Green Tea-Based Nanocomplexes for Eye Diseases

Abstract

The present invention relates to a composition comprising a self-assembled nanocomplex, the self-assembled nanocomplex comprising one or more active agents physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer, and the nanocomplex being at least partially encapsulated by a second water-soluble polymer. The present invention also relates to a method for preparing the composition and its use. The present invention also relates to a method for treating an ocular disease caused by angiogenesis, comprising administering the composition to a subject in need thereof, the composition comprising a self-assembled nanocomplex, the self-assembled nanocomplex comprising an ophthalmic anti-angiogenic drug physically bound to one or more conjugates, and each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer.

Description

[Technical field]

[0001] The present invention relates to compositions comprising self-assembled nanocomplexes, their preparation methods and uses, particularly in the treatment of eye diseases caused by angiogenesis. [Background technology]

[0002] Age-related macular degeneration (AMD) is the deterioration of the macula, the small central part of the retina that controls vision. AMD is the third leading cause of visual impairment worldwide after cataracts and glaucoma and the first in industrialized countries, with a blindness rate of 8.7%. There are two stages of AMD: (i) early non-neovascular AMD, which can be diagnosed by the accumulation of deteriorated tissue "drusen" seen as yellowish spots in and around the macula, and (ii) later "wet" neovascular AMD (nAMD), also known as choroidal neovascularization (CNV), which is associated with the growth of new blood vessels that originate from the choriocapillaris and grow into the retina. These blood vessels are abnormal and lead to leakage of blood and fluid that permanently damages the macula. The global prevalence of early, late, and all AMD has been found to be 8.01%, 0.37%, and 8.69%, respectively, and is on the rise as a result of the aging population. The number of people with AMD worldwide is projected to be 196 million in 2020, rising to 288 million in 2040. Although late-stage nAMD has a lower prevalence than early AMD, it commonly causes sudden, irreversible loss of central vision and accounts for most cases of severe vision loss.

[0003] nAMD treatment is based on angiogenesis (blood vessel formation) inhibition. The current standard of care for nAMD is anti-vascular endothelial growth factor (VEGF) drugs administered via intravitreal (IVT) injection. There are currently five approved anti-VEGF drugs: ranibizumab (Lucentis®, a monoclonal antibody fragment), pegatinib sodium (Macugen®, a pegylated aptamer), aflibercept (Eylea®, a recombinant protein), brolucizumab (Beovu®, a single-chain variable antibody fragment), and faricimab (Vabysmo™, a bispecific antibody targeting VEGF and angiopoietin-2), as well as one off-label drug approved for cancer treatment, bevacizumab (Avastin®, a monoclonal antibody). Current anti-VEGF injections improve vision somewhat. For example, monthly IVT injections of ranibizumab (0.5 mg) have been shown to improve vision in 33.8% of AMD patients. However, these treatments only slow the progression towards complete vision loss and require frequent repeated IVT injections throughout life. Due to the invasive route of administration, these injections pose high risks including tissue damage and infection, such as retinal detachment, intraocular inflammation, elevated intraocular pressure, hemorrhage, eye pain and traumatic cataract, necessitating secondary procedures, and potentially causing permanent vision loss. For example, 2 years of treatment with ranibizumab has been shown to increase the incidence of intraocular inflammation and elevated intraocular pressure to 18% and 24%, respectively, compared to untreated patients (8% and 7%, respectively). Furthermore, these injections require specialized medical intervention and incur high treatment costs of approximately US$2,000 / dose. This creates concerns for the quality of life and finances of patients, as well as a burden on clinicians with the increasing prevalence of nAMD as a result of an aging population. Therefore, there is an urgent need for effective and safe nAMD treatments.

[0004] To improve the side effect problem of IVT injections, considerable efforts have been put into developing nAMD treatment systems that reduce the injection frequency or use non-injection routes. For example, the port delivery system (PDS) with ranibizumab is a small, permanent, non-biodegradable intraocular implant with a refillable reservoir fixed to the sclera. A self-sealing septum in the center of the implant flange allows access to the implant reservoir for drug refills without the need to remove the implant from the eye. The PDS maintained sustained release of ranibizumab for more than 6 months (8.7, 13.0, and 15.0 months for 10, 40, and 100 mg / mL PDS, respectively) from device implantation to the first required refill. Visual acuity outcomes in the treatment group with high-dose PDS (100 mg / mL) were similar to monthly IVT injections of ranibizumab. The PDS is in phase 3 clinical trials. However, although PDS may reduce the frequency of IVT injections, device implantation in the sclera still causes side effects including intraocular inflammation and retinal detachment.

[0005] Another example of a system aimed at reducing injection frequency is a formulation (GB-102) of microparticles encapsulating sunitinib malate (SU, as pharmaceutical ingredient). SU is a multi-targeted tyrosine kinase inhibitor that inhibits several receptor tyrosine kinases, including the VEGF receptor (VEGFR), which has been shown to be involved in the development of choroidal neovascularization (CNV) in nAMD. In a phase 1 / 2a study, IVT injections of SU-loaded microparticles were well tolerated, with no dose-limiting toxicity, drug-related serious adverse events, or inflammation. A single dose of SU-loaded microparticles maintained evaluable patient visual acuity and central retinal thickness through 6 months in more than 80% of treated patients in all dose groups (SU 0.25, 0.5, 1, and 2 mg), and the total number of anti-VEGF injections was reduced. The best overall performance was observed with a dose of 1 mg SU, which was able to control the disease for 6 months in 7 of 8 patients and beyond 8 months in 4 of 8 patients. GB-102 has been in two Phase 2 clinical trials since 2019. However, despite the possibility of reducing injection frequency, concerns about the side effects of IVT injections remain and will increase as long as continuous therapy is required.

[0006] To avoid IVT injections, non-invasive systemic (oral) formulations have been developed. One example is borolanib, an oral formulation of a SU analog and tyrosine kinase inhibitor. In a phase 1 clinical trial, orally administered borolanib has been shown to improve visual acuity in 24 of 25 AMD patients who completed 24 weeks of treatment. However, 17% of participants had to discontinue treatment due to severe systemic adverse effects caused by the high oral dosage. Subsequently, a phase 2 study completed in 2018 showed disappointing results in which oral borolanib treatment did not improve visual acuity compared to placebo at 52 weeks.

[0007] Oral administration has been recognized as one of the most attractive systemic drug delivery routes due to its flexibility of dosage formulation and high patient compliance associated with ease of administration. However, orally administered drugs are exposed to various environmental problems in the body, including extreme pH changes, enzymatic degradation, mucus barriers, and cellular permeation through the gastrointestinal tract, which affect drug integrity and absorption and limited bioavailability. In addition, ocular delivery via systemic administration is often hindered by the blood-ocular barrier, which prevents drug penetration to the eye, resulting in drug bioavailability of less than 2%. This low bioavailability forces high doses and frequent administration to obtain therapeutic concentrations, which can result in severe systemic toxicity. Summary of the Invention [Problem to be solved by the invention]

[0008] In view of the above, there is a need for the development of compositions or methods for treating AMD that overcome or at least ameliorate one or more of the above disadvantages. [Means for solving the problem]

[0009] In one embodiment, a composition is provided comprising a self-assembled nanocomplex, the self-assembled nanocomplex comprising one or more active agents physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer, and the nanocomplex is at least partially encapsulated by a second water-soluble polymer.

[0010] Advantageously, flavonoid-based nanocomplexes (NCs) are provided in the composition and can be loaded with various active agents by taking advantage of the favorable interactions between the active agents and the flavonoid moiety of the flavonoid-polymer conjugate. Advantageously, the nanocomplexes can have a high loading amount of the active agent.

[0011] Advantageously, the second water-soluble polymer can allow the nanocomplex to have a hydrophilic surface. The presence of the second water-soluble polymer on the nanocomplex surface can promote the self-assembly of one or more active agents with one or more conjugates, resulting in a higher stability of the nanocomplex compared to a complex without a layer of the second water-soluble polymer. More advantageously, the presence of the second water-soluble polymer on the nanocomplex surface can allow proteins to be used as active agents, whereas a complex without the second water-soluble polymer cannot. More advantageously, the additional second water-soluble polymer on the nanocomplex can allow the control of the surface charge of the nanocomplex.

[0012] In another aspect, there is also provided a pharmaceutical composition or formulation comprising a composition as defined above.

[0013] In another embodiment, a) mixing a solution of an active agent with a solution of a flavonoid-first water-soluble polymer conjugate to form a mixture; b) adding a second water-soluble polymer to the mixture of step (a) to form a nanocomplex; c) allowing the nanocomplex of step (b) to self-assemble, the nanocomplex comprising an active agent physically bound to a flavonoid-first water-soluble polymer conjugate that is at least partially encapsulated by a second water-soluble polymer, the flavonoid-first water-soluble polymer conjugate comprising one or more flavonoid molecules and a first water-soluble polymer; wherein steps a) and b) may be carried out simultaneously or sequentially.

[0014] Advantageously, favorable interactions between the active agent and the flavonoid (such as epigallocatechin-3-O-gallate) can result in efficient self-assembly of the nanocomplex and encapsulation of the active agent by the first water-soluble polymer-flavonoid conjugate, followed by the second water-soluble polymer.

[0015] In another aspect, there is provided the use of a composition as defined above, or a pharmaceutical composition or pharmaceutical formulation as defined above, in inhibiting endothelial cell proliferation when activated by pro-angiogenic growth factors in vitro.

[0016] In another aspect, there is provided a composition as defined above, or a pharmaceutical composition or pharmaceutical formulation as defined above, for use as a medicament.

[0017] In another aspect, there is provided a method for treating an eye disease caused by angiogenesis, comprising administering to a subject in need thereof a composition as defined above, or a pharmaceutical composition or formulation as defined above.

[0018] In another aspect, there is provided a composition or pharmaceutical composition as defined above, or a pharmaceutical formulation as defined above, for use in the treatment of an eye disease caused by angiogenesis.

[0019] In another aspect, there is provided the use of a composition as defined above, or a pharmaceutical composition or pharmaceutical formulation as defined above, in the manufacture of a medicament for treating an eye disease caused by angiogenesis.

[0020] In another aspect, there is provided a method of treating an ocular disease caused by angiogenesis comprising administering a composition to a subject in need thereof, the composition comprising a self-assembled nanocomplex comprising an ophthalmic anti-angiogenic drug physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer.

[0021] In another aspect, a composition is provided comprising a self-assembled nanocomplex for use in treating an ocular disease caused by angiogenesis, the self-assembled nanocomplex comprising one or more ophthalmic anti-angiogenic drugs physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer.

[0022] In another aspect, there is provided a use of a composition in the manufacture of a medicament for treating an ocular disease caused by angiogenesis, the composition comprising a self-assembled nanocomplex comprising one or more ophthalmic anti-angiogenic drugs physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer.

[0023] In one example, green tea-based NCs are provided in a composition and can be loaded with a variety of protein-based and small molecule VEGF / VEGFR inhibitor drugs, formed by taking advantage of the favorable interaction between the drug and the (-)-epigallocatechin-3-O-gallate (EGCG) moiety of hyaluronic acid (HA)-EGCG conjugate. The drug-loaded NCs can inhibit in vitro VEGF-induced proliferation of endothelial cells under normal growth conditions and exhibit minimal cytotoxicity, which may be useful in the treatment of neovascular AMD (nAMD).

[0024] Advantageously, the improved inhibitory effect of drug-loaded NCs compared to drug alone may be due to the synergistic effect of the drug with the HA-EGCG carrier. Drug-loaded NCs may show improved and sustained antiangiogenic activity through both topical and intravitreal (IVT) administration compared to the current standard treatment, free aflibercept (AF) alone. The improved efficacy of NCs may be due to the efficient delivery of the drug to the diseased site in the posterior eye and the improved efficacy of the carrier. Advantageously, the composition defined above may be beneficial in both topical administration systems (as a single treatment or combined treatment with currently existing anti-VEGF therapies) and IVT administration systems, allowing sustained efficacy while reducing the dose of anti-VEGF / VEGFR drugs. Advantageously, this may overcome the problems of the current standard of care for nAMD, including injection-related adverse effects, poor patient compliance, burden on medical practices, and high treatment costs.

