more; 35 years or more; 40 years or more; or 50 years or more.

[0376] In one embodiment, a molecule consisting of a monomeric Annexin A5 protein is administered once a month for one month or longer, e.g., two months or longer; three months or longer; four months or longer; five months or longer; six months or longer; seven months or longer; eight months or longer; nine months or longer; ten months or longer; 11 months or longer; 12 months or longer; 15 months or longer; 18 months or longer; 21 months or longer; 24 months or longer; 30 months or longer; three years or longer 42 months or more; 4 years or more; 5 years or more; 6 years or more; 7 years or more; 8 years or more; 9 years or more; 10 years or more; 11 years or more; 12 years or more; 13 years or more; 14 years or more; 15 years or more; 16 years or more; 17 years or more; 18 years or more; 19 years or more; 20 years or more; 25 years or more; 30 years or more; 35 years or more; 40 years or more; or 50 years or more.

[0377] In one embodiment, a molecule consisting of a monomeric Annexin A5 protein is administered two or more times per month for one month or longer, e.g., two months or longer; three months or longer; four months or longer; five months or longer; six months or longer; seven months or longer; eight months or longer; nine months or longer; ten months or longer; 11 months or longer; 12 months or longer; 15 months or longer; 18 months or longer; 21 months or longer; 24 months or longer; 30 months or longer; three years or longer. a long period of time; 42 months or more; four years or more; five years or more; six years or more; seven years or more; eight years or more; nine years or more; ten years or more; 11 years or more; 12 years or more; 13 years or more; 14 years or more; 15 years or more; 16 years or more; 17 years or more; 18 years or more; 19 years or more; 20 years or more; 25 years or more; 30 years or more; 35 years or more; 40 years or more; or 50 years or more.

[0378] Combination therapy :

[0379] The molecule consisting of the monomeric annexin A5 protein can be administered to a subject undergoing treatment according to the invention in an amount and for a period of time to provide an overall therapeutic effect. The molecule consisting of the monomeric annexin A5 protein A5 can be administered alone (monotherapy) or in combination with other agents in a mixed form or by separate, simultaneous or sequential administration (combination therapy). For the avoidance of doubt, the other agent is not an agent comprising the sequence of human annexin A5 or a biologically active fragment thereof, and more specifically, is not dimeric annexin A5 (commonly referred to as "di-annexin" in the art), and is not pegylated annexin A5.

[0380] In the case of combination therapy, the amount and frequency of administration can be an amount and frequency that provides, for example, an additive therapeutic effect or a synergistic therapeutic effect. Further, the administration of a molecule consisting of a monomeric Annexin A5 protein (with or without one or more second or additional agents) can be used as a primary treatment, e.g., a first-line treatment, or as a secondary treatment, e.g., for a subject who has had an inadequate response to a previously administered therapy (i.e., a therapy other than a therapy with a molecule consisting of a monomeric Annexin A5 protein).

[0381] Thus, in one embodiment, a subject can be treated with a molecular therapy consisting of a monomeric Annexin A5 protein in addition to one or more therapeutic or prophylactic interventions. A subject can receive a molecular therapy consisting of a monomeric Annexin A5 protein in the same composition as one or more other therapeutic or prophylactic interventions, or one or more additional therapeutic or prophylactic treatments, separate from said interventions or treatments, simultaneously with a separate formulation containing said interventions or treatments, or sequentially with said interventions or treatments.

[0382] In some embodiments of the invention, the one or more other therapeutic or prophylactic interventions, or one or more additional therapeutic or prophylactic treatments, comprise or consist of one or more treatments selected from the group consisting of anti-VEGF therapy (e.g., anti-VEGF therapy delivered to the eye by intravitreal injection), corticosteroids, NSAIDs, pars plana vitrectomy, retinal laser photocoagulation, or anti-VEGF therapy augmented with intravitreal corticosteroids and / or retinal laser photocoagulation.

[0383] Exemplary anti-VEGF therapies may include any one or more of ranibizumab, aflibercept, bevacizumab, bricizumab, and / or faricizumab.

[0384] Exemplary corticosteroids include difluprednate, triamcinolone acetonide, triamcinolone acetonide, and dexamethasone, which can be used as monotherapy, or they may or may not be combined with immunomodulatory therapy to achieve more rapid resolution of edema.

