Methods and compositions for treating ocular neovascular diseases

Abstract

The present disclosure provides compositions and methods for treating ocular neovascular diseases and conditions using HIF1-α pathway inhibitors and PFKFB3 inhibitors. Exemplary ocular neovascular diseases and conditions treated using the provided compositions and methods include diabetic retinopathy, diabetic macular edema, age-related macular degeneration, and choroidal neovascular membranes.

Description

[Background technology]

[0001] Retinal and choroidal vascular diseases are the most common causes of moderate and severe vision loss in developed countries. They can be divided into retinal vascular diseases, in which leakage and / or neovascularization occurs from retinal vessels, and subretinal neovascularization, in which neovascularization grows into the normally avascular extraretinal and subretinal spaces. Diseases in the first category include diabetic retinopathy, diabetic macular edema retinal vein occlusion, and retinopathy of prematurity, while the second category includes neovascular age-related macular degeneration (AMD), ocular histoplasmosis, pathological myopia, and other related diseases.

[0002] Diabetic retinopathy (DR) is the leading cause of blindness in adults in developed countries. DR is a serious complication of diabetes and the most common diabetic eye disease, often leading to reduced vision or blindness. Statistics show that 50% of diabetic patients develop DR after about 10 years of disease course, and up to 80% of diabetic patients develop DR after 15 years or more of disease course. The more severe the diabetic condition and the older the patient, the higher the incidence of DR.

[0003] Both microangiopathy and neurodegeneration are involved in the pathogenesis of DR, with neuropathy preceding microvascular abnormalities, which is underappreciated in clinical practice. Traditionally, vasculopathy has been considered the main pathophysiological mechanism of diabetic retinopathy (DR). However, in recent years, the role of diabetic retinal neurodegeneration (DRN) has become increasingly evident and may replace the role of vasculopathy as the primary pathogenetic event in this disease.

[0004] Diabetic macular edema (DME) is a complication of DR that affects up to 10% of diabetic patients and is the most common cause of vision loss in DR patients.

[0005] The mechanisms of diabetic retinopathy and diabetic macular edema and therapeutic strategies for treating them have been the subject of extensive efforts. In clinical settings, antiangiogenic therapy using, for example, laser photocoagulation, corticosteroids, and drugs that bind and confine vascular endothelial proliferation (e.g., bevacizumab and ranibizumab) is the most advanced therapeutic strategy for inducing neovascular regression and reduction of DR and macular edema.

[0006] Neurodegeneration is a common pathway for a variety of processes, including activation of inflammatory pathways, loss of neuroprotective factors, DNA damage and apoptosis. Oxidative stress and the formation of advanced glycation end products amplify these processes and are elevated in the setting of hyperglycemia, hyperlipidemia and glucose fluctuations.

[0007] Damage to the neurosensory retina in diabetes is governed by various mechanisms classified as inflammatory, metabolic, and genetic / epigenetic.

[0008] Age-related macular degeneration (AMD) is a degenerative eye disease that affects the macula of the retina and is a significant cause of vision loss in the US population aged 65 years and older. Neovascular or exudative or wet AMD (nAMD, wAMD or nwAMD) is an advanced form of AMD. wAMD is characterized by choroidal neovascularization (CNVM), which is the infiltration of abnormal blood vessels from the underlying choroidal layer into the retina, causing retinal cell damage and central blindness. CNVM is also commonly found in eye diseases such as histoplasmosis, ocular trauma, and myopic macular degeneration. These abnormal angiogenic processes are usually regulated by photodynamic therapy, thermal laser treatment, and treatment with growth factors, particularly molecules that bind and sequester VEGF, such as ranibizumab (e.g., LUCENTIS®) and aflibercept (e.g., EYLEA®).

[0009] Current treatment strategies for ocular neovascular disorders (e.g., diabetic retinopathy, diabetic macular edema, age-related wet macular degeneration, and choroidal neovascular membranes), such as anti-vascular endothelial growth factor (anti-VEGF) antibodies, are aimed at treating advanced stages (diabetic macular edema and proliferative diabetic retinopathy) and do not target neuronal deterioration. Thus, prevention and treatment of the neurodegenerative elements of DR are overlooked, even though the insidious loss of neurons is irreversible. These strategies do not address the prevention or treatment of the early stages of these disorders. Furthermore, many patients do not respond to current therapeutic methods for treating ocular neovascular disorders, and prior art antiangiogenic and photocoagulation therapies are associated with significant side effects. Resistance to anti-VEGF antibodies has also been reported in association with FGF substituting for VEGF's role in promoting angiogenesis.

[0010] Thus, there is a need for new modalities for treating both vascular and neurological disorders in ocular neovascular diseases such as diabetic retinopathy, age-related wet macular degeneration, and choroidal neovascular membranes. The compositions and methods provided herein address these needs and provide other related advantages. Summary of the Invention

[0011] The present disclosure provides compositions and methods for treating ocular neovascular diseases and conditions using HIF1-α pathway inhibitors and PFKFB3 inhibitors. Exemplary ocular neovascular diseases and conditions that can be treated using the provided compositions and methods include diabetic retinopathy, diabetic macular edema, age-related macular degeneration, and choroidal neovascular membranes.

[0012] The present inventors have determined that the hypoxia-inducible factor 1 alpha (HIF1-alpha)-6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase 3 (PFKFB3) pathway plays a central role in pathological angiogenesis and neurodegeneration, and have surprisingly found that the combination of HIF1-alpha pathway inhibitor and PFKFB3 inhibitor can reduce and possibly reverse the damage caused by these pathologies.Pathological angiogenesis and neurodegeneration are two important aspects in the complications of diabetic retinopathy, and the HIF1-alpha-PFKFB3 signaling pathway is distinctive in that it is a pathological element that extends to multiple cell types in the retina in the early and late stages of DR and other ocular angiogenic disorders.

[0013] In some embodiments, the present disclosure provides:

[0014] [1] A method for treating an ocular neovascular disease or condition in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The method, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0015] [2] The method according to [1], wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor.

[0016] [3] The method of [1], wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor, and the subject has previously been administered a PFKFB3 inhibitor.

[0017] [4] The method of [1], wherein the subject is administered an effective amount of a PFKFB3 inhibitor, and the subject has previously been administered a HIF1-α pathway inhibitor.

[0018] [5] The method according to any one of [1] to [4], wherein the subject is suffering from or at risk of suffering from an ocular neovascular disease or condition.

[0019] [6] The method of any one of [1] to [4], wherein the subject is suffering from or has been diagnosed as suffering from an ocular neovascular disease or condition.

[0020] [7] The method according to any one of [1] to [5], wherein the method according to any one of 1(a) to 1(c) is administered as a prophylactic treatment of an ocular neovascular disease or condition.

[0021] [8] The method according to any one of [1] to [7], wherein the ocular neovascular disease or condition is diabetic retinopathy (DR).

[0022] [9] The method according to any one of [1] to [8], wherein the ocular neovascular disease or condition is diabetic macular edema (DME).

[0023]

[10] The method according to any one of [1] to [7], wherein the ocular neovascular disease or condition is age-related macular degeneration (AMD), for example wet AMD (wAMD).

[0024]

[11] The method according to any one of [1] to [7], wherein the ocular neovascular disease or condition is a choroidal neovascular membrane.

[0025]

[12] The method of any one of [1] to

[11] , wherein the administered HIF1-α pathway inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a dicer substrate, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0026]

[13] The method according to any one of [1] to

[12] , wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1.3, or a salt thereof.

[0027]

[14] The method according to any one of [1] to

[13] , wherein the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothiocyanate or BAY-87-2243, or a salt thereof.

[0028]

[15] The method according to any one of [1] to

[14] , wherein the administered HIF1-α pathway inhibitor is a HIF1-α inhibitor.

[0029]

[16] The method of

[15] , wherein the HIF1-α inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0030]

[17] The method according to

[15] or

[16] , wherein the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, or the nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[0031]

[18] The method of any one of [1] to

[17] , wherein the administered PFKFB3 inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3-binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0032]

[19] The method according to any one of [1] to

[18] , wherein the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0033]

[20] The method according to any one of [1] to

[19] , wherein the administered PFKFB3 inhibitor is (a) KAN0436151 or KAN0436067, or a salt thereof; (b) Formula 1 to Formula 53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A to Figure 1C or Figure 1D; (c) Formula AZ44 to Formula AZ70 or Formula AZ71, or a salt thereof, as shown in Figure 1E; or (d) AZ67 or a salt thereof.

[0034]

[21] The method according to any one of [1] to

[20] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject.

[0035]

[22] The method according to any one of [1] to

[21] , wherein the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is ocular administration.

[0036]

[23] The method according to any one of [1] to

[22] , wherein the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is ocular administration.

[0037]

[24] The method according to

[22] or

[23] , wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0038]

[25] The method according to any one of

[22] to

[24] , wherein the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intravitreal administration.

[0039]

[26] The method according to any one of

[22] to

[25] , wherein the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intravitreal administration.

[0040]

[27] The method according to any one of [1] to

[26] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered prior to the onset of one or more symptoms of an ocular neovascular disease or condition.

[0041]

[28] The method according to any one of [1] to

[26] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of an ocular neovascular disease or condition.

[0042]

[29] The method according to any one of [1] to

[26] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the non-proliferative phase of the ocular neovascular disease or condition.

[0043]

[30] The method according to any one of [1] to

[26] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the proliferation phase of an ocular neovascular disease or condition.

[0044]

[31] The method according to any one of [1] to

[27] , wherein treating an ocular neovascular disease or condition includes delaying the onset of an ocular neovascular disease or condition.

[0045]

[32] The method of any one of [1] to

[31] , wherein one or more symptoms of an ocular neovascular disease or condition are alleviated in the subject compared to a control subject or compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0046]

[33] The method of

[32] , wherein the one or more symptoms of the ocular neovascular disease or condition are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, retinal leakage area, choroidal inflammation, choroidal neovascularization, choroidal endothelial cell death, choroidal vascular permeability, choroidal ischemia-reperfusion injury, choroidal leakage area, and occludin disruption.

[0047]

[34] The method of

[32] or

[33] , wherein one or more symptoms of an ocular neovascular disease or condition are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0048]

[35] The method of any one of [1] to

[34] , wherein one or more visual acuity parameters are increased in the subject compared to the visual acuity parameters of a control subject or compared to the visual acuity parameters of the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0049]

[36] The method of

[35] , wherein the one or more vision parameters are selected from peripheral vision, night vision, low light vision, color vision, distance vision, near vision, clarity of vision, reading ability, absence of flashing lights or spots in the visual field, non-fluctuating visual acuity, pain, and eye appearance.

[0050]

[37] The method according to any one of [1] to

[36] , further comprising administering an anti-VEGF therapeutic agent to the subject.

[0051]

[38] The method according to

[37] , wherein the anti-VEGF therapeutic agent is bevacizumab, ranibizumab, or aflibercept.

[0052]

[39] The method according to any one of [1] to

[38] , wherein the subject has previously undergone treatment for an ocular neovascular disease or condition.

[0053]

[40] The method of

[39] , wherein the subject has not responded to prior treatment for the ocular neovascular disease or condition.

[0054]

[41] The method according to

[39] or

[40] , wherein the pretreatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroid and anti-vascular endothelial growth factor (VEGF) therapy.

[0055]

[42] The method according to

[41] , wherein the pretreatment is anti-vascular endothelial growth factor (VEGF) therapy.

[0056]

[43] The method according to

[42] , wherein the anti-VEGF therapy is administration of bevacizumab, ranibizumab, or aflibercept.

[0057]

[44] A method for treating diabetic retinopathy (DR) in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The method, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0058]

[45] The method of

[44] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor.

[0059]

[46] The method of

[44] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor, and the subject has previously been administered a PFKFB3 inhibitor.

[0060]

[47] The method of

[44] , wherein the subject is administered an effective amount of a PFKFB3 inhibitor, and the subject has previously been administered a HIF1-α pathway inhibitor.

[0061]

[48] ​​The method according to any one of

[44] to

[47] , wherein the subject is suffering from or at risk of suffering from DR.

[0062]

[49] The method according to any one of

[44] to

[48] , wherein the subject is suffering from or diagnosed with DR.

[0063]

[50] The method according to any one of

[44] to

[48] , wherein the method according to any one of 44(a) to 44(c) is administered as a prophylactic treatment for DR.

[0064]

[51] The method of any one of

[44] -

[50] , wherein the administered HIF1-α pathway inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0065]

[52] The method according to any one of

[44] to

[51] , wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1.3, or a salt thereof.

[0066]

[53] The method according to any one of

[44] to

[52] , wherein the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothiocyanate or BAY-87-2243, or a salt thereof.

[0067]

[54] The method according to any one of

[44] to

[53] , wherein the administered HIF1-α pathway inhibitor is a HIF1-α inhibitor.

[0068]

[55] The method of

[54] , wherein the HIF1-α inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0069]

[56] The method according to

[54] or

[55] , wherein the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, or the nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[0070]

[57] The method of any one of

[44] -

[56] , wherein the administered PFKFB3 inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3-binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0071]

[58] The method according to any one of

[44] to

[57] , wherein the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0072]

[59] The method of any one of

[44] to

[57] , wherein the administered PFKFB3 inhibitor is (a) KAN0436151 or KAN0436067, or a salt thereof; (b) has the structure of formula 1 to formula 53 or formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A to Figure 1C or Figure 1D; (c) has the structure of formula AZ44 to formula AZ70 or formula AZ71, or a salt thereof, as shown in Figure 1E; or (d) AZ67 or a salt thereof.

[0073]

[60] The method according to any one of

[44] to

[59] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject.

[0074]

[61] The method according to any one of

[44] to

[60] , wherein the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is ocular administration.

[0075]

[62] The method according to any one of

[44] to

[61] , wherein the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is ocular administration.

[0076]

[63] The method of

[61] or

[62] , wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0077]

[64] The method according to any one of

[61] to

[63] , wherein the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intravitreal administration.

[0078]

[65] The method according to any one of

[61] to

[64] , wherein the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intravitreal administration.

[0079]

[66] The method according to any one of

[44] to

[65] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered before the onset of one or more symptoms of DR.

[0080]

[67] The method according to any one of

[44] to

[65] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of DR.

[0081]

[68] The method according to any one of

[44] to

[65] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the non-proliferative phase of DR.

[0082]

[69] The method according to any one of

[44] to

[65] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the proliferation phase of DR.

[0083]

[70] The method according to any one of

[44] to

[69] , wherein treating DR includes delaying the onset of DR.

[0084]

[71] The method of any one of

[44] to

[70] , wherein one or more symptoms of DR are alleviated in the subject compared to a control subject or compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0085]

[72] The method of

[71] , wherein the one or more symptoms of DR are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, areas of retinal leakage, and occludin disruption.

[0086]

[73] The method of

[71] or

[72] , wherein one or more symptoms of DR are alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0087]

[74] The method of any one of

[44] to

[73] , wherein one or more visual acuity parameters are increased in the subject compared to the visual acuity parameters of a control subject or compared to the visual acuity parameters of the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0088]

[75] The method of

[74] , wherein the one or more vision parameters are selected from peripheral vision, night vision, color vision, distance vision, near vision, and clarity of vision.

[0089]

[76] The method according to any one of

[44] to

[75] , further comprising administering an anti-VEGF therapeutic agent to the subject.

[0090]

[77] The method of

[76] , wherein the anti-VEGF therapeutic agent is bevacizumab, ranibizumab, or aflibercept.

[0091]

[78] The method according to any one of

[44] to

[77] , wherein the subject has previously undergone treatment for DR.

[0092]

[79] The method of

[78] , wherein the subject has not responded to prior treatment for DR.

[0093]

[80] The method according to

[78] or

[79] , wherein the pretreatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy.

[0094]

[81] The method according to

[79] or

[80] , wherein the pretreatment is anti-vascular endothelial growth factor (VEGF) therapy.

[0095]

[82] The method according to

[81] , wherein the anti-VEGF therapy is administration of bevacizumab, ranibizumab, or aflibercept.

[0096]

[83] A method of treating diabetic macular edema (DME) in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The method, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0097]

[84] The method of

[83] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor.

[0098]

[85] The method of

[83] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor, and the subject has previously been administered a PFKFB3 inhibitor.

[0099]

[86] The method of

[83] , wherein the subject is administered an effective amount of a PFKFB3 inhibitor, and the subject has previously been administered a HIF1-α pathway inhibitor.

[0100]

[87] The method according to any one of

[83] to

[86] , wherein the subject is suffering from or at risk of suffering from DME.

[0101]

[88] The method according to any one of

[83] to

[87] , wherein the subject is suffering from or diagnosed with DME.

[0102]

[89] The method according to any one of

[83] to

[87] , wherein the method according to any one of 83(a) to 83(c) is administered as a prophylactic treatment for DME.

[0103]

[90] The method of any one of

[83] -

[89] , wherein the administered HIF1-α pathway inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0104]

[91] The method according to any one of

[83] to

[90] , wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1.3, or a salt thereof.

[0105]

[92] The method according to any one of

[83] to

[91] , wherein the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothiocyanate or BAY-87-2243, or a salt thereof.

[0106]

[93] The method according to any one of

[83] to

[92] , wherein the administered HIF1-α pathway inhibitor is a HIF1-α inhibitor.

[0107]

[94] The method of

[93] , wherein the HIF1-α inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0108]

[95] The method of

[93] or

[94] , wherein the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, or the nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[0109]

[96] The method of any one of

[83] -

[95] , wherein the administered PFKFB3 inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3-binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0110]

[97] The method according to any one of

[83] to

[96] , wherein the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0111]

[98] The method of any one of

[83] to

[96] , wherein the administered PFKFB3 inhibitor is (a) KAN0436151 or KAN0436067, or a salt thereof; (b) has the structure of formula 1 to formula 53 or formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A to Figure 1C or Figure 1D; (c) has the structure of formula AZ44 to formula AZ70 or formula AZ71, or a salt thereof, as shown in Figure 1E; or (d) AZ67 or a salt thereof.

[0112]

[99] The method according to any one of

[83] to

[98] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject.

[0113]

[0100] The method according to any one of

[83] to

[99] , wherein the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is ocular administration.

[0114]

[0101] A method according to any one of

[83] to

[0100] , wherein the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is ocular administration.

[0115]

[0102] The method according to

[0100] or

[0101] , wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0116]

[0103] A method according to any one of

[0100] to

[0102] , wherein the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intravitreal administration.

[0117]

[0104] A method according to any one of

[0100] to

[0103] , wherein the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intravitreal administration.

[0118]

[0105] A method according to any one of

[83] to

[0104] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered before the onset of one or more symptoms of DME.

[0119]

[0106] A method according to any one of

[83] to

[0104] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of DME.

[0120]

[0107] A method according to any one of

[83] to

[0104] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the non-proliferative phase of DME.

[0121]

[0108] A method according to any one of

[83] to

[0104] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the proliferation phase of DR.

[0122]

[0109] A method according to any one of

[83] to

[0105] , wherein treating DME includes delaying the onset of DME.

[0123]

[0110] A method according to any one of

[83] to

[0109] , wherein one or more symptoms of DME are alleviated in the subject compared to a control subject or compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0124]

[0111] The method described in

[0110] , wherein one or more symptoms of DME are selected from astigmatism, retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, areas of retinal leakage, rupture of the retinal blood barrier in retinal vascular endothelial cells or retinal pigment epithelial cells, retinal scarring, and occludin disruption.