[0025] definition As used herein, the following words and terms shall have the meanings indicated.

[0026] The term "self-organization" refers to a process in which components of a system organize into regular and / or functional structures or patterns as a result of specific, local interactions between the components themselves, without external direction, and the term "self-organization" should be interpreted accordingly.

[0027] The word "substantially" does not exclude "completely", for example a composition that is "substantially free" of Y may be completely free of Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

[0028] Unless otherwise specified, the terms "comprising" and "comprise," as well as grammatical variations thereof, are intended to denote "open" or "inclusive" language such that they include the recited elements, but also permit the inclusion of additional, unrecited elements.

[0029] As used herein, the term "about," in the context of concentrations of components of a formulation, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0030] Throughout this disclosure, certain embodiments may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Thus, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., and individual numerical values ​​within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0031] Certain embodiments may be described broadly and generally herein. Each of the narrower species and subgeneric groupings contained within the generic disclosure also form part of this disclosure. This includes the general description of the embodiments with a condition or negative limitation that removes any subject matter from the genus, regardless of whether the removed material is specifically described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] Detailed Disclosure of Optional Embodiments Composition A A composition is provided comprising a self-assembled nanocomplex, the self-assembled nanocomplex comprising one or more active agents physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer, and the nanocomplex being at least partially encapsulated by a second water-soluble polymer.

[0033] Flavonoids include (-)-epicatechin, (+)-epicatechin, (-)-catechin, (+)-catechin, (-)-epicatechin gallate, (+)-epicatechin gallate, epigallocatechin, epigallocatechin gallate, fisetinidol, gallocatechin, gallocatechin gallate, mesquitol, robinetinidol, ellagitannin, gallotannin, orolong theanin, phlorotannin, tannin, theacitrin, theadibenzotropolone, theaflavin, theanaphthoquinone, thearubigins, theasinensin, It may be selected from the group consisting of quercetin, revastrol, rutin, curcumin, isorhamnetin, kaempferol, myricetin, fisetin, hesperitin, naringenin, eriodictyol, genistein, daidzein, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, daidzein, genistein, glycitein, biochanin A, formononetin, apigenin, luteolin, biocalein, chrysin, and any mixture thereof.

[0034] The flavonoid may be a catechin-based flavonoid.

[0035] Catechin-based flavonoids have the following structure:

[0036] [ka]

[0037] (In the formula, R 1 may be H or galloyl, R 2 may have the substituents H or OH.

[0038] In catechin-based flavonoids, there may be two chiral centers at carbons 2 and 3.

[0039] The active agent may be in the form of a pharma- ceutically acceptable salt or a prodrug thereof.

[0040] The active agent may be a small molecule, a protein or an oligonucleotide.

[0041] A small molecule may be a macromolecule having a molecular weight of 1000 Da or less. A small molecule may have a molecular weight in the range of 10 Da to about 1000 Da, about 10 Da to about 100 Da, or about 100 Da to about 1000 Da.

[0042] The active agent may be a therapeutic agent selected from the group consisting of chemotherapeutic agents, anti-inflammatory agents, antioxidants, ophthalmic anti-angiogenic agents, and any combination thereof.

[0043] The therapeutic agent may be a chemotherapeutic agent selected from the group consisting of alkylating agents, anthracyclines, cytoskeletal disrupting agents, epothilones, histone deacetylase inhibitors, topoisomerase I inhibitors, topoisomerase II inhibitors, kinase inhibitors, monoclonal antibodies, antibody-drug conjugates, nucleotide analogs, precursor analogs, peptide antibiotics, platinum-based agents, retinoids, vinca alkaloids, cytokines, antimetabolites, vinca alkaloid derivatives, cytotoxic agents, and any mixtures thereof.

[0044] The therapeutic agent may be an anti-inflammatory agent selected from the group consisting of aspirin, ibuprofen, naproxen, indomethacin, diclofenac, mefenamic acid, dexamethasone, triamcinolone acetonide, rapamycin, doxycycline, tetracycline, metformin, complement component inhibitors, and any mixtures thereof.

[0045] The therapeutic agent may be an antioxidant selected from the group consisting of ascorbic acid, vitamin A, vitamin E, melatonin, lipoic acid, metformin, and any mixture thereof.

[0046] The therapeutic agent may be an ophthalmic anti-angiogenic agent selected from the group consisting of tyrosine kinase inhibitors, proteins, antibodies, sunitinib, aflibercept, bevacizumab, ranibizumab, pegaptanib sodium, brolucizumab, vatalanib, pazopanib, sorafenib, faricimab, squalamine, rapamycin, complement component inhibitors, and any mixtures thereof.

[0047] The therapeutic agent may be the protein aflibercept (AF). AF is a recombinant fusion protein composed of the second and third extracellular VEGF binding domains of human VEGFR1 and VEGFR2, respectively, fused to the Fc domain of human IgG1 immunoglobulin. AF can inhibit VEGF-induced angiogenesis by binding to extracellular VEGF. It is approved as a drug for nAMD treatment via intravitreal (IVT) administration (three times in the first month, then every two months) and is the standard of care for nAMD with a market value of $7.5 billion in 2019.

[0048] The therapeutic agent can be the small molecule drug sunitinib (SU). SU is a small molecule multi-targeted tyrosine kinase inhibitor that can block VEGF signaling by binding to the intracellular ATP-binding site of VEGF. SU requires intracellular delivery to inhibit VEGFR.

[0049] The activator may be present in an amount of about 0.1 wt% to about 90 wt%, about 0.1 wt% to about 0.2 wt%, about 0.1 wt% to about 0.5 wt%, about 0.1 wt% to about 1 wt%, about 0.1 wt% to about 2 wt%, about 0.1 wt% to about 5 wt%, about 0.1 wt% to about 10 wt%, about 0.1 wt% to about 20 wt%, about 0.1 wt% to about 50 wt%, about 0.2 wt% to about 0.5 wt%, about 0 .2wt%~about 1wt%, about 0.2wt%~about 2wt%, about 0.2wt%~about 5wt%, about 0.2wt%~about 10wt%, about 0.2wt%~about 20wt%, about 0.2wt%~about 50w t%, about 0.2wt%~about 90wt%, about 0.5wt%~about 1wt%, about 0.5wt%~about 2wt%, about 0.5wt%~about 5wt%, about 0.5wt%~about 10wt%, about 0.5wt%~ About 20wt%, about 0.5wt% to about 50wt%, about 0.5wt% to about 90wt%, about 1wt% to about 2wt%, about 1wt% to about 5wt%, about 1wt% to about 10wt%, about 1wt% to about 2 0wt%, about 1wt% to about 50wt%, about 1wt% to about 90wt%, about 2wt% to about 5wt%, about 2wt% to about 10wt%, about 2wt% to about 20wt%, about 2wt% to about 50wt%, about It may be present in the composition within the range of 2 wt% to about 90 wt%, about 5 wt% to about 10 wt%, about 5 wt% to about 20 wt%, about 5 wt% to about 50 wt%, about 5 wt% to about 90 wt%, about 10 wt% to about 20 wt%, about 10 wt% to about 50 wt%, about 10 wt% to about 90 wt%, about 20 wt% to about 50 wt%, about 20 wt% to about 90 wt%, or about 50 wt% to about 90 wt%.

[0050] The active agent may be loaded (or "physically bound") through physical interactions with the flavonoid of the conjugate in the nanocomplex. The physical interactions may be non-covalent. The physical interactions may be selected from the group consisting of ionic bonds, hydrogen bonds, dipole-dipole forces, ion-dipole forces, ion-induced dipole forces, van der Waals forces, hydrophobic interactions, pi-pi interactions, and any mixture thereof.

[0051] The first water-soluble polymer can be the same as or different from the second water-soluble polymer.

[0052] The first water-soluble polymer and the second water-soluble polymer may be selected from the group consisting of glycosaminoglycans, polysaccharides, polyacrylamides, poly(N-isopropylacrylamide), poly(oxazolines), polyethyleneimines, poly(acrylic acid), polymethacrylates, poly(ethylene glycols), poly(ethylene oxides), poly(vinyl alcohols), poly(vinylpyrrolidinones), polyethers, poly(allylamine), polyanhydrides, poly(β-amino esters), poly(butylene succinates), polycaprolactones, polycarbonates, polydioxanones, poly(glycerol), polyglycolic acid, poly(3-hydroxypropionic acid), poly(2-hydroxyethyl methacrylates), poly(vinyl alcohols), poly(vinyl pyrrolidin ...vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohols), poly(vinyl alcohol In one embodiment, the polysaccharide polysaccharide may be independently selected from the group consisting of polysaccharide polysaccharides, polysaccharides having the same structure as in the present invention ...

[0053] The first and second water-soluble polymers may be independently mucoadhesive polymers. The mucoadhesive polymers may be selected from the group consisting of glycosaminoglycans, polysaccharides, poly(hydroxyethyl methyl acrylate), poly(ethylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol) or poly(acrylic acid), and any mixtures thereof, or any derivatives thereof. The mucoadhesive polymers may be selected from the group consisting of hyaluronic acid, alginate, amylose, carrageenan, cellulose, cyclodextrin, dextrin, dextran, ficoll, gelatin, gellan gum, guar gum, heparosan, keratin, pectin, polysucrose, pullulan, scleroglucan, starch, xanthan gum, xyloglucan, chitosan, and any mixtures thereof, or any derivatives thereof.

[0054] The first and second water-soluble polymers may independently be biocompatible polymers selected from the group consisting of hyaluronic acid, alginate, amylose, carrageenan, cellulose, cyclodextrin, dextrin, dextran, ficoll, gelatin, gellan gum, guar gum, heparosan, keratin, pectin, polysucrose, pullulan, scleroglucan, starch, xanthan gum, xyloglucan, poly(ethylene glycol), poly(lactide-co-glycolide), polycaprolactone, poly(vinylpyrrolidone), poly(vinyl alcohol), poly(hydroxyethyl methacrylate), chitosan, and any mixture thereof, or any derivative thereof.

[0055] The first and second water-soluble polymers may be independently biodegradable polymers selected from the group consisting of hyaluronic acid, alginate, amylose, carrageenan, cellulose, cyclodextrin, dextrin, dextran, ficoll, gelatin, gellan gum, guar gum, heparosan, keratin, pectin, polysucrose, pullulan, scleroglucan, starch, xanthan gum, xyloglucan, poly(lactide-co-glycolide), polycaprolactone, poly(vinylpyrrolidone), poly(vinyl alcohol), chitosan, and any mixtures thereof, or any derivatives thereof.

[0056] A derivative of a first water-soluble polymer or a derivative of a second water-soluble polymer may comprise a first water-soluble polymer or a second water-soluble polymer, respectively, independently modified with one or more substituents selected from the group consisting of carboxyl, thiol, sulfonyl, carboxymethyl, phosphoryl, amino, hydroxyl, and any combination thereof.

[0057] The first water-soluble polymer and the second water-soluble polymer may be non-toxic, biodegradable and / or biocompatible, rendering them safe for use in biological systems.

[0058] The first water-soluble polymer can be hyaluronic acid.

[0059] The second water-soluble polymer may be chitosan.

[0060] The second water-soluble polymer can at least partially form a shell around a core that includes one or more active agents physically bound to one or more conjugates, each conjugate including one or more flavonoid molecules and the first water-soluble polymer. That is, the nanocomplex can have a core-shell structure. The core can be partially or completely encapsulated.

[0061] The shell can be a layer of a second water-soluble polymer that at least partially encapsulates the core comprising one or more active agents physically bound to one or more conjugates.

[0062] The one or more flavonoid molecules may be covalently attached to the first water-soluble polymer.

[0063] Advantageously, one or more flavonoid molecules can be covalently linked to the first water-soluble polymer to form a conjugate, and the intermolecular interaction between the conjugate (comprising the flavonoid molecule and the first water-soluble polymer) and the active agent can facilitate drug loading to form the nanocomplex defined above. As a result, the nanocomplex may not require the use of additional linkers for flavonoid-flavonoid or flavonoid-polymer assembly, which linkers may interfere with the interaction between the active agent and the flavonoid and / or the first water-soluble polymer conjugate.

[0064] The conjugate may be referred to as a flavonoid-first water-soluble polymer conjugate or a first water-soluble polymer-flavonoid conjugate.