[0385] Devices and kits for use in therapy :

[0386] Pharmaceutical compositions and veterinary compositions comprising molecules consisting of monomeric annexin A5 proteins can be administered with a medical device. The device can be designed to have features such as portability, room temperature storage, and ease of use, so that the device can be used, for example, by untrained subjects or by emergency personnel on site in emergency situations where there are no medical facilities or other medical equipment. The device can include, for example, one or more housings for storing pharmaceutical preparations comprising molecules consisting of monomeric annexin A5 proteins, and can be configured to deliver one or more unit doses of molecules consisting of monomeric annexin A5 proteins. The device can be further configured to administer a second agent in the form of a single pharmaceutical composition that also comprises molecules consisting of monomeric annexin A5 proteins or in the form of two separate compositions.

[0387] Pharmaceutical and veterinary compositions can be administered with a syringe. Pharmaceutical and veterinary compositions can also be administered with a needle-free hypodermic injection device, such as the devices disclosed in US Pat. Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules include: US 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; US 4,486,194, which discloses a therapeutic device for administering drugs through the skin; US 4,447,233, which discloses a drug infusion pump for delivering drugs at a precise infusion rate; US 4,447,224, which discloses a variable flow implantable infusion device for continuous drug delivery; US 4,439,196, which discloses an osmotic drug delivery system with a multi-chamber compartment; and US 4,475,196, which discloses an osmotic drug delivery system. Many other devices, implants, delivery systems and modules are also known.

[0388] Molecules consisting of monomeric annexin A5 protein can be provided in a kit. In one embodiment, the kit comprises (a) a container containing a composition comprising annexin A5 protein, and optionally (b) informational material. The informational material can be descriptive, instructional, marketing or other material related to the methods described herein and / or using the agents to achieve therapeutic benefits.

[0389] In one embodiment, the kit further comprises a second agent for treating a condition described herein. For example, the kit comprises a first container containing a composition comprising a molecule consisting of monomeric Annexin A5 protein and a second container comprising a second agent.

[0390] The form of the information material of the kit is not limited. In one embodiment, the information material may include information about the production of the compound, the molecular weight, concentration, expiration date, batch or production site information of the compound, etc. In one embodiment, the information material relates to a method of administering a molecule consisting of a monomeric annexin A5 protein, for example, in a suitable dose, dosage form or administration mode (e.g., a dose, dosage form or administration mode described herein) to treat a subject in need thereof as described herein. The information may be provided in a variety of formats, including printed text, computer readable material, video recording, or audio recording, or information providing a link or address to substantial material, for example, on the Internet. In one embodiment, the informational material provides for administering to a subject a dose of about 50 μg / kg to about 100 μg / kg, about 40 μg / kg to about 80 μg / kg, about 30 μg / kg to about 60 μg / kg, about 20 μg / kg to about 40 μg / kg, about 1 μg / kg to 30 μg / kg, less than about 20 μg / kg, such as about 0.01 μg / kg to about 10 μg / kg or about 0.01 μg / kg to about 1 μg / kg of the subject's body weight, and / or administering to a subject a dose of about 5 mg, about 4 mg, about 3 mg, about 2 mg, less than about 2 mg, such as about 1 mg, about 0.1 mg-1.5 mg, 0.001 mg to about 1 mg, or about 0.001 mg to about 0.1 mg of a molecule consisting of monomeric Annexin A5 protein.

[0391] In addition to the molecules consisting of monomeric annexin A5 protein, the composition in the kit may also include other ingredients, such as solvents or buffers, stabilizers or preservatives. The molecules consisting of monomeric annexin A5 protein can be provided in any form, for example, liquid, dry or lyophilized form, preferably substantially pure and / or sterile. When the molecules consisting of monomeric annexin A5 protein are provided in the form of a liquid solution, the liquid solution is preferably an aqueous solution. When the molecules consisting of monomeric annexin A5 protein are provided in dry form, reconstitution is usually performed by adding a suitable solvent. Optionally, a solvent, such as sterile water or a buffer, can be provided in the kit.

[0392] The kit may include one or more containers for one or more compositions containing a medicament. In some embodiments, the kit contains separate containers, separators or compartments for compositions and informational materials. For example, the composition may be contained in a bottle, vial or syringe, and the informational material may be contained in a plastic sleeve or package. In other embodiments, the individual elements of the kit are contained in a single undivided container. For example, the composition is contained in a bottle, vial or syringe with informational materials in the form of a label affixed thereto. In some embodiments, the kit comprises multiple (e.g., a pack) of separate containers of medicaments each containing one or more unit dosage forms (e.g., dosage forms described herein). The container may contain a combined unit dose, for example, a unit containing, for example, a molecule composed of a monomeric membrane annexin A5 protein and a second medicament in a desired ratio. For example, the kit comprises, for example, multiple syringes, ampoules, foil bags, blister packages or medical devices each containing a single combined unit dose. The container of the kit may be airtight, waterproof (e.g., impermeable to moisture changes or evaporation) and / or opaque.