[0125]

[0112] The method described in

[0110] or

[0111] , wherein one or more symptoms of DME are alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0126]

[0113] A method according to any one of

[83] to

[0112] , wherein one or more visual acuity parameters are increased in the subject compared to the visual acuity parameters of a control subject or compared to the visual acuity parameters of the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0127]

[0114] The method according to

[0113] , wherein the one or more vision parameters are selected from visual acuity, peripheral vision, night vision, color vision, distance vision, near vision, and clarity of vision.

[0128]

[0115] A method according to any one of

[83] to

[0114] , wherein the subject suffers from diabetes or diabetic retinopathy.

[0129]

[0116] A method according to any one of

[83] to

[0115] , further comprising administering an anti-VEGF therapeutic agent to the subject.

[0130]

[0117] The method according to

[0116] , wherein the anti-VEGF therapeutic agent is bevacizumab, ranibizumab, or aflibercept.

[0131]

[0118] A method according to any one of

[83] to

[0117] , wherein the subject has previously undergone treatment for DME.

[0132]

[0119] The method according to

[0118] , wherein the subject has not responded to prior treatment for DME.

[0133]

[0120] The method described in

[0118] or

[0119] , wherein the pretreatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy.

[0134]

[0121] The method described in

[0118] or

[0119] , wherein the pretreatment is anti-vascular endothelial growth factor (VEGF) therapy.

[0135]

[0122] The method according to

[0121] , wherein the anti-VEGF therapy is administration of bevacizumab, ranibizumab, or aflibercept.

[0136]

[0123] A method of treating age-related macular degeneration (AMD) in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The method, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0137]

[0124] The method according to

[0123] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor.

[0138]

[0125] The method of

[0123] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor, and the subject has previously been administered a PFKFB3 inhibitor.

[0139]

[0126] The method of

[0123] , wherein the subject is administered an effective amount of a PFKFB3 inhibitor, and the subject has previously been administered a HIF1-α pathway inhibitor.

[0140]

[0127] A method according to any one of

[0123] to

[0126] , wherein the subject has AMD, such as wet AMD (wAMD), or is at risk of having AMD.

[0141]

[0128] A method according to any one of

[0123] to

[0126] , wherein the subject is suffering from or has been diagnosed as suffering from AMD, e.g. wAMD.

[0142]

[0129] The method according to any one of

[0123] to

[0127] , wherein the method according to any one of 123(a) to 123(c) is administered as a preventative treatment for AMD, such as wAMD.

[0143]

[0130] The method of any one of

[0123] to

[0129] , wherein the administered HIF1-α pathway inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0144]

[0131] A method according to any one of

[0123] to

[0130] , wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1.3, or a salt thereof.

[0145]

[0132] A method according to any one of

[0123] to

[0131] , wherein the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothiocyanate or BAY-87-2243, or a salt thereof.

[0146]

[0133] A method according to any one of

[0123] to

[0132] , wherein the administered HIF1-α pathway inhibitor is a HIF1-α inhibitor.

[0147]

[0134] The method of

[0133] , wherein the HIF1-α inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or another engineered molecule, e.g., diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0148]

[0135] The method described in

[0133] or

[0134] , wherein the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, or the nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[0149]

[0136] The method according to any one of

[0123] to

[0135] , wherein the administered PFKFB3 inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3-binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0150]

[0137] A method according to any one of

[0123] to

[0136] , wherein the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0151]

[0138] The method according to any one of

[0123] to

[0136] , wherein the administered PFKFB3 inhibitor is (a) KAN0436151 or KAN0436067, or a salt thereof; (b) has the structure of formula 1 to formula 53 or formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A to Figure 1C or Figure 1D; (c) has the structure of formula AZ44 to formula AZ70 or formula AZ71, or a salt thereof, as shown in Figure 1E; or (d) AZ67 or a salt thereof.

[0152]

[0139] A method according to any one of

[0123] to

[0138] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject.

[0153]

[0140] A method according to any one of

[0123] to

[0139] , wherein administration of the HIF1-α pathway inhibitor or PFKFB3 inhibitor is ocular administration.

[0154]

[0141] A method according to any one of

[0123] to

[0140] , wherein administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is ocular administration.

[0155]

[0142] The method according to

[0140] or

[0141] , wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0156]

[0143] A method according to any one of

[0140] to

[0142] , wherein the administration of the HIF1-α pathway inhibitor or PFKFB3 inhibitor is intravitreal administration.

[0157]

[0144] A method according to any one of

[0140] to

[0143] , wherein the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intravitreal administration.

[0158]

[0145] A method according to any one of

[0123] to

[0144] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered before the onset of one or more symptoms of AMD.

[0159]

[0146] A method according to any one of

[0123] to

[0144] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of AMD.

[0160]

[0147] A method according to any one of

[0123] to

[0144] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the non-proliferative phase of AMD.

[0161]

[0148] A method according to any one of

[0123] to

[0144] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the proliferative phase of AMD.

[0162]

[0149] A method according to any one of

[0123] to

[0145] , wherein treating AMD includes delaying the onset of AMD.

[0163]

[0150] A method according to any one of

[0123] to

[0149] , wherein one or more symptoms of AMD are alleviated in the subject compared to a control subject or compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0164]

[0151] The method described in

[0150] , wherein one or more symptoms of AMD are selected from choroidal inflammation, choroidal neovascularization, choroidal endothelial cell death, choroidal vascular permeability, choroidal ischemia-reperfusion injury, choroidal leakage areas, and occludin destruction, acellular capillary formation, vascular permeability, and occludin destruction.

[0165]

[0152] The method described in

[0150] or

[0151] , wherein one or more symptoms of AMD are alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0166]

[0153] A method according to any one of

[0123] to

[0151] , wherein one or more visual acuity parameters are increased in the subject compared to the visual acuity parameters of a control subject or compared to the visual acuity parameters of the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0167]

[0154] The method according to

[0153] , wherein the one or more vision parameters are selected from peripheral vision, night vision, color vision, distance vision, near vision, and clarity of vision.

[0168]

[0155] A method according to any one of

[0123] to

[0154] , wherein the subject is suffering from or at risk of suffering from wAMD.

[0169]

[0156] A method according to any one of

[0123] to

[0155] , further comprising administering an anti-VEGF therapeutic agent to the subject.

[0170]

[0157] The method according to

[0156] , wherein the anti-VEGF therapeutic agent is bevacizumab, ranibizumab, or aflibercept.

[0171]

[0158] A method according to any one of

[0123] to

[0157] , wherein the subject has undergone prior treatment for AMD.

[0172]

[0159] The method according to

[0158] , wherein the subject has not responded to prior treatment for AMD.

[0173]

[0160] The method described in

[0158] or

[0159] , wherein the pretreatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy.

[0174]

[0161] The method described in

[0158] or

[0159] , wherein the pretreatment is anti-vascular endothelial growth factor (VEGF) therapy.

[0175]

[0162] The method described in

[0161] , wherein the anti-VEGF therapy is administration of bevacizumab, ranibizumab, or aflibercept.

[0176]

[0163] A method of treating choroidal neovascularization (CNVM) in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The method, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0177]

[0164] The method described in

[0163] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor.

[0178]

[0165] The method of

[0163] , wherein the subject is administered an effective amount of a HIF1-α pathway inhibitor, and the subject has previously been administered a PFKFB3 inhibitor.

[0179]

[0166] The method of

[0163] , wherein the subject is administered an effective amount of a PFKFB3 inhibitor, and the subject has previously been administered a HIF1-α pathway inhibitor.

[0180]

[0167] A method according to any one of

[0163] to

[0166] , wherein the subject is suffering from or at risk of suffering from CNVM.

[0181]

[0168] A method according to any one of

[0163] to

[0167] , wherein the subject is suffering from or has been diagnosed as suffering from CNVM.

[0182]

[0169] The method according to any one of

[0163] to

[0168] , wherein the method according to any one of 163(a) to 163(c) is administered as a prophylactic treatment for CNVM.

[0183]

[0170] The method of any one of

[0163] to

[0169] , wherein the administered HIF1-α pathway inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0184]

[0171] A method according to any one of

[0163] to

[0170] , wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1.3, or a salt thereof.

[0185]

[0172] A method according to any one of

[0163] to

[0171] , wherein the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothiocyanate or BAY-87-2243, or a salt thereof.

[0186]

[0173] A method according to any one of

[0163] to

[0172] , wherein the administered HIF1-α pathway inhibitor is a HIF1-α inhibitor.

[0187]

[0174] The method of

[0173] , wherein the HIF1-α inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or another engineered molecule such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0188]

[0175] The method described in

[0173] or

[0174] , wherein the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, or the nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

[0189]

[0176] The method according to any one of

[0163] to

[0175] , wherein the administered PFKFB3 inhibitor is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3-binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0190]

[0177] A method according to any one of

[0163] to

[0176] , wherein the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0191]

[0178] The method according to any one of

[0163] to

[0176] , wherein the administered PFKFB3 inhibitor is (a) KAN0436151 or KAN0436067, or a salt thereof, (b) has the structure of formula 1 to formula 53 or formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, shown in Figure 1A to Figure 1C or Figure 1D, (c) has the structure of formula AZ44 to formula AZ70 or formula AZ71, or a salt thereof, shown in Figure 1E, or (d) AZ67 or a salt thereof.

[0192]

[0179] A method according to any one of

[0163] to

[0178] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject.

[0193]

[0180] A method according to any one of

[0163] to

[0179] , wherein administration of the HIF1-α pathway inhibitor or PFKFB3 inhibitor is ocular administration.

[0194]

[0181] A method according to any one of

[0163] to

[0180] , wherein administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is ocular administration.

[0195]

[0182] The method according to

[0180] or

[0181] , wherein the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0196]

[0183] A method according to any one of

[0180] to

[0182] , wherein the administration of the HIF1-α pathway inhibitor or PFKFB3 inhibitor is intravitreal administration.

[0197]

[0184] A method according to any one of

[0163] to

[0183] , wherein the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intravitreal administration.

[0198]

[0185] A method according to any one of

[0163] to

[0184] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered before the onset of one or more symptoms of CNVM.

[0199]

[0186] A method according to any one of

[0163] to

[0184] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of CNVM.

[0200]

[0187] A method according to any one of

[0163] to

[0184] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the non-proliferative phase of CNVM.

[0201]

[0188] A method according to any one of

[0163] to

[0184] , wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the proliferation phase of the CNVM.

[0202]

[0189] A method according to any one of

[0163] to

[0185] , wherein treating CNVM includes delaying the onset of CNVM.

[0203]

[0190] A method according to any one of

[0163] to

[0189] , wherein one or more symptoms of CNVM are alleviated in the subject compared to a control subject or compared to the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0204]

[0191] The method described in

[0190] , wherein one or more symptoms of CNVM are selected from choroidal inflammation, choroidal neovascularization, choroidal endothelial cell death, choroidal vascular permeability, choroidal ischemia-reperfusion injury, choroidal leakage areas, and occludin destruction, acellular capillary formation, vascular permeability, and occludin destruction.

[0205]

[0192] The method described in

[0190] or

[0191] , wherein one or more symptoms of CNVM are alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0206]

[0193] A method according to any one of

[0163] to

[0192] , wherein one or more visual acuity parameters are increased in the subject compared to the visual acuity parameters of a control subject or compared to the visual acuity parameters of the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0207]

[0194] The method according to

[0193] , wherein the one or more vision parameters are selected from peripheral vision, night vision, color vision, distance vision, near vision, and clarity of vision.

[0208]

[0195] The method of

[0193] , wherein the subject suffers from wet wAMD, histoplasmosis, eye injury, or myopic macular degeneration.

[0209]

[0196] A method according to any one of

[0163] to

[0195] , further comprising administering an anti-VEGF therapeutic agent to the subject.

[0210]

[0197] The method according to

[0196] , wherein the anti-VEGF therapeutic agent is bevacizumab, ranibizumab, or aflibercept.

[0211]

[0198] A method according to any one of

[0163] to

[0197] , wherein the subject has undergone prior treatment for CNVM.

[0212]

[0199] The method described in

[0198] , wherein the subject has not responded to prior treatment for CNVM.

[0213]

[0200] The method described in

[0198] or

[0199] , wherein the pretreatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy.

[0214]

[0201] The method described in

[0198] or

[0199] , wherein the pretreatment is anti-vascular endothelial growth factor (VEGF) therapy.

[0215]

[0202] The method according to

[0201] , wherein the anti-VEGF therapy is administration of bevacizumab, ranibizumab, or aflibercept. [Brief description of the drawings]

[0216] [Figure 1] A-E show exemplary PFKFB3 small molecule inhibitors. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0217] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the compositions provided, but suitable methods and materials are described below.Each publication, patent application, patent, and other references mentioned herein are incorporated herein by reference in their entirety.In case of conflict, the present specification, including definitions, will take precedence.In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0218] Other features and advantages of the disclosed compositions and methods will become apparent from the following disclosure, the drawings, and the claims.

[0219] Whenever embodiments are described herein using the word "comprising," it should be understood that other similar embodiments described in terms of "containing," "consisting of," and / or "consisting essentially of" are also provided. However, when used as transitional phrases within the claims, each must be interpreted separately and within the appropriate legal and factual context (e.g., within the claims, the transitional phrase "comprising" is considered more open-ended, "consisting of" is more exclusive, and "consisting essentially of" is intermediate).

[0220] As used herein, the singular forms "a", "an" and "the" include the plural unless expressly stated or clearly evident from the context that such is not the intention. The singular forms "a", "an" and "the" also include the statistical average composition, characteristics or size of particles within a particle population (e.g., average polyethylene glycol molecular weight, average liposome diameter, average liposome zeta potential). The average particle size and zeta potential of liposomes in a pharmaceutical composition can be routinely measured using methods known in the art, such as dynamic light scattering. The average amount of therapeutic agent in a nanoparticle composition can be routinely measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

[0221] As used herein, the term "approximately" or "about" as applied to one or more values ​​of interest refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that are included within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% in either direction (above or below) of the stated reference value, unless otherwise stated or otherwise clear from the context (except where such number may exceed 100% of the possible values). For example, when used in the context of the amount of a given compound in the lipid component of a nanoparticle composition, "about" can mean + / - 10% of the stated value. For example, a nanoparticle composition that includes a lipid component having about 40% of a given compound can contain 30-50% of that compound.

[0222] The term "and / or," when used herein in phrases such as "A and / or B," is intended to include both A and B, A or B, A (alone), and B (alone). Similarly, the term "and / or," when used in phrases such as "A, B, and / or C," is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0223] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is intended to serve merely as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein.

[0224] When embodiments of the present disclosure are described in terms of a Markush group or other alternative grouping, the compositions or methods of the present disclosure not only include the entire group recited as a whole, but also each member of the group individually, all possible subgroups of the main group, and also the main group in the absence of one or more of the group members. The compositions and methods of the present disclosure also contemplate the explicit exclusion of any one or more of the group members in the compositions or methods of the present disclosure.

[0225] As used herein, terms such as "antibody" and "antigen-binding antibody fragment" include any protein- or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain, or an antigen-binding portion thereof.

[0226] The term "antibody" also includes fragments, specified portions and variants thereof, including antibody mimetics or portions of antibodies that mimic the structure and / or function of antibodies or specified fragments or portions thereof, including single chain antibodies, single binding domain antibodies and antigen-binding antibody fragments.

[0227] The term "antibody fragment" refers to a portion of an intact antibody, typically the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain (scFv) and Fv fragments, diabodies; linear antibodies; single chain antibody molecules; single Fab arm "one-arm" antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments include any protein or peptide-containing molecule that includes at least a portion of an immunoglobulin molecule, such as, but not limited to, at least one complementarity determining region (CDR) or ligand-binding portion thereof of a heavy or light chain, a heavy or light chain variable region, a heavy or light chain constant region, a framework region or any portion thereof, or at least a portion of an antigen or antigen receptor or binding protein, which can be incorporated into the antibodies provided herein.

[0228] Antibody fragments can be produced by enzymatic cleavage, synthetically, or recombinantly, as known in the art. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a combined gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CH1 domain and / or hinge region of the heavy chain. The various portions of the antibody can be joined together chemically by conventional techniques, or prepared as a contiguous protein using genetic engineering techniques.

[0229] The terms "nucleic acid" or "oligonucleotide" are used interchangeably herein and refer to at least two nucleotides covalently linked together. In some embodiments, the HIF1-α pathway inhibitor and / or PFKFB3 inhibitor administered according to the provided methods is a therapeutic nucleic acid. In some embodiments, the nucleic acid administered is ENMD-1198, shRNA, dicer substrate (e.g., dsRNA), miRNA, anti-miRNA, antisense molecule, decoy, or aptamer, or a plasmid expressing ENMD-1198, shRNA, dicer substrate, miRNA, anti-miRNA, antisense molecule, decoy, or aptamer.

[0230] Nucleic acids administered according to the provided methods are preferably single-stranded or double-stranded and generally contain phosphodiester bonds, although in some cases, nucleic acid / oligonucleotide analogs include those with alternative backbones, including, for example, phosphoramide, phosphorothioate, phosphorodithioate, O-methyl phosphoramidite bonds, as well as peptide nucleic acid backbones and bonds. Other analog nucleic acids / oligonucleotides include those with cationic backbones, non-ionic backbones, and non-ribose backbones. Nucleic acids / oligonucleotides containing one or more carbocyclic sugars are also included in the definition of nucleic acids and oligonucleotides. These modifications of the ribose-phosphate backbone can be made, for example, to facilitate the addition of additional moieties, such as labels, or to increase the stability and half-life of such molecules in physiological environments. The nucleic acid / oligonucleotide backbones of the oligonucleotides used according to the provided methods can range from about 5 nucleotides to about 750 nucleotides. Preferred nucleic acid / oligonucleotide backbones range in length from about 5 nucleotides to about 500 nucleotides, preferably from about 10 nucleotides to about 100 nucleotides.

[0231] The oligonucleotides administered according to the provided methods are polymeric structures of nucleoside and / or nucleotide monomers that can specifically hybridize to at least some region of a nucleic acid target. As described above, the "nucleic acids" and "oligonucleotides" used according to the provided methods include, but are not limited to, compounds that contain naturally occurring bases, sugars and intersugar (backbone) linkages, non-naturally occurring modified monomers that function similarly to their naturally occurring counterparts, or portions thereof (e.g., oligonucleotide analogs or mimetics), and combinations of these naturally occurring and non-naturally occurring monomers. As used herein, the term "modified" or "modification" includes any substitution and / or any change from a starting or natural oligomeric compound such as a nucleic acid. Modifications to nucleic acids include substitutions or changes to internucleoside linkages, sugar moieties, or base moieties, such as those described herein and otherwise known in the art.

[0232] As used herein, "small molecule" refers to an organic compound that is synthesized by conventional organic chemistry methods (e.g., in a laboratory) or found in nature. Typically, small molecules are characterized by containing several carbon-carbon bonds and having a molecular weight of less than about 1500 grams / mole. In certain embodiments, small molecules are less than about 1000 grams / mole. In certain embodiments, small molecules are less than about 550 grams / mole. In certain embodiments, small molecules are between about 200 and about 550 grams / mole. In certain embodiments, small molecules do not include peptides (e.g., compounds that include two or more amino acids joined by peptidyl bonds). In certain embodiments, small molecules do not include nucleic acids.

[0233] "Diabetes mellitus" is a syndrome of a series of metabolic disorders involving carbohydrates, proteins, fats, water, electrolytes, etc., caused by pancreatic islet dysfunction and insulin resistance resulting from the effects of genetic factors, immune dysfunction, microbial infections and their toxins, free radical toxins, psychological factors, and various other pathogenic factors on the body, and is clinically characterized mainly by hyperglycemia.