[0065] The first water-soluble polymer-flavonoid conjugate can be a hyaluronic acid-epigallocatechin gallate conjugate.

[0066] The flavonoid may be epigallocatechin-3-O-gallate (EGCG). EGCG may be one of the main active ingredients of green tea that has various health benefits, including inhibiting the VEGF angiogenic signaling pathway on ocular neovascularization. Advantageously, EGCG may have positive pharmacological benefits, system toxicity, short half-life, low stability and low bioavailability.

[0067] The conjugate may comprise epigallocatechin-3-O-gallate and hyaluronic acid.

[0068] The conjugate may have the formula (I), (II) or (III):

[0069] [ka] [ka]

[0070] (wherein n and m may independently be integers in the range of 1 to 30,000).

[0071] n and m are independently 1 to 100, 1 to 200, 1 to 500, 1 to 1000, 1 to 2000, 1 to 5000, 1 to 10,000, or 1 to 30,000, 100 to 200, 100 to 500, 100 to 1000, 100 to 2000, 100 to 5000, 100 to 10,000, 100 to 30,000, 200 to 500, 200 to 1000, 200 to 2000, 200 to 5000, 200 to 10,000, 200 to It may be an integer within the range of 30,000, 500 to 1000, 500 to 2000, 500 to 5000, 500 to 10,000, 500 to 30,000, 1000 to 2000, 1000 to 5000, 1000 to 10,000, 1000 to 30,000, 2000 to 5000, 2000 to 10,000, 2000 to 30,000, 5000 to 10,000, 5000 to 30,000, or 10,000 to 30,000.

[0072] The conjugate may have a molecular weight within the range of about 1 kDa to about 10,000 kDa, about 1 kDa to about 10 kDa, about 1 kDa to about 100 kDa, about 1 kDa to about 1000 kDa, about 10 kDa to about 100 kDa, about 10 kDa to about 1000 kDa, about 10 kDa to about 10,000 kDa, about 100 kDa to about 1000 kDa, about 100 kDa to about 10,000 kDa, or about 1000 kDa to about 10,000 kDa.

[0073] Advantageously, the nanocomplexes can have a hydrophilic surface, a favorable size and a favorable surface charge.

[0074] The nanocomplex may have a hydrodynamic diameter within the range of about 10 nm to about 5000 nm, about 10 nm to about 50 nm, about 10 nm to about 100 nm, about 10 nm to about 500 nm, about 10 nm to about 1000 nm, about 50 nm to about 100 nm, about 50 nm to about 500 nm, about 50 nm to about 1000 nm, about 50 nm to about 5000 nm, about 100 nm to about 500 nm, about 100 nm to about 1000 nm, about 100 nm to about 5000 nm, about 500 nm to about 1000 nm, about 500 nm to about 5000 nm, or about 1000 nm to about 5000 nm.

[0075] The nanocomplex may have a polydispersity index in the range of about 0.01 to about 0.50. The nanocomplex may have a polydispersity index between about 0.01 to about 0.50, about 0.05 to about 0.25, or about 0.09 to about 0.15.

[0076] The nanocomplex may have a surface charge in the range of about -60 mV to about 50 mV. The nanocomplex may have a surface charge of about -60 mV to about 50 mV, about -60 mV to about 30 mV, about -60 mV to about 10 mV, about -40 mV to about 50 mV, about -20 mV to about 50 mV, about -60 mV to about -10 mV, about -50 mV to about -20 mV, or about -40 mV to about -30 mV.

[0077] The nanocomplex may have a drug loading in the range of about 0.1% to about 90% by weight. The nanocomplex may have a drug loading between about 0.1% to about 90%, about 0.1% to about 70%, about 0.1% to about 50%, about 0.1% to about 30%, about 0.1% to about 10%, about 1% to about 90%, about 10% to about 90%, about 30% to about 90%, about 50% to about 90%, about 70% to about 90%, about 20% to about 70% by weight, about 20% to about 60% by weight, about 20% to about 50% by weight, about 30% to about 70% by weight, about 30% to about 60% by weight, or about 30% to about 50% by weight of the nanocomplex.

[0078] Drug loading may refer to the relative mass of active agent loaded within a nanocomplex based on the mass of the nanocomplex.

[0079] Nanocomplexes can allow for effective loading with a wide range of active agents including small molecules, proteins, oligonucleotides and any mixtures thereof.

[0080] There is also provided a pharmaceutical composition or formulation comprising the composition defined above.

[0081] A pharmaceutical composition or formulation may be a composition or formulation specifically formulated for use in the treatment of a disease or condition.

[0082] a) mixing a solution of an active agent with a solution of a flavonoid-first water-soluble polymer conjugate to form a mixture; b) adding a second water-soluble polymer to the mixture of step (a) to form a nanocomplex; c) allowing the nanocomplex of step (b) to self-assemble, the nanocomplex comprising an active agent physically bound to a flavonoid-first water-soluble polymer conjugate that is at least partially encapsulated by a second water-soluble polymer, the flavonoid-first water-soluble polymer conjugate comprising one or more flavonoid molecules and a first water-soluble polymer; Also provided is a method for producing a composition as defined above, comprising the steps a) and b), which may be carried out simultaneously or sequentially.

[0083] The nanocomplex may have a drug loading efficiency in the range of about 20% to about 100% by mass of the active ingredients mixed in step a). The nanocomplex may have a drug loading efficiency of about 20% to about 100% by mass, about 20% to about 80% by mass, about 20% to about 60% by mass, about 20% to about 40% by mass, about 40% to about 100% by mass, about 60% to about 100% by mass, about 80% to about 100% by mass, about 60% to about 99% by mass, about 70% to about 98% by mass, about 80% to about 95% by mass, or about 85% to about 95% by mass.

[0084] Drug loading efficiency may refer to the relative mass of active agent loaded into the nanocomplex based on the mass of active agent mixed in step a) for loading.

[0085] There is also provided the use of a composition as defined above, or a pharmaceutical composition or pharmaceutical formulation as defined above, in the inhibition of endothelial cell proliferation when activated in vitro by a pro-angiogenic growth factor.

[0086] The pro-angiogenic growth factor may be vascular endothelial growth factor, fibroblast growth factor, platelet-derived endothelial growth factor, angiopoietin, hepatocyte growth factor, insulin-like growth factor, interleukin, and any mixture thereof.

[0087] Advantageously, the drug-loaded nanocomplex can exhibit anti-proliferative effects under pro-angiogenic growth factor activated conditions while exhibiting minimal anti-proliferative effects under normal growth conditions. The nanocomplex exhibits a lower IC under pro-angiogenic growth factor activated conditions compared to conditions without pro-angiogenic growth factor. 50 Further advantageously, the active agent-free nanocomplex may also exhibit anti-proliferative effects under pro-angiogenic growth factor activation conditions while having minimal effects on cells under normal growth conditions.

[0088] There is also provided a composition or a pharmaceutical composition or pharmaceutical formulation as defined above for use as a medicament.

[0089] There is also provided a method for treating an eye disease caused by angiogenesis, comprising the step of administering to a subject in need thereof a composition as defined above, or a pharmaceutical composition or pharmaceutical formulation as defined above.

[0090] Advantageously, compared to a free ophthalmic antiangiogenic drug (i.e., an ophthalmic antiangiogenic drug that is not bound to any delivery vehicle and is the same ophthalmic antiangiogenic drug used in the composition), the composition can result in at least about 16-fold higher accumulation of the ophthalmic antiangiogenic drug in the retina of the eye than the accumulation of the free antiangiogenic drug. The composition can exhibit at least about 3-fold to at least about 7-fold higher accumulation of the drug along the delivery pathway to the retina, including the cornea, sclera, and vitreous humor. Even more advantageously, the composition can result in at least about 4-fold higher accumulation of the nanocomplex in the vitreous humor, suggesting enhanced delivery via the corneal pathway as well as the transscleral pathway, which is a known delivery pathway for nanoparticles, ultimately facilitating a greater amount of drug to be delivered to the retina in the posterior segment of the eye.

[0091] Advantageously, the nanocomplex in the composition can improve the bioavailability and delivery of the drug to the posterior segment of the eye via topical administration. To achieve delivery to the diseased site of the posterior segment of the eye, the drug must first be retained on the ocular surface long enough to ensure efficient penetration into the ocular tissue. The nanocomplex can increase the precorneal residence time.

[0092] Furthermore, the nanocomplex in the composition may have advantageous properties of passing through the transscleral route (conjunctiva → sclera → choroid → retina) and the corneal route (cornea → lens → vitreous humor → retina). In particular, the transscleral route may be advantageous for nanocomplexes with hydrophilic surfaces to pass through hydrophilic pores in the sclera and avoid drug clearance through conjunctival / choroidal blood vessels and lymphatic drainage, which allows for higher penetration into the choroid / retina.

[0093] Advantageously, the nanocomplexes can be used in topical eye drops for the treatment of posterior ocular diseases, which is enabled by i) enhanced accumulation in the posterior segment through mucoadhesion and high affinity of the carrier for the delivery route and pathological site, ii) high drug loading, and iii) improved efficacy due to drug-carrier synergy.

[0094] Further advantageously, the nanocomplex can have mucoadhesive properties, which can enable a longer residence time of the nanocomplex on the ocular surface compared to the free ophthalmic anti-angiogenic drug, ensuring high drug penetration into the eye.

[0095] Further advantageously, the nanocomplexes can exhibit optimal physical properties such as hydrodynamic size, surface charge, loading capacity and stability for favorable transport through the transcleral delivery route.

[0096] Advantageously, the first water-soluble polymer can be hyaluronic acid, which shows improved affinity to tissues across the delivery route and pathological area, thereby aiding in the transport of the composition to the angiogenic disease site of the retina.Furthermore, the specific binding property of hyaluronic acid to CD44, which is highly expressed in the cornea, conjunctiva, especially the retina and angiogenic vasculature, can preferentially guide the loaded drug to the angiogenic disease site of the retina via the transscleral route, due to its improved affinity to tissues across the delivery route and pathological area.

[0097] Further advantageously, the ophthalmic anti-angiogenic drug, when part of a nanocomplex as defined above, may exhibit a synergistic anti-proliferative effect compared to the combined effects of the individual components (the ophthalmic anti-angiogenic drug alone and the first water-soluble polymer and the flavonoid alone).

[0098] Further advantageously, the above defined ophthalmic anti-angiogenic drug-loaded nanocomplex may exhibit a synergistic anti-proliferative effect on VEGF-activated endothelial cells compared to the combined effect of the individual components (ophthalmic anti-angiogenic drug alone and the first water-soluble polymer-flavonoid conjugate alone).

[0099] The nanocomplex may have a combination index of less than 1, less than 0.9, less than 0.8, less than about 0.7, less than about 0.6, less than about 0.5, less than about 0.4, less than about 0.3, or less than about 0.2.

[0100] The composition may be in liquid or solution form for administration to a subject. The composition may be reconstituted from a solid formulation by using a suitable pharma- ceutically acceptable buffer, selected based on the mode of administration.

[0101] As defined herein, "compound" refers to a compound present in the composition of the present disclosure. According to the present disclosure, when used for the treatment of eye diseases caused by angiogenesis, the compound of the present disclosure may be administered alone. Alternatively, the compound may be administered as a pharmaceutical or veterinary formulation containing at least one compound according to the present disclosure. The compound may be present as a suitable salt, including a pharmaceutically acceptable salt.

[0102] Combinations of active agents, including compounds of the present disclosure, may be synergistic.

[0103] By pharma- ceutically acceptable salt is meant a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art.

[0104] For example, suitable pharma- ceutically acceptable salts of compounds according to the present disclosure may be prepared by mixing a pharma- ceutically acceptable acid, such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid, with a compound of the present disclosure.Suitable pharma- ceutically acceptable salts of compounds of the present disclosure thus include acid addition salts.

[0105] The salts can be prepared in situ during the final isolation and purification of the compounds of the present disclosure, or separately by reacting the free base function with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, Representative salts include lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine, and the like.

[0106] Dispersions of the compounds according to the present disclosure may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms.

[0107] Pharmaceutical compositions suitable for administration include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Ideally, the compositions are stable under the conditions of manufacture and storage and may contain preservatives to stabilize the composition against the contaminating action of microorganisms such as bacteria and fungi.