[0393] The kit optionally comprises a device suitable for administering the composition, such as a syringe or other suitable delivery device.The device may be provided pre-filled with one or both of the agents, or the device may be empty but suitable for loading.

[0394] F. Method for calculating the dose of Annexin A5

[0395] In a seventh aspect, the present invention provides a method for calculating the dose of Annexin A5 required for treating a condition such as macular edema and / or RVO, the method comprising the steps of:

[0396] A. Determine the number of Annexin A5-positive cells in the subject;

[0397] B. determining the number of annexin A5 binding sites on annexin-positive cells in the subject;

[0398] C. determining the number of Annexin A5 molecules required for binding to the Annexin A5 binding site, such as by multiplying the value determined for A by the value determined for B;

[0399] D. Determine the dose from the value determined for C.

[0400] Unless otherwise stated, embodiments of the invention discussed herein are applicable to the seventh aspect of the invention.

[0401] In one embodiment, the method further comprises:

[0402] C'. Determine the molar concentration of the number of molecules required to bind to the annexin A5 binding site from the value determined in C; and,

[0403] D. Also includes determining the dosage for the value determined for C'.

[0404] Preferably, D also comprises determining the dosage by multiplying the value determined for C' by the molar mass of Annexin A5. Preferably, the molar mass of Annexin A5 used is about 35,700 Da. If an Annexin A5 protein variant with a different molecular mass is used, the value used to calculate step D can be adjusted accordingly.

[0405] In one embodiment, Annexin A5 is monomeric Annexin A5, such as a molecule comprising a monomeric Annexin A5 protein as further defined herein.

[0406] In one embodiment, the method comprises:

[0407] E. administering the dose of Annexin A5 to the subject.

[0408] Preferably, the method comprises

[0409] E. administering the dose of Annexin A5 to the subject to treat the disorder.

[0410] In one embodiment, the method is an in vitro method. In another embodiment, steps AD of the method are in vitro method steps.

[0411] In one embodiment, the disorder is one or more selected from the list consisting of: viral hemorrhagic fever (such as Ebola virus hemorrhagic fever or dengue virus hemorrhagic fever); cardiovascular disorders (such as stroke, atherosclerosis, central retinal vein occlusion, myocardial infarction, stenosis or restenosis); sickle cell disease; arthritis (such as osteoarthritis) and polyuria. Preferably, the disorder is retinal vein occlusion, more preferably central retinal vein occlusion.

[0412] In an eighth aspect, the present invention provides a method for calculating the dose of Annexin A5 required to bind to the Annexin A5 binding site in a subject, the method comprising the following steps:

[0413] A. Determine the number of Annexin A5-positive cells in the subject;

[0414] B. determining the number of annexin A5 binding sites on annexin-positive cells in the subject;

[0415] C. determining the number of Annexin A5 molecules required for binding to the Annexin A5 binding site, such as by multiplying the value determined for A by the value determined for B;

[0416] D. Determine the dose from the value determined for C.

[0417] Unless otherwise stated, embodiments of the invention discussed herein are applicable to the eighth aspect of the invention.

[0418] In one embodiment of the eighth aspect of the invention, the method comprises:

[0419] E. Administering the dose of Annexin A5 to the subject for prevention; preferably, for prevention of a disorder.

[0420] **************

[0421] The invention will now be described with reference to one or more non-limiting examples.

[0422] Example

[0423] The following examples are included to illustrate specific embodiments of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention and therefore can be considered to constitute preferred modes of practice of the present invention. However, it will be appreciated by those skilled in the art, based on this disclosure, that many changes may be made to the specific embodiments disclosed and still achieve the same or similar results without departing from the spirit and scope of the present invention.

[0424] Example 1

[0425] Probe construction and validation :

[0426] Purified recombinant monomeric human Annexin A5 protein (ANXA5) was labeled with 800CW, a molecular imaging and contrast agent, to form the ANXA5-800CW agent, in which approximately 40% of the ANXA5 present was determined to be labeled with 800CW (and the remaining approximately 60% remained as unlabeled monomeric human Annexin A5 protein).

[0427] ANXA5-800CW specifically binds to phosphatidylserine (PS), a key molecule in the pathogenesis of RVO.