[0234] "Diabetic microangiopathy" refers to microangiopathy caused by various degrees of abnormality in the microcirculation of various body organs or tissues in diabetes. The process of microangiopathy formation broadly includes functional changes in microcirculation, endothelial damage, thickening of basement membrane, increase in blood viscosity, aggregation of red blood cells, and adhesion and aggregation of platelets, which ultimately lead to microthrombosis and / or microvascular obstruction. "Diabetic ocular microangiopathy" refers to ocular microangiopathy caused by diabetes. "Diabetic retinopathy" includes histological and functional changes of the retina caused by diabetes-induced diabetic microangiopathy.

[0235] As used herein, an "effective amount" refers to a dose of an agent sufficient to provide a medically desirable result. The effective amount will vary depending on the desired result, the particular disease or condition being treated (or prevented), the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent or concomitant treatment (if any), the particular route of administration, and similar factors within the knowledge and skill of the medical practitioner. An "effective amount" can be determined empirically and in a routine manner in relation to the stated purpose.

[0236] The terms "subject," "patient," "individual," and "animal" are used interchangeably and refer to mammals, such as human patients and non-human primates, as well as laboratory animals, such as rabbits, rats, and mice, and other laboratory animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as chickens, amphibians, and reptiles. As used herein, "mammal" refers to any member of the class Mammalia, including, but not limited to, humans and non-human primates, such as chimpanzees and other ape and monkey species; farm animals, such as cows, sheep, pigs, goats, and horses; domesticated mammals, such as dogs and cats; laboratory animals, including rodents, such as mice, rats, and guinea pigs; and other members of the class Mammalia known in the art. In certain embodiments, the patient is a human.

[0237] Terms such as "treating" or "treatment", "treat", or "therapy" refer to both (a) therapeutic measures that cure, slow, attenuate, alleviate, and / or halt the progression of a pathological condition or disorder, and (b) prophylactic or preventative measures that prevent and / or delay the onset of the targeted disease or condition. Thus, subjects in need of treatment include those suffering from an ocular neovascular disorder, those at risk of suffering from an ocular neovascular disorder, and those in need of prevention of an ocular neovascular disorder. A subject is identified as "suffering from or at risk of suffering from" an ocular neovascular disorder or another disorder referred to herein using well-known medical and diagnostic techniques. In certain embodiments, a subject is successfully "treated" according to the methods provided herein, for example, when the subject exhibits complete, partial, or transient remission or disappearance of symptoms associated with the disorder (e.g., diabetic retinopathy, diabetic macular edema, age-related macular degeneration (AMD), and choroidal neovascular membrane). In certain embodiments, the term "treat" or "treatment", "treating" or "therapy" refers to an improvement in at least one measurable physical parameter of an ocular proliferative disease, such as ocular neovascularization, that is not necessarily discernible by the patient. In other embodiments, the term "treat" or "treatment", "treating" or "therapy" refers to inhibiting the progression of an ocular proliferative disease, either physically, e.g., by stabilization of a discernible symptom, physiologically, e.g., by stabilization of a physiological parameter, or both. In other embodiments, the term "treat" or "treatment", "treating" or "therapy" refers to the reduction or alleviation of symptoms, reduction of inflammation, inhibition of cell death, and / or restoration of cell function, which may be achieved using the HIF1-α pathway inhibitor and PFKFB3 inhibitor compositions disclosed herein, and may be combined with additional therapeutic agents.

[0238] The term "pharmaceutical acceptable carrier" refers to an ingredient in a pharmaceutical preparation other than active ingredient that is non-toxic to subjects. Pharmaceutically acceptable carriers include, but are not limited to, buffers, carriers, excipients, stabilizers, diluents or preservatives. Pharmaceutically acceptable carriers can include, for example, one or more compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to humans or other subjects.

[0239] The "therapeutic agent(s)" used in accordance with the disclosed compositions and methods may include any agent for treating a condition of a subject. Examples of therapeutic agents that may be suitable for use in accordance with the provided methods include HIF1-α pathway inhibitors, PFKFB3 inhibitors, anti-VEGF therapeutic agents (e.g., anti-VEGF antibodies (e.g., bevacizumab and ranibizumab) and small molecule VEGF receptor inhibitors (e.g., sunitinib, sorafenib and pazopazopanib), corticosteroids (e.g., hydrocortisone, or glucocorticoids such as cortisone, etamethazoneb, prednisone, prednisolone, triamcinolone, dexamethasone and methylprednisolone). "Therapeutic agent" also refers to salt, acid, and free base forms of the above-mentioned agents. PFKFB3 inhibitors

[0240] PFKFB3 (6-phosphofructo-2-kinase-fructose-2,6-bisphosphatase 3) is a bifunctional protein involved in both the synthesis and degradation of fructose-2,6-bisphosphate, a regulatory molecule that controls eukaryotic glycolysis and is required for cell cycle progression and prevention of apoptosis.

[0241] In some embodiments, the present disclosure provides a method of treating an ocular neovascular disease or condition in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor or a HIF1-α inhibitor; The method provides said wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0242] In some embodiments, the disclosure provides a method according to any one of (a)-(c), wherein PFKFB3 is upregulated in the subject.

[0243] In some embodiments, the disclosure provides a method according to any one of (a)-(c), wherein HIF1-α is upregulated in the subject.

[0244] In some embodiments, the disclosure provides a method according to any one of (a)-(c), wherein PFKFB3 and HIF1-α are upregulated in the subject.

[0245] In some embodiments, the disclosure provides a method according to any one of (a)-(c), wherein PFKFB3 is upregulated in the subject independently of HIF1-α.

[0246] In some embodiments, the disclosure provides a method according to any one of (a)-(c), wherein HIF1-α is upregulated in the subject independently of PFKFB3.

[0247] In some embodiments, the present disclosure is a method for treating an ocular neovascular disease or condition in a subject in need of such treatment, comprising administering to the subject an effective amount of a PFKFB3 inhibitor. In certain embodiments, the PFKFB3 inhibitor is administered in combination with a VEGF antagonist.

[0248] In further embodiments, the ocular neovascular disease or condition being treated is diabetic retinopathy (DR), diabetic macular edema (DME), age-related macular degeneration (AMD), such as wet AMD (wAMD), or choroidal neovascular membrane.

[0249] The PFKFB3 inhibitor that can be used according to the provided method is not particularly limited.In some embodiments, the PFKFB3 inhibitor administered is an antibody or PFKFB3 binding antibody (e.g., single chain antibody, single domain antibody, Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or another engineered molecule, such as diabody, triabody, tetrabody, minibody, and minimal recognition unit), a nucleic acid molecule (e.g., aptamer, antisense molecule, ribozyme, dicer substrate, miRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 inhibitory binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0250] In some embodiments, the PFKFB3 inhibitor administered according to the provided methods has a concentration of PFKFB3 activity with an IC50 of 100 μM or less for PFKFB3 activity / function. In some embodiments, the PFKFB3 inhibitor has an IC50 of at least or up to about 200, 100, 80, 50, 40, 20, 10, 5 or 1 μM, or at least or up to about 100, 10 or 1 nM or less (or any range or value derivable therein). In some embodiments, the PFKFB3 inhibitor inhibits the expression of PFKFB3. Assays for determining the ability of a compound to inhibit PFKFB3 activity are known in the art. In some embodiments, the inhibition of PFKFB3 activity or expression is a decrease compared to a control level or sample. In some embodiments, a functional assay such as an MTT assay, a cell proliferation assay, a BRDU or Ki67 immunofluorescence assay, an apoptosis assay or a glycolysis assay is used to assay the ability of the composition to inhibit PFKFB3 activity.

[0251] In some embodiments, the PFKFB3 inhibitor administered according to the provided methods is an antibody or a PFKFB3-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit). In certain embodiments, the PFKFB3 inhibitor administered is a nanobody (e.g., a VHH).

[0252] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an aptamer, an antisense molecule, a ribozyme, a dicer substrate, miRNA, dsRNA, ssRNA, and shRNA. In certain embodiments, the HIF1-α inhibitor administered according to the provided methods is an siRNA or an antisense oligonucleotide.

[0253] Representative examples of human PFKFB3 coding sequences are provided in GenBank Accession Nos. NM_004566.3, NM_001145443.2, NP_001138915.1, NM_001282630.2, NM_001314063.1, NM_001323016.1, NM_001323017.1, and NM_001363545.2. The sequences associated with each of these Genbank Accession Nos. are incorporated herein by reference in their entirety for all purposes. Therapeutic nucleic acids that inhibit PFKFB3 activity can be routinely designed and prepared based on each of the above human PFKFB3 transcript sequences using methods known in the art.

[0254] Certain embodiments of the provided methods contemplate administration of a PFKFB3 inhibitory nucleic acid or any method of inhibiting gene expression of PFKFB3 known in the art. Examples of inhibitory nucleic acids include, but are not limited to, antisense nucleic acids such as ENMD-1198 (small interfering RNA), small hairpin RNA (shRNA), double-stranded RNA, and any other antisense oligonucleotides. Also included are ribozymes or nucleic acids encoding any of the inhibitors described herein. The inhibitory nucleic acid may inhibit transcription of PFKFB3 in cells or prevent translation of PFKFB3 gene transcripts. In some embodiments, the PFKFB3 inhibitory nucleic acid administered according to the provided methods is between 16 and 1000 nucleotides in length. In certain embodiments, the PFKFB3 inhibitory nucleic acid administered is between 18 and 100 nucleotides in length. In certain embodiments, the PFKFB3 inhibitory nucleic acid administered is at least or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 nucleotides, or any range derivable therein.

[0255] In some embodiments, the PFKFB3 inhibitory nucleic acid administered according to the provided methods can reduce expression of PFKFB3 by at least 10%, 20%, 30% or 40%, more particularly at least 50%, 60%, or 70%, most particularly at least 75%, 80%, 90%, 95% or more, or any range or value therebetween.

[0256] In some embodiments, the PFKFB3 inhibitory nucleic acid administered in accordance with the provided methods is 17-25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of mature PFKFB3 mRNA (e.g., a sequence disclosed in any one or more of GenBank Accession Nos. NM_004566.3, NM_001145443.2, NM_001282630.2, NM_001314063.1, NM_001323016.1, NM_001323017.1, and NM_001363545.2). In some embodiments, the PFKFB3 inhibitory nucleic acid administered is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. In some embodiments, the PFKFB3 inhibitory nucleic acid administered has a sequence (5' to 3') that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100% complementary to the corresponding 5' to 3' sequence of mature PFKFB3 mRNA (e.g., a sequence disclosed in any one or more of GenBank Accession Nos. NM_004566.3, NM_001145443.2, NM_001282630.2, NM_001314063.1, NM_001323016.1, NM_001323017.1, and NM_001363545.2), or any range derivable therein. One skilled in the art could use the portion of the probe sequence that is complementary to the sequence of the mature mRNA as the sequence of the mRNA inhibitor, and furthermore, that portion of the probe sequence could be altered so that it is still 90% complementary to the sequence of the mature mRNA.

[0257] In some embodiments, the PFKFB3 inhibitory nucleic acid administered according to the provided methods is a miRNA. In further embodiments, the administered miRNA is hsa-mir-26b-5p (MIRT028775), hsa-mir-330-3p (MIRT043840), hsa-mir-6779-5p (MIRT454747), hsa-mir-6780a-5p (MIRT454748), hsa-mir-3689c (MIRT454749), hsa-mir-3689b-3p (MIRT454749), hsa-mir-3689c-3p ...c-3p (MIRT454749), hsa-mir-3689b-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa-mir-3689c-3p (MIRT454749), hsa p(MIRT454750), hsa-mir-3689a-3p(MIRT454751), hsa-mir-30b-3p(MIRT454752), hsa-mir-1273h-5p( MIRT454753), hsa-mir-6778-5p(MIRT454754), hsa-mir-1233-5p(MIRT454755), hsa-mir-6799-5p(MIRT 454756), hsa-mir-7106-5p(MIRT454757), hsa-mir-6775-3p(MIRT454758), hsa-mir-1291(MIRT454759 ), hsa-mir-765(MIRT454760), hsa-mir-423-5p(MIRT454761), hsa-mir-3184-5p(MIRT454762), hsa-mi A member selected from r-6856-5p (MIRT454763), hsa-mir-6758-5p (MIRT454764), hsa-mir-3185 (MIRT527973), hsa-mir-6892-3p (MIRT527974), hsa-mir-6840-5p (MIRT527975), and hsa-mir-6865-3p (MIRT527976).

[0258] In some embodiments, the PFKFB3 inhibitor administered according to the provided methods is a small molecule. The small molecule PFKFB3 inhibitor administered can be any small molecule determined to inhibit the function or activity of PFKFB3. Such small molecules can be determined based on in vitro or in vivo functional assays. In some embodiments, the PFKFB3 inhibitor small molecule administered according to the provided methods is a small molecule PFKFB3 inhibitor molecule disclosed in U.S. Patent Publication Nos. 20130059879, 20120177749, 20100267815, 20100267815, and 20090074884, the disclosures of each of which are incorporated herein by reference in their entirety.

[0259] In some embodiments, the PFKFB3 inhibitor administered in accordance with the provided methods is (1H-benzo[g]indol-2-yl)-phenyl-methanone, (3H-benzo[e]indol-2-yl)-phenyl-methanone, (3H-benzo[e]indol-2-yl)-(4-methoxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanone, the HCl salt of (3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanone, (3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanone, (3H-benzo[e]indol-2-yl) -(3-Methoxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-pyridin-3-yl-methanone, (3H-benzo[e]indol-2-yl)-(2-methoxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-(2-hydroxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-(4-hydroxy-phenyl)-methanone, (5-methyl-3H-benzo[e]indol-2-yl)-phenyl-methanone, phenyl-(7H-pyrrolo[2,3-h]quinoline-8 -yl)-methanone, (3H-benzo[e]indol-2-yl)-(3-hydroxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-(2-chloro-pyridin-4-yl)-methanone, (3H-benzo[e]indol-2-yl)-(1-oxy-pyridin-4-yl)-methanone, phenyl-(6,7,8,9-tetrahydro-3H-benzo[e]indol-2-yl)-methanone, (3H-benzo[e]indol-2-yl)-(4-hydroxy-3-methoxyrutheny)-methanone, (3H-benzo[e]indol-2-yl)-(4-hydroxy-3-methoxyrutheny)-methanone, e]indol-2-yl)-(4-benzyloxy-3-methoxy-phenyl)-methanone, 4-(3H-benzo[e]indole-2-carbonyl)-benzoic acid methyl ester, 4-(3H-benzo[e]indole-2-carbonyl)-benzoic acid, (4-amino-phenyl)-(3H-benzo[e]indol-2-yl)-methanone, 5-(3H-benzo[e]indole-2-carbonyl)-2-benzyloxy-benzoic acid methyl ester, 5-(3H-benzo[e]indole-2-carbonyl)-2-benzyloxy-benzoic acid methanone,(3H-benzo[e]indol-2-yl)-(2-methoxy-pyridin-4-yl)-methanone, (5-fluoro-3H-benzo[e]indol-2-yl)-(3-methoxy-phenyl)-methanone, (5-fluoro-3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanone, (4-benzyloxy-3-methoxy-phenyl)-(5-fluoro-3H-benzo[e]indol-2-yl)-methanone, (5-fluoro-3H-benzo[e]indol-2-yl)-(4-hydroxy-3-methoxy- phenyl)-methanone, (3H-benzo[e]indol-2-yl)-(3-hydroxymethyl-phenyl)-methanone, cyclohexyl-(5-fluoro-3H-benzo[e]indol-2-yl)-methanone, (5-fluoro-3H-benzo[e]indol-2-yl)-(3-fluoro-4-hydroxy-phenyl)-methanone, (3H-benzo[e]indol-2-yl)-p-tolyl-methanone, (3H-benzo[e]indol-2-yl)-(3-methoxy-phenyl-methanol, (3H-benzo[e]indol (4-amino-3-methoxy-phenyl)-(3H-benzo[e]indol-2-yl)-pyridin-4-yl-methanol, 3H-benzo[e]indole-2-carboxylic acid phenylamide, 3H-benzo[e]indole-2-carboxylic acid (3-methoxy-phenyl)-amide, (3H-benzo[e]indol-2-yl)-(4-dimethylamino-phenyl)-methanone, (4-amino-3-methoxy-phenyl)-(3H-benzo[e]indol-2-yl)-methanone, (4-amino-3-methoxy-phenyl)-(5-hydroxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-3-methoxy-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone, N-[4-(3H-benzo[e]indole-2-carbonyl)-phenyl]-methanesulfonamide, 3H-benzo[e]indole-2-carboxylic acid (4-amino-phenyl)-amide, (4-amino-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-2-fluoro-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone,(4-amino-3-fluoro-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-2-methoxy-phenyl)-(5-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-3-methoxy-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone Methanone, (4-amino-2-methoxy-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-3-fluoro-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-2-fluoro-phenyl)-(9-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-3-fluoro-phenyl)-(3H-benzo[e]indol-2-yl )-methanone, (4-amino-2-fluoro-phenyl)-(3H-benzo[e]indol-2-yl)-methanone, (4-amino-phenyl)-(7-methoxy-3H-benzo[e]indol-2-yl)-methanone, (4-amino-phenyl)-(5-hydroxy-3-methyl-3H-benzo[e]indol-2-yl)-methanone, (7-amino-5-fluoro-9-hydroxy-3H-benzo[e]indol-2-yl)-(3-methyl- pyridin-4-yl)-methanone, (5-amino-3H-pyrrolo[3,2-f]isoquinolin-2-yl)-(3-methoxy-pyridin-4-yl)-methanone, (4-amino-2-methyl-phenyl)-(9-hydroxy-3H-pyrrolo[2,3-c]quinolin-2-yl)-methanone, and (4-amino-phenyl)-(7-methanesulfonyl-3H-benzo[e]indol-2-yl)-methanone, or at least one of their salts.

[0260] In some embodiments, the PFKFB3 inhibitor administered in accordance with the provided methods is 1-pyridin-4-yl-3-quinolin-4-yl-propenone, 1-pyridin-4-yl-3-quinolin-3-yl-propenone, 1-pyridin-3-yl-3-quinolin-2-yl-propenone, 1-pyridin-3-yl-3-quinolin-4 ... 3-yl-propenone, 1-naphthalen-2-yl-3-quinolin-2-yl-propenone, 1-naphthalen-2-yl-3-quinolin-3-yl-propenone, 1-pyridin-4-yl-3-quinolin-3-yl-propenone, 3-(4-hydroxy-quinolin-2-yl)-1-pyridin-4-yl-propenone, 3-(8-hydroxy-quinolin-2-yl)-1-pyridin-3-yl-propenone 3-quinolin-2-yl-1-p-tolyl-propenone, 3-(8-hydroxy-quinolin-2-yl)-1-pyridin-4-yl-propenone, 3-(8-hydroxy-quinolin-2-yl)-1-p-tolyl-propenone, 3-(4-hydroxy-quinolin-2-yl)-1-p-tolyl-propenone, 1-phenyl-3-quinolin-2-yl-propenone, 1-pyridin-2-yl In one embodiment, the quinolin-2-yl-propenone is at least one of 1-(2-hydroxy-phenyl)-3-quinolin-2-yl-propenone, 1-(4-hydroxy-phenyl)-3-quinolin-2-yl-propenone, 1-(2-amino-phenyl)-3-quinolin-2-yl-propenone, 1-(4-amino-phenyl)-3-quinolin-2-yl-propenone, or a salt thereof.