[0108] The term "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compound, its use in therapeutic compositions and methods of treatment and prophylaxis is contemplated. Supplementary active compounds may also be incorporated into the compositions according to the present disclosure. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The compound may be formulated for convenient and effective administration of an effective amount with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing a supplementary active ingredient, the dosage is determined by reference to the usual dose and mode of administration of said ingredient.

[0109] Also included within the scope of this disclosure are delayed release formulations.

[0110] The compounds of the present disclosure may be administered in the form of a "prodrug." A prodrug is an inactive form of a compound that is converted in vivo into an active form. Suitable prodrugs include esters, phosphonates, and the like of the active form of the compound.

[0111] In some instances, the compound may be administered topically or intravitreally. In the case of solutions, the carrier may be, for example, a solvent or dispersion medium, including water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of additives such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by including various antibacterial and / or antifungal agents. Suitable agents are well known to those skilled in the art, and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include an isotonic agent in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of an injectable composition can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0112] Sterile solutions can be prepared by incorporating the analog in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the analog into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0113] Preferably, the pharmaceutical composition may further comprise a suitable buffer to minimize acid hydrolysis. Suitable buffers are well known to those skilled in the art, including but not limited to phosphates, citrates, carbonates and mixtures thereof.

[0114] The composition may have a concentration in solution of about 0.1 mg / mL to about 100 mg / mL, about 0.1 mg / mL to about 0.2 mg / mL, about 0.1 mg / mL to about 0.3 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL, about 0.1 mg / mL to about 1 mg / mL, about 0.1 mg / mL to about 2 mg / mL, about 0.1 mg / mL to about 5 mg / mL, about 0.1 mg / mL to about 10 mg / mL, about 0.1 mg / mL to about 20 mg / mL, about 0.1 mg / mL to about 50 mg / mL, about 0.2 mg / mL to about 0.3 mg / mL, about 0.2mg / mL to about 0.5mg / mL, about 0.2mg / mL to about 1mg / mL, about 0.2mg / mL to about 2mg / mL, about 0.2mg / mL to about 5mg / mL, about 0.2mg / mL to about 10mg / mL, about 0.2mg / mL to about 20m g / mL, about 0.2 mg / mL to about 50 mg / mL, about 0.2 mg / mL to about 100 mg / mL, about 0.5 mg / mL to about 1 mg / mL, about 0.5 mg / mL to about 2 mg / mL, about 0.5 mg / mL to about 5 mg / mL, about 0.5 mg / mL to about 10 m g / mL, approximately 0.5 mg / mL to approximately 20 mg / mL, approximately 0.5 mg / mL to approximately 50 mg / mL, approximately 0.5 mg / mL to approximately 100 mg / mL, approximately 1 mg / mL to approximately 2 mg / mL, approximately 1 mg / mL to approximately 5 mg / mL, approximately 1 mg / mL to approximately 10 mg / mL , about 1 mg / mL to about 20 mg / mL, about 1 mg / mL to about 50 mg / mL, about 1 mg / mL to about 100 mg / mL, about 2 mg / mL to about 5 mg / mL, about 2 mg / mL to about 10 mg / mL, about 2 mg / mL to about 20 mg / mL, about 2 mg / mL to about The solution may be in the form of a solution within the range of 50 mg / mL, about 2 mg / mL to about 100 mg / mL, about 5 mg / mL to about 10 mg / mL, about 5 mg / mL to about 20 mg / mL, about 5 mg / mL to about 50 mg / mL, about 5 mg / mL to about 100 mg / mL, about 10 mg / mL to about 20 mg / mL, about 10 mg / mL to about 50 mg / mL, about 10 mg / mL to about 100 mg / mL, about 20 mg / mL to about 50 mg / mL, about 20 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 100 mg / mL.

[0115] The compositions may be administered once daily, twice daily, three times daily, once weekly, once every two weeks, once monthly, once every two months, once every three months, once every four months, once every five months, once every six months, once every nine months, or once every twelve months.

[0116] The composition may be administered topically to the eye of the subject.

[0117] Non-injection and non-systemic delivery of anti-VEGF / VEGFR drugs can be achieved through local administration. Advantageously, this can significantly improve treatment safety and patient and clinician convenience while avoiding side effects from intravitreal (IVT) injection and minimizing off-target toxicity. Apart from safety, local administration can have the advantage of higher patient compliance due to ease of administration and lower cost. Furthermore, it can provide treatment opportunities for patients with limited access to medical care, especially in rural and underdeveloped areas.

[0118] Advantageously, the composition defined above can be used as a single treatment without the need to administer the drug via another route of administration, such as IVT injection.

[0119] The compositions may be administered topically once daily, twice daily or three times daily in dosage volumes of 4 μL, 5 μL, 6 μL, 8 μL, 10 μL, 20 μL, 25 μL, 30 μL, 40 μL, 50 μL, 75 μL or 100 μL.

[0120] The composition, when administered topically, can provide a sustained reduction in the incidence of retinal pathology for at least 20 days, at least 25 days, at least 30 days, at least 35 days, or for the entire period that it is administered.

[0121] Advantageously, the composition, when administered topically, can exhibit at least about a 10% reduction in the incidence of retinal lesions compared to when the same ophthalmic anti-angiogenic agent is administered via intravitreal injection.

[0122] Further advantageously, the above defined compositions, when administered topically, are able to overcome problems observed with conventional topically administered drugs, such as short precorneal residence time and low penetration efficiency of the drug upon topical administration of eye drops, which prevent adequate delivery to the diseased site in the posterior segment of the eye (choroid / retina).

[0123] The composition may be administered intravitreally to the subject's eye. Intravitreal administration may be via intravitreal injection.

[0124] The composition may be administered intravitreally in a dosage of 0.5 μL, 1 μL, 2 μL, 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 40 μL, 50 μL or 100 μL once per week, once per two weeks, once per month, once per two months, once per three months, once per four months, once per five months, once per six months, once per nine months or once per twelve months.

[0125] Advantageously, the composition administered via intravitreal injection can exhibit a sustained anti-angiogenic effect compared to free anti-angiogenic drugs administered via intravitreal injection, which allows for reduced injection frequency even when lower doses of anti-angiogenic drugs are used in the medicine.

[0126] If the composition is administered topically to the subject's eye, the method may further comprise the step of intravitreally administering an angiogenic agent to the subject's eye, i.e., the compositions may be administered both topically and intravitreally to the subject's eye, either simultaneously or sequentially.

[0127] Advantageously, such combination treatment regimens can prolong the effectiveness of anti-angiogenic agents in inhibiting the growth of retinal lesions as compared to intravitreal administration of the same anti-angiogenic agent alone.

[0128] The eye disease caused by angiogenesis may be selected from the group consisting of posterior ocular disease, age-related macular degeneration, neovascular age-related macular degeneration, diabetic retinopathy, neovascular glaucoma, diabetic macular edema, retinal vascular occlusion, corneal neovascularization, and any combination thereof.

[0129] The active agent may be an ophthalmic anti-angiogenic agent. Advantageously, when the active agent is an ophthalmic anti-angiogenic agent, the ophthalmic anti-angiogenic agent, when part of the nanocomplex defined above, may exhibit a synergistic anti-proliferative effect compared to the combined effect of the individual components (ophthalmic anti-angiogenic agent alone and the first water-soluble polymer and the flavonoid alone).

[0130] Advantageously, when the active agent is an ophthalmic anti-angiogenic drug, the ophthalmic anti-angiogenic drug-loaded nanocomplex as defined above may exhibit a synergistic anti-proliferative effect on VEGF-activated endothelial cells compared to the combined effects of the individual components (ophthalmic anti-angiogenic drug alone and the first water-soluble polymer-flavonoid conjugate alone).

[0131] There is also provided a composition or pharmaceutical composition as defined above, or a pharmaceutical formulation as defined above, for use in the treatment of an ocular disease caused by angiogenesis.

[0132] There is also provided the use of a composition or pharmaceutical composition as defined above, or a pharmaceutical formulation as defined above, in the manufacture of a medicament for treating an eye disease caused by angiogenesis.

[0133] Composition B Also provided is a method of treating an ocular disease caused by angiogenesis comprising administering a composition to a subject in need thereof, the composition comprising a self-assembled nanocomplex comprising an ophthalmic anti-angiogenic drug physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer.

[0134] In the above example, the scope of "angiogenesis-induced eye disease", "ophthalmic anti-angiogenic agent", "physically attached", "conjugate", "flavonoid" and "first water-soluble polymer" are as defined above for Composition A.

[0135] In composition B, the nanocomplex may not be encapsulated by the second water-soluble polymer.

[0136] Advantageously, compared to a free ophthalmic antiangiogenic drug (i.e., an ophthalmic antiangiogenic drug that is not bound to any delivery vehicle and is the same ophthalmic antiangiogenic drug used in the composition), the composition can result in at least about 4-fold higher accumulation of the ophthalmic antiangiogenic drug in the retina of the eye than the accumulation of the free antiangiogenic drug. The composition can exhibit at least about 3-fold to at least about 9-fold higher accumulation of the drug along the delivery pathway to the retina, including the cornea, sclera, and vitreous humor. Even more advantageously, the composition can result in at least about 9-fold higher accumulation of the nanocomplex in the vitreous humor, suggesting enhanced delivery via the corneal pathway as well as the transscleral pathway, which is known as a delivery pathway for nanoparticles, which can ultimately facilitate a greater amount of drug being delivered to the retina in the posterior segment of the eye.

[0137] Advantageously, the nanocomplex in the composition can improve the bioavailability and delivery of the drug to the posterior segment of the eye via topical administration. To achieve delivery to the diseased site of the posterior segment of the eye, the drug must first be retained on the ocular surface long enough to ensure efficient penetration into the ocular tissue. The nanocomplex can increase the precorneal residence time.

[0138] Furthermore, the nanocomplex in the composition may have advantageous properties of passing through the transscleral route (conjunctiva → sclera → choroid → retina) and the corneal route (cornea → lens → vitreous humor → retina). In particular, the transscleral route may be advantageous for the nanocomplex with a hydrophilic surface to pass through hydrophilic pores in the sclera and avoid drug clearance through the conjunctival / choroidal blood vessels and lymphatic drainage, which allows for higher penetration into the choroid / retina.

[0139] Further advantageously, compared to free ocular anti-angiogenic drugs, the compositions may enable the treatment of posterior ocular diseases by i) improved accumulation in the posterior segment through mucoadhesion and high affinity of the carrier for the delivery route and pathological site, ii) high drug loading, and iii) improved efficacy due to drug-carrier synergy.

[0140] Advantageously, the nanocomplex can have mucoadhesive properties, which can enable a longer residence time of the nanocomplex on the ocular surface compared to the free ocular anti-angiogenic drug, ensuring high drug penetration into the eye.

[0141] Further advantageously, compared to free ocular anti-angiogenic drugs, nanocomplexes can exhibit optimal physical properties such as hydrodynamic size, surface charge, loading capacity and stability for favorable transport through the transcleral delivery route.

[0142] Advantageously, the first water-soluble polymer can be hyaluronic acid, which shows improved affinity to tissues across the delivery route and pathological area, thereby aiding in the transport of the composition to the angiogenic disease site of the retina.Furthermore, the specific binding property of hyaluronic acid to CD44, which is highly expressed in the cornea, conjunctiva, especially the retina and angiogenic vasculature, can preferentially guide the loaded drug to the angiogenic disease site of the retina via the transscleral route, due to its improved affinity to tissues across the delivery route and pathological area.

[0143] Further advantageously, the ophthalmic anti-angiogenic drug, when part of a nanocomplex as defined above, may exhibit a synergistic anti-proliferative effect compared to the combined effects of the individual components (the ophthalmic anti-angiogenic drug alone and the first water-soluble polymer and the flavonoid alone).

[0144] Further advantageously, the above defined ophthalmic anti-angiogenic drug-loaded nanocomplex may exhibit a synergistic anti-proliferative effect on VEGF-activated endothelial cells compared to the combined effect of the individual components (ophthalmic anti-angiogenic drug alone and the first water-soluble polymer-flavonoid conjugate alone).