[0428] A 66-year-old female with known systemic hypertension was seen in the clinic. In recent weeks, she presented with decreased vision in her right eye, which was determined to be caused by branch retinal vein occlusion (BRVO).

[0429] The presence of PS on fresh erythrocytes was determined by flow cytometry using the PS assay performed on blood samples collected before and after systemic administration of ANXA5-800CW. These results show the presence and percentage increase in PS availability compared to the negative control ( Figure 1 ). This increase in PS externalization was observed during all blood sampling time points, demonstrating the stable availability of the target for ANXA5-800CW.

[0430] Evaluation of the localization of intravenously administered Annexin A5 to the site of retinal vein occlusion :

[0431] Next, the potential to visualize the binding of ANXA5-800CW at the site of retinal vein occlusion was evaluated.

[0432] 1.0 mg ANXA5-800CW was administered intravenously as a single bolus, followed by near-infrared (NIR) fluorescence imaging at predetermined time points (up to 4 hours post-injection) using a Heidelberg Spectralis camera system.

[0433] Figure 2 The results of fluorescein angiography (FA) performed as a standard of care are shown. It shows the exact occlusion site (red arrow) and the damaged retinal area (red oval) with bleeding. When NIR fluorescence imaging was performed, we observed possible fluorescence signals of ANXA5-800CW in the blood vessels and optic nerve located in the affected retinal area 5 minutes after application. The intensity seems to increase over time, as shown in the image obtained at 229 minutes after injection.

[0434] In the peripheral retinal vessels, no fluorescent signal was detected as identified by FA (see Figure 3 ). This indicates that there is no significant ANXA5-800CW uptake in vessels not affected by BRVO.

[0435] These observations indicate site-specific accumulation of ANXA5-800CW at retinal vessels and suggest binding of Annexin A5 at sites of occlusion. We interpret these results as indicating the efficacy of systemically administered Annexin A5 (including labeled forms of ANXA5-800CW) in preferential accumulation of exposed PS (its target) at sites of occlusion in subjects with RVO.

[0436] Example 2

[0437] As discussed above, evidence suggests that several inflammatory cytokines, including IL-6, TNF-α, IL-1β, and ICAM-1, all serve to disrupt the integrity of the BRB and contribute to the development of macular edema and / or retinal vein occlusion (Haydinger et al., 2023, supra; Park et al., 2019, supra; Noma et al., 2020, supra).

[0438] Here we report experiments investigating the potential of Annexin A5 to block the action of such inflammatory cytokines.

[0439] Methods: Chondrocytes were cultured with IL-1β (10 ng / mL) or TNF-α (10 ng / mL) and with or without purified recombinant monomeric human annexin A5 (ANXA5) (100 ng / mL, 5 μg / mL, 20 μg / mL). Cells were harvested at 3, 6, 12, and 18 hours. Cell viability was determined by flow cytometry with propidium iodide (PI).

[0440] The expression of IL-6 was measured in cells stimulated with IL-1β. Cells were stimulated with 10 ng IL-1β and treated with different concentrations of ANXA5 (0.1 μg / mL, 5 μg / mL and 20 μg / mL of ANXA5) for 6 hours. The expression of genes was measured by qPCR using the SYBR green reaction kit. Gene expression was normalized to GAPDH and the relative expression of genes was measured using 2ΔΔCt.

[0441] Results: The results show that treatment with Annexin A5 reduced cell death in this in vitro study.

[0442] Figure 4 showed that in a human model, cell death promoted by TNFα application was inhibited by treatment with Annexin A5, where the cell viability of Annexin A5-treated cells was similar to that of controls not treated with TNFα.

[0443] Similar results were seen with IL-1β, e.g. Figure 5 Annexin A5 was shown to inhibit cell death in a dose-dependent manner, with treatment at doses of 5 μg and 20 μg causing cell viability comparable to that of controls not treated with IL-1β. Figure 5 It was shown that treatment with Annexin A5 protein alone did not affect cell viability compared to the control without IL-1β treatment.

[0444] In addition to inhibiting cell death, treatment with Annexin A5 also reduced key biomarkers.

[0445] like Figure 6As shown, treatment with Annexin A5 reduced the expression of ICAM1 compared to cells treated with TNFα to promote cell death and inflammation and to controls that had not been treated with TNFα.

[0446] like Figure 7 As shown, treatment with Annexin A5 reduced IL-6 expression in a dose-dependent manner compared to cells induced with IL-1β and controls that had not been treated with IL-1β. Figure 7 It was shown that treatment with Annexin A5 alone had no effect on IL-6 expression.