[0261] In some embodiments, the PFKFB3 inhibitor administered in accordance with the provided methods is 4-(3-quinolin-2-yl-acryloyl)-benzamide, 4-(3-quinolin-2-yl-acryloyl)-benzoic acid, 3-(8-methyl-quinolin-2-yl)-1-pyridin-4-yl-propenone, 1-(2-fluoro-pyridin-4-yl)-3-quinolin-2-yl-propenone, 3-(8-fluoro-quinolin-2-yl)-1-pyridinyl and at least one of 1-methyl-4-[3-(8-methyl-quinolin-2-yl)-acryloyl]-pyridinium, or a salt thereof.

[0262] In some embodiments, the PFKFB3 inhibitor administered according to the provided methods is at least one of PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), (2S)-N-[4-[[3-cyano-1-(2-methyl-propyl)-1H-indol-5-yl]oxy]phenyl]-2-pyrrolidine-carboxamide 3PO (3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-one), (2S)-N-[4-[[3-cyano-1-[(3,5-dimethyl-4-isoxazolyl)methyl]-1H-indol-5-yl]oxy]phenyl]-2-pyrrolidine-carboxamide, and ethyl 7-hydroxy-2-oxo-2H-1-benzopyran-3-carboxylate, or a salt thereof.

[0263] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is PFK15 or a salt thereof.

[0264] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is PFK158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0265] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), or a salt thereof.

[0266] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is AZ67, or a salt thereof.

[0267] In some embodiments, the PFKFB3 inhibitor administered according to the methods provided is at least one PFKFB3 inhibitor having the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1C or ID. In other embodiments, the PFKFB3 inhibitor administered according to the methods provided is a PFKFB3 inhibitor having the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E.

[0268] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is KAN0436151, or a salt thereof.

[0269] In certain embodiments, the PFKFB3 inhibitor administered in accordance with the methods provided is KAN0436067, or a salt thereof. HIF1-α pathway inhibitors

[0270] Hypoxia-inducible factor 1-α (HIF-1-α) is a subunit of the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), which is thought to be the master transcriptional regulator of the cellular and developmental response to hypoxia.

[0271] In some embodiments, the present disclosure provides a method of treating ocular neovascular diseases and conditions in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor or a HIF1-α inhibitor; The method provides said wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0272] In some embodiments, the disclosure provides a method according to any one of (a)-(c), wherein HIF1-α is upregulated in the subject.

[0273] In some embodiments, the disclosure provides a method according to any one of (a)-(c), wherein PFKFB3 is upregulated in the subject.

[0274] In some embodiments, the disclosure provides a method according to any one of (a)-(c), wherein PFKFB3 and HIF1-α are upregulated in the subject.

[0275] In some embodiments, the disclosure provides a method according to any one of (a)-(c), wherein PFKFB3 is upregulated in the subject independently of HIF1-α.

[0276] In some embodiments, the disclosure provides a method according to any one of (a)-(c), wherein HIF1-α is upregulated in the subject independently of PFKFB3.

[0277] In some embodiments, the present disclosure is a method for treating an ocular neovascular disease or condition in a subject in need thereof, comprising administering to the subject an effective amount of a HIF1-α pathway inhibitor or a HIF1-α inhibitor. In certain embodiments, the effective amount downregulates VEGF and PFKFB3. In certain embodiments, the effective amount reduces vascular damage and / or neuronal damage in the subject.

[0278] In further embodiments, the ocular neovascular disease or condition being treated is diabetic retinopathy (DR), diabetic macular edema (DME), age-related macular degeneration (AMD), such as wet AMD (wAMD), or choroidal neovascular membrane.

[0279] As used herein, the term "HIF1-α pathway-α inhibitor" refers to a composition that inhibits or reduces HIF1-α directly or indirectly through inhibiting one or more activities of the PI3K / AKT / mTOR pathway upstream of the HIF1-α pathway. The term "HIF1-α inhibitor" is used herein to refer to a composition that directly inhibits or reduces HIF1-α. Thus, for example, mTOR pathway inhibitors such as temsirolimus, everolimus, and sirolimus are considered "HIF1-α pathway-α inhibitors" and not "HIF1-α inhibitors" herein.

[0280] The "HIF1-α pathway-α inhibitor" that can be administered according to the provided method is not particularly limited. In some embodiments, the administered HIF1-α pathway inhibitor is an antibody or HIF1-α binding antibody fragment (e.g., single chain antibody, single domain antibody (e.g., VHH AG-1, AG-2, AG-3, AG-4 or AG-5, AHPC, or VHH212), Fab fragment, F(ab')2 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or another engineered molecule, such as diabody, triabody, tetrabody, minibody, and minimal recognition unit), a nucleic acid molecule (e.g., aptamer, antisense molecule, ribozyme, dicer substrate, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0281] In some embodiments, the administered HIF1-α pathway inhibitor administered according to the provided method is at a concentration of HIF1-α activity / function IC50 of 100 μM or less. In some embodiments, the HIF1-α pathway inhibitor has an IC50 of at least or up to about 200, 100, 80, 50, 40, 20, 10, 5 or 1 μM, or at least or up to about 100, 10 or 1 nM or less (or any range or value derivable therein). In some embodiments, the HIF1-α pathway inhibitor inhibits the expression of HIF1-α. Assays for determining the ability of a compound to inhibit HIF1-α activity are known in the art. In some embodiments, the inhibition of HIF1-α activity or expression is a decrease compared to a control level or sample. In some embodiments, a functional assay such as an MTT assay, a cell proliferation assay, a BRDU or Ki67 immunofluorescence assay, an apoptosis assay or a glycolysis assay is used to assay the ability of the composition to inhibit HIF1-α activity.

[0282] The HIF1-α inhibitor that can be administered according to the provided method is not particularly limited. In some embodiments, the HIF1-α inhibitor modulates one or more of HIF-1α mRNA expression, HIF-1α protein translation or degradation, HIF-1α / HIF-1β dimerization, HIF-1α-DNA binding (e.g., HIF-1α / HRE), and / or HIF-1α transcriptional activity (e.g., CH-1 of p300 / C-TAD of HIF-1α).

[0283] In some embodiments, the HIF1-α inhibitor administered according to the provided method is a small molecule.In some embodiments, the HIF1-α inhibitor administered according to the provided method is a protein or polypeptide (e.g., anti-HIF1 antibody or antibody fragment that binds to HIF1).In some embodiments, the HIF1-α inhibitor administered according to the provided method is a therapeutic nucleic acid (e.g., aptamer, antisense molecule, ribozyme, dicer substrate, siRNA, miRNA, dsRNA, ssRNA, or shRNA).

[0284] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the provided methods is a HIF1-α pathway inhibitor (e.g., a PI3K pathway inhibitor, a MAPK pathway inhibitor, an Akt pathway inhibitor, and / or an mTOR inhibitor); a HIF translation inhibitor (e.g., a topoisomerase inhibitor, a microtubule targeting drug, a cardiac glycoside, or an antisense HIF-1a mRNA); an inhibitor of HIF stability, nuclear localization or dimerization (e.g., acriflavine or an HDAC inhibitor); an inhibitor of HIF transactivation (e.g., a HIF1 coactivator recruitment inhibitor or a HIF1 DNA binding inhibitor).

[0285] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a HIF1-α pathway inhibitor (e.g., a PI3K pathway inhibitor, a MAPK pathway inhibitor, an Akt pathway inhibitor, and / or an mTOR inhibitor). In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a PI3K pathway inhibitor. In one embodiment, the HIF1-α pathway inhibitor administered is P3155, LY29, LY294002, wortmannin, or GDC-0941. In one embodiment, the HIF1-α pathway inhibitor administered is resveratrol. In another embodiment, the HIF1-α pathway inhibitor administered is glyceollin. In some embodiments, the HIF1-α pathway inhibitor administered according to the provided methods is an mTOR inhibitor. In one embodiment, the HIF1-α pathway inhibitor administered is rapamycin, temsirolimus (CC1-779), everolimus, sirolimus, or PP242.

[0286] In certain embodiments, the HIF1-α inhibitor administered is silibinin.

[0287] In some embodiments, the HIF1-α inhibitor administered according to the provided method is a HIF translation inhibitor.In one embodiment, the HIF1-α inhibitor administered is PX-478 (S-2-amino-3-[4'-N,N-bis(chloroethyl)[amino]phenylpropionic acid N-oxide dihydrochloride), NSC-64421, camptothecin (CPT), SN38, irinotecan, topotecan, NSC-644221, cycloheximide, or apigenin, or their salts. In one embodiment, the HIF1-α inhibitor administered is aminoflavone, KC7F2 (N,N'-(disulfanediylbis(ethane-2,1-diyl))bis(2,5-dichlorobenzenesulfonamide), 2-methoxyestradiol (2ME2), or an analog or salt thereof. In one embodiment, the HIF1-α inhibitor administered is ENMD-1198, ENMD-1200, or ENMD-1237, or a salt thereof. In one embodiment, the HIF1-α inhibitor administered is EZN-2208 or a salt thereof.

[0288] In a particular embodiment, the HIF1-α inhibitor administered is PX-478 or a salt thereof.

[0289] In some embodiments, the HIF1-α inhibitor administered according to the provided method is a cardiac glycoside. In one embodiment, the cardiac glycoside administered is digoxin or a salt thereof. In another embodiment, the cardiac glycoside administered is ouabain or proscillaridin A or a salt thereof.

[0290] In some embodiments, the HIF1-α pathway inhibitor administered according to the provided method is a topoisomerase inhibitor.In one embodiment, the topoisomerase inhibitor administered is camptothecin (CPT), SN38, irinotecan, or topotecan (e.g., PEG-SN38), or salts thereof.

[0291] In some embodiments, the HIF1-α pathway inhibitor administered according to the provided method is a microtubule targeting drug.In one embodiment, the microtubule targeting drug administered is 2 methoxyestradiol (2ME2), ENMD-1198, ENMD-1200, ENMD-1237, or taxotere, or their salts.

[0292] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an aptamer, an antisense molecule, a ribozyme, a dicer substrate, an siRNA, an miRNA, a dsRNA, a ssRNA, and an shRNA. In some embodiments, the therapeutic nucleic acid is an antisense oligonucleotide.

[0293] In certain embodiments, the HIF1-α inhibitor administered according to the provided methods is an siRNA or an antisense oligonucleotide.In one embodiment, the HIF1-α inhibitor administered is EZN-2968.In one embodiment, the HIF1-α inhibitor administered is RX-0047.

[0294] Representative examples of human HIF1-A coding sequences are provided in GenBank Accession Nos. NM_004566.3, NM_001145443.2, NP_001138915.1, NM_001282630.2, NM_001314063.1, NM_001323016.1, NM_001323017.1, and NM_001363545.2. The sequences associated with each of these Genbank Accession Nos. are incorporated herein by reference in their entirety for all purposes. Therapeutic nucleic acids that inhibit HIF1-A activity can be routinely designed and prepared based on each of the above human HIF1-A transcript sequences using methods known in the art.

[0295] Certain embodiments of the provided methods contemplate administration of HIF1-A inhibitory nucleic acid or any method of inhibiting gene expression of HIF1-A known in the art. Examples of inhibitory (therapeutic) nucleic acids include, but are not limited to, antisense nucleic acids such as ENMD-1198 (small interfering RNA), small hairpin RNA (shRNA), double-stranded RNA, and any other antisense oligonucleotides. Also included are ribozymes or nucleic acids encoding any of the inhibitors described herein. The inhibitory nucleic acid may inhibit transcription of HIF1-A in cells or prevent translation of HIF1-A gene transcripts. In some embodiments, the HIF1-A inhibitory nucleic acid administered according to the provided methods is between 16 and 1000 nucleotides in length. In certain embodiments, the HIF1-A inhibitory nucleic acid administered is between 18 and 100 nucleotides in length. In certain embodiments, the HIF1-A inhibitory nucleic acid administered is at least or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 nucleotides, or any range derivable therein.

[0296] In some embodiments, the HIF1-A inhibitory nucleic acid administered in accordance with the provided methods can reduce expression of HIF1-A by at least 10%, 20%, 30% or 40%, more particularly at least 50%, 60%, or 70%, and most particularly at least 75%, 80%, 90%, 95% or more, or any range or value therebetween.

[0297] In some embodiments, the HIF1-A inhibitory nucleic acid administered in accordance with the provided methods is 17-25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of mature HIF1-A mRNA (e.g., as disclosed in any one or more of GenBank Accession Nos. NM_001530.4, NM_181054.3, and NM_001243084.2). In some embodiments, the HIF1-A inhibitory nucleic acid administered is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. In some embodiments, the administered HIF1-A inhibitory nucleic acid has a sequence (5' to 3') that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100% complementary to the corresponding 5' to 3' sequence of the mature HIF1-A mRNA (e.g., as disclosed in any one or more of GenBank Accession Nos. NM_001530.4, NM_181054.3, and NM_001243084.2), or any range derivable therein. One of skill in the art would be able to use a portion of the probe sequence that is complementary to the sequence of the mature mRNA as the sequence of the mRNA inhibitor. Furthermore, that portion of the probe sequence can be modified so that it is still 90% complementary to the sequence of the mature mRNA.

[0298] In some embodiments, the HIF1-α inhibitory nucleic acid administered according to the provided methods is an miRNA mimic. In some embodiments, the HIF1-α inhibitor administered is an miR-483 mimic.

[0299] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is an inhibitor of HIF stability, nuclear localization or dimerization. In one embodiment, the inhibitor administered according to the provided methods destabilizes HIF. In one embodiment, the inhibitor administered according to the provided methods is a histone deacetylase inhibitor (HDACI). In further embodiments, the HDACI administered is LW6 / CAY10585, vorinostat, romidepsin (FK228), panobinostat, belinostat, trichostatin A (TSA), LAQ824, or phenethyl isothiocyanate, or a salt thereof. In one embodiment, the inhibitor administered according to the provided methods is PX-12 / pleurotin, HIF-1α inhibitor (CAS number 934593-90-5), cryptotanshinone, or BAY87-2243 (1-cyclopropyl-4-[4-[[5-methyl-3-[3-[4-(trifluoromethoxy)phenyl]-1,2,4-oxadiazol-5-yl]-1H-pyrazol-1-yl]methyl]-2-pyridinyl]-piperazine), or a salt thereof. In one embodiment, the inhibitor administered according to the provided methods is IDF-11774, bisphenol A / dimethylbisphenol A, or a salt thereof. Chrysin (5,7-dihydroxy-flavone), or SCH66336, or a salt thereof. In one embodiment, the inhibitor administered according to the provided methods is geldanamycin or an analog thereof, 17-AAG (Tanespimycin: allylamino-17-demethoxygeldanamycin), 17-DMAG (alvespimycin), 17AG, radicicol, KF58333, ENMD-1198, ENMD-1237, or ganetespib, or a salt thereof. In one embodiment, the inhibitor administered according to the provided methods prevents HIF dimerization. In one embodiment, the inhibitor administered according to the provided methods is acriflavine or a salt thereof. In one embodiment, the inhibitor administered according to the provided methods is TC-S7009, PT2385, or TAT-cyclo-CLLFVY, or a salt thereof.

[0300] In certain embodiments, the inhibitor administered in accordance with the methods provided is ganetespib or a salt thereof.

[0301] In certain embodiments, the inhibitor administered in accordance with the methods provided is BAY87-2243.

[0302] In some embodiments, the HIF1-α pathway inhibitor administered according to the provided method is a histone deacetylase inhibitor (HDACI). In one embodiment, the HDACI administered is LW6 / CAY10585 (methyl 3-(2-(4-(adamantan-1-yl)phenoxy)acetamido)-4-hydroxy-benzoate), vorinostat, romidepsin (FK228), panobinostat, belinostat, trichostatin A (TSA), LAQ824, or phenethyl isothiocyanate, or a salt thereof.

[0303] In some embodiments, the HIF1-α pathway inhibitor administered according to the provided methods is a heat shock protein inhibitor. In one embodiment, the HIF1-α pathway inhibitor administered is an HSP90 inhibitor. In one embodiment, the HSP90 inhibitor administered is geldanamycin or an analog thereof, 17-AAG (Tanespimycin: Allylamino-17-demethoxygeldanamycin), 17-DMAG (Alvespimycin), 17AG, radicicol, KF58333, ENMD-1198, ENMD-1237, or ganetespib, or a salt thereof. In certain embodiments, the heat shock protein inhibitor administered is ganetespib or a salt thereof. In one embodiment, the HIF1-α pathway inhibitor administered is an HSP70 inhibitor. In one embodiment, the HSP70 inhibitor administered is triptolide or a salt thereof.

[0304] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a HIF transactivation inhibitor. In one embodiment, the HIF1-α inhibitor administered according to the provided methods inhibits the recruitment of HIF coactivators. In one embodiment, the HIF1-α inhibitor administered is ketomin, YC-1 or KCN-1 (3,4-dimethoxy-N-[(2,2-dimethyl-2H-chromen-6-yl)methyl]-N-phenylbenzenesulfonamide), or a salt thereof. In another particular embodiment, the HIF1-α inhibitor administered is NSC607097 or a salt thereof. In one embodiment, the HIF1-α inhibitor administered is a proteasome inhibitor. In a further embodiment, the inhibitor administered is bortezomib or carfilzomib, or a salt thereof. In one embodiment, the HIF1-α inhibitor administered is indenopyrazole 21, FM19G11, flavopiridol, amphotericin B, actinomycin, AJM290, or AW464, or a salt thereof.In one embodiment, the HIF1-α inhibitor administered is triptolide or a salt thereof.

[0305] In certain embodiments, the HIF1-α inhibitor administered in accordance with the methods provided is YC-1, or a salt thereof.

[0306] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is an antibody or HIF1-α binding antibody fragment that binds to HIF1-α (e.g., a single chain antibody, a single domain antibody (e.g., AG1-5 VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit). In certain embodiments, the HIF1-α inhibitor administered is a VHH or a nanobody. In one embodiment, the antibody administered is AGI-5. In one embodiment, the antibody administered is AHPC.

[0307] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a HIF1 DNA binding inhibitor. In one embodiment, the HIF1-α inhibitor administered is echinomycin (NSC-13502) or the compound DJ12.162. In one embodiment, the HIF1-α inhibitor administered is an anthracycline. In a further embodiment, the inhibitor administered is doxorubicin or daunorubicin. In one embodiment, the HIF1-α inhibitor administered is a polyamide. In some embodiments, the HIF1-α inhibitor is an antibody that binds to HIF1-α, or a HIF1-α binding antibody fragment, such as a VHH or a nanobody.

[0308] In some embodiments, the HIF1-A inhibitor administered according to the provided methods is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an aptamer, an antisense molecule, a ribozyme, a dicer substrate, a MiRNA, a dsRNA, a ssRNA, and an shRNA. In some embodiments, the therapeutic nucleic acid is an antisense oligonucleotide.

[0309] In some embodiments, the HIF1-A inhibitor administered according to the provided methods is an siRNA or an antisense oligonucleotide. In some embodiments, the HIF1-A inhibitor administered is RX-0047. In some embodiments, the HIF1-A inhibitor administered is EZN-2968.

[0310] Representative examples of human HIF1-A coding sequences are provided in GenBank Accession Nos. NM_004566.3, NM_001145443.2, NP_001138915.1, NM_001282630.2, NM_001314063.1, NM_001323016.1, NM_001323017.1, and NM_001363545.2. The sequences associated with each of these Genbank Accession Nos. are incorporated herein by reference in their entirety for all purposes. Therapeutic nucleic acids that inhibit HIF1-A activity can be routinely designed and prepared based on each of the above human HIF1-A transcript sequences using methods known in the art.