[0145] Further advantageously, the ophthalmic anti-angiogenic agents, when part of the compositions disclosed herein, are capable of inhibiting endothelial cell proliferation when activated by pro-angiogenic growth factors in vitro.

[0146] Further advantageously, the ophthalmic anti-angiogenic drug, when part of a drug-loaded nanocomplex, can exhibit anti-proliferative effects under pro-angiogenic growth factor activated conditions while exhibiting minimal anti-proliferative effects under normal growth conditions. The nanocomplex exhibits a lower IC under pro-angiogenic growth factor activated conditions compared to conditions without pro-angiogenic growth factor. 50 Further advantageously, the active agent-free nanocomplex may also exhibit anti-proliferative effects under pro-angiogenic growth factor activation conditions while having minimal effects on cells under normal growth conditions.

[0147] More preferably, the ophthalmic anti-angiogenic drug-loaded nanocomplexes defined herein have a combination index of less than about 0.2 to less than about 1.

[0148] The composition may be a pharmaceutical composition or pharmaceutical formulation. The composition may be in liquid or solution form when ready to be administered to a patient. The composition may be reconstituted from a solid formulation by using a suitable pharma- ceutically acceptable buffer, selected based on the mode of administration.

[0149] As defined herein, "compound" refers to a compound present in the composition of the present disclosure. According to the present disclosure, when used for the treatment of eye diseases caused by angiogenesis, the compound of the present disclosure may be administered alone. Alternatively, the compound may be administered as a pharmaceutical or veterinary preparation containing at least one compound according to the present disclosure. The compound may be present as a suitable salt, including a pharmaceutically acceptable salt.

[0150] Combinations of active agents, including compounds of the present disclosure, may be synergistic.

[0151] By pharma- ceutically acceptable salt is meant a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art.

[0152] For example, suitable pharma- ceutically acceptable salts of compounds according to the present disclosure may be prepared by mixing a pharma- ceutically acceptable acid, such as hydrochloric acid, sulfuric acid, methanesulfonic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, phosphoric acid, acetic acid, oxalic acid, carbonic acid, tartaric acid, or citric acid, with a compound of the present disclosure.Suitable pharma- ceutically acceptable salts of compounds of the present disclosure thus include acid addition salts.

[0153] The salts can be prepared in situ during the final isolation and purification of the compounds of the present disclosure, or separately by reacting the free base function with a suitable organic acid. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethanesulfonate, Representative salts include lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, triethanolamine, and the like.

[0154] Dispersions of the compounds according to the present disclosure may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, as well as in oils. Under ordinary conditions of storage and use, pharmaceutical preparations may contain a preservative to prevent the growth of microorganisms.

[0155] Pharmaceutical compositions suitable for administration include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Ideally, the compositions are stable under the conditions of manufacture and storage and may contain preservatives to stabilize the composition against the contaminating action of microorganisms such as bacteria and fungi.

[0156] The term "pharmaceutically acceptable carrier" is intended to include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the compound, its use in therapeutic compositions and methods of treatment and prophylaxis is contemplated. Supplementary active compounds may also be incorporated into the compositions according to the present disclosure. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. The compound may be formulated for convenient and effective administration of an effective amount with a suitable pharmaceutically acceptable carrier in an acceptable dosage unit. In the case of compositions containing a supplementary active ingredient, the dosage is determined by reference to the usual dose and mode of administration of said ingredient.

[0157] Also included within the scope of this disclosure are delayed release formulations.

[0158] The compounds of the present disclosure may be administered in the form of a "prodrug." A prodrug is an inactive form of a compound that is converted in vivo into an active form. Suitable prodrugs include esters, phosphonates, and the like of the active form of the compound.

[0159] In some instances, the compound may be administered topically or intravitreally. In the case of a solution, the carrier may be, for example, a solvent or dispersion medium, including water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by including various antibacterial and / or antifungal agents. Suitable agents are well known to those skilled in the art, and include, for example, parabens, chlorobutanol, phenol, benzyl alcohol, ascorbic acid, thimerosal, and the like. In many cases, it may be preferable to include an isotonic agent in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of an injectable composition can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0160] Sterile solutions can be prepared by incorporating the analog in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the analog into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0161] Preferably, the pharmaceutical composition may further comprise a suitable buffer to minimize acid hydrolysis. Suitable buffers are well known to those skilled in the art, including but not limited to phosphates, citrates, carbonates and mixtures thereof.

[0162] The composition may have a concentration in solution of about 0.1 mg / mL to about 100 mg / mL, about 0.1 mg / mL to about 0.2 mg / mL, about 0.1 mg / mL to about 0.3 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL, about 0.1 mg / mL to about 1 mg / mL, about 0.1 mg / mL to about 2 mg / mL, about 0.1 mg / mL to about 5 mg / mL, about 0.1 mg / mL to about 10 mg / mL, about 0.1 mg / mL to about 20 mg / mL, about 0.1 mg / mL to about 50 mg / mL, about 0.2 mg / mL to about 0.3 mg / mL, about 0.2mg / mL to about 0.5mg / mL, about 0.2mg / mL to about 1mg / mL, about 0.2mg / mL to about 2mg / mL, about 0.2mg / mL to about 5mg / mL, about 0.2mg / mL to about 10mg / mL, about 0.2mg / mL to about 20m g / mL, about 0.2 mg / mL to about 50 mg / mL, about 0.2 mg / mL to about 100 mg / mL, about 0.5 mg / mL to about 1 mg / mL, about 0.5 mg / mL to about 2 mg / mL, about 0.5 mg / mL to about 5 mg / mL, about 0.5 mg / mL to about 10 m g / mL, approximately 0.5 mg / mL to approximately 20 mg / mL, approximately 0.5 mg / mL to approximately 50 mg / mL, approximately 0.5 mg / mL to approximately 100 mg / mL, approximately 1 mg / mL to approximately 2 mg / mL, approximately 1 mg / mL to approximately 5 mg / mL, approximately 1 mg / mL to approximately 10 mg / mL , about 1 mg / mL to about 20 mg / mL, about 1 mg / mL to about 50 mg / mL, about 1 mg / mL to about 100 mg / mL, about 2 mg / mL to about 5 mg / mL, about 2 mg / mL to about 10 mg / mL, about 2 mg / mL to about 20 mg / mL, about 2 mg / mL to about The solution may be in the form of a solution within the range of 50 mg / mL, about 2 mg / mL to about 100 mg / mL, about 5 mg / mL to about 10 mg / mL, about 5 mg / mL to about 20 mg / mL, about 5 mg / mL to about 50 mg / mL, about 5 mg / mL to about 100 mg / mL, about 10 mg / mL to about 20 mg / mL, about 10 mg / mL to about 50 mg / mL, about 10 mg / mL to about 100 mg / mL, about 20 mg / mL to about 50 mg / mL, about 20 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 100 mg / mL.

[0163] The composition may be administered topically to the eye of the subject.

[0164] Non-injection and non-systemic delivery of anti-VEGF / VEGFR drugs can be achieved through local administration. Advantageously, this can significantly improve treatment safety and patient and clinician convenience while avoiding side effects from intravitreal (IVT) injection and minimizing off-target toxicity. Apart from safety, local administration can have the advantage of higher patient compliance due to ease of administration and lower cost. Furthermore, it can provide treatment opportunities for patients, especially in rural and underdeveloped areas, with limited access to medical care.

[0165] Advantageously, the composition defined above can be used as a single treatment without the need to administer the drug via another route of administration, such as IVT injection.

[0166] The composition may be administered topically once daily, twice daily or three times daily in an amount of 4 μL, 5 μL, 6 μL, 8 μL, 10 μL, 20 μL, 25 μL, 30 μL, 40 μL, 50 μL, 75 μL or 100 μL.

[0167] The composition may provide a sustained reduction in the incidence of retinal pathology for at least 20 days, at least 25 days, at least 30 days, at least 35 days, or for the entire period that it is administered.

[0168] Advantageously, the composition can exhibit at least about a 10% reduction in the incidence of retinal lesions compared to anti-angiogenic drugs administered via intravitreal injection.

[0169] Further advantageously, the above defined compositions, when administered topically, are able to overcome problems observed with conventional topically administered drugs, such as short precorneal residence time and low penetration efficiency of the drug upon topical administration of eye drops, which prevent adequate delivery to the diseased site in the posterior segment of the eye (choroid / retina).

[0170] The composition may be administered intravitreally to the subject's eye. Intravitreal administration may be via intravitreal injection.

[0171] The composition may be administered intravitreally in a volume of 0.5 μL, 1 μL, 2 μL, 5 μL, 10 μL, 15 μL, 20 μL, 25 μL, 30 μL, 40 μL, 50 μL or 100 μL once per week, once per two weeks, once per month, once per two months, once per three months, once per four months, once per five months, once per six months, once per nine months or once per twelve months.

[0172] Advantageously, compositions administered via intravitreal injection can exhibit sustained anti-angiogenic effects compared to free anti-angiogenic drugs administered via intravitreal injection, allowing for reduced injection frequency and therefore greater patient compliance.

[0173] If the composition is administered topically to the subject's eye, the method may further comprise the step of intravitreally administering an angiogenic agent to the subject's eye, i.e., the compositions may be administered both topically and intravitreally to the subject's eye, either simultaneously or sequentially.

[0174] Advantageously, such combination treatment regimens can prolong the effect of anti-angiogenic drugs in inhibiting the growth of retinal lesions compared to intravitreal administration of the same anti-angiogenic drug alone, which allows for reduced injection frequency and therefore greater patient compliance.

[0175] The ophthalmic anti-angiogenic drug, when part of a nanocomplex as defined above, exhibits a synergistic anti-proliferative effect compared to the combined effects of the individual components (the ophthalmic anti-angiogenic drug alone and the first water-soluble polymer and the flavonoid alone).

[0176] Advantageously, the ophthalmic anti-angiogenic drug-loaded nanocomplex as defined above may exhibit a synergistic anti-proliferative effect on VEGF-activated endothelial cells compared to the combined effect of the individual components (ophthalmic anti-angiogenic drug alone and first water-soluble polymer-flavonoid conjugate alone).

[0177] Also provided is a composition comprising a self-assembled nanocomplex for use in treating an ocular disease caused by angiogenesis, the self-assembled nanocomplex comprising one or more ophthalmic anti-angiogenic drugs physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer.

[0178] Also provided is the use of the composition in the manufacture of a medicament for treating an ocular disease caused by angiogenesis, the composition comprising a self-assembled nanocomplex comprising one or more ophthalmic anti-angiogenic drugs physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer.