[0447] Conclusions: We demonstrate that Annexin A5 inhibits the production and / or action of several inflammatory cytokines (including IL-6, TNF-α, IL-1β, and ICAM-1) that have been implicated in dysregulation of BRB integrity and contribute to the development of macular edema and / or retinal vein occlusion.

[0448] Example 3

[0449] The inventors performed new calculations based on information previously unknown and / or overlooked by the prior art to determine a therapeutically effective dose of monomeric Annexin A5 protein for treating retinal vein occlusion, such as CRVO.

[0450] The inventors' calculations were based on various relevant parameters related to the binding of monomeric annexin A5 and the pathological features of retinal vein occlusion. Using these parameters, the inventors calculated the dose of monomeric annexin A5 that would be effective in treating retinal vein occlusion.

[0451] This calculation has taken into account the following information:

[0452] The molecular weight of monomeric annexin A5 is 35.7 kilodaltons (kDa), so 1 nanomole (nm) of monomeric annexin A5 is 0.036 micrograms / milliliter (μg / ml);

[0453] Under physiological conditions, a single monomeric annexin A5 protein is bound to approximately In other words, a single monomeric annexin A5 protein binds to a region of approximately 50 phospholipid ("PL") heads in the erythrocyte membrane, and a single monomeric annexin A5 protein binds to approximately 40 phosphatidylserine ("PS") molecules on the surface of erythrocytes;

[0454] Approximately 1.8% ± (plus / minus) 0.1% of red blood cells in patients with retinal vein occlusion (especially central retinal vein occlusion) are positive for Annexin A5, while approximately 0.7% ± (plus / minus) 0.1% of red blood cells in normal patients are positive for Annexin A5;

[0455] There are approximately 720 monomeric annexin A5 binding sites on each annexin A5-positive erythrocyte in patients with retinal vein occlusion, compared with approximately 280 monomeric annexin A5 binding sites per annexin A5-positive erythrocyte in normal patients;

[0456] There are approximately 4 liters (L) of blood in a 60 kilogram (kg) human patient;

[0457] Usually there are 5×10 12 red blood cells;

[0458] The amount of endogenous Annexin A5 produced by the body is very low, so its contribution to binding to Annexin A5 binding sites on Annexin A5-positive red blood cells is negligible; and,

[0459] Typically, CRVO patients do not have another disease that results in an increase in Annexin A5 binding sites (such as sickle cell disease).

[0460] To calculate the optimal dose of monomeric Annexin A5 for patients with retinal vein occlusion, the inventors performed the following calculations:

[0461] I. Calculate the number of Annexin A5-positive red blood cells in the blood of patients with retinal vein occlusion: (Percentage of Annexin A5-positive red blood cells × Number of red blood cells) ÷ 100 = Number of Annexin A5-positive red blood cells (AI);

[0462] II. Calculate the number of monomeric annexin A5 protein molecules required to cover the annexin A5 binding sites on the annexin A5-positive erythrocytes in the blood of patients with retinal vein occlusion: the number of annexin A5 binding sites on each annexin A5-positive erythrocyte × the number of annexin A5-positive erythrocytes (AI) = the number of monomeric annexin A5 protein molecules (AII);

[0463] III. Calculate the number of moles of monomeric annexin A5 (M) required to cover the annexin A5 binding sites on annexin A5-positive red blood cells in the blood of patients with retinal vein occlusion: the number of annexin A5 protein molecules (AII) ÷ Avogadro constant (6.022×10 23 ) = moles of annexin A5 (AIII); and,

[0464] IV. Calculation of the amount of Annexin A5 required to cover the Annexin A5 binding sites on Annexin A5-positive erythrocytes in the blood of patients with retinal vein occlusion: Number of moles of Annexin A5 (AIII) x molar mass = amount.

[0465] The information known to the applicant can be used to calculate:

[0466] The calculation is based on the average human patient having a total body weight of about 60 kg and having about 4 L of blood, with about 5 x 10 12 red blood cells (i.e. a total of about 20x10 12 The results of the study showed that approximately 1.8% of erythrocytes in patients with CRVO were positive for Annexin A5. In addition, an average of approximately 720 monomeric Annexin A5 binding sites were found per erythrocyte in patients with retinal vein occlusion.