[0311] Certain embodiments of the provided methods contemplate administration of a HIF1-A inhibitory nucleic acid or any method of inhibiting gene expression of HIF1-A known in the art. Examples of inhibitory nucleic acids include, but are not limited to, antisense nucleic acids such as ENMD-1198 (small interfering RNA), small hairpin RNA (shRNA), double-stranded RNA, and any other antisense oligonucleotides. Also included are ribozymes or nucleic acids encoding any of the inhibitors described herein. The inhibitory nucleic acid may inhibit transcription of HIF1-A in cells or prevent translation of HIF1-A gene transcripts. In some embodiments, the HIF1-A inhibitory nucleic acid administered according to the provided methods is between 16 and 1000 nucleotides in length. In certain embodiments, the HIF1-A inhibitory nucleic acid administered is between 18 and 100 nucleotides in length. In certain embodiments, the HIF1-A inhibitory nucleic acid administered is at least or up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 50, 60, 70, 80, 90 nucleotides, or any range derivable therein.

[0312] In some embodiments, the HIF1-A inhibitory nucleic acid administered according to the provided methods can reduce expression of HIF1-A by at least 10%, 20%, 30% or 40%, more particularly at least 50%, 60%, or 70%, and most particularly at least 75%, 80%, 90%, 95% or more, or any range or value therebetween. In some embodiments, the HIF1-A inhibitory nucleic acid administered according to the provided methods is 17-25 nucleotides in length and comprises a 5' to 3' sequence that is at least 90% complementary to the 5' to 3' sequence of mature HIF1-A mRNA (e.g., as disclosed in any one or more of GenBank Accession Nos. NM_001530.4, NM_181054.3, and NM_001243084.2). In some embodiments, the administered HIF1-A inhibitory nucleic acid is 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length, or any range derivable therein. In some embodiments, the administered HIF1-A inhibitory nucleic acid has a sequence (5' to 3') that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, or 100% complementary to the corresponding 5' to 3' sequence of mature HIF1-A mRNA (e.g., as disclosed in any one or more of GenBank Accession Nos. NM_001530.4, NM_181054.3, and NM_001243084.2), or any range derivable therein. One skilled in the art could use the portion of the probe sequence that is complementary to the sequence of the mature mRNA as the sequence of the mRNA inhibitor, and furthermore, that portion of the probe sequence could be altered so that it is still 90% complementary to the sequence of the mature mRNA.

[0313] In some embodiments, the HIF1-α inhibitory nucleic acid administered according to the provided methods is an miRNA mimic. In some embodiments, the HIF1-α inhibitor administered is an miR-483 mimic.

[0314] In some embodiments, the HIF1-α inhibitor administered according to the provided methods is a therapeutic nucleic acid. In some embodiments, the therapeutic nucleic acid is an antisense oligonucleotide.

[0315] VEGF antagonists In some embodiments, the present disclosure provides a method of treating an ocular neovascular disease or condition in a subject in need thereof, comprising: (a) administering to a subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; PFKFB3 inhibitors do not inhibit the PI3K / AKT / mTOR pathway or HIF1-α (i) the subject is further administered an anti-VEGF therapeutic agent; and / or The method further comprises the steps of: (ii) the subject has been previously treated with an anti-VEGF therapeutic agent;

[0316] In some embodiments, the disclosure is a method of treating an ocular neovascular disease or condition in a subject in need thereof, comprising administering an effective amount of a VEGF antagonist in combination with a PFKFB3 inhibitor.

[0317] In some embodiments, the anti-VEGF therapeutic administered to the subject is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a VEGF-binding polypeptide (e.g., an Fc fusion protein), or a small molecule VEGF pathway inhibitor.

[0318] In some embodiments, the anti-VEGF therapeutic administered to the subject according to the provided methods is a VEGF-Trap (see, e.g., U.S. Pat. No. 7,087,411), an anti-VEGF antibody (e.g., bevacizumab or ranibizumab), or a small molecule kinase inhibitor of the VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib). In some embodiments, the subject is administered bevacizumab, ranibizumab, or aflibercept. In some embodiments, the subject is administered bevacizumab. In some embodiments, the subject is administered ranibizumab. In some embodiments, the subject is administered aflibercept.

[0319] In some embodiments, the anti-VEGF therapeutic agent previously administered to the subject is a VEGF-Trap (see, e.g., U.S. Pat. No. 7,087,411), an anti-VEGF antibody (e.g., bevacizumab or ranibizumab), or a small molecule kinase inhibitor of a VEGF receptor (e.g., sunitinib, sorafenib, or pazopanib). In some embodiments, the subject has previously been administered bevacizumab, ranibizumab, or aflibercept. In some embodiments, the subject has previously been administered bevacizumab. In some embodiments, the subject has previously been administered bevacizumab. In some embodiments, the subject has previously been administered aflibercept.

[0320] Kits for administration of active agents In another embodiment, the present disclosure provides a kit comprising an HIF1-α pathway inhibitor and a PFKFB3 inhibitor, and / or other therapeutic and delivery agents. In some embodiments, a kit for preparing and / or administering the therapeutic methods described herein may be provided. The kit may comprise one or more sealed vials containing any of the pharmaceutical compositions, therapeutic agents, and / or other therapeutic and delivery agents. In some embodiments, the kit comprises a lipid delivery system. In some embodiments, the lipid is in one vial and the therapeutic agent is in another vial. The kit may include, for example, at least one inhibitor of PFKFB3 expression / activity, at least one inhibitor of HIF1-α expression / activity, and one or more reagents for preparing, formulating and / or administering the components described herein or for carrying out one or more steps of the method. In some embodiments, the kit may also include suitable container means, which is a container that will not react with the components of the kit, such as an Eppendorf tube, an assay plate, a syringe, a bottle, or a tube. The container may be made of a sterilizable material, such as plastic or glass.

[0321] The kit may further include instructions outlining the procedural steps of the methods described herein, following substantially the same procedures as described herein or known to one of skill in the art. The instructional information may be in a computer readable medium that includes machine readable instructions that, when executed using a computer, display an actual or virtual procedure for delivering a pharma- ceutical effective amount of a therapeutic agent.

[0322] In some embodiments, kits may be provided for evaluating the expression of PFKFB3 and / or HIF-α, or related molecules. Such kits may be prepared from readily available materials and reagents. For example, such kits may include any one or more of the following materials: enzymes, reaction tubes, buffers, detergents, primers and probes, nucleic acid amplification, and / or hybridization agents. In certain embodiments, these kits allow a physician to obtain samples of blood, tears, semen, saliva, urine, tissue, serum, stool, colon, rectum, sputum, cerebrospinal fluid, and supernatants from cell lysates. In another embodiment, these kits include the equipment necessary to perform RNA extraction, RT-PCR, and gel electrophoresis. Instructions for carrying out the assays may also be included in the kit.

[0323] The kits may include components that may be individually packaged or placed in containers such as tubes, bottles, vials, syringes, or other suitable container means. The components may include probes, primers, antibodies, arrays, negative and / or positive controls. Individual components may also be provided in the kit in concentrated amounts. In some embodiments, components are provided individually at the same concentration that they would be in solution with other components. Concentrations of components may be provided as 1x, 2x, 5x, 10x, or 20x or greater.

[0324] The kit may further comprise a reagent for labeling PFKFB3 and / or HIF-1α in the sample. The kit may also comprise a labeling reagent, comprising at least one of an amine-modified nucleotide, a poly(A) polymerase, and a poly(A) polymerase buffer. The labeling reagent may comprise an amine-reactive dye or any dye known in the art.

[0325] The components of the kit may be packaged in either aqueous media or lyophilized form. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe or other container means into which the components may be placed and preferably appropriately aliquoted. Where there are multiple components in the kit (labeling reagents and labels may be packaged together), the kit will generally also include a second, third or other additional container into which the additional components may be placed separately. However, various combinations of components may be included within a vial. The kit may also include means for containing the nucleic acid, antibody or other reagent containers in seal for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0326] When the components of the kit are provided in one and / or more solutions, the solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. Alternatively, the components of the kit may be provided as dry powder(s). When the reagents and / or components are provided as dry powders, the powder may be reconstituted by adding a suitable solvent. It is envisioned that the solvent may be provided in another container means. The container means will generally include at least one vial, test tube, flask, bottle, syringe and / or other container means into which the nucleic acid formulation may be placed, and preferably appropriately aliquoted. The kit may also include a second container means for containing a sterile pharma- ceutically acceptable buffer and / or other diluent.

[0327] The kits may also include a means for sealingly containing the vials for commercial sale, such as, for example, injection and / or blow molded plastic containers into which the desired vials are retained. The kits may also include instructions for using the components of the kit, as well as instructions for the use of other reagents not included in the kit. The instructions may include operable variations.

[0328] Treatment and Methods of Use Ocular neovascular disorders Intraocular angiogenesis is responsible for vision loss in several eye diseases, the most common of which are proliferative diabetic retinopathy, neovascular age-related macular degeneration, and retinopathy of prematurity. Together, these three diseases afflict people at all stages of life from birth to late adulthood, and account for the majority of cases of legal blindness. In some embodiments, the present disclosure provides methods and compositions for treating ocular angiogenesis disorders.

[0329] In further embodiments, the disclosure provides compositions and methods for treating ocular neovascular disorders (ONDs). In one embodiment, the disclosure provides a method of treating ocular neovascular disorders (ONDs) in a subject in need thereof, comprising: (a) administering to the subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, where the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, where the subject has previously been administered a HIF1-α pathway inhibitor; wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0330] In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor, and the subject has previously been administered a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a PFKFB3 inhibitor, and the subject has previously been administered a HIF1-α pathway inhibitor. In some embodiments, the ocular neovascular disease or condition being treated is diabetic retinopathy (DR). In some embodiments, the ocular neovascular disease or condition being treated is diabetic macular edema (DME). In some embodiments, the ocular neovascular disease or condition being treated is age-related macular degeneration (AMD), such as wet AMD (wAMD). In some embodiments, the ocular neovascular disease or condition being treated is choroidal neovascular membrane.

[0331] In some embodiments, the subject is at risk of developing OND. In some embodiments, the methods provided herein (e.g., any of (a)-(c) above) are performed as a preventative treatment for OND.

[0332] In some embodiments, the methods and compositions provided prevent diabetic ocular neovascular disorders in subjects at risk of developing such disorders, e.g., subjects with one or more risk factors associated with the development of such disorders. In some embodiments, the subjects have one or more risk factors selected from hyperglycemia, hypertension, hyperlipidemia, renal disease, advanced diabetes, high mean systolic blood pressure, and high hemoglobin A1c.

[0333] In some embodiments, the subject suffers from OND. In some embodiments, the subject has been diagnosed with OND. Ocular neovascular disorders are often detected during a comprehensive eye examination, including visual acuity testing (eye chart testing to measure the subject's ability to see at various distances), tonometry (measurement of pressure inside the eye), pupil dilation, and optical coherence tomography (OCT). During such an examination, a physician can look for one or more of the following: changes to retinal blood vessels, including the formation of new blood vessels, swelling, and bleeding; retinal blood vessel leakage, or dangerous signs of leaking blood vessels, such as fatty deposits, weakening of blood vessel walls, and swelling of blood vessel walls; swelling of the macula; changes in the lens, including changes in curvature or cataract formation; and damage to nerve tissue.

[0334] In some embodiments, the disclosure provides methods and compositions for preventing, inhibiting, or delaying the onset of ocular neovascular disorder (OND) by administration to a subject prior to the onset of OND, e.g., prior to the onset of one or more symptoms of OND.

[0335] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered prior to the onset of one or more symptoms of OND. OND often progresses unnoticed until vision is affected. Vision parameters affected by OND include overall vision, peripheral vision, night vision, color vision, distance vision, near vision, and clarity of vision. In some embodiments, the compositions and methods provided can reduce the incidence, severity, or level of one or more of these vision parameters. In some embodiments, treating OND according to the methods provided herein includes delaying the onset of one or more symptoms of OND.

[0336] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of OND. In some embodiments, the methods and compositions provided can be used to treat different stages of OND.

[0337] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the methods provided herein is an antibody or antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH AG-1, AG-2, AG-3, AG-4 or AG-5, AHPC, or VHH212), a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0338] In some embodiments, the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1.3, or a salt thereof.

[0339] In some embodiments, the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothiocyanate, or BAY-87-2243, or a salt thereof.

[0340] In some embodiments, the HIF1-α pathway inhibitor administered according to the methods provided herein is a HIF1-α inhibitor. In some embodiments, the HIF1-α inhibitor does not inhibit the PI3K / AKT / mTOR pathway. In some embodiments, the HIF1-α inhibitor is an antibody or an antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH AG-1, AG-2, AG-3, AG-4 or AG-5), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a MiRNA, a dsRNA, a ssRNA, and a shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0341] In some embodiments, the administered HIF1-α inhibitor is the antisense oligonucleotide EZN-2968, or nanobody AG-1, AG-2, AG-3, AG-4 or AG-5, VHH212, or AHPC.

[0342] In some embodiments, the PFKFB3 inhibitor administered according to the methods provided herein is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0343] In some embodiments, the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0344] In some embodiments, the PFKFB3 inhibitor administered is KAN0436151 or KAN0436067, or a salt thereof.

[0345] In some embodiments, the PFKFB3 inhibitor administered is AZ67, or a salt thereof.

[0346] In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1C or Figure 1D. In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E.

[0347] In some embodiments, the methods provided herein for treating OND are carried out by co-administering to a subject a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0348] In some embodiments, the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intraocular administration. In some embodiments, the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intraocular administration. In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0349] In some embodiments, the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intravitreal administration. In some embodiments, the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intravitreal administration.

[0350] In some embodiments, treating OND according to the methods provided herein includes reducing one or more symptoms of OND in a subject, compared to a control subject or compared to a subject before treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the one or more symptoms of OND that are reduced are selected from retinal inflammation, acellular capillary formation, angiogenesis, endothelial cell death, vascular permeability, ischemia-reperfusion injury, leaky areas, and occludin disruption. In some embodiments, the one or more symptoms of OND are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%, compared to a control subject or compared to a subject before treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0351] In some embodiments, the compositions and methods provided prevent or reduce the incidence, prevalence or severity of damaged and / or leaky blood vessels in the eye.

[0352] Treatment and / or prevention of ocular neovascular disorders can be measured by a variety of means. In some embodiments, treatment or prevention comprises treating one or more of apoptosis, inflammation, acellular capillary formation, angiogenesis, retinal endothelial cell death, retinal vascular permeability, ischemia-reperfusion injury, and occludin disruption in ocular neovascular disorders in a subject in need of treatment or prevention.

[0353] In some embodiments, treating OND according to the methods provided herein includes increasing one or more visual acuity parameters in a subject compared to the visual acuity parameters of a control subject or compared to the visual acuity parameters of the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the one or more increased visual acuity parameters are selected from peripheral vision, night vision, color vision, distance vision, near vision, and clarity of vision.

[0354] The present disclosure provides methods of treating ocular neovascular disorders using anti-HIF1-α and / or anti-PFKFB3 antibodies or antigen-binding fragments thereof. The efficacy of such treatments can be characterized, assessed, measured and / or monitored based on several parameters.

[0355] In some embodiments, the provided methods result in a reduction in apoptosis and / or endothelial cell death in the eye. Cell death can be monitored according to known methods. Exemplary methods for detecting cell death include, but are not limited to, nuclear staining techniques such as propidium iodide, Hoechst-33342, 4',6-diamidino-2-phenylindole (DAPI), and acridine orange-ethidium bromide staining. Non-nuclear staining techniques include, but are not limited to, Annexin V staining.

[0356] In some embodiments, the methods provided prevent ischemia-reperfusion (IR) injury. Methods for detecting IR injury are known in the art and include fluorescein analysis, the fluorescent zinc 2,2'-dipicolylamine coordination compound PSVue.RTM.794, 99mTc glucarate, and electroretinography.

[0357] In some embodiments, the methods provided reduce the levels of pro-inflammatory cytokines, such as TNFα, IL-1β, IL-6, or MCP1. Cytokine levels can be monitored via enzyme-linked immunosorbent assay (ELISA), Luminex, cytokine bead array, Proteo Plex, FAST Quant, and the like.

[0358] In some embodiments, the methods provided reduce ocular vascular permeability, ocular neovascularization, or other symptoms of ocular health. In some embodiments, the methods provided can prevent or prevent further deterioration of typical symptoms of ocular neovascular disorders in the ocular vasculature. Vascular permeability and other measures of ocular vascular health can be measured, for example, by fluorescein angiography.

[0359] In some embodiments, the provided method improves one or more visual acuity parameters or prevents the decline of one or more visual acuity parameters. Visual acuity parameters include: poor night vision, blurred vision; floating spots, black spots or flashing lights in the field of vision; fluctuating vision, color vision disorders, dark or blank areas in the field of vision, vision loss, and sudden severe painless vision loss. In some embodiments, subjects treated according to the provided method may experience one or more of the following effects: improved visual acuity, reduced vision loss, improved night vision, improved low light vision, improved reading ability, improved peripheral vision, reduced spots in the field of vision, reduced flashing lights in the field of vision, reduced pain, and improved eye appearance. Many of these parameters may be monitored by regular eye examinations.

[0360] In some embodiments, the provided method prevents, alleviates or delays OND.This method can be administered to patients who are at risk of developing OND.For such subjects, the prevention of ocular neovascular disorder can be monitored by maintaining vision or by the absence of typical characteristics of OND. For example, a subject prophylactically administered an effective amount of a HIF1-α inhibitor and an effective amount of a PFKFB3 inhibitor may not experience, or may experience a reduced incidence of, one or more of the following symptoms: microaneurysms, hemorrhages, intraretinal capillary abnormalities, beading of veins, cotton-wool spots, formation of new blood vessels (neovascularization) elsewhere and on the optic nerve, fibrous proliferation elsewhere and on the optic nerve, preretinal and vitreous hemorrhages, retinal detachment due to the formation of scar tissue, glaucoma, decreased night vision, blurred vision; floating spots, black spots or flashing lights in the field of vision; fluctuating vision, impaired color vision, dark or blank areas in the field of vision, vision loss, sudden severe painless loss of vision, traction retinal detachment, macular edema, venous dilation, and intraretinal capillary abnormalities.

[0361] In some embodiments, the methods of treating ocular neovascular disorders provided affect one or more parameters of the retinal vasculature, including, but not limited to, permeability, NFkB levels, inflammatory marker levels, incidence of apoptosis, sphingolipid metabolism, and re-endothelialization. In some embodiments, the methods of treating ocular neovascular disorders disclosed herein reduce retinal vascular permeability. Retinal vascular permeability may be monitored via fluorescence, tracer dyes, optical testing, and the like. In some embodiments, the methods of treating ocular neovascular disorders disclosed herein reduce NFkB and / or other inflammatory marker levels in the retinal vasculature. As described above, inflammatory cytokine levels can be measured via conventional means (e.g., ELISA). In some embodiments, the methods of treating ocular neovascular disorders disclosed herein reduce the incidence of apoptosis in the retinal vasculature. As disclosed above, apoptosis can be measured using conventional means, such as nuclear and non-nuclear staining techniques.