[0179] The accompanying drawings illustrate disclosed embodiments and serve to explain the principles of the disclosed embodiments, but it is to be understood that the drawings are for illustrative purposes only and are not intended as a definition of the limits of the invention. [Brief description of the drawings]

[0180] [Figure 1A] FIG. 13 is a graph showing a comparison of the hydrodynamic size of AF-NCs with and without a chitosan layer. [Figure 1B] 1 is a graph showing a comparison of the polydispersity index of AF-NC with and without a chitosan layer. [Figure 1C] 1 is a graph showing a comparison of the count rate (i.e., the number of photons detected during dynamic light scattering (DLS) measurements) of AF-NCs with and without a chitosan layer. The count rate is related to the concentration (i.e., the number of nanocomplexes) in the sample. [Diagram 2] FIG. 1 is a graph showing the hydrodynamic size of SU-NC with and without a chitosan layer during increasing dilution ratios. [Figure 3A]Figure 1 is a set of graphs showing that AF-NC inhibits VEGF-activated proliferation of endothelial cells. Graphs showing the anti-proliferative effect of AF and AF-NC on HUVECs cultured under normal or VEGF-activated growth conditions (n=5, mean±sd). *p<0.05; **p<0.01; ***p<0.005; ****p<0.001 (VEGF-activated vs. normal conditions). [Figure 3B] Figure 1 is a set of graphs showing that AF-NCs inhibit VEGF-activated proliferation of endothelial cells. Graphs showing the anti-proliferative effect of blank NCs (formed with HA-EGCG / chitosan equivalent to AF-NCs) on HUVECs cultured under normal or VEGF-activated growth conditions (n=5, mean±sd). *p<0.05; **p<0.01; ***p<0.005; ****p<0.001 (VEGF-activated vs. normal conditions). [Figure 4A] Figure 1 is a set of graphs showing that SU-NC inhibits VEGF-activated proliferation of endothelial cells. Graphs showing the anti-proliferative effect of SU and SU-NC on HUVECs cultured under normal or VEGF-activated growth conditions (n=5, mean±sd). ***p<0.005; ****p<0.001 (VEGF-activated vs. normal conditions). [Figure 4B] Figure 1 is a set of graphs showing that SU-NC inhibits VEGF-activated proliferation of endothelial cells. Figure 2 is a set of graphs showing the anti-proliferative effect of HA-EGCG on HUVECs cultured under normal or VEGF-activated growth conditions (n=5, mean±sd). ***p<0.005; ****p<0.001 (VEGF-activated vs. normal conditions). [Figure 5A] 1 is a set of graphs showing increased AF accumulation in ocular compartments. Graphs showing increased AF accumulation in the cornea following topical administration of AF and AF-NC (equivalent AF dose=4 μg) to rat eyes (n=4, mean±sem). *p<0.05; ***p<0.005; ****p<0.001. [Figure 5B]1 is a set of graphs showing increased AF accumulation in the ocular compartment. 2 is a set of graphs showing increased AF accumulation in the vitreous humor following topical administration of AF and AF-NC (equivalent AF dose=4 μg) to rat eyes (n=4, mean±sem). *p<0.05; ***p<0.005; ****p<0.001. [Figure 5C] 1 is a set of graphs showing increased AF accumulation in the ocular compartment. 2 is a set of graphs showing increased AF accumulation in the sclera after topical administration of AF and AF-NC (equivalent AF dose=4 μg) to rat eyes (n=4, mean±sem). *p<0.05; ***p<0.005; ****p<0.001. [Figure 5D] 1 is a set of graphs showing increased AF accumulation in the ocular compartment. 2 is a set of graphs showing increased AF accumulation in the retina after topical administration of AF and AF-NC (equivalent AF dose=4 μg) to rat eyes (n=4, mean±sem). *p<0.05; ***p<0.005; ****p<0.001. [Figure 6A] Figure 1 is a set of graphs showing that SU-NC results in increased SU accumulation in the ocular compartment. Graphs showing increased SU accumulation in the cornea after topical administration of SU and SU-NC (equivalent SU dose = 10 μg) to rat eyes (n = 4, mean ± sd). *p < 0.05; ***p < 0.005; ****p < 0.001 (SU-NC vs. SU). [Figure 6B] 1 is a set of graphs showing that SU-NC results in increased SU accumulation in the ocular compartment. 2 is a set of graphs showing increased SU accumulation in the vitreous humor after topical administration of SU and SU-NC (equivalent SU dose=10 μg) to rat eyes (n=4, mean±sd). *p<0.05; ***p<0.005; ****p<0.001 (SU-NC vs. SU). [Figure 6C] Figure 1 is a set of graphs showing that SU-NC results in increased SU accumulation in the ocular compartment. Graphs showing increased SU accumulation in the sclera after topical administration of SU and SU-NC (equivalent SU dose = 10 μg) to rat eyes (n = 4, mean ± sd). *p < 0.05; ***p < 0.005; ****p < 0.001 (SU-NC vs. SU). [Figure 6D] Figure 1 is a set of graphs showing that SU-NC results in increased SU accumulation in the ocular compartment. Graphs showing increased SU accumulation in the retina after topical administration of SU and SU-NC (equivalent SU dose = 10 μg) to rat eyes (n = 4, mean ± sd). *p < 0.05; ***p < 0.005; ****p < 0.001 (SU-NC vs. SU). [Figure 7A]

[0036] Figure 1 is a set of graphs showing that AF-NC inhibits retinal lesion development in Vldlr- / - mice via topical administration. Graphs showing the relative number of lesions in Vldlr- / - mice treated for 31 days with AF (2.0 μg / 0.5 μL, IVT, once on day 0), AF-NC (AF 0.2 mg / mL, topical, 10 μL, 3 times / day), or a combination of AF (2.0 μg / 0.5 μL, IVT, once on day 0) and AF-NC (AF 0.2 mg / mL, topical, 10 μL, 3 times / day) (n=4-6, mean ± sem). *p<0.05; **p<0.01; ***p<0.005; ****p<0.001. [Figure 7B] Figure 1 is a set of graphs showing that AF-NC inhibits retinal lesion development in Vldlr- / - mice via topical administration. Graphs showing relative number of lesions in Vldlr- / - mice treated with AF (2.0 μg / 0.5 μL, IVT, once on day 0), AF (0.2 mg / mL, topical, 10 μL, 3 times / day) or empty NC (equivalent HA-EGCG, topical, 10 μL, 3 times / day) for 31 days (n=4-6, mean ± sem). *p<0.05; **p<0.01; ***p<0.005; ****p<0.001. [Figure 8] Graph showing that AF-NC inhibits retinal lesion development in Vldlr− / − mice via IVT injection. Graph shows relative number of lesions in Vldlr− / − mice treated with a single IVT injection of AF (0.1 μg / 0.5 μL), AF (2.0 μg / 0.5 μL) or AF-NC (AF 0.1 μg / 0.5 μL) on day 0 (n=5-6, mean±sem). *p<0.05; ****p<0.001. [Figure 9A]A set of graphs showing that SU-NC inhibits retinal lesion development in Vldlr- / - mice via topical administration. Graphs showing the relative number of lesions in Vldlr- / - mice treated for 28 days with AF (2.0 μg / 0.5 μL, IVT, once on day 0), SU-NC (SU 0.17 mg / mL, topical, 10 μL, 3 times / day), or a combination of AF (2.0 μg / 0.5 μL, IVT, once on day 0) and SU-NC (SU 0.17 mg / mL, topical, 10 μL, 3 times / day) (n=5-6, mean ± sem). *p<0.05; ***p<0.005; ****p<0.001. [Figure 9B] A set of graphs showing that SU-NC inhibits retinal lesion development in Vldlr- / - mice via topical administration. Graphs showing relative number of lesions in Vldlr- / - mice treated with SU (0.5 mg / mL, topical, 10 μL, 3 times / day) for 28 days (n=5-6, mean ± sem). *p<0.05; ***p<0.005; ****p<0.001. [Figure 9C] A set of graphs showing that SU-NC inhibits retinal lesion development in Vldlr- / - mice via topical administration. Graphs showing relative number of lesions in Vldlr- / - mice treated with SU-NC (layered with chitosan) (SU 0.17 mg / mL, topical, 10 μL, twice daily) for 28 days (n=5-6, mean ± sem). *p<0.05; ***p<0.005; ****p<0.001. [Figure 10] Graph showing that SU-NC inhibits retinal lesion development in Vldlr− / − mice via IVT injection. The graph shows the relative number of lesions in Vldlr− / − mice treated with a single IVT injection of AF (2.0 μg / 0.5 μL) or SU-NC (SU 0.25 μg / 0.5 μL) on day 0 (n=5-6, mean±sem). *p<0.05; **p<0.01. EXAMPLES

[0181] Non-limiting examples of the invention will now be described in more detail by reference to specific examples, which should not be construed as limiting the scope of the invention in any way.

[0182] material Aflibercept (AF) used for AF-NC formulation was obtained from BOC Sciences (New York City, NY, USA). Clinical grade AF (Eylea® from Bayer, Berlin, Germany), the current standard treatment for nAMD, was used as a positive control in the in vivo study. Sunitinib malate (SU) was a product of BioVision (Milpitas, California, USA). Amicon Ultra-15 centrifugal filters were purchased from Merck Millipore Corporation (Darmstadt, Germany). All other chemicals were of analytical grade. HUVEC, EBM-2 medium and EGM-2 supplement were obtained from Lonza Bioscience Singapore Pte Ltd. (Singapore). Fetal bovine serum (FBS) was obtained from Gibco (Thermo Fisher Scientific Inc, Singapore). Chitosan was obtained from Polysciences (Warrington, Pennsylvania, USA).

[0183] method Characterization of nanocomplexes (NCs) The hydrodynamic diameter and size distribution (polydispersity index) of the NCs were assessed by dynamic light scattering (DLS) technique using a particle size analyzer (Brookhaven Instruments, USA) and the surface charge of the NCs was determined by a Zetasizer Nano ZS (Malvern, UK). Measurements were performed in triplicate at 25 °C.

[0184] The amount of SU in SU-NCs was determined by measuring the absorbance at 431 nm (UV-VIS U-2810 spectrophotometer, Hitachi, Japan) using a standard curve constructed with various concentrations of SU solutions. The amount of AF loaded in AF-NCs was determined by enzyme-linked immunosorbent assay (ELISA) according to a reported procedure with slight modifications. Briefly, 96-well Maxisorp ELISA plates (Nunc, Thermo Fisher Scientific, Waltham, Massachusetts, USA) were filled with 0.2 μg / mL recombinant human VEGF in phosphate-buffered saline (PBS). 165 (i-DNA, Singapore). The plates were blocked with PBS containing 1% bovine serum albumin (BSA) and washed with PBS containing 0.05% TWEEN® 20. Samples were then added to the plates, followed by washing with PBS containing 0.05% TWEEN® 20. Horseradish peroxidase (HRP) conjugated anti-human IgG Fc (Sigma-Aldrich, St. Louis, Missouri, USA) in PBS-BSA was then added. After washing the plates, the peroxidase activity was assayed by using SureBlue™ tetramethylbenzidine (TMB) microwell peroxidase substrate (KPL) to determine the amount of AF. The absorbance was measured at 405 nm using a microplate reader (Tecan Group Ltd., Mannedorf, Switzerland). The drug loading amount and drug loading efficiency were calculated according to the following formula:

[0185]

number

[0186] Inhibitory effect on VEGF-activated endothelial cell proliferation Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Biologics Tuas Pte Ltd (Singapore) and cultured in endothelial cell basal medium (EBM-2) supplemented with EGM-2 SingleQuots (Lonza Biologics Tuas Pte Ltd, Singapore). HUVECs were seeded (5 × 10 3 Cells were then cultured for 1 day for cell attachment (cells / well, n = 5). After starving the cells for 1 day with EBM-2 medium containing 0.1% fetal bovine serum (FBS), they were incubated with drugs, drug-loaded NCs at various drug concentrations (SU: 1.11–17.73 μM, AF: 0.001–0.206 μM), HA-EGCG or empty NCs (HA-EGCG equivalent to NCs) under two different conditions: normal medium (EBM-2 medium supplemented with 2% FBS) or VEGF-supplemented medium (0.1% FBS and 50 ng / mL recombinant human VEGF 165 The cells were treated with EBM-2 medium supplemented with 100% glycerol (i-DNA Biotechnology Pte Ltd, Singapore).

[0187] After 3 days, cell viability was measured by using AlamarBlue® reagent (Life Technologies, USA) according to the manufacturer's protocol. Briefly, the medium was replaced with phenol red-free medium containing 10% AlamarBlue® reagent. After 4 hours of incubation at 37°C, the fluorescence intensity (λ ex = 549 nm and λ em = 587 nm) was measured by a microplate reader (Tecan Group Ltd., Switzerland). The results were expressed as the percentage of viable cells relative to untreated cells.

[0188] Quantitative analysis of the combination effect of drugs and HA-EGCG on the inhibition of VEGF-activated endothelial cell proliferation of drug-loaded NCs was performed using the combination index (CI) based on the Chou-Talalay method, which has been widely used to quantify drug combinations in vitro and in vivo, where CI<1 indicates synergy, CI=1 indicates additive effect, and CI>1 indicates antagonism. The following equation was used to calculate the combination index (CI):

[0189]

number

[0190] (In the formula, (D) 薬物 and (D) HA-EGCG is the dose of drug and HA-EGCG in the drug-loaded NC to achieve x% drug activity, (D x ) 薬物 and (D x ) HA-EGCG The CI values ​​of HA-EGCG and drug combinations were calculated using the formula (where R is the dose of drug alone and HA-EGCG alone to achieve the same effect).