[0467] I.(1.8×20×10 12 )÷100=0.36×10 12 , Annexin A5-positive erythrocytes in the blood of a typical 60 kg patient with retinal vein occlusion (AI);

[0468] II.720×0.36x10 12 =259.2×10 12 , the monomeric Annexin A5 protein molecule (AII) required to cover the Annexin A5 binding sites on Annexin A5 positive erythrocytes in the blood of a typical 60 kg patient with retinal vein occlusion:

[0469] III.259.2×10 12 ÷6.022×10 23 =43.04218×10 -11 , the number of moles of monomeric Annexin A5 required to cover the Annexin A5 binding sites on Annexin A5 positive erythrocytes in the blood of a typical 60 kg patient with retinal vein occlusion (AIII); and,

[0470] IV.43x10 -11 ×35,700=1,536,605×10 -11 g, effective dose covering the Annexin A5 binding sites on Annexin A5 positive erythrocytes in the blood of a typical 60 kg patient with retinal vein occlusion.

[0471] This value is equivalent to a single monomeric Annexin A5 dose of 0.01537 mg (ie 15.37 μg).For practical purposes, this dose can be conveniently rounded up to a single therapeutic or prophylactic dose of 0.02 mg (ie 20 μg) of monomeric Annexin A5.

[0472] 1,536,605×10 -11 The value of g can also be converted to a number in mg / kg body weight, which (remember that the calculation is based on a 60 kg patient, then the amount is per kg of the patient's body weight, and then the amount can be used for patients of other weights and the total amount calculated accordingly) is 0.000256 mg / kg body weight (i.e. 0.256 μg / kg). For practical purposes, this dose can be rounded to 0.0003 mg / kg body weight (i.e. 0.3 μg / kg).

[0473] Example 4

[0474] The following is an exemplary regimen for administering monomeric Annexin A5 to a patient with retinal vein occlusion:

[0475] ·Route of administration: Infusion or intravenous

[0476] Dosage frequency: once a day;

[0477] Duration of treatment: 6 to 7 days; and,

[0478] • Dose per administration: The total dose is in the range of 0.015 mg to 0.02 mg (i.e., 15 to 20 μg dose), and / or adjusted taking into account the patient's weight, in the range of 0.000256 mg / kg to 0.0003 mg / kg (i.e., 0.256 to 0.3 μg / kg) body weight.

[0479] It is contemplated that in practice, the prophylactic and / or therapeutically effective dosage of monomeric Annexin A5 may be increased or decreased from the aforementioned exemplified values, depending on, for example, the specific characteristics of the RVO patient and / or the selected monomeric Annexin A5 protein, for example within one or both of the following ranges:

[0480] i. about 0.01 μg / kg body weight to about 10 μg / kg body weight, and more preferably about 0.01 μg / kg body weight to about 1 μg / kg body weight, and / or

[0481] ii. A total dose of about 1 μg to about 1 mg, and more preferably about 1 μg to about 100 μg.

[0482] For example, the patient may have a different total body weight than the exemplified subject; may have a greater or lesser total blood volume (e.g., having a greater or lesser total blood volume than a typical 60 kg patient having about 4 L of total blood volume); may be characterized as having an atypical proportion of Annexin A5-positive red blood cells (compared to the typical 1.8% ± 0.1% in most CRVO patients); may have an atypical number of Annexin A5 binding sites per Annexin A5-positive red blood cell (typically for CRVO patients, showing about 720 sites per cell); may have more or less than the typical number of red blood cells per liter of blood (i.e., a typical value of about 5×10 12 may contain higher or lower levels of endogenous Annexin A5 protein than a typical patient; and / or may have more or fewer Annexin A5 binding sites in non-erythroid cell types.

[0483] Furthermore, the selected monomeric Annexin A5 protein (which may be, for example, a biologically active fragment of human Annexin A5) may have a different molecular weight (compared to the naturally occurring protein having a MW of 35.7 KDa), and / or have an altered ability to bind to the Annexin A5 binding site on Annexin A5-positive erythrocytes.

[0484] Variations in any one or more of the foregoing properties or characteristics of a patient and / or a selected monomeric Annexin A5 protein may affect the amount of monomeric Annexin A5 protein useful for preventing and / or treating retinal vein occlusion (RVO) and / or one or more diseases, conditions or disorders associated with and / or caused by RVO.

[0485] **************

[0486] It is contemplated that any use, method, or composition described herein can be implemented with respect to any other use, method, or composition described herein.

[0487] When used in conjunction with the term "comprising" in the claims and / or the specification, the terms "a" or "an" may mean "one", but are also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0488] As used herein, unless otherwise stated, the term "about" may be used herein to mean a range of ±50%, ±40%, ±30%, ±20%, ±10%, ±5%, ±4%, ±3%, ±2%, or ±1% of the referenced value.