[0362] In further embodiments, the provided methods further comprise administering an anti-VEGF therapeutic to the subject. In some embodiments, the anti-VEGF therapeutic is bevacizumab, ranibizumab, or aflibercept. In one embodiment, the anti-VEGF therapeutic is bevacizumab. In one embodiment, the anti-VEGF therapeutic is ranibizumab. In one embodiment, the anti-VEGF therapeutic is aflibercept.

[0363] In some embodiments, the subject receiving the treatment provided herein has undergone a prior treatment for OND. In some embodiments, the prior treatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy. In certain embodiments, the prior treatment is an anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the prior anti-VEGF therapy included bevacizumab, ranibizumab, or aflibercept. In one embodiment, the prior anti-VEGF therapy included bevacizumab. In one embodiment, the prior anti-VEGF therapy included ranibizumab. In one embodiment, the prior anti-VEGF therapy included aflibercept. In some embodiments, the subject did not respond to a prior treatment for OND. In such embodiments, "non-responsive" refers to the prior treatment did not ameliorate and / or improve one or more symptoms of diabetic retinopathy. In such embodiments, "non-responsive" refers to the prior treatment did not ameliorate and / or improve one or more symptoms of diabetic retinopathy. In some embodiments, the prior treatment may not have resulted in an improvement in vision or retinal vascular health. In some embodiments, the prior treatment may show some results but may not achieve the desired performance or may cease to show effects after a period of time. In some embodiments, the subject may have partially responded to the prior treatment of the ocular neovascular disorder, i.e., one or more symptoms of the ocular neovascular disorder were not fully alleviated and / or the effects of the prior treatment were not fully sustained.

[0364] Existing treatments for ocular neovascular disorders include anti-VEGF therapy, steroids, laser surgery, and vitrectomy.

[0365] diabetic retinopathy In some embodiments, the present disclosure provides methods and compositions for treating diabetic retinopathy. Diabetic retinopathy refers to a medical condition in which diabetes causes damage to the retina. Chronic hyperglycemia from diabetes is associated with damage to the microvessels in the retina, leading to diabetic retinopathy. Diabetic retinopathy can cause leakage or bleeding of fluid from blood vessels in the retina, distorting vision. In the most advanced stages, new abnormal blood vessels grow on the surface of the retina, which can cause retinal scarring and cell loss.

[0366] In a further embodiment, the disclosure provides compositions and methods for treating diabetic retinopathy (DR). In one embodiment, the disclosure provides a method of treating diabetic retinopathy (DR) in a subject in need thereof, comprising: (a) administering to the subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, where the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, where the subject has previously been administered a HIF1-α pathway inhibitor, where the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0367] In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and the subject has previously been administered a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a PFKFB3 inhibitor and the subject has previously been administered a HIF1-α pathway inhibitor.

[0368] In some embodiments, the subject is at risk of developing DR. In some embodiments, the methods provided herein (e.g., any of (a)-(c) above) are performed as a preventative treatment for DR.

[0369] In some embodiments, the provided methods and compositions prevent diabetic retinopathy in subjects at risk of developing diabetic retinopathy, for example, subjects with one or more risk factors associated with the development of diabetic retinopathy. In some embodiments, the subject has one or more risk factors selected from hyperglycemia, hypertension, hyperlipidemia, renal disease, and progressive diabetes (e.g., as indicated by the use of insulin and oral antidiabetic drugs versus the use of pills alone, or the use of pills alone versus no treatment). In some embodiments, the subject has one or more risk factors selected from high mean systolic blood pressure, and high hemoglobin A1c.

[0370] In some embodiments, the subject suffers from DR. In some embodiments, the subject has been diagnosed with DR. Diabetic retinopathy and diabetic macular edema are often detected during a comprehensive eye examination, including visual acuity testing (eye chart testing to measure the subject's ability to see at various distances), tonometry (measuring pressure inside the eye), pupil dilation, and optical coherence tomography (OCT). During such an examination, a physician can look for one or more of the following: changes to retinal blood vessels, including the formation of new blood vessels, swelling, and bleeding; retinal blood vessel leakage, or dangerous signs of leaking blood vessels, such as fatty deposits, weakening of blood vessel walls, and swelling of blood vessel walls; swelling of the macula; changes in the lens, including changes in curvature or cataract formation; and damage to nerve tissue.

[0371] In some embodiments, the present disclosure provides methods and compositions for preventing, inhibiting, or delaying the onset of diabetic retinopathy by administering to a diabetic subject prior to the onset of diabetic retinopathy, e.g., prior to the onset of one or more symptoms of diabetic retinopathy. Among the most consistent risk factors, duration of diabetes is a strong predictor of the onset and progression of retinopathy. Among subjects with early-onset diabetes, the prevalence is estimated to be about 8% at 3 years, 25% at 5 years, 60% at 10 years, and 80% at 15 years. In some embodiments, the provided methods and compositions can be used to delay the onset of diabetic retinopathy, e.g., for more than 3 years, more than 5 years, more than 10 years, or more than 15 years from the onset of diabetes.

[0372] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered prior to the onset of one or more symptoms of DR. The disease often progresses unnoticed until vision is affected. Bleeding from retinal blood vessels during the early stages of the disease can cause the appearance of "floating" spots or dark lines, which may disappear spontaneously. Without prompt treatment, bleeding often recurs, increasing the risk of permanent vision loss. If diabetic macular edema occurs, blurred vision may result. In some embodiments, the provided compositions and methods can reduce the incidence, severity, or level of floating spots, retinal bleeding, vision loss, and / or blurred vision. In some embodiments, one or more vision parameters, including but not limited to, overall vision, peripheral vision, night vision, color vision, distance vision, near vision, and clarity of vision, can be improved by the provided methods.

[0373] In some embodiments, treating DR in accordance with the methods provided herein includes delaying the onset of one or more symptoms of DR.

[0374] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of DR. In some embodiments, the provided methods and compositions can be used to treat different stages of diabetic retinopathy. Diabetic retinopathy can progress through a non-proliferative stage (also called early stage) and a proliferative stage (also called late stage).

[0375] In some embodiments, the present disclosure provides methods and compositions for treating diabetic retinopathy during the non-proliferative stage. Non-proliferative diabetic retinopathy (NPDR) can be mild, moderate, or severe. Mild NPDR is characterized by microaneurysms of retinal blood vessels. These microaneurysms can leak fluid into the retina. As the disease progresses to the moderate NPDR stage, retinal blood vessels can become distorted and lose vascular transport capacity, resulting in characteristic changes in the appearance of the retina and contributing to diabetic macular edema. In the severe NPDR stage, more blood vessels become occluded, cutting off blood supply to areas of the retina. These areas then secrete pro-angiogenic growth factors. Angiogenic molecules in the eye include VEGF, FGF, P1GF, TGF-α, TGF-β, IGF, PDGF, MMP, HGF / SF, TNF-α, CTGF, IL-1, IL-8, MCP-1, leptin, integrins, and angiogenin. In some embodiments, the provided methods are characterized by preventing or reducing one or more markers of diabetic retinopathy. In some embodiments, the provided methods can prevent, reduce, inhibit, or reduce retinal microaneurysms, retinal fluid leakage, diabetic macular edema, and / or levels of retinal pro-angiogenic growth factors. In some embodiments, one or more of VEGF, FGF, P1GF, TGF-α, TGF-β, IGF, PDGF, MMP, HGF / SF, TNF-α, CTGF, IL-1, IL-8, MCP-1, integrins, and angiogenin are reduced by the provided methods.

[0376] In some embodiments, the present disclosure provides methods and compositions for treating diabetic retinopathy during the proliferative phase. Proliferative diabetic retinopathy (PDR) is an advanced stage of the disease. At this stage, pro-angiogenic growth factors secreted from the retina cause the proliferation of new blood vessels, which grow along the inner retinal surface and into the vitreous gel. The new blood vessels are fragile and prone to leakage and bleeding. The associated scar tissue may shrink and cause retinal detachment, which can lead to permanent vision loss. In some embodiments, the methods provided can prevent, reduce, inhibit, or decrease the level or severity of retinal neovascularization, retinal bleeding, retinal scarring, retinal detachment, and / or vision loss.

[0377] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the methods provided herein is an antibody or antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0378] In some embodiments, the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1.3, or a salt thereof.

[0379] In some embodiments, the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothiocyanate, or BAY-87-2243, or a salt thereof.

[0380] In some embodiments, the HIF1-α pathway inhibitor administered according to the methods provided herein is a HIF1-α inhibitor. In some embodiments, the HIF1-α inhibitor does not inhibit the PI3K / AKT / mTOR pathway. In some embodiments, the HIF1-α inhibitor is an antibody or an antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a MiRNA, a dsRNA, a ssRNA, and a shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0381] In some embodiments, the HIF1-α inhibitor administered is the antisense oligonucleotide EZN-2968, nanobodies AG-1 to AG-5, or nanobody AHPC.

[0382] In some embodiments, the PFKFB3 inhibitor administered according to the methods provided herein is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0383] In some embodiments, the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0384] In some embodiments, the PFKFB3 inhibitor administered is KAN0436151 or KAN0436067, or a salt thereof.

[0385] In some embodiments, the PFKFB3 inhibitor administered is AZ67, or a salt thereof.

[0386] In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1C or Figure 1D. In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E. In some embodiments, the methods provided herein for treating DR are carried out by co-administering a HIF1-α pathway inhibitor and a PFKFB3 inhibitor to a subject.

[0387] In some embodiments, the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intraocular administration. In some embodiments, the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intraocular administration. In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0388] In some embodiments, the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intravitreal administration. In some embodiments, the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intravitreal administration.

[0389] In some embodiments, treating DR according to the methods provided herein includes reducing one or more symptoms of DR in a subject compared to a control subject or compared to a subject before treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the one or more symptoms of DR are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, retinal leakage area, and occludin disruption. In some embodiments, the one or more symptoms of DR are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject before treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0390] In some embodiments, the compositions and methods provided prevent or reduce the incidence, prevalence or severity of damaged and / or leaky blood vessels in the eye.

[0391] Treatment and / or prevention of diabetic retinopathy can be measured by a variety of means. In some embodiments, treatment or prevention comprises treating one or more of apoptosis, inflammation, acellular capillary formation, angiogenesis, retinal endothelial cell death, retinal vascular permeability, ischemia-reperfusion injury, and occludin disruption in diabetic retinopathy in a subject in need of treatment or prevention.

[0392] In some embodiments, treating DR according to the methods provided herein includes increasing one or more visual acuity parameters in a subject compared to the visual acuity parameters of a control subject or compared to the visual acuity parameters of the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the one or more increased visual acuity parameters are selected from peripheral vision, night vision, color vision, distance vision, near vision, and clarity of vision.

[0393] The present disclosure provides methods of treating diabetic retinopathy using anti-HIF1-α and / or anti-PFKFB3 antibodies or antigen-binding fragments thereof. The effectiveness of such treatments can be characterized, assessed, measured and / or monitored based on several parameters.

[0394] In some embodiments, the provided methods result in a reduction in apoptosis and / or endothelial cell death in the eye. Cell death can be monitored according to known methods. Exemplary methods for detecting cell death include, but are not limited to, nuclear staining techniques such as propidium iodide, Hoechst-33342, 4',6-diamidino-2-phenylindole (DAPI), and acridine orange-ethidium bromide staining. Non-nuclear staining techniques include, but are not limited to, Annexin V staining.

[0395] In some embodiments, the methods provided prevent ischemia-reperfusion (IR) injury. Methods for detecting IR injury are known in the art and include fluorescein analysis, the fluorescent zinc 2,2'-dipicolylamine coordination compound PSVue.RTM.794, 99mTc glucarate, and electroretinography.

[0396] In some embodiments, the methods provided reduce the levels of proinflammatory cytokines, such as TNFα, IL-1β, IL-6, or MCP1, in a biological sample from a subject. Cytokine levels can be monitored via enzyme-linked immunosorbent assay (ELISA), Luminex, cytokine bead array, Proteo Plex, FAST Quant, and the like.

[0397] In some embodiments, the methods provided reduce retinal vascular permeability, retinal neovascularization, or other symptoms of retinal health. In some embodiments, the methods provided can prevent typical symptoms of diabetic retinopathy in the retinal vasculature or prevent further deterioration. Vascular permeability and other measures of retinal vascular health can be measured, for example, by fluorescein angiography.

[0398] In some embodiments, the provided method improves one or more visual acuity parameters or prevents the decline of one or more visual acuity parameters. Visual acuity parameters include: poor night vision, blurred vision; floating spots, black spots or flashing lights in the field of vision; fluctuating vision, color vision disorders, dark or blank areas in the field of vision, vision loss, sudden severe painless vision loss. In some embodiments, subjects treated according to the provided method may experience one or more of the following effects: improved vision, reduced vision loss, improved night vision, improved low light vision, improved reading ability, improved peripheral vision, reduced spots in the field of vision, reduced flashing lights in the field of vision, reduced pain, and improved eye appearance. Many of these parameters may be monitored by regular eye examinations.

[0399] In some embodiments, the provided method prevents diabetic retinopathy. This method can be administered to patients at risk of developing diabetic retinopathy. For such subjects, prevention of diabetic retinopathy can be monitored by vision maintenance or by the absence of typical features of diabetic retinopathy. For example, subjects who are prophylactically administered an effective amount of a HIF1-α inhibitor and an effective amount of a PFKFB3 inhibitor may not experience or experience a reduced incidence of one or more of the following symptoms: microaneurysms, hemorrhages, intraretinal capillary abnormalities, venous beading, cotton wool spots, the formation of new blood vessels (neovascularization) elsewhere and on the optic nerve, fibrous proliferation elsewhere and on the optic nerve, preretinal and vitreous hemorrhages, retinal detachment due to scar tissue formation, glaucoma, reduced night vision, blurred vision; floating spots, black spots or flashing lights in the field of vision; fluctuating vision, color vision disorders, dark or blank areas in the field of vision, vision loss, sudden severe painless vision loss, traction retinal detachment, macular edema, venous dilation, and intraretinal capillary abnormalities.

[0400] In some embodiments, provided methods prevent or reduce macular edema, which may be observed under slit-lamp biomicroscopy as a swelling and blurring of the retinal layers.

[0401] In some embodiments, the provided methods delay the onset of diabetic retinopathy.Thus, the provided methods delay the average onset of diabetic retinopathy by more than 5 years, more than 10 years, more than 11 years, more than 12 years, more than 13 years, more than 14 years, more than 15 years, more than 16 years, more than 17 years, more than 18 years, more than 19 years, or more than 20 years from the first diabetes diagnosis.

[0402] In some embodiments, the methods provided reduce, alleviate, reduce the severity, or reverse one or more symptoms of diabetic retinopathy. In some embodiments, the methods of treating diabetic retinopathy with anti-HIF1-α antibodies and / or anti-PFKFB3 antibodies or antigen-binding fragments thereof may reduce, alleviate, reduce the severity, or reverse one or more of the following symptoms: microaneurysms, hemorrhages, intraretinal capillary abnormalities, beading of veins, cotton wool spots, formation of new blood vessels (neovascularization) elsewhere and on the optic nerve, fibrous proliferation elsewhere and on the optic nerve, preretinal and vitreous hemorrhages, retinal detachment due to scar tissue formation, vision loss, glaucoma, poor night vision, blurred vision; floating spots, black spots, or flashing lights in the field of vision; sudden severe painless vision loss, traction retinal detachment, macular edema, venous dilation, and intraretinal capillary abnormalities.

[0403] In some embodiments, the methods of treating diabetic retinopathy provided affect one or more parameters of the retinal vasculature, including, but not limited to, permeability, NFkB levels, inflammatory marker levels, incidence of apoptosis, sphingolipid metabolism, and re-endothelialization. In some embodiments, the methods of treating diabetic retinopathy disclosed herein reduce retinal vascular permeability. Retinal vascular permeability may be monitored via fluorescence, tracer dyes, optical testing, and the like. In some embodiments, the methods of treating diabetic retinopathy disclosed herein reduce NFkB and / or other inflammatory marker levels in the retinal vasculature. As described above, inflammatory cytokine levels can be measured via conventional means (e.g., ELISA). In some embodiments, the methods of treating diabetic retinopathy disclosed herein reduce the incidence of apoptosis in the retinal vasculature. As disclosed above, apoptosis can be measured using conventional means, such as nuclear and non-nuclear staining techniques.

[0404] In further embodiments, the provided methods further comprise administering an anti-VEGF therapeutic to the subject. In some embodiments, the anti-VEGF therapeutic is bevacizumab, ranibizumab, or aflibercept. In one embodiment, the anti-VEGF therapeutic is bevacizumab. In one embodiment, the anti-VEGF therapeutic is ranibizumab. In one embodiment, the anti-VEGF therapeutic is aflibercept.

[0405] In some embodiments, the subject receiving the treatment provided herein has undergone a prior treatment for DR. In some embodiments, the prior treatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy. In certain embodiments, the prior treatment is an anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the prior anti-VEGF therapy included bevacizumab, ranibizumab, or aflibercept. In one embodiment, the prior anti-VEGF therapy included bevacizumab. In one embodiment, the prior anti-VEGF therapy included ranibizumab. In one embodiment, the prior anti-VEGF therapy included aflibercept. In some embodiments, the subject did not respond to a prior treatment for DR. In such embodiments, "non-responsive" refers to the prior treatment did not ameliorate and / or improve one or more symptoms of diabetic retinopathy. In such embodiments, "non-responsive" refers to the prior treatment did not ameliorate and / or improve one or more symptoms of diabetic retinopathy. In some embodiments, the previous treatment may not have resulted in an improvement in vision or retinal vascular health. In some embodiments, the previous treatment may have shown some results but may not achieve the desired performance or may have ceased to be effective after a period of time. In some embodiments, the subject may have partially responded to a previous treatment for diabetic retinopathy, i.e., one or more symptoms of diabetic retinopathy were not fully remitted and / or the effect of the previous treatment was not fully sustained.

[0406] Existing treatments for diabetic retinopathy include anti-VEGF therapy, steroids, laser surgery, and vitrectomy.

[0407] diabetic macular edema Diabetic maculopathy with diabetic macular edema, such as diabetic macular edema (DME), is considered one of the important retinal diseases in patients with diabetes. Macular edema, caused by rupture of the retinal blood-retinal barrier in retinal vascular endothelial cells or retinal pigment epithelial cells, accounts for approximately 90% of maculopathy cases and is the main cause of vision loss in maculopathy. This vision loss does not lead to blindness, but it causes an extreme decrease in vision called social blindness, making daily life difficult.

[0408] In some embodiments, the present disclosure provides methods and compositions for treating diabetic macular edema. Diabetic macular edema refers to a medical condition in which diabetes causes damage to the retina. Chronic hyperglycemia from diabetes is associated with damage to the microvessels in the retina, causing diabetic macular edema. Diabetic macular edema can cause fluid leakage or bleeding from blood vessels in the retina, distorting vision. In the most advanced stages, new abnormal blood vessels grow on the surface of the retina, which can cause retinal scarring and cell loss.

[0409] In further embodiments, the present disclosure provides compositions and methods for treating diabetic macular edema (DME). In one embodiment, the present disclosure provides a method for treating diabetic retinopathy in a subject in need of treating diabetic macular edema (DME), comprising: (a) administering to the subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, where the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, where the subject has previously been administered a HIF1-α pathway inhibitor, where the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

[0410] In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and the subject has previously been administered a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a PFKFB3 inhibitor and the subject has previously been administered a HIF1-α pathway inhibitor.

[0411] In some embodiments, the subject is at risk of suffering from DME. In some embodiments, the methods provided herein (e.g., any of (a)-(c) above) are performed as a preventative treatment for DME. In some embodiments, the subject suffers from diabetes. In some embodiments, the subject suffers from diabetic retinopathy.