[0191] Ocular biodistribution To analyze the in vivo SU distribution in the eye, SU-NC or SU solution (equivalent SU dose = 10 μg) was applied topically to the eyes of Wistar Hannover rats (InVivos Pte. Ltd, Singapore). At the indicated time points, rats were euthanized by carbon dioxide inhalation. Eyes were harvested and kept at -80 °C until further processing. The amount of SU in the ocular compartment was determined by reversed-phase HPLC (Waters 2695 Separation Module (Waters, USA) equipped with a LaChrom C18-PM column (5 μm, id 4.6 × 250 mm, Hitachi, Japan)) using a standard curve constructed with various concentrations (10-250 ng / mL) of SU in each homogenate ocular compartment from untreated rats. UV absorbance was detected at 431 nm. The entire process was carried out under minimal light exposure. Homogenates were prepared by homogenizing each ocular compartment (0.1 g tissue / mL) in phosphate-buffered saline. For extraction, the homogenate was mixed with tert-butyl methyl ether (TBME) and shaken (1,300 rpm, 24 h, 30°C). After centrifugation, the TBME phase was evaporated under vacuum (45°C) and reconstituted with HPLC mobile phase (20 mM ammonium formate (pH 2):acetonitrile = 67.5:32.5 (v / v)). The injection volume and flow rate were 20 μL and 0.8 mL / min, respectively. The amount of SU was determined from the peak integration of SU using Empower 3 software (Waters, USA).

[0192] Vldlr - / - In vivo anti-angiogenic effects in mice Very low density lipoprotein receptor gene knockout (Vldlr - / - ) Mouse (B6; 129S7-Vldlr tm1HerPairs of mice (M. moniliformes, M ... One day later (P22–P29, day 0), standard treatment consisted of AF (2.0 μg / 0.5 μL, IVT, once on day 0), SU (0.5 mg / mL, topical, 10 μL, 3 times / day), SU-NC (SU 0.17 mg / mL, topical, 10 μL, 3 times / day), AF (0.2 mg / mL, topical, 10 μL, 3 times / day), AF-NC (AF 0.2 mg / mL, topical, 10 μL, 3 times / day), blank NC (equivalent HA-EGCG / chitosan topical, 10 μL, 3 times / day), AF (2.0 μg / 0.5 μL, IVT, once on day 0) and SU-NC (SU Mice were randomly assigned to receive either AF (2.0 μg / 0.5 μL, IVT, once on day 0) or AF-NC (AF 0.2 mg / mL, topical, 10 μL, 3 times per day) in combination with AF (2.0 μg / 0.5 μL, IVT, once on day 0) and AF-NC (AF 0.2 mg / mL, topical, 10 μL, 3 times per day). To study the antiangiogenic effect of NC via IVT administration, mice were randomly assigned to receive a single IVT administration of AF (2.0 μg / 0.5 μL) as standard treatment, SU-NC (SU 2.0 μg / 0.5 μL), AF-NC (AF 0.1 μg / 0.5 μL) or AF at the same concentration as AF-NC (0.1 μg / 0.5 μL) on P22–29. Antiangiogenic activity was assessed by the ability to inhibit the development of retinal vascular leakage in terms of the relative number of lesions.

[0193] The care and use of experimental animals was regulated in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Biological Resource Centre (BRC), Biopolis, Singapore).

[0194] Fluorescence angiography Mice were anesthetized with isoflurane inhalation and pupils were dilated with 1% tropicamide (Bausch + Lomb, Singapore). Eyes were imaged with an iVivo® small animal ophthalmoscope (OcuScience, USA) after intraperitoneal injection of 0.1 mL of 10% sodium fluorescein (Sigma-Aldrich) in phosphate-buffered saline.

[0195] statistical analysis All data were expressed as mean ± standard deviation (sd) unless otherwise stated. Statistical analysis was performed by one-way analysis of variance (ANOVA) using SigmaStat 3.5 software (Systat Software Inc., USA).

[0196] Example 1 synthesis Synthesis of HA-EGCC HA-EGCG (I) was synthesized according to a previously reported procedure. Briefly, HA was first modified with a thiol group at the reducing end. HA (0.5 g) and cystamine dihydrochloride (1.2 g) were dissolved in 30 mL of 0.1 M borate buffer containing 0.4 M NaCl. Sodium cyanoborohydride (628 mg) dissolved in 20 mL of 0.1 M borate buffer was added to the solution. After stirring at 37 °C for 5 days, the resulting solution was dialyzed (1000 Da Mw cutoff) under nitrogen atmosphere against 0.1 M NaCl solution for 2 days, against 25% ethanol for 1 day, and against deionized water for 2 days. The purified solution was lyophilized to obtain thiol-end-modified HA. In the second step, EGCG (440 mg) was mixed with thiol-end-modified HA (100 mg) in PBS (70 mL). The mixture was stirred at 25 °C for 4 h. The resulting solution was dialyzed (2000 Da Mw cutoff) against deionized water under nitrogen atmosphere. The purified solution was lyophilized to obtain HA-EGCG(I).

[0197] HA-EGCG (II) was synthesized by a two-step process reported previously. First, to form the ethylamine-bridged EGCG dimer, 145 μL of 2,2-diethoxyethylamine (DA) and EGCG (2.3 g) were dissolved in methanesulfonic acid (MSA):tetrahydrofuran (THF) (1:5, v / v, 5 mL). After stirring overnight, unreacted EGCG was removed by multiple extraction cycles with ethyl acetate. In the second step of conjugating the ethylamine-bridged EGCG dimer to HA, HA (0.25 g), N-hydroxysuccinimide (89 mg), ethylamine-bridged EGCG dimer (0.205 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (150 mg) were dissolved in a mixture of MES buffer (19.8 mL) and dimethylformamide (2.5 mL). The reaction mixture was incubated overnight under a nitrogen atmosphere. HA-EGCG(II) was then purified by three cycles of ethanol precipitation, and the precipitate was then redissolved in water and dialyzed (Mw cutoff of 3500 Da) against deionized water under nitrogen atmosphere for 2 days before lyophilization.

[0198] HA-EGCG(III) conjugate was synthesized by a two-step procedure reported previously. First, to synthesize thiolated HA, HA (1 g), 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.037 g) and cystamine dihydrochloride (844.5 mg) were mixed in 110 mL of PBS. The resulting mixture was dialyzed (3500 Da Mw cutoff) against 0.1 M NaCl solution for 2 days, against 25% ethanol for 1 day and against deionized water for 2 days under nitrogen atmosphere. The purified solution was lyophilized to obtain thiolated HA. In the second step, thiolated HA (0.5 g) and excess EGCG were mixed in 100 mL of PBS under nitrogen purging conditions. After stirring at 25° C. for 4 h, the mixture was dialyzed (Mw cutoff of 3500 Da) under nitrogen atmosphere against 25% ethanol for 1 day and against deionized water for 2 days.

[0199] Formation of drug-loaded nanocomplexes (NCs) To formulate AF-NC, AF (0.10-0.50 mg / mL) solution was mixed with HA-EGCG (0.10-0.75 mg / mL) solution. Chitosan solution (0.02-0.30 mg / mL) was added to the solution simultaneously or sequentially. To generate SU-NC, SU solution (0.10-0.30 mg / mL) was mixed with HA-EGCG (0.05-0.80 mg / mL). For SU-NC containing chitosan, chitosan solution (0.01-0.3 mg / mL) was added to the solution simultaneously or sequentially. The solution was filtered through an Amicon Ultra-15 centrifugal filter (100 kDa M w The SU-NCs were filtered by centrifugation (2,000 × g for 5 min at 25 °C) using a centrifuge (cut-off).

[0200] Example 2 Drug-loaded NC AF-loaded NCs (AF-NCs) were formed by exploiting the EGCG-protein binding properties. When mixed in aqueous solution, HA-EGCG self-assembled with AF through AF-EGCG interactions, followed by layering of chitosan on the surface. AF-NC formation was systematically optimized by varying the concentrations and ratios of the components in the feed. Based on particle size, surface charge, loading capacity and stability, AF-NCs were optimized for favorable transport through the transscleral delivery route (hydrodynamic size = 204 nm, polydispersity index (PDI) = 0.097) (Table 1).

[0201] [Table 1]

[0202] Chitosan incorporation gave NCs with a narrow size distribution, whereas the chitosan-free system did not (Figure 1). It is also worth mentioning that the AF-NCs had an AF loading efficiency of 94%, indicating minimal AF loss during the NC preparation.

[0203] SU-loaded NCs (SU-NCs) were formed by the self-assembly of SU and HA-EGCG through the interaction between the SU and EGCG moieties. Notably, SU-NCs could be formulated with and without chitosan. Chitosan introduction improved NC stability while retaining its size upon 1000-fold dilution, whereas the size of NCs without chitosan increased (Figure 2). SU-NC formation was systematically optimized by varying the concentration and ratio of the components in the feed. In general, increasing the concentration of HA-EGCG led to improved loading efficiency. This indicates a greater interaction of SU with EGCG at higher concentrations of HA-EGCG. As HA-EGCG / SU (w / w) increases in relation to the loading efficiency, the loading amount of SU-NCs initially increases, followed by a decrease in loading amount with further increase in HA-EGCG / SU due to the dominating effect of increasing HA-EGCG content in the NCs. The selected SU-NCs (hydrodynamic size = 142 nm, PDI = 0.13, surface charge = -38 mV) without chitosan were used for in vitro / in vivo investigations (Table 1).

[0204] The observed negative surface charge was attributed to the negatively charged HA, indicating that HA covered the NC surface. The SU loading of the selected SU-NCs (46.9%) was significantly higher than that of reported SU-loaded nanoformulations used in cancer treatment (0.8–5.1 wt%). The favorable interaction of the SU and EGCG moieties along the HA backbone was believed to play a key role in the self-assembly of SU-NCs, resulting in the efficient encapsulation of SU. Indeed, unconjugated HA did not form particles containing SU, showing a SU content of 3.7% in the mixture. This suggests that the ionic interactions between the positively charged SU and the negatively charged HA are not strong enough to form stable NCs.

[0205] Example 3 Inhibitory effect on VEGF-activated endothelial cell proliferation The inhibitory effect of drug-loaded NCs on the proliferation of human umbilical vein endothelial cells (HUVECs) was investigated under normal or VEGF-induced growth conditions while simulating the nAMD-associated endothelial microenvironment. AF-NCs exhibited specific antiproliferative effects upon VEGF-activated conditions with minimal effects (cell viability >87.9%) under normal growth conditions (Figure 3A), and showed a more robust antiproliferative effect when the VEGF signaling pathway was activated. AF-NCs suppressed proliferation of AF (IC) cells under VEGF-activated conditions. 50 > The maximum concentration tested, 0.21 μM, significantly increased the antiproliferative effect (IC 50 = 0.15 μM). Notably, empty NCs (formed with equivalent HA-EGCG and chitosan) alone were minimally cytotoxic under normal growth conditions and showed antiproliferative effects only when they were VEGF-activated (Figure 3B). The combination effect was quantified by the Chou-Talalay method using CompuSyn software. The IC of AF-NCs 50 The combination index (CI) in was 0.156, indicating strong synergy between AF and the empty carrier.

[0206] When the inhibitory effect of SU-NC on the proliferation of HUVECs under normal or VEGF-activated growth conditions was investigated, SU-NC also showed a substantially higher antiproliferative effect when it was VEGF-activated compared to normal conditions. SU-NC showed a much higher antiproliferative effect than SU under VEGF-activated conditions, with the IC 50 IC lower than (4.32 μM) 50 (1.69 μM) (Fig. 4A). Notably, HA-EGCG alone showed an antiproliferative effect when it was VEGF-activated, but not under normal growth conditions (Fig. 4B). The combined effect of SU and HA-EGCG was examined using IC 50 When quantified at 100 μg / ml, the CI value was 0.612, indicating a strong synergistic effect between SU ​​and the HA-EGCG carrier of SU-NC. In contrast, under normal growth conditions, SU-NC exhibited a CI of 0.588, indicating a strong synergistic effect between SU ​​(IC 50 = 6.59 μM) 50(8.78 μM), which indicates lower cytotoxicity compared to SU for normal growth of endothelial cells.