[0489] It should be understood that the foregoing description, while indicating various embodiments of the present invention and many of its specific details, is given in an illustrative and non-restrictive manner. Without departing from the spirit of the present invention, many substitutions, modifications, additions and / or rearrangements may be made within the scope of the present invention, and the present invention includes all such substitutions, modifications, additions and / or rearrangements.

[0490] **************

[0491] According to the present disclosure, all compositions and / or methods disclosed and claimed herein can be prepared and performed without excessive experimentation. Although the compositions and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that the steps or sequence of steps of the compositions and / or methods and the methods described herein can be changed without departing from the concept, spirit and scope of the present invention. More specifically, it will be apparent that certain medicaments related to chemistry and physiology can replace the medicaments described herein, while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of the present invention as defined by the appended claims.

Claims

1. A molecule consisting of monomeric Annexin A5 protein for use in preventing or treating macular edema or a condition associated therewith in a subject.

2. The molecule consisting of monomeric Annexin A5 protein for use according to claim 1, wherein a dose of said molecule consisting of monomeric Annexin A5 protein is administered systemically, preferably intravenously, to said subject.

3. The molecule consisting of monomeric Annexin A5 protein for use according to claim 1 or 2, wherein the dose of the molecule consisting of monomeric Annexin A5 protein is administered to the subject daily for two or more consecutive days.

4. The molecule consisting of monomeric Annexin A5 protein for use according to claim 3, wherein said dose of said molecule consisting of monomeric Annexin A5 protein is administered to said subject daily for three, four, five, six, seven or more consecutive days.

5. A molecule consisting of monomeric Annexin A5 protein for use according to any preceding claim, wherein the daily dose of the molecule consisting of monomeric Annexin A5 protein administered to the subject is as follows: a) a total daily dose of about 5 mg, about 4 mg, about 3 mg, about 2 mg, or less than about 2 mg; or b) within a range selected from the group consisting of about 50 μg-100 μg, about 40 μg-80 μg, about 30 μg-60 μg, about 20 μg-40 μg, or less than about 20 μg per kg body weight of the subject.

6. The molecule consisting of monomeric Annexin A5 protein for use according to any one of claims 1 to 4, wherein the daily dose of the molecule consisting of monomeric Annexin A5 protein administered to the subject is as follows: a) The total daily dose is 0.1 mg to 1.5 mg; or b) within the range of 1 μg to 30 μg per kg body weight of said subject.

7. The molecule consisting of monomeric Annexin A5 protein for use according to claim 1, wherein the subject suffers from, is suspected of suffering from, or is at risk of suffering from retinal vein occlusion (RVO).

8. A molecule consisting of monomeric Annexin A5 protein for use in preventing or treating retinal vein occlusion (RVO) or a condition associated therewith in a subject, Wherein a dose of said molecule consisting of monomeric Annexin A5 protein is systemically administered to said subject daily for two or more consecutive days.

9. The molecule consisting of monomeric Annexin A5 protein for use according to claim 8, wherein a dose of the molecule consisting of monomeric Annexin A5 protein is intravenously administered to the subject daily for two or more consecutive days.

10. The molecule consisting of monomeric Annexin A5 protein for use according to claim 8 or 9, wherein said dose of said molecule consisting of monomeric Annexin A5 protein is administered systemically, preferably intravenously, to said subject daily for three, four, five, six, seven or more consecutive days.

11. The molecule consisting of monomeric Annexin A5 protein for use according to any one of claims 8 to 10, wherein the daily dose of the molecule consisting of monomeric Annexin A5 protein administered systemically, preferably intravenously, to the subject is as follows: a) a total daily dose of about 5 mg, about 4 mg, about 3 mg, about 2 mg, or less than about 2 mg; or b) within a range selected from the group consisting of about 50 μg-100 μg, about 40 μg-80 μg, about 30 μg-60 μg, about 20 μg-40 μg, or less than about 20 μg per kg body weight of the subject.

12. The molecule consisting of monomeric Annexin A5 protein for use according to any one of claims 8 to 10, wherein the daily dose of the molecule consisting of monomeric Annexin A5 protein administered systemically, preferably intravenously, to the subject is as follows: a) The total daily dose is 0.1 mg to 1.5 mg; or b) within the range of 1 μg to 30 μg per kg body weight of said subject.