[0412] In some embodiments, the provided methods and compositions prevent diabetic macular edema in a subject at risk of developing diabetic macular edema, e.g., a subject having one or more risk factors associated with the development of diabetic macular edema. In some embodiments, the subject has one or more risk factors selected from hyperglycemia, hypertension, hyperlipidemia, renal disease, and progressive diabetes (e.g., as indicated by the use of insulin and oral antidiabetic medications versus the use of pills alone, or the use of pills alone versus no treatment).

[0413] In some embodiments, the disclosure provides methods and compositions for preventing, inhibiting, or delaying the onset of diabetic macular edema by administration to a diabetic subject prior to the onset of diabetic macular edema, e.g., prior to the onset of one or more symptoms of diabetic macular edema.

[0414] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered prior to the onset of one or more symptoms of DME. In some embodiments, the provided compositions and methods can reduce the incidence, severity, or level of floating spots, retinal hemorrhage, vision loss, and / or blurred vision. In some embodiments, one or more vision parameters, including but not limited to, overall vision, peripheral vision, night vision, color vision, distance vision, near vision, and clarity of vision, can be improved by the provided methods.

[0415] In some embodiments, treating DME in accordance with the methods provided herein includes delaying the onset of one or more symptoms of DME.

[0416] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of DME. In some embodiments, the present disclosure provides methods and compositions for treating focal macular edema.

[0417] In some embodiments, the present disclosure provides methods and compositions for treating diffuse macular edema.

[0418] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the methods provided herein is an antibody or antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH AG-1, AG-2, AG-3, AG-4 or AG-5, or VHH212), a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0419] In some embodiments, the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1.3, or a salt thereof.

[0420] In some embodiments, the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothiocyanate, or BAY-87-2243, or a salt thereof.

[0421] In some embodiments, the HIF1-α pathway inhibitor administered according to the methods provided herein is a HIF1-α inhibitor. In some embodiments, the HIF1-α inhibitor does not inhibit the PI3K / AKT / mTOR pathway. In some embodiments, the HIF1-α inhibitor is an antibody or an antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a MiRNA, a dsRNA, a ssRNA, and a shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0422] In some embodiments, the HIF1-α inhibitor administered is the antisense oligonucleotide EZN-2968, nanobodies AG-1 to AG-5, or nanobody AHPC.

[0423] In some embodiments, the PFKFB3 inhibitor administered according to the methods provided herein is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0424] In some embodiments, the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0425] In some embodiments, the PFKFB3 inhibitor administered is KAN0436151 or KAN0436067, or a salt thereof.

[0426] In some embodiments, the PFKFB3 inhibitor administered is AZ67, or a salt thereof.

[0427] In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1C or Figure 1D. In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E.

[0428] In some embodiments, the methods provided herein for treating DME are carried out by co-administering to a subject a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0429] In some embodiments, the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intraocular administration. In some embodiments, the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intraocular administration. In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0430] In some embodiments, the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intravitreal administration. In some embodiments, the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intravitreal administration.

[0431] In some embodiments, treating DME according to the methods provided herein includes reducing one or more symptoms of DME in a subject compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the one or more symptoms of DME are selected from retinal inflammation, acellular capillary formation, retinal neovascularization, retinal endothelial cell death, retinal vascular permeability, retinal ischemia-reperfusion injury, retinal leakage area, and occludin disruption. In some embodiments, the one or more symptoms of DME are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0432] In some embodiments, the compositions and methods provided prevent or reduce the incidence, prevalence or severity of damaged and / or leaky blood vessels in the eye.

[0433] Treatment and / or prevention of diabetic macular edema can be measured by various means. In some embodiments, treatment or prevention includes treating one or more of apoptosis, inflammation, acellular capillary formation, angiogenesis, retinal endothelial cell death, retinal vascular permeability, ischemia-reperfusion injury, and occludin disruption in diabetic macular edema in a subject in need of treatment or prevention.

[0434] In some embodiments, treating DME according to the methods provided herein includes increasing one or more visual acuity parameters in a subject compared to the visual acuity parameters of a control subject or compared to the visual acuity parameters of the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the one or more increased visual acuity parameters are selected from blurred vision, visual distortion, and small spots in the visual field (sometimes referred to as "floaters").

[0435] The present disclosure provides methods of treating diabetic macular edema using anti-HIF1-α and / or anti-PFKFB3 antibodies or antigen-binding fragments thereof. The efficacy of such treatments can be characterized, assessed, measured and / or monitored based on several parameters.

[0436] In some embodiments, the provided methods result in a reduction in apoptosis and / or endothelial cell death in the eye. Cell death can be monitored according to known methods. Exemplary methods for detecting cell death include, but are not limited to, nuclear staining techniques such as propidium iodide, Hoechst-33342, 4',6-diamidino-2-phenylindole (DAPI), and acridine orange-ethidium bromide staining. Non-nuclear staining techniques include, but are not limited to, Annexin V staining.

[0437] In some embodiments, the methods provided prevent ischemia-reperfusion (IR) injury. Methods for detecting IR injury are known in the art and include fluorescein analysis, the fluorescent zinc 2,2'-dipicolylamine coordination compound PSVue.RTM.794, 99mTc glucarate, and electroretinography.

[0438] In some embodiments, the methods provided reduce the levels of pro-inflammatory cytokines, such as TNFα, IL-1β, IL-6, or MCP1. Cytokine levels can be monitored via enzyme-linked immunosorbent assay (ELISA), Luminex, cytokine bead array, Proteo Plex, FAST Quant, and the like.

[0439] In some embodiments, the methods provided reduce retinal vascular permeability, retinal neovascularization, or other symptoms of retinal health. In some embodiments, the methods provided can prevent typical symptoms of diabetic macular edema in the retinal vasculature or prevent further deterioration. Vascular permeability and other measures of retinal vascular health can be measured, for example, by fluorescein angiography.

[0440] In some embodiments, the provided methods improve one or more visual acuity parameters or prevent the decline of one or more visual acuity parameters. Visual acuity parameters include blurred vision, visual distortion, and speckles in the field of vision. In some embodiments, subjects treated according to the provided methods may experience one or more of the following effects: improved visual acuity, reduced vision loss, improved clarity, and reduced speckles in the field of vision. Many of these parameters may be monitored by regular eye examinations.

[0441] In some embodiments, provided methods prevent or reduce macular edema, which may be observed under slit-lamp biomicroscopy as a swelling and blurring of the retinal layers.

[0442] In some embodiments, the provided method delays the onset of diabetic macular edema.Thus, the provided method delays the average onset of diabetic macular edema from the first diagnosis of diabetes to more than 5 years, more than 10 years, more than 11 years, more than 12 years, more than 13 years, more than 14 years, more than 15 years, more than 16 years, more than 17 years, more than 18 years, more than 19 years, or more than 20 years.

[0443] In some embodiments, provided methods reduce, alleviate, reduce the severity, or reverse one or more symptoms of diabetic macular edema. In some embodiments, methods of treating diabetic macular edema with an anti-HIF1-α antibody and / or an anti-PFKFB3 antibody or antigen-binding fragment thereof may reduce, alleviate, reduce the severity, or reverse one or more of the following symptoms: microaneurysms, hemorrhage, intraretinal capillary abnormalities, formation of new blood vessels (neovascularization), vision loss, macular edema, intraretinal capillary abnormalities.

[0444] In some embodiments, the methods of treating diabetic macular edema provided affect one or more parameters of the retinal vasculature, including, but not limited to, permeability, NFkB levels, inflammatory marker levels, incidence of apoptosis, sphingolipid metabolism, and re-endothelialization. In some embodiments, the methods of treating diabetic macular edema disclosed herein reduce retinal vascular permeability. Retinal vascular permeability may be monitored via fluorescence, tracer dyes, optical testing, and the like. In some embodiments, the methods of treating diabetic macular edema disclosed herein reduce NFkB and / or other inflammatory marker levels in the retinal vasculature. As described above, inflammatory cytokine levels can be measured via conventional means (e.g., ELISA). In some embodiments, the methods of treating diabetic macular edema disclosed herein reduce the incidence of apoptosis in the retinal vasculature. As disclosed above, apoptosis can be measured using conventional means, such as nuclear and non-nuclear staining techniques.

[0445] In further embodiments, the provided methods further comprise administering an anti-VEGF therapeutic to the subject. In some embodiments, the anti-VEGF therapeutic is bevacizumab, ranibizumab, or aflibercept. In one embodiment, the anti-VEGF therapeutic is bevacizumab. In one embodiment, the anti-VEGF therapeutic is ranibizumab. In one embodiment, the anti-VEGF therapeutic is aflibercept.

[0446] In some embodiments, the subject receiving the treatment provided herein has undergone a prior treatment for DME. In some embodiments, the prior treatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy. In certain embodiments, the prior treatment is an anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the prior anti-VEGF therapy included bevacizumab, ranibizumab, or aflibercept. In one embodiment, the prior anti-VEGF therapy included bevacizumab. In one embodiment, the prior anti-VEGF therapy included ranibizumab. In one embodiment, the prior anti-VEGF therapy included aflibercept. In some embodiments, the subject did not respond to a prior treatment for DME. In such embodiments, "non-responsive" refers to the prior treatment did not ameliorate and / or improve one or more symptoms of diabetic macular edema. In such embodiments, "non-responsive" refers to the prior treatment did not ameliorate and / or improve one or more symptoms of diabetic macular edema. In some embodiments, the previous treatment may not have caused an improvement in vision or retinal vascular health. In some embodiments, the previous treatment may show some results but may not achieve the desired performance or may cease to show efficacy after a few hours. In some embodiments, the subject may have partially responded to the previous treatment for diabetic macular edema, i.e., one or more symptoms of diabetic macular edema have not been fully alleviated and / or the effect of the previous treatment has not been fully sustained.

[0447] Existing treatments for diabetic macular edema include anti-VEGF therapy and laser surgery.

[0448] Age-related macular degeneration In some embodiments, the present disclosure provides methods and compositions for treating age-related macular degeneration.

[0449] The central part of the retina, called the macula, is responsible for the vision needed for reading and other fine tasks. Damage to the macula leads to loss of vision. The most common disease process affecting the macula is AMD. In patients with AMD, the retinal photoreceptor and pigment epithelial cells in the macula die over the course of several years. The cell death and gradual loss of vision does not usually begin until the patient is over 60 years of age, hence the name age-related macular degeneration.

[0450] There are two types of AMD: dry macular degeneration and wet macular degeneration. Dry macular degeneration is more common, but is usually less severe and causes more gradual vision loss. Patients with wet macular degeneration develop new blood vessels under the retina. Patients with wet macular degeneration develop new blood vessels under the retina. Due to the slow degeneration of photoreceptors and RPE cells, blood vessels tend to grow from their normal location in the choroid to an abnormal location under the retina. This abnormal new blood vessel growth is called choroidal neovascularization (CNV). The abnormal blood vessels leak and bleed, causing bleeding, swelling, scar tissue, and severe loss of central vision. Although only 10% of patients with AMD have the wet form, it accounts for 90% of all blindness caused by AMD.

[0451] In some embodiments, the present disclosure provides methods and compositions for treating age-related macular degeneration (AMD). In some embodiments, the present disclosure provides compositions and methods for treating wet AMD (wAMD). In some embodiments, the present disclosure provides compositions and methods for treating dry AMD (dAMD). In one embodiment, the present disclosure provides a method for treating AMD (e.g., wAMD or dAMD) in a subject in need of such treatment, comprising: (a) administering to the subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, the subject having previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, the subject having previously been administered a HIF1-α pathway inhibitor, the subject having previously been administered a HIF1-α pathway inhibitor, the PFKFB3 inhibitor not inhibiting PI3K / AKT / mTOR pathway or HIF1-α.

[0452] In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and the subject has previously been administered a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a PFKFB3 inhibitor and the subject has previously been administered a HIF1-α pathway inhibitor.

[0453] In some embodiments, the subject is at risk for AMD. In some embodiments, the subject is at risk for wAMD. In some embodiments, the subject is at risk for dAMD. In some embodiments, the methods provided herein (e.g., any of (a)-(c) above) are performed as a preventative treatment for AMD.

[0454] In some embodiments, the provided methods and compositions prevent age-related macular degeneration in a subject at risk of developing age-related macular degeneration, e.g., a subject having one or more risk factors associated with developing age-related macular degeneration.

[0455] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered prior to the onset of one or more symptoms of AMD. In some embodiments, the compositions and methods provided can reduce the incidence, severity, or level of choroidal neovascularization, ocular bleeding, bleeding, or loss of central vision.

[0456] In some embodiments, treating AMD (eg, wAMD) in accordance with the methods provided herein includes delaying the onset of one or more symptoms of AMD.

[0457] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of AMD. In some embodiments, the provided methods and compositions can be used to treat various stages of age-related macular degeneration.

[0458] In some embodiments, the present disclosure provides methods and compositions for treating wet AMD.

[0459] In some embodiments, the present disclosure provides methods and compositions for treating dry AMD.

[0460] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the methods provided herein is an antibody or antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0461] In some embodiments, the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1.3, or a salt thereof.

[0462] In some embodiments, the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothiocyanate, or BAY-87-2243, or a salt thereof.

[0463] In some embodiments, the HIF1-α pathway inhibitor administered according to the methods provided herein is a HIF1-α inhibitor. In some embodiments, the HIF1-α inhibitor does not inhibit the PI3K / AKT / mTOR pathway. In some embodiments, the HIF1-α inhibitor is an antibody or an antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a MiRNA, a dsRNA, a ssRNA, and a shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0464] In some embodiments, the HIF1-α inhibitor administered is the antisense oligonucleotide EZN-2968, nanobodies AG-1 to AG-5, or nanobody AHPC.

[0465] In some embodiments, the PFKFB3 inhibitor administered according to the methods provided herein is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0466] In some embodiments, the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0467] In some embodiments, the PFKFB3 inhibitor administered is KAN0436151 or KAN0436067, or a salt thereof.

[0468] In some embodiments, the PFKFB3 inhibitor administered is AZ67, or a salt thereof.

[0469] In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1C or Figure 1D. In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E.

[0470] In some embodiments, the methods provided herein for treating AMD (e.g., wAMD) are carried out by co-administering to a subject a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0471] In some embodiments, the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intraocular administration. In some embodiments, the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intraocular administration. In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0472] In some embodiments, the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intravitreal administration. In some embodiments, the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intravitreal administration.

[0473] In some embodiments, treating AMD (e.g., wAMD) according to the methods provided herein includes reducing one or more symptoms of AMD (e.g., wAMD) in a subject, compared to a control subject or compared to a subject before treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the one or more symptoms of AMD that are reduced are selected from choroidal neovascularization, and ocular swelling and / or bleeding. In some embodiments, the one or more symptoms of AMD that are reduced are ocular swelling, bleeding, and / or reduced central vision. In some embodiments, the one or more symptoms of AMD that are reduced are selected from retinal inflammation, acellular capillary formation, choroidal neovascularization, ocular endothelial cell death, ocular vascular permeability, ocular ischemia-reperfusion injury, ocular leakage area, and occludin destruction. In some embodiments, one or more symptoms of AMD are alleviated by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0474] In some embodiments, the compositions and methods provided prevent or reduce the incidence, prevalence or severity of damaged and / or leaky blood vessels in the eye.

[0475] Treatment and / or prevention of age-related macular degeneration can be measured by various means. In some embodiments, treatment or prevention includes treating one or more of apoptosis, inflammation, acellular capillary formation, angiogenesis, retinal endothelial cell death, retinal vascular permeability, ischemia-reperfusion injury, and occludin destruction in age-related macular degeneration in a subject in need of treatment or prevention.

[0476] In some embodiments, subjects treated in accordance with the methods provided herein exhibit stable or improved vision with an improvement of 3 or more lines of vision on the ETDRS chart.

[0477] In some embodiments, treating AMD (e.g., wAMD) according to the methods provided herein includes increasing one or more visual acuity parameters in a subject compared to the visual acuity parameters of a control subject or compared to the visual acuity parameters of the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the one or more increased visual acuity parameters are selected from bleeding, swelling, scar tissue, and loss of central vision.

[0478] The present disclosure provides methods of treating AMD (e.g., wAMD) using anti-HIF1-α and / or anti-PFKFB3 antibodies or antigen-binding fragments thereof. The efficacy of such treatments can be characterized, assessed, measured and / or monitored based on several parameters.

[0479] In some embodiments, the provided methods result in a reduction in apoptosis and / or endothelial cell death in the eye. Cell death can be monitored according to known methods. Exemplary methods for detecting cell death include, but are not limited to, nuclear staining techniques such as propidium iodide, Hoechst-33342, 4',6-diamidino-2-phenylindole (DAPI), and acridine orange-ethidium bromide staining. Non-nuclear staining techniques include, but are not limited to, Annexin V staining.

[0480] In some embodiments, the methods provided prevent ischemia-reperfusion (IR) injury. Methods for detecting IR injury are known in the art and include fluorescein analysis, the fluorescent zinc 2,2'-dipicolylamine coordination compound PSVue.RTM.794, 99mTc glucarate, and electroretinography.

[0481] In some embodiments, the methods provided reduce the levels of pro-inflammatory cytokines, such as TNFα, IL-1β, IL-6, or MCP1. Cytokine levels can be monitored via enzyme-linked immunosorbent assay (ELISA), Luminex, cytokine bead array, Proteo Plex, FAST Quant, and the like.

[0482] In some embodiments, the methods provided reduce ocular vascular permeability, ocular neovascularization, or other symptoms of ocular health. In some embodiments, the methods provided can prevent typical symptoms of age-related macular degeneration in the retinal vasculature or prevent further deterioration. Vascular permeability and other measures of retinal vascular health can be measured, for example, by fluorescein angiography.

[0483] In some embodiments, the provided methods improve one or more visual acuity parameters or prevent the decline of one or more visual acuity parameters. Visual acuity parameters include blurred vision, visual distortion, and speckles in the field of vision. In some embodiments, subjects treated according to the provided methods may experience one or more of the following effects: improved visual acuity, reduced vision loss, improved clarity, and reduced speckles in the field of vision. Many of these parameters may be monitored by regular eye examinations.

[0484] In some embodiments, provided methods reduce, alleviate, reduce the severity, or reverse one or more symptoms of age-related macular degeneration. In some embodiments, methods of treating age-related macular degeneration with an anti-HIF1-α antibody and / or an anti-PFKFB3 antibody or antigen-binding fragment thereof may reduce, alleviate, reduce the severity, or reverse one or more of the following symptoms: microaneurysms, hemorrhage, intraocular capillary abnormalities, formation of new blood vessels (neovascularization), central vision loss, and intraocular (e.g., choroidal) capillary abnormalities.

[0485] In some embodiments, the method of treating age-related macular degeneration provided affects one or more parameters of choroidal vasculature, including but not limited to permeability, NFkB levels, inflammatory marker levels, incidence of apoptosis, sphingolipid metabolism, and re-endothelialization. In some embodiments, the method of treating age-related macular degeneration disclosed herein reduces choroidal vascular permeability. Choroidal vascular permeability may be monitored via fluorescence, tracer dyes, optical testing, and the like. In some embodiments, the method of treating age-related macular degeneration disclosed herein reduces NFkB and / or other inflammatory marker levels in the choroidal vasculature. As described above, inflammatory cytokine levels can be measured via conventional means (e.g., ELISA). In some embodiments, the method of treating age-related macular degeneration disclosed herein reduces the incidence of apoptosis in the choroidal vasculature. As disclosed above, apoptosis can be measured using conventional means, such as nuclear and non-nuclear staining techniques.