[0207] Example 4 Delivery to the Posterior Segment of the Eye via Topical Administration The ocular biodistribution of drug-loaded NCs via topical administration was investigated using AF-NC and SU-NC to study their delivery capability to the posterior retina (pathological site of nAMD). AF-NC showed 15.6-fold higher AF accumulation in the retina than that of free AF ​​at 1 h after administration (Figure 5). SU-NC showed 4.3-fold higher SU accumulation in the retina than that of free SU at 1 h after administration and maintained higher accumulation over the entire examination time of 4 h (Figure 6), these results indicate the superiority of NCs in delivering high doses of drugs to the retina. AF-NC and SU-NC showed higher drug accumulation along the pathway to the retina including the cornea, vitreous humor and sclera compared to the free drug. The higher distribution of NCs on the cornea compared to the free drug was believed to be a result of its increased retention time on the cornea through the mucoadhesive / CD44 binding properties of HA, which would improve the chances of transport and bioavailability in ocular tissues.

[0208] Interestingly, NCs showed higher accumulation than free drug not only in the sclera but also in the vitreous humor. This suggests improved delivery via the corneal route, rather than just the transscleral route, which is a known route for nanoparticle delivery. This would ultimately facilitate a greater amount of drug to be delivered to the retina in the posterior segment of the eye. The improved accumulation of NCs could have been due to the improved bioavailability and efficient delivery of drug to the posterior segment as a result of taking advantage of the favorably tailored NC formulation.

[0209] Example 5 In vivo anti-angiogenic effects in mice Very low density lipoprotein receptor gene knockout (Vldlr) has been shown to cause choroidal neovascularization (CNV) that recapitulates the phenomenon in nAMD. - / -We investigated the in vivo antiangiogenic activity of NCs in AF-NC mice. First, AF-NCs were examined for their antiangiogenic effect via topical administration. At the ages of postnatal days 21–28 (P21–P28), retinal vasculature was analyzed by fluorescence angiography. CNV was determined by the occurrence of retinal vascular leakage in terms of the relative number of lesions. One day later (P22–P29, day 0), mice were randomly assigned to receive standard treatment AF (2.0 μg / 0.5 μL, IVT, once on day 0), AF-NC (AF 0.2 mg / mL, topical, 3 times / day), AF (0.2 mg / mL, topical, 3 times / day), empty NC (equivalent HA-EGCG / chitosan, topical, 3 times / day), or a combination of AF (2.0 μg / 0.5 μL, IVT, once on day 0) and AF-NC (AF 0.2 mg / mL, topical, 3 times / day).

[0210] The CNV inhibitory effect of AF(IVT) was observed only in the early stages of treatment and decreased over time. This decrease in efficacy is commonly observed in patients under current anti-VEGF treatment, resulting in the need for frequent injections. In contrast, AF-NC(topical) significantly delayed retinal lesion development throughout the entire 31-day experimental period (Figure 7A), while AF(topical) failed to inhibit lesion progression at the same dose (Figure 7B). These results indicated that NC successfully delivered AF to the retina via a local route and achieved high antiangiogenic efficacy. In addition, combination with AF-NC(topical) prevented the deterioration of AF(IVT) efficacy and showed an improved and sustained antiangiogenic effect compared with AF(IVT) alone. This suggests that AF-NC(topical) in combination treatment with the current AF(IVT) treatment may prolong AF efficacy and extend the interval between IVT injections.

[0211] Notably, topically administered empty NC also significantly inhibited the progression of retinal lesions (Figure 7B). This indicates an inherent antiangiogenic effect of the HA-EGCG carrier that may contribute to the superior efficacy of AF-NC. The antiangiogenic efficacy of AF-NC was also examined via IVT administration. A single IVT administration of AF-NC at a 20-fold lower dose (AF 0.1 μg / 0.5 μL) demonstrated a much higher and sustained inhibitory effect on the progression of retinal lesions compared to AF (2.0 μg / 0.5 μL, IVT), which was shown to be effective only in the first 7 days and showed a diminishing effect over time (Figure 8). This improved and long-term antiangiogenic efficacy of AF-NC when administered via IVT injection would facilitate a reduction in injection frequency compared to current anti-VEGF therapies.

[0212] Next, SU-NC was examined for its antiangiogenic effect via topical administration. The progression of retinal lesions was significantly inhibited by topically administered SU-NC (SU 0.17 mg / mL) (Figure 9A), whereas SU(topical) showed no efficacy even at a higher dose of 0.50 mg / mL (Figure 9B). SU-NC(topical) showed sustained antiangiogenic efficacy, resulting in a significantly higher CNV inhibitory effect than AF(IVT) 28 days after the start of treatment. When administered in combination, SU-NC(topical) prevented the deterioration of AF(IVT) efficacy and showed improved sustained antiangiogenic effect compared to AF(IVT) alone. This suggests that SU-NC(topical) as a combination treatment with the current AF(IVT) treatment may prolong AF efficacy and reduce IVT injection frequency. The progression of retinal lesions was significantly inhibited by topically administered chitosan-layered SU-NCs (SU 0.17 mg / mL) (Figure 9C). When SU-NCs were administered via IVT injection, a single IVT administration of SU-NCs (SU 0.25 μg / 0.5 μL) achieved sustained antiangiogenic efficacy compared to AF (2.0 μg / 0.5 μL, IVT) (Figure 10). This suggests a high possibility of reducing the injection frequency. [Industrial Applicability]

[0213] The compositions defined above may be useful in drug delivery, in particular in the delivery of ophthalmic anti-angiogenic drugs for treating ocular diseases caused by angiogenesis, such as inflammatory eye diseases, dry eye, cataracts and eye cancer.

[0214] It will be apparent that various other modifications and adaptations of the present invention will become apparent to those skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the present invention, and that all such modifications and adaptations are intended to fall within the scope of the appended claims.

Claims

1. 1. A composition comprising a self-assembled nanocomplex, the self-assembled nanocomplex comprising one or more active agents physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer, and the nanocomplex being at least partially encapsulated by a second water-soluble polymer.

2. Flavonoids include (-)-epicatechin, (+)-epicatechin, (-)-catechin, (+)-catechin, (-)-epicatechin gallate, (+)-epicatechin gallate, epigallocatechin, epigallocatechin gallate, fisetinidol, gallocatechin, gallocatechin gallate, mesquitol, robinetinidol, ellagitannin, gallotannin, orolongtheanin, phlorotannin, tannin, theacitrin, theadibenzotropolone, theaflavin, theanaphthoquinone, thearubigins, and the 2. The composition of claim 1, wherein the hydroxybenzoate is selected from the group consisting of acinensin, quercetin, levasterol, rutin, curcumin, isorhamnetin, kaempferol, myricetin, fisetin, hesperitin, naringenin, eriodictyol, genistein, daidzein, cyanidin, delphinidin, malvidin, pelargonidin, peonidin, daidzein, genistein, glycitein, biochanin A, formononetin, apigenin, luteolin, biokalein, chrysin, and any mixture thereof.

3. 2. The composition of claim 1, wherein the flavonoid is a catechin-based flavonoid.

4. The composition of claim 1 , wherein the active agent is a small molecule, a protein, or an oligonucleotide.

5. 10. The composition of claim 1, wherein the active agent is a therapeutic agent selected from the group consisting of chemotherapeutic agents, anti-inflammatory agents, antioxidants, ophthalmic anti-angiogenic agents, and any combination thereof, or the active agent is present in the range of 0.1 wt% to 90 wt% of the total weight of the composition.

6. The composition of claim 1 , wherein the first water-soluble polymer is the same as or different from the second water-soluble polymer.

7. the first water-soluble polymer and the second water-soluble polymer are selected from the group consisting of glycosaminoglycans, polysaccharides, polyacrylamides, poly(N-isopropylacrylamide), poly(oxazolines), polyethyleneimines, poly(acrylic acid), polymethacrylates, poly(ethylene glycol), poly(ethylene oxide), poly(vinyl alcohol), poly(vinylpyrrolidinone), polyethers, poly(allylamine), polyanhydrides, poly(β-amino esters), poly(butylene succinate), polycaprolactone, polycarbonates, polydioxanone, poly(glycerol), polyglycolic acid, poly(3-hydroxypropionic acid), poly(2-hydroxyethyl methacrylate), 7. The composition of claim 6, wherein each of the polysaccharides is independently selected from the group consisting of poly(N-(2-hydroxypropyl)methacrylamide), polylactic acid, poly(lactic-co-glycolic acid), poly(orthoester), poly(2-oxazoline), poly(sebacic acid), poly(terephthalate-co-phosphate), hyaluronic acid, alginate, amylose, carrageenan, cellulose, cyclodextrin, dextrin, dextran, ficoll, gelatin, gellan gum, guar gum, heparosan, keratin, pectin, polysucrose, pullulan, scleroglucan, starch, xanthan gum, xyloglucan, chitosan, and any mixture thereof, or any derivative thereof.

8. The composition of claim 1 , wherein the one or more flavonoid molecules are covalently attached to a first water-soluble polymer.

9. The conjugate has formula (I), (II) or (III): 【Chemistry 1-1】 【Chemistry 1-2】 10. The composition of claim 1, having a structure represented by any one of the following formulas: wherein n and m are independently integers in the range of 1 to 30,000.

10. 10. The composition of claim 1, wherein the conjugate has a molecular weight in the range of 1 kDa to 10,000 kDa.

11. 10. The composition of claim 1, wherein the nanocomplex has a hydrodynamic diameter in the range of 10 nm to 5000 nm, a polydispersity index in the range of 0.01 to 0.50, a surface charge in the range of -60 mV to 50 mV, or a drug loading in the range of 0.1% to 90% by weight of the nanocomplex.

12. A pharmaceutical composition or formulation comprising a composition according to any one of claims 1 to 11.

13. a) mixing a solution of an active agent and a solution of a flavonoid-first water-soluble polymer conjugate to form a mixture; b) adding a second water-soluble polymer to the mixture of step (a) to form a nanocomplex; c) allowing the nanocomplex of step (b) to self-assemble, wherein the nanocomplex comprises an active agent physically bound to a flavonoid-first water-soluble polymer conjugate that is at least partially encapsulated by a second water-soluble polymer, the flavonoid-first water-soluble polymer conjugate comprising one or more flavonoid molecules and the first water-soluble polymer; 12. A method for producing a composition according to any one of claims 1 to 11, comprising the steps a) and b) which may be carried out simultaneously or sequentially.

14. 12. A composition according to any one of claims 1 to 11 for use in inhibiting endothelial cell proliferation when activated by pro-angiogenic growth factors in vitro.

15. 12. A composition according to any one of claims 1 to 11 for use as a medicine.

16. 12. An agent for treating an ocular disease caused by angiogenesis, comprising the composition according to any one of claims 1 to 11.

17. 17. The method of claim 16, wherein the composition is administered topically to the eye of the subject.

18. 17. The method of claim 16, wherein the composition is administered intravitreally to the subject's eye.

19. The agent of claim 17, wherein the agent is administered in combination with an angiogenic agent administered intravitreally to the subject's eye.

20. 17. The method of claim 16, wherein the ocular disease is selected from the group consisting of posterior ocular disease, age-related macular degeneration, neovascular age-related macular degeneration, diabetic retinopathy, neovascular glaucoma, diabetic macular edema, retinal vascular occlusion, and corneal neovascularization.

21. The agent according to claim 16, wherein the composition is in the form of a liquid or solution.

22. 17. The agent according to claim 16, wherein the composition is administered once a day, twice a day, three times a day, once a week, once every two weeks, once a month, once every two months, once every three months, once every four months, once every five months, once every six months, once every nine months, or once every 12 months.

23. 12. A composition according to any one of claims 1 to 11 for use in the treatment of an ocular disease caused by angiogenesis.

24. A pharmaceutical for treating an ocular disease caused by angiogenesis, comprising a composition described in any one of claims 1 to 11.

25. An agent for treating an ocular disease caused by angiogenesis, comprising a composition, the composition comprising a self-assembled nanocomplex, the self-assembled nanocomplex comprising an ophthalmic anti-angiogenic drug physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer.

26. A composition for use in treating an ocular disease caused by angiogenesis, comprising a self-assembled nanocomplex, the self-assembled nanocomplex comprising one or more ophthalmic anti-angiogenic drugs physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer.

27. ​​A pharmaceutical for treating an eye disease caused by angiogenesis, comprising a composition, the composition comprising a self-assembled nanocomplex, the self-assembled nanocomplex comprising one or more ophthalmic anti-angiogenic drugs physically bound to one or more conjugates, each conjugate comprising one or more flavonoid molecules and a first water-soluble polymer.

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