13. A molecule consisting of monomeric Annexin A5 protein for use according to any one of claims 8 to 12, wherein the use is for preventing or treating a condition in a subject having, suspected of having, or at risk of having retinal vein occlusion (RVO), and wherein the condition is selected from the group consisting of macular edema, retinal ischemia, iris neovascularization (optionally, iris neovascularization characterized by increased cytokines such as increased VEGF), retinal neovascularization (optionally, retinal neovascularization characterized by increased cytokines such as increased VEGF), neovascular glaucoma, vitreous hemorrhage, erythrosis and retinal detachment.

14. A molecule consisting of monomeric Annexin A5 protein for use according to any preceding claim, wherein the subject is an adult and / or has a body weight of about 50 kg to about 150 kg.

15. A molecule consisting of monomeric Annexin A5 protein for use according to any one of claims 7 to 14, wherein the RVO is selected from: central retinal vein occlusion (CRVO); branch retinal vein occlusion (BRVO); or hemi-retinal vein occlusion (HRVO).

16. A molecule consisting of monomeric Annexin A5 protein for use according to any one of the preceding claims, wherein the monomeric Annexin A5 protein is selected from one or more of the following: a. A protein consisting essentially of the sequence of human monomeric annexin A5 protein or consisting of the sequence of human monomeric annexin A5 protein (optionally, a protein consisting of the sequence of SEQ ID NO: 1 with or without an N-terminal methionine); b. recombinant human monomeric annexin A5 protein; c. the mammalian ortholog of the human monomeric annexin A5 protein; da), b) or c) allelic variant or genetic variant; e. A functional analog or variant of a monomeric Annexin A5 protein, which is a protein consisting of an amino acid sequence that is more than 50%, 60%, 70%, 75%, such as more than 80%, 85%, more than 90% or even more preferably more than 95% or 99% identical to a human monomeric Annexin A5 protein having a sequence defined by SEQ ID NO: 1 with or without an N-terminal methionine; and A biologically active fragment of any one of fa) to e).

17. A composition comprising a molecule consisting of monomeric Annexin A5 protein for use according to any preceding claim.

18. The composition for use according to claim 17, wherein the composition is a unit dose composition.

19. A unit dose composition for use according to claim 18, wherein the unit dose composition comprises: a) a total dose of about 5 mg, about 4 mg, about 3 mg, about 2 mg, or less than about 2 mg; or b) Total dose of 0.1 mg-1.5 mg.

20. A composition for use according to any one of claims 17 to 19, wherein the composition is a pharmaceutical composition.

21. A composition for use according to any one of claims 17 to 20, wherein the composition further comprises one or more pharmaceutically acceptable carriers.

22. A composition for use according to claim 21, wherein the composition consists of: A molecule consisting of monomeric Annexin A5 protein and one or more pharmaceutically acceptable carriers.

23. A molecule consisting of monomeric Annexin A5 protein for use according to any one of claims 1 to 16, or a composition for use according to any one of claims 17 to 22, wherein the use is as a combination therapy with another agent.

24. The molecule consisting of monomeric Annexin A5 protein or a composition comprising the molecule for use as a combination therapy with another agent according to claim 23, wherein the molecule consisting of monomeric Annexin A5 protein is used as a first line of treatment with or without the one or more other agents.

25. A molecule consisting of monomeric Annexin A5 protein or a composition comprising the molecule for use as a combination therapy with another agent according to claim 23 or 24, wherein the molecule consisting of monomeric Annexin A5 protein is administered to the subject in the same composition as the one or more other agents, separately from the one or more other agents, simultaneously with a separate formulation containing the one or more other agents, or sequentially with the one or more other agents.

26. A molecule consisting of monomeric Annexin A5 protein or a composition comprising the molecule for use as a combination therapy with another agent according to any one of claims 23 to 25, wherein the other agent is an anti-vascular endothelial growth factor (anti-VEGF) agent.

27. A molecule consisting of monomeric Annexin A5 protein or a composition comprising the molecule for use as a combination therapy with an anti-VEGF agent according to claim 26, wherein the molecule consisting of monomeric Annexin A5 protein and the anti-VEGF agent are administered in combination, simultaneously in separate administrations or sequentially in separate administrations, and preferably wherein if the administrations are separate and sequential, the time period between the sequential administrations is 28 days or less, such as 3 weeks or less, 2 weeks or less, 1 week or less, 6 days, 5 days, 4 days, 3 days, 2 days or 1 day or less, such as 24 hours, 28 hours, 12 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour or less.

28. A molecule consisting of monomeric Annexin A5 protein or a composition comprising said molecule, or a use or method for use substantially as herein described in conjunction with the specification.

Images