[0486] In further embodiments, the provided methods further comprise administering an anti-VEGF therapeutic to the subject. In some embodiments, the anti-VEGF therapeutic is bevacizumab, ranibizumab, or aflibercept. In one embodiment, the anti-VEGF therapeutic is bevacizumab. In one embodiment, the anti-VEGF therapeutic is ranibizumab. In one embodiment, the anti-VEGF therapeutic is aflibercept.

[0487] In some embodiments, the subject receiving the treatment provided herein has undergone a prior treatment for AMD (e.g., wAMD). In some embodiments, the prior treatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy. In certain embodiments, the prior treatment is an anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the prior anti-VEGF therapy included bevacizumab, ranibizumab, or aflibercept. In one embodiment, the prior anti-VEGF therapy included bevacizumab. In one embodiment, the prior anti-VEGF therapy included ranibizumab. In one embodiment, the prior anti-VEGF therapy included aflibercept. In some embodiments, the subject did not respond to a prior treatment for AMD. In such embodiments, "non-responsive" refers to the prior treatment did not ameliorate and / or improve one or more symptoms of age-related macular degeneration. In such embodiments, "not responding" refers to the fact that the previous treatment did not ameliorate and / or improve one or more symptoms of age-related macular degeneration. In some embodiments, the previous treatment may not have resulted in an improvement in vision or retinal vascular health. In some embodiments, the previous treatment may show some results but may not achieve the desired performance or may have ceased to show efficacy after a period of time. In some embodiments, the subject may have partially responded to the previous treatment for age-related macular degeneration, i.e., one or more symptoms of age-related macular degeneration were not fully ameliorated and / or the effect of the previous treatment was not fully sustained.

[0488] Existing treatments for age-related macular degeneration include anti-VEGF therapies.

[0489] Choroidal neovascular membrane In some embodiments, the present disclosure provides methods and compositions for treating choroidal neovascular membrane (CNVM).

[0490] Choroidal neovascular membranes are associated with new, harmful blood vessels that grow under the retina in an area called the choroid. The blood vessels breach the barrier between the choroid and the retina. If they leak or bleed within the retina, they cause vision loss. CNVM is often associated with leaky age-related macular degeneration and is also seen in patients with diseases including histoplasmosis, ocular trauma, and myopic macular degeneration.

[0491] In some embodiments, the present disclosure provides methods and compositions for treating choroidal neovascular membrane (CNVM). In one embodiment, the present disclosure provides a method for treating CNVM in a subject in need of such treatment, comprising: (a) administering to the subject an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject an effective amount of a HIF1-α pathway inhibitor, the subject having previously been administered a PFKFB3 inhibitor; or (c) administering to the subject an effective amount of a PFKFB3 inhibitor, the subject having previously been administered a HIF1-α pathway inhibitor, the subject having previously been administered a HIF1-α pathway inhibitor, the PFKFB3 inhibitor not inhibiting PI3K / AKT / mTOR pathway or HIF1-α.

[0492] In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a HIF1-α pathway inhibitor and the subject has previously been administered a PFKFB3 inhibitor. In one embodiment, the subject is administered an effective amount of a PFKFB3 inhibitor and the subject has previously been administered a HIF1-α pathway inhibitor.

[0493] In some embodiments, the subject is at risk of suffering from CNVM. In some embodiments, the subject is at risk of suffering from CNVM. In some embodiments, the methods provided herein (e.g., any of (a)-(c) above) are performed as a prophylactic treatment of CNVM.

[0494] In some embodiments, the methods and compositions provided prevent choroidal neovascular membrane in subjects at risk of developing CNVM, e.g., subjects with one or more risk factors associated with the development of choroidal neovascular membrane. In some embodiments, the subject suffers from AMD. In some embodiments, the subject suffers from wAMD. In some embodiments, the subject suffers from ocular histoplasmosis or pathological myopia.

[0495] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered prior to the onset of one or more symptoms of CNVM. In some embodiments, the compositions and methods provided can reduce the incidence, severity, or level of choroidal neovascularization, ocular bleeding, bleeding, or loss of vision.

[0496] In some embodiments, treating CNVM in accordance with the methods provided herein includes delaying the onset of one or more symptoms of CNVM.

[0497] In some embodiments, the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered after the onset of one or more symptoms of CNVM.

[0498] In some embodiments, the HIF1-α pathway inhibitor administered in accordance with the methods provided herein is an antibody or antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α pathway binding polypeptide, or a small molecule HIF1-α pathway inhibitor.

[0499] In some embodiments, the administered HIF1-α pathway inhibitor is silibinin, PX-478 or YC-1.3, or a salt thereof.

[0500] In some embodiments, the administered HIF1-α pathway inhibitor is ganetespib (ST-9090), phenethyl isothiocyanate, or BAY-87-2243, or a salt thereof.

[0501] In some embodiments, the HIF1-α pathway inhibitor administered according to the methods provided herein is a HIF1-α inhibitor. In some embodiments, the HIF1-α inhibitor does not inhibit the PI3K / AKT / mTOR pathway. In some embodiments, the HIF1-α inhibitor is an antibody or an antigen-binding fragment thereof (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a scFv, a dAb fragment, or another engineered molecule, e.g., a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, a MiRNA, a dsRNA, a ssRNA, and a shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, or a small molecule HIF1-α inhibitor.

[0502] In some embodiments, the HIF1-α inhibitor administered is the antisense oligonucleotide EZN-2968, nanobodies AG-1 to AG-5, or nanobody AHPC.

[0503] In some embodiments, the PFKFB3 inhibitor administered according to the methods provided herein is an antibody or antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, a F(ab')2 fragment, an Fd fragment, an Fv fragment, a scFv, a dAb fragment, or another engineered molecule such as a diabody, a triabody, a tetrabody, a minibody, and a minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

[0504] In some embodiments, the administered PFKFB3 inhibitor is BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof.

[0505] In some embodiments, the PFKFB3 inhibitor administered is KAN0436151 or KAN0436067, or a salt thereof.

[0506] In some embodiments, the PFKFB3 inhibitor administered is AZ67, or a salt thereof.

[0507] In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula 1-53 or Formula 54, PQP, N4A, YN1, PK15, PFK-158, YZ29, Compound 26, KAN0436151, KAN0436067, or BrAcNHErOP, or a salt thereof, as shown in Figure 1A-1C or Figure 1D. In some embodiments, the administered PFKFB3 inhibitor has the structure of Formula AZ44-AZ70 or AZ71, or a salt thereof, as shown in Figure 1E.

[0508] In some embodiments, the methods provided herein for treating CNVM are carried out by co-administering to a subject a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0509] In some embodiments, the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intraocular administration. In some embodiments, the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intraocular administration. In some embodiments, the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

[0510] In some embodiments, the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is intravitreal administration. In some embodiments, the administration of the HIF1-α pathway inhibitor and the PFKFB3 inhibitor is intravitreal administration.

[0511] In some embodiments, treating CNVM according to the methods provided herein includes reducing one or more symptoms of CNVM in a subject, compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the one or more symptoms of CNVM that are reduced are selected from choroidal neovascularization, and ocular swelling and / or bleeding. In some embodiments, the one or more symptoms of CNVM that are reduced are ocular swelling, bleeding, and / or reduced central vision. In some embodiments, the one or more symptoms of CNVM that are reduced are selected from retinal inflammation, acellular capillary formation, choroidal neovascularization, ocular endothelial cell death, ocular vascular permeability, ocular ischemia-reperfusion injury, ocular leaking areas, and occludin disruption. In some embodiments, the one or more symptoms of CNVM are reduced by at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% compared to a control subject or compared to a subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor.

[0512] In some embodiments, the compositions and methods provided prevent or reduce the incidence, prevalence or severity of damaged and / or leaky blood vessels in the eye.

[0513] Treatment and / or prevention of choroidal neovascular membrane can be measured by various means. In some embodiments, treatment or prevention includes treating one or more of apoptosis, inflammation, acellular capillary formation, angiogenesis, retinal endothelial cell death, retinal vascular permeability, ischemia-reperfusion injury, and occludin destruction in choroidal neovascular membrane in a subject in need of treatment or prevention.

[0514] In some embodiments, subjects treated in accordance with the methods provided herein exhibit stable or improved vision with an improvement of 3 or more lines of vision on the ETDRS chart.

[0515] In some embodiments, treating CNVM according to the methods provided herein includes increasing one or more visual acuity parameters in the subject compared to the visual acuity parameters of a control subject or compared to the visual acuity parameters of the subject prior to treatment with a HIF1-α pathway inhibitor and a PFKFB3 inhibitor. In some embodiments, the one or more increased visual acuity parameters are selected from bleeding, swelling, scar tissue, and loss of central vision.

[0516] The present disclosure provides methods of treating CNVM using anti-HIF1-α and / or anti-PFKFB3 antibodies or antigen-binding fragments thereof. The efficacy of such treatments can be characterized, assessed, measured and / or monitored based on several parameters.

[0517] CNVM can be routinely diagnosed and monitored using diagnostic tests such as optical coherence tomography (OCT) or fluorescein angiography.

[0518] In some embodiments, the provided methods result in a reduction in apoptosis and / or endothelial cell death in the eye. Cell death can be monitored according to known methods. Exemplary methods for detecting cell death include, but are not limited to, nuclear staining techniques such as propidium iodide, Hoechst-33342, 4',6-diamidino-2-phenylindole (DAPI), and acridine orange-ethidium bromide staining. Non-nuclear staining techniques include, but are not limited to, Annexin V staining.

[0519] In some embodiments, the methods provided prevent ischemia-reperfusion (IR) injury. Methods for detecting IR injury are known in the art and include fluorescein analysis, the fluorescent zinc 2,2'-dipicolylamine coordination compound PSVue.RTM.794, 99mTc glucarate, and electroretinography.

[0520] In some embodiments, the methods provided reduce the levels of pro-inflammatory cytokines, such as TNFα, IL-1β, IL-6, or MCP1. Cytokine levels can be monitored via enzyme-linked immunosorbent assay (ELISA), Luminex, cytokine bead array, Proteo Plex, FAST Quant, and the like.

[0521] In some embodiments, the methods provided reduce ocular vascular permeability, ocular neovascularization, or other symptoms of ocular health. In some embodiments, the methods provided can prevent typical symptoms of choroidal neovascularization membranes in the retinal vasculature or prevent further deterioration. Vascular permeability and other measures of retinal vascular health can be measured, for example, by fluorescein angiography.

[0522] In some embodiments, the provided methods improve one or more visual acuity parameters or prevent the decline of one or more visual acuity parameters. Visual acuity parameters include blurred vision, visual distortion, and speckles in the field of vision. In some embodiments, subjects treated according to the provided methods may experience one or more of the following effects: improved visual acuity, reduced vision loss, improved clarity, and reduced speckles in the field of vision. Many of these parameters may be monitored by regular eye examinations.

[0523] In some embodiments, the methods provided reduce, alleviate, reduce the severity, or reverse one or more symptoms of choroidal neovascular membrane. In some embodiments, methods of treating choroidal neovascular membrane with an anti-HIF1-α antibody and / or an anti-PFKFB3 antibody or antigen-binding fragment thereof may reduce, alleviate, reduce the severity, or reverse one or more of the following symptoms: microaneurysms, hemorrhage, intraocular capillary abnormalities, formation of new blood vessels (neovascularization), central vision loss, and intraocular (e.g., choroidal) capillary abnormalities.

[0524] In some embodiments, the methods of treating choroidal neovascular membrane provided affect one or more parameters of choroidal vasculature, including, but not limited to, permeability, NFkB levels, inflammatory marker levels, incidence of apoptosis, sphingolipid metabolism, and re-endothelialization. In some embodiments, the methods of treating choroidal neovascular membrane disclosed herein reduce choroidal vascular permeability. Choroidal vascular permeability may be monitored via fluorescence, tracer dyes, optical testing, and the like. In some embodiments, the methods of treating choroidal neovascular membrane disclosed herein reduce NFkB and / or other inflammatory marker levels in the choroidal vasculature. As described above, inflammatory cytokine levels can be measured via conventional means (e.g., ELISA). In some embodiments, the methods of treating choroidal neovascular membrane disclosed herein reduce the incidence of apoptosis in the choroidal vasculature. As disclosed above, apoptosis can be measured using conventional means, such as nuclear and non-nuclear staining techniques.

[0525] In further embodiments, the methods provided further comprise administering an anti-VEGF therapeutic to the subject. In some embodiments, the anti-VEGF therapeutic is bevacizumab, ranibizumab, or aflibercept. In one embodiment, the anti-VEGF therapeutic is bevacizumab. In one embodiment, the anti-VEGF therapeutic is ranibizumab. In one embodiment, the anti-VEGF therapeutic is aflibercept.

[0526] In some embodiments, the subject receiving the treatment provided herein has undergone a prior treatment for CNVM. In some embodiments, the prior treatment is a therapeutic procedure selected from vitrectomy and laser surgery, or a therapeutic agent selected from steroids and anti-vascular endothelial growth factor (VEGF) therapy. In certain embodiments, the prior treatment is an anti-vascular endothelial growth factor (VEGF) therapy. In some embodiments, the prior anti-VEGF therapy included bevacizumab, ranibizumab, or aflibercept. In one embodiment, the prior anti-VEGF therapy included bevacizumab. In one embodiment, the prior anti-VEGF therapy included ranibizumab. In one embodiment, the prior anti-VEGF therapy included aflibercept. In some embodiments, the subject did not respond to a prior treatment for CNVM. In such embodiments, "not responding" refers to the prior treatment not ameliorating and / or improving one or more symptoms of choroidal neovascular membrane. In such embodiments, "not responding" refers to the fact that the previous treatment did not ameliorate and / or improve one or more symptoms of choroidal neovascular membrane. In some embodiments, the previous treatment may not have resulted in an improvement in visual acuity or retinal vascular health. In some embodiments, the previous treatment may have shown some results but may not achieve the desired performance or may have ceased to show effect after a period of time. In some embodiments, the subject may have partially responded to the previous treatment of choroidal neovascular membrane, i.e., one or more symptoms of choroidal neovascular membrane were not fully ameliorated and / or the effect of the previous treatment was not fully sustained.

[0527] Existing treatments for choroidal neovascular membranes include laser coagulation, photodynamic therapy, and anti-VEGF therapies such as bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), and pegaptanib (MACUGEN®).

[0528] The disclosures of each of U.S. Application Nos. 63 / 189,204, 63 / 189,205, 63 / 189,206, and 63 / 189,207, each filed on May 16, 2021, are hereby incorporated by reference in their entireties.

[0529] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, an admission or any form of suggestion that they constitute available prior art or form part of the common general knowledge in any country in the world.

Claims

1. 1. A composition for use in treating an ocular neovascular disease or condition in a subject in need thereof, said use comprising: (a) administering to the subject a composition comprising an effective amount of a HIF1-α pathway inhibitor and an effective amount of a PFKFB3 inhibitor; (b) administering to the subject a composition comprising an effective amount of a HIF1-α pathway inhibitor, wherein the subject has previously been administered a PFKFB3 inhibitor; or (c) administering to the subject a composition comprising an effective amount of a PFKFB3 inhibitor, wherein the subject has previously been administered a HIF1-α pathway inhibitor; The composition, wherein the PFKFB3 inhibitor does not inhibit the PI3K / AKT / mTOR pathway or HIF1-α.

2. The composition described in claim 1, wherein the composition is administered as a preventative treatment for the ocular neovascular disease or condition.

3. 2. The composition of claim 1, wherein the ocular neovascular disease or condition is diabetic retinopathy (DR), diabetic macular edema (DME), age-related macular degeneration (AMD), such as wet AMD (wAMD), or choroidal neovascularization (CNVM).

4. The administered HIF1-α pathway inhibitor may be an antibody or an antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, an F(ab') ２ The composition of claim 1, which is a polypeptide that is a polypeptide that is a polypeptide of the present invention, the polypeptide of the present invention being a polypeptide of the present invention.

5. The composition of claim 1, wherein the administered HIF1-α pathway inhibitor is silibinin, PX-478, YC-1.3, ganetespib (ST-9090), phenethyl isothiocyanate, BAY-87-2243, or a salt thereof.

6. The HIF1-α pathway inhibitor administered is a HIF1-α inhibitor, optionally comprising an antibody or an antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody (e.g., VHH), a Fab fragment, an F(ab') ２ The composition of claim 1, wherein the antibody is selected from a polypeptide, a polypeptide chain, a polypeptide fragment, an Fd fragment, an Fv fragment, an scFv, a dAb fragment, or another engineered molecule, such as diabodies, triabodies, tetrabodies, minibodies, and minimal recognition units), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, a ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a HIF1-α binding polypeptide, and a small molecule HIF1-α inhibitor.

7. The composition of claim 6, wherein the HIF1-α inhibitor administered is the antisense oligonucleotide EZN-2968, nanobody AG-1, AG-2, AG-3, AG-4, AG-5, VHH212 or AHPC.

8. The PFKFB3 inhibitor administered may be an antibody or an antigen-binding antibody fragment (e.g., a single chain antibody, a single domain antibody, a Fab fragment, an F(ab') ２ The composition of claim 1, which is a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor, which is a PFKFB3 fragment, Fd fragment, Fv fragment, scFv, dAb fragment, or another engineered molecule such as a diabody, triabody, tetrabody, minibody, and minimal recognition unit), a nucleic acid molecule (e.g., an aptamer, an antisense molecule, ribozyme, MiRNA, dsRNA, ssRNA, and shRNA), a peptibody, a nanobody, a PFKFB3 binding polypeptide, or a small molecule PFKFB3 inhibitor.

9. The PFKFB3 inhibitor administered is (a) AZ67 or a salt thereof, (b) BrAcNHEtOP (N-bromoacetylethanolamine phosphate), PFK15 (1-(4-pyridinyl)-3-(2-quinolinyl)-2-propen-1-one), or PFK-158 ((E)-1-(4-pyridinyl)-3-[7-(trifluoromethyl)-2-quinolinyl]-2-propen-1-one), or a salt thereof. 1A-1C or 1D, or a salt thereof; or (e) the structure of Formula AZ44-Formula AZ70 or Formula AZ71, or a salt thereof, as shown in FIG. 1E.

10. The composition of claim 1, wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are co-administered to the subject, optionally prior to or after onset of one or more symptoms of the ocular neovascular disease or condition.

11. 2. The composition of claim 1, wherein the administration of the HIF1-α pathway inhibitor or the PFKFB3 inhibitor is ocular administration, and optionally the ocular administration is selected from the group consisting of topical administration, intraocular administration, subconjunctival administration, intracameral administration, injection into the anterior chamber via the lateral limbus, intrastromal administration, intracorneal administration, subretinal administration, aqueous humor injection, sub-Tenon administration, administration into the suprachoroidal space (SCS), administration into the supraciliary space, and intravitreal administration.

12. The composition of claim 1, wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the non-proliferative phase of DR or during the proliferative phase of DR.

13. The composition of claim 1, wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during a non-proliferative phase of the DME or during a proliferative phase of the DME.

14. The composition of claim 1, wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the non-proliferative phase of AMD or during the proliferative phase of AMD.

15. The composition of claim 1, wherein the HIF1-α pathway inhibitor and the PFKFB3 inhibitor are administered during the non-proliferative phase of choroidal neovascularization (CNVM) or during the proliferative phase of CNVM.

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